key: cord- -kxrzv n authors: kiselev, o. i. title: progress in the development of pandemic influenza vaccines and their production technologies date: - - journal: appl doi: . /s sha: doc_id: cord_uid: kxrzv n this article analyzes the current situation in the field of construction and production of pandemic influenza vaccines. the main task of protecting the population against influenza pandemics requires state-of-the-art approaches to the construction of influenza vaccines to be based on reassortment and genetic engineering techniques, including the analysis of primary structures of influenza viral genes, synthesis and cloning of the main viral genes, reverse genetics techniques, and banks of plasmids bearing basic viral genes. reassortant technologies are now giving way to new approaches for objective reasons. the state-of-the-art technologies provide safety not only at the laboratories where vaccine viruses are constructed but also make the production process wholly safe. we are using the following approaches to the development of industrial production: use of nanoparticles and nanoemulsions as functional adjuvants, construction of totally-safe strains for live attenuated influenza vaccines with deletions of molecular determinants of pathogenicity, application of protein and chemical chaperones to provide self-assembly of haemagglutinin molecules of the h n v- virus, and impregnation of whole-virion preparations with nanoparticles to enhance antigenicity. the level of protection against the avian flu epi zootic and the influenza pandemic caused by the swine lineage h n v virus can be characterized as insufficient for russia's population. the experience in preventing an outbreak of sars in the territory of russia has evidenced that organized contraepidemic measures produce a temporary, albeit appreciable, effect. the who recognizes the functional priority of epidemiological services, including quarantine mea sures, in localizing epicenters of infections and pre venting their cross border spread to other countries and continents, that is, the very principles, on which the activities of public health services in our country have always been based. however, the most successful part of contraepidemic measures in preventing the spread of infections with respiratory transmission mechanism is vaccinoprophylaxis [ , ] . supply of vaccines critically depends on the level of technologies used in the preparation of vaccine strains and mass production of vaccines. the technologies now in use in russia and abroad are those developed more than years ago. however, this sphere has made significant progress over the last years. the prepared ness activities related to the influenza pandemic and its global outbreak in substantially increased the demand for vaccines. according to who's estimate, the annual global demand accounts for to billion doses. russia's influenza vaccine production potential is higher than in other countries and sufficient for con ducting annual vaccinations in more than % of the population. at the same time, the problems associated with the supply and quality of influenza vaccines remain the most important for russia and the rest of the world and strongly depend on the promptness with which new technologies are adopted. vaccination is an important part in the complex of contraactions organized by public health services against influenza pandemics. like the soviet union used to be, the russian federation remains a leader in conducting mass prophylactic immunizations, in par ticular, antiinfluenza vaccinations. this must be marked as one of the most important achievements of the past. the country's complex of vaccine producers has demonstrated increased growth rates in recent years against the difficult situation in the pharmaceu tical industry. despite certain technological lagging, our country remains a leading producer of practically a whole spectrum of important vaccines in the world's sector of this industry. in fact, from the late s up to the late s, all contemporary influenza vaccines were pioneered in the soviet union. then basic tech nologies used to spread globally [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the initial impulse for scaling up the industrial supply came with the hong kong influenza pandemic of [ ] [ ] [ ] [ ] . the avian flu epidemic also brought a serious challenge to the world community, but that was already the period when a necessity to employ novel biotechnological achievements in influenza vaccine production was realized [ ] . medical services have acquired much experience in the application of influenza vaccines. in the russian federation, the population has already been vacci nated using seasonal influenza trivalent and monova lent vaccines against the pandemic h n v virus. in russia and globally, two basic types of vaccines are used for prophylaxis [ ] [ ] [ ] [ ] [ ] [ ] : inactivated influenza vaccines and live attenuated influenza vaccines. the main types of vaccine preparations with their properties and compositions are given in fig. (on the colored inset). we give also novel types of vaccines; their properties and technology are described below; generation iv represents the contribution made by nanotechnologies playing a principal role in revolu tionized vaccine production development. inactivated influenza vaccines contain influenza virus strains preliminarily subjected to uv and chem ical inactivation or fragmentation with subsequent iso lation of antigen containing components. the effi cacy of inactivated vaccines in preventing the disease onset accounts for - % for healthy recipients under years of age and - % for patients over . on the contrary, the efficacy of inactivated vaccines in preventing influenza related lethal outcomes accounts table ). for % even among such a risk group as patients over years of age [ , ] . by their production technologies inactivated vac cines can be subdivided into three groups: whole vir ion vaccines (generation i), split vaccines (generation ii) and subunit vaccines (generation iii) (see fig. ). whole virion vaccines consist of inactivated (chemical or ultraviolet inactivation) whole virions of influenza viruses and contain, apart from the required antigens for immunization, a considerable amount of ballast in the form of viral nucleocapsid. their advan tage is that the surface antigens in the composition of inactivated virions are in conformation close to the natural one. split vaccines are products of detergent fragmenta tion of virions' membranes with removal of all compo nents of nucleoid and contain, along with surface anti gens, internal antigens, such as protein m . at the same time, they are real fragments of highly purified membranes of viral particles (see fig. , phase ). subunit vaccines contain only surface protective antigens (haemagglutinin and neuraminidase, pre dominantly, the former) and are preparations with the highest degree of purification from influenza virus components. to the group of subunit vaccines, for example, belongs grippol, a polymer, which cannot be characterized as an ideal preparation (see below). the main requirements for the vaccines and their pro duction technologies were first specified under the who's special document [ , ] . live attenuated influenza vaccines, proposed at first by a.a. smorodintsev in , have successfully been applied in russia for decades. in the united states, a live intranasal vaccine was approved by the fda in june and permitted its use in age groups ranging from to years. repetitive passages on chicken embryos in decreased temperature conditions allowed researchers to obtain a weakened virus that does not replicate at high temperatures, characteristic of human lungs, but can propagate in the nasopharynx at a temperature of °c, causing a topical infection leading to the development of a secretory and general ized immune response [ , ] . since the laiv vacci nation resembles a naturally occurring transient infection, it has a sufficient efficacy and is character ized, according to different sources, by % serocon version in children, while, in combination with an inactivated vaccine, it elicits a positive immune response in % of the elderly [ , ] . the overwhelming majority of seasonal and pan demic viruses are products of reassortment [ , ] . first of all, this applies to the current h n v virus, the agent of the influenza pandemic. therefore, application of laiv for preventing the emergence of reassortants with novel properties is restricted on a rise reassortment is an exchange of viral genome fragments between two or more viruses. of morbidity and the more so in the outbreak of an influenza pandemic. the data on the age related lethality from the pan demic h n v influenza has shown that the morbidity and mortality from influenza related com plications in the period from march to december were significantly lower among patients born after . a relatively high level of antibodies to the swine lineage h n viruses was revealed in patients over to years of age, which defines a sufficiently high level of protection against the current pandemic h n v influenza [ , , ] . these data evi dence in favor of conducting a total vaccination in rel atively young groups of the population and children in the coming period prior to the second wave of the pan demic expected in - . table gives a group of influenza vaccine prep arations by domestic and foreign producers, which are now manufactured or registered in russia. construction studies on developing influenza vaccines and their production technologies have been extended in the most recent years thanks to the deployed program on influenza pandemic preparedness. the who has adopted a special technology transfer program on developing influenza vaccine production in third world countries. europe and the united states agreed to allot grants and worked out programs for developing novel technologies and vaccine preparations with improved qualities: a new generation of laiv, that is, delns vaccines with a limited replicative potential, laiv with deletions of pathogenicity factors in genes, latest variations of subunit vaccines enhanced by adju vants, and capsid nanovaccines and nanovaccines based on inactivated viruses and virus like particles [ ] . what are basic prospects opening before the vac cines constructed using state of the art genetic engi neering techniques? using the reverse genetics technique the genome of influenza type a viruses consists of segments encoding genes (table ) . vaccines against especially pathogenic strains of influenza viruses are particularly difficult to develop, since the conventional approaches, based on the reas sortment of "wild" isolates (epidemiologically impor tant viruses) and donors of attenuation, practically do not work for obtaining this type of candidate vaccine strains. usually, in reassortment, the genes that encode surface antigens in the donor are replaced by ha and neuraminidase of the epidemic's virus. a can didate vaccine strain is unable to cause a disease but in immunization it supports the development of immu the open reading frame in the pb pb f gene, which encodes the amphiphilic transmembrane protein with signaling peptide of mitochondrial local ization. ( ) mutations in the pb gene (polymerase com plex). ( ) mutations in the gene encoding the nonstruc tural ns protein-an antagonist of the interferon system of the first type. ( ) mutations in the m protein gene that controls key stages of a reproductive cycle through the so called proton pump and drug resistance to rimantadine. as was mentioned, to construct a new vaccine, the surface antigens, primarily ha, should be replaced. replacing ha in the donor of attenuation by the ha of the avian virus a (h n ) will lead to a transference of the pathogenicity determinant. a candidate vaccine strain should contain at least one sign of pathogenicity. to obtain a safe vaccine, this determinant should be removed. however, this is impossible to realize at the rna level. the viral genome should be converted to the dna form. this technique was realized for the first time by d. baltimore, a nobel prize laureate, for the polio virus rna more than years ago; relative to the rna containing influenza viruses, this approach was termed "reverse genetics" (in nature this process is realized by reverse transcriptase) and patented [ , [ ] [ ] [ ] [ ] [ ] . several research teams throughout the world construct viruses using the removal technique at the level of the dna sequences encoding pathogenicity sites (the ha cleavage site in the case of avian influ enza viruses). six basic plasmid dnas encode internal genes of a known donor of attenuation. constructing plasmids is a specific process, since, in each plasmids, bidirectional transcription is provided for the virus specific mrna and the complimentary virion rna with a negative polarity-a necessary factor for the synthesis of virus specific proteins and accumulating genomic fragments when obtaining infectious viral progeny. in researchers from the national institute for biological standards and control (nibsc), london, common cold research unit (mrc, england, salis bury), as well as from the rjc research and production therapeutic corporation, rixensart, belgium, offered the a/pr/ / strain as the master donor for con structing influenza vaccines. eight reassortants of the influenza h n and h n virus serosubtypes were prepared on the basis of this donor and then thor oughly tested in sensitive subjects from different age groups at laboratories in the united states, west europe, and australia. the a/pr/ / strain was offered as a donor of attenuation, because the reassor tants tested lacked reactogenicity and showed very high activity indicators in adults and children ( - fold excess over the generally accepted minimum pro tection level for humoral antibodies). the reverse genetics technique was successfully applied in con structing the candidate vaccine strains on the basis of the avian flu h n virus isolates having the most important pathogenicity determinant in the ha gene as a cluster of residues from lysine and arginine at the site of proteolysis with furine proteases. after the site was removed from the cloned gene its pathogenicity significantly decreased, being in conformity with the requirements for candidate vaccine strains. the a/pr/ / virus is still used as a successful donor of high reproductive activity. this very strain served as the donor for constructing important viruses for inactivated, predominantly whole virion vaccines at the research institute of influenza (st. petersburg). the vaccine passed through preliminary trials for effi cacy, genetic stability, and antigenic homogeneity; the preparation is being prepared now for clinical trials. development of inactivated vaccines or vaccines with modified ns and pb genes, replication defi cient in the human body, is recommended for obvious reasons. such vaccines are obtained by reverse genet ics techniques mastered at the international laboratory of the research institute of influenza, rams (fig. ) . a further attenuation of the virus through genetic modifications is possible by obtaining strains with lengthy deletions in the ns gene encoding the antag onist of the first type interferons and the signaling domain pdz localized at c end of this viral protein [ , ] . the level of this technology is so sophisticated that using the plasmid technique complete copies of highly pathogenic viral isolates can be constructed. in particular, within the european project fluvac, we obtained a complete copy of the kurgan h n virus isolate. the representative scheme (see fig. ) for the vac cine ni brg virus assembly shows the removal of the proteolysis site rerrrkkr and insertion of a new "attenuated" site retr. the na gene in this construction remained intact. the vaccine strain on this base has specially been prepared for the use in the territory of the russian federation. manipulations with the ns gene were put into the basis of a new technology for obtaining influenza viruses with limited replication potentialreplicatively deficient strains [ , ] -aimed to replace laiv as more safe and efficacious. the safety of these vaccines is assessed primarily by their high sensitivity to the endogenous interferon. an influenza table ; n -neuraminidase ; see explanations in the text. virus is a strong inductor of interferon and, therefore, viruses deprived of the interferon antagonist-full value protein ns -are quickly eliminated from the body under the action of interferon i and ii types after a short cycle of reproduction. this fact enables us to use the influenza virus as a safe vector. in our work the influenza virus was used as a vector for obtaining the strain expressing the esat protein of the tb caus ing bacteria [ ] [ ] [ ] . the intranasally administered modified virus manifested in animals the properties of an antitubercular vaccine [ ] . the arsenal of approaches to the technological development and solutions in constructing and manu facturing influenza vaccines has become considerably more substantial over recent years. the annual prepa ration of conventional vaccines (live attenuated, whole virion, and split and subunit vaccines) includes, in the first place, the phase of obtaining vac cine strains, which usually takes up to months. many technological techniques and innovations are oriented to the prompt preparation of antigen producers and the possibility to modify them in correspondence with the data on antigenic drifts of important seasonal or pandemic viruses. thus, the vaccines now in use can be supplemented by the following list: dna vaccines, recombinant subunit vaccines, vector based vaccines (their obtaining is connected with the use of one of the most attractive technologies for virus like particles), and a universal vaccine based on highly conservative antigens (m , m , and np proteins). in fact, the term recombinant vaccines can define any type of vaccine preparations obtained through genetic engineering. at the same time, considering the advance of these approaches to constructing influenza vaccines, it is more appropriate to specify recombinant vaccines by two types: recombinant influenza viruses used as vectors or deficient viruses for obtaining non pathogenic vaccine strains and constructs bearing individual viral genes for obtaining different types of vaccines. we considered above the first approach and now we consider the second approach. in practice, the process of constructing individual recombinant vaccines comprises the following actions: ( ) cloning and expression of individual viral genes in different vectors to obtain viral antigens in different expression systems as highly purified vaccine prepara tions. ( ) cloning and expression of individual viral anti gens to obtain virus like particles, to achieve each of the above results, one is to make a well grounded selection. the following antigen proteins are localized on a viral particle's surface: haemagglutinin (ha), neuraminidase (na), and the m protein. it is estab lished that five antigenic sites are localized in the "head" of the apical part of the ha molecule and that the antibodies' virus neutralizing activity is associated with the immune response to these sites. antibodies to ha block the interaction of the virus with cellular receptors, that is, infecting the cells. antibodies to na block the secretion of viruses by the infected cells (i.e., propagation of infection). antibodies to the m pro tein also considerably block the dissociation of infec tious viral particles from the infected cells' mem branes. we see further that, of the three main surface proteins, only ha is of a principal significance, the consideration of which the production technology for subunit vaccines predominantly containing ha is based. immunity to na is believed to be accessory. the m protein, considering its supreme conserva tiveness, became the model object for constructing a universal antiinfluenza a vaccine. thus, recombinant vaccines with individual com ponents of influenza type a viruses are mainly ori ented to the key and highly variable viral compo nent-the ha molecule. among the internal antigens of a viral particle, the np, m , and ns proteins are considered as material for obtaining vaccines. another, no less important problem consists in selecting a substrate for growing antigenic substances. scientists have been discussing for many years the option of replacing chicken embryos with cell cul tures. as previously, conventional technologies are oriented to growing viral material on chicken embryos. it applies to laiv and iiv of the current generations. this technological process consumes millions of chicken embryos (in fact, one dose of a sea sonal influenza vaccine is equivalent to one embryo). the scale of the problem is realized by everyone, if to consider that in the event of a pandemic only in russia the demand for vaccines accounts for at least mil lion doses. this scale of mass production relying on the conventional substrate cannot be promptly deployed (by the coming of a season and more so by the outbreak of a pandemic). the use of cell cultures in producing antigenic components of vaccines allows specialists to decide the above task rapidly and in sizable volumes. vaccine production based on the vero and mdck cell cultures have already been licensed in the united states and european countries. moreover, the use of this approach is in full correspondence with the complete technological chain for obtaining recombinant viruses using the plasmid technique and, in fact, it allows producers to rapidly deploy mass production from the laboratory scale capacity. in the last or years, can didate vaccine strains for a majority of seasonal viruses have already been obtained using the so called reverse genetics technique (see the above description). this approach corresponds also with the dna vaccine technology. although dna vaccines have been under development for more than years (their develop ment continues) none of such vaccines are so far per mitted for use. a marked breakthrough in the construction of recombinant vaccines is related to the use of insect cells and the obtaining of virus like particles (vlps) based on baculovirus expression vectors. this tech nique deserves our consideration in detail because of the high technological and quality indicators demon strated by vaccine preparations. best solutions on the composition of vaccines, their technologies, high immunogenicity, and safety are actually united under the project. in accordance with the classification pre sented, thanks to the absence of ballast components and preservation of the spatial structure of surface antigens, vlps excel recombinant vaccines, dna vectors based vaccines, and whole virion vaccines grown on a cell substrate. a positive advantage is, evi dently, that the sizes of vlps and virions also corre spond. we will consider, one by one, projects and achieve ments in the field of new approaches to constructing influenza vaccines and the use of different systems for their production. we will discuss the projects that are now believed to be the most important for realizing a technological breakthrough in the mass production of safe influenza vaccines. use of vectors for constructing and producing virus like particles as influenza vaccines. adenoviruses have been used as a tool in model studies on gene therapy for many years. in recent years, specialists engaged in constructing recombinant vaccines have become interested in the advantages promised by adenovirus based vectors. a particularly attractive feature of ade noviruses is their high productivity and efficiency in gene transfer and expression. vectors for vaccines are constructed with deletions by early genes e and e . in some research works, adenovirus vectors are used for the expression of individual viral antigens and, in particular, ha of influenza viruses. a serious disadvantage for these vectors is the presence in the human population of widespread immunity, which leads to a rapid limitation in the expression of target antigens. during epidemics, - % of the human population suffers from adenoviral infections. hexon is the strongest antigen among adenoviral proteins. antibodies to this protein are recorded in more than % of healthy subjects. therefore, when planning the use of these vectors for some works one should consider, first of all, their population receptiv ity. it equally refers to gene therapy and recombinant vaccines intended for a wide application against mass infections, such as influenza. one of the projects of the russian corporation of nanotechnologies is devoted to the use of adenoviruses as vectors for constructing influenza vaccines. the vectors based on alpha viruses are a relatively new tool for constructing recombinant vaccines. the venezuelan equine encephalomyelitis virus, specifi cally pathogenic for horses, is especially attractive to virologists as an object of research. the author of this survey studied the basic problems of replication and expression of the rna containing virus genome in the early s. the expression of this virus genome is supported by two operons: early and late genes. for eign genes are inserted in the reading frames of the late genes, thus providing a high level of their expression, comparable with that of the surface major proteins of alpha viruses. alphavax inc. is the world's leader in using alpha viruses as vectors for constructing vac cines. at the same time, we are underlining the safety of vectors based on alpha viruses: they are nonpatho genic for humans, and the human population has no immunity to these viruses, since they have never circu lated among people. constructing recombinant vector influenza vaccines based on alpha viruses is one of the independent and promising strategies in this field. the project offered by novavax inc. and protein science inc. is one of the most prospective for constructing a new type influenza vaccines and organizing their production. these companies use var ious vectors for the expression of ha and other influ enza virus proteins in insect cells. the latter grow at a temperature of approximately °c; the temperature regime for their growth is controlled by special equip ment. at the same time, culturing insect cells is a sim pler and less expensive process, compared to the growth of animal or human cells, and, at the same time, not associated with a risk of malignancy devel opment. human safe vectors based on baculoviruses are used to express antigens and other foreign proteins in insect cells [ ] . baculoviruses express polyhedrin protein in considerable amounts under the control of a strong promoter. thus, a polyhedrin promoter is used for the expression of foreign antigens (fig. , on the colored insert). with the above constructs, a high level of expression is achieved in insect cells for ha of different subtypes. at present, two approaches are applied for mass production of influenza vaccine components. one system (protein science) synthesizes ha, the main influenza virus component, for a vaccine. the other system (novavax) synthesizes simultaneously three proteins: ha, na, and m . synthesis of individual ha allows using this antigen, purified, as the main component of a vaccine identical in its properties to a subunit vaccine. at the same time, it remains unclear if the protein folding with the formation of ha "rosettes" is used to prepare the vaccine, which is a very important process for increasing immunogenicity and ensuring the quality of the immune response, comparable to the native ha in the composition of viral particles. the simultaneous expression of viral proteins ha, na, and m is accompanied by their self assembly in insect cells. this process results in the formation of vlps having high immunogenicity, allowing the antigenic burden to be reduced, com pared to the individual ha. the system of obtaining vlps using the sf insect cell line with a baculoviral vector expressing three main proteins ha, na, and m (see fig. ) was tested in the pandemic h n and h n viruses and the sea sonal influenza h n virus. in all cases, the vlps had high immunogenicity in conformity with the require ments for vaccine preparations. proteins. for many reasons, including economic ones, the strategy of using systems producing vaccine com ponents in plants is very attractive. attempts are now being taken to obtain plant producers for influenza virus antigens [ , ] . russia is conducting such stud ies within a few projects. there are attempts jointly it is natural that edible vaccines are important not only for controlling zoonozic pools of viruses and bac teria, but we believe that they are principally signifi cant for early vaccination in babyhood when the immune system of the intestines in neonatals is still in the process of formation. therefore, large scale works on obtaining vaccine preparations based on rice culti vars are worthy of attention [ ] . a progress in these studies may principally change our children's vaccina tion calendar and make vaccination in infancy com pletely safe. genetic properties of the influenza a viruses are characterized by extreme antigenic variability. the strains should be constantly monitored and studied as to their genetic structure and antigenic properties. a special commission of who considers, on an annual basis, data on the properties of isolates from all over the world and makes decisions on the strains in the composition of a next vaccine. at the same time, an outbreak of a pandemic brings a sharp change in the agent's antigenic structure or antigenic shift. in , an antigenic shift occurred within a subtype of h n , and a new swine lineage virus emerged with a princi pally different structure of the surface ha and na antigens. the global population in its majority proved unprotected against this virus; its relatively high pathogenicity explains the increased mortality from the infection caused by this agent (table ) . on the other hand, for the majority of other rna containing viruses, researchers have obtained vaccine strains not requiring seasonal replacement. in particu lar, it refers to the agents of measles and poliomyelitis. the natural question has been raised for years: is it possible to develop some universal anti influenza a vaccine based on the agent's conservative proteins? if we take surface antigens, only three can partici pate in the selection (see above). the m protein's principal difference from the other two is its high con servatism admitting only insignificant replacements in the gene structure. the surface of virions exposes approximately copies of the m protein tetramer that performs the functions of ion channel-proton pump. the functional activity of the proton pump is important at the onset of infection for decapsidation of the virus in endosomes and, subsequently, under final phases for gemmation and release of viral particles. thus, the m protein controls the process of infecting cells and propagation of infection. it has recently been established that the sign of contagiousness, or the virus spreading speed in human to human transmission, is associated with the m protein [ , ] . according to other data, the m protein is signifi cantly responsible for the development of oxidative stress accompanying influenza infections. therefore, induction of an immune response to this surface pro tein is believed to be very important for the protection against influenza infections. at present, the construction, preclinical trials and, in some cases, clinical trials have been completed on the following preparations: ( ) recombinant preparation of the apical (extramembraneous) m e domain in the capsid car rier of the hepatitis b protein particles. ( ) recombinant preparation, including in tan dem repetitive copies of amino acid residues of the m protein in the composition of e. coli flagellin. ( ) recombinant fused protein m e conjugated with a residue of cysteine at the position and con taining palmitine acid as an adjuvant. in fig. a (on the color insert), the m protein is represented by a physical map, where the transmem braneous domain is underlined by alpha spiral (given is the most spread sequence with indicating amanta dine and rimantadine resistant mutations; the aman tadine molecule is designated in yellow in the region of adamant binding site); given on the map's left is the extramembraneous domain at n end (positions - ), the blue arrow points to variations of this domain's sequences in different subtypes of influenza a viruses (see fig. c ). on the top right is a tetramer model of the transmembraneous domain in a lipid membrane (see fig. b ). considering the first importance of projects on cre ating a universal vaccine, we give below the m protein sequences for important pandemic viruses character ized by a high level of conservativeness, a very valuable quality for constructing a universal vaccine (fig. ) . the high conservativeness of the main the m pro tein domains used in constructing recombinant vac cines allows us to rely on reaching a sufficient efficacy of the vaccines against any possible pandemic strain, including avian flu and swine lineage h n influ enza. this type of vaccine has been created in russia on the base of the center bioengineering, russian academy of sciences [ ] . vaccines production of vaccines, as well as other biophar maceutical processes, includes two main phases: prep aration and main technological process. the preparation phase consists in obtaining a sow ing material-a virus in cell cultures-in a limited quantity, its subsequent cultivation inside voluminous reactors, and isolation of the virus from cells and main cellular components. as a result, a raw suspension of a viable virus is obtained, which is subjected to further purification. the main technological process includes the follow ing key phases of purification of the virus: concentra tion, chromatography, ultradiafiltration, isolation of separate components (for split or subunit vaccines), mixing with adjuvant (if the latter is included in the vaccine) and preparation of the final product for pack aging. below we are discussing the current level and prob lems in the influenza vaccine production realization. the key phase of the preparatory technological process is fermentation. the development of wave bioreactors used for fermentation has become a turn ing point in the field of cell cultivation and has signif icantly increased cell culture yields in growing viruses. a majority of companies trading in modern techno logical equipment recommends this type of reactors. the joint project of the research institute of influenza of the rams with the ge company (sweden) includes a series of important solutions for the key part of the production cycle. the system of cell selection, concentration, and filtration with separation of the fractions containing the end product is successfully realized using the uniflux system developed within the above project (fig. ). uniflux systems can be used for concentrating virus suspensions, vaccine preparations, protein aggregates, monoclonal antibodies, and pure recom binant proteins. concentration of virus suspensions is a necessary phase for preparing the material for chro matographic purification. the use of standard monolith columns for the chromatography of viral particles is the most impor tant technological solution (fig. ) . under a majority of technological processes, viral particles are subjected to purification through sucrose gradient ultracentrifu gation. the chromatographically purified vaccine was obtained in columns with macroporous glass. how ever, this vaccine contained hydrodynamically and mechanically destroyed virions. exposing the viral nucleotide on the surface of the destroyed viral parti cles leads to the induction of immunity against the native viral ribonucleoproteid (rnp). this phenome non is not so unsafe, since a cross reaction of antibod ies with rnp of the body's cells cannot be excluded, while this kind of phenomena may lead to autoim mune processes that may trigger systemic diseases. application of state of the art techniques for chromatography of viral particles permits one to obtain highly purified samples of both viruses and subunit preparations or vlps. chromatographic puri fication helps solve another, more important problem. electron microscopy analysis of the current vaccines as to their structure reveals that antigens in the major ity of vaccine preparations are in a denatured or heavily disorganized state (fig. ) . the research institute of influenza of the rams is now developing, within some international projects, production technologies for a new generation of vac cine preparations: ( ) live replicatively deficient vaccines del ns with additional genetic defects within a wide spectrum of molecular determinants of pathogenicity for opti mizing their safety and preserving their high immuno genicity; ( ) protein nanovaccines based on the controlled folding technology; nm nm (d) ( ) antigen and virion complexes with metalized nanoparticles (fig. ). first of all, we should underline that the principal strategy in improving influenza vaccines has ever been their increased safety and immunogenicity. this explains the transition from whole virion to split vac cines and from the latter to subunit vaccines. a high level of antigen purification generated the problem of decreased immunogenicity of vaccines and a search for immunoadjuvants. approximately years ago polyoxidonium was selected to perform this function. the polyoxidonium based vaccines were termed poly mer subunit vaccines. however, such an approach has grown old now. a number of new adjuvants based on the known mechanism of stimulating the immune response through toll receptors have been tested in recent years [ , ] . discussions are still ongoing concerning the safety of laiv. the current strategy is oriented to a search for inactivated analogues of many live vaccines. firstly, this is connected with a probable reversibility of attenuated strains of viruses towards wild types, or reassortment of strains, having a fragmentary genome, with wild viruses. using a live or even attenuated virus for mass vaccination, we, certainly, create a temporary circulatory background, considerably increasing a probability for the reassortment of viral genomes with viruses actively circulating in a current period of time in human population. despite the fact that the phe nomenon of reassortment between laiv and wild viruses has not been confirmed and viruses with signs of pathogenicity have not been isolated in the majority of complicated vaccination cases, the application of laiv is limited in the period when seasonal influenza is on the rise. mass immunization with laiv in the event of a pandemic should be conducted with maxi mum care. at the same time, improvement of laiv safety is possible through genetic techniques-in particular, we have achieved this in our studies within the euro pean fluvac project. the team led by professor a.yu. egorov has developed so called delns vac cines. vaccine strains carrying the nsi gene deletion can quickly be eliminated, since they are highly sensi tive to the endogenous interferon (the latter is the antagonist of type i and type ii interferons). there fore, this type of live vaccine was classified as replica tively deficient. a probability of recombination or reassortment is ultimately low in this case. another approach to a genetically controlled reduction in the laiv reactogenicity consists in the removal of the reading frame in the pb gene. the pb f reading frame encodes a short protein of amino acids-the factor of macrophage apoptosis causing the death of cells during translocation in mito the analysis of development strategies in vaccine production technology has shown that genetic engi neering technologies are moving ever more to the fore front of this development. on the other hand, there is an understanding, initially absent in immunologists, that the tertiary structure of the antigen is equally important for adequate immunogenicity and the func tioning of enzymes. significantly improved instru mental provision of technological processes enables creating typical multipurpose vaccine productions, including vaccine preparations based on nanotechnol ogies. the current discussions about the scale of mortality due to the h n sw pandemic are held with not quite correct argumentation. even without very strict crite ria, it is an emergency when a healthy young or mid dle aged person dies from influenza. vaccination and timely administration of efficacious antiinfluenza means, within a complex antiinfluenza therapy, offer good chances to avoid, in an overwhelming majority of cases, severe complications and, certainly, a lethal outcome. according to the latest statistics, the h n sw related mortality rate is reliably higher than the share of lethal outcomes in the - pan demic, while lower than during the asiatic influ enza. of course, medical science has made a signifi cant progress since , continuously introducing from antiviral therapeutic means into clinical practice. based on the above, we have to draw the fol lowing not very comfortable conclusion: high influ enza related mortality rates are, to a considerable degree, explained by our poor performance in orga nizing medical aid to patients. russia is also no exclu sion in this aspect. at the same time, availability of developed facilities for producing influenza vaccines allows russia, in contrast to other countries, to promptly take decisions on a national vaccination pro gram of scale. returning to the problem of genetic properties of pandemic viruses and the discussion about the molec ular determinants of pathogenicity, we have to repeat that the influenza pandemic was caused by the mixed h n v virus with a genome that included genes of swine, avian, and human viruses. based on the specific structure of its genome and pathogenicity level in animals (experimental models) and humans, we can characterize this etiological agent as a moder ately pathogenic virus. in particular, ha of the h n v virus does not contain the proteolysis sites characterizing the highly pathogenic h n viruses. the pb f reading frame in the genome's second fragment (encoding the pb protein and the supplementary pb f protein resembling bacterial toxins by structure) is three times interrupted by stop codons, which leads to the absence of protein synthe sis in the infected cells. the absence of pb f , as the most important factor of pathogenicity, in the influ enza a viruses is an additional argument in support of specifying this virus as moderately pathogenic. in addition, this very genetic sign is a principal difference of the current pandemic h n v virus from the h n virus that caused the hispanic influenza pandemic. moreover, the pandemic h n v virus is defi cient in the ns protein, the antagonist of i and ii types interferons. in the ns protein from north america and the russian federation's isolates, researchers have found c terminal deletion of an important regulatory site playing a key role in the acti vation of the signaling cell systems controlling the antiviral response of the body's cells (the deletion of so called pdz binding domain) in influenza infections. this deletion is probably related to the increased sen sitivity of the pandemic h n v influenza to interferons and, in particular, to gamma interferon. summarizing the above analysis of the functional domains of the h n v influenza virus protein, we have to state that the current pandemic influenza viruses are characterized by the following structural defects: the ha proteolysis site-psiqsr/glf gai-is a substrate for the membrane type serin pro tease tmp/sst, the presence of the three stop codons in the pb f protein, and the presence of cooh terminal deletion of the pdz binding domain of ns . the above genetic defects of pandemic viruses should additionally be characterized by the following supplementary properties: ( ) resistance to the preparation tamiflu, which is often manifested by seasonal h n viruses and which is characteristic of a pandemic virus. ( ) cooperative specific properties of the ha and na proteolysis and their dependence on membrane bound cellular proteases [ ] , a systemic induction of a "cytokine storm". ( ) ns and ns conditioned opposition of the interferon system and immunosuppression. ( ) presence of the "weakened" consensus sequence of the ebola like suppressive domain [ ] . however, it should be underlined that the above defects may in part or fully be repaired in the process of active circulation of the h n sw viruses, carrying a risk of enhanced pathogenicity. therefore, in con structing vaccines for the forthcoming pandemic sea sons we shall have to foresee the constructive specific features associated with a probable emergence of viruses with modified properties. as the most marked achievements in domestic sci ence, we should note the obtaining of a full functional copy of the kurgan isolates for the avian influenza h n virus using the recursive pcr and reverse genetics techniques ( plasmid system variation [ ]) and the technology created for producing a universal vaccine based on the m protein [ ] . gripp ptits: proiskhozhdenie infektsionnykh biokatastrof (avian influenza: the origin of infectious biocatastrophes) gripp. na poroge pandemii (influenza: on the brink of pandemia) etiologiya, immunologiya i klin ika aziatskogo grippa mezhdunarod naya konferentsiya (bull. who. hong kong flu. int. conf.), pandemiya- . novyi virus h n (pandemia- . new virus h n ) note for guidance on harmonization of requirements for influenza vaccines. (comp/bwp/ / ). euro pean agency for the evaluation of medicinal products the lan cet who guid ance on development of influenza vaccine reference viruses by reverse genetics a review of production technologies for influenza virus vaccines and their suit ability for deployment in developing countries for influ enza pandemic preparedness. world health organiza tion initiative for vaccine research proc. natl. acad. sci. usa key: cord- - gd zy authors: kim, y. c.; jarrahian, c.; zehrung, d.; mitragotri, s.; prausnitz, m. r. title: delivery systems for intradermal vaccination date: - - journal: intradermal immunization doi: . / _ _ sha: doc_id: cord_uid: gd zy intradermal (id) vaccination can offer improved immunity and simpler logistics of delivery, but its use in medicine is limited by the need for simple, reliable methods of id delivery. id injection by the mantoux technique requires special training and may not reliably target skin, but is nonetheless used currently for bcg and rabies vaccination. scarification using a bifurcated needle was extensively used for smallpox eradication, but provides variable and inefficient delivery into the skin. recently, id vaccination has been simplified by introduction of a simple-to-use hollow microneedle that has been approved for id injection of influenza vaccine in europe. various designs of hollow microneedles have been studied preclinically and in humans. vaccines can also be injected into skin using needle-free devices, such as jet injection, which is receiving renewed clinical attention for id vaccination. projectile delivery using powder and gold particles (i.e., gene gun) have also been used clinically for id vaccination. building off the scarification approach, a number of preclinical studies have examined solid microneedle patches for use with vaccine coated onto metal microneedles, encapsulated within dissolving microneedles or added topically to skin after microneedle pretreatment, as well as adapting tattoo guns for id vaccination. finally, technologies designed to increase skin permeability in combination with a vaccine patch have been studied through the use of skin abrasion, ultrasound, electroporation, chemical enhancers, and thermal ablation. the prospects for bringing id vaccination into more widespread clinical practice are encouraging, given the large number of technologies for id delivery under development. microneedle pretreatment, as well as adapting tattoo guns for id vaccination. finally, technologies designed to increase skin permeability in combination with a vaccine patch have been studied through the use of skin abrasion, ultrasound, electroporation, chemical enhancers, and thermal ablation. the prospects for bringing id vaccination into more widespread clinical practice are encouraging, given the large number of technologies for id delivery under development. the skin contains high concentrations of antigen-presenting cells, and is thus a site capable of inducing potent immune responses. the skin is composed of multiple layers, each with characteristic resident and transient immune cell subsets. outermost is the thin layer of the epidermis ( . - . mm), which is primarily made up of epithelial cells as well as langerhans cells, melanocytes, and merkel cells. beneath the epidermis, the dermis is a thicker layer ( . - mm) consisting of a network of collagen fibers. cells of the adaptive and innate system reside in or circulate through the dermis, including macrophages, mast cells, langerhans cells, and dermal dendritic cells. antigenpresenting cells in the skin perform an essential role in processing incoming antigens, resulting in immune system activation or immune tolerance of self or harmless antigens (nicolas and guy ) . for these reasons, it is possible that delivery of vaccines to the epidermis or dermis may result in superior immune responses compared to other anatomical sites (glenn and kenney ; lambert and laurent ; nicolas and guy ) . alternatively, an equivalent immune response could be stimulated by delivery of a smaller quantity of vaccine antigen to the skin. either of these mechanisms could be beneficial for developing vaccines against new disease targets, improving immune responses in hard-to-treat groups, or lowering the cost of vaccine antigens, and may be particularly valuable for improving access to vaccines in low-resource settings. while a substantial number of clinical studies evaluating intradermal (id) delivery of vaccines have been performed, the majority of studies have not been designed to evaluate whether id delivery is immunologically superior to other routes. in most cases, to simplify administration, a reduced dose ( or %) delivered id was compared to the full dose delivered either subcutaneously (sc) or intramuscularly (im) . only a few studies have compared delivery of the same dose of vaccine id and sc/im. further research will be needed to establish whether the potential for dose-sparing is unique to id delivery (path ). however, some id delivery devices in development offer additional desirable features such as needle-free delivery or improved ease of administration, which may be drivers for further adoption of id vaccine delivery even if there is no net immunologic benefit. vaccines for smallpox have been delivered to the skin dating back to edward jenner's first experiments in demonstrating that exposure to cowpox could protect against smallpox infection. a variety of scarification techniques and devices have been used to allow virus introduction, including knives, needles, scalpels, and rotary lancets. during the global smallpox eradication campaign, both multi-dose nozzle jet injectors and bifurcated needles were used for id vaccinia virus inoculation (henderson et al. ). bacille calmette-guérin (bcg) vaccine for tuberculosis is globally the most widely delivered id vaccine. id injection by needle and syringe is the most commonly used method, but in some areas bcg is also delivered to the skin using a multipuncture device. new versions of bcg are under development in an effort to improve immune protection, and are also delivered id (hoft et al. ). rabies vaccines are conventionally delivered im, but due to the high cost of cellculture-derived vaccines and the pressing need for affordable vaccination regimens in endemic regions, id delivery has been extensively studied. both post-exposure prophylaxis and pre-exposure prophylaxis id regimens induce protective titers, and who has recommended id delivery of reduced doses of rabies vaccines since (who ; . given equivalent doses of antigen, delivery to the dermis appears to be either superior or equivalent to im/sc (bernard et al. ; bernard et al. ; fishbein et al. ; phanuphak et al. ) . a detailed review on id rabies vaccination can be found elsewhere in this special volume on id immunization (warrell ). multiple studies of reduced-dose delivery of influenza vaccines have been conducted, providing some of the most informative clinical data on the potential for dose-sparing through id delivery. one study found that id delivery of lg ha per influenza strain was comparably immunogenic as the standard im dose of lg ha per strain (belshe et al. ) . a later comparison of , , and lg delivered both id and im found equivalent responses for the two delivery routes for each dose (belshe et al. ) . trials have also been conducted with influenza using novel microneedle devices to aid accurate id delivery, as discussed in sect. . . id delivery of reduced doses of hepatitis b vaccine has been evaluated in healthy infant, child, and adult populations as well as in immuno-compromised patient groups. meta-analyses have concluded that seroconversion rates are lower than full-dose im delivery, although responses are higher in children and females (chen and gluud ; sangare et al. ). when the same dose of hepatitis b antigen has been delivered id and im, immune responses were equivalent for both routes (ayoola ; milne et al. ; heijtink et al. ; coberly et al. ; rahman et al. ) . hepatitis a vaccines have also been proposed as a possible target for reduced-dose id delivery. two studies found that reduced doses delivered id produced comparable immune responses to im delivery, while a third indicated that the id route was inferior (brindle et al. ; carlsson et al. ; pancharoen et al. ) . local reactogenicity was observed for alum-adjuvanted formulations. in a few countries, id was originally the standard route of delivery for inactivated poliovirus vaccine, but injection depth was later shifted to im (weniger and papania ) . studies have found that id delivery of reduced doses is capable of inducing seroconversion, which may help make this vaccine more affordable for use in developing countries (samuel et al. ; samuel et al. ; nirmal et al. ). more recently, the who global polio eradication initiative has worked to determine the potential for this mode of delivery to be used in post-eradication settings after phase-out of oral polio vaccine. several studies have been conducted evaluating id delivery of measles vaccine, with mixed results (burland ; kok et al. ; whittle et al. ; de moraes et al. ) . however, the vaccine dose and method used to deliver the vaccine varied, and it is possible that trials using older generation delivery technology did not deliver vaccine reliably to the dermis (path ). transcutaneous immunization of measles vaccine on a coated patch has also been attempted. although a salivary siga response was observed, the key marker of immunity, an increase in neutralizing serum igg, was not detected (etchart et al. ). studies were conducted delivering the d attenuated yellow fever virus vaccine by scarification, but this delivery mode was abandoned as efficacy was low. more recently, a clinical trial compared full dose sc delivery of a d vaccine to / dose delivered id by mantoux injection, which found equivalent seroprotection between the two routes (roukens et al. ) . a more extensive description on id vaccination against yellow fever is provided elsewhere in this special volume on id immunization (roukens et al. ). a number of other vaccines have been considered for id delivery. research has shown that a reduced id dose of vaccines for diphtheria-tetanus-pertussis, tetanus toxoid, and tick-borne encephalitis can generate a comparable immune response to the standard dose and way of injection (stanfield et al. ; zoulek et al. ; zoulek et al. ; dimache et al. ). id delivery is also under investigation for a number of vaccines in development, including vaccines for tuberculosis, enterotoxigenic e. coli, and pandemic influenza, as well as dna vaccines. the traditional methods used for id delivery of vaccines have limitations which may hinder adoption of id delivery. bifurcated needles and multipuncture devices have been used successfully for delivery of smallpox and bcg vaccines, but do not deliver reproducible quantities of vaccine antigen to the dermis and are therefore unlikely to be appropriate delivery devices for new vaccines (lambert and laurent ) . the mantoux method of inserting a needle at a shallow angle into the skin can also be inconsistent, and requires additional training and skill to perform correctly (flynn et al. ) . the perceived difficulty of performing an id injection using this method may prevent development of vaccines for id delivery. new generations of devices, such as those discussed in the rest of this article, may improve the reliability of id delivery and enable adoption of the id route for more vaccines. most vaccines are administered im or sc using a hypodermic needle. to achieve id vaccination, conventional hypodermic needles can be used by employing the mantoux technique to inject into the skin. simpler and more reliable id injection is being pursued through adaptations of hypodermic needle technology, as well as novel hollow microneedle devices produced by microfabrication ). the mantoux technique is an id injection method characterized by a needle inserted at a - degree angle, approximately mm deep into the dermis, to inject a vaccine or drug (fig. a) . this method was developed by charles mantoux in the early th century, and it has been used to identify tuberculosis infection by the id injection of tuberculin (mantoux ) . however, this technique requires training and is often considered an inconsistent delivery method, thus preventing vaccine makers or medical practitioners from using id injection as a common immunization method (lambert and laurent ) . also, age or elasticity-related skin conditions have a significant effect on adequate placement of the needle in the dermis for the traditional id injection technique laurent et al. ) , thus leading to inadequate vaccination. other disadvantages of mantoux technique injection include inaccurately delivered dosage of vaccine, vaccine wastage in dead space of the needle, and variable injection success when using different gauge needles (flynn et al. ) . moreover, success rate of id injection by untrained personnel was found to be - % (howard et al. ) . in an effort to reduce training requirements and to improve the reliability of the mantoux injection technique, an intradermal adapter is under development by path (seattle, wa, usa), a nonprofit, international health agency that develops and advances health technologies for low resource settings, and sid technologies. this device fits over a conventional hypodermic needle and syringe and limits the angle and depth of penetration of the needle into the skin in order to facilitate delivery to the dermis. to overcome these limitations of conventional id injection, becton-dickinson (bd) has developed a micro-sized needle that can be inserted into skin vertically, unlike the angled injection of the mantoux method. this novel microneedle device has been studied in animals and human subjects, and is currently used in approved influenza vaccines (intanza Ò and idflu Ò ). bd's microneedle device (called soluvia tm ) uses a gauge microneedle that extends . mm beyond an insertion depth-limiting tip, which is connected to a prefilled syringe (figs. c and e). the microneedle system was evaluated versus the conventional mantoux technique to compare delivery efficiency and safety in human subjects. using ultrasound echography analysis, the distribution of fluid delivered by the microneedle was seen to be larger than the mantoux injection control. in addition, the microneedle system had a high id administration success rate ( %) and, in a study of patient compliance and safety, the microneedle device showed promising results. this system also caused fewer occurrences of injuries to the papillary dermis, lesser pain than mantoux injection and was administered easily by untrained personnel (laurent et al. ). an earlier prototype of the bd microneedle using a mm, gauge needle has been tested in rats for delivery of influenza vaccines which showed dose sparing effects compared to an im control. id microneedle administration of a low dose ( . lg) of inactivated virus vaccine induced similar serum antibody response as im injection of a dose times larger ( lg). additionally, using microneedles for id immunization with split-viron vaccine (seasonal h n strain) showed approximately ten-fold dose-sparing compared to im immunization. in the same study, id immunization using plasmid dna vaccine encoding the hemagglutinin protein of influenza a virus showed similar dose-sparing effects after multiple immunizations (alarcon et al. ). the bd microneedle was also used to deliver a live attenuated vaccine against japanese encephalitis (chimerivax tm -je) in non-human primates. in this study, id microneedle injection was compared to sc injection and transcutaneous microabrasion (see sect. . ). the microneedle id injection provided the best and most consistent immune responses (i.e., neutralizing antibodies) of the three types of immunizations . a further study compared anthrax vaccine delivery using id microneedle immunization, im injection, intranasal delivery, and epidermal delivery by microabrasion into mice ( lg dose) and rabbits ( lg dose). microneedle id vaccination showed slightly better response in the murine model than the other routes used, while all treatments in the rabbit had similar responses. a follow-up id immunization was performed to compare id injection with im injection over a range of doses in a rabbit model: , . , and . lg of anthrax vaccine (mikszta et al. ) . after prime immunization, id injection showed significantly higher immunogenicity than im injection when using and . lg dosages. interestingly, id injection with . lg showed a statistically equivalent response to im administration of the lg dose. after administration of a booster immunization, this dose-sparing phenomenon continued. furthermore, an aerosol lethal challenge with anthrax spores showed that a lg id injection completely protected the immunized rabbits, whereas im injection of the same dose protected only % of the rabbits. an early prototype of the bd microneedle system was first tested in a clinical study examining influenza vaccine delivery in healthy adults ( - yrs) and elderly adults ([ yrs) (belshe et al. ) . in this study, lg of hemagglutinin was delivered by id injection and compared to a full dose ( lg) delivered by im immunization. it was found that in younger participants, id immunization was not significantly different from immunization by im, as shown by geometric mean hemagglutination inhibition (hai) titers. however, id administration showed lower hai titers than im in elderly patients. further evaluation of id microneedle vaccination against influenza was performed in clinical studies of healthy adults ( - yrs) (leroux-roels et al. ; beran et al. ) and elderly persons ([ yrs) (holland et al. ; arnou et al. ). for healthy adults, lg of hemagglutinin (h , h , and b strains) was delivered by id injection and was compared to a lg im immunization. this study confirmed previous results that reduced-dose id injection was equally immunogenic as full-dose im injection (leroux-roels et al. ) . in a phase ii clinical trial, the effects of lower dose id immunization was investigated using , , and lg of hemagglutinin (id) and lg of hemagglutinin (im). id immunization using and lg of hemagglutinin induced inferior immune response as shown by hai titer, but a dose of lg showed comparable response compared to full-dose ( lg) im vaccination (beran et al. ). for elderly subjects ([ years old), a booster vaccine ( lg) was administered due to the inferior immune system generally found in the elderly compared to younger adults (goodwin et al. ) . therefore, lg of hemagglutinin was administered twice by either id or im routes in elderly subjects. in this phase ii clinical trial, id immunization showed significantly better immune response as determined by postimmunization gmt (geometric mean titer), seroprotection (% participants with hai titers c ), gmtr (geometric mean ratio of post-immunization titer to pre-immunization titer), and rate of seroconversion (post-immunization titer in participants with a pre-immunization titer \ ). therefore, id microneedle vaccination provided superior immunogenicity in a high priority population for protection from influenza due to high vulnerability (holland et al. ). these findings were further confirmed in a phase iii clinical trial for elderly persons ([ years old), where id immunization showed superior seroprotection, gmtr, and rate of seroconversion compared to im after prime immunization. after administration of two booster immunizations, id immunization induced consistently higher seroprotection rates than im immunization (arnou et al. ). as a final note, id immunization caused more local inflammatory-like reactions than im immunization. it is possible that because id delivery occurs in the skin, inflammatory or immunologic reactions are more easily visible than those that may occur after im immunization, which presents the antigen deep into the muscle layer where an inflammatory reaction would not be visible to the eye (belshe et al. ; holland et al. ; arnou et al. ; beran et al. ; van damme et al. ). hollow microneedles have also been developed as multi-needle arrays, which have involved shorter needles (\ \ mm) produced by novel microfabrication techniques, including laser micromachining (davis et al. ) , silicon-based mems technique using deep reactive-ion etching (gardeniers et al. ; roxhed et al. ) , integrated lithographic molding technique (luttge et al. ) , deep x-ray photolithography (perennes et al. ) , photolithography with micromolding technique ), drawing lithography with viscoelastic polymer ) and others. in addition, glass hollow microneedles have been fabricated by drawn glass micropipette techniques (wang et al. ) . a recently developed hollow microneedle array (micronjet from nanopass technologies) was used in a human clinical trial involving healthy adults ). this device consists of a row of four hollow silicon microneedles that are lm in length (fig. f) . in this study, id injection with the array using and % of the im dose ( lg) induced similar immune response as measured by gmt increase, seroconversion rate, and seroprotection rate. id delivery can also be achieved via jet injection or particle injection routes, which are needle-free methods of vaccine and drug delivery. there have been decades of clinical experience with jet injection, and more recent studies are being conducted with newer innovations in this technology. needle-free jet injectors create a fine stream of pressurized liquid that penetrates the skin. the depth of delivery-id, sc, or im-is largely determined by design variables such as the injection stream coherence, quality, and pressure; orifice size, skin and tissue thickness, and the angle of the injection relative to the skin (schramm-baxter and mitragotri ; weniger and papania ) . vaccines that have been shown to achieve immunity when administered via jet injection to conventional depths (i.e., id, sc, or im, depending on the vaccine) include typhoid, cholera, bcg, tetanus-diphtheria for adults, whole cell diphtheria-tetanus-pertussis (dtp), measles, meningococcal a and c, smallpox, yellow fever, hepatitis a, hepatitis b, influenza, plague, polio, and tetanus (weniger and papania ) . historically, multi-use nozzle jet injector (munji) devices with reusable nozzles were used successfully worldwide in the latter half of the th century to deliver countless millions, or by some estimates billions of doses of vaccines to both adults and children over the course of several decades (weniger and papania ) . in response to the risks of disease transmission due to cross contamination from reuse of injection devices, a new generation of jet injector designs were developed starting in the late s to address this safety concern. these new jet injectors utilize a sterile, disposable cartridge or syringe for each patient injection and a reusable hand-piece that relies on a power source, such as a manually powered spring or gas canister. a number of disposable-syringe jet injectors (dsjis) have been developed and approved by national regulatory authorities for a variety of applications and uses, including vaccine delivery. some of these are low-cost, manually powered dsji technologies, developed specifically for application to developing countries' immunization requirements and needs, which include design features to prevent reuse ('auto-disable') of the needle-free syringes. dsjis in clinical development for id delivery include the biojector Ò and zetajet Ò (bioject), and pharmajet Ò (pharmajet inc.). there is a long history of id delivery via the jet injector route through the use of modified syringe orifice nozzles that can either have direct contact to the skin or can involve a setback feature or 'spacer' intended to introduce a gap between the nozzle orifice and the injection site, thereby weakening the injection stream and limiting deposition to the dermal space (weniger and papania ) (figs. a, b) . munji devices provided millions of id smallpox doses during the implementation of the smallpox eradication program (millar and foege ; weniger and papania ) . jet injectors have also been utilized historically for id vaccination of rabies (bernard et al. ; bernard et al. ) , hepatitis a (williams et al. ) , bcg (paul et al. ; parker ) , dtp combination vaccine (stanfield et al. ) , measles (burland ; kok et al. ) , and influenza vaccine (weniger and papania ) . a number of studies have been or will soon be implemented to address the application of dsji id delivery to vaccines of importance to global public health. for example, the us centers for disease control and prevention is leading a study on seasonal influenza vaccine delivered id via a dsji technology in children of - months of age. this study compares full and fractional dose im with id vaccination. results-to-date indicate that injections were generally tolerable with few study-related adverse events. initial blinded assay results demonstrate comparable immune response rates. final study results and analysis can be found in gomez et al. ( ) . the who global polio eradication initiative has worked to determine the potential for dsji id delivery of inactivated poliovirus vaccine (ipv) to be used in post-eradication settings after phasing out the use of oral polio vaccine. studies have been conducted in oman, cuba, and india to evaluate reduced ('fractional') dose of ipv delivered with two different dsji devices. compared to im, inferior seroconversion rates were found when id doses were delivered at , , and ) with id spacer (white portion at end of syringe), used for investigational use only (courtesy of bioject). b jet injector applied to the skin for injection (courtesy of pharmajet). c epidermal powder immunization device for id projectile injection (courtesy of powdermed) weeks of age, but non-inferior rates of protection ([ %) were seen using a later , , and month schedule. when ipv was used as a booster to oral polio vaccine, inferior seroconversion rates were observed for id compared to im delivery (sutter ; mohammed et al. ; resik et al. ) . dsji technology has also been used for the delivery of dna vaccines for malaria in young adults (epstein et al. ; wang et al. ) and an hiv-vaccine candidate (path ). a pilot study assessment of human papillomavirus vaccine has also recently occurred (path ) . path is also working to implement a new study of purified vero cell rabies vaccine for id post-exposure prophylaxis using a dsji technology in india. results of this study are anticipated in . other vaccine trials of id vaccine delivery are planned for other applications including bcg, ipv, varicella zoster virus, h n and yellow fever (path ). epidermal powder immunization (epi) and particle-mediated epidermal delivery (pmed) utilize helium gas to deliver powdered proteins, polysaccharides, inactivated pathogens, or dna-coated particles into the epidermis at supersonic speeds (weniger and papania ) (fig. c) . companies involved in developing this technology include powderject, powdermed (acquired by pfizer in ), and iaculor injection. it is not known if this device technology class is still in active development (path ). conventional protein antigens must be specially formulated for delivery by epi, and are spray dried into powders of suitable density and size ( - lm). a clinical trial has been conducted evaluating delivery of a powdered inactivated influenza vaccine by epi injection, which found that immunogenicity was comparable to standard delivery by im needle and syringe . epi has also shown efficacy in preclinical studies with hepatitis b and hiv vaccines (chen et al. ; osorio et al. ) . in pmed, gold beads - lm in diameter are coated with vaccine and delivered by needle-free jet injection into the epidermis. this approach may be particularly suited to dna vaccines, as deposition of coated particles into the stratum corneum and epidermis may encourage dna uptake and expression by resident antigen-presenting cells. dna vaccines for hepatitis b delivered by pmed have induced protective antibodies (roy et al. ; roberts et al. ) . clinical studies have also been conducted with dna vaccines for seasonal influenza to evaluate the feasibility of this approach. results have been promising, but immune responses are not yet equivalent to standard vaccine delivery methods (drape et al. ; jones et al. ). epi and pmed delivery of dna vaccines for a variety of other diseases have also shown immunogenicity preclinically, including malaria, avian influenza, herpes simplex virus, hiv, non-small cell lung cancer, eurasian encephalitic viruses, hantaviruses, sars coronavirus, and smallpox (weniger and papania ) . for more than years, various sharp instruments have been used for vaccination by creating small holes in the skin that allow vaccine to penetrate into the body (weniger and papania ) . although most vaccine administration is currently performed by hypodermic needle injection, sharp tools such as bifurcated needles have historically been used for smallpox (frey et al. ) and bcg (darmanger et al. ) vaccination and remain in use to this day. over the past decade, new skin piercing technologies for id drug transport have been developed, and include techniques such as microneedles (prausnitz ) and tattooing (bins et al. ) . recently these methods, especially microneedles, have shown promise for delivering vaccines to the skin, thereby enabling improved immunogenicity and simpler patient administration. the bifurcated needle (fig. b) was invented by benjamin rubin in for smallpox vaccination. it consists of two sharp prongs which hold vaccine fluid by capillary action between the two tines. the use of this device is simple and does not require trained personnel (baxby ; weniger and papania ) . the needles are dipped into vaccine and then punctured perpendicularly into skin repeatedly over an area of about mm diameter by a process called scarification (who ) . although this method was effective for the smallpox eradication program, poorly controlled dosing, inefficient use of vaccine and needle-stick injuries were significant shortcomings that have limited the use of bifurcated needles for other vaccines. in addition to hollow microneedles discussed in sect. , solid microneedles can be used to pierce the skin and thereby deposit vaccine in the epidermal and/or dermal space ). techniques for vaccination using solid microneedles include the use of microneedles that penetrate the skin to make a hole through which vaccine can be transported. vaccine formulations may be placed on the skin after microneedle penetration, coated onto microneedles or embedded within microneedles and released into the skin after insertion. solid microneedles can be prepared as patches that can be easily applied to the skin, perhaps by self administration. coated microneedles have been the most extensively studied technique for id microneedle vaccination (figs. a, b) . using this approach, vaccine forms a solidstate coating on the surface of solid microneedles that dissolves off within the skin upon application. typically, this method provides a bolus delivery of a sub-milligram dose of antigen within minutes of application, which is often suitable for delivery of vaccines. an effective microneedle coating process typically involves dip-coating metal microneedles in a coating solution containing the vaccine, a surfactant to promote wetting of the microneedle surface, and a viscosity enhancer to increase coating thickness (gill and prausnitz b; gill and prausnitz a) . using this technique, compounds over a large range of sizes including small molecules, proteins, dna, and virus particles have been coated onto microneedles. novel coated microneedle designs for improved delivery have been demonstrated, such as the three-dimensional grooves-embedded microneedle (han et al. ) and the pocketed microneedle (gill and prausnitz ) . the first id vaccination using coated microneedles delivered ovalbumin as a model protein antigen to (courtesy of university of queensland). b array of solid stainless steel microneedles coated with yellow dye. each mm by mm device contains microneedles measuring lm tall. inset shows magnified view of two coated microneedles (courtesy of georgia institute of technology). c dissolving microneedles shown intact before insertion into skin, partially dissolved min after insertion into skin and fully dissolved min after insertion into skin (reproduced from (sullivan et al. ); courtesy of georgia institute of technology) hairless guinea pigs (matriano et al. ; widera et al. ) . in these studies, id microneedle vaccination showed a better immune response than an equivalent sc or im injection at low dose. the investigators also found that immune response by microneedle vaccination was dose-dependent. among the various vaccine candidates, influenza vaccine has received the most attention by id immunization using small arrays of coated microneedles measuring approximately lm in length (zhu et al. ). microneedles coated with lg of seasonal influenza h n inactivated virus vaccine induced complete protection against lethal virus infection in mice. however, subsequent studies showed that influenza vaccine lost more than % of its antigenicity during the coating process (kim et al. b) . in order to maintain antigenicity, the disaccharide trehalose was added to the coating formulation to serve as a stabilizer. this enabled successful immunizations requiring smaller doses of vaccine ( . lg) as compared to immunizations with similar immune responses by conventional im immunization kim et al. ) . coated microneedles also showed improved thermal stability of vaccine compared to the liquid form of vaccine (kim et al. b ). more detailed studies showed that coated microneedle vaccination with inactivated influence virus vaccine induced similar antibody igg response, hai titer, and neutralizing activity as conventional im immunization in mice kim et al. b ). to account for antigenic changes to the vaccine during the coating process, vaccine coated on microneedles was dissolved off the needles and then delivered im by injection. in this case, vaccination using microneedles showed a better primary immune response than corresponding im immunization using the same antigen formulation ). vaccination by coated microneedles induced robust immunity to influenza after challenge in a mouse model (kim et al. ) . notably, microneedle-immunized mice were shown to have undetectable levels of influenza virus titer in their lungs after challenge, unlike im immunized mice, which had virus titers at least -fold higher. additional assays for immune response from corresponding lung samples such as lung cytokine and lung igg also consistently showed microneedle immunization to be superior to im. as evidence for microneedle-enhanced immune system memory response, the microneedle immunized group was found to have significantly higher levels of total igg and isotypes igg and igg a postchallenge than pre-challenge, but antibody levels in im immunized mice were lower post-challenge than pre-challenge (kim et al. ). in addition to improved humoral immunity, coated microneedles also induced cellular recall response such as mhc ii-associated cd + t helper cell response ). finally, microneedle immunization performed using a different strain of influenza (h n ) virus vaccine induced similar complete protection against lethal challenge (koutsonanos et al. ). studies using virus-like particle (vlp) vaccine coated on microneedles were also performed. the vlp dose was controlled using a coating formulation including antigen concentration and a number of coating dips (kim et al. a) . when a . lg dose of vlp was delivered, microneedle vaccination induced a stronger immune response than im, as measured by igg, igg subtype (igg , igg a, igg b), hai, neutralizing activity, lung igg, lung cytokine, and more suppression of lung virus infection. microneedle immunization by vlp showed complete protection from a lethal viral challenge without major body weight loss, unlike im after the same dose, which partially protected mice from lethal viral infection ( %) and caused significant body weight loss (quan et al. ) . a novel approach to coated microneedles involved the use of polyphosphazene (pcpp), which served as both an effective coating excipient and an immune adjuvant ). id microneedle immunization with hepatitis b surface antigen (hbsag) in pigs using the pcpp coating formulation was superior in inducing antigen-specific igg compared to id injection by hypodermic needle with or without pcpp. another study demonstrated effective generation of cellular immune responses to a hepatitis c dna vaccine administered to mice using coated microneedles (gill et al. ) . other studies have sought to specifically target delivery to antigen-presenting langerhans cells using extremely short (* lm) needles that penetrate only into the epidermis. these short needles were coated using a novel coating process involving gas-jet drying (chen et al. ). in an initial study, vaccination with ovalbumin-coated needles induced similar immune response to im immunization. in a follow-up study, microneedles coated with a low dose of hemagglutinin-based influenza vaccine generated a similar immune response as im vaccination at a -times larger dose. the authors proposed that these short, densely packed microneedles could deliver more than half of the antigen directly to antigen-presenting cells such as epidermal langerhans cells and dermal dendritic cells (fernando et al. ) . methods for long-term vaccine storage without significant immunogenicity loss, especially without refrigeration, are important for vaccination campaigns. microneedles are coated with vaccine in the solid state, which is expected to confer thermal stability. in a stability study of microneedles coated with inactivated influenza vaccine, mice immunized with coated microneedles stored at room temperature for month produced similar igg responses to those of mice immunized by microneedles stored for day. furthermore, both groups were completely protected from lethal challenge after viral infection. in vitro assay of the microneedles, however, showed a decrease in antigenicity by about % (kim et al. c ). as an improvement over coated microneedles, dissolving microneedles have been developed in order to eliminate sharp, biohazardous waste after vaccination (fig. c) . unlike non-dissolving (e.g., metal) microneedles coated with a vaccine formulation, dissolving microneedles are made solely of material such as polymers or sugars that will safely dissolve in the skin after insertion, which leaves behind only the microneedle patch backing. typically, the vaccine is incorporated into the matrix of the microneedle and is released into the skin upon microneedle dissolution. dissolving microneedles have been made using a number of different materials, including polyvinylpyrrolidone (sullivan et al. ) , maltose (kolli and banga ) , carboxymethylcellulose (lee et al. ), polylactic and/or polyglycolic acid (park et al. ; park et al. ) and dextrin (ito et al. ). in a recent study, dissolving microneedles were prepared by encapsulating inactivated influenza vaccine in a polyvinylpyrrolidone matrix and used to immunize mice. the vaccine was gently encapsulated without significant damage to immunogenicity and was shown to generate similar antibody and cellular immune responses compared to im injection of the same dose and provided complete protection against lethal challenge. compared to im injection, dissolving microneedle vaccination resulted in more efficient lung virus clearance and enhanced cellular recall responses after challenge (sullivan et al. ). theraject has also developed biodegradable microneedles using carboxymethylcellulose containing various biomolecules including influenza vaccine (oh et al. ). as a simpler, albeit probably less efficient, method, microneedles can be used to pierce the skin to make it more permeable and thereby enable entry of topically applied vaccines. this method is attractive because the micro-scale pores made by microneedle insertion are generally too small for penetration of microorganisms (donnelly et al. ), yet large enough for delivery of sub-unit and possibly viral vaccines. after insertion and removal of the microneedles, vaccine can be applied using a patch or other topical formulation for slow delivery by diffusion through long-lived pores (kalluri and banga ) . this approach was investigated for transcutaneous vaccination using diphtheria toxoid and influenza vaccine (ding et al. a; ding et al. b) . when diphtheria toxoid was applied to microneedle-pretreated skin in combination with cholera toxin adjuvant, a similar immune response was induced compared to sc injection. however, microneedle pretreatment did not enhance immune response for influenza vaccine. this vaccination approach has also been studied in an ex vivo human skin model to investigate skin immune cell responses (ng et al. ). using a related approach, blunt-tipped microneedles were used to scrape the skin, thereby making microtroughs in the skin through which a dna vaccine encoding hbsag was administered (mikszta et al. ) . this approach generated stronger humoral and cellular immune responses than im or id injection. tattoo guns use high-frequency oscillating needles to make thousands of punctures in the skin, which is conventionally used to deposit tattoo ink in the dermis, but has been adapted to deliver id vaccines (fig. d) . in one study, hemagglutininexpressing dna vaccine was administered to pigs and derived humoral and protective immunity as shown by methods including hai titer and improved virus clearance from nasal swabbing (eriksson et al. ) . to overcome the slow processing of an immune response induced by dna vaccination, dna tattooing was suggested for short-interval dna vaccination (bins et al. ) . in this study, it was shown that short-interval id dna tattoo immunization generated fast and stable t cell responses to human papillomavirus and complete protection from influenza virus challenge. when compared to the im route, dna tattoo vaccination elicited much stronger and quicker humoral and cellular immune responses. in addition, studies indicated that even im immunization with adjuvant was inferior to dna tattoo immunization (pokorna et al. ). to determine the effect of the tattooing process on dna vaccine stability, the dna topology change was evaluated, including critical factors for antigen expression and immune response (quaak et al. ). it was found that the dna tattooing tool had negligible effect on dna structure and activity. other vaccines including an adenoviral vector vaccine against respiratory syncytial virus (potthoff et al. ) and a peptide vaccine against human papillomavirus (pokorna et al. ) were administrated by id tattooing. in the case of the adenoviral vector vaccine, tattooing showed similar performance to id injection. tattooing of the peptide vaccine with cpg motifs adjuvant showed better response than im vaccination with adjuvant. dna tattooing was evaluated in non-human primates, which have previously shown poor dna vaccine immunization effect, but showed remarkable enhancement of immune response by this method administering an hiv vaccine (verstrepen et al. ) . in order to advance this technique to human clinical trials, a human ex vivo skin model was tested, which showed that dna concentration was the most critical factor for effective dna vaccination by tattooing (van den ). a human clinical trial for treating melanoma is planned (quaak et al. ) . a comprehensive review on dna tattooing can be found in one of the accompanying papers in this special volume on id immunization (oosterhuis et al. ) . most of the id vaccination methods described so far involve minimally invasive needle-based methods or non-invasive jet-based methods that actively deposit vaccine within the skin. another set of approaches involve mostly non-invasive methods that increase skin permeability to enable vaccine transport into the skin in a transiently permeabilized state. the key to success using these approaches is disruption of skin's outer layer, called stratum corneum. although the stratum corneum is only - lm thick, it provides a highly effective barrier to the permeation of xenogens, including topically applied vaccine formulations (scheuplein and blank ) . a number of methods to increase skin permeability have been developed, largely for drug delivery applications, many of which have been tested for vaccination (mitragotri ; prausnitz and langer ) . a number of studies have demonstrated that the skin barrier can be broken by abrasion. a variety of abrasion methods including rough surfaces (frerichs et al. ) , tape-stripping (takigawa et al. ; peachman et al. ; inoue and aramaki ; vandermeulen et al. ), and microdermabrasion devices ) have been shown to induce adequate removal of the stratum corneum. repeated peeling by tape (for example, scotch Ò tape) effectively removes the stratum corneum. application of tumor epitope peptides on tape-stripped mouse skin primed tumor-specific cytotoxic t cells in the lymph nodes and the spleen, protected mice against a subsequent challenge with the corresponding tumor cells, and also suppressed the growth of established tumors (takigawa et al. ) . skin abrasion using a razor and a toothbrush followed by application of adenoviral vectors has yielded promising results in humans (van kampen et al. ) . skin abrasion using an abrasive paper is perhaps the most commonly used method of disrupting the stratum corneum for immunization. for example, abrasion with emery paper, after skin hydration, has been shown to induce adequate penetration of anthrax vaccine (matyas et al. ) and influenza virus vaccine (guebre-xabier et al. ) , among others. this has led to the development of a skin prep system (sps) to provide a controlled method of stratum corneum disruption for transcutaneous immunization currently under development by intercell (frerichs et al. ) (fig. a ). this technique has been shown to be effective in humans. specifically, the skin was prepared by use of two mild strokes with the skin preparation device containing a mild abrasive affixed to a pressure-controlled device. the device was a single-use, disposable system and was discarded immediately after use. following skin preparation, the patch containing vaccine against traveler's diarrhea (lt patch) was applied within the marked area and worn for h at each vaccination, then removed and discarded by the participant. lt-patch recipients were protected against moderate-to-severe diarrhea (protective efficacy of %) and severe diarrhea (protective efficacy of %). lt-patch recipients who became ill had shorter episodes of diarrhea ( . vs . days) with fewer loose stools than placebo . in another study, a similar technique was used to boost response against influenza vaccine. in this case, prior to application, the patch area was lightly abraded with ecg-grade emery paper on skin wetted with % glycerol/ % alcohol to disrupt the stratum corneum. in weeks following vaccination, hemagglutination inhibition (hai) responses in lt immunostimulatory patch recipients showed improvement over those receiving vaccine alone (frech et al. ) . microdermabrasion is a common cosmetic procedure that has been adapted to remove superficial skin layers by sandblasting and thereby enable selective removal of the stratum corneum barrier. this approach has been shown to increase skin permeability and thereby enable topical application of live attenuated vaccinia virus on microdermabraded skin to generate virus-specific antibodies in the blood ). as mentioned in sect. . . , a microneedle-based abrasion method has also been successfully used for vaccination. ultrasound, especially at low frequencies, is very effective in permeabilizing the skin (tezel et al. ) . it is now understood that acoustic cavitation, which is formation, pulsation, and collapse of gaseous bubbles under the oscillating pressure field of ultrasound, is the principal mediator for ultrasound-induced enhanced skin permeability. several studies have shown that during ultrasound exposure, transient cavitation is predominantly induced in the coupling medium (the liquid present between the ultrasound transducer and the skin) and is primarily responsible for skin permeabilization (tang et al. ; tezel et al. ; tezel and mitragotri ). an estimated bubble collapses/s/cm in the form of symmetric collapses fig. skin permeabilization methods. a skin abrasion device, in which a sandpaper device is placed on the skin ( ), scraped across the skin in a controlled fashion ( ) and then a vaccine patch is applied to the abraded skin ( ) (courtesy of intecell). b hand-held skin electroporation device, which uses microneedles as electrodes to cause highly localized electroporation in the skin to facilitate dna vaccine delivery into skin cells (courtesy of cyto pulse sciences). c heat-based device for thermal ablation of the skin. the microheater array (left side of inset) is used to ablate the skin and then a vaccine patch (right side of inset) is applied to the ablated skin (courtesy of altea therapeutics) (generating shock waves) or asymmetric collapses (producing microjets) near the surface of the skin are sufficient to explain the experimentally-observed skin permeability enhancements by ultrasound-induced skin permeabilization. ultrasound has been shown to enhance the delivery of vaccines into skin (tezel et al. ) . studies performed in mice have shown that the immune response generated by ultrasonically delivered vaccine was about -fold greater compared with sc injection per unit dose of the vaccine that entered the skin (about % of the topically applied dose entered the skin) (tezel et al. ) . compared to simple topical administration, ultrasound pretreatment showed increased vaccine delivery, thereby enabling sufficient vaccine to enter the skin to activate the immune response. furthermore, application of ultrasound resulted in activation of langerhans cells, the reasons behind which are not clear. in another study, it was shown that application of tetanus toxoid to skin pretreated with ultrasound generated anti-tetanus toxoid igg and neutralizing antibody titers (dahlan et al. ). several parameters, including concentration of co-applied sodium dodecyl sulfate and ultrasound duty cycle, impacted the magnitude of antibody titers. the authors concluded that the main mechanism of ultrasound-assisted skin immunization involved factors in addition to enhancement of skin permeability to topically applied antigen. electroporation involves the application of high-voltage, short-duration electric pulses to transiently disrupt lipid barriers in the body. for vaccination, electroporation has been used to increase stratum corneum permeability and thereby enable vaccine entry into the skin. electroporation has also been used to permeabilize cells within the skin and thereby drive, for example, dna vaccines into epidermal and dermal cells (fig. b) . electroporation has been well established as a tool for delivering molecules across the stratum corneum (prausnitz et al. ) or across the cell membranes (bilitewski et al. ) . many studies have focused on the use of electroporation for dna vaccination. this is not surprising given the long history of use of electroporation for delivery of dna into cells in vitro. however, many electroporation studies involve insertion of electrode needles into the skin. some studies have demonstrated the use of electroporation for topical vaccine delivery (zhao et al. ) . in one study, electroporation has been found to stimulate the exodus of langerhans cells from the skin, which is likely to have an adjuvant-like effect (zhao et al. ) . in this study, the efficacy of peptide delivery was found to be comparable to that of id injected with freund's complete adjuvant. further, the peptide-specific ctl response to the vaccine delivered by electroporation was equivalent to that delivered by id injection. electroporation has been shown to induce an effective immune response after delivery of dna vaccines (peachman et al. ; foldvari et al. ; medi and singh ; vandermeulen et al. ) . for example, studies in pigs have shown the ability of electroporation to deliver hbsag gene using a single-needle or a six-needle electrode (babiuk et al. ) . studies have demonstrated that in vivo skin electroporation may be used to increase transgene expression relative to naked dna injection (drabick et al. ) . transfected cells were principally located in dermis and included adipocytes, fibroblasts, endothelial cells, and numerous mononuclear cells with dendritic processes in a porcine model. transfected cells were also observed in lymph nodes draining electropermeabilized sites. a hbsag-coding plasmid was used to test skin electroporation-mediated nucleic acid vaccination in a murine model. applications for these findings include modulation of immune responses to pathogens, allergens, and tumor-associated antigens and the modification of tolerance. in another study, in vivo electroporation has shown protection against avian influenza in nonhuman primates (laddy et al. ). a number of human clinical trials testing vaccination enhanced by electroporation are currently under way. several chemicals are known to interact with the skin and disrupt the highly ordered lipid bilayer structure in the stratum corneum. this observation led to the study of chemical agents to enhance transport across skin. more than chemicals have been studied for their ability to increase skin permeability (karande et al. ) . chemical permeation enhancers are relatively inexpensive and easy to formulate, they offer flexibility in their design, are simple in application and allow the freedom of self-administration to the patient. chemical enhancers comprise a wide variety of different chemical functional groups and facilitate drug transport across the skin by a variety of complex mechanisms. they can directly exert their effect on skin structure by acting on intercellular lipids or corneocytes. chemical enhancers can extract lipids from the skin thereby creating diffusion pathways for transdermal permeation. alternatively, they can partition themselves into the lipid bilayers thereby disrupting the highly ordered lipid lamellae and causing their fluidization. chemical enhancers can also significantly increase skin transport of a drug by enhancing its thermodynamic activity in the formulation (karande et al. ) . recently, chemical enhancers have been shown to possess the ability to deliver antigens and generate immune responses. this was achieved by designing formulations that possess the ability to enhance skin permeability as well as exhibit high adjuvanticity. the rational design of such multi-functional formulations from first principles requires in-depth knowledge of interactions between chemical enhancers and skin, which exist for a very limited pool of chemicals. hence, combinatorial libraries of chemical mixtures were screened. studies have shown that in a randomly selected population of chemical formulations, certain binary mixtures of chemicals are far more potent in permeabilizing the skin as compared to single chemicals (karande et al. ) . in vaccination studies, a third chemical was added with the goal of enhancing the ability to offer adjuvanticity. the lead chemical formulations were tested in mice using the model antigen ovalbumin. the formulations that exhibited high permeation and adjuvanticity potential in in vitro screening also induced high igg titers in mice (karande et al. ). in another study, penetration enhancers and immunomodulators oleic acid and retinoic acid were used to enhance transcutaneous immunization with inactivated influenza virus across tape-stripped skin (skountzou et al. ) . pretreatment of mouse skin with oleic acid elicited increased levels of influenza virus-specific binding and neutralizing antibodies to levels equivalent to those induced by intranasal immunization with inactivated influenza virus. oleic acid and retinoic acid treatments differentially affected the pattern of cytokine production upon stimulation with influenza viral antigen and provided enhanced protection. thermal poration of skin has been used to deliver vaccines into skin. microporation systems are designed to porate the skin and are being developed by a number of companies. in this method, an array of micropores is created in the skin by removal of stratum corneum by the application of focused thermal energy based on resistive heating via the contact of electrically heated small-diameter wires to the skin surface (bramson et al. ) (fig. c ) or other methods based on radiofrequency or laser-based approaches. in this study, the microporation tip was comprised of a set of lm diameter tungsten wires with control circuitry allowing for precise control of the electrical current pulses that were passed through each wire. the software user interface was designed to enable the control of various microporation parameters including micropore density, resistive element temperature, current pulse width, number of pulses, pulses pacing, and contact pressure. the temperature of the tip that was placed in contact with the skin was calibrated by an optical calibrator device. the study showed that microporation significantly increased the penetration of topically delivered vaccine. microporation enhanced expression of luciferase upon placement of adenovirus vectors by - -fold. the same procedure led to increased ctl response and increased ifn-c secreting cells. in a related study, the same technology has been shown to deliver influenza vaccine into mouse skin. eighty micropores were created in cm area and the vaccine was placed on the porated skin. this procedure generated adequate protective response in mice (garg et al. ). id vaccination offers potential immunologic advantages to public health. the skin is known to be a site rich in antigen-presenting cells, some of which are specific to the skin, including epidermal langerhans cells and dermal dendritic cells (glenn and kenney ) . in addition, antigen may be taken up directly by lymphatic vessels for transport to antigen-presenting cells in the lymph nodes. at a minimum, the id route of vaccination appears to follow different pathways to immunity compared to im or sc routes. however, there is evidence that the id route is not only different, but is also beneficial (glenn and kenney ; lambert and laurent ; nicolas and guy ) . the possibility of dose sparing enabled by id vaccination has been suggested by previous preclinical and clinical studies; however, the successful application of this approach has yet to be definitively confirmed for many vaccines (glenn and kenney ; lambert and laurent ; nicolas and guy ) . although the conclusions vary between different studies and different vaccines, there is an indication that dose sparing may be possible. however, it is not currently clear under what conditions the skin's unique immune environment can be harnessed for optimal effect. in addition to dose sparing, there is preclinical study evidence of other beneficial differences of id vaccination. studies with microneedles showed improved influenza virus clearance from the lungs and enhanced memory responses compared to im vaccination (kim et al. ) . studies with epi showed a specific role for langerhans cells to generate robust antibody responses . studies with ultrasound-mediated vaccination suggested an adjuvant effect on the skin (dahlan et al. ). id vaccination offers potential value to public health also in terms of possible logistical advantages. for comparison, im and sc vaccination can only be carried out by hypodermic needle injection with few other options beside jet injection. id vaccination opens the door to many other technologies because the skin is readily accessible at the surface of the body. as a result, id injection may enable vaccination methods that generate no biohazardous sharp waste, can be administered by personnel with minimal training, and simplify transportation and storage logistics (table ) . mantoux technique injection requires specialized training by clinical personnel. microneedle systems and patch-based delivery (accompanied by skin permeabilization technologies) offer the promise of simplified vaccination methods that require minimal training and may permit self-vaccination by patients in certain scenarios. this not only benefits routine vaccination scenarios, but is especially important to mass vaccination campaigns associated with disease eradication programs or pandemic emergencies. in contrast, some of the novel id delivery methods, such as projectile delivery and tattoo guns, introduce new, sophisticated devices that require additional training of clinical personnel. assuming the injection is done properly, the mantoux technique can administer essentially all of the vaccine into the skin. hollow microneedles and projectile delivery can be similarly efficient. however, solid microneedles typically retain some vaccine on the device and patch-based skin permeabilization methods are extremely inefficient, such that most vaccine typically remains on the skin surface. the efficiency of vaccine utilization will be of critical importance for new, costly vaccines, as well as in developing countries where vaccine cost can be a significant barrier to access. eliminating the hypodermic needle from vaccination is a major objective of public health, given that close to one million people die each year from disease transmission from contaminated needles (miller and pisani ; kermode ) . microneedles are a step in the right direction, but still generate biohazardous sharp waste, with the exception of dissolving microneedles. projectile delivery and patchbased methods eliminate needles and therefore offer an improved safety profile. thermal ablation +++ + +++ + + ++ a +++ requires little or no personnel training, ++ requires personnel training, + requires personnel training and maintenance of a dedicated device b +++ almost % in skin, ++ [ % in skin, + \ % in skin c +++ no biohazardous sharp waste, ++ microscopic biohazardous sharp waste, + macroscopic biohazardous sharp waste d +++ in widespread clinical practice, ++ published vaccination data in humans, + preclinical e +++ no reformulation required, ++ possible new liquid formulation required, + reformulation required to produce solid-state vaccine f +++ inexpensive disposable device, ++ specialty disposable device, + reusable device. perinjection cost of reusable devices will depend on the number of times the device can be used and the cost of any disposable components however, some id delivery methods can cause added tissue trauma to the skin (bremseth and pass ) . most of the new methods of id vaccination require significant technology development. while jet injection is already in widespread clinical use, many other technologies are only in the preclinical stage of development for vaccination. that being said, many of those technologies are in much later stage of development or use for non-vaccine applications, which will facilitate their adaptation to id vaccination. most of the new id vaccination technologies also require vaccine reformulation. hollow microneedle, jet and tattoo-based methods may use standard, currently available liquid formulation, but in some cases will need to be concentrated or otherwise modified. the other methods mostly use a solid-state vaccine formulation, which offers likely advantages in terms of vaccine stability during storage, but, however, requires significant reformulation, with associated research, regulatory, and manufacturing hurdles. finally, device cost is a significant consideration, given that a hypodermic needle and syringe are extremely inexpensive, disposable devices. microneedle systems and some of the patch-based methods are expected to have low manufacturing cost in mass production. however, many of the other technologies require multiple device components, which may be engineered into disposable devices with added cost or reusable devices with disposable components that require an initial investment that can be amortized over many patients. id vaccination has already made significant impact on public health as the primary means of immunization during smallpox eradication and continues to play a role in bcg and rabies vaccination in current clinical practice (plotkin et al. ) . however, as discussed in this article, there are many more opportunities for id vaccination to potentially improve immunogenicity and simplify logistics of the administration of other vaccines. a number of new id vaccination technologies have been successful in human clinical trials. id vaccination using the bd hollow microneedle was approved in europe in for id administration of the sanofi pasteur seasonal influenza vaccine and was introduced in australia and new zealand during the influenza season (holland et al. ; beran et al. ). this microneedle device may be adapted for use to administer other vaccines as well. jet injectors have a long history of use for vaccination and are receiving renewed attention for id delivery of vaccines in clinical trials, especially to address developing countries' needs, through support from who and us centers for disease control and prevention (see sect. . . .). skin abrasion as a pretreatment before applying a vaccine patch is also in clinical trials for prevention of influenza and traveler's diarrhea (frech et al. ; frech et al. ) . projectile based delivery by epi and pmed have been studied in a number of human clinical trials for both dna and protein-based vaccines jones et al. ), although it is unclear as to what extent this technology is under continued commercial development. other id delivery devices are under advanced preclinical study. solid coated microneedles have been the subject of numerous vaccination studies in mice and larger animals to administer influenza and other vaccines (see sect. . ), and have been used in a phase ii clinical trial of a drug, parathyroid hormone (cosman et al. ). likewise, skin electroporation, in some cases in combination with microneedles, has been studied in animals for skin vaccination. as evidence for clinical feasibility, electroporation of skin for targeted delivery of chemotherapeutic agents to skin tumors is approved and used in europe (gehl ) . tattooing is of course in widespread human use, and its application to vaccination has been studied preclinically. other methods to increase skin permeability, such as ultrasound, chemical enhancers and heat, are also in clinical use or trials for transdermal drug delivery applications (prausnitz and langer ) , which compliment preclinical studies of their use for vaccination. given the large number of technologies for id vaccination under development, and the advanced clinical status of many of them, the future outlook for bringing id vaccination into more widespread clinical practice appears encouraging. the optimal delivery method will depend on the specific application and other factors, such as immunologic response, logistical needs, and financial constraints. preclinical evaluation of microneedle technology for intradermal delivery of influenza vaccines poly [di (carboxylatophenoxy) phosphazene] is a potent adjuvant for intradermal immunization intradermal influenza vaccine for older adults: a randomized controlled multicenter phase iii study hepatitis b vaccine in developing countries: problems and prospects electroporation improves the efficacy of dna vaccines in large animals smallpox vaccination techniques; from knives and forks to needles and pins serum antibody responses after intradermal vaccination against influenza comparative immunogenicity of trivalent influenza vaccine administered by intradermal or intramuscular route in healthy adults intradermal influenza vaccination of healthy adults using a new microinjection system: a -year randomised controlled safety and immunogenicity trial human diploid cell rabies vaccine. effectiveness of immunization with small intradermal or subcutaneous doses preexposure immunization with intradermal human diploid cell rabies vaccine. risks and benefits of primary and booster vaccination biochemical analysis with microfluidic systems a rapid and potent dna vaccination strategy defined by in vivo monitoring of antigen expression enabling topical immunization via microporation: a novel method for pain-free and needle-free delivery of adenovirus-based vaccines delivery of insulin by jet injection: recent observations inadequate response to intradermal hepatitis a vaccine measles vaccination by the intradermal route hepatitis a vaccination by intracutaneous low dose administration: a less expensive alternative vaccines for preventing hepatitis b in health-care workers epidermal powder immunization with a recombinant hiv gp targets langerhans cells and induces enhanced immune responses epidermal powder immunization: cellular and molecular mechanisms for enhancing vaccine immunogenicity dry-coated microprojection array patches for targeted delivery of immunotherapeutics to the skin suboptimal response following intradermal hepatitis b vaccine in infants effect of transdermal teriparatide administration on bone mineral density in postmenopausal women transcutaneous immunisation assisted by low-frequency ultrasound bcg vaccination by bifurcated needle in a pilot vaccination program hollow metal microneedles for insulin delivery to diabetic rats intradermal administration of measles vaccines epidermal powder immunization against influenza cutaneous delivery of a live, attenuated chimeric flavivirus vaccine against japanese encephalitis (chimerivax)-je) in non-human primates study of specific immune response to unadsorbed concentrated tetanus vaccine administered by intradermal route to non-immunized persons in the last ten years immune modulation by adjuvants combined with diphtheria toxoid administered topically in balb/c mice after microneedle array pretreatment microneedle arrays for the transcutaneous immunization of diphtheria and influenza in balb/c mice microneedle arrays allow lower microbial penetration than hypodermic needles in vitro cutaneous transfection and immune responses to intradermal nucleic acid vaccination are significantly enhanced by in vivo electropermeabilization epidermal dna vaccine for influenza is immunogenic in humans safety, tolerability, and lack of antibody responses after administration of a pfcsp dna malaria vaccine via needle or needle-free jet injection, and comparison of intramuscular and combination intramuscular/ intradermal routes in vivo gene transfer to skin and wound by microseeding safety and efficacy of transcutaneous vaccination using a patch with the liveattenuated measles vaccine in humans potent immunity to low doses of influenza vaccine by probabilistic guided micro-targeted skin delivery in a mouse model rabies preexposure prophylaxis with human diploid cell rabies vaccine: a dose-response study influence of needle gauge in mantoux skin testing dna delivery for vaccination and therapeutics through the skin improved immune responses to influenza vaccination in the elderly using an immunostimulant patch use of a patch containing heat-labile toxin from escherichia coli against travellers' diarrhoea: a phase ii, randomised, double-blind controlled, single-step, stratum corneum disruption as a pretreatment for immunization via a patch clinical responses to undiluted and diluted smallpox vaccine silicon micromachined hollow microneedles for transdermal liquid transport needle-free skin patch delivery of a vaccine for a potentially pandemic influenza virus provides protection against lethal challenge in mice electroporation for drug and gene delivery in the clinic: doctors go electric coated microneedles for transdermal delivery coating formulations for microneedles pocketed microneedles for drug delivery to the skin selective removal of stratum corneum by microdermabrasion to increase skin permeability cutaneous vaccination using microneedles coated with hepatitis c dna vaccine mass vaccination: solutions in the skin still-blinded safety and immunogenicity data from a trial of reduced-dose, intradermal, influenza vaccination by needle-free jet injection antibody response to influenza vaccination in the elderly: a quantitative review immunostimulant patch containing heat-labile enterotoxin from escherichia coli enhances immune responses to injected influenza virus vaccine through activation of skin dendritic cells improvement in antigen-delivery using fabrication of a grooves-embedded microneedle array low-dose ( micrograms) hepatitis b vaccination in medical students: comparable immunogenicity for intramuscular and intradermal routes smallpox and vaccinia a new recombinant bacille calmette-guerin vaccine safely induces significantly enhanced tuberculosis-specific immunity in human volunteers intradermal influenza vaccine administered using a new microinjection system produces superior immunogenicity in elderly adults: a randomized controlled trial bevel-down superior to bevel-up in intradermal skin testing toll-like receptor- expression induced by tape-stripping triggers on effective immune response with cpg-oligodeoxynucleotides feasibility of microneedles for percutaneous absorption of insulin dna vaccination protects against an influenza challenge in a double-blind randomised placebo-controlled phase b clinical trial formation and closure of microchannels in skin following microporation discovery of transdermal penetration enhancers by highthroughput screening design principles of chemical penetration enhancers for transdermal drug delivery transcutaneous immunization using common chemicals unsafe injections in low-income country health settings: need for injection safety promotion to prevent the spread of blood-borne viruses improved influenza vaccination in the skin using vaccine coated-microneedles fomulation of microneedles coated with influenza virus-like particle vaccine enhanced memory responses to seasonal h n influenza vaccination of the skin with the use of vaccine-coated microneedles formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity stability kinetics of influenza vaccine coated onto microneedles during drying and storage measles immunization with further attenuated heatstable measles vaccine using five different methods of administration characterization of solid maltose microneedles and their use for transdermal delivery transdermal influenza immunization with vaccine-coated microneedle arrays electroporation of synthetic dna antigens offers protection in nonhuman primates challenged with highly pathogenic avian influenza virus intradermal vaccine delivery: will new delivery systems transform vaccine administration? evaluation of the clinical performance of a new intradermal vaccine administration technique and associated delivery system dissolving microneedles for transdermal drug delivery drawing lithography: three-dimensional fabrication of an ultrahigh-aspect-ratio microneedle seasonal influenza vaccine delivered by intradermal microinjection: a randomised controlled safety and immunogenicity trial in adults integrated lithographic molding for microneedle-based devices tuberculin intradermo reactions in the treatment of tuberculosis: intradermituberculisation macroflux Ò microprojection array patch technology: a new and efficient approach for intracutaneous immunization needle-free skin patch vaccination method for anthrax delivery of dna into skin via electroporation improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery protective immunization against inhalational anthrax: a comparison of minimally invasive delivery platforms microneedle-based intradermal delivery of the anthrax recombinant protective antigen vaccine status of eradication of smallpox (and control of measles) in west and central africa the cost of unsafe injections low dose hepatitis b vaccination in children immunization without needles fractional doses of inactivated poliovirus vaccine in oman development of an ex vivo human skin model for intradermal vaccination: tissue viability and langerhans cell behaviour intradermal, epidermal and transcutaneous vaccination: from immunology to clinical practice immune response of infants to fractional doses of intradermally administered inactivated poliovirus vaccine intradermal influenza vaccine delivery using skin-penetrating dissolvable vaccine microneedles dna vaccines and intradermal vaccination by dna tattooing immune responses to hepatitis b surface antigen following epidermal powder immunization reduced-dose intradermal vaccination against hepatitis a with an aluminum-free vaccine is immunogenic and can lower costs biodegradable polymer microneedles: fabrication, mechanics and transdermal drug delivery polymer microneedles for controlled-release drug delivery jet gun or syringe? a trial of alternative methods of bcg vaccination intradermal delivery of vaccines: a review of the literature and the potential for development for use in low-and middle-income countries. path/who comparison of bcg inoculation by conventional intradermal and jet methods immunization with dna through the skin sharp beveled tip hollow microneedle arrays fabricated by liga and d soft lithography with polyvinyl alcohol what happens if intradermal injections of rabies vaccine are partially or entirely injected subcutaneously? vaccination with human papillomavirus type -derived peptides using a tattoo device immunogenicity and efficacy of intradermal tattoo immunization with adenoviral vector vaccines microneedles for transdermal drug delivery transdermal drug delivery electroporation of mammalian skin: a mechanism to enhance transdermal drug delivery microneedle-based vaccines gmp production of pdermatt for vaccination against melanoma in a phase i clinical trial dna tattoo vaccination: effect on plasmid purity and transfection efficiency of different topoisoforms stabilization of influenza vaccine enhances protection by microneedle delivery in the mouse skin intradermal vaccination with influenza vlps using microneedles induces superior protection to intramuscular immunization cellular and humoral immune responses induced by intradermal or intramuscular vaccination with the major hepatitis b surface antigen randomized controlled clinical trial of fractional doses of inactivated poliovirus vaccine administered intradermally by needle-free device in cuba clinical safety and efficacy of a powdered hepatitis b nucleic acid vaccine delivered to the epidermis by a commercial prototype device intradermally administered yellow fever vaccine at reduced dose induces a protective immune response: a randomized controlled non-inferiority trial intradermal vaccination to protect against yellow fever and influenza penetration-enhanced ultrasharp microneedles and prediction on skin interaction for efficient transdermal drug delivery induction of antigen-specific cd ? t cells, t helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis b virus dna vaccine immune response to intradermally injected inactivated poliovirus vaccine immune response of infants to inactivated poliovirus vaccine injected intradermally intradermal hepatitis b vaccination: a systematic review and meta-analysis permeability of the skin needle-free jet injections: dependence of jet penetration and dispersion in the skin on jet power transcutaneous immunization with inactivated influenza virus induces protective immune responses diphtheria-tetanus-pertussis immunization by intradermal jet injection minimally invasive protein delivery with rapidly dissolving polymer microneedles dissolving polymer microneedle patches for influenza vaccination status of gpei research and product development. manufacturer's meeting, geneva percutaneous peptide immunization via corneum barrier-disrupted murine skin for experimental tumor immunoprophylaxis an investigation of the role of cavitation in low-frequency ultrasound-mediated transdermal drug transport interactions of inertial cavitation bubbles with stratum corneum lipid bilayers during low-frequency sonophoresis frequency dependence of sonophoresis investigations of the role of cavitation in low-frequency sonophoresis using acoustic spectroscopy low-frequency ultrasound as a transcutaneous immunization adjuvant safety and efficacy of a novel microneedle device for dose sparing intradermal influenza vaccination in healthy adults optimization of intradermal vaccination by dna tattooing in human skin safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans effect of tape stripping and adjuvants on immune response after intradermal dna electroporation improved hiv- specific tcell responses by short-interval dna tattooing as compared to intramuscular immunization in non-human primates precise microinjection into skin using hollow microneedles hollow polymer microneedle array fabricated by photolithography process combined with micromolding technique intradermal rabies vaccination: the evolution and future of pre-and postexposure prophylaxis alternative vaccine delivery methods immunisation of - month old gambian infants with edmonston-zagreb measles vaccine who expert consultation on rabies rabies vaccines. who position paper effect of delivery parameters on immunization to ovalbumin following intracutaneous administration by a coated microneedle array patch system hepatitis a vaccine administration: comparison between jet-injector and needle injection induction of cytotoxic tlymphocytes by electroporation-enhanced needle-free skin immunization immunization by vaccine-coated microneedle arrays protects against lethal influenza virus challenge immune response to single dose, multisite, intradermal and to intramuscular administration of vaccine against tick-borne encephalitis virus different immune responses after intradermal and intramuscular administration of vaccine against tick-borne encephalitis virus key: cord- -d pd zj authors: mettu, ravinder; chen, chiang-yun; wu, chung-yi title: synthetic carbohydrate-based vaccines: challenges and opportunities date: - - journal: j biomed sci doi: . /s - - - sha: doc_id: cord_uid: d pd zj glycoconjugate vaccines based on bacterial capsular polysaccharides (cps) have been extremely successful in preventing bacterial infections. the glycan antigens for the preparation of cps based glycoconjugate vaccines are mainly obtained from bacterial fermentation, the quality and length of glycans are always inconsistent. such kind of situation make the cmc of glycoconjugate vaccines are difficult to well control. thanks to the advantage of synthetic methods for carbohydrates syntheses. the well controlled glycan antigens are more easily to obtain, and them are conjugated to carrier protein to from the so-call homogeneous fully synthetic glycoconjugate vaccines. several fully glycoconjugate vaccines are in different phases of clinical trial for bacteria or cancers. the review will introduce the recent development of fully synthetic glycoconjugate vaccine. carbohydrate-based vaccines have a long history, started from the isolation of capsular polysaccharide of streptococcus pneumonia (pneumococcus) by dochez and avery in [ ] . then, between and , avery and heidelberger at the rockefeller institute conducted a series of studies on capsular polysaccharides (cps) of pneumococcus and discovered the immunogenicity of cps [ ] . in , francis and tillett injected pure pneumococcal polysaccharides to patients and found cps-specific antibodies in those patients [ ] . later studies by finland and ruegsegger furthered the development of pneumococcal capsular polysaccharide vaccines [ ] . from to , heidelberger and his associates developed tetravalent vaccine, and the test in the us army air force was successful [ ] . after several clinical tests of pneumococcal polysaccharides, two variants of pneumococcal vaccines containing six serotypes each were first licensed in usa in [ ] . unfortunately, those two vaccines were discontinued shortly after due to the introduction of new and extremely effective antimicrobial drugs such as penicillin, chlortetracycline, and chloramphenicol [ , ] . from to , the antibiotics dominated the vaccine markets, and most research efforts focused on finding new antibiotics rather than developing vaccines. however, the field of pneumococcal vaccine research was kept alive by the persistent efforts of dr. robert austrian who was supported and motivated by the us national institutes of health (nih) towards the development of possible pneumococcal polysaccharide vaccines [ ] . meanwhile, the emergence of antibiotic resistant bacteria [ ] prompted the redirection of research efforts back to the vaccine development. the unremitting efforts of dr. robert austrian and his colleagues led to the development of -valent and valent pneumococcal cps-based vaccines that were licensed in and , respectively [ , ] . inspired by the success of pneumococcal cps vaccines, the tetravalent (a, c, w and y) meningococcal, the haemophilus influenza type b (hib) and the salmonella typhi vi cps-based vaccine were developed and licensed between and for adults and children older than years in usa [ , ] . although native cps vaccines were effective in controlling the incidence of diseases for people above years of age, there were some troublesome immunological disadvantages. for example, hib cps vaccine elicited poor immune responses in young children below years of age and immune deficient peoples whom are the more prone to infections [ ] . to overcome these issues, vaccine researchers had, then, focused on increasing immunogenicity of oligosaccharides. in , avery and goebel demonstrated that immunogenicity of a capsular polysaccharide can be enhanced by coupling to a carrier protein [ ] . unfortunately, this finding was ignored until robbins and schneerson used hib cps (poly ribosylribitol phosphate) and dt to synthesize a glycoconjugate vaccine that exhibited greater immunogenicity and efficacy in clinical trials and was the first licensed conjugate vaccine for children younger than years in the usa in [ ] . the success of the hib glycoconjugate vaccines, prompted the development of monovalentmeningococcal glycoconjugate vaccines using dt or tt as a carrier proteins to provide longer immune response and higher immunity to children younger than years against serogroup c. further extensive studies produced a quadrivalent conjugate vaccine against a, c, y and w serogroups that were licensed in the usa in [ ] . moreover, conjugation technology was applied to develop an effective vaccine against important serogroups of s. pneumoniae. prevenar™ (pcv ), the first licensed pneumococcal glycoconjugate vaccine produced by wyeth laboratories in , is composed of seven serogroups , b, v, , c, f and f and conjugated to the nontoxic mutant of diphtheria protein crm . the results of efficacy trials showed pcv was safer and more effective for children younger than years, and infections caused by s. pneumoniae significantly reduced after vaccination [ ] . but the increasing cases of infections caused by non-pcv serotypes led to the development of pcv glycoconjugate vaccine, which covers six more serotypes (pcv + , , , b, f and a) and was approved for children from weeks to months in the usa in [ ] . vaccination is an effective and safe strategy to prevent infections caused by pathogens. vaccines prepared based on the concept of conjugation generally do not display any significant disadvantages. consequently, most countries included these carbohydrate-based conjugate vaccines in their routine immunization program [ ] . following the success of antibacterial glycoconjugate vaccines, researchers further developed carbohydrate-based conjugate vaccines for viruses, protozoans, fungi and cancer. some of the vaccines are currently in preclinical and clinical evaluation stages [ ] . whereas many reviews covered the subject of carbohydrate-based vaccines and therapeutics [ ] [ ] [ ] [ ] [ ] [ ] , here we provided the latest advancement related to synthetic carbohydrate-based vaccines against most important pathogenic bacteria, viruses and cancer. over the past two decades, in addition to the traditional carbohydrate synthesis, various advanced chemical and biochemical strategies including one-pot, automated and chemo-enzymatic are being constantly developed to obtain oligosaccharides of various structures quickly in large scale with high purity for the development of carbohydratebased vaccines and drugs [ ] [ ] [ ] . the majority of the licensed carbohydrate-based vaccines such as streptococcus pneumonia, neisseria meningitides, haemophilus influenzae type b and salmonella typhi vi belongs to this category in which the carbohydrate antigens were isolated form microbial cultures and further conjugated to the carrier protein [ ] . despite their tremendous efficacy against corresponding pathogens, several major issues are associated with in vaccine manufacture including complicated purification procedures, heterogeneous composition, presence of cell components as an impurity, uncontrollable and unreproducible protein conjugation chemistry [ ] . to overcome the above issues, chemical synthesis can produce pure, homogeneous vaccines and presents a safer and more effective alternative vaccines design. advances in carbohydrate chemistry have made it possible to synthesize complex oligosaccharides on a large scale. developed in cuba, the first commercialized synthetic vaccine, quimi-hib®, is a haemophilus influenzae type b vaccine, which is comprised of a synthetically produced antigen conjugated to the known carrier protein tt through a spacer [ ] . some bacterial glycans and cancer antigens are available in limited quantities, presenting a difficulty in clinical trials. in such cases, synthetic chemistry can save the day by producing antigens in large quantities. compare to biologically isolated vaccines, the advantages of synthetic vaccines include well-defined antigen structure with spacer arm, homogeneity, highly reproducibility, higher purity and better safety profile [ ] . the third type of glycoconjugate vaccine consists of not only chemically synthetic carbohydrate antigen, but also carriers of synthetic peptides. most of the vaccines developed for cancers and viruses fall into this category [ , ] . however, there has not been any fully synthetic vaccine commercially available. the most promising candidates are still in preclinical stage. carbohydrates are the energy sources, mediate variety of biological functions and play a key role in numerous diseases in humans and animals. moreover, they are potential agents in the development of carbohydrate-based diagnostics, therapeutics and vaccines [ , ] . over the past two decades, the vaccinology has made significant progress in protection against the infections caused by bacteria and viruses. in recent days, investigations on vaccination with pathogen-derived or synthetic carbohydrate antigens do not limit to the bacteria but extended to viruses, parasites and cancers. some of those advancements are discussed in this section. carbohydrate antigens present on the cell surface of bacteria are in the form of complex glycans and often structurally unique to be differentiated from the mammalian glycans [ ] . therefore, these complex glycans became potential targets for vaccines and biomarkers. in general, long-term use or misuse of antibiotics often lead to antibiotic resistance in pathogens. while it has not yet been observed in the case of vaccines, which target the pathogens in multiple ways by inducing t-cell responses. in addition, vaccines can reduce antibiotics usage and resistance. for example, after the introduction of the pcv conjugate vaccines into the routine childhood immunization program in several countries, the invasive bacterial diseases not only controlled but also reduced antibiotic use in vaccinated populations, and in parallel the prevalence of antibiotic non-susceptible strains also decreased [ ] . therefore, vaccination is a successful strategy to overcome the evolution of resistant strains. thus, the success of s. pneumonia, n. meningitides, h. influenzae type b glycoconjugate vaccines has prompted researchers to develop vaccines for other pathogenic bacteria such as klebsiella pneumonia, acinetobacter baumannii, clostridium difficile, staphylococcus aureus and others to fight against their antimicrobial resistance that are presently not treatable by vaccination. in the following section, we will discuss some licensed glycoconjugate vaccines and promising synthetic vaccine candidates that are currently under preclinical and clinical trials. haemophilus influenzae, a gram-negative opportunistic bacterium often inhabits the nasopharyngeal region and exists either in encapsulated or unencapsulated forms. to date six encapsulated h. influenzae serogroups a-f with distinct polysaccharides are recognized. among them, hib is more virulent in nature and causes several diseases such as pneumonia, bacteremia, meningitis and otitis media in unimmunized population particularly in children younger than years old [ ] . in pro-hibit®, a glycoconjugate vaccine of polyribosyl-ribitolphosphate (prp) oligosaccharide and dt, was licensed for children younger than years of age in usa. further investigations into different types of carrier proteins offered advanced glycoconjugate vaccines with superior immunogenicity and efficacy [ ] . currently, hib glycoconjugate vaccines with different carrier proteins, including prp-crm (hibtiter® by pfizer and vaxem-hib® by novartis), prp-omp (pedax-hib® by merck), and prp-tt (acthib® by sanofi pasteur and hiberix® by gsk) are available either in single form or in combination with other vaccines. however, these vaccines exhibit inconsistency in the prp component sizes, the linkers types and coupled carrier protein; hence, the immune responses elicited are inconsistent [ , ] . since , most countries introduced the hib conjugate vaccine in the national routine child immunization programs, leading to rapid disappearance of hib diseases in vaccine-adopted countries. to cut the cost and deal with the scarce nature of native polysaccharide hib glycoconjugate vaccines, the center for genetic engineering and biotechnology (cigb), cuba, developed the first synthetic glycoconjugate hib vaccine quimi-hib® , which is comprised of an average seven repeating units of ribosylribitol phosphate conjugated to thiolated tt through -(maleimido)propanamide linker of prp ( fig. a ) [ ] . the quimi-hib® vaccine exhibited excellent safety profile and . % protective efficacy in children. hence, the vaccine was approved in cuba and included in their immunization program since . in order to identify the suitable length of prp antigen for hib vaccine design, the seeberger group synthesized prp oligosaccharides of various length using [ + ] , [ + ] , [ + ] , and [ + ] iterative size elongation strategy and successfully conjugated then to crm (fig. b) . immunogenicity studies of the synthesized conjugates - on zika rabbit model revealed that tetrameric conjugate is the sufficient epitope for the new synthetic glycoconjugate hib vaccine [ ] . neisseria meningitides, often referred to as meningococcus, is a gram-negative diplococcal bacterium and, causes various bacterial diseases, mainly meningococcal meningitis in young children and elderly people worldwide [ ] . among the meningococcal serogroups, serogroups a, b, c, w , x and y are the most pathogenic strains accounting for all meningococcal infections [ ] . these serogroups exhibits a geographical restriction. the serogroup a (mena) is predominantly found in africa and asia, and the serogroups b (menb), c (menc), and y (meny) are most common in north america and europe. the serogroup w (menw) is found in parts of africa and south america. finally, serogroup x (menx) is reported in parts of africa [ ] . to date, the development of neisseria meningitides vaccines utilizes native polysaccharides, glycoconjugates and outer membrane vesicles (omp) [ ] . at present, three licensed quadrivalent meningococcal conjugate vaccines against serotypes a, c, y and w are available with different brand names, menveo® (mena/ c/w /y-crm , gsk), menactra® (mena/c/w / y-dt, sanofi pasteur), and nimenirix® (mena/c/w / y-tt, pfizer). although the three vaccines are different in saccharide lengths, spacer, carrier protein, and conjugation methods, they showed similar immunogenicity against the vaccine serotypes and are recommended for all age groups ( months to years). furthermore, three licensed monovalent serogroup c conjugate vaccines and one licensed monovalent serogroup a vaccine (menafrivac) are available for all age groups. two of the menc vaccines menjugate® (glaxosmithkline) and meningtec® (pfizer), use crm as a carrier protein, while the third vaccine neisvac-c® (pfizer), use tt as its carrier protein [ ] . many attempts to develop a monovalent menb conjugate vaccine failed because the structural similarity between the capsular polysaccharides (comprised of α- , linked sialic acid) of menb and components of the human neuraonal cells caused autoimmune issues in clinical tests. on the other hand, the first non-glycan-based vaccine against menb was developed in cuba using outer membrane protein (omp) and the first bivalent vaccine, va-mengoc-bc, against menb and c was licensed in cuba in . later, based on reverse vaccinology, two omp/ protein-based menb vaccines, bexsero (gsk, verona, italy) and trumenba (wyeth, philadelphia, usa) were developed and approved for the age of to years [ ] . moreover, research efforts have been devoted to the development of effective synthetic glycoconjugate vaccines for meningitis. the cps structure of mena is constructed by ( → )-linked -acetamido- -deoxy-α-dmannopyranosyl phosphate repeating units with - % o-acetylation at -oh (fig. ) [ ] . the pozsgay and oscarson groups independently reported the synthesis of mena cps fragments, up to trisaccharide and cannot be further extended due to fragments instability [ , ] . correspondingly, the native mena cps also suffers from poor stability in water due to the breaking of the anomeric and phosphodiester bond by the assistance of the adjacent nac group [ ] . in order to overcome this problem, an anomeric oxygen or ring oxygen atom of pyranose with methylene group, respectively, was substituted to synthesize stable -cphosphono and carbocyclic analogues of the mena cps repeating unit (fig. ) [ , ] . adamo and lay recently reported the synthesis of crm conjugated carbocyclic monomer, dimer and trimer analogous - and evaluated their immunogenicities in mice [ ] . all the synthesized glycoconjugates - elicited carbasugar specific antibodies that recognized their respective structures, but only the conjugate trimer was able to induce specific anti-mena igg antibodies with detectable in vitro bactericidal activity although in a less extent than hexamer and pentadecamer native polysaccharide crm conjugates. similarly, -cphosphono analogues of mena cps - were synthesized, and their immunological properties were investigated. competitive elisa assays showed that all synthetic fragments with unnatural phosphonoester linkage were clearly recognized by human polyclonal anti-mena antibodies [ ] . recent studies showed that all has conjugates of -c-phosphono analogues - were able to induce both in vitro t-cell proliferation ( % proliferation at μm) and in vivo specific igg production [ ] . overall, these studies suggested that chemical modifications do not prevent an immune response. hence, carbocyclic and -c-phosphono analogues of mena cps could also serve as vaccine candidates, and its longer oligomers may induce comparable immune response to that of commercially available vaccine. the cps of menc is composed of α-( , )-polysialic acid with sporadic / -o-acetylation (fig. ). non-acetylated fragments are also immunogenic and can induce an immune response [ ] . in order to develop a synthetic vaccine against meningitis, wu and wong group synthesized a series of non-acetylated α-( , )-oligosialic acids of various lengths ranging from dimer to dodecamer - by a convergent synthetic route [ ] . later, the guo group adopted the same synthetic strategy to successfully synthesize α-( , )-sialic acid oligomers ranging from dimer to pentamer and conjugated them to klh for immunological study in a mice model. they discovered that all conjugates - were immunogenic and elicited specific antibodies that only recognized the α-( , )-polysialic acid expressing n. meningitidis cells [ ] . the same group recently reported a new type of fully synthetic vaccines - that are composed of α-( , )-oligosialic acids and monophosphoryl lipid a (mpla), which also acts as selfadjuvant [ ] . immunological studies of these conjugates in mice revealed that they alone elicited strong immune response that was comparable to corresponding klhconjugates plus adjuvant. the elicited antibodies (igg b and igg c) had strong specific binding to α-( , )-oligosialic acids and polysaccharides of menc cells. among the tested mpla conjugates, trimer and tetramer elicited highest titers of antibodies and emerged as promising vaccine candidates worthy of further investigation. the menw cps consists of a glycan repeating unit of [→ )-α-d-galp-( → )-α-d-neup ac( / oac)-( →] (fig. ). wu group reported the first synthesis of menw cps oligosaccharides in various lengths from di-to decasaccharides a- a and determined the appropriate minimal structure for the development of synthetic vaccine [ ] . oligosaccharide chain elongation was accomplished by iterative glycosylation and deprotections using disaccharide as common donor through [ + n] glycosylation strategy. the synthesized oligosaccharides were conjugated to crm for immunogenicity study in a mice model. microarray analysis and bactericidal activity assay demonstrated that immunization of vaccine candidates b- b elicited antibodies that could recognize tetra-to decasaccharides, but the vaccine candidate b did not recognize disaccharide. among the longer oligomers, the tetramer elicited antibodies with the highest bactericidal effect. these results suggested that the tetrasaccharide is the minimum saccharide length required to induce bactericidal antibodies. over the past years, incidence of meningitis caused by menx has increased in the "meningitis belt" area (sub-saharan africa). however, no available vaccines can prevent menx. recently, native cps-based glycoconjugate vaccines of various lengths and different conjugation chemistries were demonstrated to be effective in producing high igg antibody levels in mice, and the elicited antibodies showed effective serum bactericidal activity [ ] . as an alternative for the native menx polysaccharide, a tetramer-tt glycoconjugate [ ] and a trimer-crm glycoconjugate [ ] fragment of menx were synthesized (fig. ) and their immunological properties were tested. although both conjugates exhibited immunological properties, they were lower than that of natural polysaccharides. however, when oligomers were longer than three repeat units, the elicited immunogenicity that was comparable to that by native polysaccharides. recently, a longer menx oligomer with a controlled average length was generated by enzymecatalyzed one-pot elongation procedure [ ] . the prepared oligomer was conjugated to crm , for immunological study in a mice model. glycoconjugate elicited functional antibodies that were comparable to the antibodies from the controls immunized with menx glycoconjugates prepared from the natural or enzymatically prepared cps. streptococcus pneumonia are remarkable gram-positive bacteria and cause life-threating diseases such as, pneumonia, meningitis, and septicemia in pediatric and elderly populations who are not protected by the pneumococcal vaccines. based on the chemical structure [ ] . according to the recent survey, s. pneumoniae caused , , deaths ( % ui - , , ) in people of all ages worldwide in [ ] . currently, two types of vaccines against s. pneumoniae are available. one is -vlaent native polysaccharidebased pneumococcal vaccine ppv (pneumovax® ) that contains purified csps recommended for people of and above the age of years. the second type is the glycoconjugate vaccine such as pcv (synflorix®) and pcv (prevnar ®). synflorix® is a -valent glycoconjugate that contains three different carrier proteins (phid, tt and dt) and approved for children from weeks through years. and prevnar ® is a -valent glycoconjugate vaccine with crm carrier protein and was licensed to use in infant, children and adults from weeks through years [ ] . in addition, a -valent glycoconjugate vaccine developed by merck has recently completed phase clinical trials and will soon be available in the market [ ] . although existing pneumococcal conjugate vaccines (pcvs) are highly effective in preventing pneumococcal disease in infants and children, they are not without limitation. current pcvs do not cover all serotypes and only provide protection against serotypes included in vaccines. specifically, pcv exhibited lower immune efficacy against serotypes , b, and f, and pcv against f at pre-booster. none of these pcvs provided enough immune protection against serotypes , and [ ] [ ] [ ] . an alternative option to isolation is to design vaccines based on synthetic oligosaccharides providing vaccine candidate not only in pure and homogeneous form but also with lower vaccine manufacture costs. in the past few years, various methods have been developed to identify effective carbohydrate epitopes that can induce protective immunity in vivo that is generally required for vaccine development [ ] . in the development of synthetic vaccines for s. pneumonia, various research groups have reported immunogenicity, antigenicity and protective effects of synthetic oligosaccharide-protein conjugates (neoglycoconjugates) of s. pneumoniae serotypes st , st , st , st b, st , st and st f in various lengths, frameshifts, and different carrier proteins in animal models. using elisa and microarray, suitable minimum synthetic epitopes of all those bacteria were identified (fig. ) for the development of carbohydratebased third-generation pneumococcal vaccines. most of these neoglycoconjugates elicited higher titers of opsonic antibodies with prolonged memory compared to traditional conjugated vaccines in animal models [ , ] . shigella are gram-negative bacteria that belongs to enterobacteriaceae family and causes shigellosis, which is an intestinal infection that leads to severe diarrhea and abdominal cramps in humans worldwide [ ] . shigellosis is an important health problem and economic burden for developing countries. a recent study reveals that fig. structures of the minimal synthetic oligosaccharide-protien conjugates of s. pneumoniae serotypes st , st , st , st , b, st and st f ( ) ( ) ( ) ( ) ( ) ( ) ( ) shigella was the second leading pathogen that caused diarrhea and hospitalization for around . million people and , , deaths ( % ui - , ) globally in [ ] . based on the biochemical properties, around serotypes of the shigella were identified and classified into four species including s. dysenteriae ( serotypes), s. flexineri ( serotypes), s. boydii ( serotyps) and s. sonnei ( serotype). among them, s. flexineri and s. dysenteriae are more virulent in nature, whereas s. sonnei is generally least virulent [ ] . although various traditional vaccine strategies have been attempted for developing safe and effective shigella vaccines for decades, no vaccines against shigella have been licensed. most of the vaccine candidates are in various clinical stages [ , ] . in addition to these traditional efforts, a number of studies have attempted to use synthetic glycoconjugate to develop shigella vaccines, and some are currently under various clinical studies [ ] . s. dysenteriae type is a major causative pathogen of dysentery caused by the release of potent shiga toxin. the first synthetic glycoconjugate vaccine against shigellosis was reported by pozsgay group [ ] that consisted of four repeating units of the tetrasacchride [α-l-rha- o-specific polysacchride (o-sp) of the lps of s. dysenteriae type covalently bound to hsa through heterobifunctional spacer (fig. a) . the immunological studies in a mice model revealed that hexadecasaccharide conjugate with an average of nine chains of saccharides per protein molecule was the most immunogenic epitope that elicited higher level of anti o-sp related igg antibodies in mice than the isolated o-sp-has conjugate. s. flexneri serotype a is the most prevalent pathogen of s. flexneri and responsible for endemic shigellosis among children in developing countries. specifically, an important virulent factor is that s. flexneri expresses o-specific polysaccharides (o-antigen) as a part of lps. the o-antigens of all s. flexneri except serotype share a common linear tetrasaccharide re- [ ] . due to its structural similarity to other serotypes but with more pathogenicity, serotype a is considered as a suitable target for shigella vaccine design. in order to develop a synthetic glycoconjugate vaccine against shigellosis, mulard group synthesized monomer, dimer and trimer of the pentasaccharide repeating unit of o-antigen of s. flexneri a, and conjugated them to maleimide activated tt protein for immunological study in a mice model (fig. b) [ ] . and the results of the immunogenicity studies showed that when the size of the oligosaccharide increased from monomer to dimer to trimer - the igg response also improved. moreover, pentadecasaccharide glycoconjugate induced specific and long-lasting anti o-sp a antibodies in mice. further studies demonstrated that anti-osp a antibodies induced by glycoconjugate could protect the mice from shigella infection, suggesting that pentadecasaccharide is a strong candidate for vaccine development. currently, the vaccine candidate has already entered phase ii clinical trial with promising results [ ] . anthrax is an infection disease caused by spore forming, gram-positive bacterium, bacillus anthracis that exists in two forms, vegetative cells and spores. in adverse environments, the vegetative b. anthracis is able to convert into spore form (endospore), which is highly resistant to heat, radiation, ph and harsh chemicals, allowing it to persist in the soil and other environments for decades until favorable growth conditions occurs. due to its highly pathogenic nature, mortality rates, and ease of spreading, b. anthracis is considered as an agent of bioterrorism [ ] . the spores of b. anthracis can enter humans and animals by three different modes including skin lesions, inhalation and ingestion. then, the entered spores circulate through blood stream and germinate to their vegetative form that commences rapid replications and release the toxins. this entire process takes place within a few days to few weeks, and early diagnosis and treatment are unlikely [ ] . capsular polysaccharides and anthrax toxin are the main virulence factors of b. anthracis. anthrax toxin is a tripartite exotoxin composed of three proteins known as the edema factor (ef), the lethal factor (le) and the protective antigen (pa). individually, these three proteins are nontoxic, but in binary combinations, particularly pa with ef and pa with le, they produce edema toxin (et) and lethal toxins (lt), respectively [ ] . although anthrax can be treatable by antibiotics, vaccination is the best option to prevent anthrax. so far, the first and second generation of human anthrax vaccines have been developed based on the spores and anthrax toxin. however, the vaccines have several limitations, including poor immunogenicity, tedious -to primary vaccination doses with annual boosting, low efficacy, uncertain safety, and side effects [ , ] . therefore, there is a need to develop a new type of vaccines with novel formulations. in this regard, the development of wellrecognized glycoconjugate vaccines is one of the major choices. the glycans present on the surface of the b. anthracis vegetative cell and spores provide broad opportunities for the development of new vaccines and biomarkers against anthrax [ ] . many preclinical studies have focused on the tetrasaccharide expressed on the surface of b. anthracis exosporium. this tetrasacchride is composed of three rhamnose resides and one rare sugar known as anthrose at its nonreducing end [ ] . seeberger group was the first to demonstrate that synthetic anthrax tetrasaccharide bound to klh protein (fig. ) are immunogenic in mice. the resulted carbohydrate specific monoclonal igg antibodies recognized the glycan structure of native b. anthracis endospores [ ] . further studies by boon group showed that anthrose-rhamnose-rhamnose trisaccharide conjugated to klh (fig. ) was a sufficient fragment to bind to the antispore rabbit serum and the isovaleric acid substituent of anthrose played a crucial role in antibody recognition [ ] . later studies by various groups mainly focused on the role of anthrose residue and its structural requirements in immunogenicity and antigenicity. the results of these studies can be summarized to i. anthorse is the immunodominant feature of the tetrasaccharide; ii. isovaleric acid moiety at c- and methyl group at c- of anthorse are key antigenic elements and essential in recognition of anti-spore antibodies; iii. ome group at c- is not necessary, because it is not involved in recognition of antibodies; and iv. rhamnose moiety alone (without anthrose) is not crucial to antigenicity. to date most of the glycoconjugate vaccines developed against anthrax are still in preclinical stage. the gram-positive, spore forming, and toxin-producing bacterium, clostridium difficile, mainly causes nosocomial antibiotic-associated colitis and diarrhea in humans. over the past years, clostridium difficile infections (cdi) have emerged globally. in the usa alone, the estimated cdi cases reached , and cdi-attributed deaths reached , in , translating to an economic burden of $ to billion usd annually [ ] . like b. anthracis, c. difficile can also exist as spores, which are able to survive for months in all environments without loss of viability and can transmit to people via the oral route. after ingestion, spores can survive in the stomach and subsequently reach to intestine, and patient remains disease free at this stage. when the balance of natural gut microbiota is disturbed by antibiotics treatment of other diseases, the environment favors the spores to germinate into vegetative cells that can enter into the colon and secrete two enterotoxins (tcda and tcdb) that can severely damage the intestinal mucosa and lead to colitis and diarrhea [ ] . on the other hand, c. difficile strains that do not produce toxins are non-pathogenic. although cdi can be treated by antibiotics, there is still an urgent need of c. difficile vaccines due to the emergence of antibiotic resistant strains, recurrent cdis, difficulty in diagnosis and economic burden of the treatment. over the past decade, most research effort has been focused on the development of c. difficile toxoidsbased vaccines, which are currently in different stages of clinical trials [ ] . apart from that, carbohydrate-based vaccines are studied at preclinical level. although c. difficile spores don't express any surface gycans, the vegetative form of c. difficile cells do express three types of glycans (psi, psii, and psiii) on the cell surface. among them, psii is the most abundant polysaccharide and is expressed by all c. difficile ribotypes and thus, represents an important target molecule for vaccine design [ ] . two groups individually investigated the synthesis, immunogenicity and antigenicity of psii oligosaccharide of c. difficile. in order to study the role of phosphate group in immunogenicity, adamo et al. first synthesized the hexasaccharide repeating unit of psii with and without phosphate group at non-reducing end via [ + ] convergent approach [ ] . the synthetic antigens and native psii polysaccharide were conjugated to crm carrier protein, respectively fig. (hexa-crm , hexap-crm and psii-crm ), and the glycoconjugates were used to immunize balb/c mice. interestingly, igg antibodies elicited by both native psii-crm and synthetic hexap-crm glycoconjugates were able to recognize psii on the surface of c. difficile cells. however, nonphosphorylated hexa-crm did not induce either igg or igm antibodies, indicating the importance of negatively charged phosphate group for immunogenicity. concurrently, seeberger group completed another study, in which the mice were immunized with a conjugate composed of the synthetic nonphosphorylated psii hexasaccharide that bound to crm carrier protein through squaric acid [ ] . the neoglycoconjugate was immunogenic in mice and produced carbohydrate specific antibodies that specifically interacted with the synthesized glycan hapten. these results suggested that psii hexasaccharide single repeating unit with charged phosphate group is the sufficient potential epitope for vaccine design against c. difficile. in addition, the immunogenicity of psi and psiii oligosaccharides were also studied using mice and rabbit models. brucella species are non-spore-forming, gram-negative coccobacilli that causes brucellosis in humans and animals such as cattle, goats, camels, sheep, deer, swine and dogs worldwide. among the species within the genus brucella b. melitensis, b. abortus, b. suis and b. canis are the main pathogenic species in both animals and humans [ ] . brucellosis is an endemic and mostly transmitted to humans by direct contact with the infected animals or consumption of their raw milk and meat products [ ] . the emergence of human brucellosis is a serious problem and effects the economy in developing countries such as india, china, brazil and some of the african countries. the available diagnostic tools of brucella are inadequate, expensive and time consuming. moreover, the available live vaccines are limited to ruminants, and there is no vaccine for humans [ ] . furthermore, treatment of human brucellosis requires long and costly antibiotic therapy. therefore, there is an urgent need to develop a superior diagnostic tools and vaccines against brucella [ ] . the o-antigen or o-polysaccharide (ops) domain of lps of brucella is composed of a homopolysaccharide rare sugar , -dideoxy- -formamido-α-d-mannopyranose (rha nfo) that exists in two sequences, resulting in two types of antigens known as a and m antigens (fig. ) . the a antigen consists a longer inner sequence of α- , -linked d-rha nfo residues and is capped by the m type antigen, which contains one α- , -linked d-rha nfo for every four α- , -linked d-rha nfo resides [ ] . both a and m antigens are virulent in nature, and studies showed all of the investigated brucella strains have to % of m character linkages except for b. suis biovar , which only has a type antigen [ ] . in , the bundle group synthesized pentasacchride a and nonasacchride a of o-antigen and studies their antigenicity [ ] . the nonasacchride a was designed to have a and m epitopes, whereas pentasacchride a had mostly m type. after the conjugation with bsa, both conjugates b and b were coated on elisa plates, to be tested against two monoclonal antibodies (mabs) yst - and bm , specifically for the brucella a and m antigens, respectively. interestingly, nonasacchride antigen b bound to a-and m-specific mabs with equivalent avidity, whereas the pentasaccharide antigen b preferentially bound to m-specific mabs, as expected. this discrimination between m and a antibodies by pentasaccharide conjugate might improve by decreasing the number of , -linked α-d-rha nfo residues in the molecule. to study this possibility, a series of m-type oligosaccharides from di-to tetrasacchrides a- a was synthesized and subsequently conjugated to bsa to identify the smallest and largest m epitopes [ ] . surprisingly, both diand tetrasaccharide-bsa conjugates b and b (mtype) were able to detect antibodies in the sera of humans and animals infected with b. suis and b. abortus, despite of having a-dominate lps in their cell wall. moreover, the same conjugates also showed strong binding avidity to m-specific mabs and weak to negligible binding to aspecific mabs. furthermore, the anti a-antibodies elicited exclusively by α- , -linked hexasaccharide-tt conjugate , bind well to the m type disaccharide and tetrasaccharide antigens b and b [ ] . these results suggested that disaccharide antigen is the simplest structure that can detect antibodies in the sera of brucella infected animals and humans and would be a promising biomarker for the detection of brucella. cancer is a type of disease with immortalized cell growth and metastasis to other tissues of human body. vaccines for cancer treatment are classified into prevention vaccines, which prevent virus infection (e.g., hpv vaccine against human papillomavirus and hepatitis b vaccine against hepatitis b virus), and therapeutic vaccines, which are immunotherapy that train and activate immune system in human body to eliminate cancer cells (e.g., provenge® against prostate cancer). recently, immunotherapy is gaining popularity in cancer treatment due to its low side effects and high specificity [ ] . most of the immunotherapies target on the surface protein such as pd-l of the cancer cell. in addition, tumor-associated carbohydrate antigens (tacas), which are abundant on the surface of different types of cancer cells, are highly associated with tumor progression and therefore potential candidates for cancer immunotherapy [ , ] . tacas are classified into four groups (fig. ) : ( ) the globo series including globo h, ssea and ssea (gb ) which are glycolipids and overexpress in breast, prostate, lung, ovary and colon cancer cells; ( ) the gangliosides including gd , gd , gm , gm and fucosyl gm which overexpress on melanoma, neuroblastoma, sarcoma and b-cell lymphoma; ( ) the blood group including lewis x , lewis y , sialyl lewis x , and sialyl lewis a which are also gangliosides and overexpress on breast, prostate, lung colon and ovary cancer cells; ( ) the glycoprotein including thomsennouveau (tn), thomsen −friendreich (tf), and sialyl-tn (stn) which attach at the serine/threonine on the mucin and overexpress in epithelial cancer cells (breast, ovary and prostate) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . previous clinical experiences showed increasing survival rate in patients who were passively administrated antibodies recognizing carbohydrate or generated appropriate amount of antibodies after immunization with carbohydrate-based vaccine [ , ] . thus, tacas are demonstrated to be ideal targets for cancer vaccine development. tacas are poorly immunogenic and t-cell independent, similar to bacterial polysaccharides as mentioned earlier. therefore, many studies covalently conjugated tacas to carrier proteins such as bsa, klh, dt, tt, ova, and muc peptides to induce t-cell mediated immune response [ ] . interestingly, the same taca with different carrier proteins resulted in different immune response against taca. for example, helling et al. conjugated ganglioside antigen gd to different carrier proteins bsa, klh, omp, multiple antigenic peptide (map) and polylysine through reductive amination [ ] . after immunization the conjugates to mice, the strongest igg antibody titer was found in mice with gd -klh and qs- immunization. similarly, danishefsky and livingston' group synthesized several tn (consist of monosaccharide galnac) constructs: tn monosaccharide, tn-threonine trimer cluster, and tn partially or fully glycosylated muc cluster and conjugated them individually to klh or bsa carrier protein through mmalemidobenzoyl-n-hydroxysuccinimide ester [ ] . they found that tn-klh induced stronger igg titer than tn-bsa. as a part of cancer vaccine development, our group has synthesized globo h vaccines with klh, dt, tt, and bsa carrier proteins and immunized them in a mice model with different adjuvant. we found that globo h-dt with c adjuvant induced the strongest igg antibodies that specifically recognized globo series antigens (globo h, ssea and ssea ) [ ] . to conjugate tacas to the carrier protein, the reducing end of taca is installed with spacers including p-nitrophenyl, maleimide, aldehyde containing groups, which then conjugated to carrier protein through amide bond formation, michael addiction and reductive amination. although these spacers efficiently conjugate tacas and carrier protein together, they also induced immune response against itself. boon's group prepared le y conjugated klh vaccine with -(maleimidomethyl) cyclohexane- -carboxylate (mi) linker. the elisa results indicated strong igg antibody that recognized linker region was induced [ ] . based on the above results, series of carbohydrate-based anticancer vaccines have been generated and used in clinical trials including gangliosides (gd , gd , and gm ), lewis structure series, o glycans (tn, stn and tf) and globo series (globo h and ssea ) [ , [ ] [ ] [ ] [ ] [ ] [ ] . with the successful experience in monovalent vaccine development, danishefsky and livingston group developed multiple antigens in a one single taca vaccine. in their phase ii clinical trial, the patients were coadministrated with gm , globo h, lewis y , tf(c), tn(c), stn(c) tn-muc that was individually conjugated to klh and mixed with adjuvant qs as a heptavalent vaccine. eight of nine patients developed responses against at least three antigens. however, the antibodies titer was lower than the response from administration of a single corresponding vaccine [ ] . the over-dose carrier protein klh may induce a strong immune response against itself and impair the response against carbohydrate antigens. to overcome this issue, danishefsky and coworkers first synthesized unimolecular pentavalent vaccine containing globo-h, stn, tn, tf, and le y antigens, which are overexpressed on prostate and breast cancer cell surfaces (fig. ) [ ] . then, they attached these antigens to an amino acid by peptide coupling and conjugated the assembly to klh by michael addition. the immunological studies of these glycoconjugates showed that antibodies against globo-h, stn, tn, and tf were strongly induced in comparison to the pooled monovalent vaccine in the preclinical result. but antibodies against le y were not as strong, possible due to immune-tolerance caused by relatively high le y on normal cells. to improve the vaccine efficacy, the same research group developed a second-generation unimolecular pentavalent vaccine, which targets the globo h, stn, tn, tf, and gm instead of le y (fig. ) . the gm was selected because the gm induced antibodies are able to recognize cancer cell and positively correlated with patient survival in clinical trial [ ] . the vaccine induced perspective antibodies not only target each antigen but also recognize the overexpressed antigens on cancer cells [ ] . the results of the phase i study of this unimolecular pentavalent vaccine demonstrated vaccine safety and effective induction of antibody responses against five ovarian cancer cell surface antigens. specifically, igg and/or igm titers were detected against or more antigens in out of patients, or more antigens in out of patients, and or more antigens in out of patients [ ] . in short, the unimolecular pentavalent vaccines that combined several carbohydrate antigens and carrier protein conjugates could simulate immune response against the heterogeneous carbohydrate epitopes expressed on the surface of cancer cells. in comparison with combined monomeric vaccines, the unimolecular pentavalent vaccine allows higher yield of the final conjugation step, simplified carbohydrate ratio validation step, mimicking the heterogeneity of cancer cells and lower carrier protein amount to minimize the immune suppression. despite many encouraging preclinical results, many limitations have prevented the carbohydrate-protein conjugate vaccines from fda approved. first, the yield of the conjugation step is low, and the conjugation numbers are not consistent in each batch, affecting vaccine efficacy. second, both the carrier protein and the linker fig. unimolecular pentavalent vaccine containing globo h, stn, tn, ley or gm and tf between carbohydrate and carrier protein, may also be immunogenic and induce immune response against itself [ ] . the undesired antibody production that targets carrier protein and linker may affect the vaccine efficacy and decrease the desired antibody titer. lee et al. installed the phenyl no at the reducing end of glycan and conjugated it to crm [ ] . after immunization, the glycan array result showed that antiserum from the immunized mice recognized the phenylno but not the glycan. this result indicated that the strong immunogenic function group reduces the vaccine efficacy. yin et al. synthesized qβ-tn through click reaction with triazole function group [ ] . after immunization, the antiserum bound to the triazole structure and can not recognize the ta ha cancer cells. they replaced the triazole to the less immunogenic alkyl amide linker on the qβ-tn which was immunized in mice. the antiserum not only bound to the tn antigen but also recognized the cancer cells. the results indicated that the immunogenic function group at linker moiety result in reduction of vaccine efficacy. to achieve the significance of clinical trial for tacas vaccine, the strong immunogenic function group like triazole should be avoided. the less immunogenic alkyl amide may be a proper linker for covalent conjugation of tacas to carrier protein. to overcome the disadvantage brought by the carrier protein, many studies attempted to use different epitopes of immune cells to elicit immune response. agonist of toll-like receptor (tlr) on dendritic cells activates nfkb and ap- , resulting in cytokine secretion and immune activation. moreover, toyokuni et al. were the first to couple tn antigen to a tlr agonist tripalmitoyl-sglycerylcysteinylserine (pam cys) as fully synthetic vaccine (fig. a) [ ] . although only moderate igg was induced, it was the first carrier protein-free taca vaccine that could elicit immune responses against carbohydrate antigen. to induce igg antibody production and longterm memory b cells, the involvement of t cell is necessary for antibody affinity maturation in b cells. cantacuzene's group synthesized tn glycopeptide that contains pv as t cell epitope (fig. b) . the resulting vaccine induced robust igg antibodies, which recognized cancer cell line and also increased the survival rate of tumor-bearing mice [ ] [ ] [ ] . another th cell epitope, pan dr epitope (padre) installed on tacas was also able to induce robust igg antibodies titer (fig. c) [ , ] . dumy and coworkers designed clustered tn antigen conjugated on pv regioselectively using addressable functionalized templates (rafts). the raft glycoconjugates scaffold is a non-immunogenic, built-in vaccine carrier and elicits igg antibodies that recognize tn antigens (fig. d) [ ] . kunz's group connected stn glycopeptides to a th-cell peptide epitope from ovalbumin (ova - ) by a non-immunogenic amino acid spacer ( fig. e) [ ] . the resulting vaccine induced strong and specific immune response against the tumorassociated structure. later, the same group installed tn, stn and tf antigens on pam cyssk through fragment condensation (fig. f) [ ] . although antiserum titers were not as high as muc tetanus toxoid vaccine, the antibodies only recognized the muc glycopeptides with the same glycosylated site. on the other hand, to avoid the enzymatic degradation and increase the bioavailability of vaccine, benmohamed et.al conjugated tn mimetics instead of native tn on raft with an immunostimulant peptide epitope (ovapadre). this vaccine induced long-lasting and strong igg/igm antibodies, which protects mice against tumor progression [ ] . zwitterionic polysaccharides (zpss) can induce mhcii mediated immune response and replace carrier protein as a potential component of carbohydrate-based vaccine. de silva et al. modified ps-a to tn antigen by oxime formation to afford fully carbohydrate vaccine without other immune stimulant [ ] . the immunization of this vaccine evoked high titer and specific antibodies. the same group conjugated stn on ps-a and characterized the loading amount of stn to be around - % by h nmr integration and svennerholm method (fig. g) [ ] . the immunization of the vaccine with adjuvant elicited strong immune response and high titer igm/igg antibodies. these antibodies not only recognized cancer cells (mcf- and ovcar- ) but also conducted complement-dependent cellular cytotoxicity cell lines. another full carbohydrate vaccine was developed by guo's group. they individually conjugated modified gm , stn, or globo h on monophosphoryl lipid a (mpla) to form three built-in adjuvants (fig. h) . among them, globo h-mpla vaccines elicited stronger antibodies titer and higher cell toxicity activity without external adjuvant in comparison to globo h-klh with freund's complete adjuvant [ ] [ ] [ ] [ ] . the above result showed that three components, including b cell epitopes (tacas), tlr agonist (built in adjuvant) and th epitope (mhcii presenting peptides), play a crucial role for the fully synthetic vaccine to induce strong, specific and long-lasting immune response. ingale et.al synthesized three components to form fully synthetic vaccine composed of tlr ligand (pam cyssk ), th epitope (pv) and b epitope (tn glycopeptide) (fig. a) [ ] . the lipid moiety facilitates the uptake of the vaccine by macrophages and dendritic cells. impressively, the vaccine induced strong antibodies, which were able to recognize cancer cell line even without coadministration of qs- adjuvant. moreover, th epitopes induced very low antibodies, indicating that immunosuppression was tolerable. dumy and benmohamed's group developed a tetra-components vaccine by assembling a cluster of b cell epitope (tn antigen), cd + t cell epitope (pan-dr), cd + t cell epitope (ova - ) and built-in adjuvant (palmitic acid) through oxime and disulfide bond formation (fig. b) [ ] . the vaccine significantly induced strong antibodies that recognized tumor cell lines, activated cd + and cd + cells, and protected mice against lethal carcinoma cell challenge [ ] . cai et al installed different numbers of tn or stn glycopeptides into two component vaccine by the click reaction (fig. c) . the immunological study result indicated that four copies of a muc sialyl-tn antigen showed excellent antibodies titer and elicited an antiserum that killed the cancer cells by cdc [ ] . although tacas are generally ideal vaccine candidates, some of them are expressed in normal tissue or cells in the developing stage, leading to immune tolerance and lower immunogenicity of the vaccine. two types of modified taca vaccines have been studied including metabolic oligosaccharides engineering (moe) vaccine and cross-reactivity antibodies induced by modified tacas. the modification of tacas vaccine provides the following advantages, ) preventing the immune in this strategy, modified taca analogs vaccine was immunized to tumor-bearing mice. then, mice were treated with the corresponding precursor, which was processed into modified taca on the surface of cancer cells. the antibodies induced by modified taca analog vaccine were able to recognize the bio-synthesized antigen on the cancer cell and eliminated cancer cells by adcc or cdc. moreover, guo's group modified the n-acetyl group on the sialic acid of gm into different functional groups and conjugated them on klh [ ] . among them, n-phenylacetyl gm -klh showed best immunogenicity and t cell-dependent immunity. however, its antisera showed low cross-reactivity in binding to native gm . they further incubated the cancer cells with corresponding mannosamine and analyzed those cells by facs [ ] . particularly, n-phenylacetyl-d-mannosamine was used as a precursor and synthesized into nphenylacetyl gm . the modified gm expressing cancer cells could go through anti-gm pac immune serum-mediated cytotoxicity. later, they performed both in vitro and in vivo model for n-phenylacetyl gm expression. the mice treated with n-phenylacetyl mannosamine showed strong n-phenylacetyl gm expression. the n-phenylacetyl gm vaccine protected mice against tumor progression after metabolic oligosaccharides engineering. another taca stn was also modified into n-phenylacety and n-chlorophenylacetyl stn by the same group, and the vaccine immunogenicity was also stronger than native stn vaccine [ ] [ ] [ ] . these results demonstrated that moe is a powerful tool to enhance immunogenicity. most studies have focused on sialic acid modification. however, sialic acid plays many important roles in biological function. unnatural sialic acid may contribute to breakage of its original function and result in disease. hence, the investigation of moe side effects is required in the future. to overcome the shortage of moe, many studies focus on modification of tacas vaccines, which not only could generate stronger immunogenicity but also induce cross-reactive antibodies recognizing native carbohydrate antigens on the tumor cells. zheng et al. synthesized a series of gm analogs with the modification at n-acetyl group on sialic acid (fig. a) [ ] . the gm -klh vaccine with propionamide elicited higher igm and igg titer than native gm vaccine. besides, those antibodies are highly cross-reactive to native gm , indicating that modification of taca can generate not only stronger immunogenicity but also cross-reactivity to native antigen. stn antigen has also been modified and studied in many studies. ye' s group reported different modifications at nacetyl group on the sialic acid of stn [ ] . the vaccines with fluorine modified stn showed stronger igg titer and higher igg/igm ratio in comparison to native stn vaccine (fig. b) . to enhanced the vaccine stability and avoid the glycosidase hydrolysis, they also substituted the oxygen at the glycosidic bond to sulfur to generate s-linked stn derivatives with fluorine-containing modification [ ] . even though the vaccines could elicit cross-reactive antibodies to recognize native stn, the antibodies titer was not stronger than native stn vaccine. in vivo result indicated that n-fluoroacetyl modified stn vaccine was able to induce t cell-dependent immunity, increase survival in tumor-bearing mice and activate antibodies mediated cell cytotoxicity (adcc and cdc) [ ] . similar modifications were installed at n-acetyl group at tf antigen ( fig. c) [ ] . compare to native tf vaccine, nfluoroacetyl modified tf vaccine induced two-fold igg antibodies titer. although some modified vaccines showed remarkable results, and most of them targeted an amino group, which can be selectively converted to other function groups instead of majority hydroxyl on the carbohydrates. the specific modification at the hydroxyl group is more challenging because complicated protection and deprotection procedures are required for the installation of site-specific modification in numerous hydroxyl group. our group used chemoenzymatic strategy to synthesize numerous globo h analog vaccines with the modification at reducing and non-reducing end [ ] . our results indicated that azido modification at the nonreducing end of globo h-crm could elicit stronger igg titer than native globo h vaccine (fig. d) . the antiserum was able to recognize the cancer cell line and eliminate it by adcc. generally speaking, prevention is better than treatment, and vaccination is an effective and safe approach to prevent infections. since the last century most of the diseases such as polio, smallpox, rubella, influenza, mumps and other have been under controlled, and some diseases now are even completely eradicated after the introduction of traditional vaccines (live and killed vaccines) [ ] . moreover, the glycoconjugate vaccines such as s. pneumoniae, h. influenzae and n. meningitidis, which are made by poor immunogenic oligo−/polysaccharide covalently linked to carrier protein (t-cell epitope), exhibit high efficiency and effectively worked for chilfren younger than years of age. unfortunately, these vaccines are not readily available for children in poor countries due to their high cost and low supply. also, these glycoconjugate vaccines are able to protect people from vaccinated serotypes, but recently reported emergency of non-vaccine serotypes of s. pneumoniae and n. meningitidis. therefore, more studies on serotype inclusion or replacement are needed. although conjugate vaccines are effective and safe, but some issues need to be addressed. there is no general rule to predict the optimal length/size of the oligosaccharide and appropriate saccharide/protein molar ratio for vaccine development. moreover, the presence of carrier protein and linker in conjugate vaccines can lead to some disadvantages. both carrier proteins and linkers themselves can be immunogenic and elicit nonspecific immune response that can suppress carbohydrate-specific antibody production [ ] . therefore, there is a need to design and develop carrier protein free and linker free vaccines. the recent studied zwitterionic polysaccharide (zps) type vaccines are an alternative. the zps vaccines contain both positive and negative charges on adjacent monosaccharide units and were found to be able to elicit mhc ii mediated t cell response without linkage of carrier protein [ ] . this finding has important implications for the design of novel polysaccharide vaccines. the development of carbohydrate-based anti-cancer vaccine has made significant progress in the past few decades. preclinical trials of monovalent and polyvalent vaccines showed encouraging results. with more understanding about the carrier protein, many fully synthetic carbohydrate vaccines with good immunogenicity, low linker effect and optimized conjugation step between carbohydrate and immune-stimulant moiety have being developed. however, there is still a major gap between the mice models and clinical trials. so far, no tacas vaccine has been approved by the fda. the slight expression of tacas on normal tissue may result in the immune-tolerance and lead to low immunogenicity in clinical trial. although a proper model to determine the immunogenicity in human remains to be developed, modification of tacas to generate "non-self" antigen vaccine and induce cross-reactive antibody will be a good tool for the future studies. overall, with the experiences in vaccine development and clinical trials, carbohydrate-based anti-cancer vaccine seems to be closer to reach than ever. more efforts are still needed to deal with low immunogenicity issue, unsound immune system in patients, tacss expression level between cancer and normal cells in patients, and the protocol design for clinical trials. soluble substance of pneumococcus origin in the blood and urine during lobar pneumonia immunological relationships of cell constituents of pneumococcus cutaneous reactions in pneumonia. the development of antibodies following the intradermal injection of type-specific polysaccharide immunization of human subjects with the specific carbohydrates of type iii and the related type viii pneumococcus prevention of pneumococcal pneumonia by immunization with specific capsular polysaccharides the human antibody response to simultaneous injection of six specific polysaccharides of pneumococcus therapeutic strategies to combat pneumococcal disease: repeated failure of physicians to adopt pneumococcal vaccine changing interest among physicians toward pneumococcal vaccination throughout the twentieth century pneumococcus: the first one hundred years emergence of multiply resistant pneumococci pneumococcal polysaccharide vaccines vaccines in historic evolution and perspective: a narrative of vaccine discoveries update on haemophilus influenzae serotype b and meningococcal vaccines a new typhoid vaccine composed of the vi capsular polysaccharide hib vaccines: past, present, and future perspectives chemo-immunological studies on conjugated carbohydrate-proteins : ii. immunological specificity of synthetic sugarprotein antigens polysaccharide-protein conjugates: a new generation of vaccines meningococcal glycoconjugate vaccines efficacy, safety and effectiveness of pneumococcal conjugate vaccines review on the immunogenicity and safety of pcv- in infants and toddlers glycoconjugate vaccines: an update carbohydrate vaccines: developing sweet solutions to sticky situations? carbohydrates and immunology: synthetic oligosaccharide antigens for vaccine formulation recent developments in synthetic carbohydrate-based diagnostics, vaccines, and therapeutics an overview of structural features of antibacterial glycoconjugate vaccines that influence their immunogenicity synthesis and medical applications of oligosaccharides recent advances in the synthesis of glycoconjugates for vaccine development immunotherapy for cancer: synthetic carbohydrate-based vaccines automated chemical oligosaccharide synthesis: novel approach to traditional challenges one-pot" protection, glycosylation, and protection-glycosylation strategies of carbohydrates toward automated enzymatic synthesis of oligosaccharides vaccines based on the cell surface carbohydrates of pathogenic bacteria the design of semi-synthetic and synthetic glycoconjugate vaccines a synthetic conjugate polysaccharide vaccine against haemophilus influenzae type b synthetically defined glycoprotein vaccines: current status and future directions cyclopeptide scaffolds in carbohydratebased synthetic vaccines clustered carbohydrates in synthetic vaccines statistical analysis of the bacterial carbohydrate structure data base (bcsdb): characteristics and diversity of bacterial carbohydrates in comparison with mammalian glycans impact of existing vaccines in reducing antibiotic resistance: primary and secondary effects haemophilus influenzae infections in the h. influenzae type b conjugate vaccine era haemophilus influenzae type b conjugate vaccines: a review of efficacy data a modular synthetic route to size-defined immunogenic haemophilus influenzae b antigens is key to the identification of an octasaccharide lead vaccine candidate meningococcal disease description and nomenclature of neisseria meningitidis capsule locus the global meningococcal initiative: global epidemiology, the impact of vaccines on meningococcal disease and the importance of herd protection meningococcal vaccines: current status and emerging strategies. vaccines (basel) factors contributing to the immunogenicity of meningococcal conjugate vaccines meningococcal vaccinations determination of the structure and conformation of bacterial polysaccharides by carbon nuclear magnetic resonance. studies on the group-specific antigens of neisseria meningitidis serogroups a and x towards a synthetic glycoconjugate vaccine against neisseria meningitidis a synthesis of structures corresponding to the capsular polysaccharide of neisseria meningitidis group a relative stability of meningococcal serogroup a and x polysaccharides synthesis and preliminary biological evaluation of carba analogues from neisseria meningitidis a capsular polysaccharide synthesis of stable c-phosphonate analogues of neisseria meningitidis group a capsular polysaccharide structures using modified mitsunobu reaction conditions immunoactivity of protein conjugates of carba analogues from neisseria meningitidis a capsular polysaccharide synthesis and biological evaluation of phosphono analogues of capsular polysaccharide fragments from neisseria meningitidis a a synthetic disaccharide analogue from neisseria meningitidis a capsular polysaccharide stimulates immune cell responses and induces immunoglobulin g (igg) production in mice when protein-conjugated evaluation of de-o-acetylated meningococcal c polysaccharide-tetanus toxoid conjugate vaccine in infancy: reactogenicity, immunogenicity, immunologic priming, and bactericidal activity against o-acetylated and de-o-acetylated serogroup c strains efficient and stereoselective synthesis of alpha( --> ) oligosialic acids: from monomers to dodecamers synthesis and immunological study of alpha- , -oligosialic acid conjugates as anti-group c meningitis vaccines fully synthetic self-adjuvanting alpha- , -oligosialic acid based conjugate vaccines against group c meningitis synthesis of neisseria meningitidis serogroup w capsular oligosaccharides for immunogenicity comparison and vaccine development development of a glycoconjugate vaccine to prevent meningitis in africa caused by meningococcal serogroup x synthesis of a tetrasaccharide and its glycoconjugate corresponding to the capsular polysaccharide of neisseria meningitidis serogroup x and its immunochemical studies synthesis and immunological evaluation of protein conjugates of neisseria meningitidis x capsular polysaccharide fragments combined chemical synthesis and tailored enzymatic elongation provide fully synthetic and conjugation-ready neisseria meningitidis serogroup x vaccine antigens pneumococcal capsules and their types: past, present, and future estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in countries, - : a systematic analysis for the global burden of disease study review: current and new generation pneumococcal vaccines a dose ranging study of different formulations of -valent pneumococcal conjugate vaccine (pcv ) in healthy infants development and clinical evaluation of prevnar , a -valent pneumocococcal crm conjugate vaccine immunogenicity and reactogenicity of ten-valent versus -valent pneumococcal conjugate vaccines among infants in ho chi minh city direct comparison of immunogenicity induced by -or -valent pneumococcal conjugate vaccine around the -month booster in dutch infants chemical biology approaches to designing defined carbohydrate vaccines improving vaccines against streptococcus pneumoniae using synthetic glycans development of approaches to a third-generation carbohydrate-conjugate vaccine against streptococcus pneumoniae: the search for optimal oligosaccharide ligands morbidity and mortality due to shigella and enterotoxigenic escherichia coli diarrhoea: the global burden of disease study - update on: shigella new serogroups/serotypes and their antimicrobial resistance progress and pitfalls in shigella vaccine research an update on vaccines against shigella status of vaccine research and development for shigella protein conjugates of synthetic saccharides elicit higher levels of serum igg lipopolysaccharide antibodies in mice than do those of the o-specific polysaccharide from shigella dysenteriae type shigella flexneri o-antigens revisited: final elucidation of the o-acetylation profiles and a survey of the o-antigen structure diversity a synthetic carbohydrate-protein conjugate vaccine candidate against shigella flexneri a infection a synthetic carbohydrate conjugate vaccine candidate against shigellosis: improved bioconjugation and impact of alum on immunogenicity anthrax: a disease of biowarfare and public health importance toxins and vaccines: a -year journey targeting bacillus anthracis progress and novel strategies in vaccine development and treatment of anthrax glycan surface antigens from bacillus anthracis as vaccine targets: current status and future perspectives synthetic efforts towards glycoconjugate-based vaccines active against anthrax anti-carbohydrate antibodies for the detection of anthrax spores synthesis and antigenic analysis of the bcla glycoprotein oligosaccharide from the bacillus anthracis exosporium epidemiological and economic burden of clostridium difficile in the united states: estimates from a modeling approach clostridium difficile infection: epidemiology, diagnosis and understanding transmission vaccines against clostridium difficile carbohydrate-based clostridium difficile vaccines phosphorylation of the synthetic hexasaccharide repeating unit is essential for the induction of antibodies to clostridium difficile psii cell wall polysaccharide a possible oligosaccharide-conjugate vaccine candidate for clostridium difficile is antigenic and immunogenic brucellosis: a worldwide zoonosis human brucellosis brucellosis vaccines for livestock brucellosis: improved diagnostics and vaccine insights from synthetic glycans reinvestigation of the structure of brucella oantigens the epitopic and structural characterization of brucella suis biovar o-polysaccharide demonstrates the existence of a new m-negative cnegative smooth brucella serovar design and synthesis of a universal antigen to detect brucellosis molecular recognition of brucella a and m antigens dissected by synthetic oligosaccharide glycoconjugates leads to a disaccharide diagnostic for brucellosis specificity in cancer immunotherapy tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines the cancer glycome: carbohydrates as mediators of metastasis antigen structure and genetic basis of histo-blood groups a, b and o: their changes associated with human cancer the role of blood group antigens in malignant progression, apoptosis resistance, and metastatic behavior structure and function of the cell surface (tethered) mucins expression of globo h and ssea in breast cancer stem cells and the involvement of fucosyl transferases and in globo h synthesis cancer vaccines and carbohydrate epitopes stage-specific embryonic antigen- as a potential therapeutic target in glioblastoma multiforme and other cancers carbohydrate vaccines that induce antibodies against cancer. . rationale ganglioside expression on human malignant melanoma assessed by quantitative immune thin-layer chromatography improved survival in stage iii melanoma patients with gm antibodies: a randomized trial of adjuvant vaccination with gm ganglioside anti-gd antibody with gm-csf, interleukin- , and isotretinoin for neuroblastoma gd vaccines for melanoma: superior immunogenicity of keyhole limpet hemocyanin conjugate vaccines comparison of antigen constructs and carrier molecules for augmenting the immunogenicity of the monosaccharide epithelial cancer antigen tn carbohydrate-based vaccines with a glycolipid adjuvant for breast cancer the immunogenicity of the tumor-associated antigen lewis(y) may be suppressed by a bifunctional cross-linker required for coupling to a carrier protein carbohydrate antigens as targets for active specific immunotherapy carbohydrate vaccines as immunotherapy for cancer recent development in carbohydrate-based cancer vaccines carbohydrate-based vaccines: challenges and opportunities carbohydrate-based vaccines for oncotherapy recent development in carbohydrate based anti-cancer vaccines pilot study of a heptavalent vaccine-keyhole limpet hemocyanin conjugate plus qs in patients with epithelial ovarian, fallopian tube, or peritoneal cancer preparation and evaluation of unimolecular pentavalent and hexavalent antigenic constructs targeting prostate and breast cancer: a synthetic route to anticancer vaccine candidates from synthesis to biologics: preclinical data on a chemistry derived anticancer vaccine a phase i study of unimolecular pentavalent (globo-h-gm -stn-tf-tn) immunization of patients with epithelial ovarian, fallopian tube, or peritoneal cancer in first remission immunogenicity study of globo h analogues with modification at the reducing or nonreducing end of the tumor antigen significant impact of immunogen design on the diversity of antibodies generated by carbohydratebased anticancer vaccine synthetic carbohydrate vaccines: synthesis and immunogenicity of tn antigen conjugates preparation of a multiple antigen glycopeptide (mag) carrying the tn antigen. a possible approach to a synthetic carbohydrate vaccine antitumor immunity provided by a synthetic multiple antigenic glycopeptide displaying a tri-tn glycotope a fully synthetic immunogen carrying a carcinoma-associated carbohydrate for active specific immunotherapy linear padre t helper epitope and carbohydrate b cell epitope conjugates induce specific high titer igg antibody responses synthesis and biological evaluation of a multiantigenic tn/tf-containing glycopeptide mimic of the tumor-related muc glycoprotein chemoselective assembly and immunological evaluation of multiepitopic glycoconjugates bearing clustered tn antigen as synthetic anticancer vaccines a fully synthetic vaccine consisting of a tumor-associated glycopeptide antigen and a t-cell epitope for the induction of a highly specific humoral immune response fully synthetic vaccines consisting of tumor-associated muc glycopeptides and a lipopeptide ligand of the toll-like receptor a cancer therapeutic vaccine based on clustered tnantigen mimetics induces strong antibody-mediated protective immunity immunological response from an entirely carbohydrate antigen: design of synthetic vaccines based on tn-ps a conjugates sialyl-tn polysaccharide a as an entirely carbohydrate immunogen: synthesis and immunological evaluation carbohydrate-monophosphoryl lipid a conjugates are fully synthetic self-adjuvanting cancer vaccines eliciting robust immune responses in the mouse synthesis and evaluation of monophosphoryl lipid a derivatives as fully synthetic self-adjuvanting glycoconjugate cancer vaccine carriers a fully synthetic self-adjuvanting globo h-based vaccine elicited strong t cell-mediated antitumor immunity synthesis and evaluation of gm -monophosphoryl lipid a conjugate as a fully synthetic self-adjuvant cancer vaccine robust immune responses elicited by a fully synthetic three-component vaccine towards a selfadjuvanting multivalent b and t cell epitope containing synthetic glycolipopeptide cancer vaccine antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing b cell, cd + and cd + t cell epitopes synthetic multivalent glycopeptide-lipopeptide antitumor vaccines: impact of the cluster effect on the killing of tumor cells synthesis and immunological properties of n-modified gm antigens as therapeutic cancer vaccines efficient metabolic engineering of gm on tumor cells by n-phenylacetyl-d-mannosamine improving the antigenicity of stn antigen by modification of its sialic acid residue for development of glycoconjugate cancer vaccines synthetic and immunological studies of ′-n-phenylacetyl stn to develop carbohydrate-based cancer vaccines and to explore the impacts of linkage between carbohydrate antigens and carrier proteins synthetic and immunological studies of stn derivatives carrying -n-(p-substituted phenylacetyl)sialic acid for the development of effective cancer vaccines improvement of the immune efficacy of carbohydrate vaccines by chemical modification on the gm antigen enhancement of the immunogenicity of synthetic carbohydrate vaccines by chemical modifications of stn antigen synthetic and immunological studies of n-acyl modified s-linked stn derivatives as anticancer vaccine candidates a cancer vaccine based on fluorine-modified sialyl-tn induces robust immune responses in a murine model synthesis and evaluation of glycoconjugates comprising n-acyl-modified thomsen-friedenreich antigens as anticancer vaccines live attenuated vaccines: historical successes and current challenges protein carriers for glycoconjugate vaccines: history, selection criteria entirely carbohydrate-based vaccines: an emerging field for specific and selective immune responses. vaccines (basel) publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions cyw: manuscript initiation, organization, revision and submission. rm and cyc wrote the manuscript. all authors read and approved the final manuscript. availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord- -r hzazp authors: stowe, julia; andrews, nick; miller, elizabeth title: do vaccines trigger neurological diseases? epidemiological evaluation of vaccination and neurological diseases using examples of multiple sclerosis, guillain–barré syndrome and narcolepsy date: - - journal: cns drugs doi: . /s - - -y sha: doc_id: cord_uid: r hzazp this article evaluates the epidemiological evidence for a relationship between vaccination and neurological disease, specifically multiple sclerosis, guillain–barré syndrome and narcolepsy. the statistical methods used to test vaccine safety hypotheses are described and the merits of different study designs evaluated; these include the cohort, case-control, case-coverage and the self-controlled case-series methods. for multiple sclerosis, the evidence does not support the hypothesized relationship with hepatitis b vaccine. for guillain−barré syndrome, the evidence suggests a small elevated risk after influenza vaccines, though considerably lower than after natural influenza infection, with no elevated risk after human papilloma virus vaccine. for narcolepsy, there is strong evidence of a causal association with one adjuvanted vaccine used in the / influenza pandemic. rapid investigation of vaccine safety concerns, however biologically implausible, is essential to maintain public and professional confidence in vaccination programmes. vaccination is one of the most effective public health interventions successfully controlling many serious infectious diseases and saving millions of lives globally each year [ ] . however, as with any medical treatment or drug, vaccination can never be entirely risk free in terms of unwanted side effects. an important feature of vaccination is that unlike most therapeutic drugs, vaccines are given prophylactically to healthy individuals, often young children. when an event occurs shortly after vaccination in an otherwise healthy individual without an obvious cause, it is tempting to attribute its occurrence to the preceding vaccination. the assumption of a causal association with a vaccine from purely a temporal association is often incorrect as unrelated events will occur by chance irrespective of vaccination. it can be hard to disentangle these temporal associations when there is a strong perception that a temporal association is necessarily evidence of a causal association and the onset of the condition is insidious and its timing relies on patient or parental recall [ ] . even if only based on a temporal sequence of events, it is important that such safety concerns are rapidly investigated with robust epidemiological studies to allow mitigation procedures to be put in place if an association is confirmed or, if unfounded, to have the necessary evidence to sustain public confidence in the vaccination programme without which coverage drops and disease control is lost. in this article, which focusses on the evaluation of the relationship between vaccination and neurological diseases, the statistical approaches to causality assessment are first discussed and their relative merits evaluated, followed by an overview of a selection of vaccine safety studies involving neurological disease with differing conclusions; some of the included studies have shown a small elevated risk, others none, two lack evidence to draw any definitive conclusion and one provides robust evidence of causal association. to establish whether the signal seen is associated with the vaccine and to quantify the risk, a formal epidemiological study is usually needed. this requires a pre-specified protocol detailing the population under study, the period after vaccination for which an elevated risk is suspected, and the methods for case identification and statistical analysis. most importantly, the ascertainment of the condition of interest must be unbiased with respect to vaccination history [ ] . the following statistical methods have been used most commonly to address vaccine safety questions and to control for the inherent biases in the population and data under study. although these methods aim to address confounding, it can be difficult to fully control for this in an observational study. an assessment of the likelihood of residual confounding/ bias and its potential extent is an important consideration when weighing up the strength of a study and drawing a conclusion with regard to causality. in a cohort study, the risk of developing the condition is compared in the vaccinated and unvaccinated individuals in the study population. cohort studies need to be very large to detect rare vaccine adverse events and this often makes them impractical for a prospective study. retrospective cohort designs can use routinely collected data and cases identified by clinical coding but this study design may be disadvantaged by the need to collect a large number of confounding variables. factors such as underlying illnesses, sociodemographic characteristics, and propensity to consult may differ between unvaccinated and vaccinated individuals and would therefore need to be adjusted for in the analysis as they can independently determine the likelihood of the adverse event under study. the advantage is that an entire population is studied and relative and absolute incidence estimates can be reported. in addition, once the cohort is defined, several outcomes can be assessed within the same study design. when studying a vaccine that is given as part of a national schedule and high coverage is achieved, the small unvaccinated group may differ from the vaccinated group in ways that are difficult to capture and control for in an adjusted analyses. additionally, care must be taken to ensure unvaccinated cases are indeed unvaccinated and the data are not missing. this can occur when regional vaccine datasets are used and the transfer and sharing of data are not comprehensive. cohort studies are feasible for vaccine safety studies when data from a whole country or region can be used. an example of this is in denmark where danish residents contribute to a large linked dataset consisting of demographic factors that are linked to health information including potential confounding variables [ ] . the self-controlled case-series method (sccs) was designed for rapid unbiased assessment in vaccine safety studies using available disease surveillance data that may not be amenable to cohort analysis. the method only requires information on the timing of cases during a defined observation period and their vaccination status [ ] . the cases act as their own controls as the incidence of the event in pre-defined risk periods following vaccination is compared to the incidence outside the risk period generating a relative incidence (ri) measure ( fig. ) . a significant advantage of the method is that confounding factors that do not vary over the observation period, such as co-morbidities or sociodemographic status, are automatically controlled for. adjustment for timevarying confounders such as age is also possible by dividing up the observation period further into age categories. it has been demonstrated that the power of the sccs method is nearly as good as a cohort study when uptake is high and risk intervals are short, and it is superior to that of a casecontrol study [ ] . the self-controlled case-series method has been used by public health england to address many pertinent vaccine safety concerns [ ] [ ] [ ] [ ] . this design has been chosen both because of its simplicity and ability to control for individual level confounding and also because a national cohort of cases cannot be easily defined using the national hospital data as no national immunisation register is available. unlike a cohort study, the sccs method does not provide absolute risk estimates. however, if the number of doses given to the population from which the cases are derived is known and if ascertainment is complete, then absolute risks can be estimated and the cases attributable to vaccination estimated from the magnitude of the ri. a case-control study requires smaller numbers than a cohort study but the same confounding and bias can occur and it also has the added difficulty of selecting the correct controls for comparison. for vaccinations given in the short age range in the first and second year of life or during a short calendar period to target ages, close matching of the controls on date of birth is required. prior vaccination status is then compared between cases and controls using the date of onset in cases as a reference date. to obtain enough power to assess the required risk, multiple controls per case are often needed and defining appropriate criteria for the selection of controls can be problematic. while it is important to ensure that controls are similar to cases on characteristics such as age and geographical location that can independently affect vaccination status, over matching is a risk if too many extraneous variables are included in the matching, resulting in loss of efficiency and potentially introducing bias. a case-control study does not provide absolute risk estimates, rather it measures the odds of vaccination in cases compared to controls. however, as with the sccs method, if the number of doses given to the population from which the cases are derived is known and if ascertainment is complete, then absolute risks can be estimated and the cases attributable to vaccination estimated from the magnitude of the odds ratio. the case-control design has been used where controls can be selected from the same population as cases and can be readily matched on the relevant variables. as a case-control approach is more efficient than the cohort approach, it is often used on large databases that could be used for a cohort analysis. examples include the vaccine safety datalink in the usa, which accesses complete patient records from health maintenance organisations, or studies using hospital admission data bases linked to national immunisation registers such as the australian childhood immunisation register [ ] [ ] [ ] . the case-coverage design has recently been used in vaccine safety studies [ , ] . it is similar to the screening method, which until recently has been primarily used for vaccine effectiveness assessment [ ] , although it is more limited in terms of adjustment for possible confounders than the sccs method. each case is matched to a population coverage estimate and this is then used to see if the number of cases vaccinated is greater than expected. the method uses logistic regression on the odds of vaccination with an offset for the log-odds of the matched population coverage, thus it is similar to a case-control study with thousands of controls per individual. this design has been used by public health england to assess the risk between as adjuvanted h n pandemic vaccine pandemrix™ and narcolepsy. because pandemrix™ was rolled out over a short period of time in the winter season of / targeting children of different ages according to whether they had certain co-morbid conditions, it was necessary to have detailed information on dates of vaccination and dates of birth to estimate the population coverage for each narcolepsy case by age and time period. this was available from a representative subset of general practices in england, which also provided information on co-morbidities, the only other variable considered as a potential confounder [ ] . in the first study assessing the risk of narcolepsy in children [ ] , both the sccs method and the case-coverage design was used. the results from the sccs method were found not to be clear as this method requires the incidence in a pre-specified risk period after vaccination relative to the baseline incidence to be compared. because the duration of the risk period had not been defined at the time, the postvaccination interval was found to be too short and resulted in the inclusion in the baseline period of four patients with symptoms more than months after vaccination. the choice of study design to answer a vaccine safety question will depend on the hypothesis to be tested, the available data sources and the extent to which confounding variables are likely to bias the results. the sccs method has now become the gold standard design in vaccine safety studies, owing to the benefits highlighted above, but for each study question the methods should be adapted and potential biases considered in the context of the population under study, the dataset being utilised and the hypothesis being tested. it will inevitably be a trade-off between the ideal and the practical and the best designs will vary according to setting. when many studies are performed to answer the same question, the key to demonstrating causality is consistency in the results from well-designed studies [ ] . neurological conditions have a long history of causal associations with vaccination being inferred from temporally related onsets. an example is the damage that was made to the uk whole-cell pertussis vaccination programme in the late s when neurological damage was wrongly attributed to the vaccine based on case reports of infants with onset of encephalopathy shortly after vaccination. these reports of permanent brain damage following vaccination attracted intense and sustained professional and media interest causing vaccination rates to fall from % in to % in . following this, three national epidemics of pertussis occurred with an estimated hospital admissions, cases of pneumonia, cases of convulsions and deaths [ , ] . neurological vaccine safety concerns can be broadly assigned to either being biologically plausible or unsubstantiated and unexpected. the biologically plausible group are often a direct effect from a component of the vaccine. for example, in the case of a live attenuated vaccine, the adverse reaction could mimic, at a lower frequency, what the non-attenuated wild virus would do. this is demonstrated in the rare risk of acute flaccid paralysis following the oral polio vaccine after a reversion to virulence or with the risk of aseptic meningitis after the attenuated urabe mumps strain in the measles-mumps-rubella vaccine due to retention of some neurovirulent characteristics [ , ] . the unsubstantiated and unexpected group occurs usually because of the timing of the vaccine, which coincides with the diagnosis of the condition and has no immediate biologically plausible explanation. examples of this are measles-mumps-rubella and autism [ ] , gait disturbance and measles-mumps-rubella [ ] , and thiomersal and developmental delay [ ] . although a signal may not have a clear biological basis for its causation, it still needs to be fully investigated using robust epidemiological methods. neurological diseases for which a causal association with vaccination has been suspected have some common features. first, they are often serious conditions that are rare, second, their aetiology and pathophysiology are poorly understood, and third, immune stimulation is thought to play a role in the pathogenesis of the condition. because vaccines provoke an immune response, albeit targeted to a specific antigen, it can be tempting to invoke a superficially plausible causal pathway when adverse events with a suspected immune aetiology arise shortly after vaccination. universal hepatitis b vaccine was recommended by the world health organization in the early s to protect against the hepatitis b virus, which can cause chronic liver damage and cancer. following this recommendation, france carried out a mass vaccine campaign in . shortly after, reports of cases of multiple sclerosis (ms) with onset or relapse after vaccination were reported, leading to the hypothesis that the vaccine could cause an acute autoimmune reaction in susceptible persons soon after administration. with a lack of adequate background rates of ms in the vaccinated population to put the reported cases into perspective, mistrust in the vaccine soon grew and the vaccine programme was subsequently suspended. a systematic review and meta-analysis by mouchet et al. published in that included studies with a control group found no evidence of an increased risk. the overall adjusted risk ratios for ms was . ( % confidence interval [ci] . - . ) and for central demyelination was . ( % ci . - . ) [ ] . within the systematic review, there was one study that found a significant association using a primary care database from england [ ] . this study was unable to adjust for all risk factors and additionally no routine hepatitis b vaccination programme was in place at the time with most of the vaccine delivered via occupational health departments whose records may not be routinely transferred to primary care databases. france continues to have suboptimal vaccine coverage [ , ] and has the lowest level of confidence in vaccine safety in europe [ , ] . this demonstrates the need to have robust methods in place to rapidly respond to such scares because once confidence is lost in a vaccine it is difficult to restore and may generate a more general lack of confidence in vaccine safety. guillain-barré syndrome (gbs) is the most common cause of acute neuromuscular paralysis in the developed world resulting in muscle weakness and sometimes paralysis, which can lead to respiratory failure and a death rate in up to % [ ] . the strongest evidence of a causal link with a vaccine was obtained during the us swine influenza vaccine programme in military personnel, which was found to be associated with a risk of one case per , and resulted in the suspension of the vaccine programme [ ] . since then, gbs has been a potential vaccine-associated adverse event of interest particularly for vaccines given in adolescence, an age coinciding with the age at which autoimmune diseases are often diagnosed. . - . ) . the overall relative risk for gbs after seasonal vaccine was marginally increased at . ( % ci . - . ), with a somewhat larger relative risk of . ( . - . ) for the h n pandemic vaccine but this was not significantly higher than the relative risk for seasonal vaccine [ ] . the authors did not find any statistically significant differences by geographical region nor between adjuvanted and unadjuvanted vaccines. an earlier meta-analysis of studies using the sccs method also found a small elevated risk of gbs after the monovalent h n pandemic vaccine, with an ri of . ( % ci . - . ) in the days following vaccination [ ] . similarly, salmon et al. found an ri of . ( % ci . - . ) in a large study in the usa [ ] . in contrast, a strong association between gbs and a preceding influenza-like-illness was shown in a study in england using primary care data and the sccs method. no association was seen with influenza vaccine in the - days after administration (ri . [ % ci . - . ]) but a significantly increased risk was found in the days after influenza-like illness (ri . [ % ci . - . ]) [ ] . these studies show that a small overall risk of gbs after influenza vaccine probably does exist with a slightly larger risk after the monovalent pandemic vaccine. the mechanism may be multi-factored with the risk varying with the vaccine used, co-circulation of other infections and the inherent susceptibility to developing gbs. however, the small risk that exists does not outweigh the risk of developing gbs after influenza itself. human papilloma virus vaccine is given at an age when autoimmune disorders are often diagnosed. following a french study reporting a signal for gbs after human papilloma virus vaccination, a study was conducted in england identifying gbs cases in a national hospital discharge database (hospital episode statistics) [ ] . primary care practitioners were then contacted for the vaccination history and asked to confirm gbs diagnosis and provide an onset date and send supporting documentation. in a selfcontrolled case-series analysis of cases with a record of human papilloma virus vaccination, there were episodes in the -to -day risk period after any dose with no significant increased risk, ri . ( % ci . - . ). the analysis was also stratified by manufacturer (of either the quadrivalent or bivalent product); there was no difference in the ri between products and no significant increased risk for either manufacturer. the pandemic influenza vaccine, pandemrix™, was the most widely used vaccine in europe during the pandemic. it was a monovalent h n pdm vaccine containing as , a powerful oil-in-water adjuvant. uptake of the vaccine varied between countries with high coverage of % in children in finland [ ] and lower coverage in england where children in a risk group eligible for the seasonal influenza vaccine and later all children under years of age were targeted, with uptake being % and %, respectively. in england, pandemrix™ was also used in the influenza season / because of a shortage of seasonal influenza vaccine. in august , concerns were raised in finland and sweden, where vaccine coverage was high, about a possible association between narcolepsy and pandemrix ™ when a large increase in cases of narcolepsy in vaccinated cases was reported by sleep centres [ , ] . a subsequent cohort study in finland reported a -fold increased risk of narcolepsy following pandemrix™ in children aged - years, the majority of whom had onset within months of vaccination and almost all within months [ , ] . narcolepsy was a totally unexpected adverse event and the early reports were met with initial scepticism in the global vaccine community. the world health organization global advisory committee in vaccine safety issued a statement in april stating "no excess of narcolepsy has been reported from several other european states where pandemrix was used" and "it seems likely that some as yet unidentified additional factor was operating in sweden and finland". however, it was unlikely that narcolepsy would be identified by passive surveillance systems in other countries where pandemrix™ coverage was low given the low background incidence of the condition and the complexity and frequent delays in diagnosis. to assess this risk identified in finland, the health protection agency (now public health england) performed a study in sleep centres in england where the majority of children with sleep disorders are seen. this study identified a -fold increased risk in those vaccinated with pandem-rix™ [ ] with the attributable risk estimated to be . per , doses. this demonstrated that even in a country were vaccine coverage was low, the association can be demonstrated using robust epidemiological methods. the study of the relationship between narcolepsy and pandemrix™ has been an epidemiological challenge in terms of identifying the cases and their vaccine histories in a non-biased manner. not only can the diagnosis be lengthy and complex, but admitted patient care databases, which are widely used for a non-biased ascertainment of cases in vaccine safety studies, are incomplete as patients experiencing narcolepsy may not be admitted and if they are admitted, the admission date is not an accurate reflection of the onset of the narcolepsy symptoms leading to misclassification bias. an important consideration when selecting cases is the awareness of the hypothesised association. this awareness may lead to an increased reporting of cases known to be vaccinated and has two aspects; public awareness and professional awareness. first, this heightened awareness may lead to vaccinated individuals presenting to healthcare institutions and being diagnosed earlier than unvaccinated cases leading to ascertainment bias. if a condition has an insidious onset making the recall of the first symptom difficult to determine, media attention may lead to a differential recall of the symptom-onset date in the vaccinated cases. using source documents, which were created prior to any media attention in the country of study, can address this potential recall bias. professional awareness is likely to occur even if media attention is low, as health professionals in the specialty will be aware of current topics of interest through professional bodies and literature. differential misclassification bias will occur if cases known to have been vaccinated are more likely to be assigned a diagnosis of narcolepsy than unvaccinated cases. in the study from england, public awareness of the association was assessed by analysing google searches for "narcolepsy" in the period of interest and found there was little activity in the uk compared to sweden (fig. ) [ ] . even with these practical challenges, there has now been a consistent strong association demonstrated in countries that used pandemrix™ but no association has been seen with other pandemic or seasonal vaccines [ ] . as with all vaccine safety studies, but particularly in the case of narcolepsy and pandemrix™ where the association was completely unexpected, the key to demonstrating causality was consistency of results from well-designed studies in different settings. the answer to the question of whether vaccination can cause neurological disease is multifaceted. the evidence does not support an association between ms and the hepatitis vaccine, while for gbs and influenza vaccines the evidence suggests a small increased risk though it is much smaller than the risk from a natural influenza virus infection. the now established association between narcolepsy and pan-demrix™ should act as a lesson for the vaccine safety community that sometimes unexpected but serious conditions can arise and need to be investigated rapidly however biologically implausible. the neurological vaccine safety issues outlined here demonstrate that rapid assessments of safety signals are needed to ensure that public confidence is maintained in national immunisation programmes. the confirmation of a signal and estimation of the magnitude of vaccine-attributable risk will require consistent results from a number of well-designed epidemiological studies, preferably conducted in different settings. as the experience with narcolepsy has shown, not all vaccine safety concerns can be anticipated on the basis of biologically plausible and thus predictable effects. as new vaccines are introduced, the basis of discussions on vaccine safety should be the acceptance that vaccination can carry a small risk but that this risk needs to be balanced against the enormous individual and public health benefits. funding public health england, national infection service, immunisation and countermeasures division has provided vaccine manufacturers with post-marketing surveillance reports, which the marketing authorisation holders are required to submit to the uk licensing authority in compliance with their risk management strategy. a cost recovery charge is made for these reports. world health organization. the power of vaccines: still not fully utilized recall bias, mmr, and autism vaccine safety surveillance postlicensure epidemiology of childhood vaccination: the danish experience control without separate controls: evaluation of vaccine safety using case-only methods statistical assessment of the association between vaccination and rare adverse events post-licensure autism and measles, mumps, and rubella vaccine: no epidemiological evidence for a causal association guillain-barre syndrome and h n ( ) pandemic influenza vaccination using an as adjuvanted vaccine in the united kingdom: self-controlled case series idiopathic thrombocytopenic purpura and mmr vaccine the risk of intussusception following monovalent rotavirus vaccination in england: a self-controlled case-series evaluation population-based study of rotavirus vaccination and intussusception intussusception risk and disease prevention associated with rotavirus vaccines in australia's national immunization program mmr vaccine and idiopathic thrombocytopaenic purpura risk of narcolepsy in children and young people receiving as adjuvanted pandemic a/h n influenza vaccine: retrospective analysis risk of narcolepsy after as adjuvanted pandemic a/ h n influenza vaccine in adults: a case-coverage study in england estimation of vaccine effectiveness using the screening method incidence of narcolepsy after h n influenza and vaccinations: systematic review and meta-analysis pertussis immunisation and control in england and wales, to : a historical review the pertussis vaccine controversy in great britain risk of aseptic meningitis after measles, mumps, and rubella vaccine in uk children risks of convulsion and aseptic meningitis following measlesmumps-rubella vaccination in the united kingdom autism and mmr vaccination in north london; no causal relationship no evidence of an association between mmr vaccine and gait disturbance thiomerosal exposure in infants and developmental disorders: a retrospective cohort study in the united kingdom does not support a causal association hepatitis b vaccination and the putative risk of central demyelinating diseases: a systematic review and metaanalysis. vaccine recombinant hepatitis b vaccine and the risk of multiple sclerosis: a prospective study estimates of national immunization coverage european centre for disease prevention and control. measles vaccination coverage (second dose the state of vaccine confidence : global insights through a -country survey vaccine hesitancy among general practitioners and its determinants during controversies: a national cross-sectional survey in france guillain-barre syndrome guillain-barre syndrome following vaccination in the national influenza immunization program guillain-barre syndrome and influenza vaccines: a meta-analysis international collaboration to assess the risk of guillain barre syndrome following influenza a (h n ) monovalent vaccines association between guillain-barre syndrome and influenza a (h n ) monovalent inactivated vaccines in the usa: a meta-analysis investigation of the temporal association of guillain-barre syndrome with influenza vaccine and influenzalike illness using the united kingdom general practice research database no increased risk of guillain-barre syndrome after human papilloma virus vaccine: a self-controlled case-series study in england increased incidence and clinical picture of childhood narcolepsy following the h n pandemic vaccination campaign in finland risks of neurological and immune-related diseases, including narcolepsy, after vaccination with pandemrix: a population-and registry-based cohort study with over years of follow-up as adjuvanted ah n vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in finland key: cord- -elhgew x authors: spier, r.e. title: ethical aspects of vaccines and vaccination date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: elhgew x nan published by elsevier science ltd. all rights reserved printed in great britain pii: so - x( ) - - x/ $ + . r. e. spier traditional certainties no longer serve our societies as well as they might. technical developments over a broad range of engineering disciplines have radically changed the nature of the physical world in which we live. our knowledge base has expanded concurrently, enabling us to provide materially based and more convincing answers to questions which baffled our antecedents of years ago. this requires that we review and reconsider the ethical guidelines for behaviour which have been largely founded on philosophies and concepts whose origins may be traced back to between and years ago. an example of the implications of these changes may be seen in the area of vaccines and vaccination which evinces the pressing need to review traditional ethical positions to take the maximum advantage of the potential for animal and human benefit inherent in this prophylactic approach to healthcare. in this paper i examine some of the general ethical issues thrown up by recent advances in the field of vaccines and vaccination. it will touch on issues of the putative autonomy of the individual and way we will have to reassess the cost ((risk * the magnitude of the damage) + cost of manufacture and distribution + surplus) to benefit relationship. issues subtended from the effect of vaccines on the magnitude of populations will be followed by transcultural issues implicit in vaccine testing and delivery. the use of vaccines to obviate behavioural changes (technical fixes), generate transcendental concerns and provide new threats via biological warfare agents will also be treated. it is part of current medical practice when dealing with patients to extol four basic ethical principles: autonomy, beneficence, non-maleficence and justice. however, when we consider issues related to vaccination the principle of autonomy (self-determination; freedom from interference unless the act harms others (j.s. mill); as in un or cfe declarations on human rights) is challenged. while the principle of autonomy may be asailed from a number of different facets such as the competence of an individual to provide informed consent, the rights of a fetus if the pregnant mother decides to be immunized or the implicit implications of the social contract entered into when an individual chases to dwell in a particular society, i will examine in university of surrey, school of biological sciences, guildford, surrey gu xh, uk more detail a further issue involving the expression of social responsibility. it is well known that when a high proportion of a population is vaccinated, those who have not been immunized become protected by the decrease in the level of the pathogenic organism (the herd effect). the question which this poses is, do those who have opted against vaccination have the right to benefit from the expense and the risks of vaccine-induced damage accepted by those who have been vaccinated? not only that, but such unvaccinated individuals pose a threat to the vaccinees as they serve as a reservoir in which the disease can be maintained and propagated. were the society to collectively determine that all its citizens should receive the vaccine then the principle of patient autonomy is infringed. an intermediate position might be to levy a special cash buy-out dispensation to those who refuse vaccination as a contribution to the costs incurred by those who have accepted the risks of vaccine-induced damage. whatever the outcome in any particular society it is clear that vaccination poses a challenge to accepted ethical positions, the resolution of which will be dependent of the degree of social coherence and farsightedness. for much of the last years commercial companies have been required to provide products to the marketplace and society's judgement on the company has been via the acceptability of the product at the price demanded. the function of the company was to accumulate profits for its shareholders and top managers (incentives) and society did not interfere with the way the product was made, nor with the product spectrum on offer. this ethic too, is in need of review. vaccines are a clear benefit to society as a whole. however, there is a risk of vaccine-induced long-term and severely debilitating damage [well recognized for polio vaccines and less well established for any other vaccine cf. swine fever (inactivated influenza) vaccines and guillain-barre syndrome] which is especially poignant when incurred after the vaccination of a -month-old healthy infant. under present conditions the manufacturing company may be sued for damages both actual and punitive (if wilful negligence is proven). but is this the appropriate ethic? may not the society, in accepting the benefit of widespread vaccination, compensate those who suffer damage at a level communsurate with the damage? of course, were the manufacturer culpably negligent in a procedural ethical aspects of vaccines and vaccination: r. e. spier matter, then compensation would also be due from that source. resulting from this liability situation, vaccine manufacturers are reluctant to venture into vaccine projects. also, as the cost of obtaining a product license is estimated to be some $ - million, the implications for the cost of a dose of vaccine is more driven by the cost of the regulatory procedure than the production cost (which rarely exceeds the cost of the bottle plus label). the ethical issue here is whether the regulatory hurdle which has to be overcome is really operating in society's best interests. nothing we eat or do is without the risk of incurring damage. vaccines are not different in that respect. yet the cost of obtaining a license to manufacture and distribute implies that the cost of the vaccine has to be many dollars merely to recoup the costs involved. does this mean that the rich should pay a high price for a vaccine and thus subsidize the provision of cheap vaccines to the poor? or is it the job of the elected representatives of the people to act as purchaser on behalf of the poor and provide the vaccine manufacturer compensation through the tax system or other fiscal dispensation? vaccines for diseases which afflict a few are not a commercial proposition unless communal support is provided. vaccines which lead to a decrease in the need for over-the-counter or prescribed medicaments are also unlikely to be made by commercial concerns. the reluctance of pharmaceutical companies to workup a vaccine for helicobactu pylori which would prevent the recurrence of stomach ulcers and hence stomach cancers is evident from the research programmes of those companies which make anti-ulcer drugs. research on vaccines which would prevent the common cold (caused by a combination of rhinoviruses, coronaviruses and admoviruses) is also conspicuous by its absense. but prophylaxis as an approach to healthcare is also underfunded by society at large. therapeutic research receives over times the funding of prophylaxis; and this is particularly difficult to understand when there are many inexpensive ways of achieving prophylaxis, of which vaccination, which affects the immune system, is but one. other methods of prevention focus on the protection of the immune system to decrease the probability that it will be overwhelmed by an endogenous or exogenous pathogen; such procedures may be termed 'fence vaccines". a new relationship between industry and the community is indicated. the former is now required to recognize that it will be judged as much by the ethicality of its actions as the efficacy of its products. for the pharmaceutical industry, ethics is not an optional extra: it is essential. an ethical argument which is proscriptive of the use of vaccines in the developing and less developed world (containing % of the world's . billion people) is that they will lead to an increase in an already unsustainable population. this will have sequellae via an increase in suffering from malnourishment, population migrations and war. however, recent figures published by unicef refute these projections' and show that the average number of children born to a woman of the developing world throughout the period of her fertility has dropped from . to . between and . this would indicate that more, rather than fewer, vaccines are required; and indeed vaccines protective to the diarrhoeal and respiratory diseases of childhood are on test in the field, while candidate vaccines aimed at controlling malaria languish in laboratory fridges. population may not only be affected by a decrease of infant mortality but also by an increase in the average age of the community. as the level of communicable disease wanes, people live longer and society then has to adjust its working conditions such that there are productive positions for the older people to occupy. it is clearly not practicable to socially provide for protracted retirements, so the emphasis on life-long learning, skill changing, job flexibility and part-time working will become the norms of future social development. is it appropriate to use a technical fix when an almost cost-free change of behaviour will achieve the same effect? such an ethical problem is thrown up by the willingness of our communities to spend billions of dollars to provide therapeutic and prophylactic agents to control the spread and effects of the human immunodeficiency virus (hiv), while the disease would be eliminated were people to engage in safe, condom-protected, intercourse in their pre-or extramarital sexual relationships where the prospective partners had not been thoroughly tested for the presence of serum antibodies to the virus. this provision also applies to the transmission of the viruses which cause hepatitis b, genital herpes, as well as the cancers wrought by the papilloma virus. as a corollary to the practise of safe sex, it might also be expected that gonorrhoeal infections would decline, as would those caused by treponema sllphilis and the yeast candida. many of the food-and water-borne diseases caused by bacteria of the salmonella. escherichia, shigella, listeria, campylobacter and vibrio groups would be eliminated were drinking and washing water to be prepared according to the highest standards prevalent in most developed countries. however, the engineering requirements to achieve this in the short term are daunting. whereas the prospect of the development of orally deliverable vaccines which would provide protection against the diseases caused by the above pathogenic bacteria is a task which may be brought to a successful conclusion within the next decade. a further case where vaccines are used to preclude the expenditure of monies is to protect people from the effects of the diseases of propinquity; typhus and tuberculosis. both of these diseases flourish when people are housed in crowded insanitary conditions. it may be that the vaccination route is cost-effective in monetary terms but this should not be used as a way of avoiding the social improvements which would enhance ethical aspects of vaccines and vaccination: r. e. the dignity of citizens, as well as improving health. spier their infectious disease-causing organisms do not recognize national boundaries. transworld travel for tourism and business is increasing exponentially and with it are opportunities for disease-causing organisms to travel. we are also presented with a situation in which tests of vaccines in developing countries can be effected at considerably less expense than in a developed country. this has led to a series of ethical issues which are exacerbated by the different cultures of the people who may be engaged in the vaccine trials. for example, is it possible to obtain the informed consent of a person who is illiterate and who does not understand the implications of something like a vaccine with which (s)he is totally unfamiliar ? a second issue might be that the removal of blood or tissue for sampling might be regarded as an attempt to capture the spirit of the so deprived individual. additionally, there may be taboos about removal of blood via venipuncture and in some cultures the insertion of needles into bodies may have overtones not foreseen in western cultures. on removal of a sample containing cellular material from the body of an individual (generally a tissue responsible for a pathogenic effect) one obtains the opportunity to work with a highly selected and unique genome. were the genes of that cell to be used to make a pharmaceutical to particularly benefit people in the developed world, what sort of compensation should acrue to the source of the cell line from which the gene was obtained? current thinking by the nuffield ethics committee" would have it that the cell provider has not contributed to the inventive step in the drug or prophylactic development and therefore is not to be compensated. however, if advantage is taken of the uniqueness of the material derived from a person of the developing world then it would be churlish not to recognize this through some financial contribution to the individual and his/her community. one might ask, to what extent is a prophylactic trial in the developing world relevant to the circumstances prevalent in the developed world? are the conditions leading to infection and the challenge organisms relevant? are the people in whom the vaccine is tested likely to respond in an immunologically equivalent manner when the history of the exposure of their immune systems to disease is dissimilar in many ways to a person of the developed world? in the event that there is damage to an individual as a result of exposure to vaccine in a trial, what are the levels of compensation and who pays? and indeed, is it ethical that a person in the vaccine-producing country should enjoy the benefits which have been won at the expense of the risk-taking of a person in less privileged circumstances? to some extent many of these questions may have answers were the developed country vaccine producer to agree up-front to provide cost-free vaccine to all the people of the country in which the vaccine trial had been effected. in that way something of a bargain may be established such that overt exploitation has been subverted into mutual gain. in all such situations it would not be an acceptable ethic to effect trials with placebo controls which did not provide the best possible protection. similarly it would be counterproductive to use vaccines whose safety was an issue and where a less damaging vaccine could be made available, albeit at greater expense. in addition, there is the overriding consideration that if nothing is attempted then there would be a known tally of deaths and disease and our efforts to combat that embody a justification for effecting vaccine experimentation. a definition of the transcendental might be 'that which is outside the cause-and-effect system', where the latter implies that all which exists and the way it interacts is delimited by the energy and matter of its constitution. for example, ghosts, fairies, trolls, spirits, jinn and souls are described in such a way that they perform their tasks with scant regard for the properties of matter and energy, as do the panoply of deities which have been posited as having creative and control capabilities with regard to the affairs of humans. nevertheless, such considerations cannot be obviated when we review the reactions of community members to the production and use of vaccines, particularly when some of the most emphatic proscriptive reactions emanate from the leadership of recognized deitic religions. in kirkpatrick's book on inoculation published in there is the astonishing report of the abreaction of the church to vaccination because, as a result of the possibility of dying from the vaccine of the day (smallpox, occasionally contaminated with syphilis), it may be construed that the vaccinee was indeed seeking to commit suicide, which was sinful. in modern times there were a series of reports in the uk media' wherein the catholic church was seeking to prevail on its susceptible female members to forego vaccination protective against rubella, as the vaccine was prepared from the cells of an aborted human fetus: for human abortion is contrary to the teachings of that church. on a more esoteric plane, it is possible to argue that, through the use of vaccines, mankind has developed a capability which may eventually rid the world of those infectious microoganisms which have been the bane of our struggle to survive. this depletion of the deitic armamentarium may be construed as seeking to deny the deity of one of its controlling effector systems; namely, the threat of divine retribution through the causation of plagues . alternatively, it may be held with equal rectitude, that the deity ordained us to discover and use vaccines as part of its (undisclosed) master plan. consonant with this latter controversy, there are those who assert that it is unnatural to disturb the ways of nature and as vaccines are a creation of mankind, they are not natural and are therefore to be condemned. as this contention rests on the definition of what is natural it is possible to reconstrue this issue totally were we to assert that whatever exists is natural: merely by virtue of its existence. it would follow that there does not exist anything which is artificial (in the sense of unnatural) which implies that all the products of the arts (techniques, crafts, skills) of humans (and animals) are natural and are incorrectly designated as artificial unless the meaning of that term were changed to its more appropriate designation, as works which are the product of humankind's arts (hence arti-ficial; made by art). now that it is practicable to identify a defective gene in an adult, child or even embryo, techniques are in development to repair, exchange or inactivate that gene specifically. these methods imply the existence of 'genetic vaccines'. however, these self-same methods may be used not just to correct defects but to enhance characteristics we may desire to accentuate'. as the word 'disease' is defined as a state of being in which one is not-at-ease, it is not difficult to use the word to describe a situation where a person is not at ease with their height, intelligence, running or ageing, speed etc. this raises the ethical question of the use of genetic vaccines to prevent the disease resulting from such deeply held feelings. there is little doubt that the remediation and/or prevention of situations which cause physical pain is encouraged and applauded by society. the same cannot be asserted where the pain ethical aspects of vaccines and vaccination: r. e. spier may be psychological. nevertheless, this latter pain is just as actionable as the physical pain (indeed it is physical, but of the activities of the brain as opposed to muscle). indeed, not only would we relieve suffering but we might also achieve a human being who is more functionally capable of making a more extensive contribution to society; a feature which is not given to all pain-killing remedies. that such measures might be construed as interfering with some transcendental plan cannot be denied, but the ethic of beneficence might be applied to progress the use of these genetic vaccines in all their manifestations. were we to have an effective orally deliverable contraceptive vaccine' (pregnancy results from the infection of the female by a male spermatozoan) then ethical considerations will be required to determine the way in which such a powerful tool for population control might be used. the contamination of drinking water supplies with a contraceptive vaccine would act counter to the autonomy principle of ethics and could be held to be an affront to the dignity of humanity in denying individuals control over their own fertility. notwithstanding this clear ruling, it is possible to conceive of conditions in which a decrease in population levels is an urgent necessity and the use of an orally delivered contraceptive vaccine might be the only way of achieving that end without recourse to widespread sterilization. as with any other tool, we have to adopt an ethic which permits its appropriate use; what we need to do ahead of time is to discuss and debate what those circumstances might be. anthrax bacteria, botulinum toxin, the plague bacillus (yersinia pestis), measles and influenza viruses have all been proposed as agents of human destruction in the context of international and intranational conflict. both smallpox and measles have been implicated in the decimation of the indigenous populations of the americas during the colonization processes beginning in the s. vaccines protective against these diseases, therefore, become defensive devices which would have to be surmounted by a would-be aggressor. genetic engineering may be used to attempt to enhance the lethality of existing agents or make otherwise benign adding an ethical dimension to industry's external relationships legislators agents lethal. it is unlikely that an increase in lethality can be achieved through the manipulation of the genes which code for toxin structure, for the sophistication of the binding site of these toxins, coupled with the evolution of the enzyme which causes the damage, has probably reached the maximum level of efficacy as a result of the evolutionary process by which it was formed. the addition of new strain of toxin genes to a particular bacterial cell may also be assayed. but the common pattern of bacterial toxins leads to vaccine solutions which are likely to be cross-protective, irrespective of how many toxin genes are compiled in any one bacterial cell. perhaps. of more serious consequence, would be the engineering-out of the epitopes which evoke the immune system to produce toxin-neutralizing antibodies. when this occurs epitopes which heretofore were immunosuppressed become immunodominant'. this would call for the development of a new vaccine which could be made by using the newly engineered binding portion of the toxin molecule to induce neutralizing antibodies to the fully constituted toxin (binding portion plus enzymatic component). this process could be repeated many times. in addition to the manipulation of the toxin system, developments occur in the methods for the dissemination of the agents. while it is possible to conjure scenarios of contaminated water supplies and recognize that the air handling systems of large buildings may constitute a means of agent distribution, the mechanisms for the protection of such seemingly open targets are well in place already; for we do treat water supplies with inactivants and it is recognized that the circulation of air within air-conditioned buildings is fraught with problems if people's natural illnesses are circulated by air-handling equipment devoid of high performance filtration systems. to meet the contingencies of noxious agent distribution the stratagem of 'fence vaccination" may be brought into play. the cycle of measure/countermeasure with which we are familiar in the area of conventional and nuclear weapons has its echoes in the area of biological warfare. that we have a duty to defend the life of our peoples is an ethical principle which few would dispute. in the context of this paper, the pursuit of vaccines to existing and potential biological warfare agents is not just a job done in response to real or perceived threats but should become a mission whose importance ranks so highly that it has to be included with the strategic planning we have to undertake to survive. as in other areas of defensive reaction, we might expect the vaccine production techniques to be enhanced with regard to both the ability to manufacture high quality immunogenic preparations rapidly and also with respect to the engineering of those immunogens. these abilities will have spin-off effects with regard to our ability to produce vaccines protective against the biological agents which pose threats from natural sources, such as influenza viruses whose type changes require us to make immunogenically unique vaccines on a year-by-year basis. we have also to take note of the emergent infectious agents (ebola, hantavirus, hiv) which have achieved a degree of notoriety in recent years"'. our ability to respond to such agents has been slow, cumbersome, disorganized and paltry in relation to the importance of the test situation which these agents proffer. our ethics require that we improve on this performance. vaccine manufacturers must obtain a licence to enable them to market their vaccine products to the wider society. this licence is obtained on the recommendation of a body called a regulatory agency (the fda division of biologics in the usa, the committee for medicines in the uk, etc.). obtaining regulatory agency acceptability" of a vaccine product is a process which may take - years and cost $ - million dollars. as can be seen from figure , the regulatory agency is subject to being influenced by the society which it is set up to serve. in addition to this necessary connection to the regulatory agency, industry may opt to receive from universities and publicly funded research institutes information and materials which enable it to embark on new vaccine projects. this interaction between the private and public sectors is fraught with ethical problems stemming from the difference in the culture of the two types of institution". this has been rendered particularly acute in recent years when attempts have been instigated to make the publicly funded institutions behave as if they were private, profit-making bodies. the insinuation of industrialists into the peer review process for grant applications generated by academics has been one area where the bicameral functionality of the seconded industrialist leads to the funding of conservative research projects which do not compete with the industrialist's undisclosed mission. in this, society is not well served by the monies it sets aside for both academic and publicly funded research. society is not amorphous. it has structure through its institutions. the institutions which affect the way the regulatory process works may be identified as being ( ) the ethical bodies; ( ) the media; ( ) legislatures; and ( ) the educational system (figure ). the ethical bodies include recognized religions whose leaders provide ethical guidance to the members which impinges on the production and use of vaccines, while bearing in mind current social exigencies. there are secular sources of ethical guidelines which are not as well organized and overt. such individuals might recognize that ethics are not guidelines which are provided by the pronouncemnts of a deity but are rather the set points in the control system which modulates human social behaviour with the objective of promoting both the survival of the individual, society and other biotic entities as wealth and the occasion permits'". the media are informed by the religous ethicists as well as picking up on the raw nerve endings of the fears and sensitivities of their fellow socialites. they do not hesitate to evoke the image of the entity created by victor frankenstein at the prospects of the slightest opportunity of affecting a deliberate change to the genetics of the human species. our tradition encourages us to regard monsters as the avenging agents of personal wrongdoing; so such buttons do not require much pressing to evoke negative reactions to vaccine volume number ethical aspects of vaccines and vaccination: r. e. spier the prospects of experiments gone awry. the third effector on the way the regulatory agencies behave is the legislature. it is by law that pharmaceutical products have to be safe, efficacious and be made by a demonstrably consistent process. but how safe is safe? and what is efficacious? and what are the limits by which we define consistency? the answers to each of these questions pose ethical problems. it is recognized that a balance has to be struck between the urgency of the need (if we don't have the agent, people will die) and the side-effects of the product. education is a key feature when we consider how humans behave socially. some attribute the education of children to parents only, and see the education institutions as providing knowledge and capability, but not ethics. others would have us believe that we receive effective training in ethics thoughout our lives from all kinds of sources of which educational institutions are prominent. it is a matter of choice as to whether an individual believes that 'you can't teach an old dog new tricks'. it is a matter of record that modern adults have to relearn their new car's control systems and idiosyncrasies each time they change their car. learning to programme a video, access and use the internet, come to terms with computers, air travel and internationally derived food menues has come to many people late in life, yet they have made changes to their behaviours in the light of these new developments. we are indeed open to lifelong education for which our modern universities are reorganizing. these considerations provide opportunities for industry to accept a new mission. while they have accepted for many years the need for sympathetic public relations (with the media) and for lobbying activities (to affect the laws which receive legislative approval), they have not until now perceived that they have also to meet the challenge of ethics providers. this can be effected at two levels; the one, via the bodies which focus on the generation of ethical guidelines; the other, the educational system from the earliest grades to those who return to education in their third age or later. industrialists will have to recognize that ethics matters. they will have to support institutions which engage in devising an ethics which is wholly compatible with living in a modern world with ever changing technologies and concepts about the nature of life and the way the world works. and they must engage in the promotion of ethics in a society which is hungry for a new ethical synthesis (figure ) . all this has to be done at arm's length. there is no substitute for the realization that we all are members of the community and responsible in some measure for our mutual well-being: sectors cannot profit at the expense of other segments. this concurrence of aims needs a reaffirmation; it is hoped that industry will see its future success through the inclusion of this mission in its project portfolio. introducing fence vaccines the state of the world's children. unicef the analysis of inoculation comprising the history, theory and practice of it: with an occasional consideration of the most remarkable appearances in the small pocks in sickness and in health the sunday times, / / , news section p. which describes a genetically engineered anti-pregnancy oral vaccine based on salmonella evidence of cryptic v epitope(s) on native hiv- virions: immunophysicochemical analysis the coming plague; newly emerging diseases in a world out of balance on the acceptibility of biopharmaceuticals ethical aspects of the university-industry interface ethics as a control system component key: cord- - glgeft authors: possas, cristina; antunes, adelaide maria de souza; de magalhães, jorge lima; mendes, flavia maria lins; ramos, mateus pinheiro; de simone morais, juliana; homma, akira title: vaccines: biotechnology market, coverage, and regulatory challenges for achieving sustainable development goals date: - - journal: bioeconomy for sustainable development doi: . / - - - - _ sha: doc_id: cord_uid: glgeft this chapter provides an overview, from bioeconomic and global sustainability perspectives, of the main constraints to the current global vaccine innovation system for achieving sustainable development goals – sdgs. biotechnology market trends, gaps in vaccine coverage against emerging and neglected diseases, and patent protection and regulation are discussed. a structured long-term “public-return-driven” innovation model to overcome vaccine market failure is proposed. innovative preventive vaccines against emerging and neglected infectious diseases, such as zika, dengue, chikungunya, influenza, and hiv/aids, are examined here from bioeconomics and global sustainability perspectives, aiming to integrate public health and biotechnology market approaches. novel vaccines with reduced adverse effects can have an enormous impact on life expectancy and on the quality of life of the global population, significantly reducing government, individual, and business costs . nevertheless, there are significant production, technological development, market, coverage, regulatory, and governance constraints to achieving sustainable development goals (sdgs) . in this chapter we examine vaccine biotechnology market and the factors contributing to market failure, discussing policy strategies to optimize science, technology, and innovation (sti) and drastically reduce current constraints to vaccine development (singh et al. a (singh et al. , b, . for achieving sdg, it should be noted that only one of these goals, sdg , refers specifically to vaccines ( .b. ). however, in addition, we have also identified other sdg goals strongly related to vaccines and sdg goals related to vaccine, in a total of vaccine-related goals in sdgs. two of these goals are related to innovation and technological development of vaccines (sdg and sd ). we discuss the main vaccine development challenges for achieving sdg and current technological and regulatory obstacles particularly affecting developing countries. from this perspective, we propose sti governance strategies to overcome these gaps and increase global access to vaccines, focusing on institutional and regulatory perspectives, including intellectual property and ethics. policy recommendations for vaccine funding and incentives for innovation, development, and production are made. finally, we emphasize the enormous potential role that access to innovative vaccines can play on global sustainability (milstien et al. ; possas et al. ) , benefiting particularly the poorest countries in a global context permeated by sharp social inequalities. the global market for human vaccines is projected to reach usd . billion by from . billion in at a cgar of . % (markets and markets ) driven by the growing importance of vaccines in public health, reducing healthcare costs and contributing through prevention of diseases toward a more sustainable healthcare system. drastic changes in the dynamics of the global vaccine market occurred between and , with a sharp growth from usd billion in to usd billion in (access to vaccines index ) and to usd . billion in (markets and markets ) , with sales to high-income countries representing about % of the total value of this market (access to vaccines index ). in fig. . we indicate the evolution of the global human vaccine market from to and the forecast for . recently, other reports have been released anticipating an even more favorable scenario for the global human vaccines market. a recent study estimated that this market would grow from . billion in to . billion by (grand view research ) . these market forecasts also anticipate rising r&d investments in vaccine development projects by the main global players in the vaccine market. table . indicates the top pharmaceutical players according to global revenue share in . pfizer is expected to increase its participation in the market in the next decade due to the success of its pneumococcal vaccine prevnar and increasing investments in vaccine development. other important vaccine players include emergent biosolutions, csl, inovio pharmaceuticals, bavarian nordic, mitsubishi tanabe, serum institute of india pvt. ltd., alk-abelló a/s, altimmune, inc., bharat biotech international, and medimmune. the world vaccine market consists of four segments: gavi (the global alliance for vaccine and immunizations), unicef, paho revolving fund (rf), and rest of the world (row). about of the lowest-income countries in the world rely on gavi for funding of some key vaccines (who ) . unicef supply division (sd) is the procurement agent for most of these countries and for an additional approximately middle-income countries (mics) (totaling about countries). the paho rf provides financial and procurement support to about countries and territories in the americas. the row consists of self-funding and self-procuring countries spanning all income levels and receiving only marginal, mostly indirect, financial, procurement, market shaping, or other related support. these increasing investments in vaccine innovation, development, and production are guided by the growing global need for preventive vaccines and immunotherapy strategies against cancer, zika, hpv, hsv, hiv, and a broad range of infectious diseases that currently burden the healthcare system and societies worldwide (who (who , . this scenario of increasing global demand for vaccines in the next decade is supported by epidemiological indicators: annual burden of new hpv-related cancers worldwide to the tune of , ; rise of zika into a public health emergency with over countries reporting , cumulative confirmed cases of infection between and ; very high prevalence of hsv which infects approximately % of the world population under years of age; continued prevalence of tuberculosis which infects million and takes . million lives each year despite the progress made toward eliminating the disease; and rise in hiv infections worldwide over . million (who ; global industry analysts ). developments in reverse vaccinology and synthetic vaccinology are expected to help increase the rate of successful vaccine design and development (sette and rappuoli ) . emerging countries with mandatory immunization programs represent large markets with enormous potential for future growth and expansion. with large population base and relatively high proportion of young children and teen population, emerging markets including china and india represent the fastest growing markets in asia-pacific, with this region expected to grow at the fastest rate of . % in the next decade. the pharma industry is rapidly becoming a competitive player in the bioeconomy market, a new global paradigm that will introduce novel technologies such as genomics and proteomics across multiple economic sectors and industries. immunome, resulting from advances in sequencing technology and a bioinformatics resource, is also contributing to vaccine innovation and development. these advances in genomics, proteomics, immunome, bioinformatics and new information technologies and their increasing convergence are driving these new market trends. this accelerated innovation scenario is revolutionizing healthcare with new preventive and therapeutic technologies, expected to provide longer, healthier lives to the global population. physicians will eventually be able to predict a person's predisposition to a broad range of diseases and intervene appropriately, insert new genes to replace faulty ones, and tailor therapies to an individual's needs and profile. the immune system is a highly complex system, based on a coordinated expression of a wide array of genes and proteins. one of the major gaps in vaccine innovation, particularly affecting the development of new vaccines against emerging and neglected diseases, is related to the inability of scientists to explain the diversity of individual immune responses and clinical outcomes to the same vaccine and how this diversity relates to innate and acquired immunity. the human immunome, a specific set of genes and molecular structures underlying the response of the immune system to fight disease, is vast and estimated at billion times larger than the human genome project in terms of data output. because of this scale, scientists have never been able to characterize the core parts by which the immune system responds to pathogens and develops a disease. only recently, with the dramatic advances in sequencing technologies and bioinformatics, exponentially extending their informational scale, it became possible for the first time for scientists to uncover the complexity of the human immunome (soto et al. ; briney et al. ) . immunome, a bioinformatics resource, has been conceived for the characterization of the human immune system. it contains information about immunity-related proteins, their domain structure, and the related ontology terms and contains also information about the localization and mechanisms involved in the coding genes. determining the core parts of the immune system in the human immunome could drastically transform how we diagnose, prevent, and treat disease through the identification of new biomarkers while enabling highly targeted, computationally designed vaccines and therapies that reduce time and risk of product development. the immunome program of the human vaccines project is sequencing, in a global collaborative -year effort, receptors from a group of genetically diverse individuals in several continents and determines the structure and function of a key subset of receptors. through an open-source procedure, data will be made available to researchers across the world. in this program, laboratory analyses of biospecimens will be combined with an array of other genetic, lifestyle, and health information provided by volunteers to help researchers to identify individual genetic differences that contribute to diverse immune responses. the initial study will assess immune responses of ten healthy adults (ages - ) to a licensed hepatitis b vaccine (considered an ideal model to study human immunological protection), and it is expected that this study will expand to include several hundred people from neonates to the elderly in middleand low-income countries. the immunome program can thus bring crucial information to the development of more effective vaccines against emerging and neglected infectious diseases. vaccine manufacturers in developing countries, particularly affected by these diseases, should be actively involved in its international scientific and technological collaborations. in the near future, sophisticated technology will allow patients to search and manage their medical records, comparing them to current public health information based on individual genomic profiles. intelligent marketing agents will aggregate patient information from a variety of sources to provide timely and relevant responses. networks of distributed processing systems will offer new insights into data by mining all enterprise and public data sources. and supercomputing platforms and information management will enable the rigorous manipulation of genomic data. advances in remote sensing and artificial intelligence technologies have already created intelligent operating rooms with sensory control mechanisms and devices that transmit health information via telephones and personal digital assistants. the industry can now develop programmable microchips for the subcutaneous delivery of precisely timed doses of drugs and vaccines. interactive chips with built-in sensors may mimic the body's own regulatory ability. as the bioeconomy evolves, the industry faces an unprecedented era of opportunity and challenge. companies recognize that alliances are critical to their future and are now making them a major component of their strategy. virtual research organizations are now conceived searching to provide discovery technologies to scientists in pharmaceutical enterprises; providing them links to gene database, proteomics database, or high-throughput screening capabilities; and enabling fast and efficient access to vaccine and immunotherapy information. the world is facing multiple public health challenges, such as outbreaks of vaccinepreventable diseases, increasing reports of drug-resistant pathogens, climate change, and multiple humanitarian crises. the world health organization (who) included several emerging and neglected diseases among the ten global threats for (who ): influenza, dengue, hiv, and also high-threat pathogens, such as ebola, several other hemorrhagic fevers, zika, nipah, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome (sars) and disease x, which represents the need to prepare for an unknown pathogen that could cause a serious epidemic. to address these and other threats, started its new -year strategic plan: the th general programme of work. this plan focuses on a triple billion target: ensuring billion more people benefit from access to universal health coverage, billion more people protected from health emergencies, and billion more people in better health and well-being. reaching this goal will require addressing these threats to health from a variety of angles. the world will face another influenza pandemic; the only thing we don't know is when it will hit and how severe it will be. global defenses are only as effective as the weakest link in any country's health emergency preparedness and response system. who is constantly monitoring the circulation of influenza viruses to detect potential pandemic strains: institutions in countries are involved in global surveillance and response. every year, who recommends which strains should be included in the flu vaccine to protect people from seasonal flu. in the event that a new flu strain develops pandemic potential, who has set up a unique partnership with all the major players to ensure effective and equitable access to diagnostics, vaccines, and antivirals (treatments), especially in developing countries, in order to make possible the supply of required vaccines as soon as possible. dengue, a mosquito-borne disease that causes flu-like symptoms and can be lethal and kill up to % of those with severe dengue, has been a growing threat for decades. a high number of cases occur in the rainy seasons of countries such as bangladesh and india. now, its season in these countries is lengthening significantly (in , bangladesh saw the highest number of deaths in almost two decades), and the disease is spreading to less tropical and more temperate countries such as nepal that have not traditionally seen the disease. an estimated % of the world is at risk of dengue fever, and there are around million infections a year. who's dengue control strategy aims to reduce deaths from the disease by % by . hiv infections in sub-saharan africa despite being only % of the population. this year, who will work with countries to support the introduction of self-testing so that more people living with hiv know their status and can receive treatment (or preventive measures in the case of a negative test result). who has included in these ten global threats for diseases and pathogens that have potential to cause a public health emergency but lack effective treatments and vaccines. this list for priority research and development includes ebola, several other hemorrhagic fevers, zika, nipah, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome (sars) and disease x, which represents the need to prepare for an unknown pathogen that could cause a serious epidemic. international recognition of vaccines' impact and increased global demand for vaccines have stressed the need for global strategies to assure timely provision of lowprice vaccines (meissner ) through policies supporting free and universal access. in this scenario, the decade of vaccines (dov) initiative was launched at the world economic forum in davos in , signed by international agencies, such as the world health organization (who), unicef, the us national institute of allergy and infectious diseases (niaid), and the bill & melinda gates foundation, with the mission: "to extend, by and beyond, the full benefits of immunization to all people, regardless of where they are born, who they are, or where they live." this declaration was supported by a commitment by the bill & melinda gates foundation to donate usd billion to research and development and to delivering vaccines for the poorest countries. the dov initiative gained significant international support and visibility. two years later, after consultations with dov stakeholders, including industry groups, a global vaccine action plan (gvap) was launched by the member states of the th world health assembly in may , aiming to deliver universal access to immunization by . following the collaborative dov strategies, the gvap brought together multiple stakeholders to achieve the ambitious goals of the plan: the leadership of the bill & melinda gates foundation, gavi alliance, unicef, us national institute of allergies and infectious diseases (niaid), and who, mobilizing many partners (governments, health professionals, academia, manufacturers, funding agencies, development partners, civil society, media, and the private sector). if the gvap is translated into action and resources are mobilized, it is expected that between . and . million deaths could be averted by the end of the decade, with gains in billions of dollars in productivity. nevertheless, it is important to note that actions and resources will not be sufficient for the success of gvap if the plan does not conceive a global strategy to support manufacturers in the developing world to overcome the main ipr and regulatory barriers that delay and hinder vaccine development and production. the millennium development goals (mdgs) for - were incorporated by governments worldwide and had a strong global mobilization power on promoting development and social initiatives, engaging national leaders in elaborating and monitoring these goals (un ) . this mobilization was facilitated since the targets were quantifiable and could potentially be attained. although the two healthrelated goals, mdg (reduce under- mortality from to by two-thirds) and mdg (reduce maternal mortality from to by three-quarters), had not been met by and it is estimated by who that . million infants worldwide are still missing out on basic vaccines, significant progress has been made, with child and maternal mortality approximately halved, with significant global progress. in sequence to mdgs, the united nations promoted an in-depth revision of this strategy (un (un , and formulated a new global strategy, sustainable development goals (sdgs) for - with goals, with one of them (sdg ) directly related to health (un ). the target of sdg is to "ensure healthy lives and promote well-being for all at all ages." its sub-targets include ones that could be met: two-thirds less maternal mortality and a third less noncommunicable disease (ncd) mortality. they also include ending preventable newborn and under- deaths and ending hiv/aids, tuberculosis, malaria, and neglected tropical diseases, besides other non-vaccine related sub-targets. in this chapter, we argue that a major component of sdgs is crucial for attaining sdg goals and should not be minimized: innovation and technological development of vaccines. we discuss how this component should be incorporated into monitoring the sub-targets of this goal, and we emphasize the need for a new vaccine innovation model based on an expanded role of the state and incentive mechanisms to pharmaceutical companies and public manufactures to correct the current scenario of "market failure" constraining access to vaccines. it is certainly unacceptable, from ethical and sustainable development perspectives, to simply recognize this "market failure" as a detrimental and inevitable consequence of the rationale of a global market economy. on the contrary, it should be seen as a massive public health failure and a global failure to direct economic development for the benefit of societies (trouiller et al. ). the "valley of death" although vaccine candidates are in the development pipeline for neglected and emerging infectious diseases mainly affecting the poorest countries such as malaria, dengue, hiv, tuberculosis, and pneumonia, only of them have made it through the pipeline recently and are widely used in these countries: a conjugate vaccine for meningitis serogroup a diseases and a vaccine against japanese encephalitis virus (kaslo et al. ; who ) . it has been estimated by these authors that unfortunately much of this promising pipeline could go to waste and fall into the so-called valley of death, failing to move from proof-of-concept to second-phase trial due to lack of market interest in vaccines against these emerging and neglected diseases affecting only the poorest populations in developing countries. no single organization or group is interested in supporting the costly and more complex late-stage clinical trials for neglected diseases that mainly affect the poor nations. this scenario raises great concern for two reasons. first, around % of these vaccine candidates in the development pipeline target the mentioned neglected and emerging infectious diseases, a much higher problem in lower-and middle-income countries (kaslow et al. ). second, this means a significant waste of global resources in a crucial area for sustainable development, considering that these vaccine candidates received billions of dollars for the first phase of vaccine development from prestigious donors, such as the us national institutes of health (nih), the european union, the welcome trust, and the bill and melinda gates foundation. taking a vaccine candidate from a discovery at the laboratory bench to widespread deployment is a complex, lengthy, and expensive endeavor, with many financial, licensing, and regulatory barriers. no organization or group plans to support the emerging and neglected diseases vaccines from the beginning to end. therefore, it could take many decades to incorporate these vaccines into the national immunization programs in these poorest countries (kaslow et al. ) . in table . we provided a selection of promising projects for vaccines for emerging and neglected infectious diseases affecting the poorest developing countries that could significantly impact on achieving sdg targets. science and technology have made enormous progress and are now prepared to provide the innovative-intensive vaccines that the poorest populations in the world urgently need. but innovation and discovery are not the major bottleneck, which reside in technological development, production, and timely provision of vaccines to people (homma et al. ). r&d-based pharmaceutical major industry players are reluctant, due to freemarket rationale, to invest in the development of vaccines to treat the major neglected and emerging diseases affecting mainly the poorest nations, since return on their investments cannot be guaranteed. national and international policies currently support a free-market-based global order, with economic opportunities, rather than global public health needs guiding the direction and rationale of vaccines development. it is certainly unacceptable, from ethical and sustainable development perspectives, to simply recognize this "market failure" as a detrimental and inevitable consequence of the rationale of a global market economy. on the contrary, it should be seen as a massive public health failure and a global failure to direct economic development for the benefit of societies (trouiller et al. ). an urgent redefinition of priorities in vaccine development is needed. this strategy cannot rely only on fragmented contributions of researchers, funding agencies, and the pharmaceutical industry. effective national and international policies need to be urgently conceived to redirect the global economy to address the true public health needs of society (homma et al. ; røttingen et al. ) . "political will," identified as the need for a strong commitment to prioritize health considerations over economic interests, has been frequently emphasized by policy-makers as a major issue to ensure access to vaccines but is not sufficient. it is necessary to go beyond "political will," with a clear goal in mind and a realistic plan to achieve it. from this perspective, it will be necessary to promote effective global implementation of strategies to accelerate innovation, technological development, and production of new vaccines and to ensure timely global access to them. moreover, a global vaccine policy strategy should be conceived to promote the necessary enforcement of regulations and other mechanisms to stimulate vaccine development, production, and global access to these products. novel, creative, and effective strategies involving both the public and the private sector are needed to ensure low-price vaccines, accelerating innovation and technological of vaccines against emerging and neglected diseases. priority action areas should include: . advocating a preventive vaccines r&d agenda . conceiving capacity-building programs adequate to the conditions of developing countries' manufacturers . promoting technology transfer to public and private manufacturers in emerging countries . elaborating an adapted legal and regulatory framework to increase flexibility and "fast-track" procedures . prioritizing funding for vaccine development . securing availability, accessibility, and distribution of these vaccines consensus is building among the main stakeholders in the global vaccine community that the spiraling costs of risks associated with vaccine r&d are detrimental to global access to these products, particularly in the poorest developing countries. most of them agree that these vaccine r&d costs should be instead rewarded by means other than financial returns in the market from charging high product prices. novel mechanisms such as incentives, prizes, and "patent pools" for drugs and vaccine innovation and development have been proposed in the last two decades. there is now vast literature on the subject, claiming for alternative models that should be urgently implemented to meet the increasing global demand for vaccines, particularly in the poorest developing countries. the main question is: how to conceive a feasible long-term mechanism to minimize these risks faced by pharma companies? which global organizations should be responsible for this alternative model? we recommend this new vaccine incentive model should be coordinated by three international organizations: who, gavi, and unicef. these organizations would, in collaboration with the main stakeholders, identify from the list of candidates the priority vaccine candidates, identify the funding mechanisms necessary to these candidates to enter the second-phase clinical trials, and specify which organization, or alliance, would be responsible for these selected vaccine candidates from beginning to end. in this innovative global collaboration strategy, these three leading international organizations should bring together the main players and stakeholders in the vaccine market, with funding agencies such as the bill & melinda gates foundation, nih, welcome trust, and other organizations as path, iavi, and the international vaccine institute in seoul and vaccine manufacturers, in collaboration with other nongovernmental organizations in order to conceive and implement this alternative long-term model for sustainable development and provision of vaccines which are uncertain business products or require a great amount of public funding to go beyond the initial proof-of-concept phase. a novel global priority-setting strategy, driving adequate implementation, will be necessary to assess the vaccine candidates in the pipeline, trying to identify the most favorable candidates which are uncertain business cases that will require significant public funding to move into second-phase clinical trials. funding mechanisms supported by subsidies from governments, such as those of the g countries, and philanthropic organizations, such as the bill & melinda gates foundation, could remedy the market failure threatening vaccine development for lmics. gavi already provides one form of subsidy (gavi a, b). support to develop vaccines or to make them available during epidemics is also provided by public organizations, such as the coalition for epidemic preparedness innovations in oslo and the biomedical advanced research and development authority, part of the us department of health and human services. such schemes need to be expanded and rethought to give vaccine developers more certainty and upfront financial backing (kaddar et al. ) . for instance, gavi could commit to purchasing a vaccine before it has been developed, on the condition that the developers meet certain regulatory milestones. at present, the alliance buys vaccines to distribute to lmics after they have been licensed or recommended by the who for general use (gavi (gavi , b . only with this kind of leadership will the global community secure vaccines for some of the world's most debilitating diseases. there is an urgent need for a paradigm shift in global governance of health innovation systems to achieve sustainable development goals (buse and hawkes ; possas et al. ; seib et al. ; mazzucato ) . "mission-oriented" approaches have been proposed to overcome current constraints in innovation systems (mazzucato and penna ) . recently, in a new report, "the people's prescription: re-imagining health innovation to deliver public value" (mazzucato ) , the authors call for restructuring research and development innovation systems in order to create, rather than extract, value. it also calls for long-term "missionoriented" public investment and a public return on this investment. in this report the authors argue that health innovation is about making new treatments and cures available to the people that need them. profits might be earned but not at the cost of doing what the health system is meant to do: heal. this report is the outcome of result of collaboration between the ucl institute for innovation and public purpose, stopaids, and global justice now and just treatment. the report identifies gaps of the current health innovation system and sets principles for a new model. it proposes concrete policy actions that can be taken in the long term to actively shape and co-create a health system that delivers real public value. the report is structured into two sections. the first is "diagnosis" with chapters on "problems with the current health innovation system" and "principles for a health innovation model that delivers public value." the second section, "remedies," includes chapters on "immediate policy actions: getting better prices today" and "transformative proposals: re-imagining our health innovation system to deliver public value." the report focuses on the unethical and unacceptable current global scenario for health innovation, highly inefficient, with a pharmaceutical industry that makes billions in profits without providing the affordable products that people need. the report examines all those problems, and then it sets out some key principles of how a "healthy" innovation model for health would work, based on an analysis of case studies from different countries and different contexts, looking at where innovation has been done well. in vaccine development, as in drugs development, there is a tremendous waste of resources because public health is not driving the r&d agenda. we have all the money going into proof-of-concept studies instead of developing public accountability. track" the need to provide more flexible and expedite new vaccine products and processes resulting from biotechnology is challenging both developed and developing countries to accelerate the implementation of adequate regulations and intellectual property rights (crager ; possas et al. ) . ipr are granted by the state to individuals, enterprises, or organizations under temporary monopolistic conditions (patents) in order to compensate them for the investments made in their creations/innovations. in industry, a patent is clearly an instrument to guarantee the returns of the investments on r&d through the commercialization of the patented products and through the payment of property rights. patents are viewed as a crucial incentive to innovation. nevertheless, arrow ( ) recognized in his pioneer theory that in spite of its advantages, the patent system creates a suboptimal situation in economic terms: patents create a monopoly that restricts the diffusion and dissemination of innovation. the argument is that this restriction is temporary (after years the patent protection "falls" to public domain) and is compensated by the fact that the knowledge related to the patent is necessarily published in the moment that the patent is granted. nevertheless, several authors have noted the detrimental impacts of the monopoly created by the patent system on health products' innovation, particularly on the development and accessibility to new drugs for neglected and emerging diseases and proposed incentive mechanisms, such as prizes, "patent pools," and awards to compensate this "market failure." although in the vaccine sector many intellectual property and market issues affecting price remain unclear, in the current regulatory scenario, the access to new technologies in multipatented vaccines, such as adjuvants for vaccine compositions, remains a main challenge (possas et al. ) . for vaccine manufacturers in emerging countries, access to patent information on vaccine adjuvants is a crucial issue, detrimental to vaccine development. the incorporation of new adjuvants for vaccines which boost the immune response has become crucial to the development of innovative vaccines, as new antigens, with purer and smaller molecules, may have less then optimal immune responses, necessary to vaccine protection for a lengthy period of time. the malaria vaccine candidate rts provides a good example of the crucial role new adjuvants can play: this vaccine, based on the plasmodium falciparum sporozoite antigen circumsporozoite protein (csp), was successful in providing protection against clinical malaria only when combined with a powerful adjuvant (as or as ). another example are the tests using hybrid flagelins also in malaria vaccines. adjuvants have emerged thus as an alternative route for vaccine development with enormous potential in the global market (mbow et al. ) . the development of new, powerful, and safe adjuvants is therefore a key component of vaccine research. we present in table . some of licensed vaccine adjuvants, with company and class. figure . indicates the countries concentrating patent deposits for adjuvants to vaccine compositions and the diseases related to them (zika, dengue, hiv/aids, influenza), china ( %), the usa ( %), south korea ( %), and the uk ( %), with these three countries accounting for nearly % of all patent deposits. it is also observed that chikungunya had no deposit in the period from to , as indicated the espacenet base. it should be stressed that very few deposits are related to zika and dengue vaccine, only in china and the usa. it also indicates that most of these adjuvant deposits are concentrated in vaccine compositions related to just two diseases, influenza ( %) and hiv/aids ( . %), while zika, followed by dengue, is more neglected. the increasing risk of a pandemic of influenza, rapidly affecting all continents, might explain the concentration of r&d efforts on vaccine compositions and adjuvants for this disease. table . provides an overview of the patent holders of deposits for vaccine adjuvants against zika, dengue, and hiv in the - period, listed by country, number of deposits, and deposits with partnerships and partners. this table indicates that the number of partnerships is very low and should be stimulated. figure . shows the temporal evolution of patent deposits in the period - of the three largest patent depositors of adjuvants and formulation of vaccines with adjuvants for the diseases studied, showing that only in the last decade there has been a greater r&d effort. this figure also indicates the leadership of china and the increasing role played by this country in the development of adjuvants for vaccine compositions. finally, it should be noted that in addition to these intellectual property barriers to access to vaccine formulations and vaccine adjuvants, such as confidentiality and constraints to patent information sharing (possas ; possas et al. ) , other regulatory obstacles remain also a challenge to vaccine development and access to timely immunization: virtual inexistence in many countries, particularly the developing ones, of expedite and "fast-track" review processes (fda ; u.s. dept. of hhs et al. ) for evaluating priority and emergency projects; lack of flexible regulatory procedures for sharing biospecimens and samples; legal constraints in access to biorepositories and to biobank information; and the difficulties in defining the standard of care to be provided during clinical trials. we examined here, from bioeconomics and global sustainability perspectives, the current innovation system for vaccine development, providing considerations on how this system should be better designed and implemented to address sdgs' vaccine coverage targets. public investment plays a crucial role in biomedical r&d worldwide, but research and development activities supported by governments with public resources are in most cases not directed into targets that have the most public health value, such as vaccines. for this reason, there is increasing awareness and concern in the global vaccine community on the fact that public-supported vaccine products should be shared by the public and not just privately appropriated by pharmaceutical companies. this scenario evidences a need for conceiving a new innovation governance paradigm for vaccine development compatible with the market rationale, aiming profitable products with social return: an expanded role of the state, with clear procedures for planning, development, regulation, and forecast; a redefinition of the patterns of relationship between private and public players; and finally, a political agreement between the main players and stakeholders on the more adequate mechanisms to balance the risks and the rewards between those players. moreover, there is a need to conceive a more flexible intellectual property regime for emerging and neglected diseases mainly affecting the poorest populations in developing countries. the monopoly created by patent protection must be compensated by new incentive mechanisms such as awards, prizes, and "patent pools" to accelerate global access to vaccines. it is also crucial to strengthen the local capacity of vaccine r&d institutes and manufacturers in emerging developing countries in order to accelerate the incorporation of new technologies for production of innovative vaccine products. the multinational companies have the intellectual property of these new technologies, such as adjuvants for vaccine compositions, but they do not have sufficient production capacity to meet the global demand for these products, a gap that should be overcome with expanded global collaboration with developing countries' manufacturers. in addition, these players should identify gaps and priorities in infrastructure and capacity building in developing countries' manufacturers in order to facilitate technology transfer agreements with leading pharmaceutical companies and ensure a sustainable long-term supply of vaccines to the poorest populations. finally, clear expedite and "fast-track" regulatory procedures and pathways should be conceived and implemented. in other words, it will be necessary for international organizations and pharmaceutical companies to move from a short-term "shareholder-driven innovation model" to a long-term "public-return-driven innovation model", aiming global sustainability and social welfare for meeting the needs of low-income populations while searching for innovative and profitable vaccine products. access to vaccines index ( ) how vaccines companies are responding to calls for greater immunization coverage economic welfare and the allocation of resources for invention the broad socioeconomic benefits of vaccination health in the sustainable development goals: ready for a paradigm shift improving global access to new vaccines: intellectual property, technology transfer, and regulatory pathways commonality despite exceptional diversity in the baseline human antibody repertoire espacenet patent search. epo, brussels, belgium. accessed fast track, breakthrough therapy, accelerated approval and priority review for patients and patient advocates global alliance for vaccines and immunizations ( a) gavi sustainable development sustainable development goals monitoring framework human vaccines: market analysis, trends and forecasts market report on pharmaceuticals vaccine research, development, and innovation in brazil: a translational science perspective global support for new vaccine implementation in middle-income countries vaccine candidates for poor nations are going to waste vaccine markets by diseases and technologies, report the people's prescription: re-imagining health innovation to deliver public value mission-oriented finance for innovation: new ideas for investmentled growth new adjuvants for human vaccines immunization policy and the importance of sustainable vaccine pricing access to vaccine technologies in developing countries: brazil and india propriété intellectuelle et le sida dans les pays en dévéloppement: innovation et accès aux produits pharmaceutiques access to new technologies in multi-patented vaccines: challenges for brazil innovation and intellectual property issues in the "decade of vaccines new vaccines against epidemic infectious diseases policy making for vaccine use as a driver of vaccine innovation and development in the developed world reverse vaccinology: developing vaccines in the era of genomics a) intellectual property issues in biotechnology. cabi, wallingford. pages biotechnology in agriculture, medicine and industry: an overview intellectual property issues in microbiology high frequency of shared clonotypes in human b cell receptor repertoires drugs for neglected diseases: a failure of the market and a public health failure guidance for industry: expedited programs for serious conditions -drugs and biologics secretary general. the road to dignity by : ending poverty, transforming all lives and protecting the planet. synthesis report of secretary-general on the post- sustainable development agenda the millennium development goals report united nations development programme ( ) sustainable development goals who's vision and mission in immunization and vaccines - . who, geneva world health organization ( ) fact sheet v p pricing report world health organization ( ) ten threats to global health in key: cord- -vhghhvnu authors: schwartz, benjamin; orenstein, walter a. title: prioritization of pandemic influenza vaccine: rationale and strategy for decision making date: - - journal: vaccines for pandemic influenza doi: . / - - - - _ sha: doc_id: cord_uid: vhghhvnu few catastrophes can compare with the global impact of a severe influenza pandemic. the – pandemic was associated with more than , deaths in the usa and an estimated – million deaths worldwide, though some place the global total much higher. in an era when infectious disease mortality had been steadily decreasing, the – pandemic caused a large spike in overall population mortality, temporarily reversing decades of progress. the us department of health and human services, extrapolating from the – pandemic to the current us population size and demographics, has estimated that a comparable pandemic today would result in almost two million deaths. vaccination is an important component of a pandemic response. public health measures such as reduction of close contacts with others, improved hygiene, and respiratory protection with facemasks or respirators can reduce the risk of exposure and illness (germann et al. ; ferguson et al. ), but would not reduce susceptibility among the population. prophylaxis with antiviral medications also may prevent illness but depends on the availability of large antiviral drug stockpiles and also does not provide long-term immunity. by contrast, immunization with a well-matched pandemic vaccine would provide active immunity and represent the most durable pandemic response. however, given current timelines for the development of a pandemic influenza vaccine and its production capacity, vaccine is likely not to be available in sufficient quantities to protect the entire population before pandemic outbreaks occur, and thus potentially limited stocks may need to be prioritized. this chapter reviews information on influenza vaccine production capacity, describes approaches used in the usa to set priorities for vaccination in the setting of limited supply, and presents a proposed strategy for prioritization. few catastrophes can compare with the global impact of a severe influenza pandemic. the - pandemic was associated with more than , deaths in the usa and an estimated - million deaths worldwide, though some place the global total much higher. in an era when infectious disease mortality had been steadily decreasing, the - pandemic caused a large spike in overall population mortality, temporarily reversing decades of progress. the us department of health and human services, extrapolating from the - pandemic to the current us population size and demographics, has estimated that a comparable pandemic today would result in almost two million deaths. vaccination is an important component of a pandemic response. public health measures such as reduction of close contacts with others, improved hygiene, and respiratory protection with facemasks or respirators can reduce the risk of exposure and illness (germann et al. ; ferguson et al. ), but would not reduce susceptibility among the population. prophylaxis with antiviral medications also may prevent illness but depends on the availability of large antiviral drug stockpiles and also does not provide long-term immunity. by contrast, immunization with a well-matched pandemic vaccine would provide active immunity and represent the most durable pandemic response. however, given current timelines for the development of a pandemic influenza vaccine and its production capacity, vaccine is likely not to be available in sufficient quantities to protect the entire population before pandemic outbreaks occur, and thus potentially limited stocks may need to be prioritized. this chapter reviews information on influenza vaccine production capacity, describes approaches used in the usa to set priorities for vaccination in the setting of limited supply, and presents a proposed strategy for prioritization. an influenza pandemic occurs with the introduction and spread of a new influenza a virus subtype among people. although some cross-protection against antigenically different influenza viruses within a subtype occurs following prior infection or vaccination, the entire population is likely to be susceptible to an influenza a virus subtype that has not circulated (or has not circulated recently) among people. consequently, in an influenza pandemic, rates of illness are higher, severity is greater, and the distribution of mortality is more widespread compared with seasonal influenza (simonsen et al. ) . given the susceptibility of the entire population, the goal of the united states' pandemic vaccination program is to offer vaccination to everyone living in the usa. there are several potential approaches to implementing pandemic influenza vaccination when vaccine supplies are inadequate to rapidly vaccinate the entire population: vaccine could be administered on a "first come, first served" basis or could be targeted first to individuals and groups based on specified criteria. criteria for targeting in other mass vaccination campaigns have included geographic area (e.g., group a meningococcus in the african meningitis belt), exposure or proximity to a case (e.g., smallpox), age (e.g., polio), risk of infection (e.g., h. influenzae type b), risk of complications from infection (e.g., seasonal influenza), risk for transmitting infection (e.g., rubella), or (most often) a combination of these factors. targeting has been justified as providing earliest protection to those who are most vulnerable to infection, most at risk of severe or fatal disease, or whose protection may prevent or reduce further transmission (heymann and aylward ) . when vaccine supply, the capacity to administer it, or funding is limited, so that the optimal strategyrapid universal vaccination-is impossible to implement, targeting mass vaccination becomes more important to achieve the best possible outcomes. in the national strategy for pandemic influenza, the president defined a goal of establishing domestic manufacturing capacity that produces sufficient vaccine to vaccinate the entire us population within six months of the emergence of a virus with pandemic potential (the white house ). to achieve this, over $ billion has been allocated ( ) to expand domestic egg-based influenza vaccine production, ( ) to support advanced development of new vaccine production technologies, such as growth of influenza virus in cultured cells or development of recombinant vaccines, and ( ) to support the advanced development of "antigen-sparing" approaches, such as new adjuvants, that can stimulate a more robust immune response, allowing manufacturers to reduce the amount of antigen in each dose and formulating the antigen produced into more vaccine doses. until the promise of these approaches is realized, however, pandemic influenza vaccine supply is likely to be far less than pandemic response needs. for the - influenza season, most of the influenza vaccine administered in the usa was produced in other countries, and these sources of supply may not be available during a pandemic. moreover, the amount of antigen needed to achieve a protective immune response could be substantially greater for a pandemic virus compared with seasonal influenza viruses. a clinical trial of an unadjuvanted candidate h n vaccine showed that two doses containing mg of hemagglutinin antigen were needed to achieve an immune response that may correlate with protection in more than half of healthy adult recipients (treanor et al. ) . this per dose concentration is sixfold higher than the quantities of hemagglutinin antigen included for each strain in the seasonal trivalent inactivated vaccines (tiv), and twofold higher than the total hemagglutinin in a standard dose of tiv. since two doses of the h n vaccine were needed to achieve adequate immunogenicity, the quantity of antigen needed to immunize an adult would be -fold higher than the amount of antigen to vaccinate against a seasonal strain. initial trials with other candidate h n vaccines that contain alum, novel lipid-based adjuvants, or that use the inactivated whole virus documented immunogenicity with two doses of mg, . , and mg, respectively (bresson et al. ; leroux-roels et al. ; lin et al. ). while additional studies are needed, these results suggest a potentially wide range of antigen quantities needed in different vaccine formulations, which will directly impact how quickly the population can be effectively vaccinated in the event of a pandemic. vaccine supply, therefore, would depend on the production capacity for different vaccine formulations at the time a pandemic occurs. under some scenarios, vaccine supply would be very limited, whereas under others, assuming success in evaluating and licensing new formulations and producing them in the usa, supply may be robust. the time required to develop, license, and manufacture pandemic influenza vaccine is also an important variable. using current technologies, at least weeks would be required from the time the pandemic virus was identified until the first vaccine doses become available. depending on a combination of factors, including where the pandemic begins, how quickly it is detected, the effectiveness of containment measures and the season, the first us pandemic wave may occur before any pandemic vaccine becomes available or after sufficient lead time such that vaccination is already widespread (ferguson et al. ; longini et al. ). in the pandemic, the first us cases occurred in june but no community outbreak occurred until august and the first pandemic wave did not peak until the end of october; by this time almost half of the approximately million vaccine doses eventually produced had been delivered. by contrast, in , the pandemic was not recognized until later in the year, and at the time initial us outbreaks began few persons had been vaccinated (schwartz and wortley ) . because influenza vaccine production capacity, vaccine formulation, and the time from pandemic recognition to onset of us outbreaks all are uncertain for the next pandemic, we are unable to predict how many people will be vaccinated before pandemic disease is widespread. thus, prioritizing who is vaccinated earlier and who later will best target available supply to achieve national pandemic response goals. us pandemic response goals include slowing the spread of pandemic disease and reducing the health, societal, and economic impacts of the pandemic (the white house ). the approach to using a limited supply of pandemic vaccine may differ depending on which goals are considered most important. results of mathematical models suggest that vaccinating school-aged children can best reduce transmission of influenza, slowing disease spread and reducing overall community attack rates (germann et al. ) . while studies of vaccination for seasonal influenza support a strategy of vaccinating children to protect others in the community through herd immunity (monto et al. ; piedra et al. ; reichert et al. ) , uncertainty in the amount of vaccine that will be available or its timeliness make reliance on trying to induce indirect protection a risky strategy. hospitalizations and deaths from pandemic illness can be reduced by directly vaccinating those at highest risk for these severe outcomes. based on age-specific mortality rates in the and pandemics, vaccinating persons ³ years old would have prevented substantially more deaths compared with vaccinating other age groups, despite the lower vaccine efficacy among the elderly (in this would not have been the case because of the high mortality rate among young adults). another approach to reduce the health impacts of a pandemic would be to vaccinate healthcare workers so that they can continue to provide care to others. in an unmitigated pandemic, the demand for healthcare services will be overwhelming at a time when healthcare workers may be out of work due to illness, the need to care for sick family members, or because they are afraid of becoming infected at the workplace. a survey of county health department workers in maryland found that % of respondents indicated they would not report to work in a pandemic. in a multivariable analysis, confidence in one's personal safety was significantly associated with a willingness to work (balicer et al. ) . whether response to a survey is predictive of actual behavior is unclear; anecdotally, virtually all healthcare workers in toronto reported to work during the sars outbreak, despite the fear associated with a new disease and the spread that occurred within hospitals. whether vaccinating healthcare providers to maintain effective care or vaccinating those at highest risk of illness would better reduce the health impacts of a pandemic is unknown. the potential societal and economic impacts of a pandemic are associated with pandemic severity, although even in a severe pandemic these impacts cannot accurately be predicted. historical experience does not provide a guide, as a severe pandemic has not occurred for almost a century. a report by the us department of homeland security's national infrastructure advisory committee (niac) analyzed the components of critical infrastructure sectors that would be essential to society in a pandemic and the workforce needed to maintain those products and services (national infrastructure advisory council ). the report identifies significant interdependency between sectors, expresses concern about the maintenance of supply chains, many of which stretch overseas, and emphasizes the importance and challenges of implementing a targeted vaccination program. of the approximately million workers in these sectors, . million were defined by niac as essential in a pandemic. about nine million of these workers are in the healthcare and emergency services (emergency medical services, law enforcement, and fire protection) sectors. in other sectors, the proportion of the workforce defined as critical ranges from almost % in the nuclear sector to less than % of the food and agriculture sector. because the availability of pandemic vaccine before disease outbreaks is not assured, business planning includes other measures such as "social distancing," improved hygiene, use of facemasks or respirators, and possibly antiviral drug prophylaxis to protect workers in essential operations. because of the uncertainties about the severity and epidemiology of the next pandemic, vaccine supply, and the best approach to using vaccine to reduce health, societal and economic impacts, there is no scientific method to define the optimal use of pandemic influenza vaccine. in , a working group from two us advisory committees, the advisory committee on immunization practices (acip) and the national vaccine advisory committee (nvac) met to develop a pandemic vaccine prioritization strategy. the working group considered the epidemiology and impacts of pandemics, the groups at highest risk for complications and death from influenza, vaccine efficacy, critical societal functions, and ethical issues. the prioritization strategy proposed by the committees included vaccinating groups defined in tiers and subtiers, depending on vaccine supply. groups that were prioritized for earliest vaccination included healthcare workers, manufacturers of pandemic vaccine and antiviral drugs, and persons at high risk of severe illness and death. personnel in critical infrastructure sectors other than healthcare were prioritized after these groups, which include over million persons. this strategy was published in the department of health and human services' pandemic plan to provide guidance to state planners and stimulate further discussions (us department of health and human services ). shortly after publication of the plan, a federal working group was created to reassess and potentially revise pandemic vaccine prioritization guidance. factors contributing to the decision to reassess the recommendations included a shift in national pandemic planning assumptions to a more severe pandemic scenario extrapolated from the pandemic (table ); recognition that the hhs guidance did not include groups that could be considered for prioritization such as border protection personnel or the military; a broader understanding of the risk to essential services stimulated by the niac report; and a series of public engagement meetings convened by the cdc, where participants identified protecting essential community services as the most important goal for pandemic vaccination rather than protecting those who are at highest risk (public engagement pilot project on pandemic influenza ). the federal working group process included consideration of the scientific issues reviewed in the earlier prioritization process, assessment of mathematical modeling results, and discussion with public health officials, critical infrastructure providers and homeland and national security experts. recognizing that science alone cannot define the best approach to pandemic vaccine prioritization, key elements of the process were consideration of ethical issues, input from the public and stakeholders, and a formal decision analysis. ethical input into the working group process was achieved through the participation of public and private sector ethicists and an analysis conducted by the ethics subcommittee of cdc's advisory committee to the director (ethics subcommittee of the advisory committee to the director, cdc ). a strategy of targeting pandemic influenza vaccination to reduce health, societal and economic impacts was considered ethically appropriate. although a strict utilitarian principle could not be applied because of uncertainty about what strategy would provide the most benefit, targeting protection of society in a broad sense was given higher priority than protecting individuals at high risk of complications from influenza. fairness and equity are important principles where everyone is recognized to have equal value, and all table national pandemic planning assumptions. note that planning for some responses such as nonpharmaceutical community mitigation strategies is done across a range of pandemic severities, as defined by the pandemic severity index (cdc, community mitigation guidance) clinical illness attack rate of % (rates highest among school-aged children, about %, and declining with age); us national estimate: , , cases° care seeking by about half of those who are clinically ill° hospitalization of % of clinical cases; us national estimate: , , ° case fatality rate of . %; us national estimate: , , • risk groups for severe illness and death will depend on the pandemic virus and are likely to include infants, pregnant women, persons with chronic and immunosuppressive medical conditions, and the elderly • outbreaks will last - weeks in affected communities; effective use of nonpharmaceutical community mitigation strategies (e.g., social distancing) will prolong community outbreaks but reduce their overall magnitude • multiple waves of illness will occur, with each wave lasting - months persons within a targeted group should have similar access to vaccination. reciprocity, which posits that protection should be afforded to those who assume increased risk in an occupation that benefits society, also was considered important, and a reasonable corollary to healthcare providers' "duty of care" where one is committed to provide care even in settings that increase personal risk. procedural ethical principles of inclusiveness and transparency were met through a process of engaging with the public and stakeholders in meetings, and through a request for comments posted in the federal register and on the government's pandemic influenza website. the goal of the public and stakeholder meetings was to identify the objectives of a pandemic vaccination program that participants felt were most important to pursue. public meetings were held in two demographically different communities with participants recruited by community groups. stakeholder representatives from government, healthcare, business, and community organizations participated in a third meeting. each meeting included initial presentations to educate participants on influenza and influenza vaccine, pandemics, and the rationale for vaccine prioritization. participants discussed potential objectives of pandemic vaccination in small groups and then met in a plenary session where the objectives were discussed further. finally, participants rated the importance of each of ten proposed objectives using a seven-point likert scale ranging from "extremely important" (a score of ) to "not important" (a score of ). despite the differences between groups in terms of geographic location, demographic characteristics, and occupational background, the values expressed at each meeting were similar (table ) . table importance of pandemic vaccination program objectives based on scores assigned by participants at public engagement meetings in las cruces, new mexico, and nassau county, new york, and a stakeholders meeting in washington, dc. scores were assigned from a seven-point likert scale ranging from = extremely important to = not at all important key outcomes of this process included the importance of achieving multiple objectives with the pandemic vaccination program, the value given to protecting critical services and exposed workers, and the preference for vaccinating children before those who are most likely to become sick or to die-older adults and those who have underlying medical conditions. results from the public and stakeholder engagement process provide insight into the values and preferences of the population but do not translate directly into a prioritization strategy for pandemic vaccine. we therefore conducted a formal decision analysis to assess the priority of different population groups. we identified potential target groups for pandemic vaccination defined by their occupation or by their age and health status. the degree to which each group met each of the ten vaccination program objectives was then assessed and scored: how well each group met objectives related to occupational role or exposure was scored by representatives on the federal working group; for objectives where clinical trial or epidemiological data can be used to assess how well a group met an objective, scoring was done by influenza experts from cdc and academic medical centers. the score assigned to each group for each objective was then weighted by the average rating of the objective's importance from the public engagement and stakeholders meetings ( table ) . a total score was calculated for each group as the sum of the objective scores multiplied by their weights for the ten vaccination program objectives, as described by s x = o w + o w + ... + o w , where s x is the total score for group x; o - are the scores the group received for each of the ten objectives; and w - are the weights for each of the objectives. as an example, medical care practitioners received high scores from the working group for objectives of fighting the pandemic and providing care, providing an essential community service, being vulnerable due to their jobs, and being at risk of spreading infection to those who are unprotected (their patient population). because most healthcare workers are healthy adults who would respond well to vaccination, they also received high scores for the objective of being most likely to be protected by the vaccine. medical care practitioners score lower for providing essential economic services, protecting homeland and national security, and being most likely to get sick or die (as some may have underlying medical conditions or be years old or older). this group would receive no points for keeping the pandemic out of the usa or being children. based on this analysis, groups scoring highest for vaccination were front-line public health workers involved in the pandemic response (for example, providing vaccinations), medical care practitioners, emergency medical service personnel, law enforcement personnel, and emergency relief workers. occupational groups invariably scored higher than general population groups defined by their age and health status because more of the ten program objectives were relevant (i.e., they would receive some score for objectives related to one's occupational role and exposure risk as well as one's age-and health-related risk of influenza, ability to be protected by vaccination, and potential role in disease spread). by contrast, general population groups received no score for the occupationally-related objectives. to control for this difference, we stratified potential vaccination target groups into four categories: those that provide healthcare and community support services; those that provide critical infrastructure services; those that protect homeland and national security; and the general population. within these categories, target groups were clustered based on their scores, with breakpoints between clusters defined by difference between scores. groups scoring highest among each of these categories are shown in table . the us pandemic vaccine prioritization guidance incorporates both the tier structure from the guidance included in the hhs pandemic plan and the target group categorization used in the decision analysis. reflecting the similar value placed by the public on protecting persons who provide pandemic healthcare, who maintain essential community services or are at high occupational risk, and protecting children, each of the highest vaccination tiers for a severe pandemic includes groups from each category (table ) . generally, the specific groups included in each tier track closely with the results of the decision analysis. some groups, such as deployed military forces and those who provide support for their mission, are placed in a higher tier in recognition that they may be affected in a pandemic earlier than persons in the usa due to their geographical locations, their increased risk because of crowded living conditions, and the impact of illness on their ability to function effectively. in some critical infrastructure sectors, target groups are prioritized in a lower tier because their expected occupational burden would likely decrease in a pandemic (e.g., passenger transportation), they can largely be protected by changes in work practices such as teleworking, and/or the workforce or work is "fungible;" essential support and sustainment pers. , intelligence services , border protection personnel , national guard personnel , other domestic national security pers. , other active duty and essential suppt. that is, the impact of absenteeism or reduced function can be mitigated by the redundancy within the sector (e.g., trucking, food processing). workers in infrastructure sectors are targeted for early pandemic vaccination to maintain the essential services they provide in recognition of the interdependencies between sectors. healthcare, for example, relies on the sectors that provide electricity, clean water, communications, information technology, transportation, pharmaceuticals, food, and chemicals. in a less severe pandemic, however, historical experience suggests that these services are unlikely to be substantially affected. in both the and pandemics, essential services were maintained without targeting pandemic vaccination. therefore, the us strategy differs for severe, moderate, and less severe pandemics, with some of the occupational groups not targeted in moderate and less severe pandemics, and those workers being vaccinated with their age and health status group in the general population category. pandemic severity is classified using the pandemic severity index, which defines five categories based on the case fatality rate of pandemic illness (cdc ) . a category pandemic, defined by a case fatality rate of < . %, would result in a mortality only slightly greater than a severe seasonal influenza epidemic, and the proposed us vaccine prioritization guidance for less severe pandemics (categories and ) is formulated to be more similar to recommendations for annual influenza vaccination. pandemic vaccine prioritization strategies developed in other industrialized countries are generally based on similar ethical principles and target similar groups to those in the us plan. while healthcare providers and those critical to a pandemic response are the groups targeted first in many plans, workers in other infrastructure sectors may not be targeted. this may reflect national planning assumptions for a less severe pandemic, lower predicted rates of worker absenteeism, and a belief that infrastructures can be protected by planning to protect workers using nonpharmaceutical interventions and antiviral medications to treat or prevent illness. some countries, such as canada or australia, which have substantial domestic influenza vaccine manufacturing capacity and small populations, may choose not to prioritize vaccination because of the ability to vaccinate everyone over several months. to our knowledge, only the us strategy explicitly presents different vaccine targeting based on pandemic severity, although every country is likely to reassess and potentially modify their national plan based on the epidemiology of the pandemic. prioritizing pandemic vaccination addresses only a single component of planning an effective pandemic influenza vaccination program. plans are also needed on how the vaccine supply will be allocated among the states or other jurisdictions, how it will be distributed, and how the program will be implemented. key implementation issues include the method of identifying persons who are in target groups, validation at the vaccination site, vaccine administration and tracking, and monitoring for the occurrence of adverse events. a major problem could be having to turn away persons who are panicked about the severity of a pandemic yet do not meet the criteria for vaccination at that time under the prioritization strategy. currently, no comparable program exists and each step will need to be planned and tested in preparedness exercises. effective communications also will be important. while substantial public involvement in the development of the vaccine prioritization strategy increases the chance that the approach will be acceptable to the public, communications goals will be to assure the public that the entire population will have the opportunity to be vaccinated, to communicate the rationale for prioritization and the prioritization strategy, and to inform people when it is their turn to be vaccinated. rationing of healthcare is not an issue that most americans have had to face in the past. outside of military settings, healthcare services generally have not been limited by availability as much as by economic or geographic factors. prioritizing pandemic influenza vaccine introduces a new paradigm. the approach taken by us planners considering science, ethics, and public values and preferences creates a model for how such rationing can take place. nevertheless, the optimal solution is to pursue preparedness activities that will obviate the need to prioritize. ongoing programs to increase influenza vaccine production capacity, to stretch vaccine supply through the use of new adjuvants, and to develop influenza vaccines targeted at antigens that are conserved across the different influenza a subtypes may all lead to a time when pandemic influenza vaccine prioritization will be unnecessary. local public health workers' perceptions toward responding to an influenza pandemic safety and immunogenicity of an inactivated split-virion influenza a/vietnam/ / (h n ) vaccine: phase randomised trial community strategy for pandemic influenza mitigation ethics subcommittee of the advisory committee to the director, cdc ( ) ethical guidelines in pandemic influenza strategies for containing an emerging influenza pandemic in southeast asia strategies for mitigating an influenza pandemic mitigation strategies for pandemic influenza in the united states mass vaccination: when and why antigen sparing and cross-reactive immunity with an adjuvanted rh n prototype pandemic influenza vaccine: a randomised controlled trial safety and immunogenicity of an inactivated adjuvanted whole-virion influenza a (h n ) vaccine: a phase randomised controlled trial containing pandemic influenza at the source effect of vaccination of a school-aged population upon the course of an a /hong kong influenza epidemic the prioritization of critical infrastructure in a pandemic influenza outbreak in the united states: final report and recommendations by the council herd immunity in adults against influenza-related illnesses with use of the trivalent-live attenuated influenza vaccine (caiv-t) in children public engagement pilot project on pandemic influenza ( ) citizen voices on pandemic flu choices the japanese experience with vaccinating schoolchildren against influenza mass vaccination for annual and pandemic influenza pandemic versus epidemic influenza mortality: a pattern of changing age distribution national strategy for pandemic influenza safety and immunogenicity of an inactivated subvirion influenza a. (h n ) vaccine pandemic influenza plan: appendix d key: cord- -f wwn z authors: douglas, r. gordon; samant, vijay b. title: the vaccine industry date: - - journal: plotkin's vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: f wwn z nan the vaccine industry is composed of companies that are engaged in any of the following activities: research (including that performed in industry and biotech), development, manufacture, or sales, marketing, and distribution of vaccines. they receive their revenue chiefly from sales of vaccine products or expectations thereof. the vaccine industry is relatively small, compared to the pharmaceutical industry, but growing. we estimate that total infectious disease vaccine sales in were more than $ billion worldwide and expected to grow to about $ billion by . although components of the vaccine industry are found in countries worldwide, the large vaccine companies are primarily u.s.-or european-based and have the dominant share of vaccine business on a revenue basis; but regional companies are gradually growing their market share on a dose basis (table . ). in the past years, the vaccine business, a former laggard in the pharmaceutical business, has shown remarkable growth powered by new innovative vaccines coupled with superior pricing strategies ( fig. . ). specifically contributing to this spectacular growth were the varicella, hepatitis a, pneumococcal conjugate, shingles, rotavirus, meningococcal conjugate for a, c, y, w, and human papillomavirus (hpv) vaccines, as well as myriad combination vaccines. this projected growth may plateau in the early s unless the vaccine industry continues to introduce new innovative products targeting diseases that impact the western world. sustaining this growth will be a challenge because of dwindling numbers of high-value vaccine targets for which the biology of protection is well understood (see table . ). the vaccine business is a capital-intensive business that requires considerable ongoing investment in manufacturing assets, facilities, and people to maintain compliance with everincreasing regulatory directives. the recent departure of baxter and novartis from the vaccine industry is an ominous sign that reflects the continued financial pressure on the remaining four major vaccine makers. further consolidation of this business is likely. in addition, new alliances will be formed between the big four manufacturers and emerging companies in india, china, and brazil, to take advantage of increasing immunization rates in those countries as well as growth of their private markets. the united states has been extraordinarily successful in vaccine research and development (r&d). , in the past years, most new vaccines approved worldwide were developed in the united states. approximately new vaccines were approved in the united states between and . , since then, combinations of existing vaccines have been introduced for simplified pediatric vaccination resulting in a wider adoption of acellular pertussis vaccination. a polyvalent pneumococcal conjugate vaccine for infants introduced by wyeth (now a subsidiary of pfizer) has been widely adopted and has made pfizer a major force in the vaccine business. since , several new vaccines have been licensed, including a combination of measles, mumps, and rubella (mmr) and varicella, as well as new vaccines against rotavirus, herpes zoster, hpv, meningococcus, influenza, and others. the hpv vaccines developed by merck and glaxosmithkline significantly expanded the field of adolescent vaccines and confirmed market acceptance of premium pricing. in the last years, the vaccine industry in the united states and europe has considerably improved its reliability as a supplier. chronic shortages are a thing of the past; this turnaround has primarily been achieved by modernization of vaccine manufacturing and distribution infrastructure supported and funded by the profitability of the vaccine business. the centers for disease control and prevention (cdc) stockpiling of pediatric vaccines has alleviated some concerns of critical shortages in case of supply interruptions. but the industry's vulnerability because of dependence on singlesourced vaccines continues to be an unresolved concern. the regulators and the industry must proactively develop a solution to this critical challenge and avoid any future public health crisis resulting from vaccine shortages during a prolonged supply interruption. vaccine development is difficult, complex, highly risky, and costly, and includes clinical development, process development, and assay development. the risk is high because most vaccine candidates fail in preclinical or early clinical development and less than in vaccine candidates entering phase ii achieves licensure. the high failure rate is the result of a variety of reasons: . not fully understanding the biology of protection. . lack of good animal models to predict vaccine behavior in humans. . unpredictability of human immune system reactions to antigens as it relates to immunogenicity or safety. . the unpredictability of the impact of combining multiple components in a vaccine. vaccine development requires strong project management systems and controls and requisite skill sets among scientists and engineers. a key strategic document that guides the stakeholders in vaccine development is the "target product profile" (tpp). the tpp summarizes the desired characteristics and features of the product under development, the key attributes of the product that provide competitive advantage, and, finally, a topline roadmap of nonclinical and clinical studies required to evaluate the products efficacy and safety in the target population. a well-defined tpp provides all the stakeholders, including research, process development, manufacturing, clinical, regulatory, and senior management, with a clear statement of the desired outcome of the product development program. process development involves making preparations of the test vaccine that satisfy regulatory requirements for clinical testing including clinical lots, preclinical toxicology testing, and analytical assessment, and finally, scale-up methods that lead to a consistent manufacturing process at one-tenth of full scale. usually three consecutive lots are tested in the clinic for immunogenicity. assay development involves the definition of specific methods to test the purity of raw materials, stability and potency of the vaccine product, and immunologic and other criteria to predict vaccine efficacy. go/no-go decisions must be made at each stage of clinical and process development and the vaccine industry r. gordon douglas and vijay b. samant must be data driven. clinical, process, and assay development tasks must be closely integrated. clinical development involves studies of the effects of vaccines on patients for safety, immunogenicity, and efficacy through a staged process: phase , early safety and immunogenicity in small numbers; phase , safety, dose ranging, and immunogenicity in to individuals; sometimes phase b, nonlicensure, proof-of-concept trials for efficacy; and phase , safety and efficacy trials that permit licensure, which generally require thousands of subjects. "process" can be broadly divided into two categories: bulk manufacturing and finishing operations. bulk manufacturing includes cell culture and/or fermentation-based manufacturing followed by a variety of separation processes to purify the vaccine. the finishing operations include formulation with adjuvant/stabilizer followed by vial or syringe filling (including lyophilization in the case of live viral vaccines) followed by labeling, packaging, and controlled storage. process development may be as costly as clinical development and is critically important to the overall success of a vaccine development program. as development proceeds toward licensure, costs escalate as clinical studies become larger, manufacturing scales up, and facilities must be built. postlicensure studies of safety and efficacy (phase ) of vaccines are essential and represent a large additional cost. it is important to note that, unlike pharmaceuticals, vaccines that pass early proof-of-concept studies in humans have a very high probability of achieving licensure. clinical activities are more visible than bioprocess development and clearly drive the go/no-go decisions that direct progress. the two are interwoven and each has rate-limiting steps, so they must be done in concert. the first stage of vaccine development involves acceptance of a candidate from a basic research laboratory and development of a small-scale process and formulation to make material for phase i study, analytical release assays, preclinical toxicology, immunological assays to evaluate clinical responses, an investigational new drug (ind) filing, and well-designed phase i/iia studies. the second step is to complete the definition of product and process prior to initiation of phase ii dose-ranging studies, which may take a year or more. product definition includes methods of synthesis/bioprocess steps, number of components, and stability/formulation. stability, release, and raw material assays must be in place. immunologic and other assays must be established to support dose-ranging studies, and a regulatory plan for vaccine process and product submissions must be written. the third step is to define the clinical dose and arrive at the appropriate manufacturing scale, which may take years or more. it results in the identification, manufacture, filling, and release of clinical-grade vaccine-usually in a pilot plantdemonstration of safety and a dose response in a phase ii clinical study; validation of critical assays to support phase iii clinical studies; consistency of lot manufacture (ability to produce three or more consecutive production-scale lots that meet all product specifications based on validated analytical methods); and completion of technology transfer to final site material manufactured in the commercial factory. this is especially difficult if immune studies are not highly reproducible, as is the case with most cellular immune assays. such decisions pose large financial risks if the product in development fails and requires access to large amounts of capital, an attribute usually restricted to large pharmaceutical companies. estimates of cost of development of a new drug or vaccine have risen from $ million in , to $ million in , to $ billion in . [ ] [ ] [ ] these estimates take into account all costs, including r&d costs of products that fail, postlicensure clinical studies, and improvements in manufacturing processes. approximately % of the cost is for construction; the remainder is the cost of capital interest. these numbers have been debated (others estimate $ million to $ million); however, the higher estimates have been validated in two ways. first, the number of new vaccines brought to licensure annually by a company or the industry is very small compared with other products, and correlates with r&d expenditures of $ million to $ million for each new product. thus, if a company spends $ million annually for vaccine r&d, one might expect one new product every to years, and this appears to hold true. second, biotechnology companies that are focused on one vaccine and have successfully brought it to market have spent $ million to $ million on r&d as exemplified by the development of the live attenuated influenza vaccine by aviron, now medimmune. in summary, vaccine development from concept to licensure is a lengthy process as illustrated by timelines for some of the currently licensed vaccines (table . ). to understand the predominant role of major pharmaceutical companies in the development of vaccines, one must examine the role of a vaccine development company in relation to its of manufacture of full-scale lots, including process and analytical procedures. for vaccine targets for which animal studies are not predictive of efficacy in humans, such as hiv, malaria, and tuberculosis (tb), small phase iib proof-of-concept studies based on adaptive clinical trial designs may be used to gain confidence before committing significant resources for process development, analytic development, and factory construction. in general, the analytical and release assays are particularly difficult to develop because, in most cases, vaccines are considered "not well-characterized" biologicals by regulatory agencies. the release assays initially involve functional potency assays such as animal immunogenicity prior to acceptance of more robust and precise in vitro assays that correlate with these functional potency assays. in general, variability of biological assays is a major hurdle in achieving process scale-up and manufacturing consistency. the fourth stage is the completion of phase iii pivotal clinical studies and corresponding consistency lot studies, which requires to years. keys to successful phase iii clinical studies are an accurate estimate of sample size based on disease incidence, low dropout rates, precise clinical end point definitions related to future label claims, and rigorous data management to the highest standards. in addition to clinical studies, scale-up and manufacture of consistency lots, including transfer to the facility of all assays, facility validation, demonstration of consistency and real-time stability are needed to support adequate shelf-life claims. the final stage is biologics license application (bla) preparation, licensure, and vaccine launch, which requires . to years. thus the total elapsed time for development is to years, assuming all activities proceed as planned. manufacturing plants are very expensive to construct, ranging from $ million to $ million depending on the size (dose requirements) and manufacturing complexity, with an additional expenditure of approximately % of that cost for cleaning and process validation activities that are now required under the current good manufacturing practices regulations. with few exceptions, each vaccine requires a different plant because of unique manufacturing requirements and the regulatory difficulties associated with changing over to a different product. some processes are scalable, such as bacterial or yeast fermentation, so that increasing the size of the manufacturing unit (i.e., fermenter) will greatly increase the yield; unit cost will decrease with volume increase. other manufacturing processes, for example, those dependent on viral growth in embryonated hen eggs or cell lines, are not scalable. additional plants or modules within plants must be built to increase the throughput, so unit costs do not appreciably decrease with volume increases. despite the complexity of bulk vaccine manufacturing, to years post-product launch, the fully burdened bulk cost of production for most of the older vaccines declines to as little as $ . to $ . per dose, and significant elements of product cost are primarily driven by activities related to filling, vialing, and packaging (table . ). established vaccines with a limited number of suppliers can generate very high profit margins over the product life cycle. the commitment to build a plant must be made early ( to years before expected licensure) including a -to -month finished goods inventory build-up to expedite product to the market. otherwise a gap of to years between licensure and product launch will occur. furthermore, it is far better to produce consistency lots in the final vaccine production factory to demonstrate the ability to manufacture the vaccine reliably and to use those lots in the phase iii efficacy trials. otherwise, immune studies will be required for "bridging" the product used in the efficacy trial to bioshield for the procurement of advanced medical countermeasures for biological as well as other threats and has successfully developed medical countermeasures against smallpox, anthrax, and botulinum toxin. in addition, barda is funding a variety of early stage novel vaccine approaches for pandemic influenza. barda essentially is intended to overlap with and close the gap between nih-funded preclinical or initial phase i trials and the more advanced project bioshield programs that are in late stage phase iii or licensure stages of development. the u.s. agency for international development (usaid) supports limited r&d targeted toward those vaccines that potentially will have the greatest impact on children younger than age years in developing countries. the center for biologics evaluation and research (cber), a division of the u.s. food and drug administration (fda), is responsible for licensing new vaccines. cber establishes standards for manufacturing processes, facilities, and pre-and postlicensing clinical studies to ensure that licensed vaccines are safe and effective (see table . ). these standards have a profound partners. the relative contributions of the various partners to the delicate fabric of vaccine r&d is shown in table . . several branches of the u.s. government play major roles in vaccine r&d. the u.s. national institutes of health (nih) is the major funding source via intramural and extramural (largely academic) programs of fundamental research (e.g., gene-based vaccines or t-cell memory studies) and directed research on pathogens (e.g., hiv), which may lead to new vaccine candidates. the nih, through its vaccine trials network, has increased its role in clinical development domestically and internationally. in addition, the dale and betty bumpers vaccine research center at the nih was established in primarily to pursue the development of hiv vaccines. the centers for disease control and prevention (cdc) is the primary government agency responsible for epidemiological monitoring of disease trends. the cdc conducts disease surveillance and epidemiological studies to ascertain the prevalence and incidence of specific diseases; this information provides a rationale for prioritizing vaccine development. these studies by the cdc are performed in addition to studies conducted by the vaccine companies, such as phase iv studies. through the advisory committee on immunization practices (acip), the cdc recommends usage of vaccines, and is responsible for most of the public purchases (directly through the vaccines for children program for approximately %, and indirectly through other federal, state, and local government purchases for approximately %, together totaling approximately % of all childhood vaccines in the united states), thereby playing a major role in determining the demand and potential profit associated with vaccines. professional organizations such as the american academy of pediatrics and the american academy of family physicians also make recommendations for vaccine usage. there is no federal vaccine program for adults, although medicare does reimburse for influenza and pneumococcal conjugate vaccines. historically, many adults with private insurance were not covered for immunizations. however, the affordable care act of requires health plans to cover vaccines recommended by the acip prior to september with no copayments or other cost-sharing requirements when those services are delivered by an in-network provider. the department of defense (dod) does targeted vaccine r&d to help it perform its mission of protecting deployable impact on the nature and direction of vaccine development and its costs. in addition, cber maintains a strong research base internally, so it is better positioned to evaluate data from various studies. cber remains the premier vaccine regulatory agency in the world. nongovernmental organizations (ngos) are playing an increasing role in vaccine research. the bill and melinda gates foundation supports several organizations including the international aids vaccine initiative, the malaria vaccine initiative, aeras (dedicated to developing tb vaccines), and others with significant funding for development of vaccines that would have the greatest impact on diseases of developing countries. in addition, a related organization, programs for appropriate technology in health (path), is a nonprofit group that forges private sector partnerships to develop vaccine technologies suitable for the developing world. these product development partnership organizations (pdps; essentially not-for-profit biotech companies) bring together specialized knowledge, animal models, immunologic assays, and field sites for vaccine testing as well as early capital investment to reduce the scientific technical risks, opportunity costs, and financial risk to their biotech and large pharma industrial partners. they also provide opportunities for validation of novel vaccine technologies and platforms. the role of large, full-service vaccine companies ( in some limited basic research and significant amounts of targeted research regarding specific organisms, but the preponderance of activity is in clinical and process development. sufficient personnel and expertise in process development and chemical engineering reside almost exclusively in these companies; there is no other resource for such development. clinical development that will satisfy fda standards is also done mostly by the large companies, performed by academia and contract research organizations. personnel and expertise in clinical research, regulatory affairs, data management, statistics, project management, and all other required disciplines also exist within the large companies. perhaps most importantly, their management is structured to make rapid go/no-go decisions required to minimize risk and assess efficient vaccine development. many smaller organizations, often referred to as biotechnology companies, are engaged in vaccine research. they are often started by university scientists, supported by venture capitalists, and are capable of basic research on a vaccine idea. at this early stage, they usually have limited capacity in process development, manufacturing, and clinical development, and none in distribution, sales, or marketing. if research results are favorable, capacity in process engineering, clinical studies, and manufacturing must be enhanced or obtained by partnering. because of the large cost of adding new capacities and expertise, many biotech companies in advanced product development will opt to partner with large, full-scale companies. although or so small companies claim engagement in vaccine r&d, only about a dozen or so consider it a major activity, and only a very few, such as medimmune, have made it to the market or close to the market on their own. more have licensed their products or technology platforms to larger companies that have then completed development, yielding new vaccines such as those for hepatitis b and haemophilus influenzae type b. for example, the hepatitis b innovation came from the research laboratories of chiron corporation that succeeded in making hepatitis b surface antigen in yeast, and thus enabling merck and glaxosmith-kline to commercialize the modern hepatitis b vaccines. in the case of h. influenzae type b (hib), praxis biologics and connaught laboratories pioneered the development of hib polysaccharide and conjugate vaccines. these companies were eventually acquired by sanofi and wyeth-lederle, respectively. the greatest contributions of the biotechnology companies have been the introduction of multiple ideas into early vaccine development, and testing them to determine if they should be rejected or carried forward. these small companies are dependent on several factors for their success: . a vibrant basic research environment that allows for creation of new ideas, an environment that exists in wellfunded (nih) academic research programs. . a strong venture capital and investment community that views vaccine companies as potentially financially rewarding as other investment opportunities. . strong patent laws providing the intellectual property protection that is essential for commercial success. funding sources for vaccine r&d include government, profits from sales of product, risk capital, and charitable foundations. the nih competes with other federal agencies and programs for taxpayer support, and, in general, has been more successful than most. similarly, vaccine r&d sponsored through the dod, fda, cdc, and usaid is competitive with other public needs as determined by the executive and legislative branches of government. risk capital from private investors is the primary source of funds for small companies. investors are attracted to the potential profits of a new vaccine, a forecast determined in part by sales of current vaccines. large vaccine companies, which are divisions of much larger pharmaceutical companies, seek a profit by selling products. on average, pharmaceutical companies reinvest approximately % of their profits from product sales into r&d, and this proportion applies to vaccine sales as well as other pharmaceutical products (pharmaceutical research manufacturers association, personal communication, ). because vaccine companies are subsidiaries of large companies, vaccine r&d and manufacturing must compete with other product areas for resources. comparisons of the economics of the vaccine industry with the pharmaceutical industry in europe, and separately in the united states, were performed by the mercer consulting company in (fig. . ) . these studies in the united states showed that the in terms of technical feasibility, strong patent protection, and potential market size will be taken forward into development (post-phase i). in addition, other candidate vaccines might be licensed from small companies. even in the largest companies, only a few products can be in development at the same time. thus, go/no-go decisions must be made and market size is a major determinant of the choice between two candidate vaccines, otherwise equal in technical feasibility and likelihood of success (table . ). this system works extremely well for vaccines with large potential markets in the developed world when technical feasibility is demonstrated. it does not work for vaccines for diseases that exist predominantly in the poorer regions of the world (e.g., tb); it works imperfectly for diseases of the developed world that affect relatively few persons because of geographic restriction (e.g., lyme disease) or diseases limited to specific risk groups (e.g., cytomegalovirus [cmv] in transplant recipients), and it does not work when technical feasibility has not been demonstrated (e.g., hiv). the last problem has to be solved by a strong basic program in vaccine-related sciences, particularly for hiv, staphylococcus aureus, malaria, and other challenging targets. niche vaccines for developed-world markets are much more attractive to biotech than to large pharmaceutical companies as evidenced by recent biotech vaccine efforts for west nile virus, japanese encephalitis virus, the cmv-transplant indication, and dengue. to involve large companies in development and manufacturing of vaccines to meet needs such as biodefense or health needs of poorer countries, incentives must be established to convince these companies that they should develop and manufacture such products. such incentives might take the form of guaranteed purchase of certain volumes of a vaccine if specified standards are met, direct contracting by a government agency, or some other publicly funded mechanism. , the use of advanced market commitments to create a funding mechanism for vaccines needed in the developing world has been endorsed by the g and pilot projects may be starting soon. this will not solve the problem of the high technical risk and opportunity costs associated with such vaccines, but it may contribute to the solution if combined with early investment. companies may be willing to engage in such work. indeed, they may already have donated or sold vaccines at very low prices to poorer countries. however, such practices alone will not solve the enormity of the health problems worldwide. without special incentives, it is unrealistic to expect companies to engage in r&d on diseases that only, or predominantly, affect the poorer regions of the world. manufacturers in developing countries (initially in india and china, and more recently in brazil) are playing an increasing role in meeting these needs. indeed, they already supply the majority of doses of older vaccines for such countries. as their expertise and capacity in vaccine r&d increases they will perhaps evolve into major participants in supplying new vaccines to the developing world. there are numerous manufacturers in these emerging countries, but a few truly stand out. the vaccine industry has slowly mushroomed in india with several key companies emerging including bharat biotech, biological e., panacea biotec, and others, but the largest one is the privately held serum institute of india. the indian vaccine industry has significantly benefited from technology transfer from the west. despite the industry's success, the available estimates suggest that r&d spending remains relatively low as a percentage of sales. serum institute of india is the world's largest producer of vaccines by number of doses, producing . billion doses contributions to r&d, interest, taxes, and earnings after expenses were similar for the two industries ( % vs. %, respectively). however, the expenses were quite different. significantly more was spent on production and distribution ( %, which includes production, distribution, and returns of product) in the vaccine industry compared with the pharmaceutical industry ( %), whereas the pharmaceutical industry spent more than the vaccine industry on sales, marketing, and administrative expenses ( % vs. %, respectively). consequently, within companies, there is an expectation that sales-to-expense ratios for vaccines will be similar to those of other pharmaceutical products, and that revenues will increase every year. although some of this increase may be accomplished with sales volume, prices stabilize as vaccine products mature, and increased revenues are no longer possible; hence, the requirement for a steady rollout of new products. however, unlike pharmaceuticals, old vaccines continue to be profitable for a variety of reasons, including: . the absence of a regulatory pathway for generic vaccines deters potential entrants from engaging in a complex and expensive approval process. . in most cases, access to knowhow, such as proprietary cell lines, virus strains, and internally developed processes, is far more valuable than patent protection. . the birth cohort is renewable, providing an ongoing unmet need for vaccines. as a result, sole-sourced vaccines, manufactured in fully depreciated assets, are profitable for pharmaceutical companies. one such example is the mmr vaccine, which after years still has no competition in the united states. a typical vaccine company will have several vaccine candidates in early development, defined as all r&d through phase i clinical testing (table . ). [ ] [ ] [ ] [ ] those that are most promising tuberculosis productivity estimated at -to -fold higher than the measles vaccines made by merck and glaxosmithkline. this privately held vaccine company has relentlessly invested in production facilities/infrastructure that surpasses some of the best biotech manufacturing facilities in the united states. so powerful has its growth been that one out of every two children immunized worldwide get at least one vaccine produced by the serum institute. vaccines recently developed by the serum institute are nasovac (live attenuated trivalent influenza vaccine), menaf-rivac (meningococcal a conjugate vaccine), pentavac (dtp hepatitis b-hib vaccine), and inactivated polio vaccine. the institute continues to invest in r&d and is currently working on a rotavirus vaccine, a polyvalent meningococcal conjugate vaccine, a pneumococcal conjugate vaccine, and hpv vaccine, combination vaccines containing acellular pertussis, and others. china ranks as the world's largest vaccine consuming and manufacturing country, with an estimated annual output of billion doses. the original six government-owned regional biological institutes are now part of the china national biotec group (cnbg) consolidated under the china national pharmaceutical group corporation (sinopharm group co., ltd.). cnbg has a large r&d center in beijing that maximizes the synergies of the six affiliated institutions. today, cnbg/ sinopharm supplies % of the doses of the chinese national immunization program vaccines. china's vaccine manufacturing capabilities are currently intensely focused on supplying their own domestic needs for the pediatric birth cohort of million newborns annually. there are registered vaccine manufacturers in china and licensed vaccines. several of the manufacturers are members of the developing countries vaccine manufacturers' network (dcvmn). in , the world health organization prequalified the chinese-made japanese encephalitis virus vaccine made by the chengdu institute for biological products in collaboration with path. china became the first country ever a year; its products are used in more than countries. serum institute is also one of the largest suppliers of measlescontaining vaccines and the diphtheria-tetanus-pertussis (dtp) vaccines to u.n. agencies (unicef and pan american health organization [paho]). the institute makes its measles vaccine in mrc- cells instead of chick embryos and has although the first two of these factors have been consistently present in recent years, downward pressure on price is a major threat to current companies and a disincentive to new companies. freedom to price vaccines is restricted to the private market. less than % of the vaccines for children sold in the united states are sold in the private market; the rest are sold to the federal or state governments at reduced prices. controls are even greater in western europe and japan, and internationally there is strong downward pressure on prices as one moves from well-developed to less-developed regions of the world. in addition to the burden of partial price controls, the vaccine industry is subject to intense regulation. it cannot sell products until the vaccine and the facility in which it is manufactured are approved by the fda or other regulatory authorities; each batch must be released by the appropriate regulatory agency; and the usage, and therefore market size, is largely determined in the united states by the cdc and in europe by national regulatory authorities. thus, the vaccine industry does not operate in a free-market environment, and its behavior reflects these constraints. vaccine business growth in the future will have three important drivers: . new vaccines for cmv, herpes simplex virus (hsv), respiratory syncytial virus (rsv), norovirus, clostridium difficile, enterotoxigenic escherichia coli (etec), "improved influenza," and others that will gradually shift the focal point of immunization activities from the pediatric sector to the adolescent and adult sectors. . private market expansion in india and china driven by "high-income family" birth cohorts of million and million, respectively. this birth cohort roughly equals the combined birth cohort of million in the united states and europe. these high-and even middle-income individuals have shown the desire and ability to pay for vaccines at relatively high prices in relation to their incomes in these and other countries. to approve a hepatitis e vaccine, which was developed by xiamen innovax biotech. brazil has four notable vaccine manufacturing companies. bio-manguinhos/fiocruz is a government-owned entity that supplies the full demand for most vaccines under the brazilian national immunization program (nip). they also have a r&d collaboration with glaxosmithkline for a dengue vaccine. butantan institute is another government-owned institution that supplies the full demand for a smaller number of vaccines under the brazilian nip. ataulfo de paiva foundation is nonprofit private institution that primarily supplies the bcg vaccine for the brazilian market. ezequiel dias foundation (funed) is a public institution and part of minas gerais state. since , it has supplied the meningococcal conjugate vaccine after transferring the technology from novartis. the indian vaccine industry is the most advanced among these three developing countries, and is already providing a significant portion of the world's vaccine supply as well as developing new vaccines. china is on the verge of the transition from a domestic-only provider to a vaccine exporter, and is demonstrating solid progress in vaccine innovation. brazil is approaching the point of supplying its own domestic needs, largely with technology transferred from the developed world. together, these emerging players from middle-income countries will have increasing influence in the global vaccine industry during the coming years. pricing is a critical component of success for large companies and for venture funding of small companies since potential sales determine the desirability of an investment decision. the public expectation is for low vaccine prices, although this has changed somewhat in recent years with the introduction of several new, higher priced vaccines, such as varicella, rotavirus, pneumococcal conjugate vaccine, zoster vaccine, and hpv vaccine ( fig. . ) . large companies believe that vaccines should be priced according to value to society such as reduction in health care and related costs, relief from pain and suffering, and/or prevention of death, and that they should be rewarded for taking the enormous risks inherent in early vaccine development. such prices far exceed manufacturing costs, but are essential to produce the revenue streams that allow vaccines to be competitive for r&d and manufacturing resources within large pharmaceutical companies or that make biotech companies attractive investment opportunities. in general, vaccine prices have declined when more than two companies have competed in a single vaccine market and profitability has fallen sharply. the influenza vaccine market highlights this cyclical ebb and flow of competitors, most recently with the h n outbreak and shortages in leading to expanded competition and a vaccine surplus, followed by lower prices in . a vigorous large-company vaccine industry is dependent upon several factors: . a rich research environment sponsored largely by the nih and mostly carried out in academia, as the source for new creative ideas. . strong patent laws and protection of intellectual property. . freedom to price products at fair levels related to value of product to society. . well-implemented immunization practices. u.s. vaccine price evolution polio vaccine grows in developing countries, alternative approaches for local production will be explored, including access to bulk injected polio vaccine, tech transfer by big pharma as a part of their strategic alliances in developing markets, and potential introduction of alternative injected polio vaccine strains such as the sabin strain. another key driver will be the expansion of vaccine markets in india, china, and brazil. vaccine uptake rates in india, china and brazil are still low compared with western countries (e.g., india's flu vaccine uptake in was . million doses vs. million doses in the united states). , the immunization rates are also expected to increase in other low-income countries, which will increase vaccine dose requirements substantially. most of this demand in low-income countries is expected to be met by manufacturers of dcvmn network. as the dcvmn expands its role, one would expect significant downward pressure on vaccine prices. the delicate balance between innovation, government support, industrial expertise, and market forces has led to the establishment of a robust vaccine industry that will continue into the future. the industry is changing, however, with the growth of new markets in emerging economies and with the pressing need for new vaccines for the developing world. the current efforts of pdps and public creation of markets in response to this need will be successful if lessons learned from the industrial vaccine effort are incorporated into these government and philanthropically driven expectations. estimates of the total worldwide vaccine market revenue are $ billion. the top four western suppliers (see table . ) account for approximately % of these sales; the remainder comes from regional vaccine companies, the largest of which are located in middle-income countries such as india, china, and brazil (see table . ). the top four companies are slowly losing market share in doses to the dcvmn sourced doses and when polio eradication is achieved their dose share will drop to less than % of worldwide dose volume. in the coming years, as the eradication of polio becomes a reality, the developing country manufacturers will phase out their oral polio vaccine production. however, the need for inactivated polio vaccine will grow as developing countries adopt it into their pediatric immunization plans. as the demand for injected assuming such vaccines become reality, there is little doubt that the international donor community, working through organizations such as the global alliance for vaccines and immunization, will provide adequate funds for purchase of effective malaria, hiv, and tb vaccines, all of which are cost-effective references . company year-end earnings releases from evaluate pharma new scientific opportunities and old obstacles in vaccine development immunizing children: can one shot do it all? in: medical and health annual public health: u.s. vaccine supply falls seriously short lessons learned from a review of the development of selected vaccines. national vaccine advisory committee vaccine development: the long road from initial idea to product licensure cost of new drug development spending on new drug development united states vaccine research: a delicate fabric of political and private collaboration. national vaccine advisory committee influenza vaccine manufacturers testimony on vaccine policy before the u.s. house of representatives committee on commerce vaccine advance-purchase agreements for low income countries: practical issues improving vaccine supply and development: who needs what? vaccines market in india china's growing biomedical industry china's emerging vaccine industry special thanks to andrew hopkins for compositional support. key: cord- - osu hdu authors: lestari, fajar budi; vongpunsawad, sompong; wanlapakorn, nasamon; poovorawan, yong title: rotavirus infection in children in southeast asia – : disease burden, genotype distribution, seasonality, and vaccination date: - - journal: j biomed sci doi: . /s - - - sha: doc_id: cord_uid: osu hdu background: rotaviruses (rvs) are recognized as a major cause of acute gastroenteritis (age) in infants and young children worldwide. here we summarize the virology, disease burden, prevalence, distribution of genotypes and seasonality of rvs, and the current status of rv vaccination in southeast asia (cambodia, indonesia, lao people’s democratic republic, malaysia, myanmar, philippines, singapore, thailand, and vietnam) from to . methods: rotavirus infection in children in southeast asia countries was assessed using data from pubmed and google scholars. most countries in southeast asia have not yet introduced national rv vaccination programs. we exclude brunei darussalam, and timor leste because there were no eligible studies identified during that time. results: according to the – rv surveillance data for southeast asia, . % of all diarrheal disease in children were caused by rv infection, which is still a major cause of morbidity and mortality in children under years old in southeast asia. mortality was inversely related to socioeconomic status. the most predominant genotype distribution of rv changed from g p[ ] and g p[ ] into the rare and unusual genotypes g p[ ], g p[ ], and g p[ ]. although the predominat strain has changed, but the seasonality of rv infection remains unchanged. one of the best strategies for decreasing the global burden of the disease is the development and implementation of effective vaccines. conclusions: the most predominant genotype distribution of rv was changed time by time. rotavirus vaccine is highly cost effective in southeast asian countries because the ratio between cost per disability-adjusted life years (daly) averted and gross domestic product (gdp) per capita is less than one. these data are important for healthcare practitioners and officials to make appropriate policies and recommendations about rv vaccination. rv was first identified in cattle in [ ] . the virus appeared similar to those that cause diarrhea in mice [ ] , calves [ ] , and a virus identified from a rectal swab of a healthy monkey [ ] . in may , bishop, davidson, holmes, and ruck examined ultrathin sections of duodenal mucosa from children with acute gastroenteritis (age) by electron microscopy (em), and found abundant viral particles in the epithelial cell linings of the upper villous surface which were similar in appearance to the rvs discovered in animals before [ ] . em also revealed -nm particles in negatively stained fecal extracts [ , ] . the viral particle was initially identified by several names including reovirus-like, orbivirus-like, duovirus, infantile gastroenteritis virus, or a "new" virus. the wheel-like structure observed on em eventually led to the naming concensus of rotavirus (rota is latin for wheel) [ ] . rvs have now been shown to be a cause of diarrhea in the young of many mammalian and avian species [ ] . rvs are -nm, non-enveloped rna viruses belonging to the family reoviridae. the rv genome consists of segments of double-stranded rna (dsrna) surrounded by a triple-layered capsid. each genomic fragment encodes protein of different function. the outer layer proteins (viral protein [vp] and vp ) mediate attachment and penetration; the inner layer is composed of vp protein and encloses the viral genome and the minor protein vp , the viral rna-dependent rna polymerase, and vp , the viral capping enzyme. the middle layer is composed of vp which interacts with and stabilized the inner and outer layer [ ] . vp defines species/group and subgroup specificities [ ] [ ] [ ] . all rna segments, except for segment , are monocistronic, encoding either structural viral proteins (vp to vp , vp , and vp ) or non-structural proteins (nsp to nsp ). genome segment codes for two proteins: nsp and nsp [ ] . rvs can be differentiated by a dual classification system, based on the two outer capsid proteins, vp and vp , that determine the g (vp , glycoprotein) and p (vp , protease-sensitive) genotypes [ ] . at least g types and p types have so far been identified in humans and animals [ ] . a whole genome-based genotyping system was recently proposed for rv group a (rva) based on the genotype assignment of all gene segments [ ] . the genome of individual rv strains is given the complete descriptor of gx-p [x]-ix-rx-cx-mx-ax-nx-tx-ex-hx to identify the genotypes of the vp -vp -vp -vp -vp -vp -nsp -nsp -nsp -nsp -nsp / encoding rna segments, respectively. most strains demonstrate either a wa-like (g -p [ ] -i -r -c -m -a -n -t -e -h ), ds- (g -p[ ]-i -r -c -m -a -n -t -e -h ), or au- -related (g -p[ ]-i -r -c -m -a /a -n -t -e -h /h ) genotype constellation [ , ] . indirect immunofluorescence techniques targeting vp are used to differentiate rv species. rvs are currently differentiated into at least nine species, designated a to i and a tentative tenth species, j. rva infects in birds and mammals; rvb, rvc, rve, rvh, and rvi have been detected in one or more mammalian hosts; rvd, rvf, and rvg have been detected only in birds; rvj infects bats [ ] [ ] [ ] . table shows rotavirus groups and its host. rvs were recognized as a major cause of age in infants and young children in [ , ] . rv is the leading cause of diarrhea-associated mortality among children younger than years, although the burden of rv has decreased during the past decade. rv infections were responsible for approximately , deaths annually among children younger than years. rv constitutes of the diarrhea etiologic agents measured in the global burden of disease study [ ] . it is the most prevalent agent causing severe diarrhea in both developed and developing countries [ , ] . after rv vaccine introduction in developed countries, norovirus become the predominant viral pathogen that caused age in children. norovirus prevalence remained stable or increased, whereas rotavirus activity dramatically decreased [ ] [ ] [ ] . nevertheles, from to in southeast asia, [ , ] approximately . % (n = , ) of total diarrhea mortality was associated with rv disease [ ] . figure shows the prevalence and death caused by diarrheal disease and rv in children under years old from to in southeast asia. the prevalence of diarrheal diseases in southeast asia countries varies, but the mortality trend associated with diarrhea and especially rv infection has been decreasing in recent years. lao people's democratic republic [pdr] reports one of the highest death rates in this period. however, improvement in hygiene and sanitation combined with the introduction of the rotavirus vaccine has contributed to decreasing rv infection [ ] . we collected rv surveillance data from countries in southeast asia (cambodia, indonesia, lao pdr, malaysia, myanmar, philippines, singapore, thailand, and vietnam) for the years - to estimate the proportion of rv gastroenteritis (rvge) ( table ) . a total of , stool samples were collected of which , ( . %) were rv positive. acute diarrheal disease caused by rv is still a major cause of morbidity and mortality in children under years old in developing countries, which may be attributed to the regions' lower standards of living and hygiene conditions [ ] . however, our study revealed that as the gross domestic product (gdp) per capita increases, and the economic status of southeast asian countries improves, the rv mortality rate steadily declines (fig. ). higher socioeconomic status (ses) can improve sanitation, hygiene practices, and healthcare facilities to support better living conditions and decrease the rv mortality in children. among the data from southeast asia countries examined, the most predominant genotype distribution of rv has changed except in lao pdr and malaysia. in - , g p [ ] and g p [ ] were the most predominant genotypes, but starting , it changed into the rare and unusual genotypes g p [ ] , g p [ ] , and g p [ ] . several uncommon rv genotypes such as g p [ ] , g p [ ] , g p [ ] , g p [ ] , g p [ ] , and g p [ ] were identified in the surveillance data. the presence of such diversity among rv isolates provides insight into the evolution of these strains, which can arise due to point mutations, genetic rearrangements, reassortment events, and interspecies transmission [ , , ] . circulating rv strain appears diverse despite rv vaccination, which may enable the increase in the prevalence of non-vaccine strains. thus, the circulation of strains in which vaccines have lower efficacy eventually impairs vaccine effectiveness [ ] . in the philippines, where the rotarix® vaccine was introduced in july , the frequency of rvge cases caused by g p [ ] decreased while the circulation of g p [ ] increased significantly [ ] . the genotype distribution of rv in southeast asia is shown in table . differences in the predominance of rv genotypes and newly emerging strains were identified over the surveillance period in cambodia. the g p [ ] genotype was predominant in , , and ( , , and %, respectively), whereas genotype g p [ ] predominated between ( %) and ( %). the previously uncommon strain g p [ ] also emerged in ( %). the proportion of g p [ ] genotype detections increased further in ( %), in conjunction with the emergence of g p [ ] ( %). by , the detection of genotype g p [ ] had increased to %, and g p [ ] became the most prevalent genotype (responsible for % of detections) [ ] . in indonesia, the surveillance results demonstrated a changing trend for the most prevalent genotype, from g p [ ] in - to g p [ ] in - . from -to , g p [ ] was the most prevalent genotype circulating, which accounted for , . , . , . , and . % each year, respectively. g p [ ] became the most predominant strain in , and this continued to , accounting for , , . %, and , %, respectively [ , , [ ] [ ] [ ] . another study reported that g p[ ]/ [ ] was also the predominant strain during - [ ] . during the -year surveillance period in lao pdr, the most predominant genotypes identified by year were g p [ ] ( %) and g p [ ] ( %) in ; g p [ ] ( %) and g p [ ] ( %) in ; g p [ ] ( %) and g p [ ] ( %) in ; g p [ ] ( %) in ; g p [ ] ( %) and g p [ ] ( %) in ; g p [ ] ( %) in ; and g p [ ] ( %) in [ ] . from to , the most common genotype in malaysia was g p [ ] ( %). other genotypes identified were g p[ ] ( . %) and g p[ ] ( . %). approximately % of the samples were either mixed or untypeable (g p [ ] , g p [ ] , g p [ ] , g p [ ] ) [ ] . a preliminary report in sabah state showed that approximately % of samples were positive for rv, of which % were of genotype g p [ , ] . in myanmar, the most common strains in were g p [ ] ( . %) and g p [ ] ( . %). g p [ ] was detected from to , ranging from . % in to % in . g p [ ] became the most predominant strain in - , followed by g p [ ] in - . g p [ ] comprised only % of the rv strains in and increased to . % in . while in , the majority ( %) of rv strains comprised g p [ ] ( %) and g p [ ] ( %). g p [ ] emerged in myanmar in and was the most common strain in and [ , ] . in the philippines, ( . %) rva-positive stool specimens were successfully typed. the most common genotypes identified were g p [ ] and two g p [ ] strains. also, animal strains were detected in specimens from children, including g p [ ] felinelike and nine g p [ ] porcine-like strains [ ] . the predominant strain observed in singapore was g p[ ] ( , %), while g p[ ] ( , %) was the second most common type observed among children in singapore [ , ] . in thailand, g p [ ] was the most common genotype in ( , %). g p [ ] strains were g p [ ] . uncommon genotypes found were g p [ ] , g p [ ] , g p [ ] , and g p [ ] , adding to the existing list of uncommon genotypes reported to circulate in thailand [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . based on rv diarrhea data from four sentinel hospitals in vietnam from to , g p [ ] was the most prevalent strain during and , accounting for and % of total genotyped samples, respectively. g p [ ] was found in % of samples in and % in . in and , the proportion of rvge caused by g p [ ] increased to and %, respectively. g p [ ] was not detected in and , it accounted for only % of specimens in , and it became predominant ( %) in [ ] . meteorological conditions have an indirect yet important impact on the epidemiology of human rotavirus infection. weather-related low indoor relative humidity and indoor crowding may be key factors in the epidemiology of rotavirus disease. hospitalizations for rotavirus gastroenteritis tended to be more common after a cold or dry month than after a warm or wet corresponding calendar month [ ] . the temporal trend of cambodian rv infection shows substantial year-round transmission with prominent peaks during colder, dry months. peaks typically occurred between november and may [ ] . in indonesia, rv infection was present throughout the year and did not demonstrate clear annual seasonality [ ] . conversely, infection generally peaks during the rainy season in singapore and malaysia. an outbreak of rv infection was observed from january to march [ , ] . positive rv cases increased in number and proportion during the dry season (january-april) each year in lao pdr [ ] . in myanmar and the philippines, rv infection has a strong seasonal peak in colder, drier months, as seen in other asian countries. the highest rate of rv infection occurred in january and february [ , ] . rv cases in thailand were most prevalent during the cooler months, specifically from january to march, but rv was detected every month in the northern part of thailand, where the weather is relatively cooler compared to the rest of the country [ ] . in vietnam, rv was detected every month, but most rv gastroenteritis (ge) cases occurred between december and may [ ] . figure shows the rv seasonality in southeast asian countries. the seasonal pattern in rv varies by climatic region and is also associated with local weather. a reduction in rv rates was associated with increased temperature and precipitation [ ] . there is a significant association between increased numbers of estimated positive rv cases and lower humidity, rain, and temperature. in children younger than years old, rv was the pathogen most frequently identified in the winter, dry, or cool/dry seasons [ ] . in tropical climates, the higher temperature was associated with a greater decrease in rv than in humid mid-latitude climates [ ] . it had been suggested that the seasonal pattern may be driven by airborne transmission of the disease [ ] . one of the best strategies for decreasing the global burden of disease is the development and implementation of effective vaccines [ ] . rv is the most common cause of vaccine-preventable severe diarrhea [ ] . the world health organization (who) recommended that rv vaccines be included in immunization programs in the european region and the americas in . in , following efficacy studies in low-income countries (lics) and lower-middle-income countries (lmics) in africa and asia, the who recommended the use of rv vaccines in all national immunization programs (nips) [ ] . rv vaccines had been introduced in countries by the end of , and global coverage was estimated to be at % [ ] . most countries in southeast asia have not yet introduced national rv vaccination programs. among asian countries, only the philippines, and recently thailand have introduced the vaccines on a limited basis [ ] . the global alliance for vaccines and immunizations (gavi), also known as the vaccine alliance, actively supports rv vaccination by subsidizing the cost in eligible countries. in lmic, the budget impact is an important criterion for funding new interventions, particularly for large public health investments such as new vaccines. by the end of , gavi had funded rv vaccine introductions in countries [ ] . research to develop a safe, effective rv vaccine began in the mid- s when investigators demonstrated that previous infection with animal rv strains protected laboratory animals from experimental infection with human rvs [ ] . the first multivalent live oral reassortant vaccine developed was rotashield® (a rhesus rv tetravalent [rrv-tv] vaccine) in the late s [ ] . the rrv-tv vaccine was licensed in august for routine use in children in the united states at , , and months of age due to its proven efficacy [ ] . in , rotashield® was voluntarily withdrawn from the u.s. market due to an increased risk of intussusception within to days after the first dose in infants < months of age [ ] . due to the past association of intussusception and the earlier rv vaccine, large safety studies were performed both for rv and rv before market authorization [ ] . rotarix® vaccine were highly efficacious in protecting infants against severe rotavirus gastroenteritis [ , ] and were not associated with an increased risk of intussusception [ ] [ ] [ ] . the pentavalent rotavirus vaccine (rotateq®) was highly efficacious against severe rotavirus gastroenteritis and provided substantial protection against rotavirus gastroenteritis of any severity. a significantly increased risk of intussusception in vaccine recipients was not detected [ ] . in february , who reviewed global intussusception data and found that the risk of intussusception following current rotavirus vaccines remains small compared to the benefits of preventing the impact of severe diarrhea [ ] . at the end of there are four globally available who-prequalified oral vaccines (rotarix® and rotateq®, rotavac® and rotasiil®) [ ] , one rotavirus vaccine licensed in china (lanzhou lamb rv vaccine), one in vietnam (rotavin-m ), and there are several candidates in development [ ] . two rv vaccines, rotarix® and rotateq®, have been developed by glaxo smith kline and merck, respectively. rotarix® is a live attenuated monovalent vaccine derived from the most common human rv strain, g p [ ] . rota-teq® is a live attenuated pentavalent vaccine containing mono-reassortant strains with genes encoding the human g , g , g , g , and p [ ] protein in the genetic background of a bovine rv strains [ , ] . these vaccines are highly effective for the global prevention of severe diarrhea and are included in the nips or phased subnational introductions in countries by the end of [ , ] . rotavac® (bharat biotech international limited) is a monovalent human-bovine rv vaccine. the vaccine consists of the e rv strain, which is a naturally occurring reassortant strain g p [ ] , containing bovine rv gene p [ ] and human rv genes [ ] . rotavac® is the first to be introduced into a public vaccination program as of april when it was introduced in four states in india [ ] . rotasiil® is a live attenuated human-bovine reassortant pentavalent rv vaccine that contains genes encoding the vp of serotypes g , g , g , g , and g . in march and september rotavac® and rotasiil®, respectively achieved who prequalification. rotavac® has a vaccine efficacy of . % for severe rv diarrhea in india [ ] , while rotasiil® has efficacies of . to . % in india [ ] and niger respectively [ ] . rotasiil® can safely be delivered with decreased dependence on the availability of a cold chain [ ] . the lanzhou institute of biological products manufactures the lanzhou lamb rv vaccine (llr). it is a monovalent lamb vaccine strain g p [ ] , attenuated by cell passage [ ] , and was licensed in china in . when given to children between and months old, one dose of the llr vaccine conferred partial protection [ ] . vaccine effectiveness in children under years of age was recently estimated at % ( to %) against rv diarrhea and % ( to %) against moderate-tosevere rv diarrhea based on a large case-controlled study [ ] . rotavin-m vaccine is manufactured by the center for research and production of vaccines and biologicals and was licensed for use in vietnam in . the vaccine was derived from an attenuated strain, g p [ ] , isolated from a vietnamese child. a clinical trial found the vaccine to be safe and immunogenic in vietnamese infants [ ] . another candidate rv vaccine, rv -bb, was developed from a neonatal strain g p [ ] identified in australia, with ongoing early clinical studies conducted in new zealand and now underway in indonesia. it was also successfully implemented for vaccination of neonates [ ] . table describes the comparison of all oral rv vaccines developed and used so far. intussusception, neutralizing antibodies present in breast milk, as well as the lower vaccine effectiveness in less developed settings has stimulated interest in an alternative, parenteral approach to immunization [ ] [ ] [ ] [ ] . the inactivated rotavirus particles, protein sub-units or virus-like particles (vlps, structurally-similar to live virus) are being investigated as rotavirus vaccine candidates [ , , ] . three types of animal models have been used to evaluate protective efficacy of vlps: infection models (adult mice and rabbits), disease models (gnotobiotic piglets), and models evaluating passive protection (neonatal mice and calves) [ ] . gnotobiotic pig was used to assessed the immunogenicity and protection of a candidate inactivated rotavirus vaccine (irv), the human strain cdc- (g p[ ] [ ] and attenuated wa human rotavirus [atthrv] or non replicating wa / rotavirus-like particles [ ] . mice, rabbits, and piglets were used to evaluate the efficacy of vpl such as / -vlps (consisting of vp and vp ) [ ] and rf - / / -vlps [ ] . human clinical trial recently assessed in south african toddlers and infants was done for the subunit vaccine p -vp -p [ ] [ , ] . rv vaccination does not completely protect young children against infection, but it reduce the severity of rvge [ ] . rv vaccines are highly effective in preventing severe gastroenteritis in young children during the first years of their life, particularly in developed countries [ ] . the ses of a country seems to influence rv vaccine effectiveness [ ] . vaccination was predicted to prevent , , and % of severe rvge in high, middle, and low ses, respectively [ ] . analysis of the data for the asia region found median vaccine effectiveness of % in low child mortality countries, % in medium child mortality countries, and % in high child mortality countries [ ] . factors that might contribute to this phenomenon including gut microbiota, genetic factors, transplacental antibodies, enteric pathogens, and environmental enteropathy [ , ] . evidence suggests that vaccine efficacy may vary by setting, due to regional differences in circulating rv vaccine strains and reduced efficacy of oral vaccines in settings with a high prevalence of malnutrition and gastrointestinal infections [ ] . pooled efficacy estimate of rotarix® and rotateq® against severe rvge in industrialized countries is % during the first year of age and % during the second year. however, rv vaccine efficacy is much lower in countries where the mortality rate for children under years of age is high [ ] . the efficacy of rotarix® and rotateq® in the u.s. depends on the level of exposure during the rv season [ ] . it can be concluded that vaccine efficacy is affected by individual factors such as nutritional level, gut microbiota, genetic factor, transplacental antibody and environmental enteropathy and external factors including ses, circulating vaccine strain, childhood mortality rate, and rv season in each country. additionally, rv vaccination confers herd protection among infants and children under years old who had not been vaccinated [ ] [ ] [ ] . in developing countries with lower rv vaccine efficacy and coverage, indirect protection gain from herd immunity is more significant than in industrialized countries where vaccine efficacy and coverage exceed % [ ] . many countries in southeast asia have not implemented national rv vaccination programs including cambodia, indonesia, lao pdr, malaysia, myanmar, singapore, thailand and vietnam. one reason is because of uncertainties regarding the cost-effectiveness of incorporating rv vaccination into the nip. in addition, the vaccine's decreased efficacy in lic settings has discouraged its introduction. prevention of diarrhea in these countries has focused on patient treatment and the management of water quality, sanitation, and hygiene [ ] . in indonesia, the rv vaccine has been commercially available since . indonesia's national vaccine manufacturer, pt. bio farma, bandung, is developing an rv vaccine using g p [ ] strain in collaboration with the murdoch children's research institute in melbourne, australia [ ] . bio farma is currently driving clinical development, intending to introduce the vaccine into the indonesian nip by , and eventually develop a product for the global market [ ] . currently rotarix® and rotateq® are commercially available in malaysia through private health providers [ ] . however, rv vaccine is not included in malaysia nip because it is not considered permissible under islamic shariah law (halal) [ ] . the current oral rotavirus vaccines use porcine trypsin in the manufacturing process [ ] . there are also concerns about competing public health priorities and price [ ] . the health ministry recently said the rv vaccine would be included in nip if the associated mortality rate for children aged and below exceeded %. however, the childhood mortality rates in malaysia was . % in and . % in [ ] . in myanmar and lao pdr where individual incomes are relatively low, international assistance will support for the rv vaccine introduction in . the total amount of gavi support for myanmar is $ , , [ ] . lao pdr is also planning rv vaccine introductions into the nip in near future and assistance will be provided the asian development bank and gavi through an accelerated transition program [ ] . in july , the philippines became the first asian country to introduce rv vaccines into its nip. the philippines has initially focus on immunizing children living in the poorest communities, which have the highest child morbidity and mortality rates from the diarrheal disease [ ] . the target population was identified by the department of social welfare and development, but there were challenges with nationwide vaccine distribution. in , vaccine introduction was limited to the caraga region, where it was co-administered with oral polio vaccine and the pentavalent vaccine. by , vaccine coverage was close to % in the province of agusan del sur within this region but subsequently decreased due to a supply shortage [ ] . in agusan del sur, the rv vaccine became available to the poorest quintile in september ; in january , availability was expanded to all age-eligible children in two municipalities, san francisco and prosperidad; it was available to the entire province in july . rv vaccine introduction was associated with a substantial decline in diarrheal hospitalizations and outpatient consultations for diarrhea in agusan del sur, philippines [ ] . two live-attenuated, orally administrable rv vaccines rotarix® and rotateq® were licensed in singapore in october and july , respectively [ ] . to date, rv vaccination is optional in singapore [ ] . the national vaccine committee of thailand considered the introduction of an rv vaccine in in sukhothai and petchabun. sukhothai province began a routine immunization program with an rv vaccine in october . evaluation of the first introduction was completed in and concluded that rv vaccine was highly effective in preventing diarrheal hospitalizations and conferring herd protection among older children who had not been vaccinated [ ] . therefore, universal rv vaccination for infants has been implemented in thailand since january . vietnam is located in a region of high rv infection incidence and eligible for financial support to introduce vaccines into the expanded program of immunization (epi) from the gavi [ ] . in , the local vaccine manufacturer polyvac licensed rotavin-m , which is based on an attenuated g p [ ] strain. rotavin-m will be offered to children less than year through a two-dose schedule, vaccinating infants at and months [ ] . rotarix® and rotateq® are also licensed in vietnam and are available in the private sector, with approximately , doses imported since . that same year, the government approved the introduction of rv vaccination into vietnam's nip by september with gavi support. in , the national government will pay for % of the vaccine cost, while gavi will cover the remaining % and all operational costs. by , all costs will be covered by the government [ ] . in lmics, understanding the short-and long-term impact of intervention adoption on national budgets is critical for ensuring program sustainability [ ] . both budget impact and cost effectiveness are key criteria, among others, for policy makers deciding how to allocate limited resources [ ] .. vaccination would be considered a worthwhile investment for improving general childhood development and health levels in most lic. the highest reduction in burden would be achieved in countries with a high disease burden (≥ rv deaths per , children under years old), but a similar reduction would be achieved in countries with a medium burden ( - rv deaths per , children under years old) because disease burden reduction also depends heavily on population size and countryspecific vaccine efficacy adjusted for local rv serotype distributions [ ] . for gavi non-eligible countries, the price for rotarix® is $ . - . , and for rotateq® is $ . - . [ ] . for gavi-eligible countries, the price per dose will depend on the country's gross national income per capita averaged over the previous years. as such indonesia, malaysia, the philippines, singapore, and thailand are fully not eligible for gavi vaccine prices and will have to rely on self-financing [ ] . for lic in asia, introducing vaccines would halve rv-related deaths and medical visits, leading to significant cost reductions [ ] . who-choice (choosing interventions that are cost-effective) uses the gdp as an indicator to develop the following widely referenced categories of cost-effectiveness. disability-adjusted life years (dalys) averted is a widely used indicator that allows easy comparison with a 'no vaccination' strategy and with others public health interventions. the ratio between costs per daly averted and gdp per capita less than one is defined as highly cost effective. the ratio between to < and ≥ are defined as cost effective and not cost effective, respectively [ ] . the lowest and highest gdp values were in myanmar with $ and malaysia with $ , , respectively [ ] . figure shows the comparison between costs per daly averted with each country's gdp in . we excluded the philippines because the rv vaccine already included in the country's nip and singapore because singapore is a high-income country. according to categories of cost-effectiveness, rv vaccine introduction into southeast asia countries is highly cost-effective because the ratio between costs per daly averted and gdp per capita it is less than one. according to - rv surveillance data for southeast asia, . % of all diarrheal cases were causes by rv. acute diarrheal disease caused by rv is still a major cause of morbidity and mortality in children under years old in southeast asia. among all assessed fig. comparison between cost per daly averted and gdp per capita in southeast asia. introducing the rotavirus vaccine in southeast asia is highly cost-effective because the ratio is less than one [ , , , ] countries, the most predominant genotype distribution of rv changed from g p [ ] and g p [ ] into the rare and unusual genotypes g p [ ] , g p [ ] , and g p [ ] . although the predominant rv strain has been changed, but the seasonality of rv infection remains unchanged. continuous surveillance is necessary to determine whether they are regional genotype differences. epidemiological data on rv prevalence will greatly facilitate vaccine development. in the mean time, the development of new vaccines will be needed if rvs are able to evade current vaccine immunity. more effective vaccines may also further decrease rv infection in children in lic and lmic, where currently available vaccines provide moderate efficacy. rv vaccine efficacy is affected by individual factors and external factors. although most countries in southeast asia have not yet introduced national rv vaccination programs, such introduction is projected to be highly cost-effective because the ratio between costs per daly averted and gdp per capita it is less than one. authors' contributions fbl drafted the manuscript. fbl and yp wrote the article and prepared the figures. sv, nw, and yp revised and finished the final version of the manuscript. all authors read and approved the final manuscript. fajar budi lestari is a doctoral student at the inter-department of biomedical science, faculty of graduate school, at chulalongkorn university, in bangkok. her primary research interests are zoonotic diseases and molecular epidemiology and evolution of rotavirus in humans and animals. availability of data and materials not applicable. ethics approval and consent to participate not applicable. not applicable. rotaviruses in human and veterinary medicine epizootic diarrhea of infant mice: indentification of the etiologic agent calf diarrhea (scours): reproduced with a virus from a field outbreak simian virus sa and the related o agent virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis detection of a new virus by electron microscopy of faecal extracts from children with acute gastroenteritis letter: virus particles in gastroenteritis relation between viruses from acute gastroenteritis of children and newborn calves fields virology molecular interactions in rotavirus assembly and uncoating seen by highresolution cryo-em rotavirus architecture at subnanometer resolution atomic model of an infectious rotavirus particle genetic and antigenic diversity of human rotaviruses: potential impact on vaccination programs full genome-based classification of rotaviruses reveals a common origin between human wa-like and porcine rotavirus strains and human ds- -like and bovine rotavirus strains uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg) genotype constellation and evolution of group a rotaviruses infecting humans vp -sequence-based cutoff values as a criterion for rotavirus species demarcation candidate new rotavirus species in sheltered dogs candidate new rotavirus species in schreiber's bats, serbia unbiased whole-genome deep sequencing of human and porcine stool samples reveals circulation of multiple groups of rotaviruses and a putative zoonotic infection complete molecular genome analyses of equine rotavirus a strains from different continents reveal several novel genotypes and a largely conserved genotype constellation rabbit colony infected with a bovine-like g p[ ] rotavirus strain wholegenome characterization of a peruvian alpaca rotavirus isolate expressing a novel vp genotype vipr: an open bioinformatics database and analysis resource for virology research evidence for presumable feline origin of sporadic g p[ ] rotaviruses in humans complete genomic sequence analyses of the first group a giraffe rotavirus reveals close evolutionary relationship with rotaviruses infecting other members of the artiodactyla simian rotaviruses possess divergent gene constellations that originated from interspecies transmission and reassortment detection of substantial porcine group b rotavirus genetic diversity in the united states, resulting in a modified classification proposal for g genotypes evolution of rotavirus c in humans and several domestic animal species detection and characterization of group c rotaviruses in asymptomatic piglets in ireland avian rotavirus enteritis -an updated review definition of two new groups of atypical rotaviruses genomic sequence of the first porcine rotavirus group h strain in the united states cameroonian fruit bats harbor divergent viruses, including rotavirus h, bastroviruses, and picobirnaviruses using an alternative genetic code rotavirus i in feces of a cat with diarrhea rotavirus vaccination and the global burden of rotavirus diarrhea among children younger than years whocoordinated global rotavirus surveillance network. estimate of worldwide rotavirus-associated mortality in children younger than years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis rotavirus and severe childhood diarrhea noroviruses: the most common pediatric viral enteric pathogen at a large university hospital after introduction of rotavirus vaccination norovirus and medically attended gastroenteritis in u.s. children major reduction of rotavirus, but not norovirus, gastroenteritis in children seen in hospital after the introduction of rotateq vaccine into the national immunization programme in finland world health organization-coordinated global rotavirus surveillance network. global, regional, and national estimates of rotavirus mortality in children < years of age estimates of the global, regional, and national morbidity, mortality, and aetiologies of diarrhoea in countries: a systematic analysis for the global burden of disease study rotavirus vaccine efficacy: current status and areas for improvement pediatric hospitalizations attributable to rotavirus gastroenteritis among cambodian children: seven years of active surveillance detection of group a rotavirus strains circulating among children with acute diarrhea in indonesia identification of rotavirus strains causing diarrhoea in children under five years of age in yogyakarta, indonesia risk factors of rotavirus diarrhea in hospitalized children in sanglah hospital, denpasar: a prospective cohort study association between severe dehydration in rotavirus diarrhea and exclusive breastfeeding among infants at dr. hasan sadikin general hospital prevalence and clinical characteristics of rotavirus diarrhea in genotyping and clinical factors in pediatric diarrhea caused by rotaviruses: one-year surveillance in surabaya identification of the p genotypes of rotavirus in children with acute diarrhea in pekanbaru, indonesia diarrhea among hospitalized children under five: a call for inclusion of rotavirus vaccine to the national immunization program in indonesia equine-like g rotavirus strains as predominant strains among children in indonesia in - molecular characterisation of rotavirus strains detected during a clinical trial of the human neonatal rotavirus vaccine (rv -bb) in indonesia. vaccine molecular epidemiology and clinical features of rotavirus infection among pediatric patients in east java, indonesia during - : dynamic changes in rotavirus genotypes from equine-like g to typical human g /g rotavirus diarrhea in hospitalized children under years of age in vientiane rotavirus genotypes in malaysia and universal rotavirus vaccination sentinel surveillance for rotavirus in children < years of age admitted for diarrheal illness to yangon children's hospital first detection of ds- -like g p[ ] human rotavirus strains from children with diarrhoea in the philippines molecular characterization of rotavirus diarrhea among children aged under five years in the philippines is your drinkingwater safe? a rotavirus outbreak linked to water refilling stations in the philippines a hospitalbased surveillance of rotavirus gastroenteritis in children < years of age in singapore rotavirus vaccine rix efficacy sustained during the third year of life: a randomized clinical trial in an asian population prevalence and phylogenetic analysis of rotavirus genotypes in thailand between prevalence and genotypic distribution of rotavirus in thailand: a multicenter study prevalence of group a genotype human rotavirus among children with dirarrhea in thailand increasing predominance of g p[ ] species a rotaviruses in children admitted to hospital with acute gastroenteritis in thailand the prevalence and genotype diversity of human rotavirus a circulating in thailand complete genome analysis of a rare g p[ ] rotavirus isolated in thailand in reveals a prototype strain of ds- -like constellation genotype distribution and phylogenetic analysis of rotaviruses in thailand and emergence of uncommon genotypes predominant prevalence of human rotaviruses with the g p[ ] and g p[ ] genotypes with a short rna profile in and in sukhothai and phetchaboon provinces high prevalence of equine-like g p[ ] rotavirus in children and adults with acute gastroenteritis in thailand emergence of g p[ ] rotaviruses in children with acute gastroenteritis in thailand novel porcine-like human g p[ ] rotavirus identified in hospitalized paediatric diarrhoea patients in ho chi minh city epidemiology of acute diarrhea caused by rotavirus in sentinel surveillance sites of vietnam gdp per capita rotaviruses: is their surveillance needed? vaccine serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs distribution of rotavirus strains and strain-specific effectiveness of the rotavirus vaccine after its introduction: a systematic review and metaanalysis reassortment of human rotavirus gene segments into g p[ ] rotavirus strain over a four year period: - disease prevention through immunization in the western pacific region molecular epidemiology of rotaviruses in the south-east asian region from rotavirus gastroenteritis and weather rotavirus and other enteropathogens in childhood acute diarrhoea: a study of two centres in malaysia rotavirus diarrhoea among children aged less than years at mahosot hospital, vientiane, lao pdr seasonality of rotavirus disease in the tropics: a systematic review and meta-analysis the seasonality of diarrheal pathogens: a retrospective study of seven sites over three years seasonality of rotavirus in south asia: a meta-analysis approach assessing associations with temperature, precipitation, and vegetation index survival and vehicular spread of human rotaviruses: possible relation to seasonality of outbreaks global burden of childhood pneumonia and diarrhoea rotavirus vaccines: an update rotavirus immunization coverage global rotavirus vaccine introductions and coverage protection studies in colostrum-deprived piglets of a bovine rotavirus vaccine candidate using human rotavirus strains for challenge pérez-schael i. efficacy of a quadrivalent rhesus rotavirus-based human rotavirus vaccine aimed at preventing severe rotavirus diarrhea in infants and young children rotavirus vaccines: an overview rotavirus intussusception investigation team. intussusception among infants given an oral rotavirus vaccine rotavirus vaccines: a story of success efficacy, immunogenicity and safety of a human rotavirus vaccine rix in singaporean infants intussusception and monovalent rotavirus vaccination in singapore: self-controlled case series and risk-benefit study safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis an update of paediatric intussusception incidence in singapore: - , years of intussusception surveillance safety and efficacy of a pentavalent human-bovine (wc ) reassortant rotavirus vaccine update on intussusception following rotavirus vaccine administration: world health organization reevaluating the potential impact and cost-effectiveness of rotavirus vaccination in gavi countries: a modelling study improving rotavirus vaccine coverage: can newer-generation and locally produced vaccines help? world health organization. rotavirus immunization coverage efficacy of a monovalent human-bovine ( e) rotavirus vaccine in indian infants: a randomised, double-blind, placebo-controlled trial view-hub report: global vaccine introduction and implementation a randomized phase iii clinical trial to assess the efficacy of a bovine-human reassortant pentavalent rotavirus vaccine in indian infants efficacy of a low-cost, heat-stable oral rotavirus vaccine in niger stability of heat stable, live attenuated rotavirus vaccine (rotasiil®) selection and characterization of strain llr- for oral rotavirus live vaccine rotavirus gastroenteritis infection among children vaccinated and unvaccinated with rotavirus vaccine in southern china: a population-based assessment effectiveness of the live attenuated rotavirus vaccine produced by a domestic manufacturer in china studied using a population-based case-control design rotavin-m vaccine trial group. a dose-escalation safety and immunogenicity study of a new live attenuated human rotavirus vaccine (rotavin-m ) in vietnamese children human neonatal rotavirus vaccine (rv -bb) to target rotavirus from birth current and upcomming rotavirus vaccine decreased performance of live attenuated, oral rotavirus vaccines in low-income settings: causes and contributing factors correlates of protection against human rotavirus disease and the factors influencing protection in low-income settings the rotavirus vaccine development pipeline understanding reduced rotavirus vaccine efficacy in low socio-economic settings next generation rotavirus vaccine. in: who product development for vaccines advisory committee current and new rotavirus vaccines host, viral, and vaccine factors that determine protective efficacy induced by rotavirus and virus-like particles (vlps) inactivated rotavirus vaccine induces protective immunity in gnotobiotic piglets an oral versus intranasal prime/ boost regimen using attenuated human rotavirus or vp and vp virus-like particles with immunostimulating complexes influences protection and antibody-secreting cell responses to rotavirus in a neonatal gnotobiotic pig model parenteral administration of rf - / / rotavirus-like particles in a one-dose regimen induce protective immunity in mice safety and immunogenicity of a parenterally administered rotavirus vp subunit vaccine in healthy adults safety and immunogenicity of a parenteral p -vp -p[ ] subunit rotavirus vaccine in toddlers and infants in south africa: a randomised, double-blind, placebocontrolled trial cost-effectiveness of rotavirus vaccination in vietnam cost-effectiveness of rotavirus immunization in indonesia: taking breastfeeding patterns into account effectiveness of rotavirus vaccine in preventing severe gastroenteritis in young children according to socioeconomic status rotavirus vaccines: effectiveness, safety, and future directions differences of rotavirus vaccine effectiveness by country: likely causes and contributing factors causes of impaired oral vaccine efficacy in developing countries the effect of rotavirus vaccine on diarrhoea mortality real-world impact of rotavirus vaccination reduced rotavirus vaccine effectiveness among children born during the rotavirus season: a pooled analysis of case-control studies from the americas evaluating the first introduction of rotavirus vaccine in thailand: moving from evidence to policy infant rotavirus vaccination may provide indirect protection to older children and adults in the united states estimating the herd immunity effect of rotavirus vaccine household catastrophic healthcare expenditure and impoverishment due to rotavirus gastroenteritis requiring hospitalization in malaysia religious and community leaders' acceptance of rotavirus vaccine introduction in yogyakarta, indonesia: a qualitative study rotavirus: govt urged to protect kids with vaccine rotavirus disease and vaccines in asia effectiveness of monovalent rotavirus vaccine in the philippines health economics of rotavirus immunization in vietnam: potentials for favorable costeffectiveness in developing countries the decision making process on new vaccines introduction in south africa capturing budget impact considerations within economic evaluations: a systematic review of economic evaluations of rotavirus vaccine in low-and middle-income countries and a proposed assessment framework health and economic impact of rotavirus vaccination in gavi-eligible countries costs of vaccine programs across low-and middle-income countries rotavirus: common, severe, devastating, preventable economic costs of rotavirus gastroenteritis and cost-effectiveness of vaccination in developing countries guidelines for estimating costs of introducing new vaccines into the national immunization system economic evaluation of human rotavirus vaccine in thailand publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no competing interests. key: cord- - krujv m authors: bremer, paul t.; schlosburg, joel e.; lively, jenny m.; janda, kim d. title: injection route and tlr agonist addition significantly impact heroin vaccine efficacy date: - - journal: mol pharm doi: . /mp w sha: doc_id: cord_uid: krujv m [image: see text] active immunization is an effective means of blocking the pharmacodynamic effects of drugs and holds promise as a treatment for heroin addiction. previously, we demonstrated the efficacy of our first-generation vaccine in blocking heroin self-administration in rats, however, many vaccine components can be modified to further improve performance. herein we examine the effects of varying heroin vaccine injection route and adjuvant formulation. mice immunized via subcutaneous (sc) injection exhibited inferior anti-heroin titers compared to intraperitoneal (ip) and sc/ip coadministration injection routes. addition of tlr agonist cytosine-guanine oligodeoxynucleotide (cpg odn ) to the original alum adjuvant elicited superior antibody titers and opioid affinities compared to alum alone. to thoroughly assess vaccine efficacy, full dose–response curves were generated for heroin-induced analgesia in both hot plate and tail immersion tests. mice treated with cpg odn exhibited greatly shifted dose–response curves ( – -fold vs unvaccinated controls) while non-cpg odn vaccine groups did not exhibit the same robust effect ( – -fold shift for ip and combo, – -fold shift for sc). our results suggest that cpg odn is a highly potent adjuvant, and injection routes should be considered for development of small molecule–protein conjugate vaccines. lastly, this study has established a new standard for assessing drugs of abuse vaccines, wherein a full dose–response curve should be performed in an appropriate behavioral task. heroin is a highly addictive semisynthetic opioid derived from the diacetylation of morphine. indeed, heroin abuse is a significant problem that incurs large social and economic costs worldwide. , alarmingly, the number of heroin users in the u.s. has grown by % from to . furthermore, since the number of deaths due to heroin overdose has also increased. due to the severity of heroin addiction, there is a dire need for anti-heroin addiction treatments that are more effective than currently available pharmacological agents (e.g., methadone, buprenorphine, and naltrexone). the first report of a working heroin vaccine was disclosed in , however, further research on heroin immunopharmacotherapy has only been conducted in the past decade. , previously, we reported the design of a heroin−klh (keyhole limpet hemocyanin) immunoconjugate ( figure ) that showed ample promise in combination with alum adjuvant as a vaccine against heroin addiction. as a testament to the vaccine's efficacy, immunization of heroin-dependent rats prevented relapse to compulsive intake of heroin in self-administration models. herein, we sought to further improve our vaccine's performance through injection route and adjuvant exploration. vaccine administration route may influence immune response, due to a difference in physiological environments of the subcutaneous tissue versus the peritoneal cavity. interestingly, no study to date has investigated injection route of small molecule conjugate vaccines. in addition to administration route, adjuvants can also impact vaccine performance. in this capacity, tlr agonists could play an important role in vaccine design with their ability to modulate immune responses. for example, cpg odn acts as a pathogen associated molecular pattern (pamp) to stimulate the innate immune receptor tlr . a specific cpg odn, cpg (figure ), is a member of "b-class" odns which activate b-cell immune responses. impressively, vaccine trials engaging a specific cpg odn sequence (# ) in combination with alum have demonstrated its ability to safely enhance igg antibody titers against the target antigen in mice. and lymphoma. results from most clinical studies demonstrate cpg odn efficacy in enhancing immunity to the target antigen while displaying a favorable safety profile. additionally, this adjuvant is inexpensive, stable, readily obtainable and can be easily manipulated for vaccine formulation. cpg odn therefore holds promise as an adjunct to our heroin vaccine "cocktail" as a means of further potentiating opioid-neutralizing antibodies. in the research we disclose herein, both injection route and tlr agonist cpg odn significantly affected antibody titer levels and opiate affinity, translating to marked differences in mitigation of heroin-induced analgesia. full heroin dose− response curves were generated in both hot-plate and tail flick tests to clearly demonstrate differences in vaccine efficacy. our results have wide-reaching applicability for both refining and evaluating drugs of abuse vaccines. all studies were performed in compliance with the scripps insitutional animal care and use committee and were in concordance with the national institutes of health guide for the care and use of laboratory animals. − week old male swiss webster mice (taconic) were immunized ip, sc, or ip/sc with μg of heroin−klh conjugate on days , , and . the mice were bled on days and . since titers were much higher on day than , day sera were used for the reported immunochemical assays. heroin−klh ( figure ) was prepared via thiol−maleimide coupling. a heroin−bsa conjugate was prepared as a surrogate to quantify hapten loading by maldi-tof ms ( copies per bsa, supporting information) as performed previously. a single batch of the conjugate was used for all immunizations. each vaccine was prepared by shaking a : v/v mixture of her−klh and imject alum (thermo pierce) for min, and the final volume injected was μl per mouse. in the oligonucleotide adjuvant group, phosphorothioated cpg odn ( figure , eurofins mwg operon) was adsorbed to alum alongside her−klh and each mouse received μg of the odn per injection (ip). elisa. for all elisas, plates were coated with ng/ml heroin−bsa conjugate at °c overnight. a ph . pbs buffer was used throughout the assay. titers are reported at od . overall titers were determined with a igg/igm/iga secondary (southernbiotech) while igg titers were determined with secondaries specific for igg or igg a (invitrogen). for competitive elisas the limiting dilution was found by normalizing titer curves for each treatment group (pooled sera) and choosing the dilution that produced % of the maximum absorbance. competition with heroin or its primary metabolites, -acetlymorphine ( am) or morphine (cambridge isotopes), was run in triplicate for each group. dmso was used to solubilize the opioids with a final assay dmso concentration of . %. generally, competitive elisa ic values underestimate actual k d by a factor of . radioimmunoassay for determining k d was not possible because of the instability of heroin. hot plate and tail immersion antinociceptive testing. at day , mice were tested for spinal (tail immersion) and supraspinal (hot plate) antinociceptive responses, both set to °c. the hot plate test was measured by placing the mouse in an acrylic cylinder ( cm diameter × cm) and timing latency to perform one of the following nociceptive responses: licking of hindpaw, shaking of hindpaw, or jumping. mice were removed from the surface immediately following response, with typical baseline latency between and s, and a s cutoff to prevent tissue damage. the tail immersion test was administered by lightly restraining mice in a small pouch constructed from absorbent laboratory underpads and dipping cm of the tip of the tail into a heated water bath, with the time to withdrawal timed. typical baseline response was − s, and a cutoff of s was imposed to prevent tissue damage. baselines for hot plate followed by tail flick were performed, and then drug was immediately administered. following a set time ( min for heroin, min for morphine/oxycodone) the tests were repeated, and if no full analgesic response was noted, the animals continued to receive further cumulative drug injections and repeated testing until cutoff times were reached. drugs were tested in a counterbalanced manner with at least days between drug cumulative-dosing regimens. computational analysis. computational and statistical analysis was performed in graphpad prism. all values are reported as means ± sem. titers were determined by applying the one site fit logic nonlinear regression. normalization was performed by fitting titer curves with the one site fit logic regression and then by expressing values as a percentage of the maximum absorbance. competition curves generated at the % limiting dilution were first normalized and then fitted with the log(inhibitor) vs normalized response − variable slope regression to calculate ic values. titers were compared via one-way anova with fisher's lsd post hoc comparisons. antinociceptive data was transformed from time to % maximum possible effect (% mpe), which is calculated as % mpe = (test − baseline)/(cutoff − baseline) × . this data was then fit using a log(agonist) vs normalized response nonlinear regression. these produced ed values for each drug under each pain test for the individual treatment groups, allowing calculation of potency ratios. differences in potency were determined by one-way anova followed by tukey's post hoc test. antibody titers and opioid affinities. mice were immunized with our first generation heroin vaccine (heroin− klh conjugate formulated with alum adjuvant), and antibody titers were measured by elisa against heroin−bsa conjugate. a significant effect of the vaccines on antibody titers was observed [igg , f , = . , p < . ; igg a, f , = . , p < . ]. ip and ip/sc administration of the heroin vaccine gave nearly identical titers, which were -fold higher compared to sc injection (table ) . on top of a significant effect of ip administration routes compared to sc, the addition of cpg odn further increased titers -fold compared to alum alone (p < . , fisher's tests). based upon competitive elisa data, the ip group exhibited the poorest overall opioid affinities, although the ip/sc group am affinity was weakest of all the groups (table ). in contrast, affinities for heroin, am, and morphine were − fold better in the sc group. when cpg odn was added to our existing formulation, affinities improved -fold for heroin, -fold for am, and -fold for morphine. statistical comparisons were not performed because affinities were determined from pooled sera. to assess th and th -associated humoral responses, we compared titers of anti-heroin igg a and igg , respectively. as shown previously, our heroin vaccine exhibits an exclusively th immune response. however, addition of cpg odn in the current study elicited a robust th humoral response as indicated by the -fold increase in igg a titers (table ; p < . ). furthermore, cpg odn enhanced the th -associated humoral response by boosting igg titers -fold over alum alone (p < . ). antinociceptive testing. to assess vaccine performance in blocking the analgesic effects of heroin, vaccinated animals were subjected to two antinociceptive tests, hot plate and tail immersion. full dose−response curves and corresponding ed values were generated to thoroughly test vaccine efficacy in a wide range of heroin doses, then repeated for selectivity against morphine and oxycodone. results in the behavioral tests generally paralleled elisa results. the vaccines showed varying degrees of shifting the heroin antinociceptive dose−response to the right, with tail immersion being more responsive to the vaccines (figure ) . a significant effect of vaccination was observed based on the derived estimates of heroin's table . (table ; tukey's test). ip and ip/sc performances were fairly similar to one another and gave ed values that were − -fold better than those of naive animals (p < . ). addition of cpg odn increased ed values by -fold in hot plate (p < . ) and − -fold in tail immersion (p < . ) over alum alone. similar antinociceptive testing was conducted for morphine as that for heroin, and a significant vaccination anova was determined [hot plate, f , = . , p < . ; tail immersion, f , = . , p < . ]. similar to previous findings, our original vaccine formulation is primarily more effective on heroin than morphine. no significant shift of morphine potency was found compared to naive, regardless of route of administration (table s in the supporting information). however, the addition of the cpg odn provided sufficient boosts in titer and affinity to generate a significant reduction in morphine potency, though only in the tail immersion test (p < . ). as expected, and in confirmation of the selectivity of the vaccine for heroin and its primary metabolites, no heroin vaccine treatment provided protection against oxycodone, a structurally similar opioid (table s in the supporting information) . the current study highlights the impact of varying the injection route of our first generation heroin vaccine, along with enhancing immunogenicity by addition of the cpg odn adjuvant. few studies have directly compared injection routes for drugs of abuse vaccines with the exception of one study that found intradermal (id) nicotine vaccine delivery to be effective compared to intramuscular (im). however, id delivery is generally not compatible with alum adjuvant: a necessary component of our heroin vaccine. although the im route was not investigated in the current study, previous studies have shown that im is comparable to sc in terms of vaccine immunogenicity. alum has been shown to stimulate dendritic cells (dcs) both intraperitoneally and subcutaneously through the nalp inflammasome, enhancing th immune responses to the antigen. although a th response was observed for both ip and sc administration routes, titers were -fold higher for ip. the difference in physiological environments between the subcutaneous tissue and peritoneal cavity may contribute to the observed discrepancy in ip versus sc immunization. the heroin−klh immunogen in the peritoneal fluid can drain directly to lymph nodes for immune recognition, however, the large particle size of the immunogen when formulated with alum prevents immediate drainage out of subcutaneous tissue. consequently, the immunogen is taken up by dcs and transported to lymph nodes to evoke an adaptive immune response. according to our study, immunogenicity of our heroin−klh immunogen is best achieved via intraperitoneal administration possibly due to more immediate drainage to lymph nodes. in sum, these findings stress the need for testing vaccination route in studying drugs of abuse vaccines, which could be especially important at the clinical level. previously, we reported reduced immunogenicity of our heroin vaccine in the presence of cpg odn; however, these results were derived from an oligonucleotide with native phosphodiester linkages. in the current study we employed nuclease-resistant phosphorothioate linkages between base pairs to enhance in vivo stability. as a result, we obtained drastically increased titers and opioid affinities similar to previous studies that showed that cpg odn bolsters antinicotine vaccine efficacy. , interestingly, we observed a marked increase not only in igg a titers but also in igg . in a previous study, only th associated antibodies were boosted by cpg odn . , the observed dually enhanced th and th humoral responses highlight a unique compatibility between this vaccine "cocktail": heroin−klh conjugate, alum and cpg odn . comparing titers, opioid affinities, and pain curves in the current study reveals that immunochemical assay results do not always present a clear indication of vaccine efficacy (especially vaccines against heroin that target three different opioids). while the sc immunized group exhibited higher opioid affinities and lower titers, analgesia testing demonstrated that the efficacy of the sc group was inferior to all other groups. although the non-cpg groups appeared different in terms of titers and affinities, the hot plate test revealed no differences between the groups. the lack of efficacy in the hot plate test compared to tail immersion suggests a differential ability to block spinal reflexive analgesia, largely mediated by spinal μ-opioid receptors, versus more complex pain-related behavior of hot plate that involves peripheral, spinal, and supraspinal processing. the hot plate test has also been found to be less sensitive compared to tail flick in reducing morphine-induced analgesic properties in μ-opioid receptor deficient mice. it should also be noted that, compared with previous titer and hot plate evaluation in rats using the same vaccination procedure, mice exhibit lower titer responses and less substantial shifts in heroin hot plate analgesia. the large enhancement in vaccine performance from cpg odn suggests that, when assessing a vaccine, > -fold greater antidrug titers and affinities must be achieved to also present improved efficacy at the behavioral level. moreover, previous evidence demonstrates that weak blockade by drug-directed vaccines tends to actually promote increased drug seeking, , often leading to subjects adjusting dosing in order to supersede the vaccine. this necessitates rigorous behavioral testing over a wide range of doses to provide an accurate indication of vaccine efficacy in reducing drug psychoactivity. we have shown that the enhanced antidrug titers and opioid affinities from cpg odn translate directly to reduced pharmacodynamics of the drug; in our case heroin-induced latency to nociception (analgesia) was greatly diminished in hot plate and tail immersion tests. previous studies have shown that when cpg odn is used as an adjuvant in nicotine vaccines, a greater degree of drug immunoantagonism is achieved; antibodies in vaccinated animals bind a greater amount of nicotine in the blood leading to a lower concentration of nicotine in the brain. , however, it is not clear to what degree this result would manifest behaviorally. analgesia testing is a robust method of measuring to what degree heroin is generating psychoactive effects in an animal and is therefore highly relevant to screening vaccines for immunotherapeutic antagonism. we demonstrated previously that blunted analgesic responses to heroin in mice vaccinated with our first generation heroin vaccine corresponded to a blockade of compulsive heroin intake in rats. , the over -fold greater ed for heroin analgesia in the cpg odn vaccine group over the first generation vaccine group (alum alone) implies that cpg odn addition to the vaccine lends a significant increase in protection from heroin addiction. in extrapolating the tail immersion test results to humans, an kg adult when immunized with the standard vaccine would have to administer mg heroin to experience an effect compared to mg in an unvaccinated adult. when cpg odn is added to the vaccine, an adult would have to administer mg heroin to experience an equivalent effect. at this large a dose required for psychoactivity, maintaining a heroin addiction would likely become impractical both behaviorally and economically. overall, the findings from our study are expected to facilitate the development of vaccines not only against opioids but also against other drugs of abuse. tables s and s , tabulating the antinociceptive potency of morphine and oxycodone, respectively, for the various vaccinated treatments. maldi-tof spectra of heroin−bsa conjugates. this material is available free of charge via the internet at http://pubs.acs.org. the economic costs of heroin addiction in the united states the economic cost of heroin dependency and quality of life among heroin users in taiwan substance abuse and mental health services administration. results from the national survey on drug use and health: summary of national findings; nsduh series h- changes in heroin self-administration by a rhesus monkey after morphine immunisation a novel bivalent morphine/heroin vaccine that prevents relapse to heroin addiction in rodents co-administration of morphine and oxycodone vaccines reduces the distribution of -monoacetylmorphine and oxycodone to brain in rats a vaccine strategy that induces protective immunity against heroin dynamic vaccine blocks relapse to compulsive intake of heroin a toll-like receptor recognizes bacterial dna mechanism and function of a newly identified cpg dna motif in human primary b cells cpg dna induces stronger immune responses with less toxicity than other adjuvants cpg dna can induce strong th humoral and cellmediated immune responses against hepatitis b surface antigen in young mice cpg odn allows lower dose of antigen against hepatitis b surface antigen in balb//c mice immunogenicity of a receptor-binding domain of sars coronavirus spike protein in mice: implications for a subunit vaccine cpg , an immunostimulatory tlr agonist oligodeoxynucleotide, as adjuvant to engerix-b hbv vaccine in healthy adults: a double-blind phase i/ii study phase trial of ama -c / alhydrogel plus cpg : an asexual blood-stage vaccine for plasmodium falciparum malaria improving the immunogenicity of pneumococcal conjugate vaccine in hiv-infected adults with a toll-like receptor agonist adjuvant: a randomized, controlled trial vaccination with ny-eso- protein and cpg in montanide induces integrated antibody/th responses and cd t cells through crosspriming in situ vaccination with a tlr agonist induces systemic lymphoma regression: a phase i/ii study investigating the effects of a hydrolytically stable hapten and a th adjuvant on heroin vaccine performance evaluation of the endogenous cannabinoid system in mediating the behavioral effects of dipyrone (metamizol) in mice high immunogenicity of nicotine vaccines obtained by intradermal delivery with safe adjuvants are there differences in immunogenicity and safety of vaccines according to the injection method? crucial role for the nalp inflammasome in the immunostimulatory properties of aluminium adjuvants patterns of lymphatic drainage in the adult laboratory rat mechanism of immunopotentiation by aluminum-containing adjuvants elucidated by the relationship between antigen retention at the inoculation site and the immune response vaccine delivery: a matter of size, geometry, kinetics and molecular patterns enhancing immunogenicity of a ′aminomethylnicotine-dt-conjugate anti-nicotine vaccine with cpg adjuvant in mice and non-human primates cpg dna is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis b surface antigen opiate receptor knockout mice define mu receptor roles in endogenous nociceptive responses and morphine-induced analgesia a critical evaluation of a nicotine vaccine within a self-administration behavioral model immune responses to methamphetamine by active immunization with peptide-based, molecular adjuvant-containing vaccines key: cord- -haf y authors: offit, paul a.; destefano, frank title: vaccine safety date: - - journal: vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: haf y nan during the past years, pharmaceutical companies have made vaccines against pertussis, polio, measles, rubella, and haemophilus influenzae type b (hib), among others ( table - ) . as a consequence, the number of children in the united states killed by pertussis decreased from , each year in the early th century to fewer than ; the number paralyzed by polio from , to ; the number killed by measles from , to ; the number with severe birth defects caused by rubella from , to ; and the number with meningitis and bloodstream infections caused by hib from , to fewer than . vaccines have been among the most powerful forces in determining how long we live. but the landscape of vaccines is also littered with tragedy: in the late s, starting with louis pasteur, scientists made rabies vaccines using cells from nervous tissue (such as animal brains and spinal cords); the vaccine prevented a uniformly fatal infection, but the rabies vaccine also caused seizures, paralysis, and coma in as many as of every people who used it. [ ] [ ] [ ] [ ] in , the military injected hundreds of thousands of american servicemen with a yellow fever vaccine. to stabilize the vaccine virus, scientists added human serum. unfortunately, some of the serum came from people unknowingly infected with hepatitis b virus. as a consequence, , soldiers were infected, severe hepatitis developed in , , and died. [ ] [ ] [ ] [ ] in , five companies made jonas salk's new formaldehydeinactivated polio vaccine. however, one company, cutter laboratories of berkeley, california, failed to completely inactivate poliovirus with formaldehyde. because of this problem, , children were inadvertently injected with live, dangerous poliovirus; in , , mild polio developed, were permanently paralyzed, and were killed. it was one of the worst biological disasters in american history. vaccines have also caused uncommon but severe adverse events not associated with production errors. for example, acute encephalopathy after whole-cell pertussis vaccine, , acute arthropathy following rubella vaccine, - thrombocytopenia following measles-containing vaccine, , guillain-barré syndrome (gbs) after swine flu vaccine, paralytic polio following live attenuated oral polio vaccine, anaphylaxis following receipt of vaccines containing egg proteins (ie, influenza and yellow fever vaccines), , severe or fatal viscerotropic disease following yellow fever vaccine, possible narcolepsy following a squalene-adjuvanted influenza vaccine, and severe allergic reactions associated with gelatin contained in the measles-mumps-rubella vaccine are problems associated with the use of vaccines, albeit rarely. as vaccine use increases and the incidence of vaccine-preventable diseases is reduced, vaccine-related adverse events become more prominent in vaccination decisions ( figure - ) . even unfounded safety concerns can lead to decreased vaccine acceptance and resurgence of vaccine-preventable diseases, as occurred in the s and s as a public reaction to allegations that the wholecell pertussis vaccine caused encephalopathy and brain damage ( figure - ). recent outbreaks of measles, mumps, and pertussis in the united states are important reminders of how immunization delays and refusals can result in resurgences of vaccine-preventable diseases. - because vaccines are given to healthy children and adults, a higher standard of safety is generally expected of immunizations compared with other medical interventions. tolerance of adverse reactions to pharmaceutical products (eg, vaccines, contraceptives) given to healthy people-especially healthy infants and toddlers-to prevent certain conditions is substantially lower than to products (eg, antibiotics, insulin) used to treat people who are sick. this lower tolerance for risks from vaccines translates into a need to investigate the possible causes of much rarer adverse events after vaccinations than would be acceptable for other pharmaceutical products. for example, side effects are essentially universal for cancer chemotherapy, and % to % of people receiving high-dose aspirin therapy experience gastrointestinal symptoms. safety monitoring can be done before and after vaccine licensure, with slightly different goals based on the methodological strengths and weaknesses of each step. - although the general principles are similar irrespective of country, the specific approaches may differ because of factors such as how immunization services are organized and the level of resources available. vaccines, similar to other pharmaceutical products, undergo extensive safety and efficacy evaluations in the laboratory, in animals, and in phased human clinical trials before licensure. , phase trials usually include fewer than participants and can detect only extremely common adverse events. phase trials generally enroll to several hundred people. when carefully coordinated, as in the comparative infant diphtheria and tetanus toxoids and acellular pertussis (dtap) vaccine trials, important insight into the relationship between concentration of antigen, number of vaccine components, formulation, effect of successive doses, and profile of common reactions can be drawn and can affect the choice of the candidate vaccines for phase trials. , sample sizes for phase vaccine trials are based principally on efficacy considerations, with safety inferences drawn to the extent possible based on the sample size (approximately to ) and the duration of observation (often < days). typically only observations of common local and systemic reactions (eg, injection site swelling, fever, fussiness) have been feasible. the experimental design of most phase to clinical trials includes a control group (a placebo or an alternative vaccine) and detection of adverse events by researchers in a consistent manner "blinded" to which vaccine the patient received. this allows relatively straightforward inferences on the causal relationship between most adverse events and vaccination. several ways of enhancing prelicensure safety assessment of vaccines have been developed. one of these ways includes the brighton collaboration ( www.brightoncollaboration.org ), established to develop and implement globally accepted standard case definitions for assessing adverse events following immunizations in prelicensure and postlicensure settings. without such standards, it was difficult if not impossible to compare and collate safety data across trials in a valid manner. for example, in the large multisite phase infant dtap trials, definitions of high fever across trials varied by temperature ( . °c vs . °c), measurement (oral vs rectal), and time (measured at vs hours). this was unfortunate because standardized case definitions had been developed in these trials for efficacy but not for safety, even though the safety concerns provided the original impetus for the development of dtap. , the brighton case definitions for each adverse event are further arrayed by the level of evidence provided (insufficient, low, intermediate, and highest); therefore, they also can be used in settings with fewer resources (eg, studies in less developed settings or postlicensure surveillance). another of the recent advances to prelicensure safety assessments of vaccines has stemmed from the recognition of the need for much larger safety and efficacy trials before licensure. because of pragmatic limits on the sample sizes of prelicensure studies, there are inherent limitations to the extent to which they can detect very rare, yet real, adverse events related to vaccination. even if no adverse event has been observed in a trial of , vaccinees, one can only be reasonably certain that the real incidence of the adverse event is no higher than in , vaccinees. thus, to be able to detect an attributable risk of per , vaccinees (eg, such as the approximate risk found for intussusception in the postlicensure evaluation of rotashield vaccine), a prelicensure trial of at least , vaccinees and , control subjects is needed. both second-generation rotavirus vaccines (rotateq and rotarix) were subjected to phase trials that included at least , infants. , while these trials were adequately powered to detect the problem with intussusception found following rotashield, in general, the cost of such large trials might limit the number of vaccine candidates that go through this process in the future. because rare reactions, reactions with delayed onset, or reactions in subpopulations may not be detected before vaccines are licensed, postlicensure evaluation of vaccine safety is critical. historically, this evaluation has relied on passive surveillance and ad hoc epidemiologic studies, but, more recently, phase trials and preestablished large linked databases have improved the methodological capabilities to study rare risks of specific immunizations. such systems may detect variation in rates of adverse events by manufacturer , or specific lot. more recently, clinical centers for the study of immunization safety have emerged as another useful infrastructure to advance our knowledge about safety. in contrast with the methodological strengths of prelicensure randomized trials, however, postlicensure observational studies of vaccine safety pose a formidable set of methodological difficulties. confounding by contraindication is especially problematic for nonexperimental designs. specifically, persons who do not receive vaccine (eg, because of a chronic or transient medical contraindication or low socioeconomic group) may have a different risk for an adverse event than vaccinated persons (eg, background rates of seizures or sudden infant death syndrome (sids) may be higher in unvaccinated people). therefore, direct comparisons of vaccinated and unvaccinated children are often inherently confounded, and teasing this issue out requires understanding of the complex interactions of multiple, poorly quantified factors. informal or formal passive surveillance or spontaneous reporting systems (srss) have been the cornerstone of most postlicensure safety monitoring systems because of their relative low cost of operations. - the national reporting of adverse events following immunizations can be done through the same reporting channels as those used for other adverse drug reactions, as is the practice in france, vaccine manufacturers also maintain srss for their products, which are usually forwarded subsequently to appropriate national regulatory authorities. , in the united states, the national childhood vaccine injury act of mandated that health care providers report certain adverse events after immunizations. the vaccine adverse events reporting system (vaers) was implemented jointly by the centers for disease control and prevention (cdc) and the us food and drug administration (fda) in to provide a unified national focus for collection of all reports of clinically significant adverse events, including, but not limited to, those mandated for reporting. the vaers form permits narrative descriptions of adverse events. patients and their parents-not just health care professionals-are permitted to report to vaers, and there is no restriction on the interval between vaccination and symptoms that can be reported. report forms, assistance in completing the form, and answers to other questions about vaers are available on the vaers web site (vaers.hhs.gov). web-based reporting and simple data analyses are also available. a contractor, under cdc and fda supervision, distributes, collects, codes (currently using the medical dictionary for regulatory activities (www.meddramsso.com/index.asp), and enters vaers reports in a database. reporters of selected serious events are contacted by trained clinical staff on report receipt and are sent letters at year after report receipt to provide additional information about the vaers report, including the patient's recovery. approximately , vaers reports are now received annually, and these data (without personal identifiers) are also available to the public (at vaers.hhs.gov and at wonder.cdc.gov/vaers.html). several other countries also have substantial experience with passive surveillance for immunization safety. in , canada developed the vaccine associated adverse event (vaae) reporting system, , which is supplemented by an active, pediatric hospital-based surveillance system that searches all admissions for possible relationships to immunizations (immunization monitoring program-active, or impact). serious vaae reports are reviewed by the advisory committee on causality assessment consisting of a panel of experts. the netherlands also convenes an annual panel to categorize reports, which are then published. the united kingdom and most members of the former commonwealth use the yellow card system, whereby a reporting form is attached to officially issued prescription pads. , data on adverse drug (including vaccine) events from several countries are compiled by the world health organization (who) collaborating center for international drug monitoring in uppsala. with so many different passive surveillance systems that collect information on various medical events following vaccination, standardized definitions of vaccine-related adverse events are necessary. in the past, different definitions were developed in brazil, canada, india, and the netherlands. however, implementation of similar standards across national boundaries has been advanced by the international conference on harmonization and the brighton collaboration. vaers often first identifies potential new vaccine safety problems because of clusters of cases in time or space, often with unusual clinical features. for example, in , passive reports to vaers of intussusception among children vaccinated with rotashield was the first postlicensure signal of a problem, leading to epidemiologic studies that verified these findings. , similarly, initial reports to vaers of a previously unrecognized serious yellow fever vaccine-associated neurotropic disease and viscerotropic disease , have since been confirmed elsewhere. because of the success in detecting these signals, there have been various attempts to automate screening for signals using srss reports. new tools developed for pattern recognition in extremely large databases are beginning to be applied. these include empirical bayesian data mining to identify unexpectedly frequent vaccine-event combinations. vaers has provided some of the first safety data after the introduction of a number of vaccines. - vaers has also successfully served as a source of cases for further investigation of idiopathic thrombocytopenic purpura after measles-mumpsrubella (mmr) vaccine, encephalopathy after mmr, , and syncope after immunization. when denominator data on vaccine doses distributed or administered are available from other sources, vaers can be used to evaluate changes in reporting rates over time or when new vaccines replace old vaccines. for example, vaers showed that after millions of doses had been distributed, reporting rates for serious events such as hospitalization and seizures after dtap in toddlers were one third of those after diphtheria and tetanus toxoids and whole-cell pertussis (dtp). because vaers is the only surveillance system covering the entire us population with data available on a relatively timely basis, it is the major means available currently to detect possible new, unusual, or extremely rare adverse events. despite the aforementioned uses, srss for drug and vaccine safety have a number of major methodological weaknesses. underreporting, biased reporting, and incomplete reporting are inherent to all such systems, and potential safety concerns may be missed. - aseptic meningitis associated with the urabe mumps vaccine strain, for example, was not detected by srss in most countries. , some increases in adverse events detected by vaers may not be true increases, but instead may be due to increases in reporting efficiency or vaccine coverage. for example, an increase in gbs reports after influenza vaccination during the to season was found to be largely due to improvements in vaccine coverage and increases in gbs independent of vaccination. an increased reporting rate of an adverse event after one hepatitis b vaccine compared with a second brand was likely due to differential distribution of brands in the public vs private sectors, which have differential vaers reporting rates (higher in the public sector). finally, pending litigation resulted in the filing of a large number of vaers reports claiming that vaccines caused autism. perhaps the most important methodological weakness of vaers, however, is that it does not contain the information necessary for formal epidemiologic analyses. such analyses require calculation of the rate of the adverse event after vaccination and a comparison rate among unvaccinated persons. the vaers database, however, provides data only for the number of persons who may have experienced an adverse event following immunization and, even then, only in a biased and underreported manner. vaers lacks data on the denominator of total number of people vaccinated and the corresponding data on number of cases and denominator population of unvaccinated people. sometimes reporting rates can be calculated by using vaers case reports for the numerator and, if available, doses of vaccines administered (or, if unavailable, data on vaccine doses distributed or vaccine coverage survey data) for the denominator. these rates can then be compared with the background rate of the same adverse event in the absence of vaccination, if available. because of underreporting, however, vaers reporting rates will usually be lower than the actual rates of adverse events following immunization. a higher proportion of serious events, such as seizures, that follow vaccinations are likely to be reported to vaers than milder events, such as rash, or delayed events requiring laboratory assessment, such as thrombocytopenic purpura after mmr vaccination. the reporting efficiency or sensitivity of srss can sometimes be estimated if an independent source of cases of specific adverse events following immunization is available to conduct capture-recapture analyses. such an analysis was conducted to estimate that vaers reporting completeness for intussusception following rotashield vaccine was %. formal evaluation has been limited by the quality of diagnostic information on vaers reports, especially the probability that a serious event reported to vaers has been diagnosed accurately. of cases reported to vaers in which gbs developed after influenza vaccination during the to season, and for which hospital charts were reviewed by an independent panel of neurologists blinded to immunization status, the diagnosis of gbs was confirmed in ( %). intussusception was verified in % of vaers reports filed after rotashield vaccination. clinical reviews of vaers reports submitted following h n influenza vaccine were able to verify % of possible gbs reports and % of reports of possible anaphylaxis. clinical review verification rates were similar for vaers reports following human papillomavirus vaccination: % for gbs and % for anaphylaxis. these studies highlight the often crude nature of signals generated by vaers and the difficulty in ascertaining which potential vaccine safety concerns warrant further investigation. the problems with reporting efficiency and potentially biased reporting and the inherent lack of an adequate control group limit the certainty with which conclusions can be drawn. recognition of these limitations in large part has helped stimulate the creation of more population-based methods of assessing vaccine safety. vaccines may undergo clinical trials after licensure to assess the effects of changes in vaccine formulation, vaccine strain, age at vaccination, number and timing of vaccine doses, simultaneous administration, and interchangeability of vaccines from different manufacturers on vaccine safety and immunogenicity. unanticipated differential mortality among recipients of high-and regular-titered measles vaccine in developing countries (albeit lower than among unvaccinated children) led to a change in recommendations by the who for the use of such vaccines. to improve the ability to detect adverse events that are not detected during prelicensure trials, some recently licensed vaccines in developed countries have undergone formal phase surveillance studies on populations with sample sizes that have included as many as , people. these studies usually have used cohorts in managed care organizations (mcos) supplemented by diary or phone interviews. these methods were first used extensively after the licensure of polysaccharide and conjugated hib vaccines. - large postlicensure studies on safety and efficacy have also been conducted for several other vaccines, including those for dtap, varicella, and herpes zoster. , requirements for phase evaluation have even been extended to less frequently used vaccines, such as japanese encephalitis vaccine. historically, ad hoc epidemiologic studies have been used to assess signals of potential adverse events detected by srss, the medical literature, or other mechanisms. some examples of such studies include the investigations of poliomyelitis after inactivated , and oral polio vaccines, sids after dtp vaccination, - encephalopathy after dtp vaccination, , meningoencephalitis after mumps vaccination, injection site abscesses after vaccination, and gbs after influenza vaccination. , , the institute of medicine (iom) has compiled and reviewed many of these studies. , unfortunately, such ad hoc studies are often costly, timeconsuming, and limited to assessment of a single event or a few events or outcomes. given these drawbacks and the methodological limitations of passive surveillance systems (such as described for vaers), pharmacoepidemiologists began to turn to large databases linking computerized pharmacy prescription (and later immunization records) and medical outcome records. these databases derive from defined populations such as members of mcos, single-provider health care systems, and medicaid programs. such databases cover enrollee populations numbering from thousands to millions, and, because the data are generated from the routine administration of the full range of medical care, underreporting and recall bias are reduced. with denominator data on doses administered and the ready availability of appropriate comparison (ie, unvaccinated) groups, these large databases provide an economical and rapid means of conducting postlicensure studies of safety of drugs and vaccines. , [ ] [ ] [ ] [ ] the cdc initiated the vaccine safety datalink (vsd) project in to conduct postmarketing evaluations of vaccine safety and to establish an infrastructure allowing for highquality research and surveillance. selection of staff-model prepaid health plans minimized potential biases for more severe outcomes resulting from data generated from fee-for-service claims. currently, eight mcos in the united states participate in the vsd. the eight participating mcos comprise a population of more than million members. each mco prepares computerized data files using a standardized data dictionary containing demographic and medical information on their members, such as age and sex, health plan enrollment, vaccinations, hospitalizations, outpatient clinic visits, emergency department visits, urgent care visits, and mortality data, as well as additional birth information (eg, birth weight) when available. other information sources, such as medical chart review; member surveys; and pharmacy, laboratory and radiology data are often used in vsd studies to validate outcomes and vaccination data. there is rigorous attention to the maintenance of patient confidentiality, and each study undergoes institutional review board review. the vsd project's main priorities include evaluating new vaccine safety concerns that may arise from the medical literature, , from vaers, , from changes in immunization schedules, or from introduction of new vaccines. , the creation of near real-time data files has enabled the development of near real-time postmarketing surveillance for newly licensed vaccines and changes in vaccine recommendations. the size of the vsd population also permits separation of the risks associated with individual vaccines from those associated with vaccine combinations, whether given in the same syringe or simultaneously at different body sites. for example, vsd safety monitoring found that the combined mmrv vaccine carried an increased risk of febrile seizures compared with giving mmr and varicella vaccines simultaneously as separate injections. such studies are especially valuable in view of combined pediatric vaccines. more than studies have been or are being performed within the vsd project, including general screening studies of the safety of inactivated influenza vaccines among children and of thimerosal-containing vaccines. disease-or syndrome-specific investigations have been or are being performed, including studies investigating autism, multiple sclerosis, thyroid disease, acute ataxia, alopecia, rheumatoid arthritis, asthma, diabetes, and idiopathic thrombocytopenic purpura following vaccination. amid these promises, a few caveats are appropriate. although diverse, the population in the mcos currently in the vsd project is not wholly representative of the united states in terms of geography or socioeconomic status. more important, because of the high coverage attained in the mcos for most vaccines, few nonvaccinated control subjects are available. therefore, vsd studies often rely on risk-interval analyses (eg, to study the question of whether outcome "x" is more common in period "y" following vaccination compared with other periods) (table - ). this approach, although powerful for evaluating acute adverse events, has limited ability to assess associations between vaccination and adverse events with delayed or insidious onset (eg, autism). the vsd project also cannot easily assess mild adverse events (such as fever) that do not always come to medical attention. finally, because vac-cines are not delivered in the context of randomized, controlled trials, the vsd project may not be able to successfully control for confounding and bias in each analysis, and inferences on causality may be limited. despite these potential shortcomings, the vsd project provides an essential, powerful, and cost-effective complement to ongoing evaluations of vaccine safety in the united states. , in view of the methodological and logistic advantages offered by large linked databases, the united kingdom and canada also have developed systems linking immunization registries with medical files. , because of the relatively limited number of vaccines used worldwide and the costs associated with establishing and operating these large databases, it is unlikely that all countries will be able to or need to establish their own. these countries should be able to draw on the scientific base established by the existing large linked databases for vaccine safety and, if the need arises, conduct ad hoc epidemiologic studies. more recently, there has been an increasing awareness that the usefulness of srss as potential disease registries and the immunization safety infrastructure can be usefully augmented by tertiary clinical centers. well-organized, well-identified clinical infrastructures for the study of rare vaccine safety outcomes were first developed in certain regions in italy and australia. , in the united states, the cdc established the clinical immunization safety assessment (cisa) network in with the following primary goals: ( ) to develop research protocols for clinical evaluation, diagnosis, and management of adverse events following immunization (aefi); ( ) to improve the understanding of aefi at the individual level, including determining possible genetic and other risk factors for predisposed persons and high-risk subpopulations; ( ) to develop evidence-based algorithms for vaccination of persons at risk of serious adverse events following immunization; and ( ) to provide a resource of subject matter experts for clinical vaccine safety inquiries. the cisa investigators bring in-depth clinical, pathophysiologic, and epidemiologic expertise to assessing causal relationships between vaccines and adverse events and to understanding the pathogenesis of adverse . define biologically plausible risk interval for adverse event after vaccination (eg, days after each dose). . partition observation time for each child in the study into periods within and outside of risk intervals, and sum respectively (eg, for a child observed for days during which three doses of vaccine were received, total risk interval time = × person-days = persondays; total nonrisk interval time = − = person-days). birth dose dose dose days . add (a) total risk interval and nonrisk interval observation times for each child in the study (person-time observed; for mathematical convenience, the following example uses and , person-months of observation) and (b) adverse events occurring in each period to complete a × table (for illustration, the example uses and cases): events following vaccinations. the cisa investigators have published a standardized algorithm for evaluating and managing persons who have suspected or definite immediate hypersensitivity reactions such as urticaria, angioedema, and anaphylaxis following vaccines. some of the studies undertaken by cisa include an assessment of extensive limb swelling after dtap, a study of the usefulness of irritant skin test reactions for managing hypersensitivity to vaccines, the clinical evaluation of patients with serious adverse events following yellow fever vaccine administration, and evaluation of vaccine safety among children with inborn errors of metabolism. new understanding of the human genome, pharmacogenomics, and immunology hold promise for future cisa studies and may make it possible to elucidate the biological mechanisms of vaccine adverse reactions, which in turn could lead to the development of safer vaccines and safer vaccination practices, including revaccination when indicated. in mass immunization campaigns during which many people are vaccinated in a short time, it is critical to have a vaccine safety monitoring system in place that can detect potential safety problems early so that corrective actions can be taken as soon as possible. mass immunization campaigns pose specific safety challenges precisely because large populations are vaccinated during a short time and often they are conducted outside the usual health care setting. mass immunization campaigns are often conducted in developing countries, which poses a particular challenge of ensuring injection safety. in any setting in which large numbers of immunizations are being administered, more adverse events will coincidentally occur following immunization. thus, it is important to have background rates available of expected adverse events to allow rapid evaluation of whether reported adverse events are occurring at a rate following immunization that is higher than would be expected by chance alone. the resources devoted to mass vaccination campaigns also provide opportunities to enhance existing immunization safety monitoring systems or to establish a system if none exists, and these may lead to long-term improvements in immunization safety monitoring beyond the specific mass immunization campaign. the response to the h n influenza pandemic involved probably the largest and most intense immunization safety monitoring effort ever undertaken in the united states and internationally. the emergence of a novel influenza a (h n ) virus prompted the development of influenza a (h n ) monovalent vaccines. the fda licensed the first -h n vaccines in september . with potentially hundreds of millions of people expected to be vaccinated, adverse events were anticipated to occur in some recently vaccinated people. to address the question of whether the vaccine could be causing the adverse events, background rates for several adverse events were developed. to rapidly detect any unforeseen safety problems, the federal government implemented enhanced postlicensure -h n vaccine safety monitoring. first, vaers undertook special outreach efforts to encourage providers to report, and daily reviews and followup of submitted reports were conducted by medical personnel to rapidly evaluate the reports and obtain any needed additional clinical or other information. second, a new web-based active surveillance system was implemented to prospectively follow tens of thousands of vaccinees for medically attended adverse events. third, large population-based systems that link computerized vaccination data with health care encounter codes were used to conduct rapid ongoing analyses to evaluate possible associations of h n vaccination with selected adverse events, including potential associations suggested by vaers or other sources. such systems included the existing vsd project; a new collaboration involving additional large health plans covering several million people that also performed rapid ongoing analyses similar to vsd; and the databases of the department of defense, medicare, and the veterans administration. fourth, active case finding for gbs was conducted in areas of the united states with a combined population of about million. the findings from the various safety monitoring activities were regularly reviewed by government and other scientists and an independent vaccine safety review panel convened by the department of health and human services. initial safety data were provided by vaers, which found that the adverse event profile after -h n vaccine in vaers ( > , reports) was consistent with that of seasonal influenza vaccines, although the reporting rate was higher after -h n than seasonal influenza vaccines, which may be, at least in part, a reflection of stimulated reporting; death, gbs, and anaphylaxis reports after -h n vaccination were rare (each < per million doses administered). , preliminary results from the large special study of gbs found . excess cases of gbs per million vaccinations, which is similar to the increased risk found with some seasonal influenza vaccines. similar efforts to intensely monitor the safety of influenza a (h n ) vaccines occurred in other countries, primarily in north america, europe, and australia, but also included the development of new immunization safety monitoring systems in countries such as taiwan. these countries collaborated in their activities and routinely shared information among themselves and with other countries that have limited vaccine safety monitoring capabilities. these extensive international safety monitoring activities and collaborations represented an unprecedented commitment to ensuring the safety of influenza a (h n ) vaccines, as well as a model for how we might improve tracking of safety for all vaccines going forward. unfortunately, vaccine safety issues have increasingly taken on a life of their own outside of the scientific arena-arguably to society's overall detriment. liability concerns, for example, have severely limited development of maternal immunizations to protect their newborn infants against diseases such as from group b streptococcus . more worrisome, however, are various chronic diseases (and their advocates) in search of a simple cause, for which immunizations-as a relatively universal exposuremake all too convenient a hypothesized link. case studies of some of these fears are discussed in the following sections. in , kulenkampff and coworkers reported a series of cases of children with mental retardation and epilepsy following receipt of the whole-cell pertussis vaccine. during the next several years, fear of the pertussis vaccine generated by media coverage of this report caused a decrease in pertussis immunization rates in british children from % to % and resulted in more than , cases and deaths due to pertussis. media coverage of the kulenkampff report also caused decreased immunization rates and increased pertussis deaths in japan, sweden, and wales. however, many subsequent excellent well-controlled studies found that the incidence of mental retardation and epilepsy following whole-cell pertussis vaccine was similar in vaccinated children compared with children who did not receive the vaccine and that many of these children actually suffered from dravet's syndrome (a neuronal sodium channel transport defect caused by an scn a mutation). - , a in the mid- s, the antivaccine group called dissatisfied parents together raised the notion that the whole-cell pertussis vaccine could cause sids. subsequent study of children who did or did not receive dtp vaccine showed that the incidence of sids was not greater in the vaccinated group. in the early s, when the hepatitis b vaccine was recommended for routine use in newborns, a program on abc's / raised the question of whether vaccines could cause sids. again, studies failed to find any association between hepatitis b vaccine and sids. , , two recent reviews have confirmed the notion that vaccines do not cause sids. , vaccines cause mad-cow disease by july , at least people in the united kingdom developed a progressive neurological disease termed variant creutzfeldt-jakob disease that likely resulted from eating meat prepared from cows with "mad-cow" disease, a disease caused by proteinaceous infectious particles (prions). some vaccines were made with serum or gelatin obtained from cows in england or from countries at risk for mad-cow disease. two products obtained from cows may be present in vaccines: trace quantities of fetal bovine serum used to provide growth factors for cell culture and gelatin used to stabilize vaccines. however, the bovine-derived products used in vaccines are not likely to contain prions for several reasons. first, fetal bovine serum and gelatin are obtained from blood and connective tissue respectively; neither are sources that have been found to contain prions. second, fetal bovine serum is highly diluted and eventually removed from cells during the growth of vaccine viruses. third, prions are not propagated in cell cultures used to make vaccines. fourth, transmission of prions occurs from eating meat contaminated with nervous tissue obtained from infected animals or, in experimental studies, from directly inoculating preparations of brains from infected animals into the brains of experimental animals. transmission of prions has not been documented after inoculation into the muscles or under the skin (routes used to vaccinate). taken together, the chance that currently licensed vaccines contain prions is essentially zero. the notion that the origin of aids could be traced to poliovirus vaccines that were administered in the belgian congo between and was the subject of a popular magazine article and book. the logic behind this assertion was as follows: ( ) the polio vaccine used in the belgian congo was grown in chimpanzee kidney cells. ( ) the chimpanzee kidney cells used at that time contained simian immunodeficiency virus (siv). ( ) siv is very closely related to human immunodeficiency virus (hiv). ( ) people were inadvertently inoculated with siv that then mutated to hiv and caused the aids epidemic. this reasoning is problematic and based on several false assumptions. - first, siv most closely related to hiv has been demonstrated in chimps in the cameroon, far from the chimps near stanleyville that were used to make the vaccine. second, siv and hiv are not very close genetically; mutation to hiv from siv would likely require decades, not years. third, polymerase chain reaction (pcr) analysis showed that the cell substrate used to make the vaccine was monkey, not chimp. fourth, siv and hiv are enveloped viruses that are easily disrupted by extremes in ph. if given by mouth (in a manner similar to the oral polio vaccine), both of these viruses would likely be destroyed in the acid environment of the stomach. last, and most important, original lots of the polio vaccine (including those used in africa for the polio vaccine trials) did not contain hiv or siv genomes as determined by the very sensitive reverse-transcription pcr assay. unfortunately, the notion that live attenuated polio vaccine could cause aids remains an obstacle to eliminating polio in some countries in africa. simian virus (sv ) was present in monkey kidney cells used to make the inactivated polio vaccine, live attenuated polio vaccine, and inactivated adenovirus vaccines in the late s and early s. recently, investigators found sv dna in biopsy specimens obtained from patients with certain unusual cancers (ie, mesothelioma, osteosarcoma, and non-hodgkin lymphoma), leading some to hypothesize a link between vaccination and the subsequent development of cancer. however, genetic remnants of sv were present in cancers of people who had or had not received contaminated polio vaccines; people with cancers who never received sv -contaminated vaccines were found to have evidence for sv in their cancerous cells; and epidemiologic studies did not show an increased risk of cancers in people who received polio vaccine between and and people who did not receive these vaccines. taken together, these findings do not support the hypothesis that the sv contained in polio vaccines administered before caused cancers. one hundred years ago, children received one vaccinesmallpox. today, young children receive vaccines routinely. although some vaccines are given in combination, infants and young children could receive more than shots and three oral doses by years of age, including as many as five shots at one time. the increase in the number of vaccines, and the consequent decline in vaccine-preventable illnesses, has focused attention by parents and health care professionals on vaccine safety. specific concerns include whether vaccines weaken, overwhelm, , or in some way alter the normal balance of the immune system, paving the way for chronic diseases such as diabetes, asthma, multiple sclerosis, and allergies. although we have witnessed a dramatic increase in the number of vaccines routinely recommended for infants and young children, the number of immunogenic proteins and polysaccharides contained in vaccines has declined (table - ). the decrease in the number of immunogenic proteins and polysaccharides contained in vaccines is attributable to discontinuation of the smallpox vaccine and advances in the field of protein purification that allowed for a switch from whole-cell to acellular pertussis vaccine. a practical way to determine the capacity of the immune system to respond to vaccines would be to consider the number of b and t cells required to generate adequate levels of binding antibodies per milliliter of blood. calculations are based on the following assumptions: -approximately ng/ml is likely to be an effective concentration of antibody directed against a specific epitope. -approximately b cells/ml are required to generate ng of antibody/ml. -given a doubling time of about . days for b cells, it would take about days to generate b cells/ml from a single b-cell clone. -because vaccine-specific humoral immune responses are first detected about days after immunization, those responses could initially be generated from a single b-cell clone per milliliter. -one vaccine contains about immunogenic proteins or polysaccharides ( table - ). -each immunogenic protein or polysaccharide contains about epitopes (ie, epitopes per vaccine). -approximately b cells are present per milliliter of blood. given these assumptions, the number of vaccines to which a person could respond would be determined by dividing the number of circulating b cells (approximately /ml) by the average number of epitopes per vaccine ( ). therefore, a person could theoretically respond to about vaccines at one time. the analysis used to determine the theoretical capacity of a person to respond to as many as vaccines at one time, although consistent with the biology and kinetics of vaccine-specific immune responses, is limited by lack of consideration of several factors. first, only vaccine-specific b-cell responses are considered. however, protection against disease by vaccines may also be mediated by vaccine-specific cytotoxic t lymphocytes (ctls). for example, virus-specific ctls are important in the regulation and control of varicella infections. second, in part because of differences in the capacity of various class i or class ii glycoproteins (encoded by the mhc) to present viral or bacterial peptides to the immune system, some people are not capable of responding to certain virus-specific proteins (eg, hepatitis b surface antigen). third, some proteins are more likely to evoke an immune response than others (ie, immunodominance). fourth, although most circulating b cells in a neonate are naïve, the child very quickly develops memory b cells that are not available for response to new antigens and, therefore, should not be considered as part of the circulating naïve b-cell pool. fifth, the immune system is not static. a study of t-cell population dynamics in hiv-infected persons found that adults have the capacity to generate about × new t lymphocytes each day. although the quantity of new b and t cells generated each day in healthy people is unknown, studies of hiv-infected persons demonstrate the enormous capacity of the immune system to generate lymphocytes when needed. primarily because of this fifth reason, the assessment that people can respond to at least vaccines at one time might be low. within hours of birth, cells of the innate and adaptive immune systems are actively engaged in responding to challenges in the environment (eg, colonizing bacterial flora). , similarly, newborn and young infants are quite capable of generating protective immune responses to single and multiple vaccines. for example, children born to mothers infected with hepatitis b virus are protected against infection after inoculation with hepatitis b vaccine (given at birth and month of age). [ ] [ ] [ ] similarly, newborns inoculated with bacille calmette-guérin (bcg) vaccine are protected against severe forms of tuberculosis presumably by activation of bacteria-specific t cells. [ ] [ ] [ ] in addition, about % to % of infants inoculated in the first months of life with multiple vaccines, including diphtheria-tetanus-pertussis, pneumococcus, hib, hepatitis b and polio, develop protective, vaccine-specific immune responses. conjugation of bacterial polysaccharides (such as streptococcus pneumoniae and hib) to carrier molecules that elicit helper t cells circumvents the poor immunogenicity of unconjugated polysaccharide vaccines in infants and young children. , vaccines weaken the immune system infection with wild-type viruses can cause a suppression of specific immunologic functions. for example, infection with wild-type measles virus causes a reduction in the number of circulating b and t cells during the viremic phase of infection and a delay in the development of cell-mediated immunity. , downregulation of cell-mediated immunity by wild-type measles virus probably results from downregulation of the production of interleukin- by measles-infected macrophages and dendritic cells. taken together, the immunosuppressive effects of wild-type measles virus account, in part, for the increase in morbidity and mortality from measles infection. similarly, the immunosuppressive effects of infections with wild-type varicella virus or wild-type influenza virus cause an increase in the incidence of severe invasive bacterial infections. live viral vaccines replicate (albeit far less efficiently than wild-type viruses) in the host and, therefore, can weakly mimic events that occur after natural infection. for example, measles, mumps, or rubella vaccines can significantly depress reactivity to the tuberculin skin test, can cause a decrease in protective immune responses to varicella vaccine, and high-titered measles vaccine (edmonston-zagreb strain) can cause an excess of cases of invasive bacterial infections in developing countries. all of these phenomena are explained by the likely immunosuppressive effects of measles vaccine viruses. however, current vaccines (including the highly attenuated moraten strain of measles vaccine) do not seem to cause clinically relevant immunosuppression in healthy children. studies have found that the incidence of invasive bacterial infections following immunization with diphtheria, pertussis, tetanus, bcg, measles, mumps, rubella, or live attenuated poliovirus vaccines was not greater than that found in unimmunized children. [ ] [ ] [ ] [ ] [ ] vaccines cause autoimmunity mechanisms are present at birth to prevent the development of immune responses directed against self-antigens (autoimmunity). t-and b-cell receptors of the fetus and newborn develop with a random repertoire of specificities. in the thymus, t cells that bind strongly to self-peptide-mhc complexes die, while those that bind with a lesser affinity survive to populate the body. this central selection process eliminates strongly selfreactive t cells, while selecting for t cells that recognize antigens in the context of self-mhc. in the fetal liver, and later in the bone marrow, b-cell receptors (ie, immunoglobulins) that bind self-antigens strongly are also eliminated. therefore, the thymus and bone marrow, by expressing antigens from many tissues of the body, enable the removal of the majority of potentially dangerous autoreactive t and b cells before they maturea process termed central tolerance. however, it is not simply the presence of autoreactive t and b cells that result in autoimmune disease. autoreactive t and b cells are present in all people because it is not possible for every antigen from every tissue of the body to participate in the elimination of all potentially autoreactive cells. a process termed peripheral tolerance further limits the activation of autoreactive cells. , mechanisms of peripheral tolerance include the following: ( ) antigen sequestration (antigens of the central nervous system, eyes, and testes are not regularly exposed to the immune system unless injury or infection occurs.); ( ) anergy (lymphocytes partially triggered by antigen but without costimulatory signals are unable to respond to subsequent antigen exposure.); ( ) activation-induced cell death (a self-limiting mechanism involved in terminating immune responses after antigen is cleared); and ( ) inhibition of immune responses by specific regulatory cells. [ ] [ ] [ ] [ ] therefore, the immune system anticipates that self-reactive t cells will be present and has mechanisms to control them. any theory of vaccine causation of autoimmune diseases must take into account how these controls are circumvented. as discussed subsequently, epidemiologic studies have not supported the hypothesis that vaccines cause autoimmune diseases. this is consistent with the fact that no mechanisms have been advanced to explain how vaccines could account for all of the prerequisites that would be required for the development of autoimmune disease. at least four key conditions must be met for development of autoimmune disease. first, self-antigen-specific t cells or selfantigen-specific b cells must be present. second, self-antigens must be presented in sufficient amounts to trigger autoreactive cells. third, costimulatory signals, cytokines, and other activation signals produced by antigen-presenting cells (such as dendritic cells) must be present during activation of self-reactive t cells. fourth, peripheral tolerance mechanisms must fail to control destructive autoimmune responses. if all of these conditions are not met, the activation of self-reactive lymphocytes and progression to autoimmune disease are not likely. evidence that vaccines do not cause autoimmunity rigorous epidemiologic studies of infant vaccines and type diabetes found that measles vaccine was not associated with an increased risk for diabetes; other investigations found no association between bcg, smallpox, tetanus, pertussis, rubella, or mumps vaccine and diabetes. a study in canada found no increase in risk for diabetes as a result of receipt of bcg vaccine. in a large year follow-up study among finnish children enrolled in an hib vaccination trial, no differences in risk for diabetes were found among children vaccinated at months of age (followed later with a booster vaccine) and children vaccinated at years only or with children born before the vaccine trial. the weight of currently available epidemiologic evidence does not support a causal association between currently recommended vaccines and type diabetes in humans. [ ] [ ] [ ] the hypothesis that vaccines might cause multiple sclerosis was fueled by anecdotal reports of multiple sclerosis following hepatitis b immunization and two case-control studies showing a small increase in the incidence of multiple sclerosis in vaccinated persons that was not statistically significant. - however, the capacity of vaccines to cause or exacerbate multiple sclerosis has been evaluated in several excellent epidemiologic studies. - two large case-control studies showed no association between hepatitis b vaccine and multiple sclerosis and found no evidence that hepatitis b, tetanus, or influenza vaccines exacerbated symptoms of multiple sclerosis. other well-controlled studies also found that influenza vaccine did not exacerbate symptoms of multiple sclerosis. - indeed, in a retrospective study of patients with relapsing multiple sclerosis, infection with influenza virus was more likely than immunization with influenza vaccine to cause an exacerbation of symptoms. a recent review also showed that the novel h n vaccine had an attributable risk for guillain-barré syndrome of - cases per million doses administered, not higher than that found following the - seasonal influenza vaccine. allergic symptoms are caused by soluble factors (eg, ige) that mediate immediate-type hypersensitivity; production of ige by b cells is dependent on release of cytokines such as interleukin- by th cells. two theories have been advanced to explain how vaccines could enhance ige-mediated, th -dependent allergic responses. first, vaccines could shift immune responses to potential allergens from th -like to th -like. second, by preventing common prevalent infections (the "hygiene hypothesis"), vaccines could prolong the length or increase the frequency of th -type responses. , although all factors that cause changes in the balance of th and th responses are not fully known, it is clear that dendritic cells have a critical role. for example, adjuvants (eg, aluminum hydroxide or aluminum phosphate ["alum"] contained in some vaccines) promote dendritic cells to stimulate th type responses. , adjuvants could cause allergies or asthma by stimulating bystander, allergen-specific th cells. however, vaccine surveillance data show no evidence for environmental allergen priming by vaccination. furthermore, local inoculation of adjuvant does not cause a global shift of immune responses to th or th type. , the other hypothesis advanced to explain how vaccines could promote allergies is that by preventing several childhood infections (the hygiene hypothesis), stimuli that evolution has relied on to cause a shift from the neonatal th -type immune response to the balanced th -th response patterns of adults have been eliminated. , however, the diseases that are prevented by vaccines constitute only a small fraction of the total number of illnesses to which a child is exposed, and it is unlikely that the immune system would rely on only a few infections for the development of a normal balance between th and th responses. for example, a study of , illnesses performed in cleveland, ohio, in the s found that children experienced six to eight infections per year in the first years of life; most of these infections were caused by viruses such as coronaviruses, rhinoviruses, paramyxoviruses, and myxovirusesdiseases for which children are not routinely immunized. also at variance with the hygiene hypothesis is the fact that children in developing countries have lower rates of allergies and asthma than children in developed countries despite the fact that they are commonly infected with helminths and worms-organisms that induce strong th -type responses. finally, the incidence of diseases that are mediated by th -type responses, such as multiple sclerosis and type diabetes, have increased in the same populations as those that experienced an increase in allergies and asthma. although some relatively small early observational studies supported the association between whole-cell pertussis vaccine and development of asthma, more recent studies have suggested otherwise. a large clinical trial performed in sweden found no increased risk, and a very large longitudinal study in the united kingdom found no association between pertussis vaccination and early-or late-onset wheezing or recurrent or intermittent wheezing. two studies from the vsd project have also lent data to this controversy. in one study of , infants with wheezing during infancy, vaccination with dtp and other vaccines was not related to the risk of wheezing in full-term infants, and, in another study of more than , children, childhood vaccinations were not associated with an increased risk for developing asthma. finally, a study from finland also suggested that children with a history of natural measles were at increased risk for atopic illness. such findings would run contrary to the hypothesis that the increase in atopic illnesses seen in several countries is due to the reduction in wild measles resulting from immunizations. another separate concern is whether inactivated influenza vaccination may induce asthma exacerbations in children with preexisting asthma. results of studies examining the potential associations between administration of inactivated influenza vaccine and various surrogate measures of asthma exacerbation, including decreased peak expiratory flow rate, increased use of bronchodilating drugs, and increase in asthma symptoms, have yielded mixed results. most studies, however, have not supported such an association. in fact, after controlling for asthma severity, acute asthma exacerbations were less common after inactivated influenza vaccination than before, and inactivated influenza vaccination seems to be associated with a decreased risk for asthma exacerbations throughout influenza seasons. several more recent studies have also shown a lack of correlation between receipt of vaccines and the development of asthma. - autism is a chronic developmental disorder characterized by problems in social interaction, communication, and responsiveness and by repetitive interests and activities. although the causes of autism are largely unknown, family and twin studies suggest that genetics has a fundamental role. in addition, overexpression of neuropeptides and neurotrophins has been found in the immediate perinatal period among children later diagnosed with autism, suggesting that prenatal or perinatal influences or both have a more important role than postnatal insults. however, because autistic symptoms generally first become apparent in the second year of life, some scientists and parents have focused on the role of mmr vaccine because it is first administered around this time. concern about the role of mmr vaccine was heightened in when a study based on children proposed an association between the vaccine and the development of ileonodular hyperplasia, nonspecific colitis, and regressive developmental disorders (later termed by some as "autistic enterocolitis"). among the proposed mechanisms was that mmr vaccine caused bowel problems, leading to the malabsorption of essential vitamins and other nutrients and eventually to autism or other developmental disorders. concern about this issue led to a decline in measles vaccine coverage in the united kingdom and elsewhere. significant concerns about the validity of the study included the lack of an adequate control or comparison group, inconsistent timing to support causality (several of the children had autistic symptoms preceding bowel symptoms), and the lack of an accepted definition of the syndrome. subsequently, population-based studies of autistic children in the united kingdom found no association between receipt of mmr vaccine and autism onset or developmental regression. , a study in the united states in the vsd project investigated whether measlescontaining vaccine was associated with inflammatory bowel disease and found no relationship between receiving mmr vaccine and inflammatory bowel disease or between the timing of the vaccine and risk for disease. soon after the lancet published the article that ignited the controversy, two ecologic analyses found no evidence that mmr vaccination was the cause of apparent increased trends in autism over time, , while two other studies found no evidence of a new variant form of autism associated with bowel disorders secondary to vaccination. , several more recent studies have also refuted the notion that mmr vaccine caused autism. [ ] [ ] [ ] [ ] [ ] [ ] in february , the lancet retracted the original article claiming an association. because of the level of concern surrounding this issue, the cdc and the national institutes of health requested an independent review by the iom. the immunization safety review committee appointed by the iom to review this issue was unable to find evidence supporting a causal relationship at the population level between autistic spectrum disorders and mmr vaccination, nor did the committee find any good evidence of biological mechanisms that would support or explain such a link. the fda modernization act of called for the fda to review and assess the risk of all mercury-containing food and drugs. this led to an examination of mercury content in vaccines. public health officials found that infants up to months old could receive as much as . µ g of ethylmercury (thimerosal) from vaccines, a level that exceeded recommended safety guidelines for methylmercury from the environmental protection agency, but not levels recommended by the fda or the agency for toxic substance disease registry. consequently, the routine neonatal dose of hepatitis b vaccine in infants born to hepatitis b surface antigen-negative mothers was suspended in the united states until preservative-free vaccines became available, and transitioning to a vaccine schedule free of thimerosal began as a precautionary measure. currently, some multidose influenza vaccines contain preservative quantities (ie, µ g per dose) of thimerosal although thimerosal-free vaccines are available. mercury is a naturally occurring element found in the earth's crust, air, soil, and water. since the earth's formation, volcanic eruptions, weathering of rocks, and burning of coal have caused mercury to be released into the environment. once released, certain types of bacteria in the environment can change inorganic mercury to organic (methylmercury). methylmercury makes its way through the food chain in fish, animals, and humans. at high levels, it can be neurotoxic. thimerosal contains ethylmercury, not methylmercury. studies comparing ethylmercury and methylmercury suggest that they are processed differently; ethylmercury is broken down and excreted much more rapidly than methylmercury. therefore, ethylmercury is much less likely than methylmercury to accumulate in the body and cause harm. a several pieces of biological and epidemiologic evidence support the notion that thimerosal does not cause autism. first, in iraq imported grain that had been fumigated with methylmercury. farmers ate bread made from this grain. the result was one of the worst, single-source, mercury poisonings in history. methylmercury in the grain caused the hospitalization of , iraqis and killed . pregnant women also ate the bread and delivered infants with epilepsy and mental retardation. however, there was no evidence that these infants had an increased incidence of autism. second, several large studies have now compared the risk of autism in children who received vaccines containing thimerosal with children who received vaccines without thimerosal or vaccines with lesser quantities of thimerosal; the incidence of autism was similar in all groups. - the iom has reviewed these studies and concluded that evidence favored rejection of a causal association between vaccines and autism and that autism research should shift away from vaccines. denmark, a country that abandoned thimerosal as a preservative in , actually saw an increase in the disease beginning several years later. third, studies of the head size, speech patterns, vision, coordination, and sensation of children poisoned by mercury show that the symptoms of mercury poisoning are distinguishable from the symptoms of autism. fourth, methylmercury is found in low levels in water, infant formula, and breast milk. although it is clear that large quantities of mercury can damage the nervous system, there is no evidence that the small quantities contained in water, infant formula, and breast milk do. an infant who is exclusively breastfed for months will ingest more than twice the quantity of mercury that was ever contained in vaccines and times the quantity of mercury contained in the influenza vaccine. one known and unfortunate sequela from the uncertainty surrounding the safety of thimerosal was confusion surrounding administration of the birth dose of hepatitis b vaccine. following the suspension of the routine use of hepatitis b vaccine for low-risk newborns in , there was a marked increase in the number of hospitals that no longer routinely vaccinated all infants at high risk of hepatitis b. as a result, there have been cases of neonatal hepatitis b that could have been prevented but were not because of many hospitals suspending their routine neonatal hepatitis b vaccination program. the hypothesis for why vaccines might cause autism has continued to shift. in , the concern was that the mmr vaccine caused autism. the following year, the concern shifted to include the fear that thimerosal in vaccines caused autism. as data continued to be generated showing that both of these concerns were ill founded, the hypothesis shifted again-this time to include the fear that too many vaccines given too soon caused autism. to address this concern, michael smith and charles woods mined data from a previous study that had been performed by cdc researchers to determine whether thimerosal in vaccines was associated with an increased risk of autism or neurodevelopmental delays. smith and woods compared children who had received vaccines according to the cdc/american academy of pediatrics schedule with children for whom a decision was made to delay, withhold, separate, or space out vaccines, noting no difference between the two groups in neurodevelopmental outcomes. aluminum salts have safely been used to adjuvant vaccines since the s. however, by the mid- s, parents became concerned that aluminum in vaccines might be harmful. indeed, high levels of aluminum can cause local inflammatory reactions, osteomalacia, anemia, or encephalopathy, typically in preterm infants or infants with absent or severely compromised renal function who are also receiving high doses of aluminum from other sources (eg, antacids). studies have shown that children who receive aluminum-containing vaccines have serum levels of aluminum that are well below the toxic range. [ ] [ ] [ ] formaldehyde in vaccines is harmful formaldehyde has been used in vaccines to detoxify bacterial toxins (ie, diphtheria toxin, tetanus toxin, pertussis toxins) and to inactivate viruses (ie, poliovirus). because formaldehyde at high concentrations can cause mutational changes in cellular dna in vitro, some parents have become concerned that formaldehyde in vaccines might be dangerous. however, because formaldehyde is a product of single-carbon metabolism, everyone has formaldehyde detectable in serum. indeed, the level of formaldehyde in the circulation is about -fold more than would be contained in any vaccine. also, people exposed to high levels of formaldehyde in the workplace (eg, morticians) are not at greater risk of cancer than people who are not exposed to formaldehyde. finally, the quantity of formaldehyde present in vaccines is at least -fold lower than that necessary to induce toxicity in experimental animals. two cell lines, mrc- and wi- , both derived from elective abortions performed in europe in the early s, have been used as cell substrates in vaccine manufacture. four vaccines continue to require the use of these cell lines: varicella, rubella, hepatitis a, and one of the rabies vaccines. human fetal cells were valuable in vaccine research because they support the growth of many human viruses and are sterile; they were first used at around the time that researchers found that primary monkey kidney cells were contaminated with sv virus. some religious groups have become concerned about the use of cells originally obtained from elective abortions. however, the pontifical academy of life of the catholic church has deemed vaccines made using these cells worthy of continued use, despite their origins. disease prevention, especially if it requires continuous nearuniversal compliance, is a formidable task. in the preimmunization era, vaccine-preventable diseases such as measles and pertussis were so prevalent that the risks and benefits of disease vs vaccination were readily evident. as immunization programs successfully reduced the incidence of vaccine-preventable diseases, however, an increasing proportion of health care providers and parents have little or no personal experience with vaccine-preventable diseases. for their risk-benefit analysis, they are forced to rely on historical and other more distant descriptions of vaccine-preventable diseases in textbooks or educational brochures. in contrast, some degree of personal discomfort, pain, and worry is generally associated with each immunization. in addition, parents searching for information about vaccines on the world wide web are likely to encounter web sites that encourage vaccine refusal or emphasize the dangers of vaccines. , similarly, the media may sensationalize vaccine safety issues or, in an effort to present "both sides" of an argument, fail to provide perspective. , for reasons discussed earlier, there may be uncertainty if vaccines are associated with rare or delayed adverse reactions if only because the scientific method does not allow for acceptance of the null hypothesis. therefore, one cannot prove that a vaccine never causes a particular adverse event, only that an adverse event is unlikely to occur by a certain statistical probability. the combination of these factors may have an impact on parental beliefs about immunizations. a national survey found that although the majority of parents support immunizations, % to % have misconceptions that could erode their confidence in vaccines. within this context, the art of addressing vaccine safety concerns through effective risk communication has emerged as an increasingly important skill for managers of mature immunization programs and health care providers who administer vaccines. the science of risk perceptions and risk communications, developed initially for technology and environmental arenas, has only recently been formally applied to immunizations. for scientists and other experts, risk tends to be synonymous with the objective probability of morbidity and mortality resulting from exposure to a particular hazard. in contrast, research has shown that laypersons may have subjective, multidimensional, and value-laden conceptualizations of risk. among the key principles and lessons learned about public perceptions of risk are the following: -individual people differ in their perceptions of risk depending on their personality, education, life experience, and personal values; , educational materials tiered for different needs are therefore likely to be more effective than a single tier. -perceptions of risk may differ dramatically among various stakeholders, such as members of government agencies, industry, or activist groups. the level of trust between stakeholders has an impact on all other aspects of risk communication. trust is generally reinforced by open communication about what is known and unknown about risks and by providing candid accounts of the evidence and how it was used in the decisionmaking process. -certain hazard characteristics, including involuntariness, uncertainty, lack of control, high level of dread, and low level of equity, lead to higher perceived risk; only risks with similar characteristics should be compared in risk communication efforts. -for quantitatively equivalent risk that is due to action (eg, vaccination reaction) vs inaction (eg, vaccinepreventable disease caused by nonvaccination), many people prefer the consequences of inaction to action. -when there is uncertainty about risks, patients frequently rely on the advice of their physician or other health care professionals; continuing education of health care professionals on vaccine risk issues is key. -finally, different ways of presenting, or framing, the same risk information (eg, using survival rates vs mortality rates) can lead to different risk perceptions, decisions, and behaviors. , risk communication can be used for the purposes of advocacy, public education, or decision-making partnership. people care not only about the magnitude of risks, but also how risks are managed and whether they participate in the risk-management process, especially in a democratic society. in medical decision making, this has resulted in a transition from more paternalistic models to increasing degrees of informed consent. some have argued that a similar transition to informed consent also should occur with immunizations. however, immunization is unlike most other medical procedures (eg, surgery) in that the consequences of the decision affect not only the individual person, but also others in the society. because of this important distinction, many countries have enacted public health (eg, immunization) laws that severely limit an individual person's right to infect others. without such mandates, persons may attempt to avoid the risks of vaccination while being protected by the herd immunity resulting from others being vaccinated. unfortunately, the protection provided by herd immunity may disappear if too many people avoid vaccination, resulting in outbreaks of vaccine-preventable diseases. , debates in the united states have focused on whether philosophical (in addition to medical and religious) exemptions to mandatory immunizations should be allowed more universally and, if so, what standards for claim of exemption are needed. , , thus, vaccine risk communications should not only describe the risks and benefits of vaccines for individual people, but also should include discussion of the impact of individual immunization decisions on the larger community. empathy, patience, scientific curiosity, and substantial resources are needed to address concerns about vaccine safety. although each evaluation of a vaccine safety concern is in some ways unique, some general principles may apply to most cases. as with all investigations, the first step is objective and comprehensive data gathering. it is also important to gather and weigh evidence for causes other than vaccination. for individual cases or clusters of cases, a field investigation to gather data firsthand may be necessary. , advice and review from a panel of independent experts also may be needed. , , causality assessment at the individual level is difficult at best; further evaluation via epidemiologic or laboratory studies may be required. even if the investigation is inconclusive, such studies can often help to maintain public trust in immunization programs. scientific investigations are only the beginning of addressing vaccine safety concerns. in many countries, people who believe they or their children have been injured by vaccines have organized and produced information highlighting the risks of and alternatives to immunizations. from the consumer activist perspective, even if vaccine risks are rare, this low risk does not reassure the person who experiences the reaction. such groups have been increasingly successful in airing their views in electronic and print media, frequently with poignant individual stories. , because the media frequently raise controversies without resolution and choose "balance" over perspective, one challenge is to establish credibility and trust with the audience. , factors that aid in enhancing credibility include demonstrating scientific expertise, establishing relationships with members of the media, expressing empathy, and distilling scientific facts and figures down to simple lay concepts. however, statistics and facts compete poorly with dramatic pictures and stories of disabled children. emotional reactions to messages are often dominant, influencing subsequent cognitive processing. therefore, equally compelling firsthand accounts of people with vaccine-preventable diseases may be needed to communicate the risks associated with not vaccinating. clarifying the distinction between perceived and real risk for the concerned public is critical. if further research is needed, the degree of uncertainty (eg, whether such rare vaccine reactions exist at all) should be acknowledged, but what is certain also should be noted (eg, millions of people have received vaccine x and have not developed syndrome y; even if the vaccine causes y, it is likely to be of magnitude z, compared with the magnitude of known risks associated with vaccine-preventable diseases). in the united states, written information about the risks and benefits of immunizations developed by the cdc has been required to be provided to all people vaccinated in the public sector since . the national childhood vaccine injury act requires every health care provider, public or private, who administers a vaccine that is covered by the act to provide a copy of the most current cdc vaccine information statement (vis) to the adult vaccinee or, in the case of a minor, to the parent or legal representative each time a dose of vaccine is administered. health care providers must note in each patient's permanent medical record the date printed on the vis and the date the vis was given to the vaccine recipient or his or her legal representative. viss are the cornerstone of provider-patient vaccine risk-benefit communication. each vis contains information on the disease(s) that the vaccine prevents, who should receive the vaccine and when, contraindications, vaccine risks, what to do if a side effect occurs, and where to go for more information. current viss can be obtained from the cdc's national center for immunization and respiratory diseases at www.cdc.gov/vaccines and are available in more than languages from the immunization action coalition at www.immunize.org. an increasing number of resources that address vaccine safety misconceptions and allegations also have become available, including web sites, brochures, resource kits, and videos (table - ) . some studies have been conducted to assess the use and effectiveness of such materials; - however, more research in this area is needed. immunization programs and health care providers should anticipate that some members of the public may have deep concerns about the need for and safety of vaccines. a few may refuse certain vaccines or even reject all vaccinations. an understanding of vaccine risk perceptions and effective vaccine risk communication are essential in responding to misinformation and concerns. toward this end, cdc's vaccine safety website (http://www.cdc. gov/vaccinesafety/index.html) provides basic information on the safety of routinely administered vaccines, as well as responses to frequently asked questions. the website also provides more detailed information on how vaccines are tested and monitored for safety; cdc's specific projects for monitoring, evaluation, and research on vaccine safety (vaers, vsd, and cisa); detailed sections addressing common concerns (eg, autism, thimerosal); and a resource library with articles, fact sheets, and other related materials on immunization safety. parental vaccine acceptance in a new era: the role of health care providers and public health professionals one consequence of the success of vaccines is that an increasing number of parents and clinicians have little or no personal experience with or knowledge of many of the diseases that vaccines prevent. thus, vaccine-preventable diseases often are not perceived as a real threat by parents. , moreover, increasingly parents want to be fully informed about their children's medical care, thus merely recommending vaccination may not be sufficient. also in this new era, stories in the media highlighting adverse events (real or perceived) may cause some parents to question the safety of vaccines. apart from the media attention on vaccine safety issues, a confluence of factors has an influence on parents' vaccine attitudes in the present environment of a low incidence of vaccinepreventable diseases. these factors would be relatively unimportant in an environment where diseases such as polio and measles were common and people lived in fear of their children contracting disease; however, they have become predominant in the current climate for some parents. some of these factors are: ( ) lack of appropriately tailored information about the benefits of vaccines and contrary information from alternative health practitioners, ( ) mistrust of the source of the information, ( ) perceived serious side effects, ( ) not perceiving the risks of vaccines accurately, and ( ) insufficient biomedical literacy. addressing these issues is a challenge for medical and public health professionals because the typical arrangement for providing medical care does not allow full reimbursement of health care providers for educating patients and parents. nevertheless, it is important for us to try to meet the challenge because an understanding of the aforementioned factors and a proactive approach to vaccine education may prevent future concerns from escalating into widespread refusal of vaccines, with a consequent increased incidence of vaccine-preventable diseases. most people today want to be thoroughly informed about their health care. the desire for more information also applies to parents with regard to medical issues for their children. parents want to be part of the decision-making process when it comes to immunizations for their children. providing the appropriate information at the appropriate time is especially important now with the increased questioning of vaccines and with states allowing philosophical exemptions in . there is an association between information and vaccine acceptance. a recent study found that while % of parents agreed that they had access to enough information to make a good decision about immunizing their children, % of parents disagreed or were neutral. parents who disagreed they had enough vaccine information had negative attitudes about immunizations, health care providers, immunization requirements and exemptions, and trust in people responsible for immunization policy. moreover, a larger percentage of parents who reported they did not have access to enough information about vaccines also had several specific vaccine concerns compared with parents who were neutral or agreed they had access to enough information. it may be that when there is a void of accurate, trusted information, doubts about vaccines arise and misinformation is more readily accepted. other studies have demonstrated the effect of providing information on the well-being of patients. for example, information is one factor that has been shown to positively influence a sense of control in patients with rheumatoid arthritis, and perceived lack of information among mothers was one reason contributing to nonimmunization of children in india. by using the principle of audience segmentation (partitioning a population into segments with shared characteristics), a survey study identified five parent groups that varied on health and immunization attitudes and beliefs. the two audience segments identified as most concerned about immunizations ("worrieds" and "fencesitters") were chosen as the focus of a follow-up study to obtain the input of mothers in these segments in the development of evidence-based, tailored educational materials. the purpose of these materials would be to assist health care providers in busy office settings to address questions from these two groups of parents. presentation of these tailored brochures by children's health care providers to parents in an empathetic and respectful manner could aid in improving the health care provider-parent relationship, increasing vaccine acceptance, and ultimately preventing vaccine-preventable diseases. the viss are typically given to parents the day the child is scheduled for immunization. , , this often places the parent in a conflict situation of attending to the vis or attending to a frightened or upset child. not surprisingly, studies have shown that parents would rather receive the information in advance of the first vaccination visit. , [ ] [ ] [ ] suggested earlier times for vaccine education include prenatal clinic visits and just after delivery in a hospital. a national survey indicated that % of providers said that a preimmunization booklet for parents would be useful for communicating risks and benefits to parents. the use of complementary and alternative medicine (cam) has been increasing during the past years in the united states. part of this increase is due to mcos providing coverage for some cam therapies. chiropractic care is among the top most commonly used cam therapies. it is of note that some chiropractic colleges teach a negative view of immunizations. in one study, one third of chiropractors agreed that there is no scientific proof that immunizations prevent disease. the basis for the negative views of vaccine effectiveness may lie in the chiropractic doctrine that disease is the result of spinal nerve dysfunction caused by subluxation coupled with the rejection of the germ theory of disease. , it may be that some chiropractors who adhere to this belief influence parents against immunizing their children. in one study, parents who requested immunization exemptions for their children were more likely to report cam use in their families than parents who did not request these exemptions. this emphasizes the importance of a trusting physician-patient relationship and providing parents with tailored information in advance of their child's immunizations; in this manner their questions are answered and they are prepared with the facts when they encounter contrary information from other sources. reaching out to chiropractic organizations to foster a better understanding of the benefits of immunizations may be advantageous to medical and public health professionals. parental concern about immunizations has been associated with a lack of trust. for example, one of the factors influencing parents who choose not to vaccinate their children for pertussis is doubt about the reliability of the vaccine information. in another study, compared with parents of vaccinated children, parents of children with an immunization exemption were more likely to express a low level of trust in the government, in addition to other factors such as low perceived susceptibility to and severity of vaccine-preventable diseases and low perceived efficacy and safety. these parents were less likely to believe that medical and public health professionals are good or excellent sources of immunization information. the majority of parents ( %), however, report receiving immunization information from a physician. thus, having a physician who engenders trust providing immunization information and who is available to listen and answer questions is the optimal situation from the public health perspective. if trust in a child's physician is low, parents may be drawn to other, less credible sources of information. when a child experiences an adverse event following receipt of a vaccine, it often raises the question "was this vaccine necessary"? to the parent, it may seem that the risks of the vaccine are greater than the risks of not getting the vaccine. parents who sought medical attention for any of their children owing to an apparent adverse event following immunizations ( . %) not only expressed more concern about immunizations, but also were more likely to have a child who lacked one or more doses of three high profile vaccines compared with parents who reported that none of their children had experienced an adverse event following immunization. two scenarios were seen as plausible. it may be that parents who were already concerned about vaccines before their child began the vaccination schedule were more reactive and thus sought medical attention for minor side effects (eg, fever) or nonrelated problems. it is also possible that an apparent adverse event following immunization that resulted in parents seeking medical attention for their child caused the parents' perception of vaccines to become more negative. both possibilities may result in parents declining future vaccines for their children. negative attitudes could be addressed by improving communication between clinician and parent. benefit-cost analysis research has shown that physician advice can produce benefits for health issues (eg, problem drinking). moreover, positive communication behaviors such as humor and soliciting questions are associated with lowered physician's risk of a malpractice suit. it may be that in this era of low vaccine-preventable disease incidence and increased public questioning of immunizations, improved provider communication can produce a positive net benefit for parents (reduced anxiety), a cost benefit to the health care system (reduced calls and medical visits for nonserious adverse events following immunization), and an improved physician-patient relationship (more trust and fewer malpractice suits). individual people can vary in their perception of the magnitude of vaccine risks. studies have shown that various factors such as sex, race, political worldviews, emotional affect, and trust are associated with risk perception. in addition, risk perception factors such as involuntariness, uncertainty, lack of control, and high level of dread can lead to a heightened perception of risks. all of these can be seen as associated with childhood immunizations. moreover, these factors have been referred to as "outrage" factors in the risk communication literature. outrage can lead to a person responding emotionally and can increase further the level of perceived risk. it can be difficult to communicate the risk of many vaccinepreventable diseases given their low prevalence in the united states and difficult to communicate the risks of serious vaccine adverse events because they affect such a small proportion of vaccine recipients. , several factors have been studied that might help people to better understand risk; the first are comparisons. comparisons that are similar (apples to apples) are reported to be better accepted, and, thus, comparisons for vaccines should focus on things that generally prevent harm in children but could pose a small risk (such as bicycle helmets, car seats). the second are visual presentations that help people understand numerical risk, including risk ladders, stick figures, line graphs, dots, pie charts, and histograms. unfortunately, there has been little research done in either of these areas. trust in the source of the risk information is an important factor in its ability to influence people and, as discussed, is developed through listening and ongoing communications. in , american adults had an average score of . on an index of biomedical literacy designed to measure understanding of biomedical terms and constructs. people with scores less than would likely find it difficult to understand medical stories about why antibiotics are not effective in combating the common cold and the relationship between certain genes and health. the main factors associated with biomedical literacy are the following: ( ) level of formal education, ( ) number of college-level science courses, and ( ) age. some characteristics of scientific literacy include the following abilities: ( ) distinguishing experts from the uninformed; ( ) recognizing gaps, risks, limits, and probabilities in making decisions involving a knowledge of science or technology; ( ) recognizing when a cause-and-effect relationship cannot be drawn; and ( ) distinguishing evidence from propaganda, fact from fiction, sense from nonsense, and knowledge from opinion. unfortunately, parental characteristics of those least motivated to obtain timely immunizations for their children are often characterized by low educational level of either parent. there is a wide gap in the level of biomedical understanding across the us population, and this gap emphasizes the need for tailored information. the need for tailored information applies to all areas of health, including childhood immunizations. immunization educational materials aimed at a middle level or a "one size fits all" are not likely to satisfy all parents' needs. the importance of educating parents concerned about vaccines why should we care about a small number of parents who are worried about vaccines for their children? we should care because it is not only ethically the right thing to do, it is also the right thing to do from a practical viewpoint. vaccine acceptability refers to the factors that go into parents' decisions to have their children immunized. it is important not to assume that just because most parents are having their children immunized that they will continue to do so. while the host of factors contributing to parents' decisions to have their children immunized (eg, need for information, experience with adverse events) might remain stable for some time, it is possible that one or more of the factors may change so that some parents perceive the risks of vaccines to be greater than the risk of disease. this would then push the parents above a theoretical "unacceptability threshold" in which they would choose not to have their children immunized with one or more vaccines. this is especially possible as more vaccines are added to the immunization schedule. an increasing number of parents have a choice, through religious or philosophical immunization exemption laws or schooling their children at home. averting the future possibility of outbreaks of vaccine-preventable diseases will take a concerted effort by health care and public health professionals to educate and better communicate with parents concerned about immunizations. in guidance for clinicians, the american academy of pediatrics suggests that pediatricians should listen carefully and respectfully to parents' immunization concerns, factually communicate the risks and benefits of vaccines, and work with parents who may be concerned about a specific vaccine or having their child receive multiple vaccines in one visit. providers can make a huge impact on vaccine acceptance, resulting in a cascading effect in which providing information can increase trust and increasing trust can lead to greater acceptance of and confidence in vaccines. for health care providers to be able to optimally fill this important role, however, two related issues should be addressed. the first is the need for quality communication courses and training in medical schools and residencies and training programs for medical and public health professionals. , the second is for mcos and medical insurance companies to adequately reimburse physicians for health education. lack of reimbursement to physicians has been noted as a barrier to implementation of behavioral treatments for health issues such as heart disease and smoking. it is important to note that studies have shown education programs can be a cost savings to health care systems. , we live in a world already benefiting from vaccines that exist, and there is the promise of more vaccines to come. the challenge we have now is to make sure that the promise is not lost because we did not present the benefits and risks of vaccines in a meaningful way acceptable to the public. an optimal immunization safety system requires rigorous attention to safety during prelicensure research and development; active monitoring for potential safety problems after licensure; and clinical research and risk management activities, including risk communication, focused on minimizing potential vaccine adverse reactions. prelicensure activities form the foundation of vaccine safety. rapid advances in biotechnology are leading to the development of new vaccines, and novel delivery technologies, such as dna vaccines and new adjuvants, are being developed to permit more antigens to be combined, reducing the number of injections. , new technologies can also be expected to be used to detect potential safety problems throughout the research and development process (eg, adventitial agents). a challenge will be determining the proper role and interpretation of new technologies. for example, a recent study used powerful new metagenomics and panmicrobial microarray technologies to screen for adventitious viral nucleic acid sequences in a number of vaccines. the study identified the presence of dna from porcine circovirus type (pcv ) in rotarix. this finding led to a temporary suspension of the use of the vaccine while the fda evaluated the study and its implications. ultimately, it was determined that the presence of the pcv nucleic acid sequences did not represent a health concern, and use of the vaccine was allowed to resume. in the prelicensure evaluation of new vaccines, the trend is likely to continue to conduct larger phase trials enrolling tens of thousands of participants. while such larger trials are helpful in identifying more rare adverse events, even these larger trials may not be large enough to detect increased risks of rare events. for example, the rotarix preclinical trial identified no increased risk of intussusception in a study that enrolled more than , infants. the manufacturer nevertheless committed to conduct a large postlicensure safety monitoring study. a preliminary analysis of postlicensure monitoring data from mexico identified a statistically significant increased risk within days of vaccination with an attributable risk of approximately per , . the attributable risk was much less than that found for rotashield (approximately per , ), and no changes were made to the vaccine recommendations. although technological advances and more thorough evaluation of safety before vaccines are licensed should lead to the development of safer vaccines, there will continue to be a need for comprehensive postlicensure safety monitoring systems. combined with the difficulties associated with identifying rare, delayed, or insidious vaccine safety problems in prelicensure studies, the well-organized consumer activist organizations, internet information of questionable accuracy, , media eagerness for controversy, , and relatively rare individual encounters with vaccine-preventable diseases virtually ensure that vaccine safety concerns are unlikely to go away. the existence of a robust vaccine safety monitoring system is essential for providing assurance of the safety of currently marketed vaccines and for rapidly identifying and responding to potential safety problems. currently, srss, such as vaers, serve as the frontline systems for the early identification of vaccine safety problems. such systems could be improved if reporting were more complete. application of web-based and text messaging technologies could make reporting easier and more accurate and also enable more active follow-up of vaccinated persons. alerts built into electronic medical record systems could also improve reporting to vaers, as could linkages with immunization registries. some of these advances will be particularly important to enable monitoring vaccine safety in mass vaccination campaigns during which vaccinations may be administered primarily outside of the traditional health care system. an optimal vaccine safety monitoring system must also include a mechanism or infrastructure to rapidly conduct formal epidemiologic evaluations of potential safety problems identified from srss or other sources. in the united states, this function is primarily served by the vsd project. the diffusion of electronic health records and the capability to link records across data systems (such as large health insurance claims databases and immunization registries) may allow the expansion of the population that could be included in postlicensure epidemiologic evaluations of vaccine safety. for example, the fda sentinel initiative has a goal to develop a national electronic system covering million people for monitoring the postmarket safety of drugs and other medical products, including vaccines. for adverse reactions that are established to be caused by vaccines, clinical and laboratory research is essential for determining the biological mechanisms of the adverse reaction, which in turn could lead to the development of safer vaccines. clinical research is also essential for the development of protocols for safer vaccination, including revaccination of persons who have previously experienced an adverse reaction. advances in genomics and immunology hold particular promise for elucidating biological mechanisms of vaccine adverse reactions and the development of possible screening strategies for persons who may be at high risk for an adverse reaction. a challenge for such research will be identifying sufficient numbers of people who may have rare vaccine adverse reactions and enrolling them into studies in which appropriate biological samples can be collected, stored, and analyzed under a standardized protocol. scientific data are essential in the monitoring and evaluation of vaccine safety, but scientific evidence alone often is not sufficient for providing reassurance about the safety of a vaccine. although immunization levels of us children are high, a sizable fraction of parents do not have their children fully immunized, and concern about vaccine safety is the leading reason for underimmunization. these concerns persist despite the scientific evidence that vaccines do not cause autism or a host of other conditions that have been alleged to be caused by vaccines, such as asthma, diabetes, and autoimmune diseases. thus, it is critically important that public health agencies, medical organizations, and other influential authorities continue to focus on the safety of vaccines and assure public confidence by providing clear, consistent messages on vaccine safety concerns; supporting effective and transparent vaccine safety monitoring systems and research activities; providing review and recommendations by respected independent expert groups on vaccine safety controversies; and engaging advocacy groups in constructive and open dialogue about their vaccine safety concerns. although the efforts of government, medical, and other authorities are important, it is health care providers who have the greatest influence in determining the acceptance of vaccines by individual people. even among parents who believe that vaccines may not be safe, most will have their children vaccinated if they have a trusting relationship with an influential health care provider. thus, development of tools and strategies that can assist health care providers in effectively communicating with their patients on the risks and benefits of vaccines will continue to be important. vaccine safety has also become an important concern in developing countries. the high-titer measles vaccine mortality experience highlighted the importance of improving the quality control and evaluating the safety of vaccines used in developing countries. plans to eliminate neonatal tetanus and measles via national immunization days, during which millions of people receive parenteral immunizations over a period of days, pose substantial challenges to ensuring injection safety, especially given concerns about inadequate sterilization of reusable syringes and needles, recycling of disposable syringes and needles, and cross-contamination resulting from the current generation of jet injectors. the who has promoted the use of safer auto-disposable syringes and disposal boxes. these and other new, safer administration technologies are urgently needed. in addition, there is a need to establish minimal vaccine safety monitoring capabilities, such as srss, and the capability to rapidly investigate vaccine safety problems and effectively communicate the findings of the investigations. vaccines are among the most successful and cost-effective public health tools for preventing disease and death. vaccines, however, are not completely without risk of side effects or other adverse outcomes. a timely, credible, and effective monitoring system, coupled with prompt action in response to identified safety problems, is essential to preventing adverse effects of vaccination and to maintaining public confidence in immunizations. since immunizations are typically administered to healthy people and are often recommended or mandated to provide societal and individual protection, vaccines must be held to a very high standard of safety. vaccine safety monitoring and research should optimally be able to detect potentially very small levels of increased risk, especially for adverse events that can result in death or permanent disability from vaccines that are universally recommended or mandated. the ultimate goal of such research, including the application of new developments in biotechnology, is to develop safer vaccines and vaccination practices. access the complete reference deadly choices: how the anti-vaccine movement threatens us all ensuring the optimal safety of licensed vaccines: a prospective of the vaccine research, development, and manufacturing companies active surveillance for adverse events: the experience of the vaccine safety datalink project understanding the role of human variation in vaccine adverse events: the clinical immunization safety assessment network addressing parents' concerns: do multiple vaccines overwhelm or weaken the infant's immune system? addressing parents' concerns: do vaccines cause allergic or autoimmune diseases? measles-mumps-rubella vaccine and autism thimerosal in vaccines: a joint statement of the american academy of pediatrics and the public health service autism's false prophets: bad science, risky medicine, and the search for a cure addressing parents' concerns: do vaccines contain harmful preservatives, adjuvants, or residuals we are grateful to robert davis, deborah gust, robert chen, and charles hackett who contributed sections of this chapter in previous editions of this book and for the excellent assistance on this chapter rendered by the following persons: dan salmon, john iskander, susan scheinman, christine korhonen, allison kennedy, michele russell, tamara murphy, penina haber, and gina mootrey. key: cord- -z vqfq authors: herndler-brandstetter, dietmar; grubeck-loebenstein, beatrix title: the efficacy of vaccines to prevent infectious diseases in the elderly date: journal: immunosenescence doi: . / - - - - _ sha: doc_id: cord_uid: z vqfq infectious diseases still represent a major challenge to human progress and survival. especially elderly persons are more frequently and severely affected by infectious diseases and they display distinct features with respect to clinical presentation and treatment. although vaccinations are considered a vital medical procedure for preventing morbidity and mortality caused by infectious diseases, the protective effect of vaccinations is abrogated in elderly persons. this is due to a decline in the functions of the immune system referred to as immunosenescence. the first part of this chapter will therefore summarize the status quo of the efficacy of vaccines in preventing morbidity and mortality caused by typical infectious diseases in the elderly, such as influenza, pneumonia and tuberculosis. the second part will then elucidate the underlying age-related mechanisms which may contribute to the decreased efficacy of vaccines. based on the complex mechanisms involved in immunosenescence, strategies will be outlined which may be succesfful in enhancing protective immune responses following vaccination in elderly persons. with respect to the current demographic development in many countries, including the european union and the united states of america, infectious diseases in geriatric patients are becoming an increasingly important issue. infections in elderly persons are not only more frequent and more severe, but they also have distinct features regarding clinical presentation, microbial epidemiology and treatment. urinary tract infections, lower respiratory tract infections, skin and soft tissue infections, infective endocarditis, bacterial meningitis, tuberculosis and herpes roster appear to have a higher prevalence in elderly persons. in developed countries like the united states, pneumonia, influenza and septicemia are ranked among the ten major causes ofdeaths in people aged s years and older. the reasons for the increased susceptibility to infectious diseases include epidemiological elements, imrnunosenescence, malnutrition and age-dependent anatomical alterations. infectious diseases still represent a major challenge to human progress and survival as they are responsible for about % ofall deaths in the world. this is not only related to microbial and viral factors but also to social and environmental determinants, such as social upheaval, urbanization, air travel, natural disasters and climate change.' newly emerging infectious diseases include acquired immune deficiency syndrome (aids), hepatitis c, several hemorrhagic fevers, severe acute respiratory syndrome (sars) and avian influenza. the resurgence ofseveral other infectious diseases is supported by the increased occurrence ofmultiple drug-resistant microorganisms such as staphylococcus aureus, mycobacterium tuberculosis, escherichia coli and streptococcuspneumoniae. *corresponding author: beatrix grubeck-loebenstein-institute for biomedical aging research, austrian academy of sciences, rennweg , innsbruck, austria. email: beatrix.grubeck-loebenstein@oeaw.ac.at immunosenescence, edited by graham pawelec. © landes bioscience and springer science+business media. altogether, this represents an enormous economic burden on health care systems all over the world. for instance, the annual costs ofmedical care for treating infectious diseases in the united states alone is about $ billion and for treating antimicrobial-resistant infections it may be as high as $ billion. ' a great success story was the implementation oflarge-scale vaccination strategies that led to the eradication of smallpox in and to a drastic reduction ofpoliomyelitis, tetanus, diphtheria, measles, pertussis and meningitis. presently, vaccinations are still considered the most cost-effective medical procedure for preventingmorbidity and mortality caused by infectious diseases. different infectious diseases can be prevented by vaccinations and vaccines are being developed according to a survey by the pharmaceutical research and manufacturers ofamerica," the new candidate vaccines are intended to provide protection against diseases caused by rotavirus, herpes zoster and papilloma virus and will be available from onwards ( table ) . but also improved vaccines against influenza, pneumonia and tuberculosis are currently being tested in clinical trials ( table ). this chapter now outlines the relevance ofvaccines to fight infectious diseases in old age and how age-related changes within the immune system contribute to the decreased efficacy of vaccines. it also discusses the progress made in the development of vaccines with improved immunogenicity in elderly persons. outbreaks of deadly infectious diseases such as ebola, marburg, sars or the h nl avian influenza regularly alert the world, whereas there is not much public attention paid to infectious diseases that cause substantial morbidity and mortality among the elderly population. for instance, influenza, invasive streptococcus pneumoniae infection, urinary tract and skin infections have a higher prevalence in elderly persons," old individuals may also fail to respond sufficiently to therapy and frequently suffer from opportunistic infections, recurrent infections with the same pathogen or reactivation oflatent diseases, such as those caused by mycobacterium tuberculosis or the varicella zoster virus. there are no vaccines available for many infectious pathogens that are frequent in elderly subjects and existing vaccines are underused and often do not assure such an effective protection as in young subjects. the following paragraphs will highlight the most important infectious diseases which threaten the elderly population and will provide information on epidemiology, vaccine availability and efficacy,vaccination coverage and general health authority recommendations. influenza is a highly contagious, acute viral respiratory disease that causes significant morbidity and mortality. the annual outbreaks affect approximately - % ofthe population worldwide with - million cases ofsevere illness and up to one million deaths each year. especially elderly people and persons that are chronically ill or otherwise immunocompromised are at enhanced risk. for example, during influenza epidemics, barker and mullooly reported two deaths per , healthy people below years ofage compared with per , in those over with two or more high-risk conditions? in contrast to measles, smallpox and poliomyelitis, influenza is caused by viruses that undergo continuous antigenic variation and possess an animal reservoir. therefore, we are recognizingannual epidemics that have been interruptedby three pandemics (spanish influenza, hinl, - ; asian influenza, h n , - andhongkonginfluenza,h n , ), caused by new influenza virus strains with increased virulence. influenza viruses are enveloped viruses containing eight single-stranded rna segments which encode for viral proteins, such as hemagglutinin (ha), neuraminidase (na), matrix protein (ml) and nucleoprotein (np) (fig. ) . influenza viruses belong to the family orthomyxoviridae and are divided into three genera, influenza virus a, b and c, based on antigenic differences in two oftheir structural proteins, m and np. disease symptoms caused by influenza c are rare whereas influenza b often causes sporadic outbreaks, especially in residential communities like nursing homes. influenza a viruses are further divided into subtypes according to the antigenicity oftheir major envelope glycoproteins, ha and na. with at least is different hemagglutinin and different neuraminidase subtypes, there is con siderable antigenic variation among influenza viruses. the human influenza viruses are currently limited to three hemagglutinin (h i, h and h ) and two neuraminidase subtypes (n and n ), whereas birds are the predominant hosts for the other subtype strains. ha initiates viral infection by binding to sialic acid residues on the carbohydrates ofglycoproteins present on epithelial cells ofthe respiratory trace. therefore, high-affinity iga and igg antibodies against ha may preven t infection from influenza virus. in contrast, na cleavesthe sialic acid from viral and cellular proteins to promote the release of newly synthesized influenza viruses from the infected host's plasma membrane. although antiviral drugs with moderate efficacy are available, active immunization represents the most vital element in th e prophylaxis ofinfluenza disease. however, the frequently occurring antigenic driftrequires an annual modification ofthe vaccinecomponents according to the recommendations ofthe who. therefore, vaccination has to be repeated annually to ensure protection against the circulating influenza strains. but vaccination coveragediffers largelywithin european countries. in , the rate ofvaccinedistribution washighest in spain, belgium,the netherlands, united kingdom and germany (between . and . %) and lowest in poland, czech republic, lithuania and latvia (between . and . %). canada and the united states had the highest rate ofvaccine distribution, being . and . %, respectively. remarkably, % ofus citizens aged and abovehave been vaccinated against influenza."although there are severalvaccinesavailable, the efficacyofmanyvaccinesin preventing influenza diseasein elderlypersons isonly around %. especiallyveryold and frail persons show a decreased response to influenza vaccines.p'ihe reduced vaccine efficacyis due to low levelsofiga and iggantibodies, delayed peak antibody titers and shortened maintenance of titers after vaccination. nevertheless, immunization in elderly people has been shown to be safe,cost-effective and associated with reduced rates ofhospitalization and influenza-relateddeaths.i , in particular, the efficacyofinfluenzavaccination to reduce mortality in elderlypeople is greater after repeated annual vaccination than after first administration. presently, influenza vaccines can be classifiedin split-virus, subunit, virosomal and live-attenuated vaccines ( table ). split-virus vaccines are used since the s, are cheap and offer a good protection for children above months of age and adults. recently, subunit vaccines with new adjuvants have been developed (fluado, addigrip') that show an increased immunogcnicity, a favorable safety profile and may be more suitable for the vaccination of elderly persons.'! additionally, invariant antigens, such as m and np, may also play an important role in protection and could be used in vaccines to induce long-lasting immunity to a variety of different influenza strains. another strategy to enhance immunogenicity may the use ofvirosomes that are nontoxic , biodegradable lipid-basedantigen-presentation systems." virosomal influenzavaccines, such asinflexalv', infiuvac plus'and invivac'have been on the market in several european countries for a number of years. they display a high immunogenicity and a similar safety profile in elderly persons compared with inactivated influenza vaccines.f" furthermore, new live-attenuated and subunit influenza vaccines ate currently in clinical trials that promise to have an increased efficacy ofprotection (table ) . especially live-attenuated influenza vaccines ate believed to elicit strong tcel! responses and should be able to enhance antibody levels after vaccination. importantly, before administration of live-attenuated vaccines to elderly or immunocompromised persons, an acceptable safety profile has to be demonstrated. irrespective ofthe improvement ofinfluenza vaccines for elderly persons, vaccination of children is clinically effective'" and high vaccination coverage among pupils has demonstrated to induce herd immunity and to reduce mortality in older adults. streptococcus pneumoniae is an important cause of invasive clinical manifestations such as bacterial pneumonia, meningitis and septicemia, particularly in young children and the elderly. to % ofdeaths associated with streptococcus pneumoniaeinfection occur in people aged years and above. several further groups at higher risk ofinvasive pneumococcal disease have been defined, including individuals with splenic dysfunction, immunosuppression, chronic pulmonary or cardiac disease, diabetes mellitus and chronic liver disease. generally, antibiotic therapy has to be initiated as soon as possible to reduce the risk ofcomplications due to pneumonia, meningitis or sepsis. nonetheless, % ofall deaths occur within the first hours despite adequate antibiotic therapy. this may be due to the increased occurrence of multiple drug-resistant pneumococcal strains. in , the proportion ofpenicillin-resistant streptococcus pneumoniaewas reported to be % in france and more than % in israel, poland, romania and spain." after recovering from pneumococcal infection, people are not necessarily immune, because there are about different serotypes and immunity will be guaranteed only to the strain that has caused the infection. currently, pneumococcal vaccines are available that include up to strains which are responsible to cause disease in almost % ofall cases ( table ). these vaccines offer protection against invasive pneumococcal disease in % of the general elderly population . whereas in elderly persons with high risk factors, the protective effect of vaccination seems to be only moderate. although many european countries recommend the administration of pneumococcal vaccines to all those > years ofage, vaccination coverage among the elderly population is very low. this may be due to the high costs ofthe vaccine and its unsatisfying efficacy in elderly people. but more immunogenic vaccines are currently in different phases ofclinical trials (table ) and promise to be more efficient in old age. additionally, implementing pneumococcal vaccination for children may decrease the incidence ofpneumococcal disease in the elderly by reducing transmission and possibly accomplishing herd immunity.-" each year, about million people are infected worldwide with the tubercle bacillus mycobacterium tuberculosis and . million ofthem die. the eu has a tuberculosis (tb) burden ofmore than , new casesper year,with the highest incidences in latvia, lithuania and estonia ( - cases/ , ). the risk ofdeveloping a disease following tb infection is about - % during lifetime and individuals above years ofage have a four-fold increased risk ofdeveloping tb than the average population," tb is also frequently diagnosed with delay due to an atypical manifestation in old age. this may lead to an increased morbidity and mortality and to a spreading ofthe disease, in particular within institutionalized elderly persons.p further difficulties include the increased emergence of new, multiple drug-resistant strains with higher rransmissibiliry," the poor efficacy of the current bacille calmette guerin (bcg) vaccine in protecting adults and elderly people from pulmonary infection" and the increased risk oftb co-infection in hiv positive patients." however, in the past few years, several tb vaccine candidates have entered phase i clinical trials, including adjuvanted subunit vaccines as well as improved live recombinant strains of the current bcg vaccine (table ). all these vaccine candidates are supposed to induce an effective and sustainable cellular immune response which is thought to be crucial to protect the host from an intracellular pathogen such as mycobacterium tuberculosis. primary infection with the varicella zoster virus (vzv) causes chickenpox which is usually a mild disease in childhood. the virus then persists in a latent form in sensory ganglia until its reactivation which results in the clinical manifestation of herpes zoster (shingles). between and % ofpersons with herpes zoster develop complications, such as postherpetic neuralgia." postherpetic neuralgia also increases with age with a prevalence of % in people aged years and above. the incidence and severity ofherpes zoster increase with age, because vzv reactivation is associated with a progressive decline in cell-mediated immunity to vzvy.· routine vaccination ofchildren using a tetravalent vaccine that protects against measles, mumps, rubella and varicella will soon be available (table ) and may reduce the incidence of chickenpox as well as the reactivation ofvzv in later life. since , a live-attenuated oka strain vzv vaccine is on the market that has shown clinical efficacy in preventing children from chickenpox." however, the currently available vzv vaccines have not been proven to adequately boost t-cell responses in older adults and to prevent reactivation ofherpes zoster. recently, a vaccine that may prevent herpes zoster virus reactivation has been submitted for registration. this live-attenuated vzv vaccine has been developed to prevent reactivation of herpes zoster in the elderly. s. this is of particular importance, because the elderly population has not been vaccinated against but may have been frequently infected by vzv. for instance, more than % ofadults in the united states have had chickenpox. as a consequence, it is estimated that up to , people in the united states suffer from shingles each year and the incidence is expected to increase as the population ages. thus, reactivation ofherpes zoster and its clinical manifestations represents a serious health burden to the growing elderly population and could be counteracted by potent vaccines. the cytomegalovirus (cmv), a b-herpesvirus, has also been shown to persist throughout life until its reactivation as a result ofimmune suppression or deficiency. cmv infection is quite common and affects - % ofthe adult population, dependingon the area. the cmvis transmitted via person-to-person contacts but immunocompetent subjects mostly do not recognize infection as it causes no or few unspecific symptoms. however, a cmv infection represents a severe health problem in immunocompromised persons (e.g.,due to immunosuppressive disease, chemotherapy or transplantation) or in a fetus as a result of congenital infection. research results over the past decade suggest that cmv favors an accelerated aging of the immune system as cmv infection is chronic and the organism is forced to continuously prevent virus reactivation.f'" despite the high frequency of cmv-specific cd + tscells, the virus usually can not be eliminated by the immune system. this is because the virus has evolved several mechanisms to escape the host's immune defense." for instance, cmv encodes for a type ofproteins called immunoevasins that modulate the presentation ofviral peptides or directly suppress cellular immune responses. hence, the accumulation of cmv-specific t-cells substantially constricts the diversity of the t-cell repertoire" and leads to the production ofproinflammatory cytokines, such as gamma interferon and tumor necrosis factor alpha.'? this imbalance in the cytokine production profile may not only promote the pathogenesis of age-related diseasesf but leads to a decreased production of antibodies following influenza vaccination in elderly persons.fa few antiviral substances including ganciclovir, valganciclovir, foscarnet and cidofovir are available to prevent cmv infection in immunocompromised patients. but antiviral therapy is limited by its severe adverse reactions, such as neutropenia, nephrotoxicity, hypocalcemia and seizures. another strategy is the adoptive transfer ofdonor-derived cmv-specific cd + and cd + t-cells that may restore the host's immunity against cmv. despite the need ofa safe and potent vaccine that prevents cmv disease, no vaccine candidate has yet entered the market. a few vaccines against cmv are currently in phase iiii clinical trials (table ) . active immunization against cmv could reduce the incidence ofneonatal infections as well as complications in immunocompromised persons and may prevent cmv-associated premature aging ofthe immune system when applied early in life. pertussis (whooping cough) is a highly contagious respiratory system infection caused by the bacterium bordetella pertussis and rarely by b.parapertussis, b. bronchiseptica or other pathogens. each year, more than million cases ofpertussis are reported worldwide, % ofwhich occur in developing countries, with an estimated , to , fatalities. the implementation of routine childhood vaccination against pertussis has reduced the high mortality rate among children. although most infants are being immunized against pertussis in industrialized countries, immunity usually fades during adolescence. consequently, a significant rise in pertussis incidence has been noticed in adolescents and adults." however, the reported pertussis cases in adults and elderly people are likely to be underestimated because symptoms ofdisease may be characterless and make clinical diagnosis difficult. among nonvaccinated elderly people the attack rate ofpertussis is high ( %) and up to % ofelderly persons may die from intracranial bleeding while they are symptomatic for pertussis." regular booster immunizations should thus be considered for adults and elderly persons, which is indispensable to remain protected from disease. tetanus is acquired via environmental exposure to the spores of clostridium tetani,which are present in soil worldwide. the disease is caused by a potent neurotoxin produced by the bacterium in dead tissue, e.g.,dirty wounds. diphtheria is a bacterial disease caused by corynebacterium diphtheriac and is transmitted from person to person through close physical contact. the public health burden ofboth diseaseshas been low in developed countries due to routine immunization. however, outbreaks ofdiphtheria have been reported in the independent states ofthe former soviet union, algeria, china, iraq, sudan, thailand and other countries. thus, maintaining high vaccination coverage is important to prevent the outbreak of new diphtheria epidemics. although vaccines that prevent from tetanus and diphtheria have been used for routine immunization for a long time all over the world, few studies exist that document their efficacyin elderly people. the vaccination coverage among elderly subjects is decreasing in several european countries and up to % of appropriately vaccinated elderly persons do not have protective tetanus-specific antibody concentrations. - therefore, public health authorities ofsome european countries have recommended five instead often year booster vaccination intervals for people over years ofage. additionally, strategies should be developed to draw public attention to the problem ofimmunizations in the elderly, to inform general practitioners and to increase vaccination acceptance. the increasing mobility ofelderly persons recognized worldwide is accompanied by an enhanced risk to encounter new antigens. this may be ofconcern because elderly persons possess a limited t-cell repertoire that may not guarantee full responsiveness to a wide variety ofnew antigens (see below for details). nevertheless, in vitro experiments have demonstrated that naive t-cells from elderly persons can still be stimulated by neoantigens, at least to the recombinant etr protein of tbe virus and rabies virus." based on an assessment of the risks for travel-related diseases, including the destination, the type of journey and the duration, vaccination is recommended to protect from typhoid and yellow fever, hepatitis a and b, japanese encephalitis, tick-borne encephalitis (tbe) or rabies. but elderly persons should also check whether they have followed the recommended booster intervals of routine immunizations, e.g., against tetanus, diphtheria, poliomyelitis, measles or influenza. tbe is caused by a virus that is primarily transmitted to humans by infected ticks. there are three genetically closely related subtypes of the tbe virus known (european, siberian and far eastern subtype). tbe is among the most dangerous neuro-infectious diseases in europe and asia and is responsible for up to , casesoftbe annually, most ofthem occurring in russia, czech republic and the baltic states." up to % ofadults with clinically confirmed tbe infection develop meningitis or meningoencephalitis and the lethality oftbe in europe is up to %.yet,there is no specific therapy available and, therefore, active immunization with inactivated whole virus provides the only efficient protection from tbe disease ( table ). importantly, more and more tbe cases are reported in people over years ofage and vaccination coverage in this population is lower than average. therefore, future strategies should increase the vaccination coverage among elderly persons and assure that they stick to regular booster intervals. anyhow, regular boosters should be given throughout life as this may favor the maintenance oflong-lastinghumoral immunity against tbe and may decrease the risk ofimmunization failures in the elderly. hepatitis a is an acute disease ofthe liver caused by the hepatitis a virus (hay), a nonenveloped virus belonging to the picornaviridae family. each year, an estimated . million cases ofhepatitis a occur worldwide. hay infection induces life-long immunity and is usually asymptomatic in young children, whereas adults frequently experience symptomatic disease. hay is acquired directly from infected persons by close contact or by the consumption ofcontaminated drinking water, vegetables, fruits or shells. hay vaccination is recommended when traveling to tropic and subtropic countries that have an increased risk of infection. for instance, the risk ofhepatitis a infection ofpersons traveling to developing countries was estimated to be to cases per persons per month ofstay,varying with destination, living conditions and age." improved sanitary standards in developed countries have reduced the opportunity for environmental exposure to hay and have lowered the overall incidence ofinfection. paradoxically, susceptibility to the virus increased because of the decrease in natural immunity. consequently, less than % ofpersons born after have a natural immunity against hay' in contrast to hepatitis b and c that may lead to the manifestation of a chronic infection, clinical illness after hepatitis a infection is usually mild in young individuals. but increasing age represents an enhanced risk ofsevere infection and mortality rates are about % for persons over and % for those over years ofage." several vaccines against hepatitis a are available (table ) and a study of adults showed that immunogenicity and safety profiles between 'iwinrix' and havrix' are comparable. but there is some evidence oflower antibody titers with advanced age. for instance, the seroconversion rates months after two doses ofhavrix' were found to be % and % for adults < years and > years, respectively. after the recommended immunization schedule with twinrix'.seroprotection was % and % for adults < years and > years, respectively. therefore, it may be useful to measure hay antibodies in elderly persons, as in the case ofvaccination failure, boosters have shown to be effective." it is further recommended that the vaccine is given at least to weeks before travel due to a slower onset ofthe antibody response in elderly individuals. another travel vaccine is directed against yellow fever (yf), which is endemic in tropic regions ofafrica and south america. yf is transmitted by the bite ofinfective aedes aegypti and other mosquitoes that bite during daylight hours in regions below meters ofaltitude. most infections lead to an acute illness characterized by fever, muscular pain, headache, anorexia, nausea and/or vomiting, often with bradycardia. after a few days, about % ofpatients progress to a second phase, with resurgence offever,development ofjaundice, abdominal pain and haemorrhagic manifestations. halfofthese persons die - days after the onset ofillness. the who estimates that a total of , cases ofyf occur each year, with about , deaths. oyf also represents a significant risk to more than million travelers that visit yf-endemic areas each year. neonates and elderly individuals demonstrate the highest mortality when infected by the yf virus. as there is no specific antiviral treatment against yf available yet, vaccination is the only way to protect persons from yf disease. the currently available vaccine contains a live-attenuated d strain virus ( table ) and has been shown to be safe and highly potent." however, due to the increased use in international travelers, it has become evident that advanced age might be a risk factor for serious adverse effects and even deam. compared with persons aged - years, individuals aged < had an -fold greater risk to experience serious adverse events after vaccination. the rate for systemic illness requiring hospitalization or leading to death after yf vaccination was reported to be . per , among people to years of age and . per , for people more than years. furthermore, there are no studies available that demonstrated the efficacy of the yf vaccine in elderly persons. accordingly, recommendations and manufacturing standards have been modified to increase vaccine safety in elderly persons. although the benefit-risk ratio still favors the vaccination ofpeople at high risk for infection and outlines the vaccine's fundamental role in disease prevention and control, efforts to improve safety and to ensure vaccine efficacy in elderly persons are ofurgent need. the term immunosenescence refers to a complex remodeling ofthe immune system in old age and may contribute significantly to morbidity and mortality in the elderly. thymic involution, telomere shortening, t-cell signal transduction changes, alterations in the interaction of the innate and adaptive immune response, impaired dna repair and antioxidant mechanisms as well as persistent antigenic stress may all be factors contributing to immunosenescence. although perturbations ofinnate immune system components have been described, much ofthe decrease in immunoresponsiveness seen in elderly people is associated with changes in t-cell responses. this is due to the continuous loss offunctional thymic tissue with increasing age. . the thymus, the central lymphoid organ, is responsible for the maturation and selection ofso-called naive t-cells that regenerate the peripheral tcell pool and retain the capability ofthe immune system to respond to a variety ofdifferent pathogens. in old age, the number ofnaive t-celis decreases while the number ofantigen-experienced 'tcells increases. . these antigen-experienced t-celis include a substantial proportion of senescent memory-effector t-cells that accumulate in elderly persons. senescent memory-effector tcelis display phenotypic (loss ofcostimulatory molecules such as cd and cd l) as well as functional changes (altered cytokine production profile, decreased proliferative response, shortened telomeres, increased resistance to programmed cell-death and restricted t-cell diversity). . ofparticular importance, the senescent cd +cd -memory-effector t-cell population predominantly produces the pro-inflammatory cytokine gamma interferon (ifny), but does not produce interleukin (il- ) and the anti-inflammatory, b-cell stimulating cytokine il- . recent data also support the hypothesis that chronic infection with the cytomegalovirus, ã -herpesvirus, may lead to a decrease in the size ofthe naive and early memory cd + t-cell pool, but to an increase in the number of dysfunctional, ifny-producing cd +cd -memory-effector tvcells (fig. ) , one clinical consequence of the accumulation of cd +cd -t-cells is an impaired generation of protective antibody levels after vaccination. . furthermore, the age-dependent increase in the level ofpro-inflammatory cyrokines may lead to ubiquitous chronic inflammatory responses in old age and may therefore support the development of age-related chronic diseases, such as atherosclerosis," rheumatoid arthritis" and alzheimer's disease. , although individuals maintain a relatively constant total number ofperipheral b-celis during aging, each b-cell subset comprises severeperturbations in size,dynamics and repertoire. the alterations affecting the bvcell subsets are due to a decreased generation ofb-cell precursors, such asearly lymphoid precursors and pro-bvcells, cell-intrinsic as well as micro-environmental disturbances are both likely to contribute to the decreased output ofpro-bscells. furthermore, alterations in environmental factors also impair overall v(d)j recombinase activity among pro-bvcellst'whlch accounts for the limited b-cell repertoire frequendy detected in elderly persons." although no decrease in overall serum immunoglobulin levels have been observed during aging, the antibodies generated in old age are oflower affinity due to a shift in antibody isotypes from igg to igm, of particular importance, b-celis from elderly individuals are stimulated % less efficiendy by follicular dendritic cells than bvcells from young subjects," suggesting loss ofb-cell function, in part due to the decreased expression ofcostimulatory molecules, such as cd or cd , impaired t-cell-mediated immunity as well as defects in antigen presentation by antigen presenting cells there is a tremendous need to increasethe protective effect ofvaccines in the elderly. research of the last decade has provided new insights into the molecular mechanisms ofthe immune response in old age, which can now be used for th e development of potent vaccines. in the past, vaccines were primarily designed to elicit a strong humoral immune response . however, vaccines in elderly persons may be more effective ifthe stimulate innate immune components and the generation of long-lived memory t-cells. currently,severalstrategies are being pursued to increase immunogeniclry, to minimize adverse side effects and to increase vaccine acceptance by introducing needle-free injection devices. proven and promisingvaccine technologies are used to design conjugate, subunit, live vector, dna and live-attenuated vaccines (table ) . while live-attenuated vaccines (e.g.• against varicella, measles or yellow fever) stimulate numerous immune components and display enhanced immunogenicity, conjugate and subunit vaccines (e.g., against influenza) are often supplemented with adjuvants" to ensure their protective effect. generally, adjuvants can be divided into antigen delivery systems (cationic microparticles, proteasomes and virus-like particles) and immune potentiators (e.g.• cytokines). these adjuvants may overcome the proposed age-related functional decline ofinnate immune responses by targeting pattern-recognition receptors, such as the recently identified toll-like receptors or nucleotide-bindingoligomerization domain proteins." the enhanced activation ofthe innate immune system may also improve antigen processing and presentation leading to more potent t and b-cell responses and to sustained immunological memory. vaccines supplemented with the dna of cytokines (e.g., il- , il-? il-i , il-is or il- ), chemokines or costirnulatory molecules may magnify immune responses by generating more and long-lived memory t_ceiis - and may overcome immunodominance.'" in addition to improve vaccine efficacy, a modification of vaccination strategies for elderly persons has been supported by the results of several vaccination trials. for instance, a decreased response and a shortened duration ofprotective immunity following booster immunization is a characteristic feature of old age. thus , several european health authorities have recommended five-year vaccination intervals for tetanus, diphtheria, pertussis and pneumonia. increased public awareness of regular booster vaccinations in adults should be enforced, as these immunization regimes may be essential to maintain the ability to respond to recall antigens in old age. recent result s also indicate that long-lasting protection but also a good booster effect can be expected even a long time after the last vaccination, when a live-attenuated vaccine (e.g., polio vaccine) is used for primary immunization in early life. new delivery systems that make use oftiny micro-needles or non-injectable application devices (nasal , oral, transcutaneous) may further increase vaccination acceptance, especially in the case of influenza as this vaccination has to be repeated annually. infectious diseases in elderly persons are becoming an increasingly importan t issue. an utmost need represents the development ofmore immunogenic vaccines for the elderly. the improvement of specific vaccine types regarding immunogenicity and tolerability, th e addition of adjuvants. the design ofnew delivery systems as well as specific immunization regimes should all contribute to an enhanced efficacy ofvaccines in elderl y persons. further improvements may comprise the adjustment ofvaccination intervals in old age, the increase in vaccine acceptance and vaccination coverage as well as raising people's awareness to stick to the recommended booster vaccination intervals throughout life. in the distant future, vaccines may also play an important role in treating non-infectious diseases such as allergy, autoimmunity, alzheimer's disease and cancers. deaths: leading causes for social and environmental risk factors in the emetgence of infectious diseases a successful eradication campaign. global eradication of smallpox survey: new medicines in development for infectious diseases ageing and infection pneumonia and influenza deaths during epidemics: implications for prevention influenza and pneumococcal vaccination coverage among persons aged> or = years and persons aged - years with diabetes or asthma-united states the efficacy of influenza vaccine in elderly persons. a meta-analysis and review of the literature immunity and immunization in elderly the efficacy and cost effectiveness of vaccination against influenza among elderly persons living in the community influenza vaccination programs for elderly persons: cost-effectiveness in a health maintenance organization reduction in mortality associated with influenza vaccine during - epidemic evidence of increased clinical protection of an mf -adjuvant influenza vaccine compared to a non-adjuvant vaccine among elderly residents of long-term care facilities in italy immunogenicity of new virosome influenza vaccine in elderly people immunogenicity of trivalent subunit versus virosome-formulated influenza vaccines in geriatric patients randomized study to compare the immunogenicity and reactogenicity of an influenza split vaccine with an mf adjuvanted subunit vaccine and a virosome-based subunit vaccine in elderly clinical experience with inactivated, virosomal influenza vaccine inactivated influenza virus vaccines in children the japanese experiencewith vaccinating schoolchildren against influenza the -valent pneumococcal polysaccharide vaccine. part .efficacyof ppv in the elderly: a comparison of meta-analyses impact of pneumococcal vaccination on morbidity and mortality of geriatric patients: a case-controlled study changing epidemiology of invasivepneumococcal disease among older adults in the era of pediatric pneumococcal conjugate vaccine. lama stead ww: tuberculosis tuberculosis in the elderly antitb drug resistance in the world efficacy of bcg vaccine in the prevention of tuberculosis. meta-analysis of the published literature colnfection with hiv and tb: double trouble lnununology of tuberculosis population-based study of herpes zoster and its sequelae varicella-zoster virus: pathogenesis, immunity and clinical management in hematopoietic cell transplant recipients stress-induced subclinical reactivation of varicella zoster virus in astronauts live attenuated varicella vaccine vaccination boosts adult immunity to varicella zoster virus a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults long-term cytomegalovirus infection leads to significant changes in the composition of the cd + t-cell repertoire, which may be the basis for an imbalance in the cyrokine production profile in elderly persons human immunosenescence: is it infectious? dysfunctional cmv-specific cd +t-cells accumulate in the elderly cytomegalovirus misleads its host by priming of cd tvcclls specific for an epirope not presented in infected tissues cytomegalovirus seropositivity drives the cd t-cell repertoire toward greater clonaliry in healthy elderly individuals inllamm-aging. an evolutionary perspective on immunosenescence lack of antibody produ ction following immunization in old age: association with cd +cd ' t-cell clonal expansions and an imbalance in the production of thl and th cyrokines restoration of viral immunity in immunodeficient humans by the adoptive transfer oft-cell clones the epidemiology of pertussis: a comparison of the epidemiology of the disease pertussis with the epidemiology of bordetella pertussis infection an epidemic of pertussis among elderly people in a religious institution in the netherlands vaccination against tetanus in the elderly: do recommended vaccination strategies give sufficient protection no immunity for the elderly a population-based serologic survey of immunity to tetanus in the united states t-cells from elderly persons respond to neoantigenic stimulation with an unimpaired il- production and an enhanced differentiation into effector cells epidemiology and ecology of tbe relevant to the production of effectivevaccines tbe vaccination and the austrian experience vaccine immunogenicity and safety of a booster vaccination against tick-borne encephalitis more than years following the last immunisation epidemiology and prevention of hepatitis a in travelers hepatitis a and hepatitis b: risks compared with other vaccine preventable diseases and immunization recommendations a prospective,randomized, comparative us trial of a combination hepatitis a and b vaccine (twinrix) with corresponding monovalent vaccines (havrix and engerix-b) in adults immunogenicity of an inactivated hepatitis a vaccine in dutch united nations troops immunogenicity of combined hepatitis a and b vaccine in elderly persons the effect of age and weight on the response to formalin inactivated, alum-adjuvanted hepatitis a vaccine in healthy adults district guidelines for yellow fever surveillance persistence of neutralizing antibody - years after immunization with d yellow fever vaccine advanced age a risk factor for illness temporally associated with yellow fever vaccination thymic involution in aging thymic involution with ageing: obsolescence or good housekeeping? age-related loss of naive t-cells and dysregulation of t'-cell/ b-cell interactions in human lymph nodes marked increase with age of type cytokines within memory and effector/cytotoxic cd + t-cells in humans: a contribution to understand the relationship between inflammation and immunosenescence the aging of the immune system cd t-cells and aging value of immunological markers in predicting responsiveness to influenza vaccination in elderly individuals artherosclerosis as an infectious, inflammatory and autoinunune disease role of the immune system in the pathogenesis, prevention and treatment of alzheimer's disease how chronic inflammation can affect the brain and support the development of alzheimer's disease in old age: the role of microglia and astrocytes bone marrow microenvironmental changes underlie reduced rag-mediated recombination and bvcell generation in aged mice the effect of age on the b-cell repertoire ageing, autoimmunity and arthritis: senescence of the b-cell compartment -implications for humoral immunity age-related depression of fdc accessory functions and cd ligand-mediated repair of costimulation b-cells in the aged: cd , cds and cd expression ineffective humoral immunity in the elderly vaccine development strategies for improving immunization: the role of modem immunology survey of human-use adjuvants targeting the innate immune response with improved vaccine adjuvants augmentation and suppression of immune responses to an hiv-l dna vaccine by plasmid cytokine/ig administration coimmunization with an optimized il- plasmid results in enhanced function and longevity of cd t-cells that are partially independent of cd t-cell help il- influences the frequency, phenotype and affinity of the antigen-specific cd t-cell response adjuvant il or il- overcomes immunodominance and improves survival of the cd + memory cell pool insufficient protection for healthy elderly adults by tetanus and tbe vaccines immunizations in the elderly: do they live up to their promise the authors wish to acknowledge the support ofthe austrian science fund and the austrian green cross society for preventive medicine. key: cord- -h cj izx authors: roach, e. steve title: child neglect by any other name date: - - journal: pediatr neurol doi: . /j.pediatrneurol. . . sha: doc_id: cord_uid: h cj izx nan if million people say a foolish thing, it is still a foolish thing. anatole france barely three weeks old, the baby lay fighting for life due to intracranial hemorrhages resulting not from physical trauma, but from medical neglect. child neglect includes knowingly failing to protect a child from preventable harm. his parents had refused the administration of vitamin k after birth. why? because they didn't believe in doing things that are not "normal and natural." sadly, failure to administer vitamin k to newborns, typically a single injection or a series of oral doses, has become commonplace in the united states, allowing a resurgence of the deadly hemorrhagic disease of the newborn that had become almost non-existent in westernized countries. schulte and colleagues noted that % of the babies born in private birthing centers in their area and . % of the babies born at their own academic medical center failed to receive vitamin k after birth, with sometimes tragic results. at first "normal and natural" sounds like a sweetly quaint and wholesome approach, until one considers the brutal reality of what it could mean to a child's chances of survival if fully implemented. in , when things were certainly very "natural," only % of children survived to age five. a century later, that number had improved slightly to about %. in recent years, over % of children survive to age five years. much of this stunning improvement in child mortality resulted from prevention and treatment of infections through improved sanitation, the development of antibiotics, and vaccines targeting once deadly and crippling diseases. no rational person would wish to experience those terrible losses again. indeed, rather than letting nature take its cruel path no matter how devastating the consequences, we should be trying to improve on the natural course in order to optimize each child's chances to survive and thrive. refusal of vitamin k administration shares with vaccine denial an unwillingness to accept the sound scientific evidence supporting the practice. lulled into complacency by the lower frequency of deadly contagious diseases in recent decades and gullibly accepting the internet-amplified comments of prominent but misinformed celebrity "spokespeople," too many parents discount the compelling proof of the safety and effectiveness of vaccines. some parents may be merely afraid and unaware of the facts, and they need only appropriate information and respectful guidance. these parents are merely advocating for their child's well-being. others seem to be so firmly entrenched in their beliefs that no amount of proof will change their mind. like their sister skeptics, the "climate deniers," the vaccine deniers defiantly dismiss any contrary information, and the introduction of additional scientific evidence only seems to strengthen their confidence in the correctness of their own unsubstantiated beliefs. we physicians must bear some of the blame here, as do many of our elected officials. by feebly accepting vaccine denial as even approaching a rational option, we become enablers of inappropriate choices by individuals who are ill-equipped to weigh the evidence or choose to ignore it. not all physicians have been so complacent, of course. the american academy of pediatrics has consistently and strongly recommended immunizations, although they could have been more direct in discussing the ethical failure that parental denial of immunizations represents. some individual physicians have taken a strong stand on the need for immunization despite the online bullying by militant vaccine deniers that often ensues. but on the whole, our collective response has been anemic. admittedly, physicians are placed in a very difficult situation when dealing with vaccine deniers. beneficence on behalf of the child, whose welfare is their primary concern, compels physicians to advocate strongly on behalf of the benefit that vaccines convey to that child. they may have a secondary obligation to educate the child's parents about the value of immunizations, but the child's wellbeing is paramount. as surrogate decision makers for their child, parents also have an ethical duty of beneficence that obliges them to embrace scientifically established procedures that will increase the child's likelihood of health and well-being. in parallel, the physician also has an obligation to avoid the spread of preventable infections in other children in their practice. and while discharging the unimmunized child from the physician's practice may initially seem like a plausible solution, abandoning the child because of their parents' failure to act in their child's best interests may not be the best approach. physicians need to avoid "science speak." we sometimes obscure the facts with thickheaded comments such as "there is no epidemiological evidence for a causal association" when we should say simply and directly that an assertion is "blatantly false." even the somewhat euphemistic term "vaccine hesitancy" lends a noblesounding aura to a very irrational and potentially deadly thought process. trying to "engage" families in order to educate and convince them of the wisdom of immunization is fine for the parents who want information and are willing to accept guidance, but this approach is clearly wasted on the entrenched vaccine deniers. perhaps our message needs to also directly articulate the concept of vaccine denial as a form of child neglect. the vaccine deniers may continue to ignore the scientific evidence, but at least there would be no room for doubt about what we physicians recommend. there is nothing good about the current novel coronavirus pandemic that is sweeping the globe, but if hundreds of thousands of coronavirus-related deaths serve to make some of the skeptics finally grasp the deadly seriousness of infections in a world without vaccines, it will have at least achieved something. a few of us can remember the similar terror surrounding the epidemics of poliomyelitis before the advent of vaccines, and none of us want to see children die from bacterial meningitis again. one hopes that the vaccine deniers will opt to accept a coronavirus vaccine when it becomes available, although recent outbreaks of preventable illnesses such as mumps and measles among immunized individuals seem to have opened few closed minds. the administration of vitamin k is about as close as one ever gets to risk free. similarly, the safety and efficacy of vaccines have been thoroughly established. vaccines do not cause autism, a bogus but persistent notion that arose from a long-since retracted publication containing fabricated data. , about four children per , children have a febrile seizure after receiving an immunization, arguably fewer children than would experience a febrile seizure during the very illnesses prevented by the vaccines. a few children have medical reasons to avoid specific vaccines, but immunizations are overwhelmingly safe. the bottom-line question is "does the potential benefit of an immunization exceed the likelihood of an adverse effect?" if the answer to this question is unequivocally yes, which it almost always is for immunizations, then refusal to allow vaccination after being fully informed of the facts amounts to child neglect. parents are afforded broad freedom to raise their children in keeping with their own culture and values. provided that the child is not harmed, this approach is appropriate. but injuring a child, purposely denying adequate nutrition, and failing to protect a child from preventable risk, even in the name of discipline and decorum, is taboo in most civilized cultures. ultimately a child's right to exist, free of avoidable injury or illness, should supersede a parent's right to do whatever they wish when rearing their children. parents are not always allowed to deny well-validated medical treatments for their children. a parent who attempts to deny a child chemotherapy for acute lymphocytic leukemia, for example, is typically met with a court order terminating their custody of the child until after she undergoes the needed chemotherapy. chemotherapy drugs have many serious side effects, so one can easily understand how a parent might want to avoid the whole situation. , but in the end, the child's right to live, or in this example, to maximize his odds of surviving, trumps the parents' desire to avoid chemotherapy. so why are parents allowed to forbid the administration of life-saving vaccines or vitamin k but not allowed to deny cancer chemotherapy? leukemia represents a clear and present danger to the child, while vaccines reduce the risk of disease that might or might not occur. no doubt the looming certainty of death from untreated leukemia makes it easier for the authorities to summon the courage to act, but is there an acceptable threshold for allowing a preventable risk? how much avoidable risk to a child is too much to ignore? if the imminent danger argument were reasonable, then how does one explain required car seats for children? most car trips do not result in accidents, after all, but some of them end just as tragically for the unrestrained child as would untreated leukemia. the car seat requirement is designed to maximize the odds of a child's survival in the event of an accident, and it has nothing to do with whether the danger is imminent. why are vaccines not viewed in a similar fashion? of course, few things are simple. the likelihood of some preventable infections is considerably higher than others. measles and mumps outbreaks are increasingly common, for example, while polio is nonexistent in much of the world. some of the benefit of immunizations is societal rather than individual, and vaccine denial would result in far more disease were it not for the herd immunity resulting from the responsible immunization of most children. is it fair for an individual's rejection of established scientific evidence to place their child and other people at risk in the name of personal freedom or preference? is it fair to ask others to accept the human suffering and financial burden resulting from infections that could easily have been prevented? parents also have an ethical responsibility to not promulgate preventable disease in other individuals. the argument that it is acceptable to decline vaccines because they have risks is utter nonsense, because the likelihood of preventing a disease with a vaccine is higher than the risk of a complication. the family's cultural background, intentions, and level of sophistication may sometimes be relevant when defining child abuse and neglect, but never to the point of justifying a child's injury or exposure to preventable risk. while visiting the middle east, i once encountered a bedouin child with failure to thrive and hypotonia. she had numerous oval pigmented lesions on her abdomen resembling burn scars. in western countries, the sight of intentional burn marks on a malnourished child would send most of us scurrying to notify the authorities. but cautery is a commonly employed folk remedy in her culture. her burns had resulted from application of a hot spoon from the campfire to her abdomen, the site thought to be responsible for her poor weight gain. was this child abuse? the family's intent was to help the child, not to hurt her, cautery was an accepted traditional ritual in their culture, and their ability to learn about better options may have been limited. yet she suffered avoidable burns and her failure to thrive was not quickly assessed by physicians, so she was harmed, despite the family's benign intentions. one might argue that vaccine denial represents a similar situation. the vaccine deniers do not intend to harm their children, of course, and in some circles, withholding vaccines is so prevalent and so entrenched that it resembles a primitive cultural belief system. but most western families who fail to immunize their children know about vaccines and have ready access to physicians and nurses who could clearly explain their risks and benefits. yet some of them opt to deny the solid science that would give their child the best odds of staying healthy. i will at least give the bedouins some benefit of the doubt. it is time to stop the political correctness and "science speak." parents should have the right to raise their children in accordance with their own preference, culture and religious beliefs, provided that their approach does not substantially increase the child's odds of an avoidable illness or injury. but given the extremely low risk of immunization and vitamin k administration, the bar for "substantial" risk should be extremely low. vaccine denial may not cross a threshold that triggers harsh measures by the authorities, but there should be no acceptable preventable risk. no matter how wellintentioned the decision may be, the willful, informed avoidance of scientifically proven measures that would improve a child's odds of optimal health and survival amounts to child neglect. physicians must rise with one voice and say "enough!" by even considering the premise that vaccine denial can be a reasonable choice by a rational individual, we become enablers of child neglect. intracranial hemorrhage in early infancy--renewed importance of vitamin k deficiency rise in late onset vitamin k deficiency bleeding in young infants because of omission or refusal of prophylaxis at birth ileal-lymphoid-nodular hyperplasia, nonspecific colitis, and pervasive developmental disorder in children ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children febrile seizure risk after vaccination in children one to five months of age responding to parental refusals of immunization of children professional responsibility and early childhood vaccination neurological complications of the treatment of pediatric neoplastic disorders a review of chronic leukoencephalopathy among survivors of childhood cancer ancient mideast cauterization practices and developmentally delayed children: a call for advocacy key: cord- - ix c he authors: rajan, v. title: an oral vaccine for tgev immunization of pigs date: - - journal: commercial plant-produced recombinant protein products doi: . / - - - - _ sha: doc_id: cord_uid: ix c he transmissible gastroenteritis virus (tgev) is a commercially important pathogen of hog farms and causes contagious, lethal diarrhea in piglets. while orally and parenterally administered vaccines made from inactivated or attenuated tgev are commercially available, they require individual administration to piglets, which is time and labor intensive, and run the risk of reversion to pathogenicity. also, parenteral vaccines produce neutralizing serum antibodies which may be less effective against an orally transmitted pathogen, compared to an oral vaccine that would induce the production of mucosal antibodies. there has been an effort to produce subunit vaccines in an edible form in plants for convenient administration through feed. these efforts towards the expression of the s-antigen of tgev in maize seed, its effectiveness at inducing neutralizing antibody production in the colostrum of gilts, and its efficacy in protecting piglets against challenge by virulent tgev are summarized here. epidemic diarrhea virus (pedv), and porcine hemagglutinating encephalomyelitis virus (hev) (sestak and saif ) . it is pertinent to examine the structure of tgev and related viruses to explain the choices of epitopes available for the production of effective subunit vaccines. all coronaviruses have an envelope with radiating structures composed of trimers of the kda spike glycoprotein (s, also referred to as peplomer e ), as well as the smaller membrane glycoprotein (m, - kda) and envelope protein (e , kda) ( fig. . ). the m protein interacts with the nucleocapsid n protein and the viral rna to form the icosahedral nucleoprotein core (masters ) . the s-protein, specifically the n-terminal domain between amino acids - , binds aminopeptidase n receptor in the epithelium of the small intestine and mediates the fusion of the host and viral membranes and uptake (belouzard et al. ). the surface and subviral structural components of the virus have been assessed for antigenicity, and neutralizing antibodies were found to be associated with the surface components which function in recognition and binding with subviral and host proteins (garwes et al. ) . the m protein induces interferon production and also binds neutralizing antibodies (laude et al. ). the s (or e ) protein has been found to be the most effective epitope at inducing neutralizing antibodies. the s-protein has four major antigenic sites at the n-terminal: a, b, c, and d; a and d are involved in antigen neutralization (reguera et al. ; correa et al. ; fig. . schematic of the coronavirus virion, with the minimal set of structural proteins. reproduced with permission from masters ( ) jiménez et al. ). induction of cross-protection with the use of a related virus has been attempted. pcrv shows tropism towards respiratory tissues and differs from tgev in having a deletion of the n-terminal - amino acids (containing antigenic sites a and d) of the s-protein, indicating that the a and d sites may be involved in tissue specificity. immunization with pcrv was partially protective under the same conditions- % of piglets survived challenge (de diego et al. ) . the s-antigen was therefore selected as the most efficient immunogen for a subunit vaccine. high-level expression of heterologous eukaryotic proteins has been successfully attempted in both prokaryotes and eukaryotes. however, many eukaryotic proteins require posttranslational modifications such as glycosylation, which are not carried out by prokaryotes, and so eukaryotic systems are sometimes necessary to obtain functional end products. the benefits of plants as production systems are primarily the low cost of production and rapid scale-up that make it more responsive than other methods to high production targets. traits introduced by transgenic techniques into separate lines can be combined by traditional breeding methods to produce lines with a combination of these traits. the production of bulk plant material requires only space, fertile soil, and sunlight, which are less onerous to provide than sterile fermentation chambers and animal care. plants do not harbor animal pathogens, and the concern for contamination of products with animal pathogens, such as prions or viruses, is alleviated. plant material has been shown to serve to bioencapsulate proteins, permitting slow release in the gut (kong et al. ; verma et al. ; bailey ) . thus, there may be a degree of dosage flexibility with oral antigens administered in plant tissue, and the feed can be administered over a larger window to accommodate time frames for induction of secretory and humoral immunoglobulins (bailey ; daniell et al. ) . important considerations before attempting expression of heterologous eukaryotic proteins in plants are the requirements for posttranslational protein modifications, such as glycosylation, and the presence of allergens and toxins. glycosylation moieties and patterns differ between animals and plants which has raised concerns over the potential for nonfunctional proteins or an allergic reaction to the plant glycosylated protein. these differences, however, do not appear to be significant functionally (ma et al. ; suzuki et al. ) , and plant carbohydrate-specific iges in allergic patients were shown to be clinically irrelevant when the protein is orally administered (mari et al. ; bosch and schots ) . this lack of allergenicity may be partly explained by tolerance to plant glycosylation induced by oral exposure (see ghaderi et al. for an overview of glycosylation of biotherapeutic proteins). potential concerns about glycosylation-induced allergenicity may also be overcome by the removal of plant glycosylation sites, inactivation of plant glycosylases, and the expression of plant deglycosylases and mammalian-type glycosylases (sethuraman and stadheim ; desai et al. ; mamedov et al. ) . host plants should also be screened for toxic or allergenic metabolites that may co-purify or be co-administered with the target protein. edible tissues such as grain from plants with generally recognized as safe (gras) status may be directly used for feed-based vaccines (naqvi et al. ) . seeds are a natural, desiccated, storage system lacking proteases, and recombinant proteins produced in seed have been stable at room temperature for extended periods of time (naqvi et al. ) . selection and backcrossing into parent seed and hybrid lines can establish uniform concentrations of antigen expression (hood et al. ) and standard processing and formulation methods can reliably produce consistent concentrations of antigens for administration, which is not straightforward with perishable edible tissues such as lettuce leaves and bananas. bioencapsulation of the antigen in plant tissue prevents premature degradation resulting in the antigen persisting in the intestine, a key factor for the induction of a protective siga response, important for protection against ingested pathogens. a system that exemplifies the gras system is maize (or corn) grain which was used in the work described here. its structure and specific advantages are described below. the maize kernel contains three main regions: the embryo, the endosperm, and the pericarp (fig. . ). the pericarp includes the seed coat and fruit layers. the pericarp originates from the ovary wall and functions to protect the seed. the aleurone layer is the outermost layer of the endosperm and lies directly below the pericarp. aleurone grains contain enzymes involved in the breakdown of starch and proteins in the germinating seed. the embryo and endosperm are well suited to heterologous protein accumulation as both are rich in protein and have associated tissue-specific promoters. the volume of the endosperm is greater than that of the embryo allowing, in theory, more protein accumulation but, in practice, embryo-preferred promoters have been used to produce some of the highest recorded concentrations of heterologous proteins (hood et al. ; egelkrout et al. ) . desiccation of the seed provides an environment that protects proteins from enzymatic degradation (ma et al. ) , and maize seed has cystatins (yamada et al. ; massonneau et al. ) and other protease inhibitors (jongsma and bolter ) as a defense against proteolysis by insect pathogens. the ideal vaccine for tgev will provide mucosal, lactogenic, and systemic immunity, while being simple to administer to droves which may number in the many thousands. potentially, the methods to protect naïve piglets at highest risk from tgev infection are to provide immune colostrum by vaccinating sows and an oral/nasal vaccine to boost secretory neutralizing antibody levels. since tgev is an enteric disease, the induction of siga is inferred to be more protective. it has been observed that the mucosal immunoglobulin, iga, is more efficiently induced through gut-associated lymphoid tissue (galt) if the antigen persists in the intestine (foss and murtaugh ) . induction of passive immunity transmitted through colostrum can be mediated by either of the soluble immunoglobulins igg or iga. initially, igg is more abundant and protects against systemic infection, whereas igas in colostrum provide protection to the gut lumen where the enterocytes in which tgev replicates reside. iga production and mucosal immunity is thus the response desired. there have been three main routes taken to effect protection in young piglets: ( ) parenteral immunization with inactivated virus, ( ) oral/nasal administration of attenuated virus, and ( ) oral administration of viral subunit antigens in feed. parenteral immunization of young piglets with inactivated virus is ineffective as it does not induce the local immune response in the gut, nor are piglets at this age capable of mounting an immune response. however, parenteral immunization of pregnant sows can induce production of protective immunoglobulins in colostrum and milk. the disadvantage of this approach is the necessity of individually vaccinating sows, which can be time-consuming and expensive. immunization must also be done on a schedule prior to birth that maximizes immunoglobulin presence in the colostrum, and that can be difficult, especially with large numbers. nasal administration of attenuated viruses does overcome this difficulty, but each pregnant animal still needs to be isolated and the vaccine delivered separately. as with parenteral injection, the vaccination schedule is important because, while the attenuated virus does replicate in the gut, thus stimulating antibody production over an extended period, viral replication is suboptimal. while the oral vaccine can be more easily administered, the frequency of inoculation has to be modulated to maximize colostrum immunoglobulin timed with birth. the option of using the oral route to deliver viral subunit antigens in feed is fairly straightforward in terms of administration: the food is put out in feeding troughs and animals are monitored by observation to ensure all animals have had access to the food. however, dosage for orally administered vaccines in feed is particularly significant, because antigens could be ingested at greater or lesser doses depending on the amount of feed consumed, and some of the antigens would be expected to be destroyed by the digestive process. the typical vaccine dosage in feed may be up to times greater than that used for parenteral administration, but this may be reduced by the use of suitable targeting or carrier molecules (carter and langridge ) . still, common consensus is that a precise dose, taking into account degradation in the gut, must be given to all animals. interestingly, verma et al. ( ) have shown that the response from a -fold range of antigen levels given encapsulated in plant materials is comparable. finally, with any vaccine, oral or parenteral, there is a concern over the potential induction of tolerance, which has been shown to occur for orally administered antigens with repeated doses of antigens over a long term and is mediated by regulatory t-cells. a single large dose has also been shown to induce anergy-a mechanism of adaptive tolerance that cause t-cells, in an environment presumably low in co-stimulators, to become unresponsive to antigens (weiner ; weiner et al. ) . therefore, prior to widespread vaccination, tests must be conducted to ensure this is not an issue. with these caveats addressed, plants have proven to be efficient hosts for heterologous protein production. de diego et al. ( ) showed that oral immunization with tgev was more effective at inducing the presence of neutralizing antibodies in colostrum and milk compared to intranasal immunization with pcrv. they attributed this difference to galt being more effective than bronchus-associated lymphoid tissue (balt), going even to the extent of questioning if balt was indeed an integral mammalian structure (pabst ) . pigs have been shown to not have balt constitutively (delventhal et al. ; pabst and gehrke ) . thus, the use of intranasal stimulation by pcrv may have been less effective for this reason. peyer's patches in the gut have long been known to be associated with galt, and oral immunization has been shown to be effective in producing iga and igg globulins in milk and was % effective in protecting - day piglets obtained from seronegative sows against challenge. this experiment (de diego et al. ) established that persistence in the gut and therefore a longer-term mucosal response in older animals can be achieved by using an attenuated live vaccine which replicates in the gut. a variety of viruses have been used as vectors for expression of full length and truncated versions of s-antigen. baculovirus-infected cells containing the n-terminal fragment of the s-antigen induced neutralizing serum antibodies in piglets (tuboly et al. ) . furthermore, oral immunization of piglets using s-antigen-recombinant porcine adenovirus induced both neutralizing antibodies in sera and mucosal antibodies in the intestine (tuboly et al. ) . fusions of the s-protein expressed in e. coli were immunogenic and produced cognate antibodies but did not neutralize the virus. s-protein expressed in vaccinia virus produced neutralizing antibodies when injected into animals (hu et al. ) . the expression of s-antigen in nuclear polyhedrosis virus and its expression in insect s cells allowed the production of a secreted version which was immunogenic in rats despite lack of proper glycosylation (godet et al. ) . mucosal and serum antibodies were also elicited in rabbits orally inoculated with an attenuated strain of salmonella typhimurium expressing a fusion of the d-epitope of the s-antigen to e. coli heat-labile toxin b-subunit (lt-b) (smerdou et al. ) . various tgev antigens have been produced in plants. gomez et al. ( ) expressed either the n-terminal residues, or the entire s-protein, under the control of the camv s promoter in arabidopsis. the antigen was purified from leaves and administered to mice. the mice produced antibodies that reacted specifically with tgev and neutralized infective particles. subsequently, the same group expressed the n-terminal domain in potatoes and obtained a serum response to both intraperitoneally delivered tuber extracts and orally administered tubers. immunoprecipitation and elisa results showed that antibodies were detecting the native protein; however, the sera did not neutralize the virus in vitro (gomez et al. ) . tuboly et al. successfully produced neutralizing serum antibodies in piglets using tobacco plants expressing various permutations of the s gene under the control of a synthetic super promoter . in an early experiment using s-antigen, gomez et al. ( ) used a camv s promoter to drive expression of an un-optimized sequence in arabidopsis. while the protein produced was immunogenic, its levels were too low to be visualized by sds-page and could only be detected by western blotting. subsequently, used plant-optimized s-antigen sequences driven by a super promoter and increased levels to . - . % total soluble protein (tsp) s-antigen expression in tobacco. using codon-optimized sequences for maize fused to the barley alpha-amylase signal sequence (baass), lamphear et al. ( ) achieved levels of s-antigen expression in maize seed of mg/kg. baass targets protein to the cell wall and is later cleaved-a process that permits higher levels of accumulation in the apoplast than is possible in the cytoplasm. using standard plant techniques, f lines of maize that had high levels of expression were selected and backcrossed to commercial maize lines to obtain stable, increased levels of protein expression. since the protein was produced in maize, a gras plant, further purification before oral administration was not necessary. lamphear et al. ( ) studied the levels of serum neutralizing antibodies induced by administration of tgev-s corn alone in piglets. - -day-old tgev seronegative piglets were divided into three groups (normal rations, control corn, tgev-s corn) of four piglets each, with mg of the antigen administered in ground corn mixed with medicated milk replacer. days after the last antigen administration, the piglets were challenged with ml of orally administered virulent tgev. serum was collected and neutralizing antibody levels measured (fig. . a) . piglets fed tgev-s corn showed more than tenfold greater neutralizing serum response as well as milder symptoms (a geometric mean titer of . in tgev-s-corn fed piglets compared to titers of in the control groups). while the presence of serum antibodies is significant, greater protection will potentially be afforded by mucosal antibodies. pregnant gilts received the following treatments: group a (oral corn vaccine on days À to À and À to À ), group b (oral corn vaccine on days À to À and À to À ), group c (oral corn vaccine on days À and À ), group d (oral corn placebo on days À to À ), group e (intramuscular live vaccine on days À and À ), and group f (oral corn vaccine on days À to À ), where the "À" sign represents days before farrowing. reproduced with permission from lamphear et al. ( ) . see text (sect. . . ) for discussion the induction of neutralizing antibodies in both serum and colostrum was examined in gilts following the administration of oral tgev vaccine in maize comprising a subunit vaccine of the s-protein expressed in corn (lamphear et al. ) . two kg doses of corn containing mg of antigen were administered to gilts previously sensitized with three doses of modified live vaccine (mlv-tgev, intervet) with two oral administrations and days before farrowing and one intramuscular injection days before farrowing. subsequently, they were divided into six groups for testing (a-f; see fig. . b) and administered oral tgev-s corn or control corn at various schedules. tgev-s corn was given to group a (oral corn vaccine on days À to À and À to À ), group b (oral corn vaccine on days À to À and À to À ), group c (oral corn vaccine on days À and À ), group d (oral corn placebo on days À to À ), group e (intramuscular live vaccine on days À and À ), and group f (oral corn vaccine on days À to À ), where the "À" sign represents days before farrowing. serum antibody levels dropped by up to half at the time of farrowing but were still relatively high compared to control (data not shown). colostrum obtained on the day of farrowing showed neutralizing antibodies were present. antibody levels in animals in group c administered test corn for days were almost . times higher than animals administered intramuscular injection with modified live virus, indicating that corn was more effective at inducing secreted antibody than parenteral vaccine and that the window of administration may be important. that secretory antibodies were produced in colostrum is a clear indication of the stimulation of a mucosal response. because it was not clear if the antibodies induced by oral administration were protective against infection, a trial to test protection against challenge was carried out with - -day-old, specific pathogen-free piglets lamphear et al. ) . piglets were fed tgev-s corn, control placebo corn, and orally administered mlv-tgev controls. all piglets were challenged with virulent tgev on day and monitored twice daily for symptoms for days until the end of the study. following challenge, piglets were scored twice daily for signs of diarrhea (normal ¼ , creamy ¼ , watery ¼ ) and other symptoms (dehydration and depression, anorexia ¼ , vomitus ¼ , moribund or death ¼ ) to give a total clinical score. clinical symptoms for each study group were scored as follows: percent morbidity incidence [(number of animals with clinical signs scoring ! divided by total number of animals) Â ], percent morbidity incidence and duration [(total number of clinical observations ! divided by the product of the total number of pigs and days scored) Â ], or clinical severity index (total clinical score divided by the product of the total number of pigs and days scored). half of the control group and about % of the mlv-tgev group showed morbidity, compared to % of the tgev-s corn ( -day administration) group. morbidity duration and clinical severity were highest in control corn, as expected, but tgev-s corn showed better protection even than mlv-tgev. increased duration of administration showed slightly higher morbidity levels (fig. . ) , but piglets administered tgev-s corn for days showed no symptoms. this unexpected finding suggests the possible induction of oral tolerance to extended exposure of antigen for longer periods than days. the levels of humoral and secreted (serum and stool) antibodies in these piglets were not monitored, but it seems likely that the levels were diminished by the extended exposure. this is an important result, as it indicates that a shorter duration of administration of oral vaccine is more effective and more economical as well. s-antigen expression in maize seed was directed by a constitutive polyubiquitin promoter and the protein was present in endosperm, aleurone, and embryonic tissues. whole seed expressed antigen at a level of μg/g. fractionation to determine the sites of protein accumulation within the seed showed that the embryo (germ tissue) accumulated the highest concentration of recombinant protein of μg/g-double the concentration of whole grain. the levels in bran (aleurone and pericarp layers) were very low, and endosperm contained levels in between those in grain and pericarp (lamphear et al. ) . stability of antigen was examined in whole grain stored for months at ambient temperature without humidity monitoring, at c in a seed storage facility at % humidity, and in corn meal stored at c ( fig. . ). levels of antigen in stored grain were compared to freshly harvested grain, and no difference was found. this important result indicates that antigen is stable even under uncontrolled conditions at ambient temperature, making it less onerous for storage and transport over long periods and permitting elimination of the cold chain for transport and delivery. tgev-s antigen was not a novel target for expression, but several hurdles that limited its efficacy were overcome. maize, as the bioencapsulating agent, presumably prolonged exposure to galt tissues. high levels of expression and stability were achieved using a constitutive promoter and an apoplast targeted baass sequence. the antigen was stable in stored grain, overcoming a major economic hurdle. reproduced with permission from lamphear et al. ( ) in this work, it was also unexpectedly discovered that the smallest dose regimens, administered purely by the oral route, were also the most effective. significantly, when young pigs were fed a constant diet of the antigen for weeks, protection was reduced, an indication that tolerance may develop with long-term exposure (lamphear et al. ) . the duration of protection for piglets following antigen administration in feed was not tested. administration of antigen over a -day period gave the highest levels of protection against live challengeeven higher than oral vaccination with a modified live virus (fig. . ; see sect. . . ). in gilts, as well, the shortest oral regimen of two boosters showed the highest colostrum levels of serum neutralizing antibodies. thus, if passive immunity transferred by colostrum is protective in suckling pigs, then this regimen would be more economical as well as less onerous. in gilts, colostrum levels of antibodies persisted for about - days following piglet birth (lamphear et al. ) , which provides passive immunity while the piglets developed their gut flora and become suitable subjects for the administration of vaccines in feed. fig. . antigen stability in tissues from transgenic corn seed: measurement of extracted tgev-s antigen as mg antigen per g extracted soluble protein from grain or grain meal stored for months at either c, c, or at ambient temperature in a grain storage facility in iowa. values represent mean ae one standard deviation. figure reproduced with permission from lamphear et al. ( ) regulatory approval must be obtained for each step of the process, including compliance for growing transgenic plants to licensure of the vaccine product. plant-based vaccines have already been approved for use with livestock and with humans. in , the usda approved a plant-produced vaccine against newcastle disease virus for administration to fowl by dow agrosciences. in , fda approved the first pharmaceutical product produced in plants, elelyso™ (taliglucerase alfa), to protalix biotherapeutics and pfizer. thus, precedents now exist for both human and animal pharmaceuticals produced in plants. the existence of these plant-based vaccines smooths the path for the development and regulatory approval of future plant-based vaccines. some essential regulatory procedure for growth of transgenic plants and approval of veterinary vaccines in the usa are described below. similar to the requirement for other vaccines produced in yeast or eggs, the plant crop must not inadvertently be intermixed with commodity food. one important feature of the plant production system is that field-grown plants have the potential to pollinate other food or feed crops. the aphis arm of the usda issues permits for the growth of transgenic corn. these include a number of growing restrictions to prevent intermixing of the crop inadvertently with other food and feed crops. to this point, the usda has developed a highly restrictive set of isolation conditions for growing the crop, over and above the standard conditions for producing vaccines, including a one-mile isolation corridor from other corn. as maize pollen is relatively heavy, it normally only pollinates other corn plants within a few meters (luna et al. ) making this an extreme precaution. nevertheless, as the growing acreage needed for such a product is low (~ , acres, or . % of the total acreage of corn in the usa to produce two doses of mg of vaccine at mg antigen/kg corn for each of the estimated million swine and assuming a yield of bushels/acre), it is quite reasonable to find isolated areas to grow the crop. after harvesting, the corn can be processed into corn meal, blended to obtain precise dosing, and formulated to the final product. production of a vaccine is only the first step in the process towards licensing. the vaccine must be safe and effective in order to obtain usda approval for use through the veterinary biologics program. guidelines are set out by the usda under the virus-serum-toxin act ( u.s.c. - ). the cost of production is always a potential limitation for any product to be commercialized. in this case, since the product is produced in corn which is a feed source for pigs, it does not need to be purified thereby reducing a major expense. furthermore, the corn itself has value as a feed product, further lowering the effective cost of the vaccine. the actual cost of the vaccine can therefore be calculated by the cost to keep the grain segregated from food and feed crops and loss in yield over commodity crops. both of these costs are further reduced by having high levels of antigen in maize, thereby limiting the amount of corn needed to be grown. in theory and in practice, the cost should be lower than the traditional injected vaccines. the indirect benefit of not having to physically inject the animals provides another value proposition making this even more desirable. the first group that needs to accept this product is the swine producers. this is largely dependent on their perceived need for added protection from the disease. once this hurdle is overcome, there is always the concern over trying any new product. acceptance by swine farmers, however, should be relatively straightforward since this product should cost less to administer than traditional vaccines. even though farmers may accept this relatively readily, the general public is leery of transgenic crops. on a rational basis, this product would not pose a threat to the general public for several reasons: ( ) the product will be quickly digested and will not persist in the animal at the time of slaughter; ( ) the protein itself is present in the food chain when animals are infected by the virus, and the use of vaccines limits its presence; ( ) the protein itself poses no threat as it has no toxicity or enzymatic activity; ( ) as a protein, the degradation products are amino acids that are common to all living organisms; ( ) as pork is cooked, any protein would be quickly denatured prior to human consumption; and ( ) growing maize expressing s-antigen will involve very small acreage, thus a nationwide concern would not be triggered in the event of an inadvertent exposure. unfortunately, logical arguments are not always sufficient; undoubtedly a subset of the general public will fear the product for reasons other than safety arguments. this can be due to a general fear of transgenic crops, vaccines, fear of new products, aversion to technology, or other fears. while all of these are considerable hurdles, they can be overcome if the benefits of this product outweigh the risks. in this case, the benefits can be determined by how much of a threat is perceived by the disease and the cost and efficiency of this approach can eliminate the threat. while the goal of most vaccine production in plants is commercialization, the dearth of products on the market speaks to the many barriers that must be crossed before the goal is achieved: ( ) the product must be made at a level in plants that makes it commercially viable; ( ) for scale-up with plants such as corn, adequate barriers to dissemination must be incorporated and aphis approval obtained; ( ) the product must be tested in the target animal to rule out negative side effects, and approval for marketing must be obtained; and ( ) finally, a large investment to scale-up and market the product is needed. for large companies such as dow agrosciences, which take a product from the lab to the market, this is less formidable than for small biotechnology companies which need to move to market at speed lest funding wane while the process is still underway. protection with a subunit antigen expressed in corn, exclusively by the oral route, is shown for the first time to be effective in piglets, the target species for immunization. this demonstration, using corn-encapsulated s-antigen administered orally as both primer and booster, could circumvent the need for parenteral vaccinations or oral immunizations with modified live virus, making the process of vaccinating large herds much more economical and less time-consuming than using injected vaccines. this work has three significant outcomes: ( ) the demonstrated use of an oral subunit vaccine in production of neutralizing antibodies in both adults and young pigs, ( ) neutralizing antibodies from both active and passive immunity being protective to a direct challenge with live virus, and ( ) short duration of oral exposure of antigen ( days) being sufficient to develop complete protection from challenge. the use of maize seed that can be administered directly through feed clearly shows that this approach provides protection that can be as good if not better than injectable products. using antigen-expressing corn as a top dressing on feed has the additional advantage of also bioencapsulating the antigen, which extends its contact with galt in the gut and provides a greater immune response. storage stability has also been demonstrated, with both the whole seed and meal, at room temperature and with refrigeration. in short, this approach demonstrates that a practical, low-cost, heat-stable, orally delivered vaccine is achievable. production of subunit antigens in well-tolerated, edible plant tissue opens the door to a lot of possibilities. several antigens to different diseases can be combined into a single vaccine by standard breeding techniques. since antigens expressed in maize seed are tolerant to storage over long periods at ambient temperature, they can be stockpiled against zoonotic outbreaks. the elimination of the cold chain can be highly significant in rural areas. the use of the edible vaccine as both primary and booster increases convenience and lowers duration of administration to animals. adjuvants can be co-expressed as needed to improve immunogenicity. clearly, there is ample room for improvement of this technology. the use of tgev-s protein clearly shows commercial potential as described above. using more recent technology, improvements could be made to produce a higher concentration of the antigen or having it targeted specifically to the embryo. this study also represents one of the first clear demonstrations of providing protection against a pathogen in animals, paving the way for other vaccine antigens to be tested. a model system for edible vaccination using recombinant avidin produced in corn seed mechanisms of coronavirus cell entry mediated by the viral spike protein plant glycans: friend or foe in vaccine development? contact investigation of a case of human novel coronavirus infection treated in a german hospital plant-based vaccines for protection against infectious and autoimmune diseases antigenic structure of the e glycoprotein from transmissible gastroenteritis coronavirus medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants characterization of the iga and subclass igg responses to neutralizing epitopes after infection of pregnant sows with the transmissible gastroenteritis virus or the antigenically related porcine respiratory coronavirus effects of microbial stimulation on the number, size and activity of bronchus-associated lymphoid tissue (balt) structures in the pig production of heterologous proteins in plants: strategies for optimal expression enhanced expression levels of cellulase enzymes using multiple transcription units mechanisms of vaccine adjuvanticity at mucosal surfaces antigenicity of structural components from porcine transmissible gastroenteritis virus production platforms for biotherapeutic glycoproteins. occurrence, impact, and challenges of non-human sialylation processing and antigenicity of entire and anchor-free spike glycoprotein s of coronavirus tgev expressed by recombinant baculovirus expression of immunogenic glycoprotein s polypeptides from transmissible gastroenteritis coronavirus in transgenic plants oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus novel coronavirus spreads manipulating corn germplasm to increase recombinant protein accumulation studies of tgev spike protein gp expressed in e. coli and by a tge-vaccinia virus recombinant critical epitopes in transmissible gastroenteritis virus neutralization the adaptation of insects to plant protease inhibitors oral immunization with hepatitis b surface antigen expressed in transgenic plants delivery of subunit vaccines in maize seed a corn-based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine single amino acid changes in the viral glycoprotein m affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus maize pollen longevity and distance isolation requirements for effective pollen control the production of recombinant pharmaceutical proteins in plants production of non-glycosylated recombinant proteins in nicotiana benthamiana plants by co-expressing bacterial pngase f evaluation by double-blind placebo-controlled oral challenge of the clinical relevance of ige antibodies against plant glycans maize cystatins respond to developmental cues, cold stress and drought the molecular biology of coronaviruses high-value products from transgenic maize the sars wake-up call is balt a major component of the human lung immune system? is the bronchus-associated lymphoid tissue (balt) an integral structure of the lung in normal mammals, including humans? antigenic modules in the n-terminal s region of the transmissible gastroenteritis virus spike protein porcine coronaviruses challenges in therapeutic glycoprotein production a continuous epitope from transmissible gastroenteritis virus s protein fused to e. coli heat-labile toxin b subunit expressed by attenuated salmonella induces serum and secretory immunity plant-based vaccines: unique advantages expression, characterization, and biologic activity of recombinant human lactoferrin in rice immunogenicity of the s protein of transmissible gastroenteritis virus expressed in baculovirus immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants development of oral vaccine in plants against transmissible gastroenteritis virus of swine oral delivery of bioencapsulated coagulation factor ix prevents inhibitor formation and fatal anaphylaxis in hemophilia b mice oral tolerance: immune mechanisms and treatment of autoimmune diseases oral tolerance a cysteine protease from maize isolated in a complex with cystatin key: cord- -afzjx ci authors: modrow, susanne; falke, dietrich; truyen, uwe; schätzl, hermann title: vaccines date: - - journal: molecular virology doi: . / - - - - _ sha: doc_id: cord_uid: afzjx ci vaccines are predominantly used for prevention; that means they should establish a protection in immunized people or animals which will protect them from a possible infection and the subsequent illness when they come into contact with the respective pathogens. fundamentally, there are two kinds of immunization: active and passive. the latter is based on the administration of immunoglobulin preparations that can neutralize a specific virus. therefore, passive vaccination is applied only in special cases, such as when the person to be protected recently had verifiable contact with a specific virus (postexposure prophylaxis), or if the risk of exposure to pathogens cannot be ruled out in the following weeks and an active vaccination is not possible, as in short-term planned trips to third world countries (exposure prophylaxis). an example is the administration of antibodies specific for hepatitis b virus in cases of contamination with blood from people who have an acute or chronically persistent infection with this virus, and thus have high concentrations of infectious particles in the blood. such accidents occur primarily in medical personnel by needlestick injury ( . / - - - - _ ). in certain cases, the administration is performed in combination with an active vaccination (active–passive immunization). specific immunoglobulin preparations are also administered when people have been bitten by animals that may be infected with the rabies virus ( . / - - - - _ ). in the case of early application (together with an active vaccination), the antibodies can neutralize the virus, and impede its spread in the body. since the time between contact with the virus and its spread in the organism is often very short, passive immunization is limited to a period shortly before or after exposure to the infective agent (usually within days). therefore, it is reserved for cases in which the contact with the potential pathogen is well documented and the type of infection is known, and when an appropriate immunoglobulin preparation is available. the protection afforded by antibody preparations lasts just a few weeks, as immunoglobulins are rapidly degraded in the organism. therefore, postexposure administration of active vaccines is increasingly preferred, e.g. in the context of outbreak-control vaccination. in veterinary medicine, passive immunization is employed occasionally in young animals which were born in a flock with high infection pressure. this approach is applied, for example, in kennels when infections occur with canine parvovirus ( . / - - - - _ ). however, its value is controversial, as the immunoglobulins administered hinder the more advantageous active immunization. vaccines are predominantly used for prevention; that means they should establish a protection in immunized people or animals which will protect them from a possible infection and the subsequent illness when they come into contact with the respective pathogens. fundamentally, there are two kinds of immunization: active and passive. the latter is based on the administration of immunoglobulin preparations that can neutralize a specific virus. therefore, passive vaccination is applied only in special cases, such as when the person to be protected recently had verifiable contact with a specific virus (postexposure prophylaxis), or if the risk of exposure to pathogens cannot be ruled out in the following weeks and an active vaccination is not possible, as in short-term planned trips to third world countries (exposure prophylaxis). an example is the administration of antibodies specific for hepatitis b virus in cases of contamination with blood from people who have an acute or chronically persistent infection with this virus, and thus have high concentrations of infectious particles in the blood. such accidents occur primarily in medical personnel by needlestick injury (▶ sect. . ). in certain cases, the administration is performed in combination with an active vaccination (active-passive immunization). specific immunoglobulin preparations are also administered when people have been bitten by animals that may be infected with the rabies virus (▶ sect. . ). in the case of early application (together with an active vaccination), the antibodies can neutralize the virus, and impede its spread in the body. since the time between contact with the virus and its spread in the organism is often very short, passive immunization is limited to a period shortly before or after exposure to the infective agent (usually within days). therefore, it is reserved for cases in which the contact with the potential pathogen is well documented and the type of infection is known, and when an appropriate immunoglobulin preparation is available. the protection afforded by antibody preparations lasts just a few weeks, as immunoglobulins are rapidly degraded in the organism. therefore, postexposure administration of active vaccines is increasingly preferred, e.g. in the context of outbreak-control vaccination. in veterinary medicine, passive immunization is employed occasionally in young animals which were born in a flock with high infection pressure. this approach is applied, for example, in kennels when infections occur with canine parvovirus (▶ sect. . . ). however, its value is controversial, as the immunoglobulins administered hinder the more advantageous active immunization. on the other hand, the active vaccine produces a long-lasting protection against infection, which can even last for life. in this case, a protecting immune response is induced. ideally, it consists of a combination of neutralizing antibodies and cytotoxic t cells (▶ chap. ). the active immunization can be done in two ways: with live vaccines or by using inactivated vaccines. the various methods which are now used to develop vaccines or which are being attempted are illustrated schematically in fig. . . live vaccines contain attenuated, replication-competent pathogens that can replicate in the vaccinated person, i.e. they are able to infect certain cells and initiate the synthesis of viral proteins and particles, but without triggering the respective clinical picture. these viral components are recognized by the immune system of the vaccinee as foreign (exogenous), which induces the production of specific neutralizing antibodies and cytotoxic t lymphocytes (▶ chap. ). neutralizing antibodies are predominantly directed against viral surface structures, and can bind to the surface of the virus, thus preventing both attachment of the virus to target cells and infection. the immune complexes of antibodies and virus particles activate the complement system, or are phagocytosed by macrophages and neutrophils. cytotoxic t cells recognize infected cells by the t-cell receptor on their surface; these infected cells synthesize viral (foreign) proteins and present peptide fragments derived from them in complex with mhc class i antigens. recognition leads to the destruction of infected cells, and thus to the elimination of the virus from the organism. live vaccines can develop a very effective protection against a number of viral pathogens, as they effectively activate both the humoral and the cellular mechanisms of the immune system. therefore, live vaccines are generally the ideal vaccine candidates. however, the implementation of their application requires refrigeration systems for storage, which can lead to difficulties in some developing countries. attenuated viruses are similar to pathogenic viruses with respect to structure, protein composition and infection behaviour. however, they clearly differ from them with regard to virulence (▶ chap. ). in comparison with the wild-type virus, they usually cause just a limited or weakened infection, which in most cases does not cause clinical symptoms, and can easily be controlled by the immune response of the organism. the proteins produced during the infection are ideally identical to those of the virulent virus strains, or they are at least very similar to them. the fact that viral polypeptides are synthesized in the cells during an attenuated infection leads to the emergence of virus particles which induce the generation of neutralizing antibodies and cytotoxic t cells. therefore, the immune response that is triggered by attenuated viruses is suitable to induce a long-lasting, effective protection against infections with the respective pathogen. the molecular bases of attenuation are mutations in the genome of wild-type viruses. different genes may be affected in the various vaccine viruses. frequently, it is not even known why they generate an attenuation of the wild type. one way to attenuate virus strains is their continuous cultivation and passaging in cell culture. in this way, virus mutants are selected which are optimally adapted to the cell culture conditions. occasionally, they lose their virulence during this process. this method made possible, for example, the isolation of attenuated polio, measles and yellow fever viruses, which do not cause diseases in humans, in contrast to wildtype infections (table . , ▶ sects. . , ▶ . and ▶ . ). another example is the vaccine that was successfully applied to protect pigs against infections by classical swine fever virus (▶ sect. . ). in this case, the virulent virus was attenuated by continuous passages in rabbit cells. subsequently, these lapinized viruses were no longer able to trigger diseases in swine. meanwhile, the application of this originally very successful vaccine has been banned. the justification for this lies particularly in trade. on the one hand, infections with the attenuated vaccine virus could not be distinguished from infections with classical swine fever virus in blood tests; on the other hand, following a vaccination, an export ban is imposed for a limited period, which, however, has economically adverse effects. instead, it is attempted to interrupt the infection chain and to eliminate the virus from the population by culling all herds in which classical swine fever occurs (▶ chap. , ▶ sect. . ). by transgressing the species barrier, the infection can occasionally adopt a weakened character. similarly, vaccinia viruses that were originally used to produce a protective immune response against smallpox virus induced local infections in humans, which in very rare cases had a generalized or fatal course. because of this problem, further attenuation of vaccinia virus was sought by repeated passages in chicken cells. in fact, this approach resulted in modified vaccinia ankara virus (▶ sect. . ), which exhibits fewer adverse side effects, and is currently being tested as a vector system for expression of recombinant vaccines which are to be used, for example, in human immunodeficiency virus (hiv) patients in the context of therapeutic vaccinations. vaccinations with attenuated live viruses usually confer very good protection, which remains for a long time. booster vaccinations at relatively long intervals of up to years can provide continuous protection. however, attenuated viruses entail the risk that they can lose their reproduction capability and thus their efficiency because of inadequate refrigeration systems, or they can mutate back to the wild type during the weakened infection. therefore, attention is now paid to the fact that attenuation should be based on multiple independent mutations, which largely excludes back mutation to the pathogenic wild type. however, attenuated vaccine viruses should be used only in immunologically healthy people. in immunocompromised individuals, these types of viruses can persist for a long time, and result in symptomatic infections. since attenuated live vaccine viruses can sometimes be transmitted from vaccinees to other people in their immediate environment (e.g. polio vaccination; ▶ sect. . ), this restriction also applies to immunosuppressed people in the family and the surroundings. furthermore, live vaccines should not be used during pregnancy. it is also important to emphasize that attenuation is defined only for the affected host species in which it was tested, and application of a live vaccine in species other than that in which the evaluation occurred is not permitted. this has to be considered for vaccinations of zoo and wild animals, and the unquestioned use of live vaccines should be avoided as far as possible. in particular, the use of live vaccines is prohibited when infections with the pathogenic wild type are absent or occur very rarely in a population. this status has been achieved by successful vaccination programmes, e.g. worldwide for smallpox virus and in europe and north america for poliovirus. in these cases, the risk associated with vaccination is greater than that of being infected with the wild-type virus and of becoming ill. therefore, in the case of smallpox virus, vaccination has been stopped, and to protect against poliovirus, an inactivated vaccine is being used today (▶ sects. . and ▶ . ). every active vaccination entails a certain rate of known harmless side effects. however, real complications (such as permanent nerve damage) are very rare events that occur with a prevalence of approximately one in a million vaccinations in adults. therefore, every vaccination must be accompanied by corresponding obligatory medical information and documentation. today, recombinant viruses represent a controversially discussed variant of live vaccines. it is being attempted to modify well-explored, less pathogenic viruses (e.g. adenoviruses) and vaccine viruses that were used successfully in the past (usually vaccinia viruses) by using genetic engineering methods in such a way that they encode proteins of other viral species, in addition to their own gene products necessary for infection and replication (▶ sects. . and ▶ . ). after viral inoculation, these foreign genes are expressed by recombinant viruses along with their own genes in an organism during infection. this induces an immune response against both the vaccinia virus or adenovirus proteins and the heterologous polypeptides. these recombinant, replication-competent viruses offer all the advantages of live vaccines (antibodies and cell-mediated immune response) and are, therefore, also increasingly used in therapeutic immunizations, i.e. in cases where an infection has already occurred. vaccination should stimulate the immune response to influence the progression of the disease favourably. large dna fragments cannot be cloned into the genome of vaccinia virus despite its size. therefore, it is essential to know exactly which proteins of the virus are important for the elicitation of a protective immune response in order to produce a vaccine against the viral infection. the pertinent gene is cloned into the genome of the vaccinia virus in such a way that it is under the control of an early vaccinia virus specific promoter. such a vaccine cannot generate the entire spectrum of an immune response that arises during an infection with the wild-type virus or its attenuated variant. in recombinant vaccine viruses, the immune response is confined to a specific protein. such vaccines have been extensively employed in north america (usa, canada) and europe (belgium, france, spain) to protect raccoons and foxes from rabies. the genome of the recombinant vaccinia virus harbours the genetic information coding for the g protein of rabies virus (▶ sect. . ). with use of these recombinant viruses, meat-based artificial baits are prepared and scattered for foxes. the animals bite the virus-containing plastic capsule when devouring the bait, whereby they become infected with the vaccine virus, and develop an immune protection against rabies virus infections. a vaccine based on recombinant vaccinia viruses or adenoviruses has not been approved for human use yet, but is being discussed for prevention or treatment of infections with hiv, and is being tested experimentally. according to their definition, inactivated vaccines are not able to proliferate in the vaccinated organism. they induce predominantly antibody responses. because of the lack of active viral protein synthesis, a cytotoxic t-cell response occurs rarely. when inactivated vaccines are used, an initial immunization (two or three vaccinations) and booster vaccinations are necessary to maintain the immune protection. to enhance the immune response, these vaccines have to be applied with an adjuvant, which facilitates the migration of macrophages, monocytes, and b and t lymphocytes to the inoculation site. aluminium hydroxide, aluminium hydroxyphosphate sulphate in combination with deacylated monophosphoryl lipid a components and certain toxoids (e.g. tetanus toxoid) are adjuvants approved for use in humans. other adjuvants are also used in animals. a widely used adjuvant is quil-a, a saponin that is also used for the production of immune-stimulating complexes. however, fibrosarcomas have recently been observed at the injection site in the application of inactivated vaccines (rabies virus and feline leukaemia virus) in cats. whether this is elicited by the adjuvant or by the trauma of injection has not been conclusively elucidated. for experimental immunizations, only incomplete freund's adjuvant is used in animals. the simplest form of an inactivated vaccine is a preparation of wild-type viruses that have been effectively killed by treatment with chemicals. this inactivation is usually done with aldehydic or alcoholic agents. frequently, b-propiolactone is also used. the protein components must not be denatured to the extent that they lose their native configuration and are no longer similar to the genuine viral structures. since the viral nucleic acid is per se frequently infectious and can lead to the production of progeny viruses, methods must be used to eliminate the infectivity which result in the degradation of the nucleic acid of the virus. for example, the currently commonly used vaccines against influenza virus or hepatitis a virus infections (table . ) are based on inactivated pathogens. relatively new vaccines are based solely on a selected protein component of the virus. a fundamental prerequisite for the development of such a vaccine is detailed knowledge of the immunologically important components of a pathogen. if it is known against which of the viral proteins (usually surface proteins) a protective immune response is induced, the respective coding gene can be cloned into a eukaryotic expression system. from this vector, the protein can be synthesized, purified and finally administered with an adjuvant. structural particle proteins are especially suitable for the induction of a protective immune response. an example of this is hbsag, the surface protein of hepatitis b virus, which aggregates to vesicular particles when it is artificially expressed in yeast or other eukaryotic cells. hbsag particles can induce, largely independently of adjuvants, the production of hepatitis b virus neutralizing antibodies and even cytotoxic t cells (▶ sect. . ). similarly, the capsid proteins l and l of papillomaviruses assemble together into particles that resemble the infectious viruses. the newly approved vaccines against infections with oncogenic human papillomaviruses (against human papillomaviruses and ) are based on such virus-like l particles, which -like the aforementioned hbsag particles -are produced in yeast cells using genetic engineering methods (▶ sect. . ). the particle-forming gag proteins of hiv also seem to be very appropriate for immunization. another variant of this type of a subunit vaccine has been in use in veterinary medicine for many years. it is a vaccine against feline leukaemia virus, a retrovirus (▶ sect. . ). it contains only the external glycoprotein (gp ) of subtype a of this virus as an immunologically relevant component, which is produced and purified in escherichia coli cells in a non-glycosylated form by genetic engineering methods. vaccines consisting of synthetic peptides with a length of - amino acids represent an additional form of vaccine which is currently being tested. in this case, individual epitopes of viral proteins, which induce the production of neutralizing antibodies and also activation of t cells, are selected and chemically synthesized. one advantage is that these vaccines are devoid of nucleic acids and that they can be produced in large quantities with relatively little effort. in animal studies, the protective effect of synthetic peptide vaccines has been demonstrated against infections both with foot-and-mouth disease virus (▶ sect. . ) and with canine parvovirus (▶ sect. . ). prerequisite for the development of such a vaccine is also in this case detailed knowledge of the protein regions that can elicit a virus-neutralizing immune response. however, it seems rather doubtful that a single epitope is able to do that in the long term, as most viruses have high genetic variability. furthermore, single individuals have different abilities for immunological recognition of specific protein regions. this is interrelated with the distinct mhc phenotype of each individual. in a vaccine based on synthetic peptides, several different epitopes ought to be combined and applied with a suitable adjuvant. a vaccine based on synthetic peptides has not been approved yet. another new vaccine type is administered as dna. the nucleic acid contains the genes of a virus that are capable of inducing a protective immune response, i.e. usually the regions that encode surface components of a pathogen. they are cloned into a vector system together with promoter elements, which regulate their expression, and are injected intramuscularly as purified dna. particularly in muscle cells, the dna can be detected over long periods as an episome. apparently, it is degraded very slowly there. if the corresponding genes are expressed, the organism can apparently develop both a humoral and a cellular immune response. this type of vaccine has been tested predominantly in animal systems. in animal studies, a protective effect of these vaccines has been demonstrated against a variety of different animal pathogenic viruses, such as paramyxovirus, parvovirus and herpesvirus. however, large amounts of dna and a number of booster injections are necessary. a faster degradation of the nucleic acid has also been observed. however, the inherent possibility of integration of dna sequences which were applied with the vaccine into the cellular genome and the resulting endangerment to the vaccinee are additional problems to consider in the discussion of the harmlessness of dna vaccines. viruses with rna genomes present a special challenge for the development of vaccines using genetic engineering methods. inasmuch as the techniques for targeted introduction of mutations and expression of foreign genes in prokaryotic and eukaryotic systems are based on dna as the initial nucleic acid, the genetic information of rna viruses must first be transcribed into double-stranded dna. only then does it become accessible for genetic engineering manipulations. then, targeted mutations can be selectively introduced into the nucleic acid sequence of such dna constructs, e.g. to generate attenuated vaccine viruses. in this case, the genome of rna viruses, which were previously transcribed into dna, must be present in a form that subsequently allows the synthesis of infectious viruses. this process describes the methods of reverse genetics, by which it is being attempted, for example, to construct attenuated live vaccines against respiratory syncytial virus and some other paramyxoviruses (▶ sect. . ). viruses with a segmented rna genome, such as influenza viruses, represent a particular challenge. in this case, the genetic information of all segments of the viral rna genome must be transcribed into dna and cloned into appropriate eukaryotic expression vectors. after the combined synthesis of all viral proteins and the production of new genome segments, recombinant viruses can subsequently be produced and cultivated in appropriate cell culture systems. an attenuation of the virus is possible by targeted mutagenesis. in the future, this will facilitate faster adaptation of new vaccine strains to new influenza viruses (▶ sect. . ). in this case, it is also being attempted to develop novel vaccines rapidly by using prefabricated genomic libraries or universal vaccines in order to cover all new influenza virus subtypes before they have pandemic dimensions. marker vaccines are of great importance in veterinary medicine. they allow the distinction of vaccinated animals from field-virus-infected animals (wild-type virus) by using simple serological methods (▶ chap. ). these vaccines are also referred to as diva vaccines (for "differentiating between infection and vaccination"). in principle, a differentiation is made between negative and positive marker vaccines. in the first case, the vaccine virus lacks a gene and the respective protein, so the vaccinated animals do not develop immunological reactions against the respective protein. the distinction of field-virus-infected animals is performed by determining the specific antibodies directed against this protein. prerequisites for the success of this approach are that the missing protein does not exert essential functions during viral replication, and that sufficient specific antibodies against the relevant protein are produced during an infection with wild-type viruses. in veterinary medicine, such negative marker vaccines are administered in the control of two economically important herpesvirus infections, namely aujeszky's disease in swine and infectious bovine rhinotracheitis in cattle (table . , ▶ sect. . ). positive marker vaccines are vaccine viruses that are characterized by a unique marker (nucleic acid sequence). in cases of vaccination failure, i.e. in cases in which an infection is established in spite of vaccination, positive marker vaccines facilitate the easy identification of vaccine viruses. recombinant and synthetic vaccines key: cord- -oy hsrpt authors: beutels, philippe p.a. title: economic aspects of vaccines and vaccination: a global perspective date: journal: the grand challenge for the future doi: . / - - - _ sha: doc_id: cord_uid: oy hsrpt nan a basic principle of economic decision-making is that in an environment of scarce resources choices have to be made in the allocation of these resources. this principle also applies to the provision of health care. the share of health-care expenditures in the gross domestic product (gdp) of most industrialised countries has increased from %- % in the early sixties to %- % in (from % to % in the usa) [ ] this rise has been attributed to medical advances (increasing the number and technological complexity of medical interventions), population aging, sociological changes (more, but smaller families and less familial support for the elderly) and insufficient productivity increases in the services sector. in less wealthy economies, medical decision-makers are faced with a smaller margin, and such a rise in health-care spending has not been observed yet. basically, the richer a country, the more it can afford (in nominal and in relative terms) to spend on health care. the two-way interaction between health and economic development is generally explained as follows. the healthier the population, the more adults can contribute to society by productive activity (i.e., work creating a surplus value in terms of capital gains and human resources), as well as by raising children in a stable environment, thus ensuring continued economic development. the process of economic development itself creates conditions (education, employment, infrastructure (including safe water and sanitation)), which provide a basis for continued improvement in longevity and health-related quality of life [ ] . individual good health can be seen as the product of some unknown complex function to which health care is only one of the inputs. other important inputs are: life-style variables (eating habits, smoking, etc.), environmental factors (urbanisation, climatic conditions, etc.), income, education and genetic predestination. furthermore, expenditures on health are not necessarily put to use in the most efficient economic aspects of vaccines and vaccination: a global perspective way. in this respect a distinction can be made between technical efficiency (providing maximal health care for a given cost, or delivering a certain service at minimal cost) and allocative efficiency (the distribution of resources across alternative services so as to maximise health gains, in accordance with preferences). finally, though much may be spent on health care, not all people may have equal access to health care of the same quality. indeed inequities in the consumption of health care may also interfere with the overall allocative efficiency of the system, and create inequities in health per se. therefore greater expenditures on health care are no guarantee for more global health. it should be noted that these observations do not plead for a reduction or containment of health-care budgets, but rather for a way of spending that ensures that societal goals are met. in order to achieve this, welfare economists focus research on two broad topics: efficiency and equity. efficiency relates to choosing options that maximise utility from marginal expenditures (i.e., by optimising the production process of health, for which health care is one of the inputs). equity relates to the fair distribution of all aspects related to health across members in society (e.g., equal access to care). clearly, there may exist a trade-off between efficiency and equity and giving priority to either of them is a normative issue that should be decided by social and political debate. vaccination is undoubtedly one of the major contributors to health improvements in the last three centuries. during this period, the impact of vaccination on longevity is undeniable, despite the fact that its partial contribution is difficult to distinguish from that of improved hygienic conditions and nutrition, and the discovery of penicillin [ , ] . all of these combined provided the basis for the so-called "epidemiological transition" in industrialised countries. at the same time, infectious diseases remain the main cause of death in many developing countries. despite the continuing expansion of the vaccine portfolio, implementing financially sustainable basic vaccination programs in poor countries remains problematic. though this is not so much an economic as a financial aspect, we will return to this issue in the section "financing vaccines". a number of peculiar characteristics set vaccination apart from other interventions in health care [ ] : ( ) since vaccination is (usually) a form of primary prevention, it intervenes in people (often children) who are generally in good health. but unlike most other prevention programs, the interven-tion itself can cause harm to the vaccine recipient, because in rare cases vaccine-associated adverse events (vaae) occur. this means that people make trade-offs between risks of vaccine-preventable disease and risks of vaae. the perception of these risks is quintessential to the individual demand for vaccines (if left to free-market mechanisms), and dominates the influence of other factors such as price [ ] . ( ) vaccination not only protects vaccine recipients, but it also reduces exposure of unvaccinated people, due to the reduced circulation of the infectious agent (if the transmission of infection occurs from human to human). this is not always beneficial for public health as the reduced risk of transmission leads to an increased average age at infection (with many "childhood" infections being more severe if contracted in adulthood) [ , ] . together with the first characteristic, this means that people generally have an interest in having everyone else vaccinated, but not themselves (or their children). ( ) a number of infections can be eradicated in the long run if vaccination efforts are sustained at sufficiently high coverage levels around the world. in other words, sometimes vaccination has the potential of making itself redundant. choices about eradication are closely linked to the welfare of future generations and societal time preference (see also below) [ , ] . since the perceptions outlined above are usually distorted by insufficient or biased information, government intervention (in the form of subsidies, or coercion) is desirable to ensure that vaccine uptake remains optimal. indeed, as uptake increases the risk of vaae remains constant, but individuals may perceive it to increase. at the same time the absence of vaccine-preventable disease may create a false sense of security, and lure people into believing that their risk of disease has reduced to zero as well, while this is highly dependent on historical and future rates of exposure and vaccination in the rest of the population. the vaccine market represents only . % of the global pharmaceutical market, but has high growth potential (estimated at - % per year by various sources, mainly due to new combination, new prophylactic and new therapeutic vaccines) [ ] . for a manufacturer, the contribution margins of vaccines are low compared to those of other products in both the developing and industrialised world (due to price and licensing regulations). the few suppliers of vaccines now aim to limit production to the projected global needs in any given year (unicef bought about % of "traditional" vaccine supply in , compared to about % in ). thus the market is very vulnerable to capacity problems: a problem with a single batch of vaccines by a single producer can have severe knock-on effects across the globe. this may also explain why, for some of the old vaccines, the price fluctuates, and has had the tendency to rise over the last years. close co-operation between demanders (governments or agencies) and suppliers is essential to ensure continued availability at the right time. vaccines are supplied under a tiered price system, with % of sales volume in developing countries and countries in transition, but % of sales revenue in industrialised countries [ ] . it is therefore not surprising that global vaccine manufacturers (with three big producers (glaxosmithkline, aventis and merck) occupying % of the global market) tend to focus on products for the industrialised world. it is to be expected that more combination vaccines will become available and existing combinations extended. examples of this include the hexavalent diphteria-tetanus-pertussis-inactivated polio virus -haemophilus influenza type b -hepatitis b (dtp-ipv-hib-hepb) vaccine, and the quadrivalent measles-mumps-rubella-varicella-zoster (mmrvz) vaccine. the research and development costs for these vaccines are high due to technical and regulatory complexity. the technicality, the multiple patents and requirements in terms of clinical trials (all demanding great time and money investments) increase barriers to enter the vaccine market. this may lead to more monopolistic behaviour, with risks to supply, choice and price. clearly, the benefits of combination vaccines are many. for instance, reductions in the number of injections and associated administration costs (including reduced money, time and pain costs for children and their parents), and reduced transmission by contaminated needles benefit recipients and the public health bodies. free-rider effects (important and not-soimportant vaccines can hook up with established vaccines, irrespective of how recipients perceive their importance) and economies of scope benefit manufacturers and perhaps public health bodies. these benefits will have to be traded off versus the higher price of combination vaccines. because governments, health insurers or agencies (unicef, paho) typically buy vaccines directly from producers, there is also little diversity on the demand side of the market. all of this implies that there is little competition on both sides of the market and that global societal goals (development and supply of affordable vaccines for poor countries as well as rich countries) are unlikely to be met by relying entirely on free-market mechanisms (particularly since these are hampered by (necessary) regulation with regards to quality control and licensing). by using economic evaluation we are essentially trying to answer the following questions [ ] : ) is the vaccination program under study worth doing compared to alternative ways of using the same resources? in other words: should the (health care) resources be spent on such a vaccination program, and not on something else? ) more specifically, if we are deciding to vaccinate against a particular disease, whom should we vaccinate, at which age, with which vaccine and how should the vaccine be delivered and administered in order to deploy our scarce resources in the most efficient way? most economic evaluations of vaccination are model-based, because the alternative, empirical analysis, is usually impractical, very time-consuming (for most vaccines it takes decades for the full effects to unfold), very expensive and potentially unethical. a complete economic evaluation should compare different options for an intervention, in terms of economic costs as well as health consequences. there may be several options to prevent an infectious disease, some of which are mutually exclusive, while others are complementary. the relevant costs and benefits need to be collected for each option, and calculated relative (incremental) to another option. the choice of the reference strategy against which the other options are evaluated can be highly influential for the results of the evaluation. unless it is a cost-ineffective strategy, current practice is the preferred strategy of reference. when a new vaccine is introduced, the reference strategy is often referred to as "doing nothing" (no vaccination), although in this case "doing nothing" usually means the treatment of cases as they arise, with the corresponding public health measures. a generalised distinction between the costs and benefits of vaccination is presented in table . the intervention costs dominate the cost side. these are the costs necessary to implement the vaccination program. additionally there are costs incurred to receive the vaccine. the benefits of vaccination are the gains in health and the avoided costs. direct costs can be avoided because less treatment is needed for curing or nursing the disease against which the vaccination program is aimed. additionally indirect costs can be avoided because vaccination may partly prevent people having to interrupt their normal activities in society because of their illness or the illness of their relatives. from the health-care payer's point of view, only direct medical costs need to be taken into account. however, from society's viewpoint, indirect non-medical costs are also relevant. other viewpoints can be those of patients, hospitals, travel clinics, insurance companies, employers, etc. (see fig. ). for each of these perspectives different costs and benefits may be relevant. this implies that it is possible for an intervention to be relatively cost-effective for one party involved, while it is not for another. different cost categories are listed in table . the listings in italics are often not taken into account, because they can be relatively small in comparison to the other costs and/or because they are difficult to estimate. sometimes their inclusion is not relevant to express the viewpoint of the analysis. however, if they are relevant for the viewpoint of the analysis, their impact on the results could be tested in a sensitivity analysis and their existence should be mentioned when the results are presented. some diseases affect expectations and behaviour beyond one degree of separation from the pathogen. for instance the global impact of the sars outbreak in was modest in disease burden ( probable cases, deaths) and associated health-care costs, but it had an impressive impact on the global economy (us$ - bn, or $ - m per case) in macro-economic terms (when the impact on consumption and investments are considered) [ , ] . a similar situation could arise for pandemic influenza, or any other disease that affects risk perceptions of consumers and investors (e.g., variant creutzfeld jacobs disease). however, for most currently vaccine-preventable diseases, micro-economic evaluation would provide an appropriate analytical framework, preferably adopting a societal perspective. in reality, decisions about universal vaccination are often taken from the perspective of the national health service (nhs) or the ministry of health and at best from the health-care payer's perspective (which in addition to the nhs costs also includes direct co-insurance and co-payments by the patient). indeed, decision-makers in health care tend to focus primarily on direct costs since these are most indicative of their immediate budgets, even if their decision has bearings on society at large. when it comes to estimating unit costs or prices, it should be noted that costs in an economic sense are opportunity costs: they represent a sacrifice of the next best alternative application [ ] . this entails that costs in an eco- philippe p.a. beutels health gains (physical and psychological) source: [ ] nomic sense are not necessarily the same as financial expenditures, and that they can also represent goods and services that are not expressed in monetary terms. however, market prices are often used as a proxy. if particular goods and services are not traded on a market, ("shadow -") prices of a similar activity can be used instead. for example, work of volunteers can be approximated by wages of unskilled labour. similarly, patients' leisure time could be based on average earnings or average overtime earnings. average costs per unit of output are the total costs of producing a quantity divided by that quantity. marginal costs constitute the additional costs of producing one additional unit of output. since decisions are made at the margin, marginal costs should be used where they are substantially different from average costs [ ] . for vaccination, this distinction is most relevant for estimating the costs of the program [ ] . the costs of adding a particular vaccine to the existing program depends on how well the schedule of the new vaccine fits in with the other schedules, whether specific precautions need to be taken, whether potential vaccinees need to be screened prior to vaccination or whether a specific target group is envisaged. the costs that are most heavily affected by adding a new vaccine to the existing program are the variable costs of the program (time spent per vaccinee, number of vaccines bought, etc.), whereas the influence on the fixed intervention costs (buildings, general equipment, etc.) is usually small (unless a new vaccine requires a substantially different infrastructure in terms of storage and transport). a good example of this is provided by hall et al. who examined the immunisation program in the gambia (more recently these results were confirmed by a similar analysis in addis ababa, ethiopia) [ , ] . they found that the additional costs of adding hepatitis b vaccine to the existing expanded program on immunisation (epi) vaccines (measles, polio, dtp and bacille calmette-guérin (bcg)), would be for % recurrent costs (of which % for purchasing hepatitis b vaccine (hepb)). still, the introduction of a new expensive vaccine could more than double the costs of the program in some countries because of its sheer price compared to other vaccines in the program. the main objective of vaccination is to prevent disease. the most important benefits from a public health point of view are therefore the health gains (see tab. ). these are both physical (avoiding illness, suffering, mortality, etc.), and psychological (avoiding distress, anxiety, etc.). specific vaccinerelated psychological health gains include the general feeling of well-being and security of vaccine recipients from knowing that they are protected against disease. this could evidently lead to behavioural changes (e.g., a vaccine against hiv/aids could have a large influence upon the sexual behaviour of vaccine recipients). the valuation of health outcomes has far-reaching consequences for the methodology and study design of applied analyses. generally, a distinction is made between four different methods, depending on the way in which health gains are measured [ ] . a cost-minimisation analysis compares the costs of equally effective alternatives, without quantifying the health gains. it differs from a pure cost philippe p.a. beutels source: [ ] analysis in that there is always more than one option analysed and that the effectiveness of the different alternatives is known to be equal. in a cost-effectiveness analysis, health gains are measured in one-dimensional natural units (e.g., infections prevented, hospitalisations prevented, deaths averted, life-years gained…), implying that only one aspect of effectiveness is considered (e.g., postponing the time of death) and other related aspects are not (e.g., the quality of life). the results of cost-effectiveness analyses (cea) are usually presented as a ratio. a cost-effectiveness ratio (cer) is a measure of the incremental costs, which are necessary to obtain one unit of a health gain by implementing a strategy j instead of a strategy i (expressed in incremental costs per life-year gained, incremental costs per infection prevented, etc.). the lower the ratio, the more efficiently strategy j gains health compared to strategy i. the units in which health gains are expressed should represent the final results or clinical endpoints of an intervention as adequately as possible, in order to enable comparison between different interventions [ ] . if, hypothetically, the cost-effectiveness of hepatitis b vaccination were $ per infection prevented, whereas hib vaccination is estimated at $ , per infection prevented, it is wrong to conclude that hepatitis b vaccination is more cost-effective (because it is less costly to prevent one infection). to make that judgement, the avoided effects would need to be expressed in a more comparable endpoint, like life-years saved. to make the comparison even more relevant, different health states should be weighed by their quality (scaled from (meaning death) to (meaning perfect health)). this approach is used in cost-utility analysis (cua), where health gains are measured in quality-adjusted life-years (qalys) saved or another combined measure of morbidity and mortality (e.g., disability-adjusted life-years (daly)). a cost-utility ratio (cur) is similar to a cer, except that the denominator contains the difference in qalys (or dalys), instead of the difference in natural units, such as cases avoided or life-years gained. the main advantage of cua over cea is that it allows comparison of very different health-care interventions, for instance, those that predominantly extend lives (e.g., flu vaccination of the elderly) with those that improve the quality of life (e.g., drugs against erectile dysfunction). in cost-benefit analysis (cba), the health gains are converted into monetary units, which, in theory, allows the many dimensions that are associated with an improvement in health status (over and above the length and health-related quality of life) to be included. there are benefits beyond the health outcomes such as information, caring, regret, anxiety reduction, communication and process utility (benefits from health-care use). further-more, option value (i.e., benefits derived from needing care in the future) and non-use value (i.e., externalities related to caring for the health of others) can also be (potentially) elicited [ ] . the results of a cba can be presented as the difference between costs and benefits (the net costs (or net savings)) or as a ratio. the benefit-cost ratio (bcr) expresses to which extent an investment in an intervention can be recovered by the consequences of that intervention (expressed as a unitless number or a %). cost-benefit analysis allows for comparisons between totally different projects in society (e.g., comparing a vaccination campaign with the construction of a new bridge). when budgets are very limited and many urgent interventions compete, as in developing countries, such cross-sector comparisons may actually be used in practice. clearly, the potential of cba to make such comparisons possible is a major advantage to aid decision-making. the strength of cba in theory, i.e., the explicit monetary valuation of health gains, has up till now been also its weakness in practical decisionmaking. in theory it seems preferable that the valuation of health gains (and of life) is done in an explicit, transparent and representative way as in cba, instead of the implicit, inconsistent and arbitrary way it is often done in today's decision-making. however, in a health-care environment the monetary valuation of health (and particularly of life) is often rejected on an emotional basis [ ] . additionally, economists have few credible arguments to counter these objections, as the current methods which place a monetary value on health (human-capital and willingness-to-pay methods) can hardly be called consistent and reliable in practice [ ] [ ] [ ] . in view of this, most economic evaluations in health care are based on cea or cua. the literature on these has increased exponentially since the s, for vaccines at least as much as for other interventions in health care, underlining the importance of a sound theoretical framework for these analyses [ ] . individuals (and societies), in general, prefer to receive benefits as early as possible and incur costs as late as possible. this so-called time preference means that the same amount of wealth or health would have a different value to a decision-maker in the present, if this amount is gained at different points in time. note that time preference has nothing to do with inflation. a vaccination program is an investment made in the present (i.e., the costs of buying and administering vaccines) to gain benefits spread out over the future (i.e., avoided costs of treatment, avoided morbidity and mortali-ty). discounting is a technique by which future events (e.g., costs and health outcomes) are valued less the further in the future they arise. the degree to which they are valued less is determined by the discount rate (frequently assumed to be constant through time): the higher the rate the less future benefits and costs are valued. although there is general agreement on the discounting of costs, the arguments for discounting non-monetised health outcomes are contradictory [ ] . discounting costs without discounting benefits leads, amongst others, to the paradoxical situation that any eradication program will yield infinite benefits [ ] . this would imply that all current resources should be spent on research of eradicable diseases, and the implementation of eradication programs, and not a single penny on cure. such paradoxes, and the observation that individuals generally have a positive discount rate for health, clearly indicates that health too should be discounted at a positive rate. but there is no general agreement on how the discount rate for health should compare to that of wealth. there are arguments to apply an equal discount rate to both costs and health effects [ , ] . the underpinnings and relevance of these are questionable, so that a lower discount rate for health effects than for costs has also been proposed [ , ] . because of the very long time spans over which benefits accrue, the analysis of most vaccination programs is very sensitive to discounting (of costs as well as health effects). nonetheless, this is no cause for a different approach to discounting for vaccination. still, further empirical research is needed to strengthen or to change the basis for conventional discount rates (mostly %, or %) and discount models (mostly stationary) [ ] . a slight decrease in discount rate (from, say, % to %) could change the cost-effectiveness of some vaccination programs from unattractive to attractive. also, it is likely that time preference in developing countries is substantially different (i.e., higher) from that in industrialised countries, particularly for those countries that have decreasing health (e.g., life-expectancy due to hiv/aids) or wealth (e.g., real gdp) expectations [ ] . in theory, decisions are made by interpreting the results of economic evaluation as follows. in figure a new program is plotted in terms of costs and effectiveness versus the reference strategy in the origin. if the new program is less costly and more effective than the reference, then the new program (a "dominant" strategy) should be implemented. likewise, if the new program is more costly and less effective than the reference, it should be rejected. in the other quadrants the decision is more complex, because it depends on a value judgement. if the incremental cer (or cur) is smaller than a given willingness to pay (or threshold cost-effectiveness criterion), "k", it would be acceptable. the question then is, how to determine k? this could be determined by social debate or by comparing it to what is widely accept-ed in practice. the most widely cited k in industrialised countries is $ , per qaly gained. there may also be a grey zone for k in which some interventions are implemented and others are not (e.g., between $ , per qaly gained and $ , per qaly gained), whereas under and above that grey zone all and none of the interventions are implemented, respectively. however, the greater the analytical uncertainty and the burden of disease, the more decisions are likely to deviate from such clear cut-off practices [ ] . different societies should have a different willingness to pay, though there are few instances in which societies (or their decision-makers) have tried to determine what the appropriate value of k is. the world bank has suggested using gnp per capita as a benchmark for k. note that in cba, k has already been given an explicit value. in league tables, many vaccination programs rank with the most costeffective interventions in health care in industrialised countries [ , [ ] [ ] [ ] . it is tempting to try and estimate the global historical value of vaccination. however, due to scarcity of data in most parts of the world such an exercise philippe p.a. beutels figure . the cost-effectiveness plane. cer: cost-effectiveness ratio, i.e., incremental costs divided by incremental effects; k = willingness to pay, or a cost-effectiveness ratio of acceptable magnitude. all points on a line in this plane have identical cost-effectiveness ratios. would be, by necessity, extremely crude. it seems clear, though, that the smallpox eradication program and the establishment of the epi have generated enormous benefits, not only by directly protecting against important vaccine-preventable diseases, but also by providing opportunities for health education and infrastructure in developing countries [ ] . yet the associated disease reduction in smallpox, measles and tetanus alone is bound to have been a cost-saving enterprise around the world (i.e., in the lower right quadrant of fig. ) , currently averting over million deaths per annum, compared to a "never having vaccinations" situation. however, when we are making choices today, we have to consider what additional benefits we will achieve by making additional investments, and this is bound to vary between countries at different stages of economic development, different epidemiologies of disease, and different historical vaccine-uptake levels. hence data from one country cannot always be simply extrapolated to another. in practice, there are many factors that come into play when decisions are made about new health-care interventions (see fig. ). in a democracy, a decision-maker receives a temporary, renewable mandate from the public to allocate a given budget. that person is well aware of the public perceptions of public health problems, and the impact of decisions thereon. at the same time, pressure groups may try to influence decision-makers or the public's perception. these pressure groups have vested interests in the decisions (be it as sellers of vaccines, or sellers of services for the cure of vaccine-preventable diseases). societal goals with regards to the decision can only be met by considering its medical, social, ethical and cost implications. the theoretical foundation of economic evaluation (so-called "pareto opti- figure . factors influencing decision-making in practice mality") addresses efficiency, without concern for distributional aspects (equity). therefore, economic evaluation combines the medical/epidemiological and cost implications, but does not consider the social and ethical implications depicted in figure (though in cba these aspects could theoretically be included, if a willingness-to-pay approach is used, and it is possible to weight quality-of-life gains to help achieve equity goals, as is commonly performed in dalys). therefore economic evaluation should be seen as an additional type of analysis that cannot stand on its own in its current form (it is an aid to decision-making, not a decision-maker in itself). at the same time, ideally, the influence of pressure groups, and the public's perceptions (rather than the public's true preferences) should be minimised in this process. it is noteworthy that most vaccination programs are likely to be equitable according to prevailing theories of justice [ ] . indeed, an analysis for bangladesh indicated that socio-economic inequalities in mortality of under- -year-olds were eliminated by measles vaccination [ ] . in the past, vaccination interests of poor and wealthy nations seemed more in tune than today. moreover, the research and development costs of the new generation of vaccines, based on biotechnology, are greater and the regulatory hurdles higher, meaning that new vaccines are much more expensive than the basic package of "traditional" vaccines. the first new expensive vaccine for global use was the hepatitis b vaccine, which became available in . the main reason why it was not immediately included in universal vaccination programs was its price, because initially the hepatitis b vaccine cost more than the other six epi vaccines put together. with the advent of more expensive vaccines, the introduction of a new vaccine is not as straightforward as it used to be in the industrialised world. in contrast to some of the "older" vaccines (e.g., measles, pertussis), newer vaccines may not result in net savings to the health-care system. nonetheless, if considered desirable, industrialised countries have no difficulty in financing the introduction of new vaccines, and ensuring the continuing uptake of old ones. for developing countries, the main difficulty is not so much to determine whether it would be cost-effective to introduce a vaccine, but to ensure that the introduction is financially sustainable. when external donors sponsor vaccination programs the sustainability takes the form of a partnership with shared responsibility and the promise by the receiver of the financing to create the conditions to become self-sufficient in the long run, either alone or by attracting further external funding. global immunisation efforts came under pressure as the epi, which was launched in the s as a way of building on the success of the smallpox eradication program, lost its momentum in the s, and failed to attain the year goal of % global vaccination coverage. indeed, global child-hood immunisation coverage against the six main target diseases (polio, dtp, measles and tuberculosis), which was less than % in , decreased from about % in to % in [ ] . coverage for the complete schedule of dtp remains well below % in tens of developing countries, mostly in sub-saharan africa. these countries are traditionally bottlenecks in the epi because of great financial constraints, the evolution of the hiv epidemic, logistical difficulties, poor governance and general socio-economic conditions (sometimes aggravated by war). as these factors evolved unfavourably in the s, international alliances shifted their efforts from reducing general global inequalities in health ("health for all") to more selective strategies, like the polio eradication program and the introduction of new and improved vaccines. the discrepancy between the developing and the industrialised world is likely to become greater, as private vaccine development focuses primarily on diseases that affect the wealthy. indeed, only about % of world drug sales is for african countries. it has been estimated that of all expenditures on health research (over $ billion per year), % is for diseases that affect % of the world's population [ ] . using recent examples, kaddar et al. assert that financing vaccination should be affordable by all countries, at least for the basic vaccines [ ] . the cost of fully immunizing a child with the basic vaccines is $ to $ , which typically represents % to % of public health expenditures, <$ . per capita or about . % of gdp. most vaccination costs are fixed costs of personnel and infrastructure, and the marginal costs of an additional vaccine may often be bearable for the domestic budget (though still highly dependent on vaccine price). as the th century drew to a close, the landscape of external vaccine financing underwent dramatic changes with the inception of the global alliance for vaccines and immunization (gavi) and the vaccine fund, with the aims to stimulate research and development for developing world problems, strengthen immunization systems, and promote and support the introduction of new and underused vaccines. gavi is an alliance of financiers (development banks, aid agencies, foundations), agencies (unicef, who), vaccine developers and manufacturers, as well as developing country governments, whereas the vaccine fund manages private financial resources, such as those from the bill &melinda gates foundation, and public contributions from a small number of wealthy countries. the first generation of vaccines, such as measles and oral polio vaccines, was used against common and serious childhood diseases afflicting all countries in the world. few of these vaccination programs were subject to economic analysis before introduction, and for good reason: the benefits were obvious and the costs low. indeed, they were probably amongst the most effective and cost-effective public health programs of the th century. this is no longer necessarily the case with new vaccine introductions. new vaccines are generally higher priced and unlikely to fall in price to the level of the first generation of vaccines. furthermore, they are often aimed at less common or less serious diseases (particularly in the industrialised world). thus, whether these vaccines are worth introducing is less clear. vaccine financing has recently changed with important initiatives stimulating development and use of vaccines for the developing world. these are to be welcomed as they may further alleviate the disease burden in developing countries at affordable cost, correct market imperfections with regard to research and development, and reduce inequalities in health. nonetheless, the introduction of new vaccines demands cautious planning. if it comes at the expense of the uptake of the first generation of vaccines, it may have a detrimental influence on the effectiveness and cost-effectiveness of the whole program. in view of all these developments, the role of economic evaluation in vaccine program design is only likely to increase in the future. world development indicators, online publication available on cd rom commission on macroeconomics and health: investing in health for economic development the conquest of smallpox an interpretation of the modern rise of population in europe economic evaluation of vaccination programmes in humans: a methodological exploration with applications to hepatitis b, varicella-zoster, measles, pertussis, hepatitis a and pneumococcal vaccination economic epidemiology and infectious disease infectious diseases of humans -dynamics and control increase in congenital rubella occurrence after immunisation in greece: retrospective survey and systematic review economics of eradication vs control of infectious diseases lecture at the advanced course in vaccinology, international vaccine institute assessing the economic impact of communicable disease outbreaks: the case of sars globalization and disease: the case of sars methods for the economic evaluation of health care programmes the cost of integrating hepatitis b virus vaccine into national immunization programmes: a case study from addis ababa cost-effectiveness of hepatitis b vaccine in the gambia providing health care. the economics of alternative systems of finance and delivery methodological issues and new developments in the economic evaluation of vaccines the economics of health and medicine estimating costs in costeffectiveness analysis evaluation of life and limb: a theoretical approach theory versus practice: a review of "willingness to pay" in health and health care discounting of life saving and other non-monetary effects foundations of cost-effectiveness analysis for health and medical practices discounting costs and effects: a reconsideration discounting for health effects in cost-benefit and cost-effectiveness analysis does nice have a cost-effectiveness threshold and what other factors influence its decisions? a binary choice analysis cost-effectiveness analysis and the consistency of decision making: evidence from pharmaceutical reimbursement in australia a comprehensive league table of cost-utility ratios and a sub-table of "panel-worthy" studies fivehundred life-saving interventions and their cost-effectiveness the societal value of vaccination in developing countries measles vaccination improves the equity of health outcomes: evidence from bangladesh the state of the world's children. early childhood the / gap report financial challenges of immunization: a look at gavi the author is grateful to dr john edmunds (health protection agency, uk) for constructive comments on an earlier version, and to the flemish fund for scientific research (fwo g. . ) and the european union funded project polymod (eu fp ) for financial support. key: cord- -ctass hz authors: bull, james j.; nuismer, scott l.; antia, rustom title: recombinant vector vaccine evolution date: - - journal: plos comput biol doi: . /journal.pcbi. sha: doc_id: cord_uid: ctass hz replicating recombinant vector vaccines consist of a fully competent viral vector backbone engineered to express an antigen from a foreign transgene. from the perspective of viral replication, the transgene is not only dispensable but may even be detrimental. thus vaccine revertants that delete or inactivate the transgene may evolve to dominate the vaccine virus population both during the process of manufacture of the vaccine as well as during the course of host infection. a particular concern is that this vaccine evolution could reduce its antigenicity—the immunity elicited to the transgene. we use mathematical and computational models to study vaccine evolution and immunity. these models include evolution arising during the process of manufacture, the dynamics of vaccine and revertant growth, plus innate and adaptive immunity elicited during the course of infection. although the selective basis of vaccine evolution is easy to comprehend, the immunological consequences are not. one complication is that the opportunity for vaccine evolution is limited by the short period of within-host growth before the viral population is cleared. even less obvious, revertant growth may only weakly interfere with vaccine growth in the host and thus have a limited effect on immunity to vaccine. overall, we find that within-host vaccine evolution can sometimes compromise vaccine immunity, but only when the extent of evolution during vaccine manufacture is severe, and this evolution can be easily avoided or mitigated. recombinant vector vaccines are live replicating viruses that are engineered to carry extra genes derived from a pathogen-and these extra genes produce proteins against which we want to generate immunity. these vaccine genomes may evolve to lose the extra genes during the process of manufacture of the vaccine or during replication within an individual, and there is a concern that this evolution might severely limit the vaccine's efficacy. the dynamics of this process are studied here with mathematical models. the potential for vaccine evolution within the host is somewhat limited by the short-term growth of the vaccine population before it is suppressed by the immune response. we find that evolution is a problem only when the process of manufacture results in the majority of the vaccine virus being revertant. we show that increasing the vaccine inoculum size or reducing a a a a a live vaccines replicate within the host. as true of any reproducing population, these withinhost vaccine populations may evolve. for live vaccines that do not transmit, any within-host evolution is a dead end and might thus seem to be irrelevant to vaccine function. but if the process is fast enough, or the vaccine population replicates long enough, the vaccine population may evolve to a state where it is ineffective or virulent-either change would be bad. the two main types of live viral vaccines are attenuated and recombinant-vectored. most live virus vaccines in use today are attenuated, their reduced virulence typically achieved by adapting the wild-type virus to a new environment (e.g. replication in a novel cell line or low temperature), with a consequent reduced replication rate in humans. the use of attenuated vaccines is too risky for pathogens such as hiv, and a safer alternative is to develop a live, recombinant vector vaccine where one or a few pathogen genes with immunogenic activity (proteins that elicit protective immunity) are expressed from a benign virus vector. the expected consequences of within-host evolution differ between these two types of vaccines ( table ) . evolution of an attenuated vaccine is likely to be a reversion toward the wildtype state, the rate of this process depending heavily on vaccine design and the duration of vaccine virus replication in the host (reviewed in [ ] ). to a first approximation, reversion toward the wild-type state should lead to the vaccination more closely resembling natural infection [ ] , such as higher virus densities, side-effects and disease, and possibly an increased immune response. within-host evolution of an attenuated vaccine might also predispose the virus to better transmission-also reflecting the wild-type state-but this outcome is not assured: viral adaptation to different tissues within the host may hamper growth in and dissemination from tissues important in transmission (e.g., [ ] ). the expected consequences for evolution of a recombinant-vectored vaccine are fundamentally different [ ] . in most cases, the antigen against which immunity is sought comes from a foreign transgene inserted into a competent viral vector without replacing any vector genes. vectors in development include adenovirus, vsv (vesicular stomatitis virus) and cmv (cytomegalovirus). the vector genome carries out all viral amplification and transmission functions, and the transgene does not contribute to any process benefiting vector reproduction. from an evolutionary perspective, the transgene is both dispensable and potentially costly: selection may favor loss of the transgene and thus loss of vaccine's ability to elicit immunity against the antigen encoded by the transgene. this evolution therefore generates something akin to infection by the wild-type vector. as vectors are typically chosen to be avirulent for immune competent hosts, vaccine evolution will result in no more than a harmless infection that does not generate immunity to the antigen encoded by the transgene. considerable attention has recently been given to the evolution of attenuated vaccines and designs that retard their evolution. evolutionary stability of attenuated vaccines seems attainable by engineering designs, including the introduction of hundreds of silent codon changes, genome rearrangements, and some types of deletions (comparisons and reviews are provided by [ , , ] ). far less thought has gone into the consequences of evolution for recombinant vector vaccines or of strategies to minimize this evolution. although recombinant vector vaccines are not yet in widespread use, many are under development [ , ] , and their success may rest on understanding within-host evolution. here we explore how the combination of evolution during the process of vaccine manufacture and during its within-host dynamics following vaccination could affect the immune responses elicited by a recombinant vector vaccine and reduce its efficacy-the specific interaction between evolution and immunity. we consider viral vaccines and focus on vaccines that cause shortduration (acute) infections. the ideas we discuss also apply to live vaccines of bacteria and other pathogens. our overall message is that while vaccine evolution may occur, it is either unlikely to be a problem (i.e., compromise the generation of immunity), or it is easily mitigated. when vaccine evolution does limit the adaptive immune response, we identify ways of escaping such outcomes. our analysis rests on mathematical models, but most results can be explained intuitively (perhaps only in hindsight), with the main results illustrated graphically; many analyses are relegated to supporting information. our analysis assumes that vaccines replicate within the host untill cleared by host immunity; we exclude vaccines that reproduce for just a single infection cycle (e.g., modified vaccinia virus ankara), as they have no significant opportunity for evolution. our models are numerical analyses of ordinary differential equations. the equations are given in supporting information (s appendix). the were numerically evaluated and graphed in r (s file, a markdown file), sometimes also evaluated in mathematica (s file). the key question is whether evolution of the vaccine virus (henceforth just 'vaccine') meaningfully affects immunity to the antigen encoded by the foreign transgene (henceforth just 'antigen'). the potential for vaccine evolution is easy to understand. through mutation, any large vaccine population will contain mutants that inactivate or delete the foreign transgene, and those revertants will then grow amidst the vaccine. vaccine inferiority may accrue in two different ways: the transgenic insert and its expression may intrinsically impair vaccine growth, and adaptive immunity to the foreign antigen may impair the vaccine's growth but not the revertant's during an infection. it is easy to appreciate how and why the vaccine may be inferior to the revertant, and this can result in an increase in frequency of the revertant. however, the relationship between this evolution and the extent of immunity to the vaccine antigen is more complex. we thus explain some of the factors that affect how this evolution translates into a reduction in immunity to the antigen, and why in some circumstances, substantial evolution can result in little change in immunity to the antigen, while in different situations it can result in a substantial reduction. two realms of vaccine evolution. vaccine evolution can be inimical to immunization by limiting vaccine antigen levels in the host. as noted above, one realm in which evolution may occur is within the host, starting with the inoculum and ensuing until vaccine clearance. a second realm of evolution affecting antigen levels is that occuring during manufacturingduring growth of the virus to prepare the stock used for inoculation. both realms are part of a continuum, any evolution during manufacturing advancing evolution within the host (fig ) . the inoculum sets the starting point for within-host evolution, and indeed, an inoculum that is mostly revertant will limit vaccine efficacy even if no further evolution occurs within the host. from population genetics principles [ ] , even if the inoculum has a seemingly low frequency of revertant, the evolution that has occurred prior to inoculation may greatly accelerate within-host evolution (fig ) . paradoxically, when considerable evolution occurs in manufacture, such that the inoculum is primarily revertant, there is little opportunity for further evolution within the host-the damage is already done. the effect of any 'pre-host' evolution on the host immune response is potentially as important as the effect of within-host evolution. an important difference between the two realms is that pre-host evolution may be more easily mitigated than is within-host evolution. that is, controlling pre-host evolution may be a feasible way to limit within-host evolution and to limit the loss of immunity from vaccine evolution. since pre-host and within-host evolution represent different realms on a continuum, we adopt a language that attempts to distinguish them and avoid confusion. we use the following: • 'pre-host evolution' refers to evolution during manufacture that affects inoculum composition • 'within-host evolution' refers to evolution that occurs within the host after inoculation • 'evolution' refers to either or both of the above. it is easily appreciated that the specifics of evolution in the two realms may differ-vaccine growth and fitness in an in vitro environment (pre-host) will often differ from that within the host. regardless, however, any pre-host evolution will advance subsequent within-host evolution, unless the revertant is selected in opposite directions pre-host and within-host. there may even be a common molecular basis to vaccine inferiority in both the pre-host and withinhost environments that will render the two processes somewhat similar (see below). a short duration of infection limits within-host evolution. any fitness advantage of revertant means that its frequency-its abundance relative to the vaccine-will increase during active viral growth (fig ) . however, when the infection caused by the vaccine is acute, as we consider here, the magnitude of possible within-host evolution is limited by the short duration of viral growth before clearance. if the inoculum is largely free of revertant, even a moderate fitness cost of the vaccine may have little effect on vaccine evolution, such that vaccine fitness effects and evolution can be ignored. evolution versus immunity. surprisingly, vaccine evolution per se need not reduce the immune response, even when its magnitude is large. if overgrowth by revertant does not interfere with vaccine growth, then vaccine growth and antigen production are not affected (fig ) . evolution affects antigen production only to the extent that revertant superiority suppresses vaccine growth and thereby suppresses antigen production. numbers versus frequencies. models of evolution often address relative frequencies, on a scale of to . immunity develops in response to vaccine density. in addressing immunity, it is thus necessary for the model to track densities, whereas any evolution is more easily described with frequencies (the two approaches can be compared between figs and ). the challenges are thus to understand (i) when and how much vaccine evolution occurs; (ii) whether and to what extent evolution affects the abundance of vaccine virus in the host over time; and (iii) how changes in vaccine abundance affect the generation of adaptive immunity against the antigen. the arguments presented above are qualitative and only superficially identify the scope of the problem. quantitative understanding ultimately rests on analysis of mathematical models. however, as the models have many interacting processes-minimally innate immunity, adaptive immunity and intrinsic growth differences between vaccine versus revertant-we first verbally explain the biology underlying the processes that go into those models. intrinsic fitness differences. intrinsic fitness effects are considered here to be those that stem from the intracellular processes of viral gene expression and assembly, independent of host immune responses. intrinsic fitness differences between the vaccine and the revertant (wild-type vector) are plausible because the transgene is non-essential and has no evolutionary history with the vector genome. thus, the insertion may be disruptive, and the resulting antigen expression may interfere with vector functions. intrinsic fitness effects are expected to affect evolution during vaccine manufacture as well as within-host evolution, but it has largely been investigated in vitro, as would apply to manufacture and the pre-host phase. indeed, intrinsic fitness differences may be the sole or at least the most important bases of vaccine inferiority. because recombinant viruses have often been observed to evolve loss or down-regulation of engineered inserts, they are now commonly observed during in vitro growth for their 'genetic stability' (e.g., [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). some recombinant viral genomes are stable over short term transfers in culture, others not, indicating that intrinsic fitness effects of the engineering are not universal. thus, the possibility of vaccine inferiority should not be ignored, and furthermore, even when a vaccine appears to be stable over a few transfers, the short term population where the revertant has a % fitness advantage and starts at a frequency of − . the curve shows the well-known population genetic principle that, while the favored type (revertant) is rare, its absolute frequency changes very little. but the frequency eventually reaches a level at which evolution is rapid. the yellow box represents a possible period of pre-host evolution, the green box representing the period of within-host evolution. the periods of within-host evolution are drawn to be the same length in right and left panels, as if the vaccine has the same within-host duration in both cases. the arrow represents a possible point at which manufacture would end and an inoculum be created, thus defining the boundary between pre-host and within-host evolution. the left panel depicts a short period of vaccine manufacture, the right a longer period of vaccine manufacture and one in which more pre-host evolution has occurred. it is thus easy to see the potential importance of pre-host vaccine evolution on within-host evolution, for even when the revertant is not a large component of the inoculum, it can be poised for rapid evolution within the host (right panel). the curve obeys p t ¼ p w t p w t þ À p , in which p t represents the revertant frequency in generation t and w the fitness of revertant relative to vaccine. the curve is drawn for a common evolutionary process across pre-host and within-host evolution, but evolution in the within-host phase will typically experience different parameters than evolution in the pre-host phase. retention of antigen expression may mask an underlying long term instability. thus most observations of stability merely set limits on the possible magnitudes of inferiority. yet even if vaccine selective 'neutrality' turns out to be fleeting, merely a mistaken impression from shortterm observations, we will find that the phenomenon of short-term stability mirrors a solution to minimize vaccine evolution within the host-the solution of limiting vaccine growth. the revertant virus has the superior growth rate, but in the absence of interference between the two, vaccine growth is unimpeded and immunity is triggered. https://doi.org/ . /journal.pcbi. .g three mechanisms of vaccine-revertant interference. fig presented a hypothetical case in which evolutionary superiority of revertant did not suppress vaccine growth, hence evolution had little effect on antigen production. that process was one in which there was no interference between vaccine and revertant growth. evolution does become important to antigen levels if vaccine and revertant interfere so that vaccine growth is depressed by the revertant, or if the duration of infection by the vaccine strain is reduced. in either case the revertant will then suppress antigen levels. again, the problem is complicated by the limited duration of the infection: reduced antigen production due to vaccine evolution depends not only on interference between the two genomes but also on overall growth and the extent to which it affects the level of immunity to vaccine and vector. a mechanism that forces interference between vaccine and revertant can also limit the total amount of viral growth, thereby limiting evolution. evolution of vaccine versus revertant thus depends on details, in particular, the specific mechanism by which revertant interferes with vaccine growth. we describe three different mechanisms that have been proposed that may be relevant to vaccine competing with revertant: innate immunity, resource limitation, and adaptive immunity to the vector backbone shared by the vaccine and revertant virus. (these are not the only possible mechanisms of within-host parasite competition [ ] , but they capture the relevant immune processes.) for many vaccines, each mechanism will impede revertant and vaccine equally as a collective population, thus ensuring interference. it was initially believed, implicitly if not explicitly, that the adaptive immune response played the dominant role in the control of viruses and other infections. in the 's, janeway and medzhitov identified shared pathways for the control of pathogens between vertebrates and drosophila, even though drosophila lacks an adaptive immune response (reviewed in [ ] ). this led to a resurgence of interest in the role of innate immunity in the initial control of infections. later modeling studies of influenza infections suggested yet another mechanism, that the initial control of these infections could be largely described by simple resource limitation models, of the type used in ecology for population growth [ , ] . the realization that all three different processes might suppress viral infection led to more careful examination of the roles of different factors in the early control of acute infections [ ] [ ] [ ] [ ] . the relative role of each mechanism in clearing infections is the basis of ongoing discussion, but it is widely accepted that the roles differ among infections by different viruses and that each mechanism is potentially important for some viruses. • innate immunity. there are two broad arms of immunity for suppressing vaccine growth within the host, the innate and the adaptive immune responses. innate immunity is triggered by conserved molecules associated with pathogens (pathogen associated molecular patterns, [ ] ). conserved structures of pathogens targeted by innate immunity include dsrna, frequently accompanying viral replication, plus lipopolysaccharides and endotoxins of bacteria [ ] . because innate immunity involves the activation of a standing population of immune cells such as macrophages and dendritic cells, or triggering of the complement pathway, it can be elicited much more rapidly than the adaptive response; the latter requires many rounds of clonal expansion of rare antigen-specific cells to generate a population large enough to control the infection [ ] . furthermore, recent studies have shown that the innate response is required for the initial stimulation of the adaptive response [ ] . thus, innate immunity has a major role in early control of the viral population. innate immunity can control both vector and vaccine, and it is not likely to discriminate between two genomes that differ by a single, non-essential gene (the transgene). • resource limitation another mechanism by which revertant levels can suppress vaccine levels is resource limitation. both the vaccine and revertant virus use the same resource (susceptible host cells). resource limitation can control the infection if the virus depletes this resource, whereby the rate of virus output falls below its intrinsic death rate [ ] . like innate immunity, resource limitation is expected to affect vaccine and revertant similarly. resource limitation has been considered an important mechanism for competing malarial strains within the host [ , ] . • adaptive immunity adaptive immunity can be induced by the revertant and the vaccine virus. adaptive immune responses to antigens expressed by the revertant will presumably affect the vaccine and revertant equally-because the vaccine encodes a complete vector genome, and the revertant is also a complete vector/virus. as with the preceding pair of mechanisms, adaptive immunity elicited by the revertant will also depress the abundance of the vaccine virus. adaptive immunity to the vaccine antigen will be considered shortly. all three interference mechanisms will potentially operate in any vaccinated host. with all three operating, one mechanism may take precedence over the others, simply because it is activated earlier or enforces a lower limit on viral density than the others. however, there are different stages or degrees of vaccine suppression, so an early mechanism may act to control the infection without clearing it, and another mechanism may act later to clear. because of the delay in developing an adaptive response, viral suppression by adaptive immunity typically occurs later than effects of innate immunity or resource limitation and so might seem to be unimportant in vaccine evolution. yet adaptive immunity may be important in clearing the vaccine following control by other mechanisms, in which case it could have an important role in vaccine evolution. adaptive immunity to the vaccine antigen may also contribute to vaccine inferiorityand feed back to inhibit itself. the preceding paragraphs omitted adaptive immunity to the antigen. by its very nature, adaptive immunity suppresses vaccine growth. but adaptive immunity to the antigen is specific to the vaccine and is thus another reason-besides intrinsic fitness effects-that the vaccine may have lower fitness than revertant. the evolutionary consequences should be the same for both types of inferiority, reducing the long term generation of antigen levels within the host, but adaptive immunity would be irrelevant to vaccine evolution during manufacturing and during early growth within the host. an interesting twist is that adaptive immunity to the antigen might eventually feed back negatively on itself to limit its own growth-immunity against a virus is intrinsically inhibitory, so adaptive immunity against the vaccine will limit vaccine growth and thus limit antigen build-up that would fuel further immunity. one question is whether this self-inhibition is worsened with vaccine evolution. the effect is biologically complicated because adaptive immunity to the antigen does not necessarily translate into selection against the vaccine. selection against the vaccine per se operates only when adaptive immunity specifically targets the vaccine genome over the revertant genome, and this selection need not occur-either because adaptive immunity is so delayed that it is never manifest during vaccine growth, or because the antigen is physically decoupled from its genome when attacked by the adaptive response. without imposing selection on the vaccine, antigen-directed immunity will not affect vaccine evolution. we now employ quantitative models to evaluate the intuitive ideas presented above. given the high dimensionality of the problem, we are especially interested in how well intuition works and whether generalities are observed across large regions of parameter space. a flow diagram of the elements and interactions within the host reveals the complexity of the model (fig ) and facilitates understanding the dynamical equations. v and w are the respective vaccine and revertant densities, with intrinsic growth and death rates governed by four parameters (not illustrated). the model also includes variables for resources (r), innate immunity (z), adaptive immunity to vector (y), and adaptive immunity to antigen (x) that are both influenced by and influence v and w. in the following sections, we explore the dynamics of these interactions with simulations and present results graphically (the results presented do not allow resource limitation to influence dynamics; trials where resource limitation matters were conducted but are not shown). equations and parameter values are provided in s appendix. resource limitation and innate immunity yield qualitatively similar results, so trials with resource limitation are not illustrated in the main text. the equations apply only to within-host processes; any pre-host evolution is subsumed into inoculum composition. the models assist us by forcing us to specify assumptions for how the viruses and immunity interact, and by allowing us to rigorously explore outcomes in different scenarios. however, there is uncertainty in the model structure, many parameter values are unknown, and different viruses will behave somewhat differently. consequently, we focus on broad generalities that arise from many simulations and illustrate these for a few specific cases, reserving supporting information files for further details. the presentation below briefly discusses the dynamics of individual trials for illustration but then moves to contour plots that reveal differences in outcomes as the key parameters are changed. the model used here incorporates the structure of earlier models that described immune responses [ ] [ ] [ ] ; parameter values used here were chosen as described in some of these earlier studies. evolution can matter. in the trials used for illustration, we allow innate immunity to control the infection and adaptive immunity to cause final clearance. such a scenario might correspond to the dynamics of listeria or influenza infections of mice [ ] , or the early dynamics of siv infections [ ] . to get a sense of the full dynamics in the model, we show the time course of dynamics for the different variables (fig ) under conditions of no evolution (top left), just pre-host evolution-revertant abundant in the inoculum but with no fitness advantage (top right), mostly within-host evolution (bottom left), and both (bottom right). the effect of different types of evolution is seen from a comparison of the panels. our chief interest is in final immunity to vaccine, the blue curves. the most visible effect of evolution on immunity to the antigen is evident in the lower right panel, which combines pre-host evolution with a fitness advantage of the revertant. however, the log scale diminishes the visual impact of substantial evolution in other cases. when the revertant is half the inoculum but has no fitness advantage, the immune response is diminished by nearly -fold (top right). overall, the impression is that one must at least suppress either pre-host or within-host evolution to avoid a large loss in immunity (lower right versus the others). illustrations of dynamics from individual trials convey many details. however, without a specific empirical basis for the parameter values chosen, the details have little assured relevance. we therefore provide contour plots that allow easy comparison of many different trials in which parameters of specific interest are varied (fig ) . these graphs show the cumulative vaccine load (left panel) and final level of immunity to vaccine antigen (right) as a function of initial revertant frequencies and selective advantage of the revertant (c). a strong correspondence exists between vaccine load and the level of immunity generated, as is observed empirically following infection [ ] . subsequent figures therefore illustrate the level of immunity. the initial composition of the inoculum matters somewhat more to the adaptive response than does the intrinsic cost of the vaccine (as evident by the contours being closer to vertical rather than horizontal), but this pattern rests heavily on the parameter ranges chosen. indeed, unrealistically large values of initial revertant levels (w( )) are illustrated to offer contrast, as the outcomes are otherwise moderately insensitive to vaccine composition in these graphs. the good news is that, when the inoculum is mostly vaccine and revertant fitness is not high, evolution has little effect on viral load or final level of immunity (i.e., the lower left of each panel has a broad area of one color). this occurs because of the short duration of infection. over longer periods of vaccine growth, the selective advantage of the revertant would undoubtedly play an increasing role in evolution. the large number of parameters ( ) limits the degree to which we can conduct comprehensive sensitivity tests, so the trials are confined to variations in those parameters of greatest interest. vaccine evolution driven by adaptive immunity. we focus on infections of short duration-that are cleared and do not rebound once suppressed. factors that limit the duration of infection include resource limitation, and innate and adaptive immunity. for the most part these factors act equally against vaccine and revertant virus. only one factor, adaptive immunity to the vaccine antigen (x), acts specifically on the vaccine virus and not the revertant. intuition suggests that this adaptive immunity to the antigen can potentially suppress the vaccine's growth and give an advantage to the revertant. as with intrinsic fitness costs, this selection might feed back to limit vaccine growth and thus limit the development of further immunity to antigen by allowing revertant to grow and interfere with vaccine. this section considers whether these arguments are supported by the model. any real vaccine that elicits immunity against the antigen may also experience an intrinsic fitness cost. the effect of immunity on evolution would then be confounded with the effect of intrinsic fitness effects on evolution, making it difficult to isolate one from the other. the models do not face this problem, however. they can be parameterized so that the only possible selection against the vaccine comes from immunity (by setting c = ). vaccine populations can also be freed of revertant by omitting revertant from the inoculum and setting the mutation rate to . thus, we can measure the effect of adaptive immunity on vaccine growth from trials that lack revertant and then compare those results with trials that include revertant. the revertant is included at half the inoculum (representing pre-host evolution); it has no intrinsic fitness advantage over vaccine. immunity (to vaccine) is reduced to just over a third of the level with no evolution. (lower left): the revertant is a small fraction of the inoculum ( . ) but it has a % fitness advantage over vaccine. the level of immunity is % that with no evolution. (lower right): the revertant is half the inoculum and has a % fitness advantage over vaccine. the level of immunity is now less than . % that with no evolution-a depression of almost orders of magnitude. the trials are parameterized so that virus is controlled by innate immunity with final clearance due to adaptive immunity; the mutation rate is in all cases. equations, initial conditions and parameter values not shown here are given in s appendix; r code is included in s file. there are several background points to note about the model structure. first, adaptive immunity specific to vaccine (x) develops at a rate proportional to the vaccine abundance (v) and parameters s (rate of clonal expansion of adaptive immunity) and ϕ x (antigen concentration yielding half the maximum growth rate of adaptive immunity x). in contrast, the impairment specific to vaccine is due to the level of immunity (x) and the vaccine impairment parameter k x . thus, immunity can develop while imposing little or no impairment, i.e., when k x ! . second, adaptive immunity to the vector (y) develops according to its own growth rate parameter (ϕ y ) in response to vaccine plus revertant abundance (v + w), and it impairs both vaccine and revertant growth equally by impairment parameter k y . when revertant is present, it increases the level of immunity to vector backbone/revertant but does not directly affect immunity specific to the vaccine. this immunity will result in faster clearance of both revertant and vaccine, and this results in decreased immunity to the antigen; this is the 'interference' that causes a problem from vaccine evolution. trials were run that contrasted revertant absence versus revertant introduced at % of the inoculum-no evolution versus primairly pre-host evolution, respectively (fig ) . absence of the revertant is the baseline against which the effect of evolution can be compared. the horizontal axis varies k x , the parameter for impairment/killing specific to vaccine, and the vertical axis varies k y , impairment to vector, which affects vaccine and revertant equally. in both panels, increasing impairment against vaccine leads to lower levels of immunity to the vaccinethis is the self-limiting effect of adaptive immunity, which exists even in the absence of evolution. as expected, impairment of immunity to vaccine by immunity to vector is also found. a large effect of inoculum composition on vaccine immunogenicity is evident by comparing the left panel (no mutation, no evolution) and right panel (chiefly prehost evolution): introduction of revertant reduces the level of immunity against vaccine up to -fold. for the -host evolution) . the final vaccine load and immunity against the vaccine antigen depends heavily on two parameters, the inoculum composition (plotted on the x-axis as initial abundance of the revertant virus, w( )) and the growth advantage of the revertant within the host (c, plotted on the y-axis). the heat maps show how, as the composition shifts toward revertant or as vector superiority increases (as we move to the right or up), there is a reduction in the viral load of the vaccine (defined as r v dt, left panel) and in the magnitude of immunity to the vaccine antigen (x, right panel). the initial amount of vaccine virus is always v( ) = (i.e. logv( ) = ). note that the graphs span high frequencies of revertant in the inoculum that should be easily avoided (log w( ) = , i.e. w( ) = v( ))-if the researcher is alert to the possibility. we include such extremes merely to show that the outcome is relatively insensitive to small changes in vaccine composition. equations, initial conditions and parameter values not shown here are given in s appendix; r code is included in s file. https://doi.org/ . /journal.pcbi. .g within-host vaccine evolution right panel, the revertant is / the inoculum and has no intrinsic advantage over vaccine; total inoculum size is unchanged. all reduction of immunity against vaccine is thus due to revertant in the inoculum and any within-host evolution from the selective effect that stems from immunity against vaccine. of the two parameters, k y has a much larger effect than does k x : compared to the left panel, the right panel, with revertant present, has vaccine-specific immunity suppressed more than an order of magnitude along the k y axis, much less so on the k x axis. we attribute this effect of k y to interference by the revertant: the revertant elicits high levels of immunity (y) that indiscriminately also suppress vaccine, thereby suppressing vaccine-specific immunity x. the magnitude of interference depends not only on revertant abundance but also on the values of k x , k y , and the innate immune response (k z ), so interference can appear more or less important in other trials using the same revertant abundance. a question motivating this analysis was one step deeper in the complexity of these effects: does the self-limiting effect of adaptive immunity worsen when revertant is present? this question can be answered by comparing the self-inhibitory effect between left and right panels as k x is increased. by inspection of colors along the horizontal axes, it is seen that the self-inhibitory effect is actually somewhat reduced by the revertant. the revertant lowers the overall response to vaccine, but when correcting for that difference, the effect of increasing k x is slightly weaker in the right panel than in the left. we attribute this weakening of self-limitation as due to vaccine levels being increasingly controlled by immunity against revertant. in sum, therefore, immunity to the vaccine (x) is reduced by itself (depending on the immunity parameter, k x ) and by revertant. the two effects do not interact to make the problem worse than from their separate effects. the results above suggest that vaccine evolution is only likely to compromise immunity if there is substantial pre-host or within-host evolution and if this evolution depresses vaccine effect of evolution on the suppression of immunity by impairment parameters. the final level of immunity to the vaccine antigen (x) depends heavily on the inhibitory parameters k x and k y -which respectively describe how strongly immunity to the vaccine (x) and revertant (y) suppress the viral populations. the left plot considers the absence of revertant, hence no evolution. the right panel introduces revertant at / virus in the host. as the short duration of infection limits within-host evolution, one means of achieving vaccine efficacy is to control the inoculum. two ways of controlling the inoculum are to control its composition and to control its size. pre-host evolution can be reversed by purifying the inoculum after the fact or by taking care to start with a pure isolate and limiting growth (e.g., fig ) . the benefit of suppressing revertant frequency in the inoculum is evident in fig : the magnitude of immunity to the vaccine increases by orders of magnitude as the initial frequency of the revertant is decreased. the effect is strongest at low inoculum levels, pointing to the other solution-increase inoculum size. intuition also suggests that the deleterious effects of evolution can be reduced by increasing the inoculum size, provided the composition does not change: to achieve a threshold antigen level, a large inoculum requires less growth than a small one. less growth reduces the potential for evolution-in the extreme, a large enough inoculum requires no vaccine growth, as with killed vaccines. these conjectures are supported by fig : when the revertant frequency in the inoculum is high, increasing the inoculum size appreciably increases the magnitude of immunity; a much reduced benefit is seen when revertant frequency is low, likely because there is less evolutionary interference from the revertant. these results suggest parallel benefits from reducing the frequency of the revertant in the inoculum and increasing the dose. consideration of the gains from each could help choose an economically feasible strategy, since both purifying the inoculum and increasing its dose are likely to incur financial costs. whether and how well controlling the inoculum will work in practice will depend on details. solutions may be quantitative rather than absolute. intuition is useful for guidance but needs to be confirmed by formal analyses, guided by data from the specific implementation. any live viral vaccine may evolve within the host. the potential for attenuated viruses to revert to wild-type virulence is well appreciated [ , ] , even if it presents a problem for relatively few vaccines (e.g., attenuated polio, [ ] ). there is also a potential for live, recombinant vector vaccines to evolve-our focus in this paper-with the main concern being loss or reduced expression of the transgenic insert [ , ] . if such a vaccine were to evolve fast enough or long enough that it lost the insert, vaccine efficacy might well suffer. we find that evolution during manufacture (pre-host evolution) can play a more important role than within-host evolution in reducing vaccine efficacy, and furthermore that it may be the more easily mitigated. we developed and analyzed models to explore ways in which vaccine evolution could lead to a reduction in vaccine efficacy. an intrinsic fitness advantage of the revertant virus, expected because transgene expression is likely to have metabolic and other costs, will lead to vaccine being gradually overgrown by revertant. this is only likely to cause a reduction in the immunity to the vaccine antigen if it leads to a reduction in the absolute amount (as opposed to merely a reduction in relative frequency) of the vaccine virus. there are in fact several mechanisms by which an ascending revertant population may suppress vaccine: revertant can reduce the amount of the vaccine virus in the host if the revertant uses resources required for virus replication or if the vaccine virus is cleared by the innate or adaptive responses elicited by the revertant. the clear and positive message from our study is that vaccine evolution, if it proves to be a problem for immunization, should be easily mitigated by manipulating the vaccine inoculum. critical to understanding and addressing this problem is recognizing that the vaccine may evolve both within the host and also during manufacture, whereby the inoculum already carries modest to high levels of revertant. the composition of the inoculum can have a large effect on within-host evolution and immunity. by limiting the amount of revertant in the inoculum, and also by boosting the inoculum level, it should usually be possible to limit the amount of within-host vaccine evolution and ensure that immunization is effective. we emphasize, however, that this solution will typically not work for transmissible vaccines and vaccines that establish long term infections within the host. furthermore, using a large inoculum may seem to defeat the purpose of using a live vaccine. there may be cases in which vaccine evolution is so rapid that controlling the inoculum is not sufficient. the solution in this case is to develop or engineer the vaccine with less of a disadvantage. the timing and tissues of antigen expression, location of the transgene in the vector genome, and the size of the transgene may all influence intrinsic fitness effects [ , , , , ] . directed evolution approaches might also improve vaccine efficacy: one simple approach in reducing an intrinsic cost might be to adapt the vector in vitro to host cells expressing the effects of manipulating the inoculum on immunity to the vaccine. small inocula that contain vaccine plus revertant are more prone to reduced immunity levels than are large inocula with little revertant. composition of the vaccine has the larger effect for the inoculm sizes and initial revertant fractions shown, as indicated by the contours being more horizontal than vertical. an intrinsic fitness cost of c = . was set for these trials. smaller c values would lead to higher vaccine and immunity levels across the graphs. equations, initial conditions and parameter values not shown here are given in s appendix; r code is included in s file. https://doi.org/ . /journal.pcbi. .g antigen in trans, allowing compensatory mutations to evolve in response to the antigen before the transgene is cloned into the genome. this adapted vector would then be used as the vaccine backbone. another simple approach would be to compete several different vaccine designs in vitro and pick the design with highest retention of the transgene. any approach using in vitro adaptation needs to avoid adapting the vector to the extent that it compromises ability to grow in vivo. most of these possibilities are ways to reduce pre-host evolution and reduce revertant concentration in the inoculum. one may hope that vaccine designs which reduce pre-host evolution also reduce within-host evolution. measuring the intrinsic fitness effect of the transgene is likely to be an important step in vaccine design. for assessing vaccine evolution, the relevant biological realms are within the host and in vitro. in vitro growth environments are the more easily studied and may reveal much about a vaccine's intrinsic propensity to evolve loss of antigen expression. there are various ways intrinsic fitness effects and their evolutionary consequences might be studied. vaccine growth in tissue culture may reveal some aspects of intrinsic fitness effects and should be relatively easy to study. deletion of the transgene per se would be detectable by pcr, and the fitness advantage of revertant over vaccine could be measured from changes in revertant frequency. the quantitative relevance of an in vitro estimate to in vivo growth would be unknown, but the measure should allow qualitatively comparing engineering designs that improve intrinsic vaccine fitness. if vaccine reversion were due to down regulation of the transgene instead of deletion, fitness estimation would require knowing the mutations responsible and monitoring their frequencies. use of culture-wide antigen levels to measure fitness might provide a sense of whether vaccine evolution would lead to reduced antigen levels in vivo, but it would be less sensitive in measuring evolution than is measuring mutation frequencies. evolution is not the only consideration in designing a recombinant vector vaccine, and the model helps us identify vaccine properties that promote efficacy. first the vaccine should elicit an immune response that rapidly clears the pathogen (i.e. the rate constant for clearance of the pathogen, call it k p , is high). second, the vaccine should elicit a large response to this antigen. this requires that the antigen rapidly elicits immunity (i.e. has low ϕ x , and in terms of immunology it should be an immunogenic antigen), and also requires a high vaccine viral load to generate a large response. engineering this requires tackling a trade-off between avoiding vaccine clearance (i.e. having a low k x ) but allowing for rapid clearance of the pathogen (having a high k p ). vaccines designed to express the antigen in a form that is different from that in the pathogen might help solve this problem. thus, to elicit immunity to influenza, one might design secreted forms of the hemagglutinin or neuraminidase proteins. a recombinant hemagglutinin protein that is secreted rather than on the virion surface would prevent the antibody response to this protein from clearing the recombinant vector vaccine (have low k x ) without compromising the clearance of the influenza virus pathogen which has hemagglutinin on its surface (i.e. has high k p ). in this manner our model allows the identification and tuning of parameters that affect vaccine efficacy, and a comprehensive search of parameter space would identify ideal combinations of vaccine properties. in vitro assays may be useful in measuring intrinsic fitness effects, but in vivo-in the patient-is the ultimate environment for studying within-host evolution and its effects. not only are the dynamics of viral spread different between in vitro and in vivo environments, but most immune components will be in play only in vivo. furthermore, those components may vary across tissues within the host. sampling across this heterogeneity in vivo will be challenging but may be necessary to know whether, when, and where vaccine evolution is a problem. if revertant remains a minority of the population, we expect that vaccine evolution can be ignored. perhaps in vitro studies of vaccine evolution will provide most of the information relevant to in vivo evolution, but it is too early to know. we have focused on recombinant vector vaccines that cause acute infections. necessarily, our recommendations are based on simple models that are caricatures of the complex withinhost dynamics of acute infections. simple models are appropriate at this stage because of uncertainties at many biological levels, and under these circumstances simple models frequently generate more robust results than do complex models [ , ] . the generation of innate and adaptive responses can be modeled with different assumptions than used here, and those alternative processes may affect the conclusions. for example, time-lags in the activation of cells may dominate the time for the generation of an innate immune response, with virus density having a consequently smaller role than assumed here (as can be seen in [ ] and modeled in [ ] ). we have modeled that responses to different antigens are generated independently of each other and do not compete. we have done so because vaccines are likely to cause relatively mild infections during which the densities of pathogen and immune cells do not reach sufficiently high levels required for competitive interactions to be important. the adaptive immune response may be more influenced by recruitment which is followed by a period of proliferation even in the absence of antigen [ ] [ ] [ ] . both these scenarios would minimize the impact of evolutionary changes in the vaccine on the amount of immunity generated to the transgene. finally, it is easily appreciated that there are realms we do not consider, such as within-host spatial structure [ ] and recombinant vector vaccines based on viruses such as cytomegalovirus that cause persistent infections [ ] or that are transmissible. spatial structure may limit the impact of vaccine evolution on immunity (e.g., prevent mutants from taking over the entire population). in contrast, vaccines that cause persistent infections or are transmissible are likely to be more severely affected by evolution than are vaccines causing acute infections, as there is a longer timeframe for evolution to operate. with so little experience from recombinant vector vaccines, we can merely guess how commonly the neglect of within-host evolution will compromise vaccine efficacy. given that simple steps can be taken to reduce vaccine evolution, vaccine development programs should at least entertain the possibility that evolution can underlie failure. avoiding vaccine evolution may be easier than developing an entirely new vaccine. supporting information s appendix. equations and parameters. (pdf) s file. r markdown file of code to run numerical trials of equations and generate figures. this file was used to generate the figures in the paper but may also be used to run other conditions, such as the case when resource limitation controls the infection. evolutionary reversion of live viral vaccines: can genetic engineering subdue it? virus evolution the double-edged sword: how evolution can make or break a live-attenuated virus vaccine vaccine-derived poliomyelitis years after infection in minnesota transmissible viral vaccines rationalizing the development of live attenuated virus vaccines stability of rna virus attenuation approaches unique safety issues associated with virus-vectored vaccines: potential for and theoretical consequences of recombination with wild type virus strains defining the interval for monitoring potential adverse events following immunization (aefis) after receipt of live viral vectored vaccines. vaccine an introduction to population genetics theory genomic stability of murine leukemia viruses containing insertions at the env- ' untranslated region boundary transgene stability for three replication-competent murine leukemia virus vectors in vitro and in vivo genetic stability studies of a human adenovirus type recombinant rabies glycoprotein vaccine (onrab). vaccine challenges in predicting the evolutionary maintenance of a phage transgene construction and in vitro evaluation of a recombinant live attenuated prrsv expressing gm-csf a recombinant canine distemper virus expressing a modified rabies virus glycoprotein induces immune responses in mice rescue and evaluation of a recombinant prrsv expressing porcine interleukin- a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform an attenuated emcv-hb strain acts as a live viral vector delivering a foreign gene poxviral promoters for improving the immunogenicity of mva delivered vaccines generation of infectious clone of bovine adenovirus type i expressing a visible marker gene characterization of recombinant flaviviridae viruses possessing a small reporter tag going, going, gone: predicting the fate of genomic insertions in plant rna viruses parasite adaptations to within-host competition innate immune recognition modelling viral and immune system dynamics kinetics of influenza a virus infection in humans dynamics of influenza virus infection and pathology modeling within-host dynamics of influenza virus infection including immune responses what controls the acute viral infection following yellow fever vaccination? mathematical analysis of a mouse experiment suggests little role for resource depletion in controlling influenza infection within host recognition of viruses by innate immunity a model of non-specific immunity regulation of adaptive immunity by the innate immune system age-structured red blood cell susceptibility and the dynamics of malaria infections the dynamics of acute malaria infections. i. effect of the parasite's red blood cell preference within-host population-dynamics and the evolution and maintenance of microparasite virulence virus dynamics mathematical principles of immunology and virology towards a general function describing t cell proliferation roles of target cells and virusspecific cellular immunity in primary simian immunodeficiency virus infection initial viral load determines the magnitude of the human cd t cell response to yellow fever vaccination reaching the last one per cent: progress and challenges in global polio eradication. current opinion in virology evaluating the promise of recombinant transmissible vaccines. vaccine novel replicationincompetent adenoviral b-group vectors: high vector stability and yield in per.c cells modified h promoter improves stability of insert genes while maintaining immunogenicity during extended passage of genetically engineered mva vaccines the ecological detective: confronting models with data. monographs in population biology uses and abuses of mathematics in biology analysis of in vivo dynamics of influenza virus infection in mice using a gfp reporter virus memory cd + t cell differentiation: initial antigen encounter triggers a developmental program in naive cells naive ctls require a single brief period of antigenic stimulation for clonal expansion and differentiation models of cd + responses: . what is the antigen-independent proliferation program causes and consequences of spatial within-host viral spread. viruses self-boosting vaccines and their implications for herd immunity key: cord- - ronfq authors: nicholson, karlg; prestage, howard; cole, peterj; turner, georges; bauer, sallyp title: multisite intradermal antirabies vaccination: immune responses in man and protection of rabbits against death from street virus by postexposure administration of human diploid-cell-strain rabies vaccine date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: ronfq lymphocyte transformation, production of neutralising antibody, and the development of antirabies igg antibody were studied in ten healthy volunteers in response to · ml of human diploid-cell strain (hdcs) rabies vaccine administered on one occasion in divided doses in intradermal (i.d.) sites. all ten volunteers rapidly developed substantial titres of rabies antibody, and eight of the ten had t lymphocytes that were immunologically stimulated by hdcs rabies-virus antigen. postexposure treatment with · ml of hdcs vaccine given at i.d. sites completely protected fourteen rabbits from death by street virus. the results suggest that in developing countries patients could be protected with small volumes of potent tissue-culture vaccine administered intradermally shortly after exposure. lymphocyte transformation, production of neutralising antibody, and the development of antirabies igg antibody were studied in ten healthy volunteers in response to &middot; ml of human diploid-cell strain (hdcs) rabies vaccine administered on one occasion in divided doses in intradermal (i.d.) sites. all ten volunteers rapidly developed substantial titres of rabies antibody, and eight of the ten had t lymphocytes that were immunologically stimulated by hdcs rabies-virus antigen. postexposure treatment with &middot; ml of hdcs vaccine given at i.d. sites completely protected fourteen rabbits from death by street virus. the results suggest that in developing countries patients could be protected with small volumes of potent tissue-culture vaccine administered intradermally shortly after exposure. introduction in , forty-five people severely bitten by rabid dogs and wolves in iran were treated after exposure with a new rabies vaccine produced in cultures of human diploid cells. all except one also received one injection of rabies immune serum. this treatment, in contrast to past experience with other vaccines, resulted in protection of all individuals against rabies. this resounding success has been repeated in trials in germany and the u.s.a. using or doses of human diploid-cell strain (hdcs) rabies vaccine and human rabies immune globulin.', thus, almost a century after the post exposure treatment of man began, effective antirabies prophylaxis appears to have been achieved. with few exceptions, rabies is a problem of impoverished areas of the world, where the annual per-caput expenditure on health care is often far less than the cost of a single ml dose of hdcs vaccine. as a consequence, potent tissueculture vaccine is seldom used in the third world. the need for an effective but less expensive method of treatment prompted us to investigate the possibility of administering potent vaccine more economically and efficiently than at present. our previous studies have shown that substantial titres of antibody can be achieved with small quantities of hdcs vaccine administered by the intradermal (i.d.) route and that the cost of vaccination can be reduced considerably. - in this paper we report the antibody and cellmediated immune response of man to multisite i.d. vaccination and application of the method to postexposure protection of rabbits. it appears possible that the i.d. route could be applied successfully to the post exposure treatment of man. approval for the volunteer study was given by northwick park hospital ethical committee. hdcs vaccine ( - ml) was given i.d., on single occasion, to ten volunteers at sites on the medial and lateral aspects of the upper arms and thighs. blood samples for tests of lymphocyte transformation and antibody determination were taken before vaccination and , , , and days later. a further blood sample for antibody titration was taken on day . four subjects were vaccinated with a dermojet injector;* the six remaining volunteers were given vaccine with a -gauge needle and tuberculin syringe. no volunteer had received antirabies vaccine previously. the nuchal muscles of forty-two new zealand white rabbits were inoculated with rabbit ld ofa first mouse-brain-passage arcticfox rabies virus isolate in two separate sites ( - ml each side). h later fourteen rabbits were given - ml of hdcs vaccine intramuscularly (i.m.) into the left forelimb, and fourteen others received i.d. injections of - ml of vaccine into each limb. the remaining fourteen were used as controls and received no prophylaxis. each animal was then observed for months for signs of rabies. none of the rabbits had been exposed to rabies previously or had been immunised against the disease. in both studies whole-virion hdcs rabies vaccine (i'institut merieux; lot r ; antigenic value - ) inactivated with -propiolactone was used. all blood samples were titrated for rabies neutralising antibody with the mouse neutralisation test; titres in iu/ml were calculated by reference to the international standard antiserum to rabies virus (statens seruminstitut, copenhagen, denmark). in addition, the sera were assayed for igg rabies antibody by enzyme immunoassay (elisa) using a modification of the method used for the detection of coronavirus antibodiesl&deg; (k. g. nicholson, h. prestage, unpublished). absorbance values were read at nm on a flow laboratories titertek multiskan spectrophotometer , , , and min after addition of the substrate. rabies antibody was considered present when the optical-density reading of the test sample was greater than the mean + standard deviations of comparable dilutions of negative control sera. an enriched t-lymphocyte population was obtained by passing a thrice-washed mononuclear-cell suspension taken from the top of a ficoll-triosil gradient (pharmacia fine chemicals, uppsala, sweden) twice through a nylon-fibre column." the cell suspension obtained contained less than % b lymphocytes as judged by staining with polyvalent fluorescein-labelled antihuman immunoglobulin reagent. an enriched b-lymphocyte population was obtained by sedimenting rosettes formed between t lymphocytes and sheep red blood cells." lymphocytes from human cord blood and a non-vaccinated subject were used as controls. hdcs rabies-virus vaccine (l'institut merieux; lot r ) was exhaustively dialysed against phosphate-buffered saline (pbs) and adjusted to the original volume with pbs for use as antigen. phytohaemagglutinin (pha; purified grade, wellcome laboratories, beckenham) was used as control at a concentration of - mitogenic units/ml. cultures containing of antigen or mitogen and of cell suspension containing x lymphocytes were established in microtitre plates then pulsed with tritiated thymidine and harvested as described previously.' because of the ph indicator in the vaccine, needle inoculation immediately resulted in magenta-coloured skin blebs at each injection site. vaccination with the dermojet injector was generally quicker, but bleb formation was less satisfactory and in some areas where the skin was especially soft it appeared that all the vaccine had entered the subcutaneous tissue. this method of inoculation was also associated with lower titres of antibody than occurred after needle inoculation (figs. six subjects who were inoculated with a needle and syringe. by days, neutralising antibody ranged between and iu/ml (geometric mean titre [gmt] iu/ml) and antirabies igg ranged between / and / (gmt / ). peak titres of virus neutralising antibody were found on day (gmt ' iu/ml), but substantial titres were still present days after vaccination. the increments (cpm) in lymphocyte transformation are expressed as a ratio to the cpm values of non-stimulated control cultures (table). the results show that t lymphocytes from eight of ten vaccinees were significantly stimulated in vitro to days after vaccination. this blast transformation occurred with cells from three of four people given vaccine by the dermojet injector and from five ofthe six people inoculated with a needle and syringe. there was no significant difference between the transformation increments of the two groups, and no correlation was found between the transformation increment and the titre of antirabies igg or nine of fourteen rabbits developed paralysis and died after infection with rabbit ldso of street-rabies virus. in these animals, forelimb paralysis developed within to days of infection and progressed to complete paralysis and death to days later. postexposure treatment with a single dose of hdcs vaccine reduced the mortality significantly; the administration of i - ml of vaccine i.m. gave significant (p= ' , fisher's exact test) but incomplete protection, two of fourteen animals developing paralysis and dying after incubation periods of and days. none of fourteen rabbits died after receiving i.d. inoculations of . ml of the vaccine in each limb (p = ' ). britain, [ ] [ ] [ ] [ ] , france, and germanyls, that the administration of hdcs vaccine by the intradermal route is followed by substantial titres of virus-neutralising antibody with occasional mild local and systemic reactions. it has also been shown that or doses of ml given in separate sites on a single occasion rapidly induce high titres of antibody. i , the present report confirms these observations and shows in addition that the early production of virus-neutralising antibody is accompanied by high titres of antirabies igg. this early igg response may be most important, for it is now well established that neutralising antibody of the igg class, unlike igm neutralising antibody, confers protection on animals challenged with rabies and may be the key to successful postexposure treatment of man. the possible role of cell-mediated immunity in rabies infection is poorly understood, but it too may be an important component of the host's immune response. we have reported that transformation of lymphocytes occurred with cells taken from eight of ten vaccinees after the first x ml doses of an established postexposure regimen for hdcs vaccine.' in the present study we separated the lymphocyte subpopulations and have shown that the blast transformation is a t-cell response. furthermore, it occurred in the same proportion of vaccinees (eight often) as was found in the previous study, but with only a quarter of the volume of vaccine. clearly, if high titres of neutralising antibody and a cell-mediated response are both important for protection, the present study suggests that they can be obtained equally well with much smaller quantities of vaccine than are used at present. we further showed that rabbits could be completely protected by injecting x - ml doses of hdcs vaccine intradermally h after intranuchal infection with street-rabies virus. by contrast, nine of the fourteen controls ( %) died from rabies with incubation periods of to days (mean days). opponents to the administration of rabies vaccine by the i.d. route claim that it is technically difficult, especially in the elderly and the very young. nevertheless, many vaccines are routinely administered by the i.d. route with apparent success and considerable financial savings. there seems to be ample experimental evidence to justify a postexposure study of hdcs vaccine administered by the i.d. route; we believe that this would be both ethical and potentially of great importance for developing countries. k. g. n. was partly supported by an mrc-eli lilly travelling fellowship at the u.s. national center for disease control, atlanta, ga. g. s. t. was partly supported by mrc grant no. / . we thank dr c. charbonnier and l'lnstitut merieux for the gift of the vaccine requests for reprints should be addressed to k. g. n. successful protection of humans exposed to rabies infection postexposure treatment with the new human diploid cell rabies vaccine and antirabies serum post-exposure use of human diploid cell culture rabies vaccine deitch mw postexposure trial of a human diploid cell strain rabies vaccine immunization with a human diploid cell strain of rabies virus vaccine: two year results studies with human diploid cell strain rabies vaccine and human rabies immunoglobulin in man human diploid cell strain rabies vaccine rapid prophylactic immunisation of volunteers with small doses immune responses of humans to a human diploid cell strain of rabies virus vaccine: lymphocyte transformation, production of virus-neutralizing antibody, and induction of interferon l'injecteur sans aiguille "dermo-jet" presse m&eacute quantitative assay and potency test of antirabies serum and immunoglobulin macnaughton mr enzyme linked immunosorbent assay for detection of antibody in volunteers experimentally infected with human coronavirus e group viruses purification of human t and b lymphocytes immunogenicity and acceptability of a human diploid cell culture rabies vaccine in volunteers rabies prophylaxis simplified resultats de la vaccination antirabique preventive par le vaccin inactive concentre souche rabies pm/w - - m cultivee sur cellules diploides humaines developments in biological standardisation prophylactic immunization of humans against rabies by intradermal inoculation of human diploid cell culture vaccine a large scale antirabies immunisation study in humans using hdcs vaccine. who consultation on cell culture rabies vaccines and their protective effect in man immunoglobulin igg and igm antibody responses to rabies vaccine transformed the management of diabetes and it is now administered to about million diabetics throughout the world. although the frequency of side-effects is relatively low, efforts have been made over the years to improve the quality and extend the range of insulin preparations. the pace of these changes has accelerated during the past decade. introduction of high-purity insulin and of preparations of insulin from a single species of animal have been followed by developments of two kinds: the availability for therapy of insulin containing the aminoacid sequence of the natural human hormone and the construction of continuous-delivery systems to administer insulin under conditions that more closely mimic its natural secretion in the body. the changes in quality and type of insulin available have posed problems of nomenclature.recognition that the molecular structure of insulin can influence tolerance to, and the side-effects of, therapy led to the introduction in the british pharmacopoeia (bp) in ' of a requirement that all formulations be labelled with the species of origin. at that time, the bp contained no monograph for bulk insulin, but the marketing of a variety of purified insulins made such a monograph desirable, and it appeared in the bp . the monograph covers insulin of either porcine or bovine origin that has been purified beyond the stage of conventional crystalline insulin. this has had several consequences. first, the monograph required a title and "insulin" was taken. had the title been "purified insulin" or similar, to draw a distinction with crystalline insulin, the problem might arise of a name for material of yet higher quality (already available as "monocomponent" or "pro-insulin free" preparations). this is one reason why a *professor turner is vice-chairman and dr calam a member of the nomenclature committee of the british pharmacopoeia commission. monograph for a drug substance rarely contains any qualifying adjective indicating degree of purity. however, the title "insulin" had been for many years a synonym for insulin injection, and it was necessary to delete it as an alternative name for that preparation. second, a unique situation arose in that the monographs for formulations continued to specify a minimum potency of iu/mg for the insulin used, against the higher figure in the bulk monograph, thus allowing insulin not of bp quality to be incorporated into bp formulations. this was the result of decisions not to delete conventional formulations, satisfactory for many patients, from the pharmacopoeia and not to add a set of separate monographs for formulations containing the bp grade of insulin, which would require additional titles. another factor was involved in that control of six of the nine insulin formulations is exerted by monographs in the european pharmacopoeia. for these, no unilateral change in the requirements can be made by a national authority, but revision must await agreement by the european pharmacopoeia commission, with a further lapse of time before adoption. this factor illustrates the point that pharmacopoeial decisions in the united kingdom, including those involving nomenclature, cannot be taken without considering the international constraints that apply and the international implications that may result. during the past eighteen months another aspect of insulin nomenclature has been discussed by the british pharmacopoeia commission and its committees concerned with nomenclature and with hormones. this arose from availability, for testing and clinical trial, of insulin possessing the structure of the natural human hormone, the prospect of its wider use, and the eventual need for a monograph for it. that one method of production of such insulin uses geneticengineering techniques suggests that the wider implications must be considered. thus, in the following discussion, it should be remembered that the final outcome should, so far as possible, set a pattern that can be applied to other hormones and therapeutic substances (such as growth hormone and interferons) obtained by novel techniques.the structure of human insulin was established in and was found to differ from that of the porcine hormone only in the presence of threonine in place of alanine at position of the b chain. the total synthesis of insulin having the human sequence was achieved in an elegant manner by workers at ciba geigy in , but the process is not economically viable under present conditions. this material key: cord- -fbotc authors: fagone, paolo; shedlock, devon j.; kemmerer, stephen; rabussay, dietmar; weiner, david b. title: electroporation-mediated dna vaccination date: - - journal: clinical aspects of electroporation doi: . / - - - - _ sha: doc_id: cord_uid: fbotc there are many positive attributes to dna vaccination that make it a conceptually desirable platform. in clinical studies, however, standard dna injection alone generally induces low levels of transgene-specific immunity when compared to other vaccine approaches. in order to boost the immunogenicity of this platform, next-generation dna vaccines require additional techniques such as the administration of electroporation. this new method involves the generation of a brief electric field in tissue around a local injection site that results in the transient poration, or permeabilization, of the cellular membranes. as a result, antigen-specific immune responses are greatly enhanced and are likely due to increased dna uptake and antigen expression. thus, electroporation-mediated dna vaccination represents a promising new strategy for the elicitation of strong immune responses directed against the expressed antigen(s) and not the vector, and ongoing studies are currently underway to optimize the working parameters of this technique. here, we review the uses of this technology in conjunction with vaccination and suggest future directions for its further exploration. the discouraging results from the recent human hiv trial performed by merck and collaborators, named "step," raises serious questions about current vaccine approaches using replicationdefective viral vectors [ ] . this study was an international phase ii "test of concept" trial in uninfected volunteers at high risk for acquiring hiv infection which showed that vaccination using a recombinant adenovirus serotype vector was ineffective at preventing virus infection and even increased the rate of transmission in individuals exhibiting prior immunity to the viral vector. while this vaccine was safe and immunogenic in both humans and nonhuman primates, eliciting long-lasting and multifunctional cd + t cell responses that were partially protective in rhesus macaques, the discovery that the vaccine could possibly heighten hiv infection was both unexpected and alarming, resulting in the immediate discontinuation of vaccinations as recommended by the independent data safety monitoring board for step. since preexisting immunity against a vaccine vector may compromise its efficacy in humans, future vaccine approaches should aim to utilize vectors that exhibit minimal or no reactivity in immunocompetent vaccines. dna vaccination consists of the administration of genetic material encoding a desired antigen that when expressed in the vaccine, is capable of eliciting an immune response. compared with other approaches, the advantages of dna vaccination are many [ ] ; no infectious agents are involved and vaccines are unable to revert into virulent form like live vaccines. they can prime both cytotoxic [ ] and humoral responses [ ] , and dna vectors are easily manipulated, can be tested rapidly, produced at high yield in bacteria, and are readily isolated. they are also more temperaturestable than conventional vaccines, easily stored and transported, and do not require a cold chain. furthermore, dna vaccines could potentially induce immunity in newborns in situations that usually neutralize conventional vaccines via the presence of high levels of maternal antibodies [ ] . the introduction of exogenous dna into cells or tissue can be achieved using dna conjugates [ ] [ ] [ ] [ ] [ ] , virus-derived vectors [ ] , or naked plasmid dna (pdna) [ ] . naked pdna shows variable and low transfection efficacy when administered by conventional means, such as needle injection or topical application. however, several strategies aimed to improve pdna vaccine immunogenicity have been developed, including codon optimization [ ] , mrna optimization [ ] , addition of leader sequences [ ] , and construction of consensus immunogens [ ] . while these strategies help to boost the overall immunogenicity of a dna vaccine, they may not be applicable to all antigens. recently, electroporation, or electropermeabilization, has gained great interest in multiple research areas including gene therapy and vaccinology [ ] . although the precise mechanism of action has not yet been well defined, it is hypothesized that cell membranes in host tissue receiving electroporation, normally impermeable to charged molecules, form pores or functionally equivalent structural changes upon application of an external electric field which facilitate the influx of macromolecules. thus, higher transfection efficacy of naked pdna as a result of electroporation is thought to be the major contributor to the increased immunogenicity of electroporation-mediated dna vaccination. in addition, it has been shown that electroporation increases vaccine potency by activating antigenpresenting cells (apcs) via danger signals and local inflammation [ ] and by recruiting immune cells to the site of dna administration [ , ] . furthermore, direct transfection of apcs could also be facilitated by electroporation. currently, intensive investigation is focused upon utilizing electroporation of muscle and skin as an effective method for dna vaccine delivery to small and large animals, and in humans. the safety and feasibility of electroporation in humans has recently been demonstrated, but not finally proven [ ] . thus, the paramount question for dna vaccines at this time is whether a sufficient level of efficacy can be reached with the present methodology, or if further improvements or breakthroughs in vaccine design and/or electroporation delivery will be necessary. in , the first transgene expression detected in skeletal muscle after injection of naked mrna or pdna raised the possibility of using this method for certain gene therapies and dna vaccinations [ ] . subsequently, transgene expression was also obtained in the same way in a wide variety of other tissues, but transgene expression was generally too low and variable to be useful for the envisioned purposes [ ] . attempts to sufficiently enhance pdna uptake, and thus transgene expression, with cationic lipids or the gene gun have also proven unsuccessful to date. the first publications on a substantial increase in transgene expression (about -fold) when electroporation was applied in vivo after pdna injection appeared as late as [ ] , although electroporation had been used for in vitro cell transfections since [ ] . in addition, as pointed out by bettan et al. [ ] , when using gene electrotransfer, a higher interindividual reproducibility in gene transduction can be observed. skeletal muscle (fig. . ) has been the most frequently targeted tissue in both gene therapy and dna vaccine studies, either with or without electroporation. some reasons why muscle cells (also known as myocytes) and muscle tissues continue to be attractive targets for transgene expression include: muscle tissue is easily accessible, plentiful, and well vascularized; the latter facilitates circulation of the antigens produced by the transfected muscle cells. more discussion of gene electrotransfer to muscle can be found in chap. . in vivo electroporation has been used to deliver dna vaccine encoding antigens from numerous infectious agents, summarized in table . . enhanced immune responses to electroporation-mediated dna vaccination have been observed both in small and large animals such as mice [ ] , pigs [ ] , and monkeys [ ] . widera et al. [ ] demonstrated in mice that upon electroporative treatment, the delivery of a weakly immunogenic hepatitis b virus (hbv) surface antigen (hbs ag) dna vaccine resulted in an increased humoral immune response, characterized by rapid onset and higher titers of anti-hbs ag antibodies. in addition, the authors observed in the same study that the potency of an hiv gag pdna vaccine was increased as shown by the lower dosage of dna required to induce higher antigen-specific antibody levels and increased cd + t cell responses. similarly, in a study carried out with a bovine herpes virus- truncated glycoprotein d dna vaccine, tsang et al. [ ] showed that the onset of the primary humoral response was earlier in the group treated with dna followed by electroporation, and that this group produced higher antibody levels than those in the group receiving i.m. dna immunization or a recombinant protein vaccine only; similar results were obtained earlier with [ , ] an otherwise inert microparticulate adjuvant [ ] . interestingly, the efficiency of transfection by electroporation was not increased by doubling the dose of dna administered; however, the duration of the antigen-specific antibody response was increased at a higher rate in comparison to the immunization with the same dose of plasmid without electroporation. moreover, electroporation increased the degree of consistency among the individuals in the dna-plus-electroporation group as seen in the weeks of follow-up. finally, a high correlation between the duration of the primary immune response and the magnitude of the secondary antibody response was observed, implying that electroporation could represent an effective approach to elicit a longer memory antibody response. capone et al. [ ] have demonstrated that gene electrotransfer efficiently increased the cellular immune response both in mice and rhesus macaques vaccinated with a plasmid encoding a nonstructural region of hepatitis c virus (hcv). in particular, they showed by ex vivo interferon (ifn)-g elispot assay that electroporation in mice induced a fivefold more potent t cell response than dna administration alone, and that the elicited response was directed against all six of the antigen pools spanning the hcv ns -ns b region. to assess whether electroporation treatment elicited similar responses in a nonhuman primate model, they immunized rhesus macaques three times with the vaccine and collected peripheral blood mononuclear cells at periodic intervals to test the t cell effector function. the immune responses observed in the electroporation-treated group showed a faster kinetic, with all the animals responding after the second challenge and reaching a peak after the third. moreover, all animals treated with electroporation showed both cd + and cd + t cell responses, in comparison to the naked dna group which showed a weaker cd + response and no cd + response. finally, gene electrotransferimmunized macaques maintained anti-hcv effector t cells for the entire observation period of months, indicating that the gene electrotransfer efficiently elicited a strong memory t cell response. dna vaccination in association with electroporation represents an effective strategy to elicit strong, broad, and long-lasting b and t cell responses. although muscle is the most common target for dna vaccine immunizations [ ] , the presence of apcs in both the skin layers makes it an attractive target for nucleic acid vaccination, since direct transfection of apcs may be important for t cell priming upon skin dna immunization [ ] (fig. . ) . in a murine model using a viral [ ] have demonstrated that a single intradermal (i.d.) injection (without electroporation) of naked dna encoding the influenza nucleoprotein gene is sufficient to induce production of antigen-specific antibodies and cytotoxic t lymphocytes that persist for at least weeks and are protective against a lethal challenge with a heterologous strain of influenza virus. furthermore, immune responses to i.d. dna vaccination have been recorded to be significantly enhanced by in vivo electroporation [ ] ; analysis of the antibody response to an hbs ag-encoding plasmid delivered i.d. upon electroporation in mice has revealed a strong enhancement of the t h response, which is mainly characterized by a strong cell-mediated response, compared to that elicited by protein immunization, which showed an exclusively t h pattern, characterized by a dominant humoral responses. also, in a nonhuman primate model study carried out in rhesus macaques [ ] , the i.d.-plus-electroporation group developed % more ifn-g-producing cells and twice more memory t cells than the group not treated with electroporation. higher antibody responses were recorded in the i.d.-plus-electroporation group when compared to the i.m.-plus-electroporation group. altogether, these results support the idea that electroporation following dna injection, both in muscle and skin, represents an effective approach to large animal immunization. several authors have hypothesized that inflammation caused by electroporation is important to prime the immune response to dna vaccination [ , , ] . local inflammation was previously proposed to augment immune responses in studies where pdna was coinjected with bupivicaine-hcl [ ] [ ] [ ] . the localized tissue damage induced by the electric field is thought to recruit cd + cells, increasing the number of infiltrating immune cells at the injection site [ ] . indeed, electroporation caused the activation of proinflammatory signals including the expression of chemokines such as mip- a, mip- b, mip- g, ip- , mcp- , and xcl [ ] . liu et al. [ ] characterized the extent and nature of the cellular infiltrates at the site of electroporative vaccine delivery in mice and found both polymorphonuclear and mononuclear cells localized in the perivascular spaces and throughout the muscle tissue. in particular, they observed a significant increase in b cells, cd + and cd + t cells, and a dramatic increase in macrophages and dendritic cells compared to vaccination alone. no difference, however, was recorded among cell populations of blood, spleen, and draining lymph nodes of the mice treated with or without electroporation, suggesting that only local factors are involved in the augmentation of immune responses following electroporation. also, these authors observed that cell infiltrates were transient and resolved within weeks. thus, improved antigen presentation may represent one of the mechanisms by which electroporation may elicit a more potent immune response. typically, innate immune recognition of the adjuvant component of vaccine formulations has been shown to be critical for their immunogenicity [ ] . many adjuvants are ligands for toll-like receptors (tlrs), like monophosphoryl lipid a and cpg dna [ , ] , while some conventional adjuvants, such as aluminum hydroxide and incomplete freud's adjuvant are free of tlr ligands [ ] . therefore, these examples demonstrate that multiple innate immune recognition and signaling pathways are required for adjuvants to function [ ] . in the case of dna vaccines, it has been controversial as to the main vaccine component contributing most to the induction of both innate and adaptive immune responses; while cpg motifs expressed within the plasmid backbone can stimulate innate immunity through tlr , the induction of adaptive immune responses were unaffected in the absence of this innate receptor [ , ] . however, it has recently been shown that the doublestranded structure of dna, independently of cpg sequences, possesses immunomodulatory effects when administered intracellularly [ ] , which can trigger tlr-independent, tank-binding kinase i (tbk )-and inf regulatory factor -dependent innate activation of both immune and nonimmune cells to produce type i infs and their inducible genes [ , ] . recently, ishii et al. [ ] have reported that the enhancement of dna vaccine immunogenicity achieved by electroporation may be due to increased transfection rates resulting from this technique, which better contributes to local inflammation by activating cells to produce ifn through the tbk -dependent signaling pathway. these data suggest that tbk is a key signaling molecule for dna vaccination immunogenicity by regulating innate immune signaling, which is critical for the induction of adaptive immune responses, and that the enhanced immunogenicity of pdna by electroporation may be a result of more pdna interacting with intracellular tbk . in accordance with this hypothesis, peng et al. [ ] postulated that local inflammation is more important than the actual quantity of expressed transgene in determining the magnitude of the immune response, demonstrated by higher antibody titers and cd + t cell proliferation rates observed by applying electric pulses - days prior to i.m. dna immunization. in this case, it can be postulated that both increased cross-presentation and direct transfection of infiltrating apcs resulting from increased local inflammation may contribute to the augmented immune response in electroporation-mediated dna vaccination. it appears that the mechanisms by which electroporation enhance the responses to naked plasmid vaccination is by an increase in dna transfection and possibly include local inflammation, which may be augmented by the magnitude or duration of transgene expression. indeed, babiuk et al. [ ] observed that the highest level of lymphocytic infiltration was only in muscle tissue treated with electroporation, which elicited higher levels of transgene expression, as was expected considering that antigen production is critical for the retention of the cellular infiltrates at sites of local inflammation. although two dna vaccines have been recently approved in the usa and canada for the vaccination of horses against west nile virus [ ] and salmon against infectious hematopoietic necrosis virus [ ] , no dna vaccine has been approved for use in humans. however, encouraging results from preclinical trials using electroporation technology with dna vaccination in large animal models has prompted much interest in the technique and its safety. currently, tolerability in humans has been demonstrated in healthy volunteers [ ] , anti-dna antibodies have not been detected in patients electroporated after muscle dna injection, and the integration of pdna into host chromosomes following electroporation-mediated delivery has not been observed [ ] . together, these results have been sufficient for the regulatory approval of several clinical trials [ ] . as reported on clinicaltrials.gov, seven electrotransfer dna vaccine trials for cancer and three clinical trials using dna vaccine against infectious agents in association with electroporation are currently open. ongoing clinical studies using electroporation-mediated dna vaccination against infectious agents include three phase i studies involving muscle electroporation. the first will test safety and immunological effects of pennvax ™ -b, an hiv vaccine encoding gag, pol, and env, in hiv-infected individuals (vgx pharmaceuticals, inc.); the second will assess safety, tolerability, and immunogenicity of human papillomavirus (hpv) dna plasmid (vgx- ™ ) delivered by electroporation in adult females postsurgical or ablative treatment of grade or cervical intraepithelial neoplasia (vgx pharmaceuticals, inc.); the third one, a phase i/ii trial is testing tolerability and efficacy of i.m.-administered chronvac-c ™ in combination with electroporation in chronic hcv genotype infected and naïve patients with low viral load (tripep ab). the amount of pain and distress associated with electroporation in humans has been of a tolerable level for the anticipated benefit [ ] . to date, electroporation-mediated dna vaccination in humans is performed administrating an injection volume of . - . ml followed by short ( - ms), low electric field strength ( - v/cm) pulses ( - pulses) . given that these conditions are efficient for the dna vaccination of large animals, such as nonhuman primates, they should be sufficient in humans. electroporation results in a sharp, but quick pain that is comparable to receiving a short electrical shock. while this sensation is transient, administration of short-acting sedative drugs or painkillers before treatment has been considered. accordingly, as reported by daud et al. [ ] , in a clinical trial using an interleukin- -encoding plasmid delivered by electroporation in patients with metastatic melanoma, in order to limit patients' discomfort, lidocaine was either administered topically or injected around each tumor site, and intravenous analgesic and/or anxiolytic drugs were offered to the patients before electroporation. notably, previous studies have shown that pain is not a limiting factor as patient discomfort is limited to the period of electrical stimulation, and subjects have usually returned for repeated treatments without asking for sedation. also, after muscle electroporation, like muscle injection, a mild ache may be experienced at the site of electroporation for some days, and similar to that following a strenuous workout. several factors determine the strength of pain associated with electroporation, although there is a high interindividual variability in the perception of pain. among these factors are the number, length, spacing, and thickness of the electrode needles, but primarily the electric pulse parameters dictate the pain threshold [ ] . exclusion criteria for electroporation treatment may include the presence of metal implants near the site of electrical delivery, the presence of a pacemaker, and in the case of muscle electroporation, obesity, since treatment efficacy may be decreased if muscle tissue is not reached for vaccine and/ or electric pulse delivery. electroporation-mediated dna vaccine delivery requires a pulse generator that controls the parameters of individual pulses or pulse trains (amplitude, duration, number, polarity, wave form, frequency), and electrodes usually integrated into an applicator. electrodes are in direct contact with the subject to be treated and it is their geometry (shape, size, and distance from each other) that ultimately determines the shape and strength of the electric field and the electrical currents in the target tissue ( fig. . ) . thus, both the properties of the pulse(s) and the electrodes are responsible for the desired enhancement of dna delivery as well as undesirable side effects. proper design of pulses and electrodes will maximize the effectiveness of a given dna vaccine and minimize unwanted side effects, such as long-lasting histological changes, pain, and muscle contractions. further discussion of electroporation parameters and equipment can be found in chaps. and . skin is a potentially interesting target tissue for dna vaccines because of its natural role in the immune defense of the body and its ready accessibility. discussion of gene electrotransfer of dna in general and to skin can be found in chaps. and , respectively. the goal of any vaccination strategy is to prime a broad and long-lasting immune response that is capable of robust effector responses upon antigenic restimulation [ ] [ ] [ ] and protect against infectious agents, while minimizing toxicity of the vaccine. since complications may arise due to the use of viral-based vectors, such as preexisting immunity regulating vaccine effectiveness and the possibility of reversion into virulence that may decrease vaccine efficacy or safety, alternative approaches should be explored for future vaccine approaches. dna vaccination appears an even more attractive candidate for future vaccines since it is safe, immunogenic, and does not stimulate vector-specific immunity. extensive literature supports the hypothesis that electroporation represents a valid approach to vaccine administration in that it increases the consistency and potency of vaccination, inducing higher levels of both antibody-mediated and cytotoxic t cell responses. in this way, electroporation may help augment translocation of exogenous dna into the nucleus. furthermore, it has been proven to be effective in enhancing immune responses to antigens regardless of the degree of transgene expression achieved. recent evidence suggests that innate immune recognition of the adjuvant component of vaccines and the danger signals provided by the method of vaccination may be important in determining the magnitude of the resultant immune response. electroporation may enhance local inflammation at the site of immunization by facilitating the transfection of greater amounts of pdna, which may be more readily available to interact with intracellular signaling proteins that trigger the secretion of inflammatory cytokines and their . needle-type electrodes consist of a variable number of (primarily) stainless steel needles arranged in different configurations. picture depicts a -needle array firing rotating chord patterns, a -needle array fired in opposing pairs, crossing or triangular patterns, and a -needle array firing pin to pin inducible genes. while electroporation-mediated dna vaccination is currently the topic of intensive research, more theoretical studies and practical trials are required to optimize the delivery of the vaccine into the target tissue, and the electrical parameters facilitating dna uptake while calibrating local tissue damage and reducing pain associated with vaccine delivery through electroporation. the failed hiv merck vaccine study: a step back or a launching point for future vaccine development? dna vaccination: antigen presentation and the induction of immunity heterologous protection against influenza by injection of dna encoding a viral protein genetic immunization is a simple method for eliciting an immune response vaccination with hemagglutinin or neuraminidase dna protects balb/c mice against influenza virus infection in presence of maternal antibody in vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin i increased expression of dna cointroduced with nuclear protein in adult rat liver liposome mediated gene transfer direct introduction of genes into rats and expression of the genes receptor-mediated gene delivery and expression in vivo human immunodeficiency virus type vaccine development: recent advances in the cytotoxic t-lymphocyte platform "spotty business the mechanism of naked dna uptake and expression multiple effects of codon usage optimization on expression and immunogenicity of dna candidate vaccines encoding the human immunodeficiency virus type gag protein inactivation of the human immunodeficiency virus type inhibitory elements allows rev-independent expression of gag and gag/protease and particle formation induction of potent th -type immune responses from a novel dna vaccine for west nile virus new york isolate (wnv-ny ) antigenicity and immunogenicity of a synthetic human immunodeficiency virus type group m consensus envelope glycoprotein gene therapy progress and prospects: electroporation and other physical methods electric pulses applied prior to intramuscular dna vaccination greatly improve the vaccine immunogenicity increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of dna vaccines recruitment of antigen-presenting cells to the site of inoculation and augmentation of hiv- dna vaccine immunogenicity by in vivo electroporation electroporation for drug and gene delivery in the clinic: doctors go electric direct gene transfer into mouse muscle in vivo in vivo gene electroinjection and expression in rat liver gene transfer into mouse lyoma cells by electroporation in high electric fields high-level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle increased dna vaccine delivery and immunogenicity by electroporation in vivo electroporation improves the efficacy of dna vaccines in large animals anti-hbv immune responses in rhesus macaques elicited by electroporation mediated dna vaccination a single dna immunization in combination with electroporation prolongs the primary immune response and maintains immune memory for six months enhancement of the effectiveness of electroporation-augmented cutaneous dna vaccination by a particulate adjuvant modulation of the immune response induced by gene electrotransfer of a hepatitis c virus dna vaccine in nonhuman primates high expression of naked plasmid dna in muscles of young rodents predominant role for directly transfected dendritic cells in antigen presentation to cd + t cells after gene gun immunization intradermal gene immunization: the possible role of dna uptake in the induction of cellular immunity to viruses cutaneous transfection and immune responses to intradermal nucleic acid vaccination are significantly enhanced by in vivo electropermeabilization combined effects of il- and electroporation enhances the potency of dna vaccination in macaques early events of electroporation-mediated intramuscular dna vaccination potentiate th -directed immune responses inflammatory responses following direct injection of plasmid dna into skeletal muscle dna immunization with hiv- tat mutated in the trans activation domain induces humoral and cellular immune responses against wild-type tat specific immune induction following dna-based immunization through in vivo transfection and activation of macrophages/antigen-presenting cells facilitated dna inoculation induces anti-hiv- immunity in vivo in vivo electroporation enhances the immunogenicity of hepatitis c virus nonstructural / a dna by increased local dna uptake, protein expression, inflammation, and infiltration of cd + t cells translating innate immunity into immunological memory: implications for vaccine development signaling to nf-kappab by toll-like receptors the vaccine adjuvant monophosphoryl lipid a as a trif-biased agonist of tlr adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling tlr −/− and tlr +/+ mice display similar immune responses to a dna vaccine vaccination with plasmid dna activates dendritic cells via toll-like receptor (tlr ) but functions in tlr -deficient mice a toll-like receptor-independent antiviral response induced by double-stranded b-form dna recognition of cytosolic dna activates an irf -dependent innate immune response tank-binding kinase- delineates innate and adaptive immune responses to dna vaccines recombinant canarypoxvirus vaccine carrying the prm/e genes of west nile virus protects horses against a west nile virus-mosquito challenge efficacy of an infectious hematopoietic necrosis (ihn) virus dna vaccine in chinook oncorhynchus tshawytscha and sockeye o. nerka salmon clinical evaluation of pain and muscle damage induced by electroporation of skeletal muscle in humans abstracts from the american society of gene therapy th annual meeting the uptake and fate of exogenous cellular dna in mammalian cells national institutes of health phase i trial of interleukin- plasmid electroporation in patients with metastatic melanoma applicator and electrode design for in vivo dna delivery by electroporation cd + t cells are required for secondary expansion and memory in cd + t lymphocytes requirement for cd t cell help in generating functional cd t cell memory defective cd t cell memory following acute infection without cd t cell help sphingosine-mediated electroporative dna transfer through lipid bilayers increased immune responses in rhesus macaques by dna vaccination combined with electroporation persistent antibody and t cell responses induced by hiv- dna vaccine delivered by electroporation enhancement of dna vaccine potency in rhesus macaques by electroporation a therapeutic siv dna vaccine elicits t-cell immune responses, but no sustained control of viremia in sivmac -infected rhesus macaques the relative immunogenicity of dna vaccines delivered by the intramuscular needle injection, electroporation and gene gun methods protection against influenza virus infection in balb/c mice immunized with a single dose of neuraminidase-expressing dnas by electroporation protection against influenza virus infection in mice immunized by administration of hemagglutinin-expressing dnas with electroporation protection against avian influenza h n virus challenge by immunization with hemagglutinin-or neuraminidase-expressing dna in balb/c mice electroporation-mediated hbv dna vaccination in primate models non-cytolytic antigen clearance in dnavaccinated mice with electroporation a single hbsag dna vaccination in combination with electroporation elicits long-term antibody responses in sheep enhancement of immune responses to an hbv dna vaccine by electroporation skin targeted dna vaccine delivery using electroporation in rabbits. i: efficacy enhancing b-and t-cell immune response to a hepatitis c virus e dna vaccine by intramuscular electrical gene transfer search for potential target site of nucleocapsid gene for the design of an epitope-based sars dna vaccine construction of a eukaryotic expression plasmid encoding partial s gene fragments of the sars-cov and its potential utility as a dna vaccine induction of specific immune responses by severe acute respiratory syndrome coronavirus spike dna vaccine with or without interleukin- immunization using different vaccination routes in mice analysis of humoral immunity of hepatitis d virus dna vaccine generated in mice by using different dosage, gene gun immunization, and in vivo electroporation immunogenicity of a dna vaccine for coxsackievirus b in mice: protective effects of capsid proteins against viral challenge in vivo electroporation of skeletal muscles increases the efficacy of japanese encephalitis virus dna vaccine dna vaccination against foot-and-mouth disease via electroporation: study of molecular approaches for enhancing vp antigenicity comparative studies of the capsid precursor polypeptide p and the capsid protein vp cdna vectors for dna vaccination against foot-and-mouth disease virus immunogenicity of novel consensus-based dna vaccines against chikungunya virus potentiation of an anthrax dna vaccine with electroporation smallpox dna vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge intramuscular immunization with a monogenic plasmid dna tuberculosis vaccine: enhanced immunogenicity by electroporation and co-expression of gm-csf transgene improved humoral immunity against tuberculosis esat- antigen by chimeric dna prime and protein boost strategy improved cellular and humoral immune responses against mycobacterium tuberculosis antigens after intramuscular dna immunisation combined with muscle electroporation multivalent dna vaccine protects mice against pulmonary infection caused by pseudomonas aeruginosa markedly enhanced immunogenicity of a pfs dna-based malaria transmission-blocking vaccine by in vivo electroporation identification of minimal cd + and cd + t cell epitopes in the plasmodium yoelii hepatocyte erythrocyte protein kda co-delivery of plasmid-encoded cytokines modulates the immune response to a dna vaccine delivered by in vivo electroporation in vivo electroporation improves immune responses to dna vaccination in sheep we would like to thank karuppiah muthumani and shaheed abdulhaqq for critical review of the manuscript and members of the dbw laboratory for helpful discussion.competing financial interests: the laboratory of d.b.w. has grant funding and collaborations, advising, or consulting including serving on scientific review committees for commercial entities and therefore notes possible conflicts associated with this work with pfizer, inovio, bms, virxsys, ichor, merck, althea, vgxi, j&j, aldevron, and possibly others. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. key: cord- -cwzpb fu authors: haque, azizul; hober, didier; kasper, lloyd h. title: confronting potential influenza a (h n ) pandemic with better vaccines date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: cwzpb fu influenza a (h n ) viruses are strong candidates for causing the next influenza pandemic if they acquire the ability for efficient human-to-human transmission. a major public health goal is to make efficacious vaccines against these viruses by using novel approaches, including cell-culture system, reverse genetics, and adjuvant development. important consideration for the strategy includes preparation of vaccines from a currently circulating strain to induce broad-spectrum immunity toward newly emerged human h strains. this strategy would be a good solution early in a pandemic until an antigenically matched and approved vaccine is produced. the concept of therapeutic vaccines (e.g., antidisease vaccine) directed at diminishing the cytokine storm frequently seen in subtype h n –infected persons is underscored. better understanding of host–virus interaction is essential to identify tools to produce effective vaccines against influenza (h n ). infl uenza a (h n ) viruses are strong candidates for causing the next infl uenza pandemic if they acquire the ability for effi cient human-to-human transmission. a major public health goal is to make effi cacious vaccines against these viruses by using novel approaches, including cell-culture system, reverse genetics, and adjuvant development. important consideration for the strategy includes preparation of vaccines from a currently circulating strain to induce broad-spectrum immunity toward newly emerged human h strains. this strategy would be a good solution early in a pandemic until an antigenically matched and approved vaccine is produced. the concept of therapeutic vaccines (e.g., antidisease vaccine) directed at diminishing the cytokine storm frequently seen in subtype h n -infected persons is underscored. better understanding of host-virus interaction is essential to identify tools to produce effective vaccines against infl uenza (h n ). has spread with alarming speed across europe, africa, and parts of asia in which the infection was not reported earlier. establishment of the highly pathogenic avian infl uenza (h n ) as an endemic virus within duck and poultry populations and its capacity to cross species barriers increase the possibility of adaptation to humans and a pandemic. human infl uenza infections with subtype h n viruses are often fatal. as of june , , laboratoryconfi rmed cases of human infection have been reported to the world health organization (who); % were fatal, mainly in persons - years of age (www.who.int/csr/ caculatordisease/avian_infl uenza/en). if a pandemic is triggered by transmissibility of infl uenza (h n ) from person to person, millions of people could die, and economies would likely be crippled for - months. in the event of a pandemic, vaccination against infl uenza (h n ) could limit the impact of infection at a public health level. however, no evidence exists that available vaccines would be protective against the pandemic strain of the virus. we comment on some of the limitations of currently available vaccines and propose novel strategies to improve vaccine formulations against infl uenza (h n ). the host response to infl uenza (h n ) infection has not been defi ned, which has proven a considerable challenge in epidemiology and public health research. to develop effi cient vaccines, understanding how the virus interacts with the host in natural infection is necessary. having insights into the hosts' responses to infl uenza (h n ) would help defi ne targets for therapeutic intervention. whether humans can develop immunity during a primary infection that would control replication and spread of subtype h n viruses has been questioned ( ). however, marked infl ammatory responses develop after infection with infl uenza (h n ) in humans and other animals ( ) ( ) ( ) . this condition is associated with statistically signifi cant synthesis of various proinfl ammatory cytokines, such as tumor necrosis factor-α, interleukin (il)- , interferon (ifn)-γ, il- α, and chemokines, including ip- , mig, monocyte chemoattractant protein- (mcp- , il- , and rantes. i.e., regulated on activation, normal t-cell expressed and secreted). if this is the case, these observations are consistent with the possible induction of innate immune responsiveness in the persons infected with infl uenza (h n ( ) . the death rate of ≈ % was half that of previously known outbreaks, and mild or completely asymptomatic cases have been reported. one theory holds that milder cases have been occurring elsewhere but are not being recorded. recently, persons among apparently healthy volunteers from the people's republic of china, showed detectable virus-neutralizing antibody response to subtype h n before vaccination ( ) . moreover, pigs infected with subtype h n have become asymptomatic in indonesia. are these signs of development of some degree of immunity to virus, containing its replication and thus causing milder infection in naturally infected mammals? recently, clusters of bird fl u cases were reported in western java, indonesia ( ); fatal disease developed in persons there from the same family. two other family members became ill but survived. all the family members likely had similar levels of exposure because they all lived in the same household. other cases of nonfatal infections have been seen in thailand and vietnam. unfortunately, there is little information about the immune response to the virus in those who survived, which would be valuable for understanding the mechanisms of protection. indeed, following up the persons (cohort) living in the same affected villages, presumably mostly not exposed to virus, should clarify whether the maintained response refl ects boosting through natural exposure. persons with prior exposure, as measured by antibody or viral rna at recruitment, would likely have substantially higher responses to the vaccine than those naïve at recruitment if the vaccinating antigen contains homologous or cross-reacting determinants. conceivably, boosting the "natural" immunity is a desirable outcome to improve protective effi cacy of any vaccine approach. additional studies are required to evaluate the merits of priming populations in advance of an infl uenza (h n ) pandemic. after initial hesitation about using a wide-scale program of poultry vaccination, some european and asian countries have begun vaccination. inactivated vaccines are widely used in poultry but lack of critical potency testing, standardization, and quality control has led to variable and suboptimal immune responses. moreover, a legitimate concern remains that the fowl vaccinated by attenuated live viruses may survive the disease but still carry the virus; thus, they would continue to spread infl uenza (h n ) silently at the fl ock level ( ) or to humans who come into contact with them. vaccination that resulted in low levels of seroconversion facilitated the emergence of the fujian-like sublineage of infl uenza (h n ) in poultry ( ) . the immune responses elicited by subpotent vaccines may exert selection pressure that favors antigenic drift and shift ( figure) . antigenic drift relies on the accumulation of mutations within the antibody-binding sites in the hemagglutinin (ha), neuraminidase (na), or both that abrogate the binding of antibodies. this makes infl uenza a virus strains able to evade neutralizing antibody from prior infection or vaccination. antigenic shift, which is seen only with infl uenza a viruses, is a more drastic change. it results from genetic shift by reassortment exchange of the ha, and sometimes the na, with novel subtypes that have not been present in human viruses for a long time. antigenic shift leads to replacement of circulating strains with new variants that are able to reinfect hosts immune to earlier types; the result is usually a pandemic. antigenic shifts caused of the major infl uenza a pandemics in the last century, including the subtype h n and subtype h n outbreaks ( ) . most infl uenza vaccines used in the united states and europe are produced in embryonated hens' eggs and are formaldehyde-inactivated preparations ( ) . because highly pathogenic infl uenza (h n ) subtypes may kill embryonated eggs, use of viruses that are no longer pathogenic, such as h (which lacks the polybasic cleavage site), to reduce the virulence of infl uenza (h n ) vaccine strains so that these can be effi ciently propagated in eggs for vaccine production is feasible ( ) . virus particles that lack the gene for the nuclear export protein or are defective for the matrix (m ) gene were used as live vaccines in animal models ( , ) ; however, whether these replication-defective vaccines will work in humans is not known. live attenuated (cold-adapted) infl uenza vaccines have long been used in russia, and a similar product has been approved for use in the united states ( ) . these vaccines will replicate in the host, and thus lower doses may be effective; however, the preexisting antibody to the virus is more likely to diminish the value of a live vaccine. moreover, such live vaccines are reported to cause asthma-like reactivity in children ( ) . monitoring live infl uenza vaccines is important because the risk for reversion to pathogenicity remains. with the use of a technique known as reverse genetics, a prototype of infl uenza virus (h n ) has been produced for the development of an inactivated subvirion vaccine. the gene segments encoding ha and na were derived from a/vietnam/ , and all other genes were derived from the backbone (a/pr/ / ) virus, commonly used as a platform for infl uenza vaccine production. the ha gene was further modifi ed to replace the stretch of basic amino acids at the cleavage site, and the resulting virus was avirulent in chickens. in a recent trial, healthy adult volunteers were given intramuscular doses of this inactivated infl uenza (h n ) vaccine. this split vaccine induced an antibody response predictive of protection in % of healthy adults tested, but only when given intramuscularly at high doses (two -μg shots) ( ) . the large amounts needed ( doses of vaccine, each times the dosage of that used in a standard infl uenza shot) means that hundreds of millions of doses are needed to tackle a pandemic. dose-sparing approaches, including the use of an effi cient nontoxic adjuvant to boost persons' immune responses, may improve the vaccine. another trial was performed with healthy participants - years of age, in which aluminum hydroxide adjuvant was used with similar split-virus vaccine ( ) . however, the alumadjuvanted vaccines did not improve the immunogenicity or percentage of seroconversion at lower vaccine doses and only slightly improved immunogenicity at the -μg dose. this diffi culty underscores the importance of vigorous fundamental research to address the question of how to increase the immunogenicity of such vaccines, whether by better antigen presentation or by choosing alternative routes of administration, so that lesser amount of antigen could be given to induce protective response. the present annual global production capacity is ≈ million doses of trivalent vaccine containing μg ha per strain. this is equivalent to million doses of monovalent vaccine, a quantity markedly insuffi cient for the world's . billion people. clearly, dose-sparing formulations are urgently needed. to test the hypothesis that whole-virion would be more immunogenic than conventional split-virion or subunit vaccines and may be adaptable to the antigen-sparing strategy, an inactivated, monovalent infl uenza a (h n ), whole-virion vaccine was prepared from a highly virulent strain a/vietnam/ / strain by removing the polybasic amino acids at the cleavage site, making the virus no longer pathogenic. the seed virus was grown to a high titer in embryonated eggs, inactivated with formalin, and purifi ed. these viruses were then adjuvanted with aluminum hydroxide and used in a phase trial ( ) . the highest immune response of % seropositivity was observed in the group given × μg ha, which is equivalent to that elicited by higher doses of nonadjuvanted ( μg) or adjuvanted ( μg) split-virion vaccines ( , ) . not knowing which particular genetic variant will sustain human-to-human transmission makes our ability to formulate a vaccine in advance all the more diffi cult. an inactivated vaccine that induces not only high levels of neutralizing antibody to surface proteins but also cd tcell response against well-conserved antigens derived from internal viral proteins might provide superior protection in an epidemic or pandemic. in cases of established intracellular infl uenza a infection, infected cells are mainly eliminated by effector cd + t cells (ctls) ( ) . any vaccine that will induce and direct these ctls to the site of infection and generate a long-lasting memory response will be more effective for mounting protection against a pandemic form of infl uenza (h n ). inactivated vaccines need to be presented to the host's immune system with an appropriate adjuvant, but inactivated vaccines that use an adjuvant currently approved for human use (alum or mf- ) usually have lower immunogenicity than live attenuated vaccines ( ) . therefore, the pursuit for other nontoxic adjuvants, including tlr ligands and agonists that could effectively activate dendritic cells for the presentation of viral antigens to cd and cd t cells, should vigorously be continued. use of cytokines such as il- or il- may enhance the immunogenicity of antiviral vaccines. recombinant fowlpox vaccines coexpressing ha of subtype h n and chicken il- have been shown to induce complete protection in vaccinated chickens ( ) . use of adjuvants may enhance broader cross-reactive immune responses among infl uenza viruses ( ) . the evolution of many sublineages of infl uenza (h n ) with antigenic diversity in southeast asia and southern china favors the wisdom of developing broadly cross-reactive vaccines for protection against an epidemic or pandemic ( ) . genetically engineered viruses could be constructed; these would express several variant antigens or determinants, thereby generating a broader immune response. the goal would be to develop vaccines that would induce broad-spectrum immunity-conferring protection to infl uenza including subtype h n . ferrets vaccinated with a/pr/ / single-gene reassortants that differed only in their h s were protected against a lethal challenge with a/vietnam/ / virus, suggesting generation of crossprotection ( ) . vaccination of mice with a live attenuated infl uenza vaccine or an alum-adjuvanted inactivated infl u-enza vaccine based on a related h ha from a nonpathogenic avian infl uenza virus, a/duck/pottsdam/ - / (h n ), limited the disease severity and reduced deaths following challenge with a current highly pathogenic infl uenza (h n ) ( ) . such cross-protective vaccines may provide clinical protection and prevent deaths in the early stages of a pandemic. genes of highly conserved proteins such as the nucleoprotein or m proteins could be included in adenovirus vector-based vaccines because immune responses against these infl uenza viral antigens provide protection in animal models ( , ) . recently, human adenoviral vector-based ha subtype infl uenza vaccine induced protection in mice against infl uenza (h n ) viruses isolated from humans ( , ) . however, pre-existing immune response to human adenoviruses could be a potential problem in the generation of immune response against a foreign gene of interest. delivering the vaccine nasally could largely overcome this problem because there appears to be no pre-existing immunity in the upper airways. moreover, a robust cd t-cell response would likely be fl exible and able to fi ght infl uenza (h n ). ideally, we need an effective vaccine for persons of all ages. however, if the vaccine is in short supply, priming fi rst those persons at high risk (e.g., young children, persons > years of age, healthcare workers) may be justifiable. during the early stages of an emerging h pandemic, such persons at high risk might be given an adjuvanted vaccine produced from a currently circulating strain, even if it is antigenically distinct, until an optimally matched and approved vaccine is available. this strategy is to produce a vaccine from an antigenically distant infl uenza (h n ) strain that could induce broad-spectrum immunity capable of neutralizing newly emerged human h strains. vaccine development based on a cell culture system has advantages over egg-based technology because h strains are highly pathogenic for chickens and supplying large numbers of embryonated eggs could be diffi cult in a pandemic. in addition, potential allergic reactions to egg components would be avoided by growing the vaccine virus in tissue culture cells. recently, mammalian cell culture was used for propagating viruses to prepare killed infl uenza vaccine ( ) . inactivated infl uenza vaccines produced with madin-darby canine kidney (mdck) and vero cells, which served as vaccine substrates, have been licensed in the netherlands. of note, the human cell line per.c may provide a useful cell-based system because, unlike mdck and vero cell systems, it does not require a solid matrix support for the growth of cells. selecting background viruses that grow well in these cell cultures and monitoring them for antigenic changes and contaminating microbes during propagation of the virus in cell culture need to be considered. for the development of a universal infl uenza vaccine, a possible target is the relatively conserved m homotetramer. the concept is based on identifying alternative infl uenza antigens that are not as susceptible to antigenic shift and drift. some degree of protection was induced in mice by priming with an m ectodomain peptide in adjuvant ( ) . studies that used the m ea peptide conjugated to keyhole limpet hemocyanin and neisseria meningitidis outer membrane protein illustrated good immune responses not only in mice but also in ferrets and rhesus monkeys ( ) . in a recent study, m ea sequences, representing a range of epidemic strains and the (h n ) strain, were fused to a proprietary hydrophobic protein domain. the resulting fusion proteins, formulated in liposomes, stimulated a protective response in mice challenged with subtypes h n , h n , h n , or h n ( ) . previous studies have shown that when m e is linked to hepatitis b virus core (hbc) particles, it becomes highly immunogenic, eliciting protective antibody response in mice ( ) . recently, a series of m e-hbc constructs were made by increasing the copy number of m e inserted at the n terminus from to per monomer. the best protection was seen when mice were vaccinated intranasally with these constructs combined with cta -dd, a cholera toxin a -derived mucosal adjuvant ( ) . m serves as a ph-induced proton channel on the surface of all infl uenza a viruses but is present in low quantities. further studies are warranted for understanding the mechanism of immune response to m ea and for defi ning the appropriate immunization conditions for humans. the lack of established correlates of immunity in animals and humans poses challenges to developing consistent immunologic endpoints for clinical trials and appropriate criteria for vaccine effi cacy. serum antibody titers, mainly those determined by hemagglutination inhibition (hi) or virus neutralization (vn) assays, or both, are considered surrogate measures of protection. however, the hi test is insensitive for the detection of antibody to avian ha; there also are no recognized clinical correlates of immune protection for neutralization antibody ( , ) . recently, hi or vn assay failed to detect antibodies in ferrets protected by vaccination with whole-virus vaccines containing internal protein from dk/sing virus against a heterotypic virus ( ). whether the cross-protection reported is mediated by tcell response is not known. in recent years, attempts were made to improve the sensitivity of the hi test. more sensitive detection of anti-body to avian ha was seen when horse erythrocytes were used in place of turkey erythrocytes in the hi test because infl uenza virus was better able to bind to a , gal-specifi c receptor sites on these erythrocytes ( ) . the presence of asparagines at aa (h numbering) in h ha leads to improved sensitivity of the hi test ( ) . often the immunogenicity of h vaccine candidates is assessed by hi or vn assays, but the basis of protection remains unclear. nevertheless, the tests that are used to evaluate effi cacy of candidate vaccines are based on the assumption that antibody would mediate the protection against infection induced by vaccination, although this has yet to be critically established. on the basis of initial evidence, infl ammation has been proposed as a possible cause or driving force of avian infl uenza (h n ). however, components of the infl ammatory response might even be benefi cial. to address these possibilities, we need to determine whether infl ammation in avian infl uenza is an early event and a manifestation of innate immune response. if it is, some of the mediators of innate immune response, such as cytokine/chemokine levels, can be included in the evaluation of the potency of candidate vaccines. further humoral response as a correlate for protection can be fi ne-tuned by determining the titer and isotype of antibody after vaccination. several issues concerning vaccine effi cacy are unresolved: what are the consequences of vaccination for existing infl uenza (h n ) infection, the extent of serologic cross-reactivity between the most closely related types of the virus, and the role in clinical protection? vaccine administration may provide some therapeutic effects for infected persons who have not yet made an immune response but provide none for those with persistent infection associated with measurable humoral immunity. clearly, more studies are warranted to establish a highly reproducible assay to measure immunogenicity of a candidate vaccine and to determine adequate correlates of immune protection. safety and immunogenicity of adjuvanted vaccines or new formulations should be critically assessed, and any fast-track approval of marketing vaccines must not compromise safety. the marked virulence of the outbreak suggests that infl uenza a (h n ) infection may have novel pathogenic mechanisms not seen in human infl uenza strains. to attempt to understand pathogenicity of this virus, an infl uenza virus bearing all gene segments of the pandemic virus, which claimed at least - million lives, was recently generated in cultured cells. the reconstructed infl uenza viruses displayed accelerated activation of host immune response in mice with high levels of chemo-kines and cytokines in the lungs, resulting in infi ltration of infl ammatory cells and extensive damage to the lungs with severe hemorrhaging ( ) . the pathogenicity induced by the reconstructed virus showed marked similarity to that reported with infl uenza (h n ). increasing evidence from mouse models and humans suggests that certain infl ammatory mediators are potent drivers of the disease. if this is true, this could have important implications for developing new therapeutics. acute respiratory distress syndrome, hemophagocytosis, or both, develop in a substantial fraction of patients with infl uenza (h n ) infection; both of these conditions are thought to be promoted by overproduction of proinfl ammatory cytokines (known as a "cytokine storm") ( ) . consistent with these observations, cytokine release was markedly enhanced in human macrophages after infection with infl uenza (h n ) ( ) . further, marked enhancement of chemokine and cytokine levels was observed in infl uenza (h n )-infected persons, particularly in those who died, and these correlated with high and disseminated viral replication ( ). additionally, infl uenza (h n ) viruses appear relatively resistant to the inhibitory effects of host antiviral cytokines, such as interferons (ifns) ( ) . thus, the severity of human infl uenza (h n ) infection may be related to the induction of excessive proinfl ammatory responses that can accompany a primary infection and high viral shedding. increased infl ammation was associated with viral replication in the respiratory and extrarespiratory organs of cats experimentally infected with infl uenza (h n ) ( ) . mice infected with the highly pathogenic infl uenza (h n ) strain a/hk/ / , originally obtained from diseased chickens and an ill child in hong kong, china (hk), showed reduced ability to activate transforming growth factor-β (tgf-β), a potent antiinfl ammatory cytokine, compared to mice infected with less virulent a/env/hk / viruses ( ) . the reduced ability to activate tgf-β may produce greater infl ammation at the site of infection and thus cause more severe disease. alternatively, the low levels of activated tgf-β in the sera of a/hk/ / -infected mice may allow the viruses to replicate and spread unchecked in the respiratory tracts of the mice, causing more severe disease. recently, the impact of the nonstructural (ns) gene variation of hong kong (h n )/ on cytokine production was illustrated ( ) . the ns gene reassortant induced elevated pulmonary concentrations of the infl ammatory cytokines il- α, il- β, il- , ifn-γ, and chemokine kc and decreased concentrations of the anti-infl ammatory cytokine il- . this cytokine imbalance is reminiscent of the clinical fi ndings in humans infected with infl uenza (h n )/ virus and may explain the unusual severity of the disease. the ability to site specifi c engineering changes in the virus genome allows us to consider a novel vaccine approach. by engineering a virus with site-specifi c changes in the genome (for example in ns gene), we may produce infl uenza virus vaccine that favors the production of benefi cial anti-infl ammatory cytokines but remains highly immunogenic. in another approach, a human replicationincompetent, adenoviral vector-based infl uenza vaccine could be developed, in which genes of anti-infl ammatory cytokines are coexpressed, which will inhibit overproduction of proinfl ammatory cytokines. such vaccines would be considered therapeutic vaccines (e.g., antidisease vaccines), which would inhibit infl ammation at the site of infection and protect against severe disease (figure) . excessive production of anti-infl ammatory cytokines may result in an inappropriate suppression of the host immune response. further studies will validate the benefi cial effect of the anti-infl ammatory response for temporizing the cytokine storm seen in infl uenza (h n ). development of an immunization protocol that uses an adjuvant that allows selective priming of an antigen-specifi c immunoregulatory cytokines (e.g., il- , tgf-β) would be a major advance in the development of a vaccine for bird fl u with a substantial infl ammatory component. the search for potential adjuvants, such as tlr ligands and agonists that will favor the synthesis of inhibitory cytokines including il- , should be pursued. by testing whether manipulation of infl ammatory pathways changes the pathologic course, we would identify new targets for disease intervention. vaccination is the best option by which to prevent the spread of a pandemic virus and reduce the severity of disease. defi ning the host response to infl uenza (h n ) in natural infection is urgently needed to better understand the basis of protection and subsequent development of effi cacious vaccines. improved vaccine strategies, which will require less antigen and be more robust in inducing both antibody and cell-mediated immunity for neutralizing infl uenza (h n ) viruses, should be considered. to create an effective vaccine, a combination of factors must be optimized-such as number of doses, formulation without or with better adjuvant, and dose range. we also need to develop a reproducible assay that measures immunogenicity of a vaccine and to establish adequate correlates of protection. the effi cacy of potential cross-reactive vaccine candidates to induce broad-spectrum immunity to infl uenza (h n ) viruses should be assessed critically; stockpiling of such vaccines may be justifi ed in the absence of optimally matched and approved vaccine during early stages of an h pandemic. search for therapeutic vaccines (antidisease vaccines) aimed at controlling innate immune responses should be pursued, given the clinical evidence that the h n subtype elicits a cytokine storm that contributes to disease pathogenesis. vaccine development and deploy-ment need to be undertaken by a partnership of academia, government, and industry. the risk for dissemination of pandemic virus will remain if the disease is controlled in area but not in others. a global approach is vital for combating the next infl uenza pandemic, a monumental public health challenge. newer respiratory virus infections: human metapneumovirus, avian infl uenza virus, and human coronaviruses distinct pathogenesis of hong kong-origin h n viruses in mice compared to that of other highly pathogenic h avian infl uenza viruses infl uenza a virus (h n ) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts fatal outcome of human infl uenza a (h n ) is associated with high viral load and hypercytokinemia avian infl uenza. amid mayhem in turkey, experts see new chances for research safety and immunogenicity of an inactivated adjuvanted whole-virion infl uenza a (h n ) vaccine: a phase i randomised controlled trial human transmission but no pandemic in indonesia silent spread of h n in vaccinated poultry emergence and predominance of an h n infl uenza variant in china avian fl u to human infl uenza making better infl uenza virus vaccines? emerg infect dis virus-like particle (vlp) vaccine conferred complete protection against a lethal infl uenza virus challenge infl uenza a virus with defective m ion channel activity as a live vaccine immunogenicity and effi cacy of russian live attenuated and us inactivated infl uenza vaccines used alone and in combination in nursing home residents does infl uenza vaccination exacerbate asthma in children? safety and immunogenicity of a recombinant of an inactivated subvirion infl uenza a (h n ) vaccine safety and immunogenicity of an inactivated split-virion infl uenza a/vietnam/ / (h n ) vaccine: phase randomised trial infl uenza and the challenge for immunology construction and immunogenicity of recombinant fowlpox vaccines coexpressing ha of aiv h n and chicken il cross-reactivity to highly pathogenic avian infl uenza h n viruses after vaccination with nonadjuvanted and mf -adjuvanted infl uenza a/duck/singapore/ (h n ) vaccine: a potential priming strategy establishment of multiple sublineages of h n infl uenza virus in asia: implications for pandemic control role of specifi c hemagglutinin amino acids in the immunogenicity and protection of h n infl uenza virus vaccines cross-protective immunity in mice induced by live-attenuated or inactivated vaccines against highly pathogenic infl uenza a (h n ) viruses protection against multiple infl uenza a subtypes by vaccination with highly conserved nucleoprotein universal infl uenza a vaccine based on the extracellular domain of the m protein protection of mice and poultry from lethal h n avian infl uenza virus through adenovirus-based immunization development of adenoviral-vector-based pandemic infl uenza vaccine against antigenically distinct human h n strains in mice safety and immunogenicity of a trivalent, inactivated, mammalian cell culture-derived infl uenza vaccine in healthy adults, seniors, and children induction of infl uenza type a virus-specifi c resistance by immunization of mice with a synthetic multiple peptide vaccine that contains ectodomains of matrix protein preclinical study of infl uenza virus a m peptide conjugate vaccines in mice, ferrets, and rhesus monkeys protection against h , h , h and h infl uenza a infection with liposomal matrix epitope vaccines improved design and intranasal delivery of an m e-based human infl uenza a vaccine confronting the avian infl uenza threat: vaccine development for a potential pandemic crossprotectiveness and immunogenicity of infl uenza a/duck/singapore/ / (h ) vaccines against infection with a/vietnam/ / (h n ) virus in ferrets detection of anti-h responses in human sera by hi using horse erythrocytes following mf -adjuvanted infl uenza a/duck/singapore/ vaccine characterization of the reconstructed spanish infl uenza pandemic virus preparing for the next pandemic induction of proinfl ammatory cytokines in human macrophages by infl uenza a (h n ) viruses: a mechanism for the unusual severity of human disease? lethal h n infl uenza viruses escape host antiviral cytokine responses pathogenesis of hong kong h n infl uenza virus ns gene reassortants in mice: the role of cytokines and b-and t-cell responses we thank daniel mielcarz for critical reading of this manuscript and tushar paul for the fi gure.a.h. is supported by the centre national de la recherche scientifi que (france) and the department of microbiology/immunology, dartmouth medical school.dr haque is a senior staff scientist with the centre national de la recherche scientifi que and is adjunct professor of microbiology/immunology at the immunotherapy center, dartmouth medical school. his research interests are directed at understanding the mechanisms of host immunity to parasites and viruses. key: cord- -pw f asc authors: goyal, amit k.; rath, goutam; garg, tarun title: nanotechnological approaches for genetic immunization date: - - journal: dna and rna nanobiotechnologies in medicine: diagnosis and treatment of diseases doi: . / - - - - _ sha: doc_id: cord_uid: pw f asc genetic immunization is one of the important findings that provide multifaceted immunological response against infectious diseases. with the advent of r-dna technology, it is possible to construct vector with immunologically active genes against specific pathogens. nevertheless, site-specific delivery of constructed genetic material is an important contributory factor for eliciting specific cellular and humoral immune response. nanotechnology has demonstrated immense potential for the site-specific delivery of biomolecules. several polymeric and lipidic nanocarriers have been utilized for the delivery of genetic materials. these systems seem to have better compatibility, low toxicity, economical and capable to delivering biomolecules to intracellular site for the better expression of desired antigens. further, surface engineering of nanocarriers and targeting approaches have an ability to offer better presentation of antigenic material to immunological cells. this chapter gives an overview of existing and emerging nanotechnological approaches for the delivery of genetic materials. vaccine development offers an attractive and cost-effective preventive approach against deadly disease. new advances in immunology, molecular biology and biotechnology as low as for the development of unique, safe and effective against some dreadful diseases like hiv, cancer, hepatitis, tuberculosis, etc. (table ) . genetic immunization holds potential to discover new vaccines and may be an efficient vaccine delivery system. in the early s, dna vaccines burst into the scientific limelight. tang and johnston described the delivery of dna using a gene gun into the mice skin and felt that this could be a useful technique to generate antibody responses against specific transgene product (tang et al. ) . in , at the annual vaccine meeting at the cold spring harbor laboratory reported to drive both humoral and cellular immune responses against pathogens or tumor antigens in vivo by the use of dna vectors. merck pharmaceutical company reported that developed immune responses against influenza virus antigens in mice after injecting the naked plasmids intramuscularly (ulmer et al. ) . similarly, robinson proved the ability of dna plasmids against influenza virus antigens (fynan et al. ) . the capability of plasmids carrying hiv antigens or tumor antigens to generate immune responses and protection from tumor in mice has been described (wang et al. ). importantly, a dna vaccine affects humoral as well as cellular immunity. the use of the dna approach also promised to overcome the safety concerns associated with live vaccines-their reversion risks and their potential spread to unintended individuals, avoids the risks linked to the manufacture of killed vaccine (ruprecht ) . vaccines are generally composed of whole organism-either live and weakened or killed forms (first-generation vaccines). live, attenuated organisms such as smallpox and polio vaccines are able to induce killer t-cell (t c ) responses, helper t-cell (t h ) responses, and antibody immunity (fig. ) . first-generation vaccines providing maximum protection but associated with a risk that attenuated forms of a pathogen can revert to a dangerous form and may still be able to cause disease, especially in immune compromised vaccine recipients (e.g., aids patients). killed vaccines cannot generate specific killer t-cell responses and will be effective against limited diseases where cellular response is not essential (alarcon et al. ). these were the reasons which initiated the research for second-generation vaccines. second-generation vaccines were the subunit vaccines, consisting of defined protein antigens (such as tetanus or diphtheria toxoid), recombinant protein components (hepatitis b surface antigen), or surface proteins (influenza). these vaccines are able to generate th and antibody responses, but not killer t-cell responses. this reason again restricted the utility of these vaccines to limited number of diseases. today is the era of genetic immunization, which is nextgeneration vaccine (third generation), which seems to be highly effective till date. this strategy is based upon improved gene optimization, improved rna structural design, novel formulations and immune adjuvants, and more effective delivery approaches (alarcon et al. ; robinson and pertmer ) . at the cellular level, introduction of nanotechnology and the development of nanocarrier-based vaccines provide effective immunization through better targeting and by triggering antibody responses. in order to induce an effective protective immunity, these vaccines require boosting with agents called adjuvants. adjuvants and delivery vehicles have shown to protect antigens from degradation. the current trend toward many efforts to develop novel adjuvants and carrier have persistent on systems at the micro-and nanoscale. immunization by traditional vaccines requires the administration of live attenuated virus, killed organism, whereas dna vaccines can be constructed to encode specific antigenic determinants. dna vaccines are highly flexible, stable, easily stored, manufacture on large scale, encoding several types of genes including viral or bacterial antigens, and immunological and biological proteins (gengoux and leclerc ; kutzler and weiner ) . many potential advantages of dna vaccines are summarized in table . the gene of interest having the antigenic determinant is inserted into the recombinant vectors like multiple cloning region of plasmid by enzymatically, synthetically or by pcr and delivered to the inoculation site by one of several delivery methods like physical (gene gun, electroporation), viral (virosomes), or nonviral (liposomes, microspheres, nanospheres) to either skin (intradermally), subcutaneum, or muscle. the mechanisms by which dna vaccines produce antigen-specific immunity in vivo are under intense investigation, with an idealized model presented in fig. . figure represents the overview of the mechanisms of plasmid uptake and proteinaceous antigen expression by either somatic cells (e.g., myocytes, keratinocytes) at the site of injection or the resident antigen-presenting cells (apcs), the immature dendritic cells. the mechanisms include (a) direct targeting of dendritic cells (langerhans cells, i.e., skin dendritic cells) in gene gun administration of plasmid dna, which involves high-speed shooting of gold microbeads coated with plasmid dna into the upper layers of the skin or (b) "cross-priming," most likely in intramuscular injections or any parenteral injections where the somatic cells mentioned above primarily express the protein encoded by the the success of dna vaccines concerns improving their immunogenicity and safety. therefore, there is an urgent need for the development of potent and safe adjuvants and delivery systems that can be used with new generation of vaccines. as shown in fig. , there are several ways in which antigen expression and immunogenicity can be improved for the dna vaccine platform. there are lot of steps undertaken to modify immunogenicity and safety of dna vaccines (fig. ) . promoter is an important component of the plasmid that drives high levels of expression of the gene of interest. various promoters have been utilized to improve the expression of vaccine genes. the human cytomegalovirus (cmv) promoter has been extensively used for high levels of protein expression in mammalian cells (boshart et al. ) . however, there are some drawbacks associated with cmv promoters like chromatin condensation by histone deacetylase. recently, histone deacetylase inhibitors have been supplemented with cmv promoter-based plasmid that has shown increased expression of dna vaccine antigens (lai et al. immediate-early promoter one (ie ) have also demonstrated better gene expression in insect cells compared with cmv promoter (he et al. ). the porcine circovirus type capsid gene promoter has enhanced the antigen expression and immunogenicity in a hiv- plasmid vaccine (tanzer et al. ). regulation of transcriptional termination is a key element in control of gene expression within the framework of a single transcriptional promoter (barr et al. ) . so one of the most effective ways to increase protein production is through the use of codon optimization or by adopting species-specific codon changes (gustafsson et al. ) . plasmid backbone optimization has also been important contributory factor for dna vaccine. replacement of sv t polyadenylation and splicing signals of the paec plasmid vectors by synthetic intron and synthetic rabbit beta globin-based termination/polyadenylation sequences and cpg motif have enhanced the cell-mediated ifn-gamma-secreting activity. the rna polymerase ii dependent cytomegalovirus immediate early (cmv ie) enhancer/promoter and t promoter in psmcta and pshcta has been utilized to enhance the expression of antigenic substances (yu et al. ). viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. viral vector vaccines use live viruses to carry dna into human cells. it consists of a non-replicating virus that contains some defined genetic fig. factors affecting the immunogenicity of dna vaccines material from the pathogen to which immunity is desired. viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. viral vector vaccines carry dna into a host cell for production of antigenic proteins that can be tailored to stimulate a range of immune responses, including antibody, t helper cell (cd + t cell), and cytotoxic t lymphocyte (ctl, cd + t cell) mediated immunity (draper and heeney ) . retroviruses, parvoviruses, adenoviruses, lentiviruses, adeno-associated viruses, and the herpes simplex virus are being investigated for their ability to transfer dna. gene expression with high transfection efficiencies in tissues, such as kidney, heart, muscle, eye, and ovary, has been achieved by using viral vectors. advantages of viral-vectored vaccines include their ease of production, a good safety profile, ability to potentiate strong immune responses, infect a broad spectrum of cell types, triggering t-lymphocyte activation, potential for nasal or epicutaneous delivery and mucosal immunization (chamberlain ; galimi and verma ; lien and lai ; martin et al. ; mctaggart and al-rubeai ; wolf and jenkins ) . the recombinant retroviruses have the ability to integrate into the host genome in a stable fashion because it contains a reverse transcriptase that allows integration into the host genome. - kb is the typical maximum length of an allowable dna insert in a replication-defective viral vector. lentiviruses are a subclass of retroviruses. the unique feature of lentiviruses is to their ability to integrate into the genome of nondividing cells, whereas retroviruses can infect only dividing cells. when the virus enters the cell, the viral genome in the form of rna is reverse transcribed and produce dna, which is then inserted into the genome by the viral integrase (cattoglio et al. ). their primary applications are in gene therapy and vaccination but their limits use in basic research due to it does not integrate into the genome and is not replicated during cell division. respiratory, gastrointestinal and eye infections were commonly caused in humans after the contact with adenoviruses. adeno-associated virus (aav) is a small virus that infects both dividing and nondividing cells of humans and some other primate species and may incorporate its genome into that of the host cell with causes a very mild immune response. these features make aav a very attractive candidate for creating viral vectors for gene therapy (goff and berg ) . nowadays, incorporation of molecular adjuvants has been the main strategy for melioration of vaccines. co-injection of plasmids encoding cytokines, chemokines, or co-stimulatory molecules like death receptors, growth factors, adhesion molecules, toll-receptor ligands can be used individually or in combination to maximize substantial effect on the immune response in the clinic, in both prophylactic and therapeutic studies to plasmid-encoded antigen. for example, boost the humoral and cellular response when antigen co-administered with synthetic oligodeoxynucleotides containing unmethylated cpg motifs in mice (higgins et al. ) . recently, immunomodulation is based on targeting antigen-presenting cells (apc) "majorly macrophages" by using macrosialin promoter. the immune response of the constructed plasmids expressing jev envelope (e) protein under the control of aforesaid promoter and cytomegalovirus (cmv) immediate early promoter against jev have induces comparable immunity in comparison to ubiquitous promoter construct (ahsan and gore ) . nk group , member d (nkg d) is also reported as potent-activating receptor expressed by cells of the innate and adaptive immune systems. recombinant mouse cmv expressing the high-affinity nkg d ligand rae- γ has shown better expression and profound virus attenuation in vivo and could be a powerful to develop immunogenic hcmv vaccine (slavuljica et al. ) . in vivo dendritic cells (dc) targeting is an attractive approach with potential advantages in vaccine efficacy, cost, and availability. genetic targeting of the dc-specific cd c-driven active transcription factor xbp s to dc (xbp s/dc) has potentiated vaccine-induced prophylactic and therapeutic antitumor immunity in multiple tumor models (tian et al. ) . recently, heterodimeric antigen-presenting cells targeted multireceptor ligand approaches have been implemented to access the potential of more than one apc-specific targeting unit in the antigenic molecule. results revealed that heterodimeric barnase-barstar vaccine molecules were potent and provide a flexible platform for development of novel dna vaccines with increased potency (spang et al. ). some mechanisms of adjuvant action are discussed below: . vaccine adjuvants can increase the potency and immune response of small, antigenically weak synthetic or recombinant peptides in immunologically immature, immunosuppressed, or senescent individuals. . they can improve the immune response to stronger antigens in respect of speed, vigor, and persistence. for example, aluminum adjuvants adsorbed dtp elicit early and higher antibody response after primary immunization than do unadjuvanted preparations. . vaccine adjuvants can modulate antibody avidity, specificity, quantity, isotype, and subclass against epitopes on complex immunogens. . they target antigen to a cell-surface receptor on apcs by formation of multimolecular aggregates. . they can direct antigen presentations by direct peptide exchange on surface mhc molecules or by mhc class i or mhc class ii pathways by means of fusion or disruption of cell membranes (newman and powell ) . the most important characteristic of any adjuvanted vaccine is that it is more efficacious than the aqueous vaccine but unfortunately, the absolute safety of adjuvanted vaccines, or any vaccine, cannot be guaranteed. the real or theoretical risks of administering vaccine adjuvants are local acute or chronic inflammation, painful abscess, persistent nodules, ulcers, fever, hypersensitivity, anaphylaxis, chemical toxicity to tissues or organs, autoimmune arthritis, amyloidosis, anterior uveitis, glomerulonephritis or meningoencephalitis, immune suppression or oral tolerance, carcinogenesis, teratogenesis or abortogenesis and spread of a live vectored vaccine to the environment (edelman and tacket ; bussiere et al. ; goldenthal et al. ). . it must be safe, including freedom from side effects. . it should be affordable and stable. . it should be biodegradable or easily removed from the body after its adjuvant effect. . efficacy and immunogenicity should be achieved using fewer doses and/or lower concentrations of the antigen. . it should elicit a more vigorous protective or therapeutic immune response combined with the antigen than when the antigen is administered alone. . it must be defined chemically and biologically, so that there is no lot-to-lot variation in the manufactured product. freund's adjuvant is a solution of antigen emulsified in mineral oil and used as an immunopotentiator (booster). freund's complete adjuvant (fca) is composed of inactivated and dried mycobacteria whereas the incomplete form (fia) lacks the mycobacterial components. although, fca has been proved as a potent inducer of cell-mediated immunity and ability to boost the humoral immune response, but associated adverse side effects like sterile abscesses, granulomas, muscle indurations, plasma cell neoplasia, ascites and amyloidosis has limits its utility. a modified version of fca is known as freund's incomplete adjuvant (fia) in which antigen is administered in water-in-oil (w/o) emulsion but without mycobacterial components. it consists of a mixture of mineral oil (drakeol vr, bayol f, marcol ) ( % v/v) and emulsifier (mannide monooleate) ( % v/v) with an equal volume of aqueous solution of antigen. mechanism of the freund's adjuvants is allowing a gradual and continuous release of the antigen by establishment of a repository antigen-containing locus at the site of injection or interaction with mononuclear cells such as phagocytic cells, antigen presenting cells, etc. fia has been included in veterinary vaccines (rabies, hog cholera, canine hepatitis) (freund et al. ; fastier and hansen ; ott ) , as well as human vaccines (tetanus toxoid, influenza vaccines) (salk et al. ) . in general, both fia and fca are indeed very efficient in raising high antibody titers, induce cytotoxic t lymphocytes (ctl) and used in priming immunizations. morozova et al. investigated that development of inflammatory response in the rat myocardium after immunization rats with single subcutaneous injection of cardiac myosin ( ug/kg) with incomplete freund's adjuvant (ifa) (gjessing et al. ). there are very limited studies that have been conducted which signify their utility of freund's adjuvant for dna vaccination. it has been demonstrated that plasmid pv- cpg suspended in ifa has significantly enhanced both type of cellular and humoral immune responses to hbsag (luo et al. ). aluminum compounds [aluminum phosphate (alpo ), aluminum hydroxide (al (oh) ), and alum] precipitated vaccines are currently the most commonly used adjuvants with human and veterinary vaccines owing to their good track record of safety, low cost, and adjuvanticity with a variety of antigens (gupta et al. ; gupta and siber ) . however, aluminum adjuvants have certain limitations such as local reactions at the site of injection, ige antibody responses augmentation, ineffectiveness for some antigens, and inability to supplement cell-mediated immune responses . two methods are used to prepare vaccines and toxoids with aluminum compounds-in situ precipitation of aluminum compounds in the presence of antigen and adsorption of antigen onto preformed aluminum gel (aprile and wardlaw ; holt et al. ; hem and white ; gupta ) . the mechanism of adjuvanticity of aluminum compounds includes formation of a depot, efficient uptake by antigen-presenting cells, stimulation of immune competent cells of the body through induction of eosinophilia, and activation of macrophages and complement. recently, adjuvanticity of alum has been reported due to cell death and the subsequent release of host cell dna, which acts as a potent endogenous immunostimulatory signal-mediating alum adjuvant activity (marichal et al. ) . gupta et al. ( ) showed that diphtheria toxoid adsorbed aluminum phosphate induced significant antibody levels in rabbits. previously, manam et al. reported that aluminum phosphate adjuvant had shown no effect on the tissue distribution and integration frequency of delivery genetic materials (manam et al. ) . similarly, liang et al. ( ) showed the similar results indicated that there was not increase in hbsag expression when plasmid pcdna . -s mixed aluminum phosphate. however, they demonstrated the better antibody titer after intramuscular immunization of balb/c mice with pcdna . -s mixing aluminum phosphate adjuvant. this study revealed that aluminum phosphate has a potential for dna vaccination (liang et al. ) . recently, yu et al. have demonstrated the role of aluminum adjuvant for dna vaccines against botulinum neurotoxin (bonts) and shown induced protective humoral immune responses (yu et al. ) . combined use of il- with alum adjuvants for dna immunization have also demonstrated the significant change in the survival rates of the vaccinated animals against toxoplasma gondii (khosroshahi et al. ) . cytokines are a group of secreted low-molecular weight proteins by the cells of the innate and adaptive immunity that have a major role in cell-to-cell communication. cytokines play an important role in induction of immune responses during the processing and presentation of antigens. numerous cytokines including interleukin- (il- ), granulocyte-macrophage colony stimulating factor (gm-csf), and interleukin- (il- ) have been shown to significantly modulate the inflammatory process when given systemically. the local administration of il- increases local expression of major histocompatibility (mhc) class ii antigens and enhances skin antigen reactivity, but high bolus doses of il- cause hypotension, exacerbation of underlying autoimmune disease, and induce vascular leak syndrome. this studies revealed that exogenous il- could be a valuable adjunct in the treatment of immunodeficiency virus (hiv) infected human by decreases the frequency of apoptotic peripheral blood mononuclear cells (pmbcs), which may contribute to the increase in circulating cd + t cells. il- also induces b-cell activation and antibody synthesis in vitro (cordiali fei et al. ). among various improvement strategies, the incorporation of cytokine-expressing plasmids as molecular adjuvants has been widely studied in the past years, yet still without significant clinical application. this chapter reviews recent progress in the co-application of cytokine-encoding genes used for enhancement and direction of immunogenicity, as well as discusses their therapeutic potential for future applications. coadministration of pro-inflammatory agents (such as various interleukins, tumor necrosis factor, and gm-csf) plus th -inducing cytokines increase antibody responses, whereas pro-inflammatory agents and th -inducing cytokines decrease humoral responses and increase cytotoxic responses (which is more important in viral protection, for example). co-stimulatory molecules like b - , b - and cd l are also sometimes used. mpl (monophosphoryl lipid), a immunostimulant, is derived from the lipopolysaccharide (lps) of salmonella minnesota, r . an important characteristic of mpl adjuvant activity is to enhance the generation of specific immunity without being directly associated with an antigen. the choice of an mpl adjuvant formulation will depend on several factors such as the nature of the antigen, desired immune response characteristics, and level of tolerable local reactogenicity. aqueous dispersions of mpl in isotonic buffers when admixed with soluble protein antigens can provide a strong adjuvant effect. an advantage of these mpl plus antigen is that they tend to be well tolerated and induce little or no local tissue reaction at the injection site (qureshi et al. ) . mpl-a has been used to enhance immunity induced by dna vaccination against human immunodeficiency virus type (hiv- ). results indicate that mpl performances as an effective adjuvant for immunogenic dna injection despite reduced expression of encoding protein in muscle (sasaki et al. ) . combination of mpl with antigen-encoded dna has shown the enhanced protective neutralizing antibody response against glycoprotein of the cvs rabies virus (lodmell et al. ) . lipid a has also been admixed with plasmid dna (pdna)-coated nanoparticles and studied for their immunological potential. immunological results revealed that plasmid dna with lipid a have shown significant higher immunological response, especially cellular response (cui and mumper a, b) . studies indicated that la is potential adjuvants to further enhance immune responses; however, limited studies have been utilized this adjuvant for dna vaccination. several established methods have utilized for transferring plasmid dna into cells, including calcium phosphate precipitation, electroporation, particle bombardment, liposomal delivery, polymeric delivery, viral-vector delivery, and receptormediated gene delivery. however, compared to viral vectors, nonviral vectors are easy to make and are less likely to produce immune reactions (edelman and tacket ). in addition, there is no replication reaction required. the engineered novel nano-construct may deliver immunogens safely, with the appropriate kinetics, to the appropriate location, and possibly together with the adequate recognition and maturation stimuli (fig. ) . the use of nonviral particulate carriers for dna-based vaccination could provide better and safe delivery of encapsulated genetic material, circumvent the need for muscle involvement and facilitate instead the uptake of the fig. schematic representation of immunological response greeted by novel dna-loaded nanocarrier dna by apcs. however, transfection of apcs with encapsulated dna into particulate carrier systems will be dependent upon choice of carrier surface charge, size, and lipid/polymer composition, or presence of other biological [e.g., interleukin and interferon-γ (ifn-γ)]. toxicity, transfection efficiency, nucleic acid (na) degradation and free na release are challenging problems for all of the current nonviral gene delivery systems, including lipid and polymers carrier systems (pouton et al. ; cui and mumper a, b) . one current trend in dna vaccine formulation is the use of biodegradable polymeric microparticles and liposomes delivery systems for dna vaccines are excellent formulations for delivery and enhanced immunogenicity in several different hosts like mice, nonhuman primates and humans (herrmann et al. ; kaur et al. ). as noted earlier, genetic materials attached to a particulate carrier are more likely to bring about a successful immunological reaction and some, such as chitosan particles, can act as adjuvants in their own right. natural polymers such as gelatin or albumin have been used as particulate drug delivery systems, although they are of uncertain purity and certainly have the potential for immunogenicity (pouton et al. ; cui and mumper a, b; xiang et al. ; pichichero ) . plasmid dna is trapped on the surface of the polymers like polylactice-coglycolide, chitosan, polyethyleneimine, amine-functionalized polymethacrylates, cationic poly(β-amino esters), poloxamers, and polyvinylpyrrolidone (densmore ) . polymer-trapped plasmid dna is delivered systemically or directly to mucosal surfaces (orally or via the respiratory tract), where the complex is taken up by dendritic cells (dcs) and results in upregulation of dc activation markers and further augments systemic and mucosal immune responses. liposomes offer considerable flexibility towards vaccine optimization due to its structural versatility, including vesicle surface charge (both cationic and anionic liposomes can be made), size, and lipid content. liposome with other suitable adjuvants can protect dna from degradation by serum proteins during transfer of dna across membranes and after the release of genetic material following fusion with endosome (gao and huang ; nakanishi and noguchi ) . among the different approaches to drug delivery, lipid vesicles for both hydrophobic and hydrophilic drugs have attracted much attention. lipid-based gene delivery is the focus of several specialized high-technology companies, of which vical (san diago, ca, usa), genzyme (farmington, ma, usa), genemedicine (the woodlands, tx, usa) and megabios (burlingame, ca, usa) have products in clinical trials. some of the engineered liposomal and non-liposomal versions like ph-sensitive cationic and anionic liposomes, ph-sensitive immunoliposomes, fusogenic liposomes; genosomes (dna-liposomes/lipid complexes), lipofection tm (lipid-dna complex) and recently cochleates are investigated as the major gene vectors (fig. ) . however, most of the commercially available nonviral gene vectors used for transfection is cationic liposome-dna complexes (fenske and cullis ). liposomes are self-assembling structures comprising concentric amphipathic lipid (e.g., phospholipid) bilayers separated by aqueous compartments (baca-estrada et al. ; saupe et al. ) . in , first humoral immune responses observed in mice after injection of liposome-entrapped diphtheria toxoid (allison and gregoriadis ; manesis et al. ) . liposomal vaccines that have been investigated inhuman trials include malaria, hiv, hepatitis a, influenza, prostate cancer and colorectal cancer (katre et al. ). in a liposome-based drug delivery system, genetic material is encapsulated in the liposome and then administered to the patient to be treated. advantage of the use of liposomal dna is that it may be taken up directly by apcs such as dendritic cells, which results in transfection and mhc classes i and ii expression, which stimulates the cd + and cd + t cells by antigenic peptide and induces ctl responses and also b cells to produce antibodies, whereas vaccination with naked plasmid dna, the plasmid is taken up by the myocytes, which are transfected. unfortunately, there are a number of problems associated with the use of conventional liposomes as genetic vaccine delivery vehicles. the relatively low transfectivity of liposomes, particularly evident with insufficient quantities of polynucleotide within liposomal formulations, can be overcome by adding positively charged amphipathic lipid moieties to liposomal formulations. several phospholipids may be used for the preparation of liposomes entrapped vaccines include phosphatidylcholine, phosphatidic acid, triolein, phosphatidylglycerol, phosphatidylserine, distearoyl phosphatidylcholine, dioleylphosphatidylethanolamine, phosphatidylethanolamine, polyethyleneglycol etc. overall, by modification, these systems may provide high membrane fluidity, flexibility, endocytosis and fusiogenic behavior, that is making this system far better than other particulate carriers (fig. ). cationic liposomes are widely explored nowadays for the delivery of dna into eukaryotes. they are formed by simple mixing of positively charged lipid bilayers with negatively charged naked dna. the resulting cationic liposomes-dna complexes (lipoplexes) are taken up via endocytosis, followed by their release from an early endosomal compartment (duzgunes et al. ) . cationic lipid-dna complexes have been used successfully to deliver plasmid dna to the lungs, brain, tumors and skin, by local administration, or to vascular endothelial cells after systemic, intravenous injection (brigham et al. ) . in addition to different cationic lipids (fig. have also shown an important role in membrane perturbation and fusion for intracellular delivery of genetic material. liu et al. have shown that lipoplexes showed much higher transfection in the liver than naked dna alone (liu et al. ). gregoriadis et al. for the first time showed that intramuscular immunization of mice with prc/cmv hbs (encoding the s region of hepatitis b antigen; hbsag) entrapped into positively charged (cationic) liposomes leads to greatly improved humoral and cell-mediated immunity (gregoriadis et al. ). these cationic liposome-entrapped dna vaccines generate titers of anti-hbsag igg antibody isotype in excess of -fold higher and increased levels of both ifn-γ and il- when compared with naked dna or dna complexed with preformed similar (cationic) liposomes. further, modification of liposomal surface with polymer offers potential for oral administration of plasmid dna and able to elicit markedly enhanced transgene-specific cytokine production following in vitro restimulation of splenocytes with recombinant antigen (somavarapu et al. ) . modification of lipid/dna complexes by the polymer poly(d,l-lactic acid) was found to be consistently and significantly more effective than either unmodified liposomal dna or naked dna in eliciting transgene-specific immune responses to plasmid-encoded antigen when administered by the s.c. route (bramwell et al. ) . surface-modified mannosylated cationic liposomes were developed for targeted delivery of pdna to apcs, and the results verified that man lipoplex induces significantly higher pub-m gene transfection into dendritic cells and macrophages than unmodified lipoplex and naked dna and it also strongly induces ctl activity against melanoma, inhibits its growth and prolongs the survival after tumor challenge compared with unmodified liposomes (lu et al. ). an anionic lipid formulation called fluid liposomes was capable of delivering fluorescently labeled oligonucleotides into bacterial cells. it was composed of dppc and , -dimyristoyl-sn-glycero- -[phospho-rac-( -glycerol)] (dmpg). lack of further progress of these systems may be attributed to the poor association between dna molecules and anionic lipids by electrostatic repulsion between these negatively charged species (perrie and gregoriadis ) . liposomes have been prepared from mixtures of anionic and zwitter ionic lipids, dopg and dope, respectively, at a molar ratio of : (dopg:dope). efficient and relatively safe dna transfection using anionic lipoplexes makes them an alternative for gene delivery (patil et al. ) . similarly, endosomolytic bacterial protein listeriolysin o (llo) incorporated in an anionic liposome-entrapped polycation-condensed dna delivery system (lpdii) has been developed that demonstrated better condensation of the dna with improved transfection efficiency due to endosomolytic properties of llo (lorenzi and lee ) . combination of cationic lipoplexes and pegylated anionic liposomes has also been used to prepare anionic pegylated lipoplexes. studies demonstrated that the gene expression of the developed formulation was similar for the cationic formulation taken as a control and the anionic formulations prepared (mignet et al. ) . overall, anionic lipoplex formulation shown promise as a nonviral vector with high-transfection efficiency and low cytotoxicity. a growing amount of literature describes the role of ph-sensitive liposomes for targeting and/or release encapsulated genetic material within cellular compartment. ph-sensitive liposomes are designed to release their contents in response to acidic ph within the endosomal system, while remaining stable in plasma thus improving the cytoplasmic delivery of biopharmaceuticals. they can be generated by the insertion of dope into acidic lipids liposomes such as cholesteryl hemisuccinate or oleic acid (venugopalan et al. ) . it is reported that detergent removal method is a superior method for preparing glycosaminoglycan-resistant and ph-sensitive lipid-coated dna complexes. this method is produced stable, but acid activatable, lipid-coated dna complexes (lehtinen et al. ) . at the neutral cellular ph , these lipids undergo protonation and collapse into a non-bilayer structure of endosomal compartmentalization which in turn helps in the rapid release of dna into the cytoplasm. recently, citraconyl-dope (a chemical derivative of dope), deliver dna-based therapeutics to cancer cells, in this manner combining the targeting and the rapid endosome release (reddy and low ) . addition of ph-sensitive fusogenic peptide, gala (peptide composed of repeating sequences of glu-ala-leu-ala) in lipidic preparation is also promising method to enhance the expression of the desired proteins. studies demonstrated that addition of . μm gala to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of lipofectin, but higher concentration of gala decreased transfection efficiency (futaki et al. ; nakase et al. ) . similarly, ph-sensitive histidine-modified galactosylated cholesterol derivative (gal-his-c -chol) has also been synthesized that demonstrate much greater transfection activity than conventional liposomes in hepg hepatic cells (shigeta et al. ). further, ph-sensitive tat-modified pegylated liposomes are utilized for delivery of tumor-specific stimuli-sensitive drug and gene delivery systems (kale and torchilin ) . immunoliposomes are sophisticated gene delivery systems in which incorporation of functionalized antibodies attached to lipid bilayers used for cell targeting (maclean et al. ) . using immunoliposomes, tissue-specific gene delivery has been achieved in the brain, embryonic and breast cancer tissue. recently, immunoliposomes containing an antibody fragment were successfully used in targeted delivery of tumor-suppressing genes into tumors in vivo (xu et al. ) . chloramphenicol acetyltransferase (cat) gene-encoded plasmid was entrapped in ph-sensitive immunoliposomes comprising of h- kk antibody-coated liposomes with dope, cholesterol, and oleic acid. studies revealed that approximately % of the injected immunoliposomes were taken up by the target rdm- cells. uptake was much less when liposomes without antibody were used (wang and huang ) . similarly, these authors have also reported that compositions of liposomes have altered the distribution for targeted drugs. delivery was also dependent on the lipid composition of the liposome. the ph-sensitive lipid composition gave eightfold higher efficiency than the corresponding ph-insensitive composition (wang and huang ) . ligand-modified immunoliposomes has been used to efficiently deliver plasmid dna expressing ns -ns b (hcv-specific antigenic sequence) to antigen-presenting cells. results confirm that this is as a more efficient delivery system than direct intramuscular inoculations with naked dna (zubkova et al. ). overall, studies have shown that immunoliposomes are efficiently used for targeted delivery of genetic material, especially in treatment of genetic disorders; however, very limited work has been done for delivery of dna vaccines. stealth liposomes (polyethylene glycol(peg)-conjugated lipids) are sterically stabilized liposomal formulations. pegylation prevents the liposomal vesicles by opsonization and recognition from the reticuloendothelial system and conjunction with other polymeric delivery systems such as pll to achieve longer circulation half-lives (mannisto et al. ) . peg grafted liposomes carrying antigenic epitope of gp , a transmembrane protein of hiv- has shown higher immune response and prolonged persistence of antibodies than plain liposome-based antigenic formulations (singh and bisen ) . further, it is also reported that grafting of peg on cationic liposomes have resulted in enhanced lymphatic drainage, but there is no improvement in immune responses, when compared to non-pegylated liposomes (carstens et al. ) . similarly, immune cell-specific ligand anchored pegylated liposomes have been developed to provide selective uptake at immunological cell. ultrasound (us)-responsive and mannose-modified gene carriers, man-peg ( ) bubble lipoplexes, have been utilized for transfer of ovalbumin (ova)-expressing plasmid dna to selectively and efficiently into antigenpresenting cells. developed systems have demonstrated - -fold higher gene expressions in the antigen-presenting cells (apcs) selectively in vivo compared with the conventional lipofection method (un et al. ). virosomes are lipidic envelope devoid of genetic information, which retain the antigenic profile and fusogenic properties from their viral origin. reconstituted lipid vesicles equipped with viral glycoproteins seems to possess many ideal properties for delivery of immunogens such as no limitation of size of encapsulated immunogens, high efficiency for cytosolic delivery, simplicity in handling and brevity of incubation time (okamoto et al. ) . virosome-mediated delivery has low toxicity and high immunogenicity with various prospective applications for the treatment and prevention of cancer, neurodegenerative disorders, and infectious diseases. the use of immunopotentiating reconstituted influenza virosomes (iriv) as delivery system of dna appear to be a promising tool in vaccinology and gene therapy. irivs are spherical, unilamellar vesicles with a mean diameter of~ nm, short surface projections of - nm. irivs are prepared by a mixture of natural and synthetic phospholipids containing % egg yolk phosphatidylcholine (for enhancement of immune responses), % synthetic phosphatidylethanolamine (able to directly stimulate b cells to produce antibodies), and % envelope phospholipids originating from h n influenza virus. irivs were first utilized in the manufacture of hepatitis a vaccine. the adjuvant function of virosomes is based on their virus-like particle structure providing repetitive antigen presentation to b cells, partial protection from extracellular degradation, and a depot effect (gluck et al. ) . proteasomes this immunogenic delivery system generally uses a noncovalent interaction between the proteosomes and antigen to form the appropriate complexes for delivering apolar or amphiphilic antigens. in most cases, these trials have involved intranasal administration of the vaccine and qualified as safe and well-tolerated materials through various human clinical trials. proteasome-conjugated shigella flexneri a lps vaccine shows an immune response similar to that observed after immunization with the live pathogen (fries et al. ) . intranasal delivery of proteasome-based vaccines may be able to produce both systemic and mucosal immunity. another very similar category of vaccines is the conjugate vaccine. these vaccines consist of a relatively non-immunogenic (especially in infants) antigen linked to a more immunogenic carrier such as a protein or toxoid. the conjugate vaccines for h. influenzae type b (hib) were developed using hib polysaccharide conjugated to either diphtheria toxoid (prp-d), omp of neisseria meningitidis (prp-omp), mutant diphtheria toxoid crm (hboc) or tetanus toxoid (prp-t) to provide the hib antigen immunogenic (heath ). cochleates are phospholipid calcium precipitates with a unique structure consisting of a large continuous solid lipid bilayer sheet rolled up into a "jelly roll-like" structure (papahadjopoulos et al. ) . cochleate delivery vehicles composed of simple, natural materials (phosphatidylserine and calcium) are unique vaccine carrier and delivery formulations (mannino and gould-fogerite ) . they are nontoxic, noninflammatory, and biodegradable. cochleates are prepared through the calcium-induced fusion of negatively charged phospholipid liposomes to collapse into solid sheets that roll up or stack, excluding water. the entire cochleate structure is a series of solid layers, components within the interior of the cochleate structure remain intact provides protection from degradation when exposed to harmful environmental conditions or enzymes. the protection of encochleated materials and structural stability of the cochleate allows for efficient delivery of dna by various routes like mucosal (oral, intragastric, intranasal, and intraocular) and parenteral (intramuscular, subcutaneous, intraperitoneal, and intradermal). strong, long-lasting, mucosal and circulating, antibody and cell-mediated responses are generated. protection from challenge with live viruses following oral or intramuscular administration has been achieved (mannino et al. ) . cochleates efficiency can be improved by attachment of surface glycoproteins of enveloped viruses and can be integrated into the lipid bilayers. dna cochleates can be formed by trapping oligonucleotides or high molecular weight plasmids within or between the lipid bilayers (papahadjopoulos et al. ). virus-like particles (vlps) are small particles consisting of one or more viral coat proteins can act as an adjuvant by carrying peptide sequences inside the apc and feeding into the endogenous processing pathway (schirmbeck et al. ) . these are safe, highly immunogenic, no additional adjuvant is needed, well tolerated, noninfective, and can easily be handled in the laboratory. it uses nature's own mechanism and structural principles to trigger the immune system for protective effects by stimulating both cellular immunity by effectively stimulating cd proliferative responses and cytotoxic t lymphocyte (ctl) responses and humoral immunity by efficiently cross-linking the membrane-associated immunoglobulin molecules that constitute the b-cell receptor (chackerian ; jennings and bachmann ; buonaguro et al. ) . the immune-stimulating complex (iscom) is a highly versatile and effective particulate antigen delivery system that has been extensively studied as an adjuvant system for a range of viral, bacterial, parasite, and other antigens. iscoms are threedimensional "cage-like" structures, which have been shown to form upon detergent removal from mixtures of saponins, detergents, and cholesterol. the iscom (immunostimulating complex) is a complex consisting of protein antigen, cholesterol, phospholipid, and the saponin adjuvant quil a. a similar vaccine delivery vehicle and adjuvant has also been developed that uses the same material minus the antigen and is referred to as iscomatrix ® . the antigen can be added later to the iscomatrix ® during formulation of the vaccine. this material seems to work similarly to iscoms but provides for more general applications by removing the requirement for hydrophobic antigens (pearse and drane ) . iscoms potentiate both humoral and cellular immune responses to incorporated antigens (cox et al. ) . iscoms stimulate apcs to produce il- , il- , and il- and induce thelper cells of both th and th type and the cell-mediated immune response includes cd + class i restricted cytotoxic t cells in a variety of experimental animal models and have now progressed to phase i and ii human trials (claassen and osterhaus ; barr and mitchell ) . oral administration of iscom vaccines has been shown effectiveness and immune-potentiating effect, but this route requires the use of high and frequent dosing. a study in which iscom vaccines may be able to elicit strong mucosal immune responses when administered in the pelvic presacral space of sheep, which could be useful for immunization against viral infections of the female genital tract (thapar et al. ) . a quil a-containing iscom with modified cholera toxin a (cta -dd) used as a mucosal vaccine carrier system for the influenza virus pr antigen (helgeby et al. -ji et al. ) . nasal vaccinations with p dna vaccine and matrix-m (immunostimulatory complex adjuvant) have shown significant higher iga-producing cells in addition to th and th cytokine expression. this strategies may provide a new way for the induction of specific immunity at mucosal sites (kodama et al. ). archaeosomes are nanometric size liposomes made from the polar ether lipids of archaea found in eukaryotes and bacteria. polar ether lipids of archaeosomes are providing excellent physicochemical stability and self-adjuvanting properties for delivery of vaccine preparations. archaeosomes have demonstrated relatively higher stabilities to oxidative stress, high temperature, alkaline ph, action of phospholipases, bile salts, and serum proteins (patel and chen ; benvegnu et al. ). archaeosomes facilitated a strong antibody (th ) response to entrapped protein antigens. the antibody humoral response was superior to that obtained with conventional liposomes and was in some instances comparable to that obtained with the potent but toxic freund's adjuvant (patel and sprott ; patel and chen ) . sprott et al. have also been described the role of co-enzyme q into archaeosome-based antigen formulation. incorporation of coq into archaeosomes and conventional liposomes can enhance the phagocytosis of the resultant vesicles by macrophage cells that allow the alteration in targeting profiles to specific tissues when the vesicles are administered to an animal via different routes and further enhance the immune response to coadministered immunogens. recently, "cationic archaeosomes," based on mixtures of neutral/cationic bilayerforming lipids and archaeobacterial synthetic tetraether-type bipolar lipids, have shown better transfection efficiency and can be utilized for dna vaccination (rethore et al. ). among the variety of lipid delivery systems, polymeric delivery systems have emerged as a promising alternative because of their ease of preparation, purification and chemical modification as well as their enormous stability. polymeric nonviral carriers (polyplexs) are one of the effective means of delivering a therapeutic or other biologically active substance in controlled and sustained manner. polymeric particulate delivery system induces adjuvant effect on the incorporated antigen and reduces the frequency of vaccination required to establish long-term protection. both natural and synthetic polymers have been considered to encapsulate antigenic materials for vaccination (table ) . various polymeric delivery systems have been developed using these polymers like micellar systems, emulsions, polymerosomes, nanoparticles, microspheres, nanocapsules, dendrimers, and dendrosomes (fig. ) . however, there are several associated concerns for the use of polymers as vaccines delivery systems such as toxicity, irritancy, allergenicity, and biodegradability. the advantages of using natural polymers include their low cost, biocompatibility and aqueous solubility. however, the natural polymers may also be limited in their use due to the presence of extraneous contaminants, variability from lot to lot and low hydrophobicity. in contrast, synthetic polymers are more reproducible and can be prepared with desired degradation rate, molecular weight and copolymer composition. nevertheless, synthetic polymers may be disadvantageous due to their limited solubility, they are often soluble only in organic solvents and consequently may not release biologically active antigen (rice-ficht et al. ). polymeric vaccines may offer improved stability and activity of encapsulated antigen materials by avoiding exposure to organic solvents used during formulation and acidic ph conditions caused by degradation of the polymer (duncan et al. ) . effective application of a polymeric nanoparticulate delivery system is greatly dependent on the specific polymer used, as this will dictate the properties of the nanoparticle in vivo (hanson et al. ) . for example, polycationic polymers can interact with negatively charged dna, resulting in a improved intracellular dna delivery to occur. whereas noncondensing polymers are neutral or slightly negatively charged polymers that physically encapsulate materials and can be used to target apcs and m-cells in the mucosa (bhavsar and amiji ) . there are a number of factors that affects the physicochemical properties of polymeric delivery vehicles like molecular weight, degree of branching, cationic charge density buffer capacity, polyplex properties and the experimental conditions like the polyplex concentration, the presence or absence of serum during transfection, the incubation time and the transfection model chosen for the gene delivery experiment. to reduce its cytotoxicity and improve transfection efficiency, polyplexes have been modified by conjugating with polyethylene glycol (peg), histidine, and targeting ligands including polysaccharides, transferrin, and galactose. various biodegradable polymers like aliphatic polyesters such as poly(lactic acid) (pla), poly(glycolic acid) (pga), poly(e-caprolactone) (pcl), poly (hydroxybutyrate) (phb), and their copolymers being evaluated for their uses as vaccine adjuvants and delivery systems (panyam and labhasetwar ) . recently, poly(amino acid)s-based copolymers have also been employed for the delivery of protein, vaccine, and genetic materials such as poly-l-glutamic acid, poly-l-aspartic acid, poly-l-lysine, poly-l-arginine, poly-l-proline, poly-l-asparagine, and poly-lhistidine. polyamino acids have properties that mimic proteins, making them ideal for vaccines delivery. they provide better adjuvanticity, low toxicity, biodegradability and targeting into intracellular compartments (chiang and yeh ) . various type of polysaccharides, such as agarose, alginate, carrageenan, hyaluronic acid, dextran, chitosan, cyclodextrins, mannan, and pullulan, have been used for delivery of vaccines (table ) . at specific concentrations and temperatures, when amphiphilic molecules, or molecules containing hydrophobic and hydrophilic regions, are maintained, naturally form association colloids known as amphiphilic micelles as a result of hydrophobic interactions. poly (ethylene glycol) (peg) is commonly incorporated as the hydrophilic segment in both amphiphilic micelles (gaucher et al. ) . gelatin: a denatured protein obtained by acid and alkaline processing of collagen. insoluble in water to prepare hydrogel through chemical cross-linking, with water-soluble carbodiimides and glutaraldehyde lou et al. ( ) easy processability, good biodegradability poor mechanical properties, brittle capable of targeting fibronectinbearing surfaces associated with some tumors silk fibroin: silkworm bombyx mori produces silk to weave its cocoon, and its major components are fibroin and sericin. this is light weight, extremely strong and elastic and exhibits mechanical properties comparable to the best synthetic fibers produced by modern technology zhang et al. ( ) environmentally safe, biocompatibility, excellent mechanical properties less production, high brittleness model antigen enhance the stability, up to c over more than months fibrin: fibrin is a protein matrix produced from fibrinogen, providing an immune-compatible carrier for delivery of active biomolecules, antigens. fibrin naturally contains sites for cell binding and has been investigated as a substrate for cell linkage, distribution, relocation, and propagation khan et al. ( ) induce improved cellular interaction, used as a cell carrier as well as antigen carrier rapid degradation, instable, low mechanical stiffness elastin: elastin is synthesized by vascular smooth muscle cells and secreted as a tropo-elastin monomer that is soluble, hydrophobic and non-glycosylated. elastin is a potent regulator of vascular smooth muscle cells activity, regulations important for preventing fibro-cellular pathology gaudreau et al. ( ) conferring elasticity, precise molecular weight, low polydispersity become insoluble and aggregate at a critical temperature soybean: the most cultivated plant in the world is rich in proteins ( - %), carbohydrates ( - %),and lipids ( - %). it is a species of legume native that can be processed into protein-rich products moravec et al. ( ) abundant, renewable, inexpensive, environment friendly biodegradable application of soy-based polymers in this field is still very narrow iga, mucosal iga antibody response after administered orally to mice chitosan: fully/partially deacetylated form of chitin. degree of deacetylation of commercial chitosan is usually between and %, and the molecular weight between and , kda. chitosan exhibits a ph-sensitive behavior as a weak polybase due to the large quantities of amino groups on its chain verheul et al. ( ) enhanced immune response, mucoadhesive property poly(ester-amide)s: this polymer is made up of a soft peg segment, connected to a hard diester-diamide segment through an ether bond. it is a high performance thermoplastic elastomer. it is used to replace common elastomers-thermoplastic polyurethanes, polyester elastomers, and silicones-for these characteristics: lower density among tpe, superior mechanical and dynamic properties (flexibility, impact resistance, energy return, fatigue resistance) and keeping these properties at low temperature (lower than À c), and good resistance against a wide range of chemicals. it is sensitive to uv degradation li and hu ( ) enhanced cell mediated immunity, superior mechanical and thermal properties poly(lactide-co-glycolide)(plga): plga or poly(lactic-co-glycolic acid) is copolymer, is synthesized by means of random ring-opening copolymerization of two different monomers, the cyclic dimers ( , -dioxane- , -diones) of glycolic acid and lactic acid. during polymerization, successive monomeric units (of glycolic or lactic acid) are linked together in plga by ester linkages, thus yielding a linear, aliphatic polyester as a product moore et al. ( ) degradation products are naturally occurring metabolites and readily absorbed by neighboring cells generate acidic environment and effect the stability yersinia pestis, hiv gp dominant th response block copolymer micelles are colloidal particles with a size around - nm, which are currently under investigation as carriers for delivery of biopharmaceuticals. in contrast to cationic polymeric systems, nonionic polymers enhance gene expression through mechanisms, which most likely do not involve dna condensation and facilitated transport within cells. adjuvant-active nonionic block copolymers that are flexible, linear structures, flanked on both ends by hydrophilic polyoxyethylene (poe) with a core of hydrophobic polyoxypropylene (pop) with variable ratios (newman et al. ) . the block copolymers are useful as general surfactants and display enhanced biological efficacy as vaccine adjuvants. osmolarity, ph and buffer salts mainly affected the size and morphology of the particles. molecular weight and formulation mainly affected titer and isotype of antibody. jain et al. evaluated a system of combined poly(lactic acid) (pla) and poly(ethylene glycol) (peg) for the delivery of a recombinant hepatitis b surface antigen (hbsag). pla forms the hydrophobic core in an aqueous medium, which controlling the release of the antigen as it degrades into lactic acid. an outer shell form by peg allows for prolonged release patterns and enhanced mucosal uptake to occur (jain et al. ). hunter et al. ( ) showed that the adjuvant activity of block copolymers varies with the lengths of the chains of polyoxypropylene (pop) and polyoxyethylene (poe). pluronic block copolymers have been used extensively in a variety of pharmaceutical formulations like low molecular mass drugs and polypeptides. kabanov et al. ( ) described that these molecules can modify the biological response during gene therapy in the skeletal muscle, resulting in an enhancement of the transgene expression and therapeutic effect of the transgene. block copolymers were recently used to promote gene delivery of plasmid encoding a food allergen, bovine beta-lactoglobulin. tetronic based block copolymers have decreased blg-specific ige concentrations and reduced local inflammatory response (adel-patient et al. ) . similarly, triblock copolymers consisting of three alternating hydrophobic and hydrophilic segments are also used to delivery genetic materials. biodegradable and nontoxic triblock copolymers of pla-peg-pla and plga-peg-plga were also utilized micellar carriers for delivery of encapsulated plasmid pcdna . (+)-ma against hcv. developed carrier system has provided long-term better adjuvant effect with no side effects (yang et al. ) . similarly, copolymers of a hydrophilic poly(ethylene glycol) block and a cationic poly(aminoethyl methacrylate) (paem) block have been used for dna vaccine delivery. synthesized polyplexes based carrier systems have induced a modest up-regulation of surface markers for dc maturation and better uptake by dcs in the draining lymph nodes (tang et al. ). further, cationic block copolymers poly(ethylene glycol) (peg) with a positively charged poly(dimethylamino)ethyl methacrylate have been synthesized and utilized for hiv- tat dna molecules. results indicated that synthesized cationic block copolymers was safe and ability to deliver genetic material for cell machinery and promising candidate for dna vaccination (caputo et al. ) . similar to cationic block polymers, nonionic block copolymers of poly(ethyleneoxide)-poly (propyleneoxide) (peo-ppo) have also been utilized dna vaccination using a beta-galactosidase (betagal) encoding plasmid (mcilroy et al. ). herpes simplex virus type- genes specifying glycoproteins gb and gd have been also delivered by nonionic block copolymers. plasmid-encapsulated block polymers have protected the mice against lethal hsv- challenge when immunization was performed via the i.m. route (baghian et al. ) . dendrimers are a unique class of polymeric nanoconstructs having highly branched, three-dimensional, nanoscale architecture with very low polydispersity and high functionality. first discovered in the early s by donald tomalia and coworkers, these hyperbranched molecules were called dendrimers. dendrimers are highly branched, synthetic spherical macromolecules with layered architectures that can be considered analogous to a globular protein. they have the potential for high loading capacities due to small diameters ( . - . nm) through mechanisms such as complexation or formation of chemical bonds at terminal branch points or other active sites (wiwattanapatapee et al. ) . in addition, the low polydispersity of dendrimers should provide reproducible pharmacokinetic behavior in contrast to that of some polymers containing fractions with vastly different molecular weight within a given sample (parekh ) . several dendrimer-based products have been approved by the fda and successfully commercialized for treatment and diagnosis of diseases, including vivagel™ (starpharma) designed as a topical microbicide, superfect ® , (qiagen pvt ltd.) used for gene transfection, and alert ticket™ (us army research lab) for anthrax detection (merdan et al. ) . in the past decade, research has increased on polyamidoamine, polyethylenimines, polylysine, polypropyleneimine, polyaryl ether, polyester, polyglycerol and their derivatives for the design and synthesis of biocompatible dendrimers. dendrimers form complexes by electrostatic interaction with all forms of nucleic acids such as dna, rna, and antisense oligonucleotides. the nature of the dendrimer-nucleic acid complexes ("dendriplexes") is dependent on the stoichiometry and concentration of the dna-phosphates, dendrimer amines, bulk solvent properties (e.g., ph, salt concentration, buffer strength), and even the dynamics of mixing. high ionic strength interferes with the binding process and affects the nature of complexes formed by the different generations, for example, highergeneration ppi dendrimers in higher concentrations form water-soluble dendriplexes, whereas the g and g ppi dendrimers lead to the formation of electroneutral complexes (tang and szoka ) . dendrimer-dna complex is formed by simply mixing the components in an aqueous solution. transfection property can be improved by the use of an excess of cationic dendrimer because the negatively charged phosphate groups on the dna neutralize the positively charged amine groups on the dendrimer through electrostatic interaction and an overall positively charged system is important in cell uptake (bielinska et al. ) . immunogenicity and efficacy of dna vaccines can be improved by physical conjugation of the pamam dendrimer to the mhc class ii-targeting peptide. therefore, dendrimers can be further explored for dna-based vaccine development against malaria parasite (pietersz et al. ). in a recent study, dendriplexes, complexes of dendrons and condensed plasmids containing the gene for protective antigen (pa) of bacillus anthracis, were encapsulated in polylactide-co-glycolide (plg) particles using the double emulsion method. studies indicated that the plg-dendriplex particles produced superior levels of anti-pa igg antibodies when compared to animals immunized with the plg particles (ribeiro et al. ). conjugation of fifth-generation polyamidoamine (g -pamam) dendrimers, a dna-loading surface, with mhc class ii-targeting peptides that can selectively deliver these dendrimers to apcs under conditions that enhance their immune stimulatory potency. dna conjugated with this platform efficiently transfected murine and human apcs in vitro. subcutaneous administration of dna-peptide-dendrimer complexes in vivo preferentially transfected dendritic cells (dc) in the draining lymph nodes, promoted generation of high affinity t cells, and elicited rejection of established tumors. taken together, our findings show how pamam-dendrimer complexes can be used for high transfection efficiency and effective targeting of apcs in vivo, conferring properties essential to generate effective dna vaccines. multiple antigenic peptide (map) dendrimer system is being used for vaccine and immunization purposes. map-based delivery can prepare by addition of multiple immune-functional components, like b/t-cell epitopes, cell-penetrating peptides, and lipophilic moieties or by controlled synthesis of nanomaterials like micelles, dendrimers, and nanoparticles (fujita and taguchi ) . a tetravalent multiple antigen peptide (map) dendrimer with four identical branches of a c-terminal peptide sequence of the rat gh-bp (gh-bp - ) was synthesized and used as an immunogen in rabbits. the tetravalent rat gh-bp - map dendrimer served as an effective immunogenic antigen in eliciting specific antibodies (aguilar et al. ). similar to map dendrimers, glycopeptide dendrimers containing both carbohydrates and peptides can be also used in delivery of vaccine components (niederhafner et al. ; sebestik et al. ) . the encapsulated dendrimer-nucleic acid complex within a lipophilic shell known as dendrosomes. these are novel vesicular, spherical, supramolecular entities and possess negligible hemolytic toxicity and higher transfection efficiency. dendrosome are reported to be completely nontoxic both in vitro as well as in vivo. poly (propyleneimine) dendrosome-based genetic immunization found to be highly effective against hepatitis b when compared to dendrimer-plasmid dna complex, and the results indicate that dendrosomes hold great potential in dna vaccination. in dendrosomes, the poly(propyleneimine) dendrimer-dna complex is largely protected by multilamelarity of the vesicles. it has been reported that polyamidoamine dendrimer-based dendrosomes are efficient systems for the delivery of s sirna targeting e /e oncogenes in cervical cancer (pourasgari et al. ). in vitro superior transfection efficiency displayed by pamam dendrosomes as comparison to other nonviral gene delivery vectors. nontoxic self-assembled dendritic spheroidal nanoparticles (den ) have been used for the delivery of pcmv-betv loaded dendritic spheroidal nanoparticles (den ) have shown low toxicity, enhanced transfection efficiency, and improved the immune response against birch pollen allergy (balenga et al. ) . similarly, efficiency of dendrosome (a gene porter) is assessed in transferring recombinant human rotavirus vp cdna. studies revealed that dendrosome has lower cytotoxicity and better transfectivity in a , a human lung cell line (pourasgari et al. ). dendrosome has been used to deliver the dna vaccines encoding hiv- p -gp gene. studies have proved the efficacy of this carrier for the delivery of recombinant plasmids construct (roodbari et al. ) . polymersomes are self-assembled polymeric colloidal vesicular systems containing aqueous inner core. polymersomes are made up from amphiphilic block copolymers that allow polymersomes to stably encapsulate or integrate a broad range of active molecules. the aqueous core can be utilized for the encapsulation of therapeutic hydrophilic molecules and the membrane can integrate hydrophobic drugs within its hydrophobic part. further, the brush-like surface properties of the polymersome can provide better biocompatibility and blood circulation times. these systems have better loading efficiency, stabilities and provide sustained, controlled release of encapsulated therapeutics. further, these systems have also been used to deliver biotherapeutics, especially peptides, proteins, and nucleic acids to site-specific cellular environment due to escape from endolysosomes (levine et al. ; christian et al. ). amphiphilic diblock copolymer of poly (oligoethylene glycol methacrylate)-block-poly( -(diisopropylamino)ethyl methacrylate in association with tannic acid forms dna-loaded polymersomes. developed systems have demonstrated better cytosolic release of encapsulated nucleic acid materials (lomas et al. ) . further, calcein-loaded polymersomes have also observed for their cytosolic delivery within dendritic cell (scott et al. ) . similarly, poly(g-benzyl-l-glutamate)-k (pblg -k) polymersomes have been used for delivery of influenza hemagglutinin antigen. the immunogenicity and adjuvanticity of developed polymerosomes was better for administered the influenza antigen. in future, this nanostructured polymeric vesicular system may have huge potential for delivery of protein and dna vaccines. emulsions can be manufactured as water-in-oil (w/o) or oil-in-water (o/w) particulate carrier systems. emulsion carrier systems are similar in size to pathogens and taken up by epithelial or m cells in the mucosal surfaces for successive delivery of the vaccine component to apcs and lymphoid tissue. a nanoemulsion formulation of intranasal hepatitis b vaccine showed improved vaccine efficacy, stability and ease of distribution (makidon et al. ) . multiple emulsion formulations can also be used as vaccine carrier systems due to its longer stability and high entrapment efficiency of protein antigens without damage during emulsification procedures. types of surfactants, processing methods and stabilizers is requisite for making stable multiple-emulsions (hanson et al. ) . the emulsion adjuvant mf immunostimulator has been shown to result in the recruitment of antigen-presenting cells (apcs) to the site of injection and to increased uptake of soluble antigen by the apcs. it has been formulated by a simple mixing of the antigen with the adjuvant and has shown excellent compatibility with a variety of subunit antigens. mf shows strong immunogenicity as comparison to other adjuvant is clearly seen in pre-clinical data published by ott et al. they reported that when immunized guinea pigs and goat with glycoprotein d of herpes simplex virus (hsv) type in the presence of mf showed a -fold and ninefold increases in antibody titers compared to aluminum hydroxide, respectively (ott et al. ). an oil-in-water (o/w) emulsion, syntex adjuvant formulation (saf) is an effective adjuvant composed of a muramyl dipeptide derivative (threonyl-mdp). threonyl-mdp demonstrated a lack of side effects (pyrogenicity, uveitis, adjuvant-induced arthritis) and increased adjuvant activity. saf adjuvant used with a variety of antigens, such as influenza and malaria, and showed both cellmediated and humoral immune responses. saf, or a suitable equivalent, provides an excellent tool for vaccine research (lidgate et al. ; lidgate et al. ) . there are several different types of montanide™, including isa v, , . isa and have been used in human's vaccine formulations, while isa and v have been used only in veterinary vaccine formulations. they are composed of metabolizable squalene-based oil with mannide monooleate emulsifier and permit antigens to be released more rapidly. the montanide emulsions induce high antibody titers and ctl responses due to the formation of a depot at the site of injection. these emulsions have been used as vaccines against malaria, hiv and various cancers and found to be safe and fairly well tolerated (lawrence et al. ; toledo et al. ). various physical delivery methods are being heavily investigated because of direct transfection of apcs with the dna vaccine (porgador et al. ). the transcutaneous microneedle has the ability to bypass the stratum corneum layer of the skin, thus reaching langerhans cells-the apcs of the skin. jet-injection mechanical devices deliver dna vaccines into the viable epidermis and increased efficacy in the prevention and/or therapy of infectious diseases, allergic disorders and cancer (chen et al. ; imoto and konishi ; roberts et al. ) . the tattooperforating needle device has been used to puncture the skin and transfer dna into skin-associated cells. the bundles of fine metal needles that oscillate at a constant high frequency have shown better expression of reporter genes in mice and induction of immune responses. electroporation has been extensively studied to deliver therapeutic genes that encode a variety of hormones, cytokines, enzymes or antigens in large animal species such as dogs, pigs, cattle and nonhuman primates. several different strategies of this technology are being pursued. however, too little is currently known about several of these devices and much additional research in this area is warranted (van drunen littel-van den hurk et al. ; roos et al. ; hirao et al. ). nanotechnology is the development of engineered devices due to their small size at the micromolecular level in the nanometer range and large surface area, which enhances their action for early diagnosis of cancer and infectious diseases. advances in nanotechnology have also proved to be beneficial in therapeutic fields such as drug discovery, drug delivery and gene/protein delivery. this concept has been found to be useful in developing nanovaccines using different routes of administration like oral, nasal and parenteral. the oral route is the most popular and convenient route of administration. oral delivery refers to absorption from the buccal through the rectal mucosa. several barriers associated with genetic vaccination through the oral are generally attributed to (a) low permeability across biological membranes, (b) harsh gastric environment, (c) hepatic first-pass metabolism, and (d) chemical instability. the major drawback with oral route of administration is a higher concentration and is required for the vaccine to be effective due to dilution during the transport of the vaccine through the gastrointestinal tract. to date, most gene delivery strategies have concentrated on the parenteral route of delivery and oral administration has been largely ignored. different nano-and microparticulate delivery systems using natural and synthetic lipid and polymers have been utilized to improve the stability and immunogenicity of oral dna vaccines (bhavsar and amiji ) . oral vaccination with dna-chitosan nanoparticles has appeared interesting because of their great stability and the ease of target accessibility, besides chitosan immunostimulatory properties. studies demonstrated that % of protection against parasite infection after delivery chitosan nanoparticles loaded with dna encoding rho -gtpase protein of schistosoma mansoni (oliveira et al. ) . similarly, chitosan nanoparticles are used for dna vaccine against vibrio anguillarum through oral route. studies revealed that chitosan-dna (pvaomp ) complex showed moderate protection against experimental v. anguillarum infection after oral vaccination in asian sea bass (rajesh kumar et al. ) . the orally administered tresylmonomethoxypolyethylene glycol (tmpeg) grafted liposome complexes with modified vaccinia virus ankara (mva(iiib/beta-gal) is also capable of delivering the transgenes to mucosal tissues and enhances the env-specific cellular and humoral immune responses after repeated oral immunization of balb/c mice (naito et al. ) . mannosylated niosomes loaded with hepatitis dna have shown humoral (both systemic and mucosal) and cellular immune response upon oral administration (jain et al. ) . chitosan-coated and polyplex-loaded liposomes (plls) containing plasmid prc/cmv-hbs are developed for oral delivery of vaccines specifically for targeting to peyer's patch. chitosan-coated pll demonstrated better uptake of encapsulated dna to the distal intestine and provide better stability from enzymatic degradation (channarong et al. ) . the nasal route has been chiefly employed for producing local action on the mucosa. this route has a number of advantages, such as the high permeability of the nasal epithelium, which allows a higher molecular mass cut-off for permeation of approximately , da, as well as the rapid drug absorption rate. accurate and repeated dispensing of vaccine, mucociliary clearance, presence of peptidases, proteases and nuclease enzymes in the mucus or associated with nasal membrane, variation in extent of absorption with the mucus secretion and mucus turnover and deposition of the formulated vaccine to all areas of the nasal mucosa (especially lymphoid tissues), potential of uptake of vaccine formulations by the primary olfactory nerves in the nasal cavity, local irritation and unpleasant taste from concentrated drug reaching the mouth are major challenges associated with intranasal delivery of vaccines (oliveira et al. ; sharma et al. ). these problems can be overcome by design of appropriate antigen carriers. nanocarriers for nasal vaccines are able to facilitate the transport of the associated antigen across the nasal epithelium, thus leading to efficient antigen presentation to the immune system and provide the protection and stability of encapsulated genetic materials (koping-hoggard et al. ) . further, use of mucoadhesive agents offers a strategy for the facilitation of increased residence time and increased vaccine efficacy (alpar et al. ) . polycarbophil (pc) or polyethylene oxide (peo)-based in-situ mucoadhesive polymers have demonstrated better nasal absorption of plasmid dna (park et al. ) . several studies have proven that wide applicability of chitosan nanoparticles for the nasal delivery of dna vaccines like severe acute respiratory syndrome coronavirus (sars-cov) (raghuwanshi et al. ) , pneumococcal surface antigen a (psaa) (xu et al. ) , hepatitis b antigen-encoding plasmid (khatri et al. ) , and dna plasmid-expressing epitopes of respiratory syncytial virus (iqbal et al. ) . further, several modification on the chitosan polymers have also been made to improve the potential of chitosan nanoparticles for nasal administration of dna vaccines like preparation of low molecular weight chitosan, development of water soluble chitosan (n-trimethyl chitosan), etc. blends of poly(lactic-co-glycolic acid) (plga) and polyethylene oxide (peo) have exhibited the capacity to associate and release plasmid dna in a controlled manner. results showed that dna-loaded nanoparticles elicit significantly pronounced immune response compared to the naked plasmid dna for up to weeks (csaba et al. ) . dry-powder influenza virosomes-based vaccines have also been advantageous for mucosal immunization (de jonge et al. ). needle-free nasal immunization, using nanoemulsion is made of soya bean oil, alcohol, water and detergents emulsified into droplets of nm, has been reported to be a safe and effective hepatitis b vaccine (makidon et al. ) . the release of liquid or particles into the airflow enters one nostril via a sealing nozzle and exits through the other nostril and minimizes the risk and problems related to deposition of particles in the lung, which occurs during conventional inhalation from a nebulizer and increases the delivery of particles to the posterior part of the nasal mucosa. encapsulation of the antigen into bioactive nanoparticles is a promising approach to nasal vaccine delivery (slutter et al. ) . the ocular route holds immense potential for peptides/proteins intended for pathological ophthalmologic conditions. the eye mucosa is a possible route for mucosal vaccine because it is an important entry point for environmental antigens and infectious materials occupying most of the external ocular surface (streilein et al. ) . lymphoid follicles are found in close association with the epithelium of the conjunctival mucosa in humans, rabbits, guinea pigs, dogs, pigs, and many other mammals (chodosh et al. (seo et al. ) . ocular mucosal delivery of peptide epitopes of herpes simplex virus (hsv- ) glycoprotein d (gd) has mixed with oligodeoxy nucleotides containing unmethylated cpg motifs (cpg ). results suggested enhanced local and systemic immune response after multi-instillation of gd peptide epitopes with cpg adjuvants (nesburn et al. ) . ocular mucosal administration of iron nanoparticles with glutamic acid containing dna vaccine herpes stromal keratitis (prsc-gd-il- ) have confers protection against mucosal challenge with herpes simplex virus type in mice (hu et al. ) . vaginal mucosa is a portal of entry to many viral and bacterial pathogens. vaginal route serves as a potential site of drug administration for local and systemic absorption of therapeutically important molecules, proteins, peptides, small interfering rnas, oligonucleotides, antigens, vaccines and hormones (hussain and ahsan ) . it is one of alternative site for the systemic delivery of protein drugs because of the relatively high permeability of the vaginal epithelium, by passage of the hepatic first-pass metabolism, large surface area and rich blood supply (gupta et al. (gordon et al. ) . thermo-sensitive mucoadhesive vaginal vaccine delivery systems have also been tested for the local and systemic antibody responses to hpv l virus-like particles (park et al. ) . vaginal delivery of vaccines which is associated with vaginal infection could be better alternative to induce an immune response in the genital mucosa capable of controlling the entry of the pathogen. noninvasive gene delivery approaches could be able to deliver and express naked plasmid dna to tissue-specific localized delivery to skin. there are several advantages of needle-free noninvasive gene administration such as limited toxicity, potential cell receptor-independent uptake, minimal dna size restrictions, and the potential for multiple treatments via a relatively uncomplicated administration modality, thus improving patient compliance. topically applied formulation, especially nanosystems have been shown to enter skin, accumulate in hair follicles, diffuse via dendritic cells to draining lymph nodes, and elicit antigen-specific humoral and cell-mediated immunity (nasir ). a number of methods have been developed to perform noninvasive topical gene delivery, which includes passive diffusion of genetic materials between a skin patch and skin, as well as active processes such as iontophoresis, sonophoresis, electroporation, and chemically enhanced diffusion (mehier-humbert and guy ). topical vaccination has been achieved using topical application of naked dna with or without tape stripping and dna/lipid-based complex such as liposomes, niosomes, transfersomes, or microemulsion (cui and sloat ) . ethanol-in-fluorocarbonbased microemulsion has been for topically delivery of anthrax protective antigen (pa) protein-encoding dna vaccine (pgpa). pgpa-loaded microemulsion has significantly enhanced the anti-pa antibody responses (cui and sloat ) . similarly, dna delivery by novel lipid-based biphasic delivery system has significant deliver plasmid dna into the "viable" layers of skin (foldvari et al. ) . plasmid dna-encoding hepatitis b surface antigen (hbsag)-loaded cationic transfersomes are also utilized for topical immunization. results revealed that dna-loaded cationic transfersomes elicited significantly higher anti-hbsag antibody titer and cytokines level as compared to naked dna. it was also observed that topical application of dna-loaded cationic transfersomes elicited a comparable serum antibody titer and endogenous cytokines levels as produced after intramuscular recombinant hbsag administration (mahor et al. ) . -or -nm sized polystyrene nanoparticles have been studied to target active compounds to the hair follicle and may result in a better penetration and higher efficiency of compound uptake by skin resident cells. studies demonstrated that and nm nps and modified vaccinia ankara (mva) expressing the green-fluorescent protein penetrated deeply into hair follicles and uptake by apcs and transport to the draining lymph nodes (mahe et al. ). nanoengineered genetic vaccine formulation has been developed for topical immunization comprising of emulsifying wax (oil phase), ctab (cationic surfactant), mannan (dc ligand), dioleoylphosphatidylethanolamine (endosomolytic agents), and cholesterol. all pdna-coated nanoparticles, especially the mannan-coated pdna-nanoparticles with dope, have shown significant immune response (igg titers; -fold over "naked" pdna alone) (cui and mumper ) . diffusion patches and tape stripping techniques are used for delivery of small (< da) and large molecules, respectively. liquid jet injector is an approach in which dna vaccine is delivered around the langerhans cells by a high-speed injector. (chen et al. ) reported that particle-mediated gene-gun dna immunization use similar mechanical devices to deliver dna vaccines into viable epidermis (chen et al. ) . microneedle arrays is a set of needles of microscale length with their nanoscale tips coated with dna and can accurately, efficiently and safely deliver biomolecules to the viable cells of the epidermis. recently, tran et al. ( ) developed a unique nanoliposomal ultrasound-mediated device for delivering small interfering rna (sirna) specifically targeting melanocytic tumors present in the skin and they observed that decrease early melanocytic lesion development in the skin and prevent the spread of cutaneous metastases of melanoma (tran et al. ) . these results suggested that skin may provide an appealing, noninvasive route of delivery for dna vaccines and other therapeutic genes. table represents positive and negative aspects of various routes of administration, which are very helpful for selection of particular route. novel vaccine carriers, adjuvant, vehicles, and particle-based delivery strategies are being evaluated in a variety of vaccines, including those against diseases such as cancer, malaria, aids, hepatitis, etc., in which a cellular and/or mucosal immune response is desired. various immunity responses were generated by different adjuvant like mf and mpl ® generated th responses, vlps, virosomes, nondegradable nanoparticles, and liposomes generated cellular immune responses in humans. viral vectors, iscoms and montanide™ isa , and various nanoparticulate immunopotentiators and antigen delivery vehicles have shown ctl responses. the desirable responses can be achieved by using combination of various adjuvants. systemic antibodies produced in humans when viral-vectored vaccines as well as proteasomes given in. the clinical trials required for vaccine approval are often very long and difficult. furthermore, since many vaccines are often administered to healthy individuals, and frequently to infants, it is critical that they are proven safe and well tolerated in nonhuman primates before entering human trials. while the development of novel vaccine delivery systems and adjuvant has been aided by nanotechnology, it must be necessary to perceived potential problems such as their high surface area and reactivity, the ability to cross biological membranes, slow biodegradability of some materials, its safety and tolerability before its approval. many challenges must be met before new classes of vaccines become available like ability to stimulate humoral, cellular and mucosal immune responses, longer duration response, easily metabolized of vaccine components in body, cost-effective production, and lesser risk and less invasive approaches for the administration of vaccinations. as these challenges are met, the prevention and therapy of many previously untreatable diseases should become increasingly possible. block copolymers have differing adjuvant effects on the primary immune response elicited by genetic immunization and on further induced allergy map dendrimer elicits antibodies for detecting rat and mouse gh-binding proteins comparison of immune response generated against japanese encephalitis virus envelope protein expressed by dna vaccines under macrophage associated versus ubiquitous expression promoters dna vaccines: technology and application as antiparasite and anti-microbial agents liposomes as immunological adjuvants yersinia enterocolitica as a vehicle for a naked dna vaccine encoding brucella abortus bacterioferritin or p antigen biodegradable mucoadhesive particulates for nasal and pulmonary antigen and dna delivery aluminium compounds as adjuvants for vaccines and toxoids in man: a review vaccine delivery: lipid-based delivery systems carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens protective immunity against lethal hsv- challenge in mice by nucleic acid-based immunisation with herpes simplex virus type- genes specifying glycoproteins gb and gd protective efficiency of dendrosomes as novel nano-sized adjuvants for dna vaccination against birch pollen allergy iscoms (immunostimulating complexes): the first decade transcriptional control of the rna-dependent rna polymerase of vesicular stomatitis virus preparation, characterization, cytotoxicity and transfection efficiency of poly(dl-lactide-co-glycolide) and poly(dl-lactic acid) cationic nanoparticles for controlled delivery of plasmid dna new generation of liposomes called archaeosomes based on natural or synthetic archaeal lipids as innovative formulations for drug delivery polymeric nano-and microparticle technologies for oral gene delivery dna complexing with polyamidoamine dendrimers: implications for transfection alginate coated chitosan nanoparticles are an effective subcutaneous adjuvant for hepatitis b surface antigen a very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus liposome/dna complexes coated with biodegradable pla improve immune responses to plasmid encoding hepatitis b surface antigen in vivo transfection of murine lungs with a functioning prokaryotic gene using a liposome vehicle virus-like particles as particulate vaccines preclinical safety assessment considerations in vaccine development micellar-type complexes of tailor-made synthetic block copolymers containing the hiv- tat dna for vaccine application effect of vesicle size on tissue localization and immunogenicity of liposomal dna vaccines hot spots of retroviral integration in human cd + hematopoietic cells virus-like particles: flexible platforms for vaccine development gene therapy of muscular dystrophy development and evaluation of chitosan-coated liposomes for oral dna vaccine: the improvement of peyer's patch targeting using a polyplex-loaded liposomes needle-free epidermal powder immunization contribution of poly(amino acids) to advances in pharmaceutical biotechnology comparative anatomy of mammalian conjunctival lymphoid tissue: a putative mucosal immune site enhancement of t helper type immune responses against hepatitis b virus core antigen by plga nanoparticle vaccine delivery polymersome carriers: from self-assembly to sirna and protein therapeutics the iscom structure as an immune-enhancing moiety: experience with viral systems apoptosis in hiv infection: protective role of il- iscoms and other saponin based adjuvants plga:poloxamer and plga:poloxamine blend nanostructures as carriers for nasal gene delivery topical immunization using nanoengineered genetic vaccines the effect of co-administration of adjuvants with a nanoparticle-based genetic vaccine delivery system on the resulting immune responses microparticles and nanoparticles as delivery systems for dna vaccines topical immunization onto mouse skin using a microemulsion incorporated with an anthrax protective antigen protein-encoding plasmid inulin sugar glasses preserve the structural integrity and biological activity of influenza virosomes during freeze-drying and storage delivery of messenger rna using poly(ethylene imine)-poly(ethylene glycol)-copolymer blends for polyplex formation: biophysical characterization and in vitro transfection properties polyethyleneimine-based gene therapy by inhalation priming with chlamydia trachomatis major outer membrane protein (momp) dna followed by momp iscom boosting enhances protection and is associated with increased immunoglobulin a and th cellular immune responses viruses as vaccine vectors for infectious diseases and cancer dendrimer biocompatibility and toxicity cationic liposomes for gene delivery: novel cationic lipids and enhancement by proteins and peptides encapsulation of antigenic extracts of salmonella enterica serovar. abortusovis into polymeric systems and efficacy as vaccines in mice an adjuvant vaccine against infectious canine hepatitis liposomal nanomedicines gene delivery into human skin in vitro using biphasic lipid vesicles immune response to rabies vaccine in water-in-oil emulsion safety and immunogenicity of a proteosome-shigella flexneri a lipopolysaccharide vaccine administered intranasally to healthy adults current status of multiple antigen-presenting peptide vaccine systems: application of organic and inorganic nanoparticles cellulose acetate butyrate-ph/ thermosensitive polymer microcapsules containing aminated poly(vinyl alcohol) microspheres for oral administration of dna unique features of a ph-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes dna vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations opportunities for the use of lentiviral vectors in human gene therapy cationic liposome-mediated gene transfer scaffold: a novel carrier for cell and drug delivery block copolymer micelles: preparation, characterization and application in drug delivery protective immune responses to a multi-gene dna vaccine against staphylococcus aureus in vivo induction of cd + t cell responses by antigens covalently linked to synthetic microspheres does not require adjuvant long-term safety analysis of preventive hiv- vaccines evaluated in aids vaccine evaluation group niaid-sponsored phase i and ii clinical trials a sequential study of incomplete freund's adjuvantinduced peritonitis in atlantic cod immunopotentiating reconstituted influenza virus virosome vaccine delivery system for immunization against hepatitis a construction of hybrid viruses containing sv and lambda phage dna segments and their propagation in cultured monkey cells preventive hiv type vaccine clinical trials: a regulatory perspective targeting the vaginal mucosa with human papillomavirus pseudovirion vaccines delivering simian immunodeficiency virus dna liposome-mediated dna vaccination aluminum compounds as vaccine adjuvants adjuvants for human vaccines -current status, problems and future prospects adjuvants -a balance between toxicity and adjuvanticity adjuvant properties of aluminum and calcium compounds adjuvant properties of non-phospholipid liposomes (novasomes) in experimental animals for human vaccine antigens exploring novel approaches to vaginal drug delivery codon bias and heterologous protein expression nanoscale double emulsions stabilized by singlecomponent block copolypeptides wssv ie promoter is more efficient than cmv promoter to express h hemagglutinin from influenza virus in baculovirus as a chicken vaccine haemophilus influenzae type b conjugate vaccines: a review of efficacy data the combined cta -dd/iscom adjuvant vector promotes priming of mucosal and systemic immunity to incorporated antigens by specific targeting of b cells structure and properties of aluminum-containing adjuvants immune responses and protection obtained by oral immunization with rotavirus vp and vp dna vaccines encapsulated in microparticles immunostimulatory dna as a vaccine adjuvant intradermal/subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques origin and steady-state turnover of class ii mhcbearing dendritic cells in the epithelium of the conducting airways an ocular mucosal administration of nanoparticles containing dna vaccine prsc-gd-il- confers protection against mucosal challenge with herpes simplex virus type in mice adjuvant activity of non-ionic block copolymers. iv. effect of molecular weight and formulation on titre and isotype of antibody the vagina as a route for systemic drug delivery needle-free jet injection of a mixture of japanese encephalitis dna and protein vaccines: a strategy to effectively enhance immunogenicity of the dna vaccine in a murine model nasal delivery of chitosan-dna plasmid expressing epitopes of respiratory syncytial virus (rsv) induces protective ctl responses in balb/c mice release characteristics of a model plasmid dna encapsulated in biodegradable poly(ethylene glycol fumarate)/acrylamide hydrogel microspheres mannosylated niosomes as adjuvant-carrier system for oral genetic immunization against hepatitis b synthesis, characterization and evaluation of novel triblock copolymer based nanoparticles for vaccine delivery against hepatitis b the coming of age of virus-like particle vaccines pluronic block copolymers as novel polymer therapeutics for drug and gene delivery enhanced transfection of tumor cells in vivo using "smart" phsensitive tat-modified pegylated liposomes pluronic f enhances the effect as an adjuvant of chitosan microspheres in the intranasal delivery of bordetella bronchiseptica antigens containing dermonecrotoxin multivesicular liposome (depofoam) technology for the sustained delivery of insulin-like growth factor-i (igf-i) immunogenicity in mice of a cationic microparticle-adsorbed plasmid dna encoding japanese encephalitis virus envelope protein vaccine potential of cytosolic proteins loaded fibrin microspheres of cryptococcus neoformans in balb/c mice plasmid dna loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis b comparing the effect of il- genetic adjuvant and alum non-genetic adjuvant on the efficiency of the cocktail dna vaccine containing plasmids encoding sag- and rop- of toxoplasma gondii nasal immunization with plasmid dna encoding p protein and immunostimulatory complexes elicits nontypeable haemophilus influenzaespecific long-term mucosal immune responses in the nasopharynx nanoparticles as carriers for nasal vaccine delivery dna vaccines: ready for prime time? an hdac inhibitor enhances the antitumor activity of a cmv promoter-driven dna vaccine parenteral nutrition in the malnourished: dialysis, cancer, obese, and hyperemesis gravidarum patients effect of vaccination with recombinant asexualstage malaria antigens on initial growth rates of plasmodium falciparum in non-immune volunteers glycosaminoglycan-resistant and ph-sensitive lipid-coated dna complexes produced by detergent removal method polymersomes: a new multi-functional tool for cancer diagnosis and therapy assembly of electroactive layer-by-layer films of myoglobin and ionomer poly (ester sulfonic acid) enhancement of a hepatitis b dna vaccine potency using aluminum phosphate in mice formulation of vaccine adjuvant muramyldipeptides. . processing optimization, characterization, and bioactivity of an emulsion vehicle sterile filtration of a parenteral emulsion gene therapy for renal disorders: what are the benefits for the elderly? poly(cationic lipid)-mediated in vivo gene delivery to mouse liver dna immunization in combination with the immunostimulant monophosphoryl lipid a polymersome-loaded capsules for controlled release of dna enhanced plasmid dna delivery using anionic lpdii by listeriolysin o incorporation interaction between fibronectin-bearing surfaces and bacillus calmette-guerin (bcg) or gelatin microparticles pmma particle-mediated dna vaccine for cervical cancer development of an antigen-presenting cell-targeted dna vaccine against melanoma by mannosylated liposomes plasmid dna containing multiple cpg motifs triggers a strong immune response to hepatitis b surface antigen when combined with incomplete freund's adjuvant but not aluminum hydroxide immunoliposomes as targeted delivery vehicles for cancer therapeutics (review) nanoparticle-based targeting of vaccine compounds to skin antigen-presenting cells by hair follicles and their transport in mice cationic transfersomes based topical genetic vaccine against hepatitis b pre-clinical evaluation of a novel nanoemulsion-based hepatitis b mucosal vaccine plasmid dna vaccines: tissue distribution and effects of dna sequence, adjuvants and delivery method on integration into host dna incorporation of hepatitis-b surface antigen (hbsag) into liposomes lipid matrix-based vaccines for mucosal and systemic immunization targeting immune response induction with cochleate and liposome-based vaccines structure-activity relationships of poly(llysines): effects of pegylation and molecular shape on physicochemical and biological properties in gene delivery dna released from dying host cells mediates aluminum adjuvant activity gene delivery to the eye using adeno-associated viral vectors spotlight on quadrivalent human papillomavirus (types , , , ) recombinant vaccine(gardasil(r)) in the prevention of premalignant genital lesions, genital cancer, and genital warts in women dna/amphiphilic block copolymer nanospheres promote low-dose dna vaccination retroviral vectors for human gene delivery physical methods for gene transfer: improving the kinetics of gene delivery into cells prospects for cationic polymers in gene and oligonucleotide therapy against cancer anionic ph-sensitive pegylated lipoplexes to deliver dna to tumors immunization with a soluble recombinant hiv protein entrapped in biodegradable microparticles induces hiv-specific cd + cytotoxic t lymphocytes and cd + th cells production of escherichia coli heat labile toxin (lt) b subunit in soybean seed and analysis of its immunogenicity as an oral vaccine oral vaccination with modified vaccinia virus ankara attached covalently to tmpeg-modified cationic liposomes overcomes pre-existing poxvirus immunity from recombinant vaccinia immunization confocal and probe microscopy to study gene transfection mediated by cationic liposomes with a cationic cholesterol derivative application of a fusiogenic peptide gala for intracellular delivery nanotechnology in vaccine development: a step forward local and systemic b cell and th responses induced following ocular mucosal delivery of multiple epitopes of herpes simplex virus type glycoprotein d together with cytosine-phosphate-guanine adjuvant immunological and formulation design considerations for subunit vaccines use of nonionic block copolymers in vaccines and therapeutics glycopeptide dendrimers. part i polyhydroxyalkanoates: materials for delivery systems induction of antibody response to human tumor antigens by gene therapy using a fusigenic viral liposome vaccine intranasal vaccines for protection against respiratory and systemic bacterial infections oral vaccination based on dna-chitosan nanoparticles against schistosoma mansoni infection hamster cell culture rabies vaccine design and evaluation of a safe and potent adjuvant for human vaccines biodegradable nanoparticles for drug and gene delivery to cells and tissue cochleate lipid cylinders: formation by fusion of unilamellar lipid vesicles the advance of dendrimers -a versatile targeting platform for gene/drug delivery in situ gelling and mucoadhesive polymer vehicles for controlled intranasal delivery of plasmid dna enhanced mucosal and systemic immune responses following intravaginal immunization with human papillomavirus l virus-like particle vaccine in thermosensitive mucoadhesive delivery systems archaeosome immunostimulatory vaccine delivery system archaeobacterial ether lipid liposomes (archaeosomes) as novel vaccine and drug delivery systems anionic liposomal delivery system for dna transfection iscomatrix adjuvant for antigen delivery liposome-entrapped plasmid dna: characterisation studies improving vaccine delivery using novel adjuvant systems structure and design of polycationic carriers for gene delivery predominant role for directly transfected dendritic cells in antigen presentation to cd + t cells after gene gun immunization low cytotoxicity effect of dendrosome as an efficient carrier for rotavirus vp gene transferring into a human lung cell line: dendrosome, as a novel intranasally gene porter polycation-dna complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids monophosphoryl lipid a obtained from lipopolysaccharides of salmonella minnesota r . purification of the dimethyl derivative by high performance liquid chromatography and complete structural determination dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein potential use of chitosan nanoparticles for oral delivery of dna vaccine in asian sea bass (lates calcarifer) to protect from vibrio (listonella) anguillarum enhanced folate receptor mediated gene therapy using a novel phsensitive lipid formulation region specific and worldwide distribution of collagen-binding m proteins with parf motifs among human pathogenic streptococcal isolates archaeosomes based on synthetic tetraether-like lipids as novel versatile gene delivery systems plga-dendron nanoparticles enhance immunogenicity but not lethal antibody production of a dna vaccine against anthrax in mice polymeric particles in vaccine delivery clinical safety and efficacy of a powdered hepatitis b nucleic acid vaccine delivered to the epidermis by a commercial prototype device dna vaccines for viral infections: basic studies and applications immune responses against a new hiv- p -gp /pcaggs-il- dna vaccine in balb/c mice enhancement of cellular immune response to a prostate cancer dna vaccine by intradermal electroporation live attenuated aids viruses as vaccines: promise or peril? purification of human and avian influenza viruses using cellulose sulfate ester (cellufine sulfate) in the process of vaccine production the use of adjuvants in studies on influenza immunization. ii. increased antibody formation in human subjects inoculated with influenza virus vaccine in a water in-oil emulsion monophosphoryl lipid a enhances both humoral and cell-mediated immune responses to dna vaccination against human immunodeficiency virus type immunostimulatory colloidal delivery systems for cancer vaccines hepatitis b virus small surface antigen particles are processed in a novel endosomal pathway for major histocompatibility complex class irestricted epitope presentation dendritic cell activation and t cell priming with adjuvant-and antigen-loaded oxidation-sensitive polymersomes peptide and glycopeptide dendrimers and analogous dendrimeric structures and their biomedical applications eye mucosa: an efficient vaccine delivery route for inducing protective immunity pharmaceutical aspects of intranasal delivery of vaccines using particulate systems novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte-selective gene transfer in human hepatoma hepg cells effects of various adjuvants on efficacy of a vaccine against streptococcus bovis and lactobacillus spp. in cattle adjuvanticity of stealth liposomes on the immunogenicity of synthetic gp epitope of hiv- recombinant mouse cytomegalovirus expressing a ligand for the nkg d receptor is attenuated and has improved vaccine properties rational design of nasal vaccines oral plasmid dna delivery systems for genetic immunisation heterodimeric barnase-barstar vaccine molecules: influence of one versus two targeting units specific for antigen presenting cells immune deviation in relation to ocular immune privilege immunogenic properties of the salmonella atypical fimbriae in balb/c mice the influence of polymer structure on the interactions of cationic polymers with dna and morphology of the resulting complexes genetic immunization is a simple method for eliciting an immune response well-defined block copolymers for gene delivery to dendritic cells: probing the effect of polycation chain-length the porcine circovirus type capsid gene promoter improves antigen expression and immunogenicity in a hiv- plasmid vaccine methylation-dependent t cell immunity to mycobacterium tuberculosis heparin-binding hemagglutinin secretory immune responses in the mouse vagina after parenteral or intravaginal immunization with an immunostimulating complex (iscom) genetic targeting of the active transcription factor xbp s to dendritic cells potentiates vaccine-induced prophylactic and therapeutic antitumor immunity a phase i clinical trial of a multi-epitope polypeptide tab combined with montanide isa adjuvant in non-hiv- infected human volunteers targeting v eb-raf and akt using nanoliposomal-small interfering rna inhibits cutaneous melanocytic lesion development thermoresponsive polymers as gene delivery vectors: cell viability, dna transport and transfection studies heterologous protection against influenza by injection of dna encoding a viral protein development of an ultrasound-responsive and mannosemodified gene carrier for dna vaccine therapy strategies for improved formulation and delivery of dna vaccines to veterinary target species ph-sensitive liposomes: mechanism of triggered release to drug and gene delivery prospects covalently stabilized trimethyl chitosan-hyaluronic acid nanoparticles for nasal and intradermal vaccination ph-sensitive immunoliposomes mediate target-cell-specific delivery and controlled expression of a foreign gene in mouse highly efficient dna delivery mediated by ph-sensitive immunoliposomes gene inoculation generates immune responses against human immunodeficiency virus type anionic pamam dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system? gene therapy for ovarian cancer (review) pathogen recognition and development of particulate vaccines: does size matter? systemic tumor-targeted gene delivery by anti-transferrin receptor scfv-immunoliposomes intranasal vaccination with chitosan-dna nanoparticles expressing pneumococcal surface antigen a protects mice against nasopharyngeal colonization by streptococcus pneumoniae immunogenicity of multiple-epitope antigen gene of hcv carried by novel biodegradable polymers construction of dna and rna based on bifunctional replicon vector derived from semliki forest virus enhanced potency of individual and bivalent dna replicon vaccines or conventional dna vaccines by formulation with aluminum phosphate stabilization of vaccines and antibiotics in silk and eliminating the cold chain t-cell vaccines that elicit effective immune responses against hcv in chimpanzees may create greater immune pressure for viral mutation key: cord- - mzsaz a authors: wium, martha; jonker, hester isabella; olivier, adriaan jacobus; bellstedt, dirk uwe; botes, annelise title: dna vaccines against mycoplasma elicit humoral immune responses in ostriches date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: mzsaz a in ostriches, the population densities resulting from intensive rearing increases susceptibility to pathogens such as mycoplasmas. in addition to good management practices, vaccination offers an attractive alternative for controlling mycoplasma infections in food animals, instead of using antibiotics, which often leave unacceptable residues. the use of live attenuated vaccines, however, carry the concern of reversion to virulence or genetic recombination with field strains. currently there are no commercially available vaccines against ostrich-infecting mycoplasmas and this study therefore set out to develop and evaluate the use of a dna vaccine against mycoplasma infections in ostriches using an oppa protein as antigen. to this end, the oppa gene of “mycoplasma nasistruthionis sp. nov.” str. ms was cloned into two dna vaccine expression vectors after codon correction by site-directed mutagenesis. three-months-old ostriches were then vaccinated intramuscularly at different doses followed by a booster vaccination after weeks. the ability of the dna vaccines to elicit an anti-oppa antibody response was evaluated by elisa using the recombinant oppa protein of ms as coating antigen. a statistically significant anti-oppa antibody response could be detected after administration of a booster vaccination indicating that the oppa protein was successfully immunogenic. the responses were also both dose and vector dependent. in conclusion, the dna vaccines were able to elicit an immune response in ostriches and can therefore be viewed as an option for the development of vaccines against mycoplasma infections. the ostrich (struthio camelus var. domesticus) is the largest living, flightless bird species belonging to the ratite group ( ) . although ostriches are traditionally endemic to africa, parts of arabia and the middle east ( ), they are reared in several countries across the world. south africa, however, is currently the largest producer of ostrich products (meat, leather, and feathers) on international markets ( ) . here, the majority of ostriches destined for slaughter are reared in free-range systems, but when environmental conditions are unfavorable for grazing, they are reared in grow-out camps from about months of age ( ) . mycoplasma, a genus of bacteria that belongs to the prokaryotic class mollicutes, is known to be highly infectious in these intensive rearing systems ( , ) . characteristic features of mycoplasma include the lack of a cell wall, small at-rich genome and a minimal set of genes ( , ) . the species found to specifically infect ostriches are "mycoplasma struthionis sp. nov." str. ms , mycoplasma sp. str. ms , and "mycoplasma nasistruthionis sp. nov." str. ms ( , ) . they are typically associated with upper respiratory tract infections ( ) , reduced growth rates, downgrading of carcasses and in extreme cases, chick mortalities ( , ) . the severity of infections increases with exposure to environmentally induced stress, other bacterial and viral infections. of the three mycoplasma species, "mycoplasma nasistruthionis sp. nov." str. ms was found to have the highest prevalence ( ) amongst ostriches and therefore was the focus of this study. mycoplasma infections are currently managed using a combination of biosecurity systems and antibiotics. with antibiotics there is the risk of resistance ( ) as well as an accumulation of residues in meat with concomitant risks for consumers ( ) . development of whole organism vaccines for use in ostriches is limited by the slow-growing nature of these organisms and the cost of medium required to sustain growth. dna vaccines, on the other hand, do not require large scale cultivation of the pathogen, can be produced at relatively low cost and are more temperature stable than other vaccines ( , ) . it is therefore an attractive alternative for use in ostriches where the vaccine market is small, relative to e.g., poultry, and ostriches are usually farmed in semi-arid or arid regions where temperatures are frequently high and cold storage of vaccines can be problematic. similar to live vaccines, they are also able to stimulate humoral and cellular immunity as indicated by measuring antibody, t-helper cell and cytotoxic t-lymphocyte responses ( ) . but unlike live vaccines cannot revert back to an infectious form ( ) . el gazzar et al. ( ) has reported the reversion to virulence of a commonly used live mycoplasma vaccine in poultry, ts- , whilst another has reported possible genetic recombination between live vaccines and field strains upon long term use ( ) . this exchange or transfer of genes could have a negative impact on either the pathogenicity or transmissibility of field strains. chromosomal integration of dna vaccines is of some concern, but preclinical and clinical studies have shown the rate of integration of plasmid dna into the host genome to be lower than that of spontaneous mutations ( , ) . to date, dna vaccines have not been tested in ostriches or any other ratites. the aim of this study was to use the oppa protein of "m. nasistruthionis sp. nov." str. ms as antigen in the development and evaluation of dna vaccines in ostriches at different doses and using different vectors. a dna vaccine is a plasmid expression vector containing a gene that codes for a protein antigen. as a result of their parasitic lifestyle ( ), mycoplasmas possess, and rely on, a wide range of transmembrane transport systems for their survival. the extracellular components of these transporters are ideal targets for vaccine development ( , ) . in this study the extracellular oppa domain of an oligopeptide permease (opp) transporter was chosen as protein antigen ( , ) . using mouse models, oppa has to date been evaluated as antigen in subunit vaccines against brachyspira pilosicoli ( ) , moraxella catarrhalis ( ) , and yersinia pestis ( ) , as well as against haemophilus parasuis in pigs ( ) . oppa has, however, not been evaluated in any organism as part of a dna vaccine. in this study we report, for the first time, that a dna vaccine can elicit a humoral immune response in ostriches using oppa as antigen. this response was both dose and vector dependent. site-directed mutagenesis of the oppa gene cultures of "m. nasistruthionis sp. nov." str. ms (genbank: km . ) were obtained from mr j.j. gouws (faculty of veterinary science, onderstepoort, university of pretoria). genomic dna (gdna) was isolated from these cultures using a method described by hempstead ( ) . the type a oppa gene ( ) pci-neo (promega) and vr (vical inc.) were chosen as vaccine vectors (supplementary figure ) . the pci-neo vector is a mammalian expression vector that contains a cmv enhancer/promoter region, chimeric intron and sv late polyadenylation signal sequence. the vr vector also contains a cmv enhancer/promoter region, but in combination with a cmv intron, tissue plasminogen activator (tpa) signal peptide sequence and a bovine growth hormone polyadenylation signal sequence. the mutated oppa gene was sub-cloned into each vector using restriction digestion. for pci-neo, acci and mlui (fastdigest, thermo scientific) and for vr , bamhi (fermentas) restriction enzymes were used according to the manufacturer's instructions. cultures of the pci-neo_oppa and vr _oppa vaccine plasmid were prepared and the respective plasmids purified with an endotoxin-free plasmid dna purification kit (nucleobond r xtra midi plus ef, macherey-nagel, germany). yields were determined using a nanodrop spectrophotometer. prior to large scale plasmid isolation for vaccination, the nucleobond kit's ability to yield supercoiled pci-neo_oppa and vr _oppa plasmids was evaluated by digesting plasmids with the acci and bamhi restriction enzymes, respectively, followed by electrophoresis on a % (w/v) agarose gel. to prepare a sufficient amount of plasmid for vaccination, the plasmids were prepared in batches over several days. the purified plasmids were again evaluated as before to confirm the integrity and supercoiling of the plasmids prior to being used for vaccination. the isolated plasmids were diluted to , , and , µg/ml with sterile pbs ( mm nacl, . mm kcl, mm na hpo , and . mm kh po , ph . ), respectively, a day before use. dilutions were prepared under aseptic conditions, in sterile serum glass bottles, closed with inert silicone stoppers and sealed with a tear away center aluminum cap (sigma-aldrich). ethical approval was obtained from the stellenbosch university animal ethics committee (su-acum - ) as well as the south african department of agriculture, forestry and fisheries (reference: / / / / ) in terms of section of the animal disease act (act no. of ). a group of ostrich chicks of ± -months-old were randomly selected from a chick rearing unit in the fraserburg district (northern cape province, south africa). since fraserburg is outside the major commercial ostrich production region, this limits pathogen exposure during the critical - months-old rearing phase ( ) . as per standard practice, upon reaching a weight of about kg (± months of age), trial ostriches were transported back (± km) to a commercial ostrich farm in the oudtshoorn district (western cape province, south africa). three-month-old chicks were chosen for vaccination to allow sufficient time for a primary immune response to develop before being transported to a higher stress environment with concomitant increase in pathogen load and increased risk of exposure to mycoplasma. the chicks were quarantined for days after relocation and random testing was performed for avian influenza. this is required by avian influenza control measure before ostriches can be released and allowed to mix with other populations on any farm to which they have been transported. at the start of the trial, each ostrich was tagged with a unique number for identification under the right wing, in accordance with standard ostrich farming practices. the trial was conducted under similar conditions to which a commercial vaccine would be administered and therefore trial ostriches were at all times housed and treated in the same manner as non-trial ostriches on the farms. in fraserburg, the chick rearing units were open air camps ( , m ) with - chicks per camp. in oudtshoorn, ostriches were kept in open air grow-out camps ( , m ) with - ostriches per camp. the ostriches received food and water ad libitum and were only handled by trained and experienced farm personnel. trial birds were not kept separately but grouped with other ostriches of the same weight and age. the ostriches were randomly allocated to seven treatment groups of ostriches each. the pci-neo_oppa and vr _oppa vaccine groups were vaccinated at a dose of , , and , µg, respectively. the last group was the control group that did not receive any vaccine. the dose typically administered to poultry ranges from . to µg when injected intramuscularly, with a booster dose after - weeks ( - ). dunham ( ) indicated that larger animals may require a larger dose of - , µg, but that this needs to be optimized for individual vaccines since the dose required is influenced by the target species, its size and the efficiency of plasmid delivery. the different doses used in our study were therefore selected to compensate for the increase in weight of the ostriches during the course of the trial and the fact that no adjuvant was used in the vaccine formulation to limit possible skin reactions which can impact hide quality. vaccine doses were administered in a single ml volume at week and a booster injection administered at week by intramuscular injection in the upper thigh. blood samples ( ml) were drawn from the jugular vein using gx / ′′ needles (vacuette r ) and collected in vacuette r z serum sep clot activator tubes at week , , , and . serum was separated by centrifugation at low speed for min and stored at − • c. week and samples were collected in fraserburg, and week and samples in oudtshoorn. the ostriches were moved from fraserburg to oudtshoorn days prior to the week sampling and booster injections were therefore administered during the quarantine period. ostriches were monitored after vaccination for any adverse reactions. for analysis of anti-oppa antibody responses, recombinant oppa protein was produced for use as coating antigen in an enzyme-linked immunosorbent assay (elisa). to this end, the sdm corrected oppa gene was pcr amplified and sub-cloned into a pgex- t- vector (ge healthcare life science, uk) using restriction digestion. primer sequences with bamhi and noti restriction sites are shown in supplementary table . for protein expression, the pgex- t- _oppa plasmid was transformed into escherichia coli bl (de )plyss cells (promega) and freezer stocks prepared. an overnight culture (from a freezer stock) and expression cultures were prepared using terrific-broth (tb) medium. expression of the glutathione s-transferase (gst) oppa fusion protein was induced at an od between . and . by the addition of . mm iptg, and cells harvested at and h by centrifugation ( , × g at • c). the cells were resuspended in xten extraction buffer [ mm tris-hcl, mm edta, mm nacl, . % triton x- , . m dithiothreitol and % glycerol (v/v)] at µl extraction buffer per ml culture used for centrifugation. protease inhibitor was also added to the ten buffer ( tablet per ml extraction solution of complete ultra tablets, mini, easypack tablet, roche). the gst-oppa protein was isolated using glutathioneagarose chromatography (sigma-aldrich) under gravity flow at • c according to the manufacturer's instructions. a sample ( ml) was prepared for loading of the column by treating the resuspended pellets with three freeze-thawing cycles ( min at • c followed by min at − • c) and five cycles of s sonication followed by min on ice. the samples were then triturated three times through a gx / ′′ needle (avacare) into a ml injekt-f syringe (bbraun). this was repeated using a gx / " needle (nipro) followed by centrifugation at , × g for min ( • c) to obtain a clear supernatant which was loaded onto the column under gravity flow at • c. fractions ( ml) were collected and their protein concentration determined using a modified bradford assay ( ) . expression and isolation products were analyzed using sds-page ( ) and western blot analysis ( ) . microtiter plates (maxisorp, nunc) were coated overnight at • c with µl of recombinant gst-oppa protein diluted to µg/ml in carbonate buffer ( mm nahco , ph . ). to block non-specific binding, µl/well casein buffer ( mm tris-hcl, mm nacl, . % casein and . % thiomersal, ph . ) was added and the plate incubated for h at • c. serum samples were diluted : with casein-tween [casein buffer containing . % (v/v) tween r ] and µl/well added to the plate in triplicate before incubation for h at • c. biotinylated rabbit anti-ostrich ig polyclonal antibodies, prepared as previously described ( ), were next added at a dilution of : in casein-tween and incubated for h at • c. a streptavidin horseradish peroxidase (hrp) conjugate mixture [ ml streptavidin hrp (invitrogen), ml . % casein buffer, and ml % glycerol], diluted : with casein-tween, was then added ( µl/well) and the plate incubated for h at • c. finally, µl/well substrate solution ( . mg/ml abts, . µl/ml h o , . m citrate buffer, ph ) was added and absorbance measured at nm with a thermo scientific multiskan ex plate reader after min incubation at • c. after each incubation step the content of the plate was first decanted followed by washing five times with pbs-tween [ mm nacl, . mm kcl, mm na hpo and . mm kh po , ph . , . % (v/v) tween r ] and washing three times with milli-q r water. between the coating and blocking steps washing was not applied. a first-row column blank (containing all components except ostrich serum) was used on each plate to blank absorbance values. all plates contained controls to monitor plate to plate variation. the controls were serum samples representing the week , , , and sampling points of a single ostrich, randomly selected from the pci-neo_oppa , µg group based on high titers produced after vaccination. prior to the analysis of samples, the elisa was optimized with regard to coating concentration ( - µg/ml), serum dilution ( : - : ) as well as number of washing cycles between steps. non-specific binding of anti-oppa antibodies during elisa analysis was evaluated by coating the wells with carbonate buffer ( µl/well) containing no capture antigen and serum from a vaccinated bird that gave a high absorbance value at a dilution of : when using the gst-oppa protein as capture antigen. the absorbance values obtained are referred to as elisa titres, which are a measurement of the antibody levels produced when using a serum dilution of : in the elisa. the weight of trial birds was monitored and recorded at weeks , , and . to determine the possible influence of existing mycoplasma infections on immune response data, trachea swabs were collected at weeks , , , and and tested for the presence of mycoplasma infections by pcr. birds were, however, not excluded from the trial if they tested positive since ostriches are often naturally infected with mycoplasmas. swab samples were collected using dry sterile swabs (copan) which were then rinsed in µl sterile pbs buffer. the eluate was tested by pcr using a generic primer pair specific for the mycoplasma genus ( ). positive samples were further evaluated for the presence of the ostrich-infecting mycoplasmas ms , ms , and ms ( ). statistical analysis of the elisa titer data was performed using the general linear model (glm) procedure in the agrobase generation ii r (agronomix software inc.) software. analysis of variance (anova) and least significant difference (lsd) values were calculated at a significance level of . . the bp oppa gene was successfully amplified from the ms gdna and cloned into a pgem r -t easy vector as confirmed by sequencing. using sdm, all mycoplasma tga codons in this plasmid were successfully mutated to universal tgg codons in seven consecutive steps of sdm as confirmed by sequencing (supplementary figure ) . the mutated oppa gene was successfully sub-cloned into the pci-neo and vr vaccine vector as confirmed by sequencing (supplementary figure ) . large scale production of the dna vaccines was achieved and approximately mg of plasmid was isolated for each of pci-neo_oppa and vr _oppa. the / absorbance ratio of the isolated plasmids ranged from . to . . the integrity of the isolated plasmids was confirmed with agarose electrophoresis and most of the pdna was in the required supercoiled conformation. a supercoiled conformation has a higher transfection rate into mammalian cells, is less susceptible to intracellular degradation and is more effective at inducing an immune response than other plasmid conformations ( , ) . ostriches were vaccinated with the prepared dna vaccines and no adverse reactions were observed at the injection sites (redness, swelling, inflammation, or allergic reaction) during and after the trial. ostriches resumed normal behavior such as eating, walking around and exploring immediately after vaccination (no lameness or loss of appetite). sub-cloning of the mutated oppa gene into the pgex- t- vector was successful as confirmed by sequencing frontiers in immunology | www.frontiersin.org (supplementary figure ) . sds-page and western blot analyses confirmed that the recombinant oppa protein was expressed successfully as an n-terminal gst-fusion protein with the predicted size of kda ( kda due to the gst tag) (figures a,b) . bradford analysis indicated that the oppa protein was eluted between fraction and with the highest concentration obtained in the th fraction. ostriches with more than two missing sampling points were excluded from elisa and subsequent statistical analysis. there was no non-specific binding of the anti-oppa antibodies in ostrich serum and the plate control samples indicated limited plate to plate variation. the titer values resulting from vaccination with the pci-neo_oppa and vr _oppa vaccines are shown in figures a,b , respectively. the titer values of the control group, which received no vaccine, did not show significant variation over time. however, there was a slight increase in the average titer at week . vaccination with the pci-neo_oppa vaccine (figure a ) resulted in significant treatment x time interactions (p = . ), but only the and µg doses resulted in average elisa titers that differed significantly from the control. this was also only at week after a booster vaccination was administered. the average titers of the different doses were, however, not significantly different from one another. vaccination with the vr _oppa vaccine (figure b ) resulted in a significant treatment × time interaction (p < . ) and all three doses resulted in average elisa titers that differed significantly (lsd = . ) from the control. this was again only at week after a booster vaccination was administered and all the doses differed significantly from one another. the vaccinated groups as well as the control group gained weight from weeks to , but from weeks to there was an average weight loss of . kg ( table ) . there was, however, no statistically significant difference between the treatment groups and the control group over the weeks period (p = . for pci-neo_oppa and p = . for vr _oppa). the presence of mycoplasma infections was monitored with pcr during the trial ( table ) . mycoplasma infections could not be detected in any of the groups at weeks , , and . infections were, however, detected at week . at this point the ostriches had been in oudtshoorn for days, of which days were out of quarantine. in the groups that received the vr _oppa vaccine the highest percentage of infections at week was due to ms ( . %) followed by ms ( . %) and ms ( . %) ( table ) . compared to this, the groups that received the pci-neo_oppa vaccine had fewer mycoplasma infections. amongst these the highest percentage of infections was again due to ms ( . %) followed by ms ( . %) and ms ( . %). for both vaccine groups, some of the birds were infected by more than one of these mycoplasma species. the only group that had no pcr-detectable mycoplasma infections at week was the group that had received , µg of the vr _oppa vaccine and the control group. in this study, dna vaccines were developed for ostriches using the oppa gene of an ostrich-infecting mycoplasma (ms ) as vaccine antigen. the expression vectors used (pci-neo and vr ), were selected based on dna vaccine studies in other birds, and on their immunostimulatory characteristics ( ) ( ) ( ) . after vaccination of ± -month-old chicks a statistically significant anti-oppa antibody response could not be detected at week , although a general trend of increases in the average titer values was observed from week to for groups that received the vr _oppa vaccines. based on previous studies using inactivated vaccines in ostriches ( , ) , we expected a primary immune response about weeks after the first vaccination (i.e., between weeks and ). after administering a booster vaccination, both dna vaccines were able to elicit a statistically significant anti-oppa antibody response. as these responses against oppa were significant in comparison to the negative responses following the first vaccinations, this is evidence of a secondary immune response as a result of immune memory. if memory cells are produced after the initial contact with an antigen, subsequent exposure to the same antigen will allow the host to recognize the antigen faster, and with greater magnitude ( , ) . the responses elicited by the dna vaccines were, however, dose dependent as well as vector dependent since different results were produced by each vaccine when using the same dose. this highlights a possible role of the vaccine vector in the observed antibody responses. the vr _oppa vaccine, on average, elicited higher titer values compared to the pci-neo_oppa vaccine, which might be due to the tpa signal sequence of the vr plasmid, which is situated upstream of the oppa gene, and assists with protein expression in mammalian cells and export of the protein out of the cell. this would in turn increase the immunogenicity of the antigen ( ) . in addition to this, sequences in the plasmid backbone have intrinsic immunostimulatory activity which enhance the immune system's response to the expressed protein antigen ( , ) . over the whole vaccination trial, the health of the ostriches was monitored by recording weight gain. all groups gained weight up to week , but toward week their weight gain decreased. given that this trend was also observed for the control group ostriches, the weight loss cannot be ascribed to vaccination. the decrease in weight did, however, coincide with the movement of the birds to a new location. ostriches are known to be severely affected by stress and during this period of adapting to changes in social dynamic and housing environment, reduction in weight gain and even weight loss is normal amongst farmed ostriches ( ) . the presence of existing mycoplasma infections at the start of the trial, as well as changes in infection status during the trial, were also monitored by pcr analysis of trachea swabs. mycoplasma infections could only be detected at week , which was after they were moved and exposed to the farm environment in oudtshoorn for more than days. the absence of detectable mycoplasma infections at the earlier time points may be ascribed to fraserburg being outside of the commercial ostrich production region. the oudtshoorn district, on the other hand, has a higher incidence of mycoplasma infections especially during seasonal changes and our trial was conducted during late autumn. amongst the vaccinated groups, the number of detected ms and ms infections were low compared to ms , with only the µg vr _oppa and control group having no detectable ostrichinfecting mycoplasmas. in future trials, careful consideration should be given to the timing of the primary and booster vaccination relative to the time of introduction to growout conditions. it is possible that the mycoplasma infections had an influence on the observed antibody responses. in a study done by yang et al. ( ) using recombinant oppa protein as subunit vaccine against m. catarrhalis in mice, it was found that the elisa titer values were already raised at the start of the vaccination trial which they ascribed to the presence of existing systemic antibodies to oppa as a result of existing infections. despite the possible presence of antibodies due to mycoplasma infections, this did not mask the detection of a dose dependent antibody response to the oppa protein of ms during vaccination. in conclusion, this is the first study to show that dna vaccines are able to elicit an antibody response in ostriches against a mycoplasma antigen in spite of ostriches being prone to environmentally induced stress conditions that can suppress immune function. the mycoplasma oppa protein was sufficiently immunogenic to induce an anti-oppa antibody response and this response was firstly, dose dependent and secondly, required a booster vaccination. further trials are required to determine the extent of protection provided by this mycoplasma antigen relative to the timing of the primary and booster vaccination before introduction to grow-out conditions. this study (field trial) was carried out in accordance with the recommendations of the south african department of agriculture, forestry and fisheries (reference: / / / / ) in terms of section of the animal disease act (act no. of ) . the protocol was approved by the stellenbosch university animal ethics committee (su-acum - ). mw performed the sdm, prepared the dna vaccine and expression plasmids, optimized the expression of the recombinant oppa protein, and the initial optimization of the elisa. hj further optimized the elisa and evaluated the anti-oppa antibody responses during the vaccination trial. ao assisted with the vaccine trial. mw wrote the manuscript with support from ab. ab supervised the project with the assistance of db and ao. ab and db conceived the original idea. all authors provided critical feedback and helped shape the research, analysis and manuscript. this work was supported by the technology and human resources for industry programme (thrip), south africa (project reference tp ) and south african ostrich business chamber. we would like to thank vical inc., usa for supplying the vr vector for research purposes and mr. j. j. gouws (faculty of veterinary science, onderstepoort, university of pretoria) for the culturing of the mycoplasma bacteria. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material ratite nonmonophyly: independent evidence from novel loci molecular genetic relationships of the extinct ostrich, struthio camelus syriacus: consequences for ostrich introductions into saudi arabia profile of the south african ostrich market value chain bird handling, transportation, lairage, and slaughter: implications for bird welfare and meat quality ostrich diseases investigations into mycoplasma infections in south african ostriches. the third international ratite science symposium molecular biology and pathogenicity of mycoplasmas a systematic review of mycoplasma and ureaplasma in urogynaecology identification of three novel mycoplasma species from ostriches in south africa proposal for molecular tools for the epidemiology of contagious bovine pleuro pneumonia and classification of unknown mycoplasma sp. isolated from struthio camelus mycoplasmas and their host: emerging and re-emerging minimal pathogens antibiotic residues in food: the african scenario dna-antiviral vaccines: new developments and approaches-a review immunization of animals: from dna to the dinner plate dna vaccines: an historical perspective and view to the future dna vaccines: ready for prime time? characterization of a ts- -like mycoplasma gallisepticum isolate from commercial broiler chickens the development and application of a mycoplasma gallisepticum sequence database detection of integration of plasmid dna into host genomic dna following intramuscular injection and electroporation biodistribution of dna plasmid vaccines against hiv- , ebola, severe acute respiratory syndrome, or west nile virus is similar, without integration, despite differing plasmid backbones or gene inserts atp-binding cassette transporters are targets for the development of antibacterial vaccines and therapies bacterial surface proteins and vaccines oppa, the substrate-binding subunit of the oligopeptide permease, is the major ecto-atpase of mycoplasma hominis the identification of oppa gene homologues as part of the oligopeptide transport system in mycoplasmas evaluation of recombinant brachyspira pilosicoli oligopeptide-binding proteins as vaccine candidates in a mouse model of intestinal spirochaetosis characterization and evaluation of the moraxella catarrhalis oligopeptide permease a as a mucosal vaccine antigen the abc transporter protein oppa provides protection against experimental yersinia pestis infection immune response to oligopeptide permease a (oppa) protein in pigs naturally and experimentally infected with haemophilus parasuis an improved method for the rapid isolation of chromosomal dna from mycoplasma spp ostrich manual. elsenburg: western cape department of agriculture cross-protection among lethal h n influenza viruses induced by dna vaccine to the hemagglutinin protective efficacy of dna vaccines against duck hepatitis b virus infection turkeys are protected from infection with chlamydia psittaci by plasmid dna vaccination against the major outer membrane protein dna vaccination in the avian a dna prime-protein boost vaccination strategy targeting turkey coronavirus spike protein fragment containing neutralizing epitope against infectious challenge protection of chickens against infectious bronchitis virus with a multivalent dna vaccine and boosting with an inactivated vaccine induction of a protective response in ducks vaccinated with a dna vaccine encoding engineered duck circovirus capsid protein sequential dna immunization of chickens with bivalent heterologous vaccines induce highly reactive and cross-specific antibodies against influenza hemagglutinin the application of nucleic acid vaccines in veterinary medicine evaluation of dna vaccines against mycoplasma nasistruthionis sp. nov. str. ms infections in ostriches and the production of iga heavy chain proteins. master's dissertation the development of a dna vaccine against mycoplasma nasistruthionis sp. nov. for use in ostriches antibody responses to la sota strain vaccines of newcastle disease virus in ostriches (struthio camelus) as detected by enzyme-linked immunosorbent assay impact of plasmid supercoiling on the efficacy of a rabies dna vaccine to protect cats transgene expression of transfected supercoiled plasmid dna concatemers in mammalian cells vaccinia expression of mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of m. tuberculosis ag . microbes infect measuring the effects of an ever-changing environment on malaria control comparison of five expression vectors for the ha gene in constructing a dna vaccine for h n influenza virus in chickens growth rate and hatching date in ostrich chicks reflect humoral but not cellmediated immune function generating memory with vaccination vaccination to gain humoral immune memory effect of plasmid backbone modification by different human cpg motifs on the immunogenicity of dna vaccine vectors cpg dna as a vaccine adjuvant the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © wium, jonker, olivier, bellstedt and botes. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -d szap authors: permyakova, n. v.; uvarova, e. a.; deineko, e. v. title: state of research in the field of the creation of plant vaccines for veterinary use date: - - journal: russ j plant physiol doi: . /s sha: doc_id: cord_uid: d szap transgenic plants as an alternative of costly systems of recombinant immunogenic protein expression are the source for the production of cheap and highly efficient biotherapeuticals of new generation, including plant vaccines. in the present review, possibilities of plant system application for the production of recombinant proteins for veterinary use are considered, the history of the “edible vaccine” concept is briefly summarized, advantages and disadvantages of various plant systems for the expression of recombinant immunogenic proteins are discussed. the list of recombinant plant vaccines for veterinary use, which are at different stages of clinical trials, is presented. for many thousands of years plants have served humanity as a source of medicinal substances. how ever, only at the turn of the century xxi with the appearance of dna technologies, it became possible to modify plant genomes and to create new types of plants (transgenic plants), which are capable of syn thesizing and accumulating in their tissues recombi nant proteins from various heterologous systems. to date transgenic plants in which nuclear and chloro plast genomes have been transformed with genes encoding heterologous proteins that are important in the treatment of various diseases -antigens of infec tious agents, antibodies, immunomodulators, etc. have been created [ , ] . of principal importance of this work development was the creation of the "edible vaccine" concept, the essence of which is the use of genetically modified plants containing protein anti gens of infectious agents for oral delivery of relevant antigens to the mucosa of the gastrointestinal tract of warm blooded animals. vaccination based on the programming of the spe cific mechanisms of warm blooded animal protection against pathogens is the most efficient method for the struggle against infectious diseases, which often result in a mass mortality. in agriculture, there is no alterna tive to livestock vaccination, because there are no anti viral drugs that are suitable for a wide use in animal husbandry. the importance of animal vaccination indirectly affects human health, because the use of vaccines significantly reduces the amount of pharma ceuticals in the food chain. as a rule, animal immune mechanisms are acti vated by the direct introduction of infectious agents or their components. at present, most of used vaccines are preparations on the basis of inactivated agents. although these vaccines manifest the high immunoge nicity, they are not without serious shortcomings. among such disadvantages are the increased sensitiv ity of the organism to them, the large load on the immune system, the reactogenicity of vaccines (side effects), their toxicity. etc. the application of molecular biology and genetic engineering methods opened wide prospects for the manufacturing of vaccines of new generation, which immunogenic components can be biological mole cules or their fragments. dna fragments or proteins of the infectious agent cell envelopes can serve as immu nogenic components. when the gene encoding the envelope protein of the infectious agent is transferred into the genome of another organism, for example, plant, then the cells of such plant will synthesize the protein antigen capable of formation of resistance to this agent. thus, the introduction into the organism not the whole pathogen but only its part, which is not capable of inducing infection development, will pro vide for the effect of vaccination. one fourth of the total pharmaceutical market of drugs for veterinary use, and it is constantly expanding. [ ] . preparation of medicinal substances for the pro duction of veterinary products is based on various approaches, including biotechnology using genetically modified (transgenic) organisms for these purposes; such expression systems as bacteria, yeast, cells of insects and mammals are used. the application of genetically modified plants with genes encoding phar maceutic proteins inserted in the genome opens new prospects for obtaining recombinant proteins, includ ing plant vaccines [ ] . this review is devoted to the analysis of possibilities of producing recombinant immunogenic proteins for veterinary use on the basis of plant expression systems, and the history of the concept of "edible vaccines" for animal immunization. of plant vaccines the idea of the usage of plant cells for the synthesis and accumulation of recombinant protein antigens was for the first time successfully realized in by c. arntzen and his colleagues [ ] . just this team of researchers not only demonstrated a possibility of the accumulation of the surface hbsag antigen of hepati tis b virus but also its capability for self assembling in the virus like particles in transgenic tobacco plants. the virus like particles isolated from plant tissues were identical to the particles of hbsag antigen of indus trial recombinant vaccine obtained in the yeast expres sion system and also to virus like particles from the blood plasma of patients infected with hepatitis b virus. thus, it became obvious that genetically modi fied plants producing and accumulating protein anti gens of various infectious agents can be used for oral delivery of corresponding agents to the mucosa of the gastrointestinal tract of warm blooded animals, i.e., as "edible vaccines." the next important step in the development of the "edible vaccine" concept on the basis of genetically modified plants was the creation of transgenic plants producing the heat labile enterotoxin of escherichia coli [ , ] and b subunit of the cholera toxin [ ] . heat labile toxin of e. coli consists of two parts: lt a (enzyme) and lt b (pentamer of receptor binding polypeptides). lt b binds to the receptors on the sur face of membranes of epithelial cells of the mammal small intestine and transports lt a in the intestinal cells, where it induces changes in the cell metabolism and cell dehydration. when the two parts of the heat labile enterotoxin are separated, the appearance of the lt b protein complex on the surface of epitheliocytes will stimulate a strong immune response of intestine mucosa without the appearance of any disease signs. just this feature was the basis for the research of c. arntzen [ ] team on the creation of plant vaccine providing for the resistance to enterotoxigenic e. coli toxins. the authors established that lt b synthesized in transgenic tobacco and potato plants and also lt b isolated from e. coli delivered orally to mice induced similar immune responses. later, lt b sequence was optimized for expression in plant cells and transferred into the potato genome [ ] . in potato tubers, the protein assembled correctly in oligomers and was accumulated in amounts suffi cient for the induction of the immune response at oral delivery to the organism. on the basis of clinical trials of the "candidate" plant lt b vaccine, it was estab lished that the consumption of raw potato tubers con taining . - mg lt b by volunteers resulted in the formation of serum and mucosal immune responses with high titers of antibodies [ ] . the initial concept of "edible vaccine" was heavily criticized by researchers who believed that in the aggressive medium of the gastrointestinal tract the recombinant protein should be destroyed. however, later it was experimentally established that the recom binant b subunit of the cholera toxin fused with green fluorescent protein (gfp) protected by the plant cellu lose cell wall at oral delivery is capable of passing through the gastrointestinal tract and reaching the antigen containing cells of the mouse intestine [ ] . the results obtained confirmed the possibility of using plants synthesizing protein antigens of various infec tious agents for oral delivery of antigens to the mucosa of the gastrointestinal tract and experimentally con firmed a consistency of the "edible vaccine" concept. it became evident that genetically modified plants can be used for the creation of plant vaccines as a raw mate rial, and separate plant parts (fruits, roots, berries, leaves, etc.) can be used directly in food without pre liminary heat treatment. response formation in the process of evolution, mammals developed secondary lymphoid mucosal tissue capable of antigen absorption, processing them, and using for the induc tion of the mucosal response. it was established that in this case both cellular and humoral immunity were formed. it is of importance that adaptive mucosal immunity can distinguish between usual food and symbiotic antigens and infectious agents [ ] . the scheme of the mechanism of mucosal immune response formation is presented in fig. . in the digestive tract, which is one of the pathways for the penetration of a variety of pathogens into organism, the main associated lymphoid tissue is the peyer's patches. peyer's patches, inductive sites of the intestine, which contain the dome, underlying the fol licle (b zones with germinal center) and interfollicu lar region containing t cells. the surface of the dome is covered by a specialized follicle associated epithe lium containing the folded cells (m cells), which are able to absorb and transport antigens from the intesti nal lumen. after successful capture, the antigen is par tially cleaved and enters into dendritic cells (see fig. , step ). dendritic cells are especially important in the initiation of adaptive immune responses, since they migrate to the lymph nodes (see fig. , step ), and the mediators act in the development of various subpopu lations of t helper cells from naive t lymphocytes and also can interact with b lymphocytes (see fig. , step ). the activated b and t cells leave the peyer's patches and penetrate into the circulatory system. mature t helper cells then return to the mucosa sur pathogens (antigen) epithelium t helper cells face to function as effectors (see fig. , step ). t helper expressing interleukin (il ) increases the expression of the receptors of polymeric immunoglobulin (pig) and secretion of antigen spe cific iga (fig. , step ) . subsequent generation of mature plasma cells producing iga leads to the induction of antigen specific protection of local and distal mucosal surfaces. since the mucosal immune response has a general ized nature, oral (i.e., mucosal) vaccination is not only the immune response of the mucous membranes, but also the overall immune response of the organism [ ] [ ] [ ] [ ] . it is proved that vaccination via the surface of mucosa membrane can specifically activate the immune response to infection without the develop ment of such processes related to the disease as inflammation or toxicity [ , ] . of plant vaccines in comparison with traditional expression systems, plant systems are attractive to researchers in many ways, primarily due to the absence of the risk of plant cell infection with animal pathogens, viruses, prions, etc. plants are capable of the synthesis of most recom binant antigens with the same posttranslational modi fications as in animal cells [ ] . plant vaccines can play especially important role in the protection of ani mals against diarrheal diseases and diseases, which infectious agents penetrate into the organism through the mucosal tissues. modern techniques of genetic engineering allow to selectively direct the recombinant proteins expressed in plant cells to various plant organs (seeds, tubers, fruits, etc.) [ ] . this possibility greatly simplifies the large scale production of plant vaccines and reduces their cost. according to the experts, the final price of the product (recombinant protein) produced in a plant expression system will be much less than the price of a similar protein produced, for example, in mammalian cell culture [ ] . since recombinant proteins can be accumulated in the storage organs or seeds, they are able to be main tained without any changes and the loss of biological activity for a long time (months and years). it was established that the recombinant protein of cholera toxin b subunit remained stable in transgenic rice grains for at least months, when grains were stored under room temperature conditions [ ] . recombi nant protein antigens remained stable in rice grains during three years and provided for the formation of the protective immunity in mice against cholera agent or against enterotoxigenic e. coli [ ] ; in soybean seeds and soy milk, the preservation of antigen stabil ity was observed for four years [ ] .thus, grains of transgenic plants can be transported to the site of final destination without additional freezing and treatment, and this ensures the retention of activity of the recom binant protein activity, its stability, and the constancy of dosage. a somewhat different picture is observed when lyo philization is used as a method of the conservation of protein antigens synthesized by plant cells. it was established that the recombinant protein of the norovirus envelope retained its immunogenicity in tis sues of both lyophilized and air dried tomato fruits [ ] , but the tested samples differed in immunogenic ity. the immunogenicity of this recombinant protein in air dried tomato fruits was somewhat higher in comparison with lyophilized fruits. similar results were obtained in experiments on the lyophilization of potato tubers synthesizing the protein of norovirus envelope, e.g., the immunogenicity of lyophilized tubers was lower than that of fresh tubers [ ] . how ever, additional studies are required for the final solu tion of this question. despite these advantages, plant vaccines are not without disadvantages. one of them is differences in protein posttranslational modifications in plants and animals, e.g., in glycosylation of the recombinant pro tein [ ] . it is known that more than a half of proteins synthesized by eukaryotic cells are glycosylated and more than a third of currently applied biopharmaceu tics are glycoproteins [ ] . although the activity of most proteins does not depend on glycosylation, in some cases it may be critical. specific features of pro tein glycoforms can affect their folding, stability, trans portation, and changes in their functional activity and immunogenicity. examples of biopharmaceuticals, which functional activity depends on the specific gly coform, are erythropoietin, antibodies, blood anti gens, some interferons and hormones [ ] . scheme of the n glycan complex formation in plants and animals (humans, for example) is shown in fig. . the most significant difference in glycosylation is that the plant β , xylose is attached to the core mannose residue and α , fucose -to n acetylglu cosamine residue of the core glycan. in human cells xylose is not used at all in glycosylation and a proximal fucose residue is attached to glycans through the α , bond. it was established that sugar residues attached at posttranslational modifications of recom binant proteins in plant cells themselves were capable of exhibiting the immunogenicity. approximately in one quarter of patients with allergy symptoms, ige antibodies specific for complex glycans, which include xylose or fucose, were revealed [ ] . differ ences in glycosylation during posttranslational modi fications of recombinant proteins in plant and mam mal cells can be eliminated by genetic engineering techniques, which was successfully demonstrated on the moss physcomitrella patens, in which the genes encoding the enzymes β , xylosyltransferase and α , fucosyltransferase responsible, respectively, for xylosylation and fucosylation of proteins were switched off by the "knock down" method [ ] . it is known that when the foreign gene is inserted in the genome of the transgenic plant, the level of its expression depends on the site of its insertion, which determines the level of protein (antigen in particular) accumulation in plant tissues. in this connection, one of disadvantages of plant vaccines is the difficulty in the standardization of their dosage. it is at this stage the development of the "edible vaccine" concept has undergone substantial revision, since the possibility of oral delivery of the recombinant antigen with the raw plant material was untenable because of the variability of the recombinant protein content. according to the researchers involved in the development of "candi date" plant vaccines, the antigen dosage problem in plant tissues can be successfully solved by the intro duction of additional treatment of the plant material: its refinement (to equalize the concentration of the antigen), drying or lyophilization. it is also necessary to introduce an additional stage associated with the development of rapid methods for determining and monitoring the dosage of the recombinant antigen. after appropriate preparation, plant vaccines can be encapsulated, tableted, and used in practice under corresponding medical supervision [ , ] . thus, performed studies allow a suggestion that the plant vaccine based on the genetically modified plants is capable of inducing protective immunity and open new opportunities for the creation of low cost and easy handling vaccines against infectious diseases of animals. it is of importance that developed to date, highly effective methods of cultivation of agro eco nomically important plant species, as well as the seed production system for a particular culture make the plants attractive to be used as "biofactories" for man ufacturing cheap recombinant proteins for medical purposes. roplasts). among plant expression systems with stable integration of the transgene in the nuclear genome, a separate group includes the cultivated duckweed, microalgae, and cell culture systems in vitro: cell sus pensions, cultures of "hairy roots", moss protonemas, which cultivation conditions are a completely closed environment (bioreactors). promising is the transient expression system, in which the target gene is intro duced into plant cells and is expressed for a short period of time (several days), but is not integrated into the genome. each of these expression systems has its advantages and disadvantages; the main details of these systems are considered below. the most widely used system of heterologous gene expression, in particular for plant vaccine production, are transgenic plants with the stable integration of the transgene into the nuclear genome. the creation of these plants involves the transfer of foreign genes into the genome of plant cells using agrobacterium tumefa ciens or bioballistics and subsequent regeneration of transformed plants from these cells. being inserted into the nuclear genome, transgene becomes its resi dent part, is stably expressed, and is maintained in subsequent generations. using this expression system for producing recom binant proteins, including plant vaccines, is still ham pered by relatively low levels of transgene expression, which is, as a rule, less than % of total soluble protein (tsp) and also by its variability in different plant organs and tissues within a single plant and in different plants. most often, the variability in expression of the transgene is due to the random nature of its insertion into the nuclear genome (effect of position) and may be associ ated with partial or complete transgene inactivation [ , ] . experts believe that the use of plant expression sys tems for obtaining plant vaccines is economically bene ficial at the level of expression of a target gene, which allows the accumulation of recombinant protein in an amount not less than % of tsp [ ] . a lot of ways to increase the level of foreign gene expression in transgenic plants is developed; they are very fully discussed in the reviews [ , , ] . among them are the optimization of the codon composition of the target sequence, the usage of strong promoters, the addition of introns or regions of binding with the nuclear matrix (sar), and others. the search for tis sue and organ specific promoters, i.e., promoters providing for target gene expression in definite plant tissues or organs, is of a special interest. for example, the usage of promoters directing transcription of the target gene predominantly in the seeds can increase the yield of the target protein by an order or several fold: up to - % of tsp in rice grains and up to % of tsp in tobacco seeds [ ] . it is of importance that as compared with leaves, seeds contain less pro teases and much less water; therefore, recombinant protein is saved better. despite the fact that transgenic plants with stable transgene expression in the nuclear genome are used most widely, it is just this expression system that induces a cautious attitude of human society. one of the fears is associated with the possibility of transgene transfer from the cross pollinated plants into the genomes of wild relatives at growing biotechnological crops in open field. to solve this problem, researchers developed various agricultural technologies as well as fixing male sterility in transgenic plants to prevent unwanted cross pollination of cultivated and wild spe cies. examples of production of various immunogenic proteins using for this purpose transgenic plants with stable transgene integration into the nuclear genome are presented in table . chloroplasts are most attractive among plant expression systems. genetically modified plants with the stable transgene integration into the chloroplast genome were called transplastome plants. the specific organization of chloroplasts allows achieving a high dose of foreign gene in transplastome plants, which provides for the efficient production of the target pro tein. the record of the recombinant protein yield was achieved by transplastomic tobacco plants with the bacteriophage lysin gene plygbs, encoding a hydro lase of the bacterial cell wall, the level of which accu mulation in the leaves amounted to % of tsp [ ] . although in some cases, negative physiological changes were observed in plants with such high level of foreign gene expression, usually there were no devia tions in the development of such plants. problems of transplastome plant adaptation to the high level of for eign gene expression are discussed in the review of bally et al. [ ] . transgene delivery to chloroplasts is performed using bioballistics (gene gun); its integration into the chloroplast genome occurs via homological recombi nation [ ] . the advantage of chloroplast expression system is in the absence of the effect of position observed in the case of random pattern of transgene distribution in the nuclear genome. in plastids, there is no transgene splicing (inactivation); therefore, its expression is stably preserved in subsequent genera tions. due to the prokaryotic organization of chloro plast genome, there is a possibility of co expression of several genes within a single operon [ ] . an impor tant specificity of plastids is that they are inherited through the maternal line and usually are absent from pollen. therefore, as distinct from usual transgenic plants, transplastome plants are safe for environment because the uncontrolled spread of the transgene into other plants is prevented [ , ] . it should be noted that protein posttranslational modifications, e.g., assembling multimer proteins, the tmv-tobacco mosaic virus; camv-cauliflower mosaic virus; tsp-total soluble protein; (+) immune response is revealed at oral immunization; (++) immune response is revealed at oral immunization, animals did not die after virus infection. species of immunized animals are indicated, the way of antigen delivery is indicated in the cases when it was not oral; the amount of survived infected animals is indicated in the cases when the survival was less than %. no. formation of disulfide bridges, lipid modifications, etc., occur successfully in chloroplasts. it is estab lished that recombinant proteins synthesized in chlo roplasts do not differ from native ones in their func tional activities [ , ] . chloroplast attractiveness as the system of recombinant protein expression is in the fact that they are closed structures and it preserves the metabolites, which when released into the cytosol are toxic to plant cells, such as b subunit of cholera toxin [ ] and trihalose in tobacco cells [ ] . on the basis of the above works, it becomes appar ent that transplastome plants can be regarded as the most promising system for efficient production of pro tein antigens. however, the main disadvantage of such expression system at plant vaccine manufacturing is that chloroplasts cannot glycosylate proteins. at present, the creation of transplastome plants is also associated with some technological problems related to the absence of the efficient system of regeneration for most plant species and also with the absence of the efficient system of transgen delivery to the chloroplast genome providing for the high percent of transformed plant yield. the suspension cell cultures on the basis of geneti cally modified plants attract the attention of research ers as promising potential systems for biopharmaceu tics production. such cultures can be obtained from loose callus tissues induced from genetically modified explants or on the basis of co culturing of the cell sus pension and a. tumefasciens. after the assessment of growth characteristics and cell line screening to obtain promising lines capable of the accumulation of great amounts of recombinant proteins, such lines can be cultivated in bioreactors for target protein obtaining. an attractive feature of the cell cultures as expres sion systems for recombinant protein production, as compared to the use of whole plants for this purpose, is that cell culture can be unified in growth character istics, cell dimensions and types. moreover, cells are grown under strictly controlled conditions, when the product accumulation does not depend on the sea sonal weather changes and allows the permanent product obtaining in bioreactors. the additional insertion of signal peptide nucleotide sequences into the construct permits a protein secretion into the intercellular space, which allows target protein isola tion directly from the culture liquid. the addition of recombinant protein stabilizers to the suspension cul ture increases the yield of the target protein [ ] . by their capabilities, plant cell cultures are compa rable in production of therapeutic recombinant pro teins with the conventionally used mammalian cell cultures, such as chinese hamster ovary cells. how ever, as distinct from the mammal suspension cultures, they are not infected by any animal pathogens. to data, there are many examples of plant cell cultures with the yield of recombinant protein in the amount more than mg/l, which is a threshold value for starting the commercial manufacture of the product [ ] . an example of a commercially successful produc tion of veterinary vaccine products is developed by dow agro sciences company (united states) system concert™, patented as an effective and safe system for the production of vaccine proteins in cultured plant cells, cultured in a bioreactor. the first plant vaccine against newcastle disease virus of birds obtained in the tobacco cell culture was approved for use by the min istry of agriculture of the united states in . the disadvantages of this expression system are still insufficiently high yield of the target recombinant pro tein and the instability of foreign genes in cultured plant cells due to the epigenetic silencing of the trans gene transcription [ ] . advantages, disadvantages, and specific features of recombinant protein produc tion in the plant cell cultures are discussed in reviews [ , ] . there are many examples of successful use of aqueous plants, such as duckweed, unicellular algae, and mosses, which are cultivated similarly as plant cell suspensions in bioreactors, for recombinant pro tein expression. duckweed attracts the attention of researchers as a potential highly efficient system for recombinant pro tein expression due to its capability of a rapid biomass accumulation: it can be doubled for - h. geneti cally modified duckweed as a potential producer of biopharmaceutic proteins can be used by animals as a raw or dried food. duckweed is a monocotyledonous angiosperm; for foreign gene transfer into its genome, the methods of agrobacterial transformation and bioballistics are used. examples are known when genetically modified duckweed accumulated recombi nant protein in the amounts up to % of tsp, as assessed after the accumulation of gfp [ ] . sequenc ing the duckweed chloroplast genome is close to com pleting, which opens up some prospects for a signifi cant increase in the yield of recombinant proteins. the systems of biopharmaceuticals production using genetically modified microalgae are actively developed. algae combine advantages of both bacteria (rapid growth and simplicity of cultivation) and higher plants (a capability of posttranslational modifications and photosynthesis). chlamydomonas reinhardtii is most promising among algae: it has a short time of bio mass doubling (about h), it is easily subjected to nuclear and chloroplast transformation, it can be grown under photoautotrophic conditions or with the addition of acetate as the source of carbon. nuclear transformants usually give rather low yield of protein product; therefore, recombinant protein production by this alga is based on the transformation of chloro no. plast, which occupies about % of the cell volume [ ] . c. reinhardtii nuclear and chloroplast genomes are sequenced, and this simplifies substantially any genetic engineering manipulations. known examples of protein antigen production in chloroplasts of c. reinhardtii are b subunit of cholera toxin fused with the coat protein of foot and mouth disease virus [ ] or with d fibronectin binding domain of staphylococ cus aureus [ ] , as well as protein virus cryptokary osis (shrimp disease) [ ] and e protein of swine fever [ ] . the green moss physcomitrella patens is the only representative of bryophytes, the genome of which is currently completely sequenced and approaches to its transformation are developed. the peculiarity of this moss is that at the stage of the haploid juvenile game tophyte (protonema) this moss is morphologically similar to filamentous algae and easily enough culti vated in a bioreactor [ , ] . under certain culture conditions the moss can be in the stage of protonema indefinitely long. the attractiveness of this moss spe cies as the system for recombinant protein expression is that, as distinct from plants, fragments of foreign dna can be integrated in its genome through homo logical recombination, which reduces substantially a possibility of transferred gene inactivation. the p. pat ens cells are capable of postranslational modification of proteins of eukaryotic origin. since at the step of gametophyte the moss has the haploid number of chromosomes, it becomes possible to modify the func tioning of individual genes, in particular the moss lines conducting glycosylation of recombinant proteins as in mammalian cell type were obtained [ ] . the firm grenovation (germany) is developing the technology of biopharmaceutical protein production on the basis of p. patens in bioreactors. expression system for pro ducing biopharmaceuticals based on the green moss is not the part of the food chain and is characterized by a high degree of biosafety. as distinct from above described systems based on the stable expression of foreign genes integrated into nuclear or chloroplast genomes, during transient expression target proteins are synthesized in the plant cell during relatively short time (several days) without insertion into the plant genome. at present, the fol lowing approaches are used for transient gene expres sion in plants: gene delivery with the help of agrobac terium, the use of plant virus vectors, and magnifec tion [ ] [ ] [ ] . tobacco mosaic virus (tmv), potato x virus, alfalfa mosaic virus, and cowpea mosaic virus are used as virus vectors [ ] . the availability of infec tious cdna clones, the small size of the viral genomes, the short time required for the expression of a target gene, and a high level of expression provides for a high attraction of this system. the rapid development of this expression system led to substantial modifications of the first gene inser tion vectors or full virus vectors, which is a recombi nant virus that behaves as wild type virus but is capable of expressing additional genes. the next step was the creation of "disarmed vectors" (deconstructed vec tors) lacking a number of original virus genes, and gene replacement vectors, in which a portion of the viral genes is replaced by alien genes [ ] . viral vectors have several substantial disadvantages: a tendency to the loss of foreign insertion in the process of virus spreading over the plant and a potential risk for envi ronment related to the presence of infectious recombi nant viral particles. launch vectors represent an alternative to recom binant plant viruses; cdna of these viruses is deliv ered to plants within t dna region of agrobacterial ti plasmid. firstly, primary transcription of t dna occurs in the nucleus; then viral rna is released into the cytoplasm, where its further amplification, trans lation, and protein synthesis occur [ ] . by , the system of agroinfiltration based on the use of plant viruses and agrobacterial binary plasmids was upgraded and named as magnifection [ ] . at magnifection, multiple agrobacterial lines carrying different parts of the tmv genome are used simulta neously. after agrobacterial transfer into the plant cell nucleus, separate parts of viral genome are assembled in plants in the completely functional viral replicon [ ] . the substantial modification of the viral genome, including numerous point mutations for the removal of potential sites of splicing, intron insertion, and the removal of the gene encoding envelope proteins, pro vided for the highly efficient system capable of recom binant protein synthesis (up to g/kg of fresh tissue), which is more than % of tsp [ ] . among disadvantages of transient system is a necessity for recombinant protein isolation and purifi cation immediately after its accumulation in the plant, because, as distinct from seeds and fruits, plant leaves and stems cannot be stored for a long time. the systems of transient expression of recombinant proteins are rather promising in the cases when a rapid production of a small amount of proteins is required. experiments with transient expression in plants are held indoors, which reduces the risks associated with biosafety to almost zero. examples of manufacturing immunogenic proteins for veterinary using transient expression systems are presented in table . the sys tems of transient expression for the production of recombinant proteins are described in more details in reviews [ , , , , ] . "candidate" plant vaccines for veterinary use table presents examples of using various expres sion systems for the production of "candidate" plant vaccines for veterinary use. main specific immunogenic no. proteins synthesized at respective diseases (structural proteins, hemagglutinins, glycoproteins) are usually used as antigens. the most commonly used method of transgene construct delivering into plant cells is still the agrobacterial transformation. in some cases, the level of target protein expression was rather high [ ] [ ] [ ] [ ] , espe cially in the systems of transient expression [ ] [ ] [ ] , and suitable for product commercialization. in all experiments using the "candidate" plant vac cines, the formation of a specific immune response was demonstrated in vivo, and in most experiments immunogenic proteins were delivered to animals just orally. "candidate" plant vaccines were usually tested on mice, but in approximately a quarter works the ani mals subjected to the disease were tested. protein s of the transmissible swine gastroenteritis virus synthe sized in the cells of transgenic maize [ , , ] or tobacco [ ] and delivered into the body of pigs as a food supplement, provided for % survival of ani mals after infection [ ] . rabbit protein vp virus synthesized in potato [ ] and other plants (tobacco, pea, rape) [ ] and delivered orally enhanced protective immunity: after infecting rabbits with this virus all ani mals survived [ ] . the effect of plant recombinant antigen was comparable with commercially used vac cines. the use of plant vaccines for the vaccination of wild animals using edible baits (e.g., vaccine against rabies) will lead to an increase in the proportion of wild animal populations having immunity to the rabies virus. a potential possibility to reduce the cost of produc tion of biopharmaceutics using genetically modified plants served at the end of the xx century as an impe tus for more than twenty biotechnological companies to initiate commercial programs. as seen from the table , many biological products of plant origin are developed, expressed in different types of plants and plant cell cultures. for a variety of reasons, including the still skeptical attitude of the human community to the biosafety of genetically modified plants, many of these works remained in the framework of laboratory tests. at the moment three companies function on the biotechnology market of veterinary preparations, two from the united states and one from canada (table ). in the united states the dow agro sciences company presented a recombinant plant viral hn protein of the newcastle disease virus (approved by usda) and a mixture of antiviral vaccines at the first stage of clinical trials. the second american company at thomas jef ferson university has developed a plant anti rabies vaccine (completion of phase ). the canadian guardian biosciences company presented plant vac cine against chicken coccidiosis at the second phase of clinical trials. production and wide distribution of biopharmaceu ticals is hampered by a number of circumstances. the first of them is related to the problem of biosafetythe cultivation of genetically modified plants in the field can lead to the accidental introduction of foreign genes into crops grown for human consumption. therefore, most companies producing biopharma ceutics focused on plant species, which are absent from the food chain of humans and animals and also on growing of genetically modified plants preventing their cross pollination with other crops. the second difficulty is related to the necessity of plant material treatment for the removal of various undesired com pounds, such as lignin, proteases, phenolic com pounds, and pigments, especially in the case of plant species, which are not consumed. all these facts result in the requirement of additional studies. the third cir cumstance is due to the fact that until now all aspects of maintaining and growing of plants producing biop harmaceuticals are not settled at the legislative level. ambiguity and vagueness of the existing legislation in this area lead to the fact that large biopharmaceutical and biotechnology companies do not tend to invest in the development of technological lines and research programs in this area, which significantly inhibits the development of the industry. a significant problem for the development of vac cines for veterinary use, especially those used in agri culture, is the need to minimize the price of the final product. the vaccine should be inexpensive for entre preneurs engaged in commercial animal breeding and fully subsidized, if you intend to use the vaccine for mass immunization and the prevention of the disease spread in underdeveloped regions. as a result, the potential income of manufacturers of vaccines for ani mals is much less than that for vaccines intended for humans. for example, in , the volume of the mar ket for the vaccine against human papilloma virus was estimated at more than billion dollars, but the mar ket for the most popular animal vaccines (against foot and mouth disease of cattle and against mycoplasma hyopneumoniae in pigs) together amounted to only - % of this amount [ ] . animal vaccines are cheaper and the volume of market for them is less; therefore, the investments in their development are substantially less as compared with investments in the production of vaccines for humans, whereas the com plexity and diversity of both hosts and pathogens in the case of vaccines for animals is much higher. expression systems based on the use of plant cells still have a limited application or are used primarily in some laboratories. nevertheless, biopharming (the biotechnological production of various substances for medicine) in plants has attracted the attention of researchers and manufacturers in developed countries. first biopharmaceuticals of plant origin, such as anti bodies (anti hbsag required to purify the hepatitis b vaccine), therapeutic and dietary proteins ("intrinsic factor" required at vitamin b deficiency, gastric lipase), have entered the market, that is an excellent illustration of this progress [ ] . despite the fact that today the number of biophar maceutical proteins expressed in plant cells is enor mous, many questions still remain unresolved. the methods for target recombinant protein quantification and purification are still not developed for most of products. the problems of transgene silencing and increased expression of target protein genes are still at the stage of research. the important task that has yet to be solved is to achieve a stable level of expression in different batches of plant raw material. not much work appeared for judging about maintaining the sta bility of recombinant proteins after the harvest, pro cessing, and storage. all of these problems require additional expenses for further research. one of the main obstacles for the leading research groups working on the development and production of plant vaccines, given the financial constraints, is the fulfillment of the relevant official regulations govern ing the use of oral medications. to date, the purified vaccines and therapeutic proteins of plant origin must meet the same standards relating to the production, biosafety, purification, storage, dosage, etc. as any other recombinant proteins for medical purposes. nevertheless, despite these difficulties, there were first biopharmaceuticals of plant origin that passed all the necessary tests and were approved for use by the relevant authorities. some new products having spe cific advantages over similar products obtained in mammalian cell cultures were developed. such com panies as sembiosys genetics inc. (calgary, can ada), medicago inc. (quebec, canada), protalix bio therapeutics (karmiel, israel), and orf genetics (iceland) proved the possibility of quick establishing of the production of purified plant proteins, which are quite competitive in today's market. progress has been made in the formation of the legal framework related to the cultivation of transgenic plants, testing and use of plant biopharmaceuticals. several production pro cesses based on transgenic plants have already received a brand gmp (good manufacturing practice), the interest of manufacturers to this field of biotechnology began to increase again. this work was performed within the framework of the project vi. . . (no. ) development and improvement of genetic constructs to optimize the expression of target genes and the production of recombi nant proteins for medical purposes in transgenic plants and animals. plant produced vaccines: promise and reality evolution of plant made pharmaceu ticals current status of veterinary vac cines clinical trials fuel the promise of plant derived vaccines expression of hepatitis b surface antigen in transgenic plants oral immunization with a recombinant bacterial anti gen produced in transgenic plants edible vaccine protects mice against escher ichia coli heat labile enterotoxin (lt): potatoes express ing a synthetic lt b gene effi cacy of food plant based oral cholera toxin b subunit vaccine immunogenicity in humans of a recombinant bacte rial antigen delivered in a transgenic potato receptor mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chlo roplasts into the mouse circulatory system delivery of plant made vaccines and therapeutics defending the mucosa: adjuvant and carrier formula tions for mucosal immunity induction of secretory immunity and memory at mucosal surfaces, vaccine oral delivery of human biopharmaceuti cals, autoantigens and vaccine antigens bioencapsu lated in plant cells the mucosal immune response to plant derived vaccines posttranslational modifica tion of therapeutic proteins in plants seed based expression systems for plant molecular farming the economic potential of plant made pharmaceuticals in the manu facture of biologic pharmaceuticals rice based mucosal vac cine as a global strategy for cold chain and needle free vaccination secretory iga mediated protection against v. cholerae and heat labile enterotoxin producing enterotoxigenic escherichia coli by rice based vaccine stability of a soybean seed derived vaccine antigen following long term storage, processing and transport in the absence of a cold chain tomato is a highly effective vehicle for expression and oral immunization with norwalk virus capsid protein production of plant made pharma ceuticals: from plant host to functional protein post translational modifica tions in the context of therapeutic proteins current achievements in the production of complex biopharmaceuticals with moss bioreactors low dose oral immunization with lyophilized tissue of herbicide resistant lettuce expressing hepatitis b surface antigen for prototype plant derived vaccine tablet formulation rna mediated chromatin based silencing in plants transcriptional gene silencing in plants plant based production of biopharma ceuticals production of heterologous proteins in plants: strategies for optimal expression expression of heterologous genes in plant systems: new possibilities exhaustion of the chloroplast protein synthesis capac ity by massive expression of a highly stable protein anti biotic metabolic adaptation in transplastomic plants massively accumu lating recombinant proteins transplastomic plants overexpression of the bt cry aa operon a ¸n´l in chloroplasts leads to formation of insecticidal crys tals chloroplast vector systems for biotechnology applications determining the transgene containment level provided by chloroplast transformation expression of the native cholera toxin b subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts chloroplast expression of his tagged gus fusions: a general strategy to over produce and purify foreign proteins using transplas tomic plants as bioreactors accumulation of trehalose within transgenic chloro plasts confers drought tolerance plants as bioreactors for the production of vaccine anti gens towards high yield production of pharmaceutical proteins with plant cell suspension cultures position effects and epigenetic silencing of plant transgenes bioreactor systems for in vitro production of foreign proteins using plant cell cultures high expression of transgene protein in spirodela micro algae come of age as a platform for recombinant protein production foot and mouth disease virus vp pro tein fused with cholera toxin b subunit expressed in chlamydomonas reinhardtii chloroplast heat stable oral alga based vaccine pro tects mice from staphylococcus aureus infection factors effecting expression of vaccines in microalgae recombination and expression of classical swine fever virus (csfv) structural protein e gene in chlamydomonas rein hardtii chroloplasts viral vec tors for the expression of proteins in plants agrobacterium mediated transient expression as an approach to production of recombi nant proteins in plants a novel two component tobacco mosaic virus based vector system for high level expression of multiple ther apeutic proteins including a human monoclonal anti body in plants magnifec tion -a new platform for expressing recombinant vac cines in plants plant based vaccines: unique advan tages expression of the newcastle disease virus fusion protein in transgenic maize and immunological studies multimerization of peptide antigens for pro duction of stable immunogens in transgenic plants generation and immunogenicity of japanese encepha litis virus envelope protein expressed in transgenic rice induction of protective immunity in swine by recombinant bamboo mosaic virus express ing foot and mouth disease virus epitopes in planta production of two peptides of the classical swine fever virus (csfv) e glycoprotein fused to the coat protein of potato virus x expression in plants and immunogenicity of plant virus based exper imental rabies vaccine delivery of subunit vaccines in maize seed a corn based deliv ery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immu nity in swine immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants oral immunization using tuber extracts from transgenic potato plants expressing rabbit hemorrhagic disease virus capsid protein pea derived vaccines demonstrate high immunoge nicity and protection in rabbits against rabbit haemor rhagic disease virus plant made pharmaceuti cals: leading products and production platforms pro duction of immunogenic vp protein of bovine group a rotavirus in transgenic potato plants rotavirus vp expressed by pvx vectors in nicotiana benthamiana coats pvx rods and also assembles into viruslike parti cles expression of rotavirus capsid protein vp in transgenic potato and its oral immuno genicity in mice protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants bovine herpes virus gd protein pro duced in plants using a recombinant tobacco mosaic virus (tmv) vector possesses authentic antigenicity induction of a protective antibody response to foot and mouth disease virus in mice fol lowing oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein vp induction of a protective antibody response to fmdv in mice following oral immunization with transgenic stylosanthes spp. as a feedstuff additive expression of hemagglutinin protein of rinderpest virus in transgenic tobacco and immunogenicity of plant derived protein in a mouse model systemic and oral immunogenicity of hemagglutinin protein of rinderpest virus expressed by transgenic peanut plants in a mouse model expression of hemagglutinin protein of rinderpest virus in trans genic pigeon pea oral immunogenicity of the plant derived spike protein from swine transmissible gastroenteritis coronavirus cloning and sequence analysis of the korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutraliz ing epitope in plants successful oral prime immunization with vp from rabbit haemorrhagic disease virus pro duced in transgenic plants using different fusion strate gies mucosal and sys temic immunization elicited by newcastle disease virus (ndv) transgenic plants as antigens expres sion of the fusion glycoprotein of newcastle disease virus in transgenic rice and its immunogenicity in mice expression of immunogenic s glycoprotein of infectious bronchitis virus in trans genic potatoes transient expression of the ectodomain of matrix protein (m e) of avian russian immunization with plant expressed hemagglutinin protects chickens from lethal highly pathogenic avian influenza virus h n challenge infection immunogenicity study of plant made oral subunit vaccine against porcine reproductive and res piratory syndrome virus (prrsv) expression of the rabies virus glycoprotein in transgenic tomatoes immunization against rabies with plant derived antigen development of an edible rabies vaccine in maize using the vnukovo strain induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein expression of the rabies virus nucleoprotein in plants at high levels and evaluation of immune responses in mice expression of rabies virus g pro tein in carrots (daucus carota) key: cord- -ozx u t authors: sheppard, michael title: viral vectors for veterinary vaccines date: - - journal: adv vet med doi: . /s - ( ) - sha: doc_id: cord_uid: ozx u t nan numerous reviews have described the use of viral vectors for possible vaccine delivery (e.g., cavanagh, ; sheppard and fahey, ; wray and woodward, ; graham and prevec, ; boyle and heine, ; hilleman, ; martin, ; dorner, ; babiuk et al., ; perkus and paoletti, ) . however, in this review i will focus solely on the use of viral vectors for delivery of veterinary vaccines. it is without question that vaccination plays an essential role in veterinary medicine, providing the major and often the only prophylatic approach for the control of infectious diseases. in spite of the vast array of currently available vaccines veterinarians and the livestock producers continue to express the need for vaccines that not only maintain the best features of killed or subunit vaccines (such as safety) as well as the best features of conventional modified live vaccines (such as efficacy) but improve on them. as well as the need for continual improvement of vaccines there exists a need for new vaccines either to new diseases (e.g., chicken anemia virus or porcine reproductive and respiratory syndrome virus) or to old diseases for which vaccines are not available or no longer meet the requirements of the end user (e.g., bovine virus diarrhea virus vaccines). as well as new vaccines there is also need for vaccines with special features that allow potential customers to design disease control programs that suit their specific needs on top of offering greater safety and improved protection. the design and construction of these new veterinary vaccines is a major challenge facing the field ofvaccinology. with the continued demand of improving vaccines and producing new ones it is easier for potential vaccine candidates to fail to meet the increased level of requirements that are expected. the failure of some vaccines can result from problems associated with delivery, such as insufficient or no induction of the appropriate protective immune response. the development of delivery systems to produce vaccines that are more effective, offer greater safety, are convenient to administer, and are compatible with customer practices is part of the challenge for vaccinologists. the development of safe and convenient live viral vectors for the delivery of veterinary vaccines is one possible way of meeting some of these challenges. recombinant dna technology has allowed more detailed characterization of the genetic organization of many viruses to such an extent that regions suitable for insertion of foreign genetic material have been identified. this has resulted in the development of numerous types of viral vectors from a wide variety of viral families. some of these viral vectors have been developed with the potential for delivering and expressing gene(s) from a foreign pathogen and so act as a vaccine vector (table i) . the viral vector is often genetically attenuated or cannot complete its replication cycle in the animal to be immunized, and thus produces no clinical disease. although initially the majority of viral vector development centered around poxviruses, especially vaccinia (panicali and paoletti, ; macket et al., ) , it was not long before viral vector development witnessed a virtual explosion in the types of viruses developed into vectors. these included herpeviruses (post et al., ) , adenoviruses (berkner and sharp, ) , retroviruses (wei et al., ) , papoviruses (southern and berg, ) , polyoma virus (fried and ruley, ) , picornaviruses (kitson et al., ), semliki forest virus (sfv; zhou et al., ) , sindbis virus (pugachev et al., ) , and even some plant viruses (jagadish et al., ; dalsgaard et al., ) . greater understanding of the structure and function of a wide range of viruses at the genetic level has opened up ways of designing novel viral vaccine vectors which should improve the quality and effectiveness of some future vaccines as major prophylatic tools. viral vaccine vectors have really developed from a greater technological understanding of viruses at the genetic level, where today they have become a viable alternative strategy as one method for the delivery of vaccines. the concept of viral vectors was first highlighted by bernard moss and others in the early s (mackett et al., ; panicali and paoletti, ) , where they showed that vaccinia virus could be engineered to carry and express foreign genes (panicali and paoletti, ; mackett et al., ) . from the time when moss and others first demonstrated that vaccinia virus could be developed as a vector for the expression of foreign genes, the technology has been exploited to apply to a variety of virus families as well as a variety of foreign genes including those that encode antigens from pathogens. as a result both dna and rna viruses have been developed as viral vaccine vectors (table i) . to produce viral vaccine vectors it is first necessary to study the genome of the vector to a stage of understanding where at least one region suitable for insertion of foreign genetic material has been iden-tiffed. second, genes from pathogens that encode proteins that will induce an appropriate protective immune response and can be stably integrated into the vector's genome and expressed need to be identified. finally, it is necessary to insert the foreign gene(s) in such a way as to ensure the correct and sufficient expression of the foreign gene(s). the ideal viral vaccine vector would have all or at the very least some of the following features: safe and nonpathogenic for the vaccinate evoke the appropriate protective immune response single host or limited host range stable genome no integration into the host genome readily accessible region(s) for insertion of foreign genetic material able to tolerate well insertion of foreign genetic material and expression of foreign gene(s) convenient to deliver and fits with management practices relatively simple and cost effective to produce limited background gene expression by the vector live viral vectors offer several advantages for vaccine delivery compared to killed, subunit, or conventional modified live vaccines. first, because of the possibility of delivering divalent or even perhaps multivalent vaccines, using a single type of vector can result in a single manufacturing process rather than several and possibly even a single vaccination rather than several. therefore, vectored vaccines have the potential to be less expensive to the manufacturer and the end user. because the foreign gene is being expressed in the cells of its natural host, it is expected that any post-translational modifications required will be correct and produce an authentic antigen, as opposed to escherichia coli or baculovirus systems (among others) that do not always produce authentic foreign proteins. depending on the vector selected it may be possible to deliver the vectored vaccine more conveniently to the mammal or bird by spray or water or some other means rather than by needle injection. such a mass administration approach may be particularly relevant to the poultry industry. the vector could also be constructed to deliver simultaneously an immunomodulator (e.g., gamma interferon), which could modify the type or magnitude of the immune response to allow the vaccine to be successful or more successful than it would be otherwise. the vector only expresses the antigens from the pathogen that are required to elicit a protective immune response and therefore reduces or eliminates the chance of disease by being exposed to the whole pathogen as with a killed or modified live vaccine. finally, the appropriate viral vectors will induce both cell-mediated and humoral immune responses and in some cases are particularly suitable for inducing a local immune response in the mucosal surfaces. one of the main disadvantages of using viral vectors for vaccine delivery is that like subunit vaccines each vector can only deliver one or a relatively small number of foreign antigens to the host animal and therefore rely on those being able to elicit a completely protective immune response. also the only antigens that can be delivered are those that are encoded by nucleic acid. thus such things as lipopolysaccharides are not deliverable. with any vector, regardless of type, only a limited amount of foreign genetic material can be inserted into the vector's genome stably and expressed appropriately. one must always be wary of altered tissue tropisms due to the expression of the foreign gene(s). of course the effectiveness of a viral vector is limited by preexisting immune response in the animal from prior exposure to the virus used to construct the vector. finally, as with all live vaccines there is the problem of shelf life and compatibility with other vaccine preparations. to produce viral vaccine vectors successfully it is necessary to ensure that the vector itself does not pose any disease threat to the animal that receives the vaccination or to the person delivering the vaccine to the animal. most often this is achieved by attenuating the viral vector in some way. until recent times the means of generating a live attenuated virus had been entirely empirical. this process usually involved the passaging of the virus in cell culture or animals that were not the natural host, followed by testing of the resulting viruses for decreased virulence in the natural host. the basis for attenuation is most often unknown, and may be a result from as minor as a single base change, and thus the chance of reversion back to virulence is always a possibility. this type of traditional method for generating a live attenuated virus is not necessarily the most attractive method for generating a viral vaccine vector. with the advent of molecular biology and our improved knowledge of viruses at the genetic level it is now possible to generate live attenuated viruses with precise genetic changes, improving their safety and thus make them more suitable as vectors for vaccine delivery. a good example is the deletion of the thymidine kinase (tk) gene. while the deletion of the tk gene has little or no effect on virus growth in cell culture, tk deleted viruses can be significantly attenuated in vivo ((buller et al., ; kit et al., kit et al., , becker et al., ) . this feature has been exploited successfully for the development of live attenuated herpesvirus vaccines (mcgregor et al., ; kit et al., ; marchioli et al., ; moorman et al., ) as well as safer herpesvirus and poxvirus vectors (e.g., buller et al., ; bayliss et al., ; mulder et al., ; hu et al., ) . if an essenential gene is deleted from a virus, the virus can only grow if the gene or gene product is provided in trans. this virus is phenotypically normal but genotypically defective and cannot replicate in the host because the deleted gene product is not available. this type of virus can replicate in vitro with the help of a genetically engineered supporting cell line that expresses the deleted gene product. the stage of the virus life cycle of which the gene product is required will govern how far through the replication cycle a virus will proceed. in some cases (e.g., if the essential deleted gene is required for virus penetration of the cell) the virus will complete a single round of replication in the host but the progeny viruses will not be able to invade any other cell. (farrell et al., ; mclean et al., ; peeters et al., ) . however, if the deleted essential gene is an early gene that is required to activate other viral genes, then the number of viral proteins synthesized may be limited and the viral genome may not be able to complete even a single cycle of replication (chen and knipe, ; brehm et al., ; da costa et al., ) . a third alternative, which has been exploited successfully, is to use a virus that can only completely replicate in one species as a vector in another species, where it cannot complete an entire cycle of replication but can commence a replication cycle sufficiently to allow expression of the foreign gene (tartaglia et al., ) . the canarypox virus (cpv) vector, termed alvac, has successfully been exploited to the degree of commercial success. the cpv is restricted to avian cells only for productive replication but can be used to vaccinate mammals where it can elicit an immune response to the foreign gene product without completing an entire cycle of replication (tartaglia et al., (tartaglia et al., , taylor et al., ) . the human adenovirus type (hav- ) has also been exploited in a similar fashion to the cpv (table iii ) but has the disadvantage that this virus is a human pathogen and so has yet to be exploited commercially. several other strategies are also available and in some cases have been exploited successfully in order to generate safe viral vectors for vaccine delivery. table v (next section) provides a summary of some of these possible approaches. even though there are a great many examples of viral vectors reported in the literature since they were first described in , the number of publications reporting the use of viral vectors for veterinary vaccine delivery is not that large. after searching for published papers that describe viral vectors with veterinary vaccine applications, especially those that could be described as purposely developed for veterinary use, the obvious conclusion was that even though this research was first described in the veterinary side is still in its infancy. publications describing viral vectors for veterinary vaccine delivery can be divided into several groups, which are represented in the following tables: table ii, poxvirus vectors; table iii, adenovirus vectors; table iv, herpesvirus vectors; and table v , other virus vectors. although these four tables probably do not include every single publication describing viral vectors for veterinary vaccine delivery they do describe the majority of published papers and present the reader with an idea of the limited amount of research that has occurred in this field during the last years. at the time of writing this review only three viral vectored vaccines for use in the veterinary field have been licensed for release. all three are based on poxvirus vectors and the three vectors represent the gonin et al., ( ) hav- prcv spike swine callebaut et al., ( ) hav- prv gd swine adam et al., ( ) hav- rabies g dog prevec et al., ( ) hav- prv gp rabbit/mouse eloit et al., ( ) key: oav, ovine adenovirus; hav- , human adenovirus type ; prcv, porcine respiratory corona virus; tge, transmissible gastroenteritis virus; bcv, bovine corona virus; prv, pseudorabies virus. evolution in poxvirus vector development. the first vector approved was the vaccinia virus vector carrying the rabies g glycoprotein gene (e.g., kieny et al., ; blancou et al., ; brochier et al., ) . in terms of complying with the characteristics of a desirable vector for vaccine delivery in the veterinary setting, this vector has the greatest number of undesirable characteristics. however, it satisfied an unmet need and as a result was released in various parts of the world. the second vector to be licensed for release was the fowlpox virus vector. this vector delivers the newcastle disease virus hn and f genes and is designed to vaccinate poultry (e.g., boursnell et al., a,b; taylor et al., ) . while this vector has the desirable characteristic of only replicating in poultry it also has some limitations that affect its use in the field. the third vector licensed is the canarypox virus vector and represents the state-of-the-art poxvirus vector. this vector was developed to deliver the ha and f genes of canine distemper virus and is the most recently available of the three vector vaccines (e.g., stephensen et al., ) . whatever strategy is adopted for the development of viral vectors for delivery of veterinary vaccines there are several key points to consider: ( ) will the vectored vaccine give a delivery advantage compared to ( ( ) fink et al. ( pechan et al. ( ( ) starr et al. ( jagadish et al. ( ( ) porter et al. ( roy ( what's already available? ( ) will the vectored vaccine give a manufacturing advantage compared to what's already available? ( ) will the vectored vaccine provide improved safety compared to what's already available? ( ) will the vectored vaccine increase the duration of immunity compared to what's already available? ( ) will the vectored vaccine be more convenient to store compared to what's already available? ( ) is the vectored vaccine compatible with other vaccines? if there is no other alternative available then the answer to these questions is easy. however, if there are alternative vaccines available then the answers to these questions become very important because the answers will determine whether a vectored vaccine is merely a good laboratory idea or a successful vaccine. vaccination of pigs with replicationdefective adenovirus vectored vaccines: the example of pseudorabies manipulation of the semliki forest virus genome and its potential for vaccine construction novel viral vaccines for livestock induction of mucosal immunity in cotton rats to haemagglutinin-esterase glycoprotein of bovine coronavirus by recombinant adenovirus protective immune responses induced by the immunization of mice with a recombinant bacteriophage displaying an epitope of the human respiratory syncytial virus a recombinant fowlpox virus that expresses the vp antigen of infectious bursal disease virus induces protection against mortality caused in the virus a sequence in hpai p fragment of herpes simplex virus dna determines intraperitoneal virulence in mice preparation of adenovirus recombinants using plasmids of viral dna oral vaccination of the fox against rabies using a live recombinant vaccinia virus a fowlpox virus vaccine vector within insertion sites in the terminal repeats: demonstration of the efficacy using the fusion gene of newcastle disease virus a recombinant fowlpox virus expressing the hemagglutinin-neuraminidase gene of newcastle disease virus (ndv) recombinant fowlpox virus vaccines for poultry immunization with a replication-deficient mutant of herpes simplex virus type (hsv- ) induces a cd + cytotoxic t-lymphocyte response and confers a level of protection comparable to that of wild-type hsv- large-scale eradication of rabies using recombinant vaccinia-rabies vaccine decreased virulence of recombinant vaccinia virus expressed vectors is associated with a thymidine kinase negative phenotype construction of a recombinant adenovirus for the expression of the glycoprotein s antigen of porcine respiratory coronavirus an adenovirus recombinant expressing the spike glycoprotein of porcine respiratory coronavirus is immunogenic in swine viral and bacterial vectors of immunogens a dominant mutant form of the herpes simplex virus icp protein decreases viral late gene transcription construction and characterization of a replication-defective herpes simplex virus icp mutant strain and its use in immunization studies in a guinea pig model of genital disease plant-delivered vaccine protects target animals against a viral disease an overview of vaccine vectors construction of a defective adenovirus vector expressing the pseudorabies virus glycoprotein gp and its use as a live vaccine vaccine potential of a herpes simplex virus type mutant with an essential gene deleted gene transfer to neurons using herpes simplex virus based vectors a recombinant canarypox virus protects rabbits against a lethal rabbit hemorrhagic disease virus (rhdv) challenge minireview: the herpes simplex virus amplicon--a versatile defective virus vector use of polyoma virus vector feline leukemia virus envelope protein expression encoded by a recombinant vaccinia virus: apparent lack of immunogenicity in vaccinated animals immunization trial of cats with a replication defective adenovirus type expressing the env gene of feline immunodeficiency virus adenovirus-based expression vectors and recombinant vaccines expression in recombinant vaccinia virus of the equine herpesvirus gene encoding glycoprotein and protection of immunized animals recombinant vector vaccines in vaccinology raccoon poxvirus live recombinant feline panleukopenia virus vp and rabies virus glycoprotein bivalent vaccine retrovirus-expressed hemagglutinin protects against lethal influenza virus infections chimeric potyvirus-like particles as vaccine carriers myxoma virus as a vaccine vector for rabbits--antibody levels to influenza virus hemagglutinin presented by a recombinant myxoma virus expression of rabies virus glycoprotein from a recombinant vaccinia virus bovine herpesvirus- (infectious bovine rhinotracheitis virus)-based viral vectors which expresses foot-andmouth disease epitopes attenuated properties of thymidine kinasenegative deletion mutant of pseudorabies virus intramuscular and intravaginal vaccination of pregnant cows with thymidine kinase-negative, temperature-resistant infectious bovine rhinotracheitis virus (bovine herpesvirus ) modified-live infectious bovine rhinotracheitis virus vaccine expressing monomer and dimer forms of footand-mouth disease cupsid protein epitopes on surface of hybrid virus particles expression of porcine pseudorabies virus genes by a bovine herpesvirus- (infectious bovine rhinotracheitis virus) vector chimeric polioviruses that include sequences derived from two independent antigenic sites of foot-and-mouth disease virus (fmdv) induce neutralizing antibodies against fmdv in guinea pigs biological evaluation of glycoproteins mapping to two distinct mrnas within the bamhi fragment of pseudorabies virusi expression of the coding regions by vaccinia virus construction of a pigeonpox virus recombinant: expression of the newcastle disease virus (ndv) fusion glycoprotein and protection of chickens against ndv challenge vaccinia virus: a selectable eukaryotic cloning and expression vector a vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein x genes vaccine design: future possibilities and potentials vaccination of swine with thymidine kinase-deficient mutants of pseudorabies virus protective vaccination against primary and recurrent disease caused by herpes simplex virus (hsv) type using a genetically disabled hsv- inactivation of the thymidine kinase gene of a gi deletion mutant of pseudorabies virus generates a safe but still highly immunogenic vaccine strain retroviral expressed hemagglutinin-neuraminidase protein protects chickens from newcastle disease virus induced disease protection against lethal simian immunodeficiency virus sivsmmpbjl disease by a recombinant semliki forest virus gpl vaccine and by a gpl subunit vaccine virulence and pathogenesis of non-virulent and virulent strains of pseudorabies virus expressing envelope glycoprotein e of hog cholera virus induction of neutralizing antibodies against bovine leukosis virus in rabbits by vaccination with recombinant vaccinia virus expressing bovine leukosis virus envelope glycoprotein construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the dna of infectious vaccinia virus a novel 'piggyback' packaging system for herpes simplex virus amplicon vectors non-transmissible pseudorabies virus gp mutants: a new generation of safe live vaccines recombinant virus as vaccination carrier of heterologous antigens release of viruslike particles from cells infected with poliovirus replicons which express human immunodeficiency virus type gag chicken ovalbumin gene fused to a herpes simplex virus a promoter and linked to a thymidine kinase gene is regulated like a viral gene a recombinant human adenovirus vaccine against rabies double-subgenomic sindbis virus recombinants expressing immunogenic proteins of japanese encephalitis virus induce significant protection in mice against lethal jev infection protective efficacy at a recombinant herpesvirus of turkeys as an in ovo vaccine against newcastle and marek's disease in specific-pathogen-free chickens construction and properties of a turkey herpesvirus recombinant expressing the marek's disease virus homologue of glycoprotein b of herpes simplex virus sequential nucleic acid and recombinant adenovirus vaccination induces host-protective immune responses against taenia ovis infection in sheep genetically engineered particulate virus-like structures and their use as vaccine delivery systems hepatitis b virus core particles as a vaccine carrier moiety immunity to malaria elicited by hybrid hepatitis b virus core particles carrying circumsporozoite protein epitopes herpesviruses and adenoviruses as potential vectors for the poultry industry long-term expression in sensory neurons in tissue culture from herpes simplex virus type (hsv- ) promoters in an hsv-l-derived vector mammalian cell transformation with sv vector longterm persistence of defective hsv- vectors in the rat brain is demonstrated by reactivation of vector gene expression canine distemper virus (cdv) infection of ferrets as a model for testing morbillivirus vaccine strategies: nyvac-and alvac-based cdv recombinants protect against symptomatic infection nyvacp: a highly attenuated strain of vaccinia virus protection of cats against feline leukemia virus by vaccination with a canarypox virus recombinant, alvac-fl recombinant fowlpox virus inducing protective immunity in non-avian species biological and immunogenic properties of a canarypox-rabies recombinant, alvac-rg (vcp ) in non-avian species efficacy of a recombinant fowlpox-based newcastle disease virus vaccine candidate against velogenic and respiratory challenge evaluation of swinepox virus as a vaccine vector in pigs using aujeszky's disease (pseudorabies) virus gene insert coding for glycoproteins gp and gp live attenuated pseudorabies virus expressing envelope glycoprotein e of hog cholera virus protects swine against both pseudorabies and hog cholera the use of feline herpesvirus and baculovirus as vaccine vectors for the gag and env genes of feline leukemia virus construction and isolation of a transmissible retrovirus containing the src gene of harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type vaccination against feline leukemia using a new feline herpesvirus type i vector biotechnology and veterinary science: production of veterinary vaccines human adenovirus type vectors expressing rabies glycoprotein self replicating semliki forest virus rna as recombinant vaccine generation of cytotoxic and humoral immune responses by nonreplicative recombinant semliki forest virus key: cord- - u ax authors: shrestha, ashish c.; wijesundara, danushka k.; masavuli, makutiro g.; mekonnen, zelalem a.; gowans, eric j.; grubor-bauk, branka title: cytolytic perforin as an adjuvant to enhance the immunogenicity of dna vaccines date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: u ax dna vaccines present one of the most cost-effective platforms to develop global vaccines, which have been tested for nearly three decades in preclinical and clinical settings with some success in the clinic. however, one of the major challenges for the development of dna vaccines is their poor immunogenicity in humans, which has led to refinements in dna delivery, dosage in prime/boost regimens and the inclusion of adjuvants to enhance their immunogenicity. in this review, we focus on adjuvants that can enhance the immunogenicity of dna encoded antigens and highlight the development of a novel cytolytic dna platform encoding a truncated mouse perforin. the application of this innovative dna technology has considerable potential in the development of effective vaccines. vaccines represent an effective strategy in the fight against infectious diseases and recent estimates suggest that vaccination prevents - million deaths every year [ ] . the need for rapid and large scale vaccine production during epidemics against emerging pathogens is a major challenge in vaccine development [ ] , including effective vaccines for antigenically diverse and versatile pathogens that successfully subvert host immunity such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), and malaria [ ] [ ] [ ] . dna vaccines can overcome some of these challenges, as it is relatively easy to produce large number of doses within a short period of time, and they are stable at ambient temperature and do not require cold chain transportation. they are also consistent between lots and have an excellent safety profile allowing for safety evaluations by regulatory authorities and distribution in a large scale [ , ] . importantly, dna vaccines can induce both humoral and cell-mediated responses in the vaccinated host [ ] [ ] [ ] . although they are safe and well tolerated, they are often poorly immunogenic and inefficacious in humans [ ] . therefore, recent studies on the advancements of dna vaccines are focused on effective delivery and increasing the immunogenicity of the encoded antigen/s of interest [ , ] . effective immunization with dna vaccines requires efficient transfection of host cells which is highly dependent on the delivery route and use of devices. conventional delivery routes to introduce the dna vaccine include intramuscular, intradermal, subcutaneous and oral routes [ ] . the preferred delivery route depends on the requirement to activate specific immune cells. the skin is rich in immune cells including local dendritic cells (dcs) and natural killer (nk) cells, and is therefore likely to be a more favorable site for vaccine delivery [ , ] . attempts to improve dna delivery have been made through other physical methods with the use of 'gene guns' or electroporation, which transiently permeabilizes the cell membrane to efficiently transfer the dna resulting in increased vaccine uptake by skin and muscle cells [ ] . although these methods have shown to increase dna uptake [ , ] , they require optimization to achieve increased efficiency and acceptance for clinical use. an alternative approach to improve transfection efficiency includes formulation of dna with liposomes or nanoparticles [ ] . liposomal delivery can be affected by pre-systemic (epithelial) and systemic barriers (enzymatic degradation, binding, and opsonization) [ ] . encapsulation of dna with nanoparticles has been reported to increase dna uptake or transfection efficiency [ , ] . some of the challenges in the use of nanoparticles with dna include encapsulation inefficiency, endocytosis by target cells and toxicity [ ] . the use of genetic adjuvants is one approach to enhance the immunogenicity of the antigen and can be used to complement other strategies (e.g., dna delivery) also designed to improve the immunogenicity of dna vaccines. upon immunization with a dna vaccine, the target cells uptake dna by endocytosis [ ] and the transfected cells express the dna-encoded protein antigen(s). when antigen-presenting cells (apcs) are directly transfected, the intracellular proteins are processed and immunogenic epitopes are then presented by mhc class i molecules, which can directly stimulate naïve cd + t cells [ , ] . the protein immunogen released from transfected cells can be endocytosed and/or phagocytosed by other apcs and are presented by mhc class ii molecules to activate naive cd + t cells [ , ] . if the proteins are expressed by stromal cells like keratinocytes, apcs can also indirectly capture secreted antigens and cross-present by mhc class i molecules to further stimulate cd + t cells [ ] . after dna vaccination, cd + t cells specific to the vaccine antigen undergo expansion, acquire effector functions and differentiates into memory cd + t cells [ , ] . the memory cells differentiate into effector memory t cells upon re-exposure to the antigen [ , ] . the ability of a dna vaccine to elicit t cell immunity is thus dependent on activating apcs to present antigen: mhc complexes to t cells [ ] and adjuvants can serve as an important costimulatory factor to enhance this process. in this brief review, based on our experience, we discuss the progress in the development of dna vaccines, approaches to improve delivery and genetic adjuvants used to enhance immunogenicity. we focus on an innovative cytolytic dna technology developed and patented in our laboratory. the immunogenicity of dna vaccines is enhanced by cpg motifs present in the plasmid backbone, which can activate apc via toll like receptors (tlr ) [ ] [ ] [ ] . unmethylated cpg motifs have been reported to induce b cell proliferation and secretion of immunoglobulin in vitro and in vivo [ ] . activation of macrophages and dcs results in upregulation of antigen presentation and costimulatory molecules, and secretion of cytokines (il- and il- ) involved in helper t cells (th ) response [ ] . thus, cpg motifs in dna plasmids serve as a 'natural adjuvant' for dna vaccines. plasmid dna can be designed to encode additional adjuvants with the antigen(s) of interest. molecular adjuvants such as fusion proteins including heat shock protein (hsp ), and vesicular stomatitis virus (vsvg) have been developed and used to enhance vaccine immunogenicity [ ] [ ] [ ] . the gene encoding such proteins as adjuvants is either fused with the gene encoding the vaccine antigen to produce a fusion protein driven by a same promoter or as separate proteins driven by different promoters in the same or different plasmid. co-encoding of genes creates a suitable cellular micro environment such as sustained antigen release and/or upregulation of cytokines, enhancing the immunogenicity of dna vaccines [ ] . a majority of the studies on experimental dna vaccines with genetic adjuvants have been studied in animal models such as mice (table ) , and very few of these have been clinically tested ( table ) . limited published data on clinical trials pose difficulty to compare efficacy between adjuvants in animals and humans. different cytokines, such as interleukins (il- , il- , il- ), chemokines, granulocyte/macrophage colony-stimulating factor (gm-csf), costimulatory molecules (cd , cd , and cd ), and signaling molecules (interferon regulatory factor - ) have been used as genetic adjuvants with dna vaccines [ , , [ ] [ ] [ ] , ] . genes expressing ifn-γ il- , il- , il- , and il- have been used to stimulate th responses [ , , ] , and il- , il- , il- , il- , for th stimulation [ , , [ ] [ ] [ ] . the inclusion of genes encoding cytokines, like il- or il- , as adjuvants for hiv- dna vaccines is known to increase cell mediated immunity (cmi) [ , ] . however, a bicistronic hiv dna encoding gp and il- elicited weaker specific immune response than monocistronic hiv- gp dna [ ] . combinations of genetic adjuvants like il- and il- with hiv- dna vaccine have also been used but no synergistic effect on the level of total antibody to hiv- antigen was reported [ ] . a phase i/iia trial showed that coadministration of dna vaccine encoding prostatic acid phosphatase (pap) with gm-csf elicited pap-specific cd + and/or cd + t cell responses [ ] . however, gm-csf was administered as a recombinant protein. hsp , a class of molecular chaperone, is known to induce maturation of dcs and activation of the th pathway [ ] [ ] [ ] . a fusion vaccine for multiple myeloma termed hdkk -hhsp was shown to be effective in inhibiting the targeted tumor and increased survival of vaccinated mice by eliciting tumor-specific humoral and cellular immune responses [ ] . however, a dna vaccine encoding hpv e fused with hsp , targeting hpv and cervical intraepithelial neoplasia / failed to enhance significant t cell responses in a phase i clinical trial [ ] . a bicistronic dna encoding hsp as a membrane bound or secreted protein has been used to improve the immunogenicity of a hiv gag [ ] . in this case, hsp expression was driven by a weaker sv promoter and hiv gag by a stronger cmv promoter. such a vaccine design enhanced gag-specific t cell responses, providing greater protection in mice challenged with ecohiv [ ] . ecohiv is a chimeric virus containing the envelope protein gp of mouse leukemia virus rather than hiv gp that can replicate in mouse leukocytes in vivo, thus representing a viable mouse challenge model for early assessment of hiv vaccines [ ] . the proposed mechanism of hsp as an adjuvant is that tlr / on dcs interacts with secreted or bound hsp , further attracting dcs to the site of antigen expression. this is followed by dc maturation, presentation of antigens by mhc molecules and secretion of cytokines and costimulatory molecules [ ] , thus enhancing t cell immune responses against the vaccine antigen. a chimeric version of the oligomerization domain from the chicken complement inhibitor (c bp) was used to produce an oligomeric form of vaccine antigens [ , ] . this protein, termed imx , forms a heptameric structure of the vaccine protein. this has been used to develop dna vaccines for tuberculosis, malaria and hiv to enhance humoral and/or cellular responses [ , , ] . vaccination with a dna vaccine encoding secreted hiv tat (tpa-tat imx ) induced higher humoral and cellular responses and improved protection against ecohiv challenge in mice [ ] . a phase i clinical trial of tuberculosis vaccine mva a-imx evaluated the vaccine to be safe and immunogenic, but cellular (ag a-specific ifn- [ ] . the proposed mechanism of hsp as an adjuvant is that tlr / on dcs interacts eted or bound hsp , further attracting dcs to the site of antigen expression. this is by dc maturation, presentation of antigens by mhc molecules and secretion of cytokines mulatory molecules [ ] , thus enhancing t cell immune responses against the vaccine en complement inhibitor imeric version of the oligomerization domain from the chicken complement inhibitor as used to produce an oligomeric form of vaccine antigens [ , ] . this protein, termed forms a heptameric structure of the vaccine protein. this has been used to develop dna for tuberculosis, malaria and hiv to enhance humoral and/or cellular responses [ , , ] . on with a dna vaccine encoding secreted hiv tat (tpa-tat imx ) induced higher and cellular responses and improved protection against ecohiv challenge in mice [ ] . a inical trial of tuberculosis vaccine mva a-imx evaluated the vaccine to be safe and enic, but cellular (ag a-specific ifn-Ƴ ) and humoral (mva-specific igg) responses were ficant [ ] . thus, the ability of such an adjuvant to enhance immunogenicity of dna n humans is still debated. g is a type iii viral fusion envelope protein, which mediates fusion of the virus envelope ost cell membrane [ ] . the use of fusogenic membrane glycoprotein (fmg) gene from vsv vaccine encoding the h protein of human papillomavirus type was shown to enhance ell responses and effectively control growth of tumors [ ] . the vsv g protein was also induce a t-response at low doses or a t-independent response at higher doses [ ] . fmg s into large multinucleated syncytia, which are then killed by a nonapoptotic mechanism ytiosomes are released from the membrane and present antigens efficiently to dcs [ ] . thus act as an adjuvant for any antigen expressed by a dna vaccine. vaccination of mice hepatitis c virus (hcv) nonstructural protein (ns ) together with vsvg resulted in frequency of ifn-γ, but not tnf-α-or il- -producing cd + cd + cd + effector memory lls [ ] . this dna vaccine was constructed in a bicistronic vector with vsvg co-encoded e plasmid with hcv ns . vsvg expression was driven by a weaker sv promoter and stronger cmv promoter [ ] . however, others have also reported an increase in the specific n the immunogen and vsvg were expressed from different plasmids [ , ] . tic protein rin (prf) is a pore forming protein released by immune cells including nk cells and t lymphocytes (ctl) [ ] . the -kilodalton prf protein oligomerizes to form pores that channel to release granzyme into the cytosol of target cells [ ] . suicide genes inducing of target cells have been used for cancer therapies, and the role of apoptotic cell death in n, whether immune-stimulatory or immune-suppressive, is debated [ ] . recently, a novel hnology has been developed--termed cytolytic dna technology-in which a truncated f is incorporated in a bicistronic dna vector to act as a vaccine adjuvant [ ] [ ] [ ] ] . lytic dna vaccines are based on a bicistronic dna plasmid constructed on a pvax (invitrogen) with a cytomegalovirus (cmv) promoter and a simian virus (sv ) promoter gene encoding the protein of interest as an immunogen is inserted downstream of the cmv and the gene encoding a truncated version of mouse prf downstream of the sv promoter . the sv is a weaker promoter compared to cmv and has been shown to result in -fold tein expression in transfected hek t cells in vitro [ , ] . ) and humoral (mva-specific igg) responses were not significant [ ] . thus, the ability of such an adjuvant to enhance immunogenicity of dna vaccines in humans is still debated. vsv g is a type iii viral fusion envelope protein, which mediates fusion of the virus envelope and the host cell membrane [ ] . the use of fusogenic membrane glycoprotein (fmg) gene from vsv in a dna vaccine encoding the h protein of human papillomavirus type was shown to enhance cd + t cell responses and effectively control growth of tumors [ ] . the vsv g protein was also shown to induce a t-response at low doses or a t-independent response at higher doses [ ] . fmg fuses cells into large multinucleated syncytia, which are then killed by a nonapoptotic mechanism [ ] . syncytiosomes are released from the membrane and present antigens efficiently to dcs [ ] . fmgs can thus act as an adjuvant for any antigen expressed by a dna vaccine. vaccination of mice with the hepatitis c virus (hcv) nonstructural protein (ns ) together with vsvg resulted in increased frequency of ifn-γ, but not tnf-α-or il- -producing cd + cd + cd + effector memory t (t em ) cells [ ] . this dna vaccine was constructed in a bicistronic vector with vsvg co-encoded in the same plasmid with hcv ns . vsvg expression was driven by a weaker sv promoter and ns by a stronger cmv promoter [ ] . however, others have also reported an increase in the specific cmi when the immunogen and vsvg were expressed from different plasmids [ , ] . perforin (prf) is a pore forming protein released by immune cells including nk cells and cytotoxic t lymphocytes (ctl) [ ] . the -kilodalton prf protein oligomerizes to form pores that serve as a channel to release granzyme into the cytosol of target cells [ ] . suicide genes inducing apoptosis of target cells have been used for cancer therapies, and the role of apoptotic cell death in vaccination, whether immune-stimulatory or immune-suppressive, is debated [ ] . recently, a novel dna technology has been developed--termed cytolytic dna technology-in which a truncated mouse prf is incorporated in a bicistronic dna vector to act as a vaccine adjuvant [ ] [ ] [ ] ] . cytolytic dna vaccines are based on a bicistronic dna plasmid constructed on a pvax backbone (invitrogen) with a cytomegalovirus (cmv) promoter and a simian virus (sv ) promoter [ ] . the gene encoding the protein of interest as an immunogen is inserted downstream of the cmv promoter and the gene encoding a truncated version of mouse prf downstream of the sv promoter ( figure ) . the sv is a weaker promoter compared to cmv and has been shown to result in -fold lower protein expression in transfected hek t cells in vitro [ , ] . the prf gene was modified to express a truncated version of prf (~ kda) lacking the final amino acid residues of the c terminus (unstructured region of prf) [ , , ] in order for prf to become cytolytic [ ] . the final c-terminal amino acids are required to export prf protein from the endoplasmic reticulum (er) to the golgi, from where glycosylated prf is then transported to the prf gene was modified to express a truncated version of prf (~ kda) lacking the final amino acid residues of the c terminus (unstructured region of prf) [ , , ] in order for prf to become cytolytic [ ] . the final c-terminal amino acids are required to export prf protein from the endoplasmic reticulum (er) to the golgi, from where glycosylated prf is then transported to secretory granules [ ] . removal of the c-terminal abrogates the export of prf from the er and its subsequent accumulation in the er is cytotoxic to the host cell [ , ] . different recombinant cytolytic dna-prf vaccines (rdna-prf) have been shown to elicit immune responses higher than those elicited by canonical dna vaccines (without prf) and the mechanism underlying this has been established [ ] . a previous study showed that coexpression of hcv ns and prf elicited nonapoptotic cell death in transfected cells, whilst immunization with ns -prf dna vaccine increased ns -specific t cell mediated responses as evidenced by increased ns -specific ifn-γ responses in an elispot assay and increased numbers of polyfunctional cd + t em cells that simultaneously secreted ifn-γ, il- , and tnf-α [ ] . cytolytic dna platform where the expression of immunogen is driven by a stronger promoter allows for sufficient antigen expression and accumulation within the target cells followed by nonapoptotic cell death due to lesser expression of prf driven by a weaker sv promoter; thus, balancing the level of antigen expression with the timing of cell death [ ] . necrosis is considered as the mechanism of cell death by prf as evidenced by release of lactate dehydrogenase (ldh) and low caspase activity [ , , ] . ldh release occurs after the rupture of cell membrane during secondary necrosis [ , ] . in contrast, ldh was not released by cells treated with doxorubicin (a proapoptotic drug) or cells transfected with ns wild type prf or ns del a prf (mutant and nontoxic prf) [ ] . expression of prf from a cytolytic dna, e.g., ns prf vaccine, thus results in necrotic cell death mediated by receptor-interacting protein- kinase activity, as evidenced by detection of uncleaved cytokeratin in huh- cells [ ] . necrosis releases damage associated molecular patterns (damps) which in turn activate dcs to migrate to the site of vaccination [ , ] . when purified dcs including the cd α + subset from naïve c bl/ mice were exposed to hek t cells (transfected with ovalbumin-prf), it resulted in upregulation of costimulatory molecules (cd /cd ), indicating maturation of the immune cells with the cytolytic dna [ ] . a significant increase in cd c + dcs and cross-presenting cd a + dcs, and upregulation of cd has been reported in mice vaccinated with a cytolytic dna hiv gag prf compared to a canonical dna vaccine [ ] . local and migrated dcs at the site of inflammation can take up antigens by endocytosis and are also exposed to damps. activated and matured dcs can then prime naïve cd + t cells (figure ) . antigen cross-presentation by dcs to cd + t cells has been shown to increase the number of proliferating cd + t cells by~ -fold with cytolytic dna compared to the noncytolytic prf dna [ ] . thus, a cytolytic dna vaccine has an inbuilt adjuvant to enhance the immunogenicity of the vaccine immunogen. whereas, the immunogenicity of canonical dna vaccines mostly depends on direct transfection of dcs and to a lesser extent cross-presentation of antigens shed from transfected cells and/or derived from transfected cells that have undergone spontaneous cell death [ ] . several studies have established that dcs exposed to necrotic or lytic cells expressing antigens mature and cross-present more efficiently than dcs exposed to antigens derived from a cellular milieu that comprise of apoptotic cells [ ] [ ] [ ] [ ] [ ] . comparative studies evaluating the ability of proapoptotic (e.g., rotavirus nonstructural protein (nsp ) and diphtheria toxin subunit a (dta)) and necrotic proteins (e.g., truncated prf) to enhance the immunogenicity of dna when encoded in plasmid dna vaccines showed that truncated prf is the most effective for this purpose [ ] [ ] [ ] . however, a caveat is that vaccine-encoded antigens need to accumulate significantly inside the cell before necrosis occurs following expression of truncated prf in order to activate dcs to cross-present vaccine-encoded antigens [ , ] . several studies have established that dcs exposed to necrotic or lytic cells expressing antigens mature and cross-present more efficiently than dcs exposed to antigens derived from a cellular milieu that comprise of apoptotic cells [ ] [ ] [ ] [ ] [ ] . comparative studies evaluating the ability of proapoptotic (e.g., rotavirus nonstructural protein (nsp ) and diphtheria toxin subunit a (dta)) and necrotic proteins (e.g., truncated prf) to enhance the immunogenicity of dna when encoded in plasmid dna vaccines showed that truncated prf is the most effective for this purpose [ ] [ ] [ ] . however, a caveat is that vaccine-encoded antigens need to accumulate significantly inside the cell before necrosis occurs following expression of truncated prf in order to activate dcs to cross-present vaccine-encoded antigens [ , ] . rdna-prf technology has been used in the development of hiv and hcv dna vaccines [ , , ] . direct comparison of the effects of the cytolytic prf and the apoptotic protein dta on the immunogenicity of the hiv- gag protein showed that prf activated dcs more efficiently, as evidenced by the increase in frequency of cross-presenting dcs and upregulation of activation marker (cd ) [ ] . in both dna vaccines, prf and dta were driven by sv promoter. immunization of mice with a dna vaccine encoding proapoptotic dta as an adjuvant in a hiv gag dta vaccine resulted in decreased dc activation, suggesting that dta-induced apoptosis attenuated immune response [ ] . furthermore, improved protection in the mouse ecohiv challenge model was achieved with rdna-prf encoding hiv gag compared to protection levels in mice vaccinated with a canonical gag dna vaccine [ ] . a rdna-prf vaccine encoding the hcv ns protein coexpressed with prf was shown to increase ns -specific cmi in mice and pigs, compared to ns coexpression with a proapoptotic protein, the rotavirus nsp protein [ ] . nsp is an enterotoxin that elicits a proapoptotic effect by disrupting the mitochondrial membrane and activating caspase- , - , and - [ , ] . this study showed that prf coexpression induced cell death by necrosis, and thus enhanced ns -specific immune responses, whereas, proapoptotic nsp reduced ns -specific response [ ] . importantly, hcv ns prf was more immunogenic than the canonical ns vaccine in pigs, demonstrating the translational potential of the cytolytic dna vaccines in human clinical trials [ ] . likewise, we have shown that a multi-antigenic hcv dna vaccine encoding genotype a proteins ns , ns a, ns b, and ns b coexpressed with prf induced robust cmi against the range of hcv ns proteins, compared to coexpression with vsvg [ ] . we also showed that multi-antigenic and multigenotypic (hcv genotype and a) dna cocktail vaccines encoding prf can significantly increase the magnitude and breadth of cmi responses to ns and ns b against both genotypes compared to those elicited by a single-genotype vaccine [ ] . rdna-prf technology has been used in the development of hiv and hcv dna vaccines [ , , ] . direct comparison of the effects of the cytolytic prf and the apoptotic protein dta on the immunogenicity of the hiv- gag protein showed that prf activated dcs more efficiently, as evidenced by the increase in frequency of cross-presenting dcs and upregulation of activation marker (cd ) [ ] . in both dna vaccines, prf and dta were driven by sv promoter. immunization of mice with a dna vaccine encoding proapoptotic dta as an adjuvant in a hiv gag dta vaccine resulted in decreased dc activation, suggesting that dta-induced apoptosis attenuated immune response [ ] . furthermore, improved protection in the mouse ecohiv challenge model was achieved with rdna-prf encoding hiv gag compared to protection levels in mice vaccinated with a canonical gag dna vaccine [ ] . a rdna-prf vaccine encoding the hcv ns protein coexpressed with prf was shown to increase ns -specific cmi in mice and pigs, compared to ns coexpression with a proapoptotic protein, the rotavirus nsp protein [ ] . nsp is an enterotoxin that elicits a proapoptotic effect by disrupting the mitochondrial membrane and activating caspase- , - , and - [ , ] . this study showed that prf coexpression induced cell death by necrosis, and thus enhanced ns -specific immune responses, whereas, proapoptotic nsp reduced ns -specific response [ ] . importantly, hcv ns prf was more immunogenic than the canonical ns vaccine in pigs, demonstrating the translational potential of the cytolytic dna vaccines in human clinical trials [ ] . likewise, we have shown that a multi-antigenic hcv dna vaccine encoding genotype a proteins ns , ns a, ns b, and ns b coexpressed with prf induced robust cmi against the range of hcv ns proteins, compared to coexpression with vsvg [ ] . we also showed that multi-antigenic and multigenotypic (hcv genotype and a) dna cocktail vaccines encoding prf can significantly increase the magnitude and breadth of cmi responses to ns and ns b against both genotypes compared to those elicited by a single-genotype vaccine [ ] . dna vaccines are still a promising option in the development of novel vaccination strategies. although they have many advantages, the ability to induce effective immune responses in humans required for protection has been challenging. these challenges include ineffective delivery and poor uptake of dna. consequently, a recent focus has been in the development of delivery methods and/or inclusion of genetic adjuvants. such genetic adjuvants are generally coexpressed with the antigen of interest or delivered through different plasmids. in the quest to develop and identify effective genetic adjuvants, a range of adjuvants was tested (hsp , vsvg, imx , dta, and prf) and a novel and promising cytolytic dna vaccine strategy has been developed. this cytolytic dna vaccine is unique as it is based on a bicistronic plasmid with the ability to coexpress antigen and prf in a balanced mechanism causing necrosis of vaccine-transduced cells, followed by increased activation of immune cells and cross presentation of vaccine immunogen. cytolytic dna vaccines encoding nonstructural proteins of hcv have been tested to enhance immunogenicity of vaccine antigen in mice [ , ] and in a large preclinical animal model, the pig [ ] . likewise, increased immunogenicity and improved protection against ecohiv challenge in mice with hiv gag prf [ ] demonstrate the effectiveness of cytolytic dna vaccines. adjuvants that provide effective costimulation for immune responses with specific immunogens may not have a similar effect with other immunogens, and therefore these need to be tested for their efficacy. use of a genetic adjuvant such as prf produces a suitable microenvironment for multiple/different immunogens and thus improves the delivery, immunogenicity and effectiveness of dna vaccines. this strategy has considerable potential in the development of dna-based vaccines against a range of infectious agents. world health organisation. facts on immunization the complexity and cost of vaccine manufacturing--an overview editorial: why vaccines to hiv, hcv, and malaria have so far failed-challenges to developing vaccines against immunoregulating pathogens a vision for vaccines against hiv, tuberculosis and malaria dna vaccines for emerging infectious diseases: what if? emerg advancements in dna vaccine vectors, non-mechanical delivery methods, and molecular adjuvants to increase immunogenicity effective humoral immune response from a h n dna vaccine delivered to the skin by microneedles coated with plga-based cationic nanoparticles differential humoral and cellular immunity induced by vaccination using plasmid dna and protein recombinant expressing the ns protein of dengue virus type vaccines for emerging infectious diseases: lessons from mers coronavirus and zika virus clinical applications of dna vaccines: current progress dna vaccines-how far from clinical use? dna vaccines: recent developments and the future. vaccines dose sparing with intradermal injection of influenza vaccine skin-resident antigen-presenting cells: instruction manual for vaccine development what you always needed to know about electroporation based dna vaccines gene transfer into muscle by electroporation in vivo electroporation-mediated gene delivery liposomes as vaccine delivery systems: a review of the recent advances barriers to liposomal gene delivery: from application site to the target plga nanoparticles for dna vaccination-waiving complexity and increasing efficiency solid lipid nanoparticles mediate non-viral delivery of plasmid dna to dendritic cells production and clinical development of nanoparticles for gene delivery specific endocytosis and degradation of naked dna in the endocardial cells of cod (gadus morhua l.) directly transfected langerin + dermal dendritic cells potentiate cd + ; t cell responses following intradermal plasmid dna immunization chemical adjuvants for plasmid dna vaccines programming, demarcating, and manipulating cd + t-cell memory duration of antigen expression in vivo following dna immunization modifies the magnitude, contraction, and secondary responses of cd + t lymphocytes secondary memory cd + t cells are more protective but slower to acquire a central-memory phenotype immunogenicity of infectious pathogens and vaccine antigens bacterial cpg-dna and lipopolysaccharides activate toll-like receptors at distinct cellular compartments cpg motifs in bacterial dna trigger direct b-cell activation cpg-oligonucleotides in vaccination: signaling and mechanisms of action enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing pfs to imx multimerization technology a hiv-tat/c -binding protein chimera encoded by a dna vaccine is highly immunogenic and contains acute ecohiv infection in mice enhanced presentation of major histocompatibility complex class i-restricted human immunodeficiency virus type (hiv- ) gag-specific epitopes after dna immunization with vectors coding for vesicular stomatitis virus glycoprotein-pseudotyped hiv- gag particles mechanisms of action of adjuvants engineering dna vaccines via co-delivery of co-stimulatory molecule genes dna vaccine for cancer immunotherapy intracellular adhesion molecule- modulates beta-chemokines and directly costimulates t cells in vivo coimmunization with ifn-gamma or il- , but not il- or il- cdna can enhance th -type dna vaccine-induced immune responses in vivo coadministration of dna encoding interleukin- and hemagglutinin confers protection from influenza virus challenge in mice development of th and th populations and the nature of immune responses to hepatitis b virus dna vaccines can be modulated by codelivery of various cytokine genes modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with dna immunogens toll-like receptor adaptor molecules enhance dna-raised adaptive immune responses against influenza and tumors through activation of innate immunity activation of innate immunity, inflammation, and potentiation of dna vaccination through mammalian expression of the tlr agonist flagellin regulation of dna-raised immune responses by cotransfected interferon regulatory factors novel strategies to improve dna vaccine immunogenicity co-immunization with an optimized plasmid-encoded immune stimulatory interleukin, high-mobility group box protein, results in enhanced interferon-gamma secretion by antigen-specific cd t cells dlm- /zbp ) as a genetic adjuvant for dna vaccines that promotes effective antitumor ctl immunity mda can be exploited as efficacious genetic adjuvant for dna vaccination against lethal h n influenza virus infection in chickens development of a molecular adjuvant to enhance antigen-specific cd (+) t cell responses dna vaccines with single-chain fv fused to fragment c of tetanus toxin induce protective immunity against lymphoma and myeloma induction of antigen-positive cell death by the expression of perforin, but not dta, from a dna vaccine enhances the immune respons intradermal delivery of dna encoding hcv ns and perforin elicits robust cell-mediated immunity in mice and pigs a multiantigenic dna vaccine that induces broad hepatitis c virus-specific t-cell responses in mice calreticulin is an effective immunologic adjuvant to tumor-associated antigens development of a dna vaccine targeting human papillomavirus type oncoprotein e dna vaccines encoding membrane-bound or secreted forms of heat shock protein exhibit improved potency safety and immunogenicity of an hiv- gag dna vaccine with or without il- and/or il- plasmid cytokine adjuvant in healthy, hiv- uninfected adults vaccination with a plasmid dna encoding her- /neu together with low doses of gm-csf and il- in patients with metastatic breast carcinoma: a pilot clinical trial a phase i safety study of plasmid dna immunization targeting carcinoembryonic antigen in colorectal cancer patients timing of plasmid cytokine (il- /ig) administration affects hiv- vaccine immunogenicity in hiv-seronegative subjects safety and tolerability of hiv- multiantigen pdna vaccine given with il- plasmid dna via electroporation, boosted with a recombinant vesicular stomatitis virus hiv gag vaccine in healthy volunteers in a randomized, controlled clinical trial dna priming increases frequency of t-cell responses to a vesicular stomatitis virus hiv vaccine with specific enhancement of cd + t-cell responses by interleukin- plasmid dna safety and comparative immunogenicity of an hiv- dna vaccine in combination with plasmid interleukin and impact of intramuscular electroporation for delivery safety and immunological efficacy of a dna vaccine encoding prostatic acid phosphatase in patients with stage d prostate cancer a phase i trial of a human papillomavirus dna vaccine for hpv + cervical intraepithelial neoplasia / . clin antigen-specific humoral and cellular immune responses can be modulated in rhesus macaques through the use of ifn-gamma, il- , or il- gene adjuvants genetic adjuvants for dna vaccines intranasal immunization of a dna vaccine with il- -and granulocyte-macrophage colony-stimulating factor (gm-csf)-expressing plasmids in liposomes induces strong mucosal and cell-mediated immune responses against hiv- antigens cytokine molecular adjuvants modulate immune responses induced by dna vaccine constructs for hiv- and siv intranasal administration of human immunodeficiency virus type- (hiv- ) dna vaccine with interleukin- expression plasmid enhances cell-mediated immunity against hiv- enhancement of cell-mediated immunity against hiv- induced by coinnoculation of plasmid-encoded hiv- antigen with plasmid expressing il- augmentation and suppression of immune responses to an hiv- dna vaccine by plasmid cytokine/ig administration il- expression plasmid enhances cell-mediated immunity induced by an hiv- dna vaccine a mage /heat shock protein dna vaccine induces both innate and adaptive immune responses for the antitumor activity heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology human dkk and human hsp fusion dna vaccine induces an effective anti-tumor efficacy in murine multiple myeloma a mouse model for study of systemic hiv- infection, antiviral immune responses, and neuroinvasiveness encoded novel forms of hsp or a cytolytic protein increase dna vaccine potency fusion of the mycobacterium tuberculosis antigen a to an oligomerization domain enhances its immunogenicity in both mice and non-human primates the oligomerization domain of c -binding protein (c bp) acts as an adjuvant, and the fusion protein comprised of the -kilodalton merozoite surface protein fused with the murine c bp domain protects mice against malaria a first-in-human phase trial to evaluate the safety and immunogenicity of the candidate tuberculosis vaccine mva a-imx , administered to bcg-vaccinated adults vesicular stomatitis virus g protein transmembrane region is crucial for the hemi-fusion to full fusion transition combined administration with dna encoding vesicular stomatitis virus g protein enhances dna vaccine potency vesicular stomatitis virus indiana glycoprotein as a t-cell-dependent and -independent antigen fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth viral fusogenic membrane glycoproteins kill solid tumor cells by nonapoptotic mechanisms that promote cross presentation of tumor antigens by dendritic cells comparison of primary human cytotoxic t-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production the structural basis for membrane binding and pore formation by lymphocyte perforin dna vaccines and apoptosis: to kill or not to kill? cytolytic dna vaccine encoding lytic perforin augments the maturation of-and antigen presentation by-dendritic cells in a time-dependent manner systematic comparison of constitutive promoters and the doxycycline-inducible promoter protection from endogenous perforin: glycans and the c terminus regulate exocytic trafficking in cytotoxic lymphocytes perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack the roles of caspase- and bcl- in chemically-induced apoptosis but not necrosis of renal epithelial cells apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells tolerance, danger, and the extended family how dying cells alert the immune system to danger tumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression natural adjuvants: endogenous activators of dendritic cells consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the nf-kappa b pathway cross-presentation by dendritic cells induction of genotype cross-reactive, hepatitis c virus-specific, cell-mediated immunity in dna-vaccinated mice rotaviral enterotoxin nonstructural protein targets mitochondria for activation of apoptosis during infection porcine reproductive and respiratory syndrome virus nonstructural protein induces apoptosis dependent on its c-like serine protease activity we thank thrf and the donor community for supporting the development of our novel dna vaccines. ashish c. shrestha and danushka k. wijesundara are recipients of early career fellowships from thrf. the authors declare no conflict of interest. key: cord- -bhfghcby authors: zrzavy, tobias; kollaritsch, herwig; rommer, paulus s.; boxberger, nina; loebermann, micha; wimmer, isabella; winkelmann, alexander; zettl, uwe k. title: vaccination in multiple sclerosis: friend or foe? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: bhfghcby multiple sclerosis (ms) is a debilitating disease of the central nervous systems (cns). disease-modifying treatments (including immunosuppressive treatments) have shown positive effects on the disease course, but are associated with systemic consequences on the immune system and may increase the risk of infections and alter vaccine efficiency. therefore, vaccination of ms patients is of major interest. over the last years, vaccine hesitancy has steadily grown especially in western countries, partly due to fear of sequelae arising from vaccination, especially neurological disorders. the interaction of vaccination and ms has been discussed for decades. in this review, we highlight the immunology of vaccination, provide a review of literature and discuss the clinical consideration of ms, vaccination and immunosuppression. in conclusion, there is consensus that ms cannot be caused by vaccines, neither by inactivated nor by live vaccines. however, particular attention should be paid to two aspects: first, in immunocompromised patients, live vaccines may lead to a stronger immune reaction with signs of the disease against which the patients have been vaccinated, albeit in weakened form. second, protection provided by vaccination should be controlled in patients who have been vaccinated while receiving immunomodulatory or immunosuppressive treatment. in conclusion, there is evidence that systemic infections can worsen ms, thus vaccination will lower the risk of relapses by reducing the risk of infections. therefore, vaccination should be in general recommended to ms patients. over the last years, especially in western countries, vaccine hesitancy has steadily grown and poses an increasing health concern ( ). the recent upsurge of measles in europe is an impressive example. anti-vaccinationists argue that possible side effects weigh out the benefits ( ) . especially sequelaes such as autism, multiple sclerosis (ms) and various neurological syndromes have been emphasized by the anti-vaccination lobby ( , ) . this alarming development is even partly supported by healthcare providers including some ms neurologists, who are afraid of iatrogenic deterioration of preexisting ms. indeed, studies linking vaccination and disease onset have been published. although these studies were often underpowered and lacked an adequate design in order to provide evidence of the suspected link, they caught public awareness leading to a drop of public vaccination coverage rates ( , ) . epidemiological studies and pharmacovigilance data have repeatedly demonstrated safety for the vast majority of vaccines. lately, a review concluded that there is no significant evidence for a causal relationship between the onset or deterioration of ms and vaccination against measles, mumps and rubella (mmr), influenza, hepatitis a, hepatitis b, human papilloma virus (hpv), diphtheria, tetanus, acellular pertussis, or meningococcal disease ( ) . some studies have even indicated a decreased risk for ms and reduced disease activity in preexisting ms ( ) . the aim of this review is to summarize data on vaccination and disease activity of both ms and acute disseminated encephalomyelitis (adem). moreover, vaccination-induced effects on the immune system are presented and potential interactions between ms and immunizations are discussed. vaccine-induced protection is a complex issue and depends on a cascade of mechanisms and mediators (figure ) . eventually, protection is accomplished either by antibodies or t celldependent factors or by a combination of both including neutralizing or antitoxic antibodies, cd + t cells, cd + t cells and corresponding cytokines (e.g., interleukin (il)- , , , , , , , , , and ) ( ) . generally, vaccines have to be capable of activating antigen-presenting cells (apcs) of the innate immune system, which subsequently present the vaccine epitope(s) to t cells-the so-called 'immunogenic potential' ( ) . in this context, dendritic cells play a pivotal role due to their enhanced capability to stimulate naïve t cells ( ) . the nature of vaccine-induced immunity depends on several parameters, of which the biological properties of the vaccine's epitope are of high importance ( ) . live vaccines are attenuated variants of pathogens that still can activate apcs, especially immature dendritic cells, patrolling through the body. this immunogenic potential is often lost by subcellular-or subunitbased vaccines ( ) , which is why these inactivated vaccine antigens are usually combined with so-called adjuvants to increase and modulate the vaccine's immunogenicity via a longer lasting and more effective activation of immune cells. one of the most widely used adjuvants are aluminum salts, which were originally thought to create a long-lasting depot of the antigen in order to provide its slow release, but have instead been shown to act on dendritic cells via prrs (pattern recognition receptors) leading to the secretion of pro-inflammatory cytokines ( ) . similarly, novel adjuvants like squalens or monophosphoryl lipid a (mpla-a detoxified lipopolysaccharide) aim to enhance the innate immune response, but never reach the immunogenic potential of live attenuated vaccines ( ) . adjuvants have been added to vaccines for more than years and over the last decades, considerable progress has been made in understanding their mode of action and to improve safety ( ) . besides the above mentioned aluminum salts, squalene and mpla, oil emulsions, saponin, toll-like receptor (tlr) agonists, enterotoxins, polysaccharides, and glycolipid adjuvants ( ) are used, all of which stimulate the immune system as well. aluminum adjuvants have now been used for decades and lots of experience has been gained on its use, effectiveness, and safety and they still remain the most frequently used adjuvants. their effects on the immune system comprise stimulation of macrophages and dendritic cells via prrs, inflammasome activation, il- β release and activation of th lymphocytes ( , ) . however, besides increased immunogenicity, aluminum adjuvants also increase reactogenicity and based on data from animal models and reports on narcolepsy, silicosis, guillain-barré-syndrome (gbs) and macrophagic myofasciitis, they are also discussed to induce autoimmunity ( ) . the second most commonly and long used adjuvants are oil emulsions. they have a strong reactogenic potential and can cause severe inflammatory local reactions such as ulceration and granulomas. the most well-known oil emulsion is complete freund's adjuvant. however, due to its potent reactogenicity, it is not suitable for human use. a possible association between oil emulsions and autoimmunity disorders has been hypothesized from animal models. oil emulsions are potent inducers of il- β and il- ( , ) . il- plays a major role in autoimmunity and ms and may trigger the migration of peripheral lymphocytes into the cns across the bbb ( , ) . frequently, a combination of adjuvants is used to increase immunogenicity of vaccines. as is an adjuvant emulsion containing squalene, dl-αtocopherol, and polysorbate . it is e.g., used for the pandemic swine flu vaccine pandemrix r ( ) or the fda-licensed h n monovalent influenza vaccine. in animal studies, autoimmunity was observed in connection with as ( ) and in humans, cases of narcolepsy have been reported ( ) . oil emulsions are often combined with tlr agonists such as mpla. generally, tlr agonist adjuvants activate the inflammatory transcription factor nfκb as is a combination of mpla and aluminum salts and is used as adjuvant in vaccines against hepatitis b (fendrix r ) and hpv, as well as in the new recombinant vaccine against herpes zoster. most polysaccharide adjuvants activate nfκb to induce immune processes (e.g., dextran, zymosan) ( ) . however, deltainulin for instance, a polysaccharide adjuvant used for advax r , acts via nfκb-independent mechanisms to enhance humoral and cellular immune responses. although the mechanisms are not yet fully understood, advax r has so far not shown inflammatory side effects and has proven safety in hepatitis b vaccination and influenza ( ) . after activation of the immune cascade and stimulation of dendritic cells, the latter increase their expression of mhc molecules and chemokine receptors such as ccr leading to their migration toward the draining lymph nodes in order to provide co-stimulatory signals for the differentiation of naïve t cells into immune effector cells ( ) . the activation of the immune cascade has various effects on t and b cells. in short, antigen-recognition by b cells leads to their activation and migration toward the t-b cell border of the lymph node, where they can subsequently receive additional stimuli by activated t helper (th) cells. these signals include cd interaction, secretion of cytokines by th or th cells, and finally the transformation of b cells into plasma cells predominantly secreting low affinity antibodies ( ) . later, the germinal center response contributes via affinity maturation (somatic hypermutation and affinity-based selection) and isotype switch to a sustained production of high affinity antibodies by predominantly plasma cells but also memory b cells. basically, in the lymph nodes, numerous b cells with various affinity compete for the antigens presented by follicular dendritic cells. these antigens are processed and further presented via mhc ii to follicular th cells, which provide costimulatory signals (e.g., cd , icos, and il- ) leading to survival and further proliferation of b cells with highest affinity for the antigen ( ) . in conclusion, vaccination-induced immune responses, including employed cell types and mediators, vary depending on the type of vaccine administration, kind of vaccine and choice of adjuvant. while antibodies will directly prevent and reduce infections, cd + and cd + t cells rather support the organism eventually reducing, controlling and clearing the pathogens. antibodies bind to their antigen, neutralize pathogens, activate macrophages and neutrophils as well as the complement system, while cd + and cd + t cells secrete cytokines, perforins, and granzymes ( ) . the choice of adjuvant seems to be critical, since some may cause problems in autoimmune diseases. thus, monitoring side effects regarding autoimmunity is essential. in the early days of vaccine development, louis pasteur used nerve tissue of infected animals to obtain a rabies virus vaccine ( ) . although saving countless lives it was recognized that active sensitization with neuronal tissue could occasionally lead to neuroparalytic autoimmune complications ( ) with self-limiting autoimmune encephalomyelitis that fulfilled the pathological criteria of ms ( , ) . advances in processing techniques and increasing insights in immunology led to modern vaccines devoid of neuronal tissue. ms is a chronic disease thought to be caused by immune-mediated mechanisms. thus, immune responses caused by vaccinations will affect the immune system. however, their effects on immunology per se, but especially those in ms patients, are scarcely understood. the same means by which infections can induce autoimmunity also apply for vaccination-induced immune activation. possible structural similarities between microbial epitopes and epitopes of the cns could lead to cross-reaction of antibodies via molecular mimicry as shown for streptococcal antibodies in heart tissue ( ) . additionally, epitope spreading is a mechanism leading to a broadening of the immune response from the dominant epitope to cryptic (intramolecular) or neighboring molecules (intermolecular) resulting in an increased antibody repertoire and cellular response ( ) . moreover, bystander activation, a process in which activated apcs stimulate autoreactive t cells, can occur ( ) . bacterial and viral infections can trigger relapses and mri activity in ms; vaccination has been proven to protect from or weaken infections, thus providing an "indirect" protection against ms disease activity ( ) . several reports on neurological disorders developing after immunization have been published including several cases on encephalomyelitic disorders (impaired consciousness, ataxia and optic neuritis) as well as demyelinating lesions in a patient with transverse myelitis after active immunization against influenza ( ) ( ) ( ) ( ) . immunization against rubella was associated with diffuse myelitis and recurrent relapses with optic neuritis, paraparesis and impaired motor function ( , ) . transverse myelitis ( ) as well as optic neuritis ( , ) were reported in patients vaccinated against measles, mumps and rubella. further cases with symptoms suggestive for disseminated encephalitis were reported after vaccination against diphtheriatetanus-poliomyelitis (dtp) ( ) and after immunization against smallpox, rabies or typhus ( ) . exacerbations of ms and demyelinating lesions were reported in ms patients and patients without a history of neurological conditions after immunization against hepatitis b ( ) . similarly, tourbah reported on patients with demyelinating lesions and clinical symptoms after vaccination against hepatitis b ( ) . in contrast to these case series, a case-control study (evidence class ii) ( ) including more than patients with ms or optic neuritis and controls without any underlying neuroimmunological disorder did not reveal an elevated risk for the development of ms or optic neuritis after immunization against hepatitis b, tetanus, influenza, measles/mumps/rubella, measles, or rubella ( ) . while hernan came to same results for immunization against influenza or tetanus in a case-control study (evidence class ii), active immunization against hepatitis b was reported to pose a higher risk for ms ( ) . the latter finding could, however, not be confirmed by confavreux in a large case-crossover study. additionally, no increased risk was seen for vaccination against tetanus and influenza as well ( ) . similarly, other class ii case-control studies did not report on an increased risk for ms after hepatitis b vaccination ( ) ( ) ( ) . an even decreased risk for ms was reported after tetanus immunization ( ) . in a large class i study, a patient register including , females vaccinated with the quadrivalent hpv vaccine was analyzed. thereof, , patients with ms and , patients with other demyelinating disorders were studied and no increased risk for cns manifestations was seen in this large cohort ( ). miller et al. performed a prospective class ii, randomized, double-blind, placebo-controlled study, which included ms patients, who received either standard influenza vaccination or placebo. for a months follow-up period, the occurrence of neurological symptoms or influenza was monitored and no differences were seen for relapse rates ( ) . a study by langer-gould reported on an increased risk for cns demyelinating diseases within the first days after vaccination. it was concluded that there is no increased risk for ms, but it seems that the transition from subclinical to overt autoimmunity in patients with existing disease is shortened ( ) . two major questions arise on the topic of "ms and vaccination": (i) can vaccines cause ms and (ii) can vaccines provoke or trigger relapses in patients with ms? (i) overall, the anecdotal reports associating ms onset and vaccination had limited reliability, lacked validity and could not be replicated in larger studies. therefore, there is consensus that there is yet no evidence that ms can be caused by vaccines neither by inactivated nor by live vaccines ( ). (ii) it is more difficult to assess the potency of vaccines to trigger relapses in ms patients. with respect to live vaccines it seems to be plausible that they may be able to provoke a deterioration of the disease, since they fulfill the criteria of an active infection with a replicative (although attenuated) organism. there is class iv evidence that at least the yellow fever (yf) d vaccine strain, which is derived from a natural occurring yf-virus and hasn't completely lost its neurotoxicity even after numerous passages, is able to provoke relapses in ms patients. however, it has to be kept in mind that the patient cohort had received immunomodulatory treatment and the sample size of this self-controlled case series study was rather small ( ) . the underlying potential immunologic mechanisms, which are responsible for this elevated relapse rate, are not understood yet and larger studies are necessary to confirm this association. hypotheses may be generated based on observations after infections with helminths, mycobacteria and epstein-barr virus, or by the immunologic properties of this particular vaccine strain ( ) . immunological analyzes showed that after immunization against yf, ms patients had a significantly increased mbp-and mog-specific response shown by increased numbers of cells secreting interferon, il- α, il- β and tumor necrosis factor compared to unvaccinated ms patients or ms patients vaccinated against influenza ( ) . still, there is no evidence for other live vaccines such as mmr to deteriorate ms ( , ) . for inactivated vaccines, there is already more evidence available that an association between ms relapses and different kinds of vaccines does not exist ( ) . even for vaccines, which were publicly accused to be associated with ms disease or relapse rate, like hpv or hepatitis b vaccines, there is no evidence to support any association between vaccination and clinical course of ms, as well as for vaccines containing inactivated neurotropic viruses like tbe ( , ) . it still remains unclear if inactivated vaccines may accelerate an upcoming relapse in patients with active ms by non-specific stimulation besides effects of vaccines on induction and the disease course of ms, potential immunological effects of adjuvants have to be considered as well. most experience on the possible induction of autoimmunity following administration of adjuvant-containing vaccines has been gained from animal models. however, results from experimental studies cannot be transferred to humans without reservation. first, the dose ratios tested in animal models are not the same as in humans and second, human immunology differs from animals. indeed, oil emulsions, aluminum salts and squalene have shown severe side effects in animal models, while they are considered to be safe in humans ( ) . an analysis performed by the european medicines agency (ema) ( ) investigated autoimmune disorders following vaccination against pandemic influenza a/h n between october and december ( ) . thirty percent of the million doses of the distributed vaccines contained aluminum salts and squalene-based adjuvants. overall, the study did not suggest a significant difference in the risk for autoimmune disorders for adjuvant and non-adjuvant vaccinations. adem was reported for people (adjuvant vaccines: , non-adjuvant vaccines: ), ms for people (adjuvant vaccines: , nonadjuvant vaccines: ), ms relapses for patients (adjuvant vaccines: , non-adjuvant vaccines: ), and one case of relapsing remitting ms was reported for adjuvant-containing vaccination ( ) . statistical analysis revealed only a nonsignificantly increased risk for gbs ( ) . also, a favorable benefitrisk profile of the vaccines was demonstrated ( , ) . in conclusion, following the reports from literature, all of the ema/fda-approved vaccines (with exception for yellow fever) and adjuvants do not show a significantly increased risk for ms and adem. constant improvement of basic immunological knowledge and technology will further improve the safety of adjuvants. table gives an overview of the recommendations of standard vaccinations in the general population and in ms patients. while there is a lot of literature on vaccination and risk for ms or ms relapses available, reports on vaccination and adem are scarce. yet, adem has been discussed to be a sequelae of vaccinations ( ) as well as to be preceded by infections. several cases of adem have been reported to be timely related to vaccinations against rabies ( ), hpv ( , ) , hepatitis a and b, diphtheria, tetanus and poliovirus ( ) , measles, rubella and booster immunization for japanese encephalitis ( ) . adem has been reported following vaccination against influenza, including eight cases after vaccination against h n . also, four adem cases after vaccination against yf can be found in literature ( , ) . besides case reports, there have been some observational studies, albeit all having their limitations. in out of reported cases of adem, patients had infections or vaccinations prior to disease onset ( ) . also, pellegrino et al. concluded a possible relation between post-vaccination adem in children and adults. four hundred four cases of adem were analyzed based on the data of the vaccine adverse event reporting system (vaers) database and the eudravigilance post-authorization module (evpm) ( ) . about % of the cases occurred between and days after vaccination, most commonly against influenza and hpv. a case-control study on vaccination against hepatitis b, influenza, polio, diphtheria, pertussis, tetanus, measles, mumps, rubella, japanese encephalitis, meningitis, hepatitis a, varicella and rabies did not reveal an increased risk for the onset of adem in the time spans of - days and - days after vaccination, but between and days ( ) . based on these reports, the risk for adem after vaccination cannot be completely ruled out. considerations on ms exacerbation and vaccination apply only for ms patients receiving no immunomodulatory/ immunosuppressive treatment. if any kind of immunosuppression is used for ms therapy, this choice of treatment will dominate the decision whether to vaccinate or not ( ) . in recent years, consensus statements on vaccinations during immunosuppressive treatments were published by various national and international societies and expert panels ( ) ( ) ( ) ( ) ( ) . there is consensus that inactivated vaccines will do no harm ( ) even in immunosuppressed patients. however, data on the efficacy of vaccinations in combination with the various available ms medications are missing. thus, for patients either receiving more than one immunomodulatory treatment or having underlying immunomodulating condition, the outcome is difficult to predict ( ). therefore, the success of vaccination should be verified by antibody testing if a valid test is available. except for a few treatments, which only lead to mild immunosuppression, live vaccines are contraindicated under immunosuppressive treatment. in some situations, risks and benefits of a live vaccine have to be weighed against each other, e.g., in varicella zoster virus (vzv)-negative ms patients under fingolimod treatment, varicella vaccination may be considered, since severe complications from natural varicella infection may outweigh the risk from this live vaccine. however, recommendations vary between different institutions even within the same country ( , , ) . a recent case report on a lethal vzv infection in an immunocompromised patient after vzv live vaccination drives the discussion on this issue ( ) . there is consensus about the timing of vaccination in patients, who will undergo immunosuppressive treatment: vaccinations should be given well in advance to the start of treatment (at least weeks for inactivated and ≥ weeks for live vaccines) and should be distinguished between primo-vaccinations and boosters. importantly, the refractory period after immunosuppression has to be considered as well, which may be up to year depending on the type of medication (e.g., rituximab or alemtuzumab) ( ) . vaccines will have various effects on the immune system, which greatly depend on the cell types typically engaged by the respective vaccines. the impact of immunosuppression on the various cell types (and possible mitigation of effects) should be taken into consideration. protective efficacy is mostly mediated by antibodies for the following vaccines: cholera, diphtheria toxoid, hepatitis a and b, haemophilus influenzae type b, influenza, japanese encephalitis, meningococcal ps and conjugates, papillomavirus, pneumococcal ps and conjugates, polio (sabin and salk), rabies, rotavirus, rubella, tetanus toxoid, typhoid ps, and yf. effects are solely born by t cells for tuberculosis (bcg), or by a combination of antibodies and t cells for measles and intranasal influenza vaccination. besides antibody-mediated protection, effects of t cells are discussed for pertussis ( ) . for patients receiving immunosuppressive treatment, vaccination control should be performed. for diphtheria, tbe (with caution), hepatitis a, b, haemophilus influenzae type b, measles, mumps, pneumococcus, polio, rubella, tetanus, rabies and varicella, standards are available and recommended to be tested. in general, to increase the validity of vaccination control, titers should be assessed in paired samples (before and after immunization) via the same method and at highquality standards ( ) . in general, patients should have received their recommended standard vaccines according to their region-specific vaccine guidelines. before certain immunosuppressive treatments are initiated, it is mandatory to exclude former infections and if necessary, vaccination should be considered according to the regulatory agencies. table provides an overview on necessary vaccinations according to fda/ema guidelines (extended vaccination reflects the authors' suggestion). for many immunotherapies, a prior exclusion of an ongoing vzv infection is required and vaccination should be offered to those, who haven't gained any immunity yet. additionally, vzv-seropositive patients undergoing immunotherapy should be offered vaccination as well to prevent zoster reactivation and late effects. recently, a non-live subunit vaccine has been authorized for vzv-seropositive patients. it possesses a better risk-benefit profile compared to the live vaccine and has already been approved by many countries ( ) . additionally, it should be considered to offer patients with upcoming fingolimod or alemtuzumab treatment the option of vaccination against hpv, as post-market surveillance showed increased reports of warts and cervical dysplasia due to these two ms therapies [ema; ( ) ]. furthermore, pneumococcal vaccine might be considered in patients receiving b cell-depleting therapies, as severe respiratory infections during phase iii studies were seen ( , ) . vaccine hesitancy is a major problem nowadays. the usefulness of active immunization is undisputed and has saved numerous lives. however, fear of possible, but also often unconfirmed, side effects has fostered this anti-vaccine sentiment. this has led to a recent outbreak of measles ( ) and curiously some viruses and disorders, which have been assumed to be eradicated, seem to become a hot topic for western health systems again. indeed, side effects upon vaccination may occur in rare cases, however, the benefits for individual people as well as the whole population will generally outweigh adverse effects. vaccine hesitancy results in a twofold problem: ( ) the missing protection for the unvaccinated people themselves but also ( ) a risk for people, who are not able to get vaccinated. the missing herd immunity poses a major problem for a group of patients with fragile health. for ms patients receiving immunosuppressive treatment, an acute infection can have dangerous sequelae. thus, if possible, ms patients should be vaccinated beforehand. the possible benefits outweighdependent on the individual case-the possible risks. an additional perspective raises the possibility of vaccination against ms. indeed, early approaches exploring vaccination with synthetic peptides in experimental animal models were successful, but translation into clinical treatment was so far unsatisfying ( ) ( ) ( ) . interestingly, it was recently shown that an anti-typhus vaccination (typhim vaccine) might have the potential to ameliorate the disease course of ms by targeting prohibitins on th cells. tested in an experimental ms model it led to decreased levels of il and increased numbers of foxp + regulatory t cells ( ) . further investigations are needed before studies should investigate treatment options for ms patients. still, it is a good example, how immunology of vaccination might overlap with and modulate the immunology of ms. theoretically, an increased immune response against different types of vaccines, such as live attenuated viruses, inactive attenuated viruses, or portions of bacteria and viruses, could trigger increased immune response to self-antigens ( , , ) , but an increased risk for ms itself or increased relapse rates after vaccination have not been show (with exception for yf) in case-control studies ( ) . there is, however, evidence that infections can trigger relapses in ms ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , which is why vaccination of ms patients should be pursued in order to reduce the risk of infections. to assure the best vaccination success, immunization and immunosuppressive treatments have to be well timed. vaccine hesitancy: definition, scope and determinants available online at a postmodern pandora's box: anti-vaccination misinformation on the internet the "urban myth" of the association between neurological disorders and vaccinations mass vaccination against hepatitis b: the french example central nervous system demyelinating diseases and recombinant hepatitis b vaccination: a critical systematic review of scientific production vaccines and multiple sclerosis: a systematic review tetanus vaccination and risk of multiple sclerosis: a systematic review vaccine immunology in: spwopokm, editor. plotkin's vaccines vaccine adjuvants: putting innate immunity to work in vivo activation of antigen-specific cd t cells superior immunogenicity of inactivated whole virus h n influenza vaccine is primarily controlled by toll-like receptor signalling aluminum hydroxide adjuvants activate caspase- and induce il- beta and il- release immunological mechanisms of vaccination vaccine adjuvants: from to and beyond comparative safety of vaccine adjuvants: a summary of current evidence and future needs adjuvants safety project c. safety of vaccine adjuvants: focus on autoimmunity adjuvant system as containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity cell recruitment and cytokines in skin mice sensitized with the vaccine adjuvants: saponin, incomplete freund's adjuvant, and monophosphoryl lipid a human t h lymphocytes promote blood-brain barrier disruption and central nervous system inflammation cellular mechanisms of il- -induced blood-brain barrier disruption induction of lupus autoantibodies by adjuvants increased incidence and clinical picture of childhood narcolepsy following the h n pandemic vaccination campaign in finland carbohydrate-based immune adjuvants extrafollicular antibody responses control systems and decision making for antibody production rabies vaccine prepared in human cell cultures: progress and perspectives the neuro-paralytic accidents of antirabies treatment autoimmune encephalitis in humans: how closely does it reflect multiple sclerosis? an immunological relationship between the group a streptococcus and mammalian muscle spreading of t-cell autoimmunity to cryptic determinants of an autoantigen antiviral immune responses: triggers of or triggered by autoimmunity? the risk of relapses in multiple sclerosis during systemic infections relapsing encephalomyelitis following the use of influenza vaccine ocular abnormalities after influenza immunization acute transverse myelitis after influenza vaccination: magnetic resonance imaging findings optic neuritis after influenza vaccination diffuse myelitis associated with rubella vaccination diffuse myelitis associated with rubella vaccination transverse myelitis after measles, mumps, and rubella vaccine optic neuritis complicating measles, mumps, and rubella vaccination optic neuritis following measles/rubella vaccination in two -year-old children relapsing acute encephalopathy: a complication of diphtheria-tetanus-poliomyelitis immunization in a young boy multiple sclerosis and vaccination central-nervous-system demyelination after immunisation with recombinant hepatitis b vaccine encephalitis after hepatitis b vaccination: recurrent disseminated encephalitis or ms? level of evidence reviews: three years of progress vaccinations and risk of central nervous system demyelinating diseases in adults recombinant hepatitis b vaccine and the risk of multiple sclerosis: a prospective study vaccines in multiple sclerosis study g. vaccinations and the risk of relapse in multiple sclerosis. vacc multiple scler study group hepatitis b vaccination and the risk of multiple sclerosis vaccines and the risk of multiple sclerosis and other central nervous system demyelinating diseases multiple sclerosis and immunological-related risk factors: results from a case-control study quadrivalent hpv vaccination and risk of multiple sclerosis and other demyelinating diseases of the central nervous system a multicenter, randomized, double-blind, placebo-controlled trial of influenza immunization in multiple sclerosis adverse effects of vaccines: evidence and causality yellow fever vaccination and increased relapse rate in travelers with multiple sclerosis serious adverse events associated with yellow fever vaccine epstein-barr virus, cytomegalovirus, and multiple sclerosis susceptibility: a multiethnic study a controlled trial of tick-borne encephalitis vaccination in patients with multiple sclerosis vaccines and multiple sclerosis summary of product characteristics autoimmune disorders after immunisation with influenza a/h n vaccines with and without adjuvant: eudravigilance data and literature review available online at recommended adult immunization schedule, united states ständige impfkommission: empfehlungen der ständigen impfkommission (stiko) am robert koch-institut acute disseminated encephalomyelitis: an update biphasic demyelination of the nervous system following anti-rabies vaccination acute disseminated encephalomyelitis following vaccination against human papilloma virus two cases of acute disseminated encephalomyelitis following vaccination against human papilloma virus improvement of advanced postvaccinal demyelinating encephalitis due to plasmapheresis post-vaccination mdem associated with mog antibody in a subclinical chlamydia infected boy neurologic disease associated with d- yellow fever vaccination: a report of cases longitudinal myelitis associated with yellow fever vaccination acute fulminant demyelinating disease: a descriptive study of patients acute disseminated encephalomyelitis onset: evaluation based on vaccine adverse events reporting systems vaccines and the risk of acute disseminated encephalomyelitis disease-modifying therapies and infectious risks in multiple sclerosis idsa clinical practice guideline for vaccination of the immunocompromised host general best practice guidelines for immunization vaccination in immunocompromised host: recommendations of italian primary immunodeficiency network centers (ipinet) vaccination against infection in patients with multiple sclerosis tickborne encephalitis (tbe) vaccine to medically immunosuppressed patients with rheumatoid arthritis: a prospective, open-label, multi-centre study live zoster vaccination in an immunocompromised patient leading to death secondary to disseminated varicella zoster virus infection understanding the immunology of shingrix, a recombinant glycoprotein e adjuvanted herpes zoster vaccine warts and all: fingolimod and unusual hpv-associated lesions alemtuzumab versus interferon beta a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase trial ocrelizumab versus interferon beta- a in relapsing multiple sclerosis vaccination against experimental allergic encephalomyelitis with t cell receptor peptides the ups and downs of multiple sclerosis therapeutics antigen-specific tolerization approaches in multiple sclerosis targeting prohibitins at the cell surface prevents th -mediated autoimmunity immunization in patients with multiple sclerosis chlamydia pneumoniae infection of the central nervous system in multiple sclerosis epstein-barr virus and multiple sclerosis coronaviruses in brain tissue from patients with multiple sclerosis active human herpesvirus infection in patients with multiple sclerosis cerebrospinal fluid molecular demonstration of chlamydia pneumoniae dna is associated to clinical and brain magnetic resonance imaging activity in a subset of patients with relapsing-remitting multiple sclerosis multiple sclerosis, sporadic creutzfeldt-jakob disease and bovine spongiform encephalopathy: are they autoimmune diseases evoked by acinetobacter microbes showing molecular mimicry to brain antigens? the case for rhinoviruses in the pathogenesis of multiple sclerosis epstein-barr virus infection and multiple sclerosis: a review all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © zrzavy, kollaritsch, rommer, boxberger, loebermann, wimmer, winkelmann and zettl. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - owsb bg authors: leung, gabriel m.; nicoll, angus title: reflections on pandemic (h n ) and the international response date: - - journal: plos med doi: . /journal.pmed. sha: doc_id: cord_uid: owsb bg gabriel leung and angus nicoll provide their reflections on the international response to the h n influenza pandemic, including what went well and what changes need to be made in anticipation of future flu pandemics. there is general consensus that the only predictable characteristic of influenza viruses and pandemics is their unpredictability [ ] . given such uncertainty, reasonable application of the precautionary principle should prevail in the responses. indeed many of the initial responses to the pandemic went well. once isolated, the pandemic virus strain was shared immediately, specific diagnostic assays were produced and distributed worldwide, antivirals were available in many countries, vaccine development started promptly, and clinical trials demonstrating vaccine safety and immunogenicity were conducted rapidly. there were many inherently favourable features of the pandemic itself, not all of which were immediately apparent (table ) . this was not spanish flu. the impact has been mostly confined to the health sector. but that impact has been significant and heterogeneous, with pressure experienced by primary and hospital care (especially intensive care and paediatric services). distilling descriptions of the impact of a complex public health threat like a pandemic into a single term like ''mild,'' ''moderate,'' or ''severe'' can potentially be misleading [ ] . certainly the experience of hospital clinicians indicated that this pandemic, sometimes described as ''mild to moderate,'' was not limited to only mild or moderate illness. many patients were severely ill and died, and undoubtedly, high-quality clinical management of patients with severe complications in intensive care units saved many lives of the critically ill, who often required prolonged hospitalisation [ ] . the epidemiology of this pandemic is different than for seasonal influenza epidemics, but not unlike previous pandemics. young people have been disproportionately affected in terms of hospitalisation and deaths compared to seasonal influenza in which complications and mortality are predominantly borne by the elderly [ ] . similarly, the risk to pregnant women has been higher than for seasonal influenza [ , ] , which was also noted in previous pandemics. the attributable premature mortality may remain unclear for some time. recent american analyses have estimated many more deaths than those officially reported with laboratory confirmation of infection and that years of life lost were equivalent to the pandemic. the lower bound of such estimates is equivalent to the annual burden caused by a typical h n seasonal epidemic in temperate climates [ , ] . the years-of-life-lost metric captures the impact of a different agespecific mortality pattern which death counts cannot. deaths involving the young and healthy incur many more potential years of life lost compared to those of older adults and of chronically ill individuals. there are also a number of ''firsts'' for the pandemic after an interpandemic period of more than four decades (box ). these brought both opportunities and challenges. under the auspices of the world health organization (who), the process of a global review by public health specialists from around the world has recently begun. they were nominated by national authorities and are led by an elected chair who assessed the handling of the swine influenza event among us military personnel at fort dix [ ] . here we offer some initial reflections on the first months of the present pandemic. considerable effort in recent years had been dedicated to preparing for surveillance during a pandemic and to incorporating modelling in planning in some countries. the pandemic virus was detected and isolated reasonably early, although too late for any attempt at containment. it remains unclear precisely when or where it first emerged, but the earliest human infections were detected in north america and the best estimates of the timing of emergence are variously mid-february from field epidemiology in southeast mexico or mid-january from a molecular clock model [ ] . situational awareness during the early phase allowed quick assessment by countries, notably those affected first (mexico, us, canada, and southern hemisphere temperate countries). the integration of clinical, laboratory, and epidemiologic data proved essential and gave important insights into disease severity, transmission dynamics, and anticipated impact of interventions. focused local or national studies with analyses shared through who or regional bodies proved more valuable than relying on collection of primary data for analysis in some regions [ ] . although there were modelling efforts underway, only a few governments incorporated such data for policy decisions. data from seroepidemiological studies have been limited, primarily due to the lack of routine influenza serosurveys, and the essay section contains opinion pieces on topics of broad interest to a general medical audience. technical challenges with the assays, interpretation, and validation of results. available serological data on prevalence or seroincidence of humoral immunity yielded age-specific attack rates that indicated a substantial proportion of asymptomatic infections and mild illnesses, similar to or greater than past pandemics and seasonal outbreaks. this was confirmed by a recent hong kong study showing the proportion of asymptomatic infection, secondary attack rates, viral shedding, and course of illness among household members were largely similar between infections with seasonal and pandemic influenza virus strains circulating during [ ] . the few published serosurveys revealed heterogeneities in infection rates among different groups and between different places [ ] [ ] [ ] . in particular there appears to be serological evidence of substantial preexisting humoral immunity among older adults, ranging from % ( : titre by haemagglutination inhibition in those years or over) [ ] to % ( : titre by microneutralisation assay in those years or over) [ ] in different studies. further data on population susceptibility by age or the availability of a rapid and accurate serological test could allow health services to further target vaccine efforts for subsequent waves, as has been done in a few countries [ ] . early on, some airports installed thermal screening and others asked travellers to declare fever or respiratory symptoms at disembarkation. the utility of these interventions has been repeatedly challenged [ ] , although if executed well could delay the start of community transmission by a few weeks [ , ] (table ) . similarly, during the early stages of global or local spread, quarantine, isolation, school closures, and other social distancing measures were variously implemented in some populations (e.g., mexico [ ] , western japan [ ] , uk), although most have not yet been formally evaluated and published [ ] . two exceptions are in hong kong and the uk. in the former, it was estimated that transmission fell by % when schools closed [ ] . in settings like hong kong, with the infrastructure and resources to implement such measures and n decisions regarding pandemic response during the exigencies of a public health emergency must be judged according to the best evidence available at the time. n revising pandemic plans-to be more flexible and more detailed-should wait for who leadership if national plans are not to diverge. surveillance beyond influenza should be stepped up, and contingencies drawn up for the emergence or re-emergence of other novel and known pathogens. [ ] but some countries attempted containment in phases and . some countries even instituted a ''containment phase'' using case-finding and various measures such as isolation and antiviral treatment of ill suspected and confirmed cases, and quarantine of exposed persons with or without antiviral chemoprophylaxis, while others never attempted or quickly moved from resource-intensive containment to mitigation [ ] . a preliminary evaluation of intensive containment undertaken in parts of the uk during its spring/summer wave of demonstrates how resource-and labour-intensive community containment could have been and also how even with a lot of resources the measures had to be abandoned [ ] . it is now recognised that the phrase ''containment'' was unfortunate and potentially misleading since at best the actions were only mitigating impact [ ] . this pandemic virus transmitted efficiently among children and at least one study has shown that school closures were associated with reduced population transmission when implemented early [ ] . closures appear to have stopped school outbreaks in western japan and might have also mitigated impact initially on the local communities [ ] . however, decisions on this intervention were contextually specific, dependent on feasibility and their potential downsides [ ] . in europe and the us the judgement was generally that proactive school closures would not be justified as a community mitigation intervention in the context of a perceived mild-to-moderate pandemic among the general population, and reserve plans for widespread closure have not been activated in most jurisdictions. however, local decisions were made to close schools in some areas as a response to prevent transmission and high attack rates among schoolchildren or simply where there was too much illness and absenteeism to sustain teaching [ , ] . personal protective interventions such as face masks, hand hygiene, and early isolation may have been beneficial in reducing transmission at the individual level in the home [ , ] , although household secondary attack rates during the pandemic were similar to those with seasonal influenza [ , ] . their population level impact remains to be assessed. there was much debate over whether to use conventional masks or respirators in health care settings. one well-conducted canadian trial on seasonal influenza virus transmission published during the pandemic suggested no additional advantage from n respirators [ ] . oseltamivir and zanamivir (and later peramivir in some countries) played a role in the mitigation effort, sometimes drawing on national stockpiles. except for japan, widespread use of antivirals had not been the norm previously. it became standard to recommend neuraminidase inhibitors for treatment of inpatients and high-risk outpatients, and in restricted circumstances for chemoprophylaxis. innovative delivery schemes were sometimes developed. those who fell sick in england could have a telephone assessment (taking pressure off primary care) and then if appropriate receive empiric oseltamivir treatment from a local pharmacist. in norway oseltamivir was made available ''over the counter.'' however in many european settings, reluctance remained among primary care providers to prescribe a drug they were unused to. another controversy was whether to offer oseltamivir to all those with symptoms or target those at higher risk for complications. the observational data so far suggest that early treatment with neuraminidase inhibitors have worked to reduce severe disease and have not been linked to significant adverse risks [ , ] . late clinical presentation and delayed initiation of antiviral treatment have been implicated with more severe complications worldwide, indicating gaps in identifying and treating patients before disease severity increases. while sporadic cases of oseltamivir resistance have been reported in association with a specific mutation (h y in neuraminidase), such oseltamivir-resistant viruses have rarely transmitted [ ] . indeed, the pandemic virus has remained genetically and antigenically stable so far. the core pharmaceutical preventive intervention was vaccines and this has box . a series of ''firsts'' about pandemic (h n ) n the first pandemic to emerge in the twenty-first century. it has been more widespread and remains ongoing, compared to sars. n the first pandemic to occur after major global investments in pandemic preparedness had been initiated. n the first pandemic for which effective vaccines and antivirals were widely available in many countries, thus requiring public health authorities to earn and retain the confidence of health care providers through whom such are usually distributed. n the first influenza pandemic to coincide with the ongoing hiv/aids pandemic and for which preliminary data do not suggest a substantial, disproportionate impact on hiv-infected patients. n the first pandemic that took place within the context of a set of international health regulations and global governance, which had not been widely tested until the present. n the first pandemic with early diagnostic tests that led to rapid diagnosis but also an early obsession in the media and of policymakers with having reports of the numbers of those infected. n the first pandemic with antivirals available in many countries that led to a hopeful expectation that the pandemic might be containable, leading to the preparation for and implementation of a ''containment phase'' in some places. n the first pandemic in which intensive care was available in many countries to treat critically ill patients, fostering an expectation that everyone could be treated and cured. n the first pandemic with a ''blogosphere'' and other rapid social media messaging tools that challenged conventional public health communication. been a particular focus for critics citing the uneven and suboptimal uptake across countries. development of a pandemic vaccine was a scientific success, but limited availability until after the autumn/winter wave had nearly peaked in the northern hemisphere contributed to lower coverage than anticipated [ ] . vaccination coverage depended on many factors, including availability, preordering, licensing and bureaucratic hurdles, logistics, convenience, and, most crucially, public and professional perceptions. this pandemic presented a particular risk communication challenge, since while infection usually results in mild illness, occasionally it is lethal, even in the young and previously healthy despite optimal treatment [ ] [ ] [ ] . in the absence of any excess risk of serious side effects compared to annual seasonal vaccines [ ] (despite the intensive effort to look for such) the benefits of immunisation far outweighed any potential down-sides at the individual level, particularly for those at higher risk for complications. notwithstanding such evidence, the cost of pandemic vaccines was considerable and a loss of public confidence has sometimes been triggered by unsubstantiated media reports of serious side effects with a ''new vaccine'' that utilised the same manufacturing technology as for years of seasonal vaccines. uptake among health care providers as role models has been mixed, as has their expression of the need for vaccination at all. this sometimes cast doubt in the minds of the public. conversely, pandemic deaths in young healthy people abruptly changed public perception (such as in canada, romania, and finland); supply and organisational issues then became crucial. another more fundamental criticism challenges whether vaccines should have been procured at all given an eventual surplus in the developed north. the unexpected finding that a single dose was immunogenic among all persons except for younger children, which reduced the required number of doses by half from the projected number needed in most countries, but this was not known in advance of countries placing vaccine orders. had there been ''overpreparation''? the prior worry had been the reverse -would there be sufficient production capacity to meet needs [ ] ? even in retrospect, and with the observed burden of the pandemic, a vaccine was clearly justified for countries where annual vaccines for seasonal influenza are routinely recommended. field and pharmacovigilance data so far have shown that these vaccines were immunogenic, effective, and very safe [ ] . however, the frailty was timing and availability. generally supplies came in later and in smaller amounts than forecasted, in part due to lower yield in growth of the vaccine virus strain than expected. reduce and delay community spread somewhat at the earliest stage to allow better preparation for mitigation response [ ] completely prevent entry of infected individuals due to suboptimal sensitivity and asymptomatic (including infected and within incubation period) or subclinical presentation [ ] many countries did not attempt these measures because of logistics, stage of pandemic [ ] or other cost-benefit considerations [ ] china hong kong sar japan personal protective measures (e.g., face masks, hand hygiene, cough etiquette, early self-isolation when ill) reduce risk of infection to self and close contacts (if self is ill and infected) [ , ] have not been evaluated whether they can provide significant populationlevel protection virtually all countries implemented these measures to varying degrees in health care settings according to the risk of the situation. almost all encouraged hand hygiene, cough etiquette, and early self-isolation most countries recommended adoption of hand hygiene, cough etiquette, and early self-isolation when ill, but use of face masks in the community was uncommon except in east asia. antivirals for treatment and chemoprophylaxis [ , ] mitigation: reduce illness severity and complications if administered early; reduce transmission from those receiving treatment; sometimes also used as chemoprophylaxis in high-risk circumstances provide significant population-level protection or allow containment attempts at source containment were not possible, as the pandemic was effectively already in who phase when what became the pandemic virus was first identified [ ] . initial observational studies suggest antivirals were successful when early treatment was administered canada germany hong kong sar japan uk us (these populations attempted the intervention initially but effort was not sustained towards the later stages of the pandemic) vaccines mitigation (a) at individual level by conferring immunity to infection in those at higher risk of severe disease or (b) at a population level by immunizing population groups especially those who are transmitting most (i.e., children) in most countries vaccine was not available early enough and/or arrived in insufficiently large amounts to achieve mitigation at a population level. greater population benefit may occur in the next season most of the orders arrived after the peak of the autumn/winter wave in the geographic north (whose countries had received most vaccines). therefore, judgement on their impact in averting serious morbidity and deaths may come only after the second winter. perhaps then, differential use by countries will allow for comparisons where there is good surveillance for severe disease and deaths. there have been claims of extraneous influence on the independent and objective judgment of expert advice that in turn influenced decision-making [ ] . these claims have been robustly countered as they relate to who's advisory and decision-making process [ ] . as harvey fineberg, chair of who's external review, pointed out, when assessing any allegations of impropriety or bias, or the perception of such, it would be important to distinguish between financial or other conflicts with potential pecuniary gains versus predispositions arising from an individual's background and experience. rather than aiming for a complete purge of any and all experts who had worked with vaccine manufacturers and received sponsorship, as these are often the very same group who possess the most relevant and useful expertise precisely because they have been closely involved in the research and development process, the focus should be on making the declaration of such interest wholly transparent and comprehensive according to a set of robustly established procedures that can withstand the strictest scrutiny. it is reassuring that who has honoured its commitment to making public the names and declarations of interest of the pandemic emergency committee when the pandemic was declared over on august . additionally, receiving advice should be differentiated from making decisions. the people entrusted with undertaking the latter task should then judge the validity of the advice rendered by experts, having taken into account their interest declarations. the decision makers should also be prepared to justify their actions. it is important to learn from our experience through the first year and beyond as we move into the new seasonal influenza [ , ] . it is theoretically possible, although unlikely, that the second winter of this pandemic will be worse than the first, as happened for the pandemic when transmissibility increased [ ] . equally, if the pandemic virus outcompeted the a(h n ) virus strains responsible for more intense seasonal epidemics, there may even be a diminution of disease burden in older people. as of this writing, seasonal influenza a (h n ) and b virus strains continue to cocirculate. antigenic drift in the h n virus is expected to occur in the future, especially under the pressure of so many people now being immune through infection or immunisation, although the timing is unpredictable. the pandemic virus is included in the trivalent seasonal influenza vaccine composition for both hemispheres. clear communication of public health messages will remain a particular challenge and not confusing what could happen (and should be prepared for) with what is most likely to happen. in assessing the pandemic response, decisions made during the exigencies of a public health emergency must be judged according to the best evidence available at the time. hindsight always gives perfect vision and using post-hoc information to evaluate prior decisions at best confuses and often produces unfair conclusions. preparedness plans will have to be revised in due time, after the many lessons learned have been gathered. this should be done quickly in case the worst is not yet over [ ] . however, rewriting plans should best wait for who leadership if national plans are not to diverge. a strong argument exists for making future plans more flexible and having extra descriptions including the many aspects of severity when a pandemic is emerging that then determine the consequential public health actions [ ] . broadening surveillance for a range of influenza a viruses among a wide range of animals (e.g., swine), not just in avian species, as well as strengthening the monitoring of seasonal influenza virus infections in humans will facilitate identification of novel influenza a viruses of pandemic potential, and earlier detection of the emergence of a pandemic virus. more broadly we should look beyond influenza and draw up contingencies for the emergence or re-emergence of other novel and known pathogens [ ] . one challenge faced initially in this pandemic was for timely collection and sharing of clinical data to inform optimal management of critically ill patients worldwide. establishing clinical research infrastructure prior to a pandemic and a central institutional review board will facilitate data collection and analyses [ ] , whether for the next influenza pandemic, sars outbreak, or next novel respiratory pathogen of global importance. clinical management of severe influenza disease should not be limited to the current antiviral regimen, and include the development of other therapeutics (e.g., novel antivirals and immunotherapy). ongoing improvements in the routine and timely monitoring of hospital admissions and deaths attributable to influenza, as well as representative serological surveys at regular intervals can provide epidemiological data with which to reduce uncertainty around the true burden of influenza and thus inform policy choices [ ] . assessment of the humoral and cellular immune response over time in a subset of vaccinated individuals could reveal how vaccine-induced immunity differs from natural infection, and whether cross-reactive responses to other influenza virus strains are modulated by the two types of immunological response [ ] . the latter could become important as the pandemic strain has already been cocirculating with other interpandemic influenza a virus strains in some parts of the world. greater access to antivirals and influenza vaccines worldwide is an ongoing challenge. although who secured pledges of million vaccine doses and monies for operations, and more than less-resourced countries have signed agreements with who for supply of vaccines, this gap remains. it is an indefensible fact that these vaccines started to flow to the poorer countries well after they began going to the countries with advance purchase arrangements. delivering timely pandemic influenza vaccination in countries without existing seasonal vaccine programmes is proving difficult. the longterm solution has to be improved surveillance, expanded monitoring of disease burden, and better prevention and control of influenza, including the development of seasonal vaccine use and production in all regions of the world [ ] . increased coverage of available bacterial vaccines (hib, pneumococcal) will help prevent secondary invasive bacterial coinfections with either seasonal or pandemic influenza. finally accusations of ''overreaction'' can be countered by the observation that investment in fire services or insurance is usually judged against their ability to respond to conflagrations. if the first test is a lesser fire, that experience should be used for improvements rather than as a reason to scrap the fire engines and cancel the insurance [ ] . influenza pandemics of the th century experience and lessons from surveillance and studies of the pandemic in europe writing committee of the who consultation on clinical aspects of pandemic influenza ( ) clinical aspects of pandemic influenza a (h n ) virus infection severe respiratory disease concurrent with the circulation of h n influenza pandemic influenza a(h n ) virus illness among pregnant women in the united states critical illness due to a/h n influenza in pregnant and postpartum women: population based cohort study estimates of the prevalence of pandemic (h n ) , united states preliminary estimates of mortality and years of life lost associated with the a/h n pandemic in the us and comparison with past influenza seasons the swine flu affair: decision-making on a slippery disease pandemic potential of a strain of influenza a (h n ): early findings comparative epidemiology of pandemic and seasonal influenza a in households incidence of pandemic influenza a h n infection in england: a cross-sectional serological study influenza a(h n ) seroconversion rates and risk factors among distinct adult cohorts in singapore cross-reactive antibody responses to the pandemic h n influenza virus entry screening to delay local transmission of pandemic influenza a (h n ) entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation a clinical virological and epidemiological analysis what mexico taught the world about pandemic influenza preparedness and community mitigation strategies epidemiological characteristics and low case fatality rate of pandemic (h n ) in japan europe's initial experience with pandemic (h n ) -mitigation and delaying policies and practices school closure and mitigation of pandemic (h n ) , hong kong who interim protocol: rapid operations to contain the initial emergence of pandemic influenza the role of the health protection agency in the 'containment' phase during the first wave of pandemic influenza in england influenza pandemic: an independent review of the uk response to the influenza pandemic influenza (h n ) outbreak and school closure closure of schools during an influenza pandemic facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial household transmission of influenza a (h n ) virus after a school-based outbreak household transmission of pandemic influenza a (h n ) virus in the united states surgical mask vs n respirator for preventing influenza among health care workers: a randomized trial antiviral treatment for patients hospitalized with pandemic influenza a (h n ) the truth about tamiflu? neuraminidase inhibitors in pandemic a/h n flu interim results: state-specific influenza a (h n ) monovalent vaccination coverage -united states hospitalized patients with h n influenza in the united states pediatric hospitalizations associated with pandemic influenza a (h n ) in argentina mortality from pandemic a/h n influenza in england: public health surveillance study preliminary results: surveillance for guillain-barre syndrome after receipt of influenza a (h n ) monovalent vaccine -united states preparing for the next pandemic pandemic influenza a(h n ) breakthrough infections and estimates of vaccine effectiveness in germany conflicts of interest. who and the pandemic flu ''conspiracies the international response to the influenza pandemic: who responds to the critics. pandemic (h n ) briefing note how well are we managing the influenza a/h n pandemic in the uk? a new decade, a new seasonal influenza: the council of the european union recommendation on seasonal influenza vaccination estimates of the transmissibility of the (hong kong) influenza pandemic: evidence of increased transmissibility between successive waves emerging infectious diseases: a -year perspective from the national institute of allergy and infectious diseases early observational research and registries during the - influenza a pandemic studies needed to address public health challenges of the h n influenza pandemic: insights from modeling association between the - seasonal influenza vaccine and pandemic h n illness during spring-summer : four observational studies from canada global pandemic influenza action plan to increase vaccine supply we thank timothy m. uyeki of the us centers for disease control and prevention for substantial discussion and input. icmje criteria for authorship read and met: gml an. agree with the manuscript's results and conclusions: gml an. wrote the first draft of the paper: gml. contributed to the writing of the paper: gml an. contributed to the review from the experience of the pandemic in european countries: an. key: cord- - ldxvbpz authors: pleschka, stephan; ludwig, stephan; wolff, thorsten; planz, oliver title: anti-viral approaches against influenza viruses date: journal: new concepts of antiviral therapy doi: . / - - - - _ sha: doc_id: cord_uid: ldxvbpz nan influenza is a highly contagious, acute respiratory disease with global significance that affects all age groups and can occur repeatedly in any individual. the etiological agent of the disease, influenza virus is responsible e. bogner and a. holzenburg (eds.) , new concepts of antiviral therapy, - . nordufer , d- berlin, germany; friedrich-loeffler-institute (fli) , germany; ä for an average between three and five mill. cases of severe influenza leading to about , - , mortalities annually in the industrialized world according to who estimations. compared to otherwise healthy persons, death rates in patients of risk groups (s. . ) are - fold higher in patients with cardiovascular or pulmonary disease as compared to healthy individuals. annual health cost, costs, e.g. due to work absenteeism (also related to parental care of infected children) or costs related to death, increased disabilities etc. can be higher than mil. € in european countries. furthermore. for a pandemic outbreak the centers for disease control (cdc) estimates that in the usa that % of all death will be caused by % of the population which are at high-risk. this will result in a financial burden of up to . billion us$ not including the commercial impact. the death rate would be up to , accompanied by up to , hospitalizations, - million outpatient visits and - million additional illnesses wilschut and mcelhaney, ) . this clearly would overrun the capacity of current supply and management of vaccines available. since waterfowl represents the natural reservoir for the virus (lamb and krug, ; webster, ; wilschut and mcelhaney, ; wright and webster, ) and many other animal species can be infected, the eradication of the virus is impossible and a constant reemergence of the disease will continue to occur. epidemics appear almost annually and are due to an antigenic change of the viral surface glycoproteins (fig. ) . furthermore, highly pathogenic strains of influenza-a-virus have emerged unpredictably but repeatedly in recent history as pandemics like the "spanish-flu" that caused the death of - millions people worldwide (taubenberger et al., ; webster, ) . since these pandemic virus strains usually possess different antigenic characteristics, current vaccines will be ineffective once such a virus emerges. regarding the vast possibilities for such a strain to "travel" around the world (hufnagel et al., ) it becomes evident that effective countermeasures are required for the fight against these foes. in recent outbreaks of avian viruses that infected humans ( , , / / ) hatta and kawaoka, ; li et al., ) from a total of confirmed cases people died ( / ) (world health organization, ) . fortunately, until now these particular viruses have not acquire the ability to spread in the human population. however, any novel virus strain emerging in the future may have such a capability (webby and webster, ) . here, we give an overview of current and new anti-influenza strategies, such as immunization methods and drugs against the virus. since every virus depends on its host cell, cellular functions essential for viral replication may also be suitable targets for anti-viral therapy. in this respect intra-cellular signaling cascades activated by the virus, in particular mapk pathways, have recently come into focus ludwig et al., ) . influenza viruses belong to the order of the orthomyxoviridae. they possess a segmented, single stranded rna-genome with negative orientation. they are divided into three types, a, b and c based on genetic and antigenic differences. among the three types influenza-a-viruses are clinically the most important pathogens since they have been responsible for severe epidemics in humans and domestic animals in the past. thus the focus of this chapter will be on type-a influenza viruses. a detailed description of the viral proteins and the replication cycle of influenza-a-viruses can be found elsewhere (lamb and krug, ; wright and webster, ) . therefore we will only give a brief overview on these topics without referring to individual references. the influenza-a-virus particle is composed of a lipid envelope derived from the host cell and of - structural virus proteins ( figure and table ). the components of the rna-dependent rna-polymerase complex (rdrp), pb , pb and pa are associated with the ribonucleoprotein complex (rnp) and are encoded by the vrna segments - . the pb segment of many, but not all, influenza-a-virus strains also contains a + -reading frame encoding the recently discovered pb -f protein (chen et al., ) . the viral surface glycoproteins hemagglutinin (ha) and neuraminidase (na) are expressed from vrna segments and , respectively. the nucleoprotein (np) is encoded by segment and associates with the vrna segments. it is the major component of the rnps. the two smallest vrna segments each code for two proteins. the matrix protein (m ) is colinear translated from the mrna of segment and forms an inner layer within the virion. a spliced version of the mrna gives rise to a third viral transmembrane component, the m protein, which functions as a ph-dependent ion channel. employing a similar coding strategy segment harbors the sequence information for the nonstructural ns protein and the nuclear export protein ns /nep. ns /nep is a minor component of the virion and is found associated with the m protein. figure . the influenza-a-virus particle schematic representation of the spherical influenza-a-virus particle that has a diameter of about nm. the eight viral rna segments were separated by urea-polyacrylamide gel electrophoresis and visualized by silver staining (left) . the corresponding gene products and their presumed location in the virus particle are indicated (right). ns is not a structural part of the mature virion. for details see text. table summarizes details of the genome segments, the encoded viral proteins and their according function. the viral replication cycle is initiated by binding of the ha to sialic-acid (neuraminic acid) containing cellular receptors and subsequent endocytosis of the virus (figure ) (for references: (lamb and krug, ; wright and webster, ) ). the active ha molecule consists of two subunits (ha / ha ) derived from the uncleaved precursor ha , which becomes proteolytically processed after release of the virion by extra-cellular proteases. this cleavage is absolutely essential for ha-function and cell infection. virus disassembly occurs in the acidic environment of late endosomal vesicles and involves two crucial events. first, the conformation of the ha is changed to a low-ph form, which results in exposure of a fusion active protein sequence within the ha . this fusion peptide is thought to contact the endosomal membrane and to initiate fusion with the viral envelope. second, the low ph in the endosomes activates the viral m ion channel protein resulting in a flow of protons into the interior of the virion. acidification facilitates dissociation of the rnps from the m protein. the rnps are subsequently released into the cytoplasm and rapidly imported into the nucleus through the nuclear pore complexes. the viral genomic segments are replicated and transcribed by the viral rdrp associated with the rnps in the nucleus of the infected cell. the vrna is directly transcribed to mrna and, in addition, serves as a template for a complementary copy (crna), which itself is the template for new vrna. in the late phase of infection newly synthesized viral rnps are exported to the cytoplasm. ns protein functions as a regulatory factor in the virus infected cell. the na, the m and the precursor ha (ha ) proteins follow the exocytotic transport pathway from the rer via the golgi complex and the trans golgi network. the mature ha and na glycoproteins and the nonglycosylated m are finally integrated into the plasma membrane as trimers (ha) or tetramers (na, m ), respectively. m assembles in patches at the cell membrane. it is thought to associate with the glycoproteins (ha and na) and to recruit the rnps to the plasma membrane in the late phase of the replication cycle. finally the viral rnps become enveloped by a cellular bilipid layer carrying the ha, na and m proteins resulting in budding of new virus particles from the apical cell surface. replicative viral proteins enter the nucleus to amplify the viral genome. in the late stage of the infection cycle newly synthesized rnps are exported from the nucleus and are assembled into progeny virions that bud from the cell surface. the polymerase complex of influenza viruses does not possess a proof reading activity, thus numerous mutations accumulate in the viral genome during ongoing replication (lamb and krug, ) leading to changes in all proteins. this includes conformational alteration of ha-and na-epitopes against which neutralizing antibodies are generated. influenza-a-viruses are categorized by antigenic differences of the ha-and na-proteins. the high mutation rate combined with the high replication rate results in a multitude of new variants produced in each replication cycle, thus allowing the virus to rapidly adapt to changes in the environment. this results in an escape of the existing immunity and in resistance to drugs acting directly against viral functions. gradual changes of the antigenic properties that make existing vaccines less or non effective are described as antigenic drift and demand for new compositions of the yearly vaccines. due to the nature of their segmented genome influenza virus can independently recombine segments upon the infection of a cell with two different viruses. this is described as genetic reassortment. today hasubtypes (h -h ) and na-subtypes (n -n ) are known, which can mix and lead to new antigenic properties. (lamb and krug, ; webster et al., ; wright and webster, ) . not all combination will ultimately be advantageous, but can lead to the generation of a virus that combines the ability to replicate in humans with novel antigenic properties (antigenic shift). this has happened at least three times in the last century resulting in the pandemics of ("spanish flu"), ("asian flu") and ("hong kong flu") that caused up to million death. therefore, the question is not "if" but "when" will such a pandemic occur again (horimoto and kawaoka, ; webby and webster, ; webster, b) . a vaccine against such "new" viruses can not be generated in advance and as vaccine production would need significantly more time than it takes for a pandemic virus to spread around the world (hufnagel et al., ) , alternative weapons in the fight against these enemies are urgently needed. besides pandemic variants that can occur when human and avian influenza virus reassort in porcine hosts (regarded as "mixing vessels") (webster, a; webster et al., ) , avian influenza virus strains have directly infected humans, as happened in hong kong in (claas et al., ; de jong et al., ; subbarao et al., subbarao et al., ) and recently ( subbarao et al., / koopmans et al., ) during vast outbreaks of avian influenza. these viruses show an extremely high virulence in humans with case fatality rates up to %. the virus that normally causes a respiratory disease (for references: (wilschut and mcelhaney, ) ) is transmitted by aerosol droplets and contaminated hands and can already be shed before onset of symptoms . therefore, high population density and dry air leading to reduced protection of respiratory epithelium by the mucus are conditions that promote transmission of the virus. the infection with influenza viruses is normally limited to the respiratory tract. here proteases released by clara cells in the epithelium are present that activate the ha to allow further infections (s. . ) (for review (ludwig et al., ) ). innate immunity as well as the adaptive immune system will normally restrict virus propagation. therefore population groups, that have a less protective immune system, such as young children up to two years and older persons over as well as immunocompromised or chronically diseased persons are especially of risk. the replication of the virus leads to the lysis of the epithelial cells and enhanced mucus production causing running nose and cough. furthermore, inflammation and oedema at the replication site are due to cytokines released. this can lead to fever and related symptoms. bacterial super-infections of the harmed tissue can further complicate the situation. normally onset of systemic (fever, myaglia, headaches, severe malaise) and respiratory (coughing, sore throat, rhinitis) symptoms occur after about two days incubation period and can last for about seven to ten days. coughing and overall weakness can persist for up to two weeks. if the virus spreads from the bronchiolar tract to the aveolars, viral pneumonia and interstitial pneumonitis with mononuclear and haemorrhage infiltration and finally lysis of the inter-aveolar space is possible (wilschut and mcelhaney, ) . this scenario is a likely picture in case of infection with a pandemic influenza strain, where the individual has not had a prior exposure to the virus and the innate immunity reaction can lead to a strong immunpathogenesis. high virus replication will induce secretion of large quantities of cytokines by the infected epithelia and will stimulate inflammatory processes. together with the destruction of the epithelia this results in an influx of fluid into the aveolars leading to hypoxia and acute respiratory distress syndrome, that may cause the death within a short period of time ( - days after onset). this scenario might also be caused by additional viral factors enhancing pathogenicity. such factors that are yet not well defined probably have contributed to the devastating outcome of the "spanish flu" (wilschut and mcelhaney, ) . accurate and rapid diagnosis of the disease is essential for an effective treatment, especially with anti-viral substances, as virus replication and therefore illness progresses rapidly. samples can be tested serologically, by cell culture or rt-pcr for strain typing and should be done within four days after onset of symptoms (wilschut and mcelhaney, ) . there are two main methods of influenza prophylaxis: the use of antiviral drugs and vaccines. several drugs are available for influenza prophylaxis functioning either as m -ion channel inhibitors (amantadine and rimantadine) or as inhibitors of the na (zanamivir and oseltamivir). despite these anti-viral drugs, which are a useful adjunct to influenza vaccines, vaccination itself remains the cornerstone of prophylaxis. vaccination induces a good degree of protection and is in general well tolerated by the recipient. nevertheless, while resistant virus variants can emerge after antiviral drug treatment the disadvantage of vaccination is that immunization needs to be refreshed almost every year, since the vaccine must reformulated to take account of the changing virus. in the immune response to influenza infection both the humoral and cell mediated immunity are involved. from the side of the humoral immune system, both the mucosal and the systemic immunity contribute to resistance to influenza infection. the cellular immune response is involved in recovery from influenza virus infection by eliminating virus-infected cells and by providing help for antibody production woodland et al., ) . consequently, the humoral immune response is the primary target of vaccination. after influenza virus infection antibodies directed against all major viral proteins can be detected in humans and the level of serum antibodies correlate with resistance to disease (couch, ; couch and kasel, ; coulter et al., ; nichol et al., ; potter and oxford, ) . only antibodies specific for the surface glycoproteins ha and na are associated with resistance to infection. in contrast, antibodies to the conserved internal antigens m and np are not protective (de jong et al., ; tamura and kurata, ) . the mucosal tissues of the respiratory system are the main portal entry of influenza virus and consequently the mucosal immune system functions as the first line of defense against infection apart from innate immunity (see paragraph ). antibodies secreted locally in the upper respiratory tract are a major factor in resistance to natural infection. secretory immunoglobulin a (siga) and to some extent igm are the major neutralizing antibodies directed against the entering virus. furthermore, these antibodies can function intra-cellular to inhibit influenza replication. iga and igm are involved in protection of the upper respiratory titre ≥ ) can be detected in approximately % of subjects after natural influenza virus infection and correlates with protection against the flu. plasma cells producing all three major ig classes are present in the peripheral blood in normal subjects (cox and subbarao, ; laforce et al., ) . tract while serum igg acts in protection of the lower respiratory tract ). an anti-ha antibody response (haemagglutination-inhibition (hi) the immune response induced by infection protects against reinfection with the same virus or an antigenically similar viral strain. cell mediated immunity plays a role in recovery from influenza virus infection and may also prevent flu-associated complications, but it does not seem to contribute significantly in preventing infection. influenza specific cellular t cells have been detected in the blood and the lower respiratory tract secretions of infected subjects . influenza virusspecific cytotoxic t-lymphocytes (ctl) regognize both external and internal proteins of virus on infected cells. in humans a major component of this response is directed toward the np-and m -protein. even though influenza virus specific ctl's are not able to protect against the infection, these cells are important for the clearance of the virus. futhermore, cytolysis of influenza virus-infected cells can be mediated by influenza virus-specific antibodies and complement mcmichael et al., ; mcmichael et al., ; townsend et al., ) . cd + t cells function as helper cells for antibody production. moreover, it is suggested, that cd cells might act as direct effectors in protection against influenza virusinfection (brown et al., ) . inactivated vaccines (iv) are availeble for about years. because of the antigenic drift observed in influenza ha-and na-glycoproteins these vaccines need to be matched with the randomly mutating molecular structure of the new occurring "drift" strain. besides these vaccines there are various new approaches for influenza vaccines in promising developmental stages. these new stratagies include vaccines with immunomodulators, virosomes and dna-vaccines. ivs vaccines are administered world wide each year with millions of doses. these vaccines have good safety and tolerance profiles, with very low number of adverse reactions reported. these reactions are tenderness and redness that arise locally at the injection site and are more frequent in healthy (< %) than in elderly recipients ( %) . ivs are produced by propagation of the virus in embryonated chicken eggs. the currently used bacterial endotoxin-free trivalent ivs (tiv) are formulated with µg ha each from a current influenza virus a/h n , a/h n , and a b-virus strain. the seed strain is prepared by co-infecting the allantoic sac of the chicken embryo with a laboratory-adapted high-growth phenotype of h n (a/pr/ / ) and the epidemic strain. this results in viral replication and genetic re-assortment leading to high growth reassortants. thereafter the new hybrid viruses are screened for the absence of genes encoding pr/ or pr/ -like surface glycoproteins. the selected seed strain containing ha-and na-components of the epidemic strain is mass propagated in chicken eggs to obtain sufficient quantities of vaccine virus. the allantoic fluid is harvested, and the virus is concentrated and highly purified by zonal centrifugation. as a next step the virus is inactivated. depending on the nature of inactivation the vaccine is used as whole inactivated vaccine after treatment with formalydehyde or β-propiolactone or as split vaccine (chemically disrupted by ethyl either or sds). furthermore, the vaccine is used as subunit vaccines (purified surface glycoproteins). even though influenza vaccines have excellent tolerant profiles, since propagation in chicken eggs may lead to contamination of the vaccine with trace amounts of residual egg proteins, they should not be administered to persons who have anaphylactic hypersensitivity to eggs. whole inactivated influenza vaccine is more immunogenic than split vaccine or subunit vaccine, but is also associated with more frequent side reactions. consequently, split or subunit are given to children younger than age and two half doses are recommended given at least month apart for naïve persons to develop protective immunity (bridges et al., ) . protection after vaccination against influenza virus infection is dependent on the antigenic match between the vaccine strains and circulating the influenza virus strain. moreover, protection is also dependent the age and the previous exposition to influenza of the vaccine recipient. if ivs have a good antigenic match they are - % effective in the prevention of morbidity and mortality among healthy adults (beyer et al., ) . in elderly people the effect of protection is reduced to - % because of decreased immune function. since the immune system is naïve in young children, they also show a reduction in protection against influenza vaccination (nichol et al., ) . immunosuppressed individuals, elderly people and subjects with underlying chronic diseases are at increased risk for influenza and related complications. for these people conventional influenza vaccines provide only limited protection. in order to enhance the immune reaction after influenza vaccination, several adjuvants (latin verb: adjuvare -to help) that function as immunopotentiators have been evaluated. the liposomal influenza vaccine (influsome-vac) consists of liposomes containing the viral surface proteins ha-and na-derived from various influenza strains and il- or granulocyte-macrophage colonystimulating factor (gm-csf), as an adjuvant (babai et al., ) . in clinical trails with either young adults or elderly vaccination of influsome-vac appeared to be both safe and more immunogenic than the currently used vaccine (ben-yehuda et al., a; ben-yehuda et al., b) . furthermore adjuvant emulsions combined with subunit influenza antigens are in use, such as the "oil in water"-emulsion containing squalene, mf , (fluad). this commercially available product was tested in clinical trials in comparison with non-adjuvanted conventional vaccines. again in elderly individuals the addition of the mf -adjuvant to subunit influenza vaccines enhances significantly the immune response without causing clinically important changes in the safety profile of the influenza vaccine (podda, ) . other adjuvants that increase immunoreactivity after influenza vaccination are immune stimulating complexes (iscoms). they are - nm cage-like structures, which consist of glycoside molecules of the adjuvant quil a, cholesterol and phospholipids in which the antigen can be integrated. . in animal models, even in the presence of pre-existing antibodies they function as a potent adjuvant system by inducing cellular and humoral immune responses. (coulter et al., ; rimmelzwaan et al., ; windon et al., ) . as mentioned, gm-csf has a potential role as a vaccine adjuvant. it may enhance the response to vaccination in immunosuppressed individuals. gm-csf stimulates maturation of hematopoietic progenitor cells, induces class ii major histocompatibility complex antigen expression on the surface of macrophages, and enhances dendritic cell migration and maturation (jones et al., ) . nevertheless, in various clinical trails with immunosuppressed individuals and cancer patents it was shown, that it is unlikely that gm-csf improves the immune response (ramanathan et al., ) . for production of influenza vaccines in large-scale cell culture systems several continuous cell lines have been tested for the production of influenza vaccines (kistner et al., ; pau et al., ; seo et al., ; youil et al., ) . production of influenza vaccine in mammalian cell lines has some advantages but also has disadvantages compared to production in chicken egg. (tree et al., ; youil et al., ) . process controllability, scalability and supply of substrates are much easier in cell culture systems. furthermore, cell culture production reduces the risk of microbial contamination. in contrast, the greatest disadvantage of cell culture based influenza vaccine is the relative low viral yield. on the other hand and a major disadvantage of production in chicken eggs is their supply and possible bacterial contaminations. additionally the lethality of h n influenza virus to chicken embryos (s. . . ). at the present ( ) two cell line derived vaccines have been licensed in europe . estimated time for production of such vaccines is about months. the power of this time gaining approach to generate a great variety of specific influenza-vaccines under controlled safety conditions is achieved by the direct use of field strains (kistner et al., ) as well as seed strains specifically designed by reverse genetics systems and the large scale cell culture system. nevertheless, the application of these techniques largely depends on meeting the needs of high viral yield, appropriate permissiveness, and ability to support replication of all influenza virus strains to immunopotentiating reconstituted influenza virosomes (irivs) possess several characteristics defining them as vaccine adjuvants. they are a liposomal carrier system. these reconstituted virus-like particles (vlp; diameter nm) contain a lipid bilayer of phosphatidylcholine and phosphatidylethanolamine. ha and na are intercalated into the lipid bilayer and give the irivs their fusogenic activity, but lack the viral genetic material. irivs are able to deliver proteins, rna/dna and peptides to immunocompetent cells. in addition, virosomes, as vaccine delivery systems, have been shown to be safe and not to engender any antibodies against the phospholipid components. therefore, their use in vaccination of children and elderly people is recommended. the system is already registered for human use and allows a specific targeting of antigens by a cellular or a humoral immune response. a virosome vaccine, inflexal-v, is used in switzerland and italy. (gluck et al., ; langley and faughnan, ; zurbriggen, ) . dna-vaccines are non-infectious and non-replicative plasmid constructs that encode either only the proteins of interest or the protein of interest in combination with immunomodulatory proteins. this kind of vaccination by direct intra-muscular injection of dna was first demonstrated in in a mouse model system et al. (wolff et al., ) . directed intra-muscular dna-vaccination is not very common. the creation of recombinant influenza vaccines based on dna-plasmids is more appropriate. with this technique rapid and flexible construction of dna-plasmid vectors can be achieved, which can address the problems of antigenic drift induced by the circulating influenza virus strain (ljungberg et al., ) . these above described techniques have a potential for the development of live and inactivated vaccines. the efficacy of the plasmid based dnavaccines expressing the immunogenic influenza virus genes alone or in combination with dna encoding various cytokines has also been demonstrated in several animal models (for detail see: bardiya and bae, ) . during dna-vaccination, the foreign genes are endogenously high titers in short time (reviewed in: bardiya and bae, ) . expressed in the host, the proteins subsequently processed, and recognized by the immune system of the host. dna-vaccines elicit a broad-based humoral and cellular immunity against influenza virus proteins (justewicz et al., ) . in addition, alterations in the vector, dose of the dna, inclusion of cpg-odn motifs, fusion with influenza virus-specific helper t cell or ctl-epitopes, and appropriate vaccine delivery mechanisms will further improve the efficacy of these vaccines (bowersock and martin, ; joseph et al., ) . an alternative to ivs are attenuated "live" vaccines such as cold-adapted vaccines (cav: caiv-t, flumist ® ) and ns -defective strains used as intranasal influenza vaccine, that may lead to long-lasting, broader immune response (humoral and cellular) that resembles more closely the natural immunity derived from viral infection. for example, ctls, which are important for the clearance of the virus are activated during an productive infection . additional cytokines produced by the infected cells during the innate immunity response enhance and support reaction of the humoral system. compared to ivs, that are strain-and subtype-specific the cavs (that also have to be adapted to circulating strains) can provide a broader immunity against circulating viruses (belshe et al., ; king et al., ; nichol, ; stepanova et al., ; treanor et al., ; wareing and tannock, ) . cavs that already have been used successfully in russia and are now licensed in the usa kendal, ) can be administered intra-nasally for example as aerosols (abramson, ) . this results in a limited viral replication in the upper and lower respiratory tract and circumvents the need for syringes. it also supports protective mucosal immunity, which is an important property of nasally applied live influenza virus vaccines. for the generation of a cav a donor and a wild type strain are reassorted in such a way, that the ha-and na-segments are wild type (wt) derived and the remaining six segments originate from the donor strain. for this purpose two master strains are currently used as donors in the usa. one to generate a-type and one b-type influenza cavs (mendelman et al., ; murphy and coelingh, ) . these strains are cold adapted ( °c) (kendal, ; maassab and bryant, ) and therefore temperature sensitive (ts) and attenuated, meaning that these viruses will not propagate efficiently at body temperatures. to prevent easy reversion of the genetic markers, that encode the ts-defect and allowing the virus to regain full virulence, all six donor-derived segments carry mutations. for the production of such cav strains embryonated eggs are infected with both viruses (wild type and donor strain) under the selection of antibodies directed against the ha and na of the donor strain. the attenuated donor strain by itself is unable to cause significant illness in humans, but is able to donate the ha-and na-proteins of the contemporary epidemic strain to produce live attenuated vaccine by the traditional egg-based process (belshe, ; clements and murphy, ; jin et al., ) . the live attenuated vaccines were shown to be safe and effective in the general population kendal, ; langley and faughnan, ) . cav are trivalent like the ivs and are composed according to the who recommendations (mendelman et al., ) . new possibilities of reverse genetic techniques will certainly improve production of vaccine strains in time and quality (s. . . ). even though one should consider the possibility of reassortment with another human strain in the vaccinated person, which could produce an aggressive virus, cavs have been successfully used in russia without reports of severe side effects and seem to be safe. they show a comparable effectiveness to trivalent iv's (tivs) and both vaccines can also be used in combination belshe et al., ; boyce and poland, ; edwards et al., ; glezen, ; jackson et al., ; mendelman et al., ; swierkosz et al., ; treanor and betts, ; treanor et al., ) . in addition to the traditional live attenuated vaccines, production by reverse genetics (s. . . ) of replication-incompetent influenza virus-like particles (vlps) by deletion of either the entire ns gene (encoding both the ns and ns protein) or only the ns gene has also been reported. these vlps were entirely produced from cdnas (watanabe et al., b) . although, these technologies are in the very early stages of development and so far only tested in animal models, the vlp incapable of replication and spread to other cells due to deletion of a major protion of the ns or m , are expected to be good novel influenza vaccine candidates (galarza et al., ; watanabe et al., a) . a variation of the theme is presented by influenza virus strains (generated by reverse genetics (s. . . )) that express a modified ns (palese and garcia-sastre, ; palese et al., ; talon et al., ) . this non-structural protein is the major viral interferon (ifn)antagonist (s. tab. ). even though ns is a multifunctional viral protein that supports viral replication it seems to be an accessory protein as a virus without the ns -gene can replicate in ifn-deficient systems (garcia-sastre et al., a; garcia-sastre et al., b; ludwig et al., ) . ifnα/β are two important cytokines expressed in primary infected epithelia cells, that induce innate immunity. ifnα/β-induction severely reduces viral replication even in the presence of ns . therefore recombinant viruses expressing altered ns with reduced capacity to suppress cellular ifn-induction could raise protective immunity and might represent interesting attenuated live vaccine candidates (talon et al., ) . such viruses have been generated by reverse genetic techniques (s. . . ) and have been successfully tested in experimental settings . after initial experiments that implied the in vivo reconstitution of rnps from plasmid-expressed rdrp, np and vrna it became possible to generate recombinant influenza virus de novo totally from plasmid dna (fodor et al., ; , allowing complete genetic manipulation. this manipulation can either concern the combination/mixture of the genomic rna-segments and/or the genesequences themselves. the technique involves the transfection of four plasmids expressing the viral rdrp and the np together with eight plasmids (for all eight genomic rnas) that generate a vrna-like transcript. this again results in the in vivo reconstitution of active rnp-complexes, which will replicated and transcribe the vrnas. thereby all viral rnas and proteins are generated and the viral replication cycle is established resulting ; palese et al., ) . reverse genetics technique can be used to produce influenza vaccines based on recombinant virus (for detail see: bardiya and bae, ) . these methods do not require selection procedures and eliminate the need for multiple passages in eggs, thereby reducing the time required for vaccine production. it is known that interference among the vaccine viruses of type-a and -b can occur that affect the efficacy of the live attenuated vaccines by restricting their replication. to overcome that problem a chimeric virus (a/b) possessing chimeric (a/b) ha, and full-length b-type na in the background of a type-a vaccine virus was created (horimoto et al., ) . this study provided a novel method for creating live attenuated vaccine from a single donor strain. for different reasons the technique of reverse genetics has become highly relevant for anti-viral vaccine approaches. (i) for the production of regular ivs against wild type strains, that either grow poorly or are too pathogenic in eggs (s. later) one can generate strains carrying the ha and na needed in the background of an egg adapted virus. this is normally done by reassortment of the wild type with the egg-adapted strain and can not be well controlled. this problem can be circumvented by plasmid based reverse genetics that allow the controlled design of the reassortant. (ii) as mentioned the cav are composed of ha-and na-genes from the wild type in the production of infectious influenza viruses (for review: garcia-sastre, strain and a mixture of the other six segments from wild type and donor virus. by choice of the according plasmids one can compose a cav-strain that carries all six segments from the donor strain each with an adaptive mutation. this way it is less likely that a revertant virus will arise by mutation in one of the donor strain segments (s. . . ) (maassab and bryant, ; schickli et al., ) . (iii) it is possible to specifically design viruses with altered ns -genes that could be used as highly attenuated life vaccines (s. . . ), additionally modification of other viral genes (murphy et al., ; parkin et al., ) or of the replications efficiency of the genesegment (muster et al., ) can be applied to further attanuate the virus. (iv) viruses could be produced that lack an essential gene (e.g. nep) (watanabe et al., b) . the missing gene-product can be transcomplemented from an expression-plasmid in the transfected cell during virus generation. the recombinant viruses would be still infectious and lead to expression and presentation of viral proteins, but could not themselves establish a productive propagation as they are lacking the according gene. currently used ivs are prepared from egg-grown viruses (wilschut and mcelhaney, ) . this method is not without limitations but has proven to be efficient. as mentioned earlyer ( . . ), one particular problem that could arise would be production of a vaccine strain against an highly pathogenic avain influenza virus like the types that have recently infected humans. besides bio-safety questions they pose further problems. the ha of these viruses is activated within the infected cell by ubiquitous proteases allowing the virus to spread through out the organism. due to the special hacharacteristics these viruses themselves are highly pathogenic birds and eggs as well as a vaccine strain that would carry the according ha. therefore efficient virus production in embryonated eggs will be problematic (lipatov et al., ) . by plasmid based reverse genetic techniques recombinant viruses can be produced that have lost the pathogenic character of the ha and can replicate well in eggs li et al., ; liu et al., ; subbarao et al., ) . this could additionally be combined with virus production in cell culture systems (s. p. ) romanova et al., ) thereby overcoming the limitation posed by the number of embryonated eggs available at a given time (stephenson et al., ) . it should also be mentioned that not only type-a influenza viruses but also type-b influenza viruses can be generated and manipulated by reverse genetic systems and can therefore also be engineered to fit the circulating wild type strains (dauber et al., ; hatta et al., ; jackson et al., ; maassab and bryant, ) . inhibitors of viral functions (treatment and anti-viral chemoprophylaxis of influenza) anti-viral treatment is generally considered a supporting measure to prevent and control outbreaks of epidemic influenza in addition to immunoprophylaxis. however, chemotherapy is the only option to combat the disease when there is no type-specific vaccine available as for instance upon the emergence of a pandemic shift variant. two classes of substances are currently licensed in many countries for the treatment and/or prophylaxis of influenza, which includes the adamantane compounds amantadine and rimantadine, and the na-inhibitors oseltamivir and zanamivir. other small inhibitory compounds that target the viral polymerase complex are also introduced in this section, although none of them has been converted into a pharmaceutical product so far. amantadine ( -amino adamantane hydrochloride) and its derivative rimantadine (α-methyl- -adamantane methylamine hydrochloride) have potent anti-viral activity against most influenza-a-viruses, because they block the viral m ion channel protein during the early stage of viral uncoating (pinto et al., ) . specifically, the adamantane compounds inhibit the acidification of the virion inside the endosome, which prevents the intra-cellular release of the viral rnps. the % inhibitory concentration (ic ) of most natural influenza-a-virus strains against adamantane compounds is in the range of . to . µg/ml as determined by plaque reduction assay (appleyard et al., ; hayden et al., ; scholtissek and faulkner, ) . amantadine and rimantadine have proven effectiveness in the treatment of uncomplicated influenza-a-virus infection. they can reduce the duration of fever and system symptoms by approximately one day when given within two days after onset of disease signs (demicheli et al., ; tominack and hayden, ) . furthermore, both substances also have prophylactic effectiveness in reducing influenza-associated morbidity and clinical symptoms. a survey of studies undertaken with healthy adults demonstrated average effectiveness of % for amantadine and % for rimantadine in preventing laboratory confirmed influenza (demicheli et al., ) . during long-term prophylaxis amantadine was found to cause mild reversible adverse effects in a small proportion of the recipients, which involved central nervous system (cns) and minor gastrointestinal complaints. no increase in side effects was observed during treatment with rimantadine compared to placebo (n.n., ) . an early recognized limitation for widespread clinical use of m -blockers is the rapid emergence of drug-resistant viruses in tissue culture, in animal models and in patients (appleyard et al., ; hayden et al., ; oxford et al., ) . one study found that a total of % of children with laboratory-confirmed influenza shed resistant viruses after seven days of treatment with rimantadine (hall et al., ) . unfortunately, such selected drug-resistant viruses are virulent, as they can transmit to family members and cause disease even when the contact persons were treated prophylactically with rimantadine (hayden et al., ) . viruses that become insensitive to amantadine show complete cross-resistance to rimantadine and vice versa. thus, the clinical usage of adamantane amine compounds has been limited by the reported adverse effects, the induction of viral drug resistance and the inactivity towards influenza-b-viruses. nevertheless, these drugs are still recommended as a cost-effective choice particularly in influenza chemoprophylaxis (harper et al., ) . it is noteworthy, that amantadine resistance has also been detected in the highlypathogenic h n viruses currently circulating in south east asia (puthavathana et al., ) . thus, adamantane compounds are not an option to treat such infections. amantadine and rimantadine are approved for treatment of adults and children older than years at two daily mg doses. the substances should carefully be used in individuals above years in age and patients with impaired renal functions and halving of the daily doses is recommended. only amantadine is licensed for treatment of children between and years and should be dosed with mg/kg per day. in order to avoid emergence and transmission of drug-resistent viruses, treatment should be kept to a minimal time of to days until disease symptoms disappear. chemoprophylaxis can be considered for protection among high-risk groups including children and adults with chronic pulmonary or cardiac disease, immunocompromised persons with a reduced response to vaccines or in the case of a poor match between an epidemic virus strain and the current vaccine. since the adamantane compounds do not interfere with the development of neutralizing antibodies (tominack and hayden, ) , they can also be used for the protection of persons at high risk to bridge the time gap between vaccination and the establishment of an efficient immune status. for adults and children older than years two mg doses of amantadine or rimantadine per day are recommended. children between and years should receive a maximum of mg per day in two divided doses. two anti-viral drugs that inhibit both influenza-a and b-viruses, zanamivir (relenza™, glaxosmithcline) and oseltamivir (tamiflu™, roche pharmaceuticals) have recently been approved for general use in the usa, australia, europe and japan. the current knowledge suggests that nainhibitors (ni) will have a better clinical utility than the m -blockers, because these substances are broadly effective against type-a and -b influenza viruses including highly virulent avian virus strains. further, they appear to have a very low frequency of adverse effects and are less prone to induce drug resistance. zanamivir and oseltamivir function as slow binding, substrate competitive inhibitors that strongly reduce viral na-activity by interacting with five sub-sites close to the enzymatic pocket of the na. the ic values of these inhibitors were found to be in the range of . - . nm depending on the virus types and subtypes (mckimm-breschkin et al., ) . targeting of the viral na does not require the delivery of an inhibitor into the cell interior, because the enzyme is a surface glycoprotein. influenza viruses attach to the host cell through binding of the viral ha to sialic acid moieties that are conjugated to cellular glycoproteins. by the time of progeny virus budding these receptor determinants need to be removed to allow efficient release from the host cell. this is accomplished by the viral na (acylneuraminyl hydrolase, ec . . . ) that hydrolyzes glycosidic linkages adjacent to n-acetyl-neuraminic acid (neu ac, sialic acid). thus, blockade of na-activity by antibodies, temperature-sensitive mutation or inhibitory substances results in the aggregation of budding virions at the cell membrane and, hence, reduction of virus release (compans et al., ; palese and compans, ; palese et al., ) . in infected animals or humans, na probably also enhances penetration of the virion through the viscous mucus on respiratory epithelia, which contains sialic acids (matrosovich et al., ) . thus, inhibition of viral na-activity was the rationale behind several efforts to identify substances that would reduce influenza virus spread and replication. the development of the current nis was based on early characterizations of the sialic acid transition state analogue -deoxy- , dehydro nacetylneuraminc acid (neu ac en) (meindl et al., ) and the determination of the three-dimensional structure of the na by x-ray crystallography varghese et al., ; varghese et al., ) . neu ac en had been shown to inhibit viral na-acitvity but was not protective in a mouse model of influenza (palese and schulman, ) . based upon computer-assisted drug design, von itzstein et al. demonstrated that the introduction of positively charged amino-or guanidino moieties at position of neu ac en increased na inhibition by two to four orders of magnitude (von itzstein et al., ) . importantly, the inhibition of naactivity by guanidino-neu ac en that is now also termed zanamivir translated into efficient reduction of viral replication of type-a and binfluenza viruses in the nanomolar range in vitro and dose-dependent decrease of viral titers in infected animals (von itzstein et al., ; woods et al., ) . zanamivir has low oral bioavailability, but shows high antiviral activity in humans or animals when administered topically by inhalation of dry-powder aerosol (cass et al., ) . the second currently approved na inhibitor compound oseltamivir ( r, r, s- acetamido- -amino- -( -ethylpropoxyl)- -cyclohexene- carboxylic acid¸ also termed gs /ro - ) has similarly potent activities against type a and b influenza viruses . oseltamivir emerged from an independent na structure-based study and is based on a cyclohexen ring structure in which the polar glycerol side chain of the sialic acid analogues is replaced by a lipophilic -pentyloxy moiety (kim et al., ) . importantly, oseltamivir has high oral anti-viral activity when administered as its methylester pro-drug, gs /oseltamivir phosphate, that is converted to the active drug by hepatic enzymes (hayden et al., b; li et al., ; mendel et al., ) . zanamivir and oseltamivir have potent anti-viral effectiveness against community-acquired influenza and are in general safe to use in healthy adults (abramson, ; boivin et al., ; hayden et al., ; makela et al., ; monto et al., ; n.n., ) . in clinical trials the nis significantly shortened disease duration and reduced symptoms and viral loads when treatment was initiated within hours post infection (hayden et al., ; hayden et al., b) . even, when inhalation of zanamivir was begun within hours after onset of symptoms the time to alleviation of major disease signs (cough, myalgias, fever, headache) was shortened by one to two days and patients were able to resume normal activities earlier (hayden et al., ; monto et al., ) . initiation of therapy later than hours after disease onset still reduced viral loads but was less beneficial for symptom recovery. two mg daily doses of oseltamivir for five days were shown to reduce shedding of virus and the severity and duration of influenza symptoms by one to two days when therapy was begun within hours after onset of disease signs (nicholson et al., ; treanor et al., ) . some side effects that included diarrhea, nausea and nasal symptoms were observed during clinical testings of zanamivir but were similar in placebo groups (glaxowellcome, ) . the ni substances are also highly effective to prevent spread of the disease. a post-exposure protection study with zanamivir demonstrated % efficacy in preventing transmission of influenza to family members, when the index case was treated with zanamivir . oseltamivir had a comparably high efficacy in preventing laboratory-confirmed influenza by % and influenza with fever by % (hayden et al., a) . within households, one mg dose oseltamivir per day was % protective against clinical influenza even when the index cases were not treated (welliver et al., ) . thus, to prevent the spread of the flu within household contacts the nis appear to be preferable compared to the m -blockers that can induce the emergence and transmission of virulent drug-resistant viruses. during the development of nis for clinical use it was recognized that viruses with a reduced drug sensitivity could be selected in tissue culture (summarized in (mckimm-breschkin, ; tisdale, ) ). resistance can be characterized by various methods including ic -determination of the viral na, by plaque reduction assays (number and size) and yield reduction assay in tissue culture (matrosovich et al., ; tisdale, ) . under laboratory conditions several passages are usually required to select such variants, which is different to amantadine-resistant viruses that can emerge in a single cycle experiment. drug-resistant viruses were also isolated from diseased persons treated with nis (gubareva et al., ; gubareva et al., ; kiso et al., ; zambon and hayden, ) . however, the available data on the pathogenicity of these mutant viruses in animal models suggest that they have reduced replication capability in vivo and may therefore be clinically less relevant in humans. resistance to nis was found to be complex, because it can be associated with mutations in the na, the ha or synergistically in both genes. namutations that confer reduced drug sensitivity were identified at amino acid residues , , , and (based on n -na numbering) (gubareva, ; zambon and hayden, ) . these amino acids are part of or cluster around the conserved catalytic pocket and their mutation can decrease the enzymatic activity to below % and some also destabilize the enzyme (varghese et al., ) . the various ni-molecules slightly differ in their interactions with the enzyme. thus, a given na-mutant enzyme may show a range of sensitivity against different inhibitors (gubareva et al., ) . interestingly, some viruses with a reduced sensitivity to nis were found to carry mutations in the ha, which affected the receptor binding site in the globular head region, the stalk region and the ha -subunit (mckimm-breschkin, ) . apparently, the ha-mutations reduce drug sensitivity by decreasing the affinity for cellular sialic acid receptor molecules and thereby easing the release of budding viruses from the plasma membrane. these findings corroborate the concept that efficient viral replication requires a carefully balanced interplay between the strength of ha/receptor binding and the activity of the na that removes these receptor determinants . the use of zanamivir (relenza™) and oseltamivir (tamiflu™) is recommended for the treatment of uncomplicated influenza caused by type-a and b-viruses (harper et al., ) . therapy with either drug should be initiated within hours after the onset of disease signs and should be continued for five days (glaxowellcome, ; roche, ) . it is important to consider that bacterial superinfections may occur that would not be affected by these anti-virals. neither substance has been shown to prevent serious complications of influenza like pneumonia. zanamivir is approved for treatment of influenza in persons aged years and older. the recommended dosage is two inhalations of mg doses twice a day using the inhalation device provided by the manufacturer. zanamivir is not recommended for persons with underlying respiratory conditions like asthma or chronic obstructive pulmonary disease, because of the risk of precipitating bronchospasm in such patients (glaxowellcome, ) . oseltamivir can be used for treatment of patients of year or older. depending on the age, the recommended doses for children above years and adults are two mg capsules a day. two daily doses of - mg is recommended for children under kg, x mg for children between - kg and x mg for persons weighing > - kg. currently, tamiflu™ but not relenza™ is licensed for chemoprophylaxis in children older than years and in adults. for persons with creatinine clearance of - ml/min, halving of the usual dosage for therapy or prophylaxis is recommended. two approaches are possible, a seasonal prophylaxis that provides a % reduction of confirmed influenza infection in a vaccinated population of frail elderly persons (mcclellan and perry, ) , and a short-term prophylaxis for controlling institutional outbreaks by breaking the virus circulation. several further compounds that inhibit the influenza virus na were identified in independent efforts and have been evaluated as anti-influenza agents. thus, the cyclopentane derivatives bcx- (rwj- ), bcx- , bcx- and bcx- as well as the pyrrolidine-based a (from abbott laboratories) showed strong potent anti-viral activies at least in vitro (kati et al., ; smee et al., ) . thus, although development of bcx- has been halted after showing a lack of activity in a phase iii clinical trial (chand et al., ) , additional nis may emerge as anti-influenza drugs in the future. two unique properties of the trimeric rna-dependent rna-polymerase of influenza viruses, which are not shared by cellular enzymes, provide attractive opportunities for anti-viral interference with possibly little disturbances of the host cell. first, the polymerase exhibits an endonuclease activity that cleaves the first - nucleotides including the '-cap structure from nascent host rna-polymerase ii cap transcripts and use them to prime viral mrnas (lamb and krug, ) . second, the viral polymerase replicates the negative-sense viral rna-segments via unprimed synthesis of a complementary positive-strand rna-intermediate. for both of these activities, inhibitory small molecule compounds have been identified, some of which were also shown to reduce viral propagation in tissue culture and/or in infected mice. however, further clinical development has not been reported for any of those substances so far. the viral endonuclease activity is associated with the pb -subunit and depends on binding of the polymerase to the terminal ends of the vrnatemplate and the cap structures of nascent mrna-transcripts (li et al., ) . the endonuclease most likely utilizes a two metal ion mechanism for cleavage of the cellular nucleic acid (klumpp, a) . it has been shown that derivatives of the fungal metabolite flutimide as well as a class of substituted , -dioxobutanoic acids specifically inhibited the cap-dependent endonuclease, presumably by interaction with the active catalytic site of the enzyme (hastings et al., ; parkes et al., ; tomassini et al., ; tomassini, ) . the most potent compounds of these two classes had ic values in the range of . - µm when tested in virus yield assays in tissue culture experiments. further, intranasal instillation of the l- , compound was reported to inhibit viral titers in nasal washes of mice infected with influenza virus a/pr/ / virus, but the effects on disease progression were not studied (hastings et al., ) . another screening effort has identified t- ( -fluoro- -hydroxy- pyrazinecarboxamide) to have potent and selective anti-influenza activity. t- showed ic values of less than . µg/ml in virus yield assays in mdck cells against all three influenza virus types (a, b, c) with no signs of cytotoxicity (furuta et al., ) . importantly, t- was also orally active in a mouse model and shown to significantly reduce viral lung titers and enhance survival rates from % to % after infection with influenza virus a/pr/ / virus at a dose of mg/kg per day (furuta et al., ; takahashi et al., ) . although the basis for its anti-viral activity was unclear at that time, t- was found to inhibit replication of an oseltamivirresistant mutant virus in vitro suggesting that this inhibitor targets a different viral function (takahashi et al., ) . indeed, recent analyses showed that the compound is metabolized inside the cell into t- -ribofuranosyl- 'triphosphate (t- -rtp), which is a potent and selective inhibitor of apgprimed viral rna-polymerase activity (furuta et al., ) . these findings show that t- may have the potential to become a novel oral antiinfluenza drug that targets a viral function not blocked by one of the currently licensed nis or m -blockers. influenza viruses only have a limited coding capacity. thus, these viruses employ functions of their host-cell for efficient replication. these dependencies create opportunities to design novel anti-viral strategies by targeting specific host cell functions. cell fate decisions in response to extra-cellular agents, including pathogenic invaders are commonly mediated by intra-cellular signaling cascades that transduce signals into stimulus specific actions, e.g. changes in gene expression patterns, alterations in the metabolic state of the cell or induction of programmed cell death (apoptosis). thus, these signaling molecules are at the bottleneck of the control of cellular responses. in this section we will review the recent advances in the analysis of influenza virus induced signaling pathways and first attempts to use signaling mediators as targets for anti-viral approaches. mitogen activated protein kinase (mapk)-cascades have gained much attention as being critical transducers to convert a variety of extra-cellular signals into a multitude of responses (english et al., ; hazzalin and mahadevan, ; widmann et al., ) thereby, these pathways regulate numerous cellular decision processes, such as proliferation and differentiation, but also cell activation and immune responses (dong et al., ) . four different members of the mapk-family that are organized in separate cascades have been identified so far: erk (extra-cellular signal regulated kinase), jnk (jun-n-terminal kinase), p and erk /bmk- (big map kinase) (garrington and johnson, ; widmann et al., ) . these mapks are activated by a dual phosphorylation event on threonine and tyrosine mediated by mapk-kinases (mapkk also termed meks or mkks). the mapk "erk" is activated by the dual-specific mapkk mek and - that are controlled by the upstream serine threonine mapkkkinase raf. raf, mek and erk form the prototype module of a mapk-pathway and are also known as the classical mitogenic cascade. the mapk p and jnk are activated by mkk / and mkk / , respectively, and are predominantly activated by pro-inflammatory cytokines and certain environmental stress conditions. the mek /erk module is both activated by mitogens and certain stress inducers. there is evidence that all these different mapk-cascades are activated upon infection with rna-viruses, including influenza viruses. thus, these signaling cascades may serve different functions in viral replication and host cell response. another important signaling pathway, which is commonly activated upon virus infection is the iκb-kinase (ikk)/nfκb-signaling module (hiscott et al., ) . the nfκb/iκb family of transcription factors promote the expression of well over different genes, such as cytokine or chemokine genes, or genes encoding for adhesion molecules or anti-and pro-apoptotic protein (pahl, ) . the canonical mechanism of nfκb activation includes activation of iκb-kinase (ikk) that phosphorylates the inhibitor of nfκb (iκb) and targets the protein for subsequent degradation (delhase and karin, ; karin, b) . this leads to the release and migration of the transcriptionally active nfκb factors to the nucleus (ghosh, ; karin and ben-neriah, ) . the ikk-complex consists of at least three isozymes of ikk: (i) ikk /ikkα, (ii) ikk /ikkβ and (iii) nemo/ikkγ. the most important isozyme for nfκb-activation via the degradation of iκb is ikk (karin, a) . nemo acts as a scaffolding protein for the large ikk complex (courtois et al., ) that contains still other kinases such as mekk (mapkk-kinase ) (lee et al., ) , nik (nfκb inducing kinase) (nemoto et al., ; woronicz et al., ) and the dsrnaactivated protein-kinase (pkr) (gil et al., ; zamanian-daryoush et al., ) . both nfκb and the jnk mapk-pathway regulate one of the most important anti-viral gene expression events, the transcriptional induction of interferon beta (ifnβ) . ifnβ is one of the first antiviral cytokines to be expressed upon virus infection, initiating an autoamplification loop to cause an efficient and strong type-i ifn response. the ifnβ enhanceosome, which mediates the inducible expression of ifnβ, carries binding sites for transcription factors of three families, namely the ap- family members and jnk targets c-jun and atf- , the nfκb factors p and p , and the interferon-regulatory factors (irfs) (hiscott et al., ; thanos and maniatis, ) . in the initial phase of a virus infection this promoter element specifically binds the constitutively expressed and specifically activated irf -dimer (taniguchi and takaoka, ) . ap- and nfκb-transcription factors are activated by a variety of stimuli. however, a strong irf -activation is selectively induced upon infection with several rna-viruses, in particular by the dsrna, which accumulates during replication yoneyama et al., ) . thus, irf is the major determinant of a strong virus-and dsrna-induced ifnβ-response. interestingly all four so far defined mapk-family members are activated upon an influenza virus infection (kujime et al., ; ludwig et al., ; has helped to get a clearer picture of the importance of the erk-signaling pathway for influenza virus replication. the activation of the map-kinase erk upon productive influenza virus infection (kujime et al., ) appears to serve a mechanism that is beneficial for the virus . strikingly, blockade of the pathway by specific inhibitors of the upstream kinase mek and dominantnegative mutants of erk or the mek-activator raf resulted in a strongly impaired growth of both, influenza a-and b-type viruses . conversely, virus titers are enhanced in cells expressing active mutants of raf or mek olschlager et al., ) . this has not only been demonstrated in cell culture but also in vivo in infected mice expressing a constitutively active form of the raf-kinase in the alveolar epithelial cells of the lung (olschlager et al., ) . while in the wt-situation influenza viruses primarily infect bronchiolar epithelial cells, there is efficient replication in the alveolar layer most exclusively in the cells carrying the transgene. as a consequence this results in an earlier death of the transgenic animals (olschlager et al., ) . this indicates that activation of the raf/mek/erk pathway is required for efficient virus growth. noticeably, inhibition of the pathway did not significantly affect viral rna-or protein-synthesis . the pathway rather appears to control the active nuclear export of the viral rnp-complexes that are readily retained in the nucleus upon blockade of the signaling pathway. most likely this is due to an impaired activity of the viral nuclear export protein nep . this indicates that active rnp-export is an induced rather than a constitutive event, a hypothesis supported by a late activation of erk in the viral life cycle. so far the detailed mechanism of how erk regulates export of the rnps is unsolved. there are two likely scenarios: either it does occur directly via phosphorylation of a viral protein involved in rnp-transport or by control of a cellular export factor. although in the initial studies no alteration of the overall phosphorlyation status of the np, m and nep proteins was observed there are now first indications that certain phosphorylation sites of the np indeed are affected by mek-inhibition (s.p., unpublished data). it remains to be shown, whether this is of functional relevance for the rnp-export process. it is striking that mek-inhibitors are not toxic for the cell, while more general blockers of the active transport machinery, such as leptomycin-b exert a high toxicity even in quite low concentrations. this may indicate that mekinhibitors are no general export blockers but only block a distinct nuclear export pathway. indeed there are first evidences that the classical mitogenic cascade specifically regulates nuclear export of certain cellular rna-protein complexes. in lps-treated mouse macrophages mek-inhibition results in a specific retention of the tnf-mrna in the nucleus (dumitru et al., ) . this is also observed in cells deficient for tpl- , an activator of mek and erk. in these cells the failure to activate mek and erk by lps again correlated with tnf mrna retention while other cytokines are normally expressed (dumitru et al., ) . thus the erk-pathway may regulate a specific cellular export process but leaves other export mechanisms unaffected. it is likely that such a specific export pathway is employed by influenza-a and b-viruses. the finding of an anti-viral action of mek-inhibitors prompted further research showing that replication of other viruses, such as borna disease virus , visna virus (barber et al., ) or coxsackie b virus (luo et al., ) is also impaired upon mek-inhibition. requirement of raf/mek/erk-activation for efficient influenza virus replication may suggest that this pathway may be a cellular target for antiviral approaches. besides the anti-viral action against both, a-and b-type viruses , mek-inhibitors meet two further criteria which are a prerequisite for a potential clinical use. although targeting an important signaling pathway in the cell the inhibitors showed a surprisingly little toxicity (a) in cell culture planz et al., ; pleschka et al., ) (b) in an in vivo mouse model (sebolt-leopold et al., ) and (c) in clinical trials for the use as anti-cancer agent (cohen, ) . in the light of these findings it was hypothesized that the mitogenic pathway may only be of major importance during early development of an organism and may be dispensable in adult tissues (cohen, ) . another very important feature of mek-inhibitors is that they showed no tendency to induce formation of resistant virus variants . although targeting of a cellular factor may still raise the concern about side effects of a drug, it appears likely that local administration of an agent such as a mek-inhibitor to the primary site of influenza virus infection, the lung, is well tolerated. here the drug primarily affects differentiated lung epithelial cells for which a proliferative signaling cascade like the raf/mek/erk-cascade may be dispensable. following this approach it was recently demonstrated that the mek inhibitor u is effective in reducing virus titers in the lung of infected mice after local administration (o.p., s.p. and s.l., unpublished). activation of the classical mitogenic raf/mek/erk-cascade is initiated by yet other phosphorylation events. the kinase raf is known to be regulated by phosphorylation of different upstream kinases including members of the protein kinase c (pkc)-family (cai et al., ; kolch et al., ) . the pkc-superfamily consists of at least different pkc-isoforms that carry out diverse regulatory roles in cellular processes by linking into several downstream signaling pathways (toker, ) . beside a regulation of the raf/mek/erk-cascade and other downstream pathways, pkcs may have additional functions during viral replication. a role of pkcs in the process of entry of several enveloped viruses has been proposed based on the action of protein kinase inhibitors h and staurosporine (constantinescu et al., ) as well as by the calcium-channel blocker verapamil (nugent and shanley, ) . influenza virus infection or treatment of cells with purified viral ha results in rapid activation of pkcs upon binding to host-cell surface receptors (arora and gasse, ; kunzelmann et al., ; rott et al., ) . in a recent study it was shown that the pan pkc-inhibitor bisindolylmaleimide-i prevented influenza virus entry and subsequent infection in a dose dependent and reversible manner (root et al., ) . using a dominant-negative mutant approach this function was assigned to the pkcßii-isoform. overexpression of a phosphorylation-deficient mutant of pkcßii revealed that the kinase is a regulator of late endosomal sorting. accordingly, expression of the pkcßii-mutant resulted in a block of virus entry at the level of late endosomes (sieczkarski et al., ; sieczkarski and whittaker, ) . thus, a specific inhibition of pkcßii may be a suitable approach to blunt virus replication at a very early time point in the replication cycle. activation of the transcription factor nfκb is a hallmark of most infections by viral pathogens (hiscott et al., (hiscott et al., ) including influenza et al., . influenza viral nfκb-induction involves activation of iκbkinase (ikk) and is also achieved with isolated influenza virus components. this includes dsrna (chu et al., ) or over-expression of the viral ha, np or m proteins (flory et al., ) . since gene expression of many pro-inflammatory or anti-viral cytokines, such as ifnβ or tnfα, is controlled by nfκb the concept emerged that ikk and nfκb are essential components in the innate immune response to virus infections (chu et al., ) . accordingly, influenza virus-induced ifnβpromoter activity is impaired in cells expressing transdominant negative mutants of ikk or iκbα (wang et al., ; wurzer et al., ) . nevertheless, ikk and nfκb might not only have anti-viral functions as two recent studies demonstrate that influenza viruses replicate much better in cells where nfκb is pre-activated (nimmerjahn et al., ; wurzer et al., ) . conversely, influenza virus titers from different host cells in which nfκb-signaling was impaired by means of specific inhibitors or dominantnegative mutants, a dramatic reduction could be observed (nimmerjahn et al., ; wurzer et al., ) . thus, in the context of an influenza virus infection a function of nfκb to support virus replication appears to be dominant over the function as a transcription factor in the anti-viral response. on a molecular basis this was shown to be due to the nfκbdependent expression of pro-apoptotic factors, such as tnf-related apoptosis inducing ligand (trail) or fasl . inhibition of virus induced expression of these factors results in strongly impaired viral growth. this links the pro-viral action of nfκb to the induction of apoptosis, a process that will be discussed in the next section. finally, viral need for nfκb-activity suggests that this pathway may be suitable as a target for anti-viral intervention. to this end we have shown recently that several pharmacological inhibitors of nfκb act anti-viral in vivo, without toxic side effects or the tendency to induce resistant virus variants (i. mazur, w. wurzer, c. erhardt, t. silberzahn, t.w., o.p., s.p. and s.l, unpublished). another cellular signaling response commonly observed upon virus infections, including influenza virus is the induction of the apoptotic cascade. apoptosis is a morphological and biochemical defined form of cell death (kerr et al., ) and has been demonstrated to play a role in a variety of diseases, including virus infections (razvi and welsh, ) . apoptosis is mainly regarded to be a host cell defense against virus viruses (reviewed in: julkunen et al., ; ludwig et al., ; infections since many viruses express anti-apoptotic proteins to prevent this cellular response. the central component of the apoptotic machinery is a proteolytic system consisting of a family of cysteinyl proteases, termed two groups of caspases can be distinguished: upstream initiator caspases such as caspase- or caspase- , which cleave and activate other caspases and downstream effector caspases, including caspase- , - and - , that cleave a variety of cellular substrates, thereby disassembling cellular structures or inactivating enzymes (thornberry and lazebnik, ) . caspase- is the most intensively studied effector caspase. work on caspase- deficient mcf- breast carcinoma cells has revealed a caspase- driven feedback loop, that is crucial to mediate the apoptotic process (janicke et al., ; slee et al., ) . thus, caspase- is a central player in apoptosis regulation and the level of pro-caspase- in the cell determines the impact of a given apoptotic stimulus. although it is now well established that influenza virus infection induces caspses and subsequent apoptosis, the consequence for virus replication or host cell defense is still under a heavy debate (reviewed in (lowy, ; ludwig et al., ; schultz-cherry et al., ) . with the identification of pb -f , a new influenza virus protein expressed from a + reading frame of the pb polymerase gene segment, a pro-apoptotic influenza virus protein has been discovered (chen et al., it is long known that influenza virus infection with a-and b-type viruses results in the induction of apoptosis both in permissive and un-permissive cultured cells as well as in vivo (fesq et al., ; hinshaw et al., ; ito et al., ; mori et al., ; takizawa et al., ) . interestingly, viral activation of mapks or upstream kinases has been linked to the onset of apoptosis. in a mouse model for a neurovirulent influenza infection, jnkactivity correlated with apoptosis induction in the infected brain (mori et al., ) . in embryonic fibroblasts deficient for the mapkk-kinase ask- the virus-induced jnk-activation was blunted concomitant with an inhibition of caspase- activation and virus-induced apoptosis (maruoka et al., ) . as an extrinsic mechanism of viral apoptosis induction it has been noted quite early on that the fas receptor/fasl-apoptosis inducing system (fujimoto et al., ; takizawa et al., ; takizawa et al., ; wada et al., ) is expressed in a pkr-dependent manner in infected cells (takizawa et al., ) . this most likely contributes to virus-induced cell death via a receptor mediated fadd/caspase- -dependent pathway (balachandran et al., ) . another mode of viral apoptosis induction might occur via activation of tgf-β that is converted from its latent form by the viral na (schultz-cherry and . within the influenza virus infected cell the apoptotic program is mediated by activation of caspases (lin et al., ; takizawa et al., ; zhirnov et al., ) with a most crucial role of caspase- (wurzer et al., ) . although it is now well established that influenza virus infection induces although it is now well established that influenza virus infection induces caspases (for review see: cohen, ; thornberry and lazebnik, ) . ). pb -f induces apoptosis via the mitochondrial pathway if added to cells and infection with recombinant viruses lacking the protein results in reduced apoptotic rates of lymphocytes (chen et al., ) . however, most of the avian virus strains are lacking the pb -f reading frame and pb -f deficient viruses do not affect apoptosis in a variety of other host cells (chen et al., ) . these results have let to the assumption that apoptosis induction by pb -f may be required for the specific depletion of lymphocytes during an influenza virus infection, a process which is observed in infected animals (tumpey et al., ; van campen et al., a; van campen et al., b) . a recent study adds a new aspect to the open discussion by the surprising observation that influenza virus propagation was strongly impaired in the presence of caspase inhibitors (wurzer et al., ) . this dependency on caspase activity was most obvious in cells where caspase- was partially knocked-down by sirna (wurzer et al., ) . consistent with these findings, poor replication efficiencies of influenza-a-viruses in cells deficient for caspase- could be boosted -fold by ectopic expression of the protein. mechanistically, the block in virus propagation appeared to be due to the retention of viral rnp-complexes in the nucleus preventing formation of progeny virus particles (wurzer et al., ) . interestingly the findings are consistent with a much earlier report showing that upon infection of cells over expressing the anti-apoptotic protein bcl- the viral rnp-complexes were retained in the nucleus (hinshaw et al., ) resulting in repressed virus titers (olsen et al., ) . furthermore the recently identified proapoptotic pb -f (chen et al., ) is only expressed in later phases of replication consistent with a later step in the virus life cycle that requires caspase activity (wurzer et al., ) . the observation of a caspase requirement for rnp-nuclear export was quite puzzling since this export process was shown before to be mediated by the active cellular export machinery involving the viral nuclear export protein (ns /nep) (neumann et al., ; o'neill et al., ) and the anti-apoptotic raf/mek/erkcascade . caspase activation does not support, but rather inhibit the active nuclear export machinery by cleavage of transport proteins. this suggests an alternate strategy by which caspases may regulate rnp-export, e.g. by directly or indirectly increase the diffusion limit of nuclear pores (faleiro and lazebnik, ) to allow passive diffusion of larger proteins. such a scenario is supported by the finding that isolated nps or rnp-complexes, which are nuclear if ectopically expressed, can partially translocate to the cytoplasm upon stimulation with an apoptosis inducer in a caspase- -dependent manner (wurzer et al., ) . these findings can be merged into a model in which the rnps are transported via an active export mechanism in intermediate steps of the virus life cycle. once caspase activity increases in the cells, proteins of the transport machinery get destroyed, however, widening of nuclear pores may allow the viral rnps to use a second mode of exit from the nucleus (faleiro and lazebnik, ) . that would be a likely mechanism to further enhance rnp-migration to the cytoplasm in late phase of the viral life cycle and thereby support virus replication. such a complementary use of both "active" (raf/mek/erkdependent) and "passive" (caspase-dependent) transport mechanisms is supported by the observation, that at concentrations of mek-and caspaseinhibitors, which only poorly block influenza virus replication alone, efficiently impaired virus propagation if used in combination (wurzer et al., ) . thus, while both pathways do not interfere with each other (wurzer et al., ) they appear to synergize to mediate rnp-export via different routes. therefore one may conclude that influenza virus has acquired the capability to take advantage of supposedly anti-viral host cell responses to support viral propagation. this includes early induction of caspase activity but not necessarily execution of the full apoptotic process that most likely is an anti-viral response. this dual role of "early" versus "late" apoptotic events during virus replication may exclude the use of caspase-inhibitors as anti-flu agents, although in cell culture these inhibitors may have a beneficial outcome for the host cell. besides mediators of signaling and apoptosis a variety of other cellular enzymes are required for efficient virus growth. there are also some initial attempts to use these components as target for an anti-viral intervention. another important requirement for a cellular enzyme is the proteolytic cleavage of the ha by proteases. the infectivity and pathogenicity of influenza virus is based on the proteolytic cleavage of the precursor ha into ha and ha chains by an arginine-specific, trypsin-like host protease. the viral glycoproteins are glycosylated in the endoplasmic reticulum (er) and the er-α-glycosidase-i is responsible for the removal of terminal α- , glucose residues from precursor oligosaccharides in the er. a variety of viruses such as hiv, hsv and dengue-virus have been shown to be highly sensitive to inhibitors of these enzymes (mehta et al., ; mehta et al., ) . one of these inhibitors, castanopermine, has been demonstrated to inhibit replication of influenza virus a/hongkong/ / in mdck cells with an ic value of < µm (klumpp, b) . the inhibitor also acted antiviral in vivo in a mouse model and reduced lung titers of a/pr / infected mice by tenfold when administered intranasal. the compound has reached phase ii clinical trials for the treatment of hiv and has been licensed for a potential treatment of hepatitis-c-virus infections (reviewed in (klumpp, b) ). several exogenous protease inhibitors were investigated with respect to their anti-influenza activity: camostat, a serine protease inhibitor; was shown to exhibit strong anti-influenza effects in vitro and in vivo in mice and in chicken embryos. the compound also showed strong anti-influenza effects in amantadine-resistant type-a and -b virus infection in vitro (lee et al., ) . other protease inhibitors nafamostat mesilate, camostat mesilate, gabexate mesilate and aprotinin also inhibited virus replication in vitro protease inhibitors gordox, contrycal and epsilonaminocapronic acid were tested in both animal and clinical experiments. inhalation of aminocapronic acid-containing aerosols exerted the most effective therapeutic effect, reducing the duration of viral antigen in the nasopharyngeal epithelium / to fold (zhirnov et al., ) . recombinant human mucus protease inhibitor (mpi) was investigated for its anti-viral activity in rat lungs in vitro. the c-, but not the n-terminal domain of mpi was shown to inhibit the proteolytic activity of tryptase clara and of virus activation at nm concentrations (beppu et al., ; kido et al., ) . however, the current understanding is that protease inhibitors -mainly used in hiv therapy -may produce serious toxic side effects. recent investigations showed that protease inhibitors can cause diabetes, hepatic and renal failures and mutagenic (potentially carcinogenic) effects. a further disadvantage of protease inhibitors is the rapid development of viral resistance, and a variable strain sensitivity to these anti-viral agents still under evaluation as potential anti-influenza therapeutics (kido et al., ; savarino, ) . regarding the continues threat caused by seasonal flu-epidemics and the immanent danger of re-occurring pandemic outbreaks that both impose a great burden on human and animal health, and considering the fact, that influenza viruses can not be eradicated, the possibilities to fight this disease have been greatly improved by novel molecular biological techniques in recent years. vaccination is still by far the best prophylactic measure, but new drugs, which attack the virus directly, will further support to combat these foes. nevertheless the viral tactic to escape direct intervention by resistance is a major drawback of current therapeutic interventions. this (discussed in luscher-mattli, ) . nevertheless protease inhibitors are zhirnov, ) (reviewed in luscher-mattli, ) . the : : problem might be overcome by innovative methods that target cellular functions essential for efficient virus replication intranasal, cold-adapted, live, attenuated influenza vaccine amantadine-resistance as a genetic marker for influenza viruses influenza virus hemagglutinin stimulates the protein kinase c activity of human polymorphonuclear leucocytes a novel liposomal influenza vaccine (influsome-vac) containing hemagglutinin-neuraminidase and il- or gm-csf induces protective anti-neuraminidase antibodies cross-reacting with a wide spectrum of influenza a viral strains alpha/beta interferons potentiate virus-induced apoptosis through activation of the fadd/caspase- death signaling pathway visna virus-induced activation of mapk is required for virus replication and correlates with virus-induced neuropathology influenza vaccines: recent advances in production technologies current status of live attenuated influenza virus vaccine in the us correlates of immune protection induced by live, attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine the efficacy of live attenuated, cold-adapted, trivalent, intranasal influenzavirus vaccine in children safety, efficacy, and effectiveness of live, attenuated, cold-adapted influenza vaccine in an indicated population aged - years immunogenicity and safety of a novel il- -supplemented liposomal influenza vaccine (influsome-vac) in nursing-home residents immunogenicity and safety of a novel liposomal influenza subunit vaccine (influsome-vac) in young adults human mucus protease inhibitor in airway fluids is a potential defensive compound against infection with influenza a and sendai viruses cold-adapted live influenza vaccine versus inactivated vaccine: systemic vaccine reactions, local and systemic antibody response, and vaccine efficacy a metaanalysis rapid antiviral effect of inhaled zanamivir in the treatment of naturally occurring influenza in otherwise healthy adults vaccine delivery to animals promises and challenges of live-attenuated intranasal influenza vaccines across the age spectrum: a review prevention and control of influenza. recommendations of the advisory committee on immunization practices (acip) cd t cell responses to influenza infection role of diacylglycerol-regulated protein kinase c isotypes in growth factor activation of the raf- protein kinase pharmacokinetics of zanamivir after intravenous, oral, inhaled or intranasal administration to healthy volunteers comparison of the anti-influenza virus activity of cyclopentane derivatives with oseltamivir and zanamivir in vivo the evolution of h n influenza viruses in ducks in southern china generation and evaluation of a high-growth reassortant h n influenza a virus as a pandemic vaccine candidate a novel influenza a virus mitochondrial protein that induces cell death jnk and ikkbeta are required for activating the innate response to viral infection human influenza a h n virus related to a highly pathogenic avian influenza virus development and persistence of local and systemic antibody-responses in adults given live attenuated or inactivated influenza-a virus-vaccine caspases: the executioners of apoptosis protein kinases--the major drug targets of the twenty-first century? structure of the catalytic and antigenic sites in influenza virus neuraminidase effect of antibody to neuraminidase on the maturation and hemagglutinating activity of an influenza a virus effects of protein kinase c inhibitors on viral entry and infectivity an overview of serum antibody responses to influenza virus antigens immunity to influenza in man intranasal vaccination with iscomatrix adjuvanted influenza vaccine nemo/ikk gamma: linking nf-kappa b to human disease influenza virus: immunity and vaccination strategies. comparison of the immune response to inactivated and live, attenuated influenza vaccines the influenza b virus nonstructural ns protein is essential for efficient viral growth and antagonizes beta interferon induction a pandemic warning? haemagglutinin-inhibiting antibody to influenza virus the i kappa b kinase: a master regulator of nf-kappa b, innate immunity, and epidermal differentiation prevention and early treatment of influenza in healthy adults map kinases in the immune response tnf-alpha induction by lps is regulated posttranscriptionally via a tpl /erkdependent pathway a randomized controlled trial of cold-adapted and inactivated vaccines for the prevention of influenza a disease new insights into the control of map kinase pathways caspases disrupt the nuclear-cytoplasmic barrier immunogenicity and protection efficacy of replication-deficient influenza a viruses with altered ns genes programmed cell death (apoptosis) in human monocytes infected by influenza a virus influenza virus-induced nf-kappab-dependent gene expression is mediated by overexpression of viral proteins and involves oxidative radicals and activation of ikappab kinase rescue of influenza a virus from recombinant dna avian influenza a virus (h n ) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome co-expression of fas and fasligand on the surface of influenza virus-infected cells vitro and in vivo activities of anti-influenza virus compound t- mechanism of action of t- against influenza virus virus-like particle vaccine conferred complete protection against a lethal influenza virus challenge negative-strand rna viruses: applications to biotechnology the role of interferon in influenza virus tissue tropism influenza a virus lacking the ns gene replicates in interferon-deficient systems organization and regulation of mitogen-activated protein kinase signaling pathways regulation of inducible gene expression by the transcription factor nf-kappab activation of nf-kappa b by the dsrnadependent protein kinase, pkr involves the i kappa b kinase complex control of influenza influenza virosomes as an efficient system for adjuvanted vaccine delivery molecular mechanisms of influenza virus resistance to neuraminidase inhibitors selection of influenza virus mutants in experimentally infected volunteers treated with oseltamivir evidence for zanamivir resistance in an immunocompromised child infected with influenza b virus children with influenza a infection: treatment with rimantadine prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) anti-influenza virus activities of -substituted , -dioxobutanoic acid inhibitors influenza b virus requires bm protein for replication the continued pandemic threat posed by avian influenza viruses in hong kong use of the selective oral neuraminidase inhibitor oseltamivir to prevent influenza emergence and apparent transmission of rimantadine-resistant influenza a virus in families plaque inhibition assay for drug susceptibility testing of influenza viruses inhaled zanamivir for the prevention of influenza in families. zanamivir family study group efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenzavirus infections. gg influenza study group safety and efficacy of the neuraminidase inhibitor gg in experimental human influenza use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza: randomized controlled trials for prevention and treatment mapk-regulated transcription: a continuously variable gene switch? apoptosis: a mechanism of cell killing by influenza a and b viruses hostile takeovers: viral appropriation of the nf-kappab pathway triggering the interferon response: the role of irf- transcription factor influenza a viruses possessing type b hemagglutinin and neuraminidase: potential as vaccine components pandemic threat posed by avian influenza a viruses forecast and control of epidemics in a globalized world virulent influenza a viruses induce apoptosis in chickens reduced incorporation of the influenza b virus bm protein in virus particles decreases infectivity safety of a trivalent live attenuated intranasal influenza vaccine, flumist, administered in addition to parenteral trivalent inactivated influenza vaccine to seniors with chronic medical conditions caspase- is required for dna fragmentation and morphological changes associated with apoptosis multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (flumist) derived from cold-adapted a/ann arbor/ / potential role of granulocyte-macrophage colonystimulating factor as vaccine adjuvant liposomal immunostimulatory dna sequence (iss-odn): an efficient parenteral and mucosal adjuvant for influenza and hepatitis b vaccines inflammatory responses in influenza a virus infection antibodyforming cell response to virus challenge in mice immunized with dna encoding the influenza virus hemagglutinin the beginning of the end: ikappab kinase (ikk) and nf-kappab activation how nf-kappab is activated: the role of the ikappab kinase (ikk) complex phosphorylation meets ubiquitination: the control of nf-[kappa]b activity in vitro characterization of a- , a highly potent inhibitor of a and b strain influenza virus neuraminidases and influenza virus replication novel generations of influenza vaccines cold-adapted live attenuated influenza vaccines developed in russia: can they contribute to meeting the needs for influenza control in other countries? apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics the human mucus protease inhibitor and its mutants are novel defensive compounds against infection with influenza a and sendai viruses secretory leukoprotease inhibitor and pulmonary surfactant serve as principal defenses against influenza a virus infection in the airway and chemical agents up-regulating their levels may have therapeutic potential influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors safety and immunogenicity of low and high doses of trivalent live cold-adapted influenza vaccine administered intranasally as drops or spray to healthy children resistant influenza a viruses in children treated with oseltamivir: descriptive study development of a mammalian cell (vero) derived candidate influenza virus vaccine recent advances in the discovery and development of anti-influenza drugs recent advances in the discovery and development of anti-influenza drugs protein kinase c alpha activates raf- by direct phosphorylation transmission of h n avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands p mitogen-activated protein kinase and c-jun-nh -terminal kinase regulate rantes production by influenza virus-infected human bronchial epithelial cells influenza virus inhibits amiloride-sensitive na+ channels in respiratory epithelia influenza: virology, epidemiology, disease, and prevention orthomyxoviridae: the viruses and their replication prevention of influenza in the general population mekk activates both ikappab kinase alpha and ikappab kinase beta evaluation of anti-influenza effects of camostat in mice infected with non-adapted human influenza viruses genesis of a highly pathogenic and potentially pandemic h n influenza virus in eastern asia the active sites of the influenza cap-dependent endonuclease are on different polymerase subunits recombinant influenza a virus vaccines for the pathogenic human a hong kong (h n ) viruses identification of gs as an orally bioavailable prodrug of the influenza virus neuraminidase inhibitor gs caspase activation in equine influenza virus induced apoptotic cell death virus-dependent phosphorylation of the irf- transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation influenza: emergence and control preparation of a standardized, efficacious agricultural h n vaccine by reverse genetics effective construction of dna vaccines against variable influenza genes by homologous recombination influenza virus induction of apoptosis by intrinsic and extrinsic mechanisms influenza virus-induced ap- -dependent gene expression requires activation of the jnk signaling pathway influenza-virus-induced signaling cascades: targets for antiviral therapy? a fatal relationship-influenza virus interactions with the host cell mek inhibition impairs influenza b virus propagation without emergence of resistant variants coxsackievirus b replication is reduced by inhibition of the extracellular signal-regulated kinase (erk) signaling pathway influenza chemotherapy: a review of the present state of art and of new drugs in development the development of live attenuated cold-adapted influenza virus vaccine for humans clinical efficacy and safety of the orally inhaled neuraminidase inhibitor zanamivir in the treatment of influenza: a randomized, double-blind, placebo-controlled european study structure and function of the interferon-beta enhanceosome ask regulates influenza virus infection-induced apoptotic cell death overexpression of the alpha- , -sialyltransferase in mdck cells increases influenza virus sensitivity to neuraminidase inhibitors neuraminidase is important for the initiation of influenza virus infection in human airway epithelium oseltamivir: a review of its use in influenza neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir resistance of influenza viruses to neuraminidase inhibitorsa review cytotoxic t-cell immunity to influenza recognition of influenza a virus nucleoprotein by human cytotoxic t lymphocytes inhibition of hepatitis b virus dna replication by imino sugars without the inhibition of the dna polymerase: therapeutic implications imino sugars that are less toxic but more potent as antivirals inhibition of neuraminidase activity by derivatives of -deoxy- , -dehydro-n-acetylneuraminic acid oral administration of a prodrug of the influenza virus neuraminidase inhibitor gs protects mice and ferrets against influenza infection safety, efficacy and effectiveness of the influenza virus vaccine, trivalent, types a and b, live, cold-adapted (caiv-t) in healthy children and healthy adults efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenza a and b virus infections differential activation of the c-jun nterminal kinase/stress-activated protein kinase and p mitogen-activated protein kinase signal transduction pathways in the mouse brain upon infection with neurovirulent influenza a virus in vivo induction of apoptosis by influenza virus principles underlying the development and use of live attenuated cold-adapted influenza a and b virus vaccines an influenza a live attenuated reassortant virus possessing three temperature-sensitive mutations in the pb polymerase gene rapidly loses temperature sensitivity following replication in hamsters an influenza a virus containing influenza b virus ' and ' noncoding regions on the neuraminidase gene is attenuated in mice current status of amantadine and rimantadine as anti-influenza-a agents: memorandum from a who meeting coordinate regulation of ikappab kinases by mitogen-activated protein kinase kinase kinase and nf-kappab-inducing kinase influenza a virus ns protein mediates vrnp nuclear export through nes-independent interaction with hcrm genetic engineering of influenza and other negativestrand rna viruses containing segmented genomes generation of influenza a viruses entirely from cloned cdnas live attenuated influenza virus vaccines: new options for the prevention of influenza benefits of influenza vaccination for low-, intermediate-, and high-risk senior citizens efficacy and safety of oseltamivir in treatment of acute influenza: a randomised controlled trial active nf-kappab signalling is a prerequisite for influenza virus infection verapamil inhibits influenza a virus replication the influenza virus nep (ns protein) mediates the nuclear export of viral ribonucleoproteins lung-specific expression of active raf kinase results in increased mortality of influenza a virus-infected mice bcl- alters influenza virus yield, spread, and hemagglutinin glycosylation induction of virus-specific immunity by iscoms aprotinin aerosol treatment of influenza and paramyxovirus bronchopneumonia of mice in vivo selection of an influenza a strain resistant to amantadine generation of high-yielding influenza a viruses in african green monkey kidney (vero) cells by reverse genetics activators and target genes of rel/nf-kappab transcription factors inhibition of influenza virus replication in tissue culture by -deoxy- , -dehydro-n-trifluoroacetylneuraminic acid (fana): mechanism of action influenza vaccines: present and future learning from our foes: a novel vaccine concept for influenza virus inhibitors of viral neuraminidase as potential antiviral drugs characterization of temperature sensitive influenza virus mutants defective in neuraminidase negativestrand rna viruses: genetic engineering and applications use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors genetically engineered live attenuated influenza a virus vaccine candidates the human cell line per.c provides a new manufacturing system for the production of influenza vaccines . the wild-type m channel was found to be regulated by ph. the wild-type m ion channel activity is proposed to have a pivotal role in the biology of influenza virus infection mek-specific inhibitor u blocks spread of borna disease virus in cultured cells a plasmid-based reverse genetics system for influenza a virus influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade the adjuvanted influenza vaccines with novel adjuvants: experience with the mf -adjuvanted vaccine determinants of immunity to influenza infection in man molecular characterization of the complete genome of human influenza h n virus isolates from thailand randomized trial of influenza vaccine with granulocyte-macrophage colony-stimulating factor or placebo in cancer patients apoptosis in viral infections a single dose of an iscom influenza vaccine induces long-lasting protective immunity against homologous challenge infection but fails to protect cynomolgus macaques against distant drift variants of influenza a (h n ) viruses tamiflu™ patient information live cold-adapted influenza a vaccine produced in vero cell line entry of influenza viruses into cells is inhibited by a highly specific protein kinase c inhibitor b cell superstimulatory influenza virus (h -subtype) induces b cell proliferation by a pkcactivating, ca( +)-independent mechanism expanding the frontiers of existing antiviral drugs: possible effects of hiv- protease inhibitors against sars and avian influenza plasmid-only rescue of influenza a virus vaccine candidates amantadine-resistant and -sensitive influenza a strains and recombinants influenza virus neuraminidase activates latent transforming growth factor beta induction of apoptosis by influenza virus blockade of the map kinase pathway suppresses growth of colon tumors in vivo characterization of a porcine lung epithelial cell line suitable for influenza virus studies role of protein kinase c betaii in influenza virus entry via late endosomes dissecting virus entry via endocytosis serial killers: ordering caspase activation events in apoptosis cyclopentane neuraminidase inhibitors with potent in vitro anti-influenza virus activities the humoral response to live and inactivated influenza vaccines administered alone and in combination to young adults and elderly confronting the avian influenza threat: vaccine development for a potential pandemic evaluation of a genetically modified reassortant h n influenza a virus vaccine candidate generated by plasmid-based reverse genetics characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness multidose, live attenuated, cold-recombinant, trivalent influenza vaccine in infants and young children vitro and in vivo activities of t- and oseltamivir against influenza virus activation of the apoptotic fas antigen-encoding gene upon influenza virus infection involving spontaneously produced beta-interferon induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells possible involvement of double-stranded rna-activated protein kinase in cell death by influenza virus infection recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death influenza a and b viruses expressing altered ns proteins: a vaccine approach defense mechanisms against influenza virus infection in the respiratory tract mucosa the interferon-alpha/beta system in antiviral responses: a multimodal machinery of gene regulation by the irf family of transcription factors the influenza virus: a killer comes into view virus induction of human ifn beta gene expression requires the assembly of an enhanceosome caspases: enemies within monitoring of viral susceptibility: new challenges with the development of influenza na inhibitors signaling through protein kinase c inhibition of cap (m gpppxm)-dependent endonuclease of influenza virus by -substituted , -dioxobutanoic acid compounds expression, purification, and characterization of orthomyxovirus: influenza transcriptase rimantadine hydrochloride and amantadine hydrochloride use in influenza a virus infections recognition of influenza virus proteins by cytotoxic t lymphocytes evaluation of live, cold-adapted influenza a and b virus vaccines in elderly and high-risk subjects efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial. us oral neuraminidase study group evaluation of trivalent, live, cold-adapted (caiv-t) and inactivated (tiv) influenza vaccines in prevention of virus infection and illness following challenge of adults with wild-type influenza a (h n ), a (h n ), and b viruses protective efficacy of combined live intranasal and inactivated influenza a virus vaccines in the elderly comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus a vaccine strains depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h n ) virus isolated from humans destruction of lymphocytes by a virulent avian influenza a virus virulent avian influenza a viruses: their effect on avian lymphocytes and macrophages in vivo and in vitro structure of the influenza virus glycoprotein antigen neuraminidase at . a resolution the structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase rational design of potent sialidase-based inhibitors of influenza virus replication transcription stimulation of the fas-encoding gene by nuclear factor for interleukin- expression upon influenza virus infection interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics influenza a virus ns protein prevents activation of nf-kappab and induction of alpha/beta interferon live attenuated vaccines against influenza; an historical review influenza a virus with defective m ion channel activity as a live vaccine immunogenicity and protective efficacy of replication-incompetent influenza viruslike particles are we ready for pandemic influenza? influenza virus: transmission between species and relevance to emergence of the next human pandemic predictions for future human influenza pandemics spanish influenza: the secrets remain elusive evolution and ecology of influenza a viruses interspecies transmission of influenza viruses influenza: interspecies transmission and emergence of new pandemics effectiveness of oseltamivir in preventing influenza in household contacts: a randomized controlled trial mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human local immune responses to influenza antigen are synergistically enhanced by the adjuvant iscomatrix direct gene transfer into mouse muscle in vivo cellular immunity and memory to respiratory virus infections -guanidino- , -dideoxy- , -dehydro-n-acetylneuraminic acid is a highly effective inhibitor both of the sialidase (neuraminidase) and of growth of a wide range of influenza a and b viruses in vitro communicable disease surveillance & response (csr) ikappab kinase-beta: nf-kappab activation and complex formation with ikappab kinase-alpha and nik fields virology nf-kappab-dependent induction of tumor necrosis factor-related apoptosis-inducing ligand (trail) and fas/fasl is crucial for efficient influenza virus propagation caspase activation is essential for efficient influenza virus propagation direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf- and cbp/p comparative study of influenza virus replication in vero and mdck cell lines nf-kappab activation by double-stranded-rna-activated protein kinase (pkr) is mediated through nf-kappab-inducing kinase and ikappab kinase position statement: global neuraminidase inhibitor susceptibility network caspase-dependent nterminal cleavage of influenza virus nucleocapsid protein in infected cells immunostimulating reconstituted influenza virosomes we would like to thank all the colleagues who have helped in many invaluable ways in the production of this chapter, in particular, dr. c. erhardt, dr. w. wurzer, h. marjuki, b. daubner, v. oehlschlaeger, j. lampe and k. oesterle. this work is dedicated to prof. dr. c. scholtissek's th birthday. key: cord- -q s fkh authors: roth, james a. title: mechanistic bases for adverse vaccine reactions and vaccine failures date: - - journal: adv vet med doi: . /s - ( ) - sha: doc_id: cord_uid: q s fkh nan vaccines have proven to be very beneficial for controlling diseases in domestic animals. their widespread use has dramatically reduced the incidence of severe and fatal diseases in companion animals (canine distemper, canine parvovirus, infectious canine hepatitis, and feline panleukopenia). they have also enabled the intensification of livestock production, thus enabling great increases in efficiency in animal origin food and fiber production. in addition, animal vaccines have improved human health through control of zoonotic diseases such as rabies, brucellosis, and leptospirosis. indeed, it can be argued that animal vaccines have had a profound impact on modern society. without effective rabies vaccines many people would not opt to keep companion animals in their homes, and without effective vaccines for controlling major diseases in food-producing animals the availability of animal proteins for human consumption would be greatly reduced. however, in spite of the success of animal vaccines, vaccines sometimes induce adverse reactions in animals and sometimes they fail to protect animals. when making decisions regarding vaccination programs for animals, veterinarians and animal owners must weigh the risks of vaccinating vs. the risks of not vaccinating. they must also use vaccines in a manner that induces optimal protection. this article provides an overview of some of the reasons why vaccines occasionally produce adverse reactions (table i) and reasons why vaccines sometimes fail to protect animals from disease (table ii) . to produce protective immunity, a vaccine must stimulate a reaction in the animal. there usually must be a reaction both at the site of injection and systemically in order to produce an effective immune response. this reaction involves extensive activity by antigen-presenting cells, production of a variety of cytokines, and alterations in the trafficking of lymphocytes within the body. in addition, if the vaccine contains live organisms, they probably need to replicate to induce effective immunity. live viruses must infect and replicate within cells. these essential reactions to a vaccine may induce observable clinical signs. hopefully, the reaction to the vaccine will be mild and either unnoticeable or acceptable to the animal owner. to understand vaccine safety and efficacy, it is important to understand the process by which vaccines are developed and tested by vaccine producers, and licensed by the united states department of agriculture (usda), animal and plant health inspection service (aphis), center for veterinary biologics (cvb). the federal government regulations for the united states of america regarding veterinary vaccines contamination with extraneous agents failure to inactivate agent in killed vaccine residual virulence of vaccine organisms vaccination of immunosuppressed animal immune suppression induced by the vaccine excessive induction of cytokine release multiple vaccines administered concurrently hypersensitivity to vaccine antigens type i--immediate type type iimcytotoxic type type iiimimmune complex type type ivmdelayed type triggering or exacerbation of hypersensitivity to nonvaccine antigens allergies autoimmune disease induction of neoplastic changes mlv bvd vaccine triggering mucosal disease in persistently infected cattle are found in the virus serum toxin act (vsta) in title of the code of federal regulations ( cfr). the vsta gives the usda the authority to regulate veterinary vaccines in the united states. according to the cfr a usda licensed biological must be "pure, safe, potent, and efficacious, and not be worthless, contaminated, dangerous, or harmful." to understand this statement, it is important to understand what is meant by safe and efficacious. the definition found in the cfr for safe or safety regarding veterinary biologics is "freedom from proper ties causing undue local or systemic reactions when used as recom-mended or suggested by the manufacturer." this definition has two important qualifiers for the term safety. it does not state that a vaccine should produce no reaction, rather it states that a vaccine should not cause "undue local or systemic reactions." this is a recognition of the fact that stimulating a potent immune response is likely to produce at least a mild local and systemic reaction in the animal. the second important point is that according to the definition the safety of the vaccine is only ensured when it is used as recommended or suggested by the manufacturer. the recommendations and suggestions can be found on the label for the vaccine. most vaccine label statements will indicate that a particular vaccine is only for use in healthy animals of a particular species. healthy is defined as "apparently normal in all vital functions and free of signs of disease." the cfr definition for efficacious or efficacy is "specific ability or capacity of the biological product to effect the result for which it is offered when used under the conditions recommended by the manufacturer." the label found on the vaccine will indicate the "result for which the vaccine is offered" and will also indicate the conditions under which the vaccine is recommended for use. therefore, it is very important to read and follow label instructions in order to achieve maximum safety and efficacy from vaccine usage. when animals develop adverse clinical signs within a few days to weeks after vaccination it is important to determine whether those clinical signs were vaccine induced or were not due to vaccination and only coincidentally occurred after the vaccine was administered. animals commonly experience adverse clinical signs from a wide variety of causes and animals are commonly vaccinated. therefore, it is to be expected that occasionally adverse clinical signs will occur after animals have been vaccinated for reasons unrelated to vaccine administration. there are also many reasons why vaccines may induce adverse reactions in the animal. it is important to differentiate true adverse vaccine reactions from false adverse vaccine reactions. some of the causes of true adverse vaccine reactions are summarized in table i and explained next. a prominent example of this occurred when it was discovered that some lots of the live oral human poliomyelitis vaccine were contami-nated with live simian virus (sv ) in the s (pennisi, ; shah and nathanson, ) . millions of people were potentially exposed to live sv through administration of polio vaccine. to date, there is no solid epidemiologic evidence that any adverse health affects can be attributed to exposure to this agent. the sv virus had not yet been discovered when the human polio vaccine was produced. this raises the question of how does one test for all potential known and unknown viruses in each production lot of modified live virus vaccines. (gard and lycke, ; nathanson and langmuir, ) . this resulted in several cases of poliomyelitis in people that had received the vaccine. there have also been cases where formaldehyde failed to inactivate the foot-and-mouth disease virus (beck and strohmaier, ; king et al., ) and the venezuelan equine encephalitis virus (kinney et al., ) in their respective vaccines. in both of these cases the vaccine was shown to induce disease because of the lack of complete inactivation of the virus by the formaldehyde (brown, ) . an example of a failure to completely inactivate a bacterial pathogen in a killed bacterin occurred when thimerosol was used to inactivate haemophilus somnus in an h. somnus vaccine. the thimerosol failed to kill the h. somnus. approximately half the ani- mals on one farm that were injected with vaccine shortly after its production developed thromboembolic meningoencephalitis and died. modified live vaccine organisms have been attenuated to have reduced virulence. the attenuation must be shown to be stable when passaged through animals; therefore, reversion to virulence is thought to be a rare event. however, the attenuated vaccine strains may be capable of producing disease in immunosuppressed animals. induction of disease by the vaccine organism has occasionally been reported when modified live virus (mlv) vaccines have been administered to healthy animals. however, it has occurred much more frequently when mlv vaccines are administered to unhealthy animals, by a nonrecommended route of exposure, to animals younger than the intended age for use of the vaccine, or when the vaccine is used in other than the intended species. examples of mlv vaccines occasionally causing disease in healthy animals of the recommended species without apparent predisposing causes include the induction of rabies in dogs and cats after administration of an mlv rabies vaccine (bellinger et al., ; esh et al., ; erlewein, ; whetstone et al., ; pedersen et al., ) and the induction of ovarian lesions and infertility in seronegative heifers administered mlv bovine herpesvirus (bhv ) vaccine during estrus chiang et al., ; miller et al., ; van der maaten et al., ) . since most heifers already have antibody to bhv due to either vaccination or previous exposure, this is thought to be a rare occurrence. an example of vaccine-induced disease resulting from administration of vaccine to unhealthy animals is the induction of encephalitis by mlv canine distemper virus vaccine in dogs infected with canine parvovirus (krakowka et al., ) . an example of adverse vaccine reaction after exposure of an animal to an mlv vaccine by a nonrecommended route of exposure is the induction of clinical feline viral rhinotracheitis after inadvertent exposure by the intranasal route to an mlv vaccine that was intended for intramuscular administration only (povey and wilson, ) . mlv vaccines that have been shown to be safe in older animals may not be safe in neonatal animals. an mlv bhv- vaccine induced fatal bhv infection in neonatal purebred salers calves (bryan et al., ) . this may have been partially due to the breed of the animals since there are other reports that mlv bhv vaccines are apparently safe in neonatal calves (schuh and walker, ). there have been several examples of mlv vaccines inducing lethal disease when administered to a species other than the target species. an mlv pseudorabies virus vaccine produced fatal pseudorabies in lambs (clark et al., ; van alstine et al., ) . this occurred when a syringe that had been used to administer the pseudorabies vaccine to pigs was used without proper disinfection to vaccinate lambs with another vaccine days later. the mlv canine distemper virus vaccine has been shown to induce canine distemper infection in gray foxes (halbrooks et al., ) , kinkajous (kazacos et al., ) , and lesser pandas (bush et al., ). an mlv rabies vaccine has been shown to induce rabies in a pet skunk (debbie, ). an mlv feline panleukopenia vaccine induced cerebellar hypoplasia when given experimentally to neonatal ferrets (duenwald et al., ). an mlv bovine viral diarrhea (bvd) virus vaccine has been shown to suppress neutrophil function and lymphocyte blastogenesis in cattle (roth and kaeberle, ) . this correlates with the observation that cattle tend to be somewhat more susceptible to bacterial pneumonia after administration ofmlv bvd vaccines, especially if the animals are stressed at the time of vaccination. several commercially available canine vaccines have been shown to be capable of inducing lymphopenia and suppressing blastogenesis of peripheral blood lymphocytes (phillips et al., ; mastro et al., ; kesel and neil, ) . lymphopenia and suppression of blood lymphocyte blastogenesis must be interpreted with caution, however, because it may only be an indication of changes in lymphocyte trafficking between the blood and lymphatic systems rather than an indication of depressed lymphocyte function. vaccination with an mlv bhv vaccine has been shown to exacerbate the lesions of infectious bovine keratoconjunctivitis after experimental intraocular challenge with moraxella bovis (george et al., ). interleukin (il- ), il- , and tumor necrosis factor ~ (tnf-~) are potent proinflammatory cytokines that are released by macrophages and other cells in response to infection, endotoxin and other bacterial components, and some vaccine adjuvants. these proinflammatory cytokines can induce a wide range of clinical signs. they may induce acute inflammation at the local site of production, they may induce rapid synthesis and secretion of acute phase proteins by the liver, they may act on the hypothalamus to induce fever and malaise, they may reduce rate of gain and feed efficiency, and in sufficiently high concentrations they may induce hypoglycemia, reduce cardiac output, cause hypovolemic shock, and cause disseminated intravascular coagulation. lipopolysaccharide (or endotoxin) from gram-negative bacteria is one of the most potent inducers of the proinflammatory cytokines (cullor, ; ellis and yong, ; galanos and freudenberg, ) . a number of other bacterial components, listed in table iv , have also been shown to induce proinflammatory cytokine production (erdos et al., ; henderson and wilson, ; allison and eugui, ) . these components are generally the most active if they are released from the degraded bacterial cell. killed bacterins that contain excessive amounts of these bacterial components can induce clinical signs due to excessive induction of cytokine release. this is more likely to occur if multiple killed bacterins are administered at the same time and if these bacterins contain adjuvants that also induce cytokine release. the production of small amounts of proinflammatory cytokines is beneficial to the induction of a protective immune response. however, overproduction of the proinflammatory cytokines can have mild to very severe adverse side affects. (bonin et al., ; wilson et al., ; erdos et al., ) . as with all of the hypersensitivity reactions, the animal will not react on first exposure to an antigen (unless it has received passive antibody responsible for the reaction). it will only react after there has been sufficient time to produce the sensitizing antibody or memory t cells. a local type i hypersensitivity reaction may occur due to ige induced against infectious agents by the vaccine. immunization against bovine respiratory syncytial virus under experimental conditions was shown to induce ige antibodies specific for brsv which apparently contributed to the development of symptoms following aerosol challenge with brsv (stewart and gershwin, a,b) . vaccine-induced type ii (cytotoxic type) hypersensitivity reactions can occur when vaccines are used that contain normal cell antigens. for example, vaccines that contain erythrocyte antigens may induce anti-erythrocyte antibodies leading to immune-mediated hemolytic anemia. type iii (immune complex type) hypersensitivity can occur when circulating antibody specific for vaccine antigens is present at the time of vaccination. this can lead to an arthus reaction at the site of injection due to complement fixation and neutrophil recruitment to the site. this mechanism is commonly responsible for the local inflammatory reaction at the site of injection, especially when administering booster vaccinations with killed vaccines. sometimes, hypersensitivity can be one component of a more complex adverse vaccine reaction. antibody induced by the vaccine may lead to immune complex type hypersensitivity reactions after the animal becomes infected when the antibody binds to replicating infectious agents. examples include anterior uveitis and corneal edema (blue eye) after vaccination with canine adenovirus (carmichael et al., ; wright, ) and the sensitization to the effusive form of feline infectious peritonitis after vaccination with experimental killed vaccines (pedersen and black, ) . sometimes, hypersensitivity may be one component of a more complex adverse vaccine reaction. bacterins for pasteurella haemolytica which were marketed and widely sued for several years were of marginal efficacy and were even capable of increasing the severity of lesions in animals either experimentally (wilkie et al., ) or naturally exposed (bennett, ) to the p. haemolytica. there are at least two hypothesized mechanisms by which the immune response induced by the bacterin could potentiate pneumonia after p. haemolytica challenge. first, the high concentration of complement-fixing antibody in-duced by vaccination with a bacterin could rapidly activate complement if a large number of p. haemolytica organisms were introduced into the lung either naturally or artificially. this could cause a type iii hypersensitivity response leading to acute inflammation in the lung and severe pneumonia. second, antibody against cell surface antigens will opsonize the p. haemolytica in the lung and enhance phagocytosis by alveolar macrophages and neutrophils. because there may be insufficient leukotoxin-neutralizing antibody or cell-mediated immunity to activate phagocytes, the bacteria present in the alveoli and ingested by phagocytes are not efficiently killed and may produce leukotoxin that could destroy the phagocytes. this destruction would cause the phagocytes to release their hydrolytic enzymes into the lung. in the last few years concern has been expressed that vaccination may trigger or exacerbate autoimmune disease or allergies (hypersensitivities), especially in dogs and cats (see article by dr. jean dodds in this volume). vaccination has been shown to augment production of ige antibody to pollen in inbred atopic dogs (frick and brooks, ) . remember that animals with allergies or autoimmune diseases are not healthy animals, and that vaccines are only recommended for use in healthy animals. dr. harm hogenesch addresses the topic of vaccineinduced autoimmunity in another article in this volume. in recent years, an increased incidence of fibrosarcoma occurring at sites commonly used for vaccination in cats has been observed (hendrick et al., (hendrick et al., , kass et al., ) . the causal relationship and mechanistic basis for vaccine-associated fibrosarcomas in cats has not been firmly established (ellis et al., ) . shortly after mlv bvd vaccines were introduced, it was recognized that a very small percentage of cattle developed a syndrome - days after vaccination that closely resembled bvd mucosal disease (lambert, ; peter et al., ) . based on the current understanding of the pathogenesis of mucosal disease (bolin et al., ; brownlie et al., ) this was almost certainly due to the cytopathic bvd virus in the vaccine triggering mucosal disease in calves that were immunotolerant to, and persistently infected with, a noncytopathic bvd virus. the mechanistic basis for the induction of the lesions of mucosal disease is not clearly understood. this unique syndrome is primarily due to abnormalities in the animal rather than to a defect in the vaccine. vaccines are tested for safety and efficacy when administered to healthy animals in the formulation in which they are packaged to be sold. vaccines are not required to be tested for safety and efficacy when administered concurrently with other vaccines. this would not be practical since there are too many possible vaccines that may potentially be used in combination. an example of a safety problem that occurred when two different vaccines were administered concurrently involved a newly developed mlv canine coronavirus and parvovirus vaccine given at the same time as an mlv canine distemper-hepatitis virus vaccine. the evidence indicated that the other mlv components allowed the canine coronavirus in the vaccine to induce neurologic disease in some vaccinated animals (wilson et al., ) . injection site lesions are a common occurrence and are of great concern in food-producing animals. they may lead to unacceptable blemishes in, or decreased quality of, meat intended for human consumption. there are many possible causes of injection site lesions, including organisms introduced with a contaminated needle, live contaminating organisms in the vaccine, adjuvant induced reactions, cytokine release, hypersensitivity reactions (types i, ii, iii, or iv), trauma, and hemorrhage (straw et al., (straw et al., , droual et al., ; littledike, ; stokka et al., ; dexter et al., ; apley et al., ; straw, ) . vaccines that are licensed by the usda have been tested to determine that they are safe and effective. however, "effective" is a relative term. it does not mean that the vaccine must be able to induce complete immunity under all conditions which may be found in the field. this would not be realistic since the immune system is not capable of such potent protection under adverse conditions. to be federally licensed, the vaccine must have been tested under controlled experimental conditions. the vaccinated group must have had significantly less disease than the nonvaccinated control group. this testing is typically done on healthy, nonstressed animals under good environmental conditions and with a controlled exposure to a single infectious agent. vaccines may be much less effective when used in animals that are under stress, incubating other infectious diseases, or exposed to a high dose of infectious agents due to overcrowding or poor sanitation. it is important to remember that for most diseases the relationship between the infectious agent and the host is sufficiently complicated that vaccination cannot be expected to provide complete protection. the vaccine can increase the animal's resistance to disease, but this resistance can be overwhelmed if good management practices are not followed. some of the causes for vaccine failure are summarized in table ii and explained next. the host requires several days after vaccination before an effective immune response will develop. if the animal encounters an infectious agent near the time of vaccination, the vaccine will not have had time to induce immunity. the animal may come down with clinical disease resulting in apparent vaccination failure. in this situation, disease symptoms will appear shortly after vaccination and may be mistakenly attributed to vaccine virus causing the disease (mckercher et al., ) . improperly handled and administered vaccines may fail to induce the expected immune response in normal, healthy animals. modified live bacterial and viral vaccines are only effective if the agent in the vaccine is viable and able to replicate in the vaccinated animal. observing proper storage conditions and proper methods of administration are very important for maintaining vaccine viability. failure to store the vaccine at refrigerator temperatures, or exposure to light, may inactivate the vaccine. even when stored under appropriate conditions, the vaccine loses viability over time. therefore, vaccines that are past their expiration date should not be used. the use of chemical disinfectants on syringes and needles can inactivate modified live vaccines if there is any residual disinfectant. the use of improper diluent or the mixing of vaccines in a single syringe may also inactivate modified live vaccines. diluents for lyophilized vaccines are formulated specifically for each vaccine. a diluent that is appropriate for one vaccine may inactivate a different vaccine. some vaccines and diluents contain preservatives that may inactivate other modified live vaccines. for these reasons, multiple vaccines should not be mixed in a single syringe unless that particular combination has been adequately tested to ensure there is no interference. vaccine failures may occur because a vaccinated animal is not able to respond appropriately to the vaccine. vaccine failure in young animals may be due to the presence of maternal antibody which prevents adequate response to vaccination. it can also be due to immunosuppression from a variety of causes. maternal antibodies derived from colostrum are a well-known cause of vaccine failure (greene, ) . these antibodies in the young animal's circulation may neutralize or remove the antigen before it can induce an immune response. typically, virulent infectious agents are capable of breaking through maternal immunity earlier than modified live or killed vaccines. this means that even if young animals are immunized frequently, there still may be a period when they are vulnerable to infection. vulnerability occurs between the time that young animals lose their maternal antibody and before they develop their own active immune responses. this period can be shortened by the use of less-attenuated and/or higher titered modified live vaccines or the use of killed vaccines with high antigenic mass and strong adjuvants (smith-carr et al., ; larson and schultz, ) . a high challenge dose of infectious agents will break through maternal immunity sooner than low exposure to infectious agents. therefore, overcrowding and poor sanitation exacerbate the problem of inducing immunity in young animals before they come down with clinical disease. veterinarians commonly recommend that puppies and kittens be vaccinated every weeks between approximately and weeks of age. however, for large domestic animals, a single vaccination is commonly recommended to induce immunity during the first few weeks or months of life. there is no inherent difference between large and small domestic animals in their responses to vaccination in the face of maternal immunity. the frequent vaccinations recommended in pup-pies and kittens minimizes the period of vulnerability to infectious diseases. because only one vaccination is commonly recommended for large domestic animals, the timing of vaccination is important. if the vaccine is administered too soon, it may be ineffective because of the presence of maternal antibody. if the vaccine is administered after all maternal antibodies are gone from animals in the group, there may be a prolonged period of vulnerability before they develop their own immune response. the optimal age to vaccinate young animals is highly variable. it will depend on the antibody titer of the mother and the amount of colostrum ingested. it is impossible to predict an optimal age to vaccinate a young animal, unless its antibody titers are determined. most veterinarians and producers decide that because of time and expense considerations it is impractical to vaccinate young food-producing animals frequently to minimize their period of vulnerability to infection. however, frequent vaccination may be justified in cases of unusually high disease incidence in young animals. immunosuppression due to a variety of factors including stress, malnutrition, concurrent infection, or immaturity or senescence of the immune system may also lead to vaccination failure. if the immunosuppression occurs at the time of vaccination, the vaccine may fail to induce an adequate immune response. if the immunosuppression occurs sometime after vaccination, then disease may occur due to reduced immunity in spite of an adequate response to the original vaccine. therapy with immunosuppressive drugs (e.g, glucocorticoids) may also cause this to occur. most vaccines do not produce complete immunity to disease. they provide an increased ability to resist challenge by infectious agents. if a high-challenge dose of organisms is present due to overcrowding or poor sanitation, the immune system may be overwhelmed, resulting in clinical disease. the peak response to a vaccine typically occurs - weeks after vaccination. the level of immunity then begins to gradually decline. a common recommendation is to revaccinate annually. however, if the animal did not have a strong initial immune response due to stress at the time of vaccination, or if it is stressed and exposed to a highchallenge dose several months after vaccination, there may not be enough residual immunity to protect the animal. this is especially true for certain killed vaccines. under these circumstances, it may be necessary to revaccinate more frequently than once per year. for certain types of infectious agents, particularly bacteria that are vulnerable to control by the development of antibodies against surface components and viruses which use rna as their genetic material and consequently have high mutation rates, there are often several antigenic variants of each agent. for antibody-mediated protection to be effective, the antibodies formed must bind the important strain-specific antigens on the surface of the bacteria or virus. cell-mediated immunity is usually not as strain specific as antibody-mediated immunity. to determine if a vaccine's failure to protect is due to antigenic differences between the vaccine and field strains it is necessary to isolate the field strain and compare it to the vaccine strain. antigenic differences between strains leading to lack of vaccine efficacy are usually more of a problem with killed vaccines than modified live vaccines. as mentioned earlier, vaccines are tested for safety and efficacy when administered singly to animals. however, multiple vaccines are commonly administered concurrently to animals. very little published data are available concerning the efficacy of vaccines when used in combination. one study demonstrated that there was no detrimental effect on the antibody response to a bovine respiratory syncitial virus vaccine when administered in combination with up to different immunogens (carmel et al., ) . in contrast, an mlv bhv vaccine when administered in combination with an experimental pasteurella haemolytica vaccine containing outer membrane proteins and genetically attenuated leukotoxin significantly reduced the antibody response to the leukotoxin and the efficacy of the p. haemolytica vaccine in preventing morbidity and mortality due to bovine respiratory disease (harland et al., ) . mild local and systemic reactions to vaccines are to be expected as a natural consequence of vigorously stimulating the immune system. dramatic adverse reactions to vaccines are occasionally due to mistakes during the production or handling of vaccines. more often, they are due to not following label instructions, particularly the restriction to only use vaccines in healthy animals. it is important to publish welldocumented instances of adverse vaccine reactions so that producers and users of vaccines can all learn from the experience and avoid similar problems. vaccine failure to protect from disease is usually due to problems with either client education or compliance with good animal management practices. it is important for clients to understand the proper timing and method of vaccine administration, what to realistically expect for vaccine efficacy, and the importance of minimizing immunosuppressive factors and exposure to high doses of infectious agents in vaccinated animals. veterinary vaccines have produced dramatic benefits in terms of animal health, human health, and efficiency of food production. advances in research and the accumulating experience with vaccines are leading to safer and more effective vaccines. proper usage of vaccines and adherence to good management practices will continue to be essential to achieve maximal vaccine safety and efficacy. live sv in human polio vaccine (pennisi killed hog cholera virus in pseudorabies vaccine live mycoplasma in multiple live virus veterinary vaccines live border disease virus in orf vaccine live bovine leukemia virus in babesiosis and anaplasmosis vaccines live bovine viral diarrhea virus in hog cholera vaccine live border disease virus in pseudorabies vaccine live blue tongue virus in a canine vaccine live bovine viral diarrhea virus in bovine vaccines induction of cytokine formation by bacteria and their products. in 'virulence mechanisms of bacterial pathogens subcutaneous injection site comparison of two multiple valent clostridial bacterin/toxoids in feedlot cattle subtyping of european foot-and-mouth disease virus strains by nucleotide sequence determination rabies induced in a cat by high-egg-passage flury strain vaccine efficacy ofpasteurella bacterins for yearling feedlot cattle severe clinical disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus sensitization against calf serum proteins as a possible cause of allergic reactions after vaccination review of accidents caused by incomplete inactivation of viruses experimental production of fatal mucosal disease in cattle fatal, generalized bovine herpesvirus type- infection associated with a modified-live infectious bovine rhinotracheitis parainfluenza- vaccine administered to neonatal calves vaccine-induced canine distemper in a lesser panda effects of vaccination against immunogens in beef replacement heifers at weaning viral-antibody complexes in canine adenovirus type (cav- ) ocular lesions: leukocyte chemotaxis and enzyme release the effect of infectious bovine rhinotracheitis vaccine on reproductive efficiency in cattle vaccinated during estrus pathogenicity of a modified-live pseudorabies vaccine virus in lambs safety and efficacy of gram-negative bacterial vaccines vaccine-induced rabies in a pet skunk incidence of injection-site blemishes in beef top sirloin butts local reaction and serological responses in commercial layer chickens injected intramuscularly in the leg with oiladjuvanted mycoplasma gallisepticum bacterin feline panleukopenia: experimental cerebellar hypoplasia produced in neonatal ferrets with live virus vaccine systemic adverse reactions in young simmental calves following administration of a combination vaccine use of immunohistochemistry and polymerase chain reaction for detection of oncornaviruses in formalin-fixed, paraffin-embedded fibrosarcomas from cats the demonstration of the sensitizing effect of the residual animal serum content of vaccines post-vaccinal rabies in a cat vaccine-induced rabies in four cats canine fatalities associated with the use of a modified live vaccine administered during late stages of pregnancy immunoglobulin e antibodies to pollens augmented in dogs by virus vaccines mechanisms of endotoxin shock and endotoxin hypersensitivity inactivation of poliovirus by formaldehyde. analysis of inactivation curves enhancement of infectious bovine keratoconjunctivitis by modified-live infectious bovine rhinotracheitis virus vaccine infectious diseases of the dog and cat response of gray foxes to modified live-virus canine distemper vaccines the effect of subunit or modified live bovine herpesvirus- vaccines on the efficacy of a recombinant pasteurella haemolytica vaccine for the prevention of respiratory disease in feedlot calves modulins: a new class of cytokine-inducing, proinflammatory bacterial virulence factor postvaccinal sarcomas in the cat: epidemiology and electron probe microanalytical identification of aluminum comparison of fibrosarcomas that developed at vaccination sites and at nonvaccination sites in cats: cases ( - ) hog cholera antibodies in pigs vaccinated with an aujeszkyvaccine based on antigen produced in ib-rs- cells epidemiologic evidence for a causal relation between vaccination and fibrosarcoma tumorigenesis in cats vaccinationinduced distemper in kinkajous combined mlv canine parvovirus vaccine: immunosuppression with infective shedding. vm / sac biochemical identification of viruses causing the outbreaks of foot-and-mouth disease virus in the u.k molecular evidence for the origin of the widespread venezuelan equine encephalitis epizootic of to canine parvovirus infection potentiates canine distemper encephalitis attributable to modified live-virus vaccine bovine viral diarrhea: prophylaxis and postvaccinal reactions high-titer canine parvovirus vaccine: serologic response and challenge-of-immunity study variation of abscess formation in cattle after vaccination with a modified-live pasteurella haemolytica vaccine investigation of dams and their offspring inoculated with a vaccine contaminated by bovine viral diarrhea virus. vm/ sac outbreaks of border disease in goats induced by a pestivirus-contaminated orf vaccine, with virus transmission to sheep and cattle repeated suppression of lymphocyte blastogenesis following vaccination of cpv-immune dogs with modified-live cpv vaccines complications in cattle following vaccination with a combined bovine viral diarrhea-infectious bovine rhinotracheitis vaccine infertility in heifers inoculated with modified-live bovine herpesvirus- vaccinal strains against infectious bovine rhinotracheitis on postbreeding day the cutter incident. poliomyelitis following formaldehyde-inactivated poliovirus vaccination in the united states during the spring of which bvd vaccine should i use? attempted immunization of cats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus rabies vaccine virus infection in three dogs monkey virus dna found in rare human cancers characteristics of a condition following vaccination with bovine virus diarrhea vaccine effects of vaccines on the canine immune system a comparison of inactivated feline viral rhinotracheitis and feline caliciviral disease vaccines with live-modified viral vaccines bovine leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis suppression of neutrophil and lymphocyte function induced by a vaccinal strain of bovine viral diarrhea virus with and without the concurrent administration of acth outbreaks of neonatal infectious bovine rhinotracheitis human exposure to sv : review and comment nicrotic oophoritis in heifers vaccinated intravenously with infectious bovine rhinotracheitis virus vaccine during estrus canine parvovirus. part i. pathogenesis and vaccination role of ige in the pathogenesis of bovine respiratory syncytial virus in sequential infections in vaccinated and nonvaccinated calves detection of ige antibodies to bovine respiratory syncytial virus inflammatory response to clostridial vaccines in feedlot cattle injection reactions in swine comparison of tissue reactions produced by haemophilus pleuropneumoniae vaccines made with six different adjuvants in swine antibody production and tissue irritation in swine vaccinated with actinobacillus bacterins containing various adjuvants a survey of mycoplasma detection in veterinary vaccines vaccineinduced pseudorabies in lambs ovarian lesions induced in heifers by intravenous inoculation with modified-live infectious bovinerhinotracheitis virus on the day after breeding contamination of a live virus vaccine against pseudorabies (aujeszky's disease) by an ovine pestivirus pathogen for the pig bovine viral diarrhoea virus infections in piglets born to sows vaccinated against swine fever with contaminated vaccine use of monoclonal antibodies to confirm vaccine-induced rabies in ten dogs, two cats, and one fox abortion and death in pregnant bitches associated with a canine vaccine contaminated with bluetongue virus response of calves to lung challenge exposure with pasteureua haemolytica after parenteral or pulmonary immunization a neurologic syndrome associated with use of a canine coronavirus-parvovirus vaccine in dogs detection of sensitizing tissue culture components in viral vaccines canine adenovirus: its role in renal and ocular disease: a review key: cord- -cskb njm authors: ludwig, george v.; iacono-connors, lauren c. title: insect-transmitted vertebrate viruses: flaviviridae date: journal: in vitro cell dev biol anim doi: . /bf sha: doc_id: cord_uid: cskb njm the flaviviridae include almost viruses, nearly half of which have been associated with human disease. these viruses are among the most important arthropod-borne viruses worldwide and include dengue, yellow fever, and japanese encephalitis viruses. morbidity and mortality caused by these viruses vary, but collectively they account for millions of encephalitis, hemorrhagic fever, arthralgia, rash, and fever cases per year. most of the members of this family are transmitted between vertebrate hosts by arthropod vectors, most commonly mosquitoes or ticks. transmission cycles can be simple or complex depending on the hosts, vectors, the virus, and the environmental factors affecting both hosts and viruses. replication of virus in invertebrate hosts does not seem to result in any significant pathology, which suggests a close evolutionary relationship between virus and vector. another example of this relationship is the ability of these viruses to grow in invertebrate cell culture, where replication usually results in a steady state, persistent infection, often without cytopathic effect. yields of virus from insect cell culture vary but are generally similar to yields in vertebrate cells. replication kinetics are comparable between insect and vertebrate cell lines, despite differences in incubation temperature. both vertebrate and insect cell culture systems continue to play a significant role in flavivirus isolation and the diagnosis of disease caused by these agents. additionally, these culture systems permit the study of flavivirus attachment, penetration, replication, and release from cells and have been instrumental in the production and characterization of live-attenuated vaccines. both vertebrate and insect cell culture systems will continue to play a significant role in basic and applied flavivirus research in the future. the family flaviviridae consists of at least viruses that possess a similar replication strategy and are morphologically, morphogeneticaily, and biochemicaily similar. until the advent of modem techniques in molecular biology, which are used to elucidate mechanisms of replication, the flaviviruses were organized as a separate serogroup (the group b arboviruses) within the family togaviridae. in , the international committee for the taxonomy of viruses, acting on data summarizing flavivirus biology by westaway et al. ( ) , created a separate family, the flaviviridae. members of the family flaviviridae, although biologically similar, consist of a wide variety of viruses that vary in their pathogenesis and ecology. some, such as yellow fever, dengue, and russian spring-summer encephalitis viruses, are often responsible for severe disease and significant mortality. these viruses have a multifarious ecology involving complicated transmission cycles that often include wildlife reservoirs in combination with mosquito or tick vectors. other flaviviruses, like modoc, dakar bat, and cowbone ridge, fail to produce any detectable disease in humans or domestic animals, and are not transmitted by arthropod vectors. the degree of divergence of the viruses in the family flaviviridae suggests a long history, and implies that the study of these viruses will require a variety of in vivo and in vitro systems. when viewed through an electron microscope, flaviviruses are spherical particles to nm in diameter ( ) . the virus particles consist of a ribonucleoprotein core surrounded by a lipid bilayer membrane of host-ceil origin. the virion contains three separate structural proteins: the nucleocapsid or core (c) protein, to kda; the membrane (m) protein, to kda; and the envelope (e) protein, to kda. the positive-sense viral rna codes for seven additional nonstructural proteins, all of" which play a role in virus replication ( , ) . the e protein is the major surface component of the flaviviral envelope. existing as spikes on the viral membrane, the e protein contains all of the important antigenic determinants responsible for hemagglutination and neutralization and thus plays an important role in immunologic protection from infection. e protein domains are involved in the binding of virions to ceil receptors and probably play a role in intraendosomal fusion at low ph as part of the entry pathway ( ) . antigenic determinants on the e protein serve as the basis for flavivirus classification using serologic tests. for the purposes of classification, a serogroup is defined as two or more viruses, distinct from each other by quantitative serologic standards ( -fold or greater difference between homologous and heterologous titers) in one or more tests, but related to each other or to other viruses by any serologic method. viruses closely related within a serogroup but distinct from each other are considered to constitute an antigenic ( ) and ( ) . b viruses associated primarily with encephalitis syndrome. ° parentheses indicate viruses more closely related to each other than to others of the antigenic complex. d viruses associated primarily with hemorrhagic fever. e viruses associated primarily with fever, arthralgia, and/or rash. y viruses could not be assigned to an antigenic complex because hyperimmune mouse ascitic fluids made against these viruses did not cross-react significantly with other flaviviruses. complex ( ) . by these criteria, the llaviviruses are organized into eight antigenic complexes (table ) . this paper pro des a general overview of flavivirology, with an emphasis on the importance of vertebrate and invertebrate cell culture systems for the isolation and biological characterization of these agents. in addition, the importance of cell culture for producing flavivirus diagnostic reagents and developing vaccines for current and future flavivirus health threats is discussed. ecology distribution of flaviviruses into ecologic groups closely parallels their organization based on their antigenic characteristics ( table ). the members of the japanese encephalitis, ntaya, uganda s, and dengue antigenic groups are transmitted by mosquitoes, whereas members of the tick-borne encephalitis and tyuleniy antigenic groups are predominantly transmitted by ticks. members of the rio bravo and modoc antigenic groups have no known invertebrate vectors and are probably transmitted directly from infected animal to animal. blok and colleagues ( ) have made a detailed comparison of the nucleotide sequences of a variety of flaviviral polymerase and hehcase-like protein coding regions (ns and ns , respectively). genetic dendograms prepared from these sequence comparisons suggest that the primary division of the flaviviruses is between those transmitted by mosquitoes and those transmitted by ticks. these observations suggest that the evolution of each of the flavivirus antigenic groups is closely associated with their respective vector species. although the mosquito-borne flaviviruses are ecologically diverse, two examples will serve to illustrate the complexity of the systems responsible for maintaining, amplifying, and disseminating these viruses in nature. yellow fever virus is the type species for the family flaviviridae. the virus originated in africa, and the disease caused by the virus has been known for hundreds of years. the virus causes a diversity of symptoms including fever, chills, severe headache, back pain, anorexia, nausea, and vomiting. in severe cases, jaundice appears during a second episode of fever, often associated with generalized hemorrhagic symptoms. death occurs in to % of severe yellow fever cases ( ) . in africa, as well as in the americas, the virus is maintained in two basic cycles, the sylvatic and the urban forms. the sylvatic form of yellow fever virus is maintained in cycles involving arboreal monkeys and forest canopy mosquitoes. in africa, cercopithecid and colobrid monkeys are effective viremic hosts, circulating virus for several days at titers sufficient to infect vector mosquitoes. these hosts are generally resistant to disease, indicating a balanced host-virus relationship. in contrast, most newworm monkeys are susceptible to disease, and epizootics in howler monkeys (alouatta sp.), spider monkeys (ateles sp.), and owl monkeys (aotus sp.) are not unknown. only the capuchin (cebus sp.) and wooly monkeys (lagothrix sp.) display a high degree of resistance to disease in the americas. this unstable host-virus relationship is probably a result of the more recent introduction of virus into the western hemisphere ( ) . canopy-breeding mosquitoes serve as the primary vectors of yellow fever virus between susceptible monkey hosts. in africa, the primary mosquito species involved with sylvatic transmission include aedes furcifer, a. africanus, and a. luteocephalus. the main vectors in tropical america include members of the genus haemagogus. maintenance of virus during extended periods of dry weather may be facilitated by drought-resistant species, such as sabethes chloropterus, and through vertical transmission in certain african and tropical american vectors ( , cades. ethiopia experienced a large outbreak in to involving an estimated cases with approximately deaths. a. aegypti seems to be playing a major role in recent urban yellow fever epidemics in africa, although other species may also play a significant role ( ) . in eastern nigeria for instance, a. africanus, the vector responsible for enzootic maintenance, assumes the role of epidemic vector in the savannah-rainforest zones, where it is responsible for significant interhuman transmission ( ) (fig. ). urban outbreaks in africa continue to be a major public health concern. an excellent review of yellow fever virus ecology and epidemiology in the americas and africa has been published ( ) . the tick-borne encephalitis (tbe) virus antigenic group consists of tick-borne viruses that cause a variety of diseases in humans. the severe form of human disease, caused by infection with viruses such as russian spring-summer encephalitis virus, is characterized by gradual onset of symptoms which include anorexia, fever, headache, nausea, vomiting, and photophobia. these symptoms are followed by variable nettrologie dysfunction which result in death in approximately % of cases and mild-to-severe neurologic sequelae in to % of cases ( ) . members of the tbe virus antigenic complex are transmitted between vertebrate hosts by tick vectors. on the european and asian continents, the main vectors of endemic strains are lxodes ricinus and l persulcatus. in areas where these tick species do not exist, other tick species of the genera dermacentor and haemaphysails are probably responsible for the maintenance of tbe viruses ( ) . endemnicity of tbe correlates directly with the geographic distribution of the vector species. tick populations serve as natural reservoirs and provide a mechanism for viral overwintering by vertical transmission. maintenance over extended periods of time is, however, not possible due to inefficient transtadial and transovarial transmission ( ) . the tbe viruses are maintained, amplified, and disseminated in natural transmission cycles involving the ticks described above and wild vertebrate hosts, particularly small rodents and insectivores ( , ) . these cycles are required for endemnicity because of the inefficiency of vertical transmission. transmission of virus to humans is most common in individuals who work or play in tick-infested areas. domestic animals do not participate in normal transmission cycles as a result of low blood titers after infection, but do contribute to the potential for human infection. lactating sheep, goats, and cattle secrete virus into their milk and may pass virus to humans through contaminated raw dairy products (fig. ) . the ecologies of all of the arthropod-borne flaviviruses are equally complex, involving a variety of arthropod vectors, each varying in their ability to transmit virus. no conclusive evidence exists that suggests that any significant pathologic changes occur in the fi¢. . generalized transmission cycle of the tick-borne encephalitis viruses. ticks from several genera are responsible for transmission between vertebrate hosts. maintenance by vertical transmission in ticks does not occur due to inefficient vertical transmission, but may help to maintain natural reservoirs and provide an overwintering mechanism, lnsectivores, particularly shrews, moles, and hedgehogs, are believed to be important amplifying reservoir hosts. humans acquire virus by bite from infected ticks and when ingesting virus contaminated dairy products. mosquito or tick vector after infection with these viruses, which indicate a balanced host-parasite interaction and suggests a close evolutionary relationship. the close interrelationship between the ilaviviruses, their vectors, and their vertebrate hosts attests to the degree with which they have co-evolved. the scientific discipline of virology essentially began with the ability to culture cells in vitro. the first cell culture systems were developed in the late s. over the next to yr, improvements in cell and tissue culture techniques led to many rapid developments in virology, culminating with the landmark study by enders et al. in on the growth of poliovirus in cultured cells ( ) . these results initiated a series of studies that led to the isolation of a variety of viruses and finally ushered in the modern eta of virology ( ) . quantitative virology also matured at about the same time with the introduction of the cell culture-based plaque assay by dulhecco in ( ) . the use of cultured cells in the study of the basic biology of viruses has developed only during the last two decades. the ability to propagate viruses in homogeneous cultures of cells and the development of modern molecular biology and electron microscopy paved the way for the observation of virus attachment, penetration, uneoating, transcription, translation, packaging, and escape under controlled conditions. it is the ability to control carefully the environment under which a virus replicates that has led to our current level of understanding of viral biology. the arthropod-borne viruses, or arboviruses, are unique among viruses in that transmission, dissemination, and amplification require cyclic passage through vertebrate and invertebrate hosts. it is the ability of these viruses to replicate in multiple hosts that differ significantly in their biology, physiology, and ecology that makes them distinctive among all viruses. the availability of continuous insect cell cultures provides the opportunity to study how differences in the biology of vertebrate and invertebrate cells affect the biology of a particular infecting virus. recent research with a variety of arboviruses suggests that some biological characteristics of a virus are correlated to the host from which the virus was derived ( , , , ) . such characteristics may play an important role in the understanding of the mechanisms for attenuation and virulence at the molecular level, and have only recently begun to be studied comparatively in insect and vertebrate cells. cell culture is essential for the study of flaviviruses, and will continue to play a pivotal role in the isolation, characterization, and development of new vaccines against current and future flavivirus health threats. the elucidation of the complexities of flavivirus ecology and the study of flavivirus disease frequency and distribution in human and animal populations relies heavily on the development of isolation and diagnostic techniques utilizing cell mad tissue culture systems. improvements in the sensitivity and specificity of these techniques enhance the accuracy of epidemiologic studies which are essential to the identification of at-risk populations. the characteristics of flavivirus growth in cell culture determines how a particular culture system may be used to study specific viruses. cell cultures that are most sensitive to infection may be the best choice for virus isolation, but the same cells may produce relatively little cell-free infectious virus and therefore may not be useful for the preparation of stock virus or viral antigen. each cell line must be carefully examined for its usefulness in flavivirus diagnosis, both in terms of its replication kinetics and its production potential. one of the most interesting developments in flavivirus research has been the development of continuous insect cell lines to isolate virus and diagnose infection. cell lines derived from a. albopictus, a. pseudoscutellaris, toxorhynchites amboinesis, and other mosquitoes have been extremely useful for the study of a variety of flaviviruses, including dengue; yellow fever; west nile, rocio, st. louis encephalitis, and japanese encephalitis viruses ( ) ( ) ( ) , , , ) . growth characteristics of flaviviruses in culture vary between cell type, virus, temperature, laboratory, and even experiment. an excellent review of many of these variables has been compiled ( ) . in a typical one-step growth analysis of dengue- virus infection of a. albopictus cells, virus is first detectable in cell culture superuatant by h postinfection (pi) ( ) . peak titers develop by h pi, followed by a steady decline in titers through day pi. in another study, with the same cell line and dengne- virus, peak titers were also reached within approximately h, but titers decreased slightly through day pi, at which time a steadystate persistent infection was established. over the days the cells were monitored, there was little change in the titer of infectious virus from culture superuatant (fig. ) ( ) . incubation temperature plays an important role in the replication of flaviviruses in cultured cells. in one example, dengue- infection of c / cells was studied at ° and ° c. results revealed that a higher incubation temperature accelerated the onset of cytopathic effects, hastened the development of virus-specific proteins, and enhanced the titer of extracellular infectious virus ( ) . in another study, replication of kunjin virus in vertebrate and insect cells was closely related to the optimum growth temperature of each cell line. virus infection of insect cells at ° c produced similar amounts of extracellular virus as infection of vertebrate cells grown at ° c ( ) . in yet another study, a. albopictus cells infected with dengue- virus initially produced less virus when cultures were incubated at suboptimal temperatures. after days in culture, however, cells incubated at ° c produced essentially the same amount of dengue- virus as those incubated at ° c ( ) (fig. ) . infection of insect cells with llaviviruses commonly results in the development of persistent infections. such infections occur with or without the appearance of cytopathic effect (cpe). kuno ( ) showed that dengue virus infection of tra- cells (from toxorhynchites amboinensis) often resulted in an apparent, persistent infection. titers of infectious extracellular virus varied extensively over mo. (fig. ) . cpe was routinely visible in cells infected with both laboratory-adapted and -unadapted strains within the first wk pi, but gradually diminished over time in the adapted strains, and was eliminated altogether in the unadapted strains. some characteristics of viral replication in culture were altered during the course of persistent infection, as demonstrated by changes in neutralization characteristics, syncytia formation, and temperature sensitivity ( ) . ( ) showed that of three mosquito cell lines tested, the tra- line from t, amboinensis mosquitoes was the most sensitive to dengue virus and suggested that this line be used for routine isolation and identification procedures. in many cases, flavivirus isolation in insect cell culture is as, or more, sensitive than isolation techniques in animal hosts ( , ). isolating flaviviruses in cultured cells seems to have a host-specific effect on the antigenic structure of the virus. fukunaga et al. ( ) showed that dengne- virus isolated in suckling mouse brain was less sensitive to neutralization by patient convalescent sera than was virus isolated in mosquito cells. these data suggest that while flavivirus isolation in vertebrate cells or whole animals may be extremely sensitive, identification of the isolate by serologic techniques may be more accurate if virus is isolated in mosquito cells. as described, insect cell cultures are often more desirable for the isolation and identification of flaviviruses due to their -increased sensitivity to flavivirus infection. in addition, these cell lines are often more desirable in epidemiologic field studies where carefully controlled laboratory conditions are diflicuh to maintain. most insect cells used in flavivirus research are extremely hardy. they can be maintained in a wide range of temperatures and will survive for short periods under relatively severe temperature extremes. c / cells, for instance, have been maintained in our laboratory for several days at temperatures as low as ° c and as high as ° c. cell husbandry of well-established insect cell lines is often less regimented than that with vertebrate cell lines. in our laboratory, c / cells can be maintained at ° c for as long as wk postconfluency in the absence of a medium change. all of these characteristics illustrate the utility of insect cell culture for use in field studies under less than optimal conditions. although virus isolation and identification are standard techniques for flavivirus diagnosis, they are not always possible due to the laboratory conditions required to grow and maintain most cell cultures. under these conditions, serologic tests are used to diagnose disease by the identifying virus-specific antibodies from patient sera. these techniques also play the major role in epidemiologic studies. rapid serologic assays, such as the enzyme-linked immunosorbent assay (elisa), require a supply of high-quality viral antigen. this has traditionally required the production of large amounts of infectious virus, usually under stringent bioeontainment conditions, followed by some form of virus inactivation, which may or may not lead to alteration of immunologically important viral antigens. with the recent development of baculovirus expression systems, the production of large amounts of viral antigen produced in spodoptera frugiperda (sf ) and other insect cells in biocontainment-free environments has become possible. in this system, autographa californica nuclear polyhedrosis virus (baculovirus) can be genetically altered to express unusually high levels of foreign gene products when the gene is inserted into the baculovirus polyhedron gene ( , ) . utilization of this technique to produce large amounts of flavivinas antigen to be used in diagnostic tests may ultimately reduce the need for containment facilities in diagnostic laboratories. the first steps an enveloped virus takes to infect cells are those of attachment and fusion. attachment of virus to cells takes place through the interaction of virus attachment and cellular receptor molecules. it is this mechanism that directs tissue tropism and the pathogenesis of many viruses ( , , , , , , , , ) . fusion of virus to cells is a separate although not necessarily an indistinguishable event from virus attachment. fusion takes place either at the plasma membrane (herpesviruses, retroviruses, and paramyxoviruses) or in endocytic vesicles after uptake by receptormediated endoeytosis (rhabdoviruses, orthomyxoviruses, bunyaviruses, coronaviruses, and togaviruses). although it is generally accepted that the initial interaction of flaviviruses with cells is a virusreceptor interaction, the phenomenon separate from fusion has not been thoroughly studied. in contrast, viral fusion has been well studied with flaviviruses, although not without some controversy. many investigators have provided considerable evidence that flaviviruses are taken up by vertebrate cells by receptor-mediated endocytosis followed by a ph-dependent fusion event ( , , , ) f g. . entry of wnv into p d ceils by adsorptive endocytosis at ph . a, single virus particles, labeled with several gold particles, attached to cells; b, coated invagination of the plasma membrane endocytosing virus particles labeled with a single gold particle; c, coated vesicle containing a single virus particle. bar = nm. ]reproduced from ref ( ) with permission.] (fig. ). more recent research has shown that fusion of dengue, yellow fever, and japanese encephalitis viruses may in fact involve fusion of virus directly to the cell's plasma membrane ( , ) . in these studies, fusion of mosquito-passaged virus to both c / cells and human peripheral blood monocytes was observed by electron microscopy to occur in the absence of endosome formation. the authors suggest that a more accurate reproduction of natural transmission cycles by passing virus through mosquito cells is required before viral fusion activity in target vertebrate cells can be accurately assessed. the method of infectious flavivirus internalization is likely to prove to be cell specific and to rely heavily on specific viral envelope components that vary considerably, depending on the host cells from which the virions originate. once virus has penetrated the cell, replication proceeds with the transcription and translation of the flavivirus genome. the flavivirus genome consists of one single-stranded rna molecule of positive polarity with a type cap structure at the ' terminus ( ) . the genome is approximately kilobases long and does not typically possess a polyadenylated sequence at the ' terminus, but instead possesses a sequence that has the potential to form secondary structure. mandl et al. ( ) identified a ' terminal polyadenylated sequence on the central european encephalitis virus genome, suggesting that ' polyadenylation may be a characteristic of at least some flaviviruses. the full-length tlavivirus genome is the only mrna species found in virus-infected cells and encodes proteins which are expressed from the polycistronic rna to produce a polyprotein that is co-translationatly and posttranslationally processed ( ) . virion rna also serves as the template for the production of viruscomplementary rna (vcrna), which subsequently serves as the template for the production of viral (positive sense) rna. the genome organization places the coding sequences for the three structural proteins, capsid (c), precursor-membrane (prm)/membrane (m), and envelope (e), at the ' end of the rna followed by the coding sequences for the seven nonstructural (ns) proteins; ns , ns a, ns b, ns , ns a, ns b, and ns ( ) (fig. ) . although the process of flavivirus protein production is still not completely understood, there have been extensive studies to evaluate these questions in virus-infected cell cultures. excellent reviews on this subject have been published ( , , ) . briefly, proteolyric cleavage points in a number of flavivirus polyproteins have been identified by n-and c-terminal sequencing of individual viral proteins produced in infected cell culture. in addition, a number of flavivirus genomic sequences have been determined and their deduced amino acid sequences have been compared. amino acid sequences of all flaviviruses are highly conserved immediately adjacent to their predicted or known polyprotein proteolytie cleavage sites. this observation of sequence conservation suggests a common pathway for the proteolytic processing of viral proteins of all flaviviruses. both cellular and viral proteases are known to be specifically involved in viral protein processing. at least one additional, unidentified protease is also involved in flavivirus protein processing ( ) . nearly all data generated on viral polyprotein processing have been obtained by using mammalian cell culture systems. molecular expression and evaluation of flavivirus proteins from insect cell cultures have been limited. it has been reported that antigenic reactivity of some flaviviruses is altered after replication in insect cells ( ) . this suggests that differences exist in the mechanisms of viral protein processing between mammalian and insect cell culture systems. the development of recombinant baculoviruses capable of expressing foreign genes of interest to high concentrations in certain insect cell cultures (e.g., sf cells) has been used more recently in continued efforts to study the proteins of flaviviruses ( , ) . assembly of flavivirus virions occurs concurrently with genome translation and transcription. two flavivirus particles have been described: immature, which contain the precursor-membrane protein (prem), and mature, which contain the fully processed m protein ( ) . the m protein is not found inside virus-infected cells but is the predominant species found in extracellular virions. the prem protein is found typically inside virus-infected cells as part of intracellular virions but it also can be found in virions that have been released from infected cells; these are referred to as immature virions ( ) (fig. ) . as reviewed in chambers et al. ( ) the initial location of flavivirus particles in competent vertebrate and invertebrate cells is the lumen of the endoplasmic reticulum. production of flavivirus particles in infected cells occurs rapidly, less than h pi; and whole particles are associated with perinuclear cisternae of endoplasmic reticulum vesicles ( , ) . viral nucleocapsids have not been detected within infected cells ( , ) . therefore, rapid nucleocapsid assembly and membrane association are supported. an excellent review of virion assembly and maturation in infected cells and a model suggesting virus cis-and trans-aeting elements and the order of events in the process are described by chambers et al, ( ) . subcellular transport of immature flavivirus to the cell surface is thought to occur by the translocation of immature virion-containing vesicles from membranous components of the cell to the plasma membrane. lipid fusion then occurs between the vesicle and the plasma membrane, thus releasing vesicle contents extracellularly (fig. ) . concurrent with tlavivirus translocation and release from cells, a proteolytic cleavage event takes place in which the aminoterminal portion of the prem protein is released forming the mature m protein ( , ) . this proteolytic event is required to produce fully infectious flavivirus particles ( ) . recent studies on selected flaviviruses suggest that the processing of prem to m is a ph-dependent event, which takes place in post-golgi acid vesicles. this proteolytic event may be necessary for fusion of virus with susceptible cells ( , ) . some flaviviruses may, however, be released from infected cells predominantly in the immature form ( ) . additional factors may be involved in the cellular release of the immature flavivirions. techniques in molecular biology have allowed for the development of flavivirus infectious clones. these clones are full-length flavivirus cdna sequences that can be transcribed in vitro. then their product is transfected into competent mammalian cells, which results in the recovery of infectious virus indistinguishable from the parent. these techniques are being developed in an effort to define genetically flavivirus elements that are responsible for viral infectivity, enzymatic activities, viral pathogenesis, host range and tissue tropisms, latency, and attenuation ( ) . two examples of the use of this technology with flaviviruses have been described. rice et al. ( ) made an infectious clone of the d yellow fever vaccine virus in an effort to produce a homogenous population of vaccine virus and to help elucidate the mechanisms for vaccine attenuation. more recently, lai et al. ( ) developed an infectious clone of dengue- virus, also to be used to study dengue virus biology and vaccine development. the development of safe and effective flavivirus vaccines has consistently been an area of intense research. the earliest human vaccines to protect against flavivirus infections were inactivated virus vaccines prepared by passing virulent virus in mouse brains, then treating them with formalin. such mouse brain-derived vaccines against yellow fever and tbe viruses were effective at preventing disease in immunized individuals. although such vaccines were effective, severe reactigenicity and rapid decay of protective immunity were common, resulting in the need to develop improved vaccines. in , theiler and smith ( ) reported that neurovirulence of yellow fever virus for monkeys could be reduced by passing the virus in cultured cells. this observation initiated research resulting in the development of a series of live-attenuated flavivirus vaccines. vaccines derived from in vitro passage of flavivirus continues to be the major source of safe and effective vaccines. the best-described history of a live-attenuated flavivirus vaccine is that of the d yellow fever virus. the d vaccine strain dates back to a isolate of yellow fever virus from the blood of a young ghanian named asibi ( ) . the virus isolate bearing the name of the donor was subsequently passed times in rhesus monkeys and intermittently in a. ae~ypti mosquitoes. the virus was then passed times in minced mouse embryo tissue culture, times in minced whole chicken embryo tissue culture, times in minced chicken embryo without central nervous system (cns), and times in chicken embryo with or without cns. somewhere between in vitro passages and , a marked change in monkey virulence occurred, eventually leading to the present d vaccine ( , ) . differences in the deduced amino acid sequences of the asibi and d yellow fever viruses concentrate in the e, the m, ns a, and ns b proteins ( , ) . amino acids in the ns proteins are not highly conserved among flaviviruses, and substitutions within these proteins are thought not to have a significant effect on their function ( ) . identification of critical regions of the flavivirus genome responsible for attenuation has yet to be absolutely identified, but these observations suggest that structural coding regions are probably important. similar studies with other arboviruses con-firm that structural proteins play an important role in virus attenuation ( , ) . the availability of a full-length infectious clone of d virus may allow identification of specific coding changes that account for attenuation of this strain ( ) . it is possible that systematic nucleotide substitutions into the d infectious clone may provide positive information on sequences that contribute to yellow fever virus virulence. although the d yellow fever vaccine has been highly effective, complications are not unknown, and improvement of the existing vaccine continues to be a major area of research. recently it has been shown that attenuation of asibi virus in monkeys can be established in as few as six hela cell culture passages. analysis of the hela cell-passed virus suggests that attenuation is due to a genetic change in the virus population rather than to selection of a preexisting variant of asibi virus. based on changes in the reactivity of monoclonal antibodies with the parent and attenuated variant, the results suggest that antigenic changes in the viral envelope protein may also play a role in attenuation of the virus ( ). although the development and testing of the d yellow fever vaccine represent the best-known example of flavivirus vaccine development in animal cell culture, three other vaccines have been developed recently by similar strategies. these include the dengue- and vaccines, as well as the attenuated japanese encephalitis virus vaccine. research describing the current state of development and testing of these vaccines has been described ( , , , , , ( ) ( ) ( ) ( ) , , , , ) . no examples of serial insect cell passage of flaviviruses resulting in vertebrate-attenuated strains of virus have been reported. the observation that persistent dengue virus infections can be established in a non-vector mosquito cell line associated with changes in several of the important biological characteristics, such as temperature sensitivity and antigenic structure ( ) , presents the possibility that genetic selection of attenuated virus strains may be possible with insect cell lines. pilot studies looking at the feasibility of producing flavivirus vaccines in insect cells should be initiated. one of the most exciting new techniques to be introduced for the production of new live attenuated vaccines, is infectious clone technology. this technique has been used to insert systematically attenuating point mutations into alphaviruses ( , , ) . with the availability of infectious clones of yellow fever and dengue- virus, this technique may provide a method for the genetic construction of flaviviruses deliberately attenuated for use as vaccines. taking the concept one step further, flavivirus infectious clones may prove to be useful vectors to introduce genetic information that encodes protective epitopes from other viruses. such research on intertypic chimeric flaviviruses is currently focused on developing a dengue- virus vaccine candidate containing genetic information from dengue- and ( ) . such vaccines may protect against multiple dengue types in a single immunization. the selective expression of llavivirus antigens in benign microbiologic hosts provides unique sources of specific virual proteins for use in the development of a new generation of subunit flavivirus vaccines. flavivirus antigens have been expressed successfully in recombinant prokaryotic and eukaryotic cell culture systems. purified escherichia coil fusion proteins have been described and reported to be feasible flavivirus vaccine candidates, usable diagnostic reagents, and useful for defining tlavivirus antigenic sites ( , , , ) . several although the live virus vaccines are usually preferable to inactivated or subunit vaccines, alternative approaches to vaccinology still play an important role in disease control strategies. proteins prepared in baculovirus expression systems have been used to prepare antigens suitable for vaccination. most notable is the preparation of a human immunodeficiency virus (hiv ) subunit vaccine from recombinant baculovirus expressed hiv antigens ( ) , which is currently being tested in humans. recombinant baculovirus-expressed flavivirus proteins have been evaluated for their use as vaccines and can protect appropriate animal models against homologous virus challenge ( , ) . although this expression system has the potential to express recombinant proteins to extremely high levels in cell culture, the problems involved with protein purification currently limit their use in humans. each of the approaches to vaccine production listed above is currently being used to develop new and to improve existing flavivirus vaccines. each technique totally relies on an appropriate cell culture system for vaccine development, production, and evaluation. future use of these vaccines in humans will depend on the availability of cell lines certified for the development of human use products. although certified vertebrate cell hues capable of growing virus for human use vaccines are available, no comparable insect cell lines have been developed. the availability of primary and continuous cell culture systems is essential for the study of flavivirus biology, ecology, and epidemiology, and play a significant role in the development and testing of flavivirus vaccines. although the importance of cell culture systems for the study of infectious diseases cannot be overstated, the limitations of these systems must also be understood. flavivirus infection of animal hosts is an extremely complex event. complex organisms are made up of a large number of tissues, each tissue containing many cell types. each cell type has a different biology reflected by its different functions. in vitro, research of flavivirus infection is conducted most often in clonal populations of cells derived from a single tissue type. very often the biological, morphologic, and genetic characteristics of the cultured cells bear little resemblance to those of the cells of the original tissue. like the cultures used in these studies, the biology of flaviviruses in cultured cells may boar little resemblance to the biology of the viruses in functioning tissues and organs. as a result, studies on llavivirus biology conducted in cultured cells must be viewed only as a model representing a single event in the complex process contributing to the pathogenesis of viral disease. cell culture should be used as a tool for studying viral biology in animals and not as a total replacement for studies utilizing intact hosts. with these limitations in mind, further research on flavivirus biology, pathogenesis, ecology, and epidemiology utilizing mammalian and invertebrate cell cultures should be used to enhance our understanding of these viruses at the molecular, microscopic, and organismal levels. as previously discussed, the arboviruses are unique in that transmission, dissemination, and amplification require cyclic passage through vertebrate and invertebrate hosts. control of the diseases caused by these viruses can be obtained either at the level of the vertebrate host, in the form of vaccination, or at the level of the invertebrate host. an understanding of the basic biology of these viruses in the cells of the invertebrate host is essential to establishing control at the level of the arthropod vector. such research with flaviviruses is under-represented and is required for a thorough understanding of these viruses in nature. identification of mutations that occurred on the genome of japanese encephalitis virus during the attenuation process selection of a better and highly attenuated live vaccine virus strain of japanese encephalitis. ii. safety and immunogenicity of live vaccine sa - - observed in inoculated children yellow fever vaccines attenuation of wild-type yellow fever virus by passage in heea cells comparison of a dengue- virus and its candidate vaccine derivative: sequence relationships with the flaviviruses and other viruses dengue virus premembrane and membrane proteins elicit a protective immune response construction ofintertypic chimeric dengue viruses by substitution of structural protein genes mice immunized with recombinant vaccinia virus expressing dengue- virus structural proteins with or without nonstructural protein ns are protected against fatal dengue virus encephalitis replication of llaviviruses characterization of west nile virus persistent infections in genetically resistant and susceptible mouse cells. ii. generation of temperature-sensitive mutants antigenic relationship between flaviviruses as determined by cross-neutralization tests with polyelonal antisera reduction of yellow fever virus mouse neurovirulenee by immunization with a bacterially synthesized nonstructural protein (ns ) fragment the role of mammals in natural foci of tick-borne encephalitis in central europe flavivirus genome organization, expression, and replication comparison of three methods used to isolate dengue virus type the influence of higher temperature on dengue- virus infected c / mosquito cell line une poussre pizootique de fi~vre jaune selvatique du senegal oriental: is ement du virus de lots de moustiques adultes male et femelles receptors in the initiation of picornavirus infections the cd (t ) antigen is an essential component of the receptor for the aids retrovirus the growth of two togaviruses in cultured mosquito and vertebrate cells attenuating mutations in the e glyeoprotein gene of venezuelan equine encephalitis virus: construction of single and multiple mutants in a full-length cdna clone in vitro synthesis of infectious venezuelan equine encephalitis virus rna from a cdna clone: analysis of a viable deletion mutant characterization of yellow fever virus proteins e and ns expressed in vero and spodopterafrugiperda cells dengue virus envelope protein expressed by a recombinant vaceinia virus falls to protect monkeys against dengue initial stages in infection with animal viruses replication of dengue virus in cultured mosquito cells at suboptimal temperature production of plaques in monolayer tissue cultures caused by single particles of an animal virus transovarial transmission of yellow fever virus by a sylvatic vector, haemagogus equinus the association of enhancing antibodies with seroconversion in humans receiving a dengue- live-virus vaccine japanese encephalitis virus live-attenuated vaccine, chinese strain sa~ - - ; adaptation to primary canine kidney cell cultures and preparation of a vaccine for human use cultivation of the lansing strain of poliomyelitis virus in cultures of various human embryonic tissues immunization of mice with recombinant vacciuia virus expressing authentic dengue virus nonstructural protein ns protects against lethal dengue virus encephalitis genetic and molecular mechanisms of viral pathogenesis: implications for prevention and treatment flavivirus type-specific antigens produced from fusions of a portion of the e protein gene with the escherichia coli trpe gene neutralization characteristics of dengue viruses isolated in mosquito culture and suckling mouse brain from a case of laboratory dengue infection epidemiologieal models of tick-borne infections (aeari: ixodidae and argasidae) flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry the uncoating and infectivity of the flavivirus west nile virus on interaction with cells: effects of ph and ammonium chloride genetic determinants of the virulence and infectivity of la crosse virus two monoelonal antibodies against la crosse virus show host-dependent neutralizing activity crc handbook series in zoonoses, section b: viral zoonoses fusion activity of flaviviruses: comparison of mature and immature (prm-containing) tlck-borue encephalitis virions comparison of the virulent asibi strain of yellow fever virus with the d vaccine strain derived from it comparison of the asibi and d strains of yellow fever virus expression of the structural proteins of dengue virus and yellow fever virus by recombinant vaccinia viruses expression of envelope glycoprotein (e) of japanese encephalitis virus by recombinant vaccinia virus selection of attenuated dengue viruses by serial passage in primary kidney cells. i. attributes of uncloned virus at different passage levels selection of attenuated dengue viruses by serial passage in primary kidney cells. iv. characterization of a vaccine candidate in fetal rhesus lung cells selection of attenuated dengue viruses by serial passage in primary kidney cells. ii. attributes of virus cloned at different dog kidney passage levels selection of attenuated dengue viruses by serial passage in primary kidney cells. ill. reversion to virulence by passage of cloned virus in fetal rhesus lung cells flavivirus entry into cultured mosquito cells and human peripheral blood monocytes maturation process of japanese encephalitis virus in cultured mosquito cells in vitro and mouse brain cells in vivo preparation of an attenuated dengue ( carib) virus vaccine. i. safety and immunogenicity in humans growth of arboviruses in arthropod cell cultures: comparative studies. i. preliminary observations on growth of arboviruses in a newly established mile of mosquito cell (culex quinquefasciatus say) isolation ofa singh's aedes albopictus cell clone sensitive to dengue and chikungunya viruses isolation of viruses from female culex tritaeniorhynchus in aedes albopictus cell cultures high-level expression of the tick-borne encephalitis virus ns protein by using an adenovirus-based vector: protection elicited in a marine model host-adaptive antigenic variation in bunyaviruses selection for accelerated penetration in cell culture coselects for attenuated mutants of venezuelan equine encephalitis virus general characteristics and antigenic relationships the effect of ph on the early interaction of west nile virus with p d cells selective tropism of lymphadenopathy associated virus (lav) for helper-induced t lymphocytes persistent infection of a nonvector mosquito cell line (til~.- ) with dengue viruses comparative sensitivity of three mosquito cell lines for isolation of dengue viruses dengue- virus infection of human mononuclear cell lines and establishment of persistent infections infectious rna transcribed from stably cloned full-length cdna of dengue type virus the acetylcholine receptor as a cellular receptor for rabies virus trends in the development of baculovirus expression vectors vaccinia virus: a selectable eukaryotic cloning vector the construction and characterization of vaccinia virus recombinants expressing foreign genes presence of poly(a) in a flavivirus: significant differences between the ' noncoding regions of the genomic rnas of tick-borne encephalitis virus strains preparation of an attenuated dengue ( carib) virus vaccine. i. pre-clinical studies virus entry into animal cells molecular characterization of a neutralizing domain of the japanese encephalitis virus structural glycoprotein japanese encephalitis virus--vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal jev infection baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins protection of mice against lethal japanese encephalitis with a recombinant baculovirus vaccine use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein carboxy-terminally truncated dengue virus envelope glycoproteins expressed on the cell surface and secreted extracellularly exhibit increased immunogenicity in mice biological characterization of plaque-size variants of yellow fever virus in mosquitoes and mice yellow fever: victor, victoria? conqueror, conquest? epidemics and research in the last forty years and prospects for the future vaecinia virus expression vectors togavirus morphology and morphogenesis possible involvement of receptors in the entry of kunjin virus into vero cells nucleotide sequence of the virulent sa- strain of japanese encephalitis virus and its attenuated vaccine derivative construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the dna of infectious vaccinia virus preliminary analysis ofmurine cytotoxic t cell responses to the proteins of the flavivirus kunjin using vaccinia virus expression recombinant vaccinia virus producing the prm and e proteins of yellow fever virus protects mice from lethal yellow fever encephalitis persistent infection of vero cells by the flavivirus murray valley encephalitis virus protection of mice against yellow fever virus encephalitis by immunization with a vaccinia virus recombinant encoding the yellow fever virus non-structural proteins, ns , ns a, and ns b establishment and characterization of st. louis encephalitis virus persistent infections in aedes and culex mosquito cell lines acidotropic amines inhibit proteolytic processing of flavivirus prm protein transc~ription of infectious yellow fever rna from full-length cdna templates produced by in vitro ligation structure of the flavivirus genome humoral immune response to the entire human immunodeficieney virus glycoprotein made in insect cells chemical and antigenie structure of flaviviruses distinction of influenza viruses of different host cell origin replication of togaviridae and flaviviridae studies on yellow fever in ethiopia. . epidemiological study multiplication of arboviruses in cell lines from aedes albopictus and aedes aegypti isolation of dengue viruses in aedes albopictus cell cultures host cell receptors for two strains of sindbis virus arbovirus replication in mosquito cell lines (singh) grown in monolayer or suspension culture experimental transmission of yellow fever to laboratory animals the structure and function of the flavivirus and pestivirus genomes. viral hepatitis and liver disease flaviviruses can mediate fusion from without in aedes albopictus mosquito cell cultures pneumotropism of sendai virus in relation to protease-mediated activation in mouse lungs the use of yellow fever virus modified by in vitro cultivation for human immunization biochemistry and replication structural and nonstructural proteins of saint louis encephalitis virus cell-associated west nile flavivirus is covered with e+pre-m protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release susceptibility of aedes albopictus c / cells to viral infection induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express prem and e glycoproteins of japanese encephalitis virus selection of a better immunogenic and highly attenuated live virus strain of japanese encephalitis. i. some biological characteristics of sa - - mutant safety of a live-attenuated japanese encephalitis virus vaccine (sa - - ) for children expression of dengue virus structural proteins and nonstructural protein ns by a recombinant vaccinia virus the authors thank drs. joel dalrymple, mike parker, and jim leduc for critical evaluation of the manuscript. the views of the authors do not purport to reflect the positions of the u.s. department of defense. key: cord- -h uq z b authors: crank, michelle c; mascola, john r; graham, barney s title: preparing for the next influenza pandemic: the development of a universal influenza vaccine date: - - journal: j infect dis doi: . /infdis/jiz sha: doc_id: cord_uid: h uq z b nan structural biology, protein engineering, and antigen delivery amenable to platform manufacturing approaches. molecularand atomic-level information about the immune-viral interface combined with new capacities for surveillance and rapid response to pandemics are shaping a new conceptual framework for vaccine development. as a result of these advances, highlevel, broad, and durable immunity against the large universe of influenza viruses may now be within reach [ ] . this issue of the journal of infectious diseases was motivated by the confluence of the influenza pandemic centenary and the new opportunities afforded by technological advances and breakthroughs along with the improved understanding of influenza biology. we have gathered information, opinions, and ideas from thought leaders in immunology, virology, epidemiology, and vaccinology to address the challenge of developing a universal influenza vaccine and articulate some of the knowledge and technical gaps that remain. in , there were approximately . billion persons living on earth; today, there are more than billion [ ] . if a pandemic similar to that of occurred today, how would the devastation compare? because of rapid international travel, the spread of a new virus would be faster than it was in , when spread was largely related to the movement of troops at the end of world war i. today, population density is higher and cities are larger, providing a more favorable environment for a rapidly spreading virus. although we now have more sophisticated medical care, the availability of hospital beds and life support equipment would probably not be sufficient to manage an outbreak equivalent in magnitude to that of . if there were sentinel events as in and in -a small spring epidemic preceding the fall pandemic-current vaccine manufacturing approaches would not be sufficiently fast or scalable for worldwide distribution to preempt pandemic spread. therefore, development of a universal influenza vaccine that can reliably protect against drifted seasonal strains and pandemic strains without biannual reformulation is imperative. ideally, this vaccine would not need to be given every year; however, even if annual vaccination was required but antigenic components needed updating only every - years, it would still be a significant advance over the current system. s • jid : (suppl ) • crank et al there are some clear pathways to explore and knowledge gaps to fill in the immediate future using currently available technology, as described in the accompanying commentaries, outlined here: . by harnessing high-throughput sequencing and computational biology, more sophisticated algorithms based on sequence analysis, glycan patterns, and other features that may anticipate high transmissibility can be developed for predicting the next dominant strain [ ] . the prudent study of gain-of-function mutations would allow scientists to learn more about what molecular signatures to look for. . improving strain selection for seasonal vaccines would increase the likelihood of an antigenic match between the vaccine and dominant circulating strains and thereby improve the utility of current vaccine technology [ ] . the current vaccines could be further improved by better standardization of the neuraminidase content, adjustment of antigen doses, addition of improved adjuvants, and production in cell substrates that minimize the likelihood of viral adaptations and changes in protein sequences [ ] . . precisely defining the b-cell repertoire and epitope-specific phenotypes involved in the response to influenza infection and vaccination would provide insight into the problem of "original antigenic sin" described by thomas francis in and the related phenomenon of immunodominance [ ] . prior influenza immunity and poorly understood antigenicity patterns make it difficult to reshape and broaden the antibody response using current vaccines [ ] . defining all the ways antibody can bind and neutralize influenza structurally and establishing a new nomenclature for describing antigenic sites across both influenza a groups as well as influenza b would reduce confusion and improve communication between scientists [ ] . in addition, learning which features of vaccine-induced local or systemic immune responses result in sustained serum antibody responses may inform vaccine formulation and delivery approaches. . understanding more precisely the b-cell and antibody responses would allow the application of protein engineering for antigen design and display using molecular targets and antibody lineage end points to guide iterative design modifications [ ] . . the role of cd + t cells in determining the efficacy of a b-cell response is an area of active investigation; however, more work in this area may be required to solve the problem of durability and maintenance of antibody responses [ ] . . the direct role of cd + or cd + t-cell effector functions and whether those cells require localization in mucosal tissue or lymph nodes to effectively protect against respiratory viral pathogens are poorly understood. optimizing vaccine formulation and delivery route and modality is dependent on acquiring this type of knowledge [ ] . . defining the importance of including specific antigenic targets, such as the head or stem domains of hemagglutinin, neuraminidase, or the m ectodomain in universal vaccines, and determining whether they are more effective when used in combination or alone could be accomplished through both vaccine protection and natural history studies that provide a better understanding of protective immunity [ ] [ ] [ ] [ ] . . understanding the mechanistic correlates of immunity generated by immunization with live attenuated vaccines may reveal the importance of secretory immunoglobulin a and intraepithelial t cells that require induction of immunity to occur at the mucosal surface [ ] . . defining both the virological and host immune response patterns associated with transmissibility would allow better modeling of population dynamics and factors that could best interrupt transmission cycles [ ] . this could be particularly important for identifying distinct vaccination strategies for different target populations, including in societal settings in which transmission dynamics and target populations vary [ ] . . using human challenge studies and improving animal models of influenza infection and transmission may help answer some of these questions [ ] . however, the utility of animal models hinges on selecting those that are most relevant to human pathogenesis and immunity. improving the characterization of and expanding the reagents for these models would not only benefit influenza vaccine development but would also provide answers to immunological questions relevant to other respiratory virus infections and emerging infectious diseases in general. recent estimates place the cost of influenza pandemics at upward of $ billion per year [ ] . in this context, an investment of at least $ billion per year in the biomedical research effort to achieve a solution for protection against pandemic influenza seems justified. in addition, efforts to develop a universal influenza vaccine, even before reaching this goal, will likely lead to improved seasonal vaccines, with the potential to reduce morbidity rates and save tens of thousands of lives each year. solving this problem is potentially achievable with today's technology, and with an organized and sustained focus on interventions for influenza and other potential emerging infectious threats, more advanced approaches could be rapidly developed. finally, gaps remain in our understanding of influenza biology and immunity and methods to produce highly effective vaccines. to close those gaps, it will be important to align the interests of all the stakeholders preparing for the next pandemic. priorities of public health officials in lower-, middle-, and high-income countries, academic researchers, regulatory bodies, major funders, and pharmaceutical companies must be understood and collectively addressed to face the logistical and scientific challenges ahead [ ] . thanks to scientific and technological breakthroughs of the past decade, vaccinology is experiencing a revolution. may we find the resolve, political will, and new business plans to take full advantage of these new opportunities and prepare ourselves before the next pandemic arrives. updating the accounts: global mortality of the - "spanish" influenza pandemic influenza vaccines: good, but we can do better making universal influenza vaccines: lessons from the pandemic can we predict the next influenza pandemics? antibody determinants of influenza immunity the way forward: potentiating protective immunity to novel and pandemic influenza through engagement of memory cd t cells immunodominance and antigenic variation of influenza virus hemagglutinin: implications for design of universal vaccine immunogens dynamic perspectives on the search for a universal influenza vaccine universal influenza vaccine approaches employing full-length or head-only ha proteins universal influenza virus vaccines that target the conserved hemagglutinin stalk and conserved sites in the head domain the role of m e in the development of universal influenza vaccines neuraminidase, the forgotten surface antigen, emerges as an influenza vaccine target for broadened protection how live attenuated vaccines can inform the development of broadly cross-protective influenza vaccines new vaccine design and delivery technologies influenza immunization in low-and middle-income countries: preparing for next-generation influenza vaccines novel vaccine technologies: essential components of an adequate response to emerging viral diseases emerging viral diseases from a vaccinology perspective: preparing for the next pandemic advances in antiviral vaccine development a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases historical estimates of world population on the doctrine of original antigenic sin pandemic risk: how large are the expected losses? vaccine development in the twenty-first century: changing paradigms for elusive viruses we thank our colleagues at the national key: cord- -qsqaimsy authors: hankaniemi, minna m.; baikoghli, mo a.; stone, virginia m.; xing, li; väätäinen, outi; soppela, saana; sioofy-khojine, amirbabak; saarinen, niila v. v.; ou, tingwei; anson, brandon; hyöty, heikki; marjomäki, varpu; flodström-tullberg, malin; cheng, r. holland; hytönen, vesa p.; laitinen, olli h. title: structural insight into cvb -vlp non-adjuvanted vaccine date: - - journal: microorganisms doi: . /microorganisms sha: doc_id: cord_uid: qsqaimsy coxsackievirus b (cvb) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type diabetes. the licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (vlps), can offer important advantages in the manufacturing and epitope engineering. we have previously produced vlps for cvb and cvb in insect cells. here, we describe the production of cvb -vlps with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. we also resolved the cvb -vlp structure by cryo-electron microscopy imaging and single particle reconstruction. the vlp diameter is . nm on average, and it is similar to coxsackievirus a vlps and the expanded enterovirus cell-entry intermediate (the s particle), which is ~ nm larger than the mature virion. high neutralizing and total igg antibody levels, the latter being a predominantly th type (igg ) phenotype, were detected in c bl/ j mice immunized with non-adjuvanted cvb -vlp vaccine. the structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus vlp-vaccines in the future. coxsackievirus b (cvb ) is an rna virus that belongs to the enterovirus genus of picornaviridae. infections caused by the six coxsackievirus b serotypes (cvb - ) usually manifest in mild flu-like symptoms or respiratory illness. however, cvbs can infect several organs after primary infection, such as the nervous system, pancreas, and heart [ ] . such disseminated infections can lead to severe organ damage and serious diseases including aseptic meningitis, encephalitis, pancreatitis, and myocarditis. if myocarditis becomes chronic, it can lead to dilated cardiomyopathy (dcm) which is a leading cause of heart transplantation. cvb is the enterovirus most often responsible for viral myocarditis [ ] and it can also cause hand, foot, and mouth disease (hfmd) outbreaks [ ] . cvbs have also been associated with the development of type diabetes [ ] [ ] [ ] . therefore, cvbs have been considered as potent targets for the future enterovirus vaccines [ ] [ ] [ ] . besides polio [ ] and enterovirus- [ ] (ev-a ) vaccines, no clinically approved treatments or vaccines are available against enteroviruses. to meet the demand for the prevention of cvb associated diseases, the clinical development of a polyvalent cvb vaccine has recently commenced [ ] . cvb vaccines in clinical development and ev-vaccines in clinical use contain either live-attenuated viruses (oral polio vaccine, opv) or formalin inactivated viruses (cvb vaccine, inactivated polio vaccine, ev-a -vaccine). these vaccines have worked well in the prevention of enterovirus diseases. however, as a live vaccine, opv faces safety issues, due to the reversion of the attenuated vaccine virus strain towards the virulent strain in the vaccine recipients leading to vaccine-associated paralytic polio and subsequent polio outbreaks [ ] . thus, the use of opv is becoming difficult, however two of the three wild poliovirus strains have been declared to be eradicated. inactivated poliovirus vaccine has been effective and safe, but this vaccine technology does not allow flexible epitope engineering and it cannot be used for enterovirus types which do not grow well in cell cultures. with this comes an increasing demand for new vaccine technologies and cost-effective vaccine production methods that allow flexible vaccine engineering and the large-scale production of vaccines against different enterovirus types. virus-like particles (vlps) are empty particles assembled from recombinantly expressed viral structural proteins, which ensures therefore, that they are non-infectious and can be produced for viruses which do not grow well in cell cultures. thus, vlp-based vaccines are not associated with the safety concerns related to live virus vaccines and offer also important advantages in the vaccine engineering and manufacturing process. promising vlp-based vaccine candidates against evs have been produced as demonstrated by monovalent vlp-vaccines against cvb [ , ] , ev-a [ ] , cva [ ] , cva [ ] , and ev-d [ ] or polyvalent vlp-vaccines against ev-a , cva , cva , and cva [ ] . we have previously produced cvb -vlps with moderate yields (yield referring to the total amount of the pure end product protein) [ ] , while the efficient production of cvb -vlps was described based on a new construct design, use of a baculoviral (flashbac) genome, and an improved purification method [ ] . in the present study, we aimed to enhance cvb -vlp production yields and purity as well as define an industrially scalable production process. due to these aims, cvb -vlps were produced utilizing a production process similar to that described for cvb -vlp [ ] . enteroviruses are non-enveloped icosahedral viruses that are around nm in diameter. the capsid protects an approximately . kb single-stranded positive-sense rna genome, which is translated into a polyprotein that is co-translationally and post-translationally cleaved by the viral proteases a and cd to yield structural proteins vp , vp , and vp . these structural proteins assemble into the viral capsid, wherein the viral rna is encapsidated. vp is further processed into vp and vp in a viral rna-driven autocatalysis reaction yielding the mature virus [ ] . the basic building block (protomer) of the icosahedral capsid contains vp -vp proteins and the entire capsid consists of such protomers [ ] . in mature enteroviruses, vp together with the n-terminus of vp are located at the internal surface of the capsid [ ] . on the outer surface of the capsid, there are three-fold propeller-like protrusions, star-shaped five-fold plateaus (called mesa), and depressions (called canyons) surrounding each plateau [ ] . the canyon, often contains the enterovirus receptor-binding site and binding at the canyon results in a cascade of structural changes [ ] , ultimately leading to the release of the n-terminus of vp and vp [ ] to form the expanded s cell-entry intermediate (also called a-particle) [ ] . high-resolution structures of cvb-vlps have thus far remained unavailable. as such, here, we aimed to determine the native structure of cvb -vlps by cryoem and image reconstruction. structural information provides the possibility to engineer both the stability of the particle and it's immunogenic properties. we also compared the vlp structure resolved in this study to the published cvb and cva whole-virus structures to identify possible differences between the mature virus and the vlp. our previously produced cvb -vlp vaccine induced a robust neutralizing antibody response in mice, however this result was obtained by utilizing a strong freund's adjuvant [ ] which is not allowed for human use. therefore, we studied the immunogenicity of non-adjuvanted cvb -vlp vaccine in mice. the baculoviral transfer vector poet , containing separate cassettes for the cvb vp - capsid region under the control of the polyhedrin promoter and the cd-protease under a cmv promoter control was ordered from geneart (regensburg, germany). cmv promoter acts as a weak promoter in insect cells, decreasing the toxic effects by the cd protease [ ] . the fully sequenced cvb strain (genbank accession number m . ) was used as a template. the recombinant baculovirus was produced according to the flashbac baculovirus expression system manual utilizing the flashbac ultra baculovirus genome. baculovirus working stock titer was determined using bacpak baculovirus rapid titer kit (clontech, mountain view, ca, usa). cvb -vlps were produced in high-five insect cells with a multiplicity of infection (moi) value of . the vlp containing production medium was clarified by mixing it with g/l sartoclear dynamics lab filter aid (diatomaceous earth) and then filtering it through a . µm filter. cvb -vlps were concentrated from the culture supernatant by tangential flow filtration (tff), utilizing mwco hollow fiber with an Äkta flux system. the buffer was exchanged to mm tris ph . , mm mgcl , mm nacl, and . % tween with the same system, and the impurities were removed from the preparation using a hiload / q sepharose high performance anion exchange column (ge healthcare). the vlps were captured into cimmultus so cation exchange chromatography column (biaseparations) and were eluted using nacl gradient (pure vlp eluted with~ mm nacl). purified vlps were analyzed in mini-protean tgx stain-free precast gradient gels ( - %) (biorad, finland). vlp proteins and impurities were assessed by densitometry analysis of sds-page gels using the imagej software [ ] . vp and vp proteins were detected by western blotting using in-house produced rabbit anti-cvb - polyclonal antibody [ ] and irdye-labeled secondary antibody. determination of vlp total protein concentration and dynamic light scattering (dls) analysis were performed as described [ ] . the thermal stability of the vlp particle was characterized by differential scanning fluorimetry (dsf) as described in [ ] . the vlp was also characterized by transmission electron microscopy (tem). formvar coated and carbon evaporated copper grids were made hydrophilic by uv-c irradiation at nm ( . mwatts/cm ) for min. then, µl of vlp ( . mg/ml) was added on the grid and incubated for s, after which the excess vlp was removed by blotting with whatman mm paper. the vlps were negatively stained through the addition of µl % uranyl acetate on the grid for s which was also removed by blotting. the grids were dried overnight and imaged with a f s/tem (jeol) transmission electron microscope. data collection was performed using a jeol jem f tem operating at kv. briefly, a . µl of cvb -vlp containing solution with final concentration of . µg/ml was placed on quantifoil r / cu mesh grids. after a . -min incubation time, . µl of np detergent was added for s. the excess solution was removed by automated blotting and quickly plunged into liquid ethane. the frozen-hydrated cvb -vlps were transferred into the em using a gatan cryo-transferring system. the specimen was examined at , magnification and images were captured using a ccd camera with a pixel size of . Ångstroms using serialem autofocusing software with defocus range set to . - . Ångstroms. digital images with no stigmatism or drift were selected for further analysis. cryo-em micrographs of cvb -vlp were processed to generate a dataset for single particle analysis (spa). semi-automated particle selection [ ] was employed to select a total of particles for refinement and reconstruction. iterative de novo initial models were generated to determine and cross-validate the particle parameters. to optimize the d alignment and orientation determination during refinement, a polar fourier transform (pft) method [ ] with radial range pushed to Ångstroms beyond the particle edge was used in conjugation with a center search. the particle stack was further refined by euler/center, defocus, scale-factor, and astigmatism screenings using jspr. an iterative d refinement was carried out using the jspr package [ ] . to determine the final resolution of the d volume, the dataset was divided into two halves (even and odd numbered particles) [ , ] . using eotest program from jspr/eman packages [ , ] , the final resolution of the cvb -vlp was determined to be . Ångstroms based on the . fourier shell correlation cut-off (data not shown). male c bl/ j-mice ( - weeks old) were vaccinated by interscapular subcutaneous injections (i.s.) on days , , and with µg of non-adjuvanted cvb -vlp vaccine (n = ) or with vaccine buffer (n = ). experiments with c bl/ j mice were conducted at karolinska institutet, stockholm, sweden in accordance with the nih principles of laboratory animal care and national laws in sweden and were approved by the local ethics committee (permission number s - ) . blood samples were collected prior to each immunization, on day and at the experimental end point (day ) from the tail vein or by terminal heart puncture (day ) and used to isolate serum. two-fold serial dilutions from : diluted sera of individual mice were analyzed in elisa for the presence of cvb -specific igg, igg and igg a antibodies (invitrogen, saint louis, mo, usa). then, -well maxisorp plates (nunc, fisher scientific, vantaa, finland) were coated with ng of cvb -vlps per well (room temperature, overnight) in pbs. after washing, the plates were blocked with . % bsa in pbs and the serum samples diluted in pbs containing % nacl, % bsa and . % tween were applied. horseradish peroxidase (hrp)-conjugated goat anti-mouse igg (sigma-aldrich, saint louis, mo, usa), igg or igg a (invitrogen, saint louis, mo, usa) and sigma fast opd substrate (sigma-aldrich, saint louis, mo, usa) were used for the detection of total igg or igg subtype responses. absorbance (optical density, od) was measured at nm in a microplate reader (victor , perkin elmer, turku, finland). the background signal (wells without serum) was subtracted from all od readings in the plate. a sample was considered positive if the net absorbance value was above the cut-off value calculated as follows (control mice mean od + × sd). serum titres below were considered negative. cvb neutralizing antibodies were measured as previously described [ ] . according to previous studies, uninfected animals that have not been vaccinated occasionally show a neutralizing antibody titre of : but not beyond this titre [ ] . previously, we have produced cvb -vlps [ ] and cvb -vlps [ ] in insect cells. cvb -vlps have been earlier produced using the bac-to-bac baculovirus-insect cell expression system, where the p polyprotein was expressed under polh promoter and cd protease was expressed under p promoter [ ] . co-expression of cvb p and cd led to p polyprotein cleavage into vp , vp and vp by cd and subsequent vlp assembly [ ] . however, the production yields of the vlps were moderate. subsequently we produced cvb -vlps and to enhance the vlp yield, we constructed a cvb -vlp construct, in which the p polyprotein expression was under the strong polh promoter and cd protease under the weaker cmv promoter [ ] . in the new design, the cd protease was produced in low levels, which decreases the toxic effects of cd on the host cells and allows the baculovirus-infected insect cells to produce higher amounts of vlp compared to the previous design. also, the baculoviral genome (flashbac ultra) utilized with the cvb -vlp production contains deletions in the non-essential baculoviral protease genes [ ] , which increases the cell stability and lowers the burden on the baculovirus-infected cells, allowing for a more efficient production of the target protein. another improvement made which enhanced the cvb -vlps production yield involved the selection of the insect cell line. cvb -vlps were produced in spodoptera frugiperda sf insect cells [ ] , whereas cvb -vlps were produced in trichoplusia ni high five insect cells [ ] , that have been shown to out-perform sf cells for vlp production yields [ ] . during the current study, we applied the same construct design, insect cell line and purification method for producing cvb -vlps as that which we have previously used to produce cvb -vlps [ ] . cvb -vlps produced in high five insect cells were extracted from the cell culture supernatants with tangential flow filtration and subsequently purified with anion and cation exchange chromatographic steps. first, the impurities were removed from the concentrated vlp with anion exchange chromatography. then, cvb -vlps were captured into the so cation exchange chromatography column and were eluted from the column with a linear nacl gradient. the vlps eluted from the column at a nacl concentration of~ mm into two fractions ( figure a ). sds-page analysis and the subsequent detection of proteins with a stain-free staining method ( figure b , left panel) revealed three prominent proteins of approximately kda, kda, and kda corresponding to the cvb capsid proteins vp , vp and vp . vp and vp capsid proteins were also detected with rabbit anti-cvb - polyclonal antibody in western blot analysis ( figure b , right panel). according to tem-analysis, the particles were intact and had the correct morphology and an average size of nm ( figure c ). dls analysis showed, that the first and the second fraction contained % particles with average hydrodynamic diameters of . (± . ) nm and . (± . ) nm respectively and the samples were very monodisperse (polydispersity indexes, pdi: fraction : . ± . , fraction : . ± . ) ( figure d ). these sizes were comparable with the cvb -vlps [ ] and cvb -vlps [ ] we studied previously. differential scanning fluorimetry (dsf) was used to analyze the thermal stability of cvb -vlp. as we have shown previously for cvb -vlp, the thermal stability of cvb -vlp was lower than that of native viruses and comparable to the thermal stability of cvb -vlp [ ] . two melting temperatures (tm) were determined for cvb -vlp, tm ( . • c ± . ) that can be assigned to the initial capsid unfolding and expansion and tm ( . • c ± . ) that can be assigned to the denaturation of the vlps ( figure e ). (e) thermal stability profile of cvb -vlp with differential scanning fluorimetry (dsf). fluorescence intensity of the dye in the presence of vlps was plotted as a function of the temperature, and melting temperatures (tm) of the vlps were derived from the inflection points of the transition curve using the boltzmann equation [ ] . the data is shown as an average of three independent measurements ± standard deviation. high-resolution structural information is available for several enteroviruses such as cvb [ ] . however, the structure of cvb-vlps has remained unavailable. cryo-em images of cvb -vlp revealed spherical particles of uniform size with on average, a diameter of ~ nm (cryo-em field view in figure a and contrast inverted selected particles in figure b ). the overall threedimensional structure of cvb -vlps (resolved here by cryo-em single particle analysis) is similar to cva -vlp, ev-a -vlp, and cva s particles and is ~ nm larger than the mature virion of ev-a and cva , as well as the cvb x-ray structure [ ] [ ] [ ] [ ] [ ] . using a cross-correlative circular averaging technique introduced by cheng et al. in [ , ] , the icosahedral particles were sorted by a scale factor based on statistical measures ( figure c ). to ensure a homogeneous population for single particle analysis, particles with a diameter larger than ~ nm and smaller than ~ nm were withdrawn from the analysis; these particles were generally broken and/or collapsed, with irregular shape and size. the remaining set of images were further sorted based on euler orientations (θ, ω, Φ) into two-dimensional class averages ( figure d ), from which an iterative de nova initial model (e) thermal stability profile of cvb -vlp with differential scanning fluorimetry (dsf). fluorescence intensity of the dye in the presence of vlps was plotted as a function of the temperature, and melting temperatures (tm) of the vlps were derived from the inflection points of the transition curve using the boltzmann equation [ ] . the data is shown as an average of three independent measurements ± standard deviation. high-resolution structural information is available for several enteroviruses such as cvb [ ] . however, the structure of cvb-vlps has remained unavailable. cryo-em images of cvb -vlp revealed spherical particles of uniform size with on average, a diameter of~ nm (cryo-em field view in figure a and contrast inverted selected particles in figure b ). the overall three-dimensional structure of cvb -vlps (resolved here by cryo-em single particle analysis) is similar to cva -vlp, ev-a -vlp, and cva s particles and is~ nm larger than the mature virion of ev-a and cva , as well as the cvb x-ray structure [ ] [ ] [ ] [ ] [ ] . using a cross-correlative circular averaging technique introduced by cheng et al. in [ , ] , the icosahedral particles were sorted by a scale factor based on statistical measures ( figure c ). to ensure a homogeneous population for single particle analysis, particles with a diameter larger than~ nm and smaller than~ nm were withdrawn from the analysis; these particles were generally broken and/or collapsed, with irregular shape and size. the remaining set of images were further sorted based on euler orientations (θ, ω, Φ) into two-dimensional class averages ( figure d ), from which an iterative de nova initial model generation was carried out. the resolved cvb -vlp cryo-em structure has been deposited into worldwide protein data bank with id: emd- . from the cryo-em density map, the mesa (star-shaped plateau at five-fold axis), the canyon surrounding the five-fold axis, and propeller-like features at the three-fold axis are clearly resolved ( figure e ). rigid-body fitting of ev-a (pdb: vbf) revealed that the two adjacent alpha helices from vp do not fit into the density map. a depression at the two-fold icosahedral axis is observed, which illustrates an open conformation at the junction channel and is similar to cva -vlp, ev-a -vlp, and cva s particles. after detecting the above mentioned similarity of cvb -vlp to cva s particles, we further characterized the differences between cva mature virion and the cvb -vlps by carrying out computer simulations for molecular dynamic flexible fitting of atomic structures (using imodfit [ ] and pdb c w [ ] ) to calculate the expansion of the cvb -vlps. we observed that the cvb -vlp diameter is about . nm on average, in contrast to . nm of the cva mature virion, an . % increase in the overall diameter ( figure f-g) . generation was carried out. the resolved cvb -vlp cryo-em structure has been deposited into worldwide protein data bank with id: emd- . from the cryo-em density map, the mesa (star-shaped plateau at five-fold axis), the canyon surrounding the five-fold axis, and propeller-like features at the three-fold axis are clearly resolved ( figure e ). rigid-body fitting of ev-a (pdb: vbf) revealed that the two adjacent alpha helices from vp do not fit into the density map. a depression at the two-fold icosahedral axis is observed, which illustrates an open conformation at the junction channel and is similar to cva -vlp, ev-a -vlp, and cva s particles. after detecting the above mentioned similarity of cvb -vlp to cva s particles, we further characterized the differences between cva mature virion and the cvb -vlps by carrying out computer simulations for molecular dynamic flexible fitting of atomic structures (using imodfit [ ] and pdb c w [ ] ) to calculate the expansion of the cvb -vlps. we observed that the cvb -vlp diameter is about . nm on average, in contrast to . nm of the cva mature virion, an . % increase in the overall diameter ( figure f-g) . the immunogenicity of the cvb -vlp vaccine was tested in five c bl/ j-mice by immunizing the animals on three occasions (days , and ) with µg non-adjuvanted vaccine by i.s. injection. serum samples were collected on days , , , , and . control mice (n = ) received vaccine buffer alone. sera of c bl/ j-mice immunized with cvb -vlp vaccines or the vaccine buffer were analyzed for cvb -specific serum igg, igg and igg a days after the prime vaccination. all mice were positive for cvb -specific igg by elisa with geometric mean titer (gmt) ( figure a ). cvb -specific igg and igg a immunoglobulin subtypes which are indicators of th and th type immune responses were also studied. cvb -vlp vaccine primarily induced primarily a th -type (igg ) oriented response with gmt , whereas the th -type (igg a) gmt was in the sera from vaccinated animals ( figure a) . sera from cvb -vlp immunized mice were also analyzed for cvb neutralizing ability in vitro ( figure b ). all vaccinated mice generated neutralizing antibodies by day (after one vaccination) with gmt . after three vaccinations, the neutralizing gmt increased to (p < . ). the immunogenicity of the cvb -vlp vaccine was tested in five c bl/ j-mice by immunizing the animals on three occasions (days , and ) with µg non-adjuvanted vaccine by i.s. injection. serum samples were collected on days , , , , and . control mice (n = ) received vaccine buffer alone. sera of c bl/ j-mice immunized with cvb -vlp vaccines or the vaccine buffer were analyzed for cvb -specific serum igg, igg and igg a days after the prime vaccination. all mice were positive for cvb -specific igg by elisa with geometric mean titer (gmt) ( figure a ). cvb -specific igg and igg a immunoglobulin subtypes which are indicators of th and th type immune responses were also studied. cvb -vlp vaccine primarily induced primarily a th -type (igg ) oriented response with gmt , whereas the th -type (igg a) gmt was in the sera from vaccinated animals ( figure a) . sera from cvb -vlp immunized mice were also analyzed for cvb neutralizing ability in vitro ( figure b ). all vaccinated mice generated neutralizing antibodies by day (after one vaccination) with gmt . after three vaccinations, the neutralizing gmt increased to (p < . ). cvbs have constantly been among the top enteroviruses most commonly reported to the cdc by diagnostic laboratories in the us that cause significant morbidity. currently there are no treatments or vaccines available against cvbs, but the clinical development of a polyvalent formalininactivated whole-virus cvb vaccine is in progress [ , ] . thus, there is a clear need to develop new vaccine technologies against cvbs. in addition to the traditional inactivated vaccine technologies, new vlp technologies can offer solutions for cost-effective manufacturing and epitope engineering of future cvb vaccines. previously, we have produced cvb -vlp [ ] and cvb -vlp [ ] vaccines in insect cells and purified those with ion exchange chromatography. in the present study, our aim was to enhance the cvb -vlp yield by utilizing a similar expression construct design and purification method to that employed for the cvb -vlp [ ] . as with the production of cvb -vlp, here, we utilized a baculoviral (flashbac) genome containing deletions in the non-essential protease genes [ ] and the cd protease was produced in low levels by utilizing a cmv promoter. these changes reduce the toxic effects of baculoviral and [ ] enteroviral [ ] proteases on the production cvbs have constantly been among the top enteroviruses most commonly reported to the cdc by diagnostic laboratories in the us that cause significant morbidity. currently there are no treatments or vaccines available against cvbs, but the clinical development of a polyvalent formalin-inactivated whole-virus cvb vaccine is in progress [ , ] . thus, there is a clear need to develop new vaccine technologies against cvbs. in addition to the traditional inactivated vaccine technologies, new vlp technologies can offer solutions for cost-effective manufacturing and epitope engineering of future cvb vaccines. previously, we have produced cvb -vlp [ ] and cvb -vlp [ ] vaccines in insect cells and purified those with ion exchange chromatography. in the present study, our aim was to enhance the cvb -vlp yield by utilizing a similar expression construct design and purification method to that employed for the cvb -vlp [ ] . as with the production of cvb -vlp, here, we utilized a baculoviral (flashbac) genome containing deletions in the non-essential protease genes [ ] and the cd protease was produced in low levels by utilizing a cmv promoter. these changes reduce the toxic effects of baculoviral and [ ] enteroviral [ ] proteases on the production cell line. in the cvb -vlp concentration step, the peg-precipitation method used previously was replaced with tangential flow filtration and the impurities were removed from the concentrated vlp by anion exchange chromatography. the final cation exchange chromatographic capture and concentration step was performed as we have described previously [ , ] . the optimizations in the construct design, baculoviral genome, and purification method accounted for the enhanced production yield and purity of cvb -vlp, and furthermore, this purification method is also a process that is compatible with industrial scale production. since the construct design, production cell line and purification method utilized here differ to those used for the previously produced cvb -vlp [ ] , it is impossible to address how much each change in the production process improved the final yield of the pure cvb -vlp. however, we reached our objectives. firstly, the developed purification method is scalable and utilize techniques generally used in industrial settings. secondly, based on the characterization of the pure cvb -vlp and the polydispersity index (pdi) determined by dls, both pure cvb -vlp fractions were very monodisperse, demonstrating that compared to the previously developed purification method [ ] , cvb -vlps that were more homogenous were successfully purified. thirdly, the obtained yield after purification was approximately . mg/l, which is three times higher than the yield of cvb -vlp in our previous study [ ] . an atomic-resolution structure of cvb virus was resolved by x-ray crystallography years ago [ ] , however, similar structures of other cvbs are lacking. also, the structures of cvb-vlps have been unresolved until now. in the present study we determined a high-resolution cryo-em three-dimensional structure of cvb -vlp. the capsid surface structures characteristic for enteroviruses, i.e., mesa (star-shaped plateau at five-fold axis), the canyon surrounding the five-fold axis, and propeller-like features at the three-fold axis were clearly resolved from the vlp structure. according to the structure, cvb -vlps are~ nm larger than the mature virion of cvb [ ] and the size difference is also similar to that seen when comparing ev-a and cva viruses with their respective vlps [ ] [ ] [ ] [ ] [ ] . we found that the cvb -vlp diameter is about . nm on average, which is very similar to the size of cva -vlp, ev-a -vlp, and cva s particle (the expanded cell-entry intermediate, also called a-particle) [ , , ] . the structure of cvb -vlp axis closely resembles that of the expanded enterovirus a-particle. the enlargement of the two-fold axis is associated with mature virion-to-uncoating intermediate states [ ] . the fenestration of viral a-particles near the two-fold axis has been shown for several native enteroviruses including echovirus and ev-a [ , ] . we saw a similar folding in the cvb -vlp. in the a-particle, vp and the rna-genome are still in the capsid and the particle is primed for infecting cells. once the vp protein and the genome have been expulsed, the resulting empty capsid is referred to as empty particle ( s) [ ] . heating of the native enterovirus capsid results in the formation of both a-and empty-particles [ ] . however, in physiological conditions enterovirus particles resemble the a-particles [ , ] . since the structure of cvb -vlp resembles that of a-particle, the vlp may closely resemble the form of mature virion in physiological conditions. poliovirus capsid structure have been stabilized by heat selected mutants [ ] and such mutations have been applied to generate poliovirus vlps with better immunogenicity [ ] . the importance of the solved cvb -vlp structure is speeding up rational engineering of the cvb particle to be more stable by introducing point mutations and thus improving its immunogenicity similarly to poliovirus vlps. most licensed vlp-vaccines contain adjuvants in their vaccine formulas [ ] and to the best of our knowledge, most immunogenicity studies with ev-vlps have also included adjuvants [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . adjuvants improve the quality, magnitude, and duration of the immune response, and generally, it is thought that vaccines composed of vlps alone may not sufficiently trigger the innate immunity response to the level required for optimal stimulation of the adaptive immune system [ ] . however, since we observed that nonadjuvanted cvb -vlp produced a clear neutralizing immune reaction in our previous study [ ] , we wanted to explore the immunogenicity of cvb -vlps in c bl/ j mice without the use of adjuvants. we hypothesized that vlps should be able to induce robust immune responses, since the vlp structures present a repetitive arrangement of antigens [ ] , making them immunogenic without the need for immunomodulatory substances. cvb infections induce rapid and strong neutralizing antibody responses in mice [ , ] and humans [ ] and the protective efficacy of vaccines correlates well with the magnitude of the antibody response [ , , , , ] . in our previous studies with inactivated cvb whole-virus vaccines [ ] and with cvb -vlp vaccine [ ] high total igg and neutralizing antibody responses were induced in c bl/ j male mice with non-adjuvanted vaccines and the responses were primarily th -type (igg ) oriented. here, the immune response of cvb -vlp was like the previously measured immune response of inactivated whole-virus vaccines [ ] and cvb -vlp vaccine [ ] in c bl/ j male mice demonstrating the immunogenic potential of non-adjuvanted cvb -vlp. in our previous study with cvb -vlp, the immunogenicity of the vlp was studied in balb/c mice in the presence of freund's adjuvant [ ] . the total igg and neutralizing antibody responses in the previous study [ ] were slightly higher than the antibody responses obtained here without adjuvant, but it is important to note that the previous and the current experiment represent independent studies and the results obtained from different mice strains should not be compared quantitatively to each other. the structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic vlp-vaccines in the future. recent progress in understanding coxsackievirus replication, dissemination, and pathogenesis a kidnapping story: how coxsackievirus b and its host cell interact two coxsackievirus b outbreaks associated with hand, foot, and mouth disease in china and the evolutionary history worldwide coxsackievirus b is associated with induction of beta-cell autoimmunity that portends type diabetes coxsackievirus b infections are associated with the initiation of insulin-driven autoimmunity that progresses to type diabetes developing a vaccine for type diabetes by targeting coxsackievirus b rationale for enteroviral vaccination and antiviral therapies in human type diabetes a hexavalent coxsackievirus b vaccine is highly immunogenic and has a strong protective capacity in mice and nonhuman primates the problems with polio: toward eradication the current status of the disease caused by enterovirus infections: epidemiology, pathogenesis, molecular epidemiology, and vaccine development recombination among picornaviruses vaccination with coxsackievirus b virus-like particles elicits humoral immune response and protects mice against myocarditis coxsackievirus b vlps purified by ion exchange chromatography elicit strong immune responses in mice non-clinical safety assessment of repeated intramuscular administration of an ev-a vlp vaccine in rabbits virus-like particle-based vaccine against coxsackievirus a protects mice against lethal infections a virus-like particle vaccine for coxsackievirus a potently elicits neutralizing antibodies that protect mice against lethal challenge a virus-like particle vaccine confers protection against enterovirus d lethal challenge in mice a virus-like particle-based tetravalent vaccine for hand, foot, and mouth disease elicits broad and balanced protective immunity formalin treatment increases the stability and immunogenicity of coxsackievirus b vlp vaccine enteroviral proteases: structure, host interactions and pathogenicity the structure of coxsackievirus b at . å resolution human enterovirus uncoating captured at atomic resolution cell-induced conformational change in poliovirus: externalization of the amino terminus of vp is responsible for liposome binding studies on the in vitro uncoating of poliovirus enhanced enterovirus virus-like particle yield from a new baculovirus design an open-source platform for biological-image analysis optimized production and purification of coxsackievirus b vaccine and its preclinical evaluation in a mouse model eman : an extensible image processing suite for electron microscopy a model-based approach for determining orientations of biological macromolecules imaged by cryoelectron microscopy single particle cryo-electron microscopy and -d reconstruction of viruses cauliflower mosaic virus: a subunit (t = ), multilayer structure outcome of the first electron microscopy validation task force meeting a coxsackievirus b vaccine protects against virus-induced diabetes in an experimental mouse model of type diabetes genetic modification of a baculovirus vector for increased expression in insect cells trichoplusia ni cells (high five) are highly efficient for the production of influenza a virus-like particles: a comparison of two insect cell lines as production platforms for influenza vaccines the use of differential scanning fluorimetry to detect ligand interactions that promote protein stability a . -angstrom resolution cryo-electron microscopy structure and antigenic sites of coxsackievirus a -like particles the enterovirus a-particle forms a gateway to allow genome release: a cryoem study of picornavirus uncoating a sensor-adaptor mechanism for enterovirus uncoating from structures of ev structures of coxsackievirus a capsids with native antigenicity: implications for particle expansion, receptor binding, and immunogenicity picornavirus uncoating intermediate captured in atomic detail efficient and robust flexible fitting based on vibrational analysis in internal coordinates structure, immunogenicity, and protective mechanism of an engineered enterovirus -like particle vaccine mimicking s empty capsid extracellular albumin and endosomal ions prime enterovirus particles for uncoating that can be prevented by fatty acid saturation fatty acid-depleted albumin induces the formation of echovirus a particles increasing type poliovirus capsid stability by thermal selection genetically thermo-stabilised, immunogenic poliovirus empty capsids; a strategy for non-replicating vaccines adjuvant formulations for virus-like particle (vlp) based vaccines enterovirus d virus-like particles expressed in pichia pastoris potently induce neutralizing antibody responses and confer protection against lethal viral infection in mice novel chimeric virus-like particles vaccine displaying mers-cov receptor-binding domain induce specific humoral and cellular immune response in mice a virus-like particle based bivalent vaccine confers dual protection against enterovirus and coxsackievirus a infections in mice virus-like particles induce robust human t-helper cell responses a link between a common mutation in cftr and impaired innate and adaptive viral defense subclass restriction of human enterovirus antibodies a comparative study of the effect of uv and formalin inactivation on the stability and immunogenicity of a coxsackievirus b vaccine key: cord- - p n tjx authors: lambe, teresa; bowyer, georgina; ewer, katie j title: a review of phase i trials of ebola virus vaccines: what can we learn from the race to develop novel vaccines? date: - - journal: philos trans r soc lond b biol sci doi: . /rstb. . sha: doc_id: cord_uid: p n tjx sporadic outbreaks of ebola virus infection have been documented since the mid-seventies and viral exposure can lead to lethal haemorrhagic fever with case fatalities as high as %. there is now a comprehensive body of data from both ongoing and completed clinical trials assessing various vaccine strategies, which were rapidly advanced through clinical trials in response to the – ebola virus disease (evd) public health emergency. careful consideration of immunogenicity post vaccination is essential but has been somewhat stifled because of the wide array of immunological assays and outputs that have been used in the numerous clinical trials. we discuss here the different aspects of the immune assays currently used in the phase i clinical trials for ebola virus vaccines, and draw comparisons across the immune outputs where possible; various trials have examined both cellular and humoral immunity in european and african cohorts. assessment of the safety data, the immunological outputs and the ease of field deployment for the various vaccine modalities will help both the scientific community and policy-makers prioritize and potentially license vaccine candidates. if this can be achieved, the next outbreak of ebola virus, or other emerging pathogen, can be more readily contained and will not have such widespread and devastating consequences. this article is part of the themed issue ‘the – west african ebola epidemic: data, decision-making and disease control’. the - epidemic of ebola virus disease (evd), in west africa, was unprecedented in both scale and spread. the case count and death toll has surpassed the total number of cases for all previous known outbreaks. this epidemic has caused more than deaths with more than cases: figures widely thought to be underestimated [ ] . development and deployment of efficacious evd therapeutics and vaccines could have played a more significant role in limiting the - outbreak and remain a priority for the prevention of future evd epidemics [ ] . the family filoviridae includes marburg and five ebolaviruses, named for their location of recognition: zaire (now known as ebola virus, ebov), sudan (sudv), reston (restv), taï forest (tafv) and bundibugyo virus (bdbv) [ ] . the single protein expressed on the surface of filoviruses, the glycoprotein (gp), is antigenically and immunologically important and is frequently therapeutically targeted. however, bioinformatics analysis, at the structure-based and amino acid level, demarcates limited regions of homology in gp [ ] and also large areas of divergence particularly in the head domain of gp. it is therefore unclear if exposure to one member of the family filoviridae confers cross-protective immunity. it is unclear if the immune response involved in ebola virus clearance is dependent on a single aspect of the immune system (cellular or humoral immunity) or if a multifaceted response is a prerequisite and at present there is no clear correlate of protection for evd. indications from experimental infection of vaccinated non-human primates suggest a role for gp-specific igg and cd þ t cells [ , ] , and the limited analyses of convalescent samples suggest that the presence of ebola virus-specific igg and intact cell-mediated immunity (cmi) are associated with survival from natural infection [ ] [ ] [ ] . these observations are supported by immunological analysis of survivors from the current outbreak, which indicates a multi-faceted response is induced post-infection with strong humoral immunity and significantly pronounced transcriptional changes in cd þ t cells in response to several ebola virus proteins [ ] . evd vaccine development has been well established since the discovery of ebola virus and a number of different vaccine platforms have been used, resulting in at least one efficacious vaccine. these vaccine platforms include dna, recombinant or subunit proteins, virus-like particles (vlps) and recombinant viral vectors. a number of these vaccines have advanced past pre-clinical testing and are undergoing clinical testing; generally, those modalities that have demonstrated efficacy or high levels of immunogenicity in pre-clinical models. the most clinically advanced vaccines against evd are based on generating immune responses toward gp with trials ongoing in europe, the usa and africa. there are eight vaccines in clinical trials, all targeting the ebola virus gp, with some regimens employing a single dose and others using two vaccines in a heterologous prime-boost approach. prime-boost regimens have been shown to be more immunogenic than single-dose vaccinations for diseases such as malaria; however, there are financial and logistical implications associated with administering two vaccines [ ] . the ebola virus vaccines that are currently being assessed differ in the qualitative immune response postvaccination, which may be due to the use of alternate vaccine platforms. indeed, while humoral immunity may be critical for protection post vesicular stomatitis virus (vsv)-vectored vaccines in non-human primates (nhps), cmi may be key post single-shot adenovirus vectored vaccines [ ] . as both humoral and cellular immunity have been demonstrated to be protective in nhp, and are induced in man post-ebov infection, a vaccine regimen which induces long-lived and sustainable levels of cmi and antibodies is desirable. primarily, and finally, only those vaccines that are safe, efficacious and deployable will be field-effective, therefore only vaccine candidates that have progressed to published phase i studies will be reviewed here. clinical trials where results were published in peer-reviewed journals by july are summarized in table . ebolavirus disease the earliest clinical studies of vaccines against evd used plasmid dna encoding the nucleoprotein from the zaire ebolavirus and the glycoproteins from ebov and sudv [ , ] . although well tolerated with mostly mild and short-lived adverse events (aes), dna vaccines tend to be poorly immunogenic and have been largely superceded by subunit proteins, recombinant viral vectors and vlps in several vaccine development fields, including ebola virus [ ] . the vaccines most progressed for the prevention of evd use viral vector platform technology and use either the adenovirus, modified vaccinia virus ankara or vesicular stomatitis virus backbones. recombinant human serotype adenovirus (adhu ) vectors encoding gp have been evaluated in single-dose studies and have shown very acceptable safety profiles. unfortunately, adhu -seropositive volunteers demonstrated statistically significantly lower gp-specific igg titres [ ] . increasing the administered dose of vaccine appears to overcome pre-existing immunity to this serotype; however, this is associated with increased reactogenicity [ ] . unfounded concerns also still persist about a potential increase in hiv- infection rates among adhu -seropositive vaccinees, based on data from two phase ii studies of the merck rad hiv- gag/pol/nef vaccine (the step and phambili trials) [ ] [ ] [ ] . use of rarer serotypes of human adenoviruses, such as ad and adhu , can bypass pre-existing immunity [ ] and have demonstrated acceptable safety and tolerability profiles. the most widely evaluated adenoviral vector evd vaccines are based on a simian adenovirus serotype backbone (chad ) encoding either ebolavirus glycoprotein alone [ , ] and administered as a single shot or as a mixture of chad ebov and chad sudv vaccines [ ] . between september and january , four phase i studies of these vaccines were undertaken, one each in the us, uk, switzerland and mali. replication-deficient chimpanzee adenovirus vectors have been extensively evaluated as candidate vaccines for malaria, hiv, hcv, rsv, influenza and tuberculosis, in adults, children and infants, with no safety concerns [ ] . safety profiles were similar for candidate ebola virus vaccines, with higher doses eliciting more aes; however, aes were mostly graded as mild, resolving within h of vaccination. an additional vaccine modality being tested is modified vaccinia ankara (mva); both unmodified and recombinant mva vectored vaccines have been evaluated extensively as vectors for smallpox, malaria, flu and tb vaccines with excellent safety demonstrated in immunocompetent and hiv-infected participants [ , ] . in pre-clinical trials, chad was observed to provide % protection when nhps were challenged with ebola virus shortly after vaccination; however, an mva boost was required to enable uniform protection when challenge occurred months after priming vaccination. this increased durability of protection was attributed to the superior effector and memory cd þ t cell responses elicited by the mva boost [ ] . owing to the superior durability of protection demonstrated by the prime-boost regimen in preclinical trials, several phase i clinical trials were initiated to test this regimen in humans. mva-bn filo is a recombinant, replication-deficient mva vector, encoding ebov, sudv and marburg glycoproteins as well as tafv nucleoprotein. two clinical trials have assessed a chad prime with mva-bn filo boost, demonstrating acceptable safety profiles and significantly enhanced cellular and humoral immunogenicity compared with a single-shot chad vaccination [ , ] . the other vaccine modality being assessed as an ebola vaccine candidate is the recombinant vesicular stomatitis virus encoding ebov glycoprotein (rvsv-zebov), which is an attenuated replication-competent viral vector. unmodified vsv causes either asymptomatic infection or a mild, flu-like illness in humans [ ] ; however, truncation of the vsv glycoprotein results in an attenuated, avirulent form that has been shown to be safe in pre-clinical studies in macaques and mice with no evidence of viral shedding [ ] . a rvsv hiv- gag vaccine has also generated acceptable safety data in two phase i studies [ , ] . candidate vaccines using these vectors also elicited protection against both ebola and marburg viruses in a nhp model where several rvsv each encoding a single filovirus glycoprotein were mixed and injected in a single dose that elicited protection against a lethal challenge [ ] . six phase i studies of rvsv-zebov were initially performed between october and january , two in the us and one each in kenya, gabon, switzerland and germany [ , ] . almost all participants displayed transient rvsv viraemia, starting at day , and lasting beyond day in around % of participants, with mostly mild and moderate aes lasting up to h on average. in total, % ( / ) of participants across the six trials had documented fever after vaccination, significantly higher than the % observed after vaccination with chad ( / , p ¼ . , two-tailed fisher's exact (prism version , graphpad software)). although both vaccines have been evaluated across a range of doses, post-immunization fever levels are an important consideration for a vaccine that may be deployed in an outbreak of a highly febrile illness, such as evd. in the geneva trial, of participants developed arthritis lasting a median of days, with virus subsequently identified in synovial joint fluid, indicating peripheral viral replication; two cases of shorter duration were identified among participants in the other studies. a further study in geneva where a much lower dose of rvsv-zebov was evaluated in a further volunteers resulted in much lower total igg and neutralizing antibody titres and was again associated with arthritis in % of participants [ ] . transient arthritis is often observed after immunization with commonly used live vaccines, such as rubella [ ] ; however, when rvsv-zebov progressed to a large-scale phase iii cluster-randomized efficacy trial, this particular adverse event was not reported and, in addition, in an interim analysis the vaccine elicited high levels of efficacy in evd-exposed participants [ ] . incidence of post-vaccination fever in that study has not yet been reported. in summary, all of the candidate vaccines for evd that progressed to phase i studies in response to the - outbreak demonstrated acceptable safety profiles; therefore, describing the magnitude and characteristics of vaccine-induced immunity was the next scientific aim. in the absence of any gold standard for measuring humoral immunity against ebola virus, a wide variety of assays were used to evaluate antibody responses to vaccines during the recent evd outbreak. binding assays were used to quantify antibody binding to recombinant protein or inactivated whole virion while neutralization assays were used to assess the functional capacity of antibodies. binding assays, particularly enzyme-linked immunosorbent assays (elisas), are commonly used to assess the quantity of antigen-specific antibody induced post-vaccination and can additionally be used to measure qualitative aspects of the humoral immune response such as isotype profiles and in vitro avidity. ebolavirus outbreaks occur sporadically and have previously been considered too limited in size to enable assessment of vaccine efficacy prior to regulatory approval. in , the usa food and drug administration (fda) introduced the 'animal rule' enabling data from pre-clinical trials to be used to demonstrate efficacy when human trials were not possible as an alternative licensing route for drugs and vaccines against highly lethal diseases [ ] . despite the wide use of elisa assays to measure antiglycoprotein igg responses, the majority of phase i studies used an array of different antibody binding assays to assess the quantity and quality of the antigen-specific antibody response induced by vaccination (table ). the use of different assays conducted in different laboratories following different protocols hinders the comparison of humoral immunogenicity induced by different vaccine candidates. key differences in these assays that affect comparability of results include the use of glycoprotein from different strains of zaire ebola virus (kikwit, makona or mayinga), use of whole virion or recombinant protein and the use of different read-outs or units. centralized standard assays or readily available biological standards for a range of emerging pathogens would be useful tools to aid decision makers in choosing the most promising candidate to take forward for further testing and rapid deployment during outbreaks. a who reference reagent for anti-ebov igg has been established by the who expert committee on biological standardisation (ecbs) for use as a reference standard in humoral immunoassays including neutralization and elisas against ebola virus [ ] . it is now freely available from the national institute for biological standards and control (nibsc, uk) and should facilitate retrospective comparison of responses between different trials. to aid in the comparison of immunogenicity across trials, samples from a recent phase i trial of chad mva-bn filo [ ] were run on a large number of these assays and correlation analyses were conducted (table ) . a lack of correlation between some of these assays may indicate that they measure different aspects of the humoral immune response and may therefore correlate differently with protection. for example, the competition elisa used by ewer and co-workers did not correlate with neutralizing titre to live ebola virus mayinga strain or several of the glycoprotein elisas. the competition elisa measures the ability of anti-ebov antibodies in a sample to compete with the monoclonal ebov antibody (mab) g and therefore only detects activity against a single gp epitope. the lack of a correlation with neutralizing titre may indicate that antibodies binding to this epitope are non-neutralizing and that the protection that has been observed when this mab has been used in a mab cocktail in nhp models is conferred by an alternative mechanism [ ] . many of the other binding assays used in these phase i trials correlate strongly and, in particular, it is useful that the standardized glycoprotein elisa and pseudotyped lentivirus assays each correlate strongly with neutralizing titres against live ebola virus as these assays avoid the need to work at high containment levels, but may be used to indicate the presence of antibodies with neutralizing activity. in a pre-clinical nhp model, igg responses after immunization with adhu -based ebola virus vaccines were measured using an elisa against gp, where % protection against a lethal challenge was predicted by titres of or greater, while a titre of around predicted % survival. at an interim analysis of the phase iii ring vaccination study of rvsv-zebov in guinea estimated vaccine efficacy in this trial to be % [ ] . no immunogenicity data were published for this trial, due to logistical difficulties associated with collecting biological samples, therefore, it is unclear which components of the immune response to vaccination mediated this protective effect. however, phase i trials of the same vaccine at the same dose, in the usa, induced igg against zaire-mayinga gp with a geometric mean titre (gmt) of ( % ci: - ) [ ] at day post-vaccination, which is probably higher than the level achieved - days post-vaccination by which time no new cases were observed in the rvsv-zebov ring vaccination trial. a gmt of is well below the titre required to protect % of non-human primates in the experimental model. chad zebov given at a dose of  viral particles (vp) induced titres comparable to those induced by rvsv-zebov, with day gmts of ( % ci: - ) in us adults and ( - ) in malian adults [ ] . peak titres after chad is boosted with mva zebov were significantly increased to ( - ) [ ] . although the use of different assays (summarized in table ) limits comparison of immunogenicity across clinical trials, there is still much to be learnt from comparisons of groups within trials. desantis and co-workers showed a limited dose effect on anti-gp igg titres, as assessed through elisa, when chad ebov was tested at .  vp and  vp. peak titres and response rates were similar in these two groups both at peak and at day , indicating that durability was also comparable [ ] . however, in a trial by ledgerwood et. al., which compared a mixture of chad zebov and chad sudv in a : ratio, each at  or  vp, indicated that a -fold higher dose induced significantly higher geometric mean elisa titres of anti-gp igg despite similar response rates [ ] . in the same trial, % of volunteers in the high dose group developed antibody responses against the vaccine strain of zaire glycoprotein (mayinga), while only % developed responses against glycoprotein from the outbreak strain (guinea). additionally, the response rates induced against the vaccine strain sudan glycoprotein ( % in the high dose group and % in the low dose group) were lower than for zaire ( and %) despite receiving the same dose of each. this may reflect the different sensitivities of the two assays. the rapid development of multiple vaccine candidates in parallel during the recent outbreak has highlighted the need for standardization of assays that measure humoral immunogenicity. correlating responses measured by different assays and the inclusion of reference standards can aid comparisons of the quality and quantity of the humoral response induced by different vaccine candidates and support decisions on which to take forward for further development. there is a lack of discrimination between neutralizing and non-neutralizing antibody levels through elisa output and while non-neutralizing antibodies play an important role in curbing infection, there is also a concerted effort to identify neutralizing antibodies (nab) as a key determinant of infection control in many disease settings [ , ] . at present, there are data suggesting that both neutralizing and non-neutralizing antibodies are important in the evd setting and as such measuring both aspects of humoral immunity through elisa and neutralization assays will be informative. virus neutralization assays (e.g. fluorescent antibody virus neutralization (favn) assay or plaque reduction neutralization test (prnt)) can measure nab responses. however, there is a prerequisite for highly trained staff and access to containment level facilities to work with ebolavirus, which are not widely available: for example, a single laboratory based in marburg analysed samples from most of the phase i studies. additionally, these assays necessitate careful and considered planning resulting in low-throughput, expensive and timeconsuming assays to prevent accidental exposure [ , ] . to circumvent these restrictions, assays using non-pathogenic, replication-defective pseudotyped viruses (pvs) have been developed that facilitate the study of highly pathogenic viruses in standard containment level laboratories. pvs are chimeric virions, which comprise the structural and enzymatic core of one virus with a heterologous envelope protein, e.g. ebov table . relationships between different assays used to assess humoral responses to ebola vaccine candidates. spearman's rank and p-values for correlations between each of the assays tested. the same samples were run on each assay. samples were serum from healthy uk volunteers in a phase i trial of chad _mva ebo z conducted at the university of oxford [ ] . all samples were from the same time point-two weeks after mva boost. adi elisa, pseudotyped lentivirus neutralization assay and standardized elisa were carried out at the university of oxford, competition elisa at phe, neutralization assay and whole virion elisa at institute for virology, philipps university, marburg, and the nih elisa at the nih. glycoproteins [ ] . typically, retroviruses (lentiviruses and gammaretroviruses) or rhabdoviruses (vesicular stomatitis virus) are used as pseudotype cores. transduction of permissive target cells, facilitating genome transfer and subsequently reporter protein expression, allows a quantifiable read-out of transduction and produces a quantitative read-out. however, pre-incubation of pv with nab which bind an envelope protein, and can inhibit cell entry, will result in a lower level of quantifiable reporter protein expression. there are many pv assays which have been developed to assess neutralizing antibodies toward the filovirus family members and indeed a number of these assays have been used in a who collaborative study to establish a reference standard for antibodies to ebola virus [ ] . the data from this study suggest that pv assays, while offering an obvious improvement to live nab assays, may generate false positives and as such any new filovirus pv assay requires stringent standardization using validated negative and positive controls. there have been a limited number of pv assays used in recent clinical trials, which may reflect the urgent need to rank vaccine modalities during the - ebola outbreak and the concerted effort to screen using live neutralization assays. however, there are a number of informative comparisons between live ebov neutralization and pv neutralization during some of the more recent clinical trials which will help establish the usefulness and early application of these assays in screening and ranking vaccine candidates (table ) . in , neutralizing antibodies, as measured with a singleround infection assay with ebov gp-pseudotyped lentiviruses, were measured following a single im vaccination in a phase i clinical trial of adhu vaccine expressing ebov gp antigen. with the exception of one subject, sera did not inhibit virus entry [ ] . the same team also used ebov or marv gp-pseudotyped lentiviruses to measure neutralizing antibody activity after or dna vaccinations. no significant marburg virus neutralizing activity was observed following the dna vaccines, and only low-level ebov neutralizing activity was measured following the fourth dna vaccination [ ] . these data have helped evaluate vaccine regimens and have identified those vaccines and regimens which yield improved neutralizing responses. in a recent phase i clinical study in oxford, seven different assays of humoral immunity were undertaken on samples from vaccinees, providing a unique opportunity to assess the relationship between different measures of immunogenicity (table ) . volunteers were administered a single dose of chad ebov gp and a subset were then administered a booster dose of a mva strain, encoding the same ebola virus glycoprotein. two assays were used to measure neutralizing antibodies; direct neutralization of live ebov (mayinga strain) and a pseudotyped lentivirus expressing the glycoprotein from the mayinga strain, with a read-out of % inhibitory concentration (ic ). neutralizing antibody titres to live ebov (mayinga strain) were low at days after the chad dose while the levels were significantly higher days after the mva boost vaccine. again, low-level inhibition of infection was observed post-prime, using a pseudotyped lentivirus expressing the gp from the mayinga strain of ebov, and the levels of nab increased significantly post-boost. importantly, neutralizing antibody titres in the live ebov and pv assay correlated positively with each other (r ¼ . , p ¼ . ) as did titres measured by elisa and live ebov (mayinga strain) neutralization assay (r ¼ . , p ¼ . ) [ ] . nab were also assayed in vaccinees who received rvsv-zebov, through the use of vsv pseudovirions expressing the glycoprotein from the ebov kikwit strain, and a live neutralization assay with infectious ebov isolate mayinga. nab have been detected in vaccinees receiving as little as  pfu rvsv-zebov, and the two nab assays showed significant increases in neutralizing antibodies after all doses of rvsv-zebov (ranging from  pfu to  pfu) [ ] . a strong correlation between antibody titres as assessed with glycoprotein elisa and vsv pseudovirions or live virus neutralization titres were demonstrated. a significant correlation between vaccine dose and neutralizing antibody titres was also identified using the vsv pseudovirions but such correlations were not observed using whole virions or infectious ebola virus. this may reflect the lower sensitivity and thus weaker discriminatory capacity of whole virions or infectious ebola virus assays. the increased interest in measuring nab post vaccination toward highly pathogenic viruses and the suggested increased sensitivity in established and standardized pv assays will underpin the continued development and use of pv assays for broad-spectrum detection of post-vaccination humoral responses. baize and co-workers analysed samples from patients in two large outbreaks in in gabon and compared cellular and humoral responses between survivors and patients who succumbed to infection to determine the relative contribution of different immune parameters to evd outcome [ ] . survival was associated with early and rapid rises in igg directed mainly toward the viral nucleoprotein, in addition to activation of cd þ t cells with upregulation of cd , fasl, perforin and ifng. cd þ t cells specific to the ebola virus nucleoprotein are also associated with protection in a mouse model of evd [ ] and t cells induced by rad zebov gp vaccination in nhp afford protection in a lethal challenge model [ ] , while in vivo depletion of cd þ t cells using a monoclonal antibody abrogated protection in four out of five nhp, whereas passive transfer of igg did not induce protection. therefore, enumeration of antigen-specific t cell responses to candidate ebola vaccines is an important aspect of the immunological outcomes for studies in humans. many clinical trials of replicationdeficient viral vectored vaccines have reported t cell responses as measured by flow cytometry with intracellular cytokine staining (ics) using pools of peptides spanning the glycoprotein to stimulate peripheral blood mononuclear cells (pbmc). comparisons across studies are impeded by differences in sample types used (freshly isolated versus cryopreserved pbmc), variation in denominator used to report responses (memory versus total cd þ ) and differences in data analysis methodology [ , , , ] . some groups have also reported glycoproteinspecific t cell responses by ifng elispot in addition to ics; however, again differences in responses between fresh and frozen pbmc make broad comparisons difficult. although comparisons between all the trials are unreliable, limited comparisons are possible; e.g. when using fresh pbmc with ifng elispot, radhu and chad vectors encoding ebov gp induced antigen-specific t cell responses of a similar magnitude [ , ] . for the vaccine regimen where chad vectors encoding ebov and sudv gp were mixed, only the rstb.royalsocietypublishing.org phil. trans. r. soc. b : responder frequency and not the magnitude of response was reported; however, responder frequency to the mixture of vectors was similar to that observed to chad ebov gp alone. however, and possibly more importantly, comparisons between groups within the same study have yielded interesting insights into the use of viral vectors that have broader relevance beyond ebola virus vaccines. for example, a large trial in oxford comparing mva-bn filo priming and adhu zebov boosting demonstrated for the first time in humans that priming with the mva vector and boosting with an adenovirus is at least as effective at inducing antigen-specific cd þ t cells as the conventional delivery of ad-prime followed by mva boost [ ] . surprisingly, the rvsv-zebov vaccine, which to-date has been the only vaccine for which field efficacy has been assessed, has not yet been described for t cell-inducing capability [ , ] . in cynomologous macaques immunized with rvsv-zebov, cytokine-secretion from cd þ t cells stimulated with gp peptides was not detectable after immunization; however, responses were detected in vaccinated animals after ebov challenge, suggesting that immunization may have primed a response [ ] . if t cell responses to the rvsv-zebov have been assessed in phase i studies, then the absence of cmi would be of interest to the vaccine development field as maintenance of humoral immunity is obviously dependent on cmi, yet the failure to detect such a response peripherally is both surprising and interesting from the immunologist's perspective. as the immediate response to the - evd outbreak in west africa begins to wind down, scientists and policy makers involved in vaccine development can reflect on both the progress made by the field in this difficult time as well as implications for future outbreaks. notable gains and successes have been achieved, including rapid approval and completion of clinical studies for both existing and newly produced candidate evd vaccines which underpins the capacity of both ethical and regulatory authorities to expedite approvals in the context of public health emergencies. publication of data from studies undertaken in response to the outbreak has also been rapid with many journals setting aside policies relating to prior disclosure of data to allow data sharing with who and other bodies, without prejudice of future publication. that vaccines against evd existed at all is partly due to north american biodefense priorities, rather than a planned strategy of preparedness for rapid response to emerging pathogens. the efficacy of the rvsv-zebov vaccine in the phase iii trial in guinea in the context of a primarily iggmediated response suggests that an efficacious evd vaccine may be comparatively easy to produce, compared with efforts to generate vaccines for diseases such as malaria, hiv and tuberculosis. indications from the nhp vaccine model with rad ebov gp and analysis of immune responses in evd survivors indicated that a cd þ t cell component would be a prerequisite of an effective evd vaccine. yet the rvsv-zebov vaccine may not induce a significant cmi component based on pre-clinical data. an additional consideration is that passive transfer of ebola-specific antibody can fail to protect nhps from lethal ebola virus challenge, indicating that antibody titres may be a nonmechanistic correlate of protection and that qualitative aspects of the antibody response and/or cellular immunity that are not readily measured also play key roles in protection [ ] . it is likely that the mechanism of protection and protective level of a particular immune component may well differ in an experimental model in nhps, challenged in a manner that may not accurately reflect natural exposure and in humans exposed to natural infection. this highlights the importance of not being over-reliant on immunological correlates derived from pre-clinical testing. clearly, different vaccine delivery methods induce qualitatively different immune responses; induction of long-lasting humoral immunity in the absence of t cells seems unlikely, so the ability of prime-boost regimens to induce durable cmi and humoral immunity may be desirable for longevity of protection. in december , who held a workshop on the prioritization of pathogens, generating a list of seven diseases requiring urgent r&d, with a further three diseases requiring action as soon as possible [ ] . this list encompasses emerging diseases with potential to generate a public health emergency and for which no, or insufficient, preventative or curative solutions exist. crimean congo haemorrhagic fever (cchf), evd, marburg haemorrhagic fever, lassa fever, mers, sars, nipah and rift valley fever virus were identified as priorities for emerging pathogen research, with zika, chikungunya and severe fever with thrombocytopaenia syndrome (sfts) determined to be serious and necessitating further action as soon as possible. this evd outbreak has focused attention on emerging pathogens, shifting their place in the public consciousness from a theoretical emergency to a very tangible threat to global health. vaccine developers must now think in terms of addressing emerging pathogens as an entirety rather than a collection of individual highly virulent pathogens. to this end, the recent outbreak has yielded some valuable insights. firstly, in the context of an ongoing outbreak, a single dose of a viral vector vaccine could be sufficient to induce at least short-term protective immunity against filoviruses. this can usefully protect front-line healthcare workers and communities, thereby reducing the spread of an epidemic in the earliest stages [ ] . where the outbreak pathogen is readily identified a single dose of a recombinant adenoviral or vsv vector may suffice for outbreak control. however, to counter the threat of infectious outbreaks which may emerge unpredictably, large-scale prophylactic immunization of healthcare workers would be a useful first line of defence. a single-dose multivalent vaccine encoding antigens from several putative emerging pathogens could be particularly valuable in such a scenario. for example, recombinant mva would be suited to this purpose, given the potential to insert various foreign genes at numerous sites in the genome under a number of promoters [ ] , in contrast with adenovirus vectors that have a limited cloning capacity to carry foreign genes. immunity could subsequently be boosted with an adenoviral vector expressing a specific transgene, once the outbreak pathogen is identified. a study of an evd vaccine regimen employing mva as a priming vector and adhu as a boost has demonstrated that this is a feasible approach [ ] . speculatively grouping rstb.royalsocietypublishing.org phil. trans. r. soc. b : emerging pathogens according to geographical incidence may be one strategy for determining components of multivalent vaccines and an example of a potential strategy for designing vaccines using this approach is suggested in figure . almost all of the current evd vaccine candidates use gp as the antigen due to abundant expression on the surface of the virus, and there is extensive pre-clinical data reflecting promising immunogenicity and efficacy against challenge for this target antigen. studies of patients with evd in two outbreaks in gabon revealed that in survivors, igg responses were largely directed against nucleoprotein [ ] . the same observation was made in a small study of evd cases in the usa [ ] . characterization of immune responses in natural ebolavirus infection have also revealed that exposure can induce cross-reactive antibodies capable of neutralizing multiple ebolavirus species [ ] . this study of naturally acquired immunity in survivors of a bdbv outbreak in uganda in demonstrated for the first time that potent neutralizing antibodies to the glycan cap of gp could inhibit several ebolaviruses, including sudv, and protect guinea pigs from a heterologous challenge with ebov which may offer insight into immunogen design to confer heterosubtypic immunity across multiple ebolaviruses. given the very large number of survivors of the - outbreak, significant efforts should be made to characterize the immunity that afforded protection in the face of infection with such a highly lethal pathogen, to help inform antigen selection for future vaccines and advance our understanding of post-evd immunology. whether vaccines need to emulate these naturally occurring mechanisms remains to be seen, but these observations are of critical importance to help advance the field. closer examination of the priority pathogens highlighted by the who may facilitate development of broad-spectrum vaccines; viruses with small genomes that have obvious vaccine candidate antigens or where pre-clinical data are highly indicative [ ] . in this setting, data from the one health approach, which combines human, animal and environmental considerations to address global health challenges, are invaluable; for example, immunogenicity data derived from studies in zoonotic reservoirs can be applied to vaccine development [ , ] . furthermore, studies of families of viruses using technological advances such as cryo-electron microscopy, b cell cloning and antibody repertoire sequencing may yield novel targets that may be effective for tackling emerging pathogens. phylogenetically related viruses that share receptor-binding domains, and epitopes within envelope proteins may well share therapeutic and vaccine targets [ ] . as discussed above, great efforts have been made to characterize the magnitude of neutralizing antibody titres induced by vaccination, yet the detail of these entry-blocking mechanisms has not yet been fully utilized by vaccinologists. detailed understanding of host-pathogen interactions has yielded numerous opportunities for vaccines against other diseases, including malaria and hiv [ ] . in-depth understanding of the processes associated with inhibition of receptor binding, such as conformational rearrangement of glycoproteins required for viral fusion, could possibly identify ubiquitous targets for mabs that can prevent infection of multiple virus species [ ] : for example, the entry of all filoviruses into mammalian cells utilizes the same endosomal receptor [ ] , the niemann-pick c protein, presenting an obvious target for inhibition of virus entry. outbreaks of highly pathogenic diseases such as evd are devastating for the populations affected and will always focus attention on control strategies to prevent future occurrences. while it is widely acknowledged that the global public health response to the - was tragically slow to begin, immunologists and vaccinologists now have a valuable opportunity to learn as much as possible from this disaster. as well as probing the vaccine-induced responses from the many clinical trials that have taken place, detailed characterization of naturally acquired immunity in both survivors of evd and asymptomatic, ebov igg-seropositive members of communities should be systemically compiled. previous data from gabon have illustrated that substantial proportions of rural populations have evidence of ebov-specific immunity [ ] . we now have a collective responsibility to assimilate all the information that this outbreak has generated in order to be best placed when the next epidemic comes, so that we can respond effectively and robustly to curb an outbreak in its infancy. who. situation report: ebola virus disease emergency ebola response: a new approach to the rapid design and development of vaccines against emerging diseases virus nomenclature below the species level: a standardized nomenclature for filovirus strains and variants rescued from cdna structural basis for marburg virus neutralization by a cross-reactive human antibody chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge cd þ cellular immunity mediates rad vaccine protection against ebola virus infection of nonhuman primates defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in ebola virusinfected patients camouflage and misdirection: the full-on assault of ebola virus disease human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis human ebola virus infection results in substantial immune activation progress with viral vectored malaria vaccines: a multi-stage approach involving 'unnatural immunity immunology of protection from ebola virus infection a dna vaccine for ebola virus is safe and immunogenic in a phase i clinical trial a replication defective recombinant ad vaccine expressing ebola virus gp is safe and immunogenic in healthy adults safety and immunogenicity of dna vaccines encoding ebolavirus and marburgvirus wild-type glycoproteins in a phase i clinical trial safety and immunogenicity of ebola virus and marburg virus glycoprotein dna vaccines assessed separately and concomitantly in healthy ugandan adults: a phase b, randomised, double-blind, placebo-controlled clinical trial chimpanzee adenovirus vector ebola vaccine-preliminary report a monovalent chimpanzee adenovirus ebola vaccine boosted with mva a recombinant vesicular stomatitis virus ebola vaccine-preliminary report safety and immunogenicity of a chimpanzee adenovirus-vectored ebola vaccine in healthy adults: a randomised, double-blind, placebo-controlled, dose-finding, phase / a study use of chad -ebo-z ebola virus vaccine in malian and us adults, and boosting of malian adults with mva-bn-filo: a phase , single-blind, randomised trial, a phase b, openlabel and double-blind, dose-escalation trial, and a nested, randomised, double-blind, placebocontrolled trial phase trials of rvsv ebola vaccine in africa and europe-preliminary report safety and immunogenicity of novel adenovirus type -and modified vaccinia ankara-vectored ebola vaccines: a randomized rstb.royalsocietypublishing.org phil safety and immunogenicity of a novel recombinant adenovirus type- vector-based ebola vaccine in healthy adults in china: preliminary report of a randomised, double-blind, placebocontrolled, phase trial the effect of dose on the safety and immunogenicity of the vsv ebola candidate vaccine: a randomised double-blind, placebo-controlled phase / trial efficacy assessment of a cell-mediated immunity hiv- vaccine (the step study): a double-blind, randomised, placebocontrolled, test-of-concept trial preferential infection of human ad -specific cd t cells by hiv in ad naturally exposed and recombinant ad -hiv vaccinated individuals recombinant adenovirus type hiv gag/pol/nef vaccine in south africa: unblinded, long-term follow-up of the phase b hvtn / phambili study recombinant adenovirus serotype (ad ) and ad vaccine vectors bypass immunity to ad and protect nonhuman primates against ebolavirus challenge viral vectors as vaccine platforms: from immunogenicity to impact recombinant modified vaccinia virus ankara-based malaria vaccines safety and immunogenicity of modified vaccinia ankara-bavarian nordic smallpox vaccine in vaccinia-naive and experienced human immunodeficiency virus-infected individuals: an open-label, controlled clinical phase ii trial vesicular stomatitis virus in panama. human serologic patterns in a cattle raising area recombinant vesicular stomatitis virus-based vaccines against ebola and marburg virus infections live virus vaccines based on a vesicular stomatitis virus (vsv) backbone: standardized template with key considerations for a risk/benefit assessment first-in-human evaluation of the safety and immunogenicity of a recombinant vesicular stomatitis virus human immunodeficiency virus- gag vaccine (hvtn ). open forum infect. dis. , ofv single-injection vaccine protects nonhuman primates against infection with marburg virus and three species of ebola virus absence of an association between rubella vaccination and arthritis in underimmune postpartum women efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial correlates of protective immunity for ebola vaccines: implications for regulatory approval by the animal rule who collaborative study to assess the suitability of an interim standard for antibodies to ebola virus mechanism of binding to ebola virus glycoprotein by the zmapp, zmab, and mb- cocktail antibodies lyophilisation of influenza, rabies and marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation-assay-based diagnostic kit which antibody functions are important for an hiv vaccine? front management of accidental exposure to ebola virus in the biosafety level laboratory development of a fluorescent antibody virus neutralisation test (favn test) for the quantitation of rabiesneutralising antibody retroviral pseudotypes-from scientific tools to clinical utility protection from ebola virus mediated by cytotoxic t lymphocytes specific for the viral nucleoprotein human adenovirus-specific t cells modulate hiv-specific t cell responses to an ad -vectored hiv- vaccine live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses passive immunization of ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses blueprint for r&d preparedness and response to public health emergencies due to highly infectious pathogens report of the sage working group on ebola vaccines and vaccination with provisional recommendations for vaccination cross-reactive and potent neutralizing antibody responses in human survivors of natural ebolavirus infection chimpanzee adenovirus vaccine provides multispecies protection against rift valley fever emerging zoonotic viral diseases zoonoses and one health: a review of the literature inhibitory antibodies targeting emerging viruses: advancements and mechanisms a pfrh -based vaccine is efficacious against heterologous strain blood-stage plasmodium falciparum infection in aotus monkeys broadly neutralizing alphavirus antibodies bind an epitope on e and inhibit entry and egress ebola viral glycoprotein bound to its endosomal receptor niemann-pick c high prevalence of both humoral and cellular immunity to zaire ebolavirus among rural populations in gabon authors' contribution. all authors contributed to the design, drafting and revision of the article and approved the final version for publication.competing interests. we have no competing interests. funding. k.j.e. is funded by a horizon award from the european union. g.b. is funded by the wellcome trust. t.l. is funded by the mrc. key: cord- - ynsgu c authors: heldens, j.g.m.; patel, j.r.; chanter, n.; ten thij, g.j.; gravendijck, m.; schijns, v.e.j.c.; langen, a.; schetters, th.p.m. title: veterinary vaccine development from an industrial perspective date: - - journal: vet j doi: . /j.tvjl. . . sha: doc_id: cord_uid: ynsgu c veterinary vaccines currently available in europe and in other parts of the world are developed by the veterinary pharmaceutical industry. the development of a vaccine for veterinary use is an economic endeavour that takes many years. there are many obstacles along the path to the successful development and launch of a vaccine. the industrial development of a vaccine for veterinary use usually starts after the proof of concept that is based on robust academic research. a vaccine can only be made available to the veterinary community once marketing authorisation has been granted by the veterinary authorities. this review gives a brief description of the regulatory requirements which have to be fulfilled before a vaccine can be admitted to the market. vaccines have to be produced in a quality controlled environment to guarantee delivery of a product of consistent quality with well defined animal and consumer safety and efficacy characteristics. the regulatory and manufacturing legislative framework in which the development takes place is described, as well as the trend in developments in production systems. recent developments in bacterial, viral and parasite vaccine research and development are also addressed and the development of novel adjuvants that use the expanding knowledge of immunology and disease pathology are described. the industrial development of a veterinary vaccine starts once the proof of concept has been established. proof of concept is based on robust academic exploration of fundamental science. the scientific literature has reported many academic proofs of various novel vaccine concepts in recent years. industrial vaccine development programs (see table for its phases) starts once this proof of concept has been demonstrated and should eventually lead to market authorisation (ma), i.e. the permission to place a vaccine on the market, which is granted by the appropriate veterinary authorities. in contrast to academic proof of concept demonstrations, the industrial development of a vaccine should be seen in an economic context. veterinary vaccines are produced and marketed by pharmaceutical companies which are in competition with one another. for veterinary vaccines there are two main target markets that can be recognised each with its own dynamics. these markets are ( ) the agricultural or production animal sector and ( ) companion animals. in recent decades the general trend has been that the agricultural sector has become more industrial and price sensitive. in contrast, the public perception of companion animals has changed leading to increased demand for medicinal care. in general, vaccines for production animals are produced in larger quantities at low cost, whereas vaccines for companion animals are produced in lower quantities and sold at higher prices. moreover, it should be realised that it is difficult for profit-making enterprises to justify the development of vaccines for diseases of low incidence or vaccines for species that are kept in low numbers (schetters, ) . apart from operational costs there is the expense of complying with the legal and regulatory requirements for ma and manufacturing. regulatory requirements are under continuous review and vaccines which could be registered years ago may not be registrable today, owing to changes in the regulatory framework. furthermore, regulatory requirements are sometimes difficult to fulfil for a particular vaccine concept. vaccines are produced on an industrial scale in government certified manufacturing facilities. the good manufacturing practice (gmp) requirements, which have been incorporated into law, describe in detail the manufacturing standard. since the introduction of veterinary medicinal product licensing in europe some years ago, the economic environment, the increasing legal and regulatory pressure and the changed agricultural scenery have lead to a marked consolidation among the veterinary vaccine manufacturers. in this review, we first outline the regulatory and manufacturing legal context in the eu. second we discuss developments in production methods, and third we describe concepts of viral, bacterial and parasitic vaccines and immune potentiators. vaccines for animals, like all other medicinal products have to be licensed by the relevant authorities who have to ensure that the medicine is of adequate quality and purity, that it is safe and that it works in the target species as claimed for the indication for which it is intended (jones et al., ; schetters and gravendyck, ) . licensing of veterinary vaccines was formally introduced in the eu in (cd / /eec; cd / / eec; cd / /eec; cd / /eec; eudralex ph. eur, ) . all the information and test results required by the guidelines (cd / /eec; cd / /eec; cd / /eec; cd / /eec; ph. eur, ) , which demonstrate quality, purity, safety and efficacy are compiled in a dossier. the ma is based on this dossier after review and further questioning. the gathering of all the required information and test results on the vaccine (vaccine development) is usually organised in structured and well phased manner. the various development phases that can be distinguished are summarised in table and a flow chart of the key stages of the development is given in fig. . regulations may of course change during the course of the development and may have an impact on development time and cost. before marketing the vaccine, a manufacturing license and a gmp certificate of each facility involved in the manufacturing of the vaccine are required, in addition to a ma license. here we describe briefly quality, safety, efficacy and gmp (manufacturing) requirements for veterinary vaccines. soulebot et al. ( ) . to ensure consistent quality, the production method and technology underlying a vaccine must operate in compliance with the principles of current gmp, which is monitored by a quality assurance system (cd / /eec; eudralex). vaccine production under gmp is required by law (cd / /eec; eudralex). the quality of starting materials used for production is assured by testing each batch purchased or is made to ensure that it meets acceptance limits. additional requirements exist for starting materials of biological origin, which are commonly used for vaccine production (e.g. ensuring exclusion of extraneous agents). seed stocks of the vaccine strain and production cell-lines are laid down in a seed lot system, making passages from the master seed to establish a bank of working seed from which all production batches are produced. the initial master seed must have a known history of its isolation and previous passages to minimise the risk of transmission of transmissible spongiform encephalopathies (tses). in addition, the purity (absence of extraneous agents such as bacteria, fungi, mycoplasma and viruses) and identity of vaccine antigen needs to be demonstrated. to ensure that each batch of a commercial vaccine is equivalent in quality, the manufacturer must register all relevant in process tests as well as tests to be performed on the finished product, giving limits of acceptance that must be met before the batch can be released for sale. in addition, the manufacturer must prove that the quality of the vaccine when produced on a (semi) commercial scale, is guaranteed until the end of its shelf life based on at least three batches of vaccine in the final container. safety is of paramount importance. the determination of safety is fundamentally a firmness of purpose that the benefits of the product outweigh any potential risks, not only to the target species being vaccinated, but also to the administrator of the vaccine, the environment, the food test batch production (small scale): live vaccine: low passage lot for safety (glp) on target species including pregnant animals in case indication is required high passage lot for efficacy: onset of immunity and duration of immunity inactivated vaccine: high passage lot for safety (glp) and efficacy licensing batches ( % commercial scale, gmp) -consistency of production, process validation -transfer of production process and control tests to manufacturing departments and quality control departments -stability studies on antigen and final product in final container field studies (gcp) -safety -efficacy derived from treated animals from which food is derived, and the consumer. experimental data obtained with batches with the highest potency or titre (see below) must be generated in specially designed experiments carried out under the good laboratory practice (glp) regimen (cd / /eec; oecd ). in addition to the safety of a single dose, the safety of an overdose and repeated doses of the vaccine must be shown (such as injection site reaction, clinical signs). safety studies have to be designed in accordance with the recommended schedules and using susceptible target species. there are additional special requirements for live vaccines, such as (genetic) stability (i.e. the vaccine should not revert to virulence during consecutive passages) and the risks of genetic recombination and/or genomic reassortment should be minimal. also studies on the immunological functions or reproductive capacity may have to be carried out depending on the nature of the vaccine, adjuvant, or disease. additional requirements have to be met for vaccines containing genetically modified organisms (gmo). a product must be able to do what is claimed on the label (e.g. reduce virus shedding, limit typical clinical signs, disease) preferably, these data are obtained from field trials performed under good clinical practice-veterinary (vich-gl ) conditions and in laboratory studies in which, if possible, validated experimental challenge models are used. onset and duration of immunity, for instance a few days (onset) to year (duration) followed by a yearly vaccination to sustain the level of immunity, must be supported by experimental data. these data must show that year after the primary vaccination, animals are still satisfactorily protected and also that animals that receive a single booster vaccination year after initial vaccination are still protected year later. confirmation of the protective effect of a specific antigen is usually established by vaccination-challenge experiments. this protective effect is correlated to alternative laboratory animal or in vitro tests which are carried out on each batch of finished vaccine. the pass level for batch release is set at the minimum level that was shown to be efficacious in the target animal (correlate of protection). if the correlate of protection is unknown, it may take years before an accurate potency test can be developed. moreover the manufacturer must provide data that guarantee the immunising capability and thereby the protective effect of a product over its entire shelf life. in the eu, each ma is granted with an approved summary of product characteristics (spc) describing the pharmaceutical form of the product, the (categories of) target animal, contra-indications, recommended vaccination schedule and route of administration. moreover, the spc contains recommendations for use such as withdrawal period, use with other products and advice for minimisation of the risk of concurrent infections during the vaccination period, as these could interfere with the induction of the active immune response. since licensing of veterinary vaccines was formally introduced in the eu in it has been a requirement that both the active ingredient (antigen) and the finished product (vaccine) must be manufactured according to gmp (cd / /eec; cd / /eec; eudralex). apart from validated methods and tests, a preventive maintenance system should be in-place for all critical equipment and buildings. responsibilities of key personnel and key departments have to be documented in policy documents. trend analysis of different parameters (utilities, environmental monitoring) and in process controls (ipc) should provide useful information about process performance and quality. in general, all tests and processes must be validated and reported including buildings and equipment. moreover, every one of these quality related systems, procedures and policies have to be written down in a site master file. even during commercial production it can be necessary to make changes to the processes or to the tests that have been developed. with a full operational gmp quality system it is still possible to optimise the process and to make significant improvement changes. a change control system ensures that all changes are formally requested, documented and approved. the impact of these changes on the marketing authorisation and the validation status of the process need to be determined and documented in detail as this may have regulatory consequences. moreover, a vaccine producer must employ at least one qualified person who is, without prejudice to his relationship with the holder of the manufacturing authorisation, personally responsible for release of vaccine onto the market (cd / /eec; cd / /eec; cd / /ec). regular internal and external audits by competent authorities aim at surveying the quality control procedures inplace described here. the increasing regulatory, gmp and economic burden imposed on vaccine manufacturers necessitate the development of reliable, large-scale antigen production methods, such as the use of cell culture substrates. in recent years, the quest for highly productive cell culture processes has been focused on the front-end to continuous cell-line development rather than the use of primary cells such as chicken embryo fibroblasts (cef). the improvement in protein expression systems, high throughput screening methods and cell characterisation techniques have enabled the development of more productive and stable cell-lines used for antigen production. final product quality and consumer safety is already considered at cell-line development, i.e. at the very early stage of a vaccine development project. the development of and transition to animal-componentfree culture media for cells, the identification of suitable animal-free replacements for certain components such as bovine serum, and, ultimately, the development of fully chemically defined culture media, are examples of this consideration. with an increasing need for higher antigen yields in combination with shorter product development and production process times, sophisticated production process development methods are required in every stage of the process from early development to scale-up to large volumes. the current generation of miniature bioreactors, with working volumes of a few millilitres, modelling large-scale cell culture processes, provide the capability to examine different conditions within a single experiment. this offers the opportunity to understand the process at the cellular level and enables better and more predictable extrapolation to large-scale bioreactor processes. the establishment of analytical technologies enabling in-line monitoring of antigen production has the advantage of studying important immunogenic antigen properties such as glycosylation or cell metabolites influencing overall growth of cells and consequently antigen and contributes to the consistency, reliability and scalability of entire processes. early effort to produce proteins expressed by bacteria, yeast, and mammalian cells in commercial quantities required costly re-usable hardware. this involved an extensive cleaning and re-sterilisation processes and the validation of these processes as required by the rules set-out in gmp regulations. this longstanding approach placed heavy demands on standard operating procedures (sops), and usually resulted in high costs and extensive personnel training. the most significant costs involved in manufacturing are facility time (that can represent up to % of the total manufacturing costs of a plant) and validation (that can account for between % and % of a plant cost). these costs can be reduced through increasing facility throughput in terms of number of campaigns or runs per year, increasing the antigen yield per run and installing equipment that allows plants to operate both as multi-purpose and multiproduct production facilities. an option that is adopted more and more by biotech manufactures in an effort to reduce costs is moving to disposable manufacturing. ''use once, throw away" technology is used more and more by many manufacturers in various stages of production processes. it results in improvement in throughput by reducing the amount of downtime between campaigns, and it also allows the development of a multi-purpose plant design because of the flexibility in use of disposable systems in existing clean rooms. furthermore, the disposable individual components or whole systems can eliminate the long lead times in the initial manufacturing and installation of stainless steel equipment. the design of a disposable system is primarily dictated by the application and usually custom made. key factors to take into consideration during the design phase of such an application may be the volume to be processed, chemical compatibility with the product, the number of production runs per year, processing conditions (as pressure, temperature, flow rates, mixing times), sensitivity of the product to extractable materials, which are present in all plastics, and validation support from the vendors. currently, the most widely used components include containers, filtration, connections, clamps, bioreactors and tubing. nowadays many of the compounds in a biotechnology production suite can be supplied both in hard piped stainless steel or disposable systems. if components are already supplied pre-assembled and sterilised, significant reductions will be made in contamination risks, validation efforts and labour time. pre-assembled disposable components dramatically increase the manufacturer's dependence on its supplies and may jeopardise sustainable delivery of vaccine product ( table ) . as already indicated, disposable systems do contain extractables (components of the disposable material leaking into the product stream), which may interfere with antigen production or product safety. manufacturers of disposable systems undertake extractable testing using model solvents as part of their own validation protocols. however, system users must generate their own validation data to demonstrate that extractables do not adversely affect the product being stored or processed within or by the disposable. this is done with a worst-case exposure where the conditions are based on known characteristics of the product and the model solvent covering the entire disposable assembly. moreover, plastics are not, by definition, animal component-free and disposables also have to comply with tse guidelines ( table ) . the use of disposable technology still requires hardware and control panels that need initial validation and maintenance. some elements of current manufacturing processes, such as centrifugation, large-scale chromatography and large-scale tangential flow filtration systems are not yet available as disposable options. in-line or real time monitoring of production processes is still in a development stage and disposable sensor technology is also not yet available. this means cleaning in-place or discarding expensive re-usable sensors, or avoiding sensors all together, which makes the processes uncontrollable (table ) . recent improvements in chromatography and tff equipment, aseptic fluid transfer devices, as well as improvements on plastic film strength and resistance will lead eventually to the replacement of traditional stainless steel gmp manufacturing components with single-use disposable formats. thinking even further ahead, the development of a totally disposable manufacturing system can be imagined. when such disposable factories may become commonplace will depend largely on new disposable product developments and evolution of regulatory and gmp guidelines. the industry is adapting to these developments as shown by the introduction of short-term disposable mixing and monitoring systems. bacterial vaccines are mainly inactivated whole cell cultures (or bacterins), inactivated culture supernatants (or toxoids), crude extracts of the cell surface or attenuated live preparations. advances in our knowledge and biological technologies give the opportunity to develop improved vaccines. the inactivated crude preparations had the advantage that knowledge of the molecular basis of pathogenesis was not a prerequisite to vaccine development since there was a good chance they contained the antigens necessary to stimulate protective immunity. the disadvantages were the greater potential for antigenic competition and/or diversion so that immune responses to protective antigens (that were nonetheless present in the preparations) failed, and that they usually contained lipopolysaccharide, techoic acid or other cell wall constituents. these components led to the heavy stimulation of the innate immune systems so that undesirable adverse effects were likely. nonetheless, the traditional approaches remain a perfectly acceptable and an acceptable solution for certain diseases (andre-fontaine et al., ) . the early live vaccines had the advantages of the inactivated antigens combined with the potential for in vivo dependent gene expression to provide a more protective immune stimulus (feberwee et al., ) . the non-specific or unstable genetic alterations inherent in these vaccines could lead to suboptimal growth for adequate immune stimulus (i.e. over attenuated) or insufficient attenuation to prevent adverse reactions and reversions to virulence (i.e. under attenuated and in a reversible fashion). later, vaccine development took advantage of new studies of the molecular basis of bacterial pathogenesis. initially these depended on physicochemical separations of bacterial components and assessment of the interaction of these with isolated host tissues, cells and biochemistry. in addition, the effects of immunisations with purified preparations on the outcome of experimental challenges, and of the epidemiological distribution of the efficacious components amongst clinical and non-clinical isolates (moon and bunn, ) , were taken into account. these studies heralded the introduction of the first so-called subunit vaccines. later still the introduction of molecular cloning enabled the genetic isolation of characterised protective immunogens and in particular the construction of host/vector combinations with enhanced expression of the recombinant antigen to provide higher yields of less reactive vaccine components more cheaply (petersen et al., ) . commercially available vaccines developed by these methods were first introduced only in comparatively recent times. the arrival of whole bacterial genome sequencing and bioinformatic analyses (prediction of structure, function and cellular location from sequence) and the development of micro-array analysis of whole genome expression are bringing about a revolution in the design and development of bacterial vaccines. this is particularly the case for those diseases where the traditional approaches have not been efficacious (scarselli et al., ) . in simple terms, with all of the genes hypothetically identified for a bacterial pathogen it is possible to construct an array of sites on a solid sheet each containing bound synthetic dna complementary to all of the genes in the genome. the messenger rna (mrna) extracted from the bacteria living in different environments is used to prepare probes in strengths proportionate to the amounts of each mrna species present at the time of extraction. by detecting differences in the strength of probe hybridisation to the gene array it is possible to see which gene's levels of expression have changed relative to the different environments. genes upregulated in vivo, or in cultures designed to mimic in vivo environments, that bioinformatic analyses indicate are likely to be surface expressed or secreted during critical stages of host colonisation or pathogenesis (and hence accessible to interference from host immunity) enable the identification of attractive candidate vaccine antigens without necessarily knowing their function. it is then possible to delete the gene(s) in question to confirm their role in pathogenesis in challenge models and also to produce recombinant purified antigens to use in vaccination trials. provided the antigens are reasonably conserved amongst clinical isolates this strategy can provide a powerful route to efficacious vaccine development where crude preparations in the past have not achieved a desirable level of efficacy or safety. this technology now makes it possible to create live vaccines incapable of reversion to virulence, with minimal interference in the expression of important protective immunogens whilst inactivating powerful immune diverting immunogens that may interfere with the strength of efficacious responses. dna vaccination is a relatively recent consideration where genes encoding protective immunogens are inserted into non-replicating elements able to be taken up by host cells and initially express the foreign antigen intracellularly and then presented on the cell surface in association with mhc antigens to t cells. this offers the advantages ( ) that the th /th balance of immune responses can be modulated to achieve more effective immunity and ( ) that dna is highly stable at room temperature and easily quality assurance (qa) controlled during production (jechlinger, ) . unfortunately, for many projects the payload of dna required has been prohibitively expensive and new technologies are needed along the lines of adjuvants that enhance responses to antigens. one of the ironies of the molecular genomic approach is that protective subunit antigens and dna vaccines are often poor immunogens because they are not associated with other bacterial components present in crude preparations that non-specifically enhance immune responses, that is to say with inherent adjuvant properties. consequently, not only are new adjuvant strategies needed to make recombinant subunit or dna vaccines work, but bacteria may themselves provide the solution. most intensively investigated as adjuvants are cholera and escherichia coli heat labile adp-ribosylating enterotoxins (lycke, ) , the zonula occludens toxin of vibrio cholerae (de magistris, ), mycobacterium tuberculosis heat shock protein (bulut et al., ) , salmonella typhimurium fljb (simon and samuel, ) , synthetic analogs of bacterial lipoproteins (ghielmetti et al., ) , synthetic oligodeoxynucleotides containing unmethylated cpg dinucleotides (mccluskie and krieg, ; gomis et al., ) and monophosphoryl lipid a (jiang et al., ) . veterinary bacterial vaccine development can still call upon perfectly valid traditional techniques but we have only just started the era where bacterial behaviour from the gene to genome will allow us to predict what we need to do for successful control of disease. it is only a matter of time before efficacious vaccination could be a reality for almost any bacterial disease for which there is a market. viruses belonging to this subfamily cause significant diseases in horses, pigs, cattle and poultry. genomes of several members in the herpesvirus subfamily have been sequenced and molecular virology has played a significant role in the identification of virulence-associated, virus encoded genes and their functions (wittmann and rziha, ; mettenleiter, ; kimman et al., ) . this has led to the development of effective and safe, conventional as well as biotechnological marker vaccines (kit et al., ; marchioli et al., ; quint et al., ; pensaert et al., ; van oirschot, ) . the deletion mutants' biotech approach has been particularly successful for pseudorabies virus (prv) and bovine herpesvirus (bhv- ). in contrast, for equine herpesvirus- and - (ehv- ; ehv- ), deletion of non-essential virus glycoprotein and enzyme genes with the aim of deriving attenuated live virus vaccine candidates, similar to prv and bhv- , have been fruitless (patel and heldens, ) . there is, however, a clear need for improved ehv- vaccines, particularly to protect against ehv- induced abortion and paresis and also to reduce the incidence and therefore the transmission of ehv- and ehv- by unweaned passively immune foals (patel and heldens, ) . in this regard, a highly promising experimental ehv- vaccine was a temperature-sensitive (ts) strain cloned from a classically mutagenised stock of an abortigenic ehv- isolate (patel et al., a,b; patel et al., ) . marek's disease virus (mdv) occurs worldwide and is the cause of significant loss in chickens. mdv has been developed as a vector for newcastle disease virus glycoprotein recombinant vaccine. effective commercial marek's disease virus vaccines for poultry contain cell-associated, low passage variants of the original rispens isolate. quests to generate efficacious and safe cell-free mdv vaccines, using conventional or biotech approaches have been unsuccessful so far despite the veterinary medicinal and logistic need for such vaccine. porcine circovirus- (pcv- ) is a cause of multi-organ disease and significant economic loss in domestic pigs (allan and ellis, ; krakowa et al., ) . immunoprophylaxis against pcv- infection of domestic pigs is with conventional and biotech vaccines, such as a baculovirus expressed pcv-orf protein. additionally, there have been many biotech experimental pcv- vaccines investigated. examples of such vaccines are pcv- orf gene expressed in prv vector, an apathogenic pcv- /pcv- chimera vaccine, and dna plasmid vaccines (fenaux et al., ; kamstrup et al., ; chunmei et al., ) . routine prophylactic vaccination against classical swine fever virus (csfv) in the eu ceased in (westergaard, ) . outside the eu, conventional vaccines as well as biotech csfv-e glycoprotein subunit marker vaccine are widely used (lin and lee, ; van oirschot, ; de smit et al., ) . several biotech approaches have been investigated for csfv vaccines and include baculovirus expressed e glycoprotein (ahrens et al., ) , chimeric bovine virus diarrhoea virus (bvdv)-csfv vaccine (van gennip et al., ) and recombinant prv expressing csfv-e glycoprotein that was protective for diseases due to prv and csfv (van zijl et al., ) . some of these biotech vaccines are experimental but they allow serological discrimination of vaccinated and infected animals, allowing the use of csfv vaccination, where previously it has been banned. application of biotech approaches for protective bvdv vaccines include glycoprotein e expression and production in baculovirus and defective bvdv replicons. no biotech bvdv vaccines are currently available in the eu but there is a choice of live and killed conventional vaccines (straub, ) . however, only three killed vaccines claim to afford protection against bvdv abortion (brownlie et al., ; patel et al., ; salt et al., ) . over the last decade west nile virus (wnv), which causes significant disease in horses and other animal species including man, has become a potent threat. currently biotech vaccines marketed for use in horses are a canarypox virus vectored live vaccine and a chimerical wnv glycoprotein and yellow fever virus backbone vaccine (monath, ) . influenza a viruses cause greater problems in birds than mammals. in particular, some strains of h and h subtypes are highly pathogenic causing high mortality and pose a zoonotic risk to man along with some h subtypes. in the eu, vaccination against avian influenza is discouraged, but the emphasis is changing (capua and alexander, ) . the likely approach to develop vaccines would be, first, the cloning and site directed mutagenesis to turn the ha-gene into a non-pathogenic form, and, second, the production of so-called high growth re-assortants producing considerable amounts of the new ha protein, which is, among others, the protective antigen in influenza virus. the latter can be achieved by reverse genetics and transfection techniques (wood and robertson, ) . this has been the approach for the currently circulating and potentially pandemic h n avian influenza virus, which has caused more than human deaths so far. other biotech approaches, such as the cloning of the changed ha-gene into other viral vectors such as newcastle disease virus (ndv) can be carried out as well. reverse genetics has been used to alter the cleavage site of the ndv-f protein, but so far conventional live and killed vaccines dominate the markets. non-replicating vector avipoxvirus approaches, such as canarypox virus expressing canine distemper virus hf glycoprotein gene have been on the market in the usa since . modern biotech vaccine approaches do not always give better vaccines, however. for avian influenza h for instance, it has been shown in the field that classically grown, inactivated vaccines formulated with potent adjuvants can offer cross-protection against related pathogenic strains (swayne et al., ) . african swine fever (asf) is a highly contagious fatal disease of pigs caused by an iridovirus (asfv). an effective vaccine is clearly needed and there are indications that this is possible (mettraux et al., ) . commercial development of the approach has however never been undertaken for economic reasons or has been unsuccessful for reasons such as manufacturing difficulties. african horse sickness (ahs) is a highly fatal, insect transmitted disease of equidae caused by african horse sickness virus (ahsv), an orbivirus. currently, conventional mouse brain or tissue culture grown vaccines are in use but future vaccines may use ahsv vp protein as a subunit biotech vaccine (ranz et al., ) . as is the case for asfv, commercial development of the approach has never been undertaken for economic reasons or has been unsuccessful owing to manufacturing difficulties. for bluetongue virus (btv), whilst there are eggadapted polyvalent vaccines to prevalent serotypes in use, biotech approaches have been investigated which hold much promise. thus btv-like and virus core-like structures have been constructed using major (vp , vp ) and minor (vp , vp , vp ) capsid proteins. the vp proteins were produced in baculovirus multiple expression vectors. these virus-like single and double shelled particles emulsified in freund's incomplete adjuvant or montanide isa- adjuvants were highly immunogenic and protective for naïve sheep (roy, ) . the approach holds promise since baculovirus expression vectors for different btv serotypes could be prepared in advance and stored in a bank on similar lines to foot and mouth disease virus (fmdv; westergaard, ) and brought out when an outbreak occurs. conventional rabies virus vaccines, mostly killed, for immunising various mammalian species are in common use. rabies virus is one of the examples where biotech approaches have or are likely to make good progress. the latter is well exemplified by the attempts to reduce the incidence of wildlife rabies, mostly in the red fox (vulpes vulpes) in mainland europe. a replicating recombinant vaccinia virus expressing rabies virus glycoprotein has been highly effective in reducing the incidence of wildlife rabies in mainland europe pastoret and brochier, ; westergaard, ) . monovalent and subunit feline leukaemia virus (felv) glycoprotein expressed in e. coli is marketed in the eu. immunogenicity of an experimental vaccinia virus recombinant that expresses bovine leukaemia virus envelope protein (gp ) has given promising results in bovine calves and rabbits (valikhov et al., ) . it is important and relevant to point out that there are animal diseases for which there are no vaccines at present. the diseases affect domesticated and wild ruminants and deer for instance. other wildlife diseases, currently unknown or undetected, may be transmitted to domesticated animals and possibly to man. recent notable examples of animal viruses crossing into man are of retroviruses like hiv, sars coronavirus, west nile virus and avian influenza h n subtype virus. the gamma herpesviruses named alcephaline herpes virus- (ahv- ) and ovine herpesvirus- (ohv- ) cause fatal excessive lymphoid proliferation in secondary deadend secondary ruminant hosts, namely cattle and deer species (nettleton et al., ; reid and buxton, ; plowright, ; hussey et al., ) . ahv- and ohv- are innocuous in reservoir hosts, wildebeest and sheep, respectively. two retroviruses, one belonging to subfamily lentivirus (visna-maedi virus and caprine arthritis/encephalitis virus) and one to the subfamily oncovirus (sheep pulmonary adenomatosis virus), cause significant diseases in sheep, for which no vaccines are available. similarly, it is not possible at present to control some other veterinary retroviruses such as bovine leukaemia, avian type c oncovirus and reticuloendotheliosis virus. two new fatal zoonotic infections due to related paramyxoviruses, named hendra and nipah viruses, affect horses and pigs, respectively, and also man. the reservoir for both viruses are petropid bats found in australia and the far east (haplin et al., ) . currently no vaccines are available for these viruses. classically, the most important parasitic diseases in humans and animals are treated and/or controlled by using chemotherapeutics (cornelissen and schetters, ) . a series of developments (including drug resistance) has given impetus to the research into parasite vaccines. as a result, a number of vaccines against parasitic diseases are now commercially available. it is envisioned that more parasite vaccines will come to market and will aid in the control of parasitic diseases (vercruysse et al., ) . in many parasitic diseases, the host develops some level of immunity once the infection is cured. this indicates that the parasite has sufficient immunogenic potential that could be exploited as a vaccine. research aims at the development of a vaccine and/or vaccination protocol that induces protective immunity while limiting the induction of pathology. different approaches can be recognized varying from the use of live vaccines, attenuated live vaccines, killed vaccines and subunit vaccines. the most obvious examples of such a vaccine are the live vaccines against coccidiosis in chickens (williams, ) . as this type of infection is transient (the parasite ''passes" through the chicken) the infection is self-limiting and no chemotherapeutic treatment is necessary to cure the infection. to limit the induction of pathology it is necessary that a defined low-dose is given to the animals, and that this infection is initiated simultaneously in all chickens from a flock. the virulence of parasite strains derived from a single isolate can be variable. for example, using babesia bovis isolates, passage through splenectomised animals can select for strains of reduced virulence. such parasite strains are being used to vaccinate cattle in africa and australia. the infection that develops is less virulent and the animals develop immunity against subsequent challenge infection (de waal and combrink, ) . these vaccines are distributed by government institutions. similarly, strains with reduced virulence can be selected from eimeria isolates. some commercially available coccidiosis vaccines for broilers contain strains that are selected after repeated passage through chickens. these so-called precocious strains require less time to develop into oocysts, and the numbers of progeny are reduced compared to the wild-type parent population (williams, ) . some parasite strains have been selected that differ from the wild-type strains in that they cause a self-limiting infection. one such example is the temperature-sensitive strain of toxoplasma gondii. this strain, which resulted from chemically induced attenuation, can be propagated successfully in vitro at relatively low temperatures, but will not propagate successfully at the body temperature of the target animal. as a result the infection will self-cure (lindsay et al., ) . this vaccine has not been commercialised. in case the parasite has a tendency to survive in the host for longer periods of time, chemotherapeutic cure of the infection is also required. an example of this approach is the live vaccine against theileria parva infection. the vaccine is based on isolates of virulent t. parva strains which are used to infect cattle that are simultaneously treated with a long-acting tetracycline preparation to control the infection (boulter and hall, ) . this method is still being used in africa, and the vaccine is produced by centre for ticks and tick borne diseases (cttbd) in malawi. many parasite species have complicated life-cycles characterised by distinct life-cycle stages, sometimes involving more than one host. in some cases the early life-cycle stages are sufficiently immunogenic to induce protective immunity; selection for parasite strains with truncated life-cycles is another strategy to develop vaccines. a good example is the t. gondii s strain. this strain has lost the capacity to develop from the tachyzoite into the bradyzoite stage, and thus does not form tissue cysts. the tachyzoites induce a transient infection in the host, while triggering protective immune reactions (buxton, ) . irradiation of parasites has also been used as a mechanism to truncate the life-cycle. the live vaccine against lungworm infection in cattle contains l larvae of dictyocaulus viviparus that do not develop further than the l stage. vaccinated cattle are immune to challenge with l larvae (urquhart, ) . theoretically, virulent parasite strains could be genetically modified to reduce their virulence. for instance, parasite strains could be genetically manipulated such that during the production of the vaccine, parasites would be fully virulent and when administered to the host would cause a self-limiting infection. this principle has been investigated using toxoplasma gondii in which a tetracycline-dependent regulatory element was cloned in front of an essential gene. the parasite may be propagated during the vaccine production phase in the presence of tetracycline. once injected into the target animal, the parasite will not be able to continue propagation in the absence of tetracycline, as this will lead to blocking of the expression of the essential gene (van poppel et al., ) . if no live vaccine strains are available, or the use of live vaccines is undesirable, one may want to inactivate the parasites prior to the formulation of a vaccine. such preparations by themselves do not induce protective immunity and an appropriate adjuvant and formulation must be developed. examples of such vaccines are the vaccine against abortion in cattle due to neospora caninum infection (schetters, ) and a vaccine against giardiasis in dogs (olson et al., ) . a more detailed analysis of the immune response acquired after natural infection or vaccine induced immunity, can lead to the discovery of critical antigenic components of an organism that can be used in a vaccine. again such preparations require an adjuvant for the induction of protective immunity. the vaccine against babesiosis of dogs due to babesia canis infection is one such example (schetters, ) . it contains soluble antigens secreted/ excreted from the parasite. research has shown that during b. canis infection in dogs these antigens are released and cause disease. vaccination helps animals to quickly produce neutralising antibodies against these antigens. the adjuvant appears critical: saponin works, whereas oil-based adjuvants have shown little or no efficacy (schetters, ) . in some cases the antigens are produced using recombinant dna technology. the best example is a vaccine against taenia ovis in sheep, which is based on recombinantly produced parasite antigens that induce antibodies that block the attachment of oncospheres to the gut epithelium (harrison et al., ) . saponin adjuvant was shown to be most efficacious. another example is the vaccine against the cattle tick boophilus microplus (willadsen, ) . the vaccine contains recombinantly produced gut wall antigens of the tick. upon vaccination of cattle, high levels of antibodies to the gut wall of ticks are produced. during feeding of the tick on the vaccinated animal these antibodies are ingested and destroy the gut epithelium of the tick thus killing the parasite. in recent times, immunopotentiators received abundant attention in the media as critical adjuvants in novel human vaccines. examples include the prophylactic vaccines against human papilloma virus (hpv), novel pandemic influenza virus, as well as experimental allergy and tumour vaccines. indeed, vaccine adjuvants, also referred to as major platform technologies, are recognised to make the difference between competing vaccines based on identical antigens. in addition, it is recognised that vaccines designed for certain diseases require a matching combination of selected antigen(s) together with a critical immunopotentiator that selectively drives the essential immune pathway with minimal adverse reactions. the increased awareness of immunopotentiator importance is prominent in particular among human vaccinologists. for veterinary vaccine designers, the application and importance of adjuvants has always been evident. traditional veterinary vaccines, consisting of relatively crude extracts of microbial cultures, are inherently more immunogenic relative to purified subunits or peptide antigens, which are preferred in human vaccines. obviously, the constitution of the antigen of interest may contribute to immunogenicity and overall level of immune responsiveness. another trend in recent years is an increasing zscientific knowledge of mechanistic activities of many immunopotentiators, especially as a result of research focused on innate immunity receptors. these insights enable more rational adjuvant and vaccine design, which, ideally, is based on predictable immunophenotypes following vaccination. schijns et al. ( schijns et al. ( - have highlighted recent developments in immunopotentiators. mechanistically, vaccine adjuvants can be classified in two major groups: ( ) vaccine delivery systems that facilitate the timing, dosing and geography of the antigen, -also called facilitators of signal , and ( ) signal facilitators, which directly activate certain (innate) immune cells (degen et al., ) . vaccine adjuvants also influence the quality of the immune response, since they may preferably instruct for the development of t helper (th) , th , th , th , or regulatory t cells (t reg), a stimulus originally referred to as signal (kapsenberg et al., ) . immunopotentiators make the vaccine work, that is they evoke and potentiate antigen-specific t and b cell responses to both poorly immunogenic subunit vaccines and crude antigen preparations, respectively. they largely determine the immunophenotype of the response to the antigen in the vaccine. for example, vaccine adjuvants are able to influence and accelerate the onset of immunity, which may become urgent during emergency vaccination, or at early age (post-hatch or after birth) when the offspring is naïve to microbial attack. vaccine adjuvants may also increase the overall magnitude of the antigen-specific response in order to reach a minimal level of protective antibody concentration or effector t cell population. in addition, adjuvants can prolong the duration of vaccine effector immune responses, allowing for fewer or no booster immunisations, which becomes critical during mass vaccinations. moreover, certain vaccine adjuvants are able to positively affect the quality of the immunophenotype, for example induction of cell-mediated t cell immunity, considered necessary for control of many types of intracellular pathogens. antigen dose (cost) sparing is enabled by certain adjuvants, which is relevant in case of expensive or cumbersome antigen production systems. importantly, immunopotentiators may cause transient unwanted adverse reactions, either at the injection site or systemically. the level of acceptance depends on the relative benefit of the vaccine and the medical need to prevent or treat the disease of interest. unfortunately, there is no single adjuvant for all needs. instead, there are many different choices of potential immunopotentiators. rational selection of vaccine adjuvants during vaccine design is hampered by limited knowledge of the immunophenotype evoked by most classical vaccine adjuvants. selection of the best immunopotentiator has long been a matter of trial-and-error and serendipity. fortunately, in recent times mechanistic studies based on new insights of especially innate immunity receptors have allowed for more systematic and scientific investigations on immune induction in general and immunopotentiation in particular. this not only holds for mice and humans (the best studied immune systems) but increasingly for species of veterinary relevance. this new knowledge will allow for the development of new and improved immunopotentiator-based prophylactic vaccines as well as novel therapeutic vaccines. vaccines play an important role in the control of animal diseases, particularly in the food producing industries. however, this is currently largely achieved by conventionally developed and produced vaccines. progress on veterinary vaccine development in industry is largely influenced by boundaries set by regulatory, gmp and economic constraints, as outlined above. for obvious reasons regulatory requirements are strict, and will be further strengthened, to ensure delivery of safe and efficacious vaccines. gmp and glp quality systems were developed to achieve these ends. from the proof of concept of a new vaccine, these qa systems are implemented at all stages of vaccine development. in early phases only seed material is produced under a gmp regimen whereas in the final stages, a fully operational gmp system is applied. the burden placed on vaccine manufacturers caused them to develop reliable and flexible large-scale production methods. the development of more cell culture systems, animal-component-free culture media, multi-purpose facilities and disposable manufacturing materials are clear examples of this trend. against this background we have dealt with the current trends in industry, namely the use of biotechnology in vaccine development and production. examples of the success of biotech and classical vaccine approaches have been given for bacterial, viral and parasitic vaccines. with respect to virus diseases, a separate review dealing in more detail with epidemiology, pathogenesis and vaccines available or in development has been written (patel and heldens, accepted for publication). this will give a more complete and comprehensive overview of vaccine options in development and marketing. as indicated, the number of biotechnology-based vaccines presently on the market or in late stage development against bacterial, viral or parasitic pathogens is rather limited, but it is expected that products based on biotechnology/bio-engineering will increase in the future. knowledge of the working mechanism of adjuvants is increasing, based on progress in understanding animal diseases, immunology and adjuvant working mechanisms. this allows more rational vaccine design. notwithstanding the fact that the majority of current veterinary vaccines are derived and produced by conventional attenuation and/or inactivation processes in tissue culture, genetic engineering techniques have been widely used and will be used even more in the future but they have yet to fulfil the promise of improved vaccines. it is important to realise that there is no perfect vaccine and there may never be since pathogens are constantly mutating in order to survive and to evade their host's defences. basic academic research to increase our understanding of pathogen behaviour must be an ongoing activity so that we can improve our ability to protect the health and welfare of both humans and animals. to accomplish this we must continue and improve our vaccines that can be made in an industrial manner. efficacy of the classical swine fever (csf) marker vaccine porcilis pesti in pregnant sows porcine circoviruses: a review comparison of the efficacy of three commercial bacterins in preventing canine leptospirosis immunity and vaccine development in the bovine theileriosis protection of the bovine foetus from bvdv infection by means of a new inactivated vaccine mycobacterium tuberculosis heat shock proteins use diverse toll-like receptor pathways to activate pro-inflammatory signals toxoplasmosis: the first commercial vaccine the challenge of avian influenza to the veterinary community directive / /eec of september on the approximation of the laws of the member states relating to veterinary medicinal products council directive / /eec of september on the approximation of the laws of the member states relating to analytical, pharmacotoxicological and clinical standards and protocols in respect of the testing of veterinary medicinal products council directive / /eec of december extending the scope of directive / /eec on the approximation of the laws of the member states relating to veterinary medicinal products and laying down additional provisions for immunological veterinary medicinal products directive / /ec of the european parliament and of the council of july laying down the principles and guidelines of good manufacturing practice for veterinary medicinal products commission directive / /ec of march modifying de annex to council directive / /eec on the approximation of the laws of member states relating to analytical, pharmacotoxicological and clinical standards and protocols in respect of the testing of veterinary medicinal products immunogenicity of a recombinant pseudorabies virus expressing orf -orf fusion protein of circovirus type vaccines against protozoal diseases of veterinary importance vaccine adjuvant technology: from mechanistic concepts to applications zonula occludens toxin as a new promising adjuvant for mucosal vaccines the detection of antibodies against the e rns envelope protein of classical swine fever virus live vaccines against babesiosis good manufacturing practice for medicinal products (ec catalog number guidelines for the testing of veterinary medicinal products (ec catalog number co vaccination against salmonella enteritidis in dutch commercial layer flocks with a vaccine based on a live salmonella gallinarum r strain: evaluation of efficacy, safety, and performance of serologic salmonella tests a chimeric porcine circovirus (pcv) with the immunogenic capsid gene of the pathogenic pcv type (pcv ) cloned into the genomic backbone of the nonpathogenic pcv induces protective immunity against pcv infection in pigs synthetic bacterial lipopeptide analogs: structural requirements for adjuvanticity protection of chickens against a lethal challenge of eschericia coli by a vaccine containing cpg oligodeoxynucleotides as an adjuvant isolation of hendra virus from petropid bats: a natural reservoir of hendra virus duration of immunity, efficacy and safety in sheep of a recombinant taenia ovis vaccine formulated with saponin or selected adjuvants fifth international congress of veterinary virology, istituto zooprofillatico sperimentale della lombardia e dell emilia romagna, brescia fondazione iniziative zooprofilattiche e zootechniche optimization and delivery of plasmid dna for vaccination monophosphoryl lipid a analogues with varying -o-substitution: synthesis and potent adjuvant activity regulatory requirements for vaccine authorization immunisation against pcv structural protein by dna vaccination of mice the paradigm of type and type antigen-presenting cells. implications for atopic allergy round table on epidemiology and control of fox rabies role of different gene in the virulence and pathogenesis of aujeszky's disease virus second-generation pseudorabies virus vaccine with deletions in thymidine kinase and glycoprotein genes immunologic features of porcine circovirus type infection an overall report on the development of a highly safe and potent lapinized hog cholera virus strain for hog cholera control in taiwan safety and results of challenge of weaned pigs given a temperature-sensitive mutant of toxoplasma gondii adp-ribosylating bacterial enzymes for the targeted control of mucosal tolerance and immunity a vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein x genes enhancement of infectious disease vaccines through tlr -dependent recognition of cpg dna molecular biology of pseudorabies (aujeszky's disease) virus. comparative immunology and microbiology of infectious diseases approaches to the identification of non-essential genes of african swine fever virus prospects for the development of a vaccine against the west nile virus vaccines for preventing enterotoxigenic escherichia coli infections in farm animals herpes virus infections in cervidae oecd series on principles of good laboratory practice and compliance monitoring, numbers - giardia vaccination the development and use of a vacciniarabies recombinant oral vaccine for the control of wildlife rabies; a link between jenner and pasteur equine herpes viruses (ehv- ) and (ehv- ) -epidemiology, disease and immunoprophylaxis: a brief review accepted for publication. immunoprophylaxis against important virus diseases of farm animals: a review prevention of transplacental infection of bovine foetus by bovine viral diarrhea virus through vaccination derivation and characterisation of a live equid herpes virus (ehv- ) vaccine to protect against and respiratory disease due to ehv- equid herpes virus (ehv- ) live vaccine strain c : efficacy against respiratory diseases following ehv types and challenges efficacy of a live equine herpes virus - (ehv- ) strain c vaccine in foals with maternally derived antibody. protection against ehv- infection round table on control of aujeszky's disease and vaccine development based on molecular biology recombinant derivatives of pasteurella multocida toxin: candidates for a vaccine against progressive atrophic rhinitis european pharmacopoeia, fourth ed. the european department for the quality of medicines malignant catarrhal fever virus construction and characterisation of deletion mutants of pseudorabies virus: a new generation of ''live" vaccines diagnostic methods for african horse sickness virus using monoclonal antibodies to structural and non-structural proteins malignant catarrhal fever and gamma herpesviridae of bovidae from genes to complex structures of bluetongue virus and their efficacy as vaccines breite kreuzneutralisation von europäische bvdv typ- und typ- stämmen und signifikante verbesserung der fertilität nach testinfektionen the impact of genomics on vaccine design vaccine development from a commercial point of view intervet symposium bovine neosporosis vaccination against canine babesiosis regulations and procedures in parasite vaccine development immunological concepts of vaccine adjuvant activity induction and direction of immune responses by vaccine adjuvants antigen delivery systems and immunostimulation mechanisms of vaccine adjuvant activity: initiation and regulation of immune responses by vaccine adjuvants unravelling ''the immunologist's dirty little secret vaccine adjuvant technology: from theoretical mechanisms to practical approaches practical aspects of vaccination activation of nf-kappab-dependent gene expression by salmonella flagellins flic and fljb properties of vaccines advantages and disadvantages of common vaccination programs inactivated north american and european h n avain influenza vaccines protect chickens from asian h n high pathogenicity influenza virus field experience with the bovine lungworm vaccine immunogenicity of vaccinia virus (vv) recombinants, expressing bovine leukemia virus (blv) env gene, in rabbits and calves chimeric classical swine fever viruses containing envelope protein e(rns) or e of bovine viral diarrhoea virus protect pigs against challenge with csfv and induce a distinguishable antibody response present and future of veterinary viral vaccinology tight control of transcription in toxoplasma gondii using an alternative tet repressor live attenuated pseudorabies virus expressing envelope glycoprotein e of hog cholera virus protects swine against both pseudorabies and hog cholera veterinary parasitic vaccines: pitfalls and future directions good clinical practice. international cooperation on harmonisation of technical requirements for registration of veterinary medicinal products control strategies of major viral infectious diseases of live stock in europe, ten years of experience anti-tick vaccines safety of the attenuated anticoccidial vaccine paracox in broiler chickens isolated from extraneous coccidial infection anticoccidial vaccines for broiler chickens: pathways to success herpes virus disease of cattle, horses and pigs reference viruses for seasonal and pandemic influenza vaccine preparation the secretarial support from hanny van lare for typing (sometimes hand-written) manuscripts and for editing (hand-written) suggestions for improvement to the paper is acknowledged. key: cord- -uw padla authors: williams, joshua t.b.; o’leary, sean t.; nussbaum, abraham m. title: caring for the vaccine hesitant family: evidence-based alternatives to dismissal date: - - journal: j pediatr doi: . /j.jpeds. . . sha: doc_id: cord_uid: uw padla nan over one third of children are on alternative or shot-limiting vaccine schedules. measles infected , americans in -the most cases since -and the us nearly lost its measles elimination status. one in kindergartners attends school with a non-medical vaccine exemption, and many more kindergartners attend school under-immunized without plans to catch up. anti-vaccination groups spread doubt about vaccines through film and social media outlets. from - , elected officials introduced anti-vaccination bills into state legislatures more often than pro-vaccination bills. even a few physicians promote non-recommended vaccination schedules and exempt children from vaccines without medical cause. in response, pro-vaccination advocates and policy makers are rebutting anti-vaccination bills and working to tighten or eliminate vaccine exemptions. since , no us state has added or broadened non-medical exemptions and twelve states have since repealed or restricted nonmedical exemptions. in alone, maine and new york eliminated non-medical exemptions, washington repealed personal belief exemptions for the measles, mumps, and rubella vaccine, and seven more state legislatures introduced bills to eliminate non-medical vaccine exemptions. simultaneously, public health officials have proposed novel communication slogans -such as "safe vaccinations for a healthy nation" -to educate americans about vaccines, with researchers suggesting novel public health partnerships with diverse stakeholders, such as clergy. unfortunately, legislative actions meant to raise vaccination rates have been slowed or even doomed by coordinated anti-vaccine opposition and skepticism about institutional intrusion into family life. public health interventions are well-intended, but they may fail to boost vaccination rates among the vaccine-hesitant and even decrease vaccination intentions. even a global pandemic that has overwhelmingly sickened and killed frail, vulnerable, and disadvantaged people may not be transforming hesitancy into trust among vaccination skeptics. physicians are uniquely positioned to restore american confidence in vaccines because our clinical work depends upon personal encounters that engender trust. however, clinicians who care for children are adversely impacted by vaccine hesitancy, too. a survey of us primary care providers found that in providers reported that ≥ % of parents in their practice had refused a vaccine, while in said ≥ % of parents asked to spread out vaccines in a typical month. when talking with parents with substantial concerns, % of respondents spent - minutes, of a typical -minute visit, doing so. these conversations bring burnout instead of booster shots; % of pediatricians found their work less satisfying as a result of needing to discuss vaccines at length, as did % of family medicine physicians. the hard work is likely to continue: a survey of pediatricians found that % of respondents reported frequent requests for alternative vaccination schedules. how should physicians proceed? there is no clear consensus, but a concerning trend may be emerging. some providers, understandably frustrated and tired of parental pushback, are refusing to care for families who forgo vaccines. in national surveys of pediatricians in and , - % of pediatricians reported always or often dismissing families who refused vaccines from their practice, with rates rising over time. , this is occurring even though many have argued strongly against the widespread adoption of this practice on ethical, legal, and public health grounds in the u.s. and canada. [ ] [ ] [ ] [ ] in , the american academy of pediatrics' (aap) report "countering vaccine hesitancy" characterized dismissal as acceptable only after careful consideration of the situation, transparency with parents about the risks to their child, and openness about practice policies. although we are unaware of any recent studies measuring practice dismissal, pediatricians' willingness to embrace dismissal may be increasing after the measles epidemic. recently, one pediatrician in a high-refusal setting encouraged colleagues to engage vaccine hesitant families through multiple strategies of non-violent resistance, including dismissal. "so far," he said, "punishing kids by practice exclusion for their parents' folly has been limited nationally, but may need to be more formally included in the strategy of all physicians." dismissal policies likely will create more problems than they solve. first, it is unclear where children go after dismissal, especially given the shortage of primary care providers in north america. , second, practice dismissal may paradoxically increase the risk of outbreaks. as increasing numbers of unvaccinated patients cluster in practices tolerant of vaccine delay or refusal, the risks of vaccine-preventable diseases in those practices and communities only increases. , third, anti-vaccination groups are targeting the practice. in february , antivaccination proponents introduced a bill in colorado's house of representatives entitled the "vaccine consumer protection act." the bill mandated that healthcare providers or facilities "shall not limit or deny health care services or benefits to a patient […] because the patient or the patient's parent or guardian delayed or declined a vaccination." the bill mandated that providers pay fines of "up to one thousand dollars for the first violation and up to five thousand dollars for each subsequent violation." we are unaware of any such bills being enacted, but such legislation raises the potential of financial and legal difficulties for providers who continue to dismiss families. as an alternative to practice dismissal, there are several evidence-based tools in the provider-parent communication literature that can increase parental vaccine acceptance while keeping children of vaccine-hesitant parents in our practices. these tools -a presumptive approach, motivational interviewing, and persistence -may even restore physician morale. as policymakers and public health experts work to address vaccine hesitancy, we believe pediatricians also can play a role in increasing vaccine confidence. what would it look like to increase vaccine confidence, one visit at a time? first, physicians could increase vaccine acceptance with a presumptive approach to vaccine discussions. in , researchers in seattle videotaped over encounters between physicians and parents, of which % were vaccine-hesitant, in order to determine what predicted vaccine uptake at the end of the visit. they found that how we begin the conversation matters. some physicians began the conversation with what the authors called a participatory approach, offering parents latitude over vaccination decisions with opening lines such as: "what do you think about doing some shots today?" others presumed that parents intended to vaccinate their children, using phrases such as "johnnie is due for his -month vaccines today." when physicians used a presumptive approach, % of parents accepted vaccines; conversely, when physicians used a participatory approach, % of parents resisted vaccines. by emphasizing vaccination as the social norm, a presumptive approach helps parents draw confidence from provider confidence as they face the decision of vaccination. its benefits accrue with use. a longitudinal study of parent-child dyads found child immunization status at months was associated with the number of presumptive discussions parents had with providers at , , and month well-child visits. a presumptive approach also may increase adolescent vaccination uptake. a multi-arm randomized trial in pediatric and family medicine clinics across north carolina found a . % increase ( % ci; . %- . %) in hpv vaccination coverage for patients in clinics whose providers received presumptive training, compared with conversation training or usual care. one can imagine similar successes in alternative contexts (e.g. hospital care units, emergency departments, or subspecialty clinics) and with different vaccines. for example, a seattle hospital-based intervention demonstrated that a presumptive approach with parents increases child seasonal influenza vaccination. to help pediatricians adopt a presumptive approach, the aap has communication tools that generalize to diverse settings and can be built into electronic medical record systems. to build confidence in clinical settings, we should begin vaccination discussions presumptively. second, if patients or parents resist a presumptive approach, we should engage them with motivational interviewing. instead of a paternalistic approach which compels parents to vaccinate because "the doctor said so," motivational interviewing guides conversations in a noncondescending manner so that parents gradually develop their own commitment to follow vaccine recommendations. the spirit of motivational interviewing has four components, easily remembered with the acronym pace: partnership, acceptance, compassion, and evocation. the table provides conceptual demonstrated that a presumptive approach with motivational interviewing increased human papillomavirus (hpv) vaccine initiation for adolescents - years old by %, compared with a % decrease in controls. providers believed that motivational interviewing was useful; % of providers reported using the technique frequently, much more so than websites, decision-aids, or disease images. a multi-site clinical trial to train providers on how to presumptively initiate vaccines and optimize talk with motivational interviewing (pivot with mi) is ongoing. given its perceived utility and effectiveness, motivational interviewing may even counter clinician burnout. in a pilot study of the impact of motivational interviewing on obesity-related patient encounters, clinicians who received training had notable improvements in their burnout scores, compared with controls. in sum, both parental confidence and physician wellbeing may improve as we work with vaccine hesitant parents via motivational interviewing. physicians have a third tool to build vaccine confidence: persistence. in the presumptive versus participatory study discussed above by opel et al, the study team also measured the percentage of parents who ultimately vaccinated their children despite an initial reticence to do so. ultimately, % of parents agreed to follow the physician's recommendation when physicians continued to discuss their concerns and offer them a positive recommendation for vaccination. in a separate study of audio-recorded visits with unvaccinated adolescents and their parents, researchers found that parents expressed hesitancy about the hpv vaccine at least once during the visit. some providers responded with only persistence, some responded with a mix of acquiescence and persistence, and some simply acquiesced to parents' concerns. of the encounters in which providers used persistence only, adolescents received the hpv vaccine; conversely, no adolescent received the vaccine when providers simply acquiesced. furthermore, whether discussing childhood vaccines or hpv, physicians can persist over the short-term horizon of a visit through motivational interviewing, as described above. we must also persist over the long-term horizon of the physician-patient relationship. in the longitudinal study cited above, persistent use of the presumptive approach at -, -, and month well child visits was associated with a ~ % decrease in a child's percentage of days under-immunized at months. in another longitudinal study of nearly parents who had ever declined hpv vaccination for their - year old child, researchers found that secondary acceptance of the hpv vaccine -within months of an initial refusal -was associated with receiving follow-up counseling (or . ; % ci, . - . ). when asked why they ultimately accepted the hpv vaccine for their child, one-third of parents cited receiving a second recommendation. we believe persistence will pay off with other vaccines as clinicians form long-term relationships with parents and children, understand their concerns, and recommend vaccines anew. providers could also ask parents to sign a vaccine declination form, such as the one created by the aap. as of , nearly half of pediatricians and one third of family medicine physicians used such forms when parents refused vaccines. although the use of a declination form has not been tested in a randomized trial, the use of such a form offers the opportunity to clearly delineate the risks of vaccine-preventable diseases, the importance of vaccines, and the responsibility parents have for the consequences of vaccine delay. of course, we must discern the best time and context in which to introduce a declination form, but doing so may motivate parents on the fence to accept recommendations from their persistent providers. dealing with parental vaccine hesitancy has become a frustrating yet essential part of being a physician. practice dismissal may be a tempting option for burnt-out physicians to avoid hesitant parents and the public health risks their children pose to other patients. yet, as sars-cov- circles the globe and researchers race to develop a vaccine, we can develop, in parallel, our ability to talk about vaccines with concerned parents. evidence-based alternatives to dismissal exist, increase parental acceptance of vaccines, will keep under-vaccinated and unvaccinated children with pediatricians, and improve population health. as policymakers and public health experts work at system levels, we should consider what we as pediatricians might do to increase vaccine confidence. who else is better positioned to do so than the pediatricians who form long-term relationships with parents and their children? contentious conversations require care, but we believe evidence-based tools that teach a presumptive approach, motivational interviewing, and persistence can help. as we work as pediatricians to improve the state of vaccine confidence, let us consider caring for vaccine-hesitant families, one visit at a time. we avoid being the "expert", assuming the role of a partner and validating concerns. we work "for" and "with" patients and parents; we don't lecture "to" or "at" them. after hearing parental concerns, we ask permission to share information with them. "it makes sense that you're worried about vaccine safety. all parents want to keep their children safe. could i share a few things i've learned about vaccine safety with you?" we affirm the absolute value of our patients or parents, accepting them as fellow humans. we highlight their autonomy to make decisions, although we are free to disagree with them. "i strongly recommend this vaccine, but the choice is yours. thank you for continuing to have this hard conversation with me. i'm happy to continue talking with you at our next visit." we seek the good and well-being of others. we recommend vaccines because we believe they help others, not out of self-interest. adherence to timely vaccinations in the united states measles cases and outbreaks vaccination coverage with selected vaccines and exemption rates among children in kindergarten -united states anti-vaccine activists, web . , and the postmodern paradigm -an overview of tactics and tropes used online by the anti-vaccination movement trends and characteristics of proposed and enacted state legislation on childhood vaccination exemption lessons from california's discipline of a popular physician for vaccination exemptions without medical cause update on top resolutions adopted at annual leadership forum safe vaccinations for a healthy nation: increasing us vaccine coverage through law, science, and communication building trust: clergy and the call to eliminate religious exemptions how anti-vaccine activists doomed a bill in new jersey effective messages in vaccine promotion: a randomized trial prevalence of parental concerns about childhood vaccines: the experience of primary care physicians factors associated with pediatrician responses to alternative immunization schedule requests characteristics of physicians who dismiss families for refusing vaccines vaccine delays, refusals, and patient dismissals: a survey of pediatricians provider dismissal of vaccine-hesitant families professional responsibility and early childhood vaccination when is it permissible to dismiss a family who refuses vaccines? legal, ethical and public health perspectives. paediatr child health (oxford) is physician dismissal of vaccine refusers an acceptable practice in canada? a overview countering vaccine hesitancy follow gandhi's approach when responding to vaccine aversion provider dismissal policies and clustering of vaccine-hesitant families: an agent-based modeling approach - : vaccine consumer protection act. denver: second regular session, seventy-second general assembly of the state of colorado the architecture of provider-parent vaccine discussions at health supervision visits impact of childhood vaccine discussion format over time on immunization status announcements versus conversations to improve hpv vaccination coverage: a randomized trial parental vaccine hesitancy and declination of influenza vaccination among hospitalized children immunizations/practice-management/pages/vaccine-hesitancy-practice-improvement-project.aspx motivational interviewing: helping people change. third effect of a health care professional communication training intervention on adolescent human papillomavirus vaccination a cluster randomized clinical trial evaluation of the implementation of a multicomponent intervention to improve health care provider communication about human papillomavirus vaccination presumptively initiating vaccines and optimizing talk with motivational interviewing (pivot with mi) effect of teaching motivational interviewing via communication coaching on clinician and patient satisfaction in primary care and pediatric obesity-focused offices parent-provider communication of hpv vaccine hesitancy parents who decline hpv vaccination: who later accepts and why? documenting parental refusal to have their children vaccinated key: cord- -pmclolvh authors: head, katharine j.; kasting, monica l.; sturm, lynne a.; hartsock, jane a.; zimet, gregory d. title: a national survey assessing sars-cov- vaccination intentions: implications for future public health communication efforts date: - - journal: sci commun doi: . / sha: doc_id: cord_uid: pmclolvh with sars-cov- vaccines under development, research is needed to assess intention to vaccinate. we conducted a survey (n = , ) with u.s. adults in may assessing sars-cov- vaccine intentions, intentions with a provider recommendation, and sociodemographic and psychosocial variables. participants had high sars-cov- vaccine intentions (m = . / -point scale), which increased significantly with a provider recommendation (m = . ). hierarchical linear regression showed that less education and working in health care were associated with lower intent, and liberal political views, altruism, and covid- -related health beliefs were associated with higher intent. this work can inform interventions to increase vaccine uptake, ultimately reducing covid- -related morbidity and mortality. the covid- (coronavirus disease ) pandemic, caused by the sars-cov- (severe acute respiratory syndrome coronavirus ) virus, emerged in late with u.s. cases presently at . million, and > , attributable deaths (centers for disease control and prevention [cdc], b) . with no available vaccine, public health agencies like the centers for disease control have advised the public on specific behaviors to limit transmission (e.g., "social distancing," wearing a face mask, etc.; cdc, a). beyond individual behaviors, local and state governments across the country enacted various "stay-at-home" orders and closed nonessential businesses during parts of march, april, and may (lee et al., ) . despite these measures, covid- has caused a serious disease burden to the u.s. health care system. consensus among medical experts is that until a vaccine is available and we reach high-vaccine coverage, nonpharmaceutical interventions will only be able to curb the spread of the virus (corey et al., ) . several sars-cov- vaccines are in development and might be available by early , though availability will depend on successful clinical trials demonstrating efficacy and safety (lurie et al., ) . public health and medical practitioners must prepare to promote acceptance of these vaccines. vaccine hesitancy, which describes a range of stances toward vaccination, from deep skepticism about vaccine efficacy and safety to more mild concerns, has been identified by the world health organization as a major global health threat and is particularly prevalent in the united states (macdonald, ; quinn et al., ; world health organization, ) . because scholars have argued that vaccine hesitancy is driven by contextspecific factors including time and place as well as individual factors such as beliefs about threat of disease (brewer et al., ; dubé et al., ; larson et al., ) , it is important to understand perceptions related to sars-cov- vaccination and to assess what factors may contribute to higher or lower intentions to vaccinate. previous research with other vaccine-preventable diseases show that there are identifiable factors that may influence vaccination intentions and acceptance. for example, certain sociodemographic factors have played a role in adult vaccination acceptance, such as socioeconomic status, age, race and ethnicity, and geographic location (abbas et al., ; almario et al., ; galarce et al., ) . since vaccination relies on the principle of "herd immunity," prosocial motives for behaviors that benefit others, such as general altruism, prosociality, and sympathy, can play a role in some vaccination decision making (li et al., ; vietri et al., ) . additionally, theoretical models like the health belief model have long recognized that variables like perceived severity and susceptibility to a disease may predict behavioral intentions, which in turn, predict behavior (brewer et al., ; bish et al., ; champion & skinner, ; gerend & shepherd, ; yang, ) . the extended parallel process model further posits that health promotion and message design must consider the balance between addressing issues of severity and susceptibility in a way that promotes message acceptance, rather than provoking too much or not enough fear and thus causing people to reject the message (prati et al., ; quick et al., ; vorpahl & yang, ; witte, ) . vaccine communication and promotion work has long relied on theoretical models like these not only for guiding formative work with target populations (cameron et al., ; chen et al., ) but also to develop and test behavioral interventions (gerend & shepherd, ; gore & bracken, ) . finally, research demonstrates that a provider recommendation remains an important predictor of vaccination behavior in the united states reiter et al., ) . more important, strong provider recommendations are needed to maximize the effect on patient vaccination decisions (gilkey et al., ; lu et al., ) . given the novel nature of covid- , research is needed to assess the public's intentions to get the sars-cov- vaccine, when it becomes available, as well as what factors may be associated with higher or lower intent. to ensure high vaccination coverage, public health campaigns must be carefully designed based on evidence about target populations and may even need to employ targeted communication strategies based on sociodemographic and psychosocial variables dubé et al., , kriss et al., minor et al., ; stockwell et al., ) . otherwise, we risk disseminating counterproductive messaging that may reinforce hesitancy in those already hesitant (bloom et al., ) . therefore, a national survey of adults in the united states was used to address the following research questions: research question : what are the sars-cov- vaccine behavioral intentions of adults in the u.s.? research question : what are the sars-cov- vaccine behavioral intentions of adults in the u.s. when a health care provider recommends the vaccine? research question : what factors are associated with sars-cov- vaccine behavioral intentions of adults in the united states? the data for this study come from a survey assessing knowledge, beliefs, and behaviors related to the covid- pandemic. data were collected between may and may , through an online survey. participant recruitment was facilitated by dynata, a market research firm that maintains panels of million volunteer survey respondents throughout countries. panelists receive monetary incentives tailored to both the time and effort required for participation and regional preferences. email invitations were sent to members of dynata's u.s. panel who met eligibility criteria of being years or older and able to read english. the study was approved by the university's institutional review board as exempt and not requiring written informed consent. a total of , participants opened the survey and ( . %) chose not to continue after reading the informed consent welcome page. we excluded anyone who did not answer the intention outcome measures for the current study. importantly, because vaccine intent and/or need may be different for people who were previously infected with sars-cov- and perceived threat variables (discussed below) are usually only measured for future threats, only participants who answered "no" to the question "do you believe that you've had covid- " are included in the current study (n = , ). in addition to demographic information, the study team collected data on participants' vaccine behavioral intentions, sociocultural beliefs, experiences with covid- , and health beliefs regarding personal risk and threat of covid- . detailed information on variables measured at the categorical level as well as their response options can be found in table ; variables measured at the continuous level are described below. vaccine behavioral intentions. two items, adapted from previous vaccine work, assessed participants' likelihood to receive a sars-cov- vaccine (gerend & shepherd, ) . based on pretesting of our survey instruments, it was determined that using the term "covid- vaccine" in the survey was more appropriate for lay audiences, since sars-cov- is less frequently used in lay communication. these two vaccine intent items included "how likely is it that you'll get a covid- vaccine, if it becomes available?" (individual intent) and "if your healthcare provider strongly recommended a covid- vaccine in the next year, how likely is it that you'd get vaccinated?" (provider rec intent). both items were assessed using a -point likert-type scale ( = very unlikely to = very likely). because these two items were highly correlated with high reliability (cronbach's α = . ), the two behavioral intention items were averaged into a single overall intent measure (overall vaccine intent). altruism. we assessed participants' altruism using an -item scale adapted from rushton et al. ( ) . participants responded to each item on a -point likert-type scale where = never to = very often. we conducted a principal components exploratory factor analysis, which extracted two factors. we labeled the first factor, which consisted of five items (cronbach's α = . ), high commitment altruism (i.e., behaviors that require a relatively high level of personal involvement; e.g., "i have helped push a stranger's car out of the snow or mud."). we labeled the second factor, which consisted of four items (cronbach's α = . ), low commitment altruism (i.e., behaviors that require a relatively low level of personal involvement; e.g., "i have given money to charity."). covid-related worry. a three item scale adapted from liau et al. ( ) and fan et al. ( ) was used to measure participants' personal worry about covid- ("i am scared about getting infected with covid- ," "the possibility of getting infected in the future with covid- concerns me," and "i don't really worry about getting infected with covid- "). participants responded to each item on a -point likert-type scale where = strongly disagree to = strongly agree. the last item was reverse coded, and then the three items were summed and averaged to derive a single covid-related worry score (cronbach's α = . ). perceived severity of covid. a four-item scale adapted from cahyanto et al.'s ( ) work on ebola was used to measure participants' perceptions of the severity of covid- (e.g., "i am afraid that i may die if i contract covid- ."). participants responded to each item on a -point likert-type scale where = strongly disagree to = strongly agree. the items were summed and averaged to derive a single perceived severity of covid score (cronbach's α = . ). likelihood of infection. personal susceptibility was measured with a single item: "how likely do you believe it is that you will get infected with covid- ?" participants responded on a -point likert-type scale where = not at all to = extremely. threat to physical health. perceived threat to physical health was measured with a single item: "if you got infected with covid- , how threatening would it be to your physical health?" participants responded on a -point likert-type scale where = not at all to = extremely. first, the sample was described using frequency distributions or means and standard deviations, as appropriate. we then examined our two vaccination intent variables (individual intent and provider rec intent) and examined if the participant changed their likelihood of receiving a sars-cov- vaccine when they were told a provider recommended it. we then examined bivariate associations between the overall vaccine intent score and each of the potential predictor variables using linear regression. any variable that was significant at p < . in bivariate linear regression was included in subsequent analyses. we used . , rather than . as the cutoff because, with our large sample size, a cutoff level of . might identify trivial relationships. we then conducted a three-step hierarchical multiple linear regression analysis. in the first step, we included demographic characteristics, in the second step we added in health care characteristics, and in the third step we included health belief characteristics. this approach was used to determine if health beliefs influenced likelihood of receiving a sars-cov- vaccine, above and beyond demographic and health care characteristics. the final analytic sample included , participants who reported no previous covid- diagnosis. mean age was . years (sd = . ) and the majority of participants were female (n = , ; . %) and non-hispanic white (n = , ; . %). for a complete inventory of sample descriptive statistics, see table . when asked how likely they were to get the sars-cov- vaccine, the mean score was . (sd = . ). this average intention increased to a mean score of . (sd = . ) when they were asked the likelihood of receiving the vaccine if their health care provider strongly recommended it. for a categorical breakdown of responses to each of the intent variables, see table . the mean increase from individual intent to provider recommendation intent was significant, t = − . (p < . ). when examining change in intent from individual intent to intent due to provider recommendation, the majority of the sample (n = , ; . %) did not change their response to the likelihood of receiving the vaccine. however, almost one quarter of the sample (n = ; . %) became more likely to receive the vaccine if a provider recommended it and a smaller percentage (n = ; . %) became less likely to receive the vaccine if a provider recommended it; see figure . in bivariate analyses with overall intent score (individual intent and provider recommendation intent combined; m = . , sd = . ), variables that had associations at p > . included region (p = . ), knowing someone who has had covid- (p = . ), and sex (p = . ). these variables were not included in subsequent analyses. see table for all bivariate analyses. multivariable regression analyses can be found in table . the first step of the hierarchical multiple regression including only demographic variables that had an adjusted r value of . . when personal health care variables were added in step , the adjusted r value increased to . . finally, in the third step of the hierarchical multiple regression, the adjusted r increased to . when the health belief variables were included. in step of the hierarchical regression model, with all variables included, less education was associated with lower intent to receive a sars-cov- vaccine. likewise, being currently employed in health care was also negatively associated with intent to receive a vaccine as compared with those who were never employed in the health care system (Β = − . ; % ci [− . , − . ]). participants who self-identified as liberal reported the highest intent to receive a sars-cov- vaccine (Β = . ; % ci [ . , . ]), followed by moderates, and then conservatives. the health belief variables that were significant in the full regression model were all positively associated with intent to receive a sars-cov- vaccine. specifically, as low-commitment altruism increased, likelihood of receiving a sars-cov- vaccine increased (Β = . ; % ci [ . , . ]). furthermore, as perceived threat to physical health increased, likelihood of receiving a sars-cov- vaccine increased (Β = . ; % ci [ . , . ]). those who believed covid- was a major problem in their community had higher likelihood of receiving a sars-cov- vaccine compared with those who did not (Β = . ; % ci [ . , . ]). worry was most strongly associated with sars-cov- vaccine intent; as worry increased, intent likewise increased (Β = . ; % ci [ . , . ]). this article aimed to examine u.s. respondents' intentions to receive the sars-cov- vaccine when it becomes available, and investigate factors associated with those intentions. overall, participants in this study reported step : demographic variables step : including health care variables step ref. ref. ref. ref. ref. ref. ref. ref. step : demographic variables step : including health care variables step ref. ref. ref. ref. work in health care currently employed in health care ref. ref. ref. ref. household income ( ) less than $ , ref. ref. ref. ref. ref. ref. ref. ref. health care characteristics received a flu vaccine, months yes ref. ref. ref. (continued) step : demographic variables step : including health care variables step ref. step r = . (adjusted = . ); step r = . (adjusted = . ); step ref. = reference group. boldface type indicates statistical significance (p< . ). high intentions to receive a sars-cov- vaccine, which were even higher with a strong provider recommendation. several sociodemographic and health belief variables were also associated with higher and lower sars-cov- vaccine intentions. below, we discuss the implications of these findings and suggest areas for future work, including research and practical application. importantly, participants reported relatively high individual intent to receive a sars-cov- vaccine. on a -point scale, participants in this study reported an average of . . while not quite a ceiling effect, we believe this suggests strong support for a vaccine, more so because no vaccine has been fully tested and made available to the public. our findings are consistent with other recent work examining perceptions of the sars-cov- vaccine, also showing highvaccine intentions in the united states (reiter et al., ; thigpen & funk, ) . interestingly, this level of intention to receive the sars-cov- vaccine is markedly higher than what is seen for actual u.s. adult vaccination behaviors for influenza. the cdc reports that - flu vaccination coverage among adults ≥ years was only . % (cdc, ). related, research shows that the relationship between intention and actual behavior, while usually significantly positive, is not always a perfect correlation and that different predictors (e.g., perceived susceptibility, doctor recommendation) may differently predict intentions versus actual behavior (juraskova et al., ; krawczyk et al., ; schwenk & möser, ; webb & sheeran, ) . therefore, while participants in this study expressed high sars-cov- vaccine intentions, these findings should be interpreted cautiously. actual uptake of a future vaccine will likely depend on many factors, including the status of the covid- pandemic at the time of vaccine debut. of note for communication scholars, these findings suggest that social normative messaging could capitalize on the high level of vaccine intention. social norms campaigns use descriptive norms (i.e., descriptive statistics) to correct or reinforce the frequency with which others are performing a behavior, with the assumption that individuals seek to conform to the pressures of societal norms (i.e., subjective norms; burchell et al., ) . while most social norms campaigns target audiences who may be overestimating the frequency of an unhealthy behavior (e.g., binge drinking; campo et al., ) , the same normative principles have been found to significantly predict hpv vaccination intentions and uptake among young women (de visser et al., ) . for example, social norms messages can address sars-cov- vaccine hesitancy by highlighting the high intentions to vaccinate expressed by the majority of people in one's social network. this approach will require communication scientists to engage in formative research to develop and test messages with different audiences, especially given the differences in intention across subgroups of population found in this study. participants in this study also were significantly more likely to receive the vaccine if their health care provider strongly recommended it. this finding is consistent with previous work showing a doctor's recommendation is a significant predictor of vaccination behavior (gorman et al., ; rahman et al., ; sturm et al., ) , including when newer vaccines, such as the h n influenza vaccine, are being considered (coe et al., ) . a key limitation of this study is that the single-item measure only asked participants about intentions if their provider strongly recommended the vaccine; no information was gathered about what information they may want about the sars-cov- vaccine from their provider. providers are the most trusted source of health information for patients (jackson et al., ) , including information about vaccines (eller et al., ) , which may be important once a sars-cov- vaccine becomes widely available. vaccine promotion campaigns may need to emphasize the importance of talking with a health care provider about the vaccine, including asking for information to address any concerns or questions. at the same time, health care providers may need support and training such as that already offered through the cdc (cdc, ; cdc, ) to be most effective in recommending a sars-cov- vaccine. specific sociodemographic and health belief variables were associated with intentions to vaccinate, and are worthy of consideration for future work, especially for communication interventions seeking to promote a sars-cov- vaccine. demographics. participants with less education expressed a lower intention to receive a sars-cov- vaccine. education is often associated with health literacy (kutner et al., ; paasche-orlow et al., ) , suggesting the critical importance of educating the public on the role of vaccines in reducing covid- prevalence through herd immunity. these efforts may need to be done in conjunction with messages about how herd immunity works, as previous work has shown that limited understanding can undermine vaccination intentions and behavior (sobo, ) . the effective deployment of "flatten the curve"-a phrase previously not commonly used among lay audiences when discussing a disease outbreak-via social media is an example of effectively educating the public about complex health terms in accessible ways (boboltz, ) . interestingly, participants who were employed in health care indicated a lower vaccine intention. this was contrary to what was expected. previous work has shown that some health care providers express vaccine hesitancy and low-vaccine acceptance themselves (collange et al., ; verger et al., ) . additionally, our question only queried whether the individual worked in health care and did not distinguish positions entailing direct patient care or type of training. given that many health care-related positions are nonclinical (e.g., janitorial, receptionist), some participants who answered this question may have limited understanding about the role of vaccines in preventing infectious diseases. we believe further work is needed to clarify this finding. participants' self-reported political views were associated with vaccine intent, with liberals expressing the strongest sars-cov- vaccine intentions, followed by moderates, and then conservatives. the united states has a complex and often partisan political environment, which may be compounded by mass media news consumption and "echo chambers" within social media platforms (bakshy et al., ; iyengar & hahn, ). one group espousing significantly lower intentions than other groups represents a potential challenge for high vaccine community coverage; however, these media trends may also represent an arena for targeted messaging going forward. we make an especially strong call for future work on this issue and implore other health and science communication researchers and practitioners to devote particular attention to targeted work on political ideology as we inch closer to an available sars-cov- vaccine. finally, we found that as individuals' level of low commitment altruism increased, so too did their likelihood of receiving a sars-cov- vaccine. importantly, we all must remember that vaccines provide both a personal benefit and public health benefit. research on the relationship between concepts like altruism and vaccination is an area that has received increasing, but still inadequate, attention in the vaccine literature (korn et al., ; li et al., ; quadri-sheriff et al., ; vietri et al., ) . going forward, research examining individual's concern for the "other" as a potential motivating factor for sars-cov- vaccination, as well as a potential message design strategy, is an important focus. intentions. consistent with frameworks like the health belief model and the extended parallel process model, individuals who expressed fear-measured in this study as higher worry, perceived threat to physical health, and perceived covid- to be a major problem in their community-were more likely to intend to get the sars-cov- vaccine when it becomes available. the data for this study were collected in early may , when many states in the united states were still in "lock down" mode and covid- rates and hospitalizations were high but steady. if covid- rates and hospitalizations are high when the vaccine debuts, these perceived threat variables may continue to be positively associated with intention. however, if infection rates drop or individuals become numb to the threat posed by the disease, these variables may not be as strongly associated with intentions. it will be important, therefore, to do both longitudinal and cross-sectional surveys over time to monitor changes in public attitudes and perceptions about covid- disease and a sars-cov- vaccine as well as examine the potential association of other social and behavioral determinants of health such as access and cost issues. in the meantime, communication scientists can capitalize on these findings by exploring messaging strategies that address individuals' fears about covid- . a limitation of this study is that we used a national but not a population representative sample. participants were members of an opt-in panel and may not reflect all u.s. adults. furthermore, the cross-sectional survey design precludes determination of causal direction in the relationships identified and necessarily represents a snapshot in time, rather than the evolving landscape of the public's knowledge and attitudes about covid- . as previously noted, intent can be an imperfect predictor of subsequent behavior. finally, two measurement limitations worth mentioning include a mismatch in the wording of our intention measures (i.e., the provider intention measure specified a timeline of "in the next year" while the individual intention item did not) and excluding participants who believed they had a previous sars-cov- infection from the health belief items (e.g., perceived severity, worry, likelihood of infection, threat). this study examined sars-cov- vaccine intentions and factors associated with these intentions. in addition to high intentions to receive the vaccine, provider recommendation increased intentions and will likely be an important factor in achieving the level of vaccination needed for herd immunity. several sociodemographic and health belief variables were associated with demographics, perceptions, and socioeconomic factors affecting influenza vaccination among adults in the united states persistent racial and ethnic disparities in flu vaccination coverage: results from a population-based study exposure to ideologically diverse news and opinion on facebook factors associated with uptake of vaccination against pandemic influenza: a systematic review addressing vaccine hesitancy here's why everybody's talking about "flattening the curve meta-analysis of the relationship between risk perception and health behavior: the example of vaccination increasing vaccination: putting psychological science into action marketing social norms: social marketing and the "social norm approach the dynamics of travel avoidance: the case of ebola in the us using theoretical constructs to identify key issues for targeted message design: african american seniors' perceptions about influenza and influenza vaccination social norms and expectancy violation theories: assessing the effectiveness of health communication campaigns adult vaccination information for healthcare and public health professionals influenza (flu): flu vaccination coverage, united states - influenza season: summary the health belief model using the health belief model to understand caregiver factors influencing childhood influenza vaccinations the use of the health belief model to assess predictors of intent to receive the novel ( ) h n influenza vaccine knowledge, attitudes, beliefs, and behaviors of general practitioners/family physicians toward their own vaccination: a systematic review a strategic approach to covid- vaccine r&d the importance of social norms for uptake of catch-up human papillomavirus vaccination in young women strategies intended to address vaccine hesitancy: review of published reviews vaccine information sources and parental trust in their child's health care provider behavior and health beliefs as predictors of hiv testing among women: a prospective study of observed hiv testing socioeconomic status, demographics, beliefs and a(h n ) vaccine uptake in the united states predicting human papillomavirus vaccine uptake in young adult women: comparing the health belief model and theory of planned behavior provider communication and hpv vaccination: the impact of recommendation quality testing the theoretical design of a health risk message: reexamining the major tenets of the extended parallel process model theory-based predictors of influenza vaccination among pregnant women red media, blue media: evidence of ideological selectivity in media use americans' trust in health information sources: trends and sociodemographic predictors hpv vaccine promotion: does referring to both cervical cancer and genital warts affect intended and actual vaccination behavior? women's health issues vaccination as a social contract human papillomavirus vaccination intentions and uptake in college women evaluation of two vaccine education interventions to improve pertussis vaccination among pregnant african american women: a randomized controlled trial the health literacy of america's adults: results from the national assessment of adult literacy. nces - understanding vaccine hesitancy around vaccines and vaccination from a global perspective: a systematic review of published literature see how all states are reopening (and closing again stimulating influenza vaccination via prosocial motives attitudes about human immunodeficiency virus immunization: the influence of health beliefs and vaccine characteristics association of provider recommendation and offer and influenza vaccination among adults aged ≥ years-united states developing covid- vaccines at pandemic speed vaccine hesitancy: definition, scope and determinants improving influenza vaccination rates by targeting individuals not seeking early seasonal vaccination collaborative patientprovider communication and uptake of adolescent vaccines the prevalence of limited health literacy influenza vaccination: the persuasiveness of messages among people aged years and older the role of herd immunity in parents' decision to vaccinate children: a systematic review examining mechanisms underlying fear-control in the extended parallel process model measuring vaccine hesitancy, confidence, trust and flu vaccine uptake: results of a national survey of white and african american adults provider recommendation mediates the relationship between parental human papillomavirus (hpv) vaccine awareness and hpv vaccine initiation and completion among -to -year-old us adolescent children hpv vaccination among adolescent males: results from the national immunization survey-teen acceptability of a covid- vaccine among adults in the united states: how many people would get vaccinated? vaccine. advance online publication the altruistic personality and the self-report altruism scale intention and behavior: a bayesian meta-analysis with focus on the ajzen-fishbein model in the field of environmental behavior what is herd immunity, and how does it relate to pediatric vaccination uptake? us parent perspectives effect of a text messaging intervention on influenza vaccination in an urban, low-income pediatric and adolescent population: a randomized controlled trial pediatrician-parent conversations about human papillomavirus vaccination: an analysis of audio recordings most americans expect a covid- vaccine within a year; % say they would get vaccinated vaccine hesitancy among general practitioners and its determinants during controversies: a national cross-sectional survey in france vaccinating to help ourselves and others who is to blame? framing hpv to influence vaccination intentions among college students does changing behavioral intentions engender behavior change? a meta-analysis of the experimental evidence putting the fear back into fear appeals: the extended parallel process model ten threats to global health in predicting young adults' intentions to get the h n vaccine: an integrated model key: cord- -zh e w authors: dimenna, lauren j.; ertl, hildegund c. j. title: pandemic influenza vaccines date: - - journal: vaccines for pandemic influenza doi: . / - - - - _ sha: doc_id: cord_uid: zh e w since their compositions remain uncertain, universal pandemic vaccines are yet to be created. they would aim to protect globally against pandemic influenza viruses that have not yet evolved. thus they differ from seasonal vaccines to influenza virus, which are updated annually in spring to incorporate the latest circulating viruses, and are then produced and delivered before the peak influenza season starts in late fall and winter. the efficacy of seasonal vaccines is linked to their ability to induce virus-neutralizing antibodies, which provide subtype-specific protection against influenza a viruses. if pandemic vaccines were designed to resemble current vaccines in terms of composition and mode of action, they would have to be developed, tested, and mass-produced after the onset of a pandemic, once the causative virus had been identified. the logistic problems of generating a pandemic vaccine from scratch, conducting preclinical testing, and producing billions of doses within a few months for global distribution are enormous and may well be insurmountable. alternatively, the scientific community could step up efforts to generate a universal vaccine against influenza a viruses that provides broadly cross-reactive protection through the induction of antibodies or t cells to conserved regions of the virus. against influenza a viruses. if pandemic vaccines were designed to resemble current vaccines in terms of composition and mode of action, they would have to be developed, tested, and mass-produced after the onset of a pandemic, once the causative virus had been identified. the logistic problems of generating a pandemic vaccine from scratch, conducting preclinical testing, and producing billions of doses within a few months for global distribution are enormous and may well be insurmountable. alternatively, the scientific community could step up efforts to generate a universal vaccine against influenza a viruses that provides broadly cross-reactive protection through the induction of antibodies or t cells to conserved regions of the virus. influenza viruses belong to the family of orthomyxoviridae, which includes negative single-stranded rna viruses with segmented genomes. among the three genera of influenza viruses (a, b, and c), influenza a and c viruses infect humans as well as other species, while influenza b virus mainly infects humans. the most common and serious infections of humans are caused by influenza a virus. influenza a viruses are further divided into subtypes based on their hemagglutinin (ha) and neuraminidase (na) genes, which encode the two viral surface proteins. influenza a virus typically infects epithelial cells that line the respiratory tract, but may also replicate in other tissues in different hosts, including conjunctiva, intestine, brain, liver, kidney, and gut. in general, influenza a virus infections are self-limiting in healthy human adults, and mainly cause life-threatening disease in the very young and in the elderly. notwithstanding, this depends on the circulating type. aquatic birds serve as the main reservoir of influenza a viruses and carry all of the known subtypes (h - , n - ) without necessarily developing disease upon infection. the virus can adapt to other species such as poultry, pigs, horses, or humans. in humans, thus far the h , h , or h , and n or n influenza viruses have established transmittable infections. influenza viruses mutate rapidly, and these mutations affect mainly (but not exclusively) the genes encoding the surface proteins. point mutations that cause gradual changes are referred to as antigenic drift, and allow the virus to evade protective neutralizing antibody responses induced by previous infections. most annual epidemics are caused by antigenic drift variants. rearrangements of the ha-or na-encoding gene segments between viral types circulating in humans and those endemic in animals result in more dramatic changes, also called antigenic shifts, and the pandemics of with h n and with h n were caused by such new types of influenza virus. according to the world health organization (who), a pandemic is the emergence of a serious new disease caused by an agent that spreads easily among humans. who recommends three measures to lessen the impact of the next influenza virus pandemic: ( ) increased surveillance to allow for the earliest possible warning that a human pandemic has started; ( ) early intervention to stall global spread and prevent further adaptations; and ( ) development of an effective pandemic vaccine. available vaccines against influenza virus are seasonal vaccines that are updated annually to incorporate the latest circulating viruses. seasonal vaccines are composed of three different influenza viruses, which are typically two subtypes of influenza a virus and one strain of influenza b virus. the vaccine composition is generally agreed upon in spring to allow for manufacturing and distribution before onset of the influenza season in late fall to winter. seasonal vaccines are currently derived from egg-grown viruses that are either inactivated and then given systemically or attenuated by cold adaptation and given directly to the airways. pandemic vaccines, the focus of this chapter, are at this stage virtual vaccines of an unknown composition. they aim to protect against a newly evolved pandemic influenza virus. a pandemic vaccine may thus have to be manufactured at the onset of a pandemic, or alternatively one would need to devise a vaccine that induces broadly cross-reactive protection, unlike the current vaccines. influenza a viruses are enveloped spherical viruses which contain eight segments of single negative-stranded rna. segments , , and encode the transcriptase complex composed of basic polymerases (pb) (segment ) and pb (segment ) and acid polymerase (pa, segment ). segment encodes the hemagglutinin (ha), which has receptor-binding activity, promotes cell fusion, and is the major target for neutralizing antibodies. segment encodes the nucleoprotein (np) which complexes the viral rna to form the nucleocapsid. np is a major target for cross-reactive cd + t cells in mice and humans (falk et al. ) . segment encodes the viral neuraminidase (na), a cell surface protein with enzymatic activity, which also provides a target for neutralizing antibodies. drugs such as zanamivir and oseltamivir, which block the enzymatic cleavage of sialic acid residues by na, are available and can be used to treat or prevent infections (garman and laver ) . segment encodes matrix (m) protein and . m has ion channel activity, which is blocked by the antiviral drug amantadine (ison and hayden ) . m protein is also a target for cross-reactive cd + t cells in humans, while the m ectodomain is a target for nonneutralizing but nevertheless protective antibodies . segment encodes nonstructural proteins (ns) and . the twentieth century experienced three major influenza virus pandemics (table ) and several small abortive pandemics, as well as pandemic threats and numerous outbreaks in animals also called epizootics or panzootics. the first pandemic of the twentieth century started in in the usa and then spread to africa and europe, first to france and then spain, and subsequently to every part of the globe. this pandemic was caused by an h n virus and is paradoxically and unfairly referred to as the spanish flu. the pandemic that started in march of and lasted until june of killed half a million americans and somewhere between and million humans worldwide (johnson and mueller ) . this virus infected nearly % of the population and killed . % of all of those that became infected. it is estimated that million people died during the first weeks of the pandemic. death rates were high in humans between the ages of - , an age group which generally recovers easily from influenza a virus infection. during the initial stages of the pandemic, the early symptoms of infection, which included hemorrhages and lung edema followed by death within - h, were commonly misdiagnosed. the severity of the symptoms is assumed to have been caused by an excessive release of cytokines in response to the virus (kash et al. ) , which was most severe in healthy adults with sturdy immune systems. the h n virus was recently isolated from victims preserved in permafrost, and upon sequencing the virus was rederived through genetic engineering . this allowed for an extensive characterization of the virus using modern tools of science. the h n virus has several distinct features that may explain its unique virulence. most types of influenza a virus require trypsin-like enzymes for cleavage of the viral ha, which in turn restricts their cellular tropism. the na of the h n virus of can directly or indirectly cleave ha, thus rendering this virus independent of trypsin-like enzymes (steinhauer ) . increased virulence was further enabled by ns proteins, which allow the virus to disable the interferon (ifn) pathway (seo et al. ), a crucial component of both innate and adaptive immunity. human-to-human transmission, a prerequisite for a human pandemic, appears to have involved a switch in preferential binding of the ha protein from a- , sialic acid found in the avian enteric tract to a - , sialic acid present in the human respiratory tract ). this altered receptor binding activity can be achieved experimentally through a single amino acid exchange at position of the ha of the h n subtype. additional changes in the viral pb and pb proteins, which contain four amino acids that are conserved in human viruses and that differ from those prevalent in avians, are likely to have affected transmission between humans (russell and webster ) . the pandemic, also referred to as the asian flu, originated from a recombination between a circulating human virus and a virus endemic in ducks. the virus was first isolated early in in guizhou, china, and by february of had spread to singapore, and to the usa by june of that year. this virus, an h n virus, caused an estimated - million deaths worldwide (dunn ) . death rates were highest in the elderly. the pandemic, also called the hong kong flu, was caused by an antigenic shift of an h n virus to an h n virus. this pandemic was comparatively mild, causing an estimated , human deaths (cockburn et al. ; kilbourne ) . again mortality was high in those above years of age. in an h n virus that was first seen in japan and korea spread to military bases in the usa (lessler et al. ). further spread was not observed. in , an h n virus spread rapidly from china and caused epidemic disease in children and young adults (< years) worldwide. older humans were not affected, presumably due to protection from previous exposure to h n viruses. in the winter of , a novel swine influenza virus subtype was detected in military recruits at fort dix, new jersey. a total of soldiers became symptomatically infected and one died. there was only limited spread to humans living outside the military base. fearing a major pandemic, a vaccine was rapidly generated and administered to million humans. a few months after mass vaccination had started, reports of guillain-barré syndrome in vaccine recipients started to accumulate, and by early (when vaccination was stopped) more than cases of gbs had been reported, of which were fatal (langmuir et al. ). a highly pathogenic form of avian h n virus was first detected in asian poultry in (centers for disease control and prevention ) . during this year, a total of human cases were reported from hong kong, of which six were fatal. the virus rapidly caused pneumonia and multiple organ failure in infected individuals, which were mainly young adults. culling of infected flocks of poultry initially appeared to have stopped further spread, but then in additional human cases with a similar h n virus were recorded in vietnam (tran et al. (steel et al. ; cristalli and capua ) . other countries rejected the idea of bird vaccination due to fears that this may mask infections and allow for further mutations that may promote human transmissibility. similar to the h n virus, pathogenic h n virus activates ha through a trypsin-independent mechanism (hulse et al. ). pathogenic h n virus has a multibasic cleavage site that can be digested by furin and furin-like proteases, which are more ubiquitously present in human tissues than the trypsin-like enzymes that cleave ha of current human influenza viruses. the ns protein of pathogenic subtypes of h n virus renders the virus resistant to the activity of ifns and tumor necrosis factor (tnf)-a (seo et al. ) . the h n virus has changed since its first isolation in . such changes include resistance to the antiviral drug amantadine due to a m mutation first reported in from thailand (cheung et al. ) . the virus has become more lethal for humans and mice, and has gained robustness against destruction in the environment. the virus has increased its host range and has been shown to cause disease in felines such as tigers (keawcharoen et al. ) , which are otherwise resistant to influenza a virus infections. in , an h n virus, which also originated from poultry, caused illnesses in two children in hong kong. both children survived and there was no serological evidence that the virus spread to their contacts. a number of epizootics and panzootics have been caused by a wide variety of influenza viruses. in poultry, numerous outbreaks with highly pathogenic influenza viruses have been reported from all over the globe within the last years. these outbreaks were caused by a variety of subtypes, such as h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , and others. h n virus, which is currently endemic in asia, africa, and europe, has within the last eight years caused the deaths of millions of birds, many of which were culled to prevent further spread and to protect humans. influenza virus outbreaks have been observed in other species. for example, from to , several hundred harbor seals died along the coast of new england due to infection with a h n virus (geraci et al. ) . as of , h n circulates in pigs (gramer et al. ) . horses have been infected with h n and h n viruses (amonsin et al. ; oxburgh and hagström ) . the latter can also infect and kill canines. h n has caused the deaths of felines, including tigers and domestic cats (cristalli and capua ; steel et al. ) . several of these viruses have infected humans without achieving the capacity for human-to-human transmission. in , people were infected with h n influenza virus from poultry in the netherlands (koopmans et al. ) . in - , two residents of us mid-atlantic states showed serologic evidence of infection with h n . in , two poultry farm workers in british colombia became infected with h n virus (tweed et al. ) . in , egypt reported human infections with h n . any subtype of the influenza virus thus has the potential to infect humans and to evolve into a pandemic virus, which has to be taken into account when designing pandemic vaccines. more than % of deaths during seasonal influenza virus outbreaks occur in the elderly (³ years of age). immunosenescence during aging leads to impaired immune responses, which increases the susceptibility of the aged to infectious agents. the elderly are affected by primary immunological changes, which are part of the natural aging process, and secondary immunological changes caused by underlying diseases and unhealthy life styles (malaguarnera et al. ) . primary changes of the immune system in healthy elderly involve mainly t cells, though changes in natural killer (nk) cells and nk t cell function with age have been noted (ginaldi et al. c; solana and mariani ) . t cells show clonal senescence, their potential for expansion is decreased, and their ability to produce certain cytokines and to respond to cytokines decreases. the proportion of t cells with a memory cell phenotype increases while numbers of naïve t cells decrease. stimulation with new antigens appears to result in shortened immunological memory (ginaldi et al. b ). the t cell repertoire loses diversity (effros et al. ) due to chronic antigenic stimulation, leading to continued clonal expansion of some t cells, which undermines the homeostatic balance of the immune system. primary b cell responses in the elderly are commonly low and short-lived, resulting in antibodies with low affinity (ginaldi et al. a ). formation of germinal centers is decreased, antigen transport is impaired, and follicular dendritic cells show atrophy and their capacity to form antigen depots is reduced (zheng et al. ; aydar et al. ). autoantibodies are more common and the b cell repertoire becomes more restricted. many of these changes reflect secondary effects due to an age-related decline of helper functions from cd + t cells, which show reduced expression of critical costimulatory receptors that are essential for activation of b cells, germinal center formation and rearrangement, and hypermutation of immunoglobulin genes. underlying chronic diseases dramatically increase the risk of serious complications of an influenza virus infection. patients with one or two chronic diseases have or -fold (respectively) greater risk for developing pneumonia upon influenza virus infection (janssens and krause ; stott et al. ) . underlying chronic heart, lung, or liver diseases increase the risk of serious influenza virus infection in all age groups, not just the elderly. vaccines perform poorly in the elderly, commonly resulting in inadequate and short-lived titers of protective antibody responses (biro ; saurwein-teissl et al. ) . current influenza virus vaccines provide - % protection against a closely related virus in those < years of age, but only - % protection in humans above the age of . young children, pregnant women, and immunosuppressed individuals also have an increased risk for influenza a virus-associated morbidity. another risk factor is superinfection of the airways with bacterial pathogens, which can enhance virulence of the influenza virus through bacterial proteases (callan et al. ). on the other hand, influenza virus can increase bacterial infection by destroying respiratory epithelium and increasing bacterial receptor (mccullers ) . other risk factors include living in institutionalized settings such as prisons or nursing homes, or working in healthcare, where the risk of exposure and the risk of further spread are increased. vaccines aim to induce memory immune responses that, upon encountering the virus, are rapidly reactivated or recruited to either completely prevent an infection by causing so-called sterilizing immunity, or to rapidly control viral spread. it is thus important to understand which type of immune response provides reliable protection in order to specifically design immunogens that elicit this type of a response. influenza virus pandemics unfortunately have an element of surprise on their side by their very nature, and it may be unrealistic to expect that at the onset of a pandemic, which can potentially spread around the globe within less than six months, sufficient doses of a reliable vaccine or efficacious antiviral drugs will be available to protect the entire human population. other preventions, such as activation of protective innate immune responses in those at immediate risk for infection, may add to the repertoire we can call upon to combat the next influenza virus pandemic. innate immunity can provide resistance to influenza virus infection, as has been demonstrated in animals treated with immunomodulators such as baculovirus, lentidan, double-stranded rna, or modified heat-labile toxin of escherichia coli prior to infection (abe et al. ; irinoda et al. ; saravolac et al. ; williams et al. ) . clinical trials in children who were vaccinated with an attenuated influenza a virus vaccine after the onset of an influenza a virus outbreak also suggested that protection was at least in part mediated by an innate immune response to the vaccine (piedra et al. ). influenza a virus infection leads to the rapid increase of proinflammatory cytokines in nasal and pulmonary secretions (jao et al. ; gentile et al. ) . the virus causes the activation and maturation of dendritic cells and stimulates plasmacytoid dendritic cells to secrete large amounts of type i ifns (lópez et al. ; cella et al. ) . influenza virus activates macrophages to secrete il- , and and tnf-a (mak et al. ; pirhonen et al. ). il- in turn induces ifn-g production by nk cells. the early cytokine response to influenza virus can be pronounced and can result in significant pathology (van reeth et al. ) . nevertheless, early cytokines such as interferons also provide resistance to influenza a viruses (beilharz et al. ; fattal-german and bizzini ) . ns of h n renders the virus resistant to the antiviral activity of ifns and tnf-a (sekellick et al. ) . reassortant influenza a viruses carrying the ns of h n induce increased levels of cytokines in mice and decreased levels of il- (lipatov et al. a ). both macrophages and nk cells can kill infected cells and are crucial to early infection control (zychlinsky et al. ; tsuru et al. ) , as are natural igm and the early components of the classical pathway of complement, which together can neutralize influenza virus (jayasekera et al. ). inhalation infection with influenza a virus triggers a mucosal immune response in the upper respiratory tract that is initiated within nasal-associated lymphoid tissue (nalt) in mice and within waldeyer's ring (tonsils) in primates. in the lower respiratory tract, responses are induced in bronchus-associated lymphoid tissues. responses can also be detected in distant lymphoid tissues such as spleen or blood. infection causes a local secretory iga response as well as igm and igg antibodies directed mainly against the viral ha. antibody-secreting cells can be detected in mice in the respiratory mucosa and in lung tissue within five days after infection. dimeric iga (diga) antibodies which are transcytosed across epithelial cells upon binding to their receptors can bind to de novo synthesized viral antigens and block viral assembly, thus contributing to viral clearance (tamura and kurata ) . influenza virus-specific cd + and cd + t cells are induced upon intranasal application of influenza a virus (roti et al. ; swain et al. ). viral clearance following a primary infection is mediated in part by cd + t cells and in part by antibodies, which in turn require the activity of cd + t helper cells for their induction. lack of cd + t cells does not affect induction of a primary cd + t cell response to influenza a virus (yap and ada ; mozdzanowska et al. ) , although absence of cd + t cells in general reduces the magnitude of the memory cd + t cell pool and the cd + t cell recall response. neither ifn-g nor ifn-a/b appear to be essential for viral clearance (price et al. ) , although loss of both ifn pathways has been reported to exacerbate disease. perforin is essential for viral clearance, and mice lacking perforin show delayed viral clearance and increased mortality to influenza a virus infection (topham et al. ) . increased mortality was also observed in il- receptor knockout mice (szretter et al. ); these mice developed normal cd + t cell responses and viral titers were only modestly above those of normal mice. il- receptor knockout mice showed a defect in recruitment of inflammatory cells to the site of infection, most notably neutrophils and cd + t cells. a secondary infection with influenza a virus can be prevented by local siga and can be blunted by rapid activation of memory b cells. neutralizing iga antibodies are thought to primarily prevent infection of the upper respiratory tract, while serum igg plays a role in protecting against viral pneumonia (tamura and kurata ) . protective neutralizing antibody responses induced by infection or vaccination are subtype specific and do not provide protection against heterotypic challenge. their ability to provide resistance to an antigenic drift subtype depends on the degree of antigenic variation between the viruses (kaye et al. ) . it must pointed out, however, that although the role of neutralizing antibodies in providing resistance to influenza virus is not debated, it remains far from clear-cut. some mouse studies showed that adaptive transfer of neutralizing secretory iga protected the animals, while transfer of neutralizing antibodies of the igg isotype was inefficient (renegar and small ) . other mouse studies showed that protection by h -specific igg monoclonal antibodies can be achieved against h n infections (hanson et al. ). yet others reported protection by igg antibodies that bound ha but failed to neutralize the virus (mclain and dimmock ). one monoclonal neutralizing antibody was described that cross-reacted between h and h and consequently protected animals upon passive transfer against infection with either virus (okuno et al. ). in other virus infections, such as those with rabies virus, where neutralizing antibodies are known to play a dominant role in protection against infection and disease, protective titers of neutralizing antibodies have been defined. for rabies virus, a titer of or above . international units protects against challenge; this knowledge has greatly facilitated vaccination efforts. in contrast, it is still not known what titer of influenza a virus-neutralizing antibodies reliably provides protection against disease. in general, it is assumed that titers above : are protective, although numerous clinical trials have demonstrated that humans with lower titers were protected while others with higher titers developed symptomatic infections. protection against heterotypic challenge (i.e., challenge with a different subtype of influenza virus than that used for immunization) can be mediated by a number of mechanisms. as already mentioned above, some neutralizing antibodies can cross-neutralize several subtypes of influenza a virus. nonneutralizing antibodies to the ectodomain of matrix protein (m e) can protect against heterotypic challenge in animal models (mozdzanowska et al. ) . the amino acid (aa) long m e is conserved in its nine n-terminal amino acids and shows relative minor variability in the remaining sequences. this is likely to reflect a lack of selective pressure, as natural infections or traditional vaccines induce only low antibody responses to m e ). the currently circulating avian h n and h n subtypes show sequence variability with previous human isolates that affect m e antibodybinding sites. for example, they show changes in amino acids at positions - of m e (h n : pirnewg to ptrngwg, or ptrnewe) (liu et al. ) . cd + t cells induced by repeated infections appear to contribute little to natural resistance to influenza virus infection in humans. this may be linked to suboptimal stimulation of this t cell subset upon natural infection, as human volunteers with exceptionally high levels of circulating influenza a virus-specific cd + t cells showed reduced viral shedding upon an experimental infection compared to those with low levels of pre-existing influenza a virus-specific cd + t cells (epstein ; murasko et al. ) . in mice, a number of studies showed that cd + t cells protect, while other showed that they fail to protect. early studies from the group of g. ada showed that adoptive transfer of influenza virus immune cells provided protection against challenge with a heterotypic subtype of the virus (yap and ada ) . these studies were confirmed by r. dutton and colleagues, who studied the efficacy of passively transferred, in vitro activated cd + t cells isolated from mice transgenic (tg) for a t cell receptor (tcr) to the influenza a virus ha (cerwenka et al. ) . transfer of naïve tcr-tg cd + t cells failed to provide resistance to challenge. protection against a lethal infection could be provided by the transfer of rested memory-like or effector tcr-tg cd + t cells, although the latter effected more rapid viral clearance, which may indicate that the rested cd + t cells needed to expand before they assumed effector functions. protection was only mediated by cd + t cells that were able to home to the infected respiratory tissues. poxvirus vectors expressing the influenza a virus np, which induce a cd + t cell response (andrew et al. ), were shown to induce some protection against heterotypic challenge (endo et al. ; altstein et al. ) . further studies showed that although vaccinia virus vectors expressing the influenza virus np induced only limited protection in mice, adoptive transfer of t cells isolated from np-immune mice and expanded in vitro were highly effective (mbawuike et al. ). yet another group reported that a vaccinia virus vector which expressed a sequence of np that induced a sturdy cd + t cell response in mice, including in their lungs, completely failed to induce protective immunity as assessed by peak viral loads, morbidity or mortality (lawson et al. ). heterotypic t cell-mediated protection was also reported after immunization of mice with an adjuvanted influenza virus vaccine (sambhara et al. ) or with dna vaccines expressing internal proteins of influenza virus (saha et al. ; fu et al. ) . another group reported that protection upon intranasal immunization with an adjuvanted nucleoprotein vaccine was mediated by t helper cells of the th type rather than by cd + t cells (tamura et al. ). yet another group reported protection with an adenovirus vector expressing nucleoprotein (roy et al. ). in our hands, subunit vaccines expressing the nucleoprotein induced strong cd + t cell responses that could readily be detected in spleen, blood, or even lungs of vaccinated mice. nevertheless, vaccinated mice were not reliably protected against disease or death following challenge with influenza a virus (unpublished). overall t cell protection studies largely agree that adoptive transfer of in vitro expanded cd + t cells provides protection against influenza virus. results on the protective nature of in situ activated influenza virus-specific cd + t cells range from solid protection to complete absence of protection, even under circumstances where high numbers of influenza virus-specific cd + t cells were present in the airways at the time of challenge. the lack of consistency of protection through cd + t cells may reflect genetic differences in the mouse strain used for the experiments, differences in the dose or type of challenge virus, differences in the interval between vaccination and challenge, and/or differences in the functionality of cd + t cells induced by various approaches. the take-home message for developing an influenza vaccine that is useful for preventing or ameliorating a pandemic therefore remains ambiguous. neutralizing antibodies protect against ha provided there is sufficient homology between the vaccine and the infecting virus. antibodies against m e protect against a wider array of subtypes, as m e is more conserved; nevertheless, m e shows some variability, and protection through m e-specific antibodies is not as robust as protection provided by neutralizing antibodies. the rules that govern cd + t cell-mediated protection against influenza virus remain ill-defined. influenza virus was first isolated in (smith et al. ) , and effective vaccines were developed and tested by - and became available by (francis et al. a,b) . vaccines were thus not available during the spanish flu pandemic, but rapidly became available during the asian flu pandemic (gundlefinger et al. ) , when they were mainly used in military personnel. although the hong kong flu subtype was identified rapidly, vaccine production was delayed, and a vaccine was not available during the outbreak. until recently, all available influenza vaccines were trivalent inactivated (killed) virus vaccines. initially whole-virus vaccines were used, which were then replaced by by the less reactogenic split-virus vaccines. in june of , a live attenuated, cold-adapted, temperature-sensitive, trivalent influenza virus vaccine was licensed in the united states for use in humans between and years of age. multiple clinical trials have been performed in adults (demicheli et al. ), children , and the elderly (jefferson et al. ) to assess the efficacy of influenza vaccines. studies on live vaccines are still limited, but to date they suggest that such vaccines may be more effective than inactivated vaccines in some cohorts (treanor et al. ) . one manuscript published an analysis of trials involving a total of , adults (demicheli et al. ) which showed that the live attenuated vaccines reduced the number of cases of serologically confirmed influenza by % while the inactivated vaccines had a vaccine efficacy of %. the yearly recommended vaccines had low effectiveness against clinical influenza cases or time off work, the later a nonspecific outcome that included illness caused by influenza as well as other agents. the authors concluded that universal immunization of healthy adults is not supported by their results. fifty-one studies involving , children were included in an analysis of influenza virus vaccine efficacy in children ). the attenuated vaccines showed an efficacy of % in children older than two years, while inactivated vaccines had a lower efficacy of %. in children under two, the efficacy of inactivated vaccine was similar to placebo. in another study, results from randomized clinical studies covering a total of , children were analyzed and reported to show an overall vaccination efficacy of % against clinical disease, % against laboratoryconfirmed cases, and % against acute otitis media. between-study variability was related to the children's age and study quality. for example, when studies from the ussr were excluded from the analysis, the overall efficacy of the vaccine in preventing clinical cases increased from % to % (manzoli et al. ) . indirect evidence for the effectiveness of annual influenza virus vaccination of children can be gained from japan, where as of school children were vaccinated annually. vaccination became mandatory in the s and was discontinued in . during the time of mandatory vaccination, mortality among the elderly declined markedly, presumably due to reduced exposure to their infected grandchildren. sixty-four studies were analyzed to determine the efficacy of influenza vaccination in the elderly (jefferson et al. ; rivetti et al. ). in homes for elderly individuals, the effectiveness of vaccines against disease caused by influenza virus could not be demonstrated. when the vaccines were closely matched to the circulating virus subtype, they prevented pneumonia, hospital admission, and deaths. in elderly individuals living in the community, vaccines were not significantly effective against clinical influenza or pneumonia that were not laboratory confirmed. the authors concluded that vaccination was useful in long-term care facilities but not necessarily in community settings. another large analysis of community-living elderly came to the opposite conclusion. this analysis showed that vaccination was associated with a % reduction in the risk of hospitalization for pneumonia and a % reduction in the risk of death (nichol et al. ). other smaller studies showed that immunization of frail elderly did not reduce the rate of hospital admissions due to acute respiratory illnesses (jordan et al. ) , and that vaccination failed to reduce the overall mortality of the elderly (rizzo et al. ). in summary, although annual influenza virus vaccination is highly recommended, especially for high-risk populations, results of clinical trials designed to prove their efficacy remain controversial and thus far do not fully support the notion that vaccination affords reliable protection against influenza virus infection and its sequelae. who has summarized a number of global pandemic phases that have been adopted in federal and regional response plans and serve to define the type of responses required. details on these phases and suggested courses of action can be obtained online (see also as of early , the usa is currently in an interpandemic period, while parts of asia, africa, and eurasia have entered phase iii(/iv) of a pandemic alert period; pathogenic avian h n virus has repeatedly infected humans without causing proven human-to-human transmission yet. small clusters of human infections that may reflect human-to-human transmission have been observed. in anticipation of an influenza virus pandemic that would kill up to an estimated . million americans and require the hospitalization of an estimated million americans, in november of the department of health and human services issued a pandemic influenza plan, and state governments developed blueprints for local pandemic response plans. funding was provided to increase infrastructure, enhance vaccine production capability, and to augment basic knowledge on influenza virus pathogenesis and host responses. how much experience do we have with pandemic influenza virus vaccines? as mentioned above, vaccines for the spanish and hong kong flu pandemic were not available at that time, and the vaccine that was available during the asian flu pandemic was mainly used in military personnel (dull et al. ) . in summary, our experience with the global use of a vaccine for pandemic influenza virus is nonexistent. one could envision four scenarios for the role of a vaccine in the next influenza virus pandemic: (a) an ideal outcome in which the world could be vaccinated with a universal vaccine that would never allow another pandemic to strike, (b) an optimistic outlook in which sufficient doses of a vaccine are produced in advance in order to rapidly immunize those at the epicenter of the pandemic and those at high risk, before additional vaccine for global immunization could be produced and distributed, (c) a pragmatic attitude that prepares as effectively as possible for the next pandemic without necessarily expecting that a vaccine will be on hand at the start of the pandemic, and (d) a worst-case scenario, in which the next pandemic influenza virus will outsmart us. in an ideal scenario, scientists would develop a universal vaccine for influenza virus, industry would rapidly get involved in conducting large-scale trials needed for licensure, and then, with the aid of governments and philanthropic agencies, initiate a worldwide vaccination program before the next pandemic subtype of influenza virus evolves. ideally, the vaccine would be adjuvanted to induce robust, long-lasting immunity not only in healthy adults but also in high-risk populations such as the elderly, infants, or those suffering from chronic diseases. it is hoped such a vaccine would prevent the development of any future influenza virus pandemics (table ) . in experimental animals, some vaccines affect protection against heterotypic challenge with influenza virus, such as vaccines based on m e (mozdzanowska et al. ; liu et al. ; slepushkin et al. ; fan et al. ; frace et al. ; neirynck et al. ; de filette et al. ; eurekalert ) . protection through m e-expressing vaccines is mediated by humoral immunity and can be achieved by passive transfer of monoclonal m e-specific antibodies prior to virus challenge (mozdzanowska et al. ) . one vaccine developed by w. gerhard and colleagues was based on an m e peptide linked to universal t helper cell epitopes. others developed m e vaccines based on papilloma virus-like particles (ginaldi et al. a) , or fusion proteins linking m e to hepatitis b virus core protein (de filette et al. ). all of these subunit vaccines elicited antibodies to m e in animals that protected against subsequent challenge with different types of influenza a virus, and the m e-hepatitis b virus core fusion protein vaccine has now entered a phase i trial (eurekalert ) . the immunogenicity of an m e vaccine could be increased by adjuvants such as toll-like receptor ligands (huleatt et al. ). in one study, passively immunized animals were challenged with influenza a viruses that were identical or that differed in their m e sequence; animals were protected against viruses that expressed the same m e sequence but not against subtypes with m e variants, (fan et al. ) . several m e sequences corresponding to the h n , h n , and h n influenza subtypes were formulated using a liposome-based vaccine technology and evaluated as potential immunogens for the development of a "universal" influenza vaccine. mice immunized with the polyvalent liposomal m e survived challenges with different subtypes of influenza virus, and antiserum from immunized mice provided passive protection to naïve mice (ernst et al. ) . one study on a dna vaccine expressing m e fused to the nucleoprotein of influenza a virus reported increased mortality in vaccinated pigs, indicating that a poorly immunogenic vaccine (and dna vaccines are commonly poorly immunogenic, especially in larger species) may exacerbate influenza virus-associated pathology (heinen et al. ) . in most studies, vaccines expressed one sequence of m e. notwithstanding, although m e is far less variable than ha, it is not completely conserved, and mutants such as those present in recent h n variants have been observed, suggesting that a universal m e-based vaccine for influenza a virus should incorporate several common variants of m e, including those that are present (aydar et al. ). m e vaccines induce some protective immunity, although this protection wanes against high challenge doses of virulent virus. m e vaccines thus need to be optimized further, either through the use of novel adjuvants, or by their incorporation into more immunogenic vaccine carriers. once this is achieved, m e vaccines may well become part of an ideal universal vaccine for influenza virus, alleviating the need for a pandemic vaccine. under some circumstances, cd + t cells directed against conserved sequences of influenza a virus provide protection against heterotypic challenge; however, under other circumstances, they fail to protect. influenza virus antigens such as np and m proteins (which carry conserved epitopes of influenza a virus) and vaccine carriers that induce robust cd + t cell responses to such epitopes are readily available-the missing link remains a solid knowledge of what distinguishes a protective cd + t cell from one that is ineffective or, even worse, exacerbates disease. once this knowledge is gained, a universal influenza vaccine based on antigens that aim to induce t cell responses could be developed and deployed, either alone or in combination with an m e-expressing vaccine. currently there is no universal vaccine for influenza virus in the industrial pipeline, and who estimates that it will take at least another - years before such vaccines become available. the highly pathogenic h n virus that is endemic in wild birds in asia, africa, and europe, and has spread to poultry and from there to humans, is currently viewed as a major candidate to evolve into the next pandemic subtype, through mutations that allow for efficient human-to-human transmission. several entities have started to develop vaccines based on current subtypes of avian influenza virus under (a) the assumption that h n would evolve into a pandemic virus, and (b) the optimistic conjecture that the pandemic virus would have sufficient homology with currently circulating viruses to allow for cross-protective immunity ( table ) . the asian highly pathogenic avian h n virus has divided into two antigenic clades. clade includes human and bird isolates from vietnam, thailand, and cambodia and bird isolates from laos and malaysia. clade viruses include bird isolates from china, indonesia, japan, south korea, the middle east, europe and africa, and were primarily responsible for human h n infections during - . clade is further subdivided into six subclades with a distinct geographic distribution. over time, the pool of h n viruses that could potentially evolve into a pandemic form is diversifying rapidly, making it very difficult to decide on a specific virus as the basis for a vaccine. initial vaccines were developed for protection against the h n subtype that was isolated from humans in hong kong in february , but this virus has changed substantially, so these vaccines are now most likely no longer useful (suguitan et al. ) . in april , who made a h n prototype seed virus available to manufacturers. in august , who changed the prototype and now offers three new prototype viruses. future changes of the reference virus to accommodate additional mutations are expected. developing vaccines to h n based on traditional approaches was a challenging task. the highly virulent h n viruses rapidly kill embryonated chicken eggs, which are used to propagate the influenza a viruses for the annual vaccines. a number of manufacturers thus started to develop cell culture systems based for example on vero or mdck cells to propagate h n influenza virus. cell-grown influenza virus vaccines were tested in humans and showed immunogenicity and safety profiles that were comparable to those of egg-grown vaccines (halperin et al. ) . others used reverse genetics to develop reassortant viruses in which gene segments encoding ha and na were derived from highly pathogenic h n virus, and all other genes were derived from the h n virus a/pr/ / , which was isolated in puerto rico in and is commonly used in animal studies (lipatov et al. b; subbarao et al. ) . the ha gene was further modified to replace the stretch of six basic amino acids at the cleavage site that can be digested by furin (shi et al. ) , and the resulting virus is avirulent in chickens and can be grown readily in eggs. most vaccines for highly pathogenic h n tested to date were based on inactivated or attenuated virus used with or without adjuvant subbarao et al. ; lipatov et al. b; stephenson et al. ) . these vaccines achieved protection in mice, ferrets, or birds against pathogenic subtypes of influenza a virus expressing the same or a closely related ha through the induction of neutralizing antibodies. in a human clinical trial with an inactivated h n influenza virus vaccine attenuated through reverse genetics and changes of the ha cleavage site to allow propagation in eggs, protective titers of neutralizing abs could be induced in volunteers after two doses of the vaccine (treanor et al. ) . unfortunately, the dose that was needed to induce immune responses was six times that used for current influenza a virus vaccines. in a subsequent larger trial, the vaccine was adjuvanted with aluminum hydroxide, which did not improve the vaccine's immunogenicity (bresson et al. ) . others reported the opposite results (leroux-roels et al. ). additional clinical trials were conducted with inactivated whole-virus vaccine, which caused seroconversion in ~ % of vaccines that received the highest vaccine dose ( mg), again indicating that the ha of h n viruses is not a potent inducer of neutralizing antibody responses (lin et al. ) . the immunogenicity of h n vaccine could be increased by adding mf adjuvant (nicholson et al. ) . clinical trials have also been initiated with an attenuated h n vaccine. a number of groups have developed subunit vaccines for h n virus. a dna vaccine encoding h provided partial protection against challenge with h n virus (bright et al. ) , while dna vaccines encoding np or m were comparatively ineffective (epstein et al. ) . dna vaccine priming followed by a booster immunization with a replication-defective vector of adenovirus of the human serotype (adhu ), both expressing np, augmented specific t cell responses and provided superior protection against challenge (epstein et al. ) . two groups explored adhu vectors expressing h ; they were shown to induce b and t cell responses against ha which protected against challenge with a pathogenic h n virus hoelscher et al. ). nevertheless, it should be pointed out that seroprevalence rates of neutralizing antibodies to adhu are high in humans, especially those living in asia or africa, and that such antibodies strongly dampen antibody responses to the transgene product expressed by an adhu vector. fowl pox vectors (qiao et al. ) and alpha virus replicons (schultz-cherry et al. ) expressing h were also shown to induce protective immunity against h n influenza viruses. in , a h n virus caused an outbreak in poultry in the netherlands during which humans became infected and mainly developed conjunctivitis, while one died of complications due to pneumonia. the virus isolated from the fatal case showed a mutation in the polymerase gene that was similar to that of highly pathogenic h n (munster et al. ) . a reassortant vaccine expressing h and n on the a/pr background was developed, and an inactivated adjuvanted form of this vaccine induced neutralizing antibodies and protection in mice after two doses (de wit et al. ) . a low-pathogenic subtype of h n has been circulating in poultry in the northeastern usa since , while highly pathogenic avian influenza has sporadically appeared, such as in an outbreak in chile (h n ) in , an outbreak in the united states (h n ) in , and an outbreak in canada (h n ) in (senne ) . these viruses readily become pathogenic through some mutations (lee et al. ) and thus pose a pandemic threat. a reassortant vaccine has been generated against h n and was shown to induce protective immunity in mice and ferrets . thus far, only one h n influenza vaccine has been licensed by the united states food and drug administration (fda), while a number of other candidate vaccines against h n avian influenza are in clinical trials and should be licensed in the near future. whether or not these vaccines will be protective against the next pandemic virus is unknown. initiating widespread vaccination before the actual pandemic starts would thus raise ethical questions-any vaccine, even one that is well tolerated, carries risks for the recipients. without any clear indication that h n is turning into a pandemic virus, the risk of vaccination would surpass the benefit to the vaccinated individual. this was demonstrated during the swine flu vaccine debacle of , when vaccination against a virus that never spread caused a serious, crippling disease in hundreds of recipients. in addition to causing harm to these unfortunate individuals, this incident continues to provide ammunition to the vocal community of vaccine opponents that seem to have forgotten the haunting images of humans disfigured by poxviruses or wards full of polio virus-infected children on iron lungs. in contrast, a universal vaccine could be given before a pandemic, as such a vaccine would prevent seasonal influenza, thus providing a tangible benefit to its recipients. scio me nihil scire (i know that i don't know) is a famous saying attributed to the greek philosopher socrates by plato. if we take a socratic view of the form and shape of the next pandemic influenza virus, mass production of a vaccine that induces protection through subtype-specific antibodies before a pandemic virus has actually evolved makes no sense. making sure that an infrastructure is in place to rapidly and efficiently respond to a pandemic is, on the other hand, a prudent approach, and global agencies such as who in concert with governments are preparing for the next pandemic. constant monitoring of evolving subtypes, new human infections and potential human-to-human spread in order to detect a pandemic at the earliest possible time is a vital task, and this been established. the sharing of virus isolates to identify potential vaccine candidates is important and requires international collaboration. indonesia, which has the highest incidence of h n related human deaths, initially refused to provide h n samples to who in order to focus attention on their concern that while developing countries provide new viral isolates, any resulting vaccines produced by commercial companies would likely be used primarily in developed countries. by march , indonesia, which was the only country that took this stance, reversed its policy. to date, global vaccine production capacity is insufficient, as seasonal influenza vaccines are only used by a small portion of the global population. a number of vaccine manufacturers have started to increase their production capacity, and it is expected that the current capacity will double by . the long-term goal is to increase production capacity to three billion doses per year. it is also expected that manufacturing will commence in less developed countries. idiosyncrasies of the actual pandemic vaccine, such as the required dose and the potential need for repeat injections, will determine whether this capacity will suffice. this is being addressed by attempts to increase the immunogenicity of influenza virus vaccines through novel adjuvants. poorly growing vaccine subtypes could also offset the speed of production, and this is being tackled by developing cell-culture-based systems and through the use of reverse genetics to achieve rapid attenuation of influenza viruses. recent studies indicated that antibodies to currently circulating viruses show some cross-reactivity with h n . these studies must be confirmed. if indeed annual vaccinations with certain types of vaccine offer some degree of heterotypic protection a broadening of seasonal influenza vaccine coverage would certainly be warranted, especially in countries that have advanced to a "pandemic alert period." progression through all of the steps of vaccine development, from preclinical trials in rodents through to clinical trials in humans and then licensing, generally takes - years. the fda, which regulates vaccine licensure in the usa, has formulated guidelines for industry for the accelerated licensure of pandemic influenza vaccines based on the induction of neutralizing antibodies to hemagglutinin in order to ensure that regulatory aspects do not hinder the rapid deployment of a pandemic vaccine. vaccines that do not contain viral hemagglutinin are not covered by these guidelines. nevertheless, even if the production of a vaccine starts on the day that a pandemic virus has been identified, it will still take - months until the very first dose of vaccine is available. other control measures are therefore needed to limit damage until the vaccines become available for everyone. in the usa, the federal government and state governments have formulated pandemic preparedness plans to be followed in the event of a pandemic. these plans not only list the responsibilities of government entities and individuals, but also address the use of limited pharmacological agents and other types of control measures. similar to vaccines, the availability of antiviral drugs is expected to be limited. antiviral drugs may slow the pandemic if used in a timely manner at the epicenter of the pandemic. they may also be extraordinarily useful for protecting persons that provide essential healthcare and for maintaining vital infrastructure. assuming a delay of at least - months before sufficient doses of vaccine are available for global mass vaccination, vaccines will have to be rationed at the beginning of the pandemic. governments will issue lists of high-priority personnel that are to be vaccinated first. although these lists vary from state to state in the usa, they typically include hospital and health department staff, emergency medical service personnel and household members, law enforcement personnel, fire fighters, medical laboratory workers, emergency management personnel, long-term care facility staff, utility workers (gas, electric, water, waste management, etc.), communications personnel, fuel and food suppliers, public transportation and air travel personnel, corrections workers, morticians/coroners/medical examiners, pharmacists, red cross field workers, us postal service staff, persons involved with vaccine production and delivery, etc. it has been suggested that once more vaccine becomes available, healthy working adults should be vaccinated before high-risk populations such as children or the elderly. it is still to be decided who will ultimately purchase the pandemic vaccinefederal or state governments, who could clearly facilitate orderly distribution, or the private sector. if the private sector carries the cost, insurance companies will have to take a stance on cost coverage, and plans will have to be developed for the uninsured. once a vaccine becomes available, other problems will arise. some of these issues are being addressed by governments, and the examples below apply to the usa. a pandemic vaccine would not be expected to undergo the vigorous safety testing typical of other vaccines. manufacturers that develop pandemic vaccines can request indemnification from the secretary of health and human services for "an activity that involves unusually hazardous risks and for which insurance is not available or sufficient to cover those risks." a vaccine will have to be distributed rapidly and in an orderly manner. the us government ruled that it may mobilize the phs commissioned corps to distribute vaccines to federal agencies with direct patient care responsibilities, or to states, tribes, and other localities through the national disaster medical system and through agreements between the federal government, states, and localities. liability protection must be put in place. the us government has ruled that federal employee administrators are covered by the federal government and could make claims through the federal tort claims act. state employees may be covered for malpractice or tort claims coverage under state law. federal contractor and private sector employees distributing the vaccine would be expected to carry malpractice insurance or they could be covered by the volunteer protection act, state good samaritan act, or state emergency compact provisions. if a person is injured following administration of a pandemic vaccine or antiviral medication in connection with his/her employment, compensation may be available under a state's worker's compensation program. for federal employees, compensation may be available under the federal employees' compensation act. assuming that vaccines will not be available at the onset of the next pandemic, other nonpharmaceutical measures are being discussed to ( ) limit international spread, ( ) reduce spread within national and local populations, and ( ) reduce an individual person's risk for infection. influenza viruses are typically shed - h before the onset of disease, and virus titers peak within the first three days after onset of symptoms and then decline by day - . it is possible but not yet proven that the virus spreads by shedding a small amount of virions before the first symptoms occur, which could markedly reduce the effectiveness of most quarantine measures. infection occurs predominantly via droplets formed by coughing or sneezing individuals. infection by aerosolized virus is less common. the virus can also be transmitted via infected hands or surfaces. the wearing of masks and the employment of appropriate sanitizing measures to clean hands and infected surfaces are thus useful actions for protecting an individual (jefferson et al. ) . it is thought that temporary protection of populations may in part be achieved by implementing quarantine measures. in the pandemic of , some island countries enacted maritime quarantines (markel et al. ). australia and madagascar were able to delay the start of the pandemic by several months, while samoa and new caledonia remained completely free of the pandemic. quarantine measures were attempted on land, but they were unsuccessful. quarantine was tried again in , but it largely failed. quarantine was very successful in stopping the sars epidemic of (hsieh et al. ). the sars virus has a longer incubation time than influenza virus, and peak virus titers are not reached until several days after the onset of symptoms. isolating cases thus proved an effective way to prevent the further spread of sars, but it is unlikely to work against influenza virus. who recommends exit screening for international travelers that leave countries that are affected by an influenza virus pandemic. it is unclear if and to what extent this may delay the spread of the virus. however, any delay would be useful in allowing extra time for vaccine production. the us government has issued an executive order adding potentially pandemic influenza viruses to the list of quarantinable diseases, which empowers the centers for disease control and prevention (cdc) to detain, medically examine, or conditionally release individuals that are reasonably believed to be carrying a communicable disease. the intent of this order is to enable the united states to respond efficiently and effectively in the case of an outbreak by pandemic influenza viruses. this order gives legal authority to the department of health and human services to isolate a passenger arriving on board an international vessel that show evidence of infection with a novel influenza virus, even if that passenger refuses to cooperate. the federal government (such as the cdc) generally defers to the state and local health authorities in the use of their own quarantine powers. state-implemented interventions would likely include the isolation and treatment of infected individuals, voluntary home quarantine for members of the households of infected individuals, the closure of schools and universities, and the encouragement of social distancing through the cancellation of large public gatherings. who estimates that in the worst case scenario more than million people could die as a consequence of the next influenza virus pandemic. other estimates are higher: - million deaths. to put this number into perspective, million is approximately the total population of south america, or half of the population of europe. death tolls will primarily depend on the virulence of the next pandemic-current clades of highly pathogenic h n virus kill > % of those infected. it would also depend on the ease with which the virus is transmitted between humans, and last but not least on the effectiveness of control measures. late detection of a pandemic, which could evolve in a war-ravaged country that lacks surveillance, would shorten the time interval available for the development of a vaccine. vaccine production could be further delayed by problems with the seed virus, such as toxicity toward eggs or cell substrates, poor immunogenicity of the vaccine, or unacceptable reactogenicity. the modern world, with its high degree of social and economic interdependency, has not yet experienced a major pandemic that disrupts crucial aspects of local and global infrastructure. lack of available workers and restricted movement could threaten essential services, and the failure of any one system could trigger others to fail too, causing cascading breakdowns. highly pathogenic h n influenza viruses have been circulating for more than ten years and many have come to believe that they are unlikely to jump species. although this may well be the case, the consequences of relaxing our efforts to prepare for the next pandemic could be horrific. in , when smallpox virus was on the brink of extinction and polio was disappearing from developed countries, the us surgeon general, william stewart, told congress that it was time to "close the books on infectious diseases." unfortunately, congress listened and shifted federal funding from microbiology/virology to cancer and cardiovascular diseases. ever-increasing liability costs made the industry more and more reluctant to stay involved in vaccine production, and the number of companies that produce vaccines has now become so limited that annual vaccine shortages are common (markel et al. ). infectious agents have continued to take a major toll on human lives. since more than new microbes have emerged, such as hiv- , which has claimed over million lives thus far. even old microbes such as influenza virus continue to take a toll on human lives; for example, in the usa alone, seasonal influenza causes over , hospitalizations and over , death each year. the world will experience a new influenza virus pandemic. no-one can predict when it will happen, where it will start, what virus will cause it, and what the global and local impacts will be. the only aspect of the next pandemic we can predict with certainty is that it will happen. complacency, not only in the usa but worldwide, has weakened the infrastructure for efficiently combating newly emerging infectious agents, and this infrastructure needs to be rebuilt. communications with the public must be improved globally in a manner that informs accurately without alarming unduly. it is clear that this has not yet been achieved, as exemplified by a recent h n outbreak in west bengal in india, where children were reported to have unprotected contact with birds that died due to infection with h n . our continued lack of knowledge about the very basic question of the immunobiology of influenza viruses and the efficacy of vaccines in different cohorts is mind-boggling. we still do not fully understand correlates of protection against influenza virus, and debates on the role of cd + t cells in providing protection are continuing. this knowledge needs to be generated, especially in order to enable the development of a universal influenza virus vaccine, the "holy grail" that could prevent future influenza virus pandemics. baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice immunization with influenza a np-expressing vaccinia virus recombinant protects mice against experimental infection with human and avian influenza viruses genetic analysis of influenza a virus (h n ) derived from domestic cat and dog in thailand cell-mediated immune responses to influenza virus antigens expressed by vaccinia virus recombinants follicular dendritic cells in aging, a "bottle-neck" in the humoral immune response protection from lethal influenza virus challenge by oral type interferon age-related change of the absolute number of igg-, and igm-bearing b-lymphocytes and t-lymphocytes in human peripheral blood following influenza vaccination safety and immunogenicity of an inactivated split-virion influenza a/vietnam/ / (h n ) vaccine: phase randomised trial impact of glycosylation on the immunogenicity of a dna-based influenza h ha vaccine cleavage of influenza a virus h hemagglutinin by swine respiratory bacterial proteases plasmacytoid dendritic cells activated by influenza virus and cd l drive a potent th polarization isolation of avian influenza a(h n ) viruses from humans-hong kong naive, effector, and memory cd t cells in protection against pulmonary influenza virus infection: homing properties rather than initial frequencies are crucial distribution of amantadine-resistant h n avian influenza variants in asia origin and progress of the - hong kong influenza epidemic practical problems in controlling h n high pathogenicity avian influenza at village level in vietnam and introduction of biosecurity measures the universal influenza vaccine m e-hbc administered intranasally in combination with the adjuvant cta -dd provides complete protection protection of mice against lethal infection with highly pathogenic h n influenza a virus by using a recombinant low-pathogenicity vaccine strain vaccines for preventing influenza in healthy adults pandemic influenza in ; review of international spread of new asian strain monovalent asian influenza vaccine: evaluation of its use during two waves of epidemic asian influenza in partly immunized penitentiary population cd t cells and aging homotypic and heterotypic protection against influenza virus infection in mice by recombinant vaccinia virus expressing the haemagglutinin or nucleoprotein of influenza virus prior h n influenza infection and susceptibility of cleveland family study participants during the h n pandemic of : an experiment of nature dna vaccine expressing conserved influenza virus proteins protective against h n challenge infection in mice protection against multiple influenza a subtypes by vaccination with highly conserved nucleoprotein protection against h , h , h and h influenza a infection with liposomal matrix epitope vaccines universal flu vaccine being tested on humans identification of naturally processed viral nonapeptides allows their quantification in infected cells and suggests an allele-specific t cell epitope forecast preclinical study of influenza virus a m peptide conjugate vaccines in mice, ferrets, and rhesus monkeys assessment of the anti-viral effect of a short-term oral treatment of mice with live saccharomyces cerevisiae cells influenza a virus infection engenders a poor antibody response against the ectodomain of matrix protein modified m proteins produce heterotypic immunity against influenza a virus protective effect of vaccination against induced influenza a protective effect of vaccination against induced influenza b protective cellular immunity: cytotoxic t-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by dna immunization protection of mice and poultry from lethal h n avian influenza virus through adenovirus-based immunization increased interleukin- levels in nasal lavage samples following experimental influenza a virus infection mass mortality of harbor seals: pneumonia associated with influenza a virus the immune system in the elderly: i. specific humoral immunity the immune system in the elderly: ii. specific cellular immunity the immune system in the elderly: iii. innate immunity serologic and genetic characterization of north american h n swine influenza a viruses effectiveness of influenza vaccines during an epidemic of asian influenza safety and immunogenicity of a trivalent, inactivated, mammalian cell culture-derived influenza vaccine in healthy adults, seniors, and children passive immunoprophylaxis and therapy with humanized monoclonal antibody specific for influenza a h hemagglutinin in mice vaccination of pigs with a dna construct expressing an influenza virus m -nucleoprotein fusion protein exacerbates disease after challenge with influenza a virus development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human h n strains in mice impact of quarantine on the sars outbreak: a retrospective modeling study potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza m e to the tlr ligand flagellin molecular determinants within the surface proteins involved in the pathogenicity of h n influenza viruses in chickens stimulation of microbicidal host defence mechanisms against aerosol influenza virus infection by lentinan therapeutic options for the management of influenza pneumonia in the very old production of interferon in volunteers infected with asian influenza natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity efficacy and effectiveness of influenza vaccines in elderly people: a systematic review physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review updating the accounts: global mortality of the - "spanish" influenza pandemic influenza-related mortality in the italian elderly: no decline associated with increasing vaccination coverage global host immune response: pathogenesis and transcriptional profiling of type a influenza viruses expressing the hemagglutinin and neuraminidase genes from the pandemic virus studies on inactivated influenza vaccines. i. the effect of dosage on antibody response and protection against homotypic and heterotypic influenza virus challenge in mice avian influenza h n in tigers and leopards influenza pandemics of the th century transmission of h n avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands an epidemiologic and clinical evaluation of guillain-barré syndrome reported in association with the administration of swine influenza vaccines primary pulmonary cytotoxic t lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza a virus nucleoprotein peptide do not protect mice against challenge pathogenic potential of north american h n avian influenza virus: a mutagenesis study using reverse genetics antigen sparing and cross-reactive immunity with an adjuvanted rh n prototype pandemic influenza vaccine: a randomised controlled trial transmissibility of swine flu at fort dix, safety and immunogenicity of an inactivated adjuvanted whole-virion influenza a (h n ) vaccine: a phase i randomised controlled trial pathogenesis of hong kong h n influenza virus ns gene reassortants in mice: the role of cytokines and b and t cell responses efficacy of h influenza vaccines produced by reverse genetics in a lethal mouse model monoclonal antibodies recognizing evetpirn epitope of influenza a virus m protein could protect mice from lethal influenza a virus challenge sequence comparison between the extracellular domain of m protein human and avian influenza a virus provides new information for bivalent influenza vaccine design tlrindependent induction of dendritic cell maturation and adaptive immunity by negative-strand rna viruses the generation of 'cytotoxic' macrophages in mice during infection with influenza a or sendai virus immunosenescence: a review the efficacy of influenza vaccine for healthy children: a meta-analysis evaluating potential sources of variation in efficacy estimates including study quality nonpharmaceutical interventions implemented by us cities during the - influenza pandemic safety evaluation in chickens of candidate human vaccines against potential pandemic strains of influenza control of mucosal virus infection by influenza nucleoprotein-specific cd + cytotoxic t lymphocytes insights into the interaction between influenza virus and pneumococcus protection of mice from lethal influenza by adoptive transfer of non-neutralizing haemagglutination-inhibiting igg obtained from the lungs of infected animals treated with defective interfering virus induction of influenza type a virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein roles of cd + t cellindependent and -dependent antibody responses in the control of influenza virus infection: evidence for noncognate cd + t cell activities that enhance the therapeutic activity of antiviral antibodies the molecular basis of the pathogenicity of the dutch highly pathogenic human influenza a h n viruses role of humoral and cell-mediated immunity in protection from influenza disease after immunization of healthy elderly a universal influenza a vaccine based on the extracellular domain of the m protein effectiveness of influenza vaccine in the community-dwelling elderly safety and antigenicity of non-adjuvanted and mf -adjuvanted influenza a/duck/ singapore/ (h n ) vaccine: a randomised trial of two potential vaccines against h n influenza protection against the mouse-adapted a/ fm/ / strain of influenza a virus in mice by a monoclonal antibody with cross-neutralizing activity among h and h strains a pcr based method for the identification of equine influenza virus from clinical samples development and evaluation of an influenza virus subtype h n vaccine candidate for pandemic preparedness trivalent live attenuated intranasal influenza vaccine administered during the - influenza type a (h n ) outbreak provided immediate, direct, and indirect protection in children virus infection activates il- beta and il- production in human macrophages by a caspase- -dependent pathway the role of alpha/beta and gamma interferons in development of immunity to influenza a virus in mice development of a recombinant fowlpox virus vector-based vaccine of h n subtype avian influenza passive transfer of local immunity to influenza virus infection by iga antibody vaccines for preventing influenza in the elderly a case-control study of elderly patients with acute respiratory illness: effect of influenza vaccination on admission healthy human subjects have cd + t cells directed against h n influenza virus partial protection against h n influenza in mice with a single dose of a chimpanzee adenovirus vector expressing nucleoprotein the genesis of a pandemic influenza virus a fused gene of nucleoprotein (np) and herpes simplex virus genes (vp ) induces highly protective immunity against different subtypes of influenza virus heterotypic protection against influenza by immunostimulating complexes is associated with the induction of cross-reactive cytotoxic t lymphocytes immunoprophylactic strategies against respiratory influenza virus infection lack of antibody production following immunization in old age: association with cd (+)cd (−) t cell clonal expansions and an imbalance in the production of th and th cytokines influenza virus (a/hk/ / ) hemagglutinin expressed by an alphavirus replicon system protects chickens against lethal infection with hong kong-origin h n viruses transient resistance of influenza virus to interferon action attributed to random multiple packaging and activity of ns genes avian influenza in north and south america ecology and epidemiology of avian influenza in north and south america lethal h n influenza viruses escape host anti-viral cytokine responses the ns gene of h n influenza viruses circumvents the host anti-viral cytokine responses generation of an attenuated h n avian influenza virus vaccine with all eight genes from avian viruses protection of mice against influenza a virus challenge by vaccination with baculovirus-expressed m protein a virus obtained from influenza patients vaccines for preventing influenza in healthy children nk and nk/t cells in human senescence a combination in-ovo vaccine for avian influenza virus and newcastle disease virus role of hemagglutinin cleavage for the pathogenicity of influenza virus crossreactivity to highly pathogenic avian influenza h n viruses after vaccination with nonadjuvanted and mf -adjuvanted influenza a/duck/singapore/ (h n ) vaccine: a potential priming strategy influenza in old age evaluation of a genetically modified reassortant h n influenza a virus vaccine candidate generated by plasmid-based reverse genetics live, attenuated influenza a h n candidate vaccines provide broad cross-protection in mice and ferrets t cell responses to influenza virus infection: effector and memory cells role of host cytokine responses in the pathogenesis of avian h n influenza viruses in mice defense mechanisms against influenza virus infection in the respiratory tract mucosa acceleration of influenza virus clearance by th cells in the nasal site of mice immunized intranasally with adjuvant-combined recombinant nucleoprotein cd + t cells clear influenza virus by perforin or fasdependent processes avian influenza a (h n ) in patients in vietnam evaluation of trivalent, live, cold-adapted (caiv-t) and inactivated (tiv) influenza vaccines in prevention of virus infection and illness following challenge of adults with wild-type influenza a (h n ), a (h n ), and b viruses safety and immunogenicity of a recombinant of an inactivated subvirion influenza a (h n ) vaccine mechanism of protection during the early phase of a generalized viral infection. ii. contribution of polymorphonuclear leukocytes to protection against intravenous infection with influenza virus characterization of the reconstructed spanish influenza pandemic virus a two-amino acid change in the hemagglutinin of the influenza virus abolishes transmission human illness from avian influenza h n , british columbia in vivo studies on cytokine involvement during acute viral respiratory disease of swine: troublesome but rewarding innate imprinting by the modified heat-labile toxin of escherichia coli (ltk ) provides generic protection against lung infectious disease the recovery of mice from influenza virus infection: adoptive transfer of immunity with immune t lymphocytes fine specificity and sequence of antibodies directed against the ectodomain of matrix protein of influenza a virus immunosenescence and germinal center reaction a homogeneous population of lymphokineactivated killer (lak) cells is incapable of killing virus-, bacteria-, or parasite-infected macrophages key: cord- -bhe dky authors: cohen, cheryl; reubenson, gary title: influenza date: - - journal: viral infections in children, volume i doi: . / - - - - _ sha: doc_id: cord_uid: bhe dky influenza is one of the commonest infections in human populations, and causing substantial morbidity and mortality globally. the influenza virus is divided into different types and subtypes, three of which are currently circulating widely in humans: influenza a(h n ) and influenza b. the virus undergoes constant evolution, leading to annual seasonal winter epidemics in temperate countries and necessitating annual updates to the vaccine. rarely, completely new influenza viruses can emerge in human populations, giving rise to influenza pandemics. children aged < years (especially those < years) and those with underlying illness such as cardiac, respiratory and severe neurologic disease have an increased risk of severe outcomes associated with influenza. pregnant women have an increased risk of severe influenza. complications may involve the respiratory tract (e.g. otitis media or pneumonia) or, less commonly, other organ systems (e.g. encephalitis or myocarditis). specific antiviral treatment should be offered as soon as possible for hospitalized children with presumed or confirmed influenza and for influenza of any severity for children at high risk of severe complications of influenza without waiting for laboratory confirmation. antiviral treatment is usually not warranted for uncomplicated influenza as this is usually self-limiting. annual influenza vaccination should be offered to all individuals at increased risk for complications of influenza. vaccine cannot be given to children aged < months but maternal influenza immunization during pregnancy is recommended and can confer protection to the young infant. influenza is one of the commonest infections in human populations, infecting a significant percentage of the population each year and causing substantial morbidity and mortality globally. while seasonal influenza is an important cause of morbidity and mortality, novel influenza virus strains can emerge with the potential to cause global pandemics. children are a common source of inluenza transmission in the community and form an important risk group for severe influenza illness (particularly infants and children with underlying chronic illnesses). influenza viruses are enveloped viruses from the family orthomyxoviridae [ ] . influenza has a negative sense rna genome divided into eight segments. the segmented genome allows for exchange of genes between influenza viruses of the same type through genetic reassortment. two types of influenza viruses, influenza a and b, cause epidemic disease in humans. the influenza a viruses are divided into subtypes and influenza b viruses into lineages based on their antigenic structure. the influenza a subtypes are differentiated based on characteristics of the haemagglutinin (ha) and neuraminidase (na) surface antigens. haemaglutinin is responsible for virus attachment during the early stages of infection and is the main antigen against which the host immune response is directed. neuraminidase facilitates the release of mature virus from the cell surface. currently there are two influenza a subtypes (influenza a(h n ) and influenza a(h n )pdm and two influenza b lineages (yamagata and victoria) co-circulating globally in human populations. influenza a viruses of at least ha and na subtypes have been isolated from animals such as birds, pigs, horses and dogs. because many different ha and na subtypes can circulate in animals, animals such as birds and pigs may be the reservoir of emerging influenza virus subtypes which can infect humans [ ] . several of these subtypes such as influenza a(h n ) or influenza a(h n ) can cause severe illness and even death in individuals in close contact with animals but are not able to be efficiently transmitted from person-to-person [ ] . antigenic drift is the emergence of new influenza virus antigenic variants as a result of point mutations and recombination which occurs during viral replication [ ] . this frequent emergence of antigenic variants contributes to seasonal influenza epidemics and leads to the requirement for annual assessment of the need to update the viruses included in the influenza vaccine. antigenic shift is a term for larger genetic changes which occur infrequently in influenza a viruses. new, or substantially different, influenza a virus subtypes which emerge in humans, have the potential to cause pandemics if they are efficiently transmitted between humans in the presence of little or no pre-existing population immunity [ ] (fig. . ). epidemiology including pandemics it is estimated that - % of the population become infected with influenza each year and about % of these develop symptomatic illness. rates of influenza infection are highest in children aged - years [ ] . annually in children aged < years there are approximately million new cases of influenza, million cases of influenza-associated acute lower respiratory tract infection (alri) ( % of all cases of paediatric alri) and one million cases of influenza-associated severe alri ( % of all severe alri) globally [ ] . between , and , influenza-associated deaths in children < years are estimated to occur each year, with % of these occurring in developing countries. influenza is associated with approximately % of respiratory hospitalizations in children < years worldwide ranging from % in children aged < months to % in children aged - years [ ] . influenza-associated hospitalization rates are more than three times higher in developing than industrialised countries. the incidence and mortality associated with influenza can vary substantially from year-to-year as a result of different circulating types and subtypes with differing propensity to cause severe illness. years in which influenza a(h n ) predominates may typically be associated with increased risk of severe disease [ ] . people of all ages may develop symptomatic influenza infection but the highest rates of influenza-positive influenza-like illness (ili) are seen in children aged - years [ ] . school-age children are an important source of infection in the community and influenza outbreaks can occur in schools during the influenza season [ ] . during the influenza season, influenza is an important cause of school absenteeism. illness in children can cause a substantial economic burden as a result of caregiver absenteeism from work to care for ill children as well as outpatient visits in children and can lead to additional antibiotic courses being prescribed. hospitalizations and mortality during the influenza season can be substantial. in severe influenza seasons, the large number of medical care visits as a result of influenza can overwhelm health systems. the highest rates of influenza-associated hospitalizations and deaths are typically seen in individuals aged ≥ years, < years and those with underlying medical conditions that confer an increased risk for severe influenza [ ] . children aged < years and, to a lesser extent, those aged - years have increased rates of influenzaassociated hospitalization and mortality compared to older children. children with underlying illnesses, particularly cardiac, respiratory and severe neurologic disease have an increased risk of severe outcomes associated with influenza. a study from south africa, found that amongst children aged < years, malnutrition, prematurity and hiv infection were associated with increased odds of influenza-associated hospitalization [ ] . hiv-infected children have an approximately two times elevated risk of influenza hospitalization and are more likely to die of influenza once hospitalized compared to hiv-uninfected children [ , ] . pregnant women have an increased risk of severe influenza. some studies suggest that influenza in pregnancy may be associated with adverse outcomes in infants born to these women (such as low birth weight, pre-term birth and stillbirth), but others have disputed this [ ] . in temperate climates influenza typically causes annual seasonal epidemics in the winter months, between april and september in the southern hemisphere and between october and april in the northern hemisphere [ , ] . in more tropical climates influenza commonly circulates year-round with two or more peaks which may coincide with climatic events such as the rainy season [ ] . this may present challenges for decision-making around the best time to vaccinate and which vaccine formulation (the northern or southern hemisphere) should be used (see section on vaccines) [ ] . the start, peak, size and duration of the influenza season may vary substantially from year-to-year. seasonal influenza can give rise to outbreaks in closed settings such as schools, these can occur at any time of year but are more common during the influenza season [ ] . influenza pandemics are caused by the emergence and spread in human populations of a new influenza a virus with either a new or substantially altered ha or na combination against which there is little or no immunity in humans, which is easily transmitted between humans and causes clinical illness in humans [ ] . the emergence of a pandemic influenza strain is unpredictable and can occur through two mechanisms. firstly, a host could be simultaneously infected with two different influenza virus subtypes which could allow for exchange of genetic material or reassortment and the emergence of a new subtype. for example this could occur if a pig were infected by both a human and avian origin influenza subtype simultaneously with genetic exchange leading to the emergence of a virus adapted to spread in humans, but with ha and/or na not currently circulating in humans. the second way that novel subtypes can emerge is if avian or other animal adapted subtypes are directly transmitted to humans and then undergo adaptation to allow transmission between humans. currently some avian influenza virus subtypes such as influenza a(h n ) can be transmitted to humans, usually following close contact with poultry, and cause severe infections. however, these viruses are not adapted for efficient transmission from person to person and therefore have not given rise to a new pandemic strain [ ] . global surveillance for new influenza virus strains is essential for early identification of novel strains to allow a global public health response. the pandemic of influenza a(h n ) is widely acknowledged as the most severe in recent times with an estimated > million deaths worldwide. other recent pandemics ( , asian flu h n and hong kong flu h n ) have been associated with a lower death toll [ ] . a characteristic of pandemic influenza strains is the shift in the age distribution of deaths from predominantly affecting the extremes of age (young infants and the elderly) to mortality in young adults aged - years [ ] . although influenza pandemics can cause substantial mortality, the annual cumulative deaths each year, associated with seasonal mortality, far outweigh this burden. in , a novel influenza a virus, influenza a(h n )pdm emerged in the human population and caused a global pandemic. this virus, was antigenically distinct from the h n virus which had been circulating in human populations from to early and was thought to have entered the human population from pigs (hence the colloquial name "swine flu"). the overall mortality burden of this strain was estimated at between , and , deaths globally, similar to the annual mortality burden from seasonal influenza, although this strain did exhibit the characteristic pandemic age shift, disproportionately affecting individuals aged - years [ ] . subsequently, influenza a(h n )pdm has been circulating in human populations and immunity in the population has built up. influenza a(h n )pdm has become the predominant h n seasonal train, replacing those that previously circulated and behaves like any other seasonal influenza virus [ ] . influenza is predominantly spread person-to-person by large droplets and through direct contact with respiratory secretions [ ] . the contribution of airborne transmission is unclear. the incubation period for influenza typically ranges from to days (median days) [ ] . influenza virus is typically shed from the nasopharynx for up to days after illness but viral shedding may be longer in severely ill individuals, young children and immunocompromised individuals. the reproductive number for influenza is between and and the serial interval usually estimated at - days. an individual's susceptibility to infection and disease will depend on host characteristics including preexisting cellular or humoral immunity to influenza [ ] . young children may have no pre-existing immunity to influenza, but older children and adults have often been exposed to circulating influenza several times before and may also have pre existing immunity from vaccination. natural immunity is not fully protective, largely because of the variability of influenza ha and na. influenza virus replication predominantly occurs in the respiratory tract columnar epithelial cells, with infection leading to loss of cilia and cell death [ ] . damage to the respiratory tract as well as immunologic changes can lead to increased susceptibility to bacterial superinfection. viremia with influenza is relatively uncommon although constitutional symptoms are a prominent feature of clinical disease. in most children influenza infection results in acute self-limiting upper respiratory tract (urt) symptoms, however, systemic manifestations are not uncommon [ ] . factors that influence clinical presentation include: age of the child, previous influenza exposure, vaccination status, underlying disease states or co-morbidities, as well as viral factors. children are considered important influenza "vectors" and are often responsible for introducing the virus into their homes and broader social settings [ ] . influenza classically presents with the sudden onset of systemic (fever, myalgia, headache, and malaise) and urt symptoms (sore throat, cough, rhinitis). since many patients do not have all these typical symptoms, accurate clinical diagnosis is challenging particularly in the younger pre-verbal child and outside of the influenza season [ ] . a large study evaluating the clinical presentation of influenza in children found that almost all ( %) had fever; cough ( %) and rhinitis ( %) were also very common, but much lower proportions experienced headache ( %) or myalgia ( %) [ ] . younger children have not yet been exposed to influenza very often and so have yet to acquire immunity to a substantial repertoire of circulating seasonal influenza strains. they, therefore, are more likely to develop severe or complicated disease [ ] . further, they are less likely to manifest with classic symptoms, experience higher fevers (not uncommonly associated with febrile convulsions), less prominent urt involvement and more gastro-intestinal symptoms (vomiting, diarrhea, abdominal pain, loss of appetite). examination may be completely normal in some children, others may manifest with tachypnea, conjunctival injection, nasal inflammation and discharge, or cervical lymphadenopathy. oropharyngeal findings are often limited, even in those children complaining of a sore throat [ ] . symptoms of uncomplicated influenza usually start improving within a few days, but symptoms lasting more than a week are not uncommon. cough, in particular, may persist for a number of weeks, but steady improvement can be expected [ ] . the differential diagnosis of influenza largely depends on the presenting symptoms and clinical findings, but includes other respiratory viruses (rhinovirus, coronavirus, respiratory syncitial virus, human metapneumovirus, adenovirus, parainfluenza) and some bacterial urt infections (streptococcus pyogenes, mycoplasma). the clinical manifestations of these conditions are very similar, regardless of the implicated pathogen [ ] . all influenza strains may result in severe illness and knowing which infection a particular child has, is not helpful in predicting their disease course. complications may involve the respiratory tract (e.g. otitis media, pneumonia) or, less commonly, other organ systems (e.g. encephalitis, myocarditis). otitis media may occur in as many as % of cases; this may be related to the influenza virus itself or secondary infection with bacteria or other viruses [ , ] . symptoms of acute otitis media generally present a few days after onset of influenza symptoms. lower respiratory tract complications may include the following [ , ] : • laryngo-tracheo-bronchitis ("croup") • bronchiolitis • pneumonia-especially in children < years of age, often mild but may be severe, rapidly-progressive and occasionally fatal, particularly if associated with secondary bacterial infection (usually streptococcus pneumoniae or staphylococcus aureus). a variety of radiographic appearances have been described • acute exacerbation of asthma-this is the most common respiratory tract complication of influenza. central nervous system involvement can include the following [ ] [ ] [ ] : • aseptic meningitis • acute cerebellitis • transverse myelitis • guillain-barré syndrome • febrile seizure • necrotizing encephalitis • postinfectious encephalitis (also referred to as acute disseminated encephalomyelitis). neurologic complications appear to be more common in younger children and in those with underlying neurologic and neuromuscular disease. following the rapid decline in aspirin use over the last few decades, influenza-associated reye syndrome is now rare. mild transient myositis is common with influenza infection; it is more likely with influenza b and is associated with moderate elevations in creatine kinase levels [ ] . acute myositis is an important, severe, but rare, complication of influenza infection [ ] . it presents with extreme muscle tenderness, often involving the calf muscles, extreme elevations in creatine kinase as well as significant myoglobinuria. during influenza season, influenza should be considered in all children presenting with suggestive clinical features-this includes those already admitted to hospital, as nosocomial transmission of influenza is well described. influenza should still be considered outside of the influenza season, particularly in travelers and children residing in tropical and sub-tropical climates where year-round influenza transmission occurs. accurate clinical diagnosis of influenza is challenging, particularly in younger children. the lack of specific signs or symptoms results in patients receiving diagnoses of "influenza-like illness" or "viral upper respiratory tract illness" unless further diagnostic testing is undertaken. this degree of diagnostic uncertainty should be acknowledged, however, since most such cases are self-limiting and management is largely supportive, there is usually no need to obtain a precise microbiological diagnosis. currently available diagnostic tests for influenza include the following [ , ] : • rapid, point-of-care, antigen detection tests a number of different tests are currently available, although they remain unavailable in many settings. they provide results within min, and, when used appropriately, are helpful in confirming influenza infection. in general, they are insufficiently sensitive to reliably exclude the disease. further, their performance will depend on which antigens are expressed by currently circulating strains. when influenza activity is low, positive results are likely to be false-positives, however, their positive predictive value improves as influenza activity increases. conversely, during periods of high influenza activity, false-negatives are more likely and may warrant additional testing in some patients. since diagnostic confirmation seldom affects management of such children, the use of these tests is generally not recommended in low-resource settings and should be used judiciously in better-resourced areas. when used, it should be clear in the clinician's mind as to how the result is going to alter treatment: positive results can potentially reduce antibiotic usage and allow for early use of antivirals in those at high risk of complications or severe disease. however, it is important to recognize that the identification of influenza does not exclude the presence of bacterial co-infections. these tests are currently considered to be the most reliable for the diagnosis of influenza in children. amongst the available options, they are the most sensitive and specific. they can be performed on most respiratory samples, most commonly nasopharyngeal aspirates or swabs. they are also able to differentiate influenza a and b, as well as subtypes of influenza a. more recently, point-of-care pcr assays have becomes available in some developed world settings. they are performed on nasal swabs and can supply a reliable result in as little as min. while, not currently available in most developing world settings, if more affordable they could become an important new influenza diagnostic. since the live attenuated influenza vaccine contains influenza genetic material, recent receipt of this vaccine will also result in a positive pcr test. these are also performed on nasal or nasopharyngeal swabs and allow for the direct or indirect detection of influenza antigens. influenza a and b can be differentiated, however, the sensitivity of these tests is moderate and, particularly during periods of high transmission, negative tests may need to be repeated using a more sensitive methodology (pcr or culture). • viral culture viral culture takes at least - hours and so has limited utility in the routine diagnosis of influenza. however, they are helpful as part of surveillance activities and isolates can be used to inform annual vaccine planning. a variety (hemagglutination-inhibition, enzyme-linked immunosorbent assay (elisa), and complement fixation assays) of serological assays can be performed but are of limited diagnostic value as they require acute and convalescent sampling. a fourfold increase in titre allows for a retrospective influenza diagnosis to be made. their primary role is as a research tool. antivirals are available for the specific treatment of influenza. two classes of antiinfluenza drugs are available: neuraminidase inhibitors (e.g. oseltamivir, zanamivir) and m inhibitors (e.g. amantadine, rimantadine). both classes are inactive against all other respiratory viruses. resistance can emerge in circulating influenza virus strains and for this reason antiviral resistance should be constantly monitored through surveillance and recent guidance consulted for the latest antiviral resistance profiles. • neuraminidase inhibitors [ ] neuraminidase inhibitors prevent the release of new virions from influenza infected cells and are active against both influenza a and b. oseltamivir is the most widely available member of this drug class and can be used for children and adults. it is dosed orally and is approved for both treatment and prevention of influenza. it is available both in capsule form and as a powder for suspension, although the suspension has a relatively short shelf life and is often substantially more expensive than the capsule. as a result, the capsule is the most widely used formulation. when the oral suspension is unavailable the capsule can be opened and diluted with sweetened liquids to provide the appropriate dose. zanamivir is predominantly available as an inhaled formulation, although intravenous zanamivir is available for investigational use, particularly for severely ill patients or those with suspected or confirmed oseltamivir-resistant virus. the inhaled preparation is contra-indicated in children with a history of wheezing or other chronic respiratory condition. its use is not recommended for children younger than years of age. peramivir is an intravenous neuraminidase inhibitor that is not approved for use in children and is not widely available. neuraminidase inhibitors are generally well tolerated. common side effects include nausea, vomiting and rash, as well as bronchospasm with zanamivir. neuropsychiatric symptoms have been linked to oseltamivir use, particularly in japan, however, more recent evidence suggests no such causative link [ , ] . severe adverse reactions have been reported but are considered rare [ ] . the mainstay of influenza prevention remains immunization, but in high risk situations, amongst partially or unimmunized children, chemoprophylaxis can be considered. both pre-and post-exposure chemoprophylaxis have been advocated, however, concerns regarding induction of oseltamivir-resistance have tempered enthusiasm for this approach and their prophylactic use is increasingly discouraged. antiviral treatment should be offered as soon as possible for hospitalized children with presumed or confirmed influenza and for influenza of any severity for children at high risk of severe complications of influenza (table . ) [ ] . timely oseltamivir treatment can reduce the duration of fever and symptoms. there are no prospective randomized controlled trials of treatment efficacy of neuraminidase inhibitors in hospitalized children and for severe outcomes but observational data suggest that treatment does reduce the risk of hospitalization and death, although there is some controversy in the literature (table . ) [ ] . treatment should be initiated as early as possible. the benefit of any treatment is maximal early in the course of the disease and should be started within - hours of symptom onset. influenza diagnosis may not have been confirmed within this timeframe and so treatment will often be initiated empirically prior to microbiological confirmation. some evidence suggests benefit to those with very severe illness even when treatment is initiated later than hours into the disease course. therefore, in table . children at high risk of severe influenza in whom influenza antiviral treatment is recommended by the centers for disease control and prevention (cdc) and american academy of pediatrics (aap) current guidance [ , ] . children aged < years . persons with chronic pulmonary (including asthma), cardiovascular (except hypertension alone), renal, hepatic, hematologic (including sickle cell disease), or metabolic disorders (including diabetes mellitus) or neurologic and neurodevelopment conditions (including disorders of the brain, spinal cord, peripheral nerve, and muscle such as cerebral palsy, epilepsy [seizure disorders], stroke, intellectual disability, moderate to severe developmental delay, muscular dystrophy, or spinal cord injury) . persons with immunosuppression, including that caused by medications or by hiv infection . persons who are receiving long-term aspirin therapy . american indian/alaska native persons . residents of chronic care facilities patients with severe or complicated disease, treatment should be initiated even if > hours after illness onset. children meeting the clinical criteria for treatment should be treated irrespective of whether they have been vaccinated. since influenza is generally a mild, self-limiting disease in previously well children, most such children will not require antiviral treatment even when they present soon after symptom onset. treatment of such children increases the risk of adverse events, potentially increases the risk of resistance developing and may deplete medicine supply for those in greater need. • adamantanes [ , ] these agents target the influenza a m protein, which is essential for efficient viral replication. they have no activity against influenza b and are not active mg (two -mg inhalations) twice daily mg (two -mg inhalations) once daily against currently circulating influenza a strains. as such, there use is not currently recommended for the treatment or prevention of influenza. it has been suggested that they may have a role, in combination with oseltamivir, for the treatment of oseltamivir-resistant influenza a. vaccines and guidelines for vaccination influenza vaccination can be given to any person who wishes to reduce the risk of becoming ill during the influenza season. some countries such as the united states of america (usa) and united kingdom (uk) recommend influenza vaccination for all children, or all individuals. in addition, special effort should be made to vaccinate children at risk of severe influenza listed in table . . individuals such as healthcare personnel and childcare providers (especially those in contact with infants aged < months and children with underlying risk conditions) should be vaccinated to reduce the risk of transmission to high risk children. lastly, pregnant women are recommended to receive influenza vaccination, to reduce the risk of severe illness in the mother, to provide direct protection to the young infant through trans-placental transfer of maternal antibodies and to reduce the risk of transmission of influenza from the mother to the young infant [ ] . iiv has been available for many years and includes inactivated components of two influenza a (one each of influenza a(h n )pdm and influenza a(h n )) and one influenza b strains. since the s, two antigenically distinct influenza b lineages (victoria and yamagata) have been circulating globally. this is a limitation of iiv , as protection may be reduced when the circulating influenza b strain is of the lineage which is not included in iiv . the iiv includes an additional strain of the other influenza b lineage not included in tiv to make a total of four strains and thus potentially offers additional benefit of protection against both circulating influenza b lineages. inactivated influenza vaccines (iiv and iiv ) contain no live virus. standarddose iiv should contain μg of each haemagglutinin antigen in each . ml dose. iiv and iiv are available in formulations for both intramuscular (im) and intradermal (id) use but the id formulation is only licensed for use in individuals aged years and older. two formulations of iiv manufactured using technologies that do not include eggs have become available in recent years, but neither is licensed for individuals aged < years. these are cell-culture based inactivated influenza vaccine and recombinant influenza vaccine. d. hiv infection e. sickle cell anemia and other hemoglobinopathies f. diseases that necessitate long-term aspirin therapy, including juvenile idiopathic arthritis or kawasaki disease g. chronic renal dysfunction h. chronic metabolic disease, including diabetes mellitus i. any condition that can compromise respiratory function or handling of secretions or can increase the risk of aspiration, such as neurodevelopmental disorders, spinal cord injuries, seizure disorders, or neuromuscular abnormalities j. morbid obesity k. pregnancy . children aged months- months as this group (particularly those aged < months) has an increased risk of influenza-associated hospitalization and mortality b. individuals who come into contact with children at risk of severe influenza . all household contacts and out-of-home care providers of the following: children with high-risk conditions; and children younger than years, especially infants younger than months . all health care personnel (hcp) . all child care providers and staff; and . all women who are pregnant, are considering pregnancy, are in the postpartum period, or are breastfeeding during the influenza season laiv is a live attenuated influenza vaccine which is administered intranasally and licensed for use in individuals aged - years of age. since the laiv has only been available in a quadrivalent formulation. adjuvanted formulations of influenza vaccine are licensed for use in individuals aged ≥ years in the usa but not currently in children [ ] . they have been shown to have a higher efficacy then iiv in a randomized controlled trial in children [ ] . adjuvants have several potential advantages over more traditional vaccine formulations including increased immunogenicity, potentially reducing the amount of antigen required. they elicit a more robust immune response and could potentially reduce the number of doses needed in children. children aged months through years should receive two influenza doses administered ≥ weeks apart the first time influenza vaccine is administered. for young children who require two doses of influenza vaccine, vaccination should not be delayed to ensure that both doses are given with the same product. any licensed, effective influenza vaccine product may be used for each dose. it is important to document all doses of influenza vaccine administered in the child's medical records. in temperate countries, influenza vaccine should be administered as soon as possible after the influenza vaccine becomes available. the recommended dosage of influenza vaccine for patients of different age groups is described in table . [ ] . influenza vaccine effectiveness depends on characteristics of those being vaccinated (age and health), whether there is a good match between the circulating viruses and the viruses contained in the vaccine, and on influenza types and subtypes circulating each year. in general, influenza vaccines work best among children ≥ years and healthy adults. older people (≥ years), children < years and severely immunocompromised individuals often have poorer immune responses to inactivated influenza vaccine (iiv) compared with healthy adults. however, even for these people influenza vaccine still provides some protection. other products, e.g. high-dose influenza vaccine and adjuvanted vaccines, have been shown to be more effective in certain groups [ ] but these vaccines may not be available in all settings and are not licensed for use in all age groups. there have been a number of studies of iiv effectiveness in children aged - months. for seasonal iiv in young children, two doses of influenza vaccine provides better protection than one dose in the first season a child is vaccinated. estimates of iiv efficacy in young children are limited and vary by season and study design. efficacy is lower in children aged - months. data are unclear as to the effectiveness in hiv-infected children aged < years [ ] . a randomized controlled trial of laiv in healthy children aged - found a vaccine effectiveness of % ( % ci - %). several other randomized controlled trials and observational studies have demonstrated high efficacy of laiv against laboratoryconfirmed influenza. studies comparing efficacy of iiv and laiv have generally found that laiv has similar, or better efficacy, than iiv. since , laiv has replaced laiv as the available laiv. licensure was on the basis of immunogenicity studies. in , conflicting findings on the effectiveness of laiv emerged from different settings, with the usa withdrawing its recommendation for laiv use in the - influenza season based on concerns of reduced effectiveness, particularly against the a(h n )pdm component of the vaccine [ ] . studies from europe in the same seasons found moderate laiv effectiveness, similar to that of iiv for the same year [ , ] with recommendations for laiv remaining unchanged in england and europe. vaccinating individuals at risk of severe influenza may provide direct protection for these individuals. in addition, vaccinating individuals in close contact with people at risk for severe influenza may provide indirect protection through preventing transmission to high-risk individuals. vaccinating children can protect children directly and the general population indirectly. this strategy is especially important for individuals in whom influenza vaccine is not indicated, such as children aged < months (who may be protected through maternal immunization) [ ] [ ] [ ] . a randomized controlled trial conducted in south africa has shown that when pregnant women receive the influenza vaccine, their risk of developing influenza is halved, as is the risk to their infants in the first weeks of life [ ] . the vaccine has been shown not only to be efficacious for prevention of influenza in both mothers and their infants, but also safe [ ] [ ] [ ] . vaccination of healthcare workers may decrease the risk of spreading influenza to their patients. recent influenza vaccination does not preclude a diagnosis of influenza as the vaccine is not % effective. because of the large year-to-year variability in influenza vaccine effectiveness depending on the circulating influenza strains, many countries publish annual estimates of influenza vaccine effectiveness using a test-negative case-control study design. a systematic review of test-negative case-control studies found that the pooled ve was % ( % ci - ) for h n , % ( - ) for type b, % ( - ) for h n pdm , suggesting reduced protection of available vaccines against influenza a(h n ) in recent years [ ] . [ , ] the iiv is an inactivated vaccine, and has a well-established safety record. it is safe for use in pregnancy and in children ≥ months of age. minor illness, with or without fever, is not a contraindication to influenza vaccine administration. clinicians should always consult the manufacturer's package insert for current contraindications and precautions for particular products. contraindications to the administration of iiv include: • a history of severe (anaphylactic) hypersensitivity to any components of the vaccine including, egg protein, or after previous dose of any influenza vaccine. anaphylaxis is rare and a careful history will distinguish between anaphylactic reactions and allergic reactions like rashes. mild egg protein allergy is not a contraindication for influenza vaccine • infants < months of age. precautions to iiv administration include: • persons with moderate to severe illness with or without fever should preferably be immunized after symptoms have resolved • person who developed guillian-barrè syndrome within weeks of receiving an influenza vaccine. contraindications to the administration of laiv include the following: • children - years of age with a history of recurrent wheezing or a medically attended wheezing episode in the previous months because of the potential for increased wheezing after immunization • children with the diagnosis of asthma • children with a history of egg allergy • children who have received other live virus vaccines within the past weeks; however, other live virus vaccines can be given on the same day as laiv • children who have known or suspected immunodeficiency disease or who are receiving immunosuppressive or immunomodulatory therapies • children who are receiving aspirin or other salicylates • any female who is pregnant or considering pregnancy • children with any condition that can compromise respiratory function or handling of secretions or can increase the risk for aspiration • children taking an influenza antiviral medication (oseltamivir or zanamivir), until hours after stopping the influenza antiviral therapy • children with chronic underlying medical conditions that may predispose to complications after wild-type influenza infection. as for all vaccines, influenza vaccine should be administered in a setting where there is the ability to respond to acute hypersensitivity reactions. the most common adverse events following intramuscular iiv administration are pain and tenderness at the injection site. fever can occur in - % of children aged < years within hours of vaccination, but is much less common in older children. in trials, when iiv are administered, - % of those vaccinated experience local reactions in the arm, lasting for or days. short-term reactions (mild fever, malaise and muscle pains) have been reported in a much smaller proportion in the first few hours following vaccination. trials of the split and subunit vaccines show even fewer systemic reactions. there have been no strong temporal or causal associations of the current vaccines with more severe reactions. anaphylaxis is very rare but does occur as with all vaccines. more severe adverse events, like guillain-barré syndrome have been reported with a particular vaccine in the s but they are extremely rare. with the modern influenza vaccines, the causative risk is either found to be very rare ( . per million doses) [ ] or there is no causal link found at all [ ] [ ] [ ] and more association is found with influenza infection than vaccination [ ] . an increased risk of fever and febrile seizures was reported from australia in associated with the southern hemisphere iiv produced by csl biotherapeutics (now sequirus) [ ] . following this, many countries do not recommend the csl iiv for children aged < years. influenza vaccination during pregnancy has been shown to protect both the mother and her baby (up to months old) against influenza [ , , , ] . influenza vaccination is safe in pregnancy and influenza vaccines have been administered to millions of pregnant women over many years and have not been shown to cause harm to pregnant women or their babies [ ] . the most common adverse events associated with laiv include runny nose or nasal congestion, headache, lethargy and sore throat. laiv should not be administered to children with marked nasal congestion as this can reduce vaccine delivery. the safety of laiv is not established in people with a history of asthma, diabetes mellitus, or other high-risk medical conditions associated with an elevated risk of complications from influenza. the use of laiv in young children with chronic medical conditions is not recommended in the usa but is in some other countries. appropriate hand and respiratory hygiene (cough etiquette) has been shown to reduce influenza transmission in children in day-care or school [ ] . sick children and adults should remain at home and not attend school or work until symptoms have resolved to prevent transmission of influenza to others. surveillance for emerging respiratory viruses comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study global burden of respiratory infections due to seasonal influenza in young children: a systematic review and metaanalysis global role and burden of influenza in pediatric respiratory hospitalizations, - : a systematic analysis estimating deaths due to influenza and respiratory syncytial virus incidence of medically attended influenza during pandemic and post-pandemic seasons through the influenza incidence surveillance project closure of schools during an influenza pandemic prevention and control of seasonal influenza with vaccines recommendations of the advisory committee on immunization practices-united states, - influenza season risk factors for influenza-associated severe acute respiratory illness hospitalization in a high hiv prevalence setting-south africa severe influenzaassociated respiratory infection in high hiv prevalence setting, south africa mortality amongst patients with influenza-associated severe acute respiratory illness, south africa maternal influenza and birth outcomes: systematic review of comparative studies twenty-five years of outpatient influenza surveillance in south africa influenza seasonality in the tropics and subtropics-when to vaccinate? seasonal influenza vaccine policy, use and effectiveness in the tropics and subtropics-a systematic literature review pandemic versus epidemic influenza mortality: a pattern of changing age distribution global mortality estimates for the influenza pandemic from the glamor project: a modeling study transmission of influenza a in human beings inactivated influenza vaccines the underrecognized burden of influenza in young children clinical practice. prevention and treatment of seasonal influenza prevention of influenza in children clinical presentation of influenza in unselected children treated as outpatients clinical and epidemiologic characteristics of children hospitalized with pandemic h n influenza a infection duration of cough in acute upper respiratory tract infections understanding the symptoms of the common cold and influenza a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion respiratory viruses in laryngeal croup of young children neurologic complications in children hospitalized with influenza: characteristics, incidence, and risk factors acute childhood encephalitis and encephalopathy associated with influenza: a prospective -year review neurological manifestations of influenza infection in children and adults: results of a national british surveillance study influenza-associated myositis in children seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america healthcare workers handbook on influenza. the national institute for communicable diseases (nicd) the association between oseltamivir use and adverse neuropsychiatric outcomes among tricare beneficiaries, ages through years diagnosed with influenza assessment of neuropsychiatric adverse events in influenza patients treated with oseltamivir: a comprehensive review neuropsychiatric adverse effects of oseltamivir in the fda adverse event reporting system committee on infectious diseases. recommendations for prevention and control of influenza in children neuraminidase inhibitors for preventing and treating influenza in healthy adults and children vaccine against influenza who position paper oil-inwater emulsion adjuvant with influenza vaccine in young children efficacy of high-dose versus standard-dose influenza vaccine in older adults efficacy and immunogenicity of influenza vaccine in hiv-infected children: a randomized, double-blind, placebo controlled trial effectiveness of the live attenuated and the inactivated influenza vaccine in two-year-olds-a nationwide cohort study finland, influenza season / effectiveness of seasonal influenza vaccine for adults and children in preventing laboratory-confirmed influenza in primary care in the united kingdom: / end-of-season results influenza vaccination of pregnant women and protection of their infants effectiveness of maternal influenza immunization in mothers and infants maternal influenza vaccination and effect on influenza virus infection in young infants variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of testnegative design studies the guillain-barre syndrome and the - and - influenza vaccines guillain-barre syndrome and the - influenza vaccine guillain-barre syndrome and influenza vaccination in the us army, - guillain-barre syndrome in the united states, - and - . lack of an association with influenza vaccination investigation of the temporal association of guillain-barre syndrome with influenza vaccine and influenza like illness using the united kingdom general practice research database maternal influenza immunization and reduced likelihood of prematurity and small for gestational age births: a retrospective cohort study influenza vaccine given to pregnant women reduces hospitalization due to influenza in their infants safety of influenza vaccination during pregnancy effectiveness of hand hygiene interventions in reducing illness absence among children in educational settings: a systematic review and meta-analysis key: cord- - rm y authors: flower, darren r.; perrie, yvonne title: immunomic discovery of adjuvants, delivery systems, and candidate subunit vaccines: a brief introduction date: - - journal: immunomic discovery of adjuvants and candidate subunit vaccines doi: . / - - - - _ sha: doc_id: cord_uid: rm y mass vaccination, when coupled to profound improvements in general sanitation, has given rise to the most remarkable transformation in public health in human history. yet the development of vaccines remains largely trapped in the past, a hostage to the methodology of pasteur. infectious disease continues to threaten humanity, with new and renascent diseases emerging continually. the last two decades have seen a breath-taking revival in the commercial market for vaccines and the simultaneous emergence of a whole tranche of new technologies that promise to free vaccine development from the muddle of empirical thinking. in this short introduction, we set the scene for this renaissance, and explore how the combination of computational and experimental techniques promise so much for the future development of vaccines and the science of vaccinology. vaccination and sanitation are indisputably the most efficient, and thus costeffective, prophylactic treatments for infectious disease. together they are the firm bedrock upon which the modern world resides. the truth of this is often ignored by those intoxicated by the many distractions of life in the early twenty-first century, from burgeoning social media to the recondite discoveries of the large hadron collider. people say that the internet or the ipad or even facebook have transformed the world. if there is any truth in such assertions, then such transformations are at best shallow and superficial compared to the extraordinary reworking of lives that has transpired over hundreds rather than tens of years. the source of such change can be traced back, in part at least, to the discovery and exploitation of vaccines, vaccination, and vaccinology. for around the first years of the vaccine story, the story was solely that of smallpox. as recently as the late s, some - million cases of smallpox were recorded in countries, with annual deaths of million. yet today smallpox has, with the exception of a few well-guarded stockpiles, been completely eradicated: there have been no new cases for years. the story of smallpox is thus the high point of the vaccination story; no other disease has been eradicated. polio or poliomyelitis is the next nearest to full eradication, having long been targeted by a systematic, coordinated, worldwide eradication campaign. in , one such programme run by the pan american health organization effected a partial eradication of polio in the western hemisphere. subsequently, the global polio eradication program has radically reduced polio throughout the rest of the world, so that today we can count cases worldwide in the tens or hundreds instead of the hundreds-of-thousands or millions. yet, vaccine-preventable disease still kills millions. infectious and contagious disease cause approximately % of world mortality, particularly in children under five. while in developed countries, mortality for diseases such as diphtheria, polio, or measles is less than . %, in other parts of the world deaths from such infectious diseases is significant. pertussis, tetanus, influenza, hib, hepatitis b are all responsible for deaths that number in the hundreds-of-thousands. perhaps the most execrable situation is measles, which accounts for , (over ) and , (under ) deaths. however, the leading global causes of death worldwide remain tuberculosis; diarrhoeal illnesses, especially rotaviruses; hiv/aids; and malaria. in , . million contracted tb and . million died. disturbing though these numbers may seem, they nonetheless represent a significant reversal of a once ever-escalating trend. the number with latent tb peaked in at million, while deaths from tb reached their peak at . million in . however, these bald numbers are likely to be significant underestimates. let us also look at malaria. murray et al. have recently provided evidence that deaths from malaria over the thirty-year span to are much higher than previously believed [ ] . their epidemiological figures show a peaked distribution over this period, increasing from around a million in , peaking at approximately , , in , and then reducing to about , , in , with the greatest number dying in africa. these figures are roughly twice the values published by the who. it seems unlikely that the who's estimates for other major diseases are uniformly more accurate. why are these numbers so high? a principal reason is that there are no effective vaccines for either malaria or hiv, two of the who's big three diseases; nor is there expectation that such vaccines will appear in the near future, irrespective of the optimism of those working in the area. and as for tuberculosis-which is carried by around billion people worldwide-the only licensed vaccine has limited efficacy. many viral infections remain recalcitrant threats of the first order. about million people are infected with hepatitis b, million by hepatitis c, and million by human immunodeficiency virus type (hiv- ). this dire situation is further compounded by the threat from the new or previously unknown infectious diseases identified in the past years: hiv, west nile fever, ebola, dengue fever, sars, and the potentially pandemic h n influenza. every year, between and % of the global population becomes infected with a new influenza strain, causing upwards of half-a-million deaths. it is widely thought that there will be a continual emergence of new infectious diseases during the present century: emerging zoonotic infections and antibiotic-resistant bacteria prominent amongst them. in the face of declining birth rates, coupled to decades of ever-enhancing nutrition, advances in treatment regimens and medicines are leading the population of most developed and developing countries figuratively to age: that is for larger and larger proportions of the population to live to maturity and beyond. growth in life expectancy is matched by growth in the diseases of old age. these include neurodegenerative diseases, principally parkinson's or alzheimer's disease; cardiovascular diseases; and stroke. disease has altered significantly in the preceding century. it will continue to alter during the century to come. some alterations we can predict; others will escape the forecaster's eye. disease, particularly infectious disease, has been beaten, or, at least, severely restrained. many factors have conspired to effect this-improved water quality, better precautionary hygiene, improved nutrition, decreased overcrowding-as well as many interventionary measures, principally antibiotic therapy and vaccines. hitherto, vaccines have been an uncompromising success, yet, as we see, so much more needs to be done if the full potential of vaccines is to be achieved. although the licensing and use of vaccines varies between countries, - commonly licensed vaccines target a range of viral or bacterial infectious diseases, with approximately paediatric diseases targeted during the first few years of life. other than paediatric vaccination, most vaccines are used by travellers to tropical or subtropical regions; a significant minority fight infection in the developing world. vaccination also works to greatly reduce the morbidity of disease, often imbuing lifetime protection; this is particularly important for benign yet economically important infections, such as the so-called common cold. diverse sporadic or epidemic infections of the human respiratory track-as caused by an excess of distinct viruses, such as rsv or, more properly, respiratory syncytial virus, coronaviruses, influenza a and b, rhinoviruses, parainfluenza virus, and cytomegalovirus-remain a principal cause of hospitalisation and community morbidity with an estimated % of gp referrals associated with such infections, and cause the loss of enormous numbers of working days in developed countries. given the recalcitrance, and the immense investment in treatment and prophylaxis, it may be that many of the diseases alluded to above will never be eradicated, as smallpox was, and that vaccines alone will not be enough. we may instead need a complex network of prophylactic and therapeutic measures at least as complex as the diseases themselves in order to effectively reduce the prevalence of the disease and to treat those who become infected. whatever other countermeasures we may have recourse to-artemisinin-based drugs, genetically manipulated vectors, or insecticide-treated bednets-the clinical and cost effectiveness of vaccines means they remain the must-have component in the ongoing search for better means of combating endemic infectious disease. beyond infectious disease lies what is possibly the most underexplored area within the ever burgeoning field of vaccinology and vaccination: vaccines against allergy and allergic disease; vaccines that target so-called lifestyle diseases, such as those deriving from addiction; and vaccines that target chronic diseases, the most important of which is cancer. therapeutic vaccines against cancer are probably the best studied amongst the more novel, innovative, and underexplored areas at the forefront of vaccine discovery. the present overall whole-life risk from cancer, at least in developed countries, runs at or about %. the figure is currently rising. there are approximately · million new cancer cases in europe each year, resulting in around · million deaths. in the usa, over one and half million new cases are reported annually. clearly, this is an important disease burden, and thus a key target for the pharmaceutical and biotechnology industries. lifestyle vaccines are another innovation, of which much is expected in certain quarters. they target all kinds of medical problems, ranging from drug addiction through dental caries, all the way to major genetic and multifactorial diseases, including obesity. versatility and flexibility are major hallmarks of the vaccination concept. vaccines take many forms and work in many ways. this facet has been exploited in the development of life-style vaccines. let us look at serious addiction. during , the united nations office on drugs and crime estimated . - . % of the global population abused so-called illicit substances-a serious problem indeed with an enormous implicit health, economic, and behavioural burden, with the worst excesses coming from cannabis abuse and the abuse of amphetamine, cocaine, and opiates. anti-drug vaccines generate antibodies able to bind particular drugs; the drug-antibody complexes thus generated should have too high a molecular weight to penetrate the blood-brain barrier effectively, thus reducing the amount and rate of drug egress into the brain and so inhibiting psychoactive effects at the system level. anti-addiction vaccines have been with us for some time, beginning over years ago, when two proof-of-principle studies [ , ] demonstrated in rats and in rhesus monkeys that morphine could be used as a hapten in order to create an antibody against morphine addiction. today, addiction vaccines are being developed to target a range of major abused drugs, such as nicotine, cocaine, various amphetamines, and heroin. anti-allergy vaccination also offers great potential for successful commercial exploitation. the prophylaxis and treatment of allergy can now be addressed in many ways, including, notably, recombinant proteins and dna vaccines. vaccines against the common cold or anti-allergy vaccines are similar in mechanism to many lifestyle vaccines. these do not save lives directly but do help to greatly reduce the vast economic burden of disease morbidity. an array of interconnecting factors that have made the pharmaceutical and biotechnology industries re-evaluate the potential of vaccines as a commercially viable product. prior to , there were relatively few vaccines, most targeting major pandemic diseases of the developed or developing worlds. subsequently, partly as a result of enhanced technology as discussed at length in the current book, many vaccines have become available, most recently the cervical papillomavirus vaccine. likewise, there are hundreds upon hundreds of vaccines in trials. the growth rate in the sales of vaccines reflects this feverish and febrile activity: the rate of sales growth for vaccines is something like %, compared to the sluggish drugs market, meandering its desultory way at % per annum. there is profit in vaccines, clearly; what remains problematic for the profit-driven decision-making processes of big pharma is the haphazard and probabilistic nature of vaccine discovery. what the pharmaceutical industry needs is the capacity to apply the same systematic, automated, high-technology approaches used to identify new small-molecule drugs to the discovery and development of vaccines. no right-minded scientist, looking back across the last years, would wish to argue seriously with the contention that the design and development of vaccines is an innately labour-intensive process. the processes deployed to meet the objective of creating new and better vaccines are in desperate need of change. this change must be radical if we hope to simplify such processes. simple processes are hopefully also fast and efficient processes. in the search for subunit vaccine antigens, one technical development-reverse vaccinology-has proved the most profound and hopeful. just over a decade ago, rino rappuoli used the expression "reverse vaccinology" to describe development of vaccines using a genomic-based approach, rather than the ponderous empirical methods favoured then, and still in use today. reverse vaccinology seems about to deliver on its early potential: the european medicines agency is in the process of evaluating novartis's bexsero, the first commercial vaccine developed using the reverse vaccinology approach. the vaccine may become the first vaccine effectively to combat meningococcus b, a disease causing over % of global meningococcal meningitis. a decision on bexsero is expected shortly. during the development of bexsero, new protective protein antigens were identified using genomics: initially over surface-exposed proteins were predicted from the n. meningitidis proteome as molecules liable to host immune surveillance, of which about were then expressed in e. coli. this number was reduced by using these proteins to immunize sets of mice, identifying that could induce antibodies in vivo, of which killed n. meningitidis in vitro. by comparing the genomes of clinical strains, a further subset of proteins offering broad protection could be identified. reverse vaccinology has become perhaps the most famous well-developed approach amongst many advanced approaches now available within the discipline of vaccinology. indeed, a whole range of other, high-technology methods and techniques have been and are being developed to complement and optimise reverse vaccinology. capitalising on its success, these offer new hope in our constant struggle with infection; all we need is for this technology to be fostered, developed, and utilised. this book is intended to fill a gap, if not a void, in current thinking within vaccine design and development by attempting to draw together several disparate strands; and, by doing so, also identify and illuminate some important areas replete with potential. science, in much the same way that all human activities, from the most profound to the most trivial, follows fashion and progresses by tracking trends. whether we think of publically funded science or the pharmaceutical industry, similar phenomena are observed. science follows the money, and money follows consensus. the decision-making process underpinning the strategic direction that policy in both publically funded science and the pharmaceutical industry takes is only in part influenced by science. it is also regrettably in thrall to many, sometimes contradictory, voices: the fickleness of public opinion, vested interests of many hues and flavours, and the myopia of the profit margin, amongst many others. this is because the decision-makers in such organisations are seldom if ever scientists engaged in doing science directly; management and policy, at both the strategic and tactical levels, are often swayed by the prevalence of opinion. in the pharmaceutical industry, for instance, this is manifest as the next big thing: combinatorial libraries, genomics, high-throughput screening, antisense, even molecular modelling; all were hailed as transformative saviours that would remove happenstance and unpredictability from drug discovery-yet as the current parlous state of the pharmaceutical industry readily attests, while all promised much, none really delivered. no single technique can achieve everything, which is why we should always develop a large range of alternatives, both informaticsbased and experimental, all running in parallel. central to computational immunology is the capacity to make accurate predictions. yet, obtaining routes to prediction that are accurate, robust, and dependable continually eludes us. immunoinformatics deals with empirical, datadependent methods. the success and utility of such methods depends very much on the data used to propagate and parameterise them; they cannot escape the severe limitations imposed by the data used to create them. the data from which we build models forms a complex phase space of structural and property variation, which can be extremely multidimensional, with a high degree of interdimensional correlation. when the data we work with is reliable and our knowledge of it is complete, then we can create useful models by applying standard methods from computer science to build accurate and predictive models relating observed biological activity to underlying measurable or predictable properties. usually, such approaches are also much superior when used to interpolate than they are when used to extrapolate. we need complete and thorough data sets effectively and efficiently able to explore the complex relationships between structure and function, necessitating continuous improvement in all aspects of data quality. within the wider context of vaccine design and discovery, we shall in this book describe and explore a range of key alternative strategies and technologies, both informatics-based and experimental, which are, by degrees, both supportive and complementary to reverse vaccinology and more traditional approaches to vaccine discovery. this book looks in turn at reverse vaccinology and the identification of putative candidate antigens, at the discovery of a wide range of different types of adjuvants, and finally at the development of sophisticated new delivery mechanisms, such as liposomes and other applications of nanotechnology. in chap. , cafardi et al. review the present state of play with respect to reverse vaccinology, with particular emphasis on how completion of bacterial genomes impinges upon the vaccine discovery. they show how this approach allows the development vaccines that are difficult or near impossible to address with conventional approaches. they also highlight how advances in genome-based techniques and in so-called next-generation sequencing approaches and technologies will help to enhance reverse vaccinology, enabling timely identification of novel candidate antigens for new, emerging, or recrudescent infectious diseases. in chap. , flower et al. review the discovery of candidate vaccine antigens in more detail. placing their analysis in the context of emerging ideas about the possible nature of immunogenicity and how it may be propagated by elements of the immune response at the system level, the authors discuss the three main approaches to the identification of novel immunogenic antigens: sequence similarity-based approaches, whereby the antigen nature of a protein is inherited from similar sequences; methods based on identifying the subcellular location of microbial proteins, on the basis that proteins with only certain locations would be accessible to immune surveillance; and the use of empirical alignment-independent approaches to the prediction of antigens. usefully, the chapter also includes discussion of expert systems for antigen discovery. in chap. , vordermeier et al. review how genomics and the development of bioinformatics have radically transformed the cattle vaccinology of bovine tuberculosis. within the context of a generalised infrastructure of bioinformatic analytical techniques, the authors describe in detail how the application of comparative in silico transcriptome and genome analysis is able to undertake prospective prioritisation of immunogenic antigens for experimental testing, leading to the identification of candidate subunit vaccines. in chap. , he explores the use of epitope-focused immunoinformatic analysis in the prediction of optimal vaccine candidates when undertaking a genome-wide reverse vaccinology exercise. specifically, he describes the web-server vaxign, concentrating on a case study: vaccine design against the virulent bacterium francisella tularensis, where candidates were chosen using a combination of pertinent selection criteria. in chap. , dhillon et al. offer us a wide-ranging review of methods and strategies for two important areas of immunoinformatic analysis within the domain of vaccine discovery: predicting the immunogenic subcellular location of microbial proteins and identifying proteins encoded by so-called genomic islands. while in chap. , ansari et al. describe a variety of database systems that facilitate immunoinformatics and antigen selection. chapters and look at adjuvants and their discovery. in chap. , edwards describes the basis of adjuvant action, and the role played by macromolecular adjuvants. in chap. , flower explores and examines different varieties of molecular adjuvant and their discovery, concentrating on small molecule adjuvants, and their systematic identification using virtual screening technology. this topic is put into context by a thorough review of extant adjuvants, molecular mechanisms of adjuvant action, as well as macromolecular adjuvants and how various adjuvants engage pattern recognition receptors of the innate immune system. in addition to the characteristics of the antigen and the adjuvant independently, how the antigen and adjuvant are presented to the immune system has a major impact on the biological output of the vaccine. indeed, the co-delivery and continued association of antigen and adjuvant may be a prerequisite in effective immunisation. this is not a new idea; the ability of alum to promote an antigen depot effect at the site of action has been ascribed as one of its main mechanisms of action for several years. for example, since the who has recommended that over % of diphtheria toxoid needs to be adsorbed to alum for its effective use [ ] . whilst this does not ensure that the antigen remains adsorbed to alum after injection and exposure to interstitial fluid, it is thought to at least initially promote colocation of the antigen with the alum adjuvant. however, alum is not the only adjuvant able to promote the co-delivery of antigens and adjuvants in one system; a range of particulate delivery systems can offer this. examples of such delivery systems include lipid-based systems (e.g., liposomes, niosomes, iscoms) and microparticles. each of these systems offer a suite of advantages and disadvantages and when considering the choice of delivery system attributes including antigenloading capacity, antigen retention and protection both on storage and within the biological milieu, and the ability to incorporate adjuvants within the delivery system all require optimisation and this is without consideration of the ability of the delivery system to act as an adjuvant in its own right. in this book we aim to address these issues by considering several of the most commonly employed particulate vaccine delivery systems. out of these particulate delivery systems liposomes are one of the most established systems; liposomes were first reported as an effective immunological adjuvant for diphtheria toxoids by allison and gregoriadis in [ ] . since then, a large array of understanding on their design has been gathered and strong links between their formulation and function identified. of these parameters, the composition of the liposomes will play a pivotal role. for example, the surface charge of liposomes used for vaccine delivery can influence the interactions between liposomes and protein antigens, and affect how liposomes interact with cells. this manipulation of surface charge can range from the inclusion of anionic lipids such as phosphatidylserine, which may facilitate the targeting of antigen presenting cells through interaction with phosphatidylserine receptors. alternatively, the use of cationic lipids can improve the loading of anionic antigens to the liposomes and upon injection promote a depot effect at the site of injection, promoting the codelivery of antigen and adjuvant to dendritic cells (e.g., [ , ] ). however, this depot effect is more than electrostatically driven, with the choice of cationic lipids used in the formulation having an impact as explored in chap. . in addition to liposomes, there are a range of alternative surfactant-based delivery systems, such as niosomes. these are similar in many ways to liposomes; however, non-ionic surfactants form the main component of their bilayers. the most common composition of niosomes investigated for vaccine delivery is -monopalmitoyl glycerol, cholesterol, and dicetyl phosphate. one might argue this is not a niosome formulation due to the inclusion of anionic dicetyl phosphate. however, the addition of charged surfactants has proven to enhance the stability of these vesicles and their inclusion in these vesicles is common practice. generally, niosomes exhibit many similarities to liposomes; however, potential advantages cited include their lack of predisposition to oxidative degradation, as well potential lower cost of components and reduced variability compared to natural phospholipids. whilst with current manufacturing methods, the cost and reproducibility of phospholipids is less of an issue, niosomes still offer a useful alternative to liposome formulations. in particular, niosomes appear an attractive option for oral delivery of vaccines due to their ability to withstand the harsh gastrointestinal environment as outlined in chap. . with both liposomes and niosomes, immunostimulatory agents can be easily incorporated within the system and in some cases this can result in restructuring of the particulate delivery system as is the case with iscoms (chap. ). iscoms are prepared from a mixture of phospholipid, cholesterol, and a saponin (often quil a). whilst a phospholipid/cholesterol mixture would normally form liposomes, the addition of appropriate concentrations of saponin to the mixture can result in restructuring of the system to form spherical, open, cage-like structures around nm in size, as nicely shown in chap. . given that their structures are open, iscoms cannot incorporate hydrophilic antigens, and antigens need to display a degree of lipophilicity for inclusion into iscoms. if required, antigens can be modified through a range of methods to incorporate lipophilic regions within their structure, thereby promoting their incorporation into the structure. alternatively, similar to cationic liposomes, cationic iscoms (where cationic components are used to build their structure) can electrostatically bind a range of anionic antigens and enhance their delivery. in chap. the formulation, preparation, and application of iscoms as vaccine adjuvants is considered. however, lipid-based systems are only one group of particulate delivery systems, and polymeric systems have also been extensively studied. in particular the use of biodegradable polymers to formulate nano-and microparticulate delivery systems for vaccines has been widely investigated. much like the lipid-based systems, there is a wide selection of options to consider in the formulation of polymeric nanoparticles and microspheres for vaccine delivery, with polyester polymers offering advantages due to their clinical approved use in a range of medical products. as with the other systems considered, immunostimulatory agents can be incorporated within these polymer constructs and thus these systems can act as delivery systems and adjuvants for antigens. within chap. , the design of polymeric microspheres as vaccine adjuvants is considered from the choice of base polymer, through to optimisation of process parameters, and finally to considerations of their stability as a product. whilst much of current research into the development of vaccines has focused on the design of vaccines for administration of a particulate suspension, dry powder vaccines may hold considerable advantages. in chap. , the authors consider the design of dry powder vaccines. such vaccines may offer low-cost, temperaturestable products suitable for pulmonary delivery, with the added advantage that the pulmonary route avoids the use of needles (and their associated risks). furthermore, it can allow for the effective delivery of antigens to target cells of the immune system without the harsh conditions faced by orally delivered vaccines. the development of spray-drying methods outlined in chap. supports the ability of such powder vaccines to be delivered using conventional dry powder inhalers already clinically licensed for pulmonary delivery. therefore, by considering these vaccine delivery platforms in conjunction with the appropriate choices for antigen and adjuvant it is hoped that the threefold multicomponent nature of a vaccine can be considered more completely than before. when viewed conceptually, vaccines comprise an important triad. the first part of the vaccine, and in a sense the most important, is the biological component. this is the whole protein, or whole organism, or epitope-based part which confers the ability to be recognised by the immune system. it is this part which differentiates one vaccine from another, an anti-flu vaccine from an anti-tb vaccine. the second part of the vaccine is the adjuvant, which is one of many alternatives, that confers an immunogenicity to many vaccines that they would otherwise not possess. it often does this in a generic fashion, such as via agonising the innate immune system, so that the same adjuvant is quite capable of functioning in many different vaccine formulations. the final and third part of the vaccine is the delivery vehicle, as opposed to the delivery mechanism, such as oral vaccines versus injectable. the vehicle can be things as different as a viral vector or a liposome. an attenuated or heat-treated whole-organism vaccine can be thought of as combining all three parts of this triad in one supra-molecular moiety. of course, this is a gross simplification, and many other things go into deployable vaccine formulations, such as preservatives, contaminants, and other chemical or biological components that so exercise the anti-vaccine lobby. hopefully, this book will encourage us to think of vaccines in these terms, and provides the background necessary to engage with each of the three components of the vaccine triad. with this in mind, the anticipation inherent with this work is indeed simple and straightforward: to foster and foment interest in those areas of vaccinology that this far have not received the level of intense work that they richly deserve. to help achieve this, we have sought to balance optimistic positivity and cold, hard rationality. not all of the approaches described will ultimately bear fruit, but each should, nonetheless, be examined with equal diligence, sedulousness, and assiduity. global malaria mortality between and : a systematic analysis evidence for active immunity to morphine in mice changes in heroin selfadministration by a rhesus monkey after morphine immunisation world health organization. manual for the production and control of vaccines-tetanus toxoid. blg/undp/ . rev liposomes as immunological adjuvants liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen a liposome-based mycobacterial vaccine induces potent adult and neonatal multifunctional t cells through the exquisite targeting of dendritic cells key: cord- -i dyg n authors: henn, wolfram title: allocation criteria for an initial shortage of a future sars-cov- vaccine and necessary measures for global immunity date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: i dyg n nan institute of human genetics, saarland university, university clinic bldg. , homburg-saar, germany correspondence to wolfram henn (email: wolfram.henn@uks.eu) although healthcare systems around the world currently are fully absorbed with the day-today challenge of slowing down the spread of the sars-cov- virus, ongoing research makes it very likely that a protective vaccine will be developed within a rather short period of time [ , ] . it appears obvious that such a vaccine will not be available ad libitum right from the beginning [ ] , but rather that an initially short supply will meet a huge and desperate worldwide demand. although we currently enjoy impressive examples of individual generosity and altruism in the wake of the crisis, we should not overstate the role of philanthropic actors, as it is still primarily governments that invest in fundamental research. any shortage of vital goods bears severe ethical challenges, as it generates distribution conflicts that can amount to antisocial behavior at the individual as well as at the governmental level, such as bribery, black markets, or protectionist trading practices. if we do not want to face scenarios as epitomized by graham greene in "the third man" for penicillin after world war ii [ ] , we are well advised to equip ourselves with reasonable and transparent criteria for the allocation of a foreseeably scarce vaccine as long as it will not be readily available for everybody. given the unprecedented public attention to the issue, these criteria must be medically adequate, socially fair, transparent, verifiable, and easily understandable for non-experts, in order to bridge thehopefully short but anyway relevant-initial shortage of vaccine supply without creating social discomfort or even unrest. the recent promising data on antiviral therapies or convalescent plasma treatments for already affected patients may at best alleviate the pressure a bit [ ] , yet not resolve the problem as a whole. what is more, it is still unclear how long the postinfection immunity of convalescents will last [ ] . as current data clearly show that covid- mortality is strongly associated with age [ ] , it should be the leading and also easily verifiable medical parameter for the distribution of the expected vaccine during an initial scarcity. however, it goes without saying that a few specific groups of professionals must be preferred as recipients in the interest of all, namely medical and security personnel immediately involved in the fight against the pandemic. the general rule should be that those who are most needed come first, followed by those most in need. accordingly, the following preference list might be reasonable: rank : physicians and nurses in immediate patient care, plus police and comparable public security officers in immediate contact with the general public. high-ranking policymakers or security officers may argue that they are indispensable for societies as well, but as there is no sharp definition for that kind of functional importance, any subjective assignment of professional preference will be sure to diminish the societal acceptance of the chosen ones. rank : documented recipients of organ transplants under ongoing immunosuppressive medication. it might be argued that other patients receiving immunocompromising therapies can be equally endangered, but any openness to other medical indications beyond transplantation will likely give rise to illegitimate attempts of circumventing the restrictions. rank : all other persons, ordered by date of birth from old to young, without any exception, and most notably, irrespective of insurance status. although it is a well-known yet not universally accepted fact that the prosperous and well-insured have access to higher quality healthcare, social inequality in the struggle against a challenge that is obviously equal to all will most likely elicit strong adverse reactions even among otherwise indulgent patients. admittedly, this strict allocation scheme may neglect relevant medical and epidemiological issues such as reduced vaccine efficacy among immunocompromised persons [ ] and among the elderly due to immunosenescence [ ] , as well as the fact that schools and universities are major hotspots of transmission [ ] . however, any granularity in the criteria would render them less transparent and actionable. beyond this ranking, anti-bribery rules will have to be rigidly enforced, and illegitimate vaccine sales pathways such as cryptomarkets [ ] , be they real or fake, vigorously prosecuted. political precautions should also be taken against cross-border buy-outs of vaccine stocks or manufacturers [ ] . after having overcome the expected initial shortage of vaccines, the global community must take appropriate measures to rapidly generate a worldwide herd immunity against sars-cov- through implementing mandatory vaccination programs encompassing all countries and age groups. the who is uniquely experienced and equipped to meet that ambitious challenge, and any political attempt to weaken it in this critical situation is, to say the least, counterproductive. any person left behind for whatever reason will not only be in avoidable peril, but will also put the goal of eradication of sars-cov- at risk [ ] . therefore, the eu and other well-heeled supranational organisations should feel not only morally obliged but also pragmatically well-advised to participate in supplying weaker healthcare systems with sufficient amounts of vaccine and logistic support for their application even in remote areas [ ] . preliminary identification of potential vaccine targets for the covid- oronavirus (sars-cov- ) based on sars-cov immunological studies sars-cov- and covid- : the most important research questions vaccine update: recent progress with novel vaccines, and new approaches to safety monitoring and vaccine shortage fake penicillin, the third man, and operation claptrap covid- : fda approves use of convalescent plasma to treat critically ill patients immune responses and pathogenesis of sars-cov- during an outbreak in iran: comparison with sars and mers clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study influenza vaccination in immunocompromised patients: efficacy and safety vaccines to prevent infectious diseases in the older population: immunological challenges and future perspectives what are the underlying transmission patterns of covid- outbreak? -an age-specific social contact characterization. eclinicalmedicine hidden wholesale: the drug diffusing capacity of online drug cryptomarkets covid- : trump sought to buy vaccine developer exclusively for us, say german officials a classification of the aims of vaccination and its relevance to transgenerational justice challenges for nationwide vaccine delivery in african countries key: cord- - jdn kw authors: chen, juine-ruey; liu, yo-min; tseng, yung-chieh; ma, che title: better influenza vaccines: an industry perspective date: - - journal: j biomed sci doi: . /s - - - sha: doc_id: cord_uid: jdn kw vaccination is the most effective measure at preventing influenza virus infections. however, current seasonal influenza vaccines are only protective against closely matched circulating strains. even with extensive monitoring and annual reformulation our efforts remain one step behind the rapidly evolving virus, often resulting in mismatches and low vaccine effectiveness. fortunately, many next-generation influenza vaccines are currently in development, utilizing an array of innovative techniques to shorten production time and increase the breadth of protection. this review summarizes the production methods of current vaccines, recent advances that have been made in influenza vaccine research, and highlights potential challenges that are yet to be overcome. special emphasis is put on the potential role of glycoengineering in influenza vaccine development, and the advantages of removing the glycan shield on influenza surface antigens to increase vaccine immunogenicity. the potential for future development of these novel influenza vaccine candidates is discussed from an industry perspective. seasonal influenza outbreaks cause to million cases of severe illness and , to , respiratory deaths each year [ , ] . the orthomyxoviridae are a family of enveloped viruses with a genome consisting of ~ segments of negative-sense single-stranded rna, including four genera of influenza virus: a, b, c and d [ ] . influenza a and b are the main cause of annual flu outbreaks in humans, with influenza a further classified into subtypes based on their surface glycoproteins hemagglutinin (ha) and neuraminidase (na). ha subtypes (h ~h ) and na subtypes (n ~n ) are currently known, most notable today are the h n and h n subtypes that cocirculate in the human population. since the s influenza b has diverged into two lineages based on antigenicity, the yamagata and victoria lineages, with little or no serum cross-reactivity [ ] . in contrast to the severity and epidemic potential of influenza a and b, influenza c infections induce only mild flu symptoms in children, while influenza d is not known to infect humans [ ] . recurrent influenza epidemics with pre-existing immunity occurs because the influenza virus employs two mechanisms to escape recognition: antigenic drift and antigenic shift. antigenic drift is the gradual accumulation of point mutations on the influenza virus' surface glycoproteins ha and na, driven by high error rates (estimated at . × − per nucleotide per replication [ ] ) of the virus' rna-dependent rna polymerase (rdrp). mutations that allow the virus to evade the host immune system are positively selected for and become fixed, resulting in the rise of new strains that are antigenically different from what the host was vaccinated against. the second escape mechanism, antigenic shift, is the reassortment of gene segments across different strains infecting the same host, resulting in a wholesale change in antigenicity [ , ] . antigenic shift have historically been associated with influenza pandemics, the most recent example being the swine-origin h n that included segments from classical swine h n , eurasian swine h n , and a triple reassortant from [ ] . the rise of new strains through antigenic drift and shift is followed by cross-immunity mediated competition between antigenically similar strains, which results in a progressive replacement of existing strains with new variants [ , ] . unfortunately, current seasonal influenza vaccines are strain-specific and have a very narrow range of coverage, meaning extensive surveillance, accurate predictions and annual vaccination are needed as circulating strains evolve continuously over time. this task is coordinated by the world health organization (who) global influenza surveillance and response system (gisrs), which gathers year-round data from hundreds of national influenza centers around the world and issue vaccine formulation recommendations for each upcoming flu season [ ] . when vaccine strains are well-matched with circulating strains, vaccination provides healthy adults younger than years with - % protection [ ] , and reduced hospitalizations in the elderly and those with chronic illnesses by - % [ ] [ ] [ ] . however, in years when there is a mismatch between the vaccine and circulating strains, the vaccine effectiveness (ve) tends to be much lower [ ] . here we discuss various challenges the current seasonal flu vaccine is facing, and how a universal influenza vaccine approach through carbohydrate design to elicit broadly neutralizing antibodies (bnabs) targeting the influenza ha glycoprotein can potentially play a role in the future of influenza prevention. despite the first influenza vaccine being commercially available as early as , influenza outbreaks continue to be a major public health concern today. it is imperative for health authorities, researchers and the pharmaceutical industry to work together on improving the efficacy of influenza vaccines. traditional trivalent influenza vaccines include two inactivated influenza a strains (h n and h n ) and one influenza b strain, but this has recently been overtaken by quadrivalent influenza vaccine comprised of h n , h n and both influenza b lineages that offers a more complete coverage [ ] . commercially available vaccine options include egg-or cell-based inactivated influenza vaccine (iiv), a live attenuated influenza vaccine (laiv), and a recombinant ha vaccine produced in insect cells [ ] . the production of egg-based influenza vaccines has remained virtually unchanged since the advent of split (subvirion) vaccines in the s, and still commands % of the global market share in [ ] . the main advantages of the egg-based platform include an excellent production capacity that is capable of producing an estimated . billion doses annually, and a low production cost that allows global access to the vaccine [ ] . the strain-specific nature of current vaccines necessitates the annual selection of candidate vaccine viruses (cvvs), including screening the antigenicity of isolates, preparing reassortant viruses, and adaptation of the virus to eggs (fig. ) . for egg-based manufacture, the entire process from strain selection to vaccine availability typically takes ~ months with tight time constraints, and any unexpected circumstance such as a delayed who strain recommendation [ ] or unexpected low virus yield [ ] , can snowball into significant production delays and directly affect vaccine supply. this lengthy interval also gives circulating influenza viruses time to mutate, as it did during the - flu season when late-emerging h n variants rendered the recommended vaccine strain ineffective [ ] . a second drawback of using an egg-based platform stems from the adaptation process of culturing a human virus in avian tissue, where adaptive mutations may accumulate and potentially change the strain's antigenicity [ ] [ ] [ ] . ha, apart from being the primary target for neutralizing antibodies, is the main facilitator of influenza virus entry by binding to sialic acids on the surface of the host cells. human influenza ha preferentially bind to α- , linked sialic acids commonly found on epithelial cells in the human upper respiratory tract [ , ] . however, in egg-based production vaccine strains are inoculated into the allantoic cavity of embryonated chicken eggs which only contain α- , linkages [ ] . with successive passages, this becomes a selective pressure that can cause the acquisition or a total shift in receptor specificity, with its accompanying mutations and antigenic changes on ha's receptor binding site. a recent example of this occurred during the - flu season, when egg-adapted vaccine strains were found to lack a glycosylation site (t , h numbering) on h n ha antigenic site b, one of the five major antigenic sites that induce neutralizing antibodies [ ] . a third concern is the egg-based platform relies on a steady supply of embryonated eggs. this egg supply can be overwhelmed by sudden increases in demand, such as during a pandemic. laiv is generated by combining the ha and na of currently circulating strains with the internal proteins of an attenuated cold-adapted strain. this results in a reassortant vaccine virus that can be administered intranasally and has some limited replicative ability in the human upper respiratory tract. as the entire influenza replication cycle is utilized at the site of infection, laiv has also been reported to elicit cell-mediated immunity [ ] and local mucosal immunity [ ] besides the induction of a robust antibody response. clinically, laiv has shown variable but overall comparable efficacy to iiv in adults and better efficacy in children. recently however, the necessity of effective replication in human respiratory tissue has emerged as an area of concern. the us advisory committee on immunization practices (acip) recommended against laiv between to due to low efficacy of the h n component [ ] , although this phenomenon was not noted in europe and canada [ ] . the reason for this lack of efficacy is still unclear, but possibilities include viral interference of tetravalent vaccine strains resulting in reduced virus shedding for the weakest strain, strong cross-reactive antibodies from previous seasons preventing virus replication, and inherent lower replication in host tissue by the h n pandemic strain [ ] , among others. acip has since resumed recommendation for laiv in following a change in the h n vaccine component [ ] . secondly, as currently-available laiv is also produced in embryonated chicken eggs, it is plagued by many of the same concerns as egg-based iiv. in astrazeneca's laiv product flumist experienced manufacturing issues due to low yields in two strains, resulting in a reduction in shipments worldwide [ ] . in order to overcome limitations of the egg-based manufacturing process, production systems using mammalian or insect cell cultures have emerged [ , ] . the manufacturing process for cell-based iiv is similar to egg-based iivs, but has several advantages over the latter (fig. ) . viral production in a cell culture bioreactor is more flexible, more scalable and unaffected by egg shortages. additionally, recent comparisons have shown that cell-based vaccines provided a moderately higher ve for elderly individuals (≧ years old) than egg-based vaccines, possibly due to less egg-adapted mutations [ ] . for recombinant ha production in insect cells, the baculovirus expression system is utilized to manufacture recombinant ha, which is then purified and formulated into ha trimer "rosettes" [ ] . this not only has the same benefits of speed, flexibility and scalability as cellbased iiv, but also eliminates the reliance on influenza virus replication for vaccine production and the timeconsuming process of strain selection. flublok, a recombinant ha vaccine developed by sanofi pasteur, was found to be % more efficacious than traditional iiv for people ≧ years old [ ] . high yielding vaccine strains for egg-or cell-based production are generated by either classic or reverse genetic reassortment. these adapted viruses go into mass production, either in embryonated chicken eggs or mdck cells with a production timeline of approximately six to eight months. in recombinant ha (rha) vaccines, the ha sequence is cloned into baculovirus and expressed by insect cells, significantly shortening production time however, the comparatively high cost of these alternatives to egg-based influenza vaccines have prevented them from taking a bigger share of the influenza vaccine market. according to the us centers for disease control (cdc) adult influenza vaccine contract pricing for - , the cost of the cell-based vaccine flucelvax is approximately % higher than an inactivated egg-based vaccine produced by the same manufacturer. the recombinant ha vaccine flublok can be more than twice as expensive as egg-based vaccines [ ] . additionally, while cell-based and recombinant vaccines have the benefit of speed and flexibility that is critical for pandemic preparedness, it does not translate to a competitive advantage on the seasonal vaccine market [ ] . so far slow progress has been made to transition away from egg-based production, and more support from governments around the world is needed. various next-generation influenza vaccines under development aims to broaden or lengthen the human immune response with novel antigens and adjuvants, gradually expanding the strain-specific nature of current vaccines to include all strains within a subtype (eg all h strains), multiple subtypes (eg h /h /h ), or incorporating all subtypes within a group (influenza a group or group ), with the ultimate goal of creating a truly "universal" pan-influenza vaccine that can elicit lifelong immunity against all influenza a and b viruses [ ] . from a public health perspective influenza continues to be the only human disease that requires annual vaccination. it is estimated that replacing just % of seasonal vaccines with a universal vaccine would avert influenza-related deaths and save . billion us dollars in direct healthcare costs per year in the united states alone [ ] . in , the national institute of allergy and infectious diseases (niaid) in the us laid out a detailed strategic plan for the development of a universal influenza vaccine, highlighting knowledge gaps and research areas in pursuit of this common goal [ ] . in their outline, they established four criteria for a universal influenza vaccine as: % effectiveness against symptomatic influenza infection, protection against both group i and group ii influenza viruses, durable protection that last at least year, and be suitable for all age groups. it is with these criteria in mind that we discuss various vaccine candidates being developed (table ) . historically, a crucial strategy of influenza virus' escape from pre-existing immunity is the addition of nglycosylation sites on the immunodominant ha head domain [ ] . these bulky but poorly-immunogenic n-glycans allow the virus to hide antigenically-conserved domains from host immune system recognition [ ] , a mechanism known as "glycan shielding". when h n first emerged in , it carried only one conserved glycosylation site at position (h numbering) on the ha head. but as the virus continued circulating in the human population up to the s, it sequentially acquired glycans at positions , and , all at or adjacent to the major antigenic site sa on the ha head. this was followed by a -year hiatus as h n was supplanted by h n , before re-emerging in carrying the same three acquired and one conserved glycosylation sites as before. the following decades saw n replaced by n , the disappearance of n , and the acquisition of n before the glycan shield was ultimately reset due to the emergence of pandemic h n , carrying only the original conserved glycosylation site on [ ] . conversely, h n circulated in carrying two glycans on its ha head, n and n (h numbering). although the glycosylation site at position was subsequently lost, positions , , , , , and were accrued and retained [ ] . overall, the continued circulation of an influenza subtype in the human population corresponds to a steady increase in n-glycans on its ha head domain. evidence that these acquired n-glycans provide a shielding effect comes from not only the observation that they tend to appear on or near major antigenic sites, but also studies have shown the acquisition of sites and on h n slow genetic drift in the shielded areas [ ] , and mutational deletion of , and on a pre-pandemic h n strain elicited a protective immune response against the pandemic h n strain [ ] . similarly, in h n positive selection disappeared when an antigenic site becomes shielded by n-glycans [ ] , and the introduction of five recent glycosylation sites at positions , , , and allowed a h n strain to evade polyclonal human serum raised against it [ ] . these observations indicate that exposing the comparatively conserved, glycan-shielded regions of viral hemagglutinin could be a potential strategy to increase the breadth of influenza vaccine protection [ , , ] . however, previous attempts have shown complete deglycosylation of all carbohydrate moieties on influenza ha by either prokaryotic production [ ] , tunicamycin treatment [ ] or pngase f digestion [ ] does not appear to be a viable strategy. conserved n-glycosylation sites on the ha stem are essential for intracellular transport, correct glycoprotein folding and ha trimerization [ ] , and a completely unglycosylated ha would have a high chance of altered antigenicity. therefore, our group focuses on harnessing glycoengineering techniques to alter n-glycan composition on the ha, creating recombinant has that retain only a single n-acetylglucosamine (glcnac) attached to asparagine per n-glycosylation site (monoglycosylated ha, or ha mg ). to accomplish this, n-acetylglucosaminyltransferase i deficient (gnti − ) human embryonic kidney cells that are unable to synthesize complex type n-glycans were used to produce secreted, transmembrane domain truncated has that have only high mannose residues on their nglycosylation sites. these high mannose has were then further trimmed with the high mannose-cleaving enzyme endoglycosidase h leaving a single glcnac residue, dramatically decreasing the size and shielding effect of these n-glycans while still maintaining the native ha structure in its trimeric state. antibodies raised against ha mg inoculation demonstrated better binding affinity, neutralization and crossreactivity than the unprocessed ha (fully glycosylated ha, or ha fg ) [ , ] . ha mg also induced the maturation of dendritic cells, more splenic granzyme bsecreting cd + t cells, and elicited a more diverse haspecific b-cell repertoire than that of ha fg when used as a vaccine (fig. ) . in terms of cross-protection, inoculation with an h n pre-pandemic bris/ ha mg not only provided better protection in mice against laboratory strains wsn and pr , but also offered % protection against a pandemic strain [ , ] . while a recombinant ha mg vaccine would have all the advantages of a cell culture production system including speed, flexibility and safety, egg-based production remains the mainstay of influenza vaccine manufacture today. devising a simple method to apply the monoglycosylation concept to egg-based vaccines with minimal modification will allow this procedure to be integrated into established production methods. extensive testing found that kifunensine, an α-mannosidase i inhibitor, can be injected into embryonated eggs to convert influenza virus membrane glycoproteins to a uniformly high mannose composition. after harvesting these virions their high mannose n-glycans were then trimmed with endoglycosidase h to create intact monoglycosylated virus particles, and all participating reagents are removed in subsequent purification steps [ ] . like the recombinant ha mg before, monoglycosylated split inactivated influenza vaccines produced by kifunensine and endoglycosidase h treatment were shown to have higher neutralization and cross-neutralization activity, higher hemagglutination inhibition (hai), more ha stem selectivity, and higher antibody dependent cellular cytotoxicity (adcc) (fig. ) . a monoglycosylated pandemic h n split virus vaccine offered cross-protection against strains as diverse as the pre-pandemic nc/ and the laboratory strain wsn [ ] . aside from having simplified glycans, this procedure produces antigens that are virtually identical to the current influenza vaccine, and would presumably offer a similar safety profile. an adjuvanted recombinant ha trivalent nanoparticle influenza vaccine (tniv) has been developed by novavax using the baculovirus expression system to produce recombinant has, which were then purified and mixed with polysorbate to form protein-detergent nanoparticles of ~ ha trimers [ ] . the administration of this tniv with a saponin adjuvant (matrix-m) in ferrets induced higher levels of neutralizing antibodies against a panel of a (h n ) strains than a commercial inactivated vaccine (trivalent fluzone). a phase i/ii clinical trial showed similar results in patients, where tniv induced significantly greater hai responses compared to trivalent fluzone against not only previous strains, but a forward-drifted a/singapore variant [ ] . another candidate is a chimeric ha (cha) vaccine born from a collaboration between icahn school of medicine at mount sinai and gsk/nih. this strategy originates from the observation that our immune system tends to focus on the immunodominant but highly variable ha head domain, while the subdominant conserved stem region has a better ability to elicit bnabs. by sequential immunization with a cha protein consisting of a stem from circulating strains coupled to an irrelevant ha head from exotic influenzas, the strategy is devised to re-direct our immune system to better stimulate stemspecific responses [ ] . in a preclinical study, ferrets sequentially immunized with heterologous influenza strains including live attenuated influenza vaccine (laiv) bearing an h head domain and an h stem domain (ch / ) and a split-inactivated vaccine bearing an h head domain and an h stem domain (ch / ), conferred superior protection against challenge with pandemic h n virus following different prime-boost combinations and immunization regimens [ ] . this approach is currently in collaboration with gsk in a phase i study, and clinical data will be obtained by the end of . multimeric- (m- ) is a vaccine currently being developed by biondvax pharmaceuticals consisting of nine conserved b and t cell epitopes from ha, nucleoprotein (np) and matrix (m ) protein arranged in triplicate and put onto a single recombinant protein [ ] . phase i/ii clinical studies have shown the m- vaccine induced both cellular and humoral immunity to influenza a and b strains as a standalone vaccine [ ] , and also enhanced seroconversion when used as a primer for elderly patients before inoculation with inactivated trivalent vaccines [ ] . flu-v is another epitope-based vaccine developed by seek (peptcell) based on in silico multiple alignment of influenza sequences and prediction of possible t-cell epitopes. six consensus sequences from influenza np, m and matrix (m ) proteins were identified and synthesized into a candidate vaccine. flu-v has been shown to induce a specific cd + response against these conserved epitopes and confer protection against heterotypic infection in mice [ ] , and a phase ib challenge trial also showed that the blood cells from immunized subjects exhibited cross reactive immunity against different influenza viruses [ , ] . codavax is an laiv being developed by codagenix that takes advantage of inherent human codon pair bias to reconstruct the influenza viral genome with synonymous but sub-optimal codons. this results in viral proteins that have the same amino acid sequence and antigenicity as wild type strains but attenuated due to excessive use of rare codons [ , ] . in animal models, the vaccine is shown to be effective at lower doses than conventional laiv [ ] . codavax has scheduled a phase i/ii trial in the first quarter of . m sr is an m deficient single-replication laiv being developed by flugen. in this strategy the m sequence in the viral genome (critical for viral uncoating and assembly) is largely deleted, but viruses are produced in m -expressing cells to generate infective virions. therefore, after inoculation into a host the attenuated virus is unable to propagate infective progeny, limiting the infection to a single round of replication [ ] . in a ferret model m sr was found to be less susceptible to the negative effects of pre-existing immunity on drifted strains [ ] . initial results from a placebo-controlled phase ii trial indicate that the vaccine was effective against a mismatched h n challenge. inovio has made efforts to apply their syncon® synthetic dna vaccine platform to influenza. by sequence alignment and cluster grouping of ha they have generated four "micro-consensus" sequences within an influenza subtype, which were then cloned onto expression vectors and delivered to the vaccine recipient via in vivo electroporation [ ] . in mouse and ferret models these micro-consensus sequences against h n , h n and h n were found to elicit protective immunity against lethal challenges. acam-flu-a is an influenza m ectodomain vaccine developed by acambis (now sanofi pasteur). due to overlapping nucleotides with m , the m ectodomain is highly conserved in influenza a viruses, but poorly immunogenic [ ] . acam-flu-a utilizes the hepatitis b core (hbc) as a carrier to fuse three tandem repeats of the m ectodomain onto each hbc subunit, creating an immunogenic virus-like particle (vlp). initial results showed intramuscular injection of the vaccine was able to generate anti-m ectodomain seroconversion in % of healthy volunteers [ ] . however, after immunization m -specific antibody titers steadily declined over a year period [ ] , so combination with the other antigens or adjuvants might be necessary. the need for accurate surrogate markers of ve for clinical study and licensing approval precisely characterizing influenza immunity and correlates of immune protection is one of the three major areas for improvement outlined in niaid's strategic plan for a universal influenza vaccine [ ] . serological assays such as hemagglutination inhibition (hai) and single radial hemolysis (srh) have long been held by regulatory agencies as a correlate of protection for inactivated influenza vaccine licensure. european medicines agency's (ema) committee for human medicinal products (chmp) criteria indicates that for seasonal influenza vaccine approval one of three conditions must be met: seroprotection (hi titer of ≧ : or srh of mm ) rate of over %, seroconversion ( -fold increase in titer) rate more than %, or a geometric mean increase (pre-vs post-vaccination) of . times in healthy adults, and , %, . x respectively for elders [ ] . the us fda center for biologics evaluation and research fig. the production and immune response of monoglycosylated influenza vaccine. the production of monoglycosylated split virus vaccine adds two key steps to the traditional egg-based platform. kifunensine, a mannosidase i inhibitor, is added during egg inoculation to arrest viral glycoprotein processing, resulting in a uniformly high mannose composition. endoglycosidase h is added after harvest to trim high mannose residues down to a single glcnac. the resultant monoglycosylated split vaccine provides a more diverse immune response and more effective cross-strain protection than conventional egg-based vaccines. ha fg , non-modified egg-based vaccine with complex type n-glycans attached to ha; ha hm , ha with only high mannose type n-glycans; ha mg , ha with a single glcnac at its n-glycosylation sites. models of ha fg , ha hm and ha mg are created with protein data bank id code lzg and fyt by adding glycan with glyprot (http://www.glycosciences.de/modeling/glyprot/ php/main.php), coot (https://www .mrc-lmb.cam.ac.uk/personal/pemsley/coot/) and pdb of lipid bilayer from lipid bilayer membranes for rasmol (https://www.umass.edu/microbio/rasmol/bilayers.htm). the images were displayed with program pymol (www.pymol.org) (cber) follows a similar criterion for accelerated approval [ ] . however, hai and srh assays may not always be applicable when it comes to laiv or novel nextgeneration vaccines currently under development. hai measures the antibody-mediated inhibition of erythrocyte agglutination caused by ha binding to sialic acids on the erythrocyte surface. as such, the assay only detects antibodies directed at the ha head domain where its receptor binding site is located. universal vaccine strategies based on eliciting immune response against conserved epitopes on the ha stem domain, m , m or np would not be detected by the hai assay. srh detects the concentration of influenza-targeting antibodies by measuring a ring of hemolysis caused by the antibody-virus-erythrocyte complex activating the complement system [ ] . while this method measures all serum antibodies against influenza surface antigens, it still does not recognize local mucosal immunity or cellmediated immunity, such as immunization strategies that target m or np [ ] . this has led to the recognition that non-hai or srh assays need to be taken into account for regulatory approval of next-generation influenza vaccines [ , ] , though challenges in standardization of assays and reproducibility between laboratories still need to be overcome. finally, human challenge trials are gaining acceptance by regulatory agencies for universal vaccine development which may lack traditional serological correlates for protection [ , [ ] [ ] [ ] . there is increasing recognition that utilizing all aspects of our immune system are needed to control influenza outbreaks. elderly people often have more serious complications from influenza infections and a less robust immune response to vaccination [ ] . currently, high dose or adjuvanted iivs are recommended for people years and older, while laiv is only approved for healthy adults up to the age of . on the other end of the spectrum, maternally-derived antibodies generated from inoculation during pregnancy are expected to provide protection for infants < months, so vaccination that elicit a predominantly cell-mediated immune response are unlikely to be of use. novel strategies for a universal flu vaccine will have to take into account differences in immune response from specific populations that are at higher risk for influenza complications. with traditional seasonal flu vaccine human immunity wanes in - months of time, enough to last through the influenza season [ , ] . but if a universal vaccine were to break the cycle of yearly vaccinations, long-term protection will be needed. having durable protection for at least year and preferably through multiple seasons is one of the four criteria set by the niaid for a universal influenza vaccine [ ] , but how to achieve that goal is still unknown. immunization schedules, formulations, dosages, and adjuvants will all likely have to be considered. the evolution of influenza vaccine development has shown a trend of cell-based vaccines gradually taking the place of traditional egg-based manufacture. with the plethora of next-generation vaccines currently under development, who expects a universal influenza a vaccine to be in advanced clinical trials as early as [ ] . although many candidates have shown promising results in preclinical studies, demonstrating clinical safety and efficacy in a human population remains the most significant hurdle towards regulatory approval. our group has pioneered the strategy of exposing previously-shielded conserved epitopes on the ha through enzymatic trimming of n-glycans. this technique has been shown to elicit cross-neutralizing antibodies against antigenically diverse strains of influenza viruses within a subtype [ , ] , and thus hypothetically a trivalent or tetravalent monoglycosylated vaccine containing the three influenza subtypes (h , h , and influenza b) circulating in the human population would be, for all intents and purposes, a universal flu vaccine. we believe this monoglycosylated split virus vaccine strategy has three unique qualities that give it a significant advantage in the new drug developmental process: the monoglycosylated split vaccine provides multiple conserved epitopes for immune recognition due to the rapid mutation rate of the influenza virus, using only a single conserved epitope as the antigenic target for universal vaccine runs the risk of generating escape mutants [ , ] . in our previous studies we have only demonstrated the concept that monoglycosylated split virus vaccine induces more stem-specific antibodies directed against conserved epitopes on the ha stem [ ] . however, in theory by trimming off oligosaccharides on every n-glycosylation site on the ha, multiple conserved epitopes would be exposed, inducing a multi-faceted immune response that imposes a higher evolutionary barrier for escape mutant generation. another influenza glycoprotein that could potentially benefit from the monoglycosylation process is na. the preparation of monoglycosylated split virus vaccine would remove glycans from not only ha but also na, hypothetically inducing more anti-na antibodies that interfere with virus budding, disease progression and severity of symptoms [ ] . the monoglycosylated split vaccine induces a similar immune response to current iivs, meeting established surrogates of ve although a more diversified criteria encompassing cmi, neutralization assays and na antibodies is encouraged, traditional serological assays remain the gold standard for regulatory approval. by incorporating our monoglycosylation technology onto the existing inactivated split vaccine platform, we could invoke a similar humoral response as conventional iivs. serological surrogates of vaccine efficacy such as hai or srh can be measured and noninferiority comparisons with conventional vaccines can be made, opening up a well-trodden path towards licensure. the monoglycosylated split vaccine is suitable for all age groups whether novel vaccine strategies that are effective on healthy adults are equally suitable for all age groups remains a concern. due to having the same constituents as an iiv, the monoglycosylated split vaccine can be expected to offer a similar safety profile as the conventional flu vaccine. as such, it is possible that formulations suitable for different age groups, such as reduced dosage for children and high dose/adjuvanted vaccines for the elderly, can also be applied to our monoglycosylated split vaccine. furthermore, the robust humoral immunity induced by iiv assures sufficient protection for infants < months by maternal vaccination. even though recent advances in influenza vaccine manufacture such as cell-based and recombinant ha have allowed for a much quicker production timeline, using conventional strain-specific vaccines against a rapidly evolving influenza virus assures we are always playing catch-up. as our understanding of influenza pathogenesis and immune response continues to grow, developing a universal vaccine that provides long-lasting protection against divergent strains or subtypes is becoming an increasingly attainable goal. we believe our monoglycosylated split vaccine strategy that applies a simple modification step to pre-existing egg-based production platforms to provide broader immunity in the end product, is a significant step towards this goal. available from: who.int/en/news-room/fact-sheets/detail/influenza-(seasonal) estimates of global seasonal influenza-associated respiratory mortality: a modelling study influenza: old and new threats cocirculation of two distinct evolutionary lineages of influenza type b virus since characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the orthomyxoviridae family measurement of the mutation rates of animal viruses: influenza a virus and poliovirus type molecular mechanisms of variation in influenza viruses identification of hemagglutinin residues responsible for h n antigenic drift during the - influenza season antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans ecological and immunological determinants of influenza evolution predicting the evolution of human influenza a control of influenza cdc. key facts about influenza (flu) available from antibody response to influenza vaccination in the elderly: a quantitative review prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices-united states prevention of antigenically drifted influenza by inactivated and live attenuated vaccines quadrivalent influenza vaccine: a new opportunity to reduce the influenza burden age group (pediatric and adult), and route of administration (injection and nasal spray): global opportunity analysis and industry forecast the global production capacity of seasonal and pandemic influenza vaccine addendum to the recommended composition of influenza virus vaccines for use in the - northern hemisphere influenza season lessons from pandemic influenza a(h n ): the research-based vaccine industry's perspective low - influenza vaccine effectiveness associated with mutation in the egg-adapted h n vaccine strain not antigenic drift in circulating viruses contemporary h n influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains molecular changes associated with adaptation of human influenza a virus in embryonated chicken eggs evolving complexities of influenza virus and its receptors the role of receptor binding specificity in interspecies transmission of influenza viruses differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection live and inactivated influenza vaccines induce similar humoral responses, but only live vaccines induce diverse t-cell responses in young children live attenuated influenza vaccine in children induces b-cell responses in tonsils influenza vaccine effectiveness against pandemic influenza a(h n ) virus differed by vaccine type during - in the united states intranasal influenza vaccine: why does canada have different recommendations from the usa on its use? paediatr child health urgent challenges in implementing live attenuated influenza vaccine update: acip recommendations for the use of quadrivalent live attenuated influenza vaccine (laiv )-united states, - influenza season an advisory committee statement (acs): canadian immunization guide chapter on influenza and statement on seasonal influenza vaccine for - . ottawa: public health agency of canada cdc. flublok seasonal influenza (flu) vaccine safety, efficacy, and immunogenicity of flublok in the prevention of seasonal influenza in adults relative effectiveness of cell-cultured and egg-based influenza vaccines among elderly persons in the united states a fast track influenza virus vaccine produced in insect cells efficacy of recombinant influenza vaccine in adults years of age or older archived cdc vaccine price list as of statement from the press secretary on the executive order modernizing influenza vaccines in the u.s. to promote national security and public health the pathway to a universal influenza vaccine future epidemiological and economic impacts of universal influenza vaccines novel hemagglutinin nanoparticle influenza vaccine with matrix-m adjuvant induces hemagglutination inhibition, neutralizing, and protective responses in ferrets against homologous and drifted a(h n ) subtypes improved titers against influenza drift variants with a nanoparticle vaccine iscom technology-based matrix m adjuvant: success in future vaccines relies on formulation chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies a universal influenza virus vaccine candidate confers protection against pandemic h n infection in preclinical ferret studies h stalkbased chimeric hemagglutinin influenza virus constructs protect mice from h n challenge advances in universal influenza virus vaccine design and antibody mediated therapies based on conserved regions of the hemagglutinin glycans on influenza hemagglutinin affect receptor binding and immune response vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections egg-based influenza split virus vaccine with monoglycosylation induces cross-strain protection against influenza virus infections priming by a novel universal influenza vaccine (multimeric- )-a gateway for improving immune response in the elderly population epitope-based approaches to a universal influenza vaccine safety and immunogenicity of multimeric- --a novel universal influenza vaccine evaluating the immunogenicity and safety of a biondvax-developed universal influenza vaccine (multimeric- ) either as a standalone vaccine or as a primer to h n influenza vaccine: phase iib study protocol synthetic multi-epitope peptides identified in silico induce protective immunity against multiple influenza serotypes synthetic influenza vaccine (flu-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled phase i trial evaluation of the immunogenicity and safety of different doses and formulations of a broad spectrum influenza vaccine (flu-v) developed by seek: study protocol for a single-center, randomized, double-blind and placebo-controlled clinical phase iib trial a synthetic influenza virus vaccine induces a cellular immune response that correlates with reduction in symptomatology and virus shedding in a randomized phase ib live-virus challenge in humans antiviral lectins: selective inhibitors of viral entry live attenuated influenza virus vaccines by computer-aided rational design deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice liveattenuated h n influenza vaccine candidate displays potent efficacy in mice and ferrets m sr, a novel live single replication influenza virus vaccine, provides effective heterosubtypic protection in mice novel influenza vaccine m sr protects against drifted h n and h n influenza virus challenge in ferrets with pre-existing immunity a synthetic micro-consensus dna vaccine generates comprehensive influenza a h n immunity and protects mice against lethal challenge by multiple h n viruses broad crossprotective anti-hemagglutination responses elicited by influenza microconsensus dna vaccine protective immunity to h n influenza viruses elicited by synthetic dna vaccine dna-based influenza vaccines: evaluating their potential to provide universal protection universal influenza a m e-hbc vaccine protects against disease even in the presence of pre-existing anti-hbc antibodies m e-based universal influenza a vaccines. vaccines (basel) effect of priming with h n influenza viruses of variable antigenic distances on challenge with pandemic h n virus playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection glycosylations in the globular head of the hemagglutinin protein modulate the virulence and antigenic properties of the h n influenza viruses evidence for n-glycan shielding of antigenic sites during evolution of human influenza a virus hemagglutinin crossneutralization of and influenza viruses: role of glycans in viral evolution and vaccine design effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza a/h n virus hemagglutinin vaccine design of hemagglutinin glycoprotein against influenza quantification of the impact of the hiv- -glycan shield on antibody elicitation interactions of misfolded influenza virus hemagglutinin with binding protein (bip) role of conserved glycosylation sites in maturation and transport of influenza a virus hemagglutinin back to the future: immunization with m- prior to trivalent influenza vaccine in / enhanced protective immune responses against / epidemic strain the role of matrix protein ectodomain in the development of universal influenza vaccines a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases a review of the changes to the licensing of influenza vaccines in europe. influenza other respir viruses an overview of the regulation of influenza vaccines in the united states. influenza other respir viruses single-radial-hemolysis: a new method for the assay of antibody to influenza haemagglutinin. applications for diagnosis and seroepidemiologic surveillance of influenza the use of cell-mediated immunity for the evaluation of influenza vaccines: an upcoming necessity clinical, scientific and ethnographic studies of influenza in quarantine would you volunteer to be quarantined and infected with influenza virus? preexisting influenza-specific cd + t cells correlate with disease protection against influenza challenge in humans the efficacy of influenza vaccination in elderly individuals. a randomized double-blind placebo-controlled trial modest waning of influenza vaccine efficacy and antibody titers during the - influenza season intraseason waning of influenza vaccine protection: evidence from the us influenza vaccine effectiveness network who preferred product characteristics for next generation influenza vaccines two escape mechanisms of influenza a virus to a broadly neutralizing stalk-binding antibody how single mutations affect viral escape from broad and narrow antibodies to h influenza hemagglutinin contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions jrc, yml, y-ct, and cm wrote the manuscript. jrc and yct designed and illustrated table and figures. all authors read and approved the final manuscript. funding was provided by academia sinica genomics research center summit project as-summit- .availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. key: cord- -l ost q authors: oli, angus nnamdi; obialor, wilson okechukwu; ifeanyichukwu, martins ositadimma; odimegwu, damian chukwu; okoyeh, jude nnaemeka; emechebe, george ogonna; adejumo, samson adedeji; ibeanu, gordon c title: immunoinformatics and vaccine development: an overview date: - - journal: immunotargets ther doi: . /itt.s sha: doc_id: cord_uid: l ost q the use of vaccines have resulted in a remarkable improvement in global health. it has saved several lives, reduced treatment costs and raised the quality of animal and human lives. current traditional vaccines came empirically with either vague or completely no knowledge of how they modulate our immune system. even at the face of potential vaccine design advance, immune-related concerns (as seen with specific vulnerable populations, cases of emerging/re-emerging infectious disease, pathogens with complex lifecycle and antigenic variability, need for personalized vaccinations, and concerns for vaccines' immunological safety -specifically vaccine likelihood to trigger non-antigen-specific responses that may cause autoimmunity and vaccine allergy) are being raised. and these concerns have driven immunologists toward research for a better approach to vaccine design that will consider these challenges. currently, immunoinformatics has paved the way for a better understanding of some infectious disease pathogenesis, diagnosis, immune system response and computational vaccinology. the importance of this immunoinformatics in the study of infectious diseases is diverse in terms of computational approaches used, but is united by common qualities related to host–pathogen relationship. bioinformatics methods are also used to assign functions to uncharacterized genes which can be targeted as a candidate in vaccine design and can be a better approach toward the inclusion of women that are pregnant into vaccine trials and programs. the essence of this review is to give insight into the need to focus on novel computational, experimental and computation-driven experimental approaches for studying of host–pathogen interactions and thus making a case for its use in vaccine development. vaccination has been undeniably very helpful in promoting a healthy global population. it has severally saved lives, reduced healthcare costs and raised man's quality of life. it greatly reduces disease burden, disability and death. however, newly emerging and reemerging infectious diseases (erid), infectious agents with complex lifecycle and antigenic variability and the need for personalized vaccination present additional challenges in vaccine development. , for many pathogens (especially the emerging and those with antigenic variability), their genomes are known but their immune correlates of protection remain unclear. , some of these reasons are why vaccine development for erid and multi-lifecycle pathogenic diseases is a tall order. serendipitous discoveries in immunology coupled with knowledge of bioinformatics tools for epitope predictions have resulted in the emergence of new pattern of vaccine design. , the art and science of efficient and comprehensive information extraction and analysis of data deposited in relevant databases is now increasingly essential in researches related to immunology. even with this capacity (efficient information extraction), some challenges in the application of bioinformatics in immunology include structure and/or function analysis and immune process analyses as concern the immune interaction specificity. fortunately, although researches in immunology are experimentally costly and very intensive, colossal amounts of data are usually generated. such data can only be analyzed with high precision and speed using bioinformatics tools. for instance, genome sequencing as well as in vitro t-cell confirmation is done in few months as opposed to years using the conventional vaccine design. also, computational immunological methods drastically reduce both time and labor needs in epitopes screening. , with computational immunology techniques, it is possible to discover vaccine candidate epitopes simply by scanning the protein sequences in a pathogen of interest. many of these proteins are yet to be isolated or at least cloned. being pathogens specific and unique, they present ready candidates in vaccine construct. this review describes the need to use immunoinformatics-based techniques to unveil vital determinants of immunity made available in the genome sequence database and design vaccines. also, this review gives insight into the need to focus on novel computational, experimental and computation-driven experimental approaches for studying of host-pathogen interactions and thus making a case for its use in vaccine development. this review will further show the need for new approaches for effective drugs or vaccine design so as to combat the antigenic variability of some pathogens. the process of generating vaccine-induced immunity is somewhat challenging in immunology. current conventional vaccines came empirically when there were vague or no knowledge of vaccine immune system activation. a lot of research [ ] [ ] [ ] has been geared toward the understanding of this challenge, but the complexity of it requires a different dimension of approach. an approach that must accommodate many factors affecting vaccine development like pathogen antigenic variability, the emergence of infectious disease, human genetic variation is the goal of immunoinformatics [ figure ]. activation of the immune system involves, among many processes, induction of the immune memory. the strength of this induction determines the efficacy of a vaccine. hence, vaccine efficacy in the long run is influenced by the determinants of immunological memory stimulation, persisting antibodies and kind and type of immune memory cells induced. the primary vaccine-mediated immunological effectors (table ) are mainly the antibodies (from b lymphocytes/ cell) , and sometimes cd + and cd + t cells. these antibodies bind specifically to a particular kind of toxin or pathogen. vaccines and most antigens evoke humoral as well as cell-mediated immune responses. vaccines that mediate immune responses through these systems (b and t cell responses) are said to be more effective. although b cells are regarded as the primary vaccine immune effectors, t cells induce immune memory cells and high-affinity antibodies. studies in reverse vaccinology and immunomics had also proved t cells as prime immune effectors following the discovery of novel vaccine targets with epimatrix. [ ] [ ] [ ] this change of immune target has led to successful advances in vaccine design. even at the face of potential vaccine design advances, immune-related questions are now focused on specific vulnerable populations such as the young, elderly and immunocompromised. , these concerns have propelled a better understanding of the efficacy of current vaccines on this vulnerable population and have also paved way for the application of new approaches that can put into consideration the differences of population and better targets that can generate optimum immune induction [ ] [ ] [ ] with the exception of type ii t-cell-independent (ti- ) antigens (i.e., polysaccharide antigens). antigens that could provoke the b lymphocytes as well as the t lymphocyte responses stimulate the germinal centers causing antigen-specific highly efficient b-cell multiplication and eventual differentiation into antibodyforming plasma cells and memory b cells. all existing protein and dna antigens induce immunological memory b cells unlike type ii t-cell-independent (ti- ) antigens (i.e., polysaccharide antigens). these polysaccharide antigens do not generate memory b cells but can induce longlasting humoral immunity even when recall responses are lacking. vaccine efficacy may be short term if only the b cells are activated. the traditional approach for developing vaccines for infectious disease threats has shifted to include other vaccine design techniques like cloning and expressing major surface antigens although this frequently resulted in the formulation of vaccines with poor immunogenicity, requiring strong adjuvantation. this approach is particularly likely to be less specific for pathogens with complex lifecycles (e.g., parasites) or very high mutability (e.g., rna viruses). these pathogens do not depend on one route for their virulence of pathogenesis in human and thus to alter this process, increasing the specificity of the vaccine should be the aim and not just the effectiveness as seen in the current conventional vaccines. , vaccines for several neglected tropical diseases are in various stages of development, thanks to mega drug companies that have continued to demonstrate a willingness to invest money in the research and development as regards to diseases plaguing the developing nations. , [ ] [ ] [ ] it is very pertinent to invest in researches that have an interest in vaccine specificity on the pathogen antigens than totally computational vaccinology may now be applied to screen these genomes for possible vaccine target. with these tools, many proteins of virulence interest can be sequenced and the most essential gene of interest modeled for a potential vaccine candidate specific for that pathogen. immunoinformatics is the way forward in the identification of vaccine candidates for these tropical erid, for pathogens with varying antigens and for individualized therapy. immunology studies produce data in colossal quantities. also, with proteomics and genomics projects, extensive screening of pathogens and/or pathogen-host interaction, it has become increasingly necessary to store, manage and analyze these data, hence the birth of immunoinformatics. immunoinformatics deals with computational techniques and resources used to study the immune functions. statistical, computational, mathematical and biological knowledge and tools are applied in immunoinformatics in order to accurately and specifically store, and analyze data concerning the immune system and its functions. to handle evidence diversity, immunoinformatics uses tools that cut across several aspects of bioinformatics such as creation and management of databases, , use and definition of both structural and functional signatures and the formation and application of predictive tools. [ ] [ ] [ ] these strategies can synergize toward a better understanding of the immune system of both man and animals and fight against some less predictable pathogenesis. the complex nature of vertebrates' immune system, the variable nature of pathogens and environmental antigens coupled with the multi-regulatory pathways show that colossal quantities of data will be needed to unveil how the human immune systems work. conventionally, much cannot be achieved based on the complexity of the immune system and the virulent antigen but with the application of computational vaccinology, researches on vaccine design have been made easier, accurate and specific. applying immunoinformatics in disease study (table ) requires the knowledge of disease pathogenesis, the immune system dynamics, and computational vaccinology, painstaking searches of the database, sequence comparison, structural modeling as well as motif analysis. , these methods can go a long way in analyzing the pathogenesis of a disease and identification of vaccine candidates. in order to help understand complex pathogenrelated processes, computational models were developed for viral , bacterial, parasitic and fungal pathogens. the bioinformatics tools (table ) are used to identify possible epitopes for vaccine formulation. each tool can screen protein sequences and identify aggregates of mhc binding and supertype motifs for possible use in epitopebased vaccine development and for use among human populations with genetic variability. there are several databases ( table ) that can provide a wide range of information for all forms of immunological studies. generated data from the studies are further organized and stored in the databases (table ) to provide a means for the development and advance in immunotargets and therapy : immunological research. a tour on these databases will actually stimulate some interest in the vaccinology of emerging and re-surging disorders attributable to pathogen including cancer. emerging infections (eis) include infections that are entirely new in a population or that may have existed before in the population but are now gaining rapid and continued spread and/or wide geographical range. re-emerging or re-surging infections represent the infections that were previously of historical relevance but are now quickly becoming relevant because of either increasing incidence or increasing geographical and/or human host range while emerging infections represent the infections that were not originally observed in man. several factors such as human behavioral changes, environmental changes, and host/intermediate factors, animal-human switching and microbial genetic changes all affect infectious disease emergence and spread. these factors interact to promote the evolution of pathogens into new ecosystems, infect, spread and thrive in their new hosts. the overall consequences of these are continued epimatrix this is an in-silico product of epivax developed for predicting and identifying the immunogenicity of therapeutic proteins and epitopes. it is also used to re-design proteins and in designing t-cell vaccine has been applied in comparing strings from different strains of same pathogens and for pathogens identification. configuration of conservatrix allows for amino acid replacement at unusual positions. highly conserved t-cell epitopes in variable genomes such as some viruses are amenable to the algorithm , clustimer potential t-cell epitopes usually aggregate in specific immunogenic consensus sequence (ics) regions as clusters of - amino acids with - binding motifs instead of randomly distribute throughout protein sequences. in combination with epimatrix, the clustimer algorithm may be used to identify those peptides with epimatrix immunogenicity cluster scores ≥ + . such peptides are usually immunogenic and tend to make a promising vaccine candidate. blastimer using blastimer program, one may also choose to automatically blast "putative epitopes against the human sequence database at genbank". blasting screens off those epitopes with possible autoimmunity and cross reactivity questions and locates the epitopes that can safely be used in developing human or animal vaccine. blastimer can also blasts sequences against pdb, swissprot, pir, prf and non-redundant genbank cds translations. vaccine cad this algorithm evaluates junctional epitopes for possible immunogenicity and inserts "spacers and breakers into the design of any string-of-beads construct". infectious disease emergence and re-emergence, epidemics and public health challenges. emerging infections and multi-antibiotic-resistant strains of pathogenic bacteria usually surge from one geographic location from where it spreads to other places due to immigration of people. , most emerging infections originate from a specific population and can spread to a new population or become selectively advantaged that it can lead to the emergence of new strains of the pathogen. , , also, there could be microbial traffic, in which case, an infectious agent transfers from animals to humans or spreads from isolated groups to new populations. , , several factors, including ecology, are known to be associated with infectious disease outbreaks. these factors bring man into close contact with a natural disease reservoir/host. with an increasing world population and poor infection control, the emergence of infection and increased microbial populations are sure. the human growth population will only increase the spread of the infection across populations. the information provided in table is the list of remerging infections and current emerging diseases put forward during the who annual review. the review noted that these infections, if not well controlled, can cause disease outbreaks, bioterrorism and similar occurrences requiring urgent public health attention and that with the dearth of efficacious medicines or vaccines, there is a compelling demand for continuous as well as accelerated research and development in those areas. advances in genomics, proteomics, immunomics, vaccinomics and nanotechnology are being continually exploited in diagnostic, therapeutic and in rational drug and vaccine development. these advances have also served in the control of the afore-mentioned emergences. , the knowledge of the emerging pathogen's genome, protein make-up, pathogen-immune system interactions and researching the possible therapeutics will go a long way in directing the optimum path to containing the infection spread and controlling potential re-emergence or emergence in a different population. approaches in direct and computer-based structural determinations, protein-protein interactions predicting, and bioinformatics tools now exist and are used in modern-day development of drug and biologics. vaccine development has been sped up through the advance in the knowledge of the immune system of man. researches in the traditional targets of vaccines (adaptive immune response) and the less specific and fast-acting innate immune responses have been clear evidences for this advance. [ ] [ ] [ ] as our understanding of the intercourse between innate and adaptive immunity increases, reasons and opportunities for more effective vaccine adjuvants will open up. this can be a step forward in solving a critical world's health challenge per population. following the conventional approach of vaccine design, much cannot be achieved but when the knowledge of immunoinformatics is applied, population safety and disease control can be achieved through pathogen's genome sequencing leading to optimum new vaccine design or development of a novel vaccine for the infection. antigenic variability is an important mechanism pathogens use to evade their host immunity. the surface proteins of pathogens are normally variable. this assists them to escape recognition by the immune system. a successful infectious agent presents to the host immune system information that differs from that of its virulence. pathogenic organisms have organized systems of escaping destruction by the immune system of their hosts. for instance, toxoplasma invades and appropriates the host cells thereby circumventing phagocytosis and then spread within their host to establish infection. vertebrates on their own are endowed with immune system robust enough to efficiently and effectively surmount the non-self-attacks. yet the more the host's immune system elaborates, the better the organisms in their evasion of immune effector cells. antigenic variation refers to a pathogen's ability to modify its surface proteins such that it can circumvent the host's immunological attacks. it involves several mechanisms including the varying of surface protein's phase, shifting and drifting of surface protein antigens and/or any other form of alteration of antigenic protein. antigenic variation plays significant roles in the pathogenicity of microorganisms by evasion of the host immune responses and establishment of re-infection. when a pathogen alters its surface antigens, it can evade the host's adaptive immunity and so reestablishes infection. the immune system may battle to generate new immunoglobulins against the new antigen. certain bacteria like neisseria gonorrhea, neisseria meningitides, mycoplasma and species of the genus streptococcus show antigenic diversity. in eukaryotic pathogens, antigenic variation is shown by trypanosoma brucei and plasmodium falciparum. , another vital cause of antigenic variation in bacteria is horizontal gene transfer (more important than point mutation) through plasmid acquisition and transduction via bacteriophages. virulence genes are normally acquired by non-virulent organisms via these routes. once this occurs, the new bacteria may quickly get established and cause fresh epidemic outbreak. species of the genus neisseria are champions in the rapid change of surface antigens amongst bacterial pathogens. pathogenic forms exhibit an amount of phenotypic variability not found in the commensal species. the pathogenic forms are implicated in std and meningitis. they employ amazing varieties of antigenic variability mechanisms. • they can recombine their pilin genes in a similar manner that eukaryotes recombine their own genes, such that they can express variable surface protein. • some cell-surface proteins and enzymes synthesizing bacterial cell-surface carbohydrates are expressed in a variety of ways. this is as a result of replication slipping or slippage errors and repairs of simple tandem nucleotide repeats involving either the di-, or tri-or tetra-nucleotides. • neisseria is able to take up and incorporate environmental dna into its genomes. these are why an effective vaccine against neisseria infections is not yet developed. neisseria may be considered as an extreme example. however, many other bacterial pathogens like streptococcus and mycoplasma in promoting their antigenic variation tend to utilize one or more of these techniques. additionally, there are reports that dna-related defects have a much greater association with bacterial pathogen from symptomatic patients than samples of the same bacterial species isolated from environmental sources. [ ] [ ] [ ] pneumococcus streptococcus pneumoniae, gram-positive cocci bacteria that cause otitis media, bacteremia and pneumonia, are a public health concern, causing morbidity and mortality in adults and children. two forms of vaccines (polysaccharide and conjugate vaccines) are currently marketed for the prevention of pneumococcal infections. while the polysaccharide vaccines are for vaccination in the adult population, conjugate vaccines have an added immunogenic non-pneumococcal protein conjugated to the pneumococcal polysaccharides for enhanced immunogenicity in children. it is not yet known that these vaccines can evoke complete immunity against the infection. a polysaccharide capsule is a major virulence factor in the bacteria. several of these capsule types have been identified, and these form the basis of pathogen's antigenic serotyping. , current pneumococcal vaccines are combinations of various capsular (polysaccharide) antigens from the serotypes most common in a particular population. currently, over different serotypes are known but are not all covered in the available vaccines. the discovery of a common antigen(s) will produce an effective vaccine. knowledge of the genome of the organism and the different strains has led to a possible advance in driving the pneumococcal potential vaccine search through a different approach. and this consideration will help solve a lot of concerns about the current vaccines. with this knowledge, many methods are been tried to determine whether they can be a source of effective vaccine design that can accommodate all the serotypes of the organism. search for antigen that is common to all the serotypes can be achieved with the knowledge of genomics and immunoinformatics. the introduction of genomic and computational technologies has given new directions in the study of bacterial pathogenesis and vaccine design. , plasmodium plasmodium falciparum undergoes two life cycles: one in humans and the other in mosquitoes. the human host's erythrocytes and hepatocytes usually display modified parasite proteins called plasmodium falciparum erythrocyte membrane protein (pfemp ) and plasmodium falciparum hepatocyte membrane protein (pfhmp ), respectively. these proteins function to assist the parasite to evade destruction by the host immune systems. , the pfemp proteins were identified as the prime ligands responsible for cytoadherence and resetting. they cause the infected rbcs of host tissues to sequester thus helping the parasite to circumvent clearance by the host's spleen. the membrane proteins also shield infected host cells from destruction by the spleen by adhering to the endothelium. luckily, if the pfemp protein is expressed for a long while; it comes under attack by the naturally acquired immunity. , in defence to this, the parasite has expanded the var genes coding for pfemp such that the genes can exist as a polymorphic family of as much as over members in every genomic haploid. antigenic switches work well here in that members of the polymorphic family (also called antigenic-variant-protein family) can be interchanged and cannot express their proteins at the same time. in this way, only one particular protein at the surface of the infected rbc is expressed at any given time. , when studying antigenic-variant-protein families, it is pertinent to understand if grouping them into single-family results in any meaningful antigenic activity. studies have tried to understand the "languages" of the antigenic variant of pfemp proteins. , , they sought to know the pfemp proteins binding properties or search to understand the correlation between motifs and infection severity. the vardb database is a repository for protein sequences involved in antigenic variation and their associated functionalities. antigenic variant data obtained from several pathogens may be regrouped into a unified database. this will enable researches from several multicopy gene families to be accessed and compared swiftly in a single moment. updated vardb database contains close to , dna sequences, several protein translations, tens of infectious diseases and pathogens with their gene families. with a novel sequencing-based approach, pacbio, the different pfemp proteins can be sequenced and the related sequences used as potential vaccine targets. , trypanosoma for many pathogens, antigenic variability occurs during the infection pathogenesis and is to enable them to escape destruction by the host antibodies. for instance, some eukaryotic parasites take to genetic assortment and rearrangement thereby changing their surface antigens. a ready example is seen in trypanosoma brucei, the causative organism for sleeping sickness. trypanosoma brucei replicates in the bloodstream (outside the cells) of their host, but at maturity, it crosses the blood-brain barrier to cause several fatal complications. during replication in the bloodstream, the parasites are subjected to humoral as well as cellular immune responses. it evades the host defenses by encasing itself in homogeneous coat of glycoprotein called the variant surface glycoprotein (vsg). , though at initial invasion, the protein coat tends to protect the microbe from the immune system but on constant exposure, the coat will be identified as a foreign matter, and at this point the immune effectors can launch an attack against it. in a particular trypanosoma brucei, there are diversities of the vsg protein being coded by more than a thousand genes in the parasite's genome. unfortunately for the host, the expression of these genes is mutually exclusive. expressed vsg gene is normally due to genetic reassortments causing new alleles to be copied into the sites of expression. some trypanosomes with these abnormal vsg genes evade humoral immunity and multiply thereby causing re-infection and chronic recurring infections. influenza is a viral infectious disease due to infection by any of the three types of rna viruses, namely influenza types a, b and c. current vaccines contain double type a and single type b strains and induce strong antibody responses to neuraminidase and the surface glycoprotein hemagglutinin. these vaccines, however, cannot effectively protect against newly emerging viruses with antigenic shift and drift. , antigenic drift results in changes in the antigenic site (a minor change) while antigenic shift results in a new virus subtype. hemagglutinin and neuraminidase are the two enzymes dictating the antigenic properties of the viruses. while inside its host, defined host proteases break the peptide bonds in the hemagglutinin molecule to form hemagglutinin and subunits. virulence tendencies are decreased when the amino acids at the cleavage sites are lipophobic, the virus exhibits high virulence tendencies. the surface glycoprotein can be regarded as antigen and hence can serve as a target for the immune system which if sequenced, using the new immunoinformatics approach and a common site for the varying proteins identified, a potent vaccine can be developed which can accommodate the antigenic drift/shifts of the virus. influenza viruses are able to thrive for a long while in a given human population. , the virus has a high mutation rate such that a once effective vaccine can easily lose efficacy. antigenic variability is only one of the evidences of phenotypic variation in the biology of the influenza virus. the use of immunoinformatics in vaccine development has been accelerated toward the design of a multiepitope vaccine construct which has and will fully address the challenges faced with pathogens with mutagenic antigens. previous vaccines developed by conventional approaches consist of several proteins or a whole pathogen. this constitutes unwarranted antigenic load and increases the chances of inducing allergy. the use of peptide-based vaccines surmounts these challenges. the vaccines are made from short peptide fragments capable of eliciting highly specific immune responses, precision targeting and multiepitope constructs, in the case of varying antigenic peptides, which has been made feasible with the advancements in the field of computational biology. [ ] [ ] [ ] [ ] vaccines for pathogens with immune escape potentials can basically be constructed by using most, if not all, of their immunogenic peptides , because such vaccines prove to be better than single-epitope and classical vaccines. multiepitope vaccines enjoy the following advantages over single-epitope and classical vaccines: a) they are an assemblage of several epitopes obtained from distinct protein targets/antigens of an intended infection; b) the multiple t-cell receptors (tcrs) in the vaccine recipient can easily recognize vaccines with multiple hla epitopes; c); they can be easily adjuvanted to improve their immunogenicity; d) they can activate antibody-mediated and cell-mediated immunological responses because of their overlapping helper t lymphocytes (htl), cd + t-cell and b-cell epitopes; and e) unwanted protein antigens are excluded in such construct thereby reducing the chances of untoward effects and/ or immune responses likely to cause disease(s). [ ] [ ] [ ] [ ] [ ] [ ] thus producing a vaccine with these qualities can provide chances of combating most infections such as streptococcus pneumoniae and hiv infections. immunoinformatics can be employed in the docking of single and multiepitope vaccines and subsequently to predict their properties (physicochemical, allergenic and antigenic). this approach has seen the use of diverse tools and database in the analysis of ligands with their targets and has greatly helped to predict the binding score of antigenic peptides with the immune proteins like hla. peptides and hla allele modeling can be done by the d structural designing of the epitopes using pepfold (an online server), retrieving from protein data bank (pdb) the x-ray crystallographic structure of human population most occurring hla alleles (hla-drb : , : and hla-a : ) followed by filtration of previously bound ligands. the following is a step-wise detail on how to construct a single or multiepitope-based vaccine and its property prediction; • molecular docking analysis: to determine the interaction pattern of the screened out epitopes with the hla alleles by employing cluspro v. (a proteinprotein docking web server). this server performs this task by energy minimization, calculation of both the binding energy scores of the docked complex and electrostatic/shape complementarity. • target-protein comparative modeling and associated structure validation: the sequence of the amino acid in the target protein (e.g., tlr- ) can be retrieved from uniprot and the tertiary structure with raptor-x and i tasser (online comparative modeling tools). the server constructs and creates a d model immunotargets and therapy : (mathematical representation) of the target protein using hierarchical algorithms. personalized vaccine refers to vaccines "targeted" toward an optimized outcome. immunogenicity is maximized while either the risk of vaccine failure or reactogenicity and side effect is minimized. personalized are developed in the following cases; the individual level vaccines are developed to take care of haplotype and polymorphism knowing that they can retard the formation of a protective immune response or become pointers to the risk of an adverse vaccine reaction. this is needed when it is clear that females produce a higher antibody titre against a particular vaccine antigen than do their male counterparts. where it is clear that a particular human race or ethnic group has a higher or lower immune response to a particular vaccine antigen. personalized vaccines arise due to known complex interactions between host environmental, genetic and some other factors that may be influencing the vaccine immune responses. the associations between the immune response gene polymorphisms and variations in immune responses to a particular gene must be pine-pointed when it is clear that a particular drug either suppresses or augments the transcription of an immune response gene. , this could help in designing vaccines or vaccine adjuvants that can circumvent restrictions due to immunological differences arising from varying genetic compositions. , personalized vaccines stem from our understanding of how, within the human leukocyte antigen (hla) systemalso referred to as the major histocompatibility complex (mhc), the t cells are able to recognize peptides of pathogenic origin. , hla molecules enjoy the double advantages of having stable polymorphisms and being fully characterized. these advantages make good candidates for personalized vaccine design. hla polymorphism, although stable, is complex. for instance, more than , alleles of hla class i molecules and greater than class ii molecules have been identified among human populations. , hla class i and ii molecules have heterodimeric character comprising of α and β chains, three highly variable extracellular domains (α , α , and α ) and then transmembrane and intracytoplasmic domains that are less variable. , hla genes contain eight exons. exon is responsible for producing the leader peptide; exons , , produce α , α , and α extracellular domains, respectively, for mhc class or α , β and α , respectively, for mhc class ii; exon encodes transmembrane anchor; exons and encodes the cytoplasmic tail while exon encodes the ʹ-untranslated region. most of the several forms associated with the class i and ii genes are seen in α- and α- (as known as class i) and in α- and β- (as known as class ii) domains. mhc i and ii bind and present the peptide to t cells. t cell responses to viral pathogens differ from one patient to the other, basically because of the expression of differing hla (mhc) alleles which determine the several viral amino acid sequence brought to the t cells to read. , it is most likely that during an infection, diverse epitopes are usually presented to the t cells to read owing to the several forms of hla alleles and also because each human person expresses six hla class i and six hla class ii alleles. now, antibody-binding sites in a given hla (mhc) molecule are mostly prediction-servers predetermined on the basis of particular binding motifs and the anchor residues. , these residues refer to known amino acids located at defined locations in the peptide chain and which are peculiar to each mhc molecule. , prediction-server database of peptide motifs and/or of mhc ligands may be obtained from web-based and/or from prediction-servers dedicated to netmhc family. , in another example, sequence analysis of lassa fever virus (the lasv) and other viruses' immunoproteomic was used to identify the best immunogenic protein predicting t-cell as well as b-cell epitopes and also target sequence and binding sites. , the ssnlykgvy peptide sequence at aa - of glycoprotein (produced by the l segment) was the best candidate epitope for the induction of humoral as well as the cell-mediated immunity for lassa fever vaccine construct. hla-i and hla-ii molecules have been proven in sizable african populations and their combination with the ssnlykgvy peptide sequence may prove useful in such lassa fever virus endemic areas. this approach will strongly improve individualized vaccination and help combat emerging infections. the hla region is suspected to contribute, to a large extent, to genetic propensity to infections and differences in vaccine expected immune responses. , studies show that females exhibit stronger immune responses to immunization compared to males. , there are differential antibody responses to rubella and measles viral protein between males and females and that both hormonal and genetic difference may be influencing the immune responses. , , practical issues may stand in the way of achieving this new development (personalized vaccinology). having to use different vaccines for different persons based because of personal genetic composition requires more time and labor during the vaccination process. also, screening for individual factors for targeted vaccination can significantly increase vaccination cost. but with all these challenges, personalized vaccination is the new age approach in achieving an optimum immunization that takes into consideration the individual immune differences in a particular population and it is a new dawn for vaccine development. personalized vaccine development is strongly improved by vaccinomics. the field of vaccinomics looks at how immune response gene polymorphisms affect the cellmediated, humoral and innate immune responses to vaccine antigens at population and specific individual levels. "vaccinomics" encompasses both immunogenomics and immunogenetics as it concerns immune responses to vaccine antigen. the fields of personalized vaccinology and vaccinomics were the products of phase i of the international hapmap and that of the human genome project. also, modern molecular assay techniques permitting highthroughput detection of variations at gene level, in particular linkage disequilibrium maps and single nucleotide polymorphism (snp), played significant roles in the development of personalized vaccinology and vaccinomics. it has also been shown that polymorphisms at vital immune response genes can bring about differing immune responses to biopharmaceutical products including vaccines. [ ] [ ] [ ] newer, accurate, cheap and reproducible sequencing technologies; validated databases containing genotypephenotype data; statistical and bioinformatics tools are needed in order to analyze and interpret data that will help and improve vaccine adverse and immune response quantifiability and predictability. the information will enhance clinical practice and accelerate rational and directed vaccine development. safe vaccines are a critical requirement for any immunization program. conventional vaccination has been an approach targeted at all groups and individuals but has failed toward the enrolment of pregnant women into vaccination programs because of presumed fetal and maternal harms. , evidence on the safety of vaccination in pregnancy is very small because pregnant women were usually excluded from participating in vaccine trials. pregnancy can alter the maternal as well as fetal immunological responses. it is pertinent to explore research opportunities presented in advanced vaccine designs such as immunoinformatics (multiepitope vaccine docking) by studying human immune system functions and responses specific to pregnant women and their unborn children. according to a report from the dominican republic of congo, during the - zika virus outbreaks, over a thousand pregnant women were suspected of being infected with the virus and a sizable number were at their first trimester. the report further stated that fetal loss was approximately one-tenth of the pregnancies and that there were up to cases of fetus with head circumferences smaller than normal. the widespread morbidity during the epidemic showed that zika virus infection adversely affects pregnancy outcome. , currently, there is no proof that pregnancy predisposes to ebola virus infections in comparison with the non-pregnant population, but there is some evidence suggesting pregnancy to worsen the disease prognosis including fetal loss. also, evidence showed that the virus can pass the placental barriers to establish infection in the unborn child. designing, implementing and enrolling pregnant women as well as perspective pregnant women into vaccine trials and programs is imperative in order to protect this group and ensure good vaccine uptake by them during infection outbreaks and epidemics. , these recommendations will give an informed decision to be investigated using the immunoinformatic tools to determine the immunogenic responses worthy of safe vaccine development for the pregnant women and perspective pregnant women group. maternal immunization offers palpable benefits in several ways. some vaccines are primarily administered to shield these pregnant women from morbid conditions and/ or death including fetal death. , pregnant women stand the risk of being exposed to virulent pathogens and may be at a higher risk of morbidity and/or mortality when compared to the general population. there has been an explosion of new immunological data (table ) due to an increase in research to understand the immune system pathway in infectious disease pathogenesis and the application of the knowledge of bioinformatics has led to a better exposition of the immune system importance through immunoinformatics. the knowledge of immune system and the cost-effective, specific and effective approach like immunoinformatics, the concerns for emerging and re-surging diseases caused by pathogenic organisms, antigenic variability/complex lifecycle of pathogens and the need of personalized vaccination can be combated on a molecular level. the future of immunological research is sharpened by the ability to make discoveries in biologics (e.g., vaccines) more effectively and efficiently. this will mean reduction and better targeting of wet laboratory experiments and will only be possible if wet laboratory experimentation is combined with bioinformatics techniques. • immunoinformatics depends on experimental science (wet laboratory) to produce raw data for analysis. the predictions are not formal proofs of any concepts. they do not replace the traditional experimental research methods of actually testing hypotheses. • the quality of immunoinformatics predictions depends on the quality of data and the sophistication of the algorithms being used. sequence data from high-throughput analysis often contain errors. if the sequences are wrong, or annotations incorrect, the results from the downstream analysis are misleading as well. immunotargets and therapy is an international, peer-reviewed open access journal focusing on the immunological basis of diseases, potential targets for immune based therapy and treatment protocols employed to improve patient management. basic immunology and physiology of the immune system in health, and disease will be also covered. in addition, the journal will focus on the impact of management programs and new therapeutic agents and protocols on patient perspectives such as quality of life, adherence and satisfaction. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. immunotargets and therapy : time for t? immunoinformatics addresses vaccine design for neglected tropical and emerging infectious diseases vaccinology in the third millennium: scientific and social challenges antigenic variability: obstacles on the road to vaccines against traditionally difficult targets complex immune correlates of protection in hiv- vaccine efficacy trials immunoinformatics approach to design a novel epitope-based oral vaccine against helicobacter pylori immunoinformatics approach for multiepitopes vaccine prediction against glycoprotein b of avian infectious laryngotracheitis virus transcriptome and proteome: the rise of omics data and their integration in biomedical sciences neoantigen vaccine: an emerging tumor immunotherapy designing of cd + and cd + -overlapped cd + epitope vaccine by targeting late and early proteins of human papillomavirus immunization with a recombinant antigen composed of conserved blocks from tsa provides broad genotype protection against scrub typhus vaccine mediated protection against zika virus-induced congenital disease vaccination with sporozoites: models and correlates of protection novel approaches for the design, delivery and administration of vaccine technologies vaccination to gain humoral immune memory humoral and cell-mediated immune responses after a booster dose of hbv vaccine in hiv-infected children, adolescents and young adults an evaluation of the cold chain technology in south-east, nigeria using immunogenicity study on the measles vaccines a review of the immunological mechanisms following mucosal vaccination of finfish reverse vaccinology . : human immunology instructs vaccine antigen design large screen approaches to identify novel malaria vaccine candidates reverse vaccinology and subtractive genomics reveal new therapeutic targets against mycoplasma pneumoniae: a causative agent of pneumonia evolution of the immune system in humans from infancy to old age the twilight of immunity: emerging concepts in aging of the immune system pneumococcal vaccination strategies. an update and perspective progress and challenges in tb vaccine development vaccines for the elderly: current use and future challenges remembrance of things past: long-term b cell memory after infection and vaccination global practices of meningococcal vaccine use and impact on invasive disease new vaccine technologies to combat outbreak situations what are the most powerful immunogen design vaccine strategies? reverse vaccinology . shows great promise the new malaria vaccine program for african children is promising but still quite limited. quartz africa merck's ebola vaccine helps combat deadly outbreak in the congo as the virus spreads. biotech and pharma innovation for the 'bottom million': eliminating neglected tropical diseases in the americas emerging and neglected infectious diseases: insights, advances, and challenges exploitation of reverse vaccinology and immunoinformatics as promising platform for genome-wide screening of new effective vaccine candidates against plasmodium falciparum the use of databases, data mining and immunoinformatics in vaccinology: where are we? systems vaccinology and big data in the vaccine development chain biochemical functional predictions for protein structures of unknown or uncertain function reverse vaccinology: the pathway from genomes and epitope predictions to tailored recombinant vaccines universal genotyping for tuberculosis prevention programs: a -year comparison with on-request genotyping integrating whole-genome sequencing data into quantitative risk assessment of foodborne antimicrobial resistance: a review of opportunities and challenges bioinformatics and drug discovery new technologies in predicting, preventing and controlling emerging infectious diseases how genomics can be used to understand host susceptibility to enteric infection, aiding in the development of vaccines and immunotherapeutic interventions in silico analysis of epitope-based vaccine candidates against hepatitis b virus polymerase protein advances in designing and developing vaccines, drugs and therapeutic approaches to counter human papilloma virus computational approaches and challenges to developing universal influenza vaccines. vaccines (basel) experimental and computational analyses reveal that environmental restrictions shape hiv- spread in d cultures dynamic computational model of symptomatic bacteremia to inform bacterial separation treatment requirements a model of plasmodium vivax concealment based on plasmodium cynomolgi infections in macaca mulatta a computational modeling approach predicts interaction of the antifungal protein afp from aspergillus giganteus with fungal membranes via its γ-core motif. msphere an overview of bioinformatics tools for epitope prediction: implications on vaccine development bioinformatics tools for identifying class i-restricted epitopes in silico-accelerated identification of conserved and immunogenic variola/vaccinia t-cell epitopes nhlbi-abdesigner: an online tool for design of peptide-directed antibodies ivax: an integrated toolkit for the selection and optimization of antigens and the design of epitope-driven vaccines from genome to vaccine: in silico predictions, ex vivo verification designing string-of-beads vaccines with optimal spacers hiv vaccine development by computer assisted design: the gaia vaccine nerve: new enhanced reverse vaccinology environment jennerpredict server: prediction of protein vaccine candidates (pvcs) in bacteria based on host-pathogen interactions genome-wide prediction of vaccine target of human herpes simplex viruses using vaxign rv vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines vacsol: a high throughput in silico pipeline to predict potential therapeutic targets in prokaryotic pathogens using subtractive reverse vaccinology panrv: pangenome-reverse vaccinology approach for identifications of potential vaccine candidates in microbial pangenome understanding emerging and re-emerging infectious diseases. bethesda (md: national institutes of health (us) the consequences of human actions on risks for infectious diseases: a review institute of medicine (us) forum on microbial threats. microbial evolution and co-adaptation: a tribute to the life and scientific legacies of joshua lederberg: workshop summary trends in antimicrobial resistance of bacterial pathogens in harare understanding emerging and re-emerging infectious diseases. bethesda (md): national institutes of health (us) evolution and emergence of infectious diseases in theoretical and real-world networks carbapenem-resistant enterobacteriaceae posing a dilemma in effective healthcare delivery annual review of diseases prioritized under the research and development blueprint the role of nanotechnology in the treatment of viral infections proteomics for development of vaccine computational methods in drug discovery computational approaches for prediction of pathogen-host protein-protein interactions new approaches to understanding the immune response to vaccination and infection adaptive immune responses mediated by natural killer cells friend retrovirus studies reveal complex interactions between intrinsic, innate and adaptive immunity from entry to early dissemination-toxoplasma gondii's initial encounter with its host evolution and divergence of h n equine influenza viruses circulating in the united kingdom from neisseria genomics: current status and future perspectives antigenic variation in plasmodium falciparum malaria involves a highly structured switching pattern neisseria gonorrhoeae muts affects pilin antigenic variation through mismatch correction and not by pile guanine quartet binding antigenic variation in bacterial pathogens mechanisms and regulation of extracellular dna release and its biological roles in microbial communities mobile genetic elements in neisseria gonorrhoeae: movement for change. pathog dis genomic characterization of novel neisseria species the pneumococcus: epidemiology, microbiology, and pathogenesis. cold spring harb perspect med purification of capsular polysaccharides of streptococcus pneumoniae: traditional and new methods pneumococcal capsules and their types: past, present, and future whole-genome sequencing of bacterial pathogens: the future of nosocomial outbreak analysis human genomic loci important in common infectious diseases: role of high-throughput sequencing and genome-wide association studies immune response and evasion mechanisms of plasmodium falciparum parasites plasmodium falciparum pfemp modulates monocyte/macrophage transcription factor activation and cytokine and chemokine responses plasmodium falciparum proteins involved in cytoadherence of infected erythrocytes to chemokine cx cl effect of mature blood-stage plasmodium parasite sequestration on pathogen biomass in mathematical and in vivo models of malaria controlled human malaria infection with plasmodium falciparum demonstrates impact of naturally acquired immunity on virulence gene expression multiple plasmodium falciparum erythrocyte membrane protein variants per genome can bind igm via its fc fragment fcμ plasmodium falciparum malaria parasite var gene expression is modified by host antibodies: longitudinal evidence from controlled infections of kenyan adults with varying natural exposure expression of the plasmodium falciparum clonally variant clag genes in human infections vardb: a pathogen-specific sequence database of protein families involved in antigenic variation var csa binding phenotype has ancient origin and arose before plasmodium falciparum crossed to humans: implications in placental malaria vaccine design. sci rep global genetic diversity of var csa in plasmodium falciparum with implications for malaria in pregnancy and vaccine development how does the vsg coat of bloodstream form african trypanosomes interact with external proteins? variant surface glycoprotein density defines an immune evasion threshold for african trypanosomes undergoing antigenic variation towards a universal influenza vaccine: different approaches for one goal the emerging influenza virus threat: status and new prospects for its therapy and control influenza a virus hemagglutinin antibody escape promotes neuraminidase antigenic variation and drug resistance a comprehensive review on equine influenza virus: etiology, epidemiology, pathobiology, advances in developing diagnostics, vaccines, and control strategies novel platforms for the development of a universal influenza vaccine computer aided epitope design as a peptide vaccine component against lassa virus genome-wide identification of novel vaccine candidates for plasmodium falciparum malaria using integrative bioinformatics approaches. biotech in-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases antigenic variation and immune escape in the mtbc vaccinomics approach for designing potential peptide vaccine by targeting shigella spp. serine protease autotransporter subfamily protein siga designing a multi-epitope based vaccine to combat kaposi sarcoma utilizing immunoinformatics approach efficient control of chronic lcmv infection by a cd t cell epitope-based heterologous prime-boost vaccination in a murine model a novel multi-epitope vaccine from mmsa- and dkk for multiple myeloma immunotherapy evaluation of tandem chlamydia trachomatis momp multi-epitopes vaccine in balb/c mice model development of a multi-epitope peptide vaccine inducing robust t cell responses against brucellosis using immunoinformatics based approaches identification of a cd t-cell epitope in the hemagglutinin stalk domain of pandemic h n influenza virus and its antigen-driven tcr usage signature in balb/c mice pep-fold : faster de novo structure prediction for linear peptides in solution and in complex the protein model portal-a comprehensive resource for protein structure and model information exploring leishmania secretory proteins to design b and t cell multi-epitope subunit vaccine using immunoinformatics approach ap- transcription factors as regulators of immune responses in cancer vaccinomics and personalized vaccinology: is science leading us toward a new path of directed vaccine development and discovery? milieu intérieur consortium. distinctive roles of age, sex, and genetics in shaping transcriptional variation of human immune responses to microbial challenges evaluation of levamisole as an adjuvant for typhoid fever vaccine formulation hla and infectious diseases predicting antigen presentation-what could we learn from a million peptides? front immunol development of a human leukocyte antigen-based hiv vaccine hla dna sequence variation among human populations: molecular signatures of demographic and selective events major histocompatibility complex: antigen processing and presentation harnessing the power of t cells: the promising hope for a universal influenza vaccine. vaccines (basel) recalling the future: immunological memory toward unpredictable influenza viruses human leukocyte antigen (hla)-binding epitopes dataset for the newly identified t-cell antigens of mycobacterium immunogenum fundamentals and methods for t-and b-cell epitope prediction human leukocyte antigen (hla) and immune regulation: how do classical and non-classical hla alleles modulate immune response to human immunodeficiency virus and hepatitis c virus infections? front immunol amino acid signatures of hla class-i and ii molecules are strongly associated with sle susceptibility and autoantibody production in eastern asians predicting hla class i non-permissive amino acid residues substitutions major histocompatibility complex class i binding predictions as a tool in epitope discovery prediction of mhc class i binding peptides using profile motifs design of peptide-based epitope vaccine and further binding site scrutiny led to groundswell in drug discovery against lassa virus immunoinformatics approach for epitope-based peptide vaccine design and active site prediction against polyprotein of emerging oropouche virus polymorphisms of immunoglobulin receptors and the effects on clinical outcome in cancer immunotherapy and other immune diseases: a general review sex differences in vaccine-induced humoral immunity sex differences in older adults' immune responses to seasonal influenza vaccination biological sex affects vaccine efficacy and protection against influenza in mice differential antibody responses to rubella virus infection in males and females genomics of immune response to typhoid and cholera vaccines human immune system variation omic technologies and vaccine development: from the identification of vulnerable individuals to the formulation of invulnerable vaccines advances in genetics and genomics: use and limitations in achieving malaria elimination goals. pathog glob health safety evaluation in mice of the childhood immunization vaccines from two south-eastern states of nigeria prevent working group. pregnant women & vaccines against emerging epidemic threats: ethics guidance for preparedness, research, and response vaccinations for pregnant women efficacy and safety of pertussis vaccination for pregnant women -a systematic review of randomised controlled trials and observational studies beyond passive immunity: is there priming of the fetal immune system following vaccination in pregnancy and what are the potential clinical implications? front immunol zika virus epidemic in pregnant women ebola virus disease in pregnancy: clinical, histopathologic, and immunohistochemical findings the ethics working group on zikv research & pregnancy. pregnant women & the zika virus vaccine research agenda: ethics guidance on priorities, inclusion, and evidence generation maternal immunization maternal immunization: where are we now and how to move forward strengthening maternal immunisation to improve the health of mothers and infants immunoinformatics: in silico approaches and computational design of a multi-epitope, immunogenic protein while this review has not been funded directly by them, we gratefully acknowledge the drug design laboratory of faculty of pharmaceutical sciences, nnamdi azikiwe university, nigeria, and drug discovery africa. all the needed data are included in this manuscript. all authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work. the authors report no funding and no conflicts of interest in this work. key: cord- -y g ceq authors: affolder, rebecca; zaffran, michel; lob-levyt, julian title: global immunization challenge: progress and opportunities date: - - journal: maternal and child health doi: . /b _ sha: doc_id: cord_uid: y g ceq after reading this chapter and answering the discussion questions that follow, you should be able to: outline important milestones in the emergence of vaccines as a means of disease control and prevention. discuss factors that underpin the disparity in access to vaccines between rich and poor countries. identify and appraise innovative options for financing vaccine development, and for ensuring wider access to new and underused vaccines in developing countries. evaluate strategies for ensuring sustainability in vaccine development, management, and access. outline priorities for future research, policy, and practice with regard to vaccine development, procurement, and access. vaccines, having been developed over the last years to become one of the most cost-effective and successful public health interventions, are one of the most exciting technologies in the world today. yet every year, around . million children die from diseases that can be prevented by currently available or new vaccines. vaccines have the potential to erase some of the most glaring global health inequities which currently shape the lives of millions. often the most vulnerable -women, children, and adolescents in even the poorest countries, could be protected against life-threatening and debilitating disease within a generation. this chapter presents a historical perspective on the emergence of vaccines as a means of disease control and prevention over the past two centuries. beginning with discov- inequity in access to vaccines between rich and poor countries and the underpinning factors are discussed, including lack of safety and quality assurance systems in poor countries, focus of research and development on rich nations' priorities, and the diversion of scarce resources to other emerging global health priorities. various innovative options for financing wider access to new and underused vaccines in poor countries are explored, including the role of the international finance facility for immunization (iffim), the advanced market commitment (amcs), the heavily indebted poor countries (hipci) and multilateral debt relief (mdri) initiatives, and the debt buy-down program of the world bank. issues of sustainability in vaccine development, procurement, and management are discussed as are priorities for future research, policy, and practice. the first immunization -and the origin of a smallpox vaccine -is believed to have been in (table . ) when british physician edward jenner administered fluid from a cowpox lesion obtained from a milkmaid named sarah nelmes andre ( ) ; plotkin ( ) to a -year-old boy named james phipps. jenner later found that the boy was ''secure'' to smallpox virus (andre ) . louis pasteur later coined the term vaccine in reference to the latin word for cow: vacca. records of a similar medical approach can be found in chinese literature dating back to the eleventh century and linked with the fight against the smallpox virus (plotkin ) . according to the national library of medicine (u.s. national library of medicine ), the practice of variolation, where small scabs of tissue containing smallpox were inhaled causing the individual to contract the disease in a mild form, reduced the mortality rate among those exposed to the disease to - % as opposed to % when individuals contracted the disease naturally. by , the practice of variolation as a response to smallpox had expanded to india, africa, and throughout the ottoman empire. variolation was first practiced in europe by and, by , in the american colonies (u.s. national library of medicine ). the immunization field grew in the th and th centuries, with major breakthroughs in the mid-to late th century through discovery of vaccines that protect against such diseases as influenza, polio, and yellow fever (table . ) . prior to the development of such vaccines, the loss of life from disease is illustrated in some staggering figures. for example, the influenza (or ''spanish flu'') outbreak of - resulted in more deaths than enemy fire in world war i (plotkin ) . the period of - can be considered a second phase in the history of immunization. the world health organization (who) launched the expanded program on immunization (epi) in , expanding the smallpox eradication effort which was focused on one single vaccine into an infant program of six vaccines (against diphtheria, pertussis, tetanus, poliomyelitis, measles, and tuberculosis). at the time, less than % of the world's children were immunized against these six diseases. meanwhile, an increased degree of population mobility, for example, through commercial air travel, helped bring about the recognition that infectious disease prevention required a coordinated, global effort. the epi launch marked an important turning point: immunization became an international public good. in response to a world health assembly challenge (world health assembly ), immunization coverage rose over the next decade, with the united nations children's fund (uni-cef) declaring % of the world's children under the age of immunized against tuberculosis, polio, and measles by (hardon and blume ) . a number of global initiatives contributed to the progression of immunization coverage rates in the s. unicef, with the support of other international organizations, launched the ' 'child survival revolution'' in (unicef . this initiative comprised four interventions for reducing mortality: growth monitoring, oral rehydration, breastfeeding, and immunization (gobi). at the same time, who led major vertical programs to combat vaccine-preventable disease, diarrhea, and acute respiratory infections (hardon and blume ) . the universal childhood immunization (uci) goal was launched in to catalyze efforts toward universal immunization coverage. uci aimed at accelerating epi, capitalizing on the success in mobilizing support. as a result of these dedicated efforts, child mortality declined in many countries (hardon and blume ) . yet, despite the overall success of accelerating immunization coverage in the period described above, significant disparities are apparent ( fig. . ). the expansion in coverage was largely in developed countries with large populations. one hundred and seven countries did not reach the immunization coverage of %, and the declaration of success did not reflect the uneven coverage within many countries -where some of the most vulnerable children in hard-to-reach areas were missed. a great success for some masked the growing divide in access between north and south. the characteristics of the north/south divide, which remains the current global situation, developed during the s. a gap in the routine immunization schedules for children in developed and developing countries emerged as new vaccines, including those for hepatitis b, haemophilus influenzae b (hib), varicella, pneumococcal, meningococcal, and combination formulations became a routine part of the immunization schedule for children and adolescents in high-income countries (hardon and blume ) . research and development priorities favored those products targeting developed countries. vaccine quality and safety, taken for granted in many countries with robust regulatory agencies, fell behind in many countries lacking an effective quality assurance program for medical products. quality and safety issues also point to the weakness of health delivery systems in many poor countries which limited the effective rollout of routine immunization. the gap in financial commitment to maternal and child healthwhich underpins and drives the north/south divide in access to immunization -widened over the s as scarce resources were diverted to other emerging global health priorities. many developing countries struggled to improve or even maintain their immunization rates. the end of the decade saw an overall decline in global immunization and vaccine production, and particularly among the poorest populations in the poorest parts of the world. the new millennium set the stage for a major shift in the global response to the growing inequities between north and south. under the leadership of the then un secretary general kofi annan, the un millennium summit, the largest-ever gathering of world leaders, was convened at the united nations headquarters in new york, usa, in september (united nations development program . at the close of the summit, world leaders unanimously adopted the ''united nations millennium declaration'' taking on a clear obligation to act through commitment to the millennium development goals (mdgs) (united nations ) . these goal comprised a set of time-bound and measurable goals and targets for combating poverty, hunger, disease, illiteracy, environmental degradation, and discrimination against women. corresponding financial commitments from the developed world in the form of aid, trade, debt relief, and investment were made at the international conference on financing for development in monterrey, mexico (ifad ). as part of a renewed commitment to poverty reduction and human development, the international community moved to address the growing inequalities in immunization and the unacceptable toll of infectious disease in developing countries. marking the start of a ''third phase'' in the history of immunization, the global alliance for vaccines and immunization (now the gavi alliance) was launched in january to accelerate access to new and underused vaccines in the poorest countries. gavi, an innovative public/private partnership, brought together the major stakeholders in immunization in order to achieve global immunization targets. these stakeholders included national governments, unicef, who, the world bank, the bill and melinda gates foundation, the vaccine industry, public health institutions, and nongovernmental organizations (gavi alliance a). soon after gavi's launch its mandate came to include action on the child mortality target of the millennium development goals -namely, a / reduction of the under- mortality rate by (gavi alliance b). in the years since gavi's launch, overall dtp coverage increased from % in to % in in gavi-eligible countries, i.e., those with a gross national income (gni) of less - than $ , per capita. the figures are more pronounced in the who african region where dtp coverage increased from % ( ) to % ( ) and has overtaken southeast asia ( % in ), which is now the region with most unimmunized children (who b) . much of this increase in dtp coverage has been attributed, through independent evaluation, to the immunization services support provided by gavi to strengthen immunization delivery systems and infrastructure (lu et al. ) . in terms of new and underused vaccine introduction, the cumulative achievement of the poorest countries to improve coverage is impressive (gavi alliance b). over years, . million additional children were immunized against hepb ( ) ( ) ( ) ( ) ( ) ( ) . four and a half million additional children were immunized against yellow fever in , equaling a cumulative . million additional children immunized over years against yellow fever. an additional . million additional children were immunized with hib vaccine in , equaling a cumulative . million additional children immunized with hib vaccine over years. critical to these improvements has been the ability of the gavi alliance to raise new and additional resources -providing funds to introduce new and underused vaccines, improve injection safety, improve immunization delivery services, and strengthen health systems. gavi-supported countries are continuing to produce impressive results (gavi alliance a). despite the exciting results, we must not lose sight that the key challenges remain gaining better data on disease burden to stimulate demand and ensuring the affordability and long-term sustainability of new vaccine introduction. until prices become more affordable, slow uptake of new vaccines in the poorest countries remains inevitable. how this challenge can be better addressed through innovative approaches is covered in the discussion on funding challenges below. the gavi alliance is but one element of a growing complexity of agencies working on maternal and child health issues; while it maintains a niche focus, this requires close collaboration with partners in the broader global health community. the launch of the global immunization vision and strategy (givs) in (who/unicef ) provided a critical overarching framework that exhibits the need for coordinated mix of instruments and approaches. these approaches may be in the form of highly successful vertical campaign strategies for the global eradication of polio and control of measles, delivery of basic vaccines in conflict environments, or in the longer-term efforts to create sustainable markets for new and underused vaccines in the poorest countries. givs was approved by the member states of who and the executive board of unicef in . it sets out a plan to address the global immunization challenges over the decade - and strives to act with equity and gender equality, in addition to personal ownership, partnership, and responsibility. placing immunization firmly within the health system strengthening agenda, givs ''aims to sustain existing levels of vaccine coverage, extend immunization services to those who are currently unreached and to age groups beyond infancy, introduce new vaccines and technologies, and link immunization with the delivery of other health interventions and the overall development of the health sector'' (who/ unicef ). the vision and goals of givs are a world in that highly values immunization and that has equal access to immunizations for all. this world would also support sustainable interventions in diverse social situations, changing demographics and economies, as well as being a world that will put vaccines to the best global health and security use. addressing the key challenges: funding, sustainability, equity following the launch of givs in , a who/ unicef study examined the cost, financing, and impact of immunization programs in the poorest countries (who/unicef ). implementation of givs would protect more than million children in the world's poorest countries against the major childhood diseases by . the estimated total price tag for immunization activities for - in these countries is us $ billion, one-third of which would be spent on vaccines and two-thirds of which would be spent on immunization delivery systems. the study concluded that spending on immunization will need to rise from us $ . billion per year ( ) to us $ . billion by and us $ billion by (who/unicef ) . national budgets will ultimately fund vaccines and health services. the challenge will be to grow and sustain financing from domestic resources. how will the poorest countries reach this point? donor funding in the interim and the growth of poor economies will determine the ability of countries to finance their health sectors. to illustrate the additional sums required, it is worth noting that the report of the commission for africa ( ) recommended that donors spend around % of the commission's proposed us $ billion package for africa to strengthen health systems and ensure a satisfactory response to hiv and aids by . this call for additional spending is supported by analysis which shows that many countries will be able to work within a substantially increased spending envelope for health (foster ). yet donor aid remains volatile. in health, the shortcomings of traditional aid -from poor allocation to an absence of a results-focused, coordinated effort among donors -have clearly, if not tragically, been illustrated over the last decades (radelet and levine ) . innovative financing mechanisms provide a way to overcome some of the current limitations of aid while mitigating the political risks that many donors associate with significantly scaling up finance to developing countries, for example, through transfers such as budget support. global funds and partnerships such as gavi have shown that innovative solutions to development challenges, including raising additional finance for development, can be generated by bringing together public and private stakeholders, including the civil society. gavi provides the leverage so that both donor and developing country governments can employ new and innovative funding strategies -such as performance-based grants and co-financing (long-term subsidy agreements) for new vaccines -which characterize gavi as an instrument for innovative financing. while it is too early to make any conclusive statement on the long-term market-shaping impact of gavi, an independent study states that ''emerging suppliers view the gavi market as attractive and credibility-building, with the added economic advantage of alignment with domestic or middle-income markets. this is thanks to the significant size and growth of gavi, as well as the price levels it has provided'' (boston consulting group ) . as a catalyst for further innovation in finance, gavi has had a critical role in developing two further mechanisms for financing vaccine introduction and development: the international finance facility for immunization (iffim) and advance market commitments (amcs). the iffim, launched in , is a pilot of the larger international finance facility (iff) that was originally proposed by the government of the united kingdom in to double global aid for development and to accelerate the availability of funds through the gavi alliance in of the poorest countries around the world. the mechanism takes long term ( years), legally binding commitments from donors (iffim ) and borrows against them for years in the capital markets, producing upfront finance and thus stabilizing a portion of aid flow to developing countries. because of the innovative ''frontloading'' funding program, an anticipated iffim investment of us $ billion is expected to prevent million child deaths between and and more than million future adult deaths from hepatitis b-related liver disease. advance market commitments (amcs) provide legally binding promises, usually offered by governments or other financial entities, to guarantee a viable market if a vaccine is successfully developed. this ensures revenues will be generated from the newly developed vaccine that will match those of other comparable medicines. amcs speed the development of new vaccines by enabling biotech and pharmaceutical companies to successfully invest in vaccine development (iavi ) . beyond the clear benefit of providing long-term, predictable finance to countries, allowing them to make longer-term budgeting and planning decisions, the predictable funding for immunization through iffim has the potential to leverage significant market benefits by allowing bulk purchasing of vaccines. the predictability and legally binding nature of the financial commitment provides strengthened negotiating power and the ability to negotiate longer-term arrangements with suppliers, generating lower prices and therefore more vaccines for the same envelope of funds. a second market-shaping innovative mechanism -an ''advance market commitment'' (amc) pilot for a pneumococcal vaccine -was launched in february . an amc is a financial commitment to subsidize the future purchase, up to a pre-agreed price, of a currently unavailable vaccine -if an appropriate vaccine is developed and providing the demand exists when the vaccine is finally produced. by guaranteeing that the funds will be available to purchase vaccines once they are developed and produced, the amc mimics a secure vaccine market and takes away the risk that countries will not be able to afford a high-priority vaccine, addressing current market failure: vaccines that would prevent millions of deaths facing long delays before they are developed, tested, and produced for use in the poorest developing countries. by establishing a valuable market, amcs provide incentives for private investment in the development of vaccines against neglected diseases. such a ''pull mechanism'' is not an alternative, but is highly complementary to other public and philanthropic interventions in the health sector and, more generally, in development aid. amcs will be most effective when combined with push interventions because of the network effects of the increased number of scientific researchers working on the target diseases as well as the enhanced probability that scientific research swiftly translates into the production of effective and safe vaccines. push interventions include public and philanthropic funding of research through academia, public-private partnerships, and other bodies. the private resources mobilized by successful amcs would act in synergy with initiatives to expand immunization (e.g., gavi and iffim) and strengthen health systems. the success to date of raising funds through innovative financing instruments will continue to catalyze more thinking on both innovative means for raising and delivering development aid and how to better align these new instruments with more traditional aid streams. debt relief is an emerging area in innovative financing for health which could usefully be applied to accelerate sustainable vaccine introduction. the two major broad initiatives for debt relief are the heavily indebted poor countries initiative (hipc) and multilateral debt relief initiative (mdri) programs. the hipc initiative was launched by the international monetary fund (imf) and the world bank in and aims to reduce debt for heavily indebted poor countries that face unsustainable debt burdens, that are pursuing reform programs, and that have developed a poverty reduction strategy paper. the hipc estimates providing debt assistance in the amount of us $ billion dollars in debt relief, funded by bilateral creditors and multilateral lenders, to a total of countries ( taking the hipc a step further, the multilateral debt relief initiative (mdri) was launched by the group of eight industrialized countries (g ) in and will provide % cancellation of debt owed by hipcs to the international development association (ida), to the african development fund (afdf), and to the imf (international monetary fund, b) . this program enacts up-front, irrevocable debt cancellation for eligible countries (table . ). the main objective of the mdri is to enable hipcs to mobilize funding for poverty reduction programs in order to reach the millennium development goals. the intent is that additional resources made available through debt relief should be allocated to poverty alleviation programs. but as there is no formal obligation to allocate resources relieved by the mdri to any specific sector, competition between departments for the use of these extra resources is likely. potential impact of the mdri on health system strengthening and on financing immunization programs could be significant. as annual amounts of debt service relief will be significant in many hipcs, especially around - , a small percentage of these resources could have a reasonable impact on the health sector and in particular on immunization financing. the gavi alliance partners are currently exploring options for using debt relief -in the form of an international development association (ida) buy-down -to specifically support countries' vaccine programs. in addition, a number of bilateral debt relief programs may also offer an opportunity for targeted debt relief. ida buy-downs are currently being explored as new innovative financing mechanisms for vaccines. ida is member of the world bank group. it provides long-term loans (also called concessional loans or credits) and grants to the poorest of the developing countries, particularly those that are severely constrained by conflict, epidemics, and debt. a buy-down refers to a third party paying off all or part of a specific ida credit on behalf of the government upon successful achievement of pre-determined performance indicators. the world bank began an ida buy-down pilot in , when it provided the governments of nigeria and pakistan with roughly $ million in ida credits for the purchase of vaccine to help achieve the global polio eradication objective. the bill and melinda gates foundation, rotary international, and the united nations foundation agreed to pay off the ida credits upon successful achievement of the performance indicators, in this case receipt and distribution of vaccine and specified polio immunization coverage levels. innovative financing, while not a magic bullet, will nonetheless offer a range of new possibilities for countries to help reach the significant increases in finance required to meet the mdgs. ultimately, the real test will be whether the donor community is successful in working together to ensure traditional aid is aligned to a mixed instrument approach. this has been done before. bangladesh, one of the poorest countries in the world, has achieved the most radical improvements in reproductive health the world has ever seen. this has impacted significantly on women's and child mortality and morbidity, their social status and economic growth -despite poverty, poor governance, political upheaval, and an apparent lack of any potential for economic growth in the early years. the key was that for years from the mid- s, through a mixture of aid instruments, donors and multilateral agencies provided substantial, predictable but coordinated financial and technical support for salaries, a radical expansion in the workforce (notably paramedics), associated infrastructure, and ''expensive'' reproductive commodities which the government delivered through state and civil society structures. it has become clear that new technologies such as vaccines or antiretrovirals (arvs) for hiv have the potential to deliver a generational leap in achieving the mdgs. the health gains made in europe over years could be achieved in africa over a - year period (who/unicef ). of the more than million annual child deaths, an estimated % could be avoided through immunization with existing and newly developed vaccines such as pneumococcal and rotavirus vaccines. procurement of essential health commodities is an area where this can be carried forward without risk to macroeconomic stability. yet without basic health systemsessential for the sustainable availability of medical products -the poor will never access these benefits. despite evidence of the cost-effectiveness of vaccines in particular and the economic and social benefits of health in general, the track record of national and donor budget allocations to date is not good. gavi-eligible countries have very modest health budgets, with government health spending across africa, for instance, averaging $ -$ per capita and with many countries below $ . responding to the needs of poor countries by investing in the critical foundation for the delivery of basic health services requires a long-term view. while vertical approaches have been effective at raising the profile and funding levels for vaccines, countries must now be supported to move systematically to introducing the full range of vaccines in immunization programs as part of integrated maternal and child health services. with expensive new vaccines coming to market (for example, three doses each of pentavalent (dtp-hepb-hib), rotavirus, and pneumococcal conjugate vaccines could amount to more than us $ per child) it is clearly no longer appropriate to focus on financial sustainability of a single product in isolation from broader system sustainability. moving toward a truly sustainable planning framework will not be a simple endeavor, yet it represents an exciting opportunity for the gavi alliance partners. one challenge will be to gather the information on demand and future prices required by countries to inform longer-term planning and decision making. unicef's commitment and global procurement ability over the years has brought great benefits in terms of quality, security, and better prices for such long established vaccines as bcg, dpt, measles, and polio. but it has become clear that this procurement model is most effective in mature markets with overcapacity and competition, and notably capacity in countries located in emerging markets (e.g., india, brazil, indonesia, and cuba). new or combination vaccines such as dtp-hepb-hib challenge the established means of procurement, where cost limits the ability of donors to deliver affordable products to the poorest parts of the world. it is only through competition that the prices of new vaccines will become affordable to the poorest countries. clearly the key to success will be the ability to mobilize additional donor funds, but to use those funds in such a way that the vaccine market is shaped to promote competition and to bring prices within reach of the poorest countries. beginning in , gavi support shifted toward national co-financing (as opposed to gavi providing vaccines free). this is based on the intent by the gavi alliance partners to ensure that gavi financial support is seen by all stakeholders as time limited and to ensure that countries move to a fuller ownership of their immunization program, including the introduction of new vaccines. co-financing therefore aims at supporting and stimulating evidence-based priority-setting within the immunization program and within the health sector more generally. financial commitments, however small, also generally require a higher level of government engagement. through this approach, which will be evaluated in , gavi alliance partners are working to help countries to be on a trajectory of eventual independence from gavi support, acknowledging, however, that, for most of the gavi-eligible countries this is likely to require a very long time over the next decade, the ability of developing countries to achieve sustainable introduction of new technologies will be largely dependent on how donor funds are provided, particularly whether there is a shift toward long-term, predictable aid and if innovative financing instruments are appropriately aligned and taken to scale. the other key determinant will be sustained political support for health and for vaccines by developing country governments. guyana is an example of a country that has been highly successful in achieving high immunization coverage and is the first gavi-supported country to fully finance the purchase of pentavalent vaccine from its national budget (united nations ). guyana's continuing success is in part due to a very strong political commitment at the highest levels to finance the national immunization program, including efforts to protect it from economic shocks and shifts in donor priorities. more broadly, there has been a remarkable growth in the health budget from us $ . per capita in to us $ in (excluding overseas development assistance). this accounts for % of national expenditure, while the government's goal is to reach % (ministry of health, guyana ; editorial, pharmacoeconomics and outcomes news, ) . from an equity point of view, gavi's condition of support to the ministry of health, china, was that vaccines be made available at no cost (removing the previous charge). this policy was subsequently adopted across china for all vaccines. while the spread of hiv and aids has led to recent discourse on health as a global security issue, most arguments -and certainly those related to maternal and child health -have at their root the principle of equity and the belief that health is a basic human right. equity in health has been defined (for measurement and operationalization) as ''the absence of systematic disparities in health (or in the major social determinants of health) between groups with different levels of underlying social advantage/disadvantage -that is wealth, power or prestige'' (braveman and gruskin ) . the world development report, making services work for poor people, noted that ''the concern for equity is either a social choice or based on the notion that health is a human right'' (world bank ) . as an ethical or social justice issue, equity in health is therefore a critical element for consideration and measurement, particularly when looking at the trade-offs and choices made around financial sustainability issues discussed in the previous section. many of the disparities in health result from social determinants such as poverty, access to services, education, gender, and ethnicity. harnessing the potential of new medical technologies, such as vaccines, to reach underserved groups will take concerted effort and in some cases, explicitly defined political choices. new vaccines against human papilloma virus (hpv) provide the opportunity for such a political choice: to ensure that all women, rather than just those in wealthy countries, are provided with a vaccine that will prevent most cervical cancer cases. hpv vaccines, as the first vaccines to focus primarily on women's health, provide the global health community an unprecedented opportunity to tackle a key neglected women's health issue -one which especially impacts on the poorest women. cervical cancer is not difficult to prevent; yet, it affects an estimated , women each year and leads to more than , deaths (ferlay et al. ) . it is largely a disease of poor women who have limited access to health services; about % of women dying from cervical cancer live in developing countries (fig. . ) (ferlay et al. ) . the lack of effective cervical cancer prevention interventions -part of a regular medical checkup for women in wealthy countries -is a major factor in the high rates of cervical cancer among poor women. if current trends in women's health continue, there are projected to be over , , new cases of hpv annually by the year (boyle ) . many challenges must be addressed before hpv vaccine can reach the millions of girls and young women who would benefit from it, especially those living in the developing world where the need is greatest. with the right combination of scientific, educational, and financing efforts, hpv vaccine could become available globally within a few years. accelerating access to hpv vaccine could make cervical cancer -the second most common cancer among women worldwide -a rarity in just a few decades. another social determinant of health is where one lives. within large developing countries, such as india, nigeria, or china, there are significant inequities in the population's health. disparities in access to, and utilization of, services within these countries are often a result of factors such as geography, social barriers, conflict, and weak governance. of the million children that missed out of immunization in more than % live in countries (fig. . ). india and nigeria stand out as countries with the largest number of unimmunized children in the world. reaching mdg will thus require a significant increase in investment in immunization -both domestic and external -in countries with large numbers of unimmunized children who account for more than half of all vaccine-preventable deaths among children less than years of age. with some states or regions in some of these countries being equal or larger in population to many countries, a fresh state-or region-based approach will likely be required, with a focus on the poorest. for example, child and maternal mortality rates in the poorest eastern provinces of china equal or exceed those found in much of africa (world bank ) . despite economic growth, equity is worsening. national political commitment in such countries will be key. a program approach, tailored to country-specific challenges, will be required. additional long-term finance (domestic and global) will be critical to support that political commitment. new technology, including new and better vaccines, will be vital. which vaccines for the future? research and development for vaccines and other essential health commodities point to another disparity between north and south and constitute a market failure. priorities in the global allocation of resources for vaccine research and development do not match the global burden of death and disease. few resources are allocated to tackling diseases that disproportionately affect people in developing countries; new vaccines are therefore expensive and out of the reach of the poor. this discrepancy between need and reality is illustrated in table . , illustrating that normal market mechanisms do not work for the poor. among the vaccines currently under development, the three most needed today in terms of their potential public health impact are for aids, tb, and malaria. jointly, these diseases account for over million deaths per year or around % et al. ( ) of all infectious disease deaths. the total investment in vaccines against these diseases is far lower than their importance as dictated by disease burden and it will probably take at least - years before a vaccine against any of these diseases is available. in the past two decades, advances in biotechnology have resulted in the licensure of new vaccines such as hib, acellular pertussis, hepb, and attenuated varicella. most of the basic scientific breakthroughs have been generated in research institutions in the public sector whereas the cost for clinical development is borne by the pharmaceutical industry. this requires heavy investments that need to be recouped from profits. the markets needed to recoup these investments are in industrialized countries that can afford to buy. the evolving disease burden in developing countries will bring new diseases into prominence while sometimes allowing old ones to resurface. this will influence priorities for vaccine research (table . ). the severe acute respiratory syndrome (sars) epidemic, the outbreak of avian influenza, and the emergence of bioterrorism threats such as anthrax have led to new research avenues for vaccines against these infections. the threat of a reassorted influenza pandemic virus strain has highlighted the need for more resources and attention to the development and distribution of effective flu vaccines. alternative administration routes for vaccines would greatly contribute to improving immunization program safety and potentially reduce the quantity of contaminated waste which needs to be safely disposed. this could help avoid needle transmission of blood-borne pathogens and ease vaccine delivery strategies where non-professionals can administer vaccines. new administration routes such as oral, nasal, and transcutaneous are currently being explored. one option currently being explored through collaboration by who, path, and the serum institute of india is focusing on the development of a measles aerosol vaccine that could make a big difference in eliminating this disease by facilitating administration, during mass campaigns (burger et al. ) . the measles aerosol vaccine is useful in situations where the availability of trained medical personnel, who can safely administer injections, is limited. immunogenically in studies, the aerosol vaccine was proven effective > % of the time among infants < months of age and - % among infants > months and school-aged children (henao ) . this vaccine continues to be tested in clinical trials in order to find the most appropriate and effective aerosol delivery method. another interesting option is the concept of using plant-derived or edible vaccines that involve encoding protective antigens from pathogens into transgenic plants (mor et al. ). the plants are processed so that they can deliver a uniform dose of vaccines. human clinical trials have been conducted with bananas and raw potatoes, which showed encouraging antibody responses (sala et al. ) . plant-derived vaccines are formed when a gene is integrated with a plant nucleus or chloroplast genome. this transforms higher plants (e.g., tobacco, potato, tomato, and banana) into bioreactors for the production of subunit vaccines for oral or parental administration (sala et al. ). the potential advantage of this technology could include thermostability, low investment needs, multivalency, and oral administration. new technologies that strengthen vaccine delivery are under development. priority is given to such technologies that will (a) expand access, (b) improve safety, and (c) cut the cost of immunization programs. they include the following five technologies: (i) ''sharps'' processing: the increased use of autodisable (ad) syringes (syringes which lock themselves after a single injection) has greatly improved the safety of immunization programs by avoiding the reuse of contaminated syringes and reducing risks of transmission of blood-borne pathogens such as hepatitis b, hepatitis c, and hiv (lloyd ) . this success is, however, highlighting another problem which the health sector is facing, that of the handling of contaminated medical waste. in the case of immunization, this is mainly related to the disposal of used syringes and needles (these syringes represent between and % of all injections given in the health sector but nevertheless the push to introduce ad syringes is increasing the pressure on immunization programs to tackle this challenge). sharps are rarely disposed of at the point of use. since sharps are transported to the point of destruction, the risk of infection from accidental exposure to sharps must be minimized. four different technologies are being explored for this purpose: corrosive disinfectants, thermoprocessing, needle destruction, and plastic melting (lloyd ) . however, none of these options is currently sufficiently developed to be put into use in the field. (ii) monodose pre-filled devices: vaccine wastage constitutes a considerable cost to immunization programs. monodose presentations eliminate wastage and the risk of contamination. when the monodose is pre-filled into an injection device, it increases quality and safety at the point of use. uniject is one such device that has been tested with hepb and tetanus toxoid (tt) (lloyd ) . village health workers can administer it. currently, major obstacles reside in the cost of the device and the need for additional cold storage space when multidose presentation is exchanged for monodose, but ultimately, the objective would be to provide an increasing number of immunizations with monodose preparations that would not require increased cold chain capacity. (iii) needle-free injections: needle-free injectors deliver vaccine at high velocity into the skin without penetration of a needle, thereby reducing the risk of transmission of blood-borne pathogens (who c) . technologies are being developed for both mono-and multidose presentations. multidose injectors available have not been found safe and new models are under development. there are several monodose models available; however, they are not feasible for large-scale programs because of regulatory obstacles and high cost (who ) . (iv) thermostable vaccine: vaccine distribution and storage without a cold chain would considerably simplify the delivery system, reduce cost, and allow for integrated supply mechanisms. removal of vaccines from the cold chain should be the highest priority for technology research. sugar glass drying is one such technology that has shown great promise (lloyd ) . it can be used to produce multivalent vaccines that are completely heat stable, except under extreme climatic conditions. the high cost of regulation/licensing and the uncertainty about market prospects in industrialized countries have so far impeded the development and use of this technology. vaccines are delicate products that are easily destroyed if handled incorrectly. vaccine management spans a spectrum of aspects involving the use and disposal of vaccines, from the manufacturers to the end-users, for which plans must be in place and regularly updated to ensure an effective and efficient service delivery including (i) inventory and forecasting; (ii) stock control; (iii) in-country distribution; (iv) storing and handling; (v) equipment replacement; (vi) procedures for the use of vaccine; (vii) monitoring of vaccine storage; (viii) transport management; and (ix) operational management. all of these areas would benefit significantly from research efforts to find alternative and innovative approaches. for instance, the heavy reliance on the cold chain remains a major economic and logistical burden on programs. the possibility of taking greater advantage of the real thermostability of vaccines and the increasing use of the vaccine vial monitor by taking vaccines ''out of the cold chain'' is a field which has only begun but could potentially revolutionize immunization delivery (table . ) . vaccine vial monitors are heat-sensitive circular labels, no wider than a centimeter, that change color as vaccines are exposed to heat. they are time-temperature indicators used to (i) ensure that the vaccines have not been damaged by excessive exposure to heat, (ii) identify weaknesses in the cold chain, and (iii) take vaccines beyond the cold chain to reach out to children who have no access to fixed health facilities. health workers can use the vaccine vial monitor color to tell if the vaccine has been overexposed to heat and whether or not it is safe for immunization. this indicator cuts down on the uncertainty of vaccine safety due to potential temperature changes during transport along the cold chain. therefore, the vaccine vial monitor reduces waste. immunization remains one of the most cost-effective of all public health interventions. maternal and child health-related mdgs will be difficult to meet without significantly scaling up the coverage of existing vaccines and successfully introducing new pipeline products -ensuring that research and development priorities are aligned with the diseases for which preventative technologies are needed most. financing this effort, however, poses a considerable challenge. a serious commitment to closing the north/south divide and meeting mdgs will require a joint approach that involves increased investment by developing country governments and better, more stable aid flows from donors. increased investment, particularly in the social sector, will be critical to finance costs such as system building that require large amounts of sustained finance. in-kind investments in commodities can be scaled up rapidly without major concerns around absorptive capacity or macroeconomic stability. long-term, predictable aid flows are also needed to reduce volatility and provide increased certainty over future budget flows to enable better planning in countries. as a global community, we must start approaching our work from a perspective that evaluates who is taking on the burden of risk -it clearly should not be the poorest countries. risk analysis is a common tool in the private sector -companies only take decisions based on the probable level of risk it implies for them. yet the donor community consistently places the poorest countries in a position where it is very difficult for them to make choices of how or whether to radically scale up access to basic services. the donor community, including the gavi alliance and the international financial institutions, needs to develop strategies to reduce financial and political risks. this means adjusting processes and requirements to support the long-term integrated plans of developing countries. the financial risks of development strategies must be more equitably shared between donors and national governments. development will be led by developing countries when they are enabled to plan ahead; what factors account for the disparity in immunization coverage between developed and less developed countries? . what is the gavi alliance? how does its mission compare with those of global immunization vision strategy what major barriers confront the gavi alliance and givs in their efforts to ensure equity in access to new and underused vaccines in developed and less developed countries? in a narrative of about , words, describe the meaning and mission of the following initiatives: a. international finance facility for immunization advance market commitments (amcs) how successful are iffms and amcs in accomplishing their mission? vaccinology: past achievements, present roadblocks, and future promises global vaccine supply: the changing role of suppliers cervical cancer prevention: current situation. eurogin international expert meeting on hpv infection and cervical cancer prevention defining equity in health stabilizing formulations for inhalable powders of live-attenuated measles virus vaccine developing countries are providing cofinance for life-saving vaccines fiscal space and sustainability: towards a solution for the health sector. high level forum on health mdgs unfinished agendas and mixed results an overview of aerosol immunization, meeting of the who steering committee on new delivery systems international fund for agricultural development (ifad) ( ) international conference on financing for development -statement by lennart ba˚ge, president of ifad advance market commitments: helping to accelerate aids vaccine development debt relief under heavily indebted poor countries (hipc) initiative the multilateral debt relief initiative (mdri) benin: third review under the three-year arrangement under the poverty reduction growth facility, request for waiver of nonobservance of a performance criterion, and request for extension of the arrangement technologies for vaccine delivery in the st century effect of the global alliance for vaccines and immunization on diphtheria, tetanus, and pertussis vaccine coverage: an independent assessment guyana financial immunization sustainability plan . brickdam, georgetown: ministry of health/ministry of finance perspective: edible vaccines -a concept coming of age why certain vaccines have been delayed or not developed at all can we build a better mousetrap? three new institutions designed to improve aid effectiveness vaccine antigen production in transgenic plants: strategies, gene constructs, and perspectives. vaccines united nations children's fund (unicef) ( ) the state of the world's children : the s: campaign for child survival human development report: millennium development goals: a compact among nations to end human poverty developing countries join gavi alliance and who to ''co-finance'' vaccines for poor children the world development report -making services work for poor people china's progress toward the health mdgs proceedings of the first global vaccine research forum traditional medicine. the fifty-sixth world health assembly (wha . ) geneva: world health organization china immunises millions of children against hepatitis b in historic collaboration between government and gavi alliance united nations children's fund (unicef) ( ) global immunization strategy who ivb human papillomavirus & hpv vaccines: technical information for policy-makers and health professionals world health organization (who) ( b) who report on gavi progress world health organization (who) ( c) immunization safety for ensuring sustainability in procurement, access, and uptake of vaccines in less developed countries. what are the major barriers? . what should be the priorities for future vaccine research and development globally? provide justification for your position. key: cord- - evbeijx authors: pandey, rajan kumar; bhatt, tarun kumar; prajapati, vijay kumar title: novel immunoinformatics approaches to design multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: evbeijx malaria fever has been pervasive for quite a while in tropical developing regions causing high morbidity and mortality. the causal organism is a protozoan parasite of genus plasmodium which spreads to the human host by the bite of hitherto infected female anopheles mosquito. in the course of biting, a salivary protein of anopheles helps in blood feeding behavior and having the ability to elicit the host immune response. this study represents a series of immunoinformatics approaches to design multi-epitope subunit vaccine using anopheles mosquito salivary proteins. designed subunit vaccine was evaluated for its immunogenicity, allergenicity and physiochemical parameters. to enhance the stability of vaccine protein, disulfide engineering was performed in a region of high mobility. codon adaptation and in silico cloning was also performed to ensure the higher expression of designed subunit vaccine in e. coli k expression system. finally, molecular docking and simulation study was performed for the vaccine protein and tlr- receptor, to determine the binding free energy and complex stability. moreover, the designed subunit vaccine was found to induce anti-salivary immunity which may have the ability to prevent the entry of plasmodium sporozoites into the human host. increase the drug efficacy and kills the remaining parasites. the most widely used combination therapies are artemether-lumefantrine (commercial name: coartem) and amodiaquine-artesunate (commercial name: coarsucam) . while the recently approved combination therapies are artesunate-pyronaridine (commercial name: pyramax) and dihydroartemisinin-piperaquine (euartesim) . the recent emerging resistance against artemisinin urges to develop some new strategy to prevent the malaria diseases condition . therefore, in this study, we applied a novel immunoinformatics approach to design multi-epitope based subunit vaccine that may prevent the disease by maintaining the host hemostasis by the inhibition of anticoagulant and anti-inflammatory proteins present in mosquito saliva. it will also inhibit the entry of parasite within the host body by a similar mechanism. apart from this, if any way parasite enters into the host body, vaccine candidate will stop the salivary protein-mediated induction of parasitic growth. among different anopheline vectors, anopheles stephensi is a sub-tropical species that most abundantly present in the indian subcontinent and also distributed across the middle east and south asia region . a. stephensi transmit the malarial infection by injecting various plasmodium species to the human host typically via bites. salivary gland of mosquitos implement various functions for the survival of the vector and complement their feeding behavior by producing a large array of a biochemically active molecule that has immunomodulatory, anti-coagulant and anti-inflammatory properties that disable the host hemostatic response for successful blood feeding , . salivary proteins are antigenic and immunogenic in nature which helps the infectivity of parasite , . d protein and salivary apyrase are two different salivary proteins that help in binding, and inhibition of the platelets aggregation, respectively. salivary peroxidase helps in heme binding and peroxidase activity while putative til domain polypeptide functions as trypsin inhibitor . recently, it was reported that hamadarin is a kda protein present in anopheles stephensi saliva which inhibits the activation of plasma contact system and ultimately blood coagulation. another protein from anopheles gambie saliva namely gsg plays essential blood feeding , . recently, vijay s. et al. reported that salivary proteins might be utilized to develop novel antimalarial control strategies via innate immune protection against malaria . these proteins could likewise evoke a host igg response in natural conditions , . mosquito salivary gland surface (sgs) proteins are the prevalent immunogenic component present in saliva having ability to induce immunogenic responses . this is the reason why we chose the a. stephensi salivary proteins from the national center for biotechnology information (ncbi) and subjected to design multi-epitope subunit vaccine. allergenicity, antigenicity and physiochemical properties were also obtained for the vaccine protein. moreover, tertiary structure prediction followed by refinement was performed to get a refined d model having a higher number of residues in the favored region of ramachandran plot. molecular docking and molecular dynamics simulation of vaccine constructs with tlr were also performed to check the binding energy and complex stability. finally, disulfide engineering and in silico cloning was performed to increase the stability of vaccine construct and ensuring its effective expression in the microbial system, respectively. this study finally represents a novel approach to develop malaria vaccine using salivary protein instead of parasitic protein, which could be helpful to prevent the plasmodium infection to human host. sequence retrieval of salivary protein and assurance of antigenic conduct. in order to design an immunogenic multi-epitope subunit vaccine, the sum of a. stephensi salivary protein sequences was retrieved from the ncbi protein database. major proteins name is salivary lysozyme, a salivary protein precursor, salivary galectin, salivary lipase, anti-thrombin anopheline, salivary protein sg , salivary apyrase, salivary secreted serine protease inhibitor, salivary defensin and salivary cecropin. among salivary protein sequences, only proteins were found to be antigenic as predicted by antigenpro. these sequences were selected based on their score obtained for the probability of antigenicity and all these proteins having a score of ≥ . obtained score for antigenicity probability clearly denoting the antigenic nature of selected protein sequences which can be used for the subunit vaccine designing . subset of t-cell responses to kill those target cells having intracellular viral, bacterial or protozoan infection . during infection, whenever they encounter to the mhc-i mounted antigen specific to their receptor, they enter the cell cycle and perform several mitotic divisions followed differentiation into the effector cells . here, we tried to predict the ctl receptor specific immunogenic epitopes using the netctl . server and total ctl epitopes of mer length were obtained for the input of salivary protein sequences . in the next step, the immunogenicity of epitopes was determined and as per the instruction of iedb , higher score indicate greater probability to elicit an immune response; therefore total ctl epitopes with high immunogenicity score were selected and subjected to the vaccine designing (table ) . helper t-lymphocyte is the key player of both humoral and cell-mediated immune response . therefore, htl receptor specific epitopes are probably going to be a crucial part of the prophylactic and immunotherapeutic vaccine . all salivary protein sequences were subjected to iedb mhc-ii epitope prediction module and epitopes of mer length were obtained. in order to become highest immunogenic epitopes, they must have a lower percentile rank and ic value . only epitopes with lowest percentile rank ranging from . - . were selected for the vaccine designing (table ). their ic value ranging from - denoting that out of epitopes with lowest percentile rank, epitopes have intermediate affinity while remaining to have low affinity for the htl epitopes. on the other side, all epitopes were found to have ifn-γ inducing capability that was obtained from their positive score on the ifnepitope server output , (supplementary table ). all these epitopes were used for the vaccine construction. construction of multi-epitope subunit vaccine. a final vaccine construct of amino acid residues was designed using ctl and htl epitopes as described elsewhere , (supplementary figure ) . in order to attain maximum immune response tlr- agonist (rs ) was used as an adjuvant at the n-terminal site of the vaccine construct . each joint was occupied by the suitable linkers as described by nezafat et al. , for example, adjuvant and ctl epitopes were combined together by eaaak linker, intra-ctl and intra-htl epitopes joint by aay and gpgpg linker, respectively. finally, vaccine construct was obtained having adjuvant, linker, ctl, and htl epitopes in a sequence moving from n-terminal to c-terminal. as this designed subunit vaccine consisting of immunogenic ctl and ttl epitopes along with suitable adjuvant and linker, it may have the ability to inhibit the entry of malaria parasite within the human host body , . b-cell epitope mapping. b-cells are a key player of humoral immunity. an epitope corresponding to the b-cell receptor plays an important role in vaccine design following antibody production . therefore, bcpreds server was used to reliably predict the linear b-cell epitopes where bcpred was the selected prediction method . total b-cell epitopes of mer length were predicted among the primary input sequence of final vaccine construct. among them, only epitopes were selected and finalized because of their high score of . ( fig. a ). due to the selection of highest scoring b-cell epitope among designed subunit vaccine, our vaccine may have the ability to enhance humoral immunity as well as cell mediated immunity . discontinuous epitopes of amino acids long were also predicted from the final d model of vaccine construct with the probability scoring of . (fig. b) . the obtained probability score also confirming the immunogenic behavior of the designed subunit vaccine . antigenicity and allergenicity prediction of designed vaccine. a vaccine given to human host must be immunogenic in nature and capable to trigger significant humoral immune response which ultimately leads to the memory cell formation against the pathogenic epitopes. the antigenicity of designed vaccine construct was determined by using an alignment-free antigenpro server and found that it has the antigenicity probability of . , which represent the antigenic nature of vaccine construct . the antigenicity score obtained for this vaccine construct is comparable with the antigenicity of subunit vaccine reported elsewhere . allergy is an overreaction by our immune system to the previously encountered, ordinarily harmless substance that results in sneezing, wheezing, skin rash, and swelling of the mucous membrane . allergenicity of predicted vaccine construct was determined using allertop online server and found that the vaccine protein is nonallergic in nature and safe for the human use , . physiochemical properties assessment. the physiochemical properties of vaccine construct were characterized by using protparam server and evaluated for seven parameters. the molecular weight of vaccine protein was found to be kda which will favor the antigenicity of the vaccine construct . the theoretical pi was found to be . showing its slightly acidic nature while the total numbers of negative and positive charge residues were and , respectively . the estimated half-life in mammalian reticulocytes was . hours, in vitro; while and hours in yeast and e. coli, in vivo. the extinction coefficient was found to be m- cm- , at nm measured in water, assuming that all cysteine residues are reduced. the score obtained for instability index was . , showing the stable nature of vaccine construct. the value of the aliphatic index and grand average of hydropathicity (gravy) was . and − . , respectively. the estimated value of aliphatic index represents the thermostable nature of designed subunit vaccine because higher the value of aliphatic index, greater will be the thermo stability . while, negative value of gravy for the input subunit vaccine represents the hydrophilic nature of vaccine . conclusively, the designed vaccine is immunogenic, thermostable and hydrophilic in nature. tertiary structure prediction, refinement, and validation. the tertiary structure was predicted by using the raptorx server and d model was obtained as described elsewhere (fig. a) . the best template used for the homology modeling was crystal structure of a legionella phosphoinositide phosphatase (pdb id: fye). total amino acid residues were modeled as a single domain with % disorder. secondary structure information resulting in the presence of % helix, % beta sheet, and % coiled structure. p-value is a parameter of homology modeling where low p-value defines the good quality of modeled structure . the p-value obtained for the modeled structure was . e- which is low and significant. further, protein refinement using galaxyrefine leads to the increase in a number of residues in the favored region . initially, % of residues were in the rama-favored region while after refinement the number of residues in the rama-favored region reached to . %. the refinement output was also validated by plotting ramachandran plot and found the same that . % residue in rama-favored region, . % residues in allowed region and only . % residues in outlier region (fig. b ). disulfide engineering for vaccine stability. in order to stabilize the modeled structure of final vaccine constructs disulfide engineering was performed using disulfide by design v . and found that there are total pairs of residues that can be used for the purpose of disulfide engineering. but after evaluation on other parameters like energy and chi value, only four pairs of residues were finalized because their value comes under the allowed range i.e. the value of energy should be less than . and chi should be in between − and + degree . therefore, total mutations were created at the residues pairs namely ala -gln , tyr -gly , trp -glu , and gly -phe (fig. ) . codon adaptation and in silico cloning. the main purpose of in silico cloning was to express the vaccine protein epitope of anopheles mosquito origin into e. coli expression system . therefore, it was necessary to adapt the codon respective to subunit vaccine construct as per the codon usage of e. coli expression system. we adapted the codons as per e. coli k strain using jcat server and found that the gc-content of the improved sequence was . % while the value of codon adaptive index was . which is near to . that was satisfactory . later on, xhoi and ndei restriction sites were created and cloned into the pet a(+) vector (fig. ) . the target sequence in the clone is represented in blue color in between aforementioned restriction sites . the target sequence is also enclosed between -histidine residues on both ends that will be helpful to the purification purposes. the total length of the clone was . kbp. and tlr- receptor was performed using the cluspro . and total models were generated . among them, only that model was selected which properly occupied the receptor and having lowest energy score and found that model number . fulfill the desired criteria that's why selected as the best-docked complex (fig. ). the energy score obtained for the model . was found to be − which is lowest among all other predicted docked complex showing highest binding affinity. formed using gromacs . . using a gromos a force field . the potential energy obtained for the complex was − . kj/mol, while the value of temperature, pressure, and density was obtained as . k, . bar and . kg/m ( supplementary figure a-c) . the radius of gyration obtained for the docked complex showing that the distance in rotating complex from the center of mass is . nanometers that decreases up to . nanometers at the time duration of nanoseconds (supplementary figure d) . the rmsd value of protein backbone was . nanometers (fig. a) while rmsf score obtained for the protein side chain was found to be . nanometers (fig. b ). both these scores are satisfactory showing strong complex stability , . malaria is severe infection characterized by the high fever with irregularity but may also lead to brain injury and coma. it affects million people from countries, worldwide. lack of effective vaccine and emergence of resistance against artemisinin created a disastrous condition among the people living in the endemic zone. therefore, it's the need of time to search for the new options to tackle this severe problem. the vector for malaria is the anopheles stephensi in the indian subcontinent leads to the transfer of malaria parasite. literature survey reveals that salivary proteins of anopheles mosquito not only supports the pathogenesis but are also immunogenic in nature. therefore, this study was designed to reach a step ahead in the path of vaccine development. we used the primary amino acid sequence of anopheles mosquito salivary protein to design a subunit vaccine construct. the constructed vaccine has ctl, htl and bcl epitopes of varying length. it has antigenic properties in the absence of allergenic properties. it was stable and having a good binding affinity for the tlr- receptor. collectively, this study applied a series of immunoinformatics tools in a sequential manner to find an effective vaccine that may fight against the malaria infection. however, this study needs experimental validation to prove this computational work. the experimental work may include the synthesis of designed subunit vaccine followed by the in vitro and in vivo analysis to determine the immunogenicity and safety concern of the same. anopheles stephensi mosquito is the vector of malaria transmission to the human being in the indian subcontinent. while the salivary gland proteins of anopheles mosquito was reported for their role in parasite pathogenesis and having the ability to induce igg response in the natural host , . therefore, total salivary proteins of a. stephensi were obtained from the national center for biotechnology information (ncbi) protein database (retrieval date / / ) and subjected to multi-epitope vaccine designing. as the main purpose of vaccination is to induce an immunogenic response within the host body, all the retrieved protein sequences were subjected to their antigenicity prediction using antigenpro. based on the antigenicity result, only proteins were found to have an antigenic probability of ≥ . were selected and used in the next step. cytotoxic t-lymphocyte were shown to inhibit malaria parasitic growth and development, inside the hepatocytes cells . to get an immunogenic ctl epitopes having the ability to elicit cell-mediated immunity and form the memory cells, all salivary protein sequences were subjected to the netctl . server . netctl . is an online web server intended for predicting ctl epitopes among input protein sequences based on the training dataset. netctl was selected to predict the ctl epitopes because of its higher prescient execution on all execution parameters as compared to the recently developed servers namely mhc-pathway, mappp, and epijen. all the salivary protein sequences were submitted in the fasta format to predict the ctl epitope at the threshold score of . (default). those epitopes having a combined score of greater than . were selected as ctl epitope and further subjected to the immune epitope database (iedb) mhc class i immunogenicity prediction module. predicted ctl epitopes for each salivary protein was used as an input sequence and the result was obtained in the form of the score, where higher score determines that greater will be the probability of eliciting an immune response. helper t-lymphocyte (htl) epitope prediction. helper t-cell response is the major part of cell-mediated immunity and helps in pathogen clearance by the help of various cytokines and immune cells , . they have the ability to induce both ctl and humoral immune response by the secretion of lymphokines like il- , il- , il , granulocyte-macrophage colony-stimulating factor (gm-csf), and ifnγ. in view of that, we can say that htl epitopes mainly of th type are most likely going to be a crucial part of the prophylactic and immunotherapeutic vaccine. therefore, iedb mhc-ii epitope prediction module was used to predict the htl epitopes for all anopheles salivary protein sequences . the available parameters were kept default except for allele selection where the nominated alleles were h -iab, h -iad, and h -ied. output epitopes were ranked based on their percentile rank score where lower percentile rank representing that greater will be the binding affinity for htl receptor. secondly, to prove our work that the predicted htl epitopes will have ability to activate th type immune response followed by the ifn-γ production, top predicted htl epitopes were subjected to the ifn epitope server using predict option. all epitopes were submitted in the fasta format followed by the approach selection and model of prediction. motif and svm hybrid was selected as the approach and ifn-gamma versus other cytokine as model of prediction. designing of multi-epitope subunit vaccine. so as to plan an appropriate vaccine candidate, it must have the ability to induce ctl and htl immune response. in other words subunit vaccine must contain both ctl and htl epitopes along with suitable linkers. keeping in mind the end goal to effectively activate both innate and adaptive immune response, subunit vaccine must consist of a strong immunostimulatory adjuvant. in the previous decades, there is a huge headway in the adjuvant engineering, for instance, toll-like receptor (tlr) agonists have made its contribution as a part of peptide-based subunit vaccine as a functional option for present-day immunotherapy . recently, junqueira et al. have shown that cpgs oligodeoxynucleotides (cpg odns) and glycoinositolphospholipids (gipl) gotten from trypanosome cruzi having the ability to activate tlr- and tlr leads to actuate potent pro-inflammatory reaction . secondly, proteo-glycolipid complex (p glc) derived from leishmania parasite has shown its affinity for the tlr- receptor and recognized as ligand . moreover, tlrs having the capability to recognize the plasmodium ligands, for example, plasmodium falciparum primes the human tlr- response towards high proinflammatory cytokine profile . shanmugam a. et at. has reported that synthetic tlr- agonist namely rs- (sequence: apphals) can be used as a novel class of adjuvant , therefore, it was added as an adjuvant and linked with epitopes (ctl and htl) by using eaaak linker . linkers assume an imperative part in simulating the vaccine construct to work as an independent immunogen and producing higher antibody titer than that of single immunogen . total three linkers namely eaaak, aay, and gpgpg, were used to construct the final vaccine. aay and gpgpg linkers were added at the intra-epitope position to link the ctl and htl epitopes, respectively. b-cell epitope prediction. b lymphocytes, a type of white blood cells, are the key player of humoral immunity by antibody production. the identification of b-cell epitopes is an essential part in vaccine designing. bcpreds server was used to predict the linear b-cell epitopes of amino acids long. the amino acid sequence of final vaccine construct was used as an input sequence in plain format followed by the selection of fixed length epitope prediction method and length of the epitope. bcpreds (default method) was selected as the prediction method for the epitope of amino acids long . the specificity threshold was set to be by default at % to get the result in a user-friendly format. while conformational epitopes were predicted using ellipro server for the input of tertiary protein structure of vaccine construct. antigenicity and allergenicity prediction of designed vaccine. antigenicity determines the ability of an antigen to binds with the b-and t-cell receptor that may lead to the immune response and memory cell formation. therefore, the antigenic nature of predicted vaccine construct was determined to ensure its ability to interact with the immune receptor. antigenpro is a sequence based, pathogen independent and alignment-free prediction method that was used to check the antigenic behavior of vaccine protein. it uses svm classifier to summarize the probable antigenic or non-antigenic nature of proteins. antigenpro uses the existing protein antigenicity microarray files of eight feature sets for five pathogens to construct two-stage architecture; among them, the first one is multiple representations of the primary protein sequence and the second one is five machine learning algorithms. allergenicity is the potential of a material to cause sensitization and allergic reactions associated with the ige antibody response. therefore, the predicted vaccine construct must be free from the allergenic nature. allertop v. . was used to check the allergenicity of the vaccine construct based on the method that uses auto cross-covariance (acc) transformation of protein sequences into uniform equal-length vectors. input protein sequence of vaccine protein was classified by the k-nearest neighbor algorithm (knn, k = ) which is based on the training set of known allergen from different species and nonallergen from similar species. physiochemical properties assessment. the main purpose of vaccination is to induce an immune response after injecting the vaccine into the body. therefore, it is necessary to define the physical and chemical parameters associated with the vaccine. protparam web server, a part of expert protein analysis system (expasy), was used to define various physicochemical properties of predicted vaccine construct. the primary protein sequence of the vaccine was used to predict the various parameters including molecular weight (kda), estimated half-life, theoretical pi, aliphatic index, grand average of hydropathy (gravy) and so on. tertiary structure prediction. protein molecule achieves maximum stability in its lowest energy state by proper bending and twisting to form a tertiary structure. it is the interaction between the amino acids side chain residue which is responsible to stabilize the protein structure. the -dimensional structure of predicted vaccine construct was obtained by utilizing raptorx structure prediction server. raptorx is a pure ab initio method that can be used to build a d model in a template-free manner. it is the degree of likeness between the target and available template structure that determines the quality of protein model structure created by contemporary protein structure prediction techniques , . therefore, it was necessary to improve the template based predicted model beyond the accuracy by utilizing the template information. to fulfill this thought, output model of raptorx server was subjected to the galaxyrefine web server , which is based on the casp tested refinement method. galaxyrefine performs rehashed structure perturbation followed by overall structural relaxation by performing molecular dynamics simulation. disulfide engineering for vaccine stability. before proceeding to the next step, it was necessary to improve the stability of refined protein model. disulfide bonds are covalent interactions that emulate the stabilizing molecular interaction and provide a considerable stability to protein model by confirming precise geometric conformations. disulfide engineering is a novel approach for creating disulfide bonds into the target protein structure. therefore, the refined model of final vaccine construct was subjected to the disulfide by design . to perform disulfide engineering. initially, the refined protein model was uploaded and run for the residue pair search that can be used for the disulfide engineering purpose. total residue pairs were selected to mutate them with cysteine residue using create mutate function of the disulfide by design . server. in silico cloning. codon adaptation is a way to attain major expression rate of foreign genes in the host when the codon usage of the host differs from that of the organism where the gene stems from. unadapted codon may lead to the minor expression rate in the host. therefore, the primary sequence of vaccine protein was submitted to the java codon adaptation tool (jcat) to adapt their codon usage to most sequenced prokaryotic organisms (e. coli k ) . cai value and gc content of the adapted sequence was also obtained. later on, the adapted nucleotide sequence corresponding to the designed vaccine construct was cloned into the e. coli pet a(+) vector by using the restriction cloning module of snapgene tool . used to predict the preferred orientation of ligand molecule to the receptor molecule in their stable complex form . it can be also used to predict the binding affinity between these two molecules in terms of scoring function. as mentioned in the previous section, tlrs having the capability to recognize the plasmodium ligands and p. falciparum primes the human tlr- response towards high proinflammatory cytokine profile . therefore, tlr- was selected as receptor and its pdb file (pdb id: g a) was obtained from rcsb-protein data bank while the refined model of vaccine protein was used as a ligand. protein-protein docking was performed using the cluspro . : protein-protein docking server, to check the binding affinity between them . widely accepted computational approach which is used to determine the stability of protein-ligand complex at the microscopic level . the protein-protein docked complex output of cluspro was used as an input to perform the molecular dynamics simulation using gromacs v . . . initially, the crystal water of complex was removed followed by the topology generation using a gromos a force field. in the next step, protein complex was centered in a cubic boundary box and filled by water molecule using simple point charge (spc) water model and chloride ion was used for the charge neutralization of complex. moreover, energy minimization followed by canonical equilibration (nvt ensemble) and isothermal-isobaric (npt ensemble) was performed for a time duration of ps. finally, molecular dynamics simulation was executed for the time duration of ns . the root mean square deviation (rmds) for backbone and root mean square fluctuation (rmsf) for side chain was determined. cerebral malaria; mechanisms of brain injury and strategies for improved neuro-cognitive outcome exploring dual inhibitory role of febrifugine analogues against plasmodium utilizing structure-based virtual screening and molecular dynamic simulation antimalarial drug discovery -approaches and progress towards new medicines pre-travel malaria chemoprophylaxis counselling in a public travel medicine clinic in são paulo recent advances in malaria drug discovery a large focus of naturally acquired plasmodium knowlesi infections in human beings advances and challenges in malaria vaccine development the global pipeline of new medicines for the control and elimination of malaria recent clinical and molecular insights into emerging artemisinin resistance in plasmodium falciparum the biology and control of malaria vectors in india from sialomes to the sialoverse: an insight into salivary potion of blood-feeding insects an insight into the sialome of blood-feeding nematocera salivary gland lysates from the sand fly lutzomyia longipalpis enhance leishmania infectivity mass spectrometry based proteomic analysis of salivary glands of urban malaria vector anopheles stephensi a mosquito salivary protein inhibits activation of the plasma contact system by binding to factor xii and high molecular weight kininogen the anopheles gambiae salivary protein gsg : an anopheline-specific protein with a blood-feeding role molecular and immunological toxic effects of nanoparticles natural human humoral response to salivary gland proteins of anopheles mosquitoes in thailand identification of antigenic proteins from salivary glands of female anopheles maculatus by proteomic analysis members of the salivary gland surface protein (sgs) family are major immunogenic components of mosquito saliva exploring leishmania secretory proteins to design b and t cell multi-epitope subunit vaccine using immunoinformatics approach exploring dengue genome to construct a multi-epitope based subunit vaccine by utilizing immunoinformatics approach to battle against dengue infection regulation of cd (+) t cell responses to infection with parasitic protozoa the great balancing act: regulation and fate of antiviral t-cell interactions discovery of t-cell driven subunit vaccines from zika virus genome: an immunoinformatics approach properties of mhc class i presented peptides that enhance immunogenicity a novel multi-epitope peptide vaccine against cancer: an in silico approach designing of interferon-gamma inducing mhc class-ii binders intranasal vaccination against hiv- with adenoviral vector-based nanocomplex using synthetic tlr- agonist peptide as adjuvant the role of b cells and humoral immunity in mycobacterium tuberculosis infection epitope-based vaccine target screening against highly pathogenic mers-cov: an in silico approach applied to emerging infectious diseases allertop -a server for in silico prediction of allergens disulfide by design . : a web-based tool for disulfide engineering in proteins a multi-subunit based, thermodynamically stable model vaccine using combined immunoinformatics and protein structure based approach developing imidazole analogues as potential inhibitor for leishmania donovani trypanothione reductase: virtual screening, molecular docking, dynamics and admet approach molecular docking and molecular dynamics simulation based approach to explore the dual inhibitor against hiv- reverse transcriptase and integrase t cells as mediators of protective immunity against liver stages of plasmodium large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction differential expression of mirna regulates t cell differentiation and plasticity during visceral leishmaniasis infection perturbed microrna expression by mycobacterium tuberculosis promotes macrophage polarization leading to pro-survival foam cell peptide binding predictions for hla dr, dp and dq molecules advances in the design and delivery of peptide subunit vaccines with a focus on toll-like receptor agonists trypanosoma cruzi adjuvants potentiate t cell-mediated immunity induced by a ny-eso- based antitumor vaccine mechanisms of immune evasion in leishmaniasis plasmodium falciparum infection causes proinflammatory priming of human tlr responses synthetic toll like receptor- (tlr- ) agonist peptides as a novel class of adjuvants a potential protein adjuvant derived from mycobacterium tuberculosis rv enhances dendritic cells-based tumor immunotherapy new directions in nicotine vaccine design and use predicting linear b-cell epitopes using string kernels protein identification and analysis tools in the expasy server molecular modeling and virtual screening approach to discover potential antileishmanial inhibitors against ornithine decarboxylase protein structure refinement driven by side-chain repacking febrifugine analogues as leishmania donovani trypanothione reductase inhibitors: binding energy analysis assisted by molecular docking, admet and molecular dynamics simulation the cluspro web server for protein-protein docking high-throughput virtual screening and quantum mechanics approach to develop imipramine analogues as leads against trypanothione reductase of leishmania structure-based virtual screening, molecular docking, admet and molecular simulations to develop benzoxaborole analogs as potential inhibitor against leishmania donovani trypanothione reductase r.k.p. is thankful to department of science and technology for providing inspire fellowship. v.k.p. is thankful to the central university of rajasthan for providing computational facility. we acknowledge the scientific help of ms. rani soni from department of biotechnology, central university of rajasthan for this manuscript. protocol designed by r.k.p., t.k.b., v.k.p. methodology performed by r.k.p., v.k.p. manuscript was written by r.k.p., t.k.b.,v.k.p. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - q xi xx authors: wang, fuzhou; kream, richard m.; stefano, george b. title: an evidence based perspective on mrna-sars-cov- vaccine development date: - - journal: med sci monit doi: . /msm. sha: doc_id: cord_uid: q xi xx the first outbreak of coronavirus disease (covid- ) caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) occurred in wuhan, hubei province, china, in late . the subsequent covid- pandemic rapidly affected the health and economy of the world. the global approach to the pandemic was to isolate populations to reduce the spread of this deadly virus while vaccines began to be developed. in march , the first phase i clinical trial of a novel lipid nanoparticle (lnp)-encapsulated mrna-based vaccine, mrna- , which encodes the spike protein (s protein) of sars-cov- , began in the united states (us). the production of mrna-based vaccines is a promising recent development in the production of vaccines. however, there remain significant challenges in the development and testing of vaccines as rapidly as possible to control covid- , which requires international collaboration. this review aims to describe the background to the rationale for the development of mrna-based sars-cov- vaccines and the current status of the mrna- vaccine. in late , in wuhan, hubei province, china, an outbreak occurred of coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ). this outbreak was followed by the release of the first situation report from the world health organization (who) on january , , and since then, a global pandemic has resulted in an increasing number of deaths [ ] [ ] [ ] . the pandemic has resulted in uncertainty regarding when or whether the pandemic will end, and whether covid- will follow the same clinical course as severe acute respiratory syndrome (sars) did years ago. however, the current view is that this novel coronavirus infection will persist, and without a treatment, a vaccine is urgently required [ ] . review of the epidemiology of the sars epidemic of shows that it disappeared suddenly without resulting in a pandemic, and this was also the reason for the lack of development of a sars vaccine [ ] . however, covid- is different from sars based on its ability to spread globally. currently, antiviral pharmaceuticals and immunotherapies are two principal therapeutic approaches for covid- [ ] , whereas vaccines are still considered the most promising way to eradicate this virus [ ] . in general, dna-based and protein-based vaccines have been the major approaches for the development of stable and effective vaccines with less innate immunogenicity [ , ] . however, mrna-based vaccines have become a potentially preventive approach, as they are safer, more efficient, and easier to manufacture [ ] . due to the rapid spread of covid- , mrnabased vaccine development seems to be an approach to prevent infection and to prevent a second wave of this pandemic. typically, at least to months is required to develop a new vaccine, using current processes for vaccine development, clinical trials, and regulatory approval [ ] . therefore, it is unlikely that an effective vaccine will be developed in time to control the current pandemic. however, on march , the first phase i clinical trial of a novel lipid nanoparticle (lnp)-encapsulated mrna-based vaccine, mrna- , which encodes the spike protein (s protein) of sars-cov- , began in the united states (us), conducted by moderna and the vaccine research center (vrc) of the national institute of allergy and infectious diseases (niaid) [ , ] . the first patient enrolled in this phase study was a -year-old woman at the kaiser permanente washington health research institute in seattle, washington, usa [ ] . the phase i study of mrna- is being conducted to evaluate the safety and immunogenicity, with the following trials currently being planned to evaluate the efficacy and effective dose of the new mrna vaccine [ ] . this review aims to describe the background to the rationale for the development of mrna-based sars-cov- vaccines and the current status of the mrna- vaccine. the terminology for the rna virus that causes covid- , sars-cov- , has been established by the international committee on taxonomy of viruses (ictv) [ ] , due to its extensive homology with the sars coronavirus. the sars-cov- coronavirus belongs to the subfamily of coronavirinae, with a genomic structure of (+)ss-rna of kb in length that includes a '-cap structure and '-poly-a tail [ ] . from the viral rna, polyprotein a/ ab (pp a/pp ab) is synthesized in the host to form non-structural proteins (nsps) that organize the replication-transcription complex (rtc) in doublemembrane vesicles (dmvs). the nrtc synthesizes a set of minus-strand subgenomic rnas (sgrnas) discontinuously [ ] . between open reading frames (orfs), transcription terminates, and then a subsequent acquisition of a leader rna occurs. during this process, subgenomic mrnas need these sgrnas as the templates [ , ] . at least six orfs exist for a typical cov, including sars-cov- . the first orfs (orf a/b) with over % of the whole genome length encode nsps. of note, two polypeptides (pp a and pp ab) come from a - frameshift between orf a and orf b. for the other orfs on the % of the genome close to the '-terminus encode at least four main structural proteins, including spike (s), membrane (m), envelope (e), and nucleocapsid (n). all these structural and non-structural proteins are translated from the sgrnas [ , , ] . currently, more than complete and partial genome sequences of sars-cov- have been decoded and deposited in the global initiative on sharing all influenza data (gisaid) database (https://www.gisaid.org/) and in the national institutes of health (nih) genbank database (https://www.ncbi.nlm.nih.gov/nuccore/?term=covid- ). phylogenetic analysis showed that sars-cov- was closely related to two sars-like coronaviruses present in bats, including bat-sl-covzc and bat-sl-covzxc , with % identity, and showed % homology with sars-cov, and % with mers-cov [ ] . however, homology modeling disclosed that sars-cov- had a similar rbd structure to that of sars-cov, despite amino acid variation at some key residues [ , ] . these findings suggest that sars-cov- emerged from a single animal source within a short period [ ] . however, because the sequence similarity between sars-cov- and its close relatives bat-sl-covzc and bat-sl-covzxc is less than %, the bat-derived viruses may not be the direct origins of sars-cov- . vaccines based on the cytoplasmic expression of chimeric mrnas containing curated orf viral sequences possess great potential to translate directly in the cytoplasm and block chromosomal integration [ ] . once injected, the delivered mrna can be processed by immune cells and start to produce targeted protein directly through translation, of which is being followed by activating other immune cells to recognize the newly produced viral protein to make antibodies. two types of rna vaccine exist against infectious pathogens, or non-replicating mrna vaccine and self-amplifying or replicon rna vaccine [ , ] . due to different delivery methods, non-replicating mrna vaccines are further divided as ex vivo loading of dendritic cell and direct in vivo injection into various anatomical sites [ ] . the penetration of the lipid membrane barrier is the first step for exogenous mrna to reach the cytoplasm before the translation of functional protein happen [ ] . also, the uptake mechanisms of mrna vaccines show cell specificity [ ] , and the physicochemical properties of the mrna may significantly influence its cellular delivery and organ distribution [ ] . all these factors must be considered when designing an effective mrna-based vaccine. even so, an mrna vaccine is still considered the most promising candidate because it can be scaled rapidly, which can save time when the rapidly spreading covid- emerged and started to infect millions of people worldwide [ , ] . as a (+)ss-rna virus, sars-cov- possesses self-amplifying rna that can realize extreme rna replication in the cytosol [ ] . this finding supports the role of mrna-based vaccine development. however, the safety and efficacy of mrna vaccines for use in humans remain unknown. the hypothetical benefits of mrna vaccine seem strong, whereas limitations such as the delivery and stability issues related to rna degradation, and the safety concerns due to immunogenicity hinder its development [ ] . the results from the phase i trial of the mrna- vaccine are awaited [ ] . the mrna-based vaccines actively induce activation of both b cell responses and t cell cytotoxicity. first, the mrna vaccines use the mrna sequence of the target protein that recombine in vitro, rather than the sequence of the target antibody. subsequently, the recombinant target protein mrna sequence is carried by lnps and enters the somatic cytoplasm to achieve direct translation and encoding the target protein. when the target protein is released from the host cell, the antigen-presenting cells will quickly capture and process the heterologous protein. then, the presentation of mhc i and mhc ii on the surface of the antigen-presenting cell membrane [ ] . this step is important for the subsequent activation of b cells and t cells and is also the key to the humoral and cytotoxic response. figure is a schematic representation of mrnabased active immunization. targeting the sars-cov- s protein sequence in mrna vaccine development finding the most suitable target site for sars-cov- vaccine development is extremely important. the spike glycoprotein (s protein) is now a key target for vaccine development, therapeutic antibody generation, and the clinical diagnosis of covid- . sars-cov- enters the host cell by using highly glycosylated homotrimeric s protein to achieve fusion with cell membranes through its structural changes. this process includes: the s subunit binds to the host cell receptor, which triggers trimeric instability that is followed by the separation of the s subunit from the s subunit to form a highly stable fusion structure [ ] [ ] [ ] . to access host cell receptors, rbd in the s subunit undergoes hinge-like conformational changes to hide or expose key sites for receptor binding, which is very similar to sars-cov [ ] [ ] [ ] . this high homology of rbd suggests that the covid- virus shares the same host cell receptor ace as sars-cov [ ] [ ] [ ] . although there are similarities, covid- has its own characteristics. the most significant change is the rrar amino acid sequence with a s /s protease cleavage site, which is consistent with the characteristics of a furin recognition site. this common phenomenon occurs more frequently in influenza viruses rather than in sars viruses that only have a single arginine [ ] . also, sars-cov- and ratg s proteins have amino acid residues that differ, of which are located at the rbd site. the rbd of sars-cov- is much closer to the center of the trimeric s protein. one of the three rbds in the s protein will spiral upwards to form a spatial conformation that helps the virus bind to the host receptor ace easily, which suggests that sars-cov- would be more infectious than sars [ ] . a cross-reactivity test of rbd of sars-cov- was performed using the rbd monoclonal antibody of sars, and it was found that this antibody did not cross-react with sars-cov- [ ] . these results provide an important structural biological basis for designing vaccines more accurately and discovering antiviral drugs. s protein helps the virus to enter target cells, but this endocytosis simultaneously depends on both the binding of s protein to membrane ace receptors and the initiative activation of s protein by cellular proteases [ ] . therefore, a vaccine against s protein provides an approach for preventing the proliferation and spread of sars-cov- . the vaccine can prevent the initial activation of the s protein by blocking the s protein binding e - to ace . sars-cov- infects cells in a transmembrane serine protease (tmprss ) protein-coding gene-independent manner, and cathepsins b and l directly activate s protein in cells that do not express tmprss , and sars-cov- infects cells in a tmprss -dependent manner. recently, hoffmann et al. showed that the use of tmprss inhibitors significantly blocked the entry of sars-cov- into cell lines expressing tmprss and that promoting tmprss expression antagonized this inhibition [ ] . therefore, if the antibody titer of s protein is high enough to prevent the virus being engulfed into endosomes or undergoing fusion at the host cell surface, then virus infection associated with s protein activation and the intracellular release of the virus particles will be effectively inhibited. given the bioavailability of ace as a key determinant of the spread and infectious capacity of sars-cov- , if enough s protein figure . schematic diagram of the mrna-based vaccine targeted to the spike protein (s protein) of severe acute respiratory syndrome coronavirus (sars-cov- ). the mrna-based vaccine targeted to the s protein of sars-cov- works by active immunization. this technique will not use part of the virus but only recombine mrna of the s protein in vitro according to the gene sequence, which is coated with lipid nanoparticles for effective delivery. once injected into the muscle, the myocytes take up the lipid nanoparticle (lnps) and then release the mrnas into the cytoplasm for translation into the s proteins. these endogenously synthesized s proteins will be secreted to activate both humoral and cellular immune responses. s protein -spike protein; im -intramuscular, lnp -lipid nanoparticle; dc -dendritic cell; mhc -major histocompatibility complex; ag -antigen. antibodies are formed in the body, then two therapeutic effects may occur. firstly, the s protein-antibody complex will be quickly cleared by the immune system, which will subsequently result in clearance of the virus itself. secondly, ace bioavailability will be significantly reduced, which helps mitigate the spread and infectivity of the virus [ ] . although empirical data from both sars-cov and sars-cov- research have identified ace as the primary portal for cellular entry of these two viruses [ ] [ ] [ ] , the probability of a membrane-associated co-receptor complex remains. therefore, targeting of ace /sars-cov- binding may not be the rate-limiting step for inhibiting viral infection. even with hopes of an mrna-based vaccine targeting the s protein, it is important to note that the stoichiometric relationship between s protein and the immune response occurs in several ways. first, the intrinsic ratio of outer surface s protein per sars-cov- virion (s/v) may predetermine the preference of induced igg in immunological responses. the inherent ratio of nucleotides to the s protein (n/s) may determine whether there are redundant s proteins when igg effectively neutralizes the s protein, which reduces the immune efficacy of igg. therefore, both ratios will finally determine how high the antibody titer should be and how many vaccine boosters are needed so that it can effectively neutralize the virus without concerns about the existence of redundant s protein complexes that may effectively remove igg antibodies to leave unblocked s protein available for processing and binding to ace . although several previous studies have investigated this finding in human immunodeficiency viruse (hiv) infection and concluded that low spike density with wide spacing of the envelope spikes is unhelpful for the activation of b cells [ , ] , this remains unclear for sars-cov- , and it is a challenging factor for vaccine development against sars-cov- . an important consideration relates to mutations in the s protein that may affect the degree of viral pathogenesis [ ] . direct evidence of functionally meaningful s protein mutations affects the s protein sars-cov- and the host cell ace , which appears to mediate a higher binding affinity when compared with sars-cov [ ] . the reason for this increased s protein/ace affinity in sars-cov- (- . kcal/mol) versus that of sars-cov (- . kcal/mol) is mainly attributed to three main factors [ ] . first, the emergence of two loops around the rbds in sars-cov- may promote its chemical interaction with ace by increasing the number of atoms needed. second, more amino acid residues in the s protein of sars-cov- determine a higher number of protein-protein contacts between s protein and ace . third, a longer capping loop in the s protein of sars-cov- favors its interaction with the receptor. therefore, it is difficult to ensure that the new vaccine that is targeted to s protein can be used long term or in the near future, it there is the possibility that the vaccine will not be effective because of mutations in the s protein. for protein-based immunization, repeated vaccine administrations due to the clearance and indication may be required to maintain a therapeutically effective level [ ] . furthermore, some non-specific effects may result from these typical vaccines, such as cross-reactive tcrs and antibodies, bystander activation of pre-existing effector or memory cells, and classical cell-mediated immunity may function as either beneficial or unfavorable to the public health of the population [ ] . also, although there have been previous studies on the safety of live attenuated or killed vaccines, and the safety of these vaccines used in clinics is very reliable, the potential for inducing infection still exists [ ] . there are four main safety and efficacy advantages of the use of mrna-based antiviral vaccines over traditional approaches. first, mrna-based antiviral vaccines minimize the potential risk of infection and insertion-induced mutagenesis due to natural degradation of mrna in the cellular microenvironment [ ] . second, the immunogen's high efficacy due to engineered mrna structural modifications improves its stability and translation efficacy. third, the high potency of mrna-based vaccines capable of generating potent antiviral neutralizing immunoglobulins with only one or two low-dose immunizations [ , ] may induce strong immune responses by activating both cd + and cd + t cells [ ] . fourth, engineered mrna production facilitates large-scale production of sufficient vaccine doses required to treat mass populations [ , ] . all these factors make the mrna vaccine more suitable for a rapid response to the emerging covid- pandemic. although these beneficial features of mrna vaccines provide some hope for the development of the first clinically applicable sars-cov- mrna vaccine, recent reports regarding rare cases of moderate or severe reactions for different mrna vaccines have raised concerns about safety and immunogenicity, including in the primary outcome findings of the phase i trial on mrna- [ , ] . therefore, it is important to clearly understand the potential risks of this type of mrna-based vaccine, which include local and systemic inflammatory responses, the biodistribution and persistence of the induced immunogen expression, possible development of autoreactive antibodies and toxic effects of any non-native nucleotides and delivery system components [ ] [ ] [ ] . glycosylation of viral envelopes is a very common biological phenomenon. glycosylation refers to the process in which proteins or lipids are linked to sugar chains by the action of enzymes. glycosylation is one of the important forms of posttranslational modification of proteins and is an essential way to regulate protein localization, function, persistence, and e - diversity [ , ] . for viruses, their replication and invasion into host cells are closely associated with the glycosylation of their structural proteins [ ] . the main purpose of viral vaccine development is to evoke an immune response to fight the virus. however, the high glycosylation of proteins on the surface of the viral envelope works like invisible camouflage, which can help the virus to successfully escape the detection of the human immune system and achieve the purpose of survival [ , ] . therefore, the higher the degree of glycosylation, the greater the probability that the virus will escape the immune response and the lower the success rate of vaccine development. therefore, overcoming the highly glycosylated viral envelope is another challenge of sars-cov- vaccine development. sars-cov- includes highly glycosylated spherical particles and has a large structure containing at least n-linked glycan sites, of which sites show similarity with sars-cov [ ] . in contrast, the glycan sites of hiv are between three times and six times that of the influenza virus, which is one of the major reasons why an hiv vaccine has not been successfully developed at this time [ ] . however, sars-cov- has more than twice the glycan sites of hiv [ ] . this unusual degree of glycosylation means that sars-cov- may mutate quickly, making the development of a vaccine extremely difficult. however, vaccine development failures caused by glycosylation remains significant, because vaccine design is based on the traditional inactivated or attenuated live vaccines, and different traditional vaccines may induce various glycosylation patterns of antibodies that subsequently affect the protective role of the antibodies [ ] . however, the use of mrna-based vaccine technology and targeting only the s protein, rather than the entire virus particle, may lead to the human immune system producing s protein antibodies without the influence of viral glycosylation. the successful delivery of oligonucleotide-based therapies [ ] has provided new opportunities to develop mrna-based vaccines. given their beneficial properties, which include lack of persistence, lack of integration into the genome, the absence of induction of autoantibodies, the ability to produce mrna vaccines in large quantities, and their high purity [ ] , an mrna-based vaccine has become a hopeful choice for the development of a sars-cov- vaccine. currently, no clinically applicable vaccine against covid- is available, and even one of the first vaccines, mrna- , is undergoing a phase i safety and immunogenicity trial [ ] . the recently developed encapsulated rna delivery and self-amplifying rna technique assists in the ease of use of the mrna vaccine with an increased success rate. however, several issues still need to be clarified for mrna-based vaccines. concerning the worldwide spread of covid- , the development of an effective and safe vaccine in time has become the most promising way to control the current viral pandemic. if currently available technology can be used to develop a sars-cov- vaccine, which may be clinically applicable, this will give hope for controlling the covid- pandemic. however, further research and development should continue to ensure that an effective sars-cov- vaccine is developed. there are several challenges to address as new formulations are required to stabilize the mrna vaccine at higher temperatures during its distribution. a more suitable packaging formula for the mrna vaccine within the nanoparticles is required to ensure vaccine stability. the best way should be sought to anchor the rnase inhibitor into the mrna vaccine co-formulation and to develop new ways to purify or remove the mrna vaccine-related reaction components. studies are still required to investigate the mechanisms for reducing the innate immune response to the delivered mrna vaccine. also, novel in vivo delivery methods requires development to maximize the uptake efficiency of the mrna vaccine, and to determine which immune signaling pathways are the most effective in response to the exogenous mrna vaccine. in addition to the described technical difficulties related to mrna vaccines, the difficulties associated with vaccine development need to be resolved. even though clinical trials of some vaccines have begun [ ] , there remain several technical issues that affect vaccine development and efficacy that require further study. first, strict bioinformatics analysis of the probability of a membrane-associated co-receptor complex is required to find the rate-limiting pattern of antibodies that affect ace /sars-cov- binding. the stoichiometric relationship between the s protein and the strength of the immune response requires studies on the intrinsic ratio of the outer surface s protein per sars-cov- virion (s/v) and nucleotides to the s protein (n/s). the precise function of viral self-defense proteins and the spherical configuration of the virion require further research, as does the mutation probability of the s protein that affects viral tropism and receptor affinity. there is also the potential hindrance of the high glycosylated state of the viral envelope to vaccine development. therefore, further in-depth studies are needed to elucidate the structure and corresponding physiological and immunological properties of the s protein and also other structural proteins that have a potential role in vaccine development, including mrna based vaccines. this review aimed to describe the background to the rationale for the development of mrna-based sars-cov- vaccines e - and the current status of the mrna- vaccine. although mrna vaccines are commencing human clinical trials, due to the rapid global spread of this new viral pandemic, it may not be possible to develop a safe and effective vaccine for sars-cov- in time to prevent the increasing number of deaths due to this novel rna virus. the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak -an update on the status world health organization (who) world health organization (who) base medicine task force: covid- : facts and recommendations from a to coronaviruses -drug discovery and therapeutic options discovering drugs to treat coronavirus disease (covid- ) immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic dna vaccine delivery and improved immunogenicity aiming at the heart: the capsid protein of dengue virus as a vaccine candidate mechanism of action of mrna-based vaccines principles of vaccine potency assays moderna doses first patient with mrna- in coronavirus vaccine trial safety and immunogenicity study of -ncov vaccine (mrna- ) to prevent sars-cov- infection coronaviridae study group of the international committee on taxonomy of viruses: the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- the reproductive number of covid- is higher compared to sars coronavirus improved translation efficiency of therapeutic mrna naming the coronavirus disease (covid- ) and the virus that causes it understanding of covid- based on current evidence crystal structure of sars-cov- main protease provides a basis for design of improved a-ketoamide inhibitors structural basis for the recognition of the sars-cov- by full-length human ace the proximal origin of sars-cov- new vaccine technologies to combat outbreak situations nucleoside modified mrna vaccines for infectious diseases lipid-based mrna vaccine delivery systems developing mrna-vaccine technologies introduction to rna vaccines sars-cov- and covid- : the most important research questions features, evaluation and treatment coronavirus (covid- ) improved translation efficiency of therapeutic mrna mrna vaccine delivery using lipid nanoparticles the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade novel coronavirus ) -recent trends cryo-em structure of the -ncov spike in the prefusion conformation identification of novel proteolytically inactive mutations in coronavirus c-like protease using a combined approach sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor covid- , an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics. hum vaccin immunother modeling how many envelope glycoprotein trimers per virion participate in human immunodeficiency virus infectivity and its neutralization by antibody estimating the stoichiometry of human immunodeficiency virus entry structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry role of changes in sars-cov- spike protein in the interaction with the human ace receptor: an in silico analysis pharmacokinetics and immunogenicity of broadly neutralizing hiv monoclonal antibodies in macaques non-specific effects of vaccines illustrated through the bcg example: from observations to demonstrations risk of transmission associated with live attenuated vaccines given to healthy persons caring for or residing with an immunocompromised patient stability of the osmoregulated promoter-derived prop mrna is posttranscriptionally regulated by rnase iii in escherichia coli mrna as novel technology for passive immunotherapy modified tumour antigen-encoding mrna facilitates the analysis of naturally occurring and vaccine-induced cd and cd t cells in cancer patients inhibition of the inflammatory pathway enhances both the in vitro and in vivo transfection activity of exogenous in vitro-transcribed mrnas delivered by lipid nanoparticles complexities of viral mutation rates induction of an ifn-mediated antiviral response by a self-amplifying rna vaccine: implications for vaccine design type i interferons (alpha/beta) in immunity and autoimmunity new insights into the role of glycosylation in lipoprotein metabolism functional roles of o-glycosylation exploitation of glycosylation in enveloped virus pathobiology effects of glycosylation on the properties and functions of influenza virus hemagglutinin virus glycosylation: role in virulence and immune interactions emerging wuhan (covid- ) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd glycosylation profiling to evaluate glycoprotein immunogens against hiv- site-specific analysis of the sars-cov- glycan shield opportunities to exploit antibody glycosylation in vaccination oligonucleotide therapies: the past and the present vaccination with messenger rna (mrna) status of mrna sars-cov- vaccines key: cord- -n ncamqh authors: donaldson, braeden; lateef, zabeen; walker, greg f.; young, sarah l.; ward, vernon k. title: virus-like particle vaccines: immunology and formulation for clinical translation date: - - journal: expert rev vaccines doi: . / . . sha: doc_id: cord_uid: n ncamqh introduction: virus-like particle (vlp) vaccines face significant challenges in their translation from laboratory models, to routine clinical administration. while some vlp vaccines thrive and are readily adopted into the vaccination schedule, others are restrained by regulatory obstacles, proprietary limitations, or finding their niche amongst the crowded vaccine market. often the necessity to supplant an existing vaccination regimen possesses an immediate obstacle for the development of a vlp vaccine, despite any preclinical advantages identified over the competition. novelty, adaptability and formulation compatibility may prove invaluable in helping place vlp vaccines at the forefront of vaccination technology. areas covered: the purpose of this review is to outline the diversity of vlp vaccines, vlp-specific immune responses, and to explore how modern formulation and delivery techniques can enhance the clinical relevance and overall success of vlp vaccines. expert commentary: the role of formation science, with an emphasis on the diversity of immune responses induced by vlp, is underrepresented amongst clinical trials for vlp vaccines. harnessing such diversity, particularly through the use of combinations of select excipients and adjuvants, will be paramount in the development of vlp vaccines. virus-like particles (vlp) are a type of subunit vaccine based on virus-derived proteins, assembled to form a particle. vlp hold several advantages over other particulate subunit vaccines. these include a morphological resemblance to their parent virus, a highly repetitive immunogenic surface structure, and the retention of cell uptake and immune processing pathways associated with their parent virus [ ] . vlp themselves are nonpathogenic, devoid of an intact virus genome, and are incapable of infection or replication. the noninfectious nature of vlp significantly improves their safety profile over live-attenuated vaccines, while also possessing advantages when compared to other forms of subunit, killed, or particulate vaccines. commercially successful vlp vaccines include hepatitis b virus (hbv) vlp, such as recombivax hb (merck), engerix-b (glaxosmithkline), elovac b (human biologicals institute), genevac b (serum institute) and shanvac b (shantha biotechnics), hepatitis e virus (hev) vlp, such as hecolin (innovax), and human papillomavirus (hpv) vlp, including gardasil (merck) and cervarix (glaxosmithkline). vlp vaccines currently under investigation in clinical trials include influenza a virus (iav) vlp (nct , nct ) [ , ] , chikungunya virus vlp (nct ) [ ] , human cytomegalovirus (hcmv) vlp (nct ) [ ] , and human norovirus (hunv) vlp (nct , nct , nct ) [ ] [ ] [ ] . recent and current vlp clinical trials registered in the united states of america and the european union are summarized in table . vlp can be bulk expressed in bioreactor cultures, utilizing advanced in vitro protein expression systems for producing vaccine-grade vlp suitable for clinical administration. the manufacture of some vlp also includes additional processing steps, such as disassembly and reassembly of vlp. the manufacture of the hpv vlp utilizes disassembly and reassembly to improve vlp morphology and stability [ ] . similarly, the manufacture of qβ vlp includes this method [ ] . different vlp expression systems and the applications of vlp disassembly and reassembly was explored in a recent review [ ] . compatibility with commercial upscaling technology, gmp production, and with minimal postproduction manipulation or modification supports large-scale use of vlp vaccines; however, some vlp vaccines struggle in their translation from laboratory research and development, to clinical trials and routine public access [ ] [ ] [ ] . open accessibility to the molecular or genetic components of derivative constituents may place some limitations on the commercialization of specific vlp vaccines. this may be circumvented through tactical use of proprietary modification, or by utilizing the vaccine as a constituent within a composite formulation [ ] . the development of some vlp vaccines can be challenged by issues with stability and longevity, which may be alleviated with formulation excipients, or other vaccine additives that facilitate vaccine distribution and storage [ , ] . the purpose of this review is to explore some of the challenges in the translation [ ] , and negative-sense rna viruses such as human parainfluenza virus type [ ] . variety can be observed in vlp size, ranging from ms bacteriophage vlp at around . nm in diameter [ ] , to hpv vlp at around nm [ ] , and influenza vlp at around nm [ , ] . vlp also vary in structural complexity, as illustrated in figure , including mono-layer vlp such as hbv vlp formed from hbv surface antigen (hbsag) or hbv core antigen (hbcag) [ ] [ ] [ ] , and multi-layer vlp such as rotavirus vlp [ , ] . vlp can be encapsulated within a phospholipid bilayer envelope to resemble their parent virus, such as hiv [ ] or sendai virus vlp [ ] . the envelope itself can also form the primary particle structure of some vlp, with recombinantly expressed virus envelope-stabilizing proteins embedded within the membrane, such as with iav virosomes [ ] . some vlp are compatible with the formation of polyvalent or mosaic vlp, derived from multiple virus strains [ ] . while this increases the diversity of vlp vaccines beyond the variety of parent viruses, the increased complexity of polyvalent or mosaic vlp may require post-production manipulation to facilitate stable particle formation [ , ] . in addition to facilitating the development of complex vlp vaccine constructs, introduction of postproduction manipulation has also been demonstrated to improve the consistency and stability of some standard structure vlp vaccines [ , ] . the diversity of vlp can also be characterized based on the variety of diseases these vaccines have been developed to prevent or treat. these include vlp vaccines developed for both human and veterinary pathologies, with some examples including an hbv hbcag core particle-based vaccine for her + cancer [ ] , an adenovirus vlp-based vaccine for placental malaria [ ] , and a qβ vlp-based vaccine for leishmania infection [ ] . a selection of recently developed and investigated novel vlp vaccines are provided in table . vlp can be produced using a variety of expression systems, usually involving vlp assembly by spontaneous polymerization upon the expression of virus protein constituents. vlp can be expressed in cells derived from bacteria, yeast, insects and mammals, in cell-free expression systems, and in live organisms such as silkworm pupae and various plants [ ] [ ] [ ] . selection of an appropriate expression system is important, as each expression system can differ in their efficacy for expressing specific vlp, their post-translational modification (e.g. phosphorylation, glycosylation), and their potential for contamination with biologically compatible zoonotic viruses. protein expression for the production of vlp is highly amenable to modification, such as substituting strain-specific amino acid sequences [ ] , or inserting foreign peptide sequences or proteins [ , ] . manipulation of this process can facilitate the engineering of recombinant chimeric vlp containing xenogeneic antigens, inducing an immune response against targets other than the parent virus [ ] . vlp can also present self-antigens in an immunogenic context, which can be particularly useful in immunotherapeutic vaccination for cancer [ ] . a diverse range of applications for this type of modification have been investigated prophylactically and therapeutically for various conditions, including protection against infection from other organisms [ ] , autoimmune inflammatory disease [ ] , and as an antitumor immunotherapy [ , , ] . chimeric vlp have been investigated as a potential therapy for conditions not usually associated with vaccination, such as atherosclerosis [ ] , type ii diabetes mellitus [ ] , and nicotine addiction [ ] . some of these vlp vaccines utilize chemical conjugation for incorporation of antigenic sequences as opposed to recombinant insertion. vlp can serve as an effective immunogenic scaffold for chemical conjugation, compatible with a broad range of conjugation chemistries. the applications of chemical conjugation with vlp has been comprehensively reviewed in recent articles [ , [ ] [ ] [ ] ; however, it is worth noting that introducing unique forms of chemical conjugation may provide some proprietary claim over methods involving vlp vaccines already in the public domain. vlp are a form of subunit vaccine, inducing a characteristic immune response shared amongst exogenous antigens. while vlp are taken up by cells through non-specific pathways such as phagocytosis and macropinocytosis, some vlp can also utilize specialized uptake pathways inherited from their parent virus. in general, protein-based subunit vaccines like vlp are internalized into antigen-presenting cells (apcs), digested within the phagolysosome, and the resulting antigen peptides are presented loaded on major histocompatibility complex (mhc) ii molecules to cd + t helper cells (t h cells). t h cells recognize epitopes presented on mhc-ii through their specific t cell receptor (tcr), and activate dependent on the strength of this interaction, in addition to the degree of signaling through costimulatory receptors and cytokines. these additional signals are usually supplied by the apc, which is activated to increase the presentation of costimulatory receptors and the release of cytokines in response to a variety of stimulatory signals. these stimulatory signals correspond with the activation of pattern recognition receptors (prrs), such as tolllike receptors (tlrs), nod-like receptors (nlrs), and rig -like receptors (rlrs), which each recognize specific pathogenassociated molecular patterns (pamps) and damage-associated molecular patterns (damps). as vlp vaccines usually contain minimal or negligible amounts of these stimulatory molecules, these signals are instead induced by vaccine adjuvants. manipulation and selective targeting of specific apcs or other target cell populations may be an effective means of developing a novel proprietary vlp vaccine. chemical conjugation of receptor ligands onto the surface of compatible vlp should promote binding and uptake into cells expressing the corresponding receptor, while primary vlp uptake and processing pathways should continue unperturbed. this type of modification may be particular effective when targeting a specific apc population is desired for the induction of a specific type of immune response. for example, chemical conjugation of mannoside-based saccharides on the surface of rabbit hemorrhagic disease virus (rhdv) vlp selectively targets the mannose receptor expressed on the surface of apcs, inducing increased uptake and alteration of antigen cross-presentation in murine dendritic cells [ ] . chemical conjugation of unmethylated cpg oligonucleotides on the surface of rhdv vlp has also been investigated for targeting uptake through the dec receptor [ ] . similarly, coating of hiv vlp with phosphatidylserine-laced liposomes enhanced uptake into macrophages by mimicking apoptotic bodies [ ] . although the immune response to vlp may be generalized due to similarities shared with other subunit vaccines, there are some unique components of a vlp-induced immune response. the pathways for uptake of vlp into apcs vary depending on the size and morphology of each vlp, including the potential retention of receptor binding motifs. particles smaller than nm, such as norovirus p particles [ ] , can drain directly from the vaccination site into the lymph through pores in the fenestrated lymphatic vessels [ ] . trafficking of molecules within the lymphatics is likewise size dependent, with soluble proteins smaller than kda or nm transported by specialized small antigen conduits [ , ] . vlp are larger than nm, and many are instead transported passively to the lymph nodes bound on the surface of myeloid immune cells [ ] [ ] [ ] . vlp larger than nm cannot drain directly through the fenestration pores into the lymph, and instead require active transport following internalization by apcs at the vaccination site, such as in dendritic cells (dcs), macrophages and b cells [ ] . uptake of virus antigens into antigen presenting cells can occur through multiple pathways, including phagocytosis, macropinocytosis, caveolae-mediated uptake, clathrinmediated uptake and clathrin noncaveolae-mediated uptake [ ] . vlp have been identified to utilize a similar repertoire of uptake pathways, including phagocytosis [ ] , size-dependent macropinocytosis [ ] , and clathrin-dependent or independent forms of receptor-mediated endocytosis [ ] [ ] [ ] . the retention of receptor-mediated endocytosis indicates that some vlp can inherit functional receptor binding domains, and are compatible with the corresponding uptake pathways derived from their parent virus. these mechanisms of receptor-mediated uptake can also be introduced to vlp through post-expression modification, such as the chemical conjugation of superantigen on hbv vlp for internalization through mhc-ii molecules [ ] , or the chemical conjugation of transferrin on qβ vlp for internalization through the transferrin receptor, which is upregulated in some cancer cells [ ] . non-specific uptake into apcs subjects vlp to standard components of exogenous antigen processing pathways, including lysosomal fusion, enzymatic digestion, mhc-ii loading and antigen presentation (figure (a) ). parent viruses of vlp internalized through these pathways engage additional mechanisms to progress toward virus replication, including capsid disassembly or uncoating, exportation of the virus genomic material, and disruption of vesicular fusion. while the retention of intracellular processing mechanisms resembling those utilized by parent viruses has been investigated for some vlp [ , ] , it remains unclear whether vlp universally mimic the intracellular processing of their parent virus. vlp can retain access to cross-presentation pathways, facilitating presentation of antigens on mhc class i (mhc-i) for induction of a cd + t cell-mediated cytotoxic immune response. there are several known mechanisms of cross-presentation in apcs, including antigen escape from the early endosome, fusion of the endosome with the endoplastic vlp can be internalized and processed through a variety of intracellular pathways. (a) vlp internalized through non-specific pathways such as phagocytosis and macropinocytosis can be processed, with peptides presented on mhc-ii as exogenous antigen. (b) some vlp can also utilize cross-presentation pathways, facilitating the presentation of peptides on mhc-i [ ] . (c) influenza vlp, in this example an influenza virosome, is internalized through receptor-mediated endocytosis prior to fusion of its envelope with the endosomal membrane [ , , ] . (d) jcv vlp is thought to utilize the processing pathway of its parent virus to facilitate delivery of exogenous nucleic acids, including clathrin-dependent endocytosis, nuclear trafficking, and uncoating of the capsid within the nucleus [ , ] . reticulum, and recycling of mhc-i receptors from the cell surface. rhdv vlp are known to utilize the mhc-i receptor recycling pathway of cross-presentation [ ] (figure (b) ). additional examples of vlp known to induce cross-presentation of antigens include vlp derived from hbv [ , ] , hepatitis c virus (hcv) [ ] , hpv [ ] , papaya mosaic virus [ ] , and parvovirus [ ] . the ability to induce cross-presentation is important when a cellmediated cytotoxic immune response is the primary desired outcome of a vlp vaccine. the retention of receptor-mediated internalization in vlp illustrates that these mechanisms of uptake in their parent virus are autonomous, independent of the integrity of the complete virion. vlp derived from influenza a or b viruses are prime examples of this process. influenza vlp can be produced in several forms, including enveloped vlp formed by expressing the influenza matrix (m ) protein [ ] , retroviral gag proteins [ ] , or as independent virosome particles without a traditional protein core [ , ] . each of these constructs includes the expression of influenza hemagglutinin (ha) with or without neuraminidase (na). these influenza proteins are crucial for the induction of an influenza-specific immune response, and they also play essential roles the budding of influenza virus-like particles [ , , ] , and in receptor-mediated endocytosis. influenza vlp can bind to sialylated glycoproteins and glycolipids on the surface of cells from the inherent activity of ha, and are internalized through clathrin-mediated endocytosis [ , ] (figure (c)); however, alternative endocytic pathways have also been identified [ ] [ ] [ ] [ ] . as the early endosomal ph lowers, the envelope fuses with the endosome membrane. ha and na proteins distributed along the internal surface of the endosome membrane are enzymatically degraded, with peptides presented as exogenous antigens on mhc-ii (figure (c) ). specialized vlp vaccines designed to deliver a genetic payload add further complications to vlp uptake and processing. retention of the ability to deliver dna/rna requires conservation of intracellular processing pathways utilized by the parent virus for replication, or the induction of an alternative method of nucleic acid translocation. john cunningham virus (jcv) vlp is a polyomavirus vlp that has received significant attention over its ability to facilitate gene delivery and genetic manipulation [ , , ] . jcv is internalized by clathrin-dependent endocytosis upon binding to an undefined plethora of receptors and molecules known to include the serotonin receptor and sialic acid [ , ] . the virus then colocalizes to endosomes along with transferin, prior to cytosolic translocation, and subsequent trafficking through nuclear pores, mediated by the n-terminus of the vp capsid protein [ ] . the virus then uncoats, exposing the genomic material within the capsid. jcv vlp are thought to mimic this process due to their ability to deliver dna plasmids or other nucleic acids to the nucleus [ , ] (figure (d) ). vlp that demonstrate this ability to deliver nucleic acids likely utilize similar processing pathways to their parent virus [ , ] , but vlp can also be modified to utilize alternative processing pathways [ , ] . the primary desired outcome of most commercial vlp vaccines is the production of antibodies specific for the vlp parent virus. anti-vlp antibodies produced by these vaccines correspondingly neutralize the parent virus, protecting against infection. the production of anti-vlp antibodies involves the activation of a humoral immune response. b cells that specifically recognize vlp surface domains through their b cell receptor (bcr) bind and internalize vlp. binding of vlp to the bcr provides a stimulatory signal that primes the b cell for activation. most vlp consist of structurally identical capsid proteins arranged in a repetitive quasicrystalline pattern, which can crosslink multiple bcrs to provide a stimulatory advantage over other types of subunit vaccines. crosslinking of bcrs is important for inducing a robust humoral response, as b cells can ignore some monomeric soluble antigens [ ] . potent stimulation through bcr crosslinking can override inherent tolerogenic mechanisms, and can activate unresponsive or anergic b cells [ ] . highly repetitive structures also promote binding to low-affinity bcrs through multivalent, or highavidity interactions [ ] . antigen primed b cells receive additional activation signaling induced by pamps delivered with the vlp vaccine. these signals are usually provided by vaccine adjuvants, but may also be induced by other virus-associated or expressionderived endogenous molecules associated with the vlp. vlpderived antigens are presented to t h cells loaded on mhc-ii, inducing the cytokine and costimulatory receptor signaling from the t h cell that continues b cell activation. activated b cells initially become plasmablasts, a transient extrafollicular activation state that can provide some immediate antibody production [ ] . plasmablasts migrate to the follicular region of lymph nodes to form germinal centers, a specialized region where activated b cells proliferate. plasmablasts within germinal centers under affinity maturation and immunoglobulin class switching, guided by signaling from t follicular helper (t fh ) cells [ , ] . this specialized t fh cell guidance results in the development of high-affinity antibody-producing plasma cells, and long-lived memory b cells. rapid t cellindependent activation can also be induced in marginal zone b cells and b cells, providing an alternative pathway for activation and antibody production over the predominant follicular b cell population [ ] . some vlp have been identified utilizing this alternative form of b cell activation [ ] . the production of anti-vlp antibodies has been investigated and characterized amongst a broad range of vlp. this process can be influenced by various vaccine components, constituents, and even unexpected molecules, such as those derived from the vlp expression system. for example, the presence of bacterial rna packed inside qβ vlp plays a role in the induction of igg a/c antibodies in mice, and igg antibodies in humans [ , ] . qβ vlp can also induce the production of iga class antibodies in immunocompetent mice when immunized through the intranasal route [ ] , while t h cell-deficient mice required subcutaneous rather than intranasal vaccination to induce robust iga production through t-cell independent b cell activation [ ] . tlr- , simulated by singlestranded rna in endosomes, was also identified as crucial for the induction of both igg c and iga antibodies against qβ vlp in mice. this further indicates the importance of endogenous bacterial rna present in these vlp. the induction of anti-hpv vlp antibodies has also been extensively studied. although the production of secretory iga antibodies is desired for protection against viral infection at mucosal surfaces, the predominant antibody class found in secretions from the female genital tract following vaccination with hpv vlp is igg [ ] [ ] [ ] . while the presence of igg in these secretions may be due to transudation from the serum, some active transportation or local production in the mucosa may be possible [ , ] . vaccination with hpv vlp induces the production of both igg and iga antibodies in the serum and cervical secretions of humans [ ] . while serum titers of anti-hpv antibodies may initially decline post-vaccination, these levels plateau and remain stable to provide prolonged immunity [ ] . while the induction of a potent humoral immune response and the subsequent production of anti-vlp antibodies is the primary desired outcome of most commercial vlp vaccines, these is increasing appreciation for the role of vaccine-induced cell-mediated immunity [ ] [ ] [ ] . measurements of anti-vlp titers can provide an important indication of vaccine efficacy with respect to the neutralization of a virus challenge, a cellmediated immune response also plays an important role in antivirus immune defense [ , ] . activation of a cellmediated immune response can also be the primary desired outcome of vlp vaccines, particularly for chimeric or other modified vlp vaccines that target non-virus pathologies. a potent cell-mediated response to vlp vaccines is dependent upon cross-presentation of vlp-derived antigens on mhc-i. cd + t cytotoxic (t c ) cells can recognize through mhc-i antigen complexes through their tcr, and become primed for activation. these antigen-primed t c cells require additional signaling for activation, including interaction with costimulatory receptors, and cytokines from t h cells. while many vlp may be capable of cross-presentation, some may be more effective at inducing cross-presentation due to uptake and processing pathways inherited from their parent virus [ , ] . the induction of a potent cell-mediated immune response is particularly important for immunotherapeutic cancer vaccines. tumor cell-specific antibodies can enhance the inherent cytotoxic activity of natural killer cells through mechanisms such as antibody-dependent cell-mediated cytotoxicity, or directly through complement-dependent cytotoxicity; however these mechanisms tend to be restricted to passive vaccination with monoclonal antibodies, such as the her -specific monoclonal trastuzumab [ ] . a cell-mediated immune response combines target specificity with the in vivo expansion of effector cell populations, with the capacity to establish prolonged memory. rhdv vlp are particularly effective as a vector for cancer vaccines, capable of inducing the cross-presentation of vlp-derived antigens [ ] , and compatible with recombinant insertion and chemical conjugation [ ] . rhdv vlp vaccines have been investigated in models of hpvinfected cervical cancer [ ] , melanoma [ ] , lewis' lung carcinoma [ ] , and colorectal cancer [ ] . additional examples of vlp that can induce cross-presentation and a potent cellmediated immune response include qβ vlp [ ] , ms vlp [ ] , and alphavirus vlp [ ] . preexisting immunity in the form of preformed anti-vlp antibodies can be an important consideration for some vlp vaccines [ , ] . binding of anti-vlp antibodies may interfere with the normal uptake and presentation pathways of vlp vaccines. anti-vlp antibodies may be present in unvaccinated individuals due to environmental exposure to prevalent strains of the parent virus, or induced following vlp vaccination. while the presence of preexisting antibodies may decrease the efficacy or alter the immune recognition of some vlp vaccines [ , ] , they can also be associated with measures of enhanced vaccine efficacy [ ] . this may be due to accessing fc receptor-mediated uptake pathways, the formation of antibody-vlp complexes, or antigenic boosting of specific b cells. the presence of anti-vlp antibodies have also been found to have no observable effect on the intended vaccine outcome for several vlp vaccines, such as polyomavirus [ ] , and rhdv vlp [ ] . when anti-vlp antibodies present deleterious interference, alternating between different vlp vaccine vectors may be a suitable solution. alternating between rotavirus and adenovirus vlp has been found to enhance both the humoral and cell-mediated immune responses against target antigens in comparison to repeat delivery with the same vlp vector [ ] . similarly, alternating between closely related vlp such as rhdv and human norovirus (hunv) vlp can be sufficient to enhance vaccine immunogenicity [ ] . further complication can arise when vlp vaccines induce a phenomenon referred to as carrier-induced epitopic suppression (cies), in which the intended immune response is outcompeted by a strong anti-vlp response [ ] . cies may be prevented by masking vlp surface antigens, or by avoiding recognition through recombinant modification. for example, recombinant insertion of the p domain of hiv into hev vlp was identified to avoid recognition by anti-hev antibodies [ ] . the formulation of a vaccine refers to the constituents that make up the final administrable solution, including the vaccine vector, adjuvants, and excipients. for vlp vaccines, these excipients can include a variety of salts and compounds prepared as an aqueous solution or emulsion, which maintains the physical stability of vlp for enhanced shelf life of the vaccine. appropriate use of buffers can limit fluctuations in ph, while protection from fluctuations in temperature and desiccation may be provided by thermoprotectants and lyoprotectants, respectively. formulation science involves investigating the various components of a vaccine under different environmental conditions, with the intention of formulating a stable vaccine product suitable for the route of administration, and with maximized immunogenicity. the combination of formulation components, vlp vaccine preparation states and routes of administration is outlined in figure . the majority of vlp vaccines currently on the market and under clinical evaluation are liquid suspensions, ready for administration. this places strict limitations on vlp vaccine storage and distribution for safe and compliant administration. for example, the commercial hpv vaccine gardasil (merck) must be refrigerated at - °c, and protected from light. gardasil cannot be frozen, and guidelines for the use of gardasil advise that the vaccine must be used within h when removed from refrigeration at temperatures below °c, or when stored at - °c. these guidelines mirror those of many other commercial vlp vaccines, and outlines the primary stability, storage and distribution challenges for formulation science to investigate. maintaining the integrity and stability of vlp in solution is largely dependent on the combination of salts, and the buffering chemicals and compounds used. optimization of buffer ph, ionic strength, and other stabilizing components is imperative to developing a marketable, stable, liquid vlp vaccine [ ] . an investigation into the stability of ev vlp identified that sodium phosphate-based buffers were superior to citrate or tris buffered solutions, with vlp stored in sodium phosphate buffer remaining stable for month at both and °c [ ] . such prolonged stability at room and core body temperature suggests that this vlp may be suitable for importation into countries where maintaining cold-chain storage and distribution can be difficult. other important considerations for the selection of an appropriate buffer include compatibility with downstream applications, such as chemical conjugation, and the availability of scavengable nutrients that may promote the growth of potentially harmful organisms in an improperly stored vaccine. various additive molecules, particularly carbohydrates such as trehalose, sucrose and glycerol, have been investigated extensively in vlp vaccine formulations. the addition of these molecules to vaccine formulations demonstrated enhancement in vlp stability the role of formulation science in vlp vaccine manufacture includes the chemical composition of buffers, preservatives, additives and other stabilizing compounds for maintaining intact vlp. this includes protecting vlp from chemical or physical instability, and enzymatic degradation. formulations can also include targeted delivery compounds, such as muco-adhesives, and immunogenic components such as adjuvants. storage and distribution of vlp vaccines, and the subsequent route of administration are also important considerations in formulation science, critical in determining the efficacy and immunogenicity of the vaccine. within a liquid suspension of norwalk virus vlp [ ] and rotavirus vlp [ ] . other than sugar-based formulation additives, polyanionic solutions can also stabilize vlp that would otherwise be unstable at neutral ph, such as chikungunya virus vlp [ ] . the majority of commercial vlp vaccines are distributed as a liquid suspension, requiring a cold chain maintaining - °c throughout distribution and storage. even under stable cold chain conditions, the longevity of vlp can be limited. this can be prolonged by storage at temperatures at or below − °c, stabilized with the addition of a cryopreservative such as glycerol or trehalose [ ] ; however, this only further exacerbates the issue of delivering these vaccines intact where they are needed, such as in developing countries without reliable cold-chain delivery infrastructure. alternative storage methods such as freeze-drying, or lyophilization, can have variable effects on the stability of vlp. mechanical damage induced by ice crystallization, or exposure to varying salt concentrations and ph changes during the freezing process may adversely affect the integrity of vlp [ ] . exposure to these factors can be limited through the addition of specific cryoprotectants and lyoprotectants. for example, red-spotted grouper nervous necrosis virus (rgnnv) vlp [ ] reportedly retain stable particles and remain immunogenic when freezedried in the presence of sorbitol, but were adversely affected in the presence of mannitol. qβ vlp [ ] , murine polyomavirus (mupyv) vlp [ , ] , and hbv vlp [ ] have likewise demonstrated some capacity to survive varying freeze-drying methodologies. spray-drying has also gained traction as an alternative vaccine storage mechanism, avoiding the potential mechanical damage induced by freeze-drying by instead forming a dry powder formulation through a combination of nebulization and dehumidification [ ] ; however, spray-drying involves exposure to elevated temperatures, which can be similarly damaging to thermolabile vlp. hpv vlp suspended in a formulation containing mannitol, trehalose, dextran, l-leucine and inositol are capable of surviving this procedure [ ] [ ] [ ] . spray-dried ms - l vlp stored at room temperature for months were found to induce high titer anti-hpv l igg antibodies, and significantly protected vaccinated mice from challenge with hpv [ ] . air or vacuum drying methods have also been reported for coating microneedles with influenza vlp suspended in a formulation containing carboxymethylcellulose (cmc). microneedle-based delivery of dried h n (a/pr ) [ ] and h n (a/aichi) [ ] were found to induce immune responses comparable to intramuscular injection, but with the advantage of prolonged longevity in storage. investigation of similar formulation and preparation strategies with different types of vlp may uncover some universality in their application, with the potential for these methods to eradicate the cold-chain limitation imposed on many vlp vaccines. as has been previously described, many vlp possess structural or molecular features that can confer some auto-immunostimulatory properties. these properties facilitate the induction of immune responses by vlp without the need for adjuvants; however, the use of adjuvants with vlp vaccines may enhance vaccine immunogenicity, and promote the activation of a specific type of immune response. currently licensed adjuvants for use with vaccines include aluminum sulfate salts (alum), proprietary combination adjuvants such as as (glaxosmithkline), as (glaxosmithkline) and mf (novartis), thermo-reversible oil-in-water immersions, and montanide isa [ ] . while many standard vaccine adjuvants may be suitable for vlp vaccines where the generation of anti-vlp antibodies is the desired outcome, these adjuvants may fail to overcome immunotolerance and induce antitumor immunity in cancer vaccine formulations [ ] ; however, where traditional adjuvants may fail, novel tlr agonist adjuvants have had success. the tlr agonist adjuvant imiquimod was recently approved for use with vaccines for melanoma, and another clinical trial is assessing its candidacy for treatment of bladder cancer [ ] . tlr / stimulate anti-viral interferon pathways in apcs, promoting the activation of cd + t cells and nk cells [ , ] . the use of the adjuvant gardiquimod in combination with vaccination has been found to induce tumor regression in murine models of melanoma [ ] , human hepatocellular carcinoma [ ] and pancreatic cancer [ ] . tlr agonist adjuvants such as unmethylated cpg oligonucleotides have drawn considerable interest due to their inherent ability to associate with some vlp [ , ] . similarly, the presence of other nucleic acids can have downstream ramifications on the immune response induced by vlp. for example, the presence of rna inside qβ vlp can skew the humoral response induced in mice to produce igg a antibodies, while the removal of this rna results in the production of igg antibodies [ ] . qβ vlp can also contain additional adjuvant molecules derived from their expression system, encapsidated during particle formation. a recent study in mice investigated the use of various adjuvants in conjunction with a filovirus vlp vaccine, including the tlr agonist adjuvant poly-iclc (hiltonol), tlr agonist adjuvant monophospholipid a (mpla), cpg odn and alhydrogel [ ] . poly-iclc was found to induce a predominantly t h response, with an igg c subtype antibody production that correlated with protection from virus challenge. in comparison, administration of the vlp vaccine with alhydrogel elicited a strong t h response with high antibody titers, but conferred no protection from virus challenge. this study highlighted the importance of carefully considering what kind of adjuvant is suitable to accompany a vlp vaccine, as the most immunogenic adjuvant may not necessarily provide the desired vaccine outcome. vlp vaccines approved for clinical use have utilized various administration modalities, including vaccination subcutaneously, intradermally, intramuscularly or at mucosal surfaces [ ] . live attenuated vaccines are traditionally injected subcutaneously, while inactivated or subunit vaccines are often administered intramuscularly. in infants, intramuscular injection of vaccines tends to confer enhanced immunogenicity over subcutaneous administration [ ] . modern vaccine delivery technologies, such as intradermal microneedle patches, have also been investigated for their potential applications with vlp vaccines [ , , ] . while the majority of recent clinical trials involving the administration of vlp favor intramuscular injection, alternative routes of administration are being investigated based on differential immunogenicity at different delivery sites. for example, an investigation into the routes of administration of hunv vlp found that intranasal inoculation of mice induced the production of mucosal anti-hunv igg and iga antibodies, while intramuscular injection only induced igg production [ ] . the study identified that only mucosal iga was suitable for neutralization of infectious hunv virions, rendering intramuscular injection unsuitable for administration of hunv vlp. a similar finding was identified regarding the administration of respiratory syncytial virus (rsv) vlp, with the desired t h response, neutralizing mucosal antibodies and cd + t cell responses identified following intranasal inoculation, rather than intramuscular injection [ ] . vaccination with vlp at mucosal surfaces also requires higher doses of vlp in comparison to parental administration. oral delivery of virus-like particles is also being investigated as an alternative, convenient route of administration. vlp expressed in plant-based systems represents a particularly efficient means of both production and delivery, with expression in an edible plant species potentially forgoing the need for vlp purification and vaccine formulation. examples of this concept include the expression of hbv and norwalk virus vlp in solanum tuberosum potato and lycopersicon esculentum tomato plants [ , ] . nicotiana benthamiana tobacco are also used as an efficient plant expression system for production of vlp. hbv vlp purified from n. benthamiana are capable of withstanding a simulation of the acidic environment within the stomach [ ] ; however, the hbcag polypeptide of hbv vlp was digested when exposed to porcine pepsin. the protection of protein-based vlp from enzymatic digestion is a significant hurdle for oral delivery, despite the promise of this vaccination route. the translation of vlp vaccines from preclinical research to routine clinical administration is a multifaceted task combining studies into stability, formulation science, immunogenicity, and clinical vaccine efficacy. these issues are not necessarily unique to the development of vlp vaccines, encompassing the breadth of research and development necessary for bringing any vaccine candidate to market. the expression of stable vlp is only the first step toward the development of a novel vlp vaccine, which may eventually include various excipients, adjuvants and other compounds in the administrable form. advancements in virology and vaccinology that have facilitated the development of novel vlp vaccines seemingly parallel regulatory and proprietary restrictions placed upon new vaccines; however, vlp vaccines also possess unparalleled potential due to their balance between clinical vaccine efficacy and safety, and their versatility as a vaccine vector. the field of vlp vaccines has continued to experience significant growth over the past decade, both in the diversity of vaccines and in the translation of vaccines toward routine clinical administration. our research focuses upon the development of rhdv vlp as a versatile vaccine scaffold, particularly for immunotherapeutic vaccination as an alternative treatment option for cancer. the translation of vlp vaccine constructs from proof of concept models toward clinical administration is no menial feat. we have observed a corresponding shift toward focusing on clinical translation amongst our colleagues in the field. focal points of discussion highlighted by the process include the relevance and translatability of research animal models, adaptation to the established dogma within the field of human vaccination, and the establishment of a developmental and commercial niche conducive to the advancement of novel vaccines throughout the rigors of clinical trials and regulatory approval. the immune response to vlp vaccines tends to strike a desirable balance between outcomes indicative of optimal vaccine efficacy, such as the induction of high-titer mucosal iga antibodies, while also maintaining an almost unparalleled vaccine safety profile. the inherent inability of vlp to infect or replicate alleviates potential vaccine risks, such as spontaneous reversion to pathogenesis, or incomplete inactivation. retention of the ability to facilitate cross-presentation of associated antigens, and the induction of a potent cell-mediated immune response can also have significant implications for vlp vaccines, diversifying the field to cover a broader range of disorders and diseases. the intracellular processing pathways remain largely unexplored for many vlp, and elucidation of the underlying mechanisms involved in some of the more advanced applications of vlp vaccines, such as gene delivery and immunomodulation. the role of formulation science appears underrepresented amongst clinical trials and clinical reports for vlp vaccines, with at times minimal elucidation of the vaccine administration route, the excipients included, and even exclusion of adjuvant delivered with the vaccine. the importance of formulation science and the relevance of excipients and adjuvants may be expectedly underappreciated during earlier phases of research and development, where establishment of vaccine efficacy may be paramount; however, early adoption of these vital constituents may ease clinical translation, potentially expanding the repertoire of marketable vlp vaccines. novelty, proprietary design, and versatility are almost as important in the development of vlp vaccines as the actual vaccine efficacy itself. maintaining compatibility with the upscaling of production and gmp manufacture pipelines is another important component in the development of a vlp vaccine for routine clinical administration. the benefit of such foresight is clear, and the promotion of clinical translation will be advantageous across the field of vlp vaccines. upon review across the field including current trends in new and emerging research, the progression of established vaccine research, and clinical trial schedules, vlp vaccine research is experiencing a period of rapid growth. this includes both the repertoire of organisms and conditions being targeted with vlp vaccines, and the advancement of established vaccines through clinical trials, translation, and clinical applications. over the next five years, we predict that there will be a significant increase in the diversity of vlp vaccines in active circulation. we expect that vlp will be used in more complex applications than as a vaccine against the cognate virus from which they were derived. we also predict the development of novel vlp vaccine constructs for human and zoonotic pathogenic organisms and conditions with or without current vaccination options, and the incorporation of advanced vaccination methodologies and techniques into routine vlp vaccine production, distribution and administration. in particular, we predict increased incorporation of alternative vaccine-storage techniques such as freeze-dried or spray-dried methods. the development of vlp vaccines is often focused on facilitating distribution amongst populations and communities where these vaccines are most needed. we predict that over the next five years, the capability to support vlp vaccine distribution will increase by limiting the current reliance on cold-chain delivery. these predictions outline an exciting future over the next five years for vlp vaccine research and development. • virus-like particles consist of virus-derived proteins and associated molecules that spontaneously form a particulate structure. • vlp vaccines have the safety profile of a subunit vaccine, but with efficacy and vaccination outcomes that can be comparable to killed or live attenuated vaccines, depending on the particular vlp vaccine. • while the predominant desired immune response amongst the majority of vlp vaccines is a potent humoral response producing high titer antibodies, some vlp vaccines can induce a potent cytotoxic immune response for selective elimination of cells currently infected by the target virus, or target tumor cells. • formulation science plays a major role in the development of vlp vaccines, facilitating the selection of appropriate combinations of excipients and adjuvants. excipients are used to increase vlp particle stability in the vaccine formulation, while adjuvants enhance vaccine efficacy, and help to select for a specific desired immune response and outcome. this research was funded by the university of otago. interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. braeden donaldson http://orcid.org/ - - - vernon k. ward http://orcid.org/ - - - papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers antigen delivery by virus-like particles for immunotherapeutic vaccination this review covers the structural diversity of vlp, post-production modification, and the immune response to vlp vaccines with a specific focus on immunotherapeutic vaccine development immunogenicity of a quadrivalent virus-like particles (vlp) influenza vaccine in healthy adults. nct immunogenicity, safety and tolerability of a plant-derived seasonal virus-like-particle quadrivalent influenza vaccine in adults. nct national institute of allergy and infectious diseases (niaid) study to evaluate safety, tolerability, and immunogenicity of candidate human cytomegalovirus vaccine in healthy adults. nct efficacy and immunogenicity of norovirus gi. /gii. bivalent virus-like particle vaccine in adults. nct . takeda long-term immunogenicity of the norovirus gi.i/gii. bivalent virus-like particle (vlp) vaccine in adults. nct . takeda safety and immunogenicity of norovirus gi. /gii. bivalent virus-like particle vaccine in an elderly population. nct . takeda disassembly and reassembly improves morphology and thermal stability of human papillomavirus type virus-like particles this research article investigates the effects of disassembly and reassembly on the morphological consistency and thermal stability of human papillomavirus vlp. disassembly and reassembly is including as a post-production modification in the manufacture of select commercial vlp vaccines inventors; cytos biotechnology ag, assignee. vlp-antigen conjugates and their uses as vaccines gmp issues for recombinant plant-derived pharmaceutical proteins methodological issues for trials of vaccine efficacy against hpv types and raising expectations for subunit vaccine intellectual property policies in early-phase research in public-private partnerships vaccine instability in the cold chain: mechanisms, analysis and formulation strategies improved storage stability and immunogenicity of hepatitis b vaccine after spray-freeze drying in presence of sugars expression of animal virus genomes novel epstein-barr virus-like particles incorporating gh/gl-ebna or gb-lmp induce high neutralizing antibody titers and ebv-specific t-cell responses in immunized mice expression and characterization of yeast derived chikungunya virus like particles (chik-vlps) and its evaluation as a potential vaccine candidate viral protein requirements for assembly and release of human parainfluenza virus type viruslike particles cell-specific delivery of diverse cargos by bacteriophage ms virus-like particles structure of small virus-like particles assembled from the l protein of human papillomavirus influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles influenza virus-like particles comprised of the ha, na, and m proteins of h n influenza virus induce protective immune responses in balb/c mice. vaccine dynamic of immune response induced in hepatitis b surface antigen-transgenic mice immunized with a novel therapeutic formulation recombinant hepatitis b vaccine (engerix-b): a review of its immunogenicity and protective efficacy against hepatitis b [review] immunological characterization of two hepatitis b core antigen variants and their immunoenhancing effect on co-delivered hepatitis b surface antigen characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells assembly of double-shelled rotaviruslike particles by simultaneous expression of recombinant vp and vp proteins expression of human immunodeficiency virus type gag protein precursor and envelope proteins from a vesicular stomatitis virus recombinant: highlevel production of virus-like particles containing hiv envelope paramyxovirus sendai virus-like particle formation by expression of multiple viral proteins and acceleration of its release by c protein a virosomal formulated her- /neu multi-peptide vaccine induces her- /neu-specific immune responses in patients with metastatic breast cancer: a phase i study influenza virus-like particle can accommodate multiple subtypes of hemagglutinin and protect from multiple influenza types and subtypes. vaccine generating enveloped virus-like particles with in vitro assembled cores core-like particles of an enveloped animal virus can self-assemble efficiently on artificial templates human parvovirus b virus-like particles: in vitro assembly and stability engineering hepatitis b virus core particles for targeting her receptors in vitro and in vivo novel adenovirus encoded virus-like particles displaying the placental malaria associated var csa antigen virus-like particle display of the α-gal carbohydrate for vaccination against leishmania infection molecular pharming-vlps made in plants the large-scale production of an artificial influenza virus-like particle vaccine in silkworm pupae virus-like particles: the future of microbial factories and cell-free systems as platforms for vaccine development chimeric gii. norovirus virus-like-particle-based vaccines induce broadly blocking immune responses chimeric bivalent virus-like particle vaccine for h n hpai and nd confers protection against a lethal challenge in chickens and allows a strategy of differentiating infected from vaccinated animals (diva) novel recombinant chimeric viruslike particle is immunogenic and protective against both enterovirus and coxsackievirus a in mice multi-target chimaeric vlp as a therapeutic vaccine in a model of colorectal cancer an enhanced heterologous virus-like particle for human papillomavirus type tumour immunotherapy protective effect of a germline, il- -neutralizing antibody in murine models of autoimmune inflammatory disease virus-like particles from rabbit hemorrhagic disease virus can induce an anti-tumor response memory and effector cd t-cell responses after nanoparticle vaccination of melanoma patients a vlp-based vaccine against interleukin- α protects mice from atherosclerosis development of an interleukin- β vaccine in patients with type diabetes a vaccine against nicotine for smoking cessation: a randomized controlled trial virus-like particles, a versatile subunit vaccine platform bioengineering virus-like particles as vaccines construction and characterization of virus-like particles: a review this review covers the breadth of expression systems available for vlp vaccine production, while also discussing vlp structural diversity and characterization mannosylation of virus-like particles enhances internalization by antigen presenting cells coupling the adjuvant cpg oligonucleotides to rhdv vlp. dunedin: university of otago αenv-decorated phosphatidylserine liposomes trigger phagocytosis of hiv-virus-like particles in macrophages norovirus p particle: a subviral nanoparticle for vaccine development against norovirus, rotavirus and influenza virus nanoparticles target distinct dendritic cell populations according to their size conduits mediate transport of low-molecular-weight antigen to lymph node follicles the humoral immune response is initiated in lymph nodes by b cells that acquire soluble antigen directly in the follicles virus-like particle (vlp) lymphatic trafficking and immune response generation after immunization by different routes follicular shuttling of marginal zone b cells facilitates antigen transport b cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node a molecular assembly system that renders antigens of choice highly repetitive for induction of protective b cell responses. vaccine dissecting virus entry via endocytosis despite differences between dendritic cells and langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime t cells. virology cross-presentation of epitopes on virus-like particles via the mhc i receptor recycling pathway the length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes differential uptake and crosspresentation of human papillomavirus virus-like particles by dendritic cells and langerhans cells an engineered non-toxic superantigen increases cross presentation of hepatitis b virus nucleocapsids by human dendritic cells multivalent display and receptormediated endocytosis of transferrin on virus-like particles low ph-dependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the vp n-terminal sequence (n-vp ), n-vp cleavage, and uncoating of the full-length genome efficient gene transfer using the human jc virus-like particle that inhibits human colon adenocarcinoma growth in a nude mouse model cross-presentation of virus-like particles by skin-derived cd -dendritic cells: a dispensable role for tap hepatitis b virus-like particles access major histocompatibility class i and ii antigen presentation pathways in primary dendritic cells uptake and presentation of hepatitis c virus-like particles by human dendritic cells proteasome-independent major histocompatibility complex class i cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human t cells in vivo, dendritic cells can crosspresent virus-like particles using an endosome-to-cytosol pathway influenza-pseudotyped gag virus-like particle vaccines provide broad protection against highly pathogenic avian influenza challenge eleven years of inflexal ® v-a virosomal adjuvanted influenza vaccine inflexal ® v a trivalent virosome subunit influenza vaccine: production influenza virus assembly and budding in raftderived microdomains: a quantitative analysis of the surface distribution of ha, na and m proteins influenza virus m ion channel protein is necessary for filamentous virion formation infectious entry pathway of influenza virus in a canine kidney cell line studies on the mechanism of influenza virus entry into cells epsin is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway endocytosis of influenza viruses influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis gene therapy for human lung adenocarcinoma using a suicide gene driven by a lung-specific promoter delivered by jc virus-like particles in vivo sirna delivery using jc virus-like particles decreases the expression of rankl in rats the human polyomavirus, jcv, uses serotonin receptors to infect cells jc virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis contrasting roles of endosomal ph and the cytoskeleton in infection of human glial cells by jc virus and simian virus use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles human jc virus-like particles as a gene delivery vector encapsulation and delivery of plasmid dna by virus-like nanoparticles engineered from macrobrachium rosenbergii nodavirus a novel polyethyleneimine-coated adeno-associated virus-like particle formulation for efficient sirna delivery in breast cancer therapy: preparation and in vitro analysis gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified dna encapsidation capacity by addition of packaging sequences from the l or l protein of human papillomavirus type novel mir- delivery system based on ms virus like particle surface displaying cell-penetrating peptide tat for hepatocellular carcinoma neutralizing antiviral b cell responses virus-like display of a neo-self antigen reverses b cell anergy in a b cell receptor transgenic mouse model low-affinity b cells transport viral particles from the lung to the spleen to initiate antibody responses this research article investigates the transportation of vlp following intranasal delivery, mediated by low-affinity b cells. intranasal vaccination and the induction of mucosal iga anti-vlp production is an important vaccination outcome for vlp vaccines against viruses that infect through mucosal surfaces extrafollicular antibody responses germinal center b and follicular helper t cells: siblings, cousins or just good friends competence and competition: the challenge of becoming a long-lived plasma cell plasma cell development: from b-cell subsets to long-term survival niches b lymphocyte activation by human papillomavirus-like particles directly induces ig class switch recombination via tlr -myd t cell-dependent and-independent iga responses: role of tlr signalling alveolar macrophages and lung dendritic cells sense rna and drive mucosal iga responses efficient induction of mucosal and systemic immune responses by virus-like particles administered intranasally: implications for vaccine design mucosal immunity in the female genital tract mucosal immunity in the female genital tract: relevance to vaccination efforts against the human immunodeficiency virus a comparison of antibody titres in mouse uterine fluid after immunization by several routes, and the effect of the uterus on antibody titres in vaginal fluid systemic and secretory humoral immunity in the normal human vaginal tract comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women specific antibody levels at the cervix during the menstrual cycle of women vaccinated with human papillomavirus virus-like particles induction of immune memory following administration of a prophylactic quadrivalent human papillomavirus (hpv) types / / / l virus-like particle (vlp) vaccine. vaccine induction of virusspecific cytotoxic t lymphocytes as a basis for the development of broadly protective influenza vaccines influenza vaccines: t-cell responses deserve more attention this review article discusses the importance of inducing a potent cytotoxic immune response for vaccination against select virus infections t-cell-mediated and humoral approaches to universal influenza vaccines better influenza vaccines for older people: what will it take? influenza hemagglutinationinhibition antibody titer as a correlate of vaccine-induced protection efficient homologous prime-boost strategies for t cell vaccination based on virus-like particles use of chemotherapy plus a monoclonal antibody against her for metastatic breast cancer that overexpresses her antigen incorporated in virus-like particles is delivered to specific dendritic cell subsets that induce an effective antitumor immune response in vivo messenger rna vaccine based on recombinant ms virus-like particles against prostate cancer anti-tumor effect of the alphavirus-based virus-like particle vector expressing prostate-specific antigen in a hla-dr transgenic mouse model of prostate cancer universal influenza a m e-hbc vaccine protects against disease even in the presence of preexisting anti-hbc antibodies segments of puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice carrier induced epitopic suppression of antibody responses induced by virus-like particles is a dynamic phenomenon caused by carrier-specific antibodies. vaccine innate immunity mediates follicular transport of particulate but not soluble protein antigen this research article investigates the transportation and delivery of qβ vlp to follicular dendritic cells within the lymph node, and the impairment of this delivery in the presence of anti-qβ vlp antibodies effects of preexisting anti-carrier immunity and antigenic element multiplicity on efficacy of a modular virus-like particle vaccine immunomodulation and vaccination with rhdv vlp: a thesis submitted for the degree of doctor of philosophy prime immunization with rotavirus vlp / followed by boosting with an adenovirus expressing vp induces protective immunization against rotavirus in mice can different virus-like particles be used for primeboost vaccination? dunedin: university of otago chimeric hepatitis e virus-like particle as a carrier for oral-delivery. vaccine evaluation of the stability of enterovirus virus-like particle physical stabilization of norwalk virus-like particles downstream processing of triple layered rotavirus like particles development of a stable viruslike particle vaccine formulation against chikungunya virus and investigation of the effects of polyanions stability studies of hiv- pr gag virus-like particles made in insect cells after storage in various formulation media rational design of a stable, freezedried virus-like particle-based vaccine formulation stability of virus-like particles of redspotted grouper nervous necrosis virus in the aqueous state, and the vaccine potential of lyophilized particles modular virus-like particles for sublingual vaccination against group a streptococcus. vaccine virus-like particle formulation optimization by miniaturized high-throughput screening freeze-drying of plant tissue containing hbv surface antigen for the oral vaccine against hepatitis b developments in the formulation and delivery of spray dried vaccines preclinical refinements of a broadly protective vlp-based hpv vaccine targeting the minor capsid protein, l . vaccine optimized formulation of a thermostable spray-dried virus-like particle vaccine against human papillomavirus characterization of a spraydried candidate hpv l -vlp vaccine stored for multiple years at room temperature. papillomavirus res this research article demonstrates the compatibility of a recombinant ms - l vlp vaccine with spray-drying, and the retention of vaccine immunogenicity after prolonged storage at room temperature immunization by vaccine-coated microneedle arrays protects against lethal influenza virus challenge transdermal influenza immunization with vaccine-coated microneedle arrays vaccine adjuvants: from to and beyond vaccine adjuvants as potential cancer immunotherapeutics distinct indirect pathways govern human nk-cell activation by tlr- and tlr- agonists tlr / -mediated activation of human nk cells results in accessory cell-dependent ifn-gamma production the tlr agonists imiquimod and gardiquimod improve dc-based immunotherapy for melanoma in mice tlr / agonists promote nk-dc crosstalk to enhance nk cell anti-tumor effects in hepatocellular carcinoma activation of toll-like receptor inhibits the proliferation and migration, and induces the apoptosis of pancreatic cancer cells immunotherapy of type- allergies with virus-like particles and cpg-motifs a universal virus-like particle-based vaccine for human papillomavirus: longevity of protection and role of endogenous and exogenous adjuvants. vaccine adjuvant-enhanced cd t cell responses are critical to durable vaccine immunity influence of parenteral administration routes and additional factors on vaccine safety and immunogenicity: a review of recent literature immunogenicity and safety of measles-mumps-rubella-varicella (mmrv) vaccine followed by one dose of varicella vaccine in children aged months- years or - years primed with measles-mumps-rubella (mmr) vaccine long-term protective immunity from an influenza virus-like particle vaccine administered with a microneedle patch mucosal antibodies induced by intranasal but not intramuscular immunization block norovirus gii. virus-like particle receptor binding a single intranasal administration of virus-like particle vaccine induces an efficient protection for mice against human respiratory syncytial virus virus-like particle expression and assembly in plants: hepatitis b and norwalk viruses expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice plant-expressed hepatitis b core antigen virus-like particles: characterization and investigation of their stability in simulated and pig gastro-intestinal fluids key: cord- -l puqk authors: yun, cheol-heui; cho, chong-su title: nanoparticles to improve the efficacy of vaccines date: - - journal: pharmaceutics doi: . /pharmaceutics sha: doc_id: cord_uid: l puqk this editorial aims to summarize the nine scientific papers that contributed to the special issue entitled ‘nanoparticles to improve the efficacy of vaccines’. a vaccine, providing active and protective immunity against a target disease, contains an agent that originated from and/or resembles a disease-causing microorganism. it is often made from a weakened or inactivated microbe, its toxins, or one of its nucleotides, peptides or proteins. vaccines can be prophylactic to ameliorate or better prevent the effects of a wild-type pathogen, or therapeutic against likely cancers. to date, the world health organization lists twenty-seven preventable infections for which vaccines are available [ ] -far less than what our society needs. vaccination, a process of introducing foreign antigenic material(s) in order to activate a host immune system, has been a key strategy to control diseases and improve quality of life in humans and animals. despite the presence of some successful vaccines, many novel and modified diseases including ebola virus disease, zika virus disease, coronavirus diseases [middle eastern respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov), and novel coronavirus ( -ncov)], dengue fever, marburg disease, malaria, and tuberculosis are in need of effective vaccines together with qualified adjuvants. while traditional adjuvants such as alum have been exclusively employed clinically to promote humoral responses, recent developments in adjuvant research have identified molecules, which are pathogen-associated molecular patterns, a few chemical compounds, and agonists of toll-like receptors, all of which induce strong immune responses. with great advancements in the area of material science, a new era of innovative strategies for vaccine design has arrived, enabling the precise delivery of vaccines, the enhanced role of vaccine adjuvants, an increase in the sparing effect, better stabilization, and slow release at the induction site. nanomaterials that modified to trigger antigen-specific immune responses could be categorized into liposomes and lipid-based nanoparticles, polymeric nanoparticles, gold nanoparticles, inorganic nanoparticles, virus-like particles, self-assembled proteins, and carbon-based nanoparticles (carbon nanotubes and graphenes). in the special issue, entitled 'nanoparticles to improve the efficacy of vaccine' in the pharmaceutics (https://www.mdpi.com/journal/pharmaceutics/special_ issues/nanoparticles_vaccines), we draw attention to the advanced platforms and technologies using nanomaterials in order to produce the best outcomes in terms of vaccination and immunological memory. kim et al. [ ] described a method to improve the battle against intracellular bacterial or viral infections, and malignant tumors by various vaccination schemes, using physicochemical characteristics that target antigen-presenting cells (apcs). in particular, the improvement of an unconventional type of antigen presentation, called 'cross-presentation' in apcs when treated with certain nanomaterials for antigen-specific cd + t cell responses, was discussed [ ] . the authors focused on the mechanisms of two major intracellular pathways that nano-sized vaccines harness for cross-presentation, namely endosomal swelling and rupture, and membrane fusion. these processes allow exogenous antigens exported from phagosomes into the cytosol, followed by loading on major histocompatibility complex (mhc) class i, triggering the clonal expansion of antigen-specific cd + t cells. barnowski et al. [ ] discussed nano-vaccines in the form of virus-like particles (vlps), which share structural identities with their wild-type viruses, allowing b cells to face the natural conformation of the virus. the authors concentrated on the use of flagellin, a potent inducer of innate immunity via toll-like receptor , as an adjuvant to formulate human immunodeficiency virus (hiv)-based nanoparticle b cell-targeting vaccines that display either the hiv- envelope protein (env) or a model antigen, hen egg lysozyme (hel). they postulated that, in the context of vlp-based b cell nano-vaccines, flagellin may outcompete against a less immunogenic antigen, while, in combination with a strong immunogen, the adjuvanticity of flagellin dominates over its immunogenicity. kang et al. [ ] introduced a vlp vaccine containing rhoptry organelle proteins (rop) and/or rop secreted by toxoplasma gondii together with influenza m . it was intriguing that upon challenge via the oral route, mice immunized with rop( + ) vlps elicited higher levels of t. gondii-specific igg and iga responses in fecal, urine, intestine and vaginal samples in accordance with cd + t, cd + t cells, and germinal center b cell responses when compared with other vaccines, rop vlps, rop vlps, and rop +rop vlps. thus, the article shed light on important insights and potential strategies for the design of a vaccine using the t. gondii rops and vlp system. viyayan et al. [ ] discussed biomimetic nanoparticles (nps) to deliver vaccines for the treatment of diseases including hiv, malaria, some tumors and bacterial diseases due to their beneficial advantages such as improved antigen stability, targeted delivery, long-time controlled release and evasion of immune responses. they covered four kinds of biomimetic nps for the delivery of vaccines. the first was liposomes, obtained by the dispersion of phospholipids in water, because they display high antigen loading and co-delivery of both hydrophobic and hydrophilic antigens. the second was nps coated with cell membranes from red blood cells, leukocytes, cytotoxic t-cells, nk cells, platelets, macrophages or cancer cells, because they preserve the physicochemical properties of the core synthetic nps and maintain the cellular composition on their membrane shell. the third was self-assembled proteins, because they play diverse physiological roles and mimic natural microbe architectures. the fourth was virus-like nps containing noninfectious subsets of viruses, because they assemble without containing any viral rna. sartorius et al. [ ] described the possibility of filamentous bacteriophages (fbs), prepared by phage display technology, as nature-made nps to deliver therapeutic vaccines, because lytic bacteriophages have been used as antibacterial materials in several clinical trials due to their safe and inexpensive therapeutic behavior, where the fbs enter the blood vessels easily, owing to their nano size, and exhibit high-density antigen expression. the authors showed the induction of specific cytotoxic t cell responses to hla-a -restricted and hepatitis b virus epitopes due to the ability of the filamentous rod, internalized into apcs. it seems likely that the targeted delivery of these nps in new-generation vaccines against tumors such as melanoma and mastocytoma might be realized, although clinical trials are necessary to establish their safety in humans. lim et al. [ ] described nano delivery systems for the improvement of the enhanced gene expression of dna vaccines that provide potent humoral and cell-mediated responses. they pointed out the limitations of their clinical applications, which present hurdles in their delivery to targeted immune cells, although none have been approved for clinical use thus far. the authors discussed the advantages and weaknesses of polymeric nps, lipid nps, hybrid lipid-polymer nps, inorganic nps, virus-like nps and protein-based nps in the delivery of dna vaccines. dna vaccine platform technology would have a high chance to be successful in clinical application if the better adjuvants and/or the encoded antigens were selected properly. tan et al. [ ] discussed norovirus capsid protein-derived nps and polymers obtained by homotypically and/or heterotypically self-assembled mechanisms through bioengineering norovirus capsid shell (s) and protruding (p) domains, making them efficient vaccine candidates against noroviruses because they are easily produced, highly stable, they elicit strong host immune responses and several chimeric s and p nps as well as p domain-derived polymers, as shown in other virus vaccine candidates such as rotavirus, influenza virus, ev , hiv- , alzheimer, and astrovirus diseases. it may be expected that p nps and polymers function as multifunctional p domain-based oligomer vaccine platforms displaying foreign antigens. there are two articles describing the importance of adjuvants. park et al. [ ] described a novel adjuvant in the form of a single-stranded rna (ssrna) nano-structure that can stimulate both th and th responses. the authors claimed that neither adverse side effects such as hematological and serum biochemical parameters, nor ige and anti-nuclear antibodies, which are markers of autoimmune disease, was found, and postulated that this ssrna nanostructure may be an alternative choice over traditional adjuvants. sharma et al. [ ] discussed the possibility of zinc oxide (zno)-based nanocomposites as vaccine adjuvants and the application of cancer immunotherapeutics because of their biocompatibility, unique physicochemical properties, cost-effective mass production, intrinsic adjuvant-like properties and immunomodulatory function. the authors introduced the effects of diverse formulations and compositions on the efficiency of antigen delivery and immunogenicity by augmenting antigen processing, enhancing antigen stability and controlling the release of antigens. they described that the immunomodulatory effects of these innate immune cells were mediated by intracellular zn + dissolution in the lysosomes after the phagocytosis of particles and the generation of reactive oxygen species, resulting in the release of inflammatory cytokines and the activation of immune cells. furthermore, they discussed the possibility of the enhancement of anti-cancer immunity when combined with tumor-specific antigens into zno nps, although the potential toxicity and biodistribution of the zno nps during in vivo studies should be thoroughly verified. as vaccine development pushes toward less immunogenic components such as nucleotide-based, peptide-based or sub-unit vaccines because of their side effects and the life-threatening risks of live attenuated vaccines, strategies to boost both innate and adaptive immune responses are increasingly needed. in the present special issue, the authors discuss the modification of nanomaterials to provide a functional and stable interface for different applications and strategies for vaccination. we hope this special issue, highlighting the importance and use of nanomaterials as a platform technology, will direct scientists as well as manufacturers to improve the efficacy of vaccines that are currently under development against modified or novel diseases. world health organization, global vaccine action plan the role of nanovaccine in cross-presentation of antigen-presenting cells for the activation of cd + t cell responses intranasal immunization with pneumococcal surface protein a in the presence of nanoparticle forming polysorbitol transporter adjuvant induces protective immunity against the streptococcus pneumoniae infection advantages and limitations of integrated flagellin adjuvants for hiv-based nanoparticle b-cell vaccines influenza virus-like particles presenting both toxoplasma gondii rop and rop enhance protection against t. gondii infection recent advances in nanovaccines using biomimetic immunomodulatory materials arming filamentous bacteriophage, a nature-made nanoparticle, for new vaccine and immunotherapeutic strategies engineered nanodelivery systems to improve dna vaccine technologies norovirus capsid protein-derived nanoparticles and polymers as versatile platforms for antigen presentation and vaccine development comprehensive analysis of the safety profile of a single-stranded rna nano-structure adjuvant application of zno-based nanocomposites for vaccines and cancer immunotherapy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we wish to express sincere thanks to all authors for their contribution to this special issue, entitled 'nanoparticles to improve the efficacy of vaccine'. we are also pleased to acknowledge all the editorial office team members for their great help and support during the peer-review process. the authors declare no conflict of interest. key: cord- -ypefqm authors: roberts, christine c.; maslow, joel n. title: assay challenges for emerging infectious diseases: the zika experience date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: ypefqm from the perspective of vaccine development, it is imperative to accurately diagnose target infections in order to exclude subjects with prior exposure from evaluations of vaccine effectiveness, to track incident infection during the course of a clinical trial and to differentiate immune reactions due to natural infections from responses that are vaccine related. when vaccine development is accelerated to a rapid pace in response to emerging infectious disease threats, the challenges to develop such diagnostic tools is even greater. this was observed through the recent expansion of zika virus infections into the western hemisphere in – . when initial zika vaccine clinical trials were being designed and launched in response to the outbreak, there were no standardized sets of viral and immunological assays, and no approved diagnostic tests for zika virus infection. the diagnosis of zika virus infection is still an area of active research and development on many fronts. here we review emerging infectious disease vaccine clinical assay development and trial execution with a special focus on the state of zika virus clinical assays and diagnostics. the declaration by the world health organization (who) that zika is a public health emergency of international concern in february led to a global effort to support vaccine development and control the spread of zika virus (zikv). our collaborative dna vaccine consortium focused and accelerated pre-clinical, manufacturing and early clinical development efforts to bring forward the first zika vaccine, gls- , into human clinical trials [ ] [ ] [ ] . at the outset, it was clear that gaps would need to be filled as the public health and science communities learned and shared new information on zika. one of the clear gaps affecting both public health efforts and vaccine development programs was a lack of standardized reagents and methods to test for evidence of current or prior zika infection. the need for fast and accurate diagnostic tests of infection in an outbreak situation is obvious: identify the source or epicenter so that appropriate healthcare measures can be quickly instituted. expanding that concept to the public health scale and attaining accurate infectious disease diagnoses allows for better understanding of the course and severity of an outbreak and aids decision-making for population-level countermeasure implementation. the clinical assays with which the immune response and pathogen presence are measured in vaccine trials become part of the basis for licensure for all vaccine products [ ] . because vaccines are tested in healthy populations through all phases of clinical development for immune response and/or pathogen presence whereas drugs/biologics (post-phase i) are most often tested in a population with specific disease to demonstrate improvement, the selected methods to measure vaccine responses and endpoints are of the utmost importance. the identification of an immune correlate of protection for each vaccine is highly desirable, though not always attainable [ ] . here we will use the recent experience with the zikv outbreak and ensuing public health countermeasures for containment and vaccine development as an example of challenges faced during emerging infectious disease emergencies. zika virus was discovered in during a survey to map the extent of yellow fever in the entebbe region of uganda. the virus was cultured from the serum of a sentinel macaque placed in the zika forest that developed fever but was otherwise well. initially restricted to equatorial regions of africa and asia, zikv started to spread eastward across the pacific ocean with an outbreak on yap island, micronesia in [ ] , in french polynesia in [ ] , and reaching brazil in late or early [ ] . zikv infection typically causes a self-limited illness that is minimally symptomatic for most individuals. zika infection presents similarly to dengue or chikungunya with fever in most, rash, malaise, myalgia, conjunctivitis and retro-orbital pain but may present with few, if any, discernable symptoms [ ] . however, documentation in brazil of severe neurologic complications associated with zikv infection beginning around october raised worldwide alert. the most publicized and dramatic complications are those that occur during fetal development. they include microcephaly, intra-uterine growth retardation, cerebral calcifications, ocular calcifications and other ocular abnormalities-with an attack rate estimated at - % of women who become infected during pregnancy [ ] . zikv can also directly infect the placenta and can result in spontaneous miscarriage with an unknown prevalence [ ] [ ] [ ] [ ] . in adults, the most common complication of zikv infection is guillain-barré syndrome occurring at an estimated rate of in cases [ ] . zika has also resulted in deaths among adults with and without other complicating factors [ , ] . there are no approved therapies or vaccines for zikv infection. there have been a number of vaccines developed for other flavivirus infections. live virus vaccines utilizing chimeric viral constructs have been approved for use to prevent yellow fever and dengue. dna vaccines have been developed and published for dengue [ , ] , west nile virus [ ] [ ] [ ] [ ] [ ] , and japanese encephalitis virus [ ] . notably, dna vaccines targeting west nile virus [ , ] and dengue virus [ ] have been tested as part of phase i clinical trials without vaccine associated toxicity. currently, as recently reviewed elsewhere, a number of vaccine modalities targeting zikv have reached early stage clinical trials and even more are in preclinical development [ ] [ ] [ ] . zikv is spread primarily through aedes species mosquitoes, mainly aedes aegypti, but can be carried by other mosquito species [ ] and does not appear to be transmission competent except for aedes species [ ] [ ] [ ] [ ] [ ] [ ] . aedes mosquitoes also transmit other arboviruses such as dengue and chikungunya [ , ] . in fact, these infections are co-endemic in most regions and may cause concurrent infections [ ] [ ] [ ] [ ] . alternative nonmosquito-borne routes of zikv spread include: blood transfusions, breast milk [ ] , sexual transmission [ ] [ ] [ ] , and may include urine and saliva [ ] [ ] [ ] [ ] . the additional potential routes of zikv transmission have increased the need for definitive monitoring and a variety of surveillance strategies to prevent disease spread [ ] [ ] [ ] [ ] [ ] . in an outbreak situation, such as with zika, it is important to have the ability to quickly develop both diagnostic kits for public health purposes and vaccine clinical assays to support pre-clinical studies and early stage clinical trials. both were largely unavailable on a commercial scale or for widespread use at the outset of the zika outbreak, though development ensued at a rapid pace upon the declaration of a worldwide public health emergency. because there is significant homology between zikv and other cocirculating flaviviruses, detection and diagnosis has had the extra challenge of avoiding cross-reactivity without sacrificing sensitivity. while the avoidance of cross-reactivity is more easily engineered into molecular tests of virus rna because primers or probes can be designed to be virus-, antigen-and serotype-specific [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it is not as easily achieved for immunoassays [ ] [ ] [ ] [ ] [ ] [ ] . diagnostic assays are often the same style tests as those used in vaccine development, but are for the intended purpose of identifying the source of a patient's illness to enable the initiation of appropriate treatments by healthcare professionals. the need for sensitivity and specificity as it relates to clinical disease identification for patient treatment is of the utmost concern. early in the zika outbreak, the us centers for disease control and some academic laboratories studying flaviviruses had assays developed for zikv that generally supported their own research interests [ ] [ ] [ ] [ ] [ ] [ ] [ ] . making sufficient quantities of these tests available for public health use while ensuring consistency and quality was a significant challenge. additionally, cross-reactivity in a number of immunological assays and the short time frame in which viremia can be detected in bodily fluids necessitated the institution of an algorithm to confirm zikv infection that was based on a combination of risk factors, clinical symptoms and diagnostic test results [ ] . the centers for disease control (cdc) issued testing guidance for healthcare providers using algorithms that included use of molecular testing for pregnant or non-pregnant and symptomatic or non-symptomatic individuals, the types of specimens and timing of collection of specimens that would provide the most reliable results [ ] . in addition, guidance algorithms were provided for the use and interpretation of zikv igm assays to indicate recent exposure with or without accompanying molecular test results [ ] . in july of , united states (us) health and human services (hhs) sponsored a hhs summit to accelerate zika diagnostics development, recognizing the important need. at that point in time, very few tests had gained emergency use authorization (eua) from the food and drug administration (fda). the five serological zika diagnostic kits currently authorized by the us fda are shown in table and fourteen viral diagnostic kits in table [ ] . at the fda medical countermeasures webpage, links can also be found for each eua approved molecular assay's key [ ] and performance [ ] characteristics including instruments approved for use and limits of detection. although no zika diagnostic kit, serological or viral, has been fully approved by the fda to date, the fda has worked collaboratively with developers to accelerate both eua approvals and the transition to formal full approvals of zika diagnostic kits using standard review timelines [ ] . a number of studies have evaluated the various eua tests both for independent sensitivity and specificity [ , , , , , , , ] assessments of assay performance and as part of comparative studies [ , [ ] [ ] [ ] [ ] to determine those best to use for the diagnostic or epidemiological surveillance need. a large number of companies have also developed tests that remain classified as "research use only" (ruo) for which emergency authorizations have not been granted. other authorizations for emergency use have been granted to different diagnostic kits by other agencies, such as who's emergency use assessment and listing procedures (euals) [ ] . while many diagnostic kits use methods similar or identical to those that may be used to support a vaccine clinical trial, often they are focused more toward the support of a clinical patient diagnosis and may not be sensitive enough for use to support vaccine development. often there is not enough data at the outset of a vaccine program, but especially in the case of emerging infectious diseases, to understand what assays will be most useful or informative or will perhaps even provide a correlate of protection for the vaccine into the future. a few general assumptions can be made at the outset about which types of assays will be needed to detect vaccine-induced humoral and cellular immune responses and these will evolve over time. the typical go-to methods used for vaccine clinical assays are: antibody binding-enzyme-linked immunosorbent assays (elisa); functional-virus neutralization or bactericidal; cellular-interferon gamma enzyme-linked immunospot assay (ifnγ-elispot) using the target antigen or antigen-derived peptide pools; and molecular or culture methods to detect the pathogen. the technologies used to develop and run these assays have improved over the years to allow for higher throughput, multiplexing, reduced sample volumes, and automation. often, more tests of a larger variety are done early in a program and are then whittled down based on the usefulness of the data generated so that just the relevant few remain to support large trials and licensure [ ] . when the first zikv vaccine trials commenced in late summer of , even though a few zika diagnostics had achieved eua status, none were commercially available and were initially restricted to public health use only. challenges to vaccine development centered around the determination of prior zikv exposure and immunity and determination of incident infection among study participants. each of the serological assays listed in table detect igm reflective of recent infection, however many, such as the mac-elisa and the inbios assay, will detect igm against the zikv envelope which is the target antigen for many vaccines. igg-based assays have not yet been validated primarily due to cross-reactivity between zikv and other flaviviruses, principally dengue. igm immunoassays targeted to the ns antigen are generally more specific than those directed to the viral envelope [ ] . in september , we initiated the first clinical trial of the gls- zikv dna vaccine in an endemic region, puerto rico, an area also known to have high rates of exposure to dengue. because no widely accepted standardized assays nor any international reference standards or reagents existed at the time of trial initiation, individual vaccine projects needed to rely on internally developed clinical assays to understand prior exposure and vaccine related immune responses. similar to patient diagnostic tests for zikv and as mentioned above, there was no accepted "gold standard" for any of the immunoassays one might choose to develop or use in a vaccine program. the extensive experience of our collaborative dna vaccine team allowed us to develop zikv-specific tests such as elisa, virus microneutralization and elispot around the development and pre-clinical testing of our zikv plasmid dna vaccine constructs [ , , , ] . these assays performed consistently on a pre-clinical scale and we were able to quickly expand their use to support our two phase gls- vaccine clinical trials. a concern always remains, however, that the lack of highly-characterized reagents and controls in early versions of vaccine clinical assays will result in difficulties in the maintenance of the assays through its full life cycle. sourcing, batch-to-batch variability and overall quality of critical reagents, standards and controls can become an issue over time. lack of standardization across the scientific field can be confounding in that interpretation of results from multiple "home brew" assays across labs are not directly comparable in the absence of an accepted international standard or a proficiency panel of samples [ ] . the main methodologies used to detect incident zikv infection are currently molecular-based, mainly reverse transcriptase polymerase chain reaction (rt-pcr) [ , , , , , , , , ] or varieties thereof [ , , ] , since culture methods can be both difficult and laborious [ ] [ ] [ ] . those viral detection systems with eua approval are shown in table . in clinical trials, identification of newly infected subjects over the treatment and follow-up periods are necessary to determine vaccine efficacy. as discussed earlier, for most individuals zikv infection is minimally symptomatic such that few present to clinical care. zikv is detectable in serum by rt-pcr for only a short interval following the onset of symptoms, typically for only seven days to a maximum of days [ , ] , though longer periods of rt-pcr detection (up to days) have been observed in serum of pregnant women [ ] , which may contribute to the incidence of zikv-related birth defects. zikv is excreted into the urine for approximately four weeks in most individuals, though this observation was not documented until over a year into the epidemic [ , , , ] . because of this, rt-pcr-based diagnosis of incident infections in a vaccine clinical trial would require very frequent sampling. other sample types have been evaluated for rt-pcr detection including saliva, whole blood, plasma, brain tissue, amniotic fluid and vaginal secretions [ , , , , , ] . a key concern from developers of both diagnostics and of vaccines or therapeutics for zikv highlighted at the hhs summit for diagnostics in july was the lack of well-characterized human zikv specimens which groups could use to fully understand the performance characteristics of the assays being developed and, eventually, work toward some standardization across the field. the who has initiated in, july , a collaborative study effort for the development of nucleic acid testing international standard for zika [ ] . additionally, in july , a plasma sample panel became available through the us fda for use in evaluating zikv immunoassay performance [ ] . reagents for newly emergent infectious diseases like zikv were not readily available from commercial vendors that had consistent production methods and quality controls in place, thus many reagents were not well characterized early on in the development of zikv clinical assays. because validated assays supporting vaccine efficacy endpoints need to support clinical and regulatory expectations over the life of the product, it is imperative that a line-of-sight is maintained such that reliable and qualified materials in appropriate quantity are available for resolving issues that arise. assays typically require some troubleshooting over time, changes to reagent lots or instruments, potential for multiplexing, platform changes to increase testing throughput, or a desire to bridge to a new technology [ ] . the development, qualification, validation and maintenance of vaccine clinical assays should be done in close consultation with biostatisticians and bench scientists to ensure the optimal assay design, control parameters and performance characteristics for the needs of the vaccine program from beginning to end. it should be noted that vaccine assay quality is highly dependent upon clinical study execution from collection through final data reporting. sample collection & handling are critical to the quality of the specimen and its ability to be used in an assay. implementing methods to assure the following are keys to successful vaccine clinical trials: proper sample storage and shipping conditions, processing and aliquotting with methods for contamination control, proper training of site and clinical research organization lab personnel, chain of custody verification of samples from collection to final valid test result, quality control checks and good data management. in the execution of vaccine clinical trials, there is the need for testing methodology to be reliable, reproducible (accuracy, specificity, robustness), and occasionally to provide rapid diagnosis (on-site or point-of-care testing, if needed), which contributes to patient care as well as to the understanding of vaccine efficacy or disease epidemiology. the diagnostic assay development response to zikv was quite rapid with the eua approval of different molecular detection assays and five serological assays in roughly months' time (tables and ) and the initiation of efforts to build international reference standards for both assay types. however, at the time of writing, there are still no fully approved diagnostics, no established "gold standard" detection methods nor any fully characterized and accepted international reference standards for zikv. while our understanding of the immune response to zikv has greatly expanded since the start of the most recent outbreak and leverages the years of vaccine research for other flaviviruses, such as dengue, there is still no established immune correlate of protection. other flavivirus vaccines have established immune correlates that are based on neutralization titers and it has been assumed that zikv will, as well [ ] . post-vaccination serum from participants enrolled in our group's gls- zika dna vaccine phase trial protected % of interferon α/β receptor knockout mice (ifnar) in a lethal-challenge model of zikv infection, however this protection was not dependent upon neutralizing antibody titers. other vaccines in clinical development have achieved neutralizing antibody titers in humans which were similar to the titers conferring protection in pre-clinical studies of the vaccines [ , ] . serum from participants in a phase clinical study receiving a purified inactivated zikv vaccine was also able to protect mice in a passive transfer mouse model [ ] . the development and validation of sensitive, specific and standardized zikv immunoassays to better characterize the immune response to vaccination are necessary to work toward the establishment of an immune correlate of protection in humans. efforts are being made through the work of the coalition for epidemic preparedness innovations (cepi) and others to ensure that rapid response mechanisms are in place to address emerging infectious diseases. well-seasoned development teams have accepted the challenges and funding to support building the vaccine design, manufacture and clinical assessment infrastructure needed to save lives in outbreak situations. scientific and quality principles must still apply although speed may be required when developing vaccine or diagnostic assays during an outbreak of a new emerging infectious disease. eid public health and countermeasure programs often have unique challenges for diagnostic and vaccine clinical assay development purposes including: . incomplete understanding of biology or epidemiology for appropriate target selection. lack of available reagent sources; inconsistency in quantity and/or quality of those available. difficulty in obtaining relevant human sample panels for the evaluation of test methods. challenges to produce clear line of sight sourcing and quality from early vaccine development through licensure. although the speed at which the diagnostic or vaccine development field needs to move will be dependent upon the urgency of the emerging infectious pathogen and its impact on human life, biological assay standardization is a critical component and requires three separate activities: • development of relevant, sensitive, specific and preferably quantitative biological assays. use of biostatistics to analyze assay performance data from development to validation to life cycle performance management. • ability to prepare stable and reproducible results over time to support diagnostic or vaccine program needs. if assay performance consistency and quality is not demonstrated, the validity of clinical study results may be questioned. assay standardization for any newly emergent infectious disease will be challenging and will: (i) take time to develop, collect and characterize quality reagents, (ii) to achieve sufficient sources of confirmed positive samples for establishment of serological standards, and (iii) to confirm new molecular standards, though this is more straightforward for molecular assays than serology. any efforts to prepare reagents, collect characterized samples, develop research materials and international standards in advance for eids for whom alerts have already been raised will leave the field better suited to respond in case of emergency. rapid response to an emerging infectious disease-lessons learned from development of a synthetic dna vaccine targeting zika virus safety and immunogenicity of an anti-zika virus dna vaccine-preliminary report vaccines for emerging infectious diseases: lessons from mers coronavirus and zika virus utilization of serologic assays to support efficacy of vaccines in nonclinical and clinical trials: meeting at the crossroads. vaccine nomenclature for immune correlates of protection after vaccination zika virus outbreak on yap island, federated states of micronesia zika virus, french polynesia, south pacific zika virus: history, epidemiology, transmission, and clinical presentation guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study teratogenic effects of the zika virus and the role of the placenta zika virus targets different primary human placental cells, suggesting two routes for vertical transmission zika virus on a spreading spree: what we now know that was unknown in the 's miscarriage associated with zika virus infection zika virus epidemic in brazil. i. fatal disease in adults: clinical and laboratorial aspects zika virus associated deaths in colombia evaluation of a prototype dengue- dna vaccine in a phase clinical trial development of a novel dna syncon tetravalent dengue vaccine that elicits immune responses against four serotypes a west nile virus dna vaccine utilizing a modified promoter induces neutralizing antibody in younger and older healthy adults in a phase i. clinical trial a west nile virus dna vaccine induces neutralizing antibody in healthy adults during a phase clinical trial coimmunization with an optimized il plasmid adjuvant enhances humoral immunity via stimulating b cells induced by genetically engineered dna vaccines expressing consensus jev and wnv e diii induction of potent th -type immune responses from a novel dna vaccine for west nile virus new york isolate (wnv-ny ) development of vaccines against zika virus zika virus structural biology and progress in vaccine development role of zika virus prm protein in viral pathogenicity and use in vaccine development zika virus emergence in mosquitos in southeastern senegal evolutionary enhancement of zika virus infectivity in aedes aegypti mosquitoes assessing seasonal risks for the introduction and mosquito-borne spread of zika virus in europe zika virus vector competency of mosquitoes aedes hensilli as a potential vector of chikungunya and zika viruses outbreak of zika virus infection, chiapas state, mexico, , and first confirmed transmission by aedes aegypti mosquitoes in the americas autophagy and viral diseases transmitted by aedes aegypti and aedes albopictus zika virus: following the path of dengue and chikungunya? lancet spatio-temporal coherence of dengue, chikungunya and zika outbreaks in merida zika might not be acting alone: using an ecological study approach to investigate potential co-acting risk factors for an unusual pattern of microcephaly in brazil viremia and clinical presentation in nicaraguan patients infected with zika virus, chikungunya virus, and dengue virus evidence of perinatal transmission of zika virus complete genome sequence of zika virus isolated from semen probable non-vector-borne transmission of zika virus potential sexual transmission of zika virus detection of zika virus in saliva persistence of zika virus in body fluids-preliminary report comparison of test results for zika virus rna in urine, serum, and saliva specimens from persons with travel-associated zika virus disease-florida potential use of saliva samples to diagnose zika virus infection use of blood donor screening data to estimate zika virus incidence, puerto rico zika virus outbreak and the case for building effective and sustainable rapid diagnostics laboratory capacity globally comparison of four serological methods and two reverse transcription-pcr assays for diagnosis and surveillance of zika virus infection seroprevalence of arboviruses among blood donors in french polynesia unexpected outbreaks of arbovirus infections: lessons learned from the pacific and tropical america evaluation of altona diagnostics realstar zika virus reverse transcription-pcr test kit for zika virus pcr testing molecular and serological techniques to detect co-circulation of denv, zikv and chikv in suspected dengue-like syndrome patients assay optimization for molecular detection of zika virus prolonged detection of zika virus rna in pregnant women simultaneous detection of zika, chikungunya and dengue viruses by a multiplex real-time rt-pcr assay attomolar zika virus oligonucleotide detection based on loop-mediated isothermal amplification and ac susceptometry single-reaction multiplex reverse transcription pcr for detection of zika, chikungunya, and dengue viruses detection of dengue virus genome in urine by real-time reverse transcriptase pcr: a laboratory diagnostic method useful after disappearance of the genome in serum cryptic properties of a cluster of dominant flavivirus cross-reactive antigenic sites flavivirus-induced antibody cross-reactivity a multiplex microsphere immunoassay for zika virus diagnosis serodiagnosis of zika virus (zikv) infections by a novel ns -based elisa devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance dengue viruses are enhanced by distinct populations of serotype cross-reactive antibodies in human immune sera a broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of e protein detection of virus-specific antigen in the nuclei or nucleoli of cells infected with zika or langat virus detection of anti-arboviral immunoglobulin g by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay standardization of immunoglobulin m capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections evaluation of diagnostic tests: dengue approaches for the development of rapid serological assays for surveillance and diagnosis of infections caused by zoonotic flaviviruses of the japanese encephalitis virus serocomplex flaviviruses in europe: complex circulation patterns and their consequences for the diagnosis and control of west nile disease quantitative real-time pcr detection of zika virus and evaluation with field-caught mosquitoes interpretation of nucleic acid and immunoassay test results for suspected zika infection cdc zika testing guidance for healthcare providers fda zika emergency use authorizations table summary molecular assay characteristics fda zika eua table molecular assay performance characteristics fda zika virus diagnostic development high specificity of a novel zika virus elisa in european patients after exposure to different flaviviruses kinetics of anti-zikv antibodies after zika infection using two commercial enzyme-linked immunoassays diagnostic performance of commercial igm and igg enzyme-linked immunoassays (elisas) for diagnosis of zika virus infection evaluation of commercially available zika virus immunoassays advances in diagnosis, surveillance, and monitoring of zika virus: an update world health organization emergency use and listings procedures dna vaccination protects mice against zika virus-induced damage to the testes in vivo protection against zikv infection and pathogenesis through passive antibody transfer and active immunisation with a prmenv dna vaccine genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia future developments in biosensors for field-ready zika virus diagnostics electrochemical biosensors for early stage zika diagnostics role of cell culture for virus detection in the age of technology isolation of infective zika virus from urine and saliva of patients in brazil prolonged detection of zika virus in vaginal secretions and whole blood detection of zika virus in urine prolonged detection of zika virus rna in urine samples during the ongoing zika virus epidemic in brazil congenital cerebral malformations and dysfunction in fetuses and newborns following the to zika virus epidemic in french polynesia detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study collaborative study to evaluate a candidate world health organization international standard for zika virus for nucleic acid amplification technique (nat)-based assays fda fda provides new tools for the development and proper evaluation of tests for detecting zika virus infection current status of zika vaccine development: zika vaccines advance into clinical evaluation preliminary aggregate safety and immunogenicity results from three trials of a purified inactivated zika virus vaccine candidate: phase , randomised, double-blind, placebo-controlled clinical trials safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase clinical trials this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -twp bb authors: becker, pablo d.; guzmán, carlos a. title: community-acquired pneumonia: paving the way towards new vaccination concepts date: journal: community-acquired pneumonia doi: . / - - - - _ sha: doc_id: cord_uid: twp bb despite the availability of antimicrobial agents and vaccines, community-acquired pneumonia remains a serious problem. severe forms tend to occur in very young children and among the elderly, since their immune competence is eroded by immaturity and immune senescence, respectively. the main etiologic agents differ according to patient age and geographic area. streptococcus pneumoniae, haemophilus influenzae, respiratory syncytial virus (rsv) and parainfluenza virus type (piv- ) are the most important pathogens in children, whereas influenza viruses are the leading cause of fatal pneumonia in the elderly. effective vaccines are available against some of these organisms. however, there are still many agents against which vaccines are not available or the existent ones are suboptimal. to tackle this problem, empiric approaches are now being systematically replaced by rational vaccine design. this is facilitated by the growing knowledge in the fields of immunology, microbial pathogenesis and host response to infection, as well as by the availability of sophisticated strategies for antigen selection, potent immune modulators and efficient antigen delivery systems. thus, a new generation of vaccines with improved safety and efficacy profiles compared to old and new agents is emerging. in this chapter, an overview is provided about currently available and new vaccination concepts. the mucosa of the human respiratory tract represents a primary target for a large number of microbial pathogens. typically, colonization is an asymptomatic process, resulting from the interplay between bacterial factors and host clearance mechanisms. clinical illness may result from either the local release of bacterial toxins or the systemic dissemination of the pathogen after breaching the mucosal barrier. in the course of respiratory infections adaptive immune responses could be significantly impaired. this might lead to more severe forms of disease or to super-infections, which in turn complicate the clinical management of the patient. the most severe forms of respiratory infection tend to occur in very young children and among the elderly, in whom immune competence is eroded by immaturity or immunesenescence, respectively. in addition, patients who are immunocompromised, as a result of disease or therapeutic interventions, have the greatest risk of developing a fatal infection. despite the availability of new antimicrobials and effective vaccines, community-acquired pneumonia remains a common and serious illness. in fact, it is a leading contributor to the nearly million deaths occurring each year due to respiratory infections, especially in children from developing countries [ , ] . the main causative agents of pneumonia differ according to the patient age and the geographic area. in addition, there are relatively few comprehensive studies on the specific aetiology of pneumonia [ ] due to (i) overlaps in the clinical manifestations of the different syndromes, (ii) difficulties in establishing the precise aetiology, and (iii) frequent occurrence of co-infections. however, streptococcus pneumoniae, haemophilus influenzae, respiratory syncytial virus (rsv), and parainfluenza virus type (piv- ) have been identified as the main agents responsible for acute respiratory infections in children, whereas influenza virus related pneumonia is the leading cause of disease-related deaths in the elderly. in addition, the availability of new and more sensitive diagnostic tests have contributed to the identification of hitherto unknown lower respiratory pathogens, such as the human metapneumonovirus (hmpv) and novel coronaviruses causing the severe acute respiratory syndrome (sars). significant efforts have been invested in the last two decades to develop new diagnostic tools, to elucidate the molecular mechanisms of microbial pathogenesis and to understand host clearance mechanisms. this resulted in an improved knowledge on host responses to infection and immuno-pathogenesis, which in turn have facilitated the establishment of new prophylactic and therapeutic interventions. however, despite our accomplishments in vaccine development, there are many pathogens for which vaccines or adequate therapies are not available or the existent ones are suboptimal. the main approach applied for vaccine development has radically changed in recent years. whole cell vaccines are systematically being replaced by subunit vaccines, in which purified antigens or their coding genes are exploited in combination with new adjuvants and/or delivery systems. as a result, many of the vaccines under development will exhibit consistently improved stability, safety and efficacy profiles. they will also be amenable for mucosal administration, thereby mimicking natural infections. influenza a viruses are the most commonly responsible for severe respiratory illness in humans, followed by influenza b. the population's susceptibly to infection is renewed annually, because of the rapid antigenic variation of this virus. the antigenic variation is due to the accumulation of point mutations in the two major surface glycoproteins of the virus, haemagglutinin (ha) and neuraminidase (na). this can lead to an antigenic drift of the virus, which often leaves current influenza vaccines outdated and ineffective. antigenic shift can also occur due to the segmented nature of the viral genome that favours the emergence of re-assorted strains, in which an entire glycoprotein can be acquired from a different animal influenza virus. both types of variation represent a critical bottleneck for the establishment of a robust vaccination strategy against influenza. in fact, when an influenza virus with the capacity to spread from person-to-person and a complete new glycoprotein subtype suddenly emerges, a worldwide pandemic outbreak can result [ ] . the earliest vaccines against influenza were whole cell vaccines obtained in the s by inactivating viruses grown in the allantoic cavity of embryonated chicken eggs with formalin. while contemporary inactivated influenza vaccines are still produced in embryonated eggs, improvements in manufacturing have resulted in a highly purified and less-reactogenic detergentsplit product. three viral strains are selected on the basis of the previous year's surveillance data on the most prevalent subtypes, therefore, vaccine composition may vary from year-to-year. vaccination has a high benefit:cost ratio, since influenza-related illness (e.g., hospitalizations and deaths) are effectively prevented [ ] . the world's total vaccine production is approximately million doses, with a maximum capacity of million doses. however, the world health organization (who) estimates that there are about . billion people at high risk for severe influenza outcomes (e.g., elderly over years of age, infants, health care workers, children and adults with underlying cardiopulmonary disease). furthermore, the global infrastructure would not be able to handle the timely manufacturing and distribution of a vaccine for a pandemic outbreak [ ] . one alternative would be to lower the quantity of antigen per dose and add adjuvants to the vaccine formulation, but this needs to be tested in clinical trials [ ] . another solution would be to improve current vaccine production technologies (i.e., egg-derived vaccines). however, there is the limited number of egg producers and viral strains can emerge, which could not be easily adapted to embryonated eggs. to overcome these problems, several pharmaceutical companies have embarked themselves on projects for the development of vaccines produced by growing the virus on cell lines. the influenza virus can be adapted to grow on a variety of mammalian cell lines, including vero, per.c- , and madin-darby canine kidney (mdck) cells [ ] [ ] [ ] . this strategy would also improve the possibility of up-scaling vaccine production in face of a pandemic spread. alternatively, it would be possible to develop a vaccine against any influenza virus, such as the avian h n strain, by using reverse genetics techniques [ ] (see below in advances in vaccinology). cold adaptation was found to be a reliable and efficient procedure for the derivation of live attenuated viral vaccine strains for humans. cold-adapted (ca) virus strains can grow in primary chick kidney cells or embryonated eggs at - °c, however, they exhibit a reduced replication at °c. the process of genetic re-assortment with the transfer of the six internal genes from a stable attenuated ca master donor strain of influenza a or b to the new prevailing wild-type epidemic strain has been used to generate attenuated cold-reassorted vaccines with the proper level of attenuation, genetic stability and immunogenicity, which show low or absent transmissibility [ ] . medimmune and wyeth have developed along these lines a trivalent live ca vaccine (flumist) for intranasal spray delivery, which was licensed in . in contrast, to parenterally-administered vaccines, this formulation triggers immune responses resembling those observed after natural infections [ ] . despite the moderate hemagglutination-inhibiting antibody titres observed in vacinees, flumist showed % efficacy over a -year period in children, including protection against antigenic variants that circulated during the second year [ ] [ ] [ ] . this ca vaccine also stimulated the production of nasal iga, as well as t-cell and interferon responses [ ] . the cell-mediated immunity against virus matrix and nucleoprotein antigens may favour viral clearance and early recovery from illness [ ] . the advisory committee on immunization practices has recommended its use only in persons from to years of age, since side-effects were observed in young children (wheezing, nasal congestion) and there are no data available for elderly [ , ] . despite its remarkable genetic stability, this vaccine has to be kept at - °c. thus, a new heat stable derivative has recently been developed, which showed good efficacy in clinical trials [ ] . a live vaccine based on a master virus strain developed at the institute of applied microbiology (austria) by growing wild influenza virus in vero cells at °c was also demonstrated to be safe, well-tolerated and immunogenic after intranasal immunization in young adults [ ]. a number of subunit-or dna-based vaccines are also in various stages of development. an influenza vaccine formulated in virosomes has been commercialized by berna biotech (inflexal ® v); it contains the surface spikes of the three currently circulating influenza virus strains inserted in vesicle membranes of the three corresponding virus types (for more details see section "pseudoviruses as antigen delivery systems") [ ] . this company has also developed a virosome-based nasal formulation. however, it was withdrawn from the market due to the presence of sideeffects (i.e., bell's palsy), which was assumed to be linked to the presence of the escherichia coli heat labile toxin (lt) as adjuvant. two companies, yeda and bionvax, are also developing a peptide-based influenza vaccine for nasal administration, which showed protective efficacy in humanized mice [ ] . a subunit vaccine containing recombinant ha protein produced using a baculovirus system was successfully tested in a phase ii trial in to -year-old volunteers. an epidermal dna-based influenza vaccine, which contained the ha gene from a/panama/ / delivered by particle-mediated epidermal delivery was also tested in humans by powderject [ ] . serum haemagglutination-inhibition antibody responses were observed in volunteers receiving a single dose of , or g of dna, with the strongest and most consistent responses in subjects vaccinated with the highest dosage. some immunization approaches aim at the development of a universal vaccine with a broad spectrum of protective activity against different influenza strains [ ]. among them, the use of the highly conserved transmembrane m protein of the virion can be mentioned. a recombinant particulate vaccine has been engineered by genetically fusing copies of the m to the hepatitis b core antigen (hbc). the m -hbc fusion protein spontaneously assembled into virus-like particles (vlp), which provided complete protection against a lethal challenge with influenza virus a in mice [ , ] . promising results were also obtained after vaccination with a m peptide conjugated with a neisseria meningitidis outer membrane protein complex (ompc) in monkeys [ ] . the human piv (hpiv) consist of four serotypes, with hpiv- being the second leading cause of bronchitis and pneumonia in infants. no vaccine has been licensed to date against piv, however, several approaches are currently under investigation. the initial attempts to provide protection by using vaccines based on formalin-inactivated viruses failed. subsequent work demonstrated that the glycoproteins haemagglutininneuraminidase (hn) and f, which are responsible for virus attachment and fusion, are able to stimulate the elicitation of neutralizing antibodies in animals. however, their poor immunogenicity in naïve subjects led to the currently favoured approach, which is based on the use of live attenuated piv. live attenuated piv vaccines have been developed from both human and bovine strains, which are amenable for delivery by the intranasal route. candidate vaccines should be able to replicate and induce a protective immune response in young infants, even in the presence of maternally acquired antibodies. two main attenuated strains have been studied in detail. one is the hpiv- strain cp , which was selected after passages of the virus in african green monkey cells at low temperature. the other is a bovine piv (bpiv)- strain, which is antigenically related to the hpiv- , and replicates poorly in humans. both cp and bpiv- have been evaluated in phase i/ii trials in sero-positive and sero-negative children and in young infants. they were found to be over-attenuated in sero-positive children, but immunogenic in sero-negative children and infants [ ] . however, the magnitude of the anti-hn response was lower in children who received the bpiv- vaccine [ ]. this prompted the engineering of chimeric bovine/human piv- candidates (e.g., hpiv-nb strain in which the human nucleocapsid is replaced by the bovine counterpart, or a bpiv- strain that expresses the f and hn proteins of hpiv- ). attenuated, chimeric viruses that contain piv- cp internal genes with the f and hn genes from either piv- or piv- have also been tested in hamsters [ ] . berna biotech is also developing a virosomal formulation of the piv- [ ] . using the successful approach of the influenza vaccine, a formalin-inactivated candidate against the respiratory syncytial virus (rsv) was tested in children in the s. the consequence was the hospitalization of % of vaccinees and two deaths [ ] . moreover, vaccinated children also suffered more severe disease on subsequent exposure to the virus, as compared to unvaccinated controls [ ] . this demonstrated that the elicitation of a strong immune response is not sufficient to confer protection against disease, and can even lead to immuno-pathological reactions. thus, it is essential to stimulate the "right" type of immune response. in the particular case of rsv, host responses play an important role in the pathogenesis of the disease, thereby making the development of a preventive vaccine extremely difficult. in addition, naturally acquired immunity to rsv is neither complete nor long-lasting, and recurrent infections often occur [ ] . however, older children and adults are usually protected, suggesting that protection against severe disease develops after several consecutive infections. passive immunization with rsv-neutralizing immune globulins was also shown to prevent rsv infection in newborns with underlying cardiopulmonary disease [ ] . this demonstrates that antibodies play a major role in protection against this disease, whereas t-cell immunity targeted to internal viral proteins appears to contribute to clearance. although live attenuated vaccines seem to be preferable for immunization of naïve infants, subunit vaccines may be of choice for elderly, high-risk children and pregnant women. candidate subunit vaccines based on purified f proteins (pfp- , - and - ) were demonstrated to be safe and immunogenic, even during pregnancy [ ] . maternal immunization using a pfp-based vaccine could be an interesting strategy to protect infants younger than months of age [ ] . however, no significant protection was reported in a phase iii trial performed on children - years of age with cystic fibrosis after vaccination with a subunit vaccine based on pfp- [ ] . a formulation based on surface glycoproteins f and g together with the virion matrix protein m from rsv-a was tested in healthy adult volunteers in the presence of either alum or polyphosphazene as an adjuvant. short-live neutralizing antibody responses to rsv-a and rsv-b were detected in - % of the vaccinees, suggesting that annual boosting will be needed [ ] [ ] [ ] . the central domain of the g protein of rsv-a is relatively conserved among viruses from the groups a and b. thus, a recombinant vaccine candidate, bbg na, was developed by fusing the g na domain to the albumin binding region of streptococcal protein g. this candidate was shown to be moderate immunogenic in adult human volunteers, but its clinical development was interrupted due to the appearance of purpura in vaccinees [ ] . the main two difficulties associated with the generation of live attenuated vaccines against rsv are over-or under-attenuation of the virus and limited genetic stability. temperature-sensitive (ts), ca and cold-passaged (cp) mutant viral strains have been generated. despite the attenuation shown in adults and sero-positive children, cpts mutants still caused moderate congestion in the upper respiratory tract of sero-negative infants ( - months old) [ ] . recombinant rsv vaccines with deletions in non essential genes (e.g., sh, ns or ns ), which also carry cp and ts mutations in essential genes are currently being evaluated [ ] . through recombinant dna technology chimeric viruses were engineered, which contain the genes of hpiv- surface glycoproteins f and nh together with those of rsv glycoproteins f and g in a bpiv- genetic background. one of these candidates was found to be attenuated and able to induce the elicitation of immune responses against both hpiv- and rsv in rhesus monkeys [ ] . similarly, a bpiv- genome was engineered to express hpiv- f and hn proteins and either native or soluble rsv f protein [ ] . the resulting strain, which induced rsv neutralizing antibodies and protective immunity against rsv challenge in african green monkeys, needs to be tested for safety and efficacy against rsv and piv- in infants. this emerging disease was originally described in the guangdong province of china in . even when the global outbreak of sars was under control in , new infections were reported in persons who had contacts with animals in and [ ] . the typical sars-cov-like virus is not transmitted from animals to humans. however, under certain conditions the virus can evolve into the early human sars-cov, which has the ability to be transmitted from animals to humans or even humans to humans, thereby leading to localized outbreaks of mild disease. the early human sars-cov, under selective pressure in humans, may further evolve into the late human sars-cov, which can cause local or global outbreaks of typical sars [ ] . sars can be easily grown in cell cultures [ ] . thus, there is an urgent need for vaccines, not only to prevent naturally occurring epidemic outbreaks, but also as a tool against the threat of biological weapons. several structural proteins are expressed by sars-cov, including nucleocapsid, envelope and spike (s) proteins [ ] . the latter is a type i trans-membrane glycoprotein, which is responsible for virus binding, fusion and entry, and being the major target of neutralizing antibodies [ , ] . the extracelullar domain of the s protein consists of two subunits, s and s [ ] . the s subunit possess a receptor-binding domain (rbd), which is responsible for viral binding to one of its receptors [ , ] . vector-based vaccines expressing the s protein, as well as dna vaccines encoding full-length s protein have been assessed in preclinical studies [ , ] . when modified vaccinia virus ankara (mva) coding for full-length s protein was administered by either intranasal or intramuscular route, neutralizing antibodies were elicited [ ] . however, vaccination of ferrets resulted in liver damage after challenge, raising some concerns about the safety of this approach [ ] . vaccines formulated using different synthetic peptides encompassing linear b cell epitopes from the s protein, which were identified using sera from convalescent patients, stimulated high antibody titres. nevertheless, none of them triggered the elicitation of neutralizing activity. on the other hand, some studies demonstrated that although antibodies against s protein of the late sars-cov (urbani strain) exhibit neutralizing activity, they can also enhance infection by an early human sars-cov isolate (gd t ) and the civet sars-cov-like viruses. a derivative of the s protein with a truncation at amino acid (aa) fails to cause antibody dependent enhancement of infection, but retains the ability to induce neutralizing antibodies. these findings suggest that the elimination of the putative heptad repeated (hr , aa - ), which is implicated in viral fusion, might abrogate the stimulation of virus infection-enhancing antibodies [ , ] . the use of the nucleoprotein of the coronavirus in a dna vaccination protocol also led to the stimulation of a protective response [ ] . in contrast, protection was not achieved when a recombinant piv- expressing the nucleoprotein alone or together with the matrix protein was used [ ] . this demonstrates that the selection of the delivery system and immunization strategy play a critical role in vaccine efficacy. the human adenoviruses are divided into six subgroups (a-f). the adenovirus can cause large-scale epidemics of acute respiratory disease, and dissemination is especially favoured under conditions in which persons are housed communally. the subgroup a viruses, such as ad , have been associated with pneumonia in immunocompromised patients. neutralizing antibodies directed against the capsid (hexon and fiber proteins) seems to be the main effector mechanism to prevent re-infections by adenovirus [ ] . until , military recruits in usa were administered enteric-coated capsules containing live viruses from the serotypes and . the virus, which was not attenuated if delivered by respiratory route, was able to replicate in the gastrointestinal tract without causing disease, thereby stimulating protective responses in the respiratory tract [ ] . when the vaccine went out of production, outbreaks of respiratory diseases caused by adenovirus reemerged among the military recruits [ ] . since serotypes , , and cause the % of adenovirus associated respiratory disease in young children, the development of a tetravalent vaccine similar to the above mentioned might solve the problem in children [ ] . however, the implementation of a vaccine (live or attenuated) against adenovirus should be carefully evaluated, since recombinant adenoviruses are proposed both as vaccine vectors and as tools for the transfer of foreign genes in gene therapy protocols. polysaccharide-based vaccines against s. pneumoniae in , macleod et al. [ ] reported the protective efficacy of a capsular polysaccharide (ps) vaccine in military personnel during an outbreak of pneumococcal pneumonia. the immunization with purified ps showed a drastically reduced reactogenicity, in comparison with the previously used inactivated whole cell vaccines. this was a major breakthrough, not only in terms of safety, but also because it demonstrated that a specific virulence factor can be purified and effectively implemented for the prevention of an infectious disease, thereby paving the road for modern non toxoid-based subunit vaccines. although the serological correlates of immunity are poorly defined, type-specific anti-capsular antibodies are responsible for protective immu-nity. however, immunity is serotype specific, rendering extremely difficult the development of a universal vaccine. this is in part due to the elevate number of serotypes, the regional variations in dominant serotypes and the lack of updated sero-prevalence data for certain regions. these problems have been partially solved by the use of ps-based polyvalent vaccines. the currently licensed formulations contain serotypes of s. pneumoniae, which cover approximately % of serious pneumococcal disease, but only in western industrialized countries. relatively good antibody responses ( - %) are elicited in healthy adults - weeks after a single intramuscular or subcutaneous immunization [ ] . unfortunately, they are poorly immunogenic in children aged less than years, in immune compromised individuals (e.g., aids patients) and in elderly people with concomitant disease, and they do not induce good immunological memory. randomized controlled trials in healthy elderly and young men also failed to show a beneficial effect against pneumonia [ ] . however, vaccination is recommended for healthy people over years of age to confer protection against invasive disease [ ] . ps-based vaccines can be also used in pregnant women to stimulate the production of antibodies, which are transferred to the foetus via the placenta or to the newborns by breast-feeding. however, it is still a matter of controversy whether maternal vaccination can indeed protect newborns against pneumococcal infections [ ] . the second generation of ps-based conjugate vaccines stimulates stronger antibody responses, even in infants, young children and immune deficient individuals, as well as immunological memory. these vaccines also suppress nasopharyngeal carriage of the pathogen and reduce bacterial transmission in the community leading to herd immunity, which adds considerable value to their implementation. the introduction of these vaccines in usa in resulted in a dramatic decline in the rates of invasive pneumococcal disease [ , , ] . a significant reduction in the incidence rates among non vaccinated individuals was also observed as a result of herd immunity [ , ] . however, the licensed seven-valent vaccine does not contain some of serotypes that cause severe disease in developing countries (i.e., serotypes and ). new conjugate vaccines including more serotypes, such as the ninevalent vaccine (wyeth) and two -valent vaccines (glaxosmithkline and sanofi-pasteur), should provide better serotype coverage. new approaches to develop protein-based subunit vaccines against s. pneumoniae are currently being pursued by different research groups. this is expected to enable the generation of a universal vaccine conferring protective immunity against a large number of serotypes, as well as to avoid the complexity of manufacturing a conjugate vaccine [ ] . there are different pneumococcal candidate antigens, such as the pneumolysin, neuraminidase, autolysin, pneumococcal surface protein a (pspa) and adhesin a (psaa), which are in an early phase of clinical development [ ] . in addition, several promising candidates have been identified, which are currently being tested in pre-clinical experimental models [ ] . among them, the two iron uptake abc transporters of s. pneumoniae (piaa and piua), which trigger protective immunity against invasive pneumococcal disease in mice. through the screening of s. pneumoniae genomic expression libraries with sera from convalescent patients, bacterial surface proteins were identified (e.g., bvh- and bvh- ) that promote the elicitation of protective anti-pneumococcal antibodies in mice [ ] . a recombinant hybrid protein, bvh / v, has successfully been tested in toddlers and elderly volunteers. this candidate vaccine should be able to trigger serotype-independent responses, since the bvh and bvh antigens are common to all serotypes of s. pneumoniae. the major obstacle for developing an effective vaccine against h. influenzae capsular ps was related to the inherently poor immunogenicity of this t-cell-independent antigen. antibody responses against ps are age-related, with extremely poor immunogenicity in infants during the first months of life. unfortunately, this age group exhibits the highest risk for invasive infections caused by h. influenzae. a ps-based vaccine against the h. influenzae type b (hib) was licensed in the united states in , for children more than months old [ , ] . the protective efficacy after licensure studies showed the inefficacy of this vaccine not only in infants, but also in older children [ ] . this problem was solved by the generation of a conjugate hib vaccine. to this end, the hib ps (i.e., polyribosylribitol phosphate; prp) was covalently linked to an immunogenic carrier protein, thereby leading to t-cell-dependent responses against the ps. different conjugate hib vaccines currently exist. these vaccines are hboc, prp-t and prp-omp, which make use of the mutant diphtheria toxin crm , the tetanus toxoid and the outer membrane protein from group b n. meningitidis as carriers, respectively. all of them trigger similar immune responses at the recommended doses. however, the dynamic of the elicited response may vary for each of them [ , ] . efficacy studies of these vaccines showed that they confer protection not only against meningitis, but also against pneumonia [ ] [ ] [ ] . although hib vaccines are highly effective, their cost is still prohibitive for the world's poorest nations. however, with the establishment of the global alliance for vaccines and immunization (gavi), we have moved consistently ahead in making them also available for developing countries. gavi has approved the establishment of a hib initiative to support countries wishing to sustain hib vaccination, as well as those exploring whether their introduction could be considered a priority in the near future. although the introduction of conjugated ps vaccines has significantly decreased the prevalence of invasive hib disease, paediatric infections due to non typeable h. influenzae (nthi) are still highly prevalent. nthi is most often associated with otitis media, sinusitis and bronchitis. in addition, nthi is an important cause of lower respiratory infection in adults with chronic obstructive pulmonary disease (copd). thus, the development of a vaccine against nthi is considered an important goal in public health. in contrast to hib, vaccines against the non-encapsulated nthi strains must be directed against alternative virulence factors. the lipoproteins d and p are widely distributed and antigenically conserved among h. influenzae strains, and also trigger the elicitation of protective immunity in animals vaccinated by mucosal route [ ] [ ] [ ] [ ] . thus, their incorporation in vaccine candidates might facilitate the generation of a universal vaccine against all typeable and non typeable h. influenzae. even in the age of vaccine availability, b. pertussis continues to be a major cause of childhood morbidity and mortality (i.e., approximately million cases and , deaths occur annually worldwide). since the late s, the incidence of whooping cough has dramatically decreased in most developed countries, as a result of widespread immunization. the first vaccine formulations, which are still in use, consist of preparations based on killed b. pertussis. the frequent incidence of minor adverse effects (e.g., fever, protracted crying and local erythematous reactions), as well as concerns raised by reports of serious neurological side-effects, resulted in a decline in vaccine acceptance and use [ ] . this in turn led to a re-emergence of whooping cough and its complications. this serious problem prompted the development of a new generation of acellular vaccines. in japan was the first country to successfully introduce acellular vaccines against whooping cough in its immunisation programme [ ] , leading to a consistent reduction in the reported side-effects. in the mid 's a major phase iii trial of acellular vaccines was undertaken in sweden, at a time when the banning of the whole cell vaccine had resulted in a pertussis epidemic in that country [ ] . the first vaccine trials contained chemically detoxified pertussis toxin (pt) and filamentous haemagglutinin (fha), or detoxified pt alone. the results of these trials showed that whilst producing good antibody responses, the vaccines failed to give an adequate level of protection in infants. the mono-component vaccine conferred no protection against infection, whereas the use of the two component candidate only gave incomplete protection against infection [ ] . the results obtained in japan and sweden stimulated vaccine companies in the usa and europe to establish vigorous research programmes aimed at the development of a new generation of acellular vaccines with higher efficacy. currently available vaccines have incorporated chemically or genetically inactivated pt and additional virulence factors, such as fha, the outer membrane protein pertactin (prn) and fimbrial proteins (fims). the efficacy studies of this second generation of acellular vaccines have demonstrated that they confer levels of protection equivalent to the whole cell vaccines. the advent of improved techniques for antigenic characterisation and the introduction of acellular vaccines containing genetically defined components also resulted in a reduction of lot-to-lot variation in comparison with conventional whole cell vaccines and the acellular formulation originally introduced in japan. however, despite the wide implementation of vaccination campaigns in infants and children, the disease continues to be endemic. in addition, in countries with high vaccine coverage we are now observing a consistent increment in the cases of pertussis in adolescents and adults [ ] [ ] [ ] . these patients can then transmit the disease to infants, thereby now representing a primary reservoir for bacterial transmission and cycling in the community. the above-mentioned observations can be explained by one or more of the following factors: (i) improved detection techniques, (ii) major awareness on the possibility that bacteria may affect these age groups, (iii) vaccinedriven antigenic changes in circulating isolates, and (iv) reduction in vaccine efficacy over time. in this context, concerns have been raised about genetic variation between the strains used for vaccine preparation and circulating isolates. this seems to be true, since the currently used whole cell and acellular vaccines are prepared with strains that were isolated before mass vaccine introduction and show clear mismatches with respect to circulating strains. there is a steady tendency to decrease diversity in recent isolates, together with clonal expansion during epidemic outbreaks [ , ] . over time, at least two surface proteins (pt and prn) may have changed sufficiently to allow for an increase in the incidence of disease. unfortunately, our global information on antigenic variation and disease in adults and adolescent is extremely limited. thus, despite widespread introduction of pertussis vaccines, it is essential to continue surveillance studies and collection of circulating strains. the present view is that successful control of pertussis in the community may require routine immunization of adolescents and adults with the new acellular vaccines, perhaps in combination with the diphtheria and tetanus toxoids (dtap). this intervention might help in turn to reduce the burden of disease and transmission to infants. chlamydia pneumonia is an intracellular bacterium transmitted person-toperson via respiratory droplets. this pathogen is a common cause of pneumonia, with infections usually being oligosymptomatic or asymptomatic in young age groups. however, the rate of asymptomatic carriage in the normal population is unknown. there is also a tremendous gap in our understanding of host response to infections caused by c. pneumoniae. most of the studies have been focused on the development of efficient diagnostic methods. however, less work has been done on vaccine development, and there is a paucity of knowledge on the microbial components which may serve as target antigens. in fact, at present there are no licensed vaccines against c. pneumoniae. however, the potential of different antigens, such as the major omp [ ] have been assessed in experimental animal models. nevertheless, mice vaccinated with omp using a protocol based on priming with dna and boosting with recombinant vlp showed only partial protection [ ] . recent studies also suggested that ctl responses play a role in protection and clearance [ ] . animals immunized with a mini-gene encoding seven h- (b)-restricted ctl epitopes fused to a endothelial reticulum-translocation signal showed protection following intranasal challenge with a virulent c. pneumoniae [ ] . the current view is that multi-component vaccine will be required in order to induce a protective response [ ] . using the promising approach of reverse vaccinology combined with proteomics (see section "reverse vaccinology"), the whole-genome of c. pneumoniae was screened searching for vaccine candidate antigens among exposed and immune accessible surface proteins [ ] . the selected candidates were then expressed in a heterologous system and used in immunization studies. approximately proteins were able to trigger the elicitation of c. pneumoniae-binding antibodies. when tested in secondary screenings, six of them were also able to neutralize bacteria in vitro, and four inhibited systemic dissemination of c. pneumoniae in a hamster model [ ] . moraxella catarrhalis is the third most common bacterial etiologic agent of otitis media in children. furthermore, m. catarrhalis is an important cause of respiratory infections in patients with copd. thus, different studies have been carried out to characterize potential protective antigens. in this context, two major omp (cd and e) have been identified, which are considered prime candidate antigens for vaccine development. these proteins are expressed on the surface and show a high degree of conservation among circulating strains. both omp triggered the elicitation of bactericidal antibodies and protective immunity in preclinical models [ ] . additional candidates are the uspa protein [ ] , which seems to be required for bacterial colonization of the human upper respiratory tract, the iron-induced omp b and lbp, and the iron-repressed omp b [ ] . a conjugate vaccine based on detoxified lipo-oligosaccharide was also tested in mice by intranasal route with encouraging results [ , ] . some of these candidates are planned to be tested in clinical studies soon [ ] . mycoplasmas are commensal microorganisms, as well as opportunistic pathogens. mycoplasma pneumoniae is one of the causative agents of acute and chronic human respiratory diseases and the main responsible for primary atypical pneumonia, accounting for approximately - % of all community-acquired pneumonia [ ] . there is a considerable underreporting for m. pneumoniae-associated diseases. this is in part due to the wide diversity of clinical manifestations, the difficulties associated with its cultivation from clinical specimens and the lack of adequate diagnostic tools. no vaccines are currently available against this pathogen. however, studies conducted in human volunteers in the late s demonstrated that a formalin-inactivated whole cell vaccine and an acellular extract were able to confer moderately protective immunity against m. pneumoniae [ ] . unfortunately, immune pathological reactions were observed following challenge with live organisms. therefore, studies are still needed to understand the underlying mechanisms to the observed autoimmune responses [ ] . more specifically, we need to elucidate the specific role played by humoral and cellular response in protection against m. pneumoniae. m. pneumoniae is one of the smallest self-replicating prokaryotic pathogens (approximately kb). the complete genome sequence is now available. this is expected to expand our knowledge on the physiological and virulence properties of this agent, as well as new hints for vaccine development. a previously unrecognized bacterium was isolated after the outbreak of legionnaires disease in , which was designated legionella pneumophila [ , ] . the spreading of l. pneumophila is increasing due to the use of air-conditioners and humidifiers, since infections can occur by inhalation of aerosolized contaminated water sources. several approaches have been developed in the fight against this facultative intracellular pathogen. infection and immunization induce a rapid increase of antibody titres. however, antibodies do not seem to play a significant role in host resistance, particularly after aerosol challenge [ ] [ ] [ ] . some authors also suggested that these antibodies can promote bacterial phagocytosis, thereby favouring invasion and subsequent intracellular replication [ ] . in contrast, cellular responses appear to be important for protection. different vaccine candidates were tested in the past. heat-, acetone-and formalin-killed l. pneumophila vaccines were not able to confer protective immunity in guinea pigs, whereas animals immunized with l. pneumophila membranes survive an aerosol challenge with virulent bacteria [ , ] . additional work demonstrated that also purified antigens, such as the major secretory protein [ ] , the major cytoplasmatic membrane protein [ ] , the peptidoglycan-associated lipoprotein [ ] , omps [ ] and flagella [ ] can confer protection against challenge with virulent l. pneumophila. finally, different live attenuated mutants of l. pneumophila were used in animal infection models with promising results [ ] . cystic fibrosis (cf) patients are particularly susceptible to severe bacterial infections of the lung, being pseudomonas aeruginosa one of the most prominent etiologic agents. thus, significant efforts have been invested to develop a vaccine against this pathogen. surface ps are among the antigens that were most intensively assessed. berna biotech have developed an octavalent vaccine against the eight most prevalent serotypes based on o-ps conjugated with the exotoxin a [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a consistent reduction in the number of cf patients with chronic p. aeruginosa lung infection was observed in a cohort receiving the basic immunization protocol, followed by yearly boosters over a period of years [ , ] . the conjugate vaccine induced the production of specific igg antibodies and increased the number of igg memory b cells. it is still unclear if cellular responses might contribute to the overall protection conferred by this vaccine. however, strong proliferative responses of lymphocytes with a th phenotype were observed in vaccinated individuals in response to the carrier exotoxin a protein [ ] . alternative vaccination strategies are currently being tested in clinical trials. among them, formulations based on a fusion protein between the outer membrane proteins f and i, which have been administered by parenteral and mucosal routes [ , ] . these formulations were demonstrated to be safe in volunteers and conferred increased protection against p. aeruginosa in cf patients. cell-surface alginate, flagella, components of the type iii secretion system, inactivated toxins and proteases are other proposed target antigens [ ] . some of them are already in clinical trials alone or in combination [ ] . when pasteur returned from his summer holidays in to continue with his studies on chicken cholera, he inoculated chickens with an old culture of pasteurella multocida, which was left during the whole summer on his bench. the animals that received the preparation were protected against a challenge performed with a fresh isolate. thus, pasteur developed the hypothesis that pathogens could be attenuated by exposure to environmental insults (e.g., high temperature, oxygen and chemicals) [ ] . the strategy was then successfully extrapolated for developing anthrax vaccines in livestock in the s, with significant economic benefits. this was followed by the generation of attenuated vaccines against rabies and other important pathogens towards the end of the nineteenth century. pasteur's approach for "attenuating" or "inactivating" a pathogenic organisms still constitutes a cornerstone in vaccine technology [ ] . this exemplifies that until recently the major achievements in vaccinology have been facilitated by technological (e.g., adjuvants, delivery systems, reverse vaccinology, genetic engineering) rather than immunological advances [ ] [ ] [ ] . however, it is expected that the impressive knowledge accumulated in recent years in the fields of immunology, immune pathology and microbial pathogenesis will pave the road to a new golden era in vaccinology, in which knowledge and technology will enable rational vaccine design. in the th century, pertussis vaccines progressed from crude bacterial preparations to the highly purified antigens used for acellular vaccines. a similar quantum jump in technology allowed the development of subunit vaccines against influenza, hib and s. pneumoniae, as well as the production of antigens by recombinant dna techniques (e.g., genetically inactivated pt). despite the fact that these techniques enable the production of almost any foreseeable antigen, the identification of suitable targets still remained as a main bottleneck for vaccine development [ ] . the advent of genomics and its exploitation in the vaccinology field have rendered possible the implementation of a systematic and holistic approach for the screening, identification and prioritisation of candidate antigens. this new approach, called "reverse vaccinology" [ ] , does not require cultivation of the original pathogen, thereby being amenable for highlypathogenic or non culturable micro-organisms. it is possible to predict and select the most promising candidates by the analysis of genomic sequences in silico, which will then be cloned and expressed in heterologous systems. the resulting proteins are then used to perform immunological and/or functional studies to select the most promising candidates (e.g., able to induce the production of microbicidal or neutralizing antibodies, capacity to confer protective immunity). flanking studies are usually carried out, such as molecular epidemiological analysis to assess their degree of conservation among circulating strains, or transcriptional profiling to evaluate their expression during natural infections [ ] . the time-consuming process in which highly expressed components of an in vitro cultivable organism are identified (one at a time) and separated (different components between them) is one of the disadvantages that reverse vaccinology has solved. the conventional method usually requires - years to arrive to a clinical trial, whereas reverse vaccinology reduces the process to approximately years. reverse vaccinology also allows the identification of hundreds of potential candidates in a few days, in comparison with the small number of antigens that conventional approaches have provided after decades of research. moreover, reverse vaccinology offers the possibility to select potential candidates independent of their expression levels or purification easiness. the reverse vaccinology approach has proved its usefulness in the field for both viral and bacterial pathogens (e.g. hepatitis c virus, group b meningococci, group b streptococci) [ , ] . reverse vaccinology has also become an essential tool for several vaccine development projects against agents causing community-acquired pneumonia (e.g., c. pneumoniae, streptococci). the potential and speed of genomic-based approaches was also shown when the nucleotide sequence of the coronavirus causing sars was made available in less than one month. in addition, the increasing number of available genomes from bacteria and viruses would allow comparative genomic studies, thereby providing hints on conserved protein families and/or functional domains. this would facilitate the generation of vaccines using immunogens covering multiple micro-organisms [ ] . despite the incredible potential of reverse vaccinology, this approach also has some important limitations (tab. ). among them is the fact that it is not be possible to identify non-protein antigens (e.g., ps, glycolipids), which are the cornerstone for many successful vaccines (e.g., pneumococcal and hib vaccines). currently available influenza vaccines (see above) are based on inactivated viruses, and, more recently, attenuated ca viruses and virosomes. all these vaccines exploit the same starting material (wild-type virus), which is inactivated or attenuated. the last approach consists in the co-infection of chicken eggs with the new isolate and a master attenuated strain, and subsequent selection for re-assorted viruses with the desired genotype/phenotype. however, the virulence of certain virus strains, such as the h n , renders difficult the implementation of this traditional strategy. the use of reverse genetics represents a valid alternative for the generation of vaccines against rna respiratory viruses, such as the influenza virus, piv and rsv. it consists in the production of the virus from cloned dna [ ] , thereby allowing the development of vaccines against any pandemic viral strain. in some cases (e.g., avian h n ) an additional mutagenesis step would be required to attenuate its virulence [ ] . then, the new ha and na segments would be transferred into an appropriate influenza a virus master strain adapted to grow in a cell line. the final re-assorted virus will have the antigenic specificity of the pandemic strain and the growth characteristics of the master strain [ , ] . this technology would also allow production of the influenza vaccine in cells that are co-transfected with plasmids encoding for different frag- [ ] . therefore, the complete genome is inside the cell and virus can be produced and assembled. one of the main advantages is that a plasmid encoding for ha and na can be easily replaced. therefore, re-assortment and selection become unnecessary. this method would considerably reduce the time for vaccine production, from many months to only a few weeks. another advantage would be the simple manipulation of the genome (contained in plasmids), which would enable detoxification of specific virulence factors. similar approaches can be implemented for other viruses, such as rsv, piv and sars-cov. however, intellectual property and liability issues are still obstacles for the industrial development of reverse-genetics-based vaccines [ ] . furthermore, since the resulting viruses are considered genetically modified organisms, additional problems may arise from the regulatory stand point [ ] . most of the infective agents are either limited to the mucosal membranes, or need to transit across them in order to cause disease. therefore, it is highly desirable to elicit an efficient immune response at the local site in which the first line of defence is laid. the stimulation of a pathogen-specific response at the portal of entry is expected to impair infection (i.e. colonization), thereby reducing the risk of transmission to susceptible hosts. parenterally administered vaccines mainly stimulate systemic responses, whereas vaccines given by the mucosal route mimic natural infections, thereby leading to efficient mucosal and systemic responses. thus, there is a considerable interest in the development of mucosal vaccines. however, antigens administered by this route are usually poorly immunogenic. different strategies are being pursued to overcome this bottleneck, among them can be cited the use of (i) advanced synthetic delivery systems, (ii) live attenuated bacterial or viral vectors, (iii) bacterial ghosts, (iv) pseudoviruses and (v) mucosal adjuvants [ ] [ ] [ ] [ ] . advanced synthetic mucosal delivery systems particulate antigens are more immunogenic than those in solution, due to their vulnerability to degradation by enzymes and extreme ph. thus, it would be helpful to incorporate them into a protective vehicle. often, these vehicles do not serve only to protect them, but can also enhance their uptake, promote targeting to antigen presenting cells and serve as adjuvants [ ] . the most commonly exploited delivery systems are: (i) gelatine capsules, which are dissolved at alkaline ph in the intestine but not in the stomach, (ii) muco-adhesive polymers that are highly viscous inert ps, (iii) eldexomer and carboxymethyl cellulose, which have been used for oral, nasal and vaginal delivery, (iv) lipid-based structures with entrapped antigens, such as immune stimulating complexes (iscoms) and liposomes, and (v) biodegradable micro/nano-spheres based on biocompatible materials such as starch, copolymers of lactic or glycolic acid [ , ] . some of these approaches are currently being explored to develop vaccines against agents causing community-acquired pneumonia. encouraging results have been obtained, among others, using surface antigens from s. pneumoniae encapsulated in micro-spheres [ ] and a iscom-adjuvanted vaccine obtained by reverse genetics against the influenza virus, in preclinical models [ ] . attenuated viruses and bacteria can be used not only as vaccine candidates per se, but also as delivery systems for heterologous antigens. thus, many attenuated microorganisms have been exploited as a scaffold for the development of subunit vaccines against other agents, under the premise that the expression of the recombinant antigen(s) does not increase their pathogenic potential for humans or animals. the most frequently exploited bacterial vectors are attenuated derivatives of salmonella enterica and shigella spp., and the bacille calmette-guérin (bcg). for example, vaccination with an attenuated salmonella expressing the oprf-opri was also shown to be able to confer protection against p. aeruginosa in a murine experimental infection model [ ] . in addition, it was also demonstrated that a recombinant bcgbased vaccine expressing the pspa confers protection against s. pneumoniae in an infection animal model [ ] . the use of commensals represents an alternative to attenuated organisms (e.g., lactobacilli). in this context it was demonstrated that oral administration of lactobacillus expressing proteins from coronavirus can protect against a gastric infection [ ] . thus, this approach has been also proposed to combat sars. promising results were also obtained using x chlamydia psittaci [ ] . on the other hand, different attenuated viruses, such as mva, bovine or attenuated hpiv- and adenovirus can be used as delivery systems for heterologous antigens [ , ] . in fact, mva has recently been exploited for antigens of the sars associated coronavirus [ ] . an alternative approach to the use of live attenuated carriers is given by the use of bacterial ghosts. ghosts are generated by the conditional expression of the lethal lysis gene e from bacteriophage phix in gram-negative bacteria [ ] [ ] [ ] [ ] [ ] . this leads to the formation of a trans-membrane tunnel through the bacterial cellular envelope [ ] . due to the high internal osmotic pressure, the cytoplasm content is expelled through the tunnel, thereby leading to an empty bacterial cell envelope [ ] . the presence of envelope components in the ghosts provides a strong danger signal through the activation of pattern recognition receptors [ ] . in addition, bacterial ghosts are efficiently taken up by antigen-presenting cells, stimulating their maturation and activation [ ] . bacterial ghosts retain all morphological, structural, and antigenic features of the cell wall and can be used as vaccine candidates per se. ghosts can also be externally loaded with purified antigens. alternatively, ghosts can be generated from recombinant bacteria expressing heterologous antigens, hence avoiding the difficulties associated with the purification steps. this technology also offers the possibility to manipulate the topology of the recombinant antigen (e.g., the antigen can be bound to the inner membrane, secreted into the periplasmic space or associated to the surface). encouraging results has been obtained in preclinical models using ghosts expressing chlamydial antigens [ , ] . promising results have been reported using different types of pseudoviruses, such as virosomes and virus-like particles (vlp), which are non-replicating viral-like structures. virosomes are based on the principle of reconstituting empty viral envelopes through integration of viral envelope proteins in liposomes. they offer the versatility of liposomes in terms of lipid composition, with the advantage of including viral membrane proteins. virosomes are produced by disassembling the viral membrane envelope with detergents. then, the viral nucleocapsid is removed by ultracentrifugation before reconstitution (fig. ) . in contrast, vlp exploit the capacity of recombinant viral coat proteins to spontaneously self-assemble, thereby mimicking at structural level the viral capsid. vlp can be isolated after protein expression in eukaryotic cells or by in vitro assemblage from subunits produced in an heterologous system [ ] . their main advantages are the lack the viral genetic material with an "intact" envelope, and the fact that they are significantly more immunogenic than soluble proteins. they can be used as vaccines per se, as well as a delivery system for protein-or nucleic acid based vaccines, or as carriers for small molecules. foreign antigens can be expressed on their surface, or can be simply encapsulated. in addition, amphiphilic adjuvants can be incorporated into their membranes, thereby offering the advantage of combining an adjuvant and the antigen in one entity without a covalent attachment. pseudoviruses are especially attractive for mucosal vaccination protocols, since they offer the opportunity to use the natural route of transmission of the agents. induction of serum antibodies, secretory iga, t helper and ctl responses, and protection against mucosal pathogen challenge has been reported from studies in animals and humans [ ] [ ] [ ] . the virosomes generated using the influenza virus retain membrane fusion properties very similar to the naïve virus. therefore, they are able to deliver material to the cytosol of target cells, offering the possibility to access the mhc class i-restricted pathway of antigen presentation to prime ctl activity [ ] [ ] [ ] . bacterial toxins and their derivatives are among the first molecules that have been used as mucosal adjuvants. they are characterized by the presence of an a moiety with enzymatic activity, and a b moiety that mediates toxin binding to the target cells. cholera toxin and the closely related escherichia coli heat-labile toxin showed potent adjuvant activity when co-administrated with different antigens by the mucosal route [ ] [ ] [ ] . however, their use in humans is hampered by their intrinsic toxicity. thus, mutated derivatives were developed, in which the a subunit was modified to remove the adp-ribosylating activity. the resulting polypeptides retain their adjuvanticity, in the absence of detectable toxicity [ ] [ ] [ ] . however, additional studies have demonstrated that even these derivatives can lead to potential severe side-effects, such as retrograde homing of adjuvant and antigen to neural tissues [ ] . this might explain, at least in part, the side-effects observed after intranasal vaccination against influenza with a virosomes-based formulation containing heat-labile toxin (i.e., bell's palsy), which in turn led to its retraction from the market. however, chimeric derivatives lacking the targeting moiety for neural tissues (i.e., b subunit) are now available [ ] . they might allow the exploitation of the high potential of these molecules for the development of vaccines against respiratory pathogens. in fact, preclinical studies provided the proof-of-concept for the usefulness of derivatives of bacterial toxins in the generation of acellular vaccines against microorganisms, such as s. pneumonia and h. influenzae [ , ] . other bacterial components were also explored for their activity as adjuvants. the monophosphoryl lipid a retains much of the immune stimulatory properties of lps, without the inherent toxicity [ ] . on the other hand, extracellular matrix binding proteins, such as the fibronectin binding protein i of streptococcus pyogenes, also exhibit adjuvant activity [ ] . this offers the possibility of using them as dual antigen/adjuvant moieties in the same formulation. recent reports also demonstrate that vaccine formulations containing adamantylamide dipeptide, a non-toxic compound obtained by linking the l-alanine-d-isoglutamine residue of the muramyl dipeptide to the antiviral drug amantadine, confer protection against non typeable h. influenzae in preclinical models [ ] . the innate immune system plays a critical early role in host defence against pathogenic microorganisms through the recognition of pathogenassociated molecular patterns [ ] . this is achieved through the stimulation of pattern-recognition receptors (prr) that sense a broad range of exogenous and endogenous danger signals [ , ] . toll-like receptors (tlr) represent the best-characterized family of prr. natural and synthetic tlr agonists are being used as immune modulators to optimize responses after vaccination. since the identification of the tlr , many mammalian tlr homologues have been identified (i.e., in humans and in mice) [ ] . each tlr member binds specifically to different ligands (tab. ), alone or in combinations (e.g., heterodimers formed by tlr with either tlr or tlr ). an example of tlr agonist is bacterial dna, but not vertebrate dna, and synthetic oligodeoxynucleotides containing unmethylated cpg motifs. they act on tlr , thereby inducing a strong th responses by activation of dendritic cells [ , ] . cpg motifs have been successfully used as adjuvants in preclinical studies of different candidate vaccines against agents causing community-acquired pneumonia [ ] [ ] [ ] . another important adjuvant with tlr-binding capacities is the mycoplasma-derived macrophage-activating lipopeptide malp- , which act a the level of the tlr heterodimer / [ , ] . malp- promotes a global activation of cells from the innate and adaptive immune system [ , ] , such as macrophages, dc, t-and b-lymphocytes [ , ] . when coadministered with an antigen by either the parenteral or the mucosal route, malp- promotes the elicitation of humoral and cellular responses at systemic and mucosal level [ ] . preclinical studies suggested that malp- could be exploited in vaccine formulations against the sars-associated coronavirus, m. catarrhalis and influenza virus, among others (unpublished data). dna vaccination offers some advantage over the normal antigen vaccination, such as the fact that it is not necessary to express any antigen. in contrast, it is the biosynthetic machinery present in the cells of the vaccinees that takes care of this work. furthermore, since eukaryotic cells are in charge of protein synthesis, their glycosylation and folding are optimal. however, the large-scale purification of dna might be associated with high costs. this can be solved by the use of attenuated or inactivated bacteria or viruses as delivery systems [ ] . this approach can also lead to an enhanced induction of antibodies, which is otherwise poor using conventional naked dna vaccines. we have recently demonstrated that bacterial ghosts can be also exploited as a delivery system for dna vaccines for both in vivo and ex vivo applications [ ] . the potential of this approach is demonstrated by the fact that it is possible to optimize performance by a broad range of manipulations, such as (i) choice of optimal promoters, (ii) use of codon optimized genes for expression in mammalian cells, (iii) addition of nuclear localization signals or ubiquitination signals to improve expression and processing, and (iv) co-delivery of dna constructs coding for immune modulatory molecules [ ] . in addition, by the presence of immune stimulatory cpg motifs, the dna vaccine constructs has built-in adjuvant properties. this vaccination approach is particularly suited for the stimulation of cellular immune responses [ ] . interestingly, several reports suggest that dna vaccines may represent a valid alternative to prime the neonatal immune system, even in the presence of passive transferred maternal antibodies [ , ] . in fact, promising results were also obtained in preclinical models of community-acquired pneumonia, such as influenza [ ] and s. pneumoniae [ ] . furthermore, dna coding for vaccine antigens appears to induce excellent immunological memory, which can be reawakened by later immunization or exposure to the pathogen. the knowledge generated in several basic disciplines, such as immunology and microbial pathogenesis, has allowed the identification of critical bottlenecks for establishing a successful vaccination strategy. it is expected that in the coming years we will develop customized approaches to address each of them, in order to stimulate efficient protection against infective agents under specific clinical settings (i.e., newborns, aging individuals, immunocompromised patients). the importance of immunological memory b lymphocytes that have differentiated into plasma cells are the producers of antigen-specific igg antibodies. bone-marrow (bm) plasma cells have a short life, therefore, the bm reservoir needs to be replenished by the stimulation of memory b cells [ , ] . the maximal life span of bm plasma cells is still debated. only few factors have been identified that control the differentiation of antigen-specific b cells toward short-or long-life plasma cells or to memory b cells [ ] . beside the requirement of cd + t cells, the nature of the antigen [ ] and the dose are also important. higher antigen doses, as well as rapid vaccination schedules (closely spaced vaccine doses) tend to favour the rapid induction of short-term effectors, whereas lower doses of antigens preferentially support the induction of immune memory [ ] [ ] [ ] [ ] . it was demonstrated that neonatal vaccination (priming) and infant boosting might be effective even when pathogen exposure occurs very early in life. in children in whom vaccine-induced hib antibody titres have fallen to undetectable levels, memory is readily demonstrated [ ] . however, immune memory per se is not enough to protect against pathogens that required high levels of neutralizing antibodies. the delay between memory b-cell reactivation and differentiation may limit the ability to interrupt pathogen invasion. therefore, it is important to establish vaccination protocols in which the population is boosted at different ages in order to maintain the required levels of antibodies. this is particularly important in diseases in which antibodies play a central role in microbial clearance or toxin neutralization. in the particular case of community-acquired pneumonia, we should consider that aging individuals are neglected in many vaccination programs. however, the strategies proposed for elderly would be different from those used for small children, since the main factors affecting vaccine efficacy are immune senescence and immaturity, respectively. the attempts to give a rational solution to this issue are discussed in the next sections. the immune system in children immune responses to bacterial and viral antigens usually increase with age in a stepwise manner [ ] . prompt immunization after birth is required to induce active immunity against diseases that may occur early in life. unfortunately, this strategy is limited by the relative immaturity of the neonatal and infant immune system. some factors implicated in this poor response are the limited switch from igm to igg antibodies, impaired complement-mediated reactions and deficient organization of the splenic marginal zone. vaccination studies performed in newborn mice suggested that limited germinal centre reactions may results from the delayed devel-opment of follicular dc and limit plasma cell differentiation [ ] . it was also showed that the neonatal bm has a limited capacity to support the establishment of long-life antibody-secreting plasma cells [ ] . thus, the responses to glyco-conjugates and to most t-cell-dependent antigens are usually affected [ ] . therefore, only few and highly immunogenic vaccines show significant protective efficacy after a single dose in infants. the limited igg responses are extended all over the first year of life. in addition, the immune responses, particularly antibodies, elicited in the first year of life after vaccination rapidly decline [ ] . however, the problem observed in infants in terms of magnitude and duration of immune response does not seem to affect efficient priming. in fact, the immune memory generated in neonates may be recalled later in life [ ] . nevertheless, strategies to generate strong and long-lasting protective responses in infants are still needed. this is in part due to the presence of maternal antibodies, which inactivate and clear the vaccine antigens, thereby rendering difficult the stimulation of an immature immune system [ ] . in addition, the effects of adjuvants reported in adults cannot be extrapolated to neonates [ ] . a potential strategy to overcome these problems would be to implement vaccination during pregnancy, to provide the required antibodies by placenta and later by maternal feeding [ , [ ] [ ] [ ] . this could be complemented with an early priming of the "immature" immune system of the newborn by dna vaccination, followed by a boost during the second half of the first year or later in life [ ] . poly-pathology and multiple organ failure is the rule rather than the exception in aging individuals. thus, many systems are affected (e.g., endocrine, cardiovascular), and the immune system is not an exception. the mechanisms involved in the immune senescence process, which in turn may lead to poor response to vaccination, are not fully understood. however, it is clear that responses against certain vaccines are more affected by immune senescence than others (e.g., ps-based vaccines against s. pneumoniae) [ ] . in contrast, the responses to a boost dose of the anti-tetanus vaccine are hardly affected by age [ ] . a rapid decline of antibody responses, together with a relative restriction of the t-cell repertoire is characteristic of the immune senescence process. this restriction and the reduction in the pool of naïve cells can explain the poor cd + t cell responses against antigens that are cross-reacting with proteins which were seen earlier in life. in contrast, t-cells responses of healthy elderly individuals to new antigens are often unaffected. nevertheless, the overall response to vaccination in the elderly is less efficient than in young adults, making more vigorous approaches necessary (fig. ) . in the case of influenza, the actual strategy is annual re-vaccination. however, there are concerns regarding the capacity to increase antibodies with proper specificity against re-assorted viruses in aging adults who have been repeatedly infected or immunized. after exposure to a new, but cross-reacting antigenic variant, such individuals may respond by producing antibodies. however, these antibodies could be primarily directed against influenza strains, which were encountered earlier in life. figure . factors affecting the responses in young adults and aging individuals after vaccination. the process of immune senescence impairs host response to both infection and vaccination. this critical issue needs to be considered during vaccine design and will require the development of special approaches. for example, individuals previously exposed to the "old" h n influenza strain (i.e., years ago), may respond differently from naïve adults who are vaccinated with a "new" h n strain which have accumulated different mutations. the former might produce antibodies against the ha of the "old" h n strain rather than to the cross-reacting epitopes of the new strain [ ] . this is phenomenon is the so-called "original antigenic sin" [ ] . on the basis of this observations, it was proposed that variations in vaccine efficacy might be due to differences in the antigenic distance between the vaccine strains and the epidemic strains responsible for influenza outbreaks [ ] . however, this hypothesis was not confirmed by epidemiologic studies [ ] . even more, individuals aged years or older who were annually vaccinated showed a significantly reduced mortality risk. therefore, until now, it seems that the antigenic sin does not represent a major practical obstacle in influenza vaccination and additional strategies may not be required. despite the broad availability of vaccines against agents causing community-acquired pneumonia, they still represent an important cause of death, human suffering and economic losses. however, we have dramatically expanded our knowledge on the pathophysiology of diseases caused by respiratory pathogens, their virulence factors and the effector mechanisms responsible for their clearance. it is becoming clearer which microbial components are attractive as vaccine targets, as well as the type of immune response needed to confer protection against disease. thus, it is now possible to address vaccine development using rational rather than empiric approaches. this is facilitated by powerful bioinformatics tools for the accurate prediction of epitopes and proteasome trimming [ ] [ ] [ ] , as well as by the availability of a broad palette of immune modulators and delivery systems. therefore, we can predict that new and improved vaccines against the etiologic agents of community-acquired pneumonia will considerably reduce the global impact of this disease in the coming years. a review of vaccine research and development: human acute respiratory infections estimates of world-wide distribution of child deaths from acute respiratory infections respiratory viral vaccines public health. will vaccines be available for the next influenza pandemic novel generations of influenza vaccines safety, reactogenicity and immunogenicity of madin darby canine kidney cell-derived inactivated influenza subunit vaccine. a meta-analysis of clinical studies development of a vero cell-derived influenza whole virus vaccine influvac: a safe madin darby canine kidney (mdck) cell culture-based influenza vaccine structure of influenza haemagglutinin at the ph of membrane fusion influenza vaccine-live influenza virus: immunity and vaccination strategies. comparison of the immune response to inactivated and live, attenuated influenza vaccines current status of live attenuated influenza virus vaccine in the us evaluation of trivalent, live, cold-adapted (caiv-t) and inactivated (tiv) influenza vaccines in prevention of virus infection and illness following challenge of adults with wild-type influenza a (h n ), a (h n ), and b viruses safety, efficacy, and effectiveness of live, attenuated, cold-adapted influenza vaccine in an indicated population aged - years correlates of ratory syncytial virus purified fusion protein- vaccine in pregnant women sequential annual administration of purified fusion protein vaccine against respiratory syncytial virus in children with cystic fibrosis enhanced pulmonary immunopathology following neonatal priming with formalin-inactivated respiratory syncytial virus but not with the bbg na vaccine candidate safety and immunogenicity of a novel recombinant subunit respiratory syncytial virus vaccine (bbg na) in healthy young adults the immunogenicity, protective efficacy and safety of bbg na, a subunit respiratory syncytial virus (rsv) vaccine candidate, against rsv-b evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy recombinant bovine/human parainfluenza virus type (b/hpiv ) expressing the respiratory syncytial virus (rsv) g and f proteins can be used to achieve simultaneous mucosal immunization against rsv and hpiv parainfluenza virus type expressing the native or soluble fusion (f) protein of respiratory syncytial virus (rsv) confers protection from rsv infection in african green monkeys severe acute respiratory syndrome sars-associated coronavirus angiotensin-converting enzyme is a functional receptor for the sars coronavirus a -amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme a dna vaccine induces sars coronavirus neutralization and protective immunity in mice identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets interaction between heptad repeat and regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses induction of sars-nucleoprotein-specific immune response by use of dna vaccine contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity prevention of adenoviral acute respiratory disease in army recruits: cost-effectiveness of a military vaccination policy current research on respiratory viral infections: third international symposium respiratory bacterial vaccines pneumococcal polysaccharide randomised trial of -valent pneumococcal capsular polysaccharide vaccine in prevention of pneumonia in middle-aged and elderly people. swedish pneumococcal vaccination study group maternal immunization with pneumococcal polysaccharide vaccine in the third trimester of gestation a trial of a -valent pneumococcal conjugate vaccine in children with and those without hiv infection impact of the pneumococcal conjugate vaccine on otitis media pneumococcal conjugate vaccine decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine pneumococcal vaccines: an update on current strategies the epidemiology and prevention of disease caused by haemophilus influenzae type b prevention of hemophilus influenzae type b bacteremic infections with the capsular polysaccharide vaccine the albert lasker medical research awards. prevention of systemic infections, especially meningitis, caused by haemophilus influenzae type b. impact on public health and implications for other polysaccharide-based vaccines haemophilus influenzae type b vaccines: history, choice and comparisons the use of haemophilus influenzae type b-tetanus toxoid conjugate vaccine mixed with diphtheria-tetanus-pertussis vaccine in gambian infants randomised trial of haemophilus influenzae type-b tetanus protein conjugate vaccine [corrected] for prevention of pneumonia and meningitis in gambian infants haemophilus influenzae type b conjugate vaccines: a review of efficacy data large scale, postlicensure, selective vaccination of chilean infants with prp-t conjugate vaccine: practicality and effectiveness in preventing invasive haemophilus influenzae type b infections biological activity of serum antibodies to a nonacylated form of lipoprotein d of haemophilus influenzae enhanced respiratory clearance of nontypeable haemophilus influenzae following mucosal immunization with p in a rat model intranasal immunization with recombinant outer membrane protein p induces specific immune responses against nontypeable haemophilus influenzae intranasal vaccination with recombinant p protein and adamantylamide dipeptide as mucosal adjuvant confers efficient protection against otitis media and lung infection by nontypeable haemophilus influenzae dtp-associated reactions: an analysis by injection site, manufacturer, prior reactions, and dose development of a pertussis component vaccine in japan placebo-controlled trial of two acellular pertussis vaccines in sweden -protective efficacy and adverse events secondary analyses of the efficacy of two acellular pertussis vaccines evaluated in a swedish phase iii trial analysis of bordetella pertussis isolates collected in japan before and after introduction of acellular pertussis vaccines recommendations are needed for adolescent and adult pertussis immunisation: rationale and strategies for consideration polymorphism of bordetella pertussis isolates circulating for the last years in france, where a single effective whole-cell vaccine has been used for more than years comparison of acellular pertussis vaccines-induced immunity against infection due to bordetellapertussis variant isolates in a mouse model analysis of bordetella pertussis populations in european countries with different vaccination policies immunity to chlamydia pneumoniae induced by vaccination with dna vectors expressing a cytoplasmic protein (hsp ) or outer membrane proteins (momp and omp ) dna immunization followed by a viral vector booster in a chlamydia pneumoniae mouse model a cd + t cell heptaepitope minigene vaccine induces protective immunity against chlamydia pneumoniae identification of new potential vaccine candidates against chlamydia pneumoniae by multiple screenings genomic approach for analysis of surface proteins in chlamydia pneumoniae the major outer membrane protein, cd, extracted from moraxella (branhamella) catarrhalis is a potential vaccine antigen that induces bactericidal antibodies evaluation of purified uspa from moraxella catarrhalis as a vaccine in a murine model after active immunization progress toward the development of a vaccine to prevent moraxella (branhamella) catarrhalis infections specific immune responses and enhancement of murine pulmonary clearance of moraxella catarrhalis by intranasal immunization with a detoxified lipooligosaccharide conjugate vaccine synthesis and characterization of lipooligosaccharidebased conjugate vaccines for serotype b moraxella catarrhalis mycoplasmas: sophisticated, reemerging, and burdened by their notoriety inactivated mycoplasma pneumoniae vaccine. evaluation in volunteers mycoplasma pneumoniae and its role as a human pathogen classification of the legionnaires' disease bacterium: legionella pneumophila, genus novum, species nova, of the family legionellaceae, familia nova legionnaires' disease: isolation of a bacterium and demonstration of its role in other respiratory disease vaccination with the major secretory protein of legionella pneumophila induces cell-mediated and protective immunity in a guinea pig model of legionnaires' disease vaccination against legionella pneumophila: serum antibody correlates with protection induced by heat-killed or acetone-killed cells against intraperitoneal but not aerosol infection in guinea pigs induction of protective immunity by legionella pneumophila flagellum in an a/j mouse model legionella pneumophila immunity and immunomodulation: nature and mechanisms major cytoplasmic membrane protein of legionella pneumophila, a genus common antigen and member of the hsp family of heat shock proteins, induces protective immunity in a guinea pig model of legionnaires' disease comparison of responses elicited by immunization with a legionella species common lipoprotein delivered as naked dna or recombinant protein human and guinea pig immune responses to legionella pneumophila protein antigens omps and hsp a live avirulent mutant legionella pneumophila vaccine induces protective immunity against lethal aerosol challenge safety and immunogenicity of a pseudomonas aeruginosa o-polysaccharide toxin a conjugate vaccine in humans characterization of the human immune response to a pseudomonas aeruginosa o-polysaccharide-toxin a conjugate vaccine immunization of noncolonized cystic fibrosis patients against pseudomonas aeruginosa immunization of cystic fibrosis patients with a pseudomonas aeruginosa o-polysaccharide-toxin a conjugate vaccine jr ( ) safety and immunogenicity of pseudomonas aeruginosa conjugate a vaccine in cystic fibrosis effect of high-affinity anti-pseudomonas aeruginosa lipopolysaccharide antibodies induced by immunization on the rate of pseudomonas aeruginosa infection in patients with cystic fibrosis vaccination of cystic fibrosis patients against pseudomonas aeruginosa reduces the proportion of patients infected and delays time to infection cellular immunity in healthy volunteers treated with an octavalent conjugate pseudomonas aeruginosa vaccine safety and immunogenicity of an intranasal pseudomonas aeruginosa hybrid outer membrane protein f-i vaccine in human volunteers mucosal vaccination with a recombinant oprf-i vaccine of pseudomonas aeruginosa in healthy volunteers: comparison of a systemic vs. a mucosal booster schedule application of vaccine technology to prevention of pseudomonas aeruginosa infections vaccines: past, present and future six revolutions in vaccinology can successful vaccines teach us how to induce efficient protective immune responses vaccines, vaccination, and vaccinology reverse vaccinology dna content analysis on microarrays immunoinformatics: mining genomes for vaccine components the development of multi-epitope vaccines: epitope identification, vaccine design and clinical evaluation reverse vaccinology and genomics reverse vaccinology a new approach to an influenza live vaccine: modification of the cleavage site of hemagglutinin generation of high-yielding influenza a viruses in african green monkey kidney (vero) cells by reverse genetics from lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza influenza vaccines generated by reverse genetics influenza: girding for disaster. facing down pandemic flu, the world's defenses are weak mucosal immunity and vaccines bacteria as dna vaccine carriers for genetic immunization mucosal adjuvants and delivery systems for protein-, dna-and rna-based vaccines a novel recombinant multisubunit vaccine against chlamydia poly(lactideco-glycolide) microencapsulation of vaccines for mucosal immunization iscoms, liposomes, and oil-based vaccine delivery systems lipid based particulate formulations for the delivery of antigen cross-protective immunity of mice induced by oral immunization with pneumococcal surface adhesin a encapsulated in microspheres protection of mice against lethal infection with highly pathogenic h n influenza a virus by using a recombinant low-pathogenicity vaccine strain enhanced immunogenicity in the murine airway mucosa with an attenuated salmonella live vaccine expressing oprf-opri from pseudomonas aeruginosa protective humoral response against pneumococcal infection in mice elicited by recombinant bacille calmette-guerin vaccines expressing pneumococcal surface protein a intragastric administration of lactobacillus casei expressing transmissible gastroentritis coronavirus spike glycoprotein induced specific antibody production expression of chlamydia psittaci-and human immunodeficiency virus-derived antigens on the cell surface of lactobacillus fermentum br as fusions to bspa jr ( ) oral immunization with a replication-deficient recombinant vaccinia virus protects mice against influenza evaluation of modified vaccinia virus ankara based recombinant sars vaccine in ferrets new strategies for combination vaccines based on the extended recombinant bacterial ghost system online monitoring of escherichia coli ghost production extended recombinant bacterial ghost system bacterial ghosts as carrier and targeting systems bacterial ghosts as multifunctional vaccine particles dynamics of phix protein e-mediated lysis of escherichia coli the danger model: a renewed sense of self endotoxicity does not limit the use of bacterial ghosts as candidate vaccines delivery of chlamydia vaccines virus-like particles as vaccines and vessels for the delivery of small molecules recombinant norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses chimeric recombinant hepatitis e virus-like particles as an oral vaccine vehicle presenting foreign epitopes papillomavirus pseudovirus: a novel vaccine to induce mucosal and systemic cytotoxic t-lymphocyte responses induction of cytotoxic t lymphocyte activity by fusion-active peptide-containing virosomes delivery of protein antigens to the immune system by fusion-active virosomes: a comparison with liposomes and iscoms virosomes in vaccine development: induction of cytotoxic t lymphocyte activity with virosome-encapsulated protein antigens modulation of the immune response by the cholera-like enterotoxins mucosal adjuvants and anti-infection and anti-immunopathology vaccines based on cholera toxin, cholera toxin b subunit and cpg dna new generation of immune modulators based on toll-like receptors signaling mucosal vaccines: non toxic derivatives of lt and ct as mucosal adjuvants detoxification of cholera toxin without removal of its immunoadjuvanticity by the addition of (sta-related) peptides to the catalytic subunit. a potential new strategy to generate immunostimulants for vaccination mutant escherichia coli heat-labile enterotoxin [lt(r g)] enhances protective humoral and cellular immune responses to orally administered inactivated influenza vaccine cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues induction and recall of immune memory by mucosal immunization with a non-toxic recombinant enterotoxin-based chimeric protein expression and characterization of cholera toxin b-pneumococcal surface adhesin a fusion protein in escherichia coli: ability of ctb-psaa to induce humoral immune response in mice intranasal immunization enhances clearance of nontypeable haemophilus influenzae and reduces stimulation of tumor necrosis factor alpha production in the murine model of otitis media fibronectin-binding protein i of streptococcus pyogenes is a promising adjuvant for antigens delivered by mucosal route innate immune recognition the evolution of vertebrate toll-like receptors toll-like receptor mediates cpg-dna signaling the immunobiology of the tlr subfamily mucosal immunity induced by pneumococcal glycoconjugate safety and immunogenicity of cpg injection as an adjuvant to fluarix influenza vaccine the adjuvant effect of synthetic oligodeoxynucleotide containing cpg motif converts the anti-haemophilus influenzae type b glycoconjugates into efficient anti-polysaccharide and anti-carrier polyvalent vaccines mycoplasmal lipopeptide malp- induces the chemoattractant proteins macrophage inflammatory protein alpha (mip- alpha), monocyte chemoattractant protein , and mip- and promotes leukocyte infiltration in mice isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from mycoplasma fermentans acting at picomolar concentration toll-like receptor -and -mediated stimulation by macrophage-activating lipopeptide induces lipopolysaccharide (lps) cross tolerance in mice, which results in protection from tumor necrosis factor alpha but in only partial protection from lethal lps doses stimulation of human toll-like receptor (tlr) and tlr with membrane lipoproteins of mycoplasma fermentans induces apoptotic cell death after nf-kappa b activation the toll-like receptor ligand malp- stimulates dendritic cell maturation and modulates proteasome composition and activity the mycoplasma-derived lipopeptide malp- is a potent mucosal adjuvant dna vaccines: immunology, application, and optimization* bacterial ghosts are an efficient delivery system for dna vaccines polynucleotide viral vaccines: codon optimisation and ubiquitin conjugation enhances prophylactic and therapeutic efficacy preclinical efficacy of a prototype dna vaccine: enhanced protection against antigenic drift in influenza virus successful nucleic acid based immunization of newborn chimpanzees against hepatitis b virus the potential use of dna vaccines for neonatal immunization cross-reactive protection against influenza a virus by a topically applied dna vaccine encoding m gene with adjuvant protective efficacy of pspa (pneumococcal surface protein a)-based dna vaccines: contribution of both humoral and cellular immune responses protective long-term antibody memory by antigendriven and t help-dependent differentiation of long-lived memory b cells to short-lived plasma cells independent of secondary lymphoid organs on differences between immunity and immunological memory kinetics of protective antibodies are determined by the viral surface antigen haemophilus influenzae type b conjugate vaccine diluted tenfold in diphtheriatetanus-whole cell pertussis vaccine: a randomized trial a randomized trial of alternative two-and three-dose hepatitis b vaccination regimens in adolescents: antibody responses, safety, and immunologic memory dose dependency of antibody response in infants and children to pneumococcal polysaccharides conjugated to tetanus toxoid accelerated hepatitis b vaccination schedule in childhood immunological response to conjugate vaccines in infants: follow up study neonatal and early life vaccinology unresponsiveness to lymphoid-mediated signals at the neonatal follicular dendritic cell precursor level contributes to delayed germinal center induction and limitations of neonatal antibody responses to t-dependent antigens delayed and deficient establishment of the long-term bone marrow plasma cell pool during early life influence of mycobacterium bovis bacillus calmette-guerin on antibody and cytokine responses to human neonatal vaccination transfer of antibody via mother's milk maternal immunization with pneumococcal polysaccharide vaccine in the philippines pneumococcal conjugate vaccines as maternal and infant immunogens: challenges of maternal recruitment the protective efficacy of polyvalent pneumococcal polysaccharide vaccine booster effect of low doses of tetanus toxoid in elderly vaccinees vaccine-induced antibodies to heterologous influenza a h n viruses: effects of aging and "original antigenic sin variable efficacy of repeated annual influenza vaccination annual revaccination against influenza and mortality risk in community-dwelling elderly persons immunoinformatics: bioinformatic strategies for better understanding of immune function. introduction syfpeithi: database for mhc ligands and peptide motifs paproc: a prediction algorithm for proteasomal cleavages available on the www this work was supported in part by grants from the dfg (gu / - ) and the bmbf ("pathogenomik" -competence center for genome research of pathogenic bacteria "pathogenomik", u b) to cag. we are particularly grateful to d. felnerova from etna biotech, who provided us with a micrograph from a transmission electron microscopy of a virosome, and to m. höfle for critical reading of the manuscript. key: cord- - zz x li authors: day, m. j.; horzinek, m. c.; schultz, r. d. title: compiled by the vaccination guidelines group (vgg) of the world small animal veterinary association (wsava) date: - - journal: j small anim pract doi: . /j. - . . .x sha: doc_id: cord_uid: zz x li nan one of the greatest successes of modern veterinary science has been the control of infectious disease through the development and implementation of vaccination programmes. this success is typified by the rapid decline in the prevalence of key canine infectious diseases (caused by canine distemper virus [cdv] , canine adenovirus [cav] and canine parvovirus [cpv] ) following the introduction of efficacious modified live virus vaccines. similar effects relate to the introduction of feline vaccines, with clear reduction in mortality caused by feline parvovirus (fpv; feline panleukopenia) and morbidity caused by feline calicivirus (fcv) and herpesvirus (fhv) infections. however, the success of these vaccines cannot be a cause for complacency, and indeed vaccine-related issues have featured high on the agenda of the veterinary profession over the past decade. there are many challenges remaining in small animal vaccinology, and in the wsava vaccination guidelines group (vgg) was convened with the specific remit of taking a global perspective on issues surrounding the practice of vaccination of dogs and cats. the vgg has met formally on three occasions and corresponded electronically between these meetings, and this document is the result of these deliberations. the vgg guidelines are built on those developed by the american animal hospital association (aaha) canine vaccine task force and the american association of feline practitioners (aafp) feline vaccine advisory panel. based upon a consensus among experts, these recommendations reflect a combination of opinion, experience, and scientific data, published and unpublished. the present vaccination guidelines are intended for the general veterinary practice and the shelter environment; they do not represent a standard of care or set of legal parameters. they have been drafted with the objective of educating and informing the profession and to recommend rational vaccine use for individual pets and dog/cat populations. if vaccination has been so successful, then why is it necessary to continually re-evaluate vaccination practice? there is little doubt that in most developed countries the major infectious diseases of dogs and cats are considered at best uncommon in the pet population, but there do remain geographical pockets of infection and sporadic outbreaks of disease occur, and the situation regarding feral or shelter populations is distinctly different to that in owned pet animals. however, in many developing countries these key infectious diseases remain as common as they once were in developed nations and a major cause of mortality in small animals. although it is difficult to obtain accurate figures, even in developed countries it is estimated that only - % of the pet animal population is vaccinated, and this is significantly less in developing nations. in small animal medicine, we have been slow to grasp the concept of 'herd immunity'that vaccination of individual pet animals is important, not only to protect the individual, but to reduce the number of susceptible animals in the regional population, and thus the prevalence of disease. a second major concept regarding vaccination of dogs and cats has been the recognition that we should aim to reduce the 'vaccine load' on individual animals in order to minimise the potential for adverse reactions to vaccine products. for that reason we have seen the development of vaccination guidelines based on a rational analysis of the vaccine requirements for each pet, and the proposal that vaccines be considered 'core' and 'non-core' in nature. to an extent this categorisation of products has been based on available scientific evidence and personal experience -but concerted effort to introduce effective companion animal disease surveillance on a global scale would provide a more definitive basis on which to recommend vaccine usage. in parallel with the categorisation of vaccines has been the push towards marketing products with extended duration of immunity (doi), to reduce the unnecessary administration of vaccines and thereby further improve vaccine safety. both of these changes have necessitated a frame-shift in the mindset of veterinary practitioners in a culture in which both veterinarian and client have become subservient to the mantra of annual vaccination. the following vgg guidelines are prepared when considering the optimum model of a committed pet owner, willing and able to bring their animal to the veterinarian, for the full recommended course of vaccination. the vgg is aware that there are less committed pet owners and countries where severe financial constraints will determine the nature of the vaccine course that will be administered. in situations where, for example, a decision must be made that an individual pet may have to receive only a single core vaccination during its lifetime, the vgg would emphasise that this should optimally be given at a time when that animal is most capable of responding immunologically, i.e. at the age of weeks or greater. the vgg has additionally considered vaccination in the shelter situation. the guidelines that we have proposed are those that we consider provides the optimum level of protection for these highly susceptible animals. the vgg also recognises that many shelters run with limited financial support which may constrain the extent of vaccination used. the minimum vaccination protocol in this situation would be a single administration of core vaccines at or before the time of admission to the shelter. this document seeks to address these current issues in canine and feline vaccinology, and to suggest practical measures by which the veterinary profession may move towards more rational use of vaccination in these species. the most important message of the vgg is therefore encapsulated in the single strap-line: we should aim to vaccinate every animal, and to vaccinate each individual less frequently the basic immunisation schedule guidelines and recommendations for core (recommended), non-core (optional), and not recommended vaccines for the general veterinary practice are given in table . the vgg considers that a core vaccine is one that all puppies throughout the world must receive in order to provide protection against infectious diseases of global significance. the vgg recognises that particular countries will identify additional vaccines that they consider core. a particular example of a vaccine that may be considered core in only some countries is that against rabies virus. in a geographical area in which this infection is endemic all dogs should be routinely vaccinated for the protection of both the pet and human populations. in some countries, mandatory rabies vaccination is a legal requirement, and is generally also required for international pet travel. non-core vaccines are those that are licensed for the dog and whose use is determined on the basis of the animal's geographical and lifestyle exposure and an assessment of risk-benefit ratios. not recommended vaccines are those for which there is little scientific justification for their use. pup vaccination and the month booster most pups are protected by maternally derived antibodies (mda) in the first weeks of life. in general, passive immunity will have waned by to weeks of age to a level that allows active immunisation. pups with poor mda may be vulnerable (and capable of responding to vaccination) at an earlier age, while others may possess mda at such high titres that they are incapable of responding to vaccination until $ weeks of age. no single primary vaccination policy will therefore cover all possible situations. the recommendation of the vgg is for initial vaccination at to weeks of age followed by a second vaccination to weeks later, and a third vaccination given between to weeks of age. by contrast, at present many vaccine data sheets recommend an initial course of two injections. some products are also licensed with a ' week finish' designed such that the second of two vaccinations is given at weeks of age. the rationale behind this protocol is to permit 'early socialisation' of pups. the vgg recognises that this is of great benefit to the behavioural development of dogs. where such protocols are adopted, great caution should still be maintained by the owner -allowing restricted exposure of the pup to controlled areas and only to other pups that are healthy and fully vaccinated. in immunological terms, the repeated injections given to pups in their first year of life do not constitute boosters. they are rather attempts to induce a primary immune response by injecting the attenuated virus (of modified live virus [mlv] vaccines) into an animal devoid of neutralising antibody, where it must multiply to be processed by an antigen presenting cell and stimulate antigenspecific t and b lymphocytes. in the case of killed (inactivated) vaccines, mda may also interfere with this immunological process by binding to and 'masking' the relevant antigens. all dogs should receive a first booster months after completion of the primary vaccination course. the vgg redefines the basic immunisation protocol as the ensemble of the pup regime plus this first booster. the month booster will also ensure immunity for dogs that may not have adequately responded to the pup vaccination course. dogs that have responded to vaccination with mlv core vaccines maintain a solid immunity (immunological memory) for many years in the absence of any repeat vaccination. following the month booster, subsequent revaccinations are given at intervals of three years or longer, unless special conditions apply. it should be emphasised that the considerations given above do not generally apply to killed core vaccines nor to the optional vaccines, and particularly not to vaccines containing bacterial antigens. thus leptospira, bordetella and borrelia (lyme disease) products require more frequent boosters for reliable protection. serological testing to monitor immunity to canine vaccines antibody tests are useful for monitoring immunity to cdv, canine parvovirus- (cpv- ), canine adenovirus- (cav- ), and rabies virus. antibody assays for cdv and cpv- are the tests of greatest benefit in monitoring immunity, especially after the puppy vaccination series. during recent years, many laboratories have standardised their methodologies for such testing. there are legal requirements for rabies antibody testing for pet travel between some countries. in-practice testing will probably become more popular as soon as rapid, simple, reliable and cost-effective assays are more widely available. a negative test result indicates that the animal has little or no antibody, and that revaccination is recommended. some of these dogs are in fact immune (false-negative), and their revaccination would be unnecessary. a positive test result on the other hand would lead to the conclusion that revaccination is not required. this is why robust yes/no answers must be provided by any assay. with cdv and/or cpv- tests, an animal with a negative result, regardless of the test used, should be considered as having no antibody and susceptible to infection. on completion of the puppy series at to weeks of age, an animal should have a positive test result, provided the serum sample is collected or more weeks after vaccination. seronegative animals should be revaccinated and retested. if it again tests negative, it should be considered a non-responder that is possibly incapable of developing protective immunity. testing for antibody is presently the only practical way to ensure that a puppy's immune system has recognised the vaccinal antigen. vaccines may fail for various reasons: ( ) mda neutralises the vaccine virus this is the most common reason for vaccination failure. when the last vaccine dose is given at $ weeks of age however, mda should have decreased to a low level, and active immunisation will succeed in most puppies (. %). ( ) the vaccine is poorly immunogenic poor immunogenicity may reflect a range of factors from the stage of vaccine manufacture to administration to the animal. for example, the virus strain, its passage history or production errors in the manufacture of a particular batch of product may be a cause of vaccine failure. post-manufacture factors such as incorrect storage or transportation (interrupted cold chain) and handling (disinfectant use) of the vaccine in the veterinary practice, may result in inactivation of an mlv product. ( ) the animal is a poor responder (its immune system intrinsically fails to recognise the vaccinal antigens) if an animal fails to develop an antibody response after repeated revaccination, it should be considered a non-responder. because immunological non-responsiveness is genetically controlled in other species, certain breeds of dogs have been suspected to be poor-responders. it is believed (but unproven) that the high susceptibility to cpv- recognised in certain rottweilers and dobermans during the s (regardless of their vaccination history) was due to a high prevalence of non-responders. in the usa today, these two breeds seem to have no greater numbers of non-responders than other breeds, possibly because carriers of the genetic trait may have died from cpv- . this may not be true for other countries. for example, in the uk and germany, the non-responder phenotype remains prevalent amongst rottweilers. most vaccinated dogs will have a persistence of serum antibody (against core vaccine antigens) for many years. immunologically, this antibody reflects the function of a distinct population of long-lived plasma cells (memory effector b cells). induction of immunological memory is the primary objective of vaccination. for core vaccines there is excellent correlation between the presence of antibody and protective immunity and there is long doi for these products. this correlation does not exist for many of the non-core vaccines and the doi related to these products necessitates more frequent revaccination intervals. antibody tests can be used to demonstrate the doi after vaccination with core vaccines. it is known that dogs often maintain protective antibody to cdv, cpv- , cav- , and cav- for three or more years and numerous experimental studies support this observation. therefore, when antibody is absent (irrespective of the serological test used) the dog should be revaccinated unless there is a medical basis for not so doing. antibody determinations to other vaccine components are of limited value because of the short time period these antibodies persist (e.g., leptospira products) or the lack of correlation between serum antibody and protection (e.g. canine parainfluenza). important considerations in performing antibody tests are the cost and the time to obtain results. the vgg recognises that at present such serological testing has limited availability and might be relatively expensive. however, the principles of 'evidence-based veterinary medicine' would dictate that testing for antibody status (for either pups or adult dogs) is better practice than simply administering a vaccine booster on the basis that this should be 'safe and cost less'. in response to these needs, more rapid, cost-effective tests are being developed. the basic immunisation schedule guidelines and recommendations for core (recommended), non-core (optional) and not generally recommended vaccines for the general veterinary practice are given in table . a particular example of a vaccine that may be considered core in only some countries is that against rabies virus. in a geographical area in which this infection is endemic all cats should be routinely vaccinated for the protection of both the pet and human populations. in some countries, mandatory rabies vaccination is a legal requirement, and is generally also required for international pet travel. in terms of feline core vaccines it is important to realise that the protection afforded by the feline calicivirus (fcv) and feline herpesvirus (fhv) vaccines will not provide the same efficacy of immunity as seen with the feline panleukopenia (feline parvovirus; fpv) vaccines. thus the feline core vaccines should not be expected to give the same robust protection, nor the duration of immunity, as seen with canine core vaccines. although the fcv vaccines have been designed to produce cross-protective immunity against severe clinical disease, there are multiple strains of fcv and it is possible for infection and mild disease to occur in the vaccinated animal. with respect to fhv, it should be remembered that there is no herpesvirus vaccine than can protect against infection with virulent virus, and that virulent virus will become latent and may be reactivated during periods of severe stress. the reactivated virus may cause clinical signs in the vaccinated animal or the virus can be shed to susceptible animals and cause disease in them. as discussed for pups, most kittens are protected by mda in the first weeks of life. however, without serological testing, the level of protection and the point at which the kitten will become susceptible to infection and/or can respond immunologically to vaccination is unknown. this is related to the level of maternal antibody and variation in uptake of mda between litters. in general, mda will have waned by to weeks of age to a level that allows an active immunological response, and an initial vaccination at to weeks of age followed by a second vaccination to weeks later is commonly recommended. many vaccines carry data sheet recommendations to this effect. however, kittens with poor mda may be vulnerable (and capable of responding to vaccination) at an earlier age, while others may possess mda at such high titres that they are incapable of responding to vaccination until sometime after weeks of age. therefore the vgg endorses the recent recommendation made in the aafp guidelines of administering the final kitten dose at weeks or older. all kittens should receive the core vaccines. a minimum of three doses -one at to weeks of age, a second to weeks later and a final dose at weeks of age or older should be administered. cats that respond to mlv core vaccines maintain immunity for many years, in the absence of any repeat vaccination. all cats should receive a first booster months after completion of the kitten vaccination course (this will ensure adequate vaccineinduced immunity for cats that may not have adequately responded to the primary course). following this first booster, subsequent revaccinations are given at intervals of three years or longer, unless special conditions apply. adult cats of unknown vaccination status should receive a single initial mlv core vaccine injection followed by a booster vaccination one year later. cats that have responded to vaccination with mlv core vaccines maintain a solid immunity (immunological memory) for many years in the absence of any repeat vaccination. it should be emphasised that the considerations given above do not generally apply to killed core vaccines nor to the optional vaccines, and particularly not to vaccines containing bacterial antigens. thus chlamydophila and bordetella products require more frequent boosters for reliable protection. any given time. just as the vaccination strategy varies with each individual pet, there is no one-size-fits-all strategy for vaccinating shelter animals. the likelihood of exposure and the potentially devastating consequences of infection necessitate a clearly defined shelter vaccination program. shelter medicine differs from individual care in that it has to practice in an environment where eradication of infectious disease cannot be attained. it is possible, however, to minimise the spread of infections within a high-density, high-risk population and maintain the health of not yet infected individuals. when the overall purpose is to place healthy pets into welcoming homes, the time and effort dedicated to controlling infectious disease is only one of many variables in the complex shelter medicine and husbandry equation. the recommendations provided here attempt to address some shelter-unique issues as they pertain to vaccination and disease control. guidelines and recommendations for vaccines to be used in shelters are given in tables and . if unambiguous documentation of vaccination is provided for an animal at the time of admission to a shelter, there is no reason to revaccinate. the vgg discriminates between a shelter and a boarding kennel/cattery. the latter are facilities where fully vaccinated animals may be temporarily boarded for relatively short periods of time (e.g. when owners are on vacation). it should be a requirement of entry to any such facility that the individual dog or cat is fully vaccinated with core products given according to the guidelines presented herein. the use of non-core vaccines against respiratory infections is also appropriate under these circumstances. in the past, veterinary practice has benefited from the annual administration of vaccines. by encouraging owners to bring their pets yearly for vaccination, veterinarians were able to recognise and treat disease earlier than might otherwise have been the case. in addition, the annual visit provided an opportunity to inform clients of important aspects of canine and feline health care. unfortunately, many clients have come to believe that vaccination is the most important reason for annual veterinary visits. veterinarians are now concerned that a reduction in vaccination frequency will cause clients to forgo the annual visits and that the quality of care will diminish. it is therefore essential that veterinarians stress the importance of all aspects of a comprehensive individualised health care program. emphasis should be placed on a detailed vaccination interview, a comprehensive physical examination by the veterinarian, and individualised patient care. the importance of dental care, proper nutrition, appropriate diagnostic testing and the control of parasites and of zoonotic diseases should also be addressed during evaluation of each pet. behaviour concerns should be discussed, as well as the necessity for more frequent examination of young and geriatric animals. the yearly health care/vaccination interview should assess the need for non-core vaccines for the pet. the practitioner should explain to the client the types of vaccines available, their potential benefits and risks, and their applicability to the particular animal, given its lifestyle and risk of exposure. whilst an animal might not receive core vaccination every year, most non-core vaccines do require annual administration -so owners will continue to see their animal vaccinated annually. the regional incidence and risk factors for various infectious diseases should also be discussed. ways to reduce the impact of acquired disease (e.g., avoiding overcrowding, improving nutrition, and restricting access to infected animals) should also be reviewed. vaccinations should be considered as only one component of a comprehensive preventative health care plan individualised based on the age, breed, health status, environment (potential exposure to harmful agents), lifestyle (contact with other animals), and travel habits of the pet. age has a significant effect on the preventative health care needs of any given individual. puppy/kitten programs have traditionally focused on vaccinations, parasite control, and neutering. today, opportunity exists to incorporate behaviour counselling and zoonotic disease management. for the aging pet, senior care programs are becoming increasingly popular. nutritional, dental disease, and parasite control assessment and counselling should take place on an individualised basis throughout the life of the pet. certain breeds are predisposed to various diseases. early detection (particularly of neoplasia) and management of breed-associated disease can significantly improve the quality of the animal's entire life. pets with chronic medical conditions warrant periodic scheduled medical progress examinations and testing. animals receiving certain medications also warrant therapeutic monitoring of blood levels and/or organ systems. the development of recheck protocols for chronic diseases and medications, which can be included in reminder systems, can greatly improve client compliance and, accordingly, pet care. the environment in which a pet resides can profoundly affect its health status and should be assessed during annual health care visits in order to define risk factors and develop appropriate preventative measures. by determining the extent to which dogs and cats come into contact with other animals in unobserved circumstances, veterinarians can assess the need for non-core vaccinations. dogs that visit kennels, grooming salons, common areas, and wooded, tick-infested areas are potentially at greater risk from certain infectious diseases than dogs that do not frequent these areas. just as the human population has become more mobile, so has the pet population, resulting in potential exposure to infectious agents, parasites, and environmental hazards not found in the home environment. determining past and anticipated future travel during each visit allows for greater individualisation of preventative care and diagnostic testing plans. at the time of vaccine administration, the following information should be recorded in the patient's permanent medical record: d date of vaccine administration, d identity (name, initials, or code) of the person administering the vaccine, d vaccine name, lot or serial number, expiry date, and manufacturer d site and route of vaccine administration. the use of peel-off vaccine labels and stamps that imprint the medical record with the outline of a pet facilitates this type of record keeping which is mandatory in some countries. adverse events should be recorded in a manner that will alert all staff members during future visits. informed consent should be documented in the medical record in order to demonstrate that relevant information was provided to the client and that the client authorised the procedure. at the very least, this notation should indicate that a discussion of risks and benefits took place prior to vaccination. adverse events are defined as any side effects or unintended consequences (including lack of protection) associated with the administration of a vaccine product. they include any injury, toxicity, or hypersensitivity reaction associated with vaccination, whether or not the event can be directly attributed to the vaccine. adverse events should be reported, whether their association with vaccination is recognised or only suspected. a vaccine adverse event report should identify the product(s) and animal(s) involved in the event(s) and the individual submitting the report. reporting field observations of unexpected vaccine performance is the most important means by which the manufacturer and the regulatory agency are alerted to potential vaccine safety or efficacy problems that may warrant further investigation. the purpose of pre-licensure safety studies is to detect relatively common adverse events. rare adverse events will be detected only by post-marketing surveillance through analysis of reported adverse events. adverse events should be reported to the manufacturer and/or the local regulatory authority. the vgg recognises that there is gross under-reporting of vaccine-associated adverse events which impedes knowledge of the ongoing safety of these products. the vgg would actively encourage all veterinarians to participate in such surveillance schemes. if a particular adverse event is well documented, reporting serves to provide a baseline against which future reports can be compared. in addition, reported adverse events can lead to detection of previously unrecognised reactions, detection of increases in known reactions, recognition of risk factors associated with reactions, identification of vaccine lots with unusual events or higher numbers of adverse events, and can further stimulate clinical, epidemiological, or laboratory studies. therefore, veterinarians are encouraged to report any clinically significant adverse event occurring during or after administration of any licensed vaccine. reporting a vaccine adverse event is not an indictment against a particular vaccine; it facilitates review of temporally associated conditions and adds to the safety database of the product. non-core. vaccination should be restricted to use in geographical areas where a significant risk of exposure has been established or for dogs whose lifestyle places them at risk. these dogs should be vaccinated at to weeks of age, with a second dose - weeks later, and then at intervals of - months until the risk has been reduced. this vaccine is the one least likely to provide adequate and prolonged protection, and therefore must be administered annually or more often. protection against infection with different serovars is variable. this product is associated with the greatest number of adverse reactions to any vaccine. in particular, veterinarians are advised of reports of acute anaphylaxis in toy breeds following administration of leptospirosis vaccines. the administration of rabies vaccine will be determined by whether the shelter is in a country in which the disease is endemic, and by local statute the version of these guidelines printed in this issue of the journal of small animal practice includes only the main text and tables of the guidelines document. the original document also includes (as appendices) individual fact sheets for the core canine and feline vaccines, and a series of frequently asked questions on canine and feline vaccination. it is hoped to print these in a subsequent edition of the journal but, from september , the entire vgg document will be available on the wsava web-site -http://www.wsava.org these wsava vaccination guidelines are reproduced here with permission from dr brian romberg, president of the wsava. the administration of rabies vaccine will be determined by whether the shelter is in a country in which the disease is endemic, and by local statute the vgg does not recommend the use of other feline vaccines in the shelter situation aaha canine vaccine guidelines the american association of feline practitioners feline vaccine advisory panel report the work of the vaccination guidelines group has been generously sponsored by intervet. the vgg is an independent group of academic experts who have formulated these guidelines without consultation with industry. key: cord- -tfcerilc authors: rao, srinivas; kong, wing-pui; wei, chih-jen; yang, zhi-yong; nason, martha; styles, darrel; detolla, louis j.; sorrell, erin m.; song, haichen; wan, hongquan; ramirez-nieto, gloria c.; perez, daniel; nabel, gary j. title: multivalent ha dna vaccination protects against highly pathogenic h n avian influenza infection in chickens and mice date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: tfcerilc background: sustained outbreaks of highly pathogenic avian influenza (hpai) h n in avian species increase the risk of reassortment and adaptation to humans. the ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. while vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. methodology / principal findings: the ability of dna vaccines encoding hemagglutinin (ha) proteins from different hpai h n serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous hpai h n strain challenge in mice and chickens after dna immunization by needle and syringe or with a pressure injection device. these vaccines elicited antibodies that neutralized multiple strains of hpai h n when given in combinations containing up to has. the response was dose-dependent, and breadth was determined by the choice of the influenza virus ha in the vaccine. monovalent and trivalent ha vaccines were tested first in mice and conferred protection against lethal h n a/vietnam/ / challenge weeks after vaccination. in chickens, protection was observed against heterologous strains of hpai h n after vaccination with a trivalent h serotype dna vaccine with doses as low as µg dna given twice either by intramuscular needle injection or with a needle-free device. conclusions/significance: dna vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. in addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. the highly pathogenic h n influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. the greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. an effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers. dna vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, sars, siv and hiv. in addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained t cell responses [ ] [ ] [ ] , , ] . dna vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle (against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis) [ , ] , pigs (against classical swine fever virus and mycoplasmosis) [ ] , and horses (against west nile virus and rabies) [ ] . in addition, dna vaccines have been tested against avian plasmodium infection in penguins [ ] and against influenza and infectious bursal disease in chickens [ , , ] , duck hepatitis b virus in ducks [ ] , and avian metapneumovirus and chlamydia psittaci in turkeys [ , ] (reviewed in ref. [ ] ). while they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity h n strain [ ] , there is only one previous report of a monovalent dna vaccine effective against h n (and that only against a matched h n isolate) [ ] ; no protection with multivalent dna vaccines against heterologous strains has been reported. development and characterization of a dna vaccine modality for use in poultry offers a potential countermeasure against hpai h n avian influenza outbreaks. the virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species [ ] [ ] [ ] . while there is marked diversity in the host range of type a influenza viruses, many experts have speculated that a pandemic strain of type a influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment [ , ] . thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses [ , , ] . in undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals [ ] so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies. in this study, we used an automated high capacity needle-free injection device, agro-jeth (medical international technology, inc., denver, co) to explore the feasibility of dna vaccination of poultry. after optimization of injection conditions, alternative multivalent dna vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of hpai h n infection. the findings suggest that it is possible to develop a multivalent dna vaccine for poultry that can protect against multiple hpai h n strains and that could keep pace with the continued evolution of avian influenza viruses. immunogenicity and neutralizing antibody specificity of alternative ha dna vaccines in mice to evaluate the efficacy of multivalent dna vaccines, initial studies were performed in mice. expression vectors encoding has from ten phylogenetically diverse strains of influenza viruses [ ] were generated by synthesis of cdnas (see materials and methods) in plasmid expression vectors, pcmv/r or pcmv/r kb, which mediates high level expression and immunogenicity in vivo [ , , ] . animals were immunized with each expression vector intramuscularly (im) at three week intervals, and the antisera were evaluated on day after the third immunization for their ability to neutralize hpai h n pseudotyped lentiviral vectors as previously described [ , ] . we have previously shown that lentiviral assay inhibition (lai) yields similar results to microneutralization and hai analyses with higher sensitivity in mice [ , ] to determine whether immunization with multiple has simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of ha dna vaccine immunogens was administered im at proportionally lower concentration ( . mg per immunogen) into groups of mice (see materials and methods). remarkably, despite a log lower dna concentration of each component, significant neutralizing antibody titers were generated to each of the immunogens, with . % neutralization against out of h ha pseudoviruses at dilutions of up to : ( fig. a) . to evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of immunogens were selected, based on the phylogenetic diversity of ha among the avian influenza viruses [ ] and the crossreactivity of the neutralizing antibody responses of select individual immunogens (fig. ). as expected, there were substantial differences in the breadth of neutralization between these two sets of immunogen multivalent vaccines (fig. , b vs. c). in one set, while neutralization of homologous strains was comparable to the monovalent and the immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against a/iraq protection of dna-vaccinated mice against challenge with heterologous h n a/vietnam/ / influenza virus mice immunized as described above were challenged with a heterologous h n virus weeks after the final dna vaccination. animals were then challenged with ld of the highly pathogenic a/vietnam/ / virus intranasally, and morbidity and mortality were monitored for days after the viral challenge. the control animals, injected with the plasmid expression vector with no insert, died within days of infection. complete survival was observed in the groups immunized with the component and set of the component multivalent dna vaccines (fig. ) . immunization with ha derived from the a/ indonesia/ / strain or set of the component multivalent dna vaccine showed a survival rate approaching %. in contrast, animals injected with ha plasmid dna derived from a/ anhui/ / , which has diverged more from a/vietnam/ / , showed a lower percent survival ( %) after lethal viral challenge. survival differences between groups were assessed using a log-rank test and the gehan-wilcoxon test on the survival curves for pairs of groups. a test was deemed significant if the pvalue was , . . mice injected im with different has, a/ indonesia/ / , a/anhui/ / , ha, ha (set ), or ha (set ) showed a significant difference compared to control (all p values, . ). among the ha-immunized groups, there was no significant difference between any two groups (p. . for all comparisons). for example, no significant difference was observed between the a/anhui/ / group, which had the least survival among the ha immunized groups ( out of ), and other ha groups: a/indonesia/ / (p = . ), ha (p = . ), ha (set ) (p = . ), or ha (set ) (p = . ). therefore, we cannot exclude the possibility that the deaths in the a/anhui/ / group may have been due to random chance. since it is desirable to confer protective immunity in poultry and ha dna vaccination was effective in mice, we next examined the breadth and potency of single or multiple ha plasmid immunization in chickens. the ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies (a/vietnam/ / , a/anhui/ / and a/indonesia/ / ), administered individually or in combination, by different injection methods. in addition to needle injection, a needle-free repetitive injection device, agro-jeth (medical international technology, inc., denver, co), was analyzed. this device disperses the . to ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a co gas pressure plunger [ ] . the injection conditions were determined by histologic analysis of tissues that received injections of india ink; a pressure of psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues (fig. s ). immunization of chickens with the control plasmid (cmv/r) without an ha gene insert elicited minimal neutralizing antibody titers compared to ha-immunized animals week after dna immunizations. nearly all chickens immunized with either monovalent or multivalent ha dna vaccines generated significant neutralization titers ( fig. and table s ). in general, there was a progressive increase in the amount of neutralization after each successive dna vaccination (data not shown) with maximal response at week after the rd dna immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the agro-jeth device. neutralization of the indonesia ha strain was the most robust, with neutralization nearing % at titers greater than : . both the monovalent and multivalent vaccines elicited robust homologous ( vaccine (fig. ). even though one chicken ( ) in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera. to determine whether chickens immunized with single or multiple dna vaccines were protected from a lethal challenge of a heterologous hpai h n virus, vaccinated chickens were in panels b and c, mice (n = ) were immunized with mg of plasmid ( mg each) three times at week intervals. serum pools from the immunized animals were collected days after the third immunization. the antisera were tested against the indicated pseudotyped lentiviral vectors at varying dilutions. error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. in general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. doi: . /journal.pone. .g inoculated with ld of highly pathogenic a/vietnam/ / heterologous virus intranasally using standard methods [ , ] and monitored for morbidity, mortality, viral shedding and serum antibodies. while all the control animals died within days of infection, % survival was noted in the rest of the chickens (fig. a ). the animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day . there was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens - days after challenge as determined by embryonal inoculation (data not shown: egg infectious dose (eid ) limit of detection , virus particles). to compare the relative efficacy of dna vaccines delivered im by needle and syringe versus the needle-free agro-jeth device injection, a dose-response study was performed with amounts of dna vaccine ranging from to . mg with two inoculations. in these experiments, the ha derived from a/chicken/nigeria/ / was substituted for a/vietnam/ / since it represented a more contemporary isolate. the observed rate of protection was higher among the animals receiving mg by agro-jet ( / ) than by im injection ( / ) (fig. , b vs. c). both modes provided complete protection for all animals at doses higher than this, and % protection for the animals receiving . mg doses (fig. b, c) . survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. a test was deemed significant if the p-value was ,. , and marginally significant if the p-value was ,. but .. . chickens injected im showed a marginally significant difference between . and mg (p = . ). in the same group there was a significant difference between control and , and mg (p,. for all comparisons) and the difference between control and . mg was marginally significant (p = . ). chickens that were injected using agro-jeth showed a significant difference between . and mg (p = . ) and between control and , , and mg (p,. for all comparisons). there were no differences between control and . mg or between , , and mg. lastly, the survival differences between agro-jeth and im for each dose group were not significant. the neutralizing antibody response to homologous and heterologous has corresponded with protection and correlated with dose, with higher titers elicited by injection with agro-jeth compared to needle (table s ) . we assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge (week ). while we noted some embryonic lethality at the . mg dose, there was no embryonic lethality at , or mg groups (data not shown). since the hpai h n virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in asia, africa, and europe [ , [ ] [ ] [ ] [ ] . in addition to its effects on human health by crossspecies transmission [ , , ] and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. the pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. while the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission [ , ] , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. one approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. we have previously reported that dna vaccines encoding ha can confer protection against a highly lethal human pandemic influenza virus, the h n virus, in mice [ ] . dna vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models [ , , , , ] . because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. there is evidence that dna vaccination elicits cell-mediated immunity against influenza ha in addition to inducing an antibody response [ ] , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous h influenza strains. a multivalent h vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. dna vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. the mutations promote a focused and enhanced immune response [ , , ] that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. the use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains. a more broadly protective murine vaccine was developed here by including more has from varying strains in the multivalent vaccine (figs. and ) . however, it is less practical to include large numbers of different has in one vaccine due to the cost and complexity of manufacturing such a vaccine. therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. a trivalent vaccine was subsequently identified for further studies. due [ ] . while three dna immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice [ ] , we found that effective protective immunity could be induced with two dna vaccinations and as little as mg trivalent dna immunization using the id/sc route with the agro-jeth device. in addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as mg of dna. the device's capacity for rapid repetitive injection and the lower quantity and stability of dna enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals. a/vietnam/ / (h n ) (a/vn/ / ) was obtained from the repository at the centers for disease control and prevention (cdc), atlanta, georgia. the virus was propagated in -day old embryonated chicken eggs at uc and stored at uc until use. the virus was titrated by the reed and muench method to determine eid [ ] . genbank abd ) were synthesized using human-preferred codons (geneart, regensburg, germany) [ ] . ha cdnas from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pcmv/r or pcmv/r kb, which mediates high level expression and immunogenicity in vivo [ , , ] . for initial trivalent immunizations in chickens, the a/vietnam/ / , a/anhui/ / and a/indonesia/ / strains were used and in the dose response study, the vietnam strain was replaced with a/chicken/nigeria/ / . the immunogens used in dna vaccination contained a cleavage site mutation (pqrerrrkkrg to pqretrg) as previously described [ , ] . this mutation was generated by site-directed mutagenesis using a quickchange kit (stratagene, la jolla, ca). dna immunization of mice [ ] [ ] [ ] week old female balb/c mice were purchased from the jackson laboratory and maintained in the aaalac-accredited vaccine research center animal care facility (bethesda, md) under specific pathogen-free conditions. all experiments were approved by the vaccine research center animal care and use committee. the mice were immunized as previously described [ ] . briefly, mice ( animals for all test groups, animals for the the study was carried out in the aaalac-accredited animal facility at the university of maryland school of medicine. six groups of one-day-old male and female spafas white leghorn chickens, gallus domesticus, were obtained from charles river laboratories (connecticut). the animals were housed in brooder and grower cages (mcmurray hatcheries, iowa). feed (teklad japanese quail diet - , harlan-teklad, wi) and water were provided to the animals ad libitum. the study was performed in strict accordance with the ''guide'' after approvals from the animal care and use committees of the vaccine research center, nih and the university of maryland. dna immunizations were performed as described at , and weeks. a total dose of mg of one or a combination of the following dna plasmids in a volume of ml was administered to each animal: pcmv/ r, pcmv/r-ha agro-jeth is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. the device is semiautomatic and requires a small co tank or compressed air for low pressure delivery. upon trigger activation, co disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. we used an effective volume of . ml in our injectate [ ] . in this study we were able to effectively deliver . ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of - psi. sixty-eight weeks after the last immunization, female balb/c mice were lightly anesthetized with ketamine/xylazine and inoculated intranasally with ld of a/vietnam/ / virus diluted in phosphate-buffered saline in a ul volume. mice were monitored daily for morbidity and measured for weight loss and mortality for days post infection. any mouse that had lost more than % of its body weight was euthanized. all experiments involving the hpai virus were conducted in an aaalac accredited facility (bioqual inc., gaithersburg, md) under bsl conditions that included enhancements required by the usda and the select agent program. white leghorn chickens were challenged one week after the last immunization with lethal dose (ld ) of a/vietnam/ / (h n ) influenza a virus, equivalent to eid based on previous challenges [ ] . chickens were infected with ml virus intranasally. tracheal and cloacal swabs were collected days and post-challenge and stored in glass vials containing bhi medium (bbl tm brain heart infusion, becton dickinson) at uc. blood was collected days post-challenge and serum was titered by microneutralization assay. chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. chickens that survived the study were bled and humanely euthanized at day post-challenge. lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. experiments were carried out under bsl + conditions with investigators wearing appropriate protective equipment and compliant with all institutional animal care and use committee-approved protocols and under animal welfare act regulations at the university of maryland, college park, maryland. representative tracheal and cloacal swabs were chosen to run an eid assay for comparison and virus titers were determine by the method of reed and meunch [ ] . briefly, swabs were used to infect day-old embryonated chicken eggs in -fold dilutions. three eggs were inoculated per dilution and incubated for hours before titration. neutralizing antibodies were titrated from serum samples collected week and post-vaccination and day post-challenge. the microneutralization assay was performed using a -well plate format. serum was treated with receptor-destroying enzyme (denka seiken co.) and treated at uc per the manufacturer's instructions. after an overnight incubation and subsequent inactivation samples were brought to a final dilution of : using pbs and each sample was serially diluted and virus, diluted to tcid , was added to each well. the plates were then incubated at uc, % co for - hours. following incubation, supernatants were used to infect a second -well plate of mdck cells. microplates were incubated at uc for minutes and then uc, % co for minutes. supernatants of serum and virus were then discarded and ml of optimem (containing x antibiotics/antimycotics, mg/ml tpck-trypsin) was added and incubated at uc, % co for days. after days, ml of the supernatant from each well was transferred into a new -well microplate, and an ha assay was performed to calculate the antibody titers. virus and cell controls were included in the assay. two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described [ ] for the presence of antibodies that neutralized the infectivity of tcid ( % tissue culture infectious dose) of the a/vietnam/ / h n virus on mdck cell monolayers by using two wells per dilution on a -well plate. the recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [ , ] . for the neutralization assay, antisera from immunized animals were heat-inactivated at uc for minutes and mixed with ml of pseudovirus at various dilutions. the sera/virus mixture was then added to a cells in -well b&w tc isoplates (wallac, turku, finland; , cells/well). two hours later, the plates were washed and fresh medium was added. cells were lysed in mammalian cell lysis buffer (promega, madison, wi) hrs after infection and luciferase activity was measured using the luciferase assay system (promega, madison, wi). the following strains were used for the production of pseudotyped viruses: for ha we used a/thailand/ (kan- the ha/hi titers were determined as previously described [ ] . briefly, ha titers were calculated using ml of . % chicken red blood cell suspension in pbs added to ml of twofold dilutions of virus in pbs. this mix was incubated at room temperature for minutes. the ha titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. hi titers were calculated by titrating ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of a/vietnam/ / virus (four hemagglutinating doses) was added to each well. wells were incubated at room temperature for minutes and ml of a . % suspension of chicken red blood cells was added. hi titers were calculated after minutes as the reciprocal of the serum dilution that inhibited hemagglutination. table s hemagglutination inhibition (hi), microneutralization titer (nt), and lai of sera from individual chickens immunized with different vaccines. sera from immunized animals were obtained at week or , a week before or after the final boost, and neutralization was assessed by hi, microneutralization (nt) and lai (shown as ic ). individual animal serum of each group is shown and was analyzed as described in the materials and methods section. figure s characterization of needle-free (agro-jeth) dna immunization in chickens. to evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by agrojeth, or week old chickens were injected with a solution containing india ink with this needle-free device at various pressures, ranging from to mm hg. three sites (thigh, wing and breast) were used, and biopsies were taken for routine hematoxylin and eosin staining. representative sections of thigh injections are shown from week old chickens and were similar at weeks (data not shown). while the mm hg pressure deposited the injectate into the dermis/subcutaneous region (left), the higher pressure injections, and mm hg, deposited the injectate into the subcutaneous and muscle layers (middle, right). mm hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all agrojeth immunizations. found at: doi: . /journal.pone. .s ( . mb doc) the plasmid combination group ( ha) received . mg dna for each of the ha plasmids (total mg) as used in the single plasmid groups mentioned above. for the two plasmid combination groups pcmv/r kb-ha biological features of genetic immunization dna vaccines dna vaccines: immunology, application, and optimization strategies for inducing protection against avian influenza a virus subtypes with dna vaccines a dna vaccine induces sars coronavirus neutralization and protective immunity in mice immunotargeting with cd (cd ligand) enhances dna vaccine responses in ducks development and application of reference antisera against hemagglutinin subtypes of influenza virus by dna vaccination of chickens effects of dda, cpg-odn, and plasmid-encoded chicken ifn-gamma on protective immunity by a dna vaccine against ibdv in chickens principles for vaccine protection in chickens and domestic waterfowl against avian influenza: emphasis on asian h n high pathogenicity avian influenza association of serologic and protective responses of avian influenza vaccines in chickens effect of plasmid dna vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques route and method of delivery of dna vaccine influence immune responses in mice and non-human primates needle-free injection of dna vaccines: a brief overview and methodology a dna prime-mycobacterium bovis bcg boost vaccination strategy for cattle induces protection against bovine tuberculosis immune response in mice and cattle after immunization with a boophilus microplus dna vaccine containing bm gene immunization of pigs to prevent disease in humans: construction and protective efficacy of a salmonella enterica serovar typhimurium live negative-marker vaccine humoral response to west nile virus vaccination in alpacas and llamas preliminary results of an anticircumsporozoite dna vaccine trial for protection against avian malaria in captive african black-footed penguins (spheniscus demersus) recombinant influenza a virus vaccines for the pathogenic human a/hong kong/ (h n ) viruses immunization of turkeys with a dna vaccine expressing either the f or n gene of avian metapneumovirus protection of turkeys against chlamydophila psittaci challenge by dna and rmomp vaccination and evaluation of the immunomodulating effect of alpha dna vaccination in the avian cross-protection among lethal h n influenza viruses induced by dna vaccine to the hemagglutinin enhanced protective efficacy of h subtype avian influenza dna vaccine with codon optimized ha gene in a pcaggs plasmid vector characterization of h n influenza viruses that continue to circulate in geese in southeastern control and prevention of avian influenza in an evolving scenario genesis of pandemic influenza containing pandemic influenza at the source the next influenza pandemic: can it be predicted viruses and vaccines scientific barriers to developing vaccines against avian influenza viruses overview of avian influenza diva test strategies emergence and predominance of an h n influenza variant in china a human t-cell leukemia virus type regulatory element enhances the immunogenicity of human immunodeficiency virus type dna vaccines in mice and nonhuman primates immunization by avian h influenza hemagglutinin mutants with altered receptor binding specificity protective immunity to lethal challenge of the pandemic influenza virus by vaccination crossprotectiveness and immunogenicity of influenza a/duck/singapore/ / (h ) vaccines against infection with a/vietnam/ / (h n ) virus in ferrets vesicular stomatitis virus vectors expressing avian influenza h ha induce cross-neutralizing antibodies and long-term protection efficacy of the agro-jet mit-ii needle-less jet injector for iron dextran administration in piglets molecular determinants within the surface proteins involved in the pathogenicity of h n influenza viruses in chickens are we ready for pandemic influenza? breakthrough of the year. avian influenza: catastrophe waiting in the wings the threat of an avian influenza pandemic avian influenza and pandemics-research needs and opportunities public health. public health risk from the avian h n influenza epidemic host range restriction and pathogenicity in the context of influenza pandemic probable person-to-person transmission of avian influenza a (h n ) comparative evaluation of three different intramuscular delivery methods for dna immunization in a nonhuman primate animal model hiv- dna vaccines amino acid in the hemagglutinin of h n influenza viruses determines cell tropism and replication in human airway epithelial cells ab and t cell epitopes of influenza a virus, knowledge and opportunities a simple method of estimating fifty percent endpoints a new generation of modified liveattenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates eight-plasmid system for rapid generation of influenza virus vaccines replication and transmission of influenza viruses in japanese quail we thank ati tislerics, tina suhana, and catherine murray for help with manuscript preparation, michael cichanowski and brenda hartman for figure preparation, alida ault, john paul todd and vi dang for technical assistance, and members of the nabel lab for helpful advice and discussions. we are grateful to louis detolla, aruna panda, elisa luna and their staff for housing, care, and management of the chickens in their aaalac-accredited facility at university of maryland medical school, baltimore. additionally we would like to thank the influenza genome project of the nih and the depositors of the sequences in genbank. key: cord- -jd wxobz authors: běláková, jana; horynová, milada; křupka, michal; weigl, evžen; raška, milan title: dna vaccines: are they still just a powerful tool for the future? date: - - journal: arch immunol ther exp (warsz) doi: . /s - - - sha: doc_id: cord_uid: jd wxobz vaccination is historically one of the most successful strategies for the prevention of infectious diseases. for safety reasons, modern vaccinology tends toward the usage of inactivated or attenuated microorganisms and uses predominantly subunit vaccines. the antigens need to be clearly defined, pure, stable, appropriately composed, and properly presented to the immune system of the host. differing ratios of various proportions between specific cd (+) and cd (+) t cell responses are essential for conferring the required protection in the case of individual vaccines. to stimulate both cd (+) and cd (+) t cells, the antigens must be processed and presented to both antigen-presentation pathways, mhc i and mhc ii. protein antigens delivered by vaccination are processed as extracellular antigens. however, extracellularly delivered antigen can be directed towards intracellular presentation pathways in conjugation with molecules involved in antigen cross-presentation, e.g. heat shock proteins, or by genomic-dna vaccination. in this overview, current knowledge of the host immune response to dna vaccines is summarized in the introduction. the subsequent sections discuss techniques for enhancing dna vaccine efficacy, such as dna delivery to specific tissues, delivery of dna to the cell cytoplasm or nucleus, and enhancement of the immune response using molecular adjuvants. finally, the prospects of dna vaccination and ongoing clinical trials with various dna vaccines are discussed. immunization with plasmid dna (dna-based vaccination) is a relatively novel technique for the efficacious stimulation of a specific cellular and humoral immune response to protein antigens. dna vaccines deliver transgenes, which code for antigens, directly into the cells of immunized host organisms. such antigens are expressed in a similar way as antigens during viral infection [ ] . antigens are processed identically as proteins synthesized in cytoplasm [ , ] and peptide fragments are presented to the immune system on cell-surface mhc i molecules. if the dna vaccine-coded proteins are secreted from the cell, they could be processed by mhc ii and could elicit a specific antibody response [ , ] . dna-based immunization is a new and attractive strategy in the prophylaxis and treatment of infections caused by extracellular and intracellular pathogens. the method of application, dose, boosting schemes, and species used are factors that influence the strength and nature of the elicited immune response. vaccination with plasmid dna may offer several important advantages over traditional vaccines, e.g. the relative stability of dna, the specificity of the antigen produced, and the possibility of guiding the type of elicited specific immune response [ ] . one of the major benefits of dna vaccines is that host cells express a vaccine-coded antigen and thus the antigen presents epitopes which may resemble native viral epitopes more closely than is the case with other vaccination approaches. intracellularly processed epitopes are presented to the host immune system in a way similar to that of a natural viral infection (mhc i presentation followed by cd + t cell responses), but without the risk associated with the administration of infectious agents [ ] . dna vaccines encoding several glycoproteins, i.e. multivalent vaccines, can be delivered to the host in a single dose. a few micrograms to milligrams of plasmid dna are sufficient for inducing a vigorous immune response. dna vaccines can also be manufactured in a relatively cost-effective manner and stored with relative ease [ ] . the temperature-stable storage of dna in a lyophilized form is feasible and more practical for transport and distribution as well [ ] . another important advantage of dna vaccines is their therapeutic potential in ongoing chronic viral infections. dna vaccines are very promising tools for an effective induction of a protective immune response against viral infections (hbv, hcv, hiv) and parasitic infections (malaria, leishmaniasis) [ , , ] . dna vaccines are principally derived from bacterial plasmids [ ] . conventional plasmid dna vaccines consist of two different parts: ) a eukaryotic cistron coding for the target antigen and consisting of a strong promoter/enhancer, cdna coding for the target antigen(s) (full-length or truncated), and a polyadenylation/termination signal, and ) sequences necessary for the manipulation-construction and amplification of plasmid dna in a prokaryotic host (e. coli) and consisting of the origin of replication (usually from e. coli), a multiple cloning site, and an antibiotic-resistant gene used as the selection marker during bacterial amplification [ , , , ] . the majority of eukaryotic promoters used are derived from the human cytomegalovirus (hcmv) or the rous-sarcoma virus (rsv). hcmv and rsv were found to give the highest levels of antigen expression after intramuscular (i.m.) dna injection [ ] . other promoters tested are tissue-specific promoters, e.g. the myocyte-specific desmin promoter [ ] , the actin promoter, major creatine kinase promoter, alphaglobin promoter, chicken beta-actin promoter, and adenovirus promoter. these have been tested primarily for i.m. dna application. efficacious expression of protein from dna vaccines is dependent on the presence of dna vaccine in the nucleus. the effective targeting of plasmid dna to the cell nucleus is dependent on the presence of dna sequences recognized by proteins or peptides called nuclear localization signals (nlss) [ ] , which form a dna-nls complex recognized by cytoplasmic proteins called importins which dock dna-nls complexes to the nuclear membrane receptor/transporter (nuclear pore complex) as a nuclear import substrate [ ] . nuclear dna transport is typical for viral dna. the most effectively recognized dna sequence is localized within the sv early promoter enhancer region of simian virus dna. this dna sequence was shown to be effective in the nuclear transport of plasmid dna [ ] . other dna sequences have been identified and tested, but their recognition through cellular nlss followed by the transport of plasmid to the nucleus is generally poor. an alternative approach is based on the chemical linkage of plasmid dna with an nls. a variety of nlss were tested (large sv t-antigen, m sequence of heterogeneous nuclear ribonucleoprotein, adenovirus fiber protein residues), but they generally obtained unsatisfactory results in in vivo experiments [ ] . this was most likely linked to the size-exclusion limit for active nuclear transport of such artificial dna-nls complexes. studies of the viral dna nuclear transport mechanism are promising because some viral dna, although more than ten times larger than standard vaccination dna plasmids, are effectively transported to the nucleus [ ] . polya-termination signals, which provide stabilization of mrna transcripts, are commonly taken from bovine growth hormone or from sv [ , ] . dna vaccines stimulate both exogenous (mhc class ii-restricted) and endogenous (mhc class i-restricted) antigen-presentation pathways. mhc i-restricted cytotoxic t lymphocytes (ctl) may be induced after dna vaccination by: a) directly transfected somatic cells (myocytes, keratinocytes, or any mhc ii-negative cells) which present expressed antigen on mhc i, b) directly transfected professional antigen-presenting cells (apcs) such as dendritic cells (dcs) which, besides mhc i presentation of the expressed antigen, effectively stimulate naive t cells via a variety of co--stimulatory molecules and cytokines, or c) by cross--priming when protein expressed by dna-transfected somatic cells is taken up by the surrounding professional apcs and presented to t cells [ , ] . the cross--priming phenomenon, discovered by bevan in , is at present used as a general explanation for the re-presentation of exogenously derived cell-associated antigens on both mhc i and mhc ii molecules [ , ] . when the dna vaccine reaches the cell nucleus, transcription is initiated and subsequent translation of the coded protein takes place on cytosolic ribosomes. the proteasome complex processes the expressed protein and released peptides are transported to the endoplasmic reticulum through the membrane transporter complex tap- and tap- . inside the endoplasmic reticulum, the peptides are bound to mhc i molecules [ , ] , which subsequently migrate to the cytoplasmic membrane and present the peptides to the surrounding cd + t cells [ ] . professional apcs, such as macrophages, b cells, and dcs, play a central role in the regulation of the immune response to any vaccine. in contrast to somatic cells, apcs can present the antigen to both mhc i and mhc ii molecules and thus stimulate t-helper cells (cd + ), which control other t cell or b cell responses [ , ] . dcs are much more effective than other apcs because of their unique ability to prime naive t cells. thus dcs are most likely the key cells initiating the immune response to the dna vaccine [ ] . intradermal (i.d.) and gene-gun application would directly target the plasmids to langerhan's cells, which represent immature dcs located in the stratum spinosum of the epidermis. dcs can migrate to lymphatic organs (the spleen and the lymph nodes) where they activate antigen-specific t lymphocytes. apart from high levels of mhc i and mhc ii, dcs express the stimulatory molecules b . and b . (cd and cd , respectively) that are required for immune response initiation [ ] . if non-apcs such as myocytes take up the dna vaccine (following i.m. injection), the expressed antigen is delivered to t cells by cross-presenting dcs [ , , , ] . cross-presentation and cross-priming are responsible for inducing the immune response to apoptotic and/or necrotic bodies [ ] , which may also be associated with dna vaccination. thus dna immunization results in the induction of both humoral (antibody) and cellular (t cell) immune responses. the major advantage of dna vaccines is their ability to generate a strong cellular immunity with preference for mhc i-restricted cd + cytotoxic t cells and mhc ii-restricted t helper type (th ) cell responses [ ] . on the other hand, dna vaccines can induce th responses and results from a number of studies indicate that dna vaccination can be effective in inducing long-term antibody responses [ , ] . this effect may depend on the type of antigen coded by the vaccine [ ] . effective dna vaccination should generate a long--term memory immune responses. there are several possible ways for dna vaccines to induce a long-term response: a) antigen is continuously expressed at a low level sufficient for antigen presentation and b) plasmid dna as well as antigen are completely gone and the response is antigen independent. memory cells generated by dna vaccines probably differ qualitatively from those achieved by other forms of vaccination, such as protein plus adjuvant [ , ] . memory cd + t cell and b cell populations are the most relevant for vaccine development. the most critical factors for current dna vaccines are: ) the efficacy with which the dna vector reaches the target cells' nuclei (transfection efficacy) and ) the amount of actual protein synthesized in dna vaccine--transfected cells. it has been estimated that injection of microgram doses of dna plasmid results in the production of only nanograms of protein [ ] . it is difficult to quantify the number of plasmids that enter the cell nucleus and the number of plasmids that are degraded before they enter the nucleus. it is estimated that more than % of the dna plasmids never reach the cytoplasm and only about . % of cytoplasmatically localized dna plasmids enter the nucleus, where gene expression is initiated [ ] . identifying and overcoming each hurdle along the dna vaccine entry pathway (low uptake across the plasma membrane, inadequate release of dna molecules within the cell's cytoplasm resulting in reduced dna stability, and a lack of nuclear targeting) can improve dna vaccine efficacy [ ] . dna vaccination was described in when wolff et al. [ ] demonstrated induced gene expression after direct i.m. injection of naked plasmid dna into experimental mice. naked dna was then injected into various tissues with the aim of comparing the intensity of proteosynthesis and the host immune response. besides i.m. injection the most frequently tested injection routes were i.d., intravenous, subcutaneous, epidermal, intraepidermal, intraperitoneal, injection into lymphatic follicles, and injection into the thyroid gland [ , , , ] . different routes of administration lead to markedly different levels of protein expression as well as different levels of intensity and quality (th , th , antibody) of the immune response. the skin was found to be one of the best sites for immunization due to the ease of skin injection and the high concentration of dcs (in the skin langerhans cells), macrophages, and lymphocytes, which are necessary for the induction of the immune response [ ] . in contrast, muscle is generally not equipped with dcs and elicitation of the immune response probably relies on cross-presentation, the effectiveness of which is limited by the dose of available antigen [ ] . thus methods which increase antigen expression by increased uptake of the dna by muscle cells could dramatically improve the applicability of i.m. dna vaccination. dna delivery methods can be classified into two general types: ) mechanical and electrical strategies for introducing naked dna into cells, including microinjection, particle bombardment, and the use of electroporation, and ) dna delivery systems which can be classified into biological viral dna delivery systems and chemical non-viral delivery systems (dna-binding polymers and liposomes). physical methods for increasing naked dna delivery dna vaccination into skin and muscle. wang et al. [ ] tested the effect of i.m. injection followed by electroporation on plasmid uptake in mice. the electroporation increased muscle cell plasmid uptake by approximately -to -fold. direct i.d. or i.m. dna injection vaccination very often results in the induction of a th response characterized by interferon (ifn)-γ synthesis and predominately igg a antibodies in mice [ , ] . the doses applied by needle injection are normally in the range of about - µg of naked plasmid dna. in the case of gene-gun dna application, only . - µg of plasmid dna is sufficient to induce antibody or ctl responses. the gene-gun or biolistic system uses compressed heli-um to propel micrometer-sized colloidal gold particles coated with precipitated plasmid dna (dna-coated microparticles) directly into the epidermal cells. in mice, intraepidermal gene-gun dna inoculation generates a prominent th response with interleukin (il)- production and an excess of the igg isotype [ , , ] . the approximately log of the dna dose may explain the dominance of the th response because low plasmid doses result in a low cpg motif moiety, which is important for a th response elicitation [ , ] . needle-free jet injection (biojector) is another dna delivery approach which has been investigated extensively as a method of i.d. immunization of laboratory animals, such as mice, pigs, rabbits, dogs, and monkeys [ , ] . gramzinski et al. [ ] analyzed the effect of various routes for immunizing with a dna vaccine encoding hepatitis b surface antigen (hbsag). experiments confirmed that needle-free injection of a dna vaccine (biojection of dna) induces a greater immune response to hbsag antigen than i.d. or i.m. injection using the classic needle-syringe approach [ ] . similar results were obtained in the earlier experiments of baizer et al. [ ] . the needle-free injection of dna has been tested in several human clinical trials as well [ ] , e.g. a gag-pol candidate hiv dna vaccine [ ] . the delivery of dna vaccines to the liver. a high level of dna vaccine expression in liver cells was achieved by the rapid injection of naked plasmid dna in relatively large volumes via the tail vein, the portal vein, the hepatic vein, and the bile duct in mice and rats [ , ] . this liver-specific approach has been designated "hydrodynamic delivery" and it is increasingly being used as a research tool for elucidating the mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of diseases in laboratory animal models [ , , ] . the procedure has also been shown to be effective in large animals such as dogs and non-human primates [ , ] . the hydrodynamic approach is proving to be a very useful research tool not only for gene expression studies, but also more recently for the delivery of small interfering rna [ , , ] . the application of dna vaccine to the liver is associated with enormous protein expression followed by a strong antibody-and cell-mediated immune response. when the same dose of dna was administered by hydrodynamic application or by i.m. or i.d. injection, the antigen-specific antibody levels induced by the hydrodynamic application were approximately times higher (raska et al., unpublished result) . dna vaccine minimization to minicircle dna. minicircles are small circular dna molecules that are derived from parental dna plasmids by specific recombination. antibiotic resistance genes, selection markers, and bacterial origins of replication are fully removed by the specific recombination [ , ] . minicircles contain only the gene of interest, making them promising tools for dna vaccination and gene therapy. chen et al. [ ] have shown that minicircles can express high ( -and -fold more) and persistent levels of target protein in mouse liver compared with their parent plasmids. this could be attributed to a higher transfection efficacy and the low to minimal content of cpg in minicircle dna. avoidance of bacterial dna also increases the safety of dna application because no antibiotic-resistant genes can be passed to pathogenic bacteria present in host tissues. non-viral carrier systems: dna/polyplexes. dna carriers tested for dna vaccination are various molecules which complex with dna by ) electrostatic forces between negatively charged dna molecules and a positively charged carrier (or cationic ions on a negatively charged carrier), ) analogous to the natural dna-protein interaction, or ) artificial covalent linkage between dna and a carrier [ ] . the dna/carrier complexes protect dna from serum dnases, increase transmission of dna through the cytoplasmic membrane of target cells, allow targeting to specific tissues, and some of them induce the escape of dna entrapped in endosomes by promoting endosomal disruption (weak bases such as chloroquine, the proton-sponge effect of many polymers) [ ] . non-viral dna vaccine delivery systems are based on ) electrostatic complexation of dna with cationic polymers (poly-l-lysine, protamine sulfate, polyethyleneimine, chitosan, polyethylene glycol, poly--(d,l-lactide-co-glycolide)), complexes commonly termed dna/polyplexes, ) electrostatic complexation and condensation of dna with artificial cationic lipids or lipopolyamines (dc-chol, dotma, dotap, dospa, dogs) which are mixed together with zwitterionic helper lipids responsible for the fusion of complexes with the target cell membrane (chol, dope, dppc), complexes commonly termed dna/lipoplexes or dna/lipopolyplexes, ) complexation of dna with artificial anionic lipids (dmpg) through electrostatic interaction mediated by na + and k + ions which are supplemented with zwitterionic helper lipids (dppc), complexes commonly termed fluid dna/liposomes, ) association of dna with proteins or peptides (histones, peptide tyrosine-lysine-alanine-(lysine) -tryptophan-lysine, fab fragments of anti-dna antibodies, cationic viral proteins µ and vp ) which in combination with cationic polymers or lipids facilitate nuclear targeting, and ) complexation of dna with dendrimers with a very low degree of polydispersity (pamam), complexes commonly termed dna/dendrimers (table ) [ , , , , , , ] . cationic liposomes and polymers are accepted as effective vectors for gene delivery with low immunogenicity, unlike viral vectors [ ] . liposomes are often used for systemic (intravenous, i.m., i.d.) or topical (nasal, oral) dna administration [ , , , , , ] . the final dna/lipopolyplex structures, dna concentration, ratio of cationic moiety to dna, and supplements such as condensing agents, endosmolytic agents, or nuclear targeting molecules are the most critical factors in the transfection efficacy of each formulation [ , , ] . the development of modified dna/lipopolyplexes suitable for intravenous application and targeting to tissues and organs such as heart, lung, liver, spleen, or kidney is desirable and necessary. the current focus is on decreasing the toxicity of complexes and increasing transfection efficacy and tissue specificity. to target specific tissue, the carriers are modified by linkage to various ligands allowing dna complexes to be recognized by specific tissue and cell populations. targeting to the liver is possible by linking liposomes or other polymers (polylysine, pei, polyamidoamine dendrimers) with a sugar motive recognized by the liver--specific asialoglycoprotein receptor, which is expressed on hepatocytes. such ligands are composed of trimeric terminal galactose [ , , ] . lipid-based dna/complexes are a promising approach in systemic application because of the minimal toxicity of particular lipids (dppc) in contrast to cationic polymers [ ] . on the other hand, lipid-based dna complexes are larger in size and are thus limited in their penetrability into liver--specific compartments (disse spaces), which are the port for hepatocyte targeting [ , , , ] . therefore an effort has been made to decrease the size of final complexes by protamine sulfate or poly-l-lysine, which increased the transfection efficacy in some in vitro experiments by up to times [ , ] . targeting the dna/complexes to dcs, mainly by interaction with the mannose receptor on the dc surface, is immunologically very promising. a specific ligand, mannose, was linked to a variety of polymers and lipids used for dna/polymer preparation which were tested in vitro or in vivo according to their dc specificity and transfectability. unfortunately, the high transfection efficacy commonly achieved in in vitro experiments is still dramatically diminished under in vivo conditions [ , ] . viral carrier systems. one highly efficacious delivery system for dna vaccines, or, more precisely, genetic vaccines, is based on recombinant viral vectors derived either from attenuated viruses used for preventive vaccination (vaccinia, poliovirus, hepatitis b virus, measles virus) or from viruses such as human adenovirus (hadv), adeno-associated virus (aav), alphavirus, vesicular stomatitis virus (vsv), or poxviruses other than vaccinia [ ] . in contrast to plasmid dna vaccines, virally vectored genetic vaccines induce a specific immune response not only against the expressed transgene, but also against the viral capsid and/or envelope and this response is often effective even after the first immunization. therefore, repeated immunization, often necessary for elicitation of a satisfactory immune response against the transgene product, could be inefficient. virally vectored genetic vaccination is generally performed as the second immunization after dna priming, i.e. a heterologous vaccination approach [ ] . the choice of transgene, viral vector, dose, route of application, prime/boost regimen, and number of immunizations are the most important factors influencing appropriate antigen-specific humoral and cd + and cd + cellular responses. furthermore, delivery efficacy of viral vectors could be dramatically hampered by preexisting immunity to the capsid or envelope proteins in a population. only a few viral vectors (alphavirus, vsv, and some serotypes of hadv or aav) are considered to be unrecognized by preexisting immunity of vaccines. finally, similarly to dna plasmid, a protective immune response elicited in experimental animals by virally vectored genetic immunization may not necessarily be observed in subsequent human clinical trials. this could be attributed to the above-mentioned preexisting immunity, the restricted range of viral hosts, restricted viral tissue tropism, or to many other, not yet well-described factors. poxvirus-derived vectors are some of the most frequently tested. they are safe and genetically stable double-stranded (ds) dna vectors whose entire life cycle occurs in the cytoplasm of somatic cells. they do not enter the nucleus and therefore do not integrate into the host genome. poxviruses are capable of carrying large amounts of foreign genetic material. two vaccinia--derived vectors, the new york vaccinia virus [ , , ] . edible bait vaccine, raboral vr-g, is licensed worldwide for the prevention of rabies in animals. vectors derived from avian poxviruses such as canarypox virus (alvac) and fowlpox virus (fwpv- ) are expected not to be recognized by preexisting immunity in humans. poxvirus-vectored transgenes induce good specific humoral and cd + and cd + cellular responses [ , , ] . recombinant adenoviruses are extensively tested for both vaccination and gene therapy. they contain dsdna in a rigid icosahedral non-enveloped nucleocapsid. the wild-type of adenovirus does not integrate into the genome. nevertheless, hadvs are oncogenic for animals and thus the safety of hadv therapy is still an open question [ ] . replication-incompetent recombinant hadvs are able to carry transgenes of up to kb. adenoviruses are used because of effective cellular uptake and transgene expression. wild and recombinant adenoviruses induce intense inflammatory responses followed by an induction of serotype-specific immunity, which could hamper the effectiveness of a subsequent vaccination with the same serotype [ ] . among the about known hadv serotypes, hadv- is the most explored for recombinant vaccine application. hadv- has one of the weakest pathogenicities associated with mild upper respiratory disease and fever in humans. recombinant hadvs were tested in animal models as a potential vaccine for hiv, malaria, sars, and ebola. heterologous dna-prime/recombinant adeno-boost immunization induced both humoral and cd + and cd + cell responses in experimental animals. a weak response was detected in human volunteers. because the prevalence of hadv- is relatively high in the population, altered surface proteins or chimeric hadv- carrying the surface proteins of another serotype are developed for avoiding the hampering effect of preexisting immunity [ ] . aav are members of the parvoviridae family of single-stranded (ss) dna non-enveloped viruses of the genus dependovirus [ ] . recombinant aavs are nonreplicative but persist within the cells as non-episomal, mainly circular dna. the integration frequency determined in rodent and rabbit muscle tissue is less than approximately - , which is two orders lower than spontaneous mutations in human genes. recombinant aavs could package kb of ssdna, including itr (transgene < . kb) [ ] . within the cell, the ssdna genome is transcriptionally active after conversion to a dsdna template, which takes in vivo a few weeks, a necessary delay before the induction of an immune response. therefore, dsaavs were developed. the maximum capacity for transgene insertion is . kb [ ] . in mice and rhesus macaques, a single i.m. injection of serotype aav expressing hiv antigens induced robust specific antibody and cd + cell responses [ ] . a significantly lower response in humans could be attributed to many factors, including preexisting immunity, because the presence of specific neutralizing antibodies ranges from - % according to age group and geographic location. specific antibodies recognize other serotypes, but their neutralizing activity seems to be less prominent, for example aav- with an affinity for muscle and hepatocytes, aav- for airway epithelium, aav- for muscle, and aav- for hepatocytes. beside vaccination, aav vectors are one of the most frequently tested vectors for gene replacement therapy [ ] . alphavirus vectors were earlier used for in vitro recombinant protein expression. their wild precursors belong to the togavirus family of positive ssrna enveloped viruses which replicate entirely in cytoplasm. a high level of transgene expression from this viral promoter is achieved due to the self-replicating nature of viral rna and the efficient inhibition of translation of host mrna by the viral replicase. because of limited prevalence, the preexisting immunity to the vector seems to be minimal. in experimental animals, immunization with vectors based on sindbis, semliki forest, and venezuelan equine encephalitis viruses induced a cellular and humoral immune response to the transgene. in the case of hiv- antigens the immune response in non-human primates was able to significantly reduce the viral load after challenge [ , ] . the vsv belongs to the rhabdoviridae family of enveloped negative-sense ssrna viruses. vsv-derived vectors are able to generate viral particles that express foreign transmembrane protein on the membrane surface. because vsv proteins do not down-regulate the interferon response of an infected cell, vsv could exhibit promising adjuvant activity. although wild-type vsv could induce neuropathy after intranasal application to mice, recombinant vsvs were proved to be safe in non-human primates after intranasal and i.m. application. immunization of non-human primates with vsv expressing hiv and siv antigens demonstrated efficacy in subsequent challenge with the highly pathogenic shiv . p virus. thus the potential neural toxicity of vsv needs to be finally solved before entering into the clinical trials [ ] . a few additional viral vectors were designed for genetic vaccination. one viral vector of future vaccines will be probably based on the attenuated measles virus, an enveloped negative-sense ssrna virus. at present, genetically stable recombinant vaccine strains are available for cloning up to three transgens (edmonston, schwartz). in animal experiments a recombinant measles vector expressing hiv- envelope antigen induced neutralizing antibodies and envelope-specific cd + and cd + cell responses after a single dose [ ] . although about % of the population experienced a measles infection or vaccination, it is estimated that years after the measles experience the immune response does not preclude successful immunization with recombinant measles vaccines. furthermore, it is expected that within to years measles will be eradicated [ ] . dna/polyplexes for targeting dna vaccine to mucosal surfaces. mucosal immunity establishes the first line of the defense against pathogens which attack the body via mucosal surfaces [ ] . induction of the mucosal immune response, either cellular or humoral, requires local mucosal application of antigen or an antigen-coding dna vaccine. although some mucosal response is detectable after a systemic dna vaccination, i.m. injection and gene-gun delivery of plasmid dna have a limited ability to induce mucosal immune responses [ ] . therefore, various mucosal dna application routes have been tested to achieve sufficient antigen production on mucosal surfaces (intranasal and intratracheal application, inhalation of dna vaccine in aerosol form, application of dna on external genital mucosa, and oral administration) [ , , ] . complexing dna with various polymers enhances dna uptake on mucosal surfaces. dna/polyplexes adhered to the mucosal cells either through specific receptors or through electrostatic interaction between a negatively charged mucosal cell surface and positively charged dna/polyplexes. when dna/lipoplexes were delivered orally or intranasally, they induced a significant mucosal immune response, including secretory iga responses [ , ] . an alternative means of enhancing the efficiency of dna vaccines is the use of genetic adjuvants. genetic adjuvants are most often genes coding for cytokines, chemokines, or co-stimulatory molecules. the cdna can be either administered in separate dna plasmids (monocistronic dna vaccine) or the cdna is cloned into parental dna vaccine plasmid under separate promoters or under promoters shared with antigen-coding dna sequence separated by an internal ribosome entry site element (bicistronic dna plasmid) [ ] . such molecules supply t cells or dcs with the second, antigen-independent stimulatory signal. several co-stimulatory molecules have been tested for enhancement of a) apc activation (cpg, cd l, mip- α), b) ctl response (il- , il- , il- , il- , il- , gm-csf, ifn-γ, cd l, icam- , lfa- ), c) th -type antibody production: igg a in mice (il- , il- , il- , il- , gm-csf, ifn-γ, cd l) or th -type antibody production: igg in mice (il- , il- , il- , tgf-β), and d) cellular response associated with ifn-γ induction (il- , il- , il- , il- , gm-csf, ifn-γ, cd- l, icam- , lfa- ) [ , , , , , , , ] . two additional co-stimulatory molecules of the tumor necrosis factor receptor superfamily expressed on activated t cells are antiapoptotic, i.e. - bb (cd ), expressed on cd + t cells, and its cd + t cell analogue ox (cd ), which protect activated t cells from death and thus enhance the antigen-specific t cell response. stimulating these receptors may be useful in dna vaccine development [ ] . during mucosal dna vaccination, specific parameters are critical for the use of molecular adjuvants. the proinflammatory cytokines il- α, il- , and il- were tested for antibody and mucosal ctl responses. il- has the potential to increase antigen-specific ctl activity and is thus particularly interesting due to its potential role in regulating the homeostasis of memory t cells [ ] . il- and il- were shown to be able to markedly increase iga reactivity to co-expressed heterologous antigen. monocyte chemoattractant protein- was effective in increasing mucosal iga secretion and ctl responses [ ] . rantes, lymphotactin, mip- β, mip- , human neutrophil peptides (hnp- , hnp- , hnp- ), ifn-γ, and ifn-β are important candidates for use as adjuvants [ , ] . another approach used for modification of the immune responses to dna vaccines includes the addition of heterologous gene fragments encoding localization or secretory signals or fusion of the antigen-coding cdna with the sequence coding for ligands which drive the antigen to sites appropriate for immune induction. for example, a number of studies have shown that higher titers of antigen-specific igg (igg in mice) were elicited when the antigen was secreted rather than localized on the cell membrane or within the cell [ , ] . the cellular localization of heterologous antigen may play a role in modulating the immune response, although the role may depend upon the nature of the antigen and/or the model system used. another strategy consists of fusing the antigen-encoding gene with the ubiquitin-encoding gene, thereby accelerating cytoplasmic degradation of the antigen by targeting it to proteasomes and improving class i antigen presentation [ ] . enhancement of the immune response could additionally be reached by fusion of antigen with the hsp -binding viral j-domain during the construction of dna vaccines. this molecule stabilizes fusion antigen by binding to the hsp protein, which in addition serves as an instruction molecule to induce an increase in the cd + t lymphocyte and b lymphocyte response [ ] . further, dna vaccine potency may be improved through fusion of the antigen-coding dna with the endosomal/lysosomal sorting signal sequence (derived from lysosome--associated membrane protein type ; lamp- ), which directs the expressed antigen towards mhc class ii molecules. thus the cd + t cell response could be significantly enhanced [ ] . the above-mentioned use of the bicistronic vector is the most common practice for heterologous production of the binary protein complex, but these methods are primarily in the research stage [ ] . bicistronic vectors could be useful in constructing multivalent vaccines derived from two, and possibly more, different antigens originating from one or more microorganisms. an element common to the majority of plasmids are the cytosine-phosphate-guanosine dinucleotides (flanked by two ´ purines and two ´pyrimidines recognized in mice) [ ] , called cpg motifs, which are unmethylated when the plasmids are amplified in a bacterial host. hypomethylated or unmethylated cpg nucleotides are specific to bacterial dna, but are very rare in eukaryotic dna. cpgs play an important role as an immunomodulatory component of dna vaccines [ ] . they are recognized by the toll-like receptor (tlr) localized in the cytoplasm [ ] . cpgs are potent stimulators of b cell proliferation and antibody secretion. cpg induce apcs (macrophages and dcs) to secrete th -type cytokines il- and ifn-α/β, which activate natural killer cells and t cells (cd + ) [ , ] . rankin et al. showed that cpg motifs or sequences which are effective in mice are ineffective in humans [ ] . this highlights the species diversity within tlr substrate specificity. the amount of cpgs in plasmid backbones could be changed by simple recombinant technology. thus the addition of cpgs to dna plasmids could theoretically decrease the dna vaccine dose used for a single immunization. however, from murine experiments it is clear that too many cpg motifs can actually reduce immunogenicity [ ] . the major safety concerns here are: ) the integration of the plasmid dna into the host genome, thereby increasing the risk of malignancy by activating protooncogenes or inactivating oncosuppressors, ) transfer of antibiotic-resistant genes to surrounding bacteria, ) the induction of an immune response to transfected cells, resulting in the development of an autoimmune disease, ) the stimulation of cytokine responses that alter the host immune homeostasis, and ) the induction of tolerance rather than immunity [ , , , ] . at the present time there is still no evidence for plasmid dna integration into the host genome although traces of plasmid dna are detectable in host cells up to one year after dna vaccination. current methods used for the detection of plasmid integration into genomic dna are not sufficiently sensitive or specific and this question remains open [ ] . the spread of antibiotic resistance is another important question. dna plasmid could be detected far from the original site of injection (in the case of i.m. or i.d. application). the responsible carriers were transfected lymphocytes and macrophages. such cells, of course, could spread antibiotic resistance genes to bacteria. there is, however, no direct experimental evidence to date for this phenomenon [ ] . both plasmid integration and antibiotic resistance transfer could be effectively minimized by minicircle dna technology, as mentioned above [ ] . autoimmune response a) against dna plasmid, mediated by anti-dna antibodies or b) against transfected cells, mediated by a type i immune response was experimentally modeled in mice [ ] . dna vaccination was able to moderately increase the dna-specific serum antibody titers for a limited period of time, but clinical signs of autoimmunity were not confirmed. cpg motifs, on the other hand, may induce a truly harmful autoimmune response when injected together with autoantigens such as the myelin basic protein (inducing encephalomyelitis) or a chlamidia-derived antigen (inducing myocarditis) in experimental models. the above effects were not found in healthy animals treated with therapeutic dna vaccine doses and no sign of toxicity was found in human volunteers exposed to dna vaccines during clinical trials [ , , ] . the use of cytokine genes in modern multicistronic dna vaccines could theoretically disrupt the immune homeostasis and increase susceptibility to other infections. it could also be associated with exacerbation of autoimmune or allergic diseases. experimental observations have confirmed that the cytokines are released locally and that serum cytokine levels are unchanged [ ] . induction of tolerance rather than immunity after dna vaccination is a problem for both extreme age groups. very young animals (mice under days of age) and very old ones (mice older than years) respond weakly to dna vaccination. in newborn mice, dna vaccine encoding circumsporozoite protein of the malaria plasmodium induced tolerance which was long lasting [ ] . thus dna vaccination schedules for both extreme age groups would need to be separately tested, possibly modified by use of cytokines or addition of costimulatory molecules. the majority of ongoing human dna vaccination trials are focused on assessing vaccine safety and immunogenicity [ , ] . as estimated from the us database at www.clinicaltrials.gov as of june , approximately one hundred dna vaccination clinical trials have been registered (irrespective of their trial phase). a large proportion of dna vaccines use viral vectors as delivery systems. the dna vaccine gendicine, produced by sibiono genetech, uses an adenovirus vector for delivery of dna encoding the p suppressor. it is registered by the chinese fda for treatment of head and neck squamous cell carcinoma [ ] . another adenovirus-vectored p -encoding dna vac-cine, advexin, produced by introgene therapeutis, is being tested in phase iii clinical trials for the same indications in the us. if we focus on plasmid (naked) dna vaccines, approximately clinical trials had been registered by www.clinicaltrials.gov as of may . ten have attained phase ii. the majority of dna vaccines are focused on hiv- infection. other infectious diseases include hepatitis b, malaria, ebola hemorrhagic fever, west nile virus infection, and avian flu. the other trials deal with various cancers (hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, chronic lymphocytic leukemia, breast cancer, ovarian cancer, prostate cancer, bladder cancer, synovial sarcoma, leiomyosarcoma, lung cancer, and melanoma). all ongoing clinical trials have confirmed minimal toxicity and good tolerance to dna vaccines. however, the still poor immune response to the majority of clinically tested dna vaccines is a great challenge for further optimization using novel antigen, delivery systems, and prime-boost-based schedules. dna vaccines remain a promising approach for inducing both humoral and cellular immune responses. one of the more interesting aspects of dna vaccination is the mechanism of inducing antibody-and/or cell--mediated immune responses. this is associated either with antigen presentation on vaccine transfected cells or with cross-presentation of the antigen by dcs, which are the only known cells capable of inducing both cd + and cd + t cell response. dna vaccines are effective inducers of host immune protection against viral, bacterial, fungal, and parasitic infections and may be suitable for cancer therapy. multicistronic dna vaccines that, for example, coexpress cytokines may be able to modulate any autoimmunity or allergic reactions. one basic obstacle to applying dna vaccines in human medicine is their still relatively poor immunogenicity, linked to low transfection efficacy and low antigen production. thus the development of effective delivery systems is the main research challenge in this area of immunology. dna vaccination: transfection and activation of dendritic cells as key events for immunity increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes induction of immune responses by dna vaccines in large animals biojector needle-free intradermal injection enhances immune responses to a dna vaccine current strategies and future directions for eluding adenoviral vector immunity biological features of genetic immunization immune responses to gene therapy vectors: influence on vector function and effector mechanisms pulmonary dna vaccination: concepts, possibilities and perspectives reversible covalent attachment of cholesterol to oligodeoxyribonucleotides for studies of the mechanisms of their penetration into eucaryotic cells vaccine delivery methods using viral vectors mechanism of plasmid delivery by hydrodynamic tail vein injection. ii. morphological studies cross-presentation: a general mechanism for ctl immunity and tolerance optimization of plasmids for gene delivery utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems minicircle dna vectors devoid of bacterial dna result in persistent and high-level transgene expression in vivo development of th and th populations and the nature of immune responses to hepatitis b virus dna vaccines can be modulated by codelivery of various cytokine genes intradermal immunization with novel plasmid dna-coated nanoparticles via a needle-free injection device evaluation of hiv- dna vaccines containing cpg motif and fowlpoxvirus vaccines co-expressing ifnγ or il- plasmid dna expression systems for the purpose of immunization cpg motifs for optimization of dna vaccines sequence requirements for plasmid nuclear import efficient gene delivery into human dendritic cells by adenovirus polyethylenimine and mannose polyethylenimine transfection mannose receptor-mediated gene delivery into antigen presenting dendritic cells dna vaccines priming with plasmid dnas expressing interleukin- and simian immunodeficiency virus gag enhances the immunogenicity and efficacy of an experimental aids vaccine based on recombinant vesicular stomatitis virus dna vaccines safety, tolerability, and lack of antibody responses after administration of a pfcsp dna malaria vaccine via needle or needle--free jet injection recent advances in mucosal vaccines and adjuvants dna vaccines against human immunodeficiency virus type prime-boost immunization generates a high frequency, high-avidity cd + cytotoxic t lymphocyte population exploiting receptor biology for oral vaccination with biodegradable particulates dna vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations nonviral gene delivery: what we know and what is next dna vaccines: improving expression of antigens dna vaccines for biodefence dna-antiviral vaccines: new developments and approaches ensuring safety of dna vaccines immune response to a hepatitis b dna vaccine in aotus monkeys: comparison of vaccine formulation, route, and method of administration dna vaccines. naturwissenschaften dna vaccines: immunology, application, and optimization clinical experience with plasmid dna-and modified vaccinia virus ankara-vectored human immunodeficiency virus type clade a vaccine focusing on t-cell induction in vivo gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector efficient gene transfer into macrophages and dendritic cells by in vivo gene delivery with mannosylated lipoplex via the intraperitoneal route cross-presentation, dendritic cells, tolerance and immunity time course of gene expression after plasmid dna gene transfer to the liver hydrodynamic delivery of dna dna vaccination with the hantaan virus m gene protects hamsters against three of four hfrs hantaviruses and elicits a high-titer neutralizing antibody response in rhesus monkeys in vivo cross-priming of mhc class i-restricted antigens requires the atp transporter repeat administration of dna/liposomes to the nasal epithelium of patients with cystic fibrosis vaccination of macaques with siv immunogens delivered by venezuelan equine encephalitis virus replicon particle vectors followed by a mucosal challenge with sivsme two-promoter vector is highly efficient for overproduction of protein complexes enhancing dna vaccine potency by combining a strategy to prolong dendritic cell life with intracellular targeting strategies cpg dna as a vaccine adjuvant dna vaccines: safety and efficacy issues the structure of peg--modified poly(ethylene imines) influences biodistribution and pharmacokinetics of their complexes with nf-kappab decoy in mice efficient vaccination by intradermal or intramuscular inoculation of plasmid dna expressing hepatitis b surface antigen under desmin promotor/enhancer control adenovirus and adenoassociated virus vectors efficient delivery of sirna for inhibition of gene expression in postnatal mice viral vectors for malaria vaccine development hydrodynamics--based transfection in animals by systemic administration of plasmid dna cpg motif acts as a 'danger signal' and provides a t helper type -biased microenvironment for dna vaccination dna vaccines dna vaccines: recent developments and future possibilities. hum immunization of the female genital tract with a dna-based vaccine enhancement of antibody responses to a hiv- dna envelope vaccine using an expression vector containing a constitutive transport element a single injection of recombinant measles virus vaccines expressing human immunodeficiency virus (hiv) type clade b envelope glycoproteins induces neutralizing antibodies and cellular immune responses to hiv synthetic dna delivery systems nonviral gene therapy and its delivery systems synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for cellular targeting rna interference in adult mice route and method of delivery of dna vaccine influence immune responses in mice and non-human primates dna vaccines: safety aspect assessment and regulation prospects for cationic polymers in gene and oligonucleotide therapy against cancer induction of neonatal tolerance by plasmid dna vaccination of mice genetic immunization by jet injection of targeted pdna-coated nanoparticles - bb and ox stimulation enhance cd and cd t-cell responses to a dna prime, poxvirus boost vaccine enhanced cationic liposome-mediated transfection using the dna-binding peptide m (mu) from the adenovirus core helper-dependent adenovirus vectors elicit intact innate but attenuated adaptive host immune responses in vivo replicating minicircles: generation of nonviral episomes for the efficient modification of dividing cells gene therapy progress and prospects: nonviral vectors microparticles for the delivery of dna vaccines modulation of immune responses to dna vaccines by codelivery of cytokine genes dna--based therapeutics and dna delivery systems: a comprehensive review immunization with dna through the skin liposome-mediated dna vaccination: the effect of vesicle composition dna vaccines for the treatment of autoimmune disease cpg motif identification for veterinary and laboratory species demonstrates that sequence recognition is highly conserved comparison of protective effect of protein and dna vaccines hsp in murine model of systemic candidiasis recombinant protein and dna vaccines derived from hsp trichophyton mentagrophytes control the clinical course of trichophytosis in bovine species and guinea-pigs dna vaccines expressing antigens with a stress protein-capturing domain display enhanced immunogenicity degradation of cell proteins and the generation of mhc class i-presented peptides multigene, multiclade hiv- plasmid dna prime and mva boost is safe and highly immunogenic in healthy human volunteers salivary gland delivery of pdna-cationic lipoplexes elicits systemic immune responses revealing the ootential of dna-based vaccination: lessons learned from the hepatitis b virus surface antigen mechanism of plasmid delivery by hydrodynamic tail vein injection. i. hepatocyte uptake of various molecules dna vaccines: future strategies and relevance to intracellular pathogens regulation and review of dnavaccine products gene vaccines mucosal adjuvants structural impact of hydrodynamic injection on mouse liver recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid dna vaccines safety and immunogenicity of a gag-pol candidate hiv- dna vaccine administered by a needle-free device in hiv- -seronegative subjects non-viral gene therapy: polycation-mediated dna delivery molecular adjuvants for mucosal immunity tlr pathway is involved in adjuvant effects of plasmid dna-based vaccines dna vaccine constructs against enterovirus elicit immune response in mice generation of mhc class i-restricted cytotoxic t lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo detection of integration of plasmid dna into host genomic dna following intramuscular injection and electroporation tricistronic viral vectors co-expressing interleukin- (il-- ) and cd (b - ) for the immunotherapy of cancer: preclinical studies in myeloma the mechanism of naked dna uptake and expression long-term persistence of plasmid dna and foreign gene expression in mouse muscle direct gene transfer into mouse muscle in vivo oral administration of recombinant adeno-associated virus elicits human immunodeficiency virus-specific immune responses expression of naked plasmid dna injected into the afferent and efferent vessels of rodent and dog livers systemic linear polyethylenimine (l-pei)-mediated gene delivery in the mouse this work was supported by grant msm (ministry of education, youth, and sport, czech republic). key: cord- -zkawdcac authors: perrie, yvonne; kaur, randip; henriksen-lacey, malou title: vaccines date: - - journal: fundamentals of pharmaceutical nanoscience doi: . / - - - - _ sha: doc_id: cord_uid: zkawdcac vaccines continue to offer the key line of protection against a range of infectious diseases; however, the range of vaccines currently available is limited. one key consideration in the development of a vaccine is risk-versus-benefit, and in an environment of perceived low risk, the benefit of vaccination may not be recognised. to address this, there has been a move towards the use of subunit-based vaccines, which offer low side-effect profiles but are generally weakly immunogenic. this can be compensated for by the development of effective adjuvants. nanotechnology offers key attributes in this field through the ability of nanoparticulates to incorporate and protect antigens from rapid degradation, combined with their potential to effectively deliver the antigens to appropriate cells within the immune system. these characteristics can be exploited in the development of new adjuvants. this chapter will outline the applications of nanosystems in vaccine formulations and consider the mechanisms of action behind a range of formulations. virus (hiv)] and re-emerging old and/or persistent infectious diseases (e.g. tuberculosis, malaria, and foodborne infections). as we have already seen within this book, nanotechnology can play a key role in many pharmaceutical applications, and vaccines are no exception. the application of nanoscience to vaccine formulation offers the potential to enhance the effi cacy of vaccination by promoting enhanced protection and effective delivery of antigens. to effectively exploit nanotechnology in vaccine development, we must fi rst consider the possible mechanisms which support effective immunisation. the immune system comprises of many cellular and humoural components which protect the host from disease, a term which includes infection, autoimmune syndromes, injury, and mutations. to do so, the host has acquired the ability to evolve to its environment, a classic example being the gut whereby commensal microbes live in harmony with the host. the immune system has two main functions; to recognise invading pathogens and to activate mechanisms that will destroy them. such pathogens are controlled and terminated by the innate immune response which is ready to react quickly ( fig. . a ). most components of innate immunity are present before the onset of infection and constitute a set of disease-resistance mechanisms that are not specifi c to a particular pathogen. these mechanisms include cellular and molecular components that recognise classes of molecules as different to the frequently encountered pathogens (goldsby et al. ) . phagocytic cells such as neutrophils, macrophages, in addition to pattern recognition receptors, nk cells, complement, and variety of antimicrobial compounds synthesised by the host all play important roles in innate immunity (goldsby et al. ) . the adaptive immune response (fig. . b ) is made up of b and t lymphocytes that have unique receptors specifi c to various microbial antigens (sudhakar and subramani ) , in contrast to the receptors of the innate immune system which are of many different types but not specifi c to a particular pathogen (parham ). these antigen-specifi c receptors are encoded by genes generated during a complex process of gene rearrangement that occurs during the course of lymphocyte development. as each b and t lymphocyte contains a unique antigenic receptor, it allows for large and diverse population of cells capable of recognising a wide spectrum of pathogens. this is termed the lymphocyte repertoire (sudhakar and subramani ) . in response to an infection, lymphocytes-bearing receptors specifi c for the pathogen are then selected to participate in the immune response. the proliferation and differentiation of these cells, termed clonal selection and expansion, generates a large population of specifi c effector cells. to assist in future invasion by the same pathogen, some of the lymphocytes persist in the body and provide longterm immunological memory, thus resulting in a faster and stronger response (parham ). therefore, the immune system is developed to offer a wide range of protectionhowever, one issue is that during an infection the body must be able to respond quickly and vigorously enough to provide the appropriate protection without the individual suffering the potentially lethal consequences of the infection. to address this vaccines have been developed; vaccines have been defi ned as 'any preparation nonself-cells are rapidly attacked in the innate immune system. key players in the innate system are neutrophils, macrophages, pattern recognition receptors, nk cells, and complement. the desired end result is the destruction of the foreign substance, the non-self-cell. ( b ) adaptive immune response. the major players in this response are the b lymphocytes, t lymphocytes, and nk cells. the desired end result is destruction of the non-self-cell but through a more complex and tightly orchestrated series of events (based on bingham ) made from a pathogen that is used for vaccination and provides protective immunity against infection with the pathogen' (parham ). the ultimate goal of a vaccine is to develop long-lived immunological protection, whereby the fi rst encounter with a pathogen is remembered and recognised by the immune system and therefore the immune system can generate a rapid, protective response against the infection. the origins of vaccination lie with smallpox, a disease which once ravaged most, if not all parts of the world. initial descriptions of smallpox stem from as early as bc from texts originating in china. smallpox was known to spread rapidly and resulted in disfi gurement, blindness, and death. it was also known that smallpox was infectious, and early reports dating from bc describe survivors of smallpox being used to treat those infected in a process known as inoculation (gross and sepkowitz ) . over many hundreds of years the concept of inoculation began to take form and involved a small swab of infectious material (otherwise known as pus) being placed on the skin of non-infected persons, and in , , members of the english royal family were successfully immunised against smallpox using this method (riedel ) . however, it was the work of edward jenner, born in in gloucestershire, uk, that supported the development of vaccination. jenner, after overhearing a young dairymaid claim that she may never have smallpox as she had had cowpox, decided to further investigate this. in jenner successfully inoculated a young boy with infectious material from a dairymaid who had cowpox lesions; months later he exposed the boy to smallpox and no disease resulted. this was the beginning of what we now term vaccination. in their traditional organisation, vaccines are grouped into four categories: killed, attenuated, toxoids, or subunit vaccines. live attenuated vaccines consist of a live microbial agent that has mutated so that it has a reduced ability to grow in human cells and is no longer pathogenic to humans (parham ). these microorganisms are still able to infect their target cells. however, infection is ineffi cient (mild) and there are limitations in the replication of the microorganisms. vaccines produced in this manner include the bacillus calmette-guérin (bcg) and the measles, mumps, yellow fever vaccines, and rubella combination vaccine (mmr), and such vaccines are generally capable of stimulating both a humoural and cell-mediated immune response. however, there is a risk of reversion to virulence, and this type of vaccine is not considered safe for use in immunocompromised individuals. • inactivated vaccines inactivated vaccines consist of microorganisms or viruses that have been treated with, e.g. heat or chemicals such as formaldehyde, thereby removing their ability to be infectious while retaining immunogenicity. while offering advantages in terms of safety, such vaccines are generally less effective than live attenuated vaccines, usually only stimulating humoural immunity and often requiring booster doses. examples of these vaccines include: trivalent inactivated infl uenza vaccines, cholera, and hepatitis a vaccines. some microorganisms produce toxic compounds that are the responsible for causing the disease (i.e. tetanus toxin and diphtheria toxin). toxoids are inactivated forms of these toxic compounds. in addition to being successful vaccines in their own right, toxoids may also be used to increase the immunogenicity of some other vaccines, such as the haemophilus infl uenzae type b (hib), which contains a polysaccharide unit from the virus conjugated to diphtheria or tetanus toxins. subunit vaccines initiate strong immune responses using a small part of the organism that could include a gene from the genome. recombinant dna technology has greatly facilitated the development of such vaccines, a process where foreign genes are introduced into yeast or bacteria expression systems. this allows the production of large quantities of antigen that is purifi ed and used as a vaccine (arvin and greenberg ) . the principle benefi ts of subunit vaccines are their inability to revert to a pathogenic form, decreased toxicity, reproducible production, and improved antigen specifi city; however, the immune response induced by such vaccines is short-lived and thus several boosts are required to achieve protection. for hepatitis b virus for example, only the surface protein of the virus is used to generate the subunit vaccine. as is highlighted in table . , there is no clear rule as to which type of vaccine (recombinant/killed/attenuated, etc.) may be superior against a certain disease. certainly the present aim of vaccinologists is to focus on safety (including low toxicity and prevention of reversion to virulence); however, immunogenicity obviously plays a major role. with regard to smallpox, while the live attenuated composition of the vaccine led to the successful eradication of the disease, there were numerous (friede ; mbow et al. ) other factors which certainly contributed including the lack of an animal reservoir, non-zoonotic disease, stability of the vaccine formulation, and clear disease symptoms. however, it is interesting to note that the side effects from this vaccine were also suffi ciently bad for the vaccination campaign in the united states to be halted in , years prior to the declaration by the who that the world was free from smallpox (kennedy et al. ). therefore, even the only vaccine to have ever led to the eradication of a disease had its faults and it is most probable that if the smallpox vaccine was still required today, it would not be used as it would fail clinical trials. the fi rst marketed subunit vaccine, available in the united states in , was for protection against hepatitis b (hilleman ) . therefore, in terms of arrivals onto the vaccine market, the subunit vaccines are latecomers with development rather than discovery being hindered by their poor immunogenic profi le. however, they have the very important advantage over attenuated or killed vaccines in that there is no chance of reversion to a virulent form. subunit vaccines are based on solely the antigenic epitopes originally derived from a virulent organism; there is a total loss of the molecules which would have typically alerted the host to the dangerous nature of the pathogen, in addition to the loss of particulate nature. this appears to be the downfall of subunit vaccines-they lack suffi cient resemblance to pathogens. therefore in an effort to improve the immune responses to subunit antigens, adjuvants are included in the formulation. adjuvants, whose name stems from the latin 'adjuvare' meaning to aid, are defi ned as substances used in combination with a specifi c antigen that produce a robust immune response when compared to the antigen alone ( gupta et al. ; vogel ) . adjuvants come in such a wide range of shapes and sizes that even though documentation on adjuvants has existed for nearly years, there is still no universally approved grouping system. figure . summarises some of the current suggestions for mechanisms of adjuvant action. one of the fi rst papers published by ramon described the adjuvant effect of a range of compounds including tapioca, agar, and starch oil (ramon ) . following this, the use of inorganic compounds including aluminium phosphate and aluminium hydroxide was documented ( glenny et al. ) and these aluminium-based compounds became the fi rst licensed adjuvants in commercial vaccines. until late , aluminium-based compounds remained the only us-licensed adjuvants, while in europe a wider scope of adjuvants had been recognised (e.g. table . ). however, adjuvant development from bench to marketed vaccines has been slow with the fi rst non-aluminium salt adjuvant being licensed less than years ago (mbow et al. ; o'hagan and gregorio ). the slow output and lack of successful licensing is due to numerous reasons including poor scale-up and infl exibility with regard to the scope of antigens with which they can be administered (o'hagan and gregorio ). however, when the varied immunising abilities of different vaccine adjuvants bearing remarkably similar structure and composition are considered, these requirements are hardly surprising. for these reasons adjuvants are licensed in individual vaccines as opposed to being considered a separate entity ( fig. . ). 'alum' is a collective term often used to refer to a group of aluminium salts including aluminium hydroxide, aluminium phosphate, and aluminium potassium sulphate. the correct term for this group of adjuvants is aluminium salts and not alum, which correctly refers to aluminium potassium sulphate (marrack et al. ). the widespread use of aluminium salts as vaccine adjuvants is due to a combined ability to (generally speaking) improve vaccine immune responses as well as provide an excellent safety profi le. while occasional local reactions including infl ammation, erythroma, subcutaneous nodules, and allergic reactions are reported, when the numbers of people who have been vaccinated are considered, aluminium salts are exemplary adjuvants (clements and griffi ths ) . the major downfall of aluminium salts is the polarised immune response which they activate being predominate activators of th -biased immunity, i.e. aluminium salts are ideal adjuvants in vaccines requiring increase in co-stimulatory molecules adjuvant:antigen association strong humoral immunity with high levels of igg antibodies and cytokines such as il- . examples of this include vaccines against extracellular pathogens such as parasitic diseases (e.g. leishmaniasis) or toxoid-producing pathogens (e.g. diphtheria, tetanus). among the methods used to mediate the th /th balance, one has involved combining aluminium salts with adjuvants known as strong stimulators of th responses. examples include aluminium hydroxide combined with cationic liposomes (agger et al. ) or monophosphoryl lipid a (mpl), the latter of which is now the licensed adjuvant as (glaxosmithkline (gsk) biologicals). it is only recently that the mechanisms by which aluminium salt adjuvants act have started to be uncovered, challenging the previous dogma that adjuvanticity was (i) pathogens are endocytosed and activate tlrs via their pathogen-associated molecular patterns (pamps) resulting in the initiation of intracellular events including release of alarmins and activation of myd adapter proteins respectively (ii). the traf signalling pathway becomes activated leading to cleavage of iκb and activation of the transcription factor nfκb with production of pro-infl ammatory cytokines such as il- β and il- , both of which are produced with 'pro' inhibitory domains (iii). endogenous atp can act as an alarmin to activate the p x receptor leading to potassium effl ux from the cell (iv). the combination of potassium effl ux and tlr activation lead to cleavage of the active caspase component of infl ammasomes which is then able to cleave pro-domains of the cytokines il- β and il- (v) cell proliferation/ pro-inflammatory cytokines simply due to longer retention of antigen at the injection site, also known as the depot-effect (marrack et al. ). the importance and mechanisms of antigen association to aluminium adjuvants remains debated (e.g. morefi eld et al. ) ; however, it is clear that aluminium salts prevent the rapid removal and degradation of antigen normally seen upon injection of free antigen. there is now a wide range of literature on the mechanisms of aluminium action (for in-depth reviews see (brewer ; marrack et al. )) which strongly suggest a role for the infl ammasome and uric acid release upon local tissue damage (marrack et al. ; o'hagan and gregorio ) . briefl y, injection of aluminium salts is known to cause tissue damage and cell death with release of alarmins such as endogenous uric acid, infi ltration of neutrophils, and infl ammatory mediators. uric acid is capable of activating caspase , a component of the infl ammasome, which can subsequently cleave pro-units of il- β and il- to their active forms ( kool et al. , see reference within kennedy et al. ; mariathasan ; martinon et al. ; monie et al. generally speaking, adjuvants can be divided into groups depending on their physical properties, such as inorganic salts, liposomes, oil in water (o/w) emulsions, surfactants, etc. this classifi cation poses the problem that the method in which the adjuvant acts is unknown and therefore does not help with eliminating or identifying potential nanosystems as future adjuvant candidates. another classifi cation which will become more complex with time is proposed by o'hagen and de gregorio ( ) whereby adjuvants are classed as fi rst-or second-generation adjuvants. in this system a fi rst-generation adjuvant refers to one of the more traditional substances normally composed of one immunostimulatory compound. these include aluminium salts, liposomes, and mf among others. addition of further immunostimulatory compounds to these existing fi rst-generation adjuvants results in a second-generation adjuvant such as the aforementioned 'adjuvant systems' (gsk biologicals), ic- ® (intercell, austria), and iscoms. while the possibility exists to extend the system to third-generation adjuvants (and fourth and fi fth, etc.), the system becomes increasingly awkward and does not give any indication as to how the adjuvants may work. in virgil schijns proposed a system whereby adjuvants can be divided into groups depending on their method of immunostimulatory action (schijns ) . while fi ve groups were suggested, it is possible that further mechanisms may be deduced in the future, in addition to the diffi culty in classifi cation when one adjuvant has more than one mode of action. finally and possibly the simplest involves a classifi cation system based on whether the adjuvant works via tlrs or not (mosca et al. ) . in this instance only two groups exist (tlr-dependent and tlr-independent); however, for adjuvants containing two or more immunogenic components (such as gsk's as adjuvant containing aluminium hydroxide and mpl), they may act via both tlr-dependent and tlr-independent mechanisms further complicating matters. for the purposes of identifying the mechanisms of action of present and future adjuvants, the classifi cation system by schijns is the most appropriate and the basis of how these adjuvants work will be described herein. according to schijns classifi cation, there are fi ve methods in which adjuvants may be immunostimulatory. highlighted in fig. . , these methods provide an ideal way to explain the immune system with relevance to systemic delivery of vaccines. upon parental vaccine delivery there is localised tissue damage which results from the physical insertion of the needle and vaccine components into the tissue milieu. cells will inevitably be ruptured releasing their intracellular contents including mitochondria, uric acid, and heat shock proteins (hsps), all of which are termed 'alarmins' (bianchi ) . alarmins are host-derived substances released upon non-intentional cell death (in contrast to apoptosis) and, unsurprisingly signal to the host that there is a problem such as tissue destruction or stress. this can be considered the fi rst mechanism of adjuvant action (shi et al. ) and is termed the danger signal, in reference to the 'danger model' originally described by matzinger ( ) . the danger model assumes that the host can differentiate between harmless and harmful as opposed to self and non-self [the later model originally described by metchnikoff and thoroughly reviewed by janeway ( ) ]. overlapping with the danger model is the concept of signal , the second class by which adjuvants act. signal can include alarmins but with respect to adjuvant composition and formulation the signal group principally includes exogenous pathogen-associated molecular patterns (pamps). importantly the signal concept is the physical binding of alarmins or pamps [collectively termed danger-associated molecular patterns, or damps (bianchi ) ] to pathogen recognition receptors (prr's) and the resulting intracellular signalling cascade that results in activation of the cell (see fig. . for an schematic of damps and their tlrs). prrs are constitutively expressed on or within cells of the innate immune system (schijns ) and include, among others, tlrs, infl ammasomes, integrins, c-type lectins, and antibody fc receptors. the specifi c signalling cascades initiated by these damp-prr interactions will be discussed later but some of the resulting actions include the production of pro-infl ammatory cytokines, chemokines, and co-stimulatory molecules required for the productive activation of the t cell upon antigen presentation. in fact, antigen presentation without simultaneous co-stimulatory molecule interactions has been shown to lead to tolerance. co-stimulatory molecules such as cd and cd are consequently very important for successful t cell activation and while being expressed constitutively on dcs, they are only expressed on other apcs including macrophages and b cells upon activation. adjuvants which are able to activate macrophages and b cells to induce expression of cd or cd , and those able to upregulate expression of the said markers on dcs can be grouped under the third adjuvantal category, 'recombinant signal '. the principle adjuvants in this group are cytokines which can be considered as endogenous adjuvants and have also been administered with vaccines experimentally [see (boyaka and mcghee ; egan and israel ; heath and playfair ) for review articles], as well as tlr-signalling adjuvants. the fi nal two adjuvantal categories relate more to the physical structure of the adjuvant and its physical localisation in vivo. as discussed previously, there is much diversity in the structures of adjuvants, although many of them are based on emulsions or vesicular structures. the benefi ts of vesicular structures are twofold; fi rstly they can be used to 'carry' antigen, either by entrapment or adsorption; secondly vesicles are naturally occurring and appropriately sized structures that can be endocytosed by cells depending on their composition and size, making them ideal intracellular delivery systems. this paradigm of assisting antigen uptake is the fourth adjuvantal category and principally concerns liposomes, nanoparticles, microspheres, virosomes, emulsions, and iscoms, all of which are able to package antigen into a delivery vehicle. the fi nal concept is the idea that the antigen held at the injection site for a long period of time results in lengthy presentation of the antigen to innate immune cells. known as the depot-effect, it refers to localisation of antigen (with or without adjuvant) at the injection site and not in the lymphoid organs (although increased presentation of antigen in the lymphoid organs may be a direct consequence of the depot-effect and is often the desired effect). the depot-effect is therefore dependent on numerous factors such as the route of injection, the tissue found at the injection site, and characteristics associated with the formulation itself such as viscosity and particulate size. from this analysis of different 'types' of adjuvants, it is clear that there are still many overlaps between groupings; however, the system devised by schijns does help to defi ne the mechanisms by which adjuvants work. with this in mind, it is possible to envisage how the perfect adjuvant would work. firstly, it must contain a suffi cient amount of pamps to alert the immune system of danger but without causing hyper-immunostimulation which may result in anaphylactic shock or local tissue damage by excess of infl ammatory mediators. too few pamps or pamps without suffi cient toxicity may not stimulate suffi cient production of pro-infl ammatory molecules or chemokines which are vital to alert circulating apcs. it may just be that a certain degree of local tissue damage is also beneficial as it allows the release of alarmins, further promoting apc infl ux to the injection site (shi et al. ) . the adjuvant should also ideally stay associated with the antigen until uptaken by apcs so that the immune system can make a collective association of both components. free antigen, in the form of nucleic material or small peptide antigens, is rapidly degraded by extracellular enzymes and many subunit protein vaccine antigens are themselves immunogenically inert. protein and peptide antigens undergo rapid removal via the circulatory or lymphatic system and without any danger signals attached, the protein may simply be removed before antigen uptake and presentation can occur. given the key characteristics outlined above, it is no surprise that nanosystems have been extensively considered and exploited as potential vaccine adjuvants, with a wide range of nanomaterials being considered. examples of such include nanoparticles, virosomes, immune-stimulating complexes (iscoms), liposomes, and bilosomes among others. by associating antigens with such nanosystems, the antigen can be protected from the extracellular milieu thereby limiting peptide/protein and nucleic antigen breakdown by enzymes, as well as preventing the rapid removal of such small compounds by the mononuclear phagocytic system (mps). the immunological role and adjuvant properties of liposomes were fi rst identifi ed by allison and gregoriadis ( ) , where the ability of negatively charged liposomes (prepared with the inclusion of dicetyl phosphate) to deliver and potentiate immune responses against diphtheria toxoid (dt) was demonstrated. since then the immunological adjuvanticity of liposomes has been well recognised and liposomes have been extensively investigated as potential vaccine adjuvants for more than years and for a number of antigens, including, e.g. tetanus toxoid (davis and gregoriadis ) , leishmania major antigen (kahl et al. ) , hepatitis b surface antigen (brunei et al. ) , dna vaccines (perrie et al. (perrie et al. , , and tuberculosis vaccines (davidsen et al. ; smith korsholm et al. ) , with some liposomal-based vaccines (i.e. virosomes) having been licensed for human use (i.e. infl exal vaccine for infl uenza). yet while there is a wealth of research dating from the s and s based on improving immune responses with the aid of liposomes, generally more attention was given to the ability of the formulations to effectively deliver the vaccines through enhancing protection and apc uptake of the antigen. however, given current understanding of adjuvants, more recently the focus has turned not just to delivery attributes but also immunomodulation, therefore research has focussed on combining the delivery attributes of liposomes with adjuvant properties through the inclusion of immunomodulating molecules. for example, olsen et al. ( ) showed an increase and an induction of protective immunity against tuberculosis when isolated protein antigens, found in mycobacterial culture fi ltrates, were incorporated within liposomal vesicles combined with dimethyldioctadecylammonium (dda; when hydrated in an aqueous environment this cationic lipid self-assembles into closed bilayers) (olsen et al. ). yet these identifi ed proteins possess low inherent immunogenicity when injected alone (andersen ) . the authors reached a conclusion that in order for immune protection to remain high over an extended period, a depot must have been formed at the injection site by dda (holten- andersen et al. ; olsen et al. ). holten-andersen and co-workers conclude dda may act to increase antigen and immunostimulator uptake into apc when it forms the depot. the immunostimulatory properties of cationic lipids were originally documented in a screening study by gall ( ) . within this study a wide range of compounds were investigated for their ability to adjuvant diphtheria or tetanus toxoids in guinea pigs. compounds included non-ionic, anionic, and cationic surface-acting agents, amines, guanidines, benzamidines, thioureas, thiosemicarbazides, thiosemicarbazones, thiouroniums, and various nitrogenous bases. with regard to the group of surface-acting agents, or more commonly termed surfactants, gall observed increased adjuvanticity for those expressing cationic quaternary ammonium head groups and long alkyl chains (gall ) . within this group was dda, a synthetic amphiphile, which contains a quaternary ammonium group with two -carbonlong alkyl chains forming the hydrophobic moiety and two methyl groups, which together with the ammonium group form the polar head group (fig. . ) . the positively charged head group carries a monovalent counter ion, typically bromide or chloride. due to its amphiphilic character dda can form liposomal structures when dispersed in aqueous media at temperatures above its gel-to-liquid phase transition temperature (~ °c) (davidsen et al. ) . dda is known to induce cell-mediated immunity and delayed-type hypersensitivity (snippe et al. ) , and along with its cationic nature and surfactant properties, has been shown to be an effective adjuvant in numerous applications, including mucosal immunisation (klinguer et al. ) , gene delivery (esposito et al. ) , and subunit vaccine delivery (lindblad et al. ; brandt et al. ; holten-andersen et al. ; rosenkrands et al. ) . the mechanism of action behind the adjuvant effect of dda has been attributed to its positive surface charge and its ability to associate antigens (hilgers et al. ) . this was recently confi rmed and further elaborated by using ovalbumin (ova) as a model antigen ). stimulation of immature bone marrow-derived dendritic cells (bmdcs) with fl uorescently labelled ova showed that adsorption of ova onto dda enhanced the cellular acquisition of the antigen. further inhibition of active cellular processes by ova stimulation at °c or by the addition of cytochalasin d reduced the cellular uptake, suggesting that active actin-dependent endocytosis is the predominant uptake mechanism ). dda-mediated ova uptake was further associated with a functional enhancement of the apcs. this was shown by measuring the increase in ifngamma production and cellular proliferation of purifi ed autologous do . t-cells transgenic for a t-cell receptor recognising a major histocompatibility complex (mhc) class ii-restricted ova-epitope (ova - ). both proliferation and ifn-gamma production were increased upon interaction with either murine bmdcs or purifi ed b-cells, stimulated with ova adsorbed to dda christensen et al. ). more recent studies replacing dda with a neutral lipid, dspc, further demonstrate the role of the cationic lipid in the liposomal adjuvant the role of charge in the depot-effect of cationic liposomes has been further demonstrated by masking of the cationic charge in combination with manipulation of vesicle size and antigen location (fig. . ) ; results from our laboratory have shown that pegylation of dda-based liposomes (which contained an immunostimulatory lipid trehalose dibenate) with polyethylene glycol (peg) at mol% was able to signifi cantly inhibit the formation of a liposome depot and also severely limit the retention of antigen at the site, resulting in a faster drainage of the liposomes from the site of injection (soi). this change in biodistribution profi le was refl ected in the immunisation response, where lower levels of igg b antibody and ifn-γ and higher levels of il- cytokine were found. additional studies investigated the impact of a combination of reduced vesicle size and surface pegylation on the biodistribution and adjuvanticity of the formulations, in a bid to further manipulate the pharmacokinetic profi les of these adjuvants. from these biodistribution studies, it was found that with small unilamellar vesicles, % pegylation of the formulation could infl uence liposome retention at the injection site after days, while higher levels ( mol%) of peg blocked the formation of a depot and promoted clearance to the draining lymph nodes. interestingly, while the use of % peg in the small unilamellar vesicles did not block the formation of a depot at the soi, it did result in earlier antibody response rates and switch the type of t cell responses from a th to a th bias, suggesting that the presence of peg in the formulation not only controls the biodistribution of the vaccine, but also results in different types of interactions with innate immune cells (kaur et al. a , b ) . (kaur et al. a , b ) yet it is important to note that the effi cacy of dda may not be purely electrostatically driven as substitution of dda with other cationic lipids including _-[ n -( n ′ , n ′ -dimethylaminoethane)carbomyl] cholesterol (dc-chol; fig. . ) and , -dioleoyl- -trimethylammonium propane (dotap; fig. . ) were considered: while all three cationic liposomes facilitated increased antigen presentation by antigen-presenting cells, the monocyte infi ltration to the soi and the production of ifn-γ upon antigen recall was markedly higher for dda and dc-chol-based liposomes which exhibited a longer retention profi le at the soi. a long-term retention and slow release of liposome and vaccine antigen from the injection site hence appears to favour a stronger th immune response (henriksen-lacey et al. ) . similarly a modifi cation of the hydrophobic backbone of dda to a lower transition temperature lipid (dimethyldioctadecylammonium bromide; doda) demonstrated that the antigen would more readily dissociate from the less rigid bilayer, dodabased liposomes and these liposomes were also rapidly removed from the soi. this resulted in lower up-regulation of co-stimulatory cd and cd molecules on adjuvant-positive antigen-presenting cells (christensen et al. ) . furthermore, the adjuvant properties of these dda systems can be additionally supplemented by the use of a second lipid which can act as an immunomodulator. table . highlights example immunomodulators that can be incorporated into liposomal delivery systems. for example, α,α′-trehalose , ′-dibehenate (tdb; fig. . ) is a synthetic analogue of trehalose , ′-dimycolate (tdm) with two saturated fatty acid chains of carbons (behenyl), each replacing the branched mycobacterial mycolic acids of > carbons. these two behenyl chains are linked by ester bonds to carbon number of each of the two glucopyranose rings making up the trehalose head group. tdb has been shown to retain much of the bioactivity of the native form, while showing less toxicity as a result of the shorter fatty acid chains (pimm et al. ; olds et al. ) . the combination of the dda with tdb was fi rst studied by holten- andersen et al. ( ) . using esat- as a possible tb antigen they investigated the ability of seven different immunostimulators to increase the protective effi cacy of dda, which included four mycobacteria-derived immunostimulators. dda combined with mpl and/or tdb induced an effective ifn-gamma response and protection in mice was equivalent to that provided by bcg vaccination (christensen et al. ). the adjuvant activity of dda:tdb when combined with ag b-esat- was also compared to aluminium hydroxide, an adjuvant approved for human use (davidsen et al. ) . cd + t cells in mice secreted high levels of ifn-gamma and low levels of interleukin- (il- ) in response to dda:tdb, whereas the opposite pattern was observed for aluminium hydroxide (davidsen et al. ; christensen et al. ). although high levels of igg antibody titres were seen with both dda:tdb-adjuvated vaccine and aluminium hydroxide-adjuvated vaccine, higher levels of igg antibody titres were seen with dda:tdb (davidsen et al. ; agger et al. ) . dda:tdb has also been shown to induce a multi-functional cd + t-cell populations expressing several cytokines, mainly tumour necrosis factoralpha (tnf-alpha) + , il- + , and ifn-gamma + , tnf-alpha + , il- + . in mice such a population is maintained for at least year and thus are long-lived (lindenstrom et al. ). in addition to liposomes there are a wide range of alternative vesicle constructs and many of these have been investigated as potential adjuvants; these include niosomes (e.g. baillie et al. ) , surfactant polymers (e.g. polymersomes (okada et al. , see reference within mann et al. ) ), vesicles incorporating bile salts to improve stability (e.g. bilosomes (conacher et al. )), or virus components (e.g. virosomes (almeida et al. ) ) to name but a few. many of these systems use alternatives to phospholipids to circumvent potential issues related to storage instabilities and cost (e.g. synthetic-based systems), others to improve stability within harsh biological environments (e.g. bilosomes and polymersomes), or alternatively to modulate the properties of the vesicles in terms of immunological effi cacy (e.g. virosomes). in terms of niosome use for antigen delivery, the combination of -monopalmitoyl glycerol, cholesterol, and dicetyl phosphate (dcp) is often employed (bramwell and perrie ) . the inclusion of dcp within these systems helps vesicle formation and is also reported to enhance stability due to the electrostatic repulsive forces between the vesicles which restrict aggregation (bayindir and yuksel ) . the anionic nature of the vesicles has been reported to aid in uptake when delivering antigens via the oral route (eldridge et al. ). niosomes prepared from -monopalmitoyl glycerol, cholesterol, and dcp at a : : m ratio incorporating bovine serum albumin (brewer and alexander ) , ovalbumin (brewer et al. , see reference within bramwell and perrie ( ) ), or a synthetic peptide containing a known t-cell epitope (brewer et al. ) were shown to stimulate higher levels of igg a compared with freud's complete adjuvant, but the vesicle formulations were shown to be weak stimulators for igg . in addition, the adjuvant activity of niosomes was wholly dependent on the model antigen being entrapped within the vesicles; mixing free antigen with the preformed vesicles was unable to elicit a signifi cant immune responses (brewer and alexander ) . this was attributed to the ability of niosomes to retain the antigen for a prolonged period and promoting apc uptake through active or passive targeting to cells (brewer and alexander ; conacher et al. ). -monopalmitoyl glycerol (mpg)-based niosomes have also been considered for the delivery of dna vaccines. mpg-based niosomes incorporating cationic surfactants (dc-chol) rather than anionic surfactants have been shown to offer an increased stability and increased plasmid dna retention in the presence of competitive anions when compared to similarly formulated pc-based liposomes and engender transgene-specifi c immune responses comparable with their liposomal counterparts (obrenovic et al. ; perrie et al. perrie et al. , . as with the liposome systems a range of immunostimulatory agents have been considered in the design of niosomes as vaccine adjuvants. for example, mpgbased vesicles incorporating both dda and tdb were developed by vangala et al. ( ) , which resulted in an increase in the vesicle size due to the hydrophilicity of the surfactants, without altering the zeta potential of the vesicles compared to dda:tdb vesicles. these systems were used to deliver two malarial antigens (merozoite surface protein (msp ) and glutamate rich protein (glurp)), and the mpg-based vesicles, in comparison to dda liposomes, gave similarly strong th humoural responses when analysing igg titres; however, the mpg-based vesicles also showed high igg b titres unlike the dda:tdb systems (vangala et al. ) . in a further modifi cation of non-ionic-based vesicles, bile acids have been included in the formulation. these systems, known as bilosomes, have been developed to promote the oral delivery of vaccines by offering protection to antigens from the enzymes present in the git and acting as immunological adjuvants. this is achieved by incorporating bile salts such as sodium deoxycholate into the formulation thereby increasing the stability of the carrier, thus preventing premature release of the antigen via the oral route. it has been proposed that by incorporating bile salts into the vesicles this offers resistance against degradation and disruption from the digestive enzymes, therefore making the formulation more stable (schubert et al. ) . studies using bilosomes incorporating several antigens have proven to be successful in various animal models, e.g. the a/panama (mann et al. ) , tetanus toxoid (mann et al. ) , and hepatitis b (shukla et al. ). virosomes, in terms of general structural attributes, resemble liposomal systems and are often considered within the general area of liposomes. they are unilamellar vesicles (with a mean diameter < nm) built from phospholipids, but in addition, virosomes incorporate functional viral envelope glycoproteins, such as infl uenza haemagglutinin. this promotes heamaglutinin-receptor binding, cell fusion, and immunostimulation. of all the variations of liposomal systems discussed, currently only virosomes have been developed as clinical products. two examples of virosome-based vaccines are epaxal ® and infl exal ® (table . ), which are licensed in over countries for clinical use. immune-stimulating complex particles (iscoms) were initially described by morein et al. ( ) as novel structures that facilitated antigenic presentation of membrane proteins. iscoms are cage-like particles approximately nm in size that incorporate protein antigen through hydrophobic interactions and due to their particulate nature (myschik et al. ) . iscoms generally consist of a mixture of quil a saponins, cholesterol, and phospholipids. therefore, while this system contains lipids, their structural attributes are very different from those of liposomes, and it is the addition of the saponin to the phosophipid/cholesterol mixture that drives the change in structure of these nanosystems. iscoms can also be prepared to offer a cationic charge when dc-cholesterol replaces cholesterol, or the substitution of pc with dioleoyl-trimethyl-ammonium-propane. the cationic complexes formed are similar to the classical anionic iscoms and allow a more diverse range of antigens to be used in their formulation (lendemans et al. , see reference within lendemans et al. ). orally, iscoms have shown promising systemic immune responses by eliciting th , th , and mhc-restricted cytotoxic t-cell responses in addition to local induction of iga (mowat et al. ) . as a result, the introduction of immunostimulatory agents has been shown to be useful to enhance immune responses to sub-unit vaccines and offer a promising platform for further studies. solid nanoparticles have also been extensively investigated for their potential as vaccines adjuvants. like the vesicle type systems already described, they can be an analogue of trehalose , ′-dimycolate (tdm) but consists of a shorter fatty acid chains, therefore is considered to be less toxic davidsen et al. ( ) . very immunogenic as a co-adjuvant for eliciting protective immunity against tuberculosis holten-andersen et al. ( ); davidsen et al. ( ) prepared in a range of sizes, with a choice of surface characteristics and include a selection of immunomodulators. polymeric nanoparticles are generally formulated from natural or synthetic polymers with the most commonly studied polymers being those which are biodegradable such as poly(lactide-co-glycolide) (plga), poly(lactic acid) (pla), poly(caprolactone) (pcl), and polysaccharides (particularly chitosan). the advantage of these polymers is that they are well characterised and used in a range of clinical products, particularly plga. alternatively, nanoparticles can be prepared from solid (high melting point) lipids dispersed in an aqueous phase. examples of lipids used can include solid triglycerides, saturated phospholipids, and fatty acids which are well tolerated by the body. due to their composition, they are sometimes described as 'solidifi ed' o/w emulsions in which the oil globule is replaced by solidifi ed lipids. much like the solid polymeric nanoparticles, solid lipid nanoparticles can be used as vaccine adjuvants by the antigen being incorporated within the lipid matrix of the particle or by attaching it to the lipid nanoparticle surface. lipid particles normally start from around nm in size and can be prepared in a large-scale by homogenisation to disperse the lipid into an aqueous environment. the use of polymer-based particulates as vaccine adjuvants has been strongly investigated, but it has been more recent work that has refocused investigations into the potential advantage of using these systems in the nano range. for example, work by stano et al. ( ) demonstrated that by increasing the size of their polypropylene sulphide nanoparticles from to nm, the antigen was more effectively delivered into both mhc class i and mhc class ii-presentation pathways. enhancing the targeting of nanoparticles to dendritic cells has also been considered, e.g. the addition of a recombinant fusion protein to the surface of plga nanoparticles was shown to promote a twofold increase in dc uptake in vitro, and in vivo studies demonstrated these formulations promoted enhanced antigen-specifi c igg and igg subclasses, and higher cytokine responses. while not all emulsion-based systems fall within the nanoscience defi nition, it is useful to consider the role of emulsions in general in vaccine formulation. in the fi rst emulsion adjuvant mf ® (novartis, italy) was licensed in europe in the infl uenza vaccine fluad ® (o'hagan and gregorio ). mf ® is an oil-in-water (o/w) emulsion composed of % squalene (naturally occurring oil) combined with surfactants sorbitan trioleate (span ) and poly(oxyethylene) sorbitan monooleate (tween ). despite concerns regarding its safety due to the occurrence of autoimmune dysfunctions in rats (carlson et al. ) , the adjuvant has an established safety profi le in humans of good-health and immunocompromised populations (black et al. ; donatoa et al. ) . it is interesting to note that mf ® is not the fi rst emulsion to be licensed for human use; the well-known experimental adjuvant incomplete freund's adjuvant (icf) (freund et al. ) was once used in human infl uenza vaccines (chang et al. ) . in contrast to mf ® , icf is a water in oil (w/o) emulsion composed of a light mineral oil (such as bayol f) and the emulsifi er mannide monooleate (aracel a™), combined in a : volume ratio (lindblad ) . while it has been withdrawn from human use due to occasional serious local reactions, it remains an experimental gold-standard adjuvant in immunisation protocols. the success of mf ® has been attributed to the rendering of soluble antigen to particulate form, improved cell recruitment to the injection site, and antigen uptake with transport to local lymph nodes (mosca et al. ). similar to aluminium salts, no direct activation of dendritic cells has been noted, although improved traffi cking and antigen uptake by macrophages and dcs, respectively, has been noted, and expression of soluble activation factors may indeed lead to indirect dc activation (mosca et al. ). in terms of potential advantages of these solid nanoparticles over the bilayer type systems, there has been few direct comparisons made, as it is diffi cult to control the number of different parameters between the systems. similarly little work has been undertaken to understand the impact of the route of administration of such these adjuvants on the type and strength of immune response promoted. however, in a recent multi-centre study, the difference in immune responses generated in mice vaccinated by the subcutaneous, intradermal, intramuscular, and intralymphatic routes was considered with ovalbumin-loaded liposomes, n -trimethyl chitosan (tmc) nanoparticles, and poly(lactide-co-glycolide) (plga) microparticles, all with and without specifi cally selected immune-response modifi ers were directly compared (mohanan et al. ) . interestingly, neither the route of administration nor the presence of immunomodulators within the formulations made a notable difference to induced igg antibody responses. however, the administration route had a strong impact both on the kinetics and magnitude of the igg a response: a single intralymphatic administration of all the evaluated vaccine formulations (liposomes, nanoparticles, and microspheres) generated a strong igg a response, whereas subcutanteously, only the adjuvanted nanoparticles were able to promote notable igg a responses, and the intradermal and intramuscular routes generated intermediate igg a responses (mohanan et al. ) . the benefi t of the intralymphatic administration route for eliciting a th -type response was confi rmed in terms of ifngamma production. this study demonstrated that the igg a associated with th -type immune responses is sensitive to the route of administration, whereas igg response associated with th -type immune responses was relatively insensitive to the administration route of the particulate delivery systems. therefore, consideration of the vaccine formulation in combination with the route of administration should be considered when planning and interpreting preclinical research or development on vaccine delivery systems. given the range of clinically approved nanotechnology-based pharmaceutical products already available, it is interesting to note that currently only virosomal vaccines [epaxal ® and infl exal ® v (crucell)] are clinically approved for use. however, until the desirable characteristics for a nanosystem-based vaccine adjuvant are fully elucidated, it is diffi cult to identify key attributes we should be focusing on other than fi nal in vivo performance, which is a lengthy and expensive marker to rely on. similarly there continues to be issues regarding regulation. for example the who stipulates that liposomal adjuvants must be licensed as a vaccine formulation, and not as an adjuvant which could be combined with various antigens post-licensing. this adds further complications to the development and licensing processes. however, progress continues with cationic nanosystems such as the dda:tdb formulation developed by staten serum, institute, and the cationic adjuvant ic ® (intracell, austria). overall, liposomes offer a strong potential as vaccine adjuvants by combining the ability to deliver antigens to the correct cells, and also appropriately interact and stimulate such cells. while progress remains limited to-date, new advances in the understanding of effective vaccine systems and in the production and regulation of liposomes in a cost-effective manner should enhance their progress into the clinical setting. . identify the various vaccine types currently clinically available. discuss their respective advantages and disadvantages. answer: vaccines can be divided into three basic groups: ( ) live vaccines, ( ) inactivated vaccines, ( ) toxoids and subunit systems. live attenuated vaccines consist of live microorganisms or viruses that have mutated so that it has a reduced ability to grow in human cells. they are still able to infect their target cells, but the infection is mild and replication is limited. these vaccines generally give good protection, but there is a risk of reversion to virulence, and this type of vaccine is not considered safe for use in immunocompromised individuals. inactivated vaccines consist of microorganisms or viruses that have their ability to be infectious removed while retaining immunogenicity. in terms of advantages they tend to have improved safety profi les compared (continued) with live system, they are generally less effective than live attenuated vaccines, usually only stimulating humoural immunity and often requiring booster doses. the third group consists of part of the infectious agent, such as excreted toxoids or subunit proteins. subunit vaccines initiate strong immune responses using a small part of the organism. the advantage of subunit vaccines is their good safety profi le; however, the immune response induced by such vaccines is short-lived and thus several boosts are required to achieve protection. . describe the structural attributes of virosomes and discuss, with the use of examples, how these are able to be used as vaccines. answer: virosomes are bilayer vesicles around nm in size. they are prepared from phospholipids and incorporate functional viral envelop glycoproteins, such as infl uenza haemagglutinin. this promotes heamaglutinin-receptor binding, cell fusion, and immunostimulation. for example, epaxal is a virosome vaccine clinically indicated for hepatitis a immunisation. by incorporating the subunit antigen for hepatitis a, the antigen can be protected from the extracellular milieu, thereby limiting antigen breakdown by enzymes, as well as preventing the rapid removal of such small compounds by the mps. this ensures the antigen carried within the virosome delivery system to the appropriate immunological cells, and subsequently the antigen is taken by the cells due to the action of the glycoproteins. adjuvant modulation of the cytokine balance in mycobacterium tuberculosis subunit vaccine; immunity, pathology and protection liposomes as immunological adjuvants formulation of virosomes from infl uenza subunits and liposomes effective vaccination of mice against mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins new viral vaccines the preparation and properties of niosomes-non-ionic surfactant vesicles damps, pamps and alarmins: all we need to know about danger charcterisation of niosomes prepared with various non-ionic surfactants for paclitaxel oral delivery basic immunology for the non-immunologist: from pathophysiology to therapeutics safety of mf -adjuvanted versus non-adjuvanted infl uenza vaccines in children and adolescents: an integrated analysis cytokines as adjuvants for the induction of mucosal immunity the rational design of vaccines esat- subunit vaccination against mycobacterium tuberculosis how) do aluminium adjuvants work? the adjuvant activity of non-ionic surfactant vesicles (niosomes) on the balb/c humoral response to bovine serum albumin cationic lipid dc-chol induces an improved and balanced immunity able to overcome the unresponsiveness to the hepatitis b vaccine the endogenous adjuvant squalene can induce a chronic t-cell-mediated arthritis in rats adjuvant activity of incomplete freund's adjuvant liposome-based cationic adjuvant formulations (caf): past, present, and future vaccine adjuvants based on saturated quaternary ammonium lipids have different in vivo distribution kinetics and display distinct t cell-inducing capacity compared to their unsaturated analogues the global impact of vaccines containing aluminium adjuvants oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts (bilosomes) characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from m. tuberculosis (trehalose , dibehenate) a novel adjuvant inducing both strong cmi and antibody responses liposomes as adjuvants with immunopurifi ed tetanus toxoid: infl uence of liposomal characteristics imiquimod and resiquimod as novel immunomodulators safety and immunogenicity of mf -adjuvanted inuenza vaccine in the elderly the use of cytokines and chemokines as genetic adjuvants for plasmid dna vaccines measurement of chemically induced cell proliferation in rodent liver and kidney: a comparison of -bromo- '-deoxyuridine and [ h] thymidine administered by injection or osmotic pump preparation and characterization of cationic microspheres for gene delivery antibody formation and sensitization with the aid of adjuvants developing adjuvants for public use: a long and treacherous road the adjuvant activity of aliphatic nitrogenous bases immunological notes xxiii. the antigenic value of toxoid precipitated by potassium alum the myth of the medical breakthrough: smallpox, vaccination, and jenner reconsidered noninvasive vaccine delivery in transfersomes, niosomes and liposomes: a comparative study liposomal cationic charge and antigen adsorption are important properties for the effi cient deposition of antigen at the injection site and ability of the vaccine to induce a cmi response comparison of the depot effect and immunogenicity of liposomes based on dda, dc-chol and dotap: prolonged liposome retention mediates stronger th responses combination of two synthetic adjuvants: synergistic effects of a surfactant and a polyanion on the humoral response vaccines in historic evolution and perspective: a narrative of vaccine discoveries combination of the cationic surfactant dimethyldioctadecylammonium bromide and synthetic mycobacterial cord factor as an effi cient adjuvant for tuberculosis subunit vaccines the immune system evolved to discriminate infectious nonself from noninfectious self vaccination against murine cutaneous leishmaniasis by using leishmania major antigen/liposomes. optimization and assessment of the requirement for intravenous immunization pegylation of dda: tdb liposomal adjuvants reduces the vaccine depot effect and alters the th /th immune responses manipulation of the surface pegylation in combination with reduced vesicle size of cationic liposomal adjuvants modifi es their clearance kinetics from the injection site, and the rate and type of t cell response smallpox vaccines for biodefense lipophilic quaternary ammonium salt acts as a mucosal adjuvant when co-administered by the nasal route with vaccine antigens the adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes cage-like complexes formed by dotap, quil-a and cholesterol freund's adjuvants adjuvant modulation of immune responses to tuberculosis subunit vaccines tuberculosis subunit vaccination provides long-term protective immunity characterised by multifunctional cd memory t cells optimisation of a lipid based oral delivery system containing a/panama infl uenza haemagglutinin oral delivery of tetanus toxoid using vesicles containing bile salts (bilosomes) induces signifi cant systemic and mucosal immunity asc, ipaf and cryopyrin/nalp : bona fi de intracellular adapters of the caspase- infl ammasome towards an understanding of the adjuvant action of aluminium the infl ammasome: a molecular platform triggering activation of infl ammatory caspases and processing of proil-β tolerance, danger and the extended family new adjuvants for human vaccines administration routes affect the quality of immune responses: a cross-sectional evaluation of particulate antigen-delivery systems activating immunity: lessons from the tlrs and nlrs effect of phosphorylation of ovalbumin on adsorption by aluminium-containing adjuvants and elution upon exposure to interstitial fl uid effective subunit vaccines against an enveloped animal virus molecular and cellular signatures of human vaccine adjuvants immue-stimuating complexes as adjuvants for indicing local and systemic immunity after oral immunisation with protein antigens effect of dimerization of the dglucose analogue of muramyl dipeptide on stimulation of macrophage-like cells on the preparation, microscopic investigation and application of iscoms the path to a successful vaccine adjuvant-'the long and winding road plasmid dna entrapment into niosomes: characterisation studies induction of resistance to schistosoma mansoni by natural cord factor and synthetic lower homologues protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen b and esat- the immune system liposome-mediated dna vaccination: the effect of vesicle composition liposome-mediated dna vaccination via the oral route liposome-mediated dna immunisation via the subcutaneous route liposome and niosome mediated dna vaccination via the subcutaneous route viability counts on bcg vaccines for tumour immunotherapy; divergent effects on different growth media sur l'augmentation anormale de l'antitoxine chez les chevaux producteurs de serum antidiphtierique jenner and the history of smallpox and vaccination cationic liposomes containing mycobacterial lipids-a new powerful th adjuvant system immunological concepts of vaccine adjuvant activity studies on the mechanism of bile saltinduced liposomal membrane damage cell injury releases endogenous adjuvants that stimulate cytotoxic t cell responses m-cell targeted delivery of recombinant hepatitis b surface antigen using cholera toxin b subunit conjugated bilosomes the adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes delayed-type hypersensitivity in rabbits. comparison of the adjuvants dimethyl dioctadecyl ammonium bromide and freund's complete adjuvant nanoparticle size infl uences the magnitude and quality of mucosal immune responses after intranasal immunization mechanisms of bacterial pathogenesis and targets for vaccine design safety and immunogenicity of a hepatitis b vaccine formulated with a novel adjuvant system a comparative study of cationic liposome and niosome-based adjuvant systems for protein subunit vaccines: characterisation, environmental scanning electron microscopy and immunisation studies in mice immunologic adjuvants for modern vaccine formulations trehalose , '-dimycolate (cord factor) of mycobacterium tuberculosis induces foreign-bodyand hypersensitivity-type granulomas in mice key: cord- -gmitz gg authors: clemens, john d.; ochiai, r. leon title: sequential stages of clinical trials and overview of issues to be considered date: journal: the grand challenge for the future doi: . / - - - _ sha: doc_id: cord_uid: gmitz gg nan we live in an exciting era in which the fruits of molecular biology and biotechnology are yielding a profusion of new and improved candidate vaccines, delivery systems, and adjuvants that have the potential to control many of the infectious disease scourges of the developing world. indeed, it has been estimated recently that there are over vaccine candidates currently under development against nearly infectious diseases [ ] . discovery of a new vaccine candidate and demonstration of its safety, immunogenicity, and protectivity in animal models are, however, only the first steps toward licensure and introduction of the vaccine into public health practice. the longest phase of development for most vaccines before licensure, and arguably the most uncertain, laborious, and expensive phase, comprises clinical testing of the vaccine in humans [ ] . at a minimum, this clinical testing must demonstrate the vaccine to be acceptably safe and suitably protective in the population that will ultimately be targeted for the vaccine in public health practice [ ] . over the years, regulatory agencies have adopted a standard paradigm for the manner in which new vaccines are tested in humans. a key feature of this paradigm is the phased fashion in which the testing occurs. this chapter describes the phases of vaccine evaluations in humans, including several considerations for the phased testing of vaccines in developing countries. vaccines have had a remarkable track record of safety. nevertheless, even when manufactured flawlessly, some vaccines have caused serious sideeffects [ ] . the mechanisms for these side-effects are diverse and are sometimes related to such problems as vaccine-induced immunopathology, as occurred with early-generation measles and respiratory syncytial virus vaccines [ , ] . at times the mechanism may be obscure, as was the case for sequential stages of clinical trials and overview of issues to be considered intussusception induced by quadrivalent rhesus reassortant vaccine against rotavirus diarrhea [ , ] . whatever the mechanism, the point to be noted is that such severe side-effects are not always predictable. to minimize potential injuries to subjects caused by vaccine side-effects during re-licensure trials, vaccines are tested in a phased manner. in this phasing, early evaluations are conducted in small numbers of subjects, so that if reactions are observed, they will affect a minimum number of volunteers. and early evaluations are typically conducted in the least vulnerable subjects, often healthy adults, so that if the reactions occur, their severity will be minimized. successive evaluations of a particular vaccine are then conducted in progressively larger numbers of volunteers, and, as confidence in the safety-profile of the vaccine increases, in subjects in the ultimate target group, including persons who are more vulnerable (e.g., infants). with these successive studies, the complete ensemble of information about the vaccine's safety, immunogenicity, protectivity, and sometimes additional characteristics (e.g., transmissibility) is accrued. moreover, the large number of subjects ultimately studied ensures estimation of these features in a statistically precise fashion, and, for sideeffects, in a way that enables detection of relatively rare events. clinical trials for licensing a new vaccine candidate are generally planned in three phases. in the code of federal regulations (usa), the phases of clinical trials are described by using arabic numerals (phase , phase , phase ), while in world health organization publications, roman numerals are generally used (phase i, phase ii, phase iii). in this paper, we use roman numerals to describe the phases of clinical trials. phase i trials of experimental vaccines are the first human studies to be conducted after preclinical studies have demonstrated suitable safety, potency, immunogenicity, and, when possible, protectivity. the primary purpose of a phase i trial is to rule out the possibility of frequent vaccine sideeffects. additional goals include preliminary assessment of vaccine immunogenicity, determination of an appropriate dose and regimen, and, for live vaccines, measurement of vaccine shedding. phase i trials are typically small, often on the order of - subjects, usually enroll healthy adults, and may be done with preliminary formulations of the vaccine. depending on whether there are concerns about potential severe sideeffects and the need for biological containment of excreted vaccines, such studies may be done on an inpatient or an outpatient basis. for example, a genetically attenuated, live oral vaccine candidate might well be tested initially on an inpatient basis with containment if there is concern about transmission of fecally excreted vaccine organisms, or about the genetic stability of the candidate during the course of fecal shedding [ ] . often, phase i studies are designed in an uncontrolled fashion. for vaccine candidates that are found to yield promising findings in phase i trials, phase ii trials may be undertaken. the primary goals of phase ii trials are to evaluate vaccine safety and immunogenicity in larger numbers of subjects, and ultimately in the target population for whom the vaccine is intended. for live vaccines, phase ii trials may also be designed to evaluate vaccine shedding and transmissibility. phase ii trials may also further evaluate different vaccine doses and regimens. a frequently used strategy for phase ii trials of vaccine candidates intended for infants is to initiate the studies in an older age group, often adults, and to conduct successive studies in progressively younger age groups, with the transition to each younger age group contingent on satisfactory results from the study of the previous age group. in contrast to phase i trials, which are often done with preliminary formulations, phase ii trials typically evaluate the final formulation of the vaccine, since data from studies of preliminary formulations are not usually eligible for consideration by regulatory authorities in their deliberations about vaccine licensure. phase ii trials are typically larger than phase i trials, sometimes enrolling or more subjects. by the time that a vaccine candidate has reached phase ii, concerns that would mandate testing it on an inpatient basis have typically been resolved, and outpatient studies are the norm. in contrast to phase i trials, phase ii trials are conventionally designed as randomized, controlled trials, and control groups typically receive a placebo or an active agent to permit blinding. for live vaccine candidates, there may be a concern about unintended transmission of the vaccine strain from vaccinees to non-vaccinees with whom they are in contact. special phase ii trials are sometimes conducted to assess the transmissibility of such vaccine candidates. for example, a phase ii study of the live oral cholera vaccine, cvd -hgr, in jakarta, indonesia, randomly allocated pairs of sibling children within households to either vaccine or placebo, and judged transmissibility of the vaccine candidate by the rate of fecal excretion of the vaccine strain and seroconversion to the vaccine in placebo recipients [ ] . phase ii trials may also be done to address environmental concerns about genetically engineered, live vaccine candidates. for cvd -hgr, for example, open sewers outside the jakarta homes of children who had received the vaccine were sampled to evaluate whether there was any detectable persistence of the vaccine strain during the - hours after vaccination [ ] . a recent trend in vaccine evaluation is to use phase ii trials to obtain initial data on the level of vaccine protection against targeted, naturally occurring infections. what distinguishes these phase ii trials from conventional phase iii trials (vide infra) is that they are smaller in size (although usually larger than phase ii trials geared only to the assessment of safety and immunogenicity) and less able to evaluate vaccine protection with suitable statistical precision than well-designed phase iii trials. also in contrast with phase iii trials, such phase ii trials may be used to evaluate vaccine prototypes that will be later modified or augmented into final vaccines for licensure (e.g., a vaccine against a single serotype, when a multi-serotype vaccine will be required for the final vaccine), and may attempt to obtain estimates of vaccine protection based on prevention of a surrogate endpoint (e.g., the use of hiv viral load as a surrogate for the rapidity of hiv disease progression). for certain vaccines, studies are done in volunteers to evaluate the clinical protection against an intentional challenge with the target pathogen. such studies are sometimes termed phase iib trials. in these studies volunteers are typically allocated at random to receive the vaccine or a comparison agent, usually a placebo, and are then challenged at a defined interval after vaccination with an inoculum of the pathogen predicted to cause the target disease in nearly % of the control group. the comparative attack rate of the target disease in vaccinees versus controls provides an estimate of the conventional measure of vaccine protective efficacy (pe) = ( minus the relative risk of the disease in vaccinees versus controls) × %. estimates of vaccine protection in phase iib studies, which are typically small, often conducted in ca. - subjects, may be helpful in triaging vaccines that are deserving of study in larger and more expensive phase iii trials [ , ] . in addition to providing estimates of vaccine protection, phase iib trials can serve to provide data on vaccine safety, vaccine immunogenicity, vaccine shedding and transmissibility (for live vaccines), and preliminary assessments of immunological responses that correlate with protection. because such studies entail intentional challenge with pathogens, subjects for the studies should always be healthy adults. the intentional challenge also limits phase iib trials to infections for which there is no risk of severe acute complications, significant sequelae, or chronic infection if appropriate therapy is administered promptly upon recognition that the challenge has resulted in infection. examples of pathogens for which phase iib studies have been successfully carried out include cholera, diarrheagenic escherichia coli, shigella, rocky mountain spotted fever, malaria, and influenza [ ] . it is imperative that such studies be carried out by staff who are highly skilled in the diagnosis and treatment of the infections under study, and it is usually desirable to conduct the challenge phase of these studies under inpatient conditions. for vaccine candidates found to be suitably safe and immunogenic in phase ii trials, phase iii trials may be done to provide rigorous evidence about vaccine protection against naturally occurring infections, and to provide additional data on vaccine safety in larger numbers of vaccinees. phase iii studies are designed as randomized, controlled trials with clear hypotheses, and are conducted in the target group for whom vaccine licensure is desired and in a population that normally experiences the target infection. these studies thus constitute pivotal evaluations that provide the basis for decisions about whether to license a vaccine for use in public health practice [ ] . figure diagrammatically depicts the design of a phase iii trial comparing two groups. in such a trial participants are recruited from a target population and are enrolled for the study after acquisition of informed consent and ascertainment of eligibility. prior to the trial, a formal randomization scheme is developed to allocate subjects to an experimental vaccine group or a control group, and this randomization scheme is used to allocate the compared agents to consenting subjects who are eligible for participation. to ensure blinding of investigators and subjects to the identities of the compared agents, controls may receive an inert placebo or an active vaccine. if the latter is chosen, it is typically an agent that is identical in appearance to and given with the same regimen as the experimental vaccine, but does not elicit immune responses that are known to protect against the pathogen targeted by the experimental vaccine. after randomization, subjects in the compared groups are followed concurrently with uniform surveillance procedures to detect target infections and adverse events, and the target infections adverse events immune responses figure . a simplified schematic of a phase iii vaccine trial designed as a two-group, randomized, controlled trial. in such a trial, the study population is assembled from a target population and is then randomized to receive an experimental vaccine or a control agent. the experimental and control groups are followed longitudinally and concurrently to detect the comparative occurrence of target infections, adverse events, and immune responses in the two groups to assess vaccine protection, vaccine safety, and vaccine immunogenicity, respectively. comparative rates of these events in the two groups form the basis for the assessment of vaccine pe and safety. similarly, participants, or a subsample of participants, are assessed immunologically at baseline and at a defined interval after dosing. because phase iii trials must provide statistically meaningful estimates of vaccine pe (calculated in the same way as described above for phase iib trials) and because the target disease outcomes to be prevented by vaccination in the trials are typically rare in occurrence, phase iii trials are often quite large, sometimes enrolling tens of thousands of subjects. in addition, because it may be necessary to measure vaccine pe over several years following vaccination in order to provide information necessary to convince regulatory and public health authorities, phase iii trials may entail maintenance and follow-up of study populations for long durations. the large size, lengthy duration, prospective conduct, and extensive quality control and quality assurance procedures needed for phase iii trials make them extremely expensive, often costing millions of dollars. measurement of immune responses in phase iii trials is done for two purposes. first, it is necessary to document that the vaccine tested in a phase iii trial elicited the level of immune response expected on the basis of earlier studies. comparison of immune responses in the vaccine and control groups permits estimation of the proportion of immune responders among the experimental vaccine group that can be attributed to receipt of the vaccine. if vaccine pe proves unexpectedly low in the trial, this assessment will be crucial in helping to explain these results. assessments to assess whether immune responses were as robust as expected usually require evaluation only of a small subsample of subjects participating in the trial. second, it is often desirable that a trial define an immunological correlate of vaccine protection -a level of short-term immune response to vaccination that demarcates vaccinees who are protected against the target infection. immunological correlates of protection are best defined by comparing immune responses to the vaccine in vaccinees who develop the target infection (breakthroughs) versus vaccinees who do not. definition of immunological correlates of vaccine protection is very important because such correlates permit assessments of the protection of the tested vaccine and ones suitably similar to it in small, short-term studies with immunological endpoints, without resort to full-scale, phase iii efficacy trials with clinical infection endpoints. phase iii trials usually provide the only opportunity before licensure for determining an immunological correlate of protection. unfortunately, determination of immunological correlates within phase iii trials can be logistically demanding and costly. these problems result from four considerations: ) it is necessary to contrast short-term immune responses in vaccinees who develop the target infection versus those who do not in order to determine an immunological correlate of protection; ) at the time of vaccination and collection of specimens for immunological assessments it is unpredictable which vaccinees will acquire the target infection ("breakthrough events"); ) most phase iii trials have very few breakthrough events; and ) blinding of phase iii trials prevents knowledge of who received the vaccine and who received the control agent. the implication of these considerations is that it may be necessary to obtain suitable specimens from virtually every participant in the trial to ensure that immunological assessments will be available for a large enough number of vaccine breakthroughs to enable statistically meaningful assessments. moreover, even when arrangements are made to collect the necessary specimens from the required number of subjects, measurement of immune responses in these specimens may not necessarily yield an immunological correlate of the vaccine protection observed in the trial, sometimes for obscure reasons [ ] . experience has demonstrated that the performance of a vaccine in affluent populations in industrialized countries cannot be assumed to generalize to persons living in developing-country settings. examples of vaccines that performed less well in developing-country than in industrialized-country populations have included oral polio vaccine, certain live oral rotavirus and cholera vaccines, and parenteral, polysaccharide-diphtheria protein conjugate haemophilus influenzae type b (hib) vaccine [ ] [ ] [ ] [ ] . it is therefore important that new vaccines be tested in developing countries prior to their licensure in these settings. this raises the question, for vaccine candidates developed in the industrialized world, of when in the phased sequence the candidate should be tested in the developing world. traditionally, it has been argued, partly over concerns about using poor populations in the developing world as "human guinea pigs", that trials in the developing world should commence only when a vaccine has been fully evaluated and licensed in the industrialized world. recent changes in thinking have challenged this view. it is now recognized that deferring trials in developing countries to this extent carries the unwanted consequence of significantly delaying the availability of vaccines to populations in developing countries. for example, polysaccharideprotein vaccines against hib, which have been licensed for over a decade in the united states and other industrialized countries and have nearly eliminated invasive hib disease in children in many of these settings, are used only to a limited extent today in the developing world [ ] . this inequity in use of new-generation vaccines has elicited calls for parallel testing, rather than sequential testing, of new vaccines in industrialized-and developing-world populations. parallel-testing of new vaccines in the industrialized and developing worlds is now being pursued for both experimental hiv and rotavirus vaccines [ , ] . another important trend affecting the testing of vaccines in developingcountry populations is the emergence of highly qualified vaccine producers in the developing world, some capable of developing innovative new vaccines. clearly, it is appropriate that the initial groups for testing of an experimental vaccine developed by such producers be selected from the population of the country in which the vaccine is developed. conventional descriptions of phased trials often portray a seamless progression of trials from phase i to phase ii, and from phase ii to phase iii. in reality, the vast majority of vaccine candidates that are tested in phase i studies do not progress all the way through phase iii testing, because of disappointing findings or because of a lack of sufficient resources to support the increasingly expensive studies in successive phases [ ] . while unwelcome findings at any stage may lead to termination of the clinical development of a vaccine, this need not always be the case. for example, the live-attenuated, oral cholera vaccine, cvd -hgr, was shown to be safe and highly immunogenic when given as a single dose of × organisms in an extensive series of studies in north american volunteers. however, when the vaccine was subsequently tested in thai adult volunteers, it proved erratically immunogenic [ ] . to address this problem, the vaccine developer raised the dose by one log, to × organisms, and found the vaccine to be suitably immunogenic but still safe in a variety of settings in developing countries [ ] . the cvd -hgr experience illustrates that successful vaccine development may require detours in the phased sequence of clinical trials in order to accommodate necessary adjustments in vaccine dose, schedule, or even formulation in response to findings that emerge during the course of clinical studies. however, because vaccines that appear promising in developed countries not infrequently fail to perform as well in developing countries, and because such detours can greatly increase the expense of clinical development, completion of clinical testing of vaccines for developing countries may be exceptionally challenging. the conventional sequence of phases usually culminates in one or more phase iii trials that are designed to provide pivotal evidence on the clinical protection conferred by the vaccine against the targeted, naturally occur-ring, infectious disease. sometimes, however, proof of clinical efficacy in a phase iii trial may not be necessary for licensure of a vaccine. for example, cvd -hgr vaccine against cholera was licensed in europe for use in travelers on the basis of phase i-ii studies, together with phase iib trials showing a high level of protection against experimental cholera in north american volunteers [ ] . however, it is unlikely that phase iib evidence from industrialized countries will be taken as a basis for licensure of vaccines in developing countries, in part because of the discrepancies between vaccine performance in developed versus less-developed settings and in part because of the uncertainties about whether protection in phase iib studies predicts the performance of a vaccine in populations experiencing endemic disease. in addition, if there is an accepted correlation between a certain immune response (e.g., a serological antibody titer) to vaccination and clinical protection, this immune response may sometimes be used as prima facie evidence for judging whether the vaccine is likely to be sufficiently protective to warrant licensure. for example, prp-crm diptheria toxin and prp-neisseria meningitidis outer membrane protein (omp) conjugate vaccines against hib were licensed for infants on the basis of phase iii evidence of clinical protection against invasive hib disease. subsequently, prp-tetanus toxoid conjugate vaccine was licensed for infants in the united states largely on the basis of proven safety and the attainment of protective serum antibody titers to prp, without corresponding evidence of clinical protection from a phase iii trial [ , ] . serological endpoints are also commonly used as a basis for licensure of combination vaccines for which the component vaccines have already been licensed and seroresponse criteria for these components have been established [ ] . some vaccines of public health importance are directed to diseases that cannot be reliably predicted in populations at risk and that cannot be ethically studied in phase iib trials. examples include vaccines against some bioterrorism agents such as anthrax and plague, and vaccines against certain epidemic diseases such as sars and ebola. in these situations, decisions about licensure will have to depend on pre-clinical data, demonstration of safety in humans, and assessments of immune responses in humans (even if such immune responses are not known with certainty to be correlated with clinical protection of humans against the target disease). in recent years, considerable attention has been devoted to the ethics, scientific quality, conduct, documentation, and reporting of clinical trials. several organizations have produced guidelines for these activities, which are subsumed under the rubric of "good clinical practice (gcp)". guidelines for gcp have been published by the world health organization, the united states food and drug administration, and the international conference on harmonization (ich), among others [ ] . table presents the essential elements of gcp in guidelines issued by the international conference on harmonization [ ] . the promulgation of gcp has had several implications for vaccine trials in developing countries. increasingly, the world of vaccines is becoming globalized. vaccines targeted for a global market may be tested in developing countries to provide initial licensure. for example, a live oral human rotavirus vaccine developed by a major multinational vaccine producer has recently received its initial licensure in mexico. moreover, vaccines produced by manufacturers in developing countries are now being used throughout the world. indeed, the majority of all doses of measles vaccines now administered to children in the world are produced in india. with the trend toward globalization of vaccines, increasing emphasis has been placed on conducting clinical trials in developing countries in a manner that conforms to international gcp standards. while the benefits of gcp trials are unquestionable, rigid adherence to gcp standards can be a double-edged sword. the expense of clinical trials has risen rapidly in recent years [ ] . a portion of this increased expense arises from the extensive documentation and auditing requirements demanded by regulatory agencies as a component of gcp, together with the expense of proprietary products developed to ensure compliance with regulatory requirements, such as proprietary computer software for data management and electronic reporting of trials. while this increased expense can be borne by producers anticipating major markets for their vaccines in affluent markets, it constitutes a disincentive to the clinical testing of vaccines against "orphan diseases" affecting developing countries, for which lucrative markets are not foreseen. the ethical guidelines for clinical trials in developing countries have been the subject of recent controversy. the ethical principles of respect for persons, beneficence, and justice, which underlie modern clinical trials, are well accepted [ ] . nevertheless, several issues continue to be debated, of which three are touched on here [ ] [ ] [ ] . one issue concerns the threshold for the risk/benefit ratio for a vaccine to be tested in developing countries. for example, a live oral, quadrivalent rhesus rotavirus-reassortant vaccine was licensed and then withdrawn from the united states market by its manufacturer because of an association between vaccination and the rare occurrence of intussusception [ , ] . understandably, this action had a chilling effect on the testing of this vaccine in developing countries. a meeting of experts organized by the world health organization acknowledged the rationale for the company's withdrawal of this vaccine in the united states, where rotavirus diarrhea is a cause of morbidity but not of significant mortality. nevertheless, there was agreement among participants in the meeting that the burden of mortality of rotavirus in developing countries provided an ethical justification for continued evaluation of this vaccine in these settings [ ] . a second controversy over the ethics of trials in developing countries has focused on the use of placebos. as stated in the declaration of helsinki, "the benefits, risk, burdens, and effectiveness of a new method should be tested against the best current prophylactic, diagnostic, and therapeutic methods" [ ] . there is agreement that a placebo would be inappropriate as a control agent for a trial if a locally licensed standard alternative vaccine exists; there is also general agreement that if such an alternative does not exist, it would be preferable to use for the control group an active vaccine against a different target infection, provided the active control does not cross-protect against the target pathogen and that use of the active vaccine maintains blinding in the trial [ ] . however, it remains controversial whether a placebo can be ethically administered to the control groups in trials for which there is no suitable alternative active control vaccine and in which a licensed alternative vaccine against the target infection exists, but the alternative is not licensed in the country for the trial [ ] . yet a third controversy concerns what is owed to the participants at the conclusion of a vaccine trial. the declaration of helskinki states that: "at the conclusion of a study, every patient entered into the study should be table . principles of good clinical practice (gcp). from [ ] . clinical trials should be conducted in accordance with the ethical principles that have their origin in the declaration of helsinki and that are consistent with gcp and the applicable regulatory requirement(s). . before a trial is initiated, foreseeable risks and inconveniences should be weighed against the anticipated benefit for the individual trial subject and society. a trial should be initiated and continued only if the anticipated benefits justify the risks. . the rights, safety, and well-being of the trial subjects are the most important considerations and should prevail over interests of science and society. . the available nonclinical and clinical information on an investigational product should be adequate to support the proposed clinical trial. . clinical trials should be scientifically sound and described in a clear, detailed protocol. . a trial should be conducted in compliance with the protocol that has received prior institutional review board (irb)-independent ethics committee (iec) approval-favorable opinion. . the medical care given to and medical decisions made on behalf of subjects should always be the responsibility of a qualified physician or when appropriate, a qualified dentist. . each individual involved in conducting a trial should be qualified by education, training, and experience to perform his or her respective task(s). . freely given informed consent should be obtained from every subject prior to clinical trial participation. . all clinical trial information should be recorded, handled, and stored in a way that allows its accurate reporting, interpretation, and verification. . the confidentiality of records that could identify subjects should be protected, respecting privacy and confidentiality rules in accordance with the applicable regulatory requirement(s). . investigational products should be manufactured, handled, and stored in accordance with applicable good manufacturing practice (gmp). they should be used in accordance with the approved protocol. . systems with procedures that assure the quality of every aspect of the trial should be implemented. assured of access to the best proven prophylactic, diagnostic, and therapeutic methods identified by the study" [ ] . few would disagree that if the vaccine tested in a trial is found to be beneficial, it should be provided to all participants in the trial. however, controversy surrounds the boundaries for the group of persons entitled to receive the investigational vaccine at the conclusion of the study -only participants in the trial, all persons living in the same region, or all persons living in the same country? and for how long are these beneficiaries entitled to receive the vaccine? increasing expectations for demonstration of vaccine safety before licensure because vaccines are administered in public health practice to healthy individuals, most commonly children, there is a higher expectation of safety for vaccines than for many therapeutic agents. in the past, pre-licensure phase iii trials were often designed merely to evaluate the safety profile of a vaccine observed in previous studies. accordingly, the focus of such trials was on evaluating events that were expected on the basis of earlier studies, that occurred during an interval immediately following vaccination, and that were seen with appreciable frequency. indeed, a common tactic in older phase iii vaccine trials was to evaluate adverse events only in a small subsample of the total trial population, obviating the possibility of detecting rare but potentially significant side-effects. modern regulatory agencies now usually require that surveillance for adverse events be undertaken for the total study population rather than for a subsample, at least for events that can be detected passively (e.g., among persons presenting to health care centers for treatment), and that this surveillance be maintained for the duration of the trial. increasing concern about vaccine safety has also led to other changes in the design of phase iii trials. formerly, calculation of sample sizes for such trials was usually geared to enable detection of a certain level of vaccine protection against the target illness, with an acceptable level of statistical power. detection of rare side-effects was not a typical goal of such calculations. the recent experience with the live oral, quadrivalent rhesus rotavirus reassortant vaccine, which was withdrawn from the united states market because of an association with a small but statistically significant risk of intussusception [ , ] , has led to more stringent regulatory requirements for pre-licensure trials of newer-generation, live oral rotavirus vaccines. the united states food and drug administration, for example, has required that these newer-generation vaccines be tested in numbers of infants sufficiently large to be able to detect whether intussusception is a vaccine side-effect. current trials of these newer vaccines have therefore enrolled samples of infants many times the number enrolled in pre-licensure trials of the live oral, quadrivalent rhesus rotavirus reassortant vac-cine, resulting in greatly increased clinical development costs. whether the experience with rotavirus vaccines portends regulatory requirements for substantially larger phase iii trials for other new-generation vaccines remains to be seen. the experience in the united states with the live oral, quadrivalent rhesus rotavirus reassortant vaccine illustrates yet another issue confronting pre-licensure trials. data from pre-licensure trials of this vaccine showed a suggestive but non-significant elevation of the risk of intussusception [ ] . the united states food and drug administration licensed the vaccine with the proviso that the occurrence of this potential side-effect be scrupulously monitored post-licensure. this was possible because of the well-developed systems for post-licensure surveillance for vaccine side-effects established in the united states [ , ] . unfortunately, post-licensure surveillance systems capable of detecting and evaluating rare but serious vaccine adverse reactions are absent in most developing countries. the weaknesses of post-licensure surveillance systems in developing countries place an even greater onus upon pre-licensure trials in these settings to provide reassurance that a vaccine will not cause rare but serious side-effects. however, the large size and great expense of trials capable of detecting rare side-effects present a major dilemma, especially for vaccines against diseases that are primarily limited to the developing world, for which resources for clinical trials are scarce. clearly, there is an urgent need to develop improved systems of post-licensure surveillance capable of detecting rare but significant vaccine adverse reactions in developing countries. recently work has begun to evaluate the methodological challenges and practical feasibility of establishing large population-based, dynamic computerized databases for evaluating potential vaccine side-effects in developing-country settings. such databases link vaccination histories to severe disease outcomes in defined cohorts, and have been used successfully in industrialized countries for rapid and credible evaluations of putative vaccine adverse reactions [ ] . one such database has been successfully established on a pilot scale in vietnam [ ] . overcoming the challenges of establishing such databases in the diverse settings of the developing world remains a significant but important research agenda. the basic phases of testing of vaccine candidates are relatively straightforward and widely accepted. the successive phases of clinical evaluation of vaccine candidates allow for acquisition of critical information about vaccine safety, immunogenicity, excretion, transmission, and protection in an incremental fashion, while minimizing the risks to subjects who volunteer to participate in these studies. in recent years we have seen a burgeoning of vaccine candidates against diseases of developing countries, creating breathtaking possibilities for disease prevention in these settings. at the same time, this profusion of vaccine candidates for the developing world has added a layer of complexity to the seemingly straightforward phased sequence of trials. the factors underlying this complexity are multiple, and include considerations about when in the phased testing sequence a vaccine developed in an industrialized country should begin testing in developing countries; about whether and how to pursue the clinical development of a vaccine when disappointing clinical results are observed in developing countries; about how to meet the scientific, financial, logistical, and ethical challenges of conducting clinical trials in developing countries that conform to contemporary international standards of good clinical practice; and about how to ensure that a vaccine is acceptably safe before and after licensure. the manner in which these issues are addressed in the future will have a great bearing on the success of efforts to accelerate the introduction of new-generation vaccines into programs for the poor in developing countries. the jordan report: accelerated development of vaccines. the institute vaccine economics: from candidates to commercialized products in the developing world long-term evaluation of vaccine protection:methodological issues for phase iii and iv studies the hazards of immunization an epidemiological study of altered clinical reactivity to respiratory syncytial (rs) virus infection of children previously vaccinated with an rs virus vaccine measles immunization with killed virus vaccine, serum antibody titers and experience with exposure to measles epidemic intussusception among infants given an oral rotavirus vaccine population-based study of rotavirus vaccination and intussusception safety, immunogenicity, and efficacy of recombinant live oral cholera vaccines, cvd and cvd -hgr the safety, immunogenicity, and transmissibility of single-dose live oral cholera vaccine cvd -hgr in - month old indonesian children the ethical challenge of infection-inducing challenge experiments infectious disease experimentation involving human volunteers initial clinical evaluation of new vaccine candidates: investigators' perspective of phase i and phase ii clinical trials of safety, immunogenicity, and preliminary efficacy evaluating new vaccines for developing countries: efficacy or effectiveness? protective effect of two acellular pertussis vaccines in a double-blind, placebo-controlled trial factors affecting the immunogenicity of oral polio vaccine in developing countries: a review trial of an attenuated bovine rotavirus vaccine (rit ) in gambian infants limited efficacy of a haemophilus influenzae type b conjugate vaccine in alaskan native infants. the alaska h. influenzae vaccine study group efficacy of a single dose of live oral cholera vaccine cvd -hgr in north jakarta, indonesia, a cholera-endemicarea global reduction of hib disease: what are the next steps? designing hiv vaccines for developing countries the future of rotavirus vaccines: a major setback leads to new opportunities safety and immunogenicity of different immunization regimens of cvd -hgr live oral choleravaccine in soldiers and civilians in thailand randomized, double-blind, placebo-controlled, multicentered trial of the efficacy of a single dose of live oral cholera vaccine cvd -hg-r in preventing cholera following challenge with vibrio cholerae el tor inaba three months after vaccination the efficacy in navajo infants of a conjugate vaccine consisting of haemophilus influenzae type b polysaccharide and neisseriae meningitidis outer-membrane protein complex efficacy in infancy of oligosaccharide conjugate haemophilus influenzae type b (hiboc) vaccine in a united states population of , children. the northern california kaiser permanente vaccine study center pediatrics group prelicensure evaluation of combination vaccines international conference on harmonisation expert working group ( ) international conference on harmonisation guideline for good clinical practice. e . the conference the future of vaccines: an industrial perspective the national commission for the protection of human subjects of biomedical and behavioral research ( ) the belmont report: ethical principles and guidelines for the protection of human subjects of research. the commission the ethics of clinical research in the third world ethical issues in the design and conduct of clinical trials in developing countries ethical complexities of conducting research in developing countries the future of research into rotavirus vaccine declaration of helsinki: ethical principles for medical research involving human subjects. the association joint united nations programme on hiv/aids ( ) ethical considerations in hiv preventive vaccine research when are placebo-controlled trials ethically acceptable? public health considerations for the introduction of new vaccines against rotavirus: a case study of the rhesus rotavirus-reassortant vaccine the vaccine adverse event reporting system (vaers) vaccine safety datalink project: a new tool for improving vaccine safety monitoring in the united states the vaccine data link in nha trang, vietnam: a progress report on the implementation of a database to detect adverse events related to vaccinations key: cord- -k h ejjt authors: ilyinskii, p. title: aspects of microparticle utilization for potentiation of novel vaccines: promises and risks date: journal: silicon versus carbon doi: . / - - - - _ sha: doc_id: cord_uid: k h ejjt many recombinant vaccines against novel (hiv, hcv) or ever-changing (influenza) infectious agents require the presence of adjuvants/delivery vehicles to induce strong immune responses. the necessity of their improvement led to the major effort towards development of vaccine delivery systems that are generally particulate (e.g., nano- and microparticles) and have comparable dimensions to the pathogens (viruses or bacteria). the mode of action of these adjuvants is not fully understood but implies the stimulation of the innate or antigen-specific immune responses, and/or the increase of antigen uptake or processing by antigen-presenting cells (apc). moreover, enhancement of adjuvant activity through the use of micro- and nanoparticulate delivery systems often resulted from the synergistic effects producing immune responses stronger than those elicited by the adjuvant or delivery system alone. among particulate adjuvants, biodegradable micro- and nanoparticles of poly(d,l-lactide-co-glycoside) (plga) or poly(d,l-lactide) (pla) have been reported to enhance both humoral and cellular immune responses against an encapsulated protein antigen. cationic and anionic polylactide co-glycolide (plg) microparticles have been successfully used to adsorb a variety of agents, which include plasmid dna, recombinant proteins and adjuvant active oligonucleotides and are also currently tested in several vaccine applications. another approach envisions specific targeting of apc, especially peripheral dc and exploitation of particulate systems that are small enough for lymphatic uptake (polystyrene nanobeads). micro- and nanoparticles offer the possibility of enhancement of their uptake by appropriate cells through manipulation of their surface properties. still, questions regarding toxicity and molecular interaction between micro- and nano-particles and immune cells, tissues and whole organisms remain to be addressed. these risks and other possible side effects should be assessed in detail especially if mass-production and massive administration of such preparations is to be considered. prevention of infectious disease through vaccination is one of the most important achievements in the history of medicine. its effects on the mortality reduction are enormous and may be compared only to those caused by the establishment of safe water supply [ ] . adaptive immunity against infectious disease, i.e. lack of infection in previously exposed individuals has been noted for a long time, most famously by athenian historian thucydides in th century bc. it is often said that first recorded attempts of vaccination against infectious disease (smallpox) go back to ad . in china, but they cannot be verified till th- th centuries, when the procedure of variolation started to be used on a reasonably large scale. in the end of xviii pioneering works of jenner and his less-known predecessor jesty on vaccination against smallpox with cowpox paved the way to modern vaccination, whose true founder in a scientific sense was louis pasteur [ ] . interestingly, first successful vaccines were launched against two viral diseases, smallpox and rabies. smallpox became first and only infectious disease to be eradicated in - [ ] . it is commonly said that jenner's work constitutes the first deliberate use of live virus vector as a vaccine, but we should also note that it was first recorded use of nanotechnology (virion size of a poxvirus is around nm in diameter and nm in length). we should also mention that the problems of unknown side effects, possibility of human infection with new or incompletely inactivated virus and as well as initial difficulties of social acceptance of vaccination that were faced by both jenner and pasteur are not dissimilar from the risks that nanotechnology of vaccination encounters today. in fact, these problems have persisted for more than years and yet many other major diseases have been put under control with the help of vaccination. these include, to name just a few, diphtheria, tetanus, yellow fever, pertussis, poliomyelitis (currently targeted for eradication by who), measles, mumps and rubella. and this list is far from complete. at the same time, it is important to remember that a number of vaccine-related disasters and incidents were caused over this period of time, all of them due to failures in vaccine manufacturing techniques. specifically, during the st century of scientific vaccination (post-pasteur) three types of vaccines were generally used: live-attenuated (weakened), killed viruses/bacteria and subunit or extract (chemically or biochemically processed and thus detoxified, the technique that mostly important in anti-toxin vaccination, i.e. against diphtheria). it is thus of no surprise that there were instances of incomplete attenuation, sterilization or detoxification of vaccine products (see [ ] for details). while historical role of early-generation vaccines cannot be underestimated, current awareness of health and safety issues resulted in much higher standards of safety and it is true that many past vaccines would not even meet the minimum standards of today. new techniques of vaccine development that may enable a generation of safe, effective and economically feasible vaccines are being actively explored. additionally, it is now clear that successful vaccination against several newly-discovered infectious agents as human immunodeficiency virus (hiv), hepatitis c virus (hcv) or severe acute respiratory syndrome (sars) will require the development of radically new vaccine concepts and products. there are several types of novel vaccines or vaccine components the utilization of which is not mutually exclusive. in fact, their complementary use may be even required in some cases. firstly, there are recombinant molecules: proteins or nucleic acids (na; dna vaccines have been tested extensively over the last decade, rna vaccines are now actively pursued in several experimental systems, but relevant information is scarce at the moment save for rna alphaviral replicons). protein antigens induce immune response directly, while na vectors encode necessary antigens and produce them upon their introduction into organism. secondly, these molecules may be carried or delivered by biological or chemically produced vectors, separately or in combination. these vectors are known to tremendously augment the immune response induced by proteins or nucleic acids used alone. such augmentation is also called adjuvant effect. the size of these vectors is generally within - nm and it is a specific mechanism by which our immune system recognizes such particles that underlies their adjuvant potencies (in addition, many carriers protect proteins/na from rapid degradation in vivo and release them into the organism during prolonged periods of time, which also results in higher immunogenicity). needless to say, many details of this mechanism remain to be elucidated, although current vaccine development fashion is definitely of "going small", i.e. toward micro-and nanoscale [ ] . in this paper, i will briefly discuss the current state of the vaccine nanotechnology, promises of the new approaches as well as potential risks that these approaches may contain. both recombinant na and protein vaccines have been designed to be safer than traditional vaccines based on whole viruses and bacteria, but their limited immunogenicity has so far hindered their development for clinical use. this led to the sustained research effort aimed at development of specific adjuvants that may make such vaccines more potent without compromising on their safety. currently, only one adjuvant, alum (aluminum hydroxide or aluminum phosphate) is used worldwide in diphtheria, tetanus and hepatitis b vaccines. one of the proposed mechanisms for alum action is generation of small salt particles in which vaccinating agent is trapped and then preferentially taken up by cells of immune system, macrophages and dendritic cells (dc), which, in turn, leads to elevated immune response. multiple attempts to create antigen-bearing particles that will be capable to imitate this suggested avenue of immunogenicity augmentation have been undertaken over the last decade and many of them have been useful. what may be the mechanism of higher immunogenicity exhibited by particulate antigens compared to soluble ones and does the size of these particles matter? adjuvanting effects of micro-and nanoscale particulates are now known to be a consequence of them being of suitable size for uptake by antigen-presenting cells (apc), such as dc and macrophages through phagocytosis or pinocytosis. apc then stimulate t-lymphocytes constituting cellular arm of immune response, which among others is responsible for specific antiviral immunity. this apc-mediated activation of t-cells initiates adaptive immune response. antigens delivered in particulate form are preferentially processed by apc and via so-called mhc class i and ii pathways and then presented to cd + (cytotoxic lymphocytes) and cd + (t-helpers) t-cells, which first serve as effector cells of immune response and then are instrumental in generation memory t-cells, those that are responsible for the defense of the organism in case of repeated exposure to the infectious agent bearing antigen in question. those particulate antigens that are not directly taken up by dc, but by other cells of the organism (e.g., by muscle cells after intramuscular injection) may also stimulate immune response by two possible pathways. either they eventually either find their way to dc after cell death followed by phagocytosis (if they are stable enough) or they may be processed to the cell surface and then recognized by already activated t-cells thus providing further stimulus for their activation and multiplication. obviously, that particle-driven stability of delivered antigens is a very important part of such an equation. moreover, the surface of micro-or nanoparticle may be further modified to increase their targeting specificity, which can be also attained via simple size variation. it is now clear that different subtypes of dc, those residing in skin and in lymph nodes (ln) are differentially affected by nanoparticles of various size with smaller nanoparticles ( - nm) being capable of passive migration to lymph nodes and rapid activation of immature ln-resident dc, while particles of the larger sizes ( - nm) are preferentially uptaked by dc at a vaccine injection site. those skin or mucosal apc residing at those parts of the body where the contact with infectious agents is most likely constitute "the first line of defense" of the organism. it is more than probable that activation of several dc populations done sequentially or simultaneously is a process imperative for generation of potent immune response. moreover, activation of dc does also trigger non-specific immune responses, known as innate (e.g., induction of interferons and other cytokines), which in turn may further potentiate generation of prolonged and strong specific immune response against vaccinating agent. all of these developments made by fundamental research are now actively transposed into the science of vaccine manufacturing. there are two types of vectors of viral origin that have been extensively explored lately, first being viral vectors per se (replication-competent or -deficient) and second, viruslike particles (vlp). viral vectors consist of replicating or non-replicating virus that contains genetic material (in form of dna or rna) encoding the desired antigen. these vectors maintain the ability to penetrate cells and disseminate in the organism and sometimes even replicate, but are rendered harmless by specific changes or mutations. advantages of virally-vectored vaccines include their ease of production, a good safety profile (at least in some cases), ability to potentiate strong immune responses, potential for nasal or epicutaneous delivery and mucosal immunization [ ] . they are also more potent than pure dna vaccines (since dna in this case is protected from degradation by viral capsid and directly delivered to virus-susceptible cells), but are especially good if used sometime after initial dna immunization (in so-called prime-boost regimen). adenoviruses are probably among the best-studied viruses and they were heavily exploited for vaccine development over last two decades [ ] . there are many promising experimental results using either replication-competent or -incompetent (the latter, by definition, are very safe) adenoviral vectors. however, consistent presence of adenoviruses (the agents of common cold) in human population resulting in pre-exisitng immunity (the ability of human immune system to recognize adenoviral vector and expeditiously remove it from the organism) has somewhat damped the enthusiasm for their clinical use. still, experimental data, especially in the immunization against hiv and influenza were sufficiently promising to enable large-scale clinical trial of adenoviral-based hiv vaccine manufactured by merck (usa). this, as we know, has recently ended in a complete failure. the trial was aborted [ , ] . importantly, not only this vaccine was not protective, it has apparently also increased risk of hiv infection in vaccinated subjects that were previously exposed to adenovirus (those with pre-existing immunity). notably, we do not currently know the fundamental reason for the latter (and it was not foreseen by anyone based on current immunological knowledge). this example shows that vaccines of novel generation, however promising, may cause side effects of completely unpredictable nature. another type of viral vectors is based on various poxviruses, closely related to vaccinia virus that has been used for many years for vaccination against smallpox. it is known that immunization with vaccinia viruses induces potent immune response against recombinant antigen [ ] and many experimental vaccines containing proteins from hiv, influenza, malaria, etc. have been produced and tested. it is important to stress that unmodified live vaccinia virus has never been proposed as delivery vehicle for vaccines aimed at general use (application of such vaccines as anti-cancer therapeutics is outside of this review) since the risks of its utilization would have been enormous. in particular, live vaccinia virus can not be used to vaccinate people with immune deficiencies, suffering from eczema, etc. however, its replication-deficient strain, called modified vaccinia virus ankara (mva) is much safer and still very immunogenic. another approach envisions the utilization of avian poxviruses, e.g. fowlpox or canarypox viruses, which are also incapable of replication in humans, but still immunogenic. similar to adenoviral vectors, the issue of pre-existing immunity should be addressed regarding poxviral-based vaccines since nearly all humans over years old have been vaccinated against smallpox at least once and may therefore respond poorly to new poxviral vaccination. the possibility of adverse effects similar to the one observed in merck anti-hiv vaccine trial can not be discounted as well, although what is true for one group of viruses may not be automatically applied to another. many mva-vectored vaccines have been already developed, but only one of them, against malaria, is now under active clinical investigation, which at the moment appears to be highly successful. in this case prime vaccination with dna molecule is followed by boost immunization with recombinant poxvirus. currently we do not know why poxviral-based vaccine is especially potent against malaria and if these results could be translated into other infectious systems. other replicating virus vectors are based on measles and vesicular stomatitis virus and they suffer from similar drawbacks: prior immunity and safety concerns. the same could be said about two more novel non-replicating viral vectors based on alphaviruses and herpesviruses [ ] . it appears that viral vaccine vectors will require considerable breakthrough in order to enable their extensive clinical application. another approach envisions the utilization of vlps. vlps are non-infective virus particles (capsids) consisting of self-assembled viral proteins without na, which mimic the structure of native virions [ ] . these particles fall in the general size range of viruses ( - nm) [ ] , albeit it is fair to say that full viral size range is within - nm [ ] . they also maintain a specific virus-like morphology and are effectively consumed by cells similarly to infective viral particles. they may be formed by a single viral protein or by co-expression of many, in the latter case creating complex virus-like structures. it appears that particulate nature of vlps drives their efficient presentation to the cells of immune system. moreover, those vlps that closely resemble infectious viruses may trigger the same array of antiviral immune responses (e.g., via viral receptor) without causing immune suppression (as living viruses do). vlps can be produced in many expression systems, those of mammalian cells, yeast, bacteria, baculovirus (viruses of insects) and plants. they have been manufactured for many viruses including, but not limited to hiv, hepatitis b virus (hbv), hcv, ebola, influenza, rotavirus, human papillomavirus (hpv) and norwalk virus [ ] . several vlpbased vaccines have been licensed for general use, many of them against hbv, which are composed of hbv surface antigen (hbsag), which is a main component of currently used protein-based, alum adjuvant-potentiated vaccine. upon its production in vitro (e.g., in yeast) hbsag protein aggregates in nm particles (single-component vlp), which are both safe and highly immunogenic [ ] . in fact, hbsag is now actively pursued as molecular adjuvant on its own ("vlp as platform" approach). fusion of several less immunogenic proteins to hbsag, most notably, influenza m protein capable of cross-protective immunity, resulted in significant immunogenicity augmentation [ ] . similar data has been reported for hbv vlp containing proteins from the circumsporozoite stage of the malaria plasmodium [ ] . improvements of single-protein hbv vlps by inclusion of large and middle viral envelope proteins are also actively sought [ ] . the most recently approved vlp vaccine is gardasil that is protective against several types of hpv, which are known to be associated with cervical cancer and genital warts. this vaccine is composed of self-assembled particles of hpv major capsid protein [ ] [ ] [ ] and it also contains an aluminum salt adjuvant. it has been shown to reduce infection of hpv by % and is almost % effective hpv types , , and . this vaccine if administered early in life is expected to drastically reduce the occurrence of this life threatening disease in women and has generated significant excitement. therefore, it is currently recommended that girls at - years group age are vaccinated and many u.s. states will likely make this vaccination mandatory. anti-hpv vaccine is clearly the most dramatic example of recent success of recombinant vaccine developers. at the same time, there was a significant fight-back against general utilization of this vaccine stemming from different social groups that we will describe in the final part of this review. several other vlp vaccines are now close to clinical use, those against norwalk virus and rotavirus (both causing acute gastrointestinal infections). rotavirus is the leading cause of diarrhea-related deaths ( - , ) in children worldwide and clinical trials of vlp-based vaccine against rotavirus has long been advocated [ ] . vlp-based vaccine against norwalk virus is now assessed in phase i clinical trials. a variety of other vlp vaccines have been evaluated in preclinical studies include hiv, influenza, hcv, ebola and sars coronavirus [ ] . it should be added that non-vaccine nanoparticle vlp applications also exist such as using them as scaffolds to allow assembly of biopolymers (large plant virus-derived vlps are utilized, notably from tobacco and cowpea mosaic viruses), including their utilization as templates to polymerize nanowires as building blocks for semiconductors or manufacture nanowires for the construction of smaller lithium ion batteries [ ] . liposomes (phospholipids-based vesicles) are roughly of the similar size as vlp (usually - nm) and they were used extensively as drug delivery vehicle for nearly four decades. liposomes transport their contents across cellular membranes and release it following fusion with internal cellular compartments called endosomes. thus, they are capable of delivering of encapsulated or adsorbed antigen for vaccine use and were investigated as adjuvants for vaccines against influenza, hiv and malaria, among others. however, it seems that no unmodified liposome-based vaccine possesses an adjuvant effect strong enough to substantiate its clinical development. conversely, modified liposomal vaccines based on viral membrane proteins (virosomes) are sufficiently potent and are approved for use in europe as vaccines against hepatitis a virus (hav) and influenza [ ] . immunopotentiating influenza virosomes are liposomes containing main influenza surface protein hemagglutinin intercalated into their membrane. their approximate mean diameter is - nm. the glycoproteins of influenza virus stabilize the liposomal particles and maintain their receptor-binding actrivities when reconstituted into such protein lipid vesicles and are therefore actively taken up by cells in vivo and actively conveyed to immunocompetent cells [ ] . they have an excellent safety profile. other virosomal vaccines produced include not only those against hav, but also combined hav-hbv vaccine in which inactivated hav virions and hbv hbsag cores were covalently coupled to the surface of virosome [ ] . adjuvant properties of virosomes can be further increased by integration of costimulatory molecules (immune response mediators and activators) or their specific targeting to particular dc subtype. moreover, they can additionally carry dna or rna and thus provide for multifaceted immune stimulation via different pathways. therefore, virosomes are becoming more and more popular in modern vaccine concepts since they combine the safety and flexibility of subunit vaccines with the biological and immunogenic properties of vlps [ ] . another mode of imitating nature is the creation of sometime misleadingly called proteosomes (note the capital "p"), nanoparticles composed of the outer membrane proteins (omps) of neisseria meningitides and other neisseria. proteosome particles are considered to serve as potent vaccine delivery vehicles based on their nanoparticulate ( - nm size), vesicle-like nature [ ] . in addition, hydrophobicity of proteosomes may inhibit antigen degradation facilitate its uptake and processing by immunocompetent cells. omps have been used successfully in a marketed meningococcal vaccine since and are considered non-toxic and well-tolerated [ ] . meningococcal omps have been safely given parenterally to hundreds of million of children in a haemophilis influenza type b conjugate vaccine [ ] . in several cases, a novel adjuvant known as protollin consisting of proteosomes non-covalently complexed with immune stimulant lipopolysaccaride (lps) has been used [ ] . proteosome-based influenza vaccines for nasal application have been also generated using this technology and successfully tested and many others (against shigella, hiv, staphylococcal and brucella toxins are under active development). there are other bacteria-based adjuvants, but they may not be called particles in a true sense of words, rather being biomolecules (modified proteins, peptides, components of bacterial wall). it is for their efficient delivery that some of synthetic micro-and nanoparticles can be successfully used. the only nano-sized vaccine adjuvant that is approved for human use (in europe, but not in the u.s.) as a vaccine component is mf . it was the first new adjuvant to be licensed for human use (in ) years after introduction of alum [ ] . mf is an oil-in-water emulsion composed of < nm uniform and stable microparticles made by a drop of oil surrounded by a coat of water droplets held onto oil by surface detergents. specifically, these droplets are formed when squalene ( . % v/v) and two surfactants, polysorbate ( . % v/v, tween ) and sorbitan trioleate ( . % v/v, span ) are emulsified in citrate buffer [ ] . this oil is obtained from shark liver and is also found in humans as natural metabolite [ ] . the strong immunogenicity enhancement of mf has been repeatedly demonstrated with some researchers showing an effect even stronger than that of alum. the mechanism of adjuvanticity of mf is believed to be through cytokine production, although no precise information is available as of yet. in general, it is thought that adjuvant emulsions potentiate immune responses via formation of antigen depots and stimulation of antibody-producin plasms cells. as noted above, an mf -adjuvanted influenza vaccine, fluad, is licensed in europe and experimental vaccines for avian influenza have been produced. their strong immunogenicity and protectivity has been demonstrated experimentally. other vaccines strongly potentiated by mf include those against hbv, hcv, hiv, herpes simplex virus (hsv), haemophilis influenzae type b and n. meningitides. montanide is a different, water-in-oil emulsion (one modification is a water-in-oil-inwater emulsion). emulsions of montanide and are composed of a metabolizable squalene-based oil with a mannide monooleate emulsifier, which augments immunogenicity via formation of antigen depot at the site of injection [ ] . while similar to incomplete freund's adjuvant (ifa), a well-known but extremely reactogenic adjuvant that contains a mineral oil and an emulsifying agent, in its physical character, montanide is biodegradable. this eliminates many of the cytotoxic properties inherent for ifa. isa and emulsions have been in phase i and/or ii clinical trials for vaccines against malaria and hiv and various cancers and in most cases they were found to be safe and fairly well-tolerated. another particulate vaccine vehicle is immunostimulating complex (iscom). iscoms are based on triterpenoid saponins (q saponins). they form particles of approximately nm. these are essentially cage-like structures containing protein antigen, cholesterol, phospholipid and the saponin adjuvant quil a or its purified component quil , which is derived from the aqueous extract from the bark of the south american tree quillaia saponaria molina. these components are held together by hydrophobic interactions. saponins have been tested and used in both human and veterinary medicine for decades and they are known to efficiently induce t-helper responses. they have not been extensively used because of their reactogenicity (quil a composed of more than different saponins is too toxic for human use), although recently semi-synthetic saponins were created that are apparently less toxic [ ] . iscom matrix traps the protein antigens (typically hydrophobic membrane proteins) through apolar interactions. a similar vaccine delivery vehicle and adjuvant has also been developed that uses the same material minus the antigen (iscomatrix) and still possesses preferential targeting to apc [ ] . a clinical study that compared a classical trivalent subunit influenza vaccine with an iscom adjuvanted version revealed a stronger immune response with the iscom vaccine eliciting rapid antibody responses as well as t helper (cd + t-cell) and some ctl (cd + t-cell) responses [ ] . additional iscom/iscomatrix vaccines have been tested in the clinic for hiv, hsv, hpv and hcv [ ] . in all cases, these studies have shown a good safety and tolerability profile in humans as well as effective induction of both humoral and cellular immune responses. still, the actual use of iscoms in human vaccines has been deterred by concerns regarding safety since some saponins are toxic at elevated levels [ , ] . another completely new mode of biochemical modification of antigen is its attachment to so-called protein aggregating domains that form intracellular inclusions of several hundred nm. such inclusions are then digested via the process called autophagy, which is now known to be linked to effective antigen presentation via mhc class ii pathway. initial data provided evidence of dramatic increase of immunogenicity when a test soluble antigen was attached to such aggregating domain [ ] , although this very promising approach is still in the very first stages of possible development. there are several types of synthetic micro-and nanoparticles that are effective in vaccine delivery and potentiation. calcium phosphate microparticles are relatively lessknown. they can be generated by combining (while stirring) of calcium chloride, sodium phosphate and sodium citrate [ ] . since calcium phosphate is naturally occurring in the body, these materials are thought not to present a danger of significant side-effects. calcium phosphate microparticles are less than ~ . μm in diameter (< , nm according to other authors, [ ] ). phase i study showed that these microparticles are safe and non-toxic when administered subcutaneously. vaccines utilizing cap, which are currently in preclinical studies include anthrax, hbv, influenza (h n avian and seasonal) and hsv- [ ] . much better known are polymeric biodegradable microparticles. those used most frequently are poly(d,l-lactide-co-glycolide) (plg/plga) and polylactide (pla) and their copolymers as well as polyorthoesters, polyanhidrids and polycarbonates [ ] . microparticles may be loaded by many types of recombinant vaccines, i.e., dna (which is thus protected from degradation), proteins and also other adjuvants of smaller molecular size (of protein, peptide or oligonucleotide nature). these biodegradable, biocompatible polymers have been approved for use in humans (e.g., as sutures, bone implants, screws and implants for sustained drug delivery) and have been extensively studied for use in the formulation of vaccine antigens. plg-based microparticles are the primary candidates for the development of microparticles as vaccine adjuvants since they have been safely used in humans for many years. the direct uptake of biodegradable microparticles into dc has been demonstrated both in vitro and in vivo and it appears that their appropriate size is within - μm range and that cationic microparticles are particularly effective in this regard [ ] . in these formulations, antigen can be either entrapped or adsorbed to the surface of the particles. furthermore, these particles can be tailored to degrade over a range of rates, which is especially important since several of those systems degrade rather slowly. thus, additional triggering of their dissolution is necessary for efficient antigen delivery. on a positive side, they can therefore act as a depot from which the encapsulated antigen is gradually released [ ] . additionally, polymeric particles may offer high degree of protection to encapsulated antigens delivered orally and nasally and thus subjected to many degrading enzymes residing at these sites of the body. it appears that dcs respond to biomaterials via an innate immune response, which then stimulates an adaptive response to an antigen delivered by polymeric particles. biodegradable and biocompatible micro-and nanoparticles of plga or pla have been reported to enhance both humoral and cellular immune responses against an encapsulated protein antigen. plg nanoparticles can induce systemic antibody titers comparable to those of aluminum salts. it was demonstrated that plg nanoparticles loaded with mpl (an immune stimulant composed of detoxified lipopolysaccharide (lps) from salmonella minnesota) were efficiently taken up by dc. recently, it was shown that the adsorption of antigens at the surface of pla particles also leads to elevated immune response. cationic and anionic plg microparticles have been successfully used to adsorb a variety of agents, including dna, recombinant proteins and adjuvant active oligonucleotides and are also currently tested in several vaccine applications. another approach envisions specific targeting of apc, especially peripheral dc and exploitation of particulate systems that are small enough for lymphatic uptake (polystyrene nanobeads). these are in contrast non-degradable nanoparticles (represented also by gold, latex and silica beads). it appears that solid synthetic, particulate vaccines can induce strong immune responses imitating classical "danger signals" generated by infectious agents and microparticles. in this application - nm particles preferentially taken by some types of dc are often used and are known to generate potent and broad immune response [ ] . moreover, it was demonstrated that for polysterene beads even miniscule difference in size can influence the breadth and type of induced immune response [ ] . recently, ultra-small ( nm) nanoparticle systems were shown to be capable of interstitial-to-lymphatic flow to deliver antigen and adjuvant to ln-resident dc via lymphatic capillaries, whereas nm nanoparticles were only % as efficient [ ] . also, these ultra-small, ovalbumin-conjugated, polyhydroxylated nanoparticles based on pluronic (a block copolymer of polyethylene glycol and polypropylene glycol) were shown to induce antigen-specific cellular immunity since their surface chemistry has activated complement system, a pathway of innate immunity that serves as a biochemical defense system that clears pathogens nonspecifically, but can also play a role in promoting antigen-specific responses. in general, nanoparticles as vaccine vehicles might have three different advantages over microparticles. most importantly, they have increased surface area for adsorption allowing for a higher antigen/polymer ratio and are also easier to prepare and process. as for their higher immunogenicity, the jury is still out since different groups of investigators are presenting conflicting evidence [ ] . it should be also noted that sometimes the utilization of preposition "nano" to describe nearly any type of vaccine component is a bit overdone [ ] . since non-degradable nanoparticles may remain in the tissues for extended periods of time, it is thought that it will therefore enhance the time of immobilized antigen presentation and thus augment immunogenicity. gold particles have been frequently described for vaccine delivery both with and without the aid of electroporation, a method which is unlikely to be employed in humans. an alternative approach to delivering dna vaccines employing non-degradable nanoparticles is through particle bombardment also referred to as "gene gun" approach. this is essentially firing the dna-coated gold nanoparticles into the epidermis. while the delivery efficiency of this technique is quite low, only small amounts of dna are required to achieve a significant immune response. this method has been tested for vaccines against hbv, influenza and malaria. furthermore, micro-and nanoparticles offer the possibility of enhancement of their uptake by appropriate cells through manipulation of their surface properties. as delineated above, upon their inoculation, particulate compounds, microspheres or nanoparticles, must reach the secondary lymphoid organs, which are the sites of the immune response. this led to design of novel methods of dermal or transcutaneous vaccination, including the use of micro-and nano-particles to target the skin apc that will then deliver consumed antigens to the ln. finally, pulmonary delivery (which may be especially potent and useful against influenza and other respiratory viruses) requires dry forms of vaccines that are low cost, temperature-tolerant, efficiently aerosolized, and apc-directed. therefore, nanoparticles can play a critical role in the formulation, development and delivery of needle-less pulmonary vaccines and these are now actively pursued as well. additionally, nanoparticles containing vaccines for the oral delivery are also investigated. these particles are - nm in diameter and are likely needed to be targeted towards a special subtype of apc called m cells that reside within gut-associated lymphoid tissue. it is apparent that novel micro-and nanoparticle-based delivery vehicles are being actively evaluated in many vaccine systems. promising results have been reported from many directions. still, questions regarding toxicity and molecular interaction between micro-and nanoparticles and immune cells, tissues and whole organisms remain to be addressed. there are many regulatory hurdles for new adjuvants and they are there for a reason. one of the greatest hurdles is the sheer size of population that needs to be tested to prove safety of a new adjuvant or vaccine. these numbers have dramatically increased in recent years since it became apparent that some approved drugs have rare serious and even fatal side effects that were not identified because of inadequate sample sizes during their clinical development [ ] . during that time the association of adverse reactions with two vaccines resulted in their withdrawal from the market. these are nasal inactivated influenza vaccine associated with increase of cases of bell's palsy and also rotavirus vaccine, the administration of which lead to higher intussusception [ ] . the case of adenoviral-based hiv vaccine has been mentioned above. reaction of parts of the society to a new vaccine may be also caused by imaginary side-effects. we have already noted that this is a case with gardasil, a highly efficient anti-hpv vaccine, which is likely to tremendously diminish the incidence of cervical cancer and genital warts. several conservative groups and think-tanks in the u.s. have questioned if such a vaccination will spark more promiscuity in young women. statements like "what message are we sending to our elementary students when we inoculate them for a sexually transmitted disease in the third grade?" or "this means even christian children who are brought up knowing that sexual activity before marriage is a sin would still be forced to be vaccinated against this std or they could not attend school" [ ] are not uncommon. but conservatives are not the only ones, who are in opposition to a mandatory vaccination against deadly disease and throughout the history of vaccination such reactions have been noted many times. it was aptly observed that groups fighting against gardasil are very diverse and include christian conservatives, who have long argued that safe sex encourages profligate sex, the growing antivaccine movement, which objects to all school-entry requirements and the parental-rights adherents, incensed by any mandates regarding their children's health [ ] . these arguments may seem laughable or medieval to some, but they are very valid to others and it is advisable that the introduction of this great novel product of vaccine technology is done employing all possible precautions and public-relations instruments. unquestionably, there is a lesson to be drawn. no new medicinal technology can be successful unless public is educated and well-informed of it. it is apparent that nanotechnology is currently driving the development of novel vaccines and adjuvants. at the same time, some concerns regarding the toxicity of such small particles have appeared. currently, there is a keen interest in nanotoxicology research since the processing of nanostructures in biological systems could lead to unpredictable and hence unknown toxic effects [ ] . one may draw some lesson from the history of polio vaccine when during - more than million americans received one or more of its doses contaminated with polyomavirus sv , which was then simply unknown being identified and isolated only in . when it became apparent that under specific set of conditions sv may cause cancer in laboratory animals, this led to a serious public scare. fortunately, subsequent studies of vaccinated humans over many years have shown that there was no causal relationship between receipt of sv -contaminated vaccine and cancer. such a completely unforeseen event should not be discounted when we in fact are actively engaged in genetical modification of humans (immunized individual always contains cells of immune system, which genome is partially rearranged in a way that cells of a non-immunized individual are not). other potential problems include high surface area and reactivity of small particles, their involvement in catalytic and oxidative reaction, ability to cross biological membranes as well as slow biodegradability of some materials used for their manufacturing. possibility of all of these being potential toxicity issues are at least partially supported by data describing the effects of pollutants on human health [ ] . still, it seems reasonable to anticipate that in the case of vaccines, the infrequent and low-level exposure to nanoparticles that an individual will encounter during immunization is not enough to cause adverse health problems such as those potentially attributed to nanotoxicity effects. with that being said, the development of any novel vaccine adjuvant or delivery platform should undergo all the necessary safety tests. they will also need to withstand public and political scrutiny. recently, regulatory authorities and the general public start to be concerned with products that cross traditional lines, i.e. combine biomaterials with cells, dna or proteins as in non-viral polymeric carriers for vaccines [ ] . currently, the most probable reason for the micro-and nano-based vaccines not flooding the market is linked to the cost of their safety testing. necessary clinical trials required for their approval are very long and difficult. unlike animal studies, human trials often require significant waiting period before protection can be analyzed. furthermore, since many vaccines are often administered to healthy individuals, and frequently to infants, it is critical that they are proven safe and well-tolerated in nonhuman primates before entering human trials. these risks and other possible side effects should be assessed in detail especially if mass-production and widespread administration of novel preparations is to be considered. currently, there is an uncertainty about nanoparticle processing in vivo and also near-complete absence of understanding of mechanisns of their interactions with biological systems (although the latter was never necessary in the history of medicine provided that the safety of this or that useful approach is demonstrated experimentally). therefore, continued animal research dealing with nanoparticle in vivo pharmakinetics and tissue distribution, nanotoxicity investigated in the same vein as drug toxicity currently is, as well as human trials for the safety evaluation of the experimental microand nanoparticle-based vaccine regimens will be of immense importance. a short history of vacination vaccines and vaccination in historical perspective nanotechnology in vaccine delivery replicating and non-replicating viral vectors for vaccine development hiv vaccine failure prompts merck to halt trial hiv vaccine may raise risk vaccinia virus: a tool for research and vaccine development virus like particle (vlp) vaccines. in: levine virus-like particles: passport to immune recognition virus-like particles-universal molecular toolboxes hepatitis b vaccine improved design and intranasal delivery of an m e-based human influenza a vaccine phase i testing of a malaria vaccine composed of hepatitis b virus core particles expressing plasmodium falciparum circumsporozoite epitopes efficacy of a bivalent l virus-like particle vaccine in prevention of infection with human papillomavirus types and in young women: a randomised controlled trial prophylactic quadrivalent human papillomavirus (types , , , and ) l virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase ii efficacy trial sustained efficacy up to . years of a bivalent l virus-like particle vaccine against human papillomavirus types and : followup from a randomised control trial recent developments in vaccine delivery systems immunostimulating reconstituted influenza virosomes proteosome tm technology for vaccines and adjuvants protollin: a novel adjuvant for intranasal vaccines mf adjuvant emulsion the perfect mix: recent progress in adjuvant research vaccine adjuvants revisited a randomized, double blind study in young healthy adults comparing cell mediated and humoral immune responses induced by influenza iscom vaccines and conventional vaccines adjuvant potential of aggregateforming polyglutamine domains different interactions between isomeric tetrakis-(n-hexadecylpyridiniumyl) porphyrins and cds nanoparticles targeting dendritic cells with biomaterials: developing the next generation of vaccines microparticle-based technologies for vaccines type and immunity following vaccination is influenced by nanoparticle size: formulation of a model vaccine for respiratory syncytial virus exploiting lymphatic transport and complement activation in nanoparticle vaccines a practical approach to the use of nanoparticles for vaccine delivery guard against gardasil who's afraid of gardasil? nation nanotoxicity: the growing need for in vivo study interaction of dendritic cells with biomaterials key: cord- - u ot h authors: graham, barney s.; anderson, larry j. title: challenges and opportunities for respiratory syncytial virus vaccines date: - - journal: challenges and opportunities for respiratory syncytial virus vaccines doi: . / - - - - _ sha: doc_id: cord_uid: u ot h respiratory syncytial virus (rsv) causes a significant proportion of the global burden of respiratory disease. here we summarize the conclusions of a series of chapters written by investigators describing and interpreting what is known about the virology, clinical manifestations, immunity, pathogenesis, and epidemiology of rsv relevant to vaccine development. several technological and conceptual advances have recently occurred that make rsv vaccine development more feasible, and this collected knowledge is intended to help inform and organize the future contributions of funding agencies, scientists, regulatory agencies, and policy makers that will be needed to achieve the goal of a safe, effective, and accessible vaccine to prevent rsv-associated disease. in this collection of brief reviews, we have attempted to consolidate much of the existing data relevant to the development of effective rsv vaccines. in addition, we sought to capture current views and opinions of the leaders in their respective disciplines to help frame the major issues important for developing new candidate vaccines and for navigating the regulatory pathways into clinical trials. rsv has been with us for a long time and continues to fill our pediatric hospital wards during each wintertime epidemic. the pathology and clinical syndrome of epidemic rsv bronchiolitis were probably first described in by adams ( ) , the virus was first discovered as chimpanzee coryza agent in (blount et al. ) , and rsv was identified as the major cause of bronchiolitis in infants in by robert chanock . rsv is a global pathogen, causing yearly wintertime epidemics in temperate climates and more unpredictable and continuous outbreaks in tropical climates generally associating with rainy seasons (see chapter by c. b. hall et al., this volume) . despite the global disease burden and extended time since its discovery, we still have not developed an effective vaccine for rsv. the reason for assembling these interpretive reviews at this time is based on a confluence of scientific events that have created the opportunity for an effective rsv vaccine to finally be realized. there has been much recent work on the clinical and molecular epidemiology of rsv on a global level including data from developing countries. these studies have confirmed the magnitude of the rsv-associated disease burden and the scope and dynamics of rsv genetic diversity. second, the combined efforts of many groups over the last decades have resulted in a better understanding of the vaccine-enhanced disease that occurred when children were immunized with formalin-inactivated alum-precipitated whole rsv vaccine in the s. these studies, largely conducted in animal models provide immunological parameters and biomarkers that can help estimate the likely safety or potential risk of a candidate vaccine. third, advances in rsv virology, particularly the development of reverse genetics and understanding of virus assembly and architecture, have provided more precise understanding of the specific roles of individual rsv proteins in the virus life cycle and immune evasion, and have provided the basis for multiple potential vaccine approaches. fourth, new technologies used to rapidly isolate new human antibodies and breakthroughs in the structure of the rsv f glycoprotein have provided a blueprint for designing better vaccine antigens. fifth, advances in immunology have suggested new vaccine formulation strategies for achieving protective immunity in the settings of immature and senescent immune responses. understanding the immunological limitations of the very young and very old is especially critical for rsv because these groups experience the greatest disease severity. sixth, technological advances in gene delivery and the ability to construct and manufacture a variety of gene-based vaccine vectors allows more selective control over the specificity and pattern of vaccine-induced immune responses than more traditional vaccine approaches based on whole virus. physicians, epidemiologists, and virologists have always recognized that rsv was a ubiquitous pathogen and caused annual global epidemics. recently, because of efforts of a few investigators, the availability of multiplex pcr and other rapid diagnostics, and improved surveillance for respiratory viruses in general due to heightened awareness and investment fueled by outbreaks of sars, avian influenza, and pandemic influenza, there are more data documenting the importance of rsv as a respiratory pathogen in developing countries (see chapter by c. b. hall et al., this volume). these advances have been punctuated by publications estimating the global disease burden (nair et al. ) and mortality (lozano et al. ) caused by rsv. the work on rsv in developing countries has demonstrated that the seasonality and age of peak illness severity may vary based on geographic and climatic parameters and should be taken into consideration in the planning of trials and development of target product profiles. in aggregate, the data suggest that two major immunological goals should be achieved before advancing a candidate rsv vaccine into sero-negative infants. first, potent neutralizing antibody should be induced so that virus spread and antigen load are controlled, and binding antibody that does not neutralize virus should be minimized to avoid immune complex deposition in airways. secondly, th -biased cd t cell responses should be avoided to diminish the potential for allergic inflammation associated with airway hyperreactivity. with the advent of well-characterized vaccine antigens that can induce potent neutralizing activity and vaccine delivery systems that induce cd t-cells and a balanced th /th response, these goals should be achievable. the immune responses induced by new candidate vaccines will need to be carefully evaluated and found to be distinct from those induced by fi-rsv to be tested in rsv naïve children (fig. ). biological profiles of candidate rsv vaccines. when new vaccine candidates emerge they will be compared to the fi-rsv vaccine associated with enhanced disease. instead of categorizing vaccines as ''killed'' or ''live'' there should be a more precise biological profile described. there will be nuances in each product that could distinguish it from the fi-rsv. with new antigen designs that display targets for potent neutralizing antibody, modern adjuvants with established safety databases, and new vaccine antigen delivery approaches, there should be acceptable and rational avenues for moving several new rsv vaccine approaches into seronegative infants where the need for protective immunity is the greatest. the chart depicts f being expressed by gene-based vectors. it is representative of other vaccine antigens that could be chosen, just as the recombinant adenovirus vector is representative of other potential gene delivery vectors. the categories shown are not exhaustive, but illustrate some of the properties that can determine the safety and immunogenicity of a candidate vaccine. ''neutralizing epitopes'' refer to the likelihood the vaccine approach will induce antibodies against all or some of the known neutralizing epitopes. ''mhc pathway'' indicates the major antigen processing and presentation pathway engaged by the vaccine. ''cd t cell induction'' is the relative potency for the vaccine platform to generate this response. ''il- '' is a representative term for the potential for inducing th -type cytokines following rsv challenge after vaccination. ''immune modulation'' indicates the potential for the representative vaccine to contain elements that alter or avoid rsv-induced immune responses based on in vitro or animal model data. ''delivery route'' and ''replication competence'' are self-explanatory the molecular mechanisms of rsv immune evasion and modulation are better described each year, and may eventually explain why durable immunity against infection is never achieved. these advances have been driven largely by the development of reverse genetics and ability to construct molecular clones of virus that can be selectively engineered to answer questions about viral pathogenesis (see chapter by p.l. collins et al., this volume) . viral proteins like ns , ns , and g have multiple effects on cells responsible for initiating immune responses (see chapters by s. mukherjee and n.w. lukacs, and by s. barik, this volume), and suggest that vaccines that elicit responses that block or avoid the immunomodulation associated with wild-type rsv infection without enhancing disease could provide more potent and durable immunity than natural infection. improving on immunity induced by natural infection has the potential to affect transmission efficiency, and could have a profound effect on the ecology of rsv which seems to depend largely on the niche of very young infants with immature immune systems (see chapter by a.m.w malloy et al., this volume). structural biology is changing the approach to antigen design for many vaccine development programs (kwong et al. ). rsv vaccine development may particularly benefit from defining the atomic structure of antigenic sites on the fusion (f) glycoprotein (see chapter by j.s. mclellan et al., this volume). the structures for three of the four known antigenic sites on the f glycoprotein associated with neutralization have now been solved by x-ray crystallography. most recently the structure of the pre-fusion f trimer has been solved and the atomic structure revealed ''antigenic site ['' which is at the apex of the native trimer and the binding site for a new group of extremely potent monoclonal antibodies (mclellan et al. ) . antibodies to antigenic site [ may explain the observation that most neutralizing activity in human serum cannot be absorbed by the postfusion f protein (magro et al. ) . live-attenuated virus vaccines and genebased vectors expressing the full-length f will express this vulnerable antigenic site, but prior subunit protein products have exclusively been in the post-fusion form of f. it is not known whether the fi-rsv vaccine product expressed antigenic site [, but that question and many others can now be addressed with the new reagents coming from this work. advances in immunology have impacted several aspects of rsv vaccine development. studies to define the antibody repertoire in young infants responding to either rotavirus or rsv have shown that infants do not have significant somatic mutation until - months of age, which limits affinity maturation (see chapters by s.m. varga and t.j. braciale and by a.m.w. malloy et al., this volume). for infants infected with rsv early in life this would result in expanding the precursor frequency of b cells with relatively low affinity for rsv antigens. this may compromise the potency of vaccine-induced neutralizing antibodies, and raises the question of whether immunizing children beginning at months of age would be a better strategy for achieving durable immunity. the improved understanding of toll-like receptors (tlrs) and other receptors for pathogen-associated molecular patterns (pamps) has led to a better understanding of how rsv disables the innate immune system (see chapters by s. mukherjee and n.w. lukacs and by s. barik, this volume). it has also led to new adjuvants approaches that may help in overcoming the immunological immaturity of the neonate and senescence of the elderly, which have been impediments to effective immunization in these target populations. in addition, we now have the ability to quantify and characterize the immune response patterns of human t cells using a variety of flow cytometric techniques to better recognize vaccine responses likely to be safe and those that may present risk. largely driven by the investment in hiv, malaria, and tb vaccine development, a variety of approaches are now available for delivery of genes encoding vaccine antigens (see chapter by r.j. loomis and p.r. johnson, this volume). although originally designed for inducing t cell-mediated immunity, many vaccine vectors or combinations can elicit substantial antibody responses. the great advantage of this type of vaccination is that it mimics live virus infection in that vaccine antigens are produced and presented by host cells, an immunization approach known to be safe in sero-negative infants. an added advantage to gene-based vaccination over live virus infection is that virus proteins important for inducing protective immunity can be selectively included, while excluding proteins associated with immunomodulation or other undesirable properties. thus, vaccine vectors could potentially be designed to improve upon natural immunity induced by rsv infection. an illustration of this occurred in experiments performed over years ago in which chimpanzees were immunized with recombinant vaccinia vectors expressing f or g prior to rsv challenge. although the vaccinia recombinant did not induce a potent primary immune response and did not prevent infection, the neutralizing antibody responses post-rsv challenges were among the highest ever recorded (collins et al. ). this observation suggests that if a properly designed rsv vaccine antigen could be appropriately delivered prior to the first rsv infection, a much more robust and durable immunity may be achievable. there are other issues relevant to rsv that need more attention to fill our gaps in knowledge and to provide the experimental tools and intellectual framework needed to complete the job of rsv vaccine development. these include the need for improved animal models, better understanding of mucosal immunity, more definitive clinical endpoints to use in efficacy trials, alternate vaccination strategies to protect the young infant (e.g., vaccinating pregnant women) and other high risk populations for whom vaccination may have limited effectiveness, and remedies for liability concerns. animal models have been critical for hypothesis generation related to the pathogenesis of rsv disease, fi-rsv vaccine-enhanced illness, and virus-induced airway hyperreactivity. rodent models were also instrumental in the development and licensing of passive antibody prophylaxis for children at high risk of severe disease (see chapters by m.s. boukhvalova and j.c.g. blanco, and by p.j. openshaw, this volume). however, all reported animal models of human rsv infection are semi-permissive except for chimpanzees. chimps have been especially useful for gauging the attenuation of cold-adapted and temperature-sensitive strains and selection of live-attenuated virus vaccine candidates for clinical trials. african green monkeys are perhaps the next most permissive of the nonhuman primate but still require a large virus inoculum to establish a significant infection. baboons have recently been reported to be susceptible to rsv but also require relative large inocula to establish modest levels of infection (papin et al. ). the rodent models (mice and cotton rats) can be informative about the patterns of immune response and pathology following immunization and challenge, but neither system recapitulates the sequence of upper airway infection and spread to the lower airway that occurs over about days. the large amount of virus and large volume of the inoculum required to infect the lower airway in current animal models essentially bypasses the first days of natural infection. using pneumoviruses matched to their natural host (e.g., pvm in mice or bovine rsv in cattle or sheep) may be a more authentic model of natural infection, but still not necessarily a reliable model for evaluating safety and efficacy of human rsv vaccines (see chapter by g. taylor, this volume). therefore, animal model data cannot guarantee the safety, immunogenicity, or efficacy of a candidate rsv vaccine in humans. clinical trials in the target population, at this time, are the only way to determine which vaccines should be advanced to licensure. since animal models will still be used for rank ordering and for determining ''no-go'' decisions when an inappropriate immune response is encountered, there are some important factors that should be remembered in interpreting results. first, the preparation of viral stocks is critical. the cell line used for production, the level of fetal bovine serum and other factors in the final media, the quality of the stock in terms of defective interfering particles, and minimizing the amount of cytokines and other biologically active substances in the final preparation can affect immune response patterns and pathology. use of purified or semi-purified challenge virus should minimize the above noted factors that might confound results. second, the possibility of enhanced disease after vaccination cannot be adequately assessed unless challenge is associated with some virus replication. the titer of rsv at the peak of the replication post challenge in untreated animals should be at least e pfu/gram lung, or the vaccine effect will be difficult to assess. finally, it is important to consider sample type. for example, the expected type of inflammatory infiltrate is different for bronchoalveolar lavage specimens (primarily includes cells present in the larger airways) or from homogenized lung tissue (includes cells from intraepithelial and lower airways of the lung). one of the key decisions a vaccine developer has to make is whether induction of vaccine-elicited immune responses will occur systemically or mucosally. the vaccines that have advanced into sero-negative infants are live-attenuated viruses delivered intranasally. some of the other vaccine approaches in development can be delivered mucosally, but most subunit proteins or vaccine vectors will be delivered intramuscularly (im). palivizumab provides the proof-of-concept that serum antibody with a sufficient level of neutralizing activity can prevent severe disease in infants at high risk. however, palivizumab does not prevent infection of the upper airways and it is not known if it alters transmission. a vaccine that decreases transmission has the potential to be given in contacts of high risk persons to decrease their risk of infection. there is also, very little known about the role of mucins in viral clearance or in interactions with secreted antibodies. muc ac is known to be induced in airways of animals infected with rsv, and is associated with airway hyperreactivity (see chapter by m.t. lotz et al., this volume). the rsv g glycoprotein is heavily o-glycosylated and is rich in proline, serine, and threonine, so its chemical composition more resembles muc proteins than a typical viral glycoprotein. it seems likely that a better understanding of how muc proteins interact with rsv and with rsv-specific antibodies and a more complete understanding of the role of the rsv g glycoprotein in the airway may help guide vaccine development. the major goals for an rsv vaccine are to prevent severe disease in young infants, to establish durable protection against reinfection, and to reduce excess mortality in the elderly. there are several potential approaches to achieve these goals without focusing directly on the neonate as the primary target population for vaccination. in some respects, immunizing neonates may be counter-productive if ineffective immune response patterns are established that are perpetuated throughout life. more work is needed to determine whether vaccination of infants beginning at months of age, when about % of infants are still uninfected, could be a more effective strategy for achieving immunity in the general population. that would allow the vaccine to be the first rsv antigen exposure in the majority of infants at a time when effective somatic hypermutation was occurring, the th -biased tendencies of the infant are waning, and antigen presenting cells have a more adult-like phenotype. it is possible that establishing effective immunity in the majority of children or immunizing the parents and siblings of young infants could reduce the exposure of newborns to rsv resulting in a progressively larger number of uninfected children reaching the month vaccination time point. more data on the epidemiology of transmission and more sophisticated mathematical modeling of transmission are needed, especially in developing country settings. understanding transmission and mathematically modeling were a critical part of the successful immunization campaign to eliminate rinderpest, another paramyxovirus (mariner et al. ) . immunizing pregnant women is another approach to protect infants by passive transfer of antibody from mother to children transplacentally or through breast milk. vaccines for the various target populations will require different considerations and potentially different vaccine formulations as outlined in table . there is a rich pipeline of candidate rsv vaccines and other prevention approaches in various stages of development (see chapters by h.y. chu and j.a. englund , r.a. karron et al., t.g. morrison and e.e. walsh, and by r.j. loomis and p.r. johnson, this volume, and fig. ). since animal models are limited, and there are many potential target populations, the clinical development plans and clinical trial designs will be critical for achieving success. there are several issues to consider such as the seasonality of rsv which dictates when vaccine trials should be timed and makes north-south clinical collaborations appealing. in tropical regions where there is not distinct seasonality, the timing of trials will require more consideration. having clear diagnostic and disease severity endpoints to judge efficacy are essential to determine the size and expense of the studies and ultimately the safety and efficacy of the vaccine. for example, strict diagnostic criteria and establishing a composite index of illness severity will be needed for studies with multiple sites to account for site differences in managing patients. it would be helpful to also use similar criteria and composite indices across studies. endpoints will need to be tailored to the target population. for example, for the sero-negative children, hospitalization has been used for passive antibody and vaccine trials. for be rsv or chimeric parainfluenza or newcastle disease virus. pre-fusion f protein refers to either the soluble purified protein or the protein presented on a particle (e.g. ferritin or vlp). pre-fusion f is listed as the ''preferred'' and simplest antigen choice since it includes antigenic site Ø in addition to neutralizing determinants on the post-fusion f. however, this should not be interpreted as excluding the potential value of post-fusion forms of f, wt f expressed in a gene-based vector, or additional vaccine antigens that may have value. importantly, there is at least one additional target for broadly neutralizing antibody in g and in animal models antibodies to g reduce immunopathology. in addition, some vaccine approaches may benefit by the addition of genes for internal structural and regulatory proteins as a source of additional t cell epitopes or to add constructs designed to stabilize glycoprotein structure. it is reasonable to include these additional vaccine antigens if care is taken to avoid proteins that interfere with induction or maintenance of immunity or epitopes that may elicit immunopathological responses older children, medically attended lower respiratory tract illness may be used. in adults, diagnosing rsv infection is more difficult and the presence of underlying disease makes assigning causality for acute respiratory illnesses less precise. it is possible that experimental human challenge studies may help understand vaccine efficacy in adults, especially the effect of vaccination on virus shedding and mild illness. because of the legacy of fi-rsv vaccine-enhanced illness and the fact that many of the key target populations for an rsv vaccine are especially vulnerable (young infants, pregnant women, and the frail elderly), a concern about liability has been a significant part of the risk:benefit analysis for companies contemplating rsv vaccine development. if concerns present a road block to vaccine development, it seems reasonable to explore legislative solutions for diminishing the financial risks to developers, investigators, and study participants by providing assurance of indemnification for unanticipated adverse events. at this point in time, the opportunities for success far outweigh the remaining challenges for providing safe and effective vaccine-induced immunity to prevent rsv infection and to reduce the substantial disease burden that it still causes. to complete that task will require the sustained commitment of funding agencies, ongoing combined and organized efforts of government, university, and industry scientists across multiple disciplines, thoughtful guidance from regulatory agencies, and facilitation by prescient law and policy makers. primary virus pneumonitis with cytoplasmic inclusion bodies: study of an epidemic involving thirty-two infants, with nine deaths recovery of cytopathogenic agent from chimpanzees with coryza recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca). ii. epidemiologic aspects of infection in infants and young children recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca). i. isolation, properties and characterization evaluation in chimpanzees of vaccinia virus recombinants that express the surface glycoproteins of human respiratory syncytial virus the changing face of hiv vaccine research neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention rinderpest eradication: appropriate technology and social innovations structure of rsv fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis infant baboons infected with respiratory syncytial virus develop clinical and pathologic changes that parallel those of human infants acknowledgments we thank the authors of these collected chapters for their outstanding scholarship and commitment to finding vaccine approaches for preventing rsv-associated disease. we dedicate this book to the memory of dr. caroline hall whose career was committed to the care of children and prevention of viral diseases. her life embodied the mission of this book and her fingerprints will be evident in the final solution for controlling rsv. this work was supported by intramural funding from the national institute of allergy and infectious diseases. key: cord- - f vr a authors: madan, anuradha; segall, nathan; ferguson, murdo; frenette, louise; kroll, robin; friel, damien; soni, jyoti; li, ping; innis, bruce l.; schuind, anne title: immunogenicity and safety of an as -adjuvanted h n pandemic influenza vaccine in a randomized trial in healthy adults date: - - journal: j infect dis doi: . /infdis/jiw sha: doc_id: cord_uid: f vr a background. almost cases of human infection with avian influenza a/h n have been reported since . pandemic preparedness strategies include h n vaccine development. methods. we evaluated an inactivated h n vaccine in an observer-blind study in healthy adults aged – years. participants ( ) were randomized to receive of as -adjuvanted vaccines (low or medium dose of hemagglutinin with as (a) or as (b)), one nonadjuvanted vaccine, or placebo. the coprimary immunogenicity objective determined whether adjuvanted vaccines elicited an immune response against the vaccine-homologous virus, days after the second vaccine dose per us and european licensure criteria in the per-protocol cohort (n = ). results. all adjuvanted vaccines met regulatory acceptance criteria. in groups receiving adjuvanted formulations, seroconversion rates were ≥ . %, seroprotection rates ≥ . %, and geometric mean titers ≥ . % versus . %, . %, and . for the nonadjuvanted vaccine. the as adjuvant enhanced immune response at antigen-sparing doses. injection site pain occurred more frequently with adjuvanted vaccines (in ≤ . % of vaccinees) than with the nonadjuvanted vaccine ( . %) or placebo ( . %). none of the serious adverse events reported were related to vaccination. conclusions. two doses of as -adjuvanted h n vaccine were well tolerated and induced a robust antibody response at antigen-sparing doses in healthy adults. clinical trials registration. nct . periodic outbreaks of h avian influenza a virus infections occur in poultry worldwide, with sporadic transmission to humans. in , an outbreak of h n disease in the netherlands resulted in human infections and death, with evidence of limited human-to-human transmission [ ] . human infections with h n viruses were first reported in china in february ; to the present time, there have been waves of infection [ ] . as of december , a total of laboratory-confirmed cases, including deaths, had been reported to the world health organization [ , ] . the case fatality rate of h n influenza is approximately % [ , ] . the virus can cause rapidly progressive pneumonia, often complicated by extrapulmonary disease associated with hypercytokinemia [ ] . genetic changes observed in the h n virus suggest adaptation to mammals, carrying the risk of human-to-human transmission [ ] . it has been shown that h n and h n influenza viruses are capable of airborne transmission in a mammalian host (ferret), without losing virulence [ , ] . these observations suggest the potential for an h pandemic in humans, and support pandemic h vaccine development. several h inactivated influenza vaccines and live-attenuated influenza vaccines are in clinical development, but have not been highly immunogenic in humans [ ] [ ] [ ] . adjuvanted vaccines have shown improved immunogenicity [ ] [ ] [ ] [ ] . a recent mix-and-match study demonstrated that a monovalent h n vaccine adjuvanted with as induced a better immune response than the nonadjuvanted or mf -adjuvanted formulations, when administered to adults according to a -dose schedule [ ] . here, we present the findings of a study that evaluated h n vaccine formulations with hemagglutinin (ha) antigen doses of . and . µg, given with as adjuvants of different potency and a nonadjuvanted formulation. the doses of as -adjuvanted ha antigen were chosen for testing based on a clinical development program by gsk biologicals with an as -adjuvanted split virus h n marketed vaccine. this was a phase i/ii, randomized, placebo-controlled, multicenter trial evaluating an h n influenza vaccine (nct ). the trial was approved by independent ethics committees or institutional review boards and was conducted in accordance with the declaration of helsinki, the international conference on harmonisation good clinical practice guidelines, and regulatory requirements of participating countries. participants provided written informed consent. the trial was observer blind and enrolled healthy participants - years of age in the united states and canada (inclusion criteria are detailed in supplementary text ). the inactivated, split-virion vaccine, manufactured with a reverse geneticderived reassortant seed virus developed by world health organization collaborating centres and references laboratories from a/shanghai/ / (h n ) (gsk vaccines, quebec, canada), was adjuvanted with as , an oil-in-water emulsion containing . mg (as b ) or . mg (as a ) of dl-αtocopherol. participants were randomized : : : : : to of groups receiving different ha antigen doses (mixed with adjuvant in groups - ) or placebo: ( ) the antigen doses were less than the initially targeted concentrations of . and . µg, because the single radial immunodiffusion assay used to determine the antigen concentration during formulation overestimated the concentration in relation to subsequently available reagents provided by the center for biologics evaluation and research (cber) to evaluate vaccine potency. vaccines were administered twice, days apart, by intramuscular injection in the deltoid muscle. the coprimary immunogenicity objective was to evaluate whether the adjuvanted a/shanghai/ / (h n ) vaccines elicited an immune response against the vaccine-homologous virus that met us cber and european committee for medicinal products for human use (chmp) immunogenicity targets at day ( days after the second vaccine dose). the primary immunogenicity objective of the study was met if the following criteria were fulfilled for any adjuvanted vaccine formulation: the lower limit of the . % confidence interval was ≥ % for the seroconversion rate (scr) and ≥ % for seroprotection rate (spr), and chmp criteria were met if point estimates were > % for scr, > % for spr, and > . for the mean geometric increase (mgi). the coprimary safety objective was to describe the safety and reactogenicity of the vaccines up to day . immunogenicity assessments were done with hi and mn assays at baseline (day ), at days after each dose (days and ), and at months after the first vaccine dose (day ), and with hi assay only at months after the first vaccine dose (day ). hi and mn antibody titers were assessed using horse red blood cells (rbcs). to remove nonspecific agglutinin to horse rbcs and nonspecific virus inhibitors introduced by the hemadsorption step, a receptor-destroying enzyme treatment step was added after horse rbc hemadsorption. humoral immune response assays were performed by a gsk biologicals laboratory (hi) and by viroclinic biosciences (mn). the following derived parameters related to the tested vaccine virus were estimated for hi titer: spr, gmt, scr, and mgi. spr was defined as the proportion of participants with reciprocal hi titers ≥ . gmts were defined as the antilog of the mean of the log -transformed inverse titers. scr was defined as the proportion of participants with either a prevaccination reciprocal hi titer < and a postvaccination reciprocal titer ≥ or a prevaccination reciprocal hi titer ≥ and a ≥ -fold increase in postvaccination reciprocal titer. mgi was defined as the geometric mean of the within-participant ratios of the postvaccination to the prevaccination reciprocal hi titer. for mn titer, seropositivity rate and gmt were derived in a similar way as for hi. participants recorded solicited injection site and general symptoms between days and after each vaccination in diary cards, collected at the next visit. symptoms were graded by severity from (mild) to (severe). grade was defined as "significant pain at rest, preventing everyday activities" for pain, "surface diameter > mm" for redness and swelling, temperatures of "≥ . °c (≥ . °f)" for fever, and "preventing normal activities" for all other solicited symptoms. blood samples for safety evaluations were collected on days , , , , and . participants recorded unsolicited symptoms (graded by severity) until days after each vaccination. medically attended adverse events (aes), potentially immunemediated disorders ( pimds), and serious aes (saes) were followed up until the study end. participants were asked to report immediately any events perceived as serious. immunogenicity analyses were performed on the per-protocol cohorts ( participants who complied with the protocol, received vaccine, and had assay results available for antibodies against the vaccine-homologous ha antigen at the specified intervals). the safety analysis was descriptive and was performed on the total vaccinated cohort ( participants who received ≥ dose of study vaccine or placebo). statistical methods are described in detail in supplementary text . a total of participants were included in the total vaccinated cohort and in the per-protocol cohort ( figure ). demographics were similar in all study groups (supplementary table ). the mean participant age was years, % of participants were women, and most ( . %) were of white european/caucasian ethnic origin. immunogenicity cber and chmp criteria against the vaccine-homologous virus were met for all adjuvanted vaccines at day , days after the second vaccine dose. the scrs and sprs were similar in all adjuvanted vaccine groups; scrs were . %- . % and figure s ). between . % and . % of participants were seropositive before vaccination, although all baseline gmt values were low (only samples had a titer ≥ ). after the peak observed on day , seropositivity rates and antibody titers declined at day and further at day , but gmts remained above baseline levels in the adjuvanted groups, ranging from . to . (table ; supplementary figure s ). the sprs at days and were . %- . % and . %- . %, respectively. an immune response against an h n virus was also observed at day , albeit at lower levels than against the vaccine-homologous virus. in the adjuvanted groups, days after the second vaccine dose, scrs were . %- . %, mgis were . - . and gmts were . - . , with the highest response observed in the as a adjuvanted groups (table ; figure ). immune responses assessed by the mn assay showed a similar kinetic; however, titers remained . - . times higher at day compared with baseline ( figure ). the vaccine response rate is presented in supplementary table s . the non-adjuvanted hd ha vaccine elicited a considerably lower immune response against both vaccine-homologous and vaccine-heterologous viruses (table ; figure ). adjuvant effect was demonstrated in all adjuvanted groups, as the lower limit of the . % confidence interval for gmt ratio (adjuvanted over nonadjuvanted) exceeded . , and the scr difference (adjuvanted minus nonadjuvanted) exceeded % in all groups at days after the second vaccine dose (table ). at days and , no study participant had reciprocal hi titers ≥ . the immune response was low in all vaccine groups after administration of only vaccine dose (table ; figure ). in a sub-analysis of the homologous immune response by age, the adjuvanted vaccine was immunogenic in both age groups, with sprs ≥ . % in participants - years of age, despite lower gmts (table ). the spr was . % in participants - years of age who received the nonadjuvanted hd ha vaccine. the immune response was generally lower in participants who had previously received seasonal influenza vaccine than in nonrecipients. at day , gmt values in the adjuvanted vaccine groups were . - . in prior recipients and . - . in nonrecipients. spr values were . %- . % in prior recipients and . %- % in nonrecipients. pain was the most common injection site solicited symptom, occurring more frequently in the adjuvanted vaccine groups than in the hd ha nonadjuvanted group (table ). redness and swelling at the injection site occurred in . %- . % of participants receiving vaccines adjuvanted with as b and in . %- . % of those receiving vaccines adjuvanted with as a (table ) . grade injection site solicited symptoms were reported by up to . % of the participants in the adjuvanted groups and by none of the participants in the nonadjuvanted and placebo groups. fatigue, headache, and muscle ache were the most frequently reported solicited general symptoms (table ). fatigue and muscle ache occurred in . %- . % of participants in all adjuvanted groups, compared with . %- . % in the hd ha nonadjuvanted and . %- . % in the placebo group. fever occurred infrequently and at a similar rate across all study groups. most solicited aes resolved spontaneously. grade solicited and unsolicited events occurred at a low rate and few unsolicited aes were considered related to vaccination (table ). twenty saes were reported in participants up to the study end, and none of them were assessed as vaccination related. nine pregnancies occurred during the study. one participant (exposed to the vaccine during the first trimester) underwent elective abortion, not related to the vaccination. for the other pregnancies, the exposure to study vaccine occurred before the pregnancy; gave birth to live neonates, and for the outcome was unknown. one potentially immunemediated disorder assessed by the investigator as not related to vaccination-autoimmune thyroiditis-was reported days after administration of the second dose of placebo. no deaths were reported during the study. for hematological and biochemical parameters, results outside of the normal laboratory range were evenly distributed across all time points (including baseline) and vaccine groups, and no clear clinical trends were observed (supplementary tables s and s ). the results of this phase i/ii randomized, placebo-controlled trial showed that doses of the h n as -adjuvanted vaccine elicited a robust immune response in healthy adults up to years of age, with an acceptable safety profile. adjuvantation with as enabled an immune response that satisfied regulatory acceptance criteria at antigen-sparing concentrations of ha, a prerequisite for a pandemic influenza vaccine. a dose as low as . µg ha elicited a robust hi antibody response. hi responses were low after the first vaccine dose in all vaccine groups, indicating that doses are required to induce an adequate immune response. three weeks after the second vaccine dose, sprs and scrs against the vaccine-homologous virus were ≥ . % in the adjuvanted vaccine groups. the non-adjuvanted vaccine elicited a poor immune response, and an adjuvant effect was demonstrated in all adjuvanted groups in terms of gmt ratio and scr difference. the gmt and mgi for the as -adjuvanted vaccines against the vaccine-homologous virus were highest in the md ha/as a group, followed by the ld ha/as a group, the hd ha/as b group, and finally the ld ha/as b group. thus, the potency of the as adjuvant ( . or . mg tocopherol in as a or as b , respectively) seems to have more influence than antigen content on immunogenicity. this was also observed in a study of a h n ha antigen produced by a different manufacturer mixed with gsk's as a at the point of use [ ] and in a study of with the as -adjuvanted h n pandemic vaccine [ ] . immune responses in older participants (aged - years) were generally lower than in younger participants (aged - years) and lower in participants who had previously received seasonal influenza vaccine than in nonrecipients, consistent with findings in other studies of pandemic influenza vaccines [ , , , ] . up to . % of study participants were seropositive before vaccination. detectable levels of hi antibody in the general population before vaccination with pandemic influenza vaccines have been reported in several studies [ ] [ ] [ ] , suggesting either natural immune response to previous exposure or crossreactivity between virus strains. previous exposure is unlikely, because the h n virus circulated in china and no infections in humans were reported in the north american population [ ] . in addition, human antibody response to this strain has been shown to be very poor [ ] . the same study suggests cross-reactivity of h and h seasonal influenza subtypes to the h n virus, and this is the most likely explanation of our finding. the cd + t cells to seasonal influenza are reported to recognize h n epitopes and to exhibit cross-reactivity with the h n virus [ ] . in our study, cross-reactivity could originate either from previous infection or seasonal vaccination. of note, a total of study participants had received vaccination against influenza within the previous seasons, although the virus strain to which the participants were previously exposed was not documented. the study shows that the as adjuvant enhances the immune response against h antigens at antigen-sparing doses. previous studies of h vaccines that were nonadjuvanted or adjuvanted with aluminum hydroxide have shown poor immunogenicity in humans [ , ] . clinical studies of adjuvanted h n vaccines from different manufacturers have found higher immunogenicity compared to nonadjuvanted formulations [ ] [ ] [ ] [ ] . a phase ii study of an h n vaccine mixed at the point of use with mf adjuvant resulted in seroconversion in % of participants with the ld ha dose; higher antigen doses did not elicit an increase in immunogenicity [ ] . a recent phase ii study compared different doses of an h n vaccine mixed at the point of use with as a or mf or administered without an adjuvant [ ] . as in the present study, the immune response with all formulations was low after vaccine dose. after doses, the immune response was superior with the as -adjuvanted formulations compared with the mf -adjuvanted or standard or high-dose nonadjuvanted formulations [ ] . for as -adjuvanted vaccines, similar immunogenicity was attained with antigen content varying between . , , and µg of ha [ ] . the present study also demonstrated cross-reactivity of the vaccine against a vaccine-heterologous strain. phylogenetic analysis has shown a high degree of homology in the ha gene sequence of various h viruses [ ] . an as -adjuvanted h n vaccine, engineered by reverse genetics from an h n virus, has been developed by gsk vaccines, and therefore the present study evaluated cross-reactivity against an h n vaccine-heterologous virus. robust cross-reactivity was seen, with sprs ranging from . % to . %. in preclinical studies in mice, an h n /as vaccine elicited antibodies cross-reacting with h n , h n , and h n viruses [ ] , and an h n viruslike particle vaccine produced in insect cells using a baculovirus vector elicited antibodies cross-reacting with an h n virus [ ] . to our knowledge, this is the first report on the immune response against h n in humans beyond day after vaccination. based on the blood sampling schedule, hi antibody titers against the vaccine-homologous virus peaked at days after the second dose. at day , a notable decrease in gmts was observed, although of different magnitude across groups. however, seropositive rates remained above % at day and above % at day in all adjuvanted groups, compared with . % and . % in the nonadjuvanted group and . % and . % in the placebo group. in addition, recent studies seem to suggest that a robust immune response to the ha head and stalk domains, as measured with enzyme-linked immunosorbent assay, may be induced even in the absence of hi and mn response [ ] , so the clinical relevance of the decline in antibody titers is not clear. the observed kinetics of the immune response in adjuvanted groups in our study is similar to that elicited by other influenza vaccines, in particular the adjuvanted h n vaccines [ ] , for which a strong anamnestic response was elicited by a heterologous booster dose, up to years after priming [ , ] . the h n as -adjuvanted vaccine was generally well tolerated. pain was the most common solicited injection site symptom, as observed in other studies of adjuvanted influenza vaccines [ - , , , ] . tolerability seemed to be acceptable, as most participants returned for the second vaccine dose. most aes were low grade and resolved spontaneously. none of the saes was assessed as related to vaccination. in this study, a trend was observed for higher reactogenicity with as a -adjuvanted vaccines than with as b formulations. however, all formulations were well tolerated and no safety signal was identified during the study. the use of the as a adjuvant led to an improved immune response compared with that induced by as b -adjuvanted formulations, so the risk-benefit ratio in using a high potency adjuvant seems clinically acceptable. pandemic preparedness strategies include development of vaccines that are antigen sparing, because the time available to produce sufficient antigen to provide effective vaccine coverage is likely to be limited. in addition, vaccines that elicit a broad cross-reactive immune response are needed to allow pre-pandemic priming and use of prime-boost vaccination schedules. formulation of pandemic vaccines with an effective adjuvant is an accepted strategy to accomplish both goals [ ] and to ensure adequate immunogenicity for the elderly and those with reduced immune responsiveness due to concurrent illness. the assessment of immunogenicity for this study is based on hi and mn assays; no additional evaluation such as cellmediated immunity was performed which could bring additional information but for which no regulatory acceptance criteria have been established. no formal comparisons between adjuvanted vaccine groups were performed, and analyses were descriptive. these could constitute potential limitations to the study. nevertheless, the type i error was adjusted for the evaluation of the primary immunogenicity objective, as well as the secondary objective related to adjuvant effect. in conclusion, doses of an as -adjuvanted h n vaccine induce a robust anti-h immune response with an acceptable safety profile. when balancing antigen sparing with effective immunization, a . -µg antigen dose (or . µg to align with the already licensed h n vaccines made by the same process) with as a adjuvantation seems the most desirable formulation. this vaccine candidate could be beneficially deployed should the h n virus acquire the ability for sustained human-to-human transmission or to protect persons at risk. supplementary materials are available at http://jid.oxfordjournals.org. consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. transmission of h n avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands world health organization. who risk assessment of human infections with avian influenza a(h n ) virus influenza at the human-animal interface: summary and assessment as of viral lung infections: epidemiology, virology, clinical features, and management of avian influenza a(h n ) emergence of the virulence-associated pb e k substitution in a fatal human case of highly pathogenic avian influenza virus a(h n ) infection as determined by illumina ultra-deep sequencing airborne transmission of highly pathogenic h n influenza virus in ferrets novel avian-origin human influenza a(h n ) can be transmitted between ferrets via respiratory droplets a phase i clinical trial of a per.c cell grown influenza h virus vaccine a randomized clinical trial of an inactivated avian influenza a (h n ) vaccine assessment of human immune responses to h avian influenza virus of pandemic potential: results from a placebo-controlled, randomized double-blind phase i study of live attenuated h n influenza vaccine a recombinant viruslike particle influenza a(h n ) vaccine a cell culture-derived mf -adjuvanted pandemic a/h n vaccine is immunogenic in adults serological responses to an avian influenza a/h n vaccine mixed at the point-of-use with mf adjuvant: a randomized clinical trial effect of varying doses of a monovalent h n influenza vaccine with and without as and mf adjuvants on immune response: a randomized clinical trial antigen sparing and crossreactive immunity with an adjuvanted rh n prototype pandemic influenza vaccine: a randomised controlled trial immunogenicity and safety of varying dosages of a monovalent h n influenza vaccine given with and without as adjuvant system in healthy adults and older persons safety and immunogenicity of a split-virion as a-adjuvanted a/indonesia/ / (h n ) vaccine in taiwanese adults trial of influenza a (h n ) monovalent mf -adjuvanted vaccine safety and long-term humoral immune response in adults after vaccination with an h n pandemic influenza vaccine with or without as adjuvant safety and immunogenicity of an inactivated subvirion influenza a (h n ) vaccine prevalence and control of h avian influenza viruses in birds and humans human antibody responses to avian influenza a(h n ) virus human cytotoxic t lymphocytes directed to seasonal influenza a viruses cross-react with the newly emerging h n virus isolation and characterization of h n viruses from live poultry markets-implication of the source of current h n infection in humans as -adjuvanted h n detergent-split virion vaccine is highly immunogenic in unprimed mice and induces cross-reactive antibodies to emerged h n and additional h subtypes development of influenza h n virus like particle (vlp) vaccine: homologous a/anhui/ / (h n ) protection and heterologous a/chicken/jalisco/cpa / (h n ) cross-protection in vaccinated mice challenged with h n virus an h n influenza virus vaccine induces broadly reactive antibody responses against h n in humans dose-sparing h n a/indonesia/ / pre-pandemic influenza vaccine in adults and elderly adults: a phase iii, placebocontrolled, randomized study an assessment of prime-boost vaccination schedules with as a-adjuvanted prepandemic h n vaccines: a randomized study in european adults long-term booster schedules with as a-adjuvanted heterologous h n vaccines induces rapid and broad immune responses in asian adults long-term immunogenicity of an as -adjuvanted influenza a(h n )pdm vaccine in young and elderly adults: an observer-blind, randomized trial extended antigen sparing potential of as -adjuvanted pandemic h n vaccines in children, and immunological equivalence of two formulations of as -adjuvanted h n vaccines: results from two randomised trials stockpiling prepandemic influenza vaccines: a new cornerstone of pandemic preparedness plans acknowledgments. we are indebted to the participating study volunteers, clinicians, nurses, and laboratory technicians at the study sites. we are grateful to the principal investigators, alan kravitz and jack yakish. we also thank janine linden for writing the study protocol; stephanie sharp (veristat on behalf of gsk vaccines) for writing the study report; thierry ollinger for contributing to immunological data generation; andré key: cord- - sqkh m authors: schmidt, alexander c; couch, robert b; galasso, george j; hayden, frederick g; mills, john; murphy, brian r; chanock, robert m title: current research on respiratory viral infections: third international symposium date: - - journal: antiviral res doi: . /s - ( ) -x sha: doc_id: cord_uid: sqkh m nan the third international symposium on respiratory viral infections was convened by the macrae group (new york, ny) in st. lucia, windward islands, on - december . for the third time, this symposium provided a forum for virologists, vaccinologists, clinicians, pharmacologists and public health specialists to discuss recent advances in respiratory virus research in an interdisciplinary fashion (kaiser et al., ; munoz et al., ) . the spectrum of discussion ranged from basic virology and pathogenesis to vaccinology, immunology, and management strategies for respiratory viral infections. epidemiology of respiratory viral disease and possible preparations for the next influenza pandemic were also an important part of the agenda. until , influenza viruses were the only known filterable human respiratory tract pathogens. in , shope recovered an influenza a h n virus from swine, which probably was the first human influenza virus isolated. two years later smith, andrews and laidlaw recovered the first influenza isolate from humans, and soon after, efforts to develop an inactivated influenza a vaccine began. influenza b and c were isolated in and by francis and taylor, respectively. in in the laboratory of infectious diseases (lid) at the national institutes of health, and hilleman, then working at the walter reed army medical center, recovered the first human adenoviruses and established their importance in acute febrile respiratory tract disease (rowe et al., ; hilleman, ) . in the following years, robert chanock discovered most of the remaining respiratory viruses that are considered important lower respiratory tract pathogens today. the first of them, a croup-associated myxovirus, was discovered during an outbreak of croup in cincinatti in , and it was later designated human parainfluenza virus type (piv ). in , morris and colleagues recovered the chimpanzee coryza agent (cca) during an outbreak of a cold-like illness in a chimpanzee colony, and a year later chanock and colleagues recovered two similar isolates from an infant with bronchopneumonia and from another infant with laryngotracheobronchitis, and characterized the human virus now known as respiratory syncytial virus (rsv) (chanock et al., ) . the discovery in of piv and piv , the single most common cause of croup and the second most common cause of serious viral pediatric lower respiratory tract disease, respectively, broadened our understanding of the etiology of acute lower respiratory tract disease (chanock et al., ) . the discovery of piv in followed in short order. in , a double-blind prospective study evaluating the use of tetracycline in the treatment of cold agglutinin-positive atypical pneumonia led to the identification of the etiologic agent of this disease. the agent, originally recovered by eaton from patients with this form of pneumonia, was known to be filterable and thought to be a virus but its role as an etiologic agent was heavily disputed. a large double-blind study by chanock, kingston and mufson, in which antibodies to the eaton 'virus' were used to define a subset of patients with pneumonia, showed that tetracycline therapy decreased duration of the disease, thereby excluding a virus etiology. subsequently, the eaton agent was shown by chanock, hayflick and barile to be a mycoplasma that grew in cell-free medium; later it was named mycoplasma pneumoniae (chanock et al., ) . serologic analyses and studies in adult volunteers confirmed the etiologic role of this organism in cold agglutinin-positive atypical pneumonia. renewed efforts in vaccine development against respiratory viruses began in the s with the observation that infants and young children, after having recovered from respiratory tract infection with adenoviruses, shed virus from their gastrointestinal tract for an extended period of time without experiencing gastrointestinal symptoms. this led to the hypothesis that one could potentially use the gastrointestinal tract to vaccinate against respiratory tract disease caused by these viruses. wild-type adenovirus type and administered orally in enteric-coated capsules was found to protect military recruits against respiratory tract disease caused by these viruses. gastrointestinal symptoms were not observed, and although virus was shed from the intestine, it did not infect close contacts (couch et al., ) . the development of vaccines against respiratory viruses suffered a major setback in when formalin-inactivated rsv vaccine not only failed to protect infants against rsv infection but instead potentiated rsv disease upon subsequent rsv infection (kim et al., ) . the inactivated vaccine did not induce a potent neutralizing antibody response but it stimulated an exaggerated cd + t cell response without stimulating cytotoxic cd + t cells. this unanticipated failure of a non-living vaccine reoriented the research agenda of the laboratory of infectious diseases towards the development of live-attenuated virus vaccines. a cold-passaged rsv strain (cp ) was selected in as the first candidate live-attenuated rsv vaccine strain (friedewald et al., ) . this candidate vaccine was safe and immunogenic in adults and older children but was insufficiently attenuated in seronegative infants (kim et al., ) . since then, the search for a live rsv vaccine strain has been a central focus of the lid. developing a live rsv vaccine candidate that is attenuated yet immunogenic in seronegative infants has proved to be a formidable task. since incidence and morbidity of rsv are highest in the second and third month of life, a vaccine candidate has to be safe for administration to neonates, able to stimulate an immature immune system, and able to overcome the immunosuppressive and antiviral effects of passively acquired maternal rsv antibodies. initial vaccine candidates were derived in the late s and early s by passage of virus at low temperature (cold passage) or by chemical mutagenesis. several different lineages of mutants, such as temperature sensitive mutants generated by -fluorouracil ( fu) mutagenesis, were evaluated in infants and young children but were insufficiently attenuated or genetically unstable. the cold-passaged (cp) mutant that was subsequently further attenuated by the acquisition of two missense mutations that conferred temperature sensitivity (ts) to yield cpts rsv / , has provided the most promising vaccine candidate tested thus far. this candidate vaccine virus was infectious, safe and immunogenic in -month-old seronegative infants, conferred protection against challenge with a second dose of vaccine virus weeks later, and caused only mild upper respiratory tract symptoms (wright et al., ) . recent development of a method for rescue of infectious rsv from cdna by collins enhanced our ability to develop rsv vaccine candidates rapidly (collins et al., ) . site-directed mutagenesis can now be used for the first time to construct viruses with one or more additional attenuating mutations. using recombinant cdna technology, viable rsv mutants with deletion of the ns , ns , sh or m - gene have been constructed as vaccine candidates that bear genetically stable attenuating mutations. for these reasons, it is likely that a live-attenuated vaccine that exhibits an acceptable balance between attenuation and immunogenicity can be developed within the next several years. this vaccine virus might possess one or more gene deletion mutations together with or without the earlier characterized cold-passaged (cp) and temperaturesensitive (ts) mutations. the first piv vaccine candidates were also prepared by formalin-inactivation. similar to the experience with formalin-inactivated rsv, these vaccines did not protect against piv disease. in the early s, belshe attenuated a piv isolate by passages at °c (cp ) (belshe and hissom, ) . in clinical studies, the piv cp vaccine candidate has proved to be safe, genetically stable and immunogenic in seronegative in-fants (karron et al., ) . this vaccine candidate is currently being tested in phase ii clinical trials. a recombinant version of piv cp has been rescued from cdna, and the genetic basis of its attenuation (att), temperature-sensitivity (ts) and cold-adaption (ca) phenotypes has been determined (skiadopoulos et al., ) . influenza a and b virus vaccine development followed this same path of serial passage at °c (cold-adaptation) to generate mutants with att, ts and ca phenotypes (maassab, ; maassab and bryant, ) . in contrast to piv and rsv, live attenuated influenza a vaccine strains were virus reassortants that were generated by mating the attenuated donor virus with an epidemic wild type virus so that the reassortant virus vaccine was a chimera that contained the attenuating genes of the donor virus, while the ha and na genes were derived from the current epidemic virus (murphy et al., ) . this strategy, developed by john maassab, of using the cold-adapted mutant virus a/aa/ / as the donor of the six attenuating internal and non-structural genes for construction of reassortant vaccine strains was validated by the large series of consecutive reassortants that have proven to be attenuated and immunogenic. analysis of the genetic basis of attenuation showed that the influenza a pb and pb genes each consistently specified the ts phenotype, and pa specified the ca phenotype. however, all three of these genes of the viral polymerase complex contribute to the attenuation of the trivalent influenza a (h n and h n ) and b vaccine viruses (murphy, ; maassab and bryant, ) . the safety, protective efficacy and phenotypic stability were confirmed in large phase iii trials, and licensure is expected in the near future (belshe et al., ) . evidence for the prophylactic effect of serum rsv neutralizing antibodies was demonstrated in the s (prince et al., b) . passive transfer of homologous rsv convalescent serum to cotton rats protected them against rsv replication in the lungs following subsequent intranasal challenge with wild type virus. a serum rsv neutralizing titer of : in recipient cotton rats conferred almost complete protection. this amount of neutralizing antibody necessary for protection was later confirmed in clinical trials and today forms the basis for passive rsv prophylaxis in high-risk infants (groothuis et al., ) . an increased incidence and severity of rsv disease is seen in preterm infants with or without chronic lung disease (cld), in children with congenital heart disease (chd), and in immunosuppressed children and adults. respiratory disease in general is a common cause for re-hospitalization of preterm infants. cunningham and colleagues compared a cohort of preterm infants (mean gestational age at birth weeks) to a cohort of term infants and found a -fold increase in readmission for respiratory disease in preterm infants without cld, and a -fold increase for preterm infants with cld ( . , and %, respectively) (cunningham et al., ) . cld patients in a home oxygen program were at even higher risk ( % hospitalization, % icu admission) (groothuis et al., ) . children with pulmonary disorders such as cystic fibrosis, lung malformation or recurrent aspiration pneumonitis, when admitted for rsv disease, are as likely to require icu treatment ( - %) and mechanical ventilation (up to %) as children with cld ( and %, respectively) (arnold et al., ) . earlier studies of high-risk infants admitted for rsv disease yielded similar results, with icu treatment necessary in - % and mechanical ventilation necessary in - % (meert et al., ; navas et al., ) . apart from preterm infants with or without cld, children with congenital heart disease (chd) are a second group of high-risk patients, particularly when they suffer from pulmonary hypertension. in a prospective study of children hospitalized in five consecutive rsv seasons ( ) ( ) ( ) ( ) ( ) , % of patients with rsv disease and chd (compared with % of patients without chd) required icu treatment and % died (macdonald et al., ) . in a prospective study of children with chd, the incidence of rsvrelated hospitalization during one rsv season was % for children younger than years and % for infants younger than months of age (simoes et al., ) . immunocompromised children are a very diverse population and not all of them are at equal risk for severe rsv infection. children receiving corticosteroid therapy have a much lower risk for rsv-related hospitalization and death than children receiving chemotherapy for malignancies or children with primary immunodeficiencies. rsvrelated mortality was % for chemotherapy-recipients and % for children with primary immunodeficiencies compared with % for steroid-treated children (hall et al., ) . rsv grew to very high titer in children receiving chemotherapy, and more than half the patients shed rsv for weeks or longer. for immunocompromised adults, the picture is not much different from that described for children. infections are often acquired nosocomially, virus shedding is prolonged, and the incidence of pneumonia and death is high. rsv is the most important viral respiratory pathogen in these patients, followed by picornaviruses, influenza and parainfluenza viruses: % of confirmed rsv infections in leukemia and bone marrow transplant patients resulted in pneumonia, and the fatality rate was greater than % . whimbey and colleagues reported rsv case fatality rates for bone marrow transplant recipients as high as % when therapy was administered early and adequately (ribavirin and ivig), and %, when therapy was initiated late or inadequately . hiv-infected children in an urban setting in south africa also have an increased burden of viral lower respiratory tract illness (lri) although respiratory viruses are less frequently isolated from nasopharyngeal aspirates of hiv-infected children than from children without hiv infection. the relative risk for severe lri caused by rsv was twice as high in hiv-infected than in uninfected children two years of age or younger (madhi et al., ) . little information is available regarding genetic and environmental factors in susceptibility to rsv infections. most rsv epidemiologic studies are conducted in affluent countries and temperate climates although rsv is thought to be the leading cause of severe viral acute respiratory infections (ari) in infants around the globe (weber et al., ) . a -year prospective surveillance study was conducted in the yukon-kuskokwim (yk) delta of south-western alaska to determine the rate and severity of rsv infections requiring hospitalization for infants in this yupik eskimo population (karron et al., ) . the annual rate of rsv hospitalization for yk delta infants less than year of age was unusually high, i.e. - / . one in children born in the yk delta, compared with between one in to one in infants in affluent countries (sims et al., ; martin et al., ; glezen et al., ) , required ventilatory support for rsv disease. rsv infection was the single most frequent cause of hospitalization of yk delta infants. as in temperate climates, rsv epidemics in the yk delta occur annually from november through june, with peak hospitalizations for rsv disease occurring between november and february. within sub-regions of the yk delta, epidemics were as brief as month, probably because there is very limited traffic between villages in the winter months. most of the infants admitted to hospital were less than year of age and had no medical risk factors for rsv disease. surprisingly, % of the admitted infants in the yk delta and % of infants in a comparison group admitted to johns hopkins hospital (jhh) were less than one month old. of children with severe disease, % in the yk delta and % at jhh were less than months old. disease severity in non-high risk children did not differ between children admitted to jhh or yk delta regional hospital, suggesting that differences in hospitalization practices could not account for the high rates of hospitalization for rsv in yk delta infants. in the yk delta, % of those admitted for rsv disease were readmitted within a single rsv season. severity of rsv disease, age at first illness and receipt of ribavirin were all associated with readmission. in infants less than months of age, a low neutralizing antibody titer in cord blood samples was strongly associated with severe disease. a questionnaire-based case control study that was matched for age and sub-region in the yk delta detected three risk factors influencing rsv hospital admission. medical risk factors (prematurity, chronic lung disease, congenital heart disease) increased the risk of admission . -fold. more than eight people living in one household doubled the risk of hospitalization while breastfeeding had a protective effect. smoking, food-pre chewing and economic status were not significantly associated with rsv hospitalization. this study may be useful in the continued analysis of the impact of rsv in developing countries. as in the yk delta, rsv is the leading cause of viral lower respiratory tract disease in most developing countries, but lack of access to diagnostic reagents and hospital facilities has made it difficult to quantify the impact of rsv. also, the rate of severe rsv disease in term neonates was much higher than in earlier studies, both in the yk delta population and the comparison group in baltimore. these findings should be confirmed in other populations because they have important implications for rsv vaccine development. . . respiratory syncytial irus (rsv) and human rhino irus (hrv) infections in children with aids and lower respiratory tract illness (lrti) acute respiratory infections (ari) cause a great burden of disease in developing countries (de arruda et al., ) . although a growing number of children from developing nations are hiv infected, there is little knowledge about the frequency and severity of viral ari in hiv infected children. earlier studies of viral pathogens in immunocompromised adults indicated that cmv, herpes simplex, influenza, parainfluenza, rhinovirus, adenovirus, enterovirus, and rsv cause lower respiratory infection (connolly et al., ) . a recent study assessed the frequency of rsv and rhinoviruses (hrv) in hospitalized children with or without aids, who presented with lower respiratory tract infection (lrti). about episodes of lrti in children with aids and in children without hiv infection, matched by age and sample collection month, were studied in a rural area of southern brazil. the frequency of rsv infection was highest in the fall and winter, between february and july, whereas hrv was detected throughout the year. rsv was found in / ( %) and / ( %) of lrti episodes in aids and non-hiv infected children, respectively. hrv was found in / ( %) and / ( %) of the episodes in children with aids and hiv-uninfected children, respectively. no difference was detected in frequencies of hrv and rsv infections between the two groups. hrv infections, however, tended to be more frequently associated with pneumonia in children with aids ( / , %) than in the control group ( / , %) (p= . ). other clinical presentations of lrti were observed with equal frequency. these findings did not confirm hrv as a causative agent of pneumonia in children with aids, but suggest that further studies of lrti are desirable and that interventions for hrv could be considered for immunocompromised children with lrti. the human adenoviruses (ads) are a large family of over serotypes, as well as numerous variants and intermediates. they are divided into six subgroups (a-f) that exhibit different tissue tropism. the clinical manifestations of adenoviral disease are protean. the most common are respiratory syndromes in both children and adults. it is estimated that ads cause - % of all respiratory disease in children, including pharyngitis, tonsillitis and pertussis-like syndrome. in children and adults, ads are also associated with lower respiratory tract infections such as bronchitis and pneumonia. subgroup d ads are associated with both epidemic and sporadic ocular infections including conjunctivitis and keratoconjunctivitis. due to their stability in the environment, these viruses are highly transmissible, particularly in nosocomial settings. in adults, ads cause largescale epidemics of acute respiratory disease (ard) in closed populations of military recruits, dormitory residents and long-term care facility occupants, which are primarily associated with ad serotypes and a, and to a lesser extent with serotypes , , , and . while replication in the gastrointestinal tract is a feature of most ad infections, only ad and ad (subgroup f) are associated with gastroenteritis in infants and young children. ad infections in immunocompromised subjects are increasing as their numbers increase, with severe consequences. clinical manifestations of ad infection in immunocompromised patients include pneumonia, hepatitis, encephalitis, and systemic and disseminated disease, with case fatality rates from to %, depending on the nature of the immunodeficiency. nearly all ad serotypes have been associated with these infections, but the higher numbered subgroup d serotypes that are usually not associated with clinical disease in the immunocompetent host have been especially common. a number of 'new' disease associations with ad infection have recently been reported, probably due to improved highly sensitive molecular diagnostic techniques that also increase the probability of laboratory contamination. detection of viral genome in the absence of positive viral culture has been described in cases of myocarditis and pericarditis in children and adults (martin et al., ; bowles et al., ; pauschinger et al., ) , sudden infant death (shimizu et al., ) , toxic shock-like syndrome (price, ) and 'unexplained death' (perkins et al., ) . isolation of ads from patients with central nervous system manifestations of fatal acute flaccid paralysis (cardosa et al., ) and encephalitis with cerebral edema (chatterjee et al., ) has also been reported. it is, perhaps, not surprising that the clinical manifestations of ad infection are evolving because the viruses themselves are in the process of continuous evolution. the ad mutational repertoire includes homologous recombination, illegitimate recombination, and single base mutation (sbm). homologous recombination occurs in conserved regions of the genome between closely related viruses within the same subgroup, and it requires regions of homology in the two parent strands. it is the primary mechanism responsible for intermediate ads, which are mosaic viruses with shared hexon characteristics, or with the hexon characteristics of one type and fiber of another. illegitimate recombination requires only short regions of homology of one to three nucleotides (short direct repeats), and is thought to be the result of polymerase stuttering or slippage. it causes deletions, insertions and duplications of short regions of dna. in ads it occurs in noncoding regions and hypervariable regions (hvrs) of hexon capsid proteins that tolerate structural variation. the hvrs of the hexon contain the viral neutralization epitopes, so that mutations in these regions result in deletion, formation or alteration of these epitopes, leading to antigenic shift. it is the primary mechanism by which new serotypes arise, particularly among the fastest growing group, the subgroup d ads. single base mutations accumulate gradually across the viral genome but can occur at a -fold higher rate in the hvrs, where they cause incremental antigenic drift and the creation of variant strains. ad evolution is compounded by all three mechanisms and perhaps others that have yet to be defined. as a result, serological identification of subgroup b and d ads has become extremely difficult. it seems reasonable that the time has come to consider a sequence-based ad classification system, similar to that in use for papillomaviruses and enteroviruses. adenovirus has re-emerged as a leading cause of febrile respiratory disease among military recruits. large and frequent epidemics were common at trainee camps before but were eliminated with the introduction of the live enteric type and vaccines. in , the sole vaccine manufacturer discontinued production of these live enteric coated vaccines because of contractual issues. while limited vaccine stores were still available in and , vaccine stores were completely depleted by early . to monitor the effect of discontinued vaccination, weekly surveillance for febrile respiratory infections (fri), defined as oral temperature \ . °f with respiratory disease symptoms, was conducted from october -june at four military training camps. during this interval, ( %) of throat cultures yielded adenovirus. during the winter of - , adenovirus infections caused more than % of fri at each of the four camps. ad , , , and accounted for , , , and % of the isolates, respectively. three training camps experienced a high prevalence of adenovirus type and the fourth camp experienced a type outbreak. among symptomatic trainees, those who did not receive vaccine were times more likely to be infected by ad or than vaccinated subjects (gray et al., ) . surveillance was extended to eight sites in june and virus isolation was attempted for adenovirus, influenza a and b, rsv, and parainfluenza - . large ad epidemics were observed in six training camps throughout the us, while rsv and influenza a and b viruses were isolated less frequently. the impact of adenovirus epidemics on basic training can hardly be overestimated. recruit camps were forced to convert barracks into special infirmaries to care for the ill, and hospitals were forced to halt elective surgeries. at one camp, the number of trainees that had to repeat their basic training because of extended illness increased -fold. this 'recycling' has an extremely negative effect on the morale of trainees and it impacts on the military's readiness. as many as preventable adenoviral trainee medical encounters occurred during the winter months of and in ; two military trainees died with molecular evidence of acute adenoviral infection, one with encephalomyelitis and another with acute respiratory distress syndrome (ards). vaccination against ad and has proven to be extremely safe and effective, and to prevent an enormous burden of disease in military trainees (howell et al., ) . it is urgent that a new manufacturer for adenoviral vaccines be identified, and vaccine production must resume as soon as possible. in recent years, much progress has been made in understanding virus-induced modulations of the host immune response to viral infections. for ad, more than viral gene products are known to participate in the modulation of immune responses, and many of these gene products are expressed from genes clustered in the early region (e ) (wold et al., ; horwitz, ) . the overall effect of ad e gene products on immune responses in vivo can be appreciated from the results of three studies in mice that investigated transplant rejection and development of autoimmune disease. in the first study, the expression of the complete ad e cassette in pancreatic islet cells as transgenes under the control of the rat insulin promoter (rip) enabled allogeneic islet donor cells containing the h- bxd class i mhc to be accepted long-term by h- d recipient mice (efrat et al., ) , indicating that ad e gene products could potentially be used as a powerful tool in the control of transplant rejection. the second study used the lymphocytic choriomeningitis virus (lcmv) model of autoimmune diabetes mellitus, in which the lcmv proteins np or gp are expressed on the surface of islet cells, and diabetes is induced by infection with lcmv that induces cd + (gp) or cd + and cd + (np) t-cell mediated immune responses. in this model, the co-expression of rip-e with lcmv-np or gp completely prevented the onset of diabetes after lcmv infection (von herrath et al., ) . similar protective effects of ad e transgenes were seen in a third study that used the non-obese diabetic mice (nod) model of diabetes mellitus, and the underlying mechanisms are currently being investigated (efrat et al., ) . thus, as the understanding of the mechanism of action of the ad e immunoregulatory genes are being pursued in various systems, they are being utilized to control selected immune reactions that might be involved in the genesis of autoimmune diabetes. some of the better-characterized gene products of the e region are (in order of increasing distance from the e promoter) gp , . , . , . and . k. only the functions of gp and . k shall be discussed here; the other ad e gene products have been reviewed elsewhere (horwitz, ) . in vitro, gp k reduces the expression of class i major histocompatibility complex (mhc) molecules by retaining the mhc heavy chain in the endoplasmic reticulum or retrieving it back from the golgi, and also by inhibiting peptide processing (bennett et al., ) . in the cotton rat model of adenovirus pneumonia, gp k deletion mutants replicate like wild-type virus but they induce a much stronger inflammatory response (ginsberg et al., ) , whereas in c bl mice an increase in pulmonary pathology is not seen (sparer et al., ) . the ad e . k protein inhibits tnfa-induced cell death by a process that does not involve down-regulation of the tnfa-receptor. in cotton rats (ginsberg et al., ) and c bl mice (sparer et al., ) deletions of ad e . k modify the pulmonary inflammatory response, i.e. an increase of polymorphonuclear leukocytes in cotton rats and more pronounced alveolar infiltration in mice. in order to determine how the ad . k protein prevents cell death, the cell proteins that interact with this viral protein were determined. using a yeast two-hybrid system, four . k-interacting proteins (fips) were identified. three of them have been characterized and have been shown to participate in quite diverse cellular pathways (li et al., (li et al., , (li et al., , b . fip- (also known as rag-a, a ras-related small gtpase) can bind to tctel, a component of the microtubule motor protein dynein, forming . k-fip- -tctel complexes (lukashok et al., ) , and . k has been postulated to affect microtubule dependent macromolecular transport or even modulate the transport of virus. however, because . k is not a structural protein of adenoviruses and must be made de novo from early viral transcripts, it is unlikely to play a role during viral entry, even though the process is known to be microtubule dependent. the role of . k during viral exit from cells has not been studied. fip- also binds to a second gtpase (gip- ) that localizes in the centrosome and in addition to potential effects during mitosis may be involved in transporting macromolecules between the nucleus and the cytoplasm. fip- binds to abnormal huntingtin, and more specifically to the expanded polyglutamine tract that appears to be associated with cell death of neurons in huntington's disease (faber et al., ) . whether or not ad e - . k and/or fip- can prevent huntingtin-induced cell death is currently being investigated. over-expression of fip- , which is also called nf-kb essential modulator (nemo) or inhibitor of kappa kinase gamma (ikkg) causes morphologic changes and eventually apoptosis in a variety of cell lines. the amount of apoptosis induced by fip- can be reduced by % when ad e - . k is present. apart from . k, fip- seems to interact with a number of key molecules in the tnf receptor and nf-kb signaling pathways such as the receptor interacting protein (rip), the inhibitor of kappa b kinase beta (ikkb) and the nf-kb inducing kinase (nik) (li et al., b) . these few examples of the effects ad e gene products have on the pathobiology of diseases as different as autoimmune diabetes, transplant rejection and huntington's disease indicate how much remains to be learned from studying adenovirus-host interactions. the rsv (strain a ) genome is a single stranded negative-sense rna of nucleotides that is transcribed into major subgenomic mrnas. three of the eleven encoded proteins are transmembrane proteins. the g protein mediates attachment to cell surface receptors, the f protein mediates virus-cell and cellcell fusion, and the function of the sh protein is unknown. other structural proteins are the m protein, which plays a role in virion assembly, the n, p and l proteins that make up the viral polymerase, and the m orf protein that functions as a transcription anti-termination factor. the other proteins include two non-structural species, ns and ns , and the m - protein encoded by the second orf of the m mrna. using a reverse genetics system to rescue infectious rsv from cdna, five of the genes of rsv can be ablated individually and in some cases in combination without rendering the virus non-viable jin et al., b) . these five non-essential genes are ns , ns , sh, g, and m - . since all of these genes confer a selective advantage to rsv in vitro and/or in vivo, they can be described as virulence factors -the deletion of which will lead to attenuation of the virus. deletion of the small hydrophobic (sh) transmembrane protein yields a recombinant rsv called rsva dsh, which replicates in vitro as well as wild type (wt) rsv and induces plaques in hep- cells that are larger than wt rsv plaques. there is no reduction in synthesis of rna or protein associated with the deletion of sh. in chimpanzees, however, the virus is slightly attenuated (whitehead et al., a) . deletion of the ns or ns gene results in a substantial reduction in replicative efficacy in vitro, and this reduction is more pronounced in hep- cells than in vero cells (which lack interferon a and b genes), suggesting that these two genes act as antagonists to type interferon effects. deletion of ns and ns from bovine rsv provided direct evidence that ns and ns cooperatively antagonize a/b interferon-induced antiviral responses (schlender et al. ) . in chimpanzees, the level of replication of both rsva dns and rsva dns is reduced greater than -fold in the lower respiratory tract. in the upper respiratory tract, the dns virus is more attenuated than the dns virus (teng et al., ) . deletion of the m orf not only identifies a markedly attenuated rsv mutant but reveals an important role for this orf in the replicative cycle of rsv (bermingham and collins, ) . during infection with wildtype rsv, transcription appears to shut off at approximately - h post infection while rna replication increases concurrently. in contrast, this apparent switch from transcription to rna replication was not observed for the rsvdm - virus, implying that m - is a regulatory protein involved in the shift. instead, transcription continued to increase while rna replication remained low compared to wild-type rsv. overall, gene expression was increased - fold. the synthesis of the g and f proteins also was increased and resulted in increased syncytium formation. replication of the rsvdm - virus in vitro was attenuated, probably due to reduced rna replication (bermingham and collins, ; jin et al., a) . in chimpanzees, comparison of the four rsv gene deletion mutants mentioned above with wt rsv and the incompletely attenuated rsv cpts- / vaccine candidate results in a hierarchy of increasingly more attenuated viruseswt rsv b dsh b dns b / b dns b dm - . the final rsv gene deletion mutant, rsva dg, was not evaluated in chimpanzees because the absence of this major protective antigen would not be desirable in a vaccine virus. rsva dg replicates as efficiently as wt rsv in vero cells, showing that g is not essential for efficient virus replication. however, the rsvdg virus is highly attenuated in balb/c mice, indicating the importance of the rsv g protein in vivo. it is evident from the above ranking that rsv reverse genetics is able to generate mutants exhibiting gradations in their level in attenuation ). this menu of viruses with different levels of attenuation is crucial in identifying an rsv vaccine that exhibits the desired balance between attenuation and immunogenicity in seronegative infants. since clinical data indicate that rsv / is just slightly under-attenuated in the -month-old target population, the dns mutant could be exactly what is needed. in order for rsv assembly to be an efficient process, viral structural proteins must be brought together in a coordinated fashion (peeples, ; lenard, ) . compared with other paramyxoviruses rsv exhibits several unique features. the g, m - and m - proteins are found only in the pneumo irus genus of the paramyxo iridae, and the role these proteins play in rsv assembly is much less well understood than the role of the f, hn and m proteins of other paramyxo iridae (collins et al., ) . comparison of multiple human, bovine and ovine rsv strains shows that the cytoplasmic domains of the f and g proteins are well conserved amongst human rsv strains and subtypes, and conserved to some degree between the three species. in analogy to other paramyxoviruses, it is likely that the cytoplasmic domain of f and g interact in the process of virion assembly with cellular proteins involved in protein trafficking and in the polarized budding process, as well as with other viral proteins. the rsv g and m proteins co-localize in the golgi apparatus, not only in rsv-infected cells but also in cells transfected with only the g and m proteins, indicating that other viral proteins are not needed for this interaction (peroulis et al., ) . the g-m interaction is seen with fulllength g protein but not with a secreted form of g that lacks the conserved cytoplasmic domain and transmembrane domains. systematic deletion and substitution mutagenesis of the cytoplasmic domain of g has identified a sequence-specific, six amino acid motif that directly interacts with m (peroulis et al., ) . during rsv infection m protein can initially be detected in the nucleus, but later in the infectious cycle it is found in inclusions within the cytoplasm (ghildyal et al., unpublished) . the n and p proteins of rsv were earlier shown to be necessary and sufficient for these inclusions to form, and the rsv m - and l proteins were also shown to be present in these inclusions (garcia et al., ) . these same investigators were unable to identify m protein in the inclusions (garcia et al., ) . using confocal immunofluorescent microscopy of infected and cotransfected cells the m protein was shown to be present in these inclusions . m protein does not localize to the inclusions unless m - is also present . the m and m - proteins not only co-localize by confocal microscopy but also interact in a protein overlay assay (ghildyal et al., unpublished) . taken together, these data suggest that, as with other single stranded negative-sense viruses (peeples, ; lenard, ) , the rsv m protein seems to play a crucial role in rsv assembly; bringing the nucleocapsid together with the envelope proteins by binding to the cytoplasmic domains of g and f and to the nucleocapsid proteins n and p together with or via m - . the interaction between m, m - and the nucleocapsid proteins might be more complex than outlined here, and might involve additional cellular or viral proteins. to better understand rsv assembly, the domains in the m protein that interact with g, f and m - will have to be defined. while influenza viruses readily develop resistance to older antivirals such as amantadine or rimantadine, resistance to neuraminidase inhibitors occurs much less frequently. nonetheless, resistant influenza a virus mutants can be isolated from patients treated with oseltamivir (treanor et al., ) . one of the resistant mutants carries an arginine to lysine mutation at position (r k) of the neuraminidase protein, causing a reduction in substrate binding and enzymatic activity, as well as resistance to oseltamivir (mckimm-breschkin, ) . the infectivity of influenza a viruses carrying the r k mutation was earlier found to be markedly reduced in mice and ferrets. transmission of an influenza a h n clinical isolate with a r k mutation was studied in ferrets in comparison to transmission of the parent wt h n virus that was isolated from the same patient. donor ferrets (four per group) were inoculated intranasally with the r k mutant or wt virus and housed with three naïve contact ferrets per donor ferret. the four donors inoculated with wt virus were infected and transmitted virus to each of the contacts. wild type virus replicated to between and pfu/ml nasal aspirate. only two of four donor ferrets inoculated with mutant virus became infected, and the level of replication of mutant virus was reduced - -fold compared with that of wt virus. however, both infected ferrets transmitted virus to contacts. one of them transmitted the r k virus to one of three contacts only, with virus detected at very low titer on day only. the other donor transmitted virus to all three contacts, and the transmitted virus replicated to titers greater than pfu/ml in the contact ferrets. sequence analysis showed that the donor virus was a mixed population of mutant and wt virus and that only wt virus was recovered from contacts. these data confirm the reduced infectivity of oseltamivir resistant r k mutants. effective transmission of the r k mutant virus in ferrets was not observed, suggesting that the transmission of oseltamivir-resistant virus from human to human will be unlikely, even during widespread use of na inhibitors in the treatment of influenza. respiratory virus infections in immunocompromised patients are characterized by persistence of viral infection, prolonged shedding of virus, a high rate of nosocomial acquisition, and a high frequency of pneumonia and death. similarly, respiratory virus infections in the elderly are responsible for a substantial amount of morbidity and mortality. detection of respiratory virus infections in these high-risk patients is important for several reasons. it enables the initiation of specific isolation procedures, initiation of specific antiviral therapy, cessation of unnecessary therapy, tests and procedures, and it can aid in identifying and preventing potential outbreaks. respiratory virus infections can be diagnosed using serology and culture techniques, as well as newer methods such as antigen-detection by enzyme-linked immunoassay (eia) or immunofluorescence (ifa), enzymatic detection by chemical reactions, or amplification of parts of the viral genome (pcr). serology is not useful in the acute phase of most illnesses and is of limited usefulness in immunocompromised patients, the elderly or those receiving blood products. recovery of virus in cell culture is still seen as the gold standard by many but the importance of obtaining a good specimen to ensure that the culture is not falsely negative is often overlooked. a good clinical specimen is the most critical factor in ensuring a correct diagnosis regardless of the diagnostic method used, although it is perhaps most important for culture. use of nasal wash to obtain a specimen for virus isolation is well-tolerated in cooperative adults and, compared to nasal swabs or throat swabs, increases the sensitivity of cell culture for virus detection. other factors critical to laboratory success are the use of appropriate transport media, temperature of transport/incubation and time until processing (atmar and englund, ) . this is particularly important in the case of rsv infections in immunocompromised or elderly patients, who often have a relatively low viral titer such as - pfu/ml, whereas children often have a titer greater than pfu/ml of nasal wash or bronchoalveolar lavage fluid (englund et al., ) . for rsv, parainfluenza and influenza virus there are a number of commercially available rapid detection kits. multiple simultaneous rt-pcr for detection of rsv, influenza a and b, and piv , and (hexaplex ® ) was tested in pediatric samples and yielded % sensitivity and % specificity as compared with culture (fan et al., ) , with only h processing time. in a separate study, pcr for respiratory viruses in adult patients with hematologic malignancies was found to be as sensitive as culture (van kraaj and van elden, ) . for influenza, several antigen detection kits ((directigen, fluoia and quick-vue) and one neuraminidase (zstatflu) assay are available to detect virus, with sensitivities ranging from to % and specificities ranging from to %. rapid diagnosis of influenza in pediatric patients leads to a decrease in the frequency and duration of antibiotic use and an increase in the frequency of antiviral therapy (noyola and demmler, ) . rapid diagnosis kits for the detection of rsv in children range in sensitivity from to % and in specificity from to %, with some test kits performing better than others (dominguez et al., ) . in immunocompromised adults, however, the sensitivity of antigen detection kits from nasal wash or throat swab sample was only %, while endotracheal or bronchoalveolar sampling increased sensitivity to or %, respectively, (englund et al., ) . several studies in recent years have highlighted the importance of upper respiratory tract infections in the exacerbation of asthma in children. freymuth and colleagues reported human rhinovirus (hrv) ( . %) and rsv ( . %) as the most frequent pathogens detected in patients with exacerbation of asthma, and noted that pcr increased detection rates . -and . -fold in hrv and rsv infections, respectively, over that of conventional assays (freymuth et al., ) . in a study of wheezing children between months and years of age, respiratory viruses were detected in %, with rsv in infants (detected in % of subjects) and hrv in older children ( %) as the predominant pathogens. both were strongly associated with wheezing (rakes et al., ) . in a community-based longitudinal study, respiratory viruses were detected in % of episodes of acute illness with reduced peak expiratory flow, % of episodes of wheezing, and in % of episodes of upper respiratory symptoms, cough, wheezing, and a fall in peak expiratory flow (johnston et al., ) . in these settings, rt-pcr provides a fast and sensitive method to detect rna viruses. real time quantitative pcr assays can be similar to conventional pcr in specificity and speed. however, real-time pcr can also quantify viral load (quantity of virus in respiratory secretions) during asthma exacerbations, and it is sometimes more sensitive than conventional pcr. real time taq-man quantitative pcr allows estimation of the input viral genome copy number by including a fluorescence reporter on one end and a quenching molecule on the other. the reporter does not fluoresce until the quencher has been cleaved off by the exonuclease activity of taq polymerase, permitting an estimate of the quantity of pcr product. fluorescence is being measured continuously every seven seconds, and quantification of the target is based on the number of pcr cycles it takes to produce detectable fluorescence. a retrospective study was conducted in asthmatic children - years old to compare viral loads in quiescent and exacerbation periods of asthma using the taqman technology. nasal aspirates had been collected earlier and records of peak flow measurement and clinical scoring were available. a quiescent period was defined as absence of clinical symptoms for two weeks or longer. real time quantitative pcr detected respiratory viruses (mostly rhinoviruses) in % of the children with an asthma exacerbation and in % of children in quiescence. viral load was higher in children with exacerbation of asthma than in those with quiescent asthma and higher viral loads also correlated with more severe clinical disease. although the study showed that real time quantitative pcr is more sensitive than nonnested conventional pcr and also allowed esti-mation of viral load, there is the caveat that the study had a retrospective design. modalities of immunity to acute viral respiratory infections are both specific and nonspecific, humoral and cell mediated. fever, interferon (ifn), tumor necrosis factor (tnf), natural killer cells and activated macrophages are non-specific modalities stimulated by infection that are capable of mediating antiviral effects. lung collectins such as sp-a, have also been implicated in the control of viral respiratory infections (ghildyal et al., ) . specific modalities are antibody in serum and secretions, lymphocyte proliferation responses with cytokine release, and cytotoxic lymphocytes (ctls). all modalities participate, to some degree, in containing an infection and promoting recovery either via inactivation of free virus or elimination of infected cells. thus, there is considerable redundancy in the mechanisms controlling the virus infection and promoting recovery. on the other hand, protection against infection is primarily conveyed by specific antibody. sufficient data is available to conclude that serum igg neutralizing antibody is the primary mediator of resistance to influenza virus infection, presumably because infection is initiated in the lower respiratory tract and igg antibody derived from serum is the dominant antibody isotype at that site (couch and kasel, ) . in contrast, iga antibody is the primary mediator of resistance to rhinovirus and coronavirus infection because evidence indicates these infections are initiated in the nasopharynx where iga is the dominant antibody isotype (cate et al., ) . since primary infections with influenza virus, rsv, or piv induce disease in both the upper and lower respiratory tract, both igg and iga antibodies are correlates of immunity to infection and disease (crowe, ) . although adenoviruses also cause upper and lower respiratory disease, only serum antibody has been shown to correlate with immunity. reinfection with homologous rsv, piv, rhinovirus and coronavirus can occur but is generally confined to the upper respiratory tract, presumably because igg antibody in protective quantities is more durable in serum (and lower respiratory secretions) than is iga antibody in nasopharyngeal secretions. for rsv, the risk of infection declined from % for naïve subjects to or % after one or two infections, respectively; the risk of lower respiratory disease in these groups declined from to to %, respectively, (glezen et al., ) . for a rhinovirus, resistance to reinfection of the nasopharynx correlated with the titer of iga antibody in secretions (cate et al., ) . rodent models of viral respiratory disease provided much of the early data on immune modalities conferring protection (crowe, ) . both cd + and cd + t cells can effect clearance of influenza a virus, rsv and piv in mice in the absence of the other cell type (lightman et al., ) . while cd + cytotoxic lymphocytes (ctl) mediate immunity through lysis of infected cells and expression of antiviral cytokines, cd + t cells exhibit limited direct antiviral activity but play a role in activating b cells and in inducing antiviral cytokine expression (epstein et al., ) . there is a general consensus that ctls contribute significantly to the resolution of primary infections with influenza, rsv and piv in rodents. while the correlation between antibody response and protection in humans was established decades ago, cellular immune responses were studied much later, and understanding of the development of these responses in infants and immunosenescence of them in the elderly is still incomplete. whether or not ctls in humans convey immunity to respiratory virus infection in the lower respiratory tract and whether they promote clearance of virus in the upper respiratory tract of humans remains to be elucidated. however, for rsv disease in humans, a correlation has been observed between the presence of rsvspecific ctls in year and absence of severe rsv disease in year (mbawuike, in press). morever, the level of influenza-specific ctls correlated inversely with the quantity of virus in nasal secretions after challenge of humans, and the ability of ctls to function at this site was demonstrated by a reduction in influenza virus titer in nasal turbinates of mice which were adoptively immunized with purified cd + ctls before intranasal challenge (mcmichael et al., mbawuike, personal communication). knowledge regarding viral-bacterial interactions in the respiratory tract goes back at least to the influenza pandemic, and interactions have been described for later influenza pandemics as well. epidemics and pandemics of influenza have been followed regularly by an increase in the incidence of bacterial pneumonia (schwarzmann et al., ; cartwright et al., ) . associations between rsv and haemophilus influenzae, bordetella pertussis, neisseria meningitidis and staphylococcus aureus infections have also been described (patel et al., ; jiang et al., ) . interactions between viral and bacterial diseases are fairly complex and the underlying mechanisms are only beginning to be elucidated. it is known that up-regulation of tnfa and il- increases adherence and uptake of pneumococci (cundell et al., ) , and that several cellular receptors for bacterial adherence are up-regulated by viral infections. s. pneumoniae and h. influenzae interact with paf receptors (swords et al., ) , and n. meningitidis interacts with cd and cd (raza et al., ) . not all interactions are regulated at the level of cell surface receptors. in the mouse influenza model, for instance, severe damage and desquamation of the respiratory epithelium enables access of s. pneumonia to the basal membrane and thereby increases the risk for invasive disease (plotkowski et al., ) . otitis media was traditionally thought to be a purely bacterial infection, with s. pneumoniae, h. influenzae and moraxella catarrhalis as the main pathogens. more recent studies, however, indicate that the majority of acute otitis media (aom) cases are a result of mixed bacterial and viral infection. heikkinen and colleagues reported an increase of rsv, parainfluenza virus, influenza or adenovirus infection in children with otitis media (heikkinen et al., ) . all respiratory viral infections of the nasopharynx are thought to predispose to aom but some viruses, e.g. rsv, are frequently found in the middle ear during aom. the mechanism underlying this respiratory tract infection-aom sequence involves eustachian tube obstruction, leading to negative middle ear pressure and inspissation of bacteria into the middle ear (giebink et al., ) . children with rsv, adenovirus or influenza virus infections have a % risk of developing aom within weeks of the onset of the respiratory tract infection (henderson et al., ) , and coinfection with bacteria and viruses also adversely influences the outcome of aom. if aom does not respond to antibiotic therapy within h, it is more likely to involve a viral infection (arola et al., ) . the effects of viral co-infection complicate the evaluation of clinical efficacy of anti-bacterial drugs in the treatment of aom (marchant et al., ) . with bacterial-viral co-infection in aom, there is also delayed clearance of bacteria from the middle ear during anti-bacterial therapy compared with disease attributable to bacterial infection alone (chonmaitree et al., ) . it is unclear whether the decrease in efficacy of antibiotics is due to impaired host responses (poor neutrophil function) or due to poor penetration of antibiotics into the middle ear. in a recent clinical trial heptavalent pneumococcal vaccine had % efficacy for prevention of bacteremia and % efficacy for prevention of aom caused by serotypes included in the vaccine. however, the frequency of aom caused by pneumococci not included in the vaccine increased (replacement phenomenon), so that the overall effect of the vaccine was reduced (eskola et al., ) . in contrast to the pneumococcal vaccine, viral vaccines seem very effective in preventing aom. belshe and colleagues reported considerable efficacy for an attenuated live influenza vaccine in preventing aom, and rsv prophylaxis with rsv antibodies was also associated with a marked decrease in otitis media (belshe et al., ; group, a ). the human adenoviruses consists of over known serotypes which have been divided into six subgroups (a-f) with distinctly different organ tropism. although other factors may influence infectivity and replication of these viruses, high affinity attachment of virions to host cell receptors represents a key determinant of tissue tropism. examples of this distinctly different organ tropism are a predominance of subgroup a adenoviruses (ad) such as ad in pneumonia in patients with primary immunodeficiencies; the preference of subgroup b viruses such as ad , and for the urinary tract in patients with kidney transplants; and the predominance of subgroup c viruses in hepatitis in liver transplant patients. receptor binding is mediated by the ad fiber protein, a homotrimeric molecule composed of an amino terminal region that anchors the fiber to the penton base capsid protein, an elongated central shaft domain (van raaij et al., ) , and the carboxy-terminal receptor binding knob. a high resolution structure of the ad fiber knob bound to its receptor, the coxsackie-adenovirus receptor (car), has recently been obtained by x-ray diffraction (bewley et al., ) , and amino acid residues directly involved in receptor binding were defined for several adenoviruses through mutagenesis studies (roelvink et al., ) . adenoviruses differ remarkably in the isoelectric point of the fiber protein knob domain, e.g. ph . for ad versus ph . for ad , suggesting that these fiber proteins cannot use the same receptor. besides car, heparin sulfate proteoglycans (dechecchi et al., ) and sialic acid (arnberg et al., ) mediate adenovirus attachment. since car is only expressed on the basolateral but not the luminal surface of epithelium, car can probably not be used to target adenoviral vectors in the therapy of cystic fibrosis. the important role that charge plays in virus-cell interactions can be deduced from the differential effect that removal of sialic acid from the cell surface by neuraminidase treatment has on adenovirus attachment. while ad p attachment is not affected by neuraminidase treatment, ad attachment is increased and ad attachment is decreased. the difference in ph optima may well be a determinant of tissue tropism. the major pathogens in adenoviral eye infections, ad , ad and ad , belonging to subgroup d, are very similar in their knob charge. chimerization of fiber proteins, whether evolved in nature or generated by mutagenesis, can cause a significant change in adenovirus pathogenesis. in , a new ad genotype (ad h) appeared in argentina, uruguay and chile, where it caused severe respiratory tract infections in children. analysis of the ad h fiber protein revealed that it was a chimera containing ad and ad sequences. in this new genotype and ad d also emerged in japan, where ad had been absent for years, and caused outbreaks of respiratory disease. the fiber protein, however, is not the only factor that determines adenovirus tropism. virus uptake is thought to also be mediated by an interaction between the penton base protein with integrin avß or avß . adenoviruses also differ in their ability to induce inflammatory responses. while ad is a potent inducer of interleukin (il- ), a hallmark cytokine of viral pneumonia, ad is not; this might explain why ad but not ad causes significant respiratory disease. in summary, it can be concluded that neither subgroup classification alone nor fiber protein knob charge alone determine adenovirus tissue tropism. key amino acids in the knob, as well as certain motifs of the penton base protein and possibly other adenovirus proteins interact in virus attachment and internalization. avian influenza viruses of the h n subtype were found to be transmitted directly from poultry to humans in in hong kong. these viruses were highly pathogenic in chickens and also caused severe clinical symptoms in humans, leading to the death of six of infected individuals. in order to obtain a better understanding of the pathogenesis, tropism and kinetics of replication of these viruses in primates, cynomolgus monkeys (macaca fascicularis) were infected with the highly pathogenic h n a/hong kong/ / isolate that was obtained from the index case. four monkeys were inoculated with . × tcid in a ml inoculum that was administered intratracheally, orally (tonsils) and onto the conjunctiva. two of the monkeys were euthanized on day and the remaining two on day post infection. the two monkeys that were euthanized on day , post infection developed a respiratory distress syndrome with high respiratory rate and fever on day . by day , one of the monkeys was lethargic and severely ill, with central cyanosis. the other monkey was also ill and developed fever. the virus replicated to a titer greater than tcid per g lung tissue on day but could not be isolated on day . macroscopic lung pathology was dominated by peribronchial consolidation and necrotic lesions. histopathologic examination of the tissues collected and days post infection revealed extensive pathologic changes in the respiratory tract characteristic of a viral necrotizing interstitial pneumonia. although rt-pcr for h n influenza virus was positive not only in the respiratory tract but in spleen, heart and also the cerebrum and cerebellum of one monkey, virus could only be demonstrated by immunoperoxidase staining in, and isolation from, the respiratory tract. the respiratory tract seems to be the major and probably the only target for the h n influenza virus. although cynomologous monkeys have been used earlier as a model for h n influenza disease (rimmelzwaan et al., ) , this is the first time a primate model for h n viruses has been described. influenza h n clinical symptoms observed in this study correlate well with what was seen in human disease caused by these highly virulent viruses, and was more severe than what is seen with h n viruses. therefore, cynomologous monkeys may provide a useful model for studying influenza h n pathogenesis and for developing h n vaccines. . . sb- , an orally acti e, selecti e p mitogen acti ated protein (map) kinase inhibitor impro es pulmonary functions in a murine influenza pneumonia model influenza infections are responsible for significant morbidity, especially in high-risk groups with underlying cardiopulmonary disease and in the elderly. the pathology results from a vigorous inflammatory response in the respiratory tract and damage to respiratory epithelial cells. in cells exposed to inflammatory cytokines, p mitogen activated kinase (map) activation leads to the upregulation of cytokines such as il- , il- and tnfa (ono and han, ) . a recent study examined the effect of sb- , a highly selective orally bioavailable inhibitor of p map kinase, on pulmonary function in mice infected with influenza a virus. mice were infected intranasally with influenza a/pr / and treated with sb- at different time points post infection. initiation of treatment on day , or post-infection resulted in a % (p b . ), % (pb . ) or % improvement in pulmonary capacity, respectively, compared with placebotreated control animals. no effect on virus clearance, survival, or antiviral immunity was observed. the efficacy of sb- in reducing pulmonary resistance and increasing blood oxygenation was similar to that of the neuraminidase inhibitor oseltamivir, the steroid dexamethasone, and the cox- inhibitor nimesulide. sb- was superior to non-specific nsaids indomethacin, naproxen, and ibuprofen. in mice and ferrets, sb- reduced airway neutrophilia, and treatment was well-tolerated without any adverse effects. these data suggest that p map kinase is involved in influenza-induced cell signaling and that inhibition of this enzyme might reduce the severity of pulmonary disease. fortunately, the number of viruses that cause respiratory disease severe enough to require hospitalization is limited. in children younger than five years, rsv (subgroup a and b viruses), the parainfluenza viruses (piv , - and - ), influenza a and b, and adenovirus types , , and are the major respiratory pathogens. influenza a and b remain, due to antigenic drift and shift, important agents in all age groups with very severe disease most commonly occurring in the elderly. rsv is the single most important cause of severe respiratory disease in infancy and childhood but it also causes significant morbidity in the elderly and in immunocompromised individuals. kim, chanock, brandt and parrott were the first to quantify the contribution of these viruses to the severe respiratory tract disease leading to hospitalization of infants and young children. rsv, piv , piv , piv , adenoviruses and influenza b caused , , , , , and %, respectively, of respiratory disease leading to hospitalization, while influenza a was responsible for % between and (h n era) and % between and (h n era) (brandt et al., ; parrott et al., ; kim et al., ; murphy et al., ) . what are the general principles underlying vaccine development for these viruses? first, the protective antigens of the virus and mediators of immunity to reinfection are largely known. it is generally accepted that neutralizing antibodies to surface glycoproteins (g and f of rsv, hn and f of piv, and ha and na of influenza) or to capsid proteins (hexon and fiber proteins) of adenoviruses are the major mediators of resistance to reinfection. second, serum and mucosal antibodies make independent contributions to immunity against reinfection. whereas serum antibody is needed to mediate immunity in the lower respiratory tract, mucosal antibody is needed to protect the upper respiratory tract (with the exception of adenovirus, where serum antibody alone can prevent uri). third, live virus vaccines are generally more immunogenic than nonliving virus vaccines in immunologically naïve subjects, but identifying a live vaccine virus that is both safe and sufficiently immunogenic can prove to be a difficult task. fourth, different age groups might need different vaccines. infants younger than four months of age make less antibody than older children due to immunosuppression by maternal antibodies and immaturity of the immune system, and only potent immunogens, e.g. live virus vaccines, will be successful in this population (murphy et al., ) . on the other hand, a live virus vaccine that is sufficiently attenuated for use in infants might be over-attenuated in the elderly (gonzalez et al., ) . fifth, viruses differ in their ability to cause disease in the presence of maternal serum antibody. while mucosally delivered rsv, piv and influenza are infectious in the presence of maternal antibody, piv , piv and adenovirus seem to be restricted in their replication in young infants by maternal antibodies. these five principles need to be considered when determining the optimal vaccination strategy (systemic versus mucosal, vectored antigen versus live attenuated virus) and the target age for vaccination. which vaccines are available? against influenza, the vaccine currently in use in the us is mostly a non-living, subvirion vaccine made from egg-grown purified virus disrupted with detergents. for practical purposes, the only relevant protein in the vaccine is the influenza hemagglutinin. the strains to be included in the vaccine for annual vaccination are selected by the public health service based on the antigenic profile of the currently circulating strains. subivirion or subunit vaccines plus adjuvant are also being developed (minutello et al., ; gluck et al., ) . live attenuated virus vaccines based on cold-adaptation of influenza a strain a/aa/ / -h n and influenza b strain b/aa/ / have been evaluated in phase iii clinical trials and should soon become available as a mucosal vaccine administered by nasal spray (maassab and bryant, ) . to date, an rsv vaccine has not been licensed. two non-living virus vaccines against rsv are being developed. one is a purified f protein subunit vaccine and the other consists of a g protein-specific peptide conjugated to the albumenbinding site of the streptococcus g protein (cano et al., ) . the purified f subunit vaccine is being evaluated for use in seropositive populations such as the elderly or high-risk older children (gonzalez et al., ) . used by itself, the f subunit vaccine has the disadvantage of not inducing a potent mucosal antibody response. also, this antigen induces a serum antibody response that is dominated by non-neutralizing antibodies (high ratio of titer of f protein-specific binding antibody to titer of neutralizing antibody). subgroup a and b live rsv vaccine candidates are being developed . one candidate vaccine bearing multiple attenuating mutations has been evaluated in - months old infants and was found to be attenuated and immunogenic but retained mild reactogenicity for the upper respiratory tract (wright et al., ) . it is likely that recombinant cdna technology will be used to develop or improve live attenuated rsv vaccines in the near future (whitehead et al., a) . for example, a subgroup b rsv vaccine candidate was developed by substituting the g and f glycoprotein genes of rsv b for the corresponding genes in an attenuated recombinant subgroup a virus (whitehead et al., b) , thereby rapidly generating a live attenuated subgroup b rsv vaccine candidate. to date, a piv vaccine has not been licensed. subunit and vectored vaccine candidates against piv have been developed and tested in animal models, but there are no reports of ongoing clinical trials. two live-attenuated piv vaccine candidates, a bovine piv (bpiv ) and a cold-adapted piv (cp ), have been evaluated in clinical trials, and both viruses were found to be safe and immunogenic in seronegative infants (karron et al., (karron et al., , . recombinant versions of these two viruses are also being evaluated as vaccines against piv and as vectors for the expression of foreign viral glycoproteins such as rsv g or measles ha (karron et al., ; durbin et al., ; schmidt et al., ) . the recombinant piv cp vaccine candidate is currently being tested in phase ii trials in seronegative infants. for adenoviruses, a live virus vaccine against serotypes and has been used successfully in military recruits from to . the vaccine made use of site-specific attenuation, i.e. a wt virus capable of inducing respiratory disease if administered to the respiratory tract was administered orally in an enteric coated capsule. the vaccine virus replicated only in the gastrointestinal tract and did not spread to the respiratory tract. the oral vaccine was found to be com-pletely attenuated and highly efficacious in inducing serum antibodies and protecting the upper and lower respiratory tract (couch et al., ; howell et al., ) . a similar tetravalent vaccine against serotypes , , and should be evaluated for use in young pediatric patients since these four viruses are responsible for over % of the adenovirus-related respiratory tract disease in this population. in virology, reverse genetics refers to the ability to rescue infectious virus from cdna. reverse genetic systems have been developed for all major viral respiratory pathogens, i.e. influenza a virus, paramyxoviruses, adenoviruses and most recently coronaviruses. this short review will only address the impact of reverse genetics on vaccine development for selected members of paramyxoviridae. for rsv, the generation of attenuated vaccine viruses started with conventional biological methods. first, wild-type rsv was passaged extensively at low temperature, and the resulting cold-passaged (cp)rsv was demonstrated to be attenuated in chimpanzees, as well as in seropositive adults and young children (friedewald et al., ). the cprsv virus was further modified by two rounds of chemical mutagenesis and biological selection for temperature-sensitivity (crowe et al., a) . a panel of resulting mutant viruses was evaluated in mice and chimpanzees and ranked according to increasing attenuation. several promising candidate vaccines then entered clinical trials. rsv cpts / , a cold-passaged virus with two additional attenuating point mutations, is the most promising vaccine candidate tested so far, but it still causes transient nasal stuffiness in infants (wright et al., ) . the development of a reverse genetics system for rsv provided a method for expediting development of further attenuated viruses. as a first step, the biologically derived mutant viruses were sequenced completely and their mutations were identified and evaluated by introduction, individually or in combination, into wt recombinant (r)rsv. the direct identification of attenuating mutations made it possible, for example, to improve the above mentioned cpts / mutation by incorporating one or more additional attenuating mutations from other vaccine candidates. furthermore, in many cases, the amino acid substitutions can be engineered to involve two nucleotide substitutions relative to wt, which should confer increased genetic stability. similarly, a reverse genetics system for piv was established and used to analyze all mutations in the biologically derived cold passaged piv cp (skiadopoulos et al., ) . however, the capabilities of reverse genetics go far beyond the analysis of existing biological mutants. a whole new set of methods for generating attenuated paramyxoviruses has been developed as described above. gene knock-out mutations, i.e. the deletion of non-essential genes, such as the rsv sh, ns , ns , m - or g gene, generated a number of vaccine candidates with different levels of attenuation. as another example, in some cases an attenuating point mutation was found to involve a residue conserved, for example, between the l proteins of rsv and piv or between the c proteins of sendai virus and piv . transfer of the attenuating mutation between the heterologous paramyxoviruses resulted in transfer of the attenuation phenotype. host range restriction could be employed as a basis of attenuation by creating antigenic chimeric viruses that use an animal virus as a platform for the expression of human rsv or piv glycoprotein protective antigen genes schmidt et al., ) . in this manner, an hpiv vaccine candidate was generated by replacing the bpiv f and hn glycoprotein genes in rbpiv with their hpiv counterparts (schmidt et al., ) . thus, the menu of available attenuating mutations for rsv and hpiv include numerous attenuating point mutations (which in some cases specify temperature sensitivity and in other cases do not), gene deletions, and host range restriction elements. the attenuating mutations or elements from each menu can be combined as desired to develop appropriately attenuated vaccine candidates. reverse genetics also expedites vaccine development because attenuated platforms can be used to make vaccines against additional viruses. for ex-ample, attenuated derivatives of rsv a (subgroup a) can be used to generate rsv subgroup b vaccine candidates by replacing the a f and g glycoprotein genes with their counterparts from the b strain of subgroup b (whitehead et al., b) . as another example, a live-attenuated vaccine candidate was developed for hpiv by replacing the f and hn coding regions of hpiv with their hpiv counterparts. the resulting virus thus bears the antigenic determinants of hpiv in a wt hpiv backbone, which can then be attenuated by the introduction of mutations from the hpiv attenuation menu. reverse genetics can potentially help us to make vaccines that are satisfactorily attenuated but more immunogenic than wt virus. protective antigen genes can be moved closer to the genomic promoter and codon usage can be optimized for translation in mammalian hosts. cytokines and/or chemokines can be co-expressed from additional genes (bukreyev et al., ) , and reactogenicity can potentially be reduced, for example by ablating the secreted form of rsv g, which might be a decoy for antibody and might also perturb the t helper lymphocyte response. vaccine specificity can be broadened by adding additional genes to existing vaccine viruses, e.g. the measles hemagglutinin (ha) gene can be expressed from an additional orf in rhpiv , generating a vaccine candidate that protects against both hpiv and measles (durbin et al., ) . this use of recombinant piv as vaccine and as a vector has several advantages over existing vector systems. it reduces the number of viruses to be administrated to infants and enables intranasal administration and thereby mitigates neutralization and immune suppression by maternal antibodies. the eradication of measles could be facilitated by a vaccine that does not include an infectious measles virus, which might cause prolonged infection in immunocompromised hosts. in summary, vaccines can be optimized through reverse genetics in a number of ways, such as the creation of novel combinations of mutations, the fine-tuning of the attenuation phenotype, the provision of a common attenuated platforms, and the generation of multivalent vaccines. . . predicti e alue of animal models in rsv accine de elopment rsv was initially isolated from chimpanzees that developed a common cold-like disease in . since then chimpanzees and other animal models for rsv disease have been studied extensively. principally, these higher primates have been used for three different purposes: the development of live virus vaccines, the recovery of rsv antibodies, and lastly to evaluate formalininactivated and subunit vaccines, and to study enhanced disease following vaccination with formalin-inactivated vaccine. a number of animal models are used to study rsv disease. among the apes, chimpanzees have been used most extensively for several reasons. they are permissive for rsv, their core body temperature is similar to that of humans, and symptomatic scoring of uri is possible (rhinorrhea score). the permissiveness of chimpanzees makes it possible to perform quantitative virologic studies in the upper and lower respiratory tract over the time course of an acute rsv infection. also, the restriction of rsv replication in the respiratory tract of chimpanzees correlates well with attenuation in seronegative human infants. in addition, human immunologic reagents can be used in chimpanzee studies. however, only young, carefully raised chimpanzees are rsv seronegative. other primates used include old world monkeys such as african greens, rhesus, cynomologous or bonnet monkeys and new world monkeys such as marmosets, tamarins and owl monkeys. although all these monkeys are semi-permissive for rsv, their core body temperature is higher than that of humans so that the level of attenuation of temperature-sensitive rsv mutants might be overestimated. although primate studies provide essential data for vaccine development, their use can be difficult. transport and care of primates, as well as sample collection are difficult and potentially dangerous, and they require well-trained personnel. primate research centers must provide an environment similar to that of a child-care facility and, therefore, maintenance costs are very high. lambs and calves are also used to study rsv infection, mostly because ovine and bovine rsv causes significant disease in their natural host. also, these viruses are important economically. cotton rats, mice and other rodents are used as small animal models. mice have the advantage of having a body temperature similar to that of humans and immunological reagents are readily available. also, gene knockout mice, transgenic mice and various inbred strains are available to study pathogenesis. the mouse model is not without difficulties, however. mice acquire a large portion of their maternal antibodies by suckling, and, therefore, they are not an optimal model for study of immunosuppression by maternal antibodies. peak rsv titers vary -fold among different mouse strains, and subgroup b rsv is poorly infectious and immunogenic in mice. immune mechanisms may also vary among different mice strains. rodent models can be used to study quantitative virology, immunology and airway pathophysiology (a model of wheezing), and weight loss can be used as a surrogate marker for rsv disease. whichever animal model one chooses to study respiratory syncytial virus, it is essential to clearly define whether the ultimate goal of the study is to prepare for clinical vaccine trials or to understand basic mechanisms of disease. several cold-passaged (cp) live attenuated rsv candidate vaccines have been evaluated in clinical trials. most of these candidate vaccines were derived by further attenuating the original cprsv through chemical mutagenesis and selection of ts mutants. the first two vaccines evaluated in the cpts lineage -cpts- / and cpts- / , were either insufficiently attenuated or were transmitted amongst seronegative children (karron et al., ) . a study of a more attenuated vaccine candidate, cpts- / , has recently been completed (wright et al., ) . this virus initially was evaluated in chimpanzees where it replicated to a peak titer of only . pfu/ml in the upper respiratory tract. due to this restriction in the respiratory tract, it was subsequently evaluated in clinical studies. cpts- / did not replicate in adults or older children. in seronegative children - months of age, however, cpts- / replicated to a peak titer of pfu/ml in the upper respiratory tract and induced an antibody response against rsv f and g glycoproteins. the virus was well tolerated in this age group, and the frequency of upper or lower respiratory tract disease, otitis media or fever was not different from that of the placebo group. based on these data cpts- / was selected to be the first rsv vaccine to be administered to - -month-old infants, which represent the primary target group for vaccination. in this youngest age group, of vaccine recipients developed a clinical syndrome characterized by nasal congestion that occurred most typically between days and and lasted for approximately h. since young infants are obligate nose breathers, this interfered with feeding and caused fussiness and difficulty in falling asleep. most vaccine recipients shed approximately pfu of rsv per ml nasal wash. age of the infant or level of maternal antibodies against rsv did nor affect virus shedding, indicating that virus replication in the nasopharynx was independent of these variables. in these young infants, neutralizing antibody responses and igg elisa responses to rsv f or g were rarely detected, most likely because these responses were masked by the presence of maternally derived antibody. these young infants did, however, develop serum and mucosal iga responses preferentially to the rsv g glycoprotein and detection of serum iga correlated with protection from re-infection with a second dose of vaccine. although cpts- / is not an acceptable vaccine in one to two month-old infants, several lessons were learned from the study of this vaccine candidate. first, seronegative infants are a much more susceptible host to rsv than chimpanzees. this means that for further attenuated vaccine candidates, the chimpanzee model of rsv infection will not be very useful. second, only viruses that do not replicate in adults and older children are attenuated enough for vaccination of infants. and third, reverse genetics is needed to further attenuate the existing biological rsv vaccine candidates. two of these recombinant rsv vaccine candidates are currently being evaluated in clinical trials. cpts- / dsh was generated by deleting the sh gene from the recombinant cpts- / virus, and cpts- / / dsh was engineered to contain an additional ( ) mutation in the polymerase protein. preliminary data suggest that both recombinant viruses are well tolerated in - -month-old children and are not associated with lower respiratory tract illness. whereas cpts- / dsh replicates as well as its parent biological virus without the sh gene deletion, the cpts- / / dsh mutant appears to be much more restricted in its replication in the nasopharynx. the more restricted cpts- / dsh induced both neutralizing and elisa igg antibodies in this age group. however, since this virus replicates, as well as cpts- / , it is likely not to be suitable for vaccination of one to two month old infants. cpts- / / ?sh is currently being evaluated in one to two month old infants. in summary, although cpts- / is close to an ideal vaccine candidate, further modifications of the virus through reverse genetics, such as addition of the mutation, are likely to be needed to generate an rsv vaccine that is immunogenic yet attenuated enough to be given to young infants. antibody preparations that neutralize free virus have been used as passive immunoprophylaxis to prevent a number of viral diseases, including hepatitis a and b, varicella and respiratory syncytial virus (rsv) disease. the efficacy of antibody preparations in preventing rsv disease was established first in cotton rat and chimpanzee models, and later in extended clinical trials. therapy of rsv infections with rsv antibodies, however, have so far failed to prove efficacious (malley et al., ; van woensel and kimpen, ) . the most important mechanism of action of antibody preparations is probably neutralization of free virus. this can occur via aggregation of free virus by bivalent or multivalent antibody, via receptor blockade, via antibody-complement lysis, or via fusion inhibition. receptor blockade is thought to result from steric inhibition of receptor binding rather than binding of antibody directly to the receptor-binding site itself. even if receptor binding does occur, virus infectivity can be neutralized through fusion inhibition. most rsv neutralizing antibodies are thought to use this mechanism of action. although complete antibody is most effective in neutralizing free virus, antibody binding fragments (fabs) alone can also neutralize. the minimal unit necessary to ablate infectivity is probably a peptide corresponding to one loop of the complementarity-determining region (cdr ). although neutralizing and non-neutralizing antibodies against infectious virus are often distinguished, it is not clear whether there really are antibodies that bind glycoproteins in virions without neutralizing the virion (sakurai et al., ) . many so-called non-neutralizing antibodies bind purified (conformationally relaxed) rsv f glycoprotein but not the (conformationally correct) f protein on infected cells. different immunoglobulin subclasses neutralize virus with varying efficacy, and mouse and human igg subclasses also bind complement with varying efficacy. the replication of sendai virus (murine piv ) and influenza a virus can be inhibited intracellularly by iga and similar effects of iga on rsv replication may occur. a last but essential determinant of neutralizing activity is defined by the amount of antibody available to neutralize virus. to prevent rsv disease in the lower respiratory tract, a serum neutralizing antibody titer of approximately : is required (groothuis et al., ; top et al., ) . upper respiratory tract infections, however, can only be prevented with serum antibody titers as high as : - : . such titers can only be achieved in experimental settings in small rodent models. there is no single monoclonal antibody directed against rsv g protein that neutralizes completely on its own but cooperative neutralization occurs when multiple mabs are used. effective treatment for rsv illness is very limited. corticosteroids, bronchodilators, ribavirin, and, more recently, rsv mabs have been used but none of these therapeutic interventions are accepted as reproducibly efficacious. it seems that neither antivirals nor anti-inflammatory agents alone improve the outcome of rsv disease. a single dose of topically administered human fabs has been shown to clear free virus from the respiratory tract of rodents, but their effect is shortlived since infected cells release newly synthesized infectious virus within a day or two (crowe et al. b ). whereas peak virus titers in humans usually occur around day , most patients with clinical rsv disease likely present no earlier than day . at this point, virus load is already in decline and it may be too late for antivirals, and antiinflammatory drugs such as map kinase inhibitors may be more effective in reducing cell injury. are there immunological consequences of passive immunoprophylaxis? in chimpanzees therapy with rsv antibodies suppresses the primary antibody response to rsv. this effect is mostly antigen-specific but may also have a non-specific aspect mediated by the fc portion of the antibody. the secondary antibody response to rsv in chimpanzees is enhanced by prior antibody therapy. however, this effect was not observed in mice or in clinical studies. t cell responses do not seem to be as affected by antibody therapy as humoral responses, and t cells might fill in for absent primary antibody responses. whether this is a desirable effect or not, remains uncertain. without a safe and effective rsv vaccine available, monthly infusion of rsv-igiv (respigam ® ) was the first effective measure to prevent rsv-induced lower respiratory tract infection (lri) and hospitalization in infants. the evaluation of rsv-ivig in cotton rats correctly predicted the serum concentrations necessary to protect against rsv-induced lri (prince et al., a) . protective concentrations in children were achieved by monthly infusions of mg/kg of rsv-ivig. in order to increase the potency of a rsv antibody preparation, and in order to be able to replace intravenous with intramuscular administration, a humanized monoclonal anti-rsv fusion protein antibody was developed. mab , a mouse monoclonal antibody developed in the laboratory of infectious diseases at nih against the rsv f protein antigenic site a, one of two antigenic sites that are conserved amongst different rsv strains, was selected for humanization based on in vitro and in vivo studies, and the complementarity determining regions (cdr) were transferred from the mouse antibody to a human igg. this humanized igg should have pharmacokinetics similar to human igg and permit repeated administration at monthly intervals. the chosen mab, medi- , had no crossreactivity with adult or neonatal tissue, broadly neutralized rsv subgroup a and subgroup b isolates at concentrations of ng/ml, and proved to be - times more active on a weight basis than rsv-ivig in the cotton rat model (johnson et al., ) . adult volunteers tolerated doses of the humanized antibody medi- from to mg/kg well, and only some volunteers developed a low-titer, transient anti-idiotypic antibody response. medi- had a half-life of days, as is expected for igg, and serum concentration after iv and im administration were comparable except for the initial bioavailability. medi- was safe and well tolerated in phase i/ii studies in high-risk children, and had no specific immunogenicity in and of itself. monthly dosage of mg/kg maintained serum concentrations greater than mg/ml, and a single intravenous dose of mg/kg reduced rsv titers in tracheal secretions of intubated children with rsv infection (malley et al., ) . the impact-rsv study that led to fda approval of medi- , also called palivizumab, was a : randomized, double-blinded, placebo-controlled phase iii multicenter study conducted at sites in the us, canada and the uk. study subjects received five doses of medi- or placebo at intervals of days and were followed for a total of days. the primary endpoint of this study, overall rsv-related hospitalization, was significantly reduced (pb . ) from . % for placebo recipients (n= ) to . % for medi- recipients (n= ). for premature infants with chronic lung disease, the incidence of rsv hospitalization was reduced from . to . % (p= . ), and for premature infants without chronic lung disease, it was reduced from . to . % (pb . ). total rsv hospitalization days, total days with increased oxygen requirement, total days with severe lri, rate of icu admission and total days in icu were secondary endpoints that occurred less frequently in the medi- treated group (p b . ). thus, administration of mg/kg medi- by im injection was found to be safe, well-tolerated, and to lead to a % reduction of rsv hospitalization in high-risk children (group, b) . this conclusion was confirmed in an outcome survey conducted in nine centers in and , in which high-risk children received mg/kg palivizumab. although direct comparison to the impact-rsv study is not possible, rsv hospitalization rates were low and similar to those observed in the impact-rsv study: . % (vs. . % in the impact-rsv study) of premature infants without cld and . % (vs. . %) of children with cld were hospitalized in the - rsv season (sorrentino and powers, ). an alternative strategy to protect neonates and young infants against severe rsv disease is vaccination of pregnant women. transfer of high concentrations of maternal rsv-specific igg to the fetus is expected to protect the infant against severe disease (glezen et al., ) . the safety and immunogenicity of respiratory syncytial virus purified fusion protein- (pfp- wyeth lederle vaccine and pediatrics, pearl river, ny) were recently evaluated for use in pregnancy. thirtyfive pregnant women were randomized at a ratio : to receive rsv pfp- or saline placebo at - weeks of gestation and were followed until the time of delivery. infants were followed during their first year of life and their first rsv season. rsv pfp- was safe and well tolerated by pregnant women, and there were no systemic reactions or serious adverse events associated with vaccine administration. all infants were born healthy, and there were no differences in the frequencies and outcomes of neonatal events between the groups. during the first rsv season, there was no increase in the frequency or severity of respiratory tract illnesses in infants of vaccine recipients. there is ample material from which to draw lessons relevant to needed preparations for pandemic influenza. but, to date, the response, and specifically preparations for dealing with a serious pandemic of influenza remain more in the realm of academic reflection than meaningful action. although much time has been devoted to the development of a national response plan, we do not seem to be much better equipped to deal with a new pandemic of influenza than we were in the spring of , when the h n strain of influenza a virus emerged. at that time, we faced a burgeoning epidemic, whose virulence and propensity for spread were as yet unknown. a program of surveillance and field epidemiology to better define the epidemic had to be developed and, at the same time, preparations were needed for distribution and use of a new influenza vaccine, if it arrived in time. it did not. it appears today that not much progress has been made over the past years, and still no real sense of urgency in dealing with the essentials of the problem can be felt. although much has been written about the epidemic, it is still perceived by most as an interesting but questionably relevant tale of death and disease during an earlier, pre-antibiotic era of medicine. many doubt that an epidemic of this severity would be possible today. but, here are a few of the facts. first, it is important to recognize that, for the us, it dwarfed all other outbreaks of the th century. at that time, more than million died worldwide and in the united states, there were more than registered deaths. from various studies, it is thought that, overall, perhaps % of the population became ill, of whom about % died. medical services were overwhelmed but, other than supportive care, there was little that curative medicine could offer. the deaths were so numerous that burials were greatly delayed because of the lack of morticians and grave diggers. pictures from the time provide at least a pale illumination of that catastrophic period. the effect of the disease was anything but uniform. surveys revealed morbidity rates ranging from % to more than % in different parts of the country. some remote and rural areas escaped the disease entirely but in some areas, it was remarkably lethal. western samoa, then a new zealand protectorate, registered deaths in a population of . this represented % of the entire population. curiously, american samoa, only miles away but with a quarantine in effect, was one of the few political entities to escape the epidemic entirely. what was surprising and unique about the epidemic was that more than half of all deaths were in persons between and years of age -an age bracket in which death is a relatively uncommon phenomenon. pregnant women and those with cardiac problems were at highest risk but most, who died, were otherwise in good health. a substantial number in this age group died of a fulminant, rapidly progressive pneumonia marked by severe cyanosis with death occurring within a matter of - days, in brief, what seemed to be almost certainly, a primary influenza pneumonia that would have benefited little from antibiotics, had they been available. today, a better outcome might be foreseen with better ventilatory support in intensive care units; with the administration of antiviral agents; and with the administration of antibiotics for the treatment of secondary pulmonary infections. but under epidemic circumstances, the bulk of cases in a new pandemic would occur over a period of only - weeks and only a fraction of patients could be accommodated in suitable hospital settings, let alone appropriate intensive care units. what quantity of antiviral drugs is available for emergency use? where do we obtain the added quantities of the standard antibiotics that today, are produced on a just-in-time basis -when, during a period when clinical facilities are not stressed, we are regularly experiencing antibiotic shortages on a regular, rotating basis. a cogent question is whether under epidemic circumstances, our modern health care system could provide a standard of clinical care that would be better than it was in . but there is still another dimension to the problem. do we appreciate that a 'pandemic', so characteristic of the emergence of a new influenza strain, means exactly that -a worldwide epidemic. many countries -developed and developing, have made no preparations and will inevitably turn to those with resources to help them in dealing with a catastrophe -a catastrophe that poses an international security threat. the rapid production and administration of large volumes of vaccine effective against the emergent new strain has been a basic building block of the strategy for dealing with pandemic influenza and, understandably so, given the limitations of curative medicine and the capacity of clinical services. the first real test of this strategy came during the epidemic. the first notice of a major outbreak occurred in mid april; specimens were received in the us on may; and field testing of new lots of the vaccine began in july. not a bad record. it was foreseen that million doses would be required. with good fortune in adapting the virus to grow at reasonably high titer on egg membrane and provided that sufficient fertilized eggs could be obtained, it was expected that a production target of february could be met. even this was a problematical date given that the seasonal peak of epidemic influenza usually occurs between the end of december and the end of february. still, it was believed that substantial numbers would be able to be protected. however, was not a typical year, and such, one must note, was the case when the epidemic strain first appeared. widespread epidemics began occurring in mid to late september, two months before they were anticipated. more than half of all counties reported epidemics by mid-october and by the end of october, the peak incidence had past, long before any substantial quantity of vaccine was available. given these experiences, could we expect another new strain to behave differently today? over the past years, extensive studies have been conducted in the search for a satisfactory live attenuated vaccine and for various approaches in production which would permit new antigenic variants to be produced rapidly and in quantity in tissue cell culture. however, today we are still producing influenza vaccine in the allantoic cavity of hens' eggs, as we were in ; procuring adequate supplies of fertile eggs in a timely manner remains a serious problem; and difficulties in adapting new strains to eggs remain. were we able to solve the vaccine production problem for our own country, this would not be the end to the practical quandary of dealing with the pandemic. few countries have influenza vaccine production capability and the international implications of the us having all or much of the vaccine supply are profound. following the swine influenza outbreak at fort dix, new jersey, the us had embarked on a program to rapidly produce an appropriate vaccine. vaccine production capability in europe and other countries was so marginal that a world health organization committee could only recommend that the fort dix situation be carefully monitored -a strategy of wait and hope. but as those at the who meeting commented in corridor conversations, they would not wish to face the ethical and political dilemma the united states would face were there a pandemic and the us was the only nation with a vaccine. in recent meetings with national and local hospital authorities, current capabilities of the medical system in us to deal with sudden surges in demand such as might follow release of a biological weapon were explored. such a release would result in an epidemic that would stress the system not unlike the way it would be stressed by a pandemic of severe influenza. from these meetings, it was evident that the elasticity of the nation's bed supply has been significantly reduced as drives for financial efficiency and the increasing use of out-patient procedures have sharply reduced the numbers of beds in all hospitals. meanwhile, managed care-driven market pressures and federal government reimbursement reductions have driven large numbers of hospitals into operating deficits. many of the municipal hospitals, once a primary source of care for the less prosperous and uninsured have been privatized. meanwhile, the hospitals are experiencing severe labor shortages, especially for nursing and technical personnel. few have either the resources or motivation to prepare to respond to the challenges posed by mass casualties due to any cause. reserves of antibiotics, as noted earlier, are marginal to nil; the public health infrastructure needed to deal with epidemic disease is grossly understaffed, underpaid and under trained; mechanisms for the development and implementation of community-wide plans are largely unexplored. it is not unreasonable to suggest that we are today less well-prepared to deal with an epidemic of influenza than we were years ago in most parameters that one can identify. what might a new strain of influenza mean in terms of numbers of cases and deaths. estimates of past pandemics suggest that perhaps % of the population were affected in a first wave. with rising proportions of the population in urban areas, the greater and more rapid mixing of populations through travel, a figure of not less than % would seem more reasonable. thus, in a city of . million, one might expect . million cases of influenza. it is doubtful that either antibiotics or antiviral agents would be of much help given the number of cases and the dearth of reserve supplies. the number of deaths would vary greatly depending on the strain. in hong kong, a recent new strain, h n , resulted in death among six of persons infected. it did not spread readily, however. it is estimated that the strain killed % of those who became ill, while the case-fatality rate was about one-tenth as large or . %. thus, in a city of million persons, one might anticipate between and deaths over a period of - weeks. medical care would consist largely of supportive therapy given the numbers of those ill. public health measures, likewise, would consist of little more than reassurance given the likelihood that vaccine supplies would not be available and that stocks of antiviral drugs would be too small to be of significance. many would argue for the closing of schools, churches and other places of public gathering but experiences suggests that such actions produce little benefit. the wearing of masks was once a favored intervention but this, too, has been discredited. in brief, without vaccine, there would be little that could be done of practical benefit except to reassure the community that the epidemic would someday pass and to accept the criticism of the public and political leadership for failure to make reasonable preparations. at least four lessons can be derived from past experience. first, the threat of pandemic influenza caused by an especially virulent strain is a continuing threat that has to be taken seriously. second, adequate supplies of an effective vaccine, available in a timely manner, are absolutely critical to a preventive effort. solving this problem should command top priority for research and development funding. third, special plans, programs and funding are needed within the health care system to permit development of an adequate community-wide response to the occurrence of mass casualties whatever the cause. lastly, additional research in influenza is needed to better understand its pathogenesis and epidemiology with the expectation that better preventative measures might eventuate. pandemics are the most dramatic presentation of influenza a virus and they cause considerable excess mortality as a result of pneumonia and exacerbation of cardiopulmonary or other chronic diseases. the epidemiologic success of influenza a is in large part due to antigenic variation that takes place in the two surface glycoproteins of the virus, i.e. its hemagglutinin (ha) and neuraminidase (na) proteins. to date, ha and na subtypes have been defined and are used to classify influenza a viruses. antigenic variation occurs either gradually through accumulation of point mutations (antigenic drift) or more abruptly though introduction of a new ha gene into virus circulating in the human population, be it through reassortment of animal and human viruses or through a change in host-specificity of an animal virus (antigenic shift). a 'pandemic virus' can be defined as a virus with a new ha with or without a novel na gene, acquired through antigenic shift, that spreads readily from person-to-person in a population that is highly susceptible to infection. the th century saw three pandemics caused by new ha subtypes. influenza a h n caused the 'spanish flu' in , subtype h n ('asian flu') was the causative agent of the pandemic and the h n subtype ('hong kong flu') caused a pandemic in . the excess mortality for the , and pandemics in the us alone can be estimated to have been , , and deaths, respectively. if one applies mathematical modeling to estimate the effects of a future pandemic, the us alone can expect between and excess deaths in the next pandemic (meltzer et al., ) . what are the viruses with pandemic potential? in , individuals in hong kong were infected with h n influenza, an avian subtype earlier not known to infect humans (subbarao et al., ) . of the patients - years of age, six died. molecular analysis established that all eight genes of the h n virus were of avian origin and that reassortment with human viruses had not occurred (subbarao et al., ) . the reported infections were most likely acquired from poultry; human-to-human spread, however, was a rare event. sequence analysis of the hong kong h n virus suggests that it is a reassortant made up from two or three different avian parent viruses: ha from a goose h n virus (xu et al., ) , na from a teal h n virus (hoffmann et al., ) , and internal genes from a quail h n (guan et al., ) or a teal h n virus (hoffmann et al., ) . although this virus did not spread readily from person to person, deep concern was caused by the fact that this was the first known avian influenza a virus that caused disease in humans. until this outbreak it was thought that the receptor specificity of avian ha proteins limited their infectivity to avian species. this notion, however, needs to be revised. in march , h n influenza a viruses were isolated from two children with febrile upper respiratory tract illness (peiris et al., ) . this virus, again, was an avian virus that was able to infect humans without passage through an intermediate host (lin et al., ) . what are our options for vaccination against potentially pandemic viruses? in the case of the h n viruses, there are four options for generating an effective influenza vaccine. a conventional inactivated vaccine can theoretically be generated by reassortment of internal genes from influenza a/pr/ / with h n glycoprotein genes. there are, however, problems with incompatibility between certain gene segments. secondly, a cold adapted live attenuated vaccine approach could be taken. the influenza a/ann arbor/ / coldadapted strain has been used to generate an h n vaccine in which the ha cleavage site was modified (li et al., a) . this virus could also be used to produce an inactivated vaccine. thirdly, a surrogate virus that is nonpathogenic but antigenically related (takada et al., ) could be sought. one of the closest antigenically related viruses found to date was of the h n subtype, but this virus was not highly immunogenic in humans. fourthly, purified protein could be used but again limited immunogenicity may be a problem. the development of vaccines against potential pandemic viruses poses a number of challenges. to begin with, most of the work has to be conducted in biosafety level + laboratories. there is a large array of avian viruses that could potentially become pandemic viruses and those with a genotype that confers transmissibility have to be identified. h n , h n and h n viruses that were the genetic precursors of the h n and h n viruses that caused human infections in hong kong (guan et al., ; xu et al., ; hoffmann et al., ) and that continue to circulate among birds should certainly be given priority in vaccine development. an adequate strategy for vaccine development has to be selected, and safety testing must be expedited in mice, ferrets, chickens, and eventually humans. use of recently described plasmid based reverse genetics systems may lessen the technical challenges faced in generating vaccine candidates. assays to evaluate immunity have to be optimized (hemagglutination inhibition assays do not perform well with avian ha proteins, yet neutralization assays are time consuming and technically more difficult), and serologic and molecular diagnostic reagents must be made available. collaboration between veterinary and public health authorities must be maintained and new technologies such as plasmid based reverse genetics systems must be used. last but not least, alternative substrates for vaccine production and new adjuvants are urgently needed. although influenza epidemics occur every winter, we have only had three pandemics this past century - , and . while annual influenza epidemics generally cause an excess mortality of - people in the us, it is only the pandemics that remain in memory. of the three pandemics that occurred in the twentieth century, the pandemic was the most devastating and claimed the most victims. although there is wide spread public interest in the pandemic, the event is mostly remembered as an interesting event in the distant past. whether due to denial or not, few feel that an influenza pandemic is a current threat. most journalists see their role not only as educators and advocators but also as entertainers. the interest of the readership has to be captured, and it can only be maintained if their curiosity is satisfied. while the aids pandemic and its effect on people's lives found a wide audience through most of the early s, the late s were characterized by fatigue and a reduced interest in the pandemic (although it was still on the increase). as a result, editors were much less willing to include related stories. reportage on influenza pandemics suffers a similar fate: the disease itself is old, and the last pandemic occurred too long ago for most to remember. the mere warning of a coming pandemic is almost perceived as an unfulfilled promise if the event does not occur shortly after the article is published. breast cancer, in contrast, is one of a few examples, where interest in a disease process can be maintained over an extended period of time. the risk of suffering from breast cancer is felt much more acutely, it seems, and there is a sense that proactive behavior will lead to an improved outcome. whether the mass media will take on a role in changing the public's attitude toward influenza as a threat is very much in doubt as long as journalists themselves perceive the next pandemic as an event as anonymous and inevitable as an earthquake. rsv infection is initiated by g glycoprotein attachment to cell surface receptors and followed by virus-cell fusion that is mediated by the f protein. the cleavage-activated rsv f protein is thought to interact with the target cell membrane through its n-terminal fusion peptide, which is released from a shielded position within the f homotrimer through a major conformational change. insertion of the f n-terminus into the cell membrane destabilizes the cell membrane and induces lipid mixing that is followed by mixing of contents, thereby enabling the viral nucleocapsid to enter the cytoplasm. the rsv f protein was recently found to interact with a small gtpase called rhoa through a domain that lies just carboxyterminal to the f fusion peptide (pastey et al., ) . rhoa is a member of the ras superfamily that is expressed intracellularly in all cell types. it induces bundling of actin filaments into stress fibers, focal adhesion plaque formation, cell-to-cell adhesion and organization of integral membrane proteins; it has additional roles in cell morphology and motility as well as cell cycle transition from g to s. a series of overlapping rhoa peptides from the interaction domain were evaluated in rsv plaque reduction neutralization assays. rhoa peptide - has an ic of about mg/ml against an inoculum of pfu. neutralizing activity is also seen against parainfluenza virus type (piv ), but not against a variety of other paramyxo, orthmyxo, corona and filoviruses (pastey et al., ) . intranasal administration of mg of peptide - prior to intranasal infection of mice with rsv reduces rsv replication more than -fold and also diminishes weight-loss. studies with recombinant rsv expressing green fluorescent protein indicated that rhoa peptide - inhibited rsv replication at a very early stage of infection. subsequently, it was shown that fusion of cells transfected with rsv f, g and sh was inhibited by this peptide, suggesting that it exerted an effect on rsv replication at the level of membrane fusion or entry. since rhoa is an intracellular molecule, one would have to hypothesize that the interaction of the n-terminal heptad repeat and fusion peptide with the target membrane causes enough membrane disruption to allow an interaction between rhoa and rsv f. alternatively, rsv f may not interact with rhoa during the entry process, and the rhoa-derived peptides may simply disrupt the f structure or function to render the virus noninfectious. the question of whether rhoa and rsv f interact during a natural infection is not yet resolved. rhoa can be found in the cytoplasm bound to gdp and a guanine nucleotide dissociation inhibitor, and, upon gtp exchange and isoprenylation, rhoa associates with the cell membrane. if the proposed model of rhoa-rsv f interaction at the membrane is correct, then inhibition of isoprenylation should inhibit rsv infection. indeed, inhibition of isoprenylation through hmg coa reductase inhibitors, such as lovastatin, inhibits rsv infection of hep- cells at an ic of mm and reduces peak rsv titers in lungs of mice greater than -fold with oral gavage of mg per day (gower, t.l., graham, b.s., submitted). the antiviral effect of lovastatin is also seen with piv in vitro. although it has not been formally proven that rsv f interacts with rhoa in the infected cell membrane, rhoa signaling activity is triggered in rsv-infected cells (gower, t.l. et al., submitted) . while rhoa signaling is not required for rsv replication, syncytium formation is diminished when rhoa signaling activity is inhibited. in addition, rhoa signaling may play a role in other aspects of rsv pathogenesis. rhoa kinase inhibitors reduce the transcription of il- and il- mrna in rsv infected cells (cytokines abundant in the nasal secretions of rsv-infected patients), and obviate airway hyperresponsiveness, one of the cardinal symptoms of rsv disease (hashimoto, k. et al., submitted). the balb/c mouse model was used to explore whether mycophenolic acid (mpa), an inhibitor of de novo purine synthesis in t and b lymphocytes, could improve the outcome of rsv disease, which was monitored by clinical symptoms such as ruffling of fur, increased respiratory rate and weight loss. oral administration of mg/kg mmf (the prodrug of mpa) daily from day to post-infection reduced weight loss on day post-infection from % in untreated animals to % in mpa treated mice (p b . ). a reduction of weight loss ( . %) was also observed when initiation of treatment was delayed until day post-infection. virus titers in lungs of mmf treated mice were similar to those of untreated controls but histological changes were reduced. on day post-infection, ifng levels were elevated . -fold in the treatment group while il , il and il levels were unchanged, indicating a shift toward a t helper cell type response that is thought to correlate with improved rsv disease outcome. these data suggest that inflammatory responses contribute to rsv disease in mice and that an immunomodulatory approach to the treatment of human rs virus disease is worth further consideration. using a cell-based assay to identify compounds which are able to inhibit fusion of hela cells infected with rsv, more than analogues of a lead compound were synthesized and one compound (termed r ) was selected for further evaluation. the in vitro % inhibitory concentration (ic ) of this benzimidazole derivative (mw ) was pm, and thus its potency exceeds that of ribavirin almost -fold (ic = mm). r exhibits in vitro antiviral activity against human rsv (subgroup a and b) and bovine rsv but not against pneumovirus of mice or other paramyxoviruses. rsv-induced cytopathic effect was reduced by r at . nm, and concentration of nm reduced rsv titers -fold in multi-cycle growth curves. time of addition studies indicated that both virus-cell fusion and cell-cell fusion were inhibited by this compound. selection of resistant viruses in vitro yielded two mutants with single point mutations in the f-protein: one upstream of the second heptad repeat motif and another within it (s l and d n). rsv titers, determined by quantitative rt-pcr, were reduced -fold in bronchoalveolar lavage fluid and in lung tissue of cotton rats treated once by inhalation with r prior to rsv infection. rfi- , a compound that was derived by chemical optimization of the earlier described antiviral cl- , is a small molecule antiviral drug that selectively inhibits rsv (wyde et al., ) . the molecule is water-soluble and not orally bioavailable, but it proves to be efficacious when administered intranasally or by inhalation. the in vitro ic varies between and ng/ml for laboratory strains or clinical isolates of rsv subgroup a and b. viral specificity and the large therapeutic window of rfi- (\ fold) indicate that the antiviral activity of the compound is not due to adverse effects on normal cells. addition of rfi- to cell culture prior to adsorption reduces rsv yield -fold at h post infection (wyde et al., ) . temperature shift experiments suggest that the rsv f protein is the target for rfi- , and this observation is confirmed by the inhibitory effect that rfi- has on rsv b cp- / b , a viable rsv mutant in which both the g and sh open reading frames are deleted. if rfi- is added to cell culture h post infection, it inhibits syncytium formation indicating that fusion inhibition occurs both in the early and late phase of the infectious cycle. rfi- -resistant viruses can be selected, albeit much less easily than amantadine resistant variants. resistance to rfi- is acquired by point mutations solely in the f protein, mostly upstream of the second heptad repeat motif, but not by mutations in the g or sh protein. when administered prophylactically by the intranasal route, rfi- inhibits rsv replication in vivo, with mice and cotton rats exhibiting a - -fold reduction in rsv replication on day post infection. in african green monkeys, rfi- reduces peak rsv titers not only when administered prophylactically but also when therapy is initiated at h post infection and continued for a total of days. a nebulized form of rfi- has been shown to be active in monkeys. the preclinical profile of this drug supports its development for treatment and prophylaxis of rsv disease in pediatric, adult and geriatric populations. phase clinical trials confirmed that rfi- is a potent antiviral, and that the safety profile of this drug is encouraging. . . de elopment of picona irus c protease inhibitors ag is a potent, irreversible inhibitor of human rhinovirus (hrv) c protease, the enzyme that is responsible for the cleavage of the viral polyprotein into its functional protein subunits (matthews et al., ) . ag was discovered by protein structure based rational drug design, and the compound exhibited activity against a large set of different hrvs. the % effective concentration (ec ) ranges between and mm. other piconaviruses such as coxsackievirus a or b , enterovirus , and echovirus are also sensitive to the compound. in a placebo controlled challenge study in which adult volunteers were infected with hrv and treated with ag ( mg per dose intranasally, times per day), ag reduced mean viral titers, as well as mucus weight, respiratory symptom score and total symptom score significantly. in this study viral titers were determined by culture and also by quantitative pcr (taqman) to exclude ex-vivo effects of ag on the reduction of virus titers. a phase ii clinical trial with subjects was conducted to determine the efficacy of ag in naturally acquired piconavirus infections. patients selected for the study had to present within h of onset of symptoms and had to suffer from at least two mild or one moderate cold symptom. a five step symptom score was used to record severity of rhinorrhea, cough, sneezing, sore throat, chills, headache, and malaise. the treatment group was stratified into two or four daily doses of mg ag per dose intranasally, and the mean respiratory symptom score for days - was chosen as the primary endpoint. only % of all enrolled patients were infected by picor-naviruses. no significant difference in respiratory or total mean symptom score for days - was detected. however, the drug was well tolerated and safe. the lack of efficacy in this particular study might have been due to a lower than expected frequency of picornavirus infections. in a retrospective analysis, stratification for start of therapy within h detected a trend to fewer respiratory symptoms and fewer total symptoms. there was a trend to earlier onset of relief, but the difference between treatment and placebo group again did not reach significance. . . pleconaril (p) treatment reduces the incidence of acute otitis media (om) in children with iral respiratory illness: a pilot study pleconaril, an orally bioavailable picornavirus c protease inhibitor, has in vitro antiviral activity against % of all rhinoviruses. to evaluate the efficacy of pleconaril in preventing acute otitis media (aom) in an outpatient population, a double-blind, placebo-controlled pilot study was conducted in children with viral respiratory illness. eighty-seven children with a median age of years and a history of - episodes of aom were randomized to pleconaril treatment ( mg/kg or . mg/kg) or placebo thrice daily for days following presentation with upper respiratory symptoms. picornavirus rna was detected by rt-pcr in nasal samples in % of patients at baseline. the overall incidence of physician-diagnosed aom during the day follow-up were . % ( / ), . % ( / ), and % ( / ) in the placebo, low dose and high dose groups, respectively. of the children developing aom, tested positive for picornavirus rna. pleconaril treatment reduced the frequency of nasal symptoms by % in the high dose ( . mg/kg, p= . ) and % in the low dose ( . mg/kg, p= . ) group. it reduced frequency of systemic symptoms by % in the . mg/kg group (p= . ) and % in the . mg/kg group (p= . ). pleconaril was well tolerated, and adverse events did not differ from the control group. these results encourage further expanded trials of pleconaril in children with viral respiratory infections. . . oral oseltami ir reduces influenza-related complications in all age groups influenza illness is associated with the development of secondary complications, often requiring antibiotic treatment or even hospitalization. insurance data indicate that up to % of influenza related hospital admissions occur in - year old individuals, often without recognized underlying disorders. influenza complications affect all age groups but the type of complication differs among them. while acute otitis media has been reported in over % of children with influenza, lower respiratory tract infections (lrti) such as bronchitis and less often pneumonia, are the most common complication in adults. in recent years six phase iii clinical trials were completed; two of them in pediatric populations, three in healthy adults and one in the elderly. the neuraminidase inhibitor oseltamivir (o) is already approved for the treatment of influenza in adults, and it was recently approved for treatment in children aged year and older by the fda. in six phase iii trials, most of the patients were enrolled within h of onset of symptoms, and oseltamivir was administered at mg/kg twice daily for children and mg twice daily for adults. subjects with febrile ( \ . °c) influenza were randomized to a day regimen of oseltamivir or placebo, and an important endpoint of all studies was the effect of oral oseltamivir (o) on the incidence of influenza-related complications requiring antibiotics. one thousand and twenty nine children between the age of and (mean age . years) were enrolled in the pediatric trials. in the adult and elderly populations, patients with a mean age of years and elderly patients with a mean age of years were enrolled. in the pediatric, adult and elderly trials , and %, respectively, tested positive for influenza a or b by culture or fourfold increase in hai titers. physician-diagnosed secondary complications (bronchitis, pneumonia, lrti, sinusitis or otitis media) requiring antibiotics were assessed in patients with confirmed influenza infection. oseltamivir reduced the rate of complications compared with placebo by % in children (p= / , o = / ; p = . ), by % in adults (p = / , o = / ; p = . ), and by % in the elderly (p= / , o= / ; p= . ). in the pediatric population, aom was the most common complication, followed by bronchitis, pneumonia and sinusitis. the frequency of aom was reduced from % in the placebo group to % in the oseltamivir group, a significant % reduction. in adults and the elderly, bronchitis and sinusitis were the most common complications of influenza. the frequency of bronchitis in the elderly was reduced from to %. thus, oral oseltamivir reduced the incidence of secondary complications and associated antibiotic use in all age groups. when all age groups were combined, antibiotic use for any indication was also significantly reduced. twelve patients in the placebo group versus four patients in the oseltamivir group required hospitalization for probable or possible influenza-related complications, suggesting a possible effect on hospitalization. . . neuraminidase inhibitors: clinical update . . . oseltami ir oseltamivir was recently approved in the us for prophylaxis of influenza in adults and adolescents over years of age after three successful phase three trials. one was conducted in healthy adults, one in a family setting with an infectious index case, and another evaluated seasonal prophylaxis in the elderly. the efficacy of oseltamivir in all three trials ranged between and %. an application for the approval of oseltamivir for treatment of influenza in children over one year of age was recently approved by the fda. in otherwise healthy children of - years, treatment with oseltamivir reduced the median time to freedom from illness by . days (p= b . ) and also reduced the relative risk of otitis media (aom) by %. in - year old asthmatic children, oseltamivir treatment, started within h of first symptoms, decreased the median illness duration by . days (p = . ), and airway function was significantly improved ( s forced expiratory volume on day improved by . % compared with baseline values among oseltamivir recipients and . % among placebo recipients (p= . )). the incidence of development of drug resistant virus under therapy was evaluated in more than subjects. none of subjects sampled on day of treatment harbored resistant virus. on day , however, / subjects ( %) shed virus with a resistant phenotype. in children, the frequency of viral resistance was % ( / ). all of these subjects recovered normally despite this incidental finding. so far, resistant mutants have been less infectious than wild type virus; no evidence of transmission has been detected in an experimental infection model in ferrets. the impact study evaluated the benefit of early intervention in reducing the total time with symptoms starting from the time of first onset of fever. this reflects the total burden of illness from a patient's point of view. a total of patients, - years of age were enrolled and treated within h of the first onset of symptoms. treatment was initiated within h in % of those recruited. using an accelerated time to failure model, earlier intervention was shown to strongly correlate with shorter duration of illness. the total duration of illness could be halved if treatment was started within h of the onset of symptoms compared with intervention at h. in phase iii trials, zanamivir reduced the duration of illness in adults over years of age and in children between and years of age by . days ( % ci= - . days). in high risk patients ( subjects, % with asthma and % with chronic obstructive pulmonary disease) duration of illness amongst all influenza positive subjects was reduced by . days. duration of illness was reduced in both vaccinated ( . day) and nonvaccinated ( day) subjects. during therapy, pulmonary function improved slightly between days and . zanamivir was well tolerated, and there were no differences in adverse effects between treatment and placebo groups. with subjects studied in clinical trials, resistant mutants were not isolated by sampling on days and . efficacy for treatment of influenza b was assessed in subjects and found to be similar to that of influenza a. a prophylaxis study in a family setting showed % protective efficacy. finally, in a nursing home study, zanamivir exhibited a % in-creased efficacy in preventing influenza disease compared with rimantadine. rwj- , an orally available once daily neuraminidase inhibitor that is active against influenza a and b in vitro and in animals, was evaluated in phase ii trials of experimentally infected healthy adults. therapy was started approximately h post infection with influenza a/texas h n or influenza b/yamagata. viral load over time was the primary endpoint, and nasal wash titers were determined every h on day - and daily thereafter. in the influenza a study, subjects were enrolled into four treatment ( to mg per day) and one placebo arm. high dose treatment ( mg once daily or mg twice daily) reduced viral load by - %, whereas or mg per day led to a reduction by %. shedding was reduced by - days in the high dose groups, and the incidence of fever declined from % in placebo controls to less than % in all treatment groups combined. to evaluate efficacy against influenza b, subjects were enrolled into two treatments ( or mg per day) and one placebo group. a % reduction of viral load was shown for the high dose treatment ( mg per day). no evidence for the emergence of resistant virus was detected, and oral rwj was well tolerated without excess gastrointestinal side effects. world-wide, acute respiratory infections (ari) are responsible for more than million deaths per year in children under five years of age. viral respiratory infections contribute significantly to this burden of disease, not only in children but also in adults. although there is a myriad of viral respiratory pathogens, the number of virus families that cause significant burden of disease is limited. in children, the paramyxoviridae are the most important family of viruses, with respiratory syncytial virus (rsv) and the parainfluenza viruses (piv) causing most disease, followed by adeno-and influenza viruses. in adults, influenza a virus with its constantly changing antigenic composition surpasses other viral pathogens, but the impact of rsv and rhinoviruses are being increasingly appreciated. the epidemiology of respiratory virus infections very much depends on host and environmental factors. while adenovirus infections are a major concern in military training camps and in immunocompromised patients, they are not as important in the general adult population. the immunocompromised are not only at increased risk for viral respiratory disease but diagnosis and management are much more difficult in this group. newer diagnostic methods based on antigen detection or viral genome amplification make rapid diagnosis possible and thereby affect clinical management of viral respiratory disease. vaccines are still the most powerful tool in preventing viral respiratory disease but for many important pathogens there is still no vaccine available. the inactivated influenza vaccine, with its antigenic composition changing annually, is still the mainstay of influenza prevention. coldadapted live attenuated influenza virus vaccines are on the horizon but are not yet approved by the fda. adenovirus vaccines have been used successfully in the military for many years, and the recent resurgence of large outbreaks of respiratory disease after discontinuation of vaccination emphasizes the importance of maintaining a preventive strategy. live virus vaccine candidates against rsv and piv, derived biologically or by reverse genetics, are currently in clinical trials, and there is hope that safe and efficient vaccines will become available within this decade. new insight into virus-host and virus-bacteria interactions in viral respiratory illness may help us understand the complexity of these disease entities and allows a re-evaluation of management options. diseases earlier thought of as purely bacterial, such as otitis media, might be more effectively prevented by viral vaccines than by bacterial vaccines. populations at high risk for complications resulting from respiratory viral infections are now better defined and a more targeted prophylaxis is possible, be it passive prophylaxis against rsv disease with monoclonal antibody preparations or active prophylaxis with influenza-or adenovirus vaccines. in the management of influenza virus disease, much has changed for the better. neuraminidase inhibitors (ni) are now established as an effective intervention to decrease the severity and to shorten the duration of illness when therapy is initiated within days of the onset of symptoms. zanamivir and oseltamivir are now licensed in the us and elsewhere for adults and adolescents twelve years of age and older, and a license for use in children over one year of age has recently been obtained for oseltamivir. both drugs are safe and well tolerated, and development of resistance occurs much less frequently than with older antivirals such as amantadine or rimantadine. rhinovirus infections, the cause of most colds, may now be amenable to treatment with the experimental c protease inhibitors pleconaril (given orally) and ag (given intranasally), and initial studies indicate that pleconaril treatment of colds in children might reduce their risk of developing otitis media. antivirals effective against rsv and piv are still in an early phase of development, but inhibitors of the viral fusion protein look promising. despite all progress, severe deficiencies remain in the preventative efforts. our level of preparedness for an influenza pandemic does not seem much different from that years ago. streamlining of hospital infrastructure and just-in-time production of antibiotics and antivirals limit our ability to respond effectively should a pandemic strike. although potential pandemic influenza viruses have been defined and novel techniques such as reverse genetics are being employed in vaccine development, the implementation of pandemic preparedness is still a daunting task. adenovirus type uses sialic acid as a cellular receptor variable morbidity of respiratory syncytial virus infection in patients with underlying lung disease: a review of the picnic rsv database. pediatric investigators collaborative network on infections in canada respiratory virus infection as a cause of prolonged symptoms in acute otitis media viral infections of humans cold adaptation of parainfluenza virus type : induction of three phenotypic markers the efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children cutting edge: adenovirus e has two mechanisms for affecting class i mhc expression the m - protein of human respiratory syncytial virus is a regulatory factor involved in the balance between rna replication and transcription structural analysis of the mechanism of adenovirus binding to its human cellular receptor the detection of viral genomes by polymerase chain reaction in the myocardium of pediatric patients with advanced hiv disease infections in infants and children in a controlled study of respiratory tract disease. ii. variation in adenovirus infections by year and season chimeric bovine respiratory syncytial virus with glycoprotein gene substitutions from human respiratory syncytial virus (hrsv): effects on host range and evaluation as a live-attenuated hrsv vaccine interferon gamma expressed by a recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity partial protection to respiratory syncytial virus (rsv) elicited in mice by intranasal immunization using live staphylococci with surface-displayed rsv-peptides isolation of subgenus b adenovirus during a fatal outbreak of enterovirus -associated hand, foot, and mouth disease in influenza a and meningococcal disease the role of nasal secretion and serum antibody in the rhinovirus common cold recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca). i. isolation, properties and characterization newly recognized myxoviruses from children with respiratory disease growth on artificial medium of an agent associated with atypical pneumonia and its identification as pplo isolation and characterization of adenovirus from the brain of an infant with fatal cerebral edema respiratory viruses interfere with bacteriologic response to antibiotic in children with acute otitis media parainfluenza viruses rational design of live-attenuated recombinant vaccine virus for human respiratory syncytial virus by reverse genetics production of infectious human respiratory syncytial virus from cloned cdna confirms an essential role for the transcription elongation factor from the % proximal open reading frame of the m mrna in gene expression and provides a capability for vaccine development recovery of viruses other than cytomegalovirus from bronchoalveolar lavage fluid immunity to influenza in man immunization of types four and seven adenoviruses by selective infection of the intestinal tract respiratory viral infections in immunocompetent and immunocompromised persons host responses to respiratory virus infection and immunization satisfactorily attenuated and protective mutants derived from a partially attenuated cold-passaged respiratory syncytial virus mutant by introduction of additional attenuating mutations during chemical mutagenesis recombinant human respiratory syncytial virus (rsv) monoclonal antibody fab is effective therapeutically when introduced directly into the lungs of rsv-infected mice the molecular basis of pneumococcal infection: a hypothesis rehospitalization for respiratory illness in infants of less than weeks' gestation acute respiratory viral infections in ambulatory children of urban northeast brazil heparan sulfate glycosaminoglycans are involved in adenovirus type and -host cell interactions comparison of rapid diagnostic techniques for respiratory syncytial and influenza a virus respiratory infections in young children human parainfluenza virus type (piv ) expressing the hemagglutinin protein of measles virus provides a potential method for immunization against measles virus and piv in early infancy prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes rapid diagnosis of respiratory syncytial virus infections in immunocompromised adults mechanism of protective immunity against influenza virus infection in mice without antibodies efficacy of a pneumococcal conjugate vaccine against acute otitis media. new engl huntingtin interacts with a family of ww domain proteins rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human parainfluenza virus types , , and by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (hexaplex) detection of viral, chlamydia pneumoniae and mycoplasma pneumoniae infections in exacerbations of asthma in children low-temperature-grown rs virus in adult volunteers cytoplasmic inclusions of respiratory syncytial virus-infected cells: formation of inclusion bodies in transfected cells that coexpress the nucleoprotein, the phosphoprotein, and the k protein surfactant protein a binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity the respiratory syncytial virus matrix (m) protein localises to cytoplasmic inclusions in the presence of n, p, l and m proteins experimental otitis media after nasal inoculation of streptococcus pneumoniae and influenza a virus in chinchillas role of early region (e ) in pathogenesis of adenovirus disease risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level risk of primary infection and reinfection with respiratory syncytial virus safety and immunogenicity of intranasally administered inactivated trivalent virosome-formulated influenza vaccine containing escherichia coli heat-labile toxin as a mucosal adjuvant evaluation of the live attenuated cpts / rsv vaccine in combination with a subunit rsv vaccine (pfp- ) in healthy young and older adults adult adenovirus infections: loss of orphaned vaccines precipitates military respiratory disease epidemics respiratory syncytial virus infection in children with bronchopulmonary dysplasia prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. the respiratory syncytial virus immune globulin study group palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. the impact-rsv study group molecular characterization of h n influenza viruses: were they the donors of the 'internal' genes of h n viruses in hong kong? respiratory syncytial viral infection in children with compromised immune function prevalence of various respiratory viruses in the middle ear during acute otitis media a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion recovery of a new agent from patients with acute respiratory disease characterization of the influenza a virus gene pool in avian species in southern china: was h n a derivative or a precursor of h n ? adenovirus immunoregulatory genes and their cellular targets prevention of adenoviral acute respiratory disease in army recruits: cost-effectiveness of a military vaccination policy fimbria-mediated enhanced attachment of nontypeable haemophilus influenzae to respiratory syncytial virus-infected respiratory epithelial cells respiratory syncytial virus that lacks open reading frame of the m gene (m - ) has altered growth characteristics and is attenuated in rodents recombinant respiratory syncytial viruses with deletions in the ns , ns , sh, and m - genes are attenuated in vitro and in vivo community study of role of viral infections in exacerbations of asthma in - year old children development of a humanized monoclonal antibody (medi- ) with potent in vitro and in vivo activity against respiratory syncytial virus first international symposium on influenza and other respiratory viruses: summary and overview evaluation of a live attenuated bovine parainfluenza type vaccine in two-to six-month-old infants a live human parainfluenza type virus vaccine is attenuated and immunogenic in healthy infants and children severe respiratory syncytial virus disease in alaska native children respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine clinical and immunological response of infants and children to administration of low-temperature adapted respiratory syncytial virus influenza a and b virus infection in infants and young children during the years - negative-strand virus m and retrovirus ma proteins: all in a family? interaction of an adenovirus . -kilodalton protein inhibitor of tumor necrosis factor alpha cytolysis with a new member of the gtpase superfamily of signal transducers interaction of an adenovirus e . -kilodalton protein with a novel tumor necrosis factor alpha-inducible cellular protein containing leucine zipper domains recombinant influenza a virus vaccines for the pathogenic human a/hong kong/ (h n ) viruses identification of a cell protein (fip- ) as a modulator of nf-kb activity and as a target of an adenovirus inhibitor of tumor necrosis factor a-induced apoptosis do l t + t cells act as effector cells in protection against influenza virus infection avian-to-human transmission of h n subtype influenza a viruses: relationship between h n and h n human isolates an adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis complexes with dynein and a small gt-pase biologic and immunologic characteristics of cold-adapted influenza virus the development of live attenuated cold-adapted influenza virus vaccine for humans respiratory syncytial viral infection in infants with congenital heart disease increased burden of respiratory viral associated severe lower respiratory tract infections in children infected with human immunodeficiency virus type- reduction of respiratory syncytial virus (rsv) in tracheal aspirates in intubated infants by use of humanized monoclonal antibody to rsv f protein measuring the comparative efficacy of antibacterial agents for acute otitis media: the 'pollyanna phenomenon epidemiology of respiratory viral infection among paediatric inpatients over a year period in north-east england acute myocarditis. rapid diagnosis by pcr in children structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus c protease with potent antiviral activity against multiple rhinovirus serotypes cytotoxic t-cell immunity to influenza does prematurity alter the course of respiratory syncytial virus infection? the economic impact of pandemic influenza in the united states: priorities for intervention safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with mf adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons current research on influenza and other respiratory viruses: ii international symposium. antiviral res use of live attenuated cold-adapted influenza a reassortant virus vaccines in infants, children, young adults and elderly adults temperature-sensitive mutants of influenza a virus. transfer of the two ts- a ts lesions present in the udorn/ -ts- a donor virus to the influenza a/alaska/ / (h n ) wild type virus effect of age and preexisting antibody on serum antibody response of infants and children to the f and g glycoproteins during respiratory syncytial virus infection current approaches to the development of vaccines effective against parainfluenza and respiratory syncytial viruses improved outcome of respiratory syncytial virus infection in a high-risk hospitalized population of canadian children. pediatric investigators collaborative network on infections in canada effect of rapid diagnosis on management of influenza a infections the p signal transduction pathway: activation and function infection and disease with respect to age, immunologic status, race and sex rhoa interacts with the fusion glycoprotein of respiratory syncytial virus and facilitates virus-induced syncytium formation a rhoa-derived peptide inhibits syncytium formation induced by respiratory syncytial virus and parainfluenza virus type effect of respiratory syncytial virus on adherence, colonization and immunity of non-typable haemophilus influenzae: implications for otitis media detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction paramyxovirus m proteins: pulling it all together and putting it on the road human infection with influenza h n unexplained deaths due to possibly infectious causes in the united states: defining the problem and designing surveillance and laboratory approaches. the unexplained deaths working group the respiratory syncytial virus matrix protein interacts with the cytoplasmic domain of the g glycoprotein adherence of type i streptococcus pneumoniae to tracheal epithelium of mice infected with influenza a/pr virus immunoprophylaxis and immunotherapy of respiratory syncytial virus infection in the cotton rat quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses infection with respiratory syncytial virus enhances expression of native receptors for non-pilate neisseria meningitidis on hep- cells induction of protective immunity against influenza virus in a macaque model: comparison of conventional and iscom vaccines identification of a conserved receptorbinding site on the fiber proteins of car-recognizing adenoviridae isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines bovine respiratory syncytial virus nonstructural proteins ns and ns cooperatively antagonize a/b interferon-induced antiviral response bovine parainfluenza virus type (bpiv ) fusion and hemagglutinin-neuraminidase glycoproteins make an important contribution to the restricted replication of bpiv in primates bacterial pneumonia during the hong kong influenza epidemic of - molecular identification of viruses in sudden infant death associated with myocarditis and pericarditis respiratory syncytial virus immune globulin for prophylaxis against respiratory syncytial virus disease in infants and children with congenital heart disease respiratory syncytial virus infection in north-east england identification of mutations contributing to the temperature-sensitive, cold-adapted, and attenuation phenotypes of the live-attenuated coldpassage (cp ) human parainfluenza virus candidate vaccine effectiveness of palivizumab: evaluation of outcomes from the to respiratory syncytial virus season the role of human adenovirus early region proteins (gp k, . k, . k, and . k) in a murine pneumonia model characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness non-typeable haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the paf receptor avirulent avian influenza virus as a vaccine strain against a potential human pandemic recombinant respiratory syncytial virus that does not express the ns or m - protein is highly attenuated and immunogenic in chimpanzees efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial therapy for respiratory tract infections caused by respiratory syncytial virus expression of adenoviral e transgenes in beta cells prevents autoimmune diabetes respiratory syncytial virus infection in tropical and developing countries community respiratory virus infections in immunocompromised patients with cancer recombinant respiratory syncytial virus (rsv) bearing a deletion of either the ns or sh gene is attenuated in chimpanzees replacement of the f and g proteins of respiratory syncytial virus (rsv) subgroup a with those of subgroup b generates chimeric live attenuated rsv subgroup b vaccine candidates immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy cl exhibits marked and unusual antiviral activity against respiratory syncytial virus in tissue culture and in cotton rats genetic characterization of the pathogenic influenza a/goose/guangdong/ / (h n ) virus: similarity of its hemagglutinin gene to those of h n viruses from the outbreaks in hong kong key: cord- -dltsdqcm authors: siegel, frederic r. title: lessening the impacts from non-tectonic (natural) hazards and triggered events date: - - journal: mitigation of dangers from natural and anthropogenic hazards doi: . / - - - - _ sha: doc_id: cord_uid: dltsdqcm floods are a global problem. they are predictable to some degree by weather forecasting but to a greater degree and with more accuracy when drainage basin monitoring equipment is in place. this includes stream gauges that telemeter the elevation of stream/river surface in a channel and the rate of water flow to a central computing station. the computed data from the telemetered sites plus the input of stream/river channel cross-sections data allow prediction of where flooding will be a problem, when the flooding will reach an area, and to what level out of a channel (magnitude) the flood is estimated to reach. this gives the populations at risk of the flooding early warnings (hours, days) and time to prepare for the floodwaters or to gather important documents and evacuate to safe higher ground. will be issued for residences or other structure for areas proximate to rivers, whether fl ood insurance for structures will be written for a given zone, and if written, what the cost of fl ood insurance will be. floods can cause landslides and potential health problems for populations when there is torrential rain that is long lasting. first, depending on the location of a fl ooding river channel, rushing water can undermine bank material or erode base of valley walls causing landslides that could affect people living in the threatened areas. second, torrential rains can overload sewer systems so that sewerage carrying pathogens are discharged into waters that may affect water users downstream. populations warned of these potential fl ood-triggered hazards can follow advisories to evacuate if necessary and drink bottled water if their water supply is tainted. preparedness against fl oods includes warning/alert systems sent by weather bureaus and fl ood management agencies and, of course, evacuation plans showing routes to safe sites with staffs and supplies in place to help displaced citizens. an intergovernmental panel on climate change report suggests that fl ooding has been more frequent and more severe in some regions and that there is less fl ooding in other regions [ ] . the report projects that extreme precipitation events will occur over most of the mid-latitude land masses and that those over wet tropical regions will become more intense and frequent. these extreme precipitation events often cause fl ooding that endangers people and property especially if early warning systems are not in place. natural changes in river fl ow other than those that result in fl ooding can be categorized as disasters for people and for a country. for example, the semliki river marks the border between uganda and the democratic republic of congo (drc) . in recent years, and perhaps as a result of global warming/climate change, the volume of water discharged into the river from melting snow and ice from mountain peaks has been reduced as the ice has receded and less snow has fallen. in addition, there have been periods of erratic rainfall and the wet season is wetter. the result has been a meandering of the river that has fl owed through uganda fi elds and left them as part of the drc . ugandan farmers and herders have to pay drc owners of the "new land" a tribute to be able to cross m of river water to continue to work the fi elds and care for their herds on the other side of the river that was ugandan territory before the border shifted as the river meandered. there is international intervention that is working to set a fi xed border so that land that was lost can be assigned correct ownership and ugandan territory can stop shrinking [ ] . landslides also called landslips are the best known of the disaster class called mass movements. mass movements are a world wide problem that cause deaths and hundreds of millions of dollars damage each year to homes, infrastructure, and utilities. other movements in this grouping include: ( ) subsidence or a lowering of an area of the earth's surface; ( ) collapse of soil/rock into a void in the subsurface; ( ) rockfalls; and ( ) mudfl ows (debris fl ows). the latter is activated when torrential or sustained rain over a period of time loads water weight into slopes and seeps into earth materials there, lubricating them and also pushing grains apart because of increased water pressure. the heavy, lubricated, destabilized matter fi nally breaks loose and speeds downslope without warning at as much as a mile a minute ( km/ ph). the destabilization can be further abetted when some of the earth materials comprising a slope is composed of the clay mineral class called smectite (montmorillonite/bentonite), a mineral that expands when it is wetted and shrinks when it dries. a combination of the expansion and lubrication from added water and its weight can bring down a hillside. populations at the base of slopes and beyond are at risk of sudden burial with major loss of life. it should be noted that away from hillsides, soils that expand when wet and shrink when dry cause cracks in basements and foundations of buildings that rest on them, a property damage problem but a problem that is not a threat to the well being of people. avoidance of possible building sites underlain by expansive/shrinkage soils or extraction of the soils prior to construction can mitigate the potential basement/foundation problem. keeping the soils wet with an irrigation system that activates during dry weather has been used to stabilize the reactive soils. landslides occur in hilly topography (elevated terrain, > ° slopes) where the soils are well developed, where poorly consolidated sedimentary rocks comprise the hills, where the structure of the rocks dip (angle) downward and outward from the at risk slopes, and where there is considerable rainfall. these are predictive observations that suggest the possibility of a landslide prone region. landslides are a threat when water seeps into the hills adding water weight that increases the pull of gravity on the earth materials, increases groundwater (pore) pressure that pushes grains apart, and acts as a lubricant for subsurface earth materials. other observations that alert citizens that there is a downslope movement of soil are cracks in the ground and trees that tilt back into a slope. a geologist's observations of scarps in a hilly area attests to it being landslide prone. scarps are mini-cliffs often with exposed soil caused by a landslide but that may be hidden from the untrained eye by overgrowth. tilt-meters (inclinometers) are electrical driven pieces of equipment that can be installed in slopes to telemeter movement or deformation data that suggests the possible onset of a landslide. a recent approach to identify the strong probability of a landslide and thus could serve as an early warning system that allows people to evacuate to safety uses fi ber-optic strain sensors complemented by rainfall data [ ] . these sensors render continuous monitoring in time and space. fiber-optic sensors that can detect displacement, groundwater pore pressure, displacement (slow slope slippage), ground vibrations, and temperature, are glued equally spaced to the surface of a pvc (fl exible) pipe and embedded in shallow trenches in a soil. the pipe . mass movements can bend or twist with pre-slope failure tensile strain (e.g., elastic, plastic, shear, viscous volumetric) registered as d deformation. the various sensors will register the location of the deformation on monitors at an off slope location. physically, a landslide appears as a block of earth materials that breaks from a slope along a scarp and slides down along a concave surface pushing lobes of slide materials out at the base of the block. the areas affected by landslides are generally limited and distances landslide move are generally small although in areas of very steep topography the momentum of a landslide can carry the detached mass a signifi cant distance and across adjacent terrain. as noted previously, landslide moving into a river valley can act as a dam, cause fl ooding initially upstream and subsequently downstream when the temporary dam is breached. in addition to landslides starting as a response to an earthquake's shaking, jarring, rolling motions and excessive rainfall, human activity can lead to landslides as well. this can happen when toes of slopes that give stability to hills in landslide prone regions are cut away to establish a road. this disturbs a slope's equilibrium and is an example of bad land use planning that creates conditions that can lead to landslides. similarly, landslides may be caused if the head of a slope at equilibrium between gravity stress trying to pull soils and rocks down and the strength of the mass resisting slope is weakened by overloading the head with housing and welltravelled roads. the added weight (stress) and vibrations from vehicular traffi c can result in a slope failure. also, sliding can be abetted by loss of anchoring vegetation especially where logging has been active. in landslide prone areas as evaluated by geologists and geological engineers, citizens can be protected to a good degree from injury, loss of property, and even death in two ways. one is zoning that prevents habitation where conditions are conducive for landslips if a slope were to be loaded with destabilizing weight such as the earlier cited homes and well traffi cked roads. second, for existing habitation or planned habitation, geological engineering can diminish the threat of landslides. this is done by ( ) redirecting rainwater or snowmelt so that it does note invade slopes at risk; ( ) by installing retaining walls with perforations that allow in seeping water to exit a slope; and ( ) by installing concrete caissons, a few feet in diameter down to base rock along the front of a vulnerable slope. these efforts are costly so that economic constraints prevent their general use. in areas of japan where topography is hilly and where landslides threaten infrastructure including critical transportation systems, the japanese government has invested in landslide prevention . they sealed hills with shotcrete so that rainwater or snowmelt can not seep into the hills and destabilize them with added weight and lubrication at earth material slide planes. preparedness would require that earth moving equipment and search teams be available to rescue people who survived but are trapped by displaced earth materials. recurrence of landslides is associated with the amounts of rainfall an area receives over a period of time. landslide activity will increase in some global areas and in some regions as a result of increased rainfall because of climate change. as mentioned in the previous section on fl ooding, extreme precipitation events, or simply increases in annual mean precipitation is likely for the high latitudes, the equatorial pacifi c region, and mid-latitude wet regions and can provide the stimulus for landslide activity. central america and south america are especially ar risk [ ] . an avalanche is a large mass of snow that suddenly slides downslope. there are two general types of avalanches: ( ) sloughs that are small fl ows of powdery snow that are unlikely to kill people or destroy structures; ( ) full depth avalanches that are massive slabs of snow that break loose from a mountain and cause death and destruction. the latter may carry ice, soil, and rock debris. avalanches generally occur without warning on slopes > ° and < °at alpine areas worldwide (e.g., swiss mountains, western canada, new zealand, alaska, the himalayas). one or a combination of factors can contribute to an avalanche event. these include storminess, slope shape, orientation of steep slopes with respect to the sun, the rough or smooth character of the ground beneath a snowpack, vegetation, the nature of the layers of a snowpack, and vibration. thus, if there is a cm ( in.) or more snowfall in a h periods, there is likely to be an overloading and an avalanche depending on how the layers in a snowpack are bound together. most avalanches occur during snow storms and blizzards. clearly, a steeper convex slope is more conducive to avalanche activity. a rapid temperature increase can cause melting of a layer in the snowpack that results in an avalanche. the more vegetation there is deters the down fl ow of an avalanche as would a rough rocky surface beneath a snowpack. vibrations from activity on a slope (skiers, snowboarders, snowmobiles), thunder storms, low fl ying jet planes, and explosions can set off an avalanche. during wwi, thousands of soldiers in alps regions were killed by avalanches triggered by artillery fi re. avalanches can fl ow downslope at speeds of km/h ( mph) or more. depending on the mass being moved, an avalanche can kill people, and damage or destroy structures and infrastructure (homes, recreational areas, bridges, tunnels [block road and/or railway movement]), pipes and utility lines (water, natural gas, electricity), and put workmen maintaining an infrastructure at risk. hundreds of people are killed by avalanches each year. during and , avalanches in nepal , triggered by an earthquake and by unseasonal severe rain and snow blizzards, roared downslope from mount everest and other peaks in the region. many trekkers were killed, as were sherpa guides and climbers preparing for the climb to the summit of mount everest. people can be protected by zoning that evaluates the history of avalanches, the paths they follow, and their frequency and reach in an alpine area to prepare risk maps to prevent use or allow limited or full use of the zoned terrain. in areas where use of terrain is allowed, warning systems are in place so that when sounded, citizens will not enter a threatened area, or if there, evacuate it immediately. avalanches can not be prevented but defensive structures can be used in established populated areas to try to divert them such as snow fences or snow walls. avalanche sheds can be used to protect structures and transportation routes by making the fl ow of snow ride over them. dangerous buildups of snow on slopes that are known for avalanches can be set off as snow slides or snow slips using vibrations caused by controlled explosions with explosives implanted in the snowpack, dropped by helicopter, or delivered by artillery shells. excellent sources of information on avalanches can be found at www.ussartf.org/avalanches.htm and at www.conserve-energy-future. com/types-causes-effects-of-avalanches . a rockfall happens when a mass of rock from a very steep to vertical cliff detaches from the face of a cliff and free falls down. the rocks bounce off underlying rocks, often detaching them as well. the falling rocks crash onto the base of a cliff sometimes running out damaging structures and/or infrastructure, blocking roads and putting vehicles at risk. in addition to steep topography, the geological character of the rocks comprising a cliff, the climate, and sometimes vegetation are factors that infl uence the risk of a rockfall but do not predict when one may occur. fractures and fi ssures in rocks can fi ll with water during a winter day and freeze at night causing ice wedging that weakens rocks against the pull of gravity. trees that root in cracks and crevices in a rock cause root wedging that does the same. stress when an earthquakes strikes an area can loosen rocks to the point that they fall from a cliff face. a geological evaluation of an area can reveal areas that have suffered rockfalls and areas likely to have rockfalls but geologists can not reliably predict when a fall will occur. to protect an area and its inhabitants, infrastructure, and businesses from rockfalls, municipalities have options. they can install catchment fences at the base of cliffs to prevent run outs, require that rock bolts be inserted to stabilize an at risk rock face or that chain-link fencing be fastened to the rock face. subsidence of an area of the earth's surface is the result of the continuous extraction of large volumes of groundwater or petroleum from underlying sedimentary rocks without recharge or replenishment of fl uids. fluids in subsurface rocks provide buoyancy pressure that strengthens the resistance of the rocks to compaction and hence subsidence of the earth's surface . if volume loss does take place in subsurface rocks, subsidence may or may not occur depending on the strength and thickness of overlying rocks. overlying rocks may be inherently strong enough so that they do not subside. conversely, they can respond to the compaction of underlying rock by subsiding. subsidence can be arrested or even reversed somewhat in some geological settings if a degree of buoyancy pressure is reestablished by re-injecting fl uid (e.g., brine) into the rock. for example, in oil fi elds, eight barrels of brine are extracted with one barrel of crude oil. the brine can be recycled into the oil reservoir under pressure. similarly, if extraction of groundwater from aquifer rock is replenished by groundwater recharge, any subsidence that has taken place should stop. oil production at the wilmington field in southern california, usa, began in and caused a subsidence that by reached . m ( ft) at long beach harbor and extended to parts of los angeles harbor. the subsidence damaged oil wells, pipelines harbor infrastructure, railroad tracks, streets, and bridges and reversed the fl ow of sewers and storm drains. repair cost more than u$s million at that time. brine reinjection was used to arrest the subsidence and there was stabilization and a rebound of cm ( in.) [ ] . mitigation of a possible subsidence problem can be determined by geologists and geological engineers who study samples of the sequence of rocks from the surface to the oil or water reservoir. they can predict whether or not subsidence will take place and if it is realistic to plan to recharge the extraction reservoir with volumes of fl uid that equal the volumes withdrawn, thus maintaining buoyancy pressure in the reservoir/aquifer. this can prevent or at least minimize subsidence if it begins. governments can be stressed two ways economically because of subsidence. first is the cost to repair the damage done to infrastructure and structures in the areas affected by subsidence. second are the economic losses that can be incurred if a productive sector is slowed down by not being able to extract critical fl uids from the subsurface without having to invest more funding to improve extraction by drilling wells deeper. this would likely mean an increase in the price of a commodity used to make or grow a product and hence be infl ationary for the public. a case in point is the more than four year severe drought being suffered in in the central valley (especially the san joaquin valley) of california, usa where subsidence has been a recurring problem during past droughts as groundwater was extracted, but not excessively, from aquifers to make up for the shortage of surface water. however, excessive groundwater pumping during the existing extended drought, mainly by the agricultural sector, has lowered groundwater tables to ft (~ m) lower than recorded in the past. as a result some areas experienced a subsidence of up to two inches ( cm) a month as fi negrained layers in the aquifer were depleted of their buoyancy pressure and compacted. if the compaction is tight enough, part of the storage capacity (porosity) and permeability (ability to transmit fl uids) of an aquifer could be lost. the subsidence varies with location in the valley. one area subsided at / in. a month. the maximum subsidence was about a foot ( in. or cm) a month [ ] . another result of the over pumping is a reduction of fresh water pressure in the aquifer that could result in salt water intrusion where the aquifer extends to the marine continental shelf. the differential subsidence in the san joaquin valley has caused damage to infrastructure, the most important of which may be the california aqueduct comprised of canals, pipelines, tunnels and pumping stations. the aqueduct carries water from northern california rivers that receive melt from the sierra nevada snowpack and from rainwater some miles (~ km) to southern california. change in the fall (inclination) of sections of the aqueduct from subsidence and low spots on the system prevent the water from fl owing as well as it did pre-subsidence and thus needs reworking. changes in the fall of sewer lines have to be repaired to prevent sewage backup and its consequences from a reversal of slope. similarly, water pipelines have to be reset to allow effi cient fl ow of water. some bridges have subsided to the degree that they are no longer above the water surface. roads are ruptured and have to be repaired. levees in place for fl ood control when the rains come have sunk and have to be raised. building foundations sink as well and need correction. very important in this agriculturally dependent valley and its towns has been the destruction of thousands of public and private well casings. california and the agriculture industry have the economic wherewithal to make the necessary repairs once the drought breaks. the central valley grows about % of the vegetables and fruits sold in the united states. as previously indicated, if crops fail or yields drop because of the lack of water for irrigation, the cost of the produce and fruit will increase. the state is investing large sums of money to develop a capital improvement program. collapse of a small areas of the earth's surface into voids in the subsurface causes sinkholes. this is an action that takes place most often where there is a relatively high water table in terrain underlain by limestone, a rock type that houses many famous cave systems worldwide. in this scenario, groundwater moving slowly through limestone continually during geologic time (millions of years) dissolves the limestone leaving voids in the subsurface. when these are large enough and the roof rock strength cannot resist the pull of gravity, the roof rock collapses into the subsurface cavity. the areas affected are generally small, rounded or oval shaped, and tens of meters or less across. sinkholes have caused structural damage to buildings, swallowed homes and car dealerships, ruptured roads (pavements) including highways, and in rare cases have caused injury and death. sinkholes have developed as well in terrain underlain by salt-rich (evaporite) strata that are dissolved by irregular groundwater fl ow. in some cases, water pipes underlying roadways have broken and the released water has washed out the earth materials around a ruptured waterlines creating open spaces in the subsurface into which earth/road materials have collapsed. underground coal fi res can create cavities into which the overlying rock and soil can subside and ultimately collapse. to lessen the risk of suffering loss from collapse from a future sinkhole when buying a home or business or land to develop and build on, one should commission an evaluation of the terrain by experts that will predict its vulnerability for collapse. for example, a geologist will fi rst assess the geology of the area under study (especially when underlain by limestone or dolostone), the topography, and determine the position of the water table that could cause dissolution of underlying rock. he/ she will review the history of insurance claims against collapse situations in the area. in a surface analysis, the geologist will look for signs of potential surface movement that could portent sinkhole activity by examining building foundations in the neighborhood for visible cracks, especially arcuate ones, cracks in roads or sidewalk pavement, and depressions or low spots in the terrain as well as the presence of small ponds that could represent former sinkholes. this study will suggest the level of risk in an area but only an investigation of the subsurface conditions can give more accurate vulnerability information. this can be done using ground penetrating radar (gpr) that takes a short time using modern equipment that digitizes data for ready presentation and interpretation [ ] . in one florida study, the gpr data for nine m lines were generated in h. another option is to do a seismic study that will yield information as to the solid rock nature or void presence in the subsurface. studies such as these, where collapse is an endemic problem, but on a larger, perhaps county scale, can result in sinkhole vulnerability maps that can guide land use planning for small scale evaluations of potential sites for buildings or infrastructure. where there is a void in the subsurface but there is a need to use the terrain, it may be possible to fi ll the void with grout to stabilize the surface but this can be very costly. a question exists as to whether scientists can spot an incipient sinkhole as it develops so as to be able to warn inhabitants of at-risk homes to evacuate. the possibility exists using the nasa satellite system insar (interferometric synthetic aperture radar) that detects small movements on the ground. a study of an area in louisiana, usa and noted that the ground shifted horizontally in. ( cm) in a section of the survey. a sinkhole opened up there a month later and the horizontal displacement observed was towards the center of the sinkhole [ ] . scientists believe that there is the potential to use this deformation as an early warning system in other sinkhole prone areas so that people can remediate the condition or evacuate buildings that might be at risk of a calamitous collapse. this is a step towards preparedness. however, they noted that not all sinkhole sites have surface shifting before a collapse, making necessary the previously cited methods to detect subsurface cavities that could become sinkholes. mitigation of an economic loss of home or business to a collapse into sinkhole event can be achieved by having collapse/sinkhole insurance as part of a home owners policy or an insurance policy that covers a business structure and inventory. the policies should cover collapse whether the event is from natural processes (e.g., subsurface dissolution of limestone) or from the failure of a water or sewer pipe and subsequent washout of supporting earth materials that leads to a collapse of an overlying home, business structure, road, highway, or bridge. because many home buyers and home owners are unaware of their vulnerability (risk) from a collapse/sinkhole event, the state of florida, usa mandates that home owners carry catastrophic ground collapse coverage. in the united states, alabama, kentucky, missouri, pennsylvania, texas, and tennessee are states with collapse into sinkhole problems and their own insurance requirements. infectious/communicable diseases are natural hazards abetted in some instances by human actions or inactions. some of these diseases have been eradicated (smallpox) or nearly eliminated (polio, measles, guinea worm disease [dracunculiasis]). others are treatable (malaria) or can be stabilized (hiv/aids), and still others are not preventable (dengue fever) but the exposure to which can be alleviated to some degree. still others for which prevention and/or curing/stabilization medications are not available are being researched intensely such as an ebola vaccine because of the - west african outbreak of the disease. in the most recent clinical trial of an ebola vaccine, the success rate was %, encouraging but requiring more testing before it is approved for universal application. the threat or onset of infectious disease can be mitigated in several ways. first is prevention. there are vaccines available that can protect citizens against contracting specifi c infectious diseases. in the case of measles and polio, the actions of religious zealots (taliban sect) who have burned down health clinics and injured or killed health workers have prevented vaccinations for all so as to achieve global eradication of poliomyelitis in two countries where it still occurs (pakistan and afghanistan) and measles (globally), especially in asia and africa. however, this not withstanding, vaccination programs for measles are making good progress. from to the number of deaths from measles has dropped %, from , to , . mumps and measles (mmr vaccine) and whooping cough (dtap vaccine) are two other infectious/communicable diseases that can be eliminated if vaccinations are given. in the case of children, a second application of the mmr vaccine is necessary to give them lasting immunity to mumps and measles. it should be noted that vaccinations may not be close to % effective. scientists have modeled the probable effectiveness of vaccinations for endemic infections [ ] . they conclude that vaccines denominated as "leaky" provide the same degree of resistance to a disease to all who have been vaccinated and gives a partial immunity after being vaccinated. a vaccine called " all-or-nothing (aon)" is best applied when the probability of re-infection is high, transmission is likely, or when a vaccine has low power to reduce the risk of infection. in contrast to a "leaky" vaccine, an aon vaccine completely protects a major number of vaccinated persons but others in the population receive no direct benefi t from it. some infectious/communicable diseases such as tuberculosis can be cured with medicines taken over a prescribed period of time. chagas is a disease that is endemic in latin american nations. it can be cured if medication is taken early enough after the onset of the disease. if this does not happen the persons with chagas disease can look forward to a middle age with cardiac and gastrointestinal problems with the healthcare burden this implies. cholera is an infectious disease that can be controlled with access to adequate sanitation or cured by rehydrating victims with oral rehydration salts. if a cholera victim's dehydration is severe, iv plus antibiotics are used to cure the patient. a short term lasting cholera vaccine is available and used by health workers where there is a cholera epidemic. aids/ hiv is an infectious/communicable disease that is not curable and that has killed million people. the disease can be stabilized if an infected individual has access to (or the funds to pay for) a cocktail of antiretroviral medications taken during a lifetime. this allows an hiv/aids carrier to live an otherwise healthy life and be productive in his/her community. about . million of the . million people with the disease are now taking the retroviral medications. seventy percent of the global total of two million new cases are in sub-saharan africa [ ] . unless the . million infected individuals not on antiretroviral medications get access to them and follow prescription protocols, the disease will continue spreading through populations. some infectious diseases can be controlled but not eliminated. infl uenza is controlled by a seasonal vaccination that is prepared according to what scientists consider will be the dominant strains of the infl uenza virus during a coming infl uenza season. a strain was missed during the - season and more people suffered sickness as a result. although some people do contract the disease, the vast majority of the vaccinated population is protected. if some do contract infl uenza from the unaccounted for strain, its effects are generally less than they would be without the vaccination. the earlier cited ebola epidemic that raged in liberia, sierra leone, and guinea, western africa controlled and the region declared free of the disease after more than , deaths of more than , infected. the ebola epidemic shows how vulnerable many countries/regions are because of an inadequate health infrastructure that is not prepared to cope with a disease once identifi ed, its spread, and the care and treatment of large numbers of infected people. ebola is transmissible by body fl uid contact and this had to be learned by family members and others caring for infected individuals as a fi rst phase in controlling and ultimately leaving a country ebola free. the spread of this disease to other countries, regions, or around the world was limited by the general immobility of people from affl icted countries and controls at transportation centers and by immigration controls at adjacent countries for those from the disease ridden countries who were mobile and presented a transmission risk. as the disease progressed in the affl icted nations, help arrived rapidly from developed nations that had experience in disease control that experts applied on site and taught to health givers in spite of the fact that who delayed before putting out a public health emergency alert. laboratories that had been researching vaccines against ebola increased their efforts and laboratory testing on animals. test results of the promising vaccines on human subjects have been encouraging for two of the vaccines developed. although the vaccines may prove to be effective against ebola after future positive test results, they were not available at the time the epidemic was identifi ed in west africa . preparedness is the key to dealing with the outbreak of an infectious disease that could develop into an epidemic/pandemic. as noted above, initial preparedness such as protocols in the physical contact with affl icted persons that caused transmission of the disease, their treatment, and burial practices of their kin were lacking in the west african ebola outbreak. a november, report by a panel from the harvard global health institute and the london school of hygiene and tropical medicine critiqued the who for not declaring a public emergency until months after being informed of the ebola outbreak by guinea and sierra leone [ ] . the panel recommended several ways in which the who could improve its role in preparing for and dealing with an infectious disease crisis. among the recommendations were the following: ( ) the need to invest in developing a nation's core capacity, that is, its ability to detect, report, and respond rapidly to outbreaks; ( ) the strengthening of incentives for early reporting of outbreaks and science-based justifi cations for trade and travel restrictions to prevent transmission of a disease; ( ) the creation of a who center with adequate capacity to respond quickly to a disease outbreak; ( ) an assurance of access to the benefi ts of research that yield improved diagnostics, and the most effective medicines and vaccines; ( ) information on best protocols to follow in treating an infectious disease and on cultural awareness and traditions to account for when dealing with families of the affl icted or dead; and ( ) availability of funding to put these and other recommendations into practice [ ] . with respect to improved diagnostics ( above), nature published a december supplement in which contributors modeled the impact of new diagnostic and prognostic technologies for lessening the global burden of infectious diseases [ ] . they are of the opinion that new diagnostics can more rapidly direct patient treatment and limit disease transmission to the general population thus effectively reducing the spread of epidemics. the effectiveness lies in the education of on-site health providers in the new diagnostics and protocols as they become operational. the contributors also believe that research can come up with new and rapid diagnostic protocols for multiple diseases that affect populations worldwide, that are effective and reduce costs that existing protocols incur, especially in less developed and developing countries. the ebola outbreak and spread by direct or indirect contact of an infectious disease raises the question of whether the world is prepared to combat an infectious disease, natural or from bioterrorism, that is transmitted through the air we breathe. given the ease of transmission, the mobility of disease carriers within a country or internationally, the populated venues where transmission through respiration can take place (e.g., airplanes, cruise ships, theaters, arenas, subways, malls), the answer is no for many less developed and developing countries that do not have a well functioning health infrastructure … not enough doctors, nurses, well equipped clinics, hospitals, laboratories, medicines, or vaccines. thus, a virulent disease transmitted through the air could infect and kill millions of people before medical care to treat it or vaccines and medicines to cure it are found and tested so as to control and/ or eliminate a killer virus. it is possible to minimize the spread of an easily transmittable infectious disease but this requires a major investment. a proactive action would be for the health capable and economically advantaged nations to work with less prepared ones to create a good healthcare infrastructure, a mission of the who that has been neglected [ ] . this would include training personnel as deemed necessary, building clinics where hospital care may be lacking, setting up laboratories that can identify diseases at their onset rather than waiting for results from a central laboratory, and by educational programs for citizenry. response team training in all countries is essential to initially deal with a disease. obviously, there has to be open and clear lines of instant communication between health organizations locally, regionally, nationally, and internationally when disease surveillance detects an outbreak node of a known or unidentifi ed infectious disease. this allows a global response to begin on how to cope with the outbreak of a deadly viral disease in order to minimize its spread and develop a treatment protocol. diarrheal disease that affect s of millions of people each year can be controlled in two ways. first, they can be countered and their impact on society lessened by the use of antibiotics assuming medications are available and economically accessible. second, is the identifi cation and elimination of the sources of diarrheal diseases. basically this means improving sanitation conditions and food storage, preparation, and handling operations. investment by governments to provide access to advanced sanitation systems and for education with respect to food cleanliness can lessen the onset and spread of diarrheal infections and limit employee sick days, thus helping a country's productivity and economic development. there are several infectious/communicable diseases that do not fall into the categories described in previous paragraphs. vaccines and medications are being researched with some in trial stages. these diseases include malaria, dengue fever, yellow fever, west nile virus, plague, and most recently ebola because of the west african epidemic. as already noted, in the case of the ebola epidemic, preliminary tests of new vaccines being researched have had good results on human volunteers. however, it is uncertain whether the positive results are because of the vaccines or the care factor given to those with the disease. a zika vaccine is scheduled for phase clinical trials during the latter period of . lastly it should be noted that science magazine commissioned a survey to prioritize ten potential vaccines that would merit increased government and industry funding for their research and development. these vaccines do not show clear scientifi c or safety obstacles and would benefi t societal health conditions mainly in less developed and developing countries. the surveys were sent to vaccine experts globally who were asked to prioritize the listed vaccines based on scientifi c feasibility, morbidity and mortality, and societal/economic impact. fifty experts fi lled the survey. the overall priority ranking results in were ( ) ebola sudan, ( ) chikungunya, ( ) mers, ( ) lassa fever, ( ) marburg, ( ) paratyphoid fever, ( ) schistosomiasis, ( ) rift valley fever, ( ) sars, and ( ) hookworm [ ] . vaccines for some of these ( , , , , , and ) could prevent or reduce the chances of serious outbreaks in the near future. zika would be a addition. the psychological impact of living through a natural or anthropogenic hazard that injures or kills family and friends, and damages and destroys homes, business, and places of employment is a shock to citizens that puts them in a state of disbelief and distress. for that reason, the preparedness sections on hazards recommends that mental health professionals be available to the suffering populations. mental health is considered here as a health hazard that in this text is triggered by a primary event but may result from a secondary event (e.g., earthquake aftershock) or a triggered hazard. the stresses caused can affect populations for reasons other than suffering physical, biological, and chemical dangers environments can present [ ] . citizens living through a natural or anthropogenic hazard and secondary hazards that can occur suffer stress from several results of the event. the immediate stress factor is a concern for families in the degree of destruction citizens see wreaked on their environment. stress levels heighten from the loss of or injury to family and friends and loss of home and possessions. the greater the loss, the higher the level of stress. other factors that stress populations suffering through the immediate . mental stress aftermath of a major hazard event include displacement, sense of vulnerability and insecurity, apparent slowness of assistance midst the chaos a hazard can cause, fear of what might come next, and physical exhaustion. there are ongoing discussions on coping with the stress of disaster [ , ] . post hazard stress arises from loss of employment and income, apparent slowness in recovery leading to societal normalcy, and reconstruction. in a latter section of this book, different classes of insurances are discussed that can, if purchased, mitigate economic stress from hazards losses. the number of people susceptible to hazard-caused stress and their resulting mental health problems will increase in the future because of population growth and increased population density in urban centers that have experienced recurring hazards. this is especially true for major cities (perhaps greater than one million inhabitants) and for mega-cities with more than ten million people. easing or mitigation of stress levels involves having access to mental health professionals who can help relieve people's lasting anxieties, receiving social support from family and friends, focusing on the present and future rather than reliving the past, and taking care of oneself physically. climate change the shifting river that is making uganda smaller brillouin optical time-domain analysis for geotechnical monitoring subsidence, rebound and surface strain associated with oil producing operations progress report: subsidence in the central valley sinkhole detection in florida using gpr and cpt ( pp.) sinkhole: precursory deformation measured by radar interferometry vaccination programs for endemic infections: modeling real versus apparent impacts of vaccines and infection characteristics hiv/aids fact sheet n . geneva: world health organization will ebola change the game? ten essential reforms before the next pandemic ( pp.). the report of the harvard global health institute and the london school of hygiene and expanding the role of diagnostic and prognostic tools for infectious diseases in resource-poor settings vaccine priority survey the role of public health within the united nations post- framework for disaster reduction impact of natural disasters on mental health coping with the stress of natural hazards key: cord- -ldfa a authors: joung, young hee; park, se hee; moon, ki-beom; jeon, jae-heung; cho, hye-sun; kim, hyun-soon title: the last ten years of advancements in plant-derived recombinant vaccines against hepatitis b date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: ldfa a disease prevention through vaccination is considered to be the greatest contribution to public health over the past century. every year more than million children are vaccinated with the standard world health organization (who)-recommended vaccines including hepatitis b (hepb). hepb is the most serious type of liver infection caused by the hepatitis b virus (hbv), however, it can be prevented by currently available recombinant vaccine, which has an excellent record of safety and effectiveness. to date, recombinant vaccines are produced in many systems of bacteria, yeast, insect, and mammalian and plant cells. among these platforms, the use of plant cells has received considerable attention in terms of intrinsic safety, scalability, and appropriate modification of target proteins. research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over diseases, most frequently hepb and influenza. more inspiring, approximately plant-made antigens have already been tested in clinical trials, with successful outcomes. in this study, the latest information from the last years on plant-derived antigens, especially hepatitis b surface antigen, approaches are reviewed and breakthroughs regarding the weak points are also discussed. hepatitis b (hepb) is an infection with the hepatitis b virus (hbv), which attacks the liver and can cause both acute and chronic disease. the world health organization (who) estimates that million persons are chronically infected with hbv and that more than , people die every year due to complications of hepb, including cirrhosis and liver cancer [ ] . the point that needs the most attention is the high rates of chronic infection found in the sub-saharan africa and east asia, where between % and % of the adult population is infected, as well as in the amazon and the southern parts of eastern and central europe. otherwise, less than % of the population in western europe and north america is chronically infected [ ] . hbv is a hepatotropic dna virus that replicates by reverse transcription. the human hbv is a small circular dna molecule of . kb [ ] . its genome consists of four partially overlapping open reading frames (orfs), namely the envelope gene (pre-surface/surface (pre-s/s)), the core gene (pre-core/core (pre-c/c)), the polymerase gene (pol) and the transactivating protein x (x). the orf pre-s/s encodes pre-s , pre-s and surface (s) proteins that form the surface antigen (hbsag), and hbsag protein is the main antigen to elicit virus-neutralizing and protective antibodies by the immune system [ , ] . understanding the hbv genome and structure is an essential prerequisite for preventive or therapeutic vaccination against hepb. a vaccine against hepb has been available since . this first licensed anti-hbv vaccine containing subviral particles of hbv purified from the inactivated serum of carriers revealed very high efficacy [ ] , and a subsequent subunit vaccine made using the small surface antigen (s-hbsag) was developed in the early s [ ] . the yeast system for the recombinant antigen was to ensure safety and low cost. yeast-derived s-hbsag assembled into virus-like particles (vlps) were as immunogenic as natural subviral particles, and highly effective vaccines containing s-hbsag have been widely used as prophylactic vaccines against hbv infection [ ] . however, some groups of vaccines do not develop protective immunity against the virus and immunosenescence frequently occurs in adults [ ] . additionally, high cost limit and the necessity of accompanying infrastructure for the cold chain distribution and intravenous administration still constituted a barrier to vaccination approaches in developing countries. in order to successfully solve these problems, many research projects have been undertaken to develop more efficacious, easily administrated, and thermostable vaccines. a new recombinant hbv vaccine containing the pre-s/s has greater immunogenic potential than the conventional s antigen-based vaccines in terms of antibody induction and cellular immune response. middle (pre-s + s, m-hbsag) or large (pre-s + pre-s + s, l-hbsag) surface antigens [ ] have been used as components of specific immunotherapeutic vaccines for chronic hbv carriers [ , ] . additionally, chimeric protein created by fusing the hbv core antigen (hbcag) to the pre-s showed strong anti-hbc and moderate anti-pre-s immune responses [ ] . although vaccination is one of the most powerful and cost-competitive achievements, some vaccines may still have certain limitations related to maintenance of the cold chain, downstream processing costs, administration risk, and expensive scalability [ ] [ ] [ ] [ ] [ ] . from these reasons, the use of plant cells as alternative production platforms have received considerable attention in terms of intrinsic safety, scalability, and posttranslational modification of target proteins [ , ] . plant systems can be scaled up quickly to generate large quantities of the protein product, are not susceptible to contamination with known human or mammalian pathogens and are resistant to enzymatic digestion in the gastrointestinal tract. in addition, transgenic plants can be engineered to express and translate multiple proteins concurrently with appropriate folding and assembly into multimeric proteins, especially the posttranslational adjustments of antibodies. not all recombinant antigens will benefit from plant-based systems, but the best production system for each recombinant protein should be chosen using a case-by-case approach [ ] . merlin et al. [ ] proposed that plants are the most the beneficial for the production of four major categories of pharmaceutical proteins: ones that are required in large quantities, that need to be rapid-response, that require complex posttranslational modifications, or that are intended for oral delivery. within these categories, they suggested appropriate candidates to meet a spectrum of research, development, commercial needs, such as human glutamic acid decarboxylase, norwalk virus-like particles, monoclonal antibody g , and human interleukin- . those plant-made antigens have already been tested in clinical trials, with successful outcomes ( table ). the enzyme glucocerebrosidase for gaucher's disease, the first pmf-derived enzyme "elelyso™", has been approved and marketed by protalix in . elelyso™ is based on the use of carrot cells to produce recombinant taliglucerase alpha, which is used in enzyme replacement therapy to treat adult patients. this special food and drug administration (fda) approval case was fast tracked based on its applicability to a rare genetic disease and the bioreactor production under stringent conditions [ , ] . medicago has ongoing phase ii clinical trials for a plant-derived vlp quadrivalent seasonal influenza vaccine and an h pandemic influenza vaccine [ ] . they are focusing on vlp vaccine development. vlps are self-assembled structures derived from viral antigens that mimic the native architecture of viruses but lack the viral genome and thus are not infective. another important advantage as emerging vaccine is the more effective activation of key aspects of the immune response to achieve potent immune stimulation and to provide immunological memory for long-lasting protection [ , ] plant-based platforms including whole plant, organs or cell and expression technology to produce target antigens of interest are diverse [ ] [ ] [ ] . representative plant species expressing the oral vaccine are potato, tomato, and tobacco; additionally, maize, rice, carrot, and soybean are also applied in this field [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . those plants are mainly focused on traditional and usually eaten crops in human, because it is known that inexperienced plants sometimes have problems with certain plant allergies. target antigen proteins were expressed by a plant cell nuclear genome expression system in these plant species. edible plant vaccines are based on different parts of plants, such as fruits, seeds, and root vegetables. such food vaccines are prepared directly without expensive purification of the antigens, which is essentially required for parenteral administration of vaccines [ ] . therefore, the lyophilization of organs expressing stable antigens would facilitate their processing, purification and storage, reducing costs and allowing more practical vaccines. although stable transformation into transgenic plants is commonly accepted, the low production level of the resultant recombinant protein remains an issue of concern. an efficient alternative to nuclear transformation for vaccine antigens and other therapeutic proteins is plastid transformation [ ] . the highest expression of transgenes, up to more than % of total soluble protein, are reported in chloroplast transformation [ , ] otherwise, the universal expression level in most studies has been % of total soluble protein (tsp) or µg/g fresh leaf tissue [ , ] . chloroplast technology can also avoid the controversy related to transgene containment [ ] and express multigenes as single operon [ ] . waheed et al. [ ] reviewed recent vaccine antigens against human diseases expressed via plastid genome since . two plant species, tobacco ( different antigens) and lettuce (four different antigens), have mainly been used in plastid transformation. these results suggest that more industrial interest is needed to strengthen the research/academia-industry linkages in the chloroplast-based vaccine market. stable transformation has its own advantages such as reliable harvest of target proteins over multiple generations and optimized protocols for delivery of foreign genes into various plant species. although there are problems with the time required, seed resources can be grown anywhere with minimal cost and labor once the plant has been developed for the first time [ ] . most clinical trials, except for the three cases of eleyso, prx- , and recombinant human intrinsic factor, have used a tobacco-based transient expression system (table ). in recent years, interest in transient expression has increased due to the containment of the system and the possibility of rapid upscaling due to the short interval between transformation and expression, which are attractive features for the industrial scale production and approval of the expressed products, e.g., the mass production of tobacco by medicago and kentucky bioprocessing. pogue et al. [ ] reviewed plant-based transient expression systems for the production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product. transient expression provides a safe and environmentally friendly system for both indoor and outdoor application with high speed and low cost of the genetic manipulation, rapid manufacturing cycles, and economical production. transient production using an agrobacterium tumefaciens-mediated transfer-dna delivery system (agro-infiltration) and/or virus-based replicating systems, the two dominant approaches, guarantees both the quality of the resulting purified products and the speed of development [ , ] . spiegel et al. [ ] demonstrate the application of the classical nicotiana benthamiana/a. tumefaciens transient expression system to accelerate the development of a malaria vaccine candidate, with screens for expression, solubility, and stability using fluorescent fusion proteins. in marin viegas et al. [ ], a transient expression system for the production of human tg in n. benthamiana leaves was optimized, and the reactivity of plant-produced tg in a cd screening test was evaluated. hence, transient expression performed in contained facilities satisfies good manufacturing practices, and quick expression can avoid the time-consuming stable transformation [ , ] . in a comparison of productivity in terms of biomass production, hiatt and pauly [ ] reported that grams of product may take only two weeks plus a few weeks more. in large-scale biomanufacturing systems, recombinant proteins can be produced at levels of - mg/kg fresh weight tissue in as little as three months [ ] . the transient expression of human interleukin- in n. benthamiana ( . % tsp) produces -fold more than stable expression in tobacco plants ( . mg/g fresh weight (fw)) [ ]. conversely, hgad mut is expressed at higher levels in stable tobacco plants ( . µg/g fw) than in n. benthamiana ( . µg/g fw) [ ] . this result suggests that expression potential or level varies case by case, depending on the target protein. in last decade, there has been a considerable increase in the use of transgenic plants to generate recombinant proteins for medical and veterinary use ( table ) . research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over diseases, most frequently hepatitis b and influenza. in the case of hepatitis b, both stable and transient expression systems have been developed in various plants, including potato, lettuce, tobacco, tomato, carrot, and arabidopsis for stable expression and n. benthamiana for transient expression. a detailed review of hepatitis b will be presented in the next part of this article. influenza is also a main target of this field because it is a widely distributed viral infection of humans and animals, and a new epidemic strain appears every one to two years. this pattern requires the production of new vaccines at the same frequency, and a promising solution is to establish a rapid, flexible, and safe system. the production of various antigens such as hemagglutinin (ha) and the extracellular domain of matrix protein (m e) has mainly focused on transient expression systems using n. benthamiana leaves. using the medicago "proficia™" system, vaccine production can be initiated within less than three weeks from the identification of the genetic sequence of a pandemic or seasonal influenza strain [ ]. vamvaka et al. [ ] reported the development of transgenic rice plant expressing the hiv-neutralizing antibody g in the endosperm ( µg/g dry seed weight) to evaluate the potential of rice seeds as a vehicle for inexpensive microbicide production. production is higher than the initial achievement of maize-derived g ( µg/g) by rademacher et al. [ ] . rubio-infante et al. [ ] demonstrated the immunogenic potential of tobacco chloroplast-derived multi-hiv in an oral immunization scheme and proposed it as a vaccine prototype capable of inducing broad immune responses as it carries various b and t cell epitopes from several hiv strains. dengue has become a significant public health problem, and the threat of dengue fever is now increasing in temperate regions due to dramatic climate change. the rice codon-optimized consensus domain iii of dengue virus envelope glycoprotein (e) has been fused to the m cell-binding peptide via agroinfiltration with a plant virus-based expression system [ ] [ ] [ ] . carrying these results a step further, kim et al. [ ] generated an ebola ric-based denv vaccine in tobacco plants using a geminiviral vector expression system and reported its immunogenic properties as a self-adjuvanting dengue vaccine candidate. previously, phoolcharoen et al. [ ] reported that plant-expressed ebola ric protected mice against a lethal ebola virus challenge. the expression of subunit vaccines for animal viral diseases, such as avian influenza [ , ] , foot-and-mouth disease (fmd) [ ] [ ] [ ] , and diarrhea [ , , ] , which are considered to be the most important causes of economic losses in plants, has been frequently reported. the commercialization of veterinary vaccine is relatively easy compared with that of human vaccines. to date, four cases in clinical trials are ready to enter the market. [ ] suggest that the highly immunogenic vp epitopes produced in n. benthamiana are candidates for a subunit vaccine, specifically for the g p rotavirus strain. the greatest problem of plant-derived vaccine development is the extremely low expression level of the foreign protein in plants. for this reason, many researchers have studied how to improve protein expression levels in plants. in the case of plant-derived hbv vaccines, the first report was on the expression of the small hepatitis b surface antigen (s-hbsag) in transgenic tobacco plants. in this report, the hbsag produced in transgenic tobacco was antigenically and physically similar to the hbsag particles derived from human serum and recombinant yeast [ ] . afterward, many research groups attempted hbsag expression in different tissues and plant species, such as tobacco, potato, lettuce, soybean, lupine, maize, tomato, peanut and laminaria japonica (table ). in the transgenic tobacco plant transformed with the s-hbsag gene controlled by the s promoter, expression levels were very low: less than . % total soluble protein and less than ng/g fresh weight in leaf tissues. the expression levels of s-hbsag in other plant species were not significantly higher; in some species, expression levels were even lower than in tobacco. to improve vaccine production in plants, the most widely used strategies involve: ( ) suitable promoters, such as strong constitutive promoters, tissue-specific promoters and promoters that are inducible by environmental factors; ( ) targeting systems to specific organelles; ( ) optimized codon usage; ( ) alternative polyadenylation signals; ( ) increased translation efficiency using leader sequences; and ( ) different vector systems. many hbsag-overexpressing transgenic plants have been developed using strong constitutive promoters, such as the s promoter with enhancer [ ] [ ] [ ] . in addition to tissue-specific promoters, the patatin promoter for potato tuber [ , ] , globulin promoter for maize seed [ ] and fruit-specific promoters [ , ] were used. specific organelle-, endoplasmic reticulum (er)-, vacuole-and chloroplast-targeted strategies have also been tried [ , ] . the hbsag has been expressed in non-edible plants, such as tobacco, using four different expression cassettes: the hbsag gene without er retention signal (hbs), the hbsag gene with er retention signal (her), and each gene controlled by the ubiquitin promoter (ubq) or ethylene forming enzyme promoter (efe) [ ] . in this report, the maximum expression level ( . ng/g fw of leaves) was observed in efe-hbs transformed plant growth in vitro, but a higher proportion of the particulate form of the antigen was observed when it was expressed with an er retention signal. the efe promoter is more effective in in vitro-cultured plantlets, whereas the ubq promoter is more effective in greenhouse-grown plantlets. the maximum expression level was µg/g fw in the ubq-her transformed nt-l cell suspension culture [ ] . the expression level was increased up to µg/g fw using hbsag fused with the region from the soybean vegetative storage protein gene and was controlled by a chimeric ocs-mas promoter. upon transformation into a soybean cell culture using the same vector, the maximum expression level was µg/g fw [ ] . soybean cell/transgenic pmsi /eha ubq /er ng/g fw n.a. [ ] banana/transgenic pbi /eha efe er ng/g fw (leaf) n.a. [ ] lupin/transgenic prok/c s/n.a. ng/g fw (callus) oral, igg antibodies in serum (maximum miu/ml) [ ] lupin/transgenic prok/gv , eha , lba s/n.a. . - µg/g fw (callus) n.a. [ ] maize/transgenic n.a./eha xglb /cell wall . % of tsp (seed) n.a. [ ] maize/transgenic n.a./eha glob /cell wall . % of tsp (seed) oral (germ, bioencapsulated), iga and igg antibodies in serum (maximum miu/ml) [ ] maize/transgenic n.a./eha kbglb /cell wall . % of tsp (seed) oral (germ, wafer feeding), iga and igg antibodies in serum [ ] maize/transgenic n.a./eha glob /cell wall . % of tsp (seed) oral (germ, wafer feeding), iga and igg antibodies in serum [ ] cherry tomatillo/transgenic pcambia /eha s/n.a. ng/g fw (fruit) oral, igg antibodies in serum [ ] tomato/transient pbi /eha efe/er . µg/g dw (leaf) n.a. [ ] tomato/transgenic pbinplus-ars/lba s/er n.a. n.a. [ ] tomato/transgenic pbm/lba d s/n.a. . %- . % of tsp (leaf) n.a. [ ] tomato/transgenic pbinplus-ars/lba s/n.a. . µg/g dw (fruit) oral, iga and igg antibodies in serum (maximum miu/ml) [ ] carrot cell/transgenic ppcv /n.a. mas/n.a. ng/g fw n.a. [ ] laminaria japonica/transgenic pcat/n.a. sv /n.a. . %- . % of tsp n.a. [ ] peanut oral, igg antibodies in serum (maximum miu/ml), oral, iga and igg antibodies in serum (maximum miu/ml) [ , ] lettuce/transgenic pgptv-bar/eha s/n.a. - µg/g fw n.a. [ ] tomato/transgenic pbinplus-ars/lba s/n.a. . % of tsp (fruit) n.a. [ ] tomato/transgenic pbinplus-ars/aglo s/n.a. . %- . % of tsp (fruit) oral (freeze-dried material), igg antibodies in serum [ , ] carrot/transgenic pbinplus-ars/n.a. s/er ng/g fw (leaf) n.a. [ ] l-hbsag hbsag has been expressed in vegetative crops, such as potato, tomato, soybean and lettuce. the expression level of transgenic potato tubers was - µg/g fw. the highest expression in a tuber was developed using a construct driven by the camv s promoter with dual enhancers, the tobacco etch virus -utr, and the region from the soybean vegetative storage protein gene [ ] . expression level of hbv-protein in potato was little increased when controlled by the tuber specific promoter [ ] . target dna is inserted into a genomic dna as a random event when a using agrobacterium-mediated transformation. for this reason, it is difficult to conclude what is the best method for increase of hbv-protein expression level because differences in the expression level between the transgenic lines even with the same vector construction. expression in tomato fruit has been reported at . µg/g dry weight. to achieve a higher level of expression, several strong and inducible promoters, such as the enhanced dual s, ubq and efe promoters, were tested, as well as organelle targeting sequences. the greatest improvement resulted from the hbsag gene with an er retention signal controlled by efe promoter [ ] . sunil kumar et al. [ ] reported hbsag transformation in banana. the maximum expression in banana leaves has been reported at ng/g fw. the expression levels in banana fruits were not presented in this report, but the expression level was presumably lower than in the leaf tissue. as with leafy vegetables, a variety of expression technologies have not yet been applied. in lettuce, the maximum expression level was µg/g fw, which is the maximum anti-hbsag antibody titer of miu in immunized mice serum [ , , ] . upon transformation into a soybean cell culture using a construct of hbsag fused with the region from the soybean vegetative storage protein gene and as controlled by a chimeric ocs-mas promoter, the maximum expression level was µg/g fw [ ] . grains are a further option for the expression of candidate vaccine antigens. they have long stability of expressed recombinant proteins with low water content [ ] . in maize seed, the maximum expression has been reported at . % of total soluble protein (approximately µg/g fw). this level of expression was achieved using a barley alpha amylase signal sequence-fused s-hbsag gene with a × globulin promoter [ ] . all of the results suggest that the expression levels of hbsag are highly variable and depend on plant species, tissue types and culture conditions. the major recombinant hepatitis b vaccines contain s-hbsag; therefore, the expression of this protein has been the focus in plants. the proteins pres -s, m-hbsag, and pres -pres -s, l-hbsag, have been much less studied than s-hbsag. m-hbsag and l-hbsag have been transformed into potato, tomato, and tobacco (table ) . although the expression was optimized using suitable promoters, leader sequences and targeting signals, the expression levels of m-/l-hbsag were lower than for s-hbsag. however, hbcag induces a heightened immune response [ , ] and spontaneously assembles into capsid-like particles [ ] . for these reasons, efforts devoted to the production of an anti-hbv vaccine have focused on hbcag in the last few years. especially, hbcag has been abundantly produced using transient expression systems mediated by icon binary vectors [ ] or viral vector systems [ ] (table ). transgenic tobacco plants-derived hbsag was antigenically and physically similar to the human serum and recombinant yeast derived-hbsag particles [ ] . to analyze the immunological response in vivo, tobacco-expressed hbsag was purified and injected into balb/c mice. the anti-hepb response to the tobacco-derived hbsag was qualitatively similar to the response obtained by immunizing mice with commercialized yeast-derived hbsag vaccine [ ] . these results showed a possibility of developing injected vaccines using plant-expressed hbsag. due to differences of the manufacturing processes between companies, the amount of hbsag protein per dose differs among the various hbv vaccine products [ ] . for this reason, there is no international standard for the hbsag protein quantity in vaccines, but there is a standard based on protective efficacy of vaccination related to the anti-hepb antibodies induction. an anti-hbsag of ≥ miu/ml measured - months after the last dose of the vaccine are considered to be immune to hepb. although no international standard of antigen concentration is defined, considering the feasibility and cost-effectiveness of the injected vaccine, the concentration of antigen should be over µg/ml [ ] . despite many attempts to increase hbsag expression in transgenic plants, the expression level remains too low for use as an injected vaccine. the currently used hbv vaccine contains hbsag and is produced by yeast cells. the yeast-derived hbv vaccine can be supplied inexpensively ($ - per single dose [ ] ); therefore, it is difficult for plant-derived vaccines to have a competitive price. however, plant suspension cultures may be used as an alternative to yeast to produce antigens for purification. expression levels have approached µg/g fw ( mg/l culture medium) in transgenic soybean culture [ ] . although the expression level ( µg/g fw) was lower in transgenic tobacco cell suspension culture than in soybean [ ] , the former has been used to secrete hbsag into the culture medium, with a six-fold increase in secretion in response to jasmonic acid or salicylic acid treatment during cell culture, and the amount of antigen secreted was µg/l medium [ ] . another breakthrough regarding expression problems was achieved through the utilization of virus-based transient expression systems for the robust production of hbv antigens, such as s-hbsag and hbcag, with yields as high as mg/g fw [ , , ] . tobacco-derived proteins showing the maximum anti-hbsag antibody titer of miu in immunized mice serum [ ] are preferred the application of injection after purification process, rather than oral administration in order to remove many toxic alkaloids and phenolic substances which have a tobacco plant [ ] . however, improvements of several orders of magnitude are still needed for plant cell culture systems to be competitive, particularly given the slow growth rates of plant cells compared with yeast. in transgenic suspension cell culture, the formation of vlps by hbv antigens made it possible to exploit relatively inexpensive protein purification techniques, such as the sucrose gradient [ , , ] or cesium chloride gradient ultracentrifugation [ ] . the highest expressed soybean cell culture was used for antigen purification, and the antigen was suitable for injection [ ] , but the yield remained unsatisfactory and was not cost-effective. the biggest advantage of edible plant-derived vaccines is their easy application to oral delivery. the benefits of plant-derived edible vaccines are as follows: ( ) during oral delivery, plant-derived vaccines are protected in the stomach by plant cell wall and slow release in the gut; ( ) the plant tissue expressing antigen may be used as raw or dried food; ( ) capsules can also be made from partially or fully purified vaccine proteins; ( ) no need for cold chain systems for storage and delivery of the plant tissues or extracts; and ( ) the plant-derived vaccines are cost efficient compared with traditional vaccines. edible plant-derived hbv antigens have been administered by oral injection or feeding in mice with/without adjuvants [ , [ ] [ ] [ ] ]. an oral vaccine candidate has also been administered to human volunteers in small-scale clinical trials without adjuvants. the first trial was administered to three human volunteers in row lettuce leaves in two doses ( . - µg of s-hbsag/dose) without the use of an adjuvant. all volunteers responded, with two of them having serum responses in excess of the protective minimum level ( miu/ml of serum). however, the antibody levels declined rapidly [ , ] . in the second trial, previously vaccinated human volunteers were fed two or three doses of g of raw potato tubers (approximately mg of the s-hbsag/dose). more than half of the subjects showed increased antibody titers [ ] . the animal experiments and trials showed the potential for plant-derived hbv antigens to be used as an oral vaccine for the prevention of hbv, but there remain many problems to be solved for practical application, such as the administration of bulky plant material, declining long-term responses, individual differences in the immune response and the difficulty of defining the antigen dose [ ] . the expression level of plant-derived hbv antigen is only / - / of the expression of yeast-derived hbv antigen; however, the expression yield and plant production scale are still increasing [ , ] . tomato is possible intake without any processing or cooking. therefore, tomato fruit is a very attractive crop to develop an oral vaccine. according to the study to date, the expression level of hbv antigen was very low as ng/g fw ( table ). the maximum titers of anti-hbsag antibody in serum is miu using oral application. this antibody yield was high compared to the expression level of hbv antigen in tomato fruits [ ] . hbv antigen expression in maize produced much higher levels of antigen, and the palatability and digestibility were better than for potato. that is, cereal crops can easily transport or storage in dry state. in addition, the maize system induced a strong immune response with miu of maximum titer by both injection and oral administration [ , ] . this result suggests the possibility of providing a raw material for thermostable formulation at $ . per dose [ ] . plant components such as saponin, flavonoids, and plant oils also function as adjuvants [ ] [ ] [ ] and help maintain the immune response in the long term [ ] . the lyophilization method is an excellent way to increase the stability and shelf life of the plant-derived vaccines. in the previous study, the storage stability of lyophilized powder form was limited at • c [ ] . in a recent study, successful long-term storage at • c was achieved though improvements in the process [ ] . it is easier to control the concentration and standardize antigen doses and process the antigen into a tablet or capsule form using a powdered tissue instead of freeze-drying [ ] . despite over years of effort, no commercial plant-based anti-hbv vaccine has been developed. to commercialize a plant-derived hbv vaccine, several points should be considered. first, the greatest barrier is the low expression levels of hbv antigen in plants; however, expression yield and plant production scale can still be increased using plant expression vector optimization, which should be focused on the target plant. the process can also be more competitive by improving the plant-derived antigen to increase the immune response to the vaccine. second, an hbv antigen expressed in an edible plant has the advantage of being usable as an oral vaccine without processing. it is first necessary to analyze the characteristics of the target plants and the expressed protein for the development of an oral vaccine because plant components, secondary metabolites and foreign protein expression characteristics vary with plant species. to obtain feasible and cost-effective vaccines, the target plants for edible vaccines should have a long shelf life, be heat stable and be edible as a raw material. candidate grain crops are maize and rice; candidate vegetative crops are tomato and banana. third, consideration should be made of the public's acceptance of gm crops, especially plant-derived edible vaccines. for injected vaccine development, the most cost-effective method is a suspension culture in a closed environment, according to the regulations of good manufacturing practice. further safety of plant-derived vaccines can be obtained by following the same regulations established for traditional vaccines. in addition, for oral vaccines produced from gm crops, environmental risk assessment and human risk assessment should be performed. for these reasons, plant-derived oral vaccines cannot be called cost efficient compared with traditional vaccines, and the current concern over the use of gm plants is now affecting research in this field. world health organization (who) compact organization of the hepatitis b virus genome. hepatotlogy structural and functional analysis of full-length hepatitis b virus genomes in patients: implications in pathogenesis overview of hepatitis b virus mutations and their implications in the management of infection the newly licensed hepatitis b vaccine: characterics and indications for use human hepatitis b vaccine from recombinant yeast recombinant hepatitis b triple antigen vaccine: hepacare comparative immunogenicity of a pres/s hepatitis b vaccine in non-and low responders to conventional vaccine hepatitis b virus morphogenesis specific vaccine therapy in chronic hepatitis b: induction of t cell proliferative responses specific for envelope antigens induction of strong hepatitis b virus (hbv) specific t helper cell and cytotoxic t lymphocyte responses by therapeutic vaccination in the trimera mouse model of chronic hbv infection recombinant hepatitis b core antigen carrying pres epitopes induce immune response against chronic hbv infection medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants biological safety concepts of genetically modified live bacterial vaccines role of genetic factors and environmental conditions in recombinant protein production for molecular farming evolution of plant-made pharmaceuticals a perspective on the use of pleurotus for the development of convenient fungi-made oral subunit vaccines immunological aspects of using plant cells as delivery vehicles for oral vaccines comparative evaluation of recombinant protein production in different biofactories: the green perspective carrot cells: a pioneering platform for biopharmaceuticals production plants as factories for human pharmaceuticals: applications and challenges molecular pharming-vlps made in plants heat-precipitation allows the efficient purification of a functional plant-derived malaria transmission-blocking vaccine candidate fusion protein malaria vaccine candidate antigen targeting the pre-erythrocytic stage of plasmodium falciparum produced at high level in plants plant-based production of recombinant plasmodium surface protein pf and evaluation of its potential as a vaccine candidate a plant-produced pfs vlp malaria vaccine candidate induces persistent transmission blocking antibodies against plasmodium falciparum in immunized mice transgenic tomato plants expressing the antigen gene pfcp- . of plasmodium falciparum production, characterisation and immunogenicity of a plant-made plasmodium antigen the kda c-terminal fragment of plasmodium yoelii merozoite surface protein a plant-produced pfs vaccine candidate blocks transmission of plasmodium falciparum production and characterization of an orally immunogenic plasmodium antigen in plants using a virus-based expression system rice endosperm produces an underglycosylated and potent form of the hiv-neutralizing monoclonal antibody g a plant-derived multi-hiv antigen induces broad immune responses in orally immunized mice oral delivery of plant-derived hiv- p antigen in low doses shows a superior priming effect in mice compared to high doses chloroplast expression of an hiv envelop-derived multiepitope protein: towards a multivalent plant-based vaccine biochemical and immunological characterization of the plant-derived candidate hiv- mucosal vaccine ctb-mpr anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plants expression of hiv- antigens in plants as potential subunit vaccines rapid high-yield expression of a candidate influenza vaccine based on the ectodomain of m protein linked to flagellin in plants using viral vectors high-yield expression of m e peptide of avian influenza virus h n in transgenic duckweed plants oral immunization of haemaggulutinin h expressed in plant endoplasmic reticulum with adjuvant saponin protects mice against highly pathogenic avian influenza a virus infection intranasal vaccination with a plant-derived h ha vaccine protects mice and ferrets against highly pathogenic avian influenza virus challenge. hum. vaccines immunother biochemical composition of haemagglutinin-based influenza virus-like particle vaccine produced by transient expression in tobacco plants plant-derived h vlp vaccine elicits protective immune response against h n influenza virus in mice and ferrets safety and immunogenicity of a plant-produced recombinant monomer hemagglutinin-based influenza vaccine derived from influenza a (h n ) pdm virus: a phase i dose-escalation study in healthy adults human antibody response to n-glycans present on plant-made influenza virus-like particle (vlp) vaccines. vaccine a plant-based system for rapid production of influenza vaccine antigens. influenza other respir the human potential of a recombinant pandemic influenza vaccine produced in tobacco plants setting up a platform for plant-based influenza virus vaccine production in south africa formulation development of a plant-derived h n influenza vaccine containing purified recombinant hemagglutinin antigen. hum. vaccines immunother plant-produced recombinant influenza vaccine based on virus-like hbc particles carrying an extracellular domain of m protein preclinical and clinical development of plant-made virus-like particle vaccine against avian h n influenza a plant-produced influenza subunit vaccine protects ferrets against virus challenge. influenza other respir plant-expressed ha as a seasonal influenza vaccine candidate highly pathogenic avian influenza as a zoonotic agent transient expression of the ectodomain of matrix protein (m e) of avian influenza a virus in plants a transgenic plant cell-suspension system for expression of epitopes on chimeric bamboo mosaic virus particles development of a new vector using soybean yellow common mosaic virus for gene function study or heterologous protein expression in soybeans expression of vp protein of serotype a and o of foot-and-mouth disease virus in transgenic sunnhemp plants and its immunogenicity for guinea pigs immunogenicity of foot-and-mouth disease virus structural polyprotein p expressed in transgenic rice foliar extracts from transgenic tomato plants expressing the structural polyprotein, p - a, and protease, c, from foot-and-mouth disease virus elicit a protective response in guinea pigs induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes optimization of ammonium sulfate concentration for purification of colorectal cancer vaccine candidate recombinant protein ga -fck isolated from plants optimization of elisa conditions to quantify colorectal cancer antigen-antibody complex protein (ga -fck) expressed in transgenic plant expression of ga -fc fusion protein as a vaccine candidate for colorectal cancer in transgenic plants plant-derived epcam antigen induces protective anti-cancer response apple latent spherical virus vector as vaccine for the prevention and treatment of mosaic diseases in pea, broad bean, and eustoma plants by bean yellow mosaic virus generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine a plant based protective antigen [pa (div)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax recombinant plants provide a new approach to the production of bacterial polysaccharide for vaccines production of a plant-derived immunogenic protein targeting apob and cetp: toward a plant-based atherosclerosis vaccine a plant-produced antigen elicits potent immune responses against west nile virus in mice suppression of inhibitor formation against fviii in a murine model of hemophilia a by oral delivery of antigens bioencapsulated in plant cells effects of plant cultivation density and light intensity on the production of a vaccine against swine edema disease in transgenic lettuce production of double repeated b subunit of shiga toxin e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease immunogenicity of eit chimeric protein expressed in transplastomic tobacco plants towards development of an oral vaccine against escherichia coli o :h induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin b subunit vaccine without plant-associated sugar modification oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (as ) of ascaris suum fused with cholera toxin b subunit synthesis and assembly of an adjuvanted porphyromonas gingivalis fimbrial antigen fusion protein in plants transient expression of vp in nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus vp antigen produced in tobacco transplastomic plants confers protection against bovine rotavirus infection in a suckling mouse model greenhouse and field cultivations of antigen-expressing potatoes focusing on the variability in plant constituents and antigen expression oral administration of plant-based rotavirus vp induces antigen-specific igas, iggs and passive protection in mice efficacy of a bvdv subunit vaccine produced in alfalfa transgenic plants immunocompetent truncated e glycoprotein of bovine viral diarrhea virus (bvdv) expressed in nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines the effect of plant tissue and vaccine formulation on the oral immunogenicity of a model plant-made antigen in sheep use of the wound-inducible ntqpt promoter from nicotiana tabacum for production of a plant-made vaccine the release and induced immune responses of a plant-made and delivered antigen in the mouse gut plant expressed coccidial antigens as potential vaccine candidates in protecting chicken against coccidiosis immunogenicity study of plant-made oral subunit vaccine against porcine reproductive and respiratory syndrome virus (prrsv) extraction, purification and characterization of the plant-produced hpv subunit vaccine candidate e ggg plastid expression of a double-pentameric vaccine candidate containing human papillomavirus- l antigen fused with ltb as adjuvant: transplastomic plants show pleiotropic phenotypes anti-cancer activity of plant-produced hpv e vaccine production of human rotavirus and salmonella antigens in plants and elicitation of fljb-specific humoral responses in mice piri, i. isolation of ompa gene from salmonella typhimurium and transformation into alfalfa in order to develop an edible plant based vaccine expression of helicobacter pylori tonb protein in transgenic arabidopsis thaliana: toward production of vaccine antigens in plants expression of an immunogenic f -v fusion protein in lettuce as a plant-based vaccine against plague biopharmaceutical protein production under controlled environments: growth, development, and vaccine productivity of transgenic tomato plants grown hydroponically in a greenhouse plant-derived recombinant fl, v, and f -v fusion antigens of yersinia pestis activate human cells of the innate and adaptive immune system effective plague vaccination via oral delivery of plant cells expressing f -v antigens in chloroplasts a plant-produced plague vaccine candidate confers protection to monkeys plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice expression and immunogenicity of the mycobacterial ag b/esat- antigens produced in transgenic plants by elastin-like peptide fusion strategy superexpression of tuberculosis antigens in plant leaves oral immunogenicity of a plant-made, subunit, tuberculosis vaccine continued expression of plant-made vaccines following long-term cryopreservation of antigen-expressing tobacco cell cultures toward the development of a plant-based vaccine against reovirus generation of plant-derived recombinant dtp subunit vaccine analysis and stability of the respiratory syncytial virus antigen in a t generation of transgenic tomato plants immunogenic properties of plant-derived recombinant smallpox vaccine candidate pb accumulation of recombinant sars-cov spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines clinical safety and immunogenicity of tumor-targeted, plant-made id-klh conjugate vaccines for follicular lymphoma recombinant antibody g produced in maize endosperm efficiently neutralizes hiv- and contains predominantly single-glcnac n-glycans m cell-targeting ligand and consensus dengue virus envelope protein domain iii fusion protein production in transgenic rice calli expression of a cholera toxin b subunit and consensus dengue virus envelope protein domain iii fusion gene in transgenic rice callus novel vaccination approach for dengue infection based on recombinant immune complex universal platform a nonreplicating subunit vaccine protects mice against lethal ebola virus challenge immunization with plant expressed hemagglutinin protects chickens from lethal highly pathogenic avian influenza virus h n challenge infection engineering and expression of a human rotavirus candidate vaccine in nicotiana benthamiana expression of hepatitis b surface antigen in transgenic plants expression and characterization of hepatitis b surface antigen in transgenic potato plants production of hepatitis b surface antigen in transgenic plants for oral immunization oral immunization with hepatitis b surface antigen expressed in transgenic plants expression of the hepatitis b surface s and pres antigens in tubers of solanum tuberosum production of highly concentrated, heat-stable hepatitis b surface antigen in maize transient and stable expression of hepatitis b surface antigen in tomato (lycopersicon esculentum l.) expression of hepatitis b surface antigen in transgenic banana plants expression of the human hepatitis b virus large surface antigen gene in transgenic tomato plants hepatitis b surface expression in transgenic tobacco (nicotiana tabacum) plants using four different expression cassettes expression of hepatitis b surface antigen in transgenic banana plants and nt- cell line of tobacco hepatitis b surface antigen (hbsag) expression in plant cell culture: kinetics of antigen accumulation in batch culture and its intracellular form immunogenicity of transgenic plant-derived hepatitis b surface antigen analysis of transgenic tobacco plants carrying the gene for the surface antigen of the hepatitis b virus immunogenicity of parenterally delivered plant-derived small and medium surface antigens of hepatitis b virus oral administration of low doses of plant-based hbsag induced antigen-specific igas and iggs in mice, without increasing levels of regulatory t cells tissue specific expression of hepatitis b virus surface antigen in transgenic plant cells and tissue culture immunogenicity and tolerance following hiv- /hbv plant-based oral vaccine administration production of marker-free plants expressing the gene of the hepatitis b virus surface antigen virus-like particles expression and assembly in plants: hepatitis b and norwalk viruses secretion of hepatitis b surface antigen in transformed tobacco cell suspension cultures a plant signal peptide-hepatitis b surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells process options in hepatitis b surface antigen extraction from transgenic potato study of the immunogenicity of hepatitis b surface antigen synthesized in transgenic potato plants with increased biosafety a plant-derived edible vaccine against hepatitis b virus low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis b surface antigen for prototype plant-derived vaccine tablet formulation freeze-drying of plant tissue containing hbv surface antigen for the oral vaccine against hepatitis b analysis of the limitations of hepatitis b surface antigen expression in soybean cell suspension cultures obtaining tomato plants transgenic for the pres -s-hdel gene, which synthesize the major hepatitis b surface antigen agrobacterium tumefaciens-mediated transformation of yellow lupin to generate callus tissue producing surface antigen of hbv in a long-term culture bioencapsulation of the hepatitis b surface antigen and its use as an effective oral immunogen supercritical fluid extraction provides an enhncement to the immune response for orally-delivered hepatitis b surface antigen oral delivery of wafers made from hbsag-expressing maize germ induces long-term immunological systemic and mucosal responses oral immunization of animals with transgenic cherry tomatillo expressing hbsag construction of a plant effective expression vector containing the gene of hepatitis b virus surface antigen the integration of a major hepatitis b virus gene into cell-cycle synchronized carrot cell suspension cultures and its expression in regenerated carrot plants expression of hepatitis b surface antigen gene (hbsag) in laminaria japonica (laminariales, phaeophyta) transforming hbsag into peanut and detection of its immunogenecity plant expression, lyophilisation and storage of hbv medium and large surface antigens for a prototype oral vaccine formulation oral immunogenicity of potato-derived hbsag middle protein in balb/c mice retention of the ability to synthesize hiv- and hbv antigens in generations of tomato plants transgenic for the tbi-hbs gene synthesis of hepatitis b virus surface antigen in tomato plants transgenic for the pres -s gene candidate mucosal vaccine against hepatitis b based on tomatoes transgenic for the pres -s gene comparative analysis of hbv m-antigen production in leaves of individual transgenic carrot plants application of the human hepatitis b virus core antigen from transgenic tobacco plants for serological diagnosis high-level production of hepatitis b core antigen in plant leaf and its immunogenicity in mice extremely high-level and rapid transient protein production in plants without the use of viral replication a dna replicon system for rapid high-level production of virus-like particles in plants tandem fusion of hepatitis b core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins seed-based expression systems for plant molecular farming the use of viral vectors to produce hepatitis b virus core particles in plants hbv core particles as a carrier for b cell/t cell epitopes envelopment of the hepatitis b virus nucleocapsid weekly epidemiological record; hepatitis b position paper world health organization (who) toward the global elimination of hepatitis b virus transmission optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants plant-based edible vaccine against hbv oral immunization of human with transgenic lettuce expressing hepatitis b surface antigen the twenty-year story of a plant-based vaccine against hepatitis b: stagnation or promising prospects? recombinant hepatitis b vaccines: disease characterization and vaccine production. in production of recombinant proteins. novel microbial and eukaryotic systems plants as bioreactors for the production of vaccine antigens immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis b and human immunodeficiency viruses saponin-adjuvanted particulate vaccines for clinical use immunomodulatory properties of vitamins, flavonoids and plant oils and their potential as vaccine adjuvants and delivery systems activation of antigen-presenting cells by immunostimulatory plant dna: a natural resource for potential adjuvant stability of s-hbsag in long-term stored lyophilised plant tissue key: cord- -qautse a authors: liu, li; morrow, matthew p.; bagarazzi, mark title: clinical use of dna vaccines date: - - journal: handbook of electroporation doi: . / - - - - _ sha: doc_id: cord_uid: qautse a owing to their unique advantages in simplicity, safety, scalability, and possibility of repeated administrations, dna vaccines represent an appealing and competitive immunization approach for a wide array of conditions, including but not limited to infectious diseases and cancer immunotherapy. despite the exciting efficacy observed in preclinical studies, dna vaccines have faced challenges in inducing strong immune responses in humans. this unexpected poor immunogenicity has severely hampered the translation of dna vaccines from investigational medications to licensed products. to overcome this obstacle, tremendous efforts have been made to improve antigen expression and enhance immunogenicity. among these endeavors, in vivo dna electroporation (ep) has proved to be a breakthrough technology capable of mediating efficient dna uptake and resulting in enhanced antigen expression and vaccine immunogenicity. ep-mediated dna delivery has become one of the major platforms used in clinical trials to evaluate dna vaccines in humans. in this chapter, in addition to ep delivery, other progress made in dna vaccine development including plasmid optimization, antigen design, and immunologic adjuvants is also reviewed. finally, the use of dna vaccines in the context of clinical trials for infectious diseases and cancer immunotherapy is summarized. specifically, the strategies that allow dna vaccines to overcome antigenic diversity for viral infection and break immune tolerance for cancer therapy are explored. based on the advantages of dna vaccines and the immense progress, led by the electroporation-mediated vaccine delivery, dna vaccines appear to have the potential to fundamentally transform the vaccine field, providing important benefits for preventing and curing diseases. in , wolff and colleagues first demonstrated that long-term gene expression could be achieved simply by direct injection of plasmid dna into mouse skeletal muscle (wolff et al. ). two years later, tang et al. reported that in vivo delivery of plasmid dna encoding human growth hormone (hgh) resulted in the production of detectable levels of hgh in host mice. importantly, the inoculated mice developed anti-hgh antibodies, suggesting the immunological use of dna (tang et al. ) . these pioneering observations engendered great enthusiasm for using dna immunization against infectious diseases, as well as in the applications of gene therapy. the immunological potential of dna was confirmed by several independent preclinical studies demonstrating that dna immunization could result in protection from infectious diseases in immunized animals (moss ). the action of dna immunization involves intramuscular or subcutaneous injection of a dna plasmid encoding the antigen of interest expressed under the control of a eukaryotic promoter. upon administration of the plasmid dna, the in vivo expressed antigen can stimulate the host immune system to elicit a response. as a third generation platform, dna vaccination has distinct advantages over other vaccine approaches. dna vaccines can induce both humoral and cellular immune responses making them distinct from conventional inactivated vaccines and subunit vaccines. in addition, plasmid dna has a number of unmethylated cpg motifs, due to its bacterial origin, that are capable of stimulating innate immune responses via toll-like receptor (tlr ). the ability to activate all arms of the immune system (b cells, t h cells, and cytotoxic t cells) means the dna vaccine platform has the potential for not only prophylactic but also therapeutic applications. unlike the traditional prophylactic vaccines that prevent infections from occurring, therapeutic dna vaccines can be used as an immunotherapy to treat chronic viral infections and cancers by direct killing of latently infected cells and mutated tumor cells. dna vaccines have also been shown to have a benign safety profile. the injected plasmid only encodes a selected gene from a pathogen, eliminating risk of pathogenic infection or reversion to virulence, a safety concern that is associated with live attenuated vaccines. importantly, as plasmid dna vaccines are noninfectious, the elicited immune responses are solely specific to the antigen encoded by the transgene rather than to the plasmid itself. as a result, no anti-dna vector responses have been observed, thereby allowing for repeated vaccine administration without weakening the specific immune response. this feature is critical for the prime and boost regimen, a commonly used approach for enhancing vaccine immunogenicity. another advantage that makes dna vaccines a competitive option is its ease of design and manufacture. the advancement of gene synthesis and recombinant dna technology allows for flexible and rapid alterations of the expressed immunogen. large-scale production of plasmids can be completed within a short period of time, making dna vaccines particularly suitable for responding to pandemic outbreaks. finally, dna vaccines are more stable and resistant to temperature extremes than other vaccine forms which are of great benefit for storage, transport, and distribution especially in resource-limited regions. despite all the positive characteristics and the superb efficacy observed in preclinical studies, dna vaccines have been slow to demonstrate similar success in humans. the unforeseen poor immunogenicity observed in most early clinical trials was disappointing and poses a formidable challenge for dna vaccine licensure. however, several dna vaccines have been approved for animal health. the first one, developed by fort dodge laboratories against west nile virus infection in horses was approved by the us department of agriculture (usda) in . a second dna vaccine (apex-ihn, novartis animal health) was licensed in canada for prevention of hematopoietic necrosis virus infection in farmed salmon and trout. in , a therapeutic cancer vaccine (oncept, merial) designed to treat dogs with melanoma was approved by the usda. finally, in , an injectable dna plasmid (lifetide sw , vgx animal health) encoding porcine growth hormone releasing hormone (ghrh) was licensed in australia as a gene therapy for use in breeding age sows to increase the number of piglets in litters. the approval of these plasmids as commercial products in veterinary practice is very encouraging and sheds lights on human dna vaccine development. for example, the licensed therapeutic vaccine for canine malignant melanoma was developed by using human tyrosinase (a melanoma associated antigen) as the immunogen to immunize dogs. the human tyrosinase elicited a strong immune response against dog melanoma and significantly prolonged the lives of dogs with advanced disease (bergman et al. ) . the success of this vaccine supported the concept of using a xenogeneic protein to break central immune tolerance for cancer vaccine development (liu ) . additional insight gained from the success of dna vaccines in veterinary practice is that the dna scaling-up might not be a potential hindrance for dna vaccine being effective in humans. it has been extrapolated that an impractically high dose of dna vaccine would be required for use in human subjects in order to reproduce the vaccine efficacy obtained in mice (tregoning and kinnear ) . however, the clinical benefits observed in large animals, including horses and pigs, suggest that the size of humans may not be the reason for the various disappointing clinical results (liu ) . in light of the aforementioned advantages of dna vaccines and encouraged by their success in veterinary medicine, great efforts have been made to translate dna immunization into the clinic, with novel strategies being explored to enhance immunogenicity. since the first human study in (calarota et al. ) , numerous clinical trials with dna vaccines targeting a wide spectrum of diseases have been performed or are currently underway. in the following sections, various aspects related to dna vaccine development including its working mechanisms, route of delivery, advances being made in optimizing dna vaccine immunogenicity, and their usage in the applications of infectious diseases and cancer treatment will be addressed. upon intramuscular or intradermal injection, plasmid dna is internalized by myocytes or keratinocytes located at the site of vaccine administration. the plasmid encoded antigen is then expressed intracellularly using host cell machinery. however, since myocytes and keratinocytes are not professional antigen presenting cells (apc) and do not express co-stimulatory molecules, they are unable to productively stimulate cytotoxic t lymphocytes (ctl) activation. in order to prime a cellular response, the intracellularly produced antigens must be released into extracellular matrix either by secretion or by cell lysis. the released antigens are then engulfed by apcs and loaded onto major histocompatibility complex (mhc) class i molecules leading to activation of cd + t cell. this uptake mechanism is different from that which occurs with exogenous proteins and is thus termed as cross-priming (liu ) . dna plasmids can also directly transfect apcs that have migrated to the injection site. after being expressed in apcs, the antigens are cleaved to peptides and uploaded onto mhc i or ii molecules, leading to activation of both cd + ctls and cd + t h cells. additionally, antigens released from myocytes and keratinocytes can be captured by b lymphocytes leading to activation of humoral immunity. in addition to the stimulation of the adaptive immune system, dna plasmids can also trigger activation of innate immunity. plasmids derived from bacteria contain hypomethylated cpg dinucleotides motifs, which are immunostimulatory and rarely seen in eukaryotes. the cpg motifs bind to toll-like receptor (tlr ), a transmembrane protein found on a number of immune cells including dendritic cells (dc), b cells, and natural killer (nk) cells. activation of tlr leads to a cascade of pro-inflammatory responses and results in the production of various cytokines including type i interferon (ifn), interleukin (il)- , il- , and tumor necrosis factor-alpha (tnf-α). in addition to cpg-tlr pathway, the double-stranded structure of plasmids can be detected by dna sensors in cytosol, which triggers activation of the stimulator of interferon genes (sting)/tank-binding kinase (tbk ) pathway, leading to type i interferon (ifn) production. historically, various administration routes including intramuscular, intradermal, subcutaneous, intranasal, vaginal, oral, and intravenous route have been tested (liu ; tregoning and kinnear ) in order to elicit a desired immune response from dna immunization. the selection of the appropriate delivery method is dependent on multiple factors including optimum expression, immunogenicity, feasibility, and tolerability. among the considered delivery approaches, intramuscular (i.m.) injection is the principal route of choice for dna vaccination due to the fact that skeletal muscle is readily accessible and highly effective in taking up dna. furthermore, myocytes are energy rich and postmitotic cells capable of sustaining long-term transgene expression for eliciting durable immunity (aurisicchio et al. ; tregoning and kinnear ) . in addition to i.m. injection, intradermal (i.d.) delivery is also considered to be an effective pathway for vaccination not only because of its greater ease of accessibility but also the prominent presence of large amount of immune cells in the dermis including dendritic cells, langerhans cells, and migrating lymphocytes. both i.m. and i.d. are two major administration routes used in preclinical studies and clinical trials for dna delivery. some studies suggested that the nature of the immune response could be driven by the vaccine administration route. for example, due to the enrichment of antigen presenting cells in skin, the i.d. route is believed to preferentially induce cellular immune responses as a result of direct transfection of apcs. on the other hand, i.m. injection equally induces both humoral and cellular immunity via direct transfection and by the crosspriming mechanism. however, in a recent clinical trial where an hiv-specific dna vaccine was delivered via either an i.m., or i.d. route, no differences in induced immune responses were observed (enama et al. ). after administration, the plasmid dna must successfully overcome a series of barriers before mounting the desired immune response. compared to viral vectors, uptake of naked dna by somatic cells is inefficient; most of the i.m.-injected dna does not actually transfect cells (liu ) . after entering the cell, plasmid dna needs to translocate to the nucleus where dna transcription occurs. however, the efficiency of intracellular movement is very low, with less than . % of plasmid dna that enters the cytosol is eventually transcribed (tregoning and kinnear ) . moreover, prior to arrival at the nucleus, dna in the cytosol is subject to degradation by nuclease digestion. finally, during the process of protein synthesis, mrna translation efficiency can also be modulated by factors such as the presence of a kozak sequence and codon usage. to overcome these obstacles, several approaches focusing on augmenting dna uptake, maximizing protein expression, and enhancing antigen immunogenicity have been developed and tested in clinical trials. several dna delivery methods including gene gun, jet injection, electroporation (ep), cationic polymers, and liposomes have been used in clinical trials. given that the inclusion of extra chemical components may delay the licensure of the vaccine due to concerns over the inflammatory profile of the agents, the device-mediated delivery is considered to be a more universal and preferred approach for enhancing dna uptake (tregoning and kinnear ) . among the device-mediated approaches, ep has the highest transfection efficiency and has achieved the most remarkable progress in dna vaccine delivery (aurisicchio et al. ; best et al. ). the ep technology was initially devised for in vitro transfection (see also ▶ chap. , "gene delivery by electroporation in vitro: mechanisms"). the mechanism of ep involves using controlled electric pulses to open transient pores on cell membrane through which the negatively charged dna molecules gain access to the cytoplasm. the cell membrane is permeabilized for only a short period of time and rapidly closes after electrical treatment terminates, returning the cell to its original state. ep has been proven to be a very powerful technology, resulting in - -fold increases in plasmid uptake and subsequent antigen expression compared to naked dna vaccines (aurisicchio et al. ). furthermore, ep has the additional benefit of causing tissue damage that can lead to local inflammation and release of cytokines (fioretti et al. ) . this adjuvant effect of ep consequently promotes the induction of innate and adaptive immune responses, potentiating the immunogenicity of the delivered dna. the ep technology has been safe and well tolerated (bagarazzi et al. ) with the most common minor side effects observed in clinical trials being local injection-site pain and discomfort associated with short muscle contraction triggered by electric pulses. it is believed that ep represents the most promising technology in dna delivery (aurisicchio et al. ) . recently, the application of i.m. injection followed by ep delivery has become one of the major platforms used in clinical trials to evaluate dna vaccines in humans. however, it should be noted that ep technology has intrinsic limitations. the ep approach requires penetration with several sharp needle-like electrodes into the subject's body followed by introduction of transient electric pulses. as a result, the ep procedure is less convenient, more invasive, and sometimes intimidating, potentially reducing patient compliance compared to simple i.m. injection. furthermore, the ep procedure demands developing practical and affordable devices and providing adequately trained health professionals to administer the vaccine. the immune responses to dna vaccines have been shown to be dependent on antigen expression (tregoning and kinnear ) . therefore, optimizing antigen expression is key to improving vaccine performance. plasmid codon optimization is one such approach to augment expression. it refers to changing the antigen coding sequence, via synonymous substitutions, from the wild type to a selected sequence that has the most abundant trnas in the cytosol of transfected cells. through codon optimization, adverse rare codons are avoided and secondary structures in the mrna are minimized, therefore, protein expression can be increased severalfold. in addition to codon optimization, various manipulations of the dna sequence can be performed to modulate antigen intracellular processing and immune response priming. for example, adding nuclear localization signals to the plasmid helps direct dna to the nucleus for transcription. for the purpose of introducing cellular immunity, ubiquitin coding sequence can be incorporated in the dna vaccine to enhance protein degradation and antigen peptide production in the proteasome (leifert et al. ). if humoral immunity is desired, the antigen coding region can be modified by adding a leader sequence capable of guiding the expressed antigen so that it undergoes proper folding in the endoplasmic reticulum (er), export through the secretory pathway, and release into extracellular matrix for further presentation to b lymphocytes and apcs (fioretti et al. ). due to its bacterial origin, plasmid dna carries a naturally occurring adjuvant. co-injection of antigen-encoding plasmid with an empty vector carrying no insert has been shown to induce stronger immune responses than using the antigen-encoding plasmid alone (liu ) . the enhanced immunogenicity was attributed to the adjuvant effect of the hypomethylated cpg oligodinucleotides that can bind to tlr , thereby stimulating innate immunity and creating an inflammatory milieu for boosting the adaptive immune response against the encoded antigen (liu ) . in an attempt to mimic this adjuvant effect, additional cpg motifs have been used in dna vaccine platforms resulting in enhanced immune responses. alternatively, instead of using incorporated cpg sequence to trigger tlr activation, co-administration of additional plasmid expressing ligands for tlr / / or their signaling molecules has been shown to improve the immunogenicity of dna vaccines (kwissa et al. ) . one disadvantage of using the inflammatory adjuvants is that they boost the vaccine immunogenicity in a sequential fashion, i.e., innate immunity activation followed by adaptive immunity activation. it is possible that the acute inflammation induced by the adjuvant may result in clearance of the transfected cells before sufficient antigen is expressed (tregoning and kinnear ) . to avoid this timing issue, genetic adjuvants have been investigated to enhance vaccine immunogenicity. genetic adjuvants refer to immunoregulatory cytokines and proteins encoded by additional plasmids. the most commonly used genetic adjuvants include il- , il- , il- , granulocyte-macrophage colony-stimulating factor (gm-csf), intercellular adhesion molecule (icam-i), cd , and heat shock protein (hsp ) (fioretti et al. ; tregoning and kinnear ) . the plasmid encoding the genetic adjuvant is normally delivered by co-injection with the plasmid encoding the vaccine. alternatively, the adjuvant transgene can be tailored and encoded in cis with the antigen coding sequence in the same dna vector as well (fioretti et al. ) . in this way, the peak expression of the genetic adjuvant will match antigen expression, leading to a synergistic response. moreover, the effect tends to be more specific than tlr-type adjuvants, which can be reactogenic (tregoning and kinnear ) . conventional licensed influenza (flu) vaccines have traditionally been made by growing the recommended seasonal vaccine strains in eggs followed by inactivation or attenuation. the inactivated or attenuated virus in the vaccine promotes an immune response that can prevent infection from identical or very similar viruses. however, due to antigenic diversity, the annually recommended flu vaccine can become ineffective if new strains emerge. in order to avoid frequent composition adjustment for emerging influenza strains, new vaccine platforms capable of inducing a universal immune response against a broad spectrum of influenza strains are greatly needed. viral surface proteins are the primary targets for prophylactic vaccine development, because of their crucial role in initiation of infection and their prominent display on the virion surface. however, due to host immune pressure, these surface proteins are constantly evolving and have high degrees of variability, making it difficult to develop a universal vaccine. thanks to the versatility and ease of rapid alteration of the expressed immunogen, a dna vaccine strategy has great potential in overcoming influenza antigenic diversity. for example, a novel synthetic consensus sequence technology has been developed to broaden the effectiveness of the vaccine to cover variant strains of influenza displaying antigenically altered hemagglutinin (ha). specifically, using the combination of extensive genetic data and a computer algorithm, the gene sequences encoding the ha protein of a large number of circulating influenza strains were analyzed. subsequently, a new arrangement of the dna sequence is created resulting in an average gene sequence for the ha protein. the synthetic consensus dna sequence is substantially similar to but different from those encoded by naturally occurring circulating variants. upon delivery of the synthetic consensus plasmid dna, the in vivo expressed, artificial, and unmatched ha led to generation of neutralizing antibodies capable of recognizing a wide variety of related strains (choo et al. ) . the ability of synthetic consensus plasmid-generated h n dna vaccine in cross-protection has been tested in ferrets, in which the sera derived from immunized animals exhibited protective neutralization against all four subclades of h n ; this protection level had not been attained previously with strain-matched influenza vaccine candidates (choo et al. ) . a synthetic consensus dna vaccine targeting h and h variant strains of influenza has been tested in a phase i clinical trial (nct ). it is well known that the vaccine-induced influenza neutralizing antibodies invariably target the globular head region of influenza hemagglutinin (ha) and are largely strain specific. on the other hand, a localized region in the ha stem was found to be structurally conserved among type a influenza viruses; antibodies specific to this region are broadly neutralizing. although the ha stem-specific antibodies have been identified in humans, it has not been possible to specifically elicit them through vaccination. a prime-boost strategy involving dna vaccination has been developed and shown to be able to induce broadly neutralizing antibodies (wei et al. ) . specifically, vaccination with plasmid dna encoding h n influenza hemagglutinin and boosting with seasonal vaccine or replication-defective adenovirus vector encoding ha stimulated the production of broadly neutralizing antibodies specific to the conserved stem region. this prime-boost combination increased the neutralization of diverse h n strains dating from to and offered cross-protection against divergent h n viruses in mice and ferrets (wei et al. ). based on these results, the prime-boost strategy was further tested in a human study (nct ) to evaluate its ability in stimulating broadly neutralizing antibodies (ledgerwood et al. ) . it was found that h dna priming with a -week monovalent inactivated vaccine (miv) boost interval induced protective hemagglutination inhibition (hai) titers in % of individuals. moreover, as compared to another phase i trial (nct ), where an h n miv vaccine was used as the priming immunization (homologous prime-boost), dna-miv heterologous prime boost approach demonstrated a stronger ability in eliciting humoral immunity as evidenced by a more than fourfold increase in hai titers (ledgerwood et al. ) . the results obtained from these studies suggested that dna vaccine prime followed by a miv or viral vector boost is a promising approach for the development of a universal influenza vaccine for humans. in addition to surface proteins, viral antigens that are inaccessible to antibodies, such as nucleocapsid protein (np) and matrix protein (m), can also be included as the immunogen in dna vaccines. generally, these internal structural proteins are highly conserved among variant strains. ctls raised against these conserved internal proteins can confer cross-protection against diverse related strains. for example, in a preclinical study, a dna vaccine targeting the np from influenza h n strain elicited broadly effective ctls that provided cross-protection against challenge from a h n strain arising three decades later. these findings suggest that immune responses to both surface and internal viral proteins will provide optimal protection. therefore, in order to obtain the greatest efficacy, it is conceptually reasonable to include multiple plasmids in an influenza dna vaccine cocktail to induce antibody to the ha protein and cytotoxic immunity to the viral np and m protein. in the case of influenza, the traditional manufacturing process for the licensed flu vaccines is labor intensive and time consuming, which impedes an imperative response against outbreak crisis. this occurred in , when a swine-origin h n influenza caused a global pandemic. the licensed pandemic vaccine in the form of killed viruses could not be released until a half year after the world health organization (who) announcement of the global outbreak. in contrast, by virtue of its simplicity in manufacturing, an investigational pandemic h dna vaccine was quickly made available months earlier than the licensed pandemic vaccine. when tested in a phase i clinical trial (nct ), the h dna vaccine elicited both humoral and cellular immunity against the pandemic strain. the immune response could be further boosted by the licensed pandemic vaccine when it became available (crank et al. ) . the results derived from this phase i clinical trial suggests that dna vaccines are of particular value in a pandemic setting to control the outbreak of emerging infectious disease. since its initial identification in early s, the scientific community has worked diligently towards a safe and efficacious vaccine that renders sterilizing immunity against hiv infection. however, despite the tremendous efforts made during the past more than three decades, a successful hiv vaccine remains elusive. historically, for a large number of infectious diseases, the live attenuated or killed whole virus proved to be the first and second best approach in successful vaccine development (gallo ) . however, neither of them can be included in hiv vaccines due to the safety concerns that attenuated hiv would cause aids and one cannot be certain that all virus particles would be completely inactivated. to date, only two subunit vaccines, namely, the hepatitis b virus (hbv) vaccine and the human papillomavirus (hpv) vaccines are approved for clinical use. however, similar success has not been reproduced for other viral diseases including hiv infection. it was noted that the hbv recombinant protein is different from many other viral recombinant proteins in regards to its ability to form particles, a feature that may render hbv recombinant protein super immunogenic (liu ) . nevertheless, the field remains far less experienced with the recombinant subunit vaccine approach. hence, it has been reasoned that for a long period of time, the formulation of hiv vaccines might be limited to hiv proteins or their dna form (gallo ) . the first clinical trial of dna vaccination against hiv was performed in , in which the infected patients were immunized with plasmid constructs encoding the nef, rev, or tat regulatory protein. the immunization resulted in detectable but transient-specific cytotoxicity in eight of nine patients immunized (calarota et al. ). since then, clinical studies using improved formulation and delivery strategies have been conducted. in several phase i trials, in an effort to improve the immunogenicity, additional plasmids encoding il or il were given together with dna vaccine encoding hiv env, gag, and pol proteins. the inclusion of these molecular adjuvants led to an increase in magnitude and breath of cellular and humoral immunity (felber et al. ) . recently, several vaccines using dna as a prime in combination with different boosts have also been evaluated. the boosts used in these trials were either viral vectors (recombinant modified vaccinia ankara (rmva), recombinant adenovirus (rade), or recombinant vesicular stomatitis virus (rvsv))expressing env or in the form of a purified recombinant gp protein (felber et al. ). the rationale of using a dna prime and protein boost approach is based on the observations that dna vaccination is suited to eliciting strong cellular immunity, whereas protein immunization preferentially induces antibody response (felber et al. ) . from to , a large-scale efficacy trial carried out in thailand (rv trial) used a similar prime-boost schedule, in which the priming dna (hiv env, gag, and pro gene) was introduced by a canarypox vector rather than by a naked dna vaccine. although the dna uptake efficiency mediated by viral vectors could be higher than naked dna, one potential disadvantage of using viral vector is that the preexisting anti-vector immunity may dampen the effectiveness of the priming immunization, an issue that can be perceived from the failed step (also referred to as merck v - ) trial. nonetheless, until now the rv trial is the only one to demonstrate a transient and modest protection benefit. notably, the limited protection appeared to be correlated with the humoral responses. nevertheless, a consensus agreement has been reached in the field that an effective hiv vaccine should afford both humoral and cellular immunity whereby neutralizing antibody blocks virus entry at the sites of infection, and ctls eliminates any infiltrating infection (felber et al. ). an additional application of dna vaccines for hiv is the potential for use as therapy. using the simian immunodeficiency virus (siv)/macaque model, a therapeutic dna vaccine induced potent cellular responses so that the viral rebound was not observed for many months after cessation of the anti-retroviral therapy (art) (felber et al. ) . similarly, up to tenfold reduction in viremia and delay in the kinetics of viral rebound has been observed in some human studies testing the therapeutic dna vaccines (mylvaganam et al. ) . however, the clinical benefit of these positive results is still unclear. it has been hypothesized that to optimally boost the preexisting hiv-specific immune responses, therapeutic vaccines need to be combined with other therapeutic approaches such as check point inhibitors (mylvaganam et al. ) . the application of dna vaccination is not limited to influenza and hiv infection. to date, there are more than viral diseases for which dna vaccines have entered clinical trials. a list of clinical trials targeting other viral diseases is summarized in table . among these trials, a therapeutic dna vaccine targeting cytomegalovirus (cmv) reactivation in cmv-seropositive hematopoietic stem cell transplant recipients has entered the pivotal phase iii study (nct ) in . this vaccine consists of two plasmids expressing cmv antigens glycoprotein b (gb) and phosphoprotein (pp ). in a previous phase ii trial (nct ), this cmv dna vaccine provided initial evidence of the safety and revealed significant reduction in viral load and extended period of time before the onset of viremia (smith et al. ) . it has been thought for some time that t lymphocytes act as sentinels in recognizing and eliminating continuously arising neoplastic cells. indirect evidence supporting the notion that immunotherapy could be a useful alternate treatment for cancers comes from the observations that the incidence of some malignancies, such as kaposi's sarcoma, non-hodgkin's lymphoma, and invasive cervical cancer, was increased significantly in immunocompromised patients. the presence of cd + and t h cells in the tumor microenvironment was also found to be a favorable prognostic factor for many different cancers. moreover, tumor infiltrating t lymphocytes (tils) have been successfully used in adoptive t cell transfer for treatment of metastatic melanoma and resulted in tumor regression. thus, one effective approach for developing cancer immunotherapies is to activate the immune system through vaccination to generate cytotoxic t lymphocytes capable of eliminating tumor cells. dna immunization is of particular interest for developing therapeutic cancer vaccines based on the generation of t cells. ideally, the vaccination-activated t cells should be able to recognize the tumor-associated antigens (taas) and initiate targeted killing of malignant cells. however, most tumors are caused by loss of growth control in normal tissues and thus express self-antigens that are either not recognized by or are only weakly reactive to the immune system. this phenomenon is described as immune tolerance, an inherent mechanism to prevent autoimmunity in which the immune system attacks the host's own cells and tissues. therefore, one key element to improve dna vaccine efficacy is to formulate a vaccine with an immunogenic cancer antigen so that it can prime t cells for immune responses. to date, a large number of cancer antigens have been identified. based on their expression pattern, cancer antigens can be classified into four categories. the first group consists of antigens that are known as cancer-testis (ct) antigens. these antigens are only expressed on particular tumor cells and immune privileged germ line tissues but not on normal adult cells. melanoma-associated antigen (mage) and new york esophageal squamous cell carcinoma (ny-eso- ) are examples of this group. the second class of antigens is a large and diverse group that includes any protein found at increased levels in tumors compared with normal healthy cells and tissues. these antigens are termed as overexpressed self-proteins. representative members for this family are prostate-specific membrane antigen (psma), human telomerase reverse transcriptase (htert), and human epidermal growth factor receptor (her /neu). antigens that are specific to a certain type of tissue and shared between tumors and normal tissue of origin are called differentiation antigens. prototypes in this class include gp , tyrosinase, and melan-a/melanoma antigen recognized by t cells (mart- ), which are molecules expressed by both melanoma and normal melanocytes. finally, antigens that are found exclusively on tumor cells but not on any normal tissues are called tumor-specific antigens (tsa). the unique tumor antigens are generated by somatic mutations as a result of aberrant gene expression. the mutated proteins usually play an important role during the process of cancer development and thus are selected to survive (yang et al. ) . because tumor-specific antigens are only present on tumor cells, they are readily recognized by the host immune system and are ideal targets for cancer vaccine development. however, incorporating tumor-specific antigens in the vaccine design is complicated by the fact that the same tumor type can be induced by various distinct point mutations, making it difficult to identify a broadly applicable tumorspecific antigen for a vaccine (yang et al. ). viral antigens carried by oncogenic viruses such as hpv and hbv represent another class of tumor-specific antigen. for example, the two hpv viral oncoproteins, e and e , are required for the induction and maintenance of cellular transformation and are consistently co-expressed in hpv-associated cancers. their exclusive presence on virus-infected cells and their nature of foreign origin renders vaccines targeting these proteins less vulnerable to the central immune tolerance and the undesired "on-target off-tumor" cytotoxicity. although cancer antigens have the potential to activate the immune system resulting in tumor regression, their application in human cancer vaccination is hampered by their inherent poor immunogenicity. most cancer antigens are tumor related rather than tumor specific. the unresponsiveness to self-antigens caused by immune tolerance is a major roadblock for cancer vaccine development. thus, there is a pressing need for innovative cancer vaccine design so that immune tolerance can be circumvented. one way to induce immunity against a tissue specific differentiation antigen on cancer cells is to vaccinate with a xenogeneic version of antigen. as the name suggests, a xenogeneic antigen is homologous to the cancer antigen but originates from a species foreign to the host. xenogeneic dna vaccines incorporate plasmid dna encoding for a protein homologous between the two species. ideally, xenoantigens should be sufficiently different from self-antigens in order to be immunogenic; they must, on the other hand, also preserve an optimal homology range with self-proteins to secure the cross-reactivity of the induced immune responses. the most striking example of using xenogeneic antigen to break immune tolerance is evidenced by the licensed canine melanoma dna vaccine, a therapeutic agent for advanced melanoma (bergman et al. ) . this vaccine consists of dna encoding a human version of tyrosinase, which enabled the dog's immune system to break tolerance to the dog tyrosinase and mount an effective response. the concept of using xenoantigens to enhance vaccine immunogenicity is also supported by a recent preclinical study in which mice immunized with human p (hp ) dna vaccine protected against challenge with murine colon cancer mc while those immunized with mouse p (mp dna) were not (soong et al. ) . in a therapeutic model, established mc tumors were also well controlled by treatment with hp dna therapy in tumor bearing mice compared to mp dna. mice vaccinated with hp dna plasmid exhibited an increase in mp -specific cd + t-cells compared to vaccination with mp dna (soong et al. ) . associative recognition is another strategic antigen design aimed at breaking central immune tolerance. the rationale of this approach is to introduce additional gene coding sequences to a dna vaccine by which a fusion protein containing the cancer antigen and the associated peptide is expressed. the peptide moiety has epitopes that are highly immunogenic to t h cells, which help induce cd + cytotoxic t cell and cd + helper t cell responses against the weakly immunogenic selfantigen. for example, tetanus toxin (tt) contains epitopes that avidly bind to t helper cells. in several preclinical studies, tt-fused antigen has been used in dna vaccines to significantly enhance the immunogenicity of various cancer antigens including c-myb, pas domain containing (pasd ), and ny-eso- . these studies demonstrate the potential of dna vaccines incorporating tt epitopes in order to enhance immunogenicity (yang et al. ) . breaking central immune tolerance is critical for cancer vaccine efficacy. however, breaking tolerance may raise the risk of autoimmunity. many cancer antigens are also expressed on normal cells, albeit at very low level. enhancing antigen immunogenicity should not be at the expense of damaging normal tissues. the detrimental "on-target off-tumor" side effect has been reported in cancer treatment using adoptive transfer of genetically engineered t cells. until now, strategies used to break immune tolerance have not been shown to trigger harmful autoimmune attacks against normal tissues. however, more studies are needed to further evaluate the safety of breaking immune tolerance. it also should be noted that breaking immune tolerance represents only one of the greatest obstacles faced by scientists developing effective cancer vaccines. other factors in the peripheral immunosuppressive networks including, but not limited to, myeloid-derived suppressor cells (mdsc), regulatory t cells (t reg ), inhibitory cytokines, and immune exhaustion also profoundly affect the efficacy of dna vaccination. therefore, in agreement with the notion that most effective cancer therapies are multimodal, clinical development of cancer vaccines may ultimately be part of polytherapy (e.g., vaccine + checkpoint blockade mabs) to counteract central and local immunosuppressive mechanisms. numerous therapeutic dna vaccines are currently being evaluated in clinical trials to assess their translational potential. these vaccines target a wide variety of malignancies including cervical cancer, colorectal cancer, prostate cancer, breast cancer, head and neck cancer, melanoma, merkel cell carcinoma, and leukemia. a summary of these trials including their targeted cancer antigens, strategies used to break the immune tolerance, and trial stages is shown in table . the tert antigen encoded by the dna vaccine was synthesized and codon-optimized. two immunogenic mutations (r y and d y) were incorporated into the htert sequence to assist in breaking tolerance to date, the most successful and encouraging outcomes of using dna vaccine in the clinical setting were obtained from treatment of malignant diseases where the etiological agent is of foreign viral origin, such as the human papillomavirus (hpv), as these viral agents can readily induce a strong immune response against cancerous cells harboring viral antigens. in the case of hpv, more than strains have been identified with most of them causing asymptomatic infections. however, oncogenic high-risk hpv types (e.g., hpv , , , , and ) have been shown to be the etiological agents for cervical, penile, vulvar, vaginal, anal, and oropharyngeal cancers. particularly, hpv and have been regarded as the genotypes most closely associated with cervical intraepithelial neoplasia (cin) and cervical cancers. the hpv genome encodes two classes of proteins including the early (e) and late (l) proteins. the late proteins (l and l ) are structural components of viral capsid, whereas, the early proteins (e , e , e , e , e , and e ) are regulatory proteins participating in dna replication, transcriptional regulation, cell transformation, and viral assembly. among these viral proteins, e and e can inactivate tumor suppressor p and retinoblastoma (rb) protein, respectively, thereby preventing cellular apoptosis, promoting cell proliferation, and contributing to progression to intraepithelial neoplasia and carcinoma. moreover, e and e expression in hpv-infected cells is constitutive and irrespective of infection stage. in contrast, after primary infection, the expression of several early (e , e , and e ) and late (l and l ) proteins becomes invisible as a result of the deletion of the viral genes upon viral integration into host genome (lin et al. ) . therefore, owing to their constitutive expression in hpv-associated malignancies and their crucial role in tumor pathogenesis, the e and e proteins are ideal choices of hpv antigens for developing therapeutic vaccines against hpv-associated malignancies. indeed, several dna vaccines targeting the hpv / e and e proteins have been tested in clinical trials and demonstrated impressive efficacy as evidenced by regression of high grade cin to normal tissue and clearance of viral infection. during the last few years, great progress has been made in the field of dna vaccination. the advancements in plasmid construction, antigen design, delivery device optimization, and use of immunologic adjuvants have greatly enhanced the immunogenicity and efficacy of dna vaccines. among these developments, electroporation represents the most remarkable progress, which exerts the greatest impact on dna vaccine efficacy. recently, a hpv / -specific dna vaccine delivered by ep demonstrated clinical benefit in a phase iib clinical trial in the form of regression of cervical intraepithelial neoplasia (cin / ). this encouraging result has led to resurgence of the platform. it is promising that with further improvement, dna immunization will bring revolutionary transformation to the vaccine field. to that end, scientists are working diligently towards the first licensure of dna vaccines. cancer vaccination by electro-gene-transfer immunotherapy against hpv / generates potent th and cytotoxic cellular immune responses long-term survival of dogs with advanced malignant melanoma after dna vaccination with xenogeneic human tyrosinase: a phase i trial administration of hpv dna vaccine via electroporation elicits the strongest cd + t cell immune responses compared to intramuscular injection and intradermal gene gun delivery cellular cytotoxic response induced by dna vaccination in hiv- -infected patients dna-based influenza vaccines: evaluating their potential to provide universal protection phase study of pandemic h dna vaccine in healthy adults phase i randomized clinical trial of vrc dna and rad hiv- vaccine delivery by intramuscular (i.m.), subcutaneous (s.c.) and intradermal (i.d.) administration (vrc ) hiv dna vaccine: stepwise improvements make a difference dna vaccines: developing new strategies against cancer the end or the beginning of the drive to an hiv-preventive vaccine: a view from over years adjuvanting a dna vaccine with a tlr ligand plus flt ligand results in enhanced cellular immunity against the simian immunodeficiency virus dna priming and influenza vaccine immunogenicity: two phase open label randomised clinical trials targeting plasmid-encoded proteins to the antigen presentation pathways therapeutic hpv dna vaccines dna vaccines: an historical perspective and view to the future prospects for control of emerging infectious diseases with plasmid dna vaccines hiv therapeutic vaccines: moving towards a functional cure clinical development of a cytomegalovirus dna vaccine: from product concept to pivotal phase trial xenogeneic human p dna vaccination by electroporation breaks immune tolerance to control murine tumors expressing mouse p genetic immunization is a simple method for eliciting an immune response using plasmids as dna vaccines for infectious diseases induction of broadly neutralizing h n influenza antibodies by vaccination direct gene transfer into mouse muscle in vivo dna vaccine for cancer immunotherapy ▶ gene delivery by electroporation in vitro: mechanisms key: cord- - lmj op authors: ada, gordon title: overview of vaccines and vaccination date: journal: mol biotechnol doi: . /mb: : : sha: doc_id: cord_uid: lmj op of the -plus known infectious agents pathogenic for humans, there are now more than vaccines against mainly viral and bacterial infections and these greatly minimize subsequent disease and prevent death after exposure to those agents. this article describes the nature of the vaccines, from live attenuated agents to subunits, their efficacy and safety, and the kind of the immune responses generated by those vaccines, which are so effective. to date, all licensed vaccines generate especially specific antibodies, which attach to the infectious agent and therefore can very largely prevent infection. these vaccines have been so effective in developed countries in preventing mortality after a subsequent infection that attempts are being made to develop vaccines against many of the remaining infectious agents. many of the latter are difficult to manipulate; they can cause persisting infections or show great antigenic variation. a range of new approaches to improve selected immune responses, such as immunization with dna or chimeric live vectors, viral or bacterial, are under intense scrutiny, as well as genomic analysis of the agent. vaccines are designed as a prophylactic measure to induce a lasting immune response so that on subsequent exposure to the particular infectious agent, the extent of infection is reduced to such an extent that disease does not occur ( ). there is also increasing interest in designing vaccines that may be effective as a therapeutic measure, immunotherapy. there are two contrasting types of infectious processes. some organisms, including all viruses and some bacteria, are obligate intracellular infectious agents, as they only replicate inside a susceptible cell. some parasites, such as plasmodia, have intracellular phases as part of their life cycle. in contrast, many bacteria and parasites replicate extracellularly. the immune response required to control the different patterns of infections may therefore differ. in the case of an acute infection, exposure of a naïve individual to a sublethal dose of the infectious agent may cause disease, but the immune response generated will clear the infection within a period of days or weeks. death occurs if the infecting dose is so high that the immune response is qualitatively or quantitatively insufficient to prevent continuing replication of the agent so that the host is overwhelmed. in contrast, many infections persist for months or years if the process of infection by the agent results in the evasion or subversion of what would normally be an effective immune control reaction. most of the vaccines registered for use in developed countries, and discussed briefly in the next section, are designed to prevent acute human infections. . one approach, pioneered by edward jenner, is to use a virus that is a natural pathogen in another mammalian host as the basis of a vaccine in humans. examples of this approach are the use of cowpox and parainfluenza viruses in humans, and the turkey human virus in chickens. more recently, the use of avipox viruses such as fowlpox and canarypox, which undergo an abortive infection in humans, is being used in humans as vectors of dna coding for antigens of other infectious agents for which vaccines are not yet available ( ). . the measles, mumps, rubella, and yellow fever vaccines are typical of the second approach. the wild-type viruses are extensively passaged in tissue culture/animal hosts until an acceptable balance is reached between loss of virulence and retention of immunogenicity in humans. . type polio virus is a naturally occurring attenuated strain that has been highly successful. more recently, rotavirus strains of low virulence have been recovered from children's nurseries during epidemics ( ). . a fourth approach has been to select mutants that will grow at low temperatures but very poorly above °c ( ). the cold-adapted strains of influenza virus grow at °c and have mutations in four of the internal viral genes ( ). such strains were first described in the late s and have since been used successfully in russia, have undergone extensive clinical trials in the united states ( ) and the vaccine has now been licensed, but not for young children and the elderly. in contrast to these successes, bacillus calmette-guerin (bcg) for the control of tuberculosis was for many years the only example of a live attenuated bacterial vaccine. although still widely used in the world health organization (who) expanded programme of immunization (epi) for infants, it has given highly variable results in adult human trials. in general, it has proven more difficult to make highly effective attenuated bacterial vaccines, but with increasing examples of antibiotic resistance occurring, there is now a greater effort. a general approach is to selectively delete or inactivate one or more genes ( ). salmonella strain ty a has a faulty galactose metabolism, and strains with other deletions are being made. the most recent addition to this category is a vibrio cholerae vaccine. a new approach is to sequence the bacterial genome to establish the properties and hence the probable location of different proteins in the bacteria, and this has now been done for many different bacteria ( ). genetic modification can be useful for complex viruses. thus, open-reading frames, including six genes involved in nucleotide metabolism, have been selectively deleted from the copenhagen strain of vaccinia virus. the product, nyvac, has low virulence but has retained immunogenicity ( ). the same approach has been used with simian immunodeficiency virus (siv) but with limited success. it now seems very unlikely that a live, attenuated strain of human immunodeficiency virus (hiv) will ever become available. live, attenuated agent vaccines have the potential to stimulate strong humoral and cell-mediated immune responses that can be highly effective in preventing or clearing a later infection in most recipients. viruses and bacteria can be treated to destroy their infectivity (inactivation) and the product used with varying efficacy as a vaccine ( table ) . compared to attenuated preparations, inactivated preparations must be given in larger doses and sometimes administered more frequently. the vi-molecular biotechnology volume , ral vaccines are generally effective in preventing disease. the lower efficacy of the influenza viral vaccine is partly due to the difficulty of closely matching the specificity of the virus strains that are circulating when the vaccine finally becomes available. this is due to the continuing antigenic drift that characterizes this virus ( ). the only bacterial vaccine of this nature widely used is the whole cell pertussis vaccine, which is quite effective ( table ) , but has been replaced in some developed counties by the subunit (acellular) vaccine to avoid the adverse effects attributed to the whole cell vaccine ( ). inactivated whole vaccines should induce many of the desirable immune responses, particularly infectivity-neutralizing antibodies. generally, they do not induce a class i major histocompatibility complex (mhc)-restricted cytotoxic t-cell response, which is the major response required to clear intracellular infections by viruses, and by some bacteria and parasites. class ii mhc neutralizing antibodies are adequately induced. the generation of antibodies that prevent infections by both intra-and extracellular microorgan- isms has been regarded as the prime requirement of a vaccine. the epitopes recognized by such antibodies are usually restricted to one of a few proteins or carbohydrate that is exposed at the external surface of the microorganisms. isolation (or synthesis) of such components formed the basis of the first viral and bacterial subunit vaccines. the influenza virus vaccine was composed of the two surface protein antigens, the hemagglutinin and neuraminidase, and the hepatitis b virus vaccine of the surface antigen, hbsag. encapsulated bacteria have a coating of polysaccharides that is not recognized by the immune system of very young children (< yr old) and only moderately well by older individuals (mainly an immunoglobulin[ig] m response), therefore polysaccharide vaccines needed to be improved. in , it was found ( ) that immunizing with a polysaccharide/protein conjugate gave a much stronger antipolysaccharide response (mainly iggs, because t cells were now involved). much later, we now have three such conjugate vaccines ( table ) that are highly immunogenic and these are saving especially many young lives ( ). more will become available. the two bacterial toxoids, tetanus and diphtheria, represent a special situation in which the primary requirement was neutralization of the toxin secreted by the invading bacteria. whereas toxoids have traditionally been made by treatment of the toxins with chemicals, it is now being achieved by genetic manipulation ( ) . hbsag exists in the blood of hepatitis b virus (hbv)-infected people and infected blood was the source of antigen for the first vaccines. production of the antigen in dna-transfected yeast cells initiated the era of genetically engineered vaccines ( , ) . a second genetically engineered subunit preparation from borrelia burgdorferi to control lyme disease later became available in the mid- s but was withdrawn from sale in because of poor sales. all available data concerning the efficacy and safety of candidate vaccines are reviewed by regulatory authorities before registration ( ). at that stage, potential safety hazards, which occur at a frequency of about / doses, should have . this advice has been accepted by several other developed countries such as australia. after successful vaccination campaigns that greatly reduced disease outbreaks, the low levels of undesirable side effects after vaccination gained some notoriety. the evidence bearing on causality and specific adverse health outcomes following vaccination against some childhood viral and bacterial diseases, mainly in the united states, has been evaluated by an expert committee of the institute of medicine (iom) ( ). the possibility of adverse neurological effects was of particular concern, and evidence for these as well as several immunological reactions, such as anaphylaxis and delayed type hypersensitivity, was examined in detail. in the majority of cases, there was insufficient evidence to support a causal relationship, and where the data were more persuasive, the risk was considered to be extraordinarily low. measles has provided an interesting example of vaccine safety. the experience of the who epi shows that the vaccine is very safe ( ). although natural measles infection induces a state of immunosuppression, even immunocompromised children rarely show this effect after vaccination ( ) . in developing countries, the epi schedule is to give the vaccine at mo of age. this delay is meant to allow a sufficient drop in the level of maternally derived antibody so that the vaccine can take. in some infants, this decay occurs by mo, resulting in many deaths from measles infection in the ensuing to mo, "high-titer" vaccines were therefore developed that could be given at mo of age. trials in several countries showed the apparent safety and efficacy of the new vaccine, but after who authorized its wider usage, some young girls in disadvantaged countries died, leading to the withdrawal of the vaccine ( ). one possibility is that the high titer vaccine caused a degree of immunosuppression sufficient to allow infections by other infectious agents. another example is the rotavirus vaccine that was registered for use in the united states in . it was withdrawn in after administration to . million children because of an unacceptable level-about one case per , recipients in some areas-of the condition intussusception ( ). this was surprising because the disease itself does not cause this condition. fortunately, other preparations are well advanced as the need for such a vaccine is great especially in developing countries. it is particularly difficult to attribute causality to the onset of diseases that may occur many months after vaccination. when such claims are made, national authorities or who establish expert committees to review the evidence. there have been claims-rarely in the medical literature but often by antivaccination groups-that a vaccine can cause sudden infant death syndrome (sids), multiple sclerosis, autism, asthma, or a specific allergy. there is no sound medical, scientific, or epidemiological evidence to support these claims. for example, at least different investigations have found no evidence that inflammatory bowel disease and autism occur as a result of measles, mumps, and rubella (mmr) vaccination ( , ) . this claim was apparently made by the antivaccination lobby simply because the increase in austism in the united states late last century coincided with the introduction of a second mmr vaccination. many countries keep yearly records of diseases incidence and the cdc in atlanta have kept records from as early as . table compares the incidence of cases of some common childhood infectious diseases during a major epidemic before the vaccine was licensed, with levels in and , some years after the introduction of the vaccine. most show a very high level of efficacy. pertussis, the cause of whooping cough, is an exception in showing an increase in cases recently and this may lead to the introduction of an additional dose of the vaccine. the crowning achievement of a vaccination program would be to eradicate some infectious diseases. jenner ( ) had proposed that his new vaccination procedure could be used to eradicate smallpox, but a century and a half passed before this suggestion was taken seriously. having achieved control of smallpox infections in their country, the russians in proposed to the world health assembly (wha) the global eradication of that disease. a -yr unfunded, voluntary program was initiated by who. great progress was achieved in developed countries but in , there were million deaths and million disease cases in developing countries. in , a -yr funded (usd $ million) program was begun and despite many difficulties, such as vaccine shortages and wars, the last case was treated in . eradication was declared to the wha in ( ). three senior investigators, f. fenner, d. a. henderson, and i. arita shared the japan prize for this achievement. would it be possible to repeat this outstanding success with another infectious disease? poliomyelitis and measles were two considered. both ful-filled the necessary requirements but not the desirable properties ( table ) . the former was chosen as there was already some success in limiting transmission of the disease in developed countries. the global polio eradication initiative was spearheaded by who, rotary international, the united nations children's fund (unicef), and cdc in , and joined later by other groups. there were many difficulties. the infection persisted in some individuals for many months and at one stage, one of the three vaccine virus strains mutated into a virulent form. repeated vaccinations became necessary so that the deadline had to be progressively extended until to . in , the virus was still endemic (less than cases) in six countries, but especially in nigeria. in kano province, a religious group refused vaccination because of concerns that the vaccine was contaminated with hiv and other factors that could affect the fertility of their children. by the time (mid- ) such contamination claims were shown to be wrong and the concern abated, infectious cases had occurred in previously infection-free countries in west and central africa. re-vaccination of children under the age of yr in countries in this region will begin in late and extend into , provided an ongoing funding gap of usd $ million is overcome ( ). the reason for such funding is that for every case of infection found, up to million children in surrounding areas may need to be re-vaccinated. having been at the edge of successful eradication, it would be a disaster if the extra funding was not made available. as of early october , this vaccination program has begun. for yr until the measles vaccine was introduced in , the yearly incidence of infections in the united states was never less than , cases, with epidemics occurring every to yr. the maximum incidence of reported cases was , in ( table ) . introduction of the measles vaccine in led to a reduction in incidence of cases by > . %. a -yr epidemic of , cases began in and led to the introduction of a second vaccine dose for mainly chil-molecular biotechnology volume , dren about to go to school. in , the cdc concluded that measles was no longer an endemic disease in the united states. in , it was established that all cases had been brought into the country by visitors from specified countries. in , ministers of health from north and south america met and resolved to eliminate measles from all the western hemisphere by . the pan american health organization (paho) under ciro de quadros contributed greatly with catch-up and follow-up campaigns with the result that for mo in , transmission of measles in the whole of the americas was prevented. in , the paho strategy was successfully extended to seven south african countries ( ). although a major difficulty to prevent transmission of measles is the high level of vaccination coverage necessary ( %), the interruption of indigenous measles transmission was also achieved in cuba from , and in england and wales from . some countries such as japan, italy, france, and germany do not consider this a national priority ( ). but there is still hope that one day, a global eradication program will be announced: "it is not a dream to imagine a world free of measles by year " ( ). this may depend on the final result of the poliomyelitis eradication campaign. and new vaccines vaccines are not currently available for more than infectious agents pathogenic for humans. vaccine development is well advanced for some of these and is in progress for the most of the remainder. table contains a short list of agents for which improved vaccines are desirable, and a longer list of those agents that should have priority in vaccine development plans. although the current measles vaccine is highly effective, a vaccine that could be administered well before mo would save many lives. in the new category, the "terrible trio," hiv, m ycobacterium tuberculosis, and malaria, would be at the top of many lists. they are major killers and despite great efforts over many years, effective vaccines are some way off. fortunately in subtropical developed countries, and with effective drugs, each agent is largely controlled. the advantages of this approach include the fact that the anti-idiotype should mimic both carbohydrate-based and peptide-based epitopes, and the conformation of the epitopes in question. the potential advantages of the former point have dis- vaccines ( , ) . the use of this technology may be largely restricted to very special situations, such as identifying the nature of the epitope recognized by very rare antibodies that neutralize a wide spectrum of hiv- primary isolates ( ). the receptors on t cells (lymphocytes) recognize on the surface of the antigen-presenting cell (apc) or infected target cell, a complex composed of a peptide from a protein in the apc or infected cell attached to a mhc antigen. simi-larly, the ig receptors on b cells may recognize a pattern on an antigen often formed by a linear peptide sequence. therefore, it could be advantageous to link such linear sequences to form a particular antigen. the sequences may contain either b-cell epitopes or t-cell determinants, or often both (see subheading . .). sequences containing b-cell epitopes may either be conjugated to carrier proteins, which act as a source of t-helper cell determinants, or linked in different ways to achieve particular tertiary configurations. some of the obvious advantages of this approach are that the final product contains the critical components of the antigen and avoids other sequences that may mimic host antigen sequences, and thus potentially induce an autoimmune response. multiple antigenic peptide systems (maps) are more immunogenic than individual sequences ( ), and the immunogenicity of important "cryptic" sequences may sometimes be enhanced by the deletion of other segments ( ). new methods of synthesis offer the possibility of more closely mimicking the conformational patterns in the original protein. this approach is likely to be applicable, especially for some bacterial and parasitic vaccines. however, the first peptide-based candidate vaccine, despite giving encouraging results in an early small trial, gave disappointing results in an efficacy trial in malaria endemic regions ( ). a preparation composed of polymers of linked peptides from group a streptococcus, which was effective in a mouse model ( ), is currently undergoing clinical trials. this is now a well-established procedure. three cell types have been used: ( ) prokaryotes (bacteria), ( ) lower eukaryotes, mainly yeast; and ( ) mammalian cells, either primary cells (e.g., monkey kidney), cell strains (with a finite replicating ability), or cell lines (immortalized cells such as chinese hamster ovary cells [cho]). each has its own advantages. as a general rule, other bacterial proteins should preferably be made in transfected bacterial cells, and human viral an- molecular biotechnology volume , tigens, especially glycoproteins, in mammalian cells, because of the substantial differences in properties, such as post-translational modifications in different cell types. table lists the viruses and bacteria mostly used for this purpose. of the viruses, the greatest experience has been with vaccinia and its derivatives such as the highly attenuated, modified vaccinia virus ankara. these have a wide host range, possess many different promoters, and can accommodate dna coding for up to averagesized proteins. the avipox viruses, canary and fowlpox, undergo abortive infection in mammals, making them very safe to use as vectors ( ). adenovirus ( ), polioviruses ( ), and salmonella ( ) are mainly used for delivery by a mucosal route, although vaccinia and bcg have been administered both orally and intranasally. making such chimeric vectors has also been an effective way to evaluate the potential role of different cytokines in immune processes. inserting dna coding for a particular cytokine as well as that for the foreign antigens results in the synthesis and secretion of the cytokine so its maximum effect should be displayed. thus, interleukin (il)- and il- have been shown to have dominant effects in inducing a humoral or cell-mediated immune response, respectively. incorporation of dna coding for il- into the genome of ectrome-lia virus greatly increased the virulence of this virus in otherwise resistant mice, and even if the latter had been immunized to increase resistance before challenge ( ). the most fascinating and exciting of the recent approaches to vaccine development has been the injection of plasmids containing the dna coding for antigens of interest, either directly into muscle cells or as dna-coated tiny gold beads into the skin, using a "gene gun" ( , ) . in the latter case, some coated beads bind to the toll-like receptor (tlr) on langerhan's (dendritic) cells, and during passage of the cells to the draining lymph nodes ( h), the expressed foreign protein is processed. appropriate peptide sequences attach to mhc molecules and the resulting complex is expressed at the cell surface. these complexes are recognized by naïve, immunocompetent t cells in the node and this stimulates activation of the cells. a basically similar process occurs after muscle injection. in lower primates, this procedure primarily induces a type- t-cell response, resulting in both an antibody-and cell-mediated immune response, with both cd + and cd + effector t cells. in many respects, this is similar to the response following an acute infection. one advantage is that the response to dna occurs in the presence of specific antibody to the encoded antigen. our knowledge of the properties of lymphocytes-the cell type of major importance in vaccine development-has increased enormously in recent years ( ). the major role of b lymphocytes is the production of antibodies of different isotypes and of course, specificity. the other class of lymphocytes, t cells (so-called because they mature in the thymus), consist of two main types. one, with the surface marker cd , exists in two subclasses, the th- and th- cells (h = helper activity). the major role of th- cells is to help b cells differentiate, replicate, and in the form of plasma cells, secrete antibodies of a defined specificity, and of different subclasses, igg, ige, and iga. furthermore, there are different subclasses of igg. this is facilitated by the secretion of different cytokines (interleukins), which are listed in table . th- cells also have a small but important role in helping b cells produce antibody of various igg subtypes, but the overall pattern of cytokine secretion is markedly different. factors, such as interferon (ifn)-γ, tumor necrosis factor (tnf)-α, and tnf-β, have several functions, such as antiviral activity and up-regulation of components (e.g., mhc antigens on other cells). with macrophages, this can lead to their activation and if infected, greater susceptibility to recognition by t cells. th- cells can also mediate (through cytokine secretion) delayed-type hypersensitivity (dth) responses that may have a protective role in some infections. it was recognized that during an infection by a pathogenic microorganism, the immune response may be suppressed to limit immunopathology, and this has recently been shown to be due to a particular cd + t cell, now called a regulatory cell (treg). it is not expected that these cells would be activated during vaccination by a live attenuated vaccine. a second type of t cell has the cell-surface marker cd , and its pattern of cytokine secretion is similar to that of cd + th- cells, although they generally mediate dth responses rather poorly. as primary effector cells in vivo, cd + t cells recognize and lyse cells infected by a virus or by intracellular bacteria and some parasites, hence the name cytotoxic t lymphocytes (ctls). an important aspect of this arm of the immune re- molecular biotechnology volume , sponse is that susceptibility of the infected cell to lysis occurs shortly after infection, and many hours before infectious progeny is produced, thus giving a "window of time" for the effector cell to find and destroy the infected cell ( ). both cd + and cd + t cells recognize on the surface of the apc a complex between a mhc molecule and a peptide from the foreign antigen. in the first case, the peptide (average length, amino acids), which is derived from antigen being degraded in the lysosomes, complexes with class ii mhc antigens. in the second case, the peptide (average length, amino acids) is derived from newly synthesized antigen in the cytoplasm, and binds to class i mhc molecules. class ii mhc antigens are expressed mainly on cells that can act as apcs. in contrast, nearly all cells in the body may become infected and express class i mhc molecules. thus, the role of ctls has been described as performing a continuous molecular audit of the body ( ). table ascribes particular roles to specific antibody and to the t-cell subsets. some general conclusions regarding adaptive immune responses are: . specific antibody is the major mechanism for preventing or greatly limiting an infection; . ctls are the major mechanism for controlling and finally clearing most acute intracellular infections ( during an acute model infection such as murine influenza, the sequence of the appearance in the infected lung of adaptive responses is: first, cd + th cells, then cd + ctls, and finally antibody-secreting cells (ascs). the ctls are largely responsible for virus clearance and it has been thought that the subsequent decline in ctl activity that occurs shortly after infectious virus can no longer be recovered ( ), and this is because of the short half-life of these cells. the two dominant cytokines are il- , which favors a th- response ( ), and il- , which favors a th- response. the production of il- , which results in the enhancement of the antibody response, may curtail the life of the ctls. the finding that if il- is "artificially" induced very early after infection, ctl production is substantially suppressed ( ), supports this notion. one important point is that a large pool of memory ctls has already been formed by the time that ctl activity usually disappears. these memory cells persist, and are rapidly activated if the host is exposed to the same or a closely related infectious agent at a later time. it is now recognized that, as well as affecting the magnitude and persistence of immune responses to noninfectious preparations, adjuvants can also greatly influence the type of immune response ( ). some adjuvants, such as alum and cholera toxin and its b subunit, favor cd + th- responses. water-in-oil emulsions, such as freund's complete and incomplete adjuvant and lipopolysaccharide, favor a th- response. a variety of delivery systems is available for the induction of ctl responses ( ). although new technologies are making it more likely that attempts to develop vaccines to an in-creasing number of infectious agents will be successful, many other factors can influence the final outcome. some of these are: . the simpler the agent, the greater the chance that important protective antigens will be identified. . the occurrence of great antigenic diversity in the pathogen can be a major hurdle, especially in the case of rna viruses because escape mutants (antigenic drift) may readily occur. . integration of dna/cdna into the host cell genome is likely to lead to lifelong infection, which is difficult for a vaccine to prevent/overcome. . if a sublethal natural infection does not lead to protection from a second infection, it becomes necessary to understand the pathogenesis of the infection and how the normal protective responses (antibody, cell-mediated) are subverted or evaded. . the ready availability of an inexpensive animal model that mimics the human disease can be very helpful, but is rare. most early studies are done with mice but it is becoming increasingly common to then work with lower primates before initiating clinical studies. vaccine delivery is a major cost component in vaccination programs. combining vaccines so that three or more can be administered simultaneously results in considerable savings, therefore there are determined efforts to add other vaccines to longtime successful combinations (diphtheria, acellular pertussis, tetanus [dapt] and mmr) such as dapt-hepatitis b-h aemophilus influenzae type b. there must be compatibility and no interference by one component on the other. there is the risk of antigenic competition that occurs at the t-cell level, and the likelihood of such interference is difficult to predict. however, in the case of mixtures of protein/carbohydrate conjugates, using the same carrier protein should decrease this risk. the use of the same vector (e.g., the same poxvirus) in mixtures of chimeric constructs should minimize this difficulty. vaccination with dna should offer the same advantage. some antigens to be used in vaccines, mainly subunits, and many antibodies can be produced in transgenic plants. initially it was thought that simply "eating the fruit of the plant" would achieve satisfactory vaccination (i.e., edible vaccines) and that the technique would be particularly suitable for use in developing countries ( ). but the idea has evolved more recently into obtaining licensed products under strict biological regulations and subject to the same safeguards as products produced by conventional techniques ( ). in the long run, such products may be less expensive than conventional products, but not by a very large margin. most recently, although different antigens have been produced in this way, there are still substantial regulatory hurdles to be overcome in the use of these products ( ). in an effort to limit vaccine administration by needles, progress is being made in inducing immunization by direct application of the antigen, with a strong adjuvant such as cholera toxin, to prewashed skin, using a patch. with this technique, called transcutaneous immunization, the antigen rapidly contacts and is taken up by dendritic (langerhan's) cells in the epidermis and later, on arrival at draining lymph nodes, the processed antigen is presented to t cells ( ). using a gene gun is an efficient way of introducing dna coding for different antigens into the epidermis and directly into langerhan's cells and sometimes into the cell's nucleus. tiny gold beads are coated with the dna and this becomes an efficient way to induce an immune response ( ). in contrast with the time-honored approach of giving several doses of the same preparation of an antigen to obtain a stronger and longer immune response, the concept arose of priming with one formulation of an antigen and boosting with a different formulation. immunization of naïve volunteers with an hivgp / vaccinia virus construct followed by boosting with a recombinant gp preparation gave higher anti-gp antibody titers compared to using either preparation for both priming and boosting ( ). it was then shown that molecular biotechnology volume , mice immunized with a chimeric dna preparation and later boosted with chimeric fowlpox virus both expressing influenza hemagglutinin (ha), gave anti-ha titers up to -fold higher than those found after two injections of the same preparation ( ). this approach has been vigorously pursued to induce high and persistent ctl responses to hiv- , siv, ebola virus, m. tuberculosis, and plasmodia antigens with positive results in mice or monkeys ( ). two phase clinical trials using preventive aids vaccine candidates, one by an australian consortium supported by the national institutes of health, washington, and the other, an oxford-nairobi-uganda partnership supported by the international aids vaccine initiative, new york, were recently completed. both used chimeric dna followed by a chimeric live poxvirus vector to generate strong cell-mediated immunity (cmi) responses (ctls), but unfortunately they gave disappointing results ( ). the reason is not clear but at least temporally, the results cast a shadow over this technology in humans. studies are proceeding using different chimeric live vectors for priming and boosting. whole genome sequencing of complex microbes such as bacteria and parasites is poised to revolutionize the way different components are chosen to form a vaccine ( ).this enables the characterization of potential candidate proteins that might be recognized by infectivity-neutralizing antibodies, and which ones may provide important t-cell determinants. in one recent example, mice immunized with with of proteins from steptococcus pneumoniae that had been identified from the dna sequence as having appropriate structural characteristics, were protected from disease when later challenged with this organism ( ). infectious agents in his book, guns, germs and steel ( ), jared diamond retraces the history of civilization over the past , yr. (this should be of special interest to those younger than yr old.) because of the exposure, of especially europeans, to domesticated and other animals for most of this period, some of the animal infectious agents had evolved over many years to become essentially human infectious agents. these include smallpox, influenza, tuberculosis, malaria, plague, measles, and cholera. many infected people died, but the population survived and expanded. but when they traveled abroad, some of the group might be infected. beginning with columbus's voyage in , shortly followed by cortes' invasion of the aztec empire, the european invasion of north america, and in the late eighteenth century, the british invasion of australia, the mortality rate of indigenous peoples exposed for the first time to these agents was often as high as % and occasionally much higher. within the first yr, the indigenous populations in these regions had decreased by at least %, due mainly to the introduced infections. at the beginning of the twentieth century, in well-off countries, families were large because it was expected that several children in one family could die from childhood infections. parents were especially proud if they reached the biblical goal of three score years and ten. but why was the mortality rate so high? the nobel prize in physiology or medicine was awarded to peter doherty and rolf zinkernagel for research they had done in the early s at the australian national university. based on their experimental findings, they predicted that ctls could distinguish between infected and normal cells because the former expressed at the cell surface a complex between a mhc molecule and a protein (later shown to be a peptide) from the infecting virus ( ). thirteen years later, this interpretation was shown to be correct by xray crystallography ( ). the human chromosome contains loci, each containing dna coding for a particular mhc antigen. at conception, the fetus receives such genes from each parent giving a total of . dna at six loci codes for class i mhc molecules and at the other loci, the dna codes for class ii mhc molecules. yet on a population basis, there are many, sometimes > dna molecules coding for different mhc antigen specificities, anyone of which can occupy the same loci in different individuals. thus, individual humans possess only a small sample of the total number of mhc specificities. one result is that it was highly unlikely that all people would have the best protective response to all infections. person a may make a strongly protective response against infection x, but a weakly protective response against infection y. person b may make the opposite responses. the resistance or susceptibility of different inbred strains of mice to different pathogens supports this view. thus, c bl mice are relatively resistant to ectromelia (mousepox) infection, whereas balbc mice are susceptible, with a high mortality rate. the protection afforded by the vaccination programs, especially death from childhood infections in nonindigenous populations in developed countries, has dramatically changed the previous family pattern. at present, the family size is generally much smaller as parents expect all children to survive, and many adults are living much longer, well into their s or s. there are many known infectious agents for which vaccines are not yet available, but the biggest danger is emerging infectious diseases. a recent article on this topic lists about a dozen such agents ( ). table lists some of these agents where there is substantial evidence for their animal origin, showing them as zoonoses or vector-borne diseases. many induce a high mortality rate in humans. some such as sars (severe acute respiratory syndrome) has spread around the world. there were three influenza virus pandemics in the past century, the "spanish flu" causing million deaths due to the special properties of the hemagglutinin gene of the virus ( ). the appearance of a/h n in hong kong in and in neighboring countries in , and possibly the first indication (new york times, sept , ) that it may have recently spread from one person to another, has heightened concern. the maximum opportunity for a pandemic occurs when the bird virus infects a person currently infected with a human strain, allowing the different particles to enter the same cells and while replicating, to exchange genes. the hiv- , which causes aids, is also a very serious hazard as there have now been million infections worldwide. it jumped from non-human primates about yr ago, probably by the consumption of infected "bushmeat" and this form of transmission has occurred six more times since. in richer countries, it is controlled by a complex mixture of drugs. in the absence of treatment, the average time to death is yr but can be as long as yr, so it is difficult to know the exact death rate, but it is > %. there is a small group of long-term nonprogressers whose immune system keeps the hiv virus levels very low. in some african countries, which have about a % incidence of hiv infection, the average lifespan has dropped from yr to about yr. despite intensive efforts, it has so far not been possible to develop an effective vaccine. other infections listed in table have mortality rates varying from about % (sars) to between % and % (ebola and marburg hemorrhagic fevers). there is a number of infections called reemerging diseases. these are infections that were reasonably localized but have suddenly spread to other regions. although originally confined to one region, one example, west nile virus, is causing epidemics of encephalitis in the united states and russia due to migratory birds, and an abundance of both vector mosquitoes and susceptible local bird species in those two countries. as the population of the world grows and there is increasingly frequent interaction between mankind and wildlife, it is inevitable that new diseases and re-emerging diseases will continue to occur. efforts to rapidly identify, investigate, and control such outbreaks will continue to be coordinated by who, ably assisted by designated authorities in different countries such as the cdc in the united states. although special situations such as hiv/aids can be kept under control in rich countries by a cocktail of drugs, the basic global need is to be able to develop appropriate vaccines. this must continue to be a top priority if we and our descendants are to live fruitful lives vaccines advances in immunology: vaccines and vaccination rotavirus vaccine temperature sensitive mutant vaccines influenza vaccine-live current status of live, attenuated influenza virus in the us regulation of host immune responses by modification of salmonella virulence genes the use of complete genome sequences in vaccine design nyvac, a highly attenuated strain of vaccinia virus cocirculation and divergence of human influenza viruses pertussis vaccine chemoimmunologial studies on conjugated carbohydrate proteins. . immunological specificity of synthetic sugar-protein antigen preparation of polysaccharide-conjugate vaccines genetic detoxification of bacterial toxins vaccine perspectives from the vantage of hepatitis b the cellular basis for the lack of antibody responses to hepatitis b vaccine in humans assuring the quality and safety of vaccines. regulatory expectations for licensing and batch release vaccine protocols an epidemiological and clinical evaluation of guillain-barre syndrome reported in association with the administration of swine influenza virus vaccine acute encephalopathy followed by permanent brain injury or death associated with further attenuated measles vaccines vaccines today adverse events associated with childhood vaccination. evidence bearing on causality. institute of medicine indications and contraindications for vaccines used in the expanded programme of immunization increased mortality following high titer measles vaccines: too much of a good thing intussusception among infants given an oral rotavirus vaccine measles, mumps, rubella immunization, autism, and inflammatory bowel disease: an update. communicable disease intelligence mmr vaccine: the continuing saga smallpox and its eradication. world health organization new polio cases confirmed in guinea, mali and sudan. cases reported as kano, nigeria, resumes immunizations is global measles eradication feasible? perspectives for the elimination/eradication of diseases with vaccines crystal structure of a neutralizing human igg against hiv- : a template for vaccine design synthetic peptide vaccine design: synthesis and properties of a high density multiple antigenic peptide system efficacy trial of a malaria vaccine spf in gambian infants towards a vaccine for rheumatic fever: identification of a conserved target epitope on m protein of group a streptococci new multi-determinant strategy for group a streptococcal vaccine designed for the australian aboriginal population live viral vectors: vaccinia virus live viral vectors: construction of a replication-deficient recombinant adenovirus academic development of attenuated salmonella strains that express heterologous antigens expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox molecular medicine; dna vaccines dna vaccination: an update the immune system cytotoxic t cells recognise very early, minor changes in ectromelia-infected target cells unravelling the mysteries of molecular audit: mhc class i restriction the immunology of vaccination the role of antibody in controlling and/or clearing virus infections expression of cytokines by recombinant vaccinia viruses: a model for studying cytokines in virus infections in vivo the immune response to antigens; the immunological principles of vaccination the adjuvant effect of il- in a vaccine against leishmaniasis major interleukin mediates down regulation of antiviral cytokine expression and cytotoxic t lymphocyte responses and exacerbates vaccinia virus infection in vivo adjuvant formulations for experimental vaccines comparison of numerous delivery systems for the induction of cytotoxic t lymphocytes by immunization plant biotechnology: new procedures and applications oral vaccines derived from transgenic plants edible vaccines not ready for main course transcutaneous immunization: a human vaccine delivery strategy using a patch the powderjet particle-mediated epidermal delivery of dna vaccines. a new technology augmentation of human immunodeficiency virus type- neutralizing antibody by priming with gp recombinant vaccinia and boosting with rgp in vaccinia-naive adults generation of enhanced immune responses by consecutive immunization with dna and recombinant fowlpox virus hiv dodges one-two punch the use of complete genome sequences in vaccine design use of a whole genome approach to identify vaccine molecules affording protection against streptococcus pneumoniae infection guns, germs and steel. a short history of everybody for the past , years. vintage a biological role for the major histocompatibiliy antigens the foreign antigen-binding site and t cell recognition regions of class histocompatibility antigens the challenge of emerging and re-emerging infectious diseases enhanced virulence of influenza a viruses with the haemagglutinin of the pandemic virus key: cord- - gcpk x authors: koprowski, hilary title: vaccines and sera through plant biotechnology() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: gcpk x nan during the last years, more vaccines and sera have become available for prophylaxis or treatment of disease than during the preceding century. yet at the same time, the number of obstacles preventing the use of those biomedical discoveries for the benefit of mankind has risen almost proportionately. stricter regulations for safety and efficacy have led to increased cost of production of biomedicals, but secondly the price to the consumer has risen to a degree that now precludes prophylactic vaccination against some diseases on a global scale. in addition to the extraordinarily high price of new vaccines and sera, the method of administration of these products through syringe and needle presents another burden in their worldwide use. in , i organized a mass vaccination trial with my oral polio vaccine in the then belgian congo. two hundred and fifty thousand individuals were vaccinated in weeks by oral spraying. this became a model for worldwide polio vaccination-a project requiring only one oral administration of a product-to eradicate polio in the world. the global use of vaccine requires that the vaccines be inexpensive and easily maintained and distributed. after considering various alternatives of fulfilling the criteria established for a global approach to immunization, it has become clear that our only choice is the production of vaccines or other materials of biomedical importance in plants. the advantages of plants are quite clear. plants are very inexpensive to grow in mass; some can grow in almost any soil or climate in the world. various parts of plants (leaves, seeds, fruits, and chloroplasts) can also be used as vehicles for the biomedical products. production of biomedicals in plants and their administration to humans and animals also provides an added margin of safety as compared to biologicals produced in animal tissues. early concerns that plants might not be capable of producing biomedicals of animal origin, and that plant-specific glycosylation patterns might functionally alter plant-produced antibodies have proven unwarranted. basic methods to transfer foreign genese into plants are well established. in the first approach, the plant is transfected with a foreign gene in combination with the plant parasite, agrobacterium tumefaciens. in this constitutive approach, future generations of the same transgenic plant species heritably retain the capacity to express the original product. the product is isolated from the extract of leaves, seeds, etc. (table ). in the second approach, the gene is inserted into a plant virus (for example, alfalfa or tobacco mosaic virus), which is used in combination with a foreign antigen to infect plants. here, the final product is harvested from the plant in tandem with the plant virus. in both cases, purification of the vaccine or antibody is a rather simple procedure. the achievements in production of plant-derived vaccines and sera, can be classified in two categories: ( ) vaccines and antibodies for infectious diseases; ( ) vaccines and antibodies for cancer. table shows the list of infectious disease agents against which plant-derived products were made in the biotechnology foundation laboratories, in chronological order, respiratory syncytial virus (rsv), hepatitis b, hiv, rabies, anthrax, diphtheria, sars, and smallpox virus. one of the first vaccines produced in this system was against rsv, which causes disease of newborn children. we produced the vaccine antigen as a fusion protein with the capsid protein of alfalfa mosaic virus in tobacco. immunogenicity was tested in mice, which were either injected with or fed the plant-produced vaccine ( as compared to controls; high-titer antibodies against rsv were also induced. plant-derived vaccines for hiv and anthrax will be presented by dr. alexander karasev. scientists under the leadership of professor andrzej legocki in poznan, poland, have produced a vaccine against hepatitis b in transgenic lettuce. these scientists have shown that vaccines expressed in lettuce leaves can be ground, frozen, dried and powdered without loss of immunogenicity. observation of immune response in mice fed the plant-derived vaccine indicates that two feedings at -month intervals produce optimal immunogenic responses. rabies is a worldwide disease of humans and warmblooded animals. except for the time-honored pasteur type vaccine, all modern vaccines are too expensive for use on a worldwide scale. rabies antibody is essential in treating severe bites but is unavailable because it is not sufficiently lucrative for production by the pharmaceutical industry. rabies in dogs is a major, but not unique, source of infection in southeast asia and in many african countries. an inexpensive bait vaccine for dogs could lead to the eradication of rabies in those parts of the world. to express rabies vaccine in plants, we have used a recombinant alfalfa mosaic virus in spinach leaves. this virus expressed a chimeric peptide containing antigenic determinants of rabies virus glycoprotein, which elicits immune responses; and of nucleoprotein, which increases glycoprotein immunogenicity. the chimeric gene antigen was pcr-amplified and cloned for fusion with the gene encoding the coat protein of alfalfa mosaic virus. tobacco and spinach were the plants used to express the antigen. the presence of rabies virus protein in spinach leaves was checked by western blot and immunogenicity was tested in mice. then volunteers were fed g of spinach extract recombinant virus twice at -week intervals. half of the subjects responded to the oral administration of spinach as indicated by a vigorous booster response after a single injection of commercial vaccine. there is a global crisis in post-exposure treatment of rabies because of worldwide unavailability of the antirabies serum required for severe bites. thus, it was decided on an emergency basis to produce rabies antibody in plants. research conducted by dr. kisung ko, led to the production of a transgenic tobacco plant containing the heavy and light chains of human rabies antibody. the two chains recombined in the plants to produce a complete antirabies antibody, which was as effective as the original antibody in animals, before and after exposure to rabies (table ). two very interesting results emerged from the research with this antibody. first, leaf extract of the transgenic tobacco was found to be virtually free of nicotine and other alkaloids (fig. ) . second, the glycosylation pattern of the plant-derived antibody was considerably different from that of the animal tissue-derived antibody (fig. ) . mannose is the only glycan prevalent in the plantibody, yet the antibody was at least as efficacious as the native antibody produced in animals. table in vivo efficacy of mab p for post-exposure prophylaxis of hamster injected with a lethal dose of coyote rabies street viruses (dr. c. rupprecht, cdc) post-exposure treatment antibody a (iu/animal) vaccine (hdcv) b − + mab p ( ) / c / mab p ( . ) / / mab p ( . ) / / hrig ( ) / / untreated control / / ko et al. [ ] , pnas. a iu: international unit. b hdcv: (imovax, lot mo ) human diploid cell culture rabies virus vaccine, "−" and "+" indicate treatments without or with hdcv, respectively. c number of surviving hamsters/number of hamsters tested. strict guidelines regarding the amount and potency of antirabies antibody for use in post-exposure treatment has enabled calculation-based on the yield of antibody per tobacco plant-of plant acreage needed to produce a given number of antibody international units. if, as expected, kg of tobacco produces - mg of antibody, ha or . acres is needed to produce enough antibody for vaccination of , people. i leave it to accountants to calculate the production cost of such an antibody as compared to antibody produced in animal tissue. one of the many advantages of plants for production of biological products is their ability to express multiple genes in the same plant. vaccines against diphtheria, tetanus, and oral pertussis are the triad injected into infants during their first - months of life. a plan to substitute oral products for injectable vaccines was initiated through production of transgenic tobacco (as a model) and transgenic carrots expressing all three vaccines (table ). plants expressing current diphtheria toxoid have already been produced (fig. ) . the spike protein of sars coronavirus is presumed to be immunogenic. transgenic tobacco and tomato expressing the spike protein were generated (fig. ) . interestingly, only months elapsed between the introduction of the spike construct into the plant and the first observation of sars coronavirus mrna expression by the plant (fig. ) . theoretically, the vaccine could be obtained by growing large numbers of transgenic plants. it is doubtful whether other methods currently available would produce vaccine material against a newly discovered disease in such a short time. after decades of neglect, we are now witnessing a revived interest in immunological approaches to cancer treatment. thus, we sought expression of colorectal cancer anti-gen ga and of the corresponding - a antibody in plants. attempts were also made to express antibodies recognizing epidermal growth factor (egf) which is present at the surface of epithelial tumors in plants. the tobacco mosaic virus-based gene vector (p b) was used for expressing the - ga antigen. nicotiana betthamiana was infected with the construct and checked expression by western blot analysis of the leaf extract. a group of mice were then immunized with the plant-produced - antigen and, for comparison, another group with the same antigen expressed in baculovirus systems. sera obtained from both groups of mice recognized human colorectal cell lines expressing this antigen (fig. ) . a cross reactivity test capable of discriminating the two antigens, that produced in plants and that produced in baculovirus vector, ruled out any "contaminant" basis for the immune response. complement-dependent cytotoxicity assays of sera obtained from mice immunized with the plant-derived - antigen showed lysis of breast cancer cells, but not of antigenically unrelated melanoma cells (fig. ) . the plantderived - antigen also induced antigen-specific proliferation of t cells. using the same technique as for antirabies antibodies, we expressed - -reactive antibody - a in tobacco plants. purified leaf extract of these plants specifically recognized receptors present in human colorectal cancer cells. perhaps the most interesting results obtained from the study of the two plantibodies against rabies and cancer relate to the glycosylation pattern. the plant-derived antirabies heavy chain was fused to kdel (lysineasperagine-glycosamine-leucine) endoplasmic reticulum retention signal. as a result, the anti-rabies plantibody contained abundant mannose, whereas the plant-derived anticancer antibody contained less mannose and more nacetylglucosamine (fig. ) . despite the different glycosylation patterns of the two plantibodies and the difference of both patterns from those of the native antibody, their biological activity was equivalent to each other and to that of the native antibody. antibodies reactive with the epidermal growth factor were recently licensed to treat certain types of epithelial cancers. such antibodies were first developed at the wistar institute many years ago. they have recently been adapted to production in tobacco plants and within the near future might provide an inexpensive therapeutic tool in human cancer. however, plant production of vaccines and sera is not a simple procedure with assured success in each undertaking. one of the most important remaining problems is the low yield of the bioproduct in plants. to date, no magical solution to this problem has been found. codon optimization, careful approaches to harvesting and purifying plant products, use of plant parts such as chloroplasts to increase uptake of the material are but a few potential avenues to help increase the yield of the final product. at present, it is still difficult to produce sizeable amounts of plant-derived products. great strides have been made in our knowledge and expertise in the use of plants as hosts for production of biomedicals. table there are four stages of adopting new ideas the first is "it's impossible" the second is "maybe it's possible, but it's weak and uninteresting" the third is, "it's true and i told you so" and the fourth is, "i thought of it first" as shown in the last table (table ), a segment of the population remains to be convinced that the future of biologicals is linked with production in plants. some people will consider the facts and ultimately opt for acceptance. others will remain "true believers" in harmful effects caused by transgenic plants. their voices may become subdued or even silenced by increasing evidence that the future of global health improvement rests strictly in the progress made on the worldwide use of plants to combat diseases. function and glycosylation of plant-derived antiviral monoclonal antibody key: cord- -as v c h authors: wilder-smith, annelies title: dengue vaccine development: status and future date: - - journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: . /s - - - sha: doc_id: cord_uid: as v c h dengue, the most common arbovirus, represents an increasingly significant cause of morbidity worldwide, including in travelers. after decades of research, the first dengue vaccine was licensed in : cyd-tdv, a tetravalent live attenuated vaccine with a yellow fever vaccine backbone. recent analyses have shown that vaccine performance is dependent on serostatus. in those who have had a previous dengue infection, i.e., who are seropositive, the efficacy is high and the vaccine is safe. however, in seronegative vaccinees, approximately years after vaccination the vaccine increases the risk of developing severe dengue when the individual experiences a natural dengue infection. the world health organization recommends that this vaccine be administered only to seropositive individuals. current efforts are underway to develop rapid diagnostic tests to facilitate prevaccination screening. two second-generation dengue vaccine candidates, both also live attenuated recombinant vaccines in late-stage development, may not present the same limitations because of differences in the backbone used, but results of phase trials need to be available before firm conclusions can be drawn. dengue is increasingly frequent in travelers, but the only licensed dengue vaccine to date can be used only in seropositive individuals. however, the vast majority of travelers are seronegative. furthermore, the primary series of three doses given months apart renders this vaccine difficult in the travel medicine context. dengue is globally the most frequent arboviral disease, present in more than countries in the tropics and subtropics and poised to increase even further in terms of incidence and continued geographic expansion [ , ] , thereby also affecting international travelers [ , ] . the four dengue virus serotypes belong to the family of flaviviridae and are genetically distinct but still closely related. infection with any of the four dengue virus serotypes may be asymptomatic or may result in clinical manifestations ranging from a mild undifferentiated febrile syndrome to severe dengue. severe dengue is characterized by plasma leakage, hemorrhagic tendencies, organ failure, shock, and, occasionally, death [ ] . patients with a second dengue infection with a dengue serotype different from the first are at increased risk for severe dengue. the hallmark of severe dengue is capillary leakage leading to shock and, if not managed well, death. the pathomechanism of severe dengue is still poorly understood, although the most plausible hypothesis is antibody-dependent enhancement in secondary infections [ ] . because effective vector-control measures are not scalable or sustainable, community-based approaches have led to mixed results [ , ] , and promising novel strategies such as wolbachia are still under development [ ] , a dengue vaccine would appear to be the best intervention. the purpose of this review is to elaborate on the first licensed dengue vaccine and review second-generation dengue vaccines, both in the context of endemic populations as well as international travelers. according to modeling estimates, about - million dengue cases occur every year [ ] . the incidence of dengue has increased greatly, with the number of cases more than doubling every decade, from . million ( . - . million) apparent cases in to . million ( . - . million) apparent cases in , responsible for . million disability-adjusted life-years [ ] . in dengueendemic countries, approximately % of febrile episodes in children and adolescents are due to dengue, with a higher incidence in asia ( . episodes per person-years) compared to latin america ( . episodes per person-years); the percentage of dengue infections requiring hospitalization was % in asia versus % in latin america [ ] . many dengue infections lead to hospitalizations, which can overwhelm weak health care structures, in particular during times of outbreaks. given the unpredictability of outbreaks, the increasing magnitude and frequency of such outbreaks, and the current lack of highly effective and sustainable vector-control interventions, there is a clear indication for a dengue vaccine for endemic populations. several difficulties have hampered the development of a dengue vaccine. one challenge is the lack of an appropriate animal model and poor knowledge of correlates, both for protection and disease enhancement [ ] . but the biggest hurdle is the interaction among the four serotypes. as a tetravalent immune response is desired, when a mixture of all four serotypes in a tetravalent live attenuated vaccine is given, each component would need to independently result in four different monotypic immune responses that are solid to each serotype. this has, unfortunately, proven to be difficult to achieve. despite more than years of efforts using various vaccine platforms including inactivated, dna, and live vaccines, only live attenuated vaccines have entered phase trials. three live attenuated dengue vaccines are now in late-stage development, with one candidate having completed phase trials including longterm follow-up of years: cyd-tdv by sanofi pasteur, lyon, france, with the trade name of dengvaxia. cyd-tdv, a tetravalent live attenuated vaccine with a yellow fever d backbone, is the first dengue vaccine to be licensed. phase trials revealed a vaccine efficacy that depended on age, serostatus, and serotype but also showed a population-level benefit [ ] . interference manifested by asymmetric immunological responses to the mixtures of four dengue vaccine viruses was recognized as a possible reason for this varied vaccine performance [ ] . post hoc retrospective analyses of the long-term safety data using a novel nonstructural protein (ns ) antibody assay revealed an excess risk of severe dengue in those who were seronegative at baseline, which means those who were dengue-naïve at the time of administration of the first dose [ ] . this increased risk was observed starting from months after administration of the first dose. the reasons for the excess cases are not fully understood, but a plausible hypothesis is that dengvaxia may trigger an immune response to dengue in seronegative persons that predisposes them to a higher risk of severe disease, analogous to what is seen in natural secondary dengue infections. in other words, it is plausible that dengvaxia results in a "primary-like" silent infection (which live attenuated vaccines often elicit) [ ] . a subsequent infection with the first true wild-type dengue virus would then be a "secondary-like", clinically more severe dengue illness. it is not the vaccine itself that causes excess cases, but rather the vaccine's induction of an immune status that increases the risk that subsequent infections will be more severe. despite licensure in dengue-endemic countries to date, cyd-tdv has been introduced in only two subnational public health programs: those in the philippines and in brazil. after a media release in november about the safety concern for seronegative persons, the philippines decided to suspend its program, while brazil completed its program but has not expanded it. the media release resulted in a major public outcry in the philippines, with heightened anxiety and lack of confidence around vaccines in general [ ] , which led to the subsequent resurgence of measles in the philippines, reflecting the global resurgence of measles [ ] [ ] [ ] . communication around the introduction of any newvaccine, butespeciallythose vaccines with partial efficacy or complex vaccine performance, needs to be improved to avoid public distrust and lack in vaccine confidence. the world health organization recommends that for countries considering cyd-tdv vaccination as part of their dengue-control program, a prevaccination screening strategy is recommended so that only dengue-seropositive persons are vaccinated [ ] . the challenge is now to urgently develop and license rapid diagnostic tests to support such a prevac-cination screening strategy [ ] . in may , the u.s. food and drug administration (fda) approved cyd-tdv for use in seropositive individuals - years of age living in endemic areas of the united states. the european medicines agency also endorsed the use of this vaccine in seropositive individuals only. the world health organization has published guidance on evaluating the quality, safety, and efficacy of live attenuated dengue tetravalent vaccines, including the need for baseline blood samples from all participants for a priori analysis plans stratified by serostatus, as well as long-term follow-up for - years after the first dose [ ] . this document will guide vaccine developers in trial design and facilitate regulatory review to enable broader public health recommendations for second-generation dengue vaccines. indeed, the phase efficacy trials of the two second-generation dengue vaccines have incorporated all these requirements. furthermore, there is a need for the development of standardized end points for vaccine and other interventional trials, including the need for subsequent validation with prospective data sets [ ] . the complexity of developing moderate disease research end points for dengue is particularly challenging. two live attenuated dengue vaccines are now in phase trials. one such live attenuated dengue vaccine is being developed by takeda: denvax vaccine consists of an attenuated denv- (den -pdk- ), whereby three chimeric viruses containing the prm and envelope proteins of denv- , denv- , and denv- are inserted into the den -pdk- backbone. the difference from dengvaxia, therefore, is the presence of nonstructural (ns) proteins due to the denv backbone. the conserved ns proteins within the dengue backbone may well be required to generate t-cell-mediated responses to dengue infection, and antibodies against ns are associated with cross-protective humoral immune responses [ ] . this vaccine has performed well in phase and phase clinical trials, with high titers of neutralizing antibody to all four serotypes in nonhuman primates and humans, including cross-reactive t-cellmediated responses that may be necessary for broad protection against dengue fever [ , ] . the vaccine efficacy is currently being tested in approximately , recipients in phase trials in asia and latin america using a twodose regimen given months apart. efficacy data for the first months are imminent. the other tetravalent live attenuated dengue vaccine was developed by the u.s. national institutes of health (nih) and is currently in a phase trial in brazil, but it was also licensed to merck and various other vaccine manufacturers for further development outside brazil. this vaccine consists of three full-length dengue virus (denv) serotypes attenuated by one or more deletions in the ′ untranslated region with den Δ , den Δ , and den Δ , while the fourth component is a chimeric virus in which the prm and e proteins of denv- replace those of denv- in the den Δ background [ ] . this vaccine performed well and was safe in phase and phase trials [ ] . a single dose induced robust tetravalent antibody and cellular t-cell responses and resulted in % efficacy in a human challenge study [ ] . the capacity to elicit cd + cell responses closely mirrored those observed in a population associated with natural immunity [ ] . the advantages of these second-generation dengue vaccines are the inclusion of ns proteins of the dengue backbone and more convenient dosing, with reduced numbers of doses needed: while dengvaxia is licensed for three doses months apart, the takeda vaccine is currently being considered for two doses months apart and the nih vaccine for a single dose. whether these second-generation vaccines will provide balanced high protection against all four serotypes and thus overcome the serostatus-dependent problem of cyd-tdv remains unknown and can be addressed only by the longterm results of the pending phase trials. many dengue-endemic countries are commonly visited travel destinations, and therefore dengue has become a frequent cause of febrile illness among international travelers [ ] . an increase in hospitalizations and health care visits for dengue has been seen in american [ ] and european travelers [ , ] . geosentinel is an international network of travel medicine providers [ ] that has also reported an increase in dengue over the past decade [ ] . dengue can affect tourists, business travelers, expatriates [ , ] , pilgrims [ ] , and migrants, including those visiting friends and relatives [ ] , and can affect both adults and children [ , , ] . interruption of travel, premature return, hospitalization during or after travel, and outof-pocket expenses are the result [ ] . with an incidence of about - % for travelers to dengue-endemic countries [ ] , dengue is much more frequent than many of the other travel-associated vaccine-preventable diseases, such as hepatitis a, yellow fever, and japanese encephalitis [ , ] . vaccination would be of clear benefit to travelers, but the benefit versus risk needs to be clearly weighed [ ] . although vaccination is now licensed in europe by the european medicines agency and in the united states by the fda, and is also licensed in australia, it has not yet been endorsed for the travel medicine indication. furthermore, the only currently licensed dengue vaccine, cyd-tdv, should be used only in seropositive travelers [ ] . most travelers, however, are seronegative [ ] . furthermore, the dosing schedule of three doses months apart for cyd-tdv renders the use of such a vaccine difficult in the travel medicine context. a safe and efficacious vaccine that can be used regardless of serostatus would enhance the use of a dengue vaccine in travelers [ ] . travelers should be advised to take daytime personal protective measures against mosquito bites [ ] . given the unpredictability of dengue outbreaks, the increasing magnitude and fre- bundesgesundheitsbl · : - https://doi.org/ . /s - - - © springer-verlag gmbh deutschland, ein teil von springer nature abstract dengue, the most common arbovirus, represents an increasingly significant cause of morbidity worldwide, including in travelers. after decades of research, the first dengue vaccine was licensed in : cyd-tdv, a tetravalent live attenuated vaccine with a yellow fever vaccine backbone. recent analyses have shown that vaccine performance is dependent on serostatus. in those who have had a previous dengue infection, i.e., who are seropositive, the efficacy is high and the vaccine is safe. however, in seronegative vaccinees, approximately years after vaccination the vaccine increases the risk of developing severe dengue when the individual experiences a natural dengue infection. the world health organization recommends that this vaccine be administered only to seropositive individuals. current efforts are underway to develop rapid diagnostic tests to facilitate prevaccination screening. two second-generation dengue vaccine candidates, both also live attenuated recombinant vaccines in late-stage development, may not present the same limitations because of differences in the backbone used, but results of phase trials need to be available before firm conclusions can be drawn. dengue is increasingly frequent in travelers, but the only licensed dengue vaccine to date can be used only in seropositive individuals. however, the vast majority of travelers are seronegative. furthermore, the primary series of three doses given months apart renders this vaccine difficult in the travel medicine context. severe dengue · cyd-tdv · antibodydependent enhancement · travelers · live attenuated chimeric dengue vaccine das dengue-virus, das am meisten verbreitete arbovirus, stellt weltweit eine zunehmende ursache für morbidität dar, auch bei reisenden. nach jahrzehntelanger forschung wurde der erste impfstoff gegen dengue-fieber zugelassen: cyd-tdv, ein tetravalenter, attenuierter lebendimpfstoff auf basis des gelbfieber-impfvirus ("backbone"). neuste analysen haben gezeigt, dass die performance des impfstoffs vom serostatus abhängig ist. bei menschen, die bereits eine dengue-infektion hatten und seropositiv sind, ist die wirksamkeit hoch und der impfstoff sicher. bei seronegativen impflingen erhöht der impfstoff jedoch im fall einer nachfolgenden dengue-wildvirusinfektion das risiko für eine schwere dengue-erkrankung etwa jahre nach der impfung. die weltgesundheitsorganisation empfiehlt daher, den impfstoff nur an seropositive menschen zu verabreichen. derzeit wird intensiv an der entwicklung von schnelltests gearbeitet, um das screening vor der impfung zu erleichtern. zwei dengue-impfstoffkandidaten der zweiten generation, beide ebenfalls attenuierte rekombinante lebendimpfstoffe, befinden sich in der spätphase der entwicklungspipeline und könnten aufgrund der unterschiede der verwendeten "backbones" nicht dieselben limitierungen aufweisen; es müssen aber die ergebnisse der phase- -studien abgewartet werden, um dies sicher beurteilen zu können. dengue-fieber tritt immer häufiger bei reisenden auf. die überwiegende mehrheit der reisenden ist jedoch seronegativ, weshalb bei ihnen der bisher einzige zugelassene impfstoff gegen dengue-fieber nicht eingesetzt werden kann. darüber hinaus sind für die grundimmunisierung drei impfdosen nach dem schema - - monate erforderlich, wodurch der einsatz dieses impfstoffes im reisemedizinischen kontext schwierig ist. schweres dengue-fieber · cyd-tdv · antibody-dependent enhancement · reisende · attenuierter chimärer dengue-lebendimpfstoff quency of such outbreaks, and the high morbidity of this disease that can overwhelm already weak health care systems, a dengue vaccine is urgently needed. furthermore, dengue vaccines would be indicated for international travelers, in particular those who have already had one primary infection in order to reduce the risk of severe dengue as a result of a second infection during repeat travel. the second-generation dengue vaccines may overcome some of the shortcomings of the first licensed dengue vaccine; however, these vaccines will not be available for at least another couple of years. phase efficacy trials of second-generation dengue vaccines need to be analyzed and stratified by serostatus, and longterm data of at least years following the first dose should be available before such vaccines are licensed. a safe and efficacious vaccine that can be used regardless of serostatus would enhance the use of a dengue vaccine in travelers. epidemic arboviral diseases: priorities for research and public health evidence-based risk assessment and communication: a new global dengue-risk map for travellers and clinicians dengue infection in international travellers visiting bali, indonesia imported dengue fever in east london: a -year retrospective observational study camino verde (the green way): evidence-based community mobilisation for dengue control in nicaragua and mexico: feasibility study and study protocol for a randomised controlled trial mitigating diseases transmitted by aedes mosquitoes: a cluster-randomised trial of permethrin-impregnated school uniforms wolbachia and the near cessation of dengue outbreaks in northern australia despite continued dengue importations via travellers the global burden of dengue: an analysis from the global burden of disease study symptomatic dengue in children in asian and latin american countries participants in the summit on dengue immune correlates of p. immune correlates of protection for dengue: state of the art and research agenda efficacy and long-term safety of a dengue vaccine in regions of endemic disease evaluation of interferences between dengue vaccine serotypes in a monkey model effect of dengue serostatus on dengue vaccine safety and efficacy deliberations of the strategic advisory group of experts on immunization on the use of cyd-tdv dengue vaccine vaccine confidence plummets in the philippines following dengue vaccine scare: why it matters to pandemic preparedness measles cases hit record high in europe measles and human movement in europe measles: a global resurgence pre-vaccination screening strategies for the use of the cyd-tdv dengue vaccine: a meeting report clinical development and regulatory points for consideration for second-generation live attenuated dengue vaccines development of standard clinical endpoints for use in dengue interventional trials: introduction and methodology a recombinant, chimeric tetravalent dengue vaccine candidate based on a dengue virus serotype backbone immunogenicity and safety of one versus two doses of tetravalent dengue vaccine in healthy children aged - years in asia and latin america: -month interim data from a phase , randomised, placebocontrolled study robust and balanced immune responses to all dengue virus serotypesfollowingadministrationofasingledose of a live attenuated tetravalent dengue vaccine to healthy, flavivirus-naive adults development of tv /tv , a single dose, highly immunogenic live attenuated dengue vaccine; what makes this vaccine different from the sanofi-pasteur cyd vaccine? the human cd + t cell responses induced by a live attenuated tetravalent dengue vaccine are directed against highly conserved epitopes human cd + t cell responses to an attenuated tetravalent dengue vaccine parallel those induced by natural infection in magnitude, hla restriction, and antigen specificity severe dengue in travellers: pathogenesis, risk and clinical management upward trend in dengue incidence among hospitalized patients, united states attack rates of dengue fever in swedish travellers sentinel surveillance ofimporteddengueviatravellerstoeurope to : tropnet data from the denguetools research initiative sentinel surveillance in travel medicine: years of geosentinel publications ( - ) geosentinel surveillance of illness in returned travelers business travelassociated illness: a geosentinel analysis dengue fever among israeli expatriates in delhi, : implications for dengue incidence in delhi, india illness in travelers visiting friends and relatives: a review of the geosentinel surveillance network dengue epidemic in touba, senegal: implications for the grand magal pilgrimage for travelers paediatric dengue fever diagnosed through parents' epidemiologic report and preventive strategy during the acute phase of infection morbidity among israeli paediatric travellers a prospective study on the impact and out-of-pocket costs of dengue illness in international travelers travel vaccine preventable diseases-updated logarithmic scale with monthly incidence rates what proportion of international travellers acquire a travel-related illness? a review of the literature redefining priorities towards graded travel-related infectious disease research serostatus-dependent performance of the first licensed dengue vaccine: implications for travellers the first licensed dengue vaccine: can it be used in travelers? what is the prospect of a safe and effective dengue vaccine for travelers mosquito repellents for the traveller: does picaridin provide longer protection than deet? key: cord- -j assowx authors: guibinga, ghiabe h.; sahay, bikash; brown, heather; cooch, neil; chen, jing; yan, jian; reed, charles; mishra, meerambika; yung, bryan; pugh, holly; schultheis, katherine; esquivel, rianne n.; weiner, david b.; humeau, laurent h.; broderick, kate e.; smith, trevor r.f. title: protection against borreliella burgdorferi infection mediated by a synthetically engineered dna vaccine date: - - journal: nan doi: . / . . sha: doc_id: cord_uid: j assowx lyme disease is the most common vector-borne disease in north america. the etiological agent is the spirochete borreliella burgdorferi, transmitted to mammalian hosts by the ixodes tick. in recent years there has been an increase in the number of cases of lyme disease. currently, there is no vaccine on the market for human use. we describe the development of a novel synthetically engineered dna vaccine, pld targeting the outer-surface protein a (ospa) of borreliella burgdorferi. immunization of c h/hen mice with pld elicits robust humoral and cellular immune responses that confer complete protection against a live borreliella burgdorferi bacterial challenge. we also assessed intradermal (id) delivery of pld in hartley guinea pigs, demonstrating the induction of robust and durable humoral immunity that lasts at least year. we provide evidence of the potency of pld by showing that antibodies targeting the ospa epitopes which have been associated with protection are prominently raised in the immunized guinea pigs. the described study provides the basis for the advancement of pdl as a potential vaccine for lyme disease control. lyme disease is highly prevalent in europe and north america. the etiological agents are spirochetes of the borreliella burgdorferi group and related species. these bacteria are transmitted by ticks, with small mammals serving as reservoirs. in the united states, borreliella burgdorferi is the most prevalent lyme diseaseassociated species, and the number of reported cases have doubled over the past years. thirty-thousand cases of lyme disease are reported to the cdc in the us each year, and predicts the number of clinician-diagnosed cases exceeds , a year. , the majority of patients respond adequately to antibiotic treatment. however, a significant number of patients develop persistent symptoms with local and systemic manifestations that vary in severity and duration that can last for months to years, termed post-treatment lyme disease syndrome (ptlds). the armamentarium available to the medical community to tackle the increasing incidence of lyme disease, and to effectively prevent or treat ptlds is limited. to develop effective countermeasures against lyme disease several targets and mechanisms have been investigated. multiple studies have demonstrated that the transmission of borreliella burgdorferi from tick to mammalian host can be interrupted with antibodies targeting the ospa antigen. [ ] [ ] [ ] [ ] ospa mediates the attachment of the spirochetes to the tick midgut, and its expression is downregulated during the blood-feeding process when the bacteria migrate from the midgut to salivary glands; consequently, anti-ospa antibodies harbored in the host can block bacterial transmission during hematophagy. vaccines targeting ospa have been developed. the recombinant ospa protein vaccine, lymerix demonstrated efficacy, and was briefly available on the market. the efficacy of this recombinant protein ospa vaccine was modest, % after immunizations, and yearly boosts were required. furthermore, there were concerns surrounding reports of immune cross-reactivity between an epitope present in ospa and the human lfa- antigen, and the postulation that vaccination may initiate an autoreactive immune mechanism leading to lyme-associated arthritis. additionally, poor representation of the vaccine in the media and lack of public enthusiasm resulted in poor sales. the vaccine was ultimately pulled from the market in . post hoc assessment revealed the vaccine to be safe with no difference in arthritis cases between vaccinated and controls, and the postulation that the ospa vaccine-elicited cross-reactive anti-lfa- responses that could mediate arthritis was ultimately withdrawn. , currently, there is no lyme disease vaccine available for human use. newer engineered synthetic nucleic acid vaccines delivered by advanced cellectra® adaptive electroporation have proven to be highly effective in eliciting robust and long-lasting immunity in multiple clinical studies against infectious disease targets, including zika, ebola and mers. [ ] [ ] [ ] in these studies, rapid seroconversion at durable high titers, neutralizing antibodies, passive transfer protection to animal challenges from human sera, and induction of cd and cd t cells has been observed in most subjects. [ ] [ ] [ ] furthermore, in other therapeutic studies involving hpv-related cervical infections, strong t cell responses and t cell invasion into the infected tissues as well as viral clearance have been observed. , based on efficacy in clearing hpv infection in the cervix, a phase iii study is in progress. collectively these studies have established a strong safety database in over individuals, and demonstrated that this advanced platform can reproducibly drive humoral and cellular immunity in humans with relevance to the prevention of infections or clearance of infectious disease. potentially the induction of t cell responses by this vaccine platform may afford improved b cell help to generate durable humoral immunity. the current manuscript describes the development of a synthetic consensus (syncon®) ospa dna vaccine, pld in combination with cellectra® in vivo delivery technology. the data were benchmarked against an ospa and ospc chimeric recombinant protein vaccine, vanguardcrlyme, an approved veterinary vaccine, henceforth referred to as crlyme. the data show that pdl dna vaccine elicits robust humoral and cellular immune responses in mice against the ospa antigen that confers protection against borreliella burgdorferi spirochete tissue colonization following a live bacterial challenge. also, intradermal immunization of guinea pigs with pld leads to a durable and robust antibody response which persists for at least a year. moreover, antibodies from the immunized guinea pigs effectively compete for ospa binding with a borreliacidal human mab - . , human embryonic kidney t cells were cultured in dulbecco modified eagle's medium (dmem) and supplemented with % heat-inactivated fetal bovine serum (fbs), along with u/ml of penicillin and streptomycin each. cells were incubated at °c and % co . for immunofluorescence analysis, . × cells were seeded per well into a -well cell culture plate, and transfected with either gfp (transfection control), pvax or pld using the lipofectamine transfection kit (thermofisher, waltham, ma) following the manufacturer's instructions. fortyeight hours later transfected cells were washed thrice with pbs, and incubated overnight at °c with a mouse anti-ospa monoclonal antibody (clone lsbio, ls-c ). cells were washed with pbs and incubated with alexa fluor® donkey anti-mouse secondary antibody (life technologies, a- ). the nucleus was stained with ′, -diamidino- -phenylindole (dapi) at room temperature for min. images were captured using imagexpress pico automated cell imaging system (molecular devices). a proprietary synthetic consensus sequence was generated to target ospa serotype (st ). (pld ). the sequences were codon and rna optimized. , the resulting nucleotide sequences were synthesized, digested with bamhi and xhoi, and cloned into the expression vector pvax under the control of the human cytomegalovirus immediate-early promoter and a bovine growth hormone polyadenylation signal (genscript, piscataway, new jersey). female hartley guinea pigs ( - weeks) weighing - g and c h/hen female mice ( - weeks) were grouped and housed with ad libitum access to food and water. animals were housed at acculab (san diego, ca ) or the university of florida (department of infectious diseases and immunology, college of veterinary medicine, university of florida, gainesville, fl ). all housing, handling and treatment protocols were approved and handled according to the standards of the institutional animal care and use committee. µl dna vaccine pld or pvax formulations were administered by g needle intramuscular to the tibialis anterior muscle and this step was followed by cellectra® in vivo electroporation using the p array. the cellectra® ep delivery consists of two sets of pulses with . amp constant current. second pulse set is delayed s. within each set, there are two ms pulses with a ms delay between the pulses. the vaccine dose was µg of plasmid dna per injection. each group was dosed on week , and for a total of three immunizations. hartley guinea pigs were immunized either by intradermal injection (mantoux technique) of pld on the abdominal left flank followed by cellectra®-ep with the p array (as described above) or subcutaneous (sc) injection of a recombinant protein vaccine. treatments consisted of a total of three immunizations each at a dose of . mg in . ml volume of pld administrated at weeks , and . the crlyme vaccine group of animals was immunized sc with µl of vaccine, commercially available for lyme disease prevention in dogs (vanguardcrlyme by zoetis). briefly, spleens from mice were collected individually in ml of rpmi media supplemented with % fbs (r ), processed into single-cell suspensions with a gentle macs dissociator (miltenyi biotec, auburn, ca), then centrifuged at rpm for min. cell pellets were resuspended in ml of ack lysis buffer (life technologies, carlsbad, ca) for min at room temperature, and ml pbs was then added to dilute the lysis buffer. the samples were again centrifuged at rpm for min, cell pellets resuspended in r , and then passed through a um nylon filter before use in elispot assay. to assess cellular ifnγ responses, mouse elispot assays were performed using commercial mabtech pre-coated ifny elispot kits (mabtech - apw- , sweden). the -well elispot plates were washed times with sterile x pbs and blocked with r medium for h. the r was removed and , mouse splenocytes in r media were added to each well and incubated at °c in % co the presence of peptide pool , or , each consisting of -mers overlapping by amino acids, together these pools spanned the length of ospa protein, dmso (negative control), cona (positive control for mouse). after - h, plates were washed and developed according to the manufacturer's protocols, and ifn-γ positive spots were counted by an automated elispot reader (ctl, shaker heights, oh). ospa peptide-reactive responses were calculated by subtracting the number of spots in dmso from peptide wells. results (cellular responses or interferon-gamma elispot responses) are shown as individual animal spot-forming units (sfu)/ pbmcs. elisas were performed to determine sera igg antigenbinding titers. nunc elisa plates were coated with µg/ml recombinant ospa protein (immune technology, new york, ny) in dpbs overnight at °c. plates were washed three times then blocked with % bsa dpbs with . % tween for h at °c. plates were then washed and incubated with serial dilutions of mouse or guinea pig sera and incubated for h at °c. plates were again washed and then incubated with hrp conjugated-species specific secondary antibodies ( in , dilution) and incubated for h at °c. after the final wash, plates were developed using sureblue tmb component peroxidase substrate and the reaction stopped with tmb stop reagent (kpl). plates are then read at nm within min using a spectramax plus microplate reader (molecular devices). cielisas were performed to determine the presence of antibodies in the serum after ospa immunization in guinea pigs competed with - human monoclonal antibody known to bind the ospa epitope targeted by the la- antibody. , elisa plates were coated with . µg/ml recombinant ospa protein (immune technology, new york, ny) in dpbs overnight at °c. plates were washed three times then blocked with % bsa dpbs with . % tween for h at °c. plates were then washed and incubated with serial dilutions ( in ) of guinea pig sera and incubated for h at °c. plates were washed and then incubated with . µg/ml of - human antibody for h at room temperature. hrp conjugated-anti-human kappa light chain (bethyl laboratories) was added for h at room temperature. after the final wash, plates were developed using sureblue tmb component peroxidase substrate and the reaction stopped with tmb stop reagent (kpl). plates were then read at nm within min using a spectramax plus microplate reader (molecular devices). low-passage b. burgdorferi strain was maintained at °c in barbour-stoenner-kelley medium containing % normal rabbit serum (bsk media, complete; sigma aldrich, st. louis, mo; cat # b ) and then temperature-shifted to °c. increased expression of ospc as a virulence factor and presence of ospa as a target for the vaccine was confirmed by coomassie staining of whole borrelial lysates separated by sds-page. mice were challenged with × bacteria injected subcutaneously at the sternum as described previously. bacteria were confirmed ospa+ by pcr. all the mice were bled days before the challenge from a facial vein using. four weeks postinoculation, mice were anesthetized for intracardiac bleeding, and euthanized for tissue collection that included joints, heart, ear and bladder, respectively. dna was isolated from the excised tissues using a quick-dna miniprep kit (zymo research; cat # d ). bacterial burden in tissues was determined using genomic dna as previously described. briefly, ng of extracted genomic dna was along with b. burgdorferi-specific flab primers ( nm) and probe ( nm) or primers ( nm) and probe ( nm) directed against the mouse actin gene, as described elsewhere. standards were developed to quantify the number of b. burgdorferi flab and mouse actin copies across a range of °- copies. amplification data were acquired and analyzed using the mic real-time pcr system, and quantification of target dna was accomplished as previously described. freshly grown temperature-shifted b. burgdorferi strain was counted, and × bacteria incubated with µl two-fold serially diluted serum from the study animals for h in ul tubes. at this stage, we included naïve serum from c h/hen mice as a negative control as well as a heat-inactivated counterpart. µl of the aliquots (in triplicate) were inoculated in . ml microfuge tubes with . ml of bsk complete media and sealed with parafilm to create microaerobiosis. these tubes were kept at °c for weeks. the cultures were monitored daily for change in ph, and if ph drops down, the bacteria were visualized. the presence of bacteria visualized in all the tubes under a dark-field microscope. the highest dilution of the serum that prevented the growth of b. burgdorferi was recorded as a bactericidal titer of the serum. data were presented as mean ± sem for all data points. the statistical difference between individual groups was assessed using mann whitney test with statistical significance set at p < . . data that support the findings of this study are available from the corresponding author upon reasonable request. a synthetic consensus sequence was generated to target ospa serotype (st ), the vaccine is intended for north american use. thirteen full-length protein sequences from orthologous open reading frames of borreliella burgdorferi were collected. a consensus was generated by alignment of the ospa protein sequences using clustalw, sharing . %- % identity with each individual ospa sequence. three residues of the consensus were replaced with the corresponding residues of an ospa protein sequence from b. afzelii in order to eliminate an epitope homology with human leucocyte function antigen (lfa- ) (y f, v t, t k). the pld construct was generated using this modified ospa consensus sequence (figure (a) ). syncon® ospa st (design a, pld ), a potentially exposed n-terminal cys was deleted from the full length modified ospa consensus to avoid possible posttranslational modifications and covalent conjugations and the endogenous n-terminal leader sequence was replaced with an immunoglobulin e (ige) leader sequence to enhance the translation of the construct which has been demonstrated to increase the immunogenicity of the translated product. the final sequence was codon and rna optimized and cloned into the pvax plasmid (see figure (b)). expression of the ospa was confirmed in vitro using immuno-fluorescence of t cells transfected with pld (figure (c) ). we evaluated the immunogenicity of the ospa antigen expressed after pld vaccination in c h/hen mice. immunogenicity analyses revealed a robust immune response mediated by the pld vaccine after a single dose, and the response was boosted after subsequent doses (figure (a) ). on a different set of animals, we examined the cellular immune response to pld vaccination against three pools of overlapping peptides, which together spanned the length of the ospa antigen. here, groups of mice received three doses of either pld , pvax or crlyme, recombinant protein ld vaccine. all animals were female, we did not address the sex of the animals as a biological variable. two weeks after the final immunization cellular ifn-γ response was evaluated to ospa peptide recall. as illustrated on figure (c), pld elicited a significantly stronger cellular response than the control (pvax ) and crlyme groups, with the epitopes in pool being where the cellular response predominantly targeted. we evaluated the efficacy of pld in a live bacterial challenge. schematic of the challenge study is illustrated in figure (a). c h/hen mice are susceptible to borreliella burgdorferi infection, and the model recapitulates some pathophysiology features of human lyme disease, such as sub-acute arthritis, which is a consequence of the bacterial spread and colonization in the animal's organs and tissues. , , three weeks after the final immunization mice were inoculated with , of borreliella burgdorferi strain , and weeks later bacterial burden was evaluated by pcr in a double-blinded manner. as shown in figure (b) , only the pvax control group presented with detectable bacterial burden in joints, heart and bladder as well as ear tissues. in groups of mice that received one, two or three dose of the pld vaccine or three doses of crlyme vaccine, a bacterial presence in the tissues tested was not detected. we proceeded to determine the presence of functional anti-b. burgdorferi antibodies in the animals' serum employing a bacterial killing assay. this assay measures the dilution of serum at which the bacterial growth is enabled. as illustrated in figure (c) bacterial growth was enabled at a serum dilution of approximately for pld and crlyme immunized mice indicating robust bactericidal activity. these results from both bacterial tissue burden and bacterial growth inhibition assay demonstrate the efficacy of pld and crlyme vaccines against borreliella burgdorferi infection in the c h/hen mouse needle challenge model. we assessed the immunogenicity of pld in guinea pigs, an established model for intradermal vaccine delivery. , we measured the ospa antigen-binding titers of igg in the serum up to weeks after initial vaccination. animals in group one received . mg of pld vaccine via id-cellectra®ep delivery on weeks , and . group two received µl ( / dose recommended for dogs) of crlyme vaccine administered subcutaneously at weeks , and . humoral immune responses against ospa antigen were measured up to week . a robust humoral response against ospa was detected across both groups. we proceeded to evaluate the quality of the anti-ospa humoral immune response by assessing the generation of antibodies targeting the la- epitope. to accomplish this we used the la- equivalent mab - in a competition assay with the serum from immunized guinea pigs. similar assays have previously been shown to be a pre-clinical correlate of protection. the humoral response elicited by pld and crlyme vaccines demonstrated the ability to compete and inhibit the binding of human mab - ( figure ). these results underscore the potential of the pld vaccine to provide durable antibody titers to ospa epitopes associated with correlates of protection. previous studies employing dna vaccines encoding ospa have shown promise in small animal studies, - however they have yet to advance to clinical testing. here we have employed advanced delivery technology of in vivo ep to enhance the immunogenicity of the lyme disease vaccine pld after administration to the c h/hen mouse muscle or hartley guinea pig skin. such technology increases in vivo gene expression up to fold, and the subsequent cellular and humoral immune responses elicited are substantively enhanced. here, the described study is the first to employ this vaccine delivery technology as a development concept for lyme disease prevention. we show that a synthetically engineered vaccine pld elicits robust and durable anti-ospa igg levels, and confers protection against borreliella burgdorferi infection in a c h/hen mouse needle challenge model. the pld vaccine induced a robust anti-ospa immune response in c h/hen mice ( figure ). the ability to induce strong t cell immunity is likely due to the intracellular antigen expression pathway associated with dna vaccines, assuring antigen processing in the mhc class i and ii pathways, driving broad t cell responses. furthermore, the activation of toll-like receptors by the un-methylated cpg motifs contained within synthetic nucleotides, and the activation of the intracellular sting pathway may provide innate signals that lead to enhanced cellular responses. , activated t cells can provide help to b cells leading to increased antibody levels and memory cell development, enhancing the humoral immunity. in reference to lyme disease immune responses, an ospa t cell epitope that promotes ospa antibody response has been described, supporting the importance of anti-ospa t cell responses in promoting antibody production. data here show one dose of pld generates high antibody titers (see figure (a,b) ), enough to provide protection against bacterial challenge (figure (b) ). we recently reported intradermal delivery of an ebola gp synthetic dna vaccine resulted in % seroconversion and induction of t cell responses in % of subjects within weeks, by simple dose sparing id formulations. in the current study, we evaluated the immunogenicity of the pld dna vaccine in guinea pigs employing intradermal delivery. guinea pigs represent an excellent id immunization model, because unlike mice they possess a well-defined epidermis. an important aspect of targeting vaccine delivery to the skin is that it is rich in professional antigen-presenting cells that are capable of mediating host immune responses. id delivery of pld elicited a robust humoral immune response (figure ). one limitation in this current study was that it did not consider the role of ospc-targeted immune responses of the comparator vaccine. crlyme is a chimeric vaccine containing ospa st and a chimeric protein of multiple ospc antigens. unlike ospa, ospc is not downregulated and is present on spirochetes entering the host, and vaccines containing ospc antigens may have the ability to elicit responses to spirochetes which have entered the host. crlyme has a usda-approved -month duration of immunity on its label. in contrast to ospa, over different ospc genotypes have been defined, with many circulating in the usa. the inclusion of ospc antigens may be considered in formulations. the ability of serum from ospa-immunized guinea pigs to competitively inhibit the binding of ospa specific- - mab was also evaluated ( figure ). this type of competition assay can serve as a correlate of protective anti-ospa response as previously shown for the murine la- antibody. , the la- antibody has been identified as protective against borreliella burgdorferi infection in passive transfer and active immunization studies targeting ospa protein. , , - mab has demonstrated the equivalent capability of mediating protection against borreliella burgdorferi infection in antibody transfer experiments as the la- mab. , in the context of the present study, antibodies from sera of pld immunized guinea pigs provided inhibition of - mab ospa binding. these data indicate the immune response elicited by pld can generate antibodies which can bind to the same ospa epitopes as mabs which have been shown to prevent transmission of lyme disease spirochetes. while there is an obvious unmet need for a lyme disease vaccine, it is important to consider the hurdles faced by previous vaccine programs which were ultimately not successful. the efficacy of this recombinant protein ospa vaccine was only % after immunizations and required yearly boosts to maintain protective antibody titers. furthermore, there was speculation about side effects due to the vaccine containing an epitope with homology to a human epitope in the lfa- protein. with pld we have aimed to enhance the durability of the antibody titers which would potentially result a reduction in the frequency of boosts required to maintain protective serum titers. additionally, we replaced the epitope with homology to human lfa- (figure (a) ). preliminary surveys are ongoing to assess the current market for a lyme disease vaccine. with the increasing incidence and impact of this disease, we are optimistic about the demand and the level of utilization of such a medical countermeasure. future studies are planned to address the limitations of the scope of work presented here. these will focus on efficacy testing in borreliella burgdorferi infected tick challenges, which will reflect a more natural exposure route than the bacterial needle challenge model reported here. studies will be designed to test vaccine efficacy after tick challenge in the dog, a large animal model which exhibits similarities in ld pathophysiology with humans. we thank maria yang and team for the manufacturer of plasmid dna vaccines. ghg, hb, nc, jc, jy, ch, by, hp, lh, keb and trfs are employees of inovio pharmaceuticals and as such receive salary and benefits, including ownership of stock and stock options, from the company. dbw has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees and board services. remuneration received by dbw includes direct payments or stock options, and in the interest of disclosure he notes potential conflicts associated with this work with inovio and possibly others. in addition, he has a patent dna vaccine delivery pending to inovio. all other authors report there are no competing interests. this work was supported financially by inovio pharmaceuticals, inc. the emergence of lyme disease vital signs: trends in reported vectorborne disease cases -united states and territories incidence of clinician-diagnosed lyme disease therapy for lyme arthritis: strategies for the treatment of antibiotic-refractory arthritis long-term protection of mice from lyme disease by vaccination with ospa elimination of borrelia burgdorferi from vector ticks feeding on ospa-immunized mice vaccination against lyme disease: past, present, and future the outer surface protein a (ospa) vaccine against lyme disease: efficacy in the rhesus monkey a vaccine consisting of recombinant borrelia burgdorferi outer-surface protein a to prevent lyme disease. recombinant outer-surface protein a lyme disease the lyme vaccine: a cautionary tale relationship between immunity to borrelia burgdorferi outer-surface protein a (ospa) and lyme arthritis intradermal syncon(r) ebola gp dna vaccine is temperature stable and safely demonstrates cellular and humoral immunogenicity advantages in healthy volunteers safety and immunogenicity of an anti-zika virus dna vaccine -preliminary report safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase , open-label, single-arm, dose-escalation trial augmentation of cellular and humoral immune responses to hpv and hpv e and e antigens by vgx- safety, efficacy, and immunogenicity of vgx- , a therapeutic synthetic dna vaccine targeting human papillomavirus and e and e proteins for cervical intraepithelial neoplasia / : a randomised, double-blind, placebo-controlled phase b trial identification of a borrelia burgdorferi ospa t cell epitope that promotes anti-ospa igg in mice anti-ospa dna-encoded monoclonal antibody prevents transmission of spirochetes in tick challenge providing sterilizing immunity in mice pre-exposure prophylaxis with ospa-specific human monoclonal antibodies protects mice against tick transmission of lyme disease spirochetes increased immune response elicited by dna vaccination with a synthetic gp sequence with optimized codon usage multiple effects of codon usage optimization on expression and immunogenicity of dna candidate vaccines encoding the human immunodeficiency virus type gag protein cd signaling restrains chronic inflammation through induction of p -mapk/socs-dependent tolerance signaling through cd attenuates the inflammatory response to borrelia burgdorferi, the agent of lyme disease borrelia burgdorferi changes its surface antigenic expression in response to host immune responses rapid and sensitive quantification of borrelia burgdorferi-infected mouse tissues by continuous fluorescent monitoring of pcr an effective second-generation outer surface protein a-derived lyme vaccine that eliminates a potentially autoreactive t cell epitope dna vaccination and gene therapy: optimization and delivery for cancer therapy lyme borreliosis in laboratory mice gene expression profiling provides insights into the pathways involved in inflammatory arthritis development: murine model of lyme disease characterization of guinea pig t cell responses elicited after ep-assisted delivery of dna vaccines to the skin development of an intradermal dna vaccine delivery strategy to achieve single-dose immunity against respiratory syncytial virus reactivity with a specific epitope of outer surface protein a predicts protection from infection with the lyme disease spirochete, borrelia burgdorferi protective immunization with plasmid dna containing the outer surface lipoprotein a gene of borrelia burgdorferi is independent of an eukaryotic promoter an ospa-based dna vaccine protects mice against infection with borrelia burgdorferi dna vaccines expressing a fusion product of outer surface proteins a and c from borrelia burgdorferi induce protective antibodies suitable for prophylaxis but not for resolution of lyme disease electroporation delivery of dna vaccines: prospects for success cpg dna as a vaccine adjuvant cpg oligonucleotides as adjuvants for vaccines targeting infectious diseases cd + t cells promote antibody production but not sustained affinity maturation during borrelia burgdorferi infection diversity of the lyme disease spirochetes and its influence on immune responses to infection and vaccination identification of borrelia burgdorferi ospc genotypes in canine tissue following tick infestation: implications for lyme disease vaccine and diagnostic assay design incomplete protection of hamsters vaccinated with unlipidated ospa from borrelia burgdorferi infection is associated with low levels of antibody to an epitope defined by mab la- monoclonal antibodies specific for the outer surface protein a (ospa) of borrelia burgdorferi prevent lyme borreliosis in severe combined immunodeficiency (scid) mice key: cord- - wv s authors: viret, jean-francois; glück, reinhard; moser, christian title: development of a sars vaccine: an industrial perspective on the global race against a global disease date: - - journal: expert rev vaccines doi: . / . . . sha: doc_id: cord_uid: wv s nan the constant threat of novel emerging diseases is well recognized by the scientific community, public health authorities and the private sector. scenarios for efficient crisis management and rapid effective disease control, for example, against an influenza pandemic, have been developed by the world health organization (who global influenza program [ ] ), the european community [ ] and the us center of disease control, atlanta [ ] . however, prior to the current situation, few countries had implemented strategic plans at the national level and such scenarios have fortunately never before been validated on a global scale. importantly, next to the undisputed practicality of strategic planning against the next influenza pandemic, which has to start during the current interpandemic period, a good level of preparedness of appropriate national and supranational structures may positively affect the speed and efficiency of the response to any emerging disease with pandemic potential. the current outbreak of severe acute respiratory syndrome (sars) is an unprecedented example of an emerging viral disease on a global scale. beside the dramatic consequences of this crisis, important lessons will be learnt on how the world community is capable of appropriate reactions. in november , a novel pathogen, of then unknown origin and characteristics, started spreading among the human population in china reaching at least three continents within a short period of time. efficient control of the outbreak during the initial phase was impeded by the lack of knowledge about the etiology of the disease and consequently by the reluctant or inadequate measures taken by local and national authorities. the high profile of sars in the international news media contributed to early public disease awareness but also caused fear in both affected and unaffected populations, placing additional political and economic pressure on authorities to act on the threat despite of an insufficient basis for informed decisions. international organizations, most prominently the who, played a crucial role in international co-ordination and local support of control measures as well as in information and know-how management. once the global scale of the outbreak became fully apparent, the scientific community, supported by the who, committed to '...the development of a vaccine against the pathogen is severely impeded by the current fragmentary information on viral pathogenicity and the lack of adequate animal models and correlates of protection in humans.' an unparalleled collaborative effort. within a very short period of time a novel coronavirus (sars-cov) was established as the cause of sars [ , ] and the knowledge about this novel virus continues to expand at breathtaking speed on an almost daily basis. as a consequence, more focussed measures could be taken to prevent further spread of the virus and treat sars patients. however, travel restrictions, improved diagnostic tools and hygiene measures remain the main instruments in controlling the spread of sars. currently ( th june, ), days after the outbreak of the disease, the who have reported over sars cases and over deaths, the short-term measures proved instrumental in controlling the pandemic. the rapid availability of diagnostic tests, as well as the development of specific therapeutic drugs and vaccines, depends on the commitment of the pharmaceutical industry, which became seriously involved at the time the causative agent was identified. on april , , the us health authorities held a meeting on invitation in washington dc, usa. individuals present at this gathering of selected global vaccine manufacturers were urged to use every endeavour in the rapid development of candidate vaccines against the sars agent. indeed, in view of the reported rapid and largely uncontrolled spread of the disease to three continents, the situation appeared to request a drastic emergency plan including the implementation of a broad range of technologies to maximize the chances of success. whereas the necessity of a global response to such a global threat appears painstakingly obvious to everybody, including private industry's decision-makers, the situation vaccine manufacturers have to deal with on such occasions is rather unusual. what is currently expected from the private sector in the race against sars is the immediate investment of considerable resources in the fast-track development plan of candidate vaccines against a largely uncharacterized pathogen of unknown origin and with unknown perspectives. the classical industrial decision-making process to initiate a new development project for a therapeutic drug or a vaccine relies on the careful analysis of a series of premises, such as: • the quality of the relevant results collected during the exploratory research phase, particularly on the identification of the correct immunogen; the formulation; the eventual adjuvant; the vaccine's effectiveness in established animal models for the appropriate route of application; and on the chances that it will be safe and effective in humans • profitability calculations -for example net present value, break-even point, return on investment -based upon estimations of the potential market and time to reach it, existing competition, likelihood of success, uniqueness of strategy, existing intellectual property (ip) or potential to generate new ip a particular critical aspect of the initiation of any new project is the necessary competition for resources and infrastructure with the ongoing research and development portfolio. this especially holds true for the use of pilot gmp facilities (i.e., as soon as clinical lots are to be manufactured). in the case of sars, the risks associated with the development of a vaccine increase under time pressure and due to the very limited basis of knowledge on the pathogen. it may be appropriate to remember that the usual time-to-market for a new drug or vaccine is - years, starting from the early feasibility research phase and including preclinical (process and immunological profile) development, clinical development and registration, as well as manufacturing plant planning and set-up [ ] . therefore, even if due to a very special emergency situation the timelines for preclinical and clinical development may be shortened, the question remains whether there will be any need for these products at the time they are ready for the market. more specifically, in spite of the fortunate capability to propagate the sars-cov on a well-accepted cell substrate (vero), the development of a vaccine against the pathogen is severely impeded by the current fragmentary information on viral pathogenicity and the lack of adequate animal models of persistent infection and correlates of protection in humans. the origin of sars-cov is still unclear but animals in close contact to humans are a likely source. in that sense, sars-cov is possibly not really a novel virus but rather a newly discovered one following its adaptation to a new host species, namely humans. despite the potential of coronaviruses to mutate rapidly and to cross species barriers the known strains are generally species-specific and genetically stable. aside from humans, coronaviruses have been found in other mammals (pig, cat, dog, cattle, mouse) and birds (chicken), causing a wide variety of diseases (respiratory, gastrointestinal, hepatitis, immunopathology) depending on the species [ ] . the pathogenic potential of coronaviruses varies from moderate to deadly and mutations occurring during the course of infection can dramatically alter the virulence, as documented for feline coronavirus (fecv). several veterinary vaccines against animal coronaviruses are on the market and the experience with these products provides useful information for the design of a sars-cov vaccine. for instance, a number of vaccines against infectious bronchitis virus (ibv) are widely used in chicken farming. in cattle, bovine coronavirus (bcv) vaccines are administered to pregnant cows in order to protect new-born calves from severe diarrhea, presumably via maternal antibodies [ ] . cats are vaccinated via the mucosal route with a live-attenuated fecv. on the other hand, pre-existing antibodies against fecv in cats are not necessarily protective and can even aggravate the disease via antibody-dependent enhancement (ade) of the infection [ ] . the existing veterinary vaccines are all based on the classical vaccine approach. namely, live-attenuated or whole inactivated viruses are administered either via the parenteral or mucosal route. '...despite the numerous hurdles waiting on the road, the threat of a globally emerging disease certainly justifies unconventional and highrisk (in terms of investment and outcome) approaches.' for a sars vaccine this approach may seem attractive at first sight, both with regard to speed and costs of development and production, and it has been used successfully in the past (e.g., live attenuated smallpox, oral polio, measles, rubella, mumps, yellow fever; or killed hepatitis a and influenza). however, when considering the current regulatory, safety and cgmp requirements for vaccines, a whole-virus vaccine approach is associated with significant drawbacks, both for safety reasons (delicate balance between attenuation and immunogenicity; genetic stability, i.e., risk of vaccinecaused disease as observed for sabin's oral polio vaccine and vaccinia-based smallpox vaccine) and for manufacturing issues (scaleup production at bl safety level). for this reason, a subunit-or nucleic acid-based approach would be more favorable. due to the early availability of the full viral genome sequence, the quick development of prototype vaccine candidates based on strategies similar to hepatitis b surface antigen produced in yeast or the expression of sars antigens in well-characterized, live-attenuated viral and bacterial vectors may be envisaged. however, even if such vaccines prove to be immunogenic they may not be protective, or may even lead to ade-mediated pathology. in order to meet the urgent demands for a sars vaccine, a fasttrack development may be supported by all parties involved but bares the risk that not all safety aspects can be assessed prior to administration to a large population, especially not for the longterm. thus, the main issues to be critically addressed are dealing with vaccine safety and may be summarized in two questions: how to achieve an acceptable balance between requested speed and safety aspects? how to assess quickly but reliably immunogenicity and efficacy of candidate vaccines or more specifically, in the absence of a relevant animal model, what is the minimal acceptable fast-track toxicity program ethically acceptable before going to phase i trial? it has to be stressed that safety remains a major concern in vaccinology, not only for the well-being of the vaccinees themselves but also due to the fact that the public perception of vaccines in general would be strongly affected by negative news on the safety of a new vaccine, being one with a high benefit/risk ratio as an efficacious sars vaccine would be. in conclusion, the global scientific community reacted with unprecedented speed and efficiency to the news that an unknown agent causing life-threatening disease had emerged in asia. essential information, for example the genome sequencing, was rapidly generated and immediately made publicly available. the industry in general is certainly ready to rise to the challenge posed by sars with regard to a vaccine, particularly as health authorities have urged it to invest a lot of effort in commercially high-risk fasttrack research programs. indeed, despite the numerous hurdles waiting on the road, the threat of a globally emerging disease certainly justifies unconventional and high-risk (in terms of investment and outcome) approaches. however, due to the exceedingly high possibility of failure, appropriate incentives are urgently needed from major national and international organizations. grants contributing to the early high-risk phase of the projects (feasibility studies up to clinical phases i and ii) may allow an increase in the number of approaches investigated and thereby maximize the overall chances of success. undoubtedly, sars will provide a number of valuable lessons for the management of future emerging diseases with pandemic potential. a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome vaccine development: the long road from initial idea to product licensure enhancement of passive immunity with maternal vaccine against newborn calf diarrhea antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever world health organization eurosurveillance project, a european tribune to exchange information on communicable diseases the authors would like to thank c griot for scientific advice and ian c metcalfe for his editorial skills and critical reading of this manuscript.'undoubtedly, sars will provide a number of valuable lessons for the management of future emerging diseases with pandemic potential.' key: cord- -p ysi authors: davis-wurzler, gina m. title: update on current vaccination strategies in puppies and kittens date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: p ysi vaccines remain one of the practitioner’s greatest tools in preventing disease and maintaining individual and population health. this article is an update to “current vaccination strategies in puppies and kittens” published in veterinary clinics of north america, small animal practitioner, in may . there are now comprehensive guidelines readily available for small animal practitioners regarding canine and feline pediatric (and adult) vaccination recommendations. perhaps more importantly, there is an increased dialogue regarding all aspects of preventive medicine, of which vaccination is only a small, yet significant portion; and an increased drive to provide scientific evidence for developing vaccination recommendations. recommendations, and adverse events; as such, a comprehensive discussion is not possible here. as veterinarians we should look forward to the ongoing growth of this area of interest within clinical practice and within the research community, to eventually provide practitioners with answers currently sought by pet owners and veterinarians alike. it is far better to prevent than experience disease. this tenet should be the philosophy and goal of every veterinarian and every pet owner. for decades the veterinary profession has diligently educated pet owners about the benefits of preventing infectious disease, so well that there has been a significant decline in many of these diseases, in large part attributable to the development and use of effective vaccines. veterinary practice staff members have done remarkable jobs sending reminder cards to ensure that canine and feline patients are current on their vaccinations. in fact, vaccines have become such a priority that many pet owners are inclined to forfeit other, indicated medical care in lieu of vaccines lest their beloved pets fall behind on their vaccine schedule. veterinarians should commend themselves on a job well done, and commend pet owners for such conscientious stewardship of their pets. now, however, the veterinary community must reflect on what has been accomplished, and make decisions for current and future patient care based on scientific, rational merit. with the advent of knowledge on demand (ie, the internet), pet owners have access to information regarding all issues of animal care. however, such information may not be accurate. it is our duty to educate pet owners; in fact, it should be seen as an opportunity. who better to disseminate knowledge about veterinary medicine to the general public than veterinarians? no other group of individuals is as equipped with knowledge, skills, and insight as the veterinary community. to adequately discuss and understand how to make appropriate choices regarding pediatric vaccinations, a brief review and discussion of terms relative to basic immunology are warranted. passive transfer of immunity occurs when maternal antibody is transferred by the dam or queen to the fetus via the placenta, which occurs minimally in dogs and cats. it also occurs during initial suckling through the ingestion of colostrum, which has more significant effects in these species. this maternal immunity does provide initial protection against many pathogens, but of course depends on the health and immune status of the mother and the health of the fetus and neonate. although this may result in temporary protection for the neonate, in the long term it may be deleterious to that individual's health by essentially keeping the animal naïve to different antigens (eg, maternal antibody interference with vaccination of the neonate). maternal or passive immunization is effective in protecting neonates for the first several weeks of life, but begins to decline and lose the ability to protect against diseases rapidly as the maternal antibodies are degraded through natural catabolic processes. between the ages of and weeks, depending on multiple factors (including species, amount of maternal antibody produced, transferred, and absorbed, and the individual health status of the neonate), most puppies and kittens have maternal antibody levels below protective levels. however, if present at high enough levels, maternal antibodies can interfere with the neonate's ability to respond to vaccination, as the circulating maternal antibody within the puppy or kitten may effectively respond to and neutralize the vaccine antigen, or render it ineffective by preventing recognition of the antigen by the immune system. this is one reason why multiple, sequential vaccines are recommended in kittens and puppies until davis-wurzler they are at least to weeks of age. , of importance is that maternal antibodies can interfere with immunization, although the level of maternal antibody present may not be protective against pathogens. a functioning immune system is composed of multiple parts. innate immunity is the oldest (evolutionarily), least specific, and most immediate (in terms of response to potential invaders/pathogens) form of immunity. macrophages, neutrophils, dendritic cells, and natural killer (nk) cells combined with numerous products produced by these cells comprise the innate immune system. examples of some of the chemical components produced and released by these cells in response to microbial invasion include lysozyme, complement, various cytokines such as tumor necrosis factor a and interleukins, and various vasoactive molecules such as histamine. active immunization is the process of the individual responding to an antigenic stimulus appropriately, by either natural infection or vaccination. active immunization is processed through the acquired immune system. the main types of acquired immunity are cell-mediated immunity and antibody, or humoral, immunity. cell-mediated immunity is predominantly directed against pathogens that typically are obligate, intracellular organisms. examples include viruses, some obligate intracellular bacteria, some fungi, and protozoa. t lymphocytes are the predominant effector cells, and depend on foreign protein (antigen) being presented to them before they can take effect against the pathogens; thus, multiple cell types are involved in forming cellmediated immunity. antibody or humoral immunity is predominantly directed against pathogens that can survive outside the host, or at least survive extracellularly. examples include most bacteria, fungi, protozoa, and helminths. multiple cells act in concert to confer humoral immunity as well, but the primary effector cell is the b lymphocyte. having stated this, in actuality humoral immunity is extremely important in protection against viral infections, and is intricately and definitively dependent on competent cellmediated immunity. kittens and puppies will have varying degrees of ability to respond to antigens, whether resulting from natural or vaccine exposure, based on antigen load, route of exposure, antigenic virulence, genetics of the individual animal, and levels of persistent maternal immunity. in naïve animals whose maternal immunity has declined sufficiently so as not to interfere with an immune response; the first vaccine should stimulate a primary immune response (priming of the immune system). this initial exposure and recognition process and the ability to produce antibody to respond to the antigen typically takes to days; however, the maximum response takes up to weeks. this primary response must not be confused with the animal having been immunized. a subsequent dose of vaccine (exposure) will lead to immunologic memory. subsequent exposures to the same antigen elicit a stronger response: a greater amount of antibody is produced and the subsequent response is more rapid. this process is known as the secondary or anamnestic immune response, which results in immunity. although multiple cell lines are involved in this response, subsets of t and b lymphocytes known as memory cells preserve the host's ability to recognize and respond to antigens to which the animal had previously been exposed. to design, recommend, and actuate an effective plan for each patient, a practitioner must have familiarity with multiple variables. those variables include duration of protection conferred on the neonate by the mother; the typical length of time maternal antibody may persist and pose interference with the young animal's ability to respond fully to a vaccine; and the length of time needed for an appropriate response. in vaccination strategies in puppies and kittens addition, knowledge of the various diseases that pose risks to pediatric patients and knowledge of available safe, efficacious vaccines is critical. in essence, each patient must be assessed as an individual within the population to provide optimal wellness over the lifetime of each individual, as well as the population. this rationale has led to the concepts of core and noncore vaccines, terms commonly used when discussing vaccination within the veterinary field. criteria for assigning vaccines into these categories, and a third category, "generally not recommended," are based on: ( ) morbidity and mortality associated with the specific disease (does the organism cause serious illness or does it cause a mild, transient disease that may pose only minimal risk to the individual or population?); ( ) the prevalence and/or incidence rate of the disease (although a specific disease may not commonly be seen, the organism is ubiquitous in the environment and therefore poses risk to the individual or population); ( ) the risk of the individual for exposure to the disease (indoor-only animal vs free-roaming individual, regional variations of occurrence); ( ) the efficacy of the vaccine (does the vaccine prevent infection or simply ameliorate some signs or length of disease?); ( ) the risks associated with administering the vaccine (are the risks associated with that vaccine greater than the risk of the disease?); ( ) the potential for zoonotic disease; ( ) the route of infection or transmissibility. , , - when these criteria are assessed, general guidelines may be generated for the individual practitioner and the veterinary community at large. again, guidelines are not to be thought of as absolutes, nor are they to be used to establish standard of care. simply stated, they are tools for each of us to use to promote optimal wellness for our patients when considering all factors affecting the individual's health (environmental, organismal [both pathogen and host], owner concerns, and current vaccine technologies). , , - multiple vaccines are available for canine and feline patients, although most fall within basic categories. assignment of vaccine products (which are considered biological agents, not drugs, and are therefore assessed and approved under the united states department of agriculture [usda] animal and plant health inspection service rather than the food and drug administration) into these categories is based on how the product is created. simply stated, modified live virus (mlv) vaccines are vaccines created by altering (attenuating) the pathogen in some way so that it is no longer able to cause serious or clinical disease in the targeted species. killed vaccines are vaccines produced by inactivating the pathogen completely, rendering it incapable of reproducing and thereby unable to cause disease. the third category of vaccines consists of recombinant vaccines, of which there are multiple types, and this category itself has subcategories. these vaccines use genetic technologies to either introduce genetic material directly into the host (no vector, eg, purified subunit vaccines or type i recombinant, is used), alter the genetic material to change its virulence (gene deletion, type ii recombinant), or incorporate genetic material from the desired pathogen into an attenuated vector organism (eg, feline rrabies [r recombinant], type iii recombinant). , within the near future, multiple new technologies are likely to provide even more choices, potentially providing patients with better protection against disease with minimal vaccine-associated risks. a more recent discussion for categorizing vaccines has evolved, and assigns vaccines to of groups: infectious or noninfectious. simply stated, infectious vaccines include those biologics that have the ability to enter host cells and undergo replication within the host (ml, rcanary poxvectored vaccines). noninfectious vaccines do not have the ability to undergo replication within the host. for a comparison between vaccine types, the reader is referred to table . vaccines are available in single-dose and multiple-dose (tank) vials. the use of singledose vial vaccines is highly recommended in these species. conversely, the use of multiple-dose vials is discouraged because of the increased risk of contamination and the inability to assure consistent levels of antigen and adjuvant in individual doses from a single vial. , multivalent vaccines are not recommended in cats other than the core feline vaccine designed to protect against feline panleukopenia, feline herpesvirus i, and feline calicivirus. owing to increased inflammation at the site of multivalent vaccines, all other vaccines should be given as a separate vaccine, at the indicated site (see later discussion on feline core and noncore vaccines). , allowing vaccines to acclimatize to room temperature before administration, particularly in cats, is recommended, as the administration of cold vaccines was found to have an increased association for tumorigenesis in cats. use of sterile, single-use syringes is also recommended, as vaccines may become inactivated and/or ineffective with exposure to various products used to clean and sterilize syringes. mixing of more than vaccine within a syringe should not be performed because of the potential for inactivation of vaccine material, in addition to increasing the amount of antigen deposited within a single site. moreover, administration of reconstituted vaccines (mlv r) should be done within hour of reconstitution or otherwise discarded, owing to the potential inactivation of product and loss of efficacy. the practitioner is advised to always follow the manufacturer's directions for dose and route of administration. using a topical product parenterally or splitting doses should never be done. a full dose is required to stimulate the immune system; there is no medical basis for giving a smaller dose to a toy breed dog, and this practice could lead to vaccine failure in that animal. if done with a rabies vaccine the practitioner is not following federal requirements, which carries potential legal implications. , the interval between various vaccines, whether using the same product serially in the initial series or whether using different products in an adult animal, should never less than to weeks. interference between the first product administered and a second vaccine product may lead to failure to optimally respond to the second vaccine. the exact mechanism of this interference is unknown, but may be associated with interferon produced by cells processing an mlv agent, or by transient immunosuppression by an mlv agent. multiple vaccines administered at the same time do not appear to elicit this interference and is therefore an acceptable practice. , the reader is referred to tables and for comparison between pediatric canine and feline core, noncore, and generally not recommended vaccines. the diseases that fall within this category carry high rates of morbidity and/or mortality, are of public health concern, or are readily transmissible or may be ubiquitous in the environment. in addition, safe, efficacious vaccines are available and either provide sterile immunity (prevent infection) or confer a high degree of protection (do not prevent infection, but may confer protection such that the animal will not develop clinical signs of disease). , essentially, the vaccines that fall within this category are recommended for each individual within the population regardless of the animal's lifestyle or locale. canine distemper virus (cdv), an enveloped morbillivirus, has been well controlled because of the widespread vaccination programs over the last several decades. however, the disease still persists and, in addition to high virulence, it is readily vaccination strategies in puppies and kittens transmissible. infection with the virus causes respiratory, gastrointestinal, and neurologic signs, and is often fatal. the distemper vaccine is commonly administered as part of a multivalent product. the general recommendation is to use a modified live or recombinant, multivalent product (cdv, canine adenovirus type ii [cav-ii], canine parvovirus [cpv]) beginning at to weeks, and to give serial vaccines every to weeks until the puppy has reached to weeks of age. , , many studies support the improved ability of recombinant vaccines to overcome maternal antibody interference in comparison with modified live virus vaccines. , most puppies will receive or distemper vaccinations, depending on the age at which they are first presented to the veterinarian. however, it is the interval between or the timing of the vaccinations, rather than the number, that is important. serial vaccinations help to increase the likelihood of a complete response of the patient and thereby decrease the risk of vaccine failure that may occur when only vaccine is administered. in addition, by eliciting a secondary immune response, they may help to increase the level of circulating antibody and decrease the lag time between exposure to an antigen and achievement of maximal antibody level. potential causes for vaccine failure include: a modified live vaccine that was improperly stored and therefore has lost its efficacy; the vaccine was improperly administered (wrong route or accidental loss of vaccine onto the skin of the patient); the patient's immune system did not respond (the immune system may have been responding to another antigenic challenge or the vaccine may have been given too soon after a previous vaccine); or maternal interference. in theory, if a puppy were kept sequestered from exposure to this virus, modified live distemper vaccine administered after weeks of age would confer protection for at least year. , however, in reality most pet owners are not inclined to isolate their puppies for the first months of life, nor should they. early socialization is an important part of families bonding with their puppies. exposure to various people, other dogs, and new places helps decrease behavioral problems in the young adult and mature dog. as long as the last distemper vaccine is administered after weeks of age, the puppy should be able to mount a strong active response and fully overcome any residual maternal antibody. the current recommendation is to have the puppy return year later (when approximately months old) for another distemper vaccine. after the first annual vaccination, triennial immunization is recommended, regardless of vaccine type used. , , canine adenovirus there are types of adenovirus that cause disease in canine patients. canine adenovirus type i (cav-i), a nonenveloped virus in the family adenoviridae, causes the potentially fatal disease infectious canine hepatitis. clinical signs include fever, depression, vomiting and diarrhea, and potential petechiation and ecchymotic hemorrhage secondary to hepatic dysfunction. in addition, uveitis and renal disease are associated with infection with this virus. cav-ii causes respiratory tract disease. cav-i is associated with severe, potentially fatal disease, and protection against this disease is recommended. transmission is via the oronasal route and exposure to infected secretions. cav-ii infection typically results in mild self-limiting disease and is therefore considered to be a noncore disease; however, the modified live vaccine designed for prevention of cav-i has been associated with adverse effects such as uveitis and corneal edema (an arthus reaction, similar to effects caused by natural infection). , the current recommendation is to use the cav-ii modified live virus product, as it stimulates the immune system to protect against both cav-i and cav-ii, without the associated adverse reaction caused by the type i vaccine. , , the modified live adeno-type ii virus is typically included in a multivalent injection (as mentioned earlier) and is therefore usually administered at intervals of to weeks, beginning between and weeks of age and ending between and weeks old. a vaccination year later is recommended before instituting triennial vaccinations. cpv is a nonenveloped type parvovirus. the predominant form currently causing infection in the united states is type b, but other subtypes exist and cause disease elsewhere. because the virus is nonenveloped, it may exist (outside of a host) under certain environmental conditions, and is somewhat resistant to many disinfectants. transmission is via the fecal-oral route, and clinical signs include lethargy, anorexia, pyrexia, vomiting, and diarrhea (typically hemorrhagic). young animals appear to be at highest risk for developing severe, life-threatening disease. the current recommendation for vaccination is to use a multivalent mlv vaccine beginning at to weeks and to repeat the vaccine at intervals as already stated (every - weeks, until the puppy is - weeks old). in the past there was concern that certain breeds may have been at increased risk for contracting and developing severe parvoviral disease (doberman pinschers, rottweilers), but it is generally agreed that these breeds will mount an appropriate response to a quality product if the last vaccine is given between and weeks of age. , , there is, however, a small population of dogs that is genetically unable to respond to vaccination against cpv , regardless of the number of vaccinations (nonresponders). one benefit of having a well-vaccinated population is that even those nonresponders are at decreased risk of exposure and subsequent infection by parvovirus, based on strong herd immunity. studies using mlv cpv b strains showed a higher antibody response to cpv and cpv b, and were better able to overcome maternal antibody interference than the cpv -strain vaccines used , ; however, all cpv vaccines currently available should produce strong immunity in immunocompetent dogs. immunization year after completing the initial puppy series is recommended, with subsequent triennial vaccinations. , , there is also emerging evidence that weimaraner puppies are at increased risk of developing a severe form of hypertrophic osteodystrophy (hod) in association with vaccination with mlv distemper, adenoviral, and parvoviral products. the exact vaccination strategies in puppies and kittens mechanism is unknown, but the current recommendation is to use killed products in this breed for their pediatric vaccinations, and consider starting vaccinations when they are slightly older. rabies virus, an enveloped virus in the rhabdoviridae family, is capable of infecting all mammals. because it is an enveloped virus, it is not stable in the environment and is readily inactivated by most common disinfectants. the virus is transmitted through infected saliva, most commonly from a bite by an infected animal. clinical signs range from anxiety or other vague behavioral changes to pica, dysphagia, photophobia, and paralysis. because of the zoonotic potential and implications regarding public health, canine vaccination programs are strongly regulated and enforced. the current recommendation is to vaccinate puppies using a killed-virus vaccine at a minimum of or weeks of age. state regulations vary as to the minimum age for canine rabies vaccination: in california the legal minimum age of canine vaccination against rabies is weeks. a second rabies vaccine (killed product) is administered year later and then annually or triennially thereafter, depending on local regulations. , it is the practitioner's professional responsibility for knowledge of and adherence to regional laws regarding rabies vaccination frequency. vaccines in the noncore category may have limited efficacy, or the organism causing disease is not readily transmissible or may have limited geographic distribution or prevalence. in addition, the diseases these vaccines are designed to prevent may be so mild or self-limiting that the risks associated with administering the vaccines may be greater than the actual disease. lastly, some vaccines may interfere with common screening methods for disease detection, and are therefore not recommended unless absolutely warranted for a specific individual. it is the burden of the practitioner, along with the pet owner, to make decisions regarding which, if any, of the noncore vaccines should be administered to a puppy. , [ ] [ ] [ ] leptospirosis a bacterial pathogen that causes acute hepatic and renal disease, leptospirosis is typically transmitted through urine of infected animals (reservoir hosts include dogs, rats, wildlife, and livestock), and in contaminated water. there are at least different species (leptospira interrogans and leptospira kirschneri) that can infect dogs, with multiple serovars (variants of the same species) of l interrogans causing disease in dogs. although these organisms have the potential to cause serious disease, dogs are not likely to be at risk in a mostly urban, controlled environment (housed in a fenced yard with no exposure to wildlife or livestock). however, a dog that frequents rural environments or has exposure to waterways or livestock is definitely at risk of infection and should therefore be protected against the disease. again, the initial puppy appointments should involve a through history and include the owner's plans for the dog's future use. if an owner brings a labrador retriever puppy to the veterinarian for "whatever vaccines he needs," it is up to the practitioner to ask "will he be a hunting dog, will he be used in field trials, will he be exposed to wildlife and waterways?" the border collie who lives on a working sheep ranch surely should be vaccinated appropriately against leptospirosis. conversely, a long-haired miniature dachshund who will spend her days on her owner's lap in an urban setting will be at minimal risk of exposure and, therefore, vaccination is most likely not warranted. in essence, regional distribution, seasonality (increased prevalence during and immediately following the rainy season), and lifestyle of the puppy will be factored into the decision as to whether the puppy should be vaccinated. if the decision is made to vaccinate against leptospirosis, the general recommendation is to wait until the puppy is at least weeks old, at which time a killed or purified subunit vaccine is administered. infection is serovar specific, and no cross-protection is seen between different serovars; therefore, vaccination with as many serovars known to cause disease in a given region is recommended. an initial series of vaccinations should be administered to weeks apart and repeated at least annually thereafter, as long as the risk of exposure to the agent exists. the recommendation to wait until the puppy is at least weeks old before administering the leptospirosis vaccine is based on the increased potential for adverse events associated with killed vaccines, and to increase the likelihood of a complete immune response. , bordetella bordetella bronchiseptica is a bacterial agent that causes infectious tracheobronchitis. infection with this agent may occur in concert with other agents infecting the respiratory tract (canine parainfluenza virus [cpiv], cav-ii). transmission occurs via direct contact or through aerosolized microdroplets from infected dogs, and is most likely to occur under crowded conditions such as boarding and grooming facilities and dog-show venues. the current recommendation is to vaccinate puppies at risk a minimum of week before potential exposure with a combination vaccine containing both an avirulent live bacterin for b bronchiseptica and a modified live cpiv. the vaccine can be administered to puppies as young as to weeks of age, but is generally not indicated unless the puppy is in a kennel environment. many organized puppy socialization and obedience classes commonly require proof of vaccination against bordetella at the time of enrollment or before beginning the course. the general consensus is that intranasal vaccines are superior to parenteral vaccines, as they stimulate rapid local immunity (which is not affected by persistent maternally derived antibody). , intranasal vaccines should never be given subcutaneously, owing to the potential for severe (in some cases fatal) reactions (fig. ) . if the puppy will be intermittently exposed throughout the year (traveling to shows, boarding or grooming facilities) the vaccine should be repeated every months to annually. as already stated, cpiv may occur in concert with other respiratory tract agents. the vaccine recommendations are as stated for b bronchiseptica if indicated. there are vaccination strategies in puppies and kittens multiple products available, but the product currently recommended is the combination of intranasal vaccine containing a modified live parainfluenza virus with an attenuated b bronchiseptica bacterin. intranasal vaccines can be used in puppies aged to weeks for individuals at high risk of exposure (depending on vaccine manufacturer label restrictions). for optimal protection, the vaccine should be administered every months to annually if indicated. alternatively many multivalent, parental products containing modified live cdv, cav-ii, cpv, and parainfluenza are available and appropriate for use. , borreliosis borrelia burgdorferi is a vector-borne, spirochete bacterium responsible for lyme disease (borreliosis). transmission occurs when an infected tick (various species within the ixodes genera, also referred to as hard ticks) bites and remains attached to a host, in this case a puppy. direct, horizontal transmission is not likely to occur, so the risk to humans and other pets is thought to be minimal. if a puppy has a significant burden with infected ticks, it of course increases the exposure to others in the household but, as ticks typically do not reattach once they have taken a complete meal, the risk is considered to be fairly small unless appropriate tick control is not instituted. vaccination to protect against lyme disease is controversial, as the duration of immunity and degree of protection provided by vaccination is unknown, and vaccination with some vaccines interferes with standard screening diagnostics. therefore, vaccination against lyme disease is warranted only if a puppy will be expected to be at high risk for tick exposure, and only if it lives in a borrelia-endemic area. there are killed and recombinant (ospa subunit) vaccines available for use against b burgdorferi, and if vaccination is deemed warranted, the current recommendation is to use one of the subunit vaccines before exposure to ticks. the vaccine can be given as early as weeks and should be repeated to weeks later. the best prophylaxis is likely achieved by using appropriate tick prevention, such as fipronil with methoprene spray or spot-on products (eg, frontline top spot; merial ltd, iselin, nj), amitraz collars (eg, preventic collar; virbac animal health, fort worth, tx), or an imidacloprid/permethrin topical product (eg, canine advantix; bayer animal health, shawnee mission, ks). , these products should be chosen and recommended carefully by the veterinarian based on household situations, owner concerns, and the age of the puppy. this virus, also a morbillivirus, can stimulate an immune response that is crossprotective against cdv. the indication for using this vaccine is for puppies that may have maternal antibody to distemper virus sufficient to cause interference with distemper vaccination but inadequate to protect against infection. if indicated (see later discussion on special circumstances), a single vaccination with a modified live vaccine should be given intramuscularly as early as weeks of age. subsequent immunizations with mlv cdv vaccines should be given serially as recommended (see cdv section). , , canine measles vaccines should never be administered to female puppies older than weeks, as they may develop an acquired immune response to the virus, which could be problematic if a female puppy vaccinated against measles at weeks of age later became pregnant. if she developed antibodies to the measles virus and maintained immunologic memory, she would confer measles antibody to her puppies via passive transfer, thus rendering measles vaccination in those puppies ineffective. a more appropriate alternative to administering a measles vaccine to a young puppy thought to be at risk for infection but too young to receive an mlv cdv vaccine would davis-wurzler be to use a recombinant cdv vaccine, thereby decreasing the likelihood of maternal antibody interference. , canine influenza virus canine influenza virus has been seen in various countries, most notably in enzootic outbreaks. this virus is typically seen in puppies and dogs in shelter, boarding, and day-care facilities, and often occurs as a coinfection in canine infectious respiratory disease (cird) with bacterial pathogens. there is a commercially available inactivated vaccine available for use in puppies as young as weeks. vaccination with this product should be used only in puppies with a high risk of exposure, such as to shelters and areas known to be dealing with current/recent outbreaks (typically not in client-owned puppies). in addition, some countries now require an initial vaccination series ( doses, given - weeks apart) before importation. , rattlesnake vaccine a vaccine designed to protect against envenomation by crotalus atrox, the western diamondback rattlesnake, was released onto the market several years ago. the original provisional licensure was granted to provide possible protection against this single species of snake, and was granted for use only in california. the company was later granted extended licensure for multiple states, and has extended its claim for potential protection against multiple species of members of the crotalidae (pit vipers). to date, no challenge studies have been performed in the canine species to validate efficacy claims. all claims are based on antibody titer to the venom component included in the vaccine, to murid challenge studies, and to field reports of protection of naturally occurring envenomation. no controlled, independent studies exist concerning the impact of prior vaccination on therapeutics after envenomation. the manufacturer does not claim that vaccination with this product will completely protect against effects of envenomation; rather, they claim it may slow the onset of clinical signs and decrease the severity of signs. immediate veterinary care is still the gold standard for any snake bite. because of the great potential for variability in envenomation (site of bite on animal, size and age of snake, amount of venom injected into animal, and species of snake), field observations and anecdotal reports of protection are difficult to substantiate. challenge studies conducted under controlled conditions will likely be necessary to validate the efficacy of this product. at present, owing to the preceding statements, this vaccine is not recommended for general use. aversion training and keeping dogs out of areas known to favor rattlesnake habitation, and immediate veterinary evaluation and care are still the standard recommendations for preventing and treating disease associated with rattlesnake envenomation. if an owner is extremely concerned about the potential for exposure and envenomation by a western diamondback rattlesnake, the decision to vaccinate should be made after a discussion between the veterinarian and owner, with full disclosure of vaccine efficacy and a risk/benefit analysis, understanding the potential for adverse events from vaccination. this vaccine has been shown to be safe for use in puppies as young as months. an enveloped virus belonging to the family coronaviridae, this virus is transmitted via the fecal-oral route. vaccination against this disease is generally not recommended because the vaccines provide questionable protection, and the actual prevalence vaccination strategies in puppies and kittens and severity of the disease are unknown. those most likely to be infected and develop clinical disease are neonates younger than weeks. clinical signs may include diarrhea, possibly hemorrhagic, but typically self-limiting. the general recommendation is to vaccinate puppies against cpv (as recommended in the section on cpv), as this practice appears to confer protection against coronavirus in addition to preventing infection with cpv . , canine adenovirus type i as stated in the canine core vaccine section, cav-i causes serious disease in dogs; however use of the cav-i is associated with a high incidence of adverse events. vaccination with cav-ii induces an immune response that is protective against both cav-i and cav-ii without the adverse effects. the recommendation is to use cav-ii as part of the canine core vaccination program; cav-i should not be used. feline panleukopenia, a nonenveloped parvovirus closely related to canine parvovirus, causes serious, often fatal disease in kittens. transmission typically occurs from direct contact with infected animals, although in utero infection and fomite transmission also occurs. clinical signs typically include pyrexia, anorexia, lethargy, vomiting, and diarrhea. kittens may be immunosuppressed subsequent to pancytopenia associated with this viral infection. kittens infected in utero may exhibit cerebellar disease. prevention is achieved by using modified live virus vaccines beginning between and weeks of age. the standard recommendation is to use a parenteral product (as opposed to intranasal products, which have higher incidences of postvaccinal viral shedding and potential for clinical disease induced by the more virulent viruses in these vaccines). , , as is the case for canine distemper, adenovirus, and parvovirus, the core feline diseases, with the exception of rabies, are typically administered in a multivalent product in series. there are numerous vaccine products containing feline panleukopenia virus, herpesvirus i, and calicivirus (see later discussion). the current recommendation is to choose an mlv or killed product from a reputable manufacturer. vaccines are administered subcutaneously in the distal aspect of the right thoracic limb (elbow or distally) and given every to weeks until the kitten is at least to weeks old. repeat administration is recommended year later before instituting a triennial schedule. , feline herpesvirus i feline herpesvirus i (fhv-i), also known as feline viral rhinotracheitis virus, is an enveloped virus causing respiratory tract disease in cats. clinical signs include sneezing, nasal congestion and discharge, conjunctivitis, and ocular discharge. in addition, kittens may exhibit pyrexia, anorexia, and lethargy along with oral/lingual ulcerations and associated hypersalivation. in some cases ulcerative, crusting dermatitis occurs, which may mimic other dermatologic disease. the virus typically causes upper respiratory disease but the lower respiratory tract may become involved, especially in neonates or debilitated animals. infection with this virus is lifelong, although many cats will "recover" and not show clinical signs. however, cats infected with fhv-i may have recurrent outbreaks, especially under times of stress or if their immunity is otherwise compromised. cats may persistently shed the virus and act as a source of infection in shelters, catteries, and multiple-cat households. therefore, prevention before exposure is key to controlling this disease. , vaccination with a modified live virus (or killed product) beginning as early as to weeks is recommended, this being commonly administered as part of a multivalent product, given subcutaneously, in the right thoracic limb. the current recommendation is for kittens to receive a second vaccination weeks later. the last vaccine in the series should be given when kittens are to weeks of age. a vaccine should be given year later before beginning the triennial schedule. feline calicivirus causes respiratory tract disease in kittens and cats. because it is a nonenveloped virus, it is more resistant to disinfectants and may therefore persist in the environment. signs are similar to those associated with fhv-i, but lameness and stomatitis are also commonly seen. transmission of both fhv-i and calicivirus is through direct contact, exposure to contaminated secretions, aerosolization, and fomites. , another, highly virulent, strain of feline calicivirus was identified several years ago and carries a higher incidence of mortality. transmission is through either direct contact or via fomites. prior vaccination against feline calicivirus does not appear to be protective against this strain, and adult cats appear to be more severely affected than kittens. , the current recommendation is as for panleukopenia and fhv-i: administering a modified live virus inactivated-virus parenteral vaccine beginning at or weeks with a subsequent vaccine weeks later (the last vaccination should be when the kitten is at least to weeks old). a booster vaccine should be administered year later, and then every years. as stated earlier, rabies virus affects all mammals and in the united states, with most documented cases of rabies in pet animals occurring in cats. because of the significant risk to pets, wildlife, and humans, vaccination against rabies virus is highly recommended for all kittens and cats, even those kept inside. , local requirements vary, but the general recommendation is that all kittens should be vaccinated beginning at weeks of age with either the recombinant rabies vaccine (preferable) or a killed rabies virus vaccine. , , the recombinant product uses gene-splicing technology: reverse transcriptase is applied to rabies viral rna to create complementary dna. the segment of rabies virus dna that codes (a codon) for the immunogenic protein associated with the virus (glycoprotein g) is then spliced from the rabies dna and inserted into a canarypox virus. the canarypox virus, which is attenuated, is nonpathogenic to mammalian cells and therefore carries no potential to cause disease in this species. because the vaccine is essentially a modified live product, the canarypox virus can enter cells, delivering the codon for rabies virus glycoprotein g to its targeted site. once inside the cell the canarypox virus is unable to replicate, but the rabies glycoprotein g codon is preserved, leading the host cell to express the glycoprotein on its surface; this stimulates both cell-mediated and humoral immune responses. besides the benefit of stimulating both types of immunity, because this product is adjuvant-free there may be a decreased risk of local inflammation associated with vaccination, thereby potentially decreasing the risk of subsequent vaccine reactions and tumorigenesis. the current recommendation is to use either the rrabies virus vaccine (preferred when possible) or a killed-virus vaccine in a case where increased duration of immunity is required (not pertinent to kittens because all pediatric/initial rabies vaccinations provide only months of protection, regardless of label claims). , rabies vaccines should be administered subcutaneously in the right pelvic limb, as distally as is reasonably possible: the level of the stifle is acceptable and areas distal to the tarsus are difficult to inject, and therefore not really feasible or appropriate. administering vaccines (or any injections for that matter) in the tail should be avoided. giving injections in the tail is difficult because of the scant amounts of loose skin and subcutaneous tissue, which is vaccination strategies in puppies and kittens likely to cause more discomfort in patients during vaccination. more importantly, if a tumor does arise proximally on the tail, the potential for complete resection and cure are decreased because of the potential for tumor infiltration into the vertebral column. at present there is only one recombinant rabies vaccine approved for use in cats (pure-vax feline rabies vaccine'; merial ltd, duluth, ga). the current usda approval/label states that this product should be administered annually. there are multiple killed-virus rabies vaccines approved for use in cats, with initial vaccination occurring at weeks of age with a subsequent vaccination year later. because regulations vary depending on state or region, the veterinary practitioner must be familiar with local laws regarding rabies vaccination in this species. feline leukemia virus (felv) is a retrovirus primarily affecting cats of any age, but kittens and juvenile cats appear to be most susceptible to infection. clinical signs are numerous and nonspecific, and include pyrexia, failure to thrive, chronic or recurrent respiratory tract, and gastrointestinal disease. infection in kittens occurs via vertical transmission from the queen to the fetus, but may also spread horizontally from queen to kitten during lactation and grooming. transmission also occurs through direct and usually prolonged contact with other infected cats from behaviors such as grooming and sharing food, and water bowls, and litter boxes. viral screening using an enzymelinked immunosorbent assay (elisa) test designed to detect antigenemia should be performed on all kittens, even if their owners plan to house them strictly indoors. because the elisa test detects antigen, maternal antibody and vaccination do not interfere with test results. therefore kittens of any age may be tested, and the current recommendation is to test every kitten (and adult cats with an unknown viral status) prior to felv vaccination. if a kitten is antigen negative, the current recommendation is to administer either a killed or a recombinant vaccine on the first or second kitten visit. a second vaccine should be administered weeks later followed by vaccination year after the last felv kitten vaccine. , the recommended site for administration of any felv vaccine is the left pelvic limb, as distally as is reasonably possible. at present there is only one recombinant felv vaccine available (purevax recombinant leukemia vaccine; merial ltd, duluth, ga). although felv is considered a noncore vaccine in adult cats because kittens are most vulnerable to infection and may be exposed if outdoors, and immunity increases with age, it is rational to vaccinate all kittens against this disease with a repeat vaccination year later. if the cat is subsequently housed strictly indoors and does not live with an infected (felv) cat, additional vaccinations are not indicated. chlamydophila felis, formerly known as chlamydia psittaci, is a bacterium that causes upper respiratory tract disease in kittens and cats. the most common sign is conjunctivitis, but sneezing and nasal discharge may also be present. transmission is typically through direct contact with infected cats. kittens are most commonly affected, but usually recover fully with appropriate antibiotic therapy: either topical oxytetracycline (terramycin ophthalmic ointment) or systemic tetracycline (panmycin aquadrops) or doxycycline (vibramycin). vaccination against this agent typically does not prevent infection but may prevent clinical signs of disease. because the vaccine does not fully prevent infection and carries an association with adverse events that may be greater than the actual disease, routine vaccination of household pets with this product is davis-wurzler generally not recommended. however, it may be of use in some environments where the risk of infection is high, such as shelters or catteries with recent outbreaks. , if vaccination is deemed appropriate by the practitioner, an attenuated parenteral vaccine can be given to kittens beginning at weeks, with a second dose given to weeks later. bordetella this bacterial agent causes respiratory tract disease in cats, and cats affected by stress, poor nutrition, or overcrowding seem more susceptible. many kittens infected show mild, self-limiting disease with signs including pyrexia, sneezing, and nasal and ocular discharge, although bronchopneumonia has been documented. there is a topical, modified live bacterin vaccine designed for use in this species, but it is generally not recommended for routine use. if the practitioner feels protection against b bronchiseptica is warranted based on the kitten's risk of exposure, such as attendance at cat shows or visiting a boarding facility, or is in a shelter with potential contact with dogs (with a recent b bronchiseptica outbreak), administration of the vaccine designed for use in cats may be considered. a single dose of the modified live intranasal vaccine can be given to kittens as young as weeks of age. the product designed for use in canines should not be used in cats. there are multiple vaccines in addition to those described and recommended here; however, many of these diseases pose a minimal risk to most of the feline population or the vaccines are minimally efficacious at preventing infection or disease, and therefore are generally not recommended. additional reasons not to use some of these products are vaccine interference with screening tests and adverse events associated with some vaccines. a retrovirus, feline immunodeficiency virus (fiv) primarily affects cats by compromising their immune system, leaving them vulnerable to opportunistic infections. in addition to immunosuppression, with most of the effect targeted against the cellmediated (t-cell) immune response, infection with fiv also carries an increased risk for development of certain types of neoplasia, b-cell lymphoma being the most common. transmission occurs most commonly from breeding and fighting. the virus is not spread through casual contact between housemates not engaging in the behaviors stated, nor is it spread through casual encounters between nonbreeding, nonfighting cats outside. naturally occurring infection of kittens from queens is rare; however, kittens can become fiv-antibody positive via passive transfer from ingestion of colostrum of fiv-positive queens or queens previously vaccinated against fiv. , fiv-antibody levels acquired from maternal transfer in kittens who are actually fivvirus negative decline over the first several months of life. the standard screening test for fiv is an elisa test designed to detect fiv antibody. the elisa was designed to detect antibody rather than antigen, because infected cats produce high levels of circulating antibody in contrast to low levels of circulating virus. because kittens may have circulating fiv antibody although actually may be fiv-antigen negative, it is generally not recommended to test kittens younger than months. if a kitten is tested and a positive result is obtained, the test result should be repeated with a different methodology (western blot or polymerase chain reaction [pcr] ) and should be repeated once the kitten is more than months old. if a kitten is truly not infected, the maternal antibody will wane by months of age, leading to seroconversion. if, however, a kitten or cat remains seropositive, the recommendation is made to keep the cat indoors only from that point, both to prevent infection of other cats and to decrease exposure to potential environmental pathogens. fiv-infected cats can live for years and, unless otherwise indicated by concurrent disease, euthanasia is generally not indicated for most owned pets. there is a killed fiv vaccine available, but the efficacy of this product is still unknown. there are known subtypes of fiv virus, and the vaccine has been formulated to protect against subtypes a and d; however, the predominant subtype infecting cats in north america and europe appears to be subtype b. it is unknown whether cross-protection exists between the different subtypes. because the vaccine elicits a strong antibody response, vaccinated kittens and cats will become seropositive on both elisa and western blot tests, as both tests detect antibody. a pcr test is available but is currently only performed at certain laboratories, and results and reliability vary with testing centers. because of the increased technological needs and increased costs of this test, it is not considered the standard screening test. if done under specific conditions it can detect virus, and therefore may be of benefit in differentiating between cats with viremia (truly infected cats) and kittens or cats with circulating antibody, attributable either to maternal transfer or vaccination. because of the nature of transmission of the virus and interference with the standard screening methods for infection, the vaccination against fiv is not currently recommended. keeping cats indoors if possible, neutering all cats going outside, and preventing exposure to stray or feral cats that may be more likely to engage in fighting behaviors remain the gold standards for preventing this disease. , feline infectious peritonitis the disease feline infectious peritonitis (fip) is caused by a member of the coronaviridae. feline enteric coronavirus (fecv) and fip virus are phenotypes of the same virus. fecv transmission occurs through the fecal-oral route where it typically infects intestinal epithelium, but the organism can be transmitted via fomites and persists for long periods of time in the environment. most cats infected with fecv either do not show clinical signs of disease or may have transient diarrhea, and some will persistently shed the virus in their feces. fecv can, however, undergo random mutations within a host, creating fip virus, although in most cats the virus does not mutate into this form and most cats will not develop fip. the fip virus enters and replicates within macrophages where it can then be disseminated throughout the body. clinical signs are numerous, but commonly include weight loss, failure to thrive, diarrhea, pyrexia, and chronic respiratory tract disease. two main types of the disease exist, the dry (noneffusive) and the wet (effusive) forms. both are ultimately fatal diseases. although there is a vaccine available, its efficacy and indication for use is believed to be minimal, if at all. the current recommendation is not to use this vaccine, based on efficacy concerns and the minimal risk of infection in most kittens and cats. infection with fecv and mutation with subsequent development of disease occurs most commonly in multiple-cat households ( ), catteries, and shelters. the standard screening test for fip is a serologic, indirect immunofluorescent antibody (ifa) test designed to detect antibody. this test may be of some value, but results need to be interpreted with caution, and concomitantly with signalment, clinical signs, and other laboratory data. prior vaccination against fip will yield positive ifa results, further posing potential complications in routing screening of this disease. in general, kittens are most vulnerable to this disease, with greater than % of cats with fip being younger than years. prevention is directed toward decreasing stress in kittens and cats in multiple-cat households, preventing exposure of naïve kittens and cats in environments known to have high endemic levels of feline enteric corona virus, and at depopulating catteries known to have high prevalence rates of fecv and fip. , because of the complexity of this disease and the limited space and objectives of this discussion, readers are encouraged to review infectious diseases of the dog and cat, rd edition, by greene, and textbook of veterinary internal medicine, th edition, by ettinger and feldman, for a more comprehensive review of this disease. vaccines are potent biological agents designed to prevent disease. any foreign product administered to an animal has the potential to be associated with an unexpected response by that animal. while vaccines must meet usda requirements for safety, efficacy, potency, and purity, there still exists the potential for adverse events with products that have met these standards. veterinarians should always report adverse events associated with vaccination to the vaccine manufacturer. some adverse events are more likely to occur with certain agents, whereas others appear to have an increased rate of occurrence in certain breeds. still others may be idiosyncratic and are not predictable. the following is offered as a brief overview of some types of adverse events associated with vaccination, with suggestions as to how a practitioner might best respond to and prevent such events from recurring. the reactions seen most commonly are local inflammation at the site of the injection or general malaise, pyrexia, and anorexia for to days after vaccination. most of these reactions are self-limiting and require nothing more than monitoring by the animal owner. it is appropriate for the practitioner to note any reaction along with a description of signs documented in the medical record, and offer supportive care if indicated. in some instances administration of an mlv vaccine will cause transient mild clinical disease. supportive care and isolation from unvaccinated animals is recommended, as the vaccinated animal showing clinical disease will shed the vaccinal organism and is potentially infectious to other animals. contact information for vaccine manufacturers, support agencies, and disease-reporting organizations is included in table . feline injection-site sarcomas (fiss), formerly known as feline vaccine-associated sarcomas or fibrosarcomas, develop secondarily to local inflammation at injection sites. originally it was thought that there was an increased risk for development of these tumors associated with specific adjuvants and vaccines; however, it is now accepted that all vaccines and repositol agents such as long-acting penicillin and corticosteroid injections, in addition to other injections, can be associated with the formation of fiss. measures to prevent these tumors are aimed at decreasing the local inflammatory response by avoiding the use of adjuvants in this species and administering only those vaccines indicated for the individual animal. , multiple vaccines should not be administered in one site, as this may increase the amount of inflammation in that site. following the recommended sites for injection is strongly recommended (see individual vaccine sections for specific sites) , and avoiding adjuvanted products when there is a reasonable alternative (mlv or recombinant) product available is ideal, as a recent study confirmed an increased association of tumor formation with adjuvanted vaccines compared with recombinant vaccines, although no vaccine was risk free. , there are specific guidelines as to how a practitioner should proceed if a cat develops a swelling at the site of a vaccine or injection. the practitioner is advised to monitor the patient closely, documenting -dimensional measurements and temporal association if a mass or swelling develops at the site of a vaccine. the - - rule developed by the table feline vaccine-associated sarcoma task force should be closely applied. "three" refers to persistence of the mass for months or greater; " " refers to a size of cm or greater; and " " applies if the mass increases in size after month. if any of these criteria are met, the mass should be biopsied with wedge technique or needle biopsy allowing for complete resection of the biopsy margins in the future, and subsequent referral to an oncologist or surgical oncologist if fibrosarcoma is confirmed. fine-needle aspiration is not recommended for evaluation of potential injection-site sarcomas. , most vaccine manufacturers have programs established to help defray the medical and surgical costs associated with these tumors, and the practitioner is advised to always notify the vaccine manufacturer any time an adverse event is seen. type i hypersensitivity, also known as immediate hypersensitivity and, in some cases, anaphylaxis, is mediated by immunoglobulin e antibody. the host's immune system may react to anything contained within the vaccine product, including cellular products used for culture, adjuvant, preservative, and the antigen itself, and reaction typically occurs within to hours after the administration of a vaccine. in the dog, signs range from urticaria, angioedema, and pruritus ( fig. ) to respiratory distress and fulminant vascular collapse (anaphylaxis). in the cat, acute onset of vomiting and diarrhea with associated hypovolemia and respiratory and vascular shock may be seen. if an animal develops any of these signs within the first several hours after vaccination, it should be presented to the veterinarian immediately for emergency medical care and support. it is not the goal of this review to offer therapies for shock, so the reader is referred to emergency veterinary literature for recommended therapies. the point here is to advise the practitioner to proceed with caution when using vaccines that may have a higher incidence of these reactions, or in breeds that may be at increased risk for immediate hypersensitivity. the increased association between killed bacterin vaccines and type i reactions is well documented, and there are reports that toy breeds may be at increased risk for type i reactions associated with these vaccines. if an animal does have a type i reaction to a vaccine, the signs shown by the patient, interval between vaccine and onset of signs, and therapeutics administered should be well documented in the medical record, as well as plans for future vaccination of the patient in question. once an animal has this type of reaction to a vaccine, ideally the product should not be used again in that patient. all subsequent vaccines should be administered after a complete physical examination, and the vaccine should be given early in the day to allow monitoring of the patient in the hospital for several hours. however, if this is not possible the patient should remain in the veterinary hospital for monitoring for at least minutes followed by subsequent monitoring by the owner at home for several hours. pretreatment with diphenhydramine (benadryl) is an option, given parenterally (subcutaneous or intramuscular routes) at the dose of . mg/kg to minutes before vaccination if hypersensitivity is a concern. however, administration of corticosteroids concurrently with vaccination to prevent a hypersensitivity reaction is neither appropriate nor recommended because of potential immunosuppression and vaccine interference. the patient's medical record should be identified, outside and inside, to prevent future accidental readministration of the product. advising the owner that the patient should never receive that product again is important. type ii hypersensitivity reactions (autoimmune reactions) are suspected to occur in dogs secondarily to vaccine administration. although this theory is yet unproved, there are reports of dogs developing immune-mediated thrombocytopenia and immunemediated hemolytic anemia temporally associated with recent vaccination. if a dog develops either of these conditions within to months after vaccine administration, the practitioner is advised to strongly consider the risk/benefit ratio of subsequent use of that product in the patient. , type iii hypersensitivity type iii hypersensitivity reactions are immune complex reactions. examples include the anterior uveitis associated with use of the cav-i vaccine and the complement-mediated rabies vaccine induced vasculitis-dermatitis seen in dogs. other examples include glomerulonephritis and polyarthritis. antihistamine administered at the time of vaccine will do nothing to prevent the reaction, nor is it recommended to administer corticosteroids concurrently with vaccination. once an animal has had this type of reaction, subsequent use of the product should be avoided in that patient. , type iv hypersensitivity type iv hypersensitivity reactions are cell-mediated responses occurring locally or systemically. examples include sterile granulomas at the sites of vaccine administration or polyradiculoneuritis. many sterile granulomas resolve without any intervention, but for more severe reactions the practitioner is referred to various medicine texts for recommendations. , the foregoing discussion applies mainly to puppies and kittens owned by individuals. puppies and kittens housed in shelters face unique challenges, as do orphaned animals. these animals may not have received colostrum, and it is more likely that their mothers were not adequately vaccinated. the implications are that these animals are less likely to have received maternal antibodies, leaving them more vulnerable in the earliest stages of life. in addition, they frequently are malnourished, have an increased parasite burden, and are placed in crowded environments possibly with high numbers of endemic pathogens. the american animal hospital association canine vaccination task force and american association of feline practitioners have developed recommendations specifically designed for puppies and kittens in these environments. in general, neonates who may not have vaccination strategies in puppies and kittens received colostrum or who are housed under the aforesaid conditions may be vaccinated at an earlier age, and ideally should be vaccinated before or at the time of entry into the shelter. use of recombinant products may be of benefit in these animals, as well as additional vaccines (noncore vaccines). husbandry is extremely important in these animals: providing proper nutrition, anthelmintics, and clean, dry housing is paramount. in general, these animals are special subsets of the general population facing challenges most young animals do not experience. fiscal considerations and overall population health applies in these cases much more so than to individual, client-owned pets. vaccines are perhaps one of the practitioner's greatest tools in preventing disease and maintaining individual and population health. vaccination is to be used with forethought based on the risk of disease to the population and the individual, balanced with assessment of the risks associated with individual vaccines. it is the practitioner's role to educate pet owners regarding actual risks associated with both undervaccination and overvaccination. the goal is to reach the highest level of overall animal health with the minimum number of adverse events, based on scientific and epidemiologic merit. immunity in the fetus and newborn aafp feline vaccination advisory panel report american animal hospital association canine vaccination task force the defense of the body cells and their response to antigen avma council on biologic and therapeutic agents' report on cat and dog vaccines vaccines & vaccinations: guidelines vs. reality vaccines and vaccinations: the strategic issues infectious diseases of the dog and cat vaccines and their production multicenter case-control study of risk factors associated with development of vaccine-associated sarcomas in cats the use of vaccines canine viral diseases canine vaccination vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection using recombinant attenuated poxvirus vaccines quedgeley (gloucester): british small animal veterinary association infectious canine hepatitis and canine acidophil cell hepatitis infectious diseases of the dog and cat seroconversion of puppies to canine parvovirus and canine distemper virus: a comparison of two combination vaccines canine parvovirus (cpv) vaccination: comparison of neutralizing antibody responses in pups after inoculation with cpv or cpv b modified live virus vaccine evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus, infectious canine hepatitis virus, and distemper virus experimental challenges duration of serologic response to five viral antigens in dogs national association of state public health veterinarians. compendium of animal rabies prevention and control prevalence of and risk factors for leptospirosis among dogs in the united states and canada: cases ( - ) leptospirosis: a re-emerging zoonotic disease st louis (mo): elsevier saunders infectious diseases of the dog and cat canine borreliosis saunders infectious diseases of the dog and cat protection of dogs against canine distemper by vaccination with a canarypox virus recombinant expressing canine distemper virus fusion and hemagglutinin glycoproteins efficacy of the canine influenza virus h n vaccine to decrease severity of clinical disease after cochallenge with canine influenza virus and streptococcus equi subsp rattlesnake vaccine to prevent envenomation toxicity in dogs. presented at the dr ross o. mosier th annual western veterinary conference use of serologic tests to predict resistance to feline herpesvirus , feline calicivirus, and feline parvovirus infection in cats other feline viral diseases infectious diseases of the dog and cat update on feline calicivirus: new trends an outbreak of virulent systemic feline calicivirus disease rabies surveillance in the united states during epidemiologic evidence for a causal relation between vaccination and fibrosarcoma tumorigenesis in cats feline leukemia virus american association of feline practitioners' feline retrovirus management guidelines infectious diseases of the dog and cat feline immunodeficiency virus infection feline immunodeficiency virus infection infectious diseases of the dog and cat feline infectious peritonitis and feline coronavirus infection vaccine-associated feline sarcoma task force. vaccine-associated feline sarcomas vaccine-associated feline sarcoma task force. the current understanding and management of vaccine-associated sarcomas in cats resistance to tumors comparative vaccine-specific and other injectable-specific risks of injection-site sarcomas in cats vaccine-associated adverse events immune complexes and type iii hypersensitivity key: cord- -exbs tz authors: pumchan, ansaya; krobthong, sucheewin; roytrakul, sittiruk; sawatdichaikul, orathai; kondo, hidehiro; hirono, ikuo; areechon, nontawith; unajak, sasimanas title: novel chimeric multiepitope vaccine for streptococcosis disease in nile tilapia (oreochromis niloticus linn.) date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: exbs tz streptococcus agalactiae is a causative agent of streptococcosis disease in various fish species, including nile tilapia (oreochromis niloticus linn.). vaccination is an effective disease prevention and control method, but limitations remain for protecting against catastrophic mortality of fish infected with different strains of streptococci. immunoproteomics analysis of s. agalactiae was used to identify antigenic proteins and construct a chimeric multiepitope vaccine. epitopes from five antigenic proteins were shuffled in five helices of a flavodoxin backbone, and in silico analysis predicted a suitable rna and protein structure for protein expression. f and e were identified as the best candidates for a chimeric multiepitope vaccine. recombinant plasmids were constructed to produce a recombinant protein vaccine and dna vaccine system. overexpressed proteins were determined to be kda and kda in the e. coli and tk systems, respectively. the efficacy of the chimeric multiepitope construct as a recombinant protein vaccine and dna vaccine was evaluated in nile tilapia, followed by s. agalactiae challenge at × ( ) cfu/ml. relative percentage survival (rps) and cumulative mortality were recorded at approximately – % and – %, respectively. these chimeric multiepitope vaccines should be applied in streptococcosis disease control and developed into a multivalent vaccine to control multiple diseases. immunogenic protein characterization. proteins bound to a s. agalactiae antibody were eluted from protein a agarose and divided into two fractions. the first fraction was subjected to - % gradient sds-page to observe the protein features and compare the protein profile from serotypes ia and iii. the second fraction was subjected to lc-ms/ms mass spectrometry to identify the immunogenic proteins. the protein profile from the immunoprecipitation on d-sds-page demonstrated that the major protein (approximately kda) corresponded to rabbit immunoglobulin. however, several bacterial proteins could not be bound to rabbit immunoglobulin and were removed through the flow-through fraction (ft), whereas the protein that specifically bound to the anti-s. agalactiae antibody could be detected in the eluted fraction (fig. ) . comparative immunoproteomics analysis of s. agalactiae serotypes ia and iii was determined by lc-ms/ ms and assessed by a venn diagram ( supplementary fig. ). one hundred proteins were matched and identified between serotype ia and serotype iii via in-house protein databases, resulting in shared proteins between serotype ia and serotype iii. the protein expression levels of the common proteins were determined by hierarchical clustering (hcl). two groups of immunogenic proteins were demonstrated based on their abundance, and proteins were overexpressed in serotype iii, whereas there was a lower abundance of immunogenic proteins in serotype iii than in serotype ia (fig. ) . regarding specific antigen-antibody interactions, and proteins were uniquely identified in serotypes ia and iii, respectively (supplementary figs. , ) . linear β-cell epitope prediction and chimeric vaccine design. the epitopes of immunogenic proteins were predicted by the bcpreds server based on b cell epitopes to be used in chimeric multiepitope vaccine construction. in this study, not only immunogenic proteins from the immunoproteomics analysis were used but also other subunit vaccine candidates were subjected to epitope prediction and combined to produce a chimeric multiepitope vaccine. the amino acid sequences of the c-β protein (bac), surface protein rib (rib), lpxtg cell wall anchor domain-containing protein (spb ), surface immunogenic protein (sip), and cell surface protein heat map with hierarchical clustering (hcl) of normalized protein abundance reveals the differentially expressed immunogenic proteins. the expression value showed in the relative intensities ranges from the highest protein abundance (red) to the lowest protein abundance (green) expression value. alternatively throughout the backbone likely provides potential bioactivity . considering α/β fold structure, flavodoxin from escherichia coli [pdb accession code: chy] was utilized as a linker to combine the epitope fragments from five antigenic proteins. predicted epitopes were randomly displayed on the α-helix structure of flavodoxin, generating , designed models due to the variance of epitopes of bac and of epitopes of sip protein. after joining, protein conformation was examined by molecular modeling with , constructs. i-tasser and stereochemical qualitative allowance manifested from f and e showed appropriate potential tertiary structure with optimal c-scores between − and . f and e also demonstrated the highest score of the amino acid allowance region in the ramachandran plot. the f multiepitope model represented . %, . %, and . % of residues located in the most favored, allowed, and disallowed regions, respectively. meanwhile, the ramachandran plot regions for the e designed model comprised . %, . %, and . %, respectively, of the residues ( supplementary fig. ). the epitope arrangements in f and e were represented in a d structure of chimeric proteins, showing that all chosen epitopes were exposed to the protein surface. the five epitopes were displayed as α-helical layers surrounding chy linkers, which appeared as five-stranded parallel β-sheets at the structure's center, with the order (fig. ). codon optimization of chimeric multiepitope vaccines and plasmid construction. the ectopic expression of bacterial protein in the fish cells may not be achieved due to different codon utilization in the bacterial system. subsequently, codon optimization of the chimeric multiepitope vaccine was analyzed by geneart ™ 's gene optimization according to iso standards (registration no. ) to apply the codon bias of oreochromis niloticus. the region of an ideal gc content range-between % to %-was well optimized. moreover, negative cis-acting sites included internal tata-boxes, chi-sites and ribosomal sites; at-rich or gc-rich sequence stretches; rna instability motifs; repeat sequences; rna secondary structures; and splice donor and acceptor sites in higher eukaryotes, which were successfully removed from these chimeric multiepitope dna vaccine sequences. the best two predicted chimeric multiepitope vaccines were designated f and e . codon adaptation index (cai) presented f and e scores that matched in codon utilization with that of nile tilapia of . and . , respectively. the codon quality distribution index of f and e demonstrated that the codons within the dna sequence were distributed frequently in - positions at % and % ( supplementary fig. a-d) . the average gc content of both chimeric multiepitope vaccines was % ( supplementary fig. e ,f). single-stranded rna-folding prediction revealed the minimum free energy (mfe) secondary structure of f and e ( supplementary fig. the protparam server demonstrated a theoretical pi of . and a molecular mass of kda for f and e . the total number of negatively (asp and glu) and positively (arg and lys) charged amino acid residues of f was and residues, while for e , there were and residues, respectively. the estimated half-life of both chimeric multiepitope constructs was approximately h in mammalian reticulocytes (in vitro), more than h in yeast (in vivo), and over h in e. coli (in vitro). f showed aliphatic index and grand average of hydropathicity values of . and − . , respectively, whereas e showed values of . and − . , respectively. the f and e proteins were indicated to be stable proteins, as represented by instability indexes of . and . , respectively. antigenicity of the f and e chimeric multiepitope vaccines was predicted as . % and . % at a . % threshold for the bacterial model, consistent with antigenpro server prediction by representing . and . , respectively. these results indicate that both vaccine candidates have high potential antigenic www.nature.com/scientificreports www.nature.com/scientificreports/ properties. conformational b cell epitopes from the d protein structure computed by the discotope server demonstrated b cell epitope residues in both f and e at a − . threshold ( table ) . interestingly, the number of epitopes was reduced when computed at the − . and − . thresholds, with f showing and b cell epitope residue regions, respectively, while e contained only and b cell epitope residue regions, respectively (table ). recombinant plasmids harboring e and f were constructed, namely, pet a (+)_ e or _ f and pcdna . (+)_ e or _ f , which were used to determine the recombinant chimeric multiepitope vaccine expression (fig. ) . chimeric multiepitope protein expression was tested in a bacterial expression system and a fish cell (tk- ) culture expression system. these results demonstrated that both chimeric multiepitope proteins could be expressed in both systems, with the expression detectable within h in e. coli ( kda) and within days post-transfection in tk- cells ( kda) (fig. ). larger-sized chimeric multiepitope proteins in the e. coli expression system resulted from an additional tag at the n-terminus, which was contained in the pet expression vector. vaccine efficacy. after vaccination, fish were challenged with s. agalactiae, and infected fish showed clinical signs of streptococcosis disease, such as swirling swimming, opaque eye, exophthalmia and abscess. these moribund fish were collected, and bacteria were re-isolated, showing that they were infected with s. agalactiae serotype iii ( supplementary fig. s ) . www.nature.com/scientificreports www.nature.com/scientificreports/ dna vaccine efficacy testing showed that fish immunized with either f or e had cumulative mortality rates of . ± . % and . ± . %, respectively, which were not significantly different from those of the fkc-vaccinated fish (p > . ). however, in the control group [empty vector; pcdna . (+)], . ± . % mortality was observed at days post-challenge (fig. a ). the recombinant chimeric multiepitope protein vaccination showed that f and e produced cumulative mortality rates of . ± . % and . ± . %, respectively, which were significantly lower than those of the negative control group, at % (p < . ) (fig. a) . the f and e dna vaccines demonstrated similar patterns of rps, with . ± . % and . ± . %, respectively, which were not significantly different from those of the fkc-immunized fish ( . ± . %). however, they were significantly higher than those of the recombinant protein vaccines, which showed . ± . % and . ± . % for e and f , p < . , respectively (fig. b ). immune response. to determine the immune response, dot blot analysis of serum prepared from e -or f -vaccinated fish was used. it was demonstrated that the dna vaccine could gradually activate the production of fish antibodies from the st to the th week. the pattern of antibody response differed from that for the recombinant protein vaccine, with the highest activation of antibody production being significantly produced in the nd - rd week and suddenly dropping in the th week. the highest induction was observed in fkc-immunized fish (fig. ) . dot blot analysis of vaccinate fish sera against whole cell lysate of s. agalactiae serotype ia and iii demonstrated that fish vaccinated with recombinant protein vaccine e and f showed cross-reactivity to whole cell lysate of s. agalactiae serotype ia and iii ( supplementary fig. s ). for the reverse vaccinology approach, computational analysis using a variety of bioinformatics tools is robust and beneficial when identifying appropriate vaccine candidates . bacterial genomics and proteomics analysis indeed help researchers analyze proteins, short domains, and pathogenic epitopes that provide high immunogenicity and high antigenicity for multimeric vaccine development . therefore, immunoproteomics should be applied as a preliminary process to screen antigenic proteins and minimize potential candidates for vaccine development . several immunogenic proteins in this study were described previously, such as c a peptidase and laminin-binding surface protein (lmb), which are cell surface proteins that have an important function in www.nature.com/scientificreports www.nature.com/scientificreports/ chemoattractant activities and are proteins promoting invasion of group b streptococcus (gbs) , . however, the current immunoproteomics analysis from this study identified new immunoreactive proteins, such as bacteriocin transport accessory protein, dihydrofolate reductase, ssu ribosomal protein s p, transposase tnpa, , -alpha-glucan, cell wall surface anchor family protein, and the gtp-binding protein era. as expected, most of these are cell surface proteins, which are suggested to be associated with bacterial virulence , . subsequently, the identified immunoreactive proteins may be used in further vaccine development. multiepitope vaccines are an interesting issue since constructed vaccines designed by in silico analysis may elicit cellular immunity and provide effective responses , . it is known that immunodominant b cells could strongly induce both cellular and humoral immunity; thus, evaluation of b cell epitopes was performed to identify potent epitopes before integrating them to produce a multiepitope vaccine. moreover, this vaccine type is more efficient than whole antigens for controlling staphylococcus spp. infections , . from the present study, table . predicted conformational b-cell epitopes from d structure of designed chimeric multiepitope vaccines using discotope . server. www.nature.com/scientificreports www.nature.com/scientificreports/ linear b cell epitope prediction was assessed and identified potent epitopes from common immunogenic and virulence proteins that were present in serotypes ia and iii. the previous studies supported that one of the chosen proteins, sip, represented a highly conserved protein among gbs isolates and showed cross-protective immunity against gbs infections , , , . prediction of candidate antigenic proteins can be used to select the bacterial strains that carry antigenic genes, as well as to determine high expression levels in the target host and the accessibility of particular antigens in host organisms . therefore, these selected immunogenic proteins might be suitable for consideration in a rational vaccine design. rational chimeric multiepitope vaccine design was achieved by randomly combining epitopes from immunogenic proteins and conjugating with core structures of flavodoxin (β- - - chy) to produce a secondary structure with α/β folding. in addition to the α/β-type folding of flavodoxin, it was also useful to construct our chimeric multiepitope vaccine by forcing the chosen epitope segments to fit within α-helix loops and protrude out of the d-folded structure since that configuration benefits protein solubility by exposure to water molecules . additionally, this linker may promote the solubility of the constructed vaccine and help enhance the recognition of the vaccine by the host's immune system, which contributes to vaccine efficacy. f and e presented the most favored region of protein folding, with the stereochemical quality representing the disallowed region at only . %, which is acceptable since the minimum quality should be less than % . it is suggested that in silico analysis could design a chimeric multiepitope vaccine that could probably manifest effective properties , . to achieve a high level of protein expression in nile tilapia, codon optimization was conducted to improve the transcription and translation capability by removing all possible cis-acting sequence motifs, which may have a negative impact on protein expression. both proteins had a cai > . and a codon with frequent distribution (cfd) > %, which are acceptable for high expression in the target organism , . the gc content of f and e was optimized between - % and had a suitable thermodynamic ensemble free energy, which allowed rna folding and thermodynamic stability , . the overall points suggested that the modeled f chimeric multiepitope vaccine was clearly the best candidate vaccine. numerous effective single-serotype gbs vaccines have been reported, including vaccines for controlling streptococcosis in tilapia , , , , , , . however, it is known that single-serotype whole-cell inactivated vaccines have limitations during cross-prevention against different serotypes. for instance, a s. iniae vaccine (serotype i) could not protect atlantic salmon from infection by s. iniae (serotype ii) . meanwhile, mixed-serotype vaccines (serotypes iv and vii) could promote antiserum levels and enhance the survival rate of newborn pups against streptococcal infection . although formalin-killed vaccines generally provide highly protective effects compared with those of subunit vaccines and dna vaccines, the subunit and dna vaccines may replace the original formalin-killed vaccines or inactivated vaccines due to their promising efficacy, which are similar to those of inactivated vaccines, and longer shelf life , . evidence suggests that dna and subunit vaccines can efficiently trigger the immune system and promote protective efficacy, with an rps value greater than % , , , . nevertheless, these vaccines have limitations, such as their mass production costs, and they may require various optimizations to obtain the highest stable storage conditions , . regarding this idea, a chimeric multiepitope vaccine composed of different epitopes from different proteins common in both serotypes ia and iii was generated to achieve broad protection against different serotypes and increase their stability. interestingly, the designed chimeric multiepitope dna vaccine and protein vaccine exhibited effective prevention in nile tilapia against s. agalactiae, with efficacy similar to that of the whole-cell inactivated vaccine. this evidence supports the strategy of rational vaccine design through b cell recognition using in silico analysis. importantly, immunoproteomics analysis could assist the preliminary determination of suitable immunogenic proteins for vaccine development due to the distinct antigenic determinants that can mediate dissimilar immune responses. the criterion in immunogenic protein selection for vaccine development has focused on the ability of a particular protein to induce an immune response. among identified proteins shared in both serotypes, in addition to providing the highest bcpred scores (table ) , the proteins chosen were also reported as virulence proteins and used as vaccine candidates for streptococcosis disease prevention . for example, c-β protein (bac) can lead to antibody production through fc region binding of human iga . sip protein has been shown to mediate protection against streptococcal infection , , . additionally, the chosen immunogenic protein should be conserved among streptococcus spp., so it would be suitable for application in cross-reactive prevention among s. agalactiae serotypes , . moreover, it should be mentioned that peptide vaccines or epitopes with only amino acid residues may trigger immune responses through binding directly to mhc-i or mhc-ii molecules. these molecules localize to nonprofessional antigen-presenting cells. vaccines containing proteins with longer amino acid sequences can enhance the presentation of epitopes to dendritic cells due to t cell induction , , . herein, the comparative efficacy of both the f and e dna and recombinant protein vaccines indicated that the dna vaccine provided a higher efficacy than the recombinant protein vaccine. this result suggests that the dna vaccine can prolong the activation of the immune response by triggering both humoral and cellular immune responses , . moreover, the clearance rate of the recombinant protein vaccine in the host system may be faster than that of the dna vaccine. this difference implies that the dna vaccine can enter the host cell to produce chimeric multiepitope protein, with that protein existing in the host system for longer than the recombinant protein vaccine, thus enhancing its bioavailability. taken together, these data indicate that the antigen combination has shown promise for streptococcosis disease control in nile tilapia. this research demonstrated a novel platform for rational vaccine design based on chimeric vaccine development that used flavodoxin with a tim-barrel structure as a template. our chimeric protein backbone is suitable for presenting epitopes to be recognized by the host immune system. with epitopes, it could activate antibody production and demonstrated promising protection against bacterial disease similar to that of a whole-cell inactivated vaccine. this platform will promote the production of multivalent vaccines to control multiple diseases and for other applications in the future. experimental fish, bacterial strain and antibody. all male s. agalactiae-free nile tilapia (oreochromis niloticus linn.) were obtained from a commercial gap farm in thailand. the experiments were conducted in accordance with guidelines approved by the national research council of thailand. the experimental fish were anesthetized with clove oil to minimize stress during vaccination and challenge testing. s. agalactiae serotypes ia and iii were cultured as described previously . s. agalactiae serotype iii was used for polyclonal antibody (pab) production, which was kindly provided by prof. ikuo hirono, tumsat, japan. antibody against igm of nile tilapia was kindly provided by assist. prof. eakapol wangkahart. mahasarakham university, thailand. immunoproteomics analysis. s. agalactiae was grown in bhi broth at °c with agitation until reaching exponential phase. bacterial cells were collected by centrifugation, lysed in µl of lysis buffer [tris-buffered saline (tbs) with % tween- and . % lysozyme] and incubated at °c for min following sonication on ice. protein a agarose beads (cell signaling, usa) were added to the bacterial protein lysate, and nonspecific proteins were removed by min of centrifugation at , × g at °c. clarified supernatant was supplemented with % glycerol and then with a pab specific to s. agalactiae serotype iii ( : dilution). then, µl of protein a agarose beads were added to separate bound immunogenic proteins, and the bound proteins were separated by acetone precipitation [ : (v/v)]. precipitated proteins were solubilized in mm tris-hcl with . % sds, and a lowry assay was used to measure the protein concentration. the protein profile was assessed by fractionating µg of protein on a nupage - % bis-tris protein gel (thermofisher, usa). µg of immunogenic protein was mixed with a lysis buffer ( . % rapidgest sf in mm ammonium bicarbonate) and mm dtt in mm ammonium bicarbonate at °c for h. this step was followed by incubation with mm iodoacetamide (iaa) in mm ammonium bicarbonate at room temperature for min in the dark. the protein solution was cleaned up by a zeba spin desalting column before digestion with ng of sequencing-grade trypsin (promega, germany) at °c for h. tryptic peptides were dried at °c under a vacuum and then protonated with . % formic acid in lc water before injection into an lc-ms/ms. the tryptic peptides' immunoproteomics profiles were analyzed using an ultimate ™ nano/capillary lc system (dionex, uk) and hybrid quadrupole q-tof impact ii ™ (bruker daltonics gmbh, germany) equipped with a nano-captivespray ion source. first, nl of extracted peptide was subjected to a trapping column (thermo scientific, pepmap , c , μm i.d. × mm) through a full loop injection before being resolved in an analytical column (pepswift c nano column, μm × cm, i.d.) at °c. the linear gradient method was used to elute peptides with mobile phase a ( . % formic acid in water) and mobile phase b ( . % formic acid in % acetonitrile) at a . µl/min constant flow rate into the mass spectrometer. electrospray ionization was conducted at . kv using captivespray. mass spectra (ms) and ms/ms spectra were fully acquired in positive ion mode (compass . for otofseries software, bruker daltonics). mass accuracy was assessed using positive detection mode after internal calibration with sodium trifluoroacetate (na-tfa) within . ppm. raw lc-ms/ ms spectra were collected using compassxport version . . . (bruker daltonics gmbh, germany) to convert all spectra into the mzxml data format. the mzxml files of the lc-ms/ms datasets for label-free quantification of peptides were evaluated based on the ms profile by maxquant software. chimeric multiepitope vaccine design. the linear b cell epitope was predicted by bcpred . the scop and cath databases were used to design an appropriate chimeric multiepitope vaccine structure (an: wp_ ). a d structure was rendered by i-tasser (iterative threading assembly refinement) using the qualifying c-score value as a confidence score . to refine the tertiary structure, the derived i-tasser results in the pdb files were prepared using the galaxyrefine server, which performed a repeated structure perturbation, and the best structural relaxation candidates were chosen . moreover, to obtain the best chimeric multiepitope vaccine candidates, the residues were determined according to residue stereochemical quality for all the refined chimeric multiepitope models and validated by the procheck program v. . . to generate ramachandran plots . codon optimization. amino acid sequences were reverse-translated to nucleotide sequences using nile tilapia codon usage (oreochromis niloticus [gbvrt]: ). the codon adaptation index (cai) of the designed vaccine candidates' nucleotides was analyzed by an optimizer program (http://genomes.urv.es/optimizer/) and combined with geneart tm 's gene optimization process (thermo fisher scientific, usa). the secondary structure of the single-stranded rna folding and free energy of the thermodynamic ensemble were calculated by the rnafold web server . the optimized dna sequence was synthesized by geneart ® gene synthesis (thermo www.nature.com/scientificreports www.nature.com/scientificreports/ to verify the ectopic expression of the chimeric multiepitope dna vaccine, pcdna . (+)_ e or _ f was transfected into tk (tilapia kidney ) tilapia cells using effectene transfection reagent (qiagen, germany). the transfected fish cell cultures were maintained with leibovitz's l- media containing % fbs and penicillin-streptomycin at °c, and dna vaccine expression was determined after week. recombinant chimeric multiepitope protein was purified by ni-nta agarose beads (qiagen) with a gradient concentration buffer of imidazole ranging from mm to mm. subsequently, the gel filtration chromatography method was performed by fast protein liquid chromatography (fplc) incorporated with a hiprep / & / sephacryl s- high-resolution column (ge healthcare, usa) using a × pbs buffer with a ml/min flow rate. recombinant protein detection was confirmed by sds-page analysis and western blot analysis using an anti-his tag antibody (recombinant protein vaccine) or an anti-flag (rabbit igg) (dna vaccine) and anti-rabbit antibody conjugated to ap (alkaline phosphatase). vaccine efficacy analysis. to evaluate vaccine performance, nile tilapia (o. niloticus) were immunized with chimeric multiepitope vaccines (recombinant protein and dna vaccines), followed by bacterial challenge. a total of experimental groups, namely, ) the f recombinant protein vaccine, ) e recombinant protein vaccine, ) f dna vaccine, ) e dna vaccine, ) formalin-killed (fkc) s. agalactiae vaccine , and ) pcdna . (+) [empty vector], were conducted in triplicate. before vaccination, streptococcosis-free nile tilapia ( ± g) were transferred into glass aquarium tanks containing l of water for one week. after a week of acclimatization, fish were vaccinated according to above mentioned groups. all fish were maintained under running and aerated water at ± °c and fed with commercial pellet feed twice a day. for the chimeric multiepitope protein vaccination, purified f and e proteins were mixed with montanide isa (seppic, france) in a : ratio prior to intraperitoneal injection with µg of protein per fish. for the chimeric multiepitope dna vaccine, plasmid dna of f and e were purified by ultracentrifugation using a cscl gradient and dissolved in te buffer (ph . ) to obtain a concentration of . µg/µl. the dna vaccine was applied to the fish with µg of dna through intramuscular injection. fkc and pcdna . (+) were used as positive and negative controls, respectively. the schedule of vaccine efficacy analysis and immune response analysis was demonstrated in supplementary fig. . for the immune response analysis, blood was drawn from the caudal vein to separate serum for the immunoblotting assay, and those fish were transferred to another separate tank. the analysis was performed every week, using fish in each treatment from the st week to the th week. after one month of vaccination, vaccinated fish in each treatment group were taken from among the remaining fish for serum collection and anesthetized with eugenol before challenge with s. agalactiae (serotype iii) at × cfu/ml through ip administration. mortality and clinical signs of infected tilapia were recorded daily for weeks. the brain, head kidney, and liver were collected from moribund fish for bacterial isolation and identification . cumulative mortality and relative percentage survival (rps) were calculated . a one way analysis of variance (anova) was used for statistical analysis and p < . was considered significant. to detect the antibody response after immunization, antibody production was evaluated through dot blot analysis using the minifold ® i dot blot system (ge healthcare, germany). briefly, µl of purified e , f proteins, or a whole-cell lysate of s. agalactiae ( µg/ml) were spotted on a nitrocellulose membrane and blocked with blocking solution ( . % bsa in tbst) before adding µl of serum of the different treatment groups as above mentioned. then, the membrane was probed with a primary antibody (anti-igm at : , ) for . h, followed by washing times with tbst buffer and min of incubation with an anti-mouse igg hrp-linked ab ( : , ). subsequently, the signal was detected with a chemidoc ™ imaging system (bio-rad) after adding a substrate reagent (perkinelmer, usa). the integrated density of the dot blot was analyzed by imagej (version .x) . five different piscidins from nile tilapia, oreochromis niloticus: analysis of their expression and biological functions prevention and control of viral disease in aquaculture development of a quantitative pcr assay for monitoring streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia parasites and diseases streptococcus agalactiae serotype distribution and antimicrobial susceptibility in pregnant women in gabon microevolution of streptococcus agalactiae st- from australia indicates dissemination via imported tilapia and ongoing adaption to marine hosts or environment molecular serotyping, virulence gene profiling and pathogenicity of streptococcus agalactiae isolated from tilapia farms in thailand by multiplex pcr a microwave-irradiated s. agalactiae vaccine provides partial protection against experimental challenge in nile tilapia oreochromis niloticus efficacy of an experimentally inactivated s. agalactiae vaccine in nile tilapia (oreochromis niloticus) reared in brazil development of live attenuated streptococcus agalactiae vaccine for tilapia via continuous passage in vitro a recombinant truncated surface immunogenic protein (tsip) plus adjuvant fia confers active protection against group b streptococcus infection in tilapia development and efficacy of feed-based recombinant vaccine encoding the cell wall surface anchor family protein of s. agalactiae against streptococcosis in oreochromis sp safety and immunogenicity of an oral dna vaccine encoding sip of streptococcus agalactiae from nile tilapia oreochromis niloticus delivered by live attenuated salmonella typhimurium protective efficacy of cationic-plga microspheres loaded with dna vaccine encoding the sip gene of s. agalactiae in tilapia assembly and role of pili in group b streptococci dentification of universal group b streptococcus vaccine by multiple genome screen protection of nile tilapia (oreochromis niloticus l.) against streptococcus agalactiae following immunization with recombinant fbsa and alpha-enolase designing an efficient multi-epitope peptide vaccine against vibrio cholera via combined immunoinformatics and protein interaction based approaches immunoinformatics analysis and in silico designing of a novel multi-epitope peptide vaccine against staphylococcus aureus multiple b-cell epitope vaccine induces a staphylococcus enterotoxin b-specific lgg protective response against mrsa infection designing of complex multi-epitope peptide vaccine based on omps of klebsiella pneumonia: an in silico approach evaluation of β-amino acid replacements in protein loops: effects on conformational stability and structure crystal structure of escherichia coli chey refined at . -a resolution reliable b cell epitope predictions: impacts of method development and improved benchmarking a novel multi-epitope peptide vaccine against cancer: an in silico approach structure of the streptococcal cell wall c a peptidase enhanced expression of lmb gene encoding laminin-binding protein in streptococcus agalactiae strains harboring is in scpb-lmb intergenic region adhesion, invasion and evasion: the many functions of the surface proteins of staphylococcus aureus multi-epitope vaccines: a promising strategy against tumors and viral infections novel targeted immunotherapy approaches for staphylococcal infection identification of group b streptococcal sip protein, which elicits cross-protective immunity the i-tasser suite: protein structure and function prediction designing a novel multi-epitope peptide vaccine against pathogenic shigella spp. based immunoinformatics approaches novel immunoinformatics approaches to design multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein exploring dengue genome to construct a multi-epitope based subunit vaccine by utilizing immunoinformatics approach to battle against dengue infection on the normalization of the minimum free energy of rnas by sequence length efficacy of feed-based adjuvant vaccine against streptococcus agalactiae in oreochromis spp growth, immune responses and protection of nile tilapia oreochromis niloticus immunized with formalinkilled streptococcus agalactiae serotype ia and iii. vaccines recovery of streptococcus iniae from diseased fish previously vaccinated with a streptococcus vaccine conjugate vaccines against group b streptococcus types iv and vii. the journal of infectious diseases development of streptococcus agalactiae vaccines for tilapia streptococcal β protein has separate binding sites for human factor h and iga-fc identification of group b streptococcal sip protein, which elicits cross-protective immunity immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines a web resource for designing subunit vaccine against major pathogenic species of bacteria prospects for control of emergimg infectious dyseases with plasmid dna vaccines dna vaccines: ready for prime time? predicting linear b-cell epitopes using string kernels scope: structural classification of proteins extended, integrating scop and astral data and classification of new structures aqua and procheck-nmr: programs for checking the quality of protein structures solved by nmr viennarna package . . algorithms for molecular biology dna synthesis and biological security prediction of n-glycosylation sites in human proteins precision mapping of the human o-galnac glycoproteome through simplecell technology protein identification and analysis tools on the vaxijen: a server for prediction of protective antigens, tumor antigens and subunit vaccines high-throughput prediction of protein antigenicity using protein microarray data first report of streptococcus agalactiae isolated from oreochromis niloticus in piura, peru: molecular identification and histopathological lesions dna extraction from . µm sterivex filters and cesium chloride density gradient centrifugation potency and efficacy test of a vaccine in addition with adjuvant against koi herpesvirus in koi (cypronus carpio) nih image to imagej: years of image analysis the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to s.u. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -ycdaw vh authors: maslow, joel n. title: zika vaccine development—current progress and challenges for the future date: - - journal: trop med infect dis doi: . /tropicalmed sha: doc_id: cord_uid: ycdaw vh zika virus is an emergent pathogen that gained significant importance during the epidemic in south and central america as unusual and alarming complications of infection were recognized. although initially considered a self-limited benign infection, a panoply of neurologic complications were recognized including a guillain–barré-like syndrome and in-utero fetal infection causing microcephaly, blindness, and other congenital neurologic complications. numerous zika virus vaccines were developed, with nine different vaccines representing five different platforms entered into clinical trials, one progressing to phase ii. here we review the current landscape and challenges confronting zika virus vaccine development. the zika virus, discovered in uganda in [ ] , was shown to be endemic through sub-saharan africa and tropical areas of southeastern asia in studies through the second half of the th century [ ] . isolated outbreaks occurred in yap island in [ ] and on french polynesia in [ ] . starting in mid- , zika virus infection achieved epidemic status, spreading rapidly through south america, central america, and the caribbean islands [ ] . it was soon recognized that zika virus infection occurring during pregnancy caused microcephaly and other congenital disorders in the developing fetus, the latter being the primary reason for the world health organization (who) labelling zika as an international threat in early . beginning in late , numerous academic labs and pharmaceutical companies initiated work to develop a vaccine against zika, however, by the time the first vaccines entered clinical trials, the zika epidemic had started to wane creating significant challenges to vaccine assessment [ ] that has engendered discussion of other regulatory pathways to licensure [ ] . in this paper, we provide a brief update of current progress in zika virus vaccine development and explore the challenges to vaccine assessment and eventual licensure. zika virus infection presents with a symptom complex consisting of a diffuse maculo-papular rash, fever, asthenia, myalgias, arthralgias, headache, and retroorbital pain. the frequency and degree of symptomatology has, however, varied between studies. a retrospective study immediately following the outbreak on yap island found that only % of survey participants reported symptoms consistent with zika virus infection [ ] , a figure that has been cited to suggest that zika virus infection is asymptomatic in as many as % of individuals. however, of individuals who provided blood samples, representing % of households, % were symptomatic [ ] . a second retrospective study of the larger outbreak in french polynesia [ , ] , similarly appeared to have a low rate of symptomatology based on a sample of blood donors [ ] . however, a subsequent seroprevalence study found that % of those with evidence of prior infection had symptoms consistent with zika virus infection [ ] . a more recent meta-analysis of studies noted that between and % of cases were reported as asymptomatic, although as the authors note, assessment of the prevalence of symptoms was not the goal of many studies [ ] . in adults, the most common reported complication of zika virus infection is a guillain-barré-like illness that had a prevalence of . cases per cases of infection in the french polynesian epidemic [ ] . of interest, a recent meta-analysis has questioned this causal association [ ] . other less common reported neurologic complications in adults include meningoencephalitis [ ] and an adem illness. in contrast, infection during pregnancy has been associated with fetal microcephaly and a number of other congenital illnesses including visual deficits, hearing disorders, neural calcifications, learning disabilities, and arthrogryposis that may affect as many as % children born to mothers infected during pregnancy [ ] [ ] [ ] [ ] [ ] [ ] . additionally, zika can directly invade the placenta and has been associated with prolonged maternal viremia [ ] [ ] [ ] . zika virus can also be transmitted through sexual contact, first reported for a researcher returning from senegal in [ ] . numerous subsequent case reports were published among travelers as part of the epidemic affecting the americas [ ] . the frequency of sexual transmission in endemic regions is unknown and could not be differentiated from mosquito-borne transmission. zika virus carriage in the male urogenital tract is common through days post infection and may persist in some to months [ ] . in mice, zika virus infection causes testicular atrophy and significantly decreases spermatic function and fertility rates [ ] [ ] [ ] . in one study of a zika virus dna vaccine, the adverse effects in the male reproductive system were prevented by vaccination [ ] . the question of whether zika virus can adversely affect human reproductive potential and, if so, whether such effects would be age-related is unknown. females may also excrete zika virus rna for extended times. a prospective study of five women showed that zika rna was detected in vaginal fluid for as long as months and a month or more in three of the five [ ] . murine studies showed that zika virus caused infection of the ovaries of non-immunosuppressed c bl/ mice and induced a t-cell inflammatory reaction, but without affecting reproductive potential [ ] . in contrast to the above human study, female macaques rapidly cleared virus from the genital tract [ ] . thus, while zika virus infection is mildly symptomatic and self-limited for the vast majority of individuals, with infrequent neurologic complications in adults, vaccination of the general population may not be warranted. however, vaccination of females at or entering reproductive age and their male partners is prudent. as discussed previously, vaccination during pregnancy is non-ideal due to the time to generate protective immunity and unknown vaccine safety [ ] . the one unknown aspect is whether vaccination of males of any age may be beneficial to protect against testicular complications. calls for generalized vaccination programs have, however, been put on hold due to the decreasing incidence of zika infection rates, as discussed in the next section. in late and through mid- , global concern for this spreading epidemic was increasing, however, the experience in the two prior epidemics was predictive of the subsequent epidemiology in the americas. the zika virus outbreaks in yap island and french polynesia were characterized by rapid onset and rapid resolution over a span of - months [ , ] . the zika epidemic in the americas was characterized by a longer time to reach peak incidence, taking approximately one year for brazil [ ] presumably related to the larger and more varied geography, with attack rates decreasing greater than -fold the next year [ ] . data published by the pan american health organization (paho) found similar patterns through south and central america (https://www.paho.org/). the almost complete disappearance of zika has created significant barriers to ongoing vaccine studies as attack rates have fallen below those need to meet reasonable sample sizes [ ] . the who removed the designation of zika a virus of global concern in as case rates have fallen. in march , the who and the national institutes of allergy and infectious diseases (niaid) convened a meeting of academics, representatives from industry, and regulators to discuss the approach to zika virus vaccine development in an era of waning incidence [ ] . although zika virus remains endemic in asia, africa, and central and south america, current transmission rates, coupled with high background herd immunity, would require an extremely large and logistically difficult study. as population immunity wanes, future outbreaks are likely. the ability to conduct a meaningful future clinical trial may be dependent upon having pre-existing site and regulatory approvals with an established vaccine network to enable rapid response to new reports suggesting increased disease activity. vaccine development against the zika virus began in earnest in late following the reports of microcephaly in fetuses and infants in brazil. of note, the first demonstration of immunoprotection was as part of a study to define the ultrastructural characteristics of zika virus, that found intramuscular vaccination of mice with infectious viral filtrates protected against cerebral infection [ ] . poland et al. provide a comprehensive listing of almost zika virus vaccines in development as of [ ] . diamond et al. discuss potential immunoreactive epitopes on the zika envelope and provide further information on those vaccines that progressed into clinical trials [ ] . the large number of delivery platforms include live attenuated and inactivated whole-viruses; viral-vectored vaccines utilizing adeno-associated virus, vesicular stomatitis virus, measles virus, and dengue or yellow fever chimeric vaccines; dna and mrna vaccines; and peptide and protein subunit vaccines. most have been assessed in animal models utilizing non-human primates and/or lethal challenge experiments involving immunosuppressed mice [ ] . as of , six vaccines had advanced to phase i studies [ , , ] . since that time, two additional vaccines have entered into clinical trials (table ) . a total of three dna vaccines have entered into human testing [ , ] , including one that has advanced to phase ii (table ) . gls- , a dna vaccine encoding for the zika virus prme genes designed as a consensus based on available zika virus sequences through december [ ] , was the first to enter into clinical trials. in pre-clinical studies, vaccinated mice and non-human primates were shown to develop b and t-cell immune responses against the zika virus envelope and protected against development of neurologic disease and death in immunosuppressed, interferon α, β receptor deficient (ifnar) mice [ ] . moreover, histologic sections of brain tissue showed that vaccinated mice were without inflammatory infiltrates evident in non-immunized mice [ ] . subsequent studies showed that the vaccine protected against testicular damage, testicular atrophy, spermatozoal damage, and infertility in mice [ , ] . a phase i study evaluated gls- administered via intradermal injection (id), followed by electroporation (ep) at doses of either or mg per vaccinations followed immediately by electroporation at baseline, weeks and weeks [ ] . there were no serious adverse events (sae) reported as part of the study, with the most frequent adverse events related to discomfort, pain, or swelling at the injection site. seroconversion was observed in % of individuals after two vaccinations and % after three vaccinations with gls- [ ] . neutralizing antibodies were detected for % of participants in a vero-cell (monkey kidney cell) assay, however, % of participants demonstrated the ability to neutralize infection of u mg neuroblastoma cells [ ] . vaccine responses were maintained through a year of follow-up. there was no difference in responses based on dose level. notably, passive transfer of immune serum from vaccinated participants was able to protect % of ifnar mice against lethal infection independent of the presence of vero cell neutralizing antibodies [ ] . gls- is being evaluated as part of a second double-blind, placebo-controlled clinical trial (nct ) performed in puerto rico. analysis of the latter study is in progress. two additional dna vaccines, based on the french polynesian h/pf/ strain were developed as chimeras that included the jev prm signal sequence followed by the zika envelope (e) gene (vrc ) or a similar construct with the terminal amino acids of e, representing the stem and transmembrane regions, exchanged for the analogous jev sequence (vrc ) [ ] . both vaccines were immunogenic for mice and nhps and protected > % of nhps against viremia at a dose of either or mg given twice [ ] . both vaccine candidates were advanced into clinical trials with mg of administered intramuscularly at weeks , and with vaccine vrc administered by needle and syringe while vrc was administered either as a single dose or split dose by needle and syringe or as a split dose given by the pharmajet needle-free device [ ] . seroconversion was % in the group administered vaccine with the needle-free device, less for vaccine administered as split-dose by needle and syringe, and lowest for vaccine given as a single injection with needle and syringe. the vrc vaccine was advanced into phase ii studies in the americas that utilized clinical sites with clinical site selection guided by epidemiologic modeling [ ] . long-term follow-up has not been reported. three inactivated vaccines have entered into clinical studies, of which clinical data for only zpiv vaccine has been published [ ] . zpiv is a whole inactivated virus vaccine of puerto rican strain prvabc [ ] . studies in mice showed that a single vaccination given intramuscularly with alum generated antibody titers of approximately . log and fully protected balb/c immunocompetent mice from viremia, whereas unvaccinated mice were unprotected and subcutaneously vaccinated mice were only partially protected against the zika brazil strain [ ] . a subsequent study in non-human primates vaccinated twice at four-week intervals with alum generated binding and microneutralization antibody titers of . and . log , respectively, and complete protection against viremia and viruria following challenge with either brazilian or puerto rican strains of zika virus [ ] . zpiv safety and immunogenicity was tested in three clinical trials to assess zika vaccine responses relative to dose level, vaccination schedule, or following vaccination with either the yellow fever yf-vax or japanese encephalitis virus (jev) ixaro vaccines to assess responses in a flavivirus-exposed population [ ] . there were no vaccine-associated saes reported. the most common adverse events were pain and tenderness at the injection site; no neurologic events were reported. seroconversion was % using a cutoff for peak geometric mean titer of : and % using a titer of : . response rates after a single immunization were % and . % using cutoffs of : or : , respectively. vaccine responses were observed through day . passive transfer of purified igg derived from zika immunized participants to groups of balb/c mice per participant provided sterilizing immunity against viremia for % ( of ) of mice overall, with viremia observed for one or more mice per group inoculated with sera from five ( %) individuals [ ] . clinical trial data for the other vaccines has not yet been reported as of the date of this monograph, however, pre-clinical data has been reported for three candidate vaccines. an mrna vaccine that incorporates the prm-e genes of a micronesian strain of zika virus was created incorporating with or without four-point mutations in the fusion-loop segment of the dii region of the envelope gene that abolished binding of antibodies directed against the fusion-loop region to reduce the potential risk for antibody-dependent enhancement of infection [ ] . immunization of ag mice with un-modified lipid nanoparticle-encapsulated vaccines mrna was immunogenic and protective against lethal infection; immunization of c bl/ immunocompetent mice followed by treatment with anti-ifnar blocking antibody showed protection against viremia in approximately % of animals [ ] . pizv, an inactivated vaccine derived from puerto rican strain prvabc selected as without passage-related mutations, protected against lethal zika virus challenge in ag mice when administered with alum adjuvant [ ] . a measles-vectored vaccine encoding the zika virus prme was shown to lessen viremia in pregnant ifnar mice and prevented clinical disease in mouse pups [ ] . preclinical data for vla- inactivated viral vaccines and the rzikv/d ∆ - live virus vaccine has not yet been reported. a number of animal models have been assessed for vaccine development and have been reviewed elsewhere [ ] . immunocompetent mice can develop transient viremia following infection and may represent models to test sterilizing immunity. interferon α/β receptor knockout mice (ag or ifnar -/-) develop lethal infection following zika virus infection and have been used as a more stringent measure of protection. non-human primates develop a self-limited mild illness associated with viremia and can transmit virus in utero to primate fetus [ ] . the logistical difficulties in pursuing standard vaccine evaluation have created significant interest in the possibility of controlled human infection models (chim). the conduct and planning for human challenge trials raises unique medical and ethical considerations. for zika virus trials, one must balance the relative benign and self-limited infection experienced by the vast majority against the risk for less benign complications and transmission risks. as reviewed above, published studies provide estimates that zika virus infection is relatively asymptomatic and self-limited for the majority of individuals, however, methodology in these studies varies widely. in adults, two sets of complications warrant consideration for a proposed chim study. as reviewed above, a guillain-barre-like syndrome occurs in approximately of zika virus infections [ ] , whereas other neurologic complications such as meningoencephalitis, myelitis [ , , ] , and acute disseminated encephalomyelitis [ ] are rare. second, and perhaps more important, is the risk of transmission to a sexual partner and the potential for infection during pregnancy. zika virus commonly persists in the male urogenital tract for months, and may persist in some individuals for up to months [ ] . some have considered limiting studies to non-pregnant females as zika virus colonization of the female genital tract may be temporally limited. the fact that zika virus is known to cause testicular atrophy in mice [ ] [ ] [ ] , raises yet another as yet theoretical concern for humans. these questions as well as the theoretical potential for vector-borne transmission were debated in detail in late with the conclusion that the benefits of a human challenge infection did not outweigh the risks [ ] , however, this analysis was performed just as the initial epidemic wave in the americas was ending. the group published a follow-up in [ ] . despite the recognition that conducting a placebo-controlled vaccine trial had become significantly more difficult due to declining case rates, the group's conclusion was essentially unchanged. to address safety concerns, there has been work to develop attenuated viral strains deleted for potential neurotropic regions [ ] with a goal to prevent viremia. whether the attenuated viral strain (rzikv/d ∆ - ) being tested in a phase i study will serve as putative challenge strain is as yet undetermined. in summary, zika vaccine development continues with multiple candidate vaccines in clinical trials. because of the significant decline in incidence, evaluation of vaccine efficacy is increasingly difficult. there has been renewed interest in animal model and human infection models of infection. the author is an employee of geneone life science, inc., a developer of dna-based vaccines including a vaccine against the zika virus. walter reed army institute of research niaid national institutes of allergy and infectious disease vrc vaccine research center zika virus: (i). isolations and serological specificity zika virus: following the path of dengue and chikungunya? lancet zika virus outbreak on yap island, federated states of micronesia european centre for disease prevention and control. rapid risk assessment: zika virus infection outbreak, french polynesia zika virus in the americas-yet another arbovirus threat steep drop in zika cases undermines vaccine trial demonstrating vaccine effectiveness during a waning epidemic: a who/nih meeting report on approaches to development and licensure of zika vaccine candidates bilan de l'épidémie à virus zika survenue en polynésie française entre octobre et mars . de la description de l'épidémie aux connaissances acquises après l'évènement potential for zika virus transmission through blood transfusion demonstrated during an outbreak in french polynesia zika virus seroprevalence prevalence of asymptomatic zika virus infection: a systematic review guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study zika virus infection and risk of guillain-barre syndrome: a meta-analysis leparc-goffart, i.; et al. zika virus associated with meningoencephalitis congenital cerebral malformations and dysfunction in fetuses and newborns following the to zika virus epidemic in french polynesia zika virus infection in pregnant women in rio de janeiro european centre for disease prevention and control. rapid risk assessment: zika virus epidemic in the americas: potential association with microcephaly and guillain-barre syndrome birth defects among fetuses and infants of us women with evidence of possible zika virus infection during pregnancy hearing loss in infants with microcephaly and evidence of congenital zika virus infection-brazil risk factors associated with the ophthalmoscopic findings identifed in infants with presumed zika virus congenital infection zika virus infection with prolonged maternal viremia and fetal brain abnormalities prolonged detection of zika virus rna in pregnant women zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? probable non-vector-borne transmission of zika virus frequent zika virus sexual transmission and prolonged viral rna shedding in an immunodeficient mouse model zika virus shedding in semen of symptomatic infected men zika-induced male infertility in mice is potentially reversible and preventable by deoxyribonucleic acid immunization zika virus infection damages the testes in mice dna vaccination protects mice against zika virus-induced damage to the testes prolonged shedding of zika virus rna in vaginal secretions zika virus causes acute infection and inflammation in the ovary of mice without apparent defects in fertility zika virus persistence in the central nervous system and lymph nodes of rhesus monkeys vaccine development for emerging virulent infectious diseases pan american health organization/world health organization. zika-epidemiologic report brazil comparison by electron microscopy of the ntaya and zika viruses development of vaccines against zika virus zika virus vaccine development: progress in the face of new challenges vaccines for emerging infectious diseases: lessonso from mers coronavirus and zika virus an update on zika vaccine developments. expert rev safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase clinical trials safety and immunogenicity of an anti-zika virus dna vaccine-preliminary report in vivo protection against zikv infection and pathogenesis through passive antibody transfer and active immunization with a prmenv dna vaccine rapid development of a dna vaccine for zika virus preliminary aggregate safety and immunogenicity results from three trials of a purified inactivated zika virus vaccine candidate: phase , randomised, double-blind, placebo-controlled clinical trials vaccine protection against zika virus from brazil protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys modified mrna vaccines protect against zika virus infection purified inactivated zika vaccine candidates afford protection against lethal challenge in mice a measles virus-based vaccine candidate mediates protection against zika virus in an allogeneic mouse pregnancy model animal models of zika virus infection, pathogenesis, and immunity zika virus infection during pregnancy in mice causes placental damage and fetal demise increased hospitalizations for neuropathies as indicators of zika virus infection, according to health information system data guillain-barré syndrome, acute disseminated encephalomyelitis and encephalitis associated with zika virus infection in brazil: detection of viral rna and isolation of virus during late infection ethical considerations for zika virus human challenge trials; national institutes of health bystander risk, social value, and ethics of human research zika vaccines: role for controlled human infection key: cord- -zfh im authors: saxena, jyoti; rawat, shweta title: edible vaccines date: - - journal: advances in biotechnology doi: . / - - - - _ sha: doc_id: cord_uid: zfh im in recent years edible vaccine emerged as a new concept developed by biotechnologists. edible vaccines are subunit vaccines where the selected genes are introduced into the plants and the transgenic plant is then induced to manufacture the encoded protein. foods under such application include potato, banana, lettuce, corn, soybean, rice, and legumes. they are easy to administer, easy to store and readily acceptable delivery system for different age group patients yet cost effective. edible vaccines present exciting possibilities for significantly reducing various diseases such as measles, hepatitis b, cholera, diarrhea, etc., mainly in developing countries. however, various technical and regulatory challenges need to overcome in the path of this emerging vaccine technology to make edible vaccine more efficient and applicable. this chapter attempts to discuss key aspects of edible vaccines like host plants, production, mechanism of action, advantages and limitations, applications, and different regulatory issues concerned to edible vaccines. vaccine is a biological preparation intended to produce immunity to a disease by stimulating the production of antibodies. dead or attenuated organisms or purified products derived from them are generally used to produce various vaccines. over the past decade, scientific advances in genetics, molecular biology, and plant biotechnology have improved the understanding of many infectious diseases and led to the development of vaccination programs. the most common method of administering vaccines is by injection but some are given by mouth or nasal spray. though immunization is the safest method to combat the diseases worldwide but there are many constraints regarding its mode of production, distribution, delivery, cost, and lack of enough research. hence it is desirable to look for an effectual and powerful yet cost effective, easy for storage and distribution yet safe method of immunization. it should also be readily acceptable to all sociocultural groups around the globe. research underway is dedicated to solving these problems by finding ways to produce edible vaccines in the form of transgenic plants which have been investigated as an alternative means to produce and deliver vaccine. edible vaccines are called by several alternative names such as food vaccines, oral vaccines, subunit vaccines, and green vaccines. they seem to be a viable alternative especially for the poor and developing countries. they have come up as great boon in medicinal science for which biotechnologists should be given all credit. the concept of edible vaccines lies in converting the edible food into potential vaccines to prevent infectious diseases. it involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. it has also found application in prevention of autoimmune diseases, birth control, cancer therapy, etc. edible vaccines are currently being developed for a number of human and animal diseases. this new technology hopefully will contribute positively toward the global vaccine programs and have a dramatic impact on health care in developing countries. many people in developing countries do not have access to the vaccines they need, as the traditional vaccines are costly and require skilled medical people for administration and are less effective in inducing mucosal immune response. it was these needs which inspired hiatt et al. ( ) who attempted to produce antibodies in plants which could serve the purpose of passive immunization. the first report of edible vaccine (a surface protein from streptococcus) in tobacco, at . % of total leaf protein level, appeared in in the form of a patent application published under the international patent cooperation treaty. by conceiving the idea of edible vaccine dr. charles arntzen tried to realize it (arntzen ) . in , arntzen and coworkers introduced the concept of transgenic plants as a production and delivery system for subunit vaccines in which edible tissues of transgenic crop plants were used (mor et al. ) . they found that this concept could overcome the limitations of traditional vaccines, thereby triggering the research on edible vaccine. in s, streptococcus mutans surface protein antigen a was expressed for the first time in tobacco. the same group also pioneered the field with work on hepatitis b and heatlabile toxin, b subunit in tobacco plants and potato tubers. in the same year, the successful expression of hepatitis b surface antigen (hbsag) in tobacco plants was also achieved (mason et al. ) . to prove that plant-derived hbsag could stimulate mucosal immune responses via oral route, potato tubers were used as an expression system and were optimized to increase the accumulation of the protein in plant tubers (richter et al. ) . parallel to the evaluation of plant-derived hbsag, mason and arntzen explored plant expression of other vaccine candidates including the labile toxin b subunit (lt-b) of enterotoxigenic escherichia coli (etec) and the capsid protein of norwalk virus. the plant-derived proteins correctly assembled into functional oligomers that could elicit the expected immune responses when given orally to animals (mason et al. ). in a new era was opened in vaccine delivery when researchers supported by the national institute of allergy and infectious diseases (niaid) have shown for the first time that an edible vaccine can safely generate significant immune responses in people. the report by collaborators from the university of maryland in baltimore, the boyce thompson institute for plant research in ithaca, n.y., and tulane university in new orleans appeared in the may issue of nature medicine. according to the then director of niaid ''edible vaccines offer exciting possibilities for significantly reducing the burden of diseases like hepatitis and diarrhea, particularly in the developing world where storing and administering vaccines are often major problems,'' mor et al. ( ) also discussed the rapid increase of research in the edible-vaccine field and pointed out that plants could be used to create multicomponent vaccines that can protect against several pathogens at once. this is an aspect of the edible-vaccine approach that further strengthens its impact. later, in sala and research group reported that proteins produced in these plants induced the mucosal immune response which was the main aim behind this concept. research into edible vaccine is still at a very early stage and scientists have a long way to go before it will become a major part of immunization program world wide. to date, many plant species have been used for vaccine production. the choice of the plant species is important. an edible, palatable plant is necessary if the vaccine is planned for raw consumption. in case of vaccine for animal use, the plant should preferentially be selected among those consumed as normal component of the animal's diet. some food vehicles are discussed below: the concept of edible vaccine got impetus after arntzen and coworkers expressed hbsag in tobacco. the first edible vaccine was produced in tobacco in in which . % recombinant protein (a surface protein from streptococcus) of the total soluble leaf proteins was found. it appeared in the form of a patent application published under the international patent cooperation treaty. transgenic tobacco is successfully engineered for the production of edible vaccines against hepatitis b antigen using 's' gene of hepatitis b virus (hbv). the optimum level of recombinant protein was obtained in leaves and seeds. since acute watery diarrhea is caused by enterotoxigenic e. coli and vibrio cholerae that colonize the small intestine and produce one or more enterotoxin, an attempt was made toward the production of edible vaccine by expressing heat-labile enterotoxin (lt-b) in tobacco. besides, antibodies against dental caries, expressed in tobacco, are already in preclinical human trials. italian researchers have now developed an immunologically active, cost-efficient vaccine against human papilloma viruses (hpv). hpv are the causative agents for cervical cancer, and are also involved in skin, head, and neck tumors. cervical cancer is one of the main causes of cancer-related deaths. genetically modified potatoes are also a viable option and seem to be the desired vector. many of the first edible vaccines were synthesized in potato plants. in serum antibodies at some point after immunization, and of the ( %) also showed fourfold mount in intestinal antibodies. the potatoes were well tolerated and no one experienced serious adverse side effects. vaccine development has successfully tested a potato-based vaccine to combat the norwalk virus, which is spread by contaminated food and water. the virus causes severe abdominal pain and diarrhea. a research team led by william langridge of the loma linda university in california has reported that transgenic potatoes engineered with a cholera antigen, ctb can effectively immunize mice. mice fed transgenic potatoes produce cholera-specific antibodies in their serum and intestine; iga and igg antibodies reach their highest levels after the fourth feeding. in yet another experiment genetically engineered potatoes containing a hepatitis b vaccine have successfully boosted immunity in their first human trials. attempts have also been made to boil the potatoes as raw potatoes are not very appetizing but unfortunately the cooking process breaks down about % of the proteins in the vaccine. while some proteins are more tolerant to heat, for most proteins it will be necessary to amplify the amount of protein in the engineered foods if they are to be cooked before consumption. tomatoes are an excellent candidate because they are easy to manipulate genetically and new crops can be grown quickly. moreover, they are palatable and can be eaten raw. while tomatoes do not grow well in the regions in which the edible vaccines are most needed, the engineered tomatoes can be dried or made into a paste to facilitate their delivery. the anti-malaria edible vaccines in different transgenic tomato plants expressing antigenic type(s) have been proposed by chowdhury and bagasara in . they hypothesized that immunizing individuals against - antigens and against each stage of the life cycle of the multistage parasites would be an efficient, inexpensive and safe way of vaccination. tomatoes with varying sizes, shapes, and colors carrying different antigens would make the vaccines easily identifiable by lay individuals. tomatoes serve as an ideal candidate for the hiv antigen because they unlike other transgenic plants that carry the protein, are edible and immune to any thermal process, which help to retain their healing capabilities. scientists have claimed that tomatoes could be used as a vaccine against alzheimer's disease. the work is in progress to genetically modify the fruit to create an edible vaccine that fires up the immune system to tackle the disease by attacking the toxic beta-amyloid protein that destroys vital connections between brain cells, causing alzheimer's. researchers have engineered tomato plants (lycopersicon esculentum mill var. uc b) to express a gene for the glycoprotein (g-protein), that coats the outer surface of the rabies virus. the recombinant constructs contained the gprotein gene from the era strain of rabies virus, including the signal peptide, under the control of the s promoter of cauliflower mosaic virus (camv). a common fruit-the banana-is currently being considered as a potential vehicle for vaccines against serious as well as too common diseases. the advantage of bananas is that they can be eaten raw as compared to potatoes or rice that need to be cooked and can also be consumed in a pure form. furthermore, children tend to like banana and the plants grow well in the tropical areas in which the vaccines are needed the most. hence, the research is leaning toward the use of banana as the vector since a large number of third-world countries, who would benefit the most from edible vaccines have tropical climates. on the negative side, a new crop of banana plants takes about months to bear fruit. after fruiting, the plants are cut down and a new crop of vaccine-bearing plants must be planted. researchers have also developed bananas that deliver a vaccine for hbv. the banana vaccine is expected to cost just cents a dose, as compared to the $ for the currently available injectable vaccine. maize has also been used as a vector for various edible vaccines. egyptian scientists have genetically engineered the maize plants to produce a protein known as hbsag which elicits an immune response against the hepatitis b virus and could be used as a vaccine. if human trials are successful more than billion people infected with hepatitis b, and about million of these at high risk of serious illness and death from liver damage and liver cancer would be benefited. researches are in offing at iowa state university with the aim to allow pigs and humans to get a flu vaccination simply by eating corn or corn products. it is quite likely that corn vaccine would work in humans when they eat corn or even corn flakes, corn chips, tortillas, or anything that contains corn. genetically modified maize could provide protection to chickens against a highly contagious and fatal viral disease affecting most species of birds. mexican researcher octavio guerrero-andrade and his colleagues at the centre for research and advanced studies in guanajuato, central mexico, genetically modified maize to create an edible vaccine against newcastle disease virus (ndv). they inserted a gene from the ndv, a major killer of poultry in developing countries, into the maize dna and found antibodies against the virus in chickens that ate the genetically modified maize. one pig vaccine has also been produced in corn successfully. efforts are being made by us company prodigene to genetically modify maize to contain a key protein found on the surface of the monkey form of hiv. according to us national institute of health this development brings an edible, more effective, hiv vaccine for people a step closer. transgenic maize expressing the rabies virus glycoprotein (g) of the vnukovo strain has also been produced using ubiquitin maize promoter fused to the whole coding region of the rabies virus g gene, and a constitutive promoter from camv. maize embryogenic callus were transformed with the above construct by biolistics. regenerated maize plants were recovered and grown in a greenhouse. the amount of g-protein detected in the grains was approximately % of the total soluble plant protein. rice is another potential crop which has been used for developing vaccines. it offers several advantages over traditional vaccines; it does not require refrigeration. in fact, the rice proved just as potent after months of storage at room temperature and the vaccine did not dissolve when exposed to stomach acids. in an attempt, predominant t cell epitope peptides, which were derived from japanese cedar pollen allergens, were specifically expressed in rice seeds and delivered to the mucosal immune system (mis); the development of an allergic immune response of the allergen-specific th cell was suppressed. furthermore, not only the specific ige production and release of histamine from mast cells were suppressed, but the inflammatory symptoms of pollinosis, such as sneezing, were also suppressed. these results suggest the feasibility of using an oral immunotherapy agent derived from transgenic plants that accumulate t cell epitope peptides of allergens for allergy treatment. the transfer of genetic material from the microbe responsible for producing cholera toxin into a rice plant has been achieved. the plants produced the toxin and when the rice grains were fed to mice they provoked immunity from the diarrhea-causing bacterium. genetically modified spinach has also been considered for the development of edible vaccine. spinach is being investigated as a plantderived, edible vehicle for anthrax vaccine, as well as a vehicle for the hiv- tat protein (a prospective vaccine candidate). in an experiment a fragment of protective antigen (pa) that represents most of the receptor-binding domain was expressed as a translational fusion with a capsid protein on the outer surface of tobacco mosaic virus, and spinach was inoculated with the recombinant virus. the plant-expressed pa is highly immunogenic in laboratory animals. among other food crops with potential to be developed as edible vaccine; sweet potato, peanuts, lettuce, watermelon, and carrots are on the top priority. the development of plant-based vaccines to protect against many other diseases, such as hiv- , hepatitis b, rabies, and non-hodgkin's lymphoma are ongoing throughout the globe using one of these edible plants. the advantages and disadvantages of various plant host systems are given in conventional subunit vaccines are expensive and technology-intensive, need purification, require refrigeration, and produce poor mucosal response. in contrast, edible vaccines would enhance compliance, especially in children, and because of oral administration would eliminate the need for trained medical personnel. their production is highly efficient and can be easily scaled up. for example, hepatitis b antigen required to vaccinate whole of china annually, could be grown on a -acre plot and all babies in the world each year on just acres of land. they are cheaper, sidestepping demands for purification (single dose of hepatitis b vaccine would cost approximately . cents), grown locally using standard methods and do not require capitalintensive pharmaceutical manufacturing facilities. mass-indefinite production would also decrease dependence on foreign supply. fear of contamination with animal viruses-like the mad cow disease, which is a threat in vaccines manufactured from cultured mammalian cells, is eliminated as plant viruses do not infect humans. edible vaccines activate both mucosal and systemic immunity, as they come in contact with the digestive tract lining which is not possible with subunit vaccines which provide poor mucosal response. this dual effect of edible vaccines provides first-line defense against pathogens invading through mucosa, such as mycobacterium tuberculosis and agents causing diarrhea, pneumonia, stds, hiv, etc. the specific advantages are stated below: . edible means of administration. . no need of medical personnel and syringes. . sterile injection conditions are no more required. . economical in mass production by breeding compared to an animal system. . easy for administration and transportation. . effective maintenance of vaccine activity by controlling the temperature in plant cultivation. . therapeutic proteins are free of pathogens and toxins. . storage near the site of use. . heat stable, thus eliminating the need of refrigeration. potential for outcrossing in field; deep root system problematic for cleaning field (mason et al. ) . antigen protection through bioencapsulation. . subunit vaccine (not attenuated vaccine) means improved safety. . seroconversion in the presence of maternal antibodies. . generation of systemic and mucosal immunity. . enhanced compliance (especially in children). . delivery of multiple antigens. . integration with other vaccine approaches. . plant-derived antigens assemble spontaneously into oligomers and into virus like particles. . no serious side effect problems have been noticed until now. . reduced risk of anaphylactic side effects from edible vaccine over injection system is one benefit reported by the bio-medicine.org. they reported that the edible vaccine carries only part of the allergen compared to injection methods which reduce anaphylactic risk. . administration of edible vaccines to mothers to immunize the fetus-in utero by transplacental transfer of maternal antibodies or the infant through breast milk. edible vaccines have a potential role in protecting infants against diseases like group-b streptococcus, respiratory syncytial virus (rsv), etc., which is under investigation. . edible vaccines would also be suitable against neglected/less common diseases like dengue, hookworm, rabies, etc. they may be integrated with other vaccine approaches and multiple antigens may also be delivered. with advancement come many hurdles and problems, so is true for edible vaccines. like, one could develop immunotolerance to the vaccine peptide or protein, though a little research has been done on it. one of the key goals of the edible-vaccine pioneers was to reduce immunization costs but later many limitations were reported as given below: . consistency of dosage from fruit to fruit, plant to plant, lot to lot, and generation to generation is not similar. . stability of vaccine in fruit is not known. . evaluation of dosage requirement is tedious. . selection of best plant is difficult. . certain foods like potatoes are generally not eaten raw and cooking the food might weaken the medicine present in it. . not convenient for infants as they might spit it, eat a part or eat it all, and throw it up later. concentrating the vaccine into a teaspoon of baby food may be more practical than administering it in a whole fruit. . there is always possibility of sideeffects due to the interaction between the vaccine and the vehicle. . people could ingest too much of the vaccine, which could be toxic, or too little, which could lead to disease outbreaks among populations believed to be immune. . a concern with oral vaccines is the degradation of protein components in the stomach due to low ph and gastric enzymes. however, the degradation can be compensated by repeating the exposure of the antigen until immunological tolerance is accomplished (mason et al. ) . . potential risk of spreading the disease by edible vaccine delivery is a concern of many. potential contamination of the oral delivery system is a possible danger. foreign proteins in plants accumulate in low amounts ( . - % of total protein) and are less immunogenic, therefore the oral dose far exceeds the intranasal/parenteral dose. for example oral hepatitis b dose is - times the parenteral dose and g potato expressing b subunit of labile toxin of etec (lt-b) is required in three different doses to be immunogenic. attempts at boosting the amount of antigens often lead to stunted growth of plants and reduced tuber/fruit formation as too much mrna from the transgene causes gene silencing in plant genome. techniques to overcome these limitations are given below: . optimization of coding sequence of bacterial/ viral genes for expression as plant nuclear genes . expression in plasmids . plant viruses expressing foreign genes . coat-protein fusions . viral-assisted expression in transgenic plants . promoter elements of bean yellow dwarf virus with reporter genes gus (b -glucuronidase) and green fluorescent protein (gfp), substituted later with target antigen genes. . antigen genes may be linked with regulatory elements which switch on the genes more readily or do so only at selected times (after the plant is nearly fully grown) or only in its edible regions. exposure to some outside activator molecule may also be tried. development of edible vaccines is a possible high-volume, low-cost delivery system for thirdworld countries to fight against fatal maladies like aids, hepatitis, and diarrhea. researches by the niaid and the university of maryland showed no significant side effects in a small study using genetically engineered potatoes to make toxin of the e. coli, a diarrhea-causing bacterium. volunteers reported no serious adverse reactions to genetically altered potatoes used to deliver edible vaccine toxin, according to the national institutes of health (nih). the nih reported that - volunteers who ate the raw potato bites developed four times the antibodies against e. coli without obvious side effects. long-term reactions to edible vaccines are yet to be determined and possible delayed reactions have not yet been discovered. an organized large scale study is required before edible vaccines are put into large scale production. creating edible vaccines involves the genetic engineering approach for the introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. this process is known as ''transformation'' and the plants altered with new characteristics are called ''transgenic plants''. like conventional subunit vaccines, edible vaccines are composed of antigenic proteins and are devoid of genes responsible for pathogenicity. thus, they have no way of establishing infection, assuring its safety, especially in immune-compromised patients. the gene which codes the active antigenic protein is first isolated from the pathogen and is incorporated in a suitable ''gene vehicle''. this gene vehicle is integrated into the genome of the plant and is allowed to express the corresponding antigen. then these plant parts are fed to animals and humans to run their course. two main strategies are used for the production of candidate vaccine antigen in plant tissues (fig. . a, b) . . stable genomic integration: this is the most popular method used for the published edible vaccine clinical trials to date. under this method the genetic line is produced that can be propagated either by vegetative (stem cuttings) or sexual (seeds) reproduction methods. the stable expression strategy provides an opportunity to introduce more than one gene for possible multicomponent vaccine production. furthermore, the choice of genetic regulatory elements allows organ and tissue-specific expression of foreign antigens. stable transformation causes the desired gene to be incorporated either in nucleus or chloroplast. agrobacterium mediated gene transfer is used for transforming the plants in which the gene is integrated in nucleus. besides, direct delivery of dna into the tissue can also be applied, biolistic being the most popular method. however, chloroplast engineering has also got impetus in last decade due to its various advantages over nuclear engineering. gram-negative soil bacterium, which infects the wound sites in dicot plants causing the formation of the crown gall tumor. this bacterium is capable of transferring a particular dna segment (t-dna) of the tumor-inducing (ti) plasmid into the nucleus of infected cells where it is subsequently integrated into the host genome and transcribed. the t-dna usually contains cancer-causing oncogenic genes and genes that synthesize opines which are excreted by infected crown gall cells and are a food source for bacterium. during the genetic manipulation, the ti plasmid is engineered to carry the desired gene for vaccine and the virulent genes that cause tumor growth in plants are deleted. the transgene is integrated, expressed, and inherited in mendelian fashion. the whole plant can be then regenerated from individual transformed plant cell. it has been studied that genes are successfully expressed in experimental model plants and when given orally to animals, the extract of transgenic plant containing the antigen induced serum antibodies, thus can be used to produce the edible vaccine. the application of agrobacterium mediated transformation is at present possible to most species of agronomic interest, including members of family graminae and leguminosae. this opens interesting new aspect for the development of edible vaccines for both human and veterinary uses. the second approach for nuclear transformation is based on the microprojectile bombardment method, also known as the gene gun or biolistic method. this method is especially beneficial for those plants which can not be transformed by a. tumefaciens mediated gene transfer method. selected dna sequences are precipitated onto metal microparticles and bombarded with a particle gun at an accelerated speed in a partial vacuum against the plant tissue placed within the acceleration path. microparticles penetrate the walls and release the exogenous dna inside the cell where it will be integrated in the nuclear genome. thus, this method effectively introduces dna. the cells that take up the desired dna, are identified through the use of a marker gene (in plants the use of gus is most common), and then cultured to replicate the gene and possibly cloned. this method has various advantages including ( ) thousands of particles are accelerated at the same time causing multiple hits resulting in transferring of genes into many cells simultaneously, ( ) since intact cells can be used, the difficulties encountered with the use of protoplast are automatically circumvented, and ( ) the method is universal in its application so that cell type, size, and shape or the presence/absence of cell wall do not significantly alter its effectiveness. another important use of the gene gun involves the transformation of organelles such as chloroplasts, and yeast mitochondria. the biolistic particle delivery system ''shoots'' adequately processed dna particles, which penetrate into the chloroplast and integrate with its genome. the chloroplast's transformation is an interesting alternative to nuclear transformation which has come up in recent past. all plant cells have chloroplasts that capture light energy from the sun to produce free energy through a process called photosynthesis. in chloroplast genetic engineering, the recombinant dna plasmid is bound to small gold nanoparticles that are injected into the chloroplasts of the leaf using a gene gun as described above. this device uses high pressure to insert the plasmid coated particles into the cells. these plasmids contain multiple genes of importance such as the therapeutic gene, a marker gene (may or may not be for antibiotic resistance), a gene that enhances the translation of therapeutic gene and two targeting sequences that flank the foreign gene. the foreign genes are inserted through homologous recombination via flanking sequences at a precise and predetermined location in the organelle genome. the gene expression level in plastids is predominately determined by promoter and -untranslated regions ( -utr elements) (gruissem and tonkyn ) . therefore, suitable -utrs including a ribosomal binding site (rbs) are important elements of plastid expression vectors (eibl et al. ) . in order to obtain high-level protein accumulation from expression of the transgene, the first requirement is a strong promoter to ensure high levels of mrna. most laboratories use the strong plastid rrna operon (rrn) promoter (prrn). besides gene gun, peg mediated transformation and galistan expansion femto syringe microinjection techniques are also used for gene delivery in chloroplast. some of the advantages of chloroplast transformation technology are its low cost, natural gene containment, site specific insertion, very high level of stable expression, generation of production lines with a competitive timeline, elimination of gene silencing, and high accumulation of the recombinant protein. precise steps are given below: step : a heteroplasmic diploid plant cell (first round of selection) a homoplasmic diploid plant cell (second round of selection) step : multiple gene expression step : reproductive organs disintegration of paternal plastids step : maternal inheritance of transgenic traits. the most common entry point for pathogens is at mucosal epithelia lining the gastrointestinal, respiratory, and urino-reproductive tracts, which are collectively the largest immunologically active tissue in body. the mis is the first line of defense and the most effective site for vaccination against those pathogens; nasal, and oral vaccines being the most effective. the goal of oral vaccine is to stimulate both mucosal and humoral immunity against pathogens. edible vaccines have plant parts which are fed directly and the outer tough wall of plant cell acts to protect the antigens against attack by step : single gene step an antigen in a food vaccine is taken up by m cells in the intestine and passed to various immune system cells, which then start a defensive attack, as antigen is a true infectious agent, not just part of one. the response leaves longlasting ''memory cells'' able to promptly neutralize the real infectious agent. the whole procedure can be explained in two stages (fig. . a, b) antibodies and antibody fragments produced against specific antigens are given in table . . there are numerous therapeutic and diagnostic applications of edible vaccines which are summarized in table . . some of the diseases on which the work is going on are described below: malaria is a disease of humans transmitted by the bite of an infected mosquito. it remains one of the most significant causes of human morbidity and mortality worldwide. according to who's world malaria report there are more than million cases of malaria killing around , people. three antigens are currently being investigated for the development of a plant-based malaria vaccine, merozoite surface protein (msp) , msp from plasmodium falciparum and msp / from p. yoelli. wang et al. ( ) have demonstrated that oral immunization of mice with recombinant msp , msp / , and msp , co-administered with ctb as a mucosal adjuvant, induces antibody responses effective against blood stage parasite. measles is an infection of the respiratory system caused by a virus. in an experiment mice fed with tobacco expressing mv-h (measles virus haemagglutinin from edmonston strain) antibody titers five times the level considered protective for humans could be attained and secretory iga was found in their feces. prime boost strategy by combining parenteral and subsequent oral mv-h boosters could induce titers times the human protective levels. these titers were significantly greater than with either of the vaccine administered alone. mv-h edible vaccine does not cause atypical measles, which may be occasionally seen with the current vaccine. thus, it may prove better for achieving its eradication. the success in mice has prompted similar experiments in primates. transgenic rice, and lettuce, and baby food against measles are also being developed. when given with ctb (adjuvant), - g mv-h lettuce is enough; however, an increased dose would be required if given alone. rabies is a deadly viral infection that is transmitted to humans from animals. tomato plants expressing rabies antigens could induce antibodies in mice. alternatively, tmv may also be used. transformed tomato plants using camv with the glycoprotein (g-protein) gene of rabies virus (era strain) was shown to be immunogenic in animals. hepatitis b is a potentially life-threatening liver infection caused by the hepatitis b virus. it is estimated to have infected million people throughout the globe, making the virus one of the most common human pathogen. first human trials of a potato-based vaccine against hepatitis b have reported encouraging results. since immunization is the only known method to (das ) prevent the disease of the hepatitis b virus, any attempt to reduce its infection requires the availability of large quantities of vaccine hbsag. the amount of hbsag needed for one dose could be achieved in a single potato. levels of specific antibodies significantly exceeded the protective level of miu/ml in humans. when cloned into camv, the pcmv-s plasmid encoding the hbsag subtype ayw showed higher expression in roots as compared to leaf tissue of the transgenic potato. furthermore, expression of the antigen was found to be higher in roots of transgenic potato than in leaf tissues. however, the expression of hbsag in transgenic potatoes is not sufficient for using as oral vaccine. further studies are underway to increase the level of hbsag by using different promoters e.g., patatin promoter, and different transcription regulating elements. cholera is an infection of the small intestine that causes a large amount of watery diarrhea. it causes up to million deaths per year in the developing world, primarily among children. studies supported by who have demonstrated possibility of an effective vaccine for cholera, which provides cross protection against enterotoxic e. coli. to address this limitation, plants were transformed with the gene encoding b subunit of the e. coli heat liable enterotoxin (lt-b). transgenic potatoes expressing lt-b were found to induce both serum and secretory antibodies when fed to mice; these antibodies were protective in bacterial toxin assay in vitro. this is the first ''proof of concept'' for the edible vaccine. since people eat only cooked potatoes, the effect of boiling on the properties of ctb expressed in transgenic potatoes was examined. after boiling for min, over half of the vaccine protein survived in its biologically active form, providing evidence that cooking does not always inactivate edible vaccines. thus, the spectrum of plants for producing edible vaccines may be expanded beyond raw food plants such as fruits. co-expression of mutant cholera toxin subunit (mct-a) and lt-b in crop seeds has been shown to be effective by nasal administration and is extremely practical. the prevalence of diabetes is increasing globally and india is no exception. more than million people are affected with diabetes worldwide. type-i diabetes, also known as insulindependent diabetes mellitus (iddm) or juvenileonset diabetes, primarily affects children and young adults and accounts for - % of the diagnosed diabetes in north america. research by ma and hein ( ) at the university of western ontario showed that diabetes can be prevented in mice by feeding them with plants engineered to produce a diabetes related-protein. the idea is based on 'oral tolerance' where the autoimmune system is selectively turned off early by teaching the body to tolerate the ''antigenic proteins''. the pancreatic protein, glutamic acid decarboxylase (gad ) is linked to the onset of iddm, and when injected into mice it is known to prevent diabetes. the canadian group developed transgenic potato and tobacco plants with the gene for gad , fed them to nonobese diabetic mice, which developed insulin-dependent diabetes spontaneously. the results were intriguing, only % of the prediabetic mice fed with transgenic plants developed the diabetes, while % nontreated mice developed the disease. the treated mice also showed increased levels of ig , an antibody associated with cytokines, which suppresses harmful immune responses. thus, the antigen produced in plants appears to retain immunogenicity and prevent diabetes in an animal model. according to canadian scientists, this is the first proof of principle for the use of edible vaccines in the treatment of the autoimmune diseases. human immunodeficiency virus (hiv) is a retroviral that causes acquired immunodeficiency syndrome (aids), a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancer to thrive. in order to produce edible vaccine initial success in splicing hiv protein into cpmv has been achieved. two hiv protein genes and camv as promoter were successfully injected into tomatoes with a needle, and the expressed protein was demonstrable by polymerase chain reaction (pcr) in different parts of the plant, including the ripe fruit, as well as in the second generation plants. recently, spinach has been successfully inoculated for tat protein expression cloned into tmv. each gram of leaf tissue of spinach was shown to contain up to - lg of tat antigen. mice fed with this spinach followed by dna vaccinations resulted in higher antibody titers than the controls, with the levels peaking at weeks post-vaccination. it is still unclear whether the edible vaccines would be regulated under food, drugs. or agricultural products and what vaccine component would be licensed-antigen itself, genetically engineered fruit or transgenic seeds. they would be subjected to a very close scrutiny by the regulatory bodies in order to ensure that they never enter the food supply. this would include greenhouse segregation of medicinal plants from food crops to prevent outcrossing and would necessitate separate storage and processing facilities. although edible vaccines fall under ''genetically modified'' plants, it is hoped that these vaccines will avoid serious controversy, because they are intended to save lives. edible vaccines are future vaccines and some challenges are yet to be overcome before these can become a reality. like all products regulated by food and drug administration, edible vaccines undergo a rigorous review of laboratory, and clinical testing that are conducted to get information regarding safety, efficacy, purity, and potency of these products. these trials can take place only after satisfactory information has been collected on the quality of the nonclinical safety. successful expression of antigens in plants has been demonstrated in the past. the vaccines have also been checked for their efficacy in humans. results from the primary phase of the first-ever human clinical trial of an edible vaccine were published in the journal nature medicine in (blaine p. friedlander, boyce thomson institute of plant research), which indicated that consumption of servings of raw potatoes resulted in immunity to specific diseases. the human clinical study was conducted under the direction of dr. carol tacket at the center for vaccine development, university of maryland school of medicine in baltimore. in the first phase of human testing, the potatoes eaten by volunteers contained a vaccine against travelers' diarrhea, a common condition resulting from intestinal infection by the bacterium e. coli, which contaminates food or water supplies. the clinical trials were approved in advance by the food and drug administration. encouraged by the results of this study, scientists started exploring the use of this technique for administering other antigens. in thanavala's group has developed a potato vaccine booster for use in conjunction with injected hepatitis b vaccine. it is currently in phase ii clinical trial and phase i for patients who have previously been vaccinated. in , tacket and his team mates studied the human immune response to the norwalk virus capsid protein expressed in potatoes. overall, % ( out of volunteers) developed some kind of immune response, although the antibody increase in some cases was modest. in same year, pogrebnyak's lab developed an effective vaccine against the coronavirus which causes severe acute respiratory syndrome (sars). tomato and tobacco plants are used for high expression of the coronavirus spike protein (s ). first, lyophilized tomato fruit was fed to mice and then boosting occurred with s protein expressed in tobacco roots; high igg immune responses and significant igg a and igg b responses were observed in their sera. research is also going in the direction to engineer the plants to produce a variety of functional monoclonal antibody (ma et al. ) . in the first human study of transgenic plant vaccine designed to induce active immunity, adult volunteers were given either g of transgenic potato, g of transgenic potato or g of wild type potato, each transgenic potatoes containing from . to . lg/g of lt-b. the variable dose per gram of potato was due to the tissue specificity of the promoter, therefore, that lt-b was expressed to a different degree in the different tissues of the potatoes. the potatoes in this study were ingested raw; however, subsequent studies have shown that transgenic potatoes expressing the b subunit of cholera toxin could be boiled for min until the tissue becomes soft with loss of only about % of the ct-b pentameric gm binding form. serologic responses were also detected after vaccination. totally out of the volunteers ( %) who ingested transgenic potatoes developed igg anti-lt and in half of them responses occurred after the first dose. there are of the ( %) volunteers developed fourfold rise in serum iga anti-lt. researchers supported by the niaid have shown for the first time that an edible vaccine can safely trigger significant immune responses in people. the goal of the phase proof-ofconcept trial study was to demonstrate that an edible vaccine could stimulate an immune response in humans. volunteers ate bite-sized pieces of raw potato that had been genetically engineered to produce part of the toxin secreted by e. coli, which causes diarrhea. the trial enrolled healthy adults, were chosen at random to receive the genetically engineered potatoes and received pieces of ordinary potatoes. the investigator periodically collected blood and stool samples from the volunteers to evaluate the vaccine's ability to stimulate both systemic and intestinal immune responses. the potatoes were well tolerated and no one experienced serious adverse side effects. niaid supported scientists are exploring the use of this technique for administering other antigens. edible vaccines for other intestinal pathogens are already in the pipeline. potatoes and bananas that might protect against norwalk virus, a common cause of diarrhea, and potatoes and tomatoes that might protect against hepatitis b are being developed. thirty million children throughout the world do not receive even the most basic immunizations each year. as a result, at least three million of these children die from diseases that are fully vaccine-preventable. the solution to vaccinate these children might seem simple with the idea of large scale production of edible vaccines for various diseases. as a recent progress, the first human clinical trials for plant-based vaccine have been performed; it brings many challenges like optimization of expression levels, stabilization during post harvest storage, etc. long-term reactions to edible vaccines are yet to be determined. possible delayed reactions not yet discovered may be the point of consideration. in addition to that, edible vaccines can be further improved for their oral immunogenicity by the use of specific adjuvant which can be applied either as a fusion to the candidate gene or as an independent gene. some of the diseases to which edible vaccines have shown promising application may be elaborated in the veterinary as well as human spectrum. these studies conclude plant-derived vaccines as a new hope and promise for more immunogenic, more effective, and less expensive vaccination strategies against both respiratory as well as intestinal mucosal pathogens. research in the field of edible vaccines holds immense potential for the future and every advancement made in this direction is bringing the dream of edible vaccine one step closer. there is hope that in coming future edible vaccines will conquer all serious diseases and make the planet beautiful to live in. edible vaccines an edible vaccine for malaria using transgenic tomatoes of varying sizes, shapes and colors to carry different antigens plant derived edible vaccines in vivo analysis of plastid psba, rbcl and rpl utr elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency control mechanisms of plastid gene expression production of antibodies in transgenic plants immunotherapeutic potential of antibodies produced in plants production of biologically active human interleukin- in transgenic tomato and potato transgenic plants as vaccine production systems expression of hepatitis b surface antigen in transgenic plants edible vaccine protects mice against escherichia coli heat-labile enterotoxin (lt): potatoes expressing a synthetic lt-b gene edible plant vaccines: applications for prophylactic and therapeutic molecular medicine edible vaccines: a concept comes of age plant biotechnology and in vitro biology in the st century production of hepatitis b surface antigen in transgenic plants for oral immunization human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes immunogenicity in humans of an edible vaccine for hepatitis b oral immunization with a combination of plasmodium yoelii merozoite surface protein and / enhances protection against lethal malarial challenge key: cord- -k uby n authors: nabel, gary j. title: the development of gene-based vectors for immunization date: - - journal: vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: k uby n nan the development of dna vaccines has evolved since the initial description of the ability of naked dna to support gene expression after intramuscular injection. , the concept behind these vaccines is that expression of specifi c viral genes under the control of eukaryotic enhancer-promoters and polyadenylation signals allows appropriate expression of specifi c viral gene products which can be processed and presented as foreign antigens. the genes encoded by dna vaccines can be readily modifi ed and regulatory sequences can be adjusted to optimize level, duration and potency of the immunogen. when injected into muscle, dna is taken up by cells surrounding the injection site and internalized. after uptake and transport to the nucleus, transcription, translation, and post-translational modifi cation allow for the synthesis of a specifi ed gene product. in contrast to inactivated virus particles or recombinant protein vaccines produced in bacteria, yeast or mammalian cells, proteins expressed from gene-based dna vaccines are more likely to assume a native conformation, and their expression within cells allows for more native processing and presentation of antigens that can stimulate cd and cd responses in vivo. because they are in native form, the antibodies generated against these immunogens are theoretically more likely to be cross-reactive with native viral gene products from the pathogen. in addition, because dna is rapidly degraded in the body, the plasmid dna vaccines can provide an advantage in terms of safety, in contrast to live-attenuated viruses, with the possibility of chronic infection and immune stimulation. the application of dna-based genetic immunization has now been demonstrated in a variety of animal models. [ ] [ ] [ ] in addition, in animals it has been shown to be effective in inducing protective immunity against infl uenza virus, malaria, [ ] [ ] [ ] [ ] tuberculosis, ebola virus, rabies, lymphocytic choriomeningitis virus, , herpes simplex virus and lentiviruses in addition to other pathogens. studies in nonhuman primates and humans have indicated that the approach is effective in inducing ctl responses ( and graham et al, unpublished data) . dna vaccines have also been used successfully alone or in combination with other gene-based approaches to develop protective immunity against pathogenic shiv and siv challenge. , - various prime-boost strategies have utilized gary j. nabel vaccines can confer immune protection against infectious agents through divergent arms of the adaptive immune response. the elaboration of antibodies through the humoral immune system has been highly effective in the neutralization of many bacteria, viruses, fungi and parasites. the cell-mediated immune response also plays a major role in containment of infectious agents. t lymphocytes comprise a diverse set of cells, and their functional activity is dependent upon helper t cells, which elaborate a variety of cytokines and stimulate b cells to produce antibodies and induce the formation of cytolytic t lymphocytes (ctl). ctls recognize processed antigen on major histocompatibility complex (mhc) molecules and lyse infected cells. both humoral and cellular immunity are the targets of vaccine-induced immunological responses, each with its own effector functions that can inactivate pathogens in different ways (table - ) . while the humoral immune response is wellknown to confer protection, the role of ctl in protective immunity against viral infections has been recognized more recently. the function and specifi city of these cells has provided the foundation for understanding mhc restriction and its importance in protection against viral infection. , such cellular immune responses help control infectious diseases, particularly when it is diffi cult to generate neutralizing antibodies, as in hiv/aids, malaria and tuberculosis. humoral immunity is more readily induced with purifi ed proteins or inactivated viruses together with appropriate adjuvants; gene-based vaccines appear to be particularly effective at inducing t cell responses, both cd and cd . at the same time, some genebased vaccines can induce humoral immune responses when used with specifi c vectors or in specifi c prime-boost combinations. the majority of adjuvants that have been utilized in vaccine development have affected humoral immunity and appear to enhance antibody responses without inducing cellular immunity. in contrast, the gene-based delivery of vaccine vectors can stimulate both humoral and cellular immunity, thus providing greater selective pressure on infectious agents in vaccines. in this chapter, the major genebased vaccines progressing into clinical trials are summarized, together with the advantages and disadvantages of each individual vector and their infl uence on different effector arms of the immune system. while there is considerable experience with inactivated viruses and protein-based vaccines, the development of gene-based vaccine vectors is only beginning. the advantages of their ability to induce cellular immunity, immunogenicity, safety, mode of antigen presentation, and other attractive features are countered by limitations in knowledge about clinical effi cacy, production methodologies, dna vaccination as the initial vaccine constituent and replication-defective viral vectors, including modifi ed vaccinia ankara virus (mva), , rad , , , or proteins to boost the initial response. this approach avoids repeat exposure to the same viral vector and takes advantage of the ability of dna vaccines to evade anti-vector immunity and to induce immune responses to subdominant t cell epitopes that might otherwise not be stimulated. in the case of dna/rad prime-boost vaccination, this vaccination approach induces greater breadth of the cd response which in turn supports a greater magnitude cd response that does not change in specifi city. there is one phase ii study with a dna prime-rad boost vaccine for hiv infection that has been conducted internationally. a potential limitation of dna vaccine technology is its low immunogenicity in humans. though immune responses can be induced in primates, their potency appears reduced relative to rodent species. in part, this may be due to the relatively lower dose of dna in mass per body weight or surface area; however, improvements in expression vector technology and in the development of dna adjuvants offer the potential for improvements in this area ( fig. - ). one successful approach has involved improvement of transcriptional and translational effi cacy using modifi ed codons preferred in the host species. , in addition, the development of improved enhancer/ promoter regions can allow for even higher expression and these vaccines have advanced into multiple human phase i studies, alone or in combination with other gene-based vectors. advancements of this approach for human use will require further improvements, both in delivery technology and dna adjuvants, of which some representative approaches are described ( fig. - ). advances in molecular virology have facilitated an understanding of the regulation of viral replication, gene expression, and molecular pathogenesis. at the same time, this understanding has enabled the development of novel viral vectors useful for vaccination. a variety of such vectors have now been advanced in preclinical and clinical studies ( fig. - ). depending on their ability to target antigen presenting cells, ability to develop packaging lines, inherent immunogenicity of both the vector and insert, and other factors (table - ), these viral vectors are helping to improve vaccine effi cacy in a variety of infectious disease models. the properties of the more promising vectors and current progress in their development are summarized in the following sections. among the viral vectors that have shown promise for their ability to elicit protective immunity, recombinant adenoviral vectors (rad) have now demonstrated immunogenicity and protective immunity in a variety of animal models. similar to dna vaccines, these vectors transduce cells which can synthesize native gene products and appear to be quite potent in their ability to induce not only helper but specifi cally cytolytic t cell immunity; from - % in various human studies. the majority of clinical vectors have been derived from adenovirus serotype (ad ), although there are more than known human serotypes in six subfamilies (a-f). ad is derived from the c subfamily and is the most common and best-studied serotype; however, the relatively high prevalence of immunity to ad in human populations may pose limitations to the use of these vectors. pre-existing anti-ad immunity may inhibit the response to rad vaccine immunization. for this reason, alternative serotypes and chimeric vectors have been developed to circumvent this potential limitation. the attraction to rad for immunization has followed from its success with a variety of preclinical animal models and phase i/ii human trials. with respect to animal models, the replication-defective adenovirus has been shown to elicit potent immune responses and protection against ebola virus, either administered alone as a single injection or in prime-boost combinations. , it is interesting to note that the prime-boost approach induces more potent and durable immunity suitable for a preventive vaccine, while a single rad vaccination induces a more rapid response that is suffi cient for protection ( fig. - ). this latter approach may be useful in containing acute outbreaks of ebola infection and could be applicable to other pathogens. in addition, both recombinant ad vaccines, as well as dna prime/recombinant ad boost combinations, have been shown to confer partial protection in rhesus macaques against multiple hiv isolates, including shiv- . p, , sivmac and sivmac . , , replication-defective adenovirus has also been used in a variety of additional animal models of infectious disease, including plague, anthrax, infl uenza and malaria. phase i and ii clinical studies with replication-defective adenoviral vectors for hiv- have undergone analysis independently by the merck research laboratories and the nih vaccine research center in niaid. the clinical utility of these vaccines has yet to be defi ned; however, the preliminary data suggest that rad vaccines elicit potent cellular immune responses in humans. in addition, the dna prime-rad boost combinations appear to promote even further stimulation, which has proven more effi cacious in animal models of siv challenge. a more comprehensive phase iib clinical trial has begun and should provide information regarding potential effi cacy of the merck vaccine. in addition, the vrc dna-rad vaccine has completed phase ii testing and may undergo effi cacy testing in the near future. the effect of pre-existing antivector immunity and alternative adenovirus serotypes despite the ability of rad to induce potent and sustained immune responses against a variety of infectious pathogens, concerns remain that preexisting immunity against rad may compromise its effi cacy. this immunity has been found in particular in certain regions of africa, where ad seroprevalence is greater than % with a high degree of neutralizing antibody. while both cellular and humoral immune responses contribute to anti-ad immunity, it is likely that the ad neutralizing antibodies play the major role in suppressing rad -induced immunogenicity, and such immune responses have been observed in humans. this pre-existing immunity can reduce the immunogenicity of ad vaccines in mice, , rhesus monkeys and potentially in humans. , but it is not clear that pre-existing immunity in humans will block vaccine immunogenicity. the reduction in the gag-specifi c response induced by rad in ad seropositive recipients seen in the initial merck rad hiv vaccine trial was less striking when the expression and immunogenicity of the vector were improved. similarly, in vrc trials of dna priming followed by rad boosting, signifi cant immune responses are observed in rad seropositive individuals. several strategies have been developed to overcome the potential problem of rad immunity. novel methods to deliver existing recombinant ad vectors are being explored. for example, it is possible that the administration of higher doses of recombinant ad vectors may overcome anti-ad immunity, although this strategy may be limited by increased toxicity with dose escalation. ad boosting after dna priming may potentially overcome its immunosuppression, too. , the effi cacy of this approach in humans remains to be determined. finally, the administration of ad vectors through mucosal routes may help to circumvent this problem. however, the safety of this approach, particularly for intranasal delivery, has yet to be determined. in addition, several investigators have explored the possibility of coating rad particles with chemicals such as polyethylene glycol that may block access of antibodies to the viral surface. a alternative approaches to evasion of ad immunity include engineering of the vectors to evade dominant ad immune responses. a variety of chimeric fi ber or hexon proteins have been described that maintain immunogenicity and can evade neutralizing antibodies, both against the fi ber [ ] [ ] [ ] [ ] or through the use of hexon chimeras which appear to be the targets of the major neutralizing antibody response. , another approach to antivector immunity involves the development of novel vectors from alternative serotypes. to develop such vectors, investigators have evaluated rad vectors from low seroprevalence human adenoviruses, as well as from nonhuman primates. recombinant ad vectors from human serotypes have been well described. [ ] [ ] [ ] seroprevalence of the ad serotypes suggests that the ad and ad subfamilies as well as adenoviruses from subfamily d, including ad , are uncommon in humans and may therefore offer advantages over ad as vectors. novel vectors based on rad and rad have been developed, and preclinical studies suggest that they are resistant to anti-ad immunity in mice. , the utility of these vectors has been compared to rad . while some of the alternative vectors show less seropositivity, they are often also less immunogenic in preclinical animal studies; how they are able to perform in human studies compared to ad vectors in the presence of rad immunity has yet to be determined. in addition to the replication-incompetent ad vectors, replicationcompetent vectors from ad and ad have been used as vaccine vectors, either for immunization against adenovirus infection or as recombinant vectors, for example, against hiv. , these vaccines offer not only alternative serotypes but also deliver the immune stimulus to the gut mucosa, which may have potentially desirable effects in protection against some diseases. finally, recombinant ad vectors have been developed from alternative species, including sheep, pigs, cows and chimpanzees. [ ] [ ] [ ] [ ] [ ] [ ] in conclusion, the immunogenicity of rad vectors has prompted their development as candidate vaccines for a variety of infectious diseases. these vectors are well tolerated and highly immunogenic at moderate doses. whether the frequency of preexisting ad immunity may compromise their utility in humans remains to be determined; however, a variety of strategies are under development to overcome this effect should it be found. novel delivery vectors, as well as molecularly engineered rad with development of alternative ad serotypes from humans or other species should provide a number of options to expand their use in the future. the effi cacy of vaccinia virus as a vaccine vector represents one of the most well-documented examples of a vaccination against infectious diseases. based on safety issues observed in the use of vaccinia strains against smallpox, - a number of alternative vaccinia virus strains have been developed as immunization vehicles. to avoid these complications, several highly attenuated virus vaccine vectors have been described, as well as avipox and fowlpox vectors. these strains are listed in table - . the development of such attenuated vaccinia viruses also promoted their use as delivery vectors for gene products against specifi c pathogens other than smallpox and the use of these vectors has now been explored extensively in a variety of infectious disease models. one of the two major attenuated strains of poxvirus is modifi ed vaccinia ankara (mva), developed by repeated passaging of the ankara strain on primary chicken embryo fi broblasts. this resulted in the ability of the virus to replicate effi ciently on a variety of non-avian cell types because of multiple genetic changes, which facilitates its propagation and use as a vector. an alternative attenuated strain, the new york vaccinia strain, nyvac, was developed by genetic modifi cation of the viral genome, including the deletion of open reading frames associated with virulence and host range in the copenhagen strain. - nyvac, like mva, is attenuated in animal models and shows favorable safety and immunogenicity in animals and humans. , , this virus also shows block at an early stage of replication, though it is able to replicate productively in african green monkey kidney cells and primary chicken embryo fi broblasts (cef). the avipox vectors include fowlpox and canarypox as well as alvac. alvac is derived from a plaque-purifi ed virus isolated from an existing canarypox strain, canapox. alvac is able to express inserted transgenes and has been shown to be immunogenic in both animal and early clinical trials. , , [ ] [ ] [ ] [ ] these vectors have been evaluated both alone and in primeboost combinations in a variety of infectious disease and cancer models (reviewed in ref. ). poxviruses are notable for their large genome size and their ability to express recombinant genes without an effect on their replication capacity. polyvalent recombinants have been used to immunize experimental animals and have proven useful in a variety of infectious disease models, including rabies, measles, siv, canine distemper, rsv, in addition, these vectors have been studied in a variety of hiv challenge models, both in preclinical studies and in humans [ ] [ ] [ ] [ ] [ ] [ ] and human studies have been undertaken with vaccinia, - nyvac - and alvac. , , [ ] [ ] [ ] [ ] [ ] [ ] to date, these vectors have shown marginal effi cacy that has limited their ability to be tested for effi cacy in human studies, with ctl response rates generally < %, although alvac-env(clade e)gag/pol(clade b) is currently under evaluation in a phase iii study in thailand. such poxvirus vectors have also been evaluated in cancer immunotherapy protocols. while attenuated poxvectors have been evaluated in a variety of human studies, it is clear that it has been more challenging to develop these vaccines for human studies. in part this may be due to the fact that recombinant transgenes represent a small minority of gene products expressed in this otherwise large vector. thus, there is no certainty that the immune response will be focused to the foreign transgene rather than to gene products synthesized endogenously by the poxvirus. in addition, similar to rad, the concern of antivector immunity remains for this virus as well, though it may be a lesser concern for canarypox vectors. although poxvirus vectors show thermostability, ability to incorporate a large foreign transgene, a lack of persistence or genomic integration, and success in smallpox eradication, the diffi culties in manufacturing virus in high yields from primary chicken embryo fi broblast cells, as well as their antigenic complexity, reactogenicity and poor immunogenicity has limited their utility in human trials. whether additional modifi cations of these vectors can be made to facilitate human trials remains unknown. if such modifi cations of the vector platform can be achieved, this vector may have an opportunity to contribute to the development of a variety of successful vaccines. the adeno-associated viruses were defi ned initially by their presence as 'helper' viruses that facilitated the propagation of wild-type adenovirus in cell culture. in contrast to the large genome sizes of rad and vaccinia vectors, this virus is much more limited in size, with insert size of approximately kb. similar to other replication-defective viruses, these particles can be produced in packaging lines that provide complementary structural proteins made constitutively by the cell rather than the virus. a variety of serotypes have been defi ned, and an hiv vaccine expressed in aav has been analyzed in phase i human studies, without evidence of strong immunogenicity. alternative serotypes, including aav , are currently under development and may be assessed both alone and in primeboost combinations for effi cacy in humans. the alphaviruses represent negative-stranded rna viruses that can be modifi ed to express foreign recombinant genes rather than produce pathogenic infections often seen with prototypes such as venezuelan equine encephalitis virus (vee), , sindbis virus , and semliki forest virus (sfv). replication-defective hsv can be produced using packaging cell lines similar to those described for replication-defective rad , aav, or alphavirus vectors. these vaccines have been developed not only to deliver foreign genes as potential immunogens but also as vectors against hsv itself, including both hsv and hsv . more recently, vesicular stomatitis virus, dengue virus type , and yellow fever virus have been modifi ed to express heterologous viral genes for vaccines for infectious disease targets including hiv, west nile virus, fi loviruses and other pathogens. [ ] [ ] [ ] [ ] [ ] [ ] [ ] cell substrates the progress of more recent viral vectors has been dependent upon the development of appropriate packaging cell lines and cell substrates for viral production. changes in regulatory requirements that allowed the advancement of transformed cell lines for virus production have proven invaluable in facilitating this effort. for recombinant adenoviral production, the perc and gv cell lines have supported production of clinical-grade adenovirus type that have progressed into trials for hiv, ebola virus and malaria, and are under study for other infectious agents, such as marburg virus and tuberculosis. once approved, these cell lines can be used for diverse vectors, and the perc cell line has now been used to develop a number of vaccines, including those for west nile and infl uenza viruses. in these latter cases, the propagated virus is subsequently inactivated before administration to humans. for the generation of replication-defective viral vectors, these cell lines allow the production of vectors that can be used in human vaccine studies. of the viruses developed for such vaccines, representative members, summarized in figure - b, include recombinant ad, poxviruses, measles, venezualan equine encephalitis (vee) virus and aav, all of which have progressed into human trials. the development of transformed and continuously propagatable cell lines, in contrast to the previous standard, avian leucosis free primary chick embryo fi broblasts, represents a major advance in vaccine production technology, largely because such cell lines facilitate the production of replication-defective viral vectors in stably transfected cell lines. such lines also offer potentially improved yields and stable production capacity. the development of such lines has taken years to implement because of regulatory concerns regarding adventitious agents, tumorigenicity and other safety/consistency considerations. such oversight and evaluation of the strengths and limitations of these cell substrates continues, based on guidelines several years ago, , with an increasing number of such lines becoming better characterized and available. because many infectious agents replicate at mucosal membranes and transit through the gastrointestinal tract for primary infection, the ability to elicit effective immune responses at these sites is desirable. a variety of bacteria are able to replicate at mucosal sites of natural infection, and it has been proposed that attenuation of these microorganisms and modifi cation to facilitate the delivery of antigen might allow the development of improved vaccines to protect against pathogens that enter through the mucosa. development of live bacterial vectors has therefore focused on both their ability to induce mucosal iga responses as well as cytolytic t cell responses at mucosal sites. the delivery of antigens into mammalian cells to stimulate antibody responses does not require the types of novel gene-based vaccines summarized in this chapter. on the other hand, the synthesis of proteins within mammalian cells delivered by bacterial vectors has the potential to induce the cellular immunity that is the goal of many gene-based viral and nonviral vaccines. these approaches have been reviewed in detail elsewhere [ ] [ ] [ ] and are summarized briefl y here. among the live bacterial vectors used for antigen delivery, there are attenuated mucosal pathogens, such as listeria monocytogenes, salmonella, vibrio cholera, shigella, mycobacteria bovis, yersinia enterocolitica and bacillus anthracis. in addition, there are commensal strains such as s. gordonii, lactobacilli and staphylococci that have been used for the induction of humoral chapter and cellular responses. for gene-based vaccination, listeria monocytogenes has been a particular focus of research. this gram-positive intracellular pathogen has been studied as a model to understand class i mhc-restricted immune responses. these responses are normally seen against the bacterial proteins or co-expressed antigens. this microorganism utilizes a specialized system to introduce proteins into cells and facilitate processing and presentation through mhc class i, and different mutations have been used to develop attenuated strains that retain the ability to deliver antigens. similarly, salmonella bacterial strains are intracellular pathogens that become restricted to the endosomal compartment of eukaryotic cells where they are resistant to lysis. a variety of mutations have been introduced into salmonella to generate several different live vaccine carriers, and these vaccine prototypes have undergone further development for vaccine delivery. among the other bacterial carriers, mycobacteria bovis calmette-guerin (bcg) has been a widely used bacterial vaccine; for example, recently this organism has been used to express hiv antigens. , in some instances, expression of mammalian genes has required modifi cation of codons more consistent with the host cell type, which has improved immunogenicity. at the present time, however, the ability of such microorganisms to induce cellular immunity has been limited. an area of intense interest has been the use of live bacterial vectors for the delivery of dna vaccines. in this instance, the aim is for the bacteria to deliver plasmid dna into the cytoplasm of infected cells; such organisms as shigella and listeria have been used for this purpose. , in addition, attenuated salmonella has been evaluated and has shown some promise in both infectious disease and tumor models in experimental animals. [ ] [ ] [ ] while the use of such bacterial vectors has been attractive in theory, it has been more diffi cult to reduce this method to practice. among the concerns is the possibility of reversion or reactogenicity of these potentially pathogenic bacteria to wild type forms, the stability of the recombinant bacteria, as well as the possibility that pre-existing immunity from exposure to natural pathogens may limit their infectivity. a variety of host genetic factors can modulate the immune response induced by the bacterial carrier, and variability in the innate immune responses to such pathogens may limit their consistency in vivo. finally, perhaps the most challenging problem has been the ability to effect a gene transfer from bacteria into mammalian cells. it is likely that very specialized transport pathways are required for the successful implementation of this technology, and additional improvements in the future will be necessary to improve the effi cacy of this approach, which remains limited in its present form. while substantial work has progressed in animal models of vaccine effi cacy, the ultimate value of gene-based vaccination has yet to be shown in human studies. several trials using the poxvirus technology have advanced into clinical evaluation. these include canarypox, which has progressed through phase ii studies in the united states for hiv vaccine evaluation, and has advanced into a proof-of-concept effi cacy trial currently in progress in thailand. in addition, both modifi ed vaccinia ankara and nyvac have been evaluated in phase i human studies. because the production technology for poxviruses is well known, and gmp procedures for amplifi cation of these viruses followed protocols similar to those developed for vaccinia virus, the path into clinical studies has been relatively straightforward, as have the several trials of modifi ed vaccinia ankara, which has been evaluated both as a vaccine for hiv, alone or in prime-boost combinations, and as a potentially safer next-generation vaccine for smallpox. other and a dna vaccine for infectious hematopoietic necrosis virus, developed by merial for use in farm-raised fi sh. an additional vaccine is being developed against viral hemorrhagic septicemia virus in farmed salmon. in these studies, a single injection of microgram amounts of dna induces rapid and long-lasting immune protection. a recombinant yellow fever vaccine has advanced into effi cacy studies as well. the precedent set by these studies provides hope that additional gene-based vaccines will become available for human use and may contribute to the development of protective immunity for a variety of challenging infectious diseases that have thus far eluded the grasp of vaccine-induced immunity. major transplantation antigens, viruses, and specifi city of surveillance t cells mhc-restricted cytotoxic t cells: studies on the biological role of polymorphic major transplantation antigens determining t-cell restriction-specifi city, function, and responsiveness direct gene transfer into mouse muscle in vivo heterologous protection against infl uenza by injection of dna encoding a viral protein a human t-cell leukemia virus type regulatory element enhances the immunogenicity of human immunodefi ciency virus type dna vaccines in mice and nonhuman primates genetic immunization is a simple method for eliciting an immune response dna inoculation induces protective in vivo immune responses against cellular challenge with hiv- antigen-expressing cells vaccination of mice and nonhuman primates using hiv-gene-containing dna circumventing genetic restriction of protection against malaria with multigene dna immunization: cd + cell-, interferon gamma-, and nitric oxide-dependent immunity dna vaccines against malaria: immunogenicity and protection in a rodent model protection of mice against plasmodium yoelii sporozoite challenge with p. yoelii merozoite surface protein dna vaccines vaccination against tuberculosis by injection immunization for ebola virus infection overcoming immunity to a viral vaccine by dna priming before vector boosting plasmid chemokines and colony-stimulating factors enhance the immunogenicity of dna priming-viral vector boosting human immunodefi ciency virus type vaccines comparative immunogenicity in rhesus monkeys of dna plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodefi ciency virus type gag gene development of an hiv- vaccine based on replication-defective adenovirus. presented at the keystone symposium on hiv vaccine development impact of pre-existing immunity on the immunogenicity of ad -based vaccines. presented at aids vaccines oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier enhanced mucosal immunoglobulin a response of intranasal adenoviral vector human immunodefi ciency virus vaccine and localization in the central nervous system pegylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo adenovirus type and capsid chimera: fi ber replacement alters receptor tropism without affecting primary immune neutralization epitopes improved adenovirus vectors for infection of cardiovascular tissues exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease an adenoviral type vector carrying a type fi ber as a vaccine vehicle: dc targeting, cross neutralization, and immunogenicity circumvention of immunity to the adenovirus major coat protein hexon construction and characterization of hexonchimeric adenoviruses: specifi cation of adenovirus serotype circumventing the immune response to adenovirus-mediated gene therapy seroswitch' adenovirus-mediated in vivo gene transfer: circumvention of anti-adenovirus humoral immune defenses against repeat adenovirus vector administration by changing the adenovirus serotype circumvention of anti-adenovirus neutralizing immunity by administration of an adenoviral vector of an alternate serotype replication-defi cient human adenovirus type vectors for gene transfer and vaccination: effi cient human cell infection and bypass of preexisting adenovirus immunity immunogenicity of recombinant adenovirus serotype vaccine in the presence of preexisting anti-ad immunity immunogenicity of heterologous adenovirus prime-boost regimens involving ad and ad . presented at aids vaccines protection against mucosal simian immunodefi ciency virus siv(mac ) challenge by using replicating adenovirus-siv multigene vaccine priming and subunit boosting live recombinant vectors for aids vaccine development ovine adenovirus vectors overcome preexisting humoral immunity against human adenoviruses in vivo replication-defective bovine adenovirus type as an expression vector porcine adenovirus- as a helper-dependent expression vector replicationdefective vector based on a chimpanzee adenovirus construction and characterization of recombinant porcine adenovirus serotype expressing the transmissible gastroenteritis virus spike gene novel, chimpanzee serotype -based adenoviral vaccine carrier for induction of antibodies to a transgene product complications of smallpox vaccination disseminated vaccinia in a military recruit with human immunodefi ciency virus (hiv) disease smallpox and its eradication. world health organization live attenuated vaccinia and other poxviruses as delivery systems: public health issues nyvac: a highly attenuated strain of vaccinia virus safety and immunogenicity of recombinants based on the genetically-engineered vaccinia strain poxvirus-based vectors as vaccine candidates p as a target for cancer vaccines: recombinant canarypox virus vectors expressing p protect mice against lethal tumor cell challenge poxvirus-based vaccine candidates for cancer, aids, and other infectious diseases applications of pox virus vectors to vaccination: an update highly attenuated poxvirus vectors immunisation with canarypox virus expressing rabies glycoprotein genetically engineered poxviruses for recombinant gene expression, vaccination, and safety vaccine therapy in early hiv- infection using a recombinant canarypox virus expressing gp mn (alvac-hiv): a double-blind controlled randomized study of safety and immunogenicity canarypox virus-based vaccines: prime-boost strategies to induce cell-mediated and humoral immunity against hiv gene gun intradermal dna immunization followed by boosting with modifi ed vaccinia virus ankara: enhanced cd + t cell immunogenicity and protective effi cacy in the infl uenza and malaria models a group specifi c anamnestic immune reaction against hiv- induced by a candidate vaccine against aids poxvirus-based vectors as vaccine candidates vaccination of vaccinia-naive adults with human immunodefi ciency virus type gp recombinant vaccinia virus in a blinded, controlled, randomized clinical trial. the aids vaccine clinical trials network potential improvement for poxvirus-based immunizations vehicles multienvelope hiv vaccine safety and immunogenicity in small animals and chimpanzees containment of simian immunodefi ciency virus infection in vaccinated macaques: correlation with the magnitude of virus-specifi c pre-and postchallenge cd + and cd + t cell responses largescale production and purifi cation of a vaccinia recombinant-derived hiv- gp and analysis of its immunogenicity removal of cryptic poxvirus transcription termination signals from the human immunodefi ciency virus type envelope gene enhances expression and immunogenicity of a recombinant vaccinia virus recombinant virus vaccine-induced siv-specifi c cd + cytotoxic t lymphocytes immunization with a modifi ed vaccinia virus expressing simian immunodefi ciency virus (siv) gag-pol primes for an anamnestic gag-specifi c cytotoxic t-lymphocyte response and is associated with reduction of viremia after siv challenge reduction of simian-human immunodefi ciency virus . p viremia in rhesus monkeys by recombinant modifi ed vaccinia virus ankara vaccination enhanced simian immunodefi ciency virusspecifi c immune responses in macaques induced by priming with recombinant semliki forest virus and boosting with modifi ed vaccinia virus ankara effect of vaccination with recombinant modifi ed vaccinia virus ankara expressing structural and regulatory genes of siv(macj ) on the kinetics of siv replication in cynomolgus monkeys induction of simian immunodefi ciency virus (siv)-specifi c ctl in rhesus macaques by vaccination with modifi ed vaccinia virus ankara expressing siv transgenes: infl uence of preexisting anti-vector immunity comparison of vaccine strategies using recombinant env-gag-pol mva with or without an oligomeric env protein boost in the shiv rhesus macaque model immunogenicity and protective effi cacy of a human immunodefi ciency virus type recombinant canarypox (alvac) vaccine candidate in cynomolgus monkeys mature dendritic cells infected with canarypox virus elicit strong anti-human immunodefi ciency virus cd + and cd + t-cell responses from chronically infected individuals potentiation of simian immunodefi ciency virus (siv)-specifi c cd (+) and cd (+) t cell responses by a dna-siv and nyvac-siv prime/boost regimen cross-protection against mucosal simian immunodefi ciency virus (sivsm) challenge in human immunodefi ciency virus type -vaccinated cynomolgus monkeys induction of cytotoxic t lymphocytes by recombinant canarypox (alvac) and attenuated vaccinia (nyvac) viruses expressing the hiv- envelope glycoprotein memory cytotoxic t lymphocyte responses in human immunodefi ciency virus type (hiv- )-negative volunteers immunized with a recombinant canarypox expressing gp of hiv- and boosted with a recombinant gp clade b-based hiv- vaccines elicit cross-clade cytotoxic t lymphocyte reactivities in uninfected volunteers induction of neutralizing antibodies and gag-specifi c cellular immune responses to an r primary isolate of human immunodefi ciency virus type in rhesus macaques alvac-sivgag-pol-env-based vaccination and macaque major histocompatibility complex class i (a* ) delay simian immunodefi ciency virus sivmacinduced immunodefi ciency aav vectors: is clinical success on the horizon? venezuelan equine encephalitis virus vectors expressing hiv- proteins: vector design strategies for improved vaccine effi cacy vaccination of macaques against pathogenic simian immunodefi ciency virus with venezuelan equine encephalitis virus replicon particles sindbis virus vectors for expression in animal cells evaluation of recombinant alphaviruses as vectors in gene therapy replication-defective viruses as vaccines and vaccine vectors an effective aids vaccine based on live attenuated vesicular stomatitis virus recombinants west nile virus/dengue type virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective effi cacy live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses priming with plasmid dnas expressing interleukin- and simian immunodefi ciency virus gag enhances the immunogenicity and effi cacy of an experimental aids vaccine based on recombinant vesicular stomatitis virus a single-cycle vaccine vector based on vesicular stomatitis virus can induce immune responses comparable to those generated by a replication-competent vector a live, attenuated recombinant west nile virus vaccine a single intranasal inoculation with a paramyxovirusvectored vaccine protects guinea pigs against a lethal-dose ebola virus challenge vaccine cell substrates cell line issues: historical and future perspectives points to consider in the characterization of cell lines used to produce biologicals. united states food and drug administration, department of health and human services use of live bacterial vaccine vectors for antigen delivery: potential and limitations progress towards the use of listeria monocytogenes as a live bacterial vaccine vector for the delivery of hiv antigens novel bacterial systems for the delivery of recombinant protein or dna phagolysosome formation, cyclic adenosine ′: ′-monophosphate and the fate of salmonella typhimurium within mouse peritoneal macrophages bacterial vaccine vectors and bacillus calmette-guerin protective immune responses induced by secretion of a chimeric soluble protein from a recombinant mycobacterium bovis bacillus calmette-guerin vector candidate vaccine for human immunodefi ciency virus type in small animals attenuated shigella as a dna delivery vehicle for dna-mediated immunization delivery of antigen-encoding plasmid dna into the cytosol of macrophages by attenuated suicide listeria monocytogenes oral somatic transgene vaccination using attenuated s. typhimurium gene transfer in dendritic cells, induced by oral dna vaccination with salmonella typhimurium, results in protective immunity against a murine fi brosarcoma wild-type hfe protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis a multiclade hiv- dna vaccine elicits humoral and sustained cellular immune responses in humans in a randomized phase i clinical trial a dna vaccine for ebola virus is safe and immunogenic in a phase i clinical trial a west nile virus vaccine induces neutralieing antibody in healthy adults in a phase i clinical trial west nile virus recombinant dna vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays dna vaccines for aquacultured fi sh prospects for development of a vaccine against the west nile virus poxviruses as immunization vehicles key: cord- -cot vx authors: jackson, nicholas a. c.; kester, kent e.; casimiro, danilo; gurunathan, sanjay; derosa, frank title: the promise of mrna vaccines: a biotech and industrial perspective date: - - journal: npj vaccines doi: . /s - - - sha: doc_id: cord_uid: cot vx mrna technologies have the potential to transform areas of medicine, including the prophylaxis of infectious diseases. the advantages for vaccines range from the acceleration of immunogen discovery to rapid response and multiple disease target manufacturing. a greater understanding of quality attributes that dictate translation efficiency, as well as a comprehensive appreciation of the importance of mrna delivery, are influencing a new era of investment in development activities. the application of translational sciences and growing early-phase clinical experience continue to inform candidate vaccine selection. here we review the state of the art for the prevention of infectious diseases by using mrna and pertinent topics to the biotechnology and pharmaceutical industries. continued growth in the vaccine business is expected based on expanded coverage, improved existing products, and new vaccines. among other factors, manufacturing must change to support growth. capital-rich investment in fixed facilities that commit to a given form of production for a given target poses significant costs and challenges to vaccine manufacturers if there is a major change in strategy. long lead times in manufacturing, and potentially hundreds of complex process steps, all complicate capabilities. vaccine companies, like the biotech industry, desire novel production methods out of a need for efficiency that reduce the cost of goods, shorten time to licensure, and respond quicker to disease outbreaks. mrnabased vaccines hold the promise to revolutionize the field by addressing current manufacturing challenges and offering novel vaccine compositions. assuming that mrna vaccines will be proven clinically efficacious and safe, one of the central advantages hinges on rapidity of manufacture. within weeks, clinical batches can be generated after the availability of a sequence encoding the immunogen. the process is cell-free and scalable. of paramount advantage, a facility dedicated to mrna production should be able to rapidly manufacture vaccines against multiple targets, with minimal adaptation to processes and formulation. in addition, new targets requiring multi-antigen approaches will benefit from the speed in which mrna can render multiple constructs. beyond manufacturing advantages, mrna technology is impacting vaccine discovery and research. expression may be possible for complex proteins that are difficult or impossible to generate with current expression systems. mrna constructs can also be used to express potent monoclonal antibodies for novel immunoprophylaxis. here we review the state of the art in mrna constructs and delivery technologies for the prevention of infectious diseases, and a review of pertinent topics to the biotechnology and pharmaceutical industries. "state-of-the-art" mrna constructs and delivery technologies the core principle behind mrna as a technology for vaccination is to deliver the transcript of interest, encoding one or more immunogen(s), into the host cell cytoplasm where expression generates translated protein(s) to be within the membrane, secreted or intracellularly located. two categories of mrna constructs are being actively evaluated: non-replicating mrna (nrm) and self-amplifying mrna (sam) constructs (fig. ). both have in common a cap structure, ′ and ′ untranslated regions (utrs), an open-reading frame (orf), and a ′ poly(a) tail. sam differs with the inclusion of genetic replication machinery derived from positive-stranded mrna viruses, most commonly from alphaviruses such as sindbis and semliki-forest viruses. , generally, the orf encoding viral structural proteins is replaced by the selected transcript of interest, and the viral rna-dependent rna polymerase is retained to direct cytoplasmic amplification of the replicon construct. the potential merits of nrm versus sam will be addressed later. the manufacturing process begins with the generation of a plasmid dna (pdna) containing a dna-dependent rna polymerase promoter, such as t , and the corresponding sequence for the mrna construct. the pdna is linearized to serve as a template for the dna-dependent rna polymerase to transcribe the mrna, and subsequently degraded by a dnase process step. the addition of the ′ cap and the ′ poly(a) tail can be achieved during the in vitro transcription step , or enzymatically after transcription. enzymatic addition of the cap can be accomplished by using guanylyl transferase and ′-o-methyltransferase to yield a cap ( n me gpppn) or cap ( n me gpppn ′-ome ) structure, respectively, while the poly-a tail can be achieved through enzymatic addition via poly-a polymerase. purification is a crucial next step, which can be achieved with the application of high-pressure liquid chromatography (hplc). the resultant drug substance is then formulated into drug product and released based on sterility, identity, purity, and potency testing. these processes allow good manufacturing practise (gmps) facilities to switch to a new vaccine within a very short period of time, given that the reaction materials and vessels are the same. the design of a mrna construct for vaccination, once released into the cytoplasm of a cell, is to efficiently utilize the translational machinery of the host cell to generate a sufficient quantity of the encoded immunogen that is presented appropriately to the immune system. across the field, several critical quality attributes have been, and continue to be, the focus of efforts to maximize gene expression (fig. ) . first, the purity of the mrna is a crucial determinant of yields, and it is known that the dna-dependent rna polymerases yield smaller oligoribonucleotide impurities as a result of abortive initiation events, as well as double-stranded (ds) rna generated by self-complementary ′ extension, which can result in type i interferon and inflammatory cytokine production through pattern recognition receptors. karikó et al. demonstrated that removal of contaminants in mrna -aaaaaa ' -aaaaaa modificaƟon of sequence, such as codon opƟmizaƟon, have contributed to improved expression. fig. critical quality attributes (cqas) have been identified that dictate the performance of the mrna construct to express the gene of interest efficiently. five principal cqas include ′ capping efficiency and structure; utr structure, length, and regulatory elements; modification of coding sequence; poly-a-tail properties; mrna purity. preparations reduced innate immune responses and resulted in significantly higher levels of reporter protein expression in vitro. second, the ′ and ′ utr regions are important for maximizing gene expression. the length of the ′ utr, ′ utr structures, and regulatory elements in both utrs all impact efficiency. third, the ′ -methylguanosine (m g) cap of the mrna molecule, linked via a triphosphate bridge to the first transcribed nucleotide, is essential for efficient translation, and blocks ′- ′ exonucleasemediated degradation. the specific cap structure plays a critical role in both protein production and immunogenicity, with incomplete capping ( ′ triphosphate) and cap structures shown to stimulate rig- . [ ] [ ] [ ] in addition, -o′-unmethylated capped rna can be sequestered by cellular ifn-induced proteins with tetratricopeptide repeats (ifit ) that prevent the initiation of translation, or detected by the cytoplasmic rna sensor mda . manufacturers of mrna vaccines pay careful attention to the choice of enzyme and reaction conditions, in order to catalyze the highest percentage of cap formation. fourth, the poly (a) tail and its properties such as length, are crucial for translation and protection of the mrna molecule. , last, codon optimization and modification of nucleotides have contributed to translation efficiency. for example, optimization of guanine and cytosine (gc) content can have a significant impact, and has been well established with dna vaccines. the innate immune activation to mrna can also influence its utility as a delivery system. the use of modified nucleosides, such as pseudouridine or n- -methylpseudouridine to remove intracellular signaling triggers for protein kinase r (pkr) activation, resulted in enhanced antigen expression and adaptive immune responses. [ ] [ ] [ ] it has been demonstrated that successful protein production, minimal undesired inflammatory responses, and systemic adaptive immune responses could be achieved preclinically by using unmodified mrna through a combination of optimizing the coding sequence and removal of any unwanted inflammatory impurities. , ultimately, comparative immunogenicity between these approaches require studies that control all potential factors, including the delivery system. human studies "head-to-head" comparing modified and unmodified nucleoside mrna constructs would confirm clinically relevant differences, if any. similarly, controlled comparisons of the aforementioned nrm and sam are needed to determine any distinctions. until then, all the approaches-modified, unmodified, nrm, sam, and combinations thereof-appear feasible, and are supported by preclinical data, although unmodified nucleoside constructs may be desirable for manufacturing efficiency and transcriptional fidelity. , in addition to optimizing an mrna construct, and of quintessential importance, is the delivery of the mrna vaccine from the bolus at the injection site into the cytoplasm of cells for the initiation of translation. as mrna is a transient molecule by nature that is susceptible to degradation primarily through nuclease activity, efficient protection is required. , this has been an intense area of research in the field, for which lipid nanoparticle (lnp) formulations are currently emerging as a leading category. lnp delivery systems serve multiple purposes in their applications. in addition to the aforementioned sustained stability imparted through protection from nuclease degradation, they also facilitate organ specificity, efficient cellular uptake, and provide endosomal escape properties that can enhance the successful delivery of the mrna cargo to the cytoplasmic site of action. [ ] [ ] [ ] there have been numerous examples of successful delivery of mrna by using lnps for therapeutic [ ] [ ] [ ] [ ] as well as vaccine applications. [ ] [ ] [ ] [ ] much of the focus of the continued development of such lnp carrier systems involves optimization of the ionizable lipid component, with particular focus on the acid dissociation constant (pka) and fusogenic properties (both of the ionizable component as well as helper lipid[s]), which have been demonstrated to play key roles in efficient cytoplasmic entry and release of cargo. [ ] [ ] [ ] [ ] next-generation lnps may include specific targeting motifs for homing and uptake by professional antigen-presenting cells, such as dendritic cells (dc). ligands for dc receptors could be embedded on the surface of the lnps to target these cells and promote antigen presentation to the immune system. perspective # : improved understanding of the molecular mechanisms of action will guide further improvements in mrna constructs and formulations continual optimization and improvements toward developing the next generation of mrna-based drugs are undoubtedly occurring. sequence optimization within utrs and coding regions of mrna providing greater stability and/or potency can result in higher production of the desired antigen, potentially leading to a more favorable therapeutic index. , additional sites within the mrna construct are available for optimization as well. novel cap structures focused on base or sugar modifications have resulted in greater translational properties through increased ribosomal interaction or enhanced stability. improved stability of mrna can also be achieved through various modifications of the triphosphate bridge within the cap structure. [ ] [ ] [ ] optimization of the carrier system can provide significant benefit as well. substantial attention has been placed on the development of novel ionizable lipids and formulations, with improvements in cellular uptake, endosomal release, potency, and biodegradability. [ ] [ ] [ ] the combinations of all of these areas for optimization are near limitless, and success within any of these parameters can allow for beneficial effects and can provide novel approaches for vaccines to successfully prevent disease. perspective # : translational sciences will inform preclinical and clinical studies to promote rapid downselection of constructs and formulations a key aspect of vaccine development efforts is the goal of making early informed decisions, based on objective data that favor or disfavor a particular candidate. it is underappreciated in the field that multi-antigen vaccine approaches are a significant challenge in the decision-making process. for example, the lnp:mrna mass ratio can be around : - : . thus, multi-antigen candidates necessitate a significant amount of lnp for a given dose. lnps are known to have inherent adjuvant properties. therefore, safety and tolerability may limit multi-antigen approaches, and here translational sciences are crucial for development. there are a variety of new translational medicine tools that can be leveraged in evaluating immunity, which include in vitro human immune system models and the related organoids to improve the predictability of clinical results. systems biology techniques can help in the understanding of fine differences between various nrm and sam vaccine sequences, as well as serving as useful tools to frame sequence optimizations during iterative development schemes. further, the use of functional assessments, like human challenge models, where the immunologic profiles of the participants and the specific details of the challenge strain(s) are well characterized-an approach mostly used to date in the evaluation of more traditional vaccinesprovides an important and powerful method to obtain early decision-making data regarding the performance of an mrna vaccine candidate. these tools are likely quintessential for development because current data have demonstrated a poor translation between preclinical and clinical studies with an mrna pandemic influenza vaccine. in ferrets (id, × / mcg), nonhuman primates (id/im, × mcg), and humans (im, × mcg), an h n -derived ha mrna vaccine formulated with lnp elicited hai titers in the range of - , , , and , respectively. , whether this lack of translation is a function of mrna not having optimized quality attributes, suboptimal delivery or an inherent limitation of preclinical or in vitro translational models is not known. understanding this is going to be key for further development. perspective # : challenges ahead for clinical trials while specific regulatory guidelines are lacking for the clinical development of mrna-based vaccines, the following general principles, as outlined in overarching guidance documents, are generally sufficient to help facilitate the entry of candidate vaccines into early-phase clinical trials. at the time of writing, clinical trials for mrna-based infectious disease vaccines have been completed, or are at various stages of progression, building experience (see table ). all studies assess viral targets, many have been reviewed elsewhere recently. the current focus from a clinical perspective is to optimize the benefit (immunogenicity and efficacy) while reducing the risk (safety) profile of a candidate mrna vaccine by optimizing the quality attributes that dictate expression and/or augmenting delivery. it is clear that immune activation can be both advantageous and potentially detrimental, and has to be titrated accordingly. thus, early-phase clinical trials need to be designed in a way to appropriately capture the inflammatory component intrinsic to all mrna vaccines, given that several intracellular innate immune response sensors are activated by rna. elements include measuring administration site reactions such as pain, tenderness-associated systemic reactions such as fever and malaise, and routine biochemical laboratory parameters (e.g., serum electrolytes, liver function test, and cbc). these parameters, when followed closely, can be designed to develop enrollment pause rules in the event that severe tolerability issues are observed in a clinical trial. detailed characterization of the immune response fully leveraging modern techniques such as transcriptomics and systems biology, in addition to traditional methods of immune monitoring, needs to be implemented. several agency guidelines developed for the study of novel adjuvants in human subjects provide sufficient guidance that can be applied to mrna candidate vaccines on how to monitor safety in early-phase clinical studies. the data from early-phase clinical studies, particularly around local and self-limiting systemic reactogenicity, have been mixed. , in fact, reporting of human trials has generally concluded that new formulations are required to optimize the profile. however, these early claims need further confirmation, and in many cases, complete datasets are still awaited. as mentioned above, multi-antigen approaches will only complicate the issue of establishing acceptable tolerability. humoral elicited responses have been generally underwhelming, compared with the established potency in the field of protein or live attenuated vaccines. , this indicates that much formulation work is still needed to achieve sufficient immunological potency of different vaccine candidates, while maintaining acceptable tolerability-but we can be encouraged by incremental progress to date. furthermore, very limited data exist on repeat administration of mrna vaccines in humans. these data are important as most vaccines generally require a booster dose. as the field accrues more data from early-phase human studies, the focus of mrna vaccines will shift from documenting local and systemic tolerability to capturing potential long-term safety. unfortunately detecting safety signals for uncommon adverse events requires thousands of subjects. as with novel adjuvants, an adequate safety database to assure safety for candidate mrna vaccines is likely to be in the tens of thousands range. given that different manufacturers are pursuing different strategies to optimize their candidate vaccines, conclusions from one candidate may not be generalizable. therefore, it is likely that each candidate vaccine will have to independently prove its risk/benefit profile that is favorable. the potential advantages of mrna as a vaccine range from the discovery of immunogens to rapid response manufacturing. currently, the field is pursuing two approaches: non-replicating and self-replicating constructs. a number of quality attributes, that dictate stability and efficiency of expression, continue to be an intense area of development. it is widely recognized that the delivery of the mrna into the cytoplasm is equally important to successfully elicit a robust and durable immunity. as a result, much progress has been achieved with considerable focus on novel ionizable lipid formulations and the next generation of delivery systems. the nature of mrna technology allows rapid refinement with almost limitless combinations of derivatives in the pursuit of optimization. this necessitates the application of translational sciences to accelerate selection of the optimal construct and formulation for subsequent development. clinical experience in the last years is building upon the plethora of preclinical data generated. these trials have informed our understanding of the need to find the optimal balance between immune and inflammatory activation to establish an acceptable risk/benefit profile for a given vaccine. whether clinical results from different sponsors will be generalizable for mrna technologies and formulations remains to be determined. overall, with significant advances in mrna biology, delivery, and manufacturing, the biotechnology and vaccine industries are poised for further investment in the development of novel products. multi-antigenic human cytomegalovirus mrna vaccines that elicit potent humoral and cell-mediated immunity a lipid-encapsulated mrna encoding a potently neutralizing human monoclonal antibody protects against chikungunya infection mrna vaccines-a new era in vaccinology enhancement of sindbis virus self-replicating rna vaccine potency by linkage of herpes simplex virus type vp protein to antigen self-replicating alphavirus rna vaccines promoter specificity determinants of t rna polymerase synthesis and properties of mrnas containing the novel "anti-reverse" cap analogs -methyl( ′-o-methyl) gpppg and -methyl( ′-deoxy) gpppg synthesis of anti-reverse cap analogs (arcas) and their applications in mrna translation and stability modification of rna by mrna guanylyltransferase and mrna (guanine ) methyltransferase from vaccinia virions hplc purification of in vitro transcribed long rna. in synthetic messenger rna and cell metabolism modulation oligoribonucleotide synthesis using t rna polymerase and synthetic dna templates self-coded ′-extension of run-off transcripts produces aberrant products during in vitro transcription with t rna polymerase generating the optimal mrna for therapy: hplc purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mrna translational efficiency is regulated by the length of the untranslated region functional ʹ utr mrna structures in eukaryotic translation regulation and how to find them structural basis for m g recognition and ′-o-methyl discrimination in capped rnas by the innate immune receptor rig-i rig-i: a multifunctional protein beyond a pattern recognition receptor discrimination of cytosolic self and non-self rna by rig-i-like receptors sequestration by ifit impairs translation of ′o-unmethylated capped rna ribose ′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda multifunctional deadenylase complexes diversify mrna control short poly(a) tails are a conserved feature of highly expressed genes high guanine and cytosine content increases mrna levels in mammalian cells incorporation of pseudouridine into mrna enhances translation by diminishing pkr activation n -methylpseudouridine-incorporated mrna outperforms pseudouridine-incorporated mrna by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice nucleoside-modified mrna vaccines induce potent t follicular helper and germinal center b cell responses unmodified mrna in lnps constitutes a competitive technology for prophylactic vaccines sequence-engineered mrna without chemical nucleoside modifications enables an effective protein therapy with large animals efficacy and immunogenicity of unmodified and pseudouridine-modified mrna delivered systemically with lipid nanoparticles in vivo base modifications affecting rna polymerase and reverse transcriptase fidelity effect of mrna modifications on translational fidelity stability of endogenous and added rna in blood specimens, serum, and plasma the many pathways of rna degradation delivery the messenger: advances in technologies for therapeutic mrna delivery current status of messenger rna delivery systems lipid-based mrna vaccine delivery systems improved efficacy in a fabry disease model using a systemic mrna liver depot system as compared to enzyme replacement therapy therapeutic efficacy in a hemophilia b model using a biosynthetic mrna liver depot system systemic messenger rna therapy as a treatment for methylmalonic acidemia & arginase, i. mrna therapy-a novel approach to rescue arginase enzyme deficiency results of the first phase i/ii clinical vaccination trial with direct injection of mrna protective efficacy of in vitro synthesized, specific mrna vaccines against influenza a virus infection a novel, disruptive vaccination technology. self-adjuvanted rnactive vaccines zika virus protection by a single low-dose nucleoside-modified mrna vaccination a novel amino lipid series for mrna delivery: improved endosomal escape and sustained pharmacology and safety in non-human primates optimization of lipid nanoparticle formulations for mrna delivery in vivo with fractional factorial and definitive screening designs structural and fusogenic properties of cationic liposomes in the presence of plasmid dna cationic lipids, lipoplexes and intracellular delivery of genes improving mrna-based therapeutic gene delivery by expression-augmenting ′-utrs identified by cellular library screening codon optimality is a major determinant of mrna stability locked nucleic acid (lna)-modified dinucleotide mrna cap analogue: synthesis, enzymatic incorporation, and utilization phosphorothioate cap analogs stabilize mrna and increase translational efficiency in mammalian cells novel cap analogs for in vitro synthesis of mrnas with high translational efficiency mrna cap analogues substituted in the tetraphosphate chain with cx : identification of o-to-ccl as the first bridging modification that confers resistance to decapping without impairing translation bioinspired alkenyl amino alcohol ionizable lipid materials for highly potent in vivo mrna delivery non-viral delivery of nucleic acids: insight into mechanisms of overcoming intracellular barriers nanoscale platforms for messenger rna delivery a novel lipid nanoparticle adjuvant significantly enhances b cell and t cell responses to sub-unit vaccine antigens human organoid cultures: transformative new tools for human virus studies moving from empirical to rational vaccine design in the human challenge models: tools to accelerate the development of malaria vaccines preclinical and clinical demonstration of immunogenicity by mrna vaccines against h n and h n influenza viruses mrna vaccines against h n and h n influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase randomized clinical trials mrna as a transformative technology for vaccine development to control infectious diseases extracellular rna sensing by pattern recognition receptors guidance for industry: toxicity grading scale for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials guidelines on adjuvants in vaccines for human use moderna reported pipeline safety and immunogenicity of a mrna rabies vaccine in healthy adults: an open-label, non-randomised, prospective, first-in-human phase clinical trial safety and immunogenicity of a mrna-based chikungunya vaccine in a phase dose-ranging trial curevac announces positive results in low dose - μg -rabies vaccine clinical phase study curevac reported pipeline moderna announces positive interim phase data for first combination vaccine against the respiratory viruses hmpv and piv protective efficacy of nucleic acid vaccines against transmission of zika virus during pregnancy in mice award from barda for million with potentialofupto million to accelerate development of zika messenger rna (mrna) vaccine author contributions n.a.c.j. was an employee and shareholder of sanofi pasteur at the time the paper was written, and changed employment to cepi (a not-for-profit organization) during the peer review of the paper. k.e.k., d.c. and s.g. are employees and shareholders of sanofi pasteur. f.d. is an employee and shareholder of translate bio. correspondence and requests for materials should be addressed to n.a.c.j.reprints and permission information is available at http://www.nature.com/ reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -i z tdrk authors: dangi, mehak; kumari, rinku; singh, bharat; chhillar, anil kumar title: advanced in silico tools for designing of antigenic epitope as potential vaccine candidates against coronavirus date: - - journal: bioinformatics: sequences, structures, phylogeny doi: . / - - - - _ sha: doc_id: cord_uid: i z tdrk vaccines are the most economical and potent substitute of available medicines to cure various bacterial and viral diseases. earlier, killed or attenuated pathogens were employed for vaccine development. but in present era, the peptide vaccines are in much trend and are favoured over whole vaccines because of their superiority over conventional vaccines. these vaccines are either based on single proteins or on synthetic peptides including several b-cell and t-cell epitopes. however, the overall mechanism of action remains the same and works by prompting the immune system to activate the specific b-cell- and t-cell-mediated responses against the pathogen. rino rappuoli and others have contributed in this field by plotting the design of the most potent and fully computational approach for discovery of potential vaccine candidates which is popular as reverse vaccinology. this is quite an unambiguous advance for vaccine evolution where one begins with the genome information of the pathogen and ends up with the list of certain epitopes after application of multiple bioinformatics tools. this book chapter is an effort to bring this approach of reverse vaccinology into notice of readers using example of coronavirus. it compelled us to apply the well-known reverse vaccinology (rv) approach on available proteome of coronavirus. rv approach has been successfully applied on many prokaryotes, but there are very few known applications on eukaryotes and viruses. so, it is worthwhile to explore the potential of this approach to identify potential vaccine candidates for coronavirus. rv basically does the in silico examination of the viral proteome to hunt antigenic and surface-exposed proteins. this approach was initially applied successfully to neisseria meningitidis serogroup b (kelly and rappuoli ) against which none of the prevailing techniques could develop a vaccine. the present book chapter is intended to explore the potential of rv approach to select the probable vaccine candidates against coronavirus and validate the results using docking studies. undoubtedly, the traditional approaches for vaccine development are fortunate enough to efficiently resist the alarming pathogenic diseases of its time. however, the traditional approach suffers from certain limitations like it is very timeconsuming, the pathogens which can't be cultivated in the lab conditions are out of reach, and certain non-abundant proteins are not accessible using this approach (rappuoli ) . consequently, a number of pathogenic diseases are left without any vaccine against them. all these limitations are conquered by reverse vaccinology approach utilizing genome sequence information which ultimately is translated into proteins. hence all the proteins expressed by the genome are accessible irrespective of their abundance, conditions in which they expressed. the credit of fame of reverse vaccinology should go to the advancements in the sequencing strategies worldwide. accordingly, improvement in the sequencing technologies has flooded the genome databases with huge amount of data which can be computationally undertaken to reveal the various crucial aspects of the virulence factors of the concerned pathogen. reverse vaccinology is based on same approach of computationally analysing the genome of pathogen and proceeds step by step to ultimately identify the highly antigenic, secreted proteins with high epitope densities. the best epitopes are selected as potential vaccine candidates (pizza et al. ) . this approach has brought the unapproachable pathogens of interest in spotlight and is evolving as the most reassuring tool for precise selection of vaccine candidates and brought the use of peptide vaccines in trend (sette and rappuoli ; kanampalliwar et al. ). bexsero is the first universal serogroup b meningococcal vaccine developed using rv, and it has currently earned positive judgement from the european medicines agency (gabutti ) . whether it is discovery of pili in gram-positive pathogens which were thought to not have any pili or the sighting of factor g-binding protein in meningococcus (alessandro and rino ), the reverse vaccinology steals all the credits from other conventional approaches. most of the applications of rv are against prokaryotes and very few against eukaryotes and viruses because of complexity of their genome. corynebacterium urealyticum (guimarães et al. ) , mycobacterium tuberculosis (monterrubio-lópez et al. ) , h. pylori (naz et al. ) , acinetobacter baumannii (chiang et al. ) , rickettsia prowazekii (caro-gomez et al. ) , neospora caninum (goodswen et al. ) and brucella melitensis (vishnu et al. ) are the examples of some pathogens that are recently approached using this in silico technique in order to spot some epitopes having potential of being a vaccine candidate. herpesviridae (bruno et al. ) and hepatitis c virus (hcv) (kolesanova et al. ) are the examples of the viruses that are addressed using this approach. (altschul et al. ; okonechnikov et al. ; golosova et al. ) . multiple sequence alignment (msa) was done via clustalw, and the phylogenetic tree was constructed using nj method from unipro ugene . . bioinformatics toolkit (okonechnikov et al. ). analysis of secondary structure of the proteins of seed genome was done by means of expasy portal. the aim is to forecast the solvent accessibility, instability index, theoretical pi, molecular weight, grand average of hydropathicity (gravy), aliphatic index, number of charged residues, extinction coefficient etc. (http://web. expasy.org/protparam/; gasteiger et al. ) . virus-mploc was used to identify the localization of proteins of virus in the infected cells of host (http://www.csbio.sjtu.edu.cn/bioinf/virus-multi/; hong-bin shen and kuo-chin chou ) . this information is important to understand the destructive role and mechanism of the viral proteins in causing the disease. in total six different subcellular locations, namely, host cytoplasm, viral capsid, host plasma membrane, host nucleus, host endoplasmic reticulum and secreted proteins, were covered. these predictions could help in formulation of better therapeutic options against the virus. as per the protocol of rv, secreted and membrane proteins are of special interest, therefore, filtered for further analysis. to predict the number of transmembrane helices tmhmm server v. . (http://www.cbs.dtu.dk/services/tmhmm/; krogh et al. ) was used. signal peptides are known to impact the immune responses and possess high epitope densities. moreover, most of the known vaccine candidates also possess signal peptides. hence, it is worthwhile to predict signal peptides in proteins prior to epitope predictions. signal-blast web server is used to predict the signal peptides without any false predictions (http://sigpep.services.came.sbg.ac.at/signalblast.html; frank and sippl ) . the prediction options include best sensitivity, balanced prediction, best specificity and detect cleavage site only. we choose to make the predictions using each option, and the proteins predicted as signal peptide by all the four options were preferred for further investigation. the most appropriate targets as vaccine candidates are those which possess the adhesion-like properties because they not only mediate the adhesion of pathogen's proteins with cells of host but also facilitate transmission of virus. adhesions are known to be crucial for virulence and are located on surface which makes them promptly approachable to antibodies. the stand-alone spaan with a sensitivity of % and specificity of % was used to carry out the adhesion probability predictions, and the proteins with having adhesion probabilities higher than or equal to . were selected (sachdeva et al. ). betawrap motifs are dominant in virulence factors of the pathogens. if the proteins are predicted to possess such motifs, then they are appropriate to be taken under reverse vaccinology studies. betawrap server is the only online web server to make such predictions. the proteins having p-value lower than . were anticipated to contain betawraps (http://groups.csail.mit.edu/cb/betawrap/betawrap.html; bradley et al. ). for added identification of the antigenic likely of the proteins, they were subjected to vaxijen server version . . it is basically an empirical method to hunt antigenic proteins. so, if the proteins are not found antigenic using other sequence-based methods, then they can be identified using this method. this step confirms the antigenicity of proteins selected using above-mentioned steps (http://www.ddgpharmfac.net/vaxijen/vaxijen/vaxijen.html; doytchinova and flower ). for being a probable vaccine candidate, the protein should not exhibit the characteristics of an allergen as they trigger the type- hypersensitivity reactions causing allergy. therefore, to escape out such possibilities, the proteins were also subjected to allergenicity predictions using allertop (http://www.pharmfac.net/ allertop; dimitrov et al. ) and algpred tools (http://www.imtech.res.in/ raghava/algpred/submission.html; saha and raghava a, b). to check whether the filtered proteins possess any similarity to host proteins or not, the standard blastp (http://blast.ncbi.nlm.nih.gov/blast) searches were performed. in case of sequence similarity, there is a feasibility of generation of immune responses against own cells. predicting the epitopes binding to mhc class i is the main decisive phase of the rv to carry out valid vaccine predictions. the predicted epitopes were docked with receptor that is hla-a* using cluspro (http://cluspro.bu.edu/login.php; kozakov et al. ) that is an automated protein-protein docking web server. the literature searches provided the information of conserved residues of the receptor site. the default parameters were used for docking (comeau et al. a, b; kozakov et al. ). a total of different sequenced strains of coronavirus are available at ncbi. among them strains are pathogenic to humans. various information regarding source, host and collection of these strains are presented in table . and . . this information can be obtained from ncbi's genome database, the virus pathogen database and analysis resource and genomes online database (liolios et al. ; pickett et al. ) . the mers strain is taken as seed genome as it is the most prevalent and disastrous strain among others. its proteome consists of total proteins as shown in table . . the results of sequence similarity to reveal orthologs using blastp are shown in table . . the sequences with greater than % identity score are considered as homologs. the phylogenetic tree is depicted in fig. . and the mers-cov, taken as seed genome, found clustered with different bat coronaviruses. the results of analysis of secondary structure of the proteome using expasy tools are shown in the table . . from the analysis of charge on the residues and ph values, it is concluded that six of the proteins are basic and positively charged unlike allergens which are acidic in nature. however, five proteins are acidic and show negative charge. the negative gravy score of five proteins justify them to be of hydrophilic nature with majority of the residues positioned towards the surface. for the rest of six proteins, the gravy score is positive; it means that these are the accession number and identity of orthologs obtained in different strains is shown in the table hydrophobic proteins. the proteins with less than value of instability index are quite stable than those with higher values. all the proteins are having the molecular weight less than kda except (yp_ . , yp_ . and yp_ . ). this exhibits the effectiveness of lightweight proteins as targets as they can be easily purified because of their low molecular weights. the protein yp_ . is reported as a spike glycoprotein. it is acidic with prominent negative charge, with negative gravy score which suggests its hydrophilicity and figure . depicts the subcellular localization of proteins of the seed genome, i.e. mers-cov. only one protein was predicted to be localized in host cytoplasm, four in host membrane, two in both host cell membrane and endoplasmic reticulum (er) while two in only er, and two are left unrecognized. the known spike protein is predicted to be localized in host er. from these results we decided to pick the proteins which are located in host membrane or were predicted to be localized in both host membrane and er. the two are known envelop protein and membrane protein from bibliographic studies, and along with that, the known spike protein was also included in the filtered results. out of the filtered proteins, only two (yp_ . and yp_ . ) contain more than two transmembrane helices, therefore filtered out. the results of transmembrane helices prediction are tabulated in table . . figure . depicts the subcellular localization of proteins of all the four selected genomes using virus-mploc prediction tool. the proteins that are predicted to possess the signal peptides by signal-blast web server are yp_ . and yp_ . . the results of signal-blast web server are tabulated in the table . . this step takes into account the concept of adhesion-based virulence. adhesions cause pathogen recognition and initiation of inflammatory responses by the host. spaan predicted (yp_ . and yp_ . ) out of proteins of mers strain as adhesive (table . ). only one protein (yp_ . ) was predicted to contain betawrap motifs within it (table . ). hence, it is considered virulent and might be responsible for initializing the infection in the host. a total of out of proteins of mers strain were predicted antigenic (prediction values greater than . ). the protein with accession number yp_ . and yp_ . were among the filtered proteins, however, not predicted antigenic, therefore filtered out. as a result, only four proteins (yp_ . , yp_ . , yp_ . and yp_ . ) were kept for further analyses. none of the proteins of mers-cov possessed any clue of allergenicity as per prediction results from algpred and allertop tools; it means that no vigorous immune responses will be mounted if the epitopes from these proteins will be adopted as vaccine candidates. none of the protein of mers strain shows similarity with the proteins of host that demonstrates that the epitopes from these proteins can safely elicit the required immune response without the hazard of autoimmunity. in total different -mer epitopes with potential to bind to receptors of both b-cell and t-cell were predicted. the list of the predicted epitopes can be found in the table . and are specific for mers-cov strain. all these epitopes displayed no conservancy with proteins of other human and non-human pathogenic strains. docking permits to reveal the binding energy or potency of connection among epitopes and the receptor in appropriate orientation. the cluspro docking server was used to dock the predicted epitopes against hla-a* . the structure of the receptor was available from pdb and was optimized before docking to free it from the complexed self-peptide ( u y, resolution . Å, bouvier et al. ). pepstr (peptide tertiary structure prediction server; kaur et al. ) was used to derive the tertiary structure of the predicted peptides. figure . depicts the quaternary structure of the receptor hla-a* with its conserved active site known to form complex with the peptides (bouvier et al. ). the binding energy results obtained after performing docking analysis are listed in table . . the -mer epitope vvcaitllv at site of protein yp_ . docked to the receptor with smallest amount of binding energy (À . ) and hydrogen bonds. the next epitope in the list was also from the same protein yp_ . at site , i.e. tllvcmafl. the predicted structure of the top potent epitopes on the basis of docking energy and the snapshots of docking results are displayed in figs. . , . , . , . and . . the most chief restriction for developing a safe and sound vaccine against any of the virus is to identify the protective antigens. the present study is an effort of application of reverse vaccinology approach to investigate a choice of coronavirus proteomes to identify possible vaccine targets. this technique has demonstrated to be a competent way to forecast different epitopes from the selected seed genome. these epitopes are from spike glycoprotein, ns protein, ns b protein and envelope protein. unfortunately none of the epitope is found conserved in other strains, and all are specific to mers-cov. the docking analysis studies revealed perfect binding between hla-a* receptor and epitopes. the conserved residues of the receptor site are also involved in h-bonding with epitope residues. further, the selected antigenic epitopes must be validated using in vitro and in vivo studies to confirm their potential as vaccine candidates. review: reverse vaccinology: developing vaccines in the era of genomics basic local alignment search tool the middle east respiratory syndrome coronavirus -a continuing risk to global health security crystal structures of hla-a* complexed with antigenic peptides with either the amino-or carboxyl-terminal group substituted by a methyl group betawrap: successful prediction of parallel beta -helices from primary sequence reveals an association with many microbial pathogens geminiviridae. in: virus taxonomy-ninth report of the international committee on taxonomy of viruses lessons from reverse vaccinology for viral vaccine design discovery of novel cross-protective rickettsia prowazekii t-cell antigens using a combined reverse vaccinology and in vivo screening approach identification of novel vaccine candidates against acinetobacter baumannii using reverse vaccinology cluspro: a fully automated algorithm for protein-protein docking cluspro: an automated docking and discrimination method for the prediction of protein complexes middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group allertop v. -a server for in silico prediction of allergens vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines high performance signal peptide prediction based on sequence alignment techniques meningococcus b: control of two outbreaks by vaccination protein identification and analysis tools on the expasy server unipro ugene ngs pipelines and components for variant calling, rna-seq and chip-seq data analyses discovering a vaccine against neosporosis using computers: is it feasible? genome informatics and vaccine targets in corynebacterium urealyticum using two whole genomes, comparative genomics, and reverse vaccinology reverse vaccinology: basics and applications pepstr: a de novo method for tertiary structure prediction of small bioactive peptides reverse vaccinology and vaccines for serogroup b neisseria meningitidis way to the peptide vaccine against hepatitis c piper: an fft-based protein docking program with pair wise potentials the cluspro web server for protein-protein docking predicting transmembrane protein topology with a hidden markov model: application to complete genomes an integrative approach to ctl epitope prediction, a combined algorithm integrating mhc-i binding, tap transport efficiency, and proteasomal cleavage prediction improved method for predicting linear b-cell epitopes the genomes on line database (gold) v. : a monitor of genome projects worldwide identification of novel potential vaccine candidates against tuberculosis based on reverse vaccinology identification of putative vaccine candidates against helicobacter pylori exploiting exoproteome and secretome: a reverse vaccinology based approach database resources of the national center for biotechnology information unipro ugene: a unified bioinformatics toolkit scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide side-chains virus pathogen database and analysis resource (vipr): a comprehensive bioinformatics database and analysis resource for the coronavirus research community identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing reverse vaccinology spaan: a software program for prediction of adhesins and adhesin-like proteins using neural networks algpred: prediction of allergenic proteins and mapping of ige epitopes prediction of continuous b-cell epitopes in an antigen using recurrent neural network reverse vaccinology: developing vaccines in the era of genomics virus-mploc: a fusion classifier for viral protein subcellular location prediction by incorporating multiple sites propred : prediction of promiscuous mhc class-i binding sites identification of potential antigens from non-classically secreted proteins and designing novel multitope peptide vaccine candidate against brucella melitensis through reverse vaccinology and immunoinformatics approach key: cord- -zhwr h authors: oxford, john; gilbert, anthony; lambkin-williams, robert title: influenza vaccines have a short but illustrious history of dedicated science enabling the rapid global production of a/swine (h n ) vaccine in the current pandemic date: - - journal: influenza vaccines for the future doi: . / - - - - _ sha: doc_id: cord_uid: zhwr h vaccines for the swine flu pandemic of have been produced in an exquisitely short time frame. this speed of production comes because of years of hard work by virologists worldwide in pharma groups, research laboratories, and government licensing units. the present chapter presents the background framework of influenza vaccine production and its evolution over years. isolation of the causative virus of influenza in , followed by the discovery of embryonated hen eggs as a substrate, quickly led to the formulation of vaccines. virus-containing allantoic fluid was inactivated with formalin. the phenomenon of antigenic drift of the virus ha was soon recognized and as who began to coordinate the world influenza surveillance, it became easier for manufacturers to select an up-to-date virus. influenza vaccines remain unique in that the virus strain composition is reviewed yearly, but modern attempts are being made to free manufacturers from this yolk by investigating internal virus proteins including m e and np as “universal” vaccines covering all virus subtypes. recent technical innovations have been the use of vero and mdck cells as the virus cell substrate, the testing of two new adjuvants, and the exploration of new presentations to the nose or epidermal layers as dna or antigen mixtures. the international investment into public health measures for a global human outbreak of avian h n influenza together with a focus of swine influenza h n is leading to enhanced production of conventional vaccine and to a new research searchlight on t-cell epitope vaccines, viral live-attenuated carriers of influenza proteins, and even more innovative substrates to cultivate virus, including plant cells. when the influenza a virus first emerged from a presumed avian reservoir at the end of the ice age , or so years ago, there was a distinct difficulty in finding new human victims. for example, at that time, only a few hundred settlers were in the london region near the royal london hospital, now a community of four million people. at that time a traveler would have to walk a miles to find another small settlement, perhaps at stonehenge near salisbury. nowadays we have a truly global community of six billion people, linked so that two million people are moving each day by plane, while perhaps ten million are journeying in their homelands. influenza, like all viruses, is opportunistic. in , it had the unprecedented opportunity to spread at the end of the first global war. ten million soldiers began the move homewards, and every steamship was packed as they fanned out from france to england, europe, the usa, canada, australia, india, and se asia [ ] [ ] [ ] . how perfect for a virus spread by aerosol droplets, close contact, and contamination of towels, cups, and every day utensils. a virgin population, which had never before encountered the avian virus (h n ), was on the stage of this theater of infection. perhaps, a billion people were infected in the next months, and - million died, making this by far the biggest outbreak of infectious diseases ever recorded, with an impact many times greater than the so-called bubonic plague outbreaks in medieval europe. however, more than two billion people survived. the overall mortality was less than %, although in a few semi-closed societies of hunter-gatherers in the arctic, the mortality from the disease and subsequent starvation as young hunters died and husky dogs attacked and ate the survivors exceeded % [ ] [ ] [ ] [ ] . it is well to remember that when h n emerged in / and became pandemic in everyone except for the over s were fully susceptible. this is different from today where most people on planet earth have immune memory to the h n family of viruses and by definition to a/swine flu. this explains why the current h n vaccine is immunogenic. while most people in the world were infected, we are forced to view the innate protective power of our immune system with awe [ , ] . we are equipped with , genes, seven million years of evolution, and , years of specialization since our emergence from africa. in contrast, influenza is a miniscule eight-gene vehicle. a recent study [ ] of the reproductive number (r ) of the virus suggests that, unexpectedly, it may have been quite low, not exceeding three persons infected with a single case. the current pandemic a/swine h n virus is not so different. this would place pandemic influenza not far above the lowly group of viruses such as small pox and sars and not reaching the heights that measles has attained. however, this unexpected theoretical analysis, if it is not flawed, gives us more practical opportunities to break a chain of infection of a pandemic with antivirals, hygiene, and vaccines [ ] [ ] [ ] . we are experimenting with these approaches at the present moment. the new world of the twenty-first century, although harboring in some countries a few old-fashioned attitudes, akin to "influenza and pneumonia is the old person's friend" nevertheless has the capability for the first time to defend itself against mother nature and her threat of influenza. for the first time in history, intense surveillance by the world health organization (who), early identification of a new pandemic influenza virus by molecular diagnostics, application of vaccination and antiviral chemoprophylaxis, and possible quarantine and masks could actually prevent a pandemic arising. for the expressed intention of who and the world community of infectious disease researchers is to deflect the first wave of the first pandemic of the twenty-first century. in this endeavor, our huge resources of natural innate immunity, assisted by new vaccines, are already helping us. the formulation of the vaccines and their stockpiling alongside antineuraminidase (ni) antivirals has needed significant investment of time and money, and this started with a three billion euro investment from the usa and eu. we are presently gathering the fruits of this investment with the outbreak of a/swine (h n ) virus. baroness findlay of glandaff put the epidemiology of influenza h n situation succinctly in the house of lords report of pandemic influenza [ ] "we believe the risk of a pandemic of human-to-human transmissible virus is to be taken very seriously. we believe that it may not happen in the very short time. to explain why we came to this stance; we believe that the problem, if it does emerge is more likely to emerge in asia. asia is where fire fighting must be done today." the baroness had just heard the background science that china alone holds million domestic ducks, a possible trojan horse of virus persistence, which approximates to % of the world's domestic duck population. expert evidence from fao had summarized that china, indonesia, and vietnam represented the core of the problem, but only million dollars were available at that point in / to help, and biosecurity is not imposed strictly, while veterinary services are haphazard. the current pandemic virus emerged from pigs but a continuing threat is another reassortant event with h n most likely in a coinfected child in egypt or se asia where h n viruses are endemic and where swine h n viruses are spreading. we are not the first generation of virologists to recognize the influenza pandemic threat, but we are the first to have the knowledge of the avian and pig reservoir and the tools to deal with the problem in a scientific manner. the world capacity for influenza vaccine today of one billion doses did not arrive by accident: it came to us from the hard work and dedication of four generations of dedicated scientists and doctors. the intention here is to give just tribute to these pioneers and their new discoveries. using the vaccine methods developed over six decades, we can for the first time confront influenza as it emerges, surround it, and actually prevent a pandemic. we no longer need to be passive observers at a theater of infection. churchill coined the phrase "give us the tools and we will finish the job." well, we now have them and we will. such is the essence and spirit of this chapter. the serendipitous discovery of infection of ferrets, which produce clinical signs, and the cross-infection of a student from a ferret was the first technology foundation stone [ ] . ferrets are used today as a key model to investigate new vaccines. the two most important technologies, which form the granite-like foundation of influenza vaccine research, are the hemagglutination inhibition test (fig. ) and the cultivation of virus in embryonated hen's eggs (fig. ) , first reported in and , respectively [ , ] . if one adds two other vital scientific observations that of hobson et al. [ ] who correlated a hi titer of with protective efficacy in volunteers in and then the discovery of a single radial diffusion for standardization of the hemagglutinin (ha) content of vaccines by schild in , it is quite apparent that the technologies are all now well tried and tested [ ] . the elucidation of the structure of the fragmented influenza genome [ ] has quickly led to techniques, genetic reassortment, and correlation of functions with certain genes (fig. ) . from a practical viewpoint, some old much passaged viruses such as a/pr/ / (h n ) grew to extraordinary infectious titers in the egg allantoic cavity, exceeding a new wild-type virus by fold or more. why not create a reassortant in the laboratory with six replicative genes of a/pr/ / to give high replication while having the two new ha and neuraminidase (na) genes of the new epidemic virus? this technique proved to be a masterstroke and in the last quarter of a century three laboratories, csl in fig. the classic hemagglutination inhibition test. the test depends upon interaction of eight ha units of virus that would normally agglutinate . % turkey red blood cells. preincubation of this standard virus with dilutions of serum antibody abrogates the agglutinating property of the virus (vertical rows and ). no antibody is detectable in rows - , [ ] [ ] [ ] melbourne, nibsc in london, and ed kilbourne's laboratory in new york, have rushed each year to produce the new candidate vaccine viruses prefixed ivr-, nib-, and x-, respectively. the almost made-to-order technique of gene reassortment fig. inoculation of embryonated hen's eggs to grow influenza virus for vaccine. virus is inoculated through the shell of a -day-old embryonated hen's egg and more rarely in the research laboratory into the amniotic cavity (top). after days of incubation at c, the clear fluids are removed and titrated for ha by hemagglutination fig. the influenza genome is in eight fragments. the genome could be labeled with p extracted and separated on polyacrylamide gels with influenza was also central to producing host range mutants with attenuation genes for live vaccines. some of the starter and seed viruses for the current production of a/california/ / h n (swine) vaccine used this biological technology, while others used reverse genetics to make a gm starter virus. undoubtedly the simultaneous discovery of the reverse genetics [ , ] by the three laboratories in new york, wisconsin, and oxford was a masterstroke in technical advance, which has enabled mutations to be placed, at will, into the genomes of the negative-strand viruses. the conjunction of older and newer techniques with the licensing of the mammalian cell lines from monkey kidney (vero) [ ] , dog kidney (mdck) [ ] , or human tissue (per- ) has led directly to the newly emerging influenza vaccines of the twenty-first century. we are using all these techniques of the last years to produce the a/swine h n vaccines of for the current pandemic. the first experiments on the attempted immunization of animals were made in the usa by francis magill [ ] and in england by andrewes and smith in [ ] . the model is still vital today and the first experimental assessment of a/california/ / h n vaccine was made in this model. mouse lung suspensions or filtrates were used after inactivation with formaldehyde, and it was found relatively easy to protect mice against intranasal infection with influenza. immunization experiments in man were accelerated when allantonic fluid preparations of virus formed the starting material soon after the technique of allantoic inoculation of fertile hen's eggs was discovered [ ] . the first field trial demonstrating short-term protection by inactivated vaccine took place in the usa during a sharp epidemic of influenza in (commission influenza ) [ ] . progress with the development of purer, more potent vaccines has proceeded steadily since those early days, and technical advances with ultracentrifugation and chromatography, by methods producing richer cultures and chemical inactivation avoiding too great a modification of the surface ha and na antigens have all helped. to avoid the relatively high rate of local and general systemic reactions caused by the older egg-grown inactivated whole-virus vaccines, chemical treatment to disrupt the particle and to separate the wanted antigens (ha and na) from other constituents of the virus has led to a variety of different split or subunit vaccines . ether extraction [ , ] , deoxycholate treatment [ ] , and treatment with other detergents have been introduced. some methods have provided subunit vaccines causing fewer clinical side reactions than the older whole-virus particle vaccines, but drawbacks have appeared, including that of reduced antigenicity. adjuvants of oily emulsions promised potent vaccines with excellent antibody responses, and a few reactions were first encountered. however, a rare abscess at the site of inoculation caused much distress and this early approach had to be abandoned. in spite of attempts to develop safer materials, none have yet the whole virus is disrupted with detergent, which dissolves the lipid membrane releasing ha, na, and internal np, seen as "lamb tails" been developed commercially until very recently when mf and a have been formulated. thus, after years of work, the hope of an ideal inactivated vaccine free from the induction of clinical reactions and yet potent immunogenically has just been fulfilled with pandemic h n vaccines and swine h n vaccines. in , a major antigenic deviation of influenza a virus occurred with the appearance of a/cam/ (h n ) virus in australia. in the usa and europe, outbreaks of influenza occurred early in , which were due to the same virus; some communities previously receiving vaccine containing pr and weiss viruses (h n in the old classification and now reclassified h n ) were attacked. this time the vaccine did not protect against the new virus typified by the prototype a/fm/ / (h n ) [ , ] , and this led to realization of the enormous importance of the updated antigenic make-up of inactivated vaccine. yet other difficulties have become appreciated, one of which is the inappropriate antibody response occurring sometimes after inoculation, when the vaccine induces cross-reacting antibody to heterologous viruses or the first virus in the subtype which the vaccine first experienced, rather than that appropriate to the specific antigen, ha, of the vaccine virus. this response is probably allied to the phenomenon of "original antigenic sin." sometimes this aberrant response can be useful as with a/swine h n vaccine. it is likely that the over s will produce recall antibody to h n viruses which infected them in the s and that this virus is somewhat related to the current a/swine virus. the starting materials for almost all types of inactivated vaccine are allantoic fluids from fertile hen's eggs previously inoculated with a seed culture, the yield of which is enhanced using a recombinant virus, one parent of which is a high-yielding laboratory strain (a/pr / ) and the other acts as the donor of the requisite surface ha and na antigens from a wild-type virus [ ] . the a/pr/ / virus donates six genes and the wild-type virus two genes: the ensuing reassortant high growth viruses are called / reassortants. purification from unwanted egg material is accomplished by ultracentrifugation on a zonal ultracentrifuge [ ] . whole-virus particles thus separated are inactivated by formalin or b-propiolactone, the ha content being as high as possible commensurate with the necessity to avoid febrile reactions after inoculation. children were sensitive to the older egg-grown wholevirus vaccines; as many as % under years developed fever after . ml of vaccine and up to % of -year-old children were similarly affected after . ml [ ] . the precise constituent producing the fever was not clearly identified, but the viral proteins were believed to be concerned [ , ] . more modern whole-cell virus vaccines produced in cell culture are more purified and produce fewer side reactions. separation of the ha and na by means of detergents such as tween or triton n produced split-virus or subunit vaccine, and general experience suggested that these materials are less pyrogenic, but less immunogenic, than whole-virus vaccine [ ] . this was particularly well demonstrated by studies during the swine influenza campaign in the usa in , when many observers reported results, which ultimately led to the recommended use in children of two doses of split-type rather than whole-virus vaccines. such recommendations continue at the present time. in adults, too, the older egg-grown whole-virus vaccines gave a higher proportion of febrile reactions than split virus [ ] . however, this situation is changing as whole-virus vaccines produced in vero cells for example come to the fore. former methods for assays of the potency of inactivated vaccine depended on measuring the ha activities of the vaccines with erythrocyte suspensions using the salk pattern technique of miller and stanley [ ] . in retrospect, this technique was not hugely accurate especially for subunit and split viruses. in a major technological breakthrough, schild et al. [ ] proposed a method of assay based on single radial immunodiffusion (srd) (fig. ) . the ha antigen content of vaccines was estimated using srd tests in agarose gels containing specific hi antibodies. the srd method was modified and refined by wood et al. [ ] . it may be gradually replaced now by hpmc technologies. the srd technique was valid for both whole-virus and split-virus vaccines and was quickly adopted for international use and is still the gold standard. in this test, vaccine virus preparations and reference antigen calibrated in terms of micrograms of ha are disrupted with detergent, and dilutions of the treated antigens are introduced into wells in srd immunoplates. the size of the precipitation ring obtained for the vaccine is compared with that obtained with a reference antigen of calibrated ha content titrated on the same plate. the vaccine potency is measured in terms of micrograms of ha per vaccine dose. inactivated influenza vaccines frequently contain two or more virus strains and the ha content of each component ( mg) is assayed independently. it has been known for many years that the serological response to inactivated vaccine depends on the previous experience of the recipient to infection by viruses of the same subtype of influenza a virus as that present in the vaccine. although a single subcutaneous injection of (h n ) vaccine gave as good a response as two doses prior to , the advent of the new pandemic a/asian (h n ) virus produced a different effect. thus, holland et al. [ ] demonstrated that two doses at an interval of two or more weeks produced a better response to one dose and in this regard the vaccine-induced immune response was much inferior to that noted single radial diffusion (srd) test to standardize ha. vaccine antigen is pipetted into -mm wells in an agar plate containing specific anti-ha, -na, and -np antibodies. after a few hours incubation, a zone of precipitation is quantified and the area is proportional to the quantity of ha in the vaccine before the change in virus subtype. such an experience was again noted during the first year of circulation of a/hong kong (h n ) virus and also when the a/new jersey/ (hsw n ) vaccine was used in children and young adults. also, in the circumstances of - , when most persons under years of age had no previous antibody to the recirculating h n virus, a two-dose regimen for children and young adults produced a more satisfactory response than a single injection [ ] . to reiterate, in an "old" h n virus from the s was accidentally released from a laboratory and established itself as an epidemic virus. it was called a "pseudo pandemic." everyone over years had previous immunity. the contrast between the effects of a single dose of vaccine in persons infected with h n viruses at least years earlier was very striking. these data have immediate relevance today in terms of h n vaccine and of course with the a/swine vaccine. the world is full of immune virgins as regards h n but not in the case of a/swine h n . most persons have immune memory to the h n family, and therefore it comes as no surprise that vaccines induce high levels of hi antibody. several factors are of importance in the determination of the quantity and the precise composition of the antibody response to the surface antigens of the virus present in inactivated vaccine. first and foremost, the quantities of hi and ni antibodies induced by vaccine are broadly related to the quantity of antigen present in a single dose. second, the precise composition of the antibodies formed in response to influenza a virus is important. thus, reinforcement of previously acquired antibodies by the orientation of the b-lymphocyte response to the first infection by the particular subtype of virus experienced in childhood or later may take precedence over the strain-specific antibody response to the vaccine virus. third, the precise response is influenced by the route by which the vaccine is presented to the body's immune system. first then, several earlier studies reported a graded relationship between the quantity of antigen inoculated and the antibody response that results. this was so in the study of mostow et al. [ ] , who gave increasing doses of vaccine in a single injection containing - , chick cell agglutination (cca) units containing a/japan/ (h n ) virus groups of volunteers. the serum hi response was tested with four different h n viruses isolated - and also the homologous virus. with more than a tenfold increase in ha from the least to the highest dose, the geometric mean titer (gmt) of antibody increased only fivefold. similar results were obtained by potter et al. [ ] , who inoculated student volunteers with vaccines ranging in dosage from to iu and containing a/port chalmers/ (h n ) virus. the vaccine was a surface-antigen detergent-treated material [ ] adsorbed to aluminum hydroxide gel. gmt hi serum titers increased against homologous virus from -to -fold with the increase in dose of vaccine ha. three other h n strains and a/singapore/ (h n ) virus were also tested, and all three h n viruses showed graded hi antibody responses proportional in magnitude to increase in antigen dose, as did the homologous virus. the pandemic working group of the mrc committee on influenza vaccine [ ] gave graded doses of whole-virus vaccine containing the a/new jersery/ (hsw n ) strain to groups of volunteers in . those less than years of age, who did not possess significant serum hi antibody to the virus before immunization, showed a postvaccination antibody titer ranging from to gmt with a nearly eightfold increase in dose from to mg of ha. above this age, in those - with preexisting hsw antibody, there was an increase in antibody titer from to times (gmt) with a change in ha concentration from to mg. thus, the effect of increasing the potency of this vaccine on the antibody response was much greater in those sera, which indicated that they had been exposed to the antigen, presumably by infection with a related virus, than in those with no such exposure. both whole and detergent-split-virus vaccines showed a relatively poor hi response in volunteers less than years of age whose initial serum had no significant amount of prevaccination or postinfection hi antibody. in this group of subjects, two doses of vaccine gave a better antibody response than did one, but the resultant postvaccination gmt was half that obtained with a single dose of the vaccineover years of age. this historical data is very relevant to us today as we analyze the hi data from the current batches of a/swine h n vaccine where in the over years a single dose of vaccine is sufficient because of wide preexposure to members of the h n family of viruses. the younger groups had no prior immune memory to the h n family of virus, their experience being more orientated to h n and h n families. these examples underline the practical importance of a considerable degree of antigenic drift within a subtype comprising hi antibody response. also, the recall of antibodies induced by previous infection illustrates the general rule that an up-todate monovalent vaccine reinforces antibodies against former members of the subtype, while also inducing specific antibodies to the vaccine virus. this was clearly shown by direct comparison of monovalent and polyvalent vaccines such as the mrc committee on influenza vaccine's trials [ ] [ ] [ ] [ ] . the quantitative dose response already described for hi is also found with ni antibody but is less consistent. thus, potter et al. [ ] noted that there was a two-to sixfold increase in ni antibody as vaccine potency was increased from to iu of ha. yet the trial of a/new jersey/ (hsw n ) vaccine conducted by the pandemic working group of the mrc influenza vaccine committee [ ] found only a slight increase in ni antibody after an increased dose from to iu using iu of ha in the vaccine. nicholson et al. [ ] gave a whole-virus vaccine of the a/ussr/ (h n ) virus, which ranged in potency up to sixfold, and found, in those under , a threefold increase in ni antibody. however, in those over years of age, an increase in dose of vaccine had a less constant effect on ni antibody formation. one possible reason for the variation in the effect of different vaccines on the ni antibody is the lack of consistency in the na content [ ] ; however, another possibility may be that immunological priming to the ha in the vaccine can in some way suppress the immunogenicity of the na antigen, which may be physically associated with the ha. the second important variable in the immune response to inactivated vaccine arises from the relative amounts of cross-reactive and strain-specific antibodies that are generated. the differentiation of these require special techniques such as srd and the adsorption studies. webster et al. [ ] compared, in adults, the response to an a/port chalmers/ (h n ) subunit vaccine to homologous and heterologous h n viruses. most of the antibody was cross-reactive with a/hong kong/ virus but when higher doses of the vaccines were used, strain-specific a/port chalmers/ antibody was produced in addition to that against heterologous virus. oxford et al. [ , ] compared whole-and split-virus vaccines containing a/victoria/ or a/scotland/ viruses and using single radial hemolysis and adsorption techniques showed that in an immunized adult, cross-reactive antibody was induced much more frequently than specific antibody against homologous virus. they showed the same phenomenon in adults during infection with a/port chalmers/ virus, who frequently also developed antibody rises to a/hong kong antigens from . oxford et al. [ ] used similar techniques to analyze sera from children aged - years immunized with a surface-antigen vaccine containing a/victoria/ (h n ) antigens. most children produced a strain-specific serum antibody to the vaccine antigens, whereas adults similarly vaccinated tended to produce antibody cross-reacting with all variants of the h n subtype tested. postepidemic sera from those of various ages recently infected by a/texas/ -like strain showed crossreactive antibody in adults but in contrast mostly strain-specific responses in children. strain-specific antibody is considered to be more protective. the influence of the route of immunization with inactivated vaccine has been studied in the past by many observers. the chief alternative to the subcutaneous-intramuscular route is intradermal injection using a reduced amount of vaccine. the advantages of this route are economy and the avoidance of febrile reaction. the principal disadvantage is the fact that the antibody response is less consistent. it was shown by appleby et al. [ ] that the gmt after intradermal vaccine was less than half that obtained with subcutaneous vaccine, and this seemed logical in that only one-tenth of the vaccine dose was given intradermally. mccarroll and kilbourne [ ] found little difference in the antibody responses to intradermal and subcutaneous vaccines in equivalent doses. tauraso et al. [ ] reinvestigated the question using a two-dose regime before the arrival of the a/hong kong/ (h n ) epidemic. in the equivalent amount of . ml of vaccine, antibodies formed in higher titer after intradermal than subcutaneous vaccine. however, the titers after . ml of vaccine subcutaneously were little different from intradermal injection of . ml. it is considered advisable, however, in practice to limit intradermal vaccination when the vaccine is in short supply or when, in children or the aged, reactions after subcutaneous vaccine might pose problems. the nasal route of inoculation either by instillation of drops or by spray was first studied in detail by waldman et al. [ ] . compared with the subcutaneous vaccine in a dose of . ml, antibodies capable of neutralizing the virus a/taiwan/ (h n ) increased to a greater extent in sputum and nasal secretions after repeated nasal inoculation with a total volume of . ml vaccine. in contrast, the intranasal vaccine produced a much lower rise in serum antibody, the gmt being only onesixth that after subcutaneous vaccine. waldman et al. [ ] , using an aerosol spray, found that a better serum antibody response occurred with a small-sized particle spray than a larger one, but the nasal antibody response was better after the latter or with nasal drops. absorption studies showed that a majority of the secretory antibody (iga) response in nasal secretion was cross-reactive with heterologous viruses (a/hong kong/ h n ). phillips et al. [ ] compared subcutaneous or intradermal vaccine in nurses with vaccine dropped intranasally. the subcutaneous route produced the best serum antibody rises, and intradermal vaccine was superior to the intranasal route in terms of antibody response. the nasal antibody titers after immunization by either subcutaneous or respiratory routes paralleled those in serum. the fact that nasal antibodies increase after subcutaneous vaccine [ , ] is important because the lack of a good response in serum antibody in those given the same vaccine intranasally is a limitation hardly offset by local nasal secretory changes. challenge of immunized groups of persons by live-attenuated virus also supports the view that nasal antibodies play a supplementary role to serum hi antibody [ ] . [ ] . after a second dose of the same vaccine, fewer volunteers experienced reactions than seen after the first dose. later studies of the endotoxin content of various pools of inactivated type a or b vaccines using the limulus lysate test gave no hint of a parallel between the occurrence of general reactions and the endotoxin content [ ] . neurological illness is a recognized sequel to immunization with a variety of vaccines but had not previously been observed with any frequency after influenza virus vaccines. wells [ ] noted the rare instance of guillain-barré syndrome (gbs), which appeared in excess among the persons vaccinated with a/swine vaccine compared with the numbers in unvaccinated individuals. of , persons with gbs reported from october , , to the january , , had received vaccine before the onset of neurological symptoms. the overall risk of gbs was calculated as ten cases per million vaccinated. the rate of occurrence during the -week swine vaccine period was five to six times greater than in unvaccinated persons. however, the excess in number was greater in the second and third weeks after inoculation than either the first or subsequent weeks. as reported by langmuir [ ] , gbs was not associated with a particular variety of vaccine or age group. however, that numbers were slightly greater in those aged - than in middle aged or elderly persons, which appears to rule out the possibility that the syndrome was, in some way, related to the absence of antibodies to the swine virus before immunization, for most of those aged over would have been exposed to antigens of this virus many years before. after the swine influenza campaign was terminated, surveillance was continued, and during the period - , when . million doses of ordinary inactivated vaccine were estimated to have been used, the related risk of gbs was . times the incidence in unvaccinated persons. this risk was regarded as not significant [ ] . no clue to the cause of the marginally increased risk of gbs in immunized persons in has yet been obtained but could be virus strain related. no untoward effects have been noted in the billions of vaccines used since to the present day. at the time when a/hong kong/ (h n ) virus was spreading in asia, plans were made by the mrc committee on influenza vaccine to protect children in residential schools and other groups in a controlled manner. inactivated polyvalent vaccine containing two h n viruses (a/england/ and a/england/ ) and a b strain were compared with an h n a/hong kong whole or deoxycholate-treated virus vaccine in initial serological trials. antibody formation even in those without detectable serum hi antibody gave gmts over in those receiving a/hong kong vaccine intramuscularly. however, controlled trials in two boarding schools showed no convincing evidence of protection. in uncontrolled trials in other schools either the polyvalent or the a/hong kong vaccine were given or no vaccine at all. there were schools where epidemics of influenza occurred in january and february but no evidence of protection was found in those receiving a/hk vaccines. the only clue obtained concerning the vaccine failure was first that only one dose of vaccine had been given, and this is known to be inadequate to give a satisfactory antibody response in previously seronegative persons, and second, there was an interval between vaccine administration and infection of - months. these two factors may have combined to explain the absence of protection because of the inadequacy of the antibody response at the time of challenge. it would be fair to add that others [ , ] did obtain protection from a/hong kong/ whole-virus vaccine during the first outbreak of influenza due to this virus in the usa. the use of modern adjuvanted h n vaccine in two doses is anticipated to give protective effects. the current a/swine vaccines produce protective hi antibody (> ) in most persons over years of age following a single dose. as emphasized above, this reassuring situation is because most persons have prior immunity to the h n family of viruses which circulated between and and then again from to the present day. the use of living but attenuated virus as an immunizing agent developed slowly from the initial studies of mawson and swan [ ] in australia and the ussr. the major difficulty of the lack of a laboratory test to indicate that cultured virus had lost its pathogenicity, while retaining infectivity for man, meant that deliberate intranasal inoculation of volunteers furnished the only way to select a suitable strain for infection without causing clinical reaction. in spite of the widespread adoption of live vaccines selected by this method and given as an intranasal spray in the ussr, little interest was exhibited in most other countries. from onwards, trials took place in volunteers in england and wales to provide evidence of safety and immunogenicity of cultured viruses and the drawback of a reduced infectivity of well-attenuated viruses handicapped progress. the necessity to observe a match between the antigens of epidemic viruses and those present in the vaccine was a further drawback until the technique of reassortment of characters between two strains, one of which was of proven attenuation, was utilized to yield seed viruses with the desirable clinical and antigenic properties. other disadvantages of live viruses appeared during the intensive researches of the s particularly in the usa and in england [ , ] . it cannot yet be claimed that the ideal live-attenuated virus vaccine has been formulated, but reverse genetics and increased knowledge of virulence genes have now lead to a resurgence of interest. in the s, genetic studies were intensively pursued in attempts first to define the particular gene or combination of genes, donated by the attenuated virus that confers the property of attenuation upon the reassortant strain. it was found that the biological properties of excreted virus may be altered compared with those of the original virus in the vaccine and the manner of this alteration was also studied genetically. such work is essential in achieving the goal of an effective and safe vaccine virus for human use. experimental inoculations were carried out initially in small-scale tests in volunteers under semi-isolation to permit close observation (see below). multiple cultivation and passage of viruses either in animal hosts, such as ferrets and mice, or in developing chick embryos or tissue cultures had been practiced even before the use of temperature-sensitive (ts) or cold-adapted (ca) mutants was suggested. early workers in britain used the pr / virus as a host range mutant, which, although noninfective for man, has retained animal pathogenicity even after many passages in eggs. as a donor parent with good powers of multiplication in the laboratory, pr was mated with various strains of wild-type influenza a viruses to obtain recombinants with up-to-date surface ha and na antigens. this method was preferable to simple laboratory cultivation because some viruses failed to alter in pathogenicity after as many as serial passes in cultures [ ] , although other virus strains appeared to become attenuated with only a few passages in eggs. pr virus was chosen also by workers in belgium who prepared reassortants from a number of viruses, some of which were licensed for human use [ ] . to select recombinants with as high proportion of rna components as possible derived from the host range mutant pr , florent et al. [ ] used rna-rna hybridization to identify gene origins. later the gene constellation of four of the candidate vaccine viruses was determined, and florent [ ] found that some clones of beare and hall's [ ] recombinants of pr and a/englannd/ (h n ) containing five genes from pr were satisfactorily attenuated. however, one clone though containing six pr genes was nevertheless clinically virulent to volunteers. a further genetic study of pr host range recombinants using viruses tested clinically by beare and reed [ ] was made by oxford et al. [ ] . it was again found that recombinants from pr and a/england/ viruses could contain only the surface ha and na genes from wild-type virus and yet retain virulence for man. additional attempts to stabilize the attenuation of candidate viruses were made both by beare at the medical research council's laboratories at salisbury and the rit workers by rendering the virus resistant to an inhibitor present in normal horse serum. this property was present in the rit series of recombinants. it seems strange that stabilization has not been pursued since nor has cultivation of host range mutant viruses, such as pr , at abnormally low temperatures, such as c. this method was found by sabin [ ] to be preferable to normal temperatures when attenuating polio viruses, and it was exploited by both workers in the usa and ussr. marker tests, which can be equated with attenuation of virulence for man, were sought with relatively variable results. one such test used weanling rats that were inoculated intranasally first with virus and later with cultures of haemophilus influenzae. virulent virus induces bacteremia and meningitis, and using this method jennings et al. [ ] successfully separated a number of reassortant viruses and obtained some correlation with clinical virulence. yet the host range mutant parent pr / and rit , which are both attenuated in man, were classed as virulent by the rat. a new approach at that time used an avian (duck) virus, which was found to have only low pathogenicity for squirrel monkeys inoculated intranasally and was proposed as a donor of attenuation. a reassortant with a virulent human a/udorn/ (h n ) virus behaved as did the avian parent in the squirrel monkey, although immunizing the latter against the virulent parent. clinical trials have suggested that this virus is attenuated for man and is immunogenic but has not been investigated since [ ] . most work on the development of viruses with restricted multiplication at temperatures above the normal range for cultivation has been affected by chanock, murphy, and associates at the national institutes of health, bethesda [ ] . the technique used chemically produced mutation in virus rna by cultivation in the presence of the mutagenic agent -fluorouracil. after cultivation and plaquing at c, c, and c, mutant viruses with the requisite temperature sensitivity were obtained. intranasal inoculation of hamsters confirmed temperature restriction, in that much lower titers of virus were found in the hotter lungs than in the cooler upper respiratory tract. spread from inoculated volunteers to adults in contact was not observed, and no evidence of a change in virulence was found in viruses recovered from adult recipients of vaccine [ ] . however, in seronegative children, the a/hong kong/ -ts-l [e] virus produced mild febrile reactions and a virus that had lost its properties was recovered from some who were infected. a second series of ts- a was then developed by combining two defective ts viruses, each of which belonged to a different complementation group in respect of the genetic defect. the progeny exhibited greater temperature restriction than the ts- [e] line of viruses. it was termed a/udorn/ ts- a , and it was recombined with three further viruses; wild-type a/victoria/ / , a/alaska/ (h n ), and a/hong kong/ (h n ). these ts- a viruses were highly immunogenic and exhibited temperature restriction of multiplication in cell cultures and reduced replication in the hamster lung. the a/victoria/ / -ts- a recombinant retained its ts properties after inoculation into doubly seronegative children. unfortunately, when the a/alaska/ -ts- a virus was similarly tested in a single child after tests in adults had shown genetic stability, the nasal secretions of the vaccine yielded a ts-positive virus that produced plaques at c even though the child had shown no symptoms or fever. the recombinant a virus with a/hongkong/ (h n ) parent exhibited a capacity to infect % of doubly seronegative adults and was attenuated compared with the wild-type parent. nevertheless, it appeared possible that a virus such as the a/alaska-ts- a might, if transferred to contacts from an inoculated child, result in clinical illness, and clinical studies with this particular virus were not pursued. beginning with a strain of h n virus recovered in ann arbor, michigan, in by cultivation of throat washings in tissue cultures at c, maassab [ , ] evolved a virus, a/ann arbor/ / (h n ), which has acted as a donor of attenuation to other viruses by genetic reassortment. earlier passages were made in chick kidney tissue cultures followed by intranasal passages in mice and then a gradual adaptation to lower temperatures, in tissue cultures and in developing hens' eggs inoculated allantoically, led to a virus with good powers of multiplication at c. the ca variant was found to retain the infectivity of the original strain for both the mouse and the ferret, although it produced no deaths in mice and no fever or turbinate lesions in ferrets, whereas the original virus was pathogenic for both species. the virus proved to be temperature sensitive with a shut-off temperature of c [ ] . recombinants with wild-type viruses of both h n and h n subtypes were prepared, studied in the laboratory and in volunteers, and analyzed genetically. the original a/ann arbor/ / (h n ) virus was not, however, tested in fully susceptible persons presumably because of the difficulty in that period of finding seronegative adults. a few persons with low titers of serum neutralizing antibodies ( : to : ) were inoculated and as judged by antibody responses, became infected without undergoing clinical illnesses. more rigorous clinical studies have been pursued with recombinants, in particular, those with h n antigens, and details of the results have been brought together and earlier data summarized by kendal [ ] . the donor ca parent has been more recently reassorted with h n genes. it is clear that infectivity and immunogenicity were fully retained for seronegative adults of whom received h n recombinants. among those receiving three of four recombinants, clinical reactions were minimal or negligible but with the fourth, derived from the a/scotland/ parent, in of volunteers receiving . and in receiving . tcid , there were clinical illnesses. viruses reisolated from the vaccines retained ts properties and so did those given recombinants of a/victoria/ (h n ) and a/alaska/ (h n ). however, some loss of ca restriction was found in virus re-isolated from volunteers given the a/scotland/ recombinant. cold-adapted recombinants with a/ussr/ (h n )-like virus have also been studied in adult volunteers and found to be less immunogenic as judged by hi antibody responses. a better response was obtained by wright et al. [ ] in children in nashville given . tcid of strain cr (h n ) and none of children developed adverse clinical reactions even though eight became infected. all reisolated viruses retained the ts phenotype. the failure to elicit serum antibody response in adults given this same virus recombinant is puzzling. using the elisa enzyme-linked assay, murphy et al. [ ] found that by this more sensitive method antibody rises could be demonstrated and the results tallied better with the ability to re-isolate viruses from the inoculated volunteers than did the serum hi responses. the leningrad group of workers led by smorodinstev [ ] was the first to obtain a virus indirectly attenuated by cultivation at c. the group used strains selected by inoculating volunteers with several viruses derived from cultures repeatedly incubated at - c to speed up attenuation. approximately - months were required for the preparation and production of new strains even using genetic recombination to incorporate new surface ha and na antigens. although alexieva et al. [ ] found that cold cultivation was not successful in producing reliably attenuated viruses for use in children, the technique was adopted for general use. genetic studies of the leningrad viruses are described briefly by kendal et al. [ ] , and these parent ca viruses are currently the center of new interest for attenuated h n vaccines. usually, preliminary studies were made in the ussr in - -year-old seronegative adults who receive virus twice at intervals of - days administrated by nasal spray. viruses were attenuated by passage for varying periods at c and both donor viruses and recombinants proved temperature sensitive. in - , when h n viruses were circulating, , children aged from to received the ca a/leningrad/ (h n ) virus. some febrile reactions occurred but only in less than % of the children. further studies of recombinants with h n or h n antigens and the same leningrad h n parent after serial passages under cold conditions of cultivation ( c) were conducted in children, half of whom had no detectable serum antibody to the vaccine strain. no reactions occurred and over % of the children responded with antibody production. it is clear from the earlier papers by alexieva et al. [ , ] that intranasal administration of children aged - were too reactogenic and that this is the reason why the peroral route has been chosen for routine administration in the ussr. a japanese virus recovered in , a/okuda/ (h n ), was found to be attenuated for children and served as a donor of attenuation both in japan and in england. zhilova et al., japanese workers, [ ] developed a recombinant virus (ko- ) from ultraviolet-irradiated a/okuda/ and wild-type a/kumamoto/ / (h n ). serial passaging in eggs in the presence of normal horse serum was followed by plaque purification and later clinical tests in a few children. the m (membrane) gene was found to have been donated by the okuda parent. from reassortants with other human viruses, a candidate wrl virus was selected and underwent clinical trials without harmful clinical effects [ ] but has been little investigated since that time. cultivation of influenza viruses in mammalian cells rather than eggs initially encouraged two manufactures to invest in cell culture fermenters for vaccine production [ , ] . many more groups are now using these technologies to produce the current a/swine h n vaccine. capacity can be increased to cope with a surge in demand for a pandemic virus vaccine. moreover, the final vaccine has the theoretical advantage of the absence of egg proteins. the cell culture vaccine virus is also easier to purify. where clinical isolates of influenza viruses are cultivated in mammalian cells and eggs in parallel, different antigenic variants may be selected [ ] . the biological variants have amino acid substitutions in the receptor binding site in proximity to an antigenic site on the ha, and an amino acid change in this region can alter antigenicity. of the two virus subpopulations that can be selected, the virus which is grown on mdck (or vero) cells rather than in eggs appears more closely related to the wild-type clinical virus. there is some indication that cell-grown virus vaccines offer greater protection in animal models than the corresponding egg-grown vaccine. these are all powerful arguments in favor of the new generation of influenza vaccines being cultivated currently in vero [ ] or mdck [ ] or per cells. alongside years of experience producing an immunogenic and safe vaccine, the world capacity for influenza monovalent vaccine manufacture has expanded to the present two billion doses. the preparation work and investment for h n are showing rewards with the current pandemic of a/swine h n . most manufacture is still located within the eu, but production is increasing in the usa, korea, japan, china, and most recently, india. the international collaboration in face of the outbreak of a/california/ / (h n ) in mexico around christmas to the present, the exchange of clinical data and viruses enabled vaccine manufacture to start production by may/june . by october , the production of hundreds of millions of doses of a monovalent vaccine containing mg of ha and immunogenic after a single ion dose in the over -year olds is a quite remarkable achievement. many countries have started to immunize at-risk groups, namely younger people < years of age with diabetes, obesity, chronic heart, or lung problems, and the immunosuppressed including pregnant women. it is forecast that up to - % of some countries could volunteer for the vaccine. it is especially important that medical and nursing staff take the vaccine to protect both themselves and their patients. however, such large vaccination campaigns open schisms in modern societies,which on the one hand become very concerned about young persons dying but on the other hand have prejudices about vaccines in general. in the first winter wave the over s, unusually for a pandemic virus, are protected by prior experience of the h n family and hence overall mortality is likely to be less than a seasonal year but the mortality is likely to be in younger persons, thus exposing our achilles heel. additionally nearly half the deaths to date have been in young persons without comorbidities. finally, within a year the virus is likely to mutate to allow it to infect the over group, so, paradoxically, mortality in the second pandemic year could easily exceed the first. twenty-first century vaccines induce protection across the different virus subtypes? there are known subtypes of the ha of influenza a virus. only three subtypes have caused pandemics in humans, h , h , and h , while h , h , and h predominantly circulating in birds have crossed the species barrier into humans and caused human outbreaks. we do not know whether these latter three subtypes could mutate into human-to-human transmitters and thereby acquire pandemic potential. at present, h n is causing considerable concern in se asia. an important question therefore is whether a vaccine could be engineered to give socalled heterotypic or cross-subtype immunity to protect against all these potentially pandemic viruses. it is well known that the internal proteins of influenza a virus such as m , m and np are shared by all influenza a viruses. these internally situated proteins are certainly immunogenic (particular np) but could the immunity induced, either t cell or antibody, be broadly reacting? to back up the central core of this approach, it has been known for years that mice infected with an influenza a (h n ) virus would later resist a lethal challenge from an influenza a (h n ) virus. given the lack of genetic and antigenic relatedness between the h and h proteins, or indeed the corresponding n and n proteins, this strong cross-immunity was attributed to an internal protein such as np or m. however, it has been difficult to construct a solid database and there has been a lingering doubt about this so-called cross-protective immunity. most virologists deduced, virtually by elimination, that a cross-reactive portion of the ha (ha ) could have provided the cross protection. furthermore, this cross protection is particularly seen in the mouse model, leading some to conclude that the mouse recognized cross protection epitopes that perhaps humans did not. fundamental studies to correlate the genetics and immunology of np and m established the cytotoxic t-cell response to portions of these proteins. however, the work clearly showed that m could be a cross-reactive immunogen, although a relatively weak one [ ] . the m protein is an integral membrane protein of influenza a viruses that is expressed at the plasma membrane of virus-infected cells and is also present in small amounts on virions. the important extracellular domain, potentially targeted by antibodies and t cells, is conserved by virtually all influenza a viruses. even the pandemic virus differs only in one amino acid. the first indication that the m was immunologically active was the observation that an anti-m monoclonal antibody reduced the spread of virus cell culture. not unexpectedly, the antibody reacted with the extracellular domain of m . even more excitingly, the antibody reduced the replication of virus in mouse lungs. immunization studies with m constructs, however, have given more mixed results. immunization of mice with dna plasmid of m and m gene gave protection mainly via t-helper cell activity. an alternative approach utilized a hepatitis b core and m fusion protein. the cross protection resided in antibodies, although m specific antibodies did not neutralize the virus in vitro. presumably, protection was mediated by an indirect mechanism such as complement-mediated cytotoxicity or antibody-dependant cytotoxicity. however, the protection induced in the mouse model was considerably less than that induced by a conventional sub unit ha/na vaccine. it could be argued that weak heterotypic immunity may be present already in the community and that this is helping to prevent the emergence of chicken influenza a (h n ) in se asia [ ] . certainly with evidence of tens of millions of domestic birds infected since late in countries in se asia, with only a handful of human infections and only human-to-human transmission in family groups, there is a possibility that the unique cocirculation since of two influenza a viruses (h n and h n ) may have enhanced heterotypic immunity in most communities, which in turn abrogates the emergence of chicken influenza a (h n ) into humans. it would be foolhardy, though, to take this argument to a fuller conclusion and relax preparations for a new pandemic influenza a virus. at present, with the unprecedented research investment into influenza vaccines, there are new discoveries of adjuvants and vaccine formulations to be tested as well as fundamentals of virus transmission, infectiousness, and pathogenicity. the ultimate test is in influenza-infected volunteers. this specialized work was initiated over years ago. during the great pandemic of , when the precise nature of the causative microbe of the spanish influenza had not been established, a group of american scientists asked for young volunteers from the army and navy. the quest was to probe the nature of the microbe that was already causing devastation in their own country and where, by , , young people were to die. however, this was not the first study into the precise nature of the microbe. the infection had first been documented a year earlier as a herald wave in the great city-sized military base and encampment of etaples [ , ] . here the british army constructed the largest establishment [ ] in its history, where , newly recruited soldiers each day intermingled with thousands of wounded soldiers, pigs and, in the nearby villages and markets, with ducks, domestic chickens, and geese. these are now recognized as the necessary biological features of an epicenter for the creation of a pandemic virus. we surmise, in retrospect, that an avian virus from a silently infected goose or duck could have crossed species either to a pig or to a soldier already infected with a human strain of influenza. this is the mixing bowl hypothesis. indeed, common epidemic influenza was known to be circulating in the winter of - in etaples. another factor in etaples could have been the hundreds of tons of gases of varieties contaminating the landscape of the nearby somme battlefield, as well as many of the wounded soldiers brought by the night trains into the hospitals on site and causing respiratory distress. a group of pathologists there and at abbeville, led by g. gibson, raised the question of the nature of the microbe. could it be a gram-negative bacterium such as h. influenzae, already described by pfeiffer as the cause of the previous influenza pandemic of ? or could it be a virus? viruses were rather unknown entities at that time but had been identified by their filter-passing nature. hence, gibson's experiment was quite simply to take sputum from a soldier victim and filter it through a berkefield candle filter, which would hold back any known bacterium but allow the passage of the much smaller ultrafilterable virus. but what then? gibson had not even considered that a human volunteer would receive the filtrate. in fact, he gave it to a series of macaques and, inadvertently, to himself. he died and the macaques became ill. his premature discovery of new virus influenza has lain undiscovered and hitherto unquoted in the archives of the first world war [ ] . meanwhile, in the usa, a more vigorous decision had been taken, and army and navy volunteers were infected intranasally with filtered material from spanish influenza victims. some volunteers were placed . m from dying servicemen, who coughed in their faces. the incredible result of this heroic endeavor is that not a single volunteer became ill, whereas all around the usa their companions were dying. it is more than possible that the volunteers had already been subclinically infected in the early summer outbreak of , which was less virulent than the autumn virus and would be expected to give cross-immunity. unit in salisbury (uk) as soon as the second world war was over, the medical research council in the uk established the common cold unit in salisbury at the harvard hospital. the hospital was a donation from the usa to cope with expected bomb casualties from london. in the event, this fully equipped multibuilding facility was used as an acute surgical hospital for servicemen. with christopher andrewes as its first chief scientist, the unit recruited volunteers to unravel the virological mysteries of respiratory disease. for the next years, a small team of virologists and clinicians infected volunteers and discovered the first human coronavirus, the common cold virus, and were the first to describe the clinical effects of interferons. essentially similar units were set up in the usa and ussr. the considerable difficulties encountered in mounting field trials led to experiments in which immunized volunteers were subjected to deliberate inoculation with live virus in the form of either attenuated strain or modified wild-type strain. this protocol was suggested by henle et al. [ ] , who immunized a group of children with inactivated influenza a (h n ) virus vaccine and then inoculated them with egg-cultured virus of the same subtype but recently isolated, by inhalation of an aerosol. high rates of infection ( %) were produced in unimmunized children of whom became ill. those receiving vaccine either escaped subsequent infection or developed serological changes; only child of the vaccinated children thus challenged became ill. although this study illustrated the outstanding success of the immunized protocol, there are probably few observers today who would be prepared to submit their children to a similar risk of deliberately induced illness. ideally young adults - are used for quarantine experiments. such a risk is, of course, experienced during epidemics and bell et al. [ ] undertook a similar experiment in adult volunteers some of whom were immunized with a single dose of inactivated a/japan/ / (h n ) virus vaccine soon after the a/asian epidemic began. the volunteers were isolated before being given intranasally pooled nasopharyngeal washings from patients with influenza and this caused clinical illness in % of volunteers previously given a placebo. as % of the vaccinated volunteers developed fever after challenge in this experiment, the single injection of inactivated vaccine proved relatively ineffective, presumably because of its inadequate immunogenicity. the information obtained by deliberate challenge of immunized volunteers has been explored in the past using modified attenuated virus strains. beare et al. [ ] did this in their comparison of inactivated or live influenza b vaccines in which a challenge from the live virus b strain was used to assess the comparative efficacy of the two vaccines. reinoculation with live virus was resisted better by those receiving the same material a month previously than by those injected with inactivated vaccine. couch [ ] has reported a number of trials in volunteers after inactivated vaccine using a low dose of an essentially unmodified h n virus that had received one or two passages in human embryonic kidney culture. it was first established by greenberg et al. [ ] that previous infection by homotypic h n virus gave protection against deliberate exposure for up to years after the original infection. comparison of inactivated vaccine a/hongkong/ (h n ) given intranasally or subcutaneously showed that following challenge with live virus only those who had developed a serum antibody response after vaccine by either route resisted infection. in a further trial of an anti-na inactivated vaccine made from an heq n virus, it was shown that a reduced frequency of illness and a reduced titer of virus in nasal wash specimens resulted following live h n virus challenge compared with the findings in control subjects. the number of those who contracted infection was also reduced somewhat by the inactivated na vaccine, thus supporting the suggestion of schulman et al. [ ] that na antibody, although incapable of neutralizing viral infectivity, could limit the extent of viral replication. beutner et al. [ ] also immunized children with an na-specific vaccine and noted that antibody to na had a role protecting against illness rather than against infection. slepushkin et al. [ ] and monto and kendal [ ] came to similar conclusions with regard to na vaccine and the clinical evidence of protection from illness. a series of experiments on volunteers, designed to obtain evidence of protection from vaccines containing viruses that were homotypic or heterologous to the challenge virus, is important in relation to the determination of the best composition of inactivated vaccine. potter et al. [ ] gave one of four inactivated monovalent h n virus vaccines to groups of students, measured their pre-and postimmunization antibodies by hi and ni tests, and later challenged all the groups with a live intranasal h n virus (wrl ). this virus was antigenically nearest to the a/port chalmers/ virus and vaccine from this latter strain and also that containing a/scotland/ virus gave better protection against infection than earlier h n virus vaccines; the result thus correlated with the induced hi antibody titers. larson et al. [ ] also challenged the immunity produced by inactivated vaccine made from a/port chalmers/ (h n ) virus with that from a strain developed by the pasteur institute [ ] . this virus ( c) with an antigen closely similar to a/england/ (h n ) was selected in the laboratory by a method analogous to natural selection by antigenic drift, and thus represents the first human attempt to anticipate antigen variation in nature. challenge of those immunized with one or the other vaccines showed that protection by the heterologous c virus was about one-quarter as effective as that produced by the homologous a/port chalmers/ virus. experiences related by couch also confirm [ ] that antibody effective against the homologous ha of the challenging virus is more protective than that formed by heterologous antigen. protection was also compared after inactivated vaccine by intranasal or subcutaneous routes, which showed that the important mediator of immunity was the serum igg content of anti-ha rather than the respiratory secretion content of specific iga. we have established a new quarantine unit, based in london (http://www.retroscreen. com), but very much centered upon the experience and ethos of the common cold unit of the past [ ] . in a series of experiments over the past years, we have infected over young volunteers with influenza a (h n ), influenza b, and influenza (h n ) virus and more recently respiratory syncytial virus, and we now have fully characterized virus pools [ ] . in the usa, a quarantine unit had already been established in virginia and also at baylor and pioneered work into the new na inhibitors of influenza using an influenza a virus isolated in [ ] . so far our own unit has focused on evaluating new influenza vaccines [ ] . we use groups of young volunteers and quarantine them in a student hostel or hotel or phase i clinical unit along with clinicians and scientists (fig. ) . the mrc common cold unit was rooted strongly in the postwar era with deck chairs, free run rabbits, country walks, afternoon cream teas, and two-course english meals. our new unit reflects a more diverse community, so chicken tikka is as common on the menu as roast lamb and baked potatoes, but the wish of many of the volunteers is the same: to contribute to knowledge. influenza a virus has a proven record as a "bioterrorist" virus but driven not in churchill's words by the "evil forces of perverted science" but by the vast unfathomable laws of nature and emergence, reemergence, and resurgence of natural disease. we are experiencing the attacks on pregnant women and younger persons at the present moment with a/swine h n [ ] [ ] [ ] . information from the human genome project, whereby a significant proportion of the , active genes are already known to be involved in innate and acquired immunity, provides reassurance that the immune system will continue to provide some protection against new fig. a volunteer room at the common cold and influenza unit, harvard hospital, salisbury, in the s. volunteers would stay for weeks in this country-placed unit to be infected and carefully studied for clinical symptoms viruses. this is excellently illustrated with a/swine where most of the population has immune memory to this h n family of viruses. gauguin in his last great painting "who are we, where have we come from, where are we going?" asks crucial questions about the future of humankind. but it was the medieval painter breugel who asked the major question, yet to be answered in the twenty-first century. his medieval painting "the triumph of death" shows a horseman on a white charger scything at random and gathering souls during an outbreak of pasteurella pestis in medieval times. the question haunting the painting is "why do some persons survive while others die." even in in most communities % of persons infected with the virus survived. but why did some die and exactly how were they killed by such a minute and fragile form of life that we know as the orthomyxovirus influenza? was the immune reaction and ensuing cytokine storm overwhelming or was virus replication in the endothelial cells of the air sacs more important? an extraordinary clear message is emerging, which tells us to build our public health infrastructure and continue and expand our epidemiological vigilance and surveillance against all these infectious viruses and bacteria. the virus cannot be permanently dislodged from its avian and swine reservoir. for pandemic influenza, every country needs a detailed and practical plan and a supply of antiviral drugs and new vaccines at hand for an emergence of h n . this virus will be a lot more difficult to deal with than a/swine h n . we would then be "at the end of the beginning" as regards protection of all citizens. influenza was the twentieth century's weapon of mass destruction. nature is the greatest bioterrorist of our world and emerging viruses could do for us all, as easily and as quickly, or even more so, than the great influenza of , except for the fact that we now have the ammunition to fight back: knowledge of virus transmission and how to break it with disinfectants and social distancing, and effective antivirals and vaccines. the current a/swine h n pandemic has exposed flaws in pandemic plans and also has exposed many countries that have no preparation whatsoever. the spanish influenza pandemic of - : new perspectives the great war america's forgotten pandemic studies of influenza in hospitals of the british armies in france reports on the pandemic of influenza - . reports on public health and medical subjects influenza a pandemics of the th century with special reference to : virology, pathology and epidemiology medical services diseases of the war human virology: a text for students of medicine influenza: the viruses and the disease strategies for containing an emerging influenza pandemic in southeast asia the great influenza, the epic story of the deadliest plague in history preparing for the first influenza pandemic of the st century a hypothesis: the conjunction of soldiers, gas, pigs, ducks, geese and horses in northern france during the great war provided the conditions for the emergence of the "spanish" influenza pandemic of - quantative aspects of the red blood cell agglutination test for influenza virus growth of influenza virus in the allantoic cavity of the chick embryo the role of serum hi antibody in protein against challenge infection with influenza a and b viruses a single radial immunodiffusion technique for the assay of haemagglutinin antigen mapping of the influenza virus genome: identification of the haemagglutinin and neuraminidase genes a dna transfection system for generation of influenza a virus from eight plasmids plasmid-only rescue of influenza a virus vaccine candidates development of a mammalian cell (vero) derived candidate influenza virus vaccine immunogenicity and reactogenicity of influenza subunit vaccines produced in mdck cells or fertilised chicken eggs immunological studies with the virus of influenza influenza: further experiments on the active immunisation of mice commission on influenza, board of influenza and other epidemic diseases in the arm ( ) a clinical evaluation of vaccination against influenza comparisons of serological and febrile responses in humans to vaccination with influenza viruses or their haemagglutinins respiratory virus vaccines. iii. some biological properties of sephadex-purified ether-extracted influenza virus antigens antibody response in humans to deoxycholate-treated influenza virus vaccines experience with vaccination against influenza in the spring of results of vaccination against influenza during the epidemic of future influenza vaccines and use of genetic recombinants purification of large quantities of influenza virus by density-gradient centrifugation a field evaluation of inactivated, zonal-centrifuged influenza vaccines in children in chapel hill influenza in the ussr in : recurrence of influenza virus a subtype h n reactions to concentrated influenza vaccines the antibody response and immunity to challenge infection induced by whole inactivated and tween-ether split influenza vaccines correlation of laboratory studies with clinical responses to a/new jersey influenza vaccines application of an improved singleradial-immunodiffusion technique for the assay of influenza haemagglutinin antigen content of whole virus and subunit vaccines a serological trial of asian influenza vaccine after the autumn epidemic clinical studies of monovalent inactivated whole virus and subunit a/ussr/ (h n ) vaccine serological responses and clinical reactions studies on inactivated influenza vaccines. ii. effect of increasing dosage on antibody with resistance to influenza in man immunity to attenuated influenza virus wrl infection induced by heterologous, inactivated influenza a virus vaccines a surface antigen influenza vaccine. . purification of haemagglutinin and neuraminidase proteins. . pyrogenicity and antigenicity pandemic working group of medical research council's committee on influenza and other respiratory virus vaccines ( ) antibody responses and reactogenicity of graded doses of inactivated influenza a/new jersey/ whole-virus vaccine in humans medical research council committee on influenza vaccine ( ) clinical trials of influenza vaccine clinical trials of influenza vaccine trials of an asian influenza vaccine clinical trials of oiladjuvant influenza vaccine, - dose-response relationship after immunisation of volunteers with a new surface-antigen-adsorbed influenza virus vaccine further studies of the neuraminidase content of inactivated influenza vaccines and the neuraminidase antibody responses after vaccination of immunologically primed and unprimed populations influenza virus subunit vaccines. ii. immunogenicity and original antigenic sin in humans the specificity of the antihaemagluttinin antibody response induced in man by inactivated vaccines and by natural infection strain specificity of serum antibody to the haemagglutinin of influenza a (h n ) viruses in children following immunisation or natural infection immunisation with influenza virus a vaccines: comparison of intradermal and subcutaneous routes immunisation with asian strain influenza vaccineequivalence of the subcutaneous and intradermal routes effect of dosage and route of inoculation upon antigenicity of inactivated influenza virus vaccine (hong kong strain) in man influenza antibody in human respiratory secretions after subcutaneous or respiratory immunisation with inactivated virus specificity of respiratory secretion antibody against influenza virus purified influenza vaccine; clinical and serological response to varying doses and different routes of immunisation antibody in respiratory secretions following immunisation with influenza virus vaccines humoral and secretory antibody responses to immunisation with low and high dosage split influenza virus vaccines the production of neutralising activity in serum and nasal secretions following immunisation with influenza b virus endotoxin content and clinical reactivity to influenza vaccines a neurological note on vaccinations against influenza guillain-barré syndrome: the swine influenza virus vaccine incident in the united states of america, - guillain-barré syndrome and the - influenza vaccine immunogenicity of polyvent and hong kong influenza vaccines serological responses and results of natural infectious challenge of recipients of zonal ultracentrifuged influenza.a /aichi/ / vaccine intranasal vaccination of humans with living attenuated influenza virus strains present status of live influenza virus vaccine development of cold-adapted recombinant live attenuated influenza a vaccines in the usa and ussr investigation into attenuation of influenza viruses by serial passage live attenuated influenza virus vaccine in vitro and in vivo properties rna's of influenza virus recombinants derived from parents of known virulence for man gene constellation of live influenza a vaccines recombinant influenza a viruses as live vaccine for man the study of antiviral compounds in volunteers analysis of virion rna segments and polypeptides of influenza a virus recombinants of defined virulence poliomyelitis congresses ( - ) papers and discussions at st, nd, rd, th and th international poliomyelitis congresses the replication of type a influenza viruses in the infant rat: a marker for virus attenuation the basis of attenuation of virulence of influenza virus for man genetic approaches to control of influenza temperature-sensitive mutants of influenza a virus xii. safety, antigenicity, transmissibility and efficacy of influenza a/udorn/ -ts- [e] recombinant viruses in human adults adaptation and growth characteristics of influenza virus at c biological and immunologic characteristics of cold-adapted influenza virus cold adapted variants of influenza a. ii. comparison of the genetic and biological properties of ts mutants and recombinants of the cold-adapted a/ann arbor/ / strain cold-adapted recombinant influenza a virus vaccines in young seronegative children use of the enzyme-linked immunosorbent assay to detect serum antibody responses of volunteers who received attenuated influenza a virus vaccine obtaining of an additionally attenuated vaccinating cryophilic influenza strain studies on some biological properties of vaccinal influenza strains cultivated at low temperatures some problems with modern influenza prophylaxis with live vaccine recombinant wrl strain live attenuated influenza vaccine. immunogenicity, reactivity and transmissibility evidence for host-cell selection of influenza virus antigenic variants a universal influenza a vaccine based on the extra cellular domain of the m protein induction of protective immunity against influenza virus in a macaque model: comparison of conventional and iscom vaccines testament of youth: an autobiographical study of the years - the etiology of influenza: a filterable virus as the cause (with some notes on the culture of the virus by the method of noguchi) demonstration of the efficacy of vaccination against influenza type a by experimental infection of human beings artificially induced asian influenza in vaccinated and unvaccinated volunteers assessment of immunity to influenza virus using artificial challenge of normal volunteers with influenza virus duration of immunity to type a influenza protective effects of specific immunity to viral neuraminidase on influenza virus infection of mice evaluation of a neuraminidase-specific influenza a virus vaccine in children. antibody responses and effects on two successive outbreaks of natural infection neuraminidase and resistance to vaccination with live influenza a hong kong vaccine effect of neuraminidase antibody on hong kong influenza immunity to challenge in volunteers vaccinated with an inactivated current or earlier strain of influenza a (h n ) sélection par pression immunologique de mutants dominants du virus de la grippe a (hong kong) cold wars: the fight against the common cold fluinsure tm , an inactivated trivalent influenza vaccine for intranasal administration, is protective in human challenge with a/panama/ / (h n ) virus. in: kawaoka y (ed) options for the control of influenza volunteer challenge studies dna vaccination protects against an influenza challenge in a phase b double blind randomised placebo controlled clinical trial the emerging influenza pandemic: estimating the case fatality ratio assessing the severity of the novel a/h n pandemic cdc. novel h n influenza vaccine acknowledgments we are pleased to receive grant income from the eu to develop new influenza vaccines. key: cord- -p vqpazu authors: tsai, theodore f.; rao, raman d.s.v.; xu, zhi yi title: immunization in the asia-pacific region date: - - journal: plotkin's vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: p vqpazu nan and inactivated vero cell-derived je vaccines from china, japan, and korea, regionally; live attenuated hepatitis a vaccine from china, regionally; live attenuated and inactivated pandemic and seasonal influenza vaccines from india and china, internationally; and oral cholera vaccine from vietnam, internationally). previously, asian manufacturers did not themselves market novel vaccines in europe or the united states, choosing to distribute their innovative products, such as acellular pertussis and live attenuated varicella vaccines, through multinational companies. however, an increasing global integration is taking place, as multinational companies acquire asian manufacturers (e.g., sanofi-aventis, france, acquired shantha biotechnics, india); asian companies acquire or obtain technologies and distribution rights from european countries (e.g., inactivated polio vaccine by serum institute of india ltd. acquiring bilthoven biologicals, netherlands; astellas, japan, acquiring recombinant influenza hemagglutinin from protein sciences, u.s.; thai government pharmaceutical organization acquiring chimeric je vaccine from sanofi-pasteur, france; and biological evans, india, acquiring je vaccine from intercell ag, austria); and vaccine codevelopment is agreed between entities in developed and asian countries (e.g., genetically modified, inactivated hiv vaccine codeveloped by sumagen, korea, and the university of western ontario, canada; mycobacterial proteinag a candidate tuberculosis vaccine codeveloped by tianjin cansino biotechnology, china, and mcmaster university, canada; universal influenza vaccine codeveloped by xiamen wantai and sanofi-pasteur, france; and novel pneumococcal conjugate vaccine codeveloped by sk chemicals, korea and sanofi-pasteur, france). , , the role of asian companies as developers and providers of neglected and improved vaccines for the region and, for developing countries more generally, is an emerging trend as illustrated by the joint research activity agreement between the national research council, canada, and the chinese national biotec group that covers development of h. influenzae type a and hib bivalent conjugate vaccine, novel mucosal adjuvants and therapeutic vaccines against helicobacter pylori infection, and cell culture manufacturing platforms for viral and vectored vaccines. a korean-manufactured biosimilar (generic) biological, infliximab, now is licensed in europe, a step toward commercial expansion of asian region-manufactured biologicals to developed countries. the emergence of asia as the base of new multinational vaccine companies with broad development, production, and distribution capabilities is on the horizon, even as consolidation of existing companies occurs elsewhere. [ ] [ ] [ ] the broad income range within countries in the region results in large population segments that have sufficient means to pay for vaccines out-of-pocket. even among countries that otherwise qualify economically for gavi funding (e.g., india), substantial numbers of families can avail themselves of vaccines not covered by the national epi, resulting in a two-tiered system of vaccination, paralleling the public-private dichotomy of healthcare delivery in general. practitioners serving these and expatriate families generally follow current u.s., european, or australian vaccine recommendations, or some modification of those schedules. five je vaccines have been developed and licensed in asian countries. the widely used first-generation inactivated suckling mouse brain (smb)-derived vaccine is being replaced rapidly in economically disadvantaged countries by the chinese developed and manufactured live attenuated or inactivated vaccine (sa - - strain) grown in primary baby hamster kidney (phk) cells and in higher-income countries with vero cell-derived inactivated vaccines (licensed in the united states, australia, canada, and europe, as well as several asian countries) or a replicating chimeric yellow fever-je virus recombinant vaccine (manufactured in thailand). details are provided in chapter . to control cases and occasional outbreaks of the far eastern subtype of tickborne encephalitis virus in northeastern china, the changchun biologicals institute developed a formalininactivated vaccine, derived from a human isolate, senzhang strain, and grown in phk cell cultures. related vaccines prepared from central european strains and distributed in europe are described in chapter . the kyasanur forest disease virus (kfdv) is a highly pathogenic member of the family flaviviridae causing a zoonosis, kfd, that is transmitted by the bite of infective ticks (haemaphysalis spinigera) primarily in its nymphal stage, and characterized by acute febrile illness with severe hemorrhagic manifestations. it was first described from outbreaks centered in karnataka state, india, among herders and villagers with forest exposure and was considered to be localized in the shimoga district area of the state. however, since first being reported in , the virus has been found in other areas of india including the kutch and saurashtra parts of gujarat state, andaman islands and west bengal. it is estimated that close to cases of kfd occur in india every year and, from to , among confirmed cases, were fatal. following the outbreak in india various vaccines including a formalin inactivated russian spring summer encephalitis virus, a russian spring-summer encephalitis virus-based mouse-brain vaccine, and a live attenuated vaccine that was serially passaged in tissue culture were tried but with limited success. finally, a formalin inactivated vaccine with the kfd virus grown in chicken embryo fibroblasts was tested in a large field trial from to among inhabitants of affected villages. the disease attack rates reported were . % ( / ) among persons receiving one dose and . % ( / , ) among recipients of two doses, respectively, compared to an attack rate of . % ( / , ) in unvaccinated persons, for vaccine efficacies of . % and . %, respectively. the vaccine was subsequently commercialized and is produced by the state institute of animal health and veterinary biologicals, hebbal, bangalore, and has been central to kfd prevention efforts in the state of karnataka. annual vaccinations have been done since in the shimoga and adjacent districts wherein two doses of the vaccine were administered in individuals to years of age at an interval of month. periodic boosters were also administered after to months. however, recent observations suggest a lower field effectiveness than had been reported previously, especially following a single dose, while overall coverage has also been low. between and , effectiveness among individuals perceptions of the value of vaccines and their risks also range widely, regionally and within individual countries, from largely enthusiastic acceptance and even demand for additional routine vaccinations (e.g., for je vaccine in southern and southeast asia) to a degree of skepticism equal to, if not more deeply and widely held, than vaccine hesitancy in europe and the united states. within the last years, japan discontinued routine childhood vaccine programs for combination measles-mumps-rubella, influenza, and je, and withdrew recommendations for the human papillomavirus (hpv) vaccine for adolescents, owing in several of instances, to incorrectly thinking that coincidental adverse events were causally related. the requirement for subcutaneous, as opposed to im administration for all vaccines, exemplifies the misattribution of adverse reactions, arising in this case from an extrapolation of muscle contractures resulting from repeated im administration of antibiotics, to other intramuscularly administered products, including vaccines. , the extrapolation has had unintended consequences of impeding the licensure of vaccines with newer adjuvants with mechanisms of action that require im administration. with the global spread of information, concerns over the thimerosal content of childhood vaccines and vaccine-associated autism have been as active a parental concern among middle-class families in developing countries as elsewhere. parental refusal of routine je vaccination in korea and significant declines in vaccine coverage occurred in a different context after seven cases of temporally related cases of anaphylactic shock and neurological disease, including five deaths, occurred in . the cases could not be excluded as causally related to administration of the mousebrain-derived vaccine, prompting a national debate and establishment of a vaccine adverse events reporting scheme, a national vaccine injury compensation system, and introduction of a live attenuated je vaccine derived from a nonneural tissue substrate. from this mosaic, we describe some common themes, highlighting representative approaches and unique issues that hold a wider interest. because they are covered elsewhere in this volume, we have not reviewed specific vaccines of regional concern (e.g., pandemic influenza and je vaccines) or vaccination topics common to developing countries (e.g., initiatives surrounding injection safety, measles and neonatal tetanus elimination, and polio eradication, nor financing mechanisms). we concentrate, instead, on other aspects of vaccine development and implementation, organized by the steps of vaccine development, approval, production, recommendation, and delivery. we also focus on childhood vaccines and vaccination and on selected countries in the region. japan is acknowledged as the innovator of several vaccines now used internationally, including acellular pertussis and live attenuated varicella vaccines, but other novel vaccines have been developed by japan, china, india, australia, and vietnam for region-specific needs (table . ). these include vaccines for je, hantaan (htn)-and seoul (seo) virus-related hfrs, russian spring-summer encephalitis, kfd, cholera, severe acute respiratory syndrome, and q fever. in addition, novel attenuated strains of measles, mumps, hepatitis a, rotavirus, and intranasally delivered pandemic h n virus have been derived for products distributed principally within the region. additional novel vaccines for hepatitis e and ev-a have potential for broader use internationally, an indicator of the region's transition from a provider of fill-finish and manufacturing capacity to a full-fledged participant in biotechnology research and clinical development. text continued on p. the reasons for low vaccine efficacy and coverage rates need to be investigated and the appropriate vaccine regimen for effective control requires further definition. newer vaccine approaches (e.g.., chimeric or virus protein subunit vaccines) are being investigated to potentially replace the current vaccine. elsewhere, a nearly identical strain to the kfdv was isolated from a patient suffering from acute febrile illness from yunnan province, china in . seroprevalence studies indicate that kfdv (or the nanjianyin virus or a related tickborne flavivirus) may be present in various parts of southwestern china. in a virus similar to kfdv called alkhurma hemorrhagic fever virus was isolated from patients with febrile illness in saudi arabia. overall, cases with two deaths occurred in sheep and camel handlers exposed to a tick ornithodoros savignyi. the disease has now been confirmed to be more widespread in the country than previously considered. as tickborne diseases are "diseases of place," kfd virus itself, if it spreads, is likely to disseminate locally. nevertheless, the discovery of antigenically related viruses elsewhere, such as alkhurma hemorrhagic fever virus, suggests a potential for more widespread use of kfd vaccine, depending on public health needs. hfrs, a widespread rodent-borne bunyaviral zoonosis in asia, is a pantropic infection with prominent capillary hemorrhages, interstitial nephritis, and a % to % case-fatality ratio that, until the last decade, caused more than annual cases in the republic of korea and more than , cases in china. although the disease had been well known in parts of russia and asia as a sporadic and occasionally epidemic disease among farmers, soldiers, and others exposed to campestral and sylvatic habitats, it was largely unknown in the west until thousands of military cases and deaths occurred during the korean war, when the disease was described as korean hemorrhagic fever. the etiologic agent eluded investigators until , when a novel bunyavirus, htn virus was isolated from the striped field mouse, apodemus agrarius, which proved to be the principal viral reservoir in most areas of asia. later, antigenically related seo virus was isolated from rattus rattus and rattus norvegicus, explaining the occurrence of sporadic hfrs cases and outbreaks in urban areas. subsequently, sin nombre and related hantaviruses were discovered in the western hemisphere, where rare encounters with infected rodents lead to small numbers of cases that feature prominent pulmonary involvement. a multitude of hantaviruses now have been described globally. the widespread impact of hfrs in china led public health authorities in the s and s to pronounce the disease second only to hepb as a public health menace, and, beginning in , several chinese vaccine manufacturers used smb, primary baby gerbil kidney cells (gkcs) or phk cells to produce inactivated, monovalent vaccines against htn or seo viruses. the gkc vaccine was inactivated by β-propiolactone and the other two by formalin. subsequently, vero cell linederived vaccines have been developed. these vaccines were evaluated in nine chinese provinces hyperendemic for hfrs during to . the gkc-derived vaccine against htn virus produced seroconversions to putatively protective titers of neutralizing antibody in . % of subjects after three primary doses at , , and days, the proportion rising to . % after a booster at year, and declining to . % at years and . % at years. similar immunogenicity results were reported for the phkderived vaccine and the purified smb vaccine. in a randomized, controlled, three-arm trial of gkc vaccine in which vaccinated subjects received three primary doses and a booster at year, hfrs cases were observed in the age-, sex-, and residence-matched controls, and cases in the , unvaccinated subjects of similar age ( to years), compared with none in vaccinees during months of follow-up, for a protective efficacy of % ( % lower confidence limit of . %, p = . , cumulative binomial probability). efficacy of the three primary doses alone was shown in the year between administration of the three primary dose series and the booster dose: with zero cases in the vaccinated, and nine and cases in the unvaccinated and control groups respectively. among cases in the control and unvaccinated groups, were caused by htn virus, were caused by seo virus, and four by a virus of indeterminate serotype. thus, the monovalent gkc-derived htn virus vaccine was protective not only against the homologous virus, but also cross-protective against seo virus. no vaccine-related serious adverse event was reported during the trial, and mild local and systematic reactions were reported in . % of vaccinees. the efficacies of the phk vaccine and the purified smb vaccine were similar: in nonrandomized trials, one hfrs case was found in , recipients of phk vaccine, compared with in , unvaccinated subjects, a reduction of . %; for the purified smb vaccine, the rates were . per , ( / , ) versus . per , ( / , ) for vaccinees and unvaccinated subjects, respectively, a reduction of . %. the observed reductions were maintained through years of follow-up. nonsevere reactions were found in . % of phk vaccine recipients and in . % of smb vaccine recipients. , bivalent htn and seo gkc-and phk-derived vaccines were developed and improved by purification procedures through gradient density ultracentrifugation or chromatography to be more immunogenic and less reactogenic. the purified bivalent gkc vaccine induced neutralizing antibody seroconversion against htn virus and seo viruses in . % ( / ) and . % ( / ) of volunteers, respectively, after two doses with an interval of days, and . % ( / ) and . % ( / ), respectively, after a booster dose at months. only mild reactions were observed; local reactions in . % ( / ) and systemic reactions in . % ( / ) of the vaccinees. the purified bivalent phk vaccine induced neutralizing antibody seroconversion against htn virus and seo virus in . % ( / ) and . % ( / ) of subjects, respectively, after two doses separated by days, and . % ( / ) and . % ( / ), respectively, after a booster dose at months. no systemic reaction was found among vaccinees and mild local reactions were observed in two ( . %). the purified, bivalent gkc vaccine was tested for protective efficacy in a nonrandomized trial among , subjects, to years of age; , persons received the two primary doses with an interval of days and a booster dose at months; , persons were unvaccinated. the two groups were similar in age distribution. during years of follow-up, hfrs cases were found in , person-years among the unvaccinated, a rate of . per , , compared with none in the vaccinated , person-years, a reduction of %. several manufacturers have adapted their processes from primary gerbil or hamster cells to continuous vero cells. the purified, bivalent vero cell-derived vaccine administered in two doses separated by days, induced neutralizing antibody against htn virus and seo viruses in . % ( / ) and . % ( / ) adult volunteers, respectively. mild systemic reactions were observed in . % ( / ) and mild local reactions in . % ( / ) of vaccine recipients. the immunogenicity and safety profiles of the vero cell-derived, purified bivalent vaccine were similar in children and older adults. based on the above data, a schedule of two primary doses with an interval of days, plus a booster at months, has been recommended for the purified bivalent gkc-, phk-, and vero-cell-derived vaccines. a postlicensure, retrospective study was conducted to measure the long-term effectiveness of the gkc vaccine among , adults to years of age, in villages located in a hyperendemic area of shaanxi province. hfrs incidence rates were compared between the vaccinated and the unvaccinated adults: . % ( / ) versus . % ( / ), respectively, for the first years after vaccination; . % ( / ) versus . % ( / ) in years to ; . % ( / ) versus . % ( / ) for years to ; and . % ( / ) versus . % ( / ) at to years. the vaccine's effectiveness was thus estimated at . %, . %, . %, and . %, respectively, for the four study periods. the effectiveness was underestimated because the year of onset of hfrs was unknown for cases, all of whom belonged to the unvaccinated group and were not included for analysis. the overall hfrs attack rate was . % ( / ) in the vaccinees and . % ( / ) in the unvaccinated subjects, a reduction of . %. a long-term study of the monovalent phk-derived seo virus vaccine also was conducted among adults to years old in a seo virus-predominating area, from through . only three primary doses were given at , , and days without a booster. seven hfrs cases were found in , , subjects in the vaccine group, a rate of . per , , and cases were found in , , controls, a rate of . per , , with an overall reduction of . % ( % ci, . % to . %) during the years of the study. the vaccine's effectiveness was estimated at % for the first year, . % for the second year, and . % for the th year. the rate reductions in other years were approximately %. a smb-derived htn virus vaccine also was developed in the republic of korea and is available for at-risk individuals. the incidence of hfrs in china and korea has declined in the last years with the introduction of vaccination and probably, more importantly, because of urbanization, rural economic development leading to improved (cement) houses, and grain harvesting and storage practices, resulting in reduced exposures to the rodent reservoir. this trend has been most evident in rapidly developing areas of southeastern china and likely will continue in other regions, leading to a diminution of disease incidence and, potentially, discontinuation of routine vaccination in endemic provinces. see table . . two similar live attenuated hepatitis a vaccines, based on the h and la- strains, and measles vaccines, based on the shanghai s- and changchun- strains, have been licensed in china and are used domestically and exported. the national institute of hygiene and epidemiology in vietnam developed an oral bivalent o -o killed whole-cell cholera vaccine that now is produced and distributed by vabiotech, company for vaccine and biological production no. , in hanoi; another oral bivalent o -o vaccine based on the vabiotec vaccine but with improved production design is produced in india by shantha (sanofi, france). both vaccines are used domestically and also exported. similarly, live rotavirus vaccines based on local strains have been developed in india and china for local use. ev-a and related enteroviruses have emerged in major seasonal epidemics in asia and australia, leading to millions of cases and extensive social disruption as daycares and schools are closed. the extent and impact of seasonal outbreaks stimulated vaccine development in china, taiwan, malaysia, singapore, and japan, with government prioritization and support in some countries, analogous to mechanisms that facilitated pandemic influenza vaccine development. an escherichia coli-expressed capsid peptide virus-like particle hepatitis e vaccine, approved by the china food and drug administration, is the first novel recombinant vaccine developed and licensed in asia. its potential use in africa, south asia and, possibly, even in developed countries in immunocompromised or other risk groups could be envisioned. implicit in the region's progress toward novel vaccine development is a maturing capacity to conduct clinical trials and improvements toward more robust regulatory processes and capacity, including pharmacovigilance systems. in addition, multinational companies increasingly have turned to countries in asia to conduct clinical trials because of lower costs and more streamlined regulatory approvals of clinical trial applications. international contract research organizations operate in many countries, and a growing local infrastructure to conduct clinical trials in compliance with the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use and good clinical practices standards will improve clinical research conducted in the region. unlike europe, asian countries are not unified in a central regulatory approval process. nor is there a regional public health presence as in latin america, where the pan american health organization (paho) leads regional vaccination programs and also provides central purchasing of certain qualified vaccines. however, the -nation association of southeast asian nations (asean; includes brunei-darussalam, cambodia, indonesia, lao pdr, malaysia, myanmar, philippines, singapore, thailand, and vietnam) in initiated efforts for a subregional regulatory harmonization scheme to reduce differences in technical requirements and regulatory procedures for pharmaceuticals. a harmonization initiative, under auspices of a pharmaceutical product working group, aimed to remove barriers to regional commerce and to eliminate technical barriers to trade without compromising product quality, efficacy, and safety. eventually, a subregional central or mutual-recognition procedure similar to that of the european union could be envisioned. importantly, local clinical trials are not required for registration under abbreviated pathways specified by the asean common technical dossier if the vaccine was approved and licensed by a benchmark regulatory agency, resulting in a certificate of pharmaceutical product. by contrast, the national regulatory authorities of china, india, japan, korea, and taiwan have required local clinical trials before or after registration, and in other countries, while data in local populations may not be required for registration, those data are important in deliberations on a vaccine's inclusion in the national schedule. for dengue, a disease of special public health urgency regionally, the global debut of candidate vaccines in the region is being considered, with individual country vaccine registrations ahead of approval by a benchmark agency and provision of a certificate of pharmaceutical product. the text continued on p. various countries in the region have had the effect of delaying the registration of proven vaccines that otherwise could have prevented significant morbidity and mortality with more timely introductions. descriptions of individual regulatory requirements for clinical trial applications and new product approvals are beyond the scope of this chapter; see the previous edition for a more detailed introduction. governments have had a greater role in vaccine manufacturing in the region than elsewhere, although devolution toward privatized or state-owned enterprises (i.e., government-owned corporations) has occurred (e.g., commonwealth serum laboratories in australia was privatized, and the six major government vaccine institutes in china now operate as a state-owned enterprise, china national biotech group; see table . ). although a growing number of private manufacturers have emerged, especially in china and india, in other countries, national and local government manufacturers continue to be important sources of certain vaccines and biologicals for domestic needs (e.g., the government pharmaceutical office in thailand, research institute for tropical medicine in the philippines, biofarma in indonesia, the national institute of hygiene and epidemiology in vietnam, and the central research institute and local government institutes in india). these and other facilities also fill and distribute bulk vaccines supplied by international manufacturers. several private and state-owned enterprise manufacturers in the region are members of the dcvmn, a consortium that seeks to identify and develop solutions to common challenges faced by manufacturers in developing countries. , , , a number of manufacturers (including in five asia-pacific countries) operate under practices and procedures that have prequalified them to produce certain vaccines for unicef, paho, and gavi purchases (e.g., pentavalent dtp combinations, oral polio vaccine, inactivated polio vaccine, hepb, rabies, influenza, oral cholera, and measles-containing vaccines) or that allow them to export vaccine to other countries in the region. a reliable supply of inexpensive diphtheria and tetanus toxoids combined with whole-cell pertussis (dtwp)-hib-hepb combination vaccines, made possible largely by indian and korean manufacturers, has facilitated the introduction of hib antigen into schedules of economically disadvantaged countries that otherwise would not have adopted the monovalent vaccine. similarly, provision of measles and measles containing vaccine by indian manufacturers was key to the elimination of that disease in latin america and the current state of polio elimination could not have been achieved without supplies from asian regional manufacturers. the provision of oral cholera vaccine for outbreak control in haiti, pakistan and other countries is an important example of the increasing ability of and global dependence on these manufacturers. who prequalification requires that the manufacturers and plants not only must satisfy who good manufacturing practices inspections, but, in addition, that national notifications of adverse events following immunization are captured and analyzed satisfactorily. this last requirement has been the principal impediment to prequalification of products from some countries and prequalification aided by who blueprint and other vaccine safety-related guidelines have facilitated the improvement of vaccine-related pharmacovigilance in the region. in china, the state-owned china national biotec group is the dominant supplier of vaccines in the country, providing % of doses used in the public program and % taken up initiative is a collaboration among the nongovernmental organizations, the dengue vaccine initiative, and the world health organization (who) developing countries vaccine regulatory network (dcvrn). the who through the dcvrn has been actively working toward harmonizing procedures in affiliated countries, including china, india, and indonesia, to bring those regulators under the who prequalification umbrella and to facilitate approval and supply of their products for gavi and united nations children's fund (unicef). the requirement of some national regulatory authorities for clinical data in local populations is based on a concern that racial, ethnic, or environmental differences could affect responses of the local population, both immunologically and in their risk for adverse events. genetically based differences in drug pharmacokinetics and pharmacodynamics, as well as disease risk, increasingly have been recognized, including immune responses to vaccines. studies of antibody responses to pneumococcal conjugate vaccines in asia, for example, have found higher prevaccination and postvaccination antibody titers among philippine and taiwanese infants compared with european or historical control subjects, and in korea, a considerably higher proportion of subjects were seropositive to meningococcal serogroup w polysaccharide at baseline than in the united states. [ ] [ ] [ ] while the basis for these differences may be an earlier exposure in life to cognate or crossreacting antigens (e.g., because of regional differences in host microbiomes), genetically restricted responses, as have been observed with hepb, measles, vaccinia, rubella, hib, and other antigens, or, in the case of oral rotavirus vaccine, in genetically determined viral attachment or receptor binding molecules, have been described. [ ] [ ] [ ] [ ] from the perspective of adverse events following immunization, the example of narcolepsy occurring in some recipients of an adjuvanted pandemic h n vaccine illustrates the role of genetic background as a cofactor in risk. in many examples, regulatory systems and processes in the region have had the effect of markedly slowing or effectively blocking the introduction of novel vaccines developed externally. in china, the introduction of an internationally registered and otherwise widely used product nevertheless necessitates recapitulating the entire clinical development program in china, including phase i studies, despite an abundance of previously scrutinized evidence. this requirement introduces a delay of a decade or more for registration of internationally developed, as opposed to domestically developed, vaccines. specifications in national pharmacopeias that deviate from established compendia, for example, exclusion of well-accepted excipients or methods, also have seriously impeded or prevented registration of foreign products or, when imposed with a revision of the pharmacopeia, have led to withdrawal of a previously registered product. clinical trial processes also have hindered local introduction of established or novel products (e.g., a indian supreme court ordered suspension of ongoing clinical trials and reexamination of previously approved trials was followed by a wholesale revision of clinical trial guidelines, leading to a temporary cessation of all industry-sponsored clinical trial activity). the potential inclusion of video recording of the informed consent process, newer insurance requirements and further proposed but unclear amendments to the drug and cosmetics act that could impose criminal penalties against trial investigators for poorly defined violations may further limit trial activity. china and indonesia place severe restrictions on the exportation of clinical samples from study subjects, thereby requiring that validated laboratories and procedures are established locally, adding a barrier that has led to delays of or avoidance of clinical studies in those countries. whether resulting from inexperience, a dearth of trained personnel, trade protection, or other reasons, administrative mechanisms in in most countries in the region, public health authorities now draw on external advisors to help formulate national vaccine recommendations in national immunization technical advisory groups (nitag), resulting, in part, from activities of the supporting independent immunization and vaccine advisory committees initiative (at the agence de médecine préventive). [ ] [ ] [ ] [ ] the advisory committee on immunization practices (acip) in taiwan and korea, expert committee on immunization in singapore, chinese expert committee on epi, hong kong scientific committee on vaccine preventable diseases, immunization committee of the indonesian pediatric society, national technical advisory group for immunization in india, and the australian technical advisory group on immunization are examples of such medical advisory groups. in china, vaccine recommendations are made through the national centers for disease control based on recommendations of the chinese expert committee on epi under the ministry of health and family welfare; however, provincial or local centers for disease control may issue independent recommendations for specific vaccines or modify the national recommendation for routine vaccines (see tables . the issues considered by asian nitags in formulating vaccine recommendations parallel those of other nitags, focusing on medical need, vaccine safety and efficacy, national resources, as well as implementation issues, including supply, cold-chain, fit within the national schedule, vaccine presentation, etc. health economic analyses are considered in the deliberation of some committees or are provided by an independent body (e.g., the health intervention and technology assessment program in thailand); although, in general, the use of health technology assessments in the region lag behind the united states and united kingdom. in some cases, industry sponsors, in providing such analyses to nitags in their justifications to include new vaccines into national programs, have played a role in introducing cost-to-benefit analyses to the recommendation process. in indonesia and malaysia, the halal status of vaccines is an important factor in public acceptance of a product and also is a consideration in the vaccine recommendation process, although there is movement to remove this consideration from debate. in certain asian countries, as well as in latin america, the approval process to include a new vaccine into the national program is used to leverage multinational companies to foster local manufacturing expertise. in brazil, technology transfer of the vaccine production process is required in turn for the vaccine's inclusion into the national schedule while, in indonesia, all epi vaccines are locally produced by biofarma, and no new vaccine has been introduced into the national schedule unless it was produced locally. technology transfer of some element of the manufacturing process also is a factor in introduction of new vaccines to thailand and malaysia. such requirements may be tested as costly vaccines manufactured by more complex technologies are introduced to the region. the recommendation process in japan illustrates how, even after registration, organizational and administrative processes can result in a lengthy interval before a new vaccine is introduced to the national schedule. although, since , japan has recovered from a "vaccine gap"-the self-acknowledged interval during which antigens such as hib and pneumococcal conjugate, rotavirus, hpv, inactivated polio and various combination vaccines were not introduced into japan despite their widespread use in other developed countries-adoption of new vaccines into the national immunization program after their registration still lags several years. a number of sequential approvals lengthens the process: the immunization policy and vaccination committee provides an initial recommendation whether the newly registered vaccine should privately. the group comprises manufacturing sites, which produce some products, including the first who prequalified vaccine produced in china (sa - - je vaccine). other private companies compete principally to provide vaccines for out-of-pocket sales at local centers for disease control and prevention and hospitals. within the asean community, comprised principally of low-and middle-income countries, regional vaccine security has been a focus of discussion, reflected in the establishment of the asean-network for drug, diagnostics and vaccines innovation that focuses on a broad agenda of health technology development and collaborations on vaccine manufacturing and plans for regional vaccine purchasing-similar to paho's revolving fund. similarly, the eight-nation south asian association for regional cooperation includes biotechnology in its agenda for cooperative research. a goal to achieve self-reliance in vaccine supply also has been articulated in korea, in its horizon-setting. to a growing extent, multinational companies are acquiring or partnering with local companies in the region, with the result that manufacturing standards and their regulation should improve toward meeting international specifications. table . lists the region's principal vaccine manufacturers and their licensed products. the list is not intended to be comprehensive, as the sometimes rapid emergence or disappearance of pharmaceutical and vaccine companies in china and elsewhere is difficult to track. vaccines that are manufactured elsewhere and refilled and distributed by local manufacturers are not listed. countries in the region can be divided broadly into countries with a single national schedule and countries in which a basic schedule of free epi vaccines is supplemented by recommendations of a professional organization (such as the national pediatric society) for additional antigens that are paid for outof-pocket. countries in the first group include, on the one hand, mainly developing countries offering a basic epi schedule and, on the other, countries like australia, new zealand, and taiwan that provide a universal vaccination program that includes an array of antigens or combination vaccines paralleling those of european and u.s. schedules. the continued introduction of new and frequently expensive vaccines is an ongoing tension for vaccine recommending and funding entities that must weigh the relative value of such innovations against other preventive and therapeutic health expenditures. even for low-middle-income countries in the region, the total per capita expenditure for all healthcare may be less than the cost of a full course of a novel vaccine! on the other hand, national schedules in the region can be as comprehensive as to include the hpv vaccine (australia) and influenza and varicella vaccines (e.g., korea, taiwan). at the same time, hib vaccine still is not recommended in some jurisdictions with high per capita income (hong kong, singapore). to some degree, the seemingly paradoxical recommendations of relatively high-income countries in the region reflect different social expectations of personal responsibility in healthcare purchases (see subsequent text). as shown in table . , some national schedules provide optional recommendations for some antigens; in many countries where government tenders choose specific manufacturer products, specific combinations are recommended in the national schedule. in addition, for some antigens, provincial-specific recommendations address regional differences in risk (e.g., for routine group ac meningococcal vaccine in china; for je vaccine in sarawak, malaysia, and for the torres straits, australia; and for rabies vaccine [preexposure] in areas of the philippines). be classified as either "routine" or "voluntary," based on available data; the technical recommendation is considered by the tuberculosis & infectious diseases control division which makes the administrative decision for the vaccine's inclusion in the national schedule; however, that decision requires additional legislative approval whether the disease (category a or b) qualifies for full or partial vaccine funding (up to circa %), respectively. the recommendation process is even lengthier than appearances suggest, as the immunization policy and vaccination committee does not convene a deliberative vaccine working group until after the product is registered, unlike the parallel activities of the u.s. acip and food and drug administration. only then does the committee assemble a dossier (fact sheet) that establishes the epidemiology of the disease and its local burden; if insufficient data are available, de novo studies might be required to establish need. the overall interval between vaccine approval and issuance of a recommendation typically is years. other asian countries have similar or even lengthier intervals between vaccine registration and full epi implementation. in thailand, for example, after a preliminary nitag recommendation, a new vaccine is implemented in a pilot program to establish effectiveness and to collect additional safety experience. such a program may be gradually extended to other localities over a period of as long as a decade before the antigen is provided nationally. for diseases with regional differences in disease burden, high-risk provinces may be covered first (e.g., je vaccine initially was introduced in thailand to eight high-incidence provinces and progressively, from to , to all provinces, while local production was established and expanded). for new, often costly vaccines, phased introduction provides a mechanism to accommodate their full epi coverage costs over time. in the interim, local governments of wealthier provinces or municipalities have issued their own recommendations for vaccines to be reimbursed (e.g., shanghai provides pneumococcal polysaccharide vaccine free of cost to older adults and bangkok established a school-based hpv vaccination program, while neither vaccine is included in respective national schedules). innovative financing mechanisms have played an important role in the introduction of vaccines to low-income countries, and their extension to graduating gavi will enable more rapid adoption of new vaccines in those countries. at the same time, tiered pricing, negotiated between sponsors, local government and other entities will aid middle income countries to accelerate vaccine adoption, as exemplified by introductions of pcv and rotavirus vaccines. during the interval between a vaccine's registration and its inclusion in the national schedule, after which it is available without cost, out-of-pocket sales still may result in considerable uptake. while rotavirus vaccine is still considered a voluntary vaccine in japan, coverage among infants is estimated to be approximately %. in korea, although almost all pediatric vaccines are self-paid by parents, vaccine coverage for antigens such as hib and pneumococcal conjugate vaccine rapidly reached coverage rates of approximately % that, with herd effects, led to disappearance of the respective diseases as quickly as in other countries. although in japan, the "voluntary" vaccine recommendation emanates from a government committee, in other countries, academic societies play the principal role in recommending vaccines that are not included in the epi schedule. the malaysian pediatric association, the pediatric society of thailand, and the philippines foundation for vaccination not only advise their respective ministries and nitags in formulating national recommendations, but also promulgate recommended schedules of administration for other approved but not epi-covered antigens, emulating in large part or entirely from u.s., australian, or european schedules. vaccines are delivered in varying proportions through public or private channels, depending mainly on local income levels and accessibility to private practitioners. in general, vaccines on national schedules are available at no cost in primary health centers or their equivalent (e.g., puskesmas in indonesia; polyclinics or government hospital clinics in singapore, malaysia, and thailand; village and county level centers for disease control in china; village communes in vietnam; public health centers and clinics in india and japan; and at general practitioner offices in japan, and australia). as vaccines generally are available free in public clinics, even in affluent countries, families may obtain them in government clinics or hospitals (e.g., in singapore, ≈ % of families obtain vaccines through the government system of polyclinics and hospitals). however, to avoid long waiting times and rotating staff at public clinics, many families opt to obtain these otherwise free vaccines privately and to pay out-of-pocket at pediatric, general practitioner, or other private clinics. in addition, as newer vaccines may be delayed in their introduction to the national reimbursement scheme, it is common for parents to pay voluntarily for these vaccines (see earlier). as might be expected from the distribution of income, the proportion of children vaccinated in government primary health centers is higher in rural areas. overall, approximately % of children in thailand and % in malaysia are vaccinated through public channels. in china, all vaccinations are under control of centers for disease control and prevention; therefore, nearly all chinese children receive free epi vaccines, as well as payable optional vaccines (e.g., hib, pneumococcal conjugate vaccines, varicella, rotavirus, and others) at public clinics. fig. . summarizes the coverage for epi vaccines for selected countries. supplementary immunization activities have played a critical role in the elimination of polio from the who southeast asian region that was achieved in , and in ongoing efforts to eliminate measles and congenital rubella syndrome. routine and supplementary immunization activities tetanus vaccinations have eliminated maternal and neonatal tetanus in all but four countries in the region: cambodia, indonesia, papua new guinea, and pakistan. economic growth and development in asia and secular trends in population structure and the evolution of healthcare systems are forces that inevitably will change various aspects of immunization in the region, if in as-yet unforeseeable ways. , the population of asia, as in other regions, is aging and shifting toward a structure with a larger proportion of adults and elderly persons. between and , the birth cohort of asia will decrease slightly from . to . million, and the population of children to years old will hold nearly constant while the number and proportion of adults from to years will increase dramatically, and the number of people older than years of age will nearly double, from . to . million. a demographic crossover point with more adults + years of age than children younger than years of age was reached in europe in the s, and will occur within another generation in asia (fig. . ) . with the exception of almost universal epi programs of tetanus toxoid vaccination of pregnant women, adult vaccination has been viewed mainly in the context of travel, as in group a meningococcal vaccine for the hajj, and in tropical asia, influenza vaccine alliance for vaccines and immunization-eligible countries (shaded) have similar coverage rates of basic vaccines as countries at higher levels of economic development, illustrating the success of expanded programme on immunization. hepatitis a, interestingly, is now principally a risk in the cohort of young adults who were raised in an era of economic development and improved sanitation and who therefore lack natural immunity but were born before routine childhood vaccination was implemented. a catch-up program to address this epidemiological shift has been recognized by adult vaccination recommendations in some countries (see table . ). in china, adult measles vaccination is under discussion, as more than , cases have occurred annually in recent years, in equal proportion in adults older than years and in infants who had not received their first vaccine dose. growing awareness of adult vaccination is reflected in an increasing number of countries with adult vaccination recommendations (see table . ). two other population trends that will influence the demand for vaccines and channels for their delivery are urbanization and income disparity. the urban-dwelling population in asia is projected to increase by almost a billion persons between and , from . billion to . billion, while the rural population will decline only slightly. urban crowding is likely to affect the transmission patterns of certain person-to-person transmitted diseases and even of infections acquired from environmental sources. dengue, for example, is transmitted by mosquito vectors that are more prevalent in urban environments; the already great need for a dengue vaccine will almost certainly increase with the growth of urban centers. while the growing size and number of large cities may increase transmission of certain infections, delivery of vaccine and of healthcare in general is better organized in cities than in rural areas. specific interventions are needed to ensure that the existing disparity in access to healthcare between urban and rural dwellers does not widen. associated with urbanization is the increasing income gap in many countries that, in the health arena, has translated into a two-tiered system of healthcare, including preventive medicine. while vaccines are regarded by many as a public good to be provided as a government service, as mentioned, access to the increasing number of new vaccines is likely to be stratified by income level and ability to pay, as governments must choose among increasingly costly vaccines and other health interventions. as shown in table . , pediatric societies in a number of countries promulgate recommendations emulating those of the u.s. acip, and these schedules, aimed at practitioners serving private-paying families, may diverge increasingly from the national epi schedules benefiting the majority of children in those countries. how the public and governments will respond to an increasing disparity of what has been perceived as a basic medical service remains to be seen. in coming years, more novel vaccines are likely to be developed in asia or licensed first in asia for a regional, developing world, or international market. governments and asean have expressed increased interest in providing for national and regional vaccine security. the collaboration of industry sponsors with nongovernmental organizations and government in public private partnerships for new product development has been highlighted by the successful introduction of vaccines and drugs for several neglected diseases, for which the dcvmn view a responsibility. for example, the japan international cooperation agency and kitasato daiichi sankyo provided technical assistance to establish domestic measlesrubella vaccine production in vietnam's public corporation, center for research and production of vaccines and biologicals, polyvac. at the same time, the entry of nongovernmental organizations as actual sponsors of novel vaccine development for certain target diseases introduces competition with dcvmn manufacturers and multinational companies that might also consider similar development programs. asian academic institutions and companies possess elements the crossover point when the population of adults older than years of age exceeded the population of children younger than years of age was crossed in europe around ; that crossover is projected to occur in asia around , within a generation from now. for travelers to temperate locations. however, the severe acute respiratory syndrome and pandemic h n outbreaks and the regional threat of h n influenza have focused attention on routine seasonal influenza vaccination for the first time in many countries, beginning with elderly populations, and the role of children in influenza transmission is being recognized while it is rediscovered in japan. as a result of high pediatric vaccination coverage in developed countries in the region, je has become almost exclusively a disease of adults older than years of age, reflecting the intrinsic biological susceptibility of older adults to neurotropic flaviviruses and suggesting a of the scientific and technical expertise needed to develop vaccines for current and emergent needs and, seemingly, the will to establish themselves on the global stage and contribute to their development. regional institutions responded rapidly to threats of middle east respiratory syndrome virus and ebola virus with candidate vaccine development even when transmission was geographically remote. further participation of regional institutions in global responses in the future is likely. trends toward increasing local development and manufacturing in the region and the accompanying need to strengthen respective regulatory agencies have been recognized by the who and local national regulatory authorities. revising and harmonizing guidelines and procedures to international standards and enforcing procedures in a consistent and predictable manner will improve the timely regional introduction of vaccines developed internationally. as important, compliance with international standards will be required of regional manufacturers hoping to license locally developed vaccines more broadly. indian and chinese manufacturers currently export a limited number of vaccines, mainly regionally and to african and latin american countries, but their horizons undoubtedly will expand. in the six-component framework of product development capability-manufacturing; national and international distribution systems; private and public r&d capabilities; intellectual property system; and drug and vaccine regulation-regional manufacturers are at different stages of maturation. in its ascendance to an advanced country producing complex biologicals as well as other high technology products, korea followed a path that might be emulated by others in the region, highlighted by its arrival at a stage with a national system of innovation in science and technology, linking government, universities and industry, a strong regulatory system and observation of intellectual property rights, including adherence to trade-related aspects of intellectual property rights (trips agreement). a specific area of regulatory control needing particular attention is the strengthening of national control laboratories. many countries lack the laboratory capacity to test samples for lot release, and because manufacturing and testing technologies change rapidly, keeping up with new procedures and purchasing needed equipment are ongoing challenges. continuous support also is needed to produce working quantities of reference standards, validation of new assays, staff training, and proficiency testing. as resources are unavailable in many countries to establish and maintain a fully functioning national control laboratory, a regional network has been proposed as an approach to share expertise and to divide workload, while at the same time standardizing methods and criteria. field surveillance of adverse events following immunization is another area requiring strengthening. investigations of adjuvanted h n and h n pandemic and prepandemic vaccines administered in korea and taiwan, respectively, illustrated the interest in and epidemiological capacity of local investigators but also the limitations of existing systems and databases. japan is establishing a database of clinical encounters that if linked to immunization records could be used as a future adverse events surveillance system. regulatory oversight of clinical trials and human subjects protection are other areas that are under growing pressure for improvement. multinational companies have increased the number of clinical trials in asian countries to reduce costs and to obtain local registration of products. their activities serve an important role in strengthening local compliance with good clinical practices, as many groups conducting trials in the region have limited experience with these precepts and procedures. countries in the region have an interest to establish and enforce clear guidelines, not only as hosts to an increasing number of trials but also because their manufacturers, as future sponsors of new products, will be accountable internationally to uphold recognized standards. references for this chapter are available at expertconsult.com. immunization in the asia-pacific region .e references . country hub. gavi, the vaccine alliance role of vaccine manufacturers in developing countries towards global healthcare by providing quality vaccines at affordable prices asia's ascent-global trends in biomedical r&d expenditures emergence of biopharmaceutical innovators in china, india, brazil and south africa as global competitors and collaborators developing countries can contribute to global health innovation the indian and chinese health biotechnology industries: potential champions of global health? chinese health biotech and the biollion-patient market indian vaccine innovation: the case of shantha biotechnics safety concerns regarding combination vaccines: the experience in japan tracking the global spread of vaccine sentiments: the global response to japan's suspension of its hpv vaccine recommendation japanese encephalitis immunization in south korea: past, present and future kyasanur forest disease kyasanur forest disease: an epidemiological view in india field evaluation of formalin inactivated kyasanur forest disease virus tissue culture vaccine in three districts of karnataka state coverage and effectiveness of kyasanur forest disease (fkd) vaccine in karnataka isolation of kyasanur forest disease virus from febrile patient isolation of a flaviirus related to the tick-borne encephalitis complex from human cases in saudi arabia a global perspective on hantavirus ecology, epidemiology, and disease quality control of vaccines against hemorrhagic fever with renal syndrome a randomized, controlled field trial of gerbil kidney cell derived-, mono-valent vaccine against hantaan virus of hemorrhagic fever with renal syndrome in jiende city a study of mass immunization against hemorrhagic fever with renal syndrome in china guidelines for control of hemorrhagic fever with renal syndrome observation of purified gerbil kidney cell derived-vaccine against hemorrhagic fever with renal syndrome on human volunteers immunization effect of purified, bivalent primary hamster kidney cell derived vaccine against hemorrhagic fever with renal syndrome study of immunization effectiveness in people vaccinated with a bivalent hfrs vaccine clinical study on safety and serology of a bivalent, purified vaccine against hemorrhagic fever with renal syndrome investigation of immunogenicity of purified, bivalent hemorrhagic fever with renal syndrome vaccine e (vero cell) in children and the elderly people long-term epidemiologic effects of vaccination against hemorrhagic fever with renal syndrome (hfrs) in areas of shaanxi province endemic for hfrs. zhonghua liu xing bing xue za zhi long-term epidemiological effect of vaccine against hemorrhagic fever with renal syndrome in a large population harmonization of standards and technical requirements in asean dengue vaccines regulatory pathways: a report on two meetings with regulators of developing countries greater antibody responses to an eleven valent mixed carrier diphtheriaor tetanus-conjugated pneumococcal vaccine in filipino than in finnish or israeli infants safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in taiwanese infants immunogenicity and safety of a novel quadrivalent meningococcal conjugate vaccine (menacwy-crm) in healthy korean adolescents and adults identification of antigen-specific b cell receptor sequences using public repertoire analysis profiling of measles-specific humoral immunity in individuals following two doses of mmr vaccine using proteome microarrays both lewis and secretor status mediate susceptibility to rotavirus infections in a rotavirus genotype-dependent manner genetic polymorphisms of cxcr and cxcl are associated with non-responsiveness to the hepatitis b vaccine india's proposed amendments to the drug and cosmetics act: compensation for injuries to clinical trial participants and the criminalization of clinical research. life sciences law and industry report. lslr , / / bloomberg bna vaccines, our shared responsibility immunization policy development in thailand: the role of the advisory committee on immunization practice an overview of the national immunization policy making process: the role of the korea expert committee on immunization practices progress in the establishment and strengthening of national immunization technical advisory groups: analysis from the who/unicef joint reporting form, data for recent progress and concerns regarding the japanese immunization program: addressing the "vaccine gap who-unicef estimates of dtp coverage world health organization department of economic and social affairs population division vaccine preventable diseases surveillance program of japan. japanese encephalitis: surveillance and elimination effort in japan from to seroprevalence of hepatitis a and associated socioeconomic factors in young healthy korean adults urbanization and geographic expansion of zoonotic arboviral diseases: mechanisms and potential strategies for prevention development of health biotechnology in developing countries: can private-sector players be the prime movers? we are grateful for the help of john o'shea, jason humphries, takashi sugimoto, and hyun-ah chang. key: cord- - cf j authors: ahmad, sajjad; navid, afifa; farid, rabia; abbas, ghulam; ahmad, faisal; zaman, naila; parvaiz, nousheen; azam, syed sikander title: design of a novel multi epitope-based vaccine for pandemic coronavirus disease (covid- ) by vaccinomics and probable prevention strategy against avenging zoonotics date: - - journal: eur j pharm sci doi: . /j.ejps. . sha: doc_id: cord_uid: cf j the emergence and rapid expansion of the coronavirus disease (covid- ) require the development of effective countermeasures especially a vaccine to provide active acquired immunity against the virus. this study presented a comprehensive vaccinomics approach applied to the complete protein data published so far in the national center for biotechnological information (ncbi) coronavirus data hub. we identified non-structural protein (nsp ), c-like proteinase, and spike glycoprotein as potential targets for immune responses to covid- . epitopes prediction illustrated both b-cell and t-cell epitopes associated with the mentioned proteins. the shared b and t-cell epitopes: drdaamqrk and qarsedkra of nsp , edmlnpnyedl and eftpfdvvr of c-like proteinase, and vnnsyecdipi of the spike glycoprotein are regions of high potential interest and have a high likelihood of being recognized by the human immune system. the vaccine construct of the epitopes shows stimulation of robust primary immune responses and high level of interferon gamma. also, the construct has the best conformation with respect to the tested innate immune receptors involving vigorous molecular mechanics and solvation energy. designing of vaccination strategies that target immune response focusing on these conserved epitopes could generate immunity that not only provide cross protection across betacoronaviruses but additionally resistant to virus evolution. a recent outbreak of pneumonia in wuhan, china, is associated with betacoronavirus of group b from family coronaviridae and the order nidovirales [ ] [ ] . the viruses are positive-sense rna, enveloped and non-segmented [ ] . this coronavirus disease (covid- ) is known as a third human zoonosis of the st century and is caused by a new strain not previously identified in humans [ ] . the coronaviruses causing minor infections of the respiratory tract in humans are nl , oc , hcov- e, and hku while, the lethal coronavirus infections that emerged in this century are the middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov) and the recent sars-cov or covid- [ ] . the source of the covid- is still not confirmed but some evidence suggests that the source may be in the seafood market of huanan in wuhan, china [ ] [ ] . the center for disease control and prevention (cdc) reported that the recent covid- is caused by betacoronavirus just like the previous two outbreaks of coronaviruses; mers and sars, the source of which is camels and bats, respectively [ ] . the first transmission of cov from animals to humans was notified in causing sars-cov with a % mortality rate [ ] . it was suggested that the virus needs some intermediate reservoir to infect humans efficiently as confirmed later by a thorough investigation revealing palm civets and raccoon dogs of the wet market carried sars-cov viral rna and might act like intermediate reservoirs [ ] . the covid- rna virus carries a high mutation rate and ability to transfer from person to person as compared to other coronaviruses. according to the world health organization (who), till rd february , the covid- affected a total of , individuals across globally of which, , were reported in china while were reported in other countries. the death toll in china is and deaths have been reported in the rest of the world. according to the reports till nd january , patients have been admitted to the hospital of which most of the patients were men and % of them had exposure to the huanan seafood market and the median age of patients was years [ ] . the health-care workers are also diagnosed with the infection including those working in similar wards [ ] . the time taken by covid- to infect other individuals is similar to that of sars. it is estimated that on average each infected person infects - persons and this occurrence increases two-folds, every . days [ ] . it was observed that people with mild infection are more actively spread the infection [ ] . due to critical nature of the outbreak, the virus was sequenced on urgent basis and the first sequence was available on th january, online at virological.org. it was noted that covid- has much resemblance to sars-cov at the genomic level [ ] . symptoms of covid- include fever, dry cough, shortness of breath and dyspnea, sore throat and leukopenia. to date no vaccine covid- is available and is need of an hour to develop a vaccine to prevent further spread of the disease. to this end, immuno-informatics can be applied to a complete protein data set of the virus for deep antigen analysis and thus can save time and cost for designing a vaccine against covid- . this will ease the early development of a vaccine and proposed design can be subjected immediately to experimental trials. the stepwise flow of the methodology followed to design a vaccine against covid- is illustrated in fig. . fig. . designed workflow for in silico vaccine engineering against covid- . the complete dataset of proteins available in (ncbi) [ ] coronavirus data hub (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?seqtype_s=nucleotide&viruslineage_ss =wuhan% seafood% market% pneumonia% virus,% taxid: &utm_campaign=w uhan_ncov&utm_source=insights&utm_medium=referral) was retrieved and subjected to screening phase to identify potential vaccine candidates. first, host non-similar proteins of the pathogen were filtered that show no homology to the human host (taxonomic id: ). proteins having sequence e score < . e - , bit score > , and sequence identity ≤ % were selected [ ] . again, a blastp search against the mouse (mus musculus, taxonomy id: ) was performed using host non-similar proteins keeping the input parameters e score cut-off . , bit score > , and identity < % [ ] . the screened mouse non-similar proteins were then subjected to tmhmm . [ ] and hmmtop . [ ] for observing the number of transmembrane helices. proteins having less than two transmembrane helices were subjected to spaan [ ] for predicting the adhesive proteins as they have the potential to facilitate attachment to the host tissues [ ] . selected vaccine candidates were then used in blastp tool to align the selected adhesive protein candidates with the probiotic bacteria proteome including three lactobacillus species: l. rhamnosus (tax id: ), l. johnsonii (tax id: ), and lactobacillus casei (tax id: ) to avoid accidental inhibition of the useful gut bacteria [ ] . selected vaccine candidates were then subjected to the immune epitope database (iedb) bepipred linear epitope prediction . [ ] [ ] [ ] . the threshold of . was used for the prediction of linear b-cells epitopes which were then utilized in t-cell epitopes mapping to identify subsequences with the potential to bind reference set of major histocompatibility complex (mhc) class i and ii alleles [ ] . epitopes were ranked according to their percentile score, the ones with the low percentile were considered as high affinity binders. the selected b-cell derived t-cell epitopes were then subjected to mhcpred . [ ] analysis for interpreting their binding affinity potential. the cut-off criterion was set to ic values < nm for drb* [ ] . following this, the virulentpred [ ] and vaxijen . [ ] were utilized to validate the virulence and antigenicity of the selected epitopes, respectively. allertop . [ ] was applied to remove the allergic epitopes. clc main workbench was employed to check the conservation of non-allergic epitopes required for designing an effective broad-spectrum vaccine. overlapping immunodominant epitopes were used to construct a multi-epitope peptide (mep) which is considered a promising strategy to stop viral infections [ ] . one of the key issues with the design of peptide vaccine is its weak immunogenicity that can be resolved by designing a mep with appropriate adjuvants [ ] . in the current study, mep was designed using aay linkers to combine screened multiple epitopes [ ] . to the n-terminal of mep, an adjuvant in the form of b subunit of cholera toxin was linked to the mep thus creating a multi-epitope peptide vaccine construct (mepvc) [ ] . the tertiary structure of the mepvc was created through a software called dpro of scratch protein predictor [ ] , i-tasser [ ] , and swiss-model [ ] . the best model was further loop modelled using galaxyloop [ ] and refined using galaxyrefine [ ] of galaxyweb. to improve the construct's stability, disulfide bonds were introduced in the structure [ ] using design . [ ] . the sequence of the mepvc was translated in reverse and then optimized for codon usage according to the escherichia coli, which will end up in the increased expression of the mepvc sequence cloned in the mentioned expression system [ ] . the entire activity was accomplished using java codon adaptation tool (jcat) server [ ] . in order to assess the expression of sequence that have been cloned, the gc content and codon adaptation index (cai) were measured. the value of cai is contemplated ideal [ , ] whereas the appropriate gc content should be fluctuated between - % due to favorable transcriptional and translational efficiencies [ ] . there were other input factors carefully calculated to prevent rho-independent transcription termination, the binding sites of the prokaryotic ribosome, and the cleavage sites of restriction enzyme. as the final step of this phase, the cloning of the engineered construct was carried out into pet- a (+) expression vector using snapgene (https://www.snapgene.com/). protparam tool [ ] was applied for analyzing the physical and chemical properties of mepvc such as amino acid composition, estimated half-life, instability index, extinction coefficient, theoretical pl, atomic composition, molecular weight, and grand average of hydropathicity (gravy) to assist experimental studies. instability index is one of the key parameters that is significantly considered as it helps in discarding the unstable protein candidates (protein instability index > ). in this step, the vaccine construct underwent immune response profiling and immunogenicity classification, which was done using the c-immsim server [ ] . in order to predict the immune epitopes, a position-specific scoring matrix (pssm) employed by c-immsim was used. whereas, the different machine learning procedures were used to forecast the immune connections. this server is concurrently used to execute an immune simulation for compartments such as bone marrow, tertiary lymph nodes, and thymus [ ] . default simulation parameters were used which are as follows: random seed ( ), simulation steps ( ), simulation volume ( ), host hla selection (a mhc class i a allele, b mhc class i b , dr mhc class ii drb _ allele), and time step of injection was set to . the technique of molecular docking was utilized to predict conformation of the mepvc with respect to a suitable innate immune receptor. this analysis plays a significant role to determine the high-affinity contacts amid the vaccine construct and the immune receptor. the pdb id: g a and tlr pdb id: a z were retrieved from the protein data bank (pdb) for the tlr and tlr , respectively. blind docking employed to calculate the regular pose of the vaccine construct with the mentioned receptors using an online patchdock server [ ] . the resulting structures were further refined using the fast interaction refinement in molecular docking (firedock) [ ] . the top ranked complex with the minimum global energy was selected and used to analyse the binding pose and intermolecular connections using ucsf chimera . . [ ] , discovery studio (ds) visualizer . . [ ] . , and visual molecular dynamics (vmd) . . software [ ] . in order to gain insights into the dynamics of the vaccine construct with the receptors, molecular dynamics (md) simulations have been carried out. the simulation analysis was also important to validate the exposure of epitopes towards the host structure for identification and handling of a substantial outcome. the md simulations took place in three different phases: system preparation, pre-processing and production [ ] with an assistant model building with energy refinement (amber) [ ] . the antechamber program [ ] was used to build the libraries and parameters for the tlr and vaccine construct. the tip p solvation box (size Å) was inserted to solvate the construct. to study the intermolecular interactions, force field, ff sb [ ] was used whereas, the system was neutralized by the addition of na + counter ions. in the second phase of md simulations, the energy minimization of the complexes was carried out. each complex was minimized using the following steps: the energy minimization of hydrogen atoms ( cycles), energy minimization of water box ( cycles, control of kcal/mol -Å on rest of the system), minimization of the whole atoms of the system ( cycles with the restraint of kcal/mol -Å on cα atoms), and the rest of the system was subjected to non-heavy atoms minimization with cycles and restraint of kcal/mol -Å . in the next step, the system was gradually heated from k to k with the time step of femtoseconds and restraints of kcal/mol -Å on cα atoms. in order to sustain the temperature of the system, langevin dynamics [ ] with the gamma value of . was castoff. the shake algorithm [ ] was used to put constraints on the hydrogen bonds of the system for heating. in the next step, systems were equilibrated for ps with a time step of fs followed by pressure equilibrium, which was attained using the npt ensemble with restraints of kcal/mol -Å on cα atoms. the same step was extended for ps with the scale down on restraints on carbon atoms. however, the step of system equilibration was carried out for a time scale of nanosecond followed by the production run of ns with a time scale of fs. for the production run, the berendsen algorithm [ ] with the nvt ensemble cast off with a cut-off of . Ǻ. simulation trajectories were calculated to investigate the strength of a complex via the cpptraj module [ ] of amber. however, the visualization of simulation trajectories was done with ucsf chimera [ ] , ds visualizer [ ] and vmd [ ] . in order to estimate the mmpbsa binding free energies for the receptors and multi-epitope peptide vaccine construct, the mmpbsa.py module [ ] of amber was castoff. the program generated the input files for the complex, receptor and mepvc molecule using the ante-mmpbsa.py module. to compute the variance between the solvated and un-solvated phases, frames of simulation trajectories were picked and analyzed [ ] . for the precise values of binding free energies, the two different conformations were matched to the binding energies of significant residues. to estimate the free binding energy of the anticipated complex, Δg bind, solv was resolved using the three equations given below: the net free binding energy was then decomposition into each residue to highlight the interacting and stable residues. the ncbi most recently dedicated a coronavirus disease data hub containing all nucleotide and protein sequences information published from across the world. in this study, we aimed at in silico prioritization potential vaccine candidates and designing a chimeric peptide vaccine for covid- based on all available protein sequences in the data hub. several bioinformatic and immunoinformatics techniques are employed with the aim to assist experimentalists in vaccine development against the virus. prioritization of potential vaccine candidates could help in minimizing time, labor cost and resources for developing and optimizing the success of getting an effective vaccine against the pathogen. in total, protein entries were retrieved (s- table ) and analyzed first for sequence homology with the human host proteome. this was significant to evaluate as homology between virus protein (s) to be used in vaccine designing and the host is likely to cause strong autoimmune reactions in the host [ ] . this check identified two proteins: (orf a polyprotein (accession id, yp_ ) and nsp (accession id, yp_ ) as host homologous thus discarded from further evaluations. experimental studies of the human vaccines are usually done in mice because of the many practical advantages they provide compared to vaccine research in higher animal models [ ] . the appropriateness of mice as an animal model for vaccine research is defined by its ability to reproduce relevant human physiology [ ] . considering this, a homology check was devoted to the pipeline and applied to the filtered human non-similar proteins ( in number) to ensure the selection of non-similar mice proteins. this assessment resultant into shortlisting of orf ab polyprotein (accession ids, qhq , qhd , qhr , qhr , qhq , qho , qhq , qhr , qho , qho , qhn , qhr , qhr , qhn , qhr , and qho ) and nsp (accession id, yp_ ) as mice similar proteins. the mice nonsimilar proteins will aid in discouraging false positive results during in vivo experimentations and accurate interpretation of immune protective efficiency of the prioritized vaccine candidates against the virus [ , ] . next, enumeration of transmembrane helices in the pooled mice non-similar proteins was accomplished. this transmembrane topology characterization is deemed vital in relatedness to the afterward experimental expression studies of the proteins. protein with transmembrane helices less than in numbers are often considered as best vaccine candidates as multiple helices make recombinant proteins purification and expression difficult in vaccine development [ ] . this result proteins spanning across five types, and include: orf a polyprotein (accession id, qhq , qhr , qhd , qhq , qhq , qho , yp_ , qhn , qhn , qho ), nsp (accession id, yp_ ), membrane glycoprotein (accession id, qhd , qhq , qhq , qhq , yp_ , qho , qhn , qhn , qho , qhr ), membrane protein (accession id, qhr , qhr , qhr , qhr , qhr ), matrix protein (accession id, qho , qho ), and nonstructural protein ns (accession id, qhr , qhr , qhr , qhr , qhr ) containing multiple helices therefore not proceeded further. the creation of adhesin-based vaccines is considered an attractive and effective strategy and is being explored as a solution to number of infectious pathogens [ ] . the idea behind exploiting adhesin for a vaccine is based on the promising preclinical findings. the aim is to confer protective immunity via two main mechanisms: (i) opsonization driven by opsonising antibodies that is capable of binding the target antigen as an immunological tag leading to activation of other components of the host immune system for enhance recognition of the pathogen and subsequent complement system activity and virus killing by phagocytosis, (ii) neutralization driven by adhesin-specific antibodies that block virus binding ability to host tissues. the adhesion probability computation revealed protein to have adhesion probability value greater than a threshold as tabulated in s- table and can be ideal putative vaccine candidates against covid- . the adhesin probability of protein ranges from . to . (mean, . ).antigenicity of proteins was predicted to reflect their ability of binding to products of adaptive immunity: antibodies or t-cell receptors. in total, proteins: nsp , nsp , nsp , c-like proteinase, spike glycoprotein, surface glycoprotein, and orf ab polyprotein were recognized as antigenic and scored higher than the threshold. coronavirus nsp suggested having diverse activities, including template-dependent rna polymerase activities, canonical rna-dependent rna polymerases, cofactor function of nsp for nsp -mediated rna-dependent rna polymerase activity, and metal ion-dependent rna ′ polyadenylation activities [ ] . nsp is a non-structural protein , key to coronavirus replication and is a single-stranded rna-binding protein [ ] . nsp is a critical cofactor that switches on multiple enzymes in replication cycle. it is known to interact with nsp and nsp subunits activating their respective ′- ′ exoribonuclease and ′-o-methyltransferase functions [ ] . the c-like proteinase is main cysteine protease and is nonstructural protein number (nsp ) and essential in mediating cleavage of nsp to nsp [ ] . the trimeric transmembrane coronavirus spike glycoprotein initiates infectious cycle by binding to a specific receptor on the host membrane followed by viral fusion [ ] . the surface glycoprotein was analyzed to be different in a sequence patch (leu -phe ) at the start of the spike glycoprotein. the orf ab is replicase polyprotein cleaved by papain-like protease and c-like protease at specific cleavage sites to yield to non-structural proteins (nsps) [ ] . the final numbers of potential vaccine candidates obtained in this step by step subtraction phase are presented in fig. . identification of epitopes in given antigens is vital for a number of practical reasons, including understanding etiology of a disease, monitoring of immune system, development of diagnostic assays, and epitope-based vaccines designing [ ] . from vaccine designing point of view, host adaptive immunity is highly specific and is able to recognize and destroy the invading pathogen [ ] . additionally, adaptive immunity is able to remember the pathogens, creating long-lasting pathogen-specific protective memory enabling stronger attacks against the pathogen reencountered on successive times [ ] . this arm of host immune system is driven by lymphocytes of two types: b and t-cells responsible for the humoral and cell-mediated immunity, respectively [ ] . both cells recognize pathogen molecular components called antigens. the antigens interact with specific receptors present on the surface of b and t-cells. the activation of both these cells required antigen recognition by these receptors, in addition, to the second activation signals from the innate system. the vaccine candidates prioritized in the first phase were deeply investigated for b-cell epitopes. different lengths of linear b-cell epitopes were predicted for each protein and only recurrent epitopes simultaneously predicted by different servers were selected for chimeric vaccine designing. the b-cell epitopes predicted for the vaccine candidates were in the following order: nine for nsp and c-like proteinase, five for nsp , eight for nsp , for spike glycoprotein and surface glycoprotein, and four for orf ab polyprotein| partial. these b-cells epitopes are recognized as solvent-exposed antigens through b-cell receptors (bcr) and upon activation, b-cells secrete antibodies. antibodies have different functions including neutralizing pathogens, toxins and labeling pathogens for destruction [ ] . the proteins were also analyzed for t-cell epitopes through very stringent criteria of p-value less than . . the epitopes were of different lengths and interact with several different alleles of mhc-i and mhc-ii. for nsp , epitopes predicted whereas nsp , nsp , c-like proteinase, spike glycoprotein, surface glycoprotein and orf ab polyprotein| partial protein were mapped for , , , , , and , respectively t-cell epitopes. these epitopes are presented on the surface through specific receptors known as t-cell receptor (tcr) allowing recognition of these antigens when displayed by antigen-presenting cells bound to mhc molecules [ ] . epitopes presented by mhc i are recognized by cd (cytotoxic t lymphocytes) [ ] whereas those presented by mhc ii are recognized by cd t-cells [ ] . the cd t-cells later become helper t-cells that amplify the immune responses against the pathogen. comparative analysis of the predicted epitopes was further carried out to select epitopes that are common to b-cell, cd t-cell and cd t-cell alleles in order to design a specific, effective, and strong vaccine. on this basis, three epitopes from nsp (drdaamqrk, qarsedkra, eqavangdsev), none for nsp and orf ab polyprotein| partial protein, four for nsp (gcscdqlrep, ylasggqpit, ylasggqpi, tvtpeanmdqesfg), three for c-like proteinase (edmlnpnyedl, kynyepltqdhv, eftpfdvvr), five for spike glycoprotein (rvystgsnvfq, vnnsyecdipi, ladagfikqygdclg, gqskrvdfc, rnfyepqiittd) and surface glycoprotein (rvystgsnvfq, vnnsyecdipi, ladagfikqygdclg, gqskrvdfc, rnfyepqiittd). the epitopes were then reevaluated in antigenicity check to make sure their binding potential of binding to immune cells. for nsp , drdaamqrk and qarsedkra were found antigenic with score higher than the default threshold of . whereas the third epitope eqavangdsev was found non-antigenic hence removed. all the four epitopes of nsp revealed non-antigenic therefore not processed further. in case of c-like proteinase, kynyepltqdhv was found non-antigenic whereas edmlnpnyedl and eftpfdvvr were antigenic therefore considered in afterward analysis. the spike glycoprotein contains epitopes vnnsyecdipi, and rnfyepqiittd as antigenic whereas none of the surface glycoprotein provided epitopes were antigenic. following, the affinity of the filtered antigenic epitopes for the most prevalent drb* allele in humans was evaluated through ic value and those with value < nm were classified as high affinity binders. all the pooled antigenic epitopes were found to have great ability of binding to the mentioned allele. similarly, these epitopes were evaluated in allergenicity and virulent potential check and only virulent and nonallergen were selected. virulent check was significant in ensuring selection of epitopes mediating infectious pathways in the host. the final selected epitopes that cleared all these checks are tabulated in table . a mep was constructed first comprising epitopes finalized in the previous phase. mep based vaccines are considered an ideal approach to prevent and treat viral infections [ ] . the epitopes shown in table were linked to each other through flexible aay linkers as such it allows efficient separation required for the effective working of each epitope. once the mep was designed, to its n-terminus an adjuvant of cholera toxin subunit b (ctb) was added [ ] . the schematic representation of the mepvc is shown in fig. . ctb is nontoxic part of cholera toxin and is considered an accelerator in protective immunity and a break in auto-immunity. it shows high affinity for monosialotetrahexosylganglioside displayed on variety of cell types, including gut epithelial cells, antigen-presenting cells (dendritic and macrophages) and b-cells [ ] . ctb is a preferred choice as an adjuvant because of its ability of self-expression in variety of organisms and can be coupled to antigens through several approaches involving chemical manipulation and genetic fusion resulting in strong immunological responses against the antigens to which it is attached. the vaccine construct was then used in a comparative d structure prediction to ensure confidentiality in selection of the most suitable model for the construct with minimum structural errors. several different physicochemical properties of the mepvc were deduced from its sequence. the vaccine construct is amino acids long with total number of atoms. the molecular weight of the construct is ideal i.e. . kd as small size construct is easy to handle and purify during experimental evaluation. the construct has instability index value of . , signifying its high stability. the aliphatic index computed for the construct is . , reflecting high thermostability. the estimated half-life in mammals, yeast, and escherichia coli is hours, > hours, and > hours, respectively. the grand average of hydropathicity (gravy) score is - . which highlights hydrophilic nature of the construct. the theoretical pi is . pointing to construct slightly acidic nature. the secondary structure elements of the vaccine construct can be divided into the following order: alpha helix ( . %), helix ( %), pi helix ( %), beta bridge ( %), extended strand ( . %), beta-turn ( . %), bend region ( %), and random coil ( . %). compared to the itasser, phyre , and swiss-model, the dpro predicted structure was determined as the most suitable structure based on the complete modeling of the given length of the amino acid sequence. loop modeling was done at leu -gln , ser -gln , glu -gln , glu -ser , val -thr , and glu -asp . the model was refined to minimum rmsd of . Å and molprobity score of . that is quite low compared to the original structure score of . , reflecting good quality of the modelled structure. similarly, the clash score in contrast to the original structure is . times lower demonstrating steric clash free structure. the galaxy energy of the structure is very stable (- . ) and ramachandran favored distribution increased from . to . %. the top refined models of the mepvc are tabulated in table . the d models of the vaccine construct after loop modelling and refinement is presented in fig. a . the overall z-score of the modelled structure is - and the score is within the range of same size proteins in the pdb illustrating good quality as depicted in fig. b . refinement of the structure ramachandran plot demonstrated the construct to contain . % of its residues in the most favored regions, while . %, . %, . % residues are in additional allowed region, generously allowed region, and disallowed regions, respectively (fig. c) . the overall average g-factor of the construct is . . ramachandran plot for the mepvc illustrating distribution of torsion angles (blue squares) comparative to the core (shown in red) and allowed (shown in brown) regions. residues in the generously allowed region are shown in dark yellow whereas disallowed regions are depicted in pale yellow. enhancing protein stability is important in many biomedical applications and is an appealing approach to emulate nature stabilizing molecular interactions [ ] . the covalent disulfide bonds provide substantial stability to target proteins and disulfide engineering had achieved considerable success in broad range of applications [ ] . in total, pairs of residues were selected for the purpose of disulfide engineering. these include met -ser , lys -ala , val -asp , ser -asp , thr -ser , val -gln , ala -ala , met -ala , ala -asp , leu -leu , and asn -asp . the average chi and energy value for the pairs is . (max, . and min, - . ) and . (max, . and min, . ). the disulfide engineered mepvc structure is presented in fig. . in the follow up experimental studies, the maximum expression of mepvc is highly desirable [ ] . one requirement for that is the codon usage of mepvc that must be adapted according to the expression system, for instance, here we used e. coli k as a mepvc expression system. the codon adaptation index (cai) and gc content revealed for the improved sequence are highly satisfactory with value of . and . , respectively strongly indicating high mepvc expression. the mepvc then enclosed on both sites by x histidine tag to ease its purification process and inserted at appropriate sites of pet a(+) vector as shown in fig. . fig. . in silico restriction cloning of the mepvc into pet a(+) vector. the insert is shown in red. molecular interactions and binding conformation of the designed mepvc with tlr and tlr innate immune receptors were deciphered via a protein-peptide docking approach. both tlr and tlr belong to toll-like receptor family of pattern recognition receptor and function to activate intracellular signaling nf-κb pathway and production of inflammatory cytokines responsible for the development of effective innate immunity [ , ] . these receptors recognize viral associated molecular patterns and induce the production of interferon leading to activation of strong host defense responses. also, the specific adaptive immunity takes time to establish against antigens therefore it's important to evaluate mepvc affinity for the innate immune receptors. in case of mrpvc-tlr complex, the patch dock predicted best solutions sorted based on the docking geometric shape complementarity score (s- table ) . a high score implies enhanced affinity of the interacting molecules and best docked conformations of the molecules with respect to each other. solution was visualized for docked conformations and intermolecular forces responsible for such high affinity of the molecules. the selection was based on the firedock analysis (s- table ) which is an efficient package for refinement and reassigning procedure of docking scores to rigid body docking solutions. the global binding energy of solution is better i.e. - . kj/mol compared to the rest of predicted solutions. the contribution to the total score form attractive van der waals (vdw) energy is - . kj/mol, repulsive (vdw) energy ( . kj/mol), hydrogen bond (hb) energy (- . kj/mol), and atomic contact energy (ace) ( . kj/mol). the mepvc, within Å, was noticed to posed right in the center of the tlr receptor (fig. ) interacting via hydrogen and hydrophobic bonds with his , asp , lys , glu , ser , phe , asp , asp , ser , ser , asp , ser , tyr , asp , asp , glu , tyr , phe , glu , tyr , arg , glu , his , lys , tyr , p , his , iso , gly , his , glu , pro , asp , phe , gln , and glu . similarly, among predicted mepvc-tlr complexes (s- table ), solution (s- table ) was affirmed as best with total global energy of - . kj/mol whereas the rest of complexes were noticed as highly unstable with score in positive. the attractive vdw, repulsive vdw, hb and ace contribution to the global energy is - . kj/mol, . kj/mol, - . kj/mol, and . kj/mol, respectively. visual analysis of the complex revealed binding of the mepvc at the interface of chains b and d (fig. ) . the mepvc is surrounded by chain b residues: pro , glu , ser , asp , lys , asp , asp , arg , arg , glu , pro , gln , asp , his , asp , ser , and lys and chain d residues: lys , phe , and asp . the dynamic simulations of the human immune system in response to the designed vaccine construct were deciphered through c-immsim server [ ] . the vaccine construct upon administration revealed to generate robust primary immune responses. as can be seen in fig. a that combine igm and igg antibodies has a titer scale close to , /ml followed by igm antibody (> antibody titer per ml). the combined igg and igg and igg were seen to generate high titer scale of around /ml, /ml, and respectively. the igg antibody response revealed to be low throughout post vaccine administration period. the dimerized soluble cytokine ifn-g produced against the antigen is > ng/ml (fig. b ). in silico simulation of the host immune system using mepvs as an antigen. a. antibodies titer (a) and cytokines and interleukins (b) in response to the antigen. the stability and dynamics of the designed vaccine construct ensemble docked to innate immune receptors were disclosed through -ns of md production run and interpreted through the root mean square deviation (rmsd) [ ] , root mean square fluctuation (rmsf) [ ] , the radius of gyration (rg) [ ] and beta factor (β-factor) [ ] assays as depicted in fig. . the average cα atomic distance over , frames of tlr -mepvc and tlr -mepvc was decoded through rmsd assay. an average rmsd of . Å (maximum, . Å) and . Å (maximum, . Å) were estimated for tlr -mepvc and tlr -mepvc, respectively. the tlr and tlr receptors are observed more compact than the mepvc and as a result, continuous movements of the vaccine construct through its length are noticed at the exposed regions though rooted stable at the docked position. this seems to be responsible for bringing small structural deviation as probed by rmsd. visual inspection of the trajectories illustrated no major global and local secondary structure conformation changes in the receptor tlr and tlr structure. the mepvc movements with respect to the tlr are shown at different snapshots as illustrated in fig. . the mepvc seems responsible for the deviations in the receptor tlr at positions glu -ser , hie -asn , gln -gln , gln -pro , and lys -lys . continuous flexibility of the loops of mepvc exposed region is observed and is highly flexible responsible for the movements of the mepvc at the docked site though rooted stably (fig. ) . hydrogen bonding results when a hydrogen atom attached to a highly electronegative atom is attracted by another electronegative atom [ ] . in a biological system, these hydrogen bonds are vital in determining specificity and directionality fundamental in molecular recognition [ ] . the patterns of hydrogen bonds for both complexes were illustrated in each frame within Å in order to probe the strength of intermolecular association across the simulation period. the maximum number of hydrogen bonds of mepvc with tlr and tlr are and , respectively demonstrating the high strength of interactions. the number of hydrogen bonds for both complexes are shown in fig. . fig. . the number of hydrogen bonds formed during simulation between mepvc and tlr and tlr receptors. in a protein molecule, salt bridges are formed between charged side chains of amino acids at neutral ph. the residues mainly involved in these interactions include negative full electron charge glutamine and aspartate and opposite positive full electron charge arginine and lysine [ ] . the presence of salt bridges between the interacting molecules is a clear sign of strengthening interaction stability. for tlr -mepvc complex high numbers of salt bridges were estimated within . Å between receptor glu , glu , arg , glu with mepvc lys , lys , glu , arg , respectively. in case of tlr -mepvc complex, receptor residues arg , asp , arg are involved in salt bridging with asp , arg and glu of the mepvc, respectively. the binding free energies of both tlr -mepvc and tlr -mepvc complexes were computed using continuum solvation mmpbsa and its complementary mmgbsa. the binding free energies of the vaccine construct for the receptors were estimated considering molecular mechanics energies as well as solvation energies. the net binding free energy for both complexes revealed to be very high illustrating the high interacting affinity of the molecules. for tlr -mepvc, the total free energy of binding is - . kcal/mol in mmgbsa while in mmpbsa it is - . kcal/mol ( after performing the immune system simulation to mepvc and a thorough peptide dynamics analysis, a novel mepvc is proposed as a probable solution to the widely spread viral coronaviruses outbreak. meanwhile, this seems necessary to have a deep down lesson as covid- viral outbreak is propagating to the human species on this part of the universe called earth. as we are already living under a serious threat of antibiotic and antimicrobial resistance [ ] which if ignored can cause havoc and the current spread of covid- is a clear example of even a viral resistance coming in line. referring to the introduction section where the probable cause of this viral attack is discussed, this is mandatory to mention that there is retaliation from animals and animal-associated viruses or bacteria observed against homo sapiens quite frequently in the recent past [ ] . this not only alarms the way of consuming animals in our food in the form of livestock but at the same time explains the advancing defense system of various species around. either it is horizontal gene transfer among bacteria or transient nature of viral transmission throughout the world as one way or the other this is a form of advanced defense of organisms. it is meant to state that even before and after the cognitive revolution, homo sapiens cannot be exempted from biological laws. while considering under the realm of these biological laws this is much expected from all other species to have the right of keeping and exercising a defense mechanism [ ] . instead of spending on wars among humans there is a dire need to be equipped for all wars to come against human species under the disguise of either environmental hazards or antimicrobial resistance. even viruses are finding new ways of infecting hosts. under this perspective the role of avian has been observed as much dominating in the recent past [ ] . after having insights from comparative advancing mechanism we mention here and thereafter the preliminary existence of a "theory of retaliation" on the basis of currently available facts and observations. this theory of retaliation will play its role in coming future. the continuous attacks in the form of outbreaks by them have raised many challenges and threats to human beings. a recent mechanism study by bazel et al reported h n influenza viruses is posing threat to human and animal health and is currently unclear what restricts these interspecies jumps on the host side. it is further signified that pb -f (a short viral peptide) assists h n bird influenza viruses to overcome a human restriction factor of the viral polymerase complex hax- . it is also evidenced that a functional pb -f aids in direct transmission of viruses from birds to humans [ ] . additionally, there is already a mechanism existing for the antimicrobial defense of avian eggs which illustrates their efficacy in defense mechanisms [ ] . furthermore, discovery of avian antimicrobial peptides, classified as bdefensins, present in chicken and turkey found active against bacteria, fungi, and yeast. this is another example of advance mechanism showed by avian and the possibility of a common ancestral gene between avian and other mammalian peptides seems obvious [ ] . who alarmed quite often that influenza viruses with a vast silent reservoir in aquatic birds are impossible to eradicate while avian influenza is proved to be a threat to human health [ ] . even having the phylogenetic identity between sars-cov- and sars-cov, some clinical characteristics differentiate sars-cov- from sars-cov, mers-cov, and seasonal influenza infections [ , ] . this leads to the fact that viruses are attacking humans with different clinical features every time. the complex relationships between the human and animal species never faced a halt in evolving giving rise to numerous infectious pathogens. whatsoever is the case, the dramatic impact of infectious diseases affecting the modern human population worldwide is evident and unexpected rise of coronavirus infections raised to while writing this research and getting beyond the normal control (https://www.nature.com/articles/d - - -w). after all we belong to the kingdom animalia with even ten on ten embarrassing similarities to chimpanzees [ ] . probable prevention in this context is to somehow avoid the excruciating utilization and consumption of other mammals as edibles from homo sapiens and stop becoming a reason for the extinction of certain species. avenging from these species from time to time by utilizing the microbes associated with them against human has become obvious and vaccine design and discovery is of utmost importance. this is crucial to strategize a preventive control not only for symptomatic relief but in general taking steps to prevent hunting and strategy required to maintain a balanced ecosystem where specifically avian will not dwell under threat by sapiens. the advent of various new mechanisms of viral survival has remained sapiens perplexed and a broader strategy is required to circumvent this problem. in this study, we used available immunoinformatics approaches for the purpose to prioritize potential vaccine candidates against covid- considering their ease of use in experimental investigations. bearing in mind, the wide immunological applications of peptide vaccines only highly antigenic, virulent, conserved and non-allergic epitopes targeted by both b and t-cells were disclosed. a multi-epitope peptide was constructed and an appropriate adjuvant was added to allow suitable delivery and efficient immune processing of the epitopes. these promising computational findings might deliver preliminary epitopes set for a vaccine against the covid- notwithstanding the experimental testing in appropriate animal models to unravel real effectiveness. meanwhile, probable prevention discussed in this study for homo sapiens is to avoid becoming a reason for the extinction of various species either by hunting and/or over utilizing other mammals as this may be a reason for resistance both from microbes and other animal species of this kingdom. there must be strategic studies keeping in view the advancements in defense mechanism of avian and avian related microbes avenging humans. preventive use of animals or avian in human diet and avoiding hunting can be preventive options self-defense in this connection and/or maintenance of a balanced ecosystem should be reinvestigated supplementary files s-table . complete protein data set of covid- at ncbi coronavirus data hub adhesive proteins shortlisted for covid- top patchdock solutions of the mepvc-tlr complexes s-table . firedock refinement of tlr -mepvc complexes top patchdock solutions of the mepvc-tlr complexes s-table . firedock refinement of tlr -mepvc complexes authors are highly grateful to the pakistan-united states science and technology cooperation program (grant no. pak-us/ / ) for granting financial assistance a novel coronavirus from patients with pneumonia in china clinical features of patients infected with novel coronavirus in return of the coronavirus: -ncov the continuing -ncov epidemic threat of novel coronaviruses to global health &#x ; the latest novel coronavirus outbreak in wuhan, china novel coronavirus ( -ncov) situation summary | cdc a novel coronavirus outbreak of global health concern sciencedaily: your source for the latest research news database resources of the national center for biotechnology information exoproteome and secretome derived broad spectrum novel drug and vaccine candidates in vibrio cholerae targeted by piper betel derived compounds combating tigecycline resistant acinetobacter baumannii: a leap forward towards multi-epitope based vaccine discovery predicting transmembrane protein topology with a hidden markov model: application to complete genomes the hmmtop transmembrane topology prediction server spaan: a software program for prediction of adhesins and adhesin-like proteins using neural networks adhesins as targets for vaccine development the immune epitope database (iedb): update improved method for predicting linear b-cell epitopes bepipred- . : improving sequencebased b-cell epitope prediction using conformational epitopes mhcpred: a server for quantitative prediction of peptide--mhc binding identification of putative vaccine candidates against helicobacter pylori exploiting exoproteome and secretome: a reverse vaccinology based approach virulentpred: a svm based prediction method for virulent proteins in bacterial pathogens vaxign: the first web-based vaccine design program for reverse vaccinology and applications for vaccine development allertop-a server for in silico prediction of allergens multi-epitope vaccines: a promising strategy against tumors and viral infections peptide vaccine: progress and challenges development of a multi-epitope peptide vaccine inducing robust t cell responses against brucellosis using immunoinformatics based approaches exploring dengue genome to construct a multi-epitope based subunit vaccine by utilizing immunoinformatics approach to battle against dengue infection scratch: a protein structure and structural feature prediction server i-tasser server for protein d structure prediction homology modelling of protein structures and complexes others, galaxy: a platform for interactive large-scale genome analysis galaxyrefine: protein structure refinement driven by side-chain repacking disulphide bonds and protein stability disulfide by design . : a web-based tool for disulfide engineering in proteins codon usage: nature's roadmap to expression and folding of proteins jcat: a novel tool to adapt codon usage of a target gene to its potential expression host novel immunoinformatics approaches to design multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein in-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases computational immunology meets bioinformatics: the use of prediction tools for molecular binding in the simulation of the immune system patchdock and symmdock: servers for rigid and symmetric docking firedock: fast interaction refinement in molecular docking rrdistmaps: a ucsf chimera tool for viewing and comparing protein distance maps discovery studio visualizer vmd: visual molecular dynamics a one-pot multicomponent facile synthesis of dihydropyrimidin- ( : h)-thione derivatives using triphenylgermane as a catalyst and its binding pattern validation antechamber: an accessory software package for molecular mechanical calculations the ff sb force field langevin stabilization of molecular dynamics a fast shake algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations on the berendsen thermostat cpptraj: software for processing and analysis of molecular dynamics trajectory data ucsf chimera-superimposing and morphing mmpbsa.py: an efficient program for end-state free energy calculations assessing the performance of the mm_pbsa and mm_gbsa methods. . the accuracy.pdf hosen, others, application of the subtractive genomics and molecular docking analysis for the identification of novel putative drug targets against salmonella enterica subsp. enterica serovar poona what is the predictive value of animal models for vaccine efficacy in humans? rigorous simian immunodeficiency virus vaccine trials can be instructive immunoinformatics design of a novel multiepitope peptide vaccine to combat multi-drug resistant infections caused by vibrio vulnificus adhesion, invasion and evasion: the many functions of the surface proteins of staphylococcus aureus the sars-coronavirus nsp + nsp complex is a unique multimeric rna polymerase capable of both de novo initiation and primer extension severe acute respiratory syndrome coronavirus nsp dimerization is essential for efficient viral growth lécine, others, coronavirus nsp , a critical co-factor for activation of multiple replicative enzymes others, biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus c-like proteinase cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer genetic diversity and evolution of sars-cov- fundamentals and methods for t-and b-cell epitope prediction panrv: pangenome-reverse vaccinology approach for identifications of potential vaccine candidates in microbial pangenome dna-based adaptive immunity protect host from infection-associated periodontal bone resorption via recognition of porphyromonas gingivalis virulence component adaptive immunity antibodies, viruses and vaccines the major histocompatibility complex and its functions the mhc class i antigen presentation pathway: strategies for viral immune evasion the ins and outs of mhc class ii-mediated antigen processing and presentation cholera toxin subunit b as adjuvant----an accelerator in protective immunity and a break in autoimmunity cholera toxin b: one subunit with many pharmaceutical applications protein disulfide engineering a comparative review of toll-like receptor expression and functionality in different animal species zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor tlr significance of root-mean-square deviation in comparing three-dimensional structures of globular proteins determination of ensemble-average pairwise root mean-square deviation from experimental b-factors radius of gyration as an indicator of protein structure compactness molecular dynamics simulation studies of novel $β$-lactamase inhibitor the hydrogen bond in molecular recognition hydrogen bonds in proteins: role and strength an intramolecular salt bridge linking tdp rna binding, protein stability, and tdp -dependent neurodegeneration the evolution of antibiotic resistance, science ( -. ) behavioural defences in animals against pathogens and parasites: parallels with the pillars of medicine in humans horizontal gene exchange in environmental microbiota the challenge of emerging and re-emerging infectious diseases h n influenza a virus pb -f relieves hax- -mediated restriction of avian virus polymerase pa in human lung cells antimicrobial activity of the anseriform outer eggshell and cuticle avian antimicrobial peptides: the defense role of $β$-defensins avian influenza virus (h n ): a threat to human health clinical characteristics of coronavirus disease in china he, others, differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development key: cord- -cbelsymu authors: gross, peter a. title: current recommendations for the prevention and treatment of influenza in the older population date: - - journal: drugs aging doi: . / - - sha: doc_id: cord_uid: cbelsymu influenza is a major cause of morbidity and mortality in the elderly. influenza vaccine is recommended for people aged years and older and those in long term care. currently only % of high risk persons are vaccinated. vaccination generally stimulates an adequate immune response, is well tolerated and is to be encouraged. prophylactic amantadine mg/day should be given for weeks with influenza vaccine in the aged population when they have not been previously immunised. broad application of these preventive measures would have a significant impact on reducing influenza prevalence in the elderly and other high risk groups. influenza is a major cause of morbidity and mortality in the elderly. influenza vaccine is recommended for people aged years and older and those in long term care. currently only % of high risk persons are vaccinated. vaccination generally stimulates an adequate immune response, is well tolerated and is to be encouraged. prophylactic amantadine mgjday should be given for weeks with influenza vaccine in the aged population when they have not been previously immunised. broad application of these preventive measures would have a significant impact on reducing influenza prevalence in the elderly and other high risk groups. if we were more aware of the full impact of influenza on our lives, we would be more concerned about preventing it. in the united states alone more than deaths are attributed to influenza virus in an average epidemic year (recommendations of the immunization practices advisory committee ). in some epidemics, the death toll has risen to more than odd. the majority of deaths - to % -occur in persons years of age and older . the estimated economic loss from influenza in the united states is estimated to be more than influenza is a major cause of morbidity and mortality in the elderly. influenza vaccine is recommended for people aged years and older and those in long term care. currently only % of high risk persons are vaccinated. vaccination generally stimulates an adequate immune response, is well tolerated and is to be encouraged. prophylactic amantadine mg/day should be given for weeks with influenza vaccine in the aged population when they have not been previously immunised. broad application of these preventive measures would have a significant impact on reducing influenza prevalence in the elderly and other high risk groups. if we were more aware of the full impact of influenza on our lives, we would be more concerned about preventing it. in the united states alone more than deaths are attributed to influenza virus in an average epidemic year (recommendations of the immunization practices advisory committee ). in some epidemics, the death toll has risen to more than odd. the majority of deaths - to % -occur in persons years of age and older . the estimated economic loss from influenza in the united states is estimated to be more than $usi billion each year (schoenbaum ) . in reality, the cost is probably to times greater. while much has been written on the cost-benefit ratio for influenza vaccine, in persons years of age and above the benefit clearly outweighs the cost. despite the favourable impression gained from economic models, however, individual patients and their physicians do not as a rule perceive the benefit. as a result, only a small percentage of the elderly population are vaccinated each year. what is influenza? is it easy to diagnose? how effective is influenza vaccine? are the side effects a frequent concern? what efforts have been and can be made to improve vaccine acceptance? these are some of the important questions we attempt to answer in this article. i influenza virus was first isolated from humans in the s (ruben ) . the clinical syndrome of a typical case of influenza begins with fever, cough and myalgias that last for a few days to a week . the clinical course may be complicated by laryngotracheobronchitis and pneumonia. secondary bacterial pneumonia may occur simultaneously or a few days later. hospitalisation is more likely to occur in individuals who are elderly or have pre-existing cardiac or pulmonary disease. children aged under years taking aspirin during the early phases of infection are more likely to get reye's syndrome. late complications of infection such as fatigue, chronic cough, and small-airway disease may last for weeks. making a diagnosis of influenza virus infection is difficult because the acute respiratory syndromes may be confused with other viral respiratory agents such as parainfluenza viruses, adenoviruses, respiratory syncytial viruses as well as the more common rhinoviruses and coronaviruses. laboratory i educational materials on influenza are available from the centers for disease control [technical information services, center for prevention services, mailstop e , cdc, atlanta, ga , usa; phone ( ) - as well as from state and local health departments in the usa. drugs & aging ( ) tests can distinguish among these agents, and rapid, sensitive tests are now available to diagnose influenza virus (reichelderfer et al. ) . a specific diagnosis of influenza can be made in a few hours to a few days, rather than in a week or more as was the case several years ago. while influenza virus infection may be difficult to distinguish from other respiratory viruses on clinical grounds, influenza virus is unique among the respiratory viruses for the high mortality associated with it. since the sixteenth century, people have recognised the increased mortality resulting from an influenza epidemic (monto ) . excess mortality from influenza has been documented since . on occasion, influenza outbreaks reach pandemic proportions, as occurred in . influenza epidemics occur almost every winter. quantifying the mortality from influenza begins with describing the known seasonal variation in mortality from all causes, as shown in figure . to reduce the chance of error a dashed line equal to . standard deviations (the 'epidemic threshold') is drawn above the known season incidence line. when the mortality rate exceeds the standard deviation, it is considered significant. this so-called excess mortality is invariably associated with an outbreak of influenza. excess deaths, therefore, are considered to be due to epidemics of influenza virus. quantifying morbidity from influenza is more difficult. while excess mortality occurs in the elderly and those with certain chronic diseases, morbidity is generally more common among healthy children and young adults. the severe morbidity associated with influenza, such as hospitalisation, is most common among the elderly and chronically ill. a number of studies have documented the toll influenza exacts among the infirm and the elderly. the most recent large scale study on influenza-related mortality is by barker and mullooly ( ) . in influenza a (h n ) epidemics, they report that to excess deaths occurred per persons. when they examined those aged $us billion each year (schoenbaum ) . in reality, the cost is probably to times greater. while much has been written on the cost-benefit ratio for influenza vaccine, in persons years of age and above the benefit clearly outweighs the cost. despite the favourable impression gained from economic models, however, individual patients and their physicians do not as a rule perceive the benefit. as a result, only a small percentage of the elderly population are vaccinated each year. what is influenza? is it easy to diagnose? how effective is influenza vaccine? are the side effects a frequent concern? what efforts have been and can be made to improve vaccine acceptance? these are some of the important questions we attempt to answer in this article.! influenza virus was first isolated from humans in the s (ruben ) . the clinical syndrome of a typical case of influenza begins with fever, cough and myalgias that last for a few days to a week . the clinical course may be complicated by laryngotracheobronchitis and pneumonia. secondary bacterial pneumonia may occur simultaneously or a few days later. hospitalisation is more likely to occur in individuals who are elderly or have pre-existing cardiac or pulmonary disease. children aged under years taking aspirin during the early phases of infection are more likely to get reye's syndrome. late complications of infection such as fatigue, chronic cough, and small-airway disease may last for weeks. making a diagnosis of influenza virus infection is difficult because the acute respiratory syndromes may be confused with other viral respiratory agents such as parainfluenza viruses, adenoviruses, respiratory syncytial viruses as well as the more common rhino viruses and coronaviruses. laboratory drugs & aging ( ) tests can distinguish among these agents, and rapid, sensitive tests are now available to diagnose influenza virus (reichelderfer et al. ) . a specific diagnosis of influenza can be made in a few hours to a few days, rather than in a week or more as was the case several years ago. while influenza virus infection may be difficult to distinguish from other respiratory viruses on clinical grounds, influenza virus is unique among the respiratory viruses for the high mortality associated with it. since the sixteenth century, people have recognised the increased mortality resulting from an influenza epidemic (monto ) . excess mortality from influenza has been documented since . on occasion, influenza outbreaks reach pandemic proportions, as occurred in . influenza epidemics occur almost every winter. quantifying the mortality from influenza begins with describing the known seasonal variation in mortality from all causes, as shown in figure . to reduce the chance of error a dashed line equal to . standard deviations (the 'epidemic threshold') is drawn above the known season incidence line. when the mortality rate exceeds the standard deviation, it is considered significant. this so-called excess mortality is invariably associated with an outbreak of influenza. excess deaths, therefore, are considered to be due to epidemics of influenza virus. quantifying morbidity from influenza is more difficult. while excess mortality occurs in the elderly and those with certain chronic diseases, morbidity is generally more common among healthy children and young adults. the severe morbidity associated with influenza, such as hospitalisation, is most common among the elderly and chronically ill. a number of studies have documented the toll influenza exacts among the infirm and the elderly. the most recent large scale study on influenza-related mortality is by barker and mullooly ( ) . in influenza a (h n ) epidemics, they report that to excess deaths occurred per persons. when they examined those aged years and older, the incidence of excess deaths increased to between and per . these estimates should in reality be even higher because pneumonia or influenza is often omitted from the diagnoses listed on death certificates . showed that the risk ofhospitalisation for acute respiratory disease (ard) during influenza epidemics was . per persons with high risk conditions for which influenza vaccine is indicated, compared to . per for persons without these conditions. chronic pulmonary disorders followed by chronic cardiac conditions were the most common highrisk conditions. renal failure, diabetes and long term care residence are other risk factors. for persons over years of age with a concomitant chronic pulmonary condition the hospitalisation rate for ard soared to . per . the peak of hospitalisations for ard typically followed by one week the peak of influenza virus isolations (perrotta et al. ) . the disease severity varies according to the influenza strain and the age ofthe patient. the h n subtype of influenza a causes the most severe ilness, while the hi n subtype of influenza a is associated with the mildest illness (monto et al. ) . type b influenza causes an illness intermediate in severity between the a subtypes. a third type of influenza, type c, is rarely responsible for epidemic disease. but why is influenza a problem year after year? becoming infected with each of the strains of influenza does not confer long-lasting immunity; in contrast, becoming infected naturally with, for example, measles does confer long-lasting immunity. why the difference? there is only i strain of measles and its nonsegmented genome preserves its prevention and treatment of influenza years and older, the incidence of excess deaths increased to between and per . these estimates should in reality be even higher because pneumonia or influenza is often omitted from the diagnoses listed on death certificates . showed that the risk of hospitalisation for acute respiratory disease (ard) during influenza epidemics was . per persons with high risk conditions for which influenza vaccine is indicated, compared to . per for persons without these conditions. chronic pulmonary disorders followed by chronic cardiac conditions were the most common highrisk conditions. renal failure, diabetes and long term care residence are other risk factors. for persons over years of age with a concomitant chronic pulmonary condition the hospitalisation rate for ard soared to . per . the peak of hospitalisations for ard typically followed by one week the peak of influenza virus isolations (perrotta et al. ) . the disease severity varies according to the influenza strain and the age of the patient. the h n subtype of influenza a causes the most severe ilness, while the hi n subtype of influenza a is associated with the mildest illness (monto et al. ) . type b influenza causes an illness intermediate in severity between the a subtypes. a third type of influenza, type c, is rarely responsible for epidemic disease. but why is influenza a problem year after year? becoming infected with each of the strains of influenza does not confer long-lasting immunity; in contrast, becoming infected naturally with, for example, measles does confer long-lasting immunity. why the difference? there is only i strain of measles and its nonsegmented genome preserves its singularity. influenza virus, on the other hand, has a segmented genome. the multiple rna segments -there are in all -predispose to the genetic instability of the influenza virus (table i, fig. ). the nucleotide sequences in the rna segments are inherently unstable, and when different strains of the same subtype infect the same host the rna segments can reassort, giving rise to progeny that are different from the parents. the naming of influenza viruses is straightforward. for example, influenza a/shanghai/ / (h n ) is so named because this strain is a type a influenza virus first isolated in shanghai in . it was the sixteenth isolate of the subtype h n detected in that year. haemagglutinin (h) and neuraminidase (n) proteins exist in types a, b, and c influenza virus. but only haemagglutinin and neuraminidase proteins of influenza a are sufficiently diverse to warrant subtype designations. for the type a influenza viruses, haemagglutinins (h\, h and h ) and neuraminidases (n \ and n ) have been described. currently, only subtypes of influenza a are circulating and causing disease in humans, and they are the h n and the hin\ subtypes. the genetic changes that occur have been referred to by the terms antigenic drift and antigenic shift. antigenic drift is a minor change that occurs (h n ) is an example of this . minor changes also occur when antigens are glycosylated and camouflage the previously exposed antigenic site. antigenic shift is a major change that occurs by gene reassortment resulting in a new subtype. the change from the influenza a (h n ) to the influenza a (h n ) subtype that occurred in is such an example. gene reassortment probably occurs when different subtypes coinfect the same host. serum antibody to the surface haemagglutinin and neuraminidase proteins is important in protection against infection and the development of disease. humoral antibody to the haemagglutinin component is the most critical, but the importance of cell-mediated immunity is less clear . the humoral immune response is highly specific for certain epitopes on the surface of the virus. the cellular response, in contrast, is less specific and more cross-reactive. the b cells which produce humoral antibody recognise antigenic determinants, or epitopes, adjacent in space but not in sequence, while t cells recognise epitopes adjacent in sequence but not necessarily in space. the singularity. influenza virus, on the other hand, has a segmented genome. the multiple rna segments -there are in all -predispose to the genetic instability of the influenza virus (table i, fig. ). the nucleotide sequences in the rna segments are inherently unstable, and when different strains of the same subtype infect the same host the rna segments can reassort, giving rise to progeny that are different from the parents. the naming of influenza viruses is straightforward. for example, influenza a/shanghai/ / (h n ) is so named because this strain is a type a influenza virus first isolated in shanghai in . it was the sixteenth isolate of the subtype h n detected in that year. haemagglutinin (h) and neuraminidase (n) proteins exist in types a, b, and c influenza virus. but only haemagglutinin and neuraminidase proteins of influenza a are sufficiently diverse to warrant subtype designations. for the type a influenza viruses, haemagglutinins (hi, h and h ) and neuraminidases (n i and n ) have been described. currently, only subtypes of influenza a are circulating and causing disease in humans, and they are the h n and the hin i subtypes. the genetic changes that occur have been referred to by the terms antigenic drift and antigenic shift. antigenic drift is a minor change that occurs . minor changes also occur when antigens are glycosylated and camouflage the previously exposed antigenic site. antigenic shift is a major change that occurs by gene reassortment resulting in a new subtype. the change from the influenza a (h n ) to the influenza a (h n ) subtype that occurred in is such an example. gene reassortment probably occurs when different subtypes coinfect the same host. serum antibody to the surface haemagglutinin and neuraminidase proteins is important in protection against infection and the development of disease. humoral antibody to the haemagglutinin component is the most critical, but the importance of cell-mediated immunity is less clear . the humoral immune response is highly specific for certain epitopes on the surface of the virus. the cellular response, in contrast, is less specific and more cross-reactive. the b cells which produce humoral antibody recognise antigenic determinants, or epitopes, adjacent in space but not in sequence, while t cells recognise epitopes adjacent in sequence but not necessarily in space. the implication here is that b cell epitopes are intact external molecules such as haemagglutinins, while t cell epitopes appear to be linear sequences exposed by enzymatic breakdown of the original molecule. a killed (inactivated) vaccine against influenza virus was first developed in the s. since then the manufacturing process has resulted in a more pure vaccine. the virus is grown in embryonated eggs. most of the egg protein is now eliminated and the side effects have been reduced markedly. killed vaccines are referred to as either whole virus or split virus: whole virus vaccines are inactivated by formalin; split or subunit vaccines use whole virus which is treated with a detergent to free up the haemagglutinin and neuraminidase subunits from the viral surface, then formalin is added. in children who have not previously been vaccinated, split virus vaccines cause fewer side effects such as fever and myalgias than whole virus vaccines. live attenuated vaccines have been in the development stages for over years and are currently being tested; they are not commercially available. a genetically stable method of production was the initial problem but this has since been solved. however, it has been difficult to show a clear advantage of live over killed vaccines. the currently licensed influenza vaccine contains strains: influenza a/shanghai/i / (h n ); influenza a/taiwan/i/ (hinl); and influenza b/ yamagata/i / . there are ilg of haemagglutinin for each strain in the vaccine. it should be given intramuscularly in the deltoid in adults and in the anterolateral thigh in infants and young children. administration in the buttock is not recommended because it may result in a poorer immune response due to inadequate absorption from the subcutaneous fat. the strains to be included in the vaccine change from year to year according to world health organization (who) recommendations. the primary target groups are as follows (recommendations of the immunization practices advisory committee ). i. persons aged years or older. . residents of nursing homes and other chronic-care facilities housing persons of any age with chronic medical conditions. of the pulmonary or cardiovascular systems, including children with asthma. . adults and children who have required regular medical follow-up or hospitalisation during the preceding year because of chronic metabolic diseases (including diabetes mellitus), renal dysfunction, haemoglobinopathies, or immunosuppression (including immunosuppression caused by medications). i. physicians, nurses and other personnel in both hospital and outpatient-care settings who have contact with high-risk persons in all age groups, including infants. . employees of nursing homes and chronic-care facilities who have contact with patients or residents. . providers of home care to high risk persons (e.g. visiting nurses, volunteer workers). other groups merit consideration (recommendations of the immunization practices advisory committee ): (a) in the general population, anyone who wishes to reduce the chance of acquiring influenza infection; (b) anyone who provides essential community services and persons living or working in an institutional setting; (c) persons infected with hiv should be vaccinated, although they may not respond as well if their disease is far advanced; and (d) foreign travellers should be immunised when travelling to the trop-prevention and treatment of influenza implication here is that b cell epitopes are intact external molecules such as haemagglutinins, while t cell epitopes appear to be linear sequences exposed by enzymatic breakdown of the original molecule. a killed (inactivated) vaccine against influenza virus was first developed in the s. since then the manufacturing process has resulted in a more pure vaccine. the virus is grown in embryonated eggs. most of the egg protein is now eliminated and the side effects have been reduced markedly. killed vaccines are referred to as either whole virus or split virus: whole virus vaccines are inactivated by formalin; split or subunit vaccines use whole virus which is treated with a detergent to free up the haemagglutinin and neuraminidase subunits from the viral surface, then formalin is added. in children who have not previously been vaccinated, split virus vaccines cause fewer side effects such as fever and myalgias than whole virus vaccines. live attenuated vaccines have been in the development stages for over years and are currently being tested; they are not commercially available. a genetically stable method of production was the initial problem but this has since been solved. however, it has been difficult to show a clear advantage of live over killed vaccines. the currently licensed influenza vaccine contains strains: influenza a/shanghai/i / (h n ); influenza a/taiwan/i/ (hin j); and influenza b/ yamagata/i / . there are j.lg of haemagglutinin for each strain in the vaccine. it should be given intramuscularly in the deltoid in adults and in the anterolateral thigh in infants and young children. administration in the buttock is not recommended because it may result in a poorer immune response due to inadequate absorption from the subcutaneous fat. the strains to be included in the vaccine change from year to year according to world health organization (who) recommendations. the primary target groups are as follows (recommendations of the immunization practices advisory committee ). i. persons aged years or older. . residents of nursing homes and other chronic-care facilities housing persons of any age with chronic medical conditions. . adults and children with chronic disorders of the pulmonary or cardiovascular systems, including children with asthma. . adults and children who have required regular medical follow-up or hospitalisation during the preceding year because of chronic metabolic diseases (including diabetes mellitus), renal dysfunction, haemoglobinopathies, or immunosuppression (including immunosuppression caused by medications). i. physicians, nurses and other personnel in both hospital and outpatient-care settings who have contact with high-risk persons in all age groups, including infants. . employees of nursing homes and chronic-care facilities who have contact with patients or residents. . providers of home care to high risk persons (e.g. visiting nurses, volunteer workers). other groups merit consideration (recommendations of the immunization practices advisory committee ): (a) in the general population, anyone who wishes to reduce the chance of acquiring influenza infection; (b) anyone who provides essential community services and persons living or working in an institutional setting; (c) persons infected with hiv should be vaccinated, although they may not respond as well if their disease is far advanced; and (d) foreign travellers should be immunised when travelling to the trop-ics at any time of the year or from the southern to the northern hemisphere between august and march and the northern to the southern hemisphere between april and september. the vaccine is contraindicated in persons with anaphylaxis to eggs and in those with an acute febrile illness. a protocol is available for giving vaccine to allergic patients at high risk of influenza complications (recommendations of the immunization practices advisory committee ). other vaccines can be given along with influenza vaccine, such as pneumococcal, measlesmumps-rubella, haemophilus injluenzae type b, and oral polio vaccines. pertussis vaccine, however, should not be given within days of influenza virus vaccine. for optimal effect, the vaccine should be given in november in the northern hemisphere or may in the southern hemisphere, before the winter influenza season begins. effective antibody levels develop within weeks in most previously immunised persons. the levels decline a few months later, when they may be suboptimal. studies investigating vaccine efficacy are difficult to conduct and to evaluate. they are hard to conduct because randomisation to control and vaccinated groups raises ethical questions in high risk patients. evaluation is difficult because other respiratory infectious agents also cause colds, pneumonia, occasionally death, and often result in hospitalisation -the criteria used to determine efficacy. vaccine immunogenicity and protective efficacy in the elderly have had mixed reviews. immunogenicity, the vaccine's ability to stimulate protective levels of antibody, appears to be adequate in many studies. protective levels of serum antibody are considered to be equal to or greater than the reciprocal of serum dilutions at : or : , depending on the dilution method. protective levels are usually achieved in to % of vaccine recipients. reviewed papers on this subject published between and . a total drugs & aging ( ) of comparisons were available in the papers. in , the immune response was superior in young subjects; in , the elderly responded better, and in , no significant difference was noted between the young and old subjects; in this instance, the lack of differences may have been due to a type error because the numbers studied were low. pointed out methodological limitations. the studies included, firstly, subjects with illnesses or taking drugs, which affect the immune system; secondly, patients immunised previously with influenza vaccine; and finally, those with protective antibody levels before immunisation. inclusion of patients with any of these factors would tend to underestimate the ability of elderly subjects to respond to immunisation. the association between increasing age and a poor immune response to influenza vaccine has thus not yet been convincingly made. perhaps a small subgroup of the elderly population respond poorly while most respond normally. this was originally suggested in a study by phair et al. ( ) examining the t cell response of infirm elderly to influenza antigens. more recently, in a study by , bedridden, infirm elderly patients appeared to respond less well to influenza vaccine than did healthy, ambulatory elderly patients. alternative approaches to the standard immunisation have been tried. two doses month apart did not improve the immune response , neither did and times the standard dose . from currently available information we can conclude that the recommended single standard dose of killed influenza vaccine will stimulate an adequate immune response in most elderly persons. finally, and most importantly, in a number of recent studies influenza vaccine has been shown to reduce mortality from influenza virus infection gross et al. a ). recent studies on the adverse effects of influenza vaccine show that the vaccine is typically well ics at any time of the year or from the southern to the northern hemisphere between august and march and the northern to the southern hemisphere between april and september. the vaccine is contraindicated in persons with anaphylaxis to eggs and in those with an acute febrile illness. a protocol is available for giving vaccine to allergic patients at high risk of influenza complications (recommendations of the immunization practices advisory committee ). other vaccines can be given along with influenza vaccine, such as pneumococcal, measlesmumps-rubella, haemophilus injluenzae type b, and oral polio vaccines. pertussis vaccine, however, should not be given within days of influenza virus vaccine. for optimal effect, the vaccine should be given in november in the northern hemisphere or may in the southern hemisphere, before the winter influenza season begins. effective antibody levels develop within weeks in most previously immunised persons. the levels decline a few months later, when they may be suboptimal. studies investigating vaccine efficacy are difficult to conduct and to evaluate. they are hard to conduct because randomisation to control and vaccinated groups raises ethical questions in high risk patients. evaluation is difficult because other respiratory infectious agents also cause colds, pneumonia, occasionally death, and often result in hospitalisation -the criteria used to determine efficacy. vaccine immunogenicity and protective efficacy in the elderly have had mixed reviews. immunogenicity, the vaccine's ability to stimulate protective levels of antibody, appears to be adequate in many studies. protective levels of serum antibody are considered to be equal to or greater than the reciprocal of serum dilutions at : or : , depending on the dilution method. protective levels are usually achieved in to % of vaccine recipients. reviewed papers on this subject published between and . a total drugs & aging ( ) of comparisons were available in the papers. in , the immune response was superior in young subjects; in , the elderly responded better, and in , no significant difference was noted between the young and old subjects; in this instance, the lack of differences may have been due to a type error because the numbers studied were low. pointed out methodological limitations. the studies included, firstly, subjects with illnesses or taking drugs, which affect the immune system; secondly, patients immunised previously with influenza vaccine; and finally, those with protective antibody levels before immunisation. inclusion of patients with any of these factors would tend to underestimate the ability of elderly subjects to respond to immunisation. the association between increasing age and a poor immune response to influenza vaccine has thus not yet been convincingly made. perhaps a small subgroup of the elderly population respond poorly while most respond normally. this was originally suggested in a study by phair et al. ( ) examining the t cell response of infirm elderly to influenza antigens. more recently, in a study by , bedridden, infirm elderly patients appeared to respond less well to influenza vaccine than did healthy, ambulatory elderly patients. alternative approaches to the standard immunisation have been tried. two doses month apart did not improve the immune response , neither did and times the standard dose . from currently available information we can conclude that the recommended single standard dose of killed influenza vaccine will stimulate an adequate immune response in most elderly persons. finally, and most importantly, in a number of recent studies influenza vaccine has been shown to reduce mortality from influenza virus infection gross et al. a) . recent studies on the adverse effects of influenza vaccine show that the vaccine is typically well tolerated. for example, margolis et a . ( a,b) reported that in elderly chronically ill patients fever and significant disability could not be attributed to the vaccine compared to a control group. a flu-like illness, however, was attributable to vaccine in . %. they concluded that the overall frequency of adverse effects was low. another area of concern is the effect of vaccine on drug metabolism. the mechanism postulated is vaccine suppression of oxidative hepatic metabolism. interferon production induced by vaccination is supposed to block the oxidative cytochrome p system, resulting in accumulation of certain drugs. metabolism of theophylline, chlordiazepoxide, lorazepam, warfarin, phenytoin and phenobarbital (phenobarbitone) has been studied, with variable results. for theophylline, in particular, most studies seem to indicate that the effect of vaccine is minor (gomolin et a . ; grabowski et a . ; meredith et a . ) . less than % of high risk persons are vaccinated annually. consequently many studies have been undertaken to determine the best strategies for immunising appropriate groups. the current strategies for implementing the influenza vaccine recommendations include immunising persons in the following settings (recommendations of the immunization practices advisory committee ). . in outpatient clinics and physicians' offices label medical records of high risk persons; use mail or telephone reminders; begin vaccinating in september (northern hemisphere) [buchner et a . ] and march (southern hemisphere). . in facilities providing episodic or acute care (e.g. emergency rooms, walk-in clinics) healthcare providers be aware of high risk groups; provide written information in appropriate languages. . in nursing homes and other residential long term care facilities vaccinate all residents with the prior aggreement of attending physicians; do not require individual physician orders; obtain permission for vaccination on admission to facility. . in acute-care hospitals encourage vaccination before discharge between september and april (northern hemisphere), and march to august (southern hemisphere) of all persons aged years or over, or with high risk conditions at any age . . in outpatient facilities providing continuing care to high risk patients (e.g. haemodialysis centres, hospital speciality care clinics, outpatient rehabilitation programmes); facilities providing services to people years and older (e.g. retirement communities, recreation centres); clinics and other centres providing healthcare for travellers; healthcare workers, visiting nurses and others providing home care to high risk persons provide educational materials; encourage vaccination; emphasise to staff in intensive care units, medical/surgical units, employees in nursing homes and long term care facilities; make vaccine available for all work shifts, including night and weekend. two antiviral agents are effective in the prevention and treatment of influenza type a infections -amantadine and rimantadine. only amantadine is currently available commercially in the united states. it has no effect against influenza type b (douglas ). the primary use of amantadine is in preventing influenza a infection. preventive efficacy varies between and over %. when influenza a is in the community, an unvaccinated elderly person should receive vaccine plus weeks of amantadine at a dose of mg/day (nicholson & wiselka ) . amantadine does not interfere with the immune response to vaccine, and after weeks the vaccine-induced immune response should be sufficient to prevent influenza. amantadine treatment should not be stopped if the high risk person cannot be vaccinated or is immunodeficient. it should be continued for as long as influenza is epidemic -usually about to weeks. amantadine has also been used for the treatment of influenza a. when the drug is started within hours of the onset of illness it produces prevention and treatment of influenza tolerated. for example, margolis et al. ( a,b) reported that in elderly chronically ill patients fever and significant disability could not be attributed to the vaccine compared to a control group. a flu-like illness, however, was attributable to vaccine in . %. they concluded that the overall frequency of adverse effects was low. another area of concern is the effect of vaccine on drug metabolism. the mechanism postulated is vaccine suppression of oxidative hepatic metabolism. interferon production induced by vaccination is supposed to block the oxidative cytochrome p system, resulting in accumulation of certain drugs. metabolism of theophylline, chlordiazepoxide, lorazepam, warfarin, phenytoin and phenobarbital (phenobarbitone) has been studied, with variable results. for theophylline, in particular, most studies seem to indicate that the effect of vaccine is minor . less than % of high risk persons are vaccinated annually. consequently many studies have been undertaken to determine the best strategies for immunising appropriate groups. the current strategies for implementing the influenza vaccine recommendations include immunising persons in the following settings (recommendations of the immunization practices advisory committee ). . in outpatient clinics and physicians' offices label medical records of high risk persons; use mail or telephone reminders; begin vaccinating in september (northern hemisphere) and march (southern hemisphere). . in facilities providing episodic or acute care (e.g. emergency rooms, walk-in clinics) healthcare providers be aware of high risk groups; provide written information in appropriate languages. . in nursing homes and other residential long term care facilities vaccinate all residents with the prior aggreement of attending physicians; do not require individual physician orders; obtain permission for vaccination on admission to facility. . in acute-care hospitals encourage vaccination before discharge between september and april (northern hemisphere), and march to august (southern hemisphere) of all persons aged years or over, or with high risk conditions at any age . . in outpatient facilities providing continuing care to high risk patients (e.g. haemodialysis centres, hospital speciality care clinics, outpatient rehabilitation programmes); facilities providing services to people years and older (e.g. retirement communities, recreation centres); clinics and other centres providing healthcare for travellers; healthcare workers, visiting nurses and others providing home care to high risk persons provide educational materials; encourage vaccination; emphasise to staff in intensive care units, medical/surgical units, employees in nursing homes and long term care facilities; make vaccine available for all work shifts, including night and weekend. two antiviral agents are effective in the prevention and treatment of influenza type a infections -amantadine and rimantadine. only amantadine is currently available commercially in the united states. it has no effect against influenza type b (douglas ). the primary use of amantadine is in preventing influenza a infection. preventive efficacy varies between and over %. when influenza a is in the community, an unvaccinated elderly person should receive vaccine plus weeks of amantadine at a dose of mg/day (nicholson & wiselka ) . amantadine does not interfere with the immune response to vaccine, and after weeks the vaccine-induced immune response should be sufficient to prevent influenza. amantadine treatment should not be stopped if the high risk person cannot be vaccinated or is immunodeficient. it should be continued for as long as influenza is epidemic -usually about to weeks. amantadine has also been used for the treatment of influenza a. when the drug is started within hours of the onset of illness it produces a therapeutic effect. the duration of illness decreases by %, virus shedding decreases more rapidly, and peripheral airway resistance is reduced faster. serum antibody still develops despite the use of amantadine. amantadine treatment for the acute illness should be continued for days. studies on the therapeutic efficacy of amantadine have been done in healthy adults; there are no data on its efficacy in high risk persons (recommendations of the immunization practices advisory committee ). resistance of influenza a virus to amantadine has been reported . the adverse effects of amantadine at a dose of mg/day involve the central nervous system (nervousness, anxiety, insomnia, difficulty concentrating and lightheadedness) and the gastrointestinal tract (anorexia and nausea). they usually decrease or stop after the first week of use or when amantadine is discontinued. using a smaller dose, mg/day, reduces the adverse effects, apparently without diminishing efficacy. the manufacturer's recommendations should be read carefully to adjust the dose for age, renal function, weight, and interactions with other drugs and diseases (see table ii) . rimantadine is a structural analogue of amantadine. it has advantages over amantadine: it has fewer central nervous system side effects at doses of mg/day, and, because most of the drug is drugs & aging ( ) metabolised, elimination from the body is not dependent on renal excretion. the use of influenza vaccine in the elderly is no longer an option"to be exercised only occasionally. administration of influenza vaccine and the use of amantadine when indicated should become a standard part of the care given our elderly population (nicholson ). a therapeutic effect. the duration of illness decreases by %, virus shedding decreases more rapidly, and peripheral airway resistance is reduced faster. serum antibody still develops despite the use of amantadine. amantadine treatment for the acute illness should be continued for days. studies on the therapeutic efficacy of amantadine have been done in healthy adults; there are no data on its efficacy in high risk persons (recommendations of the immunization practices advisory committee ). resistance of influenza a virus to amantadine has been reported . the adverse effects of amantadine at a dose of mg/day involve the central nervous system (nervousness, anxiety, insomnia, difficulty concentrating and lightheadedness) and the gastrointestinal tract (anorexia and nausea). they usually decrease or stop after the first week of use or when amantadine is discontinued. using a smaller dose, mg/day, reduces the adverse effects, apparently without diminishing efficacy. the manufacturer's recommendations should be read carefully to adjust the dose for age, renal function, weight, and interactions with other drugs and diseases (see table ii) . rimantadine is a structural analogue of amantadine. it has advantages over amantadine: it has fewer central nervous system side effects at doses of mg/day, and, because most of the drug is drugs & aging ( ) metabolised, elimination from the body is not dependent on renal excretion. the use of influenza vaccine in the elderly is no longer an option to be exercised only occasionally. administration ~f influenza vaccine and the use of amantadine when indicated should become a standard part of the care given our elderly population (nicholson ) . experiences in the use and efficacy of inactivated influenza vaccine in nursing homes impact of epidemic type a influenza in a defined adult population underestimation of the role of pneumonia and influenza in causing excess mortality effectiveness of inactivated influenza vaccine among non-institutionalized elderly persons resistance of influenza a virus to amantadine and rimantadine: results of one decade of surveillance antibody induced by influenza vaccines in the elderly: a review of the literature influenza vaccination in community elderly: a controlled trial of postcard reminders clinical manifestations and consequences of influenza antimicrobial agents: antiviral agents immunizations for health care workers and patients in hospitals survey of underlying conditions of persons hospitalized with acute respiratory disease during influenza epidemics in houston lack of effect of influenza vaccine on theophylline levels and warfarin anticoagulation in the elderly the effect of split virus influenza vaccination on theophylline pharmacokinetics association of in fluenza immunization with reduction in mortality in an elderly references experiences in the use and efficacy of inactivated influenza vaccine in nursing homes impact of epidemic type a influenza in a defined adult population underestimation of the role of pneumonia and influenza in causing excess mortality effectiveness of inactivated influenza vaccine among non-institutionalized elderly persons resistance of influenza a virus to amantadine and rimantadine: results of one decade of surveillance antibody induced by influenza vaccines in the elderly: a review of the literature influenza vaccination in community elderly: a controlled trial of postcard reminders clinical manifestations and consequences of influenza antimicrobial agents: antiviral agents immunizations for health care workers and patients in hospitals survey of underlying conditions of persons hospitalized with acute respiratory disease during influenza epidemics in houston lack of effect of influenza vaccine on theophylline levels and warfarin anticoagulation in the elderly the effect of split virus influenza vaccination on theophylline pharmacokinetics association of in fluenza immunization with reduction in mortality in an elderly population: a prospective study immunization of elderly people with high doses of influenza vaccine relation of chronic disease and immune response to influenza vaccine in the elderly immunization of elderly people with two doses of influenza vaccine effect of influenza vaccine on serum anticonvulsant concentrations epidemiologic implications of changes in the influenza virus genome t-cell recognition of influenza viral antigens impact of influenza epidemic on mortality in the united states from frequency of adverse reactions to influenza vaccine in the elderly frequency of adverse reactions after influenza vaccine effects of influenza virus vaccine on hepatic drug metabolism infleunza: quantifying morbidity and mortality tecumsah study of illness. xiii: influenza infection and disease influenza vaccination and the elderly amantadine for influenza a acute respiratory disease hospitalizations as'll.ijleasure of impact of epidemic influenza failure to respond to influenza vaccine in the aged: correlation with b-cell number and function prevention and control of influenza. and addendum clarification economical laboratory support system for influenza virus surveillance prevention and control of influenza: role of vaccine economic impact of influenza: the individual's perspective prevention and treatment of influenza population: a prospective study immunization of elderly people with high doses of influenza vaccine relation of chronic disease and immune response to influenza vaccine in the elderly immunization of elderly people with two doses of influenza vaccine effect of influenza vaccine on serum anticonvulsant concentrations epidemiologic implications of changes in the influenza virus genome t-cell recognition of influenza viral antigens impact of influenza epidemic on mortality in the united states from frequency of adverse reactions to influenza vaccine in the elderly frequency of adverse reactions after influenza vaccine effects of influenza virus vaccine on hepatic drug metabolism key: cord- -y l xg authors: gruber, marion f.; marshall, valerie b. title: regulation and testing of vaccines date: - - journal: plotkin's vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: y l xg nan vaccines are one of the most significant achievements of science and public health. as a result of successful vaccination programs and campaigns, many vaccine-preventable diseases are now uncommon in the united states. vaccines for prevention of infectious diseases are regulated by the u.s. food and drug administration (fda) and the legal framework for regulation is derived from section of the public health service act and from certain sections of the federal food, drug, and cosmetic act (fd&c act). , the fd&c act defines drugs, in part, by their intended use as "articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease." thus, vaccines are a unique class of pharmaceutical products that meet the statutory definition of both a drug and biological product. prophylactic vaccines differ from many other drugs and biologicals primarily in how they are administered to a large population, in particular, young healthy people to prevent rather than treat disease, their mechanism of action, and their risk/benefit profile. although subject to the same regulations as other biological products, vaccines are inherently more difficult to develop, characterize, and manufacture than most pharmaceutical products. current u.s. licensed vaccines are listed in tables . and . . regulation of biologics has historically been initiated in response to issues of safety. over time, legislative authorities have evolved to strengthen and modernize the regulation of vaccines and other biologics. prior to , manufacturing and product standards for biologics were not federally mandated. however, in , the u.s. congress passed an act to regulate the sale of viruses, serums, toxins, and analogous products (later referred to as the biologics control act) following the deaths of children who received contaminated products. this act authorized the hygienic laboratory of the public health and marine hospital service to issue regulations that governed all aspects of commercial production of vaccines, serums, toxin, and antitoxins and similar products with the objective of ensuring their safety and purity. the regulations under this legislation contained the primary concepts for regulation of biologicals, such as labeling, mandatory facility inspections, and batch-certification guidelines. in , the hygienic laboratory was reorganized, expanded, and renamed the national institutes of health (nih). in , congress recodified the biologics control act as part of the u.s. public health service act (phs act) of . the phs act incorporated the biologics control act into section of the phs act ( u.s.c. ). as with the act, the phs act focused primarily on extensive control over manufacturing methods to ensure safety and purity. unique to the phs act was congress' explicit addition of the requirement that biologics manufacturers demonstrate potency as a measure of clinical usefulness. the phs act created the laboratory of biologics control to facilitate testing and licensure of biologicals products and manufacturing establishments. after , the authority of the laboratory of biologics control was derived from section of the phs act and from certain sections of the fd&c act. in , the laboratory of biologics control joined the nih division of infectious diseases and division of tropical diseases to form the national microbiological institute (later renamed the national institute of allergy and infectious diseases). administrative authority for regulation of biologics was originally granted to the national microbiological institute. although important regulations had been enacted to improve product safety, by the s, the only legal requirement for vaccine licensing was submission of written protocols for vaccine production and safety testing to the laboratory of biologics control. regulations were dramatically expanded in , when more than cases of polio were attributed to incompletely inactivated polio vaccine manufactured by the cutter laboratories. as a result of the "cutter incident," administrative authority for the regulation of biologicals was transferred by congress to the division of biologics standards, a newly created division within the nih. regulations were strengthened that required more precise experimental testing to assess the safety of vaccines. congress passed the consumer safety act of and transferred regulatory authority from nih to fda for the administration of the phs act. in , the division of biologics standards, which was charged with administering and enforcing section of the phs act, was transferred by the secretary of health, education and welfare to the fda, and became the bureau of biologics. once administrative responsibility for the regulation of biologicals was transferred from nih to fda, the fda announced its intention to require that all new biologicals satisfy the additional standards of safety and efficacy mandated in the drug amendments act of . this resulted in the transfer of the regulations pertaining to biologics from part of chapter i of title atric subpopulation for which the product is safe and effective. the fdaaa of includes titles that added many new provisions to the fd&c act. it reauthorized and amended several drug and medical device provisions, and provided the fda with additional responsibilities and new authorities. the provisions of fdaaa that have had a significant impact on the regulations of vaccines and the review process are contained in title iv, the prea, and title ix, enhanced authorities regarding postmarket safety of drugs. the fdaaa reauthorized and revised the prea, primarily to enhance fda oversight and applicant accountability for the agreed-upon the pediatric research equity act (prea) of amended the fd&c act by adding section (b) to address product development for pediatric subjects from birth to years of age. it requires that manufacturers submit a pediatric assessment with every application submitted under section of the fd&c act or section of the phs act for a new active ingredient, new indication, new dosage form, new dosing regimen, or new route of administration unless the applicant has obtained a waiver or deferral from the fda. the pediatric assessment must contain data adequate to assess the safety and effectiveness of the drug or the biological product for the claimed indications in all relevant pediatric subpopulations and data to support dosing and administration for each pedi- section of the fd&c act or section of the phs act ( usc § ) . section of the fdaaa also created new sections - and (o)( ) of the fd&c act, authorizing the fda, under certain circumstances, to require risk evaluation and mitigation strategies and safety-related labeling changes, respectively. the fdaaa also specifies adverse event reporting requirements for products with labeling changes that are a result of a pediatric assessment. specifically, during the months from the date that such a labeling change is made, all adverse event reports are reviewed by the fda pediatric advisory committee. following review, the pediatric advisory committee makes recommendations regarding whether pediatric assessments. of note, a new provision directed the fda to establish the pediatric review committee (perc), an internal review committee with pediatric expertise. this committee is required to provide consultation to fda review divisions on all pediatric plans and assessments and on all deferral and waiver requests. thus, early in the application review process the review team must assess whether prea applies. if prea applies, then a pediatric assessment must be presented to the perc. section of title ix of the fdaaa authorizes the fda to require certain postmarketing studies and clinical trials for prescription drug and biological products approved under the fda should take action in response to such reports and whether the current pharmacovigilance plan is adequate. the fdasia was signed into law in and expanded the fda's authority by strengthening the agency's ability to safeguard and advance public health by promoting innovation, increasing stakeholder involvement in fda processes, and enhancing the safety of the drug supply chain. in addition to reauthorizing the prescription drug and medical device user fee programs, fdasia established new user fee programs for generic drugs and biosimilar biological products. the provisions of fdasia that impact the regulation of vaccines are contained in pediatric drugs and devices (title v) and drug approval and patient access (title ix). title ix expands the scope of products that qualify for accelerated approval and creates a new "breakthrough therapy" program, among other things (see below). fdasia also revised prea to include a provision that requires vaccine manufacturers to submit a pediatric study plan early in the drug development process. this initial pediatric study plan must contain an outline of the pediatric study or studies that the sponsor plans to conduct including, to the extent practicable, study objectives and design, age groups, relevant end points, and statistical approach, as well as any request for a deferral, partial waiver, or waiver. the fda internal perc must be consulted for the review of the initial study plan, the agreed initial pediatric plan, and certain amendments to such plans. both sponsors and the fda must comply with prescribed timelines regarding submission, review, responses, and agreements reached apply to vaccines, regardless of their indication or intended target population. section of the phs act ( usc § ) states that a bla can be approved based on a demonstration that "…(a) the biological product that is the subject of the application is safe, pure, and potent; and (b) the facility in which the biological product is manufactured, processed, packed, or held meets standards designed to assure that the biological product continues to be safe, pure, and potent…" some of the more pertinent operational definitions for biologics contained in the statutes and cfr are as follows: • section of the phs act defines a biological product as any virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood component or derivative, allergenic product, or analogous product applicable to the prevention, treatment, or cure of diseases or conditions of human beings. thus, vaccines clearly are regulated as biological products. • safety is defined as the relative freedom from harmful effect to people affected directly or indirectly by a product when prudently administered, taking into consideration the character of the product in relation to the condition of the regarding the pediatric study plan, which are also described in applicable fda guidance. of note, these amendments to the fd&c act also renewed the prescription drug user fee act (pdufa) that was first enacted in , and authorized the fda to collect user fees from companies. these fees enabled fda to hire additional reviewers and support staff and upgrade its information technology systems. in return for these additional resources, the fda agreed to certain review performance goals, such as completing reviews of new drug applications and biologics license applications (blas) and taking regulatory actions on them in predictable timeframes. these changes revolutionized the drug approval process in the united states and enabled fda to speed the application review process for new drugs and biologics without compromising the fda's high standards for demonstration of safety, efficacy, and quality. the fda's cber is the national regulatory authority in the united states charged with the regulation of biological products including vaccines. these regulations cover not only the methods and establishment standards pertaining to the manufacture of a biological product to assure that the product is safe and meets the quality and purity characteristics that are claimed by the manufacturer, but also requirements for performing clinical trials (i.e., cfr § ). a single set of basic regulatory requirements applies to all vaccines, regardless of the technology used to produce them. the regulatory approval criteria contained in title cfr also recipient at the time. thus, the property of safety is relative and cannot be ensured in an absolute sense. • purity is defined as the relative freedom from extraneous matter, regardless of whether it is harmful to the recipient or deleterious to the product. usually, the concepts of purity and safety coincide; purity most often relates to freedom from such materials as pyrogens, adventitious agents, and chemicals used in manufacture of the product. • potency is defined as the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through administration of the product in the manner intended, to effect a given result. potency, as thus defined, is equivalent to the concept that the product must be able to perform as claimed, and, if possible, this must correspond with some measurable effect in the recipient or correlate with some quantitative laboratory finding. • standards mean specifications and procedures applicable to an establishment or to the manufacture or release of products that are designed to ensure the continued safety, purity, and potency of biological products. the word standard is also used with a secondary meaning, usually in the sense of a reference preparation, such as a bacterial or viral antigen that can be used in evaluating potency or, in some cases, safety and purity. • the regulations regarding biological products, in addition, define effectiveness as the reasonable expectation that, in a significant proportion of the target population, pharmacologic or other effects of the biological product, when administered under adequate directions for use and warnings against unsafe use, will serve a clinically significant function in the diagnosis, cure, mitigation, treatment, or prevention of disease in humans. • cgmps define a quality system that manufacturers use as they build quality into their products. the regulations outline the minimum manufacturing, quality control, and quality assurance requirements for the preparation of a drug or biological product for commercial distribution. for example, approved products developed and produced according to cgmps are safe, properly identified, of the correct strength, pure and of high quality. the fda also periodically publishes various guidelines and guidance documents with regard to the manufacture and clinical evaluation of biologicals. these documents published by the fda do not have the force of law, but are intended to provide useful and timely recommendations; table . lists those applicable to vaccines. guidance documents are particularly useful as a means for the fda to provide recommendations that are current with areas of rapidly progressing science, and for specifying a degree of detail beyond what is included in the regulations. in the past few years, several fda regulations and guidance documents have had a direct impact on the review of vaccines for licensure by the fda, such as the "guidance for industry: expedited programs for serious conditions-drugs and biologics ( )." some of these regulations and guidance documents evolved from an effort to streamline the regulatory process, while others-such as "guidance the fda's cber regulatory review staff consists of an internal multidisciplinary team of scientists, medical officers, and regulatory and public health professionals. to stay current on scientific advances and biotechnologies, the team is involved in a dynamic exchange of information with the outside scientific community through laboratory research and collaborations, participation in workshops and seminars, and engagement with national partners. the fda also relies on the expertise of formal advisory committees, which include experts in the fields of vaccinology, microbiology, infectious diseases, immunology, biostatistics, epidemiology, and clinical trial design. in addition, cber works closely with its counterparts in other u.s. government agencies within the department of health and human services and the u.s. public health service (phs), such as the national vaccine program office, the centers for disease control and prevention (cdc), the nih, and the health resources and services administration. the cdc is responsible, among its other duties, for epidemiologic surveillance of disease and for support of immunization programs. its advisory committee on immunization practices makes recommendations for vaccine use. the director of the national vaccine program office coordinates vaccine efforts throughout the phs and other governmental agencies. the nih is responsible for conducting and providing funds for a wide variety of biomedical research. the health resources and services administration is responsible for managing the national vaccine injury compensation program. other important collaborators inside the u.s. government involved in vaccine activities include the u.s. department of defense, and the department of veterans affairs. additionally, cber works closely with its multilateral partners, notably the pan american health organization and the world health organization (who), to provide assistance in regulatory capacity building. cber has been very active in supporting the who's developing countries' vaccine regulators network and the who's african regional office-led african vaccine regulatory forum. as a who collaborating center, cber contributes to a range of activities, including establishment of physical and written standards, implementation of who international standards, strengthening global regulatory systems, and serving as the national regulatory authority (nra) of reference for who's vaccine prequalification program. cber experts also contribute as members of several who advisory committees including the global advisory committee on vaccine safety, the polio research committee, the hiv vaccine advisory committee, and the expert committee on biological standardization. cber is an essential regulatory laboratory in who's global influenza surveillance and response system. in addition, the fda has confidentiality arrangements with many nras around the globe, allowing it to share information as part of its regulatory processes. these arrangements have strengthened interactions between the regulatory authorities and have contributed to improving the promotion and protection of public health globally. the regulatory review in cber incorporates a managed and integrated regulatory process that is continuous from the regulatory requirements for biological products cover the entire life-cycle of the product from the pre-ind stage, through the premarketing (consisting of the various ind phases and prelicensure) and postmarketing stages. the pre-ind stage consists of laboratory development, preclinical testing of candidate vaccines, and development of the manufacturing process. the clinical development of a new drug in the united states usually begins with a sponsor approaching the fda for permission to conduct a clinical study with an investigational product through submission of an ind application form. these requirements can be found in the ind regulations. sponsors are encouraged to request a pre-ind meeting with fda to discuss preclinical studies, clinical study design, and data requirements that require resolution prior to the initiation of clinical trials. in the application, the sponsor (a) describes the composition, source, and method of manufacture of the product and the methods used in testing its safety, purity, and potency; (b) provides a summary of all laboratory and preclinical animal testing; and (c) provides a description of the proposed clinical study and the names and qualifications of each clinical investigator. the fda has a maximum of days to review the original ind application and determine whether study participants will be exposed to any unacceptable risks. as part of the ind process, each clinical investigator files information describing the investigator's qualifications for performing clinical trials, details of the proposed study, and assurance that a number of conditions specified by the regulations will be met. a signed informed consent must be obtained from each study participant. approval for the study must be obtained in advance from a local institutional review board. the regulations also cover the evaluation of the preclinical laboratory animal studies undertaken to support the use of the product in humans. only licensed vaccines may be shipped from one state to another; however, during the premarketing phase, interstate shipment of products for investigational use is allowed under the law and regulations. there are generally three separate phases in the clinical evaluation of experimental biologicals at the premarketing stage ( fig. . ). these phases may overlap, and the clinical testing may be highly iterative because discovery to postmarketing, and is designated as the managed review process. cber's managed review process is designed to effectively review all regulatory submissions to reach informed evidence-based regulatory decisions to ensure safe and effective biological products. cber uses a team-based approach with substantive involvement of discipline team leaders and management. the managed review process begins when a sponsor requests a pre-investigational new drug (ind) meeting that may result in the submission of an ind and, eventually, a bla. the review process in cber begins with an initial review of a submission for scientific content and compliance with the regulations. members of a multidisciplinary review team are selected based on their expertise with the type of product and its method of manufacture. it is the responsibility of cber's review component to evaluate submissions and recommend appropriate regulatory action to facilitate the approval of safe and effective biological products. the review includes an evaluation of chemistry, manufacturing, and controls information; the manufacturing facility and equipment; preclinical and clinical data on the safety, efficacy, pharmacology, and toxicology; the suitability of clinical trial design; and analysis of clinical data derived from such trials. in addition, reviewers monitor for conformance with fda regulations in all phases of biological product development, including postmarketing. cber scientists also perform research in the areas of statistical and epidemiologic analysis, clinical trial design, and chemistry, manufacturing and control specific to product issues, and contribute to policy development. surveillance activities are performed to ensure that the safety of biological products is not compromised. these activities ensure the rapid availability and approval of safe and effective biological products. the fda encourages meetings with sponsors to the extent that they aid in the evaluation of the vaccine and in resolving scientific issues concerning the product. the general principle underlying the conduct of such meetings is that there should be free, full, and open communication about any scientific or medical question that may arise. agreements reached at pdufa meetings (e.g., pre-ind, ind, pre-bla, and bla meetings) are recorded in official minutes taken by fda personnel and provided to the sponsor. they serve as a permanent record of any agreements reached. detailed information on the conduct of regulatory meetings is described in seven-valent polysaccharide conjugate vaccine (pcv ) was successful in preventing a low incidence of invasive pneumococcal disease caused by the streptococcus pneumoniae capsular serotypes included in the vaccine enrolled close to , children who were randomized equally to receive the pneumococcal conjugate vaccine or an unrelated control vaccine. in contrast, licensure of the pneumococcal -valent polysaccharide conjugate vaccine (pcv ) was based on noninferiority comparative studies to pcv . effectiveness of pcv was inferred from measuring anti-polysaccharide binding and functional opsonophagocytic antibodies because clinical end point disease efficacy studies were no longer feasible owing to the further decline of invasive pneumococcal disease as a result of introduction of pcv in the united states. in some situations, human challenge studies have been conducted during early clinical development or in lieu of clinical trials in an endemic area. these studies served to demonstrate "proof of concept" of the vaccine early in clinical development (e.g., plasmodium falciparum sporozoite challenge of malaria-naïve u.s. volunteers previously administered a candidate malaria vaccine). human challenge studies may also be considered to demonstrate the efficacy of the vaccine. for example, the agency convened the vaccines and related biologics products advisory committee (vrbpac) to consider whether data from human challenge studies in u.s. subjects could be sufficient to demonstrate efficacy of a cholera vaccine in travelers to endemic areas who are at high risk for contracting the disease. in , the vrbpac agreed that human challenge studies could suffice to demonstrate efficacy of a cholera vaccine provided that studies were adequate and well-controlled and conducted under the provisions of good clinical practices. in , the fda approved vaxchora, a live, attenuated vaccine for the prevention of cholera in adults traveling to cholera-affected areas. efficacy of vaxchora was demonstrated in a controlled human challenge study in adult u.s. volunteers. safety is one of the most important considerations when evaluating new vaccines and modifications to currently multiple phase i or phase ii trials may be performed as new data are obtained. phase i trials are intended primarily to provide a preliminary evaluation of safety and immunogenicity. these trials are typically conducted in a small number (e.g., to ) of closely monitored adult volunteers. if the ultimate target population for the vaccine is infants or young children, as is commonly the case, the product is usually evaluated in a stepwise progression from older to younger age groups down into the first year of life. phase ii studies can involve up to several hundred participants, are often randomized and wellcontrolled, and provide further information on safety and immunogenicity and optimal dose. in some cases, phase ii studies may provide preliminary data on the vaccine's activity against the infectious disease of interest. phase iii studies are large-scale trials to provide a more thorough assessment of safety as well as a definite assessment of efficacy. the general considerations for clinical studies to support licensure of a vaccine include demonstration of safety, efficacy (immunogenicity may be sufficient in some cases), and evaluation of simultaneous administration with other licensed vaccines. vaccine efficacy should be demonstrated, ideally in randomized, double-blind, well-controlled trials. the end points are product specific, and may be clinical disease end points or immune response end points if efficacy against clinical disease had been previously established and there are immune correlates or surrogates of that protection. in recent years, efficacy trials for various vaccines have involved a broad range in the number of study participants, from thousands to tens of thousands. this broad range is related to a number of interconnected variables such as study design and the incidence of the disease to be prevented. for example, clinical disease end point studies that are designed to demonstrate that a new vaccine is noninferior to an already existing product of the same type generally require larger numbers than one in which a new vaccine can be compared with a control that has no activity against the clinical disease. the incidence of the disease to be prevented in the study population is also important. as an example, a trial to show that pneumococcal submission, including methods for presenting the data; (c) information on the status of needed or ongoing studies; and (d) any other information for discussion at the meeting. the primary purpose of this exchange is to uncover any major unresolved problems; identify those studies that the sponsor is relying on as adequate and well-controlled to establish the product's effectiveness; identify the status of ongoing studies; acquaint fda reviewers with the general information to be submitted in the bla (including technical information); review methods used in the statistical analysis of the data; and discuss the best approach for the presentation and formatting of data in the application. at the time that an applicant submits a bla to the director of cber's office of vaccines research and review precise production methods and procedures should be defined, and the manufacturing process should be standardized. critical information to be contained in the bla include data derived from nonclinical laboratory and clinical studies that demonstrate that the manufactured product meets prescribed requirements for safety, purity, and potency. the bla should contain information that supports compliance with standards addressing requirements for (a) organization and personnel; (b) buildings and facilities; (c) equipment; (d) control of components, containers, and closures; (e) production and process controls; (f) packaging and labeling controls; (g) holding and distribution; (h) laboratory controls; and (i) records to be maintained. furthermore, a full description of manufacturing methods; data establishing stability of the product through the dating period; sample(s) representative of the product for introduction or delivery for introduction into interstate commerce; summaries of test results performed on the lot(s) represented by the submitted sample(s); specimens of the labels, enclosures, and containers; and the address of each location involved in the manufacture of the biological product should be included in the bla. an application for a biologics license is not considered as filed (or accepted by the agency for review) until cber determines that it has received all pertinent information and data from the applicant. in this regard, cber can refuse to file a bla if it deems the submission to be incomplete. additionally, the manufacturing facility must be inspection-ready at the time the bla is submitted. the applicant is also required to include either an environmental assessment or a claim for categorical exclusion from the requirement to submit an environmental assessment or an environmental impact statement. other components of the bla review include product labeling, which describes the indications for use, contraindications, dosage and possible adverse effects; protocols for the manufacturing and testing of the number of product lots specified to establish the consistency of the process; and confirmatory testing results within cber of samples of in-process material or product in final containers and conformance to existing regulations. an internal cber multidisciplinary committee performs the scientific review of the bla. this process occurs for each bla or supplement to a bla in which significant changes are proposed. during the review, there are discussions and exchanges of correspondence between the sponsor and the cber review committee concerning issues that may arise. during the fda review of the bla an announced prior approval inspection (pai) of the manufacturing facility is performed. this inspection is designed as an in-depth review of the facilities, records, total production process, methods, equipment, quality control procedures, and personnel. with the implementation of the bla process, changes have occurred in the scope of issues reviewed during the pai. instead of the manufacturer submitting detailed records with the bla regarding studies on cleaning validation, monitoring data for licensed vaccines. the initial responsibility for determining vaccine safety starts with clinical investigators and vaccine manufacturers. the fda is responsible for assuring that clinical trials are done under good clinical practices, a requirement essential for the evaluation of safety data intended to support a license application. in general, when evaluating safety, one must compare the risk of the vaccine-preventable disease with the risk of the adverse event(s) potentially associated with the vaccine, and these may change over time. as an example, the reported association between rotashield (rotavirus vaccine, live, oral, tetravalent, manufactured by wyeth) and intussusception resulted in the additional requirement for the evaluation of the safety of rotateq (live, oral pentavalent human-bovine reassortant rotavirus vaccine, manufactured by merck) with respect to intussusception. this clinical trial enrolled more than , infants divided equally between rotateq and placebo. the primary safety hypothesis was that the oral rotateq would not increase the risk of intussusception relative to placebo within days of any dose. the intended target population should be taken into consideration in assessing the adequacy of the safety database. for routinely administered childhood vaccines in the united states, the target population would be the birth cohort in the united states (approximately million/year). this is generally a healthy population, and a government body (e.g., state or local governments) may mandate vaccination. common reactions can be studied adequately in hundreds of individuals, but many thousands will be required to define low-incidence adverse reactions. for vaccines evaluated in clinical end point efficacy trials, a large safety database likely will derive from a double-blind, randomized, well-controlled efficacy study. however, for vaccines evaluated in immunogenicity end point studies, additional studies likely will be needed to obtain an adequate safety database. additional controlled safety studies are often requested when the numbers of subjects included in the efficacy studies are deemed insufficient to provide adequate safety data. safety studies may be unblinded if the number of injections, route of administration, or schedule differs between groups, in particular when infants and young children are involved. phase ii safety studies should provide data on common local and systemic reactions to the study vaccine. phase ii clinical development also should include immunogenicity and preliminary safety data on the concurrent administration of the study vaccine with other vaccines, if relevant. phase iii safety studies are designed to evaluate less common reactions, may be unequally randomized, and may have a simplified trial design for assessing less-common adverse events in large trials. if a vaccine is recommended on the same schedule as other routinely recommended vaccines, safety and immunogenicity data should be obtained in prelicensure studies to support simultaneous administration. following completion of ind studies demonstrating the safety and efficacy of the vaccine for a specific use and population the sponsor can submit a bla to obtain a license for a new vaccine under section of the phs act for commercial manufacture and distribution of the product. prior to the submission of a bla, a pre-bla meeting with the fda is strongly encouraged to discuss the sponsor's product development plan. for the fda to provide sponsors with advice regarding the adequacy of information to support a bla, the following information in advance of the pre-bla meeting should be submitted, depending on the type of meeting: (a) an executive summary of the clinical studies to be submitted in the application; (b) a proposed format for organizing the strate safety and efficacy in humans are required at the time of use. under new section (o) of the fd&c act, the fda is authorized to also require postmarketing studies or clinical trials at the time of approval or after approval if the fda becomes aware of new safety information. section (o)( ) (b) states that postmarketing studies and clinical trials may be required to (a) assess a known serious risk related to the use of the drug involved, (b) assess signals of serious risk related to the use of the drug, and (c) identify an unexpected serious risk when available data indicate the potential for a serious risk and when the adverse event reporting system is not adequate. the fda has defined a clinical trial as any prospective investigation in which the sponsor or investigator determines the method of assigning treatment or other intervention to one or more human subjects. a study is all other investigations, such as investigations using humans, that are not clinical trials as defined above (e.g., observational epidemiologic studies), animal studies, and laboratory experiments. the fda has issued guidance for industry to describe the type of studies and clinical trials that are required (post marketing requirement [pmr]) under the fdaaa , and those that will remain agreed-upon postmarketing commitments. a pmr describes all required postmarketing studies or clinical trials including those required under accelerated approval, prea, the animal rule, and fdaaa. examples of required studies are pharmacoepidemiologic studies designed to assess a serious risk, trials with a primary safety end point, preclinical studies investigating specific end organ toxicities, as well as pharmacokinetic studies in the indicated population at potential risk for high drug exposure that could result in toxicity. studies that generally would not be considered required postmarketing studies or clinical trials are agreed-upon studies (postmarketing commitments) and include biologic quality studies (such as manufacturing, stability, and immunogenicity studies that do not have a primary safety end point), trials in which the primary end point is related to further defining efficacy, and pharmacoepidemiologic studies designed to examine the natural history of disease or background rates for adverse events. since passage of fdaaa , several new vaccines have been approved with either pmrs or postmarketing commitments. the fda has the authority to monitor the progress of postmarketing studies or trials by requiring the applicant to submit an annual status report. applicants are required to provide a timetable for study completion, a periodic status report on the status of the study including whether enrollment has begun, the number of participants enrolled, the expected completion date, and whether any difficulties in completing the study have been encountered. the fda is responsible not only for approving vaccines but also for monitoring their safety postlicensure. because of the relatively small size of most prelicensure trials, rare adverse events are unlikely to be detected. consequently, postlicensure or postmarketing surveillance (i.e., the continued monitoring of vaccine safety in the general population after licensure) is critical for identifying and evaluating rare or uncommon adverse events. an adverse event refers to any untoward medical occurrence associated with the use of a drug in humans, whether or not considered drug related. the vaccine adverse event reporting system (vaers) is a national system for passive surveillance of adverse events following vaccination. established in as a result of the national childhood vaccine injury act of , vaers is administered jointly by the fda and the cdc and, in recent years, has received more than pharmaceutical-grade water, facility support systems (e.g., clean steam, compressed air, and building management systems), and other facility-related systems, a more detailed review of this type of data is done onsite during the pai. pais tend to require longer periods of time for the fda inspectors to be in the facility because of the increased scope of issues that are reviewed onsite. if licensure is denied following inspection for the original license application, reinspection will occur after receipt of assurance that all deficiencies that were the basis of the denial were corrected. with the implementation of fdaaa ( ) and fdasia ( ), in addition to completing the discipline reviews during the pdufa v mandated timelines, the review committee must complete numerous additional tasks. these include, but are not limited to, review committee assignments, internal information exchange meetings, filing decisions, presentation of the application to the perc, midcycle review meetings and midcycle communication with the applicant, late-cycle meetings with the applicant, and presentation of planned or required postmarketing studies to an internal fda safety committee. after cber reviews the entire package of information in the bla, its advisory committee (the vrbpac) and consultants, if needed, are asked to review and comment on the adequacy of the data to support safety and efficacy in the target population. the standards for safety and efficacy are relative; that is, the benefit-to-risk ratio of a biological product is considered. the vrbpac's advice is considered in cber's decision regarding licensure, and in developing recommendations for use to be given in the package insert. the committee may recommend additional studies to be performed either before or after approval. once cber determines that the data and information from the applicant are satisfactory and support the safety and efficacy of the product, the product is licensed. if the manufacturer wishes to significantly modify the approved manufacturing process or directions for vaccine use, prior approval must be obtained from the fda before these changes can be implemented. the applicant is required to submit an account of these changes to the appropriate license applications. modifications to the manufacturing process may occur post licensure, such as scale-up or change in equipment to optimize the production process. furthermore, clinical studies with the product also may be performed after licensure as the manufacturer seeks additional indications for product use (e.g., new target populations that would benefit from vaccination). for most new approvals, manufacturers may be asked to commit to completing specific postmarketing or so-called phase iv studies, for example, to provide additional assessments of less-common or rare adverse events or further assess the duration of vaccine-induced immunity. these studies may also be designed to collect additional safety data in large numbers of vaccine recipients, as well as focus on issues that were identified during the prelicensure testing. submission of status reports for certain postmarketing studies are required by regulation. in particular, this requirement for status reports pertains to postmarketing studies for clinical safety, efficacy and pharmacokinetics, and nonclinical toxicology to which an applicant committed in writing prior to licensure. prior to fdaaa , the fda required postmarketing studies in the following situations (a) accelerated approvals for products approved under (b) of the fd&c act or section of the phs act, respectively, which require postmarketing studies to demonstrate clinical benefit, (b) deferred pediatric studies, where studies are required under prea, and (c) animal efficacy rule approvals, where studies to demon-lesser importance for which the manufacturer must provide notification days before distribution of product made using the change; and (c) changes for which the manufacturer need only notify the agency by submission of an annual report. the guidance document, "changes to an approved application: biological products" ( ) , provides examples of changes that fall into these categories. following approval, there is continued surveillance of the product and of the manufacturer's production activities. for most licensed vaccines, samples are submitted along with protocols for each lot prepared by the firm that provide the details of production and a summary of test results. although not required by law or regulation, cber often performs selected laboratory tests. the type and extent of confirmatory testing performed by cber depend on several factors, such as the newness of the product or the difficulties that may have arisen with manufacture or use of the product. release or rejection is based on a review of all test results, including those done by the manufacturer and those performed by cber. alternatives to official lot release are allowable under the provisions outlined for extensively characterized products having a track record of continued safety, purity, and potency. a manufacturer must be able to produce a vaccine that repeatedly meets the standards for potency, purity, and stability of bulk and final container material while using a consistent process. important factors to be considered are the nature of the product with respect to correlation between the measure of potency and biological activity and effectiveness. surveillance samples and protocols may be required to be submitted to cber at predetermined intervals. licensed establishments are inspected at least every years. the purpose of the inspection is to determine whether licensed products are manufactured and tested as described in the license application and in accordance with applicable regulations. manufacturers who fail to meet product standards or who are not in compliance with cgmps may have their licenses suspended or revoked, depending on the nature of the potential health hazards created. the major issues observed during inspections can be categorized in three major areas: ( ) process-related issues, ( ) quality unit-related issues, and ( ) facility-and production environment-related issues. some examples of process validation issues include lack of documentation of time limits for major steps in the production process, lack of validation of rework or reprocessing steps in the manufacturing process, and lack of data to support in-process specifications. quality unit-related issues include the appropriate reporting of out-of-specification results and process deviations (including adequate investigations into causes), appropriate documentation of product release, and adequate training of personnel. facility and production monitoring concerns include controlling production environments by appropriately monitoring heating, ventilation, and air conditioning (hvac) system performance and microbial quality (e.g., pressure differentials, appropriate sampling sites, and frequency of sampling). other concerns pertaining to the facility include adequate cleaning, sanitization, storage, and changeover procedures for multiproduct areas and equipment. if the inspection team finds cgmp deficiencies in an already licensed facility, the team may remain in the facility until they have achieved an audit that provides confidence in the ability of the firm to reproducibly manufacture a safe and potent product. mechanisms for providing earlier access to vaccines to prevent or treat severe and life-threatening illness have been developed. , reports per year. the purpose of vaers is to detect possible signals of adverse events associated with vaccines to help ensure the safety of u.s.-licensed vaccines. vaers collects and analyzes information from reports of adverse events that occur after the administration of u.s.-licensed vaccines. reports are submitted by healthcare providers, vaccine recipients or their parents or guardians, vaccine manufacturers, and other interested parties. fda medical officers review all serious reports (defined as events that are fatal, disabling, or lifethreatening; require or prolong hospitalization; result in congenital anomalies; require medical intervention to prevent such outcomes; or are deemed to be other medically important conditions). the vaers system is not limited to routinely recommended pediatric vaccines; voluntary reports of adverse events occurring after administration of any vaccine are also accepted. fda and cdc continually monitor vaers reports for any unexpected patterns of adverse events. another important mechanism used by cber to monitor adverse events is the vaccine safety datalink, a collaborative effort between cdc's immunization safety office and nine healthcare organizations. the vaccine safety datalink uses electronic health data from participating sites and conducts vaccine safety studies based on questions or concerns raised from the medical literature and vaers reports. when there are new vaccines that have been recommended for use in the united states, or if there are changes in how a vaccine is recommended, the vaccine safety datalink will monitor the safety of these vaccines. the fda's sentinel initiative was launched in in response to a congressional mandate in the fdaaa of . the sentinel initiative aims to develop and implement a proactive system that will complement existing systems that the fda has in place to track reports of adverse events linked to the use of its regulated products. it is a national electronic system that is transforming the fda's ability to track the safety of drugs, biologics, and medical devices once they reach the market. the post-licensure rapid immunization safety monitoring system (prism), a component of the fda's sentinel initiative dedicated to vaccines, uses the fda's sentinel distributed database, which includes a population exceeding million. prism monitors the largest u.s. general population cohort designated for active surveillance of vaccine safety by linking data from health plans with data from state and city immunization registries. the fda structured prism as a program that includes specific vaccine evaluations. for example, several vaccines including a human papillomavirus vaccine, gardasil, and two rotavirus vaccines, rotateq and rotarix, were chosen for surveillance because their evaluations would benefit most from prism's large cohort size. in , the fda published a final rule, changes to an approved application, which amended cfr § . and § . to simplify and categorize manufacturing reporting requirements for changes in testing methods, equipment, facilities, or personnel. proposed changes in manufacturing methods that have a substantial potential to have an adverse effect on the safety or effectiveness of the product may not become effective until notification is given of cber's approval. the changed created the following categories: (a) those sufficiently significant with regard to safety, purity, potency, and effectiveness of the product to require preapproval of a supplemental application before product distribution; (b) those of feasible. this rule, referred to as the "animal rule," allows the use of animal efficacy data in lieu of human efficacy data when human challenge studies cannot be conducted ethically and field efficacy studies are not feasible because of infectious disease epidemiology (in the case of vaccines). in these situations, certain drug and biological products (e.g., vaccines) that are intended to reduce or prevent serious or life-threatening conditions caused by lethal or permanently disabling toxic chemical, biological, radiologic, or nuclear substances may be approved for marketing based on evidence of effectiveness derived from appropriate studies in animals and additional supporting data. safety, pharmacokinetics, and immunogenicity data are still necessary in humans. under the animal rule, the fda licensure of a product for which safety has been established and the requirements of cfr § . have been met is based upon adequate and well-controlled animal trials, when results of these animal studies establish that the product is reasonably likely to provide clinical benefit to humans. the fda can rely on the evidence from animal studies to provide substantial evidence of the efficacy of these products when: . there is a reasonably well-understood pathophysiological mechanism for toxicity of the chemical, biological, radiologic, or nuclear substance and its amelioration or prevention by the product. . the effect is demonstrated in more than one animal species that is expected to react with a response that is predictive for humans, unless the effect is demonstrated in a single animal species that represents a sufficiently wellcharacterized animal model (in other words, the model has been adequately evaluated for its responsiveness) in predicting the response in humans. . the animal end point is clearly related to the desired benefit in humans, which is generally the enhancement of survival or prevention of major morbidity. . the data or information on the pharmacokinetics and pharmacodynamics of the product or other relevant data or information in animals and humans is sufficiently well understood to allow selection of an effective dose in humans, and it is reasonable to expect the efficacy of the breakthrough therapy designation provides for increased interaction with fda to expedite the development and review of the application. in contrast to fast track designation, a breakthrough therapy designation requires evidence of substantial improvement over current treatments. products regulated by cber are eligible for priority review if they provide a significant improvement in the safety or effectiveness of the treatment, diagnosis, or prevention of a serious or life-threatening disease. the fda has months to complete the review of a new bla it designates as a priority, as opposed to months for the completion of the review of a standard bla submission. under the fda's traditional approval pathway, a demonstration of vaccine effectiveness is based on a clinical disease end point (e.g., prevention of disease) or, alternatively, an accepted correlate of protection. in addition to these programs, the fda's regulations provide for expedited pathways for licensure. accelerated approval, cfr § . , may be granted for certain biological products that have been studied for their safety and effectiveness in treating a serious or lifethreatening disease or condition and that provide meaningful therapeutic benefit over existing treatments. such an approval is based on adequate and well-controlled clinical trials establishing that the product has an effect on a surrogate end point that is reasonably likely to predict clinical benefit or on a clinical end point that can be measured earlier than irreversible morbidity or mortality that is reasonably likely to predict an effect on irreversible morbidity or mortality or other clinical benefit. approval under this pathway is subject to the requirement that the sponsor study the biological product further, to verify and describe its clinical benefit, where there is uncertainty as to the relation of the surrogate end point to clinical benefit. of note, the fdasia of provided that evidence to support an end point is reasonably likely to predict clinical benefit to include "epidemiological, pathophysiological, therapeutic, pharmacologic, or other evidence developed using biomarkers, for example, or other scientific methods or tools." in other words, fdasia expanded the scope of available end points that can be used to demonstrate that a product qualifies for accelerated approval, but do not affect the quantity and quality of evidence needed to demonstrate substantial evidence of effectiveness or safety. two vaccines to protect against meningococcal b diseases were licensed using the accelerated approval provisions and were designated breakthrough therapy. while the incidence vaccines are tested during both the prelicensure and the postlicensure phases. testing procedures are developed with the goals of controlling and minimizing the potential for productrelated adverse events by taking into consideration the experience gained from the same or related products. as an example, for inactivated vaccines, a clear understanding of the kinetics of inactivation is critical. for live vaccines, attenuation must be stable both to avoid reversion to virulence and to avoid the vaccine becoming over attenuated and, as a consequence, less potent. for example, during the first decade of its widespread use, yellow fever d vaccine virus was serially propagated in eggs, as required to meet demand. however, it soon became obvious that the level of attenuation of the vaccine from one passage to the next could vary considerably. some lots were excessively neurovirulent, especially in infants and young children, whereas successive lots might by chance be overattenuated and, consequently, not immunogenic. the who formulated a solution to this problem, which was to adopt a "seed-lot system" wherein the vaccine is prepared from a master seed virus at a specified passage number in eggs. working seed virus is prepared by one passage of the master seed and is, in turn, used to generate all production lots. all d vaccines and all other live virus vaccines now adhere to a seed-lot system for manufacture. the fda requires that cell substrates and vaccine viral seeds used in production be appropriately selected and tested to ensure that they do not introduce any unintended risks. this document provides manufacturers of viral vaccines with guidance for the characterization and qualification of cell substrates, viral seeds, and other biological materials used for the production of viral vaccines for human use to assure that they meet the highest safety standards achievable using modern technology. characterization of cell substrates should address certain general issues that might affect the safety and purity of vaccine products. for example, in the early s, exogenous and endogenous contamination product in animals to be a reliable indicator of its efficacy in humans. the animal rule does not apply if the product can be approved based on standards described elsewhere in fda regulations (e.g., accelerated approval based on surrogate markers or clinical end points other than survival or irreversible morbidity). emergency use authorization (eua) is another regulatory mechanism by which the fda can accelerate the availability of vaccines and other pharmaceutical products. under an eua, the fda can authorize the use of an unapproved product or the unapproved use of an approved product when an emergency or a potential emergency exists. section (b)( ) of the federal fd&c act was amended by the project bioshield act of to allow the secretary of health and human services (secretary) to authorize the introduction into interstate commerce of a drug, device, or biological product intended for use in an actual or potential emergency. before an eua may be issued by fda, the secretary must declare an emergency justifying the authorization based on: • a determination by the secretary of homeland security that there is a domestic emergency or a significant potential for an emergency that involves a heightened risk of attack with a specified biologic, chemical, radiological, or nuclear agent or agents; or • a determination by the secretary of defense that there is a military emergency or a significant potential for an emergency that involves a heightened risk of attack with a specified biologic, chemical, radiological, or nuclear agent or agents; or • a determination by the secretary of health and human services of a public health emergency under section of the phs act that affects or has the significant potential to affect national security and that involves a specified biological, chemical, radiological or nuclear agent or agents or a specified disease or condition that may be attributable to such agent(s). once the secretary declares an emergency, the fda can authorize the emergency use of a particular product if the other statutory criteria and conditions are met. based on the particular circumstances, the process for authorization can be expected to range in duration from hours to days. the secretary has delegated the authority to issue an eua under section of the fd&c act to the fda commissioner. facture of many biotechnology-derived biological products. thus, the fda has published a final rule to remove this requirement. in addition to the tests required by regulation, other tests tailored to the specific product may be required (e.g., neurovirulence testing and cell culture and animal tests for extraneous viruses). once the product is licensed, the manufacturer's testing must be conducted according to the exact specifications in the manufacturer's license application, and the results of these tests must be within the specified prescribed limits. prescription drug labeling, also known as the package insert, package circular, or prescribing information, is the primary mechanism through which the fda and drug manufacturers communicate essential, science-based prescribing information to healthcare professionals. labeling provisions contained in cfr § § . and . require that prescribing information must summarize the essential information on the safe and effective use of the product; that information contained in the labeling must be accurate and not false and misleading; and that there must be no implied claims or suggestions for use if evidence of safety or effectiveness is lacking. whenever possible, data contained in labeling should be derived from human experience. in the united states, the fda regulates the format and content of labels for product containers, cartons, and the package insert that accompanies the product. in january , the fda issued a final drug labeling rule, commonly referred to as the physicians' labeling rule, amending the content and format of prescribing information for human drug and biologic products. the new format is intended to provide healthcare professionals with clear and concise prescribing information by reorganizing critical information into a streamlined format. moreover, these revisions make it simpler for healthcare professionals to access, read, and use prescribing information, and enhance the safe and effective use of prescription drug products. new sections were added to the label, such as the highlights section, which contains key benefit and risk information, and a table of contents for the full prescribing information. on december , , the fda issued the pregnancy lactation and labeling rule, which revises the content and format of the pregnancy subsection of labeling for prescription drugs and biologics (sections . to . of the prescribing information). previous regulations required that each product be classified under one of five pregnancy categories (a, b, c, d, or x) based on the risk of reproductive and developmental adverse effects or, for certain categories, such risk weighed against potential benefit. the most significant change proposed by the rule is the replacing of these letter risk categories with a narrative summary of the risks and benefits of using a drug during pregnancy, based on the available human and/or animal data and a discussion of the data. additionally, the rule requires that prescription drug labeling include relevant clinical information to help healthcare providers make prescribing decisions and counsel women about the use of drugs during pregnancy and/or lactation. the structured product labeling defines the content of human prescription drug labeling in xml format. it is a document markup standard approved by health level seven and adopted by the fda as a mechanism for exchanging product and facility information. structured product labeling documents contain both the content of labeling (all text, tables, and figures) for a product and additional machine-readable information (drug listing data elements). drug listing data elements include information about the product (product and generic names, ingredients, ingredient strengths, dosage forms, of primary monkey kidney cells with simian virus and chick embryo fibroblasts with avian leukosis virus were reported. although chick embryo fibroblasts are still used for the production of viral vaccines, these cell substrates must be well-characterized and tested to assure absence of potentially infectious agents. issues related to cell substrates have been discussed in a variety of forums. [ ] [ ] [ ] furthermore, if a vaccine is manufactured in a cell substrate that is derived from a tumor, or that has developed a tumorigenic phenotype through an unknown mechanism, it was considered to carry a higher theoretical risk of containing oncogenic substances such as oncogenic viruses and cellular dna, derived from that cell substrate. this was the main reason tumorigenic cells or cells derived from human tumors had been considered unsuitable as cell substrates. however, at a meeting of the vrbpac, experts recognized that cell lines derived from human tumor are an important tool for manufacturing of vaccines and could provide a wider repertoire of cell substrates for vaccine production. they indicated that risk-mitigation strategies are the same for vaccines generated using human tumor-derived cell lines as for other cell substrates. a thorough characterization of the cell substrate with respect to adventitious viruses, which includes oncogenic viruses, should be done using new virus-detection technologies such as massively parallel sequencing, virus microarrays and broad-range polymerase chain reaction to complement existing assays. the manufacturing process should lower the amount and reduce the size of the dna. residual dna for continuous nontumorigenic cells, such as low-passage vero cells, should be limited to less than ng/dose for parenteral inoculation and to less than µg/dose for oral vaccines. cells with tumorigenic phenotypes or other characteristics that give rise to special concerns may require more stringent limitation of residual dna quantities and size to assure product safety. the regulation of biologicals includes requirements for testing of licensed products ( cfr § ). these tests include those for bacterial and fungal sterility, general safety, purity, identity, suitability of constituent materials and potency, thereby this specific testing performed may vary depending on the vaccine. for example, tests for potency may be based on studies of immunogenicity or, for some vaccines, protection from virulent challenge in laboratory animals. however, in vitro tests, including virus titration (e.g., live vaccines such as polio, measles, mumps, and rubella), antigen content (e.g., influenza and inactivated poliovirus vaccines), and biochemical and biophysical measurements (e.g., meningococcal conjugate vaccines) have been used. tests for purity are designed to determine that the product is free of extraneous material, except that which is unavoidable in the manufacturing process described in the approved license application. tests for residual moisture and pyrogenic substances may also be included. final-container material must be identified by a test specific for each product (e.g., neutralization of each of the components of live measles, mumps, and rubella vaccine with specific antisera). with regard to constituent materials, the manufacturer must ensure that all ingredients used in the product, such as diluents, preservatives, or adjuvants, meet generally accepted standards of purity. an adjuvant may not be used unless there is adequate proof that it does not adversely affect the safety or potency of the product. of note, the fda periodically evaluates the appropriateness of testing requirements. an example is the general safety test (required under cfr § . ) used to detect extraneous toxic contaminants that may be present in the product in the final container from every final filling of each lot of the biological product. technological advances have increased the ability of manufacturers to control and analyze the manu-unless there is satisfactory evidence that it does not adversely affect the safety or potency of the product." from a regulatory perspective, adjuvants are not considered active ingredients as defined in cfr § . (b)( ) and vaccine adjuvants are not licensed separately. it is the adjuvanted vaccine formulation, in toto, that is tested in nonclinical and clinical trials and licensed. there is a requirement that the adjuvanted vaccine formulation, as with any vaccine, must be both safe and effective, with its benefits outweighing the risks of adverse events that may occur. however, there is no explicit requirement for demonstrating the added safety and effectiveness of the adjuvanted vaccine formulation over that of the unadjuvanted vaccine formulation in comparative clinical trials. the regulatory considerations for the nonclinical and clinical development of preventive vaccines, as well as pathways to licensure described elsewhere in this chapter, are largely also applicable to vaccines formulated with adjuvants. however, adjuvants can exhibit a range of properties that invoke complex immune responses, the mode of action is not always known or fully understood, and animal models that are relevant for evaluating both the safety and the efficacy of an adjuvantantigen combination are frequently not available. thus, there are some unique issues to be addressed during preclinical and clinical development of the adjuvanted vaccine formulation. a who guideline published in describes the nonclinical, quality, pharmacological, toxicological, and other information needed to support initiation of clinical trials with a vaccine combined with a novel adjuvant. in , cber published two guidance documents ("guidance for industry: clinical data needed to support licensure of pandemic influenza vaccines" and "guidance for industry: clinical data needed to support the licensure of seasonal inactivated influenza vaccines" ). each of these documents discusses the development of adjuvanted inactivated influenza vaccines and notes that "[d]ata to support the safety of the adjuvanted formulation and added benefit over the unadjuvanted formulation must be submitted in the bla…" , sponsors were advised that "at an early stage of development, clinical data supporting the value of adding the adjuvant should be provided…" , the seasonal influenza vaccine guidance notes further that "[i]f an adjuvant is added to a licensed seasonal vaccine without antigen sparing effects…the immune response elicited by the adjuvanted formulation should be substantially better than that elicited by the unadjuvanted vaccine…" as the fda's experience with novel adjuvants has grown, the agency continues to reexamine guidance and engages with sponsors and applicants regarding the clinical development of adjuvanted vaccines. vaccine manufacturers should provide a rationale for the use of an adjuvant in the vaccine. the "added benefit" of an adjuvant may be defined as evidence of enhanced immune response, antigen-sparing effect, dose sparing, increased breadth of immune response, or superior clinical efficacy. information to support the "added benefit" of the adjuvant may be derived from preclinical studies, for example, in vitro assays and/or proof-of-concept studies in animal models conducted prior to the initiation of clinical trials or early phase clinical trials. there is no regulatory requirement to demonstrate the "added benefit" of an adjuvant in clinical comparative phase iii effectiveness trials unless the applicant plans to make a claim of superiority of the adjuvanted product over unadjuvanted product. the benefits from incorporating or adding an adjuvant to any vaccine formulation need to be balanced with the risk of adverse reactions. adjuvants have their own pharmacological activity, which may affect both the immunogenicity and the safety of vaccines. adverse reactions may include local routes of administration, appearance, drug enforcement agency schedule) and the packaging (package quantity and type). during the bla review, the agency considers the draft labeling and clinical studies submitted by the manufacturer, and the proposed indication for the licensed product, which is based on clinical data submitted by the sponsor demonstrating the safety and effectiveness of the product for its intended use and target population. subsequently, significant changes in labeling, including new indications for use, new dosage forms or regimens, expanded patient populations who receive the product and additional information regarding safety and effectiveness, require manufacturers to submit a supplemental filing for review and approval by cber. unlike other product labeling, the promotional labeling and advertising are not subject to preclearance; however, they are monitored for misleading claims. these documents must also meet the standard of fair balance, that is, claims of efficacy are balanced with information about the product's safety. labeling changes are usually initiated by the manufacturer but may be initiated by cber. historically, manufacturers have had to obtain prior approval from cber before the labeling changes were made. the changes to cfr § . , mentioned previously, also apply to labeling changes and allow exceptions for a change that adds or strengthens a contraindication, warning, precaution, or adverse reaction; adds or strengthens instructions about dosage and administration intended to increase safe use; or deletes false, misleading, or unsupported indications for use or effectiveness claims. under this regulation, a manufacturer could effect such changes and, at the same time, submit them and the supporting data to cber without preapproval. as described earlier, the fdaaa amended the fd&c act by adding new section (o) authorizing fda to require and, if necessary, order labeling changes if the fda becomes aware of new safety information that the fda believes should be included in the labeling of the drug. section (o)( ) of the fd&c act imposes timeframes for application holders to submit and for fda staff to review such changes, and gives the fda new enforcement tools to bring about timely and appropriate safety labeling changes. strategies and approaches for the development and delivery of vaccine antigens have expanded over the last several decades, leading to a broad range of novel products comprised of purified subunit antigens or subunit proteins. these antigens may require the presence of adjuvants to enhance the immune response to the vaccine antigens, reduce the dosing frequency, induce cross-protective effects, direct the immune response and/or achieve antigen sparing. the number of investigational vaccines containing novel adjuvants evaluated in clinical trials has increased in recent years and some vaccines containing novel adjuvants have been licensed by the fda. for example, the human papillomavirus vaccine manufactured by glaxos-mithkline, cervarix, contains as , an adjuvant system comprised of an aluminum hydroxide and monophosphoryl lipid a. glaxosmithkline's pandemic influenza vaccine, q-pan, contains as , an adjuvant system comprised of an oil-inwater emulsion. the cfr defines adjuvants, together with ingredients, preservatives, and diluents as constituent materials ( cfr § . ). these regulations state, "all ingredients…shall meet generally accepted standards of purity and quality" and that, "an adjuvant shall not be introduced into a product infectious diseases (eids)-from pandemic influenza to novel pathogens like severe acute respiratory syndrome and ebola, to biological threats that have the potential to be intentionally released into the general population by humans-also pose a threat to global public health. vaccines will continue to be an important medical countermeasure against a broad range of infectious diseases from anthrax, smallpox, and influenza to newly emerging infectious diseases. moreover, infectious diseases, such as tuberculosis and malaria present global public health challenges and increasing resistance to currently available treatments by common bacteria such as staphylococci, underscores the importance of the development of safe and effective vaccines. the development of safe and effective vaccines to protect against global infectious diseases (e.g., tuberculosis, malaria, hiv/ aids), enteric diseases, and other neglected diseases of the developing world is of critical public health importance. development and availability of such vaccines, particularly for use in the developing countries most affected by these diseases, will benefit u.s. and global health. in , the fda published a revised guidance document to assist sponsors in developing vaccines targeted against infectious diseases or conditions endemic in areas outside the united states. the fdaaa of revised the fd&c act by adding section , recognizing the importance of having products to treat and prevent tropical diseases that disproportionately affect poor and marginalized populations and for which there is no significant market in developed nations. under section , the agency can grant priority review of applications under section (b)( ) of the fd&c act or section of the phs act for the treatment and prevention of specified tropical diseases, including tuberculosis, malaria, cholera, and "any other infectious disease for which there is no significant market in developed nations and that disproportionately affects poor and marginalized populations, designated by regulation by the secretary." consequently, this guidance provides general recommendations for regulatory pathways to use in the development of vaccines to protect against global infectious diseases for u.s. licensure and clarifies applicable regulations: (a) the fda can license vaccines to protect against infectious diseases or conditions that are not endemic or have not been reported to occur in the united states; (b) the regulatory pathways to u.s. licensure for the development of vaccines to protect against infectious diseases that are not endemic or have not been reported to occur in the united states are the same as for vaccines to protect against diseases that are endemic in the united states; (c) a sponsor may submit data from clinical trials conducted outside the united states to support product licensure, (d) noting that the accelerated approval regulations may be used in appropriate cases; and (e) that when pivotal studies are conducted outside the united states, in some instances, it may not be necessary to conduct studies in the united states. this guidance also responded to the congressional mandate in section of the fiscal year appropriation act (agriculture, rural development, food and drug administration, and related agencies appropriations act, , public law - ) by requiring the fda to make recommendations on appropriate preclinical, trial design, and regulatory paradigms to prevent, diagnose, and treat rare diseases and neglected diseases. the u.s. military has implemented vaccination programs to protect troops against several biological threats; however, the risk-to-benefit ratio for protecting civilians against agents of bioterrorism is more difficult to assess. as of this writing, there is one licensed smallpox vaccine in the united states, sanofi pasteur biologics' acam . new smallpox vaccines are being developed under ind applications, with the goal to seek licensure, and new vaccinia immunoglobulin reactions such as pain, swelling, injection site necrosis, and granulomas. systemic reactions may include nausea, fever, arthritis, as well as potential immunotoxic reactions. unexpected, rare events may also occur. for example, during the h n influenza pandemic in - , an increased risk of narcolepsy, a chronic neurological disorder caused by the brain's inability to regulate sleep-wake cycles normally, was observed in several european countries following vaccination with an as adjuvanted, monovalent h n influenza vaccine, pandemrix. [ ] [ ] [ ] [ ] [ ] the finding of narcolepsy in several european countries following vaccination with pandemrix caused the european medicines agency to recommend restricting use of pandemrix. pandemrix was not licensed for use in the united states and no adjuvanted influenza vaccines were used in the united states during the influenza pandemic. the cdc published a study on a possible association between u.s.-licensed unadjuvanted h n influenza vaccines, - seasonal influenza vaccines, and narcolepsy. the analysis included more than , people who received the pandemic influenza vaccine in and more than , people who received the seasonal influenza vaccine in - . the study found that neither vaccine was associated with an increased risk for narcolepsy. additional studies including those conducted by the vaccine manufacturer will help discern whether this finding is attributable to the adjuvant, the h n vaccine antigen, or both. the safety of an adjuvanted vaccine formulation has to be demonstrated in adequate and well-controlled prelicensure safety studies. safety information supporting licensure of an adjuvanted vaccine may include the safety experience obtained from domestic or foreign trials conducted using the adjuvanted vaccine formulation. in addition, safety experience with the same adjuvant formulated with other vaccine antigens may also contribute to the safety evaluation of the adjuvant. early in clinical development (e.g., phase i and phase ii clinical trials), supportive safety data may be derived by comparing the adjuvanted vaccine to a placebo or the unadjuvanted vaccine antigen, if feasible. safety follow-up of human subjects administered vaccine with novel adjuvant is typically longer than for nonadjuvanted vaccines (e.g., months rather than months) and includes specific inquiries regarding symptoms consistent with autoimmune and neuro-inflammatory diseases. furthermore, the safety database required to support licensure of a vaccine formulated with novel adjuvant may be larger than for unadjuvanted vaccines. in summary, regulatory pathways supporting development and approval of vaccines formulated with novel adjuvants are similar to those for unadjuvanted vaccines. efficient planning of the development pathway for any adjuvanted vaccine requires careful attention to preclinical testing, study design, dosing decisions, and safety monitoring. although manufacturers are not required to demonstrate the "added benefit" of adjuvanted versus unadjuvanted vaccines in clinical comparative phase iii studies, manufacturers should provide a justification for including an adjuvant in the vaccine. lastly, evaluation of safety of an adjuvanted vaccine needs to include special safety considerations. immunization programs in the united states have been remarkably effective at reducing morbidity and mortality from the most common, naturally transmitted infectious diseases, such as polio, measles, and diphtheria. however, emerging conducted according to applicable regulations including the requirement for nonclinical testing and protection of human subjects. these efforts resulted in rapid availability of safety and immunogenicity data for the leading ebola vaccine candidates, initiation of u.s. government-sponsored phase iii clinical studies to demonstrate the safety and effectiveness of these vaccines in individuals residing in outbreak areas, and collection of data supporting licensure of these products. to enable an initiation of these pivotal studies in an expedited manner, the fda engaged in joint reviews with vaccine manufacturers, the who, regulatory agencies in europe and canada, and west african regulators to discuss study designs, ethical considerations, and required product information to reach international regulatory convergence, thus facilitating trial initiation. it is not always possible to test whether a vaccine or treatment will work against a new or emerging infectious disease, or against a biothreat, because the threat may be rare or even nonexistent at the time the vaccine or therapy needs to be developed. moreover, many vaccines against eids and biothreats pose difficult challenges with regard to obtaining clinical efficacy data. for many of these infectious agents or toxins, human efficacy trials are not feasible because natural exposure no longer occurs (e.g., smallpox), occurs at a very low incidence, or occurs in an unpredictable manner. the animal rule described earlier (see "accelerating availability of vaccines and pathways to licensure") is one regulatory mechanism that allows the fda to address the challenges of obtaining clinical efficacy data for these products if the results of adequate and well-controlled animal studies establish that the product is reasonably likely to provide clinical benefits to humans. animal testing is often the only available option, but many diseases lack even good animal models, and animal studies are technically difficult to conduct and typically limited in size. consequently, regulatory science is needed to develop and validate improved predictive models. regulatory science can also support the identification and validation of surrogate measures of product efficacy. biomarkers that predict efficacy are not yet available for most terrorism threats, emerging pathogens or major global infectious diseases. efforts to develop, refine, and validate new biomarkers may lower development costs and improve and speed the development of safe and effective products for unmet public health needs. in summary, for licensure, an eid vaccine product, just as for any product, must have an acceptable quality, safety, efficacy, and potency profile. likewise, production and quality control also must be in compliance with cgmps. however, vaccines against eids and biothreats agents present unique issues for clinical development and evaluation by the fda. overall planning and coordination among the fda and its national and international partners is necessary to move these products toward licensure and into distribution. fda guidance and engagement with partners is critical to make sure these products can move from the future into the present. the primary responsibility of nras is to ensure the quality, safety, and effectiveness of pharmaceutical products. the implementation of a strong regulatory system will facilitate these goals, which are especially critical for vaccines that are inherently more difficult to develop, characterize, and manufacture than most pharmaceutical products. the fda has developed a managed review process that provides regulatory oversight through all phases of vaccine development. advances across a wide range of scientific disciplines have enhanced the preparations to treat certain complications of smallpox vaccination have been approved. there is one licensed anthrax vaccine in the united states, anthrax vaccine adsorbed (emergent biosolutions' biothrax). there are significant scientific and regulatory challenges associated with developing and testing new vaccines against eids and biothreat agents. vaccines against eids are more likely to use novel technologies, and the science behind these technologies may be more complex; for example, use of novel cell substrates, the need to develop alternative potency assays, and the need to identify surrogate markers in humans or animals that predict vaccine effectiveness. to respond to this challenge, the fda collaborates with interagency groups within the department of health and human services, such as the biomedical advanced research and development authority, the cdc, and the nih, as well as the department of defense and department of homeland security to prepare for responding to an emergency. a committed, continuous investment in regulatory science is essential to producing medical countermeasures against public health threats. as noted in the public health emergency medical countermeasures review, "enhancement and ultimate application of updated regulatory science and scientific review capacity will help strengthen the mcm [medical countermeasure] regulatory process and thus streamline the mcm development process. [the] fda will undertake a new initiative designed to focus on augmenting the tools used to assess the safety, efficacy, and quality of medical products, with a particular focus on mcms, and to get them from concept through the approval process efficiently." an example of how the fda's regulatory science efforts have assisted the agency in facilitating the licensure of vaccines against emerging diseases and biothreats is the successful public-private partnership during the h n influenza pandemic, which resulted in the development and approval of safe and effective vaccines against the pandemic in record time. this included creating vaccine strains needed for vaccine manufacturing within weeks of the very first pandemic h n influenza cases appearing, developing reagents and tests through international collaborations to measure the vaccine's potency, consulting the fda's expert vaccine advisory committee to review the agency's approach to approval of the h n vaccine s as well as extensive in process quality control and product testing. licensure of vaccines against the h n influenza virus occurred in september based on the fda's determination that standards to ensure the safety and potency of these vaccines had been met. in parallel to these efforts, nih and vaccine manufacturers initiated clinical trials to determine the optimal vaccine dosage and number of doses needed to induce a protective immune response against pandemic h n virus. another example illustrating collaboration both within the department of health and human services and between the department and international partners is the response to the ebola virus disease outbreak in west africa in - that caused more than , cases of ebola virus disease and claimed the lives of more than , people, representing the most widespread epidemic of ebola virus disease in history. to address this need, the fda engaged with federal partners, sponsors of medical products, and international organizations to facilitate development of vaccines. to advance promising vaccine candidates to phase i clinical trials to obtain preliminary human safety data, the fda worked closely with manufacturers, clinical trial sponsors, and international regulators to rapidly assess product characterization data and protocols for these first human clinical trials and granted permission for the initiation of these phase i studies in an expedited manner. guidance provided by the fda assured that these studies were references for this chapter are available at expertconsult.com. prospects of developing new and better vaccines. novel vaccine approaches such as recombinant vaccines and novel adjuvants and delivery systems pose regulatory challenges for nras. however, nras should be dynamic and flexible entities, as they strive to develop regulatory requirements to address the evolving science. further, nras must be prepared to address public health emergencies that will require expedited approval mechanisms, such as biological terrorist events, pandemic influenza, and other eids. for industry: characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications draft guidance for industry: clinical considerations for therapeutic cancer vaccines guidance for industry: clinical data needed to support the licensure of pandemic influenza vaccine guidance for industry: clinical data needed to support the licensure of trivalent inactivated influenza vaccines guidance for industry: toxicity grading scale for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials draft guidance: emergency use authorization of medical products draft guidance for industry: characterization and qualification of cell substrates and other biological starting materials used in the production of viral vaccines for the prevention and treatment of infectious diseases guidance for industry: reports on the status of postmarketing studies-implementation of section of the food and drug administration modernization act of guidance for industry: clinical studies section of labeling for prescription drugs and biologics-content and format draft guidance for industry: labeling for human prescription drug and biological products: implementing the new content and format requirements guidance for industry: adverse reactions section of labeling for human prescription drug and biological products-content and format draft guidance for industry: inds guidance for industry: considerations for developmental toxicity studies for preventive and therapeutic vaccines for infectious disease indications guidance for industry: fast track drug development programs-designation, development, and application review guidance for industry: quality systems approach to pharmaceutical current good manufacturing practice regulations guidance for industry: providing regulatory submissions to the center for biologics evaluation and research (cber) in electronic format-lot release protocols guidance for industry: considerations for plasmid dna vaccines for infectious disease indications guidance for industry: development and use of risk minimization action plans draft guidance for industry: how to comply with the pediatric research equity act guidance for industry: fda review of vaccine labeling requirements for warnings, use instructions, and precautionary information guidance for industry: sterile drug products produced by aseptic processing current good manufacturing practice draft guidance for industry: vaccinia virus-developing drugs to mitigate complications from smallpox vaccination draft guidance for industry: postmarketing safety reporting for human drug and biological products including vaccines draft guidance for industry on recommendations for complying with the pediatric rule guidance for industry: formal meetings with sponsors and applicants for pdufa products guidance for industry: submitting and reviewing complete responses to clinical holds guidance for industry: content and format of chemistry, manufacturing and controls information for a vaccine or related product guidance for industry: providing clinical evidence of effectiveness for human drugs and biological products draft guidance for industry: stability testing of drug substances and drug products guidance for industry: implementation of section of the food and drug administration modernization act of -elimination of certain labeling requirements guidance for industry: environmental assessment of human drug and biologics applications guidance for industry for the evaluation of combination vaccines for preventable diseases: production, testing and clinical studies guidance for industry: changes to an approved application: biological products guidance on alternatives to lot release for licensed biological products food drug and cosmetic act. usc § title iii, § title iii, sec. , stat. , currently codified at usc § food and drug administration modernization act of food and drug administration amendments act of us fda review and regulation of preventive vaccines for infectious disease indications: impact of the fda amendments act food and drug administration safety and innovation act (fdasia) pediatric study plans: content of and process for submitting initial pediatric study plans and amended pediatric study plans. guidance for industry center for biologics evaluation and research. guidance for industry: expedited programs for serious conditions-drugs and biologics guidance for industry: clinical data needed to support the licensure of seasonal inactivated influenza vaccines guidance for industry: clinical data needed to support the licensure of pandemic influenza vaccines guidance for industry: characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications considerations for developmental toxicity studies for preventive and therapeutic vaccines for infectious disease indications food and drug administration a primer on cber's regulatory review structure and process code of federal regulations. title , part . . washington, dc, office of the federal register, national archives & records administration office of the federal register, national archives & records administration vaccines and related biological product committee (vrbpac) meeting transcript postmarketing studies for approved human drugs and licensed biological products; status reports vaccine safety: vaccine safety datalink (vsd) food and drug administration food and drug administration. cfr parts , , , , and . changes to an approved application. final rule food and drug administration guidance on alternatives to lot release for licensed biological products guidance for industry: characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications continuous cell lines-an international workshop on current issues recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks meeting of the vaccines and related biological products advisory committee (vrbpac) (transcript) office of the federal register, national archives & records administration guidance for industry: providing regulatory submissions in electronic format -content of labeling office of the federal register, national archives and records administration office of the federal register, national archives and records administration guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines guidance for industry: clinical data needed to support licensure of pandemic influenza vaccines guidance for industry: clinical data needed to support the licensure of seasonal inactivated influenza vaccines risk of narcolepsy in children and young people receiving as adjuvanted pandemic a/h n influenza vaccine: retrospective analysis increased childhood incidence of narcolepsy in western sweden after h n influenza vaccination the adjuvant component α-tocopherol triggers via modulation of nrf the expression and turnover of hypocretin in vitro and its implication to the development of narcolepsy narcolepsy in association with pandemic influenza vaccination: a multi-country european epidemiological investigation as adjuvanted ah n vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in finland european medicines agency recommends restricting use of pandemrix, ema/chmp/ . press release vaccine safety datalink. narcolepsy and influenza a(h n ) pandemic vaccination in the united states department of health and human services. the public health emergency medical countermeasures enterprise review: transforming the enterprise to meet long-range national needs i would like to acknowledge the employees in the office of vaccines research and review, office of biostatistics and epidemiology, and the office of compliance and biologics quality/cber for their assistance in the preparation of this manuscript. key: cord- - ggaqf authors: nan title: abstracts of the papers presented in the xix national conference of indian virological society, “recent trends in viral disease problems and management”, on – march, , at s.v. university, tirupati, andhra pradesh date: - - journal: indian j virol doi: . /s - - - sha: doc_id: cord_uid: ggaqf nan patients showed rashes on face, hand and foot. ev detection carried out in vesicular fluid, stool, serum and throat swab specimens by rt-pcr of ncr gene. serotyping was carried out by using rt-pcr of viral protein of vp / a junction region followed by sequencing and phylogenetic analysis using neighbor-joining-algorithm and kimura- parameter model of mega- software. overall ev positivity detected in hfmd patients from kerala, tamil nadu, west bengal and orissa states was found to be . %, . %, . % and . % respectively. typing of vp gene sequences indicated presence of ca- , ev- , echo- strains in kerala and ca- in west bengal, orissa and tamil nadu. phylogenetic analysis indicated ca- , ev- , echo- strains showed . - . % and - . % homology with japanese, australian and french strains. however, ca- strains were closer to malaysian strains with . - . % nucleotide homology. the present study documents the association of multiple types of ev's i.e., ca- , ev- , echo- and ca- strains contributing as prime viral pathogens in hfmd epidemics in the reported regions with new emergence of ca- circulating strain in kerala, india. tasgaon september . sera were collected from suspected hepatitis cases and there contacts and tested for anti hev igm/igg antibodies (elisa) and liver enzymes like alanine aminotransferase (alt). anti hev igm antibodies were detected in . % ( / ) of the suspected cases. the overall attack rate was . %. male to female ratio was : . majority ( . %) of the cases were in the age group - years and recovered without any clinical complications. weekly distribution of cases showed that the majority ( . %, / ) cases occurred between nd and rd week of june. dark urine ( . %), jaundice ( . %), fatigue ( . %), abdominal pain ( . %), anorexia ( . %), vomiting ( . %), fever ( . %), giddiness ( . %), diarrhoea ( . %) and arthalgia ( . %) were the prominent symptoms. sera collected from antenatal cases (ancs) showed anti hev igm antibody in . affected pregnant women had a normal outcome. a death of year, male hepatitis e case was reported during the outbreak period that had cirrhosis of liver with oesophageal varices. sanitary survey revealed that water pipelines were laid down in close proximity of sewerage system, and water posts were without tap. these are the likely sources of faecal contamination of water supplies. among water samples collected from various places, were found to be unfit for drinking based on the routine bacteriological tests conducted at state public health laboratory, pune. no case occurred after the pipelines were repaired. this typical outbreak of hepatitis e re-emphasizes need for proper water supply/sewage disposal pipelines and adequate maintenance measures. jayanthi shastri, nilima vaidya, sandhya sawant, umesh aigal department of molecular biology, kasturba hospital for infectious diseases, mumbai, india dengue and dengue haemorrhagic fever are amongst the most important challenges in tropical diseases due to their expanding geographic distribution, increasing outbreak frequency, hyperendemicity and evolution of virulence. the gobal prevalence of dengue has grown dramatically in recent decades. who estimates - million cases of dengue virus infections worldwide every year resulting in , to , cases of dhf and , deaths each year. public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. laboratory diagnosis of dengue virus infection can be made by the detection of specific virus, viral antigen, genomic sequence and/or antibodies. currently basic methods used by laboratories for diagnosis of dengue virus infection are virus isolation and characterisation, detection of genomic sequence by nucleic acid amplification technology assay and detection of dengue virus specific antibodies/antigen. molecular diagnosis based on reverse transcription (rt)-pcr s.a. one step or nested pcr, nucleic acid sequence based amplification (nasba), or real time rt-pcr, has gradually replaced the virus isolation method as the new standard for the detection of dengue virus in acute phase serum samples. several pcr protocols for detection have been described that vary in the extraction method, genomic location of primers, specificity, sensitivity and the methods to determine the products and the serotype. pcr-based dengue tests, due to the specificity of amplification, enable a definitive diagnosis and serotyping of the virus. in addition dna sequencing of the amplification product enables the virus to be genotyped, providing important information on the sources of infection. more recently tests have incorporated flurogenic probe, so called taq man technology for the specific real time detection of dengue - amplicons. product is detected by a specific oligodeoxy nucleotide probe that is labelled with carboxy-fluorescein (fam). this technology offers the advantage of being both rapid and potentially quantitative. second, the detection of product by hybridisation of flurochrome labelled probes increases specificity. third, as the product is detected without the need to open the reaction tube, the risk of contamination by product carry over is minimised. the advantages of speed, contamination minimisation and reduced turn around time justify application of this assay over the currently used nested pcr assay. during the period january to october , molecular laboratory received samples from patients presenting with acute onset fever for dengue . %) samples were tested positive by this method. the disease peaks in the monsoon season with a percentage of . %. rapid tests, igm and igg capture elisa are popularly used tests for diagnosis of dengue infection. its utility is limited for diagnosing dengue in convalescecce ( - days) . specificity is also compromised due to infections with flaviviruses: japanese encephalitis and chikungunya. dengue ns ag elisa with its cost effectiveness, specificity and sensitivity should be considered as the test of choice for diagnosing dengue in the acute phase of illness in the developing countries. molecular diagnosis enables confirmatory diagnosis of dengue in the acute phase of the illness and is suitable for further typing methods. assistant general manager and r&d coordinator, division of quality control and r&d, bharat immunologicals and biologicals corporation ltd., village chola, bulandshahr, up vaccine development in india, though slow to start, has progressed by leaps and bounds in the past years. it was dependent on imported vaccines but now it is not only self-sufficient in the production of vaccines conforming to international standards with major supplier of the same to unicef. the role of drug authorities is to enhance the public health by assuring the availability of safe and effective a indian j. virol. (september ) (suppl. ):a -a vaccines, allergenic extracts, and other related products. vaccine development is tightly regulated by a hierarchy of regulatory bodies. guidelines provided by the indian council of medical research (icmr) set the rules of conduct for clinical trials from phase i to iv studies as well as studies on combination vaccines. these guidelines address ethical issues that arise during a vaccine study. a network of adverse drug reaction (adr) monitoring centers along with the adverse events following immunization (aefi) monitoring program provide the machinery for vaccine pharmacovigilance. genetic modifications have been developed to develop effective and cheaper vaccines by the use of recombinant technology. to ensure safety of consumers, producers, experimental animals and environment, governments all over the world are following regulatory mechanisms and guidelines for genetically modified products. as with other industrializing countries undergoing rapid shifts, india clearly recognizes the need to restructure its regulatory system so that its biopharmaceutical industry can compete in international markets. genetic engineering approval council (geac), recombinant dna advisory committee (rdac), review committee on genetic manipulation (rcgm), institutional biosafety committees (ibsc) are responsible for development, commitment for parameters and commercialization of recombinant vaccines. to centralize and coordinate the whole system, government has taken to form two agencies to regulate the regulation laws to develop recombinant pharmaceuticals products including vaccines. the first is the creation of the national biotechnology regulatory authority (nbra), under the department of biotechnology (dbt), as part of india's long-term biotech sector development strategy. the second major initiative will affect the entire indian pharmaceutical industry. this is the replacement of most state, district, and central drug regulatory agencies with a single, central, fda-style agency, the central drug authority (cda). the cda is expected to have separate, semi-autonomous departments for regulation, enforcement, legal, and consumer affairs; biotechnology products; pharmacovigilance and drugs safety; medical devices and diagnostics; imports; quality control; and traditional indian medicines. it will set up offices throughout india and will be paid for inspection, registration, and license fees. its enforcement powers will be strengthened by a new law increasing the criminal penalties for illegal clinical trials. in the manufacturing area, though, the country has been tightening the rules and enforcement. an amendment to the regulations, ''schedule m'' of the drug and cosmetics act, now specifies the good manufacturing practice (gmp) requirements for factory premises and materials. these requirements were modeled after us fda regulations, to improve regulatory coordination between indian and us regulators. india has realized the importance of regulations in pharmaceutical specially in vaccine field but it will take several years to implementation of these. india has coordinated some of its regulatory functions with western organizations. the us pharmacopoeia established an office in hyderabad in . a representative of the indian pharmaceutical lobby also recently has expressed openness to an expansion of the fda's oversight of indian manufacturing. as india expands its global drug and biologicals production, us and europe, as the world's largest drug importers, will likely expand their regulatory support in the development of the country's regulatory systems. rapid diagnosis of japanese encephalitis virus (jev) infections is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. however, the speed and accuracy of diagnosis must be balanced against test cost and availability, especially in developing countries. an antigen capture enzyme-linked immunosorbent assay (elisa) for detection of circulating jev specific nonstructural protein (ns ) was developed by using monoclonal antibodies (mabs) specific to recombinant (ns ). the applicability of this jev ns antigen capture elisa for early clinical diagnosis was evaluated with acute phase serum/ cerebrospinal fluid (csf) specimens collected from different epidemics during [ ] [ ] [ ] . jev ns antigen was detected in circulation from day to . the sensitivity and specificity of jev ns detection in serum/csf specimens with reference to reverse transcriptase pcr was %, and . % respectively. no crossreactions with any of the other closely related members of the genus flaviviruses (dengue, westnile, yellow fever and saint louis encephalitis (sle) viruses) were observed when tested with either clinical specimens or virus cultures. these findings suggested that the reported jev specific mab-based ns antigen capture elisa will be a rapid and reliable tool for early confirmatory diagnosis as well as surveillance of je infections in developing countries. manmohan parida the recent emergence of a novel human influenza a virus (h n ) poses a serious global health threat. the h n virus has caused a considerable number of deaths within a short duration since its emergence. a two-step single tube accelerated rapid real-time and quantitative swine flu virus specific h rtlamp assay is reported by targeting the h gene of the novel h n hybrid virus. the feasibility of swine flu h rtlamp for clinical diagnosis was validated with a panel of suspected throat wash samples comprising confirmed positive and confirmed negative cases of ongoing epidemic. the comparative evaluation of h specific rtlamp assay with real-time rt-pcr demonstrated exceptionally higher sensitivity by picking up all the h n positive and additional positive cases amongst the negatives that were sequence confirmed as h n . none of the real-time rtpcr positive samples were missed by rtlamp system. the comparative study revealed that rtlamp was -fold more sensitive than rtpcr with a detection limit of copy number. these findings suggested that rtlamp assay is a valuable tool for rapid, real-time detection as well as quantification of h n virus in acute phase throat swab samples without requiring any sophisticated equipments. because of its recurrent nature. despite considerable progress in understanding of the virus at cellular and molecular levels, the proper management of the disease in its different stages is still a dilemma particularly whether to use antiviral or steroids or both. the risk of using steroids with its attendant complications has to be weighed against the risk of progression of the disease if avoiding the use of steroids. this dilemma can be reduced to a considerable extent if basic principles of virology and pathogenesis are kept in mind. this article reviews current concepts of virological and clinical aspects of hsv keratitis to enable a broad understanding of the disease process. it is recognized several influential host factors including the fact that hsk is more common in men than women. it is observed that the ability of hsv to establish latent infection in sensory neurons and possibly cornea, but have as yet been unable to use this knowledge to prevent the disease limitations. acknowledging limitations may further stimulate application of laboratory knowledge in coping with hsk which constitutes to present major challenge in terms of management. mvo- study on effect of human bhsp in immunity of hcv core protein and hbv hbsag there are more than million individuals with hepatitis b and c in the world. in spite of vaccination in the different areas there are several reports about patients who got vaccine before. also there is not efficient vaccine against of hepatitis c and one of the important problems in vaccine project is development of effective and suitable adjuvant in human vaccines. at present research we applied human bhsp protein as adjuvant and chaperon. this protein injected to balbc mice as adjuvant together with recombinant proteins of hcv core and hbv hbsag. then humoral and cellular immune systems of the mice were studied. core and hbsag genes were cloned into petduet- vector and thermal vector of pgp - was used for human heat shock protein expressions. the different combination of these three proteins was injected to mice and we evaluated the total igg and igg a of mice serums after a week. two weeks after booster injection, we studied the proliferation and cytokine secretion of spleen, inguinal and popliteal lymph nodes lymphocytes in vitro and ex vivo conditions. so the core/hbsag + hsp and core + hbsag + hsp complexes induced total igg and igg a secretion. the spleen lymphocytes proliferation were increased equal to serum igg a level that was constant in second time bleeding with significant different to complexes with freund's adjuvant. at first il- and il- cytokines were increased and then decrease of il- meaned no hypersensitivity. the chaperon effect of hsp on structure of core and hbsag proteins was studied by cd and flourometer. it could fold the proteins after heating and unfolding. hepatitis b virus (hbv) infection is vaccine preventable global public health problem. all commercially available vaccines contain one or more of the recombinant hepatitis b envelope protein or surface antigen (hbsag). measurement of antigen responsible for immunogenicity of vaccine is central to quality assessment. the problems associated with the use of a polyclonal antibody in an assay with regard to its poorly defined nature and batch-to-batch variation has been mitigated by the use of mabs as described in this paper. the initial capture of hbsag by the mab could orientate it such that the same antibody could bind to it as a detection antibody after labeling with out steric hindrance. the development of an immuno-capture elisa (ic-elisa) to measure the hbsag content using a monoclonal antibody (mab) specific to determinant ''a'' of hbsag in the experimental vaccine formulations is being discussed. murine mabs developed against hbsag, subtype adw were found to cross-react with the other subtypes viz. ad and ay too. the mabs have been characterized following which, one mab hbs was chosen for developing ic-elisa format for the quantification of the hbsag in the final algel adsorbed vaccines. the unadsorbed hbsag was used to establish the standard curve of hbsag/a. the elisa had a sensitivity of ng/ml of hbsag. the recovery rate of hbsag/a was found to be around % in the vaccines treated to desorb the antigen from algel. twenty seven experimental batches of monovalent hepatitis b vaccines were analyzed for the hbsag content, both by ic-elisa and a commercial kit (axsym kit, abbott laboratories, usa). the statistical analysis of ic-elisa results indicated that an experimental equation f(x) = . (x) + . , could precisely estimate the amount of hbsag in the adsorbed vaccines. the amounts of hbsag recovered from the adsorbed vaccines as estimated by the ic-elisa format had a good correlation with the estimates derived from a commercial kit, which is being used by several vaccine manufacturers in india for the quality control of vaccine antigen. the varying amounts of vaccine antigens that could be recovered seemed to depend on the quality of the hbsag and the methods of hbsag adsorption to the alum gel during vaccine manufacture. epidemiology of the spread of h n virus. children of school going age have become victim of this deadly virus as evident from the reporting data generated in the past few weeks. the mortality rate has also been slightly increased. the disease spread in wave pattern and presently the world is passing through the second wave of pandemic with more severity in young and otherwise health people with a predilection for lungs leading to viral pneumonia and respiratory failure. now the pandemic gained hold in the developing world affecting more severely as millions of people live under deprived conditions having multiple health problems, with little access to basic health care. current data about the pandemic from developed counties need to be very closely watched in relation to shift in virus sub type, shift of the highest death rate to younger populations, successive pandemic waves, higher transmissibility than seasonal influenza, and demographic differences etc. presently the world appears to be better prepared. vaccine is available in market in many countries. even vaccine trials are actively going on in indian population. effective antivirals are available. although till now h n diagnostic centers worked with cdc/who recommended h n specific primer, probes with taqman chemistry by real time pcr, efforts on the development of indigenous diagnostics, vaccines and chemoprophylaxis is going on to have a better combat against this highly infectious virus. were positive for rotavirus infection by either page or elisa methods. the available data highlights the importance of rotavirus as a cause of diarrhea in children, which is severe enough to deserve specialized care. the observed proportion of . % of all diarrhea cases being associated with rotavirus falls within the range of values reported by other workers. the reported positivity varies from . to . %. in our study a complete concordance of elisa and page results were observed in ( %) of the tested specimens. this finding closely correlates with the findings of other authors who found a . - . % concordance results between elisa and page methods. some authors found rna-page method that is as sensitive and rapid as elisa for detecting rotavirus in stool samples of cases of diarrhea and some others proposed elisa is more sensitive than page method fond to be % specific. the remaining ( %) samples showed conflicting results. in a lone sample in which the od value of elisa test was . , this value was almost at the cutoff level, the possibility of this sample being positive by elisa test is doubtful. negative result of the same sample in page method is difficult to explain, the possibility of presence of lot of empty virus particles or due to low concentration of viral rna in the fecal specimen and insufficient extraction of viral rna could be possible. on the other hand, of the samples which gave positive results by page method were negative by elisa test. these samples had a typical - - - rna pattern. the reason for their being elisa negative thus remains unexplained, however blocking factors or the presence of inhibitory substance in stools might have been responsible. the samples containing predominantly complete particles can also give false negative results. since, the group antigen is not exposed. earlier studies have also reported page to be the most sensitive technique although some are of view that it is laborious procedure. how ever, the page system used in this study was very simple to perform and the results were available on the same day. the main requirement was of trained personnel and proper standardization of the technique. most reports states that the greatest advantage of page and silver stain method are its lack of ambiguity and the fact that it provides information about viral electropherotypes. the modified page system was thus found to be reliable, simple and rapid, no expensive reagents were required. locally available reagents from hi media were used. the cost of the chemical for page per specimen was rs. approximately as compared to rs. per test by confirmatory elisa. a locally produced slab gel electrophoresis system with power pack was the only equipment required. this method could be used for the routine diagnosis of rotavirus infection in the laboratory. vaccine, rapid diagnosis plays an important role in early management of patients. in this study a qc-rt-pcr assay was developed to quantify chikungunya virus rna by targeting the conserved region of e gene. a competitor molecule containing an internal insertion was generated, that provided a stringent control of the quantification process. the introduction of -fold serially diluted competitor in each reaction was further used to determine sensitivity. the applicability of this assay for quantification of chikungunya virus rna was evaluated with human clinical samples and the results were compared with real-time quantitative rt-pcr. the sensitivity of this assay was estimated to be rna copies per reaction with a dynamic detection range of to copies. specificity was confirmed using closely related alpha and flaviviruses. the comparison of qc-rt-pcr result with real-time rt-pcr revealed % concordance. these findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of chikungunya virus in acute-phase serum samples. in india, measles vaccine was introduced as part of expanded programme of immunization in . measles, mumps and rubella (mmr) vaccine is still not part of the national immunization schedule of india. the indian association of paediatrics (iap) recommends measles vaccine at months of age and mmr vaccine at - months. however, in a recent policy update, iap committee on immunisation opined that there is a need for a second dose of mmr vaccine for providing adequate immunity against mmr. the aim of the present study was to assess the extent of sero-protection against mmr at - years of age in children who have received one dose of mmr between and months of age. an attempt has also been made to assess the sero-response to the second dose of mmr vaccine in - years old children. a total of consecutive children between the ages of - years who had received mmr vaccine between and months of age and attending the immunization clinic of gtb hospital, delhi were enrolled. the vaccination status, anthropometry and physical examination findings were recorded. three ml of venous sample was again withdrawn for estimation of post vaccination antibody titre. it was observed that . %, . % and . % children were seroprotected for mmr respectively after . - . year of receiving first dose of mmr vaccine. seroprotection rose to . %, % and % for mmr respectively after - weeks of receiving second dose of mmr vaccine. geometric mean concentration of antibody also rose significantly in all three diseases. in view of low seroprevalence of mmr and hence high susceptibility to infection at - years of age, who have already received mmr vaccine, there is need to boost the immune responses against these three diseases by giving a second dose of mmr vaccine. baseline information on the epidemiology of viral agents causing stis and types of risk behaviour of affected persons are essential for any meaningful targeted intervention. the present study documents the pattern of viral stis in patients attending a tertiary care hospital, correlating the syndromic approach and the laboratory investigations to determine the aetiology. three hundred consecutive patients attending the sti clinic were diagnosed and categorized according to the syndromic approach of the who along with detailed history and demographic data. majority of the patients were men ( . %) with a mean age of years. men received education up to middle school. half of the female subjects were illiterate. sixty percent of the patients were married and among these, % were regular condom users. first sexual contact at or before years of age was more in men ( % vs. . % in women). promiscuity was more among male patients who had contact with csw. genital herpes was the commonest viral sti ( / ) followed by genital wart ( / ). concomitant infection with more than one virus was seen in % of patients. hiv was prevalent in . % of sti patients. hepatitis b, hepatitis c, herpes simplex type and molluscum contagiosum were the other viral agents seen in sti clinic attendees at our centre. this disease currently prevalent in more than countries world wide and annually - million people are infected with dengue virus among which . - lakhs cases were dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) which are serious forms of dengue virus infection and due to this condition , deaths might occur annually world wide and approximately million children were hospitalized for the fast decades. this disease is characterized by sudden onset of high fever with sever headache, pain in the back and limbs, lymphadenopathy macuolo-papulur rash over the skin and retro-bulbar pain. early diagnosis can be established with simple and rapid lgg/ gm antibodies detection in the blood samples of the patients based on the bi-directional immunoassay system for its management and control to reduce morbidity and mortality. details will be presented. myocarditis and dilated cardiomyopathy (dcm) are common causes of morbidity and mortality both in children and adults. the most common viruses involved in myocarditis are coxsackievirus b or adenovirus. recently, the coxsackievirus and adenovirus receptor (car), a common receptor for coxsackieviruses b , b and adenoviruses , has been identified. increased expression of car has been reported in patients with dcm suggesting utilization of car by these viruses for cell entry. the present study was designed to study the expression of car in myocardial tissue of patients with dcm. formalin fixed myocardial tissues were obtained from autopsy cases. a total of cases of dcm and cases of controls which included non-cardiac (group-a) and cardiac disease other than dcm (group-b) were included in the study. expression of car was studied by immunohistochemical staining of myocardial tissue using car specific rabbit polyclonal antibody and biotin conjugated secondary antibody. the tissue sections were considered positive when[ % of the cell showed brown color staining by immunohistochemistry (ihc). the car positivity in dcm cases was found to be % ( / ) as compared to % in control group a and % in control group b respectively. the car positivity was significantly higher in the test group as compared to both the control groups. further car positivity in all the cellular types (myocytes, endothelial cells and interstitial cells) was found significantly higher in test group as compared to both the control groups. the expression of car was significantly higher in myocytes as compared to both endothelial and interstitial cells in all the groups. however, no significant difference was observed in car positivity between endothelial and interstitial cells. the present study highlights the increased expression of car in dcm cases with further significance of car expression in myocytes and endothelial cells. this may help further in understanding the tropism of viruses or cellular susceptibility, which in turn will help in appropriate diagnostic and therapeutic approach in management of viral myocarditis and dcm cases. food security and safety vary widely around the world, and reaching these goals is one of the major challenges, raising public concern for the wellbeing of mankind, in particular. industrialized production and processing as well as improper environmental protection have clearly shown severe limitations such as worldwide contamination of the food chain and water. contaminated water and food during the processes of production, processing and handling are essentially responsible for food and water borne viral infections/diseases. the cases of viral food borne outbreaks are on the rise, creating a threat to human health. recent researches indicate that epidemiological studies are meager to focus the frequently contaminated foods and food borne viral diseases. current paper projects the etiology of select food borne viral diseases, probable reasons for non availability of appropriate methods to detect the viruses responsible for the diseases, routes of water and food borne transmission of enteric viral infections, currently available methods of detection of select viruses and bio safety measures to prevent food borne viral infections. dietary/nutritional management in food borne viral diseases is crucial to control weakness and gastro enteric intolerance due to disease condition and antibiotic therapy. it will principally improve food intake, resulting in better nutritional status leading to optimum immune response. food borne viruses are mainly belong to rotaviruses, enteropathogenic viruses, astroviruses, adenoviruses and caliciviruses, causes acute gastroenteritis (ag) which is an important health problem. the frequency of rotavirus as a cause of sporadic cases of ag ranges between . % and . %. astroviruses cause ag, with a frequency ranging between and %: outbreaks have been described in schools and kindergartens, but also in adults and the elderly. the frequency of identification of adenoviruses and as causes of sporadic ag in non-immuno suppressed children ranges between . % and . %, although there is probably underreporting because the sensitivity of conventional techniques is low. caliciviruses are separated phylogenetically into two genera: norovirus and sapovirus. norovirus is frequently associated with food-and water-borne outbreaks of ag. it is estimated that % of cases of ag due to norovirus are food borne. in sweden and some regions of the united states, norovirus is the first cause of outbreaks of food borne diseases. sapovirus outbreaks due to person-to-person and food borne transmission affecting both children and adults have recently been reported in countries such as canada and japan. it has been predicted that the importance of diarrhoeal disease, mainly due to contaminated food and water, as a cause of death will decline worldwide. evidence for such a downward trend is limited. this prediction presumes that improvements in the production and retail of microbiologically safe food will be sustained in the developed world and, moreover, will be rolled out to those countries of the developing world increasingly producing food for a global market. sustaining food safety standards will depend on constant vigilance maintained by monitoring and surveillance but, with the rising importance of other food-related issues, such as food security, obesity and climate change, competition for resources in the future to enable this may be fierce. in addition the pathogen populations relevant to food safety are not static. food is an excellent vehicle by which many pathogens (bacteria, viruses/prions and parasites) can reach an appropriate colonization site in a new host. although food production practices change, the well-recognized food-borne pathogens, such as salmonella spp. and escherichia coli, seem able to evolve to exploit novel opportunities, for example fresh produce and even generate new public health challenges, for example antimicrobial resistance. in addition, previously unknown food-borne pathogens, many of which are zoonotic, are constantly emerging. awareness and surveillance of viral food-borne pathogens is generally poor but emphasis is placed on norovirus, hepatitis a, rotaviruses and newly emerging viruses such as sars. it is clear that one overall challenge is the generation and maintenance of constructive dialogue and collaboration between public health, veterinary and food safety experts, bringing together multidisciplinary skills and multi-pathogen expertise. such collaboration is essential to monitor changing trends in the well-recognized diseases and detect emerging pathogens. it is also necessary to understand the multiple interactions between these pathogens and their environments during transmission along the food chain in order to develop effective prevention and control strategies. to analyse the effectiveness of these sirnas targeting rabies virus l gene, the bhk- cells expressing sirnas in shrna form were produced by transduction of cells with radv-l. the transduced bhk- cells expressing sirna were infected with rabies virus pv- strain. there was reduction in rabies virus multiplication as analysed by reduction in fluorescent foci forming unit (ffu) count by . % ( ffu in bhk- cells expressing sirna-l compared to ffu in bhk- cells expressing negative sirna). the expression of l gene mrna was reduced by . fold in rabies virus infected radv-l transduced cells compared to radv-neg transduced cells (negative control) as detected using real-time pcr. after analyzing the effectiveness of radv-l in vitro, its effectiveness was also evaluated in vivo in mice after virulent rabies challenge. the mice were inoculated with plaque forming units (pfu) of radv-l in masseter muscle (i/m route) and challenged with ld rabies virus challenge virus standard (cvs) strain. the results indicated % protection with improved median survival from to days compared with group of mice treated with radv-neg. the results of this study indicated that sirnas targeting rabies virus polymerase (l) gene delivered through adenoviral vector inhibited rabies virus multiplication in vitro and in vivo. and were successfully produced and purified from the infected spodoptera frugiperda (sf- ) cells using these recombinant baculovirus. the morphology of the vlps was validated by electron microscopy in comparison to the authentic bt virions. the vlps produced here were stable and were highly immunogenic with intact outer layer which is rapidly lost during normal infection of btv. these btv-vlps elicited long lasting protective immunity in vaccinated sheep against virulent virus challenge. with the use of btv-vlps it was also possible to differentiate the infected and vaccinated animals (diva). vlp-based btv vaccine has potential advantages with regard to controlling the spread of btv with multiple serotypes. it is possible to produce milligram quantities of correctly folded and processed protein complexes using this baculovirus expression system and hence it is a more promising system for producing new generation vaccines like vlp subunit vaccine against any viral diseases in large scale. peste des petits ruminants (ppr), goatpox and orf are oie notifiable diseases of small ruminants especially goat and sheep. these diseases are economically important, in enzootic countries like india and cause significant loss and are major constraints in the productivity. considering the geographical distribution of ppr, goat pox and orf infections and prevalence of mixed infection, in the present study, safety and potency of the experimental triple vaccine comprising attenuated strains of thermostable-ppr virus (pprv jhansi, p- ) grown at °c, high passaged goat poxvirus (gtpv uttarkashi, p ) and attenuated orf virus (orfv mukteswar, p ) was evaluated in sub-himalayan local hill goats. goats simultaneously immunized with ml of vaccine consisting of either tcid or tcid of each of pprv, gtpv and orfv were monitored for clinical and serological responses for a period of - weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to pprv, gtpv and orfv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, snt, respectively following immunization. all the immunized animals resisted infections when challenged with virulent strains of either gtpv or pprv or orfv on day dpi, while in contact control animals developed characteristic signs of respective disease. further, ppr viral antigen could be detected by using ppr sandwich elisa kit in the excretions (nasal, ocular and oral swab materials) of unvaccinated control animals after challenge but not from any of the immunized goats. triple vaccine was found safe at dose as higher as tcid and induced protective immune response even at lower dose ( tcid ) in goats, which was evident from sero-conversion as well as challenge studies. the study indicated that these viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this triple vaccine in combating these infections simultaneously. toll like receptors (tlrs), primary sensors of microbial origin, plays a crucial role in the innate immunity. till now mammalian tlrs have been identified, while there is no information available on tlrs of yak. this study is part of world bank funded-icar project. yak, named bos grunniens for its distinctive vocalization and relationship with cattle, is natural habitant of extremely cold environment. when these animals comes to a lower altitude grazing land, adjacent to villages, become susceptible to the diseases of cattle, buffalo etc. thus, present study was undertaken to with genetic characterization and evolutionary lineage analysis of yak tlrs. we worked on tlr gene, which plays an important role in recognition of ssrna viruses. total rna was extracted from mitogen stimulated pbmcs of yak. the rt-pcr conditions were standardized for full length amplification of tlr gene using specific self designed primers. the expected amplicon of bps was obtained. it was cloned in pgemt-easy vector followed by transformation in e. coli top strain. the recombinant clones were screened, picked up for plasmid isolation and release of tlr was confirmed by restriction digestion. the cloned tlr product was sequenced and analyzed for the nucleotide and deduced amino acid sequences, and d structure analysis. the results revealed that yak shows more than % sequence homology with other bos indicus breeds and bos taurus breeds. however, identity was less than % with other animal species (equine, murine, feline, canine etc.). the evolutionary lineage findings cluster yak more closely with bovine species. point mutations revealed changes at nucleotide positions with corresponding amino acid change at positions. smart analysis of yak protein domain architecture revealed toll-interleukin i receptor (tir), leucine rich repeats (lrr) and signal peptide region. the variations in yak mainly lie in the lrr region. homology modeling revealed horse shoe shaped structure with alpha helix. the additional alpha helix present in bos indicus was not detected in yak. the present study shows existence of genetic variability in tlr gene of yak, in particular the lrr region, which plays an important role in the pathogen recognition and the evolutionary lineage analyses shows its closeness with other bovine species. a.p. aquaculture and fisheries, tirupati in this new millennium, aquatic animal health management strategies in asia expanded and adjusted to the current disease problems faced by the aquaculture sector. this presentation will briefly discuss some of the most serious trans-boundary pathogens affecting asian aquaculture including a newly emerging disease and highlight recent regional and national efforts on responsible health management for mitigating the risks associated with aquatic animal movement. a regional approach is fundamental since many countries share common social, economic, industrial, environmental, biological and geographical characteristics. capacity and awareness building on aquatic animal epidemiology, science-based risk analysis for aquatic animal transfers, surveillance and disease reporting, disease zoning and establishment of aquatic animal health information systems to support development of national disease control programs and emergency response to disease outbreaks are needed. molecular diagnostics with emphasis towards standardization and harmonization, inter-calibration exercises and quality assurance in laboratories, accreditation program and utilization of regional resource centres on aquatic animal health will also be needed. whilst most of these strategies are directed in support of government policies, implementation will require pro-active involvement, effective cooperation and strategic networking between governments, farmers, researchers, scientists, development and aid agencies, and relevant private sector stakeholders at all levels. their contributions are essential to the health management process. generally, aquaculture plays an important role in economy as harvests from natural waters have declined or, at best, remained static in most countries. fish and shrimp, the main aquaculture product sources, have gained the most attention. many factors can cause losses in yields of fish products and infectious disease in fish and shrimp is the biggest threat to the fishery industry. shrimp and fish aquaculture has grown rapidly over several decades to become a major global industry that serves the increasing consumer demand for seafood and has contributed significantly to socio-economic development in many poor coastal communities. however, the ecological disturbances and changes in patterns of trade associated with the development of shrimp and fish farming have presented many of the pre-conditions for the emergence and spread of disease. shrimp and fish are displaced from their natural environments, provided artificial or alternative feeds, stocked in high density, exposed to stress through changes in water quality and are transported nationally and internationally, either live or as frozen product. these practices have provided opportunities for increased pathogenicity of existing infections, exposure to new pathogens, and the rapid transmission and trans boundary spread of disease. not surprisingly, a succession of new viral diseases has devastated the production and livelihoods of farmers and their sustaining communities. this review examines the major viral pathogens of farmed shrimp and fish, the likely reasons for their emergence and spread, and the consequences for the structure and operation of the shrimp farming industry. in addition, this review discusses the health management strategies that have been introduced to combat the major pathogens and the reasons that disease continues to have an impact, particularly on poor, smallholder farmers in asia. btv isolates from the same geographic region have been termed as 'topotypes' and initial observation on segment nucleotide sequences identified a correlation between topotypes and genetic information. later topotyping was proposed based on segment , on the premise that the encoding protein ns , which is involved in virus egress from insect cells, would lead to evolutionary fitness in parallel with the geographic distribution of the different culicoides species. further studies attempted to extend this to nucleotide sequence homology in segments and , but failed to identify clear cut correlations or any evidence for positive selection. for example, south african isolates were found not to cluster into separate african lineage. in this study, we carried out a more extensive analysis of segment sequences. our analysis showed no segregation of isolates into topographically distinct groups. instead we observed topological clustering of the clades, and we attribute this to genetic bottleneck resulting in genetic drift and founder effect leading to homogenous gene pool in a geographical area. we hypothesize that when a new virus enters a geographical area where local btv strains are already circulating, the new genes/segments would enter into a bigger gene pool. consequently, the newer incursions into a heavily endemic area tend to get diluted and disappear from the population because the rate of drift is inversely proportional to the population size, unless they are positively selected. use of live attenuated vaccine in israel, europe, south africa and usa also led to more homogenous population similar to the vaccine strains due to continuous infusion of the vaccine type genes into the gene pool. we conclude that restriction of specific strains to certain geographical areas could generate uniquely imprinted genotypes which would not only indicate origin but also predict movement of viral strains to new areas. vvo- viral diseases of zoonotic importance: indian context k. prabhudas pd-admas, ivri, campus, hebbal, bangalore zoonoses are generally defined as animal diseases that are transmissible to humans. they continue to represent an important health hazard in most parts of the world, where they cause considerable expenditure and losses for the health and agricultural sectors. the emergence of these zoonotic diseases are very distinct, hence their prevention and control will require unique strategies, apart from traditional approaches. such strategies require rebuilding a cadre of trained professionals of several medical and biologic sciences. the article discusses virus infections that have significant zoonotic implications for india. buffalopox is a contagious viral disease affecting milch buffaloes and rarely, cows, with a morbidity rate up to % in the affected herd. although the disease is not responsible for high mortality, it adversely affects the productivity of the animals, resulting in large economic losses. furthermore, the disease has zoonotic implications, as outbreaks are frequently associated with human infections, particularly in the milkers. the causative agent, buffalopox virus (bpxv), is closely related to vaccinia virus. the outbreaks of febrile rash illness among humans and buffaloes were investigated in the villages of districts solapur and kolhapur of western maharashtra. clinico-epidemiological investigations of humans and buffaloes were carried out and representative clinical samples were collected respectively. the samples include vesicular fluid, scab, and blood. laboratory investigations for buffalo-pox virus (bpxv) was done by pcr on blood samples, scabs and vesicular fluid. in vitro virus isolation attempts were carried out by using vero e- cells. negative staining electron microscopy was also employed for detection of virus particles. a total of human cases with pox lesions on hand and other body parts from village kasegaon, district-solapur and cases from different villages of kolhapur district were reported. besides pox lesions patients were having fever, malaise, pain at site of lesion and axillary and inguinal lymphadenopathy. in kasegaon village, attack rate in human cases was . % and in buffaloes . % ( / ). whereas in kolhapur area attack rate in buffaloes was . % ( / ). bpxv was confirmed in blood, vesicular fluid and scab specimens from human cases and scab specimen from buffalo by polymerase chain reaction (pcr) method. the bpxv was also isolated from different clinical specimens and further identified by pcr and electron microscopy. clinical manifestation of the disease in buffaloes from solapur district was as reported earlier like common pox lesions on teats and udders whereas the buffaloes from kolhapur district had lesions on hairless parts of ears and on the eyelids with purulent discharge. bpxv from human and buffalo cases showed similarity. vaccines have been made against several diseases and used for controlling the afflictions. however a few of them were not effective for successfully controlling the disease. the reasons for the failure are many, the major being, either the pathogen is not completely cleared from the vaccinated animal or it reemerges after changing its antigenic structure, thus making the vaccination programme less effective. in addition to this, emergences of newer diseases such as hiv the development of suitable vaccines have become a challenging task. this is especially true in the case of viral diseases. these challenges have warned the researchers ''that protection by vaccination is not that simple and strait forward approach'', and lot need to be understood in terms of host virus interaction and role of environment in perpetuating the disease. so the immediate step that was considered was the environmental safety by way using non infectious materials as vaccines. with the understanding that has been developed in molecular immunology and molecular biology and with the availability of molecular tools that have been developed through recombinant dna technology the field of vaccinology has changed dramatically to emerge as modern vaccinology. this presentation deals with the modern approaches that are being used to produce effective vaccines in the case of foot and mouth disease of cloven footed animals. the similar approach may be worked out for other viral diseases also. despite the availability of an inactivated vaccine that is noted to provide solid immunity against the disease over a short period of time, the search for an ideal vaccine, the criteria for which are; safety of the vaccine for environment, easy in its preparation, does not require a cold chain for its storage, provides longer lasting immunity, economically viable and may be able to clear the virus in case of persistent infection is on. the advent of recombinant dna technology together with the information available on the molecular biology of viruses has enabled to design the development of newer vaccines that can induce strong cellular and humoral responses. the underlying principal in the present vaccine development strategy world over is the virus antigen gene has to be expressed in the tissue and the vaccine backbone has to trigger the immune system for eliciting desired immune response. bangalore campus of ivri has been vigorously pursuing research to develop ideal vaccines for foot and mouth disease keeping above principal in mind to achieve the previously mentioned criteria. the approaches selected are to see that the virus antigen/s replicate transiently in the host. the self replicating vaccines that have been developed are pox virus vectored vaccines, alpha virus replicase based vaccines and fmdv vectored vaccines. the approach and the result obtained so far will be discussed. silkworm, bombyx mori is affected with various diseases caused by viruses viz., nuclearpolyhedrosis (bmnpv), densosnucleosis (bmdnv) and infectious flacherie (bmifv). silkworm viral diseases form major constraints for the silk cocoon production in all the sericultural countries. the losses due to silkworm diseases is estimated about - % and among them viral diseases are most common. in sericulture, prophylactic measures play a vital role in the management of silkworm diseases. these include disinfection of silkworm rearing house and appliances, rearing area, rearing surroundings, silkworm egg and body, and rearing bed disinfection associated with maintenance of general hygiene and personnel hygiene. all these activities are generally carried out as rituals by using general disinfectants often with partial success. recent trends in complete management of silkworm diseases include development of silkworm hybrids evolved from disease resistant/tolerant breeds, effective eco-and user-friendly disinfectants, anti-microbial feed-supplements and use of transgenic silkworms. biotechnological breakthrough in this regard is through rna interference (rnai) approach involving dsrna mediated nuclear polyhedrosis management and this is presently pursued by apssrdi, hindupur in collaboration with centre for dna fingerprinting and diagnostics (cdfd), hyderabad. nadu and karnataka. the disease appears to be more severe in rural flocks than organized farms. our investigations revealed the morbidity, mortality and case fatality rates among rural and organised farms as . %, . %, . % and . %, . %, . % respectively. higher morbidity and mortality in rural areas may be due to stress factors like poor nutrition, parasitic burden, fatigue due to long walks and non availability of veterinary aid. kulkarni et al. also reported the severe bt outbreaks in rural areas of maharashtra with overall morbidity, mortality and case fatality of %, % and % respectively. all the south indian sheep breeds were found to be susceptible and clinical farm of the disease is evident in all of them though saravanabava ( ) reported variations in susceptibility among the indigenous sheep. trichy black and ramnad white sheep were found to be more susceptible than the vambur and mecheri sheep of tamil nadu. prevalence of bluetongue in sheep, goat and cattle appears to be high in the region. serological surveys conducted in andhra pradesh during revealed the prevalence of btv antibodies in sheep ( . %) goats ( . %) cattle ( %) and buffaloe ( %). similar high prevalence of btv antibodies in sheep and goats were also reported from the other states in the region. clinical disease has not been recorded in kerala though btv antibodies were recorded in sheep ( . %) and goats ( . %) (ravi sankar ) . culicoides are the known biological vectors of btv. all the culicoides species are not capable of transmitting the btv. the occurrence of the disease is related to the presence of the competent vectors in the area. jain et al. ( ) established the involvement of the culicoides in transmitting the btv by isolating the virus from culicoides at haryana, the north indian state. c. imicola and c. oxystoma were found to be prevalent in andhra pradesh and tamil nadu. narladakar et al.( ) reported the presence of c. schultzei, c. perigrinus and c. octoni in marathwada region of maharastra. culicoid vectors are significantly affected by the climate and annual variations in the climate reflects the outcome of the disease. the monsoon season (june to dec) with the temperature ranging from . to . °c appears to be favourable period for the multiplication of culicoides. the maximum no of outbreaks were recorded during the north east monsoon period (oct-dec) followed by south west monsoon period (june to sep) in the region. however, details on the distribution of the competent vectors, feeding habits and their dynamics in the region is lacking multiple btv serotypes were found to be circulating in the region. (kulkarni and kulkarni ; janakiraman etal. ; mehrotra et al. ) a total of serotypes viz. - , , , , , and were identified based on the virus isolations. sreenivasulu et al. isolated btv serotype from an outbreak of bt in native sheep of andhra pradesh. btv serotype , and were also isolated from the outbreaks occurred in andhra pradesh. some of the isolates need to be serotyped. deshmukh and gujar ( ) isolated btv type from maharashtra. following is the summary of the distribution of btv serotypes in this region. clinical picture of bt in native sheep appears to be slightly different, the major difference being that swelling of lips and face was less conspicuous. mucocutaneous borders appeared to be very sensitive to touch and bleed easily upon handling. the classical signs of cyanosis of tongue and reddening of coronary band are not the common features of the disease in native sheep. the disease was also confirmed by the virus isolation and identification. clinical disease has not been reported in cattle, buffaloes and goats in spite of high seroprevalence. in conclusion bt is established in native sheep and causes severe economic losses to the farmers. the disease is concentrated in the southern peninsula of the country. the disease is seasonal and is associated with the rain fall. multiple serotypes appear to be circulating in this region. the btv serotypes were of virulent in nature as evident by severe outbreaks. s. janardana reddy*, d. c. reddy department of fishery science and aquaculture, sri venkateswara university, tirupati in less than three decades, the penaeid shrimp culture industries of the world developed from their experimental beginnings into major industries providing hundreds of thousands of jobs, billions of u.s. dollars in revenue, and augmentation of the world's food supply with a high value crop. concomitant with the growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. in india viral diseases have become an important limiting factor for growth of shrimp aquaculture industry. although more than different viral pathogens have been identified in different species of shrimp world wide, only a few viruses have identified which are causing disease problems in cultured tiger shrimps in india, east coast of andhra pradesh, in particular. diagnostic methods for these pathogens include the traditional methods of morphological pathology (direct light microscopy, histopathology, and transmission electron microscopy), enhancement and bioassay methods, traditional microbiology, and the application of serological methods. while tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. the need for rapid, sensitive diagnostic methods led to the application of modern biotechnology to penaeid shrimp disease. the industry now has modern diagnostic genomic probes with nonradioactive labels for viral pathogens like infectious hypodermal and hematopoietic necrosis (ihhnv), hepatopancreatic virus (hpv), taura syndrome virus (tsv), white spot syndrome virus (wssv), monodon baculo virus (mbv), and bp. highly sensitive detection methods for some pathogens that employ dna amplification methods based on the polymerase chain reaction (pcr) now exist, and more pcr methods are being developed for additional agents. these advanced molecular methods promise to provide badly needed diagnostic and research tools to an industry reeling from catastrophic epizootics and which must become poised to go on with the next phase of its development as an industry that must be better able to understand and manage disease. within this field, shrimp immunology is a key element in establishing strategies for the control of diseases in shrimp aquaculture. research needs to be directed towards the development of assays to evaluate and monitor the immune state of shrimp. the establishment of regular immune checkups will permit the detection of shrimp immunodeficiencies but also to help monitor and improve environment quality. for this, immune effectors must be first identified and characterised. in the end, however, the assumption may be made that the sustainability of aquaculture will depend on the selection of disease-resistant shrimp, i.e. to develop research in immunology and genetics at the same time. the development of strategies for prophylaxis and control of shrimp diseases could be aided by the establishment of a collaborative network to contribute to progress in basic knowledge of penaeid immunity. however, to improve efficiency, it appears essential also to open this network to complementary research areas related to shrimp pathology, physiology, genetics and environment. bluetongue is an important viral disease of sheep causing severe economic losses to the farmers. lack of effective vaccine is the major impediments in controlling the disease. multiple serotypes were found to be circulating in the state. attempts are being made to develop the vaccine employing the available serotypes to control the disease. hence, it is essential to identify the antigenic relationship among the serotypes to identify the candidate vaccine strains to be incorporated in the preparation of vaccine. reciprocal cross neutralization test was employed to find out the r% values between btv- , - and - which indicated the extent of antigenic relationship between the serotypes. r% value between btv- and btv- was recorded as . r% value of . and . were observed between btv- and - and btv- and - respectively. the r% values recorded in the present study revealed a weak antigenic relationship between the btv serotypes. the extent of antigenic relationship between the btv serotypes was also determined by multiple sequence alignment of the nucleotide and amino acid sequences of the reference btv serotypes , and . the sequence analysis of the vp gene revealed a homology of - % and - % at the nucleotide and amino acid levels respectively. r% values obtained using reciprocal cross neutralization test with the btv- , and serotypes isolated in native sheep of andhra pradesh and the genomic analysis of the reference serotypes of btv- , and revealed very weak antigenic relationship and were highly divergent. diseases especially those by viral pathogens cause greater economic losses in most horticultural crop species throughout the world as compared to agricultural crops. non-genetic methods of management of these diseases include quarantine measures, eradication of infected plants and weed hosts, crop rotation, use of certified virus-free seed or planting stock and use of pesticides to control insect vector populations implicated in transmission of viruses. however, none of these measures is likely to provide an enduring solution against these diseases especially those caused by viruses due sometimes to the huge expenditure involved, but mostly to the questionable effectiveness and reliability of those methods. as key control pesticides are getting increasingly abandoned, development of alternative methods to control diseases has been a felt-need in the recent past. though breeding for disease resistance generally provides a reliable security in a long run, introgression of host plant resistance did not materialise in most important crops. non-availability of an appropriate source of resistance in inter-fertile relatives, linkage to undesirable traits, or often times polygenic nature of such sources of resistance are the stumbling blocks in breeding programs. the limitations of conventional breeding and routine cultural practices prompted the need for the development of other approaches of virus control that could be fully incorporated into traditional methods. in this perspective, the concept of pathogen-derived resistance offers an attractive strategy to evolve newer methods of virus management, by transforming crop plants with nucleotide sequences derived from the pathogen's genome. an increasing number of molecular characterisation of plant virus genomes and the stable transformation of a number of horticultural crop species have in fact opened an avenue for molecular breeding against virus pathogens. successful field-testing of genetically modified crop cultivars renders proof of their supremacy over existing cultivars. it also contributes to demonstrate their capability with regard to environmental safety with a view to winning over public concern and scepticism. in general, the eventual commercialisation transgenic lines expressing virus resistance will rely upon a host of factors including their field performance, genetic stability, public acceptance and the resolution of environmental concerns and patent related issues. as such, elaborate field trials and allied studies are now required to adapt genetically engineered horticultural crops expressing virus resistance for their implementation into practical agriculture. a few examples from current research at tnau, in india or elsewhere will be discussed in this presentation. virology unit, division of plant pathology, iari, new delhi in recent times there has been greater emphasis on vegetatively propagated crops in india to help diversify the indian agriculture. fruit, flower, spice and plantation crops are important vegetatively propagated horticultural crops, which have become a driving force for economic development in several parts of india. however, most of the vegetatively propagated crops are threatened by biotic stress caused by plant pathogens in general and plant viruses in particular. plant viruses produce specific and non specific symptoms and in some cases no symptoms are produced. correct identification and diagnosis of viral diseases is first step in the management of any disease including viral diseases. there have been two major breakthroughs in virus diagnostics during last four decades. the first one was serological assay using monoclonal or polyclonal antibodies in enzyme linked immunosorbent assay (elisa) and the other one was the use of in vitro amplification of dna in polymerase chain reaction (pcr). a significant development in serological assays has been its simplification in form of user's friendly quick strip/dip stick method. the one-step lateral-flow (lf) tests have been developed for the on-site detection and identification of several plant viruses. rapid advancement in virus genome characterization has led to the development of novel approaches of nucleic acid based diagnostics which include conventional pcr, real time pcr, multiplex pcr, micro/macro arrays and biochips. pcr protocols already exist for many plant viruses of citrus, banana, apple, papaya, vegetables, ornamental and spice crops. a further advancement has led to development of realtime pcr assay which is relatively easy but requires training for diagnosticians. in real-time pcr assays, results can be available within min. the nucleic acid template preparation in pcr has been simplified. membrane based dna template protocol and co-isolation of nucleic acid template preparation are novel approaches in pcr detection of virus and virus like pathogens. since many of the horticultural crops are often infected by more than one virus, their individual detection by pcr is not only expensive but also time consuming. therefore, multiplex pcr has been developed where in genome of more than one virus could be amplified and detected in the same reaction mixture. development of nucleic acid based chip is now one of the fastest and recent growing areas in the field of pathogen detection. these nucleic acid based chips have been named as dna/rna chips, biochips, genechips, biosensors or dna arrays. when it comes to applications of microarray technology for plant viruses, it is not too difficult to see the value of a method that could potentially detect a whole range of viruses using a single test. however, microarrays are unlikely to become the only method in use in a diagnostic laboratory. processing of germplasm including transgenic planting material imported for research purposes into the country. during the last two decades, a total of , samples of wheat including transgenics were imported from cimmyt (mexico), icarda (syria) and many other countries. these were grown in post-entry quarantine nursery each year at nbpgr, new delhi and the transgenic samples were grown in national containment facility of level- (cl- ) since its inception to ensure that no viable biological material/pollen/pathogen enters or leaves the facility during quarantine processing of transgenics. in addition, post-entry quarantine inspections of the transgenic wheat grown by indenters are also undertaken by nbpgr quarantine scientists. virus-induced gene silencing (vigs) is a technique in which viral genomes are used, usually after appropriate modifications, for transient gene silencing in plants. the mechanism behind vigs is the phenomenon called rna-interference (rnai), which is widespread in many organisms and is believed to be form of inherent defence system against intracellular pathogens, such as viruses and transposons. double-stranded rna or rna containing strong secondary structures, commonly produced during viral infections, are believed to cause triggering of rnai, which employs a battery of proteins and nucleoprotein complexes to identify and degrade specific viral transcripts. in vigs, viral genomes not causing severe symptoms, but which can accumulate and spread efficiently in the host plant are used as vectors in which a host gene is cloned and introduced into the plant. upon replication, the viral vector triggers rnai response in the host plant, which also targets the host gene, leading to its silencing and subsequently, the silenced phenotype revealing gene function in vivo. vigs has been used extensively to study gene functions in dicot plants, such as tobacco, tomato, pea, soybean, etc., using vectors derived from reference genes are commonly used as an/the endogenous normalisation measure for the relative quantification of target genes. the expression (characteristics) of seven potential reference genes was evaluated in tissues of healthy, physiologically stressed and barley yellow dwarf virus (bydv) infected cereal plants. these genes were tested by rt-qpcr and ranked according to the stability of their expression (characteristics) using three different methods (two-way anova, genorm and normfinder tools). in most cases, the expression (characteristics) of all genes did not depend on the abiotic stress conditions or on the virus infections. all the genes showed significant differences in expression (characteristics) among plant species. glyceraldehyde- -phosphate dehydrogenase (gapdh), beta-tubulin (tubb) and s ribosomal rna ( s rrna) always ranked as the three most stable genes. on the other hand, elongation factor- alpha (ef a), eukaryotic initiation factor a (eif a), and s ribosomal rna ( s rrna) for barley and oat samples; and beta-tubulin (tubb) for wheat samples were consistently ranked as the less reliable controls. the bydv titre was determined in two oat varieties by rt-qpcr by three different quantification approaches. statistically, there were no significant differences between the absolute and the relative quantification, or between quantification using gapdh + tubb + tuba + s rrna and ef a + eif a + s rrna. the geometric average of gapdh, s rrna, tuba and tubb is suitable for normalisation of bydv quantification in barley and oat tissues. for wheat samples, a combination of gapdh, s rrna, tubb, eif a and e fa is recommended. department of microbiology, yogi vemana university, vemanapuram, kadapa large scale production and import of propagative material poses potential risk of introducing several destructive pathogens particularly viruses and mycoplasma like organisms in our country. this demands adequate quarantine safe guards such as growing them under approved post entry quarantine facility for specific period so as to facilitate virus detection, thereby curtailing risk. when such facilities are coupled with propagation by tissue culture will ensure virus free propagative plant material. the requirement of nationwide network of post entry quarantine facility working in close collaboration with crop institutions are very much emphasized for considering import of high risk plant genera for agriculture development. present paper discusses about virus disease of quarantine importance affecting ornamental and fruit plants such as chrysanthimum, dahlia, dianthus, rosabengalensis, cattleya, cymbidium, dendrobium, lilium, citrus, vitis etc. the paper also discusses on immunodiagnostic methods of detection and methods of obtaining virus free propagative material. rice tungro occurs as epidemics in regular cycles and has been reported in the last years from all the major rice growing regions of india, especially prevalent in the southern and eastern states. development of the durable resistant varieties to tungro is crucial for the management of the disease. molecular breeding, involving the use of dna markers linked to the resistant gene(s) for selection, can overcome the difficulties encountered in conventional resistant breeding programs. for successful marker-assisted selection (mas), the identification of closely linked markers through the process of gene tagging and mapping is a prerequisite. attempts have been initiated for identification of tungro resistance genes through molecular mapping and their introgression into the target varieties using marker-assisted selection at drr, hyderabad. the inheritance of resistance to rice tungro virus disease was studied in seven resistant rice cultivars with field evaluation at hot spot locations. the microsatellite markers linked to rice tungro resistance in utri merah was studied and found that resistance genes were linked to rm on chromosome . through molecular mapping two qtl were identified controlling rtv resistance on chromosomes and in 'utri rajapan' explaining . % and . % of the phenotypic variance. in variety 'vikramarya', another two qtl for rtv resistance were detected on chromosomes and explaining . % and . % of the phenotypic variance. the closely linked markers identified in this study flanking the gene of interest through mapping will improve the efficiency and precision of introgression programs in marker assisted breeding for rtv resistance. functional characterization of these qtl for rtv resistance is under progress. there is only a limited pool of natural virus resistance in cassava against cassava mosaic geminiviruses and cassava brown streak ipomovirus hence the development of transgenic resistance in this significant crop might present an option. rna mediated resistance through the expression of inverted-repeat dsrna sequences derived from the virus genome and the modification of plant microrna to produce antiviral artificial microrna are strategies that have recently been proven very effective for induction of virus resistance (immunity) against a number of rna viruses. results from rna interference strategies against geminiviruses never resulted in immunity of transgenes. however, it suggest that viral mrna are targets of rna silencing and that the success of the strategy depends on the relevance of the target gene in the systemic spread of the virus. we have generated a number of rna silencing constructs to induce resistance against cbsv and the indian cassava mosaic viruses icmv and slcmv. due to the serious problems inherent with transformation of cassava and subsequent resistance screening, these constructs were tested for efficiency either by transient-or by transgenic expression in n. benthamiana. complete immunity was reached in transgenic n. benthamiana against cbsv using inverted repeat or amirna constructs. using different species of cbsv for resistance screening, immunity was broken, to show the minimum context for broad spectrum resistance. similarly, highly specific resistance was reached in expression of amirna. in contrast, virus resistance against icmv/ slcmv using single amirna constructs was not successful. results from the experiments to generate virus resistance against cbsv and icmv/slcmv will be shown; methods to evaluate efficiency of rnai gene constructs by transient gene expression in n. benthamiana and strategies to develop efficient resistance against rna and dna viruses in cassava will be discussed. bitter gourd (momordica charantia l.) which is also called bitter melon, balsam apple and balsam pear belongs to family cucurbitaceae. it is an important traditional vegetable of nutritive and medicinal value that is cultivated in tropical and sub-tropical asia, but is considered as a weed host reservoir for viruses in jamaica. viral disease-like symptoms were observed occurring naturally on the crops of bitter gourd grown in the fields of northern india during - . an incidence of . % of diseased plants was recorded which showed chlorotic spots and mosaic ranging from mild mottling to green blisters along with leaf smalling, leaf and fruit deformations, bud necrosis and stunted growth whereas . % plants exhibited leaf curling alone or in combination with mosaic-type disease. a reduction of . % in fruit yield was recorded in mosaic-like disease which could be attributed to lesser fruit setting due to bud necrosis, smaller fruit size and stunted plant growth. such plants produced deformed, notched, irregularly shaped fruits wherein pre-mature yellowing and necrosis on the anterior and posteriors ends made . % fruits unfit for marketability. the dwindling yield and production of unmarketable fruits posed a major constraint for profitable cultivation of this economically important crop, thus warranting for studies on etiology and management of these diseases. the mosaic-like disease was transmitted to healthy seedlings of bitter gourd at -leaves stage by sap inoculation as well as by aphid viz., myzus persicae sulz. and aphis gossypii glov. initially studies were carried out to optimize protocols for efficient plant regeneration and agrobacterium-mediated transformation for nagpur sweet orange, which is a popular and elite citrus cultivar in india. organogenesis was induced in etiolated epicotyl explants of one-month-old axenically raised polyembryonic seedlings by culturing them in mt medium supplemented with g/l sucrose with varying concentrations of plant hormones. it was found that bap at mg/l without auxin was best for efficient shoot regeneration in citrus using epicotyl explants. a % regeneration frequency was obtained and multiple shoot formation was obtained from both the cut ends of all the explants. an average of . well-differentiated shoots per explant were obtained, all of which rooted normally under the influence of mg/l iba. this improved regeneration protocol was utilized in standardizing agrobacterium-mediated transformation of citrus using a. tumefaciens strain eha , containing binary plasmid pcambia that harbors gus reporter gene and npt-ii plant selection marker gene. one-month-old epicotyl explants infected with over-night grown agrobacterium (a . - . ) for min and co-cultured for days were found to be optimum for transformation as assessed on the basis of pcr analysis and gus activity displayed by the stem and leaf sections of putative transgenics. overall transformation frequency ranged from to %. current study focuses on the generation of citrus transgenics for ctv resistance using a. tumefaciens strain eha containing binary plasmid pbinar harboring portion of coat protein gene of ctv and npt-ii gene employing the standardized protocols. several putative transgenic shoots were recovered on selection medium and they are being utilized for molecular analyses and resistance against ctv. work is also in progress on the generation of citrus transformants using rnai construct harboring ctv cp and p genes, singly and in conjunction. our lab was also involved in developing rice transgenics for resistance against rice tungro disease, which is one of the most important and widespread virus diseases of rice in south and southeast asia, causing an annual estimated loss in crop yield of economic losses worth millions of rupees are caused due to these diseases annually. virus diseases are frequently less conspicuous than those caused by other plant pathogens and last for much longer. this is especially true for perennial crops and those that are vegetatively propagated. one further problem with attending to assess losses due to various diseases on a global basis is that what most of the data are from small comparative trials rather than wide scale comprehensive surveys, even the small trials do not necessarily give data that can be used for more global estimates of losses. this is for several reasons, including: ( ) variation in losses by a particular crop from year to year; ( ) variation from region to region and climatic zone to climatic zone: ( ) differences in loss assessment methodologies; ( ) identification of the viral etiology of the disease; variation in the definition of the term 'losses' and ( ) chilli is the major vegetable and spice crop grown in thar desert areas of rajasthan. leaf curl disease (chlcd) is one of the major constrains in chilli cultivation faced by farmers and cause yield loss up to %. a survey was conducted in major chilli growing areas of thar desert; bikaner, nagur, jodhpur and jalore districts of rajasthan during november, to understand the present status of leaf curl disease in chilli. among the four district surveyed for chlcd, the disease incidence was recorded maximum (up to %) in jodhpur district followed by jolore district (up to %). no relation was found between the disease incidence and varieties. the major varieties grown in these area are; mehsana, rch (mandoria), haripur raipur, mathania and local cultivars. the number of whitefly was also counted in top, middle and bottom leaf of chilli grown in these areas. the average number of whitefly per plant ranged from . to . . more number of whitefly ( . ) was recorded in jodhpur district and lowest ( . ) in jalore district. total dna was extracted from three leaf curl infected samples from each district and tested for the presence of begomovirus using coat protein (cp) and dna-b specific primers. all the samples were positive for cp and dna-b amplifications by pcr. the cloning and sequencing of selected cp gene and dna-b fragments are in progress. the preliminary investigations shows that the leaf curl disease of chilli is widespread in the arid region of rajasthan and may be caused by begomovirus associated with satellite dna-b. bittergourd (momordica charantia) is an important vegetable crop of kerala. the crop is affected by several diseases of which mosaic is a prominent one. a field experiment was conducted to evaluate the efficacy of potentised resistance inducing substances (ris) viz., mosaic affected bittergourd plant tissue, ash of mosaic affected bittergourd plant tissue, plumbago and salicylic acid for control of bittergourd mosaic in march . ris were applied as drench and foliar spray at three potency levels twice, before flowering of the crop. the experimental crop was grown as per the package of practice recommendations in split plot design with five replications per treatment. the disease incidence, disease severity and yield of the crop were recorded. the result of the experiment shows that spraying was more effective than drenching of treatments for reducing mosaic incidence and severity. among treatments, infected plant extract at potency was the most effective one for reducing mosaic incidence and it showed the maximum incubation period and minimum disease severity. the spray application of treatments produced significantly higher yield than drenching. among the treatments, ash of infected plant at and potency and infected plant extract at potency were on par and produced comparatively higher yield. elephant foot yam (amoprhophallus paeoniifolius), colocasia (colocasia esculenta) and tannia (xanthosoma sagittifolium) are the major edible aroids cultivated in india. the elephant foot yam cultivation is gaining importance due to its high production potential, nutritional and medicinal values and good economic returns. all these aroids are vegetatively propagated and viral diseases are spreading through planting materials. ctcri has the mandate of producing healthy planting materials of these edible aroids. accurate diagnosis and identification of the virus is essential for production of healthy planting material and effective management of the disease. though occurrences of viral diseases on edible aroids in india were known in s, not much attention was given for detection and identification of the virus involved. in case of elephant foot yam - % mosaic incidence was observed with varying symptoms of mosaic, puckering, filiformy etc. in colocasia and tannia, - % incidence was noticed. rt-pcr amplification with potyvirus group specific primers and subsequent cloning and sequencing of the amplified product has confirmed the association of dasheen mosaic virus (dsmv) with all the three edible aroids cultivated in india. the complete full length coat protein gene of dsmv infecting elephant foot yam was cloned in pgem-t vector and sequenced. further sequence analysis revealed that the cp of dsmv consisted of nucleotides and the utr comprised of nucleotides. blast and phylogenetic analysis showed highest similarity of % with that of dsmv isolate af , reported from usa. the deduced amino acid sequence of cp had . - . % identity with other dsmv isolates. blast analysis of the partial cp gene sequences of colocasia and tannia also confirmed that the virus involved is dsmv. rt-pcr analysis of large number of samples from all the three crops confirmed that the potyvirus group specific primers (mj and mj ) are good for rapid detection of dsmv in these crops. dsmv specific biotinylated cdna and digoxigenin labelled crna probes were also prepared and dsmv in elephant foot yam was detected through nucleic acid spot hybridization. yellow leaf disease (yld) caused by sugarcane yellow leaf virus (scylv) is a recently recorded disease in india and is found wide spread throughout country. in popular varieties, the disease incidence varied from to . % and attained epidemic levels under field conditions. detailed studies on the impact of yld on sugarcane revealed that the virus infection significantly reduces various cane growth parameters, cane yield and juice quality. sequence comparisons of the coat protein (cp) and movement protein (mp) of scylv isolates from india and database sequences showed a significant variation between indian isolates and the database sequences both at nt and aa level in the cp/mp coding regions. the significant variation in our isolates with the database isolates, even in the least variable region of the scylv genome showed that the population existing in india is different from rest of the world. further, comparison of partial sequences encoding for orf and revealed that yld in sugarcane in india is caused at least by three genotypes viz., cub, ind and bra-per, of which a majority of the samples were found infected with cuban genotype (cub). the genotype ind was identified as a new genotype and this was found to have significant variation with the reported genotypes. we have identified specific primers from cp region of the virus and optimized rt-pcr conditions to diagnose the virus. this assay has been found efficient in detecting the virus in asymptomatic plants and tissue culture derived seedlings. elimination of the virus through meristem culture has been demonstrated to purify the virus from the infected planting materials and this technique needs to be adopted to supply disease-free planting materials for effective management of the disease. studies are also in progress to identify the yld-resistant sources in sugarcane germplasm to initiate breeding for yld-resistance in sugarcane. mycoviruses are viruses that infect fungi. they have been identified in all major fungal families. in the present scenario, mycoviruses are the important means of biocontrol of plant fungal pathogens. most identified fungal viruses have double stranded rna genomes, often with more than one dsrna present per virus particle, and have been spherical in shape. these viruses are mostly vesicle bound, as other viruses have protein coatings. to be a true mycovirus, they must demonstrate an ability to be transmitted-in other words be able to infect other healthy fungi through anastomosis and spores. mycoviruses lead 'secret lives', reduce the ability of their fungal hosts to cause disease in plants. this property, known as hypovirulence (hypovirulence is a term used to describe reduced virulence found in strains of pathogens), this phenomenon was first observed in cryphonectria (endothia) parasitica (chestnut blight fungus) on european castanea sativa in italy, where naturally occuring hypovirulent strains were able to reduce the effect of virulent ones. these slower growing hypovirulent strains of c. parasitica contain a single cytoplasmic element of double-stranded rna (ds rna) similar to that found in mycoviruses that was transmitted by anastomosis in compatible strains through natural virulent populations of c. parasitica. hypovirulence has also been reported in many other fungal plant pathogens, including rhizoctonia solani, gaeumannomyces gramini var. tritici, ophiostoma ulmi, sclerotinia homoeocarpa, diaporthe ambigua alternaria alternata, and fusarium sp. etc. hypovirulence has attracted attention owing to the importance of fungal diseases in agriculture and the limited strategies that are available for the control of these diseases. it reduces the use of toxic fungicides which also affect the plant growth. the symptoms resulted by the mycoviruses are reduction in growth, reduction in pigmentation and sporulation, excessive sectoring and aerial mycelial collapse. these are the consequences of alteration in complex physiological and biochemical processes involving interaction between host and virus. cassava (manihot esculenta crantz.) is the major tuber crop in peninsular india, it is grown in an area of . lakh hectares with the annual production of . million tonnes both for direct consumption and the starch grain (sago) producing industries, mainly in the southern states of tamil nadu, kerala and andhra pradesh (fao ) . in tamil nadu, cassava primarily produced for sago producing industries where it is considered as an industrial crop rather than food crop, so the resource rich farmers are cultivating the cassava as irrigated crop in their fertile land and the poor farmers are raising the crop under rainfed conditions. in south india in addition to cassava there is a practice of intercropping important vegetable crops like, tomato, brinjal, legumes and gourds in cassava fields since all the above mentioned crops are short duration and are money spinners for the farmers. unfortunately, the major production constraint in these vegetable crops including cassava is the geminiviruses belonging to the family of in recent years there has been growing concern regarding the standard of scientific researches in india. the strengths, weaknesses, opportunities and threats (swot) analysis on indian scientific research reviewed the progress of science during the last six decades. although the 'strengths' were highlighted in good measure, it was the list of 'weaknesses' that called for attention to upgrade the standard of research and 'opportunities' that provide scope for overall scientific growth. a comparison between india and other countries in terms of research papers published revealed that india's contribution to science has come down enormously. what ails indian science? should we compare the growth of indian science with other developed countries? what criteria should be adopted to judge the quality and standard of scientific research? how to motivate the scientists to improve their scientific output? how do motivate the scientists to improve their scientific output? how do indian journals perform in maintaining quality? this paper analyses critically the scientific journals around the world, based on the scores allotted by the national academy of agriculture sciences (naas) in and for and journals respectively. in general, the indian journals performed poorly irrespective of the disciplines with only - % in the high standard. the paper dealt with the reasons for low impact factor, the anomalies in the allotment of scores to wide spectrum of the journals and the disadvantages the scientists face with the scoring system. a case study was presented of an institute with over scientists whose publications were analyzed to discuss the merits and demerits of the system. the performance of the journals published by prestigious academics, societies and councils was also projected. the paper concluded with the need for enhancing the image of the country through research publications in high standard journals and the role of various scientific bodies with shore and long term measures. poster session herpes simplex virus (hsv) keratitis is a leading cause of corneal blindness throughout the world. the infection can be diagnosed by clinical manifestations but in case of atypical ocular cases, laboratory diagnosis is more helpful in timely management of disease. collection of corneal scrapings in all cases of stromal and epithelial keratitis may not be possible, but collecting tear fluid is a convenient procedure causing less discomfort to the patients. therefore, the present study was intended to evaluate the suitability of tear specimens for detecting hsv by polymerase chain reaction (pcr) and immunofluorescence (ifa). tear fluid and corneal scrapings were collected from patients of suspected herpetic keratitis. hsv- antigen was detected by ifa using rabbit anti-hsv antibodies. pcr was performed to amplify bp region of thymidine kinase (tk) coding gene and bp region from dna polymerase coding gene of hsv. out of patients hsv antigen was detected in ( . %) of corneal scrapings and ( . %) of tear specimens and in ( . %) patients from both the specimens. hsv gene could be amplified in ( . %) of corneal scrapings and ( . %) of tear fluids and in ( . %) patients from both the specimens. although, corneal scraping seemed to be marginally superior material for detection of hsv, tear fluid may also serve as an appropriate alternative clinical specimen, due to ease of collection and least discomfort to the patients. in either cases pcr detected higher number of hsv cases than ifa. therefore if and when feasible, both ifa and pcr should be used simultaneously on each specimen to obtain best results. cytokines play a key role in the regulation of immune responses. in hepatitis c virus infection (hcv), the production of inappropriate cytokine levels appears to contribute to viral persistence and to affect response to therapy. il- is produced by a variety of cells including t cells, phagocytes and fibroblast. cytokine genes are polymorphic at specific sites, and certain mutations located within coding/regulatory regions have been shown to affect the overall expression and secretion of cytokines in patients with hcv infection. to correlate the serum levels and polymorphism of il- gene in chronic hepatitis c patients and healthy controls. forty patients positive for hcv rna attending the medicine out patient department and wards of lok nayak hospital, new delhi as well as forty healthy controls were enrolled for the study. the serum level of il- was detected by using elisa. genomic dna was extracted from whole blood of hcv infected patients and healthy controls by using accuprep genomic dna extraction kit according to manufacture's instruction. the genotyping of il- promoter (- variant) was carried out by pcr and direct sequencing using the method of patricia woo et al. . the serum level of il- was significantly down regulated in hcv infected chronic patients as compared to the healthy controls. genotyping of - promoter variant of il- was performed by pcr and direct sequencing. il- polymorphism in the g/g, g/c and c/c allele was non significant when compared to hcv patients and healthy controls. the il- serum levels were significant among hcv infected patients when compared to healthy controls. the polymorphism in the promoter region of il- (- ) was found nonsignificantly associated in hcv patients compared to healthy controls. in conclusion, the present study suggests that the host il- polymorphism alone may not play a significant role in the outcome of hcv infection. acute gastroenteritis (age) is a global health problem and has been associated with multiple etiological agents, which include bacteria, protozoa and viruses. viral gastroenteritis is considered as the second most common illness in children after upper respiratory tract infection. among enteric viruses, rota, noro, enteric adeno, astro and enterovirus are found to be associated with gastroenteritis. although, association of enteric viruses has been established in children hospitalized for age no such data is available from hospitalized children other than enteric infections. to determine the prevalence of enteric viruses circulating in hospitalized children. fecal samples, n = ( symptomatic and asymptomatic for age) were collected from children \ year of age from three different hospitals across the city of pune from june to feb. . detection of group a rotavirus was carried out by using antigen captured elisa. rt-pcr and pcr was carried out for the detection of norovirus, enterovirus, astrovirus and enteric adenovirus detection by using primers targeted to rdrp gene, ncr gene and consevered gene for serine protease and hexon gene respectively. out of fecal samples tested for enteric viruses in age cases, the prevalence of rota, entero, noro, enteric adeno and astrovirus were . % ( ), . % ( ), . % ( ), . % ( ) and . % ( ) respectively. however, the presence of these viruses in the asymptomatic cases (n = ) was detected at . % ( ), . % ( ), . % ( ), . % ( ) and . % ( ) levels respectively. mixed infections of enterovirus and rotavirus were found in both symptomatic . % ( ) and asymptomatic cases . % ( ). however, mixed infection of enterovirus with adenovirus were found only in asymptomatic cases . % ( ). no marked difference was observed in the seasonal pattern of all viruses in the patients with or without gastroenteritis. the findings of this study document highest circulation of rotaviruses in patients symptomatic and asymptomatic for age. the entero and noroviruses remain second most important enteric viruses in these patients. influenza in humans is a major public health concern and the understanding of its evolution in the light of its ''antigenic drift'' helps prediction of epidemics and update of yearly influenza vaccine. to antigenically characterize influenza a (h n ) isolates and study antigenic drift during to in pune city. patients with influenza like illness were identified using a strict case definition from dispensaries located in different areas in pune and clinical samples (ns/ts) were collected after obtaining informed consent. these clinical samples were processed in vivo (in fertile eggs) and in vitro ( overall, an additional ( . %) positive cases of dengue could be detected when ns antigen assay was also used in the study. highest ns antigen positivity was encountered among the samples collected on the rd day of fever whereas mac elisa for anti igm antibody was positive after th day and gradually there was an increase in the positivity towards the convalescent phase of the disease. the results of this study indicate that ns antigen based elisa test can be an useful tool to detect the dengue virus infection in patients during the early acute phase of disease since appearance of igm antibodies usually occur after fifth day of the infection. concurrent use of both diagnostic assays namely ns antigen as well as mac elisa will improve the overall detection of dengue infection. early detection of acute dengue virus infection is crucial to provide timely information for the management of patients. human parvovirus b , a member of the parvoviridae family, is a pathogen associated with a wide variety of diseases. most commonly, it causes childhood rash erythema infectiosum, but in some cases more serious symptoms such as persistent arthropathy, critical failures of red cell production causing transient aplastic crisis, this infection in pregnancy causes hydrops fetalis and myocarditis. traditional immunosuppressive therapy being unsuccessful, anti-viral therapy might be worthy of consideration. functional annotation would provide role of viral proteome in its survival and pathogenic mechanisms. svmprot functional family annotations of vp protein had deciphered its zincbinding, coat protein, outer membrane, chlorophyll biosynthesis, dna repair and calcium-binding nature. vp protein is having a key role in viral assembly of b virus and being non-homologous to human proteome, it was identified as an attractive molecular target for structure based drug discovery. the vp protein crystal structure was energy minimized using charmm. a structure based virtual screening method was applied using ligandfit to identify potential inhibitors of vp protein from chembank database and ten potential human parvovirus b vp inhibitors were proposed. prism genetic analyzer. the drafting of the sequences was performed using bioedit software and were submitted in genbank. for phylogenetic interpretation denv representing the full extent of genetic diversity in denv- , denv- and denv- were collected from genbank. neighbor joining algorithm was implemented with bootstrap value of , replicates for phylogenetic inference using mega . . . the genomic region to (c-prm gene junction) of denv were amplified directly from patient serum. twelve of samples were positive for dengue viral rna. of these were dengue type , was dengue type and were dengue type . for molecular epidemiological survey and genotyping of the sequences more than sequences from different geographical areas including sequences form previously reported north indian isolates were compared with our present data set. the critical analysis of the sequences revealed: dengue type sequences were clustered within sub-type of genotype iii and all the sequences of den- clustered along with genotype iii. thus, among the dengue types , and currently circulating in north india, dengue type , genotype iii, being the predominant one followed by, genotype iii of dengue type . although there is no specific treatment or vaccine available currently, the confirmative rapid diagnosis based on detection of viral nucleic acid or igm antibodies in serum, an indication of recent infection, helps in epidemiological monitoring, symptomatic treatment of patients and determining prognosis. serological detection of anti-cgv igm antibodies was performed using rapid immuno-chromatographic assay (rica) and igm-antibody capture enzyme linked immunosorbant assay (mac-elisa). eighty convalescent sera were tested by rica and of them were found positive for anti-cgv igm antibodies. twenty-five anti-cgv igm antibody rica positive sera were further assayed using mac-elisa. more sera from the patients are currently being tested to compare the sensitivity of these two serological assays in anti-cgv igm antibody based early serological diagnosis of cgv infection and the findings will be presented. thus the present study was designed to evaluate the utility of multiplex pcr (mpcr) for simultaneous and rapid detection of dengue and chikungunya viral infections. seventy-two acute phase blood samples from clinically suspected dengue cases were subjected for dengue and chikungunya uniplex pcr using dengue genus specific primers and e gene specific primers for chikungunya virus as well as multiplex pcr was developed for simultaneous detection of dengue and chikungunya infection. standard strains of dengue and chikungunya virus were used as controls. of the clinically suspected dengue samples were found to be positive for dengue viral rna by dengue uniplex pcr as well as dengue chikungunya mpcr whereas none of the samples were positive for chikungunya virus infection by both uniplex chikungunya pcr and dengue chikungunya mpcr. the result of dengue and chikungunya uniplex pcr was found to be % concordant with dengue chikungunya multiplex pcr. dengue chikungunya multiplex pcr was found to be a potential rapid test to detect dengue and chikungunya viral infections simultaneously in clinical samples. sheetal malhotra, neelam marwaha, karan saluja, ratti ram sharma department of transfusion medicine, pgimer, chandigarh transmission through blood and blood products can be reduced to a great extent by efficient and reliable testing of the blood. the newer fourth generation elisa assays simultaneously detect antibodies against hiv- and and the presence of p antigen and thus shorten the window period to about days, as compared to days with third generation elisa. to compare the hiv seroprevalance among blood donors using fourth generation elisa (antigen-antibody) versus third generation elisa (antibody) assay. this was a prospective study involving blood donors of which were voluntary donors ( being students and being non students) and were replacement donors. sex workers are one of the core group for transmission of sti/hiv and as a ''bridge group'' to the general population. accordingly, highest priority is given to this group in targeted intervention for prevention of hiv/aids. here we are describing one such female sex worker who was harbouring concomitant sti including viral sti. a year old female sex worker was brought to the sti clinic of a tertiary care hospital by ngo with complaint of genital discharge for days. on per speculum examination, cervix was slightly erythematous, tender with mucopurulent discharge. there was no vaginal discharge or ulcer in anogenital area. however, there was a wart at lateral wall of vagina. as per naco syndromic management guideline, treatment was given for n. gonorrhoeae, c. trachomatis and hpv. cervical swab was taken and subjected to various microbiological investigation for the detection of sti viz n. gonorrhoeae, c. trachomatis, t. pallidum, candida spp., t.vaginalis, hsv- , hsv- , hiv, hbv, hcv, hpv and m. contagiosum. saline wet mount showed pus cells, but no yeast cells or trophozoite of trichomonas vaginalis. gram stained smear showed more than four polymorphonuclear leucocytes in the absence of gramnegative intracellular diplococci and a presumptive diagnosis of non gonococcal urethritis was made. no organism was isolated on any culture media after appropriate incubation. cervical swab was negative for antigen of c. trachomatis. serum was tested positive for hbv, hcv, hsv- and t. pallidum though it was seronegative for hiv. in the present case, the female sex worker was harbouring four viral sti viz hsv- , hbv, hcv and hpv alongwith t. pallidum. however clinically she was diagnosed and treated accurately only for genital wart while cervical discharge due to hsv- was misdiagnosed. it is necessary to try to test alternative approaches such as periodic presumptive therapy of viral sti, because this will not only boost up the efforts of sti control in the target group but also help in hiv control. alternatively, regular clinical and laboratory screening for viral sti may be tried. densonucleosis viruses (dnv) belong to parvoviridae family. they are the etiological agents of insect's disease known as densonucleosis, which leads to death or loss of vital functions of the infected insect. densonucleosis virus of mosquitoes has generated lot of scientific interests because of its tremendous potential in biological control and its application as a transducing vector. earlier, we have reported the isolation and characterization of a dnv from aedes aegypti mosquitoes and its prevalence among different ae. aegypti populations from india. there are reports suggesting that when aedes albopictus mosquitoes co-infected with dengue- and dnv, the multiplication of den- is suppressed. the present study focus on the effect of coinfection of ae. aegypti mosquitoes with dnv and chikungunya virus (chik). the first instar mosquito larvae were infected with dnv and the emerging dnv infected females were then infected with chikv by oral feeding. thus obtained chik infected female mosquitoes were analyzed by real time pcr for both dnv and chikv on alternate days post-infection, up to the th day. the data showed no significant difference in the multiplication of either of the viruses after co-infection. results suggest that chikv neither stimulates the replication of dnv nor is its own replication suppressed due to co-infection. this study forms an initial step in understanding the role played by such endogenous viruses on the vector dynamics. chandipura virus pathogenesis is manifested as encephalitis in young children with a very high mortality rate. this damage could be due to direct replication of the virus in brain parenchymal tissue or immune system mediated. this study aims at elucidating the role of brain infiltrating lymphocytes in pathogenesis using mice as the model system. mice were inoculated intracerebrally with the virus and the perfused brain tissue was used to isolate the lymphocytes. control mice were inoculated with an equal amount of media. in order to standardize the procedure for isolation of lymphocytes from brain tissue, splenocytes were processed to isolate the lymphocytes using histopaque density gradient method. methods to isolate lymphocytes from brain tissue as described by earlier workers were tested for the ease and efficiency of procedure using known suspension of lymphocytes from spleen. percoll density gradient method provided optimum yield of lymphocytes with an ease of handling. in this, brain cell suspension used to prepare % percoll is layered over % percoll prepared using media in : ratio. density gradient centrifugation is carried out at g for min at °c to obtain lymphocyte layer at the interface. leishman staining was performed to analyze the morphological characteristics of isolated lymphocytes. normal lymphocytes showed dark blue stained nucleus. some bigger sized cells with diffused nucleus characteristic of atypical lymphocytes were observed and some of the cells were surrounded by hair like structures. phenotypic characterization was carried out using flow cytometry. the presence of cd + , cd + and cd + cells was observed. the percentages of cd + , cd + and cd + cells was found to be . %, . % and . % respectively in the lymphocytes isolated from infected animal and . %, . % and . % respectively from control animal. hence, cd + cells showed maximum infiltration after infection. (santosh et al. ; pradeep et al. ). in the present study chikv suspected blood samples were collected and the acute phase samples were subjected to rt-pcr for the presence of virus specific rna by using the primer pair dvrchk-f/dvrchk-r as described by us earlier (naresh kumar et al. ). the convalescent phase samples were screened for chikv specific antibodies by using sd bioline chikungunya igm rapid test. six sets of primers were designed to amplify the complete nsp and complete structural genes of chikungunya virus. the products were further gel purified, cloned in ptz r/t vector and the recombinant clones were sequenced and submitted to the genbank. the complete ns gene and structural genes were compared with other available sequences in the genbank. sequence analysis results will be presented. the present study discusses these aspects in detail. . some of these phages (viz. v , v ) showed plaques at °c but not at °c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v , v , v , v and v . also, phage dilution medium (pdm) as described by chaterjee et al. ( ) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p . the various passages of the phage isolates v , v , v , v and v (i.e. original, p , p and p ) were stored at - °c. the four major routes of transmission are unsafe sex, contaminated needles, transmission from an infected mother to her baby at birth (vertical transmission) and breast milk. screening of blood products for hiv has largely eliminated transmission through blood transfusions or infected blood products in the developed world. in , globally, about million people died of aids, . million were living with hiv and . million people were newly infected with the virus. hiv infections and aids deaths are unevenly distributed geographically and the nature of the epidemics vary by region. more than % of people with hiv are living in the developing world. there is growing recognition that the virus does not discriminate by age, race, gender, ethnicity, socioeconomic status-everyone is susceptible. however, certain groups are at particular risk of hiv, including men who have sex with men (msm), injecting drug users (idus), and commercial sex workers (csws). the present study indicates the prevalence of hiv infection among the people residing in the northern region of india predominantly among the foothills of the himalayas. the study was carried out on the patients visiting herbertpur christian hospital (a unit of emmanuel hospital association) under the integrated counselling and testing centre scheme at the respective hospital during the - . the study indicates the screening of people groups residing in the respective area through community health schemes. the diagnosis of the hiv infection is done by three types of assays namely. the tridot method which is the rapid method of diagnosis followed by the. hiv coombs test which involves the dot immunoassay principle. the third assay is the enzyme linked immunosorbent assay (elisa). the number of patients screened during the period of september to march is which include patients coming from four different states namely haryana uttarakhand uttarpradesh and himachal pradesh. the number of people who were tested positive are and the number of people who were tested negative are . the people tested positive are sent to the higher centre for other confirmatory tests such as pcr and western blot analysis. these patients are sent for treatment and prophylaxis at a respective recognised centre in dehradun. the present study determines a consistent community hiv screening and treatment approach through diagnostics counselling and awareness programmes. classical swine fever (csf) also known as hog cholera is a highly contagious and fatal disease of swine. csf became rapidly a major issue of pig industries. it still causes important economical losses worldwide. it is considered as a major health problem of swines in india. during the month of august to october there was an outbreak of classical swine fever in bihar. from three districts darbhanga, patna and supol, total numbers of different infected tissue samples like kidney, spleen and lymphnode were collected from the dead morbid/pigs. total rna was isolated from % homogenate of infected tissues in sterile pbs by tri-reagent (sigma, usa) according to the manufacturer's instructions and cdna was prepared by using commercial available kit. the cdna was stored frozen at - °c until used. for the molecular detection of classical swine fever virus specific nested pcr amplification of e and ntr was done along with ns b and e rns amplification. primarily these samples were found positive with these primers. further confirmation by sequencing was done by cloning of these pcr products in pgem-t easy vector. e and ntr sequences were considered for phylogentic analysis along with complete available sequences of csfv. nucleotide sequence alignments were carried out using the clustalw program (dnastar) and phylogenetic tree analysis (dnastar) showed that ntr have close proximity with taiwan strain (accession no. ay ) and e shows close proximity with chinese isolate csfv- (accession no. af ). peste des petits ruminants (ppr) and sheeppox are oie notifiable diseases of small ruminants especially sheep and goat. both the diseases are economically important, in enzootic countries like india and are major constraints in the productivity of animals. considering the geographical distribution of both ppr and sheep pox infections and prevalence of mixed infection, in the present study, safety and potency of the experimental duel vaccine comprising attenuated strains of thermostable-ppr virus (pprv-revati, p- ) grown at °c and attenuated sheep poxvirus (sppv-srinagar, p ) was evaluated in local non-descript sheep. experimental animals were grouped into four groups and each group was comprising six animals, received doses ( tcid ), dose ( tcid ) and / th dose of vaccines and normal saline as control in ml volume subcutaneously, respectively. serum samples were collected on , , , and th day post vaccination. sheep simultaneously immunized with ml of vaccine consisting of either or doses of each of pprv and sppv were monitored for clinical and serological responses for a period of - weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to both pprv and sppv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, respectively following immunization. all the immunized animals' resisted infection when challenged with virulent strain of sppv (srinagar isolate at p- ) on day dpi, while in contact control animals developed characteristic signs of sheeppox. the challenge of the sheep against ppr was not carried out, however, the antibody titre after immunization determined by snt and elisa, indicated that protective titre, as per earlier report on the goats. dual vaccine was found safe at higher dose and induced protective immune response even at lower dose ( tcid ) in sheep, which was evident from sero-conversion as well as challenge study with sppv. the study indicated that both the viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this dual vaccine in combating both infections simultaneously. goatpox is one of the highly contagious, oie notifiable and economically important viral diseases of goats. the disease is caused by goatpox virus (gtpv) is classified of the genus capripoxvirus in the family poxviridae. the disease incurs severe economic losses in terms of high morbidity in adults and heavy mortality in young kids and is a major constraint in goat farming in india. considering the enzotic nature and economic impact of the disease, it is all important to control the infection by developing an effective vaccine. recently, vero cell based a live attenuated goat pox vaccine; using gtpv uttarkashi isolate (p ) has been developed in authors' laboratory and evaluated in goats. the vaccine was found safe, potent and immunogenic experimentally and even at field trials. the vaccine has been evaluated at large-scale at different regions of the country and found suitable for mass vaccination. however, the longevity of potency was not evaluated. therefore, a long term potency trials were studied for a period of years with annual challenge by using virulent goatpox virus and sero-monitoring. a sufficient number of hill goats has been vaccinated with dose of vaccine ( . tcid /ml) and monitored for clinical and serological response. every year, significant number of vaccinated (n = ) and control animals (n = ) were used for challenge with virulent strain ( . srd /ml, gtpv mukteswar). sera of pre-and post-challenged ( dpc) animals including controls have been collected and monitored for serological response in the form of specific antibody production by snt and indirect elisa. all the vaccinated animals were protected on challenge, whereas, all unvaccinated controls developed infections. the same has been reflected in sero monitoring of collected sera. so the developed live attenuated goat pox vaccine was found safe, immunogenic and potent for a period of years of immunization and suitable for mass scale vaccination in control and eradication of goat pox along with a/are suitable diagnostic tool/s in goatpox enzootic country like india. rotavirus infection in avian species varies from subclinical infections to outbreaks of diarrhea. the economic significance of rotaviral enteritis to the poultry industry has not yet been defined, but by analogy to the situation in mammals, it is likely to be significant. unlike the extensive studies performed on rotavirus infection in humans and animals, limited studies have been carried out to determine the extent of exposure of poultry birds to rotaviruses. to determine the prevalence of avian rotavirus antibodies in commercial broiler chickens. a total of chicken serum samples were collected from the lairage of a poultry slaughter house where birds from four different broiler farms in and around pune city were supplied to. the serum samples were tested by an igg antibody capture elisa wherein purified chicken rotavirus ch was used as coating antigen. sera from specific pathogen free (spf) chick (n = ) served as negative control in the test. cut off was calculated as mean negative control ? sd (standard deviation). s/co (mean sample od /cut off) values above ( . - . ) in % ( / ) serum samples were indicating positivity to rotavirus antibodies. the result of the study indicates exposure of the birds to avian rotavirus or similar agent that is circulating in pune city. bluetongue has become established in south india causing regular outbreaks in sheep. btv serotypes , , and were isolated from native sheep of andhra pradesh. the other serotypes circulating in the state need to be identified. however the major constraint is the serotype identification. to overcome the difficulties of traditional serotyping methods (neutralization tests), nucleic acid based tests are being tried. rt-pcr for serotyping was standardized using primers specific to vp gene of btv- , and serotypes. rt-pcr resulted in bp product of btv- , bp product of btv- which was defined by specific primers. however non specific amplification at two different sites i.e. bp and bp was noticed for btv- . specificity of rt-pcr was evaluated. btv- and btv- specific primers could amplify only btv- and btv- respectively where as btv- type specific primers amplified not only btv- but also btv- and btv- . nucleic acid sequence data obtained from btv- pcr product and btv- cloned products were specific to vp gene of btv- and btv- respectively. however, and bp products of btv- were identical to vp gene of btv- , , , , and and vp gene of btv- , and respectively, indicating the non specific amplification of btv- . foot and mouth disease is the most contagions and highly economically impotent disease of cloven footed animals. the disease is controlled by regular vaccination using the vaccine produced from the virus grown in the cell culture. the vaccine strain used for vaccine production is selected from the field isolates based on the adaptability and growth kinetics in bhk cells and antigen coverage. however the field viruses need to be passaged several times to adapt in tissue culture. passage of field viruses in tissue culture may results in development of mutants whose genetic makeup may differ from the field samples also some of the field strains may fail to adapt or may grow poorly in the tissue culture, thus the efficiency of the vaccine gets affected. structural proteins of fmdv carry the sequences which determine the serotype specificity and immunogenicity. thus one may replace the gene coding for structural proteins from the full length cdna copy of the vaccine strain that has been adapted to the tissue culture with the poly-structural protein gene (pi) so that the chimeric virus gets the serotype specificity of the field strain besides retaining the other characteristics that are needed for a vaccine virus. we have made replication competent fmdv asia i full length genome and cloned under t and cmv promoter separately in plasmid vectors. bam h sites were created for inserting pi- a gene of other field strains. the p - a of type 'o' vaccine strain was amplified directly from the cattle tongue material, cloned in plasmid vector and studied the specificity by sequence analysis and gene expression. we have introduced 'o' p - a gene into the full length construct devoid of asia structural protein gene, p - a. the in vitro transcribed rna in case of t promotered construct and plasmid dna in case of cmv promotered construct were transfected into the bhk cells. after the passaging the virus obtained was studied for the speciality. this approach may be used not only for rapid selection of vaccine strain and also as a repository of the cdna copy of the virus. the p is composed of a, b, c and d (vp , vp , vp , and vp ) respectively of which the vp is the most immunogenic and subunit vaccine produced with vp alone was able to induce high level of neutralising antibodies. thus to control the disease in india polyvalent vaccine consisting of the inactivated virus of all the three serotypes are in use. however the conventional vaccines have several drawbacks which include safety and temperature sensitivity. hence alternatively sub-unit vaccines consisting of vp protein has been tried. however this showed limited success due to the antigenic variations occurring in the field viruses thus escaping the neutralization from the antibodies generated from single cloned protein. hence the present study was undertaken with an objective to include all the neutralizing epitopes present in the three serotypes by linking vp ( d) genes and produce a poly valent protein for using as poly subunit vaccine. in this study we have constructed a cassette by linking the genes of three serotypes 'o' ( bp), 'a' ( bp) and 'asia ' ( bp). these genes were cloned individually in commercially pbsk vector and confirmed by sequence analysis before linking in pc dna vector. the linked gene construct was sub-cloned in pet expression vector. the expression of the protein gene from the pet vector was induced with iptg and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page). a fusion protein of size kda was observed in page gels. since the protein contains his residues from the vector at the n-terminal end, affinity purification was carried out using nickel nitrilo-tri-acetic-acid (ni-nta) agarose matrix. the immunoreactivity of the purified protein was assayed by western blot with the anti fmdv type 'o' and 'asia ' specific sera. the may be used as a subunit vaccine. silkworm diseases caused by viruses, bacteria, fungi and protozoans form major constraints for the silk cocoon production in all the sericultural countries and among these silkworm viral diseases viz., nuclear polyhedrosis and infectious flacherie caused by bmnpv and bmifv cause severe crop loss. the traditional disease management strategies include prophylactic measures and use of disease free silkworm eggs. the prophylactic measures such as disinfection of silkworm rearing house and appliances, egg surface, silkworm bed disinfection and rearing surroundings. the disinfectants used presently in sericulture are either formaldehyde or chlorine based products, but these chemicals are neither eco-nor user-friendly. the awareness about health hazards caused by formaldehyde and environmental pollution caused by cl necessitated the development of eco-and user-friendly disinfectant products for use in sericulture. these include alternative disinfectant products developed using biodegradable chemicals and plant based ingredients by apssrdi, hindupur and central silk board for the management of silkworm diseases in india. the ideal disinfectant for sericulture would be the one which can inactivate silkworm pathogens of diverse origin and economical for sericulture. the paper discusses on the disadvantages of hcho and cl based disinfectants and advantages of eco-and user-friendly disinfectant for the management of silkworm diseases especially the ones caused by viruses. the baculovirus expression vector system (bevs) is widely used for the production of high levels of properly post-translationally modified, biologically active and functional recombinant proteins and has facilitated basic biomedical research on protein structure, function, drug discovery and the roles of various proteins in disease. bevs is based on the introduction of a foreign gene into nonessential for viral replication genome region via of homologous recombination with a transfer vector containing target gene. the resulting recombinant baculovirus lacks one of nonessential gene (polh, v-cath, chia etc.) replaced with foreign gene encoding heterologous protein which can be expressed in cultured insect cells and insect larvae. insect cell-bev system is widely used to produce recombinant proteins. bevs also eliminates concerns regarding pathogens that could potentially be transmitted to humans as it is non-infectious to vertebral animals. these features make silkworm system an ideal expression and delivery package for producing proteins of medicinal importance. the efficiency, low cost and large-scale production of proteins using bevs represents breakthrough technology that is facilitating highthroughput proteomic studies. the bevs has become a core technology for cloning and expression of genes for study of protein structure, processing and function; production of biochemical reagents; study of regulation of gene expression; commercial exploration, development and production of vaccines, therapeutics and diagnostics; drug discovery research; exploration and development of safer, more selective and environmentally compatible biopesticides. utilization of silkworm larvae and pupae as bioreactor with recombinant bmnpv producing foreign proteins extends the usages of silkworms. due to its large-size and high protein synthesis ability as well as the expediency in mass culture, silkworm is considered as good candidate for producing recombinant proteins. wssbv is the causative agent of a disease, which has recently caused high shrimp mortalities and severe damage to shrimp culture. wssbv has been found across different penaeid shrimp species. in order to develop a effective diagnostic tool, a wssbv genomic library was constructed by cloning wssbv genomic dna extracted from purified virions. in the present study wssv disease free (confirmed by pcr analysis) were collected from hatcheries from different areas of guntur and prakasam districts and analysed to study the effect of various physical parameters like temperature, p h , salinity and turbidity on the prevalence of above disease. the studies on the surface water temperature revealed fluctuations in the ponds ranging between to . °c in diseased ponds and . to . °c in healthy ponds. these results show definite influence of temperature on the prevalence of wssv. present day strategy in vaccine development is to include marker facility that helps in distinguishing antibody response due to vaccination vis-à-vis infection in vaccinated animals. such information becomes relevant for effective disease control programmes especially when using inactivated virus vaccines like foot and mouth disease (fmd). the antibodies generated in the animals, only through vaccination, is the measure of vaccine efficacy and safety. presently inactivated fmd virus (fmdv) vaccines are used to control the disease in the endemic countries like india. the quality assurance of the vaccine depends on the efficacy of the vaccine in generating protective antibody without causing subclinical disease due to improper inactivation. since protective antibody response in vaccinated animals can not be distinguished from that of infected animals one needs to assay the antibody response against non structural proteins (nsps) and the vaccine must be free of contaminated nsps. production of vaccine free of nsps requires the cumbersome method of virus purification which adds to the cost of the vaccine. alternatively one may develop a positive marker vaccine by including a foreign protein or epitope which is not expected to be present in the vaccine and the antibodies generated against which helps in detecting the vaccine related response. here we report a molecular approach by which we introduced a immuno-dominant epitope of green fluorescent protein (gfp) into the structural protein gene of foot and mouth disease virus vaccine strain asia ( / ). our laboratory has produced a mini-genome of fmdv asia that lacks structural protein gene (p - a) coding for all the structural proteins (vp - ) of fmdv asia as a vector (pcfl dasia ). the p - a of the asia vaccine strain was cloned separately into a plasmid vector and by successive pcr mutagenesis and cloning we have introduced nucleotide sequence corresponding to amino acid epitope of gfp into p - a gene. gfp epitope was inserted by replacement at n-terminal region of vp- which is not immunogenic. the modified p - a was expressed in e. coli and studied. the modified p - a gene with gfp epitope was inserted into the pcfl dasia to get full length replication competent cdna cloned under cmv promoter in pcdna (pcflasiagfp). this can be used to produce synthetic virus with gfp epitope that can generate antibodies not only against neutralizing epitopes but also against gfp epitope. presence of antibody against gfp epitope in the vaccinated animal will reveal vaccine efficacy. elisa against gfp can be used as a companion test not only for safety evaluation but also for quick evaluation of efficacy. further absence of nsp antibodies in the serum may reveal the quality of the vaccine in respect of safety. self replicating dna vaccines are developed to achieve robust immune response through enhanced antigen production and gamma interferon expression in vaccinated animals. since self replicating dna vaccines induce gamma interferon expression which helps in viral clearance such vaccines are expected to be useful to cure even the carrier and persistently infected animals. understanding the events that help in the elicitation of both the arms of immune response in vaccinated animals is necessary to understand the effectiveness of the vaccine. the work presented here deals with the immunological evaluation of a sindbis virus replicase based dna vaccine carrying linked fmdv vp genes in vaccinated guinea pigs. we have constructed self replicating dna vaccine vector and to the down stream of a sub genomic promoter we have inserted secretory signal followed by linked-vp genes of fmdv serotypes (o-a-asia ) with glycine and proline bridge in between. guinea pigs were vaccinated with the construct and the sera at days post vaccination were evaluated both for cellular response by studying the cd levels and by mtt and cytokine profiles by real time assays. the humoral response was evaluated by studying cd levels in the whole blood by facs analysis and serum antibody levels by snt and elisa. the animals were challenged with gp infective dose of fmdv type 'o' virus lesions were scored. further the replicative efficiency of the challenge virus was studied by ab elisa. the results showed that all the assays except antibodies against ab protein have positive correlation with the protection. as expected the titre of the antibodies against ab protein was lower indicating that the challenge virus replication was inhibited in the vaccinated animals. the limited studies conducted by us showed that self replicating vaccine has a potentiality to emerge as potent vaccine for fmd. ganjam virus (ganjv) belongs to the genus nairoviruses (family bunyaviridae). these viruses cause diseases in livestock. it has been isolated from different animal hosts and tick vectors from india. genus nairoviruses includes a total of tick-borne viruses, classified into serogroups. the important serogroups are crimean congo hemorrhagic fever (cchf) and the nairobi sheep disease (nsd). the main members of the nsd group are nsd and dubge viruses. their genome consists of three segments of single stranded rna, viz. s, m and l that encodes viral nucleocapsid protein, viral glycoprotein g and g and the viral polymerase respectively. ganjv is very closely related to (nsdv). nsdv is found in east and central africa, causes very high morbidity and mortality in livestock. the present study involves phylogenetic comparison of ganjv isolates from india with other nairoviruses based on complete n gene. it will help to understand the kind of nucleotide (nt) and amino acid (aa) changes that have occurred in ganjv strains from different geographical areas. eight strains of ganjv isolated at niv during - from different parts of india were used in this study. virus stocks were prepared in vero e cell line these were used as the source of viral rna. the n gene was amplified either as a complete gene in one reaction or in fragments whenever necessary. thus obtained sequences were analyzed; annotated to get a consensus sequence, aligned against the sequence of prototype strain of ganjv and other representative nairoviruses. the nt sequences were converted to aa sequences and analysis was done at both nucleotide and amino acid levels. based on what nt or aa phylogenetic tree was constructed and compared with other nairoviruses (cchf, dugv, hazv, kupv and nsdv) where complete s segment sequences were available gen-bank database (ncbi). the phylogenetic data at both the nt and aa levels showed that all the strains of ganjv form monophyletic lineage with the nsdv. cchfv and hazv together formed another clade, whereas dugv and kupv made a separate branch in the tree. the different ganjv strains showed - % difference with nsdv at the nucleotide level and - % difference at the amino acid level. hazv showed - % difference at the nt level and % difference at the aa level with ganjv as well as nsdv. the present data obtained suggests that ganjv and nsdv are minor variants of the same virus. diarrhoeal syndrome is one of the major concerns of the livestock industry. most of the diarrheic cases in animals go unnoticed and limited attention is paid on viral etiology. presence of large amount of fecal matter in animal shed acts as a source of infection for calves via drinking water, feed, or contaminated soil. keeping this in view, investigation was planned to detect the association of rotaviruses with diarrhea in dairy calves and to observe the genomic diversity among the circulating viruses in tarai area of uttarakhand. a total of diarrheic fecal samples collected from instructional dairy farm, nagla, pantnagar, uttarakhand were screened during the study. samples were collected from both cow calves and buffalo calves in - months of age. for the diagnosis of rotavirus, all the fecal samples were subjected to rna-electrophoresis after nucleic acid extraction. viral genome segments were visualized by silver staining. out of the total samples tested, seven were found positive in rna-page showing typical genome segments migration pattern of bovine rotavirus. in the given samples prevalence of bovine rotavirus was . % and % in cow and buffalo calves, respectively. on the basis of migration patterns of rotavirus in rna-page, group a were identified with typical : : : pattern. variation within movement of various genome segments among isolates of bovine rotaviruses was observed during the study that may be indicative of emergence of mutants in the circulating isolates. the vp gene based group a specific rt-pcr was standardized and all the isolates in this area were confirmed to be of group a type. work is in progress to genotype the bovine rotaviruses of this region based on vp and vp genes. this study emphasizes the need to explore the prevalence of bovine group a rotaviruses in different places of uttarakhand and their genetic characterization which could help in selection of control strategies for rotavirus infections. foot-and-mouth disease (fmd) is endemic in india causing enormous economic loss to the animal keepers and trade embargo with fmd free countries in livestock and animal products. rapid diagnosis of fmd is of immense importance in prevention and control of the disease. fmd is initially diagnosed clinically and confirmed by laboratory tests. virus isolation in cell culture and sandwich elisa for antigen detection are commonly practiced in laboratories. the virus isolation though is very sensitive but it can be slow and analytical sensitivity of the elisa is lower and can not be used with certain sample types. the use of molecular techniques in the diagnostic laboratory has greatly increased the speed, specificity and sensitivity of fmd diagnostic tests. molecular techniques like rt-pcr, pcr-elsa and dot hybridization can be used with more success for detecting carrier animals and animals harboring sub-clinical infection and can be applied in a wide range of clinical sample types. these techniques can be used as genus and serotype specific test including detection of particular lineage/genotypes with in the serotype. multiplex pcr has been used to differentiate serotypes of fmdv and the technique is sensitive, experimentally simpler, cost effective and less time consuming. the assay can be used for serotyping on elisa negative samples. the molecular techniques not only help in diagnosis but also useful for epidemiological studies. lineage differentiating rt-pcr has been useful in identifying different lineages of serotype asia (lineage b, c and d) before proceeding with sequencing of d region. similarly genotype differentiating rt-pcr has been developed and used in differentiating two different genotypes of serotype a (genotype vi and vii). these assays have the potential to be applied on clinical samples directly, thereby saving much time needed for sample processing and nucleotide sequencing. recent development of real time rt-pcr methodology has allowed the diagnostic potential of molecular assays to be realised. advancement in real time pcr technology made it possible to combine several assays within a single tube which is in the progress in our laboratory. integration of these assays onto automated high throughput platforms provides diagnostic laboratories with the capability to test large numbers of samples. microarray technology was provided greater screening capabilities for pathogen detection. the microarray allows the addition of large number of oligonucleotide probes for identification of mutant pathogen and also for subtype determination. the combined properties of high sensitivity and specificity, low contamination risk, and speed has made realtime pcr and microarray technology a highly attractive alternative to conventional methods in increasing percentage of outbreaks confirmed and analyzed and for tracing the origin of fmd virus responsible for outbreaks. dna vaccines are expected to elicit both humoral and cellular responses, cellular response being long lasting. however the approach has several limitations like poor stability of dna, poor expression and risk of integration. poor expression becomes the major limitation in the case of fmd as fmdv proteins are poor immunogens. also dna vaccine vectors carrying only eukaryotic promoters elicit strong cmi response and weak humoral response. the methodology to achieve humoral response involves the expression and secretion of the expressed protein so that the antigen presenting cells will be able to process the antigen and produce humoral response. in case of fmd humoral response is as important as cellular response. the present project aims at addressing these issues; achieving higher expression and getting the protein secreted out by constructing self replicating gene vaccines for fmd and studying their efficacy. the vector for humoral immune response contains eef promoter, sindbis virus polymerase gene and secretory and anchoring signals. the integrity of the vectors was confirmed by sequence analysis. the linked polyvalent protein genes of fmdv serotype a, o and asia were cloned into the vectors and the presence of the insert was confirmed by restriction enzyme digestion. the functionality of the constructed dna vaccine vector (pvac self rep ) was assayed by transfecting the dna into bhk cell monolayer and studying the s labeled proteins in immuno-precipitation assays. the studies showed high level of expression in case of constructed vector as compared to infected virus for the specific protein. the secretion of the expressed protein was assayed by immuno-fluorescence assay and found to be positive. encouraged with these studies the preliminary studies were conducted on vaccine efficacy studies in guinea pig model. the immunized guinea pigs showed high antibody titres by snt and elisa, as compared to conventional dna vaccines (pup cd) even at / th of the dose. this approach of constructing self replicating dna vaccine for humoral response is the first report. genetically engineered microorganisms are important sources of industrial and medicinal proteins. over the past decade, plant host system has been investigated as potential host system for expressing proteins of therapeutic and diagnostic use. however concerns regarding the stability and environmental safety need to be addressed. chloroplast engineering is expected to resolve some of these issues since, plastids/chloroplasts are inherited maternally and are not disseminated through pollen. this makes plastid transformation a valuable tool for transgenic creation besides offering biological containment. since foot and mouth disease (fmd) of cloven footed animals is a major concern in the world over. foot and mouth disease (fmd) is the most feared, viral disease of the cloven footed animals causing heavy losses to the live stock industry. the disease is enzootic in many parts of the world including asia. the conventional vaccines for fmd have several limitations which include safety, temperature sensitivity and duration of immunity. attempts have been made to overcome these limitations using recombinant dna technology. amongst the newer vaccines, edible vaccines are cost effective and easy to administer. since the stability of the gene of interest is the major concern in the case of plant transgenics, marker genes are used for regular selection. the detection methods based on the available marker proteins like b glucoronidase (gus) protein/antibiotic selection are cumbersome and cost intensive. however selection based on herbicide resistance is much simpler and easy. hence in the present study, the -enolpyruvylshikimate- -phosphate synthase (epsp) gene was used as a marker along with the immunogen gene of fmdv. epsp is the key enzyme in the shikimate biosynthesis pathway necessary for the aromatic amino acids production. in order to investigate the mechanism of long term immunity and the effect of protective immunity induced by cationic plg micro particle coated dna vaccination. we constructed the expression plasmid containing a foot-and-mouth disease virus (fmdv) id gene sero type a. intramuscular vaccination of guinea pigs with the micro particles coated plasmid dna induced a strong antibody response and neutralization antibodies, cellular mediated immune response which lasted year. we further analyzed the persistence and expression of id gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction and quantitative pcr. the results showed that id gene was present and expressed in the muscle cells up to year after days post vaccination. furthermore, guinea pigs vaccinated with micro particles coated plasmid dna were protected against a challenge with fmdv virus. therefore the micro particles coated plasmid dna vaccination dose induce a protective immunity and long term humoral, cellular immuno responses against fmdv, which could be maintained by persistent expression of id gene in muscle cells. foot and mouth disease virus (fmdv) causes a highly contagious viral disease of cloven hoofed animals, which has a considerable socioeconomic impact on the countries affected. interleukin- (il- ) enhances the il- driven th immune response that is important in immunity against intracellular pathogens. the multiple roles of il- in many physiological and pathological processes have generated a great deal of interest in recent years. antiviral effects of il- have been reported. we evaluated the effects interleukin- (il- ) on the replication of fmdv in vitro in bhk- cells. bovine il- mature protein coding sequence was amplified from the bovine pbmc cells and cloned into prokaryotic expression vector pet a. protein expressed was purified and specificity was confirmed by immunoblotting. bhk- cells were treated with purified expressed il- protein with ( lg/ml) h prior to fmd infection. cell culture supernatants were collected at h post infection were subjected for elisa and virus titration assay. rna extracted from the cells was subjected to real time pcr for viral rna quantification. log titer reduction was observed in the fmd virus titer in il- treated cells compared to the untreated cells where as virus antigen quantified by elisa has shown a reduction of -folds. -fold reduction in the fmd viral rna copy number was observed in the il- treated cell compared to the untreated measured by qpcr. current study demonstrated the potent anti viral activity of il- on fmdv by inhibiting the viral replication. these results further suggests that il- has the potential role of il- as molecular adjuvant in fmd vaccine development and development of therapeutic for fmd. foot and mouth disease is the most contagious viral disease of farm animals. control of the disease in animals is by vaccination and slaughtering of infected animals. conventional oil adjuvant vaccine has its own limitation. alternate to this genetic vaccines where the dna encoding viral antigen may be a promising approach. naked dna vaccine has limitations like poor uptake of dna by cells and more importantly by nucleus. as a result delivery of naked dna through calcium phosphate nanoparticle was attempted. calcium phosphate nanoparticle is a potential delivery agent which proved to enhance the immune response. fmdv p - cd ''o'' vaccine gene constructs in pcdna . ? entrapped by the nanoparticles was prepared by using different molarity of calcium chloride and disodium hydrogen orthophosphate. the nanoparticles entrapping fmdv p - cd ''o'' and naked dna were presented to the guinea pigs through intramuscular injection to study the mrna expression of antigen by rt-pcr. animals were sacrificed at defined time to collect different organs and total rna was extracted. each time blood was collected to analyse the fmdv specific serum antibodies. dna vaccines presented through calcium phosphate produced transcripts in the injected muscle up to days whereas naked dna up to days. serum antibody levels of naked dna vaccine showed antibody titre till days. whereas nanoparticle injected animals showed serum antibody till days. serum neutralization titres of . were observed in calcium phosphate dna vaccines at about - days, where as naked dna sn titers were observed for short period of - days. the study clearly showed calcium phosphate nanoparticle entrapping fmdv vaccine dna may be a better delivery system for dna vaccines as it confirms availability of the antigen and persistence of antibody for longer duration than naked dna. capripox is highly infectious, contagious, and oie notifiable disease of small ruminants, caused by sheeppox and goatpox viruses which are members of capripoxvirus genus of the family poxviridae. in the present study, we analyzed the partial gene sequences of p protein, an immunogenic envelope protein of capripox viruses (capv) to assess the genetic relationship among different sheep pox and goat pox virus isolates from different geographical areas of the country. product of this gene has been shown to be important in attachment of capv to host cell surface receptors during viral entry and host immune response. the following virus isolates have been used in the analysis: gtpv-uttarkashi, p , vaccine virus; gtpv mukteswar, p , challenge virus; gtpv (akola), gtpv bareilly/ , gtpv ladakh/ and gtpv sambalpur/ , field isolates and sppv srinagar, p ; sppv ranipet, p ; sppv-rf, p , vaccine viruses and sppv makdhoom/ , sppv cirg/ , sppv pune/ , sppv bareilly, sppv / and sppv / , field isolates. in this study, all virus isolates were confirmed by pcr amplification and analysed in pcr-restriction fragment length polymorphism (pcr-rflp) using ecori enzyme to confirm their specificity. further, the amplicons were cloned and sequenced commercially. nucleotide and the deduced amino acid (aa) sequences were compared with published sequences of the members of the genus capripox virus. sequence analysis of partial bp sequence has shown high sequence identity among all indian sppv and gtpv isolates at both nt and aa levels. it revealed a . - % and . for gtpv field isolates where as, % for sppv field isolates at both the nt and aa levels. in general, capv isolates in this study shown . - . and . % homology between gtpv and sppv at nt and aa levels as reported earlier. further, it revealed a unique change of g a in all gtpv isolates resulting in formation of drai site in place of ecori and possible development of restriction enzyme specific pcr-rflp for differentiation of sppv and gtpv from field isolates. orf or contagious ecthyma is considered as non-contagious, proliferative disease and is caused by orf virus of the genus parapox virus of the family poxviridae. it is reported most commonly in sheep and goats and also a zoonotic agent. camels are also infected by orf virus and reported in camel rearing countries as a mixed infection with camel pox, the later is caused by an orthopox virus. in india, there are few reports of the orf virus infection in camels and identified by clinical signs and pcr. in this study, we identified the presence of orf virus from clinical samples of suspected case of sporadic infection in camels by serological and molecular techniques. viral dna isolated from processed scabs used initially in nested polymerase chain reaction as diagnostic pcr which successfully amplified bp fragments and also sequenced to check the fidelity of the product. after confirming the infection by pcr, some of the structural and non-structural genes were amplified for sequence analysis. out of the five genes characterized, the major important one selected for sequence and phylogenetic analysis is b l gene which is homologous to a major envelope protein p k of vaccinia virus. full open reading frame of bp from orf b l was amplified by pcr, cloned and sequenced commercially. nucleotide and deduced amino acid sequences of b l were compared with other published sequences of the members of the genus papapox virus. sequence analysis shows a maximum percent identity of . and (indian orf virus isolates); . and . (other orf isolates); . and . (orf-camel/jodhpur/ ); and . (bovine popular stomatitis virus) and finally . and . (pseudo cowpox virus) respectively at nt and aa levels. phylogenetic analysis of the isolate was also performed using the neighbour joining method in mega program to know the phylogeny relatedness of the virus, which revealed that the isolate is well grouped with the jodhpur isolate and closely related to pseudo cowpox virus. it warrants further analysis of other potential genes to confirm the causative agent of the contagious ecthyma in camels as pseudo cowpox virus. chikungunya an arboviral disease is transmitted through the bite of an infected aedes mosquito. it causes a self limited febrile illness along with arthralgia and myalgia. in some cases neurological and severe hemorrhagic manifestations has been observed. chikv epidemic has been reported in africa, india, south east asian countries and during the current out break imported cases of chikv has been encountered in most of the european countries. the causative agent belongs to the genus alphavirus family togaviridae. human beings serve as the chikungunya virus reservoir host during epidemic periods. outside these periods the main reservoirs are monkeys, rodents, birds, and other unidentified vertebrates. antibodies to chikv have been detected in domestic animals. in the present study we surveyed madanapalli, palamaner, b. kotta kota and tirupati and collected a total of rodent samples, bovine samples; sheep samples and canine samples. total rna was isolated from all these samples and subjected to rt-pcr using a primer pair dvrchk-f/dvrchk-r which could amplify a bp e gene product specific to chikungunya virus (naresh kumar et al. ). all the serum samples were further screened for chikv specific igm antibodies using commercially available ctk biotech strips. none of the samples were found positive either for chikv specific rna or chikv specific igm antibodies. more number of samples from domestic animals as well as rodents are being screened to study their possible role if any in the maintenance of chikv in nature and during the inter epidemic periods. the present study discusses these aspects in detail. petunia hybrida is widely used as experimental host plant for begomovirus identification and its characterization. hitherto, natural infection of begomovirus on petunia has not been reported in india. recently, petunia hybrida grown in and around ludhiana were found to be depicting typical symptoms caused by begomovirus. the symptoms include severe reduction in leaf size, downward curling and distorted leaves. severely infected plant became bushy, stunted and produces no flower. total genomic dna was extracted from the plants showing symptoms of begomovirus, by ctab method. the presence of virus was confirmed by using degenerated primers, designed to identify all the begomovirus prevailing in the world. to identify the strain associated with the disease, the positive samples along with healthy control were tested against different strain specific primers of tomato leaf curl virus, so far reported in india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus. among these, only tomato leaf curl new delhi virus specific primer was able to give the desired amplicon of * bp. hence, it is confirmed that the leaf curl disease of petunia hybrida is associated with tomato leaf curl new delhi virus. this disease of petunia can become a sever production constraint in coming years. from last years ( and ) it was observed that some varieties of brinjal grown in rainy season, showed typical leaf curl type of symptoms. the symptoms include upward curling of the leaves, cupping, vein thickening, reduction in leaf size and distortion of leaves. the severely infected plant remains stunted and bushy, became unproductive or produces only few fruits. the disease was experimentally transmitted from naturally infected brinjal to healthy seedlings by whiteflies (bemisia tabaci) and grafting, but not by mechanical or aphid transmission. to detect the begomovirus associated, total genomic dna was extracted from the plants showing disease symptoms. the presence of virus was confirmed by using pcr based begomovirus geneus-specific primers designed by deng et al., wyatt and brown and rojas et al. these degenerated primers give the expected product size of * , * and * bp, respectively. core coat protein (cp) gene and dna-b was also amplified in the samples using specific primers. to identify the strain associated with leaf curl virus, dna was subjected against primers of different indian tomato leaf curl virus strain i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl new delhi virus primer was able to show the desired product size of * bp. therefore, it was confirmed that leaf curl disease of brinjal is caused by tomato leaf curl new delhi virus in association with satellite b-dna. to identify the strain associated with the disease, all samples were further subjected to the specific primers, designed to amplify all the tomato leaf curl virus strains, so far reported from india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl palampur virus specific primer was able to give the expected product size of * bp. this shows the association of tomato leaf curl palampur virus with leaf curl disease of calendula and marigold. thus, calendula and marigold can act as a reservoir for the tomato leaf curl palampur virus and may cause severe constrain in the production of these important ornamental plants. groundnut bud necrosis virus (gbnv) belongs to serogroup iv of the genus tospovirus in bynayaviridae family and infects several economically important crops all over india. the nucleocapsid protein (np) encoded by the small rna of gbnv encapsidates the viral rnas. apart from this structural role, the np has also been implicated in the replication, transcription, maturation and cell to cell movement. with a view to study the structure and function, the np of gbnvtomato isolate from karnataka was over expressed in e. coli and purified by ni-nta chromatography. the purified np was present as ribonucleoprotein complex and as heterogeneous mixture containing monomers, tetramers and higher order multimers. in order to determine the regions involved in oligomerization and nucleic acid binding, mutational approach was taken. n-and c-terminal deletion clones were generated (n np, n np, c np and c np), over expressed in e. coli, and were purified by a procedure identical to that used for the wild type protein. initial studies on oligomeric status suggested that in addition to n-and c-terminal regions there may be additional regions or residues which contribute to multimerization of np. the amount of rna bound to the truncated proteins was reduced in case of n np, n np and c np. interestingly removal of amino acid residues (natively unfolded region) from the c terminus resulted in complete loss of nucleic acid binding suggesting that the rna binding domain was located in c-terminal region of np. further np was observed to get phosphorylated in in vitro kinase assays by a kinase present in the soluble fraction of tobacco plant sap. both atp and gtp were utilized as phosporyl donors and mn ? was the preferred metal ion which suggests that np might be phosphorylated by a ck -like protein kinase. phosphorylation studies with n-and c-terminal truncated proteins revealed that the site of phosphorylation lies within the amino acid residues - . by mass spectrometric analysis of the protein threonine- and serine- were identified as possible phosphorylation sites. a naturally occurring isolate of virus infecting gherkin (cucumis anguira l.) showing mosaic symptoms of mosaic, leaf distortion and dark green islands in the lamina was identified in the export cultivars of gherkin grown in commercial fields of kuppam rural, chittoor district, andhra pradesh. the virus infection was deadly prevalent among the field that caused a lot of economic damage to the crop that resulted in yield losses and reduced quality of fruits meant for export. symptoms of the infected fruit included blistering and malformation of the fruit. the virus infected leaf samples were collected and initial host range tests were conducted with different cucurbit species showed that the host range include propagation hosts like cucumis anguira (gherkin), cucumis sativus, cucurbita pepo, cucumis melo, langeneria vulgaris, momordica charantia and local assay host like chenopodium amaranticolor. the virus host range was only restricted to cucurbit species and chenopodium. the virus was maintained for further studies on cucurbita pepo by sap or mechanical inoculation. the virus induced mosaic, vein clearing symptoms on pumpkin. electron microscopy of the leaf dip preparations stained with % uranyl acetate from the pumpkin leaves showing symptoms revealed the presence of a long flexuous filamentous particle measuring nm. the virus positively reacted to the polyclonal antisera of papaya ringspot virus-w, potato virus y, tobacco etch virus and also strongly reacted with the polyclonal antiserum of zucchini yellow mosaic virus in direct antigen coated-enzyme linked immunosorbent assay (dac-elisa). because of very strong reaction to polyclonal antisera of zucchini yellow mosaic virus, we tried to amplify the partial nib and cp genes of the virus along with the utr by using two primers zy gctccatacatagctgag acagc and zy taggctttttgcaaacggagtcta at c . total rna from gherkin infected leaves was isolated using trizol ls reagent (sigma). rt-pcr was performed to obtain an amplicon of * . kbp, cloned into fermentas ptz r/t vector and sequenced at mwg biotech, bangalore. sequence analysis revealed that the virus was isolate of zucchini yellow mosaic virus and was showing % of homology to that of the zucchini yellow mosaic virus strain b genome ay and zucchini yellow mosaic virus nat genome ef which were strains reported from israel. the sequence of the present study was submitted to the genbank gq . the results state a suspicion that the virus could have been mobilized by some infected source brought by the commercial israeli based companies into india due to poor quarantine regulations as the gherkin cultivation in these regions is chiefly supported, purchased, exported and marketed by these private companies that are based from israel. this is the first report on molecular characterisation of zucchini yellow mosaic virus infecting cucumis anguira (gherkin) from india. they also exhibited synergism with other virus which was region specific. fifty percent of the total symptomatic plant population was found be positive only for carla while remaining showed mixed infection of carla with tospo in some regions while in others carla virus was found to be associated with cmv. presence of only carlavirus was up to - % incidence, without association of tospo, cmv, poty or tobamo viruses was also observed in some fields. avijit tarafdar, raju ghosh, k. k. biswas plant virology unit, division of plant pathology, indian agricultural research institute, new delhi citrus tristeza virus (ctv), a brown citrus aphid (toxoptera citricidus) transmitted closterovirus under family closteroviridae, is one of the major limiting factors in cultivation of citrus worldwide. ctv is a longest known plant virus having flexuous particle of nm in size. ctv genome is a positive sense ssrna of about kb nucleotide containing open reading frames (orfs) encoding proteins. several biological as well as genetic variants of ctv are reported in all the citrus growing countries in the world. ctv causes decline and death of millions of citrus trees in the world. in india, ctv is a century old problem, and has killed an estimated one million citrus trees till today. in molecular and genetic level, ctv isolates from india were not fully characterized. genetic diversity and sequence divergence in ctv isolates of india are not fully established. further, evidence of recombination and causes of evolution of ctv variants in india have not been studied till date. therefore, in the present study, effort has been made to characterize several indian ctv isolates in genetic level, examine their genetic diversity, identify recombination events and analyze evolution of divergent ctv. a total number of ctv isolates from different regions of india ( from darjeeling hills, five from bangalore, from delhi and from vidarbha) were under taken for genetic study. two genomic regions of ctv, i.e., entire cp gene (cpg) ( nt) and a gene fragment of orf a (orf a) ( nt) were amplified, cloned, sequenced and nucleotides were analyzed. based on cpg, indian isolates shared - % nucleotide identity, and based on orf a they shared - % identity, among them. incongruence of phylogenetic relationship was observed as on sequence analysis five phylogenetic clades based on cpg, and eight clades based on orf a, were generated suggesting the recombination events have been occurred between the sequences of indian ctv isolates. thus, to identify the potential recombination events, and determine the parental sequences in ctv isolates, six recombination detecting algorithms, namely, rdp, genconv, bootscan, maxchi, chimera and siscan were used. out of indian ctv, cpg of and orf a of isolates of ctv showed recombination events suggesting orf a was more prone and fragile to rna recombination as compared to cpg. this findings indicated that high degrees of genetic diversity and incongruent relationships of indian ctv isolates are due to genetic recombination occurred, which may be the important factors in driving evolution ctv variants in india, that was also supported by a splittree decomposition analysis. b. v. bhaskara reddy, y. sivaprasad, k. rekha rani, k. raja reddy department of plant pathology, regional agricultural research station, acharya n.g. ranga agricultural university, tirupati, andhra pradesh sunflower (helianthus annus l.) is one of the most important oil seed crops in the world which ranks third in area after soyabean and groundnut. the sunflower necrosis disease (snd) is characterized by necrosis of leaves, necrosis streaks on petioles, stem, floral parts and stunted growth. the causal agent of the disease has been identified as tobacco streak virus (tsv) which belongs to genus ilarvirus of the family bromoviridae. the suspected tsv infected sunflower samples collected from chittoor district in andhra pradesh were found positive for tsv-dac elisa. total rna was extracted from sunflower using rnaeasy isolation kit (qiagen). the tsv coat protein (cp) gene, movement protein (mp) gene and replicase (rep) gene were amplified by rt-pcr with specific primers, cloned in ptz r/t vector, sequenced and deposited in genbank (gu , gu and gu ). the size of cloned cp gene was bp and codes for amino acids. the cp gene sequence analysis revealed that the tsv-tpt infecting sunflower has - % homology at nucleotide level with soybean, tagietus-tpt and okra-tn isolates and - % homology at amino acid level. the movement protein gene was bp and codes for amino acids. the mp gene sequence analysis showed that it has - % homology at nucleotide level and - % at aminoacid level. chilli (capsicum annuum), the important commercial vegetable/spice of himachal pradesh, is affected by several viral diseases; of them cucumo, tospo, poty and gemini viruses are the most common genera. however, these viruses are not identified clearly and characterized fully, which are foremost needed to formulate the management strategy. therefore, in the present study, effort has been made to identify and characterize the important viruses causing diseases in chilli. in this study, several farms in major chilli growing areas of bilaspur and kangra districts in himachal pradesh were surveyed and infected plant samples were collected randomly. virus infection in these samples were detected by das-elisa using antisera to cucumber mosaic virus (cmv) and potyvirus (group specific) and through slot-blot hybridization (sbh) using cmv, iris severe mosaic poty virus (ismv), tomato spotted wilt tospo virus (tswv) and chilli leaf curl gemini virus (clcuv). based on das-elisa and sbh, the incidence of disease was estimated and ranged from . to . % by cmv and . to . % by potyvirus. to detect tospo and geminivirus in the infected chilli, sbh test was carried out. infected samples showed maximum virus titer in both das-elisa and sbh test were further confirmed by pcr using specific primers. desired sizes of amplicons; * bp, * bp, * bp and * bp of cmv, poty, gemini and tospo viruses, respectively, were obtained. as the present study clearly indicated that cmv appeared as a major one among the viruses infecting chilli in the hilly region of himachal pradesh, two isolates of cmv were characterized in genetic level. thus the amplified products (* bp) of cmv, palampur and palampur were cloned in pgemt cloning vector, sequenced and the sequences were submitted to ncbi database (palampur : acc-fm and palampur : acc-fm ). the sequences were then analyzed and compared with other sequences available in the data base. based on sequence analysis, it was found that present cmv isolates shared % nucleotide identity between them, are closely related with australian cmv isolate cmv-ly (acc-af ) by % nucleotide identity. in phylogenetic tree analysis, it was observed that indian cmv isolates formed same cluster along with cmv-ly. as it is known that cmv subgroup ii comprises cmv-ly, it is concluded that the cmvs of this hilly region of himachal pradesh belong to subgroup ii. chilli is essentially a crop of the tropics and grows better in hotter regions. chlii (capsicum annuum), a member of family solanaceae is an important vegetable and spice crop of immense commercial importance. the pungency in pepper is due to an alkaloid known as capsaicine and peppers are characterized as sweet, hot or mild depending on capsaicine content. the present investigation were conducted to find out the highly resistant cultivars of capsicum annuum against cmv and tylcv among ten cultivars of chilli in agroclimatic condition of aligarh. the highest ( and ) percentage of infection was observed in hc- and kalyanpur type- by showing the positive reaction to cmv by elisa test. no symptoms was recorded in case of bc- , lca- and jca- and showed negative reaction to cmv by elisa. bc- and lca- also showed negative reaction to tylcv by elisa and these were symptomless. maximum infection ( and ) was registered in hc- and c , cultivar. so, the bc- , lca- and jca- has proved highly resistant varieties against cmv and tylcv and these may be used in breeding programmes against viruses. cotton leaf curl virus belongs to the family geminiviridae, genus begomovirus. the members of this family contain circular single stranded dna molecules as their genomes. there are two kinds of begomoviruses-bipartite viruses with genomes consisting of two dna molecules designated dna-a and dna-b and the monopartite viruses which contain only dna-a but not dna-b. in monopartite viruses, the dna-a is accompanied by a small circular dna molecule called dna-b which is essential for the development of typical disease symptoms. cotton leaf curl virus is a monopartite virus and causes the cotton leaf curl disease which has emerged as a major disease of cotton in the indian subcontinent. the non-structural protein ac of cotton leaf curl kokhran virus-dabawali isolate (clcukv-dab) was cloned into pgex x vector and overexpressed in bl (de )plyss e. coli cells. the overexpressed gst-ac protein was purified by glutathione sepharose chromatography. the purified gst-ac protein was found to possess atpase activity. the optimum temperature and ph for the activity were °c and . respectively. the atpase activity was inhibited in presence of edta, showing that it is dependent on divalent metal ions. the activity was supported by magnesium, manganese and zinc ions but inhibited in presence of calcium ions. it was also inhibited by the non-hydrolyzable atp analogue adenosine-b, c-imido triphosphate and in the presence of other nucleotides like ctp and gtp. the k m and the v max of the reaction for atp as the substrate are . mm and . nmol/min/ mg of the protein respectively. the enzyme could also utilize gtp as the substrate. the fact that ac is specifically an ntpase and not a general phosphatase is revealed by the finding that it does not hydrolyze p-nitrophenyl phosphate to yield yellow colour while a similar reaction carried out in parallel with alkaline phosphatase readily yields the colour. it has been suggested earlier that ac may be involved in cell to cell movement of the virus (rojas et al. ) . it is possible that by its ability to hydrolyze atp, ac serves to power viral movement in the plant. thirteen sugarcane yellow leaf virus isolates causing yellow midrib and irregular yellow spot pattern from six states of india were characterized by rt-pcr assays. scylv- f and scylv- r primers were used as forward and reverse primer pairs and the amplified products were cloned and sequenced. comparative coat protein sequence analysis confirmed that all the scylv-indian isolates were clustered into two major groups confirming the existence of two strains of scylv affecting sugarcane crops of india. in a separate experiment, the member of both of the phylogenetic groups were found to be transmitted by the sugarcane aphid, melanaphis sacchari from infected to healthy sugarcane suggesting its secondary spread in nature. the symptoms produced by the virus causing cotton mosaic disease were little bit different in both sap inoculation and under natural field condition. in natural field condition it has shown clear chlorosis type of symptoms on major leaves of plants but in sap inoculated plants veinal chlorosis and mosaic type of symptoms are found to be common. in field conditions infected plants grows erect and have less boll formation. there is no effect found on seed shape or seed size. the initial symptoms produced on cotton leaves after inoculation were wonderful. local lesions observed in second week from inoculation and then they changes to chlorotic type of symptoms and some are necrotic symptoms also. the plants at early stage are found to be affected, has less lateral branch development and hence reduction in yield production. the naturally field infected plants showing good symptoms are also difficult to identify in lateral stage of plant. because they disappear with time. the virus is very easily sap transmissible. the virus is found to be transmitted by thrips palmi and thrips tobacci in persistent manner. no seed transmission is observed. virus showed same physical properties as it shows in stem necrosis of peanut or sunflower necrosis disease. the physical properties are found to be thermal inactivation point (tip) - °c, dilution end point (dep) - to - and longevity in vitro (liv) h, virus infecting nineteen different host plants are identified belonging to five different types of families viz. malvaceae, chenopodiaceae, compositae, leguminaceae and solanaceae. however they found to produce same types of symptoms as in most of the host that have been tested before. in elisa test report it is found that the virus showing positive test only with anti serum of tsv of a cowpea and cotton but negative reaction with pbnv of cowpea and cotton which clearly denied possibility of presence of pbnv in cotton producing these kinds of symptoms elisa report clearly shows that tsv antiserum of cowpea is showing positive results with clear chlorotic types of symptoms. a powerful approach to functional genomics, and an alternative to the massive generation of transgenic plants, is the use of the recently described virus induced gene silencing (vigs) process, which allows viral vectors to knock out the function of a gene-of-interest. vigs is based on a silencing mechanism that regulates gene expression by the specific degradation of rna. as a tool for reverse genetics, vigs has many advantages over other common ways to study gene function because of the ability of viruses to replicate and move systemically within a plant. vigs can generate a phenocopy of a mutant without all the troubles of traditional methods of mutagenesis. geminiviruses with their small dna genomes and ease of inoculation through agrobacterium, are excellent candidates for vigs vector development. as a first step, the geminivirus bhendi yellow vein mosaic virus, characterized in our lab (jose and usha, virology : [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) has been chosen. the satellite b dna associated with this virus has a single open reading frame (bc ). bc is essential for symptom development but not for replication. therefore, bc has been replaced by a multiple cloning site harbouring sali, xbai, bamhi, bsrgi and xhoi, initially in a cloning vector and then in the binary vector containing the partial tandem repeat of the b dna. in the place of the bc orf, the plant phytoene desaturase gene has been cloned and the resulting construct was used for agroinfiltration along with the partial tandem repeat clone of the begomovirus (dna a component chilli (capsicum annuum l.) plants exhibiting prominent symptoms of begomovirus like: leaf curl, vein swelling, shortening of petioles, crowding of leaves and stunting of plants were collected from rorkee, uttarakhand and dhaulpur, rajasthan, india. total genomic dna was isolated from naturally infected chilli samples and pcr was carried out with coat protein (located in dna-a) gene specific primers. as expected to the primers, * bp dna fragments were amplified from the infected chilli samples. to know the bipartite nature of the virus isolates, nuclear shuttle protein (located in dna-b) gene specific primers were employed which also resulted in positive amplification of * bp dna bands with all the coat protein tested positive samples. to ascertain the association of dnab component with the virus isolates, a set of dna-b specific primers were used which resulted in positive amplification of full length (* . kb) dna bands in the chilli samples collected from rorkee, uttarakhand, however, multiple sizes bands were resulted with the samples collected from dhaulpur, rajasthan. these findings confirmed that both the virus isolates under study are bipartite begomovirus associated with dna-b satellite. the sequencing of the pcr products is under progress which analysis will be discussed. groundnut bud necrosis virus (gbnv) belonging to the genus tospovirus, which is a unique member of the family bunyaviridae, infects several economically important crops. the virus has three genomic ssrna segments namely s (ambisense), m (ambisense) and l (negative sense). the s rna codes for nucleoprotein (np) and non-structural protein (nss) from viral complimentary and viral strands respectively. many viral nonstructural proteins such as ns of hepatitis c virus, yellow fever virus, dengue virus, sv large t antigen and cytoplasmic inclusion protein of tamarillo mosaic potyvirus are known to exhibit rna/dna stimulated ntpase, dntpase and helicase activity. nss of gbnv does not have any sequence similarity with any of the above mentioned viral rna/dna helicases but has a ntp binding domain. however, it has been implicated as suppressor of gene silencing in vivo. with a view to elucidate the mechanism by which nss could act as a suppressor of gene silencing and examine the other potential roles of nss in the life cycle of the virus, the gbnv (to) nss was over-expressed in e. coli and purified by ni-nta chromatography. in vitro studies with the purified rnss suggest that it exhibits an rna stimulated ntpase activity. many of the proteins that possess the rna/ dna stimulated ntpase and datpase activity, are also shown to have atp dependent nucleic acid unwinding activity. it was therefore of interest to examine whether nss has the nucleic acid unwinding activity. the helicase assays revealed that nss has dna/rna helicase activity. helicase activity of nss was absolutely dependent on atp and mg ? ion. nss could unwind dsdna substrate with overhang, or overhang. mutation of the crucial lysine in walker motif a (k ) severely affected the unwinding activity where as mutation of aspartate residue in walker motif b (d ) resulted in only % loss of activity. in this regard, rnss is a unique enzyme which does not have the canonical helicase motifs but can catalyze dsdna/dsrna unwinding in an atp and mg ? dependent manner. the rnss might act as a suppressor of by unwinding the dsrna, the substrate for dicer. in addition to being a suppressor of ptgs, nss may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. this new research finding on nss might pave way for further studies on its role in viral replication and transcription. yellow vein mosaic disease of pumpkin (cucurbita moschata) poses a serious threat to the cultivation of this crop in india. the disease was found to be associated with whitefly-transmitted bipartite begomoviruses were detected in varanasi field using polymerase chain reaction (pcr) with primer design through coat protein conserved region of begomoviruses from ncbi database. all plant samples showing symptoms were infected with begomovirus. the virus species were provisionally identified by sequencing * bp of the viral coat protein gene (av ageratum conyzoides is commonly known as billygoat-weed, chick weed, goatweed and whiteweed. in india it is popularly known as bill goat weed. it is an annual herbaceous plant with a long history of traditional medicinal uses in several countries of the world and also reputed to possess varied medicinal properties including the treatment of wounds and burns. in cameroon and congo, it is used traditionally to treat fever, rheumatism, headache, and colic. during survey in and around gorakhpur in , ageratum plants were found affected with the symptoms of leaf curling, mosaic mottling and leaf yellows. the infected leaf samples were processed for virus identification and association with pcr assays. total dna was extracted and pcr were performed with begomovirus specific primers (tlcv-cp). a * bp band was consistently amplified on % agarose. the pcr products were directly sequenced and sequence was submitted in genbank with the accession no. gq . the blast search analysis showed highest similarity of % with the ageratum enation virus. vernonia cinerea leaves with yellow vein symptoms were collected around crop fields in madurai. a bp product amplified from total dna extracted from symptomatic leaves with degenerate primers designed to amplify a part of the av gene from begomoviral dna a component was cloned and sequenced. based on the above sequences, specific primers were designed and the full length dna a of nucleotides with typical genome organization of begomoviral dna a was obtained and was submitted to embl data base (acc no: am ). the sequence comparison with other begomoviruses revealed the closest identity ( %) with emilia yellow vein virus from china and less than % with all known begomoviruses. the international committee on taxonomy of viruses (ictv) has therefore recognized vernonia yellow vein virus (vyvv) as a distinct begomovirus species. conventional pcr could not amplify the dna b or dna b from the infected tissue. however, the b dna ( bp) associated with the disease was obtained (acc no: fn ) by the rolling circle amplification-restriction fragment length polymorphism method (rca-rflp) using phi dna polymerase. sequence analysis shows that dna b of vyvv has the highest identity ( %) with dna b of ageratum leaf curl disease and - % with the b dna associated with other begomoviruses. infectious clones of vyvv dna a and dna b as dimers were made using the products of rca-rflp. these infectious clones will be used for agroinfection of vernonia and the results will be discussed. this is the first report of the molecular characterization of vernonia yellow vein virus (vyvv) from vernonia cinerea in india. production of bulb and seed crop of onion (allium cepa l.) is hampered by onion yellow dwarf virus (oydv) and iris yellow spot virus (iysv) with an incidence of . % and . % in bulb crop and . % and . % in seed crop, respectively in the popularly grown cv. hisar- . four symptom-based variants of oydv designated as grade a, b, c and d produced varied types of symptoms in onion crop incurring heavy losses in bulb and seed production. iysv caused tiny hay coloured spots of different shapes and sizes on leaves and scapes which later coalesced and led to drying and lodging of scapes. the plant height, bulb weight and bulb size were . cm, . g and . cm in plants infected with oydv, . cm, . g and . cm in iysv infection, . cm, . g and . cm due to their combined infection, as compared to . cm, . g and . cm respectively, in healthy plants of bulb crop. in plants infected with oydv grade a the plant height was minimum ( . cm) whereas the number of umbels was maximum ( . umbels/pl.) but other yield parameters viz., weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were recorded to be the lowest. the minimum reduction in plant height ( . cm), weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were recorded in oydv grade d. the plant height was . cm with . umbels per plant, . g weight/umbel, seeds/umbel, . g seed weight/umbel and . g seed yield/plant in iysv infected plants. the plant height ( . cm), umbels/plant ( . ), weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were found to be the lowest in combined infection of oydv and iysv diseases in comparison to higher values in healthy controls ( . cm, . , . g, , . g, . g, respectively). a minimum reduction in the test weight, germination and seed vigour index were found ( . g, . % and ) due to oydv grade a infection, whereas these were . g, . % and in iysv disease infected plants and . g, . % and in combined infection of oydv and iysv diseases in comparison to . g, . % and in healthy plants. the maximum hampering of seed vigour parameters was recorded due to iysv infection. lodging of scapes caused by this disease was responsible for heavy losses in seed production and seed quality. cotton leaf curl disease is one of the major threats to cotton cultivation from northern india. survey conducted during , observed the disease incidence ranged from to % from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar. in order to study genetic variability in the virus, twelve clcuv isolates were partially characterized ( bp common region, full length av gene and partial sequences of ac and av gene). full length characterization of representative isolates from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar is under progress. partial sequence analysis of clcuv isolates revealed that, the virus isolates collected during cropping season are closely related to cotton leaf curl burewala virus from pakistan and results were discussed. pratibha singh, h. s. savithri department of biochemistry, indian institute of science, bangalore tospoviruses, belonging to the family bunyavirideae, infect economically important plants such as groundnut, tomato, watermelon etc. they have a tripartite genome, with l, m and s segments of rna, in pseudo circular (panhandle) form. the viral genomes encode four structural proteins (l, n, g and g ) in the antisense orientation, and two non structural proteins nss and nsm in the sense orientation. the nsm is the only protein unique to tospoviruseses that infect plants in the bunyaviridae family and hence is proposed to be important for cell to cell movement. ground nut bud necrosis virus (gbnv), a member of the tospovirus genus, is the most prevalent virus infecting several species of leguminosae and solanaceae plants in india. total rna was isolated from gbnv infected tomato leaves and rt-pcr was performed using appropriate primers to amplify the nsm gene. the pcr product was cloned in pgex x vector. the recombinant nsm clone was transformed into bl (de ) e. coli cells and over-expressed by induction with . mm iptg. sds-page analysis of induced and uninduced fraction revealed the presence of overexpressed protein of expected size. the soluble gst-nsm was purified by gsh sepharose affinity chromatography. purified gst-nsm was shown to interact with in vitro transcribed rna transcript by electrophoretic mobility shift assay. further nsm was shown to interact with viral encoded proteins np and nss using elisa and yeast two hybrid system. nsm was also shown to be phosphorylated in vitro by pellet fraction of plant sap. thus the recombinant gbnv nsm possesses the characteristic features of a movement protein such as nucleic acid binding, interaction with nucleocapsid protein, and ability to undergo posttranslational modification. solanum melongena, commonly called as egg plant is one of the most important vegetable crop in the world. it is cultivated widely in the tropical and sub tropical regions. several viruses such as cucumber mosaic cucumo virus (cmv), potato virus-y (pvy), potato virus-x (pvx) and tobacco ring spot virus (trsv) infect egg plant under natural conditions. in india major crop losses due to cmv infection in brinjal is % (fao stat- ) . in the present study the infected leaf samples were collected from local fields of ramapuram, chandamama palli, chandragiri, madanapalli, yadhamari, durgasamudram villages in and around tirupati, were tested for cmv infection by dac-elisa with cmv antisera. the resulting positive samples were further inoculated to the raised brinjal seedlings of selected varieties through mechanical sap inoculation. different varieties of brinjal like mullabadhine, ankhur, ravya, mattigulla, casper and easter egg were used for monitoring the susceptibility to cmv infection. the mosaic symptoms were observed after weeks of inoculation in all varities of brinjal except mullabadhina. among all these susceptible varities ankhur variety is selected to study induced biochemical changes such as chlorophylls, carbohydrates, proteins, nucleic acids and polyphenol oxidases in cmv infected brinjal leaves. in the infected leaves considerable reduction in chlorophyll and starch and increase in total proteins, sugars, rna and polyphenol oxidases was observed when compared to healthy leaves. the amount of total starch, protein and dna decreased to about , and lg/g respectively in infected leaves, where as sugars ( lg/g), rna content ( lg/g) and polyphenol oxidase activity was increased as compared to healthy leaves. the above results suggests that there is an altered concentrations of chlorophyll, proteins, nucleic acids, carbohydrates and polyphenol oxidase activity in the brinjal leaves due to the effect of cucumber mosaic cucumo virus infection. leaf analysis was found to be used as widely accepted diagnostic tool to assess the nutritional status of the vegetables. the present study deals with these aspects in detail. the total rna and dna was isolated from infected leaf samples. rt-pcr assays were performed using sugarcane yellow leaf virus (scylv) specific primers (scylv- f and scylv- r). the infection of scylv was detected in all the collected samples, which showed the expected size (* bp) amplicon during rt-pcr. in another experiment with nested pcr analysis, a phytoplasma characteristic . kb rdna pcr product were amplified from dnas of all infected samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs p /p and fu /ru . dna extracts from plants with yellow mid rib and leaf yellows produced products of bp, which gave typical phytoplasma profiles when digested with hae iii and hha i. no pcr amplifications were produced using dna from symptomless plants. our results suggest that the yellow mid rib and leaf yellows symptoms on sugarcane varieties in uttar pradesh and uttarakhand states of india exhibiting midrib yellowing and leaf yellows symptoms is mainly caused by mixed infection of scylv and scylp. the affected clumps showed reduction in stalk height as compared to healthy fields. thirty-one sugarcane mosaic isolates belonged to sugarcane mosaic virus (scmv) and sugarcane streak mosaic virus (scsmv were collected from china and india), confirmed in indirect elisa and rt-pcr amplification with scmv and scsmv-specific primers. the amplicons ( . kb) from the coding region of coat protein (cp) were cloned, sequenced and compared to each other as well as to the sequences of scmv isolates from sugarcane (australia, usa, china, brazil, mexico and south africa), maize (australia, china, iranian) and one scsmv isolate from sugarcane (india) in genbank. maximum likelihood and maximum parsimony analyses robustly supported two major monophyletic groups that were correlated with the host of origin: the scmv subgroup that included isolates from china and only isolates from india, and the scsmv subgroup that contained all isolates from india. maize dwarf mosaic virus (mdmv) and johnsongrass mosaic virus (jgmv) were not detected in any of the samples tested. a strong correlation was observed between the sugarcane groups and the geographical origin of the scmv isolates. the millable sugarcane samples from china contained a virus tentatively described as sorghum mosaic virus (srmv). three isolates from nine chewing canes in fujian, yunnan and guizhou provinces of china also contained srmv, and the other samples including five isolates from india was found infected with scmv. no srmv infection has been detected in sugarcane mosaic samples from india. sequence comparisons and phylogenetic analysis indicated that srmv can be considered as the most common and prevalent potyvirus infecting sugarcane in china, however in india sugarcane streak mosaic virus is dominant in causing mosaic symptoms on sugarcane. dig-labeled dna probe complementary to coat protein (cp) region of tobacco streak virus (tsv) sunflower isolate was designed for the sensitive and broad-spectrum detection of tsv isolates, the most devastating virus in india. dot-blot and tissue print hybridizations with the digoxigenin labeled probe were performed for the tsv detection at field levels. here, dot-blot hybridization was used to check a wide number of tsv isolates with a single probe and sensitivity with different sample extraction methods. the probe with cp conserved region prepared from sunflower pcr amplicon was hybridized with the tsv field isolates of gherkin, pumpkin, sunflower, marigold and globe amaranth samples because of highly conserved with little variability in cp region. the sensitivity limits were decreased from total nucleic acid to partially purified and crude extract preparations. in particular, tissue blot hybridization offers a simple, reliable procedure as dot-blot, but requires no sample processing. because there is minimal sample preparation, tissue-print hybridization could be an important component of tsv management programs. thus, the above non-radioactive labeled probe techniques can facilitate in screening the samples during tsv outbreaks and in quarantine services. savita patil, rupali sawant*, k. banerjee virology group, agharkar research institute, macs, g.g. agarkar road, pune two mycobacterium smegmatis strains (ari lab nos. v and v ) were employed for the isolation of mycobacteriophages from soil and sewage samples. mycobacteriophages were isolated from soil samples collected from an area surrounding the tuberculosis (tb) ward, naidu hospital, pune, against m. smegmatis strain v . these were numbered as v , v and v and were isolated by using washed-cell preparation method. the bacteriophages against the other m. smegmatis strain, i.e. v , were isolated from soil samples (collected from around tb ward, sassoon hospital, pune). some of these phages (viz.v , v ) showed plaques at °c but not at °c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v , v , v , v and v . also, phage dilution medium (pdm) as described by chaterjee et al. ( ) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p . the various passages of the phage isolates v , v , v , v and v (i.e. original, p , p and p ) were stored at - °c. pvp- effect on pigments due to geminivirus infection on cowpea (vigna unguiculata) shail pande*, naveen pandey, k. shukla mahatma gandhi p. g. college gorakhpur, d.d.u. gorakhpur university, gorakhpur geminiviruses are one of the most important group of viruses causing economic losses in tropics. the symptom produced are yellowing of leaves which directly affect the pigments of diseased plants it in turn affects productivity and yield of diseased plant. cowpea vigna unguiculata is one of the important crop cultivated throughout india for its green pods which are used as vegetables and seeds are used as pulse. cowpea is affected by many viruses amongst them geminiviruses are one of the important virus on the cowpea plant. in the present study total chlorophyll content was studied in leaf of cowpea of diseased and healthy plants using arnon's method. carotenoids were also studied using ikan's method. it was found that chlorophyll content in diseased plants were lower compared to healthy plant similar results were found with carotenoids so the geminivirruses infection lowers the chlorophyll and carotenoid content in diseased plants which reduces yield of diseased cowpea plant. shweta sharma , amrita banerjee , j. tarafdar , r. rabindran , indranil dasgupta * department of plant molecular biology, university of delhi, south campus, new delhi; bidhan chandra krishi vishwavidayalaya, kalyani, nadia, west bengal ; tamil nadu agricultural university, coimbatore, tamil nadu rice tungro disease is an important disease of rice, caused by a joint infection by two viruses: rice tungro spherical virus (rtsv) and rice tungro bacilliform virus (rtbv) in south and southeast asia. the complex of rtbv and rtsv is transmitted by an insect vector green leaf hopper (glh). previously we reported complete genomic sequences of two geographically distinct isolates of rtbv; rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) collected from the field in mid- s. both the sequences showed high homology all along the genome but showed divergence from previously reported southeast asian isolate i.e. rtbv-phil (philippines). to check whether a time period of a decade has resulted into variability in the genomic sequence of different isolates of rtbv in india, we cloned and sequenced the complete genome of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari collected from the field in . the complete nucleotide sequence of the dna fragments covering the whole genome of rtbv was determined using universal primers m f and m r and by primer walking, without any ambiguities remaining. the nucleotide sequences of overlapping clones were assembled and analyzed using the dna analysis software generunner and blastn program of ncbi. homology search at the nucleotide and amino acid level were performed using the blastn and blastp (respectively) programs of ncbi. multiple sequence alignments were performed using clus-tal-w software. sequence analysis results thus obtained showed that both the recently obtained complete genomic sequences of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari showed very high homology (both at the nucleotide and amino acid levels) with the two previously reported rtbv isolates from india i.e. rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) all along the genome. as observed earlier both the sequences diverged significantly from the southeast asian isolates. this suggests that even after the spatial and temporal difference (a time gap of approx years) between the two previously reported rtbv isolates and the recently reported one, there is very little sequence variability between them. this further strengthens the earlier reports that the rtbv genomes in india are highly conserved. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of % with the rtbv west bengal isolate and % with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. complete genomic rna sequences of two geographically distinct isolates of rice tungro spherical virus (rtsv), a member of the genus waikavirus, family sequiviridae, were determined from india. out of the two previously reported sequences, the indian isolates were closer to the resistance breaking strain rtsv-[vt ] than rtsv- [phila] . between them, the indian sequences showed nucleotide as well as amino acid identities of %. a moderate homology was observed between the leader peptide and a putative helper component protein involved in insect transmission of the maize chlorotic dwarf virus, a closely related waikavirus, indicating its possible transmission-related function. unlike rice tungro bacilliform virus, which causes rice tungro disease jointly with rtsv, and is significantly different between isolates from india and philippines, rtsv genomes were observed to be much more conserved between isolates from the two countries. rice tungro bacilliform virus (rtbv) are believed to be the joint causative agents for the devastating tungro disease of rice prevalent in south and southeast asia [ ] . rice tungro disease has become the major cause of production losses in rice during last three decades in several rice growing states of india. here, we report, for the first time the complete sequence analysis of two geographically distinct indian isolates of rtsv. we analyze the deduced protein sequences and their phylogenetic relationship with the two complete rtsv sequences from philippines as well as with other members of sequiviridae family. we provide molecular evidence that the indian isolates of rtsv are closely related to those from the philippines. we had earlier reported that rtbv isolates between india and philippines differ significantly from each other [ ] . this study was undertaken in order to see whether rtsv isolates from india also show similar difference from those reported from the philippines. frequent outbreaks of tungro were reported near kanyakumari in the last - years. the present work was undertaken to clone and sequence the full-length rtbv and rtsv genomes from the infected rice plants collected from above region and to analyze the similarity of its genetic material with the existing indian isolates of rtbv and rtsv. a . kb dna fragment encoding the reverse transcriptase gene of rtbv genome was amplified and cloned in t/a vector and was sequenced commercially. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of % with the rtbv west bengal isolate and % with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. similarly, the cp region of rtsv was amplified by rt-pcr and was cloned in t/a vector. recently, rice tungro disease has been reported from kanyakumari district of tamil nadu. it is important to determine the genetic nature of this isolate in order to develop resistance strategies. it is thus necessary to clone and characterize the viruses from kanyakumari and to determine the mechanism of virus resistance in transgenic lines. rice tungro disease is an important viral disease of rice. rice tungro is caused by infection by two viruses: rice tungro bacilliform virus (rtbv) and rice tungro spherical virus (rtsv). rtsv is a plant picornavirus with a kb single stranded rna genome. it belongs to genus waikavirus in the family sequiviridae and is necessary for transmission of the two viruses by the leafhopper vector nephotellix virescens. rtsv rna is translated to form a large polyprotein, which is then self cleaved to form the viral proteins, including the three coat proteins, replicase, protease. studies have been conducted on rtsv from philippines. correct information of sequence variability of viral isolates to check whether different geographical conditions like those present in india select for genotypically variable strain and to design for transgenic resistance strategy, information on rtsv from india is absolutely essential. the objective of this study was to clone rtsv isolates from india and compare the genetic diversity of indian isolates from other southeast asian isolates and amongst each other. also develop strategy to impair the attack of virus-complex on rice. the achieve this, complete genomes of two isolates from india were cloned by amplifying different genes by rt-pcr and subsequently cloned in ta vectors, followed by sequencing. subsequently constructs containing cp - , antisense replicase, sense replicase and double stranded replicase were cloned in plant transformation vector. these constructs were used to transform aromatic rice variety from indian-pusa basmati (pb ). pcr analysis of the above plants was done to check the stable insertion of insert in the transgenics. jatropha (jatropha curcas) of the family euphorbiaceae is being grown in india as a major commercial fuel (bio-diesel) crop. jatropha is cultivated in districts of potential states of india. unfortunately, the cultivation of jatropha is limited by the severe mosaic disease. recently, a severe mosaic disease with significant disease incidence was observed in - on j. curcas grown in experimental plots of nbri and j. gossypifolia, a weed growing road side around lucknow and kathaupahadi, madhya pradesh. the disease consisted of the symptoms of severe mosaic, blistering, leaf distortion and stunting of whole plant and no fruit/seed production in severely affected plants. symptomatology and whitefly population observed on them suggested the occurrence of begomovirus infection. to detect the begomovirus infection, the total dna from leaf samples of infected jatropha plants was extracted and polymerase chain reaction (pcr) were performed using three sets of begomovirus genus specific (cpit-i/cpit-t, paliv /paric and paliv /palic ) primers and the expected size * bp, . kb and . kb amplicons were obtained which confirmed the begomovirus infection. further to identify the begomovirus/es and investigate the genetic diversity among them exists if any, the * . kb amplicons were cloned and sequenced. the sequence data were deposited in the genbank database under accession nos.: gq and fj (from j. curcas) and eu and fj (from j. gossypifolia). during blast analysis gq and fj shared highest % sequence identity with each other and - %% with sri lankan cassava mosaic virus (aj , aj , aj , aj and aj ) and indian cassava mosaic virus from india (ay ) therefore, designated as two strains of jatropha mosaic india virus-lucknow. blast analysis of eu showed maximum % similarities with croton yellow vein mosaic virus (aj ), % with tomato leaf curl new delhi virus (dq ) and - % with papaya leaf curl virus (aj and y ), therefore, identified as strain of croton yellow vein mosaic virus. blast analysis of the virus isolate (fj ) showed highest % identities with tomato leaf curl virus-bangalore ii (tolcv-b ii-u ) and - % with tomato leaf curl karnataka virus (tol-ckv, ay , fj ), therefore, considered as new begomovirus species ''jatropha yellow mosaic india virus''. the phylogenetic analysis of gq and fj (from j. curcas) and eu and fj (from j. gossypifolia) was performed along with some selected isolates of begomovirus which showed [ % sequence identities during blast analysis. the isolate eu showed closest relationship with croton yellow vein mosaic virus while fj showed separate clustering of all the four begomovirus from jatropha species. during phylogenetic analysis these isolates formed three separate clusters, therefore, they were considered as three distinct begomoviruses. the above data clearly show that some genetic diversity exists among the begomoviruses infecting jatropha species in india. bitter gourd (momordica charantia l.) of the family cucurbitaceae, also known as bitter melon is extensively cultivated in north eastern region of uttar pradesh, india. it is regarded as one of the world's major vegetable crops and has great economic importance. a severe yellow mosaic disease on bitter gourd (momordica charantia) with a significant disease incidence was observed during the survey of different locations of eastern up, india in the year . the whitefly (bemisia tabaci) population was also observed in the vicinity. the characteristic disease symptoms and whitefly population indicated the possibility of begomovirus infection. total dna were isolated from infected as well as healthy leaf samples. two primer pair (tlcv-cp and roja's primer) were used to study, which resulted * bp with tlcv-cp in / samples and * . kb amplicons with roja's primer in / samples. for further identification of the begomovirus, the pcr amplicons were cloned and sequenced (genbank accession no. eu and eu , respectively). the blastn search analysis of eu indicated - % identity with several isolates of tomato leaf curl new delhi virus (tolcndv). the phylogenetic analysis also showed closest relationships of the isolate (eu ) with tolcndv isolates. based on highest sequence identity and closed relationships with tolcndv the virus isolated from bitter gourd was considered as an isolate of tomato leaf curl new delhi virus. while, blastn search analysis of eu isolate, shared highest - % identites with pepper leaf curl bangladesh virus (peplcbv) isolates. the phylogenetic analysis of the virus isolate with selected begomovirus isolates revealed a closest relationship with peplcbv. these results confirmed the association of peplcbv on bitter gourd. study revealed the variability of viruses on bitter gourd in eastern up, india. tobacco streak virus groundnut isolate was characterized biologically by taking six cultivars (jl , tmv , k , k , k ) and one pre-release culture (k ) using seedlings of - days old under glasshouse conditions. there were clear differences were observed among cultivars tested regarding incubation period, percent seedling wilt and time taken to death of seedlings. k- was least susceptible among all the cultivars tested and it supported least virus titer (a nm: . - . ). both localized (necrotic lesions on leaf, veinal necrosis, leaf yellowing, wilting) and systemic (petiole necrosis, necrotic lesions on young leaves, death of top growing buds not only on main stem but also on all primaries (side shoots), followed by stem necrosis, stunted growth, axillary shoot proliferation with small leaves having general chlorosis, peg necrosis, pod necrosis, pod size reduction, wilt of plants) symptom were observed in all cultivars tested. biological differentiation of tsv and gbnv was made by sap inoculation of both viruses separately using susceptible groundnut cultivar jl under glasshouse conditions. there were certain similarities and differences were observed between these viruses infecting groundnut. seed infection of tsv ranged from . to . % in seeds collected from naturally infected and sap inoculated groundnut cultivars/pre-releases (jl , tmv , k- , k- , k- and k- ) belonging to spanish and virginia types. tsv was detected both in pod shell and seed testa from pod samples produced by sap inoculation under glasshouse conditions. however, seed transmission of tsv was not observed in groundnut. coat protein (cp) gene of three groundnut tsv isolates (gn-ap- - ; gn-ap - ; gn-ap - ) were sequenced and all the three isolates contained a single open reading frame (orf) of bp nucleotide and could potentially code for amino acids (aa). cp gene of tsv isolates originating from different hosts shared high degree of sequence identity both at nucleotide ( . - %) and amino acid ( . - %) levels respectively. tones grown in an area of . . ha (fao stat ). in india papaya is grown in nearly , ha with an annual production of , , tones (fao stat ) and occupies fourth place in the world. the crop is severely affected by a number of viruses. papaya ring spot virus (prsv-p) is the most important virus. the detection of virus infection in plants has traditionally involved either bioassay on indexing plants and or immunological methods (hill , torrence and jones ) . use of nucleic acid probes has improved the detection and sensitivity of viruses. the most common non-radioactive probes are biotynilated probes, which are very specific and sensitive. papaya ring spot virus (prsv-p) is a positive sense ssrna virus belonging to the genus potyvirus family potyviridae and transmitted by aphids. prsv-p coat protein gene region was used as template cdna for probe preparation. dot-blot hybridization with the biotin labeled probe were performed for prsv-p detection. the clarified sap of healthy and infected plants were serially diluted and spotted onto the nitrocellulose membrane, hybridized to biotin labeled probe. biotin labeled rna's are employed as probes, with a subsequent detection based on streptavidin-alkaline phosphatase conjugates. the sensitivity for viral detection of the biotin labeled probe was found to be sensitive than enzyme linked immunosorbent assay (elisa). in recent years tospovirus is causing devastating damage to the yield of vegetables in india. it infects economically important crops viz., tomato, chilli, peppers, groundnut, watermelon and various legumes. now it is emerging as severe disease in brinjal also. in order to monitor the natural occurrence and distribution of tospovirus in vegetable, surveys were conducted in the predominant brinjal growing areas of gujarat, karnataka, maharashtra and andhra pradesh during - incidence ranging from to %, to %, to %, and to . % respectively. samples collected from different places of india were found positive to pbnv in direct antigen coating-enzyme linked immunosorbent assay (dac-elisa). pbnv infected brinjal plants showed mosaic mottling of leaves with leaf distortion, longitudinal streaks on the stem and necrotic rings on leaves and fruits. early infection led to severe stunting and abnormal fruiting. biological and molecular characterization of pbnv-brinjal isolates were compared with other isolates and results are discussed. for identification of virus causing mosaic symptoms on soybean various host plants were tested. plants species belonging to the different families viz. caricaceae, graminae, leguminosae, malvaceae and solanaceae were tested. the virus produced symptoms on diagnostic plant species like chenopodium album, c. quinoa, helianthus anus, phaseolus vulgaris and vigna ungiculata. among tested families the leguminosae that were the host of virus included arachis hypogea, the virus causing mosaic symptoms in soybean is inactivated between and °c and between dilution of - to - . all the inoculated plants of assay host showed the symptoms at °c but not at °c. similarly local lesions produced at - but not at - . the virus in crude sap was infectious up to h but not at h at room temperature. however, the percentage infectivity decreased progressively as the aging of the sap was increased at room temperature. on the basis of reactions on diagnostic hosts pvp- identification and characterization of potyvirus infected chilli (capsicul annum l the virus under study caused mild mosaic and severe mottling symptom in leaves of infected plants. the dilution end point (dep) of the virus was found to be - to - , longevity in vitro (liv) - days at room temperature ( °c), thermal inactivation point (tip) - °c. electron microscopy of purified virus preparation revealed the presence of flexuous particle of size nm long and nm in width with characteristic cytoplasmic inclusions: pinwheels and scrolls. the virus was transmitted by sap and by aphid myzus persicae. the host range study revealed that the host species were restricted to family chenopodiaceae and solanaceae. on the basis of above characteristic, the virus under study was identified as potyvirus associated with mild mosaic and severe mottling symptom in capsicum. phytoplasma causing grassy shoot disease and sugarcane yellow leaf viruses are important pathogens of sugarcane. these pathogens are causing severe losses in sugarcane productivity. with a view to producing virus and phytoplasma free planting material of sugarcane, experiments were undertaken using infected varieties of sugarcane growing at the farms of sugarcane research institute. apical meristems measuring about mm in length, were dissected out, surface sterilized and cultured on agar gelled murashige and skoog's (ms) medium containing growth regulators for shoot induction. the established shoot cultures were multiplied through repeated subcultures on fresh media at - days interval. elimination of gsd and scylv was confirmed through molecular analysis of regenerated plants using specific primers of scylv and gsd. results revealed that apical meristem culture technique is effective in eliminating the pathogens like scylv and phytoplasma (gsd) from the infected clones. this is probably the first report on elimination of grassy shoot disease in sugarcane through meristem culture. papaya ringspot virus (prsv), which causes the most widespread and devastating disease in papaya, isolates originating from different geographical regions in south india were collected and maintained on natural host papaya. the entire coat protein (cp) gene of papaya ringspot virus-p biotype (prsv-p) was amplified by reverse transcription-polymerase chain reaction (rt-pcr). the amplicon was inserted into pgem-t vector by t-a cloning method, sequenced and sub cloned into a bacterial expression vector prset-a using directional cloning strategy. the prsv coat protein was over expressed as fusion protein in e. coli. sds-page gel revealed that cp expressed as a * kda protein. the recombinant coat protein (rcp) fused with his-tag was purified from e. coli using ni-nta resin. the antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified prsv. the purified rcp was used as an antigen to produce high titer prsv specific polyclonal antiserum. the resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (ic-rt-pcr) assay and compared its sensitivity levels with elisa based assays for detection of prsv isolates. ic-rt-pcr was shown to be the most sensitive test followed by dot-blot immunobinding assay (dbia) and plate trapped elisa. key: cord- -xcx c da authors: sahai, aastha; shahzad, anwar; shahid, mohd. title: plant edible vaccines: a revolution in vaccination date: - - journal: recent trends in biotechnology and therapeutic applications of medicinal plants doi: . / - - - - _ sha: doc_id: cord_uid: xcx c da plants have been used as a source for many pharmaceutical since long. however, utilization of plant systems for production of edible vaccines has been a comparatively recent phenomenon. there are several potential advantages of plant derived vaccines over other conventional systems of vaccine production such as mammalian or avian cell culture. the cost of vaccines is one factor preventing further use of vaccination, leaving hundreds of thousands of children susceptible to preventable diseases. especially for developing world this novel technique proved to be a boon for its low cost of production, convenient administration, easy storage and negligible chances of infection whereas the conventional system of vaccine production limits the applicability of vaccines in many parts of the world. these vaccines are prepared by introducing selected desired genes into plants and inducing these genetically modified plants to manufacture the encoded proteins. transgenic plants may provide an ideal expression system, in which transgenic plant material can be fed directly as oral dose of recombinant vaccines. expression of vaccines in plant tissue eliminates the risk of contamination with animal pathogen, provides a heat stable environment and enables oral delivery thus eliminating infection related hazards. identification of transgenic material, containment of the transgenes and control of recombinant protein may be potential problems for large scale production of vaccines in plants. factors like scaling up production as well as distribution and handling of transgenic plant material must comprise the future consideration in this field. since time immemorial, plants besides providing food and shelter have also been used for basic preventive and curative health care by human beings. plants were the fi rst and only source for every kind of medication in primitive times. gradually, animal derived products were also started being used as medicines for some ailments like snake venom has been used for medical purposes for thousands of years. in chinese medicine snake skin was used to treat superfi cial diseases, including skin eruptions, eye infections or opacities, sore throat, and hemorrhoids. however, plants still remained to be the primary source of medication till now for their ease of availability and safe nature. with the advent of modern science and technology, pharmacology has shown remarkable advances with the artifi cial production of plant and animal based medicines in laboratories. apart from this some novel medicinal compounds were also synthesized to combat life threatening diseases. all these advancements were aimed to increase average human life span and to decrease the child mortality rate. a signifi cant step taken in this regard was the universal immunization programme (uip) launched by who in s in which children all over the world are vaccinated against six deadly diseases (bcg, opv, diphtheria, tetanus, pertussis, and measles) with the aim of immunizing % of all children by . however, even after all these efforts % of infants are still left unimmunized; responsible for approximately two million unnecessary deaths every year, especially in remote and impoverished parts of the globe ( langridge ) . moreover, immunization remains an unfi nished agenda with an estimated . million children were not reached with three doses of dtp vaccine in . those who are not immunized -about every fifth child -are mostly among the poorest and the most vulnerable. vaccination is an important tool for prevention of many diseases. it is the administration of vaccine to produce immunity to a disease. vaccination involves the stimulation of the immune system to prepare it for the event of an invasion from a particular pathogen for which the immune system has been primed (arntzen ) . on recognizing the foreign substance (live/ weakened microbes, antigen or proteins) which enters the body through vaccine, the immune system activates, producing antibodies to destroy the invader. not only this but this attack leaves behind the 'memory' t and b cells which guards body in case of any future attack by that pathogen. some vaccines provide lifelong protection; others (such as those for cholera and tetanus) must be re-administered periodically (langridge ) . classically, this has been achieved by presenting the immune system with whole viruses or bacteria that have been killed or made too weak to proliferate much. however, these conventional vaccines poses threat of actually acquiring the infection from weakened or attenuated pathogenic strains. thus newer approach uses sub unit vaccines and recombinant vaccines. these vaccines consist of specifi c macromolecules that induce a protective immune response against a pathogen rather than administering the whole pathogen in the body. a "subunit vaccine" refers to a pathogen-derived protein (or even just an immunogenic domain of a protein, i.e. "an epitope") that cannot cause disease but can elicit a protective immune response against the pathogen. very often the subunit-vaccine candidate is a recombinant protein made in transgenic production-hosts (such as cultured yeast cells), then purifi ed, and injected as vaccines to immunize against a specifi c disease (mor and arntzen ) . these sub-unit vaccines increases vaccine safety by circumventing the need to use live viruses or microbes and has thus made them the preferred approach for vaccine manufacturers (national institute of allergy and infectious diseases ). unfortunately, traditional subunit vaccines are expensive to produce and not heat stable which limits their availability and use in developing countries. although these vaccines have an undue advantage over traditional conventional vaccines but they are expensive and their storage and transportation pose a problem as many of them require refrigeration. this is a disadvantage in many of the developing countries where the vaccines were needed most. thus, to ensure a successful global immunization a whole new idea of plant edible vaccines was presented by arntzen and co-workers in , by introducing the concept of transgenic plants as a production and delivery system for subunit vaccines (mason et al. ; mason and arntzen ) . they envisaged that production would be as cheap as agriculture, that distribution would be as convenient as marketing fresh produce and that administration would be as simple and safe as eating (mor et al. ). plants have long been considered an ideal expression system for many of the animal derived proteins, antibodies and pharmacologically important compounds. factors in favor of plant systems as sources of animal derived proteins include: the potential for large-scale, low-cost biomass production using agriculture; the low risk of product contamination by mammalian viruses, blood borne pathogens, oncogenes and bacterial toxins; the capacity of plant cells to correctly fold and assemble multimeric proteins; low downstream processing requirements for proteins administered orally in plant food or feed; the ability to introduce new or multiple transgenes by sexual crossing of plants; and the avoidance of ethical problems associated with transgenic animals and the use of animal materials (doran ) . the fi rst pharmaceutically relevant protein made in plants was human growth hormone, which was expressed in transgenic tobacco in (barta et al. ). since then, many other human proteins have been produced in an increasingly diverse range of crops. in , the fi rst antibody was expressed in tobacco (hiatt et al. ) . the idea of suitability of plants as vaccine production and delivery system was presented by research group of charles arntzen in s. this idea was quite promising in terms of providing a successful tool for mass immunization in developing countries where till now vaccination is hampered by high cost of production and delivery of vaccines. basic concept of edible vaccines involves the production of plants containing antigens required to stimulate the immune response in human body. thus by simply eating the plant product people will get vaccination against diseases. however the concept sounds quite simple but its development was equally complex. to produce edible vaccine, plants are engineered to contain desired gene of interest which codes for the antigen. this process is accomplished by transformation and the altered plant containing foreign gene is called transgenic plant. thus, the process remains the same as with subunit vaccine preparation because it contains only desired antigen and not the whole pathogen. these vaccines basically work in the same way as the injected dna vaccine, since a peptide sequence similar to an infectious part of a pathogen is synthesized, by itself, and is used to prime t and b cells in the body. the big difference in this case is that the protein sequences are encoded in a plant to form the desired protein. this protein is then ingested, as the plant or its fruit is eaten. one becomes immune against the ingested protein, as t and b cells become stimulated to proliferate and differentiate (mor et al. ). expression of vaccines in plant tissues eliminates the risk of contamination with animal pathogens, provides a heat-stable environment, and enables oral delivery, thus eliminating injection-related hazards (walmsley and arntzen ) . for production of edible vaccines, it is desirable to select a plant whose products are consumed raw to avoid degradation during cooking. thus, plants like tomato, banana and cucumbers are generally the plants of choice. vaccines made from plant material have enormous potential for use, in the developing world. also it may be much easier to persuade people to eat protective vegetables than to accept injections or take pills. vaccines made from mashed potato, designed to protect against travellers' diarrhoea, have already been tested in humans. vaccines made from dried tomatoes have also been developed. both have been developed by the cambridge-uk-based biotechnology company axis genetics, who pioneered the techniques involved. edible vaccines are mucosal-targeted vaccines that stimulate both the systematic and mucosal immune network, activating the fi rst line of defense of human body through mucosa. the mucosal surfaces are found lining the digestive tract, respiratory tract and urinoreproductive tract. mucosal immune system (mis) is the fi rst line of defense and the most effective site of vaccination, nasal and oral vaccines being the most effective (mor et al. ; korban et al. ) . before understanding the mechanism of action of edible vaccines it is important to understand the functioning of mis. induction of a mucosal immune response starts with the recognition of an antigen by specialized cells called m-cells. these cells are localized in the mucosal membranes of lymphoid tissues such as peyer's patches within the small intestines. the m-cells channel the antigen to underlying tissues where antigenpresenting cells internalize and process the antigen. the resulting antigenic epitopes are presented on the apc surface, and with the assistance of helper t cells activate b cells. the activated b cells migrate to the mesenteric lymph nodes where they mature into plasma cells and migrate to mucosal membranes to secrete immunoglobulin (ig) a. on passing through the mucosal epithelial layer towards the lumen, the iga molecules complex with membrane-bound secretary components to form secretary iga (siga). transported into the lumen, the siga interacts with specifi c antigenic epitopes and neutralize the invading pathogen (walmsley and arntzen ) . when edible vaccines are eaten they degrade majority of the plant cells in the intestine as a result of the action of digestive and bacterial enzymes. this degradation results in the release of antigens present in the plant product. the whole process occurs near the peyer's patches (rudzik et al. ) where m cells recognize the released antigen and lead to the production of iga antibodies in mucosal lymphoid tissues through the above mentioned mechanism. these iga antibodies get transported across the epithelial cells into the lumen where they interact with released antigens depicting immunogenic response. plant edible vaccines are basically prepared using two strategies. either through the introduction of antigen producing gene in plant genome via agrobacterium species; the process known as transformation. this can also be done by directly inserting the target gene in plant genome without the help of any vector. in this case gene gun eliminates the requirement of vector by directly bombarding the target gene in plant tissue, through the technique known as bolistic method. second strategy involves the use of plant viruses as vectors which results in transient expression of the foreign gene. the effect of the added genes is to make the plant or plant virus produce antigens which are normally found only on the surfaces of the pathogen. however, both the techniques require introduction of genes taken from the pathogenic microbe that infect humans but transformation technique offers certain advantages. there are three types of plant transformation methods: . agrobacterium mediated transformation . micro projectile bombardment/bolistics method . electroporation first method involves the use of agrobacterium tumefaciens as a carrier or vector system for introduction of antigen producing gene in plant genome. agrobacterium tumefaciens is a soil bacterium responsible for producing crown gall tumor in plants via its ti (tumor inducing) plasmid. a. tumefaciens and a. rhizogenes has the property to integrate their plasmid dna with nuclear genome of host (plants) cells. this property is exploited for introducing foreign gene in plants and the process is called 'transformation' while the plants produced through this process are 'transgenic plants'. during transformation the bacterial plasmid is fi rst disarmed by removing tumor inducing gene and the target gene for a selected immunogen is introduced. along with the target gene, antibiotic resistance genes were also added in the plasmid which acts as marker genes to identify the transformed plant tissues containing bacterial plasmid. the bacterium is then co cultivated with the wounded plant tissues so that the bacterial dna penetrate the plant cells. following this the target gene randomly incorporates in the plant nuclear genome. after selection through antibiotic resistance successfully transformed plant tissues are identifi ed and regenerated into whole plants under in vitro conditions. these transformed plant lines show varying degree of expression for introduced gene thus producing varying amount of desired antigen (shah et al. ) . plant line showing highest expression is identifi ed and propagated on large scale to be used as edible vaccine. the drawback of this method is that it gives low yield and the process is slow. it requires about weeks to months for whole plants to be formed through this process depending on regeneration ability of various plant species under in vitro conditions. this method is more successful in dicotyledonous plants like potato, tomato and tobacco which are easy to transform through agrobacterium in comparison to monocotyledons. second technique is capable of directly introducing the gene in plant cells through gene gun. this method is called 'bolistic method'. in this method selected dna sequences are precipitated onto metal microparticles and bombarded against the plant tissue with a particle gun at an accelerated speed. microparticles penetrate the walls and release the exogenous dna inside the cell where it will be integrated in the nuclear genome through mechanisms that have yet to be clearly understood (mishra et al. ). this method is quite attractive because dna can be delivered into cells of plant which makes gene transfer independent of regeneration ability of the species. but the chief limitation is the need for costly device particle gun (shah et al. ) . in another technique called 'electroporation', dna is introduced into cells by exposing them for brief period to high voltage electrical pulse which is thought to induce transient pores in the plasma lemma (singh ) . the cell wall presents an effective barrier to dna, therefore, it has to be weakened by mild enzymatic treatment so as to allow the entry of dna into cell cytoplasm. transformation technique results in stable expression of the foreign gene allowing production of subsequent generations of large numbers of transgenic plants, either by vegetative or sexual means. the seeds collected from these transgenic lines could be stored, and used as and when necessary for the molecular farming (malabadi et al. ) . it also provides the opportunity to introduce more than one gene for possible multi-component vaccine production. in addition, judicious choice of genetic regulatory elements allows organ and tissue-specifi c expression of foreign antigens . furthermore, plant dna is not known to interact with the animal dna and plant viral recombinants do not invade mammalian cells (sala et al. ). another approach of plant edible vaccine production utilizes plant viral expression systems which addresses the low yield issue of transformation approach. the rationale of this approach is based on the notion that during viral replication, gene copy number becomes amplifi ed, resulting in a much higher level of transgene expression than observed with stable transformation. among the potential advantages of transient viral expression of transgenes over stably transformed transgenic plants are the shorter time for cloning of foreign genes in the viral genome as compared with the time required to transform plants, the ease at which antigen production can be scaled up, and the wide host range of plant viruses that allows the use of multiple plant species as biofactories (koprowski and yushibov ) . capsid proteins of many types of viruses can assemble into virus-like particles (vlps). devoid of the viral genetic material, vlps often resemble the native virions in their morphology, antigenic properties and stability. these vlps are genetically engineered to express the desired peptides/proteins. two techniques namely 'overcoat technology' and 'epicoat technology' are used to design vlps that contain antigen dna on their surface. some plant viruses like cpmv (cowpea mosaic virus), alfalfa mosaic virus, tmv (tobacco mosaic virus), camv (caulifl ower mosaic virus), potato virus x and tomato bushy stunt virus can be used effectively for this purpose. overcoat technology permits the plant to produce the entire protein, whereas epicoat technology involves the expression of only foreign protein (lal et al. ). the recombinant virus is then inoculated into the host plant which produces several antigenic proteins (mor et al. ). these particulate antigens generally elicit stronger mucosal immune responses than soluble antigens, which can often repress the immune response by inducing immunotolerance (garside and mowat ) . however, each single antigen expressed in plants must be tested for its proper assembly and can be verifi ed by animal studies, western blot; and quantifi ed by enzyme-linked immunosorbent assay (elisa) (haq et al. ) . this method offers rapid onset of expression, and the systemic spread of virus so that protein is produced in every cell (desai et al. ) . vlps are therefore predicted to make excellent mucosal vaccines (estes et al. ) . however, the method poses certain limitations such as transient expression is less easy to initiate. also the subsequent plant generations do not inherit the foreign gene as it is not incorporated into the plant genome. thus the production of a plant virus-derived vaccinogen requires an extra step of inoculation of the host plants with the chimeric virus. before administration, chimeric virus particles are often purifi ed from host-plant tissues that are unpalatable, containing toxins or are not practical for direct consumption. nevertheless, the high level of foreign protein expression (up to g/kg of plant tissue) within a short period ( - weeks after inoculation) makes this an attractive alternative for vaccine production (walmsley and arntzen ) . plants and plant viruses were not used until , however, when an epitope from the foot-and-mouth disease virus (fmdv) was expressed on the surface of cowpea mosaic virus (cpmv) (usha et al. ) . chimeric plant viruses were proven effective as carrier proteins for vaccinogens in after rabbits raised an immune response against purifi ed chimeric cpmv particles expressing epitopes derived from human rhinovirus (valenzuelz et al. ) . majorly tmv and cpmv were used initially as plant viral expression systems because of advantages of high yield (l- g of virus per kg of host tissue), thermostability and ease of virus purifi cation, but now a days other plant viruses like alfalfa mosaic virus (amv), camv (caulifl ower mosaic virus) etc. are also being used. an increased interest in using plant virus vectors was witnessed for development of vaccines against many animal and human diseases (adams et al. ; clarke et al. ; dedieu et al. ; mastico et al. ; lomonossoff and johnson ) . in , who estimated that three to fi ve million children's lives could be saved each year if new vaccines were available to control or prevent commonly occurring infectious diseases. cvi emphasized on the need to create technologies that would make vaccine cheap and more reliable, especially for the developing world. cvi (children's vaccine initiative) also presented idea of needle free immunization to save millions of children from the pain of needles and to avoid risk of death due to needle carried infections. the attention was laid on oral vaccines as they fulfi ll all the requirements and are an effective means of vaccination by activating the mucosal immunity where most of the pathogens attack fi rst. development of heat stable oral vaccines were demanded which could serve the purpose of mass immunization in developing countries. being heat stable they can eliminate the requirement of cold chains which are very costly and generally not found those parts where vaccination is needed most. subunit vaccines based upon recombinant cell culture expression systems are feasible but, for commercial-scale production, these systems require fermentation technology and stringent purifi cation protocols so that suffi cient amounts of recombinant protein can be obtained for oral delivery. even with technological improvements, fermentation-based subunit vaccine production may be a prohibitively expensive technology for developing countries where novel oral vaccines are urgently needed . plant derived edible vaccines are the answer to all these requirements. these vaccines offer all those advantages which make them an ideal candidate to create a disease free world. following are the advantages of plant edible vaccines: (a) plant derived edible vaccines are cheap as they eliminate the expenses associated with fermenters, purifi cation, adjuvant, cold chain logistics, storage/transportation, needle free-administration, and sterile delivery (davoodi-semiromi et al. , walmsley and arntzen ; pascual ; malabadi ; daniell et al. ; gomez et al. ). (b) plant derived vaccine offer higher safety because they are needle/syringe free that is why risk of infections occurring from syringes is eliminated. (c) these vaccines do not require trained medical personnels for its administration unlike conventional vaccines which uses syringes as delivery system (d) plant derived vaccines are safe and have less chances of contamination with human and animal pathogenic microorganisms, because plants are not hosts for human infectious agents (giddings et al. ; ma et al. ). (e) as edible vaccine can be administered orally, they elicit both mucosal and systemic immunity which is not observed in traditional vaccines. (f) these vaccines are heat stable and can be stored at room temperature, unlike traditional vaccine which need cold chain storage which increases the yearly cost to preserve vaccines and limits their availability to areas having cold storage facility (nochi et al. ) (g) a concern with oral vaccines is the degradation of protein components in the stomach (due to low ph and gastric enzymes) and gut before they can elicit immune responses (daniell et al. b ) but in plant edible vaccines bioencapsulation of antigen in the rigid plant cell walls could provide protection from intestinal degradation (webster et al. ) . (h) for production of edible vaccines, costly equipments and machines are not necessary as they could be easily grown in farms as compared to cell culture grown in fermenters. (i) plant derived vaccines can be designed to contain numerous antigens for various disease. these multicomponent vaccines are called second generation vaccines and provide immunization against many diseases in a single dose. (j) an edible vaccine provides higher safety of individual as compared to traditional vaccine as edible vaccines are subunit preparation and do not involve attenuated pathogens which sometimes causes disease when administered as vaccine. (k) they can be ingested by eating the plant/part of the plant. so, the need to process and purify does not arise. (l) if the local/native crop of a particular area is engineered to produce the vaccine, then the need for transportation and distribution can be eliminated. (m) plant cells are suitable for vaccine production due to their capability to correctly fold and assemble, not only antibody fragments and single chain peptides, but also full-length multimeric proteins. (n) low downstream processing requirements for proteins administered orally. (o) as the antigen is present in edible plant product and can be administered by directly eating the plant tissue, it surpasses the purifi cation requirement which otherwise is a costly process. (p) new or multiple transgenes can be introduced by sexual crossing of plants thus creating novel vaccines against multiple diseases. (q) as plants are a safe biological system and harbor no pathogen of human infections, there are no ethical problems associated with plant edible vaccines unlike the vaccines produced from animal cell cultures and transgenic animals. (r) expression of antigen in plant seeds provides a convenient system of vaccine storage for long time duration thus reducing storage and shipping costs under ambient conditions. (s) plant cells are able to perform complex posttranslational modifi cation of recombinant proteins, such as glycosylation and disulfi de bridging that are often essential for biological activity of many mammalian proteins, allowing for the retention of native biological activity (lienard et al. ; rybicki ; tremblay et al. ) the fi rst report of the concept of using a plant expression system for the production of an edible vaccine appeared in a patent application published under the international patent cooperation treaty (curtiss and cardineau (mason et al. ). in parallel with evaluation of plant-derived hepatitis b surface antigen, mason and arntzen explored plant expression of other vaccine candidates including the labile toxin b subunit (lt-b) of entertotoxigenic escherichia coli (etec) and the capsid protein of norwalk virus (nvcp). it was found that the plant derived proteins correctly assembled into functional oligomers that could elicit the expected immune responses when given orally to animals (haq et al. ; mason et al. mason et al. , . subsequently, a number of attempts were made to express various antigens in plants (table . ). a. sahai et al. hepatitis b virus (hbv) is one of the major causes of chronic viremia in humans (purcell ) . the hepatitis b virus is estimated to have infected million people throughout the globe, making it one of the most common human pathogens. since immunization is the only known method to prevent the disease of hepatitis b, any attempt to reduce its infection requires the availability of large quantities of vaccine, hepatitis b surface antigen (hbsag) (malik et al. ) . mason et al. demonstrated the expression of hbsag at levels equal to . % of total soluble protein in tobacco. however, the low level of expression of the hbsag in transgenic tobacco ( . % of soluble protein) and the alkaloids present in the crude plant extract prevent direct feeding studies. thus, mason et al conducted further studies with tobacco-derived recombinant hbsag (rhbsag) protein that was recovered from leaf extracts as a vlp with an average size of nm, which are similar to those found in the sera of infected humans and in the commercial vaccine (cabral et al. ) . these plant-derived vlps mimic the appearance of recombinant yeastderived hbsag particles (scolnick et al. ) , which is the material that is used in commercially available recombinant vaccine for hepatitis b (recombivaxm; distributed by merck, sharpe, and dohme) and can be injected as vaccinogen directly. in addition, the plant-derived material had similar buoyant density and antigenicity to human and yeast-derived hbsag, indicating faithful preservation of protein folding characteristics in the plant system'. thus a crude extract of rhbsag from plants was used in parenteral immunization studies with mice (thanavala et al. ) . the extract caused an immune response that was similar to the one achieved with recombivaxm, so the studies concluded that the rhbsag from plants demonstrate that b-and t-cell epitopes of hbsag are preserved when the antigen is expressed in transgenic plants, and that the recombinant antigen is produced as a vlp that mimics the currently available commercial vaccine. subsequently many papers were published characterizing the recombinant product which assembled into virus like particles (vlps), and could invoke specifi c immune responses in mice upon parenteral delivery. further studies were made by arntzen group to prove that plant-derived hbsag can stimulate mucosal immune responses via the oral route, for which they employed potato tubers as an expression system and optimized it to increase accumulation of the protein in the plant tubers (richter et al. ) . the resulting plant material proved superior to the yeast-derived antigen in both priming and boosting of immune responses to oral immunogen in mice (kong et al. ; richter et al. ) . the hbsag has also been expressed in banana (may et al. ) and lettuce (kapusta et al. ) . recently kumar et al. ( ) transformed embryogenic cells of banana with the 's' gene of hepatitis b surface antigen (hbsag) using agrobacterium mediated transformation. the expression levels of the antigen in the plants grown under in vitro conditions as well as the green house hardened plants were estimated by elisa. maximum expression level of ng/g f.w. of leaves was noted in plants transformed with pefehbs grown under in vitro conditions, whereas pher transformed plants grown in the green house showed the maximum expression level of . ng/g f.w. of leaves. hbsag obtained by them from transgenic banana plants was found to be similar to human serum derived one in buoyant density properties. kumar et al. ( ) advocated that although expression levels of the antigen are low in banana fruits, the expression levels of the vaccine antigens can be increased by the use of promoter of abundant pulp protein (clendennen et al. ) , or promoters of the proteins found in abundance in the ripe banana fruits (peumans et al. ) . mcgarvey et al. ( ) have reported the expression of rabies virus (rv) glycoprotein in transgenic tomatoes. although mcgarvey et al. did not reported on the immunogenicity of the tomato rv glycoprotein, yusibov et al. ( ) have reported the induction of neutralizing antibodies in mice that were parenterally vaccinated with a peptide of rv glycoprotein fused to a plant virus coat protein. antigen-capsid fusion is one of the alternative strategies of producing a plant-based vaccine where plants are infected with recombinant viruses carrying the desired antigen in viral coat protein. the infected plants have been reported to produce the desired fusion protein in large amounts in a short time. it should, however, be kept in view that recombinant viruses need to be highly purifi ed for parenteral administration or partially purifi ed for oral administration. modelska et al. ( ) reported that immunization of mice intraperitoneally or orally by gastric incubation or by feeding of plants infected with the recombinant alfalfa mosaic virus (aimv) carrying rabies peptide cpdrg showed local as well as systemic immune response. after immunization, % of the mice were protected against the challenge with a lethal dose of the virus. this report also demonstrated stronger immune responses in mice that consumed the chimeric viruses in planta rather than after purifi cation (modelska et al. ). recently, loza-rubio and coworkers developed transgenic maize expressing the rabies virus glycoprotein of the vnukovo strain and they evaluated its immunogenicity in mice by the oral route. animals were fed once with μg of protein and challenged -days later with a rabies virus isolated from vampire bats. the edible vaccine induced viral neutralizing antibodies which protected mice % against challenge (loza-rubio et al. ). the vp capsid protein of the foot-and-mouth disease virus (fmdv) was also successfully expressed in transgenic arabidopsis thaliana (carrillo et al. ). carrillo and co-workers have shown that mice injected intraperitoneally with the partially purifi ed vp protein are totally resistant to a challenge with a virulent strain of the virus. oral administration of the plant-derived vp subunit of fmdv has not been demonstrated. because fmdv virions are complex structures containing several subunits therefore it is unlikely that the vp peptide alone will assemble into stable vlps. another important study was regarding enterotoxigenic escherichia coli (etec) and vibrio cholera which are the primary pathogens responsible for acute watery diarrhea. cholera is a devastating diarrheal disease that has caused recurrent pandemics throughout the world since (yu and langridge ) . the heatlabile enterotoxin (lt) of etec is closely related to cholera toxin (ct). haq et al. ( ) proved that a bacterial antigen (lt-b) could be expressed in edible tissues of transgenic plants and could assemble into pentameric ring structures (as shown by its ability to bind gangliosides). in addition, and most importantly, lt-b expressed in plants was shown to induce the production of both serum and mucosal antibodies in mice fed with transgenic potato tubers. however, the low level of expression of lt-b in potato tubers, implied that people would have to eat an unreasonably large amount of tuber to receive the desired dose. mason et al. ( ) improved expression levels of lt-b in transgenic plants by the construction of a 'plant-friendly' synthetic lt-b gene. the protein product of this synthetic gene accumulates to ~ . % of the soluble protein, which is . -fold higher than the best levels obtained previously by haq et al. ( ) . these higher expression levels have allowed this 'edible vaccine' to be tested in humans, representing the fi rst human clinical trial of a plant-derived vaccine where g of raw potato tubers expressing lt-b of etec in three doses had to be consumed in order to overcome digestive losses of the antigen and to elicit a signifi cant immune response (tacket et al. ) . these results showed, for the fi rst time, that food plant-based vaccines are immunogenic in humans. cholera toxin, which is very similar to e. coli lt, has also been expressed in plants. hein et al. ( ) generated tobacco plants expressing ct-a or ct-b subunits of the toxin. ct-a produced in plant was not cleaved into a and a subunits, which happens in epithelial cells. while ct-b undergone similar processing in plants as the ct-b derived from v. cholerae , and was thus recognized by mouse anti-ct-b antibody. antigenically it was found to be similar to the bacterial protein. even after boiling transgenic potato tubers till they became soft, approximately % of the ct-b was present in the pentameric gm ganglioside-binding form (arakawa et al. (arakawa et al. , . higher expression levels (up to . % tsp) were obtained when ctb- l fusion protein was expressed in transgenic chloroplasts (molina et al. ). according to the who, hiv-induced acquired immunodefi ciency syndrome (aids) kills - million people annually (unaids ) . stable chimeric cpmv particles that express epitopes derived from human rhinovirus and hiv- were described by porta et al. in . the inserted epitopes were immunogenic in rabbits. initial success was also achieved in splicing hiv protein into cpmv by prakash ( ) . two hiv protein genes and camv as promoter were successfully injected into tomatoes with a needle and the expressed protein was demonstrable by polymerase chain reaction (pcr) in different parts of the plant, including the ripe fruit, as well as in the second generation plant. norwalk virus is known to cause acute gastroenteritis in humans. norwalk virus capsid protein (nvcp) from the diarrhea causing norwalk virus, expressed in transgenic tobacco and potato with . % of total soluble protein, also assembled vlps and stimulated serum igg and gut iga specifi c for nvcp when fed to mice (mason et al. ) . the clinical trial was conducted at the center for vaccine development with nvcp potatoes ( tacket et al. ) . twenty adults ingested either two or three doses each of g raw potato containing - lg nvcp. nineteen of twenty adults showed signifi cant increases in the numbers of specifi c anti-nvcp antibody-secreting cells of the iga subtype, and six developed increases in igg antibody secreting cells. this study proved that orally delivered plantexpressed vlps could stimulate immune responses and further that gm binding activities not required for oral immunization. these results suggest a new strategy for the development of plant vaccines. individual soluble protein antigens are relatively ineffective for oral immunization because of intestinal digestion and lack of antigen tropism for galt (gut associated lymphoid tissues). in contrast, vlps are stable in the acidic environment of the stomach and resistant to enzyme digestion in the small intestine. most important, vlps preserve conformational epitopes located on the surface of viral particles, which are recognized by the host immune system. compared with other viral antigens, the plant-produced vlp may be the more effective antigen for protection against infectious enteric viral diseases (yu and langridge ) . attempts are underway to engineer bananas and powdered tomatoes expressing norwalk virus. over two billion individuals reside in the malaria endemic areas and the disease affects - million people annually. as a result of malarial-infection, an estimated three million lives are lost annually, among them are over one million children (majority under years of age).the world malaria situation has become signifi cantly worse in recent years as the main forms of malaria control, spraying programmes and chemotherapy, becoming less effective in the development of vector and parasite resistance. in a study by beachy et al. ( ) a -amino-acid epitope of zona pellucida , zp , protein and another epitope from malarial sporozoites have been expressed as fusion proteins with tmv capsid protein with the idea of developing anti-fertility and anti-malarial vaccines. the antigenicity of the products has been found to be positive. currently, three malarial antigens are under investigation for the development of plant-based malaria vaccine, merozoite surface protein (msp) ,msp from plasmodium falciparum, and msp / from p. yoelli. wang et al. ( ) has demonstrated that oral immunization of mice with recombinant msp , msp / and msp , co-administered with ctb as a mucosal adjuvant, induced antibody responses effective against blood-stage parasite. the study however involved complexity as the protein was expressed in e. coli and protection was only evident when high dose antigen was administered. thus it is still uncertain if the oral delivery of a plant-derived malaria vaccine would induce signifi cant immune responses in humans. it has been suggested that antigen expression level in plants are so low that an unrealistic quantity of plant material would have to be consumed to achieve meaningful immunity. for this approach to become realistic improve antigenic expression has to be achieved. moreover, due to high levels of antigen anticipated to be necessary, it is likely that strong adjutants will also be required (wang et al. ). hence, appropriate adjuvants have to be identifi ed and tested. finally, in the face of reports showing induction of tolerance or immunity through comparable oral immunizations vaccination regimens must be rigorously tested in preclinical studies (arakawa et al. ). applications of edible vaccines are expanding to autoimmune diseases where the body's own proteins recognized as foreign by the immune system. autoimmune diseases include arthritis, myasthenia gravis, multiple sclerosis and type i diabetes. it was established by arakawa et al. ( ) that food plants are feasible production and delivery systems for immunotolerization against autoimmune diseases. they found cholera toxin b to be useful as a carrier molecule for specifi c targeting to the gut-associated lymphoid tissue (galt). a cholera toxin b-insulin fusion protein produced in transgenic potato plants successfully protected non obese diabetic mice from development of autoimmune diabetes mellitus type i. ma and jevnikar ( ) expressed glutamic acid dehydrogenase in potatoes and fed them to non-obese diabetic mice, in which the reduced pancreatic islet infl ammation suggested immuno-tolerization of cytotoxic t-cell-mediated autoimmune disease. an optimal oral dose of a plant-derived auto antigen can potentially inhibit development of the autoimmune disease (carter and langridge ; sala et al. ) . edible vaccine development for the prevention or treatment of cancer is diffi cult since tumor antigens are also auto-antigens (zhang et al. ). recently, a poly-epitope isolated from a human melanoma tumor was integrated into the nuclear and chloroplast dna of tobacco in an attempt to develop a plant-derived melanoma vaccine (sala et al. ) . mccormick et al. ( ) expressed a scfv antibody fragment of the immunoglobulin from a mouse b-cell lymphoma in tobacco with a viral vector and showed that mice injected with this vaccine were protected from challenge by a lethal dose of tumor. another scfv fused to the potato virus x coat protein generated protection against lymphoma and myeloma (savelyeva et al. ). there were many questions regarding plant edible vaccines when the concept was given by arntzen and group in s. some of them were answered in the course of successive development in this fi eld while some still remain unanswered. even after all the research and developments made in plant derived edible vaccines, there are some limitations and issues which are still to be addressed. a major impediment in the successful application of these vaccines is the low expression level of antigen and uncertain dosage. in all the studies mentioned above the expression level of antigen in plant tissue was not of practical utilization as to achieve the required immunity very large amount of plant product is need to be consumed. in addition, not all vaccine candidate proteins are highly immunogenic in plant tissues and secondary metabolites found in plants may compromise the ability of the vaccine candidate protein to induce immunity (teli and timko ) . two solutions to overcome this limitation are being explored. first, techniques to enhance antigen accumulation in plant tissues are being explored. a number of factors, including genes encoding vaccine antigens can be optimized for plant codon use; plant promoters can be engineered to increase transcription levels; rna splice sites and intron sequences can be removed codon usage, the type of ′-untranslated sequence incorporated, the presence of specifi c intra-and extracellular targeting or compartmentalization sequences present, the site of gene integration into the genome, etc. affect transgene expression and ultimately vaccine epitope accumulation in plants. optimization of coding sequences of bacterial or viral genes for transient expression, as well as defining the best subcellular compartment for product accumulation to obtain optimal quantity and quality, is also being studied (teli and timko ) . to enhance the immunogenicity of the orally delivered antigens, the use of carrier proteins may also be required, especially for small, non-particulate subunit vaccine antigens (walmsley and arntzen ) . another approach is to use bacterial enterotoxins such as ct or lt , mammalian and viral immunomodulator, or plant-derived secondary metabolites (yu and langridge ) . although the constitutive expression of foreign genes may lead to the accumulation of foreign proteins to levels toxic in the plant, inducible promoters, which stimulate gene expression at specifi c points in plant development, may not only prevent accumu lation of toxic levels of the foreign gene product but may conserve the plant's photosynthate for generation of maximum plant growth, resulting in higher yields of recombinant proteins (arakawa et al. (arakawa et al. , . thus, temporal and organ-and tissue-specifi c promoters activated in fruit, tubers, or seeds must be explored (fiedler and conrad ) . another problem is that the glycosylation of trans proteins in plants differs slightly from those produced in transgenic animals or animal cells in vitro (lerouge et al. ). the addition of xylose and change from a b ! to a b ! linkage of fucose are typical in plants. a signifi cant difference with transprotein production in plants is their inability to add sialic acid to glycoproteins (lerouge et al. ) . this sugar has been implicated in longer clearance times for proteins in the blood and therefore is a major factor for a select group of pharmaceutical proteins. however, plant glycan patterns may not represent a problem in terms of human health; they may affect conformational epitopes, or clearance of plant derived antibodies (bakker et al. ; ma et al. ) . this potential problem is likely to be overcome as we learn more about the requirements for these processes in both plant and animal cells (bakker et al. ) . another concern is about the potential contamination of plant-derived recombinant proteins with potentially toxic factors due to the fact that some plant species contain numerous toxic alkaloids and other secondary metabolites. careful selection of appropriate plant materials for heterologous expression can help alleviate this potential problem (teli and timko ) . another major concern is the possibility of development of immunotolerance to the vaccine protein or peptide. also there will be a challenge in controlling transgene escape as the identifi cation of "vaccine" fruit vs a normal fruit would be diffi cult. fruit vaccines should be easily identifi able to avoid the misadministration of the vaccine, which may lead to complications such as immunotolerance. also the oral vaccines are complicated by the need to protect the antigen from the effects of the acidic and proteolytic environment of the gut. hence, to be effective, oral subunit vaccines generally require higher doses than oral replicating vaccines (walker ; mestecky et al. ) allergic reactions to plant protein glycans and other plant antigens is a challenging issue. it has been suggested that plant derived recombinant proteins or antibodies may have increased immunogenicity or allergenicity as compared to mammalian counterparts (ma et al. ; penney et al. ) . this is well explained by the fact that a state of tolerance or energy has been gained by the daily consumption of plant glycolproteins in our food (ma et al. ) . some other potential problems related to plant edible vaccines include: (a) plant and product contamination by mycotoxins, pesticides, herbicides and endogenous metabolites. (b) regulatory uncertainty, particularly for proteins requiring approval for human drug use (doran ) . (c) consistency of dosage from fruit, plant to plant, generation to generation is not similar (d) stability of vaccine in fruit is not known and differs from plant to plant. (e) some food cannot be eaten raw (e.g. potato) and needs cooking which will denature or weaken the protein present in it (moss et al. ) (f) variable conditions for edible vaccine are also a major problem. potatoes containing vaccine to be stored at °c and could be stored for longer time while a tomato does not last long. thus these vaccines need to be properly stored to avoid infection through microbial spoilage. thus while the plant edible vaccines are a lucrative option in the fi eld of vaccination, there is much remains to be done in this fi eld. with many potential issues to be addressed this area of health care is open for exhaustive research and development. transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event (chebolu and daniell ). the highest level of protein expression through transgenic tobacco chloroplast was obtained for bacillus thuringiensis (bt) cry aa protein ( . % tsp) (de cosa et al. ) . as chloroplasts are genetically semi autonomous having their independent genome, many self replicating copies of transformed plastids can be produced in a single plant cell thus enhancing the antigen/ protein yield considerably. there are several other advantages of chloroplast transformation system over nuclear transformation. environmental concerns about mixing genetically modifi ed pollen with other crops or weeds have been continually raised (stokstad and vogel ) . this objection may be partially addressed by engineering the foreign gene into the chloroplast dna (ruf et al. ) . as chloroplast genome is maternally inherited it does not follow mendelian pattern of inheritance (zhang et al. ) so gene pollution caused by transgene escape through pollen can be controlled. even if the pollen from plants that exhibit maternal inheritance contains metabolically active plastids, the plastid dna is lost during pollen maturation and is not transmitted to the next generation . therefore, the chloroplast expression system is an environmentally friendly approach. chaperones present in chloroplasts facilitate correct folding and assembly of monoclonal antibody in transgenic chloroplasts ) and also result in fully functional human therapeutic proteins, as seen in interferon alpha and gamma (falconer ; leelavathi and reddy ) . chloroplasts have the ability to process eukaryotic proteins, including correct folding of subunits and formation of disulfi de bridges (daniell et al. b ). chloroplast-synthesized cholera toxin-b subunit binds to the intestinal membrane gm -ganglioside receptor, thereby confi rming the correct folding and disulfi de bond formation through functional assay (daniell et al. a ; molina et al. ) . also, chloroplasts have the ability to express multiple genes in a single transformation event. expression of polycistrons in transgenic chloroplasts is a unique feature, which facilitates the expression of entire pathways in a single transformation event (de cosa et al. ; daniell and dhingra ) . de cosa et al. ( ) for the fi rst time expressed a complete bacterial operon in transgenic chloroplasts, resulting in the formation of stable cry aa crystals. this should facilitate expression of polyvalent vaccines or multisubunit proteins in transgenic chloroplasts (chebolu and daniell ). problem of gene silencing is also eliminated in chloroplast expression systems. inspite of higher expression level gene silencing was not observed in transgenic chloroplast derived plants (de cosa et al. ) . chloroplasts can be a good place to store the biosynthetic products that could otherwise be harmful when accumulated in cytosol (bogorad ) . this was demonstrated when cholera toxin b subunit was accumulated in large quantities in transgenic chloroplasts and it had no toxic effect (daniell et al. a ) , whereas when accumulated in the cytosol in very small quantities, ctb was toxic (mason et al. ) . similarly, trehalose, was toxic when accumulated in cytosol but was nontoxic when compartmentalized within chloroplasts (lee et al. ) . however chloroplast transformation is also not untouched with certain limitations and issues. as with any fresh tissue molecular farming system, protein stability over time will change even with refrigeration. extraction and purifi cation must be performed at very specifi c times following harvest. tobacco is currently a highly regulated crop and is not edible. large volume products and edible vaccines would not appear to be feasible using this system (horn et al. ). tobacco appears to be the only species in which plastid transformation has been established as routine (svab and maliga ; . however, recently kanamoto et al. ( ) developed plastid transformation system for lettuce. currently the researches in plant edible vaccine production are emphasizing on good manufacturing practices (gmp) with increased antigen expression being the everlasting objective of this technology. recently a novel technique developed by icon genetics (bayer crop science, germany), based on the magnifection, has additional advantageous than the routine methods used for the production of subunit vaccines in plants. this system allows very fast production, high recombinant protein expression levels for example hepatitis b virus (hb core) with an accumulation level exceeding % of total soluble protein in tobacco (gomez et al. ; yusibov and rabindran ; gleba et al. ; huang et al. ) . recently alvarez et al. ( ) conducted an important study on enhancing recombinant protein expression in transgenic plants. they suggested that in maize, γ-zein is the major storage protein synthesized by the rough endoplasmic reticulum (er) and stored in specialized organelles called protein bodies (pb). zera® (γ-zein er-accumulating domain) is the n-terminal proline-rich domain of c-zein that is suffi cient to induce the assembly of pb formation. fusion of the zera® domain to proteins of interest results in assembly of dense pb-like, er-derived organelles, containing high concentration of recombinant protein. to confi rm this, they expressed f -v antigen protein from yersinia pestis (causal pathogen of plague) in three plant models ( ncotiana benthamiana , medicago sativa (alfalfa) and nicotiana tabacum nt cells) with and without a fused zera® domain. they found that f -v protein with fused zera® domain showed three times more accumulation in plant cells than f -v protein alone. the list of diseases that could potentially be prevented with plant-based, edible vaccines is keeping on increasing. clinical trials have already succeeded in increasing the production of antibodies against hiv in mice (karasev et al. ) on the other hand, soybean has been genetically modifi ed to produce monoclonal antibo dies that act as carriers of cancer-attacking compounds. the possibility of developing hiv and cancer vaccines through transgenic plants could be a major step in the fi ght against these devastating diseases. highly pathogenic h n avian infl uenza responsible for global pandemics and with mortality rates exceeding % encouraged global efforts to develop vaccines against this highly pathogenic avian infl uenza (hpai). shoji et al. ( ) recently described the production of ha from the a/indonesia/ / strain of h n infl uenza virus by transient expression in nicotiana benthamiana plants. they demonstrated that immunization of mice and ferrets with this plant-derived ha protected ferrets against challenge infection with a homologous virus. sexually transmitted diseases are also now included in the domain of plant based vaccination. maclean et al. ( ) have reported the expression of l capsid protein from hpv- . by a transient expression assay, the authors determined not only that l protein was capable of assemble into vlps, maintaining its immunological properties, but that a human instead of a plant codon usage and the protein-direction to the chloroplast, were the best conditions to achieve high yields of recombinant l protein ( % of tsp in transgenic tobacco). these results showed an effi cacious way of producing vlps onward hpv vaccine, maybe a cheaper one than the new hpv vaccine produced in insect cells. immunizing experiments are needed to better prove this hypothesis. obregon and coworkers showed a new strategy for increasing recombinant protein production in hiv by improved the stability of p core protein. this stable protein formed dimmers that were retained within the cell resulting in an enhanced expression, apparently related to protein folding processing and assembly, subcellular targeting and protein stability (obregon et al. ) . recently, saejung et al. ( ) reported, for the fi rst time, the production of a dengue vaccine in plants. takagi et al. ( ) reported gm rice expressing two t-cell epitope peptides of cry j i and cry j ii allergens of japanese cedar ( cryptomeria japonica ) pollen. multicomponent edible plant vaccines providing immunologic protection simultaneously against several infectious diseases are under construction. recently in this context (shchelkunov et al. ) demonstrated a bivalent vaccine synthesis through a synthetic chimeric gene, tbi-hbs, encoding the immunogenic env and gag epitopes of human immunodefi ciency virus (hiv- ) and the surface protein antigen (hbsag) of hepatitis b virus (hbv), was expressed in tomato plants. tomato fruits containing the tbi-hbs antigen were fed to experimental mice and, on days and post-feeding, high levels of hiv and hbv-specifi c antibodies were present in the serum and feces of the test animals. intraperitoneal injection of a dna vaccine directing synthesis of the same tbi-hbsag antigen boosted the antibody response to hiv in the blood serum; however, it had no effect on the high level of antibodies produced to hbv. although no plant-based edible vaccines are currently commercially available, a secretory antibody vaccine was approved in the eu, a poultry vaccine against newcastle disease was approved by the usda, and a hepatitis b virus vaccine using a tobacco plant has been approved in cuba (kim and yang ) . vaccines for diarrhea, hepatitis b and rabies, and antibodies for non-hodgkin's lymphoma, colorectal cancer and dental caries, have been submitted for phase i or phase ii clinical trials in humans (ma et al. ). korban and colleagues at the university of illinois (urbana-champaign, ill.) have reported a plant-based oral vaccine against respiratory syncytial virus (rsv) in tomato fruit (korban et al. ) with the ultimate aim of moving the product into apple. hsv is a viral pathogen that causes respiratory diseases and is a leading cause of viral lower respiratory tract illness in infants and children worldwide. aziz et al. ( ) paved way for developing edible vaccines against anthrax by successfully expressing the protective antigen against anthrax in potato tubers. plant edible vaccines have the potential to change the whole scenario of vaccination. upto few years ago vaccination was only limited to six deadly diseases in chil dren but now not only human but animal diseases of bacterial, viral even protozoan origin are being successfully studied for vaccination. not only this but the researches are ongoing to provide plant based vaccines for autoimmune diseases like diabetes and cancer which has risen the need of these vaccines for developed countries also which till now were emphasized to be important majorly for developing countries. apart from many advantages of these vaccines there are some very relevant social, environmental and ethical issues concerning them which are need to be addressed. besides future research is needed to overcome limitations like low expression, immunotolerance, glycosylation, immunogenicity, and stability of the transproteins if the practical application of these vaccines is to be realized. one very important point is the proper coordination between academia and industry to help these vaccines reach people. both technical and regulatory hurdles have to be overcome. it will be a challenge to create a positive public perception regarding safety and effi cacy of these vaccines after all the fuss created over the safety issues of transgenic crops during last few years. lastly timely funding for this research and participation of corporate giant will certainly help in making this dream a reality soon. the expression of hybrid hiv-ty virus-like particles in yeast higher accumulation of f -v fusion recombinant protein in plants after induction of protein body formation expression of cholera toxin b subunit ologomers in transgenic potato plants a plant-based cholera toxin b subunit-insulin fusion protein protects against the development of autoimmune diabetes edible vaccines expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax galactose extended glycans of antibodies produced by transgenic plants the expression of a nopaline synthase human growth hormones chimeric gene in transformed tobacco and sunfl ower callus tissue use of plant viruses for delivery of vaccine epitopes engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products neutralising immunogenicity of a polyepitope antigen expressed in a transgenic food plant: a novel antigen to protect against measles cellular and humoral immunity in guinea pigs to two major polypeptides derived from hepatitis b surface antigen in vitro selection and characterization of human immunodefi ciency virus type variants with increased resistance to abt- , a novel protease inhibitor plant-based vaccines for protection against infectious and autoimmune diseases chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality presentation and immunogenicity of viral epitopes on the surface of hybrid hepatitis b virus core particles produced in bacteria the abundant -kilodalton banana pulp protein is homologous to class iii acidic chitinases world intellectual property organization multigene engineering: dawn of an exciting new era in biotechnology expression of native cholera toxin b subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology chloroplast derived antibodies, biopharmaceuticals and edible vaccines plant-made vaccine antigens and biopharmaceuticals the green vaccine: a global strategy to combat infectious and autoimmune diseases chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery over expression of the cry aa operon in chloroplasts leads to formation of insecticidal crystals poliovirus chimeras expressing sequences from the principle neuralising domain of human immunodeficiency virus type production of heterologous proteins in plants: strategies for optimal expression foreign protein production in plant tissue cultures virus-like particle vaccines for mucosal immunization expression of interferon alpha b in transgenic chloroplasts of a low-nicotine tobacco high-level production and long-term storage of engineered antibodies in transgenic tobacco seeds mechanisms of oral tolerance transgenic plants as factories for biopharmaceuticals magnifection-a new platform for expressing recombinant vaccines in plants oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus developments in plant-based vaccines against diseases of concern in developing countries oral immunization with a recombinant bacterial antigen produced in transgenic plants expression of cholera toxin subunits in plants production of antibodies in transgenic plants plant molecular farming: systems and products rapid, high level production of hepatitis b core antigen in plant leaf and its immunogenicity in mice cholera toxin b protein in transgenic tomato fruit induces systemic immune response in mice effi cient and stable transformation of lactuca sativa l. cv. cisco (lettuce) plastids high-level expression of the neutralizing epitope of porcine epidemic diarrhea virus by a tobacco mosaic virus based vector. protein expression and purifi cation a plant-derived vaccine against hepatitis b virus plant based hiv- vaccine candidate: tat protein produced in spinach synthesis and assembly of anthrax lethal factor cholera toxin b-subunit fusion protein in transgenic potato current trends in edible vaccine development using transgenic plants oral immunization with hepatitis b surface antigen expressed in transgenic plants the green revolution: plants as heterologous expression vectors foods as production and delivery vehicles for human vaccines expression of hepatitis b surface antigen in transgenic banana plants edible vaccines: current status and future edible vaccines accumulation of trehalose within transgenic chloroplasts confers drought tolerance chloroplast expression of his-tagged gus-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors n-glycoprotein biosynthesis in plants: recent developments and future trends pharming and transgenic plants eukaryotic viral expression systems for polypeptides development of an edible rabies vaccine in maize using the vnukovo strain auto antigens produced in plants for oral tolerance therapy of autoimmune diseases induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (gad) and il- antibody processing and engineering in plants, and new strategies for vaccine production optimization of human papillomavirus type (hpv- ) l expression in plants: comparison of the suitability of different hpv- l gene variants and different cell-compartment localization production of edible vaccines for oral immunization in transgenic plants, current and future prospective recent advances in plant derived vaccine antigens against human infectious diseases edible vaccine-vegetables as alternative to needles transgenic plants as vaccine production systems expression of hepatitis b surface antigen in transgenic plants expression of norwalk virus capsid protein in transgenic tobacco and protein and its oral immunogenicity in mice edible vaccine protects mice against e. coli heat-labile enterotoxin (lt): potatoes expressing a synthetic lt-b gene multiple presentation of foreign peptides on the surface of an rna-free bacteriophage capsid generation of transgenic banana ( musa acuminata ) plants via agrobacterium -mediated transformation rapid production of specifi c vaccines for lymphoma by expression of tumor-derived single-chain fv epitopes in tobacco plants expression of the rabies virus glycoprotein in transgenic tomatoes routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans edible vaccines: a new approach to oral immunization immunization against rabies with plant-derived antigen high yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts plants and human health: delivery of vaccines via transgenic plants edible vaccines: a concept come of age implications of human immunodefi ciency eradications of measles development of a plant derived subunit vaccine candidate against hepatitis c virus rice based mucosal vaccine as a global strategy for cold chain and needle free vaccination hiv- p -immunoglobulin fusion molecule: a new strategy for plant-based protein production vaccines are for dinner plant-made vaccines in support of the millennium development goals the abundant class iii chitinase homolog in young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as an important supply of amino acids for the synthesis of ripening-associated proteins development of cowpea mosaic virus as a high yielding system for the presentation of foreign peptides edible vaccines and antibody producing plants hepatitis virus: changing pattern of human disease production of hepatitis b surface antigen in transgenic plants for oral immunization repopulation with iga-containing cells of bronchial and intestinal lamina propria after transfer of homologous peyer's patch and bronchial lymphocytes stable genetic transformation of tomato plastids and expression of a foreign protein in fruit plant-made vaccines for humans and animals production of dengue envelope domain iii in plant using tmv-based vector system vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-f protein induces a systemic immune response plant viral genes in dna idiotypic vaccines activate linked cd (+) t-cell mediated immunity against b-cell malignancies clinical evaluation in healthy adults of a hepatitis b vaccine made by recombinant dna edible vaccine: a better way of immunization immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis b and human immunodefi ciency viruses plant-derived hemagglutinin protects ferrets against challenge infection with the a/indonesia/ / strain of avian infl uenza agrobiotechnology. mixed message could prove costly for gm crops high-frequency plastid transformation in tobacco by selection for a chimeric aaada gene immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes a rice-based edible vaccine expressing multiple t cell epitopes induces oral tolerance for inhibition of th -mediated ige responses recent developments in the use of transgenic plants for the production of human therapeutics and biopharmaceuticals immunogenicity of transgenic plant-derived hepatitis b surface antigen new advances in the production of edible plant vaccines: chloroplast expression of a tetanus vaccine antigen tetc tobacco, a highly effi cient green bioreactor for production of therapeutic proteins report on the global hiv/aids epidemic expression of an animal virus antigenic site on the surface of a plant virus particle antigen engineering in yeast: synthesis and assembly of hybrid hepatitis b surface antigenherpes simplex gd particles new strategies for using mucosal vaccination to achieve more effective immunisation plants for delivery of edible vaccines plant cell factories and mucosal vaccines oral immunization with a combination of plasmodium yoelii merozoite surface proteins and / enhances protection against lethal malaria challenge oral immunogenicity of human papillomavirus-like particles expressed in potato successful boosting of a dna measles immunization with an oral plant-derived measles virus vaccine oral immunization with rotavirus vp expressed in transgenic potatoes induced high titers of mucosal neutralizing iga novel approaches to oral vaccines: delivery of antigens by edible plants a plant-based multicomponent vaccine protects mice from enteric diseases recent progress in the development of plant derived vaccine antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and hiv- antibodies can eradicate cancer micrometastasis examination of the cytoplasmic dna in male reproductive cells to determine the potential for cytoplasmic inheritance in angiosperm species acknowledgements dr. anwar shahzad gratefully acknowledges the fi nancial support provided by ugc and up-cst in the form of research projects (vide no. - / sr and vide no. cst/ d respectively). dr. aastha sahai is also thankful to csir, new delhi, for the award of senior research fellowship for providing research assistance. key: cord- -q y fewk authors: lemaire, d.; barbosa, t.; rihet, p. title: coping with genetic diversity: the contribution of pathogen and human genomics to modern vaccinology date: - - journal: braz j med biol res doi: . /s - x sha: doc_id: cord_uid: q y fewk vaccine development faces major difficulties partly because of genetic variation in both infectious organisms and humans. this causes antigenic variation in infectious agents and a high interindividual variability in the human response to the vaccine. the exponential growth of genome sequence information has induced a shift from conventional culture-based to genome-based vaccinology, and allows the tackling of challenges in vaccine development due to pathogen genetic variability. additionally, recent advances in immunogenetics and genomics should help in the understanding of the influence of genetic factors on the interindividual and interpopulation variations in immune responses to vaccines, and could be useful for developing new vaccine strategies. accumulating results provide evidence for the existence of a number of genes involved in protective immune responses that are induced either by natural infections or vaccines. variation in immune responses could be viewed as the result of a perturbation of gene networks; this should help in understanding how a particular polymorphism or a combination thereof could affect protective immune responses. here we will present: i) the first genome-based vaccines that served as proof of concept, and that provided new critical insights into vaccine development strategies; ii) an overview of genetic predisposition in infectious diseases and genetic control in responses to vaccines; iii) population genetic differences that are a rationale behind group-targeted vaccines; iv) an outlook for genetic control in infectious diseases, with special emphasis on the concept of molecular networks that will provide a structure to the huge amount of genomic data. braz j med biol res ( ) infectious agents remain a major cause of morbidity and mortality worldwide in spite of advances in hygiene, drug development, and vaccines. the emergence of drugresistant strains of infectious agents (including bacteria, viruses and parasites) and emerging diseases caused by either newly identified infectious agents or newly identified pathogen strains (e.g., severe acute respiratory syndrome corona virus, h n avian flu, h n swine flu) are a global health concern. vaccine development therefore appears to be essential to limit the spread of emergent and re-emergent diseases. however, the development of vaccines faces major difficulties highlighted by the absence of efficient vaccines against major health concern infectious diseases, such as malaria, tuberculosis, and aids ( ) . the difficulties range from i) the incapacity of inducing host protective responses due to inadequate adjuvant, dose, route of immunization, or candidate vaccine antigens with low immunogenicity, high rate of genetic variation, or limited accessibility to the immune system, to ii) high interindividual variability in the response to the vaccine observed as highly variable adverse event risk or protective efficacy, which are partially determined by the genetic background and the immune status of vaccine recipients. new high-throughput technologies, new statistical and bioinformatics tools, and new biological concepts such as molecular networks should help in overcoming some of these difficulties. in particular, the availability of complete microbe genome sequences, including the sequences of multiple strains of a pathogen, may help in identifying new candidate antigens ( ) . furthermore, new high-throughput technologies may lead to a better understanding of genetic variation in resistance to infectious disease and in response to vaccination. here, we will present recent advances in genomic-based vaccine approaches, genetic control in www.bjournal.com.br braz j med biol res ( ) infectious diseases, and we will discuss the possibility of vaccines for target groups or personalized vaccines. the first vaccines resulted from empirical studies and were mainly composed of whole organisms (live attenuated or killed) or inactivated toxins. the majority of vaccine studies developed later also used conventional approaches to select for appropriate antigens. antigen selection was a key step in vaccine development in the pre-genomic era but was time-consuming and had many limitations. the properties aimed for in these molecules were: ) being accessible to the immune system and inducing protective immunity and ) being conserved within the species and not containing regions of high variability ( ). the selection of good candidate vaccine antigens was primarily based on the capacity of induction of protective antibodies, which probably explains why effective vaccines currently available mainly induce a protective humoral immune response ( ). more recently, molecular biology methods have been applied to the development of new vaccines, primarily to enable large-scale production of subunit or peptide-based vaccine antigens. nevertheless, only a small number of available vaccines are based on recombinant antigens. the strategies of conventional vaccinology pose limitations for the development of effective vaccines against a variety of infectious diseases. the development of vaccines against pathogens that show important antigenic variation or that cannot be cultivated in the laboratory remains a great challenge. currently, new approaches using high-throughput technologies are being used for the identification of new vaccine candidate molecules ( ) . the publication of the haemophilus influenzae genome, the first pathogen to have its complete genome sequence published as a result of an approach to genome analysis using new technologies of high-throughput sequencing ( ) , has opened the mind of scientists to a range of new possible approaches to the study of microorganisms and has marked the beginning of a new era in vaccine development: the identification of pathogen candidate antigens based on the knowledge of the genome of the pathogen and on the understanding of microbial biology and host-pathogen interactions, an approach called reverse vaccinology ( ) . this approach has been favored in recent years by the use of high-throughput technologies in the fields of genomics, proteomics and immunomics, among other "omics", which has expanded enormously. the number of available viral and parasitic genome sequences is growing continuously ( ) , and important advances have been made to make new biostatistics and bioinformatics tools available for candidate gene prospection. the current "genomic era" is marked by a major impact on the ability to find new candidate vaccine antigens and to develop effective vaccines. one of the approaches that may be used to analyze the dna data sequences in order to identify vaccine candidate molecules is the in silico analysis of complete genomes. it does not require cultivation of the pathogen and it uses a variety of computer resources to process dna sequence data and the available information about protein functions, cell transport and localization, antigen processing, and immunogenicity. computational analysis may then enable the identification of which dna sequences encode the proteins that have some relevant features of potential vaccine targets ( ) . the impact of in silico analysis on vaccine development can be exemplified by the first study published using this approach ( ) . a capsular polysaccharide-based vaccine against neisseria meningitidis serogroups a, c, w , and y is available, but not against serogroup b as its structure is identical to the polysialic acid structure present on human cells ( ) . the entire genome sequence of a virulent n. meningitidis serogroup b strain, which accounts for the majority of meningococcal disease burden in the united states and europe ( ) , was analyzed to identify candidates for a new vaccine. the bacterial genome was screened using bioinformatics tools to identify relatively conserved, surface-exposed outer membrane protein antigens: of open reading frames (orfs) were thus selected. immunoproteomics methods were then used to select the most immunogenic meningococcal antigens that would elicit a protective immunity. five of genome-derived candidate antigens are components of a rationally designed vaccine against n. meningitidis serogroup b ( ) and a formulation of four of these antigens ( cmenb, novartis, switzerland) has induced high titers of bactericidal antibodies in a phase i/ii clinical trial ( ) . the multivalent vaccine formulation has been proposed to overcome the issue of antigenic diversity between strains of n. meningitidis, and it would be expected to stimulate immune responses capable of recognizing multiple antigenic variants. the promising results of this new vaccine candidate may be the critical "proof of concept" stage for this novel approach that has significantly shifted the paradigm of vaccine development from microbiological to sequence-based approaches. genome-wide screening using functional selection methods such as dna microarrays, gene knockout libraries, or screening of cdna expression libraries using patients' sera has been employed in the prospection of vaccine candidates (reviewed in ref. ) . these approaches allow the evaluation of the possible immunogenic and immunoprotective roles of all molecules (either surface expressed or secreted by pathogens), thereby enabling the identification of candidate antigens that were not identified by the methodologies applied in conventional vaccinology. this means that even molecules that are expressed at very low levels or only under certain conditions can be tested for their potential as candidate vaccines. in brief, genomics coupled with other new "omics" fields of study such as www.bjournal.com.br braz j med biol res ( ) transcriptomics, proteomics, and immunomics enables the identification of vaccine candidate molecules based on pathogen genome analysis. high-throughput sequencing technologies have also been used to understand the molecular biology of several viruses and parasites. recently, genomic-based approaches have allowed the identification of potential novel viral and parasitic targets, which will be included in vaccines against infectious diseases, such as severe acute respiratory syndrome (sars; ), aids ( ) , trypanosomiasis ( ) , and malaria ( ) . pathogens have developed multiple strategies to evade the immune response, and one of the most commonly used is antigenic variation of immune response targets. the genetic variability observed in most microorganisms, especially those that are pathogenic, accounts for the capacity of microbial pathogens to adapt to genetically distinct hosts, different antibiotics, or immune system defense mechanisms ( ) . the high intra-species variability is an important limiting factor to the development of vaccines against several diseases such as hiv, hepatitis c virus (hcv), plasmodium, and meningococcus. genotypic differences among infecting mycobacterium tuberculosis strains have also been pointed out as potential factors influencing the efficacy of the bacillus calmette-guérin (bcg) vaccine against tuberculosis ( ) . this vaccine has been extensively used and studied, and shows wide variation in efficacy among populations and geographic regions worldwide ( ) . entire microorganism genome sequences can be obtained relatively inexpensively and quickly, a fact that makes it feasible to have whole genome data available from multiple isolates of a single pathogen. this technological advance has provided the basis for the exciting, newly proposed concept of pangenome ( ) . according to this concept, a bacterial species could be characterized by its global gene repertoire, which contains genes shared by all the strains (core genome) and genes shared by some strains but not all (dispensable genome). additionally, genetic variants in genes that are found in all strains can be characterized by dna sequence analysis. this concept has been used to characterize intraspecies diversity in a novel approach for the discovery of new vaccine candidate molecules: pangenomics reverse vaccinology ( ) . thus, the development of effective, multivalent and broad coverage genome-based vaccines will probably result from approaches that include genomes from multiple isolates from each microbial pathogen. yet, underrepresented strains not targeted by the multivalent vaccine may become a matter of concern after broad vaccine coverage is established. serotype replacement, a phenomenon by which minor bacterial serotypes become predominant over time, has been documented after the introduction of the heptavalent anti-pneumococcal conjugate vaccine. it can be attributed to the fact that strains not targeted by the vaccine are able to escape vaccine selective pressure ( ) . the ongoing efforts of data mining for all the information in pathogen genomes already made available should effectively help to identify new potential vaccine targets for most infectious diseases ( figure ). however, these new approaches using powerful technologies (genomic and pangenomic reverse vaccinology; functional and structural genomics) have advantages and limitations that should be considered in the rational design of a vaccine development project. in general, the methodologies used for identification and analysis of vaccine candidate antigens are based on the assumption that surface and secreted proteins will induce a protective humoral immune response. that is true for almost all extracellular pathogens, but the presence of antibodies is not always associated with protection against intracellular pathogens. then, eliciting both antibody and t-cell immune responses can be an important property of the candidate antigens to be evaluated. in fact, despite the amount of available data about the genome of pathogens and the human genome, the genes, molecules, and mechanisms involved in pathogen virulence or in host defense are still not clear. understanding the mechanisms of microbial infections and the immune response against them remains a major challenge in vaccine development. this could explain why relatively slow progress has been made in the development of new efficient vaccines in spite of the emergence of high-throughput sequencing technologies and advances in bioinformatics. much research is still needed for novel vaccine development: the contribution of each microbial gene expressed to the disease and to the activation of a protective immune response should be understood. the current data show that there is a wide variety of new molecules that can potentially constitute new vaccine targets; the choice of the selection methods for these new vaccine antigens is, thereby, a critical step and depends on the accumulated knowledge in the fields of bioinformatics, genomics, immunology, pathology, microbiology, parasitology, and virology. host-pathogen interactions are also influenced by the host background. thus, different animal models or human populations, age, specific nutrient deficiencies, co-infection/ co-morbidities, among other host conditions can influence the immune responses to a given pathogen or vaccine. there is accumulating evidence for a genetic control of infectious diseases. rare primary immunodeficiencies have been shown to cause susceptibility to either multiple infectious agents or to a single type of infectious agent; these are very rare and mendelian inherited. twin studies have provided further evidence of a genetic control, and complex segregation analyses that explicitly model the effect of genes and the environment have revealed either multigenic predisposition or a genetic predisposition based on major genes having a big influence on the phenotypes related to the disease ( ) . several candidate genes have been associated with infectious diseases ( ) . the candidate gene approach further supports the role of human genetics in resistance or susceptibility to infectious diseases. more recently, rna interference has been used to exploit human cell models of infection with various agents to elucidate pathogen virulence mechanisms capable of subverting host pathways involved in immune regulation and pathogen killing response ( ) , an approach that can be used to discover and associate differences in preferential immunoregulatory networks involved in host-pathogen interactions in susceptible versus resistant hosts. these approaches are, however, restricted to well-known genes. several genome-wide linkage analyses have been performed to map new resistance or susceptibility genes. major genes have been mapped to chromosomes q -q ( ) and q ( ) for schistosomiasis, chromosomes q ( ) and q/xq ( , ) for tuberculosis, chromosomes p ( , ) and q ( ) for leprosy, and chromosome q for visceral leishmaniasis ( ) . blood infection loads of plasmodium falciparum or mild malaria have been genetically linked to chromosomes q -q ( ), p ( ), p - ( ) , and q - ( ) . the identification of genes underlying complex diseases remains a challenging task, and few genes have been identified. nevertheless, il (chromosome q -q ), tnf and ncr (chromosome p -p ), park and pacrg (chromosome q ) polymorphisms have been associated with schistosomiasis ( ), mild malaria ( , ) , and leprosy ( ), respectively. the first genome-wide association study in infectious disease was published by fellay et al. ( ) , who found an association of hla-b and hla-c with hiv virus load, and of rnf and znrd with disease progression. more recently, several genome-wide association studies have been published for malaria, hepatitis b, and hepatitis c. the best signals of association were found at the hbb locus for severe malaria ( ) , at the hla-dp locus for hepatitis b ( ) , and at the il b locus for hepatitis c ( , ) . all the studies were based on single nucleotide polymorphism (snp) arrays: , snps were genotyped in the hiv, malaria, and hepatitis b studies, while , snps were analyzed in the hepatitis c study. twin studies have recently established that genetic variation influences the response to hepatitis a and b, diphtheria, tetanus, measles, mumps, rubella, polio, h. influenzae type b, pertussis, and bcg vaccines ( ) . such studies have led to estimate the heritability, defined as the ratio of genetic variance to total variance, of immune responses ( ) . early papers have reported a heritability of and % for the antibody response to the hepatitis b surface antigen vaccine in adults ( ) and young children ( ) , respectively; it should be stressed that high levels of antibody against hepatitis b surface antigen correlate with protection against infection and persistent carriage ( ) . the heritability of vaccine responses is generally high ( ) , reaching % for the antibody response against measles vaccine ( ) . it should be stressed that the immune mechanisms responsible for protection are largely unknown for a variety of infectious diseases. also immunological correlates of protection are needed to monitor the efficacy of vaccines and to study the genetics of protective responses to vaccines ( ) . the influence of the genetic background has been further supported by twin-based and population-based association studies, which have revealed the association of hla and non-hla candidate genes with the response to hepatitis a and b, influenza, measles, rubella, and bcg vaccines ( , ) . however, few candidates have been simultaneously tested by each research group, except for the study by hennig et al. ( ) , who analyzed candidate genes. it is likely that there are genes involved in both resistance to a particular infectious disease and response to the corresponding candidate vaccines. therefore, genes that have been associated with resistance or susceptibility to infection or disease can be considered candidates potentially involved in the response to the vaccine. the genes that will be identified through genome-wide approaches in infectious diseases will be of particular interest; conversely, the genes that affect the response to vaccination may be analyzed for their potential role in resistance or susceptibility to the corresponding disease. it may be the time to move from the candidate gene approach to genome-wide linkage and/or association approaches in order to identify genes controlling the response to vaccination (figure ). it is very likely that populations with different genetic backgrounds will differ in their genetic control of infectious diseases: for a particular infectious disease, the involvement of some loci may be evidenced in a population living in africa and may not be detected in another one living in asia. ethnic groups living in the same area can also differ in their ability to control infection or disease. for example, the fulani, whose genetic background is clearly different from that of other ethnic groups in west africa, have been compared to sympatric ethnic groups for several phenotypes related to malaria resistance. the fulani produce higher antibody levels than the mossi and the rimaibe, and have lower parasite densities and fewer and milder malaria attacks, indicating that the fulani are more resistant against malaria than the mossi and the rimaibe ( ) . similarly, it is known that populations differ in their response to vaccination, suggesting that populations differ in the frequency of the protective alleles. poland et al. ( ) have reported that native (innu and inuit) and caucasian schoolchildren differed in their production of antibodies against measles antigens after vaccination in canada. additionally, malawi and uk populations strikingly differed in the level of prowww.bjournal.com.br braz j med biol res ( ) tection following bcg vaccination (i.e., % in malawi and % in the uk), and in their ifn-γ production in response to mycobacterium antigens ( ) . furthermore, native children (navajo, white mountain apache and alaska) and children in the general us population differed in the incidence of disease due to h. influenzae type b both before and after vaccination, indicating that native children were more susceptible than other children in the us, and that their response to vaccination was also weaker ( ) . further understanding of the genetic basis of variation in vaccine response may lead investigators to consider different vaccine strategies for groups having different genetic backgrounds. the combination of high throughput methodologies and multifactorial analyses may help in the identification of genetic markers that affect immune and physiological responses leading to serious adverse effects, thus allowing the identification of groups at risk of vaccine-induced adverse events, which would be entitled to preventive therapies or alternative vaccine formulations ( ) . in the same way, the exposure to the infectious agents, current immune status (e.g., immunocompromised subjects), gender, and age could be taken into account to minimize the rate of vaccine failure or vaccine adverse events. such a targeted-group vaccination approach requires more information about the human groups of interest. efforts are particularly needed to study various populations in africa, where genetic diversity is the most important in the world, and data are lacking. in these populations, the international scientific and medical community would be able to screen for millions of polymorphisms within each potential target group. it has been also suggested that profiles based on individual information, such as genetic information, age, and gender, would allow prediction of the need to receive a given vaccine (i.e., being susceptible to infection), the number and amount of doses needed, the likelihood of response, the likelihood of adverse events, and so on, thereby making it possible to develop a personalized vaccination approach. at this stage, developing a health system in which each individual would be genotyped at the genome scale would become a political and economic choice. before coming to the era of personalized vaccination, however, we should develop methods that will eventually allow us to predict the individual response to vaccination on the basis of individual information, including genome-wide genotypes. the challenge is less technological than conceptual. the emergence of new technologies, such as the microarray technology and more recently the next generation sequencing technology, gives the illusion that genes controlling a complex phenotype would be easily identified. this is due to the fact that we can obtain a huge amount of biological data for one sample. for example, the whole-genome transcriptome can be analyzed by using one array for an individual, and more than , snps can be simultaneously genotyped by using snp arrays for the same individual (the last generation of affymetrix chips allows the analysis of , million snps). the nextgeneration sequencers (solexa/illumina and solid/abi) allow the analysis of the transcriptome, of small rnas, of dna methylation, of snps, and of copy number variation at the genome scale ( ) . all these technologies can produce a huge amount of data that need to be stored, transferred, carefully analyzed and interpreted. the statistical analysis is a significant challenge because a number of statistical tests are performed; to tackle this multi-testing problem, new approaches have been proposed ( , ) . such approaches have been used in genome-wide studies and have provided significant results from the statistical point of view. however, it is clear that most biological information is missing. this lack is highlighted by the results obtained in genome-wide linkage and association in infectious diseases. for instance, fellay et al. ( ) estimated that the polymorphisms identified explained % of the virus load variance in the study population, suggesting that the effect of other genes had not been detected. strikingly, a genome-wide association study only detected the association between protection against severe malaria and hemoglobin s after correcting for multi-tests ( ) , while several other genes and genetic variants have been associated with severe malaria in independent populations ( ) . it is clear that the biological interpretation of such data needs an integration of various biological data stored in different databases, and statistical approaches that make use of these data. the central concept that can drive the development of new approaches is the molecular network. figure represents this concept in the context of infectious diseases. this concept has stimulated the development of gene prioritization tools to propose candidate genes lying within a chromosomal region linked to the disease. the approaches used imply that most genes involved in a disease share functional properties and/or can be mapped in the same gene or protein network. the most frequent approach is to rank candidates on the basis of their similarity to a set of training genes that have been demonstrated to be involved in the disease (reviewed in ref. ) . the biological concept of the gene network implies the existence of gene-gene interactions that are generally not taken into account in genome-wide linkage and association analyses. genegene interaction can be explicitly included in the models used in linkage and association analyses. statistical approaches have been proposed to detect such interactions also in studies of vaccine responses ( ) . furthermore, the influence of tlr and tirap polymorphisms on clinical disease caused by m. tuberculosis has been found to depend on pathogen genotypes, suggesting the existence of bacterial gene-human gene interactions ( ) . obviwww.bjournal.com.br braz j med biol res ( ) ously, the gene-gene interaction studies at the genome scale pose additional multitest problems that must be tackled. in addition, such approaches are generally limited to low order interactions, and efforts are now needed to develop statistical approaches that make use of the whole biological network. the analysis of genetic variation and gene expression levels could also provide help in understanding how polymorphisms could perturb a transcriptional network in infectious diseases ( ) . microarray studies that are based on mice infected with either virulent or nonvirulent pathogens have identified gene expression patterns associated with some infectious diseases, indicating the existence of at least one transcriptional network perturbed by a virulent strain. moreover, gene expression profiles have been shown to discriminate between genetically cerebral malaria-resistant versus genetically cerebral malaria-susceptible mice at early and late stages of infection with p. berghei anka ( , ) . this further indicates that genetic variants direct at least one transcriptional network towards a state conducting to cerebral malaria and death in susceptible mice after infection. in humans, blood cell gene expression profiles have been associated with the clinical status of patients infected with different pathogens ( ) , and have been used to monitor the response to bcg vaccination in children ( ) . although very few genomic studies have been performed in the field of vaccination it has been anticipated that the biological network concept would be useful. in this way, poland et al. ( ) proposed "the immuneresponse network theory", whereby the response to the vaccine can be viewed as the cumulative result of gene interactions. the findings in naturally infected individuals and in mouse models of infectious diseases support this hypothesis. this urges the effort to perform genome-wide studies in vaccinated mice and humans, and to compare the results with those obtained in natural infections. the same authors have suggested that the network combined with individual genomic profiles may lead to predicting the response to vaccination. this is a big challenge because it will require biological networks that capture all the relevant information, including the effect of genetic and other biological information (age, gender, etc.). this further implies that one would be able to predict the effect of single or combined perturbations such as polymorphisms on molecular phenotypes (figure ). for this pur-pose, regulatory network modeling methods would be useful: models constructed on available data to predict molecular and cellular phenotypes will provide working hypotheses that can be tested, leading to the improvement of the initial model. interestingly, immune response system-level tools have been made available ( ) . thus, systems biology is a new rational approach that gives a more comprehensive understanding of networks or interacting components in the immune response to pathogen antigens. this concept has been recently reviewed ( ) . it can be applied to identify signatures of protection that would be evaluated in the de- vaccinomics) . this emergent research strategy in vaccine development was already applied to identify signatures of protection to influenza ( ) and yellow fever vaccines ( ) . nevertheless, the current tools can manage small biological networks; the challenge is to develop new approaches that allow the modeling of large regulatory networks. in conclusion, genomics of infectious agents will accelerate the identification of new relevant vaccine candidate antigens, including antigens that are conserved between strains or collectively cover the diversity of strains and that are the targets of protective immune responses. the genome-based approaches should help in developing vaccines against old, but so far uncontrolled, infectious diseases that are expected in developing countries, and may play a critical role in the design of vaccines against emerging infectious diseases as well as non-infectious diseases. the identification of relevant molecular targets will require further research on protective immune responses and the development of immunological tools. recent investigations that have systematically searched for viral protein-human protein interactions open new avenues for understanding host-pathogen interactions and therapeutic strategies. further research on the mechanisms of action of adjuvants and the use and restraints of relevant animal models will be mandatory. the human genetic variation should also be taken into account when analyzing the response to vaccination. vaccine strategies may have to be adapted to target groups in order to optimize vaccination efficacy and reduce the risk of adverse events. similarly, the possibility of a personalized vaccination strategy (appropriate dose, schedule and even molecular components of the vaccine?) has been suggested, which would be based on individual information, including genome-wide genetic information. this implies the ability to predict an individual response to the vaccine. the challenge is to develop new tools that are necessary to accurately construct and model molecular networks, the perturbation of which causes either the absence of response or adverse events. a vision for vaccines against hiv, tuberculosis and malaria the genomes on line database (gold) in : status of genomic and metagenomic projects and their associated metadata genome-based vaccines the top five "game changers" in vaccinology: toward rational and directed vaccine development whole-genome random sequencing and assembly of haemophilus influenzae rd reverse vaccinology and genomics comparative analysis of epitope predictions: proposed library of putative vaccine candidates for hiv identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing epidemiological profile of meningococcal disease in the united states a universal vaccine for serogroup b meningococcus immunogenicity and safety of a multicomponent meningococcal serogroup b vaccine and a quadrivalent meningococcal crm conjugate vaccine against serogroups a, c, w- , and y in adults who are at increased risk for occupational exposure to meningococcal isolates vaccinology in the genome era hla supertype-, genome-wide scanning and biochemical validation polyvalent vaccines for optimal coverage of potential t-cell epitopes in global hiv- variants utility of the trypanosoma cruzi sequence database for identification of potential vaccine candidates by in silico and in vitro screening genome-wide variation and identification of vaccine targets in the plasmodium falciparum genome vaccine-induced immunity circumvented by typical mycobacterium tuberculosis beijing strains variation in protection by bcg: implications of and for heterologous immunity identification of a universal group b streptococcus vaccine by multiple genome screen the use of genomics in microbial vaccine development evidence that pneumococcal serotype replacement in massachusetts following conjugate vaccination is now complete human genetics of infectious diseases: a unified theory genome-wide association studies and infectious disease functional gene discovery using rna interference-based genomic screens to combat pathogen infection genetic localization of a locus controlling the intensity of infection by schistosoma mansoni on chromosome q -q severe hepatic fibrosis in schistosoma mansoni infection is controlled by a major locus that is closely linked to the interferon-gamma receptor gene an autosomal dominant major gene confers predisposition to pulmonary tuberculosis in adults genetic susceptibility to tuberculosis in africans: a genome-wide scan a major susceptibility locus for leprosy in india maps to chromosome p chromosome q is linked to susceptibility to leprosy in a vietnamese population a major susceptibility locus on chromosome q plays a critical role in the control of kala-azar innate immunity genes as candidate genes: searching for relevant natural polymorphisms in databases and assessing family-based association of polymorphisms with human diseases genetic determination and linkage mapping of plasmodium falciparum malaria related traits in senegal linkage of mild malaria to the major histocompatibility complex in families living in burkina faso genome-wide and fine-resolution association analysis of malaria in west africa analysis of the q -q locus shows an association between il - c/t il- - a/g polymorphisms and schistosoma haematobium infections tnf as a malaria candidate gene: polymorphism-screening and family-based association analysis of mild malaria attack and parasitemia in burkina faso association analyses of ncr polymorphisms with p. falciparum mild malaria susceptibility to leprosy is associated with park and pacrg a whole-genome association study of major determinants for host control of hiv- a genome-wide association study identifies variants in the hla-dp locus associated with chronic hepatitis b in asians genome-wide association of il b with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis c genetic variation in il b and spontaneous clearance of hepatitis c virus heterogeneity in vaccine immune response: the role of immunogenetics and the emerging field of vaccinomics differential genetic determination of immune responsiveness to hepatitis b surface antigen and to hepatitis a virus: a vaccination study in twins genetic regulation of immune responses to vaccines in early life host genetic factors and vaccine-induced immunity to hepatitis b virus infection the evolutionary genomics of pathogen recombination vaccine immunogenetics: bedside to bench to population the lower susceptibility to plasmodium falciparum malaria of fulani of burkina faso (west africa) is associated with low frequencies of classic malaria-resistance genes measles antibody seroprevalence rates among immunized inuit, innu and caucasian subjects bcg-induced increase in interferon-gamma response to mycobacterial antigens and efficacy of bcg vaccination in malawi and the uk: two randomised controlled studies reemergence of invasive haemophilus influenzae type b disease in a well-vaccinated population in remote alaska adversomics: the emerging field of vaccine adverse event immunogenetics a framework for variation discovery and genotyping using next-generation dna sequencing data a tutorial on statistical methods for population association studies how malaria has affected the human genome and what human genetics can teach us about malaria prioritizing genes for pathway impact using network analysis ectopic kidney with varied vasculature: demonstrated by ct angiography the influence of host and bacterial genotype on the development of disseminated disease with mycobacterium tuberculosis interactome networks and human disease gene expression analysis reveals early changes in several molecular pathways in cerebral malaria-susceptible mice versus cerebral malaria-resistant mice gene-expression profiling discriminates between cerebral malaria (cm)-susceptible mice and cm-resistant mice assessing the human immune system through blood transcriptomics transcriptional profiling of mycobacterial antigen-induced responses in infants vaccinated with bcg at birth curating the innate immunity interactome systems biology approaches to new vaccine development systems biology approach predicts immunogenicity of the yellow fever vaccine in humans systems biology of vaccination for seasonal influenza in humans key: cord- -oc zd h authors: pagliusi, sonia; ting, ching-chia; khomvilai, sumana title: quality vaccines for all people(): report on the th annual general meeting of the developing countries vaccine manufacturers' network, – th october , bangkok, thailand date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: oc zd h the developing countries vaccine manufacturers network (dcvmn) assembled high-profile leaders from global health organisations and vaccine manufactures for its th annual general meeting to work towards a common goal: providing quality vaccines for all people. vaccines contribute to a healthy community and robust health system; the ebola outbreak has raised awareness of the threat and damage one single infectious disease can make, and it is clear that the world was not prepared. however, more research to better understand emerging infectious agents might lead to suitable vaccines which help prevent future outbreaks. dcvmn members presented their progress in developing novel vaccines against dengue, hpv, chikungunya, cholera, cell-based influenza and other vaccines, demonstrating the commitment towards eliminating and eradicating preventable diseases worldwide through global collaboration and technology transfer. the successful introduction of novel sabin-ipv and oral cholera vaccine in china and korea respectively in was highlighted. in order to achieve global immunisation, local authorities and community leaders play an important role in the decision-making in vaccine introduction and uptake, based on the ability of vaccines to protect vaccinated people and protect non-vaccinated in the community through herd immunity. reducing the risk of vaccine shortages can also be achieved by increasing regulatory convergence at regional and international levels. combatting preventable diseases remains challenging, and collective efforts for improving multi-centre clinical trials, creating regional vaccine security strategies, fostering developing vaccine markets and procurement, and building trust in vaccines were discussed. the developing countries vaccine manufacturers' network (dcvmn) convened its th international meeting, co-organised by queen saovabha memorial institute (qsmi) and bionet asia, under the auspices of the thai red cross society. nearly professionals working in research, development, manufacturing and supply of vaccines, from countries, gathered in bangkok. dcvmn is the largest alliance of corporate manufacturers, supplying over vaccine types in various presentations to immunisation programmes, and contributing significantly to global public health efforts to eradicate polio, eliminate and control the spread of known and emerging infectious diseases around the world. local health authorities and global health organisations represented at this meeting included united nations international the dcvmn to improve quality of life for every child, family and community globally. prof. s. khomvilai invited dr. j.-m. okwo-bele, director at who, to speak on the achievements and challenges of the global vaccine action plan (gvap). j.-m. okwo-bele highlighted the achievements of global immunisation: polio eradication is close, with only two countries currently reporting wild poliovirus; measles vaccination has reduced the disease by over % from to [ ] ; regional meningitis a epidemics have been eliminated in africa, with half of the population vaccinated [ ] ; in all, around million deaths have been averted by vaccination [ ] . still, challenges remain. limitations in vaccine affordability and supply, failures in healthcare integration, disruption of immunisation by geopolitical conflict and poor-quality health data have resulted in stagnant coverage and repeated failure to reach immunisation goals. for instance, in , . million children missed their rd dose of dtp in the eastern mediterranean region, mostly in security-compromised countries [ ] . solutions expected from manufacturers include measures to assure the controlled temperature chain (ctc) where cold-chain facilities are lacking, implementation of improved delivery technologies for ease of use in the field, and assistance in reducing risk of shortages. the monitoring and accountability framework of gvap identifies and responds to immunisation challenges. a. batson, chief strategy officer at path, provided an overview of the developing vaccine markets. over the past decade, demand for higher-value vaccines has grown. more antigens are incorporated into multi-valent combination vaccines (pentavalent vaccine replacing dtp, and mmr/mr replacing measles vaccine) and new, complex vaccines are being introduced. a robust vaccine development pipeline is emerging from both industrialised and developing countries. from the latter, the serum institute of india's meningitis a vaccine reached million people in [ ] , and it is anticipated that japanese encephalitis vaccine from chengdu institute, china, will reach million people by . dcvmn members play an essential role in global vaccine supply, contributing to improving health and driving new markets. they are diverse in terms of ownership (state or private), scale, portfolio and market (local providers or emerging new multinational companies, fig. ). it is important to understand their role and plan for anticipated changes in global versus national financing and procurement of vaccines. s. chunsuttiwat from the ministry of public health, thailand, discussed health security and sustainable development goals (sdg). thailand introduced universal health insurance coverage (uhc) in , and reached . % insurance coverage in . regionally, the association of southeast asian nations (asean, representing % of the world's population [ ] ), has initiated collaboration for regional vaccine security and self-reliance, seeking to understand vaccine needs from asean countries' perspectives, and to identify areas for cooperation. the challenge of harmonising regulatory pathways to improve access to vaccines in developing countries was discussed by m. ward, from who. increasing regulatory convergence at regional and international levels can be achieved by adopting common standards and practices for vaccine regulation, without requiring harmonisation of laws [ ] . such convergence and/or harmonisa- dtp = diphtheria + tetanus + pertussis combination vaccines. tion needs to be implemented consistently and intentionally to succeed. who's role in promoting access to quality medical products is to develop norms and standards and promote regulatory convergence and harmonisation. through who initiatives, benefits and challenges can be identified, efficient collaboration among regulators fostered and in-country regulatory decisions supported. collaboration should replace duplication. h. deehan from unicef briefed the audience on prevention and remediation strategies to manage vaccine supply constraints. unicef's procurement and supply of vaccines to countries needing assistance with vaccination programmes has increased significantly over the years. since , more than % of doses procured annually have been from suppliers in developing countries. recent supply constraints have included bcg , yellow fever vaccine, and ipv vaccine to support the polio endgame strategy. early and transparent communication with manufacturers and close monitoring of stock levels within countries is required to allow prompt mitigation actions and ultimately reduce risk of interrupted supply. deehan invited dcvmn members to work with unicef to achieve sustained global supply of affordable vaccines of assured quality. m. malhame, head of market shaping for gavi, presented gavi's recent accomplishments and activities. pentavalent, pneumococcal and rotavirus vaccines have been steadily introduced in a growing number of countries, surpassing gavi expectations. the volume of vaccines from dcvms continues to grow, although the value of vaccines procured from dcvms remains relatively low. to secure global supply, gavi has fostered procurement from multiple countries and manufacturers ( fig. a and b ). in terms of activities, gavi introduced two major changes in their co-financing policy in : (i) linking co-financing to prices for all countries in transition phases and (ii) developing payment plans to help countries get out of default. gavi's strategic goals for - include a vaccine goal: to accelerate equitable uptake and coverage of vaccines, a health system goal: to increase effectiveness and efficiency of immunisation delivery as a part of strengthened health systems, a financing goal: to improve sustainability of national immunisation programs, and a market-shaping goal: to shape markets for vaccines and other immunisation products [ ] . with these goals, gavi hopes to immunise million additional children around the world by the end of . d. rodriguez, from the paho revolving fund for vaccine procurement took a moment to celebrate the americas region's success, in , as the first region to eliminate rubella. this was enabled by the commitment of member countries to regional goals, the paho revolving fund and vaccine manufacturers. he then outlined challenges faced with vaccine shortages and strategies to overcome these. latin american populations are under the threat from yellow fever virus because vaccine demand exceeds the number of doses available annually by - million [ ] . to avoid supply shortages, countries need concrete demand planning, secured budgets for vaccine purchases and careful stock management. prevention of shortages starts at country level, while paho acts regionally to facilitate timely and sufficient supply, and vaccine manufacturers worldwide are essential for assured global supply. d. steele, bill & melinda gates foundation, emphasised the importance to address global challenges for diarrheal disease vac-cines. international partnerships with globally funded product development partnerships and dcvms have already delivered two vaccines for the prevention of leading causes of diarrheal mortality, demonstrating the power of partnerships. first, a cholera vaccine was developed through partnership with vabiotech, shantha biotechnics, and the ivi [ ] . the vaccine is who prequalified and is widely utilised in countries with endemic cholera. second, a rotavirus vaccine was developed in partnership with bharat biotech, indian department of biotechnology and path [ ] . the vaccine is licensed in india for introduction in early and a dossier is ready for who prequalification. c. egerton-warburton elaborated on the goal of the global health investment fund (ghif): to invest in quality by supporting late-stage clinical trials and early-stage commercialisation of vaccines, drugs, diagnostics and devices required for control of infectious diseases in low-income countries, as to the world bank classification . an example is the investment in the eubiologics manufacturing facility for oral cholera vaccine, prequalified by the who in december , testifies the impact of financial support to start up manufacturers. egerton-warburton noted that new and innovative financing mechanisms, which complement the newlyproposed revolving fund at unicef, may be required to support immunisation in non-gavi countries. while a large portfolio of new vaccines is under development by manufacturers in developing countries, the conference focused on a few novel and relevant results, due to time limitations. a. precioso, director of clinical trials and pharmacovigilance at butantan, reviewed the clinical development of a live, attenuated dengue vaccine. the tetravalent vaccine is expected to protect against all dengue serotypes, inducing both humoral and cellular immune responses, and requires only one or two doses to provide life-long immunity. phase i clinical trials in the us by nih showed that one dose of vaccine was safe and immunogenic in both flavivirus-naïve and seropositive adults. butantan is currently finalizing a phase ii clinical trial, and ethical and regulatory approval for a phase iii trial in brazil is underway [ ] . h.t. pham, ceo of bionet-asia, outlined the development of a recombinant pertussis vaccine in response to the resurgence of pertussis around the world. bionet has constructed new proprietary bordetella pertussis strains which produce non-toxic, genetically-inactivated pertussis toxin (rpt) [ ] . in a phase i/ii clinical trial, the rpt-containing vaccines were safe and immunogenic in healthy adults, and elicited significantly higher anti-pt titres than the conventional dtap vaccine. another phase i study will soon evaluate rpt vaccine with an epi-cutaneous delivery system [ ] . l. shi, ceo of shanghai zerun biotech, summarised the progress made in recombinant hpv vaccine development. he reviewed the strong causal relationship between hpv infection and cervical cancer. zerun's bivalent hpv ( / ) recombinant vaccine candidate, produced in the yeast, pichia pastoris, is currently in a phase iii clinical trial, and is expected to be on the chinese market by , and to obtain who pq thereafter. in addition, a novel recombinant hpv vaccine candidate based on escherichia coli expression systems, developed by another manufacturer in china, is also in phase clinical trials, as previously reported [ ] . l. castello-branco, scientific director at fundaç ão ataulpho de paiva (fap), presented an overview of the bcg vaccine (moreau sub-strain). this vaccine was developed in brazil in the s and used as an oral vaccine in the national program on immunisation until [ ] . genetic data shows that the sub-strain is similar to the japanese and russian sub-strains [ ] . safety and efficacy of intradermal bcg-moreau vaccine has been demonstrated in large clinical trials. it is the only bcg administered to babies born to hiv positive mothers in brazil and it is licensed for use for intravesical therapy of bladder cancer [ ] . fap is now expanding the manufacturing capacity to supply bcg globally. g. liao described development of the sabin ipv at the institute of medical biology, chinese academy of medical sciences (imbcams). to accelerate eradication of circulating polioviruses from live polio vaccines, who launched an initiative to develop ipv. imbcams started sabin-ipv development . a successful phase iii clinical trial was completed in march , and the world's first sabin-ipv vaccine was launched on the chinese market in july . imb-cams is planning to increase production capacity from the current million to million doses per annum, to develop a combination vaccine and to obtain who pq to enable global supply [ ] . k. ella, bharat biotech, presented their chikungunya vaccine, an inactivated, whole-virion chikv derived from an indian isolate [ ] . chikv was established to high titre in vero cells. pre-clinical toxicity studies demonstrated safety with times the dose proposed for phase i testing. phase i dose ranges were based on a comparative study of protective correlates in convalescent patients and data from immunogenicity testing in laboratory animals. a phase i study is proposed in india. vaccine methodology is protected by patents in several countries. m. bottazzi, from sabin vaccine institute, reviewed novel technologies for vaccines against neglected tropical diseases. seventeen tropical diseases, including leprosy, trematodiasis, leishmaniasis, dengue, chagas disease, filariasis and helminth infections are highly prevalent among the poor, and are endemic in countries, affecting . billion people [ ] . the sabin vaccine institute focuses on product development partnerships to develop effective, lowcost vaccines. the human hookworm vaccine initiative illustrates these efforts against a leading cause of maternal and childhood anaemia, which afflicts million people. two candidate vaccines have been manufactured and are currently being tested in brazil and gabon. the institute is seeking partnership with manufacturers from developing countries to advance the product and clinical development of various candidate vaccines. the eubiologics oral cholera vaccine (ocv) was presented by s. park. eubiologics has a contract manufacturing agreement with ivi for production and supply of cholera vaccines. since , investments from green cross corporation, ghif and others have supported the project. the goal in was to maintain a milliondose global stockpile, and thereafter, to gradually increase this to million doses annually, financed through gavi. the stockpile is expected to aid global response to outbreaks, improve overall availability and supply of ocv, attract new manufacturers and incentivize new developments. clinical trials were completed in august [ ] . this vaccine was approved in in korea. a prequalification dossier has been approved by who demonstrating the impact of partnerships among manufacturers. delivery of ocv was discussed by j. [ ] , and is now using the recombinant technology platform to produce, potentially, the world's rd commercialised hpv vaccine. finlay is developing a -valent pneumococcal vaccine. verez noted that, as % of the world's infants are not yet vaccinated against pneumococcal disease, it may be useful to have an affordable valent vaccine that protects against regional disease strains, while waiting for higher valency vaccines to become accessible to middle and low-income populations. head-to-head studies may help understand the impact of multivalent vaccines compared to regionally relevant formulations. leroy asked how a manufacturer such as innovax could accelerate who pq and how dcvmn members could support their clinical development. huang stated that the dcvmn is able to break barriers between countries and facilitate collaboration and knowledge sharing; it is a matter of calling for such an initiative. harmonisation of the quality of clinical trials and dcvmn's role in calling for convergence between regulators was encouraged. a. precioso commented that dcvmn has several initiatives, including workshops on clinical trial management to harmonise practices and support who prequalification of vaccines. dcvmn needs time to develop such complex initiatives, and collaborations between companies can contribute, by enabling joint-clinical development and international multicentric clinical trials. d. curry (centre for vaccine ethics and policy), whose expertise lies in ethical and legal requirements for and oversight of clinical trials, suggested that one area of support to dcvms might be development of ethical frameworks to guide clinical trial design, conduct and assessment. a discussion on vaccine security was moderated by j. kim, director general of ivi, with h. deehan of unicef, m. makhoana of biovac, n. premsri and t. mahmoud as contributors. unicef and gavi are steadily increasing vaccine funding and procurement for developing countries in response to increasing demand. h. deehan noted that, while sourcing vaccine from multiple suppliers helps prevent global vaccine shortages, it also creates fierce competition, and noted the difficulty of finding the balance in having an ideal number of suppliers and fostering a sustainable, competitive market. kim turned attention to the recent ebola outbreak in africa, which was brought under control without a vaccine [ ] . he posed a question on how manufacturers can prepare for re-emergence. m. makhoana agreed that future outbreaks may come, and emphasised that the understanding of the infectious agent must precede product development. vaccines then need to be part of broader strengthening of health systems. ultimately, responses tackling african diseases may need to come from within africa. kim queried vaccine security in asia in the context of the regional threats with bird flu, sars and mers . n. premsri related thailand's philosophy of self-reliance, with the ideal to have capacity to produce essential vaccines and an adequate stockpile from multiple regional manufacturers in order to respond to outbreaks or shortages. t. mahmoud described vaccine-supply challenges in saudi arabia, where the objective of high vaccination coverage is undermined by vaccine shortages and lack of expertise in vaccine manufacturing in the region. technology transfer with the appropriate partners and a competent team of manufacturers will accelerate efforts to secure high vaccination coverage regionally. the discussion on developing markets was moderated by j. chu, senior director of vaccine markets at the clinton health access initiative (chai). he noted that vaccine markets are unique: it requires a high investment for manufacturers, and the lengthy development times, can often result unintentionally in supply monopoly. changes in regulatory standards can cause disruption and delays in lot release and registration. despite the challenges, dcvmn members have been contributing significantly to increasing access to vaccines globally. in , the human-vaccine market was estimated at us$ billion and it is forecasted to reach us$ billion by [ ] . to seize the opportunities and compete successfully in this rapidly growing global market, manufacturers need to continuously innovate. m. malhame (gavi) raised the balance between affordability and sustainability of vaccine supply, explaining that gavi evaluates any new vaccine that has passed phase ii clinical trials for inclusion in the vaccines' portfolio financed by gavi. this evaluation is data-driven, and includes parameters such as disease burden, feasibility and cost-effectiveness of disease prevention. however, she noted that coordination and alignment between global stakeholders need to improve and ensure development of the most needed vaccines. she also commented that the high costs of vaccine development, relative to those of drugs or diagnostics, could be overcome in part by technologies to improve manufacturing processes. k. ella (bharat biotech) shared insights on building successful international partnerships and balancing private and public contributions. privatisation of manufacturing has resulted in stable vaccine supply in many countries, and private enterprise can enhance supply of public-sector. l. shi (zerun institute, walvax) discussed corporate motivation to enter international markets while also serving the needs of domestic markets. walvax is prioritizing product-portfolio investments to allow supply of vaccines internationally. large manufacturing capacity is required to satisfy both domestic and international markets and many chinese companies have this capacity, thus the world is likely to see more vaccines from chinese manufacturers in future. f. lobos described sinergium's journey into the vaccine market in south america. sinergium was a spin-off from a veterinarian vaccine manufacturer in . it established technology transfer agreements with large multinationals, primarily to satisfy the national needs for influenza, pneumococcal and hpv vaccines. importantly, partnerships with government accelerated their progress: technical support was received from the local regulatory agency in building a world-class facility complying with international standards; also, a multi-year governmental supply contract secured stability for the company. both local and international partners were critical for rapid success. now, with modern facilities and equipment, the company aims to expand partnerships with dcvms. s. inglis, director of nibsc, moderated the panel that included s. jadhav (serum institute of india), a. batson (path), p. carrasco (iaim), and k. sampson (apaci) on trust in vaccines. s. jadhav described the experiences of the serum institute of india, one of the largest and most successful manufacturers from emerging countries, with who-prequalified vaccines. still reluctance to purchase products from developing countries on the basis of perceived higher reactogenicity or preference for known brands is a barrier to acceptance of new products. measles vaccine was prequalified by who in , however unicef did not immediately take it up. serum institute supplied more than % of the measles-containing vaccines used to achieve measles and rubella eradication in latin america. for the introduction of meningitis a vaccine in african countries, community leaders were informed about benefits, resulting in high acceptance and very successful prevention of disease. the lesson is that industry needs to invest considerable effort into informing stakeholders of their products and intentions. a. batson noted that vaccine hesitancy is often specific to a country or situation, rather than a global issue, and strongly agreed that engaging the local community leaders can have significant impact on uptake. interestingly, mobile phones have proven useful to advocate for vaccines for teenagers, such as hpv vaccines. k. sampson discussed the confidence in influenza vaccines, where there is a risk that the strains that make up the vaccine may not fully match those causing disease, due to antigenic drift. he noted that asia has a large ethnically and socio-culturally heterogeneous population and a vaccine may not suit all patients. decision-making on influenza vaccination is driven by whether it is likely to protect the vaccinated individual, and also, whether is it likely to help protect others through herd immunity. ready availability of disease data that can be communicated to medical professionals would assist in highlighting value. p. carrasco elaborated further on the role of health workers delivering vaccines, noting that they build patient trust by imparting their knowledge of the vaccines and vaccination, but also inspire confidence when they clearly trust the intervention themselves. educational programs, organised by professional associations, need to provide health professionals with the right information about why and how vaccines are used, in their own language. new tools, such as smart phones, are within reach of most health workers, even in low-income countries. the dcvmn annual general meeting gave rich insights into advances in vaccine technologies and development of new vaccines, and provided valuable opportunity for vaccine manufacturers, regulatory authorities and international organisations to have face-to-face interactions. to achieve global immunisation, local authorities and community leaders play an important role in the decision-making in vaccine introduction and uptake, based on the ability of vaccines to protect vaccinated people and protect non-vaccinated in the community through herd immunity. reducing the risk of vaccine shortages can be achieved by increasing regulatory convergence at regional and international levels. combatting preventable diseases outbreaks remains challenging, and collective efforts for improving multi-centre clinical trials including various populations, creating regional relevant vaccine manufacturing and stockpiling security strategies, fostering stable vaccine markets and procurement, and building trust in vaccines are recommended approaches. the dcvmn members will continue to collaborate with one another, with partners and with regulators towards the goal of improving global access to immunisation, through sustainable supply of vaccines to match demand, as well as greater capacity to rapidly respond to outbreaks. the aim is to provide high quality vaccines for all people. who fact sheet meningococcal conjugate vaccination in burkina faso: analysis of national surveillance data gavi alliance sets out opportunity to save up to six million lives through immunization. gavi pr of media centre of world health organisation. immunization leaders call for increased political support for immunization in pakistan. media centre of world health organisation eliminating epidemic meningitis as a public health problem in sub-saharan africa. a public health breakthough regulatory convergence sought for global pharma market about gavi. gavi's business model: working together for healthy vaccine markets the yellow fever initiative: providing an opportunity for a lifetime safety and immunogenicity study of a killed bivalent (o and o ) whole cell oral cholera vaccine, shanchol, in bangladeshi adults and young children as young as year of age team science and the creation of a novel rotavirus vaccine in india: a new framework for vaccine development clinical evaluation strategies for a live attenuated tetravalent dengue vaccine construction of bordetella pertussis strains with enhanced production of genetically-inactivated pertussis toxin and pertactin by unmarked allelic exchange needle-free and adjuvant-free epicutaneous boosting of pertussis immunity: preclinical proof of concept dcvmn executive committee group. vaccines, our shared responsibility rio de janeiro: an oral vaccine against tuberculosis-review genome sequence of mycobacterium bovis bcg moreau, the brazilian vaccine strain against tuberculosis bcg immunotherapy for bladder cancer-the effects of substrain differences safety and immunogenicity of inactivated poliovirus vaccine made from sabin strains: a phase ii, randomized, positive-controlled trial chikungunya virus and prospects for a vaccine the disease next door. foreign policy a randomized, non-inferiority trial comparing two bivalent killed, whole cell, oral cholera vaccines (euvichol vs shanchol) in the philippines stockpiling oral cholera vaccine hepatitis e vaccine: who position paper ebola and its control in liberia world preview : outlook to . vaccines market to we are grateful to all speakers and moderators whose contribution made the conference possible. we thank corporate partners for supporting dcvmn with unrestricted educational grants: the merck group, temptime corporation, ge healthcare, bioengineering, gea, bosch, ompi (stevanato group), alfa wasserman, pall. this conference was partly supported by a grant from the bill & melinda gates foundation, grant no. opp . we are grateful to m. dennehy for editorial support. the authors are employees of the respective indicated organisations, and have no conflict of interest to declare. dcvmn international did not provide any financial support to speakers or moderators to participate at this meeting. key: cord- -a vtc rp authors: awasthi, raghav; guliani, keerat kaur; bhatt, arshita; gill, mehrab singh; nagori, aditya; kumaraguru, ponnurangam; sethi, tavpritesh title: vacsim: learning effective strategies for covid- vaccine distribution using reinforcement learning date: - - journal: nan doi: nan sha: doc_id: cord_uid: a vtc rp a covid- vaccine is our best bet for mitigating the ongoing onslaught of the pandemic. however, vaccine is also expected to be a limited resource. an optimal allocation strategy, especially in countries with access inequities and a temporal separation of hot-spots might be an effective way of halting the disease spread. we approach this problem by proposing a novel pipeline vacsim that dovetails actor-critic using kronecker-factored trust region (acktr) model into a contextual bandits approach for optimizing the distribution of covid- vaccine. whereas the acktr model suggests better actions and rewards, contextual bandits allow online modifications that may need to be implemented on a day-to-day basis in the real world scenario. we evaluate this framework against a naive allocation approach of distributing vaccine proportional to the incidence of covid- cases in five different states across india and demonstrate up to , additional lives potentially saved and a five-fold increase in the efficacy of limiting the spread over a period of days through the vacsim approach. we also propose novel evaluation strategies including a standard compartmental model based projections and a causality preserving evaluation of our model. finally, we contribute a new open-ai environment meant for the vaccine distribution scenario, and open-source vacsim for wide testing and applications across the globe. all countries across the globe are eagerly waiting for the launch of an effective vaccine against sars-cov- . the operation warp speed [ ] aims to deliver million doses of a safe, effective vaccine for covid- by january , however, the pace of development continues to be punctuated by the safety concerns [ ] . as potential candidates start getting ready to enter the market, there will be an urgent need for optimal distribution strategies that would mitigate the pandemic at the fastest rate possible [ ] [ ] . center for american progress estimated that million doses of covid- vaccine along with accompanying syringes will be needed for the us alone to reach herd immunity [ ] . here we summarize the key factors that will need to be considered for effective mitigation: • scarcity of supply: despite large scale production efforts, it is expected that the vaccine will still be a scarce resource as compared to the number of people who would need it. in addition to the vaccine itself, there may also be scarcity in the components leading to its delivery, e.g syringes. the white house director of trade and manufacturing policy stated earlier this year that the us would need million syringes to deliver the vaccine en-masse. this highlights the next challenge of the optimal distribution of scarce resources related to the vaccine. arxiv: . v [cs.ai] sep • equitable distribution: a truly equitable distribution will not just be defined by the population or incidence of new cases alone, although these will be strong factors. other factors ensuring equity of distribution include quantum of exposure e.g. to the healthcare workforce that needs to be protected. in this paper, we assume that the exposure is proportional to the number of cases itself, although the proposed methodology allows more nuanced models to be constructed. there may also be unseen factors such as vaccine hoarding and political influence, which are not accounted for in this work. • transparent, measurable and effective policy: the design of policy would need to be guided by data, before, during and after the vaccine administration to a section of the population. since the viral dynamics are rapidly evolving, the policy should allow changes to be transparent and effects to be measurable in order to ensure maximum efficacy of the scarce vaccine resource. on the larger scale of states and nations, this would imply continuous monitoring of incidence rates vis-a-vis a policy action undertaken. although the aforementioned factors seem straightforward, the resulting dynamics that may emerge during the actual roll-out of the vaccine may be far too complex for human decision making. the daunting nature of such decisionmaking can be easily imagined for large and diverse countries such as india and the united states, especially where health is a state subject. artificial intelligence for learning data-driven policies is expected to aid such decision making as there would be limited means to identify optimal actions in real-world. a "near real-time" evaluation as per the demographic layout of states and consequent initiation of a rapid response to contain the spread of covid- [ ] will be required. furthermore, these policies will need to be contextualized to the many variables governing demand or 'need' for the vaccine distribution to be fair and equitable [ ] . therefore, ground testing of these scenarios is not an option, and countries will have to face this challenge. in this paper, we introduce vacsim, a novel feed-forward reinforcement learning approach for learning effective policy combined with near real-time optimization of vaccine distribution and demonstrate its potential benefit if applied to five states across india. since real-world experimentation was out of question, the change in projected cases obtained via a standard epidemiological model was used to compare the vacsim policy with a naive approach of incidence-based allocation. finally, our novel model is open-sourced and can be easily deployed by policymakers and researchers, thus can be used in any part of the world, by anyone, to make the process of distribution more transparent. actor-critic methods are temporal difference (td) learning-based methods that have a policy structure(actor) which select actions, and an estimated value function(critic) that critiques the actions made by the actor. a markov decision process is represented as a function of (x, a, γ, p, r), at a given time t. an agent performs an action ∈ a following a certain policy π θ (a|x) to receive a reward r(x, a) and a transition to the consequent state x with a probability p (x |x, a). the objective of this task is to maximize the expected γ-discounted cumulative return (given the policy parameters θ). policy gradient methods optimises a policy π θ (a|x) with respect to its parameters and update θ, to maximise j(θ) [ ] , [ ] . as defined in [ ] , policy gradient is expressed as follows: where ψ t is representative of the advantage function a p i(x, a). an advantage function provides a relativistic idea of how useful a certain action is, given a certain state. a good advantage function provides low variance and low bias gradient estimates. in our work, we refer to [ ] , which uses the a c (asynchronous advantage critic) method proposed by [ ] and suggests the following advantage function: where v π φ (x t ) represents the value network, whose parameters are further trained by performing temporal difference updates. the variable k corresponds to the k-step return from the advantage function. trpo [ ] is an on-policy algorithm that is well suited for environments with both discrete and continuous action spaces. considering we make use of the latter, this optimization scheme can be employed in our model. updating policy using trpo involves taking the largest possible step to improve model performance, whilst also satisfying a constraint setting the proximity between the old and the new policies. the constraint is expressed as kl-divergence. if π θ is a policy (θ being the parameters), then the trpo update is performed as follows: where l(θ k , θ) is a surrogate advantage function comparing how the current policy performs with respect to the old policy, and d kl (θ||θ k ) is the average kl-divergence metric. natural gradient descent (ngd) [ ] is an efficient realization of second-order optimization and is based on information geometry. in contrast to first-order methods such as stochastic gradient descent, ngd converges faster by approximating the loss landscape using the fisher information matrix (fim) [ ] as the curvature matrix corresponding to the loss function. kronecker-factored approximate curvature [ ] provides an efficient way to approximate the inverse of fim, which is otherwise a computational challenge associated with this approach. acktr is a scalable and sample efficient algorithm to apply the natural gradient method to policy gradient for actorcritic methods. proposed in [ ] , it is used to calculate and apply the natural gradient update to both actor and critic. it uses a critic to estimate the advantage function. training the model amounts to solving the least-squares problem of minimizing the mean squared error (mse) between the model predictions and the target values, i.e., (r(x) = estimated(x) − target(x)). the contextual bandits algorithm is an extension of the multi-armed bandits approach [ ] which contextualizes the choice of the bandit to its current environment. this serves to circumvent the problem where a multi-armed bandit may simply end up playing the same action multiple times even though the environment (context) may have changed, thus getting stuck at a sub-optimal condition. contextual bandits play an action based on its current context, given a corresponding reward, hence are more relevant to real-world environments such as the vaccine distribution problem attacked in this work. given, for time t = ...n, a set of contexts c and a set of possible actions x and reward/payoffs p are defined. at a particular instant, based on a context c t ∈ c, an action x t ∈ x is chosen and a reward or a payoff p t = f (c t , x t ) is obtained. regret [ ] is a conceptual function to understand and optimize the performance of the bandits problem. since we don't know if an action played was the most "reward-fetching", rewards against all actions that can be played are sampled, and the difference between the action chosen and the action against the maximum reward is defined as 'regret'. therefore, minimizing regret achieves the goal of maximizing reward. for an optimal action x * ∈ x such that the expectation of reward against this action is maximum, p * = max xt∈x (e(p | x t )), the regret and cumulative regret can be expressed as z = [p * − e(p | x t )] and z * = n t= z respectively. in order to derive the exact posterior inference in linear models, a bayesian linear regression [ ] may be performed, and several computationally-efficient versions are available [ ] . linear posteriors assume that the data were generated as per p = c t β + ( ) where ∼ n ( , σ ), p represents the reward or payoff and c is the context. the joint distribution of β and σ for each action is modeled. sequentially estimating the noise level σ for each action allows the algorithm to adaptively improve its understanding of the volume of the hyper-ellipsoid of plausible β's which generally leads to a more aggressive initial exploration phase (for both β and σ ). the posterior at time t for action x t after observing c and p, is where we assume σ ∼ ig(a t , b t ), and β | σ ∼ n (µ t , σ Σ t ), an inverse gamma and gaussian distribution, respectively. their parameters are given by we set the prior hyperparameters to µ = , and Λ = λ id, while a = b = η > . it follows that initially, for . note that we independently model and regress each action's parameters, β i , σ i for i = , . . . , k [ ] . for practical purposes, a and b are initialized as integer values. in this paper, we concatenate the acktr sub-model and contextual bandits sub-model in a feedforward manner. this was done to select the optimal policy through the supervised contextual bandits approach from the ones generated by the reinforcement learning-based acktr model as shown in figure . this was done to address the following challenges that need to be tackled in real-world problems such as optimal vaccine distribution: • solving in real-time: a vaccine distribution problem is expected to be fast-paced. thus an overwhelming amount of brainstorming with constrained resources and time would be required to develop effective policy for a near future. this calls for the development of a prompt and an easily reproducible setup. • lack of ground truth: this is one of the key challenges addressed in this paper. since roll out of the vaccine will not give us the liberty of testing various hypotheses, a lack of ground truth data is generally expected. this is analogous to zero-shot learning problems thus precluding a simple supervised learning based approach. • absence of evaluation with certainty: lack of ground testing naturally implies nil on-ground evaluation. in that case, it often becomes challenging to employ evaluation metrics that offer a significant amount of confidence in results. in order to solve this problem, we rely upon the simulated decrease in number of susceptible people as vaccine is administered. • model scaling: we ensured that the learning process of the models simulates the relationship between different objects in the real world environment accurately, and at the same time can be scaled down in response to computational efficiency and resource utilization challenges. this is done by choosing the right set of assumptions that reflect the real world. while reinforcement learning approaches replicate human decision-making processes, the absence of evaluation makes them less trustworthy, especially when real lives may be at stake. therefore, we pipelined the acktr model with a supervised learning based contextual bandits approach where recommendations for vaccine distribution policy were used as training data for the latter. we extracted the state-wise time series data of covid- characteristics from the website https://mohfw.gov.in/ and use them in the experiments as described below. the five states chosen for this study, i.e., assam, delhi, jharkhand, maharashtra and nagaland are representative of possible scenarios for the vaccine distribution problem, i.e., high incidence(maharashtra and delhi), moderate incidence (assam) and low incidence (jharkhand and nagaland) as on august , . this particular date was chosen following the announcement that india may launch its indigenous vaccine on this date. in choosing the five different states, we hope to generalize our predictions to other states across the spectrum while minimizing the bias introduced into the learning by a widely variant covid- incidence across the country. we also enhanced our modeling context with: population share: the percentage ratio of the population of the state to the population of all the five states. predicted death rate: the percentage ratio of the predicted deaths in the state to the total predicted cases in that state calculated using projections obtained from a fitted standard compartmental model, i.e. a susceptible, exposed, infected and recovered(seir) model. predicted recovery rate: the percentage ratio of the predicted recoveries in the state to the total predicted cases in that state using projections obtained from the seir model. the implementation of acktr and contextual bandit sub-models of vacsim are detailed henceforth: acktr model: open-ai [ ] stable-baselines framework was used to construct a novel and relevant environment suited to our problem statement for acktr to learn in this environment. a. input: with both the observation space and action space being declared as box-type, a context vector describing the situation of the state in terms of total predicted cases, predicted death rate, predicted recovery rate, susceptible cases and population, at a given time, was fed as input. predictions were obtained from seir projections [ ] . the action space was a one-dimensional vector with its size equal to the number of recipients of the vaccine in one round of distribution. notably, we accounted for the cumulative time it would take for vaccine distribution, administration and generation of the immune response by the body i.e. the total time between dispatch of vaccine from the distribution centre to achievement of immunity in the vaccinated individual. this introduces a lag of days between the distribution date of vaccines at day 't', to the gathering of context at day 't+ ' by vacsim using seir projections. b. model working: following are the assumptions used while building the environment: vaccine efficacy % number of recipients per day table : hyper-parameters used during policy learning • the nature of the vaccine is purely preventative and not curative, i.e., it introduces immunity when administered to a susceptible person against future covid infection, but plays no role in fighting against existing infections (if any). • the vaccine has % efficacy i.e. all people who are vaccinated will necessarily develop immunity against future sars-cov- infection. this assumption is easily modifiable and is expected to have little consequence in deciding the optimal allocation, unless data reveal differential immunity response of certain populations within india. however, we leave scope for it to be modified as per the situation by taking this as a hyperparameter for vacsim. • each person requires only dose (vial) of the vaccine to be vaccinated completely and successfully. this too may be treated as a hyperparameter. reward function: the reward function was designed to maximize the decrease in the susceptible population count with the minimum amount of vaccine, considering it to be a limited resource. where r i was the reward given to vaccine recipient i s i was the susceptible population count of the same state days from the day of distribution. q i was the amount of vaccine given to the state by the model. flow: vacsim gives us a recommendation for the distribution ratio between various recipients (indian states) of the vaccine as its output. following this distribution set, it awards the corresponding number of vials to each state and converts a corresponding number of individuals in that the state from susceptible to recovered, thus short-circuiting the seir model. the explore-exploit tradeoff: we tap into the explore-vs-exploit dilemma, allowing the model to reassess its approach with ample opportunities and accordingly, redirect the learning process. we set the exploration rate at %. however, this too is flexible and can be treated as a hyperparameter. hyperparameters: a complete list of the hyperparameters is given in table . c. output: the output of the first sub-model was a distribution set dictating the share of each recipient in the batch of vaccines dispatched at the time of delivery. the output of the first sub-model spanned over days ( september- september ) with each state having a total of episodes being run for each day. for every episode, the distribution sets so obtained (one per episode) were normalised to get the percentage distribution ratio for all states. normalised here refers to the percentage ratio of a given distribution set of a state and the sum of the distribution sets of the states over an entire episode for a given date. since the time period is fifteen days, this amounts to a total of episodes. a. training: these episodes, which comprised of the normalised distribution sets along with the corresponding set of rewards, were then fed to the second sub-model, i.e., contextual bandits as training data set. the action space in this model was assumed to lie in the range [ , ] (both inclusive) to represent the percentage of vaccine that went to each state during the distribution process. the features in the context were the same as in sub-model , except population, which was now replaced by population share (fraction of population of a state with respect to the total recipient population). this was done merely for scaling purposes. c. testing: using the context, normalised actions and the corresponding set of rewards as the training dataset, we tested the model day-wise for a period of fifteen days ( september- september) with each day having fifty possible actions for each state as output, similar to what was done in acktr model. the unadjusted actions (which were not normalised) obtained after testing the model were first adjusted day-wise for each state by taking the mean of all possible fifty actions for the state on that particular day. these were then normalized to obtain the percentage vaccine distribution for the five states for the period under consideration ( days . results indicate that out of the five states, maharashtra, which had the highest number of cases, saw a gradually decreasing trend whereas nagaland, which has the lowest number of cases, sees a gradually increasing trend with respect to the distribution of the vaccine figure (right). we also calculated that if the vaccine were to be distributed on since there is no way that the evaluation of distribution policy can be done in the absence of vaccine and real-world distribution, we defined the naive baseline distribution policy as % of vaccine given to a state = % of infected people in that state and compared it with our model's learned distribution. with doses and states, we simulated the distribution of the available vaccine on september for the naive and vacsim policies. the number of resulting (projected) infections for days after the vaccine distribution were calculated using the seir model. day-wise total cases of all states for both policy models were summarized. our results indicate that the vacsimbased policy would additionally reduces a total of infected cases, with % ci [ , ] , in the next days. as seen from figure (right) and figure (left), in the case where distribution was done using naive approach, vaccine distribution for all the fifteen days was nearly the same with maharashtra, receiving the highest amount of vaccine and nagaland, receiving the least amount of vaccine as expected as per their infection rates. on the other hand, in case of the results obtained through vacsim, each state would get a variable and sufficient amount of vaccine during the fifteen-day period including nagaland, which received a negligible amount under the naive approach. unlike the naive approach, vacsim, therefore was not biased towards any state, thus ensuring equitable distribution while mitigating the epidemic. the ultimate goal of vaccine distribution is to reduce mortality and morbidity. since our model relies entirely upon simulations, in the absence of a vaccine, we checked if the data generated by such an approach follow the cause-andeffect relationships as expected in the real world data. structure-learning was carried out using a data-driven bayesian network [ ] approach using hill climbing algorithm [ ] with akaike information criterion as a scoring function, and ensemble averaging over bootstrapped networks. these models were learned using wiser package [ ] . state-wise time series data of death, recovery, infected people, susceptible people and the amount of vaccine obtained from our model were used to learn the structure. blacklisting among nodes was done such that vaccine percentage cannot be the child node of covid- trajectory indicators (susceptible, recovery, infected people, death). the resulting structure shows a causal relationship between the vaccine amount(parent node) and susceptible count (child node), thus confirming the technical correctness of the vacsim model through an external evaluation approach (refer figure ). researchers worldwide are working around the clock to find a vaccine against sars-cov- , the virus responsible for the covid- pandemic. once available, distribution of the vaccine will have numerous logistical challenges (supply chain, legal agreements, quality control, application to name a few) which might slow down the distribution process. in this paper, we have developed a novel distribution policy model, vacsim using reinforcement learning. we have pipelined an actor-critic using kronecker-factored trust region (acktr) model and bayesian regression-based contextual bandit model in a feed-forward way such that the output (action and rewards) of acktr model are fed into the contextual bandit model in order to provide a sensible context comprising actions and rewards. contextual bandits then optimized the policy considering demographic metrics such as population share of state with respect to the chosen states and time series-based characteristics of the covid- spread (susceptible population, recovery rate, death rate, total infected cases) as context. while distributing the vaccine, identifying the part of the population who needs it the most is a challenging task, and in our case, we addressed it by the usage of the aforementioned context. rather than using the present-day count of infected and susceptible people, we have used seir-based projections, which makes our predicted policy more robust and trustworthy. evaluation of model-driven policy is a tough assignment due to unavailability of ground truth, and we proposed a novel causality-preserving approach to evaluate such models. the open-source code will enable testing of our claims by other researchers. vacsim may have some limitations shared by all rl models, i.e. the transparency of their learning process and the explainability of their decisions. secondly, the development of vacsim has been carried out while observing the pandemic in the past few months. however, the dynamic nature of pandemic may require change in actions, thus calling for common-sense working alongside artificial intelligence. in conclusion, we believe that artificial intelligence has a role to play in optimal distribution of the scarce resources such as vaccines, syringes, drugs, personal protective equipment (ppes) etc. that the world will see in the coming months. we provide a novel, open source, and extensible solution to this problem that policymakers and researchers may refer to while making decisions. astrazeneca covid- vaccine study is put on hold a proposed lottery system to allocate scarce covid- medications: promoting fairness and generating knowledge if a coronavirus vaccine arrives, can the world make enough? a comprehensive covid- vaccine plan a survey of agent-based intelligent decision support systems to support clinical covid- vaccine: development, access and distribution in the indian context policy gradient methods for reinforcement learning with function approximation simple statistical gradient-following algorithms for connectionist reinforcement learning high-dimensional continuous control using generalized advantage estimation scalable trust-region method for deep reinforcement learning using kronecker-factored approximation asynchronous methods for deep reinforcement learning trust region policy optimization new insights and perspectives on the natural gradient method optimizing neural networks with kronecker-factored approximate curvature deep contextual multi-armed bandits deep bayesian bandits showdown: an empirical comparison of bayesian deep networks for thompson sampling thompson sampling for contextual bandits with linear payoffs pattern recognition and machine learning, ser. information science and statistics gym: a toolkit for developing and comparing reinforcement learning algorithms global stability for the seir model in epidemiology a tutorial on learning with bayesian networks learning bayesian networks by hill climbing: efficient methods based on progressive restriction of the neighborhood wiser: a shiny application for end-to-end bayesian decision network analysis and web-deployment key: cord- - m bsrw authors: shaw, alan r.; feinberg, mark b. title: vaccines date: - - journal: clinical immunology doi: . /b - - - - . - sha: doc_id: cord_uid: m bsrw nan vaccines represent one of the most effective and cost-effective medical and public health achievements of all time. worldwide, vaccination programs are currently estimated to save over million lives each year. in addition to having such a major beneficial impact on vaccine-preventable disease morbidity and mortality, the direct and indirect impacts of vaccination programs translate into economic savings of many billions of dollars each year. in what is considered to be one of the most significant medical successes of all time, a collaborative and comprehensive vaccination campaign against smallpox resulted in the global eradication of the disease in . similarly, efforts to eradicate poliomyelitis have made tremendous progress in reducing the global disease burden, and will hopefully soon overcome certain residual societal and programmatic obstacles to provide the second successful example of elimination of a major health threat by vaccination. concerted global efforts to provide measles vaccine have resulted in the control and elimination of the disease in many countries, including substantial reductions in mortality in a number of developing countries where the residual disease burden is greatest. these and other examples provide clear evidence of the power of vaccines in favorably manipulating host immunity to confer dramatic public health benefits, at both the individual and population level. as vaccines are administered to healthy individuals (often to entire age cohorts or populations), to prevent diseases caused by infectious agents to which they might be exposed in the future, they differ in important ways from pharmacologic agents that are used to treat individuals in whom a disease process is already manifest (or who display predispositions to disease). for this reason, vaccines are unique in the way that they impact on societies and in the way that societal commitment to vaccination determines their ultimate impact. as a result, vaccination efforts provide an informative window on challenges that need to be successfully navigated at the interface between scientific opportunity and societal capacity and commitment. indeed, current limitations in realizing the full global potential of available vaccines relate more to existing inadequacies in health care financing and infrastructure (especially as they are manifest in developing countries), and the relative value that societies place on disease prevention, than they do to any inherent biological limitations of vaccines themselves. fortunately, recent acceleration of new vaccine introductions in developing countries through public and private initiatives to build immunization infrastructure and provide funding of vaccine purchase offers hope that vaccines will one day be equitably available to all who need them. the importance of vaccines extends beyond their use as public health tools to include their role as drivers of immunologic discovery. the history of vaccine development is rich with immunologic insights that emerged from careful observations of how diseases spread in populations and how such spread differs in disease-naïve and experienced populations, as well as of how innovative experimental approaches revealed fundamental aspects of immune system function. the general concept of immunity induced by prior exposure to a disease (including its specificity and potential lifelong duration) was appreciated by the ancient greeks. use of the word 'immunity' itself dates to the th century when it was applied to describe the relative susceptibility and resistance of populations to plague. the subsequent successes of edward jenner and louis pasteur in the development of effective smallpox and fowl cholera immunization strategies, respectively, provided a foundation for modern immunology; pasteur himself coined the term 'vaccine' in recognition of jenner's use of vaccinia virus. jenner's smallpox immunization studies also provided early experimental support for the concept of immune memory. pasteur's efforts provided the first demonstration of the attenuation of pathogens by their propagation in culture (or by passage in nonnatural animal hosts), while robert koch demonstrated that killed pathogens could also engender immunity. the discovery of bacterial exotoxins by emile roux and alexandre yersin facilitated the discovery of antibodies and their potential use in passive immunotherapy with antitoxin antibodies by emil von behring and shibasaburo kitasato. these discoveries enabled the development of active immunization against diphtheria and tetanus using toxin-antitoxin mixtures. paul ehrlich's development of accurate methods for antibody quantitation made passive immunotherapy and active toxin-antitoxin immunization far more reliable and effective, and provided a stimulus for significant advances in immunologic theory. in each of these instances, vaccine development illuminated central mechanisms of immune system biology. vaccine development today has transitioned from an approach that was once largely empirical to one that is based on the hypothesis-driven application of techniques in molecular biology and immunology. evidence for this synergy can be seen in recent studies of vaccine-elicited immune responses to illuminate primary and memory t-and b-cell responses in humans, as well as the strong discovery stimulus provided by ongoing efforts to develop new vaccines for major infectious diseases for which vaccines are not currently available. vaccine development today faces a number of significant challenges. there exist tremendous public health needs to address major well-known pandemic diseases, including acquired immunodeficiency syndrome (aids), tuberculosis, and malaria, for which no vaccines currently exist and for which natural immunity does not provide a helpful guide for vaccine development. furthermore, there exists a need to confront effectively newly emerging and re-emerging diseases, ranging from the well-known, but constantly changing, threats from influenza pandemics to the appearance of previously unknown zoonotic infections such as the coronavirus that causes severe acute respiratory syndrome (sars). with changes in population density, mobility, and social constructs, along with alterations in the global climate, ecological circumstances, and the proximity of humans to animal reservoirs for previously confined infectious agents, the concept of new infectious agents entering human populations and spreading rapidly around the world is no longer novel. in confronting prevalent or newly emerging diseases, vaccines are looked to as the most promising line of defense. however, the speed at which new infectious disease threats have been shown to emerge and spread, and the fact that the pathogens that now need to be confronted may display tremendous genetic variability (e.g., human immunodeficiency virus (hiv)) or an identity that cannot be predicted in advance (e.g., avian influenza or agents like sars) places unprecedented demands on the vaccine development process. in addition to these new challenges, there remain unmet needs in the derivation of vaccines that can achieve the greatest public health benefit. these needs include the development of new ways to achieve more effective vaccine-elicited immune responses in neonates whose immune systems are immature (or are impacted by maternal antibodies) (chapter ) and in the elderly whose immune system function may be waning as a result of immune senescence (chapter ). fortunately, the scientific foundation provided by basic and applied immunology and the use of new methods for pathogen identification, antigen discovery, vaccine production, adjuvant development, and novel vector derivation afford important opportunities for vaccine development and additionally present the possibility of improving on natural immunity. success in vaccine development will be predicated on continuing the historical synergy between advances in vaccine technology and basic immunologic discovery. toward that end, this chapter focuses on preventive vaccines for infectious diseases and how they are developed. although current routine vaccine recommendations are reviewed, given the active state of new vaccine introduction and evolving vaccine recommendations, as well as differences in recommendations in different countries, readers are encouraged to refer to up-to-date national resources for the most current information. while vaccine approaches are being actively explored to modify beneficially malignant and immunologic diseases (autoimmunity and allergy), these are beyond the scope of the current discussion. n impact of vaccination programs n unlike other medical interventions, vaccines confer benefits to both individuals and populations. , while individuals may be protected from infection or disease by vaccine-induced immune responses, decreasing the number of susceptible hosts in a population also helps break the chain of transmission that pathogens require to spread and persist in human populations by induction of 'herd immunity. ' the benefits of herd immunity depend on achieving sufficiently high immunization rates in a population to impact pathogen transmission dynamics (including the potential for extinction of ongoing interhost transmission). the requisite level of vaccination coverage of a population needed to compromise pathogen spread significantly varies between pathogens, and is influenced both by vaccine efficacy (and its duration) and by the reproductive characteristics and infectiousness of the pathogen. analysis of the impact of vaccination programs in the usa provides an example of the beneficial impact of vaccines when used routinely and when high coverage levels are achieved. as shown in tables . and . , vaccination programs in the usa dramatically decreased the annual morbidity of many vaccine-preventable diseases. in many instances, the disease burden from several vaccine-preventable diseases of childhood has been reduced by over % since vaccine introduction (e.g., diphtheria, tetanus, measles, mumps, rubella, and polio). the somewhat lower rate of decline of pertussis (the annual morbidity of which has been reduced by a nonetheless impressive %) relates to the limited duration of vaccine-induced immunity, which is estimated to wane within - years after childhood vaccination. it is anticipated that recent availability of pertussis booster vaccines for use in adolescents and adults will lead to significant further declines in pertussis morbidity. even for diseases targeted by vaccines that have been in widespread use for less time (< years), impressive decreases in disease morbidity have been seen (e.g., varicella, hepatitis a, and pneumococcal disease). in a notable recent demonstration of the population benefits of vaccines, introduction of the -valent pneumococcal conjugate vaccine resulted in a decrease of % in disease morbidity in children under years of age within the first years of its introduction. interestingly, the rate of meningitis and bloodstream infections caused by antibiotic-resistant streptococcus pneumoniae also fell by % in this age group. in a striking related finding illustrating how vaccines can impact pathogen transmission dynamics, rates of antibiotic-resistant pneumococcal infections also declined by % in individuals over the age of who had not received the vaccine. thus, direct protection by vaccination of children who represent a reservoir of infection provided, via herd immunity, significant indirect benefits to those who did not themselves receive the vaccine. in addition to their benefits in preventing disease morbidity and mortality, routine vaccination programs are also impressively cost-effective. evaluation in the usa of the impact of ten vaccines routinely given as part of the childhood immunization schedule (diphtheria, tetanus, pertussis, haemophilus influenzae b (hib), polio; measles, mumps, rubella, hepatitis b and varicella) found that more than million cases of disease and more than deaths were averted over the lifetime of the immunized birth cohort of children. when the cost of the vaccination program was compared to the economic impact of diseases prevented, these vaccines alone are estimated to save nearly $ billion each year. when including indirect economic benefits (such as the time parents take off from work to care for sick children), the annual savings to society exceed $ billion. when preventive services were ranked based on clinically preventable disease burden and cost-effectiveness, childhood immunization received the highest score. progress in the development of new vaccines accelerated significantly towards the end of the th century, with the development of vaccines against diseases that were not previously preventable by vaccination, but also with the development of improved versions of existing vaccines. thus, the number of diseases that can be prevented by vaccines included in the us centers for disease control and prevention's (cdc) routine childhood and adolescent immunization schedules grew from seven in to in (table . and fig. . ) . moreover, in the past several years, new vaccines have been introduced for adolescents and young adults (e.g., pertussis booster (tdap), meningococcal conjugate, and human papillomavirus (hpv) vaccines), and older adults (e.g., tdap and zoster vaccines) have shown that the value of vaccines extends across the human lifespan (figs . and . ). new combination vaccines have been developed to increase the simplicity and acceptability of vaccination regimens, as well as to improve overall compliance with the recommended series of vaccines. such combinations include either those that contain multiple inactivated or recombinant antigens (such as a combination diphtheria, pertussis, tetanus, hib, and hepatitis b vaccine) or multiple live attenuated viruses (such as a combination measles, mumps, rubella, and varicella vaccine (mmrv)). the development of a combination vaccine is often more complicated than simply combining individual antigens, for when antigens are administered in combination, immunologic interference is sometimes seen. this necessitates titration of antigen combinations (and in the case of combinations of inactivated and/or recombinant antigens, adjuvant selection) to achieve immune responses that are not inferior to each of the antigens administered individually. despite their readily demonstrable public health impact, the value of vaccines is often not appreciated, for when vaccine programs are successful the diseases that they cause become less prevalent and may disappear. however, to prevent resurgence of an infectious disease that has been brought under control, vaccination programs need to be continued. the difficulties facing current efforts to eradicate poliomyelitis have demonstrated that failure to maintain high immunization coverage rates can lead to prompt re-emergence and spread of the disease. even in developed countries, maintenance of strong immunization programs with high degree of coverage is needed where infectious diseases can travel with remarkable speed -and do so even before the extent of spread is evident. n principles of immunization n the terms vaccination and immunization are often used interchangeably. however, vaccination specifically refers to efforts to induce protective immune responses by administration of a vaccine, whereas immunization more generically refers to interventions -either active or passive -that seek to confer immune protection. active immunization describes the induction of immune responses by administration of a specific antigen or antigens, while passive immunization involves the administration of exogenous immunologically active substances (historically, antibodies present in sera obtained from immune individuals or animals) to confer temporary protection from an infectious pathogen or toxin. although the approaches for passive immunization waned in the later half of the th century, the advent and increasing robustness of monoclonal antibody technology have led to a resurgence of interest in passive immunization. vaccines seek to engender immune responses similar to those that confer immunity to re-infection in individuals who experience (and survive) natural infection with a given pathogen. in lieu of formal demonstration of a specific type of antibody or cellular immune response that contributes to prevention or accelerated clearance of an infection, most often vaccine efficacy is demonstrated first in the course of a placebo-controlled trial. in some instances, specific immune effector mechanisms, such as a specific level or type of antibody response, can be identified that correlate with immune protection. in this case, the 'correlate of immunity' provides a benchmark against which similar vaccines can be compared. in the case of most inactivated vaccines, subunit vaccines, and recombinant vaccines that produce antibody responses, but generally meager cd t-cell responses, it is likely that humoral immune responses are the primary or sole protective immune mechanism. in the case of live attenuated vaccines that induce both cellular and humoral immune responses against the pathogen, it is likely that both arms of the immune system act in concert to confer immunity. however, the actual mechanisms of immune protection induced by either a natural infection or a vaccine are generally not understood in detail for many infectious diseases. similarly, although vaccines depend on the induction of immunologic memory, the magnitude, character, and duration of immune memory differ between vaccines, as can the actual mechanism of immune protection. for certain vaccines, such as those that protect against bacterial diseases induced via production of toxins (e.g., diphtheria or tetanus), protection induced by toxoid-based vaccines is clearly dependent on persistent antibody (igg) and memory b-cell responses, ensuring that sufficient antitoxin antibodies are present at the time of toxin exposure to inactivate and clear the toxin. in other cases, such as long-lived protection against hepatitis b, if sufficient levels of antibodies are achieved in the initial immunization period, even hosts who may with time lose detectable levels of antibody responses remain protected. in this instance, given the relatively long incubation period of hepatitis b, memory antiviral b-cell responses induced by the vaccine can be activated, facilitating neutralization and clearance of the infection before clinical disease is manifest. although it is popularly believed that vaccines confer protection by inducing 'sterilizing immunity' -wherein an infectious agent is blocked from even infecting one cell in an exposed host -this is clearly not the case for a number of vaccines. for example, the inactivated poliovirus and live attenuated rotavirus vaccines do not prevent some degree of local replication of their pathogenic counterparts in the gastrointestinal tract of exposed hosts. however, they are both effective in preventing clinical disease. in the case of poliovirus vaccine, this is mediated by elicitation of antibody responses that block dissemination of the infection to the central nervous system; while in the case of rotavirus, as yet unidentified immune effectors limit local virus replication so that significant gastrointestinal damage does not occur following infection. , the major types of vaccines licensed for use include live attenuated organisms, killed or inactivated organisms, subunit vaccines consisting of purified (or partially purified) components of an organism, and subunit vaccines produced by recombinant dna technologies. the use of live attenuated vaccines dates back to the early work of jenner and pasteur on smallpox and fowl cholera vaccines, respectively. , the fundamental concept of live attenuated vaccines is to mimic the effective host immune responses that follow natural infections. most live attenuated vaccines currently in use were derived by propagation of initially pathogenic organisms in culture on cells from different (nonhuman) species, or at nonphysiologic temperatures, for prolonged periods. driving pathogen evolution in culture to select for variants adapted to growth in heterologous cell types ex vivo often leads to the derivation of pathogen variants that grow poorly in vivo in humans and are unable to cause clinical symptoms. vaccines developed via this approach include those used to prevent a number of viral and bacterial infections, including yellow fever, measles, mumps, rubella, polio (the 'sabin vaccine'), varicella-zoster (used both for the prevention of chickenpox and shingles) and rotavirus (one version of the available vaccines), tuberculosis, and cholera. more recent technologies being applied to live attenuated vaccine development include the application of reverse genetic strategies ( fig. . ) and those involving genetic reassortment with attenuated viral variants, as have been used to develop polyvalent live attenuated vaccines against influenza and rotavirus ( fig. . ) . , the live attenuated vaccines currently in use are highly efficacious (> %) and protection is frequently durable. the efficacy of many live attenuated vaccines likely reflects the ability of the attenuated vaccine to replicate within vaccinated hosts, and to expose the immune system to pathogen-derived antigens in a manner that closely resembles the nature, location, and effects of natural infection. because live attenuated vaccines replicate within immunized individuals, they can induce both cellular (cd and cd ) and humoral (b-cell) effector responses and immunologic memory. in addition, as the live attenuated vaccines likely activate the host innate system in a manner similar to their pathogenic parents, they provide inherent adjuvant effects in augmenting adaptive immune responses. a key consideration in the development of any live attenuated vaccine relates to the relative balance between the ability to induce sufficient immune responses in vivo to confer protection (often associated with level of preserved replicative ability in vivo), and the ability to cause symptoms (which may also relate to the extent of in vivo replication). as such, an effective but also safe and well-tolerated vaccine needs to strike a specific balance between level of attenuation and level of immunogenicity. in addition, depending on the nature and number of genetic mutations responsible for the attenuated phenotype, a potential risk of reversion to a pathogenic form exists for certain vaccines. for most live attenuated vaccines, this has not been observed to be a problem in clinical practice -likely because the attenuating mutations are sufficiently numerous or genetically stable. one vaccine where reversion to pathogenic form was seen involved specific components of the live attenuated oral poliovirus vaccine (opv; the 'sabin vaccine'). in this instance, vaccine reversion to wild-type was shown to lead rarely to cases of paralytic polio (approximately one case per million doses administered). based on these observations and the elimination of endogenous polio transmission in many developed countries, the inactivated polio vaccine (ipv; the 'salk vaccine') was substituted for opv. however, in light of a favorable cost-benefit ratio, high degree of efficacy, and ease of administration, opv continues to be the mainstay of polio vaccination efforts in developing countries. the use of physical or chemical methods to kill or otherwise inactivate a pathogenic organism represents a second major approach to vaccine production. , in most cases, treatment with chemical agents such as β-propiolactone and formaldehyde is used to eliminate pathogen infectivity. while this approach has the benefit of presenting most of a pathogen's antigenic repertoire to the immune system of the immunized host, it can only be used in instances where the inactivated pathogen does not possess constituents that would confer significant toxicity. vaccines based on killed pathogens are believed to exert their protective effects via elicitation of pathogen-neutralizing antibodies and the induction of memory b-cell responses (likely in concert with cd t-cell memory). however, because inactivated pathogens cannot accomplish de novo synthesis of pathogen-derived gene products in antigen-presenting cells (apcs), they do not typically induce cd t-cell responses (chapter ). in addition, killed vaccines are generally less immunogenic than live attenuated vaccines. as a result, they are commonly administered with an adjuvant (most often alum: see section on adjuvants, below) to augment their immunogenicity. a number of viral and bacterial vaccines currently in use are killed/inactivated vaccines, including whole-cell bordetella pertussis vaccine and the influenza virus, rabies virus, and hepatitis a virus vaccines. a number of bacteria produce toxins that represent the major pathogenic components responsible for disease in infected humans. examples include corynebacterium diphtheriae and clostridium tetani. detoxified versions of these toxins are referred to as 'toxoids,' and represent the purified components of vaccines preventing diphtheria and tetanus, respectively. toxoids have historically been produced by chemical inactivation of toxins, but more recently, genetic inactivation via targeted mutagenesis has been employed. the acellular pertussis vaccine is also a purified subunit vaccine composed of a defined set of protein constituents prepared from cultured bordetella pertussis. the mechanism of immune protection conferred by purified subunit vaccines is the antibody response elicited by vaccination. antibodies directed against the capsular polysaccharides present on encapsulated bacteria also confer protective immunity in a number of important instances by inducing antibodies that exert opsonophagocytic effects (promoting phagocytosis of antibody-coated bacteria) and, in some instances, bactericidal effects. initial successful vaccine efforts against streptococcus pneumoniae and neisseria meningitidis utilized purified preparations of capsular polysaccharides. although such purified polysaccharides can induce protective levels of antibody responses in adults, they are poorly immunogenic in children under years of age (as a function of the relative immaturity of their immune systems). in addition, t-independent antibody responses elicited by purified capsular polysaccharides are less durable than those that are produced in the presence of cd t-cell help. as a means of both augmenting antibody responses against polysaccharide antigens in young children and facilitating their persistence, the development of conjugate vaccines represented an important advance. in this approach, purified polysaccharides are chemically conjugated to a carrier protein (such as diphtheria toxoid or an outer-membrane protein complex (ompc) derived from n. meningitidis). the carrier protein augments cd t-cell helper responses to the polysaccharide antigens, and enables elicitation of durable protective antibody responses even in young children. polysaccharideconjugate vaccines have been produced that protect against haemophilus influenzae b, streptococcus pneumoniae, and n. meningitidis infections. if two such segmented viruses with different genetic characteristics are used to infect one cell, the progeny viruses from this mixed infection will carry a range of mixtures of the genes of the two parent viruses. using either genetic or immunologic screening methods, reassorted viruses carrying the precise gene composition of interest can be selected. this approach has recently been employed to generate live attenuated vaccines against rotavirus and influenza virus. the strategy for generation of the pentavalent bovine-human reassortment rotavirus vaccine is shown above. rotaviruses have a segmented double-stranded rna genome comprising independent rna elements. the outer shell of the virus comprises two proteins vp and vp that are involved in cell binding and entry and that specify the viral serotype (p type for vp and g type for vp ). vp and vp also represent the targets of virus-neutralizing antibodies. the pentavalent bovine-human rotavirus vaccine was generated by a 'modified jennerian' approach in which the bovine rotavirus wc (which is attenuated in humans as a result of host range restriction) serves as the gene donor for the backbone on to which gene segments encoding four common human rotavirus g types (g - ) as well as one very common p type (p ) (derived from individual rotavirus isolates) were reassorted via a process of cell co-infection and subsequent selection of the recombinant viruses with the desired composition of bovine and human gene segments. an analogous genetic reassortment approach has also been used to generate live attenuated influenza vaccines. in this instance, three attenuated 'cold-adapted' viral strains (two a types and one type b) are used in co-infections in tissue culture with recent circulating wild-type influenza strains to derive vaccine strains that include the two relevant hemagglutinin (ha) and neuraminidase (na)-encoding gene segments admixed with the six 'backbone' genes from the attenuated master donor virus for use in annual influenza vaccines. the first recombinant vaccine developed, the recombinant hepatitis b surface antigen (hbsag) prepared in yeast, was developed in hopes of avoiding safety concerns related to the plasma-derived hbsag vaccine. the knowledge that immune sera could provide protection by passive immunization of naïve hosts, and that purified inactivated plasmaderived hbsag vaccine could elicit protective antibodies, laid the groundwork for development of this recombinant vaccine. the recombinant vaccine, when combined with adjuvant (alum), elicits favorable immune responses, is highly efficacious and is well tolerated -all features that recombinant vaccines are now expected to deliver. the second recombinant vaccine developed targeted prevention of borrelia burgdorferi infection (the cause of lyme disease), and was based on a purified recombinant version of the ospa protein. this vaccine, although conferring some degree of efficacy, faced implementation challenges, and was not widely embraced. as a result, it was withdrawn from the market. more recently, recombinant technology-derived purified subunit vaccines have been developed that consist of virus-like particles (vlps) that self-assemble when the l protein of hpv is produced in isolation of other viral proteins ( fig. . ). the l protein is the target of virusneutralizing antibodies and vaccines consisting of a mixture of types and (the cause of ~ % of cases of cervical cancer) and and (the cause of ~ % of cases of genital warts) or of hpv types and alone have been shown to be highly efficacious and well tolerated. interestingly, hpv vlps induce antibody responses that exceed those that follow natural hpv infections. in light of these successes, and the power and versatility of recombinant antigen production methods, a major proportion of new vaccine development efforts involves the use of protein subunit vaccines produced by recombinant technologies. vaccines produced by this method are those that depend largely or exclusively on the induction of antibodies against individual or a selected subset of pathogen proteins. because a number of proteins produced in isolation by recombinant methods have been observed to elicit lower immune responses than do natural infections or live attenuated vaccines, the development and use of adjuvants to optimize recombinant vaccine immunogenicity represent an important parallel area for future exploration. n vaccine development and evaluation n as a necessary prelude to clinical evaluation of candidate vaccines in humans, extensive preclinical research and development activities are undertaken to establish that the vaccine candidate has the desired properties. toward this end, a number of key issues need to be addressed. first, animal studies must show that the vaccine candidate raises the desired type and magnitude of immune response against the infectious agent. second, the vaccine needs to protect animals against death or disease in an appropriate challenge model, when feasible. ideally, in the course of these studies, a specific type or level of immune response, referred to as a correlate of immune protection, can be identified. third, the vaccine should be relatively free of serious discernible toxicities and side effects in animals when administered by the route intended for humans. fourth, it is necessary to demonstrate that the vaccine can be produced in a consistent manner by a process that is consistent with the current good manufacturing practices (cgmp) process by which the first clinical trial materials will be produced (www.fda.gov/cber/gdlns/indcgmp.pdf ). bioengineered l proteins ( ) l pentamer self-assembled virus-like particle in specific instances, vlps can be produced via a process of self-assembly of individual viral capsid proteins produced by recombinant dna methods in cell culture systems. this approach has a number of attractive aspects, including the ability to produce vlps that accurately display conformationally correct epitopes recognized by neutralizing antibodies and the absence of pathogen-derived nucleic acids. in addition, recombinant vlps have been employed to derive safe and effective vaccines for pathogens, such as hepatitis b virus (hbv) and human papillomavirus (hpv), that cannot be grown in culture (and are thus refractory to standard vaccine approaches of attenuation or inactivation). the generation of the vlps that comprises newly developed hpv vaccines is shown. the hpv l proteins (which represent the major capsid protein and target of virus-neutralizing, protective antibodies), derived from hpv types of interest (e.g., types , , , and ) are produced via recombinant methods. under appropriate conditions, individual bioengineered l proteins first self-assemble into pentamers, and then into vlps that are comprised of pentamers and that are almost identical, both morphologically and antigenically, to infectious hpv virus particles. vlps prepared from individual hpv types are then combined with specific adjuvants to prepare the final vaccine products. even before preclinical studies are completed, vaccine developers typically begin an initial dialog with regulatory authorities (such as the food and drug administration (fda) or the european medicines agency (emea)) to set expectations about what will be necessary and sufficient for advancement to clinical studies in humans (www.fda.gov/cber/ genetherapy/isct sh.pdf ). phase i studies primarily focus on detailed assessment of the safety and tolerability of a vaccine, but evaluation of its immunogenicity is also frequently conducted. generally, a phase i study includes fewer than healthy volunteers divided unequally between those who receive vaccine or placebo ( or vaccinees per placebo recipient). phase i studies typically employ escalating doses of the candidate vaccine, with a dose range progressively increasing in steps of three-to fivefold often being used. blood samples are taken at prescribed intervals and analyzed for laboratory evidence of potential toxicity, as well as for evidence of vaccine-elicited immune responses. a phase i study is considered successful if it demonstrates that the candidate vaccine is well tolerated or identifies any immediate safety concerns that will need to be closely monitored in potential future clinical studies. ideally, phase i studies also provide an initial indication of the optimal dose level and number of doses required. a phase ii study typically includes several hundred to a few thousand volunteers (randomized between vaccine and placebo) and can assume two general design types. phase iia studies provide additional safety data on a larger number of individuals of the intended age who receive the intended vaccine dose (who are more representative of the general population intended for vaccine use than the very healthy individuals included in the phase i study), as well as provide additional data on vaccine immunogenicity. even larger phase iib studies can provide additional data on vaccine safety and immunogenicity in subjects generally representative of those for whom the vaccine might be recommended, but importantly, also provide the first opportunity to address to answer the question, 'does this vaccine work in humans?' the size of a phase iib study needed to detect a signal of vaccine efficacy depends on the attack rate of the infection being targeted by the vaccine. the development of new vaccines depends on the convergence of public health need, biological plausibility, and practical feasibility. vaccine development programs are influenced by multiple considerations, including: >> what are the major unmet medical and public health needs today? >> what is known about the natural history and pathogenic mechanisms of the infection of interest? >> is immunity to a given antigen associated with protection against disease following re-exposure in the context of natural infection? >> if natural immunity capable of preventing re-infection follows an initial infection with the pathogen, can a specific host immune effector mechanism (e.g., antibody, cd t-cell) be identified as the likely agent (or 'correlate') of immune protection? if so, can a threshold level of this specific immune correlate needed for protection from re-infection be defined? >> can the pathogen be grown in culture? if so, does the pathogen cause such a life-threatening disease that an attenuated version of the virus would face an impossible barrier for demonstration of safety? >> can a specific antigen (or antigens) be identified that represents the target of protective host immune responses? >> if the protective immune response is mediated by antibodies, can the target antigen (be it a protein or polysaccharide) be produced in scalable quantities in a form that mimics its native structure so that it can effectively elicit antibody responses that can block the key functional role(s) of the target molecule in the pathogen lifecycle or otherwise lead to the clearance of an incipient pathogen infection? >> having chosen an antigen and presentation system, what is the best way to produce it on a large scale? choices will be limited by the nature of the antigen and delivery system, but definition of an optimal system for producing the vaccine (prokaryotes like escherichia coli, or diverse eukaryotic hosts including yeast, insect cells, plants, or cultured plant cells, mammalian cells) is a central consideration. >> what is the most effective way to present the antigens of the pathogen of interest to the immune system? modern molecular biology and biochemistry have provided numerous options for vaccine immunogen presentation, including recombinant proteins (and recombinant virus-like particles (vlps)), synthetic proteins, protein-polysaccharide conjugates, and gene delivery systems (recombinant viral vectors, or dna vaccines) >> is the antigen of interest sufficiently immunogenic on its own, or is augmentation of the desired immune response by conjugation to a specific carrier or addition of an adjuvant necessary to elicit a sufficient and sufficiently durable immune response in individuals in the target population for vaccination? >> what types of potential safety concerns can be anticipated for the vaccine in question? >> what is the attack rate of the infection in the general population? if the infection occurs relatively rarely in an overall population, can a subset of the population be identified that has a higher risk of infection so as to accelerate the achievement of statistically significant protection? is this subset sufficiently similar to the rest of the population to enable extrapolation of the clinical results to the broader target population as a whole? >> what tests to evaluate vaccine immunogenicity will need to carried out on clinical samples obtained from participants in the clinical trials? will measurement of antibody titers, t-cell responses, pathogen presence and quantity, pathogen serotype, and any other parameter peculiar to the disease in question represent the primary criteria for vaccine effect? development and validation of theses tests represent an essential component for the feasibility and success of a vaccine clinical study phase ii studies also present the first opportunity to identify a potential laboratory immunological correlate of protection from disease -if nature and prior experience have not already done so. in order to do so, the placebo recipients in the phase ii trial must experience a sufficient number of cases of disease while vaccine recipients need to exhibit significant evidence of decreased risk of infection or disease. in addition, immunological measurements in the vaccinees need to capture the relevant protective immune responses (e.g., the type and level of antibody and/or cellular immune response that predict protection) and measure them with sufficient precision and reliability. if laboratory measurements of immunity correlate with vaccine protection, subsequent refinements of the vaccine, its adjuvant, its manufacturing process, or its regimen may be assessed by simple immunogenicity studies, rather than repeating efficacy studies. once efficacy is established for a vaccine, it is very difficult to carry out a double-blinded, placebo-controlled efficacy study. vaccines that have been shown to be immunogenic and well tolerated in phase ii studies can then advance to pivotal phase iii studies required for vaccine licensure by regulatory authorities. phase iii studies are intended to expand further the safety database in a larger number of individuals (who are representative of the specific populations for which the vaccine will ultimately be used), establish definitive evidence of protective efficacy, and to establish clinical consistency of the vaccine made by the process run in the facility intended for licensure and commercialization (www.fda.gov/cber/genetherapy/ isct jcr.htm). typically, phase iii studies include or more subjects in a blinded, placebo-controlled design. this size trial allows the identification of less frequent safety events. it also provides an opportunity to capture data on health care utilization, cost, and impact of the vaccine on these parameters. as a new vaccine will ultimately be included in a vaccine program where multiple vaccines may be administered at the same time, it is also necessary to conduct concomitantuse studies. the developer of the new vaccine must show that the new vaccine does not impact on the immunogenicity of the existing vaccines, and that the existing vaccines do not impact on the immunogenicity of the new vaccine. in contrast to drugs, where licensure by the fda is the primary determinant of how a new product is implemented in medical practice, vaccine use in the usa includes an additional process that evaluates how best to employ a new vaccine to optimize its implementation and public health impact. the us cdc has responsibility for making recommendations about the use of licensed vaccines, and it relies on its advisory committee on immunization practices (acip) for guidance. the acip considers several aspects in addition to a vaccine's safety and efficacy, including the anticipated costeffectiveness and practical feasibility of potential alternative vaccine deployment strategies and consideration of how a new vaccine may be successfully implemented in clinical practice to achieve the greatest public health impact. once the cdc has received, reviewed, and accepted the recommendation of the acip, the recommendation is published in its final official form in morbidity and mortality weekly report (mmwr; www.cdc.gov/nip/publications/acip-list.htm). the recommended immunization schedule for children and adolescents ( fig. . ) is updated on an annual basis and can be accessed at www.cdc. gov/nip/recs/child-schedule.htm. the recommended adult immunization schedule (fig. . ) is also updated on an annual basis and can be accessed at www.cdc.gov/nip/recs/adult-schedule.htm. the recommended adult immunization schedule includes information concerning use in special populations (such as health care workers and pregnant women) and individuals with specific conditions associated with altered or impaired immune function (such as individuals with congenital and acquired immunodeficiency syndromes, recipients of immunosuppressive therapies, malignancies, asplenia, liver disease, and renal disease). readers are encouraged to check to ensure that they are following current recommendations. pregnancy registries currently exist for four vaccines in the usa. health care professionals are encouraged to report exposures of pregnant women to the appropriate registry: hbv vaccine ( - - ), hpv vaccine ( - - ), meningococcal vaccine ( - - ), and varicella vaccine ( - - ). n vaccine safety n unlike drugs that are utilized to treat individuals suffering from a given disease state, vaccines are administered to normal, healthy infants, adolescents, and adults. consequently, standards for the safety and tolerability of vaccines are set at a very high level. when developing a new vaccine, a graded process of clinical studies is employed that involves increasingly larger numbers of volunteers and that typically progresses from individuals who are selected to be free of any identifiable health problems to those who are selected to be representative of the overall population for whom the vaccine is being developed. if phase i studies reveal no evidence of safety concerns and the desired evidence of immunogenicity, a major focus of the series of larger randomized double-blind, phase ii placebo-controlled studies that are then conducted is to explore the safety and tolerability of a vaccine in increasingly vulnerable populations (such as those who may have identified pre-existing health problems or asymptomatic abnormalities detected on screening laboratory studies). reflecting the importance of documenting the safety of a new vaccine, phase iii studies to assess the safety and efficacy of a new vaccine now typically involve large numbers of volunteers. indeed, as a result of needing to provide evidence for safety, it is now common to have the size of the phase iii trial be significantly larger than would be necessary to document vaccine efficacy. the ability of a study to identify an increased risk of any given adverse event with sufficient statistical power is directly related to the size of the population in the study. as a general rule, a study of - subjects is needed to measure the risk of an event that happens in one out of individuals. for one in , - subjects are needed. even in studies of this size, very rare events may not be identified, and if a specific safety concern exists substantially larger trials may be needed. the recent experience with the development of rotavirus vaccines provides an illustrative example of the importance placed on documenting vaccine safety. rotavirus is an important cause of serious gastroenteritis in infants and young children, and the associated diarrhea and vomiting can lead to life-threatening dehydration. in developing countries where health care resources and effective rehydration options are limited, over infants die of rotavirus gastroenteritis each year. given the global importance of rotavirus gastroenteritis, the first licensure of an orally administered rotavirus vaccine in was a very welcome advance. however, as the vaccine entered routine pediatric practice, it was recognized that a low, but increased incidence of intestinal intussusception was seen after the first and second doses (with about one case of intussusception seen per vaccinees.) upon recognition of this association, the vaccine was withdrawn from the market. with the evident public health need for a safe and effective rotavirus vaccine, it was hoped that alternative rotavirus vaccines then in development (both oral vaccines based either on a combination of bovine-human reassortant viruses (fig. . ) or an attenuated human rotavirus strain) might differ from the first licensed rotavirus vaccine and not result in an increased rate of intussusception. however, to demonstrate that these alternative rotavirus vaccines were safe, and that an increased risk of intussusception was not inherent to rotavirus vaccines as a class, very large-scale safety studies were required. toward this end, the safety of each of these vaccines was evaluated in studies involving about infants -just to evaluate whether the rate of intussusception in vaccinees was discernibly increased compared to the normal background rates seen in the placebo recipients. , fortunately, both vaccines were found to be well tolerated and no increase in intussusception was observed in vaccine as compared to placebo recipients. in light of the documented efficacy of these vaccines determined in earlier and significantly smaller phase iii trials, both have now been licensed in a number of countries. however, even with the large phase iii studies conducted for these newer rotavirus vaccines, they will still be studied in large postlicensure active surveillance safety studies and closely monitored in active and passive vaccine safety surveillance systems (see below). following vaccine licensure, safety is tracked via a number of means, including both active and passive surveillance studies of adverse events. active surveillance includes phase iv postmarketing studies of vaccine safety in larger populations in real-world use. formal postmarketing studies can include tens of thousands of individuals or more. an alternative type of postmarketing safety study is carried out by the us fda and the cdc within the context of the vaccine adverse event reporting system (vaers) database (www.vaers.hhs.gov or by telephone: - - ). the vaers database accepts spontaneous reports of adverse experiences from health care providers, patients, parents, vaccine manufacturers, and other sources. the best use of the vaers database is to identify signals in a population that may appear following the introduction of a new vaccine. a newer vaccine safety surveillance system, known as the vaccine safety database (vsd), has been developed by the cdc in cooperation with seven large health maintenance organizations (hmos) around the usa. the vsd contains the complete medical records of all the members from the participating hmos, and the information used to populate the database is entered by health care professionals using relatively consistent terminology, improving the quality, uniformity, and usefulness of the data. particularly important is that the vsd construct allows comprehensive epidemiological analyses to determine if the incidence rate of a specific adverse event is higher among vaccinees than nonvaccinees. in addition to vaers and the vsd, the cdc has also created a clinical immunization safety assessment network that reviews patterns of clinical syndromes that may follow vaccination. while the safety profile of a vaccine can be relatively well defined through the efforts described above, confidence in vaccination programs has often been challenged by public perceptions, either real or unsubstantiated, about vaccine safety. in some instances, specific vaccines have been associated with increased incidence of a specific adverse experience, such as the association between the first-generation rotavirus vaccine and an increased risk of intussusception following vaccination. however, a number of other safety concerns that have emerged are not supported by scientific evidence. an example of this can be found in the case of concerns about the association of whole-cell pertussis vaccines with permanent brain damage -concerns that were later shown to be unfounded. nevertheless, public concerns about the safety of the whole-cell pertussis vaccine resulted in decreased levels of pertussis vaccination coverage that were soon followed by epidemics of whooping cough in the uk and japan. another example is the allegation that certain vaccines, such as the combination measles, mumps, rubella (mmr) vaccine, are associated with autism. highlighting how perceptions of temporal association can give rise to public concerns, mmr vaccines are generally given around year of age, and autism is generally diagnosed in the second year of life. although the alleged causal association between mmr and autism has been refuted by thorough scientific analyses, reports in the popular media in the uk resulted in a dramatic drop in vaccination rates, followed by an increased rate of new infections. , n vaccines not yet available n although an impressive armamentarium of vaccines is now available, safe and effective vaccines have yet to be developed for a number of very important infectious diseases. the reasons underlying the lack of effective vaccines for an array of important pathogens include biological considerations, safety concerns, and practical constraints. of these, the biological considerations are often the most important barrier. as discussed above, vaccines have been successfully developed for pathogens whose natural infections give rise to natural immunity wherein the infected host (at least those who survive initial infection) is no longer susceptible to re-infection (such as measles, yellow fever virus, or smallpox) or who experiences significantly less severe clinical sequelae upon re-infection (such as rotavirus). in instances where natural immunity follows natural infection, not only is a precedent for immune protection established, but the nature of protective host responses can be studied, providing a correlate of protection to guide vaccine development efforts. however, for many of the pathogens for which vaccines remain elusive, natural immunity does not follow natural infection. in the absence of natural immunity, not only is a precedent for successful immune containment lacking, but no potential correlates of protection are available to inform vaccine development. in some instances where natural immunity does not follow natural infection, persistent infections are established and maintained by active virus replication that cannot be controlled or cleared by host immune responses (such as hiv and hepatitis c). alternatively, other pathogens are able to persist in the host through establishment, via diverse mechanisms, of latent infections that are resistant to host immune clearance (such as tuberculosis or herpes viruses (such as herpes simplex virus (hsv) or epstein-barr virus (ebv))). in other instances, even when the host is cleared of an infection via drug vaccines not yet available treatment, the host remains susceptible to re-infection and disease in the future (such as malaria). although different pathogens have evolved diverse strategies for evasion of host immune responses -ranging from manifestation of tremendous genetic diversity and propensity for immune escape; to sequestration of critical structural domains that might be susceptible to antibody neutralization; to the utilization of specific mechanisms to evade host innate and adaptive immune effectors -the common end result is frustration of vaccine development. while failure of host clearance of an infection is a common theme underlying the lack of vaccines, additional obstacles to vaccine development include other immunologically related considerations as well as both practical and safety considerations. examples of immunologically related obstacles include instances where prior exposure to a given pathogen predisposes the host to more severe disease manifestations upon re-infection (as has been proposed in the case of dengue virus) or where earlier vaccine development efforts inadvertently lead to severe adverse events following infection with the targeted pathogen (such as respiratory syncytial virus (rsv )). in each of these cases, the adverse events that follow a secondary immune exposure are believed to be the result of immunopathologic responses that result from the nature of the immune response elicited by the initial exposure to pathogen-derived antigens (by either infection or vaccination). given that the mechanisms underlying these immunopathologic processes are incompletely understood, the development of vaccines that are highly immunogenic but not similarly inclined to elicit immunemediated adverse consequences represents a substantial challenge (especially given the very high expectations for vaccine safety). an additional immunologically related challenge relates to the observation that certain organisms encode antigens that resemble constituents of the human host. for example, in the case of neisseria meningitidis group b, the bacterial polysaccharide resembles those found on certain human cell lineages, thus raising concerns about whether polysaccharide-based vaccines successfully developed for group b n. meningitidis might yield undesirable autoimmune responses. an additional distinct, but important, practical barrier to new vaccine development relates to the prevention of diseases that are threats to pregnant women or their offspring (where immunization of the pregnant woman might be able to protect the neonate). although a number of inactivated vaccines are either routinely recommended for use in pregnant women (e.g., inactivated influenza vaccine) or can be used in pregnant women for pre-or postexposure prophylaxis for those at risk of infection (e.g., inactivated hepatitis a vaccine and recombinant hbsag vaccine), the development of new vaccines specifically for use in pregnant women or the study of new vaccines in pregnant women has been impeded by concerns arising from potential litigation that might follow the appearance of a congenital abnormality in a child born to a mother who was vaccinated while pregnant. given the - % prevalence of congenital abnormalities, the practical difficulties in proving the safety of a new vaccine specifically administered to pregnant women, and the current litigious environment surrounding vaccines, the development of new vaccines to address important infections of pregnant women and their neonates (e.g., group b streptococcus: gbs) faces significant challenges. there remain a number of important infectious diseases for which no effective preventive vaccines exist. below, we list the major 'missing' vaccines, comment on why they are not yet available, and highlight the major approaches currently being explored to develop them. at the end of , an estimated million people were living with hiv infection, and in the preceding year approximately . million people became newly infected, and approximately million individuals died of aids. as the most promising biomedical intervention to contain the aids pandemic, the development of an hiv vaccine is a top global health priority. yet, hiv infection represents a vexing challenge to vaccine development. , hiv infection does not result in clearance of the virus due to a host immune response. following infection of target cells, the genome of hiv -a retrovirus -is transcribed into a dna copy via the action of reverse transcriptase. the newly formed dna copy of the hiv genome then integrates into the host cell chromosomes (referred to as a provirus) as a requisite step in the viral lifecycle. once integrated into the chromosome of an infected cell, the hiv provirus can alternatively be actively transcribed, leading to the synthesis of viral mrnas and subsequently to production of new virus particles, or it can remain in a transcriptionally silent, functionally latent state in a small percentage of infected cells. as infected cells harboring latent hiv proviruses do not produce hiv protein antigens, they cannot be recognized by host antiviral immune responses and can thereby persist undetected. upon subsequent activation of latently infected cells at some later time, viral rna transcription can be coincidently activated leading to production of progeny virions. as hiv targets activated cd t cells for infection and consequent depletion, the host's ability to mount both hiv-specific and non-hivspecific immune responses is progressively impaired. the ability of the host to clear hiv infection is further complicated by the extensive genetic diversity of virus populations that emerge, and progressively diverge, within infected individuals as a function of a replicative cycle that is accomplished by the inherently error-prone reverse transcriptase and the numerous cycles of replication that occur in infected individuals. as a result of these influences, genetically diverse populations of hiv variants are established in infected persons that facilitate the outgrowth of genetic variants that can escape from selective pressures -be they effective host cellular or humoral immune responses, or the inhibitory effects of antiretroviral drugs. an extraordinary degree of genetic diversity is also manifest in the hiv variants seen in different individuals and in different geographic regions. as successful vaccines for other infectious agents have historically had to protect against pathogens exhibiting only limited genetic diversity, hiv represents an unprecedented challenge. as many successful vaccines protecting against viral infections are predicated on the induction of neutralizing antibody responses against the viral surface proteins that mediate attachment to and entry into target cells, significant efforts have focused on the potential of the hiv surface envelope (env) glycoprotein, gp , to elicit infectionneutralizing antibodies. unfortunately, hiv gp is highly resistant to the action of antibodies by virtue of its heavy glycosylation and its native conformation that shields functionally critical structural domains from antibody binding. as a result, candidate gp -based vaccines have failed to elicit meaningful levels of neutralizing antibodies in immunized human volunteers and have not protected from hiv infection in two large phase iii studies. given the inability, to date, of candidate hiv env-based vaccines to elicit appreciable levels of neutralizing antibodies, current vaccine strategies are largely focused on the induction of cd cytotoxic t-cell responses against the more constrained and conserved antigens, such as vaccines not yet available gag, pol, and nef. it has been hypothesized that induction of high levels of hiv-specific cd t-cell responses prior to infection may not prevent infection, but may enable infected individuals to control virus replication better. should this hypothesis be valid, individuals immunized with such vaccines may exhibit lower levels of ongoing hiv replication, progress to aids more slowly, and potentially be less likely to transmit hiv infection to others. much of this work involves vectored gene delivery systems (such as adenoviral vectors, described below). however, the recently announced results of a phase iib 'test of concept study' failed to demonstrate a beneficial effect on either prevention of infection or reduction of viral load among volunteers who received the vaccine despite the induction of appreciable levels of hiv-specific ctl responses by the recombinant adenovirus-based vaccine employed. while this study result does not, in and of itself, refute the 'ctl hypothesis', it represents a significant disappointment for the aids vaccine research effort, and raises important questions about the ability of vaccine-elicited cell mediated immune responses to favorably alter the outcome of hiv infection. a there are also efforts under way to utilize the recently solved three-dimensional structure of the hiv env glycoprotein to guide the derivation of nonnative structures that might serve as better immunogens to elicit broadly cross-reactive neutralizing antibodies. in addition, relatively conserved and functionally essential sequences of the extracellular domain of the hiv transmembrane env protein, gp , are being explored as immunogens to elicit broadly neutralizing antibodies. malaria is the world's most common vector-borne disease -estimated to cause approximately million clinical cases and million deaths annually. , the disease hits hardest in africa, and is especially severe in children under years of age. in addition to direct morbidity and mortality, malaria is responsible for debilitating illness with enormous social and economic consequences. of the four malaria-associated protozoal species, plasmodium flaciparum and p. vivax represent the two major agents. these parasites have a three-stage lifecycle taking place both within the mosquito, and in the liver and blood of the infected host, and each cycle is largely distinct from the others from an immunological perspective. as a result of the multiple strategies for evasion of host immune response that the parasite has evolved, parasite replication proceeds at high levels despite active host immune responses. , either as a result of these specific immune evasion strategies or the inability of the infected human host to mount immune responses that clear the parasite, prior infection does not protect an individual from repeated subsequent infections. although the severity of disease is often attenuated following repeated infection, the mechanism of disease modulation is incompletely understood, and the limited relative immunity engendered by prior infection is easily lost if an individual leaves a malaria-endemic region. as such, the limited impact and duration of host immune responses to malaria parasites suggest that any successful vaccine strategy will need to do far better than natural immune responses -a high bar for efforts to develop an effective vaccine. roughly two dozen antigens have been cloned and tested as potential vaccine immunogens, and with a few exceptions the results have been disappointing. one antigen, the circumsporozoite antigen, presented as a fusion with hbsag (rts,s), has shown modest promise in human studies. this vaccine is now undergoing larger-scale clinical efficacy testing to determine if the magnitude of protection would justify largescale implementation efforts. should 'proof of concept' be supported in these studies, but the absolute magnitude of efficacy be insufficient, future efforts will likely focus on the identification of an appropriate adjuvant to improve the magnitude and duration of immune responses. alternative approaches include immunization with irradiated or genetically attenuated sporozoites. definition of novel antigens expressed at specific stages of the parasite lifecycle, and evaluation of combinations of multiple parasite antigens. mycobacterium tuberculosis is an intracellular mycobacterial pathogen that represents one of the world's most common and most serious infectious diseases. over billion people are believed to harbor latent m. tuberculosis infections, and approximately million active cases of tuberculosis and over million deaths occur each year. furthermore, the interface of hiv infection and its attendant immune system damage both increases the severity of m. tuberculosis infection and increases the infectiousness of infected individuals. the emergence and dissemination of m. tuberculosis isolates that are resistant to multiple antimicrobial drugs represent a growing public health threat. however, while the need for a vaccine to prevent tuberculosis is clear, a significant number of challenges face vaccine development efforts. most individuals infected with m. tuberculosis can control the acute phase of mycobacterial replication, and mount vigorous innate and adaptive immune responses to the infection. however, the infection is often not cleared by the host's immune response, and the mycobacteria are able to persist and multiply within vacuoles inside macrophages. longterm latency is established in fibrotic cysts in the lung. the recrudescence and dissemination of m. tuberculosis occur at a later time in a number of infected individuals, likely as a result of waning host immune control. although the ability of m. tuberculosis to persist despite active innate and adaptive immune responses represents a major challenge to vaccine development, the fact that most individuals can contain (if not clear) m. tuberculosis infection suggests that a vaccine that can alter the course of the natural infection by limiting early dissemination and decreasing the risk of later recrudescence could provide major public health benefits. efforts to develop a vaccine against tuberculosis date back many decades. bacille calmette-guérin (better known as bcg), based on mycobacterium bovis, was first introduced in . currently, bcg is provided as a component of the routine expanded programme for immunization (epi) schedule and is administered to a significant majority of the world's children. although some protective efficacy ( - %) has been reported against miliary infection and m. tuberculosis meningitis in children, conflicting results have been obtained in different studies regarding the ability of bcg to protect against pulmonary tuberculosis in adults. one explanation for the overall limited efficacy of bcg emerges from formal genome sequencing studies that have disclosed significant differences between m. tuberculosis and of the vaccine strain of bcg. the variability in the results of bcg efficacy studies in different populations and geographies may derive from variations in the geographic prevalence of cross-reactive mycobacterial species (that may themselves confer partial protection), or the fact that bcg vaccines used throughout the world do not represent a homogenous preparation -with the root strain of bcg having been widely distributed and passaged extensively under diverse conditions. vaccine efforts against tuberculosis have primarily focused on the evaluation of specific mycobacterial antigens (e.g., esat , ag , and hsp ) that have been tested as vaccines in animal models with variable success. , some of these strategies are now being advanced into human clinical trials. an alternative strategy is based on improving the performance of the bcg vaccine by insertion of genes encoding specific potential protective antigens that it normally lacks. in addition, the development of auxotrophic mutants of m. tuberculosis is being explored as a potential immunogenic and specifically attenuated live vaccine. the determination of the sequence of the m. tuberculosis genome nearly a decade ago helped identify numerous previously unknown gene products, and increased the repertoire of antigens to be evaluated for their ability to induce protective immune responses. the pathogen sequence is also being used to elucidate virulence determinants and thereby help guide efforts to attenuate m. tuberculosis rationally. together with influenza virus, rsv and piv account for a substantial majority of pediatric upper respiratory illness and consequent acute otitis media. a variety of influenza vaccines are licensed for pediatric use, but vaccines to prevent infection with the paromyxoviruses rsv and piv remain elusive. a significant impediment to vaccine development for rsv and piv traces back to unanticipated untoward results obtained in clinical studies of inactivated rsv vaccines in the early s. these early-generation rsv vaccines -based on cultured virus that had been inactivated with formalin -raised a potent antibody response in immunized children. however, on subsequent natural exposure to rsv, vaccine recipients exhibited more frequent and significantly more severe lower respiratory tract rsv infections than did unimmunized children. as a similar phenomenon was also seen with a formalin-inactivated measles vaccine in the same era, a common immunopathologic mechanism may be operative. while the mechanism of exacerbation of rsv disease by the early inactivated vaccines is incompletely understood, it has been suggested that chemical inactivation of rsv and measles resulted in modification of a critical neutralizing structure on the surfaces of these viruses, thereby limiting the induction of the most potent neutralizing antibodies and favoring nonneutralizing and potentially immunopathologic antibody responses. (passive protection against rsv is available for premature infants in the form of monoclonal antibodies that target the rsv f protein (one of the viral envelope glycoproteins); certain anti-rsv antibody responses can clearly mediate protective as opposed to deleterious effects. ) alternatively, or in addition, it has been proposed that inactivated rsv vaccines may have preferentially induced a th -type immune response when a th -type response may be needed to effect protection of the lower respiratory tract from rsv infection and damage. while excellent live attenuated measles vaccines have been developed, rsv and piv have so far resisted the approach used for measles and mumps (these are all members of the paramyxoviridae family of viruses). based on the successful precedent provided by the live attenuated measles vaccine, an attenuated or reverse genetics-engineered rsv is considered the most promising approach. however, stable attenuation of rsv has been difficult to achieve and vaccine safety concerns result in their cautious advancement through clinical evaluation. effective vaccines for meningococcus types a, c, y, and w are available as straight capsular polysaccharides and as conjugated polysaccharides. the group b polysaccharide shares chemical similarity with a shorter sugar found on the surface of neuronal tissue. while it is possible to make highly immunogenic conjugates with the group b polysaccharide, theoretical concerns about cross-reactivity with self antigens has impeded the development of this type of vaccine. current work centers on a handful of relatively well-conserved surface proteins of meningococcus. gbs is a common component of the flora of the female genital tract, and transfer to the neonate is the cause of severe infections that are fatal or have serious sequelae. short-course intrapartum antibiotics are recommended for culture-positive women, and this approach has cut the incidence of neonatal infections by about two-thirds, thus reducing somewhat the urgency of vaccine development. however, short-course antibiotics could ultimately drive the emergence of antibiotic-resistant gbs. candidate vaccines have been shown to elicit a protective response. however, aside from a reduced market, the main impediment to development of a gbs vaccine is concern over vaccination of pregnant women or women of childbearing age. any birth defect might be attributed to the vaccine, and in a litiginous society, this would be problematic for a vaccine producer. prior to the advent of effective polymerase chain reaction methods for screening blood donations, hcv was a significant cause of transfusionrelated hepatitis. currently, transmission of hcv among the normal population is quite low; transmission among injection drug users remains high. hcv is another pathogen where infection does not typically result in an immune response that clears the infection. however, a minority of hcv patients do spontaneously clear their infection, suggesting that an appropriate immune response could do the job. current vaccine work is concentrated on vectored gene delivery vaccines, primarily adenoviruses, intended to raise antiviral cytotoxic t-cell responses. with the exception of the live attenuated varicella-zoster virus (vzv) vaccine used for the primary prevention of chickenpox and reactivation of latent vzv infections (the cause of shingles and postherpetic neuralgia in older individuals), there are no other vaccines available for use in humans to prevent infection with members of the herpes virus family. hsv types and cause recurrent vesicular eruptions "above or below the belt," respectively. like other herpes viruses, hsv infections are not cleared by the immune system and the virus can persist, remaining in a latent state that is functionally inaccessible to immune recognition and clearance. in addition, like other herpes viruses, hsv encodes a number of gene products that promote evasion of host immune responses. recent attempts to make hsv vaccines have used virus glycoproteins produced by recombinant dna methods. a recent clinical efficacy trial of this vaccine approach showed partial protection of women, but not men, who were seronegative for hsv . the reasons for this curious result are not clear, but efforts to develop this type of vaccine continue. in addition, a number of preclinical studies are exploring the ability of cell-mediated immune responses to hsv antigens induced by recombinant vaccine vectors (e.g., adenoviruses: see novel vaccine vectors, below) to prevent or ameliorate hsv infections. genetically engineered attenuated hsv variants have also been studied in experimental animal models. it is not clear when these new strategies may advance to clinical evaluation in humans. another herpes virus, cmv is a very common infection in humans, with - % of individuals being infected by adulthood. cmv is a cause of severe infections in neonates, causing debilitating neurological sequelae. following initial infection, cmv persists in infected humans, despite the fact that anti-cmv antibodies are present and that a very sizeable proportion of the overall host cd and cd immune responses are specific for cmv antigens. ongoing virus persistence and replication in the face of active host immune responses are likely explained by cmv's sophisticated repertoire of host immune evasion functions (including those that inhibit antigen presentation mechanisms and immune effector responses). for these reasons, to be successful, vaccine development efforts will need to elicit immune responses that are significantly more effective than the quantitatively impressive, but functionally limited, immune responses that are generated in the course of natural cmv infections. live attenuated vaccines have been investigated sporadically since the s. an attenuated strain, the towne strain, showed some effect, but was judged to be insufficiently immunogenic. hybrids of the attenuated towne strain and the virulent toledo strain remain in development. recent work has included recombinant dna (rdna)-derived proteins (via either dna vaccine approaches or recombinant viral vectors, such as attenuated poxviral vectors). , ebv is a herpes virus that represents the causative agent of infectious mononucleosis and is widespread among the human population. in concert with incompletely understood environmental (and perhaps additional host) factors, ebv is also etiologically associated with burkitt's lymphoma. the ability of ebv to establish persistent infections in humans (along with latent infections at the cellular level) despite readily detectable antiviral immune responses suggests that, like other herpes viruses, the development of effective ebv vaccine will likely be challenging. ebv vaccines have been in development since the s with the coat protein, gp / , as the most common vaccine antigen studied. dengue fever virus is a mosquito-borne flavivirus (the virus family that includes japanese encephalitis virus and yellow fever virus -for which successful vaccines exist). dengue virus is endemic in a substantial portion of tropical and subtropical areas and causes febrile disease as well as hemorrhagic fever. there are four distinct serotypes of dengue fever virus. prior infection with one serotype has been implicated in predisposing for more severe disease following infection with a second dengue fever virus serotype, although the evidence supporting this concept has been questioned and the underlying pathogenic mechanisms are incompletely understood. the processes of vaccine development have changed significantly in recent years -a process facilitated by substantial improvements in understanding of human immune system function, as well as the advent of powerful new technologies for vaccine development. as a result of these advances, vaccine development is now commonly pursued in a hypothesis-driven manner and is a far less empiric pursuit than in the past. however, at the same time, the infectious diseases for which no effective vaccines currently exist represent more challenging targets than those diseases that have yielded to vaccine development efforts in the past. furthermore, global changes that influence the emergence and rate of spread of infectious diseases place unprecedented challenges on the productivity and pace of new vaccine development efforts. vaccines not yet available >> the challenge of responding rapidly and effectively, with powerful new technologies, to newly emerging infectionsincluding those that haven't been seen in humans before (e.g., severe acute respiratory syndrome (sars)) or for which novel antigenic variants are anticipated but cannot be predicted (e.g., pandemic influenza) >> maximizing the value of innovative new approaches while ensuring the safety of new vaccines so derived one hypothesis proposes that antibodies against the initial infecting serotype bind to the surface of virus particles of the novel infecting serotype, but do not neutralize the infection. in a process referred to as "immune enhancement" of infection, still infectious complexes of antibody virus particles are then envisioned to be preferentially taken up by cells of the reticuloendothelial system that represent primary target cells for virus replication. although the veracity of this hypothesis in not established, it does present certain theoretical concerns about what type of antibody responses will need to be induced by vaccines to exert beneficial rather than detrimental effects. the general belief is that a vaccine providing equivalent immunity against all four serotypes will be required. vaccines based on inactivated virus, engineered chimeric viruses based on the yellow fever virus vaccine platform, engineered deletion mutant viruses, and rdna-derived proteins are in various stages of development. n new antigen discovery methods n historically, vaccine antigens were not discovered in the literal sense. rather, whole organisms were inactivated by either heat or chemistry or organisms were attenuated by forcing growth in nonphysiological conditions. the entire antigenic repertoire of the organism was delivered to the immune system. the isolation of tetanus, diphtheria, and pertussis toxins, along with chemical detoxification schemes, allowed the production of more refined vaccines. the isolation and purification of polysaccharide capsules from a range of important bacterial pathogens enabled the development of additional vaccines. with the advent of molecular biology in the late s, a new set of tools allowed a more directed approach for the discovery of pathogen virulence factors, vaccine antigen discovery, and vaccine development. the tools of molecular biology enabled for the first time the development of vaccines against pathogens that could not be propagated in culture, including the successful development of recombinant hbv and hpv vaccines. development of these vaccines was enabled by clinical and animal model studies showing that antibodies directed against a specific viral target antigen (e.g., the hbv surface antigen or the hpv l protein) were implicated in protection. in addition, molecular biologic approaches enabled the derivation of fully recombinant vaccine antigens (such as those developed using a limited set of defined antigens of bordetella pertussis), including genetically modified versions of bacterial toxins that maintain their proper antigenic structures but are no longer toxic. however, for many of the pathogens for which vaccines do not currently exist, application of these recombinant dna technology-enabled strategies are insufficient due to incomplete understanding of the pathogen antigens that would elicit a protective host immune response. as such, the development of additional techniques to discover protective antigens was needed. fortunately, several important technological advances that facilitate discovery of previously unknown protective antigens from even very complex microorganisms have opened a new era in vaccine development. the earliest rdna technology-enabled methods of antigen discovery involved the expression of individual pathogen-derived gene products (or fragments thereof ) in bacterial hosts (typically escherichia coli) using rdna expression vectors. here, the genome of a pathogen is broken up, and the fragments are inserted into a plasmid or a viral vector, typically a lambda bacteriophage. colonies, or plaques, are spread on a membrane, allowed to grow, and hopefully express the cloned gene fragments. the ability of these recombinant gene products (now isolated in individual colonies) to react with antibodies present in the serum of individuals who had recovered from infection with that pathogen could then be directly assessed. antibodies present in the immune sera are assessed for their ability to identify antigens by immunochemical reactivity. in this way, the entire genome of a given pathogen could be scanned for potential immunoreactivity. such reactivity would both indicate the in vivo expression of that gene product, as well as document its antigenicity. however, additional studies are needed to demonstrate whether antibody responses against a newly defined antigen have any protective potential. to document the ability of an antigen to elicit protective immune responses, it is necessary to immunize an experimental animal (most commonly, mice) and then, following experimental pathogen challenge, evaluate infection outcomes in immunized versus nonimmunized animals. as this approach has most often been used to identify antigens recognized by host humoral responses, sera from animals immunized with a candidate antigen can then be transferred to a naïve host to provide evidence that the antibody response to the antigen represents the relevant agent of immune protection. more recently, as dna sequencing became more efficient and scaleable, determining the entire sequence of the genomes of viruses, bacteria, and parasites has become routine, , allowing identification of previously unknown genes (and predicted gene products) that can be evaluated as vaccine immunogens. scanning the entire pathogen genome via specific computer analysis programs, genes that exhibit specific characteristics can be identified (e.g., predicted expression on the cell surface by virtue of possession of a leader sequence for secretion or membrane anchor sequences). in addition, the relative conservation of the gene within the pathogen population can be determined by assessment of gene sequences from multiple distinct isolates. once potential vaccine antigens are identified, each candidate gene is expressed in an appropriate rdna system, and the protein product is tested in an animal model. the first bacterial genome sequenced in its entirety was that of haemophilus influenzae, marking the beginning of a new approach to vaccine antigen discovery. since this initial bacterial genome sequence determination, genomic sequencing of pathogens has advanced exponentially. over bacterial genomes have now been sequenced, and hundreds more are currently in process. genome-based antigen discovery is being applied to a wide range of bacteria, including streptococci, pneumococci, staphylococci and chlamydia, as well as nonbacterial pathogens such as plasmodium falciparum. an alternate, promising approach to novel antigen discovery has been built on technological advances in proteomics. , these advances include development of high-resolution two-dimensional gel electrophoresis techniques and mass spectrometry methods that enable separation, identification, and purification of individual proteins from the complex mixture of proteins expressed by a pathogen. in proteomic analyses, a small culture of bacteria, preferably taken directly from an infected person, or otherwise grown in physiologically similar conditions, is subjected to physical or enzymatic treatment with specific proteases to generate peptide fragments that are then fractionated by a micro-high-performance liquid chromatography method and sequenced by molecular mass-by-mass spectrometry. an overlapping set of peptides of approximately - amino acids is sufficient to identify an antigen and provides the means to find the gene. although proteomic analysis is, in some ways, more involved than genomic analysis, it provides an important new approach to antigen identification, and offers a direct way to document that the specific protein identified is actually expressed by the pathogen (including, for example, demonstration that a protein of interest is expressed on the external surface of the pathogen). , a combination of proteomic and serologic methods to select potential novel vaccine immunogens, called serological proteome analysis, or serpa, , can be used to screen the pathogen proteome for expressed proteins that are recognized by antibodies present in sera obtained from individuals who have recovered from an infection with the pathogen. the proteomic approach to antigen discovery has been applied to identify novel vaccine candidates for a number of human pathogens, including helicobacter pylori, chlamydia pneumoniae, staphylococcus aureus, bacillus anthracis, haemophilus influenzae, and plasmodium falciparum. as in new genomic methods for antigen discovery, having identified a gene encoding a candidate antigen by proteomic methods, it is then necessary to show that an immune response of the desired type can be raised against the protein. in addition, it is necessary to show that immune responses elicited following immunization with the candidate antigen engender some degree of protection. often, pure protein antigens produced by recombinant methods are not very immunogenic. as such, many of the emerging recombinant vaccines so produced will likely require enhancement of their immunogenicity by means of an adjuvant. the term 'adjuvant' (derived from the latin adjuvare, to help) refers to any substance added as a component of a vaccine preparation -in addition to the vaccine antigens themselves -that improves the immunological response to the antigen. as such, 'adjuvant' is a catch-all term including a broad range of molecular entities that act via diverse -and, in a number of instances, yet to be elucidated -pathways. until recently, most adjuvants were derived empirically and the mechanisms by which they augmented immune responses were unknown . as a result, there were few, if any, principles available to guide the improvement of known adjuvants or the development of new ones. however, recent advances in understanding of the mechanisms by which dendritic cells sense the presence of pathogens and their constituents, and translate this information to shape the quantity, quality, and durability of host cellular and humoral adaptive immune responses, have transformed adjuvant discovery and optimization. what was once a process of trial and error now represents an area of hypothesis-driven research and mechanism-based discovery. a particularly promising advance emerged from the discovery that pathogen sensing by the innate immune system is mediated by recognition of specific pathogen-associated molecular patterns (pamps) by pathogen recognition receptors (prrs) such as the toll-like receptors (tlrs) that are expressed on dendritic cells (dcs) and other hematolymphoid and some epithelial cells (chapter ). the pathogen-derived pamps recognized by tlrs consist of structures that are found only in or on pathogens (including bacteria, viruses, and parasites) and are not part of normal vertebrate biology. following binding of a specific pamp to a specific prr, a specific cellular activation and response cascade is triggered that can directly confront an intruding pathogen and/or lead to the activation of specific host adaptive immune response mechanisms. these breakthroughs in basic immunology have been readily translated into what can now be considered the science of adjuvant biology. , such progress has occurred at an especially opportune time as new vaccine development strategies have transitioned from traditional approaches using attenuated or killed pathogens to highly defined and purified recombinant proteins (so-called "subunit" vaccines) or nonreplicating vectored antigens. although these newer approaches are promis-ing from the perspective of vaccine safety and the opportunity they afford to design the structures of vaccine immunogens, recombinant or synthetic vaccines are often inherently less immunogenic than traditional vaccines based on attenuated live viruses or intact killed organisms. in the context of current vaccine development and regulatory approval processes, an adjuvant is developed as part of a vaccine, not as an independent product. consequently, there are currently no adjuvants licensed by regulatory authorities as stand-alone products. most contemporary efforts to develop novel adjuvants are focused on the targeted activation of tlrs that are expressed on specific cells critical for the generation of innate and adaptive host responses to specific pathogens. a family consisting of distinct tlrs has been identified to date in humans (chapter ). tlrs are expressed in a number of innate immune cells, including dcs, macrophages, neutrophils, endothelial cells, and fibroblasts. given the importance of dcs as critical antigen-presenting cells, most studies of the biology of tlr signaling have focused on these cells. different tlrs are expressed on distinct subpopulations of dcs, and, depending on the tlr, in distinct cellular compartments. tlrs expressed on the surface of human myeloid dcs include tlr (which is heterodimerized with tlr or ), as well as tlrs , , , and ( fig. . ), while these same cells express tlrs and within endoplasmic reticulum (er) and phagolysosomes. plasmacytoid dcs (pdcs) express tlr and within er/phagolysosomes. the tlrs expressed on the cell surface are primarily activated by pamps encountered in the extracellular environment, while tlrs expressed in the er/phagolysosomes are activated by pamps (including viral pathogen-derived rna or dna) that tend to be routed through these endosomal compartments. activation of tlrs on innate immune cells leads to their production of specific cytokines, as well as their expression of co-stimulatory molecules, leading to induction of adaptive immune responses. given that different dcs express different tlrs, and that signaling via different tlrs results in the expression of a distinct pattern of cytokines, it is believed that activation of specific tlrs can variously favor the induction of th -or th -biased immune responses, or can differentially augment either direct or cross-presentation pathways for antigen presentation ( fig. . ). although most data on induction of specific types of immune responses by engagement of specific tlrs have emerged from murine studies (and have not yet been validated in humans), the ability to tailor an adjuvant preparation to achieve a desired type of immune response with a specific vaccine immunogen is a promising notion. naturally occurring ligands for tlrs include lipopolysaccharide (lps) from bacterial cell walls (recognized by tlr ), triacyl lipopeptides (recognized by tlrs + ), diacyl lipopeptides (recognized by tlrs + ), peptidoglycan (recognized by tlr ), flagellin (the monomer that makes up flagella, recognized by tlr ), singlestranded rna (recognized by tlr ), double-stranded rna (recognized by tlr ), and unmethylated dna containing the dinucleotide pair cpg (recognized by tlr ). based on these insights, a variety of approaches to develop adjuvants predicated to activation of specific tlr pathways are being actively pursued. one interesting aspect of adjuvant development is how it is revealing the mechanisms of action of adjuvants that were originally identified via a process of trial and error, as well as delineating important aspects by which certain empirically derived vaccines are able to induce high-level, long-lasting immune responses. one illustrative example can be found in the case of complete freund's adjuvant (cfa), which has long served as the benchmark for laboratory studies of adjuvants. cfa is a mixed emulsion of mineral oil, mannide monooleate, and killed mycobacteria. however, it is far too reactogenic for use in humans, causing significant pain and abcesses at the site of injection -reactions that would be exacerbated if cfa were to be used repeatedly. an alternative preparation termed incomplete freund's adjuvant (ifa) lacks the mycobacterial component, but it too is associated with injection site reactions that are severe enough to limit its use to experimental therapeutic cancer vaccines. although cfa's toxicity precludes its use as a vaccine adjuvant in humans, many of its constituents (including liposaccharides, dna, and specific bacterial cell wall components) are now understood to exert their adjuvant effects on vaccine-induced immune responses via engagement of specific tlrs. similarly, the live attenuated mycobacterium bovis strain, bcg, long widely employed as a vaccine for the prevention of tuberculosis, includes cell wall, peptidoglycan, and dna components that activate specific tlrs. interestingly, the highly effective yellow fever vaccine d has been shown to activate multiple tlrs as part of its induction of antiviral immune responses. it is quite likely that other live attenuated viruses that transiently replicate in immunized hosts also activate innate immune responses via engagement of tlrs. in yet another example involving a nonreplicating vaccine immunogen, one version of the hib polysaccharide conjugate vaccines now licensed for use in children for the prevention of invasive hib disease includes the meningococcal outer-membrane protein complex (ompc) as its protein carrier. ompc conjugates have favorable immunogenic properties that correlate with the ability of ompc to activate dcs via tlr . hundreds of different adjuvant formulations have been tested in animal models, and a few have been advanced into human studies. with a few α α ( ) toll-receptor agonists. alum, the classical adjuvant most often used in vaccines in humans, includes a range of salts of aluminum precipitated under basic conditions, usually aluminum sulfate mixed with sodium or potassium hydroxide plus a variable amount of phosphate. the relative proportions will determine the size, charge, and solubility of alum. the composition of alum used as an adjuvant varies in currently available vaccines and may influence vaccine immunogenicity. alum is utilized as an adjuvant in many of the currently available vaccines composed of inactivated toxins or recombinant proteins (live attenuated vaccines do not include alum or other adjuvants). alum serves two main purposes as an adjuvant. first, it acts as an antigen depot. vaccine antigens adsorb to alum and elute from it following injection into the host. second, alum acts a mild irritant, causing the recruitment of leukocytes necessary for generation of an immune response to the site of injection. adsorption of antigens on to alum routinely improves immunogenicity, particularly the antibody response. alum does not typically enhance cd t-cell responses. alum has been a component of many vaccines for decades and has an excellent safety record. as new adjuvants are developed, alum may remain as a component of combination adjuvant mixtures (as is the case with some newer adjuvants now approaching clinical use), or it may eventually be supplanted by other agents that more effectively provide favorable depot and local inflammatory responses to accentuate host immune responses. using lipids with polar head groups (e.g., triglycerides) and differing types of hydrophobic tails, one can form either micelles (spheres) or multilamellar sheets in aqueous environments. under the right conditions, antigens can be incorporated into the spheres or between layers of the sheets, providing a potential slow-release depot system. immunopotentiators such as qs or detoxified lps derivatives (such as monophosphoryl lipid a (mpl)) may be added to the lipid mix. iscoms are a proprietary form of liposomes made of cholesterol, saponins from quillaia bark (various members of the qs-x family of triterpene glycosides), and phospholipids that form cage-like structures into which antigens can be entrapped or intercalated. iscom complexes may provide a depot function, as well as facilitate the delivery, uptake, and processing of vaccine immunogens by apcs. purified influenza virus hemagglutinin (ha) and neuraminidase mixed with phosphatidyl choline and phosphatidyl ethanolamine (polar lipids) will form empty particles that have the surface properties of influenza virus. adding an antigen in solution before mixing the lipids results in the incorporation of the antigen inside the particle. this provides a vehicle for delivering antigens to the interior of a cell, via the influenza ha membrane fusion process, thereby enabling antigen processing and presentation via both major histocompatibility complex (mhc) class and pathways. emulsions numerous oil-in-water and water-in-oil emulsions have been tested as adjuvants. one such emulsion, mf , is used in a licensed influenza vaccine. mf consists of squalane, a metabolizable shark oil and two surfactants, polyoxyethylene sorbitan monooleate and sorbitan trioleate, in an oil-in-water emulsion. cytokines are host-produced immunomodulators that regulate immune cell action (chapter ). several cytokines are being tested as potential vaccine adjuvants, including granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin- (il- ), and il- . of the defined tlr agonists being explored as vaccine adjuvants, lps and its partially detoxified form, mpl, which activate tlr , have been most thoroughly explored in clinical trials. with evidence of enhanced ability to increase the percentage of individuals responding with protective antibody levels to hepatitis b as compared to a standard hepatitis b vaccine, one hepatitis b vaccine that employs an adjuvant formulation (termed as ) consisting of a combination of alum and mpl has been licensed for use in high-risk individuals. in addition, a vaccine against hpv that uses the same adjuvant formulation may be licensed soon, and candidate hsv- and malaria vaccines currently being studied in latestage clinical trials also include this alum-mpl combination adjuvant. a wide variety of tlr -specific agonists consisting of oligodeoxynucleotides containing unmethylated cpg motifs (cpg-odn) are being evaluated in preclinical studies. these cpg-odns resemble bacterial dna, modified to include a phosphorothioate backbone to increase their stability. two cpg-odn adjuvants have been evaluated in recent phase i and ii trials and shown to increase the timing and magnitude of induction of protective antibody levels, as well as the proportion of responding individuals, to recombinant hbsag vaccine as compared with the current commercially available version of the vaccine. one of these cpg-odn adjuvants also elicits protective antibody responses in immunized hiv-infected individuals who had previously failed to respond to the hepatitis b vaccine. this approach is now being studied as a way of inducing protective immune responses to hepatitis b earlier after initiation of the vaccination regimen or with fewer doses of the vaccine. in addition to the cpg-odn-based tlr adjuvants described above, small chemical compounds with structures that resemble nucleic acid bases have been identified that activate tlr (e.g., imiquimod) or both tlr and (e.g., resiquimod). these compounds are being evaluated as vaccine adjuvants in preclinical studies. flagellin, a tlr agonist, is also being explored as an adjuvant. recently, attention has also been focused on coupling, rather than mixing, tlr agonists to antigens. cpg oligonucleotides conjugated to antigens have been tested in preclinical studies of hepatitis b vaccines and in human clinical trials for treatment of allergy. ligands for tlr / have been coupled to hiv antigens, and the ligand for tlr (flagellin) has been fused to a variety of antigens. , in some instances, coupling a tlr ligand to an antigen resulted in a substantial improvement of the immune response compared to mixtures -potentially the result of enabling the antigen and the tlr ligand to co-locate in the same dc compartments. numerous preclinical studies have confirmed that many natural and synthetic tlr agonists possess adjuvant activity. importantly, early human clinical trials of tlr-predicated adjuvants have supported the promise of this approach to mechanism-based strategies to augment vaccine immunogenicity. an important challenge is to define the most potent and best-tolerated variants, and to define rules by which activation of specific tlr pathways might translate into predictable augmentation of desired types of immune responses. it is hoped that general rules will emerge to suggest which of an increasing number of novel adjuvants in development performs best with which type of vaccine immunogen, and if results obtained with a specific type of immunogen-adjuvant combination can be extrapolated to predict the likelihood of enhanced immunogenicity with other vaccines. although beyond the reach of available experimental results, the ability to tailor, titrate, and otherwise optimize immune responses to vaccines by manipulation of specific tlr pathways appears a realistic future possibility. however, important challenges remain. in particular, a primary challenge for nextgeneration adjuvant development is finding a combination that retains immunopotentiating action while minimizing vaccine-associated adverse experiences. short-term adverse experiences, such as local injection site reactions, represent undesirable side effects that may disqualify candidate adjuvants early in clinical development. however, given that vaccines are administered to healthy people to prevent potential future infectious diseases, the potential for rarer adverse experiences (such as autoimmunity) that may only be manifest with much longer latency from the time of vaccine adjuvant administration will undoubtedly be important considerations for use in prophylactic vaccines. as induction of cell-mediated immune responses is considered an important component of vaccine strategies for many diseases for which no vaccines are currently available (many of which are caused by intracellular pathogens), there is a need to develop safe and readily scalable approaches to elicit durable cd t-cell responses in immunized humans. further, given the critical role that cd t cells play in induction, differentiation, and maintenance of cd t-cell responses, any such novel vaccine strategy will likely also require appropriate cd tcell responses. as elicitation of cd t-cell responses against a foreign antigen usually depends on the de novo expression of the antigen within a host cell and its subsequent processing and presentation via class i mhc pathways (chapter ), most novel vaccine strategies are predicated on the need to achieve synthesis of pathogen-derived antigens within apcs of immunized human hosts. with an increasing appreciation of the role that cross-presentation pathways can play in elicitation of class i-restricted cd t-cell responses, such de novo antigen synthesis may not need to occur within apcs themselves (which may be an advantage for potential vaccine delivery strategies that do not directly target apcs). one of the many attractive attributes of effective live attenuated vaccines is their ability to recapitulate (to various degrees) many of the processes that lead to the generation of potent immune responses following natural infection. these processes include the fact that replication of all viruses depends on gaining access to host cells for genome replication and for the synthesis of essential components of virus particles that permit further propagation of the infection within and between hosts. one immunologic benefit of this requirement is that de novo synthesis of viral gene products within infected cells provides a key opportunity for viral antigen presentation (via mhc class i pathways) and elicitation of antiviral cellular immune responses. along with the processing and presentation of intact virus proteins via mhc class ii pathways leading to production of antiviral antibody responses, live attenuated viral vaccines have a strong track record for induction of broad cellular and humoral immune responses that likely both contribute to conferring protective immunity. however, despite their track record of success, it is likely that few, if any, new live attenuated viral vaccines will be derived in a manner that resembles previous successful efforts (e.g., the empiric derivation of live attenuated polio, yellow fever, or varicella-zoster vaccines). important reasons for this change include the desire for safe and well-characterized vaccines whose mechanisms of attenuation are defined and that can be monitored in the course of vaccine production and use. indeed, most of the recently developed live attenuated vaccines were derived using new approaches for genetic reassortment (fig. . ) wherein genome segments encoding pathogen-derived antigens of interest are recombined with a common set of genome segments that carry attenuating mutations (derived either by use of attenuating viral passage under specific conditions in cell culture (e.g., the cold-adapted influenza vaccine or use of a virus obtained from a nonhuman host that is itself inherently unable to replicate to high levels in humans, e.g., the reassortant rotavirus vaccine prepared via genetic reassortment between human and bovine rotavirus strains (fig. . ) ). although such approaches have proven successful, they are limited in that they can only be applied to homologous viruses (e.g., those derived from the same virus type) whose genomes are segmented and capable of ready genetic reassortment in culture, or to viruses that can be manipulated by reverse genetics. in response to the desire to produce vaccines that can safely and reliably elicit desired immune responses, especially t-cell responses, several approaches are being explored to develop novel vector systems that permit the expression of pathogen-derived antigens. as many of these approaches are based on viruses distinct from the viral pathogen targeted for induction of host immune responses, the inserted pathogen-derived gene products are expressed via recombinant methods as heterologous antigens. alternatively, in nonviral expression systems, such as dna vaccines, the pathogen-derived antigen is expressed in isolation and does not depend on virus-mediated antigen delivery to apcs following host inoculation. collectively, such recombinant heterologous expression systems are commonly referred to as 'vaccine vectors.' in some instances, such recombinant vectors express only a specific antigen (in the case of dna vaccines or certain viral vectors, e.g., adenovirus), while in others both the inserted pathogen-derived antigens and antigens encoded by the viral vector 'backbone' are expressed (e.g., poxvirus vectors). most new approaches employ expression systems that are inherently nonreplicating (e.g., dna vaccines) or that employ viral vectors that can replicate at high levels in tissue culture but not in vivo (e.g., complemented adenovirus deletion variants or host range-restricted poxviruses). while numerous approaches are being pursued to develop novel vaccine vectors, they will all need to meet certain common criteria to emerge as vaccine approaches applicable for widespread use. in particular, any successful approach must be safe in healthy and immunodeficient humans (given their increased representation in the population as a result of hiv infection and therapeutic immunosuppression), desirably immunogenic (including in individuals who may have been previously exposed to the virus from which the vector was derived, e.g., vaccinia or adenovirus), and able to be produced in large quantities and in a stable manner. a limited number of vaccine vector approaches now being pursued are likely to meet these criteria. should successful approaches emerge, there will likely be interest in applying them for use in vaccines targeting diverse pathogens. thus, while definition of promising, broadly applicable, vaccine vector approaches may help simplify certain aspects of vaccine regulatory review and manufacture, they may also present challenges to prioritize use for specific applications should administration of a given vaccine vector on one occasion compromise or preclude successful administration at a later time. nevertheless, the development of novel vaccine vectors is laying essential groundwork for the development of next-generation vaccines. several novel vaccine vectors currently being studied in preclinical studies and human clinical trials are described below, all of which depend on the delivery and expression of a candidate pathogen-derived gene sequence. in a number of ways, dna vaccines represent the simplest approach to deliver pathogen-derived genes. viral vectors similarly serve to deliver pathogen gene sequences to host apcs, either directly or indirectly, but do so in a manner that depends on and takes advantage of the lifecycle and tropism of the virus that is being adapted to express the exogenous pathogen gene products. the ability of purified plasmid dna containing heterologous antigens expressed under the control of eukaryotic transcriptional regulatory and rna processing signals to elicit immune responses when injected into experimental animals was discovered serendipitously. however, since the initial, quite surprising, description, the development of so-called dna vaccines has become an active area of preclinical and clinical vaccine development. reasons for this enthusiasm include the attractive simplicity and facile preparation of vectors that encode only the defined antigen of interest (which can itself be manipulated via recombinant methods to assume a desired configuration), a reasonably straightforward method for vaccine production, and the inherent stability to temperature (which is much greater than most currently live or subunit vaccines). although the dna vector is most commonly injected intramuscularly, the generation of specific immune responses depends on the uptake of the vector dna by apcs followed by the expression, processing, and presentation of vector-encoded antigens. as tissue and tissue fluids present a hostile environment for purified dna, and the process of dna uptake by apcs appears to be relatively inefficient, much of the dose of injected dna is degraded before it can be reached by an apc that can initiate the desired immune response. most dna vaccine research has been pursued in mice, although studies have now been performed in numerous animal species. studies have usually utilized intramuscular injection of vaccine vector dna, but various intradermal and transdermal approaches have also been explored. murine studies have shown that administration of antigen-encoding plasmid dna can elicit appreciable cellular and humoral immune responses that may confer protection against experimental challenge. however, translation of these promising results in animal models to humans has proven frustrating. while dna vaccines have been generally well tolerated in immunized volunteers, in most human studies of dna vaccines, administration of even substantial quantities of dna vaccine vectors has elicited relatively low-level immune responses. it is not yet known whether these disappointing results reflects fundamental differences in the immunogenic behavior of dna vaccines in humans and mice, or the fact that the dna doses administered to humans do not match those administered to mice (dna per weight of the immunized host). given the substantial size differences between humans and mice, it would likely be impractical (for reasons of both vaccine supply and the actual process of administration of sufficiently high doses of dna) to administer the relative murine dose to humans. as such, a variety of approaches are being explored to prolong dna survival in tissue, promote more efficient targeting of dna to apcs, or to develop novel adjuvants that might specifically amplify immune responses to dna vaccines. , dna vaccines are currently being used as candidate preventive vaccines for a wide variety of infectious diseases, including hiv, tuberculosis, malaria, and cmv. poxviruses represent the family of viruses that are physically the largest viruses and that possess the largest genomes. much of the poxvirus genome encodes gene products that serve to evade host immune responses, and that are not required for virus replication in tissue culture. further, facile techniques for the insertion and deletion of specific viral genes have been developed. the ability to accommodate sizeable foreign gene inserts is, in part, a function of the large size of the poxvirus genome (and the large packaging capacity of poxvirus virions). as a result of these favorable attributes, poxviruses have been utilized extensively in laboratory studies of virus biology, recombinant protein production, and host immune responses. although poxviruses encode multiple gene products that help the virus evade host immune responses, they are, nevertheless, potent immunogens. studies of individuals immunized decades ago with vaccinia virus (in the course of smallpox eradication efforts) have shown that this virus induces long-lasting memory t-and b-cell immune responses. in contrast to most of the other viral vectors currently being developed, poxviruses can replicate readily in culture and do not require an engineered host cell to support propagation ex vivo. one important limitation of all poxvirus vectors developed to date is that, given the large size of the poxvirus genomes and the multitude of gene products they naturally express, even large inserts derived from foreign pathogens of interest will present only a minority of the vaccine vector antigens delivered to and recognized by the host immune system. to be effective, approaches to focus immune responses on the antigen of interest will need to be developed. toward this end, a variety of so-called 'prime-boost' approaches are being explored where the host immune response is primed with one type of recombinant vaccine vector (such as a dna vaccine or adenovirus vector) and then boosted with subsequent delivery of poxvirus vectors encoding the same antigen. in this manner, immune responses to antigens of interest have been significantly augmented in a number of preclinical studies. vaccinia virus represents the prototypic vaccine vector. this virus is the same one that was employed in the successful smallpox eradication campaign, and has been used as a laboratory tool for decades. however, given current high expectations for vaccine safety, and the increased number of immunodeficient individuals present in the population (as a result of the new antigen discovery methods emergence of the hiv pandemic and the increased use of immunosuppressive therapies in clinical medicine) at high risk of serious adverse events, and potentially fatal consequences, from vaccinia immunization, the original vaccinia strains used in smallpox eradication efforts are not considered safe for general use. however, studies of vaccinia-based vaccine vectors have provided a strong basic foundation for research on other more highly attenuated poxvirus variants. modified vaccinia ankara (mva) is an attenuated vaccinia virus that was originally derived by prolonged passage of a vaccinia virus isolate on chicken embryo fibroblasts in culture. in the course of extensive passage in culture, a viral variant emerged that had fortuitously deleted large sections of the viral genome, including those that encode important poxvirus immune evasion genes and those that determine the ability of the virus to replicate on cells obtained from different animal species. specifically, while mva grows well on chicken cells, it cannot replicate in human cells in culture or in vivo, conferring an inherent safety feature. mva was safely administered to over individuals at high risk of adverse consequence for vaccinia immunization toward the end of the smallpox eradication effort. more recently, it has garnered renewed interest as a potential safer smallpox vaccine in the wake of concerns about bioterrorism threats. even though mva cannot replicate in mammalian cells, the virus demonstrates favorable immunogenic properties. mva has been used as a vector expressing genes for a wide variety of genes, including hiv and malaria antigens either alone or, as described above, in 'prime-boost' regimens, where mva has been administered following initial priming immunizations with other vaccine vectors. a concerted effort is under way to improve further the performance of mva by manipulating a series of poxvirus genes that dampen the human immune response to the virus (and to any antigens inserted in it). avipox is a family of poxviruses that infect birds and cause respiratory diseases in poultry. canarypox, a member of the avipox group, has been adapted as a vaccine vector. canarypox replicates well on avian cells in culture but cannot replicate on human cells in culture or in humans in vivo. as a result, canarypox, like mva, provides an interesting vector system with inherent safety features. canarypox vectors carrying hiv genes have been tested in several clinical studies, either alone, or in 'prime-boost' regimens following priming with adenovirus vectors and recombinant protein antigens. in a large ongoing phase iii hiv vaccine trial, a recombinant canarypox vector is being used as a priming vector, followed by boosting with a recombinant version of the hiv gp surface env protein. to date, the results from human clinical trials of canarypox vectors have been disappointing, with only low-level specific immune responses generated in human volunteers. adenoviruses, one of the common causes of upper respiratory and gastrointestinal infections, have seen extensive use in clinical trials and were one of the first gene therapy vectors. most adenovirus vectors currently being studied in preclinical and clinical settings are disabled by deletion of the early e genes that are necessary for replication in an immunized host. most adenovirus vaccine vectors developed have used the well-characterized and readily produced adenovirus serotype (ad ) as the vector 'backbone.' disabled adenovirus vectors are grown in cells that express the e genes artificially inserted into the cell's genome. once these disabled vectors, encoding a heterologous pathogen-derived antigen of interest, enter a cell, the pathogen gene product is expressed, processed, and presented by host apcs. as adenoviruses can directly infect dendritic cells, they promise to provide efficient vaccine vectors. robust antibody and cd t-cell responses to heterologous antigen genes expressed by adenovirus vectors have been observed in preclinical animal models. furthermore, in early-phase human clinical trials, adenovirus vectors have been generally well tolerated, and proven to be the most effective of any recombinant vector system studied to date in eliciting high-level cd t-cell responses. the main potential drawback to widespread use of adenovirus vectors in humans is that, depending on the adenovirus type and the geographic location, variable levels of pre-existing immunity are found in humans as a result of prior naturally acquired adenovirus infections. high levels of antibody against the adenovirus vector might blunt the immunogenicity and efficacy of an adenovirus vector-based vaccine, but it remains to be seen if this will be a significant limitation. should pre-existing immunity to adenovirus vectors derived from epidemiologically prevalent serotypes (e.g., ad ) limit vaccine immunogenicity, current efforts to develop vaccine vectors based on serotypes that are rare in human populations or novel adenovirus vectors specifically designed to avoid preexisting antibody responses may yield effective alternative approaches. adenovirus vectors are currently used in clinical trials for vaccines against hiv, malaria, influenza, and a range of other pathogens. alphaviruses are rna viruses that cause zoonotic diseases, such as venezuelan equine encephalitis. these viruses do not normally circulate in humans, so immunity to these viruses is quite rare in humans. alphaviruses have a strategy for overexpressing the proteins that make up the virion by making a separate subgenomic rna specifically encoding these gene products. current recombinant alphavirus vaccine vector strategies take advantage of this subgenomic transcript, replacing the viral genes with selected genes for other antigens, but maintaining the signals for translation and protein production. in addition, through use of genetic complementation, it is possible to generate virus particles that only contain this heterologous antigen-encoding expression cassette. such virus particles can efficiently mediate infection of host cells, but because they lack other alphavirus genes needed for virus replication cannot spread beyond the initial target cell infected. , alphavirus vectors rival the adenoviruses in efficiency of protein production in tissue culture and have induced robust antibody and t-cell responses in preclinical studies. one current limitation of the alphavirus vector system is the difficulty of scaling the production system; however, this is a technical matter that should be addressable. in addition, ample safety data will be needed before widespread use of alphavirus vaccines achieves endorsement by regulatory authorities for use in healthy populations. adeno-associated viruses (aav ) belong to a family of single-stranded dna viruses (parvoviruses) that include the b parvovirus that causes a rash in children known as 'fifth disease' (measles, mumps, rubella, and varicella make up the first four). aav is transmitted in conjunction with adenovirus infection, and is not known to cause any significant disease. it is poorly immunogenic in the course of natural infections. aav can integrate into the genome of the infected cell, usually in a particular place on chromosome , although integration does not appear to be efficient or site-specific when replicationdefective adenoviruses of the type being developed as vaccine vectors are used. the propensity for chromosomal integration and poor immune response to the virus made aav a good candidate for gene therapy; cells with an integrated viral genome could deliver a gene product for a long time without the immune system killing the infected cell. recently, efforts have been made to adapt replicationdefective aav as a vaccine vector. although encouraging results have been reported in preclinical studies, phase i studies in humans have demonstrated disappointing immunogenicity. n summary n the challenges to optimizing the full public health potential of existing vaccines largely relate to programmatic considerations. in contrast, the terrible impact of infectious diseases that cannot now be prevented by vaccines (such as the 'big three' killers of hiv, tuberculosis, and malaria) pose direct challenges to the scientific community to develop new generations of vaccines that overcome the largely biological obstacles to control and elimination of these diseases. the nature of the challenges posed by such pathogens necessitates that future vaccine efforts will not simply recapitulate the immune responses engendered by natural infection (as has been the premise of traditional vaccine development efforts), but rather, substantially improve upon them. as the development of vaccines to prevent infections with the so-far refractory pathogens is pursued, improved understanding of the immune response to natural infection, as well as delineation of the reasons why host immune responses fail either to clear incipient infections or prevent future new ones, will be essential. fortunately, early empiric approaches have now been replaced with hypothesis-driven strategies enabled by improved insight into the functioning of the human immune system, as well as new technologies, including higher-resolution tools to describe and quantitate pathogen-specific immune responses; novel methods for antigen discovery and targeted optimization of immunogenicity; the development of new, mechanism-based adjuvants; and the advent of innovative methods for vaccine vector-mediated antigen delivery. thus, although the challenges may be vexing, the scientific and technical foundations on which vaccine development efforts rest have never been stronger. n references n for many important infectious diseases for which no vaccines are currently available, successful derivation of effective vaccines will depend on improving upon natural immunity, especially in those instances where natural immunity does not follow natural infection (such as human immunodeficiency virus (hiv) and malaria) or where safety concerns limit the development of specific protective antigens (neisseria meningitidis group b). in addition, for other pathogens that typically manifest significant genetic (and antigenic) diversity (such as influenza, hiv, and bacteria such as streptococcus pneumoniae), a need exists to develop novel vaccines that can protect against a wide range of variants with a limited number of vaccine immunogens. towards these ends, a number of new approaches, enabled by new vaccine technologies, are being pursued, including: >> targeted alteration of protective antigens to increase their ability to elicit protective immune responses (e.g., efforts to alter the structure of the hiv env glycoproteins gp and gp so that they elicit higher-level, more potent, neutralizing antibody responses than their native counterparts) >> the development of synthetic consensus antigens able to elicit broader immune responses than would sequences obtained from individual pathogen isolates (e.g., efforts to develop consensus immunogens able to elicit cytotoxic t-lymphocyte (ctl) responses against genetically diverse hiv- variants) >> techniques for new antigen discovery to identify novel conserved antigens within otherwise genetically diverse pathogens (e.g., streptococcus pneumoniae) or those for which currently known protective antigens cannot be developed as vaccines (e.g., neisseria meningitidis group b) >> the use of novel adjuvants or vaccine vectors to enable generation of higher-level and/or more functional immune responses to pathogen antigens by vaccination than are seen following natural infection, and to enable high-level, fully functional memory immune responses to be activated at the time of initial infection (e.g., efforts to elicit high-level hivspecific or hepatitis c virus-specific ctls by recombinant viral vectors) >> use of novel methods to shift relative immunodominance of specific pathogen gene products to increase the immunogenicity of conserved antigens from otherwise diverse pathogen genomes that are typically poorly immunogenic in the course of natural infections (e.g., efforts to augment the antibody response to the influenza a virus m protein via the use of potent adjuvants or conjugation to immunogenic carrier proteins) ten great public health achievements -united states principles and lessons from the smallpox eradication programme advance market commitments for vaccines against neglected diseases: estimating costs and effectiveness mass vaccination: when and why immunisation and herd immunity standards for immunization practice for vaccines in children and adults economic evaluation of the -vaccine routine childhood immunization schedule in the united states priorities among recommended clinical preventive services development of an anti-core lipopolysaccharide vaccine for the prevention and treatment of sepsis safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis safety and efficacy of a pentavalent human-bovine (wc ) reassortant rotavirus vaccine the history of the smallpox vaccine grease, anthraxgate, and kennel cough: a revisionist history of early veterinary vaccines polio eradication: the opv paradox review of current preclinical testing strategies for bacterial vaccines standardization of acellular pertussis vaccines vaccines against polysaccharide antigens conjugate vaccines newer directions in vaccine development and utilization the development of novel hepatitis b vaccines papillomavirus l major capsid protein self-assembles into virus-like particles that are highly immunogenic quadrivalent vaccine against human papillomavirus to prevent anogenital diseases immunologic responses following administration of a vaccine targeting human papillomavirus types , , , and the rotavirus vaccine saga global illness and deaths caused by rotavirus disease in children effect of rotavirus vaccination programme on trends in admission of infants to hospital for intussusception rotashield: the ill-fated rhesus-human reassortant rotavirus vaccine a review of the vaccine adverse event reporting system database vaccine safety surveillance using large linked databases: opportunities, hazards and proposed guidelines permanent brain damage and pertussis vaccination: is the end of the saga in sight? absence of detectable measles virus genome sequence in blood of autistic children who have had their mmr vaccination during the routine childhood immunization schedule of uk the mmr vaccination and autism controversy in united kingdom - : inevitable community outrage or a failure of risk communication? molecular mimicry of host structures by lipooligosaccharides of neisseria meningitidis: characterization of sialylated and nonsialylated lacto-n-neotetraose (galbeta - glcnacbeta - galbeta - glc) structures in lipooligosaccharides using monoclonal antibodies and specific lectins prospects for prevention of childhood infections by maternal immunization an hiv vaccine -evolving concepts prospects for an aids vaccine: three big questions, no easy answers update of the drug resistance mutations in hiv- : hiv gp vaccine aidsvax -vaxgen, hiv vaccine aidsvax -vaxgen recent advances in the development of hiv- vaccines using replication-incompetent adenovirus vectors one step forward, two steps back -will there ever be an aids vaccine? child mortality and malaria transmission intensity in africa the past, present and future of childhood malaria mortality in africa the immune response to plasmodium falciparum malaria longevity of the immune response and memory to blood-stage malaria infection malaria vaccines in development irradiated sporozoite vaccine induces hla-b -restricted cytotoxic t lymphocyte responses against two overlapping epitopes of the plasmodium falciparum sporozoite surface protein global epidemiology of tuberculosis advances in tuberculosis vaccine strategies modern vaccines. mycobacterial diseases tuberculosis vaccines: current progress preclinical testing of new vaccines for tuberculosis: a comprehensive review tuberculosis: from genome to vaccine respiratory virus immunization. i. a field trial of two inactivated respiratory virus vaccines; an aqueous trivalent parainfluenza virus vaccine and an alum-precipitated respiratory syncytial virus vaccine sensitizing versus immunizing properties of inactivated measles vaccine recombinant bovine/ human parainfluenza virus type (b/hpiv ) expressing the respiratory syncytial virus (rsv) g and f proteins can be used to achieve simultaneous mucosal immunization against rsv and hpiv the new meningococcal conjugate vaccine. a profile of its safety, efficacy, and indications for use antigenic similarities between brain components and bacteria causing meningitis. implications for vaccine development and pathogenesis prevention of perinatal group b streptococcal disease. revised guidelines from cdc current status of a hepatitis c vaccine: encouraging results but significant challenges ahead prophylactic vaccine strategies and the potential of therapeutic vaccines against herpes simplex virus clinical trials of prophylactic and therapeutic herpes simplex virus vaccines cytomegalovirus vaccine prepared in wi- a canarypox vector expressing cytomegalovirus (cmv) glycoprotein b primes for antibody responses to a live attenuated cmv vaccine (towne) vaccine strategies against human cytomegalovirus infection phase i/ii studies to evaluate safety and immunogenicity of a recombinant gp epstein-barr virus vaccine in healthy adults understanding dengue pathogenesis: implications for vaccine design prospects for a dengue virus vaccine a method for the screening of fusion protein expression by lambda-gt recombinant clones without the preparation of lysogens antibody screening of bacteriophage lambda gt- dna expression libraries vaccinology at the beginning of the st century reverse vaccinology, a genome-based approach to vaccine development the pan-genome: towards a knowledge-based discovery of novel targets for vaccines and antibacterials genomics and proteomics in reverse vaccines haemophilus influence: the impact of whole genome sequencing on microbiology genome sequence of the human malaria parasite plasmodium falciparum bacterial proteomics and vaccine development proteomics approaches towards antigen discovery and vaccine development contribution of mass spectrometry-based proteomics to immunology structural proteomics and computational analysis of a deadly pathogen: combating mycobacterium tuberculosis from multiple fronts identification of in vivo-expressed immunogenic proteins by serological proteome analysis of the bacillus anthracis secretome identification of tumor antigens in renal cell carcinoma by serological proteome analysis innate immune recognition on regulation of phagosome maturation and antigen presentation translating innate immunity into immunological memory: implications for vaccine development innate immune induction of the adaptive immune response liposomes and iscoms understanding the role of innate immunity in the mechanism of action of the live attenuated yellow fever vaccine d haemophilus influenzae type bouter membrane protein complex glycoconjugate vaccine induces cytokine production by engaging human toll-like receptor (tlr ) and requires the presence of tlr for optimal immunogenicity structure and properties of aluminum-containing adjuvants immunoactivating potential of multilamellar liposome vesicles (mlv) in murine popliteal lymph node (pln) test adjuvant and antigen delivery properties of virosomes safety and immunogenicity of nonadjuvanted and mf -adjuvanted influenza a/h n vaccine preparations new hepatitis b vaccine formulated with an improved adjuvant system cpg adjuvant improves hepatitis b virus vaccine seroprotection in antiretroviral-treated hivinfected adults testing of cpg-optimized protein and dna vaccines against the hepatitis b virus in chimpanzees for immunogenicity and protection from challenge immunostimulatory sequences of dna and conjugates in the treatment of allergic rhinitis hiv gag protein conjugated to a toll-like receptor / agonist improves the magnitude and quality of th and cd + t cell responses in nonhuman primates vaccination with recombinant fusion proteins incorporating toll-like receptor ligands induces rapid cellular and humoral immunity a west nile virus recombinant protein vaccine that coactivates innate and adaptive immunity the development of multivalent bovine rotavirus (strain wc ) reassortant vaccine for infants intramuscular injection of plasmid dna heterologous protection against influenza by injection of dna encoding a viral protein dna vaccines: recent developments and future possibilities from plasmids to protection: a review of dna vaccines against infectious diseases poxvirus-based vaccine candidates for hiv: two decades of experience with special emphasis on canarypox vectors drug evaluation: dna/mva primeboost hiv vaccine improving recombinant mva immune responses: potentiation of the immune responses to hiv- with mva and dna vectors expressing env and the cytokines il- and ifn-gamma applications of canarypox (alvac) vectors in human and veterinary vaccination high-dose recombinant canarypox vaccine expressing hiv- protein, in seronegative human subjects biology of adenovirus and its use as a vector for gene therapy adenovirus-based expression vectors and recombinant vaccines established immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. potential for immunosuppression and vector engineering to overcome barriers of immunity attenuation of simian immunodeficiency virus sivmac infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing gag venezuelan equine encephalitis virus vectors expressing hiv- proteins: vector design strategies for improved vaccine efficacy an alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector recombinant alphaviruses as vectors for anti-tumour and anti-microbial immunotherapy immune responses to adeno-associated virus and its recombinant vectors key: cord- - yyv vuy authors: rybicki, ed title: history and promise of plant-made vaccines for animals date: - - journal: prospects of plant-based vaccines in veterinary medicine doi: . / - - - - _ sha: doc_id: cord_uid: yyv vuy plant-made vaccines are now a well-established and well-tested concept in veterinary medicine—yet the only product so far licenced was never produced commercially. this is puzzling, given the breadth of exploration of plant-made animal vaccines, and their immunogenicity and efficacy, over more than twenty years of research. the range of candidate vaccines that have been tested in laboratory animal models includes vaccines for e. coli, salmonella, yersinia pestis, foot and mouth disease virus, rabbit haemorrhagic disease virus, rabbit and canine and bovine papillomaviruses, mink enteritis and porcine circovirus, and lately also bluetongue virus, among many others. there are many proofs of efficacy of such vaccines, and regulatory pathways appear to have been explored for their licencing. this review will briefly explore the history of plant-made vaccines for use in animals, and will discuss the unique advantages of plant-made vaccines for use in a veterinary medicine setting in detail, with a proposal of their relevance within the “one health” paradigm. plant-produced vaccines for veterinary medicine are an exciting prospect, largely because of the possibilities of producing protein-based vaccines' including edible vaccines' at low cost, at almost any scale, and potentially locally and on demand. they have also been controversial because of the very real possibilities of contamination of the human food supply with vaccine-producing transgenic plants, and because of concerns around the possibility of immunological tolerance developing to oral or edible vaccines. however, one set of problems that many foresawregulatory and production problems-has not eventuated, and in fact the environment now seems primed very favourably for their introduction. the main justifications for plant-made vaccines are that vaccine antigen production in plants is safe; that it is both cheap and highly scalable; that plants produce and process eukaryote-derived proteins much better than can bacteria or even yeasts; that use of plants would allow for production of vaccines in the developing world where they are needed most; and of vaccines or therapeutics that will never be produced economically by other technologies. however, despite more than twenty years of development, there are still no plant-produced vaccines or biologics available for animals-although there are in fact products licenced for and in use in humans. this review will explore the early history of plant-produced vaccines with an emphasis on proofs of principle and of efficacy, what the recent development of robust, stable transient plant production systems for vaccine antigens could mean for veterinary medicine, and the potential of plant-produced vaccines to advance both animal and potentially human health' under the banner of the one health movement. while viral proteins have probably been the most common vaccine candidates made in plants (reviewed in rybicki ) , it was expression of a bacterial protein-escherichia coli heat labile enterotoxin (lt-b)-that first proved that veterinary-relevant antigens could be produced in plants, and provided the first proof of principle for edible vaccines. lt-b produced in transgenic tobacco or potatoes (haq et al. ) was functionally equivalent to e coli-produced protein in specific assays, and immunisation of mice by oral gavage with plant material elicited systemic and mucosal toxin neutralising antibodies. moreover, fresh potato containing lt-b was immunogenic in mice when eaten. an early virus vaccine candidate was one against mink enteritis virus (mev) disease: this was novel in that it comprised chimaeric cowpea mosaic virus (cpmv) virions incorporating a short linear epitope from mev vp capsid protein and displaying it on the surface of virions, produced by inoculation of bean plants with an infectious cdna clone of rcpmv (dalsgaard et al. ) . this conferred protection against clinical disease and virtually abolished virus shedding-and given that the epitope sequence used is found in mev, canine parvovirus, and feline panleukopenia virus, the same vaccine could potentially also protect against these viruses. another early virus vaccine candidate was against rabbit haemorrhagic disease virus (rhdv): this was made by expressing the whole rhdv vp capsid protein in transgenic potatoes; parenteral immunisation with plant extracts was protective in rabbits (castanon et al. ) . subsequently, another study demonstrated that an edible vaccine consisting of leaves of transgenic plants containing presumably partially-assembled vp subunits, was an effective priming vaccine for later baculovirus-derived parenterally-delivered vaccine (gil et al. ). the first report of a foot and mouth disease virus (fmdv) plant-made antigen was of expression in plant protoplasts of a vp -derived peptide of fmdv as an insertion into the minor coat protein of a replicating cpmv as a demonstration of antigen presentation (usha et al. ) . however, the first proof of efficacy was done using transgenic arabidopsis thaliana expressing whole vp : parenteral immunisation of mice with leaf extracts elicited antibodies that bound to vp and to intact fmdv particles, and all immunised mice were protected against virulent fmdv challenge (carrillo et al. ) . the wigdorovitz group went on to demonstrate that mice could be protected against fmdv challenge after oral or parenteral vaccination with extracts of transgenic alfalfa expressing vp (wigdorovitz et al. ) , or immunisation with leaf extracts of tobacco plants expressing vp via a recombinant tobacco mosaic virus vector (wigdorovitz et al. ) . a refinement of these achievements included transgenic expression in alfalfa of amino acid residues - of vp (vp - ) fused to glucuronidase (gus), which both allowed selection of strongest expressers by assay of enzyme activity, and was protective in mice (dus santos et al. ) . another novel application of carrier technology was the insertion of vp amino acids - (g-h loop) in an interior region of the hepatitis b virus core antigen gene (hbcag), and expression of the chimaera in transgenic nicotiana tabacum. the chimaeric protein formed virus-like particles (vlps) in the tobacco leaves, and mice immunised intraperitoneally with a soluble extract were protected against viral challenge (huang et al. ). an early attempt at showing the feasibility of making an anthrax vaccine was the expression in transgenic n. tabacum of the protective antigen (pa) protein of bacillus anthracis, possibly the best target for a subunit vaccine because it alone is protective (aziz et al. ) , although it went no further than showing cytolytic activity of the protein. soon after, the same group went on to express pa in transplastomic n. tabacum, with significant yield increases but still no efficacy trial (aziz et al. ) . another investigation of transplastomic tobacco by henry daniell's group was more thorough: yields were high ( . g/kg in fresh leaf tissue), the protein was protected in chloroplasts from protease cleavage and was stable when stored in leaves or as crude extracts, and was biologically active (watson et al. ). while they did not show immunogenicity or protection, the authors speculated that "with an average yield of mg of pa per plant using an experimental transgenic cultivar grown in a greenhouse, million doses of vaccine (free of contaminants) could be produced per acre". the daniell group subsequently showed that chloroplast-derived pa was equal in potency to the natural product from b anthracis, and that mice immunised subcutaneously with partially purified chloroplast-derived pa with adjuvant produced high igg titres and survived challenge with lethal doses of toxin (koya et al. ) . a different sort of approach to anthrax, and one of the first attempts at making a therapeutic antibody in plants, was taken by vidadi yusibov's group, who used the technique of transient agrobacterium infiltration-mediated expression in n. benthamiana to produce a human-derived pa-specific monoclonal antibody (hull et al. ) . the antibody neutralised toxin activity both in vitro and in vivo at a comparable level to hybridoma-produced antibodies. the yusibov group at what became fraunhofer usa center for molecular biotechnology later used the same transient expression technology to separately express artificial antigens comprising domain of pa or domain of b anthracis lethal factor (lf), fused in-frame with lichenase (lickm), a thermostable enzyme from clostridium thermocellum (chichester et al. ) . mice immunised with a combination of the two antigens produced high titres of mainly igg , and sera could neutralise the effects of anthrax lethal toxin (letx) in vitro. rabies vaccines made in plants included an early yet highly sophisticated candidate that was composed of the alfalfa mosaic virus (amv) cp fused to an artificial polypeptide containing rabies virus g protein amino acids - , and n protein amino acids - , and expressed either in n. tabacum plants transgenic for amv replicase, or via rtmv in either n. benthamiana or spinach (yusibov et al. ) . the plants made particles containing amv-derived rna, encapsidated with chimaeric cp: raw spinach leaves were orally immunogenic in mice and in human volunteers. a simpler candidate was the g protein alone, with plant signal peptide and er retention signal, made in transgenic n. tabacum (ashraf et al. ) . while yields were relatively low ( . % of total soluble leaf protein), purified protein injected peritoneally in mice elicited protective immunity against lethal intracerebral challenge with live rabies virus-an excellent proof of both principle and efficacy. plant-made animal rotavirus vaccines were an early target, with a stand-out study by yu and langridge ( ) providing evidence that transgenic potato could produce fusion proteins consisting of cholera toxin (ct) b and a subunits fused with murine rotavirus enterotoxin and enterotoxigenic e coli fimbrial antigen, respectively. fusion antigens assembled in potato tubers into cholera holotoxin-like structures that bound enterocytes, and elicited serum and intestinal antibodies after oral immunisation in mice. moreover, passively immunised mouse neonates were partially protected against diarrhoea after rotavirus challenge, demonstrating that combination vaccines for viral and bacterial pathogens may be made in plants. a simpler approach to rotavirus prevention was expression of a his-tagged vp * fragment of bovine rotavirus (brv) vp in n. benthamiana via recombinant tmv, purification of the antigen by ni + chromatography, and intraperitoneal immunisation of adult female mice ). eighty-five percent of suckling mice born from these mothers were protected from brv challenge, compared to % immunised with an irrelevant control antigen. the same group also showed that a fusion protein made in transgenic alfalfa consisting of a short peptide derived from brv vp fused to gus was immunogenic both when given intraperitoneally and orally to adult female mice, and their sucklings were protected against challenge ). another group used transgenic alfalfa to produce human rotavirus vp , and showed that female mice gavaged with alfalfa extract containing oligocpg as an adjuvant developed high titres of antibodies both systemically and mucosally, and their pups were partially protected against simian rotavirus challenge. the same animal model first used to show the efficacy of insect cell-made papillomavirus virus-like particle (vlp)-based vaccines (breitburd et al. ) was also used to demonstrate the efficacy of two very different plant-made papillomavirus vaccines, a few years after the demonstration that human papillomavirus l major capsid protein virus-like particles could be produced in transgenic tobacco or potato (biemelt et al. ; varsani et al. ; warzecha et al. ) . cottontail rabbit papillomavirus (crpv), the cause of the famous "jackalope" sightings in the usa, provides an excellent model system in domestic rabbits for investigation of prophylactic and therapeutic papillomavirus vaccines (breitburd et al. ) . accordingly, in the first study crpv l major capsid protein-containing extracts were prepared either from transgenic n. tabacum or n. benthamiana infected with recombinant tmv, and used with freund's incomplete adjuvant to immunise rabbits that were subsequently challenged with live virus (kohl et al. ) . although the vaccines appeared to contain small aggregates of crpv l rather than intact vlps, and immune rabbit sera failed to neutralise crpv infectivity in an in vitro assay, the rabbits were protected from wart development (kohl et al. ). in the second study, infectious recombinant tmv was used to surface display, via fusion to the capsid protein, a peptide consisting of amino acids - of the l minor capsid protein from either crpv or the rabbit oral papillomavirus (ropv) (palmer et al. ) . groups of rabbits received either or both vaccines, and were challenged with live crpv or ropv. immune rabbit sera reacted with whole l protein, and crpv-specific sera neutralised crpv pseudovirion infectivity. rabbits receiving the crpv or crpv + ropv vaccines were completely protected against crpv infection, and those receiving ropv alone were weakly protected against crpv. these studies demonstrated for the first time that plant-made papillomavirus vaccines based on l protein or l -derived peptide had real potential as prophylactic vaccines, for use in animals as well as in humans. strangely, given that bovine papillomaviruses (bpv) had been used for many years as model systems for anti-wart vaccination, it was not until , with transient agroinfiltration-mediated expression of bpv- vlps in n. benthamiana, that a candidate plant-made bpv l vlp-based vaccine was successfully made, although no efficacy trials were done (love et al. ) . expression of animal vaccine components in seeds of transgenic plants was attempted quite early on, with lamphear et al. ( ) in reviewing their own earlier work on maize seed expression of the b subunit of e coli heat-labile enterotoxin and the tgev s protein, with data on the potency, efficacy, and stability of these vaccines. another report followed in on the expression in maize seed of the s envelope protein of transmissible gastroenteritis coronavirus (tgev) of swine, and its protective efficacy in piglets fed with the seed (jilka ) . this followed an earlier demonstration of oral immunogenicity of the s protein n-terminal domain in transgenic potato tubers (gomez et al. ) . rabies too was a target for maize seed expression, with a report of g protein expression in transgenic maize seed to % of total soluble protein, and complete protection in a heterologous rabies strain challenge of mice orally immunized with one dose of * lg of g protein in seed extract (loza-rubio et al. ) . the same group later showed that sheep orally given a single dose of the transgenic maize seed containing * mg of the g protein were protected to the same extent as those immunized with a commercial inactivated vaccine ). the authors claimed that "this is the first study in which an orally administered edible vaccine showed efficacy in a polygastric model", which was an important development. maize was a popular target for both production and storage of recombinant proteins in early molecular farming times (see streatfield et al. ) ; however, other hosts were used too. for instance, the haemagglutinin (h) protein of rinderpest virus was expressed in transgenic pigeon pea to . % of total soluble protein (satyavathi et al. ) , and also in peanuts for a product that was both orally and parenterally immunogenic in mice (khandelwal et al. ); so too was glycoprotein b (gb) of human cytomegalovirus in seeds of transgenic tobacco (tackaberry et al. ) , the fusion (f) glycoprotein of newcastle disease virus in transgenic rice seed (yang et al. ) , and the serotype-specific vp protein of bluetongue virus in transgenic peanuts (athmaram et al. ) . most of these efforts were negated, however, by the one big scandal to have hit molecular farming as far as the use of food plants for vaccine production is concerned. in , aphis inspectors found volunteer tgev cp-expressing maize growing in soybean fields in two locations that were used to grow prodigene inc's tgev transgenic maize in the previous season (aphis )-and in one, the soybeans were harvested with the maize plants still standing and sent to a storage facility, where they were mixed with a large volume of other seeds. the company was fined and paid substantial cleanup costs, had to develop a new compliance implementation programme, and the us dept of agriculture issued new guidelines for trials of such products. this had an unfortunate knock-on effect for molecular farming, in that it resulted in an effectively voluntary moratorium on the use of food crops for recombinant protein production worldwide. the one major success story of early work on veterinary vaccines was the approval by the us department of agriculture's center for veterinary biologics of dow agrosciences' injectable newcastle disease virus (ndv) haemagglutin-based vaccine for poultry, that had been made in a suspension cultured n. tabacum cell line. sadly, the product was never sold: the company only wanted '… to demonstrate that our concert™ plant-cell-produced system is capable of producing a vaccine that is safe and effective and to demonstrate that it meets the requirements for approval under the rigorous usda regulatory system. ndv is well known and understood by the regulatory agency, so it served as an excellent model to prove this new technology' (rybicki ). the early historical account of molecular farming for veterinary vaccines given above gives an idea of the array of technologies available and used up to the mid- s: transgenic and transplastomic expression of subunit proteins; recombinant plant viruses either used to express whole vaccine candidate genes, or to display chosen peptides fused to their capsid proteins; fusion of vaccine protein genes to carrier proteins to improve immunogenicity, including by inherent adjuvant properties; candidate parenteral and oral vaccines to both viruses and bacteria; therapeutics for animals made in plants; use of plant cell cultures to make antigens. many proofs of principle were obtained, for candidate vaccines against a wide range of viral and bacterial disease agents; and proofs of efficacy for vaccines delivered orally or parenterally, in whole plant material or as extracts. while all of these aspects are still currently used in molecular farming, developments that have revolutionised the field were first, the widespread adoption of agrobacterium-mediated transient expression (agroinfiltration) of recombinant proteins; and second, the use of "deconstructed" plant virus-derived vectors delivered via agrobacterium to amplify expression (reviewed in rybicki ). these innovations enabled the advent of high-throughput testing of expression constructs, coupled with very rapid and generally higher yield production of vaccine antigens once optimal construct design had been determined. for example, our group investigated, via agroinfiltration techniques, three different codon usage schemes and three different intracellular localization strategies for optimization of human papillomavirus type l protein expression in n. benthamiana, in one large experiment over only days (maclean et al. ) . use of deconstructed tmv-based vectors delivered by agrobacterium routinely has allowed significant increases of antigen yield, up to grams per kilogram fresh tissue weight (gleba et al. ; klimyuk et al. ). the so-called tmv-based "launch vectors" of fraunhofer usa have also allowed significant yield increases and rapid production of antigens (chichester et al. ; shamloul et al. ) . improved non-replicating hyper-translational (ht) expression vectors derived from cowpea mosaic virus rna have also allowed significantly higher yields via agroinfiltration (sainsbury et al. (sainsbury et al. , and the possibility of multiple genes from the same vector (saxena et al. ) ; so too has the use of a ssdna geminivirus-derived set of vectors by different groups (huang et al. ; regnard et al. ) , and other ssdna plant (or other host) virus-derived vectors (rybicki and martin ) . the number of peptide display vectors/chimaeric protein fusion partners has multiplied: while self-replicating rtmv was once state of the art, now one may choose between tmv-and potato virus x (pvx)-based vectors (lico et al. ) , cucumber mosaic virus (cmv) cp (nemchinov and natilla ; zhao and hammond ) , bamboo mosaic virus (yang et al. ), pvx-vectored alternanthera mosaic virus (altmv) cp gene (tyulkina et al. ) , lichenase (lickm), cholera toxin b subunit (ctb), amv cp, and gus, as mentioned earlier. plant virus virions in particular are now seen as easily-made nanoparticles suitable for a number of vaccine-relevant purposes (steele et al. ) , including as selfadjuvanting peptide-based vaccine display vehicles (lebel et al. ; leclerc ) , and excellent inducers of cross-presentation by mhc receptors (hanafi et al. ) . the use of tags or small peptide fusion partners is now also considerably more sophisticated, with a variety of specialized tags to choose from. these include the now-ubiquitous xhis tag, used for ni + or other immobilised metal affinity chromatography (imac) protein purification technique; a new "cysta-tag" for the same purpose ; the n-terminal proline-rich domain of maize seed gamma zein (zera) that induces the formation of er-located protein bodies (torrent et al. ); elastin-like polypeptides (elps) with repeating pentapeptide 'vpgxg' sequences, or hydrophobins-small fungal proteins which alter the hydrophobicity of the fusion partner-both of which also form protein bodies (conley et al. ) . as examples, our group has recently successfully used elp fusion to the cp of beak and feather disease virus (bfdv) of parrots to aid in both accumulation and purification of the protein as a candidate vaccine (duvenage et al. ) . we have also used the zera tag as a protein body display vehicle for an ectopic m e moiety common to all influenzavirus a types, which could serve as a universal vaccine for these viruses (mbewana et al. ) . another potentially veterinary use of zera was in the enhancement of yersinia pestis f -v antigen fusion protein accumulation: this was *  higher than f -v alone in three different host plant systems-namely, n. benthamiana, alfalfa and n. tabacum nt suspension-cultured cells (alvarez et al. ) . the expression vehicles themselves have also been subject to engineering: it is now possible to precisely control glycosylation of plant-made proteins. this can be done by knock-out modification via rna interference (rnai) technology of the plant glycosyltransferases beta , -xylosyltransferase (xylt) and core alpha , -fucosyltransferase (fuct). these enzymes are responsible for the transfer of beta , -linked xylose and core alpha , -linked fucose residues to glycoprotein n-glycans, which are plant-specific modifications not found in mammalian glycoproteins (strasser et al. ) . it is also possible to use transient co-expression technologies to modify glycosylation (castilho and steinkellner ) , as well as to achieve almost completely native sialylated recombinant proteins by expression of whole mammalian glycosylation pathways in plants (castilho et al. ; steinkellner and castilho ) . it is possible to abolish n-glycosylation entirely, by co-expression of bacterial pngase f (mamedov and yusibov ) . one can also control endogenous plant proteases that may limit recombinant protein accumulation: for example, transient co-expression of secreted a /s protease inhibitor tomato cathepsin d inhibitor (slcdi) significantly lowered a and s protease activities in the n. benthamiana apoplast, while increasing recombinant protein content by * % (goulet et al. ) . it was found that co-expression of tomato cystatin slcys , which inhibits c a proteases, increased the transient expression yield of a monoclonal antibody in n. benthamiana by nearly % (robert et al. ) . it is also possible to reduce protease activity in cell suspension cultures by expression of specific antisense rnas, resulting in significantly increased accumulation of recombinant antibodies (mandal et al. ) . while suspension-cultured plant cells have been used for many years for molecular farming-and in fact were used for the only usda-licenced plant-produced animal vaccine, against ndv-new developments have made them an even more attractive prospect for low-cost vaccine production. use of flow cytometry with cell sorting, formerly the province of mammalian cell culture work only, has allowed high-expressing mab-producing tobacco by- cell lines from a heterogeneous population of cells by selecting the co-expressed fluorescent marker protein dsred (kirchhoff et al. ). however, one of the most exciting recent developments with this technology is the advent of the "cell pack": this is a technique for getting highly efficient (up to %) agrobacterium-mediated transient transformation of suspension-cultured cells that have been captured by suction onto a filter (rademacher ) . cell packs can be tiny (eppendorf tube tips) or large (e.g.: centimetres deep in a cm buchner funnel); protein expression occurs in immobilised cells in the presence of minimal liquid media, and can continue for days (https://tinyurl.com/k da q). the technology is ideal for rapid and high-throughput screening of expression-and the possibility exists for taking cells back into culture and selecting for permanent transfection. these are important developments, because of the acceptability of the products of plant cell cultures for production of biologics to regulatory bodies (see below). another production host highly suited to industrial-scale production is microalgae: they are easier to establish and use than plant cell cultures, and share all the same advantages of scalability, contained growth, and consistent transgene expression levels (specht and mayfield ) . a very important development for molecular farming has been the development of protocols for increasing yields and implementing industrial-scale production and downstream processing of vaccines and biologics, without which no large-scale trials could take place, or routine manufacturing occur. a useful development was use of a transgenic n. tabacum/n. glauca hybrid that does not synthesize alkaloids, is highly vigorous, can easily be propagated by vegetative cuttings and does not produce viable pollen, which greatly aids biocontainment (ling et al. ). the application of techniques more familiar to chemical engineers is also advantageous: for example, it proved possible, by sequential use of fractional factorial designs and response surface methodology, to optimize culture media for mab production in transgenic tobacco by- cells, and to increase mab yields up to -fold after days of culture compared to use of standard media (vasilev et al. ). the fraunhofer ime group have described generic chromatography-based strategies focusing on the binding behaviours of host cell proteins to chromatography resins under varying conditions of ph and conductivity (buyel and fischer ) . another useful technique from that group is a comprehensive description of the use of heat treatment of either intact leaves or of plant extracts to facilitate the industrial-scale removal of host cell proteins, optimised by a design-of-experiments approach that will also be familiar to engineers (buyel et al. ). many of these and other strategies used to optimise yields in molecular farming are reviewed here (twyman et al. ) . the establishment by various companies and institutes of facilities suitable for manufacture of animal and clinical trial material is also a very welcome development. as examples, the long-established kentucky bioprocessing inc (kbp) is a contract manufacturer capable of production from transgenic plants or transiently transfected plants, using the u.s. food and drug administration's current good manufacturing practices (cgmp) for pharmaceuticals, at scales up to thousands of kilograms of plants per week (https://www.kentuckybioprocessing.com/). they have recently produced and stockpiled mabs against ebolaviruses. another contract manufacturing firm with large production capacity is ibio inc: like kbp, they have a wide range of patents on their proprietary gene expression technology (holtz et al. ) . they are also partnering with a range of agencies and companies, including with the brazilian oswaldo cruz foundation for plant-made yellow fever vaccine, and the us dept of defense and the bill & melinda gates foundation for influenza vaccines (http://www.ibioinc.com/). the fraunhofer usa center for molecular biotechnology (http://www.fhcmb. org/) is a not-for-profit research and development organisation, that offers "… plant-based protein production, purification, scale-up and gmp manufacturing to support the development of vaccines, therapeutics and diagnostics", also with proprietary expression platforms, and can take products right through to fill and finish. the fraunhofer ime in aachen also has a state-of-the-art mechanised plant production facility still under construction as of . the regulatory environment has changed for the better, even though it was not in truth as inimical as first supposed: this was borne out by the fact that as early as , the cuban regulatory agencies and the usda had approved plant-made mabs for the purification of an already-licenced yeast-made hepatitis b vaccine, and the tobacco cell-made ndv vaccines, respectively (rybicki ). as another early example, the fraunhofer ime molecular farming group published in that use of whole plants for biologics production lacks intrinsic benefits of cell culture techniques, such as precise control over growth conditions, batch-to-batch product consistency, sterile containment, and it being much harder to be in compliance with good manufacturing practice (gmp) (hellwig et al. ). they pointed out that plant cell suspension cultures have all the merits of microbial and animal cell cultures, have an established track record for secondary metabolite production, and are far cheaper to use. these justifications notwithstanding, the same group later noted, in a review on gmp issues for plant-made proteins in whole plants, that: "when [plant-derived] recombinant proteins are intended for medical use… they fall under the same regulatory guidelines for manufacturing that cover drugs from all other sources, and when such proteins enter clinical development this includes the requirement for production according to [gmp] . in principle, the well-characterized gmp regulations that apply to pharmaceutical proteins produced in bacteria and mammalian cells are directly transferrable to plants" . they subsequently were able to get gmp manufacturing authorisation from german authorities for making mabs from transgenic n. tabacum for a phase i clinical trial (ma et al. ) . other entities have also scaled and regularised production to allow production of materials for animal and clinical trial-and one of the most successful has been medicago inc., who presently has routine large-scale production of influenzavirus a haemagglutinin (ha)-based vlps for use in advanced human clinical trial (d'aoust et al. ) . in , medicago inc. succeeded in manufacturing million doses of an h n vlp-based influenza vaccine candidate in one month, by phase -appropriate cgmp, as part of the us defense advanced research projects agency (darpa)-funded challenge (darpa ) . a group in japan has also recently developed a gmp-compliant production process for a transgenic rice seed-based cholera vaccine-mucorice-ctb-which is simply polished, powdered seed, now in clinical trial (kashima et al. ) . as evidence of the increasing maturity of veterinary molecular farming, one of the editors of this book has co-authored a recent article on regulatory and commercial hurdles hampering the advance to market of plant-produced veterinary vaccines, covering developing business plans, assessing market opportunities, manufacturing scale-up, financing, protecting and using intellectual property, and regulatory approval (macdonald et al. ) . at first sight, molecular farming appears the ideal way to make recombinant protein-based veterinary vaccines: production of active ingredients is markedly cheaper per unit mass than by use of any animal tissue-culture system, and generally cheaper than yeast or bacterial culture (rybicki ); partially-purified or unprocessed extracts are highly unlikely to contain any animal pathogens; edible and oral vaccines appear highly feasible; the financial barrier to entry for manufacture appears far lower than for conventionally-made vaccines. it is possible to efficiently make bacterial proteins using bacterial-derived translational machinery in chloroplasts in transplastomic plants, as well as to make other proteins at very high yield; conventional transgenics have been used to make many vaccine candidates, with many proofs of efficacy; transient expression technologies have revolutionised the field in terms of providing high yields and very rapid development times from concept to product. and yet, only one product-dow's ndv vaccine-is registered for use, and that is not sold. it is possible that heavy investment by big industry players in conventional manufacturing technologies has stalled their uptake of molecular farming technology for veterinary vaccines and biologics: this has certainly been true for human biologics. however, perhaps developments from the human field could be used as a spur for uptake of veterinary vaccines and biologics: an example here is the licencing of protalix biotherapeutics' elelyso ® or glucocerebrosidase, a therapeutic for a genetic mitochondrial enzyme deficit called gaucher disease, made using transgenic carrot cell lines in litre plastic bag fermenters (http://protalix.com/ about/elelyso/). a contamination of genzyme's mammalian cell production facilities in with a mammalian calicivirus led to the fda allowing protalix to supply the drug to patients who needed it, and to accelerated licensure (bethencourt ). the company has also successfully tested oral administration of drugs in plant cells, which would be a highly welcome development: they claim that "oral delivery of protein therapies [is] possible due to the unique cellulose wall of plant cells that makes them resistant to degradation when passing through the digestive tract" (protalix ). another apposite example was the fortuitous availability of a plant-made anti-zaire ebolavirus mab cocktail known as zmapp™, at the height of the recent west african ebola disease outbreak (reviewed in rybicki ). this was made by transient expression in n. benthamiana, and only a few clinical trial doses were available: these were used under the humanitarian principle, and later the mabs were cleared for use by the fda in an efficacy trial just before the end of the epidemic (leafbio ). both these examples are of niche products that were not being made at large scale or for a large market by conventional techniques, and for which there was a sudden, pressing need that could not be supplied by other means. this could provide motivation for small companies to either develop inexpensive vaccines for emerging diseases, or to target niche vaccines or niche therapeutics, in the knowledge that large established entities are unwilling to take the risk. one example for the former possibility comes from the recent emergence of bluetongue virus (btv) disease in sheep and small ruminants in europe, due to northward spread of the insect vector with climate change (purse et al. ) : while attenuated live vaccines are available-south africa presently uses a cocktail of such viruses-concerns in europe about reassortment of virus dsrna genome components between vaccinated and naturally diseased animals, as well as of the safety of the vaccines in terms of possible under-attenuation which may result in disease development in certain sheep breeds (niedbalski ) , mean these are not being used. the irregular occurrence of outbreaks, and the limited number of strains involved, mean that stockpiling vaccines is desirable. however, killed vaccines still require growing potentially dangerous viruses, and while it is possible to make vlps in cell cultures and these are effective (pearson and roy ; roy et al. ) , the technology is too expensive for farm animal use. it is fortunate, therefore, that it is also possible to make btv- vlps via transient expression in n. benthamiana, and these are as effective in a single injected dose as the commercial vaccine (thuenemann et al. ) . there are currently no plans to manufacture this or other plant-produced btv vaccines for the european or other markets; however, this may soon change. an example for a niche vaccine product comes from ours and others' work on beak and feather disease virus (bfdv) vaccines: psittacines are highly valued companion animals; however, there are very few vaccines for their diseases, and none yet available for bfdv. while some recent work in this area has shown that recombinant cp can be made in e coli and in insect cells (heath et al. ; patterson et al. ; stewart et al. ) , that it appears to be protective (bonne et al. ) and that this can apparently form vlps (sarker et al. ) , it still appears that the protein is too expensive to produce for use as a vaccine. while initial work with plant production of bfdv cp was disappointing due to low yields, recent work from our group (duvenage et al. ) showed a significant increase in bfdv yield due to fusion with elastin-like polypeptide (elp), and good immunogenicity in mice. this, coupled with a very simple purification protocol enabled by elpylation (conley et al. ), could allow scalable, cheap production of bfdv vaccines. while therapeutics such as mabs or other biologics for veterinary use are generally limited to high-value companion animals, plant production could open up a hitherto neglected market niche. one excellent example is the manufacture in japan of canine interferon-a (tabayashi and matsumura ) : this is done via transgenic strawberries in a completely enclosed gmp-compliant facility, and the product is powdered strawberry extract given orally, to combat canine periodontal disease. another very recent example in dogs, albeit with them being used as a model for human disease, was the proof that lyophilised transplastomic lettuce leaves expressing ctb fusions of coagulation factor ix (fix) could be used orally in feed for > days in haemophilia b dogs with no ill effects-and that this treatment resulted in robust suppression of igg/inhibitor and ige formation against intravenously-provided fix, and a marked shortening of blood coagulation times (herzog et al. ). an example for agricultural use is the oral dosing of pigs with transgenic arabidopsis thaliana seeds containing designer igas against enterotoxigenic e coli (etec) (virdi et al. ) : this product consisted of dimeric llama-derived heavy chain variable region fused to the fc portion of a porcine iga and the porcine iga j chain and secretory component, which allowed production of dimeric secretory iga-like antibodies (vhh-iga). in a piglet feed-challenge experiment with etec, dosing piglets with mg/d per pig vhh-iga produced a progressive decline in bacterial shedding and a significantly higher weight gain than seen in control or other experimental pigs. a highly novel plant-made therapeutic product was the receptor binding domain of the tailspike protein gp from the p bacteriophage: this is known to reduce salmonella colonisation in the chicken gut (miletic et al. ) . purified elp-fused gp bound to salmonella enterica serovar typhimurium in vitro, and feeding lyophilized leaves containing gp -elp to newly hatched chickens showed that it has the potential to control salmonella contamination in commercially-raised fowl. these and other experiments are reviewed here (juarez et al. ; topp et al. ) , in articles that make an excellent case for plant-made immunotherapeutics for veterinary use. the one health concept has as one of its central themes the integration of opportunities for vaccine-based approaches for the prevention of zoonotic and emerging diseases across veterinary and human medicine (monath ) a set of disease agents which exemplify the potential strength of the one health approach are influenza viruses, and they have in fact been the focus of a number of international meetings and planning sessions (chien ; dwyer and kirkland ; kahn et al. ; ludwig et al. ; powdrill et al. ; short et al. ) . the unique mix of hosts that occurs in intensive agricultural environments that could give rise to pandemics-swine, birds and humans-is a major cause of international concern; so too is the development of suitable vaccines for the prevention of infection in domesticated birds, farmed swine, and humans. plants have been shown to be highly useful for the production of influenza vaccines, and indeed possibly the fastest ever production at scale of an influenzavirus a strain vaccine- month for million doses-was done by medicago inc. for h n pdm ha vlps in (rybicki ) . medicago also managed in , as an exercise to demonstrate preparedness, to produce grams of cgmp-grade plant-made h ha-only vlps only days after accessing the h ha gene cdna sequence, in response to an outbreak in china in the same year. the fact that plant-made influenza vaccines have worked very well in animal models means that they should be trialled extensively in domestic fowl and swine, to see if the maintenance of the viruses in these hosts can be curbed. as for companion animals, there is even a canine influenza vaccine candidate: following a h n outbreak in the us, a group in canada used the plant-derived filamentous malva mosaic virus (mamv) nanoparticles as a vaccine platform to display the highly conserved ectopic m e peptide and to increase its immunogenicity. together with the adjuvant ompc derived from salmonella typhi, the vaccine was protective against both the homologous virus and a heterosubtypic strain of influenza in mice, as well as eliciting antibodies reactive with m e peptides derived from h n , h n and h n strains and being immunogenic in dogs (leclerc et al. ) . given that brucellosis is listed as a one health priority, it is worth noting that a transgenic plant-produced b abortus outer membrane protein (u-omp ) was an effective oral vaccine in mice against a systemic challenge, eliciting an adaptive il- immune response (pasquevich et al. )-and that the protein has significant adjuvant activity, and oral vaccination of mice with u-omp plus salmonella antigens was protective against virulent challenge with s typhimurium (risso et al. ) . it is important to realise that, while vaccines are the target of this review, one health products can also be reagents to be used in more effective or cheaper diagnostic kits, and in particular for point-of-care devices, or for research laboratory use-and especially proteins that could be both a reagent and used as a candidate vaccine in animals and possibly humans. a few of the best potential one health targets for plant-made dual-function proteins would be proteins from middle eastern respiratory syndrome (mers) coronavirus (wirblich et al. ) , nipah and hendra viruses (landford and nunn ; mackenzie et al. ) , diagnostic/ vaccine candidate proteins from rift valley fever and crimean-congo haemorrhagic fever viruses (kortekaas ; monath ) . inexpensive and abundant proteins made from these agents could first serve as reagents in the development of cheap point-of-care diagnostics, and then as vaccine candidates in animals, if appropriate, and then possibly in humans. a useful example here is of the expression both by agroinfiltration in n. benthamiana as a reagent, and in transgenic n. tabacum roots and leaves as a vaccine, of a fused gcgn envelope glycoprotein-encoding gene from crimean-congo haemorrhagic fever virus (ghiasi et al. ). the protein yield was - mg/kg fresh plant weight. transgenic material was orally immunogenic, and elicited humoral and mucosal antibody responses, and antibodies bound inactivated virus used as a vaccine booster in some experiments. agroinfiltration-produced gngc was used as a reagent in elisa to detect immune responses. another study from our group was of the production of cchfv n protein in n. benthamiana by agroinfiltration specifically as a reagent for use in diagnostic tests (atkinson et al. ): a plant codon-optimised and xhis tagged n protein gene was found to accumulate best as a soluble protein in the cytoplasm, from which it could be easily purified by ammonium sulphate fractionation and immobilised ni + column chromatography. purified np was used in a validated indirect elisa to detect anti-cchfv igg in sera from convalescent human patients: this was successful for / samples, with no readings for samples from patients with no history of cchfv infection. the results were % concordant with those from a commercially available immunofluorescent assay. given that soluble n protein is hard to produce and difficult to purify from insect cell cultures, the plant-made product would seem to be a desirable replacement. while the same has been said in many venues over more than twenty years now, the field of molecular farming really does seem to be near to meeting its initial promise for veterinary use. all of the technology that is required for efficient, high-yield production of biologics is in place; downstream processing modalities have been well worked out by a number of near-and cgmp-compliant facilities; many candidate vaccines for a wide variety of pathogens have been tested; therapeutic biologics too for veterinary use are now feasible; regulatory agencies seem agreeable to considering plant-made products. the generally shorter regulatory path, the possibility of using less stringently purified products, and the very real possibility of using oral vaccines and therapeutics, should also be highly attractive for product developers. i sincerely hope, then, that realisation of the promise comes very soon. higher accumulation of f -v fusion recombinant protein in plants after induction of protein body formation noncompliance history high level expression of surface glycoprotein of rabies virus in tobacco leaves and its immunoprotective activity in mice integration and expression of bluetongue vp gene in somatic embryos of peanut through particle bombardment method plant-produced crimean-congo haemorrhagic fever virus nucleoprotein for use in indirect elisa expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax transformation of an edible crop with the paga gene of bacillus anthracis virus stalls genzyme plant production of human papillomavirus type virus-like particles in transgenic plants assessment of recombinant beak and feather disease virus capsid protein as a vaccine for psittacine beak and feather disease immunization with viruslike particles from cottontail rabbit papillomavirus (crpv) can protect against experimental crpv infection the rabbit viral skin papillomas and carcinomas: a model for the immunogenetics of hpv-associated carcinogenesis generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts immunization with potato plants expressing vp protein protects against rabbit hemorrhagic disease virus in planta protein sialylation through overexpression of the respective mammalian pathway transient expression of mammalian genes in n. benthamiana to modulate n-glycosylation immunogenicity of a subunit vaccine against bacillus anthracis a plant-produced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with bacillus anthracis ames spores how did international agencies perceive the avian influenza problem? the adoption and manufacture of the 'one world, one health' framework optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants protein body-inducing fusions for high-level production and purification of recombinant proteins in plants the production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza plant-derived vaccine protects target animals against a viral disease a novel methodology to develop a foot and mouth disease virus (fmdv) peptide-based vaccine in transgenic plants expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastin-like polypeptides influenza: one health in action gmp issues for recombinant plant-derived pharmaceutical proteins mice orally immunized with a transgenic plant expressing the glycoprotein of crimean-congo hemorrhagic fever virus successful oral prime-immunization with vp from rabbit haemorrhagic disease virus produced in transgenic plants using different fusion strategies plant viral vectors for delivery by agrobacterium oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus a protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast two distinct chimeric potexviruses share antigenic cross-presentation properties of mhc class i epitopes oral immunization with a recombinant bacterial antigen produced in transgenic plants the capsid protein of beak and feather disease virus binds to the viral dna and is responsible for transporting the replication-associated protein into the nucleus plant cell cultures for the production of recombinant proteins oral tolerance induction in hemophilia b dogs fed with transplastomic lettuce commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals immunogenicity of the epitope of the foot-and-mouth disease virus fused with a hepatitis b core protein as expressed in transgenic tobacco a dna replicon system for rapid high-level production of virus-like particles in plants human-derived, plant-produced monoclonal antibody for the treatment of anthrax an oral vaccine in maize protects against transmissible gastroenteritis virus in swine biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications swine and influenza: a challenge to one health research good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants systemic and oral immunogenicity of hemagglutinin protein of rinderpest virus expressed by transgenic peanut plants in a mouse model monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting production of recombinant antigens and antibodies in nicotiana benthamiana using 'magnifection' technology: gmp-compliant facilities for small-and large-scale manufacturing plant-produced cottontail rabbit papillomavirus l protein protects against tumor challenge: a proof-of-concept study one health approach to rift valley fever vaccine development plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge delivery of subunit vaccines in maize seed good governance in 'one health' approaches leafbio announces conclusion of zmapp™ clinical trial plant viruses as nanoparticle-based vaccines and adjuvants a novel m e based flu vaccine formulation for dogs plant viral epitope display systems for vaccine development the two-faced potato virus x: from plant pathogen to smart nanoparticle an interspecific nicotiana hybrid as a useful and cost-effective platform for production of animal vaccines in planta production of a candidate vaccine against bovine papillomavirus type induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein influenza, a one health paradigm-novel therapeutic strategies to fight a zoonotic pathogen with pandemic potential regulatory approval and a first-in-human phase i clinical trial of a monoclonal antibody produced in transgenic tobacco plants bringing plant-based veterinary vaccines to market: managing regulatory and commercial hurdles managing emerging diseases borne by fruit bats (flying foxes), with particular reference to henipaviruses and australian bat lyssavirus optimization of human papillomavirus type (hpv- ) l expression in plants: comparison of the suitability of different hpv- l gene variants and different cell-compartment localization in vivo deglycosylation of recombinant proteins in plants by co-expression with bacterial inhibition of protease activity by antisense rna improves recombinant protein production in nicotiana tabacum cv. bright yellow (by- ) suspension cells production of h n influenza virus matrix protein ectodomain protein bodies in tobacco plants and in insect cells as a candidate universal influenza vaccine a plant-produced bacteriophage tailspike protein for the control of salmonella vaccines against diseases transmitted from animals to humans: a one health paradigm transient expression of the ectodomain of matrix protein (m e) of avian influenza a virus in plants bluetongue vaccines in europe protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing l capsid epitopes an oral vaccine based on u-omp induces protection against b. abortus mucosal challenge by inducing an adaptive il- immune response in mice differential expression of two isolates of beak and feather disease virus capsid protein in escherichia coli genetically engineered multi-component virus-like particles as veterinary vaccines passive protection to bovine rotavirus (brv) infection induced by a brv vp * produced in plants using a tmv-based vector one health approach to influenza: assessment of critical issues and options invasion of bluetongue and other orbivirus infections into europe: the role of biological and climatic processes high level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector u-omp from brucella abortus is a useful adjuvant for vaccine formulations against salmonella infection in mice protection of recombinant mammalian antibodies from development-dependent proteolysis in leaves of nicotiana benthamiana long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection plant-produced vaccines: promise and reality plant-made vaccines for humans and animals plant-based vaccines against viruses virus-derived ssdna vectors for the expression of foreign proteins in plants expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus rna- peaq: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants a chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants an efficient approach for recombinant expression and purification of the viral capsid protein from beak and feather disease virus (bfdv) in escherichia coli expression of hemagglutinin protein of rinderpest virus in transgenic pigeon pea [cajanus cajan (l.) millsp.] plants virus-derived vectors for the expression of multiple proteins in plants optimization and utilization of agrobacteriummediated transient protein production in nicotiana algae-based oral recombinant vaccines synthetic plant virology for nanobiotechnology and nanomedicine n-glyco-engineering in plants: update on strategies and major achievements baculovirus expression of beak and feather disease virus (bfdv) capsid protein capable of self-assembly and haemagglutination generation of glyco-engineered nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like n-glycan structure corn as a production system for human and animal vaccines forefront study of plant biotechnology for practical use: development of oral drug for animal derived from transgenic strawberry increased yield of heterologous viral glycoprotein in the seeds of homozygous transgenic tobacco plants cultivated underground a method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles the case for plant-made veterinary immunotherapeutics protein body induction: a new tool to produce and recover recombinant proteins in plants optimizing the yield of recombinant pharmaceutical proteins in plants new viral vector for superproduction of epitopes of vaccine proteins in plants expression of an animal virus antigenic site on the surface of a plant virus particle expression of human papillomavirus type major capsid protein in transgenic nicotiana tabacum cv optimization of by- cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method orally fed seeds producing designer igas protect weaned piglets against enterotoxigenic escherichia coli infection oral immunogenicity of human papillomavirus-like particles expressed in potato expression of bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein vp protection of mice against challenge with foot and mouth disease virus (fmdv) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the fmdv structural protein vp protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants one-health: a safe, efficient, dual-use vaccine for humans and animals against middle east respiratory syndrome coronavirus and rabies virus induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes expression of the fusion glycoprotein of newcastle disease virus in transgenic rice and its immunogenicity in mice a plant-based multicomponent vaccine protects mice from enteric diseases expression in plants and immunogenicity of plant virus-based experimental rabies vaccine development of a candidate vaccine for newcastle disease virus by epitope display in the cucumber mosaic virus capsid protein key: cord- -z w wkm authors: beeler, judy a. title: human and animal viruses date: - - journal: maintaining cultures for biotechnology and industry doi: . /b - - / - sha: doc_id: cord_uid: z w wkm this chapter provides an overview of the human and animal viruses. viruses held to a low number of passages in animals or cell cultures represent a viral population that is similar to that found in nature, and freezing these pools guards against genetic mutations that occur during subsequent passage. aliquots of viral stocks frozen at a designated passage level can then be used for multiple and repeatable experiments with the same viral population. furthermore, it is important that consistency should be maintained during the production of viral vaccines; new lots of final product are prepared with frozen viral seed stocks that consistently reproduce the desired immunogenic and attenuation characteristics. to better appreciate the requirements for freezing and freeze drying of human and animal viruses, some consideration is given to understanding the structural and functional organization of this diverse group of microorganisms. the classification of viruses is based on morphological and physiochemical properties. thus, viruses are divided into those with dna or rna genomes and subdivided into families based on size and structural properties. several methods for preservation of viruses are included in the chapter. shackell's successful treatment of rabies virus in represents the first published account of preservation of an animal virus using a process that was an early version of freeze-drying (shackell, ) . prior to this, viruses were maintained by passage in animals. however, freeze-drying provided a way to maintain virally infected material over long periods of time with relative ease when compared to serial passage in a susceptible animal host. the first experiment to demonstrate that this method could provide long-term stability was reported for a bovine virus that had been freeze-dried in and was shown to be viable after being maintained at room temperature for years (fasquelle and barbier, ) . later, as mechanical freezers became more available, freezing viruses at - ~ or less was adopted as a practical and reliable method of virus preservation. influenza virus-infected mouse lung tissue and yellow fever virus were successfully frozen at - ~ by turner ( ) , and horsfall ( ) , using turner's methods, demonstrated successful storage of influenza, mouse pneumonitis, and canine distemper viruses. following these early successes, viral repositories, diagnostic and research laboratories, and vaccine manufacturing facilities have relied on freezing and freeze-drying for preservation of viruses and viral vaccines. the reasons for preserving viruses are similar to those for the preservation of other microorganisms and cells: successful preservation allows for long-term maintenance of consistent stocks and for reproducibility in the testing of viral samples and manufacture of vaccines. for example, field and clinical isolates need to be preserved for transport to testing laboratories; harvested virus from animals or cell cultures may not be processed immediately and require preservation until used. viruses held to a low number of passages in animals or cell cultures represent a viral population that is similar to that found in nature, and freezing these pools guards against genetic mutations that may occur during subsequent passage. aliquots of viral stocks frozen at a designated passage level can then be used for multiple and repeatable experiments with the same viral population. furthermore, it is extremely important that consistency be maintained during the production of viral vaccines; new lots of final product can be prepared with frozen viral seed stocks that consistently reproduce the desired immunogenic and attenuation characteristics. for example, the world health organization (who) maintains aliquots of the original attenuated poliovirus strains developed by dr. albert sabin in the s. these aliquots are frozen and, when amplified properly and kept to the prescribed low passage level, should produce a vaccine today with the same properties that characterized the original sabin vaccine. similarly, viruses that are currently under development for use as vectors for gene therapy must be highly characterized and homogeneous for safe and effective use and need to be maintained as frozen stocks. in order to better appreciate the requirements for freezing and freezedrying of human and animal viruses, some consideration must first be given to understanding the structural and functional organization of this diverse group of microorganisms. the classification of viruses is based on morphological and physiochemical properties. thus viruses are divided into those with dna or rna genomes and further subdivided into families based on size and structural properties. there are families of dna viruses and families of rna viruses (see table ). the genomes of viruses within a given family are similar in terms of their polarity, genome organization, and method of replication and have morphologic features in common, including size, shape, nucleocapsid symmetry, and presence or absence of an envelope. other characteristics that help to classify viruses include sensitivity to low ph, heat, and lipid solvents. sensitivity to ph is determined by subjecting a virus to a ph of . . loss of titer greater than one log indicates that the virus is ph sensitive and acid labile. heat treatment of a virus for min at ~ is a test of thermal stability. if the virus survives this treatment, with no more than a one log loss of infectivity, it is considered to be thermostable. treatment with lipid solvents, commonly ether and despite the similarities among the physical properties of these families of viruses, relatively few general rules for freezing or freeze-drying viruses can be made. for example, it is common to use membrane-stabilizing agents to preserve viruses with a lipid envelope. picornaviruses, which lack a lipid membrane, maintain infectivity in the presence of mgc . however, the optimal conditions for freezing specific viruses need to be determined empirically. significant reduction of morbidity and mortality for many viral infections has been accomplished by the successful application of viral vaccines. in the case of smallpox, eradication of this important human pathogen has been realized. polio has now been targeted as the next viral disease that can be eradicated by immunization. vaccine production requires the use of the "seed lot system" for consistent reproduction and quality control of vaccine lots. vaccine virus seeds are usually frozen at two passage levels prior to vaccine lot production and these stocks of virus are designated the master seed and the production or working seed, respectively. multiple aliquots of these seeds are prepared for reference as well as for characterization and standardized testing for possible contaminants. finally, it is also important to distribute the storage of the master virus seed and working seed among multiple freezers in order to be assured of preserving part of the stock in the event of a mechanical failure. the seed lot system establishes a viral stock that can be standardized and provides a source of viruses so that vaccine lots manufactured over decades will be closely related to the original seed lot. this is especially important for live-attenuated vaccines that in most cases have been derived by multiple serial passage of a clinical isolate of a virus. deviation from the attenuated passage number may result in undesirable phenotypic changes of the attenuated vaccine virus. this was the case with yellow fever vaccine when uncontrolled passage of this virus resulted in a vaccine with a higher than normal incidence of side reactions (fox et al., ) . presently, researchers are using the tools of molecular biology to produce a new class of recombinant viral agents that will be used for immunization or genetic therapy. for example, mammalian retroviruses that integrate into the host cell genome are being developed as vectors for delivery of human dna sequences. the newly acquired dna is expressed in the form of protein(s) that may be deficient in the host cell or may require de novo synthesis. this form of genetic therapy has been demonstrated routinely in vitro and in vivo in animal experiments and is being proposed as therapy for some human genetic diseases (for review, see roemer and friedmann, ) . in addition, recombinant viral vectors are also paving the way for a novel type of therapy in which cancer cells can be specifically targeted for infection by a recombinant virus; following infection the inserted gene is expressed and the gene product selectively makes the infected cancer cells more susceptible to subsequent chemotherapy or to natural immune surveillance in the host (collins et al., ; culver et al., ) . alternatively, recombinant vectors may express antisense rna that can bind to and block the activity of the host gene sequences. successful experiments of this kind require viral vectors that are thoroughly characterized and safe. seed stocks of the recombinant viral vectors constructed to express the desired nonviral gene sequences must be frozen or freeze-dried in order to be preserved for future experiments and clinical trials. a somewhat similar approach is being developed for vaccination using viral vectors as delivery vehicles for genes important for immunization. for example, one approach uses a highly attenuated vaccinia virus that carries, as part of its genome, various immunogenic proteins that would evoke protection against infection (moss and flexner, ) . other viral vectors used similarly include adenovirus and canarypox. antigens used for viral diagnostic purposes must also be quality controlled and, in general, would also be produced from a virus seed lot system. variation of quality or quantity of viral antigens in diagnostic kits and reagents may directly influence results in the diagnostic laboratory. regulation of the manufacture of diagnostics is similar to those for vaccine manufacture. regulations concerning the manufacture of biological products are published by the united states public health service ( cfr ) and outline the requirements for control of reagents and raw materials in the manufacture of diagnostics to screen blood and blood products. for these purposes, it is essential to use viral seed stocks that are appropriately characterized and preserved by freezing. viability of viruses is usually determined by the use of infectivity assays. these include assays whereby viral cytopathology is measured in cell cultures or, when this is not possible, potency may be determined by inoculation of animals or eggs. by far the most accepted and accurate quantitative measure of viral infectivity is the plaque assay. most viruses form plaques in susceptible host cells. generally, after virus inoculation on suitably sensitive cell monolayers, an agar overlay is applied so that viral cytopathology is localized. after an incubation period, viable cells are stained with a vital dye, revealing areas of cell death, the plaque-forming unit. most viral freezing and recovery experiments use the plaque assay to titer virus before and after treatment as a measure of viral activity. in this way, one can determine the efficiency of the freezing or freeze-drying process. electron microscopy (em) has also been used on occasion to visualize the effects of freezing and freeze-drying. these em experiments are limited by the purity and titer of the viral preparation as well as the subjective interpretation of observations. the success of freezing or freeze-drying of viruses is dependent on the proper preparation of the harvested virus. the source of virus may be animal tissues or organs, animal sera, virus-infected cell cultures, and, on occasion, egg allantoic or amniotic fluids. the harvested tissue will depend on the particular host-range of the virus being grown. harvesting methods are also dependent on the properties of viral replication. some viruses, e.g., herpes viruses, stay cell associated whereas other are readily released from the cell into the culture fluids. in general, viruses grown after passage in vivo require disruption of the infected animal tissue or organ by homogenizers or aerosol-controlled blenders. diluent consisting of buffer or cell culture media is added for release of the virus into the aqueous phase during cell disruption. depending on the virus, serum may be required as an additive to the diluent for optimal freezing ( table ). viruses that are found in blood may be harvested and frozen directly in the serum after separation from blood cells or by cocultivating infected peripheral blood lymphocytes (pbls) with susceptible cell lines. these preparations may be frozen without further treatment, although it is generally recommended that serum or pbls be frozen and thawed only once to achieve optimal virus recovery. similarly, cell culture harvests may require disruption of the cell monolayer as a first step. cells may be scraped from the culture vessel surface into the culture fluids or these fluids may be removed and the cells treated separately. in either case, cells are disrupted and virus is released. in certain cases, virus-infected cells are harvested for a seed preparation. gentle scraping and harvesting of these cells into the culture fluids or into flesh buffer or cell culture medium is required. the specific details for optimal harvesting procedures for individual viruses may be found in the literature. usually animal serum is used as a component of cell culture medium. fetal bovine serum (fbs) is the most frequently used additive by cell ( ) culturists and virologists because it is nontoxic to most cells and the protective effect during freezing is consistent. therefore, fbs doubles for cell nutrition as well as a cryoprotectant for freezing and freeze-drying. concentrations used for cell culturing and growing viruses range from . to % in cell culture medium. in many cases the cell culture medium containing serum constitutes the cyroprotective diluent that will be used for freezing. however, further addition of serum is required for many viruses (see table ). infectious materials should be handled according to the biosafety guidelines that apply for each virus. the rate of freezing and the final temperature must be considered for virus preparations being frozen for long-term storage. to maintain viral infectivity throughout this process, the integrity of the viral capsid, the viral envelope (if there is one), and the viral nucleic acid must be preserved. dimmock ( ) demonstrated that viral infectivity may be reduced by heat damage to both viral proteins and nucleic acid. he showed that the stability of these components varies with elevated temperature so that inactivation at a particular temperature takes place through whichever component is least stable at that temperature. in general, most viruses may be "snap frozen" in a dry ice/ethanol bath prior to storage at temperatures _<- ~ it may be preferable to freeze some viruses, such as cytomegalovirus or varicella-zoster virus, as viable infected cells. in this case, a slow controlled rate of freezing is recommended: cooling should be done at l~ to- ~ and then at ~ to - ~ (simione and brown, ) . it has also been reported that influenza and measles virus may benefit from this two-step freezing process (rowe and snowman, ) . storage temperature is also critical for some viruses with envelopes, such as influenza virus, respiratory syncytial virus, and measles virus. these all require storage at - ~ or lower for optimal recovery after long-term storage (rightsel and greiff, ; greiff et al., ; law and hull, ) . it is common practice for today's virologist to store frozen viruses in ultralow-temperature freezers at temperatures of - to - ~ freezing in liquid nitrogen or its vapor phase, a common practice for preservation of mammalian cells, is not necessary for most viruses. liquid nitrogen storage may be used as a "backup" system for viral seed repositories. no known conventional virus requires - ~ (liquid) or - ~ (vapor) nitrogen for short-or long-term storage. cryoprotectants in one of the first comprehensive studies of freezing and freeze-drying with representative members of most of the rna and dna virus families, rightsel and greiff ( ) demonstrated the importance of the suspending medium used for freezing and freeze-drying. medium containing skim milk, calcium lactobionate, normal serum albumin, dimethyl sulfoxide (dmso), glycerol, or magnesium chloride promoted better freezing and freeze-drying recovery with a range of viruses, especially enveloped rna viruses. wallis and melnick ( ) further demonstrated that dmso or serum acted as a cryoprotectant for four different enveloped viruses, herpes virus, measles virus, sindbis virus, and vesicular stomatitis virus. under the conditions described dmso performed somewhat better than serum as a cryoprotectant. the nonenveloped viruses, vaccinia, adenovirus, and poliovirus, did not require dmso or serum for retention of viability during freezing. the authors concluded that enveloped viruses were similar to mammalian cells and that dmso stabilizes the viral envelope much as it stabilizes the mammalian plasma membrane. however, another member of the herpes virus family, varicella-zoster (vz), is especially sensitive to freezing and freeze-drying. grose et al., ( ) used sucrose, potassium phosphate, sodium glutamate, and albumin in a cryoprotective "cocktail" for vz virus. for both cell-associated and cell-free virus, the cocktail was fully protective, with % recovery of infectivity after freezing to - ~ and freeze-drying. additionally, these authors found that glutamate and albumin could be removed from the cocktail without loss of protection. in contrast, scott and woodside ( ) showed that sodium glutamate was essential for the stability of freeze-dried herpes virus. certain viruses are stabilized by the addition of divalent cations. for example, respiratory syncytial virus was found to be stable for up to month at ~ following the addition of magnesium sulfate, and the titer of virus was maintained through several cycles of freezing and thawing when mgso was added (fernie and gerin, ) . likewise, poliovirus was stabilized in the presence of mgc (melnick et al., ) . alternatively, mm sodium phosphate at ph . stabilized poliovirus at - ~ for up to months (mauler and gruschkau, ) . cryoprotectants and the rate of freezing minimize the formation of ice crystals that can damage the viruses; similarly, it is common practice to rapidly thaw frozen viral stocks at a previously determined optimal temperature to minimize ice crystal formation. freeze-drying, or lyophilization, is a method for preserving viruses and other biological materials by removing water from frozen samples via sublimation. the result is a dried preparation that is usually more stable than wet-frozen preparations at various temperatures and for longer periods. the process is usually divided into three stages: prefreezing, sublimation drying under vacuum (primary drying), and desorption or secondary drying. prefreezing can be accomplished in special freezers or in the freeze-dryer itself. a freeze-dryer has a vaccuum pump that removes air from a chamber that holds vials of the material to be freeze-dried. water vapor generated through this process is condensed into a refrigerated trap and is not allowed to enter the vacuum pump. the rate of sublimation can be controlled by regulating the heat transfer to the frozen material. after final drying, inert gas is used to fill the vials and protect the freeze-dried material from the deleterious effects of oxygen. the freeze-drying cycle requires optimization for each virus. a typical cycle that has been used successfully for dengue liveattenuated virus vaccines (all four serotypes) consisted of freezing to - ~ for hours and allowing the condenser to reach a temperature of - ~ (k. eckels, unpublished data). vacuum was drawn to /zm of hg, at which time heat was applied at a controlled rate overnight (approximately hours). final drying occurred at - ~ for hours followed by backfilling the vials with dry, sterile nitrogen and capping. vaccine vials were finally stored at - ~ another cycle that preserves viral infectivity has been used for dengue candidate vaccines; this consisted of freezing to - ~ overnight followed by raising the temperature to - , , and finally to ~ over a -day period while vaccuum was applied (k. eckels, unpublished data). in both cases, care was taken to avoid subjecting this thermolabile virus to prolonged periods of high temperatures that could result in inactivation of viral infectivity. greiff and rightsel ( ) have successfully freeze-dried influenza and measles virus with cycles extending over a -hour period with shelf and product temperatures not exceeding ~ greiff ( ) has outlined an approach for the development of a successful freeze-drying cycle. similarly, by varying drying time, shelf temperature, and vacuum pressure an optimal lyophilization cycle for varicella-zoster vaccine was determined (bennett et al., ) . freeze-drying methods and cycles used by the american type culture collection (atcc) for their virus stocks can be found in simione and brown ( ) . stabilizers added to the viral suspension medium for freeze-drying are many of the same used for freezing. these additives promote preservation of viral infectivity by acting as antioxidants and by providing "bulk" to the virus preparation to allow easier reconstitution. also, they should be nonhygroscopic to avoid moisture contamination of the freeze-dried mate-rial. for vaccines, the stabilizers must also be nonimmunogenic and nonreactive in the vaccine recipient. one of the first stabilizers used for viral freeze-drying was . % gum acacia to preserve the infectivity of vaccinia virus (rivers and ward, ) . peptone and normal horse serum were also successfully used for freeze-drying of vaccinia virus (collier, ; sparkes and fenje, ) . calcium lactobionate and human serum albumin were cryoprotective for measles virus during freeze-drying (greiff and rightsel, ) , whereas cell culture medium alone was less protective. a combination of sucrose, phosphate, sodium glutamate, and albumin was used for freeze-drying pseudorabies and herpes viruses (scott and woodside, ; calnek et al., ) . varicella-zoster virus was also freeze-dried successfully in this combination of stabilizers, but grose ( ) found that he could eliminate glutamate and albumin from the mixture and still retain % recovery of this very labile virus. dmso cannot be used for stabilization in freeze-dried preparations because it becomes concentrated to toxic levels (greiff and rightsel, ) . a group of viruses that requires special attention to stabilizing media is the enteroviruses, for example, poliovirus and hepatitis a virus. early work with poliovirus demonstrated that it had a high degree of lability when freeze-dried, even with stabilizers. the lability is due to the presence of inorganic salts that may be present in the culture medium. when these are removed by dialysis prior to freeze-drying, losses do not occur (berge et al., ) . the atcc uses ultrafiltration, a more rapid type of dialysis to remove salts prior to freeze-drying of enteroviruses (simione and brown, ) . vaccines that require freeze-drying have special stabilizer requirements that allow them to be used safely in humans. animal serum cannot be used as a stabilizer and must be completely removed from the vaccine prior to bottling. when a protein substitute is required, human serum albumin has been used successfully for many vaccines. more recently, hydrolyzed gelatin has been used as a replacement for albumin (table ). both of these stabilizers are acceptable for human vaccines because they usually do not stimulate deleterious immune responses in the vaccinee. sugars such as sucrose, lactose, and sorbitol are also used either alone or in combination with a protein stabilizer. cell culture medium is often used as a buffer because many live vaccines are harvested in their culture medium without further processing. optimal moisture content of the freeze-dried product needs to be empirically determined for each viral preparation. a good freeze-drying cycle can attain a moisture content of % or less in the final product. however, depending on the virus, a higher moisture content may be desirable. a varicella virus vaccine with - % moisture following freeze-drying was found to be more stable than vaccines containing less moisture (bennett et al, ) . various viruses have been freeze-dried to approximately % moisture and shown to be stable over periods of time at ~ but better long-term stability can be achieved by storage at - ~ stability assays for retained infectivity following freeze-drying are done on rehydrated vials of freeze-dried virus. a volume of sterile, distilled water is used to reconstitute the vial, with titration of the virus done immediately in the appropriate plaque or infectivity assay. the best control for baseline infectivity would be the virus harvest that was assayed prior to freezedrying. often, frozen and thawed specimens are used for baseline titers. freeze-dried viruses are normally stored at ~ or - ~ accelerated stability studies can be done to test thermostability for shorter periods at elevated temperatures. often an ambient temperature of ~ or temperatures of - ~ are used for accelerated studies. these temperatures are chosen to study thermostability for viruses, mainly vaccines, that may not have continuous refrigeration available up to the time of use. this is a problem for many live viral vaccines that are being used in developing countries (widdus et al., ) . the largest application of freeze-drying of viruses occur for liveattenuated viral vaccines that are thermolabile. the first viral vaccine to be freeze-dried was yellow fever vaccine (penna, ) . recent advances have resulted in more stable, freeze-dried yellow fever vaccines (robin et al., ; burfoot et al., ) . the who standard for stability of this vaccine is no more than one log loss of potency held at ~ for weeks. a recent collaborative study of yellow fever vaccines from manufacturers demonstrated that of vaccines met this requirement. similar who stability standards are in place for live, freeze-dried,measles vaccine. a method to freeze-dry and stabilize trivalent, live, attenuated, oral polio vaccine is being sought so that this vaccine can be delivered and used in developing countries without significant loss in potency. it has been proposed that thermoresistant mutants of polioviruses might serve as the prototype for the development of heat-stable vaccine strains (kew, ) . alternatively, organic compounds such as win and r , which bind to polivirus vp , have been found to increase the half-life of poliovirus antigen -to -fold at temperatures up to ~ (rombaut et al., ) . these drugs stabilize the conformation of the viral capsid and exhibit a potent antiviral effect that would not make them suitable as stabilizers for a live virus vaccine. in the future, similar compounds could be designed to stabilize but not inhibit the replication of live poliovirus strains. other licensed, freeze-dried live-viral vaccines are those for mumps and rubella, whereas vaccines for varicella (chicken pox) and cytomegaloviruses are being developed that will require stabilization by freeze-drying. table lists licensed vaccines as well as those still being tested and the available published data on freeze-drying of these vaccines. american type culture collection parklawn drive rockville, maryland telephone: - - - (united states and canada, only) or - - - fax: - - - request for international reference materials should be made with a statement of intent from the investigator. the catalogue of animal viruses and antisera, chlamydiae, and rickittsiae atcc preservation methods: freezing and freeze-drying diagnostic procedures for viral, rickettsial, and chlamydial infections freezing and drying diagnostic procedures for viral, rickettsial, and chlamydial infections diagnostic procedures for viral, rickettsial, and chlamydial infections biological product freeze drying and formulation diagnostic procedures for viral, rickettsial, and chlamydial infections diagnostic procedures for viral, rickettsial, and chlamydial infections temperature-stable vaccines for developing countries world health organization edwards freeze-drying handbook diagnostic procedures for viral, rickettsial, and chlamydial infections atcc preservation methods: freezing and freeze-drying temperature-stable vaccines for developing countries: significance and development strategies the author would like to thank dr. kenneth eckels for guidance and sharing data on freeze-drying dengue viruses and drs. karen goldenthal, ron lundquist, and hana golding for reviewing the manuscript. key: cord- - d vd authors: phillips, b. c.; bauch, c. title: echo chambers as early warning signals of widespread vaccine refusal in social-epidemiological networks date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: d vd sudden shifts in population health and vaccination rates occur as the dynamics of some epidemiological models go through a critical point; literature shows that this is sometimes foreshadowed by early warning signals (ews). we investigate different structural measures of a network as candidate ews of infectious disease outbreaks and changes in popular vaccine sentiment. we construct a multiplex disease model coupling infectious disease spread and social contact dynamics. we find that the number and mean size of echo chambers predict transitions in the infection dynamics, as do opinion-based communities. graph modularity also gives early warnings, though the clustering coefficient shows no significant pre-outbreak changes. change point tests applied to the ews show decreasing efficacy as social norms strengthen. therefore, many measures of social network connectivity can predict approaching critical changes in vaccine uptake and aggregate health, thereby providing valuable tools for improving public health. low vaccine rates stemming from vaccine refusal result in outbreaks of vaccine-preventable diseases in some populations [ ] , and the high costs of intervention and treatment incurred by public health systems [ ] motivate us to find tools warning of epidemics. the connection between social network activ- ity and health issues in populations has long been exploited by researchers [ ] , especially relating to disease spread [ ] , with the assertion that firm understanding of social network structure is important to the implementation of effective change is driven primarily by exposure to news sources and contrasting views from neighbours, communities can support the reinforcement of sentiments already held [ ] . echo chambers, described as well connected groups of people promoting and reinforcing the same bias [ ] , have recently come under media scrutiny since these groups can facilitate vaccine scares [ ] , support political candidates [ ] , lead to skewed evaluation of objective fact and decreased accuracy of opinion [ ] . furthermore, some studies indicate that in some cases these homogeneous sub-networks may reinforce bias [ ] , in some part due to avoidance of cognitive dissonance [ ] . given that interaction between dislike agents in a network can sometimes correct false beliefs [ ] , these echo chambers may be seen as drivers of polarisation [ ] . this is especially since much anti-vaccine content is shared without thought of its veracity [ ] , with facebook anti-vaccine groups serving the dual purpose of opinion-reinforcing echo chamber and "fake news" source in a time where a large number of people draw on social media sites for their health information [ ] . in the same vein, much work has focused on the modularity of social networks. modularity is a graph theoretic measure of the segregation of a graph [ ] ; a high degree of modularity may indicate increasing segregation of a network into clusters [ ] , with other work showing that modularity is not a "direct nodes, where effective communication between disagreeing persons can lead to change of opinion [ ] . the occurrence of opinion change within opinion-based communities has seen much attention in political studies, with some work asserting the ability of the 'wisdom of the crowd' to overcome bias [ ] , while other work shows that group phenomena reinforce the opinions held [ ] . others have argued that a commonly held belief within a community can still become more accurate even as the homogeneity of the group increases [ ] . this leads to our interest in the rate of opinion change in the network as yet another potential indicator of dynamical regime change. systems moving from one polarised state to another undergo phase transition through a sole critical point [ ] . called critical transitions, they sometimes result in a demonstration of characteristic system behaviours such as critical slowing down [ ] . these events give us easily recognisable 'hints' of approaching transitions called early warning signals [ ] . we show that trends in all of the measurements described above (modularity, global clustering coefficient, census and sizes of communities and echo chambers) provide early warning signals of epidemic and vaccine crisis events for a coupled disease-behaviour model of childhood disease. we use a binary vaccine opinion dynamic and an sirv p disease process occurring on a random network to model a childhood infectious disease. by quantifying and comparing their performance, we find that trends in the sizes of anti-vaccine communities were the best-performing signals, with the modularity and clustering coefficients of communities also performing well. all in all, we verify that changes to fundamental graph structure driven solely by opinion dynamics are good predictors of disease events and aggregate sentiment towards vaccination. this paper is organised as follows: in sec. , we will describe the disease- behaviour model used in the study and a description of the warning signals used. section will show the change in trends of the signals with respect to perceived risk of adverse vaccine effects and the social pressure of an injunctive norm, as well as a quantification of the warning provided by each measure through the use of the standard normal homogeneity change point test (snht). we will elaborate on the limitations of the measures used and the implication of the results in sec. . information showing the results obtained through the application of other change point tests are given in the appendix. for simulation, we use an abm identical to that described in [ ] , shown in fig. . a perfectly effective vaccine with no effect on mortality is immediately available to susceptible agents upon gaining pro-vaccine status s → v p . as shown in fig. a , the disease follows the sirv p model; each agent physically interacts with all their neighbours per time step. if a susceptible agent becomes (a) schematic of the infection dynamics of the model. effective contacts occur between susceptible s and infected i agents with probability p per time step ( week). upon deciding to vaccinate (with probability pn(n → vs)), a susceptible agent n becomes physically vaccinated (s → vp). infection lasts � = weeks after which agents recover (i → r). upon death (with probability µ per week), an agent is "rebirthed" with either vaccinated (probability α · µ) or susceptible (probability ( − α) · µ) status. representation of the opinion dynamics of the model. per time step, each agent switches between pro-(vs) and anti-vaccine (n ) opinion with probabilities pn(n → vs) and pn(vs → n ) respectively upon interaction with a dissenting neighbour. α gives the probability of being birthed with pro-vaccine opinion vs. become both ill and infectious for � = time steps (each time step represents a week); recovery i → r and vaccination s → v p are both permanent. injunctive social norms ≤ σ ≤ and a cost of − . ≤ κ ≤ . represent peer pressure and adverse vaccine effects respectively [ ] . figure features a binary social (opinion) dynamic, where agents demonstrate either pro-vaccine (v s ) or anti-vaccine (n) sentiment. change of sentiment occurs through imitation of neighbours, where a randomly chosen neighbour is sampled each time step; an effective interaction with a disagreeing neighbour (with different sentiment than the agent sampling) prompts a reevaluation and change of sentiment with probability p n (n → v s ) for anti-vaccine agents and p n (v s → n ) for pro-vaccine agents. any susceptible agent that adopts pro-vaccine sentiment (n → v s ) is immediately vaccinated (s → v p ); for agent n, these changes in sentiment depend on the perceived vaccine risk kappa and . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint neighbours i n : indices u n →vs n and u vs→n n in equation ( ) are utility functions defined as where d * n represents the number of neighbours of n with sentiment * , with d n representing the total number of neighbours. four of the six ews explored in this paper are related to the detection of community structure in networks; the global clustering coefficient, echo chamber, opinion-based community and modularity score are all important tools that enable biological modelling [ ] . the topological phenomenon of community refers not to a single central construct, but rather a general notion of variation in connection density. communities in social networks are vaguely defined in the literature as groups with the following basic property: members of the community are more connected to each other than with non-members [ ] . vagueness in science usually leads to artistic licence and the specific treatment of context and purpose; by the above definition, communities can then be alternately conceptualised in different areas and studies as modules, clusters, groups and so on [ ] . such refinements then lead to tighter and more technical definitions. here, we conceptualise topological communities as (connected) components on the network. components in undirected networks are maximal disjoint groups of agents such that there is a path between every pair of agents in the group. [ ] . even more specifically, the concept of a giant connected com- ponent (gcc) describes a component that contains a "significant fraction of all the nodes" [ ] . the role of components (both non-giant and giant) in spreading processes can be conceptualised as such: where some infection can be spread from person to person, gccs are formed by historical person-to-person contacts (as opposed to current contacts) [ ] . practical indirect analysis and exploitation of this component structure in policy design and epidemiological intervention is facilitated through contact tracing [ ] . recent political upheaval has thrust the phenomenon of the echo chamber into social consciousness and modern parlance, with many lay articles arguing for and against their existence , though many articles do not directly define the concept before exploiting it. there exist different definitions of echo chambers in . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . literature; also called tribes [ ] , they can be described either as a community where at least some percentage of the members hold a particular sentiment [ ] , or else a subset of community members overwhelmingly likely to restrict their neighbourhood communication to contacts with shared opinion [ ] . as such, echo chambers are usually conceptualised as closed subsystems of social networks containing members with a single orientation [ ] , and are therefore considered synonymous with homophily and strongly associated with the quick spread of misinformation [ ] , polarisation and the insulation and reinforcement of belief despite their veracity [ ] . one example is the concept of the 'filter bubble', detailing the biased filtering of information unfortunately created by personalisation algorithms [ ] . given the described ubiquity on social networks and their importance in information diffusion (and therefore decision making) [ ] , we test whether observations of the size and number of communities z * and echo chambers j * give early warnings of approaching vaccine scares and crises, as well as falls in vaccination rates. we retrieve the echo chambers j * by first listing the agents in the network with the desired vaccine opinion; the members of the network are then sorted into two primary classes. the peripheral members maintain links with at least one disagreeing neighbour and core members only maintain links with agreeing neighbours (these are direct analogies of the boundary and interior of a topological space respectively). we then characterise echo chambers as communities of core members. clustering has been shown to greatly facilitate the spread of such phenomena as political extremism [ ] and infectious disease [ ] . specifically, clustering refers to the propensity of connection between two persons if they have a mutual friend [ ] . as an indicator of this organisation, the global clustering coefficient indicates the prevalence of clusters, dense highly-connected groups of nodes in a graph [ ] . this done by finding the density of triplets on the network. an open triplet is a group of three nodes connected by edges, while a closed triplet is a group of three nodes joined by three unique edges (also called triangles for this reason). the global clustering coefficient (gcc) is then calculated as the modularity measure is similar to the global clustering coefficient as a measure of organisation; highly modular networks possess many modules, which feature dense interconnectivity between nodes similar in some way and sparse connectivity between dislike nodes. specifically, this measures the correlation between the probability of connection of two nodes and their membership of . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . the same module [ ] . the modularity score is calculated as follows [ ] ; let an undirected network be divided into two disjoint groups Λ and Ξ, with each node n given the score with an adjacency matrix a of the network, so that a ij gives the number of edges between nodes i and j. let k * represent the degree of node * , so that gives the number of edges in the network and the expected number of edges between nodes i and j is the network modularity (q) is then given as in the model investigated, vaccination occurs only through the first adoption of pro-vaccine opinion n → v s (sec. . ); as such, the number of changes of opinion Θ * undertaken by agents of either opinion can conceivably describe both the social and infection dynamics of the model and thereby yield warning signals of sudden transitions. moreover, equation ( ) shows that the probability of switching sentiment is sensitive to the number of infected agents in the individual neighbourhood; this dependence makes it highly likely that the probability of having an infected neighbour Γ * is correlated not only with vaccine coverage both also with the rate of change of opinion throughout the length of each simulation. both (infection and social) layers of the network have an erdős-rényi random network structure g ( , . ); network size n = and mean degree � d n � = were chosen for alignment with studies of similar coupled behaviour-infection models [ , ] , as well as for computational tractability. each simulation starts with proportion α = . of vaccinated pro-vaccine agents (states represented by pair (v s , v p )), with all others being susceptible anti-vaccine agents (pairs (n, s)). the probability of death per time step is µ, so that α · µ gives the probability of reset with initial state v s and ( − α) · µ the probability of reset with initial state s. ξ = × − represents the probability of switching sentiment randomly. noise parameter ξ = . represents unsystematic fluctuations commonly seen in empirical studies [ ] . the birth/death probability µ = . × − affords an average life span of years to each agent . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint [ ] . this is due to the permanence of the vaccine; an agent can change their vaccine opinion throughout their lifetime, but they cannot become 'unvaccinated' should their stances change. the chosen parameter space (κ, σ) ∈ [− , ] × [ , ] was sufficiently broad to capture transitions in both the infection (fig. a. a) and social (fig. a. b) dynamics. the contours in each panel of fig. (a. ) show the obvious correspondence between social (k s ) and infection (k p ) transitions and substantial changes in the clustering coefficient of the pro-vaccine sub-network � c vs � (fig. a. c ) and the mean size of anti-vaccine communities � |z n | � (fig. a. d ). . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . we again define the infection transition k p as the value of perceived vaccine risk κ value at which the mean number of recovered agents in the model surpasses that of the vaccinated agents and vice versa, so that this is marked by the dotted vertical line in fig. a showing the approximate intersection of (a) changes in trend in the κ-series of output variables the distance between the two transitions (i.e. k p − k s ) is called the intertransition distance [ ] ; as with similar models, we find that this intertransition distance decreases with increasing strength of the social norm σ. this is ex- plicitly demonstrated by fig a, where an increase in the social norm from σ = (fig. a, left) to σ = . (fig. a, right) brings the two vertical lines (representing transitions k s and k p respectively) together. figure b gives a full picture of the intertransition distance over the investigated parameter range ≤ σ ≤ . ; its decreasing trend with strengthening social norm σ is shown by the purple curve, with the inset panel showing the locations of the social k s (blue) and infection k p (red) transitions. the positivity of the graph tells us that k s < k p for all strengths of the social norm σ ≤ . , so that the social transition always precedes the disease transition. since physical vaccination is driven primarily by changes in sentiment, this is to be expected; strengthening social norms increases the alignment of behaviour and vaccine uptake, resulting in decreasing intertransition distance k p − k s . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint (a) mean community size; (b) mean number of communities; #z n (anti-vaccine), #z v (pro-vaccine). (c) mean echo chamber size; warning signals can potentially indicate both transitions, or maybe only one of the two; this subtlety is lost with shrinking intertransition distance and so may impact the predictive power of any early warning signals tested. figure shows the trends in the means of the numbers and sizes of communities and echo chambers (both pro-and anti-vaccine) at equilibrium. as for the number of echo chambers � #j * � (fig. d) , there is not much resemblance to the trend of the mean number of connected components � #z * � (fig. b) ; this is partly due to the low occurrence of echo chambers in this network. for σ = (fig d, left) , there is no warning given by the mean number of pro-vaccine echo chambers � #j v � ≈ , whereas the mean number of anti-vaccine echo chambers � #j n � increases before k s , reaching a maximum between the two transitions . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint and reaching zero value approaching k p . at first glance, the change in trend as the social norm strengthens to σ = . (fig d, right) � (fig. a) , the number of pro-vaccine communities � #z v � (fig. c ) and the number of pro-vaccine echo chambers � #j v � (fig. c) ; they all give very little warning of the social transition k s for all strengths of the social norm σ, as well as giving the best warning (of all the ews) for only ≈ % of the tested range of the social norm σ. they all demonstrate significantly lower lead distances than those of the other ews, giving the highest lead distance of all ews for only % of σ values (as compared to ≥ % for other ews shown in fig. ). the performance of these ews under different change point detection tests is shown in figs. (c. -c. ). as social norm σ increases, pressure on each agent to conform to surrounding opinion becomes the main driver of self-organisation of the social dynamics. since it also increases the . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . speed of this transition between these opposing organised states, lead distances k s − snht{ * } will decrease as the social morn σ increases. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . this behaviour disappears when social norm σ → . (fig. b. c, right) due to the shrinking intertransition distance for σ = and σ = . are similar ( . and . respectively), showing that the distances between the warnings and the transitions k p are similar in both cases. as σ increases to . , k s 'moves closer' to k p ; this suggests that any warning of k s occurs incidentally, rather than being directly caused by the model dynamics. this is intuitive; any measure of the probability of having an infected neighbour is an observation of the infection dynamics, therefore an assumption of some direct for all the ews in fig. (b. ) , we can see that lead distance decreases with strengthening social norm σ, with all the ews eventually failing (giving negative lead distances, so that warnings follow k s -these are useless); modularity scores for the sub-networks formed by pro-vaccine � q v � (fig. b. a ) and anti-vaccine � q n � (fig. b. a ) agents give useful warnings for all social norms σ ≤ . , while both the number of opinion changes � Θ * � (fig. b. b ) and the probability of having an infected neighbour snht �� Γ * �� give useful signals for σ ≤ . . as stated, the global clustering coefficient of the entire social network snht{ � c Σ � } (fig. b. d ) was undefined for most σ and negative for quite a few others, resulting in the worst performance of all the ews tested and giving the highest lead distance for only % of the total range of the social norm σ. the sub-networks generated by pro-vaccine agents gave often unsubstantial though positive leads snht{ � c v � } (fig. b. d) , while the sub-networks formed by anti-vaccine agents (fig. b. a ) gave very little lead distance over most of the range of σ. the performances of these ews under different change point detection tests are shown in appendix c. we compare the ews by finding the proportion of σ values for which each ews gives the largest warning; this is shown in fig. . we specify a good warning as one that gives the highest lead distance of all warnings for a single σ value and a bad warning as one that gives either a minimal, negative or undefined lead distance. for many σ values, the largest and smallest lead distances were not unique, so that the ratios on neither side sum to %. comparison of the panels of fig. gives a notion of 'dependability'; were a restricted set of ews to be employed, we would prefer good ews (ones that . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . give the largest lead distances) that aren't also bad (giving the smallest lead distances). some of the foremost ews best satisfying this criterion are the counts of all different opinion changes � Θ * � and the mean size of anti-vaccine communities � |z n | � ( % best, % worst). the best performers are the modularity scores of the opinion sub-networks � q v � , being the best ews for % of σ values, with % bad warnings. conversely, fig. suggests that the numbers of echo chambers � #j * � both bring up the rear, providing good warnings for only % of σ values while giving bad warnings for % of social norm σ values tested. overall, all observations of the sizes of anti -vaccine echo chambers |j n | on the network are poor ews. conversely, the sizes of pro-vaccine echo chambers |j v | perform generally well (≥ % best, % worst). also, the mean and maximum sizes of anti-vaccine communities ( � |z n | � and � max(|z n |) � respectively) give two of the highest ratios of best warnings (both %) and not many bad warnings (≤ %), but observations of the minimum size � min(|z n |) � give only % good warnings and % bad warnings. the global clustering coefficient � c * � performed particularly badly as an ews, with ≤ % best and % − % worst warnings. another observation made from fig. would be the relationship between the percentages of good and bad warnings; they are actually strongly anti- correlated, with a coefficient of − . . the previously shown behaviours of the lead distances eliminate the possibility of an ews that densely alternates between good and bad warnings, but some ews do neither; for example, the total modularity of the network � q Σ � is trivially neither good ( %) nor bad ( %) by virtue of being everywhere undefined. nontrivially, the clustering coefficient of the anti-vaccine sub-network � c n � gives intermediate warnings (neither good nor bad) for % of social norm strengths σ. therefore, no choice need be made between minimising the percentage of bad warnings and maximising the percentage of good warnings; both strategies yield largely identical results. figure show the performance of each ews per σ value. red tiles show where the ews gave the smallest positive lead distance of all its peers, while green tiles represent the σ values for which the ews gave the largest lead distance. yellow columns show where all ews gave equal lead distances and black tiles represent failed warnings (either undefined or negative lead distances). therefore, the relative length of an ews' red bar in fig. represents the per- centage of that ews' red tiles in its row in fig. ; the same correspondence holds between the length of an ews' green bar in fig. and the percentage of green tiles in the related row in fig. . the main insight provided by fig. deals with patterns of performance; for instance, the overwhelming red colouration of the rows corresponding to the mean number of pro-vaccine communities and anti-vaccine echo chambers ( � #z v � , � #j n � respectively) and all ews related to anti-vaccine echo chambers j n show that these ews are quantitatively the worst of the group. also, there is no detectable pattern in performance visible on the grid; in other words, the effectiveness of the ews cannot be broken down by ranges of σ value. for . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . higher values of the social norm σ ≥ . , the prevalence of yellow columns provides the observation that performance seems not to vary as much among the ews as it does for smaller σ values, but nothing else is immediately apparent. for social norms σ = . and σ = , none of the ews give valid warnings; lead distances are all negative, except for the total clustering coef- ficient � c Σ � , the number of opinion changes by anti-vaccine agents � Θ n � and the network's total modularity score � q Σ � which are undefined. this confirms behaviour seen for large σ in figs. and (b. ); indeed, � c Σ � is undefined for most social norms σ because of the disconnection in the social network. total modularity � q Σ � is everywhere undefined (row of white tiles) for this reason. in this paper, we tested the use and effectiveness of different network measures as early warning signals (ews) of sudden transitions in the social and infection dynamics of a multiplex model of disease. for the parameter values used, we found that observations of the mean and maximum sizes of anti-vaccine communities appear to be the most effective ews of all tested, unlike the size of the smallest anti-vaccine community (though it does give warnings signals); trends in the global clustering coefficient of the sub networks formed by pro-and anti-vaccine agents respectively also marked these events, as well as the number of both respective communities and echo chambers preceding both transitions. this reflects the breakup of connected components on a network preceding a . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . critical transition, an observation well supported by literature on percolation thresholds in random graphs. a phenomenon of particular interest in this study was the formation and breakup of pro-and anti-vaccine echo chambers; we found that all observations of the sizes of pro-vaccine echo chambers (maximum, minimum and mean) performed well as warning signals, while observations of the sizes of anti -vaccine echo chambers performed poorly compared to other ews. the modularity measure of the social network and the rate of opinion changes also warn of transitions of the social and disease transitions, representing changes in aggregate vaccine opinion and vaccine uptake crises respectively. as a direct observation of the infection dynamics of the model, the probability of having an infected neighbour (for both pro-and anti-vaccine agents) performed well as an ews of vaccine crisis. through our proposal and study of effective graph connectivity measures, this study complements others in the field of early warning signals. a potential limitation to the study is our strict definition of an echo chamber; it remains to be seen whether different descriptions such as those featured in other studies of social media networks will result in a more effective or dependable ews. also, the graph connectivity measures seem suitable for tracking the dynamics of an evolving network; the inclusion of preferential link formation in the dynamics, as well as social and 'on the ground' interventions for different strengths of the social norm, present other interesting avenues of research. finally, the inclusion of directionality of communication in the network may render the model more realistic [ , , , ] . together with other markers of spatial correlation and aggregation, the graph connectivity measures presented here contribute to the set of tools allowing us to leverage the ubiquity of social media involvement and the resulting data sets in the pursuit of adaptive strategies for maintaining public health. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint cambridge ¡england¿ ;, . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint association between vaccine refusal and vaccine-preventable diseases in the united states: a review of measles and pertussis estimating the cost of vaccine development against epidemic infectious diseases: a cost minimisation study part -social and political challenges: . western democracy in crisis epidemic spreading in multiplex networks influenced by opinion exchanges on vaccination the role of community structure in opinion cluster formation vaccine rejection and hesitancy: a review and call to action early warning signals in plant disease outbreaks an opinion-driven behavioral dynamics model for addictive behaviors a comparative analysis of community detection algorithms on artificial networks social network community detection using strongly connected components community detection algorithms: a comparative analysis people opinion topic model: opinion based user clustering in social networks statistical physics of social dynamics opinion dynamics driven by various ways of averaging continuous opinions and discrete actions in opinion dynam- ics problems sociophysics: a physicist's modeling of psycho-political phenomena the echo chamber: strategic voting and homophily in social networks polarization of the vaccination debate on facebook how social influence can undermine the wisdom of crowd effect the law of group polarization back to the future of dissonance theory: cognitive consis- tency as a core motive the wisdom of polarized crowds going to extremes: how like minds unite and divide the impact of search engine selection and sorting criteria on vaccination beliefs and attitudes: two experiments manipulating google output fake news and advertising on social media: a study of the anti-vaccination movement modularity and community structure in networks, proceedings of the national academy of sciences political polarization on twitter a measure of polarization on social media networks based on community boundaries on modularity of social network communities: the spectral characterization acm international conference on web intelligence and intelligent agent technology clustering coefficients for correlation networks epidemic dynamics on complex net- works the small world yields the most effective information spreading the wisdom of partisan crowds network dynamics of social influence in the wisdom of crowds condensed-matter physics theory of early warning signals of disease emergence and leading indicators of elimination anticipating the emergence of infectious diseases spatial early warning signals of social and epidemiological tipping points in a coupled behaviour-disease network the influence of social norms on the dynamics of vaccinating behaviour for paediatric infectious diseases defining and identifying communities in networks social network analysis : methods and applications, structural analysis in the social sciences networks, crowds, and markets: reasoning about a highly connected world networks and the epidemiology of infectious disease, interdisciplinary perspectives on infectious diseases chains of affection: the structure of adolescent romantic and sexual networks galaxyscope: finding the "truth of tribes" on social media tweeting from left to right: is online political communication more than an echo chamber? mitigating the spread of fake news by identifying and disrupting echo chambers users enjoy the opportunities for political debate and engagement that social media facilitates, but many more express resignation, frustration over the tone and content of social platforms echo chamber? what echo chamber? reviewing the evidence comparing extremism propagation patterns in continuous opinion models dynamics and control of diseases in networks with community structure clustering and preferential attachment in growing networks clustering in weighted networks collective dynamics of 'small-world' networks detecting complex network modularity by dynamical clustering finding and evaluating community structure in networks spatial autocorrelation as an early warning signal of regime shifts in a multiplex disease-model behaviour network stochastic formulation of ecological models and their applications table - - - life expectancy and other elements of the life table trend: non-parametric trend tests and change-point detection social media mining: an introduction what is twitter interest communities and flow roles in directed networks: the twitter network of the uk riots information network or social network? the structure of the twitter follow graph key: cord- -i o rs authors: pagliusi, sonia; ting, ching-chia; lobos, fernando title: vaccines: shaping global health() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: i o rs the developing countries vaccine manufacturers’ network (dcvmn) gathered leaders in immunization programs, vaccine manufacturing, representatives of the argentinean health authorities and pan american health organization, among other global health stakeholders, for its th annual general meeting in buenos aires, to reflect on how vaccines are shaping global health. polio eradication and elimination of measles and rubella from the americas is a result of successful collaboration, made possible by timely supply of affordable vaccines. after decades of intense competition for high-value markets, collaboration with developing countries has become critical, and involvement of multiple manufacturers as well as public- and private-sector investments are essential, for developing new vaccines against emerging infectious diseases. the recent zika virus outbreak and the accelerated ebola vaccine development exemplify the need for international partnerships to combat infectious diseases. a new player, coalition for epidemic preparedness innovations (cepi) has made its entrance in the global health community, aiming to stimulate research preparedness against emerging infections. face-to-face panel discussions facilitated the dialogue around challenges, such as risks of viability to vaccine development and regulatory convergence, to improve access to sustainable vaccine supply. it was discussed that joint efforts to optimizing regulatory pathways in developing countries, reducing registration time by up to %, are required. outbreaks of emerging infections and the global polio eradication and containment challenges are reminders of the importance of vaccines’ access, and of the importance of new public-private partnerships. the developing countries vaccine manufacturers' network (dcvmn) is the world's largest vaccine-industry alliance. in , corporate members are working to provide high-quality vaccines, and contribute to global health initiatives, ensuring uninterrupted vaccine supply to countries, to advance eradication of polio and facilitate response to emerging infectious diseases (eids) or outbreaks like the zika outbreak [ ] . and pharma from pakistan, hll biotech, green signal bio pharma and zydus cadila from india, and instituto biologico argentino (biol) from argentina -the th member. he emphasized the importance of collaboration and partnerships among members for achieving sustainable supply of high-quality, affordable vaccines and to accomplish global health initiatives. one such initiative is the switch from trivalent (topv) to bivalent oral polio vaccine (bopv) and eventually inactivated polio vaccine (ipv), in order to contain polioviruses better and, ultimately, eradicate polio. j. lemus, minister of health in argentina, began with the reminder that vaccines and potable water are the most effective ways to reduce morbidity and mortality globally. the role of the ministry of health is to protect the argentinian population and promote healthy living. the h n outbreak in argentina underscored the value of local vaccine manufacture. he also emphasized the importance of supporting international supply, and affirmed the argentinian government's and vaccine manufacturers' commitment to work with paho to provide high-quality vaccines globally. m-p. kieny reviewed evolving collaborative innovation in vaccine development. historically, one scientist, like edward jenner, could develop a vaccine. then came small groups, such as louis pasteur and colleagues. innovation subsequently became the work of a large team, sometimes spanning several corporations. now, after decades of intense competition for high-value markets, collaboration with developing countries has become critical, and involvement of multiple manufacturers as well as public-and private-sector investments are essential. the -year meningitis a project and the -year malaria vaccine development illustrate the efforts for developing vaccines for african epidemics. the accelerated ebola vaccine development in required urgent collaboration between manufacturers, regulatory authorities and funding agencies to respond to and contain the emerging outbreak [ ] . following the registration of the first dengue vaccine in , the most advanced dengue vaccine, a collaboration between butantan and national institutes of health (nih) [ ] , encourages partnerships with dcvms and public-sector entities. full participation of dcvmn members in future innovative collaboration will require increased development capability, novel production processes and regulatory skills, along with equal partnerships, justified investments and national regulatory agencies (nras) capabilities. she concluded that commitment to public health as a joint responsibility is essential. h. sigman, chairman of group insud and sinergium welcomed meeting participants. he introduced sinergium's technology transfer capabilities, providing an example of partnership between public and private sectors, and confirmed the importance of collaboration among vaccine manufacturers. the director of paho, c.f. etienne, presented via video. paho has undertaken to make national health programs sustainable and affordable, encouraging improvements in quality and availability of vaccines from both private and public sectors. rubella was eliminated from the who americas region in , and it is now the first region to be declared free of measles [ ] . she thanked dcvmn members for their contribution to national immunization programs. paho is committed to continue collaboration with dcvmn members and welcome innovative suggestions, to ensure that the americas have sustainable access to life-saving vaccines. i. danel, deputy director from paho, outlined achievements of public health goals in the americas, including extension of the reach of national immunization programs, new vaccine introductions, strengthening of regulatory pathways and improving financing and forecasting mechanisms. elimination of measles from the americas is a result of countries working together successfully. this was made possible by timely supply of measles-rubella (mr) and measles-mumps-rubella (mmr) vaccines by dcvmn members. in , dcvm's vaccines represented % of the total volume of million doses procured through paho's revolving fund. paho is constantly seeking alternative sources of vaccines and adopting strategies to enable manufacturers to find new opportunities. she encouraged dcvmn members to get involved in fast-track development of vaccines, to enhance sustainability of supply and to introduce required vaccines. k. owen from bmgf reviewed the number of deaths averted through immunization by [ ] , then discussed the challenge of creating stable vaccine supply in developing countries. stable supply requires high-quality manufacturing capacity, committed financing, strong regulatory systems and predictable demand. bmgf's efforts have focused on partnering with manufacturers, stimulating procurement, and optimizing regulatory pathways in developing countries, to reduce registration time by up to %. challenges around predictability remain. a recent example of challenge is the transition in demand from opv to ipv. it is essential that dcvms have strategies to sustain their business in a competitive market. r. bright presented barda's role in tackling emerging and reemerging infectious diseases. the large number of potential eids and unpredictability of emergence requires multi-hazard strategies and investment in a broad spectrum of vaccine platforms. emerging threats require a rapid and coordinated response which integrates diagnostics for early detection, therapeutics and vaccines. collaboration between multiple groups to develop and evaluate vaccines and new supply and funding models are becoming increasingly necessary to respond efficiently. there are currently companies developing zika vaccine, including dcvmn members bharat, butantan, and sinergium. he urged dcvms who are working on zika candidate vaccines to meet and foster possible partnerships. f. kristensen provided an overview about the new coalition for epidemic preparedness innovations (cepi) [ ] . cepi aims to prioritize, stimulate, finance, coordinate and advance vaccine development against eids with epidemic potential, especially where market incentive alone will not achieve this. the ebola response showed that it is possible to advance the clinical development of vaccines in an emergency. manufacturing capability and capacity for vaccines is a bottle-neck during crises, thus the effort will focus on driving vaccine manufacturing pipelines, while also funding diagnostics and therapeutics. cepi seeks multi-year donor contributions of up to one billion dollars between and from those aligned with the strategic objectives and mission. the operating principles are: equitable access, cost coverage and shared risks and benefits. funding from cepi will be available to vaccine producers or consortia with a track record of bringing vaccine candidates to human clinical trials. the founders of cepi are the governments of india and norway, the bill and melinda gates foundation, wellcome trust and the world economic forum (wef). cepi was, in fact, launched at the wef meeting in davos, in january . j. kalil discussed emerging and re-emerging diseases in tropical regions. butantan is developing dengue and zika vaccines, currently in phase iii clinical trials, and shows results comparable to the nih vaccine [ ] . development of a zika candidate vaccine was undertaken as an urgent response to the recent outbreak in south america. the ideal situation would have been to develop a combination vaccine against both zika and dengue. butantan is willing to collaborate with other companies developing zika vaccines to test candidate vaccines in an endemic area. m. malhame reviewed the gavi - strategy, which was informed by lessons of previous periods. it includes fostering of innovative products, further strengthening of collaboration though engagement with industry, clearer inclusion criteria for vaccines and improved, consistent market analysis and forecasting. securing multiple vaccine suppliers has a positive impact on the effectiveness of immunization programs. gavi's new strategy has three priorities: . taking a long-term view of markets to help countries and manufacturers prepare for transition to self-financing. . aligning product innovation priorities across market-shaping partners to match needs. . adopting a higher tolerance of risk in markets that require it. lastly, m. malhame extended gavi's gratitude to dcvmn members contributing to the supply of vaccines to poor countries. gavi will continue to shape healthy vaccine supply globally. s. rautio noted that more than % of the total unicef procurement of $ . billion was dedicated to vaccines in . a total of . billion doses of vaccines were procured by unicef, reaching % of the world's children. % of the doses were sourced from dcvms at a total value of $ million, providing an important market opportunity for dcvms. vaccine security is the sustained, uninterrupted supply of affordable vaccines of assured quality. to achieve it, accurate forecasting, availability of funding and appro-priate contracting with a diverse supplier base is critical. therefore, continued engagement with dcvms is essential. beyond securing expanded programme on immunization (epi) vaccines, unicef is engaged in supply of pipeline vaccines, during outbreaks or health and humanitarian emergencies, and improving access to affordable new vaccines in middle-income countries (mics). unicef supports the development of healthy markets through leveraging its procurement, transparency on pricing and building procurement capacity in countries. pentavalent vaccine is an example of a market, which is now considered healthy, years after procurement of the first dose. j. fitzsimmons provided an overview of paho's technical cooperation strategies with countries in the americas, including considerations for national decision-making on introducing new vaccines in national immunization programs. paho's sustainability model for immunization consists of national vaccine forecasting, national budgetary financing, legislation and the revolving fund mechanism which together facilitate access to vaccines. after four decades of experience, this model has proven to be effective: the americas enjoy high immunization coverage for traditional and new vaccines. the success of the paho revolving fund has been enabled by the high-level commitment from member countries, its business model and the high-quality vaccines supplied by dcvms and other suppliers (fig. ). a panel discussion moderated by j. chu, from chai, focused on management decisions that can contribute to increased success and greater commercial viability of vaccines. vaccine manufacturing is a critical component of global health, but it comes with risks. one example is the decline in success rate of clinical trials for pharmaceuticals, including vaccines, over the past years [ ] . both scientific and management factors contribute to four risk categories: -large potential return/high feasibility e.g. innovative respiratory syncytial virus (rsv) and group b streptococcus (gbs) vaccines; -small potential return/ high feasibility; -large market opportunity/ low feasibility; -small market opportunity/ low feasibility (fig. ) and emphasized that a balanced portfolio is critical to ensure that companies remain viable. m. datla said it is increasingly important to choose the vaccine portfolio wisely. biological e, for example, takes pride in supporting epi needs of india as well as gavi countries. there are also few follow-on vaccines and limited new product introductions, so many companies end up with similar pipeline. both under-capacity and over capacity of supply influence sustainability for customers and manufacturers respectively. pentavalent vaccine is a prime example of over-capacity, as the demand is now divided among a larger group of manufacturers. reduction in market shares will challenge manufactures' sustainability as their business models depend on reasonable market shares. mics have unique characteristics, such as size of birth cohort, ability to procure, local registration requirements and reluctance to join pooled procurement. however, they are seeking vaccines at gavi prices posing another challenge to manufacturers' sustainability. high-income countries have different barriers to entry, requiring heavy investments, due to the need for more clinical trials. access to funding for vaccine development is also getting increasingly difficult as it takes about years for a vaccine to reach the market, and most investors prefer faster and higher returns. j. khalil noted that partnerships help to reduce the financial risks. butantan has a history of forging successful partnerships with multinational companies with the primary objective of domestic supply. access to hpv vaccines was only possible after local government enabled a technology transfer agreement. butantan now prioritizes innovative products and products for valuable markets. if manufacturing capacity exceeds demand, lower pricing would be possible. m. malhame explained how gavi assists suppliers to understand the competitive and commercial risks involved by publishing strategic demand forecasts and roadmaps, and analyzing how countries and donors select products. companies also need to develop their own strategy. gavi also analyzes the cost of administration and calculates the weighted average prices of vaccines. there is a need to expand the conversation from price per dose to cost per child immunized. s. rautio commented that unicef's focus is on security of supply. unicef hosts a conference for vaccine manufacturers annually at which market forecasts are provided. unicef works closely with ministries of health in various countries to estimate demand. no supplier, however, should rely only on unicef; similarly, no market should be reliant only on one supplier. j. fitzsimmons commented that another risk for manufacturers is the different product requirements in different markets, such gavi and paho markets. networking opportunities, such as this meeting, offer a forum to discuss these issues and develop a more common target product profile relevant across public-health markets. g. widmyer acknowledged that bmgf is in a unique position to shape global health. while financing agencies seek the lowest pricing, suppliers need support for vaccine development. bmgf has a diverse portfolio, funding projects on various diseases. agendas may overlap, as is the case where investment in a manufacturer may result in excess capacity available for gavi markets, in addition to the domestic market. companies must focus on financial viability. the foundation is keen to engage with suppliers seeking investment who can show a portfolio that reflects a strategic and balanced vision. trust and transparency in interactions are critical. j.l. di fabio opened the session on regulatory convergence efforts to facilitate registration and improve access to health products. p. aprea, director of biologicals evaluation and control at the national administration of medicines, food and technologies, argentina (anmat), remarked that convergence is currently the biggest challenge for regulatory authorities. they require confidence, clarity and competitiveness, and must apply regulatory d. decina presented the draft who good regulatory practices (grp) guidelines [ ] for nras. the guideline is intended to assist regulators and regulated organizations, irrespective of resource levels. it provides a framework for improving practices and principles on which regulatory systems can be established. key concepts are impact analysis and stakeholder consultation. the nine major principles: legality, impartiality, consistency, proportionality, flexibility, effectiveness, efficiency, clarity and transparency are important for sharing information. she presented the types of international regulatory cooperation that help to avoid redundancy in regulatory work, for example, reliance or mutual recognition. a. porrás shared the regional approach to regulatory harmonization used by the pan american drug regulatory harmonization (pandrh) network. the technical cooperation approach for regulatory system strengthening (rss) in the americas encompasses three work streams: . facilitating development of contextspecific national regulatory systems; . promoting regulatory convergence and harmonization; . supporting efficient use of resources by leveraging the work of others. the program relies on peer-assessment of nras using standardized indicators of regulatory capacity, and the adoption of institutional development plans based on identified gaps. participation implies commitment to undergo assessment. assessment results are shared with participating nras, and aggregated data is published online on the regional platform on access and innovation for health technologies (prais [ ] ). she concluded that this effort represents a new trend towards convergence in the regulatory systems in the americas. n. dellepiane discussed how manufacturers can contribute to dialogue on global regulatory convergence. she reviewed existing approaches to regulatory harmonization, including reliance within regulators' networks, provision of mutual support, and alignment of requirements, procedures and standards among regulatory agencies. continued reliance and mutual recognition of nras through networking initiatives, improving technical and scientific expertise through joint review activities and partnerships between nras remain important. alignment of nras, by agreeing and following a common list of essential documents to fulfil countryspecific requirements for registration files may also foster regulatory convergence. she encouraged dcvmn members to collect data related to various registration approaches and contribute to the dialogue between regulatory groups and industry. j. iyer presented the view from access to medicine foundation on the role of manufacturers in promoting access. their goal is to gather insights into how pharmaceutical companies improve access to medicines, by ranking corporations based on criteria such as research and development capacity, affordability, manufacturing and supply. an access to vaccines index [ ] has been developed to help understand how companies make vaccines more accessible in low and middle-income countries. vaccine manufacturers can assess, monitor and track progress based on the analysis provided by the index and benchmark their international business strategy. d. atherly presented a study by path's center for vaccine innovation and access on the dynamics of vaccine uptake in developing-country markets. the project goal is to accelerate the development and introduction of products with high health impact. path collaborated with dcvmn member chengdu institute of biological products (cdibp) on development and introduction of japanese encephalitis (je) vaccine. this collaboration helped to obtain who prequalification, the first whoprequalified vaccine from china. from to , production capacity increased by %. approximately million children outside of china will be vaccinated by cdibp's je vaccine. it is envisioned that by , eight additional countries will introduce je vaccine as part of the national immunization program. y. lim from chai discussed the risks and potential of future vaccine markets. she advised companies to understand the market and existing competition, then compare and select candidate products based on potential risks and returns. the resultant optimized and balanced product portfolio should be responsive to changes in vaccine market dynamics. j. fournier-caruana gave an update on the who global action plan for containment of polioviruses (gapiii, [ ] ) and the containment certification scheme (ccs) endorsed by the strategic advisory group of experts (sage) in and october [ ] , respectively. eradication of poliovirus is a global endeavour and can only be achieved by engaging all stakeholders. the globallysynchronized switch from trivalent (type , , ) opv to bivalent opv (type , ) in april [ ] started the poliovirus type- containment period to eliminate vaccine-derived type poliovirus (vdpv ) infection. however, there have been delays: poliovirus production facilities, research facilities and repositories should have demonstrated implementation of gapiii, and national authorities for containment (nacs) should have certified containment. this has not been realized in many countries due to lack of national regulatory frameworks to implement containment. who and other stakeholders are supporting national containment certification efforts. unicef's role in the endgame, outlined by a. ottosen, includes securing the supply of polio vaccines as well as synchronized withdrawal of topv and roll out of bopv. from december to march , million doses of opv (topv and bopv) were delivered in a timely manner for planned activities. towards topv cessation, however, perceptions of risk increased among both suppliers and programme managers. an industry survey emphasized the necessity for involving suppliers from early stages of decision-making for final opv cessation to allow for integration into their strategies and ensure sufficient supply. the recent polio outbreak in nigeria required million vaccine doses. dcvms provided a large quantity through flexibility, stockpiles and response capacity. due to a shortage of ipv, unicef is not currently able to meet the who recommendation of at least one ipv dose in all countries. therefore, in october , sage recommended a fractional-dose ipv (fipv: one-fifth of the normal ipv intramuscular dose delivered intradermally) [ ] , and catch-up on ipv vaccination when it becomes available. currently countries do not have access to ipv [ ] . s. boyle from bmgf elaborated on ipv use and challenges, and the rationale behind the switch from topv to bopv. since , no indigenous transmission of type- wild poliovirus has been detected, while the type- component in the topv makes up more than % of the vaccine-derived polioviruses, and causes % of vaccine-associated paralytic polio cases [ ] . further, it also interferes with immune responses to type and polioviruses. in response to the shortage of ipv, who has recommended two doses of fipv delivered intradermally at and weeks, along with routine bopv as well as campaign immunization. ipv supply constraints are expected to remain an issue until . bmgf priorities include reliable supply of bopv, introducing and maintaining ipv in routine immunization, and assessing the potential of new mucosal-immunity enhancing adjuvants and more genetically stable opvs. b. giersing presented the role of who's initiative for vaccine research (ivr) in accelerating vaccine development and access to vaccines in lmic. challenges around new vaccine development include investments being driven by high-income countries, lack of robust regulatory pathways and differences in health priorities among stakeholders. since , pathogens, including rsv, were evaluated by ivr. ivr also evaluates new delivery strategies. an example is the microarray patch (map, [ ] ) for administration of mr vaccines. the map requires minimal training, is light-weight and thermo-stable, yet provides the same efficiency as traditional vaccine. ivr wishes to provide neutral evidence on the value of new technologies to overcome challenges in vaccination. d. zehrung reviewed new vaccine delivery technologies, including maps for ipv or mr, blow-fill-seal tubes for rotavirus, integrated reconstitution packaging and intradermal delivery technology. new delivery technologies are likely to reduce commodity and system costs, resulting in greater access and health impact worldwide. m. zaffran reported on the current situation of the global polio endgame strategy. in , the lowest number of cases of polio was reported [ ] , with cases in pakistan and afghanistan. four new cases of wild-type in nigeria and vdpv events caused concern. restricted access to conflict zones contributed to lack of surveillance and access to vaccines. in april , who announced the switch from topv to bopv, removing type- opv. by the end of august , countries had introduced ipv [ ] . priorities for the next six months are to interrupt transmission in the three endemic countries, maintain active surveillance in security-and ipv-deprived countries, and containment to maintain a polio-free status. the th dcvmn annual general meeting provided an occasion for professionals in the vaccine industry and global health organizations to discuss challenges, innovations, risks and gain deeper understanding of regulatory practices and convergence opportunities. the zika outbreak and polio endgame are reminders of the importance of vaccines, but also the importance of collaboration, collective effort and global partnerships. the dcvmn aims to foster networking opportunities to support members in shaping global health. ihr ) emergency committee on zika virus and observed increase in neurological disorders and neonatal malformations. media centre of world health organization outbreak news. regional office for africa of world health organization clinical evaluation strategies for a live attenuated tetravalent dengue vaccine la región de las américas es declarada libre de sarampión. paho/who regional office for the americas the vaccine alliance. results & evidence new vaccine coalition aims to ward off epidemics the productivity crisis in pharmaceutical r&d good regulatory practices: guidelines for national regulatory authorities for medical products. who working document qas/ . regional platform on access and innovation for health technologies. prais. paho/who regional office for the americas human anthelminthic vaccines: rationale and challenges access to vaccines index who global action plan to minimize poliovirus facility-associated risk after type-specific eradication of wild polioviruses and sequential cessation of oral polio vaccine use. gap iii. who/polio/ . . global polio eradication initiative polio today. global polio eradication initiative replacing trivalent opv with bivalent opv. vaccines and diseases. immunization, vaccines and biologicals -conclusions and recommendations inactivated polio vaccine: supply update. unicef supply division. unicef preparing for the withdrawal of all oral polio vaccines (opvs): replacing trivalent opv with bivalent opv. frequently asked questions opportunities and challenges in delivering influenza vaccine by microneedle patch cessation of use of trivalent oral polio vaccine and introduction of inactivated poliovirus vaccine worldwide we are grateful to all speakers and moderators whose contribution made the agenda invaluable. we thank corporate partners for supporting dcvmn with unrestricted educational grants: merck (milliporesigma), temptime corporation, ge healthcare, bioengineering, gea, bosch, ompi (stevanato group), applikon biotechnology, alfa wasserman, munters. we are indebted to the experts who graciously served as rapporteurs of specific sessions: d. kristensen, s. sobti, and j. hendriks. we thank s. villasenor for administrative assistance and m. dennehy for editorial support. this conference was partly supported by a grant from the bill & melinda gates foundation, grant no. opp . the authors are employees of the respective indicated organizations, and have no conflict of interest to declare. dcvmn international did not provide any financial or travel support to speakers or moderators to participate at this meeting. n. dellepiane is a consultant, partly with dcvmn. key: cord- - xu hq authors: taylor, deborah r. title: obstacles and advances in sars vaccine development date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xu hq the emergence of the severe acute respiratory syndrome (sars) that resulted in a pandemic in spurred a flurry of interest in the development of vaccines to prevent and treat the potentially deadly viral infection. researchers around the world pooled their scientific resources and shared early data in an unprecedented manner in light of the impending public health crisis. there are still large gaps in knowledge about the pathogenesis of this virus. while significant advances have been made in the development of animal models, the practicality of their use may be hampered by a lack of pathological similarity with human disease. described here are issues related to progress in vaccine development and the obstacles that lie ahead for both researchers and regulatory agencies. severe acute respiratory syndrome (sars) was first reported as a disease of unknown etiology in guangdong * tel.: + ; fax: + . sars is characterized by fever, cough and flu-like symptoms. severe cases resulted in alveolar damage, interstitial mononuclear cells and heavy fibrin deposition in the lungs. respiratory distress resulted in atypical pneumonia, requiring ventilation for approximately % of patients [ ] . the aim of this review is to describe the advances made in the development of animal models for sars and to identify gaps in scientific understanding that need to be filled. by addressing and possibly overcoming these challenges and making use of the advances made, a safe and effective vaccine may be attainable. the information here may provide the scientific basis for facilitating future regulatory decisions related to the licensing of a sars vaccine. the causative agent for sars is a novel member of the coronavirus family, termed sars-cov [ ] . coronaviruses are large enveloped rna viruses, so named for the radiating spike proteins found at the surface of the virion ( fig. ; [ ] ). there are three groups of related coronaviruses and sars may be a member of a new fourth group [ ] . classification of the sars coronavirus has been controversial, although phylogenetic similarities may place the virus in a subgroup of the group coronaviruses [ ] . human coronaviruses include three members that cause common respiratory infections [ ] . nonhuman coronaviruses include those that cause respiratory infections in birds, and enteric infections in cattle, pigs, dogs and cats [ ] . some of these viruses affect multiple organs, for example, both mouse hepatitis virus (mhv) and feline infectious peritonitis (fip) virus are capable of causing respiratory, enteric, neurologic and hepatic infections [ ] . coronaviruses are positive-sense rna viruses that replicate by a unique mechanism whereby the structural genes are expressed as a nested set of subgenomic mrnas, characterized by shared common ends and a conserved, capped leader sequence [ ] . the nonstructural genes are transcribed from the end as a polyprotein that is processed by viral proteases ( fig. ; [ ] ). proteins are translated from the open reading frame of each mrna [ ] . sars cov has eight open reading frames of unknown function, but has structural proteins found in all coronaviruses that include the envelope (e), the matrix or membrane protein (m), spike (s) and nucleocapsid (n) [ ] . the s protein is glycosylated and required for viral attachment and possibly entry. the nucleocapsid protein coats the viral genomic rna. viruses that belong to group , such as mhv, also contain a hemagglutinin-esterase (he) protein, which is not present in other groups [ ] . the flu-like symptoms and atypical pneumonia, characteristic of sars-cov infection, was also frequently accompanied by lymphopenia [ ] . alveolar macrophages were prevalent in patients with fatal sars [ ] and contributed to the immune-mediated nature of the disease. in situ hybridization showed that viral infection was present in alveolar epithelial cells and viral rna could also be detected the structural genes (spike, envelope, membrane, and nucleocapsid) are located in the end, which includes as many as eight genes of unknown function [ ] . in alveolar macrophages and bronchiolar epithelial cells [ ] . several studies have suggested that the immune system may be impaired by sars cov. t-cell lymphopenia was observed in % of patients observed, with a decline in cd + and cd + cell types [ ] . two weeks after disease onset, th cell-mediated immunity and inflammatory response was noted by the marked elevation of cytokines, ifn gamma, the neutrophil chemokine il- , il- , - and - , but not tnf, il- , - , or - . accumulation of monocytes/macrophages and neutrophils was also observed [ ] . li et al. [ ] noted that a rapid decline of t-cell subsets in the periphery was observed in patients during the acute phase of sars infection, but they observed restoration of t cells during recovery. the presence of proinflammatory cytokines may result from activated alveolar macrophages, suggesting that they may play a role in the pathogenesis of sars [ ] . antibodies to the sars coronavirus were found retrospectively in . % of samples collected in , indicating that the outbreak was not the first time that sars had entered the human population [ ] . most infected patients developed a humoral response to sars-cov and antiviral antibodies (igg and igm) were detected at days post-onset of symptoms [ ] . igm antibodies declined after days and igg antibodies persisted up to day [ ] and antiviral neutralizing antibodies were obtained from convalescent patients [ ] . morbidity rates were greater for older individuals, while children under years of age did not develop the severe disease that was seen in adults [ ] . taken together these data may suggest that the quality of the immune response may play a role in the outcome of virus infection. an additional challenge related to the containment of sars-cov is the lack in identification of its natural host. virus has been detected in wild and domestic animals [ ] . in , the first people to be infected were animal handlers in a food market in guangdong province, china, suggesting a role for zoonotic transmission [ ] . the sars strain observed in animals varies only slightly from the human virus and may represent a recent jump across species. the development of good animal models will not only be useful for identifying the natural host, but will be invaluable for determining correlates of immunity, for testing therapeutics and vaccine development. a remarkable advance in sars research came with the discovery that mice were susceptible to infection with sars-cov [ ] . balb/c mice were infected with or % tissue culture infective doses (tcid ) and by day after infection, yielded and tcid per gram, respectively, from lung tissue. although no clinical disease was observed, mild and focal peribronchiolar mononuclear inflammatory infiltrates were observed upon microscopic examination of the respiratory tract on day [ ] after infection. the presence of these infiltrates may suggest some mimicry with human clinical features, although much milder. the respiratory tracts of the mice were cleared of the virus by day after infection. wentworth et al. found sars-cov in the stomach, intestine, and duodenum, in addition to the respiratory tracts of infected mice [ ] . subbarao et al. also protected mice from infection by passive administration of sars-cov neutralizing antibody from previously infected mice, suggesting that neutralization in vivo is possible [ ] . mice clear the virus by day post-infection, while humans begin to clear the infection by days - [ ] . a small animal model will allow researchers to test therapeutics and vaccines, and because the mice recover from virus infection so efficiently, also identify host factors that contribute to virus resolution. hamsters have also been shown to be a good model for sars-cov infection, reaching similar titers to those seen in mice [ ] . surprisingly, immunodeficient mice can clear a sars-cov infection. mice (c bl/ background) that lack nk-t cells (cd −/− ), nk cells (beige) or those that lack t and b cells (ragl −/− ) cleared the virus by day after infection [ ] . the mice displayed high induction of proinflammatory cytokines, suggesting that the adaptive immune response and nk cells were not required for viral clearance in mice. furthermore, it indicates that the involvement of the innate immune response is important in controlling the virus. it is interesting to speculate that interferon pathways may be important in viral clearance. more evidence for the importance of innate immunity was provided through the infection of stat -deficient mice with sars-cov [ ] . stat is important to the regulation of interferons and stat -deficient mice produced a two log increase in viral titer over control mice. additionally, the mutant mice developed interstitial pneumonia, not seen in the control mice [ ] but not alveolar damage as seen in the lungs of human patients. it is unclear at this time if the observed pathological differences between human and stat -deficient mouse lungs were due to time of sampling or differences in host responses [ ] . domestic cats and ferrets have also been tested for use as a sars-cov animal model. cats and ferrets were inoculated with tcid sars-cov. cats showed no clinical symptoms [ ] , while three out of six ferrets became lethargic and one died. virus was recovered from the lungs of infected cats ( tcid / gram of tissue) and ferrets ( tcid / gram of tissue) [ ] . experiments also showed that horizontal transmission of the virus may have occurred between cats that were housed together or ferrets that were housed together, although the kinetics and mode of transmission are still unknown. the non-inoculated infected ferrets became lethargic, developed conjunctivitis and died at and days post-infection. while the ferrets did not show evidence of pneumonia, they did exhibit hepatic lipidosis and emaciation [ ] . ter meulen et al. showed that ferrets that were infected with sars-cov showed signs of multifocal pulmonary lesions affecting about - % of the lung [ ] . alveolar damage and lymphocyte infiltration was also observed upon histological examination of infected ferret lung tissue. three species of monkeys have been tested for infectivity with sars-cov: cynomolgus, rhesus and african green. african green monkeys supported the highest level of viral replication, yielding a viral titer of approximately tcid /ml from nasal swabs [ ] . some researchers working with cynomolgus macaques reported signs of dis-ease resembling sars after the monkeys were infected with sars-cov [ , ] . from day post-infection, macaques became lethargic, had a temporary skin rash, and one animal showed signs of respiratory distress [ ] . two of the macaques had interstitial pneumonia with lesions present, similar to autopsy tissue obtained from sars patients [ ] . other groups have reported that cynomolgous macaques show limited pathology, mild disease and upper respiratory symptoms [ , ] . there is no apparent explanation for the discrepancy between these groups, possibly virus strain differences or monkey subspecies differences may account for the differences in outcome, but to conclude that non-human primates most similarly mimic human disease is still controversial. vaccine efficacy is measured by the ability of the antigen to raise a protective immunologic response from b and t cells after exposure to the viral agent. ideally, by creating memory within the immune system, individuals will be protected from infection for decades. several veterinary coronavirus vaccines are currently available, but their efficacy is variable. the vaccine for prevention of infectious bronchitis virus (ibv), which infects chickens, is effective [ ] , but the canine and porcine vaccines are only partially effective [ ] . the feline infectious peritonitis (fip) vaccine is actually deleterious to the health of the animal and is discussed in further detail below [ ] . vaccines can be produced by inactivation of the virus, by using an attenuated or weak form of the virus, or by using recombinant forms of viral components. inactivated virus vaccines are relatively safe because they cannot revert back to the live form. they are also relatively stable and may not even require refrigeration. this is important in developing countries and for ease in mobilization during outbreak or emergency situations. however, there are limitations to their use. inactivated vaccines usually require several doses and some are weakly effective at stimulating an immune response. the vaccine to prevent hepatitis a is an example of an inactivated viral vaccine [ ] . live attenuated viral vaccines may require special laboratory development and cannot always be obtained. to reach effective levels, the virus must be capable of robust replication, but must have lost the ability to cause disease. several problems are associated with the use of a live attenuated vaccine. these vaccines must be kept refrigerated or frozen, and have safety issues related to the possibility of reversion to the wild-type form. additionally, they are almost never given to immunocompromised individuals for fears that the attenuated form may cause disease in the absence of an effective immune response [ ] . recombinant dna or viral vectors have been constructed in the lab for use as potential vaccines or to study the tissue tropism of the sars virus [ , ] . the vectors can be used to deliver foreign antigens using attenuated or nonpathogenic organisms. the safety of these types of vaccines [ ] centers around persistence of expression in vivo, possible genomic integration of the foreign dna and possible evolutionary changes that may cause instability of the viral vector. the potential transmission of the viral vector, including its introduction into the environment should also be evaluated. an ideal recombinant vaccine might be engineered to include the inherent ability of the foreign substance to be cleared by drug treatments that are proven safe, such as an antibiotic. antibiotic sensitivity introduced into a recombinant vector may allay fears of future adverse events although these designs may raise additional safety concerns. recombinant proteins can also be used to stimulate the immune response. these proteins are purified from yeast or bacteria and currently used in the manufacture of a licensed hepatitis b vaccine [ ] . the cell substrate used to manufacture all of these vaccines is also a concern so vaccine production must be performed in a well-characterized cell substrate. vero e cells have been used to produce the licensed poliovirus vaccine [ ] and may be appropriate for use in the development of a sars vaccine as sars grows well in vero cells. the fda center for biologics evaluation and research (cber) issued a letter to sponsors using vero cells as a cell substrate for investigational vaccines which can be found on the cber website [ ] . another consideration is the use of fetal bovine serum and bovine derivatives in the growth of cells. bovine tissues may contain the bovine spongiform encephalitis (bse) agent. guidelines on the use of bse-free blood products appear on the fda website [ ] as do guidelines on the use of bse-free bovine derivatives in the production of vaccines [ ] . for viruses that have variable strains, a combination vaccine may be effective. a combination vaccine is one that consists of two or more live organisms, inactivated organisms or purified antigens combined [ ] . this type of vaccine may be useful to prevent multiple organisms or strains of organism. each component must make a contribution to the whole and compatibility of the components is necessary. rna viruses replicate through rna-dependent rna polymerase encoded by the virus. this type of polymerase has no proof-reading mechanism associated with it, which results in a high rate of uncorrected mutations. these mutations may or may not be lethal to virus replication and may even persist, resulting in rapid evolution of the virus. for this reason, many rna viruses have multiple genomic strains, or quasispecies, present at one time in an individual. quasispecies may arise in response to selective immune pressure, thus allowing for escape mutants. the existence of quasispecies during sars-cov infection is just coming to light [ ] and their importance in escape from immune surveillance is still unknown. most coronaviruses are thought to have the ability to recombine due to homologous sequences in the and ends of the mrnas. evidence suggests that the sars-cov originated by recombination between coronaviruses [ , ] and that there was an additional host-species drift [ ] . both large and small animals can be infected with sars-cov, a giant advance for vaccine research. examining infected animal models will provide information that will lead to an understanding of the correlates of immunity. mice have been used to further the understanding of virus neutralization, cytokine upregulation and the minimum requirements for viral clearance, but have yet to show disease that mimics the atypical pneumonia seen in adult humans. while there have been some reports of disease in cynomolgous macaques [ , ] , many groups have not reproduced these findings [ ] . a promising animal model may be the domestic ferret. ferrets show elevated liver enzymes, lymphocytic infiltration and alveolar damage, which has also been observed in humans [ ] . despite the usefulness of these animal models, many challenges lie ahead. first, animal models of sars-cov infection do not mimic human disease. in mice, the virus is cleared in less than week and minor pathology in the lung is observed [ ] . histopathology performed on necropsy samples suggests that lung epithelial cells are involved, although the absence of pneumonia and infiltrating macrophages [ ] is disappointing. stat -deficient mice may prove promising as an animal model that most similarly mimics human disease [ ] . second, in order to test efficacy, large human populations must be tested in areas where the virus is endemic. finally, if sars fails to return, how will vaccine manufacturers test candidate vaccines for efficacy? while the animal rule has been provided for just this type of case, an animal model should mimic human disease in order to be applicable. the final rule was published in the federal register and can be found on the federal register website [ ] . additionally, coronaviruses may induce a short-lived immunity. this may be the reason that humans are subject to multiple infections with coronaviruses that cause the common cold. long-term immunity studies for sars-cov are currently underway. antibody-dependent enhancement (ade) has been observed in vaccinated and wild-type infections of fip. ade is thought to potentiate viral infection through the infection of macrophages. viral entry into macrophages occurs when antibodies bind the virus and attach to macrophages via the fc region of the antibody and its interaction with cell surface expressed fc receptors [ ] . neutralizing antibodies can also be enhancing antibodies if antibody titer is low or is of the igg class [ , ] . because macrophages increase with viral disease, this cell type may provide an abundant reservoir for the virus and thus expansion of the virus in the host. some similarities between fip and sars exist. first, in both cases macrophages can be infected with the virus [ , ] , and in the case of sars, the etiology of disease is contributed by infiltrating alveolar macrophages leading to pneumonitis [ ] . second, the treatment with corticosteroids and/or interferon alpha ameliorates sars disease [ ] , suggesting an inflammatory, immune-mediated disease. while there has been no observation of ade during sars infection, it is worth noting that one coronavirus, fipv, is capable of eliciting ade and in the evaluation of vaccines, we may want to consider this possible outcome. however, the difficulty in testing animal models for ade bears the caveat that if ade is not observed; it has not proved that vaccines are safe with regard to ade in humans. in contrast, if an animal model for ade is developed, we may learn more about the mechanism of sars-induced ade, which may help form the basis for developing guidelines for safe vaccine development. a potential sars vaccine might target the virus specifically through humoral or cell-mediated immune responses. alternatively, therapeutic vaccines may be useful in the treatment of viral infection. spike-specific monoclonal and polyclonal antibodies that neutralize the virus have been developed [ , ] and passive transfer of immune serum into naive mice protected them from infection with sars-cov [ ] . this suggests that neutralizing antibody alone can prevent viral infection. neutralization may not require host recognition of the fc region of the antibody, but the need to develop humanized forms of these types of antibodies may be critical if they are to be considered for use as a treatment. a human monoclonal antibody, derived from a phage display library, was administered to ferrets and protected the ferrets from lung disease and the shedding of virus in pharyngeal secretions [ ] . neutralizing antibodies from convalescent patients have been identified and characterized. usually neutralizing epitopes are located in the spike protein of the virus [ , ] . recent evidence has determined that virus neutralization is sensitive to deglycosylation of the spike protein, suggesting that conformational epitopes are important in antibody recognition [ ] . sars-cov can be efficiently inactivated by ultraviolet (uv) irradiation [ ] . mice immunized with uv-inactivated sars-cov develop humoral and cell-mediated immune responses [ ] . both t cell proliferation and cytokine upregulation was observed after boosting with the inactivated virus. beta-propiolactone-inactivated virus also elicited neutralizing antibodies when administered to balb/c mice [ ] formalin-inactivated sars-cov yielded potent humoral responses in balb/c mice as well [ ] . recombinant viruses may be used to elicit responses to introduced sars-cov genes. the first type of recombinant virus is a defective or non-pathogenic vector that expresses sars-cov proteins. the second type is one that is stimulated to assemble virus-like particles (vlp) in vitro. vlps containing the structural envelope proteins including spike (s), envelope (e) and membrane protein (m) have been assembled by coinfecting insect cells with three baculoviruses expressing one of the three structural proteins [ ] . structural proteins expressed by the live attenuated bovine parainfluenza virus type (bhpiv ) were evaluated for efficacy in hamsters [ ] and african green monkeys [ ] . high titer neutralizing antibodies were obtained after only one intranasal immunization with this vector. single immunization with bhpiv expressing s alone provided complete protection upon challenge with sars-cov [ , ] . recombinant live attenuated modified vaccinia virus ankara (mva) was used to deliver the sars spike protein (rmva-s) into balb/c mice [ ] . neutralizing antibodies were obtained and a reduction in the viral titer was observed after challenge with live sars-cov [ ] . only ferrets that were challenged with sars-cov after vaccination with rmva-s showed enhanced liver disease as demonstrated by increases in alt values and the presence of mononuclear hepatitis upon histological examination [ ] . these data suggest enhanced disease due to vaccination with a sars protein. adenoviruses expressing the s, m or n proteins were used in combination to vaccinate rhesus macaques [ ] . the immunized animals all had antibody responses to the s protein and t-cell responses to the n protein [ ] . highly infectious hiv particles expressing the s protein have been made, primarily to study the host-cell distribution of the putative sars-cov receptor [ ] . additionally, investigating the requirements for viral receptor binding and entry will also enhance our understanding of the requirements for viral control. recombinant hiv particles that express the sars spike protein may provide insight into cell tropism and receptor expression profiles [ ] . another retrovirus, murine leukemia virus, was used to generate infectious particles containing most of the s protein. convalescent serum was able to neutralize infection of the recombinant virus in vero cells [ ] . high cytotoxic t-lymphocyte (ctl) and antibody responses were observed after mice were injected three times with a recombinant plasmid vector expressing the n protein [ ] . mice immunized with a plasmid containing the s protein produced anti-sars-cov igg [ ] and developed neutralizing antibodies and a t-cell mediated response resulting in a six-fold reduction in viral titer in the lungs [ ] . plasmids encoding either the s or s regions of the spike protein elicited antibody production in mice [ ] . neither the s or s antibodies alone were capable of neutralizing the virus; how-ever, cooperatively they enabled neutralization of the virus, suggesting that both regions of the spike protein are important for host-cell viral entry [ ] . the nucleocapsid protein may also stimulate an effective immune response. dna vaccination with calreticulin fused to the n protein generated sars-specific humoral and cellular immunity in c bl/ mice [ ] . calreticulin was used because it was found to enhance major histocompatibility complex class i presentation of fusion proteins to cd (+) t cells [ ] . recombinant viruses may be generated from the fulllength infectious cdna clone of sars-cov [ ] . this clone may provide a source for genetic manipulation of the genome [ ] . once the viral virulence factors are understood, attenuated strains may be obtained by engineered mutation of the virus. vaccine development may proceed through the undertaking of a systematic approach to understanding the correlates of immunity raised by sars-cov. much of the focus has centered towards the humoral response and neutralizing epitopes, but cell-mediated immunity may also be important. ctl epitopes within sars-cov that may be presented by % of the human leukocyte antigen supertypes were identified by advanced bioinformatics [ ] . further characterization of these epitopes, including their recognition by convalescent serum, should advance the understanding of important immunological features in the control of sars-cov. while there has been intense study of sars, there is still much that is unknown about the pathology of the virus. many research questions will need to be answered, thus providing the resources necessary to develop an effective and safe vaccine. to be sure that a vaccine will provide coverage for a potentially variable virus, we need to know what the occurrences of viral quasispecies are and how variable are the important epitopes. what are the viral virulence factors and can we use this information to develop an attenuated strain? can the virus establish persistent infection and can humans be repeatedly infected? we also need to determine the innate, humoral and cell-mediated immune responses in recovered sars patients in order to understand how viral clearance comes about. to understand viral pathogenesis we must know how the immune system mediates disease. what is the role of lymphocytic infiltrates and do alveolar macrophages enhance disease or control infection? if we define the ability of the virus to replicate in monocytes/macrophages can we be assured that antibody-dependent enhancement will not be a problem for a sars vaccine? most importantly, identification of the natural animal host may launch the further development of large and small animal models for correlates of immunity and drug and vaccine screening. development of an animal model that mimics human disease will be the single most important advance in the development of a sars vaccine. development of a vaccine for sars-cov is imperative and research headed in a for-ward direction will enable the public health community to be ready. toronto sars critical care group. critically ill patients with severe acute respiratory syndrome sars working group. a novel coronavirus associated with severe acute respiratory syndrome sars-beginning to understand a new virus severe acute respiratory syndrome coronavirus phylogeny: toward consensus identification of a new human coronavirus coronaviridae: the viruses and their replication lung pathology of fatal severe acute respiratory syndrome sars coronavirus-infected cells in lung detected by new in situ hybridization technique expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome sars-related virus predating sars outbreak dissection study on the severe acute respiratory syndrome c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors sars in newborns and children isolation and characterization of viruses related to the sars coronavirus from animals in southern china role of china in the quest to define and control severe acute respiratory syndrome prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice mice susceptible to sars coronavirus contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat virology: sars virus infection of cats and ferrets human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome macaque model for severe acute respiratory syndrome replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys protection of chickens after live and inactivated virus vaccination against challenge with nephropathologenic infectious bronchitis virus safety and efficacy of a modified-live canine coronavirus vaccine in dogs antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever inactivated hepatitis a vaccine: active and passive immunoprophylaxis in chimpanzees live attenuated varicella vaccine highly infectious sars-cov pseudotyped virus reveals the cell tropism and its correlation with receptor expression genetically engineered vaccines: an overview developing new smallpox vaccines current status and future trends in vaccine regulation-usa sars-associated coronavirus quasispecies in individual patients sars associated coronavirus has a recombinant polymerase and coronaviruses have a history of host-shifting mosaic evolution of the severe acute respiratory syndrome coronavirus antibody-dependent enhancement of virus infection and disease the role of igg subclass of mouse monoclonal antibodies in antibody-dependent enhancement of feline infectious peritonitis virus infection of feline macrophages localization of antigenic sites of the s glycoprotein of feline infectious peritonitis virus involved in neutralization and antibody-dependent enhancement antibody-dependent enhancement of feline infectious peritonitis virus infection in feline alveolar macrophages and human monocyte cell line u by serum of cats experimentally or naturally infected with feline coronavirus interferon alfacon- plus corticosteroids in severe acute respiratory syndrome: a preliminary study development and characterisation of neutralising monoclonal antibody to the sars-coronavirus identification of an antigenie determinant on the s domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov subcutaneously injected uv-inactivated sars coronavirus vaccine elicits systemic humoral immunity in mice inactivated sars-cov vaccine prepared from whole virus induces a high level of neutralizing antibodies in balb/c mice immune responses in balb/c mice induced by a candidate sars-cov inactivated vaccine prepared from f strain assembly of human severe acute respiratory syndrome coronavirus-like particles severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets effects of a sars-associated coronavirus vaccine in monkeys retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein induction of sars-nucleoprotein-specific immune response by use of dna vaccine dna vaccine of sars-cov s gene induces antibody response in mice a dna vaccine induces sars coronavirus neutralization and protective immunity in mice characterization of humoral responses in mice immunized with plasmid dnas encoding sars-cov spike gene fragments generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus sars ctl vaccine candidates; hla supertype-, genome-wide scanning and biochemical validation kanta subbarao, edward tabor, miriam darnell, robin levis and hira nakhasi are gratefully acknowledged for comments on the manuscript. key: cord- - rmzaau authors: rhee, joon haeng title: current and new approaches for mucosal vaccine delivery date: - - journal: mucosal vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: rmzaau mucosal surfaces are the interface between the host’s internal milieu and the external environment, and they have dual functions, serving as physical barriers to foreign antigens and as accepting sites for vital materials. mucosal vaccines are more favored to prevent mucosal infections from the portal of entry. although mucosal vaccination has many advantages, licensed mucosal vaccines are scarce. the most widely studied mucosal routes are oral and intranasal. licensed oral and intranasal vaccines are composed mostly of whole cell killed or live attenuated microorganisms serving as both delivery systems and built-in adjuvants. future mucosal vaccines should be made with more purified antigen components, which will be relatively less immunogenic. to induce robust protective immune responses against well-purified vaccine antigens, an effective mucosal delivery system is an essential requisite. recent developments in biomaterials and nanotechnology have enabled many innovative mucosal vaccine trials. for oral vaccination, the vaccine delivery system should be able to stably carry antigens and adjuvants and resist harsh physicochemical conditions in the stomach and intestinal tract. besides many nano/microcarrier tools generated by using natural and chemical materials, the development of oral vaccine delivery systems using food materials should be more robustly researched to expand vaccine coverage of gastrointestinal infections in developing countries. for intranasal vaccination, the vaccine delivery system should survive the very active mucociliary clearance mechanisms and prove safety because of the anatomical location of nasal cavity separated by a thin barrier. future mucosal vaccine carriers, regardless of administration routes, should have certain common characteristics. they should maintain stability in given environments, be mucoadhesive, and have the ability to target specific tissues and cells. mucosal surfaces are the interface between the host's internal milieu and the external environment, and they have dual functions, serving as physical barriers to foreign bodies and pathogenic microbes and providing the foundation for crucial survival functions such as uptake of air and nutrients, reproduction, and perception of signals. the protection of mucosal surfaces is ensured by the specialized mucosal-associated lymphoid tissues (malts). mucosal vaccines, in contrast to parenteral vaccines, generally induce more efficacious protective immune reactions by inducing secretory iga responses and cell-mediated immunity in mucosal tissues and portals of entries of mucosal pathogens. for food components and inert materials in breathing air, the malt should remain tolerant so as not to cause unnecessary inflammatory responses. despite the many advantages of mucosal vaccines, there are only limited numbers of licensed mucosal vaccines. almost all licensed mucosal vaccines are composed of whole components of pathogens, either live or dead. there is no successful subunit mucosal vaccine so far. live attenuated or whole cell (wc) killed vaccines are not formulated with any specific adjuvant or delivery system. in those vaccines, pathogen-associated molecular patterns (pamps) play the role of built-in adjuvants, and cell corpuscles serve as delivery systems for protective antigens. there could be many reasons for the sluggish progress of development of mucosal vaccines. concerns about safety are the most prominent reason. mucosal surfaces are continuously exposed to environmental and food antigens and allergens, and inflammatory immune responses against mucosal vaccine antigens would result in sustained pathologic inflammation. in the case of nasal vaccination, the nasal cavity is separated from the central nervous system by a thin partition, and olfactory nerves are directly projected from the brain to cavity. the scarcity of optimal delivery systems is another reason for the slow progress. in the mucosal environment, there are many physicochemical conditions that would interfere with proper stimulation of immune cells by antigens and adjuvants. in the case of oral administration, to be taken up microfold (m) cells in the distal jejunum and ileum, antigens should be able to accommodate very low ph in the stomach and a sudden alkaline surge in the duodenum, and they should be able to resist proteolytic attacks of digestive enzymes. in contrast to the oral route, antigens delivered intranasally do not experience that dramatic fluctuation in ph, but they should be able to survive profuse mucosal secretions, mucociliary clearance, and the relative inefficiency of antigen uptake by antigen-presenting cells (apcs). for more efficient delivery of mucosal vaccines, many new delivery systems based on nanotechnology and biomaterials have been studied, but very few of them have been approved for clinical use. more vigorous clinically oriented research is needed [ , ] . in vaccine formulations currently approved or under clinical trials, nanoscale (, nm) carriers are already in use [ ] . current nanotechnology and nanocarriers on the market or in the literature are summarized in immunostimulating complexes (vlps), emulsions, liposomes, immunostimulating complexes (iscoms), polymeric and nondegradable nanoparticles (nps), and nanogels [ ] . some of the nps are able to enter apcs by diverse pathways, thereby differentially modulating downstream immune responses. moreover, the nano-based delivery systems are also able to carry antigens and specific adjuvants such as tlr ligands simultaneously in the same carriers; carriers by themselves sometimes exert adjuvant activities. the nanoscale vaccine carrier systems generally constitute three key components: an antigen, against which adaptive immune responses are induced; an adjuvant, to potentiate the interaction between innate and adaptive immune systems in reacting to the antigen(s); and a delivery or targeting system to ensure that the antigen(s) and adjuvant(s) are delivered together to the right location at right time [ ] . in this context, many effective mucosal delivery systems using the nano/microscale carriers have been very actively researched up to clinical trial levels in recent years. viral-vectored vaccines and live or killed virus vaccines by themselves are already nanocarriers with built-in pamp adjuvants. vlps and virosomes behave similarly to viruses in stimulating immune responses and carrying antigens in nanoscales. some adjuvant formulations are already composed of nanoscale structures. formulation of adjuvants with vaccine antigens became inevitable in modern vaccine development to enhance the immunogenicity of highly purified antigens that have insufficient immunostimulatory capabilities. while early adjuvants (e.g., aluminum, oil-in-water emulsions) were used empirically, rapidly increasing knowledge of how the immune system interacts with pathogens allowed better understanding figure . different micro/nanocarriers that could be applied to the development of mucosal vaccine delivery systems. iv. current and new approaches for mucosal vaccine delivery of the role of adjuvants and how the formulation of modern vaccines can be better tailored for the desired clinical benefit [ ] . of interest, currently licensed oil-in-water emulsion adjuvants such as mf , as , and as comprise nanoscale structures in the formulation. squalene is popularly incorporated in oil-inwater emulsions because of its physical and immunostimulatory properties [ ] . mf (novartis vaccines & diagnostics), as (gsk biologics), and glucopyranosyl lipid adjuvantstable emulsion (gla-se) (infectious disease research institute (idri)) have a squalene content of around %À % (w/w) with additional emulsifying agents [ ] . mf was the first oilin-water emulsion on the market produced by microfluidization and contains sorbitan trioleate and polysorbate- (ps ) as surfactants. mf has a particle size of around nm [ ] . as contains α-tocopherol and ps as a surfactant. as has a particle size of about nm [ ] . gla-se (idri) consists of a squalene emulsion in combination with gla, which is a synthetic form of monophosphoryl lipid a (mpla) and a potent immunopotentiator. gla-se has a particle size of about nm [ ] . it has been suggested that emulsions with particle sizes ranging from to nm are efficiently taken up by dendritic cells (dcs) and hence effectively stimulate immune responses against coadministered vaccine antigens [ ] . tlr ligands and immunostimulatory agents such as qs are formulated into oil-water emulsion or liposomes to make adjuvants for vaccines against diverse infectious agents and tumor immunotherapy. iscoms are approximately -nm cage-like particles produced by combining protein antigens, cholesterol, phospholipid, and the saponin adjuvant quil a [ ] . iscom is composed of matrix serving traps for protein antigens. typically, membrane antigens containing hydrophobic domains are well trapped in iscom through apolar interactions [ ] . liposomes are spherical carriers composed of one or more phospholipid membranes with aqueous core. thanks to the structure, liposomes provide a wide range of options for vaccine formulation design. proteins, peptides, dna, rna, and adjuvant components can be readily encapsulated inside the aqueous core, embedded within the lipid layer, or attached to the surface by adsorption, hydrophobic anchor insertion, or covalent fusion. liposomes by themselves are considered to be nontoxic and biodegradable when mainly phospholipids are used, since they are normal components of mammalian cell membranes and lipids are relatively nonimmunogenic. liposomes could be designed and manufactured to have the desired physicochemical characteristics optimal for inducing desired immune responses against vaccine antigens: vesicle size, lamellarity (number of lipid layers), surface charge, bilayer fluidity, and incorporation of immunostimulatory components [ a] . polymeric nps are have also been robustly studied to deliver vaccine antigens and adjuvants. polymeric nps are submicron-sized colloidal systems of natural or synthetic polymers used as delivery carriers of chemical drugs, proteins, peptides, and nucleic acids, owing to their high bioavailability, controlled release, biodegradable and biocompatible properties, and low toxic profiles [ ] . compared with liposomes, polymeric nps can more easily incorporate both hydrophilic and hydrophobic biomolecules and have better storage stability. the most commonly studied polymers are poly(d,l-lactide-co-glycolide) (plg), polylactide (pla), and poly(d,l-lactide-co-glycolide) (plga) [ , ] . these biodegradable, biocompatible polymers are well characterized and have been approved by the u.s. food and drug administration (fda) for use in humans because of their excellent safety profiles. they have been extensively studied for the formulation of vaccine antigens (proteins, peptides, dna, etc.) [ ] . in these formulations, antigens can be either entrapped or adsorbed to the surface of the particles and are protected from proteolytic degradation conferring longer half-lives in vivo. by not-so-difficult additional engineering, these particles can be regulated to degrade or to release cargos (adjuvant and/or antigens) over a wide range of rates. additionally, polymeric particles more easily pass m cells and reach to apcs in the malt after surviving harsh physicochemical conditions in many mucosal compartments [ , ] . plg polymers have been evaluated for drug delivery since the early s and have been used widely for pharmaceutical and medical device applications with excellent safety profiles in humans [ ] . generally, plg forms microparticles rather than nps. plg microparticles received attention for vaccine delivery by the world health organization (who) special program for vaccine development from the late s [ ] . triggered by this program, many antigens, such as tetanus toxoid, hepatitis b antigen, and diphtheria toxoid, were formulated with plg microparticles and evaluated in comparison with aluminum salt adjuvants [ ] . although some promising results with plg-based vaccines in small animal models were reported, challenges concerning antigen stability and insufficient immune responses compared with alum-adjuvanted vaccines prevented the use of plg microparticles in commercial vaccines [ ] . one more disadvantage of plg polymer-based vaccines is their inefficiency in translocating to lymph nodes where apcs present antigens to t lymphocytes. the particle size can influence transport to specific location and cell types in the draining lymph nodes [ , ] . nps ( À nm) drained to the lymph nodes and localized in the lymph-node-resident dcs and macrophages, whereas larger particles ( À nm) were mostly associated with dcs at the injection site. plg particles would be inefficient in presenting antigens to dcs in draining lymph nodes. in this regard, plg microparticles have handicaps to being more widely applied to the development of vaccine delivery systems. pla is a linear aliphatic polyester composed of lactic acid building blocks that are naturally occurring organic acids derived from sugarcane and cornstarch [ ] . pla's physical properties can be tuned through combining racemic mixtures of these enantiomers: poly-l-lactide and poly-dl-lactide semicrystalline and amorphous polymers, respectively [ ] . thanks to their good safety profiles, pla-based products have been approved by the fda and the european medicines agency (ema) for multiple biomedical applications. this polymer can be easily chemically modified with different ligands to improve their specificity to targeted cells [ ] . in vivo, pla is hydrolyzed into α-hydroxy acid, which is easily metabolized in the body via the krebs cycle [ ] . pla polymers can be fabricated into both microparticles and nps [ ] . despite its physicochemical and pharmaceutical advantages, pla has been less intensively applied than other polymers, such as its copolymer plga, in clinical stage vaccine developments. although a considerable number of reports have shown the usefulness of pla polymer nps as versatile vaccine carriers, the scaling-up of these laboratory methods to industrial production has faced hurdles, which are mostly related to particle size and size distribution. for further development of pla polymers as clinical-grade vaccine delivery systems, these practical problems need to be solved in advance. biodegradable nano/microparticles of plga and plga-based polymers are more widely explored as carriers for controlled delivery of macromolecular therapeutics such as proteins, peptides, vaccines, genes, antigens, and growth factors. plga is one of the most successfully developed biodegradable polymers. among the different polymers developed to formulate polymeric nps, plga has won strong attention, owing to its attractive properties: biodegradability and biocompatibility, fda and ema approval in drug delivery systems for parenteral administration, well-described formulations and methods of production adapted to various types of drugs such as hydrophilic or hydrophobic small molecules or macromolecules, protection of the drug from degradation, the possibility of sustained release, the possibility of modifying surface properties to provide stealthiness and/or better interaction with biological materials, and the possibility of targeting nps to specific organs or cells [ ] . the plga-based carriers' cargo release characteristics could be relatively well controlled by modulating encapsulation, particle size, formulation additives, molecular mass, ratio of lactide to glycolide moieties in plga, and surface morphology [ ] . the most widely used plga, with a monomer composition of : , has the fastest biodegradation rate; it completely occurs in approximately À days. the polyglycolide acid is more hydrophilic than polylactide, owing to the absence of a methyl side group [ ] . a higher glycolic acid percentage causes more water uptake and consequently faster degradation of plga polymers. plga is hydrolyzed into the original monomers lactic acid and glycolic acid, which are by-products of various metabolic pathways and are not associated with any significant toxicity except lowered ph. next to release characteristics, various other physical traits of plga particles can be manipulated, including particle size, size distribution, zeta potential, polydispersity index, encapsulation efficiency, and cargo loading [ ] . among a myriad of choices in nano/microcarrier polymers, plga has more advantages other than those listed above: plga particles can be administered via diverse routes; plga particle formulation may dampen toxicities of vaccine components; plga particles could protect the antigen from degradation and allow controlled release; plga particles could be made to target apcs and increase cross-presentation of the antigen; and plga particles allow concomitant delivery of multiple vaccine components with dose sparing. while many properties of plga polymers are favorable and controllable as vaccine delivery tools, there are drawbacks as well. plga has a negative effect on the stability of encapsulated protein antigens during preparation and storage, primarily owing to the acid-catalyzed nature of its degradation. its hydrolysis leads to the accumulation of lactic and glycolic acids in the microenvironment, which will denature encapsulated protein antigens and consequently compromise immunogenicity [ ] . in addition, processing conditions used in manufacturing plga carriers have negative effects on protein secondary structures [ ] . to overcome problems associated with protein degradation, many efforts have been made to optimize the manufacturing process and to add excipients that would protect the protein antigens being encapsulated. protein antigens adsorbed to plga particles are relatively more protected from those physicochemical insults. adsorbed antigen would offer improved stability and activity over encapsulated antigen by avoiding exposure to organic solvents used during formulation and acidic ph conditions caused by degradation of the polymer. but this may result in premature high burst release of the antigen before uptake by apcs, which can lead to deficient immune responses [ ] . natural polymers are attractive vaccine delivery vehicles, owing to their low toxicity and biocompatibility. synthetic polymers such as plga or pla are also reported to be safe and biocompatible. their degradation products affect the microenvironment by lowering ph which may be detrimental to functions of apcs and compromise immunogenicity of vaccine antigens. on the other hand, natural polymers are generally composed of biological components, making them physiologically resorbable with few to no adverse effects [ ] . the major drawback of natural polymers as vaccine delivery systems is reproducibility, which should be overcome by further technological researches. there are two major groups of natural polymers that are used to manufacture particulate carriers: peptide/proteins and polyelectrolytes including alginate, chitosan, and dextran [ ] . chitosan and chitosan derivatives are cationic polymers, which, owing to their structure, have excellent mucoadhesive and absorption-promoting properties. chitosan is manufactured by alkaline deacetylation of chitin (e.g., derived commercially from exoskeleton of crustaceans or fungi) and is a linear copolymer of β - linked monomers of d-glucosamine and n-acetyl-d-glucosamine [ ] . it is biodegradable and biocompatible. the pka of the primary amine group of chitosan is approximately . , and the nascent polymer at neural ph carries no charge; hence chitosan is insoluble in water. this solubility characteristic should prevent nascent chitosan from being able to deliver antigens that are soluble and stable at neutral ph. structural modifications have been made to chitosan to produce derivatives that are soluble at neutral ph yet retain the positive charge and unique properties of nascent chitosan. because chemical modifications make it possible to substitute both amine and hydroxyl functional groups of chitosan, various chitosan derivatives have been produced by introducing hydrophilic groups such as hydroxyalkyl, carboxyalkyl, succinyl, thiol, and sulfate or by grafting solubility enhancer polymers such as polyethylene glycol and poloxamer [ ] . of all the water-soluble derivatives, n-trimethyl and carboxymethyl derivatives of chitosan have been studied most extensively, owing to their relative ease of synthesis, ampholytic character, and ample application possibilities. soluble n-trimethyl chitosan has both mucoadhesive properties and excellent absorption-enhancing effects even at neutral ph because of its cationic charge at neutral ph [ ] . n-trimethyl chitosan is rather widely applied to the development of mucosal vaccine delivery systems, since it is mucoadhesive and has penetration-enhancing ability through the paracellular route even at neutral ph. chitosan particles can be fabricated to successfully deliver both adjuvant and antigen to dcs in the tissue or draining lymph nodes [ ] . adjuvants and antigens could be either incorporated inside or adsorbed on the surface of chitosan microparticles or nps on purpose. in addition to being a carrier, chitosan can be used to coat other polymer particles to enhance their immunogenicity, bioadhesiveness, and surface adsorption potential [ ] . n-trimethyl chitosan is freely soluble over a wide ph range as compared to other chitosan derivatives and bears positive charges, independently of the environmental ph. methylcarboxy chitosan is a polyampholytic polymer that is able to form viscoelastic gels in aqueous environments or with anionic macromolecules at neutral ph values. on the basis of these characteristics, the complexation of two chitosan derivatives without using any cross-linker could generate a vaccine delivery carrier that has high loading efficiency and can maintain integrity of a protein antigen [ ] . alginate is a linear, anionic polysaccharide found in the cell walls of brown algae. it has a high affinity for water and forms an inert and highly aqueous environment within the particle, which limits its ability to carry hydrophobic vaccine antigens and adjuvants. it is also biocompatible, biodegradable, and easily eliminated from the body [ , ] . as with chitosan particles, adsorption of adjuvant onto the surface of microspheres allows differential release of the antigen and adjuvant for temporally controlled stimulation of immune cells [ ] . alginate itself, besides its role as a vaccine carrier, has immune-stimulatory activities though stimulating nf-κb signaling pathway [ ] . dextran is a polysaccharide composed of repeating branched glucose molecules. its most commonly used form is dextran sulfate [ ] , which is biocompatible and hydrophilic and decomposes into natural byproducts. anionic dextran sulfate is often fabricated with cationic poly-l-arginine to make layer-by-layer antigen-adjuvant carriers [ ] . because this type of particle is assembled layer by layer, multiple antigens and adjuvants can be incorporated in multiple layers to maximize targeting and activation of immune cells. self-assembled peptides have been reported to be useful candidates for future vaccine delivery systems [ ] . peptide molecules can be rationally designed to self-assemble into specific nanoarchitectures in response to changes in their assembly environment, including ph, temperature, ionic strength, and interactions between host and guest molecules. they could be manufactured in the forms of nanomicelles, nanovesicles, nanofibers, nanotubes, nanoribbons, and hydrogels and would have a diverse range of mechanical and physicochemical properties [ ] . peptide delivery systems may have potential advantages over liposome or nps, since they can be composed of amphiphilic molecules with high loading efficiency, low antigen leakage, biodegradability, and high permeability to biomembranes of target cells. these molecules can be designed for cellspecific targeting by including adhesion ligands, receptor recognition ligands, or peptide-based antigens in their design, often in a multivalent display [ ] . these molecules can also act as intracellular transporters and respond to changes in the physiological environment. generally, self-assembling peptides are nonimmunogenic, serving as built-in adjuvants for fused antigenic peptides [ ] . the adjuvant activity is closely related to nanostructures, since the mutation of key amino acid residues in the self-assembling domain demolishes the immunogenicity of the self-assembled peptide vaccines [ ] . the adjuvanticity in a nanofiber self-assembled peptide vaccine was reported to be t-cell-and myd -dependent, but specific interactions with tlr and tlr as well as nalp were not noted, suggesting a novel immunomodulating mechanism. although peptide nanofiber vaccines are more efficiently taken up by dcs and subsequently activate them, these vaccines do not cause reactogenicity and nonspecific inflammatory reactions at the administration site [ ] . nanogels became more prominent recently as a vaccine delivery system. the term "nanogel" defines refers to nanoscale particles (, nm in diameter) composed of physically or chemically cross-linked bifunctional networks having good swelling capacity in aqueous environments [ ] . nanogels have a high cargo loading capacity, biocompatibility, and biodegradability. cationic nanogels are adhesive to epithelial cell surface and serve as artificial chaperones protecting antigens from aggregation and denaturation. loaded antigens are subsequently released in native forms and captured by appropriate apcs nearby [ ] . the surfaces of nanogels are relatively easy to modify by specific ligands, enabling targeted delivery to specific cells or tissues. nanogel vaccine formulations can be delivered via a wide range of routes, such as parenteral, oral, nasal, pulmonary, or ocular administration [ ] . nanogels can be formulated by various polysaccharides such as chitosan, mannan, hyaluronic acid, dextrin, cycloamylose, pullulan, and enzymatically synthesized glucogen [ ] . in recent years, pullulan has played a critical role in the development of nanogel systems for vaccine and drug delivery [ ] . pullulan is an aqueous polysaccharide synthesized by the yeast-like fungus aureobasidium pullulans. it consists of hundreds of repeated units of a maltotriose trimer. pullulan is widely used in diverse biomedical industries because it is easily modified by rather simple chemical reactions that are nontoxic, nonmutagenic, noncarcinogenic, and, most important, nonimmunogenic [ , ] . pullulan hydrophobized by cholesterol becomes amphiphilic and forms self-aggregates [ ] . the cholesterol-bearing pullulan (chp) can form complex nps with various protein antigens. chp can self-assemble in water into the nps and encapsulate protein antigens in the internal space through hydrophobic interactions. the complex np protects internal protein antigens against physicochemical or enzymatic degradation, serves as an ideal delivery vehicle, and releases payloads in a controlled fashion [ ] . the most valuable characteristic of chp nanogels is its artificial molecular chaperon activity [ ] . protein antigens are captured in denatured form in the chp nanogel under reversible denaturation temperature or in the presence of reversible denaturation reagents [ ] . in the nanomatrix, the nanogel protects denatured protein antigen as an artificial molecular chaperone and helps in proper refolding after release [ ] . another advantage of chp is its targeting ability to apcs. the chp nanogels and protein antigens could form colloidally stable nps nm in diameter, which is a relevant size allowing effective uptake by epithelial cells and apcs [ ] . moreover, chp nanogels can be modified to have cationic charge (cchp) by adding amine groups to the chp nanogels [ ] . the cchp nanogels could be well formulated with protein antigens and effectively carry vaccine antigen to the negatively charged nasal epithelium after intranasal administration [ ] . the positive charge of cchp nanogel provides more efficient adhesion to negatively charged nasal mucus and epithelia, leading to higher level and more sustained delivery of antigens to dcs inhabiting underneath mucosa. more important, the cchp vaccine formulation could induce significantly higher immune responses without adjuvant addition, while the cchp nanogel itself could not activate immature dcs, suggesting no biologically active adjuvant-like activity [ , ] . the reason why cchp, having no direct stimulatory effect on innate immune cells, could significantly enhance the immunogenicity of cargo antigens was thought to be the improved antigen residence time in the nasal cavity, which leads to better antigen transport to the nasal dcs. the -nm cchp nanogels carrying clostridium botulinum type a neurotoxin heavychain c-terminus (bohc/a) were bound by nasal epithelial cells and subsequently endocytosed. the bohc/a antigen was separated from the nanogel by protein exchange and sustainably released by exocytosis, which was subsequently taken up by cd c dcs in the mucosa [ ] . epithelial cells served as a reservoir for the cargo antigen, while no overt cytotoxicity was observed. neither the cchp nanogel nor cargo bohc/a antigen was taken up by the olfactory bulb or brain tissue, suggesting that the cchp nanogel system is safe for nasal administration (chapter : nanodelivery for mucosal vaccines). past, present and future gastrointestinal (gi) infection is a significant global health challenge, especially in developing countries. most gi infections are spread via the fecalÀoral route, primarily through contaminated water and food due to poor sanitation and social infrastructure. an efficacious vaccination policy is the most economical way of solving gi infection problems from a public health perspective. the oral vaccination is generally the best way to induce secretory immunoglobulin a (siga) in the gi tract and igg antibody responses in the systemic compartment. in fact, the only oral vaccine that has been widely used globally in infants and children in a national immunization program is the oral polio vaccine (opv) developed by albert sabin in the s. since sabin's opv vaccination, several oral vaccines against rotavirus, salmonella typhi, and vibrio cholerae have been licensed and marketed [ ] . those vaccines are made of live attenuated organism or killed microbial cells. there is as yet no licensed subunit oral vaccine on the market. there have been continuous efforts to develop oral vaccines because of the advantages of oral vaccination. as was noted during the haitian cholera epidemic, oral vaccination is a faster way of containing circulating infections and prevention of further outbreaks [ ] . after the september , , terrorist attacks, the threat of biological warfare became highlighted worldwide. the potential bioterrorism agents are likely to be disseminated by either aerosol or in food or water supplies targeting the wide mucosal surfaces in the respiratory or gi tracts, respectively. considering that the bioterrorism agents invade from the gi mucosa, oral vaccines, inducing protective immune responses at the route of entry, have generated the most interest as a frontline tool in biodefense [ ] . oral vaccination has several advantages, such as better patient compliance, mass immunization capability, easy administration or selfdelivery, simplified production and storage, lower production cost, and no needleassociated risks such as injuries and carryover infections (table . ) [ ] . the most important virtues of oral vaccination are its needle-free painless administration and that there is no need for trained personnel for administration. two major mucosal vaccination routes, oral and intranasal, are compared in table . . the most widely used oral vaccine is sabin's opv, which contributed enormously to the eradication of poliomyelitis worldwide. but recently, despite its efficacy, opv has been replaced by injectable poliovirus vaccine (ipv) in developed countries. opv is likely to be replaced by ipv globally over several coming years. live poliovirus was discovered in the stool of opv vaccines, possibly spreading infectious material in the environment [ ] . another serious concern about opv is the rare event of reversion to virulent strains in vivo, which could cause a serous iatrogenic vaccine-associated paralytic poliomyelitis [ ] . the same concerns apply to other mucosal vaccines using live attenuated organisms. but generally, oral vaccines are regarded as a better choice than injectable parenteral vaccines from production, economic, and regulatory perspectives [ ] . oral vaccines have better compliance and fewer adverse reactions. oral vaccines are better for large-scale production and mass vaccination campaigns in developing countries, since no needles are required and self-administration is possible. thermostabilization technologies would enable successful cold-chain-free vaccination of killed as well as live attenuated formulations in resourcepoor settings such as developing countries [ ] . thanks to the many virtues of oral vaccines and the success of opv, research into oral vaccines has rather a long history, but only a very limited number of oral vaccine products have become available. many oral vaccines that proved to be efficacious in preclinical studies have failed in clinical trials. live-attenuated vaccines against rotavirus and s. typhi have been successfully introduced into the market. in the case of cholera, two types of oral cholera vaccines (ocvs) are currently available: wc-rbs, which are killed wc monovalent (o ) vaccines with a recombinant b subunit (rbs) of cholera toxin (ct) (dukoral), and killed modified wc bivalent (o and o ) vaccines without the b subunit (shanchol, euvichol, and morcvax) (who cholera vaccine position paper-august at www.who.int) (chapter : cholera immunity and development and use of oral cholera vaccines for disease control). the three wc vaccines are based on the same cholera strains and dosage. although the wc killed cholera vaccines listed proved to be efficacious in multiple clinical trials, many other prototype killed and subunit vaccines could not be put on the market because of suboptimal immunogenicity. oral vaccines should have strong immune-stimulatory adjuvants and optimal delivery strategies to drive effective innate and adaptive immune responses against vaccine antigens [ ] . to achieve optimal immunogenicity, dukoral has a huge number of killed emulsions such as mf and as are extensively tested for many vaccines and immunization routes. however, in the literature, it is rather rare to find successful test results for oral vaccines against infectious diseases employing an oil-in-water or water-in-oil emulsion formulation or adjuvant system. a successful oral tumor vaccine study showed that an antigen complex (melanoma antigen mage , heat shock protein , and staphylococcal enterotoxin a) incorporated in a nanoemulsion with a small size of À nm induced efficacious protective immune responses comparable to those of subcutaneous administration [ , ] . vaccine antigen can be delivered inside the core or attached on the outside to the shell of micelles, depending on the electrochemical properties of the vaccine formulation [ ] . oral immunization of pla-peg-pla and plga-peg-plga copolymer micelles loaded with dna encoding hcv multiple epitope antigen could induce satisfactory immune responses [ ] . the copolymers showed an innate adjuvant activity and caused no significant adverse reactions [ ] . micelles can be synthesized as nanocarriers and engineered to penetrate mucus and be taken up by mucosal apcs [ ] . however, micelles may have a propensity to dissociate when diluted, leading to a loss of loaded antigen. while most clinical trials and delivery system developments employing liposomes have focused on the parenteral routes, there are still continued efforts to develop liposome-based oral delivery tools [ ] . conventional liposomes are vulnerable to acidic gastric juice and are easily digested by pancreatic lipase [ ] . also, intestinal bile salts can destroy the phospholipid membrane integrity and lyse the liposomes, resulting in the premature release of vaccine antigens [ ] . to tackle these problems, researchers have investigated different lipid moieties such as archaeal lipids or bile salts in the liposomal membrane [ , ] . an important determinant of the adjuvanticity of liposomes is the surface charge. positively charged (cationic) liposomes have been demonstrated to possess the strongest adjuvanticity compared to neutral and negatively charged liposomes [ ] . mucoadhesion is promoted by the cationic surface charge that would have a stronger interaction with negatively charged gi mucus. cationic liposomes also better adhere to negatively charged membranes of m cells and enterocytes, limiting flushing by peristalsis and providing a better chance to be internalized [ ] . however, the greater toxicity of cationic than anionic liposomes is of concern. recently, a study reported that cationic liposomes induce necrosis to release damageassociated molecular patterns and cause inflammation in vivo [ ] . to extend the clinical applications of liposomes as carriers of oral vaccines by improving stability and sustainability in gi tract mucosa, the surface modification of liposomes has been rigorously investigated [ À ]. the addition of cholesterol and quil a saponin to liposomes results in the generation of self-assembling pentagonal dodecahedrons that have intrinsic adjuvant properties and thus earned the name iscoms. two types of iscoms exist. the first, "classical" type was manufactured to entrap protein antigens, making them act as both a vaccine antigen delivery and an adjuvant system. the second type, referred as iscom matrices, contains no entrapped antigen and serves as a codelivered adjuvant, which is later formulated with vaccine antigens. early iscom formulations could not be evaluated in humans and were limited to veterinary use, owing to the reactogenicity of quil a. quil a was later replaced with qs , and iscoms could be then given clinical trials. qs is more purified in nature and shows an improved safety profile while remaining active as an adjuvant [ ] . while iscoms were widely tried with very diverse antigens for parenteral and intranasal vaccination studies, trials with oral vaccines have been relatively scarce [ , ] . one major challenge in working with iscoms is the difficulty associated with antigen incorporation. in this context, many trials used iscom matrices purely as adjuvants and formulated with vaccine antigens rather than incorporating them, which elicited immune responses as beneficial as those elicited by classical iscoms [ ] . the use of unmodified iscoms as adjuvants would significantly simplify production; however, the benefit of antigen encapsulation, which is an important key to success for the oral route, might be lost. for this reason, iscom matrix adjuvants in combination with enteric vaccine antigens are used for boosting through intranasal or parenteral routes after oral priming with antigens only or antigens with other adjuvants [ , À ] . to use iscoms for the induction of efficacious immune responses in the gi tract, the immunization protocol employing intelligent oral or injectable prime-boost regimens and incorporating additional coatings or adjuvants should be tried. vlps imitate the three-dimensional conformation of real virus and mimic infection by authentic viruses. moreover, vlps can be engineered to express additional antigens and target epitopes in repeated array. vlps have been studied as oral vaccines against virus or tumor antigens [ À ]. those vlp oral vaccines induced humoral and cellular immune responses in both systemic and mucosal compartments. significant antigen-specific siga responses were also observed. vlps could also be expressed in plant tissue successfully [ , , ] and could be purified from plant tissue at lower cost. on the other hand, rather crude freeze-dried plant tissue containing vlps could be directly administered orally to induce protective immune responses in animals, which suggests the possible development of human edible vaccines using vlps expressed in plant tissues such as tomato and potato [ , À ] . synthetic particles can serve as the most versatile oral delivery tools in which vaccine antigens and adjuvant could be loaded by encapsulation, conjugation, or adsorption. with advances in polymer material chemistry and formulation technology, different types of natural and synthetic nps are being actively tested for possible use for effective oral vaccine delivery. these recent technologies enable overcoming previous barriers in oral vaccination and allow better targeting of antigens and adjuvants to the desired tissue location and cells. particles can be engineered to release antigens and adjuvants upon degradation, swelling, and diffusion from the polymer, or change in electrostatic interactions. the production of particles in defined sizes, architectures, and chemical properties would enable oral delivery, which is the most difficult vaccination route in terms of targeted delivery, thanks to the major development in nanotechnology and biomaterial science. depending upon the polymer choices, the delivery systems by themselves provide adjuvant activity along with biocompatibility and biodegradability. the most extensively studied biodegradable polymers for the development of oral vaccine nanocarriers are pla, plga, β-glucans, alginate, and chitosan [ , ] . in the case of oral delivery, the greater surface area of nps, owing to their small size, would allow increased absorption across the intestinal epithelium, which will furnish reduced dosage and administration volume [ ] . hydrophilic nps are generally transported through enterocytes, whereas hydrophobic polymeric nps are better transported through m cells [ , , , ] . orally administered pla nps ( À nm) reached the peyer's patches (pps) through a three-step process. most particles are first entrapped in the mucus. then crossing of the epithelial barrier takes place exclusively through m cells, leading to an accumulation in pps. finally, the nps interact with underlying b cells and dcs in the pp tissue. all three steps can occur within minutes. furthermore, dcs engulfing nps were induced of tlr expression [ ] . to be licensed and clinically used for mucosal vaccine delivery, nanocarriers should be able to protect the payload from degradation, to penetrate the mucus barriers, and to control the release of both antigens and adjuvants at targeted sites. in principle, these properties could be tuned by altering their particle size, surface chemistry, and three-dimensional architecture [ ] . it is widely accepted that particulate antigens are more efficiently trafficked across the mucosa and delivered to mucosal apcs than are soluble antigens [ ] . uptake of particles in the gi tract occurs primarily via m cells. despite some controversies, particles with a diameter smaller than μm are thought to have a better chance of being taken up by m cells [ ] . to meet requisites for delivering diverse vaccine antigens and adjuvants to a specific mucosal target, plga surface characteristics have been extensively modified by coating with ionic surfactants or polymers such as polyethylene glycol (peg), sodium dodecyl sulfate, aminodextran, chitosan, polyethylene imine, poly-llysine, protamine, or cetyltrimethylammonium [ , À ] . the surface chemistry of plga particles can also be altered to increase diffusion through mucus and uptake by m cells through oral delivery [ , ] . other methods, such as coating antigen/adjuvant-loaded plga nps with methacrylate-based polymer eudragit fs d, produced gastric-acid-resistant microparticles ($ μm) that released payloads from the terminal ileum, where the ph level reaches . or higher [ ] . the ph-sensitive polymers derived from methyl methacrylate, methacrylate, methacrylic acid, acrylate, and/or dimethacrylate have been blended with plga for the protection of payloads in the np against enzymatic attacks in the stomach and small intestine [ ] . the antigen encapsulated by plga nps sequentially coated with phase-transitional shielding layer, poly[(methyl methacrylate)-co-(methyl acrylate)-co-(methacrylic acid)]Àpoly(d, l-lactide-co-glycolide) (pmmma-plga), was found to protect antigens in the gi tract and achieve targeted vaccination in the large intestine [ ] . hydroxypropyl methylcellulose phthalate (hpmcp), another enteric coating agent, was shown to make acid-resistant b nm nps with plga carrying helicobacter pylori antigen effectively induce th /th protective immune responses [ ] . to induce enhanced protective immune responses against antigens delivered by plga carriers, plga particles are functionalized by m-cell-targeting ligands in combination with stabilizing agents. incorporation of m-celltargeting lectins such as uea or lta into plga nps would enhance antigen-specific immune responses [ , ] . arginine-glycine-aspartate (rgd) ligand binding to β integrin can also target m cells. pegylated plga nps grafted with rgd or rgd peptidomimetic ligand showed significantly increased uptake by m cells and enhanced specific igg responses [ , ] . claudin , one a member of the integral membrane protein family expressed primarily in tight junctions, also serves as a target for developing m-cell-binding nanocarriers [ ] . in one study, an antigen-loaded porous plga microparticle was successfully coated with water-soluble chitosan conjugated with an m-cell-targeting peptide (cks ). the resulting microparticles effectively reached pps through m cell transcytosis to induce balanced th /th protective immune responses [ , ] . the phsensitive polymeric delivery systems employing hydroxypropyl methylcellulose phthalate (hpmcp) could be attuned by adding thiol groups to be selectively released under ileal ph condition. by formulating m-cell-homing peptide (cksthplsc) conjugated bmpb antigen with attuned hpmcp, delivery to pps and subsequent adaptive immune responses could be significantly enhanced [ , ] . recent research into oral vaccine delivery of nps has been directed toward the incorporation of mucoadhesive polymers. the mucoadhesive polymers prolong retention time of the particles in the mucus by steric or adhesive interactions. coating of nonbioadhesive nanospheres with poly(butadiene-maleic anhydrideco-l-dopa) increased the particle uptake by -fold in the small intestine [ ] . conjugation of immunostimulatory ligands to bioadhesive polymers should induce longer-lasting mucosal and systemic immune responses against entrapped antigens. bioadhesive poly(anhydride) nps ( À nm) coated with mannose or salmonella flagellin induced more potent and balanced th /th immune responses compared with noncoated particles [ ] . chitosan and its derivatives have been tried for the oral delivery of protein and dna vaccines [ À ] . the limited solubility of chitosan at alkaline and neutral ph has been circumvented by fabrication of chitosan by graft copolymerization with acyl, alkyl, monomeric, and polymeric moieties. modifications through quarterization, thiolation, acylation, and grafting resulted in copolymers with higher mucoadhesion strength, increased hydrophobic interactions (advantageous in hydrophobic antigen entrapment), and increased solubility in alkaline ph, higher solubility, and controlled/ extended release profiles, which consequently confer wider application of chitosan derivatives for oral vaccine delivery [ , ] . chitosan and its derivatives are mucoadhesive and have the ability to stimulate immune cells either by directly interacting with the m cells or by opening the tight junctions between the epithelial cells [ ] . because of the advantages mentioned above, chitosan has been applied to the manufacture of orally deliverable nps or coating of micro/nanocarriers made of other synthetic or natural biopolymers [ , ] . alginate has been used to make oral vaccine carriers utilizing its acid resistance and immunostimulatory properties [ ] . in order to overcome chitosan's instability in low-ph environments, cationic chitosan nps can be coated with acid-resistant alginate to make composite carriers [ , ] . alginate encapsulation of chitosan nps entrapping protein antigens was proved to protect payload protein antigens and dna from acidic attack in the stomach after oral administration. owing to its acid resistance property, alginate is also used to encapsulate bacterial cells to develop oral vaccines. a single oral dose of alginateencapsulated bcg elicited effective long-lasting mucosal and systemic immune responses [ ] . cold-chain-free ocv could be developed by encapsulating heat-inactivated bacterial cells with alginate [ ] . oral vaccines against edwardsiella, brucella, and aeromonas infections were also developed by encapsulation with alginate [ À ]. glucan particles are porous -to -micron cell wall shells manufactured by treating baker's yeast (saccharomyces cerevisiae) with a series of alkaline, acid, and solvent extractions [ ] . by in situ layer-by-layer synthesis through electrostatic interactions, dna could be encapsulated at high density [ ] . protein antigens could be encapsulated in glucan particles through hydration and lyophilization and could induce significant intestinal siga and th /th cellular responses to encapsulated antigens following oral vaccination [ ] . glucan microparticles target enterocytes and m cells for uptake and activate them to secrete and express cytokines and β-glucan receptors [ À ]. β-glucans, the major component of glucan microparticles, are fungal pamps, signaling through receptors such as dectin- and complement receptor expressed on dcs, monocytes, and neutrophils [ , ] . although the efficacy of glucan particles as an oral vaccine carrier was well proved with model antigens such as ova or bsa, the application to pathogenic microorganisms has been relatively rare [ ] . one reason for the limited use of glucan particles is that their manufacture is currently limited to liquid formulations, which require cold-chain storage and therefore are not optimal for the use in poorer regions [ ] . plant-based oral vaccines have advantages over the traditional vaccines in cost, safety, and scalability. since , researchers have manufactured edible plant-based vaccines in carrot, soybean, tomato, rice, potato, and tobacco against microbial pathogen antigens such as the heat-labile toxin b subunit (ltb) of enterotoxigenic escherichia coli, cholera toxin b subunit (ctb), and antigens from yersinia pestis and viruses, such as hepatitis b virus, rotavirus, and norwalk virus [ ] . many conventional vaccines are not widely distributed in developing countries where those vaccines are urgently needed, because of high production costs and the requirement of better infrastructure. one more problem standing in the way of wider distribution of desperately needed vaccines is that the conventional cell fermentation systems for producing recombinant protein vaccine antigens are often expensive and are not easily scalable [ ] . another emerging infectious disease field is one health, dealing with zoonotic diseases spreading in both animals and humans. a solution to this may be the use of plants or plant cells as bioreactors. molecular farming has become well established for the production of vaccines, and many proofs of principle and important proofs of efficacy are accumulating continuously [ ] . mucorice should be one of the most innovative approaches for oral vaccine delivery using edible rice as a carrier (chapter : plant-based mucosal vaccine delivery systems). rice seeds have stability and resistance to digestion in the stomach, making mucorice an attractive oral vaccine delivery system. in , it was first reported that cholera toxin b subunit (ctb) could be expressed in the rice seed. as much as μg of ctb per seed was stored in the storage organelle protein body. when orally ingested, rice seeds expressing ctb induced ctb-specific serum igg and mucosal iga antibodies with neutralizing activity, while no rice storage-specific immune response was noted. when expressed in rice, ctb was protected from pepsin digestion in vitro [ ] . rice-expressed ctb also remained stable and thus maintained immunogenicity at room temperature for more than years, and it provided more than months of protection against ct-or lt-induced diarrhea after primary immunization [ ] . these results show that the mucorice vaccine could be stockpiled longer at room temperature and could be widely used for oral vaccination without cold-chain management. rice-based oral vaccine developments are under way against many infectious diseases and noninfectious diseases such as allergy, autoimmunity, and alzheimer's disease [ ] . the most widely used form of whole bacterial cell vaccines for cholera and typhoid fever was liquid suspension. because of the lack of shelf stability, the liquid format is unsuitable for storage and distribution in developing countries. in this regard, a stable solid dosage vaccine platform is required for those vaccines. formulation in tablets or capsules would provide more stability and ease of handling. in comparison to microparticles and nps, capsules are significantly larger in size and could serve reservoir for multiple vaccine/ adjuvant formulations [ ] . while no subunit or wc killed oral vaccine is currently delivered by capsule or tablet, the live attenuated salmonella vaccine vivotif is routinely delivered in an enteric-coated format [ ] . capsules could be manufactured in appropriate physical sizes (the average size of capsules and tablets ranges from to mm) suitable for administration to target populations. with enteric coatings, tablets and capsules could be protected from gastric acid and endowed with controlled release properties, which will provide facilitated delivery to discrete locations in the intestine. in principle, capsules allow the incorporation of many previously introduced delivery technologies in one primary delivery format. recently, a tablet-based oral avian influenza vaccine was shown to elicit strong antiviral antibody and ifnγ t-cell responses. this approach utilized a nonreplicating adenovirus type vector expressing avian flu hemagglutinin antigens together with a dsrna tlr agonist [ ] . among the choices of mucosal routes for vaccine administration, nasal delivery has been the most widely employed for innovative research because of the ease of approach and less harsh physicochemical conditions in the nasal cavity compared with the gi tract. furthermore, nasal vaccines could be administered without professional training. there are at least three nasal vaccines licensed worldwide. the fda approved flumist, a live attenuated trivalent/quadrivalent influenza vaccine. fluenz is an ema-approved quadrivalent influenza vaccine. these two intranasal vaccines are manufactured by the same company, medimmune. the serum institute of india licensed the monovalent nasovac against pandemic a/california/ / h n influenza (chapter : nasal influenza vaccines). besides influenza vaccines, the nasal route has been widely studied for development of many prophylactic and therapeutic vaccines against other infectious diseases, such as allergy, cancer, alzheimer's disease, and lifestyle-related diseases [ À ]. the human nasal cavity is an attractive route of mucosal immunization, having a total surface area of cm with a volume of À ml [ , , ] . the nasal cavity is divided into five anatomical and functional regions: the nasal vestibule, the atrium, the respiratory region, the olfactory region, and the nasopharynx [ ] . the respiratory region is where nasal delivery of drugs and vaccines occurs, since it is the most permeable region, having a large surface area and a rich vascular bed [ ] . the respiratory region is covered by a pseudostratified epithelium composed of columnar cells interspersed with goblet cells, which are interconnected by tight junctions (zonae occludens). the tight junctions are relatively resistant to paracellular passages of particulate materials in the breathed air [ ] . the respiratory region is where mucus production actively takes place. the mucus layer in the nasal tract is relatively thinner ( μm) than other mucosal surfaces. the nasal cavity is equipped with nasopharyngeal-associated lymphoid tissue (nalt), which is highly similar to peyer's patches in the ileum [ ] . nalt is also covered with m cells that have active antigen sampling capacity [ ] . intraepithelial dcs project dendrites toward mucosal lumen and sample antigens. particulate antigens are preferentially sampled by m cells, and small soluble antigens have access to epithelium, where they are captured by intraepithelial dcs [ ] (chapter : anatomical uniqueness of the mucosal immune system (galt, nalt, ibalt) for the induction and regulation of mucosal immunity and tolerance). the mucociliary clearance mechanism should have negative effects on nasal vaccination. the rapid turnover of mucus ( À minutes) and fast mucus flow (b mm/min) in the nasal cavity limit the length of residence of administered vaccine. continuous outward movement of cilia on the epithelial apical surface accelerates the clearance of mucus-trapped substances. to make matters worse, nasal enzymes and local ph negatively affect the stability of nasally administered vaccine antigens [ ] . this could be why only live attenuated influenza vaccines proved effective in clinical trials and were approved by the fda and ema. a live influenza virus should be able to survive in the nasal mucosa and be harnessed with built-in adjuvants. an inactivated split influenza vaccine was also tested for nasal delivery but proved ineffective without coformulation with appropriate mucosal adjuvants [ À ] . to achieve equivalent antibody responses without adjuvant, an inactivated split antigen should be given at least three times more, or an inactivated whole virus should have been immunized [ À ] . given that even an inactivated virus antigen requires potent mucosal adjuvants to achieve optimal immune responses in the systemic and mucosal compartments, protein antigens should employ even stronger mucosal adjuvants to be effective by nasal vaccination. many mucosal adjuvants are suggested as formulation partners of nasal vaccine antigens [ , ] . ct and related e. coli heat-labile toxin (lt) and their mutant derivatives are the most widely tried mucosal adjuvant in preclinical studies [ ] . although those enterotoxins served as potent adjuvants for nasal vaccination of diverse antigens in animal studies, they have seldom been adopted for the development of human nasal vaccines. the use of enterotoxins as nasal vaccine adjuvants has a very serious failure history. the subunit influenza nasal vaccine nasalflu berna adjuvanted with e. coli lt had been significantly connected with bell's palsy with an odd ratio of in an epidemiological study in switzerland. the vaccine was consequently withdrawn from the market [ ] . the adverse effect could be related with the capacity of ltb and ctb subunit to bind to gm ganglioside expressed on neuronal cell and retrograde translocation toward the brain [ , ] . since the nasal cavity and brain are separated by a thin anatomical structure and are directly connected by the olfactory nerve, binding of any vaccine component to the olfactory nerve should contribute neurotoxicity. in this context, any nasal vaccine, adjuvant, or delivery system must clear the safety concern to be introduced to the market. given the versatility and ease of nasal vaccination, numerous research groups are studying safe nasal adjuvants that could replace ct and lt. pamps are most widely studied as alternative nasal adjuvants. recently published literature shows that ligands of tlr , tlr , and tlr , sting agonist, and flt ligand could be used as effective and safe nasal adjuvants [ , À ] . vaccine antigens should remain sufficiently stable in the nasal mucosa and should be able to reach to antigen-capturing cells surviving the mucociliary clearance mechanism. to overcome those hurdles, micro/nanocarriers for nasal vaccine delivery have been actively researched. to increase the residence time at mucosal surfaces, several strategies have been developed to increase adhesiveness of antigen delivery systems to the nasal mucus [ , ] . however, the mucus is not a static barrier; it is continuously secreted and cleared from the nasal cavity by the cilia beating on columnar epithelial cells. mucoadhesion ability of delivery carrier would cause earlier removal of vaccine antigens when mucociliary clearance mechanism is intact. to cope with this problem, nasal vaccine carriers should cross the mucus layer rapidly and deliver antigens to m cells and dcs rather than strongly adhering to it [ À ] . strategies that prevent vaccine car-rierÀmucus interactions and hence allow for free diffusion by mucopenetration should be more effective in inducing efficacious immune responses [ ] . however, many reports claimed that mucoadhesive nps effectively enhanced the efficacy of mucosal vaccines [ ] . one study showed that mucoadhesive nps disrupted the protective human mucus barrier by altering its microstructure [ ] . the disrupted microstructure resulted in a % increase in mucus mesh pore size. this disrupted mucus mesh would allow increased passage of other nps to reach to the epithelium. the comparative advantages and disadvantages of intranasal vaccination are summarized in table . . the most outstanding advantage is the ease of administration, while the safety issue is the most essential problem to be resolved. nanoemulsions, owing to their stability, small droplet size, and optimal solubilization properties, have great potential in nasal drug delivery. furthermore, they may act as an active adjuvant in nasal vaccine formulations. despite the promising results of in vitro and animal studies, the application of nanoemulsions for nasal delivery in humans appears to be hindered mainly by the lack of detailed toxicology studies and the lack of extensive clinical trials [ ] . a cationic nanoemulsion formulation could have facilitated cellular uptake of model antigen ovalbumin in the nasal epithelial cell line [ ] . the intranasal vaccination of hiv gp immunogen formulated in oil-inwater nanoemulsions induced robust serum anti-gp igg and th -polarized systemic cellular immune responses [ ] . a large number of studies have investigated the potential of liposomes as a delivery system for nasal vaccination. those [ ] . peptides, proteins, and dna can be successfully carried by liposomes having neutral, negative, and positive charges. cationic liposomes were shown to interact more efficiently with epithelial cells and dcs [ , ] . induction of cell-mediated immunity is another important feature of liposomemediated adjuvanticity [ ] . intranasal administration of dna vaccine formulated with cationic liposomes, together with il- -and/or gm-csf-expressing plasmids, resulted in both high levels of hiv- -neutralizing antibodies in feces and serum and high levels of hiv-specific ctl responses [ ] . using this characteristic of the liposomal delivery system, a successful antitumor cellular immune response could be induced by intranasal immunization of dctargeting liposomes carrying a tumor antigen [ ] . similarly, intranasal immunization with liposome-encapsulated plasmid dna encoding influenza virus hemagglutinin elicited both mucosal cell-mediated and humoral (iga and igg) immune responses [ ] . intranasal chitosan solution formulations were reported to enhance protective immune responses against many antigens, including diphtheria, pertussis, and influenza [ À ] . chitosan solutions seem to induce balanced th and th responses with neutralizing antibodies [ ] . whole influenza virus formulated with trimethylated chitosan showed much closer interaction with the epithelial surface, with the potential to generate enhanced uptake and induction of immune responses with minimal local toxicity in terms of ciliary beat frequency in the nasal cavity [ ] . chitosan dry power in salt form enables a thermally stable vaccine formulation that does not require cold chains. chitosan power formulations were shown to outperform solutions in eliciting humoral responses against diphtheria, anthrax, and norovirus [ ] . chitosan microparticles and nps are being robustly studied for the intranasal delivery of vaccines. chitosan particles are basically mucoadhesive and able to deliver adjuvants and antigen cargos to efficiently promote humoral and cellular immune responses. protein and peptide antigen-loaded chitosan particles are taken up by apcs in the administration site and eventually trafficked to draining lymph nodes, where t-cell activation occurs [ ] . to be used for better intranasal delivery, chitosan should be chemically modified for better solubility, stability, mucoadhesiveness, safety, and resilience against degradation [ ] . chitosan itself shows strong adhesion to mucosal surfaces, providing a longer retention time at the nasal mucosa, and disrupts the tight junctions between nasal epithelial cells, which leads to enhanced paracellular transport of antigens [ ] . the paracellular transport of the vaccine formulation into the nasal mucosa would lead to enhanced antigen uptake and presentation by apcs, with consequently augmented adaptive immunity [ ] . this was demonstrated in a subunit influenza vaccine study showing protection against a heterologous viral challenge [ ] . ntrimethyl chitosan nasal vaccine formulation was superior to plga nps in inducing nasal iga responses [ ] . improved mucosal (iga) and humoral (igg) antibody responses are generally observed in mice as well as in other animal models such as rat and rabbit [ ] . the cationic chitosan enhanced th and th responses as well as dc maturation through type i interferon induction by the cgas-sting pathway, suggesting the involvement of multiple immune components [ ] . modified chitosan particles were generated to improve delivery efficiency and targeting. pegylation improved water solubility and stability of conjugated antigens [ ] . chitosan coated poly-(ε-caprolactone) nps induced enhanced mucosal immune responses against coformulated influenza antigen [ ] . protein antigen-loaded pluronic f- /chitosan microparticles showed improved antigen release and induced higher antigen-specific secretory iga responses after intranasal vaccination [ ] . ova-loaded trimethyl chitosanÀhyaluronic acid nps demonstrated superior immunogenicity after intranasal immunization [ ] . mannosylation of chitosan particles enhanced macrophage targeting and antigen-specific secretory iga responses in mucosal secretions after intranasal immunization [ ] . chitosan application to intranasal vaccination has already reached clinical trial stages (phases and ) in the form of nps [ ] and antigen-conjugates [ ] . norovirus vlp formulated with mpla was administered intranasally twice to healthy volunteers, inducing specific iga responses in % of vaccinated individuals [ ] . a clinical study testing intranasal vaccination of neisseria meningitidis serogroup c polysaccharide (mcp)-crm conjugate antigen mixed with chitosan showed specific iga responses in nasal washes and balanced igg /igg responses in serum [ ] . despite several clinical studies with promising results, no chitosan-based product for intranasal vaccination has yet reached the market. influenza viral antigens encapsulated within bioadhesive starch and propylacrylic acid mixtures induced significant systemic antigen-specific igg responses but not mucosal iga after intranasal delivery of the influenza vaccine in rabbit [ ] . inactivated influenza antigens in positively charged nps have been tested in a phase clinical study. significant mucosal iga antibodies were produced in individuals who received two-dose nasal immunizations [ ] . a cationic maltodextrin np (cationic surface with an anionic lipid core) showed longer nasal residence time after nasal administration than liposomes and plga nps [ ] . the plga nps are the most used synthetic polymer for nasal vaccine delivery studies. cationic modification of plga enhanced residence time in the mucosa and resulted in better immune responses with higher serum antibody and siga levels [ ] . the surface modification of plga carriers with chitosan can increase mucoadhesion through a change of zeta potential from negative to positive without affecting particle size and dispersion. moreover, the clearance rate in the nasal cavity was reduced, resulting in enhanced systemic and mucosal antibody responses [ ] . many antigens encapsulated in plga nano/microparticles were immunized through the nasal route to show enhanced immune responses in both systemic and mucosal immune compartments; they were ovalbumin, bovine serum albumin, bovine parainfluenza virus, bovine syncytial virus, hbsag, malaria, swine fever virus, y. pestis, and streptococcus equi [ ] . targeting efficiency to m cells could also be achieved by functionalization of the particle surface [ ] . although many types of nanogels were tested as vaccine delivery systems, the cholesteryl group-bearing pullulan (chp) is the most extensively studied one for mucosal vaccine delivery [ ] . the cationic chp (cchp) nanogel binds better to epithelial cells and is subsequently taken up with high efficiency into the cells. the cchp nanogel itself did not activate dcs and did not have biologically active adjuvant activity. however, vigorous neutralizing serum igg and siga responses were noted without coadministration of mucosal adjuvants. the cchp nanogel was suggested as a universal protein-based antigen delivery vehicle for adjuvant-free intranasal vaccinations [ ] (chapter : nanodelivery vehicles for mucosal vaccines). to further potentiate immune responses against cargo antigens, cytokines or adjuvant could be coencapsulated in the chp nanogel. tnfα encapsulated in the nanogel acted as a vaccine adjuvant for a nasal influenza vaccine [ ] . in an antiobesity vaccine study, cchp was engineered to carry the adjuvant cyclic di-gmp along with self-origin antigen ghrelin peptide hormone conjugated to a carrier protein pspa [ , ] . the cchp-based nasal vaccines were successfully tested for use against several infectious diseases and lifestyle-related diseases: influenza, streptococcus pneumoniae, c. botulinum, obesity, and hypertension [ , , , , , ] . another very promising virtue of the cchp nanogel nasal delivery system is safety. it was shown reiteratively that protein antigens carried by the nanogel did not accumulate in the olfactory bulb and brain, thus excluding the risk of neurotoxicity or brain damage [ , ] . when all the physicochemical, biological, and immunological characteristics are considered, the cchp nanogel platform seems to be the most promising nasal delivery system to be translated into more aggressive clinical applications. although mucosal vaccination has many advantages, a very limited number of mucosal vaccines have been licensed. the most widely tested vaccination routes are oral and intranasal. currently licensed oral and intranasal vaccines are composed predominantly of wc killed or live attenuated microorganisms, where cell bodies serve as delivery systems and whose cell components act as built-in adjuvants. future mucosal vaccines should be made with more purified antigen components, which will require safe and efficacious adjuvants and delivery systems. recent developments in biomaterials and nanotechnology have enabled many innovative mucosal vaccine trials. for oral vaccination, the vaccine delivery system should be able to stably carry antigens and adjuvants and resist the harsh physicochemical conditions in the stomach and intestinal tract. besides many nano/microcarrier tools generated by using natural and chemical materials, the development of oral vaccine delivery systems using food materials should be more robustly researched to expand vaccine coverage of gi infections in developing countries. for intranasal vaccination, the vaccine delivery system should survive the very active mucociliary clearance mechanisms and provide safety, given the anatomical location of the nasal cavity, which is separated from the central nervous system by a thin barrier. future mucosal vaccine carriers, regardless of administration routes, should share common characteristics. they should maintain stability in given environments, be mucoadhesive, and have targeting ability to specific tissues and cells. progress in nanomedicine: approved and investigational nanodrugs nanoparticle-based medicines: a review of fda-approved materials and clinical trials to date nanotechnology in vaccine delivery vaccine adjuvants: from to and beyond an updated review of iscomstm and iscomatrixtm vaccines development of a nanogel-based nasal vaccine as a novel antigen delivery system recent advances in self-assembled peptides: implications for targeted drug delivery and vaccine engineering vaccine delivery using nanoparticles poly(lactic acid)-based particulate systems are promising tools for immune modulation squalene emulsions for parenteral vaccine and drug delivery an update on safety and immunogenicity of vaccines containing emulsion-based adjuvants safety of mf adjuvant development and evaluation of as , an adjuvant system containing alpha-tocopherol and squalene in an oil-in-water emulsion in vitro evaluation of tlr agonist activity: formulation effects design and evaluation of a safe and potent adjuvant for human vaccines iscoms and other saponin based adjuvants liposomal vaccine formulations as prophylactic agents: design considerations for modern vaccines colloidal nanocarriers: a review on formulation technology, types and applications toward targeted drug delivery biodegradable nanoparticles for drug and gene delivery to cells and tissue peptide/protein vaccine delivery system based on plga particles a practical approach to the use of nanoparticles for vaccine delivery the long-term potential of biodegradable poly(lactide-co-glycolide) microparticles as the next-generation vaccine adjuvant revisiting pla/plga microspheres: an analysis of their potential in parenteral vaccination protein delivery from poly(lactic-coglycolic acid) biodegradable microspheres: release kinetics and stability issues nanoparticles target distinct dendritic cell populations according to their size vaccine delivery: a matter of size, geometry, kinetics and molecular patterns poly-lactic acid synthesis for application in biomedical devices À a review pla micro-and nano-particles sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers plga-based nanoparticles: an overview of biomedical applications nano/micro technologies for delivering macromolecular therapeutics using poly(d,l-lactide-co-glycolide) and its derivatives how to achieve sustained and complete protein release from plga-based microparticles plga micro and nanoparticles in delivery of peptides and proteins; problems and approaches improving stability and release kinetics of microencapsulated tetanus toxoid by co-encapsulation of additives optimization of encapsulation of a synthetic long peptide in plga nanoparticles: low-burst release is crucial for efficient cd ( ) t cell activation natural polymers for gene delivery and tissue engineering micro and nanoparticle-based delivery systems for vaccine immunotherapy: an immunological and materials perspective chitosan-based particulate systems for the delivery of mucosal vaccines against infectious diseases n-trimethylated chitosan chloride (tmc) improves the intestinal permeation of the peptide drug buserelin in vitro (caco- cells) and in vivo (rats) chitosan microparticles and nanoparticles as biocompatible delivery vehicles for peptide and protein-based immunocontraceptive vaccines tmc-mcc (n-trimethyl chitosan-mono-ncarboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines protein release from alginate matrices modular injectable matrices based on alginate solution/microsphere mixtures that gel in situ and co-deliver immunomodulatory factors effect of alginate on innate immune activation of macrophages dextran sulphate: an adjuvant for cell-mediated immune responses surface-engineered polyelectrolyte multilayer capsules: synthetic vaccines mimicking microbial structure and function biomedical applications of selfassembling peptides a selfassembling peptide acting as an immune adjuvant modulating adaptive immune responses to peptide self-assemblies the use of self-adjuvanting nanofiber vaccines to elicit high-affinity b cell responses to peptide antigens without inflammation nanogel as a novel platform for smart drug delivery system nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines hydrogel nanoparticles and nanocomposites for nasal drug/vaccine delivery research progress of self-assembled nanogel and hybrid hydrogel systems based on pullulan derivatives nanogel engineering for new nanobiomaterials: from chaperoning engineering to biomedical applications safety studies of a novel starch, pullulan: chronic toxicity in rats and bacterial mutagenicity synthesis and characterization of self-assembled nanogels made of pullulan self-aggregates of hydrophobized polysaccharides in water. formation and characteristics of nanoparticles nanogel dds enables sustained release of il- for tumor immunotherapy nanogel-based antigen-delivery system for nasal vaccines comparison of refolding activities between nanogel artificial chaperone and groel systems protein refolding assisted by selfassembled nanogels as novel artificial molecular chaperone self-assembled cationic nanogels for intracellular protein delivery nanogel-based pspa intranasal vaccine prevents invasive disease and nasal colonization by streptococcus pneumoniae recent progress in mucosal vaccine development: potential and limitations considerations for oral cholera vaccine use during outbreak after earthquake in haiti mucosal vaccines for biodefense oral delivery of nanoparticle-based vaccines achieving and maintaining polio eradication À new strategies epidemiology of poliomyelitis in the united states one decade after the last reported case of indigenous wild virus-associated disease immunogenicity and efficacy of oral vaccines in developing countries: lessons from a live cholera vaccine long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass paxvax cvd -hgr single-dose live oral cholera vaccine a randomized, placebo-controlled, double-blind phase trial comparing the reactogenicity and immunogenicity of a single $ x colony forming units [cfu] standard-dose versus a $ x cfu high-dose of cvd -hgr live attenuated oral cholera vaccine, with shanchol inactivated oral vaccine as an open label immunologic comparator vaxchora: a single-dose oral cholera vaccine the antitumor immune responses induced by nanoemulsion-encapsulated mage -hsp /sea complex protein vaccine following different administration routes the antitumor immune responses induced by nanoemulsion-encapsulated mage -hsp /sea complex protein vaccine following peroral administration route block copolymer micelles for drug delivery: design, characterization and biological significance immunogenicity of multiple-epitope antigen gene of hcv carried by novel biodegradable polymers delivery strategies to enhance oral vaccination against enteric infections lipid-based nanocarriers as an alternative for oral delivery of poorly water-soluble drugs: peroral and mucosal routes antigen delivery to mucosaassociated lymphoid tissues using liposomes as a carrier exploring the fate of liposomes in the intestine by dynamic in vitro lipolysis bilosomes: a novel approach to meet the challenges in oral immunization archaeal lipids in oral delivery of therapeutic peptides positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins cationic liposomes as vaccine adjuvants cationic nanocarriers induce cell necrosis through impairment of na( )/k( )-atpase and cause subsequent inflammatory response recent advances in liposome surface modification for oral drug delivery polysaccharide coated liposomes for oral immunization À development and characterization lectin-bearing polymerized liposomes as potential oral vaccine carriers a human norovirus-like particle vaccine adjuvanted with iscom or mlt induces cytokine and antibody responses and protection to the homologous gii. human norovirus in a gnotobiotic pig disease model induction of protective and mucosal immunity against diphtheria by a immune stimulating complex (iscoms) based vaccine a novel non-toxic combined cta -dd and iscoms adjuvant vector for effective mucosal immunization against influenza virus immunological characterization of two hepatitis b core antigen variants and their immunoenhancing effect on co-delivered hepatitis b surface antigen high titers of circulating maternal antibodies suppress effector and memory b-cell responses induced by an attenuated rotavirus priming and rotavirus-like particle-immunostimulating complex boosting vaccine regimen an oral versus intranasal prime/boost regimen using attenuated human rotavirus or vp and vp virus-like particles with immunostimulating complexes influences protection and antibody-secreting cell responses to rotavirus in a neonatal gnotobiotic pig model chimeric hepatitis e virus-like particle as a carrier for oral-delivery new approaches in oral rotavirus vaccines quantitative analysis of the yield of avian h influenza virus haemagglutinin protein produced in silkworm pupae with the use of the codon-optimized dna: a possible oral vaccine oral and intraperitoneal immunization with rotavirus / virus-like particles stimulates a systemic and mucosal immune response in mice humoral, mucosal, and cellular immune responses to oral norwalk virus-like particles in volunteers bovine papillomavirus-like particles presenting conserved epitopes from membrane-proximal external region of hiv- gp induced mucosal and systemic antibodies freeze-drying of plant tissue containing hbv surface antigen for the oral vaccine against hepatitis b induction of mucosal and systemic immune responses against human carcinoembryonic antigen by an oral vaccine virus-like particles produced in plants as potential vaccines plant-derived virus-like particles as vaccines an efficient plant viral expression system generating orally immunogenic norwalk virus-like particles oral immunogenicity of human papillomavirus-like particles expressed in potato tomato is a highly effective vehicle for expression and oral immunization with norwalk virus capsid protein virus-like particle expression and assembly in plants: hepatitis b and norwalk viruses nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach synthesis of a novel kind of carbon nanoparticle with large mesopores and macropores and its application as an oral vaccine adjuvant pegylated plga-based nanoparticles targeting m cells for oral vaccination traffic of poly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to subepithelial immune competent cells microparticle vaccine approaches to stimulate mucosal immunisation stable cationic microparticles for enhanced model antigen delivery to dendritic cells poly(ethylene glycol) as stabilizer and emulsifying agent: a novel stabilization approach preventing aggregation and inactivation of proteins upon encapsulation in bioerodible polyester microspheres the preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated plga microparticles biodegradable nanoparticles for oral delivery of peptides: is there a role for polymers to affect mucosal uptake the manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (plga) devices large intestine-targeted, nanoparticle-releasing oral vaccine to control genitorectal viral infection encapsulation of low molecular weight heparin (bemiparin) into polymeric nanoparticles obtained from cationic block copolymers: properties and cell activity controlled and targeted release of antigens by intelligent shell for improving applicability of oral vaccines oral helicobacter pylori vaccine-encapsulated acidresistant hp /plga nanoparticles promote immune protection lectin anchored plga nanoparticles for oral mucosal immunization against hepatitis b targeted delivery of gp antigen of prrsv to m cells enhances the antigen-specific systemic and mucosal immune responses targeting nanoparticles to m cells with non-peptidic ligands for oral vaccination targeted oral delivery of bmpb vaccine using porous plga microparticles coated with m cell homing peptide-coupled chitosan targeted delivery of chitosan nanoparticles to peyer's patch using m cell-homing peptide selected by phage display technique attuning hydroxypropyl methylcellulose phthalate to oral delivery vehicle for effective and selective delivery of protein vaccine in ileum combinatorial approach of antigen delivery using m cell-homing peptide and mucoadhesive vehicle to enhance the efficacy of oral vaccine can bioadhesive nanoparticles allow for more effective particle uptake from the small intestine? immunoadjuvant capacity of flagellin and mannosamine-coated poly (anhydride) nanoparticles in oral vaccination intraperitoneal immunization with urease loaded n-trimethyl chitosan nanoparticles elicits high protection against brucella melitensis and brucella abortus infections nanoconjugation of bicistronic dna vaccine against edwardsiella tarda using chitosan nanoparticles: evaluation of its protective efficacy and immune modulatory effects in labeo rohita vaccinated by different delivery routes inactivated enterovirus with poly-gamma-glutamic acid/ chitosan nano particles (pc nps) induces high cellular and humoral immune responses in balb/c mice preparation and characterization of microencapsulated dwpt trivalent vaccine using water soluble chitosan and its in-vitro and in-vivo immunological properties eudragit(r) l -coated mannosylated chitosan nanoparticles for oral protein vaccine delivery functionalized and graft copolymers of chitosan and its pharmaceutical applications design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review oral delivery of peptide drugs using nanoparticles self-assembled by poly(gamma-glutamic acid) and a chitosan derivative functionalized by trimethylation development of orallydeliverable dna hydrogel by microemulsification and chitosan coating chitosan/alginate microparticles for the oral delivery of fowl typhoid vaccine: innate and acquired immunity a new strategy based on smrho protein loaded chitosan nanoparticles as a candidate oral vaccine against schistosomiasis alginate coated chitosan microparticles mediated oral delivery of diphtheria toxoid. part a. systematic optimization, development and characterization immunization with single oral dose of alginateencapsulated bcg elicits effective and long-lasting mucosal immune responses an approach to a cold chain free oral cholera vaccine: in vitro and in vivo characterization of vibrio cholerae gastro-resistant microparticles oral vaccination with microencapsuled strain vaccine confers enhanced protection against brucella abortus strain challenge in red deer (cervus elaphus elaphus) the immune response and protective efficacy of vaccination with oral microparticle aeromonas sobria vaccine in mice identification of an edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain assessment of different formulations of oral mycobacterium bovis bacille calmette-guerin (bcg) vaccine in rodent models for immunogenicity and protection against aerosol challenge with m. bovis recent advances in oral vaccine development: yeast-derived beta-glucan particles characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for dna delivery beta-glucan microparticles are good candidates for mucosal antigen delivery in oral vaccination distinct patterns of dendritic cell cytokine release stimulated by fungal beta-glucans and toll-like receptor agonists immune recognition. a new receptor for beta-glucans oral hepatitis b vaccine: chitosan or glucan based delivery systems for efficient hbsag immunization following subcutaneous priming novel transgenic rice-based vaccines recent development and future prospects of plantbased vaccines plant-made vaccines and reagents for the one health initiative rice-based mucosal vaccine as a global strategy for cold-chain-and needle-free vaccination secretory iga-mediated protection against v. cholerae and heat-labile enterotoxin-producing enterotoxigenic escherichia coli by rice-based vaccine oral immunisation against typhoid fever in indonesia with ty a vaccine oral administration of an adenovirus vector encoding both an avian influenza a hemagglutinin and a tlr ligand induces antigen specific granzyme b and ifn-gamma t cell responses in humans intranasal vaccination against angiotensin ii type receptor and pneumococcal surface protein a attenuates hypertension and pneumococcal infection in rodents intranasal formulations: promising strategy to deliver vaccines nanogel-based nasal vaccines for infectious and lifestyle-related diseases nanogel-based nasal ghrelin vaccine prevents obesity intranasal drug delivery: how, why and what for? anatomy, physiology and function of the nasal cavities in health and disease nasal drug delivery: new developments and strategies nanoparticulate systems for nasal delivery of drugs: a real improvement over simple systems? design of a liposomal candidate vaccine against pseudomonas aeruginosa and its evaluation in triggering systemic and lung mucosal immunity nalt m cells are important for immune induction for the common mucosal immune system the eurocine l adjuvants with subunit influenza antigens induce protective immunity in mice after intranasal vaccination intranasal administration of a flagellinadjuvanted inactivated influenza vaccine enhances mucosal immune responses to protect mice against lethal infection synthetic double-stranded rna poly (i:c) combined with mucosal vaccine protects against influenza virus infection intranasal administration of whole inactivated influenza virus vaccine as a promising influenza vaccine candidate characterization of neutralizing antibodies in adults after intranasal vaccination with an inactivated influenza vaccine intranasal vaccination with an inactivated whole influenza virus vaccine induces strong antibody responses in serum and nasal mucus of healthy adults modes of action for mucosal vaccine adjuvants mucosal vaccine adjuvants update use of the inactivated intranasal influenza vaccine and the risk of bell's palsy in switzerland cholera toxin, lt-i, lt-iia and lt-iib: the critical role of ganglioside binding in immunomodulation by type i and type ii heat-labile enterotoxins nasal administration of cholera toxin as a mucosal adjuvant damages the olfactory system in mice a cpg-adjuvanted intranasal enterovirus vaccine elicits mucosal and systemic immune responses and protects human scarb -transgenic mice against lethal challenge intranasal immunization with recombinant pspa fused with a flagellin enhances cross-protective immunity against streptococcus pneumoniae infection in mice a molecular mucosal adjuvant to enhance immunity against pneumococcal infection in the elderly sustained protective immunity against bordetella pertussis nasal colonization by intranasal immunization with a vaccine-adjuvant combination that induces il- -secreting trm cells nasal mucoadhesive drug delivery: background, applications, trends and future perspectives bioadhesive delivery systems for mucosal vaccine delivery mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues selective permeability of mucus barriers barrier properties of mucus nanoparticles that do not adhere to mucus provide uniform and long-lasting drug delivery to airways following inhalation mucoadhesive nanoparticles may disrupt the protective human mucus barrier by altering its microstructure opportunities and challenges for the nasal administration of nanoemulsions formulation and characterization of nanoemulsion intranasal adjuvants: effects of surfactant composition on mucoadhesion and immunogenicity nasal immunization with a recombinant hiv gp and nanoemulsion adjuvant produces th polarized responses and neutralizing antibodies to primary hiv type isolates liposomes as delivery systems for nasal vaccination: strategies and outcomes interaction of dendritic cells with antigencontaining liposomes: effect of bilayer composition the surface charge of liposomal adjuvants is decisive for their interactions with the calu- and a airway epithelial cell culture models liposome-mediated delivery stimulates a class i-restricted cytotoxic t cell response to soluble antigen intranasal immunization of a dna vaccine with il- -and granulocyte-macrophage colonystimulating factor (gm-csf)-expressing plasmids in liposomes induces strong mucosal and cell-mediated immune responses against hiv- antigens galactosylated liposome as a dendritic cell-targeted mucosal vaccine for inducing protective anti-tumor immunity intranasal immunization with liposome-encapsulated plasmid dna encoding influenza virus hemagglutinin elicits mucosal, cellular and humoral immune responses a mucosal vaccine against diphtheria: formulation of cross reacting material (crm( )) of diphtheria toxin with chitosan enhances local and systemic antibody and th responses following nasal delivery relationship between structure and adjuvanticity of n,n,n-trimethyl chitosan (tmc) structural variants in a nasal influenza vaccine stimulation of mucosal and systemic antibody responses against bordetella pertussis filamentous haemagglutinin and recombinant pertussis toxin after nasal administration with chitosan in mice intranasal immunization with genetically detoxified diphtheria toxin induces t cell responses in humans: enhancement of th responses and toxin-neutralizing antibodies by formulation with chitosan role of trimethylated chitosan (tmc) in nasal residence time, local distribution and toxicity of an intranasal influenza vaccine chitosan-based delivery systems for mucosal vaccines chitosan modification and pharmaceutical/biomedical applications effect of chitosan on epithelial permeability and structure co-delivery of viral proteins and a tlr agonist from polysaccharide nanocapsules: a needle-free vaccination strategy nasal vaccination with n-trimethyl chitosan and plga based nanoparticles: nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen nasal nanovaccines the vaccine adjuvant chitosan promotes cellular immunity via dna sensor cgas-sting-dependent induction of type i interferons development and characterization of chitosan coated poly-(varepsilon-caprolactone) nanoparticulate system for effective immunization against influenza pluronic f enhances the effect as an adjuvant of chitosan microspheres in the intranasal delivery of bordetella bronchiseptica antigens containing dermonecrotoxin covalently stabilized trimethyl chitosan-hyaluronic acid nanoparticles for nasal and intradermal vaccination the potential of mannosylated chitosan microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization norovirus vaccine against experimental human norwalk virus illness induction of protective serum meningococcal bactericidal and diphtherianeutralizing antibodies and mucosal immunoglobulin a in volunteers by nasal insufflations of the neisseria meningitidis serogroup c polysaccharide-crm conjugate vaccine mixed with chitosan spray-dried powders of starch and crosslinked poly(acrylic acid) as carriers for nasal delivery of inactivated influenza vaccine synthetic biomimetic supra molecular biovector (smbv) particles for nasal vaccine delivery residence time and uptake of porous and cationic maltodextrin-based nanoparticles in the nasal mucosa: comparison with anionic and cationic nanoparticles strong systemic and mucosal immune responses to surface-modified plga microspheres containing recombinant hepatitis b antigen administered intranasally intranasal and oral vaccination with protein-based antigens: advantages, challenges and formulation strategies cholesteryl pullulan encapsulated tnf-alpha nanoparticles are an effective mucosal vaccine adjuvant against influenza virus cationic pullulan nanogel as a safe and effective nasal vaccine delivery system for respiratory infectious diseases mucosal or systemic administration of re glycoprotein antigen loaded plga microspheres nasal and pulmonary vaccine delivery using particulate carriers transient facial nerve paralysis (bell's palsy) following intranasal delivery of a genetically detoxified mutant of escherichia coli heat labile toxin intranasal delivery of nanoparticles encapsulating bpi v proteins induces an early humoral immune response in mice induction, function and regulation of il- -producing t cells induction of mucosal immune responses and protection of cattle against direct-contact challenge by intranasal delivery with foot-and-mouth disease virus antigen mediated by nanoparticles synergistic action of cholera toxin b subunit (and escherichia coli heat-labile toxin b subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues key: cord- -ipv npdy authors: torreele, els title: business-as-usual will not deliver the covid- vaccines we need date: - - journal: development (rome) doi: . /s - - - sha: doc_id: cord_uid: ipv npdy governments must become active shapers of medical innovation and drive the development of critical health technologies as global health commons. the ‘race’ for covid- vaccines is exposing the deficiencies of a business-as-usual medical innovation ecosystem driven by corporate interests, not health outcomes. instead of bolstering collective intelligence, it relies on competition between proprietary vaccines and allows the bar on safety and efficacy to be lowered, risking people’s health and undermining their trust. in the early weeks of , the world was alerted to the emergence of a new deadly infectious respiratory disease, covid- , caused by severe acute respiratory syndrome coronavirus (sars-cov- ). first reported in china, it rapidly spread across the globe. without effective treatments or vaccines available, the response to the pandemic has so far largely relied on infection control measures such as hand hygiene, personal protective equipment including masks, physical distancing and restrictions on movement which have included variable periods of lockdown of cities, countries or regions. by late september, over one million people had died from covid- , among over million people diagnosed. given the enormous health, social and economic impact of the continued spread of covid- , which is nowhere near being under control, it is no surprise that a lot of hope is set on finding a vaccine. with massive financial support from governments, in particular the us, the uk, other european countries, as well as china and russia, researchers and companies engaged in what soon became a frantic race to develop vaccines against covid- . so far, an unprecedented amount of public funds (estimated at over bn us$) has been poured into vaccine research and development (r&d) and manufacturing, resulting in over vaccine candidates in clinical trials and many more in the pipeline. touted by many as a major tour de force, the ongoing 'race' towards a vaccine is also exposing the intrinsic deficiencies of relying on for-profit pharmaceutical companies, that are governed by trade rules, financial speculation and market competition, to ensure the development of essential health technologies. in fact, it risks delivering vaccines that are neither adequate nor widely accessible and may stand in the way of a truly effective global response to the pandemic. vaccinating against infectious diseases has proven to be a highly cost-effective public health intervention, combining individual and population-wide health benefits, if certain conditions are met. these are in the first place that the vaccine(s) must be safe and effective, and widely available to vaccinate the populations that can most benefit from it. depending on the disease, how it is transmitted and how contagious it is, an effective vaccine would ideally protect against infection, or else, it may only protect from getting (seriously) ill, or dying, or from further spreading the disease. an additional condition is that vaccines be deployed through an appropriate vaccination strategy. who and when to vaccinate, including priority allocation if vaccine availability is limited, are informed both by the epidemiology and the vaccine's properties, and may need to be adapted over time as the pandemic evolves and more vaccines become available. vaccine development and optimal use as public health intervention therefore depends on continuous data-driven assessment of benefits and risks in the context of the evolving pandemic, with the view of maximizing the public health impact of vaccination strategies. this is particularly important during a public health emergency like covid- , where vaccine development, regulatory review of candidates and their deployment will occur under intense clinical, economic, and political pressure (avorn and kesselheim ) . the way in which the commercial and geopolitical 'race' for a vaccine is playing out, however, risks side-lining these critical public health objectives in an r&d process that hinges on the privatization and commercialization of knowledge, and is focused on being first to get a vaccine to market (torreele a ). there currently is no mechanism to ensure that the best possible vaccines are being developed and deployed. the classic approach to vaccine development is that private companies invest in r&d based on their own proprietary platform technologies (vectors, delivery systems, adjuvants), in which they integrate a specific antigen to adapt to the target disease. despite a pipeline of nearly vaccine r&d projects, the 'race' to get a vaccine to market fastest does not incentivize the best science for public health interest. instead, it favours fragmentation and secretive competition, and precludes the free exchange of knowledge and learning from each other's successes and failures in real time, or a public health-driven and collaborative portfolio approach. none of the individual elements of each proprietary platform is necessarily the best suited for a covid- vaccine, but each company will only research within the boundaries of the its proprietary technology (covered by patents), hands tied from using other and possibly better fit elements that are owned by competitors. this is antithetical to a collective intelligence effort that would allow scientists all over the world to creatively combine the best elements of our medical knowledge and technological advances into a diverse and innovative portfolio of vaccine candidates with the best chance to achieve our common public health goal (torreele b) . failing moreover to compare the performance of the different candidates directly, the current process is bound to create a portfolio of suboptimal candidates that are neither the best in their class, nor diverse or complementary. in our supply-driven innovation system (in which the market is considered the ultimate arbiter), there is no public health mechanism to demand or impose that companies develop products according to pre-set and public health-driven performance criteria. as a result, less than optimal candidates can move through the pipeline, if sufficient resources are poured into it. in april , the world health organization (who) published a target product profile (tpp) for covid- vaccines with minimal and ideal vaccine characteristics to guide developers. it lays out safety and efficacy targets, to be demonstrated in people of all age groups, ethnicities and including subpopulations with certain co-morbidities. the who target product profile also outlines preferred features to make the vaccine well-suited for a large-scale interventions, for instance good temperature stability and a single dose regimen, or scalable and low-cost manufacturing. unfortunately, who's target product profiles are only aspirational and vaccine developers are under no obligation to comply with such criteria and ensure the products they are developing will be adequate as a public health intervention. and regulatory authorities are not empowered to demand that either. while there exists no absolute threshold for vaccine efficacy, who's tpp proposes that vaccines should have a minimum efficacy of % to be a useful tool against covid- . to effectively curb the pandemic and reach adequate population immunity, it has been calculated that vaccines should be - % effective in preventing infections (bartsch et al. ) . it remains uncertain how useful vaccines with much lower efficacy can be, or vaccines that only reduce disease severity, or only work in certain subpopulations (avorn and kesselheim ) . in any case, the tpp and the different ways in which vaccines can work must be considered when designing clinical trials for covid- vaccine candidates. in the current r&d model, it is left at the discretion of companies to set the vaccine efficacy targets they will measure in the clinical trials, which moreover they design, conduct and analyze themselves, notwithstanding the conflict of interest given their vested interest in the outcome (quigley ) . moreover, the study protocols that detail what is being compared in such trials, and how meaningful differences are going to be measured, are generally kept confidential for the public, as are the full study data and analyses. a milestone resolution on transparency around medical r&d was passed at the world health assembly (fletcher ), yet governments so far have failed to implement these commitments, despite huge financial investments in covid- r&d that could have been used as leverage to demand transparency on scientific methods and data, as well as clinical trial costs, and set performance targets for the vaccines. as companies' primary goal is to obtain marketing approval from regulators, they will design trials in ways that gives the fastest and easiest way to success, which does not necessarily coincide with asking the clinically most relevant questions. when front running covid- vaccine developers moderna, pfizer, and astrazeneca ceded to public pressure and shared their phase iii trial protocols, we learnt that they are indeed designed to get quick answers, not to demonstrate the vaccines are truly effective (doshi and topol ) . and while the three protocols differ in the details of how they measure efficacy (which conveniently will make the results impossible to compare) they all look at reducing the number of mild covid- cases as primary endpoint. the more clinically relevant endpoint of reducing severe disease and death would take longer to achieve (given that only a minority of those infected develop severe disease), as would demonstrating that the vaccine protects against infection altogether-which is the most desirable form of efficacy. who has proposed a collaborative efficacy trial, called 'solidarity', that would allow to directly compare the performance of different vaccines in light of the tpp. however, commercial vaccine developers prefer to set up their own placebo-controlled trials rather than have their vaccines compared to other candidates by independent researchers and held up to stringent public health targets. and while contrary to good scientific practice, governments too have chosen speed and political expediency over quality, generously supporting company trials as part of their geopolitical vaccine race through funding and access to public clinical trial infrastructure and capabilities. meanwhile, leading scientists advising who are left to plead in scientific journals for trials to look for 'worthwhile efficacy' (krause et al. ) , lacking ways to impose public health imperatives to commercial developers. only radical transparency of clinical trial protocols and data, and a robust and independent review of the results will allow to ensure we fully understand the performance, and limitations, of each vaccine, and restore trust that study conclusions are valid (torreele c) . amidst growing antiscience and anti-vax tendencies, it is critical that commercial and other vested interests are being removed from the assessment of covid- vaccines, allowing public health scientists, vaccination experts and other relevant stakeholders full access to the data and analyses to redress the already shaken public confidence in public health interventions to control covid- (jha ) . a further risk of focusing on speed is that researchers may not take the time to work with communities to educate them about covid- , its risk, the promise of vaccines and the mechanics of developing them, including doing trials, and get communities on board for trials and use of the vaccine. in addition to the risks of suboptimal research ethics practices, this may also create barriers and delays for rollout afterwards, because of distrust, vaccine refusal, science misconceptions, etc. the vaccine r&d playing field is not designed to enable the best covid- vaccines to move forward based on scientific and public health merit. instead, it is shaped by wealthy countries and powerful actors like pharmaceutical corporations who place their bets (knaus )-allotting large amounts of money to propel a chosen candidate forward towards (possible emergency use) authorization. financial and industrial backing such as through operation warp speed, more than desirable product characteristics, will determine the likely winners of this race, for which the primary finish line is obtaining marketing approval-typically at the united states food and drug administration (fda) and/or the european medicines agency (ema). yet the criteria used by regulators to allow a vaccine on the market are not necessarily responding to the critical question at hand: which vaccine has the potential to significantly impact global public health outcomes for covid- ? the primary role of regulatory authorities is to safeguard the public against exposure to potential harmful products. regulators will assess the potential benefits and risks of individual vaccines based on the company's data and determine whether the presented benefit/risk balance justifies commercialization of that vaccine. it is not the role of regulators to determine if a vaccine is adequate to control the pandemic, nor prioritizing which one has better safety and efficacy. notably, in their decisions to give marketing authorization, regulators are not taking into consideration a company's intention to produce a vaccine at scale or make it available widely, nor what price they are planning to charge. this means that companies have no incentive nor obligation to prioritize anything else but speed in obtaining marketing approval, based on self-chosen measures of efficacy. in addition, as the race for a vaccine became fuelled by political, financial and populist pressures, regulatory authorities may not even be able to protect us from harm or futility. concerns among scientists and experts are growing over the political pressure the us administration is putting on the fda. especially the announcement that the fda would consider emergency authorization for covid- vaccines before phase trials are complete, has caused concern. if that happens, european countries will be hard pressed to follow suit. yet, as vaccine and public health experts keep emphasizing, robust phase efficacy trials are the only way to establish whether a vaccine is effective in protecting against infections and disease, and safe to administer to large groups. having become increasingly financialized (lazonick et al. ) , pharmaceutical corporations will not invest in r&d for products that do not constitute guaranteed and profitable business opportunities. despite the excellent public health value of vaccines, producing and selling vaccines is considered unattractive from a commercial perspective. mass manufacturing and distribution of low-cost products, with only marginal profits per unit, compares poorly to the growing medicines market with its uniquely profitable lowvolume specialty drugs for which companies can charge very high prices. at the same time, the r&d process for vaccines is typically lengthy and costly, with large clinical trials required to demonstrate that it will be safe to administer a product to many millions essentially healthy people, and also effective in protecting against a given disease. only a handful of companies dominate the global vaccine market, selling essentially variations of the same - existing vaccines, with relatively little innovation in new disease areas, despite many infectious diseases that could benefit from a vaccine. emerging infectious diseases are a case in point. as exemplified again with the - west african ebola outbreak that killed over , people, commercial r&d fails to deliver needed health technologies for outbreaks of infectious diseases. despite knowing the ebola virus and its lethal epidemic potential since the s, and vaccine research having advanced in the public sector, there was neither treatment nor vaccine available when a long expected major outbreak devastated guinea, liberia and sierra leone, and caused a global panic (torreele and olliaro ) . and while a consortium of mainly public partners came together to conduct a clinical trial in that demonstrated the safety and efficacy of a vaccine candidate, initially developed by canadian public health researchers and later licensed to merck, there was still no registered vaccine available when a new ebola outbreak hit drc in - . affirming the need for public responsibility to drive r&d to improve outbreak preparedness, a group of countries and donors came together in the wake of the west african ebola crisis to create the coalition for epidemic preparedness innovations (cepi), with the mandate to accelerate development of vaccines against outbreak diseases and ensure access. with a massive injection of public and philanthropic funds, in cepi started financing public-private partnerships aiming to bring candidate vaccines for mers-cov (also a coronavirus), lassa fever and nipah forward. in parallel, cepi also invested in generic vaccine technology platforms that could be quickly adapted to any emerging infection of a so far unknown pathogen, the socalled 'disease-x' (simpson et al. ) . as soon as sars-cov- emerged, cepi mobilized funds to apply existing vaccine technologies to covid- , piggybacking on earlier investments in disease-x and mers-cov programmes, thus quickly moving several candidates into clinical trials. taking advantage of the massive public subsidies that started flowing towards covid- r&d, both small biotechs and major pharmaceutical companies jumped on the opportunity to adapt or reorient their proprietary vaccine technology platforms towards covid- , allowing to fast-track what otherwise would require many years of research. for instance moderna's and biontech/pfizer's leading mrna vaccine candidates built on years of public and private research into the potential of rna and dna vaccines (yet none of them made into a vaccine approved for human use) (akpan ) . similarly, oxford university (who later partnered with astrazeneca), j&j, cansino and gamaleya have rapidly repurposed for covid- their adenovirus-based vaccine platforms which had been explored for many years and a variety of diseases, including most recently mers-cov, zika and ebola. despite each of these r&d efforts building on a wealth of earlier research by the global vaccine research communitymuch of which is traditionally done and funded by the public sector-the basis of our commercial r&d model is that universities and companies are allowed to appropriate such technology platforms as their own. governed by the world trade organization's trips agreement that obliged countries to grant and enforce patents on pharmaceutical 'inventions', medical knowledge and technologies have largely been privatized, owned and traded as commodities, even when of critical public health importance. as a result, vaccine candidates essentially move through the pipeline as speculative commercial assets, whose market valuation can be followed through the share price of the companies owning them. while governments generously subsidize the covid- vaccines candidates of a handful of companies, it is unclear whether the financing agreements are structured to recognize this co-investment and ensure there will be commensurate sharing of the resulting outcomes in terms of access and pricing (the agreements have remained confidential). realizing that providing global access to an eventual covid- vaccine would require manufacturing at unprecedented scale, governments also provided upfront investments in the companies' manufacturing capacity and infrastructure. not only have they agreed to massively finance the expansion of private vaccine manufacturing infrastructure, with seemingly little or no strings attached, they also agreed to pay for the actual manufacturing of large volumes of selected vaccines before their safety and efficacy is proven and committed to buy large volumes once they were approved (at undisclosed prices) through so-called advance purchase commitments (apcs) . on top of this, companies have negotiated confidential liability transfers to governments, in case the vaccines would exhibit side effects that were not observed in the accelerated r&d process (halabi et al. ) . in contrast to generic drug manufacturing, there is relatively little vaccine manufacturing capacity able to produce at large scale outside of the major (western) vaccine corporations, with the notable exception of the serum institute of india, that has taken many years to build its meanwhile state-of-the-art capability. while strictly speaking there is no such thing as generic vaccines, vaccine manufacturing is technologically much more complex than small chemicals, and setting up the production of an existing vaccine typically requires lengthy technology transfer, including know-how sharing, for a newcomer to become operational. the massive investments of governments into scaling up manufacturing capacity for the covid- candidates seem to have all gone into private companies under license from the 'originator' companies, which is a missed opportunity for the global health community to have invested in expanding the global technological capacity to produce vaccines as commons, and start challenging the oligopoly that now exists among major vaccine producers. taken together, the unprecedented public investments in r&d and manufacturing capacities for covid- vaccines, and the purchase commitments and liability transfers, all directly benefit companies that 'own' these technologies, and have come with little or no strings attached, de facto privatizing all those public investments and the control over potentially hugely important public health interventions, which essentially should have been global health commons. vaccines are a public health intervention which, more than any other, require people's collective buy-in and trust. while some people argue that having a covid- vaccine that works a little, and for some, is better than having no vaccine at all, there are major risks and opportunity costs for prioritizing speed over adequate efficacy and safety. roll-out of a weakly effective vaccine in fact could worsen the pandemic in multiple ways (krause et al. ) . if authorities wrongly assume there is a substantial reduction in risk in the population, they may choose to scale down other covid- control measures and re-open the economy. if the pandemic continues to grow despite having massively invested in vaccines, they may not be willing to continue investing in a potentially better next generation vaccines. similarly, if vaccinated individuals wrongly believe they are protected against infection, compliance with other protective measures such as wearing masks or physical distancing may be lowered. when people realize they get infected despite being vaccinated, their confidence in vaccines may drop, leading them to refuse other more effective vaccines that would become available later on. deployment of a marginally effective vaccine could also interfere with the evaluation of other vaccines. it may become challenging to find enough unvaccinated trial volunteers to enrol in new vaccine trials, while conducting a trial in a population that is partially protected due to prior (poorly effective) vaccination may lead to results that are very difficult to interpret. additionally, once a covid- vaccine gets approved, subsequent vaccine candidates will have to be compared to it rather than to a placebo. as this necessarily will be done via what is called 'non-inferiority' trials, there's a risk that vaccines with even worse performance might still get approved, a methodological quirk which has been referred to as 'bio-creep' (everson-stewart and emerson ). a final collateral effect of poorly effective vaccines is that the legitimacy of scientists and the scientific process might be undermined if researchers cannot prevent commercial interests to overtake quality science. from the early days of the pandemic, influential voices including un secretary-general antónio guterres, european president ursula von der leyen and political leaders and academics from all continents have argued that the exceptional nature and huge impact of the pandemic justifies that covid- vaccines must be considered 'people's vaccines', or 'global public goods', or 'global health commons' and be available and affordable (for free) to all. however, this voluntarist discourse has not been followed with consequent actions. instead, we've seen unapologetic vaccine nationalism (kamradt-scott ) from the us, soon followed by other wealthy countries including the european commission, signing bilateral deals with companies to reserve the majority of early productions for their own populations in what essentially is an unequal scramble for vaccines (callaway ) . a multilateral effort coordinated by who in the context of their accelerated access to covid technologies (act-a) initiative to also ensure vaccine availability for poorer countries in parallel (covax) has met with mixed success-lots of verbal support but so far limited concrete financial commitments. it is also being criticized for lack of transparency and representation from the countries for whom covax is supposed to deliver solutions. the main strategy used by governments to 'ensure' access is through signing advance purchase commitments with the front-runner vaccine developers (often in combination with significant investments in r&d and even manufacturing). while initially designed as a market fixing pull mechanism to incentivize companies to do r&d in directions they would not otherwise go, governments have perverted apcs to guarantee supply and buy up the first in line positions for once the vaccines become available, without even putting demands in terms of desired product profile or pricing. this further shifts the power imbalance between governments and vaccines companies, who successfully turned the covid- crisis to their advantage and positioned themselves as key to the solutions, while largely dictating the terms of engagement, not only for availability and access to vaccines in wealth countries but also globally. at the same time, acknowledging that there will likely be a period during which supply will not be able to meet demand, difficult discussions are ongoing about (principles for) fair and equitable allocation frameworks, both globally and within countries (samuel ) . while it is beyond the scope of this article to go into detail on these, it is important to highlight how allocation frameworks and vaccination strategies cannot be seen independently of the exact profile and characteristics of the available vaccines-which we still don't know. most critically however, having left ownership and control over the vaccines to pharmaceutical companies, we do not have a collective and public-health driven governance mechanism that is able to organize and steer vaccine allocation and access in ways that maximize health impact (mazzucato and torreele ). the race for a covid- vaccine exposes the many ways in which a proprietary and market-based r&d model is illsuited, by design, to deliver appropriate vaccines to implement effective vaccination strategies to tackle this pandemic. vaccines and other health technologies are particularly suited to be considered global commons and benefit from open scientific collaboration, especially when they protect us against infectious diseases that do not care for national borders, private (intellectual) property, shareholder value or market dynamics. policy makers all over the world can and must assume responsibility for delivering such global health commons and shape the r&d ecosystem accordingly. driving medical innovation in that direction requires a major shift in how governments see and exercise their role, from by-stander market fixing to proactive entrepreneurial states (mazzucato ) . instead of handing out subsidies and market commitments without strings attached, this means the active steering of innovation towards the desired product characteristics and mobilizing the collective intelligence of researchers globally (mazzucato ) . it includes shaping the incentives and rules for public and private sector collaboration, in particular knowledge management, to optimally work together towards achieving the public interest goals. the scientific community, with its wealth of public health and infectious diseases expertise could be a key driver to this effect, redefining health innovation to address public health needs, not commercial success. these ideas are not as far-fetched as one might thinkthe us department of defence, in particular through their defence advanced research projects agency (darpa), has long understood how to steer public and private sector innovators towards national strategic objectives, with distinctive success. darpa's strategic investments underly the us strength in military and space technologies (medeiros ) , and the rise of silicon valley (cameron ). as exorbitant drug pricing and lack of critical r&d for life threatening conditions, including the growing threat of antibiotic resistance, exposed the deficiencies of our pharmaceutical r&d ecosystem, alternatives rooted in the fundamental responsibility of states to actively shape medical innovation for the public interest have been proposed, for instance in the context of the un high level panel on access to medicines (torreele ). in the people's prescription report, mazzucato calls for a darpa for health, harpa: health innovation aimed at public value, while more recently, a fully publicly owned pharmaceutical 'industry' was proposed for the us (brown ) . while that may sound radical, especially in a us context, it is important to keep in mind that until recently, many countries including in europe had kept critical parts of the pharmaceutical value chain under the control of the public sector, in particular vaccines (blume ) . countries like brazil and thailand still have significant government involvement in their pharmaceutical production, contributing to their relative resilience and autonomy for the production of essential health products, for instance antiretroviral therapy for hiv/ aids (ford et al. ). deprived of access to the global commercial market, cuba has developed a strong public innovation system for health, responding to the country's health needs (pérez et al. ) . calls for stronger public leadership in medical r&d and access, and for transparency, are gaining traction in the context of covid- . from the early days, there have been calls to ensure that commercial monopolies do not stand in the way of access, and to consider the covid-related knowledge and technologies as knowledge commons. concrete proposals have been put forward to either share and pool knowledge, for instance through the meanwhile established who covid- technology access pool (c-tab), or else to not grant or enforce intellectual property rights on them, as the proposal of south africa and india to the wto for a covid- waiver on certain provisions of the trips agreement (silverman ) . open access to knowledge and technologies needed to respond to covid- would be a major step towards more autonomy for countries or regions to produce needed health technologies. it will also require establishing adequate infrastructure and capability and strategic government interventions (finance, science and technology, health systems, industrial policy etc.) to generate such health technologies and make available for the population. the announcement of eu president von der leyen to create a european barda may be an important step in that direction, at least for the region. however it will be important to ensure that its focus is squarely on improving people's health outcomes and deliver health technologies that are widely available and accessible. success must be measured in value for health, not for business. to that effect, this initiative must be firmly rooted in the existing european health innovation infrastructure, for instance through the proposed biomed europa (florio ) that could be adapted after the us national institutes of health, except focused of global health commons, not just de-risking the biomedical industry. collaboration with the private sector is welcomed, but the terms and conditions of the public-private partnerships must be governed by public health benefit (mazzucato and torreele ) , and include open collaboration, sharing of know-how, technologies and infrastructure, and building collective intelligence. instead of secrecy and competition, this approach embraces radical transparency, both on the scientific methods and data, and financially, detailing each one's contributions in investment and risk taking. to ensure truly global health commons, technology transfer to third countries who wish to develop their own capabilities should be built in from the start. humankind is facing a global health crisis, perhaps the biggest crisis of our generation. the decisions people and governments take now, and the values on which they rely, will likely shape the world for years to come, including our healthcare systems, our economy and our culture. the time has come for global leaders to consider vaccines and other health technologies as global health commons that must be available for all and reshape the medical r&d ecosystem towards delivering that. pooling knowledge: private medicine vs. public health? moderna's mrna vaccine reaches its final phase. here's how it works regulatory decision-making on covid- vaccines during a public health emergency vaccine efficacy needed for a covid- coronavirus vaccine to prevent or stop an epidemic as the sole intervention the erosion of public sector vaccine production. the case of the netherlands medicine for all: the case for a public option in the pharmaceutical industry the unequal scramble for coronavirus vaccines-by the numbers the government agency that made silicon valley these coronavirus trials don't answer the one question we need to know bio-creep in non-inferiority clinical trials world health assembly approves milestone resolution on price transparency, health policy watch may biomed europa: after the coronavirus, a public infrastructure to overcome the pharmaceutical oligopoly sustaining access to antiretroviral therapy in the less-developed world: lessons from brazil and thailand no-fault compensation for vaccine injury -the other side of equitable access to covid- vaccines we need more transparency in covid- vaccine development why 'vaccine nationalism' could doom plan for global access to a covid- vaccine covid- vaccines: pressure is on to ensure they go to the most needy, not the highest bidder, the guardian covid- vaccine trials should seek worthwhile efficacy capitalism is broken. the fix begins with a free covid- vaccine the entrepreneurial state how to develop a covid- vaccine for all, project syndicate this economist has a plan to fix capitalism. it's time we all listened science and technological innovation in health in cuba. results in selected problems remove the for-profit variable from clinical drug trials, health and human rights journal may who should get the covid- vaccine first? the equality vs. equity debate south africa and india urge wto to waive ip rights, widen access to covid- drugs and vaccines disease x: accelerating the development of medical countermeasures for the next pandemic the rush to create a covid- vaccine may do more harm than good collective intelligence, not market competition, will deliver the best covid- vaccines as politics trumps science in the race for a vaccine, who will protect public health? ebola in west africa is a wakeup call. what the ebola crisis tells us about our failing drug development system health innovation as a public good, submission to the un high level panel on access to medicines conflict of interest the author declares no conflicts of interest. key: cord- -lj us dq authors: flower, darren r.; davies, matthew n.; doytchinova, irini a. title: identification of candidate vaccine antigens in silico date: - - journal: immunomic discovery of adjuvants and candidate subunit vaccines doi: . / - - - - _ sha: doc_id: cord_uid: lj us dq the identification of immunogenic whole-protein antigens is fundamental to the successful discovery of candidate subunit vaccines and their rapid, effective, and efficient transformation into clinically useful, commercially successful vaccine formulations. in the wider context of the experimental discovery of vaccine antigens, with particular reference to reverse vaccinology, this chapter adumbrates the principal computational approaches currently deployed in the hunt for novel antigens: genome-level prediction of antigens, antigen identification through the use of protein sequence alignment-based approaches, antigen detection through the use of subcellular location prediction, and the use of alignment-independent approaches to antigen discovery. reference is also made to the recent emergence of various expert systems for protein antigen identification. of smallpox were reported annually from across the globe, leading to about million deaths a year. yet, today, the disease has been completely eradicated. in the last years, there have been no known cases. poliomyelitis or polio is the other largescale disease which has come closest to eradication. its success too has been formidable: in , the pan american health organization effectively eradicated polio from the western hemisphere, since when the global polio eradication programme has significantly decreased the overall incidence of poliomyelitis through the rest of the world. in , there were approximately , cases spread through countries; in the past years, global figures amounted to less than , annually. yet, in spite of such remarkable success, death from vaccine-preventable diseases remains unacceptably high [ ] . there are over common infectious diseases responsible for one in four deaths globally. rotavirus and pneumococcus are pathogens causing diarrhoea and pneumonia, the leading causes of infant deaths in underdeveloped countries. in the next decade, effective, widespread vaccination programs against such pathogenic microbes could save the lives of . million children under years of age. hepatitis b causes , deaths in adults and children aged over . seasonal, non-pandemic influenza kills upwards of half a million globally each year. for those aged under in particular, a series of diseases causes an extraordinary and largely preventable death toll. for example, tetanus accounts every year for , deaths, pertussis is responsible for over , deaths, hib gives rise to in excess of , deaths, diphtheria accounts for , deaths, and yellow fever over , deaths. arguably, the most regrettable, the most lamentable situation is that of measles. measles accounts for the unneeded deaths of , under-fives and over , adults and older children. despite this, the situation is by no means bleak. by the close of , approximately million had been vaccinated against hib and million children against hepatitis b. during its first decade, vaccinations against polio, hep b, hib, measles, pertussis, and yellow fever funded by gavi had prevented the unnecessary loss of over million lives. there are approximately vaccines licensed for use in humans, around half of these are widely prescribed. yet, most of these vaccines target the prevention of common childhood infections, with the remainder addressing tropical diseases encountered by travellers to the tropics; only a relatively minor proportion combat endemic disease in under-developed countries. balancing the persisting need against the proven success and anticipated potential, vaccines remain an area of remarkable opportunity for medical advance, leading directly to unprecedented levels of saved and improved lives. from a commercial perspective, the vaccine arena has long been neglected, in part because of the quite astonishing success limned above; today, and in comparative terms at least, activity within vaccine discovery is feverish [ , ] . during the last years, tens of vaccines and vaccine candidates have moved successfully through clinical trials, and vaccines in late development number in the hundreds. in stark contrast to antibiotics, vaccine resistance is negligible and nugatory. despite the egregious and outrageous success enjoyed by vaccines, many major issues persist. the world health organisation long ago identified tuberculosis (tb), hiv, and malaria as the three most significant life-threatening infectious diseases globally. no vaccine has been licensed for malaria or hiv, and there seems little realistic hope for such vaccines appearing in the immediate future. bacille calmette guérin (bcg), the key anti-tb vaccine, is of limited efficacy [ ] . levels of morbidity and mortality generated by diseases already targeted by vaccines remain high. influenza is the key example, with a global annual estimated death toll in the region of half a million. in the twenty-first century, the world continues to be threatened by infectious and contagious diseases of many kinds: visceral leishmaniasis, marburg's disease, west nile, dengue, as well as sars potentially pandemic h n influenza, and over human and emerging zoonotic infections, as well as the persisting threat from hiv, tb, and malaria mentioned above. all this is further compounded by the additional risk arising from antibiotic-resistant bacteria and bioterrorism, not to mention major quasi-incidental issues, such climate change, an accelerating growth in the world's population, increased travel, and the overcrowding seen within the burgeoning populations concentrated into major cities [ ] . for reasons we shall touch on below, the discovery of vaccines is both more urgent and more difficult than it has ever been. in an era where conventional drug discovery has been seen to fail-or at least as seen by cupiditous investors, for whom the current model of pharmaceutical drug discovery is broken-vaccines are one of a number of biologically derived therapies upon which the future economic health of the pharmaceutical industry is thought to rest. the medical need, as stated above, is clear. set against this is the unfortunate realisation that vaccines exist for most easily targeted diseases, those mediated by neutralising antibodies, and so outstanding vaccine-targets are those of more intractable diseases mediated primarily by cellular immunity. to address those properly requires what all discoveries required: hard work and investment; but they also need new ideas, new thinking, and new vaccine discovery technology. amongst, these are computational techniques, the most promising of which are those targeting the discovery of novel vaccine antigens: the candidate subunit vaccines of tomorrow see fig. . . vaccines are agents-either molecular (epitope-or antigen-based vaccines) or supramolecular (attenuated or inactivated whole pathogen vaccines)-which are able to create protective immunity against specific pathogenic infectious microorganisms and any diseases to which they might give rise. protective immunity can be characterised as an enhanced but highly specific response to consequent re-infection-or infection by an evolutionarily closely related micro-organismsmade by the adaptive immune system. such increased or enhanced immunity is facilitated by the quantitative and qualitative augmentation of immune memory, which is able to militate against the pernicious effects of infectious disease. vaccines synergise with the herd immunity they help engender, leading to reduced transmission rates as well as prophylaxis against infection. the term "vaccine" derives from vacca (latin for cow). the words vaccine and vaccination were coined specifically for anti-smallpox immunization by the discoverer of the technique, edward jenner ( - ). these terms were later extended by louis pasteur ( - ) to include a far more extensive orbit or remit, including the entire notion of immunisation against any disease [ , , ] . several fundamentally distinct varieties of vaccine exist. these include inter alia inactivated or attenuated whole pathogen-based vaccines; subunit vaccines are based on one or more protein antigens, vaccines based upon one or more individual epitopes, carbohydrate-based vaccines, and combinations thereof. hitherto, the best-used and, thus, the most successful types of vaccine were built from attenuated-"weakened" or non-infective or otherwise inactivated-pathogenic whole organisms, be they bacterial or viral in nature. well-known examples include the following: the bcg vaccine which acts prophylactically against tuberculosis and albert sabin's anti-poliomyelitis vaccine based on attenuated poliovirus. the vast majority of subunit vaccines are immunogenic protein molecules, and are typically discovered using a somewhat haphazard search process. concerns over the safety of whole-organism vaccines long ago prompted the development of other kinds of vaccine strategy, including those based upon antigens as the innate or immanent active biological constituent of either single or composite vaccines. the vaccine which targets hepatitis b is a good exemplar of a so-called subunit vaccine as it is based on a protein antigen: the viral envelope hepatitis b surface antigen. other types of as-yet-unproven vaccines include those based on epitopes and others based on antigen-presenting cells; many have entered clinical trials, but none have fulfilled their medical or commercial potential. whole antigen discovery. when looking at a reverse vaccinology process, the discovery of candidate subunit vaccines begins with a microbial genome, perhaps newly sequence, progresses through an extensive computational stage, ultimately to deliver a shortlist of antigens which can be validated through subsequent laboratory examination. the computational stage can be empirical in nature; this is typified by the statistical approach embodied in vaxijen [ ] . or this stage can be bioinformatic; this involves predicting subcellular location and expression levels and the like. or, this stage can take the form of a complex mathematical model which uses immunoinformatic models combined with mathematical methods, such as metabolic control theory [ ] , to predict cell-surface epitope populations it is often difficult to capture the proper scientific meaning and use of recondite terms, often borrowed from common usage or archaic language. so, let us be more specific. an immunogen-a molecular moiety exhibiting the property of immunogenicity-is any material or substance capable of eliciting a specific immune response. an antigen, on the other hand, is a molecular moiety exhibiting the property of antigenicity. it is a substance or material recognised by a primed immune system. such a persisting state of immune readiness may be mediated by humoral immunity (principally via the action of soluble antibodies) or by cellular immunity (as mediated by t-cells, antigen presenting cells (apcs), or other phagocytic cells), or a combination of both, in what is often referred to as a "recall" response. immunogenicity is vital: it is the signature characteristic or property that prompts a certain molecular moiety to evoke a significant immune response. here, we shall strictly limit use of "immunogen" and "antigen" to a sole meaning. here, an "antigen" or an "immunogen" will mean a protein that is capable of educing some kind of discernible response from the host immune system. specifically, and for practical reasons, we will almost exclusively be referring to proteins derived from a pathogenic micro-organism. at present, the prophylaxis engendered by all current effective vaccines-all except bcg-is primarily mediated by the humoral immune system, via soluble antibodies. however, the disease mechanisms of most serious diseases for which vaccines are not available are usually mediated by cellular immunity. thus, for untreated disease, we seek to identify immunogenicity generated principally by cellular responses or by a combination of cellular and humoral responses, rather than by humoral immunity alone. to some extent, subunit vaccines can be thought to represent something of a compromise between vaccines based on attenuated or otherwise inactivated wholeorganisms and the many more recent and more innovative vaccine strategies typified by epitope or poly-epitope vaccines. vaccines based around whole pathogens have long engendered safety concerns [ ] [ ] [ ] . from the lubeck disaster and the cutter incident [ ] [ ] [ ] to the recent mmr debacle, issues over safety, real or imagined, have always dogged the development of vaccines [ , ] . indeed, during the eighteenth century the pre-vaccination practice of variolation against smallpox prefigured much of the current debate over the perceived danger of vaccines [ ] . while the case for vaccines is unanswerable, we should not be complacent. any live vaccine, however extensively attenuated, can revert to a pathogenic, diseaseinducing form. this is currently an on-going issue for polio vaccination [ ] . other issues, particularly the chemical or biological contamination of vaccines during manufacture, remain enduring and persistent problems. undesired immunogenicity, the type leading to severe and pathological immune responses, rather than enduring immune memory, is a concern for both whole-organism and subunitbased vaccines, as well as putative biologics [ ] . immunologists and vaccinologists have thus long sought alternatives to the use of whole organisms as vaccines. subunit vaccines and conjugate vaccines are one such. vaccines based on epitopes, singly or in combination, are another. the diversity of innovations in vaccine design holds much potential for success, but, thus far at least, has proved spectacularly unsuccessful in a clinical context. logically, a vaccine that relies solely on, at most, a few well-chosen epitopes, should be effective, efficacious, and, above-all, safe. epitopes, as peptides, may be cytotoxic and might possibly prompt some kind of inopportune immune response but cannot be infective or revert to infectivity. in many ways, epitopes are closer in size and share many properties with synthetic small molecules; possibly dealing with their pharmacokinetics as such may be better than thinking of them as biologic drugs. in practice, of course, epitope-based vaccines, like subunit vaccines, suffer from poor immunogenicity, necessitating the use of a complex combination of adjuvants and complicated delivery systems. for diverse reasons, including immunogenicity, stimulating protective immune responses against intracellular pathogens remains problematic when using nonreplicating vaccines. why should this be? first, the immune response is very complex, involving both the innate and adaptive immunity, and significant interaction between them. in all probability, and particularly when viewed in the context of the whole population, many epitopes and danger signals are involved; likewise, the many different immune actors, be they acting at the cellular or molecular levels, interact with each other and are subject to complex mechanisms of genetic, epigenetic, and system-level control and regulation. it may be that only the large and complex organism-sized vaccines can induce the range of immune responses necessary across the population to induce protection, since they comprise a potential host of immunogenic molecular moieties, not just a single immunodominant epitope see fig. in that which follows, we shall seek to explore the availability and accessibility of informatic techniques and informatic tools used to identify candidate subunit vaccines of microbial origin. yet, we shall start by adding context with an examination of experimental approaches to antigen discovery: so-called reverse vaccinology. reverse vaccinology already relies on informatics, but, in a sense at least, what we would like to do using informatics is to reproduce as much as is possible the steps inherent in successful reverse vaccinology in silico rather than in vitro. reverse vaccinology, and the necessary computational support, is a much more prevalent means of identifying subunit vaccines [ ] . see fig. . . even today, many experimentalists retain a deep and atavistic distrust of all computation. experimentalists seldom trust the reliability and dependability of computational methodology, choosing to trust instead in what they believe to be infallible, if actually rather elusive, empirical reliability of observations, experiments, and the whole paraphernalia of laboratory experimentation. yet, things are in the process of changing, and this change is likely to accelerate as we move forward into a future that looks more parsimonious and uncertain by the day. vaccines have come a long way from the days when they were prepared directly from the fluids of smallpox pustules or extracts of infected spinal cords. yet vaccine discovery and development remains firmly empirical. many modern vaccines still comprise entire inactivated pathogens. while vaccines targeting papillomavirus, tetanus, hepatitis b, and diphtheria are subunit vaccines, few are recombinant proteins devoid of contaminants. some would argue that the only molecular vaccines are glycoconjugates: oligosaccharides conjugated to immunogenic carrier proteins. conventional empirical, experimental, laboratory-based microbiological ways to identify putative candidate antigens require cultivation of target pathogenic micro-organisms, followed by teasing out their component proteins, analysis in a series of in-vitro and in-vivo assays, animal models and with the ultimate objective of isolating one or two proteins displaying protective immunity. unfortunately, in reality, the process is more complex, and more confusing, and much more confounding as this brief synopsis might suggest. cultivating pathogens outside the environment offered by their host organism can be difficult, even impossible. not every protein is readily expressed in adequate quantities in vitro, and many proteins are only expressed in an intermittent basis during the time course of infection. thus, a considerable number of potential, putative, and possible vaccine candidate antigens could be missed by conventional experimental approaches. reverse vaccinology [ ] [ ] [ ] [ ] has the potential to analyse genomes for potential antigens, initially scanning "open reading frames" (orfs), then selecting proteins because they are open to surveillance by the host immune system. this usually involves some complex combination of informatic-based prediction methodologies. recombinant expression of the resulting set of identified molecules can overcome their reduced natural abundance, which has often prevented us recognising their true potential. by enlarging the repertoire of native antigens, this technology can help to foster the development of a new cohort of vaccines. reverse vaccinology was originally established and has been established by studying neisseria meningitidis, which is responsible for meningococcal meningitis and sepsis. vaccines are currently available for all serotypes, except that serogroup b. n. meningitidis orfs were found initially [ , ] ; proteins were then identified, expressed in vitro and found to be surface exposed. seven proteins elicited immunity over many strains. the culmination of this work was a "universal" vaccine for serogroup b based on five antigens [ ] . this protovaccine, when used with alum as adjuvant, induced murine bactericidal antibodies versus % of meningococcal strains drawn from the world population of n. meningitidis. strain coverage increases to over % when used with cpg or mf as adjuvant. another key illustration is porphyromonas gingivalis, an anaerobic gramnegative bacterium found in the chronic adult inflammatory gum disease periodontitis. initially, orfs were identified [ ] ; of these, protein sequences were open to immune surveillance and were positive for several sera. two antigens were found to be protective in mice. yet another fascinating instance is provided by streptococcus pneumoniae, a prime cause of meningitis, pneumonia, and sepsis [ , ] . in this study, potential orfs were initially identified, with of these proteins being readily expressed. finally, six proteins were seen to induce protection against the pathogen. more recently, other and more advanced experimental techniques, such as microarrays, are beginning to come on-stream, opening up a gallimaufry of possible technologies to the new but maturing field of reverse vaccinology. the following gives but a taste of what is to come. using ribosome display to undertake in-vitro protein selection, weichart et al. [ ] identified within the methicillin-resistant col strain of the virulent human pathogen staphylococcus aureus genes, the majority of which were secreted or surface-localized proteins; of these, % had cell envelope function, % were transporter proteins, and % were virulence factors or toxins. using an ingenious combination of advanced proteomics techniques and in-vitro assays, giefing et al. [ ] identified novel vaccine candidates which prevented infections in children and in the elderly caused by a variety of pneumococcus serotypes; four demonstrating major protection versus sepsis in animals. two leads-stkp (a serine/threonine protein kinase) and pcsb (a structural protein with a role in cell wall separation of group b streptococcus)-showed clear cross-protection as potential candidate vaccines against four separate pneumococcal serotypes. using a whole proteome microarray, and in order to identify protein antigens, eyles et al. [ ] probed serum from balb/c mice previously immunized with a vaccine comprising: killed francisella tularensis and two immunomodulatory adjuvants. eleven out of the top twelve immunogenic antigens were known already as immunoreactive, although further proteins were discovered using this experimental approach. in further work from this consortium, titball and co-workers [ ] constructed a protein microarray of , burkholderia pseudomallei proteins, treated it with patient samples, identifying antigens. this smaller set was treated with a further distinct sera from groups of patients, identifying putative candidate antigens. this survey, brief though it is, helps to highlight the potential power of reverse vaccinology for vaccine discovery. however, since the number of antigens is high, given all the potential difficulties in characterising and expressing them, it is important to note that both computational and experimental techniques and methodologies will doubtlessly omit important and interesting proteins from further analysis, though not necessarily for the same or similar reasons. thus, with the burgeoning discipline of reverse vaccinology, both computational and experimental techniques are in need of constant development and improvement. compared to its role to drug discovery, genomics, and a host of other bioscience sub-disciplines, bioinformatics support for the preclinical discovery and development of vaccine is in its infancy; yet, as interest in vaccine discovery increases, the situation changes. there are two key types of bioinformatics support for vaccine design, discovery, and development. at the technical level, the first of these cannot be properly or meaningfully distinguished from general support for target discovery. it includes the annotation of pathogen genomes, more conventional host genome annotation, and the statistical analysis of immunological microarray experiments. the second form of support concentrates on immunoinformatics, that is, the informatics analysis of immunological problems, principally epitope prediction. b-cell epitope prediction remains defiantly basic or is largely dependent on a sometimes unavailable knowledge of three-dimensional protein structure. both structure- [ ] and data-driven [ ] prediction of antibody-mediated epitopes evince poor results. however, methods developed to predict t-cell epitopes now possess considerable algorithmic sophistication. moreover, they continue to develop and evolve, as well as extend their scope and remit to address new and ever larger and more challenging epitope prediction problems. presently, accurate and reliable t-cell epitope prediction is restricted to predicting the binding of peptides to the major histocompatibility complex (mhc). class i peptide-mhc prediction can be reasonably accurate, or is for properly characterised, wellunderstood alleles [ ] . yet a number of key studies have demonstrated that class ii mhc binding prediction is almost universally inaccurate, and is thus erratic and unreliable [ ] [ ] [ ] . a similar situation persists for structure-driven prediction of mhc epitopes [ , ] . irrespective of poor predictive performance, several other problems exist for epitope prediction. for t cell prediction in particular, a prime concern is with the availability or rather lack of availability of relevant data. it is now known that immunogenic t cell epitopes, thought previously to be peptides no more than amino acids in length, can be or more residues long. longmer epitopes now greatly expand the number of possible peptides open to inspection by t cells [ ] [ ] [ ] [ ] . the inadequate results generated by b cell epitope prediction algorithms may indicate that a fundamental reinterpretation of extant b cell epitope data is necessary before improved methods become feasible. these factors, when taken together, are consistent with the notion that methods relying only on the possession of certain epitopes will not be fully effective when tasked with antigen or immunogen identification. this is supported by information indicating a lack of correspondence between selected antigens and experimentally verified protective proteins. there are many means of identifying antigenic proteins. most focus on the properties of protein sequence and structure, but arguably one of the most insightful is instead to examine properties, both local and global, of the underlying nucleic acid. one notable way is to look for evidence of the horizontal or lateral transfer of so-called pathogenicity islands or pais. horizontal transfer, such as transformation, conjugation, or transduction, is distinct from the vertical transfer of genetic material from an ancestor within its lineage. it typically involves an organism incorporating genetic material from an evolutionarily distant organism without being its offspring. pais are a specific type of genomic island; that is, part of a genome acquired through direct transfer between microbes. a genomic island can occur in distantly related species and may be mono-or multi-functional; there are many sub-classes classified by function. other examples include antibiotic resistance islands, metal resistance, and secretion system islands. the gene products of pais are crucial to the propagation of disease pathogenesis, much as the pais themselves are key to the evolution of pathogenesis. pathogen-associated type iii and type iv secretion systems are, for example, often found together in the same pai. detecting such large (> kb) and discrete clusters of genes clusters, habitually possessing a characteristically atypical g/c content, at least when compared with the remainder of the genome, leads, in turn, to the individual identification within clusters of virulence-associated protein antigens. prokaryotic pais are frequently associated with trna-encoding genes, many are flanked by repeat structures, and many contain fragments of mobile genetic elements such as plasmids and phages. pais can be identified by combining analysis of nucleotide composition and phylogeny, amongst others. composition-based approaches rely on the natural variation between genome sequences from different species. regions of the genome with abnormal composition, as demonstrated by nucleotide or codon bias, may be potentially transferred horizontally. such methods are prone to inaccuracies; these result from inherent genomic sequence variation, such as is seen in highly expressed genes, and the observation that over time the sequences of genomic islands alter to mirror the composition of host genomes. evolution-based approaches seek regions that may have been transferred horizontally by comparing related species. put at its simplest: a putative genomic island present in one species, but absent from several related species, is consistent with horizontal transfer. of course, the island may have been present in the last common ancestor shared by the species compared and subsequently been lost from the other species. a less likely explanation would be that the island arose by mutation and selection in this species and no other. to decide, a body of extra evidence would need to be explored, such as the size of the pai, the mechanistic ease of deletion, the consistent presence of the island in more distantly related species, the relative pathogenicity of island-less species, and the divergence of the genome relative to that of other related species. many methods, which seek to quantify and leverage these somewhat vague notions, are now available [ ] [ ] [ ] . such analysis at the nucleic acid level shares many features in common with approaches used to identify cpg islands in eukaryotic genomes [ ] [ ] [ ] [ ] . recently, langille et al. tested six sequence-composition genomic island prediction methods and found that islandpath-dimob and sigi-hmm had the greatest overall accuracy [ ] . island path was designed to help identify prokaryotic pais, through the visualisation of common pai characteristics such as mobile element-associated genes or atypical sequence composition [ ] . sigi-hmm is a very accurate sequence composition-based genomic island predictor, which combines a hidden markov model (hmm) and codon usage measurement to identify genomic islands [ ] . in another work, yoon et al. coupled heuristic sequence searching methods, which aimed simultaneously to identify pais and individual virulence genes, with composition and codon-usage bias [ ] . exploiting a machine learning approach, vernikos and parkhill sampled the structural features of genomic islands using a hypothesis-free, bottom-up search, with the objective of explicitly quantifying the contribution made by each feature to the overall structure of different genomic islands [ ] . arvey et al. sought to identify large chromosomal regions with atypical features using a general divergence measureable to quantify the compositional difference between genomic segments [ ] . islandpick is a comparative genomic island predictor, rather than a composition-based approach, that can identify very probable genomic islands and very probable non-genomic islands within investigated genomes but does require that several phylogentically related genomes are available [ ] . observing pais as having a g + c composition closer to their host genome, wang et al. used so-called genomic barcodes to identify pais. these barcodes are based on the fact that the frequencies of -mers to -mers, and their reverse complement, are very stable across a whole genome when using a window size of over , bps and that this constituted a characteristic signature for genomes [ ] . the ready detection of pais, as a tool in computational reverse vaccinology, has been greatly aided by the deployment of several web-based resources. a key example of a server that successfully integrates several accurate genomic island predictors is islandviewer [ ] , which combines the methods: islandpick [ ] , islandpath [ ] , and sigi-hmm [ ] and is available at the url: http://www. pathogenomics.sfu.ca/islandviewer/query.php. the gui facilitates the visualisation of genomic islands and downloading of data at the gene and chromosome levels in a variety of formats. another important, web-accessible resource is paidb or the pai database. this is a wide-ranging database of pais, containing distinct pais and genbank accessions present in strains of pathogenic bacteria [ ] . paidb may be accessed via the url: http://www.gem.re.kr/paidb. thus, alternative techniques and methodologies are required in order to select and to rank proteins likely to be protective antigens and thus candidate vaccines. below, we shall explore three key approaches: subcellular location prediction, alignment-dependent sequence similarity searching, and alignment-independent empirical statistical approaches. in this section, we consider, perhaps, the clearest and cleanest way to identify potential new antigens in any microbial genome to alignment-dependent sequence similarity searching. there are two complimentary but distinct ways of identifying the immunogenicity of a protein from its sequence. one is to look for significant similarity to proteins of known immunogenicity. this idea seems so straightforward as to be almost facile. the other approach is somewhat less obvious conceptually but almost as straightforward logistically and involves seeking to identify antigens as proteins without discernible sequence similarity to any host protein. let us turn to the first of these two alternatives. let us begin by stating or rather reiterating the obvious. if we know the sequence of an existing antigen or antigens, we can use sequence searching to find similar sequences in the target genome [ , ] . any candidate antigens selected by this process can then be selected for further verification and validation. the same old, familiar caveats apply here: are chosen thresholds appropriate? are high-scoring matches an artefact or are they real and meaningful? the litany of such conditions is all too familiar to anyone well versed in sequence similarity searching. clearly, when a sequence search is run, using blast or fasta , for example, an enormously long list of nearly identical proteins might ensue, or one that does not get any hits at all, or almost any intervening result might be obtained. as reflective practitioners, we must judge which result can be classified as useful and which cannot, and in so doing, identify sets of suitable thresholds, above which we expect usefulness and below which we might anticipate little or no utility. thresholds are contingent upon the sequence family studied, as well as being dependent solely on the problem investigated. thus heuristically identified cut-offs are desirable, but much thinking and empirical investigation are required to select appropriate values. of course, the process adumbrated above presupposes that sufficient antigenic protein sequences are known. compilation of this data is the role of the database. recently, extensive literature mining, coupled with factory-scale experimentation, has created many functional immunology databases, although databases, such as syfpeithi [ , ] , focussing on cellular immunology-primarily mhc processing, presentation, and t cell recognition-have existed for - years. arguably, the best extant database is the hiv molecular immunology database [ ] , although clearly the depth of the database is at the expense of generality and breadth. other recent databases include mhcbn [ , ] and epimhc [ ] , amongst many others. two databases, warrant particular attention: antijen [ ] , formerly known as jenpep [ , ] ; and iedb [ ] . implemented as a relational postgresql database, antijen integrates a wideranging set of data items, much of which is not stored by other databases. in addition to the kind of cellular immunological information familiar from syfpeithi, such as mhc binding and t cell data, antijen additionally archives b cell epitopes and also includes a significant stockpile of quantitative data: kinetic, thermodynamic, as well as functional, including measurements of immunological peptide-protein and protein-protein interactions. the iedb database is considerably more extensive than other equivalent database systems, benefiting from the input of dedicated epitope sequencing projects. iedb has come to eclipse other work in this area. although both antijen and iedb are full of epitope-focussed information of many flavours, they remain incomplete concerning immunogenic antigens. fortuitously, specific antigen-orientated-rather than epitope-focusseddatabases are starting to be available. arguably, the most obvious and most unambiguous example of an antigen is virulence factor (vf): proteins, such as toxins, able to induce disease directly by attacking a host. analysis of known pathogens has allowed recurring vf systems of + distinct proteins. often, sets of vfs exist as discrete, distinct genome-encoded pais, as well as being more widely spread through the genome. clearly, antigens do not need to be vfs in order to be immunogenic and thus candidates for subunit vaccines. instead, they need only be accessible to the immune system. they do not need to directly or indirectly mediate infection. thus, other databases are needed which capture, collate, and archive the burgeoning plethora of antigen-orientated data. recently, we have helped developed a very different database: antigendb [ ] . it contains over antigens collated from the primary scientific literature, as well as other sources. another related database system has been christened violin (vaccine investigation and online information network) [ ] , which allows straightforward curation and the analysis and comparison of research data across diverse pathogens in the context of human medicine, animal models, laboratory model systems, and natural hosts. as we outline above, in addition to identifying sequence similarity to known antigens, another idea gaining ground is that the immunogenicity of an antigen is solely determined by the absence of similarity to host proteins. some think this is the prime determinant of potential protein immunogenicity [ , ] . such ideas are supported by the belief that immune systems are actively educated to lack reactivity to self-proteins [ ] , a process-often termed "immune tolerance"-which is generated via epitope-specific mechanisms [ , ] . what we really want is a meaningful measure of the "foreignness" of a protein correlating with its immunogenicity. usually, "evolutionary distance" substitutes for "foreignness." clearly, such an evolutionary distance must be specified in terms of biomacromolecular structures or sequences. but, is this practically useful for selecting candidate vaccines? another way to formulate this idea is to say that the probability that a protein is immunogenic is exclusively a product of its dissimilarity, at the whole-sequence or sequence-fragment level, to each and every protein contained within the host proteome. most search software is well matched to this problem. in terms of fragment length, the typical length of an epitope might seem logical, since the epitope is the molecular moiety typically recognised during the initial phase of an immune response. yet, even at the epitope level-say a peptide of - amino acid residues-even a single conservative mutation or mismatch in an otherwise identical match might prove significant. single sequence alterations may totally abrogate or significantly enhance neutralising antibodies binding or recognition by the machinery of cellular immunology. we have attempted to benchmark sequence similarity and correlate it with immunogenicity in order to explore the potential of this idea in a quantitative fashion. to that end, we examined the differences between sets of antigens and non-antigen using sequence similarity scores. we looked specifically at sets of known non-antigenic and antigenic protein sequences from six sources: bacteria, viruses, fungi, and parasites, as well as allergens and tumours [ ] [ ] [ ] , comparing pathogen sequence to those from humans and mice using blast [ ] . most non-antigenic and antigenic sequences were non-redundant; implying a lack of homologues between pathogens and host proteomes, although certain parasite antigens, such as catalases and heat shock proteins, had a much greater level of similarity. we were not able to determine a suitable and appropriate threshold based on the hypothesis of non-redundancy to the host's proteome, suggesting that this is not a viable solution to vaccine antigen identification. however, rather than looking at nucleic acid sequences, or at protein sequences using an alignment-based approach, a new set of techniques, based upon alignmentfree techniques, has been and is being developed; as this approach begins to show significant potential, we shall examine it next. proteins accessible to immune system surveillance are assumed to lie external to the microbial organism or be attached to its surface rather than being sequestered and sequestrated within the cell. for bacteria, this means being located on-or in-the outer membrane surface or being secreted. thus, being able to accurately predict the physical location of a putative antigen can provide considerable insight into the likelihood that a particular protein will prove to be an immunogenic and possibly protective. there are two basic kinds of prediction method for identifying subcellular location: manual rule construction and the application of data-driven machine learning methods. data used to discriminate between compartments include sequence-derived features of the protein, such as hydrophobic regions; the amino acid composition of the whole protein; the presence of certain specific motifs; or a combination thereof. accuracy differs significantly between different methods and different compartments, mostly resulting from the deficiency and inconsistency of data used to derive models. gross overall sequence similarity is unable to predict protein sub-cellular location reliably or accurately. even nearly identical protein sequences may be found in distinct locations, while there are many proteins which exist simultaneously at several distinct locations within the cell, often having equally distinct functions at these different sites [ ] . eukaryotes and prokaryotes have quite distinct subcellular compartments. the number of such compartments used in prediction studies varies. a common schema reduces prokaryotic to three compartments (cytoplasmic, periplasmic, and extracellular) and eukaryotic cells to four compartments (nuclear, cytoplasmic, mitochondrial, and extracellular). other structural classifications evince in excess ten eukaryotic compartments. ten compartments maybe a conservative estimate, such is the complex richness of sub-cellular structure. any prediction method must account for permanent, transient, and multiple locations, and, in addition, multi-protein complexes and membrane-bound organelles as possible sites. numerous signal sequences exist. several methods predict lipoproteins. the prediction of proteins translocated via the tat-dependent pathway is important but has yet to be addressed properly. however, amongst binary, single-outcome approaches, signalp is probably the most accurate and reliable method available. it uses neural networks to predict the presence and probable cleavage sites of type ii or n-terminal spase-i-cleaved secretion signal peptides [ ] [ ] [ ] . this signal is common to both prokaryotic and eukaryotic organisms. signalp has recently been enhanced with a hmm intended to discriminate cleaved from uncleaved signal anchors. a limitation of signalp is its proclivity to over-predict: it cannot properly discriminate reliably between a number of very similar yet functionally different signal sequences, regularly predicting lipoproteins and integral membrane proteins as type ii signals. many methods have been devised capable of dividing a genome or virtualproteome between the various subcellular locations of a eukaryotic or prokaryotic cell. psort is a good example; it is a multicategory prediction procedure, comprising many different programmes [ ] [ ] [ ] [ ] . psort i predicts subcellular compartments, while psort ii predicts ten different locations. ipsort deals with several compartments: chloroplast, mitochondrial, and proteins secreted from the cell, while psort-b focuses solely on predicting bacterial sub-cellular locations. another effective programme is hensbc [ ] . hensbc can assign gene products to one of four different types (nuclear, mitochondrial, cytoplasmic, or extracellular) with an accuracy of about eight out of ten for gram-negative bacteria. another programme, subloc [ ] , predicts prokaryotic subcellular location divided between three compartments. another programme is gpos-ploc [ ] , which integrates several basic classifiers. other methods include phobius [ ] , lipop . [ ] , and tatp . [ ] . a comparison of several such programmes, using mycobacterial proteins as a gold standard [ ] , showed subcellular localisation prediction and possessed high predictive specificity. we have developed a set of methods which predict bacterial subcellular location. using a set of methods for lipoprotein, tat secretion, and membrane protein prediction [ ] [ ] [ ] [ ] [ ] [ ] [ ] , three different bayesian network architectures were implemented as software pipelines able to predict specific subcellular locations, and two serial implementations using a hierarchical decision structure, and a parallel implementation with a confidence-level-based decision engine [ ] . the soluble-rooted serial pipeline performed better than the membrane-rooted predictor. the parallel pipeline outperformed the serial pipeline but was significantly less efficient. genomic test sets proved more ambiguous: the serial implementation identified more of the proteins of known location yet more accurate predictions are made overall by the parallel implementation. the implications of this work are clear. the complexity of subcellular structures must be integrated fully into sub-cellular location prediction. in extant studies, many important cellular organelles are not considered; different routes by which proteins can reach the same compartment are ignored; and proteins existing simultaneously at several locations are likewise discounted. clearly, combining high specificity predictors for each compartment appropriately must be the way forward [ ] . many difficulties, problems, and quandaries persist; the most keenly felt is the lack of high-quality, verified, and validated datasets which unambiguously established the location of well-characterised proteins. this dearth is particularly serious for certain types of secreted protein, such as type iii secretion. in a similar manner, considerably more work is required to accurately predict the locations for proteins of viral origin; while certain studies are encouraging [ , ] , the complexity of viral interaction with host organisms continues to confound attempts at analysis. predicting antigens in silico typically utilise bioinformatics tools. such tools can identify signal peptides or membrane proteins or lipoproteins successfully, yet the majority of algorithms tend to depend on motifs characteristic of antigens or, more generally, sequence alignment as the principal arbiter of definitive and meaningful sequence relationships. this is potentially a problem of some magnitude, particularly given the wide range of evolutionary rates and mechanisms amongst microbial proteins. certain protein families do not, however, show obvious or significant sequence similarity, despite having common biological properties, functions, and three-dimensional structures [ , ] . thus alignment-based approaches may not always produce useful and unequivocal results, since they assume a direct sequence relationship that can be identified by simple sequence search techniques. immunogenicity, as a signature characteristic, may be encrypted within the structure and/or sequence instead. this may be encoded so cryptically or so subtlety as to completely confound or at least mislead conventional sequence alignment protocols. discovery of utterly novel and previously unknown antigens will be totally stymied by the absence of similarity to known antigenic proteins. alignment-dependent methods tend to dominate bioinformatics and, by extension, immunoinformatics. several authors have chosen to look at alternative strategies, implementing so-called alignment-independent or alignment-free techniques. the first authors to do so were mayer et al., who reported that protective antigens had a different amino acid composition compared to control groups of nonantigens [ ] . such a result is unsurprising since it has long been known that the structure and sequence composition of proteins adapted to the different redox environments of different sub-cellular compartments [ ] . mayer's analysis was formulated primarily in terms of univariate comparisons of antigens versus controls for different properties. subsequently, we explored bivariate comparison in terms of easily comprehensible scatter-plots. see fig. . for representative examples. what their results ably demonstrate is the potential for the discrimination of antigens and non-antigens by the appropriate selection of orthogonal descriptors. the challenge, of course, is to identify a robust choice of descriptors which are capable of extrapolating as well interpolating when used predictively. progressing beyond this type of analysis, and synergising with our other work on alignment-independent representation [ ] [ ] [ ] [ ] [ ] , we have initiated the development of new methods to differentiate antigens-and thus potential vaccine candidates-and non-antigens, using more sophisticated alignment-free approach to sequence representation [ , ] . rather than focus on epitope versus nonepitope, our approach utilises data on protective antigens derived from diverse pathogens to create statistical models capable of predicting whole-protein antigenicity. our alignment-independent method for antigen identification uses the auto cross covariance (acc) transformation originally devised by wold et al. [ , ] to transform protein sequences into uniform vectors. the acc transform has found much application in peptide prediction and protein classification [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in our method, amino acid residues are represented by the well-known and well-used z descriptors [ ] [ ] [ ] , which characterise the hydrophobicity, molecular size, and polarity of residues. our method also accounts for the absence of complete independence between distinct sequence positions. we initially applied our approach to groups of known viral, bacterial, and tumour antigens, developing models capable of identifying antigen. extra models were subsequently added for fungal and parasite antigens. for bacterial, viral, and tumour antigens, models had prediction accuracies in the - % range [ , , ] . for the parasite and fungal antigens, models had good predictive ability with - % accuracy. these models were incorporated into a server for protective antigen prediction called vaxijen [ ] (url: http://www.darrenflower.info/ vaxijen). vaxijen is an imperfect but encouraging start; future research will yield significantly more insight as well-characterised protective antigens increase significantly in number [ ] . as we have said, a number of bioinformatics problems are unique to the discipline of immunology: the greatest of these is the accurate quantitative prediction of immunogenicity. this chapter has in its totality been suffused and pervaded by the idea of immunogenicity and the challenge of predicting this property in silico. such an endeavour is confounding, yet exciting, and, as a key instrument in developing better, safer, more effective vaccines, is also of undisputed practical utility. successful immunogenicity prediction is at its simplest made manifest through the identification of b cell or t cell epitopes. epitope recognition, when seen as a chemical event, may be understood in terms of the relationships between apparent biological function or activity and basic physicochemical properties. delineating structure-activity or property-activity relationships of this kind is a key concern of immunoinformatics. at the other end of the spectrum, immunogenicity can be viewed is a cohesive, integrated, system property: a property of the entire and complete immune system and not a series of individual and isolated molecular recognition events. thus, the task of predicting systems-level immunogenicity is in all likelihood manifold more demanding than predicting peptide-binding say. the clinical manifestation of vaccine immunogenicity arises from the complex amalgam of many contributing extrinsic and intrinsic factors, which includes pathogen-side and host-side properties, as well as those just coming directly from proteins themselves. see fig. . . protein-side properties include the aggregation state of candidate vaccines and the possession of pamps. pathogen-side properties are clearly properties intrinsic to the pathogen, including expression levels of the antigen, the time-course of this expression, as well as its subcellular location. socalled host-side properties are innate recognition properties of host immunity, and most obviously include t cell epitopes or b cell epitopes. a bona fide candidate antigen should be available for immune surveillance and thus highly expressed, constitutively or transiently, as well as having several epitopes. a protein without immunogenicity would logically lack all or some of these characteristics. as a prediction problem, this is, to say the least, not uncomplicated; clearly consisting of a great variety of difficult-to-compute stages. in terms of mechanism, many of these stages are poorly understood. yet, each can be addressed using standard computational and statistical tools. they can all be predicted, however, presupposing, of course, the presence of relevant data in sufficient quantity. one of the strongest messages to emerge from this review is that immunogenicity is a strongly multi-factorial property: some protein antigens are immunogenic for one reason, or set of reasons, and other immunogenic proteins will be so for another possibly tangential reason or set of reasons. each such causal manifold is itself complex and potentially confusing. thus, the prediction of immunogenicity is a problem in multi-factorial prediction, and the search for new antigens is a search through a multi-factorial landscape of contingent causes and discombobulating decoys. some of the evidence will be highly precise and quantitative. the kind provided by predictive immunoinformatics, for example. this typically yields exact values for, say, the binding affinity of a peptide to a protein component of the immune system, or an unequivocal yes or no answer to the question: is this peptide sequence an epitope? however, for each such exact prediction, we have some notional associated probability concerning how reliable we regard this result. different methods evince a range of accuracy, which, in practice, equate to probabilities of reliability: we naturally have more confidence and assume a greater reliability for a highly accurate prediction versus one of average predictability, though it can still give wrong predictions and generally inaccurate predictors may work well for a specific subset of the data. other types of forms of evidence will have a distinctly more anecdotal flavour. take, for example, the case of bacterial exotoxins. together with endotoxins, such as lps, and so-called superantigens, exotoxins form the principal varieties of toxin secreted by pathogenic bacteria. exotoxins have evolved to be the most toxic substances known to science: in terms of the median lethal dose, botulinum toxin-the active ingredient of botox and causative agent of botulism, amongst others-is about ten times as lethal as radioactive isotope polonium- and a million times more deadly than mainline poisons, such as arsenic or potassium cyanide. virtually, all such potent bacterial exotoxins comprise two functionally distinct subunits, either separate proteins or distinct domains, usually denoted a and b. the a subunit is habitually an enzyme, such as a protease, which modifies specific protein targets, thus disrupting key cellular processes with host cells. the b subunit is a protein which binds to host cell surface lipids or proteins, enabling the toxin to be internalised efficiently. the high specificity of this dual action lends exotoxins much of their remarkable lethality. exotoxins are also extremely immunogenic, inducing the immune systems to produce high-affinity neutralising antibodies against them, and thus make excellent targets for vaccinology. a toxoid-a toxin which has been treated or inactivated, often by formaldehyde-is in essence a form of subunit vaccine and, as such, requires adjuvant to induce adequate immune responses. vaccines targeting tetanus and diphtheria, which usually need boosting every decade, are based on toxoids, albeit typically combined with pertussis toxin acting as an adjuvant. poisoning by exotoxins, on the other hand, requires treatment with antitoxin comprising preformed antibodies. however, and say that we were offered a newly sequenced pathogen genome, is such a classification for ab toxins helpful when trying to identify a potential exotoxins? the answer is neither yes nor is it no, but lies somewhere between these extremes. assuming we had extant knowledge or a reliable method predicting the presence of structural and functionally distinct domains, this very simple ruleof-thumb would become a useful tool for eliminating large numbers of possible toxin molecules. it would not directly identify an antigen but would enormously reduce the workload inherent in their discovery. as well as needing more and more reliable predictors, we also need a way of combining the information we gather from any set of reliable predictors to which we have access. thus, when analysing a pathogen genome, what we seem to need, at least in order to identify immunogenic proteins, is both a set of reliable and robust tools and a cohesive expert system within which to embed them. such systems, albeit still at a relatively crude and faltering level, do exist. because there is an implicit hierarchy of one prediction being based on others, there is a need to balance and judge different pieces of probabilistic evidence. an effective expert system should be capable of such a feat. to a first approximation, an expert system is a computer programme that undertakes tasks that might otherwise be prosecuted by a human expert ostensively by simulating the apparent judgement and behaviour of an individual or organization with expertise and experience within a particular discipline. an expert system might make financial forecasts, or play chess; it might diagnose human illnesses or schedule the routes of delivery vehicles. to create an expert system, one first needs to analyse human experts and how they make decisions, before translating this into rules that a computer can follow. such a system leverages both a knowledge base of accumulated expertise and a set of rules for applying such distilled knowledge to particular situations in order to solve problems. sophisticated expert systems can be updated with new knowledge and rules and can also learn from the success of its prediction, again mirroring the behaviour of properly performing experts. at the heart then of an expert system is the need to combine evidence in order to reach decisions. combining evidence, and reaching a decision based on that combined evidence, is no easier in the laboratory, be that virtual or actual, than it is in the court room. the problem of combining evidence is encountered across the disciplines, and various solutions have arisen in these different areas. within bioinformatic prediction, a particular variety of evidence combination, so-called meta-prediction, is a now a well-established strategy [ , ] . this approach seeks to amalgamate the output of various predictors, typically internet servers, in an intelligent way so that the combined result is more accurate than any of those coming from a single predictor. indeed, combining results from multiple prediction tools does often increase overall accuracy. a consensus strategy was first proposed by mallios [ ] , who combined syfpeithi [ , , ] , propred [ , ] , and the iterative stepwise discriminant analysis meta-algorithm [ ] [ ] [ ] . multipred [ ] integrates hmms and artificial neural networks (ann). six mhc class ii predictors were combined by dai and co-workers [ ] [ ] [ ] basing its overall prediction on the probability distributions of the different scores. trost et al. have used a heuristic method to address class i peptide-mhc binding [ ] . wang et al. [ ] applied a consensus method to calculate the median rank of the top three predictive methods for each mhc class ii protein initially evaluated so as to rank all possible -, -, and -mers from one protein. this rank was used to identify the top % of peptides from each protein. in probabilistic reasoning, or reasoning with uncertainty, there are many ways to represent espoused beliefs-or, in our domain, predictions-that effectively encode the uncertainty of propositions. these include fuzzy logic and the evidential method, among many others. for quantitative data, information fusion, in its various guises [ ] , is one robust route to effective combination. another requires us to enter the world of bayesian statistics, or, at least, a special thread within it. bayes theory, and the ever-expanding strand of statistics devolving from it, is concerned primarily with updating or revising belief in the light of new evidence, while so-called dempster-shafer theory [ ] is concerned not with the conditional probabilities of bayesian statistics but with the direct combination of evidence. it extends the bayesian theory of subjective probability, by replacing bayesian probabilities with belief functions that describe degrees of belief for one question in terms of probabilities for another and then combines these using dempster's rule for merging degrees of belief when based on independent lines of evidence. such belief functions may or may not have the mathematical properties of probabilities but are seemingly able to combine the rigor of probability theory with the flexibility of rule-based approaches. several expert systems of different flavours and hues have now become available within the vaccinology arena. sundaresh et al. developed a specialist software package for the analysis of microarray experiments that could easily be classified as an expert system and used it in the area of reverse vaccinology. this package, which was written in the open-source statistical package r, was used to help analyse a variety of complex microarray experiments on the bacteria f. tularensis, a category a bio-defense pathogen [ ] . this programme implements a two-stage process for diagnostic analysis: selection of antigens based on significant immune responses coupled with differential expression analysis, followed by classification of measured antigen responses using a combination of k-means clustering, support vector machines, and k-nearest neighbours. we have already discussed vaxijen [ , , ] , and the related server epijen [ ] , which combines various methods for identifying epitopes within extant proteins. these two servers can also be classified as vaccine-related expert systems. nerve is another expert system, which has been developed to help automate aspects of reverse vaccinology [ ] . using nerve, the prioritisation of potential candidate antigens consists of several stages: prediction of subcellular localisation; is the antigen an adhesion?; identification of membrane-crossing domains; and comparison to pathogen and human proteomes. candidates are filtered then ranked and putative antigens graded by provenance and its predicted immunogenicity. the web-based expert system, dynavacs [ ] , was developed to facilitate the efficient design of dna vaccines and is available in the url: http://miracle. igib.res.in/dynavac. it takes a structured approach for vaccine design, leveraging various key design parameters, including the choice of appropriate expression vectors, safeguarding efficient expression through codon optimization, ensuring high levels of translation by adding specific sequence signals, and engineering of cpg motifs as adjuvant mechanisms exacerbating immune responses. it also allows restriction enzyme mapping, the design of primers, and lists vectors in use for known dna vaccines. vaxign is another expert system developed to help facilitate vaccine design [ ] . vaxign undertakes dynamic vaccine target prediction from sequence. methodologically, it combines protein subcellular location prediction with prediction of transmembrane helices and adhesins, analysis of the conservation to human and/or mouse proteins with sequence exclusion from the genomes of nonpathogenic strains, and prediction of peptide binding to class i and class ii mhc. as a test, vaxign has been used to predict vaccine candidates against uropathogenic escherichia coli. however, nerve and its various and varied siblings are tasked with such a confounding and difficult undertaking that they are obliged to fall somewhat short of what is required. an obvious first step in tackling the greater problem is to address first subcellular location prediction. then, we can look at antigen presentation, modelling for each component step, before building these into a fully functional model. we can also develop empirical approaches-such as vaxijen [ , , ] . we must also factor in antibody-mediated issues, properly address pamps, post translational danger signals, expression levels, the role of aggregation, and the capacity of molecular adjuvants to enhance the innate immunogenicity to usable levels. see fig. . . the value of vaccines is not yet unchallenged. however, most reasonable people would, in all probability, agree that they are a good thing, albeit with a few minor provisos. the idea underlying all vaccines is a strong and robust one: it is in the reification-that is, the realisation, manifestation, and instantiation-of this abstract concept that the trouble lies, if indeed trouble there is. existing vaccines are by no means perfect; again, most sensible and well-informed people would no doubt acknowledge this also. one might argue that their intrinsic complexity, and the highly empirical nature of their discovery over decades, and the fraught nature of their manufacture, has much to answer in this regard. why should this be? in part, it is due to the extreme complexity of immune response to an administered vaccine, which is largely specific to each individual or at least is different in different sub-groups within the totality of the vaccinated population. the immune responses is comprised, at least for whole-pathogen vaccines, of the adaptive immune response to multiple b cell and t cell epitopes as well as the responses made by the innate immune responses to diverse molecular structures, principally pamps. when one considers also the degree to which such a repertoire of responses is augmented and modified by the action of additives, be they designed to increase the durability and stability of vaccines or be they adjuvants, which are intended to raise the level of immune reactions. add in stochastic and coincidental phenomena, such as reversion to pathogenicity, and we can see immediately that navigating our way through the vaccine minefield is no easy task. all such problems engendered by this intrinsic complexity are themselves compounded by our comparatively weak understanding of immunological mechanisms, since, if we understood the mechanism of responses well enough, we could and would have designed our vaccines to circumvent these issues. part of the answer to this cacophony of conflicting and confounding quandaries is the newly emergent discipline of vaccinomics. a proper understanding of the relationships between gene variants and vaccine-specific immune responses may help us to design the next generation of personalised vaccines. vaccinomics addresses this issue directly. it seeks to identify genetic factors mediating or moderating vaccine-induced immune responses, which are known to be extremely variable within population. much data indicate that host genetic polymorphisms are key determinants of innate and adaptive response to vaccination. hla genes, non-hla genes, and genes of the innate immunity all contribute, and do so in many ways, to the variation observed between individuals for immune responses to microbial vaccines. vaccinomics offers many techniques that can help illuminate these diverse phenomena. principal amongst these are population-based gene/snp association studies between allele or snp variation and specific responses, supplemented by the application of next-generation sequencing technology and microarray approaches. yet, and for all this nay-saying and gainsaying, vaccines and vaccination have demonstrated their worth time after time; yet, to justify the continuing faith we invest in them, new and better ways of making safer and more focussed vaccines must be found. most current vaccines work via antibody-mediated mechanisms; and most target viruses and the diseases they cause. unfortunately, the stock of such disease targets is dwindling. low-hanging fruit has long since been cut down. only fruit that is well out of reach remains. vaccines based on apcs and peptides are new but unproven strategies; most modern vaccine development relies instead on effective searches for vaccine antigens. one of the clearest points to emerge from such work is that there are many competing concepts, thoughts, and ideas that may confound or help efficient identification of immune reactive proteins. certain such ideas we have outlined. some are indisputably persuasive, even compelling, yet many strategies-and the technical approaches upon which they are based-have singly failed to deliver on their promise. long ago, and based on his lifetime's experience of all things immunological, professor peter cl beverley sketched out a paradigm for protein-focussed vaccine development, which we have formalised further, and which schema is summarised in fig. . . some of his factors overlap with the factors from fig. . . he identified many of the factors that potentially contribute to the immunogenicity of proteins, be they of pathogen origin or another source entirely, and also other features which might make proteins particularly suitable for becoming candidate vaccines. of these, some are as-yet beyond prediction, such as the attractiveness for apcs or the inability to down-regulate immune responses. the status of proteins as evasins is currently only possibly addressable through sequence similarity-based approaches and likewise for the attractiveness for uptake by apcs is again, though possible there exist motifs, structural or sequence, which could be identified. currently, the dearth of relevant data precludes prediction of such properties; and, while it is possible to predict some of these properties with some assurance of success, and others are predictable but only incidentally, overall, we are still some way from realising the dream embodied in fig. failure occurs for simple reasons: we deal with simplified abstractions and cannot hope to capture all that which is required for prediction by looking superficially at a single factor. protein immunogenicity comes instead from the dynamic combination of innumerable contributing factors. this is by no means a facile or easily solved informatics conundrum. a vaccine candidate should have epitopes that the host recognises, be available for immune surveillance, and be highly expressed. factors mediating protein immunogenicity are many; possession of b or t cell epitopes, post-translational danger signals, sub-cellular location, protein expression levels, and aggregation state amongst them. predicting such diverse, complex, confounding properties is-and remains-a challenge. vaccine antigens, once discovered, should, ultimately, and with appropriate manipulation, together with an apt, apposite, and appropriate delivery system and the right choice of adjuvant, become first a candidate for clinical trials, before, hopefully, progressing to regulatory approval. we require an integrative, systemsbiology approach to solve this problem. no single approach can be applied universally and with success; what we crave is the full integration of numerous equally wakefield's article linking mmr vaccine and autism was fraudulent computer-aided biotechnology: from immuno-informatics to reverse vaccinology harnessing bioinformatics to discover new vaccines new vaccines against tuberculosis bioinformatics for vaccinology lessons learned concerning vaccine safety vaccines: the real issues in vaccine safety target the fence-sitters an american tragedy'. the cutter incident and its implications for the salk polio vaccine in new zealand - the cutter incident, years later poliomyelitis following formaldehyde-inactivated poliovirus vaccination in the united states during the spring of . ii. relationship of poliomyelitis to cutter vaccine bioinformatics for vaccinology vaccine-derived poliovirus (vdpv): impact on poliomyelitis eradication advances in predicting and manipulating the immunogenicity of biotherapeutics and vaccines the use of genomics in microbial vaccine development post-genomic vaccine development microbial genomes and vaccine design: refinements to the classical reverse vaccinology approach biotechnology and vaccines: application of functional genomics to neisseria meningitidis and other bacterial pathogens complete genome sequence of neisseria meningitidis serogroup b strain mc identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing a universal vaccine for serogroup b meningococcus identification of vaccine candidate antigens from a genomic analysis of porphyromonas gingivalis use of a whole genome approach to identify vaccine molecules affording protection against streptococcus pneumoniae infection identification of a universal group b streptococcus vaccine by multiple genome screen functional selection of vaccine candidate peptides from staphylococcus aureus whole-genome expression libraries in vitro discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies immunodominant francisella tularensis antigens identified using proteome microarray a burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens antibody-protein interactions: benchmark datasets and prediction tools evaluation benchmarking b cell epitope prediction: underperformance of existing methods prediction of mhc-peptide binding: a systematic and comprehensive overview in silico tools for predicting peptides binding to hlaclass ii molecules: more confusion than conclusion on evaluating mhc-ii binding peptide prediction methods evaluation of mhc-ii peptide binding prediction servers: applications for vaccine research a critical cross-validation of high throughput structural binding prediction methods for pmhc limitations of ab initio predictions of peptide binding to mhc class ii molecules t cell receptor recognition of a 'super-bulged' major histocompatibility complex class i-bound peptide high resolution structures of highly bulged viral epitopes bound to major histocompatibility complex class i. implications for t-cell receptor engagement and t-cell immunodominance have we cut ourselves too short in mapping ctl epitopes? a long, naturally presented immunodominant epitope from ny-eso- tumor antigen: implications for cancer vaccine design identification and characterization of pathogenicity and other genomic islands using base composition analyses a novel strategy for the identification of genomic islands by comparative analysis of the contents and contexts of trna sites in closely related bacteria mobilomefinder: web-based tools for in silico and experimental discovery of bacterial genomic islands cpgcluster: a distance-based algorithm for cpg-island detection cpgif: an algorithm for the identification of cpg islands identifying cpg islands by different computational techniques cpg_mi: a novel approach for identifying functional cpg islands in mammalian genomes evaluation of genomic island predictors using a comparative genomics approach islandpath: aiding detection of genomic islands in prokaryotes score-based prediction of genomic islands in prokaryotic genomes using hidden markov models a computational approach for identifying pathogenicity islands in prokaryotic genomes resolving the structural features of genomic islands: a machine learning approach detection of genomic islands via segmental genome heterogeneity prediction of pathogenicity islands in enterohemorrhagic escherichia coli o :h using genomic barcodes islandviewer: an integrated interface for computational identification and visualization of genomic islands towards pathogenomics: a web-based resource for pathogenicity islands identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis functional genomics of pathogenic bacteria syfpeithi: database for searching and tcell epitope prediction syfpeithi: database for mhc ligands and peptide motifs hiv sequence databases mhcbn . : a database of mhc/tap binding peptides and t-cell epitopes mhcbn: a comprehensive database of mhc binding and non-binding peptides epimhc: a curated database of mhcbinding peptides for customized computational vaccinology antijen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data jenpep: a novel computational information resource for immunobiology and vaccinology jenpep: a database of quantitative functional peptide data for immunology the immune epitope database . antigendb: an immunoinformatics database of pathogen antigens violin: vaccine investigation and online information network epitopic peptides with low similarity to the host proteome: towards biological therapies without side effects peptimmunology: immunogenic peptides and sequence redundancy primer: mechanisms of immunologic tolerance recent advances in immune modulation cutting edge: contributions of apoptosis and anergy to systemic t cell tolerance discriminating antigen and non-antigen using proteome dissimilarity iii: tumour and parasite antigens discriminating antigen and non-antigen using proteome dissimilarity ii: viral and fungal antigens discriminating antigen and non-antigen using proteome dissimilarity: bacterial antigens gapped blast and psi-blast: a new generation of protein database search programs single proteins might have dual but related functions in intracellular and extracellular microenvironments locating proteins in the cell using targetp, signalp and related tools improved prediction of signal peptides: signalp . a comprehensive assessment of n-terminal signal peptides prediction methods wolf psort: protein localization predictor secreted protein prediction system combining cj-sphmm, tmhmm, and psort psort-b: improving protein subcellular localization prediction for gram-negative bacteria psort: a program for detecting sorting signals in proteins and predicting their subcellular localization predicting protein subcellular locations using hierarchical ensemble of bayesian classifiers based on markov chains subloc: a server/client suite for protein subcellular location based on soap gpos-ploc: an ensemble classifier for predicting subcellular localization of gram-positive bacterial proteins advantages of combined transmembrane topology and signal peptide prediction-the phobius web server prediction of lipoprotein signal peptides in gram-negative bacteria prediction of twin-arginine signal peptides validating subcellular localization prediction tools with mycobacterial proteins toward bacterial protein sub-cellular location prediction: single-class discrimminant models for all gram-and gram+ compartments multi-class subcellular location prediction for bacterial proteins alpha helical trans-membrane proteins: enhanced prediction using a bayesian approach beta barrel trans-membrane proteins: enhanced prediction using a bayesian approach a predictor of membrane class: discriminating alpha-helical and beta-barrel membrane proteins from non-membranous proteins tatpred: a bayesian method for the identification of twin arginine translocation pathway signal sequences lippred: a web server for accurate prediction of lipoprotein signal sequences and cleavage sites combining algorithms to predict bacterial protein sub-cellular location: parallel versus concurrent implementations predicting the subcellular localization of viral proteins within a mammalian host cell virus-ploc: a fusion classifier for predicting the subcellular localization of viral proteins within host and virus-infected cells structure and sequence relationships in the lipocalins and related proteins structural relationship of streptavidin to the calycin protein superfamily analysis of known bacterial protein vaccine antigens reveals biased physical properties and amino acid composition adaptation of protein surfaces to subcellular location hierarchical classification of g-protein-coupled receptors with data-driven selection of attributes and classifiers gpcrtree: online hierarchical classification of gpcr function optimizing amino acid groupings for gpcr classification on the hierarchical classification of g protein-coupled receptors proteomic applications of automated gpcr classification vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines identifying candidate subunit vaccines using an alignment-independent method based on principal amino acid properties dna and peptide sequences and chemical processes multivariately modeled by principal component analysis and partial least-squares projections to latent structures principal property-values for nonnatural amino-acids and their application to a structure activity relationship for oxytocin peptide analogs peptide binding to the hla-drb supertype: a proteochemometrics analysis proteochemometrics mapping of the interaction space for retroviral proteases and their substrates proteochemometrics analysis of substrate interactions with dengue virus ns proteases generalized modeling of enzyme-ligand interactions using proteochemometrics and local protein substructures rough set-based proteochemometrics modeling of g-protein-coupled receptor-ligand interactions improved approach for proteochemometrics modeling: application to organic compound-amine g protein-coupled receptor interactions melanocortin receptors: ligands and proteochemometrics modeling proteochemometrics modeling of the interaction of amine g-protein coupled receptors with a diverse set of ligands peptide quantitative structureactivity-relationships, a multivariate approach multivariate parametrization of coded and non-coded amino-acids new chemical descriptors relevant for the design of biologically active peptides. a multivariate characterization of amino acids bioinformatic approach for identifying parasite and fungal candidate subunit vaccines jafa: a protein function annotation meta-server metamqap: a meta-server for the quality assessment of protein models a consensus strategy for combining hla-dr binding algorithms prediction of hla-a -restricted ctl epitope specific to hcc by syfpeithi combined with polynomial method propred analysis and experimental evaluation of promiscuous t-cell epitopes of three major secreted antigens of mycobacterium tuberculosis propred: prediction of hla-dr binding sites predicting class ii mhc/peptide multi-level binding with an iterative stepwise discriminant analysis meta-algorithm class ii mhc quantitative binding motifs derived from a large molecular database with a versatile iterative stepwise discriminant analysis meta-algorithm iterative stepwise discriminant analysis: a meta-algorithm for detecting quantitative sequence motifs neural models for predicting viral vaccine targets building a meta-predictor for mhc class ii-binding peptides a probabilistic meta-predictor for the mhc class ii binding peptides a meta-predictor for mhc class ii binding peptides based on naive bayesian approach strength in numbers: achieving greater accuracy in mhc-i binding prediction by combining the results from multiple prediction tools a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach combination of fingerprint-based similarity coefficients using data fusion connectionist-based dempster-shafer evidential reasoning for data fusion from protein microarrays to diagnostic antigen discovery: a study of the pathogen francisella tularensis epijen: a server for multistep t cell epitope prediction nerve: new enhanced reverse vaccinology environment dynavacs: an integrative tool for optimized dna vaccine design vaxign: the first web-based vaccine design program for reverse vaccinology and applications for vaccine development enzymes, metabolites and fluxes key: cord- -xh cw ao authors: papadopoulos, nikolaos g.; megremis, spyridon; kitsioulis, nikolaos a.; vangelatou, olympia; west, peter; xepapadaki, paraskevi title: promising approaches for the treatment and prevention of viral respiratory illnesses date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: xh cw ao viral respiratory tract infections are the most common human ailments, leading to enormous health and economic burden. hundreds of viral species and subtypes have been associated with these conditions, with influenza viruses, respiratory syncytial virus, and rhinoviruses being the most frequent and with the highest burden. when considering prevention or treatment of viral respiratory tract infections, potential targets include the causative pathogens themselves but also the immune response, disease transmission, or even just the symptoms. strategies targeting all these aspects are developing concurrently, and several novel and promising approaches are emerging. in this perspective we overview the entire range of options and highlight some of the most promising approaches, including new antiviral agents, symptomatic or immunomodulatory drugs, the re-emergence of natural remedies, and vaccines and public health policies toward prevention. wide-scale prevention through immunization appears to be within reach for respiratory syncytial virus and promising for influenza virus, whereas additional effort is needed in regard to rhinovirus, as well as other respiratory tract viruses. discuss this article on the jaci journal club blog: www.jacionline.blogspot.com. the respiratory system is one of the main portals of entry for human pathogens. although precise calculations are challenging because of methodology and inherent variability, the number of potentially infectious viruses we breathe every day can be in the range of many thousands. thus it is not surprising that viral respiratory tract infections (vrti) are the most common human diseases, leading to enormous health and economic burden. a wide variety of conditions fall within the spectrum of vrtis. many of these are by themselves major public health concerns: influenza, acute bronchiolitis, viral pneumonia, and common colds. together with their downstream effects (ie, acute exacerbations of asthma and chronic obstructive pulmonary disease [copd] ), all result in vast amounts of morbidity, mortality, and health care costs, including primary care visits, hospitalizations, and deaths but also inappropriate use of antibiotics, loss of productivity, and effects on quality of life. [ ] [ ] [ ] respiratory tract viruses have been isolated and characterized during the last century, starting from influenza virus (ifv) in the s and followed by respiratory syncytial virus (rsv), coronaviruses, adenoviruses, and rhinoviruses in the to s; nevertheless, ''new'' viruses or subtypes, such as human metapneumovirus or rhinovirus c, are still being identified. , even though several of these viruses are typically associated with a clinicopathologic entity (eg, ifv with influenza, rsv with bronchiolitis, and rhinovirus with the common cold), there is also extensive overlap, and it is often difficult to identify the etiologic agent based on clinical grounds alone. consequently, when considering prevention and treatment of vrti, potential targets include specific pathogens, the immune response, disease transmission, or just symptoms. here we provide an overview of the options and highlight some of the most promising approaches in vrti treatment, including symptomatic medication, immunomodulatory drugs, antiviral agents, and natural products, as well as in vrti prevention, ranging from vaccines to immunostimulators and public health policies. this is a vast field, and thus we emphasize advances that might be relevant in tackling the virus-induced aspects of allergic disease, such as asthma exacerbations. most mild viral respiratory illnesses are managed symptomatically with over-the-counter medications, such as nasal decongestants, antipyretics/analgesics, antitussives, or expectorants, on which no major improvements are foreseen. although generally well tolerated for short-term relief, some agents can have adverse effects, especially in young children. therefore the us food and drug administration has issued a warning against the use of over-the-counter cough and cold products in children younger than years of age. furthermore, the use of decongestants should be minimized, especially in children, whereas codeine has been restricted in children by the european medical agency since . selective cox inhibitors, such as celecoxib and mesalazine, have been widely used in clinics for their antipyretic, analgesic, and antiinflammatory properties in patients with airway diseases, whereas their combination with neuraminidase inhibitors (nais) has significantly improved the survival of ifv-infected mice. recent studies have revealed a new genus of specialized proresolving lipid mediators (spms), including lipoxins, resolvins, protectins, and maresins, enhancing anti-inflammatory, antiviral, and proresolving mechanisms. medications interfering with prostanoid and lipoxygenase biosynthesis and signaling, thus affecting resolution and spm switching, such as aspirin and nonsteroidal anti-inflammatory drugs, have been suggested as potential agents modulating antiviral immunity, , whereas several spm resolution agonists are in clinical development programs. symptomatic relief can also be sought in severe cases. noninvasive ventilation can reduce respiratory distress in patients with acute viral bronchiolitis. very recently, new devices delivering totally conditioned gas ( c at % relative humidity) through a very high-flow nasal cannula (up to l/min) have been indicated for bronchiolitis mainly as rescue therapy to reduce the need for admission to the intensive care unit. immune and antiviral pathway modulators although vrtis are most often short-lived events, impaired antiviral clearance and/or activation of inflammatory pathways lead to important downstream complications, such as exacerbations of asthma or copd. the immune and antiviral mechanisms leading from infection to exacerbation have been scrutinized, and medications targeting these pathways are being evaluated as promising candidates to reduce disease burden. impaired interferon production has been observed in patients with various obstructive respiratory diseases, potentially contributing to enhanced susceptibility to and/or severity of virus-induced acute airway exacerbations. although inhaled ifn-b supplementation has not shown a clear effect in preventing virus-induced symptom worsening in patients with mild asthma, subanalysis in patients with severe asthma showed a protective effect. interestingly, in an experimental model exogenous administration of ifn-l induced a strong and more prolonged antiviral state than ifn-b. moreover, experimental studies in an allergic asthma model showed that ifn-l supplementation enhanced t h immunity by inducing ifn-g and suppressing t h and t h responses through modulation of lung cd c dendritic cell function. , novel antibody-based drugs with antirhinovirus and immunomodulatory effects act through ifn-b induction and suppression of t h responses in experimental models. the prototype synthetic toll-like receptor (tlr) antagonist eritoran (e ) and anti-tlr igg therapy have been shown to block ifv lethality in mice by suppressing lung pathology, clinical symptoms, and viral titers. , other innate immune receptors, such as tlr , also have potential for host-targeted therapeutic approaches. interestingly, omalizumab, an anti-ige mab, prevents asthma exacerbations either by decreasing the duration and shedding of rhinovirus infection or by blocking the synergistic effect of rhinovirus infection on allergy. , because high-affinity ige receptor (fcεri) cross-linking on plasmacytoid dendritic cells reduces ifn-a responses after viral infections, it is plausible that omalizumab enhances virus-induced ifn-a production in asthmatic patients, thus limiting virus spreading and infection severity. ''severe cytokine storm,'' an entity associated with markedly higher levels of proinflammatory cytokines, has been associated with severe influenza infections; immunomodulatory agents have been proposed as potential therapeutic strategies. peroxisome proliferator-activated receptor g agonists (eg, rosiglitazone and pioglitazone) are critical regulators of inflammation and have been promising in improving the clinical outcome of severe influenza infections. their development slowed down from to because of possible cardiovascular side effects; however, in , the us food and drug administration lifted restrictions based on new safety data. moreover, sphingosine- -phosphate receptor agonists , which are located mainly on pulmonary endothelial cells, exhibit cytokine storm-blunting activity by suppressing both innate cellular and cytokine/chemokine responses, particularly when combined with antiviral agents. there is increasing interest in the use of macrolides to treat or prevent virus-induced asthma exacerbations, although microbial resistance remains a major hurdle, and therefore they are not currently indicated. early in vivo evidence suggested that azithromycin has anti-inflammatory and antiviral effects through induction of interferon-stimulated gene mrna expression and reduced viral replication and release in patients with asthma and chronic obstructive lung disease. , in a randomized clinical trial including wheezing preschool-aged children, early azithromycin administration significantly reduced the likelihood of a severe lower respiratory tract infection. novel macrolides (mycobacterium avium complex ) with anti-inflammatory, antibacterial, and, more importantly, interferon-augmenting activity in airway epithelium have been identified. finally, in vitro models have demonstrated that a -antitrypsin exerts anti-inflammatory effects in airway epithelial cells from rhinovirus-infected patients with copd, potentially through inhibition on caspase- activity, suggesting a -antitrypsin as a potential anti-inflammatory agent. antivirals vrtis are usually characterized by an acute and self-limiting course, which means that the peak of viral replication usually precedes or parallels the appearance of clinical symptoms. as a result, the time window from verification and/or typing of the pathogen, allowing a specific therapeutic intervention, is extremely narrow. additional challenges need to be overcome, such as the structural variation of viral proteins, multiple genotypes, and high mutation rates. accordingly, only a very limited number of specific antiviral drugs are currently licensed, and promising approaches mostly aim to control severe complications, reduce disease burden, or transmission. antiviral strategies aim to block particular stages of the viral lytic cycle, including attachment and entry to the host cell, replication, transcription, and translation (fig ) . in principle, preventing a viral pathogen from entering the host cell represents the ideal antiviral strategy because the virus is not allowed to ''hack'' the host: ifv nais have been successfully used to competitively bind the sialic acid-binding pocket of neuroaminidase and are good examples of this approach. oseltamivir and zanamivir have been used as anti-flu therapies, whereas laninamivir and peramivir show antiviral activity against wild-type but also against oseltamivir-resistant and nai-resistant strains, respectively. , the nonenveloped rhinoviruses use viral capsid structures to bind their receptors (intercellular adhesion molecule [icam- ], low-density lipoprotein receptor, and cadherin-related family member ). even though more than % of rhinovirus strains use icam- for cell entry, an icam- competitor, tremacamra, did not make it into the clinic despite initially promising results, and no anti-icam- drugs are currently available. another strategy is to prevent capsid uncoating and further assembly of new virions. this strategy has been successfully used against ifv and severe acute respiratory syndrome (sars)coronavirus, which use a class i fusion mechanism. das (fludase, nexbio, inc, san diego, calif) is a fusion construct that cleaves the sialic acid receptors on host cells, and its antiviral spectrum includes ifv and parainfluenza viruses (pivs). nonenveloped viruses, such as rhinovirus, release their genomes through a conformational shift of the capsid proteins accompanied by an expansion of the viral shell along with the opening of symmetry-related channels (pores) from which the genome is released (virus uncoating). , various capsid-binding compounds against rhinoviruses have been tested (r and win series) without ultimate success. pleconaril, bta (vapendavir), and pocapavir (v- ) are still under clinical evaluation. of note, a major drawback of capsid binders is the rapid emergence of resistance. several fusion inhibitors are being developed for the treatment of rsv and have been reviewed elsewhere. , because of their limited coding capacity, viruses rely on the production of polyproteins that need to be cleaved into functional subunits by viral proteases. the enterovirus polyprotein is cleaved by a family of cysteine proteases, which are highly conserved among different subtypes but lack homology with human proteases. unfortunately, after failed attempts with ruprintrivir (ag ) and ag , which showed antiviral activity in vitro but not in vivo, no similar agents are being pursued currently. the use of hiv protease inhibitors, such as lopinavir and ritonavir, in patients with sars has not been associated with any proved benefit, although retrospective studies reported that severe outcomes (acute respiratory distress syndrome or death) occurred less often in those receiving a combination of lopinavir/ritonavir and ribavirin with corticosteroids. polymerase inhibitors (nucleoside/nucleotide analogs) act by leading to termination of the polynucleotide chain elongation. ribavirin has been used for the treatment of severe rsv-related disease in high-risk infants and in combination with protease inhibitors in patients with sars, but its use has been limited because of cost and unconfirmed efficacy toward severe outcomes. als- is a promising orally bioavailable prodrug of the novel rsv replication inhibitor als- (a cytidine nucleoside analogue), which inhibits rsv replication. other promising polymerase inhibitors include amiloride (competitive inhibitor of coxsackie virus b rna polymerase) and gpc-n (multiple genera in picornaviridae) but are still in the early stages. favipiravir (t- ) is an antiviral drug that selectively inhibits the rna-dependent rna polymerase of ifv, as well as several other viruses. umifenovir has been shown to inhibit various human respiratory rna viruses, including several strains of ifv-a and ifv-b, rsv, piv , and rhinovirus b . it also demonstrates inhibitory activity against other viruses, enveloped or not, responsible for emerging or globally prevalent infectious diseases. finally, a most promising but also challenging antiviral approach is through use of antisense oligonucleotides. antisense oligonucleotides are single-stranded deoxyribonucleotide oligomers with a sequence complementary to a target mrna transcript. thus viral genomic rna or viral mrna can be targeted directly. antisense technology and rna interference have been experimentally explored in targeting measles virus, sars-coronavirus, coxsackievirus, enteroviruses and rhinoviruses, piv, human metapneumovirus, ifv, and rsv genomes. , the rna inhibition-based therapeutic that is furthest advanced in clinical development at this time is against rsv. aln-rsv is an unmodified, naked, small interfering rna designed to inhibit the replication of rsv by interrupting the synthesis of the viral n protein. the sequence of the target is well conserved throughout naturally occurring rsv a and b genotypes. in all, new antivirals are being explored continuously, particularly for life-threatening viruses, such as ifv (influenza) and rsv. rhinoviruses, even though simple in terms of genome organization and protein coding, have proved extremely difficult to target, mostly because of their high diversity and immuneevading strategies but also to some extent to the underestimation of rhinovirus infection clinical consequences. within the past few years, scientific communities all over the world have shown renewed interest in the search for novel immune-stimulating or antiviral agents of plant origin for either treatment or prevention, often using ethnopharmacologic approaches. natural compounds are widely recognized as privileged structures trimmed by evolutionary processes to interact with macromolecular targets. plants use a diverse set of biochemical pathways to generate several secondary metabolites representing ecosystem adaptations to help plants to survive various environmental stresses and protect them from infections and infestations. the antiviral potential of plant extracts or compounds varies among viruses. natural compounds occupy an equally large and complex chemical space as synthetic compounds. in the case of antiviral agents, % of entities registered in the last approximately years ( - ) can be classified as natural product botanicals, synthetic but natural product mimics, natural product pharmacophores, or a combination of the latter . oseltamivir, a success story in ifv drug synthesis, has its roots in nature: the abundant plant constituents quinic acid and shikimic acid are used as its starting materials. a screening strategy was applied to investigate crude extracts from plant species on their inhibiting potential toward nais of clostridium perfringens. moreover, bioactive compounds from cleistocalyx operculatus buds were discovered by using an anti-ifv screening approach. the chinese academy of medical sciences tested more than , plants. among them, a pronounced neuroaminidase-inhibiting effect was observed for the herb extract of elsholtzia rugulos. some extracts from agrimonia pilosa, echinacea purpurea, and prunus mume or the multicomponent mixtures polyphenol fractions from punica granatum and secoiridoid glucosides from ligustrum lucidum have shown a significant reduction of virus-induced cytopathic effects and in general antiviral or anti-influenza activity. a % to % risk reduction of common cold incidence with the use of echinacea species supplements has been shown. moreover, a recent meta-analysis demonstrated benefit on long-term ( - months) prevention with echinacea species on recurrent respiratory tract infections (rtis). another promising compound is bno , a fixed combination of herbal substances that significantly reduced symptoms and led to faster recovery in patients with acute viral rhinosinusitis. reported antiviral effects from natural products, regardless of whether obtained from clinical trials or empiric knowledge, can only give clues for further research. however, it appears that we are entering a new golden age of natural product drug discovery. prevention of viral respiratory illness is attempted by either avoiding exposure or strengthening immune defenses, either nonspecifically with immunostimulators or specifically with vaccines. often, but not always, interventions are targeted toward high-risk groups for a particular infection (eg, rsv in infants and the elderly and ifv in patients with asthma). a variety of compounds (of microbial, herbal, or synthetic origin) have been used and are still being developed as nonspecific immunostimulatory agents to enhance or modulate the immune response against respiratory pathogens in a preventive or sometimes also therapeutic context. the effectiveness of these agents is usually moderate, and therefore they are only used as secondary supportive measures. as such, however, their potential should not be underestimated. among several agents based on bacterial components (om- bv, lw , pmbl, d , and ru ), om- bv, a lyophilisate of water-soluble fractions of bacteria commonly detected in patients with rtis, has been extensively studied, and a role in the prevention of both acute and recurrent rtis has been shown. , mechanistic studies have confirmed pleiotropic immunomodulating effects on both innate and adaptive immunity. , pidotimod, a synthetic dipeptide molecule, induces a variety of immunomodulatory effects , and has shown some efficacy in preventing rtis, although this was not always confirmed. , probiotic supplementation has been shown to reduce the incidence, duration, and severity of upper respiratory tract infections through immune modulation and in particular rhinovirus infection through altering nasal innate inflammatory responses. vitamin d ( -hydroxyvitamin d) has a modulatory role in host defense, inflammation, immunity, and epithelial repair after respiratory tract infections. a recent meta-analysis has confirmed that vitamin d supplementation reduces the overall risk of acute respiratory tract infections. data from in vitro rhinovirus-infected human primary bronchial epithelial cells showed that exogenous vitamin d can reduce rhinovirus replication through increasing interferon and cathelicidin gene expression. a significant amount of research is still dedicated to the efficacy of vitamin d supplementation, although not without controversy. hopefully, specific indications will be consolidated soon. despite widespread use and a multitude of studies, the role of vitamins c or zinc supplements is still inconclusive in relation to their action against the common cold. interestingly, meditation and exercise might significantly contribute to the reduction of rti burden, suggesting that the immunostimulatory capacity of nonpharmacologic measures should also be considered. the high transmission rate and epidemic nature of respiratory tract viruses indicate that effective public health measures to reduce transmission can have a substantial role in the overall prevention of these infections. a plethora of studies and metaanalyses delineated the important contribution of health policies in reducing transmission of epidemic respiratory tract viruses. in an elegant randomized control trial, an automated web-based intervention that maximized handwashing intention was associated with fewer episodes of influenza-like illness, shorter duration of symptoms, and fewer antibiotic prescriptions in the intervention group. although similar results regarding handwashing have been confirmed in a cochrane meta-analysis, hand hygiene interventions in educational settings were not as unequivocally effective. , low adherence to hand hygiene recommendations was correlated with higher incidence of ifv infection among health care workers during the pandemic. the use of face masks has been shown to be highly effective in the interruption of respiratory viral spread. this has been demonstrated further in a cluster randomized trial in which a reduced odds ratio of influenza infection secondary attack was observed in the intervention group. face masks are now regularly worn in some communities, especially in asia, but much less so in western societies. taken together, it seems that public health measures might provide a valuable ally in decreasing the burden of respiratory tract infections in the community. both vaccines and mabs (passive immunization) are relevant interventions. vaccines for ifv, rhinovirus, and rsv were initially developed as long ago as the s to s, although with mixed success, mostly because of rapid virus evolution. improved understanding of vaccine immunology and technologic developments place us now closer than ever to developing highly effective vaccines against the major respiratory tract viruses. mab therapies to viral infections, such as ebv (rituximab) or rsv (palivizumab), provide passive immunization and are licensed, whereas similar agents targeting influenza and other viruses are in preclinical development. neutralizing antibodies can bind and inactivate viruses, inhibit viral cell entry (blocking receptor binding or conformational changes), prevent the release of virions from the cell, or modulate immune effector functions. , engineering and production strategies to produce antibody fragments, higher-affinity binding, and longer half-life are contributing to a lower overall cost for therapy, although vaccines are still considered preferable in most cases. it is notable that effective neutralizing mab epitopes can also inform the rational design of vaccines. different types of vaccines to respiratory viruses exist, and these are shown in fig . traditionally, either live attenuated or inactivated viruses are used. more recently, subunit vaccines made of detergent-disrupted whole viruses or purified viral proteins are also common. furthermore, promising approaches use microparticle/nanoparticle material and recombinant technologies to produce broadly immunogenic, often self-adjuvanting, reproducible, and safe vaccine responses. these delivery systems include synthetic polymers, virosomes, virus-like particles (vlps), liposomes, lipid nanoparticles, proteins, emulsions, and immune-stimulating complexes. currently, naturally occurring particles are favored because of safety concerns, even though synthetic polymers, such as poly lactic-co-glycolic acid, are in use, and gold nanoparticles have shown promising results. self-assembling protein nanoparticles, such as ferritin cages and vaults, have also shown promising preclinical data. , layer-by-layer peptide-fabricated vaccine containing alternately charged poly-l-glutamic acid and poly-l-lysine layers with rsv peptides added have been efficacious in animals. a virosomal adjuvanted vaccine composed of reconstituted ifv envelope, effectively removing the core proteins and rna, has been available for years with excellent tolerability and efficacy. several vlp vaccines based on hepatitis b virus surface antigen have been approved for viral infections, such as human papilloma virus and other microbes (eg, malaria ), although an ifv candidate has not progressed. nevertheless these and other vlps offer promise because of their valency, similar immune presentation to pathogens, and antigenic preservation. adjuvants form a vital part of many vaccines; however, only aluminum hydroxide and oil in water emulsions are currently approved. a number of novel adjuvants, such as microcrystalline tyrosine, matrix m, pathogen-associated molecular patterns, and chitosan, are in development. [ ] [ ] [ ] [ ] dna and rna vaccines induce an immune response to the nucleic acid-encoded antigen. impressive results have been reported in animals for a single low-dose intradermal, nonreplicating dna vaccine for rsv; however, whether this will translate effectively to human subjects is not yet known. to enhance immunogenicity, rna vaccines have been encapsulated in nanoparticles, achieving sterilizing immunity for zika virus in mice, as well as being incorporated into virus-based self-replicating constructs known as replicons. , active ifv vaccination already forms the core of the global strategy against severe seasonal and pandemic influenza. trivalent and more recent quadrivalent vaccines are largely efficacious in healthy adults provided an adequate match between circulating and vaccine strains. higher-dose ( mg) and mf -adjuvanted vaccines are available for elderly patients. , similarly, pandemic vaccines can offer greater cross-clade protection because of the presence of improved (as or mf ) adjuvants. the current frontier of ifv vaccine development is ''universal'' vaccines (table i) . ideally, these would protect not only from circulating and pandemic strains but also from novel epitopes that might evolve in the future. many such vaccines are currently in preclinical and early clinical stages. heterosubtypic cross-reactive antibodies to ifv-a against the hemagglutinin (ha) stalk have been isolated from immune subjects, leading to mabs now in phase . , similar multilineage ha-stalk antibodies to ifv-b have also been reported. other conserved proteins have also been targeted, and an anti-m e antibody is in development. therefore passive immunization or postinfection treatment might soon become another tool to combat ifv. , , ha-stalk and chimeric head/stalk-based vaccines have also shown encouraging preclinical results. , [ ] [ ] [ ] a further vaccine strategy based on conserved epitopes in proteins, such as m , np, and pb , involves induction of cd and cd t-cell immunity, leading to development of a promising mva viral vector vaccine. other vaccines use multiepitope peptides to induce ifv-specific t-cell responses, reducing viral shedding in human subjects. , self-replicating rna nanoparticles also encoding multiple proteins and hepatitis b virus-based vlps expressing m e and ha epitopes also appear promising. there are currently no licensed vaccines and only mab (palivizumab) approved for the prevention of rsv infection. however, there are numerous candidates in clinical trials, as recently reviewed. suptavumab, an anti-f mab, has reached phase iii trials in preterm infants. medi offers -fold greater potency than palivizumab and has extended half-life in primates, suggesting a once per season dosing. candidate vaccines are based on live attenuated strains, subunit, vector, and nanoparticle technologies with a range of adjuvants. chimeric and combination vaccines using expression vectors in vlps show much promise. recent preclinical results exhibit effective neutralization of rsv. [ ] [ ] [ ] [ ] [ ] the most advanced of these is the novavax f-protein vlp nanoparticle vaccine with aluminum hydroxide adjuvant, which is in phase iii for maternal vaccination. , transplacental transmission of neutralizing antibodies has been demonstrated in preclinical studies, although this has not conferred significant protection from rsv. recombinant dna vaccines are also promising because of their apparent ability to induce a balanced t h /t h response, with a broad igg/iga profile mimicking live rsv challenge. intranasal and oral vaccine formulations are now in the early stages of clinical studies. fig . vaccine types. a, live attenuated vaccines are grown in culture to make them less virulent but can have the problem of reversion. b, inactivated vaccines are treated with uv or formaldehyde to crosslink proteins and make them nonviable. c, proteins can be purified, extracted, or dissolved by using detergents. d, naked nucleic acids are also used as vaccines. e, nanoparticle vaccines encompass natural and synthetic materials. membranes can be used to make liposomes to contain and deliver an antigen to a target cell. f, viruses can have nucleic acid and core protein removed to form virosomes. g, viral proteins, such as ha stalks or antigens, can be engineered onto immunogenic core proteins (eg, ferritin or vaults). this example is ha on ferritin adapted from pbd codes bve and c s. h, viruses, such as the vaccinia virus ankara, with coat proteins and genetic material removed can be engineered to express other antigens, such as influenza m ion channel protein. i, vlps can be engineered to express antigens and naturally glycosylated proteins and have adjuvants incorporated into the coat. pamp, pathogen-associated molecular pattern. j, synthetic nanoparticles made from polymers (polystyrene or poly lactic-co-glycolic acid), gold, or carbon nanotubes can have peptides adsorbed, admixed, or encapsulated. ag, antigen. initial vaccination attempts and more recent preclinical experiments show that inactivated rhinovirus vaccines are type specific and not cross-neutralizing. however, although in animals rhinovirus antibody responses might be weakly cross-neutralizing, data from human subjects suggest that responses are mainly misdirected to internal epitopes. understanding the full extent of rhinovirus diversity would probably be required to develop a panspecies vaccine. multiple strategies are being developed to reduce the burden of viral respiratory illnesses. it is likely that many of these strategies will find a relevant indication: antiviral strategies will most probably make sense in severe life-threatening situations or when a window of opportunity is clearly present, such as in specific virus seasons and susceptible populations. ideally, prevention at a wide scale through immunization will be able to reduce the overall burden of respiratory infections with a huge effect. this appears to be within reach for rsv and ifv, whereas additional effort is needed toward rhinovirus. in the meantime, symptomatic and immunostimulatory measures provide relief, and they hold promise in relation to postviral reactive airway disease. public health measures should be expanded because they can be critical in reducing the effect and contain potential epidemics. drivers of airborne human-to-human pathogen transmission global epidemiology of non-influenza rna respiratory viruses: data gaps and a growing need for surveillance lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics estimates of hospitalization attributable to influenza and rsv in the us during - , by age and risk status asthma research in europe: a transformative agenda for innovation and competitiveness influenza: exposing the true killer identification of new respiratory viruses in the new millennium a polyvalent inactivated rhinovirus vaccine is broadly immunogenic in rhesus macaques over-the-counter medications: update on cough and cold preparations limits and danger of ephedrine and pseudoephedrine as nasal decongestants comprehensive evidence-based review on european antitussives cyclooxygenase : its regulation, role and impact in airway inflammation delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h n virus the resolution code of acute inflammation: novel pro-resolving lipid mediators in resolution targeted prostaglandin e inhibition enhances antiviral immunity through induction of type i interferon and apoptosis in macrophages role of the lipoxygenase pathway in rsv-induced alternatively activated macrophages leading to resolution of lung pathology non-invasive ventilation improves respiratory distress in children with acute viral bronchiolitis: a systematic review high-flow warm humidified oxygen versus standard low-flow nasal cannula oxygen for moderate bronchiolitis (hfwho rct): an open, phase , randomised controlled trial pathogenesis of viral infection in exacerbations of airway disease viruses and bacteria in acute asthma exacerbations-a ga( ) len-dare systematic review novel strategies for targeting innate immune responses to influenza the effect of inhaled ifn-beta on worsening of asthma symptoms caused by viral infections. a randomized trial efficacy of ifn-lambda to protect human airway epithelial cells against human rhinovirus b infection il- a (ifn-lambda ) modulates lung dc function to promote th immune skewing and suppress allergic airway disease host dna released by netosis promotes rhinovirus-induced type- allergic asthma exacerbation efficacy of novel antibody-based drugs against rhinovirus infection: in vitro and in vivo results the tlr antagonist eritoran protects mice from lethal influenza infection the toll-like receptor antagonist eritoran protects mice from lethal filovirus challenge preseasonal treatment with either omalizumab or an inhaled corticosteroid boost to prevent fall asthma exacerbations omalizumab is associated with reduced acute severity of rhinovirus-triggered asthma exacerbation interferon at the crossroads of allergy and viral infections the cytokine storm of severe influenza and development of immunomodulatory therapy immunomodulatory therapy for severe influenza fda drug safety communication: fda eliminates the risk evaluation and mitigation strategy (rems) for rosiglitazone-containing diabetes medicines dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection azithromycin induces anti-viral responses in bronchial epithelial cells azithromycin induces anti-viral effects in cultured bronchial epithelial cells from copd patients early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: a randomized clinical trial identification of novel macrolides with antibacterial, anti-inflammatory and type i and iii ifn-augmenting activity in airway epithelium the anti-inflammatory effect of alpha- antitrypsin in rhinovirus-infected human airway epithelial cells current progress in antiviral strategies influenza neuraminidase inhibitors: synthetic approaches, derivatives and biological activity japanese surveillance systems and treatment for influenza detection and management of antiviral resistance for influenza viruses viral entry pathways: the example of common cold viruses efficacy of tremacamra, a soluble intercellular adhesion molecule , for experimental rhinovirus infection: a randomized clinical trial viral membrane fusion drugs to cure avian influenza infection-multiple ways to prevent cell death viral uncoating is directional: exit of the genomic rna in a common cold virus starts with the poly-(a) tail at the '-end picornavirus rna is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes selective inhibitors of picornavirus replication replication and inhibitors of enteroviruses and parechoviruses challenges and opportunities in developing respiratory syncytial virus therapeutics advances in antivirals for non-influenza respiratory virus infections. influenza other respir viruses als- for respiratory syncytial virus infection favipiravir (t- ), a novel viral rna polymerase inhibitor arbidol as a broad-spectrum antiviral: an update the promise, pitfalls and progress of rna-interference-based antiviral therapy for respiratory viruses gene knockdown in human rhinovirus b using '-ome-modified sirnas results in the reactivation of the interferon response traditional use of medicinal agents longevity extension by phytochemicals a review on antiviral activity of the himalayan medicinal plants traditionally used to treat bronchitis and related symptoms influenza neuraminidase: a druggable target for natural products neuraminidase inhibitors from reynoutria elliptica structure-activity relationship of flavonoids as influenza virus neuraminidase inhibitors and their in vitro anti-viral activities c-methylated flavonoids from cleistocalyx operculatus and their inhibitory effects on novel influenza a (h n ) neuraminidase anti-viral properties and mode of action of standardized echinacea purpurea extract against highly pathogenic avian influenza virus (h n , h n ) and swine-origin h n (s-oiv) in vitro inhibition of human influenza a virus infection by fruit-juice concentrate of japanese plum (prunus mume sieb. et zucc) pomegranate (punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir in vitro evaluation of secoiridoid glucosides from the fruits of ligustrum lucidum as antiviral agents echinacea for preventing and treating the common cold echinacea reduces the risk of recurrent respiratory tract infections and complications: a meta-analysis of randomized controlled trials herbal drug bno is safe and effective in the treatment of acute viral rhinosinusitis impact of a mixed bacterial lysate (om- bv) on the immunogenicity, safety and tolerability of inactivated influenza vaccine in children with recurrent respiratory tract infection clinical and immunological benefits of om- bacterial lysate in patients with allergic rhinitis, asthma, and copd and recurrent respiratory infections om- is an immunomodulator of interferon-beta production and inflammasome activity bacterial lysate increases the percentage of natural killer t cells in peripheral blood and alleviates asthma in children pidotimod: the state of art metabolomic profile of children with recurrent respiratory infections pidotimod for the prevention of acute respiratory infections in healthy children entering into daycare: a double blind randomized placebo-controlled study efficacy and safety of pidotimod in the prevention of recurrent respiratory infections in children: a multicentre study probiotics for preventing acute upper respiratory tract infections effect of probiotic on innate inflammatory response and viral shedding in experimental rhinovirus infection-a randomised controlled trial vitamin d modulation of innate immune responses to respiratory viral infections vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data vitamin d increases the antiviral activity of bronchial epithelial cells in vitro prevention and treatment of the common cold: making sense of the evidence meditation or exercise for preventing acute respiratory infection: a randomized controlled trial an internet-delivered handwashing intervention to modify influenza-like illness and respiratory infection transmission (primit): a primary care randomised trial physical interventions to interrupt or reduce the spread of respiratory viruses effectiveness of hand hygiene interventions in reducing illness absence among children in educational settings: a systematic review and meta-analysis can a school-based hand hygiene program reduce asthma exacerbations among elementary school children? risk factors for influenza among health care workers during pandemic the role of facemasks and hand hygiene in the prevention of influenza transmission in households: results from a cluster randomised trial monoclonal antibodies for prophylactic and therapeutic use against viral infections the growth and potential of human antiviral monoclonal antibody therapeutics converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play antibody recognition of a highly conserved influenza virus epitope harnessing nanoparticles for immunomodulation and vaccines assessing toxicity of fine and nanoparticles: comparing in vitro measurements to in vivo pulmonary toxicity profiles current advances in research and clinical applications of plga-based nanotechnology consensus m e peptide conjugated to gold nanoparticles confers protection against h n , h n and h n influenza a viruses hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h n antibodies layer-by-layer nanoparticle vaccines carrying the g protein cx c motif protect against rsv infection and disease eleven years of inflexal v-a virosomal adjuvanted influenza vaccine human papillomavirus and hpv vaccines: a review as malaria vaccine with or without a booster dose in infants and children in africa: final results of a phase , individually randomised, controlled trial m e-based universal influenza a vaccine virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development comparison of a novel microcrystalline tyrosine adjuvant with aluminium hydroxide for enhancing vaccination against seasonal influenza matrix m h n vaccine induces cross-h clade humoral immune responses in a randomized clinical trial and provides protection from highly pathogenic influenza challenge in ferrets safety and immunogenicity of a recombinant m e-flagellin influenza vaccine (stf . xm e) in healthy adults chitosan-a versatile semi-synthetic polymer in biomedical applications genetic immunization is a simple method for eliciting an immune response development of an intradermal dna vaccine delivery strategy to achieve single-dose immunity against respiratory syncytial virus modified mrna vaccines protect against zika virus infection dna and rna-based vaccines: principles, progress and prospects replicon rna viral vectors as vaccines transforming growth factor-beta promotes rhinovirus replication in bronchial epithelial cells by suppressing the innate immune response efficacy and safety of high-dose influenza vaccine in elderly adults: a systematic review and meta-analysis effectiveness of mf -adjuvanted seasonal influenza vaccine in the elderly: a systematic review and meta-analysis a systematic review and meta-analysis of cross-reactivity of antibodies induced by oil-in-water emulsion adjuvanted influenza h n virus monovalent vaccines preclinical pharmacokinetics of mhaa a, a human monoclonal antibody to influenza a virus, and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza a efficacy and safety of treatment with an anti-m e monoclonal antibody in experimental human influenza safety and upper respiratory pharmacokinetics of the hemagglutinin stalk-binding antibody vis support treatment and prophylaxis based on population modeling of seasonal influenza a outbreaks priming by a novel universal influenza vaccine (multimeric- )-a gateway for improving immune response in the elderly population a highly potent extended half-life antibody as a potential rsv vaccine surrogate for all infants safety and immunogenicity of a sf insect cell-derived respiratory syncytial virus fusion protein nanoparticle vaccine immunogenicity and safety of a respiratory syncytial virus fusion protein (rsv f) nanoparticle vaccine in older adults evaluation of the immunogenicity and safety of different doses and formulations of a broad spectrum influenza vaccine (flu-v) developed by seek: study protocol for a single-center, randomized, double-blind and placebo-controlled clinical phase iib trial caparr os-wanderley w. synthetic influenza vaccine (flu-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled phase i trial a synthetic influenza virus vaccine induces a cellular immune response that correlates with reduction in symptomatology and virus shedding in a randomized phase ib live-virus challenge in humans a t cell-inducing influenza vaccine for the elderly: safety and immunogenicity of mva-np m in adults aged over years a phase iia study to assess the safety and efficacy of a new influenza candidate vaccine mva-np m in healthy adults-flu clinical study report evaluating the immunogenicity and safety of a biondvax-developed universal influenza vaccine (multimeric- ) either as a standalone vaccine or as a primer to h n influenza vaccine back to the future: immunization with m- prior to trivalent influenza vaccine in / enhanced protective immune responses against / epidemic strain safety and immunogenicity of multimeric- -a novel universal influenza vaccine implication of respiratory syncytial virus (rsv) f transgene sequence heterogeneity observed in phase evaluation of medi- , a live attenuated parainfluenza type vectored rsv vaccine phase-i study medi- , of a live, attenuated intranasal vaccine against respiratory syncytial virus and parainfluenza- virus in seropositive children a randomized, blinded, controlled, dose-ranging study of a respiratory syncytial virus recombinant fusion (f) nanoparticle vaccine in healthy women of childbearing age efficacy of motavizumab for the prevention of respiratory syncytial virus disease in healthy native american infants: a phase randomised double-blind placebo-controlled trial motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial trivalency of a nanobody specific for the human respiratory syncytial virus fusion glycoprotein drastically enhances virus neutralization and impacts escape mutant selection efficacy, safety, and pharmacokinetics of a new % liquid intravenous immunoglobulin containing high titer neutralizing antibody to rsv and other respiratory viruses in subjects with primary immunodeficiency disease treatment with novel rsv ig ri- controls viral replication and reduces pulmonary damage in immunocompromised sigmodon hispidus structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses broadly cross-reactive antibodies dominate the human b cell response against pandemic h n influenza virus infection highly conserved protective epitopes on influenza b viruses human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza a viruses tackling influenza with broadly neutralizing antibodies influenza virus vaccine based on the conserved hemagglutinin stalk domain chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies chimeric hemagglutinin constructs induce broad protection against influenza b virus challenge in the mouse model epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza a virus influenza vaccine research funded by the european commission fp -health- -innovation- project antibodies to watch in protective efficacy and immunogenicity of an adenoviral vector vaccine encoding the codon-optimized f protein of respiratory syncytial virus immunogenicity of rsv f dna vaccine in balb/c mice rsv fusion (f) protein dna vaccine provides partial protection against viral infection chimeric virus-like particles containing a conserved region of the g protein in combination with a single peptide of the m protein confer protection against respiratory syncytial virus infection baculovirus-expressed virus-like particle vaccine in combination with dna encoding the fusion protein confers protection against respiratory syncytial virus co-immunization with virus-like particle and dna vaccines induces protection against respiratory syncytial virus infection and bronchiolitis respiratory syncytial virus: infection, detection, and new options for prevention and treatment immunogenicity and efficacy of codon optimized dna vaccines encoding the f-protein of respiratory syncytial virus mucosal vaccines against respiratory syncytial virus prevention of colds by vaccination against a rhinovirus: a report by the scientific committee on common cold vaccines challenges in developing a cross-serotype rhinovirus vaccine misdirected antibody responses against an n-terminal epitope on human rhinovirus vp as explanation for recurrent rv infections key: cord- -m too ob authors: guzmán, carlos alberto title: next generation influenza vaccines: looking into the crystal ball date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: m too ob influenza infections are responsible for significant number of deaths and overwhelming costs worldwide every year. vaccination represents the only cost-efficient alternative to address this major problem in human health. however, current vaccines are fraught by many limitations, being far from optimal. among them, the need to upgrade vaccines every year through a time-consuming process open to different caveats, and the critical fact that they exhibit poorer efficacy in individuals who are at high risk for severe infections. where are we? how can knowledge and technologies contribute towards removing current roadblocks? what does the future offer in terms of next generation vaccines? seasons. on the other hand, they presented the pros and cons of protein-based vaccines produced using a baculovirus-based approach, which eliminates the constraints resulting from relying on influenza virus replication. nevertheless, practical considerations, like manufacturing capacity to serve the huge demand for influenza vaccines and considerably higher production costs still represent a bottleneck. in their review article, the authors also presented a comprehensive summary of the current efforts to improve the influenza vaccines produced using standard existing technologies. in this regard, the use of adjuvants to improve the overall efficacy of vaccines for specific subpopulation groups is discussed, as well as the exploitation of nanotechnologies to increase the immunogenicity of protein-based vaccines. an elegant approach enables the engineering of recombinant viruses for vaccine production, which co-expresses the vaccine relevant hemagglutinin together with a helper hemagglutinin [ ] . this strategy prevents the emergence of egg-adaptive mutations and reduces the overall production times. they also address the use of peptide-based vaccines as a potential alternative. although promising, the presented drawbacks suggest that considerable time and effort will be required to make it a viable approach for implementation in the influenza vaccine field. the review by clemens et al. further elaborates on the potential and limitations of harnessing t cells for the development of influenza vaccines [ ] . in this particular context, animal studies support the potential of exploiting this strategy to generate universal or at least broader next generation vaccines providing heterosubtypic protection by stimulating long-lasting t cell resident memory cells. nevertheless, this approach faces several major constraints. as described in the review, a universal vaccine based on peptides should mimic more closely the immune responses observed after natural infections by inducing both cross-reactive peripheral and tissue resident cd t cells in addition to cross-reactive antibodies and cd t follicular and helper cells. however, as described in the review, special vaccination strategies will be needed to promote the seeding of tissue resident memory t cells in the respiratory tract and prevent their attrition. furthermore, universal protection will require addressing human leucocyte antigen (hla)-restriction by the inclusion of appropriate peptides. such vaccines should not only stimulate multiple cross-reactive epitope-specific t cells, but also encompass epitopes recognized by rare hla types from minor ethnicities. finally, current influenza vaccines are approved dependent on the accepted correlates for protection based on the stimulation of functional antibodies recognizing the hemagglutinin. large-scale clinical studies will be needed to define similar robust correlates for a conceptually novel class of t cell influenza vaccines. new disruptive technologies, such as the use of nucleic acid vaccines (e.g., dna or rna), which render unnecessary the production of proteins, can be also exploited to develop next generation influenza vaccines. in this regard, rna vaccines represent a cutting-edge approach, which dramatically reduces production times, ensure the fidelity of the encoded sequences, and are insulated from shortages of eggs or the constraints attached to cell factories. this renders this technology particularly appealing for potential emerging influenza pandemics. preclinical studies in different animal species and first-in-man studies also demonstrated the intrinsic value of this approach [ , ] . however, these studies, as well as some of the trials carried out in the context of the covid- pandemic [ , ] showed that the higher reactogenicity of rna vaccines respect to what observed for influenza vaccines developed using conventional standard technologies might be an issue for the global acceptance of an rna-based prophylactic vaccine. in the review by scorza and pardi, the authors summarize the current state of the art in terms of preclinical and clinical studies, as well as the major roadblocks that need to be overcome for implementation [ ] . in this context, they describe the strategies exploited to increase the half-life, stability, cellular uptake and translatability of both self-amplifying and non-replicating mrna. a distinctive strength of this class of vaccines is their rapid scalable egg-independent production. of particular interest is the analysis presented in this review benchmarking the strengths and limitations of this class of vaccines in terms of the world health organization guidelines for the preferred product characteristics of next generation influenza vaccines [ ] . the availability of innovative technologies will certainly positively affect the development of next generation influenza vaccines. however, influenza vaccines based on both traditional and emerging technologies face the major constraint represented by the immune history of the individual vaccinees. the review article from lewnard and cobey analyze the specific constraints and provide examples on how the dynamics of immune memory can affect vaccine effectiveness [ ] . it is critical to understand the underlying mechanisms driving these processes, which go far beyond the potential impact of the original antigenic sin during immune imprinting. this will allow dissecting differential patterns of vaccine effectiveness in vaccinees depending on immune imprinting and memory. immediately after birth, human beings lose their naivety, and previous infection and vaccination events condition responsiveness, for good or bad, from season to season, according to age and the antigen contact history. the rational design of next generation influenza vaccines able to confer protection in all age and vulnerable groups is an ambitious and challenging venture. different approaches can be exploited to stimulate the production of broadly protective vaccine responses [ ] . among them, the more conserved structures of the virus are used as an antigenic target, such as the stalk region of the hemagglutinin, or the matrix protein , which is highly conserved between influenza subtypes. an alternative approach relies on the exploitation of in silico tools to develop consensus sequences for computationally optimized broadly reactive hemagglutinin antigen variants (cobra), which are able to stimulate broadly reactive antibodies [ ] . the use of some of these approaches might require the development of alternative tools to assess vaccine efficacy. current surrogated correlates of protection, which are accepted for the standard influenza vaccines, may not be relevant for next generation vaccines. in fact, the stimulated clearance mechanism might go far beyond traditional neutralization by blocking the virus binding to the receptor (e.g., the inhibition of viral membrane fusion and maturation, antibody-dependent and -independent cytotoxicity, the inhibition of viral replication or spreading). interestingly, the adjuvantation of conventional influenza vaccines has also proven effective, not only for improving responsiveness in certain subpopulation groups [ ] , but also at broadening responsiveness to other influenza subtypes [ , ] . a critical aspect for the development of a vaccine conferring broad cross-protective immunity against influenza is the determination of vaccine efficacy. the clinical trials required to achieve this goal may prove a roadblock. however, the availability of controlled human infection models for the influenza virus is an effective alternative to address this issue [ ] . whether these approaches can lead to a true universal influenza vaccine or to next generation vaccines with a broader efficacy profile, rendering possible to boost against seasonal influenza every - years instead of every year, or to be prepared for a broad range of strains with a potential to cause pandemics, remains to be elucidate. however, the crystallization of any of these two scenarios will represent a significant improvement with respect to the current situation. it is important to highlight that regardless of the fact that upcoming next generation vaccines are formulated with well known standard antigens or innovative components promoting cross-protective immunity, or that they will be based on well established or emerging technology platforms for vaccine formulation or delivery, the key problem in vaccine-mediated immunity against influenza will remain. namely, key influenza-specific issues of interference due to pre-existent immunity and overall poor responsiveness to vaccines in vulnerable individuals (e.g., the elderly, the very young, those affected by co-morbidities) often result in unpredictable poor responsiveness to vaccination. therefore, the problem of poor protection against influenza infection post vaccination is, by and large, defined by a knowledge gap in key aspects of human immunology, such as the underlying mechanisms involved in poor individual responsiveness, and how to tailor vaccines according to the needs of subpopulation groups to generate broadly protective and long-lasting immunity. this current knowledge gap can be addressed by performing comprehensive and standardized profiling at baseline and post immunization of responders and non-responders to influenza vaccination in different age groups, both in healthy individuals as well as in those affected by co-morbidities known to affect responses to vaccination. the in-depth analysis and comparison of the obtained immunological and molecular signatures using artificial intelligence tools will enable the identification of the putative underlying mechanisms. these mechanisms will provide intervention targets to develop more efficient tailored vaccination strategies, as well as potential predictive biomarkers of responsiveness to develop innovative diagnostics for the stratification and follow-up of vaccinees. funding: this work was supported in part by grants from the eu (transvac - ; incentive- ). the author declares no conflict of interest. global seasonal influenza-associated mortality collaborator network. estimates of global seasonal influenza-associated respiratory mortality: a modelling study drugs for influenza treatment: is there significant news? front factors that influence the immune response to vaccination immunogenicity and safety of an adjuvanted influenza vaccine in patients with decompensated cirrhosis humoral immune response to influenza vaccination in patients from high risk groups efforts to improve the seasonal influenza vaccine the global production capacity of seasonal and pandemic influenza vaccine molecular changes associated with adaptation of human influenza a virus in embryonated chicken eggs the characteristics and antigenic properties of recently emerged subclade c. a and c. a human influenza a (h n ) viruses passaged in mdck cells. influenza other respir rationally designed influenza virus vaccines that are antigenically stable during growth in eggs harnessing the power of t cells: the promising hope for a universal influenza vaccine protective efficacy of in vitro synthesized, specific mrna vaccines against influenza a virus infection preclinical and clinical demonstration of immunogenicity by mrna vaccines against h n and h n influenza viruses phase / study to describe the safety and immunogenicity of a covid- rna vaccine candidate (bnt b ) in adults to years of age: interim report an mrna vaccine against sars-cov- -preliminary report new kids on the block: rna-based influenza virus vaccines who preferred product characteristics for next-generation influenza vaccines; world health organization immune history and influenza vaccine effectiveness roads to advanced vaccines: influenza case study a computationally optimized broadly reactive antigen (cobra) based h n vlp vaccine elicits broadly reactive antibodies in mice and ferrets effectiveness of mf -adjuvanted seasonal influenza vaccine in the elderly: a systematic review and meta-analysis seeking help: b cells adapting to flu variability mucosal administration of cycle-di-nucleotide-adjuvanted virosomes efficiently induces protection against influenza h n in mice the human viral challenge model: accelerating the evaluation of respiratory antivirals, vaccines and novel diagnostics vaccines , , key: cord- -p oomzn authors: moss, william j.; griffin, diane e.; feinstone, w. harry title: measles date: - - journal: vaccines for biodefense and emerging and neglected diseases doi: . /b - - - - . - sha: doc_id: cord_uid: p oomzn abstract measles is a highly contagious disease characterized by a prodromal illness of fever, cough, coryza, and conjunctivitis followed by the appearance of a generalized maculopapular rash. measles virus is a nonsegmented, single-stranded, negative-sense rna virus and a member of the morbillivirus genus in the family of paramyxoviridae. although rna viruses have high mutation rates, measles virus is an antigenically monotypic virus and the surface proteins responsible for inducing protective immunity have retained their antigenic structure. the public health significance is that measles vaccines developed decades ago from a single measles virus strain remain protective worldwide. prior to the development and widespread use of measles vaccine, million cases of measles were estimated to occur each year, resulting in more than million deaths. several live, attenuated measles vaccines are available, either as single-antigen vaccines or in combination with rubella and mumps vaccines (mr and mmr vaccines). most of the currently used measles vaccines were derived from the edmonston strain of measles virus that was isolated by enders and peebles in . measles vaccines are recommended for all susceptible children and adults for whom the vaccine is not contraindicated. despite progress in reducing measles mortality, measles remains a major cause of vaccine-preventable death and an important cause of morbidity and mortality in children, particularly sub-saharan africa and in asia. the ideal measles vaccine would be inexpensive, safe, heat-stable, immunogenic in neonates or very young infants, and administered as a single dose without needle or syringe. a number of vaccine candidates with some of these characteristics are undergoing preclinical studies, including dna vaccines and various viral and bacterial vectored vaccines. the high infectivity of measles virus is a characteristic suitable to a biothreat agent. however, increasingly high levels of measles vaccination coverage throughout the world as part of accelerated measles control efforts would protect many from the deliberate use of measles virus as a biothreat agent. genetic engineering of a measles virus strain that was not neutralized by antibodies induced by the current attenuated measles vaccines would likely have reduced infectivity, as suggested by the fact that wild-type measles viruses have not mutated to alter their neutralizing epitopes. measles virus meets many of the biological criteria for disease eradication. measles virus has no nonhuman reservoir, can be accurately diagnosed, and measles vaccination is a highly effective intervention. where measles virus differs from smallpox and polio viruses is that it is more highly infectious, necessitating higher levels of population immunity to interrupt transmission. it remains unclear whether the threat from bioterrorism precludes stopping measles vaccination after eradication, but provision of a second opportunity for measles vaccination likely could be stopped following eradication. measles is a highly contagious disease characterized by a prodromal illness of fever, cough, coryza, and conjunctivitis followed by the appearance of a generalized maculopapular rash. deaths from measles are due largely to an increased susceptibility to secondary bacterial and viral infections, attributed to a prolonged state of immune suppression. despite the development of an effective attenuated vaccine, measles remains a leading measles vaccines are recommended for all susceptible children and adults for whom the vaccine is not contraindicated. despite progress in reducing measles mortality, measles remains a major cause of vaccine-preventable death and an important cause of morbidity and mortality in children, particularly sub-saharan africa and in asia. the ideal measles vaccine would be inexpensive, safe, heat-stable, immunogenic in neonates or very young infants, and administered as a single dose without needle or syringe. a number of vaccine candidates with some of these characteristics are undergoing preclinical studies, including dna vaccines and various viral and bacterial vectored vaccines. the high infectivity of measles virus is a characteristic suitable to a biothreat agent. however, increasingly high levels of measles vaccination coverage throughout the world as part of accelerated measles control efforts would protect many from the deliberate use of measles virus as a biothreat agent. genetic engineering of a measles virus strain that was not neutralized by antibodies induced by the current attenuated measles vaccines would likely have reduced infectivity, as suggested by the fact that wild-type measles viruses have not mutated to alter their neutralizing epitopes. measles virus meets many of the biological criteria for disease eradication. measles virus has no nonhuman reservoir, can be accurately diagnosed, and measles vaccination is a highly effective intervention. where measles virus differs from smallpox and polio viruses is that it is more highly infectious, necessitating higher levels of population immunity to interrupt transmission. it remains unclear whether the threat from bioterrorism precludes stopping measles vaccination after eradication, but provision of a second opportunity for measles vaccination likely could be stopped following eradication. iii. viral vaccines figure . countries reporting measles cases (indigenous or imported) in [ ] [ ] since its emergence thousands of years ago. measles virus most closely resembles rinderpest virus, a pathogen of cattle, and likely evolved as a zoonotic infection in communities where cattle and humans lived in close proximity. measles virus is believed to have become established in human populations about - , years ago when human populations achieved sufficient size in middle eastern river valley civilizations to maintain virus transmission. abu becr, an arab physician also known as rhazes, is generally credited with distinguishing smallpox from measles in the th century. he dated the first description of measles to the th century. however, epidemics identified as measles were not recorded until the th and th centuries, and measles was first mentioned as a childhood disease in . the name " morbilli " was derived from the italian meaning " little diseases " to distinguish it from plague, " il morbo. " sanvages in defined morbilli as measles, but called it rubeola, leading to confusion with rubella. introduction of measles into previously unexposed populations has been associated with high mortality. one quarter of the population on the fiji islands died after the introduction of measles virus in . millions died as a result of european exploration of the new world, largely due to the introduction of diseases such as smallpox and measles into native amerindian populations. the high mortality from these diseases facilitated european conquest of the americas ( mcneill, ) . many of the basic principles of measles epidemiology and infection were elucidated by peter panum, a danish physician who was sent to the faroe islands in during a large measles epidemic ( panum, ) . panum deduced the highly contagious nature of the disease, the -day incubation period, the lifelong immunity following infection, and postulated a respiratory route of transmission. measles virus first was isolated from the blood of david edmonston and others by enders and peebles ( ) . the development of vaccines against measles soon followed. measles virus is a spherical, nonsegmented, singlestranded, negative-sense rna virus and a member of the morbillivirus genus in the family of paramyxoviridae. other members of the morbillivirus genus are rinderpest virus and canine distemper virus. although rna viruses have high mutation rates, measles virus is an antigenically monotypic virus, meaning that the surface proteins responsible for inducing protective immunity have retained their antigenic structure. the public health significance is that measles vaccines developed decades ago from a single measles virus strain remain protective worldwide. measles virus is killed by ultraviolet light and heat. attenuated measles vaccine viruses retain these characteristics, necessitating a cold chain for transportation and storage. the measles virus rna genome consists of approximately , nucleotides and is enclosed in a lipidcontaining envelope derived from the host cell. the genome encodes eight proteins, two of which (v and c) are nonstructural proteins and are transcribed from the phosphoprotein (p) gene. of the six structural proteins, p, large protein (l), and nucleoprotein (n) form the nucleocapsid housing the viral rna. the hemagglutinin protein (h), fusion protein (f), and matrix protein (m), together with lipids from the host cell membrane, form the viral envelope. the h protein interacts with f to mediate fusion of the viral envelope with the host cell membrane ( malvoisin and wild, ) . the primary function of the h protein is to bind to the host cellular receptors for measles virus. the two identified receptors are cd and cd (slam). cd is a complement regulatory molecule expressed on all nucleated cells in humans. slam, an acronym for signaling lymphocyte activation molecule, is expressed on activated t and b lymphocytes and antigen-presenting cells. the binding sites on h for these receptors overlap and strains of measles virus differ in the efficiency with which each is used. wild-type measles virus binds to cells primarily through the cellular receptor slam whereas most vaccine strains bind to cd ; however, most measles virus strains can use both cd and slam as receptors during acute infection ( schneider et al., ) . additional, as yet unidentified receptors for measles virus exist on human endothelial and epithelial cells ( andres et al., ) . other measles virus proteins are involved in viral replication. the p protein regulates transcription, replication, and the efficiency with which the n assembles into nucleocapsids ( spehner et al., ) . the m protein links ribonucleoproteins with envelope proteins during virion assembly. the functions of v and c proteins have not been clearly defined, but both appear to contribute to the virulence of measles virus by regulating transcription and sensitivity to the antiviral effects of interferon (ifn) α / β ( valsamakis et al., ; patterson et al., ) . variability within the genome is sufficient to allow for molecular epidemiologic investigation. genetic characterization of wild-type measles viruses is based on sequence analysis of the genes encoding the measles virus fatal disease ( good and zak, ) . monkeys depleted of cd ϩ t-lymphocytes and challenged with wildtype measles virus had a more extensive rash, higher measles virus loads, and longer duration of viremia than control animals, further confirming the importance of cellular immunity in measles virus clearance ( permar et al., ) . cd ϩ t-lymphocytes also are activated in response to measles virus infection and secrete cytokines capable of modulating the humoral and cellular immune responses. plasma cytokine profiles show increased levels of ifn-γ during the acute phase, followed by a shift to high levels of interleukin (il)- and il- during convalescence ( moss et al., ) . the initial predominant th response (characterized by ifn-γ ) is presumed to be essential for viral clearance, while the later th response (characterized by il- ) promotes the development of measles virusspecific antibodies. the duration of protective immunity following wild-type measles virus infection is generally thought to be lifelong. the immunologic mechanisms involved in sustaining high levels of neutralizing antibody to measles virus are not completely understood, although general principles of immunologic memory probably govern this process. immunologic memory to measles virus includes both continued production of measles virus-specific igg antibodies and the circulation of measles virus-specific cd ϩ and cd ϩ t-lymphocytes ( ovsyannikova et al., ) . although immune protection is assessed by measurement of antimeasles virus antibodies, long lasting cellular immune responses almost certainly play an important role in protection from infection and disease. young infants in the first months of life are protected against measles by maternally acquired igg antibodies. an active transport mechanism in the placenta is responsible for the transfer of igg antibodies from the maternal circulation to the fetus starting at about weeks gestation and continuing until birth ( crowe, ) . three factors determine the degree and duration of protection in the newborn: ( ) the level of maternal antimeasles antibodies; ( ) the efficiency of placental transfer; and ( ) the rate of catabolism in the child. although providing passive immunity to young infants, maternally acquired antibodies can interfere with the immune responses to the attenuated measles vaccine by inhibiting replication of vaccine virus. in general, maternally acquired antibodies are no longer present in the majority of children by months of age, the time of routine measles vaccination in many countries. women with vaccine-induced immunity tend to have lower antimeasles virus antibody titers than women with naturally acquired immunity, and their children may be susceptible to measles at an earlier age. n and h proteins. one of the most variable regions of the measles virus genome is the -nucleotide sequence at the carboxy-terminal of the n protein, with up to % variability between wild-type viruses. the world health organization (who) recognizes clades of measles virus (designated a through h) and genotypes ( world health organization, ) . new genotypes likely will be identified with enhanced surveillance and molecular characterization. as measles control efforts intensify, molecular surveillance of circulating measles virus strains can be used to document interruption of measles virus transmission and to identify the source and transmission pathways of measles virus outbreaks ( rota and bellini, ) . molecular epidemiologic tools also would be important in documenting deliberate bioterrorist introductions of wild-type or genetically modified measles virus strains. host immune responses to measles virus are essential for viral clearance, clinical recovery, and the establishment of long-term immunity. the early nonspecific (innate) immune responses that occur during the prodromal phase of the illness include activation of natural killer (nk) cells and production of ifn-α and β . these innate immune responses contribute to the control of measles virus replication before the onset of more specific adaptive immune responses. the protective efficacy of antibodies to measles virus is illustrated by the immunity conferred to infants from passively acquired maternal antibodies and the protection of exposed, susceptible individuals following administration of antimeasles virus immune globulin ( black and yannet, ) . the first measles virus-specific antibodies produced after infection are of the igm subtype, followed by a switch to predominantly igg and then to igg and igg isotypes ( isa et al., ) . iga antibodies to measles virus are found in mucosal secretions. the most abundant and rapidly produced antibodies are against n, and the absence of antibodies to n is the best indicator of seronegativity to measles virus. antibodies to h and f proteins contribute to virus neutralization and are sufficient to provide protection against measles virus infection. evidence for the importance of cellular immunity to measles virus is demonstrated by the ability of children with agammaglobulinemia to fully recover from measles, whereas children with severe defects in t-lymphocyte function often develop severe or epidemiology prior to the development and widespread use of measles vaccine, million cases of measles were estimated to occur each year, resulting in more than million deaths. despite progress in reducing measles mortality, measles remains the most frequent cause of vaccinepreventable death and an important cause of morbidity and mortality in children, particularly in sub-saharan africa and in asia ( henao-restrepo et al., ) . the disease burden due to measles decreased over the past several decades due to a number of factors. measles mortality declined in developed countries in association with economic development, improved nutritional status and supportive care, particularly antibiotic therapy for secondary bacterial pneumonia. remarkable progress in reducing measles incidence and mortality has been, and continues to be, made in resource-poor countries as a consequence of increasing measles vaccine coverage, provision of a second opportunity for measles vaccination through supplementary immunization activities, and efforts by the who, the united nations children's fund (unicef) and their partners to target countries for accelerated and sustained measles mortality reduction. specifically, this targeted strategy aims to achieve Ͼ % measles vaccination coverage in every district of the countries and to ensure that all children receive a second opportunity for measles immunization ( world health organization, ) . provision of vitamin a through polio and measles vaccination campaigns has contributed further to the reduction in measles mortality ( world health organization, ) . in , the world health assembly endorsed a resolution urging member countries to reduce the number of deaths attributed to measles by % by the end of compared with estimates. overall global measles mortality in was estimated to be , deaths (uncertainty bounds , and , deaths) ( wolfson et al., ) . this estimate represents a % decrease from , when the global number of measles deaths was estimated to be , (uncertainty bounds , and , , deaths). the largest decrease in measles mortality was in africa, contributing % of the global reduction in measles mortality. measles incidence has a typical temporal pattern characterized by annual, seasonal epidemics superimposed upon longer epidemic cycles of - years or more. in temperate climates, annual measles outbreaks typically occur in the late winter and early spring. these annual outbreaks are likely the result of social networks facilitating transmission (e.g., congregation of children at school) and environmental factors favoring the viability and transmission of measles virus ( fine and clarkson, ) . measles cases continue to occur during the interepidemic period in densely populated communities but at low incidence. the longer cycles occurring every several years result from the accumulation of susceptible persons over successive birth cohorts and the subsequent decline in the number of susceptibles following an outbreak. in the absence of a vaccination program these longer epidemic cycles tend to occur every - years. measles vaccination programs that achieve coverage rates in excess of % extend the interepidemic period to - years by reducing the number of susceptible individuals. humans are the only reservoir for measles virus, a characteristic important for the potential eradication of measles. nonhuman primates may be infected with measles virus and develop an illness similar to measles in humans, with rash, coryza, and conjunctivitis. however, populations of wild monkeys are not of sufficient size to maintain measles virus transmission. measles virus is transmitted primarily by respiratory droplets small enough to traverse several feet but too large to remain suspended in the air for long periods of time. the symptoms induced during the prodrome, particularly sneezing and coughing, enhance transmission. measles virus also may be transmitted by the airborne route, suspended on small particles for a prolonged time. direct contact with infected secretions can transmit measles virus, but the virus does not survive long on fomites as it is quickly killed by heat and ultraviolet radiation. the average incubation period for measles, the time from infection to clinical disease, is approximately days to the onset of fever and days to the onset of rash (range - days). the incubation period may be shorter in infants and following a large inoculum of virus, and longer in adults. during this seemingly quiescent period, the virus is rapidly replicating and infecting target tissues. measles virus is one of the most highly contagious infectious agents and outbreaks can occur in populations in which less than % of persons are susceptible. chains of transmission commonly occur among household contacts, school children, and health care workers. generally, persons with measles are infectious for several days before and after the onset of rash, when titers of measles virus in the blood and body fluids are highest. as with many other acute viral infections (sars-coronavirus being an exception), the fact that measles virus is contagious prior to the onset of recognizable disease hinders the effectiveness of quarantine measures. measles virus can be isolated in tissue culture from the urine as late as week after rash onset. detection of measles virus in body epidemiology maculopapular rash appears first on the face and behind the ears, and then spreads in a centrifugal fashion to the trunk and extremities. the rash lasts for - days and fades in the same manner as it appeared. in uncomplicated measles, clinical recovery begins soon after appearance of the rash. complications occur in up to % of measles cases, and the risk of complication is increased by extremes of age, malnutrition, and vitamin a deficiency ( morley, ) . complications of measles have been described in almost every organ system. the respiratory tract is a frequent site of complication, with pneumonia accounting for most measles-associated deaths ( duke and mgone, ) . pneumonia is caused by secondary viral or bacterial infections, or by measles virus itself. pathologically, measles virus infection of the lung is characterized by multinucleated giant cells, formed when measles virus proteins on the cell surface allow cells to fuse. other respiratory complications include laryngotracheobronchitis and otitis media. mouth ulcers, or stomatitis, may hinder children from eating or drinking. many children with measles develop diarrhea, which further contributes to malnutrition. keratoconjunctivitis is common after measles, particularly in children with vitamin a deficiency, and was a frequent cause of blindness. because the rash of measles is a consequence of the cellular immune response, persons with impaired cellular immunity, such as those with the acquired immunodeficiency syndrome (aids), may not develop the characteristic measles rash. these persons have a high case fatality and may develop a giant cell pneumonitis due to measles virus ( moss et al., ) . t-lymphocyte defects due to causes other than hiv- infection, such as cancer chemotherapy, are also associated with increased severity of measles. rare but serious complications of measles involve the central nervous system. postmeasles encephalomyelitis complicates approximately in cases, mainly older children and adults. encephalomyelitis occurs within weeks of the onset of rash and is characterized by fever, seizures, and a variety of neurological abnormalities. the finding of periventricular demyelination, the induction of immune responses to myelin basic protein, and the absence of measles virus in the brain suggest that postmeasles encephalomyelitis is an autoimmune disorder triggered by measles virus infection. other central nervous system complications that occur months to years after acute infection are measles inclusion body encephalitis (mibe) and subacute sclerosing panencephalitis (sspe). in contrast to postmeasles encephalomyelitis, mibe and sspe are caused by persistent measles virus infection. mibe is a rare but fatal complication that affects fluids by a variety of means, including identification of multinucleated giant cells in nasal secretions or the use of reverse transcriptase-polymerase chain reaction (rt-pcr), suggests the potential for a prolonged infectious period in persons immunocompromised by severe malnutrition or human immunodeficiency virus type (hiv- ) infection ( dossetor et al., ; permar et al., ; riddell et al., ) . however, whether detection of measles virus by these methods indicates prolonged contagiousness is unclear. in densely populated urban settings with low vaccination coverage rates, measles is a disease of young children. the cumulative distribution can reach % by year of age, with a significant proportion of children acquiring measles virus infection before months, the age of routine vaccination. as measles vaccine coverage increases, or population density decreases, the age distribution shifts toward older children. in such situations, measles cases predominate in school-age children. infants and younger children, although susceptible if not protected by immunization, are not exposed to measles virus at a rate sufficient to cause a large disease burden in this age group. as vaccination coverage increases further, the age distribution of cases may be shifted into adolescence and young adulthood, as seen in measles outbreaks in the united states, brazil, and australia ( hutchins et al., ; de quadros et al., ; lambert et al., ) , necessitating targeted measles vaccination programs for these older age groups. the high infectivity of measles virus is a characteristic suitable to a biothreat agent. however, increasingly high levels of measles vaccination coverage throughout the world as part of accelerated measles control efforts would protect many from the deliberate use of measles virus as a biothreat agent. genetic engineering of a measles virus strain that was not neutralized by antibodies induced by the current attenuated measles vaccines would likely have reduced infectivity, as suggested by the fact that wild-type measles viruses have not mutated to alter their neutralizing epitopes. clinically apparent measles begins with a prodrome characterized by fever, cough, coryza, and conjunctivitis. koplik's spots, small white lesions on the buccal mucosa inside the mouth, may be visible during the prodrome and allow the astute clinician to diagnose measles prior to the onset of rash. the prodromal symptoms intensify several days before the onset of rash. the characteristic erythematous and individuals with defective cellular immunity and typically occurs months after infection. sspe is a slowly progressive disease characterized by seizures, progressive deterioration of cognitive and motor functions followed by death that occurs - years after measles virus infection, most often in persons infected with measles virus before years of age. there are conflicting and inconclusive data suggesting that measles virus infection causes or contributes to the development of chronic diseases, including multiple sclerosis, paget's disease, inflammatory bowel disease, and otosclerosis ( perry and halsey, ) . however, no causal association has been established between measles and these conditions. the characteristic clinical features of measles are of sufficient sensitivity and specificity to have high predictive value in regions where measles is endemic. however, laboratory diagnosis is necessary where measles virus transmission rates are low or in immunocompromised persons who may not have the characteristic clinical manifestations. infection with rubella, parvovirus b , human herpes virus , and dengue viruses may mimic measles. detection of igm antibodies to measles virus by enzyme immunoassay (eia) is the standard method of diagnosing acute measles ( bellini and helfand, ) . alternatively, seroconversion using igg-specific eia, hemagglutinin inhibition, complement fixation, or virus neutralization assays can be used to diagnose acute measles based on testing serum or plasma obtained during the acute and convalescent phases. measles virus can be isolated in tissue culture from white blood cells, respiratory tract secretions, and urine, although the ability to isolate measles virus diminishes quickly after rash onset. amplification and detection of measles virus rna by rt-pcr from blood, urine, and nasal discharge is highly sensitive in detecting measles virus rna and allows sequencing of the measles virus genome for molecular epidemiologic studies. no specific antiviral drug is used routinely to treat measles virus infection, although the broad antiviral agent ribavirin has been used to treat immunocompromised persons or persons with sspe, alone or in combination with ifn-α or intravenous immunoglobulin ( forni et al., ; solomon et al., ) . the major components of case management include provision of vitamin a, prompt treatment of secondary bacterial infections, and nutritional support. several placebo-controlled trials have demonstrated marked reductions in morbidity and mortality in hospitalized children with measles treated with vitamin a. the who recommends administration of two daily doses of , iu of vitamin a to all children with measles months of age or older. lower doses ( , iu) are recommended for children less than months of age. overall, this regimen resulted in a % reduction in the risk of mortality (rr ϭ . , % ci . - . ) ( d'souza and d'souza, ) . pneumonia-specific mortality is reduced, and the impact is greatest in children less than years of age ( d'souza and d'souza, ) . secondary bacterial infections are a major cause of morbidity and mortality following measles ( duke and mgone, ) , and effective case management involves prompt treatment with antibiotics. various strategies have been used to guide antibiotic therapy in children with measles. antibiotics are indicated for children with measles who have clinical evidence of bacterial infection, including pneumonia, otitis media, skin infection, eye infection, and severe mouth ulcers. streptococcus pneumoniae and haemophilus influenza type b are the most common causes of bacterial pneumonia following measles, and antibiotic therapy should be directed against these pathogens. whether all children with measles, or all hospitalized children with measles, should be given prophylactic antibiotics remains controversial. limited evidence suggests that antibiotics administered as prophylaxis to all children presenting with measles may reduce the incidence of pneumonia but not mortality ( duke and mgone, ) . the potential benefits of antibiotic prophylaxis need to be weighed against the risks of accelerating antibiotic resistance. vitamin a has been widely distributed through polio and measles supplemental immunization activities as well as through routine child health services. pooled analysis of community-based studies of vitamin a supplementation of apparently healthy children resulted in a % reduction in measles-associated mortality ( villamor and fawzi, ) . thus, vitamin a is not only effective in reducing mortality when used to treat hospitalized children with measles but community-based supplementation programs also can result in measles mortality reduction. respiratory droplets from infected persons serve as vehicles of transmission by carrying infectious virus to epithelial cells of the respiratory tract of susceptible hosts. during the - day incubation period between infection and the onset of clinical signs and symptoms, measles virus replicates and pathogenesis children following measles virus infection. functional abnormalities of immune cells have also been detected, including decreased lymphocyte proliferative responses ( hirsch et al., ) . dendritic cells, major antigenpresenting cells, mature poorly, lose the ability to stimulate proliferative responses in lymphocytes, and undergo cell death when infected with measles virus in vitro ( servet-delprat et al., ) . the dominant th response in children recovering from measles can inhibit th responses and increase susceptibility to intracellular pathogens ( griffin et al., ; griffin and ward, ) . the production of il- , important for the generation of th -type immune response, decreases following binding of the cd receptor ( karp et al., ) and is low for several weeks in children with measles ( atabani et al., ) . this diminished ability to produce il- could further result in a limited th immune response to other pathogens. furthermore, engagement of cd and cd on monocytes induces production of high levels of il- and transforming growth factor (tgf)-β , an immunomodulatory and immunosuppressive cytokine profile characteristic of regulatory t cells ( kemper et al., ) . the role of these cytokines in the immune suppression following measles is supported by in vivo evidence of elevated levels of il- in the plasma of children after measles virus infection ( moss et al., ) . attenuation of measles virus was achieved primarily by serial passage in chick embryo cells. the first attenuated measles vaccine licensed in the united states was edmonston b. this vaccine was immunogenic and widely used between and , but was frequently associated with fever and rash. the schwarz and moraten (more attenuated) strains were derived from the original edmonston strain but further attenuated through additional passage in chick embryo fibroblasts. despite differences in their passage history, these vaccine strains have identical genomic sequences ( parks et al., ) . the moraten vaccine (merck) is the only measles vaccine used in the united states, whereas the schwarz vaccine is used in many countries throughout the world. other attenuated measles vaccines have been produced from locally derived wild-type strains, particularly in russia, china, and japan. one vaccine strain, the edmonston-zagreb vaccine, was also passaged in human diploid cells after attenuation in chick embryo fibroblasts, which may account for its increased immunogenicity and reactogenicity. measles vaccines spreads within the infected host. initial viral replication occurs in epithelial cells at the portal of entry in the upper respiratory tract and the virus then spreads to local lymphatic tissue. replication in local lymph nodes is followed by viremia (the presence of virus in the blood) and the dissemination of measles virus to many organs, including lymph nodes, skin, kidney, gastrointestinal tract and liver, where the virus replicates in epithelial and endothelial cells as well as monocytes and macrophages. although measles virus infection is clinically inapparent during the incubation period, the virus is actively replicating and the host immune responses are developing. evidence of these processes can be detected. during the incubation period, the number of circulating lymphocytes is reduced (lymphopenia) . measles virus can be isolated from the nasopharynx and blood during the later part of the incubation period and during the several days prior to the onset of rash when levels of viremia are highest. the prodrome ends with the appearance of the measles rash. the rash results from measles virusspecific cellular immune responses and marks the beginning of viral clearance from blood and tissue. histologic examination of the rash reveals infected capillary endothelial cells and a mononuclear cell infiltrate ( kimura et al., ) . clearance of infectious virus from the blood and other tissues occurs within the first week after the appearance of the rash, although measles virus rna can be detected in body fluids of some children for at least several months using a rt-pcrbased assay ( permar et al., ; riddell et al., ) . the intense immune responses induced by measles virus infection are paradoxically associated with depressed responses to unrelated (nonmeasles virus) antigens, lasting for several weeks to months beyond resolution of the acute illness. this state of immune suppression enhances susceptibility to secondary bacterial and viral infections causing pneumonia and diarrhea, and is responsible for much measlesrelated morbidity and mortality ( beckford et al., ; greenberg et al., ) . delayed-type hypersensitivity (dth) responses to recall antigens, such as tuberculin, are suppressed ( tamashiro et al., ) and cellular and humoral responses to new antigens are impaired following measles virus infection ( coovadia et al., ) . reactivation of tuberculosis and remission of autoimmune diseases have been described after measles and are attributed to this state of immune suppression. abnormalities of both the innate and adaptive immune responses have been described following measles virus infection. transient lymphopenia with a reduction in cd ϩ and cd ϩ t-lymphocytes occurs in are relatively heat stable in the lyophilized (dry) form, but rapidly lose potency when exposed to heat after reconstitution. in the s, a formalin-inactivated, alum-precipitated measles vaccine (fimv) was licensed and administered to children in the united states. three doses of inactivated vaccine elicited a protective antibody response but antibody titers waned within months ( carter et al., ) . up to % of immunized children exposed to measles developed atypical measles, characterized by high fever, pneumonitis, and a petechial rash on the extremities ( fulginiti et al., ; nader et al., ) , leading to withdrawal of the fimv in . in a rhesus macaque model, atypical measles was shown to be associated with immune complex deposition in affected tissues and a systemic and pulmonary eosinophilia ( polack et al., ) . the antibody response consisted of high levels of complement fixing antibodies with low avidity for measles virus, characteristics that may have promoted exaggerated immune complex formation and disease. seroconversion rates with attenuated measles vaccines in young infants are low because of immunologic immaturity and the interference of transplacentally acquired maternal antibodies with replication of vaccine virus ( gans et al., ) . to protect young infants against measles, high-titer preparations containing - times the standard dose of vaccine virus were evaluated in several countries. seroconversion rates in - month old infants immunized with high-titer measles vaccine were comparable to those of - month old children vaccinated with standard-titer measles vaccine, and the protective antibody response persisted for over years. however, high-titer measles vaccine resulted in a poorly understood increase in mortality in immunized girls - years after vaccination compared to girls immunized with standard-titer vaccine at months of age ( holt et al., ; aaby et al., ) . the increased mortality was attributable to infections commonly associated with measles, such as diarrhea and pneumonia. although these studies were carried out in countries with different levels of socioeconomic development, excess mortality was observed only in countries with poor socioeconomic conditions and frequent malnutrition (senegal, haiti, and guinea bissau). the basis for the increased mortality in girls is not understood. several live, attenuated measles vaccines are available worldwide, either as single-antigen vaccines or in combination with rubella and mumps vaccines (mr and mmr vaccines). recently, a combined measlesmumps-rubella-varicella vaccine was licensed by the united states food and drug administration (proquad, merck and co., inc.). licensed combination vaccines do not reduce the immunogenicity of the measles component. most of the currently used measles vaccines were derived from the edmonston strain of measles virus that was isolated by enders and peebles in . these vaccines have undergone different passage histories in cell culture, but nucleotide sequence analysis shows less than . % genetic differences between the vaccine strains. vaccines in widespread use that were derived from the edmonston measles virus stain include the schwarz, edmonston-zagreb, and moraten strains. vaccines derived from other measles virus strains include cam- , and shanghai . the live, attenuated measles vaccines are typically cultured in primary chick embryo or human diploid (e.g., edmonston-zagreb) cells for several days. the supernatant fluid is harvested and frozen. vaccine stocks are quality and safety tested before they are lyophilized. measles vaccines may contain sorbitol or gelatin as stabilizers and the antibiotic neomycin, but do not contain thimerosal. traces of the reverse transcriptase of an avian retrovirus can be found in vaccines cultured in chick embryo fibroblasts but there is no evidence that this is harmful to vaccine recipients. prior to use, the vaccine must be reconstituted in sterile diluent. measles vaccines lose about half of their potency after reconstitution if stored at ° c for h, and lose almost all potency if stored at ° c for h. measles vaccines are recommended for all susceptible children and adults for whom the vaccine is not contraindicated ( table . ). adolescents and young adults may constitute a susceptible population, recommendations of the advisory committee on immunization practices for the united states • a two-dose schedule with mmr vaccine is recommended. • the first dose is recommended at - months of age. • the second dose is recommended at - years of age. • adults at increased risk should receive special consideration for measles vaccination, including international travelers, students attending colleges and other post-high school educational institutions, and persons who work at health care facilities. • measles vaccine is not indicated for persons with severe hypersensitivity (anaphylaxis) to neomycin or gelatin, or who are severely immunocompromised. • measles vaccine should be administered at months of age. • a second opportunity for measles vaccination should be provided through mass measles vaccination campaigns. • hiv-infected children should receive measles vaccine at and months of age, unless severely immunocompromised, because of their increased risk of severe measles. the proportion of children who develop protective antibody titers following measles vaccination depends on the presence of inhibitory maternal antibodies and the immunologic maturity of the vaccine recipient, as well as the dose and strain of vaccine virus. frequently cited figures are that approximately % of children develop protective antibody titers when given measles vaccine at months of age and - % respond when vaccinated at months of age ( cutts et al., ) . concurrent acute infections may interfere with the immune response to measles vaccine, although this is probably uncommon ( scott et al., ) . polymorphisms in human immune response genes also influence immune responses to measles vaccine ( ovsyannikova et al., ) . the duration of immunity following measles vaccination is more variable and shorter than following wild-type measles virus infection, with an estimated % of children developing secondary vaccine failure at - years after vaccination ( anders et al., ) . immunologic boosting from repeated exposure to measles virus may play a role in maintaining protective antibody levels in communities with measles virus transmission ( whittle et al., ) . the who encourages measles vaccination of all susceptible children and adults. because measles can be more severe in hiv-infected persons and the risk of serious adverse events appears to be small, the who recommends that asymptomatic hiv-infected persons, and even those who are symptomatic but not severely immunocompromised, receive measles vaccine. the american academy of pediatrics recommends that severely immunocompromised children and adults, defined by low cd ϩ t-lymphocyte cell counts or percentage, should not receive measles vaccine because of the potential risk of vaccine-related pneumonitis. measles vaccine is contraindicated in persons with a history of anaphylactic reaction to neomycin, gelatin, or other vaccine components. fever occurs in approximately - % of recipients - days following measles vaccination, and a rash occurs in approximately % of recipients. these signs and symptoms are a consequence of the host immune response to replicating measles vaccine virus, but do not result in serious morbidity or mortality. rarely, thrombocytopenia may occur. although assumed to be rare, the risk of disease caused by attenuated measles vaccine virus in hivinfected persons is unknown. the only documented case of disease induced by vaccine virus in an hiv-infected person was in a -year-old man who died months after receiving his second dose of measles vaccine ( angel et al., ) . he had a very low cd ϩ t-lymphocyte cell count but no hiv-related symptoms at the time of including university students, health care workers, and military recruits. a single dose of measles vaccine provides lifelong immunity in the majority of vaccine recipients. however, a second opportunity for measles vaccination provides protection to those with primary vaccine failure and a chance to reach unvaccinated children. the second opportunity may be delivered through routine health services at school entry or through supplementary immunization activities such as national immunization days. measles vaccines are usually injected subcutaneously but can be administered intramuscularly. each . ml dose of vaccine contains at least infective units of measles vaccine virus. the optimal age of measles vaccination is determined by consideration of the age-dependent increase in seroconversion rates following measles vaccination and the average age of infection. in regions of intense measles virus transmission, the average age of infection is low and the optimal strategy is to vaccinate against measles as young as possible. however, both maternally acquired antibodies and immunologic immaturity reduce the protective efficacy of measles vaccination in early infancy ( gans et al., ) . in many parts of the world, months is considered the optimal age of measles vaccination ( halsey, ) , and is the age recommended by the expanded program on immunization (epi). most countries following the epi schedule administer measles vaccine alone, although more countries are introducing combined measles and rubella vaccines as rubella control programs expand. under some circumstances, provision of an early dose of measles vaccine at months of age (e.g., in outbreaks or to hiv-infected children) is appropriate. in regions that have achieved measles control or elimination, where the risk of measles in infants is low, the age of measles vaccination is increased to ensure that a higher proportion of children develop protective immunity. for example, in the united states the first dose of measles vaccine is administered at - months of age, as the combined mmr vaccine. measles vaccine induces both humoral and cellular immune responses. antibodies first appear between and days after vaccination and peak at - days. igm antibodies appear transiently in blood, iga antibodies are predominant in mucosal secretions, and igg antibodies persist in blood for years. vaccination also induces measles virus-specific t-lymphocytes ( ovsyannikova et al., ; wong-chew et al., ) . although both humoral and cellular responses can be induced by measles vaccine, they are of lower magnitude and shorter duration compared to those following wild-type measles virus infection ( ward et al., ) . vaccination. ten months later he developed giant cell pneumonitis and measles vaccine virus was identified in his lung. fatal, disseminated infection with measles vaccine virus has been reported rarely in persons with other impairments of immune function ( monafo et al., ) , and mibe due to vaccine virus was reported in a child with an uncharacterized immune deficiency ( bitnun et al., ) . much public attention has focused on a purported association between mmr vaccine and autism following publication of a report in hypothesizing that mmr vaccine may cause a syndrome of autism and intestinal inflammation ( wakefield et al., ) . the events that followed, and the public concern over the safety of mmr vaccine, led to diminished vaccine coverage in the united kingdom and provide important lessons in the misinterpretation of epidemiologic evidence and the communication of scientific results to the public ( offit and coffin, ) . the publication that incited the concern was a case series describing children with a regressive developmental disorder and chronic enterocolitis. nine of the children had autism. onset of the developmental delay was associated by the parents with mmr vaccination in eight children. this simple temporal association was misinterpreted and misrepresented as a possible causal relationship, first by the lead author of the study and then by the media and public. subsequently, several comprehensive reviews and additional epidemiological studies rejected evidence of a causal relationship between mmr vaccination and autism ( destefano and thompson, ) . one of the most conclusive studies was a large retrospective cohort study of over half a million danish children that found the relative risk of mmr vaccine for autistic disorder to be . ( % confidence interval, . - . ) ( madsen et al., ) . aerosol administration of measles vaccine was first evaluated in the early s in several countries, including the former soviet union and the united states. more recent studies in south africa ( dilraj et al., ) and mexico ( bennett et al., ) have shown that aerosol administration of measles vaccine is highly effective in boosting antibody titers, although the primary immune response to aerosol measles vaccine is lower than following subcutaneous administration ( wong-chew et al., ) . administration of measles vaccine by aerosol has the potential to greatly facilitate measles vaccination during mass campaigns, and the who plans to test and bring to licensure an aerosol measles vaccine by . the ideal measles vaccine would be inexpensive, safe, heat-stable, immunogenic in neonates or very young infants, and administered as a single dose without needle or syringe. the age at vaccination should coincide with the epi schedule to maximize compliance and share resources. finally, a new vaccine should not elicit atypical measles upon exposure of immunized individuals to wild-type measles virus and should not be associated with prolonged immunosuppression, adversely affecting immune responses to subsequent infections. a number of vaccine candidates with some of these characteristics are undergoing preclinical studies ( table . ). naked cdna vaccines are thermostable, inexpensive, and could theoretically elicit antibody responses in the presence of passively acquired maternal antibody. dna vaccines encoding either or both the measles h and f proteins are safe, immunogenic, and protective against measles challenge in na ï ve, juvenile rhesus macaques ( polack et al., ) . a different diagnosed, and measles vaccination is a highly effective intervention. although measles virus displays sufficient genetic variability to conduct molecular epidemiologic analyses, the antigenic epitopes against which protective antibodies develop have remained stable. measles virus differs from smallpox and polio viruses, however, in that it is more highly infectious, necessitating much higher levels of population immunity to interrupt transmission. potential barriers to measles eradication include: ( ) lack of political will; ( ) difficulties of measles control in densely populated urban environments; ( ) the hiv epidemic; ( ) waning immunity and the potential transmission from subclinical cases; ( ) transmission among susceptible adults; ( ) the risk of unsafe injections; and ( ) unfounded fears of disease due to measles vaccine ( orenstein et al., ) . whether the threat from bioterrorism precludes stopping measles vaccination after eradication is a topic of debate, but, at the least, a single-dose rather than a two-dose measles vaccination strategy could be adopted ( meissner et al., ) . the elimination of endemic measles virus transmission in large geographic areas, such as the americas, suggests that global eradication is feasible with current vaccination strategies ( de quadros, ) . many believe this to be a realistic and morally imperative goal, but, as polio eradication efforts have shown, the endgame may be full of challenges. • measles is highly contagious viral infection. • great progress has been made in measles control and elimination through accelerated efforts, including increased routine vaccination coverage rates and supplementary immunization activities. • despite the reduction in measles cases and deaths, measles remains a leading cause of vaccinepreventable death worldwide. the high infectivity of measles virus is a characteristic suitable to a biothreat agent. • increasingly high levels of measles vaccination coverage throughout the world would protect many from the deliberate use of measles virus as a biothreat agent. • measles vaccine virus can be used as a vector to deliver genes derived from other pathogens. • genetic engineering of a measles virus strain that was not neutralized by antibodies induced by the current attenuated measles vaccines would likely have reduced infectivity, although this is not certain. construct, containing h, f, and n genes and an il- molecular adjuvant, provided protection to infant macaques in the presence of neutralizing antibody ( premenko-lanier et al., , . alternative techniques for administering measles dna, such as alphavirus ( pan et al., ) , parainfluenza virus ( skiadopoulos et al., ) , or enteric bacterial ( pasetti et al., ) vectors, also are under investigation. immune responses to intranasal administration of measles virus vaccines is enhanced by the use of adjuvants ( chabot et al., ) . novel oral immunization strategies have been developed using plant-based expression of the measles virus h protein in tobacco ( webster et al., ) . these vaccines induced humoral immune responses following both primary immunization and as a booster dose after a dna prime. in addition, attenuated recombinant measles virus vaccines have been used as vectors for the delivery of genes from other pathogens ( tangy and naim, ) . immune globulin can be given intramuscularly to susceptible persons within days of exposure to prevent or lessen the severity of disease. postexposure immunoprophylaxis is indicated for those at high risk of severe disease, including susceptible household contacts between months and year of age, pregnant women, and immunocompromised persons. where resources are not limited, hiv-infected children, or those exposed to hiv but whose infection status is unknown, should receive postexposure immunoprophylaxis. persons who are not immunocompromised and who have received at least one dose of measles vaccine at months of age or older should not receive postexposure immunoprophylaxis. if given with h of exposure, measles vaccine may provide protection against disease. the possibility of measles eradication has been discussed for almost years ( sencer et al., ) . serious consideration of measles eradication began in the late s as smallpox eradication was nearing completion and the effective, long-term immunity induced by measles vaccine became apparent. measles virus meets many of the biologic criteria for disease eradication ( moss and griffin, ) . measles virus has no nonhuman reservoir; infection can be accurately a comparison of vaccine efficacy and mortality during routine use of high-titre edmonston-zagreb and schwarz standard measles vaccines in rural senegal secondary failure rates of measles vaccines: a metaanalysis of published studies cd -and cd -independent endothelial cell infection with wild-type measles viruses vaccine-associated measles pneumonitis in an adult with aids natural measles causes prolonged suppression of interleukin- production measles virus infection in rhesus macaques: altered immune responses and comparison of the virulence of six different virus strains factors associated with fatal cases of measles. a retrospective autopsy study aerosolized measles and measles-rubella vaccines induce better measles antibody booster responses than injected vaccines: randomized trials in mexican schoolchildren the challenges and strategies for laboratory diagnosis of measles in an international setting measles inclusion-body encephalitis caused by the vaccine strain of measles virus inapparent measles after gamma globulin administration serologic response of children to inactivated measles vaccine a novel intranasal protollin-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cellmediated immunity in mice alterations in immune responsiveness in acute measles and chronic post-measles chest disease influence of maternal antibodies on neonatal immunization against respiratory viruses the effect of dose and strain of live attenuated measles vaccines on serological responses in young infants can measles be eradicated globally? measles eradication: experience in the mmr vaccine and autism: an update of the scientific evidence response to different measles vaccine strains given by aerosol and subcutaneous routes to schoolchildren: a randomised trial persistent measles infection in malnourished children vitamin a for the treatment of children with measles: a systematic review measles: not just another viral exanthem propagation in tissue cultures of cytopathic agents from patients with measles measles in england and wales -i: an analysis of factors underlying seasonal patterns severe measles pneumonitis in adults: evaluation of clinical characteristics and therapy with intravenous ribavirin altered reactivity to measles virus: atypical measles in children previously immunized with inactivated measles virus vaccines deficiency of the humoral immune response to measles vaccine in infants immunized at age months disturbances in gamma globulin synthesis as " experiments of nature measles-associated diarrhea in hospitalized children in lima, peru: pathogenic agents and impact on growth changes in plasma ige levels during complicated and uncomplicated measles virus infections differential cd t cell activation in measles the optimal age for administering measles vaccine in developing countries cellular immune responses during complicated and uncomplicated measles virus infections of man differential mortality by measles vaccine titer and sex plantderived measles virus hemagglutinin protein induces neutralizing antibodies in mice measles outbreaks in the united states attenuated salmonella enterica serovar typhi and shigella flexneri a strains mucosally deliver dna vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats v and c proteins of measles virus function as virulence factors in vivo role of cd ( ϩ ) lymphocytes in control and clearance of measles virus infection of rhesus monkeys prolonged measles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptasepolymerase chain reaction the clinical significance of measles: a review production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody successful dna immunization against measles: neutralizing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles dna vaccination of infants in the presence of maternal antibody: a measles model in the primate protection against challenge with measles virus (mv) in infant macaques by an mv dna vaccine administered in the presence of neutralizing antibody slow clearance of measles virus rna after acute infection update on the global distribution of genotypes of wild type measles viruses efficiency of measles virus entry and dissemination through different receptors mild illness at or after measles vaccination does not reduce seroresponse in young children epidemiologic basis for eradication of measles in consequences of fas-mediated human dendritic cell apoptosis induced by measles virus a chimeric human-bovine parainfluenza virus type expressing measles virus hemagglutinin is attenuated for replication but is still immunogenic in rhesus monkeys treatment of subacute sclerosing panencephalitis induced by measles virus in vaccinated and naturally infected individuals mechanism of suppression of cell-mediated immunity by measles virus activation of human cd ϩ cells with cd and cd induces a t-regulatory cell phenotype measles rash. i. light and electron microscopic study of skin eruptions measles outbreak in young adults in victoria a population-based study of measles, mumps, and rubella vaccination and autism measles virus glycoproteins: studies on the structure and interaction of the haemagglutinin and fusion proteins measles vaccines and the potential for worldwide eradication of measles disseminated measles infection after vaccination in a child with a congenital immunodeficiency severe measles in the tropics implications of the human immunodeficiency virus epidemic for control and eradication of measles global measles elimination differential regulation of interleukin (il)- , il- , and il- during measles in zambian children atypical exanthem following exposure to natural measles: eleven cases in children previously inoculated with killed vaccine communicating science to the public: mmr vaccine and autism measles eradication: is it in our future? frequency of measles virus-specific cd ϩ and cd ϩ t cells in subjects seronegative or highly seropositive for measles vaccine variation in vaccine response in normal populations inaugural article: modulation of disease, t cell responses, and measles virus clearance in monkeys vaccinated with h-encoding alphavirus replicon particles observations made during the epidemic of measles on the faroe islands in the year comparison of predicted amino acid sequences of measles virus strains in the edmonston vaccine lineage characterization of immune responses induced by intramuscular vaccination with dna vaccines encoding measles virus hemagglutinin and/or fusion proteins the assembly of the measles virus nucleoprotein into nucleocapsid-like particles is modulated by the phosphoprotein prospective study of the magnitude and duration of changes in tuberculin reactivity during uncomplicated and complicated measles live attenuated measles vaccine as a potential multivalent pediatric vaccination vector recombinant measles viruses with mutations in the c, v, or f gene have altered growth phenotypes in vivo vitamin a supplementation: implications for morbidity and mortality in children ileal-lymphoid-nodular hyperplasia, nonspecific colitis, and pervasive developmental disorder in children cellular immunity in measles vaccine failure: demonstration of measles antigen-specific lymphoproliferative responses despite limited serum antibody production after revaccination successful boosting of a dna measles immunization with an oral plant-derived measles virus vaccine the development of a plant-based vaccine for measles effect of subclinical infection on maintaining immunity against measles in vaccinated children in west africa has the measles mortality reduction goal been achieved? a natural history modelling study induction of cellular and humoral immunity after aerosol or subcutaneous administration of edmonston-zagreb measles vaccine as a primary dose to -month-old children world health organization progress in reducing measles mortality -worldwide world health organization global distribution of measles and rubella genotypes -update world health organization united nations children's fund. measles mortality reduction and regional elimination strategic plan key: cord- -fn b y authors: mudgal, rajat; nehul, sanketkumar; tomar, shailly title: prospects for mucosal vaccine: shutting the door on sars-cov- date: - - journal: human vaccines & immunotherapeutics doi: . / . . sha: doc_id: cord_uid: fn b y the sudden emergence of a highly transmissible and pathogenic coronavirus sars-cov- in december from china and its rapid global spread has posed an international health emergency. the rapid development of an effective vaccine is imperative to control the spread of sars-cov- . a number of concurrent efforts to find an effective therapeutic agent or vaccine for covid- (coronavirus disease ) are being undertaken globally. oral and nasal mucosal surfaces serve as the primary portal of entry for pathogens like coronaviruses in the human body. as evidenced by studies on similar coronaviruses (sars-cov and mers-cov), mucosal vaccination can provide a safe and effective means for the induction of long-lasting systemic and mucosal immunity to confer protection against sars-cov- . this article summarizes the approaches to an effective mucosal vaccine formulation which can be a rewarding approach to combat the unprecedented threat posed by this emerging global pandemic. in the st century, we have seen a worldwide spread of three previously unknown coronaviruses. the first outbreak of severe acute respiratory syndrome (sars) occurred in november in the guangdong province, china. the causative agent of the sars outbreak was identified as sars coronavirus (sars-cov). another coronavirus, middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in . on december , , several cases of pneumonia were reported in wuhan, china. the etiological agent of the outbreak was later identified as sars coronavirus (sars-cov- ) because the genomic sequence was closely related to that of the sars-cov from . , on february , , the world health organization (who) named the disease caused by the novel coronavirus (sars-cov- ) as coronavirus disease . coronaviruses belong to the coronaviridae family. the family members are enveloped, positive-stranded rna viruses which appear as crown-like entities under the electron microscope due to spikes of glycoproteins protruding from their viral envelopes, thus exhibiting a corona-like appearance. the coronaviridae family is divided into two subfamilies: letovirinae and orthocoronavirinae. the latter consists of the genera alphacoronvirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. the pathogenic coronaviruses: sars-cov, mers-cov, and sars-cov- are all betacoronaviruses. among known rna viruses, coronaviruses have the largest genomes size in the range of to kb in length. two-third of the viral genome at ˊ end encodes up to nonstructural replicase proteins which are translated as two polyproteins: pp a and pp ab. the genes encoding structural proteins, including spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins are present at ˊ end of genomic rna. , the s protein of coronaviruses is one of the most important targets for the development of sars vaccines and therapeutics because it is involved in receptor recognition, as well as virus attachment and entry. the s protein is made up of s and s subunits. s subunit has the receptor-binding domain (rbd) which binds with host receptor and then the s subunit mediates the fusion of viral and host membranes. host receptor for sars-cov is angiotensinconverting enzyme (ace ), whereas mers-cov recognizes dipeptidyl peptidase (dpp ) as its receptor. , the common symptoms of coronavirus infection are fever, cough, and sore throat. clinically, patients with sars suffer from atypical pneumonia. , clinical presentation of sars-cov- patients is similar to patients infected with sars-cov. covid- manifests itself with symptoms including fever, dry cough, fatigue, and acute respiratory distress syndrome. , these clinical features are a direct consequence of massive alveolar epithelial cell and vascular endothelial cell damage which is also accompanied by an exuberant release of proinflammatory cytokines and chemokines. the disease severity and lung damage in the case of sars-cov- infection can be directly correlated with the dysregulated immune response at - days after symptom onset and is characterized by exuberant production of cytokines including il- , il- , il- , mip- a, ip- , and tnf-α. , this 'cytokine storm' was also reported in animal studies with sars-cov infection and was responsible for the dampening of adaptive immunity of patients in the later phase of infection. as of now, the spread of sars-cov- is being quelled by strict policy measures such as travel restrictions, social distancing, patient isolation, and nationwide lockdown in several parts of the world. there are no approved vaccines available against any of the coronaviruses. this emerging global pandemic has instigated an unprecedented search for an effective prophylactic or therapeutic intervention against covid- . [ ] [ ] [ ] [ ] in this review, we have discussed the potential and challenges for the development of a successful mucosal vaccine against sars-cov- . vaccines have been one of the major contributors in the eradication of most of the infectious diseases in the last century. vaccination of a population interrupts the transmission chain of a communicable pathogen by not only protecting the immunized subjects but also by halting the transmission of the virus by 'breaking the chain' within a population by induction of herd immunity in the vaccine recipients. vaccines can be administered by either intramuscular or subcutaneous injection to introduce them into the systemic circulation. vaccination through systemic routes elicits strong systemic immune response but is not effective in generating efficient mucosal immunity. mucosal vaccines are advantageous not just in evoking strong immune response at both mucosal sites and systemic circulation but also offer greater practicality in terms of cost and administration. mucosal vaccines can be produced at considerably low cost for mass immunization, the administration is needle-free and convenient compared to systemic vaccines. majority of mucosal vaccines are administered through oral or intranasal routes while rectal, vaginal, ocular, and sublingual routes can also be used. the selection of administration route depends on the nature of the antigen and the desired site for induction of immune response. upon oral immunization, immune responses are induced strongly in gastrointestinal (gi) tract, mammary glands, and salivary glands while intranasal vaccination induces marked antigen-specific immune response in respiratory, gi, and genital tracts. the primary mode of transmission of sars-cov is through mucosal membranes of the eyes, nose, or mouth. studies with s protein of sars-cov- have revealed that it also recognizes human ace (hace ) receptor on the host cells similar to sars-cov. [ ] [ ] [ ] ace is abundantly expressed in nasal and oral mucosa rendering them the primary targets for the viral entry and dissemination of sars-cov- . concomitant gastrointestinal symptoms are reported in confirmed covid- patients along with pulmonary pathology, characteristic of sars-cov- infection. the occurrence of acute hemorrhagic colitis in a few cases and the presence of sars-cov- rna in fecal samples of covid- patients indicate enteric involvement in covid- reasserting the similarity in tissue tropism with sars-cov. [ ] [ ] [ ] considering the prominent role of nasal and gastric mucosa in the transmission and the clinical progression of sars-cov- , mucosal immunization using oral or intranasal vaccine could be an effective strategy for immunoprophylaxis against sars-cov- . vaccination at the entry site such as intranasal or oral immunization induces a strong local immune response in the case of sars-cov. , consequently, vaccination at the mucosal sites reduces the risk of antibodydependent disease enhancement (ade) by blocking the virus at the entry site. administration of mucosal vaccine has also been shown to elicit strong systemic humoral immunity thereby neutralizing any virus particle that evades the primary immune response at the mucosal site. currently, several strategies are being examined for the development of a mucosal vaccine against sars-cov- . a unique mucosal system exists independent of the systemic immune system to protect exposed mucosal surfaces against environmental antigens and downregulation of systemic immune responses. it acts as the first line of defense against most of the antigens and prevents them from evoking systemic immune system. the mucosal system serves as the most common portal for pathogen entry in the human body. the mucosal immune system consists of a complex network of tissues, non-lymphoid /lymphoid cells, and effector molecules including cytokines, chemokines, and antibodies. the mucosal immune system is compartmentalized into immune inductive sites-where antigen sampling from mucosal surface occurs and then priming of b cells and t cells takes place, and immune effector site-where the activated immune effector cells move after extravasation and secrete cytokines to promote iga class-switch recombination. , inductive site for the mucosal immune system is constituted by mucosa-associated lymphoid tissue (malt). malt comprises of highly organized structures such as appendix and peyer's patches in the intestine and tonsils in the upper airway. the effector sites of the mucosal immune system include lamina propria of various mucosae, surface epithelia, and exocrine glands. gut-associated lymphoid tissue (galt) and nasopharyngeal-associated lymphoid tissue (nalt) represent the major mucosal inductive sites. waldeyer's ring and oropharyngeal lymphoid tissues (paired palatine tonsils) in humans are considered anatomical equivalent of murine nalt. larynx-associated lymphoid tissue (lalt), salivary duct-associated lymphoid tissue (sdalt), lacrimal ductassociated lymphoid tissue (ldalt), and conjunctiva-associated lymphoid tissue (calt) are also considered part of human malt. these mucosal inductive sites are covered with a layer of enterocytes and microfold cells (m cells). m cells are specialized thin epithelial cells that move soluble antigen from the gut lumen to the underlying lymphoid tissues via transcytosis. exogenous antigens can activate t cells directly or are handed off to dendritic cells (dcs) that can act as antigen-presenting cells (apcs). , these primed t cells move to the germinal centers and secrete cytokines to promote b-cell isotype switching to iga production. secretory iga (siga) antibodies are the most essential effector molecule in the mucosa. it is recognized as the first line of protection against foreign toxins, pathogens, and overgrowth of commensal microbes. it is secreted as a dimer joined together by a joining chain and is actively transported exclusively across the mucosal surface via a polymeric iga receptor. the activated t and b cells retain immunological memory and contribute to long-lasting protective immunity against the pathogen in systemic and mucosal systems. siga neutralizes toxins or pathogens in the mucosal environment using three mechanisms: immune exclusion, antigen excretion, and intracellular neutralization ( figure ). in addition to siga, transudated iga and igg, which are generated in response to both systemic and mucosal vaccination, also contribute to local surface defense in the genitourinary mucosa and in the lower respiratory tract which are more permeable to serum-derived antibodies than intestine. these antibodies employ a diverse range of effector functions to protect against pathogens. they neutralize toxins; can mediate opsonization and facilitate internalization of invading pathogens by phagocytes. transudated igg exert its immunopathological effect when siga-dependent elimination of pathogen is unsuccessful. serum igg antibodies protect against viremia and are crucial for virus clearance from systemic circulation. a mucosal vaccine can be developed based on one of the several vaccine platforms including viral vectors, virus-like particle (vlp)-based, dnas, subunit, inactivated whole virus, or live-attenuated vaccine. , , each of these platforms has its own advantages and disadvantages. vlp-based vaccines are composed of viral structural proteins capable of selfassembly. dna vaccine comprises viral immunogens encoded by a recombinant plasmid that is delivered to the site of administration where the plasmid expresses itself and elicits the desired immune response. both these safe vaccine platforms preserve the inherent antigenic structure of viral immunogens and are noninfectious but often suffer from weak immunogenicity. viral vectors encode the antigenic protein and are delivered to the host cell where the antigen is expressed and is presented on the surface of apcs to induce a cellular and humoral immune response. these vaccines are highly efficient but preexisting immunity against the viral vector might cause a harmful immune response in some recipients. live-attenuated vaccines or inactivated vaccines are based on pathogens that have been attenuated or inactivated by heat or chemical treatment. as of now, all the mucosal vaccines licensed for human use are based on liveattenuated approach including oral polio vaccine (opv) and intranasal influenza vaccine (flumist®). safety concerns are often associated with attenuated vaccines due to the possibility of incomplete inactivation of harmful pathogens. subunit vaccines consist of specific antigenic fragments of virus capable of eliciting strong antibody-mediated and cellular immune response. inactivated whole virus vaccines and subunit vaccines are relatively inexpensive, inert, and nontoxic but the possibility of alteration of immunogenicity of antigens needs to be confirmed consistently. few sars-cov and mers-cov vaccines based on the different platforms are summarized in table . s protein is the main antigenic component of sars-cov, sars-cov- , and mers-cov among four structural proteins (s, e, m, and n proteins). vaccines using s protein elicit a potent immune response and inhibit viral infection. , , but the use of fulllength s protein as an antigen for vaccine design has raised few safety issues due to ade of viral infection in vaccinated subjects. a study on the chinese macaque models for sars-cov showed greater lung damage in vaccinated animals (vaccinated with fulllength s proteins) relative to unvaccinated subjects upon virus challenge. anti-s protein igg antibodies have been implicated in this acute alveolar damage on exposure to the virus. similar symptoms have been observed in critical patients with sars-cov infection. it is speculated that ade of viral infection is mediated through the binding of virus-neutralizing antibody complex to the fc receptors on the monocytes/macrophages leading to increased production of pro-inflammatory cytokines. additionally, this virus-antibody complex might activate the classical pathway of complement system or antibody-mediated cytotoxicity leading to cellular damage. taking a cue from studies with sars-cov and mers-cov, caution must be exercised in selecting an antigen target that minimize ade while inducing a potent immune response against future exposure to sars-cov- . the use of rbd of s protein as an antigen in the case of sars-cov or mers-cov has been extensively explored. , rbd of s protein is highly immunogenic and confers neutralizing capability against multiple strains of sars-cov and mers-cov. rbd-based vaccine minimizes the risk of ade upon exposure to virus in vaccinated individuals as it lacks the non-neutralizing immunodominant region of s protein. other fragments of s protein of sars-cov and mers-cov that have been used for vaccine design include s and s fragments ( figure ). , a mucosal vaccine based on n protein of sars-cov has shown the induction of both cellular and humoral immunity in mice. several immunogenic domains of n protein are highly conserved in coronaviruses. n protein of sars-cov- shares high sequence identity (~ %) with n protein of sars-cov and hence can be considered for the development of a broad spectrum coronavirus vaccine. a challenging alternative that can warrant protection against future outbreaks of similar coronaviruses is to identify a universal epitope in the whole virus family or genera. this strategy is being undertaken for influenza viruses and can be extended to coronaviruses as well. immunoinformatics can also be a powerful tool in prediction of epitopes on the viral surface proteins. traditionally, mucosal immune induction needs a higher dose of antigen in comparison to parenteral immunization as antigenic preparation may get diluted in mucus in the nasal cavity or get expelled by mucus and ciliary movement in the respiratory tract. effective intranasal vaccination requires the antigen to reach mucosal sites, cross the mucus layer, and induce local iga production. a vaccine administered through the oral route has to endure the low ph environment in the upper gi tract and a variety of nucleases and proteases present in the digestive tract before it can reach the immunological sites. mucosal antigens when administered alone lead to a weak induction of immune response. to overcome these physical and biochemical obstacles in priming mucosal immune cells, vaccines delivered through oral or nasal routes are often administered complemented with an adjuvant. adjuvants are the supplementary materials (natural or synthetic) in a vaccine formulation that potentiates the immune-induction capacity of a vaccine. adjuvant in a vaccine formulation plays a critical role in maintaining the structural integrity of the antigen, augmenting antigen bioavailability, and enhancing antigenic stimulation. adjuvants are broadly divided into two classes: adjuvants that can serve as carrier systems to facilitate antigen delivery to immune induction sites and adjuvants that can act as immunostimulators through enhanced internalization, presentation, and processing of the antigen in the apcs. a number of polymers including chitosan and poly lacticco-glycolic acid (plga) have been used as carriers in various vaccine formulations for immunostimulation due to their high affinity toward mucosal surfaces. , emulsions and liposomes are the other carriers routinely tested for mucosal vaccine. dc and m cells are a major determinant for induction of mucosal immune response at immune inductive sites and thus represent an ideal site for antigen presentation. surface markers at the surface of epithelial cells and dcs can be strategically targeted for antigen delivery with specialized molecules such as lectins or nanoparticles for increased antigen uptake by apcs. , pathogen recognition receptor agonists such as synthetic poly-(i:c), or toll like receptor agonists such as cpg oligodeoxynucleotides, engage these receptors present on the surface of apcs to potentiate the immunogenicity of the vaccine. , other commonly used adjuvants including cholera enterotoxin (ct) and heat-labile enterotoxin (lt) from e. coli exert their adjuvanticity by interacting with gm gangliosides on the surface of follicular dendritic cells and in turn increase the induction of b-cell clones. the use of immune-stimulating complexes (iscoms) has also proven to be highly effective for administration with mucosal vaccines. one of the major factors influencing the rational design of a mucosal vaccine is immunotolerance at the mucosal sites. immunotolerance is the ingenious modulation of the mucosal microenvironment to avoid unnecessary induction of host immune cells against foreign antigens or commensal microbes. mucosal surfaces are exposed continuously to environmental, food, or self-antigens which lead to the development of a tolerogenic microenvironment, especially in gastric mucosa. some vaccination strategies fail to develop effective immunity and can induce immunotolerance. this suppression of immune response is affected mainly by vaccine formulation, antigenic dose, and frequency of administration. administration of antigen at low doses for a long time leads to low-dose immunotolerance. contrastingly, high dose of antigen administered at a low rate induces high-dose tolerance instead of immunostimulation. this hypo-responsiveness at the mucosal induction sites can be overcome by strategically determining the antigen dose, vaccine formulation, and timing of vaccine delivery. the design of a novel mucosal vaccine also aims at mimicking the kinetics of pathogen infection for increasing the clinical efficacy of the vaccine. optimal release timing of antigen at the inductive site helps in dodging the mucosal immunotolerance. to this end, the antigen is conjugated with adjuvants like tlr ligands or cd- specific antibodies. , immunosenescence several issues are needed to be addressed for the design and development of an efficacious mucosal vaccine against sars-cov- . it has been observed in human challenge studies that immunity acquired with coronavirus infection is often shortlived and in some cases, re-infection with the same virus was possible after an extended period. it was reported in some cases that immunity acquired with sars-cov or mers-cov also declines considerably - years after viral infection. , another concern for designing a vaccine against sars-cov- is that the viral infection is associated with severe pathology specifically in patients of higher age group (typically > years). people in this age group do not respond very well to vaccination in terms of neutralizing antibody titers and require a higher amount of antigen to produce sufficient immunogenicity. specialized dose-regime in terms of the amount of antigen, use of specific adjuvants, or the immunization effects of follow-up doses after a single prime dose can be investigated in vulnerable age groups and immunocompromised people before extending vaccination to them. for most parenterally administered vaccines and mucosal vaccines, correlates and precise mechanism of their efficacy remain poorly defined. most of the licensed vaccines rely on the measurement of a single parameter that is statistically correlated with protection afforded by the vaccine. there is no absolute method of sampling, and the choice of sampling method varies according to the parameter to be evaluated. conventionally, quantification and qualification of secreted antibodies is performed using seroimmunoassays in body secretions such as saliva, tears, nasal, blood samples or genital secretions, and gut or organ lavages. cellular correlates of immunity are analyzed using assays such as enzyme-linked immunospot assays (elispots), reverse-transcriptase pcr (rt-pcr) or cell-sorting techniques. a modern system biology approach can also be used that utilizes functional genomics to identify molecular signatures that correlate well with traditional biological markers for evaluating vaccine efficacy. correlates of protection differ greatly based on whether the objective of vaccination is to prevent a mucosal or a systemic infection. several animal models including mice, ferrets, macaques, hamsters, and non-human primates have been used for evaluating safety and efficacy of sars-cov and mers-cov vaccines. , [ ] [ ] [ ] [ ] [ ] ferrets are a suitable animal model for sars-cov vaccine evaluation as they support viral replication in the respiratory tract, develop similar disease symptoms, and display severe lung pathology. , smaller animal models like rabbits and mice are a preferred choice for vaccine evaluation in animals because of inexpensive maintenance, ease of genetic manipulation, and standardized methods of testing. wild type mice are nonpermissive to sars-cov- viral replication. a transgenic mice model expressing hace has recently been developed by genetic manipulation to make the mice susceptible to sars-cov- infection. this mice model mirrors the pathological features of sars-cov- infection in human patients albeit at a moderate level as compared to sars-cov. moreover, a young animal/mice model can effectively exhibit excellent neutralizing efficiency induced by a vaccine candidate but it cannot effectively mimic the clinical manifestations of covid- in an elderly population. hence, robust lethalchallenged mice models using senescent mice that can recapitulate the clinical disease in the aged human population are needed to be developed for assessing the efficacy of a covid- vaccine candidate. following animal modelbased preclinical studies, good manufacturing practices are employed for the scale-up of the vaccine production. currently, mucosal vaccines form a small proportion of licensed vaccines for humans. limited choice of adjuvants for human mucosal vaccines, immunotolerance, and differential degree of vaccine-induced immune response in different populations are some of the impediments associated with the development of mucosal vaccines. efficacies of different mucosal vaccines depend on myriad of factors such as age, environment, host genetics, the microbiome of the recipient, and the regimen of immunization. it ranges from % to % for rotavirus vaccines to %-to % for oral vaccines against influenza and polio viruses. cholera, polio, and rotavirus oral vaccines are found to be less efficacious in developing countries. this weak induction of immune response can be attributed to nutritional deficiencies such as vitamin a or zinc, concomitant bacterial, helminth, or viral infections, and the presence of high levels of maternal antibodies in the breast milk. , intranasal immunization using live attenuated or live vectors is also associated with the risk of the harmful antigens and adjuvants (such as ct and lt) accessing the central nervous system through the cribiform plate. a number of infectious diseases have been successfully controlled with the use of parenterally administered vaccine that may or may not lead to the induction of mucosal immune response. administration of the vaccine through systemic circulation might not necessarily recapitulate the immune response generated by mucosal administration but is adequate for evoking immune response against mucosal pathogens such as human papilloma virus (hpv), influenza virus, or polio virus. all three licensed vlp-based hpv vaccines are administered intramuscularly and induce a high level of durable igg antibody response against the virus. , these antibodies are also detectable at mucosal sites of infection such as oral cavity and cervix after vaccination and serve as the primary effectors of protection against hpv. , currently, there are two types of licensed influenza vaccines: parenterally administered inactivated influenza vaccine and live attenuated influenza virus vaccine (laiv) which is delivered intranasally. inactivated influenza vaccine elicits some degree of local iga antibodies and high levels of systemic igg antibodies to mediate protection by diffusing into the mucosal sites. laiv, in turn, mimics natural infection and induce highly cross-reactive serum igg, mucosal iga, and cellular immunity in the recipients. , yearly influenza vaccines are adjusted according to the prevalent strains in the coming flu season. in clinical settings, inactivated vaccine is found to be more effective than laiv in preventing symptomatic disease in healthy adults while laiv offers greater protection against influenza disease in children. [ ] [ ] [ ] similarly, two types of vaccines are available for polio virus: an injectable polio vaccine (ipv) and opv. sabin opv, with its enormous impact in disease control worldwide, is considered to be the prototype oral vaccine. superiority of opv over ipv is attributed to the induction of high titers of mucosal iga antibodies. , despite the generation of higher serum antibody titers, ipv provides limited intestinal immunity. due to the risk of rare cases of vaccine associated paralytic polio, most developed countries have switched to either ipv or a sequential combination of ipv and opv. , based on the substantial experience in control of mucosal pathogens using parenterally administered vaccines, several parenteral vaccines are being developed or evaluated against sars-cov- . notably, dna-based vaccine by inovio pharmaceuticals (ino- ), mrna-based vaccine by moderna (mrna- ), adenovirus-vectored vaccine (chadox ), and an inactivated virus vaccine (picovacc) are currently under investigation in human clinical trials. [ ] [ ] [ ] [ ] rapid licensure and production of a mucosal vaccine time is the most valuable asset in the rapidly emerging covid- pandemic, especially in high-mortality areas. rapid development and testing of a viable vaccine can be achieved by adopting a number of novel strategies. instead of developing a novel vaccine development platform, utilizing an existing vaccine platform will accelerate the design and production of vaccines against sars-cov- and will enable a quicker future response against newly emerging viruses. since the outbreaks of sars-cov and mers-cov, several vaccine candidates were developed using different production platforms. these established platforms are being used for the development of vaccine candidate against sars-cov- . , , to expedite the process for licensure and use of the vaccine against sars-cov- in the wake of the current pandemic, eyal et al. have suggested an alternative model for accelerated rollout of an effective sars-cov- vaccine by skipping phase clinical trials. this study proposes a controlled human challenge model wherein nonsusceptible adult volunteers from a healthy group will be challenged with an increasing dose of live virus to determine a dose that induces clinical symptoms similar to the natural infection in the individuals of similar age (preparatory phase). after this dose for controlled human challenge model has been optimized, a randomized cohort of volunteers will receive the candidate vaccine or placebo before being challenged with the controlled dose of the live virus (phase ). the successful vaccine candidates can then be administered to a large cohort of individuals for evaluation of shortterm safety prior to vaccine rollout (phase ). this accelerated licensure of the vaccine can be followed up with post-approval surveillance to monitor the occurrence of rare side effects and long-term efficacy for future regulatory approvals. the overwhelming demand for a sars-cov- vaccine in the current pandemic by far exceeds the existing production capacity. to deliver required quantities of the successful vaccine, production process will have to be ramped up to an enormous scale. the necessary infrastructure can be developed simultaneously with the continued production on existing capacity. alternatively, rapid manufacturing of a successful vaccine can be facilitated by adopting a new pandemic paradigm, with a fast start and many steps of vaccine development and production executed in parallel before confirming a successful outcome of another step. in recent years, rapid production of live attenuated vaccines is being facilitated by utilizing reverse genetics process that involves the precise deletion of specific genes of viral genome. deletion of pathogenic components of the pathogen is advantageous over traditional methods of viral attenuation in terms of safety and efficacy of a vaccine. the current covid- pandemic has virtually brought most world economies to a halt and severely impacted the lives of a large proportion of the world population. development of a viable sars-cov vaccine is imperative to reduce mortality and morbidity associated with this novel virus outbreak. mucosal vaccines offer better patient compliance in terms of the physical and psychological comfort due to absence of needlestick injury and thus are highly compatible for mass immunization in a pandemic scenario. vaccine administration using injection involves the cost of injection device, it's safe disposal, and the employment of trained medical staff which adds a considerable cost for mass vaccination especially in developing countries. mucosally administered vaccines also abrogate the risk of transmission of infections by injection devices. despite our detailed understanding of mucosal system in mice, the complex cellular and molecular interplay of different component of innate immune response to mucosal vaccination in humans is still poorly deciphered. additionally, the emergence and rapid global spread of sars-cov- has provided a very small window for basic and translational studies that propel the development and evaluation of vaccine against a pathogen. while the knowledge gained from previous studies on sars-cov and mers-cov can be used for sars-cov- vaccine development, it is yet uncertain as to what extent it will work for sars-cov- or whether correlates of protection used will faithfully predict protective efficacy. potential ade and waning of vaccine-induced immune response represent other obstacles in the development of a mucosal vaccine against sars-cov- . the currently licensed mucosal vaccines against viral pathogens are exclusively live-attenuated. the development of a safe and immunogenic live-attenuated vaccine is an attractive possibility for mucosal vaccine against sars-cov- . live attenuated vaccines, by definition establish a mild infection at the immunization site and exhibit some level of in-built adjuvanticity thereby ensuring the delivery of a higher antigen dose and better targeting of mucosal inductive sites for immunostimulation. this candidate vaccine can be easily administered orally as encapsulated antigens or can be delivered through the intranasal route via droplets and aerosol spray. covid- pandemic demands for adopting nonconventional approaches for a viable mucosal vaccine development, hastening the vaccine licensure process, utilizing existing vaccine development and manufacturing platforms, and ensuring global distribution of the licensed vaccine in time to minimize the impact of covid- across the globe. it will also need an unprecedented scale-up of the manufacturing process and concerted efforts of the supply chains to make the vaccine available before this pandemic is over. learning from the covid- pandemic, sustained investments in vaccine development and production infrastructure should be made to save human lives and curtail the economic impact associated with a looming viral pandemic. moreover, continued global surveillance and vigilance in the post-pandemic scenario should be practiced to help the world counter a second wave of covid- or other possible future coronavirus outbreaks. the authors declare no potential conflict of interest. the compilation of this review was financially supported by department of science and technology, india science and engineering research board (serb) govt of india [ipa/ / ]. epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features of patients infected with novel coronavirus in wuhan the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov- vaccines: status report origin and evolution of pathogenic coronaviruses coronaviruses: an overview of their replication and pathogenesis the spike protein of sars-cov-a target for vaccine and therapeutic development dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc angiotensin-converting enzyme is a functional receptor for the sars coronavirus a cluster of cases of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada clinical and immunologic features in severe and moderate coronavirus disease zhang w viral dynamics in mild and severe cases of covid- . the lancet infectious diseases dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice covid- , an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics in silico guided drug repurposing to combat sars-cov- by targeting mpro, the key virus specific protease tomar s identification of sars-cov- cell entry inhibitors by drug repurposing using in silico structure-based virtual screening approach sars-cov- e protein is a potential ion channel that can be inhibited by gliclazide and memantine immune response of sows vaccinated with attenuated transmissible gastroenteritis virus (tgev) and recombinant tgev spike protein vaccines and protection of their suckling pigs against virulent tgev challenge exposure recent progress in mucosal vaccine development: potential and limitations mucosal vaccines: strategies and challenges the severe acute respiratory syndrome functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses structural basis of receptor recognition by sars-cov- . nature. sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis prolonged presence of sars-cov- viral rna in faecal samples detection of sars-cov- in different types of clinical specimens enteric involvement of severe acute respiratory syndrome-associated coronavirus infection intranasal immunization with inactivated sars-cov (sars-associated coronavirus) induced local and serum antibodies in mice intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines the mucosal immune system: from fundamental concepts to vaccine development immunity against mucosal pathogens mucosal dendritic cells antiviral immune responses in the genital tract: clues for vaccines normal structure, function, and histology of mucosa-associated lymphoid tissue terminology: nomenclature of mucosa-associated lymphoid tissue interactions of the invasive pathogenssalmonella typhimurium, listeria monocytogenes, and shigella flexneri with m cells and murine peyer's patches intestinal villous m cells: an antigen entry site in the mucosal epithelium induction of gut iga production through t cell-dependent and t cell-independent pathways new concepts in the generation and functions of iga the role of secretory antibodies in infection immunity mucosal immunity and vaccines recent advances in the vaccine development against middle east respiratory syndrome-coronavirus mucosal vaccine design and delivery dna vaccines: a key for inducing long-term cellular immunity recombinant viruses as tools to induce protective cellular immunity against infectious diseases modern subunit vaccines: development, components, and research opportunities comparative evaluation of two severe acute respiratory syndrome (sars) vaccine candidates in mice challenged with sars coronavirus mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (sars-cov) spike protein induces strong mucosal immune responses and provides long-term protection against sars-cov infection singledose, intranasal immunization with recombinant parainfluenza virus expressing middle east respiratory syndrome coronavirus (mers-cov) spike protein protects mice from fatal mers-cov infection single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against mers-cov infection superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length spike protein of middle east respiratory syndrome coronavirus a recombinant vsv-vectored mers-cov vaccine induces neutralizing antibody and t cell responses in rhesus monkeys after single dose immunization expression of sars-coronavirus nucleocapsid protein in escherichia coli and lactococcus lactis for serodiagnosis and mucosal vaccination intranasal immunization with plasmid dna encoding spike protein of sars-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein effect of mucosal and systemic immunization with virus-like particles of severe acute respiratory syndrome coronavirus in mice combination attenuation offers strategy for live attenuated coronavirus vaccines immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study understanding sars-cov- -mediated inflammatory responses: from mechanisms to potential therapeutic tools. virol sin. receptorbinding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets for development of antiviral vaccines structure of the sars-cov- spike receptor-binding domain bound to the ace receptor immunogenicity and protection efficacy of monomeric and trimeric recombinant sars coronavirus spike protein subunit vaccine candidates elicitation of immunity in mice after immunization with the s subunit of the severe acute respiratory syndrome coronavirus the coronavirus nucleocapsid is a multifunctional protein structural genomics of sars-cov- indicates evolutionary conserved functional regions of viral proteins a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov- compared to sars-cov mucosal vaccines: the promise and the challenge vaccine delivery: a matter of size, geometry, kinetics and molecular patterns vision for vaccines against hiv, tuberculosis and malaria the development of mucosal vaccines for both mucosal and systemic immune induction and the roles played by adjuvants enhanced mucosal and systemic immune response with intranasal immunization of mice with hiv peptides entrapped in plg microparticles in combination with ulex europaeus-i lectin as m cell target the basics and underlying mechanisms of mucoadhesion differential expression of lectin-binding sites defines mouse intestinal m-cells pre-clinical evaluation of a novel nanoemulsion-based hepatitis b mucosal vaccine vaccine adjuvants: putting innate immunity to work cpg dna as a vaccine adjuvant heat-labile enterotoxins as adjuvants or anti-inflammatory agents dendritic cells and immune responses to orally administered antigens oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens in vivo targeting of antigens to maturing dendritic cells via the dec- receptor improves t cell vaccination triggering tlr signaling in vaccination the time course of the immune response to experimental coronavirus infection of man two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome duration of antibody responses after severe acute respiratory syndrome evaluation of events occurring at mucosal surfaces: techniques used to collect and analyze mucosal secretions and cells correlates of protection induced by vaccination systems biological approaches for mucosal vaccine development middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques pneumonia from human coronavirus in a macaque model sars virus infection of cats and ferrets prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice severe acute respiratory syndrome coronavirus infection of golden syrian hamsters severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques the pathogenicity of sars-cov- in hace transgenic mice enhanced immunogenicity of an oral inactivated cholera vaccine in infants in bangladesh obtained by zinc supplementation and by temporary withholding breast-feeding factors affecting the immunogenicity of oral poliovirus vaccine in developing countries mucosal immunization: a realistic alternative hpv- l vlp vaccine elicits a broad-spectrum of cytokine responses in whole blood evaluation of the long-term anti-human papillomavirus (hpv ), , , and immune responses generated by the quadrivalent hpv vaccine quadrivalent human papillomavirus (hpv) vaccine induces hpv-specific antibodies in the oral cavity: results from the mid-adult male vaccine trial long-term persistence of systemic and mucosal immune response to hpv- / as -adjuvanted vaccine in preteen/adolescent girls and young women live and inactivated influenza vaccines induce similar humoral responses, but only live vaccines induce diverse t-cell responses in young children development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza a virus vaccine comparative efficacy of inactivated and live attenuated influenza vaccines live attenuated versus inactivated influenza vaccine in infants and young children current status of live attenuated influenza vaccine in the united states for seasonal and pandemic influenza mucosal immunity induced by enhanced-potency inactivated and oral polio vaccines immunoglobulin response in serum and secretions after immunization with live and inactivated poliovaccine and natural infection vaccine policy changes and epidemiology of poliomyelitis in the united states polio vaccination: past, present and future immunogenicity of a dna vaccine candidate for covid- an mrna vaccine against sars-cov- -preliminary report chadox ncov- vaccination prevents sars-cov- pneumonia in rhesus macaques development of an inactivated vaccine candidate for sars-cov- . science. microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development human challenge studies to accelerate coronavirus vaccine licensure developing covid- vaccines at pandemic speed an improved reverse genetics system for influenza a virus generation and its implications for vaccine production key: cord- -rtac hh authors: bhatia, saurabh; dahiya, randhir title: chapter edible vaccines date: - - journal: modern applications of plant biotechnology in pharmaceutical sciences doi: . /b - - - - . - sha: doc_id: cord_uid: rtac hh abstract vaccines are considered primary tools for health intervention in both humans and animals. vaccines can be used more widely, especially in developing countries, if their cost of production can be reduced and they can be preserved without refrigeration. in developing countries certain limitations, like vaccine affordability, the need for “cold chains” from the producer to the site of use of the vaccine, and the dependence on injection, are barriers to health care services. plant-derived vaccines do not face such limitations. research under way is dedicated to solving these limitations by finding ways to produce oral (edible) vaccines from transgenic plants. plant-derived vaccines offer increased safety, envisage low-cost programs for mass vaccination, and propose a wider use of vaccination for veterinary use. with the advent of modern molecular biology techniques in the s, new strategies were developed for the production of vaccines. these vaccines are comprised of proteins derived from pathogenic viruses, bacteria, or parasites (generally proteins are produced not by the pathogens themselves, but by expression of the gene encoding the protein in a "surrogate organism") [ ] . in the last decade, it was found that green plants can also be used as the "surrogate production organism" to produce antigens of human pathogens (including hb-sag). these proteins can elicit priming and also can boost the immune response in humans when administered orally. in addition, unlike almost all other cell lines used for production of vaccines, components of plant cells have always been an important part of the normal human diet. plants, therefore, offer significant new opportunities for making safe and effective oral vaccines [ ] . the introduction of selected desired genes into plants and then inducing these altered plants to produce the encoded proteins is the primary condition for the development of edible vaccines. this process is known as transformation and altered plants are called transgenic plants. selection of important epitope region(s) from the pathogen of interest is the one of the key factors that determines the success of potential edible vaccines. edible vaccine development has been challenged by low expression levels of foreign proteins in transgenic plants. reported expression rates range from . % to % total soluble protein (tsp), which can render edible vaccine proteins less immunogenic. selection of strong plant-specific super promoters to improve expression levels is another key factor that can determine the success of edible vaccines [ ] . after transformation of tobacco, great efforts have been made to develop efficient methods for genetic transformation and optimizing expression of foreign genes in plants. the techniques used to introduce foreign genes into plants have been extended to major crops, vegetables, and ornamental and medicinal plants. various foreign proteins including serum albumin, human a-interferon, human erythroprotein, and murine igg and iga immunoglobulins have been successfully expressed in plants. in recent years, several attempts have been made to produce various antigens and antibodies in plants. antigens or antibodies expressed in plants can be administered orally as any edible part of the plant, or by the parenteral route (such as intramuscular or intravenous injection) after isolation and purification from the plant tissue. the edible part of the plant to be used as a vaccine is fed raw to experimental animals or humans to prevent possible denaturation during cooking, and avoid cumbersome purification protocols [ ] . while agrobacterium-mediated transformation still remains the method of choice for dicots, a general method, the biolistics method, of transformation of plants, including monocots, has come into existence. other strategies for expression of foreign genes in plants include use of strong and organ-specific plant promoters, targeting of the protein into endoplasmic reticulum (er) by incorporating er-targeting and er-retention signals, creation of an optimized translation start site context, as well as alteration of codons to suit the expression of prokaryotic genes in a plant. for the production of edible vaccines or antibodies, it is desirable to select a plant whose products can be consumed raw to avoid degradation during cooking. thus, plants like tomato, banana, and cucumber are generally the plants of choice. while expression of a gene into the genome allows maintenance of the material in the form of seeds, some virus-based vectors can also be used to express the gene transiently to develop the products in a short period ( fig. . ). this may have the additional advantage of allowing expression of the product at a very high level; not always attainable in transgenic systems [ ] . plus-sense, single-stranded plant rna viruses have been proposed as an effective alternative to produce vaccine antigens in plants. in this technique, the epitope of interest is engineered into a plant virus, usually within the coat protein gene. infection of a susceptible non-gm-plant results in intracellular production and accumulation of the epitope. the epitope sequence, as well as the viral genome, never become integrated into the plant genome and hence are only expressed by the generation of infected cells. a recombinant cowpea mosaic virus has shown to elicit protective immunity in mink when engineered to express the antigenic epitope against mink enteritis virus. recombinant alfalfa mosaic virus (almv) has enabled expression of significant quantities of rabies virus and hiv epitopes upon integration of their respective coding sequence into the almv coat protein. the extra sequences were found to protrude from the virion surface without interfering with virus assembly. recombinant almv coat protein molecules have also demonstrated the ability to assemble into particles containing three different epitopes from hiv and rabies. this demonstrates the ability of plant viruses to produce multicomponent vaccines [ ] . claimed advantages of transient viral expression of transgenes over transgenic plants are: lesser time for cloning of the foreign gene in the viral genome as compared to time required for transformation of plants; the ease at which antigen production can be scaled up; and the wide host range of plant viruses, which allows use of multiple plant species as biofactories. each single antigen expressed in plants must be verified by animal studies and western blots, and quantified by enzyme-linked immunosorbent assay (elisa). most pathogens enter at mucosal surfaces lining the digestive, respiratory, and urinoproductive tracts, which are collectively the largest immunologically active tissue in the body. the mucosal immune system is the first line of defense and the most effective site for vaccination against pathogens. nasal and oral vaccines are most effective for mucosal infections. the goal of oral vaccine is to stimulate both mucosal and humoral immunity against pathogens. edible vaccines when taken orally undergo mastication, and degradation of plant cells occurs in the intestine due to the action of digestive enzymes. peyer's patches are an enriched source of iga producing plasma cells and have the potential to populate mucosal tissue and serve as mucosal immune effector site. the breakdown of edible vaccine occurs near peyer's patches, which consist of - lymphoid nodules on the outer surface of the intestine and also contain follicles from which the germinal center develops after antigenic stimulation. these follicles act as a site for the penetration of antigens in intestinal epithelium. the antigen then comes in contact with m-cells. m-cells express class- major histocompatibility complex molecules, and antigens transported across the mucous membranes by m-cells can activate b-cells within these lymphoid follicles. the activated b-cells leave the lymphoid follicles and migrate to diffuse mucosal associated lymphoid tissue where they differentiate into plasma cells that secrete the iga class of antibodies. these iga antibodies are transported across the epithelial cells into secretions of the lumen where they can interact with antigens present in the lumen (figure . ). • edible vaccines are effective as a delivery vehicle for immunization because adjuvants that enhance the immune response are not required. • edible vaccine can elicit mucosal immunity, which is not observed in traditional vaccines. • edible vaccines are also cost effective in availability, storage, preparation, production, and transportation. vaccines produced by biotechnological methods are stable at room temperature, unlike traditional vaccine, which needs cold chain storage, which multiplies the yearly cost to preserve vaccines. moreover, the seeds of transgenic plants could be dried as there is less moisture content in seeds and the plants with oil or their aqueous extracts possess more storage opportunities. manufacturing cost is low as there is no need for special premises to manufacture them. edible vaccine can be easily produced at mass level in comparison to an animal system. • edible vaccines are well tolerated, as they do not require administration by injection unlike traditional vaccines. thus, there is also a reduced need for medical personnel and risk of contamination is low. the feasibility of oral administration compared to injection is also an advantage. • plant-derived vaccines could be the source for new vaccines combining numerous antigens. these multicomponent vaccines are called second generation vaccines as they allow for several antigens to approach m-cells simultaneously. • edible vaccines are subunit preparations, do not involve attenuated pathogens, and improve the safety of individuals as compared to traditional vaccine since there is no possibility of proteins reforming into infectious organisms. • the separation and purification of vaccines from plant materials is very easy and pathogenic contamination from animal cells can be effectively prevented. • there is the possibility of development of immunotolerance to the vaccine protein or peptide. • consistency of dosage form differs from plant to plant and generation to generation. • protein content varies from plant to plant and generation to generation. • ripeness also affects the proteins that are present in form of antigens in the fruits. • limitations of methods for standardization of plant material/product. • stability of vaccine differs from plant to plant. • some food cannot be eaten raw (e.g., potato) and needs to be cooked, which will denature or weaken the protein present in it. • variable conditions for edible vaccine are also a major problem. potatoes containing vaccine can be stored at °c for longer time, while tomato does not last long at this temperature. thus, these vaccines need to be properly stored to avoid infection through microbial spoilage. • another concern regarding edible vaccine is the need for proper distinguishing characters to identify between "vaccine fruit" and "normal fruit" to avoid maladministration of vaccine, which could lead to tolerance. • the glycosylation pattern of plants and humans is different, which could affect the functions of vaccines. the first report of the production of edible vaccine (a surface protein from streptococcus) in tobacco, at . % of total leaf protein level, appeared in in the form of a patent application published under the international patent cooperation treaty. subsequently, a number of attempts were made to express various antigens in plants (table . ). since acute watery diarrhea is caused by enterotoxigenic escherichia coli and vibrio cholerae that colonize the small intestine and produce one or more enterotoxin, an attempt was made to produce edible vaccine by expressing heat-labile enterotoxin (lt-b) in tobacco. transgenic potatoes were created and grown by charles arntzen hugh s. mason and their colleagues at the boyce thompson institute for plant research, an affiliate of cornell university. transgenic potatoes containing enterotoxin stimulated strong immune responses in animals. an edible vaccine could stimulate an immune response in humans. volunteers ate bite-sized pieces of raw potato that had been genetically engineered to produce part of the toxin secreted by the e. coli bacterium, which causes diarrhea. ten of the volunteers ( %) who ingested the transgenic potatoes had four-fold rises in serum antibodies at some point after immunization, and six of the ( %) developed four-fold rises in intestinal antibodies. the potatoes were well tolerated and no one experienced serious adverse side effects (table . ). tomatoes serve as an ideal candidate for this hiv antigen because unlike other transgenic plants that carry the protein, tomatoes are edible and immune to any thermal process, which helps retain its healing capabilities. even more importantly, compared to bananas tomatoes were found to grow at a high rate of success in russia. for vaccines or subunit vaccinations, bananas seem to be the desired vector. the advantage of bananas is that they can be eaten raw as compared to potatoes or rice, which need to be cooked, and bananas can also be consumed in a pure form. research is leaning toward the use of bananas as the vector since most third world countries, which would benefit most from edible vaccines, are in tropical climates that are suitable for growing bananas. egyptian scientists have genetically engineered maize plants to produce a protein used to make the hepatitis-b virus vaccine. a team of researchers led by hania el-itriby, director of cairo's agricultural genetic engineering research institute, developed genetically modified (gm) maize plants that produce the protein known as hbsag, which elicits an immune response against the hepatitis-b virus and could be used as a vaccine (table . ). when predominant t-cell epitope peptides, which were derived from japanese cedar pollen allergens, were specifically expressed in rice seed and delivered to the mucosal immune system, the development of an allergic immune response of the allergen-specific th cell was suppressed. furthermore, not only were specific ige production and release of histamine from mast cells suppressed, but the inflammatory symptoms of pollinosis, such as sneezing, were also suppressed. these results suggest the feasibility of using an oral immunotherapy agent derived from transgenic plants that accumulate t-cell epitope peptides of allergens for allergy treatment (table . ). when the seed expression system is used as a platform for foreign protein production, substantial amounts of recombinant proteins can be accumulated, because the seed is a natural storage organ for accumulating the starch, protein, and oil required for seedling growth. also, artificial peptides or proteins accumulate in seed, which is in remarkable contrast with other tissues. plant-derived vaccines are free from animal pathogen contaminants. furthermore, plant dna is not known to interact with animal dna and plant viral recombinants does not invade mammalian cells. further safety of plant-derived vaccines can be achieved by following similar regulations established for traditional vaccines. nevertheless, the present concern over the use of gm plants is now affecting research in this important field, especially in europe. one of the fears is that gm pollen may outcross with sexually compatible plants (related crops or weeds) and affect biodiversity. in order to address this alarm, several pollen containment approaches have been developed. these are essentially based on the exploitation of different forms of male sterility (suicide genes, infertility barriers, apomixis). an alternative way of solving the problem is engineering vaccines into the chloroplast dna (cpdna), which is not transmitted to the sexual progeny through the pollen grains. an additional safety feature would be the recognition of gm plants that produce vaccines by the addition of genes encoding colored plant pigments. it is important to recognize that plants that produce vaccines are medicinal plants and should be grown, processed, and regulated as pharmaceutical products. in the majority of earlier papers, level of antigen accumulation in the plant organ was in the order of . - . % of total soluble protein, while the more recent developments on cpdna integration promises to increase this value to % or more. at the latter value, land requirements for industrial plant-derived vaccine production will be in the order of a few thousand square meters. this will definitely enable vaccine-producing plants to be set apart from field grown crop plants and offer added safety when engineered plant viruses are used for transient antigen expression. a further point of public concern in gm plants is the presence of antibiotic resistance genes (used as selective marker in most transgenic plants). approaches have now been developed to generate gm plants (with both nuclear or cpdna integration) that do not carry these genes. edible vaccines edible vaccines: current status and future transgenic plants as factories for biopharmaceuticals using transgenic plants as bioreactors to produce edible vaccines edible vaccines and antibody producing plants medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants expression of chimeric hcv peptide in transgenic tobacco plants infected with recombinant alfalfa mosaic virus for development of a plantderived vaccine against hcv a review of oral vaccination with transgenic vegetables edible vaccines: a concept comes of age food as production and delivery vehicles for human vaccine key: cord- -lldbjm authors: soni, dheeraj; bobbala, sharan; li, sophia; scott, evan a.; dowling, david j. title: the sixth revolution in pediatric vaccinology: immunoengineering and delivery systems date: - - journal: pediatr res doi: . /s - - -y sha: doc_id: cord_uid: lldbjm abstract: infection is the predominant cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. however, very young infants fail to respond optimally to most vaccines currently in use, especially neonates. in , stanley plotkin proposed that new delivery systems would spur a new revolution in pediatric vaccinology, just as attenuation, inactivation, cell culture of viruses, genetic engineering, and adjuvantation had done in preceding decades. recent advances in the field of immunoengineering, which is evolving alongside vaccinology, have begun to increasingly influence vaccine formulation design. historically, the particulate nature of materials used in many vaccine formulations was empiric, often because of the need to stabilize antigens or reduce endotoxin levels. however, present vaccine delivery systems are rationally engineered to mimic the size, shape, and surface chemistry of pathogens, and are therefore often referred to as “pathogen-like particles”. more than a decade from his original assessment, we re-assess plotkin’s prediction. in addition, we highlight how immunoengineering and advanced delivery systems may be uniquely capable of enhancing vaccine responses in vulnerable populations, such as infants. impact: immunoengineering and advanced delivery systems are leading to new developments in pediatric vaccinology. summarizes delivery systems currently in use and development, and prospects for the future. broad overview of immunoengineering’s impact on vaccinology, catering to pediatric clinicians and immunologists. the impact of vaccination on the health of the world's peoples is hard to exaggerate. with the exception of safe water, no other modality has had such a major effect on mortality reduction and population growth (dr. stanley a. plotkin, md, vaccines, ) . so what is my choice for the sixth revolution?… i suggest that it will be new delivery systems (dr. stanley a. plotkin, md, pediatric academic societies meeting, may, ). the goal of vaccination is to trigger an immune response that reduces the risk of infection and prevents disease. initially, delivery systems were redundant since the majority of vaccines employed live attenuated organisms, which were often particulate in nature and inherently carried the abundant and necessary immune-stimulating signals. however, as vaccinology moved towards the development of defined antigens such as inactivated, subunit, and purified recombinant proteins and peptides, which are inadequate to trigger an immune response alone, the use of novel delivery systems became crucial. historically, antigen stabilization (i.e., adsorption onto alum) guided vaccine formulation design. therefore, the particulate nature of materials used in many early vaccine formulations was empiric. in this context, inclusion of alum adjuvants has been key to the acceptable efficacy of these subunit vaccine formulations, even though alumadjuvanted vaccines usually require multiple doses for optimal protection. second-generation efforts employed more characterized materials, such as the biodegradable synthetic polymer poly (d,l-lactic-co-glycolic acid) (plga), which is a widely investigated nanoparticle adjuvant for controlled and effective delivery of vaccine antigens, including synthetic peptides. these are typically produced as solid-core nanoparticles ranging from to nm in size, with antigens entrapped or adsorbed on the surface of the particles. more recently, advances in the field of immunoengineering, which are developing alongside vaccinology, have begun to greatly influence vaccine formulation design. , due to the wide differences in mechanisms of action of various non-adjuvanted and adjuvanted vaccines, vaccine formulation development has become a major consideration for vaccinologists and pharmaceutical companies. these considerations include ( ) physicochemical characteristics of the formulation, ( ) adjuvant chemical structure, ( ) proposed route of administration, and ( ) short-and long-term formulation stability. specifically, vaccine delivery systems can now be engineered to mimic the size, shape, and surface chemistry of pathogens, which are often referred to as "pathogen-like particles". nanoparticle formulations can now be manufactured to target subsets of immune cells and specific subcellular compartments. [ ] [ ] [ ] one aspect consistent across current and future novel vaccines is the need to determine their ability to instruct adaptive immunity through the manipulation of antigen-presenting cells (apcs). as a part of their function as professional apcs, dendritic cells (dcs) can integrate information from extrinsic stimuli (e.g., components of pathogens or vaccines) and orchestrate these signals into appropriately regulated adaptive immune responses. as such, immunoengineering, adjuvant and antigen discovery, vaccine delivery, and increased knowledge of human immune responses are fueling a revolution in vaccinology. more than years ago, stanley plotkin identified the five crucial technical advances that revolutionized the field of vaccinology. these were attenuation ( s; e.g., live attenuated rabies), inactivation ( s onwards; e.g., killed vaccines for typhoid and cholera), cell culture of viruses ( s; cultured poliovirus), genetic engineering ( s; recombinant proteinbased hepatitis b vaccine), and the induction of cell-mediated immunity (i.e., via adjuvantation) (fig. ). dr. plotkin identified six candidate areas that may start a possible sixth revolution (fig. ) . these ranged from the combinatorial employment of older vaccine strategies to newer methods such as reverse vaccinology. ultimately, the pioneering and burgeoning science driving the use of "delivery systems" was his choice to lead the way. interestingly, there has also been remarkable progress in vaccine design and technologies including structure-guided antigen design, broadly neutralizing antibodies and promising novel vaccine platforms (dna, mrna) (fig. ) . many of these advances are reliant, at least in part, on optimized delivery. after nearly a decade and a half, in this review we reevaluate dr. plotkin's vision to assess whether his prediction has been validated or is yet unanswered. recent advances in the nascent field of immunoengineering may allow for the design of vaccine delivery systems that can potentially accelerate the development of novel and effective early life vaccination strategies. immunoengineering may guide vaccine design , by enabling the targeting of dcs, specifically via synthetic vaccine delivery systems that mimic the size, shape, and surface chemistry of pathogens. from an immunoengineering stance, the ideal vaccine formulation is (a) customizable, (b) based upon a scalable synthetic vaccine platform with (c) tunable release kinetics and can (d) stably encapsulate controllable amounts of molecules possessing diverse chemistry and water solubility. such macro-and nanoscale-theranostic (i.e. therapeutic and diagnostic) materials would enable sustained and targeted delivery of complex antigen/adjuvant combinations via nanocarriers for hours, days, weeks, or months. by providing control over intracellular delivery of antigens and adjuvants to apcs, immunoengineering can more effectively activate innate immunity and moreover apcs to boost the adaptive immune response. nanoparticle delivery systems can increase intracellular delivery to apcs by mimicking the morphology of viruses and by s; hepatitis b vaccine (hbv), the st recombinant-antigen-based vaccine, incorporated the viral surface proteins, derived from molecular biology production. combination vaccines: simultaneous administration of vaccines to target multiple diseases. the adjuvant toolbox: ranging from small-molecule adjuvants to combination adjuvants. vaccines for non-infectious diseases: new treatments for tumors, allergy, or noninfectious disorders (e.g., prevention of drug overdose). systems vaccinology: systems biology approaches to identify predictors of vaccine efficacy and explore new insights about protective immunity. reverse vaccinology: bioinformatics aided vaccine design from pathogenic genetics. immunoengineering and delivery systems: delivering precise materials for specific activation of immune system (right time, right place, right size, right shape, etc.). incorporating targeting ligands. , in addition, the size, charge, and morphology of nanoparticles also affect lymph node trafficking. [ ] [ ] [ ] control of nanoparticle delivery to intracellular compartments can be used to modulate the immune response post-vaccination. delivery of antigens to the cytosolic or endocytic compartments respectively leads to antigen presentation via major histocompatibility complex (mhc) class i or mhc class ii to prime distinct t cell subsets. this can be achieved by engineering antigen-loaded nanoparticles to induce endosomal or lysosomal escape, leading to antigen cross-presentation. , furthermore, the targeting of nanoparticles carrying toll-like receptor (tlr) agonists to endosomes leads to activation of endosomal tlrs, promoting strong th -type responses. [ ] [ ] [ ] activation of the inflammasome through cytosolic nod-like receptors (nlrs) can lead to a self-adjuvant effect by the nanoparticle vehicle itself that promotes adaptive immunity. [ ] [ ] [ ] lastly, nanoparticles containing vaccines can be used to control the duration of delivery through synchronous and sustained release of antigens and adjuvants. , delivery kinetics can be further modified using strategies such as incorporating nanoparticles into hydrogels or intradermal microneedle delivery systems. [ ] [ ] [ ] these sustained release systems can allow for the development of single-dose vaccines. thus, pathogen-mimicking nanoparticles can be engineered to enhance the immune response by controlling when and where vaccine components are delivered intracellularly to apcs. a plethora of particulate delivery systems for immunoengineering have been developed, which are summarized further in this review. liposomes have been considered a dominant vaccine delivery platform because of their superior adjuvant properties and versatility in accommodating vaccine components. , liposomes are spherical vesicle structures comprising a phospholipid bilayer shell and aqueous lumen with sizes ranging from a few nanometers to several microns. , these vesicles have been engineered to optimize their immunological role using several approaches including modulation of size and charge, , surface decoration with immune cell recognition epitopes, , and pegylation to enhance in vivo circulation times. an adjuvanted-liposomal vaccine formulation as b is marketed by glaxosmithkline as a component of shingles vaccine (shingrix) and is also in human clinical trials for malaria and hiv vaccines. of note, cationic liposomes have shown high adjuvanticity, and a cationic liposomal formulation cfa is currently being tested in human clinical trials for hiv and tuberculosis vaccines. virosomes virosomes are nanosized phospholipid vesicles with membranes incorporating viral envelope proteins, typically produced from reconstituted empty envelopes of influenza viruses. they are utilized as both a vaccine carrier system and as an adjuvant, where the antigen of interest is either adsorbed or encapsulated within the lumen. , virosomes have been reported to induce both cellular and humoral immunity through efficient presentation of antigen via both mhc class i and ii proteins; however, their exact mode of action is still unclear. virosome-based influenza vaccines are licensed in europe (as inflexal) and as adjuvants for hepatitis a vaccine (as epaxal). immune-stimulating complexes (iscoms) are spherical cage-like nanoparticles (~ nm) formed via self-assembly of a mixture of the saponin adjuvant quil a, cholesterol, phospholipids, and antigens. iscoms in the absence of an antigen are called iscomatrix and can be mixed with any antigen of interest. iscoms have been reported to stimulate enhanced cellular responses with lower antigen doses through enhanced antigen cross-presentation. however, the role of individual components in generating these immune responses is unknown. at present, iscom technology has been approved only for veterinary vaccines; however human clinical trials are currently ongoing for the development of melanoma vaccines. modern-day vaccine research is highly dependent on flexible engineering strategies such as tunable immune cell recognition epitopes, morphological diversity, and precise intracellular delivery. polymer-based systems have been considered frontrunners for incorporating these strategies as compared to their lipid-based counterparts. polymeric delivery vehicles are mainly classified as solid-core or self-assembled. these delivery vehicles have an additional advantage of greater physicochemical and in vivo stability as compared to lipid-based systems. solid-core particles plga micro-and nanoparticles have been widely explored for vaccine delivery applications because of their ease of fabrication, amenability to surface modification, encapsulation efficiency of both hydrophilic and hydrophobic vaccine components, and modifiable release properties. that the depot forming ability of plga microspheres at the injection site could be a potential single-shot vaccination strategy. , the ability of plga particulate vaccines to generate strong cytotoxic responses make them great candidates for the development of vaccines against infectious diseases , and cancer. another interesting solid-core vaccine delivery vehicle, pluronicstabilized polypropylene sulfide (pps) nanoparticles, have been developed. , these particles contain a hydrophobic pps core, which becomes hydrophilic under oxidative conditions of cell endosomes, promoting intracellular release. reddy et al. showed highly efficient transportation of the pps nanoparticles ( nm) through lymphatic capillaries to target half of the lymph node resident dcs and unveiled the role of pps surface chemistry in activating the complement cascade. further, pps nanoparticles conjugated with a model protein antigen ova stimulated both cellular and humoral responses. , self-assembled particles the self-assembly of polymeric structures is often achieved using block copolymers, which comprise linked hydrophobic and hydrophilic polymer blocks. the application of diverse block copolymer chemistries and self-assembled morphologies has been evaluated for immunomodulation. , the simplest morphology that can be attained is a micelle, which contains a hydrophobic core and a hydrophilic corona. polylactide (pla)-based block copolymer micelles have been widely explored for vaccine delivery. specifically, polyethylene glycol (peg)-b-pla block copolymer micelles have been reported to show excellent biocompatibility, immunomodulatory ability, and success in inducing cellular immunity. recently, environment-responsive micelles consisting of ph- or oxidation-responsive block copolymers have shown great promise in releasing vaccine components inside cell lysosomes. there is also great interest in developing polymersomes, which are self-assembled polymeric vesicles analogous to liposomes, for delivering vaccines and inducing cellular immunity. peg-b-pps, an oxidation-responsive block copolymer, has been reported to self-assemble into monodisperse polymersomes and encapsulate a wide range of antigens and adjuvants. , stano et al. reported that peg-b-pps polymersomes loaded with antigen (ova) and adjuvant (cpg) induced enhanced cd + t cell responses in the spleen and lymph nodes. furthermore, peg-b-pps block copolymers have been engineered to self-assemble into other diverse morphological structures. these block copolymers with peg weight fractions (f peg ) of . , . , . , and . selfassemble to form bicontinuous nanospheres (polymeric cubosomes), vesicles (polymersomes), cylindrical micelles (filomicelles), and micelles, respectively. recent studies revealed that these structures have differential organ biodistributions and immune cell uptake in vivo, , which makes them strong contenders for the development of new rationally designed immunotherapies and vaccines. aluminum salts (alum) including aluminum hydroxide and aluminum phosphate are the most commonly used adjuvants in human vaccines. in aluminum salt-based vaccine formulations, antigens are adsorbed to the highly charged aluminum hydroxide or aluminum phosphate gel. the molecular mechanisms by which alum interacts with the human immune system continues to be studied and involves multiple pathways, both direct and indirect. the majority of studies suggest that the adjuvant action of aluminum salts is mediated by activation of the nlrp inflammasome. alum may directly or indirectly trigger innate immunity via activation of inflammasome complexes, required for the processing of il- family pro-inflammatory cytokines. this process is most likely nlr-mediated, since the adjuvant effects of alum are not impaired in the absence of key tlr-dependent signal transduction adaptor molecules myd and trif in knockout mice. secondly, alum enhances delivery of antigen to apcs as particulate vaccine formulations more readily interact with dcs and macrophages than soluble formulations of antigens alone. crystalline alum binds lipids on the surface of dcs and triggers a cellular activation cascade leading to initiation of an immune response, but without itself being internalized by the cells. thirdly, alum-induced cell death seems to modulate the local milieu in favor of enhanced adaptive immune stimulation. the release of damage-associated molecular patterns, such as uric acid and dsdna, act as autologously derived auto-adjuvants. induction of humoral immunity is a hallmark feature of aluminum-containing adjuvants. for instance, alum-adjuvanted vaccines often require multiple doses for induced protection, and drive th -over th -polarized immunity. presently, there are multiple licensed pediatric vaccines, such as diphtheria, tetanus, and hepatitis vaccines, listing alum as essential to produce effective antibody (ab) titers. several new strategies have been considered to modify these alum particles for induction of cellular responses. for example, liu et al. encapsulated alum colloid inside a yeast-derived β-glucan particle, which induced greater cellular immune responses. another group, wang et al., made phospholipid bilayer-coated aluminum nanoparticles that were readily taken up by the apcs and stimulated antigen-specific cellular and humoral responses in vivo. however, there are also vaccinal antigens for which addition of alum may not be necessary for effective immunogenicity. for example, alum was excluded from the recent vaccine menveo (menacwy) due to failure of alum to enhance improvement in serum bactericidal ab titers during infant clinical trials. gold nanoparticles are versatile systems that can be easily synthesized into different morphologies and are able to accommodate a diverse range of antigens and adjuvants onto their surface. in several studies, gold particles have been reported to have efficient accumulation in dcs and b cells, playing a critical role in generating cellular and humoral responses. , recent findings on gold nanoparticle-based cancer vaccines demonstrated that the photothermal ablation ability of gold particles alongside cellular cytotoxic responses can have a synergistic impact in cancer treatment. additionally, inorganic particles like carbon nanotubes, mesoporous silica, and iron oxide nanoparticles have been widely explored as vaccine delivery vehicles. however, more understanding of their toxicity and safety profiles may be required for the advancement of these nanoparticles. diverse water-in-oil emulsions, of which incomplete freund's adjuvant is the best-known, were originally evaluated in human trials during the mid-twentieth century. muscle after intramuscular injection, the mechanism of action of which is tlr-independent. for example, the adjuvant mf does not directly mature apcs, but rather induces the production of chemokines and immune modulatory proteins from monocytes, macrophages, granulocytes, and muscle cells that indirectly leads to increased migration of apcs to/from the site of injection and into draining lymph nodes. this cell recruitment is greater than that induced by alum. mf may also instruct peripheral site monocyte differentiation into dcs and possibly induce the release of endogenous tlr agonists. therefore, one major advantage of o/w adjuvants is the antigen dose-sparing potential. since vaccine development pipelines rarely tailor formulations (adjuvants, delivery systems, etc.) rationally for use in early life, it is critical to understand the optimization of vaccine efficacy by taking into account early life immune ontogeny. [ ] [ ] [ ] for example, early life vaccination against intracellular pathogens has proven difficult. due to functionally distinct and delayed t cell-mediated immunity, newborns and young infants are highly susceptible to infection with intracellular pathogens, including bacteria such as listeria spp. and salmonella spp., viruses such as herpes simplex virus and respiratory syncytial virus, and intracellular pathogens of global significance such as hiv, tuberculosis, and malaria. here, nanoparticle-based formulations may hold great promise for pediatric vaccine development. targeting the key deficiencies of newborn dcs relative to adult dcs may enable development of age-specific vaccine formulations to overcome sub-optimal immunization responses. recently, we have combined such engineering and rational vaccine design approaches to develop a nanoparticle-based adjuvant and antigen-delivery system designed to be active in human newborns and infants. the design mimicked, and in some instances, exceeded the immunostimulatory effect of live attenuated vaccines. we employed peg-b-pps polymersomes, which are significantly more stable than liposomes, as the effective adjuvantand antigen-delivery system. these polymersomes can be effectively engineered for bioresponsive intracellular payload delivery, making them highly advantageous for the specific targeting of endosomal receptors. this is notable, since the activation of endosomal pattern recognition receptors (prrs), as compared to surface prrs (e.g. tlr and tlr ), instructs more adult-like innate immune responses in newborn dcs. smallmolecule tlr agonists (e.g. imidazoquinoline) robustly activate newborn dcs but can result in systemic responses when delivered in soluble form. to overcome this and minimize off-target effects, we developed tlr -agonist-encapsulating polymersomes, which demonstrated a preference for uptake by dcs relative to other cell populations following subcutaneous administration. furthermore, such formulations hold substantial potential for early life immunization by serving as a dual antigen/adjuvant delivery system that mimics the enhanced neonatal innate and adaptive immune responses elicited by the live bacille calmette-guerin (bcg) vaccine. strikingly, when co-loaded with the mycobacterium tuberculosis antigen b peptide , the tlr -agonist containing polymersomes were comparable to bcg in inducing antigenspecific immune responses in human tlr -expressing neonatal mice in vivo. this is promising, since bcg reduces the risk of disseminated early life tuberculosis by safely eliciting th -type neonatal immune responses and requires only a single dose at or shortly after the time of birth. another promising approach is the use of microstructures designed for controlled release of vaccine formulations in vivo. mchugh et al. recently developed a microstructure called stamped assembly of polymer layers (seal). seals are small (≤ - μm) plga-based polymeric microdevices with complex geometries, which can be filled with soluble drug solutions and then completely sealed. such structures can be rationally designed with additive manufacturing processes (i.e., threedimensional ( d) printing). importantly, depending on the formulation and chemical composition, these materials can be tuned to achieve continuous or delayed/pulsatile release kinetics. this was achieved by tuning the degradation of the materials (i.e., the copolymer ratio). as opposed to solidparticulate delivery systems formulated to deliver antigen continuously, which demonstrate an initial burst and then slow release thereafter, such "pulsatile vaccines" more closely resemble traditional vaccine schedules, which are based around the concept of priming followed by multi-booster injections, but with the advantage of requiring only a single immunization. this may be especially promising for combinatorial vaccination approaches that require multiple boosters, as commonly used in the pediatric setting, or induction of protective immunity that may rely on persistent pathogenic antigen exposure. these and other such technologies may ultimately open the way for single-injection vaccine strategies. , reassessing vaccination schedules and expanding vaccine target populations most vaccination schedules throughout the world are designed for the pediatric age group. notably, most schedules recommend children to be immunized against hepatitis b virus (and often tuberculosis through the bcg vaccine) starting in the neonatal period. immunization to rotavirus, diphtheria, tetanus, pertussis, h. influenzae type b, streptococcus pneumoniae, poliovirus, influenza, measles, mumps, rubella, varicella, hepatitis a virus, meningococcal, and human papillomavirus extend from months of age up to adolescence. as the twenty-first century progresses, the current immunization schedule should remain flexible to incorporate newly invented vaccines as they become available with emerging and reemerging infectious diseases, such as meningococcus, influenza virus, group a streptococcus, helicobacter pylori, and respiratory syncytial virus (possibly the most important infant vaccine still missing from this clinical schedule). in addition, expanding the current schedule to cover more vaccine-preventable infectious diseases and including vaccine formulations incorporating novel immunoengineered delivery systems may be key tools to allow for an accelerated schedule approach ( table ) . as outlined above, with the exception of the hepatitis b virus vaccine and bcg (which in some countries are given at birth), present immunization schedules start mostly after months of age. consequently, the present immunization schedules do not induce protection against the majority of these diseases until the fifth-sixth months of life or later. this creates a period of vulnerability during the first months of life, which is associated with significant mortality and morbidity. therefore, current aspirational efforts often focus on the development of (a) single-dose immunization strategies for newborns that instruct lifelong protection to subsequent challenges or (b) optimizing maternal immunization as means to cover this susceptibility gap. the sixth revolution in pediatric vaccinology: immunoengineering and. . . d soni et al. heterologous prime-boost vaccination strategies (e.g. an attenuated vaccine followed by an inactivated vaccine targeting the same pathogen) may also be advantageous in early life, especially if they incorporate the same antigen but distinct delivery systems. current prime-boost regimens incorporating viral vectors or dna vaccines, followed by a boost with a protein-based vaccine may be able to overcome distinct immune ontogeny in early life while also taking advantage of the relatively lower rates of reactogenicity in the neonatal and early infant periods. furthermore, increased appreciation of immune ontogeny may inform development of rationally designed age-specific vaccine formulations. future studies need to focus not only on safety and efficacy, but also on potential vaccine-vaccine interactions that can lead to interference, and possibly include adjuvants that more effectively enhance immune responses in early life. innate immune memory, i.e. an alteration of reactivity in innate immune cells previously exposed to diverse stimuli, may also confer heterologous immunity that could be leveraged by some adjuvanted vaccine formulations in the future. vaccination holds promise for primary prevention not only for neonates and infants but also for diverse age-and target groups, including adults, elderly, adolescents, pregnant women, preterm infants, fetuses, and people suffering from chronic and immunecompromising diseases. these groups represent heterogeneous populations, separated by genetic background, gender, pregnancy, microbiome, diet, lifestyle, poverty, risk of infectious diseases, and geography (those in the developed and developing world where future vaccines would be deployed) among others. therefore, considering these broad differences, it is critical to understand how novel delivery systems may be utilized effectively to optimize vaccine efficacy. two of foremost the target populations are pregnant women and preterm infants. notably, immunity of preterm infants is distinct from newborns, rendering them particularly susceptible to infection. , every year,~ million newborns are born preterm worldwide, which is~ % of all live births. preterm infants demonstrate impairments in innate and acquired immunity, significantly less maternal-derived ab than term infants, and a higher risk of infection-induced disability and death. early life immunization to close the window of disease vulnerability may be key to preventing these infections, but immune responses to subunit vaccines are impaired among preterm infants. for example, although administered at birth to term infants, hepatitis b virus immunization is often delayed in preterms due to reduced immunogenicity in this population. similarly, preterm infants demonstrate impaired serotype-specific immune responses to pneumococcal conjugate vaccine (pcv)- . novel use of preexisting and clinical licensed vaccine formulations may shed light on areas of optimization. co-administration of bcg with hbv, for example, significantly enhanced anti-hbv igg titers in mouse models of both term and preterm birth, equally, but not in adult mice. accordingly, there is an unmet need for safe and immunogenic vaccines for preterm infants, including those targeting hbv and pneumococcus. maternal vaccination has emerged as a favorable public health approach in the past decade. this approach is highly promising as it may decrease maternal, fetal, and neonatal susceptibility to infections. while implementation may vary by region, currently a number of vaccines are universally recommended/indicated during pregnancy. these include vaccines against tetanus, influenza, and pertussis, along with new vaccines for group b streptococcus and respiratory syncytial virus that are currently being developed to prevent neonatal infections. as the efficacy of maternal vaccines significantly relies on active transport of antibodies at the maternal-fetal interface (through the placenta using an fc receptor and concentrated in the fetus), antigen/ table . potential uses and benefits of "novel delivery systems" to future early life vaccination strategies. • instruct an accelerated, targeted, potent, and durable immune response in humans against pathogens (e.g. overcome pathogen diversity and immune evasion) • allow for dose sparing and reduced vaccine manufacturing costs, thereby increasing the global access to pediatric vaccines • dramatically increase the number of antigens per formulation/immunization • modulation of antigen delivery and persistence (i.e., single bolus vs. slow release formulations) • act as immunomodulators to enhance th (e.g., t helper [th ] cell versus th ) or achieve qualitative alteration of the immune response (cd + versus cd + t cells) • allow cell-mediated t cell vaccination strategies • induction of un-natural immunity (i.e., broadly protective universal vaccines) • instruct heterologous immunity, thereby reducing overall mortality rates the sixth revolution in pediatric vaccinology: immunoengineering and. . . d soni et al. adjuvant delivery systems may become key tools in expanding these efforts to cover a broader range of diseases. in addition, adjuvanted vaccine formulations may be specifically designed to polarize maternal responses to minimize interference with infant responses to subsequent vaccination or infection. lastly, passive immunization (via the use of monoclonal abs) may also be considered a relevant tool in areas where no vaccine currently exists. this includes the mab palivizumab (brand name synagis), produced by recombinant dna technology for the prevention of rsv infections, especially in at-risk preterm infants. modern vaccine design and development strategies endeavor to integrate knowledge of formulation composition and understanding of immunostimulatory mechanisms to generate immune responses otherwise unachievable in relevant target populations. , rational vaccine design approaches, employing novel immunoengineering strategies, and targeted delivery systems may allow for the controlled preparation of vaccine formulations of the desired immunostimulatory properties, particulate size, and antigen load, all of which can greatly improve safety and limit systemic toxicities due to their targeted nature. these potential advances may be key to tailored vaccines capable of matching unique characteristics of the developing immune system during the neonatal period and infancy. plotkin highlighted various candidates for the sixth revolution of vaccinology, including the expanded use of combination vaccine strategies, new adjuvants, vaccines for non-infectious diseases, and the advent of systems vaccinology. ultimately, he settled on new delivery systems. interestingly, just as the breakthrough in cell culture expanded the role of attenuation and killed vaccine strategies, breakthroughs in immunoengineering and novel delivery systems have led to multitudes of promising vaccine platforms with great potential to advance innovation in pediatric vaccinology. immune response to vaccine adjuvants during the first year of life ontogeny of early life immunity recent progress in adjuvant discovery for peptide-based subunit vaccines materials engineering for immunomodulation engineering approaches to immunotherapy vaccine adjuvant formulations: a pharmaceutical perspective vaccine delivery: a matter of size, geometry, kinetics and molecular patterns precision intracellular delivery based on optofluidic polymersome rupture tailoring nanostructure morphology for enhanced targeting of dendritic cells in atherosclerosis celastrol-loaded peg-b-pps nanocarriers as an antiinflammatory treatment for atherosclerosis immunological mechanisms of vaccination accelerating next-generation vaccine development for global disease prevention six revolutions in vaccinology mrna vaccines-a new era in vaccinology applications of nanotechnology for immunology targeting dendritic cells with biomaterials: developing the next generation of vaccines cd -targeted dendritic cell delivery of plga-nanoparticle vaccines induce potent anti-tumor responses nanoparticles target distinct dendritic cell populations according to their size plant viral nanoparticles-based her vaccine: immune response influenced by differential transport, localization and cellular interactions of particulate carriers nanoparticle surface charge impacts distribution, uptake and lymph node trafficking by pulmonary antigen-presenting cells exploration of antigen induced caco nanoparticles for therapeutic vaccine rapid endo-lysosomal escape of poly (dl-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery multivalent polymer nanocomplex targeting endosomal receptor of immune cells for enhanced antitumor and systemic memory response in vivo characterization of the physicochemical properties of polymer-linked tlr agonists that enhance vaccine immunogenicity dendritic cell activation and t cell priming with adjuvant-and antigen-loaded oxidation-sensitive polymersomes activation of the nlrp inflammasome by vault nanoparticles expressing a chlamydial epitope the role of inflammasomes in the immunostimulatory effects of particulate vaccine adjuvants aminated nanomicelles as a designer vaccine adjuvant to trigger inflammasomes and multiple arms of the innate immune response in lymph nodes poloxamer -chitosan grafted thermoresponsive hydrogels achieve synchronous and sustained release of antigen and adjuvant from single-shot vaccines hollow microneedle-mediated intradermal delivery of model vaccine antigen-loaded plga nanoparticles elicits protective t cellmediated immunity to an intracellular bacterium sustained micellar delivery via inducible transitions in nanostructure morphology is there an optimal formulation and delivery strategy for subunit vaccines? designing liposomal adjuvants for the next generation of vaccines a malaria vaccine adjuvant based on recombinant antigen binding to liposomes liposome-encapsulated human immunodeficiency virus- gp induces potent v v -specific antibodies in humans liposomes and polymersomes: a comparative review towards cell mimicking effect of surface charge on the sizedependent cellular internalization of liposomes the role of surface charge density in cationic liposome-promoted dendritic cell maturation and vaccine-induced immune responses mannose derivative and lipid a dually decorated cationic liposomes as an effective cold chain free oral mucosal vaccine adjuvant-delivery system liposomes coated with isolated macrophage membrane can target lung metastasis of breast cancer comparison of two different pegylation strategies for the liposomal adjuvant caf : towards induction of ctl responses upon subcutaneous vaccine administration shingrix: the new adjuvanted recombinant herpes zoster vaccine phase / a trial of plasmodium vivax malaria vaccine candidate vmp /as b in malaria-naive adults: safety, immunogenicity, and efficacy long-term follow-up of hiv- -infected adults who received the f /as b hiv- vaccine candidate in two randomised controlled trials efficient eradication of established tumors in mice with cationic liposome-based synthetic long-peptide vaccines therapeutic vaccination using cationic liposomeadjuvanted hiv type peptides representing hla-supertype-restricted subdominant t cell epitopes: safety, immunogenicity, and feasibility in guinea-bissau a novel liposomal adjuvant system, caf , promotes longlived mycobacterium tuberculosis-specific t-cell responses in human liposome technology monophosphoryl lipid a-adjuvanted virosomes with ni-chelating lipids for attachment of conserved viral proteins as cross-protective influenza vaccine influenza t-cell epitope-loaded virosomes adjuvanted with cpg as a potential influenza vaccine adjuvants enhancing cross-presentation by dendritic cells: the key to more effective vaccines impact of mixed equine influenza vaccination on correlate of protection in horses low-dose cyclophosphamide enhances antigen-specific cd + t cell responses to ny-eso- /iscomatrix™ vaccine in patients with advanced melanoma overcoming immune dysregulation with immunoengineered nanobiomaterials plga particulate delivery systems for subunit vaccines: linking particle properties to immunogenicity synthetic nanoparticles for vaccines and immunotherapy combined poly (lactide-co-glycolide) microspheres containing diphtheria toxoid for a single-shot immunization efficacy of plga microparticle-encapsulated formalin-killed aeromonas hydrophila cells as a single-shot vaccine against a. hydrophila infection plga particulate subunit tuberculosis vaccines promote humoral and th responses but do not enhance control of mycobacterium tuberculosis infection nanoparticle vaccines against infectious diseases polymeric nanoparticles encapsulating novel tlr / agonists as immunostimulatory adjuvants for enhanced cancer immunotherapy in vivo targeting of dendritic cells in lymph nodes with poly (propylene sulfide) nanoparticles exploiting lymphatic transport and complement activation in nanoparticle vaccines tunable t cell immunity towards a protein antigen using polymersomes vs. solid-core nanoparticles self-assembly of block copolymers toll-like receptor agonist nanoparticles mimic immunomodulating effects of the live bcg vaccine and enhance neonatal innate and adaptive immune responses influences of nanocarrier morphology on therapeutic immunomodulation block copolymer micelles in nanomedicine applications micelle-based adjuvants for subunit vaccine delivery ph-responsive micelle-based cytoplasmic delivery system for induction of cellular immunity induction of mycobacterium tuberculosis lipid-specific t cell responses by pulmonary delivery of mycolic acid-loaded polymeric micellar nanocarriers flash nanoprecipitation permits versatile assembly and loading of polymeric bicontinuous cubic nanospheres facile assembly and loading of theranostic polymersomes via multi-impingement flash nanoprecipitation benchmarking bicontinuous nanospheres against polymersomes for in vivo biodistribution and dual intracellular delivery of lipophilic and water-soluble payloads towards an understanding of the adjuvant action of aluminium mechanisms of antigen adsorption onto an aluminum-hydroxide adjuvant evaluated by high-throughput screening molecular mechanisms regulating nlrp inflammasome activation alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity dna released from dying host cells mediates aluminum adjuvant activity aluminum hydroxide colloid vaccine encapsulated in yeast shells with enhanced humoral and cellular immune responses phospholipid bilayer-coated aluminum nanoparticles as an effective vaccine adjuvant-delivery system immunogenicity of a tetravalent meningococcal glycoconjugate vaccine in infants: a randomized controlled trial gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo different-sized gold nanoparticle activator/antigen increases dendritic cells accumulation in liver-draining lymph nodes and cd + t cell responses immunological properties of gold nanoparticles synergistic immuno photothermal nanotherapy (symphony) to treat unresectable and metastatic cancers and produce and cancer vaccine effect dual stimulation of antigen presenting cells using carbon nanotube-based vaccine delivery system for cancer immunotherapy mesoporous silica nanoparticles as antigen carriers and adjuvants for vaccine delivery iron oxide nanoparticles-based vaccine delivery for cancer treatment vaccines th edn the sixth revolution in pediatric vaccinology: immunoengineering and rheology of simple and multiple emulsions subunit vaccine delivery key roles of adjuvants in modern vaccines old and new adjuvants vaccine responses in newborns pediatric vaccine adjuvants: components of the modern vaccinologist's toolbox early life immune ontogeny-understanding how we build and sustain immunity to infection protecting the newborn and young infant from infectious diseases: lessons from immune ontogeny vaccines for the twenty-first century society age-specific adjuvant synergy: dual tlr / and mincle activation of human newborn dendritic cells enables th polarization fabrication of fillable microparticles and other complex d microstructures single-injection vaccines: progress, challenges, and opportunities development of newborn and infant vaccines beyond antigens and adjuvants: formulating future vaccines innate immune memory: implications for development of pediatric immunomodulatory agents and adjuvanted vaccines adjuvant effect of bacille calmette-guerin on hepatitis b vaccine immunogenicity in the preterm and term newborn innate immunity in human newborn infants: prematurity means more than immaturity global, regional, and national causes of child mortality: an updated systematic analysis for with time trends since national, regional, and worldwide estimates of preterm birth rates in the year with time trends since for selected countries: a systematic analysis and implications hepatitis b vaccination: long-term follow-up of the immune response of preterm infants and comparison of two vaccination protocols immunogenicity and induction of immunological memory of the heptavalent pneumococcal conjugate vaccine in preterm uk infants maternal vaccination: moving the science forward maternal immunization as a strategy to decrease susceptibility to infection in newborn infants effectiveness of palivizumab in high-risk infants and children: a propensity score weighted regression analysis designing tomorrow's vaccines vaccines for the st century the authors d.s. and d.j.d. would like to thank dr. ofer levy for his mentorship, and boston children's hospital precision vaccines program for publication support. this work was supported by u.s. national institutes of health grant r ai - a and adjuvant discovery program n c . all figures for this review have been created with biorender. d.j.d. contributed to initial article conceptualization. d.s. and d.j.d. contributed to outline/framework, initial drafting, and editing/finalization for publication. s.b. and s.l. contributed to preparation of initial and advanced drafts. e.a.s. reviewed the manuscript and provided editorial input. all authors gave their final approval of this paper for publication. competing interests: the authors declare no conflict of interest.patient consent: patient consent was not required for this review.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- - rsc x authors: kaslow, david c. title: certainty of success: three critical parameters in coronavirus vaccine development date: - - journal: npj vaccines doi: . /s - - - sha: doc_id: cord_uid: rsc x vaccines for viral pathogens have been licensed for use in humans. previously, two critical biological parameters of the pathogen and the host–pathogen interaction—incubation period and broadly protective, relative immunogenicity—were proposed to account for much of the past successes in vaccine development, and to be useful in estimating the “certainty of success” of developing an effective vaccine for viral pathogens for which a vaccine currently does not exist. in considering the “certainty of success” in development of human coronavirus vaccines, particularly sars-cov- , a third, related critical parameter is proposed—infectious inoculum intensity, at an individual-level, and force of infection, at a population-level. reducing the infectious inoculum intensity (and force of infection, at a population-level) is predicted to lengthen the incubation period, which in turn is predicted to reduce the severity of illness, and increase the opportunity for an anamnestic response upon exposure to the circulating virus. similarly, successfully implementing individual- and population-based behaviors that reduce the infectious inoculum intensity and force of infection, respectively, while testing and deploying covid- vaccines is predicted to increase the “certainty of success” of demonstrating vaccine efficacy and controlling sars-cov- infection, disease, death, and the pandemic itself. in the absence of an existing, safe, effective vaccine for a pathogen (i.e., absent proof of clinical efficacy and safety), the risks and uncertainties in developing a new vaccine can be broadly divided into two categories: "biologic uncertainty"-the inherent biological ability of the candidate vaccine to elicit a protective immune response in humans with a safety profile that results in a positive benefit-risk balance; and, "execution uncertainties and risks"-the successful performance of literally thousands of tasks required to develop the vaccine. the subfactors that determine "biologic uncertainty" are perhaps the most difficult to overcome because most are largely immutable. two dominant parameters that underlie "biologic uncertainty" are the safety/tolerability and the efficacy of a candidate vaccine. unlike execution risks, such as the underlying failure rate of a production run under a given set of operational conditions, biologic uncertainties have a relatively binary outcome-the candidate vaccine does or does not have a favorable benefit-risk profile-that remains largely unchanged under a given set of epidemiologic conditions. relatively because the outcome measures, such as efficacy and effectiveness, have inherent variability around the point estimate. this article revisits previously described key parameters of biologic feasibility proposed to determine the "certainty-of-success" (also referred to as the "probability of success") in developing prophylactic vaccines , but now in the context of coronaviruses. oftentimes safety and efficacy are inversely related, the socalled double-edged sword of attenuation : an improvement in one resulting in a loss of performance in the other. of the two, safety is often given priority over efficacy in regulatory review because the target populations of prophylactic vaccines are usually healthy. the risk tolerance for safety in the midst of an outbreak for a pathogen with a high r and a high case fatality rates may be higher than for vaccine use in routine immunization for relatively low-prevalence endemic diseases, particularly those with a low case fatality rate. despite the paramount importance of safety, this article will focus on estimating the "certainty of success" from the efficacy/effectiveness side of the benefit-risk balance. as noted above, the importance (and difficulty) of accurately estimating the performance of a candidate vaccine relative to the efficacy threshold in humans has been reviewed previously . herein the previously proposed paradigm that effective vaccines have been developed mainly for pathogens with lengthy incubation periods is re-explored as it pertains to active prophylactic immunization for coronaviruses, particularly sars-cov- . whereas in the original analysis, discussion of populationbased effects of immunization (e.g., herd effects) were considered, here the focus is mainly at the level of the individual vaccinee, with the notable exception of the population-based effect of force of infection (see glossary of terms), and the continued circulation of virus in the population on the durability of individual immunity. several simplifying assumptions are made, including that vaccinees are immunocompetent and share similar underlying condition profiles, that the kinetics of an effective acquired and anamnestic immune response is similar for different vaccine modalities, and that the immune responses elicited by vaccination prior to any previous pathogen exposure is at least similar to that acquired during natural infection. the previously proposed paradigm specifically considered, as part of the "certainty-of-success" analysis of biological feasibility, two critical properties of the many inherent biological properties of viral pathogens and the dynamic interaction with the human host: incubation period; and, broadly protective, relative immunogenicity. the combination of these properties appeared to have accounted for much of the successes so far achieved in vaccine development for viral pathogens (see fig. a ) . in the original analysis conducted more than a dozen years ago, an effective, durable vaccine against sars-cov was predicted to be on the cusp of biological feasibility when applying the -day incubation period rule. in the current specific analysis for sars-cov- vaccine development, infectious inoculum intensity is elevated to a critical parameter because of its putative implications in assessing the "certainty of success" of developing an efficacious covid- vaccine for use in an outbreak setting and in the design, conduct, and interpretation of covid- vaccine efficacy and effectiveness studies. incubation period and infectious inoculum intensity incubation period, defined as the time between exposure to the pathogen and onset of signs and/or symptoms of clinically apparent disease (see box . glossary of key terms), incorporates, in an empirically derived unit of time: ( ) multiple inherent biological properties of the pathogen; ( ) the dynamic interaction between the virus and the host; and ( ) real world conditions of transmission (see force of infection below). factors that determine the incubation period include the amount of infectious virus in a typical inoculum, the infectivity of the viral pathogen, the rate of viral replication, the rate of viral clearance by a variety of host mechanisms, including innate and adaptive immunity, the impact of viral immune evasion tactics, and the viral load that results in signs or symptoms of disease. the clinical signs and/or symptoms that define the endpoint of the incubation period also impact the reported value. not surprisingly, the incubation period can be quite variable, and retrospectively, difficult to precisely and accurately measure. in its simplest iteration, the incubation period can be viewed as a race between: ( ) the immune system's ability to generate a sufficient and appropriate innate and/or adaptive response; and ( ) the replication of the pathogen to a viral load that results in symptoms. as noted previously, an important inflection point occurs around days when considering incubation periods for viral pathogens. the "certainty of success" for viruses, such as influenza (median incubation period days, with a range of - days), that have short incubation periods do not benefit from the opportunity of an anamnestic response (see box . glossary of key terms). in addition, for vaccines against viral pathogens, particularly those with a short incubation period, protection against mild symptoms is often a much more difficult endpoint to achieve than protection against severe disease. in the absence of vaccine-induced, persistent, high-level immune effector function (e.g., circulating high-titer neutralizing antibodies and/or cytotoxic t-cell lymphocytes), early protection against lower level viral replication (i.e., early mild disease) may be more difficult to achieve than an anamnestic response (e.g., newly activated memory b-and t-cell responses) to protect against higher-level viral loads (i.e., more severe disease) that occur much later during infection. this model of being able to elicit high-level protection against severe disease, but not mild clinical symptoms (e.g., observed for rotavirus vaccines), will likely apply to sars-cov- . infectious inoculum intensity, at an individual-level, and force of infection, at a population-level, are factors that may inversely contribute to the length of the incubation period, the latent period, fig. "certainty of success" of vaccine development as a function of incubation period and broadly protective, relative immunogenicity. a two-dimensional analysis of major human viral pathogens based on incubation period (x-axis; time, in days or weeks, from exposure to clinical signs or symptoms) and broad, relative immunogenicity (y-axis; high, moderate or low-see reference for definition). the viral pathogens for which vaccine efficacy have been established are depicted in boxes with gray backgrounds; those for which vaccine efficacy has yet to be established are depicted in ellipses with white backgrounds. the area of graph associated with higher "certainty-of-success" for vaccine development (light gray) and lower "certainty-of-success" (dark gray) are separated by a thick black line. (reprinted from an open-access article licensed under a creative commons attribution-noncommercial . unported license . b effect of low and high infectious inoculum intensity on the assessment of the "certainty-of-success" (cos) of sars-cov- vaccines. interval bar (white) reflect the uncertainty in the inherent broadly protective, relative immunogenicity (see glossary of key terms) associated with sars-cov- natural infection. double-headed arrow (white with black outline) reflects the effect of infectious inoculum intensity higher (light gray) and lower (dark gray) "certainty-of-success" for vaccine development. anamnestic response: a secondary or subsequent immune response that yields a faster, greater, and longer lasting immune response upon re-exposure to an immunogen than that induced during the preceding primary immune response. broadly protective, relative immunogenicity: a semi-quantitative term that captures the genetic diversity of the virus, and two aspects of the host immune responses: ( ) the quality of the immune response that is elicited during and immediately after a primary infection; and ( ) the ability and duration of that elicited immune response to protect against subsequent symptomatic reinfection. certainty of success: an estimate of the confidence in successfully demonstrating biological activity of a candidate vaccine based on pre-defined endpoints of efficacy/effectiveness. force of infection: rate at which susceptible individuals in a population acquire an infectious disease in that population. incubation period: time interval between infectious agent exposure moment in an individual and appearance of first sign or symptom of disease in that individual. infectious inoculum intensity: magnitude (or area under the curve) in an individual of the infectious agent exposure at the exposure moment(s) associated with the duration of the incubation period in that individual. latent period: time interval between infectious agent exposure moment in an individual and onset of period of infectious transmissibility to others in the population, which may be shorter or longer than the incubation period. and directly contribute the severity of symptoms associated with infection , . equally, if not more relevant to "certainty of success" for vaccine development is the relationship between infecting dose and severity of disease , as demonstrated by influenza inoculum dose-related rate of mild-to-moderate disease in a controlled human infection model . with respect to coronaviruses, an inverse correlation between the length of the incubation period and the severity of disease was recently evaluated from data collected during the sars outbreak in hong kong. comparing the length of the incubation period between fatal cases and non-fatal cases suggested a correlation between shorter incubation and greater severity, allowing for potential confounding by age, sex and occupation . a similar finding was observed between the estimated incubation period of middle east respiratory syndrome cov (mers-cov) cases and mortality during the mers outbreak in south koreapatients who died had a shorter incubation period than patients who survived . with respect to sars-cov- , jiang et al. and amodio et al. have both noted that a longer incubation time may lead to a high rate of asymptomatic and sub-clinical infection among immunocompetent individuals; however, an inverse relationship between incubation period and severity of disease has yet to be demonstrated. while the relationship between incubation period and perhaps more importantly, the infectious inoculum intensity of srs-cov- and severity of covid- requires further validation, data consistent with an inverse relationship was highlighted by sanche et al. when noting that a potential caveat of their estimation of a shorter incubation period [ . days ( % ci . - . days)] for sars-cov- than most other published reports is because most of their case reports were collected from the first few persons detected in each province, which may have biased case detection toward patients with more severe symptoms. a noteworthy exception to the inverse relationship between incubation period and severity of disease comes from the observation that a longer incubation period among human influenza h n cases was associated with a greater risk of death. virlogeux et al. noted that h n virus infection differs from h n , sars, and mers coronaviruses in several respects, including tropism limited to the human upper airways, the absence of a cytokine storm, and the stronger association of severe h n disease with exacerbation of other underlying diseases, while h n , sars, and mers coronaviruses cause severe disease in otherwise healthy persons. as such, it is proposed here that the exception to the inverse relationship between incubation period and severity of disease is unlikely for sars-cov- . admittedly confounded by multiple other factors, the population-based force of infection appears, in at least two recent examples, to be inversely associated with vaccine efficacy/ effectiveness (ve/vef). in the case of rotavirus vef, a review of the first decade of post-licensure data from countries showed a gradient of median vef of %, %, and % in countries with low, medium, and high child mortality, respectively, for the monovalent vaccine based on a single human rotavirus strain, and a vef difference of % and % in countries with low and high child mortality, respectively, for a pentavalent vaccine based on five bovine-human reassortant rotavirus strains . prelicensure rotavirus vaccine ve data demonstrate a similar gradient , and, when analyzed by the pre-existing rotavirus disease burden (mortality) as an indicator of the force of infection, are consistent with an inverse association with ve, as suggested by feiken, et al. this inverse association was also recently suggested during the regulatory evaluation of the malaria vaccine, rts,s/as e . the european medicines agency (ema) noted that "ve tends to be lower in high transmission areas" when analyzing the pivotal mal- efficacy trial conducted in eleven research centers in seven sub-saharan african countries, where the force of infection (categorized by annual mean p. falciparum parasite rate, age-standardized in to -year olds ) differed by two orders of magnitude. lastly, this notion of an association between force of infection and severity of disease is consistent with multi-year observations of seroconversion rates for the four human endemic coronavirus and frequency of virus in hospitalized children . dijkman et al. reported that the frequency of infection observed via seroconversion in the study population, presumed to be associated with the relative force of infection in that population, had the same rank order of hcov-oc ≥ hcov-nl > hcov-hku ≥ hcov- e as the frequency of virus in hospitalized children, presumed to be associated with the severity of disease. given the extensive spread of sars-cov- , it should be possible to determine if a similar association between force of infection and severity of disease is observed during the current pandemic. broadly protective, relative immunogenicity a composite of additional biological features has been incorporated into a single semi-quantitative term, broadly protective, relative immunogenicity (see box . glossary of key terms; nominally categorized as high, moderate or low; also see table ), to provide a needed second dimension to refine the estimate of "certainty of success". specifically broadly protective, relative immunogenicity incorporates both genetic diversity of the virus, and two aspects of the host immune response: ( ) the quality of the immune response that is elicited during and immediately after a primary infection; and ( ) the ability and duration of that elicited immune response to protect against subsequent symptomatic reinfection. factors relevant to coronaviruses, particularly sars-cov- , that could contribute to categorizing broadly protective, relative immunogenicity might include the number of circulating coronavirus strains that cross-react or cross-protect against other coronaviruses , the propensity for coronaviruses to propagate mutants during the incubation, latent, disease and recovery periods, the frequency of asymptomatic infections due to viral clearance by an adaptive immune response during the primary infection and during reinfection, and the durability of the protective immune response. as to genetic diversity, coronaviruses have the largest positivesense, single-stranded rna (+ss rna) genomes known to cause disease in humans, from up to kilobases (kb), with the genus betacoronavirus sars-cov- at . kb (see table ). the long coronavirus genome displays a high degree of plasticity, particularly the spike or s protein (see below), which can adapt with relative ease to exploit different cellular receptors, likely underlying the propensity of the four genera of animal coronaviruses to jump hosts . while the bat-and rodentderived alpha-and beta-coronaviruses have likely attempted to jump into humans frequently, three cross-species transmission events, over the past dozen and a half years, have resulted in outbreaks of sars-cov, mers-cov, and sars-cov- in the human population. four well adapted "common cold"-type coronaviruses also widely circulate in humans (i.e., hcov- e, -nl , -oc , and -hku ) . of the nine open-reading frames encoded in the coronavirus genome and the four structural proteins, the spike or s protein, particularly the receptor-binding domain (rbd) in the s subunit and the s subunit, is of specific interest because this essential structural protein, expressed in multiple copies on the lipid bilayer envelope of the virus, determines in part the host range through its role in host cell attachment, fusion, and entry - . in the case of sars-cov- , evolution within the modular structure of the s protein, the main target of protective immunity, may be driven by the known error rates of coronavirus rna-dependent rna polymerases, and the marked capacity of coronaviruses to employ homologous recombination in the context of coinfections. while not nearly as genetically diverse as the +ss rna hepatitis c virus nor as stable as the +ss rna hepatitis a virus, sars-cov- is likely susceptible to moderate genetic diversity, despite the sars-cov- rbd already significantly higher binding affinity to the human angiotensin-converting enzyme (ace ) receptor than sars-cov rbd . so, although sars-cov- outbreak appeared after sars-cov, phylogenetically, sars-cov- appears to be an "older" virus more closely related to the progenitor bat cov than sars-cov . one surrogate for the level of broadly protective, relative immunogenicity is the frequency of reinfection. reinfection with the four circulating human "common cold"-type coronaviruses appears to be a frequent event. examples from the two human coronaviruses studied since the s, include a -year study of hcov-oc infection in tecumseh, michigan following an hcov- e outbreak in % of the study population . the incidence of hcov-oc infection in children < years of age was high, yet subsequent symptomatic reinfection, albeit mild except chronic bronchitis in some, was quite frequent in older children and in adults. when analyzed immunologically, > % of these subsequent symptomatic infections occurred despite prior neutralizing antibodies, calling into question the protective value of circulating neutralizing antibody (or the assays used) . reinfections were also frequently observed, commonly associated with respiratory symptoms, for these two human coronaviruses in young children , as well as in a longitudinal study of working adults . similar but less robust epidemiological findings have been reported from the more recently described endemic human coronaviruses, hcov-nl (first described in ) and hcov-hku (first described in ) (see also table for references). controlled human infection model (chim) studies provide another source of evidence to inform categorization of broadly protective, relative immunogenicity. in the case of hcov- e, chim studies in adults document susceptibility to symptomatic reinfection despite the presence of detectable antibodies, although homologous re-challenge a year later led to only asymptomatic reinfection . as noted by callow et al. the human challenge data are consistent with the notion that adults have human coronavirus infections on a - year cyclic pattern and that "protective amounts of antibody may have disappeared by years, and that if we had been able to reinoculate the volunteers after a further year, the reinfection rate would have been even higher". the totality of the findings from natural and controlled challenge infections, in conjunction with a moderate degree of genetic diversity in these four endemic human coronaviruses, led to a "low" broadly protective, relative immunogenicity categorization in table . similar to the four endemic coronaviruses, the quality and the durability of the protective immune response after natural infection with the three human epidemic coronaviruses appear to be "low" or at best "moderate", the difference being that severe disease has been observed more frequently for sars-cov, mers-cov, and sars-cov- than the common cold human coronaviruses. several longitudinal sero-epidemiology studies after the sars-cov outbreak reported a high post-convalescent seroconversion rate with igg peaking at - months in patient serum samples. in a small sample size study evaluating neutralizing activity in serial serum samples from patients with sars, > % contained neutralizing antibodies (nab) against sars-cov and most of the nab activities could be attributed to immunoglobulin g (igg) . however, the duration of circulating igg appeared relatively short-lived as the longest longitudinal study reported that at years, the igg positivity had declined to . % . if the incubation period of sars-cov is sufficiently long to allow a protective anamnestic response, then subsequent re-exposure would likely lead to an asymptomatic infection. similar seroconversion and nab rates have been published for mers-cov, with several studies suggesting that antibody levels and longevity following mers-cov infection are correlated with disease severity [ ] [ ] [ ] . okba et al. reported that all fifteen severe mers-cov incubation period: median (range); italicized are published but unconfirmed incubation periods. route of transmission: dc direct non-genital contact of secretions, fo fecal-oral, r respiratory droplets. rna genome: +ssr positive single-stranded rna, -ssr negative single-stranded rna, α alphacoronavirus, β betacoronavirus. broadly protective, relative immunogenicity: using a delphi-type approach, incorporates in a single, semi-quantitative term [high; moderate (mod); low; or to be determined (tbd)]: (a) the genetic diversity of the virus (see column labeled "genetic stability"); and (b) two aspects of the host immune response: ( ) the quality of the immune response that is elicited during and immediately after a primary infection (see first term in column labeled "immune response"); and ( ) the ability and duration of that elicited immune response to protect against symptomatic reinfection (see second term in column labeled response"). cases tested positive in all tested platforms up to year after disease onset, indicating a robust immune response of high antibody titers in severe cases; however, low or undetectable seroconversion rates and undetectable neutralizing antibodies were observed after most asymptomatic and some mild mers-cov infections. early data from the current sars-cov- pandemic suggest a similar pattern of immune responses in severe and mildly symptomatic sars-cov- patients . given these data, it is tempting to speculate that a lower infectious inoculum intensity leads not only to a longer incubation period and less severe disease, but also to a less robust broadly protective, relative immunogenicity after natural infection. active immunization that optimally balances efficacy with reactogenicity/tolerability may represent the best of both worlds-robust broadly protective, relative immunogenicity without the severity of disease by administration of a high inoculum intensity without infectiousness. while it is too soon to have significant empiric data on the durability of sars-cov- immune responses to protect against symptomatic reinfection, the durability after natural infection as well as the long-term efficacy of active immunization may be influenced by the extent to which sars-cov- and/or related cross-reacting human coronaviruses continue to circulate in the population or "herd". under conditions in which insufficient herd immunity exists to curtail or even eliminate circulation of these viruses in the "herd", repeated sub-clinical infections may serve to maintain a protective immune response in an individual of the "herd". as the prevalence of these viruses diminishes in the "herd" as a result of adequate vaccine coverage and/or naturally acquired immunity, likely so will durability of protective immunity in the individuals in the "herd" as subsequent sub-clinical infections no longer occur and no longer serve to naturally "boost" an adequate protective immune response. in such situations where circulation of relevant coronaviruses significantly diminish, re-vaccination must be considered if a high "certainty-of-success" for long-term protection against future outbreaks is sought. a limitation of this rudimentary approach taken herein to assign a qualitative value to this composite biological feature of pathogens (fig. a) and sars-cov- (fig. b) is that it did not employ nor benefit from more powerful tools such as system biology analyses or mathematical models, which have been shown to provide important non-intuitive insights into host-virus interactions. such tools would need to be brought to bear on this topic for a more rigorous evaluation and for a more accurate and precise placement in the broad categories of high, moderate, and low levels of broadly protective, relative immunogenicity depicted in fig. a, b. estimating certainty-of-success by simultaneously considering the incubation timeline with the genetic diversity and the quality and durability of the host immune response, an approach to estimating the "certainty of success" based on biological feasibility emerges ( fig. ) . the model predicts an inverse relationship between the length of the incubation period and the level of the broadly protective, relative immunogenicity needed to achieve an equivalent "certainty of success". that is, for those pathogens that have a short incubation period and less opportunity for protection through an anamnestic response, a higher broadly protective, relative immunogenicity is needed to have a high "certainty-of-success"; likewise, for those pathogens having a long incubation period that benefit from protection through an anamnestic response, a lower broadly protective, relative immunogenicity is needed to have a high "certainty-ofsuccess". the association between incubation period and "certainty-ofsuccess" is just that-an association. although the proposed model may well accommodate the dataset presented in fig. a , cause and effect clearly has not been demonstrated. in fact, many of the viral pathogens that have short incubation periods also cause hit-andrun, local mucosal infections. these pathogens (e.g., rhinovirus, influenza, rsv, piv, and mpv) cause relatively brief illnesses and have limited tropism. whether the latter is the key parameter of biologic feasibility that determines "certainty-of-success" for developing prophylactic vaccines for these pathogens remains to be determined. as described in detail previously , given its short incubation period and low broadly protective, relative immunogenicity (see table ), influenza is a particular outlier in estimating "certainty of success" because of a relatively predictable transmission season, and the availability of annual immunization with an updated vaccine just prior to exposure in high-resource settings. in many low-resource settings, the latter is not an option and a fit-forpurpose influenza vaccine that does not require annual updating and annual administration has yet to be developed. predictions for the biological feasibility of developing effective covid- vaccines in the end, "predictions ought to count more than accommodations, because of the risk of 'fudging' that accommodations run and predictions avoid ". with this mind, four guiding principles and three implications on the design, conduct and interpretation of vaccine clinical trials (see box ) are offered for pressure-testing the three factors identified herein (see fig. b) , as sars-cov- candidate vaccines advance into proof-of-efficacy studies. while many other factors that are not easily controlled will affect the robustness of the principles and implications proposed, some box proposed guiding principles and implications for covid- vaccine clinical trials proposed guiding principles in determining "certainty of success" . reducing the infectious inoculum intensity will: a. lengthen the incubation period. b. lengthen the latent period. c. increase vaccine efficacy. . lengthening the incubation period (see "note" below) will: a. reduce the risk of severe disease. b. increase the opportunity for anamnestic responses upon subsequent infectious inoculum exposure. . lengthening the latent period will: a. increase the herd effect of naturally acquired immunity and/or vaccine-induced protective immunity. . increasing the opportunity for anamnestic responses will: a. increase vaccine efficacy beyond that predicted by circulating antibody levels. b. increase durability of protective immunity while the pathogen still circulates in the population. implication for vaccine trial design, conduct, and interpretation: . assessing/estimating the incubation period during vaccine efficacy trials could provide insights into the infectious inoculum intensity and could provide insights into benefit-risk assessments for different use cases (e.g., high-risk first responders and healthcare workers with high-level exposure vs. general use to protect against low-level exposure during reopening after mitigation vs. routine use during interpandemic period). . determining vaccine efficacy for specific use cases and comparing vaccine efficacy of different vaccines should account for the infectious inoculum intensity in that specific use case setting and in the vaccine efficacy trial setting, respectively. . evaluating the correlates of protection (particularly in infectious inoculum intensity settings in which the incubation period is ≥ days) should include measures of anamnestic responses, in addition to circulating functional antibody levels. note: by reducing the infectious inoculum intensity [through individual measures (e.g., handwashing, other hygiene practices, and personal protective equipment) and/or population-based measures to reduce the force of infection] or by naturally acquired or vaccine-induced protective immunity. consideration to the factors under human control would seem prudent. the one factor that emerges for consideration in sars-cov- vaccine development and implementation is reducing the infectious inoculum intensity (and force of infection, at a populationlevel) to lengthen the incubation period, reduce the severity of illness, and increase the opportunity for an anamnestic response upon exposure to the circulating virus. successfully implementing individual-and population-based behaviors that reduce the infectious inoculum intensity and force of infection, respectively, while testing and deploying covid- vaccines may be a critical human-controlled factor in assuring the "certainty of success" through immunization in controlling and eliminating sars-cov- infection, disease, death, and the pandemic itself. received: april ; accepted: may ; biological feasibility of developing prophylactic vaccines for viral pathogens: incubation period as a critical parameter the double-edged sword: how evolution can make or break a liveattenuated virus vaccine extraordinary diseases require extraordinary solutions sars-cov- viral load and the severity of covid- inoculum size, incubation period and severity of malaria. analysis of data from malaria therapy records the relationship between infecting dose and severity of disease in reported outbreaks of salmonella infections validation of the wild-type influenza a human challenge model h n pdmist: an a(h n )pdm dose-finding investigational new drug study brief report: incubation period duration and severity of clinical disease following severe acute respiratory syndrome coronavirus infection association between severity of mers-cov infection and incubation period does sars-cov- has a longer incubation period than sars and mers? outbreak of novel coronavirus (sars-cov- ): first evidences from international scientific literature and pending questions high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus . emerg association between the severity of influenza a(h n ) virus infections and length of the incubation period effectiveness of rotavirus vaccination: a systematic review of the first decade of global postlicensure data a systematic review of the effect of rotavirus vaccination on diarrhea outcomes among children younger than years global, regional, and national estimates of rotavirus mortality in children < years of age use of vaccines as probes to define disease burden mosquirix: public assessment report. mosquirix h-w- a world malaria map: plasmodium falciparum endemicity in the dominance of human coronavirus oc and nl infections in infants molecular evolution of human coronavirus genomes common human coronaviruses | cdc recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission molecular pathogenesis of middle east respiratory syndrome (mers) coronavirus adaptive evolution of mers-cov to species variation in dpp zoonotic origins of human coronaviruses characterization of the receptor-binding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine community-wide outbreak of infection with a e-like coronavirus in tecumseh, michigan the tecumseh study of respiratory illness. vi. frequency of and relationship between outbreaks of coronavims infection epidemiology of coronavirus respiratory infections coronavirus infections in working adults. eight-year study with e and oc identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia controlled human infection model (chim) studies: summary of a workshop the time course of the immune response to experimental coronavirus infection of man neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection duration of antibody responses after severe acute respiratory syndrome antibody response and disease severity in healthcare worker mers survivors transmission of mers-coronavirus in household contacts mers-cov antibody responses year after symptom onset sensitive and specific detection of low-level antibody responses in mild middle east respiratory syndrome coronavirus infections severe acute respiratory syndrome coronavirus −specific antibody responses in coronavirus disease patients testing hypotheses: prediction and prejudice an outbreak of influenza aboard a commercial airliner the experimental production of combination forms of virus realities and enigmas of human viral influenza: pathogenesis, epidemiology and control complete genome sequence of the first h n avian influenza virus isolated from chickens in lebanon in h influenza, a global update kinetics of neutralizing antibodies in patients naturally infected by h n virus adaptive evolution of human-isolated h nx avian influenza a viruses probable person-to-person transmission of avian influenza a (h n ) probable limited person-to-person transmission of highly pathogenic avian influenza a (h n ) virus in china who influenza fact sheet (avian-and-other-zoonotic human cases of avian influenza a (h n past, present, and possible future human infection with influenza virus a subtype h specificity, kinetics and longevity of antibody responses to avian influenza a(h n ) virus infection in humans specific memory b cell response in humans upon infection with highly pathogenic h n avian influenza virus comparison of the first three waves of avian influenza a(h n ) virus circulation in the mainland of the people's republic of china signs and symptoms in common colds human coronaviruses e and nl : close yet still so far effects of a 'new' human respiratory virus in volunteers human coronavirus nl : a clinically important virus? human coronavirus-nl infections in korean children new human coronavirus, hcov-nl , associated with severe lower respiratory tract disease in australia human coronavirus nl infection in canada a previously undescribed coronavirus associated with respiratory disease in humans human coronavirus nl understanding human coronavirus hcov-nl identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection epidemiologic linkage and public health implication of a cluster of severe acute respiratory syndrome in an extended family lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study duration of serum neutralizing antibodies for sars-cov- : lessons from sars-cov infection sars incubation and quarantine times: when is an exposed individual known to be disease free? multiple contact dates and sars incubation periods molecular evolution of the sars coronavirus during the course of the sars epidemic in china severe acute respiratory syndrome coronavirus isolate wuhan-hu- , complete genome the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application european centre for disease prevention and control. outbreak of severe acute respiratory syndrome coronavirus (sars-cov- ): increased transmission beyond china -fourth update incubation period of novel coronavirus ( -ncov) infections among travellers from wuhan recovery in tracheal organ cultures of novel viruses from patients with respiratory disease an outbreak of coronavirus oc respiratory infection in normandy, france epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method prevalence and clinical characteristics of human cov-hku in children with acute respiratory tract infections in china clinical and molecular epidemiological features of coronavirus hku -associated community-acquired pneumonia comparative analysis of coronavirus hku genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku coronavirus hku and other coronavirus infections in hong kong hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans preliminary epidemiological assessment of mers-cov outbreak in south korea middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide d.c.k. conceived, wrote, reviewed, approved, and is accountable for this paper. d.c.k. is an employee of path (a not-for-profit organization), has no financial interest in any for-profit organization, and declares no competing interests. correspondence and requests for materials should be addressed to d.c.k. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- - xh cd authors: serwer, philip title: optimizing anti-viral vaccine responses: input from a non-specialist date: - - journal: antibiotics (basel) doi: . /antibiotics sha: doc_id: cord_uid: xh cd recently, the research community has had a real-world look at reasons for improving vaccine responses to emerging rna viruses. here, a vaccine non-specialist suggests how this might be done. i propose two alternative options and compare the primary alternative option with current practice. the basis of comparison is feasibility in achieving what we need: a safe, mass-produced, emerging virus-targeted vaccine on – week notice. the primary option is the following. ( ) start with a platform based on live viruses that infect bacteria, but not humans (bacteriophages, or phages). ( ) isolate phages (to be called pathogen homologs) that resemble and provide antigenic context for membrane-covered, pathogenic rna viruses; coronavirus-phage homologs will probably be found if the search is correctly done. ( ) upon isolating a viral pathogen, evolve its phage homolog to bind antibodies neutralizing for the viral pathogen. vaccinate with the evolved phage homolog by generating a local, non-hazardous infection with the phage host and then curing the infection by propagating the phage in the artificially infecting bacterial host. i discuss how this alternative option has the potential to provide what is needed after appropriate platforms are built. when a distant goal is to be reached, strategies of two types typically evolve. ( ) establish the overall objective and then establish matching subsidiary objectives, with little regard for how difficult the subsidiary objectives are. the point is that, if the subsidiary objectives are reached, then the overall objective is reached. ( ) establish readily doable subsidiary objectives and delay concern about fitting them together to reach the overall objective. some enterprises begin with a type strategy and are then infiltrated by what might be called a creeping type strategy. one reason is that a type strategy has the most immediate appeal, especially when setting fundable goals and trying to maintain organizational status quo. an example of a creeping type strategy is provided by the ranger program to photograph the moon in the early days of the us space program. the first six missions failed [ ] . although the details differed for the different failures [ ] , one can summarize the failures as the products of deficient systems integration, unrealistic testing conditions and haste. the deficiency in systems integration is basically a synonym for a creeping type strategy. haste and lack of real-world orientation are corollary to a type strategy. better adherence to a type strategy yielded more success with the initially-believed-beyond-reach, year targeted apollo program [ , ] ; people walked on the moon ahead of schedule [ , ] . closer to the current topic is a non-specialist critique of research on curing metastatic cancer. in brief, this discussion outlines a creeping type strategy in that field [ ] . the central point here is that the situation is analogous for developing vaccines for pandemic viruses. that is to say, as discussed in more detail below, vaccine-producing efforts have the highest chance of success via disciplined use of a type strategy. more than one type strategy option exists for reaching the overall objective. the overall objective is a safe, mass-produced, emerging virus-targeted vaccine on - week notice. here, i describe details of the subsidiary objectives of my primary type strategy option. all subsidiary objectives are based on platforms that are ( ) rapidly, but without haste, used after being built and ( ) initially, at least, difficult to build and dependent on highly trained, highly motivated personnel. i compare the prospects with the prospects of current work in other directions. when viewed from the perspective of the overall objective, most to all of the latter are creeping type strategy options. i also present an alternative type strategy option. for the main type strategy option, the following are the initial subsidiary objectives for the overall objective. ( ) start with a platform based on live viruses that infect bacteria, but not humans (bacteriophages, or phages [ , ] ). ( ) isolate phages (to be called pathogen homologs) that resemble and provide antigenic context for membrane-covered, pathogenic rna viruses; coronavirus-phage homologs might be found if the search is correctly done. ( ) upon isolating a pathogenic virus, evolve its phage homolog to bind neutralizing antibodies to the pathogenic virus. ( ) vaccinate with the evolved phage homolog by generating a local, non-hazardous infection with the phage host and then curing the infection by propagating the phage in the artificially infecting host. the last subsidiary objective of any strategy is mass vaccine production. if this subsidiary objective is unachievable, not much point exists to working on the others. live vaccines are the most easily adapted to meet this requirement because their constituents propagate post-vaccination. smallpox vaccine [ ] and the sabin [ ] and koprowski [ ] polio vaccines are examples; the salk inactivated virus vaccine was the first vaccine for polio [ ] . indeed, most currently approved anti-viral vaccines are either attenuated, live virus vaccines or inactivated virus vaccines [ ] [ ] [ ] [ ] . in the case of phages, a single preparation typically has over phage particles (sometimes over ) when the phages have been propagated, concentrated and purified from a l lysate. this is more than enough to vaccinate everyone in the world with a phage dose, if the phages can be made to propagate post-vaccination. making a l lysate takes approximately one day. bulk-purifying phages by ultracentrifugation takes approximately two days. to achieve post-vaccination phage propagation, we have to give the phages a bacterial host. one way to do this is to start an intra-dermal infection with the bacterial host. then, cure the infection by infecting the bacterial host with the antigenic phage. this is upside-down phage therapy so to speak. the phages will multiply until either the bacteria are gone or until immune systems prevent further multiplication. thus, the characteristics of the bacterial host are key components of this platform. the host must not itself become a problematic pathogen, yet it must continue to propagate enough to yield enough phages to stimulate innate and adaptive immune systems to produce immunity to the phage and, by homology, immunity to the virus that is causing the problem in the first place. indeed, this proposed procedure is not all that far from the procedure of a smallpox vaccination and might leave a similar scar. some of us can still see scars from smallpox vaccination. the main difference is that the replicating phage antigen needs externally supplied host cells in order to produce immunity. without going into details concerning live vaccine production via eukaryotic viruses, i think it reasonable to assume that eukaryotic virus production is more difficult, more expensive and less rapid than the production of phages. further, there are fewer safety concerns with phages. to understand the principle of how this type of evolution might be achieved, i use the following analogy. suppose one wanted to maximize the speed of the non-mechanized elevation of a person to a ledge feet high. jumping that high is out of the question, no matter how many tries are made. more realistically, one would make and use a series of less incremental ledges, steps so to speak. the situation with phages is analogous, as we found when evolving phage t to propagate in . m nacl. this had to be done in steps [ ] . parenthetically, this nacl concentration is so high that, in petri plates, our only indication of host growth was the observation of plaques of our step-evolved phage. this example emphasizes the basic principle. it is not intended to indicate the complexity of what is proposed below. thus, the basic idea is to build the foundation by step-evolving phages to react with the neutralizing antibodies of the target pathogen. i assume here that the antibodies are already in hand. meeting the overall objective implies that some of the step evolving has to be done before the pathogen exists. that, in turn, can be done by antibody-selecting phage homologs for each of the multiple known and potential pathogens isolated from the environment. for example, an antibody-selected sars-cov- phage homolog might serve as the starting point for an antibody-selected sars-cov- phage homolog. that is to say, some of the inter-pandemic activity would be devoted to improving vaccines to current pathogens. recommendations to do this have already been made, in the context of other vaccine-generating options [ ] . several procedures exist for performing the antibody-selection of phages. affinity, antigen-loaded columns are already used via chromatography to select antibody-displaying phages for antibodies with increased affinity to antigen [ , ] . using this procedure, while reversing the role of antibody and antigen (antibody bound to the column), is one way to antibody-select phages. an independent approach is indicated by the fact that antibody-generated phage multimers are observed as discrete, more origin-proximal-than-monomeric-phage bands after reacting phages with antiserum and performing native gel electrophoresis [ ] . selecting phages near or at the origin is an alternative selection of antibody-reactive phage. the same is the case for phages that are in the void volume after molecular sieve chromatography. selecting rapidly sedimenting phages after rate zonal centrifugation in a sucrose density gradient is yet another procedure. then arises the question of which phage to use at the beginning. in theory, any stable phage could be used as a starting point. time to objective might be decreased by using a phage displaying a peptide from the spike protein (or other protruding protein) of a potential target virus. however, current efforts to human-engineer improved antigens for anti-rna virus vaccines have shown that neutralizing antibodies typically react with viral proteins that are in states that are context dependent and unstable [ , , , ] . the context includes assembly with other viral proteins, which results in the use of artificial scaffolds in structural vaccinology [ ] . much work has been devoted to stabilizing protein antigens in "appropriate" states and masking epitopes that interfere with the epitope that reacts with neutralizing antibody [ , , , ] . rna viruses have evolved sophistication to the point that they make most accessible the epitopes that change the most rapidly [ ] . the context also includes embedding in a lipid bilayer membrane [ ] [ ] [ ] [ ] . the context dependence of neutralizing antibody antigenicity leads to the conclusion that, in practice, the best starting point is a phage structurally homologous to the known or anticipated pathogenic virus. i take the liberty of responding here to the obvious objection that no membrane-covered, single-stranded rna phage has ever been isolated [ ] and that the pandemic viruses include influenza, zika-type and coronaviruses, all in this category. this non-isolation does not imply non-existence. membrane-covered, double-stranded rna phages have been isolated [ ] . already shown is that membrane-coated dna phages are dramatically under-isolated with the current most popular isolation procedures. chloroform treatment is a major culprit [ ] . the time is now to make the appropriate isolation attempts. i note that some propagating, bacteriolytic particles are, structurally, far from the phages isolated in the past; an example is phage phi - in [ ] . the underlying thesis for this section is that genetic selection is the most reliable way to achieve the systems engineering needed to achieve appropriate antigens for needed vaccines. when performed with phages, genetic selection is also rapid to the point that human engineering, even if enough was known to do it reliably, would probably not be as rapid. my preferred procedural advance for rapidly and unbiasedly isolating phages has already been described [ , ] . in brief, the phage isolation techniques include both initial isolation and subsequent propagation in a gel. all transfers are done with a needle. no liquid culture is involved. one advantage of a procedure of this type is that dry environmental samples (often in a powdered state) are used, thereby increasing the probability that the phages isolated can be dried for transport. presumably, further development of these procedures is possible. organization and inter-laboratory coordination will be a key feature of any effort, whatever procedures are used for phage isolation. the reason is that the phage collection will be large enough so that computerization will be needed for access to both data and the phages themselves. organization begins with a naming system that makes impossible the giving of the same name to two different phages [ , ] . any therapeutic use of phages begins with two advantages in the area of safety. ( ) although phage uptake into human cells occurs, no evidence exists that phages replicate in human cells [ ] [ ] [ ] . for example, phages use transcription and translation signals that are different from those of eukaryotic cells. ( ) the experience with phage therapy of infectious disease is that the phage particles themselves are never found to have negative effects in humans [ , ] . of course, that does not indicate zero danger. perhaps the greatest danger is the induction of non-neutralizing antibodies that aggravate an infection. this is one explanation of vaccine aggravation of dengue fever, a phenomenon preferentially encountered with young recipients of dengue fever vaccine [ , ] . however, selection against induction of non-neutralizing antibodies occurs when the phage is selected for binding to neutralizing antibody. one cannot be so sanguine about any vaccine that has either eukaryotic rna or eukaryotic dna with transcription and translation signals. dna vaccines are the more serious concern because of possible chromosome insertions [ ] . in the case of an un-encapsulated rna vaccine, a secondary concern is destruction of the vaccine by rnases [ ] . assuming that eukaryotic dna and rna vaccines will always have to be safety tested, they are not likely to meet the - week constraint that is imposed by a novel, rapidly spreading pandemic virus. many years are likely to be required to test for cancer-generating chromosomal alterations. a non-specialist observer reasonably concludes that dna and rna vaccines, when viewed in the context of our overall objective, are examples of type strategy options. furthermore, in spite of a~ year history [ ] , no dna vaccine has ever been fda approved for use with humans [ , ] . pathogen homolog rna phage vaccines, on the other hand, would have an a priori and reasonable projection (not certainty) of safety. even some dna phage vaccines might eventually be placed in this category. the importance of this latter characteristic becomes apparent when one intellectually removes the blinders that human emotion places on us. specifically, sars-cov- is not as deadly as sars-cov- . the problem is that sars-cov- transmits more actively and does so on inanimate surfaces, although i have not yet seen a claim that sars-cov- can transmit on dust particles. can a virus transmit even more actively than sars-cov- ? a combination thought/real-world experiment suggests that this can happen. first, in the real world, one of the best sources of phages is dry soil around horse watering troughs (our circumstantial observations). this soil typically resembles a powder and would be easily dispersed as dust in the atmosphere by wind. that is to say, some phages are reasonably assumed to survive on dust particles. if so, the thought part of this experiment tells us that eukaryotic viruses can also evolve to survive on dust particles. similar, more dramatic dust-generating effects occur in deserts. we were apparently lucky that the mers coronavirus [ ] did not create greater problems than it did via transmission on sand particles, given that it originated in saudi arabia. can more active transmission be accompanied by higher virulence? i will not make a projection. however, we have to be prepared for this possibility. either way, i think that the case for vaccine development speed, and therefore the case for vaccines with a priori, rational expectation of safety, needs no further justification. i do not speculate here on what the details should be for safety standards. intuitively obvious is that no current, platform-ready technology can meet the time constraint in the overall objective used here. otherwise, we would already have a vaccine and we would not be told that we have to wait over a year for one. my use of this time constraint is closely connected to the real world of both mass fatalities and economic collapse-both of which are now beginning and can be compounded by criminal misuse of sars-cov- . that is to say, realistic conditions are not being currently used for testing vaccine strategies. the analogy with the early stages of the ranger program is too close to miss. thus, i have trouble conceiving a good reason why a call for new options has not been made. i am reminded of two folk sayings (i am not sure who are the originators). ( ) "most people would rather fail conventionally than succeed unconventionally." ( ) when the tide goes out, you discover who is swimming naked." the tide is going out. the preferred option for anti-viral vaccine response depends, to some extent, on the history and agenda of the responder. the option proposed above is obviously a product of the author's history. indeed, the phage isolation aspect overlaps the author's previous proposals for resolving current crises in the therapy of multi-drug-resistant bacterial infections [ ] and metastatic cancer [ ] . investigation of natural phage bioengineering has produced a framework for understanding and curing neurodegenerative diseases [ ] . the underlying thesis is the following: make use of the systems engineering of perhaps two billion years of phage evolution. given that the platforms presented here for the above type strategy option are not (and may never be) built, i present an alternative to this option. the alternative option is a live eukaryotic virus vaccine of atypical type. this option is designed for the time constraint of the overall objective, - weeks. given that eukaryotic viruses have doubling times much greater than those of phages ( - min for typical coliphages), meeting this objective implies that a live virus vaccine has to be already present in the environment. that this presence is a realistic expectation was shown by pasteur's demonstration of the absence, for all practical purposes, of spontaneous generation of microbes [ ] . thus, any emerging pathogenic virus has ancestors. given the emerging pathogen character, most ancestors do not have the pathogenicity of the current pathogen. however, at least some ancestors are likely to have sufficient antigenic overlap to act as live vaccines. that use of such ancestor vaccines can work has been shown via finding that porcine respiratory coronavirus infection of pigs immunizes naturally against the more dangerous transmissible gastroenteritis coronavirus [ ] . further, the original smallpox vaccine was also of this type [ ] . thus, the first platform for this alternate type strategy option is a library of live viruses in each of the classes that also contain viral pathogens. recommendation to build this platform has already been made for other reasons [ ] . the second platform is enough interpreted data so that both pathogenicity and antigenicity of any potential live vaccine strain can be predicted from the base sequence of its genome. this platform is not here at present and is faced with significant difficulties. for example, even though the sars-cov- spike protein (i.e., the protein that makes the initial contact with a cellular receptor) is structurally homologous to the sars-cov- spike protein, neutralizing antibodies to the sars-cov- spike protein do not react with the sars-cov- spike protein [ ] . further, one might find that predictions made always had some level of uncertainty. one of the reasons for building/sequencing/interpreting an extensive library of phages is to assist interpretation of the sequences of eukaryotes and their viruses. the decision to make use of fast-tracked live vaccines would presumably depend on the state of the pandemic being faced. as a thought experiment, imagine that ( ) you are years old today, ( ) a fast-tracked live-virus vaccine has been developed for sars-cov- and ( ) a reasonable projection is that you have a / chance of being seriously ill from the vaccine. the bias toward or against vaccination would depend on the projection for how socio-political measures will go in the future. as the economy progressively pressures the easing of these measures, i suspect that the bias for most people will progressively become toward vaccination. the real world of "needed research" can be (and, in my opinion, is) different from the world of "fundable research". currently, the latter typically implies a type strategy, often with tenuous links to the overall objective. i am talking about both government and private foundation funding. i am also talking about bait-and-switch tactics implicit in some funding announcements. specifically, an announcement will ask for something novel, paradigm changing, realistic and needed, while embedding a requirement that precludes some or all of the above. if this requirement is not stated, the process can be reasonably called corrupted. the experience with generating polio virus vaccines [ ] suggests that non-specialist scientific influence will be needed to manage the conditions described in this paragraph. the "silver lining" is that the recent crisis might help make improvements in research on therapies for diseases other than pandemic virus infections. the following examples will, perhaps, convince those who are doubters of the value of a type strategy. paul ehrlich used a type strategy to plough through compounds until he found the first usable antibiotic, salvarsan (reviewed in [ ] ). schatz and waksman did the same during the discovery of streptomycin [ ] . early anti-cancer drug discovery was a repeat performance (reviewed in [ ] ). furthermore, the current situation with sars-cov- has been described as analogous to war [ ] . of course, in this war, everyone is on the same side, whether or not apparent. military strategy competency credentials aside for the moment, i could not help noticing that a type strategy appears to have been the more successful strategy in war. one illustrative example is the multi-easy victory, but lack-of-a-central objective and ultimately losing, type strategy of hannibal in the second punic war [ ] . (this example was recommended to me by an in-house reader of this manuscript, richard ludueña.) given its historical closeness, i also compare the poorly coordinated, low-training, take-the-easy-path, ultimately disastrous type strategy of the us at the beginning of the battle of the bulge with the initially-thought-impossible-to-execute, focused, rapid, highly coordinated, successful type strategy used by the highly trained, highly motivated us third army at the end of the battle of the bulge [ ] . let us hope that, in the contest with sars-cov- , the outcome for people resembles the latter us outcome. impact: a history of project ranger; scientific and technical information office contrary, and ugly: the lunar landing research vehicle nasa history division office of external affairs failure is not an option: mission control: from mercury to apollo and beyond the truth in small doses: why we're losing the war on cancer-and how to win it phages for phage therapy: isolation, characterization, and host range breadth bacteriophage genomics the evolution of poxvirus vaccines sabin monovalent oral polio vaccines: review of past experiences and their potential use after polio eradication administration of an attenuated type poliomyelitis virus to human subjects lessons from the salk polio vaccine: methods for and risks of rapid translation emerging vaccine technologies new vaccine technologies to combat outbreak situations emerging viral diseases from a vaccinology perspective: preparing for the next pandemic structural vaccinology for viral vaccine design length quantization of dna partially expelled from heads of a bacteriophage t mutant monoclonal antibodies and antibody like fragments derived from immunised phage display libraries antibody phage display detection of bacteriophage-antibody complexes by agarose gel electrophoresis deceptive imprinting and immune refocusing in vaccine design half a century of research on membrane-containing bacteriophages: bringing new concepts to modern virology a major lineage of non-tailed dsdna viruses as unrecognized killers of marine bacteria single-particle light microscopy of bacteriophages improved isolation of undersampled bacteriophages: finding of distant terminase genes propagating the missing bacteriophages: a large bacteriophage in a new class internalization of a polysialic acid-binding escherichia coli bacteriophage into eukaryotic neuroblastoma cells a bacteriophages journey through the human body phage therapy: what factors shape phage pharmacokinetics and bioavailability? systematic and critical review phage therapy: what have we learned? viruses bacteriophage therapy: developments and directions new dengue vaccine performs well in large trial, but safety remains key concern a review of dengvaxia ® : development to deployment dna vaccines: technology and application as anti-parasite and anti-microbial agents dna vaccines-how far from clinical use? mers coronavirus: diagnostics, epidemiology and transmission restoring logic and data to phage-cures for infectious disease phage capsids as gated, long-persistence, uniform drug delivery vehicles electron microscopy of in-plaque phage t assembly: proposed analogs of neurodegenerative disease triggers free lance of science; little, brown and co cryo-em structure of the -ncov spike in the prefusion conformation friends and partners: the legacy of franklin d. roosevelt and basil o'connor in the history of polio the contributions of paul ehrlich to pharmacology: a tribute on the occasion of the centenary of his nobel prize effect of streptomycin upon mycobacterium tuberculosis and related organisms the art of war" in the era of coronavirus disease (covid- ) the punic wars the story of the bulge acknowledgments: i thank richard ludueña and benjamin jurewicz for review of drafts of this manuscript. the author declares no conflict of interest. key: cord- -e xae authors: day, m. j.; crawford, c.; marcondes, m.; squires, r. a. title: recommendations on vaccination for latin american small animal practitioners: a report of the wsava vaccination guidelines group date: - - journal: j small anim pract doi: . /jsap. sha: doc_id: cord_uid: e xae the world small animal veterinary association vaccination guidelines group has produced global guidelines for small companion animal practitioners on best practice in canine and feline vaccination. recognising that there are unique aspects of veterinary practice in certain geographical regions of the world, the vaccination guidelines group undertook a regional project in latin america between and , culminating in the present document. the vaccination guidelines group gathered scientific and demographic data during visits to argentina, brazil and mexico, by discussion with national key opinion leaders, visiting veterinary practices and review of the scientific literature. a questionnaire survey was completed by veterinarians in five latin american countries and the vaccination guidelines group delivered continuing education at seven events attended by over veterinarians. the vaccination guidelines group recognised numerous challenges in latin america, for example: ( ) lack of national oversight of the veterinary profession, ( ) extraordinary growth in private veterinary schools of undetermined quality, ( ) socioeconomic constraints on client engagement with preventive health care, ( ) high regional prevalence of some key infectious diseases (e.g. feline leukaemia virus infection, canine visceral leishmaniosis), ( ) almost complete lack of minimal antigen vaccine products as available in other markets, ( ) relative lack of vaccine products with extended duration of immunity as available in other markets, ( ) availability of vaccine products withdrawn from other markets (e.g. giardia vaccine) or unique to latin america (e.g. some leishmania vaccines), ( ) accessibility of vaccines directly by pet owners or breeders such that vaccination is not delivered under veterinary supervision, ( ) limited availability of continuing education in veterinary vaccinology and lack of compulsion for continuing professional development and ( ) limited peer‐reviewed published scientific data on small companion animal infectious diseases (with the exception of leishmaniosis) and lack of support for such academic research. in this document, the vaccination guidelines group summarises the findings of this project and assesses in evidence‐based fashion the scientific literature pertaining to companion animal vaccine‐preventable diseases in latin america. the vaccination guidelines group makes some recommendations on undergraduate and postgraduate education and academic research. recognising that current product availability in latin america does not permit veterinarians in these countries to vaccinate according to the global world small animal veterinary association guidelines, the vaccination guidelines group makes a series of “pragmatic” recommendations as to what might be currently achievable, and a series of “aspirational” recommendations as to what might be desirable for the future. the concept of “vaccine husbandry” is addressed via some simple guidelines for the management of vaccine products in the practice. finally, the vaccination guidelines group emphasises the global trend towards delivery of vaccination as one part of an “annual health check” or “health care plan” that reviews holistically the preventive health care needs of the individual pet animal. latin american practitioners should transition towards these important new practices that are now well embedded in more developed veterinary markets. the document also includes frequently asked questions and their answers; these were posed to the vaccination guidelines group during our continuing education events and small group discussions and should address many of the issues surrounding delivery of vaccination in the latin american countries. spanish and portuguese translations of this document will be made freely available from the on‐line resource pages of the vaccination guidelines group. the world small animal veterinary association (wsava) vaccination guidelines group (vgg) was established in with the remit of providing globally applicable evidence-based advice for small companion animal veterinary practitioners on best practice for vaccination of pet dogs and cats. the vgg first released global vaccination guidelines for veterinarians in and these were updated in and (day et al. ) and translated into multiple languages. the main body of the wsava vaccination guidelines are most applicable to pet cats and dogs living predominantly in and around their owners' homes (rather than living % outdoors or in large, closely-packed groups), but advice is also given on vaccination in a shelter setting. the and revisions were accompanied by a separate document providing information on vaccination for the owners and breeders of dogs and cats and by a series of infectious disease "fact sheets" designed to be used by veterinarians during consultation with clients (https://www.wsava. org/guidelines/vaccination-guidelines). from to the vgg worked on a regional project focussing on the vaccination requirements of small companion animals in asia (day et al. ). following from the success of that project the vgg embarked on a second regional project in latin america (latam) between and . the present paper represents the final outcome from this latam project. it summarises the key challenges faced by small companion animal veterinary practitioners in latam and makes a series of recommendations for future actions that might benefit the profession, pet owners and dogs and cats in these countries. the manuscript will be made available in spanish and portuguese translation via the vgg webpages (see above). the vgg recognises that latam is a vast and diverse region comprised of numerous countries with distinctly different geography, climate, culture and socioeconomics; all of which may impact on the keeping of companion animals, the prevalence and distribution of key companion animal infectious diseases and the accessibility of veterinary preventive health care for those animal populations. the vgg could not visit every country in the region, but, as described below, gathered extensive data on which to base our comments and recommendations. we believe that the majority of these recommendations will have applicability across the latam region. the membership of the vgg was changed for the latam project. emeritus professor m. j. day and professor r. a. squires were joined by professors c. crawford and m. marcondes; the latter recruited as a regional expert in small companion animal infectious disease and vaccinology. the principle aim of the project was to gather as much information and scientific evidence concerning small companion animal practice, vaccine-preventable infectious diseases and vaccination of dogs and cats as possible, to form a firm basis for the recommendations to be made subsequently. to that end, the vgg undertook three fact-finding visits to argentina (buenos aires and rosario in ), brazil (são paulo and rio de janeiro in ) and mexico (mexico city, guadalajara and monterrey in ). each of these visits was similarly structured and involved formal small group discussions with key opinion leaders (kols) including ( ) first opinion veterinary practitioners, ( ) representatives of small animal veterinary associations, ( ) academic veterinarians involved in companion animal infectious disease research and the teaching of microbiology, immunology, clinical medicine and vaccinology, ( ) government officials responsible for the assessment and licensing of small companion animal vaccines and ( ) representatives of national and international vaccine manufacturers and distributors. the formal meetings were supplemented with site visits to veterinary practices in each of the seven cities; these were purposely selected to show a range of sizes and standards. scientific literature relevant to the vgg mission was collected by on-line database searching and directly from academics participating in kol meetings. during , the vgg met to discuss findings and draft this final report. in order to expand the information gained from these face-to-face meetings, the vgg developed a questionnaire for distribution among first opinion practitioners in the target countries (appendix). the questionnaire was designed using "google forms" (https:// www.google.com/intl/en-gb/forms/about/) and was accessed and completed on-line. the questionnaire was made available in portuguese and spanish and was completed anonymously with instruction that only one veterinarian from each practice should undertake the survey. the responses were analysed (using tools in the google survey programme) and summarised. through the survey, the vgg gathered information about ( ) the demographics of the responding practitioners, ( ) veterinary practices and their access to diagnostic laboratories, ( ) canine and feline infectious diseases seen in the practices and ( ) canine and feline vaccines and vaccination protocols used in the practices. responses to the surveys were received from practitioners in argentina, in brazil and there is little doubt that latam is experiencing remarkable growth in pet ownership and the associated pet care industry. a survey by gfk global of , consumers in different countries revealed that latam has the highest level of global pet ownership. eighty percent of the online population surveyed in argentina and mexico, and % of the surveyed population of brazil, owned a pet. of the pet owners, % in argentina, % in mexico and % in brazil owned dogs and , and %, respectively, owned cats (https://www.gfk.com/fileadmin/user_upload/country_one_pager/nl/documents/global-gfk-survey_pet-ownership_ . pdf ). there are no accurate figures for national dog and cat populations. in , there were an estimated . million dogs and . million cats in brazil (conceição ) and argentinian kols estimated up to million dogs and million cats in argentina. in some regions, many of these animals will be free roaming rather than being owned-housed pets. with such growth should come an increasing demand for veterinary services and preventive health care for companion animals, including providing protection from infectious disease for the individual animal and animal population ("herd immunity") by vaccination. the vgg discussed these demographics with academic and association colleagues in argentina, brazil and mexico. we met with academic administrators and teacher/researchers from several veterinary schools in each country. a general observation was that it appeared challenging to provide accurate and up-to-date data on the demographics of the profession in the absence of national (as opposed to provincial or state) professional regulatory authorities who might ensure the quality of veterinary education, register veterinary graduates, maintain registers of practicing veterinarians and ensure that they undertake continuing professional development. some estimates were given for the numbers of veterinarians in argentina ( , ), brazil ( , active veterinarians) and mexico ( , ) . the numbers of veterinary schools were estimated at in argentina and in mexico and these are within a mixture of public and private universities. the most extraordinary growth in veterinary schools has occurred in brazil where a marked rise in the number of private institutions offering a veterinary curriculum means that there are currently thought to be over schools in the country (brazilian federal council of veterinary medicine, personal communication). sixty-three of these are in state-or federallyfunded public universities with the remainder being in the private sector. there are no centralised national curricula and the content and standard of teaching appears to be very variable. there were wide differences in the proportions of the curricula devoted to teaching of companion animal infectious diseases, immunology and vaccinology. similarly, approaches to teaching the clinical application of vaccination in the consultation room setting were inconsistent. ce is not mandatory for veterinarians and there is no scheme for recording or recognising participation in professional development. opportunities for ce are provided through association congresses, private commercial congresses and lectures (physical and by online webinar) provided by industry. in brazil, in particular, industry has an active programme for delivering ce in vaccinology by supporting lectures on the subject. in latam, many veterinary practices are small and run by single veterinarians. this creates a challenge for those veterinarians to be able to leave their practice in order to attend ce events. many of the academic colleagues with whom the vgg met were engaged in and publishing scientific research on companion animal infectious diseases. these studies form the evidence-based scientific literature for latam and where appropriate they are referenced in this document. the global challenge of obtaining research funding for companion animal studies applies equally in latam, but because of the zoonotic significance of canine visceral leishmaniosis, this is a particularly well-investigated disease in brazil. at the practice level there are many issues with the diagnosis of companion animal infectious diseases. most practices have access to point-of-care serological infectious disease diagnostic test kits, but not to diagnostic laboratories offering alternative methodologies. there is often misunderstanding of the limitations of the test kits used and the most appropriate methods for confirming a diagnosis of infectious disease. latin america encompasses a vast land mass (over million km in area) comprising countries and a human population of over million. more importantly, when considering infectious disease frequencies, it includes parts of north, south and central america as well as some caribbean islands. it extends a vast distance from north to south, spanning the equator and including temperate, subtropical and tropical climatic zones. the region includes ecosystems ranging from desert to high mountains to tropical rain forest. it would therefore be expected that infectious disease frequencies would vary markedly from region to region within this vast, diversified area. disease frequencies in some parts of latin america have been studied thoroughly (and shown, indeed, to vary widely) while many other areas have not been studied at all. the vgg obtained information on the nature and prevalence of vaccine-preventable canine and feline infectious diseases in latam via three methods: ( ) review of the peer-reviewed scientific literature, ( ) by discussion with key opinion leaders in small group meetings and ( ) by questionnaire survey of veterinarians as described above. the results of the questionnaire survey clearly showed that, in the five countries surveyed, the major canine vaccine-preventable infectious diseases are recognised by veterinary practitioners, in particular infections caused by canine distemper virus (cdv) (recognised by to % of respondents), canine parvovirus type (cpv ) (recognised by to % of respondents) and leptospira spp. (recognised by to % of respondents) ( table ). the canine infectious respiratory disease complex (cirdc; "kennel cough") is also widely recognised (by to % of respondents). although canine rabies is variably controlled in the latam countries (see below), cases are still recognised in all countries (by to % of respondents). not surprisingly, cases of canine visceral leishmaniosis (cvl) are most often recognised in brazil (by % of respondents) but appear to occur also in the other four countries (recognised by to % of respondents). the data in tables and are of uncertain accuracy. they represent responses from veterinarians as to whether they recognise, clinically, these diseases in their practices and do not explore how robustly these diseases might have been diagnosed or confirmed microbiologically. for the major vaccine-preventable feline infectious diseases, veterinarians in the five countries clearly reported recognising infections caused by feline parvovirus (fpv) ( to % of respondents), feline herpesvirus type (fhv ) ( to % of respondents), feline calicivirus (fcv) ( to % of respondents) and chlamydia felis ( to % of respondents). there appears to be widespread recognition of feline retroviral diseases and elsewhere we describe the high prevalence of these infections in certain regions. respondents to the survey recognised feline leukaemia virus (felv) infection ( to % of respondents) and feline immunodeficiency virus (fiv) infection ( to % of respondents) in their practices. although a vaccine against feline infectious peritonitis (fip) is not available in most parts of the world, including latam, we also gathered data on fip infection, which was recognised by to % of respondents. cases of rabies in cats were reported by to % of respondents. the same qualification concerning the robustness of these data (see above) applies to the information on feline infectious diseases. a summary of the relevant latam published scientific literature on these canine and feline infectious diseases is given in the sections below. high-quality epidemiological studies evaluating the distribution of infectious diseases in latam are scarce and although there are some reports, just a few published studies defined the diseases based on clinical presentation with confirmatory laboratory diagnostics. confirmatory tests are not always available in many parts of latam, especially in places where there is no veterinary diagnostic laboratory and practitioners may use human diagnostic laboratories. moreover, diagnostic tests, especially molecular analyses (i.e. reverse transcriptase polymerase chain reaction; rt-pcr), are sometimes too expensive, making the practitioner rely only on physical examination and sometimes also simple haematological examination. although diseases such as those caused by cdv and cpv infection are preventable by vaccination, in many latam countries they are still a problem because vaccination rates (i.e. "herd immunity") are too low and there is a high number of free-roaming dogs that have never been vaccinated (hartmann et al. ) [eb ] . another problem is that in some latam countries there is no requirement that vaccination be performed only by veterinarians. therefore, vaccination without clinical examination or without consideration of the quality and viability of the vaccine product is a common practice. it is also possible for a pet owner to purchase a vaccine from an agricultural merchant, without proper storage and handling, and administer it in their own home, without clinical examination by a veterinarian and without adequate transportation or maintenance of the product. regular clinical examination by a veterinarian including adequate vaccination of dogs with core vaccines is still an uncommon procedure among dog owners in many parts of latam. although there are no formal prevalence studies in most of the latam countries, there are theses and abstracts of studies in university library repositories, and some publications, showing that infectious diseases preventable by vaccination are still present in most of the countries. a meta-analysis of cross-sectional studies addressing the global prevalence of cdv showed that most of the articles from latam were from brazil, argentina and chile (costa et al. ) [eb ]. the prevalence of canine distemper in brazil was < % to - %, and in argentina from - % to > % when diagnosis was based on molecular studies. when studies were based on serology, seroprevalence ranged from - % to - % in chile and from - % to > % in brazil (costa et al. ) [eb ] . in the south of brazil, the seroprevalence of cdv was reported to be . % ( / ) in non-vaccinated dogs (dezengrini et al. ) [eb ] . a study conducted between and in argentina found . % of cases ( / ) were confirmed by rt-pcr in dogs with clinical signs of the disease. most of the dogs in that study were reportedly vaccinated against cdv, but had likely not received a full course of vaccination (calderon et al. ) [eb ] . other studies confirm that cdv is present in brazil (budaszewski et al. , monteiro et al. , alves et al. , chile (acosta-jamett et al. , colombia (espinal et al. ) , cuba (gonzález-chávez et al. ), ecuador (digangi et al. including the galápagos islands (levy et al. (calderón et al. (calderón et al. , , brazil (alves et al. , headley et al. , chile (acosta-jamett et al. ) , colombia (duque-garcía et al. ) , cuba (pino-rodríguez et al. ) , ecuador (levy et al. , aldaz et al. , de la torre et al. , digangi et al. , mexico (ortega et al. ) and uruguay (pérez et al. , puentes et al. , maya et al. [eb ]. although many veterinarians report that they see cases of canine infectious hepatitis (caused by canine adenovirus type ; cav ) in latam countries, case reports with confirmation of the diagnosis are rare. one case report from argentina made a diagnosis based on history, macroscopic and microscopic evaluation and presence of hepatic inclusion bodies (lértora & burna ) (damián et al. ) [eb ]. in the galápagos islands, where canine and feline vaccines are prohibited, a seroprevalence for cav of . % ( / ) and for cpiv of % ( / ) was reported in dogs (levy et al. ) there is little information regarding the prevalence of bordetella bronchiseptica infection in dogs in latam countries. b. bronchiseptica strains were isolated in . % ( / ) of the nasal swabs obtained from dogs in mexico (gonzález et al. ) [eb ]. although there are many studies showing a high seroprevalence of leptospirosis in dogs in latam countries, there are few publications where the agent was isolated in order to identify the serovar causing the disease. the microscopic agglutination test (mat) is the diagnostic test of choice for canine leptospirosis; however, it has poor ability to confirm the infecting serovar. studies involving isolation of leptospires from dogs are recommended for epidemiological purposes, as well as for selection of antigens for diagnostic assay development and vaccine design (sykes et al. ) [eb ] . during the vgg visits to latam countries, another commonly reported situation was the diagnosis of leptospirosis based on testing a single blood sample, sometimes considering the serovar with the highest titre as that causing the infection. although, in the presence of clinical signs, a single titre ≥ can suggest infection, it cannot confirm a diagnosis. mat must be performed with paired serum samples collected to weeks apart. a fourfold change in titre supports a recent infection (sykes et al. ) [eb ] . the serogroup with the highest titre has been interpreted as the infecting serogroup; however, the highest mat titre can vary over time, indicating that the mat does not reliably predict the infecting serogroup in acutely infected animals (schuller et al. ) [eb ]. another problem in latam countries is the lack of standardisation and quality control in laboratories performing mat for diagnosis of leptospirosis, resulting in variation in results. leptospirosis in dogs is caused primarily by leptospira interrogans and leptospira kirschneri (sykes et al. ) [eb ]. however, leptospira noguchii (silva et al. ) [eb ] and leptospira santarosai (miotto et al. ) [eb ] were also isolated from dogs in brazil. leptospira interrogans serovars most frequently isolated from both sick and apparently healthy dogs in brazil were canicola and copenhageni (yasuda et al. , rodrigues et al. , miraglia et al. , hagiwara et al. [eb ]. l. interrogans serovar pomona was isolated from a number of dogs in one report (yasuda et al. ) [eb ] . l. interrogans serovar copenhageni was also the predominant serovar in isolates from suspected canine leptospirosis cases in trinidad and tobago (suepaul et al. ) . cases of human and canine rabies have been reduced by nearly % over the past years in latam countries following active mass vaccination programmes (schneider et al. ) [eb ] . although costa rica, french guyana, guyana, panama, suriname and uruguay are free of dog rabies, other countries still report cases (velasco-villa et al. ) [eb ] . according to the pan-american health organisation (paho) epidemiologic surveillance system for rabies, during to , bolivia and haiti had the highest incidence of human rabies transmitted by dogs in the western hemisphere: % ( / ) and % ( / ) of all cases, respectively (vigilato et al. ) [eb ]. canine visceral leishmaniosis (cvl) is widespread from mexico to argentina, with autochthonous cases reported in many countries. the number of infected dogs in latam is estimated in millions, and there are high infection rates, especially in brazil (marcondes & day ) . most epidemiological studies are conducted using serology, but the application of pcr in endemic areas has confirmed that the prevalence of infection in dogs is much higher than the seroprevalence (baneth et al. ) . the seroprevalence of cvl in endemic areas of brazil ranges from . to . % (rosypal et al. , belo et al. , marcondes & day the seroprevalence of cvl increased in paraguay between to , with values ranging from % to % (miret et al. , portillo et al. . most cases were concentrated around the capital of the country, asunción, where the seroprevalence reached % in stray dogs in (miret et al. ) [eb ] . in , when the first case of cvl was reported in argentina, the prevalence of cvl (based on serology and/or pcr) was . % (cruz et al. ) [eb ] . in uruguay, a survey in salto, a city on the border with argentina, found % seroprevalence for leishmania spp. (satragno et al. ) [eb ]. studies of the seroprevalence of cvl in mexico, venezuela and colombia have reported levels between . and . % (arjona-jiménez et al. , . and . %, (zerpa et al. , , feliciangeli et al. and . and . % (fernández et al. , cortés , rosypal et al. , paternina-gómez et al. , respectively [eb ]. few studies have been published in peer-reviewed journals with a confirmed diagnosis of this disease. in the south of brazil . % ( / ) of cats tested were seropositive for fpv; % ( / ) of vaccinated cats, . % ( / ) of unvaccinated cats and . % ( / ) of cats with unknown vaccination history (johann et al. ) [eb ] . from to , cats had necropsy examination at a university hospital in southern brazil. of these, ( . %) had a diagnosis of fpv infection confirmed by immunohistochemistry (castro et al. ) [eb ]. a study conducted in brazil ( brazil ( to of faecal samples from diarrhoeic and five asymptomatic unvaccinated domestic cats, confirmed fpv infection by pcr in six ( . %) cats (garcia et al. ) [eb ] . few studies have been published in peer-reviewed journals with a confirmed diagnosis of fhv , fcv or c. felis infection. a study of cats from the south of brazil with and without clinical signs of respiratory disease, reported isolation of fhv and fcv with pcr confirmation in . % ( / ) and . % ( / ) of the cats, respectively (henzel et al. ) [eb ] . in another study of unvaccinated kittens with and without conjunctivitis in brazil, . % ( / ) had infection with fhv , . % ( / ) with fcv and . % ( / ) with c. felis, confirmed by pcr (baumworcel et al. ) [eb ]. c. felis was also identified by pcr in . % ( / ) (seki et al. ) [eb ] and in % ( / ) (gonsales et al. ) [eb ] of cats with clinical signs in two studies in brazil. most studies on the prevalence of felv and fiv infection come from brazil. although studies are available from university repositories in countries such as argentina, chile, guatemala and mexico describing high prevalence of felv infection, few of these have been published in peer-reviewed journals. in developed countries the prevalence of felv infection is usually low, but in some latam countries the prevalence appears to be high. the prevalence of felv infection reported in brazil varies according to the region studied, with values of . % ( / ) by enzyme-linked immunosorbent assay (elisa) (sobrinho et al. ), . % ( / ) by elisa (marcondes et al. ) , . % ( / ) by immunochromatography (lacerda et al. ) , . % ( / ) by immunofluorescence antibody test (ifat) (almeida et al. ) , . % ( / ) by elisa (biezus et al. ) , . % ( / ) by elisa (teixeira et al. ) , . % ( / ) by ifat (meinerz et al. ) and . % ( / ) by pcr (coelho et al. ) [eb ] . leukaemia was associated with felv infection in . % ( / ) of the cases in a study conducted in brazil (cristo et al. a,b) [eb ]. fiv prevalence in brazil appears to be lower than that of felv, with studies showing values of . % ( / ) by pcr (caxito et al. ) , . % ( / ) by pcr (teixeira et al. ) , . % ( / ) by elisa (marcondes et al. ) , . % ( / ) by elisa (sobrinho et al. ) , . % ( / ) by elisa (biezus et al. ) , . % ( / ) by immunochromatography (lacerda et al. ) , . % ( / ) by elisa and pcr (teixeira et al. ) and . % ( / ) by pcr (lara et al. ) in argentina, cats with clinical signs compatible with retrovirus infection were evaluated and fiv infection was confirmed by immunochromatography in . % ( / ) and by pcr in . % ( / ) of the cats, while felv prevalence was . % ( / ) by immunochromatography and . % ( / ) by pcr [eb ]. in the yucatan peninsula of mexico, the seroprevalence of retrovirus exposure in a population of cats was reported to be . % ( / ) for fiv and . % ( / ) for felv (ortega-pacheco et al. ) [eb ]. a study conducted on a chilean island found a prevalence of infection, by pcr, for felv of % ( / ) (mora et al. ) [eb ]. seroprevalence of retroviral exposure in a cross-sectional survey of a convenience sample of domestic cats from costa rica's greater metropolitan area was . % ( / ) for felv and . % ( / ) for fiv (blanco et al. ) [eb ] . seroprevalence of retrovirus exposure for cats living in a shelter in venezuela was . % ( / ) for felv and . % ( / ) for fiv (pino et al. ) [eb ]. a study conducted in colombia found a seroprevalence of . % ( / ) for fiv (molina et al. ) and a study in guatemala reported a seroprevalence of . % ( / ) for felv (lickey et al. ) [eb ]. information on vaccines and vaccination practice was derived from our kol meetings, the questionnaire survey and practice visits. it is recognised that there is under-vaccination of the companion animal populations in the latam countries; e.g. our argentinian kols estimated that only to % of owned pets were vaccinated and kols from brazil suggested that % of dogs and to % of cats had an annual veterinary visit (and vaccination). small companion animal vaccines are available to practicing veterinarians throughout latam. there are two major sources for these products. the majority of vaccines are produced by the major global manufacturers and are either the same or related products to those marketed in other regions and countries by those manufacturers. such products are supported by north american and/or european licensing dossiers describing their quality, safety and efficacy, and often by independent peer-reviewed scientific literature. in this document, we will refer to such products as "international vaccines" or "quality assured vaccines." the second source of vaccines is, less commonly, national manufacturers of specific products. the vgg was unable to assess the quality, safety and efficacy of such products, which are generally unsupported by independent peer-reviewed scientific literature. for that reason, all of the recommendations made in this document (with the single exception of leishmania vaccines in brazil, which will be discussed specifically below) relate only to international quality assured vaccine products. however, although the majority of vaccine products are derived from the international manufacturers, there are different and much lesser product ranges available in the latam countries compared with markets in, for example, the united states or europe. there are: ( ) fewer products from a manufacturer's range made available, ( ) unique products from an international manufacturer that are unavailable in other regions (e.g. the giardia vaccine, which has been removed from most global markets with the exception of latam and will be discussed specifically below), ( ) a trend to large multiantigen vaccines rather than the lesser antigen combinations that are now widely available elsewhere and ( ) distinct differences with respect to licensed duration of immunity of the same vaccine product between markets elsewhere and markets in latam. all of these factors make it very difficult for veterinarians in latam to vaccinate in accordance with the current wsava global vaccination guidelines. in particular, the delivery of core vaccines to adult dogs and cats no more frequently than every years is challenging when it is not possible to obtain three-component core vaccines (e.g. a combination of cdv, cav and cpv or a combination of fpv, fhv and fcv) and these are admixed with multiple non-core antigens in large multicomponent combinations. the challenge is compounded when the licensed duration of immunity (doi) for individual core antigens is -year, when the identical products in other markets carry a minimum doi claim of years. as we have found elsewhere, there was a reluctance to accept that a -year licensed product might be used "off label" every years (with informed client consent) even though the identical product is authorised in this way in other regions. this "transition stage" in the use of core vaccines was more readily embraced by veterinarians in north america and europe than it has been, or likely will be, in markets such as latam. as we found in asia, there are also challenges around rabies vaccination of individual pet animals in the practice setting (as opposed to government-run mass vaccination campaigns). rabies vaccination is mandated by law to occur on an annual basis and currently the international inactivated rabies vaccines carry a -year licensed doi in most latam countries. however, the identical products in other global markets now have a -year licensed doi. in order to progress in latam, manufacturers and regulators will need to work to extend the label claim for doi of these products, and at the same time, the veterinary professional associations will need to lobby for changes in the law (as has already happened in many other countries) so the law becomes consistent with the science. moreover, there remains a fundamental ethos in latam that a veterinary practice derives an important proportion of its income from the sale of vaccines to clients. repeatedly on our practice visits, we noted large sign boards above the reception desk in practices that listed individual dog and cat vaccines and their prices. it was clear that clients, with or without the advice of the veterinarian, would need to select the vaccines that their pet received on the basis of what they could afford to pay. the concepts of the "annual health check" and incorporating vaccination into a preventive health care programme for the individual pet (a practice "health care plan") were novel to many members of the veterinary community in latam. because of the perceived reliance on vaccine sales for underpinning practice income, there is also a current culture of "more is better." veterinarians almost exclusively deliver vaccination on an annual basis, using the vaccine product that contains the greatest antigen combination. clients have been accustomed to visiting their veterinarian annually for a "vaccine booster." annual administration of large multicomponent vaccines are deemed preferable because the client has been led to expect that this approach is superior. we were told repeatedly that veterinarians would "lose their clients" if they did not offer annual multicomponent vaccination with the highest number of antigens possible. there is something of a "vicious cycle" to this concept, because manufacturers continue to supply and promote multicomponent products that may include (for the dog) anything up to different antigens. a further issue faced by veterinarians in latam is that companion animal vaccination is not restricted to the veterinary practice. pet stores are able to vaccinate puppies and kittens before sale and owners may obtain vaccines directly for administration to their pets using poorly-considered vaccination schedules. as the vgg had seen during our asian project, there are also common and simple issues related to "vaccine husbandry" in latam veterinary practices. these largely relate to the in-practice storage of vaccines which often takes place in multiuse domestic refrigerators with no temperature monitoring and storage of multiple medicines (and often food and drink for human consumption). during our asian project, the vgg produced some simple guidelines for effective vaccine husbandry and these are replicated and extended in the current document (table ) for the benefit of latam practitioners. although the wsava global vaccination guidelines have been translated into spanish and portuguese, and are freely available from the wsava website, it was clear that most veterinarians (except for the kols) were not aware of these and had not read them. during our country visits there was often publicity in the veterinary press concerning the wsava project and the translations were promoted at our ce events. contemporaneous with the vgg latam project has been a project run by the federación iberoamericana de asociaciones de pequeños animales veterinarios (fiavac). the project is managed by the comité latinoamericano de vacunología en animales de compañía (colavac) and aims to produce vaccination guidelines that consider the epidemiological and cultural idiosyncrasies of veterinary practice in latam. the establishment of colavac is entirely within the spirit of wsava vaccination guidelines. the wsava guidelines clearly state that "these guidelines are not a mandatory edict, but rather should be used by national associations and individual veterinary practices to develop vaccination schedules relevant to the local situation." colavac is, therefore, an example of precisely that recommendation and we congratulate fiavac on this important initiative. colavac guidelines have now been released for brazil, argentina and mexico (http://www.fiavac.org/guias.php) and recently, in a scientific journal, for peru (rubio et al. ) ; however, readers will note that the guidelines differ in their recommendations for use of the same vaccines in the three countries, and sometimes differ from the recommendations made by the vgg in the present manuscript. there are some important reasons that might account for the latter observation and these relate to the ideal working practices for an expert group that produces practice guidelines. firstly, such expert groups must be completely independent of industry and should not include industry representatives on committees or have input or right of veto from industry sponsors. as readers may see from the conflict of table . vaccine husbandry: key points for veterinary practitioners • vaccines should be kept in a designated refrigerator that is only used for storage of drugs and vaccines (not foodstuff or drinks) • the electricity supply to a vaccine refrigerator should be safeguarded against inadvertent breaks by use of a switchless electric socket or a plug clearly marked "do not switch off" • vaccines (and particularly adjuvanted vaccines) have an optimum storage temperature that is usually between and °c (domestic refrigerators should be maintained at °c). these products should not be frozen or positioned adjacent to the freezer compartment of the refrigerator, and refrigerator temperature should be monitored regularly by use of a maximum-minimum thermometer located in the main body of the refrigerator. ideally, the temperature of the refrigerator should be charted in a log book on a daily basis • vaccines should be stored in the refrigerator with adequate room for air to circulate enabling a constant temperature to be maintained around the products • vaccines should be stored in the refrigerator within the manufacturer's packaging • certain shelves should be designated for specific vaccines and the location of vaccines listed outside the refrigerator. this will minimise the time the door is kept open while accessing vaccines • correct stocks of vaccines should be maintained, without overstocking • stock should be rotated so new stock is placed at the back • vaccines transported into the field should also be subject to continuation of the "cold chain." they should be transported in a cold box, but not put in direct contact with ice or ice packs • freeze-dried vaccines should be reconstituted immediately before use with appropriate diluent or liquid vaccine given concurrently (as per manufacturer's recommendations). it is bad practice and contraindicated to make up first thing in the morning the vaccines anticipated to be used during the day. some vaccine components (e.g. cdv, fhv- ) are particularly labile in this regard and so these vaccines may not induce adequate immunity if not reconstituted just before use • vaccines should only be mixed together in the same syringe if this is specified as acceptable in the manufacturer's data sheets • syringes and needles for vaccines should not be reutilized • vaccine injection sites should not be sterilised with alcohol or other disinfectant as this may inactivate infectious (mlv) vaccines • vaccines should be "in date" and precise details of batch numbers, components and site of injection should be noted in the animal's medical record interest statement that ends the present manuscript, the vgg is considered an entirely independent academic committee. secondly, guidelines should be evidence-based and supported wherever possible by peer-reviewed published scientific literature. the vgg developed an evidence-based hierarchy for the science of vaccinology (day et al. ) , which is applied to its global and now regional guidelines. finally, guidelines documents themselves should undergo scientific peer-review and be published in a creditable scientific journal rather than an industry magazine. vgg documents have always undergone such independent scrutiny and are published in the official scientific journal of the wsava, the journal of small animal practice. it is challenging to make recommendations regarding undergraduate education in an environment where there is such diversity in resources and standards between state-funded and private veterinary schools and a lack of any form of national curriculum. there is concern from the veterinary profession itself in the standards that might apply to the rapidly growing market in private veterinary education. the vgg can only emphasise the importance of solid grounding in the traditional teaching of veterinary immunology and microbiology as it pertains to small companion animal infectious disease and vaccinology, and to the development of tuition in client communication and delivery of vaccination in a clinical setting that must occur during the clinical years of a veterinary programme. those academics who teach such curricular elements should be encouraged to be well-versed in the wsava global vaccination guidelines. there is also clearly a huge need for postgraduate ce in small companion animal vaccinology. this is, to a large extent, the responsibility of the professional associations and veterinary industry and it is encouraging to see some of the initiatives for these subjects being incorporated into congress programmes and forming the content for postgraduate seminars in latam countries. the use of electronic delivery of tuition (i.e. via webinars) is also becoming more widespread in latam and vaccinology should comprise a valuable component of such programmes. the wsava itself hopes to further develop an on-line platform for delivery of ce in the near future and vaccination guidelines will form part of that content. it would be beneficial if discussions could be held in latam about moving towards compulsory continuing professional development for re-registration purposes for veterinary professionals. as can be seen from the summary of peer-reviewed scientific data related to small companion animal infectious diseases and vaccinology presented above, there is a marked lack of relevant information available to the veterinary profession in latam. given the very large numbers of academic institutions and academic staff devoted to tuition in microbiology and immunology, it is disappointing that there are few active research programmes in this important area of veterinary medicine. it is likely that this reflects a number of possible contributing factors: ( ) that in private institutions there is a focus on teaching rather than research, ( ) that it is challenging to obtain research funding for investigation of small companion animal infectious disease that does not have zoonotic potential (e.g. cvl, leptospirosis and canine rabies), ( ) that greater academic kudos might be derived from investigating diseases of production animals and ( ) that diagnostic laboratory infrastructure for undertaking infectious disease surveillance programmes is lacking. although much of the scientific literature reviewed by the vgg was generated in brazil, it is alarming to see, in that country, the current climate of governmental cuts in support for tertiary education and research, in particular for programmes that support the training of the next generation of veterinary research scientists via msc and phd studies. the vgg strongly supports the performance of clinically relevant research into small companion animal infectious diseases. in particular, developing a clear understanding of the regional prevalence of different infectious agents and the classification of infectious agents circulating in the field (e.g. specific serovars of pathogenic leptospires). there are clearly some distinctive aspects of infectious disease epidemiology in latam, in particular the observed higher prevalence of feline retroviral infections. only by generating current surveillance and molecular phylogeny data will there be advances in clinical diagnostics, vaccine availability and disease control. as we suggested in asia, there may be mutual benefit to academic researchers in veterinary schools working more closely with industry in order to generate clinically relevant data that might be used to introduce new vaccine products into latam for the benefit of the profession and the animals for which we care. as will become clear from the recommendations made below, there is a particular challenge facing the implementation of global vaccination guidelines in latam. this relates very simply to the lack of minimal-antigen product ranges that are widely available in north america, europe and other markets, and enable veterinarians in those countries to vaccinate in accordance with wsava guidelines. until there is a shift away from large multi-antigen vaccines (containing anything up to different antigens) towards trivalent or bivalent core vaccines with monovalent or bivalent non-core vaccines, it will be challenging for latam practitioners to embrace the new standards in vaccinology that are now well-embedded in many other markets. once such product ranges are more widely available (currently only in argentina), it will then require significant education as to how they are best used in practice and a radical change in ethos to embrace the concept of preventive health care delivered through an annual health check or health care plan, as opposed to the deliberate marketing of vaccine products as commercial drivers of central importance in veterinary practice. an important challenge in making such changes lies in identifying who has responsibility for driving change. arguably, veterinary industry should lead in bringing revamped product ranges to latam, but this cannot happen without the support of the veterinary profession via the professional associations and without some flexibility in vaccine licensing requirements. with respect to the latter, the vgg supports the acceptance of licensure studies performed for other markets in obtaining new licences in new markets. at very least, not having to perform additional studies for products that are already licensed in north american or european markets would provide significant animal welfare benefits. there remains the significant challenge of veterinary vaccines being available directly to owners and breeders out with the veterinary practice. a change in emphasis from the veterinary practice simply selling vaccines to selling a holistic preventive health care package based in professional advice will be required in order to re-educate the pet-owning public and attract them back to the veterinary practice. the vgg recommends that veterinarians in latam implement the basic principles of evidence-based companion animal vaccinology presented in the wsava global vaccination guidelines (table ; day et al. ) [eb ] . understanding the concept of core versus non-core vaccines is fundamental to application of the vaccination guidelines in practice. core vaccines are those that every dog, regardless of location or lifestyle, must receive for protection against infections that cause significant morbidity or severe or fatal disease. core vaccines contain cdv, cav and cpv , preferably as modified live viruses (modified live vaccine; mlv). in countries where canine rabies remains an endemic disease, inactivated rabies vaccine is also regarded as a core vaccine for every dog. non-core vaccines are those considered for individual animals whose geographical location or lifestyle puts them at risk for specific infections. non-core vaccines are not required for every animal and should not be used where there is no evidence for existence of a related disease or when the risk for exposure is minimal. non-core vaccines include leptospira vaccines and vaccines designed to protect against elements of cirdc, which usually contain b. bronchiseptica with or without cpiv. the wsava global guidelines classify some vaccines as not recommended for any dog because there is insufficient scientific evidence to justify their use. these include the inactivated canine enteric (non-pantropic) coronavirus (enteric ccov) vaccine and the giardia vaccine when used to either prevent or treat infection. non-core vaccines are generally given annually unless the datasheet specifically recommends otherwise. felv vaccines may be given every or years to adult cats (and some qualityassured felv vaccines carry a licensed -or -year doi in markets outside of latam) [eb ] not recommended vaccines these include vaccines against coronavirus (canine or feline), giardia and microsporum canis the generic information in this table should be read in conjunction with the more detailed recommendations provided in the current wsava vaccination guidelines (day et al. ) . vaccination according to wsava guidelines is possible only where available product ranges separate core from non-core vaccine components. note that these recommendations apply only to quality-assured vaccines, most of which are produced by large, international companies. ccov is considered of minor clinical importance as a primary enteric pathogen, causing only mild diarrhoea in puppies. more severe enteric disease occurs with ccov and cpv coinfection (decaro et al. ) [eb ] . published studies have demonstrated that commercial inactivated enteric ccov vaccines induce only transient serum antibody responses and do not reduce viral infection or faecal shedding of virus compared with non-vaccinated dogs (pratelli et al. , de castro et al. [eb ]. moreover, injectable ccov vaccine does not lead to elevation of faecal ccov-specific iga antibody concentration, which is believed to underlie immune protection (decaro et al. ) similarly, dogs vaccinated with a commercial inactivated giardia vaccine were not protected from giardia infection in that there were no differences in parasite cyst or antigen detection rates or the occurrence of diarrhoea between vaccinated and unvaccinated dogs (anderson et al. , lund et al. [eb ]. in addition, treatment of giardia-infected animals with a commercial inactivated giardia vaccine was not effective in eliminating cyst production (anderson et al. ) the wsava guidelines recommend revaccination of puppies with international mlv core vaccines at timed intervals over the first months of age to overcome interference by maternally-derived antibody (mda) [eb ] . the guidelines also recommend that a final mlv core vaccine is given between months to year of age in order to ensure that all puppies receive at least one dose of vaccine that is able to confer immunity in the absence of mda [eb ]. development of protective immunity is not dependent on the number of mlv core vaccines given during the puppy vaccination series, but rather when they are given. for adult animals, there is ample evidence supporting revaccination with quality-assured international mlv core vaccines no more frequently than every years (abdelmagid et al. , bohm et al. , mouzin et al. , gore et al. , schultz , larson & schultz , mitchell et al. , killey et al. [eb ]. while regulatory authorities in latam countries require annual revaccination with international mlv core vaccines licensed elsewhere for use at -year intervals, this practice is regarded as inappropriate use of client financial resources that are better applied to annual wellness examinations, routine parasite prophylaxis, and treatment of medical issues. increasing the frequency of vaccination with mlv core vaccines does not confer greater protection to an individual animal. increasing the number of animals that are properly vaccinated is much more important to ensure protection through population or "herd" immunity than vaccinating each animal more often. even though the -year doi datasheets for quality-assured international mlv core vaccines are not currently accepted by latam countries, the vgg encourages the national and local regulatory authorities to permit practitioners to use these vaccines according to the wsava guidelines as "off-label use" products with informed client consent. this approach has been used successfully in other countries pending acceptance of the quality-assured international vaccine datasheets by the national and local authorities. issues with vaccine product availability, product licensed doi, and knowledge of disease prevalence and exposure risks hampers adoption of the wsava global vaccine guidelines by practitioners in latam countries. in many of these countries, there is limited availability of quality-assured multicomponent and single-component international vaccines allowing separate use of mlv core antigens versus non-core antigens. the paucity of peer-reviewed studies on specific disease prevalence in latam countries presents challenges for practitioners in making evidence-based decisions on what non-core vaccines are appropriate for individual animals in different regions. latam practitioners and their national associations should lobby industry and government regulators for access to quality-assured international canine vaccines that contain just the mlv core components (cdv, cpv , cav ) or the non-core components (leptospira, cpiv, bordetella). this will allow for administration of the mlv core vaccine every years and for separate annual vaccination with non-core vaccines for individual dogs at risk. currently, most quality-assured international vaccines available in latam countries contain mlv core antigens (cdv, cav , cpv ) combined with non-core antigens (leptospira) and non-recommended antigens (i.e. enteric ccov). practitioners can follow some pragmatic recommendations presented in table to transition from giving these multicomponent vaccines every year to every dog to using the core and non-core vaccines separately according to the wsava guidelines. included in this transitional pragmatic protocol is the "off-label" administration with client consent of mlv core vaccine components every years to adult dogs instead of annually. the vgg recognises that practitioner use of this pragmatic protocol is limited by local product availability. for clients that can only afford one vaccine for their dog, the recommended approach is to choose a quality-assured international vaccine containing the mlv core components and give that vaccine at a time when the single dose can induce long-lasting protective immunity in the absence of maternal antibody interference (i.e. at months of age or older). canine visceral leishmaniosis (cvl) caused by leishmania infantum is one of the most significant zoonotic diseases in latam and the geographical distribution of cvl is expanding in the region. cvl is widespread from mexico to argentina, with autochthonous cases reported in many countries (marcondes & day ) [eb ]. although vaccines can prevent active infection and the risk of development of clinical disease in some dogs, some vaccinated dogs can become progressively infected and transmit the parasite to the sand fly vectors even in the absence of clinical signs (bongiorno et al. , fernandes et al. , oliva et al. , regina-silva et al. [eb ]. therefore, for animals living in endemic areas, from an epidemiological viewpoint, it is more important to use insecticides, especially collars, to prevent sand fly bites, than it is to vaccinate (sevá et al. , lopes et al. . whenever possible, the two measures should be combined, in order to provide a high level of protection not only for dogs, but for other animals and people that share the same environment. it is important to highlight that a previous history of vaccination does not exclude a diagnosis of cvl in dogs with clinical signs or clinicopathological abnormalities suggestive of the disease. currently in latam there are only two licensed vaccines against cvl. one contains the recombinant a protein of l. donovani in an adjuvant, licensed in brazil and paraguay, and the other consists of purified excreted-secreted proteins of l. infantum (liesp) in an adjuvant, and is licensed in paraguay and argentina. the vaccination protocol for puppies includes three doses given weeks apart and an annual booster. the a vaccine can be used in dogs from months of age and the liesp vaccine from months of age. adult dogs that have never been vaccinated receive the same protocol. vaccines against cvl should only be considered for dogs living in endemic areas, where they are at risk of being infected. according to the manufacturer's recommendations, only seronegative dogs should be vaccinated; however, many dogs can be infected without seroconversion, and therefore, be improperly vaccinated. it is clear that large-scale mass vaccination programmes conducted over recent decades have succeeded in controlling canine rabies virus infection in dogs and cats (and therefore the human population) in many latam countries. there are, however, remaining "hot spots" for disease and low numbers of individual cases are recorded in countries with good overall control. in most latam countries there is ongoing surveillance and legally-mandated annual canine rabies vaccination. this may be delivered by mass vaccination field campaigns run by government or non-governmental organisations or through the veterinary practice for individual client-owned pet animals. vigilance and continued vaccination to maintain herd immunity are essential at this time for maintaining control of canine rabies. as discussed above, there is a disconnect between the law and the science pertaining to rabies vaccines. there is no doubt that non-core vaccines are generally given annually unless the datasheet specifically recommends otherwise; felv vaccines need not be administered to adult cats on an annual basis (see table ) not recommended vaccines these include vaccines against coronavirus (canine or feline), consider whether there is sufficient scientific evidence to support their use the generic information in this table should be read in conjunction with the more detailed recommendations provided in the current wsava vaccination guidelines (day et al. ) . note that these recommendations apply only to quality-assured vaccines, most of which are produced by large, international companies. in mass vaccination field campaigns (particularly where these aim to vaccinate free roaming stray or community owned dogs) annual revaccination is essential to account for population turnover. however, for a client owned, veterinarian-visiting individual pet animal, vaccination with an international quality-assured canine rabies vaccine should confer a minimum year duration of immunity (day et al. ) [eb ] . a move to licensing those vaccines with a -year doi as the same products have in the usa, canada and europe would help address this anomaly. feline vaccination: aspirational protocols when aspiring to produce an optimised vaccination protocol for cats in a particular locale, the immensity and diversity of latin america must be borne in mind. nevertheless, it is possible to provide broad advice to latam veterinarians based on what has been learned about feline infectious diseases in the region and beyond, and by considering what vaccines are commercially available in latam. veterinarians in all latam countries are encouraged to follow the fundamental advice provided in the latest wsava vaccination guidelines (day et al. ) (table ). as these guidelines make clear, there are core vaccines that, in an ideal world, all pet kittens should receive and all adult cats should receive sufficiently frequently to ensure protection throughout life. these vaccines protect against infectious agents that can cause severe illness or death, especially in kittens. in all countries, vaccines against fpv, fhv and fcv are considered core (scherk et al. , day et al. . in countries where rabies is endemic, rabies vaccines are also regarded as core (scherk et al. , day et al. [eb ]. in addition, there are non-core vaccines. not every kitten or cat need necessarily receive every one of the non-core vaccines. use of these vaccines should be based on an informed risk-benefit analysis, based on knowledge of local disease frequencies and the lifestyle of the individual cat (day et al. ) [eb ]. non-core vaccines protect against infectious agents that may be encountered frequently in some areas, but are known to be rare or absent in other places (e.g. felv). some non-core vaccines (e.g. those against c. felis infection) protect against disease agents that are generally less pathogenic than those covered by the core vaccines or are treatable using antibiotics. the vgg categorises one feline vaccine (against fip) as "not recommended." although this is a commercially-available vaccine in some countries (not in latam), the vgg has judged that there is insufficient evidence of benefit to recommend its routine use. in some latam countries (e.g. brazil) felv has been reported to be highly prevalent in some regions (i.e. the southeast and deep south of the country) and much less prevalent in others, such as the north (almeida et al. , lacerda et al. , biezus et al. , cristo et al. a [eb ]. overall, felv prevalence in brazil seems to be considerably higher than in many other countries where its prevalence has been studied (galdo novo et al. ) [eb ]. in mexico, felv prevalence has been shown to exceed that of fiv in merida, tropical mexico, but does not match the very high prevalence values reported from various parts of brazil (ortega-pacheco et al. ) [eb ]. therefore, it is recommended that latam veterinarians seek to establish felv prevalence in their local area, to allow evidence-based decisions to be made about the recommended use (or otherwise) of felv vaccines. this is the essence of how non-core vaccines should be used. high-quality, mlv fpv vaccines have been shown to provide long-lasting, robust immunity to a large majority of vaccinated cats when used in accordance with wsava vgg guidelines , barrs [eb ]. as a precaution, revaccination every years is generally recommended. vaccinating more frequently than every years with high-quality mlv fpv vaccines is unlikely to provide any improvement in the degree of protection provided by these vaccines and may increase the risk of adverse reactions. it is far more important to ensure that a large proportion of the target population is vaccinated (i.e. to increase the overall herd immunity) than it is to increase the frequency of revaccination of individual animals in the population at risk. indeed, the unhelpful annual revaccination of cats against fpv with products, known to provide many years of protection, should be viewed as poor use of potentially limited client financial resources. these could be better applied to address other health issues in the pet and perhaps could be used to purchase non-core vaccines, if use of one or more of those is supported by evidence and hence justifiable in that locale. high-quality mlv vaccines against fcv and fhv do not provide such robust or long-lasting protection as do the fpv vaccines just mentioned (jas et al. ) [eb ]. the immunity conferred by these vaccines cannot prevent infection or development of the carrier state. nevertheless, for cats living "low risk" lifestyles (i.e. indoor only cats that do not visit boarding catteries) vaccination every years is considered to provide sufficient protection (scherk et al. , day et al. . for cats at higher risk of fcv or fhv infection (i.e. cats with outdoor access or cats regularly visiting a boarding cattery), annual revaccination is recommended [eb ] . in some countries, it is possible to purchase vaccines that contain only fcv and fhv , so that trivalent vaccines can be used every years (fcv, fhv , fpv) and a bivalent vaccine (fhv , fcv), if needed, in each of the intervening years. unfortunately, such products are not, as yet, consistently available throughout the world. rabies vaccines must be used in accordance with local regulations. notably, some international quality assured rabies vaccines for use in cats provide protection for at least years (jas et al. ) [eb ]. in the usa, regulations requiring annual revaccination of cats, despite evidence of much longer-lasting protection by some vaccines, were challenged and changed as a consequence of effective political lobbying by the veterinary profession and pet owners. a crucial feature of an idealised vaccination protocol for cats in any country would include a finish to the kitten series no earlier than weeks of age. this is because evidence has accumulated in recent years indicating that a sizeable minority of kittens have significant amounts of interfering maternal antibodies against some of the vaccine components, even at up to weeks of age (digangi et al. , jakel et al. a [eb ]. a weeks or later finish is consistent with this scientific evidence and with the current wsava vaccination guidelines, as well as with guidelines from other organisations. vaccine products available in latam countries may not carry datasheet recommendations for use as described in this section. it would be helpful if local regulations and guidance from veterinary professional organisations enabled practitioners to use vaccines "off label" with informed client consent. this approach was used for years by veterinarians in other countries, before datasheet recommendations were eventually updated. the vgg hopes that, in due course, quality assured core mlv vaccines produced by large, international pharmaceutical companies will have datasheet changes made in the latam countries. feline vaccination: pragmatic protocols small animal practitioners in latam countries are currently unable to adopt the wsava vaccination guidelines in full. this is for a number of reasons. firstly, rational use of non-core vaccines is hampered in many parts of latam by a lack of information about disease frequencies. conversely, for some regions, excellent, detailed information is available. where evidence is lacking, veterinarians often decide to take a precautionary approach. this can lead to unnecessary overuse of non-core vaccines. more research and surveillance would enable more informed, selective use of non-core vaccines. secondly, in many latam countries there is limited product availability. in particular, core vaccines licensed and approved for biennial, triennial or less frequent use are unavailable in many latam countries. in part, this may be because of a lack of locallygenerated evidence for extended doi and a requirement for such evidence from local regulatory authorities. yet multiple strands of evidence, generated in numerous countries, support a view that feline core vaccines can be used in latam countries similarly, and with as much confidence, as in other parts of the world. although current datasheets for many quality assured mlv core vaccines recommend annual revaccination of adult animals, the very same vaccines are given triennially in many other countries, including some with high infectious disease pressure. another challenge concerning product availability in latam is a paucity or lack of monovalent non-core vaccines. for example, in some countries, c. felis vaccines are only available in combination with the core fpv, fhv and fcv components and felv is only available in combination with the preceding four. therefore, there exist -component, -component and -component feline vaccines, but few or no monovalent, non-core products. a veterinarian who wished to protect against felv as well as the core agents, but perceived no need to protect against c. felis, might thus be forced to administer the c. felis component, even if it was judged superfluous. veterinary practitioners and regional associations should therefore continue to lobby industry and government regulators for changes that would bring recommendations concerning use of quality assured vaccine products into line with those that apply and are used in many other parts of the world. table presents some pragmatic recommendations concerning use of feline vaccines that are intended to assist latam practitioners to head in the recommended direction. application of preventive health care plans with an annual health check to latin america as discussed earlier in this document it was clear from our discussions and practice visits in latam that the dominant culture in veterinary practice is that veterinarians sell vaccines to clients, that the sale of vaccines is the main driver for client attendance at the veterinary practice, and that vaccine sales underpin a large component of veterinary practice income. indeed, some years ago, these were global principles that also applied to veterinary practice in north america, western europe, australia, new zealand, south africa and other developed markets. in the latter markets, in , this culture has now been substantially replaced by a new way of promoting veterinary services (including vaccines) to clients. there has been a progressive move away from the concept of the "annual vaccination booster" or the "vaccine booster consultation" towards the implementation of holistic preventive health care packages delivered in part by an "annual health check" consultation. in more developed markets, this has now been extended to delivery of a practice "health care plan" for which a client might pay a regular monthly fee to cover numerous elements of preventive health care for their pets. the annual health check consultation is considered to require a longer period of time than a general consultation and provides the opportunity for the veterinarian to engage with the client to discuss in detail the overall health and wellbeing of the companion animal family member. elements of the health check consultation (or of an annual health care plan) might include consideration of nutrition, dental health, behavioural issues, endo-and ectoparasite control, vector-borne diseases testing and which vaccines (core or non-core) might be delivered during this annual visit. indeed, in many mature markets, annual assessment of the need for core revaccination (cdv, cav and cpv for dogs and fpv for cats) is now determined by in-practice serological testing ("titre testing") to determine whether an animal is already protected and therefore need not be revaccinated. the wsava global vaccination guidelines mention the value of serological testing and provide strong support for this approach. there is increasing literature to support the use of such serological tests in veterinary practice (e.g. killey et al. ) [eb ]. moreover, there is a substantial literature that evaluates the annual health check consultation and advises on the content, timing and approach to such a consultation (e.g. belshaw et al. ) [eb ] . implementing this new approach to delivery of companion animal preventive health care may be daunting for many latam practitioners. however, these changes need to be embraced in order for the profession in latam countries to keep pace with colleagues in more developed markets. whilst there may be a longer transition period for veterinarians working with economically constrained clients, these new concepts should be more readily embraced by those working in areas of relative prosperity. the vgg gratefully acknowledges the numerous kols in argentina, brazil and mexico who travelled to meet with us, often over long distances, to share their knowledge and expertise. we also thank the veterinarians in those countries who allowed us to visit their practices and the many veterinarians who completed our questionnaire survey. we are grateful to our colleagues from msd animal health, at the global, regional and national levels, who undertook all of the logistics for our country visits and we particularly thank them for the work involved in organising the ce events in each country. the work of the vgg has been supported financially by msd animal health who are global partners with the wsava. the vgg is an entirely independent group of academic experts who have authored this manuscript without consultation with industry. representatives of the sponsoring company do not attend vgg meetings. the company does not have the right of veto over vgg recommendations. questions about vaccine products the vgg does not recommend this vaccine because there is insufficient scientific evidence to justify its use. the evidence that canine enteric coronavirus is a primary pathogen leading to intestinal disease in adult dogs is weak; the diarrhoea associated with infection is mild unless there is concurrent infection with cpv . experimentally, the virus causes only mild diarrhoea, if any, in dogs over weeks of age and vaccination against cpv alone appeared to protect against challenge by both viruses. there is no evidence that available vaccines would protect against pathogenic, mutant forms of the virus that occasionally arise and have been described. there is even less evidence that the vaccine can protect against infection in the field and injectable vaccine does not appear to induce protective faecal iga antibodies (decaro et al. ) . brazilian data shows no difference in the identification of canine enteric coronavirus by pcr from the faeces of normal dogs and dogs with diarrhoea (gizzi et al. ) [eb ]. the vgg does not recommend this vaccine because there is insufficient scientific evidence to justify its use. the evidence that the vaccine can prevent either shedding or infection is weak. one large field study of dogs showed that vaccinated puppies were actually more likely to have diarrhoea than unvaccinated puppies, and there was no difference between these groups with respect to cyst or antigen detection (lund et al. ) [eb ]. the disease in dogs is not life-threatening, is rarely zoonotic, is of low prevalence and responds to therapy; for these reasons the vaccine is not recommended for client-owned dogs. it is not known whether the vaccine can cross-protect against strains of giardia other than those used in challenge studies. brazilian data shows no difference in the identification of giardia by pcr from the faeces of normal dogs and dogs with diarrhoea (gizzi et al. ) [eb ] . it is notable that the vaccine has been withdrawn from all markets globally with the exception of those in latam. although published studies addressing this question do not always agree, the vgg is of the belief that, immunologically, vaccination via a mucosal route is more likely to generate relevant protective immunity (specifically the production of mucosal iga and igg antibodies as opposed to systemic igg antibodies) for pathogens that infect via the same mucosae (larson et al. ) [eb ]. intranasal vaccination may have the added benefit of a rapid onset of immunity that may relate to a non-specific stimulation of innate immunity (via toll-like receptor engagement and local cytokine/chemokine production) [eb ]. this may be beneficial when a dog is to be exposed in the short term to an environment where there is a risk of exposure to elements of the canine infectious respiratory disease complex (cirdc). intranasal vaccines may be used in puppies as early as weeks of age as a single dose, with annual revaccination required. intranasal vaccines are available (depending on the market and not all in latam) that are specific for b. bronchiseptica (bb) alone or bb in combination with cpiv, or bb in combination with cpiv and cav . oral vaccines against bb are available in australia, north america and europe. these products have the advantage of ease of application, but contain only a single antigen (bb) and may not share the same rapid onset of immunity as intranasal products. they may be given to puppies from weeks of age and require annual revaccination. there is variation in the scientific literature as to whether the oral and intranasal bb vaccines confer equivalent protection, or whether protection is better with intranasal products (larson et al. , ellis et al. a , scott-garrard et al. [eb ]. as to why these products are not available in latam, you would need to ask the manufacturers and regulatory authorities responsible for vaccine licensure. the vgg classifies leishmania infantum vaccines as non-core, meaning that their use should be restricted to dogs at risk in areas endemic for the infection. leishmania vaccines are only available in brazil, argentina and paraguay. these vaccines should be regarded as one tool in the prevention of canine visceral leishmaniosis. control of the access of susceptible dogs to sand flies (e.g. by housing indoors during the times of greatest sand fly activity) and the use of sand fly preventives (e.g. collars) is far more important than vaccination (sevá et al. , lopes et al. [eb ]. vaccines do not produce sterilising immunity; they may prevent or lessen the severity of clinical signs in infected animals, but do not always prevent infection and so even vaccinated dogs may act as a reservoir for leishmania (regina-silva et al. ) [eb ]. dogs should be tested before vaccination as vaccination of a dog that is already infected is of no benefit in the prevention of infection and a waste of vaccine. borrelia of different species are associated with infection and disease of dogs and cats in north america and europe through to asia. borrelia are transmitted by ixodes spp. ticks and have wild mammal and avian reservoirs. in order to introduce a borrelia vaccine to latam, there would need to be robust research studies into whether the pathogen exists in this region, whether there are competent tick vectors and wildlife reservoirs, and whether dogs and cats could become infected and go on to develop clinical signs of disease. in brazil, studies have identified borreliosis in human patients with brazilian lyme-like disease or baggio-yoshinari syndrome (yoshinari et al. , mantovani et al. , and borrelia burgdorferi sensu lato in asymptomatic humans (gonçalves et al. ) and ticks from the genus dermacentor (gonçalves et al. ) [eb ]. few reports relate to companion animals, and although anti-borrelia antibodies have been found in dogs, to our knowledge the pathogen has not been isolated from sick dogs (spolidorio et al. , nascimento et al. [eb ]. on that basis, there is currently no basis on which to justify introduction of a companion animal borrelia vaccine to the region. modern high-titre international quality-assured vaccines are more likely to be able to do this, which is why the vgg recommends use of such products. where available, the use of high-titre combination cdv and cpv vaccines designed for use in young puppies is also recommended where core vaccination is started earlier than weeks of age (see tables and ). however, there is no guarantee that every puppy will make an early, active immune response to each vaccine antigen and so the global wsava guidelines should be followed: by giving the final early-life dose of core vaccine at weeks of age or older with a follow-up vaccine between and months of age. this situation was faced by veterinarians globally in the last two decades when guidelines recommendations were for adult core revaccination no more frequently than every years, but products all had a licensed duration of immunity of year. at that time, veterinarians were able to use the available products in accordance with guidelines simply by obtaining informed client consent (and documenting this in the medical record) for "off label" use of the product. there was never any legal claim brought successfully against a veterinarian for doing this, nor examples of dogs contracting infection because of extending vaccination intervals. subsequently, in many markets globally, the identical core vaccines were relicensed with a -year doi. until this relicensing occurs in latam, veterinarians can adopt the same strategy that was used very successfully over the last to years in north america, europe and other regions. over the decades since the first identification of cpv in , new biotypes of the virus (cpv a, cpv b and cpv c) have emerged in many parts of the world, including latam. these virus variants are characterised by subtle changes in amino acid sequence in the vp protein. most vaccines contain either cpv or cpv b and questions have been raised as to whether these provide adequate cross-protection against new virus variants (specifically cpv c). there are numerous studies that show such crossprotection occurs and that all current cpv vaccines remain efficacious in the field (e.g. spibey et al. , wilson et al. [eb ]. occasional reports occur of clinical parvovirosis in vaccinated dogs, but this scenario generally relates to failure to vaccinate according to guidelines recommendations or vaccination of puppies that are already incubating the virulent virus. while there is no doubt that leptospirosis occurs in dogs in latam, there is minimal high-quality scientific evidence about the geographical distribution, causative serovars and clinical manifestations of the disease. the major weakness in many published studies is that the gold-standard for confirming clinical diagnosis (i.e. paired serology weeks apart by the microscopic agglutination test [mat] ) and identifying the infecting serovar (isolation of the organism) has not been used. available studies do suggest that the dominant serovars circulating in the field in latam may still be l. interrogans serovars canicola and copenhageni [eb ] , and that consequently, the traditional canine "l " vaccines containing these organisms might confer adequate protection. in north america and europe, "l " and "l " vaccines have been marketed to help address a greater diversity of causative serovars in those regions. at present, there is insufficient evidence on which to formulate a specific latam vaccine or to recommend adoption of the north american l products in an evidence-based fashion. where there is solid scientific evidence (see question ) that leptospirosis is a significant clinical problem then it makes perfect sense to routinely vaccinate at-risk dogs to prevent a serious and potentially zoonotic infectious disease. however, it is simply not possible for the vgg to classify leptospira vaccines as core in our global guidelines, because there are parts of the world in which the infection does not exist or is of very low prevalence. moreover, the lifestyle of some individual dogs does places them at lower risk of encountering this infection. this is why the vgg promotes the use of non-core vaccines based on regional disease surveillance data coupled with a lifestyle history of the individual pet. latam practitioners must do their best to obtain reliable local data on leptospira infection to help inform decision making about use of this vaccine. decision making about whether or not to use non-core leptospira vaccines would be facilitated by robust surveillance data indicating whether leptospira was prevalent in your geographical area and which serovars were circulating in the field. unfortunately, such data are not available in most parts of the world. consequently, the decision to vaccinate needs to be taken based on the lifestyle of the individual dog. apartment dwelling dogs with limited and controlled access to outdoors are unlikely to require leptospira vaccination. however, dogs with outdoor access and particularly those with access to water that might be contaminated via exposure to rodents or domestic livestock should be vaccinated. even dogs kept in a yard may be at risk if small wildlife (e.g. rodents) can also access the yard. the prevalence of felv was much higher in europe to years ago than it is today. it is thought that the combination of ( ) diagnostic testing for felv (which has become considerably more convenient and accurate over those decades), ( ) suitable management of cats found to be infected and ( ) widespread vaccination against felv, have together led to the substantial decrease in felv prevalence in some countries (studer et al. ) [eb ]. the first step in regions of latam where felv has not been well studied would be to determine local felv prevalence. if testing is considered impractical or too expensive, then it may help to recall that high prevalence of feline multicentric lymphoma, cranial mediastinal lymphoma and very severe non-regenerative anaemia are strong clues that felv may be prevalent in the area. in regions where many cats are known to become infected each year, client education and widespread vaccination should be practiced. ideally, cats should be tested prior to first vaccination because there is no benefit to vaccinating a cat that is already infected when that dose of vaccine may benefit another animal. this decision should be based on a risk-benefit analysis. if felv is known or highly suspected on the basis of good information to be prevalent in the town or region where you work, use of the non-core felv vaccine can be justified. for example, if there is a high prevalence of strongly felv-associated diseases, such as multicentric or cranial mediastinal lymphoma, increased pressure on owners to permit testing and vaccination against felv (in addition to use of core vaccine) is justified. each has its own advantages and disadvantages. if it is judged necessary to vaccinate a pregnant or immunosuppressed cat (e.g. a retrovirally-infected cat), an inactivated vaccine is considered safer from first principles (although the evidence for this is limited and recent studies suggest this may not be the case; bergmann et al. ) [eb ] . similarly, where there may be a multi-cat establishment without a history of upper respiratory tract infection, use of a killed product (from first principles) would reduce the risk of transfer of live vaccine virus. in some countries, inactivated vaccines are used in more cats than are mlv vaccines. in many other countries, mlv feline vaccines are used in far more cats than are inactivated vaccines. although it is considered uncertain and controversial by some experts, there is evidence that adjuvant (present in inactivated and subunit vaccines but generally not in mlv vaccines) is implicated as being associated with development of the feline injection site sarcoma (fiss) (abdelmageed et al. , kass [eb ]. this would be an important disadvantage of adjuvanted vaccines. there is some limited evidence that inactivated fhv vaccines provide more rapid onset of protection than do mlv fhv vaccines (lappin ) [eb ]. finally, where canine rabies is an endemic disease and cats should receive rabies vaccination as core, use of inactivated adjuvanted vaccine is the only option, unless there is access to a recombinant product. the vgg does not make recommendations about specific commercial brands of vaccine or generally about classes of vaccine. in some parts of the world there are recombinant virus-vectored vaccines available to protect against cdv, felv and rabies. in some circumstances these do have advantages: for example for use in wildlife species and as a means of avoiding use of adjuvanted rabies vaccine in cats, as these are one possible injectable product (of many vaccines and non-vaccine injectables) that have been associated with fiss (see questions , and ). to our knowledge, such monovalent products are not available in latam. rabies vaccines are routinely administered to individual pet animals visiting the veterinarian only every years in north america and europe. in those regions the legal requirement is for triennial revaccination of adult dogs and cats. the international qualityassured rabies vaccines used in those regions all carry a licensed doi of years. the licence is based on firm scientific evidence and licensing studies (e.g. lakshmanan et al. ) [eb ]; without such evidence the laws would not have been changed to allow -yearly revaccination. unfortunately, the identical rabies vaccine products, used in asia, africa and latam are administered annually. this is because the regional or national laws have not changed and industry has not relicensed the products with a -year doi as has occurred in north america and europe. in latam, you are still legally obliged to give rabies vaccines on an annual basis; however, the veterinary profession should be lobbying for change in the laws and relicensing of the vaccine products. you should also note that this only applies to individual pet animals visiting the veterinarian for vaccination. in the context of mass vaccination campaigns in the field (as might be conducted by government authorities or non-governmental organisations), rabies vaccines are still administered annually to as many dogs as possible (including to free-roaming dogs). this is because there is generally high population turnover in free-roaming dog populations and annual revaccination is required to maintain herd immunity levels of % [eb ]. this is a frequently asked question throughout the world. vaccines are unlike pharmacological drugs and are not administered on a mg/kg basis. vaccines contain a defined amount of antigen, which is the amount required to stimulate a primary or secondary immune response in an animal. each individual person and animal has an "immunological repertoire" of antigen-specific t and b lymphocytes defined by t-cell and b-cell receptors (tcrs and bcrs). burnett's "clonal selection theory" proposed that each t and b cell carried a unique receptor specificity, but we now know that any one receptor is capable of recognising multiple epitopes (tcr "degeneracy" and bcr cross-reactivity). any vaccine must therefore contain antigenic epitopes capable of being processed and presented to tcrs, or recognised conformationally by bcrs, and the aim of the vaccine is simply to be recognised by relevant antigen-specific lymphocytes such that these cells are stimulated to generate active immunity and immunological memory. it is therefore irrelevant how large or small the target animal might be; the vaccine simply needs to be able to activate the correct cells in the immunological repertoire. vaccines are formulated with a sufficiency of antigen to achieve that aim. having said that, there is some evidence that dogs of low bodyweight do tend to make higher serological responses to some antigens (kennedy et al. ) and do have a higher incidence of post-vaccinal adverse events than larger dogs [eb ]. however, at this time, there is no suggestion that vaccines will be formulated on the basis of bodyweight. in north america, some vaccines are available in . ml rather than . ml volumes, but the antigenic content of these products is similar. you should never split a vaccine dose between animals or administer anything less than a full dose of vaccine to an animal. this is "off label" use of the product and you would be liable if that animal subsequently developed infection post vaccination. in the st century vaccination is no longer simply about inducing protection from infectious disease. therapeutic "vaccines" are used to stimulate or modulate immune responses in cancer (e.g. use of the canine melanoma vaccine) or allergy (use of allergenspecific immunotherapy in atopic dermatitis) and in human medicine there is much active research into therapeutic vaccines for autoimmune diseases. the vgg does not consider these alternative uses for vaccination, which are generally the domain of veterinary specialists. our focus is always on vaccines available globally for the prevention of infectious diseases in dogs and cats, and on products that are used widely in first-opinion veterinary practice. the vgg does not recommend the use of a vaccine to prevent dermatophytosis in dogs and cats since there are very few efficacy studies published. although there are reports of success of anti-dermatophyte vaccines in cattle and fur-bearing animals, the response does not appear to be the same in cats, as these vaccines do not protect against challenge infection (deboer & moriello , deboer et al. , frymus et al. . a commercial vaccine consisting of killed m. canis was licensed in the usa for treatment of cats; however, this vaccine did not provide a more rapid cure of an established infection in vaccinated cats compared with unvaccinated controls. the product was withdrawn from the market (frymus et al. ) [eb ] . questions about vaccine delivery . can i give core vaccination to puppies at weeks of age if they are due to be sold at weeks of age? the first thing to say about this practice is that weeks of age is really too young for puppies to be weaned, removed from their mothers and sold. in europe it is now illegal to sell puppies that are under weeks of age. the veterinary profession in latam should take ownership of this welfare issue and educate dog breeders as to appropriate times for weaning. bringing together litters of puppies aged to weeks at weekend "puppy markets" is also a "recipe for disaster" in terms of infectious disease transmission and it should also be beholden on the veterinary profession to address this welfare issue. if the bitch that has produced the litter was well-vaccinated, it is likely that she will have a high concentration of serum antibodies against core antigens (cdv, cav and cpv ) and that these will transfer to the puppies in colostrum. in such a circumstance it is unlikely that either a -week-old or -week-old puppy will respond to core vaccination, although the chances of this are improved if high-titre combination cdv and cpv vaccines designed for use in young puppies are applied. however, in latam, it is perhaps more likely that the bitch will not have been well vaccinated and it is therefore correct to attempt to provide protection to puppies as early as possible. the most appropriate way to do this would be with a vaccine containing cdv and cpv and designed for use in young puppies as described above. such a product might be administered from weeks of age; however, mlv vaccines should never be given any earlier than then as they may produce infection and malformation in neonatal animals. after weeks of age, puppies may be vaccinated every to weeks (switching to a trivalent cdv, cav and cpv vaccine at weeks old). the most important dose of core vaccine is actually that given at weeks of age or older, when all puppies should have lost maternally-derived antibodies and be capable of responding to vaccine. this should be followed by a fourth core vaccination given between and weeks of age (ideally at the earlier end of that range). it is difficult to be absolutely certain that a litter of puppies or kittens did not obtain colostrum; or that certain individuals within the litter did not obtain colostrum. however, if this is suspected, the first advice would be to implement excellent husbandry by providing as clean and isolated an environment as possible. the use of "artificial colostrum" formulated of milk replacer and serum or plasma from a well-vaccinated adult animal might also be considered in the first hours of life. it is known experimentally that colostrum-deprived animals are capable of making an immune response to core vaccine very early in life (chappuis ) [eb ]; however, mlv vaccines should never be used any earlier than weeks of age as they may induce infection or developmental defects in the neonatal animal. core vaccination in this situation might start at weeks of age. although in theory, a colostrum-deprived animal should be capable of responding to a single canine core vaccine or a single dose of fpv vaccine (because there is no inhibition from mda), it would be pragmatic to proceed through the recommended wsava protocol for puppies or kittens. even in a colostrumdeprived kitten, at least two doses of fhv and fcv vaccine would be recommended. at weeks of age, the use of core cdv and cpv vaccine designed for young puppies would be recommended. this depends on the circumstances and the age the puppy is first presented for core vaccination. initial vaccination would ideally be given whilst the puppies were still with the bitch and arranged by the breeder. in europe, for example, where puppies cannot be sold until after weeks of age and where bitches are likely to be well vaccinated, a breeder might arrange for core vaccination at to weeks of age and then responsibility for subsequent vaccines passes to the new owner. in latam, where a puppy might be obtained much earlier in life, core vaccination might begin as early as to weeks of age (see recommendations above). according to wsava guidelines, puppies may receive core vaccines every to weeks, with the final early-life dose of vaccine being given at weeks of age or older. so the actual number of core vaccines will depend on the age of starting and the frequency of administration. a "standard" protocol in north america or europe might involve core vaccination at , and weeks of age with a fourth core vaccine at weeks of age. in latam this protocol might be adapted to account for an earlier onset of core vaccination. there are in-practice serological test kits available on the market that can detect the presence of serum antibody to cdv, cav and cpv . however, these are designed to inform decision making about revaccination of adult dogs, rather than determine the optimum time for vaccination of puppies. it is simply not practical (and has welfare implications) to repeatedly blood sample very young puppies and, more importantly, until weeks of age, it is not possible to discriminate between mda and antibody produced endogenously by the puppy's own immune system in response to vaccination. so, the answer to the question is "no" it is not possible to use these test kits to determine the optimum time for puppy vaccination. however, the test kits could be used to determine the need for a core vaccine given between and weeks of age (according to wsava guidelines). if a puppy is tested at weeks of age (i.e. weeks after receiving the last core vaccine at weeks of age or older) and is seropositive (for cdv, cav and cpv ), then those antibodies must reflect the puppy's own immune response and indicate that immunological protection has been induced. in that circumstance, the puppy would not require another vaccine at to weeks of age and may go straight into the adult revaccination schedule. there is no evidence that transfer of mda is breed associated or related to body size. transfer does depend on husbandry factors such as the bitch herself being a "good mother" and being able to suckle all her puppies in the crucial window of the first hours of life (although some studies suggest that "gut closure" to absorption of mda may occur even earlier than this). however, the level of mda may vary between different bitches, depending on how well they have been vaccinated. concentrations of mda might also vary for the three major vaccinal antigens (cdv, cav and cpv ) for any one bitch. there are, however, some breeds of dog (e.g. rottweilers, dobermanns) that may more likely include genetic "low responders" or "non-responders" to individual vaccine antigens (day et al. ) . it is well recognised in north america and europe that certain individual rottweilers are more susceptible to cpv infection (houston et al. ) , which is suggested to reflect inadequate vaccinal immune responses; however, in one study dogs of this breed responded adequately to vaccination (coyne ) . rottweilers also make lesser serological responses to rabies vaccine (kennedy et al. ) [eb ] . serological testing might be used to identify non-responder animals and ideally they would not be used for breeding purposes as they would not be able to transfer adequate mda to any puppies. in an ideal world this would be the case, but it is clear that within a large litter, individual puppies must proactively find a teat in order to take in an adequate amount of colostrum within the first hours of life. smaller or weaker puppies within a litter may not be able to achieve this and will therefore have taken in less mda. these animals will be protected from infection for a lesser period of time during early life, but, in contrast, should be capable of making an endogenous immune response to core vaccines earlier than littermates that sucked more successfully and acquired a greater volume of colostrum. there is no evidence that this is the case. adult dogs that receive triennial core revaccination are known to have stable protective antibody titres during each -year revaccination cycle and experimental data have shown that puppies appropriately vaccinated in early life (and then never again as adults) maintain a plateau of protective antibody titres against cdv, cav and cpv (schultz ) [eb ]. there is a wealth of serological data that shows that annual core revaccination of adult dogs is unnecessary and that protective antibody titres are maintained perfectly adequately with triennial (or longer) core revaccination (abdelmagid et al. , bohm et al. , mouzin et al. , gore et al. , schultz , larson & schultz , mitchell et al. , killey et al. [eb ]. some veterinarians like to vaccinate breeding bitches just before they are mated, but there is no evidence that this provides higher quality mda than in bitches receiving a standard triennial core revaccination protocol. . how can we safely socialise puppies if they are potentially susceptible to infection (because they are in the "window of susceptibility") at the optimum time for socialisation? the window for effectively socialising puppies overlaps with the "window of susceptibility" to infectious disease (i.e. the short period when puppies no longer have sufficient mda to confer full protection against infection, but still have sufficient mda to block the ability of mlv core vaccines to induce an endogenous immune response) (cutler et al. ) [eb ] . there is therefore a theoretical risk that engaging puppies in socialisation activity (e.g. attending a puppy class, exposing a puppy to the outdoors) might allow them to acquire infectious disease. this is a dilemma for the veterinarian; however, we must encourage socialisation as behavioural issues are a major factor in the relinquishment of pet dogs later in life. there are certain practical measures that can be taken to minimise risk: ( ) holding puppy classes in relatively clean environments, ( ) ensuring that all puppy and adult participants are vaccinated and ( ) ensuring the puppy receives the full wsava recommended course of core vaccination. one study from the usa monitored puppies attending puppy classes and did not record a single incidence of infection (stepita et al. ) [eb ]; so with common sense the risks are small. the worst case scenario might be that a puppy had blocking mda that prevented generation of an endogenous immune response to core vaccine until the vaccination given at weeks of age or older. within days of that core vaccination the puppy will have generated some immunity, with maximal serum antibody titres likely being achieved around weeks post vaccination. of course many puppies will have responded at an earlier age to core vaccination and in latam where the process of core vaccination might begin even earlier, one might expect protection to be conferred at an earlier age. as walking on the street is part of the socialisation process, the answer overlaps with the question above. common sense should prevail and exposure risk should be minimised during early life. for example, taking such a young puppy to a dog area in a park would not be recommended. there is no evidence that delivering a vaccine to an animal during neutering will fail to engender an active immune response. anaesthetic agents per se are not immunosuppressive and the transient stress and inflammation associated with the surgical procedure will not affect the induction of vaccinal immunity [eb ] . the only reason for avoiding vaccination during neutering relates to the possible very rare occurrence of a vaccine-associated type i hypersensitivity reaction, which might potentially involve an unstarved animal vomiting and aspirating during surgery. this has nothing to do with the efficacy of the vaccine in inducing a protective immune response. one study has shown that vaccines administered to kittens during early neutering did not affect the immune response to the vaccines (reese et al. ) [eb ]. . why should shelter puppies be vaccinated every weeks from weeks of age to weeks of age if it has been shown that puppies may have persistent maternally-derived antibody until weeks of age? shelters are generally high risk environments with high population density of animals of unknown vaccination and infectious disease exposure history. in the case of puppies entering a shelter, the vaccination history of the dam will also generally be unknown. in order to optimise protection for puppies in such an environment, the vgg recommends this core vaccination schedule where it can be afforded by the shelter. the shelter environment is usually distinctly different from that of a breeding establishment and, subsequently, the new home of an individual puppy. no, this is not necessary. these products are formulated with an "overage" of antigen content to allow for the possible loss of some of the product during administration. if in any doubt, you should contact the manufacturer of the vaccine. the immune system is capable of responding to (or actively tolerating) many thousands of different antigens at any one time. the mucocutaneous surfaces of the body are naturally interacting with very large numbers of antigens (e.g. from the microbiome, dietary antigen, inhaled antigen) in a continual process. therefore, immunologically speaking, the delivery of multiple vaccine antigens on one occasion poses no problem to the immune system [eb ] . for multicomponent vaccines, a requirement of licensing is that it be demonstrated that each component is able to induce a protective immune response. manufacturers also often demonstrate "compatibility" of their own product ranges which are licensed to be co-administered [eb ] . for these reasons, there is no immunological sense in staggering vaccine delivery over different weeks. this would necessitate multiple visits by the client and may increase the likelihood of crucial vaccinations being "missed" from the schedule. one piece of practical advice is that where multiple different injections are to be given (e.g. core vaccine with a separate rabies vaccine), that these be given into different subcutaneous sites so that different draining lymph nodes are targeted for immune priming. two studies from the usa counter this, in that they show for both dogs (particularly of low bodyweight) and cats that there is a greater likelihood of adverse reactions post vaccination when increasing numbers of antigens are delivered at any one time (moore et al. , [eb ]. vaccinating according to wsava guidelines minimises the number of antigens that might be delivered on one practice visit. a fundamental principle of vaccination is that any animal that is clinically ill should not be vaccinated and vaccination should be delayed until the animal has recovered. if a dog has really recovered from a cdv infection, then it will have natural immunity to reinfection; probably better immunity than might be induced by a vaccine. consequently, that dog could be tested and if seropositive would not require cdv revaccination. however, because the cdv antigen is generally mixed with other core vaccinal antigens, that dog will likely receive standard core revaccination in the future. if it is essential to revaccinate a dog recovered from cdv infection, then a period of weeks post recovery should allow immune function to recover. the situation with ehrlichia canis infection is more complex because the disease may have acute and chronic stages and treated dogs may still harbour the infectious agent such that disease can recur following any future stressful event. again, if a dog has been diagnosed and appropriately treated, it should have been clinically normal for at least weeks before any consideration of vaccination is made. in both circumstances, performing a simple haematological and serum biochemical examination might also indicate that an immunological recovery has been made (i.e. normalisation of leucocyte counts and serum gamma globulin concentration). the simple answer is "no." as above (question ), if a dog has naturally recovered from cdv infection it will have robust natural immunity against reinfection and in reality does not require cdv vaccine (but will likely receive it as part of a multiantigen core vaccine). remember that the viruses in core vaccines are attenuated and therefore incapable of inducing tissue pathology and clinical disease. there is one recent report and some historical cases of post vaccinal cdv encephalitis in puppies (fairley et al. ) [eb ], but this is a very rare occurrence. a dog rescued from the street is taken into a house where there are young puppies still undergoing their early life vaccinations. the dog shows signs of cdv infection after a few days in the new home. should the puppies be vaccinated immediately, rather than waiting until their next scheduled vaccination day? it would always be good practice to isolate such a newly introduced adult dog from the puppies wherever possible (until the puppies had completed their core vaccination schedule). remembering that it is impossible to know precisely when each puppy might have a "window of susceptibility", some puppies might already be immune and others not at the time of introduction. if the puppies are within a to week interval between core vaccines, then in this scenario, there is no harm in revaccinating them earlier than the schedule might have otherwise been and then readjusting the schedule up to the vaccine given at weeks of age or older. however, until the week or older vaccine, there is no guarantee of protection in all cases. a dog that has recovered from natural infection with either cdv or cpv will have developed robust immune protection and will be seropositive for the relevant virus. this natural immunity is actually even better than the immunity that might be achieved by vaccination. so, while a recovered dog would likely not require vaccination against that particular pathogen, because core vaccines are formulated as a trivalent product, it will still require vaccination to ensure protection against the other two pathogens (e.g. a dog recovered from cdv infection would still require vaccination against cav and cpv ). this depends on the dose of glucocorticoids given. an anti-inflammatory dose (e.g. . to . mg/kg of prednisolone) will not impair the ability of the immune system to respond to vaccination. an immunosuppressive dose (e.g. to mg/kg of prednisolone), particularly if combined with other immunosuppressive agents, is designed to impair immune function. consequently, vaccination should not be delivered until at least weeks after the glucocorticoid has been tapered and then stopped [eb ] . the dog should, of course, also be clinically healthy following the cessation of such therapy. although there are no formal studies of the effects of glucocorticotherapy on canine vaccination, there is a study of the effect of ciclosporin treatment on feline vaccinal immune responses. whilst being treated with ciclosporin, cats were able to make adequate protective immune responses to previously seen vaccinal antigens (fpv, fhv , fcv, felv and rabies), but the drug impaired the immune response to first-time vaccination with fiv vaccine (roberts et al. ) [eb ]. dogs receiving powerful immunosuppressive chemotherapeutic drugs should not be vaccinated. these drugs impair immune function by targeting rapidly-dividing immune cells in addition to the target cancer cells. at least weeks should elapse after stopping such therapy before any vaccines are given. the dog should be clinically recovered and ideally a haematological and serum biochemical evaluation would indicate recovery in immune function. although chemotherapeutic drugs will affect immune function (see question ), they do not ablate the immune system or destroy memory lymphocytes. therefore, there is no need to routinely revaccinate dogs after completing chemotherapy other than in their normal cycle of core or non-core revaccination. a dog finishing chemotherapy could be serologically tested for the presence of protective antibodies against core vaccine antigens if there was any concern over its level of protection. human patients receiving chemotherapy are not revaccinated at the end of their treatment protocols. there is nothing about the process of surgery and administering an anaesthetic agent per se that would interfere with the ability of the immune system to respond to vaccination [eb ] . however, depending on the nature of the surgery, in the post-surgical recovery period dogs may be clinically unwell and receiving a variety of medical treatments. from first principles it makes sense to wait until the dog is clinically healthy and finished post-operative medical treatment before revaccinating. . until which age should i vaccinate an elderly dog? should elderly dogs be vaccinated every year with core vaccines because their immune system might not function as well as when they were younger? for core vaccines (cdv, cav and cpv ) there is good evidence that appropriate puppy vaccination induces lifelong protective immunity without regular adult revaccination. there are also studies that show that geriatric dogs (i.e. dogs over years of age) maintain protective levels of antibody against these three core viral antigens and that these antibody levels do not decline with age as part of the phenomenon of "immunosenescence" (hogenesch et al. ) [eb ]. in contrast, it is also known that delivering a new vaccine (i.e. one not previously given) to an older dog leads to a less effective primary immune response than might have been made earlier in life (day ) [eb ] . therefore, there is no evidence that geriatric dogs require any more frequent core revaccination than younger adults; geriatric dogs can be safely maintained on the standard triennial core revaccination programme. where core revaccination is determined by serological testing (titre testing), the vgg recommends that this might be performed annually (rather than triennially) in geriatric dogs; simply to provide reassurance that revaccination is not required [eb ]. rottweilers are a breed that is well recognised to contain a higher than average frequency of genetic low responders and nonresponders to cpv and rabies vaccines. there is no reason to vaccinate rottweilers any more frequently than other breeds of dog. if they are of this genetic type, that means that the lack the immunological ability to ever respond to the particular antigen (e.g. cpv ); that means that no matter how frequently they are vaccinated, they will not respond to vaccination. this situation is one in which serological testing is of practical benefit. test kits will be able to determine whether a rottweiler is seronegative to cpv after vaccination (note that this does not apply to rabies). such a dog would therefore be at risk of contracting infection and appropriate measures might be taken to minimise that risk. more importantly, such dogs should not be used for breeding purposes. there is no evidence that any canine breed or breed group requires any specific vaccination protocol. there has been discussion about small breed dogs (see question ) and whether rottweilers might require a different core vaccination protocol given the possibility of breed-related poor response to cpv and rabies vaccines (see questions and ). related young dogs of the weimaraner breed are susceptible to a complex syndrome involving hypertrophic osteopathy, chronic recurrent infection and serum igg deficiency. there is a suggestion that onset of this clinical syndrome might be influenced by vaccination (harrus et al. ) and there has been some discussion about vaccination protocols for dogs of this breed. however, overall there is insufficient evidence to advise anything except standard core and non-core vaccination, according to guidelines recommendations, for all breeds of dog. if a dog has missed its annual leptospira booster vaccine by a period of up to months, a single "booster" dose of vaccine should be sufficient. if the annual revaccination is delayed by more than months (i.e. a -month interval since the last vaccine) then two doses of vaccine (given to weeks apart) should be given to re-establish immunity and then annual boosters thereafter. some manufacturers may advise that protection may extend to months before a new primary course of vaccination is required, but the vgg adopts a more cautious outlook when considering all vaccines in a generic fashion. yes, vaccination will lead to generation of an antibody response post vaccination; however, this may not persist for very long and post vaccinal titres may decline or even disappear by months post vaccination, even though the dog remains protected for the full months cover of the vaccine. although post vaccination titres tend to be low, they may persist for more than months at high levels if the dog is exposed to field strains. also, cross-reactivity with non-vaccinal serovars can occur (sykes et al. ) [eb ] . because of this, if attempting to confirm a diagnosis of leptospirosis in a clinically ill dog, the timing of any previous vaccination must be considered. this is one of the major reasons why the clinical diagnosis of leptospirosis can only be achieved properly by assessment of mat testing on paired serum samples taken weeks apart. vaccinal antibodies will not show an increased titre, but antibodies against a potentially infecting serovar should show a four-fold elevation in titre. it is important to highlight that dogs can develop titres against serovars not included in vaccines, and sometimes the highest titre is against a non-vaccinal serovar. positive titres to non-vaccinal serovars should be interpreted with caution if a vaccinated dog develops clinical signs consistent with leptospirosis (barr et al. , martin et al. [eb ]. a dog at high-risk for leptospirosis might be one that has regular access to water environments where there may be contamination by rodents or farming environments with livestock. even urban dogs may therefore be at risk. in early versions of the wsava global guidelines, the vgg made the recommendation that -monthly revaccination against leptospirosis be considered for high-risk dogs. we subsequently removed that recommendation as there was insufficient scientific evidence to support it. therefore, even high-risk dogs require only annual revaccination against leptospirosis. the vgg would always recommend the use of small antigen combination vaccines (e.g. a trivalent or bivalent core vaccine with separate non-core vaccines) in order to have the flexibility to vaccinate individual animals according to wsava guidelines. as described above, such product ranges allow delivery of the minimum essential antigenic components for that individual animal based on a lifestyle assessment of its exposure risk. giving multiple injections of smaller antigen component vaccines is far preferable to giving a single injection of large multicomponent vaccine containing antigens that are not required or not recommended for that animal. this is one of the greatest challenges in latam; ensuring that the small antigen component product ranges available in other markets are brought to latam, in order to allow veterinarians to vaccinate according to wsava guidelines. for core vaccines and rabies vaccine, a single dose of mlv international quality assured vaccine will induce protective immunity in an adult animal. remember that adult animals do not have blocking mda like puppies and kittens. the one exception to this rule might be for adult cats in the case of fhv and fcv vaccines, where to be sure of the best response, two doses ( to weeks apart) might be given. this is easy to achieve where there is access to a trivalent (fpv, fhv and fcv) and bivalent (fhv and fcv) vaccine, but in latam, where only trivalent products are marketed, this would necessitate giving an additional (unnecessary) fpv vaccine. moreover, serological testing could be used to determine whether an adult dog actually required any vaccination against cdv, cav and cpv or whether an adult cat required any vaccination against fpv (note that at the time of writing the feline test is not available in latam). in the case of non-core vaccines, these will all require two doses given to weeks apart, followed by boosters at the recommended interval. in latam, it is far more important to try to increase herd immunity than it is to increase the vaccination load of individual animals. the more dogs and cats that are vaccinated within the population, the more difficult it is for infectious disease to spread within that population. veterinarians must understand that giving a core vaccine to an animal induces protective immunity. there are not degrees of protective immunity. the presence of antibody against core vaccine antigens, no matter what the titre, indicates that the animal has immunological protection and immunological memory and any exposure to the pathogen results in a rapid secondary (memory) immune response. it is simply not possible to make an individual animal more immune by giving more frequent vaccination. in fact, immunologically speaking and from first principles, repeated vaccination above the recommended levels is more likely to induce immunological "tolerance" (failure to respond) than immunological protection [eb ] . therefore, triennial core revaccination according to wsava guidelines is perfectly adequate, even for the most high-risk of dogs. precious vaccine doses would better be used to improve herd immunity than be wasted on an already well protected animal. here, the veterinary professional needs to ask the question "who is making this regulation"? in many countries (including in north america and europe) the lay owners of boarding kennels and catteries make these rules on the basis that historically, that is what they have always done. these well-meaning individuals are not scientifically trained veterinarians and are generally unaware of the scientific advances in veterinary vaccinology over the past decades. it should be up to the veterinary profession to educate this community and assist them with developing regulations that are consistent with modern science. it is generally accepted that adult cats do develop some level of natural resistance against felv infection; therefore, it is considered more important to establish immunity by vaccination of kittens than it is adult cats [eb ] . any kitten that may have an at-risk lifestyle (i.e. indoor-outdoor access, living in a multi-cat environment) might benefit from felv vaccination, especially where that kitten also lives in an area known to have high prevalence of infection (see main text of this document for where this is known in latam). for adult cats, the vgg currently recommends felv revaccination only every or years, rather than annually. that should be continued lifelong. no. in-practice diagnostic tests for felv detect viral antigen. so a true positive result indicates that the cat is currently infected with felv or fighting off a recent infection. some cats successfully rid themselves of felv. others become persistently or progressively infected. so the cat that tests positive should be retested immediately using a test from a different manufacturer to rule out a false positive result. if a second positive result is obtained, the cat should be retested in to months' time. if the cat remains positive to months later, progressive infection is likely. it is important to highlight that negative antigen tests can occur in infected cats without viraemia, which means that sometimes you may be vaccinating an asymptomatic but felv-infected cat. in this case the vaccine will not cause any harm, but is also unlikely to confer any benefit to the cat. the risk factors for felv infection include outdoor access and exposure to other cats where virus might be transmitted via salivary secretions (e.g. licking, mutual grooming, shared food and water bowls, or biting as part of fighting behaviour). an indoor only cat that may only leave the indoor environment for an annual veterinary visit would not be a candidate for non-core vaccination, including against felv. of course, considering that felv vaccination is most effectively used in kittens, making this decision about the predicted future lifestyle of the cat is sometimes difficult for the owners. if there is any suggestion that the cat might have outdoor access during its future lifetime, or that it will live with other cats that have outdoor access, there is sense in considering felv vaccination in early life; particularly in areas of high prevalence of the infection. ideally, the known felv-infected cat would be housed indoors in isolation and the owners should be advised against introducing any other cat to the household. however, in the situation described, if unavoidable, the new kitten should certainly be vaccinated against felv as soon as possible and ideally before introduction into the household. standard kitten felv vaccination with two doses given to weeks apart beginning at weeks of age and then a -month booster is required. ideally, pregnant animals should not be vaccinated. in the case of core vaccines where transfer of mda is required, normally vaccinated adult dams should have adequate titres of antibody for transfer and it should not even be necessary to revaccinate immediately before the dam becomes pregnant. unless indicated specifically by the manufacturer that vaccination is safe during pregnancy (and some products do carry this claim), there are also theoretical risks to the foetus with respect to the use of mlv vaccines in the pregnant animal. non-core vaccines should also not be given during pregnancy; non-core vaccines tend not to induce protective antibody transferred in colostrum in that way that occurs with core vaccines. there is absolutely no evidence to support this procedure even though it remains widely practiced. there is a risk that alcohol may inactivate a proportion of mlv virus particles in a vaccine and so such swabbing is actually contraindicated. although the skin carries a normal microflora, needle injection is highly unlikely to result in any subcutaneous infection by "carriage" of organisms into the skin microenvironment. you should also note that in human medicine, sites of injectable vaccine delivery are no longer alcohol swabbed, according to who and cdc recommendations (e.g. who best practices for injections and related procedures toolkit; accessible on-line) [eb ]. there is no harm in lightly warming a dose of vaccine (i.e. by holding it in the hand) immediately before use, but this is really not required. one study of feline injection site sarcoma suggested that delivery of cold vaccine may be a risk factor for the tumour (kass et al. ) [eb ]. however, vaccines should never be over-warmed or kept at room temperature for more than hour. this is because some of the viral components of mlv vaccines are thermo-intolerant and the efficacy of the vaccine will be impaired by warming. vaccines should never be reconstituted early in the morning for use throughout the day. vaccines should always be stored appropriately (refer to table ). for mlv vaccines even to hours at room temperature (and room temperature may fluctuate widely depending on geographical region and season) is sufficient to begin to inactive some of the virus components of the vaccine. more problematic to the veterinary practice is when there may be a power cut and the vaccine refrigerator may be without power for prolonged periods of time. in this context, the recommendation would always be to contact the manufacturer of the vaccines to ask for advice. recent studies suggest that international killed rabies vaccines may actually be quite thermotolerant, but these studies are done against the background of mass vaccination in the field and not from the perspective of practicing quality veterinary medicine in the veterinary clinic (lankester et al. ) [eb ]. this is a commonly-held belief that is perpetuated in many parts of the world. virtually all puppies and kittens are born with endoparasites and current recommendations are that regular deworming starts at weeks of age in puppies and weeks of age in kittens (e.g. esccap guidelines; https://www.esccap.org/guidelines/), which is naturally earlier than the administration of core vaccines (from to weeks of age). however, if a puppy or kitten is presented for vaccination, but clearly carries a massive parasite burden to the extent of being severely anaemic and clinically ill, then of course vaccination might best be delayed until the animal is healthy. however, most parasitized puppies and kittens remain apparently healthy. there is currently no evidence to suggest that carrying a "normal" burden of parasites interferes with the ability of a puppy (or kitten) to respond to vaccination. in fact, it may be more important to provide protection against life-threatening viral disease than to worry about any possible effect of parasitism on vaccination. however, immunologically speaking, there is now much research that shows that parasite infestation can influence the nature of immune responses by skewing immunity towards a t regulatory response associated with immunological suppression and a t helper type response dominated by humoral rather than cell-mediated immunity and this has also been shown for the dog (junginger et al. ) [eb ] . there are experimental studies in mice and pigs that show that endoparasitism impairs vaccinal immune responses (urban et al. ) [eb ] , but what is currently lacking is any evidence that endoparasite infestation impairs canine or feline vaccinal immune responses. deworming is clearly an important part of preventive health care for dogs and cats (and has public health implications), but there is currently no basis for delaying vaccination for susceptible animals until deworming has been completed. any veterinarian who practices in a rabies endemic country, who deals with animals that may be imported from rabies endemic countries or who deals with wild animals (particularly bats) should be routinely vaccinated against rabies according to current recommendations for people. rabies is a fatal disease and no veterinarian at risk of exposure should be unprotected. from the perspective of companion animal practice, there are no other zoonotic diseases for which human vaccination is available or recommended. this relates to what is now known as "vaccination hesitancy" and is of increasing concern in both human and veterinary medicine throughout the world. active lobby groups with strong internet presence promote a culture of fear related to the potential adverse events associated with vaccination of children and pets. the tragedy in human medicine is that such activity has seen a decline in herd immunity for common childhood diseases in recent years, with outbreaks and deaths of once-controlled diseases such as measles. there is no doubt that veterinarians are now also regularly confronted by clients who will refuse vaccination for their pets. it is our professional responsibility to calmly explain the rationale for vaccination, the importance of individual and herd immunity, and the safety of vaccines with very low prevalence of adverse events. this is one reason why the vgg has produced a companion document to the global veterinary guidelines, written in lay language for the pet owner and breeder (unfortunately only in english). this is a factual source of information to which clients concerned about vaccines might be directed. at the time of writing, the vgg is also conducting a global survey of vaccination hesitancy in small companion animal practice. the results of the survey will be presented during . a wide range of adverse events has been recognised post vaccination. most of these are transient and mild (e.g. type i hypersensitivity reactions immediately post vaccination), but some may induce more severe disease (e.g. immune-mediated haemolytic anaemia, feline injection site sarcoma). the single most common "reaction" is mild lethargy, anorexia and pyrexia for to days post vaccination. this is actually not an adverse reaction, but more an indication that the vaccine has stimulated immune and inflammatory pathways as part of generating the protective immune response. it is difficult to obtain accurate data on the frequency of adverse reactions post vaccination. reviewing all of the global information, we can say that there are somewhere between and adverse reactions (mostly mild and transient) for every , vaccinations given in practice , miyaji et al. [eb ]. the risk of contracting life-threatening infectious disease (particularly in environments such as in latam) far outweighs the risk of adverse events. theoretically, if a reaction was due to an ige-mediated type i hypersensitivity reaction, the animal is immunologically sensitised and is likely to be affected by the same type of reaction on subsequent exposure to the same antigen. in reality, this does not always happen and sometimes such reactions occur only once. if the reaction has occurred in a puppy or kitten that has not yet received the full course of early life vaccines, then that animal must be revaccinated in order to receive the full schedule of core vaccines. you might consider whether non-core vaccines are justified for that animal, and as an adult, serological testing might be used to inform the need for core vaccines. there are certain practical measures that might be taken to avoid the occurrence of such reactions for second or subsequent times. switching brand of vaccine may or may not have an effect. there is no harm in giving a dose of antihistamine or anti-inflammatory dose of glucocorticoid immediately before vaccination; this will not interfere with the efficacy of the vaccine. the animal may best be kept in the clinic and monitored for several hours post vaccination, rather than being sent home. yes, glucocorticoid at an anti-inflammatory dose may be used, as may a dose of antihistamine. there is no guarantee that this will prevent the reaction occurring again, but there should be no interference with the efficacy of the vaccine. . if a dog has recovered from an immune-mediated disease that may have been triggered by vaccination; how should i revaccinate it in the future? immune-mediated or autoimmune diseases are complex multifactorial disorders involving triggering factors acting on a background of immune dysregulation in a genetically susceptible individual. recognised trigger factors for canine immune-mediated diseases include underlying infection, neoplasia, chronic inflammation or recent exposure to drugs or vaccines. the evidence for vaccine-associated autoimmunity in the dog is limited and restricted to clinical observations of immune-mediated disease beginning to weeks post vaccination in the absence of any other possible triggers. the most common disorder linked to vaccination is immune-mediated haemolytic anaemia (imha) with conflicting evidence related to immune-mediated thrombocytopenia (imtp) or immune-mediated polyarthritis. that said, because vaccination is a potential trigger, and because immune-mediated diseases have a relapsing and remitting pattern, careful consideration should be given to revaccination protocols for any dog that has been successfully treated and recovered from an immune-mediated disorder. core revaccination might be avoided if the dog is serologically tested and shown to already have immunity. non-core vaccines should be carefully selected, weighing the balance between disease exposure risk and risk of relapse of immune-mediated disease. it may not be possible (legally) to avoid rabies revaccination; however, in some us states it is possible for the veterinarian to authorise "medical exemption" from rabies revaccination. although these precautions are sensible (from first principles), two recent studies actually show no significant ill-effect when dogs recovered from imha or imtp were revaccinated (ellis et al. b , moon & veir [eb ]. vaccines are just one of a long list of injectable products that have been associated with fiss. there is something unique about cat (versus dog) subcutis, which means that repeated injection (of anything) may establish a state of chronic inflammation that might at some point transform into neoplastic disease. the cat, in general, appears more susceptible to developing sarcomas following localised trauma or chronic inflammation (morrison ) [eb ]. whether adjuvant in vaccines makes vaccines any more likely to induce this reaction is a contentious debate. there is no doubt that adjuvant induces a long-lasting localised chronic inflammatory reaction at the injection site (much more so than non-adjuvanted vaccines) (day et al. ) [eb ], but it is suggested that any vaccine is capable of inducing this reaction. in practical terms, the advice is to minimise the use of all vaccines in cats (i.e. by vaccinating according to wsava guidelines) and wherever possible to minimise the use of adjuvanted vaccine as a precaution. consideration should also be given to the site of administration of vaccines to cats (see question ). there is no specific anatomical location in which a cat might be injected that will reduce the risk of sarcoma developing. however, there are strategies that might be adopted that will aid in the management of sarcoma, should it occur. there are several different options recommended by different organisations and authors. these include administration as far distal as possible subcutaneously into the hindlimbs and forelimbs, administration into the skin of the lateral abdomen and administration as far distal as possible into the tail. the wsava does not recommend a single option, but suggests that at very least the scruff of the neck is not used and that vaccination sites may be rotated each time and that those sites be recorded in the animal's medical record. yes, the vgg is supportive of the use of in-practice serological testing to determine whether adult dogs are protected (i.e. have serum antibody) against cdv, cav and cvp , and whether adult cats are protected against fpv (at the time of writing the feline test is not available in latam). note that serology cannot predict protection against fhv or fcv, or against any non-core vaccine antigens. serological testing for the efficacy of rabies vaccination is often required for the purposes of pet travel, but in this instance, testing is generally performed within a defined period post vaccination (e.g. weeks) and serological testing is not used in general practice to demonstrate protection from rabies because titres can decline after the recommended testing interval. you should select a well-validated test kit supported by peer-reviewed scientific literature. serology might be performed annually or triennially during the annual health check consultation. many owners, particularly those concerned about the safety of vaccines (see question ) will prefer this option for their pets. yes, one manufacturer makes an in-house test kit to measure fpv, fhv and fcv antibodies in the serum of cats (mende et al. ), but at the time of writing this test is not available in latam [eb ] . note that only antibody against fpv is predictive of protection; antibody against the upper respiratory tract viruses does not correlate with protection. feline vaccine-associated sarcomagenesis: is there an inflammation-independent role for aluminium? veterinary and comparative oncology evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus, infectious canine hepatitis virus, and distemper virus experimental challenges urban domestic dog populations as a source of canine distemper virus for wild carnivores in the coquimbo region of chile epidemiology of canine distemper and canine parvovirus in domestic dogs in urban and rural areas of the araucanıa region in chile high local genetic diversity of canine parvovirus from ecuador prevalence of feline leukemia virus infection in domestic cats in rio de janeiro identification of enteric viruses circulating in a dog population with low vaccine coverage impact of giardia vaccination on asymptomatic giardia infections in dogs at a research facility prevalence of antibodies against three species of leishmania (l. mexicana, l. braziliensis, l. infantum) and possible associated factors in dogs from mérida canine leishmaniosis -new concepts and insights on an expanding zoonosis: part one serologic responses of dogs given a commercial vaccine against leptospira interrogans serovar pomona and leptospira kirschneri serovar grippotyphosa feline panleukopenia: a re-emergent disease correlation between clinical signs of feline conjunctivitis and molecular detection of felid herpesvirus- , feline calicivirus, chlamydophila felis and mycoplasma felis in cats from shelters in rio de janeiro reliability of techniques used in the diagnosis of canine visceral leishmaniasis by the national control program in brazil: a survey in an area of recent transmission developing practical recommendations for preventative healthcare consultations involving dogs and cats using a dephi technique antibody response to feline panleukopenia virus vaccination in cats with asymptomatic retrovirus infections: a pilot study prevalence of and factors associated with feline leukemia virus (felv) and feline immunodeficiency virus (fiv) in cats of the state of santa catarina seroprevalence of viral infections in domestic cats in costa rica serum antibody titres to canine parvovirus, adenovirus and distemper virus in dogs in the uk which had not been vaccinated for at least three years vaccination with liesp/qa- (canileish®) reduces the intensity of infection in phlebotomus perniciosus fed on leishmania infantum infected dogs: a preliminary xenodiagnosis study genotyping of canine distemper virus strains circulating in brazil from detection of feline immunodeficiency provirus in domestic cats by polymerase chain reaction detection by rt-pcr and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in argentina evolution of canine parvovirus in argentina between years and : cpv c has become the predominant variant affecting the domestic dog population resurgence of canine parvovirus a strain in the domestic dog population from argentina pathologic and immunohistochemical findings of domestic cats with feline panleukopenia feline immunodeficiency virus subtype b in domestic cats in minas gerais, brazil. veterinary research communications neonatal immunity and immunisation in early age: lessons from veterinary medicine occurrence of feline leukemia virus in felis cattus in belo horizonte veterinary dermatology in brazil foco de leishmaniasis en el hobo, municipio del carmen de bolívar molecular and serological surveys of canine distemper virus: a meta-analysis of cross-sectional studies efficacy of two canine parvovirus vaccines for inducing seroconversion in rottweiler and doberman pinscher pups with various levels of maternally derived antibodies feline leukaemia virus associated with leukaemia in cats in santa catarina feline lymphoma and a high correlation with feline leukaemia virus infection in brazil a canine leishmaniasis pilot survey in an emerging focus of visceral leishmaniasis puppy socialization practices of a sample of dog owners from across canada and the united states immunohistochemical detection of antigens of distemper, adenovirus and parainfluenza viruses in domestic dogs with pneumonia ageing, immunosenescence and inflammageing in the dog and cat a kinetic study of histopathological changes in the subcutis of cats injected with non-adjuvanted and adjuvanted multi-component vaccines recommendations on vaccination for asian small animal practitioners: a report of the wsava vaccination guidelines group guidelines for the vaccination of dogs and cats canine coronavirus (ccov) in dogs vaccinated and unvaccinated domiciliated in pelotas, rs, brazil molecular characterization of canine parvovirus variants (cpv- a, cpv- b, and cpv- c) based on the vp gene in affected domestic dogs in ecuador the immune response to microsporum canis induced by fungal cell wall vaccine. veterinary dermatology , - journal of small animal practice • © world small animal veterinary association safety and immunologic effects after inoculation of inactivated and combined live-inactivated dermatophytosis vaccines in cats fecal immunoglobulin a antibodies in dogs infected or vaccinated with canine coronavirus seroprevalence of parvovirus, adenovirus, coronavirus and canine distemper virus infections in dogs of santa maria dog overpopulation and burden of exposure to canine distemper virus and other pathogens on santa cruz island effects of maternally-derived antibodies on serologic responses to vaccination in kittens prevalence and risk factors for the presence of serum antibodies against canine distemper, canine parvovirus, and canine adenovirus in communities in mainland ecuador prevalence and molecular epidemiology of canine parvovirus in diarrheic dogs in colombia, south america: a possible new cpv- a is emerging? comparative efficacy of intranasal and oral vaccines against bordetella bronchisepctica in dogs evaluation of the risk of relapse of canine immune-mediated thrombocytopenia after routine vaccination phylogenetic evidence of a new canine distemper virus lineage among domestic dogs in colombia post-vaccinal distemper encephalitis in two border collie cross littermates the burden of the leishmania chagasi/infantum infection in a closed rural focus of visceral leishmaniasis in lara state, west-central venezuela comparison of two commercial vaccines against visceral leishmaniasis in dogs from endemic areas: igg, and subclasses, parasitism, and parasite transmission by xenodiagnosis prevalence of canine visceral leishmaniasis in municipalities of huila, colombia dermatophytosis in cats: abcd guidelines on prevention and management viral diagnostic criteria for feline immunodeficiency virus and feline leukemia virus infections in domestic cats from characterization of parvoviruses from domestic cats in brazil presence of infectious agents and co-infections in diarrheic dogs determined with a real-time polymerase chain reaction-based panel first record of borrelia burgdorferi b strain in dermacentor nitens ticks in the northern region of parana (brazil) borrelia burgdorferi sensu lato in humans in a rural area of paraná state occurrence of chlamydophila felis in a cattery in osasco city, são paulo state isolation and characterization of bordetella bronchiseptica strains from canine origin clinical characterization of the canine distemper in two municipalities of havana three-year duration of immunity in dogs following vaccination against canine adenovirus type- , canine parvovirus, and canine distemper virus three-year duration of immunity in cats following vaccination against feline rhinotracheitis virus, feline calicivirus, and feline panleukopenia virus development of hypertrophic osteodystrophy and antibody response in a litter of vaccinated weimaraner puppies neutralizing antibodies to distemper and parainfluenza viruses in dogs in shelter kennels in the municipalities of novo hamburgo and porto alegre, rs, brazil concomitant canine distemper, infectious canine hepatitis, canine parvoviral enteritis, canine infectious tracheobronchitis, and toxoplasmosis in a puppy canine morbillivirus (canine distemper virus) with concomitant canine adenovirus, canine parvovirus- , and neospora caninum in puppies: a retrospective immunohistochemical study disseminated melanized fungal infection due to cladosporium halotolerans in a dog coinfected with canine adenovirus- and canine parvovirus- isolation and identification of feline calicivirus and feline herpesvirus in southern brazil effect of age on immune parameters and the immune response of dogs to vaccines: a cross-sectional study risk factors associated with parvovirus enteritis in dogs: cases ( - ) infectious canine hepatitis: cases immunohistochemical aspects of canine infectious hepatitis maternal antibodies interfere with vaccine protection vaccination against feline panleukopenia: implications from a field study in kittens three-year duration of immunity in cats vaccinated with a canarypox-vectored recombinant rabies virus vaccine three-year duration of immunity for feline herpesvirus and calicivirus evaluated in a controlled vaccination-challenge laboratory trial serum survey for antibodies to coronavirus, herpesvirus, calicivirus, and parvovirus in domestics cats from rio grande do sul, brazil zoonotic intestinal helminths interact with the canine immune system by modulating t cell responses and preventing dendritic cell maturation prevention of feline injection-site sarcomas: is there a scientific foundation for vaccine recommendations at this time? veterinary clinics of north america multicenter case-control study of risk factors associated with development of vaccine-associated sarcomas in cats factors influencing the antibody response of dogs vaccinated against rabies long-lived immunity to canine core vaccine antigens in ukdogs as assessed by an in-practice test kit feline immunodeficiency virus and feline leukemia virus: frequency and associated factors in cats in northeastern brazil three-year rabies duration of immunity in dogs following vaccination with a core combination vaccine against canine distemper virus, canine adenovirus type- , canine parvovirus, and rabies virus thermotolerance of an inactivated rabies vaccine for dogs feline panleukopenia virus, feline herpesvirus- and feline calicivirus antibody responses in seronegative specific pathogen-free kittens after parenteral administration of an inactivated fvrcp vaccine or a modified live fvrcp vaccine occurrence of feline immunodeficiency virus infection in cats three-year serologic immunity against canine parvovirus type and canine adenovirus type in dogs vaccinated with a canine combination vaccine a comparative study of protective immunity provided by oral, intranasal and parenteral canine bordetella bronchiseptica vaccines hepatitis infecciosa canina en la ciudad de corrientes. reporte de un caso infectious diseases of dogs and cats on isabela island serologic survey of domestic felids in the petén region of guatemala vaccine effectiveness and use of collar impregnated with insecticide for reducing incidence of leishmania infection in dogs in an endemic region for visceral leishmaniasis leishmania spp. epidemiology of canine leishmaniasis in the yucatan peninsula assessment of risk for diarrhea in young dogs after giardia vaccine administration amplification of the flge gene provides evidence for the existence of a brazilian borreliosis current status and management of canine leishmaniasis in latin america infection by mycoplasma spp., feline immunodeficiency virus and feline leukemia virus in cats from an area endemic for visceral leishmaniasis vaccine-associated leptospira antibodies in client-owned dogs phylodynamics analysis of canine parvovirus in uruguay: evidence of two successive invasions by different variants frequency of the virus of the feline leukemia (felv) in domestic felines (felis catus) semi-domiciliates in the municipalities of pelotas and rio grande evaluation of an in-house dot enzyme-linked immunosorbent assay to detect antibodies against feline panleukopenia virus molecular and serological characterization of the first leptospira santarosai strain isolated from a dog molecular characterization, serotyping, and antibiotic susceptibility profile of leptospira interrogans serovar copenhageni isolates from brazil situación epidemiológica de la leishmaniosis canina en el paraguay (años - ) canine visceral leishmaniosis in stray dogs of asunción, paraguay duration of serological response to canine parvovirus-type , canine distemper virus, canine adenovirus-type and canine parainfluenza virus in client-owned dogs in australia large-scale survey of adverse reactions to canine non-rabies combined vaccines in japan frequency of feline immunodeficiency virus (fiv) in southern aburrá valley, colombia detection of respiratory viruses in shelter dogs maintained under varying environmental conditions vaccination and associated adverse events in dogs previously treated for primary immune-mediated hemolytic anemia adverse events diagnosed within three days of vaccine administration in dogs adverse events after vaccine administration in cats: , cases feline immunodeficiency virus and feline leukemia virus infection in free-ranging guignas (leopardus guigna) and sympatric domestic cats in human perturbed landscapes on chiloé island inflammation and caner: a comparative view duration of serologic response to five viral antigens in dogs serosurvey of borrelia in dogs, horses, and humans exposed to ticks in a rural settlement of southern brazil viral diagnostic criteria for feline immunodeficiency virus and feline leukemia virus infections in domestic cats from a randomised, double-blind, controlled efficacy trial of the liesp/qa- vaccine in naive dogs exposed to two leishmania infantum transmission seasons infectious canine hepatitis in naturally infected dogs: pathological findings and immunohistochemical diagnosis identification of co-infection by rotavirus and parvovirus in dogs with gastroenteritis in mexico seroprevalence of feline leukemia virus, feline immunodeficiency virus and heartworm infection among owned cats in tropical mexico high prevalence of infection with leishmania (kinetoplastea: trypanosomatidae) in dogs in northern colombia first detection of canine parvovirus type c in south america prevalence of felv, fiv and feline heartworm in cats from an animal shelter in maracaibo seroprevalence of canine parvovirus in dogs of boyeros municipality prevalence of canine visceral leishmaniasis in the area of influence of the health unit family marín ka'aguy efficacy of an inactivated canine coronavirus vaccine in pups isolation and characterization of canine parvovirus type c (cpv- c) from symptomatic puppies effects of anesthesia and surgery on serologic responses to vaccination in kittens field randomized trial to evaluate the efficacy of the leish-tec® vaccine against canine visceral leishmaniasis in an endemic area of brazil effect of high-dose ciclosporin on the immune response to primary and booster vaccination in immunocompetent cats isolation of leptospira spp. from dogs with clinical suspect of leptospirosis in são paulo (brazil) combined distemper-adenoviral pneumonia in a dog guías para la vacunación de perros (caninos) y gatos (felinos) en perú autochthonous outbreak and expansion of canine visceral leishmaniasis aafp feline vaccination advisory panel report elimination of neglected diseases in latin america and the caribbean: a mapping of selected diseases european consensus statement on leptospirosis in dogs and cats duration of immunity for canine and feline vaccines: a review comparative onset of immunity of oral and intranasal vaccines against challenge with bordetella bronchiseptica dna detection and evaluation of antibodies against chlamydophila felis in domestic cats from the northeast of the state of são paulo canine-based strategies for prevention and control of visceral leishmaniasis in brazil leptospira noguchii and human and animal leptospirosis, southern brazil serofrequency of feline immunodeficiency virus and feline leukemia virus in cats of araçatuba canine parvovirus type vaccine protects against virulent challenge with type c virus survey for tick-borne zoonoses in the state of espirito santo, southeastern brazil frequency of cpv infection in vaccinated puppies that attended puppy socialization classes pan-european study on the prevalence of the feline leukaemia virus infection -reported by the european advisory board on cat diseases (abcd europe) serovars of leptospira isolated from dogs and rodents acvim small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention ocorrência do vírus da imunodeficiência felina e do vírus da leucemia felina em gatos domésticos mantidos em abrigos no município de belo horizonte feline immunodeficiency virus in northern ceará infection with parasitic nematodes confounds vaccination efficacy successful strategies implemented towards the elimination of canine rabies in the western hemisphere rabies update for latin america and the caribbean vaccination of dogs with canine parvovirus type b (cpv- b) induces neutralising antibody responses to cpv- a and cpv- c the isolation of leptospires from stray dogs in the city of são paulo coexistence of antibodies to tick-borne agents of babesiosis and lyme borreliosis in patients from cotia county, state of são paulo canine visceral leishmaniasis on margarita island isolation of leishmania infantum, zymodeme mon- from canine and human visceral leishmaniasis on margarita island, venezuela. memórias do instituto oswaldo cruz epidemiological aspects of human and canine visceral leishmaniasis in venezuela dear colleague, i am sure that you are aware of the wsava vaccination guidelines group (vgg) and the work we have done since in introducing global recommendations for the vaccination of dogs and cats. in the vgg starts an exciting new project that focuses on endemic infectious diseases and vaccination in latin america. the vgg will be making a series of fact-finding visits to countries in the region to meet with a spectrum of veterinary professionals (practitioners, industry, academia and governmental) to formulate recommendations for best-practice vaccination of pet dogs and cats in this part of the world.in advance of these visits, the vgg would like to obtain and analyse information on the perceptions of small animal practitioners working in these countries with respect to infectious disease and vaccination of dogs and cats.accordingly, we have prepared the attached short questionnaire and would be very appreciative of your time in completing this to help us in our endeavours. the data will remain confidential to the vgg and would only be published in anonymous and aggregated form.i thank you for your assistance with our work and look forward to meeting you at one of the continuing education events that the vgg will be hosting in latin america during this phase of our work. triennially (every years) less often (> years) is rabies vaccination for dogs a legal requirement in your area?yes no at what age do you give the first dose of rabies vaccine to a puppy? __________________________ do you give a second dose of rabies vaccine to a puppy? if so, at what age?____________________. do you recommend revaccinating adult dogs against rabies? if so, how frequently should they be revaccinated? _____________________. do you use other vaccines in addition to the core vaccines (cdv, cav and cpv ) in adult dogs? if so, which ones and how frequently do you give them (i.e. annually? more or less often?) _________________________________________________________________________________. approximately what proportion (%) of your feline patients has ever been vaccinated with the core feline vaccines (fpv, fhv and fcv) these wsava vaccination guidelines have been published as submitted to jsap and have been through an abridged peer-review process. key: cord- - mw kpmr authors: mcvey, scott; shi, jishu title: vaccines in veterinary medicine: a brief review of history and technology date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: mw kpmr the use of vaccines in veterinary medicine has progressed from an experimental adventure to a routine and relatively safe practice. the common and aggressive use of efficacious vaccines has been responsible for the control and eradication of several diseases. despite progress in research technologies, diagnostic capabilities, and manufacturing methods, there remain many infectious diseases for which no effective vaccines exist. global availability, field compliance, effectiveness, and safety are also significant concerns. this review addresses the history, current practices, and potential future improvements of vaccine use in veterinary medicine. become a routine component of medicine. problems with consistent potency, available supply, purity, and safety were common. nevertheless, both the effectiveness and imperfections of vaccination lead to the eventual global eradication of smallpox, and was the inspiration for development of the products and programs for immunization against several diseases in humans and animals. louis pasteur first used the term vaccine in for immunogens directed at other diseases besides smallpox. pasteur directed many investigations that demonstrated the feasibility of attenuating or inactivating microbes. studies with fowl cholera and anthrax led to the concepts of chemical inactivation as a means to reduce the virulence of microorganisms. , studies with erysipelas and rabies explored serial passage in animals (lapinization or passage through rabbits) or other animal derived tissues as an alternative strategy to reduce or eliminate virulence. thus, the virulence of infectious microbes could be completely or partially reduced. these studies have led to the eventual successful control of anthrax and rabies in particular. the work of salmon and smith ( ) clearly demonstrated that some microbes could be completely inactivated (killed). these developments eventually led to successful immunization programs against typhoid fever, tuberculosis, rinderpest, and foot and mouth disease (fmd). attenuation and inactivation principles were extended to microbial toxins by the work of gaston ramon at the pasteur institute. a tetanus toxoid was developed in through heat and formalin inactivation of the toxin to form an ''anatoxin.'' also, enhanced efficacy was provided by absorbing the toxoid to an aluminum hydroxide, providing an adjuvant effect. these process and formulation improvements were developed and refined in the early twentieth century, first through production of equine sera with antidiphtheria and antitetanus toxin-neutralizing antibody for prophylactic use. in the modern era of vaccine use, these same basic technologies are still the mainstays of vaccine production. however, new generations of recombinant, nucleic acid and subunit vaccines have become available. it is remarkable that the principles of developmental research, registration, and manufacture still follow the techniques of the grand heritage. during the early years of the modern era of vaccine production, infected tissues were often used as a source of microbial antigens through grinding, inactivation (typically with formaldehyde solutions), and subsequent filtration or clarification. more often than not these vaccines were produced in regional research institutions. industrialization of the processes began in the s and s when large-scale, controlled processes were used to produce fmd antigens in germany by waldmann and colleagues. the development of first primary and subsequently clean cell lines occurred in the s and s. development of high-volume roller bottle methods and later large-scale bioreactors has made possible the production of millions of doses of vaccines. further, production has been maintained in secure, closed systems, enhancing the security for the environment as well as the technical staff. in like manner, improvements in inactivation technologies (cyclized binary ethyleneimines), purification and concentration of antigens, storage of bulk antigens, improved aluminum gels, and oil suspension adjuvants in the formulation of polyvalent antigens have been critical achievements in the steady advancements in vaccinology. as these technical advances were employed in the industry, independent and collaborative efforts by numerous governmental authorities created regulatory frameworks that have established regulations and guidelines for registration of new biologicals as well as consistent manufacture of pure, safe, and potent vaccines. under these regulations all released lots of vaccines are tested to ensure consistent formulation characteristics and potency (immunologic strength), safety, and purity (sterility and freedom from contamination with extraneous biologic agents). development of good manufacturing practices guidelines and master seed and master cell stock concepts has further ensured consistent manufacture of vaccines that will provide consistent immunogenicity and efficacy. a veterinary clinician therefore may use with confidence any approved vaccine as recommended by the manufacturer to achieve the anticipated clinical outcome of protection. as the vaccine manufacturing processes improved with regard to consistency of biologic activity, robustness, and efficiency, routine clinical use of vaccines became more practical and economical. there is no doubt that widespread use of efficacious vaccines has been associated with the global eradication of smallpox in humans and the regional control of fmd and rabies. the routine use of processed immunoglobulins (usually in the form of processed horse serum) preceded the use of vaccines. although passive protection by the immunoglobulins is still employed (particularly for rabies and tetanus post exposure prophylaxis), the advantages of active immunity (immunologic memory and reduced risk of infection) have significantly reduced the use of passive immunity. in the mid- s, veterinarians were commonly using rabies vaccines of brain tissue origin in dogs. the principal biologic products used in practice at that time were rabies vaccines, ''viabilized'' canine distemper/hepatitis virus vaccine and antisera, hog cholera and erysipelas vaccines and antisera, leptospirosis bacterins, and clostridial toxoids (fig. ) . as the development and manufacturing capacity increased with time, vaccination of companion animals expanded to include rabies for cats, feline herpesvirus, parvovirus in cats and dogs, and feline calicivirus. table describes the types of vaccines currently available to companion animal practitioners in most regions of the world - (http://www.aphis.usda.gov/animal_health/vet_biologics/ vb_licensed_products.shtml) these vaccines include very traditional inactivated antigen formulations, multiple attenuated agents, and new technologies such as poxvectored vaccines, defined subunit vaccines, and nucleic acid vaccines (see table ). the term vaccine is now used to describe many therapeutic or prophylactic formulations and products that stimulate active immunity in the vaccinated animal. this discussion focuses on vaccinations associated with infectious diseases. routine clinical use of these vaccines usually includes immunization of puppies and kittens at approximately -week intervals after maternal-derived antibody decreases to noninterfering titers. these immunization series are usually administered between the fourth and th weeks of life. , puppies and kittens associated with unusual risk may be vaccinated at younger ages or at more frequent intervals. rabies vaccination is usually first given at months of age. it is a common and efficacious practice to provide booster doses at year of age for most vaccines. these immunization practices will provide a solid duration of immunity of at least to years and longer in some cases. general recommendations (world small animal veterinary association) are to vaccinate every third year after the initial immunization series, and these recommendations are consistent with product label guidelines. these initial immunization guidelines are derived from the initial registration immunogenicity and efficacy studies for any individual vaccine product. the efficacy studies define the minimum immunologic strength for the vaccine (the potency that must be present when the vaccine lot goes out of date). these same types of studies also define the minimum age of animals that can be successfully immunized as well as the specifics of the initial and booster immunization regimens (part , code of federal regulations). it has vaccines in veterinary medicine become very clear that many vaccines provide effective and long-term immunity for an extended period of time. over the past decades, cumulative evidence for extended duration of immunity has been provided to support the -year booster intervals for most vaccines in dogs and cats. however, as described in table , the relative efficacy of some vaccines is less than ideal. ''ideal'' immunity would be not only protection from clinical disease (morbidity and mortality) but also blocking the infection/replication/spread or progression of infectious agents. some vaccines do achieve this degree of protection. some, however, may only reduce morbidity and/or mortality without generating a sterilizing immunity. based on clinical and microbiological outcomes of an efficacy study challenge of immunity, various degrees of protection may be achieved and therefore claimed. the united states department of agriculture (usda) has recognized these differences through a hierarchy of efficacy claims that may be allowed for a vaccine based on the outcomes of efficacy studies (box ). the degree of efficacy and claim structures are usually derived from direct investigations of efficacy and challenge of immunity studies in their respective host animal species. vaccinated and nonvaccinated animals are challenged with fully virulent organisms, and the degree of protection (efficacy) is determined under controlled settings. these classic studies are adequate to establish the efficacy of the vaccine but are not always sufficient to estimate the field effectiveness of a vaccine, or, in other words, the ability of a vaccine to control disease in the field. effective control of infectious disease should result in reduced incidence and prevalence. , this would be true of not only clinical disease but also of infection and spread of the infectious agent. it is very clear that use of efficacious products has reduced incidence of rabies, particularly in dogs. immunization of dogs has reduced the incidence of canine rabies to essentially nil in the united states and western europe. the rabies immunization programs in these countries have been so effective that most manufacturers of rabies vaccine for dogs and cats have switched to master seeds from canine street strains of virus to other types of terrestrial rabies (bat strains, for instance) to protect from the most significant current threats in these regions. vaccination has also greatly reduced the incidence of canine distemper, canine parvovirus, infectious canine hepatitis, feline panleukopenia, and feline herpes virus infections as well as other diseases. when these diseases do occur, there are usually issues with vaccine dose compliance, vaccination of sick or immunocompromised animals, exposure to wildlife, or problems associated with vaccine handling and/or administration. in situations where vaccines do not provide prevention of infection, concurrent infections may exist and vaccine failures are therefore more common. there are often issues with type-specific protections. for instance, it is not clear that available vaccines can protect cats against all types of calicivirus infections. continual vigilance is required to ensure continued protection of animals in the face of potential newly evolving and emerging pathogens (eg, rabies and other lyssaviruses, canine distemper and parvoviruses, and feline calicivirus). the vaccines used in veterinary medicine generally fall into of categories: inactivated vaccines (in which antigens are typically combined with adjuvants); attenuated, live vaccines; and recombinant technology vaccines, which may include subunit antigens or genetically engineered organisms. in practice, combination and multivalent vaccines may employ all approaches. all of these technologies have been used successfully, and each approach has inherent advantages and disadvantages. the protective mechanisms associated with vaccines are also becoming clearer. historically, the most common correlate of immunity to derive from vaccination has been measurements of antibody responses. , antibodies have several functions including facilitating opsonization, complement-mediated cellular lysis, neutralization-blocking adherence or replication, and facilitating cytotoxic cells. however, mature, well-differentiated immune responses are the consequence of cumulative, regulated interactions between phagocytic cells, antigen-presenting cells, and both b and t lymphocytes. therefore, a well-differentiated antibody response with isotype switching, affinity maturation to high avidity, and memory requires some effective initial stimulation involving dendritic cells and expansion of regulatory t lymphocytes a claim that it is intended to prevent disease may be made only for products shown to be highly effective in preventing clinical disease in vaccinated and challenged animals. the entire % interval estimate of efficacy must be at least %. if so, a label statement such as ''for the prevention of disease due to [specific microorganism]'' may be used. - . . aid in disease prevention. a claim that it is intended to aid in disease prevention may be made for products shown to prevent disease in vaccinated and challenged animals by a clinically significant amount which may be less than that required to support a claim of disease prevention (section . . ). if so, a label statement such as ''as an aid in the prevention of disease due to [specific microorganism]'' may be used. - . . aid in disease control. a claim that it is intended to aid in disease control may be made for products which have been shown to alleviate disease severity, reduce disease duration, or delay disease onset. if so, a label statement such as ''as an aid in the control of disease due to [specific microorganism]'' or a similar one stating the product's particular action may be used. (likely cd ) and b lymphocytes. this stimulation phase is followed by a phase of differentiation into effector/memory t cells, b cells, and plasma cells. with respect to the nature of pathogenesis of many infectious agents, the adaptive immune response to the vaccine often blocks or interferes with a specific segment of the infection process. for instance, antibody-mediated neutralization of rabies virus in extracellular spaces inhibits transmission to neurons and subsequent axonal progression of the virus to the central nervous system. in this case the presence of preformed, neutralizing antibody is critical for protection. a summary of protective characteristics of the immune responses to vaccines (as potential correlates of protection and disease prevention) is provided in table . although antibody responses are good correlates of protection, they do not always reflect all available protective mechanisms provided by a well-differentiated immune response. in some cases, other correlates are available. it is clear that the presence of neutralizing, vaccine-derived antibody will reduce mucosal virus replication, virus shedding, and viremia in kittens vaccinated with modified live feline herpes vaccines. [ ] [ ] [ ] however, regulated cd and cd cellular responses are required to control tissue damage and reactivation of disease. in this case, antibody may be a protective correlate of infection while cellular immunity is a protective correlate of disease. the ability of modified live vaccines to generate a very rapid onset of cytokines and interferons (and rapid antigen focusing in dendritic cells in lymphoid tissues) is associated with a rapid onset of protection, even though antibody responses may not be detectable in serum for up to weeks. [ ] [ ] [ ] therefore, the early response of multiple cytokines and concurrent activation of the innate immune system may serve as early correlates of protection. there are also documented cases in which functional immunity outlasts detectable circulating antibody; this is true with many herpesvirus infections. however, the presence of detectable neutralizing serum antibody is correlated with protection against recrudescent disease. in situations where vaccinated animals may be exposed to heterotypic viruses or bacteria, the presence of immune cd t cells specific to conserved antigens may be very important for protection. it is possible that the effective mechanisms for development of protection associated with a vaccine may be specific to the nature of the disease and infectious process. recent studies have provided important information regarding this phenomenon. a common hypothesis is that vaccine-induced immunity should reflect convalescent immunity following natural infection. for example, it is known that recovery from primary poxvirus infections requires robust cytokine responses, natural killer cells, and antibodies as well as t helper (cd ) and cytotoxic t (cd ) lymphocyte effector functions. however, recovery from a secondary infection requires only t-and b-lymphocyte interaction and an anamnestic antibody response. again, neutralizing antibody will reduce infection, viremia, and spread of a virus (and may do so to the extent of blocking infection) while t-cell-mediated responses will allow survival and recovery. it seems clear that balanced antibody and cellular responses are necessary for complete protection from infection and disease as well as spread to other animals. it should be mentioned that not all antigen-binding antibodies are protective. in some cases, such as with influenza virus, canine distemper virus, and herpesvirus vaccines, nonneutralizing antibody may be produced that does not contribute to the blocking of infection or enhancing clearance of the infectious agent. , for this reason, correlates or surrogates of protection should be linked to protective mechanisms; this can be done through retrospective analysis of data from efficacy and immunogenicity studies or through associational studies in immune populations (such as with primary vaccinates in an efficacy study). veterinary vaccinology has realized significant successes that have affected human and animal well-being, and the ability to coexist. the virtual elimination of canine rabies in north america and western europe has indirectly led to human-animal bonding at a very intimate level that was not feasible when canine rabies was relatively common. however, there remain many diseases for which no efficacious or effective vaccine exists. many parasitic diseases as well as diseases of a chronic, intracellular nature are not covered by any available vaccine. in some cases safety profiles or efficacy characteristics of existing vaccines are not acceptable. fortunately, there are promising technologies that may close the technical gaps for prevention of these challenging diseases. the processes of absorption of antigens such as chemically inactivated toxoids or viruses to aluminum gels, or the creation of water-in-oil emulsions of antigen particles have been the principal methods used for veterinary vaccine formulations. in some cases compounds such as crude or purified saponins (quil a), squalenes, or pluronic block copolymers have been added to enhance immune stimulation. , although these practices have been successful, newer technologies such as cpg dna, defense peptides, imidazoquinolones, and polyphosphazenes may enhance both safety and vaccines in veterinary medicine efficacy. , further, additional cholesterol and phospholipids may be combined with antigens and saponins to create immunostimulating complexes (iscoms) particles. similar adjuvant particles can be generated with no antigen (iscomatrix) that can be admixed directly with antigen suspensions. these advanced formulations may be used to provide very efficient adjuvants to in turn allow development of microvolume formulations as well as transdermal applications. also, as better understanding of immune genotypes and phenotypes in animal populations emerges, individualized formulations of vaccines may be developed and produced that may enhance safety and efficacy. proteomic technologies may very well provide methods to identify antigen subsets from among complex organisms and infectious agents such as bacteria and protozoa. these organisms contain large, complex genomes. antigen expression is often dependent on growth conditions, and the medium may be very complex. these conditions are difficult to reproduce and regulate in vitro. the combination of transcriptional and proteomic analysis may provide a means to identify key antigens associated with tissue or cellular persistence and potential virulence. such analyses could provide means to simplify vaccine formulations to include only protective antigens and reduce the presence of nonprotective, potentially interfering bacterial proteins. not only would this potentially improve efficacy, but it could also improve safety profiles by reducing the antigenic mass in a vaccine dose. the continued use of alternative expression systems has many potential advantages. transgenic expression of protein antigens and plant-based systems may provide access to oral vaccines as well as enhanced stability of antigens. expression of antigens in avirulent viruses, bacteria, and yeast and insect cells may provide both manufacturing and user safety by eliminating the need to use a virulent or partially virulent microbe to provide immunity. further development of nucleic acid vaccines may provide even greater formulation simplicity and biosecurity. viral particles such as capsids from avirulent viruses may serve as building blocks to deliver nucleic acids, protein subunit antigens, and microadjuvants directly to secondary lymphoid tissue. not only would these biologically engineered vaccines provide targeted immunity and eliminate the need to work with dangerous microbes, they very likely would reduce the time required for the onset of immunity, with excellent safety characteristics. one of the most pressing problems associated with manufacturing vaccines is the requirement to rapidly modify antigen formulations as new diseases emerge or as older pathogens mutate and reemerge. transcriptomics and proteomics combined with established recombinant or synthetic approaches could potentially provide antigens that could be rapidly formulated with approved new-generation adjuvants to produce novel and efficacious vaccines. these technologies are commonplace in experimental laboratories. using combinations of proteomics, reverse genetics, recombinant or molecular syntheses, and stable, consistent adjuvant platforms will allow development of ''first line of defense'' vaccines for a rapidly emerging disease in a short time. such a use-inspired approach to vaccines would allow the use of assembly-line techniques to manufacture vaccines. as new antigens are required they could be selected, evaluated, and produced in a short period of time, and inserted directly into an established production system. this process would greatly reduce the time required for exploratory research and early development. classic development cycles may require to years and sometimes may require even longer times for unusual or new types of pathogenic microbes. a reduction of the development time by % to % may be achievable using newer research and development technologies. it is clear that new methods to assess efficacy and definitive, direct correlates of immunity also need to be identified. it is also clear that use of the many new technical achievements and discoveries will require advances in the regulatory framework to ensure more efficient but adequate evaluation of new biologicals. vaccine development faces many technical, political, and ethical challenges. the history of vaccine research and development as well as the continued use of immunization as the principal method to prevent infectious disease predict that the innovative experimental procedures of today will lead to common clinical applications tomorrow. china and the origins of immunology a brief history of vaccines and vaccination an inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western counties of england. particularly gloucestershire, and known by the name of the cow pox. london: samson low; de l'atttenuation du virus du cholera des poules compte rendu sommaire des experiences faites a pouilly-le fort, pres melun, sur la vaccination charbonneuse. comptes rendus de l'academie des method pour prevenir la rage apres morsure. comptes rendus de l'academie des on a new method of producing immunity from contagious diseases sur la toxine et l'anatoxine diphtheriques. pouvir floculant et proprietes immunoantes die aktive immunisierung des rindes gegen maul und klauenseuche vaccines and vaccination: the principles and the polemics duration of immunity for canine and feline vaccines: a review guidelines for the vaccination of dogs and cats. compiled by the vaccination guidelines group (vgg) of the world small animal veterinary association (wsava) review of companion animal viral diseases and immunoprophylaxis current vaccination strategies in puppies and kittens emerging aspects of rabies infection: with a special emphasis on children use of molecular epidemiology in veterinary practice rabies re-examined vaccines: correlates of vaccine-induced immunity: an official publication of the infectious diseases society of america vaccination and antigenic drift in influenza onset of immunity in kittens after vaccination with a non-adjuvanted vaccine against feline panleucopenia, feline calicivirus and feline herpesvirus feline panleukopenia virus, feline herpesvirus- , and feline calicivirus antibody responses in seronegative specific pathogenfree cats after a single administration of two different modified live fvrcp vaccines antibody and cell-mediated immune responses to feline herpesvirus following inactivated vaccine and challenge long-term immunity in cats vaccinated with an inactivated trivalent vaccine correlates of protection: novel generations of influenza vaccines correlates of protective immunity in poxvirus infection: where does antibody stand? identification of two proteins associated with virulence of streptococcus suis type strategies to link innate and adaptive immunity when designing vaccine adjuvants innate immunity and new adjuvants vaccine immunopotentiators of the future personalized vaccines: the emerging field of vaccinomics proteomic technology in the design of new effective antibacterial vaccines plant-based oral vaccines: results of human trials innovative vaccine production technologies: the evolution and value of vaccine production technologies trends affecting the future of vaccine development and delivery: the role of demographics, regulatory science, the anti-vaccine movement, and vaccinomics key: cord- -b s stvz authors: guimarães, luísa eça; baker, britain; perricone, carlo; shoenfeld, yehuda title: vaccines, adjuvants and autoimmunity date: - - journal: pharmacological research doi: . /j.phrs. . . sha: doc_id: cord_uid: b s stvz abstract vaccines and autoimmunity are linked fields. vaccine efficacy is based on whether host immune response against an antigen can elicit a memory t-cell response over time. although the described side effects thus far have been mostly transient and acute, vaccines are able to elicit the immune system towards an autoimmune reaction. the diagnosis of a definite autoimmune disease and the occurrence of fatal outcome post-vaccination have been less frequently reported. since vaccines are given to previously healthy hosts, who may have never developed the disease had they not been immunized, adverse events should be carefully accessed and evaluated even if they represent a limited number of occurrences. in this review of the literature, there is evidence of vaccine-induced autoimmunity and adjuvant-induced autoimmunity in both experimental models as well as human patients. adjuvants and infectious agents may exert their immune-enhancing effects through various functional activities, encompassed by the adjuvant effect. these mechanisms are shared by different conditions triggered by adjuvants leading to the autoimmune/inflammatory syndrome induced by adjuvants (asia syndrome). in conclusion, there are several case reports of autoimmune diseases following vaccines, however, due to the limited number of cases, the different classifications of symptoms and the long latency period of the diseases, every attempt for an epidemiological study has so far failed to deliver a connection. despite this, efforts to unveil the connection between the triggering of the immune system by adjuvants and the development of autoimmune conditions should be undertaken. vaccinomics is a field that may bring to light novel customized, personalized treatment approaches in the future. vaccines have been a preventive treatment option available for over years. they have been proven to be effective in preventing infections that previously had high morbidity and mortality. an example of this is the eradication of small pox, which was mainly attributed to successful vaccination programs. preventing a high burden disease has since proven to be a cost effective measure and, as such, vaccines have become a part of multiple national health programs. these promising results led to the development of more and more vaccines and to the study of its applicability in other fields such as cancer prevention and treatment. vaccines are drugs administered to healthy individuals, and much like other drugs, vaccines are associated with adverse events. usually the described adverse events are transient and acute, but may rarely present with hypersensitivity and induction of autoimmunity that may be severe and fatal. these adverse events play an important role in the life of the vaccinated patients. immune mediated diseases arise from various different sources; these include environmental, genetic, hormonal and immune defects. the combination of these defects can be described as the mosaic of autoimmunity [ ] . patient background can be used as a clue to determine the response that may be elicited following drug administration. it has been proven that infectious agents may elicit an autoimmune disease in a prone subject through various mechanisms, including, but not limited to, molecular mimicry, epitope spreading and polyclonal activation [ ] . scientific findings suggest that autoimmunity may be triggered by vaccine adjuvants, of which aluminum compounds (aluminum hydroxide and phosphate) have been the most studied and the most widely used. adjuvants are molecules, which, in combination with antigens, enhance immunological response. this enables an easier and more effective recognition of "non self", which in turn permits the triggering of adaptive and innate immune responses [ ] . recently a new syndrome was described: "autoimmune/inflammatory syndrome induced by adjuvants" (asia). this embodies a spectrum of reactions, which are usually mild but may also be severe. these reactions are attributed to adjuvant stimulation, which can include chronic exposure to silicone, tetramethylpentadecane, pristane, aluminum, infectious components and other adjuvants. all of these environmental factors have been found to induce autoimmunity and inflammatory manifestations by themselves both in animal models and in humans. the mechanisms of this disease will be described in further detail [ ] . this review will focus on general mechanism of vaccines, adjuvant-induced autoimmunity, and on vaccines and the specific autoimmune diseases that they may trigger. adjuvants approved to date for human vaccines are: aluminum, mf in some viral vaccines, mpl, as , as b and as a against viral and parasitic infections, virosomes for hepatitis b virus (hbv), human papilloma virus (hpv), hepatitis a virus (hav), and cholera toxin for cholera. adjuvants may be composed of several different compounds. currently, oil based adjuvants, virosomes, toll-like receptors (tlrs) related adjuvants, mpl, adjuvants made of unmethylated cpg dinucleotides and tuftsin have all been described. it is of great interest the understanding of the mechanisms related to the adjuvant effect, as well as to aluminum salts. aluminum acts through multiple pathways, which do not depend solely on tlrs signaling. each of these pathways leads to an enhanced host immune response [ ] . there are many oil based adjuvants. one is incomplete freund adjuvant (ifa), which contains water in oil emulsion. another is complete freund adjuvant (cfa), which is the same as ifa, except that it also contains killed mycobacteria in addition to water in oil emulsion. usually, cfa is used for primary vaccination and ifa for boosting. recent oil based adjuvants that have been developed are mf (novartis ® ), as (glaxosmithkline ® ), advax tm which are based on inulin compounds (vaxine tm pty) and qs- /iscoms, which are immune stimulating complexes composed of cholesterol and phospholipid with or without antigen (table ) . virosomes are adjuvants that contain a membrane-bound hemagglutinin and neuraminidase obtained from the influenza virus. both components facilitate the uptake into antigen presenting cells (apc) and mimic the natural immune response [ ] . leucocyte membranes have membrane bound pattern recognition receptors (prrs) called tlrs, which are responsible for detecting most (although certainly not all) antigen-mediated infections. their activation leads to adaptive immune responses. for this reason, many adjuvants that are used today are directed to prrs. these adjuvants are called tlrs related adjuvants [ ] . mpl is a series of 'monophosphoryl lipid a obtained from the purification of a modified lipopolysaccharide (lps) of salmonella minnesota. bacterial deoxyribonucleic acid (dna) is immunostimulatory due to unmethylated cpg dinucleotides. vertebrate dna has relatively low amounts of unmethylated cpg compared to bacterial dna. the adjuvant effect of cpg is enhanced when conjugated to protein antigens. this adjuvant is being tested in vaccines directed at infectious agents, allergens and tumor cells [ ] [ ] [ ] . another type of adjuvant is tuftsin. tuftsin is an auto adjuvant, which is a natural self-immunostimulating tetrapeptide (thr-lys-pro-arg). this tetrapeptide is a fraction of the igg heavy chain molecule produced by enzymatic cleavage in the spleen [ ] . its functions include: binding to receptors on neutrophils and macrophages to stimulate their phagocytic activity, increasing tumor necrosis factor alpha (tnf␣) release from human kupffer cells enhancing secretion of il by activating macrophages, activation of macrophages expressing nitric oxide (no) synthase to produce no and enhancement of murine natural cell mediated cytotoxicity in vitro [ ] [ ] [ ] . in summary, it is an adjuvant with minor side effects with a promising effect in restoring innate immune mediated response. adjuvants may exert their immune enhancing effects according to five immune functional activities: . translocation of antigens to the lymph nodes where they can be recognized by t cells. . antigen protection enabling longer exposure. . enhanced local reaction at the injection site. . induction of the release of inflammatory cytokines. . interaction with prrs, specifically tlrs [ ] . a adjuvant effect the term "adjuvant effect" refers to the co-administration of an antigen with a microbial specific factor to enhance an antigenspecific immune response in vivo. the microbial components of adjuvants activate apcs to produce pro-inflammatory cytokines ("non-specific" signal ) and to up-regulate molecules essential for antigen presentation. these molecules include major histocompatibility complex (mhc) class ii (antigen-specific signal ) and b - / . these innate immune events allow a more effective presentation to the adaptive immune system, resulting in an augmented activation and clonal expansion of t cells [ ] . in accordance to this effect, if self-antigens are used, an autoimmune response can be elicited [ ] . it has been shown that auto-reactive t-cells that surpass tolerance mechanisms can be triggered by exogenous adjuvants to become auto-aggressive [ ] . infectious agents are able to naturally generate their adjuvant effect and can induce autoimmunity [ ] . an example of this is the causality between viral infection and myocarditis. half the cases of myocarditis are preceded by an acute viral infection. infectious myocarditis in humans can be reproduced in experimental murine models of myocarditis [ ] . it has also been shown that the autoimmune reaction elicited by an infectious agent can be effective in treating cancer. an example of this is that bladder administration of bcg (bacille calmette-guérin) has been shown to be effective against superficial bladder cancer development [ ] . it can be inferred that the adjuvant effect can be used against specific tumor derived molecules, so that these molecules can be recognized as "non self". prr-pamp (pattern recognition receptor-pathogen-associated molecular patterns) interactions activate the apcs to promote antigen-specific lymphocytic responses [ ] . the definition of pamps has now broadened, in that the recognized structures do not need to be pathogens. thus the concept of "microbe-associated molecular patterns" (mamps) and of "danger/damage-associated molecular patterns" (damps) were defined based on the notion that the endogenous host molecules signal danger or damage to the immune system [ ] . tlrs are single-transmembrane prrs localized on cell surface and endosomal membranes. from all the prrs, these are the most studied. tlrs play a crucial role in innate immune response to "non self" and are biosensors of tissue damage. the interaction between the four known tlrs adapters: myd , tirap/mal, tram and trif, in tlr signaling, shape the innate immune response. besides prrs the innate immune system also detects proteolytic enzymes generated during infection [ ] . merging the response to different prrs signaling may be the pathway for developing customized responses to different aggressions [ ] . b experimental models of adjuvants many animals have been used in experimental models of adjuvant-related autoimmune conditions [ ] . these include primates, salmons, rabbits and swine; however, the most common are murine models. murine models include autoimmune prone strains, models of autoimmune disease and autoimmune resistant strains ( table ). an interesting model is that described by lujan et al. the authors described that a commercial sheep, inoculated repetitively with aluminum-containing adjuvants vaccinations, developed an acute neurological episode with low response to external stimuli and acute meningoencephalitis few days after immunization. an excitatory phase, followed by weakness, extreme cachexia, tetraplegia and death appeared. this was suggested to be part of the spectrum of asia syndrome. moreover, the biopsy of the nervous tissue of experimental animals indicated the presence of alum [ ] . c toxicity of aluminum adjuvants aluminum nanoparticles have both a unique capacity of surpassing the blood brain barrier (bbb) and of eliciting immune inflammatory responses. these are probably the reasons why aluminums' most sensitive target is the brain, and also why documented side effects are mostly neurologic or neuropsychiatric [ , ] . aluminum is present in nature, not only as a vaccine adjuvant, but also in food, water and cosmetics. it has been described as a neurotoxin because even when a relatively small amount of aluminium reaches the brain [ ] , is can act as a genotoxin [ ] , a prooxidant [ ] , it can be proinflammatory [ ] , act as an immunotoxin [ ] and also as an endocrine disruptor [ ] . aluminum interferes with many essential cellular processes. memory, concentration, speech deficits, impaired psychomotor control, reduced seizure tolerance and altered behaviour are manifestations of aluminium neurotoxicity. moreover, alzheimer's [ ] , amyotrophic lateral sclerosis, parkinsonism dementia [ ] , multiple sclerosis [ ] , and neurological impairments in children have been linked to aluminum neurotoxicity [ ] . brain susceptibility to aluminum compounds is possibly due to the brain's high metabolic requirement, to the fact that it possesses a large area of biological membranes and to the relatively low concentration of antioxidants [ ] . aluminum adjuvants exert their immunostimulatory effect through many different pathways that activate both the innate and adaptive immune systems. one of the most significant is the activation of the nlrp inflammasome pathway [ ] . nlpr activation has been shown to trigger type diabetes. by using nlpr knockout mice it has been demonstrated that the absence of inflammasome components leads to a better maintenance of glucose homeostasis and higher insulin sensitivity [ ] . on the other hand, activation of the inflammasome and its downstream components: pro-inflammatory cytokines il- ␤ and il- are strongly implicated in the development of several central nervous system (cns) disorders [ ] . the vast majority of people are consuming higher amounts of aluminum through dietary and parenteral intake than what expert authorities consider safe. upper limits set by us food and drug administrations (fda) for aluminum in vaccines are set at no more than g/dose. these values were not based on toxicity studies, but on the minimum amount needed for aluminum to exert its effect as an adjuvant [ ] . the quantities of aluminum to which infants, in their first year of age are exposed, have been considered safe by the fda. however the scientific basis for this recommendation does not take into account aluminum persistence in the body. the concern about aluminum in dietary intake has been reinforced by the food and agriculture (fao) who expert committee, which lowered the provisional tolerable weekly intake of aluminum from mg/kg/bw ( mg/week, for an average kg human) to mg/kg/bw ( mg/week) [ ] . the amount of dietary intake of aluminum has risen in urban societies to up to mg/day considering the widespread use of processed convenience foods. however, only about . % of dietary aluminum is absorbed into systemic circulation and most of it is thereafter eliminated through the kidneys [ ] . absorption of aluminum by the skin from ointments and cosmetics containing aluminum has been shown. moreover, the presence of aluminum in breast tissue was associated with breast cancer [ ] . aluminum compounds persist for up to - years post vaccination in human body. this fact, combined with repeated exposure, may account for a hyper activation of the immune system and subsequent chronic inflammation [ ] . the clinical and experimental evidence collected so far identify at least three main risks associated with aluminum in vaccines: . it can persist in the body. . it can trigger pathological immunological responses. . it can pass through the bbb into the cns where it can trigger immuno-inflammatory processes, resulting in brain inflammation and long-term neural dysfunction. there is a link between allergies and autoimmunity since both are the result of an abnormal immune response [ , ] . metals such as mercury, aluminum, nickel and gold are known to induce immunotoxic effects in humans. the immunologic effects of these metals include immunomodulation, allergies and autoimmunity. they may act either as immunosuppressants or as immune adjuvants. metals bind firmly to cells and proteins and thus have the ability to modify autologous epitopes (hapetenization). t-cells then recognize the proteins as foreign and trigger an autoimmune response [ ] . hypersensitivity caused by metals may be referred to as type iv delayed hypersensitivity. the reaction is considered delayed because the first symptoms appear - h after exposure, because it is mostly t-cell mediated and the gold standard for diagnosis of delayed type hypersensitivity is patch testing [ ] . in mercury-sensitized patients, even mercury concentrations within the normal range might provoke neuroallergic reactions in the brain [ ] . identifying metal sensitivity and removal of the sensitizing metals, such as dental amalgam, have been proved successful by showing symptom improvement in patients with previous autoimmune diseases. these diseases included fibromyalgia, autoimmune thyroid diseases and orofacial granulomatosis [ ] [ ] [ ] [ ] (table ). the timeline regarding the field of vaccinology has been divided in two generations, the first regarding the administration of inactivated pathogens in whole or live attenuated forms (e.g., bacillus calmette guerin (bcg), plague, pertussis, polio, rabies, and small- pox) and the second regarding vaccines assembled from purified microbial cell components, also referred as subunit vaccines (e.g., polysaccharides, or protein antigens) [ ] . this latter approach there are obstacles to conventional vaccine development methods such as non-cultivable in vitro pathogens (e.g., hepatitis c, papilloma virus types and , and mycobacterium leprae), antigen hypervariability (e.g., serogroup b meningococcus, gonococcus, malaria), opportunistic pathogens (e.g., staphylococcus aureus) and rapid evolving pathogens such as human immunodeficiency virus (hiv) [ ] . vaccine research gained a new perspective as the genomics field emerged over the last decades. bacterial genomes have been sequenced and analyzed making it possible to choose the best candidate vaccine antigens by using the concept of reverse vaccinology [ ] . the main known factors influencing the observed heterogeneity for immune responses induced by vaccines are gender, age, ethnicity, co-morbidity, immune system, and genetic background. the interaction between genetic and environmental components will dictate the response to vaccines. studying the vaccine and the host will enable the development of customized treatment options. the combination of genetics, epidemiology and genomics in vaccine design has been denominated "vaccinomics" [ ] . the importance of genetic influence is supported by twins and siblings studies, which show familial aggregation. this suggests that genomics is crucial in inter-individual variations in vaccine immune responses [ ] . both human leukocyte antigen (hla) and non-hla gene markers have been identified as markers for immune response to vaccines. multiple studies have shown connections between hla gene polymorphisms and non-responsiveness to the hbv vaccine [ ] . hla region is divided in three sub regions: class i is associated with the induction and maintenance of cell-mediated immune response, class ii is associated with presentation of exogenous antigens to helper t cd + cells and class iii, where immune non hla related genes are located. normal human tissue has at least hla antigens, and although new recombinant haplotypes may occur, it is inherited mostly intact from progenitors [ ] . hla allelic differences are associated with different responses to vaccines, either by hyper or hypo responsiveness. we can infer that a similar response may be associated with different safety in relation to the development of autoimmune reactions to vaccines, particularly in the patients with genetic predisposition to an enhanced response to vaccine inoculation [ ] . furthermore, patients that share the same hla, for instance siblings, have been diagnosed with asia following similar environmental stimuli [ , ] . autoantibodies help to diagnose certain autoimmune diseases, however, they can also be found in healthy individuals. thus, autoimmune diseases cannot be diagnosed based solely on antibody detection [ ] . inoculation of vaccines triggers autoimmune responses that result in the development of autoantibodies. many studies have been carried out in animals, healthy subjects and patients with autoimmune diseases to understand if this development is of clinical significance [ ] [ ] [ ] [ ] . a difference in eliciting the production of autoantibodies in healthy humans has been observed between adjuvanted and non-adjuvanted influenza vaccines [ ] . the annual influenza vaccine has been the most heavily researched vaccine, along with hpv and pneumococcal vaccines as far as their relationship with patients who have previously been diagnosed with an autoimmune disease [ ] [ ] [ ] . autoantibody induction after hpv vaccination was also shown in adolescent girls with systemic lupus erythematosus (sle) [ ] . although induction of autoantibodies was proven following vaccine administration, there have been no proven relation with disease diagnosis in either of the specific groups studied so far [ , ] . it has been widely demonstrated that autoantibodies can develop years before the manifestation of a full-blown autoimmune disease [ ] . moreover, the development of a specific autoantibody is also genetically determined, and the link between genetic, autoantibodies and vaccines may become an even more intriguing area of research [ ] . silicones are synthetic polymers that can be used as fluids, emulsions, resins and elastomers making them useful in diverse fields. they were thought to be biologically inert substances and were incorporated in a multitude of medical devices such as joint implants, artificial heart valves, catheters, drains and shunts. of all the silicone-containing products, the most famous are most likely breast implants. silicon is one of the substances suspected to induce asia [ ] . it is currently believed that exposure alone is not enough to trigger the disease but that it requires the presence of additional risk factors (e.g., genetic susceptibility, other environmental factors) [ ] . silicone exerts local tissue reactions. some of these reactions are considered para-physiological, such as capsular tissue formation around an implant. other reactions are viewed as abnormal, like when capsular contractures and allergic reactions to silicone or platinum (catalyst used in silicone polymerization found in minute concentrations in implants) occur [ ] . cutaneous exposure to silicone with cosmetics or baby bottles could potentially sensitize patients [ ] . there is also a systemic component of silicone exposure related to diffusion of silicone through the elastomer envelope, commonly termed "bleeding". it may arouse systemic effects as it degrades and fragments in tissue, it can also spread throughout the body and lead to the development of cancer or autoimmune phenomena [ ] . patients with ruptured implants complain more frequently of pain and chronic fatigue when compared to patients with intact implants [ ] . anti-silicone antibodies were found to be present in human sera more frequently in patients who have undergone silicone breast implants, however, their pathological significance remains uncertain [ ] . the same was seen for other antibodies such as autoantibodies directed against dsdna, ssdna, ssb/la, silicone and collagen ii, which were found to be present in increased levels in patients after exposure to silicone [ ] . it has also been shown that the formation of autoantibodies is directly related to implant duration. several autoimmune diseases have been linked to silicone exposure including rheumatoid arthritis (ra), systemic lupus erythematosus (sle), polymyositis, systemic sclerosis (ssc) and fibromyalgia. although asia symptoms may arise years after the onset of exposure to silicone implants [ ] , most of the follow-up periods are short and concluding evidence is yet to come regarding this causality. there have been published case reports, epidemiologic and research studies that suggest a connection between several vaccines and certain autoimmune conditions, notwithstanding that, overall the benefits of vaccination outweigh the risks. thrombocytopenia has been reported as the main adverse event following mmr vaccine. after mmr vaccine the onset of immune thrombocytopenic purpura (itp) usually occurred within weeks at a risk rate of : , - , mmr vaccine doses, while the incidence of itp following infections is : for measles and : for rubella [ ] . as the risk of thrombocytopenia is higher in patients who experience natural infection with measles, mumps or rubella than in those receiving the vaccine, vaccination is encouraged. arthralgia complaints have also been reported and they may present as transient arthralgia, acute arthritis and rarely chronic arthritis [ ] . some risk factors have been found to be associated with the development of arthritis in vaccinated patients such as: female gender, older age, prior seronegativity and specific hla alleles [ ] . yf vaccine is only advisable to people in, or going to endemic areas. the risk of developing yf vaccine-associated neurologic disease (yel-and) is inversely proportional to age [ ] . this is why children aged < months cannot be vaccinated and < months, except during epidemics [ ] . vaccination is not advisable to people > years because of possible higher risk of severe adverse effects (saes) even though the incidence remains low [ ] . besides being a vaccine for mycobacterium tuberculosis (tb), the bcg has proved effective as immunotherapy for bladder cancer. although the mechanism is yet to be fully understood, it is thought that bcg binds to fibronectin forming complexes that enable the recognition as "non-self" by the innate immune response of th cells. ultimately the pathways result in the apoptosis of tumor cells [ ] . because of its effect in treating non-muscle-invasive urothelial carcinoma, as well as superficial bladder tumors, it was expected that bcg could play a role in treating other types of cancer, despite data having not corroborated this hypothesis so far. adverse events vary according to the site and method of administration. intradermal administration of bcg has been reported to elicit arthritis [ ] , dermatomyositis [ ] and takayasu's arteritis (ta) [ ] among others. intravesical treatment for bladder cancer can cause reactive arthritis (rea) [ ] . the risk relies on a systemic reaction composed of an early infective phase (pcr positive and response to anti-tb treatment) and a late hypersensitivity reaction [ ] . hbv is a dna virus of the hepadnaviridae family, responsible for acute and chronic liver disease. hbv vaccines are considered the first efficient vaccines against a major human cancer. hbv vaccines have reduced the risk of developing chronic infection and they also have proved to reduce the incidence of liver cancer in children [ ] . the vaccine has been associated mainly with autoimmune neuromuscular disorders. they include, but are not limited to: optic neuritis, guillain-barre syndrome (gbs), myelitis and multiple sclerosis (ms), systemic lupus erythematosus (sle), arthritis, vasculitis, antiphospholipid syndrome (aps) and myopathy [ ] . hbv vaccine is the most common immunization associated with acute myelitis. there are studies that indicate that the pathogenicity behind such vaccine and autoimmunity might be based on cross-reactivity between hbv antigen (hbsag) epitopes, yeast antigens, as well as other adjuvants contained in the vaccine itself [ ] . up to % of cervical cancer deaths, occur in developing countries that lack the ability to fully implement the papanicolau (pap) screening programs. hpv poses a special challenge in vaccine safety. hpv is necessary for the development of cervical cancer. however, most women infected with hpv will not develop the disease since % of infections will resolve within a year and up to % within years without specific treatment. over the course of decades, cancer may result in a small proportion of the remaining infected women. death rate from cervical cancer in - year old girls is zero and long-term benefits are yet to be proven. in this specific case, short term risks to healthy subjects can prove to pose a heavier burden than cervical cancer [ ] . there are at least types of hpv strains, of which have been pathologically associated with cancer. two vaccines, gardasil tm and cervarix tm , are commercially available against hpv. both contain the l capsid proteins of several hpv strains as antigens. gardasil tm contains serotypes , , , . these antigens are combined with aluminum (al) hydroxyphosphate sulphate as an adjuvant. cervarix tm contains a combination of the oil-based adjuvant monophosphoryl lipid a (mpl) and al hydroxide (aso ) as adjuvant and is directed at strains and [ ] . there have been several reports of post-licensure adverse events, some of which have even been fatal [ ] . compared to other vaccines, an unusually high proportion of adverse drug reactions has been reported associated with hpv vaccines [ ] . in , australia reported an annual adr rate of . / , , the highest since . this increase was almost entirely due to adrs reported following the commencement of the national hpv vaccination program for females aged - years in april ( out of a total of adrs records). the numbers only decreased after the cessation of the catch-up schedule. although the percentage of convulsions attributable to the hpv vaccine decreased, the overall report remained comparable between and ( % and % respectively). these reports do not prove the association, but show that there is a higher frequency of adrs related to hpv vaccines reported worldwide, and that they fit a consistent pattern (i.e., nervous system-related disorders rank the highest in frequency) that deserves further investigation [ ] [ ] [ ] . indeed, several autoimmune diseases have been linked to hpv immunization. examples include gbs, ms, acute disseminated encephalomyelitis (adem), transverse myelitis (tm), postural orthostatic tachycardia syndrome (pots), sle, primary ovarian failure (pof), pancreatitis, vasculitis, immune thrombocytopenic purpura (itp) and autoimmune hepatitis (ah) [ ] . influenza is an acute viral infection that affects the respiratory tract and is caused by influenza type a-c viruses of the orthomyxoviridae family [ ] . h n mortality rates in the outbreak showed high risk in those aged years and older, presence of chronic diseases and delayed admission. risk of infection was lower in those who had been vaccinated for seasonal influenza with / trivalent inactivated vaccine [ ] . studies have demonstrated that influenza vaccine is safe and immunogenic in patients with sle or rheumatoid arthritis (ra), diminishing the risk of respiratory infections [ ] . it has been shown that adjuvanted vaccine had more local reactions but did not increase systemic adverse reactions [ ] . molecular mimicry has been suggested as a mechanism to explain an autoimmune response following influenza vaccination. however, a causal relationship between influenza vaccines and induction of autoimmune diseases remains unproved [ ] . diseases or symptoms reported after influenza vaccination include mostly neurological syndromes such as gbs [ref] . nonetheless, influenza vaccines should be recommended for patients with ms, because influenza infection is associated with increased risk of exacerbations. that being said, influenza vaccinations showed increased risk of autoimmune responses suggestive of asia [ ] , vasculitis [ ] and aps [ ] among others. meningococcal disease is caused by neisseria meningitidis. one of the following five serogroups causes almost every invasive disease: a-c, y, and w- . vaccines available so far for its prevention encompass either pure polysaccharide vaccines that use purified bacterial capsular polysaccharides as antigens, or protein/polysaccharide conjugate vaccines, which use the polysaccharide molecule plus diphtheria or tetanus toxoid as tcell-stimulating antigens. n. meningitidis serogroup b (menb) menb glycoconjugate vaccines are not immunogenic and hence, vaccine design has focused on sub-capsular antigens [ ] . menb capsular polysaccharide is composed of a linear homopolymer of ␣( → ) n-acetyl-neuroaminic acid (polysialic acid; psa). menb psa and psa found on neural cell adhesion molecules are structurally identical. as a result of this, it has been proposed that infection with menb or vaccination with psa may be associated with subsequent autoimmune or neurological disease [ ] . no evidence of increased autoimmunity was found to be associated with meningococcal serogroup b infection [ ] . regarding vaccination, the inoculation does not cause autoimmune diseases but may unmask autoimmune phenomena in genetically predisposed individuals. local reactions are more frequent in individuals vaccinated with quadrivalent meningococcal conjugate vaccines compared to plain polysaccharide vaccines. the intramuscular administration of the conjugate vaccine (versus subcutaneous for that of polysaccharide) may, in part, explain the higher reactivity [ ] . diseases previously associated with meningococcal vaccines are gbs [ ] , henoch-schönlein purpura (hsp) [ ] and bullous pemphigoid (bp) [ ] . streptococcus pneumoniae (pneumococcus) is the main cause of bacterial community-acquired pneumonia and meningitis in western countries, as well as the cause of more than , children deaths in developing countries [ , ] . there are three anti-pneumococcal vaccines commercially available. two of these are conjugated to a protein carrier (pcv and pcv ) and one is not conjugated (ppv ). ppv was licensed in and consists of the capsular polysaccharides of twentythree different streptococcus pneumoniae serotypes ( - , b, f, , n, v, a, a, f, , b, f, c, f, a, , f, f, and f). it does not elicit immunological memory because the immune response it triggers is t-cell independent. it is usually administered to the elderly (above years), as it is believed to be less effective in children. pcv is composed of the most frequent serotypes , b, v, , c, f, and f. pcv is directed at serotypes , - , a, b, f, v, , c, a, f, and f. contrary to ppv both pcv and pcv have an aluminum adjuvant in their composition that elicits a t-cell mediated response [ ] . ever since vaccines were introduced in the healthcare system, prevalence, fatality and admissions for invasive pneumococcal disease have decreased significantly [ ] . vaccine adverse events vary depending on whether the vaccine is adjuvanted or not. in a non adjuvanted vaccine, local reactions are present in % of people vaccinated intra muscularly and in % of those immunized sub-cutaneously [ ] . in conjugated vaccines, this percentage rises to % [ ] . systemic reactions such as fever, irritability, decreased appetite and sleep disturbances occur in - % of recipients of pcv or ppv. symptoms like arthralgia, arthritis, myalgia, paresthesia and fatigue are more frequent in patients post ppv. this may be related to the fact that the vaccines are administered to different age groups. autoimmune risk following ppv vaccine is very low. only case reports were found after ppv vaccine. six of these referred to reactivation of a previous autoimmune disorder. studies directed to access vaccine safety in subjects with autoimmune diseases showed immunization was safe [ , ] . tetanus toxoid (tt) is a potent exotoxin produced by the bacteria clostridium tetani. the toxin has a predominant effect on inhibitory neurons, inhibiting release of ␥-aminobutiric acid (gaba). when spinal inhibitory interneurons are affected the symptoms appear [ ] . the vaccine against c. tetani contains deactivated tetanus toxoid plus an adjuvant (usually aluminium hydroxide). the most studied and prevalent disease associated with tt is antiphospholipid syndrome (aps), but cns complications have also been reported such as optic neuritis, acute myelitis and encephalomyelitis [ ] . in mice, the immune response to tt depends on genetic background and to the specific adjuvant used for immunization. naive balb/c mice, immunized with tt, developed antibodies directed to tt, dsdna and ␤ gpi and were extremely sick [ ] . aps is an autoimmune disease characterized by the occurrence of thrombotic events. patients suffering from this condition have recurrent fetal loss, thromboembolic phenomena, thrombocytopenia as well as neurological, cardiac and dermatological involvement [ ] . the serological marker of aps is the presence of antiphospholipid antibodies (apl), which bind negatively charged phospholipids, platelets and endothelial cells mainly through the plasma protein beta- -glycoprotein-i (b gpi). the presence of igg and igm anti-cardiolipin antibodies (acl) and lupus anticoagulant is associated with thrombosis in patients with aps [ ] . ␤ gpi was identified as the most important antigen in aps. ␤ gpi has several properties in vitro which define it as an anticoagulant (e.g., inhibition of prothrombinase activity, adenosine diphosphate-induced platelet aggregation, platelet factor ix production) [ ] . passive transfer of anti-␤ gpi antibodies induce experimental aps in naïve mice and thrombus formation in ex vivo model [ ] . evidence suggests that the molecular mimicry mechanism between ␤ gpi and tt is one of the possible causes for aps. besides tt, aps has also been reported following hbv and influenza virus vaccination, although data are scarce [ , ] . sle is a multisystem autoimmune disease characterized by the production of a variety of autoantibodies. igg isotype antibodies to double-stranded dna (dsdna) are thought to be diagnostic markers and their presence correlates with disease pathogenesis. several factors including genetic, hormonal, environmental and immune defects are involved in the induction of autoantibodies in this disease [ ] . post vaccination manifestations of sle or lupus like syndrome have been reported and range from autoantibody induction to full blown clinical disease. reports have been published associating sle to hbv, mmr, dtp, hpv, influenza, bcg, pneumococcal and small pox vaccinations [ ] . vaccination in sle diagnosed patients is associated with disease exacerbation and decreased antibody response, which may be due to the underlying disease and the frequent use of immunosuppressive drugs [ ] . a temporal link between sle and hbv vaccination is the only relation that has been demonstrated [ ] . several studies have demonstrated an increased prevalence of hpv in individuals with lupus compared to the general population, which has increased awareness for the need to vaccinate this high-risk population [ ] . to do so, the association between immunization with hpv vaccines and sle like symptoms, as well as the higher incidence of flares in known lupus patients must be taken into account. vasculitis is the name given to a group of autoimmune mediated diseases, which involve blood vessels of different types and sizes. they can be categorized according to several disease features indluding: the type of vessel affected, organ distribution, genetic predisposition and clinical manifestation [ ] . so far, cases of large vessel vasculitis have been detected. this includes cases of giant cell arteritis (gca) following influenza vaccination, cases of takayasu disease (td), and one case of large cell arteritis involving subclavian and renal arteries following hbv vaccines. two of these patients had previous received the diagnosis of ankylosing spondylitis and polymyalgia rheumatica (pmr)-like illness [ ] . one case of polyarteritis nodosa (pan) following the administration of tetanus and bcg vaccine is described. all other cases of pan in adults follow the administration of hbv vaccine [ ] [ ] [ ] . case reports of medium vessels vasculitis -both polyarteritis nodosa and kawasaki disease (kd) -have also been published in pediatric patients. kd has been described one day after the second dose of hbv vaccine and following yellow fever vaccine [ , ] . two cases of pediatric patients with pan have been reported two months after receiving the hbv vaccine [ , ] . eosinophilic granulomatosis with polyangiitis (egpa) after tetanus vaccination [ ] and following hbv vaccine [ ] have been reported. there are also cases of microscopic polyangiitis (mpa) and cases of granulomatosis with polyangiitis (gpa) following influenza vaccines in the literature [ , ] . henoch schönlein purpura (hsp) is the most common vasculitis of childhood. it is generally benign and self-limited. it is mediated by iga immune complex deposition in various tissues as well as in small-sized blood vessels. genetic risk factors play an important role in the pathogenesis of the disease: it is associated with hla-drb* , and . hsp was associated with seasonal influenza, influenza a (h n ), pneumococcal and meningococcal disease, hepatitis a virus (hav), hbv, anti-human papilloma virus (hpv) vaccines, and following multiple combinations of vaccines, such as typhoid, cholera and yellow fever [ , [ ] [ ] [ ] . leukocytoclastic vasculitis has been associated with several vaccines, including influenza vaccine [ ] , hav vaccine [ ] , hbv vaccine [ ] , pneumococcal vaccine [ ] , varicella [ ] , rubella, smallpox [ ] and the anthrax vaccine [ ] . dermal vasculitis with pan uveitis has also been described following mmr vaccine [ ] . ra is the most prevalent chronic inflammatory arthritis affecting the synovial membrane of multiple diarthrodial joints. although its etiology has not been completely clarified, deregulation of the immune system is evident with a preponderance of inflammatory cytokines and immune cells within the joints. ra has an estimated heritability of %, leaving a substantial proportion of risk to environmental factors. immunizations have previously been proposed as potential environmental triggers for ra. in the norfolk arthritis register database, of the first patients reported receiving a tetanus vaccination within weeks prior to the onset of arthritis. similarly, a transient rise in rf titer was recorded in out of military recruits - weeks after receiving concomitant immunization against tetanus, typhoid, paratyphoid, mumps, diphtheria, polio and smallpox. however, only showed a persistent elevation in titer and none developed arthritis [ ] . several mechanisms have been proposed to explain the putative association between vaccination and the initiation of ra, the most prominent of which are molecular mimicry and non-specific immune system activation [ ] . vaccines who have been associated with ra include rubella vaccine in which reactive arthritis occurs in % of recipients. controlled studies failed to show persistent arthritis or arthralgia in these patients [ ] . patients following hbv vaccine showed an increase of arthritis in a vaers study, but this was not seen in a large retrospective epidemiological study [ ] . data so far suggest that vaccines carry an insignificant role in the pathogenesis of ra. several mechanisms are being studied to produce vaccines mainly targeting inflammatory cytokines as "antigens" such as tnf, aiming to induce high titers of endogenous neutralizing anticytokine antibodies with the goal of breaking natural th tolerance to auto antigens. other cytokines, namely il- il- , mif, rantes, il- , mcp- are also being tested [ ] . another vaccine related therapy uses autologous t cell lines to induce a specific immune response by the host's t cells directed against the autoimmune (vaccine) t cells [ ] . this strategy has been successful in mouse models and has shown encouraging results in a small pilot study of ra patients, where patients showed a clinical response, defined by acr improvement criteria [ ] . uctd is a clinical condition characterized by signs, symptoms and laboratory tests suggestive of a systemic autoimmune disease but that does not fulfill the criteria for any defined connective tissue disease (ctd). such patients with clinical manifestations suggestive of systemic connective tissue disease but not fulfilling any existing criteria are quite frequent: - % of the patients initially asking for a rheumatologic evaluation may at least temporarily be diagnosed as affected by 'undefined' or 'undifferentiated' connective tissue disease. comparing studies on these diseases is unfeasible because of the inexistence of defined criteria for diagnosis [ ] . within years of follow-up, patients usually evolve to defined ctds, which include sle, systemic sclerosis (ssc), primary sjögren's syndrome (pss), mixed connective tissue disease (mctd), systemic vasculitis, poly-dermatomyositis (pm/dm) and ra. maintaining an undefined profile for years makes evolving into ctds less probable and the diagnosis of "stable uctd" reliable [ ] . disease etiology is a concern and it has been associated with vitamin d deficiency and silicone implants, both of which lead to an imbalance in proinflammatory and anti-inflammatory cytokines [ ] . vaccines have also been associated with this disease, namely the hbv vaccine [ ] . etiopathogenesis of uctd is unknown and it has been suggested it might fall on asia spectrum since symptomatic similarities are striking and uctd etiopathogenesis has been associated with adjuvants [ ] . aa is an autoimmune disease, characterized by one or more well demarcated oval and round non-cicatricial patches of hair loss. the disease may affect any hair bearing part of the body and has a great impact on a patient's self-esteem and quality of life. depending on ethnicity and location, aa is the most prevalent skin disease. aa prevalence varies and is estimated to be between . - . % in the united states and . % in singapore [ , ] . as with any other autoimmune disease, the development of aa encompasses genetic and environmental factors. environmental factors associated with aa development are emotional and/or physical stress, infections and vaccines [ ] . secondary syphilis is one of the most well studied examples, however epstein barr virus [ ] and herpes zoster [ ] infections have also been related to the development of the disease. as far as vaccines go, hbv vaccine has been associated with aa development. in one study of patients, developed aa after vaccination with hbv vaccine. of those patients, were rechallenged, and the reappearance of disease was witnessed [ ] . in mice this association failed to be established [ ] . one case of aa was witnessed following tetanus toxoid, as well as two case reports following hpv and mmr vaccine [ ] [ ] [ ] . itp is an autoimmune disease defined by a platelet count of less than platelets/l without overlapping diseases. it can present with or without anti-platelet-antibodies. thrombocytopenia is relatively common and the overall probability of developing itp was , % in a cohort of patients. it was also found that % of patients developed an overlapping aid other than itp [ ] . the etiology of the disease is yet to be fully understood but it has been detected following infectious diseases, such as helicobacter pylori, hepatitis c virus (hcv), novel influenza a infection, rotavirus infection and human immunodeficiency virus (hiv) [ ] . itp onset has also been reported, although rarely, as a severe adverse event following vaccine administration. this was more often observed after measles-mumps-rubella (mmr), hepatitis a and b, diphtheria-tetanus-acellular pertussis (dtap), and varicella vaccinations [ ] . molecular mimicry has been suggested as a possible mechanism for the development of itp, namely following helicobacter pylori infection. its eradication has been shown to increase platelet count and diminish the levels of anti-caga antibody in a subset of h. pylori infected subjects with itp [ ] . these data point towards a beneficial role of h. pylori eradication in chronic itp. two cases of itp following anti-rabies vaccine have been reported and one after hpv vaccine. reactivation of itp was reported two weeks after a tick-borne encephalitis vaccination [ ] . the most consistent association with itp is with the mmr vaccine [ ] . however, it should be emphasized that the number of cases are fewer than expected without vaccination. t d is due to antigen specific reactions against insulin producing beta cells of the pancreas. much like other autoimmune diseases, t d results from a combination of genetic, environmental, hormonal and immunological factors. environmental factors such as pathogens, diet, toxins, stress and vaccines are believed to be involved in the beginning of the autoimmune process [ ] . although the mechanisms by which viral infections cause autoimmune diabetes have not been fully clarified, there is some evidence to suggest a role for natural infections in the pathogenesis of t d mellitus in susceptible individuals [ ] . it has been hypothesized that vaccination could trigger t d in susceptible individuals. although post-vaccination t d may be biologically plausible, cumulative evidence has not supported an increased risk of t d following any vaccine [ ] . several experimental data have suggested that, depending on the timing, vaccination might exert a protecting or aggravating effect on the occurrence of diabetes [ ] . a study suggests that haemophillus influenza type b vaccine might be a risk factor in the induction of islet cell and anti-gad antibodies measured at one year of age [ ] but there are previous studies that show no association between hib and t d [ ] . in a cohort of american military officers diagnosed with t d, there was no association found between vaccination and t d diagnosis [ ] . available data about a relation between the mumps vaccine and t d are still incomplete and their interpretation is difficult because of miscellaneous confounding factors associated with the development of t d [ ] . association between hemagglutinin neuraminidase (h n ) vaccines and t d is so far unproven [ ] . in humans, it has been hypothesized that early-age bcg vaccination is associated with the risk of t d. the few studies conducted to date provided no consistent evidence of an association. there are, however, studies showing a possible temporary boost of the immune function after vaccination [ ] . studies also show that among bcg-vaccinated children who test positive for islet autoantibodies, there is a higher cumulative risk of t d [ ] . in animal experiments it has been observed that bcg seems to have a protective effect against diabetes, however researchers have yet to translate this benefit to humans [ ] . in all, studies results do not support any strong association between vaccination and t d. narcolepsy is a sleep disorder described as excessive sleepiness with abnormal sleep pattern characterized by uncontrollable rapid eye movement (rem) events which occur at any time during the day. these event and may or may not be accompanied by a loss of muscle tone (cataplexy) [ ] . a plethora of data indicates that narcolepsy is caused by the lack of orexin (also known as hypocretin), an important neurotransmitter, which is involved in the regulation of the sleep cycle. in narcolepsy patients, a loss of orexin producing neurons in the hypothalamus and low levels of orexin in the cerebrospinal fluid (csf) has been reported [ ] . narcolepsy has been shown to have an autoimmune background. antibodies against tribbles (trib ) have been found in these patients, which may be related to the pathogenesis of disease. an experimental model of narcolepsy in mice has been made by passive transfer of total igg from narcolepsy patients into the animal's brains through intra ventricular injection [ ] . environmental factors like influenza a virus and streptococcal infections have been associated with disease onset. interestingly, fever by itself without the diagnosis of an infectious etiology was found to be a risk factor for narcolepsy [ ] . several groups have studied and found an increase in the incidence of narcolepsy diagnosis following the introduction of influenza vaccination, specifically, aso -adjuvanted pandemrix tm vaccine. this association was shown in finland especially in [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] year-olds, but also in case reports from other countries [ ] . other studies failed to find an association. the actual infection with h n has been associated with disease development in china, however no such relationship has been noted in europe [ ] . the above-mentioned associations are specifically related to the aso -adjuvanted pandemrix tm vaccine. the same association has not been reported for other h n adjuvanted or non-adjuvanted vaccines. the major difference between the aso and the mf adjuvants is the presence of the ␣-tocopherol. ␣-tocopherol is unique in that it can achieve the highest and longest antibody response by producing an enhanced antigenspecific adaptive immune response. in vitro it was shown that ␣-tocopherol could increase the production of orexin as well as increase the proteosome activity. this increased production of orexin fragments may facilitate antigen presentation to mhc class ii, thus triggering an autoimmune process [ ] . all these data together support the relationship between the h n pandemrix tm vaccine and the development of narcolepsy. gluten induced enteropathy, gluten sensitive enteropathy, or more commonly called celiac disease (cd) is a life-long autoimmune condition mainly of the gastrointestinal tract, specifically affecting the small intestine. the abnormal immune response crates autoantigens which are directed towards tissue transglutaminase (ttg). the two main autoantibodies and the most widespread serological markers to screen for the disease are anti ttg and anti endomysium. two additional auto-antibodies, namely: anti deaminated gliadin peptide and anti-neoepitope ttg were found recently to be reliable for cd screening as well [ ] . cd is an autoimmune disease induced by well-known nutritional environmental factors. the non-dietary ones are less studied and established. several infectious disease have been linked to its development, the so-called infectome [ ] . a clear cause-effect relation is yet to be established for most of the pathogens associated with cd. what has been shown, however, is that in countries with low economic status, inferior hygiene conditions and higher infectious load, cd prevalence is lower [ ] . an epidemiologic relationship was established in between rotavirus infection and cd. data showed that in genetically predisposed individuals, rotavirus infection was related to childhood cd development [ ] . in subsequent research studies, a celiac peptide was recognized and proved to share homology with rotavirus major neutralizing protein vp and with the cd autoantigen ttg. the antibodies directed against the viral protein vp were shown to predict the onset of cd and induce typical features of cd in the intestinal epithelial cell-line t [ ] . it has also been suggested that rotavirus vaccine alters b and t behavior, as the percentage of b-cells was higher in the vaccinated infants [ ] . rotavirus vaccine as an inducer of cd is still in discussion and warrants further study. pmr is an autoimmune inflammatory rheumatic disease characterized by raised inflammatory markers with pain and morning stiffness of shoulders and pelvic girdles and synovitis of proximal joints and extra-articular synovial structures. its diagnosis is clinical and it is typically a disease of the elderly occurring mainly in subjects above . etiopathogenesis of pmr remains unknown, but genetic and environmental factors play a role [ ] . a close temporal relationship has been ascertained concerning epidemics of mycoplasma pneumoniae, chlamydia pneumonia, parvovirus b and peaks of cases of pmr and giant cell arteritis, however this is not clearly proven [ ] . cases of pmr following vaccination have rarely been reported. however, it is believed that post vaccination pmr may be underreported due to its symptomatic similarities with the transient effects of vaccines, namely: arthralgia, myalgia and low-grade fever. this leads to failure in establishing a chronological relationship when the disease is diagnosed. most of the reported cases are associated with seasonal influenza vaccine (inf-v). often, the time interval between vaccine administration and symptoms onset varies from one day, to three months. three cases were reported with associated giant cell arthritis. a case report of relapsing pmr after four years of remission following tetanus vaccination has also been reported [ , ] . acute disseminated encephalomyelitis (adem) is an inflammatory demyelinating disease of the central nervous system (cns). adem is usually poly-symptomatic with encephalopathy (i.e., behavioral change or altered level of consciousness). it affects mostly children and young adults and has higher prevalence in males. its incidence is . - . per per year [ ] . although there is no concrete evidence of a clear pathogenic association, adem has been associated with immunization or previous viral infection. post-vaccination adem accounts for only - percent of all cases, while post-infectious adem accounts for percent of all cases of adem [ ] . the hypothesis that better describes these associations is molecular mimicry. t-cells targeting human herpesvirus- (hhv- ), coronavirus, influenza virus and epstein-barr virus (ebv) have been shown to cross-react with myelin basic protein (mbp) antigens. anti-mbp t-cells were detected in patients following vaccination with simple rabies vaccine [ ] [ ] [ ] . in a post experimental therapy for alzheimer's disease with a vaccine that contained aggregates of synthetic a␤ fragments of amyloid precursor protein, adem was shown to develop in mice [ ] . the experimental model of ms, eae mice, may be induced with injection of a␤ , but only when the latter is administered together with the complete freund's adjuvant [ ] . this observation points to the importance and central role of the adjuvants in induction of adem and autoimmunity in general [ ] . the overall incidence of post vaccination adem is estimated to be . - . per and a higher risk has been reported following immunization against measles. other vaccines accountable for post-vaccination adem include vaccines against the varicella zoster, the rubella, the smallpox and the influenza viruses [ ] . surprisingly, certain vaccines such as anti-tetanus vaccine were shown to have a negative correlation with adem (statistically significant decreased risk) [ ] . hbv immunization has been studied as a possible cause for adem but was later associated with clinically isolated syndrome (cis) (a first time occurring demyelinating episode that may, or not develop to ms) and complete conversion to ms [ ] . as far as case reports are concerned, adem was associated with vaccination with influenza, hepatitis a and b, mmr, hpv and tetanus [ , , ] . bullous dermatoses are characterized by the presence of blisters and autoantibodies against structural components of the skin: desmosomal proteins (in pemphigus), adhesion molecules of the dermal-epidermal junction (in pemphigoid diseases), and epidermal/ tissue transglutaminase (in dermatitis herpetiformis). the most frequent autoimmune bullous diseases are bullous pemphigoid (bp) and pemphigus vulgaris (pv). bp is more frequently observed in the elderly, while the age of onset of pv is between and years. neither of the diseases have any gender preference [ ] . bp and pv etiology is, so far, poorly understood. both diseases have been associated with various environmental factors, which include emotional and/or physical stress, infections and vaccinations [ ] . genetic predisposition has also been studied with overexpression of certain hla class ii alleles. these include hla-dqb * , drb * , drb * , and dqb * . these alleles have been found to be more prevalent in bp patients than in the general population [ ] . pv is associated with certain hla class ii loci such as hla-dr and hladr alleles (drb * and drb * , which is prevalent in ashkenazi jews, iranian and sardinian patients). other loci include drb * (common among japanese and italian patients) and two dqb alleles (dqb * and dqb * ), which are strongly associated with pv. bp and pv patients' sera were found to have significantly higher prevalence of antibodies to hepatitis b virus, hepatitis c virus, helicobacter pylori, toxoplasma gondii and cytomegalovirus [ ] . as far as vaccination is concerned, bp developed in patients following influenza, diphtheria, tetanus, pertussis, hepatitis b, bcg, polio and herpes zoster vaccines [ , , ] furthermore, reactivation of bp following influenza vaccination was reported in one case report [ ] . new onset pv was associated with: influenza vaccine, hepatitis b vaccine, anthrax vaccine, typhoid booster and rabies vaccination. in addition, exacerbation of pv after vaccination was also reported following influenza vaccine and tetanus vaccine [ ] . iim compose a group of skeletal muscles diseases in which myositis without a recognized cause occurs. iim is usually subdivided in entities: dermatomyositis (dm), polymyositis (pm), inclusion body myositis (sibm) non-specific myositis (nsm) and immune mediated necrotizing myopathy (iam) [ ] . iim prevalence is around . × − cases, with a bimodal age of distribution that peaks in childhood and again between and years. dm is the most common inflammatory myopathy while pm is the least frequent. despite exhibiting similar clinical symptoms, the subsets of iim exhibit significant immunopathological variation. dm begins with the activation of the complement and formation of membrane attack complexes (mac). in pm and sibm the fundamental process is related to cd + t cells mediated cytotoxicity [ ] . it is unclear what breaks the tolerance and drives the immune response to induce iim. so far, dm, pm and sibm have been linked to vaccination. several cases have been reported in the literature associating different vaccines with the development of idiopathic inflammatory myopathies. cases of iim had been reported to vaers database up to june . out of these cases, were classified as pm, as dm and an only one as a sibm. dm has been reported after almost any vaccine, however only a few studies have attempted to clarify the possible relationship between dm and vaccination. pm is a frequent misdiagnosed disorder. some reports have associated previous immunization, especially hepatitis b vaccine with pm [ ] . despite being recently differentiated from other iim, sibm has already been related to hbv vaccine [ ] . some vaccines associated with myositis are mmr vaccine, smallpox vac-cine, poliomyelitis (ipv), diphtheria and tetanus toxoid, influenza, hpv and bcg [ ] . fms is an entity that is related to the inability of the cns to modulate pain. the conditioned pain modulation process in the cns appears to be compromised among many fms patients, which might explain the enhanced pain sensation experienced by these patients [ ] . the etiology of fms is yet to be unveiled. genetic predisposition, physical trauma (particularly to the cervical spine), emotional stress (to various stressors) as well as a variety of infections have been linked with fms. vaccines have been associated with the triggering of fms namely rubella and lyme disease vaccines [ ] . there are several reports of fibromyalgia-like disease after vaccination, specifically hpv (martinez-lavin journal of clinical rheumatology ). the medical community and regulatory agencies should be aware of these possible adverse effects aiming at defining their magnitude. chronic fatigue syndrome (cfs) is a disease characterized by disabling fatigue, headaches, concentration difficulties and memory deficits ( %). other symptoms such as sore throat ( %), tender lymph nodes ( %), skeletal muscle pain and feverishness ( %), sleep disruption ( %), psychiatric problems ( %) and rapid pulse ( %) are often observed. it more frequently affects women and has a prevalence of . - . % [ ] . although disease etiology is still unknown, there are several pathogens, such as epstein-barr virus (ebv), which have been associated with cfs. patients often have higher titers of igm to the ebv viral capsid antigen. cytomegalovirus and human herpes virus antibodies were also detected more often in cfs patients, although other reports failed to replicate these results. parvovirus b infection has also been suggested as a trigger to cfs [ ] [ ] [ ] . vaccine inoculation has also been appointed as a probable cause. vaccinations against rubella, q fever and hepatitis b were found to be associated with higher risk of developing cfs while meningococcal vaccine, poliovirus and influenza vaccine were not. surprisingly, staphylococcus toxoid vaccine appeared to have a protective effect [ , , ] . defined in by shoenfeld and agmon-levin asia syndrome is characterized by hyperactive immune response to adjuvants [ ] . as previously stated, asia incorporates four known medical conditions: siliconosis, gws, mmf, and post-vaccination phenomena [ ] . recently, the sick building syndrome (sbs) was proposed as a candidate for the asia spectrum [ ] . all of these diseases satisfy several criteria for fms and seid [ ] . a macrophagic myofasciitis (mmf) mmf has been described as an emerging condition of unknown cause characterized by a pathognomonic lesion in muscle biopsy mixing large macrophages with submicron to micron-sized agglomerates of nanocrystals in their cytoplasm and lymphocytic infiltrates. these lesions were related to aluminum deposits in muscle following immunization with aluminum containing vaccines [ ] . mmf lesion is now universally recognized as indicative of a long-lasting persistence of aluminum adjuvant at the site of prior intramuscular immunization. the long-lasting mmf lesion should be considered as a biomarker of aluminum bio persistence in a given individual. patients with mmf have higher reported myalgia with incidence being up to %. its etiology is not clear but genuine muscle weakness is rare and the diagnosis of fibromyalgia is also rare. higher prevalence of chronic fatigue syndrome (cfs) in patients with mmf has been reported as well. cognitive impairment has been associated with mmf: in one series of mmf patients, up to % had attention and memory complaints and neuropsychological tests were abnormal in % [ ] . b gulf war syndrome (gws) gws is a clinical entity specifically related to a certain time and place in history. it was described among veterans of the military conflict occurring in - in the persian gulf. the syndrome is characterized by chronic fatigue, musculoskeletal symptoms, malaise and cognitive impairment. it clinically overlaps with post traumatic stress disorder (ptsd), fms, cfs and other functional disorders [ ] . the unique conditions that have been associated so far with disease development are the exposure to extreme climate in the persian gulf, exposure to various chemicals (pesticides, depleted uranium), stress provoked by prolonged waiting without actual combat and the intense exposure to vaccinations of the soldiers for fear of biological weaponry [ ] . comparing gulf war veterans and veterans of the bosnian conflict, multiple vaccinations administered to servicemen in the gulf war was identified as a unique exposure [ ] . the mechanism through which vaccination exposure may lead to the development of functional symptoms is not completely understood. the possibility that a shift from th to th type reactions could be of pathogenic significance was raised and is supported by an increased frequency of allergic reactions, low natural killer cell activity and low levels of interferon ␥ and il- in these patients [ ] . one study with gws patients showed a connection between anti-squalene antibodies and symptoms development. this was refuted by a larger study that found no association between antisqualene antibodies and chronic multi-symptom illness [ ] . c asia registry a registry is a collection of data related to patients with the same specific characteristic. it is often the first approach in the study of an area of inquiry. in rare diseases, registries are often the way to get a sufficiently sized sample of patients which can be used either for epidemiological or research purposes. asia syndrome may be underreported because of unawareness and failure to connect the syndrome with the exposure. this registry was created to fully understand the clinical aspects of disease and compare patients from all over the world in order to have fully validated criteria for disease diagnosis and also to define demographic and environmental history of disease. the asia syndrome registry website can be found on the following link: https://ontocrf.costisa.com/en/web/asia. only cases reported by physicians are accepted. to make an informed decision in medicine, there is always a need to weigh the pros and cons. ards may play an important role in deciding whether vaccination is or is not appropriate to a patient. in these cases, patients are immunosuppressed on account of their diagnosis and even more so if they are under specific immunomodelatory medication [ ] . if the efficacy of vaccination is reduced, there is a potential for development of disease flares following vaccination. in the case of live vaccines, its inoculation may even be enough to trigger disease in the host. for these specific reasons, live vaccines are generally contraindicated in patients receiving immunosuppressant medication. there is a need for screening and treatment of latent tuberculosis infection (ltbi) before starting anti-tnf-alpha therapy. the same is true for vaccination. preferably, even recommended vaccination (see table ) should be administered before the initiation of disease-modifying anti-rheumatic drugs (dmards) because these may reduce vaccine efficacy [ ] . immunosuppression equals high risk of infection and lower vaccine efficacy. taking into account safety concerns and efficacy, the eular recommendations for immunizations in aiird patients are: • assess vaccination status in initial investigation. • administer vaccines in a stable disease phase. • live attenuated vaccines are to be avoided especially if immunosuppressive agents are being administered. bcg is not recommended. • administer vaccines ideally before starting dmards and anti-tnf␣ agents. • influenza and -valent polysaccharide pneumococcal vaccination is recommended. • tetanus toxoid vaccination is recommended following recommendations of general population, in case of major and/or contaminated wounds in patients receiving rituximab in the previous weeks tetanus ig is indicated. • hpv and herpes zoster should be considered. • in hyposplenic/asplenic patients, influenza, pneumococcal, haemophilus influenza b and meningococcal c are advisable. • hepatitis a and b is recommended in patients at risk. • travel patients should be immunized according to general population guidelines except for live attenuated vaccines, which are to be avoided [ ] . vaccines have many beneficial effects in combating infectious diseases and preventing mortality and morbidity. they have also proved to be effective cancer treatments by immunomodulation, as demonstrated by the intravesical administration of bcg to treat superficial bladder cancer [ ] . vaccines are however, linked to autoimmunity. beneficial outcomes, like the adjuvant effect are based on immunity triggering and enhanced immunity mechanisms. these same responses account for autoimmunity exertion. vaccines induce the production of autoantibodies, but their pathologic effect is yet to be unveiled. although vaccines are widely considered safe, there are subjects with predispositions to whom vaccines pose a bigger threat. an example is the fact that animal models with autoimmune predispositions develop autoimmune disease following adjuvant exposure. as many as % of recipients of aluminum containing adjuvants may be sensitized to future exposure [ ] . silicon-induced inflammatory fibro proliferative response is irrefutable and well documented. the presence of anti-silicone antibodies and silicone-associated autoimmune phenomena seems very plausible. asia syndrome and aluminum safety studies show that the use of aluminum containing "placebo" in control groups in vaccine safety studies should be carefully evaluated. new studies must be performed using a proper placebo to adequately test vaccine safety. another evident failure in vaccine safety studies are the short-term periods which are evaluated. continued immune system activation has been observed to be a potential mechanism of disease. a disease which is poorly understood so far. vaccine recommendations should be reassessed frequently in different subsets of the population. this does not invalidate the need for vaccines, however, the lower the possibility of exerting adverse events, the easier it will be for the potential benefits to outweigh the risks. vaccinomics represents a major breakthrough in vaccine development and can lead to the development of targeted vaccines to peptides most likely to be immunogenic [ ] . a predictable response to vaccine can be achieved by differentiating the host variability. this can be achieved namely in genetics and pathogen variability. developing a vaccine accordingly will lead to increased specificity in treatment and leave less room for adverse events. by using immunomodulation, vaccinomics can also give rise to novel therapies for autoimmune diseases. there are several reports of cases of autoimmunity diseases following vaccines but despite in vitro positive results and due to both the limited number of cases and the long latency period of the diseases, every attempt for an epidemiological study has failed to deliver a connection. classification as asia syndrome, in detriment of classic specific autoimmune diseases, could be the key to finding effective preventative therapeutic strategies. it will enable the study of bigger patient clusters with earlier diagnoses. future studies that could help clarify the association between vaccinations, adjuvants and autoimmunity should ideally have a different design, more long-term data and should include autoimmune phenomena as well as large-scale epidemiological studies of autoimmune diseases. the mosaic of autoimmunity infections and autoimmunity-friends or foes? autoimmune/inflammatory syndrome induced by adjuvants (asia) : unveiling the pathogenic, clinical and diagnostic aspects asia-autoimmune/inflammatory syndrome induced by adjuvants adjuvants and autoimmunity adjuvant activity of immunopotentiating reconstituted influenza virosomes (irivs) interfaces questions and possibilities in toll-like receptor signalling immunotherapy with a ragweed-toll-like receptor agonist vaccine for allergic rhinitis immunotherapeutic strategies for the treatment of malignant melanoma does pegylated interferon alpha- b confer additional benefit in the adjuvant treatment of high-risk melanoma? tuftsin on the -year anniversary of victor najjar's discovery tuftsin a naturally occurring immunopotentiating factor. i. in vitro enhancement of murine natural cell-mediated cytotoxicity tuftsin and tuftsin conjugates potentiate immunogenic processes: effects and possible mechanisms the novel adjuvant ic strongly improves influenza vaccine-specific cellular and humoral immune responses in young adult and aged mice improving vaccine delivery using novel adjuvant systems prototype alzheimer's disease epitope vaccine induced strong th -type anti-abeta antibody response with alum to quil a adjuvant switch cpg oligodeoxynucleotides trigger protective and curative th responses in lethal murine leishmaniasis tuftsin-azt conjugate: potential macrophage targeting for aids therapy enhanced immune response induced by a potential influenza a vaccine based on branched m e polypeptides linked to tuftsin construction of a synthetic immunogen: use of the natural immunomodulator polytuftsin in malaria vaccines against resa antigen of plasmodium falciparum stimulating effect of tuftsin and its analogues on the defective monocyte chemotaxis in systemic lupus erythematosus immunological concepts of vaccine adjuvant activity recent advances in the discovery and delivery of vaccine adjuvants using eae to better understand principles of immune function and autoimmune pathology toll pathway-dependent blockade of cd + cd + t cell-mediated suppression by dendritic cells viruses as adjuvants for autoimmunity: evidence from coxsackievirus-induced myocarditis pathogenesis of myocarditis and dilated cardiomyopathy bacillus calmette-guerin immunotherapy of superficial bladder cancer pattern recognition receptors and control of adaptive immunity how dying cells alert the immune system to danger role of protease-activated receptors in inflammatory responses, innate and adaptive immunity novel signaling interactions between proteinase-activated receptor and toll-like receptors in vitro and in vivo autoimmune (auto-inflammatory) syndrome induced by adjuvants (asia)-animal models as a proof of concept the endogenous adjuvant squalene can induce a chronic t-cell-mediated arthritis in rats percutaneous exposure of adjuvant oil causes arthritis in da rats exacerbation of collagen-induced arthritis in rats by rat cytomegalovirus is antigen-specific comparative study of adjuvant induced arthritis in susceptible and resistant strains of rats. i. effect of cyclophosphamide aloh -adjuvanted vaccine-induced macrophagic myofasciitis in rats is influenced by the genetic background induction of plasma cell tumours in balb-c mice with , , , -tetramethylpentadecane (pristane) paraffin oil injection in the body: an obsolete and destructive procedure induction of lupus autoantibodies by adjuvants adjuvant immunization induces high levels of pathogenic antiphospholipid antibodies in genetically prone mice: another facet of the asia syndrome induction of the asia syndrome in nzb/nzwf mice after injection of complete freund's adjuvant (cfa) manifestations of systemic autoimmunity in vaccinated salmon comparison of three adjuvants used to produce polyclonal antibodies to veterinary drugs comparison of tissue reactions produced by haemophilus pleuropneumoniae vaccines made with six different adjuvants in swine delayed acquisition of neonatal reflexes in newborn primates receiving a thimerosal-containing hepatitis b vaccine: influence of gestational age and birth weight autoimmune/autoinflammatory syndrome induced by adjuvants (asia syndrome) in commercial sheep slow ccl -dependent translocation of biopersistent particles from muscle to brain aluminum hydroxide injections lead to motor deficits and motor neuron degeneration nanomolar aluminum induces pro-inflammatory and pro-apoptotic gene expression in human brain cells in primary culture the pro-oxidant activity of aluminum regulatory role of zinc during aluminium-induced altered carbohydrate metabolism in rat brain aluminum and alzheimer's disease: after a century of controversy, is there a plausible link? exposure to aluminium and the subsequent development of a disorder with features of alzheimer's disease elevated urinary excretion of aluminium and iron in multiple sclerosis aluminum-induced entropy in biological systems: implications for neurological disease the immunobiology of aluminium adjuvants: how do they really work? fatty acid-induced nlrp -asc inflammasome activation interferes with insulin signaling inflammasome signaling at the heart of central nervous system pathology summary and conclusions of the sixty-seventh meeting of the joint fao/who expert committee on food additives if exposure to aluminium in antiperspirants presents health risks, its content should be reduced macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle structural basis of metal hypersensitivity diagnosis and treatment of metal-induced side-effects mercury and multiple sclerosis the beneficial effect of amalgam replacement on health in patients with autoimmunity metal-induced inflammation triggers fibromyalgia in metal-allergic patients the role of environmental factors in autoimmune thyroiditis orofacial granulomatosis associated with hypersensitivity to dental amalgam, oral surg nephrotic syndrome due to ammoniated mercury endemic clustering of multiple sclerosis in time and place mercury exposure and antinuclear antibodies among females of reproductive age in the united states: nhanes, environ. health perspect gold nephropathy: serologic data suggesting an immune complex disease nickel-induced allergy and contact dermatitis: does it induce autoimmunity and cutaneous sclerosis? an experimental study in brown norway rats aluminum in the central nervous system (cns): toxicity in humans and animals, vaccine adjuvants, and autoimmunity six revolutions in vaccinology from pasteur to genomics: progress and challenges in infectious diseases systems vaccinology vaccinomics, adversomics, and the immune response network theory: individualized vaccinology in the st century studies of twins in vaccinology vaccinomics current findings, challenges and novel approaches for vaccine development the hla genomic loci map: expression, interaction, diversity and disease the link between genetic variation and variability in vaccine responses: systematic review and meta-analyses development of polyarthritis after insertion of silicone breast implants followed by remission after implant removal in hla-identical sisters bearing rheumatoid arthritis susceptibility genes autoimmune/auto-inflammatory syndrome induced by adjuvants (asia) after quadrivalent human papillomavirus vaccination in colombians: a call for personalised medicine anticardiolipin and anti-beta( ) glycoprotein i antibodies in sera of apparently healthy children at regular preventive visits autoimmunity and hepatitis a vaccine in children bacterial induction of autoantibodies to beta -glycoprotein-i accounts for the infectious etiology of antiphospholipid syndrome vaccination-induced systemic autoimmunity in farmed atlantic salmon autoimmune response following annual influenza vaccination in apparently healthy adults high non-specific t lymphocyte response to the adjuvanted h n vaccine in comparison with the h n /h n /b-brisbane vaccine without adjuvant short and long-term effects of pandemic unadjuvanted influenza a(h n ) pdm vaccine on clinical manifestations and autoantibody profile in primary sjögren's syndrome pneumococcal vaccination of patients with systemic lupus erythematosus: effects on generation of autoantibodies immunogenicity and safety of a quadrivalent human papillomavirus vaccine in patients with systemic lupus erythematosus: a case-control study safety and immunogenicity of the quadrivalent hpv vaccine in female systemic lupus erythematosus patients aged to years anti-phospholipid antibodies following vaccination with recombinant hepatitis b vaccine development of autoantibodies before the clinical onset of systemic lupus erythematosus genetics and autoantibodies silicone and autoimmunity platinum in the environment: frequency of reactions to platinum-group elements in patients with dermatitis and urticaria silicone and scleroderma revisited rupture of silicone gel breast implants and symptoms of pain and fatigue the association between silicone implants and both antibodies and autoimmune diseases a comparison of autoantibody production in asymptomatic and symptomatic women with silicone breast implants silicone implant incompatibility syndrome (siis): a frequent cause of asia (shoenfeld's syndrome) management of chronic childhood immune thrombocytopenic purpura: aieop consensus guidelines rubella-associated arthritis. i. comparative study of joint manifestations associated with natural rubella infection and ra / rubella immunisation risk of chronic arthropathy among women after rubella vaccination. vaccine safety datalink team efficacy and duration of immunity after yellow fever vaccination: systematic review on the need for a booster every years risk of yellow fever vaccine-associated viscerotropic disease among the elderly: a systematic review bacillus calmette-guérin immunotherapy for genitourinary cancer arthritis after bcg vaccine in a healthy woman dermatomyositis after b.c.g. vaccination aetiopathogenesis of takayas's arteritis and bcg vaccination: the missing link? reactive arthritis induced by intravesical bcg therapy for bladder cancer: our clinical experience and systematic review of the literature systemic granulomatosis and hypercalcaemia following intravesical bacillus calmette-guérin immunotherapy universal hepatitis b vaccination in taiwan and the incidence of hepatocellular carcinoma in children. taiwan childhood hepatoma study group vaccines and autoimmunity hepatitis b vaccination and undifferentiated connective tissue disease: another brick in the wall of the autoimmune/inflammatory syndrome induced by adjuvants (asia) adverse reactions to human papillomavirus (hpv) vaccines sustained efficacy and immunogenicity of the hpv- / as -adjuvanted vaccine up to . years in young adult women detection of human papillomavirus l gene dna fragments in postmortem blood and spleen after gardasilreg. vaccination-a case report human papillomavirus (hpv) vaccine policy and evidence-based medicine: are they at odds? annual report: surveillance of adverse events following immunisation in australia annual report: surveillance of adverse events following immunisation in australia influenza vaccine and autoimmunity infection and death from influenza a h n virus in mexico: a retrospective analysis altered response to a(h n ) pnd vaccination in pregnant women: a single blinded randomized controlled trial adverse events following immunization with vaccines containing adjuvants antineutrophil cytoplasmic antibody vasculitis associated with influenza vaccination hughes syndrome) met the autoimmune/inflammatory syndrome induced by adjuvants (asia) meningococcal group b vaccines comparative long-term adverse effects elicited by invasive group b and c meningococcal infections immune response, antibody persistence, and safety of a single dose of the quadrivalent meningococcal serogroups a, c, w- , and y tetanus toxoid conjugate vaccine in adolescents and adults: results of an open, randomised, controlled study vaccines and guillain-barré syndrome henoch-schölein purpura and polysaccharide meningococcal vaccine infantile bullous pemphigoid developing after hexavalent, meningococcal and pneumococcal vaccinations the risk of sequelae due to pneumococcal meningitis in high-income countries: a systematic review and meta-analysis burden of disease caused by streptococcus pneumoniae in children younger than years: global estimates fda. pneumococcal -valent conjugate vaccine (diphtheria crm protein) prevnar ® decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine comparative reactogenicity and immunogenicity of valent pneumococcal vaccine administered by intramuscular or subcutaneous injection in elderly adults safety and immunogenicity of a -valent pneumococcal conjugate vaccine pneumococcal polysaccharide vaccination in rheumatoid arthritis patients receiving tocilizumab therapy eular recommendations for vaccination in adult patients with autoimmune inflammatory rheumatic diseases tetanus a review of the literature optic neuritis and myelitis after booster tetanus toxoid vaccination antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of patients crystal structure of human beta -glycoprotein i: implications for phospholipid binding and the antiphospholipid syndrome induction of anti-phospholipid syndrome in naive mice with mouse lupus monoclonal and human polyclonal anti-cardiolipin antibodies characterization of antiphospholipid antibodies in chronic hepatitis b infection influenza vaccination and the production of anti-phospholipid antibodies in patients with systemic lupus erythematosus unraveling the soul of autoimmune diseases: pathogenesis, diagnosis and treatment adding dowels to the puzzle vaccines and autoimmune diseases of the adult effect of belimumab on vaccine antigen antibodies to influenza, pneumococcal, and tetanus vaccines in patients with systemic lupus erythematosus in the bliss- trial ten cases of systemic lupus erythematosus related to hepatitis b vaccine prevalence of cervical human papillomavirus infection in women with systemic lupus erythematosus revised international chapel hill consensus conference nomenclature of vasculitides large artery vasculitis following recombinant hepatitis b vaccination: cases etiology and precipitating factors of necrotizing angiitis with respiratory manifestations. case reports cutaneous polyarteritis nodosa in a child following hepatitis b vaccination systemic polyarteritis nodosa following hepatitis b vaccination kawasaki disease in an infant following immunisation with hepatitis b vaccine yellow fever vaccination and kawasaki disease churg-strauss vasculitis with brain involvement following hepatitis b vaccination anca-associated vasculitis following influenza vaccination: causal association or mere coincidence? influenza vaccination in anca-associated vasculitis henoch-schönlein purpura after hepatitis a vaccination henoch-schönlein purpura following influenza a h n vaccination henoch-schönlein purpura following influenza vaccinations during the pandemic of influenza a (h n ) leukocytoclastic vasculitis after influenza vaccination vasculitis related to hepatitis a vaccination hypersensitivity vasculitis after hepatitis b vaccination leukocytoclastic vasculitis after pneumococcal vaccination allergic reaction to varicella vaccine vasculitides associated with infections, immunization, and antimicrobial drugs lymphocytic vasculitis associated with the anthrax vaccine: case report and review of anthrax vaccination panuveitis and dermal vasculitis following mmr vaccination can immunisation trigger rheumatoid arthritis? risk of rheumatoid arthritis following vaccination with tetanus, influenza and hepatitis b vaccines among persons - years of age anti-cytokine vaccination in autoimmune diseases exploiting t. cell crosstalk as a vaccination strategy for rheumatoid arthritis vaccination with selected synovial t cells in rheumatoid arthritis undifferentiated ctd a wide spectrum of autoimmune diseases analysis of the evolution of uctd to defined ctd after a long term follow-up undifferentiated connective tissue disease after silicone-gel testicular implantation connective tissue disease following hepatitis b vaccination prevalence of alopecia areata in the first national health and nutrition examination survey tracing environmental markers of autoimmunity: introducing the infectome onset of alopecia areata after epstein-barr virus infectious mononucleosis hair loss after varicella zoster virus infection hair loss after routine immunizations recombinant human hepatitis b vaccine initiating alopecia areata: testing the hypothesis using the c h/hej mouse model alopecia universalis in an adult after routine tetanus toxoid vaccine telogen effluvium following bivalent human papillomavirus vaccine administration: a report of two cases acute-onset madarosis following mmr vaccination long-term outcome of otherwise healthy individuals with incidentally discovered borderline thrombocytopenia immune thrombocytopenic purpura (itp) associated with vaccinations: a review of reported cases epidemiology of paediatric immune thrombocytopenia in the general practice research database effect of eradication of helicobacter pylori in children with chronic immune thrombocytopenia: a prospective, controlled, multicenter study, pediatr immune thrombocytopaenic purpura: an autoimmune cross-link between infections and vaccines vaccination may be associated with autoimmune diseases viral infections and molecular mimicry in type diabetes childhood immunizations and type diabetes: summary of an institute for vaccine safety workshop. the institute for vaccine safety diabetes workshop panel stimulation of the developing immune system can prevent autoimmunity vaccinations may induce diabetes-related autoantibodies in one-year-old children lack of association between receipt of conjugate haemophilus influenzae type b vaccine (hboc) in infancy and risk of type (juvenile onset) diabetes: long term follow-up of the hboc efficacy trial cohort vaccination and risk of type diabetes mellitus in active component u. s. military difficulties in assessing the relationship, if any, between mumps vaccination and diabetes mellitus in childhood, pubmed,ncbi neurological and autoimmune disorders after vaccination against pandemic influenza a (h n ) with a monovalent adjuvanted vaccine: population based cohort study in bacille calmette-guérin vaccination and incidence of iddm in montreal, canada neonatal bacille calmette-guerin vaccination and type diabetes bacille calmette-guérin/dnahsp prime-boost is protective against diabetes in non-obese diabetic mice but not in the streptozotocin model of type diabetes narcolepsy: autoimmunity, effector t cell activation due to infection, or t cell independent, major histocompatibility complex class ii induced neuronal loss? passive transfer of narcolepsy: anti-trib autoantibody positive patient igg causes hypothalamic orexin neuron loss and sleep attacks in mice is narcolepsy a classical autoimmune disease? the pandemrix-narcolepsy tragedy: how it started and what we know today a novel algorithm for the diagnosis of celiac disease and a comprehensive review of celiac disease diagnostics infections may have a protective role in the etiopathogenesis of celiac disease rotavirus infection frequency and risk of celiac disease autoimmunity in early childhood: a longitudinal study a subset of anti-rotavirus antibodies directed against the viral protein vp predicts the onset of celiac disease and induces typical features of the disease in the intestinal epithelial cell line t influence of early environmental factors on lymphocyte subsets and gut microbiota in infants at risk of celiac disease; the proficel study cervical human papillomavirus infection in mexican women with systemic lupus erythematosus or rheumatoid arthritis giant cell arteritis and polymyalgia rheumatica after influenza vaccination: report of cases and review of the literature postvaccine vasculitis: a report of three cases clinical study of childhood acute disseminated encephalomyelitis, multiple sclerosis, and acute transverse myelitis in fukuoka prefecture inflammatory/post-infectious encephalomyelitis high level of cross-reactivity in influenza virus hemagglutinin-specific cd + t-cell response: implications for the initiation of autoimmune response in multiple sclerosis cross-reactivity with myelin basic protein and human herpesvirus- in multiple sclerosis myelin basic protein-reactive autoantibodies in the serum and cerebrospinal fluid of multiple sclerosis patients are characterized by low-affinity interactions tauopathy-like abnormalities and neurologic deficits in mice immunized with neuronal tau protein acute/relapsing experimental autoimmune encephalomyelitis: induction of long lasting, antigen-specific tolerance by syngeneic bone marrow transplantation autoimmune/inflammatory syndrome induced by adjuvants (shoenfeld's syndrome): clinical and immunological spectrum post-vaccination encephalomyelitis: literature review and illustrative case vaccinations and risk of central nervous system demyelinating diseases in adults acute disseminated encephalomyelitis cohort study: prognostic factors for relapse neuromyelitis optica following human papillomavirus vaccination hbv vaccine and dermatomyositis: is there an association? autoimmune blistering diseases of the skin pemphigus: etiology, pathogenesis, and inducing or triggering factors: facts and controversies does influenza vaccination induce bullous pemphigoid? a report of four cases bullous pemphigoid after herpes zoster vaccine administration: association or coincidence? reactivation of bullous pemphigoid after influenza vaccination pathophysiology of autoimmune polyneuropathies a review on the association between inflammatory myopathies and vaccination fibromyalgia and overlapping disorders: the unifying concept of central sensitivity syndromes etiology of fibromyalgia: the possible role of infection and vaccination infection and vaccination in chronic fatigue syndrome: myth or reality? review part : human herpesvirus- in central nervous system diseases pathogenesis of parvovirus b infection: host gene variability, and possible means and effects of virus persistence effects of staphylococcus toxoid vaccine on pain and fatigue in patients with fibromyalgia/chronic fatigue syndrome vaccination as teenagers against meningococcal disease and the risk of the chronic fatigue syndrome the sick building syndrome as a part of the autoimmune (auto-inflammatory) syndrome induced by adjuvants selective elevation of circulating ccl /mcp levels in patients with longstanding post-vaccinal macrophagic myofasciitis and asia cognitive functioning in gulf war illness health of uk servicemen who served in persian gulf war gulf war syndrome: is it due to a systemic shift in cytokine balance towards a th profile antibodies to squalene in us navy persian gulf war veterans with chronic multisymptom illness vaccination in adult patients with auto-immune inflammatory rheumatic diseases: a systematic literature review for the european league against rheumatism evidence-based recommendations for vaccination in adult patients with auto-immune inflammatory rheuma eular recommendations for vaccination in paediatric patients with rheumatic diseases vaccination against yellow fever among patients on immunosuppressors with diagnoses of rheumatic diseases updated consensus statement on the use of rituximab in patients with rheumatoid arthritis safety and efficacy of meningococcal c vaccination in juvenile idiopathic arthritis unexpectedly high incidence of persistent itching nodules and delayed hypersensitivity to aluminium in children after the use of adsorbed vaccines from a single manufacturer key: cord- - mu yp authors: syomin, b. v.; ilyin, y. v. title: virus-like particles as an instrument of vaccine production date: - - journal: mol biol doi: . /s sha: doc_id: cord_uid: mu yp the paper discusses the techniques which are currently implemented for vaccine production based on virus-like particles (vlps). the factors which determine the characteristics of vlp monomers assembly are provided in detail. analysis of the literature demonstrates that the development of the techniques of vlp production and immobilization of target antigens on their surface have led to the development of universal platforms which make it possible for virtually any known antigen to be exposed on the particle surface in a highly concentrated form. as a result, the focus of attention has shifted from the approaches to vlp production to the development of a precise interface between the organism’s immune system and the peptides inducing a strong immune response to pathogens or the organism’s own pathological cells. immunome-specified methods for vaccine design and the prospects of immunoprophylaxis are discussed. certain examples of vaccines against viral diseases and cancers are considered. immunoprophylaxis has long been used to control infectious diseases exerting an enormous impact not only on the global health but also on the safety of numerous populations of agricultural animals. in the past decades, the focus on vaccine production technologies has changed from handling intact pathogens to producing recombinant subunit vaccines based on isolated target antigens [ ] . intensive development of recombinant vaccines began in the s when it became possible to clone the desired dna sequences into expression plasmids and produce the target proteins. the construction of a recombinant vaccine takes advantage of the knowledge of the nucleotide sequences of genes encoded by a pathogen, and involves identification of the antigenic determinant, synthesis of the nucleotide sequence encoding the antigen, its cloning into an expression vector, and production of the target peptide in a certain expression system. the expression systems for individual heterologous proteins has been developed based on the prokaryotic and eukaryotic cells, which allow us to produce target proteins both on laboratory and industrial scales [ ] . the interest in recombinant vaccines is a consequence of the emergence of new infectious diseases, most often zoonotic ones. among them are the outbreaks of human diseases caused by the ebola virus, zika virus, marburg virus, the middle east respiratory syndrome, and severe acute respiratory syndrome coronaviruses [ ] . oncology also places great hopes in recombinant vaccines, which may help to overcome immune tolerance in the case of cancer treatment [ ] . moreover, there exists a constant threat of the emergence of new highly virulent strains of well-known viruses due to unceasing mutational processes in virus genomes [ ] . against this background, a new scientific concept, vaccinomics [ ] , which consists of identifying the minimum subset of antigens, which are able to induce a competent immune response to a pathogen or tumor by their specific interaction with the b and t immune cells, is being actively developed. using this approach it appears possible to design vaccines based on the minimum subset of antigens which most specifically characterize the pathogen in its interaction with the immunome (the set of all immune receptor sequences, which are present in the individual organism) [ ] . target detection of epitopes, the regions located on the surface of the protein, is possible with the aid of x-ray analysis, which is rather labor-consuming since it requires obtaining protein crystals. presently, the -d protein structure modeling has found broad use. this approach is based on identification of the physicochemical and electrostatic characteristics of the polypeptide chain regions and correlation of these characteristics with antigenic properties. two strategies are currently utilized, namely, modeling by homology and abbreviations: vlp, virus-like particles. udc . + : : - . de novo modeling. in the first case, the protein data bank (http://www.ncbi.nlm.nih.qov/genbank/) database of -d protein structures is used to model the protein. the commonly used modeller [ ] , as well as other software such as i-tasser [ ] , swiss-model [ ] , esypred d [ ] , d-jigsaw [ ] , phyre [ ] , and cphmodels [ ] , can be used as homology modeling tools. the limitation of this approach lies in the requirement that the structures of homologous proteins should demonstrate more than % identicality [ ] . the de novo modeling strategy is based on the computer simulation of the protein folding process (rosetta [ ] and tasser [ ] software). in this case, all possible variants of polypeptide chain folding are considered, and the most energetically favorable conformation, i.e., the one with the lowest potential energy, is chosen. predicted antigenic determinants are synthesized using genetic engineering vector techniques and heterologous protein expression systems, and then are experimentally tested using, for example, the antibody neutralization assay. using protein expression systems it is possible to produce virus-like particles (vlps), which are made up of monomers, which are able to multimerize into vlps, and display the antigenic determinants of target pathogens on their surface. this point will be addressed further in the review, we will just observe here that even complex epitopes represented by trimers may be obtained using heterologous expression systems. this has been demonstrated, for instance, for the trimeres of the human immunodeficiency virus (hiv- ) envelope glycoprotein [ , ] and influenza virus haemagglutinin [ ] . in order to choose the most specific antigens of the pathological agent of a new infectious disease, it appears inevitable to work with the infected material if only to isolate and sequence the nucleic acids containing the genetic information of the pathogen. however, in order to produce a vaccine based on epitopes characteristic for the target pathogen, there is no need to work with the pathogen itself. therefore, apart from all other advantages of the vaccines of this kind, they appear to be improved in terms of biological safety. vaccines based on the presentation of a subset of antigenic determinants of the infectious agent are characterized by a high level of reproducibility when commercially manufactured and are highly effective [ ] , since in this case, the immune response is directed exclusively against the most significant antigenic elements of the pathogenic microorganism or tumor. a number of vaccines specifically interacting with certain immune system receptors have been tested to date. for example, a vaccine containing only a single epstein-barr virus epitope specifically recognized by cd + t-cells is proposed for the prophylaxis of mononucleosis in human [ ] . this vaccine prevents the development of the disease, although it does not protect the organism from the entry of the virus. another example is classical swine fever virus (csfv). it has been demonstrated that the e viral protein produced in the baculovirus expression system induces the synthesis of csfv-neutralizing antibodies in pigs [ ] . vaccines are designed to produce immunity to a disease. although a single epitope is able to induce a strong immune response, it appears usually insufficient to induce protective immunity. the in vitro synthesized peptide properly representing the main antigenic determinant of the pathogen is highly likely not to be itself a strong immunogene primarily because of its small size (< nm) and high risk of proteolytic degradation. this problem may be overcome by the use of nanoparticles. it has been shown in a considerable number of works that in order to induce a strong immune response, antigenic determinants should be exposed on the surface of nanoparticles whose shape and size ( - nm) mimic those of the virus (fig. ) . in this respect also, vlps, the nanoparticles composed by viral proteins capable of self-assembly (multimerization) into structures morphologically resembling viruses, are the choice selection for the role of a strong immunogene. antigenic determinants multiply repeatedly on the vlp surface and promote complement fixation and b-lymphocyte receptor clusterization leading to the activation of the immune response. additionally, the same antigen multiply displayed on the surface of the particle promotes the multiplication of the pool of autoreactive b-cells, which is the primary objective when designing vaccines against autoimmune diseases and tumors [ ] . natural sources for vlp bioengineering and the specific features of their assembly structural proteins of various viruses are capable of autonomous self-assembly into vlps. they interact with the formation of globular, icosahedral, or rodlike structures. so far, the structural proteins of several dozen viruses have been obtained in heterologous expression systems, and almost all of them proved able to form vlps. the vlp size varies from to nm and is similar to the size of the corresponding viruses [ ] . being similar to viruses allows vlps to penetrate into the lymph and to be efficiently entrapped by antigenpresenting cells [ ] . currently, several heterologous expression systems and the corresponding expression vectors which can be used with them are available. these systems are based on both the bacterial cells (the pet system based on escherichia coli cells is often exploited) and different eukaryotic cells. in the case of eukaryotic cells, the most commonly used systems are yeast expression systems and the ones which rely on drosophila [ ] and spodoptera frugiperda insect cell lines [ ] . mammalian cells are also utilized, the most pop-ular among them being cho and hek [ ] . the cost of heterologous protein production in bacterial systems is lower than in eukaryotic cells. however, when studying the functional activity of the protein or vlp associated, for example, with the potential glycosylation sites or interaction with ubiquitin, or other ubiquitin-like peptides [ ] , no alternative to eukaryotic expression systems is available. it should be noted that the choice of the expression system is up to the researcher and is driven by the research task. for example, in different laboratories different eukaryotic systems for viral protein expression, including plant cells, are used to produce vlps which are used for vaccination against the hepatitis c virus (hcv) [ ] . in most cases, vlps assembled from a virus protein are considered as a candidate vaccine against this very virus, since protein monomers in multimeric configuration (vlp) induce a more potent immune response than protein monomers [ , ] . the strong immunogenic properties of vlps are determined by several factors. first of all, the dominant epitope of the structural protein is displayed as a part of the particle and is present in a multimeric form as is the case with a native virion [ ] . second, vlps stimulate b-lymphocytes and induce t-lymphocytes in the same manner as does the virus infecting the host organism [ ] . for example, vlps formed by the main capsid protein l of the human papilloma virus of several serotypes are successfully used as vaccines against cervical cancer [ ] . a vaccine against hepatitis b virus has been produced which contains vlps in the lipid membrane envelope [ ] . the vaccine against coxsackievirus a contains vlps assembled from the virus capsid proteins produced in the baculovirus expression system [ ] . scientists from china [ ] have demonstrated that the capsid protein (vp ) of the rabbit hemorrhagic disease virus (rhdv) efficiently multimerize into vlps, and a single intramuscular injection of the obtained vlp preparation completely protects the immunized rabbits against rhdv infection for at least days. the porcine circovirus capsid protein is also able to efficiently assemble into vlps when synthesized either in the human embryonic kidney cell culture (hek ) [ ] , or yeast [ ] and baculovirus [ ] expression systems, as well as in bacteria [ ] . footand-mouth disease vlps are assembled from three structural polypeptides vp , vp , and vp (naturally produced as a result of processing the p - a precursor polypeptide), simultaneously produced in e. coli [ ] . it has been recently demonstrated that the polyprotein ) liposome-based particles [ ] , ( ) nondegradable spherical nanoparticles (for example, metal nanoparticles) [ ] , ( ) polymer nanoparticles [ ] , ( ) graphene nanosheets [ ] and nanotubes [ ] . antigen of the duck hepatitis a virus produced in the baculovirus expression system assembles into vlps immediately in the cultured spodoptera frugiperda (sf ) cells, while immunization of ducklings with the obtained vlps induces a high level humoral immune response and protects them from developing the disease [ ] . the abilities of vlps resulting from multimerization of the cloned virus protein to play the role of an immunogen are not limited just to the presentation of their proper epitopes but may also be taken advantage of to display heterologous proteins. multimerization of capsid proteins into a particle requires the presence of nucleic acid, while for in vitro assembly, a short oligonucleotide ( - nt) is sufficient [ ] . therefore, vpls formed by capsid proteins are free from the infectious virus rna or dna. moreover, the above-mentioned property, which triggers protein monomer multimerization by nucleic acid, may be taken advantage of to package rna or dna into a particle with two different objectives. hence, antisense rna can be used to suppress virus expression. for example, in the case of the vaccine against foot-and-mouth disease, researchers from china [ ] not only displayed the vp epitope of the foot-and-mouth disease virus on the surface of vlps but also packaged the antisense rna complementary to the fragment of the viral genomic rna into a particle. another goal of nucleic acid packaging into a particle lies in the presentation of viral nucleic acids leading to the activation of specific immune receptors which induce the synthesis of type i interferons (inf) and other cytokines triggering the antivirus response [ ] . it has been demonstrated that long dna and rna molecules may be incorporated into vlps. for example, mrna for the reporter protein, red fluorescent protein, was packaged into particles representing the hepatitis e capsids [ ] . the plasmid containing the green fluorescent protein (gfp) gene was encapsidated into the vlps assembled in vitro from the main capsid protein of the hamster polyoma virus [ ] . a strategy for the packaging of up to kbp doublestranded circular dna into the particles was developed using the structural protein of the sv simian retrovirus expressed in baculovirus system [ ] . there exist two fundamentally different approaches for nucleic acid incorporation into vlps: ( ) in vitro vlp assembly from protein monomers in the presence of rna or dna, which is to be encapsidated into the vlp; ( ) exposure of the assembled vlps to osmotic shock in the presence of nucleic acids. osmotic shock is produced by using a solution with a low ionic strength; nucleic acid enters into a vlp as a result of the shift in the surface structures [ ] . however, the incubation of vlps in the presence of nucleic acids without exposure to osmotic stress results in a certain part of the nucleic acids becoming associated with the particles [ ] . a far more predictable approach is the in vitro assembly of vlps from the mixture of protein monomers and nucleic acids which should be encapsidated in them. in this case, after synthesis in a heterologous system, the obtained protein monomers are purified to completely remove the contaminating nucleic acids from the protein preparation; then the proteins are denatured in - m urea, nucleic acids which should be packaged are added, and the proteins are assembled into the particles by eliminating urea from the solution. the use of this strategy was demonstrated, for instance, in the works [ , , ] . protein association into a particle is a reversible process. vlp dissociation can be easily achieved by the addition of a denaturing agent. further, it may be removed and vlps may be reassembled in vitro [ ] with the encapsidation of the target rna or dna into the particle. this approach was implemented for the p protein of the hepatitis b virus. virus proteins produced in the heterologous expression system were denatured in m urea solution, and their assembly into vlps in the presence of the nucleic acid was further induced [ ] . protective immunity is controlled by specific humoral and cellular mechanisms activated by the antigen. posttranslational protein modifications and covalent bonds between the modifying molecules and the functional groups in the polypeptide chains are of great importance for immune homeostasis during the antiviral response. the balance between phosphorylation, ubiquitination, methylation, acetylation, sumoylation, adp-ribosylation, and glutamilation of a certain antigen performs the fine adjustment of the host's antiviral response [ , ] . the structural proteins for vlp production when synthesized in the eukaryotic expression systems undergo a number of posttranslational modifications. in particular, the gag gypsy monomer proved to be a natural substrate for type casein kinase [ ] . moreover, while produced in the eukaryotic cell, gag gypsy monomers become associated with ubiquitin and sumo, the cellular protein partners of the viral structural protein [ ] . generally, ubiquitin and sumo can bind with any lysine residue within the protein molecule, although there are preferable binding sites; it should also be noted that phosphorylation determines which of these two partners binds with the protein [ , ] . additionally, binding with a limited number of the above-mentioned signal peptides, such as monoubiquitination [ ] , usually plays a regulatory role and controls the transport of the protein itself, or the particle formed by it. in the case of polyubiquitination, the protein is destined for proteasome degradation [ , ] . by constructing recombinant polypeptides based on the viral capsid proteins it is possible to obtain vlps bearing several antigens. for example, dna encoding the vp capsid protein of the porcine parvovirus was fused with the dna fragment encoding amino acid residues of the main antigen of porcine circovirus (pcv ) nucleoprotein. the resulting hybrid dna was used to produce protein in heterologous cells which further formed vlps. these vlps induced a significantly stronger immune response against pcv than the recombinant adenovirus encoding the open reading frame (orf ) of pcv [ ] . another interesting example is represented by the vlps formed by the influenza virus matrix protein and displaying a toxoplasma gondii antigen on their surface [ ] . there exists another approach to designing therapeutic vaccines based on vlps. in this case, the vlp surface displays the variable fragment of an antibody specific to the antigen of the target virus [ ] . moreover, knowing which receptor is bound by the virus proteins renders it possible to produce particles possessing tropism to the cells of certain tissues. for example, the pre-sprotein of the hepatitis b virus exposed as a ligand on the vlp surface provided for their specific binding with hepatocytes [ ] . vlps assembled from the structural proteins of bacteriophages are also considered as carriers for human and animal vaccine production. for example, the dna fragment encoding foreign protein was inserted into the dna region encoding the n-terminal β-hairpin of the coat protein of the e. coli ms phage. it has been demonstrated that the expression of the obtained construct in bacterial cells resulted in the production of the recombinant protein in which a foreign peptide was present in the central part of the hairpin. the obtained chimeric protein monomers were able to self-assemble into particles morphologically similar to the phage capsid both in vivo and in vitro [ ] . using the mentioned property of the ms coat protein, vlps displaying the ep - epitope of the foot-and-mouth disease vp structural protein on their surface were obtained. these chimeric vlps induced string immune response in the animals, which allowed regarding them as a promising base for the development of a prophylactic vaccine [ ] . it is worth noting that the exposure of ep - on the vlp surface resolved a long-standing problem of how to make use of the beneficial properties of this peptide. researchers from several laboratories have demonstrated that this antigenic determinant of the footand-mouth disease not only induces the production of neutralizing antibodies but also stimulates t-lymphocytes [ ] . however, when isolated, ep - is not able to induce the immune response protecting animals against the foot-and-mouth virus infection [ ] . many attempts were made to solve this problem, for example, by combining the antigenic determinant with large molecules, such as for instance, t cell-specific molecules [ ] . however, only integration of ep - into vlps resulted in the possibility of using this antigenic determinant as a strong immunogen for vaccine production [ ] . practically all structural proteins of phages infecting various species of bacteria are able to autonomously form vlps. for example, salmonella typhimurium phages are able to autonomously multimerize into vlps on whose surface the epitopes of eukaryotic viruses, including the epitopes of the human influenza virus may be exposed [ ] . at the same time, however, bacteriophages are apparently incapable of presenting large epitopes, whose length exceeds amino acid residues [ ] . structural proteins of retroviruses and retrotransposons have a higher capacity compared to bacteriophage proteins. this means that protein monomers forming the particle may be fused with a longer peptide and still retain the ability to multimerize. in particular, it has been shown for the gag gypsy capsid protein [ ] , that at least % of its amino acid sequence (more than amino acid residues) is of little importance for multimerization into vlps [ ] . therefore, the truncated form of this protein may be fused with a heterologous peptide with the length similar to the length of the deleted fragments and a recombinant protein may be obtained which will retain its ability to self-assemble into vlps. in such a way, the truncated gag gypsy fused with the heterologous peptide formed particles when it was synthesized in bacterial cells. we were also able to assemble particles from the purified protein monomers in vitro [ , , ] . the obtained particles resembled the native gypsy virus by their morphology [ ] . the substitution of the deleted region in the dna encoding gag gypsy by the nucleotide sequence encoding the main antigenic determinant of the foot-and-mouth virus vp protein (ep - ) and his -tag resulted in the formation of particles which displayed ep - as the target antigen. the advantage of the technique used is that vlps formed by the protein containing his -tag can be readily isolated and purified by affinity chromatography [ ] . chimeric vlps may be obtained through the construction of recombinant dna molecules encoding both the corresponding virus protein and a foreign peptide or protein. another way is vlp pseudotyping. this approach was elaborated using the hamster polyomavirus. the v capsid protein of this virus first forms pentamers which further assemble into vlps comprised of pentamers. when v is expressed together with the minor capsid protein v , the latter binds with the central part of each v pentamer. it has been demonstrated that the n-terminal part of v is not involved in this interaction and therefore may be removed and substituted by the target epitope [ ] . another approach is also known. antigen is immobilized on the vlp via covalent binding between the reactive groups of the amino acid residues of the antigen and vlp. this approach proved to be ineffective due to the disruption of the native conformation of the antigen attached to the particle surface [ , ] . this problem was countered in the following way. glygly-lysglygly sequence was inserted into the monomer subunits for vlp assembly. in this environment, the reactive ε-amino group of lys residue is exposed and ready to interact with the cys-group of any protein in the presence of the cross-linking agent, such as m-maleimidobenzoyl-n-hydroxysulfosuccinimide ester (sulfo-mbs) [ ] , or succinimidyl- -[(β-maleimidopropionamido)hexanoate] (smph) [ ] . although the authors claimed that the described system of molecular assembly may be used to induce strong blymphocyte response against most antigens and prospective vaccine prototypes were suggested, this platform became the prototype only for a single prospective preparation based on vlps, the medication for lowering blood pressure in patients with hypertension [ ] , due to the development of the new improved techniques for antigen immobilization of the vlp surface, which will be discussed below. one of them takes advantage of the ability of his tag to bind with multivalent tris-nitrilotriacetic acid (trisnta) which in turn binds with a broad range of molecules, in particular, with biotin. the possibilities of this elegant technique for the functionalization of noninfectious viral nanoparticles were demonstrated in the case of vlps formed by the norovirus (nov) structural proteins. using the baculovirus expression system, the authors obtained nov vlps, displaying his -tag on their surface, which was first used to purify vlps, and further to bind trisnta molecules conjugated with a fluorescent dye, or biotin which in turn successfully bound streptavidin [ ] . notwithstanding some authors suggesting the described technique to be promising for vlp vaccine production, this approach continues to be only a prototype and has not advanced further than model experiments. the search for techniques providing efficient and stable immobilization of the antigen on the vlp surface led to the development of a versatile platform which allows us to covalently attach large antigens to the vlp surface [ ] . this molecular assembly system uses the tag-catcher conjugation system which was derived from the cnab domain of fibronectin-binding protein fbab from streptococcus pyogenes. as a result a highly reactive spytag peptide ( amino acid residues) was obtained which efficiently interacted with the spycatcher protein with the formation of isopeptide bonds in a broad range of buffer solutions [ ] . the spytag peptide was inserted into the vlp-forming polypeptide of the acinetobacter-infecting phage (these particles were called ap ), so that each monomer forming ap displayed two spytag peptides. a recombinant antigen consisting of the target protein and the spycatcher peptide was produced using bacterial or baculovirus expression systems [ ] . the obtained antigen efficiently bound with the vlp surface and induced a very strong immune response. almost the same effect was observed in the case of the reverse combination of the conjugating peptides, that is when spycatcher was incorporated into vlps, and spytag was fused to the antigen. this system is already utilized to design different types of vaccines, including the vaccines against tuberculosis and malaria [ , ] . a recent report has demonstrated that it was successfully used to develop a vaccine against breast cancer [ ] . this work is remarkable due to the fact that the high density of the her antigen (human epidermal factor receptor ) synthesized in the cultured drosophila melanogaster cells could be obtained on the ap vlp surface [ ] . the obtained particles induced a strong immune response to the antigen in the patients, which rendered it possible to overcome the immune tolerance to her known for patients with the her -dependent breast cancer [ , ] . in summary, the analysis of the discussed published data allows us to conclude that the nature of the vlp-forming protein is not as important for vlp functionalization as the technique which makes it possible to obtain high antigen density on the surface of vlps (table ) . the product range a number of vlp vaccines are available on pharmaceutical markets in many countries. the most wellknown are the cervarix® and gardasil® vaccines against cervical cancer, which have been successfully used for prophylaxis of this disease in girls for more than ten years. both vaccines are based on vlps formed by the main capsid protein l of the human papilloma virus belonging to several serotypes [ ] . engerix and recombivax hb vaccines against the hepatitis b virus representing vlps enveloped in a lipid membrane [ ] , as well as hecolin and xiamen innovax vaccines against hepatitis e [ ] , were developed and commercialized. the anti-malaria vaccine malaria rts has been certified, which represents the c-terminal part of the cs protein of plasmodium falciparum attached to the surface antigen of the hepatitis b virus (hbsag), which is also used in the certified vaccines against hepatitis b. to stabilize recombinant vlps, the fused protein is expressed together with the hbsag protein in saccharomyces cerevisease [ ] . a vaccine against the porcine circovirus representing vlps assembled from the vp pcv capsid protein synthesized in a heterologous system has also been commercialized [ ] . an increasing number of works have appeared that report on the development of vaccines based on nanoparticles, including vlps and/or replicationinactive viruses. among the apparent advantages of vaccines of this kind are their high specificity, efficiency, and good pharmacokinetic characteristics. it has been demonstrated that in an organism vlps reach lymphatic nodes in less than min, while the particle mixture bearing different antigens may be pro-cessed by the same antigen-presenting cells simultaneously [ ] . it should be noted that nanoprticle-based vaccines offer new possibilities in the development of immunoprophylaxis strategies, especially, the development of injection-free formulations, in particular, intranasal vaccines and inhalers. the administration of these vaccines may not involve humans, which is especially important in the case of industrial animal breeding in large agricultural enterprises characteristic of presentday russia [ ] , since it proves to be extremely difficult to administer identical injections to a large number of animals at the same time. this problem may be resolved by nebulizing aerosol vaccines in the area where animals are kept. the potential of this approach was demonstrated for the first time in with mice [ ] ; then, in the s- s, attempts were made to introduce this approach into animal and poultry farms in many countries. starting from the s, attempts were also made in the soviet union [ ] . however, at that time, the method of inhaled administration of vaccines was not introduced in practice since protective immunity could only be obtained when the tolerable dose was exceeded many times. for example, the inhaled administration of the vaccine against the newcastle disease led to - % lethality in birds [ ] . however, due to the introduction of vaccines based on the exposure of the epitopes of a pathogen on the surface of nanoparticles, including recombinant vlps, together with the development of innovative approaches to aerosol production, inhaled vaccines again became a topical issue. for example, biodegradable polymer nanoparticles formed by poly(glyceroladipate-co-ω-pentadecalactone), pga-co-pdl, proved themselves as an effective antigen carrier for inhaled vaccination [ ] , while the vaccine based on the adenovirus with the particles ranging from to µm induced stable protective immunity when administered via inhalation to monkeys [ ] . lastly, a method for vlp-vaccination via inhalation was proposed as protection against the human papilloma virus [ ] . one of the most important properties of vlps is mimicking virus particles and the consequent ability to induce a strong immune response to the antigen which they demonstrate irrespective of the source of the monomers which multimerize into vlps, these being either insect viruses, in particular the gypsy virus [ , ] , or plant viruses [ ] . vlps based on the structural proteins of plant viruses produced in plants [ ] make it possible to obtain vaccines with another nonconventional way of administration, edible vaccines [ ] . vaccines of this type may be synthesized directly in plant forage, with the oral vaccination of this kind inducing an immune response. expression vectors for foreign protein production in plants have been developed based on plant viruses, which allows obtaining plant-producing recombinant viruses or vlps displaying the target antigen on their surface [ , ] . vlps assembled from the structural proteins of plant viruses are also used to design functionalization platforms for antigen binding. these platforms are based on the strategies described above. in particular, they take advantage of the reactivity of the lys ε-group to immobilize the antigen on the surface of the plant's vlps [ ] [ ] [ ] [ ] . using the tobacco mosaic virus [ ] and potato x virus [ ] , it has been shown that a considerably large antigen can be immobilized on the surface of the corresponding vlps via the formation of the streptavidin-biotin complex. finally, based on the structural protein of the plant virus, the platform for antigen immobilization using the tag-catcher conjugation system is being developed [ ] . it should be noted that among the drawbacks of vlp vaccines manufactured using proteins which are not specific for mammals, for example, the plant virus proteins, include the development of the immune response to the structural component of the particles, which is the plant virus protein. undoubtedly, there is no therapeutical benefit in developing such immunity; however, whether there are adverse effects will be demonstrated by further studies. model molecules including biotin, alexa (fluorescent dye), and gfp protein conjugated with trisnta [ , ] isopeptide bond formation between spytag (integrated into vlp monomers) and spy-catcher (fused with antigen) peptides vaccine against breast cancer (her antigen presentation). it is also implemented to develop vaccines against malaria and tuberculosis [ ] [ ] [ ] [ ] [ ] the use of vlps for vaccination in medicine is becoming increasingly widespread. this point is reinforced by the list of clinical trials of vlp vaccines prepared by the united states national institute of health. currently, more than hundred vlp vaccines, which are directed against human and avian influenza viruses, the norwalk virus, norovirus, hiv- , and the chikungunya virus, the foot-and-mouth disease virus, as well as against melanoma, adenocarcinoma, papillomatoses, and cervical cancer, are undergoing clinical trials (http://clinicaltrials.gov/ct /search/index). it may be expected that the variety of nanoparticle vaccines against different cancers and viral infections will grow steadily, and vlps which can be used for immunization against microorganisms belonging to different taxons as well helminthes will also be developed. we thank the designers oleg vasil'ev and galina podzolkova (institute for statistical studies and economics of knowledge (issek), national research university higher school of economics) for their assistance in preparing the illustrations. the article was prepared within the framework of the basic research program at the national research university higher school of economics (hse) and supported within the framework of a subsidy by the russian academic excellence project - . the authors declare that they have no conflict of interest. this article does not contain any studies involving animals or human participants performed by any of the authors. assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery identifying and engineering promoters for high level and sustainable therapeutic recombinant protein production in cultured mammalian cells going to bat(s) for studies of disease tolerance mesothelin virus-like particle immunization controls pancreatic cancer growth through cd + t cell induction and reduction in the frequency of cd + foxp + icos-regulatory t cells quasispecies and virus vaccinomics approach to tick vaccine development immunome-derived vaccines modeller: generation and refinement of homology-based protein structure models template-based modeling and free modeling by i-tasser in casp proceedings: basic considerations on the requirements to be met by disinfection procedures: the swiss model esypred d: prediction of proteins d structures enhancement of protein modeling by human intervention in applying the automatic programs d-jigsaw and d-pssm exploring the extremes of sequence/structure space with ensemble fold recognition in the program phyre protein distance constraints predicted by neural networks and probability density functions a historical perspective of template-based protein structure prediction high-resolution structure prediction and the crystallographic phase problem scoring function for automated assessment of protein structure template quality a comparative immunogenicity study of hiv- virus-like particles bearing various forms of envelope proteins, particles bearing no envelope and soluble monomeric gp stabilized hiv- envelope glycoprotein trimers for vaccine use overexpression of a virus-like particle influenza vaccine in eri silkworm pupae, using autographa californica nuclear polyhedrosis virus and host-range expansion viruslike particle engineering: from rational design to versatile applications phase i trial of a cd + t cell peptide epitopebased vaccine for infectious mononucleosis pigs immunized with a novel e subunit vaccine are protected from subgenotype heterologous classical swine fever virus challenge liposome: composition, characterisation, preparation, and recent innovation in clinical applications vaccine delivery using nanoparticles polymers in vaccine formulation alum-functionalized graphene oxide nanocomplexes for effective anticancer vaccination immunotherapy applications of carbon nanotubes: from design to safe applications moving towards a new class of vaccines for non-infectious chronic diseases vaccine delivery: a matter of size, geometry, kinetics and molecular patterns interaction of viral capsid-derived virus-like particles (vlps) with the innate immune system. vaccines (basel) drosophila melanogaster s cells for expression of heterologous genes: from gene cloning to bioprocess development expression of the retrovirus gypsy gag in spodopterafrugiperda cell culture with the recombinant baculovirus activation of human somatostatin receptor type causes inhibition of cell growth in transfected hek but not in transfected cho cells regulation of cellular antiviral signaling by modifications of ubiquitin and ubiquitinlike molecules preclinical development and production of virus-like particles as vaccine candidates for hepatitis c. front. microbiol. , relationship between humoral immune responses against hpv , hpv , hpv and hpv in - year old girls receiving cervarix ® or gardasil ® vaccine development of imaged capillary isoelectric focusing method and use of capillary zone electrophoresis in hepatitis b vaccine recombivax hb ® virus-like particle-based vaccine against coxsackievirus a protects mice against lethal infections development of a vlpbased vaccine in silkworm pupae against rabbit hemorrhagic disease virus development of recombinant porcine parvovirus-like particles as an antigen carrier formed by the hybrid vp protein carrying immunoreactive epitope of porcine circovirus type oral application of freeze-dried yeast particles expressing the pcv b cap protein on their surface induce protection to subsequent pcv b challenge in vivo virus-like particles of chimeric recombinant porcine circovirus type as antigen vehicle carrying foreign epitopes immunogenicity and immunoprotection of porcine circovirus type (pcv ) cap protein displayed by lactococcus lactis large-scale production of foot-andmouth disease virus (serotype asia ) vlp vaccine in escherichia coli and protection potency evaluation in cattle duck hepatitis a virus structural proteins expressed in insect cells selfassemble into virus-like particles with strong immunogenicity in ducklings effect of nucleocapsid on multimerization of gypsy structural protein gag protection of a novel epitope-rna vlp double-effective vlp vaccine post-translational regulation of antiviral innate signaling recombinant hepatitis e virus like particles can function as rna nanocarriers hamster polyomavirusderived virus-like particles are able to transfer in vitro encapsidated plasmid dna to mammalian cells high cloning capacity of in vitro packaged sv vectors with no sv virus sequences cell-free assembly of a polyoma-like particlefrom empty capside and dna gene transfer using human polyomavirus bk virus-like particles expressed in insect cells the structural protein gag of the gypsy retrovirus forms virus-like particles in the bacterial cell in vitro assembly of retroviruses preparation by alkaline treatment and detailed characterisation of empty hepatitis b virus core particles for vaccine and gene therapy applications sumoylation-disrupting was mutation converts wasp from a transcriptional activator to a repressor of nf-κb response genes in t cells homologous and heterologous type casein kinases have the same effect on the affinity for rna of the gag structural protein of gypsy (mdg ) sumo-nonclassical ubiquitin the functional motifs that are revealed in the gypsy gag amino acid sequence analysis of human immunodeficiency virus type gag ubiquitination global landscape of hiv-human protein complexes viruslike nanoparticle vaccine confers protection against toxoplasma gondii construction of polyomavirus-derived pseudotype virus-like particles displaying a functionally active neutralizing antibody against hepatitis b virus surface antigen construction and immunological evaluation of multivalent hepatitis b virus (hbv. core virus-like particles carrying hbv and hcv epitopes multiple presentation of foreign peptides on the surface of an rna-free spherical bacteriophage capsid promising ms mediated virus-like particle vaccine against foot-and-mouth disease plasmids encoding foot-and-mouth disease virus vp epitopes elicited immune responses in mice and swine and protected swine against viral infection adenovirus-mediated rna interference against foot-andmouth disease virus infection both in vitro and in vivo promising multiple epitope recombinant vaccine against foot-and-mouth disease virus type o in swine symmetry controlled, genetic presentation of bioactive proteins on the p virus-like particle using an external decoration protein drosophila melanogaster endogenous retrovirus gypsy can propagate in drosophila hydei cells bacterially expressed polyprotein gag of retroelement gypsy (mdg ) is able to form multimeric complexes presence of gypsy (mdg ) retrotransposon in the extracellular virus like particles a novel strategy of veterinary vaccines production conditioned by the development of vaccinomics cellular and humoral immunogenicity of hamster polyoma virus-derived virus-like particles harboring a mucin cytotoxic t-cell epitope reengineering viruses and virus-like particles through chemical functionalization strategies functionalizing nanoparticles with biological molecules: developing chemistries that facilitate nanotechnology immunodrugs: therapeutic vlp-based vaccines for chronic diseases histagged norovirus-like particles: a versatile platform for cellular delivery and surface display a molecular assembly system that renders antigens of choice highly repetitive for induction of protective b cell responses bacterial superglue enables easy development of efficient virus-like particle based vaccines peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin improving the malaria transmission-blocking activity of a plasmodium falciparum / based vaccine antigen by spytag/spycatcher mediated viruslike display virus-like particle display of her induces potent anti-cancer responses structural analysis and insertion study reveal the ideal sites for surface displaying foreign peptides on a betanodavirus-like particle hepatitis e vaccine development: a year odyssey the rts, s/as malaria vaccine in children to months of age at first vaccination delivering adjuvants and antigens in separate nanoparticles eliminates the need of physical linkage for effective vaccination diversity of research publications: relation to agricultural productivity and possible implications for sti policy. scientometrics immunization of mice against pneumococcal pneumonia by inhaled polysaccharide anatomical and physiological features of the human and animal respiratory system in the genesis of immunity after aerosol vaccination: . role of airway structure in mechanical retention of inhyaled material dry aerosol vaccination against newcastle disease: . safety and activity controls on chickens bovine serum albumin adsorbed pga-co-pdl nanocarriers for vaccine delivery via dry powder inhalation unique cellular and humoral immunogenicity profiles generated by aerosol, intranasal, or parenteral vaccination in rhesus macaques spect/ct study of bronchial deposition of inhaled particles. a human aerosol vaccination model against hpv immunology of viruslike particles different applications of virus-like particles in biology and medicine: vaccination and delivery systems a preliminary study of a lettuce-based edible vaccine expressing the cysteine proteinase of fasciola hepatica for fasciolosis control in livestock a launch vector for the production of vaccine antigens in plants rotavirus vp expressed by pvx vectors in nicotiana benthamiana coats pvx rods and also assembles into virus-like particles chemical conjugate tmv-peptide bivalent fusion vaccines improve cellular immunity and tumor protection potato virus x as a novel platform for potential biomedical applications incorporation of tetanus-epitope into virus-like particles achieves vaccine responses even in older recipients in models of psoriasis, alzheimer's and cat allergy. npj vaccines. , modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications use of plant viruses and virus-like particles for the creation of novel vaccines key: cord- - qku ml authors: billington, john; deschamps, isabelle; erck, stanley c.; gerberding, julie l.; hanon, emmanuel; ivol, sabrina; shiver, john w.; spencer, julia a.; van hoof, johan title: developing vaccines for sars-cov- and future epidemics and pandemics: applying lessons from past outbreaks date: - - journal: health secur doi: . /hs. . sha: doc_id: cord_uid: qku ml the covid- pandemic is a stark reminder of the heavy toll that emerging infectious diseases (eids) with epidemic and pandemic potential can inflict. vaccine development, scale-up, and commercialization is a long, expensive, and risky enterprise that requires substantial upfront planning and offers no guarantee of success. eids are a particularly challenging target for global health preparedness, including for vaccine development. insufficient attention has been given to challenges, lessons learned, and potential solutions to support and sustain vaccine industry engagement in vaccine development for eids. drawing from lessons from the most recent ebola epidemic in the democratic republic of the congo, as well as the h n influenza, - ebola, and - zika outbreaks preceding it, we offer our perspective on challenges facing eid vaccine development and recommend additional solutions to prioritize in the near term. the recommendations focus on reducing vaccine development timelines and increasing business certainty to reduce risks for companies. the global health security community has an opportunity to build on the current momentum to design a sustainable model for eid vaccines. up call for the global health security community, showing that even outbreaks in remote villages can have worldwide impact. indeed, the unpredictable, heterogeneous, and fastpaced nature of some highly transmissible eids makes them a particularly challenging target for global health preparedness, including for vaccine development. unfortunately, today's vaccines cannot be developed ''on demand'' in response to a surprise threat. under ideal circumstances, it will take at least to months to bring a safe and efficacious vaccine for sars-cov- to market. vaccine development, scale-up, and commercialization is a long, expensive, and risky enterprise that requires substantial upfront planning and offers no guarantee of success. , nevertheless, there is reason for optimism. the licensure of the world's first ebola virus vaccine by european, us, and some african regulatory authorities in late , along with prequalification by the world health organization (who) in record time, marked a groundbreaking milestone for global preparedness. , regulatory authorities have made extraordinary efforts to help bring safe and effective vaccines to market faster in response to emergencies. the who launched a new health emergencies division that includes eid monitoring response, and it drafted a research and development (r&d) blueprint that prioritizes pathogens with epidemic potential. gavi, the vaccine alliance, adapted its model to include stockpiling of ebola vaccines. at the same time, national and regional government programs like the us biomedical advanced research and development authority (barda) and the european union's innovative medicines initiative (imi) have sustained their commitment to r&d for eid response. finally, new entities like the coalition for epidemic preparedness innovations (cepi) are galvanizing global pandemic preparedness and response for sars-cov- and future epidemics and pandemics, using innovative approaches, including investments in novel platform technologies. the vaccine industry has the experience, capabilities, and capacities required to develop and help deliver critical vaccines to the people who need them under accelerated timelines. a new ''golden age'' in vaccinology opens doors to greater speed, versatility, and more efficient vaccine platforms as well as more nimble manufacturing capabilities for vaccine development. , vaccine manufacturers have a demonstrated track record of responding to eids when called upon. however, responding to immediate requests to develop vaccines for emergencies often requires manufacturers to put other essential, lifesaving programs on hold. it also carries considerable business, reputational, and liability risks, including both direct and indirect financial impacts. much has been written about how we might be better prepared for the next epidemic or pandemic. however, insufficient attention has been given to challenges, lessons learned, and potential solutions to support and sustain vaccine industry engagement in vaccine development for eids. drawing from lessons from the most recent ebola epidemic in kivu, democratic republic of the congo (drc), as well as the h n influenza, - ebola, and - zika outbreaks preceding it, we offer our perspective on challenges facing eid vaccine development and recommend additional solutions to prioritize in the near term. a complete solution set must also include considerations for manufacturing, scale-up, vaccine distribution, and access, which are equally if not more challenging for eid response. however, these important aspects of eid preparedness and response are beyond the scope of this article. in this section, we draw on prior experience to show how critical factors work against vaccine development in an emergency: time and risk. in the event of a widespread epidemic or pandemic, a delay in the availability of a vaccine can result in substantial human and economic loss. in past outbreaks, vaccines came too late to have a significant impact on the epidemic curve despite extraordinary measures taken to accelerate the responses ( figure ). when the h n influenza pandemic emerged, the vaccine industry had a head start because the seasonal influenza vaccine business and the requisite manufacturing infrastructure were well established. notwithstanding these advantages, and despite strong multisectoral collaboration and communication involving companies, vaccines were not available in time (the delay was in part due to the wait for the bsl- reassorted virus, import permits, and a -month production cycle). although vaccines based on the new strain were approved by the us food and drug administration (fda) and shipped globally just months after who had declared h n a public health emergency of international concern (pheic), they came too late and in insufficient quantities to prevent the estimated , to , deaths that occurred worldwide in the first year of the pandemic. while seasonal influenza has a predictable and sufficient annual market volume to motivate sustained company involvement, it has been nearly impossible to predict the need or demand for eid vaccines. in , when the ebola epidemic erupted from a remote village in west africa, there were no approved or late-stage ebola vaccines available to use. ebola was a familiar pathogen, but the incidence and geographic spread of this epidemic was unprecedented, demonstrating that even the most isolated outbreaks cannot be ignored. within months, the epidemic had spread from guinea to neighboring liberia, nigeria, and sierra leone, marking the largest ebola outbreak to date. when who declared a pheic in august , several companies acted to expedite the development of early-stage vaccine candidates that were financed in part by public research funding, especially from canada, the united states, and the european pan american health organization and world health organization. zika -epidemiological update. union. companies advanced vaccine candidates through coordinated phase safety trials starting in september over several sites across africa, europe, and north america at unprecedented speed with help from who and other partners. but the epidemic began to wane before most could complete phase efficacy trials. one company completed positive interim analyses of phase efficacy trials in guinea just prior to the end of the pheic in december . by this time, more than , people were infected and , had died. like the ebola virus, the zika virus was discovered decades ago but was thought to cause only sporadic, mild, and self-limited symptoms in africa and asia. a vaccine was not considered a public health priority. in the well-documented outbreak in , it was the rapid rise of microcephaly in babies born to zika-infected mothers in brazil that brought this disease to global prominence. as the virus spread, fear of this previously unknown complication of infection motivated who to declare a pheic in february . again, vaccine researchers and developers responded to the call, leveraging what scientists knew about zika from work they were already doing with dengue virus, another flavivirus transmitted by the same species of mosquito that transmits zika virus. one company partnered with the us government to advance the first zika virus vaccine candidate to a phase clinical trial in just months. shortly thereafter, a second company began the development of its candidate with support from barda, and other companies started development programs at their own expense. in a story that repeats itself, however, vaccines could not be developed in time to reduce the disease burden before who ended the pheic in november of that year. the disease became endemic-acute disease declined as populations were inoculated by the disease. by then, more than , babies, most of them in brazil, had been diagnosed with irreversible microcephaly or other neurological disorders. although several zika virus vaccine programs continue today, the low transmission rates and high population-level immunity pose significant obstacles for clinical development. vaccine development involves a substantial investment and a high risk of failure. typical vaccine development programs from discovery to licensure can cost companies upwards of a billion dollars, can take over a decade to complete, and on average have a % chance of failure. , while responding to global infectious disease emergencies is central to the vaccine industry's public health mission, business leaders may not always act, and shareholders may not always approve the necessary investments, because the business risks of eid vaccine development tend to outweigh any return on investment. companies have capacities but tend to be sized for the capacity they need to support their in-line portfolio and their pipeline, running at capacity across the value chain. as such, companies that respond to an outbreak have had to divert resources from core business lines, often for many years, to develop a vaccine that tends to lack a commercial market and may only be needed in limited supply and during limited periods of time. this diversion can cripple all but the largest and most well-supported efforts and creates a major disincentive. the ebola and zika experiences illustrate problems with how governments, philanthropic donors, and nongovernment organizations (ngos) currently contribute funding for eid vaccine development. first, there are only limited public financial resources to support development in advance of outbreak. the us national institutes of health (nih) funds research on diseases that pose a potential us threat, such as zika virus, but funding dedicated to accelerating product development tends to be made available only after who has declared a pheic. similarly, other government-led initiatives like barda in the united states and the imi in europe played a substantial funding role in response to recent eids like ebola virus and zika virus, but this funding is cyclical (eg, dependent on us annual appropriations) and subject to political influence and competing priorities. the creation of cepi, as discussed further below, is a promising step forward in this regard. second, the funding tends to cover only a fraction of the direct vaccine development costs and little to none of the opportunity costs. importantly, opportunity costs that result from delayed commercially viable programs, diverted manufacturing lines, and diversion of scientists, regulatory team members, and other resources can quickly outweigh direct costs. finally, funding tends to be short-term or redirected after the emergency is over. these decisions are made without consideration for the ongoing costs companies will incur in completing development, securing licensure, fulfilling post-market regulatory obligations, and scaling manufacturing appropriately to meet uncertain demand. this short-term funding may also favor firstcomer candidates over second-and third-line innovations that may ultimately provide greater value to those at risk. companies also face legal and reputational risks when responding to emergencies. for example, during all outbreaks described above, there were concerns about liability risks during clinical stages and emergency use. manufacturers are often asked to make the vaccine candidates available on preliminary safety data based on the principle that the benefits of expediting clinical development typically outweigh the risks. however, this principle is not uniformly reflected in regulatory policy and public opinion. and under the best circumstances will take at least to months, and there is a need to appropriately inform public expectations. epidemic fluctuations also may make it impossible to demonstrate vaccine efficacy before approval and implementation. despite this uncertainty, companies must continue development without a clear regulatory pathway. these factors negatively affect company risk/ benefit assessments for engagement and increase uncertainty for managers and investors, potentially discouraging companies from responding to the next eid. to ensure we are prepared to meet the challenge of eids, the world needs sustainable solutions that consider the substantial complexity, public health burden, and business risks posed by these unpredictable pathogens. experience with h n , ebola, and zika exposed critical flaws in the vaccine development ecosystem for eids but also triggered new initiatives that may provide a more coordinated global response framework for the future. here, we offer recommendations for the near term that will improve the global preparedness ecosystem in advance of the next pandemic (table ) . despite good faith efforts by industry, governments, and ngos to expedite vaccine development during prior out-breaks, a recurring lesson was that ''faster-than-ever'' is still not fast enough. the experience with recent eids suggests opportunities to speed things up: reduce regulatory timelines and complexity, improve company and partner coordination, and accelerate manufacturing. define and streamline the eid/pandemic regulatory pathway. despite considerable progress, there is still only licensed ebola virus vaccine and no licensed zika virus vaccines. after the ebola virus disease pheic in , both stringent regulatory authorities and local regulators expedited review and approval of the clinical trial protocols for candidate vaccines. however, they did not address other structural gaps that slowed progress, including the lack of an explicit pathway for local regulators to leverage recommendations from stringent regulatory authorities like the fda. such a pathway would enable local regulators to benefit from the stringent regulatory authority expertise while reducing review and approval times at the country level. ideally, having pandemic vaccines licensed and inspected for quality by key regional stringent regulatory authorities would help facilitate the process. in addition, other methods to infer likelihood of field efficacy (eg, animal models, observational studies) need to be developed, included in guidelines, and accepted with stakeholder support. in addition, the virus outbreak underscored the importance of pre-aligned protocols and data requirements for phase trials to accelerate clinical studies. in a pheic setting, conducting clinical trials is challenging for reasons: ( ) nontraditional (ie, not placebo-controlled) trial designs may be favored by local authorities, despite ethical and regulatory controversies; and ( ) access to participants and clinical sites is limited. for the ebola virus outbreak, some regulators from nonaffected countries required placebo-controlled design, whereas regulators in affected countries prohibited this on ethical grounds. over time, who and other key stakeholders developed a protocol with the manufacturer of the leading vaccine candidate, which could be tested without a placebo. , in an encouraging development, the who solidarity vaccine trial protocol for sars-cov- vaccines would create a multisite, randomized, adaptive trial, designed to accelerate the evaluation period. this important innovation could help speed up initiation of trials, enable comparisons between multiple sites and products, streamline data collection and processing, establish surrogate endpoints, and improve integration for regulatory submissions. finally, consideration is needed to ensure that nationallevel access and benefit-sharing laws do not delay timely access to virus strains and other genetic resources needed for rapid testing and vaccine development. the critical need for timely data sharing was demonstrated most recently in china with the sars-cov- strain. build partnership models that enable more cooperation and collaboration, end-to-end. existing partnership models such as the tb drug accelerator and the collaboration for aids vaccines discovery (cavd) demonstrate the potential value of establishing legally secure constructs that foster company collaboration, data-sharing, and sharing of know-how to tackle public health product development challenges. mechanisms through which companies can align with each other and with other partners on actionable information-sharing plans outside of outbreak and epidemic periods might help reduce redundancy and improve efficiency, thus enabling manufacturers to leverage all existing data. this collaborative approach carries both potential benefits for eid preparedness and some potential risks to the companies' core businesses. the approach also may require differentiated application of competition law, tailored specifically to the eid context. to encourage company participation, all partners involved must agree to, respect, and value the intellectual property that individual companies bring to the partnerships. ''germ'' games and other simulations like the event tabletop exercise are one promising way to continue this dialogue and find collaborative solutions. invest in rapid-response platforms and continue to evaluate and fund next-generation approaches. retooling existing manufacturing facilities or building new capacity to respond to eid demand for vaccines is a significant driver of cost and time. the bill and melinda gates foundation and other public and private organizations are investing through cepi and path and directly in novel manufacturing technologies and platforms that support rapid change over relatively small batches at comparatively low cost without disruption to other vaccines. as demonstrated by current efforts to rapidly develop sars-cov- vaccines, eid presents a potentially ideal application for these technologies, most of which are still at early stages. vaccine technology platforms that prove successful as eid rapidresponse platforms could also be used for more commercially viable disease targets. efforts to define regulatory expectations and pathways will be needed, in addition to buy-in from regulators. in the meantime, shared financial commitments from industry, governments, and ngos are needed to maximize the utility of existing production and stockpiling capacity. epidemic and pandemic preparedness is a high-risk business with uncertain returns. steps to reduce perceived and actual risk and ensure sustainability could increase company engagement. ensure proactive, predictable, and sustainable vaccine development and lifecycle funding. the business case for eid vaccine development can be improved by changing the way government and other funders finance these programs. funding must be proactive, early, and based on clear criteria for prioritization. the who research and development blueprint for eid is an important step forward for pathogen prioritization: prior work on sars and mers coronavirus vaccines (both listed in the blueprint) has proven beneficial in the race for a sars-cov- vaccine. however, the existence of many different (and long) lists illustrates the difficulty of defining and anticipating which pathogens need a vaccine first and for which population. , the launch of cepi in marked a watershed moment in the history of global pandemic preparedness. for the first time, there is a multisectoral, global organization dedicated to funding and managing eid early-phase vaccine development for prioritized pathogens drawn from the who blueprint. cepi has secured substantial investments from a global coalition of donors and is accessing innovative financing mechanisms to accelerate grant making. industry partners played an active role in the conceptualization and design of cepi, fully supportive of the vision for a stronger, more nimble, well-resourced publicprivate partnership. this vision is now materializing as cepi responds to sars-cov- with several vaccine programs in development. although cepi is currently resourced only to advance candidates to phase b, it has the ambition and potential to eventually support end-toend solutions, including manufacturing and scale-up. to fully realize this goal, however, it is important that the vaccine industry retain an equal seat at the table with other global stakeholders to set strategy and deliver on these promises. improve forecasting for demand and manufacturing needs. supply and demand forecasting are challenges for routinely used vaccines and are far more complex for eid vaccines because of the highly unpredictable demand, across unpredictable markets, over time. lessons from the h n influenza pandemic and ebola virus outbreaks highlight the importance of establishing priority pathogen lists and terms for business transactions between industry and vaccine purchasers before there is an emergency. for vaccine manufacturers, it is difficult to anticipate potential demand and align those forecasts with manufacturing plans. for h n influenza, this risk was compounded by hastily negotiated supply contracts, many of which went unfulfilled when the early waves of the pandemic waned. manufacturing capacity is planned during the development process, since the facilities-not just the vaccine itself-are a component of the licensure. any additional capacity that is needed must be built and approved by all countries receiving vaccine before it can be used. in , gavi, the vaccine alliance, announced an advanced purchase agreement to help secure a stockpile of an ebola virus vaccine candidate that had not yet been licensed. even though this procurement underestimated the volume needed for the drc epidemic, lessons learned from this construct could inform how large-scale, permanent solutions for other future eid vaccines might be designed. in response to sars-cov- , gavi is exploring the use of its innovative financing mechanisms to incentivize r&d and reduce manufacturing risk. create a global indemnification mechanism. liability exposure is a serious risk that can deter investment in pandemic vaccines. without protection from some types of broad liability claims, companies may not be willing to make these vaccines available without complete development data, as these claims could lead to a substantial confidence loss, business loss, and even bankruptcy. global legal mechanisms are needed to indemnify companies during emergencies when regulatory requirements are followed. who, through consultations facilitated by the world economic forum with cepi and harvard global health institute, recently developed an insurance-based indemnification model to facilitate emergency deployment of experimental vaccines. this is a promising step forward and should be expanded beyond the clinic, in collaboration with industry and other stakeholders. as inventors and producers of vaccines that prevent diseases, vaccine companies are essential partners in all efforts to prepare for and better respond to epidemics, pandemics, and emerging infectious diseases. this work is expensive, risky, and time and labor intensive, and it incurs substantial opportunity costs. a deeper understanding of the ongoing technical, policy, manufacturing, and field-level challenges is needed to inform policy, funding, and program decisions. critically, the solution must include support for manufacturing scale-up, new regulatory models, and strong end-to-end collaboration with all partners sharing risks and benefits. the licensure and roll-out of the first ebola virus vaccine is a testament to progress made over the past decade. while the trajectory of sars-cov- remains uncertain, the mobilization of governments, industry, and funders to develop vaccines and therapies demonstrates a common, global commitment to respond to pandemics. the global community has an opportunity to build on this momentum to design a sustainable model for eid vaccines. rolling recessions are the likely economic impact of new coronavirus and covid- innovation for pandemics the complexity and cost of vaccine manufacturing-an overview the complex journey of a vaccine: the steps behind developing a new vaccine. geneva: ifpma food and drug administration. first fda-approved vaccine for the prevention of ebola virus disease, marking a critical milestone in public health preparedness and response first-fda-approved-vaccineprevention-ebola-virus-disease-marking-critical-milestonepublic-health a research and development blueprint for action to prevent epidemics: funding and coordination models for preparedness and response. geneva: who gavi, the vaccine alliance. ebola vaccine purchasing commitment from gavi to prepare for future outbreaks department of health and human services. hhs funds development of two filovirus vaccines for biodefense innovative medicines initiative. ebola +: ebola and other filoviral haemorrhagic fevers novel vaccine technologies: essential components of an adequate response to emerging viral diseases platform technologies for modern vaccine manufacturing the economic and social burden of the ebola outbreak in west africa update: novel influenza a (h n ) virus infections-worldwide ebola outbreak in west africa epidemic curves pan american health organization; world health organization regional office for the americas. zika-epidemiological update unpublished ah n pdm case study first global estimates of h n pandemic mortality released by cdc-led collaboration efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola c xa suffit!) centers for disease control and prevention. - ebola outbreak in west africa. last reviewed march unpublished zika case study zika situation report: zika virus, microcephaly, guillain-barré syndrome zika vaccine development-current progress and challenges for the future risk in vaccine research and development quantified estimating the cost of vaccine development against epidemic infectious diseases: a cost minimisation study who will answer the call in the next outbreak? drug makers feel burned by string of vaccine pleas unpublished ebola case study improving vaccine trials in infectious disease emergencies implementation of an ebola virus disease vaccine clinical trial during the ebola epidemic in liberia: design, procedures, and challenges ebodac consortium organizes symposium on community engagement, communications and technology in ebola clinical trials- / world health organization. an international randomised trial of candidate vaccines against covid- regulatory policy for research and development of vaccines for public health emergencies infectious disease threats in the twenty-first century: strengthening the global response novel coronavirus -ncov exposes a flaw in the nagoya protocol cooperative development of antimicrobials: looking back to look ahead unprecedented pace and partnerships: the story of and lessons learned from one ebola vaccine program the pandemic pipeline vaccine platforms: state of the field and looming challenges national institute of allergy and infectious diseases. niaid emerging infectious diseases/pathogens. last reviewed coalition for epidemic preparedness innovations coalition for epidemic preparedness innovation turns to iffim to accelerate funding for new vaccine development response to covid- french move fuels fears for flu vaccine sales. reuters gavi board calls for bold engagement to respond to covid- the authors thank the following for their helpful com- key: cord- - f guyea authors: chandler, rebecca e. title: optimizing safety surveillance for covid- vaccines date: - - journal: nat rev immunol doi: . /s - - - sha: doc_id: cord_uid: f guyea evolution of the current infrastructure for surveillance of vaccine safety will be essential to meet our commitments to the public in the deployment of a vaccine (or vaccines) to covid- . the incorporation of concepts and tools within the fields of data science and systems immunology can be used to propel vaccine safety monitoring into the twenty-first century. vaccines have had a profound impact on public health, with their benefits enjoyed by populations around the globe. however, even after widespread vaccine acceptance and decades of use, including in countries with high vaccination rates, public concerns over the safety of vaccines have been documented . citing measles outbreaks, the world health organization (who) last year declared 'vaccine hesitancy' one of ten threats to global health . the deployment of a vaccine (or vaccines) to covid- will be ground-breaking, given the immediacy of its need in relation to the current development phase of the candidate vaccines. some initial work on coronavirus vaccines had been completed before the covid- pandemic, and challenges to the development of effective and safe vaccines against coronaviruses have been detailed in the literature . multiple vaccines to covid- are currently being investigated, some in traditional systems using known adjuvants and others using currently unlicensed technologies , with regulatory bodies promising to fast-track approval procedures . the imbalance between our knowledge of the safety of a vaccine candidate and the extent of potential postapproval use of that vaccine at the time of licensure will be very large indeed. public confidence in vaccination programmes is therefore at high risk if the systems for monitoring vaccine safety do not perform optimally. improved coordination and the incorporation of new methodologies and technologies into current vaccine safety systems will be crucial if we are to meet our obligations to safeguard from potential harms caused by vaccination. the covid- pandemic could be the catalyst that propels vaccine safety surveillance into the twenty-first century. infrastructure for safety surveillance guidance for the clinical evaluation of vaccines is provided by large public health and regulatory authorities such as the who . clinical development programmes are designed to explore the benefit of vaccines, with demonstration of efficacy being the primary objective of pivotal trials. assessment of safety is typically a secondary objective, and pivotal trials are not powered to support statistical analyses of end points of specific adverse events following immunization (aefis). the safety data collected within these trials are sufficient to characterize the more common adverse events -the local and systemic reactions related to the immunogenicity of the vaccine -that occur within a short time after vaccination. only after the vaccine is administered within large populations after licensure is it possible to detect any rare adverse events that were not observed in clinical trials. monitoring of vaccine safety after licensure relies upon a combination of passive and active surveillance. passive surveillance systems, which are the foundation of pharmacovigilance, are databases into which spontaneous reports of aefis are collected, such as the vaccine adverse event reporting system (vaers) in the united states and eudravigilance in the european union (eu). routine surveillance for safety signals is based upon a statistical pair-wise analysis that detects disproportionality between the number of observed reports and the number of expected reports of a single adverse event for a single vaccine (such as febrile seizure for pneumococcal vaccine), followed by clinical validation and assessment of the case series for that vaccine and that aefi. by contrast, active surveillance systems seek to ascertain all reports of pre-specified aefis from a representative sample, such as sentinel sites. an advantage of such systems is that the 'denominator' , or size of the population from which the aefi arose, is known. this is followed by comparative incidence analyses of aefis in subpopulations who have not received the vaccine (or a pre-vaccination time period for subjects experiencing an aefi after vaccination), using standardized case definitions and large, linked networks of health insurance claims or electronic health record data, such as vaccine safety datalink (vsd) in the united states and advance (accelerated development of vaccine benefit-risk collaboration in europe) in the eu. global variation in safety surveillance however, there is large variation in the capacity to carry out post-licensure vaccine safety surveillance between countries . the system described above is largely applicable only to high-income countries. within these countries, aefi reports are collected in the databases of national regulatory authorities (nras). these reports are mainly received from physicians and patients themselves, either directly or through vaccine manufacturers. within such databases, the detection of safety signals and causality assessment are approached in a similar manner for all medicinal products. furthermore, advanced diagnostics and electronic health-care data sources are available to carry out epidemiological studies for signal evaluation and/or active surveillance studies using standardized case definitions. by contrast, within many low-income and middleincome countries, vaccines are largely administered by national immunization centres, which are also responsible for collecting data on aefis. support to the national immunization programmes for safety surveillance has been provided by the global vaccine safety blueprint (gvsb) of the who . the tools and guidance provided through the gvsb prioritize the detection of aefis that are suggestive of programmatic errors, such as immunization errors or quality-related problems, and of well-characterized aefis that are known to occur rarely after certain vaccines, such as thrombocytopenia or intussusception. in these countries, nras capture a smaller number of aefi reports, such as those reported directly by health-care providers and/or reports transferred to them by vaccine manufacturers. only reports handled by nras are shared with vigibase, the global database of the who programme for international drug monitoring. furthermore, limi ted resources within the health-care systems of these countries inhibit the ability to routinely carry out surveillance beyond passive reporting into spontaneous databases, and the separation of aefi reports between national immunization centres and nras is suboptimal. to ensure the safety of covid- vaccines, global coope ration in a robust system for the timely detection and elucidation of any safety signals will be crucial. real-time global data exchange is essential as the pooling of reports of aefis into larger databases will allow for the earlier detection of safety signals. any gaps in communication between local, regional and international public health authorities handling aefi data must be filled, and any weaknesses in existing networks of health data must be strengthened. a roadmap for international collaborative epidemiological monitoring of vaccine safety in low-income and middle-income countries has been described and should be put into practice as a matter of urgency . preparation for ' aefi xʹ, the unexpected adverse event, requires the consideration of different approaches to signal detection. new vaccine technologies, such as those being explored for covid- vaccines, could cause aefis that are more complex and difficult to recognize. approaches such as syndromic surveillance could be considered; this was originally developed for the early detection of release of a biological weapon with an objective to identify illness clusters early, before diagnoses are confirmed, and to mobilize a rapid response. elucidation of the mechanisms by which vaccines might cause harm requires collaboration with immunologists. with the growing evidence of inter-individual variation in vaccine responses based on differences in innate immunity, microbiomes and immunogenetics, there is an emerging field within vaccinology, known as adversomics, that acknowledges that aefis might be indivi dually determined . if and when a vaccine safety concern is identified, a commitment by regulatory authorities to determine how and why an aefi, however rare, occurred is essential both to further our knowledge of the immune system and to ensure public trust in immunization programmes. state of vaccine confidence in the eu world health organization. ten threats to global health in covid- vaccine design: the janus face of immune enhancement the covid- vaccine development landscape covid- : how ema fast-tracks development support and approval of medicines and vaccines annex : guidelines on clinical evaluation of vaccines: regulatory expectations world health organization. global vaccine safety blueprint . background research world health organization. global vaccine safety blueprint roadmap for the international collaborative epidemiologic monitoring of safety and effectiveness of new high priority vaccines adversomics: the emerging field of vaccine adverse event immunogenetics the author declares no competing interests. key: cord- - ces rn authors: tondella, m. l.; carlone, g. m.; messonnier, n.; quinn, c. p.; meade, b. d.; burns, d. l.; cherry, j. d.; guiso, n.; hewlett, e. l.; edwards, k. m.; xing, d.; giammanco, a.; wirsing von könig, c. h.; han, l.; hueston, l.; robbins, j. b.; powell, m.; mink, c. m.; poolman, j. t.; hildreth, s. w.; lynn, f.; morris, a. title: international bordetella pertussis assay standardization and harmonization meeting report. centers for disease control and prevention, atlanta, georgia, united states, – july date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ces rn abstract an international meeting on bordetella pertussis assay standardization and harmonization was held at the centers for disease control and prevention (cdc), atlanta, ga, – july . the goal of the meeting was to harmonize the immunoassays used for pertussis diagnostics and vaccine evaluation, as agreed upon by academic and government researchers, regulatory authorities, vaccine manufacturers, and the world health organization (who). the primary objectives were ( ) to provide epidemiologic, laboratory, and statistical background for support of global harmonization; ( ) to overview the current status of global epidemiology, pathogenesis and immunology of pertussis; ( ) to develop a consensus opinion on existing gaps in understanding standardization of pertussis assays used for serodiagnosis and vaccine evaluation; and ( ) to search for a multicenter process for addressing these priority gaps. presentations and discussions by content experts addressed these objectives. a prioritized list of action items to improve standardization and harmonization of pertussis assays was identified during a group discussion at the end of the meeting. the major items included: ( ) to identify a group that will organize, prepare, maintain, and distribute proficiency panels and key reagents such as reference and control sera; ( ) to encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; ( ) to define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; ( ) to develop guidance on quality of other reagents, e.g., pertussis toxin and other antigens, and methods to demonstrate their suitability; ( ) to establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; ( ) to create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and ( ) to seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines. pertussis (whooping cough) is a highly contagious, acute respiratory illness caused by the bacterial pathogen bordetella pertussis. despite high vaccination coverage, pertussis continues to be a major cause of morbidity and mortality in the united states, with , and , probable or confirmed cases reported in and , respectively [ ] [ ] [ ] [ ] [ ] . among the reportable bacterial vaccine-preventable diseases in the united states with universal childhood vaccination, pertussis is the least well controlled [ ] . pertussis also remains a substantial public health problem in other developed regions, such as the european union and canada [ ] [ ] [ ] [ ] [ ] . studies suggest that there are approximately . mil-ଝ the findings and conclusions in this report are those of the author(s) of each section and do not necessarily represent the entire group of participants or the official position of the centers for disease control and prevention/agency for toxic substances and disease registry. the findings and conclusions in this report have not been formally disseminated by the u.s. food and drug administration and should not be construed to represent any agency determination or policy. lion annual cases of pertussis worldwide, with , deaths [ ] [ ] [ ] . however, the total burden of pertussis is likely underestimated, especially among adolescents and adults, in whom typical pertussis symptoms are often absent making diagnosis difficult [ , , , [ ] [ ] [ ] [ ] . the laboratory diagnosis of pertussis is challenging [ , ] . culture is highly specific but requires a long incubation and sensitivity can be low [ , ] . rapid, sensitive, and specific polymerase chain reaction (pcr) assays have been designed to detect b. pertussis [ , [ ] [ ] [ ] ; however, these assays are not well standardized and problems have occurred with specificity, mainly during outbreaks [ , [ ] [ ] [ ] . serologic testing with b. pertussis antigens, pertussis toxin (pt) in particular, can be sensitive and specific, but the tests are not widely available, have not been fully standardized and no universally accepted serologic correlates for protection are available [ ] [ ] [ ] [ ] . the meeting sessions covered the following topics: global epidemiology of b. pertussis disease; pathogenesis and immunology of b. pertussis; assay standardization and harmonization; serodiagnosis of b. pertussis; current and future b. pertussis vaccine composition; and issues in vaccine evaluation with perspectives from regulatory authorities and vaccine manufacturers. group discussions included serodiagnostics, vaccine evaluation, correlates of protection, and harmonization of assays. a list of action items was developed to identify existing knowledge gaps and to coordinate international efforts to standardize pertussis assays for immunologic and diagnostic use. the meeting was opened by m. lucia tondella, phd (cdc), followed by nancy messonnier, md (cdc), who stressed the importance of having laboratory, epidemiologic and statistical groups working together to facilitate and support global harmonization. dr. messonnier also highlighted the necessity of improving pertussis diagnostics, pointing out that in alone, cdc experienced numerous occasions when problems with diagnostics impeded epidemiologic investigations, resulting in a substantial public health outcry [ ] . cdc's meningitis and vaccine preventable diseases branch has an extensive experience working on standardization and harmonization of assays on a variety of organisms, and this experience can be brought to bear at this meeting to determine the current status of pertussis serologic technologies in key laboratories, to facilitate discussion of the key issues in pertussis serology and to establish consensus on the action plan needed to address these issues. presentations and discussions were summarized below. presenters: james d. cherry, md (david geffen school of medicine, university of california, los angeles) and nicole guiso, phd (institute pasteur, paris, france) in the prevaccine era in the united states, pertussis was a disease with cyclic peaks averaging every . years. the average rate of reported cases was per , population. more than % of reported cases occurred in children younger than years of age. by the s, pertussis was well controlled by immunization, and only - cases were reported per year; % of those cases occurred in infants. at present, more than % of pertussis reported cases are in persons older than years of age. since there has been a modest increase in reported pertussis from < to cases per , population. in recent years ( ) ( ) the reports have increased considerably; however, the number of cases is still approximately -fold less than in the prevaccine era. the cycles of reported pertussis today are essentially the same as they were in the prevaccine era [ , ] . examination of reported pertussis in several countries between and the present found two different patterns. in some countries (argentina, bangladesh, bolivia, brazil, cambodia, ghana, pakistan, peru, sudan, vietnam), there was a reduction in total cases, similar to that seen in the united states between and . a similar pattern was also noted in the united kingdom, where pertussis was reasonably well controlled by vaccination in the s and early s, but then concerns for vaccine safety resulted in a marked decline in immunization and pertussis became epidemic. it was then brought under control in the early s by increased vaccine usage. in italy, where vaccination was not being consistently offered until the efficacy trials of , a marked reduction in pertussis was related to the use of diphtheria and tetanus toxoids and acellular pertussis component (dtap) vaccines. success in the control of reported pertussis in all of the above countries with the exception of italy was secondary to use of doses of dtwp (whole-cell vaccines) in the first year of life. in france, because of high vaccination coverage among children with dtwp vaccine during the last years, pertussis predominantly affects children who are too young to be vaccinated and adults who are no longer protected by vaccine or disease-induced immunity [ ] [ ] [ ] [ ] [ ] [ ] . a survey undertaken in and in a pediatric hospital network demonstrated an increase in the number of children hospitalized with pertussis suggesting the importance of introducing booster dose(s) to prolong vaccine immunity and reduce the exposure to b. pertussis of infants too young to be immunized [ ] . therefore, a fifth dose of acellular pertussis (ap) vaccine was introduced for - -year olds in , followed by the introduction of the so-called "cocoon strategy" in . the current vaccine schedule consists of primary immunization for children aged , and months and boosters at - months, at - years old, and for young adults and healthcare workers in contact with infants [ ] . no dtwp vaccine has been commercially available since . the national hospital-based surveillance (renacoq network) has not shown any resurgence of pertussis. as observed in other countries, cycles of the disease have occurred every - years; however, the number of pertussis cases in did not reach the values observed in the earlier peak years which could be linked to the impact of the - -year booster introduced years previously [ , ] . reported pertussis cases in australia, chile, israel, japan, norway, and poland have increased in recent years. in australia, israel, and norway, the increase followed the introduction of dtap vaccines. possible reasons for the resurgence of reported pertussis are ( ) greater awareness of pertussis; ( ) waning of vaccine-induced immunity; ( ) lessened potency of pertussis vaccines; ( ) genetic changes in b. pertussis; and ( ) the general availability of better laboratory tests for the diagnosis of pertussis. of most importance is a general greater awareness of pertussis. the availability of better laboratory tests (pcr) and single-serum serology for diagnosing adolescent and adult pertussis has also contributed to the increase in reported cases. as evidence also suggests that in general dtap vaccines may have less efficacy in children than the more potent dtwp vaccines [ , , ] , it is plausible that decreased efficacy or duration of protection could possibly contribute to the outbreaks that have been seen in pre-adolescence and adolescence. investigators in the united states and other areas of the world have used various laboratory methods to diagnose pertussis in subjects with prolonged cough illnesses. one of the most commonly used diagnostic markers is high single-serum titers to pt. results of these studies suggest that about % of prolonged cough illnesses are due to b. pertussis infection. however, because not all adults have a pt antibody response following infection, the percentage is likely to be higher [ , ] . in summary, b. pertussis infections in adolescents and adults are very common. rates of reported pertussis may be as much as to -fold lower than actual illness rates, and unrecognized infections (e.g., asymptomatic or with nonspecific symptoms) are likely to be more common than symptomatic infections. today, symptomatic adolescents and adults appear to be the major sources of infection for nonvaccinated children. much of the discussion relating to epidemiology focused on the increase of pertussis in recent years despite adequate or increasing vaccine use in young children. in general, the most likely cause was thought to be greater awareness of pertussis. however, data from australia (presented later in the meeting) suggested that falsepositive laboratory tests contributed to the increase in reported pertussis in at least one setting. discussion included the possibility that in some instances genetic change in the pertussis organism may have contributed to increases. in countries using dtwp, changes in vaccine manufacturers and even lots may have a significant relationship to efficacy. the increase in observed pertussis associated with the introduction of acellular vaccines should be studied more carefully. presenter: erik l. hewlett, md (university of virginia school of medicine, charlottesville, virginia) multiple virulence factors produced by b. pertussis are able to disrupt the normal physiology of the host, causing the disease of pertussis [ ] . this disease consists of a localized infection of the respiratory tract, with secondary systemic effects resulting from the actions of several bacterial products and from the host response to those factors. the molecular basis for the characteristic paroxysmal cough is not known, but is not mimicked by intravenous pt [ ] . one likely role of pt is to impair the innate immune response of the host, causing leukolymphocytosis and blocking the migration of murine macrophages [ ] . reduced expression of l-selectin on the surface of leukocytes from infants infected with b. pertussis has also been demonstrated [ , ] . the leukolymphocytosis and several other effects of pt are mediated by the adp-ribosyl transferase activity of the catalytic domain. the pt binding to its receptor elicits a separate set of responses, known as b-subunit effects. recently it has been shown that pt b-subunit activates signaling through the t-cell receptor, without any contribution from adp-ribosylation [ ] . similarly, pt and an enzymatically inactive (non-adp-ribosylating) mutant stimulate cytokine production and dendritic cell maturation [ ] . evidence indicates that multiple virulence factors from b. pertussis have immunomodulatory effects. in several instances the effects appear to be antagonistic to one another. for example, it has been shown that filamentous hemagglutinin (fha) is both pro-inflammatory and able to induce apoptosis in macrophages [ ] . similarly, adenylate cyclase toxin (act), which is known for its inhibitory effects and cytotoxicity on neutrophils and other phagocytic cells [ ] [ ] [ ] also has pro-inflammatory effects, inducing il- from respiratory epithelial cells [ ] and cyclooxygenase- (cox- ) in cells expressing the integrin, cd b/cd [ ] . the disease process of pertussis appears to be multifactorial, rather than the function of a single toxin/virulence factor. therefore, the ideal immunologic response from the host consists of antibodies directed against multiple virulence factors, resulting in inhibition of bacterial adherence, neutralization of the actions of the toxins and clearance of the infecting organisms. in addition, a cell-mediated component in the host response to immunization and infection has been studied, but the relevant target antigen(s) to which this cellular response is directed and the contribution that it makes to bacterial clearance are unknown [ ] [ ] [ ] . several major issues in pertussis pathogenesis remain unresolved. despite the numerous known biological effects of individual virulence factors, especially in vitro, the in vivo target tissue(s) of these molecules and the mechanism by which the characteristic cough is generated are mysteries. the molecular details of how bvg, the two-component regulatory system, controls production of the virulence factors are not known. in summary, pertussis is a multicomponent disease, which requires more than a simple approach to its recognition, treatment and control. recent analyses of the effects of known toxins and other virulence factors on the immune system have revealed that they are often multifunctional: cytotoxic, stimulatory, inhibitory, pro-and anti-inflammatory. much of the discussion focused on the potential neurophysiologic effects of the multiple virulence factors; animal modeling, including new methods of infection through inhalation techniques; and novel imaging analysis that could help in understanding the pathogenesis of pertussis in humans. recent research studies have shown presence of b. pertussis dna from clinical specimens of children several months after infection. the possibility that the remaining dna could be triggering the cough by an unrecognized mechanism was speculated. presenter: kathryn m. edwards, md (vanderbilt medical center, nashville, tennessee) standardization and harmonization are necessary for several reasons: to assess the immunogenicity of new pertussis vaccines or new combination vaccines that include pertussis antigens, to compare the serologic responses to these new vaccines with those evaluated in earlier vaccine efficacy studies for the purposes of vaccine licensure, to establish serologic criteria for the clinical diagnosis of pertussis disease, and to conduct seroepidemiologic studies to assess the circulation patterns of pertussis in the community. multiple ap vaccines were licensed for use in infants, adolescents and adults [ , , ] . combination vaccines incorporating various antigens into a single injection have been designed. these combination vaccines may be associated with reduced serologic responses to one or more vaccine antigens. therefore, antibody titers induced by these products must be compared with those seen after the separate administration of the individual vaccines before combination vaccines can be considered for licensure. safety and immunogenicity of ap and two dtwp vaccines have been assessed in a single large clinical trial [ ] . although the trial evaluated different pertussis antigens administered in various concentrations, all the vaccines were found to be safe and immunogenic. several of these vaccines were then evaluated in efficacy trials in the european union and africa and were found to be efficacious in preventing culture-confirmed pertussis. since placebo-controlled efficacy trials would now be considered unethical, new vaccines must be evaluated by comparisons with the immunogenicity data of the vaccines evaluated in earlier efficacy trials. thus, the need for standardized assays is critical. standardized serologic assays are also needed to diagnose pertussis disease, particularly in adolescents and adults. a study was conducted to determine population-based antibody levels to three pertussis antigens, pt, fha, and fimbrial proteins (fim), for the purpose of establishing diagnostic cutoff points in adolescents and adults in the united states [ ] . enzyme-linked immunosorbent assays (elisas) were performed on sera from more than us residents aged - years. quantifiable (> elisa units [eu]/ml) anti-fha and anti-fim igg antibodies were common but quantifiable anti-pt igg antibodies were less frequent. an anti-pt igg level of ≥ eu was proposed as the diagnostic cutoff point in subjects years of age and older. application of this cutoff point to culture-confirmed illness yielded a high diagnostic sensitivity ( %) and specificity ( %). anti-pt igg assays with a single serum sample appeared to be useful for identification of recent b. pertussis infection in adolescents and adults. however, these assays must be standardized to remain useful in the diagnosis of pertussis. finally, standardized serologic methods for b. pertussis are needed to assess epidemiologic trends in pertussis disease in the population. this is particularly relevant with the universal recommendations for adolescent and adult pertussis vaccine in many developed countries. how universal vaccination will affect disease burden in the various age groups remains to be determined. in addition, comparisons of seroepidemiologic results from one country to another are dependent on standardized assays. presenters: bruce d. meade, phd (meade biologics llc, hillsborough, nc), dorothy xing, phd (national institute for biological standards and control (nibsc), potters bar, uk) and anna giammanco, phd (department of hygiene and microbiology, university of palermo, italy) important insight on strategies for international standardization of pertussis immunoassays is provided by the results of an international collaborative study performed in [ ] . in that study, participating laboratories were asked to quantify specific antibody to pt, fha, pertactin (prn), and fim in a panel of blinded samples by using those elisas routinely performed in that laboratory. the laboratories employed a variety of procedures, antigens, conjugates and other reagents, and calculation methods. results indicated an overall consistency among the laboratories. a reasonable quantitative agreement was achieved among the laboratories with assays calibrated using the us reference pertussis antisera. this agreement demonstrated that use of a common reference serum had been successful at harmonizing results. to promote a higher level of international standardization of pertussis immunoassays, the highest priority recommendations would be to improve the availability of reference reagents and methods, including international reference sera, a proficiency panel of well-characterized sera, and well-characterized antigens. in recent years, significant progress in the preparation and standardization of international reference sera has been made. the who collaborative study, "evaluation of proposed international reference preparations for pertussis antiserum (human),"was a multicenter collaboration between nibsc, uk; the center for biologics evaluation and research (cber) at the food and drug administration (fda), us; the institut für infektiologie (ifi), germany; and a number of international participants. reference sera from the united states (us reference pertussis antiserum [human] lot and lot for igg-antibodies and lot for iga-antibodies) were prepared by fda, cber more than years ago, and since then have been commonly used as reference standards for pertussis serologic assays [ ] [ ] [ ] [ ] [ ] [ ] . currently, only limited quantities of these sera remain. with an increasing number of vaccine and diagnostic studies, more widely available international standard preparation(s) are needed. the who ad hoc pertussis working group recommended the preparation and standardization of an international human reference antiserum to pertussis antigens to replace the current widely used cber preparations before the supply is exhausted [ ] . it was agreed that as far as possible, continuity with the existing cber reference preparations should be maintained. these new international reference materials were intended for the following uses: ( ) vaccine studies of products in current distribution, as well as those under development; ( ) studies of serologic responses to infection; ( ) epidemiologic surveillance; ( ) future assessment of antibodies to other antigens apart from pt, fha and prn; and ( ) evaluation of new generation vaccines. serum pools were kindly donated by dr. wirsing von könig, ifi, krefeld, germany. they were prepared after recalcification of plasma samples selected from a blood bank during a pertussis outbreak in germany. these sera were then lyophilized at nibsc, resulting in four batches of freeze-dried preparations of ampoules of higher titer and , ampoules of lower titer igganti-pt. preliminary assessment of the freeze-dried materials in nibsc showed that in comparison with the materials before freezedrying, all freeze-dried material maintained their original elisa binding activities. furthermore, comparison of the candidate materials with the us reference sera showed that the dose-response lines did not deviate significantly from parallelism. an international collaborative study to evaluate the candidate preparations was initiated in march . the aims of the study were to ( ) characterize candidate international reference preparations for pertussis antiserum (human); ( ) compare candidate references with the us reference preparations, lots , , and ; ( ) compare candidate reference preparations with other available reference preparations, e.g., in-house preparations; and ( ) define unitage for the candidate preparations for anti-pt, anti-fha and anti-prn, maintaining continuity with widely used reference preparations. samples provided to the participants were the four candidate reference preparations: the three us reference sera plus a negative control serum. all participants were quested to ( ) carry out three independent assays for igg anti-pt, fha and prn by elisas; ( ) use their own methodology, reagents and calculation methods; and ( ) include their in-house references and controls. participants were also encouraged to perform other assays that could be of interest, e.g., igg anti-fim / , iga antibodies to the antigens, ptneutralizing antibodies (cho-cell assay). a total of laboratories worldwide participated in this study, including from europe, from north america, from south america and laboratory from each of asia and australia. all participants were requested to submit their raw results to nibsc, where the statistical analysis will be performed. a study report will be submitted to the expert committee on biological standardization (ecbs) of who. another multilaboratory effort to standardize pertussis serologic assays was the european sero-epidemiology network (esen) project, coordinated by the health protection agency (hpa), communicable disease surveillance center (cdsc), london [ , ] . the aim of the esen project was to coordinate and harmonize serologic surveillance for a variety of infections in europe. age-stratified serum banks of a recommended sample size of individuals were collected by each participant country and one laboratory was selected to serve as a reference laboratory for each disease. all laboratories tested sera from their own country plus a shared panel of approximately serum samples [ ] . the standardization process allowed a comparison of national serosurveys for pertussis [ ] . because of the difficulty with standardization of cell extracts, the esen project emphasized purified antigens, focusing on igg anti-pt assays because of the higher sensitivity and specificity for serodiagnosis [ ] . since most individuals had been exposed to pertussis vaccine or infection, the study focused on an estimation of the incidence of recent infections, as defined by the presence of igg anti-pt antibodies above defined thresholds [ ] . to accomplish this, quantitative assays were required and standardization efforts were based on analytical performance in the assay range near the diagnostic thresholds. in conclusion, the esen project demonstrated that standardization of pertussis igg anti-pt assay was possible [ , ] . appropriate application of a diagnostic laboratory test requires an understanding of the test as well as knowledge of the patient. relevant criteria for the test include test format; specificity and sensitivity; and reference systems. clinical interpretation of a test result concerning b. pertussis infection also requires an understanding of ( ) the distinction between primary and secondary infection; ( ) the differential diagnoses, including other agents; ( ) the kinetics of immune responses to infection; and ( ) an understanding of the information provided by the test beyond that provided by the clinical history alone. the performance of laboratory tests can be displayed by cutoff values as shown in a reporter-operator characteristics (rocs) curve, as well as by positive and negative predictive values. the differential diagnosis for b. pertussis as an agent of prolonged cough includes infectious agents such as adenovirus, respiratory syncytial virus, rhinovirus, human parainfluenzavirus, influenzavirus a and b, mycoplasma pneumoniae, human metapneumovirus, and human coronavirus. several alternate approaches have been used for serodiagnosis. when paired sera were used, one study defined a case if there was % or more increase in antibody concentration or % or more decrease in antibody concentration. for single-sample serology, various cutoff-values have been proposed, such as >∼ eu/ml igg-anti-pt in the netherlands; and > eu/ml igg-anti-pt or igg-anti-pt > eu/ml and iga-anti-fha > in germany. in most diagnostic laboratories, a so-called "grey-zone" is defined for sera that are positive and above a specified threshold but between the diagnostic cutoffs. this "grey-zone" is set in the netherlands and sweden to eu/ml of igg-anti-pt. the sensitivity and specificity of single-sample serologic assays have been determined. one study was done in a population of children - years of age, mostly nonvaccinated (germany), using a cut-off of ∼ eu/ml ( th percentile of population). with a specificity of %, a sensitivity of % was found, which increased to % when iga-anti-fha was added [ ] . another study was done without age limit in a mostly vaccinated population (netherlands) using a cutoff of ∼ eu/ml ( th percentile of population). the study found a sensitivity of . %, whereas a cutoff ∼ eu/ml (∼ th percentile of population) had a sensitivity of . % [ ] . a recent german study evaluated persons in hospital departments, pediatrician's offices, and child-care facilities, who received one injection of ap vaccines. after informed consent, blood was sampled before vaccination, and month, year, years, years and years after vaccination. igg-and iga-antibodies to pt, fha and igg-anti-prn were measured. in this population, when the distribution of igg anti-pt levels prior to immunization was examined, the th percentile was eu/ml, the th percentile was eu/ml, and the th percentile was eu/ml. applying a cutoff of eu/ml to a seroepidemiologic study in a population of persons aged to > years in various countries in the european union resulted in the following percentage of the cohort showing values above the cutoff: netherlands ( . %), finland ( . %), germany (former gdr) ( . %), france ( . %), germany (former frg) ( . %), uk ( . %), and italy ( . %) [ ] . seroepidemiologic studies can also demonstrate the cyclic nature of b. pertussis infections. in germany, a population of - -year-old blood donors was screened in different years. the percentage of this cohort with igganti-pt ≥ eu/ml was as follows for a diagnostic laboratory, it is important to define how the customer will use the serologic information, specifically if it is used for clinical decisions and patient management or if it is used for epidemiologic studies. similarly, clarity is needed regarding whether the definition should include all infections, symptomatic infections, infections with medical resource use, or severe infections. diagnostic serology is also strongly influenced by the time when patients seek medical attention relative to onset of symptoms. in german studies performed among persons with pcr-positive cases, the median number of days of coughing before seeking medical attention was found to be . days for schoolchildren, aged - years, . days for adolescents, aged - years, and . days for adults, aged - years. in france, serologic testing is performed for epidemiologic studies and surveillance. serology consists of measurement of igg anti-pt antibody titers using the reference elisa and purified pt provided by the manufacturers [ ] . a positive case is defined by either a twofold change in the titers between two serum samples obtained at a -month interval or a titer > eu/ml in a single serum sample, only if the serum is collected after more than weeks of cough and years after a vaccine booster. in some french laboratories, an "in house" western blot assay using purified pt, as well as various commercial tests have been used for routine diagnoses. these assays are not validated and have also been responsible for false-negative or false-positive results. for this reason, microbiologists have preferred real-time pcr for routine diagnoses. serology based on measurement of anti-pt titers may be less useful in the future, since children, adolescents and now adults are vaccinated with ap vaccines containing pt. the proportion of laboratory tests performed to diagnose pertussis in france has changed over the years, according to the renacoq database [ ] . in , % of pertussis cases were diagnosed by culture, % by pcr and % by serology, whereas in ; % of cases were diagnosed by culture, % by pcr and only % by serology. in the united states, the massachusetts state laboratory institute has been using a single-serum anti-pt igg eia on patient samples since . other versions of the current assay have been used in the past, including measurements of iga and igm to pt, igg to fha, and anti-pt igg in paired sera. ultimately, all these other assays were discontinued when data indicated that they did not contribute much additional information beyond that obtained from the single anti-pt igg alone. plates are coated with commercially prepared pt. standards are derived from pools of positive patient samples and are calibrated against the us reference lot . a concentration (g/ml) is assigned, calculated from a five-point standard dilution curve. the cutoff point for positivity was established on adults, including blood donors and healthy volunteers with anti-pt igg levels ranging from . to g/ml [ ] . a cutoff point was chosen based on the % upper tolerance limit, such that there is % confidence that % of the population falls below the cutoff. the cutoff point was set at g/ml, which is equivalent to cber units per ml. this is actually quite high relative to what other groups have chosen [ , ] . clinicians are asked to order a serologic assay only for patients who are older than years of age and only when coughing has been present for more than days. specimen volumes have been increasing steadily over the last years, so that in , more than specimens were tested. seropositivity has been fairly constant at approximately % of the submitted samples. many of the challenges associated with the assay arise from manipulations needed to produce a quantitative test result. it may make sense, at least in massachusetts, to consider some modification of the assay to make it more qualitative, simple, and robust, and able to be performed consistently and accurately with less effort than is currently expended. at least two knowledge gaps prevent wider application of the assay. first, the impact on assay results of adolescents and adults having recently received tetanus toxoid, reduced diphtheria toxoid and acellular pertussis (tdap) vaccination is not fully understood. the massachusetts immunizations program contacts every seropositive patient and asks about recent vaccination history. in the last years, since tdap has been available, only two seropositive patients with recent tdap vaccination have been identified. one person was vaccinated the day before blood was drawn for serology, so the antibodies detected were unlikely to have been due to vaccine. in massachusetts, tdap-associated igg may be less of a problem than was originally anticipated. possibly, post-tdap immunization igg levels do not frequently exceed the relatively high assay cutoff value. alternatively, clinicians may not be submitting samples from recent vaccine recipients, recognizing that those individuals are likely to be protected from disease. a second knowledge gap is the uncertain applicability of igg eia results to children younger than years of age. results are considered uninterpretable for that age group, because of the possible presence of vaccine-associated antibody. massachusetts state laboratory institute data on seropositivity by age demonstrate a peak in seropositivity among adolescents and young adults, and also a peak at - years of age. admittedly, no clinical information is available for the - -year-old patients because they are not considered to be pertussis case-patients and are not investigated. in addition, their vaccination status is unknown. nevertheless, the peak in seropositivity that occurs at this age may correspond to the fifth dose of dtap. interestingly, the peak is not very wide, spanning only - years, so the application of the assay could likely be extended down to some ages less than years. australia has a -year experience of using commercial serologic kits for the routine diagnosis of pertussis. iga has been used rather than igg kits because iga is believed to be short-lived ( - weeks) in comparison to igg ( - weeks) , and, in children at least, iga is not produced in response to the vaccine. unusually large numbers of pertussis iga-positive tests were reported to public health authorities during . at the time, only two iga commercial serologic tests were used, elisa kit ( % of laboratories) and elisa kit ( % of laboratories). this prompted a widespread evaluation, comparing various commercial available elisa kits with complement fixation (cf), iga immunofluoresence (if) and iga western blot (wb) kits. ninety healthy adults with no clinical history of respiratory illness in the preceding weeks and negative by cf, if and wb formed the negative group for the evaluation. the positive group consisted of children and adults with a clinical history of pertussis of less than weeks' duration and positive by cf, if and wb. the respective antigens, sensitivity and specificity of each commercial iga elisa kit were as follows: ( ) in , cidms laboratory tested serum samples for pertussis iga using the elisa kit . following the evaluation of various commercial tests, over % of these samples were retested and % were found to be false-positives. the false-positive specimens reacted with the fha antigen band only, suggesting that pt would be a better choice for an elisa antigen. however, the only kit that used pt as an antigen performed poorly, which emphasizes the need for better standardization. in addition, a group of negative samples was tested by the esen igg elisa [ ] using a eu/ml cutoff; samples had igg levels > units, which would have been interpreted as evidence of recent infection when clearly these individuals were not symptomatic. this highlights the need for caution in determining a cutoff for recent infection in adults, particularly in highly vaccinated populations. in general, diagnostic laboratories prefer commercially available assays. two studies so far have evaluated selected commercially available elisas for b. pertussis serology: kösters et al. used paired sera from patients (children), sera from vaccinees, interlaboratory comparison samples, and reference preparations [ ] . schellekens et al. used paired sera from pcr-positive patients (median age years) and control patients with respiratory symptoms (median age years) [ ] . another study with various currently existing serologic tests is ongoing and results are expected to be available in early . in summary, b. pertussis serodiagnosis still suffers from many unsolved problems, such as that diagnostic testing is performed in immunologically non-naive populations with antigens that are components of ap vaccines. generally an anamnestic immune response develops more rapidly than symptoms, and immune responses to vaccine antigens cannot be distinguished from response to infection. problems also exist with serodiagnosis of b. parapertussis infections. serologic interpretation requires knowledge of pertussis immunization, information not typically known by the testing laboratory. reliable, standardized elisa systems are not commercially available at this time. additionally, the clinical course appears to differ between primary and non-primary infections. for most subjects, clinical case definitions for pertussis remain nonspecific. there are currently no suitable serologic tests for recently vaccinated patients, since tests based on nonvaccine antigens have not been satisfactory for diagnosis. finally, when using single-sample serology, population-based cut-offs will need re-verification after any change made in a vaccination schedule. discussion focused on the use of antigens not present in vaccines for serologic diagnosis of pertussis. cherry et al. [ ] showed that patients with vaccine failure responded poorly to the act antigen, suggesting an induced tolerance due to the phenomenon called "original antigenic sin." in this phenomenon, vaccinated patients respond to antigens that they have been primed with and do not respond to new antigens (e.g., act) associated with infection. therefore, it was suggested that nonvaccine antigens may not be as good as pt for serologic diagnosis of pertussis in vaccinated populations. other points of discussion included the potential use of reverse vaccinology approach to identify new antigen candidates to be used in serologic diagnostics and lack of specificity of other antigens other than pt, such as fha and act. the lack of sensitivity and specificity of commercial tests currently available worldwide was broadly discussed. it was pointed out that one commercial igg and iga anti-pt test available in the united states has been standardized to provide adequate sensitivity and specificity for the diagnosis of pertussis in persons older than years of age. presenter: james d. cherry in studies of whole-cell pertussis vaccines, agglutinin titers of : or greater were found to be protective against pertussis when children were exposed within the household [ ] . agglutinating antibodies were thought to be primarily directed against the agglutinogens fim- / , prn and lipopolysaccharide. only two of the nine ap vaccines efficacy trials conducted during the s and s were done in such a way that data relating to serologic correlates could be developed. these trials were centered in erlangen, germany, and stockholm, sweden [ , , , ] . to determine serologic correlates, antibody titers to specific antigens were determined at the time of exposure. both studies indicated that prn was most important in protection and that antibody to fim was next most important. also noted in both studies was an apparent antagonism between antibody to pt and fim. in clinical trials in which children with both mild and severe disease were included in the analyses, efficacy jumped considerably for vaccines that contained prn as well as pt and fha when compared with pt and pt/fha vaccines. in the erlangen trial, the imputed titers at the time of exposure in dtp and dtap vaccine failures were examined. in the majority of the failures, subjects had high levels of antibody to prn, which is difficult to explain in relation to the overall data on serologic correlates. this indicates that there is much yet to be learned about serologic correlates. of interest is the fact that when a dtwp vaccine (evans vaccine) was compared with a two-component dtap vaccine, a three-component dtap vaccine and a five-component dtap vaccine, the greatest efficacy was noted with the dtwp vaccine. this vaccine elicited minimal antibody responses to pt and fha ( and eu/ml, respectively) and high values to prn and fim ( and eu/ml, respectively). discussion centered on diagnosing pertussis serologically using antigens contained in the vaccines and antigens not contained in the vaccines. although still controversial, if acute-phase serum is collected early, titer increases against both vaccine antigens and nonvaccine antigens are useful for diagnosis. further discussion related to cell-mediated responses associated with infection, use of animal models in studies of infection and consideration of additional antigens that might be included in vaccines. presenter: john b. robbins, md (national institutes of health, bethesda, maryland, us) dr. robbins presented evidence supporting the hypothesis, based on the classic article by pittman [ ] , that an inactivated and immunogenic pertussis toxoid (ptox) is both essential and sufficient for a pertussis vaccine [ ] . furthermore, he discussed data suggesting that multicomponent pertussis vaccines include nonprotective antigens [ , ] and may be more difficult to standardize [ ] . us pertussis cases reported to cdc have increased despite a high immunization rate among infants and children [ ] [ ] [ ] , ] . booster doses, starting in adolescence and offered every years, will maintain a high level of immunity in the population, and dr. robbins recommended that a genetically inactivated toxin should replace the chemically inactivated toxoids for mass immunization of infants, adolescents and adults [ , ] . with respect to the goals of the meeting, the importance of assays to measure serum pt igg was emphasized because pt igg is the only reliable assay for serologic diagnosis of pertussis after the acute phase of pertussis has passed [ , ] , and because the concentration of vaccine-induced igg anti-pt correlated with the efficacy of monocomponent ptox [ ] . the correlation indicates that the level of igg anti-pt may predict the efficacy of ap vaccines [ ] . a number of participants raised questions related to the hypothesis that ptox alone is sufficient for a pertussis vaccine. it was mentioned that the study supporting the hypotheses did not include serology. other studies, including serologic ones, showed evidence that addition of fha in the vaccine provides some protection against infection. data (not included in this report) based on extensive investigations in sweden and presented at the meeting in a supplemental presentation by rose-marie carlsson, md, swedish institute for infectious disease and control, contradicted many of the conclusions presented in this session. some participants also questioned the appropriateness of the analogy between diphtheria and ptoxs. the presentation on pathogenesis and immunity as well as the presentation on correlates of protection at this meeting are also at variance with the hypothesis that ptox alone is sufficient for a pertussis vaccine. the discussion was cut short to allow adequate time for consideration of issues related to assay standardization, the main focus of the meeting. some conclusions previously reached by a who-convened working group on the clinical evaluation of new ap vaccines served as an introduction to discussion. one such conclusion was that additional placebo-controlled protective efficacy studies were not feasible since withholding pertussis vaccine was no longer a possibility. relative efficacy studies of adequate size and statistical power also seemed unlikely. the approval of new ap vaccines would have to rely on comparative immunogenicity data. all the ap-containing vaccines that have been licensed thus far in the united states and european union incorporate the same pertussis antigens made by manufacturer-specific processes as were included in at least one of the successful vaccine efficacy trials in infants. therefore, immunogenicity data have been used to link the demonstration of efficacy for the tested ap vaccine to the new ap vaccine even when the total antigen compositions of the two vaccines differ. comparisons of immune responses between the new and reference ap-containing vaccines raise several difficult questions. for example, with no identified immunologic correlates of protection against pertussis, it is not known which antigen(s) elicits immune responses that are important for prevention of clinically apparent infection. it is also not known whether the primary comparison should be in terms of percentages with postvaccination titers above the assay cutoff, on seroconversion rates (which are open to various definitions) or to geometric mean antibody titers. whichever immunologic parameter is chosen for the primary comparison, it is difficult to decide what might constitute a clinically important difference. in addition, the predefined criteria for noninferiority might be met for one or more, but not all immunologic parameters, with unknown implications for protection. because of these uncertainties, the overall judgment of the likely protective efficacy of an ap-containing vaccine should take into account all aspects of immune responses. thus the immunogenicity data should be described in terms of percentages of subjects reaching assay cutoffs, percentages achieving fourfold increments in antibody concentrations (or a similar definition of seroconversion), geometric mean antibody titers and reverse cumulative distributions. the uncertainty regarding the predictive value of these immunologic comparisons supports the importance of postlicensure surveillance programs to assess the effectiveness of ap vaccines against pertussis disease. however, it is very unlikely that vaccinespecific estimates of effectiveness could be generated, since this would require data from a region or country in which the vaccines used all contain the same ap antigens from the same manufacturer. because of the complexities of such programs and the infrastructure needed to generate reliable data on disease, such data are most likely to come from surveillance conducted by public health agencies. in the future, new ap-containing vaccines might include the same range of ap antigens previously shown to be efficacious, but some or all of these may be made by a different manufacturer and/or by a different process. new ap-containing vaccines could also have a different composition of pertussis antigens (in type and/or amount) compared with vaccines that were previously evaluated for protective efficacy. there is no agreed regulatory position on the minimal data that would be required to support approval of these products; however, practical limitations point to the need to consider how immunogenicity data could be best used to provide reassurance regarding likely efficacy. if a new ap-containing vaccine contains only antigens that were included in at least one vaccine evaluated in previous protective efficacy studies then immune responses could be compared between the new vaccine and an approved vaccine that has the most similar ap antigen content. if the new ap-containing vaccine contains fewer antigens, different amounts of antigens and/or additional antigens as those in vaccines previously shown to provide protective efficacy there is a need for a careful justification of all the differences between the new and past efficacious vaccines, which would likely have to be based on nonclinical studies. in particular, the response to the pt component should be fully assessed for efficacy in animal models and by estimating the functional antibody to pt (for example, by using the cho cell neutralization assay). the purity, integrity and functional activity of all antigens should be assessed by physical-chemical evaluation, measurement of residual toxicity (if applicable) and assessment of immunogenicity based on binding and functional assays and protective effects in relevant to begin with a brief overview of pertussis vaccine evaluation in the united states, ap vaccines were first licensed for toddlers in , for infants in , and for adults and adolescents in . serologic responses to ap vaccination have been used as a measure of lot-to-lot clinical consistency, to bridge populations, and to evaluate booster immunizations, new combinations and concomitant vaccination. relevant parameters measured have included geometric mean concentrations as well as percentage of responders. in addition, reverse cumulative distribution curves have proved to be an informative way to display serologic response data. the composition and formulation of ap vaccines vary widely between manufacturers and products, and no globally accepted reference preparations and release criteria have been established; thus, current reference materials and release criteria are productspecific. this causes difficulty in standardization of the laboratory tests for control of these vaccines. the basis of current regulatory approaches is to demonstrate that newly manufactured lots are comparable either to lots shown to have acceptable clinical trial performance or to lots considered equivalent to these clinical trial lots. current routine control tests for ap vaccines include characterization of antigens, assay of immunogenicity, and assay of residual pt bioactivity by the histamine sensitization test and identity testing of antigenic components. a modified intracerebral challenge assay (mica, modified kendrick test) has been used in japan, korea and china as the potency assay for release with a specification ≥ unit/dose; vaccines regulated using this approach have been shown to be effective in controlling pertussis. in other countries, no nationally defined official potency test has been used at present. an immunogenicity test has been used for monitoring consistency of production in routine control procedures with comparison with a clinical trial lot (or equivalent). however, the criteria for evaluating equivalency must be defined and no defined common specification has been set. recent evidence suggests that both antibody and cell-mediated immunity contribute to the protective process to a variable extent. following the decision made at the who ad hoc working group meeting ( , nibsc, uk), international collaborative studies on protection models for ap vaccines were initiated in , and the outcome of these studies was discussed in subsequent who working group meetings. the harmonized procedure for the intranasal challenge assay (inca), defined in , has been shown to be effective for monitoring the activity of different vaccines and to be transferable between laboratories [ , , ] . future work on optimizing experimental conditions to allow calculation of a relative potency is needed and a reference vaccine needs to be agreed upon. this model should be useful for development and characterization of new products or formulations, performing preclinical evaluation or stability and lot consistency monitoring. the current safety tests for ap vaccines include assays for residual active pt and reversibility of detoxification based on histamine sensitization activity, an endotoxin assay and a general toxicity test. the absence of other toxins at significant levels (heat-labile toxin, act, tracheal cytotoxin) has been controlled through process validation. problems associated with the histamine sensitization test include variation in test performance among laboratories and the absence of an internationally defined acceptance criterion for the assay. the influence of significant interactions between pt and other vaccine components over histamine sensitization assay is unknown. the assay could be improved by ( ) defining assay sensitivity by including reference groups; ( ) narrowing the range of permissible sensitivity; ( ) expressing the results in international unit (iu) of pt activity relative to a common reference; and ( ) establishing limits based on a panel of products with a known safety history. in summary, current quality control tests and specifications for ap vaccines are product-specific, and the clinical relevance of this approach remains uncertain. an effective potency assay for new products and formulations should be defined, and suitable reference preparations should be identified. for the current histamine sensitization test, an upper limit for active residual pt should be defined and use of a reference preparation should be encouraged. finally, a specific assay for residual active pt that does not require use of animals should be identified. the challenge in testing of ap-based combination vaccines still remains. for the immunologic evaluation of dtap combinations and coadministrations with dtap-based vaccines, gsk referred back to the original dtap efficacy trials. the cornerstone of comparisons among combined vaccines to their separately administered licensed counterparts is the evaluation of the antibody responses to pt, fha and prn [ ] . noninferiority of the percentage of vaccine responders as well as of geometric mean titers forms the basis for the licensure of new combinations and co-administrations. reverse cumulative antibody distribution curves are also useful for such comparisons. further characterizations of new dtap combinations by gsk involve the evaluation of clinical t-cell responses as well as preclinical evaluations in a mouse lung clearance model [ ] . with respect to vaccine composition and protection, the available data suggest potent dtwp and dtap containing pt and prn induce high levels of protection against whooping cough [ , , , ] . the mechanisms linked to prn-mediated protection relate to the observations that prn expression affects act activity [ ] and induces opsonophagocytic antibodies [ ] . the dtap efficacy trials have been contradictory with regard to the efficacy of dtap (pt + fha without prn). initially the efficacy of dtap was reported to be % in senegal, according to who criteria, but this was later corrected to % since the original diagnostic criteria were different from other trials [ ] . this % efficacy of dtap in senegal probably is similar to the % efficacy observed with another dtap in sweden because of the finding that pt immune responses in senegal are more than . times higher than in western europe [ ] . in conclusion, dtwp and dtap with ≥ components are the preferred options for immunization against whooping cough. dtap has demonstrated efficacy comparable with dtap , although it is uncertain if fim is a protective antigen [ ] . the duration of protection with dtwp and dtap can be estimated at years or more [ ] . to further control pertussis in newborns, adolescents and adults, dtap booster immunizations can be recommended [ ] . sanofi pasteur has been using immunoassays for b. pertussis -igg elisas for pt, fha, prn, and fim antigens -to support the evaluation of vaccine-induced immune responses. the assays used are essentially those identified by manclark et al. [ ] . key reagents are qualified and standardized, coating antigens are highly purified and assessed both by physical chemistry and immunologic methods to ensure purity, antigenicity, and specificity. assay reference sera are bridged to international references, and the performance has demonstrated to remain stable. control sera are shown to be specific and represent suitable ranges across the range of the assay. all components are qualified, with performance panels of sera to ensure consistency. all assays have been fully validated by precision (inter-and intralaboratory), accuracy (using spike recovery), specificity (assessing matrix effects and performing competition studies), linearity/dilutability, limit of detection (lod), limit of quantitation (loq), and robustness. from the perspective of a vaccine manufacturer such as sanofi pasteur, issues and concerns for the task of global standardization should focus on critical reagents and data reduction methods. for coating antigens, consideration needs to be given to the impact of antigen source as well as the criteria for quality, and consumption rate. for references, most laboratories have been forced to develop internal references that are bridged to international standards; guidance is needed on how best to select these internal references and the sustainability of international references to support frequent calibrations. similarly, guidance is needed on the best characteristics for controls and implementation of quality control and assay acceptance criteria. finally, for data reduction, guidance is needed to establish which data reduction systems are most suitable in today's laboratory. chairpersons: freyja lynn, bs (national institute of allergy and infectious diseases, national institutes of health, bethesda, maryland, us) and annette morris, bs, bed (canadian center for vaccinology, dalhousie university, nova scotia, canada) to achieve harmonization of assays for the clinical diagnosis of pertussis and evaluation of immunity postvaccination, several issues will have to be addressed. first, consensus on methodologies, assay validation, and analysis of results is required to facilitate comparisons among data generated in different laboratories. the requirements for a diagnostic assay will likely differ in some aspects from assays used to perform postvaccination immunity evaluation. stability of assay results over time will be dependent on the consistent availability of appropriately characterized reagents and materials. studies are required to determine the effect on assay results of different sources and lots of antigen, conjugate and other reagents. a proficiency panel of human sera is vital to harmonization of methods between laboratories, although creating a panel with sufficient volume for worldwide use will be a challenge. a reference laboratory or a series of connected reference laboratories will be essential as resources for methodology, change control and management and evaluation of the proficiency panel. finally, a repository for the distribution of standardized materials, such as antigens, conjugates, and reference materials, will be crucial for standardization and harmonization of b. pertussis assays. a steering committee should be established for the purpose of identifying and prioritizing the steps toward harmonization, including identification of resources required for implementation. if harmonization is to be accomplished, it must be a global effort with the collaboration of many research, government and corporate organizations. the who is currently evaluating a collaborative study of proposed international reference preparations for human pertussis antiserum. if these are shown to be acceptable, the availability of international reference material will be valuable for promoting harmonization. additionally, the collaborative study data, available mid- , will provide information on comparability of results in participating laboratories and will help point to where priorities should be focused. actionable items to accomplish standardization and harmonization of b. pertussis immunoassays identified during group discussion included the following: ( ) identify a group that will organize, prepare, maintain, and distribute a common pertussis performance serum proficiency panel that can be used to help laboratories to develop their assays and monitor stability; ( ) assemble a working group to assist with the details of the panel; ( ) encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; ( ) focus on key reagents such as reference and control sera; ( ) define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; ( ) develop guidance on quality of other reagents, e.g., pt and other antigens, and methods to demonstrate their suitability; ( ) establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; ( ) create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and ( ) seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines. preventing tetanus, diphtheria, and pertussis among adults: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine recommendations of the advisory committee on immunization practices (acip) and recommendation of acip, supported by the healthcare infection control practices advisory committee (hicpac), for use of tdap among health-care personnel cdc. preventing tetanus, diphtheria, and pertussis among adolescents: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccines recommendations of the advisory committee on immunization practices (acip) outbreaks of respiratory illness mistakenly attributed to pertussis overview of pertussis: focus on epidemiology, sources of infection, and long term protection after infant vaccination historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states pertussis of adults and infants pertussis immunization in the global pertussis initiative european region: recommended strategies and implementation considerations new pertussis vaccination strategies beyond infancy: recommendations by the global pertussis initiative reemergence of pertussis in the highly vaccinated population of the netherlands: observations on surveillance data the control of pertussis- and beyond molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to bordetella pertussis and other bordetella subspecies how best to estimate the global burden of pertussis? pertussis immunization in the global pertussis initiative international region: recommended strategies and implementation considerations health burden of pertussis in adolescents and adults the epidemiology of pertussis: a comparison of the epidemiology of the disease pertussis with the epidemiology of bordetella pertussis infection diagnosis of pertussis: a historical review and recent developments defining pertussis epidemiology: clinical, microbiologic and serologic perspectives laboratory diagnosis of pertussis: state of the art in nucleic acid amplification tests for diagnosis of bordetella infections laboratory diagnosis of pertussis infections: the role of pcr and serology development and evaluation of dual-target real-time polymerase chain reaction assays to detect bordetella spp issues associated with and recommendations for using pcr to detect outbreaks of pertussis external quality assessment for molecular detection of bordetella pertussis in european laboratories prevalence and sequence variants of is in bordetella bronchiseptica: implications for is -based detection of bordetella pertussis how to make sense of pertussis immunogenicity data cellular immunity in adolescents and adults following acellular pertussis vaccine administration levels of anti-pertussis antibodies related to protection after household exposure to bordetella pertussis a search for serologic correlates of immunity to bordetella pertussis cough illnesses epidemiology of pertussis thirty-five years' experience with the whole-cell pertussis vaccine in france: vaccine strains analysis and immunogenicity epidemiology of pertussis in french hospitals in and : thirty years after a routine use of vaccination influence of vaccination coverage on pertussis transmission in france evidence of bordetella pertussis infection in adults presenting with persistent cough in a french area with very high whole-cell vaccine coverage transmission of bordetella pertussis to young infants temporal analysis of french bordetella pertussis isolates by comparative whole-genome hybridization avis du haut conseil de la santé publique pertussis surveillance in french hospitals: results from a year period efficacy of acellular pertussis vaccine in early childhood after household exposure efficacy of a two-component acellular pertussis vaccine in infants a commentary on the pathogenesis of pertussis effects of islet-activating protein (iap) on blood glucose and plasma insulin in healthy volunteers (phase studies) lymphocytosis-promoting factor of bordetella pertussis alters mononuclear phagocyte circulation and response to inflammation a marked decrease in lselectin expression by leucocytes in infants with bordetella pertussis infection: leucocytosis explained? marked increase in l-selectin-negative t cells in neonatal pertussis. the lymphocytosis explained? pertussis toxin utilizes proximal components of the t-cell receptor complex to initiate signal transduction events in t cells bordetella pertussis toxin induces the release of inflammatory cytokines and dendritic cell activation in whole blood: impaired responses in human newborns proinflammatory and proapoptotic activities associated with bordetella pertussis filamentous hemagglutinin phagocyte impotence caused by an invasive bacterial adenylate cyclase bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin inhibition of monocyte oxidative responses by bordetella pertussis adenylate cyclase toxin bordetella pertussis adenylate cyclase-hemolysin induces interleukin- secretion by human tracheal epithelial cells bordetella pertussis adenylate cyclase toxin (act) induces cyclooxygenase- (cox- ) in murine macrophages and is facilitated by act interaction with cd b/cd (mac- ) t-cell immune response assessment as a complement to serology and intranasal protection assays in determining the protective immunity induced by acellular pertussis vaccines in mice a murine model in which protection correlates with pertussis vaccine efficacy in children reveals complementary roles for humoral and cell-mediated immunity in protection against bordetella pertussis evaluation of efficacy in terms of antibody levels and cell-mediated immunity of acellular pertussis vaccines in a murine model of respiratory infection pertussis vaccination: use of acellular pertussis vaccines among infants and young children. recommendations of the advisory committee on immunization practices (acip) comparison of acellular pertussis vaccines: overview and serologic response establishment of diagnostic cutoff points for levels of serum antibodies to pertussis toxin, filamentous hemagglutinin, and fimbriae in adolescents and adults in the united states collaborative study for the evaluation of enzymelinked immunosorbent assays used to measure human antibodies to bordetella pertussis antigens characterization of serological responses to pertussis specificity and sensitivity of high levels of immunoglobulin g antibodies against pertussis toxin in a single serum sample for diagnosis of infection with bordetella pertussis european sero-epidemiology network: standardisation of the assay results for pertussis pertussis in massachusetts, - : incidence, serologic diagnosis, and vaccine effectiveness ad hoc working group on acellular pertussis vaccines, world health organisation the european sero-epidemiology network the seroepidemiology of bordetella pertussis infection in western europe evaluation of a single-sample serological technique for diagnosing pertussis in unvaccinated children evaluation of an immunoglobulin g enzyme-linked immunosorbent assay for pertussis toxin and filamentous hemagglutinin in diagnosis of pertussis in senegal comparison of five commercial enzyme-linked immunosorbent assays for detection of antibodies to bordetella pertussis serodiagnosis of pertussis with commercial elisa's determination of serum antibody to bordetella pertussis adenylate cyclase toxin in vaccinated and unvaccinated children and in children and adults with pertussis studies on pertussis immunization comparison of values of antibody to bordetella pertussis antigens in young german and american men a comparative efficacy trial in germany in infants who received either the lederle/takeda acellular pertussis component dtp (dtap) vaccine, the lederle whole-cell component dtp vaccine, or dt vaccine pertussis toxin: the cause of the harmful effects and prolonged immunity of whooping cough. a hypothesis primum non nocere: a pharmacologically inert pertussis toxoid alone should be the next pertussis vaccine immunity to pertussis. not all virulence factors are protective antigens mass vaccination of children with pertussis toxoid-decreased incidence in both vaccinated and nonvaccinated persons who working group on standardisation and control of acellular pertussis vaccines-report of a meeting the rise in pertussis cases urges replacement of chemically-inactivated with geneticallyinactivated toxoid for dtp the diphtheria and pertussis components of diphtheria-tetanus toxoidspertussis vaccine should be genetically inactivated mutant toxins correlation between pertussis toxin igg antibodies in postvaccination sera and subsequent protection against pertussis who working group on the standardisation and control of pertussis vaccines-report of a meeting informal consultation with manufacturers and who ad hoc working group on mouse protection models for acellular pertussis vaccines. national institute for biological standards diphtheria-tetanuspertussis (dtp) combination vaccines and evaluation of pertussis immune responses pertussis antibodies, protection, and vaccine efficacy after household exposure low levels of antipertussis antibodies plus lack of history of pertussis correlate with susceptibility after household exposure to bordetella pertussis role of adhesins and toxins in invasion of human tracheal epithelial cells by bordetella pertussis crucial role of antibodies to pertactin in bordetella pertussis immunity pertussis vaccines priming effect, immunogenicity and safety of an haemophilus influenzae type b-tetanus toxoid conjugate (prp-t) and diphtheria-tetanusacellular pertussis (dtap) combination vaccine administered to infants in belgium and turkey acellular pertussis vaccines and the role of pertactin and fimbriae pertussis vaccines-who position paper serological responses to bordetella pertussis support for the meeting was graciously provided by the centers for disease control and prevention, atlanta, ga. the authors thank barbara slade, md, jackie goolsby, brandy kimble, shoranda ifill (cdc) and logistics health incorporated for their contribution to the scientific and administrative organization of the meeting. we thank lynne mcintyre for critical editing of this manuscript. key: cord- -i uuf ck authors: sarkar, bishajit; ullah, md. asad; araf, yusha; rahman, mohammad shahedur title: engineering a novel subunit vaccine against sars-cov- by exploring immunoformatics approach date: - - journal: inform med unlocked doi: . /j.imu. . sha: doc_id: cord_uid: i uuf ck as the number of infections and deaths caused by the recent covid- pandemic is increasing dramatically day-by-day, scientists are rushing towards developing possible countermeasures to fight the deadly virus, sars-cov- . although many efforts have already been put forward for developing potential vaccines, however, most of them are proved to possess negative consequences. therefore, in this study, immunoinformatics methods were exploited to design a novel epitope-based subunit vaccine against the sars-cov- , targeting four essential proteins of the virus i.e., spike glycoprotein, nucleocapsid phosphoprotein, membrane glycoprotein, and envelope protein. the highly antigenic, non-allergenic, non-toxic, non-human homolog, and % conserved (across other isolates from different regions of the world) epitopes were used for constructing the vaccine. in total, fourteen ctl epitopes and eighteen htl epitopes were used to construct the vaccine. thereafter, several in silico validations i.e., the molecular docking, molecular dynamics simulation (including the rmsf and rmsd studies), and immune simulation studies were also performed which predicted that the designed vaccine should be quite safe, effective, and stable within the biological environment. finally, in silico cloning and codon adaptation studies were also conducted to design an effective mass production strategy of the vaccine. however, more in vitro and in vivo studies are required on the predicted vaccine to finally validate its safety and efficacy. . the step-by-step procedure adapted in the vaccine designing study. (https://blast.ncbi.nlm.nih.gov/blast.cgi) tool was used in the human homology determination, where homo sapiens (taxid: ) was used as the comparing organism, keeping all other parameter default. an e-value cut-off of . was set in the experiment and the epitopes that had no hits below the e-value inclusion threshold were selected as non-homologous peptides (mehla and ramana, ) . and gpgpg), padre sequence, adjuvant (human beta-defensin- ), and epitopes (ctl- , ctl- , ctl- , as well as htl- , htl- , htl- , and so on) in sequential and appropriate manner. figure s , we can see that the potential energy of the structure had quickly reduced below the order of for the protein complex. from this, we inferred that the complex system was stable enough to temperature) equilibration was done for ps and pressure as well as density were calculated. the crystallized protein structure was also prepared in the same way. then the resulting the mhc class-i and class-ii epitopes were predicted from the target protein sequences for constructing the vaccine. the epitopes that were found to follow all the previously mentioned criteria, were finally selected for vaccine construction considering as the most promising epitopes ( table ). the % conservancy of the epitopes among the selected isolates from ifn-gamma, il- , and il- inducing capacity prediction of the htl epitopes showed that many of the selected htl epitopes had the capability of inducing at least one of these cytokine. moreover, all the most promising epitopes were also found to have at least one cytokine production capability (supplementary table s , s , s , and s ). the vaccine has been constructed using the most promising epitopes which could be used to the cv candidate vaccine was predicted to be a potent antigen as well as a non-allergen. in the physicochemical property analysis of the vaccine, a high (basic) theoretical pi, half-life of h in the mammalian cells, and more than h in the e. coli cell culture system were predicted. moreover, the vaccine construct was also found to be quite stable as well as soluble upon table s and supplementary figure s ) . the d structure of the cv vaccine construct was predicted by the online server raptorx. the vaccine had domains with quite low p-value of . e- , which declared that the quality of the figure s ) . since potential disulfide bonds, therefore, it can be considered as a quite stable vaccine construct. table s ) . ~ . nm. the black line of the graph refers to the rmsd relative to the structure present in the minimized, equilibrated system and the red line is the rmsd relative to the crystal structure. since both these plots are almost highly correlated, so it can be declared that the structure remained quite stable during the experiment. the rmsf and radius of gyration graphs also point to the fact that the cv-tlr- complex was quite stable during the experiment (figure b & c ). like the complex structure, the single vaccine crystal structure also generated very good results in the md simulation. in the rmsd graph of figure d , it can be noticed that the structure also pointed towards the fact that the crystallized structure didn't have any significant region with the possibility to undergo deformation, so it can be deduced that the crystalized vaccine cv might be quite stable in the biological environment (figure e & f) . so, overall, by these antigen-presenting cells (apcs) (figure j and figure k ). the cv vaccine was also predicted to produce different types of cytokines (figure l) . henceforth, the overall immune simulation study revealed that, the cv vaccine might be able to stimulate strong immunogenic response after its administration. finally, the codon adaptation and in silico cloning were performed to design a recombinant plasmid which might be used for the mass production of the cv vaccine in the e. coli strain k . in the codon adaptation study of the vaccine, the obtained results were found to be satisfactory sars-cov- vaccines: status report. immunity firedock: fast interaction refinement in molecular docking codon usage: nature's roadmap to expression and folding of proteins design of the linkers which effectively separate domains of a bifunctional fusion protein designing a fusion protein vaccine against hcv: an in silico approach cd + regulatory t cells: mechanisms of induction and effector function atypical mhc class ii-expressing antigen-presenting cells: can anything replace a dendritic cell? protective immunity against sars subunit vaccine candidates based on spike protein: lessons for coronavirus vaccine development exploring leishmania secretory proteins to design b and t cell multi-epitope subunit vaccine using immunoinformatics approach immunoinformatics approaches to explore helicobacter pylori proteome (virulence factors) to design b and t cell multi-epitope subunit vaccine. scientific reports large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction procheck: validation of protein-structure coordinates recent advances of vaccine adjuvants for infectious diseases recent advances of vaccine adjuvants for infectious diseases. immune network cd + t cells: differentiation and functions identification of epitope-based peptide vaccine candidates against enterotoxigenic escherichia coli: a comparative genomics and immunoinformatics approach virus contaminations of cell cultures-a biotechnological view a novel design of a multi-antigenic, multistage and multi-epitope vaccine against helicobacter pylori combating covid- pandemic in bangladesh: a memorandum from developing country. preprints susceptibility of the iranian population to severe acute respiratory syndrome coronavirus infection based on variants of angiotensin i converting enzyme . future virology synonymous codon usage pattern in glycoprotein gene of rabies virus stereochemical quality of protein structure coordinates abcf atpases involved in protein synthesis, ribosome assembly and antibiotic resistance: structural and functional diversification across the tree of life computer-aided designing of immunosuppressive peptides based on il- inducing potential evaluation of predictions in the casp model refinement category adejumo sa, ibeanu gc. immunoinformatics and vaccine development: an overview. immunotargets and therapy recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production physicochemical characterization and functional analysis of some snake venom toxin proteins and related non-toxin proteins of other chordates cell differentiation and plasticity during visceral leishmaniasis infection. front. microbial. , another decade, another coronavirus amino acid neighbours and detailed conformational analysis of cysteines in proteins ellipro: a new structure-based tool for the prediction of antibody epitopes more than one reason to rethink the use of peptides in vaccine design computational immunology meets bioinformatics: the use of prediction tools for molecular binding in the simulation of the immune system reverse vaccinology . : human immunology instructs vaccine antigen design therapeutic and vaccine strategies against sars-cov- : past, present and future. future virology soluble expression of recombinant proteins in the cytoplasm of microbial cell factories algpred: prediction of allergenic proteins and mapping of ige epitopes characterization of common epitope-based peptide vaccine for nipah and hendra viruses. asian pacific journal of tropical medicine coronaviruses: candidate drugs for sars-cov- treatment? virus like particles-a recent advancement in vaccine development. 미생물학회지 immunoinformatics-guided designing of epitope-based subunit vaccine against the sars coronavirus- (sars-cov- ) a systematic and reverse vaccinology approach to design novel subunit vaccines against dengue virus type- and human papillomavirus- blueprint of epitope-based multivalent and multipathogenic vaccines: targeted against the dengue and zika viruses designing novel epitope-based polyvalent vaccines against herpes simplex virus- and exploiting the immunoinformatics approach patchdock and symmdock: servers for rigid and symmetric docking coronavirus envelope protein: current knowledge in-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases codagenix raises $ million for a new flu vaccine and other therapies subtractive proteomics to identify novel drug targets and reverse vaccinology for the development of chimeric vaccine against acinetobacter baumannii hla-dr: molecular insights and vaccine design study team: safety and efficacy of mva a, a new tuberculosis vaccine, in infants previously vaccinated with bcg: a randomised, placebo-controlled phase b trial mhccluster, a method for functional clustering of mhc molecules possible therapeutic options for covid- exploiting the reverse vaccinology approach to design novel subunit vaccine against ebola virus new additions to the c lus p ro server motivated by capri the immune epitope database (iedb): update subunit vaccines against emerging pathogenic human coronaviruses. frontiers in microbiology peptide binding predictions for discovery and investigation of o-xylosylation in engineered proteins containing a (ggggs) n linker hawkdock: a web server to predict and analyze the protein-protein complex based on computational docking and mm/gbsa prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins improving therapeutic hpv peptidebased vaccine potency by enhancing cd + t help and dendritic cell activation improving therapeutic hpv peptidebased vaccine potency by enhancing cd + t help and dendritic cell activation poiani gj. an update on current therapeutic drugs treating covid- multi-epitope vaccines: a promising strategy against tumors and viral infections diagnostics, vaccines and therapeutics. microbes and infection a novel human coronavirus oc genotype detected in mainland china. emerging microbes & infections cd t cells: fates, functions, and faults mfold web server for nucleic acid folding and hybridization prediction key: cord- - zu w a authors: ino, hiroyasu title: vaccine mandate in long‐term care facilities date: - - journal: geriatr gerontol int doi: . /ggi. sha: doc_id: cord_uid: zu w a nan telehealth consultations have helped our institution provide continuity of care to older adults who would otherwise decline healthcare attendances due to fears of contracting covid- . this has enabled us to identify problems early to institute care and avoid unnecessary admissions to prevent strain on the healthcare system. notably, telehealth consultations decreased rapidly after reaching a peak of % of total consultations when a partial lockdown was instituted. the rate of telehealth usage returned to approximately the same levels as those seen in the general medicine clinic. this may have occurred as community transmission of covid- was brought under more control with the partial lockdown, and patients and caregivers were not as fearful of venturing out as compared with the start of partial lockdown. however, this shows that with the right incentives and strategies or during times of resurgence of pandemics, a significant proportion of older adults are able to engage in the use of telehealth to minimize risks and maximize benefits. as more older adults and their caregivers become familiar with telehealth, it is likely that use of this service will only increase. further study into the factors for and against use of technology in older adults is warranted to tailor interventions for this group better. however, there are drawbacks and limitations of telehealth. clinical examination and assessment are important in caring for older adults who may present atypically. if during the teleconsultation the attending physician decides on the need for an in-person review, the teleconsultation fee is waived and an in-person review is arranged. urgent cases are referred to the emergency department for timely assessment. not all older adults have blood pressure machines at home. interval, longitudinal measurement of vital signs and parameters such as weight and body mass index are important in the holistic management of older adults. opportunities for such baseline measurements are curtailed with teleconsultations. risks of privacy and data breaches exist for all modes of telecommunication and regulations tend to play catch up to the rapid pace of technological development. in recognition of the need for education and instruction of physicians using telehealth platforms, the ministry of health (singapore) recently launched an online course and guidelines to ensure uniformity of standards across telehealth practices. careful patient selection is thus important to accrue maximum benefits of teleconsultation to older adults. while telehealth services have been developed before the covid- pandemic, the fears and public health impact of the pandemic have fortuitously led to the increased uptake of telehealth services among older adults. this group is likely to benefit greatly from telehealth due to the convenience, time and travel savings. the greater uptake has led to greater familiarity among staff on the workflows and benefits of telehealth consultations. a growing critical mass of telehealth utilization allows institutions to devote more resources to developing telehealth services and would enable us to take advantage of the rapid gains and benefits of technology. this would only bring greater benefits to patients and the healthcare system. up to half of those who have died from covid- in countries within the who european region were estimated to be residents in ltcfs. similar outbreaks were also seen in other countries. when faced with several elderly patients with fever, it is important to examine whether they were infected with covid- , as high mortality is observed in the older population. however, various infectious diseases cause fever in the population, such as aspiration pneumonia, influenza, pneumococcal pneumonia and urinary tract infection. to make a diagnosis, patients, their families or facility nurses are asked if the elderly patients are vaccinated against influenza or pneumococcus. the answers are not always "yes." apparently, there are not many cases in which the patients were vaccinated against both. influenza and pneumococcus vaccine are proved effective for lowering mortality in the elderly. making vaccination a standard part of the ltcf admission process does increase vaccination rates in nursing homes, and it is recommended by the department of health and human services. in a survey, % of us citizens indicated that nursing homes should definitely require influenza vaccination for residents. vaccine mandates for children, youths and healthcare workers have been discussed, but in contrast ethical grounds of vaccine mandates for the elderly in ltcfs have not been widely discussed. underlying health conditions, decline of cognitive ability and even their advanced age make residents of ltcfs at most risk for severe symptoms or death from infectious diseases. there are many interactions between the residents and the staffs, and among residents. residents dine at the same table and have a conversation, and participate in daily group activities. even in facilities such as assisted living, which have separated flats or bungalows, residents interact through activities. these interactions make the facility vulnerable to infectious diseases, but one must not forget that living in ltcfs provides the residents with a comfortable environment, which makes them feel like being in one's own home. ltcfs are not only facilities for care, but also living space for residents. ltcfs are also referred to as nursing homes and in the case of japan, they are called "roujin-houmu," which means "homes for elderly." in this sense, ltcfs are different from hospitals in a way that they are similar to a small community, which is characterized by physical proximity and daily interactions of the vulnerable members. when entering this community, newcomers are required to take existing residents' health into consideration. we can apply the discussion of vaccine mandate for immigrants. when entering the united states, immigrants need to be vaccinated against airborne infectious diseases and this requirement is ethically justified. the harm principle should also be applied to the ltcf setting. many elderly people are exempt from vaccine on medical grounds. therefore, if some residents are not vaccinated, the vaccine rate will not be enough to keep herd immunity. thus, we need to make sure to avoid free-riders within the ltcf population. vaccines for covid- are undergoing clinical trials, but we still do not know if these vaccines are going to be effective. some researchers point out that the covid- vaccine may not work on the elderly because of their immunosenescence. , the elderly are typically given a larger dose of the flu vaccine, so there is a possibility that larger doses of covid- vaccines are required for the elderly. however, when any vaccines that can prevent infection in elderly people are developed, vaccinating ltcf residents against covid- should be a requirement. there are some issues to be kept in mind before implementation of vaccine mandates. first, legal aspects should be examined in some countries where vaccine refusals in ltcfs are permitted. second, even if vaccinations in ltcfs become mandatory, providing education regarding the benefits and potential side effects of the immunization is important. trust between doctors and residents holds the key to success. third, vaccinations for care workers should be required, and vaccinations for visitors should be recommended. whether covid- will become a seasonal infectious disease is not clear at the moment. however, there is an urgent need to discuss vaccination mandates for residents in ltcfs. the mmr vaccine before school enrollment has saved many children. we should consider vaccine requirements for the elderly in ltcfs. clinical characteristics and prognostic factors in covid- patients aged ≥ years telemedicine in nursing homes during the covid- outbreak: a star is born (again) acceptance and use of health information technology by community-dwelling elders determinants of clinical presentation on outcomes in older patients with myocardial infarction for telehealth to succeed, privacy and security risks must be identified and addressed epidemiology of covid- in a long-term care facility in king county, washington they're death pits': virus claims at least , lives in u.s. nursing homes. the new york times statement -invest in the overlooked and unsung: build sustainable people-centred long-term care in the wake of covid- . who regional office for europe, statement to the press mortality associated with covid- outbreaks in care homes: early international evidence. ltccovid.org, international long-term care policy network, cpec-lse nursing home vaccination: reaching healthy people goals. office of inspector general: department of health and human services flu vaccine for nursing home staff and residents. university of michigan national poll on healthy aging recent vaccine mandates in the united states, europe and australia: a comparative study a proposed ethical framework for vaccine mandates: competing values and the case of hpv time to mandate influenza vaccination in health-care workers immigration justice and the grounds for mandatory vaccinations can an effective sars-cov- vaccine be developed for the older population? trained immunity: a tool for reducing susceptibility to and the severity of sars-cov- infection how to cite this article: ino h. vaccine mandate in long-term care facilities letters to the editor -research studies no funding was received for this manuscript. the authors declare no conflicts of interest.li feng tan, vanda ho wen teng, santhosh kumar seetharaman and alexander wenjun yip the authors declare no conflict of interest. the university of tokyo hospital, tokyo, japan key: cord- -v wnmg authors: flanagan, katie l.; best, emma; crawford, nigel w.; giles, michelle; koirala, archana; macartney, kristine; russell, fiona; teh, benjamin w.; wen, sophie ch title: progress and pitfalls in the quest for effective sars-cov- (covid- ) vaccines date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: v wnmg there are currently around sars-cov- candidate vaccines in preclinical and clinical trials throughout the world. the various candidates employ a range of vaccine strategies including some novel approaches. currently, the goal is to prove that they are safe and immunogenic in humans (phase / studies) with several now advancing into phase and trials to demonstrate efficacy and gather comprehensive data on safety. it is highly likely that many vaccines will be shown to stimulate antibody and t cell responses in healthy individuals and have an acceptable safety profile, but the key will be to confirm that they protect against covid- . there is much hope that sars-cov- vaccines will be rolled out to the entire world to contain the pandemic and avert its most damaging impacts. however, in all likelihood this will initially require a targeted approach toward key vulnerable groups. collaborative efforts are underway to ensure manufacturing can occur at the unprecedented scale and speed required to immunize billions of people. ensuring deployment also occurs equitably across the globe will be critical. careful evaluation and ongoing surveillance for safety will be required to address theoretical concerns regarding immune enhancement seen in previous contexts. herein, we review the current knowledge about the immune response to this novel virus as it pertains to the design of effective and safe sars-cov- vaccines and the range of novel and established approaches to vaccine development being taken. we provide details of some of the frontrunner vaccines and discuss potential issues including adverse effects, scale-up and delivery. there are currently around sars-cov- candidate vaccines in preclinical and clinical trials throughout the world. the various candidates employ a range of vaccine strategies including some novel approaches. currently, the goal is to prove that they are safe and immunogenic in humans (phase / studies) with several now advancing into phase and trials to demonstrate efficacy and gather comprehensive data on safety. it is highly likely that many vaccines will be shown to stimulate antibody and t cell responses in healthy individuals and have an acceptable safety profile, but the key will be to confirm that they protect against covid- . there is much hope that sars-cov- vaccines will be rolled out to the entire world to contain the pandemic and avert its most damaging impacts. however, in all likelihood this will initially require a targeted approach toward key vulnerable groups. collaborative efforts are underway to ensure manufacturing can occur at the unprecedented scale and speed required to immunize billions of people. ensuring deployment also occurs equitably across the globe will be critical. careful evaluation and ongoing surveillance for safety will be required to address theoretical concerns regarding immune enhancement seen in previous contexts. herein, we review the current knowledge about the immune response to this novel virus as it pertains to the design of effective and safe sars-cov- vaccines and the range of novel and established approaches to vaccine development being taken. we provide details of some of the frontrunner vaccines and discuss potential issues including adverse effects, scale-up and delivery. the recent emergence of severe acute respiratory syndrome coronavirus- (sars-cov- ), the cause of coronavirus disease , is wreaking havoc due to widespread dissemination throughout the world. on march , who formally declared that a global pandemic and by the end of august , almost million cases and over , deaths had been reported worldwide involving all continents, except antarctica ( ) . strategies to identify cases and limit spread by widespread testing and physical distancing have been challenging to implement, healthcare and public health systems have been overwhelmed, resulting in continued escalation in many countries and profound effects on lives and livelihoods. while the majority of people are either asymptomatic or experience a mild respiratory infection, ∼ % of cases are more severe and require hospital admission ( ) . reported mortality rates vary by geographic region, ranging from almost % in france to < % in many other countries ( ) . this wide discrepancy suggests selection bias due to differences in local testing strategies and capacity, consistent with differences in health system capacity, population demographics and other health determinants. certain groups such as the elderly and those with particular comorbidities are more likely to die of covid- ( ) . healthcare workers in particular have experienced significant morbidity and mortality from covid- ( ), causing clear psychological impacts and threatening delivery of healthcare services ( ) . there is no known effective treatment for this virus and currently no available vaccine, with the result being that sars-cov- continues to spread throughout the virus naïve population of the world. the urgent need for effective sars-cov- vaccines cannot be overstated. immunization can not only protect individuals but also, if provided to enough people in a timely way with (even) partially protective vaccines, induce sufficient herd immunity to curtail the spread of this virus and reduce morbidity and mortality across the globe. the race to develop safe and effective vaccines has seen sars-cov- candidate vaccines developed at a scale and pace never imagined before: currently almost potential vaccines are in various stages of development ( ) ( ) ( ) . a range of vaccine design approaches and platforms have been employed. however, since > % of candidate vaccines typically fail it is expected that the eventual number of successful vaccines may be only be a handful. they may also become available in different time frames and suitable for use in different populations. most vaccine candidates are currently in preclinical trials, but a number have entered phase or phase / studies, with plans to rapidly scale up to phase and . trials are being conducted at "pandemic speed" ( ) and using novel designs. this early success has already seen cooperation and collaboration as well as significant funding across the globe. for example, the coalition for epidemic preparedness innovations (cepi), a notfor-profit global coalition launched in to deal with the worldwide threat of epidemic outbreaks, is playing a pivotal role in supporting many of the frontrunner vaccines ( ) . herein, we review what is currently known about the immune response to sars-cov- and the various vaccine platforms being used to develop the sars-cov- vaccines. understanding the mechanism of action of the various candidate vaccines is the key focus of this review. we also discuss potential challenges at the immunological level, assessment of vaccine safety and scale-up and delivery. coronaviruses are enveloped positive-sense single stranded rna viruses belonging to the coronaviridae family. they infect birds and mammals causing a range of symptoms from respiratory to gastrointestinal disease ( ) . a number of relatively common seasonal coronaviruses are known to infect humans ( ) , causing mild respiratory illness ("the common cold"). two previous lethal human coronavirus diseases, namely severe acute respiratory syndrome (sars caused by sars-cov- ) ( ) and middle east respiratory syndrome (mers caused by mers-cov) ( ) arose in and , respectively. they have a high mortality rate of ∼ and %, respectively, but fortunately, neither reached pandemic levels. sars-cov- was able to be contained by public health measures to prevent human-to human transmission and then disappeared before vaccine development had progressed significantly. the genome of sars-cov- encodes for the structural proteins spike (s), envelope (e), membrane (m) and nucleocapsid (n) as well as a number of accessory and non-structural proteins (figure ) ( ) . the m and n proteins give the virion its shape, and the s proteins appear as spikes on the viral surface giving it a solar corona appearance. the s glycoprotein (spike) exists in trimeric form and is the structure by which binding to the host cells occurs. the virus receptor, angiotensin-converting enzyme (ace ), expressed on the figure | structure of sars-cov- and key antigenic components. illustration of sars-cov- which is a single stranded rna virus. the key antigenic components being targeted in vaccine design are shown on the right, consisting of the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. the main emphasis for human vaccines is based on the spike (s) protein, consisting of an s binding region and s fusion and cell entry region. the s domain contains the receptor binding domain (rbd) responsible for binding to the ace receptor on the surface of host cells. following fusion, the s protein sheds the s region and undergoes a dramatic structural change to its post-fusional state in order for the virus to enter the host cells. surface of multiple human cells is engaged via the receptor binding domain (rbd), which is part of the s subunit of the s glycoprotein ( ) (figure ) . the s subunit consists of two heptad repeat fusion regions, hr and hr , responsible for membrane fusion and cell entry. the rbd domain can either be down and buried or rotated up and primed for ace binding ( ) . the hidden down state is the predominant state of the s protein trimer in sars-cov- , and likely a mechanism the virus uses to hide the entry epitopes and evade the host immune response ( ) . two s trimers can concurrently bind to an ace dimer. following s protein binding in its prefusion state, the s subunit is cleaved and shed permitting the s dramatic conformational changes which constitute the post-fusion state required for viral entry ( , ) . innate immunity to covid- : protection or hyper-activation? sars-cov- stimulates an innate immune response via pattern associated molecular patterns (pamps) expressed by the virus, which in the case of sars-cov- consists of viral rna and its intermediates produced during replication ( ) . these conserved pamps stimulate multiple immune response pathways via pattern recognition receptors (prrs), including sensing by endosomal toll-like receptors (tlrs) and , cytosolic retinoic acid-inducible gene (rig- ) and melanoma differentiationassociated protein (mda ). local tissue damage in the lungs also releases damage-associated molecular patterns (damps) further contributing to local inflammation. the resultant inflammatory response provides immediate antiviral immunity via activation of antiviral type and interferon (ifn) pathways leading to an upregulation of inflammatory cytokines such as interleukin (il- ) and il- β, further recruiting neutrophils, other innate immune cells and stimulating anti-sars-cov- adaptive memory t cells and b cells ( ) (figure ) . there is still much to understand about the immune response to sars-cov- and the immunological differences in those with mild as compared to severe infection. emerging data suggest that the innate response to sars-cov- is aberrant ( , ) . for example, the early type i and iii interferon responses are relatively suppressed by sars-cov- , an immune evasion strategy employed by the virus, leading to early failure to control the virus. furthermore, uncontrolled local inflammation, or what has been described as "cytokine storm, " leading to tissue damage with inflammatory cell infiltration and the acute respiratory distress syndrome (ards) is thought to characterize late stage, severe manifestations of covid- ( ) ( ) ( ) . for example, patients with severe covid- had higher levels of il- , il- r, il- , and tumor necrosis factor alpha (tnf-α) than those with moderate disease, the former of which correlated with clinical severity and death ( , ) . those covid- patients requiring icu admission demonstrated greater plasma levels of il- , il- , il- , granulocyte colony-stimulating factor (gcsf), interferoninducible protein (ip- ), monocyte chemoattractant protein- (mcp- ), macrophage inflammatory protein- α (mip- α) and tnf-α than the non-icu covid- patients, all supporting enhanced innate immunity in the sicker patients ( ) . of note, the il- levels described in covid- patients are a log-fold lower than those described in classic cytokine storm ( ) . furthermore, il- is an immunosuppressive cytokine suggesting dysregulated immune homeostasis rather than pure inflammation. upregulated chemoattractant chemokines further recruit inflammatory cells including neutrophils, macrophages, natural killer (nk) cells and t cells resulting in further immunopathology ( ) (figure ). antibodies (abs) produced by activated b cells play a key role in anti-viral immunity via several mechanisms including viral neutralization, antibody-dependent cellular cytotoxicity (adcc), antibody-dependent cellular phagocytosis (adcp), and antibody-dependent complement activation (adca) (figure ) . it is thought that generation of high levels of neutralizing antibodies (nabs) against sars-cov- are required for a successful human vaccine ( ) . however, some patients recover without producing high levels of nabs and those with severe disease may experience an early rise in nabs ( ) . nevertheless, most vaccine efforts are focused on the induction of nabs to the s protein in order to block attachment of rbd to the ace receptor on host cells ( ) . as mentioned before, this domain is hidden in the s protein's prefusion state, presenting challenges to the success of rbd-based vaccines ( ) . for this figure | key components of the innate immune response to sars-cov- . antigen presenting cells (apcs), such as monocytes, macrophages, and dendritic cells (dcs), recognize pattern associated molecular patterns (pamps) expressed by sars-cov- via their pattern recognition receptors (prrs), such as toll-like receptor (tlr) and . this activates intracellular signaling pathways leading to the expression of type and interferons (ifns), which in turn activate innate immune cells to produce pro-inflammatory cytokines and chemokines. this leads to an influx and activation of neutrophils, further apcs and other innate immune cells, such as natural killer (nk) cells. reason, some groups have focused on eliciting nabs to the less immunogenic s subunit of the spike protein ( ) and sars-cov- vaccines based on other antigens, including the n protein, are also being developed. a recent report from the us describes outcomes among , covid- patients, many of whom had critical illness, transfused with the plasma from people who have recovered from covid- (convalescent plasma [cp]) ( ) . early cp infusion within days of illness had a lower -day mortality than those treated after or more days ( . % vs. . %). in this uncontrolled study, those who received higher levels of igg abs in the transfused plasma had a better mortality outcome. these data, alongside results from another uncontrolled study showing recovery in severely unwell covid- patients treated with cp, lends support to the notion that naturally acquired abs can be protective ( ) . however, other plasma factors such as cytokines, defensins and other non-specific abs may also play a protective role in these studies ( ) . sars-cov- -specific nabs recovered from infected humans also protected syrian hamsters and rhesus macaques in challenge studies ( , ) . ideally, a sars-cov- vaccine would induce long-lasting abs, but it is not yet known how long specific abs persist in sars-cov- infected individuals or how long they would persist after vaccination. indeed, several recent studies indicate rapid waning of sars-cov- nabs in some individuals following natural infection, although others maintained high levels to - days ( , ) , raising concerns about the persistence of nabs post-immunization; whether nabs plateau or continue to decline over time is yet to be determined. by contrast, nabs to sars and mers have been detected up to - years after infection in human survivors ( ) and a recent study reports nabs years after sars infection, suggesting that long-lasting coronavirus-specific nabs can be induced in some instances ( ) so hopefully ab mediated immunity to sars-cov- will be equally long-lasting. importantly, class-switched igg memory b cells to s and rbd have been demonstrated in covid- patients confirming the generation of b cell memory which can provide a rapid recall response on subsequent sars-cov- exposure ( ) . furthermore, we do not yet know what level of nabs are required for protection ( ) . the standardization of a range of assays to support vaccine studies, such as viral neutralization assays, to enable comparison of different vaccine candidates in different populations will be key to facilitating vaccine development, an issue which represents a current focus of the who ( ). t cell adaptive immunity: protection, immune suppression or disease enhancement? processing and presentation of sars-cov- epitopes by antigen presenting cells (apcs) via human leukocyte antigen (hla) class i leads to activation of naïve cd + t cells which differentiate into cytolytic effectors (cytotoxic t lymphocytes [ctl]) that kill virus-infected cells. activation of t helper (th ) cd + t cells via viral peptides presented on hla class ii alleles further enhances the cd + t cell response, while hla class figure | key components of the adaptive immune response to sars-cov- . the adaptive immune response is activated following viral uptake and antigen processing by a range of apcs. the apcs present viral antigen to b cells which then differentiate into antibody producing plasma cells. the neutralizing antibodies (nabs) then bind to key viral proteins, such as the spike protein, and neutralize their activity. other ab-mediated antiviral functions include antibody dependent cellular cytotoxicity (adcc), antibody dependent cellular phagocytosis (adcp), and antibody dependent complement activation (adca). cytotoxic cd + t cells kill virally infected cells via the production of granzymes and perforin and the expression of fas ligand (fasl), all of which mediate cellular apoptosis. a series of cd + t cell populations are involved in the adaptive cellular response to sars-cov- . follicular helper t cells (t fh ) and th cd + t cells both provide help for b cell antibody production. th and th cd + t cells are also thought to play a role in the inflammatory response and viral killing. cd + regulatory t cells have been implicated with an immunoregulatory role in sars-cov- infection via the production of anti-inflammatory cytokines and contact-mediated cellular suppression. whether cd + tregs and bregs play a role is not currently known. ii-restricted follicular helper (t fh ) and th cells enhance virusspecific antibody production (figure ) . effective viral clearance therefore requires a combination of cd + ctl and cd + t cell mediated enhancement of b cell and cd + t cell responses ( ) . pro-inflammatory th also play a role as evidenced by their high frequency in lung biopsy specimens of covid- patients ( ) (figure ) . severe viral disease, associated with an over exuberant t cell response, leads to local inflammation and toxicity, thus activation of immune modulatory factors or checkpoints is crucial in limiting the damage. indeed, regulatory t cells (tregs) play a key role in the control of inflammation and immunopathology in other coronavirus infections ( ) , but this has yet to be investigated for sars-cov- (figure ) . most activated t cells subsequently apoptose and die, but some persist and provide long-term virus-specific t cell immunity. natural infection with sars-cov- induced long-lasting memory t cells up to years after infection ( ) and memory t cells persisted to years after mers infection ( ) , supporting the potential for long-term persistence in sars-cov- infection. these should provide protective immunity on subsequent exposure to the pathogen, the ultimate goal of an effective vaccine. severe covid- patients exhibit immunological features associated with t cell anergy or exhaustion. for example, the lungs of severe covid- patients contain high levels of cd + t cells expressing classic markers and genes of exhaustion ( ) , while patients who have recovered from moderate or severe covid- seem to have robust memory t cell formation ( ) . these infiltrating cells also express high levels of markers of activation and cytotoxicity. other studies have similarly demonstrated that more activated and, in some cases, more exhausted t cells develop in symptomatic covid- patients compared to levels during the prodromal stage ( ) . those who experience mild-moderate disease maintain their lymphocyte counts and have more polyfunctional t cells; while studies in those with severe disease are contradicting, reporting both lower and higher cd + t cell cytotoxicity ( ) . severe disease is associated with profound lymphopaenia lending support to the theory that the disease also causes immunosuppression, although lymphocyte trafficking to the site of infection may also be responsible ( ) . a biphasic t cell response has therefore been proposed, characterized by an early cd + cytotoxic t cell response to control the virus followed by t cell exhaustion and depletion as a result of viral persistence over many weeks in some patients. this model could account for poorer outcomes in the elderly who have diminished t cell repertoires and better outcomes in children who have a diverse and abundant naïve t cell pool ( ) . nevertheless, the likely early protective role for cd + and cd + t cells in sars-cov- clearance suggests that their induction should be exploited in sars-cov- vaccine development strategies. no vaccine is licensed or available for sars or mers, however, considerable preclinical development studies have been performed. a range of vaccine development approaches were explored, including live attenuated vaccines and inactivated vaccines (such as inactivated whole virus), soluble protein vaccines, viral vectors, nanoparticles, and dna vaccines; most of these platforms are also being utilized for sars-cov- , as discussed below ( , ) . most of the sars and mers subunit candidate vaccines have targeted the s glycoprotein and been examined in studies conducted in animal models, although n, m, and e protein-based vaccines have also been tested ( ) . however, the fact that animal models, including mice and nonhuman primates, display only limited clinical manifestations of sars and mers, that it is not usually lethal, severely limit the translation of results into humans. to overcome this, animal adapted strains and transgenic animal models of severe disease have been developed ( , ) . in virus challenge studies in animals, high titers of nabs to both sars-cov- and mers virus correlated with protection ( , ) . relatively few studies have investigated the protective role of t cells, and while some correlate cd + and cd + t cell responses with protection, adoptive transfer and t cell depletion assays in mice suggest they are not necessary for protection in mice immunized with a dna-based sars vaccine ( ) . furthermore, vaccination of mice with sars and mers candidate vaccines has been shown to result in th lung pathology with eosinophilic infiltration following wild-type virus challenge ( ) ( ) ( ) ( ) . this immunopathological effect has been associated with whole virus vaccines with and without adjuvant and linked with responses to the n protein in particular; s protein-based vaccines may have a lesser effect. an n protein-based dna sars vaccine also elicited a delayed type hypersensitivity reaction in mice, further cautioning against using this protein for sars-cov- vaccines ( ). furthermore, s-based vaccines appear more immunogenic and protective in these studies and the duration of protection has been shown in challenge studies to last - months in mice immunized with a vesicular stomatitis virus (vsv)-based sars vaccine and animals immunized with dna and/or protein-based mers vaccines ( , ) . of note, in the former study, protection against viral challenge was complete in younger mice but limited in senescent mice, highlighting potential issues with successful vaccination in older individuals ( ) . the only sars vaccines to reach human phase clinical trials were based on inactivated virus, soluble s glycoprotein and dna approaches ( ) . mers vaccines tested in human phase clinical trials include a dna-based vaccine alone ( ) ; dna in combination with adenovirus or modified vaccinia ankara (mva) viral vectors as a prime-boost strategy ( , ) ; and a chimp adenovirus vaccine ( ) . efficacy against disease has not been tested in humans since it has not been deemed ethical to perform challenge studies with lethal viruses for which there is no effective treatment. the first sars-cov- genome was isolated from patients with pneumonia from wuhan, china in early and identified as a novel human coronavirus ( , ) . since that time, multiple viruses have been isolated and sequenced from patients throughout the world providing an understanding of the evolutionary capacity and diversity of this pathogen ( ) . structural genomics and molecular interaction analysis (interactomics) can be used to rapidly identify putative functional sites, facilitating rational vaccine design by identifying potential b and t cell epitope regions of key viral proteins ( ) . several groups have used computational programs and immunoinformatics to predict cd + and cd + t cell and b cell epitopes across multiple sars-cov- antigens from pathogen sequence data to design novel sars-cov- vaccines ( , ) . the genetic diversity and stability of these regions over time can then be characterized, and cross-reactivity predicted and examined to ensure a vaccine provides cross-strain immunity. the s gene of human coronaviruses is well-known to undergo genetic drift which could compromise sars-cov- vaccines based on this region ( , ) . reassuringly, comparisons between the sequences of known sars-cov- b and t cell epitopes from the n and s proteins have been shown that some of the epitopes map identically to proteins from sars-cov- ( ). importantly, these epitopes showed no mutations among available sars-cov- sequences suggesting that they reside in stable parts of the s protein. the t cell epitopes also covered a broad range of hla alleles and could therefore be future targets for vaccine design. one issue with this approach is that the epitopes will be specific for human hla alleles, making it difficult to test them for protective efficacy in animal models. one way to overcome this problem is to use humanized mouse models which have functional human immune systems ( ) , bearing in mind that mice can also be modified to express the ace receptor ( ) . also, there are often animal homologs of human cd and cd t cell epitopes so it may still be possible to screen for efficacy. however, it should be borne in mind that due to the urgent need to develop an effective sars-cov- vaccine, animal challenge data are not a mandatory pre-requisite to progression to clinical trials; nevertheless, ideally these should be conducted as part of the vaccine development strategy. the recent development of a laboratory-adapted sars-cov- strain that is pathogenic in mice, provides an ideal animal challenge model for testing the efficacy of sars-cov- candidate vaccines in mice ( ) . vaccine technology has been advancing rapidly and there is a plethora of approaches to constructing sars-cov- vaccines. these range from the original technique employed centuries ago in the smallpox vaccine development of using modified or killed whole organism, to protein and peptide-based vaccines, nucleic acid dna and rna vaccines and nanoparticle constructs. each approach has unique advantages and disadvantages including varying time to development and scalability, cost, stability, as well as anticipated safety and immunogenicity profiles; all approaches are represented among preclinical and clinical trials ( - ) (figure ). some vaccine candidates, particularly protein-based vaccines, require an adjuvant to boost their immunogenicity ( ) . to date, very few adjuvants have been licensed for human use since the original adoption of aluminum salts (alum) from the early s, which bias immunity toward a th response ( ) . several of the newer adjuvants consist of toll-like receptor agonists to stimulate innate immunity combined with alum, such as as which is based on the tlr ligand monophosphoryl lipid a (mpl). oil-in-water emulsion adjuvants including mf and as are stronger adjuvants than alum and have been adopted in several licensed influenza vaccines. the adjuvant as , present in the malaria vaccine rts,s, combines mpl and a saponin qs- ( ) . a wide variety of adjuvant approaches are being taken with the various sars-cov- vaccines in development, including those mentioned above, some of which are described in the relevant sections below ( ) . many vaccines are given via the intramuscular route, including the majority of the current covid- vaccines in development ( table ) . however, since sars-cov- causes infection via the respiratory tract, a vaccine targeting the mucosal immune system might be preferable ( ) . mucosal vaccines have the advantage of being needle-free and thus practical for mass-administration, but immune tolerance induction can be an issue. the formulation, including the use of mucosal adjuvants, is therefore important to ensure stimulation of adequate local and systemic immunity ( ) . several groups have developed mucosal sars-cov- vaccines. a deoptimized live attenuated whole virus vaccine is being developed for intranasal (i.n.) administration, and several adenovirus-based vaccines will also be administered via the i.n. route ( table ). an oral probiotic pill-based sars-cov- dna vaccine (bactrl) is also planned for clinical trials ( table ) . the sublingual route is also considered an attractive route for inducing mucosal immunity ( ) . an alternative approach is to combine parenteral vaccines with adjuvants such as retinoic acid and caf which are known to induce protective iga mucosal responses ( , ) . researchers are also investigating self-administered mechanisms for sars-cov- vaccines to overcome the need for a healthcare worker to administer the vaccine. for example, inovio tm have developed a novel hand-held cellectra r intradermal delivery device which uses a brief electrical pulse to reversibly open skin cell pores to allow the entry of their ino- dna plasmid vaccine ( ) ( table ). the university of pittsburgh school of medicine has developed an intradermal microneedle patch that can deliver sars-cov- protein antigens, either s or rbd (pittcovacc), through the skin which will enter human trials soon ( ) ( table ) . this approach delivers antigen and danger signals to the high-density dermal apcs stimulating a highly effective immune response, even in the absence of adjuvant. it requires only low doses of antigen thus reducing production costs. indeed, this method induced more potent s -specific nabs in mice than the traditional needle injections, with or without the inclusion of tlr ligand sequences ( ) . the development of live attenuated vaccines requires the organism to be modified via passage multiple times under unique conditions in the laboratory until it loses enough key virulent protein and peptide subunit vaccines are usually combined with an adjuvant in order to enhance immunogenicity. the main emphasis in sars-cov- vaccine development has been on using the whole spike protein in its trimeric form or components of it, such as the rbd region. multiple non-replicating viral vector vaccines have been developed, particularly focused on adenovirus; while there has been less emphasis on the replicating viral vector constructs. nucleic acid-based approaches include dna and mrna vaccines, often packaged into nanocarriers such as virus-like particles (vlps) and lipid nanoparticles (lnps). nanoparticle and vlp vaccines can also have antigen attached to their surface or combined in their core. the immune cell therapy approach uses genetically modified sars-cov- -specific cytotoxic t cells and dendritic cells expressing viral antigens to protect against sars-cov- infection. each of these vaccine approaches has benefits and disadvantages in terms of cost and ease of production, safety profile and immunogenicity, and it remains to be seen which of the many candidates in development protect against factors so as to not cause disease, but retains its ability to replicate in the vaccine recipient, and stimulate a robust immune response that protects against infection ( ) ( ) ( ) . these vaccines tend to be highly immunogenic, do not require an adjuvant and provide long-lasting immunity. however, they are usually contraindicated in those immunocompromised or pregnant. it is difficult to develop a live attenuated sars-cov- vaccine quickly because of the time and knowledge required to ensure that it is suitably attenuated and all virulence factors removed. reverse genetics can be employed for the rational design of the ideal attenuated sars-cov- vaccine virus, for example by determining key virulence factors, as has been done for other coronaviruses ( ). long-term maintenance of consistent live vaccine stocks is also problematic ( , ) . despite the challenges, three codon-pair deoptimized live attenuated vaccines are currently undergoing pre-clinical testing, but none have yet progressed to clinical trials ( ) ( table ) . these three candidates are being developed by codegenix/serum institute of india, indian immunologicals ltd/griffith university and mehment ali aydinlar university in turkey, respectively ( ). this approach relies on using a single virus strain which may not cross-protect against other strains, particularly as the virus continues to spread worldwide and mutates as selection pressure increases once immunity becomes more widespread. inactivated vaccines are produced by growing the virus and then killing or "deactivating" it so that it can no longer replicate. the whole virus can be used, although alternative approaches include splitting the virus with a detergent to disrupt it or purifying antigenic components to create a subunit vaccine ( ) . these vaccines are safer than live-attenuated vaccines but less immunogenic and often require an adjuvant. there are currently four inactivated vaccines in human clinical trials and a further nine in the preclinical stages of development (tables , ). an alum adjuvanted purified formaldehyde inactivated whole virus vaccine developed by sinovac biotech., called picovacc, has been shown to be immunogenic in mice, rats and non-human primates ( ) . it induced nabs to both vaccine and non-vaccine strains and provided partial or complete protection of macaques in challenge studies and has now entered human clinical trials in china ( pre-clinical data confirming induction of high levels of nabs in several animal models and protection against intra-tracheal sars-cov- challenge in rhesus macaques after doses of vaccine ( ) . the institute of medical biology/chinese academy of medical science has also developed an inactivated whole virus construct that is in a phase / trial; and bharat biotech international ltd also has one in a phase / trial ( table ) . rather than using the whole or a split fragment of the virus as the vaccine, antigenic proteins (or peptides) of the virus can be generated using recombinant technology in the laboratory instead ( ) . this is the most popular approach in the sars-cov- vaccine development field, with protein/peptide constructs in preclinical trials and that have entered clinical trials ( table ) . they are relatively simple vaccines to make and thus cheap to produce. a major advantage over whole virus vaccines is their relative safety since unnecessary reactogenic components such as lipopolysaccharides and toxins can be excluded. however, protein/peptide vaccines often require adjuvants and multiple doses in order to stimulate an adequate immune response. peptide vaccines usually consist of - amino acid b cell and t cell epitope regions of key antigens allowing for a precise and targeted immune response. peptidebased vaccines have been developed as candidates against several viral pathogens, but few have been licensed except for use in veterinary practice ( ) . this is in part due to their inherent limitations including a narrow breadth of immune response, potential for incorrect confirmation for epitope recognition, lack of b cell receptor cross-linking for b cell epitopes, hla restriction for t cell recognition, failure to induce cross-reactive immunity against different viral strains and failure to elicit longlasting immunity. the use of whole proteins in vaccines broadens their immunogenic potential, however the protein can also lose its native structure and thereby lose immunogenicity. in addition, as knowledge of the immune response to sars-cov- is still in its early stages, so too is the understanding of precisely what may be the most conformationally optimal, immunogenic and safe protein or peptide to utilize; this will be discovered as vaccine candidates using this approach are examined in clinical trials. the majority of sars-cov- candidate vaccines in development are based on the s protein or its rbd region (figure ) ( ) . indeed, five of the current protein/peptide vaccine candidates in clinical trials target the full s protein and the other two target rbd only ( table ) . some novel technologies have been employed to overcome the issue of the s glycoprotein losing its immunogenic conformational form. the university of queensland has developed a molecular clamp technique that uses proteins to stabilize the s protein in its coiled shape. this seeks to ensure that functional nabs are produced that will tightly bind to virus in its native form and thus prevent cell entry ( ) ( table ) . other groups have focused on using powerful adjuvants to ensure immunogenicity, for example novavax's recombinant s protein sars-cov- nanoparticle vaccine, nvx-cov , combined with an adjuvant called matrix-m tm . this is a saponin-based adjuvant that enhances apc recruitment and action and stimulates high levels of nabs and cell-mediated immune responses ( ) . the s protein in nvx-cov is present its native trimeric form and has been shown to stimulate high levels of nabs in mice and baboons in preclinical studies. primary phase clinical trial results have just been reported from a randomized, observer-blind, placebo-controlled trial of nvx-cov conducted in healthy adults ( ) ( table ) . safety and immunogenicity were assessed for doses of vaccine ( and µg) with (n = ) or without (n = ) the matrix-m tm adjuvant (a µg dose). a strong correlation was observed between nab titers and anti-spike igg to s protein with the adjuvanted, but not the unadjuvanted, vaccine. levels were comparable to those in convalescent sera (r = . ). both and µg adjuvanted doses elicited responses of similar magnitude and every participant seroconverted by both assays. t cell responses measured in participants showed that nvx-cov /matrix-m induces antigen-specific polyfunctional cd + t cell responses (ifn-γ, il- , and tnfα) in response to spike protein stimulation; there was a strong bias toward this th phenotype. the safety data are discussed in section comparative safety and tolerability results for current sars-cov- vaccine candidates in clinical trials and this vaccine is now progressing to phase trials. clover biopharmaceuticals's protein trimer tag© vaccine is combined with as adjuvant; the uq vaccine uses mf adjuvant; the vaxine pty ltd. s protein vaccine has a special advax tm adjuvant; and the medigen s based construct is combined with the adjuvant cpg ( table ) . generex biotechnology, in partnership with epivax, have developed a sars-cov- vaccine (epv-cov ) based on li-key technology with undisclosed sars-cov- peptides ( ). this uses synthetic peptides chemically linked to the -amino acid ii-key to ensure robust immune activation of th and th cd + t cells in particular, which in turn facilitate antibody production ( , ) . multiple viral vectors have been used for vaccine delivery due to their ability to infect cells and deliver the gene product encoding antigenic proteins for production inside the host cell following administration, usually via the intramuscular (i.m.) route ( ) . vector viruses are usually genetically modified to reduce their virulence and render them replication defective. adenoviruses and poxviruses are the most widely used non-replicating viral vectors, but many other viruses can also be adapted for vaccine delivery. replicating viral vectors, such as measles virus and vesicular stomatitis virus (vsv), can also be used for sars-cov- vaccine design; they mimic natural infection, rendering them self-adjuvanting and therefore more potent, and can be used in lower doses ( ) . while subunit vaccines are more focused on induction of humoral immunity, viral vector vaccines are able to induce robust cell mediated immunity (cmi) in addition to abs and have been shown in animal models to protect against challenge with pathogenic coronaviruses ( ) . a modified vaccinia ankara (mva) vector expressing a mers-cov recombinant protein induced t cell responses and nabs in mice, while a rabies virus-based sars-cov- vaccine protected mice against sars-cov- challenge ( ). human adenoviruses are the most common non-replicating viral vectors being used for sars-cov- vaccine development; constituting of the in clinical trials and of the in preclinical development ( table ) . the three in clinical trials include cansino's ad -ncov, janssen's ad covs ( ) and gamelaya's ad -based gam-covid-vac lyo vaccine ( table ) . all three encode the s protein. while adenovirus vectors are well-tolerated and highly immunogenic in most people, preexisting immunity to the viral vector can hamper the induction of immunity, particularly after repeated doses. animal adenoviruses can be used as vectors instead of human ones to overcome this problem, which is the approach used for the oxford university chimpanzee adenovirus-based candidate sars-cov- vaccine chadox ncov- (also called azd ) and the simian adenovirus-based reithera vaccine grad, both of which are in clinical trials ( table ) . anti-vector immunity can still develop after the first dose of vaccine even if a simian adenovirus vector is used. despite the concern about anti-vector immunity, of the adenovirus-based sars-cov- vaccines in clinical trials are planned to be given as a single dose, whereas almost all the other vaccines in clinical trials require at least doses ( table ) . phase and trials have now been completed and published for the cansino (ad -ncov) and oxford (chadox ncov- ) , the safety aspects of which will be discussed later in this article. the ad -ncov randomized, double-blind, placebo-controlled phase trial results involving volunteers ≥ years of age showed > % seroconversion by rbd-elisa, but only % seroconversion of nab responses in the higher dose group and % in the lower dose group after a single immunization ( ) . fifty two percent of volunteers had pre-existing anti-ad nabs, and those with higher anti-ad immunity had approximately half the rbd-specific elisa and sars-cov- nabs as compared to those with low pre-existing anti-vector immunity, supporting interference with vaccine immunogenicity. preliminary findings from a phase / , single-blind, randomized controlled chadox ncov- vaccine trial conducted in , healthy volunteers aged - years induced nabs in % of participants after a single dose of vaccine, rising to % after a booster dose ( ). neutralization titers were comparable to those observed in convalescent plasma from people who had recovered from covid- . both vaccine constructs elicited t cell responses which peaked at days post-immunization, as determined by interferongamma (ifn-γ) enzyme linked immunospot (elispot) assay ( , ). day responses appeared higher with chadox ncov- (median per million peripheral blood mononuclear cells [pbmc] ) as compared to the highest dose used of ad -ncov (mean per million pbmc) in the phase trial ( ); ad -ncov elicited a median of - sfu/million pbmc at day in the phase trial ( ). intracellular staining confirmed production of ifn-γ, tumor necrosis factor (tnf) and interleukin- (il- ) by both cd + and cd + t cells following ad -ncov immunization, with more cytokine detected in cd + t cells as compared to cd + t cells ( ) . institute pasteur replicating viral vector vaccine candidate tmv- , based on measles virus, is the only viral vector vaccine that has entered phase clinical trials to date ( table ) , but there are more at the pre-clinical stage of development. these include each based on influenza virus or vsv, more measles virusbased candidates, and each based on avian paramyxovirus, horsepox (called tnx- ), newcastle disease virus, and yellow fever virus ( table ) . the majority target the s protein or rbd, although one measles virus-based candidate also targets the n protein. two of the influenza-based candidates are designed for intranasal administration. a live attenuated yellow fever vector-based vaccine (yfs ) expressing the prefusion form of the s protein recently showed that a single dose protected most hamsters in a sars-cov- challenge model ( ) . nucleic acid-based vaccines (dna or mrna) offer a costefficient and scalable approach to sars-cov- vaccine development ( ) . the major nucleic acid-based approaches are described below. dna-based vaccines are stable and safe to handle. however, while there are multiple dna vaccine constructs targeting numerous viral infections in animal and human studies, to date none have been licensed for human use. naked dna can be injected and taken up by apc or apc can be directly transfected with plasmid dna encoding the target antigen. subsequent expression and presentation of the target antigen leads to the induction of antigen-specific cd + and cd + t cells and antibody production. however, dna vaccines tend to be poorly immunogenic, necessitating various strategies to improve immunogenicity such as the use of viral promotors, immunostimulatory sequences and adjuvants. nanocarriers such a virus like particles (vlps) can also be used to improve dna delivery, avoid its degradation and be immunostimulatory. there are safety concerns about potential integration into the host dna causing dysregulated gene expression although the risk of this is thought to be very low. a series of animal studies with sars-cov- nucleic acidbased vaccines have shown induction of nabs and cellular immunity, with partial protection against turkey cov challenge ( ) and vaccine-induced ab-mediated protection to the s protein in mice ( ) . twelve dna-based sars-cov- vaccines are currently in preclinical development and more have entered phase / clinical trials ( table ) . two dna-based candidates in clinical trials are administered i.m., whereas the other are administered via the intradermal (i.d.) route ( table ) . inovio pharmaceuticals is testing their cellectra r electroporation i.d. delivery device to administer their s protein-based dna plasmid vaccine ino- which induces nabs and t cells in mice and guinea pigs ( ) ( table ). an alternative approach currently in pre-clinical trials is being taken by symvivo with their dna b. longum vaccine bactrl which is administered as an oral pill ( ) . messenger rna (mrna) vaccines consist of an rna strand that codes for a target antigen ( , ) . the two main types are those packaged and delivered in non-replicating form or those packaged with other rna as an in vivo replicating vaccine. they offer a promising alternative to conventional vaccine approaches due to their high potency, capacity for rapid development and potential for low-cost manufacture, which could prove crucial for global accessibility for a covid pandemic vaccine. they also have a good safety profile, since they are not made with pathogen and do not integrate into host dna. several challenges encountered by mrna vaccines include the need for packaging, for example into particles or liposomes, as naked rna is otherwise rapidly broken down and the need for freezing or refrigeration for wide scale delivery. there are currently rna-based vaccines in pre-clinical trials and have entered clinical trials ( table ) . the clinical trial candidates include two lipid nanoparticle encapsulated rna sars-cov- vaccines that have already progressed to phase trials ( table ) . moderna/niaid have published preliminary results from their phase dose-ranging, safety and immunogenicity trial of doses of mrna- encoding the s protein conducted in healthy adults aged - years ( ) . the s protein in mrna- is stabilized in its prefusion state by proline substitutions in the s subunit; and the lipid nanoparticle coat consists of lipids. all participants developed rbd abs by elisa and sars-cov- -specific nabs by neutralization assay after doses in the upper range of those who have recovered from covid- (convalescent serum). t cell responses measured by intracellular staining for th (tnf, il- , ifn-γ) and th (il- , il- ) cytokines following stimulation with an s protein peptide pool showed a th cytokine bias and low-level cd t cell responses. an interim report from the phase / dose-ranging study of doses of biontech's mrna s protein vaccine candidate bnt administered to adults aged - years elicited rbdbinding igg and sars-cov- nabs in the order of . - . fold that of convalescent human plasma. t cell responses were not reported in this paper. this vaccine encodes trimerized rbd which is modified by adding a "foldon" trimerization domain to increase immunogenicity ( ) . both mrna- and bnt are now in phase clinical trials in adults ≥ years ( table ). the safety data for both will be reviewed in a later safety section. the remaining rna-based candidates in clinical trials include three in phase and one in phase / ( table ) . the imperial college london lnp-encapsulated rna vaccine consists of an s protein-based self-amplifying rna construct (lnp-ncovsarna); designed because sarna vaccines induce a more stable dna product and are more immunogenic than conventional nucleic acid vaccines ( ) ( table ) . nanoparticle-based vaccine approaches have received increasing interest in recent years due to their good safety profile and high immunogenic potential, with an ability to efficiently target dendritic cells (dcs) for processing and presentation, providing a clear advantage over less immunogenic dna and rna vaccines ( ) . materials used to construct nanoparticle vaccines include self-assembling viruses (virus like particles [vlps]), lipids (as liposomes), proteins, metals (e.g., gold) and polymers which also act as their own adjuvant ( ) . many nanoparticles are highly stable and less prone to degradation than other constructs such as "naked" protein, peptide, dna and rna vaccines ( , ) . selected antigens, which may be proteins or nucleic acid, can either be attached to the surface of the nanoparticles or combined in the particle core. they can be synthesized in a variety of shapes and sizes to induce robust innate as well as adaptive immunity. other modifications include altering the nanoparticle surface to target certain cells or enhance immunogenicity and packaging with tlr ligands and other immune modulators. one of the most popular approaches for viral vaccine development is engineering vlps consisting of self-assembled viral membrane in a monomeric complex which display the viral epitopes but lack multiple key viral components, ensuring they have no replication capacity ( ) . the widely used human papillomavirus (hpv) vaccines are an example of this approach. a nanoparticle polypeptide vaccine displaying sars-specific b cell epitope repeats from the c terminal heptad repeat region of the s protein in its native coiled formation was shown to elicit nabs in mice ( ) . a number of the protein and nucleic acid-based sars-cov- vaccine candidates use nanocarriers to package the vaccine product to improve stability and ensure efficient antigen processing ( ) . as mentioned above, novavax's full length s protein construct nvx-cov is a nanoparticle vaccine and the majority of mrna-based sars-cov- vaccines are packaged lnps, including mrna- and bnt . there are sars-cov- vlp vaccines in preclinical development and just one in a phase clinical trial ( table ) . the latter vaccine developed by mendicago inc. consists of plant-derived recombinant vlps made by transfecting viral genes into the cell nuclei of leaves permitting transient expression of viral proteins which form into vlps which are extracted and purified for clinical use ( ) ( table ) . sars-cov- vaccine approaches based on car-t cell concepts are also being tested in clinical trials. the shenzhen genoimmune medical institute in china is using a lentiviral vector system to create viral minigenes which express viral proteins (s, m, e and n proteins) and immune modulatory genes (p polyprotein protease) to modify dendritic cells (dcs). the lv-dc presenting sars-cov- specific antigens will activate cytotoxic t cells. novel dc (lv-smenp dc) and antigenspecific cytotoxic t cell vaccines are currently being tested by s.c. injection or i.v. infusion in a phase / multicenter therapeutic clinical trial in participants ranging from months to years of age ( ) ( table ) . the same company has also developed artificial dcs expressing sars-cov- mini-genes for various viral proteins which are being tested in a phase clinical trial as a s.c. injection in sars-cov- infected individuals in the same age range ( table ) . autologous dcs expressing sars-cov- antigens have also been developed by aivita biomed inc. in usa and are being tested as a s.c. administered vaccine in phase clinical trials in healthy adults > years of age ( ) ( table ) . whilst not suitable for large-scale delivery and use, immune cell therapy might be used in the context of pre-emptive therapy in high-risk patients such as the elderly and immunocompromised. there is increasing evidence that immunization with live vaccines can improve survival against non-vaccine targeted infections, a phenomenon termed "non-specific, " "off-target, " or "heterologous" effects of vaccines ( , ) . some of the best evidence comes from studies of immunization with the tuberculosis vaccine bacillus calmette-guérin (bcg); for example, bcg has been shown in three randomized trials to markedly reduce all-cause mortality in low birthweight neonates ( ) . bcg causes epigenetic modification of innate immune cells (monocytes and natural killer (nk) cells) thereby enhancing innate responses in a process called innate immune training ( , ) . experiments in both murine models and humans have further shown that bcg protects against certain viral pathogens ( ) . reports that people living in countries where childhood bcg is routinely given have lower covid- mortality have been posited as further evidence for protective effects of bcg ( ). however, these observations are fraught with confounders, including difficulty ascertaining covid- infection rates in these countries, the time the virus first entered the country and differences in national control strategies ( ) . furthermore, other studies contradict these findings, such as a geographic regression discontinuity analysis along the east and west german border showing that the covid- infection rates do not vary according to the bcg vaccination strategies employed during the cold war ( ) ; and a study from israel showing that sars-cov- infection rates in adults did not differ by bcg at birth status ( ) . even if childhood bcg immunization does not protect against covid- , a bcg dose as an adult may provide some short-term protection. indeed, it has been widely postulated that the nonspecific beneficial effects of bcg might protect against covid- , resulting in an explosion of interest in this vaccine as a protective strategy in recent months ( , ) . this has led to a number of clinical trials being established in north and south america, australasia, africa and europe, all testing the ability of bcg vaccine to protect against sars-cov- infection, mostly in high-risk exposed healthcare workers ( ) ( table ) . these trials collectively aim to recruit > , participants, with the largest trial of , healthcare workers currently recruiting across multiple sites in australia and europe. non-specific protective effects of live vaccines have also been postulated for oral polio vaccine (opv) ( ) and measles vaccine ( ) . as a result, a study of the protective effects of opv against covid- is due to commence in usa in june ( ) , and a clinical trial of measles-mumps-rubella (mmr) immunization and covid- protection is being conducted in egypt ( table ) . a consistent feature of the off-target effects of live vaccines has been their sex-differential nature; females often showing greater susceptibility to these immunomodulatory effects ( , , ( ) ( ) ( ) . indeed, sex differences in targeted vaccine immunogenicity and adverse events have also been widely described ( , ) . this raises the possibility that any off-target protective effect of live vaccines may differ between the sexes and that the sars-cov- vaccine candidates may show sex differences in immunogenicity and reactogenicity. it is possible, but uncertain, that strategies to use these already licensed vaccines may provide a modest level of protection against covid- , including those at highest risk, such as healthcare workers. it is important to recognize, however, that data from these studies will still need to accrue whilst we are waiting for sars-cov- targeted vaccines to come through the pipeline. given the unprecedented speed at which targeted vaccines are being developed, this approach may not be necessary or required; furthermore, these existing live vaccines are not currently recommended for this indication, so should only be utilized in this context in the setting of a clinical trial. even if an efficacious vaccine is developed there are a number of immunological challenges that need to be considered. furthermore, there is the enormous challenge of mass production and rapid and equitable delivery ( table ) . it is extremely unlikely that any of the sars-cov- vaccines will be % effective; while they may not prevent becoming infected, it is hoped that they will prevent progression to severe disease. indeed, the seasonal influenza vaccine is generally about % protective against infection but does decrease disease severity and hospitalization rates ( ) . a recent study in which macaques were vaccinated with the oxford university and astrazeneca adenovirus vaccine, chadox ncov- , found that the primates were protected from sars-cov- -induced pneumonia ( ) . however, the macaques still had high levels of virus replicating in their upper respiratory tract. it is hoped that even if the vaccines do not prevent infection in the upper airways, they may reduce viral load and disease severity and in turn, the amount of virus a vaccinated person transmits to others. even a modestly efficacious sars-cov- vaccine could mitigate the severity of this pandemic and be highly beneficial in a world struggling to contain this novel virus and its devastating social and economic effects. most vaccines are tested in healthy young adult males and non-pregnant women and, if safe, they are then tested in healthy children prior to licensure. this therefore raises the issue that any vaccines may initially have less empiric data available on use in certain key vulnerable populations such as the elderly, immunocompromised groups and pregnant women. it is plausible that vaccines may be considerably less immunogenic in older and frail elderly who experience the most severe outcomes from covid- , hence the importance of adjuvants in many of the vaccine candidates, both for dose sparing and enhancing immunogenicity ( ) . efficacy in this population is also likely to vary by the type of vaccine construct and adjuvant used. this has been seen for other diseases such as influenza, where the adjuvanted and high dose vaccines are more immunogenic than the standard inactivated vaccines in the elderly ( , ) . similarly, for herpes zoster, the efficacy of the live-attenuated vaccine in the elderly is approximately % compared with > % for a subunit adjuvanted vaccine (glycoprotein e and liposomebased as b adjuvant) ( ) . thus, ensuring studies can be conducted in these priority target groups, either pre-or earlypost deployment, will be critical and special formulations may be required to ensure adequate immunogenicity. throughout life, humans are repeatedly exposed to the endemic human seasonal coronaviruses and develop human cov (hcov) antibody repertoires that can potentially cross-react with sars-cov- -specific antigens ( ) . this early immune imprinting leads to preferential expansion of cross-reactive abs when a related antigen is encountered, in this case sars-cov- ( ). this long-recognized process is called original antigenic sin (oas) ( ) and is known to occur with several common viruses, such as influenza and respiratory syncytial virus ( , ) . oas can either lead to a less effective immune response or cause enhanced immunity and immunopathology ( , , ) . oas is one of the mechanisms responsible for the phenomenon of antibody dependent enhancement (ade) of immunity which is thought to occur in covid- , whereby non-neutralizing cross-reactive abs against other coronaviruses enhance host cell entry and viral infectivity and worsen disease severity ( , , ) . this was seen to occur in cats vaccinated against feline infectious peritonitis coronavirus ( ) ( ) ( ) and certain sars and mers vaccine platforms also appear to have shown worsened immunopathology in animal challenge studies compared with placebo ( ) . recently, it was shown in vitro using human cells that nabs to coronavirus s protein can also trigger ade by causing a conformational change of the s protein ( ) . antigen-experienced older individuals are more susceptible to the phenomenon of ade, while less antigen-experienced younger individuals mount more targeted antibody responses to viral neoantigens. this may in part account for the greater immunopathology of covid- in older individuals. indeed, it has been shown recently that covid- -naïve children and elderly have quite different cross-reactive sars-cov- -specific antibody signatures, which would play differing roles upon challenge with the wild-type virus ( ) . theoretically, a sars-cov- vaccine could skew the immune system toward production of less effective or even harmful cross-reactive hcov abs via these processes of oas and ade, rather than induce highly-targeted sars-cov- -specific abs as intended ( ) . indeed, anti-spike abs taken from critically unwell covid- patients with severe lung injury skewed macrophages in vitro into a pro-inflammatory phenotype, implicating the abs in driving the surge in lung-resident proinflammatory macrophages and subsequent pro-inflammatory cytokine release in the lungs ( ) . the authors attribute an aberrant igg glycosylation pattern in severe covid- patients with their more pro-inflammatory properties so hopefully the iggs induced by vaccination will not have this problem. indeed, none of the current sars-cov- vaccines in human clinical trials have reportedly caused ade to date, although the number of human subjects studied to date is still relatively small. since animal challenge data are not required to progress to clinical sars-cov- vaccine trials, the opportunity to screen for potential adverse events including ade at the pre-clinical stage of development may be missed. having said that, many of the sars-cov- vaccine candidates have undergone considerable pre-clinical testing, including challenge studies. there was no evidence of ade in pre-clinical sars-cov- challenge studies in rhesus macaques following immunization with dose of chadox ncov- ( ) or doses of the inactivated whole virus vaccine candidate bbibp-corv ( ), both of which were protective in these studies. it is postulated that basing the vaccine on the s rbd terminal subunit of the s glycoprotein should overcome this problem by inducing nabs only, although this has not been proven and, notably, only a few vaccine candidates have taken this approach. furthermore, the in vitro conformational changes causing ade described above occur in the rbd domain, so this approach may not be successful. aberrant manifestations of t cell responses were observed in the s for other viral vaccine candidates, such as inactivated vaccines against measles and respiratory syncytial virus (rsv), resulting in vaccine associated enhanced respiratory disease (vaerd) upon subsequent wild-type pathogen exposure ( , ) . a major driver of this was accentuation of th cytokines, with resultant allergic (eosinophilic) and airway hyperesponsiveness. this was compounded by another mechanism related to the conformation of the vaccine antigen, resulting in the generation of excessive non-neutralizing ab. having a high amount of binding, but not neutralizing, ab caused immune complex deposition and complement activation with local inflammation in the presence of a high viral load. it will therefore be important for vaccine candidates to mitigate the risk of vaerd by considering the t cell profile induced by vaccination, avoiding th biased cd + t cell immunity and biasing toward cd + cytotoxic t cell responses. the alum adjuvanted inactivated vaccine candidate picovacc could theoretically drive a th skewed immune response. while this was not observed in macaque studies when analyzing th and th cytokine profiles, it is not clear from the paper what samples were used to measure these cytokines, clarification of which will be crucial in vaccine safety assessment ( ) . many of the other vaccine constructs favor th immunity, as demonstrated in the published human trial results for several of the viral vector ( - ) and mrna vaccines ( ) and the novavax nanoparticle matrix m adjuvant vaccine ( ) . original antigenic sin also applies to t cell responses and therefore pre-existing cov-specific t cell memory may be enhanced by immunization with a sars-cov- vaccine leading to either suboptimal t cell responses or immunopathology ( , ) . vaccine safety assessment is integrated into all stages of the clinical development pipeline for candidate vaccines and continues once vaccines are deployed into the population. randomized controlled clinical trials examine safety, as well as immunogenicity and efficacy. decisions on the participant numbers to be included in phase studies will likely be powered on efficacy endpoints, but these studies also need to be of sufficient size and planned duration to ensure comparison of injection site, systemic and unanticipated or "unsolicited" adverse events between groups. comprehensive safety studies are particularly critical because some candidate vaccines use platform technologies that have not been examined extensively in human subjects to date, including some of the viral vectors, mrna and nanoparticle constructs, and because of the potential for enhanced disease and adverse events related to aberrant immune responses to be seen upon infection pre-and post-licensure. a list of adverse events of special interest (known as aesi) across all body systems, including immunological, cardiovascular, neurological, musculoskeletal and dermatological manifestations, have been agreed by the brighton collaboration in conjunction with cepi and the who with input from regulatory agencies and other experts, as have the associated case definitions and surveillance strategies ( , ) . listed conditions include anaphylaxis, vasculitides, myocarditis, generalized convulsions and meningoencephalitis, among many others. comprehensive data on safety are essential to ensure that the benefit:risk ratio of vaccination is favorable, to support decision-making by policy makers on wide-scale program implementation among healthy people, and to ensure that individuals are sufficiently confident to accept vaccination. some of these aesi will require post marketing (phase ) studies but are also undergoing evaluation in some of the preclinical and early phase evaluations, particularly the larger phase studies. interim phase / safety results have recently been published for two leading adenovirus-based vaccines, chadox ncov- ( ) and ad -ncov ( , ) ; two mrna based vaccine candidates, mrna- ( ) and bnt ( ) ; an aluminum adjuvanted inactivated whole virus vaccine ( ) ; and an adjuvanted recombinant protein nanoparticle vaccine, nvx-cov /matrix-m ( ) . all candidates exhibited acceptable safety profiles, and while local and systemic reactogenicity was common for the mrna, adenovirus and adjuvanted protein nanoparticle constructs, the inactivated whole virus vaccine was considerably less reactogenic ( table ) . reactions were generally mild to moderate and resolved within days. importantly, there were no serious adverse events reported for any of these vaccine candidates. pain at the injection site was a particularly common feature with the mrna vaccines (approaching % in some groups), and while it was the commonest reaction to the inactivated vaccine, it was still lower for this candidate than for the other vaccines (table ) . headache, myalgia and fatigue were the other most commonly reported symptoms for the mrna, adenovirus and adjuvanted protein nanoparticle candidate vaccines ( table ) . documented fever was relatively common for mrna and adenovirus candidates but ≤ % for the inactivated virus and adjuvanted protein nanoparticle candidates ( table ). the chadox ncov- trial underwent a protocol amendment during the trial allowing several sites to administer paracetamol prior to vaccination and hourly for h. this slightly reduced reports of pain, feeling feverish, chills, mylagia, headache and malaise but had no effect on confirmed fever. pre-existing adenovirus immunity, increased age and male sex were all associated with decreased fever following immunization with ad -ncov ( ) . in terms of blood parameters, immunization with chadox ncov- was associated with transient neutropaenia and bnt b with lymphopaenia. in the ad -ncov phase and trials reactogenicity was dose-dependent for pain, headache and fever; while it was dosedependent for almost all parameters for the mrna vaccines ( table ) . as a result of the high reactogenicity after a single dose of bnt b , it was decided not to give a second dose. interestingly, the ten participants given a booster dose of the chadox ncov- vaccine had less reactogenicity after the second dose. the opposite was found for the bnt- b mrna and nvx-cov /matrix-m vaccines for which reactogenicity was greater after the second dose ( , ) . these favorable safety results (alongside the good immunogenicity reported earlier in this article) have allowed the progression of the adenovirus and mrna candidate vaccines to phase clinical trials. whilst the sars-cov- vaccine pipeline is progressing at unprecedented speed, there is a concern that suppression of viral transmission in many countries will make evaluation of vaccine efficacy (ve) difficult, as phase studies need sufficient infection rates to compare disease incidence in vaccinated with control individuals. reassuringly, planned studies are currently expanding to higher burden countries where infrastructure to conduct complex adaptive clinical trials exists. nevertheless, the time needed to accrue sufficient data on efficacy will be a critical determinant impacting vaccine availability. in the setting of this uncertainty "human challenge models" for sars-cov- have been proposed. this methodology involves the intentional infection of research participants, providing safety data and pointing to immune correlates of protection. this approach has recently advanced vaccine development for infections like salmonella typhi (typhoid vaccine) and group a streptococcus ( , ) . the difference between these bacterial human challenge models and sars-cov- is that these pathogens have been researched for decades and have an effective antibiotic "rescue" treatment available. infecting a human with sars-cov- to test vaccine efficacy raises numerous ethical issues ( ) , including those around informed consent of the healthy volunteer, noting that while severe covid- is less common in young adults, deaths have still occurred in this age group. strict infection control and personal protective equipment (ppe) measures would also be required to limit "third-party" risk to staff co-ordinating any studies. while it is uncertain if this approach will be required, the who is progressing a framework, including key criteria, that will be required by ethical review boards in order to facilitate "human challenge" trials ( ). as noted by stanley plotkin, "extraordinary diseases require extraordinary solutions" ( ) . it normally takes decades for a vaccine to be developed and licensed for use. however, the race is on to develop a sars-cov- vaccine for worldwide distribution in an unparalleled timeframe to vaccinate the world's population and provide widespread herd immunity. this is already being facilitated by rapid progression through the normally slow bureaucratic regulatory and approval processes and unprecedented worldwide collaboration between governments, universities, pharmaceutical companies and philanthropic organizations; such efforts must continue. in addition, ensuring good public communication regarding the safety and effectiveness of the vaccine will be key to gaining public trust and acceptance of a vaccine that could be seen as rushed. additionally, having carefully designed post-marketing (phase ) surveillance is vital to detect rare or unexpected safety signals and aesi which may only be detected once the vaccine is rolled out on a mass scale. a key player in global access to a sars-cov- vaccine is the not-for-profit coalition for epidemic preparedness innovations (cepi) which is working with global health authorities and vaccine developers worldwide to support sars-cov- vaccine development ( , ) . cepi was founded in with the consensus that new and sustainable models of partnership are needed to respond to worldwide infectious diseases threats. founding members include the bill & melinda gates foundation (bmgf), wellcome trust uk, world economic forum, india department of biotechnology and the government of norway. cepi's goal is not just to advance development, but to also advance licensing, manufacturing, delivery and stockpiling of vaccines once an effective vaccine has been developed ( ). it has raised approximately $ . billion for sars-cov- vaccine development and initiated a series of partnerships in a bid to advance frontrunner candidate sars-cov- vaccines. cepi is investing across a range of vaccine technologies including novavax's nanoparticle vaccine (nvx-cov and matrix-m tm ), clover biopharma's s-trimer protein-based vaccine (scb- ), university of queensland's molecular clamp s protein vaccine, university of oxford's adenovirus vector vaccine (chadox ncov- ), inovio's dna plasmid vaccine (ino- ) and moderna's and curevac's mrna vaccines ( ) . many other government and philanthropic organizations are injecting large sums of money into the effort to develop effective sars-cov- vaccines. the european commission has registered an impressive $ billion in donations toward the development and deployment of vaccines, treatments and diagnostics against sars-cov- ( ). the us government agency biomedical advanced research and development authority (barda) has pledged almost $ million to accelerate the development of moderna's mrna- vaccine, with phase trials expected to begin soon ( ) . barda has also funded janssens's adenovirus (ad ) i.n. vaccine, astra zeneca's azd vaccine (formerly chadox ncov ), merck and iavi's rvsvg-cov and sanofi's recombinant sars-cov- protein vaccine and novavax's nanoparticle matrix m adjuvant vaccine ( ) , demonstrating the range of vaccine strategies being supported. importantly, these phase / studies are being conducted in countries with high disease burden (e.g., north and south america and south africa), so the vaccine efficacy (ve) and safety results can be obtained as rapidly as possible whilst meeting the vaccine regulators' requirements. the logistics of scaling up to produce the billions of doses required to immunize the world's population is an onerous task. it is not yet clear how long immunity will last and therefore whether booster doses or annual immunization will be required. furthermore, multiple initial doses may be needed and the vaccine may have to change as the virus evolves naturally. barda have pledged that they will scale up to million annual sars-cov- vaccine doses in the us each year under operation warp speed, while bmgf have recently awarded $ million to inovio's dna based vaccine ino- with the intention of providing over million doses by the end of . novavax has completed phase studies and is progressing rapidly to phase studies, with the aim of producing up to million doses by the end of and entering large-scale manufacturing of billions of doses in ( ) . curevac has a gmp-certified production facility that can produce million doses of their mrna vaccine in a single production run and million doses of azd (chadox ncov- ) will be available by july ( ) . even if sufficient sars-cov- vaccine doses can be manufactured, worldwide delivery presents another major logistic and financial hurdle. storage requirements will be enormous and the vaccine may need to be either frozen or refrigerated, presenting cold-chain issues. the decision about whether to prepare the vaccine in single-dose or multi-dose vials will impact manufacturing, storage, delivery and potential infection risks ( ) . infrastructure and manpower will be required to distribute and administer the vaccine. equity will be a major issue since richer countries may procure the vaccine for their citizens while poorer countries may not be able to afford it. cepi is negotiating global access upfront in order to ensure equitable access, hopefully averting "vaccine nationalism" ( ) . in early may, who launched the access to covid- tools (act) accelerator, which aims to handle the logistics of vaccine procurement and allocation ( ) . covax, co-led by cepi, gavi and who, is the vaccine pillar of act charged with accelerating vaccine development and manufacture and guaranteeing fair and equitable worldwide access. in an open letter, more than world leaders have united and called for a sars-cov- vaccine to be freely available to all people in all countries of the world in what they call "the people's vaccine, " which is surely the fairest way to tackle this unprecedented global pandemic ( ) . by contrast, pneumococcal conjugate vaccine which has been available for years, is still not included in the immunization programs of many countries due to lack of affordability, many children are left unprotected and about half a million deaths from pneumococcal disease each year. initially, as vaccine production commences, it will not be possible to deliver a sars-cov- vaccine to the entire world population and it is anticipated that key vulnerable populations will need to be targeted. these would likely include healthcare workers, the elderly and those with significant risk factors such as those with co-morbidities or the immunocompromised. it might even be used as a travel vaccine as borders re-open across the world, but is unlikely to be incorporated into infant schedules for some time given the very low risk of severe disease in that age group and time needed to conduct pediatric studies. while the world eagerly awaits effective sars-cov- vaccines as the solution to ending this pandemic, it should be borne in mind that there are many caveats even if robust sars-cov- specific nabs, cd + and cd + t cell responses can be induced by vaccination. the number of doses and dosing frequency will need to be determined, and, particularly if repeated or annual vaccination is required, the financial burden and logistics of delivery will have to be supported. a better understanding of what level of nabs correlate with protection and development of standardized viral neutralization along with other assays to compare vaccine candidates is eagerly awaited. potential disease enhancement and other theoretical safety concerns related to each type of vaccine need to be understood and carefully monitored for, while potential suboptimal immunity may need to be overcome. certain vulnerable populations may respond poorly to vaccination, or the vaccine may predominantly protect against severe disease, rather than infection. if t cells prove critical for protection, it will be necessary to ensure that the included t cell epitopes are recognized by enough hla types to ensure worldwide coverage. scale-up of production to billions of doses and delivery to all regions of the world is a massive logistic challenge. in the short-term the strategy will likely need to be targeted vaccination toward those most at risk (e.g., healthcare workers) with the strategy reviewed as vaccine production increases. on the positive side, given the multiple candidates being developed, there is considerable optimism that some of the vaccines currently in trials will prove effective and be available for use in with delivery scaled-up thereafter. kf proposed the project and coordinated the study group. kf, nc, and fr wrote the first draft. eb, mg, ak, km, bt, and sw reviewed and edited the manuscript, provided comments, and suggested references substantially contributing to the content. all authors approved the final submitted version of the manuscript. this project was supported by the australasian society for infectious diseases (asid) vaccination special interest group (vacsig). covid- in the usa epidemiology of covid- : a systematic review and meta-analysis of clinical characteristics, risk factors, and outcomes global covid- case fatality rates deaths in healthcare workers due to covid- : the need for robust data and analysis factors associated with mental health outcomes among health care workers exposed to coronavirus disease the milken institute. covid- treatment and vaccine trackers. ( ) available online at draft landscape of covid- candidate vaccines covid- vaccine development pipeline cepi-a new global r&d organisation for epidemic preparedness and response origin and evolution of pathogenic coronaviruses coronavirus diversity, phylogeny and interspecies jumping severe acute respiratory syndrome coronavirus pandemic-therapy and vaccines structure of the sars-cov- spike receptor-binding domain bound to the ace receptor molecular structure analyses suggest strategies to therapeutically target sars-cov- cell entry mechanisms of sars-cov- distinct conformational states of sars-cov- spike protein structure, function, and antigenicity of the sars-cov- spike glycoprotein the trinity of covid- : immunity, inflammation and intervention t cell-mediated immune response to respiratory coronaviruses imbalanced host response to sars-cov- drives development of covid- comparative replication and immune activation profiles of sars-cov- and sars-cov in human lungs: an ex vivo. study with implications for the pathogenesis of covid- histopathologic changes and sars-cov- immunostaining in the lung of a patient with covid- covid- in critically ill patients in the seattle region -case series clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan the laboratory tests and host immunity of covid- patients with different severity of illness clinical and immunological features of severe and moderate coronavirus disease clinical features of patients infected with novel coronavirus in wuhan the many faces of the anti-covid immune response dissecting antibody-mediated protection against sars-cov- sars-cov- spike protein: an optimal immunological target for vaccines effect of convalescent plasma on mortality among hospitalized patients with covid- : initial three-month experience treatment of critically ill patients with covid- with convalescent plasma convalescent plasma in covid- : possible mechanisms of action isolation of potent sars-cov- neutralizing antibodies and protection from disease in a small animal model a human neutralizing antibody targets the receptor-binding site of sars-cov- longitudinal evaluation and decline of antibody responses in sars-cov- infection. medrxiv clinical and immunological assessment of asymptomatic sars-cov- infections twoyear prospective study of the humoral immune response of patients with severe acute respiratory syndrome a sars-cov- surrogate virus neutralization test based on antibody-mediated blockage of ace -spike protein-protein interaction potent neutralizing antibodies from covid- patients define multiple targets of vulnerability questions remain following first covid- vaccine results available online at covid- : an immunopathological view. msphere regulatory t cells in arterivirus and coronavirus infections: do they protect against disease or enhance it? viruses lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses immune cell profiling of covid- patients in the recovery stage by single-cell sequencing immunology of covid- : current state of the science molecular basis of coronavirus virulence and vaccine development sars vaccines: where are we? importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on the middle east respiratory syndrome coronavirus spike glycoprotein to avoid neutralization escape a dna vaccine induces sars coronavirus neutralization and protective immunity in mice prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus evaluation of candidate vaccine approaches for mers-cov vaccine efficacy in senescent mice challenged with recombinant sarscov bearing epidemic and zoonotic spike variants safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase , open-label, single-arm, dose-escalation trial safety and immunogenicity of a modified vaccinia virus ankara vector vaccine candidate for middle east respiratory syndrome: an open-label, phase trial safety and immunogenicity of a candidate middle east respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, open-label, non-randomised, uncontrolled, phase trial a new coronavirus associated with human respiratory disease in china a novel coronavirus from patients with pneumonia in china structural genomics of sars-cov- indicates evolutionary conserved functional regions of viral proteins design of a peptide-based subunit vaccine against novel coronavirus sars-cov- bioinformatic prediction of potential t cell epitopes for sars-cov- analysis of human coronavirus e spike and nucleoprotein genes demonstrates genetic drift between chronologically distinct strains genetic drift of human coronavirus oc spike gene during adaptive evolution preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies antigenspecific human t-cell responses and t cell-dependent production of human antibodies in a humanized mouse model a mouse model of sars-cov- infection and pathogenesis adaptation of sars-cov- in balb/c mice for testing vaccine efficacy vaccine adjuvants: smart components to boost the immune system correlates of adjuvanticity: a review on adjuvants in licensed vaccines potential adjuvants for the development of a sars-cov- vaccine based on experimental results from similar coronaviruses development of sars-cov- vaccines: should we focus on mucosal immunity? mucosal vaccines: strategies and challenges caf liposomes as a mucosal vaccine adjuvant: in vitro and in vivo investigations retinoic acid as a vaccine adjuvant enhances cd + t cell response and mucosal protection from viral challenge safety, tolerability and immunogenicity of ino- for covid- in healthy volunteers. ( ) available online at prickly patch delivery of experimental covid- vaccine shows promise in animal study microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development overview of vaccines and vaccination key steps in vaccine development potential of live-pathogen vaccines for defeating the covid- pandemic: history and mechanism reverse genetic systems: rational design of coronavirus live attenuated vaccines with immune sequelae opinion: making inactivated and subunit-based vaccines work effect of an inactivated vaccine against sars-cov- on safety and immunogenicity outcomes: interim analysis of randomized clinical trials safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind, randomised controlled trial safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, non-randomised, firstin-human trial immunogenicity and safety of a recombinant adenovirus type- -vectored covid- vaccine in healthy adults aged years or older: a randomised, double-blind, placebo-controlled, phase trial an mrna vaccine against sars-cov- -preliminary report phase / study to describe the safety and immunogenicity of a covid- rna vaccine candidate (bnt b ) in adults to years of age: interim report. medrxiv development of an inactivated vaccine for sars-cov- development of an inactivated vaccine candidate, bbibp-corv, with potent protection against sars-cov- peptide-based vaccines: current progress and future challenges recent advances in vaccine technologies subunit vaccines against emerging pathogenic human coronaviruses making a vaccine for covid- creating tomorrow's vaccines today press release: generex signs contract with epivax to develop ii-key peptide vaccines to address the coronavirus pandemic increasing the potency of mhc class ii-presented epitopes by linkage to ii-key peptide generex biotechnology. covid- . ( ) available online at developments in viral vector-based vaccines replicating and non-replicating viral vectors for vaccine development a study of ad .cov .s for the prevention of sars-cov- -mediated covid- in adult participants (ensemble). ( ) available online at a single-dose live-attenuated yf d-vectored sars-cov vaccine candidate dna vaccines-how far from clinical use? immunogenicity of a dna vaccine candidate for covid- available online at mrna vaccinesa new era in vaccinology an evidence based perspective on mrna-sars-cov- vaccine development amplifying rna vaccine development nanoparticle vaccines nanoparticle vaccines against infectious diseases covid- vaccine development and a potential nanomaterial path forward major findings and recent advances in virus-like particle (vlp)-based vaccines peptide nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine covid- : medicago's development programs china's shenzhen geno-immune medical institute pursues a covid- vaccine sex-differential heterologous (nonspecific) effects of vaccines: an emerging public health issue that needs to be understood and exploited the non-specific and sex-differential effects of vaccines rapid protective effects of early bcg on neonatal mortality among low birth weight boys: observations from randomized trials bacille calmette-guerin induces nod -dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes bcg-induced trained immunity in nk cells: role for non-specific protection to infection miyasaka m. is bcg vaccination causally related to reduced covid- mortality? bcg vaccine protection from severe coronavirus disease (covid ) the spread of covid- and the bcg vaccine: a natural experiment in reunified germany sars-cov- rates in bcgvaccinated and unvaccinated young adults bcg-induced trained immunity: can it offer protection against covid- ? considering bcg vaccination to reduce the impact of covid- two randomized controlled trials of bacillus calmette-guerin vaccination to reduce absenteeism among health care workers and hospital admission by elderly persons during the covid- pandemic: a structured summary of the study protocols for two randomised controlled trials national immunization campaigns with oral polio vaccine reduce all-cause mortality: a natural experiment within seven randomized trials association of bcg, dtp, and measles containing vaccines with childhood mortality: systematic review polio global eradication initiative. the use of oral polio vaccine (opv) to prevent sars-cov sex differences in immune responses sex and gender differences in the outcomes of vaccination over the life course effect of sex on vaccination outcomes: important but frequently overlooked effectiveness of influenza vaccines in preventing severe influenza illness among adults: a systematic review and meta-analysis of test-negative design case-control studies chadox ncov- vaccination prevents sars-cov- pneumonia in rhesus macaques vaccination in the elderly effectiveness of mf -adjuvanted seasonal influenza vaccine in the elderly: a systematic review and meta-analysis. vaccine efficacy and effectiveness of high-dose versus standard-dose influenza vaccination for older adults: a systematic review and meta-analysis herpes zoster and the search for an effective vaccine covid- : are neutralizing antibodies neutralizing enough? transfusion antibody dependent enhancement due to original antigenic sin and the development of sars original antigenic sin: a comprehensive review original antigenic sin: how first exposure shapes lifelong anti-influenza virus immune responses original antigenic sin and respiratory syncytial virus vaccines what about the original antigenic sin of the humans versus sars-cov- ? med hypotheses antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages evaluation of modified vaccinia virus ankara based recombinant sars vaccine in ferrets molecular mechanism for antibody-dependent enhancement of coronavirus entry distinct systems serology features in children, elderly and covid patients. medrxiv original antigenic sin response to rna viruses and antiviral immunity anti-sars-cov- igg from severely ill covid- patients promotes macrophage hyper-inflammatory responses atypical measles and enhanced respiratory syncytial virus disease (erd) made simple rapid covid- vaccine development avoiding deceptive imprinting of the immune response to hiv- infection in vaccine development safety platform for emergency vaccines. priority list of adverse events of special interest: covid- . ( ) available online at developing a covid- vaccine, q&a with task force vaccine safety expert a controlled human infection model of group a streptococcus pharyngitis: which strain and why? msphere world health organization. key criteria for the ethical acceptability of covid- human challenge studies. who working group for guidance on human challenge studies in covid- ( ) extraordinary diseases require extraordinary solutions plotkin sa. vaccines for epidemic infections and the role of cepi coalition for epidemic preparedness innovations (cepi) coronavirus global response: e . billion raised for universal access to vaccines time is of the essence to provide a vaccine against this pandemic virus barda's rapidly expanding covid- medical countermeasure portfolio novavax to receive up to $ million funding from cepi for covid- vaccine development and manufacturing precision vaccinations. azd sars-cov- vaccine. ( ) available online at logistical challenges for potential sars-cov- vaccine and a call to research institutions, developers and manufacturers access to covid- tools (act) accelerator. ( ) available online at world leaders unite in call for a people's vaccine against covid- we would like to thank claudio rosa (www.claudiorosa.com) for medical illustration. key: cord- -c aw jkz authors: privor-dumm, lois; vasudevan, prarthana; kobayashi, kana; gupta, jaya title: archetype analysis of older adult immunization decision-making and implementation in countries date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: c aw jkz the global population of adults over years of age is growing rapidly and is expected to double by . countries will face substantial health, economic and social burden deriving from vaccine-preventable diseases (vpds) such as influenza, pneumonia and herpes zoster in older adults. it will be essential that countries utilize several public health strategies, including immunization. understanding the different approaches countries have taken on adult immunization could help provide future learnings and technical support for adult vaccines within life-course immunization strategies. in this study, we describe the priorities and approaches that underlie adult immunization decision-making and implementation processes in high-and-middle-income countries and two territories (“ countries”) who recommend adult vaccines in their national schedule. we conducted an archetype analysis based on a subset of two dozen indicators abstracted from a larger database. the analysis was based on a mixed-methods study, including results from key informant interviews in six countries and a landscape review of secondary data from countries. we found four distinct archetypes: disease prevention-focused; health security-focused; evolving adult focus; and, child-focused and cost-sensitive. the highest performing countries belonged to the disease prevention-focused and health security archetypes, although there was a range of performance within each archetype. considering common barriers and facilitators of decision-making and implementation of adult vaccines within a primary archetype could help provide a framework for strategies to support countries with similar needs and approaches. it can also help in developing context-specific policies and guidance, including for countries prioritizing adult immunization programs in light of covid- . further research may be beneficial to further refine archetypes and expand the understanding of what influences success within them. this can help advance policies and action that will improve vaccine access for older adults and build a stronger appreciation of the value of immunization amongst a variety of stakeholders. the global population of adults over years of age is growing rapidly and is expected to double by . countries will face substantial health, economic and social burden deriving from vaccine-preventable diseases (vpds) such as influenza, pneumonia and herpes zoster in older adults. it will be essential that countries utilize several public health strategies, including immunization. understanding the different approaches countries have taken on adult immunization could help provide future learnings and technical support for adult vaccines within life-course immunization strategies. in this study, we describe the priorities and approaches that underlie adult immunization decision-making and implementation processes in high-and-middle-income countries and two territories ('' countries") who recommend adult vaccines in their national schedule. we conducted an archetype analysis based on a subset of two dozen indicators abstracted from a larger database. the analysis was based on a mixed-methods study, including results from key informant interviews in six countries and a landscape review of secondary data from countries. we found four distinct archetypes: disease prevention-focused; health security-focused; evolving adult focus; and, child-focused and cost-sensitive. the highest performing countries belonged to the disease prevention-focused and health security archetypes, although there was a range of performance within each archetype. considering common barriers and facilitators of decision-making and implementation of adult vaccines within a primary archetype could help provide a framework for strategies to support countries with similar needs and approaches. it can also help in developing context-specific policies and guidance, including for countries prioritizing adult immunization programs in light of covid- . further research may be beneficial to further refine archetypes and expand the understanding of what influences success within them. this can help advance policies and action that will improve vaccine access for older adults and build a stronger appreciation of the value of immunization amongst a variety of stakeholders. Ó elsevier ltd. all rights reserved. older adults are a heterogeneous group in the second half of life [ ] . studies demonstrate that vaccine-preventable diseases (vpds), including influenza, pneumonia and herpes zoster, account for a substantial portion of premature death and disability in older adults [ , ] . vpds also have the potential to cause disability that may lead to additional issues, such as declines in functional ability and quality of life [ ] . the economic burden of vpds is also substantial [ , [ ] [ ] [ ] [ ] [ ] [ ] . the global population of adults over the age of is growing rapidly and is expected to double by [ ] . although adults are expected to live longer, they are not necessarily living in good health [ ] . to be prepared for this demographic change, countries must establish plans and infrastructure that support healthy aging. aging populations will impact countries' economies, social security policies, and health systems, as well as affect many aspects of daily living for both the individual and broader society [ ] . at a national level, the consequences of an aging population extend beyond the health sector and solutions must be viewed across the life course [ , ] . it will be essential that countries utilize several strategies to ensure that their older populations age in a healthy manner, including adult immunization [ , ] . as they have for children, vaccines have the potential to significantly reduce burden of disease and disability, dependence, healthcare costs, and more in older populations [ ] [ ] [ ] [ ] [ ] [ ] . given the imminence of a growing, worldwide adult population, anticipation of increased health costs has spurred multiple global, https://doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. regional and national calls for action for policymakers and practitioners to prioritize adult immunization programs and improve uptake [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . many high-and middle-income countries have adopted influenza vaccines [ ] , however few have adopted more than one vaccine for older adults or have comprehensive adult immunization strategies. countries that have adopted adult vaccines appear to have taken different pathways to policy adoption [ ] [ ] [ ] [ ] [ ] . further, to be prepared for healthy aging, countries must think holistically. they will need systems and an appreciation for prioritizing health in older adults to support delivery of vaccines and other crucial interventions [ , , ] . particularly in the context of covid- , preventing further strain on the health system is paramount [ ] . the global vaccine action plan [ ] and the immunization agenda [ ] call for a life-course approach to immunization, which some countries have begun to implement. despite similarities in disease burden, demographic profile, and geographic proximity, these countries have taken different approaches and achieved different results in adult vaccine adoption. to understand those differences and explain the factors influencing country decision-making and uptake, we conducted an archetype analysis. this analysis aims to describe the country priorities and approaches that underlie adult immunization decision-making and implementation. by characterizing groups of countries by features other than disease burden, geography or demographics, the analysis seeks to support global efforts to address country needs in strengthening processes for vaccine decision-making and implementation; facilitating sharing of best practices amongst countries with similar characteristics; and providing evidence, system or advocacy support to help countries succeed within their specific context. the archetype analysis does not replace the need for individual country strategies, but groups needs in a way that enables the global community to provide meaningful support across a broader group of countries. thirty-two countries and two territories (herein referred to as countries) were selected for analysis. countries selected had high proportions of older adults, were geographically diverse, and represented a range of potential archetypes based on their adult vaccine adoption status, financing models, degree of health system centralization, and vaccine coverage. all countries were included in a literature review and data abstraction. six countries (argentina, australia, canada, germany, japan, and the united kingdom (uk)) were selected for further qualitative research to provide additional depth in a diversity of contexts, approaches and performance. the insights gained in each case country helped characterize archetypes. each case country, to some degree, prioritizes adult vaccination and recommends and finances one to three adult vaccines, but varies in terms of government and/or healthcare system centralization and adult vaccine coverage rates. the united states (us) was not selected as a case study country, due to ivac's working knowledge of the american public immunization system and the abundance of publicly available peerreviewed articles and government documentation on older adult immunization. the study plan was reviewed by the johns hopkins bloomberg school of public health institutional review board and deemed to be non-human subjects research. domains (table ) were identified as part of a framework of potential barriers and facilitators for adult vaccine decisionmaking: country characteristics, adult vaccine/aging policies and decision-making, health immunization systems, uptake, and stakeholders and champions. these domains were the subject of key informant interviews conducted in six case countries and a concurrent landscape analysis of the countries. a series of indicators were subsequently identified, informed by previous research [ ] , an adult vaccine situational analysis [ ] , and the case study interviews. a team of abstractors conducted the indicator research. we searched peer-reviewed and grey literature, including government and professional society websites, disease burden and vaccine introduction and program status databases, reports from countries, the world health organization (who), non-governmental organizations (ngos), and media articles. we reviewed minutes from national technical advisory groups on immunization (nitags) as well as official recommendations and policies for both key informant interviews were conducted in in argentina, australia, canada, germany, japan and the uk. informants included vaccine experts, government and former government officials, healthy aging advocates, economists, and civil society. respondents were selected using a snowball approach. one-hour face-to-face interviews were conducted by - people using an interview guide. for each respondent type -technical respondents, economic respondents and health advocacy-focused respondentsunique guides were prepared to ensure the focus of the questions was on their area of expertise. a combination of open-ended questions and probes were used. topics included respondent background; country health priorities; key players and stakeholders; robustness of the process; drivers of decisions and uptake. we used scales of very important, moderately important, not important, and don't know/not sure to ascertain respondents' perceptions about drivers or degree of agreement with certain statements and to map responses. interviews were transcribed and entered into atlas.ti for mac os x (a qualitative data analysis software) to conduct a thematic analysis. no identifiers were included to keep the identities of respondents confidential. descriptive categories of their function (e.g., technical, economic, civil society organization, etc.), were included instead. based on the case study findings, we selected indicators that most differentiated countries and created a scoring of - to rate each country, with meaning that it fit the criterion well and that it did not fit the criterion and/or there was no data available to assess fit ( table ). each indicator was ascribed a score based upon a qualitative description. following validation, we found some of our scoring was insufficient in describing qualitative nuance and added an intermediate score of . to one indicator and . to four indicators. we scored all countries on indicators related to decision-making ( indicators) and implementation ( indicators). each country received a score and a ranking for both domains (i.e., decision-making and implementation). we recorded the scores in a table, organizing countries by pneumococcal vaccine coverage (high to low). we mapped all countries on a grid using graph pad prism version . . we used the case study insights to identify characteristics that could describe the primary driver of a country's approach to decision-making and implementation (the ''archetype"). based on the qualitative insights, we found four distinct archetypes and placed each country into one archetype. although countries could fit in more than one archetype, we selected the archetype that most closely fit each country's primary driver. thus, country performance does not define each archetype's description. we transcribed each country's data into individual country profiles, providing the qualitative description associated with each quantitative score. we asked respondents to indicate their level of agreement (agree/disagree) with each score. we requested the respondents to identify missing data or inaccurate scores, that if corrected, could substantially move a country's score higher or lower. if there was disagreement, we asked respondents to provide an explanation and publicly available source(s) that support the change they suggested. we reached out to - experts per country, including a ministry of health representative, wherever possible. we provided a slide set describing the project, including objectives, methodology and the archetype map to enable countries to provide meaningful input. countries that responded are listed (table ). thirty of the countries analyzed had more than % of their total population adults over years of age. twenty-four countries had over % of their population over years of age. high-income countries and upper middle-income countries, had older populations than lower-middle and low-income countries (table ), but the age of populations did not predict the number of vaccines adopted for their adult populations nor uptake of influenza or pneumococcal vaccine (fig. ). one hundred and twenty key informant interviews were conducted in the six case countries, including a range of respondents covering health, immunization, government policy, aging and economics. overall, respondents in australia and the uk had the greatest degree of confidence in their country's ability to make decisions about new vaccines for adults and implement programs. the uk respondents attributed their country's ability to make decisions on the broadest variety of factors and only the influence of advocacy and bringing a variety of perspectives into decision-making was ranked lower. respondents in canada expressed a high degree of confidence in their country's ability to make decisions but reported lower confidence in their government's level of priority for adult vaccines, access to providers and financing. germany had similar perceptions to canada in that the decision-making process was strong, but respondents questioned the ability to implement, ease of access and the variety of advocacy efforts. argentine and japanese respondents had lower composite ratings on their perception of their country's capability to make decisions and implement adult immunization than the other countries but rated their country's commitment to adult health and the variety of perspectives highly. argentine respondents also rated their nitags as capable. both argentine and japanese respondents had less confidence in ease of access, financing, surveillance for adults. we also noted varying approaches to how cost-effectiveness (c-e) data were used both amongst case study countries and other countries that the respondents described. a few countries, including the uk, used c-e thresholds in their adoption decisions. other countries considered c-e data but did not use it. in some countries, childhood vaccination was viewed as a higher priority, particularly for funding. some respondents in germany even went as far as to state that putting adult health before child health would be unethical. in japan, we saw no evidence of c-e studies used in their nitag decisions, although safety studies were a requirement (fig. ). similar to decision-making, factors influencing implementation varied by country (fig. ). in the case countries, we saw table scoring. early or late adopter of adult vaccines = no or late decision-making and adoption of either pneumococcal or herpes zoster vaccine = follower in decision-making and adoption of at least one vaccine = leader in decision-making and adoption of one or more vaccines country-specific policy requirements of manufacturers = multiple = one = none disease burden surveillance = no surveillance = some surveillance (mostly of flu and pneumococcal disease) = national surveillance of flu, pneumococcal disease, and herpes zoster nitag's prioritization of health security in decision-making = small to no priority on health security, or no evidence available = nitag considers health security, but it is not a main driver of decisions = nitag considers health security as a main driver of decisions nitag's utilization of cost-effectiveness (c-e) data in decision-making = small to no focus on c-e, or no evidence available = nitag considers c-e data, but it is not a main driver . *= nitag mostly considers c-e data as a main driver = nitag considers c-e data as a main driver nitag has adult vaccine working group(s) = such working groups . * = nitag has such working groups, but is involved in other recommending bodies where government is engaged = such working group = multiple such working groups (as part of a broader vaccine-specific working group or a standalone) public policy -pneumococcal vaccination for older adults = pcv or ppsv not recommend, unknown if considered by nitag = pcv or ppsv considered by nitag, but not recommended . * = pcv or ppsv was recommend by nitag, but not yet implemented = pcv recommended by nitag public policy -herpes zoster vaccine (hzv) for older adults = hzv not recommended, unknown if considered by nitag = hzv considered, but not recommend by nitag . *= hzv recommended by nitag, but not yet implemented = hzv recommended by nitag publication of health aging strategies = no healthy aging strategy publicly available = aging strategy available at the sub-national or national level . *= sub-national or national aging strategy available that mentions adult immunization, but is over ten years old = national aging strategy available that mentions adult vaccines publication of national immunization strategies = no immunization strategy publicly available = only pediatric immunization strategy publicly available = national immunization strategy published and covers both pediatric and adult vaccines vaccine financing -level of public financing (for each vaccine) = older adults must pay out of pocket = vaccine covered by private insurance, requires co-pay, or limited coverage is provided in certain geographic areas or at-risk populations . *= mixed system of payment (covered) = vaccine is fully funded by the government for all vaccine registry (for pediatric and adult populations) = countries were assessed on their adult vaccine implementation and decision-making. decision-making was scored upon indicators and implementation upon indicators. each indicator was ascribed a quantitative score, as described above. recommendations were not universally implemented (particularly in canada and germany). the exceptions were the uk, which has a very centralized immunization program, and australia, which was decentralized but took a more centralized approach to monitoring and promotion. access to immunization was a factor reported to influence older adult immunization uptake. lower uptake may be due to limited mobility or lack of awareness of the need for adult vaccines. in some countries, such as germany and japan, respondents stated that vaccines were still viewed as something only for children. additionally, health system complexity was reported as an important factor contributing to access, and ultimately uptake, with some respondents describing receiving vaccination as an older adult as an overly cumbersome process. in canada, respondents' general perception was that there are not many places or providers of adult vaccination. restrictions on who could administer vaccines was also reported as a perceived barrier. improvements in access were described in japan, where nurses can now vaccinate; in france, where pharmacists can offer influenza vaccines; and in the uk, where pharmacists were allowed to administer influenza and pneumococcal vaccines. despite some countries expanding their range of adult vaccine providers, respondents noted that uptake does not always correspondingly increase. this gap offers a role for advocacy efforts in improving uptake. the influence of advocacy can be difficult to assess and is sometimes subjective. nonetheless, we used the number of national advocacy organizations as a proxy for influence in database countries. in case countries, we were able to gather insights. each respondent was asked about advocacy efforts in their country, yet there was a wide range of level of familiarity of country efforts. australia is a clear leader and uses a variety of approaches to advocate for adult immunization. australian respondents described influential advocacy efforts that impacted both the national adult vaccination decision-making and implementation processes. for example, in the country citizens, government, medical societies, and health aging specialists have organized large groups to advocate for better recommendations and financing as well as influence uptake and equal access to vaccines. in japan, respondents were not familiar with any major advocacy effort, although could describe small-scale patient-advocacy groups or activism against the government regarding vaccine injuries. based on the findings from the case studies, ten indicators emerged as descriptors of countries' older adult immunization decision-making capacity, specifically whether countries: were early adopters; had country-specific barriers such as requirements for technology transfer (brazil, india, japan, korea); preferences for indigenous product (china, indonesia, brazil, india, russia); halal vaccines (malaysia); safety study requirements (japan); or c-e requirements (uk, netherlands); prioritized surveillance on vpds in older adults (emerging as a factor in many of the countries concerned about health security); were prompted in their vaccine decisions by health security concerns; considered c-e a major driver in vaccine decisions; had working groups in their national immunization technical advisory groups (nitags) specific to adult vaccine issues; adopted pneumococcal conjugate vaccine (pcv) ; adopted herpes zoster vaccine (hzv); published a national healthy aging policy (including whether there is a mention of vaccines); and included adult vaccines in their national immunization policy. for implementation and uptake, indicators that emerged included whether there was government financing for influenza vaccines, pcv and hzv; centralized vaccine registries for children and adults; coverage data for all three vaccines; advocacy (measured by the number of advocacy organizations); documented influence of organizations or leaders; access (easy to get vaccinated by expanded list of providers or lack of bureaucracy or cumbersome process); equity focus; centralization of the adult vaccine program; and centralization of the health system. few countries that had strong decision-making also had strong uptake and vice-versa. further, the scoring table shows that countries varied significantly in the indicators that drove their performance on adult vaccine decision-making (table ) and implementation (table ). to determine how composite scores corresponded to performance, we ordered countries based on their pneumococcal vaccine uptake and compared that to rankings of the composite score on both decision-making and implementation/uptake. countries with the highest coverage of pneumococcal vaccines did not always perform the highest on decision-making, with the exception of australia, us, uk, and canada. we did a similar exercise for influenza vaccine uptake and found that top ten countries with highest influenza coverage were similar to those with the highest decision-making and implementation scores. the us had the highest ranking, compared to other countries, and scored points when assessed for the robustness of policies and decision-making. australia (score: . ), the uk (score: . ) and italy (score: ), had the next set of highest scores. countries with the least robust policies and decision-making were denmark, table countries responding to validation survey. received survey results responded with feedback the results of the archetype analysis was shared with experts representing all countries. countries responded and participated in the validation process. india, peru, the philippines, switzerland, and saudi arabia (all ranking last; scoring: ) (see table ). when assessed for the promotion of implementation and uptake, the uk (score: out of a possible ), new zealand ( ), and australia ( ) had the highest rankings compared to other countries. in contrast, russia (score: ), peru ( ), and india ( ) had the lowest ranking (see table ). based on a synthesis of case study data, we found four distinct archetypes with unique characteristics: ''disease preventionfocused"; ''health security-focused"; ''evolving adult focus"; and ''child-focused and cost-sensitive" (fig. ) . some countries could belong to multiple archetypes, but we selected the archetype most closely aligned with primary driver of approach. we also noted that the highest performing countries in terms of both decision-making (australia, us, uk, canada, italy, netherlands, germany and mexico) and implementation (uk, new zealand), australia, us, korea, canada, italy and norway) belonged to either the ''disease prevention-focused" or ''health securityfocused" archetype (see fig. ). disease prevention-focused: countries in the ''disease prevention" archetype included canada, france, germany, netherlands, uk, and us. these countries valued the use of data and process. most countries' nitags in this archetype had considered most of the adult vaccines and performed fairly high on decision-making. we noted use of their own disease burden/impact evidence in decision-making, as well as use of other countries' data. most of these countries had their own adult surveillance and formal adult vaccine working groups on their nitag. the uk and the netherlands also placed high importance on economics. most countries considered economics, but it was not a primary driver. germany, and to a lesser extent the us, considered economics, but disease burden was the primary driver in decisions. there was significant variation in implementation performance. reasons varied within this archetype, and included lack of national adult registries, equity focus, sufficient advocacy and centralization. health security-focused: the ''health security" archetype was the largest of the four and also the most diverse in terms of performance. it included argentina, australia, china, greece, hong kong, italy, japan, mexico, new zealand, saudi arabia, taiwan, and turkey. the characteristic of this archetype is that outbreaks (h n in australia and argentina; pneumonia in japan), vpd threats (due to migration or in refugees), and natural disasters (tsunami in japan) were viewed as an important country motivation for action. we saw wide variation of performance within this archetype, with australia, italy, new zealand and mexico performing highest and china, japan, hong kong, taiwan and greece lagging behind. degree of centralization and registries contributed to performance. australia, for example, reported that data use was strengthened during the h n outbreak in . in argentina, surveillance and epidemiologic response was also strengthened as a result of an h n outbreak. it also led to strategies to establish mass vaccination centers to improve access to vaccines more widely. in , the great east japan earthquake struck northeastern japan and destroyed the local healthcare system. at the time, pneumococcal vaccine coverage in japan was only % and many healthcare workers feared the occurrence of pneumonia outbreak at evacuation shelters [ ] . to avoid future outbreaks, pneu-mococcal vaccine was provided free of charge in the three prefectures most affected by the earthquake. in , they reached record levels of uptake with miyagi and iwate at %, and fukushima at % coverage [ ] , leading to significant reduction in deaths attributed to pneumonia in the area [ ] , and eventually to the ministry of health, labour, and welfare's decision in to include pneumococcal vaccine to the routine immunization schedule for adults aged and older. the national coverage increased from % in to % in [ ] . evolving adult focus: countries in the ''evolving adult focus" archetype include belgium, brazil, colombia, ireland, korea, spain, sweden and malaysia. these countries had moderate to strong systems and decision-making ranged from weak (denmark, malaysia) to moderate (korea, belgium). many of the countries in this archetype lack a strong nitag for adult vaccine decisions, but exhibit some elements of the ''disease prevention" archetype. some have healthy aging policies or immunization strategies, but only brazil has both. some countries were early adopters for some adult vaccines, including pcv in ireland and belgium, and hz vaccine in uae and norway. belgium has published an adult immunization strategy recently [ ] . financing for recommended adult vaccines, varies as well. although vaccines were recommended in some countries, they were not always publicly financed or financed for risk groups. child-focused and cost-sensitive: finally, some countries, including russia, peru, india, switzerland and the philippines remain ''child-focused and cost-sensitive" in their public markets. the countries in this archetype have not prioritized adult immunization programs and have generally lacked focus on decisionmaking for older adults. we found no adult vaccine working groups on the three vaccines analyzed (influenza, pneumococcal and herpes zoster) or policies around adult immunization at the time of our study. in terms of implementation, these countries may require patients to pay out of pocket for adult vaccines (russia, india) although there were some exceptions for influenza vaccine like the philippines and switzerland (although in switzerland vaccines were covered through insurance). we did not find a focus on implementation. these five countries have no registries or policies around adult health or adult immunization. additionally, advocacy for adult immunization was not strong and, in most cases, seemed to have little influence on the outcomes of the government. lastly, for most countries in this archetype, there is a cost-based argument: given limited resources, public investments in child health and vaccines are prioritized, as they are seen as necessary to further national economic development (russia, peru, india, philippines). we were able to validate our data and scoring in of countries ( %) ( table ) . where there was disagreement and documented sources, we corrected data and in five instances, adjusted scoring to reflect a more accurate picture of the parameters which had to do with timing of decisions or policies, the importance of c-e data in decision-making, nitag working groups, and a mixed financing system. importantly, the act of classifying countries into a primary archetype has its own set of limitations. firstly, there is a level of subjectivity that comes with archetyping, which we have tried to ameliorate by taking an evidence-based approach. secondly, archetyping elevates a certain set of common characteristics above others and there are variations between and within countries that get under-accounted. countries may not fit perfectly in an archetype and may actually belong to other archetypes simultaneously; we therefore classified countries into primary archetypes, according to the indicator that they scored the highest on. our analysis was also limited by data availability and quality. in some cases, a zero score was given due to a lack of data or clarity on an indicator. we tried to address this through the validation process, but little additional information was provided. additionally, each indicator's scoring was based only on data that was publicly available through october (unless otherwise provided by experts during validation) and may be impacted by timing. changes in scoring of - points is unlikely to impact score mapping, but larger changes may shift archetype categorization. archetypes can be useful in identifying factors that are most influential in improving decision-making and implementation for adult vaccines in the context of a country's approach and priorities. this will enable various countries to learn from experiences amongst countries within the same archetype. there has been some use of archetypes, mainly in health technology assessment [ ] [ ] [ ] [ ] [ ] , but experiences have not yet been widely documented. the archetype describes the primary driver of decisions and implementation as well as country priorities or approach rather than performance. there can be significant variation in perfor- mance within an archetype, which we saw in this exercise. top performing countries belonged to two different archetypes, which were ''disease prevention-focused" (us, uk, canada) and ''health security-focused" (australia, italy), suggesting that there are multiple ways to achieve adoption and uptake. it is likely that countries within the ''evolving adult focus" archetype could improve their performance by learning from countries in the ''disease prevention" or ''health security" archetypes. the improvements needed within the ''child-focused" archetype may require both stronger advocacy as well as additional data. while we provide broad-based archetypes, we also saw different approaches within the archetypes; this is important as each country will need to take lessons based on what works for them. understanding the success factors of the highest performing countries within a particular domain (e.g., decision-making) can also help other countries move up their performance in the context of their structure and priorities. the countries in the ''disease prevention" archetype all value data and its use, albeit to different levels and strategies can focus on data use and advocacy for disease prevention. at the one extreme of this archetype is the uk, where data use permeates through the process of both decision-making and implementation. decisions are evidence-based, with little influence of advocacy or champions. on the implementation side, there was also strong use of data with information on the immunization status of individual patients, which is all mapped to their general practitioner (gp). for countries who will consider a centralized approach, the uk provides a good model. however, many countries have decentralized systems, including the us and canada, who also have strong use of data for decision-making. they also have greater degrees of advocacy, which could perhaps also ensure certain groups have a voice in the process. use of data for implementation in decentralized settings may also benefit from learnings from table for score descriptors. generally, the higher the score the better that country meets the indicator; scores either mean the country doesn't meet the indicator or no data were found. countries that have centralized registries in a decentralized system (e.g., australia, although a different archetype). health security concerns provide countries with an opportunity to overcome a wide array of issues, from financing to barriers that may slow down decisions (e.g., tech transfer requirements as seen in brazil or requirements/strong preference for local products such as in china or india, requirements for local studies as in japan, etc.) and may be a motivator to strengthen surveillance, use of data, communications and platforms. the current context of covid- highlights likely opportunities to leverage the importance of developing a strong system. in doing so, it is important for countries to consider how all people, including older adults and/or marginalized populations such as migrants or refugees, can access vaccines. for example, in the us, adult immunization access increased as a result of the influenza an h n pandemic, with states granting pharmacists the authority to vaccinate against influenza [ ] . also, during that pandemic, a study demonstrated that american indian and alaska native persons have a higher risk for death from the h n influenza [ ] supporting prioritization of seasonal influenza vaccines amongst native americans that year [ ] . we saw wide variation of performance within this archetype and note that while emergencies may provide motivation to take action, other table for score descriptors. generally, the higher the score the better that country meets the indicator; scores either mean the country doesn't meet the indicator or no data were found. contextual factors must be taken into account. degree of centralization of adult immunization is one important factor that influences success of implementation. registries and stronger use of data are an important element in both ''health security" and ''disease prevention" archetypes, but the motivator to get them done may be different -in times of emergency, data are essential, but it is prior to an emergency that data are needed [ ] . after the severe acute respiratory syndrome (sars) epidemic, china was challenged to improve its public health emergency management systems (phems) [ ] . the system was significantly improved within ten years after the outbreak [ ] . more recently, china was able to contain covid- within the country [ ] , but the virus has caused more than . million cases and , deaths worldwide and continues to spread as of early april [ ] . there is now more than ever a strong need for systems to detect risk, transparently communicate the risk, and have infrastructure in place to address both existing and emerging infectious threats [ ] . engaging civil society and building accountability mechanisms can help motivate action more urgently. this current pandemic provides a window of opportunity to build urgency for the need for adult immunization and systems to deliver them. efforts, however, must not focus solely on covid- , but on the platforms needed to build strong health systems for all ages, including older adults. in countries that have taken action in the face of an emergency this message will resonate; for others, particularly without the resources or capacity to address older adults, different approaches may be needed to build focus on the needs of this group. in countries with an evolving adult focus, a strategy could be to emphasize nitag strengthening and sharing experiences with the ''disease prevention" and/or ''health security" archetypes. this will be particularly important should a vaccine become available for covid- and in considering vaccines that can address vpds in an older population now. in countries who follow other countries' recommendations, stronger global guidance is needed to emphasize preparedness, highlight the importance a growing adult population, and the consequences of doing nothing. in countries that need to improve uptake, access, financing, registries, and monitoring are all important. one factor that correlates to uptake is expansion through pharmacists [ ] , although with covid- , perhaps other ways of delivery that don't require contact with people could be considered. additional provider-focused strategies could be implemented to improve uptake of current vaccines, such as financial incentives to vaccinate older adult patients. proactive outreach (e.g., actively sending reminders to patients to get their vaccines) may also be important post-pandemic to remind patients of vaccines' importance. finally, even in the ''child-focused and cost-sensitive" archetype there may be opportunities to take advantage of synergies and address immunization through broader issues such as universal health coverage and antimicrobial resistance. central to these opportunities is building the links between child and adult health, and economic development [ ] . the economic pressure placed on countries through covid- may make adult immunization seem unreachable and studies that capture the broader value of vaccines may help in justifying investments [ , ] . stronger global guidance synthesizing healthy aging, universal health coverage, antimicrobial resistance and immunization recommendations and agendas can also be helpful. importantly, strong individual champions, coalition building across the vaccine and aging communities, and more resonant messaging could all help elevate routine older adult vaccination as a priority [ ] . a critical, momentumbuilding milestone would be when global institutions, like who, commit to leading the coordination of implementation [ ] , empowering countries to think about implementing older adult immunization in a more timely manner. combining two separate analyses, this graph plots countries' performance in implementation and decision-making as a point estimate, overlaid with each country's primary archetype denoted by a symbol (within an archetype, countries share similarities around vaccine decision-making; (see table ). countries are plotted according to their adult vaccine implementation score (see table ) on the x axis and their adult vaccine decision-making score (see table ) on the y axis. for example, the uk scored in implementation and . in decision-making, and is plotted at ( , . ) . some countries share the same coordinate, with two names next to a single point. countries with the highest implementation and decision-making scores appear in the top right corner of the graph. primary archetype (disease prevention focused; health security focused; evolving adult focus; or cost-sensitive and child focused) is designated by the color of the country's name and the symbol overlaid each point. most countries (n= ) belong to the health security primary archetype. within and across all four archetypes, vaccine confidence plays a role as both a facilitator and barrier of countries' performance. although fear of side effects, spread of misinformation, lack of provider recommendation, and reduced belief that vaccines are valuable are common reasons for hesitancy across all ages, a recent review of the literature yielded few insights into describing how adult vaccine hesitancy varied from that for children [ ] . this is an area of further research that may help explain country uptake and better address implementation gaps. specifically, once the research gap is more clearly described, tailored communication and outreach strategies could be developed that encourage older adults to seek immunization services. countries take different approaches to adult immunization. drivers and facilitators of primary adult immunization archetypes should be considered when developing global guidance for countries. experiences and lessons learned should be shared within archetypes to improve performance of countries falling behind on decision-making or implementation. the results of this study may inform strategies in countries with similar contexts and priorities. further research may be beneficial to further refine archetypes and expand the understanding of what influences success within an archetype. this can help advance policies and action that will improve vaccine access for older adults and build a stronger appreciation of the value of immunization amongst a variety of stakeholders. the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: lpd has received grants from glaxosmithkline, merck and pfizer and an honorarium from pfizer. the other authors declare no competing interests. the world report on ageing and health: a policy framework for healthy ageing global, regional, and national disability-adjusted life-years (dalys) for diseases and injuries and healthy life expectancy (hale) for countries and territories, - : a systematic analysis for the global burden of disease study estimated human and economic burden of four major adult vaccine-preventable diseases in the united states t-cell immunity to influenza in older adults: a pathophysiological framework for development of more effective vaccines herpes zoster epidemiology, management, and disease and economic burden in europe: a multidisciplinary perspective cost-effectiveness of adult vaccinations: a systematic review modeling the economic burden of adult vaccine-preventable diseases in the united states cost-effectiveness of adult pneumococcal vaccination policies in underserved minorities aged - years compared to the us general population cost of shingles: population based burden of disease analysis of herpes zoster and postherpetic neuralgia a review of the cost-effectiveness of adult influenza vaccination and other preventive services united nations department of economic and social affairs population division population ageing in europe. facts, implications and policies. brussels: european commission directorate-general for research and innovation socioeconomic sciences and humanities adult vaccination as part of a healthy lifestyle: moving from medical intervention to health promotion vaccines for the elderly: current use and future challenges the public health value of vaccination for seniors in europe moving beyond traditional valuation of vaccination: needs and opportunities influenza vaccination in older adults: recent innovations and practical applications effectiveness of influenza vaccination on hospitalizations and risk factors for severe outcomes in hospitalized patients with copd report on who meeting on immunization in older adults the full benefits of adult pneumococcal vaccination: a systematic review identifying barriers to adult pneumococcal vaccination: an nfid task force meeting adult vaccination: now is the time to realize an unfulfilled potential national vaccine advisory c. a pathway to leadership for adult immunization: recommendations of the national vaccine advisory committee: approved by the national vaccine advisory committee on focusing on the implementation of st century vaccines for adults european geriatric medicine society (eugms) and the world association for infectious diseases and immunological disorders (waidid) first mexican consensus of vaccination in adults adult vaccination: a key component of healthy ageing -the benefits of life-course immunisation in europe a global review of national influenza immunization policies: analysis of the who/unicef joint reporting form on immunization adult immunization policies in advanced economies: vaccination recommendations, financing, and vaccination coverage variation in adult vaccination policies across europe: an overview from venice network on vaccine recommendations, funding and coverage challenges in adult vaccination vaccine myopia: adult vaccination also needs attention what isn't measured isn't done -eight years with no progress in aboriginal and torres strait islander adult influenza and pneumococcal vaccination a blueprint for improving the promotion and delivery of adult vaccination in the united states the world health organization. the global vaccine action plan the world health organization. immunization agenda : a global strategy to leave no one behind evidence-to-policy gap on hepatitis a vaccine adoption in countries: literature vs. policymakers' beliefs situational assessment of adult vaccine preventable disease and the potential for immunization advocacy and policy in low-and middle-income countries an activity report of physicians of the department of general medicine, juntendo university school of medicine against the great east japan earthquake relationship between public subsidies and vaccination rates with the -valent pneumococcal vaccine in elderly persons, including the influence of the free vaccination campaign after the great east japan earthquake pneumococcal vaccine (ppsv ) advies -basisvaccinatieschema development of archetypes for non-ranking classification and comparison of european national health technology assessment systems different paths to high-quality care: three archetypes of topperforming practice sites segmentation of seven asia-pacific health technology assessment (hta) agencies into different evolutionary hta archetypes integrating pharmacies into public health program planning for pandemic influenza vaccine response deaths related to pandemic influenza a (h n ) among american indian/alaska natives - states prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) the public health emergency management system in china: trends from evaluation of the effectiveness of surveillance and containment measures for the first patients with covid- in singapore coronavirus resource center early response to the emergence of influenza a (h n ) virus in humans in china: the central role of prompt information sharing and public communication covid- : too little, too late? pharmacy-based interventions to increase vaccine uptake: report of a multidisciplinary stakeholders meeting life-course immunization: building the consensus for adult vaccination in ifpma generation of political priority for global health initiatives: a framework and case study of maternal mortality characterizing global vaccine hesitancy: how adult and pediatric hesitancy may differ glaxosmithkline (gsk) biologicals, belgium provided funding for the archetype analysis. merck and gsk provided funding for the landscape analysis and case studies. the authors thank nina martin, geervani daggupati, nobutoshi nawa, so yoon sim, dexter waters, and gatien de broucker, for their valuable contributions; members of the international council on adult immunization (icai) for their review and input; the in-country experts who participated in case study interviews and provided input in the validation process; and haley budigan for her careful proofreading. key: cord- - tre rn authors: premanand, balraj; zhong wee, poh; prabakaran, mookkan title: baculovirus surface display of immunogenic proteins for vaccine development date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: tre rn vaccination is an efficient way to prevent the occurrence of many infectious diseases in humans. to date, several viral vectors have been utilized for the generation of vaccines. among them, baculovirus—categorized as a nonhuman viral vector—has been used in wider applications. its versatile features, like large cloning capacity, nonreplicative nature in mammalian cells, and broad tissue tropism, hold it at an excellent position among vaccine vectors. in addition to ease and safety during swift production, recent key improvements to existing baculovirus vectors (such as inclusion of hybrid promoters, immunostimulatory elements, etc.) have led to significant improvements in immunogenicity and efficacy of surface-displayed antigens. furthermore, some promising preclinical results have been reported that mirror the scope and practicality of baculovirus as a vaccine vector for human applications in the near future. herein, this review provides an overview of the induced immune responses by baculovirus surface-displayed vaccines against influenza and other infectious diseases in animal models, and highlights the strategies applied to enhance the protective immune responses against the displayed antigens. the existing battle between the human immune system and pathogens has a long-lasting history and is destined to continue. due to the advancement in the field of infectious diseases and the discovery of effective vaccines, therapies in recent history have aided in controlling the spread of diseases. however, the looming danger of re-emerging pathogens poses a serious issue and threatens to tilt the newfound equilibrium. among the types of vaccines available, inactivated vaccines are generally considered safe and stable. given their inactive nature, these vaccines can be given to people with weakened immune systems without the serious complication of opportunistic infection. however, the immune responses induced by inactivated vaccines are mainly humoral, and repeated immunization is required to improve immunity to the targeted disease. moreover, an inactivated vaccine may not retain the correct antigenic conformation and thus induces poor immune response against the targeted antigen [ ] . in contrast, live attenuated vaccines are more effective in inducing protective immune responses in vaccinated individuals. the major risks of live attenuated vaccines are the possibility of reversion and recombination with circulating strains, as well as the incompatibility of the vaccine with the immunocompromised, the elderly, the chronically ill, and the pregnant [ ] . recombinant subunit vaccines, on the other hand, can generally be used regardless of health status but possess other disadvantages. some of their disadvantages include the need for supporting adjuvants to improve immunogenicity, and purification issues due to the hydrophobic nature of antigens, which complicates the purification process, reducing the cost-effectiveness of vaccine production [ ] . dna vaccine utilizes genetically engineered dna capable of inducing humoral and cell-mediated immune responses against parasites, bacteria, and viruses [ , ] . modification of elements in the dna, such as promoters or enhancers, can enhance the expression of the encoded protein in vaccine recipients. moreover, a mixture of plasmids encoding a plethora of immunogenic genes can potentially be used to generate broad-spectrum vaccines. however, dna vaccine may induce antibodies against dna, resulting in autoimmune responses and the development of immunologic tolerance in recipient host [ ] . since , viral vaccine vectors have emerged, and such vectors have a more favorable safety profile than live attenuated virus vaccines, while having better immunogenicity than inactivated vaccines. viral vaccine vectors present the desired antigens in their native conformation, resulting in a stronger immunogenic response, while maintaining a higher level of foreign gene expression in vivo compared to dna vaccines [ , ] . however, some considerations exist for the use of viral vectored vaccines in humans: ( ) pre-existing vector immunity may have a serious impact on vaccine vector efficacy and ( ) increased transgene size could cause genetic instability and decrease viral yield. to overcome the issues of pre-existing vector immunity, vaccine vectors utilizing recombinant viruses of nonhuman origin have been developed to avoid neutralization of viral vector by pre-existing antibodies. a class of virus infecting insect cells, known as baculoviruses, has been developed for use as nonhuman viral vectors. among the various baculoviruses, autographa californica multicapsid nucleopolyhedrovirus (acmnpv) is the most widely studied. acmnpv is a large, double-stranded dna virus with a genome size of . kbp and contains open reading frames (orfs) [ ] . acmnpv has a biphasic life cycle resulting in the production of two forms of virus, budded virus (bv) and occlusion-derived virus (odv). the odv is responsible for the primary infection of the host insect, while bv is released from the host cells for cell-to-cell infection. moreover, the bv is produced during early stages of infection, whereas the odv is produced during late stages of viral infection, becoming concentrated in the nucleus and occluded within polyhedra [ ] . generally, acmnpv expresses each gene at specific time phases during its replication cycle. the genes encoding proteins that are responsible for viral replication and/or late gene expression (e.g., dnapol, lef - ) are expressed at an early time; structural protein genes (e.g., vp , p . , and e ) are expressed at late times; some genes are expressed at both early and late phases (e.g., ie , pp , and gp ); and p and polyhedrin (polh) are highly expressed at late and very late times of infection [ ] . based on published reports, the acmnpv promoters polyhedrin (polh) or p have been extensively used for expression of foreign proteins. in addition to the conventional acmnpv and other viral promoters, polh and p showed high levels of recombinant protein expression [ ] . interestingly, the combination of some of these promoters exhibited higher levels of protein expression compared to standard late promoters alone [ ] [ ] [ ] [ ] . moreover, it is well known that the polyhedrin promoter is active in the late stage of infection, and budding baculoviruses in the early stage of infection are unable to efficiently incorporate the target protein on the baculovirus envelope. hence, the use of immediate-early promoter may improve incorporation of target protein into the viral envelope. its salient features, including the ease of high-titer virus production, capability for simultaneous delivery of multiple genes, potential transduction ability, as well as its nonpathogenic and nonreplicative nature in humans, have led to the use of acmnpv in the production of complex eukaryotic protein in vitro, ex vivo, or in vivo [ ] [ ] [ ] and also for the vaccine development using surface display technology [ ] . in this review, we describe the immune responses elicited by recombinant baculovirus displayed vaccines against various infectious diseases and explore the unique strategies used to enhance the protective immunity against baculovirus displayed antigens (table ) . nt-not tested. since , influenza virus has been considered as an ever-present threat to humans, causing annual epidemics and infrequent pandemics, resulting in emergence of new virulent strains that pose a sustained alarm as a public health emergency [ ] . currently, the best method for the prevention and control of influenza virus infections is via vaccinations. however, a traditional influenza virus vaccine, while effective at managing infections, faces severe challenges. limited vaccine production capacity, long production time, poor growth properties of selected vaccine strains, and necessity of high-level biocontainment facilities are some major issues limiting vaccination production. particularly, uncertainty in prediction of emerging pandemic viral subtypes poses an obstacle in the prompt production of vaccines during an influenza pandemic. thus, a novel platform for the rapid development of vaccines, which overcomes limitations of currently available traditional vaccines, is required for swift response to influenza outbreak. hemagglutinin (ha) is a major surface glycoprotein of the influenza virus and is the main target for generating protective immunity against influenza virus [ ] . known to be a key determinant of host specificity, introduction of new ha subtypes was found to be associated with influenza strains responsible for pandemic outbreaks [ ] [ ] [ ] [ ] . hence, ha could serve as an important ideal target for development of influenza vaccines. recombinant subunit vaccines present one such method for targeting ha. recombinant subunit vaccines targeting ha were developed using an insect cell expression system and have been extensively evaluated in humans and animals as influenza vaccines. previously, a trivalent recombinant ha influenza vaccine-flublok ® , produced in insect cells using a baculovirus expression vector-was approved by the us fda. however, the need for a high dose with adjuvants and vaccine purification issues due to the hydrophobic nature of ha pose major setbacks in the development of recombinant subunit vaccines. an alternative strategy in vaccine development is the use of the baculovirus surface display technique. with baculoviral vaccines, targeted protein can be expressed on the surface of the virus without affecting the replication of the virus. moreover, insect cells possess the necessary cellular machinery to perform extensive post-translational modifications, such as glycosylation [ ] , phosphorylation [ ] , and disulfide bond formation [ ] , essential for the expression of many complex eukaryotic proteins, which require such modifications to maintain structural integrity and biological activities. furthermore, it has been reported that baculoviruses have strong adjuvant properties and are capable of inducing humoral and cellular immune responses against vaccine antigens [ ] . glycoprotein (gp ) is a major envelope protein of acmnpv which is essential for viral infection in insect cells. through the addition of gp domains (signal sequences, transmembrane and cytoplasmic regions), foreign proteins such as glutathione-s-transferase, human immunodeficiency virus (hiv) gp protein, rubella virus envelope protein, and synthetic igg binding domains can be displayed on the baculoviral envelope [ ] [ ] [ ] . alternatively, certain membrane proteins can be displayed on the budded virus itself without the need for gp domains. for example, vesicular stomatitis virus (vsvg) protein and measles virus receptor can be independently displayed on the viral envelope [ , ] . similarly, ha protein can be incorporated into the viral envelope without gp domains. the different regions of gp , such as the signal sequences, transmembrane domain, and cytoplasmic domain, have been widely used for the expression of secretory proteins in baculovirus/insect cell expression systems, and have been exploited to display foreign proteins or peptides on viral envelopes [ , ] . in a case study, the efficiency of h ha incorporation into the baculoviral envelope was shown to differ between recombinant baculoviruses carrying ha with different cytoplasmic domains (ctds), despite equal expression of recombinant ha. bac-ha , which expresses histidine-tagged ha with gp -ctd (cytoplasmic domain of gp ), incorporates ha to the viral envelope more efficiently than bac-ha, which expresses ha with ha-ctd (cytoplasmic domain of ha). moreover, bac-ha elicited higher hemagglutination inhibition titer (hi) compared to bac-ha. the result indicates the importance of the cytoplasmic domain and the degree of palmitoylation on the domain in the incorporation of ha on the viral envelope and on vaccine potential [ ] . in a second study, a modified bac-ha vaccine was developed by employing baculovirus p and cytomegalovirus (cmv) promoters with ha, which demonstrated surface display and endogenous expression of ha after transduction. an immunization study on the vaccine vector system revealed superior or equivalent immune responses against ha compared to other vaccine forms. the immune response is mediated by interaction with antigen-presenting cells (apcs) via the major histocompatibility ii (mhc-ii)-mediated antigen presentation pathway [ ] . in general, vaccines targeting respiratory viral infections, like influenza, act by inducing neutralizing antibodies that neutralize virions or block viral attachment and entry into host cells. however, currently available conventional vaccines induced humoral responses against only homologous strains and provided inadequate protection against heterologous strains. in contrast, t-cell-mediated cellular immune responses against conserved internal regions of antigens mediate protective immunity against heterologous strains [ , , ] . in previous studies, stimulation of helper t-lymphocytes (th cells) and cytotoxic t-lymphocytes (ctls) by nucleoprotein amino acids np - and np - provided complete cross-protection in challenge studies [ , ] . sridhar et al. [ ] observed that individuals having a higher number of pre-existing cd + t-cells against conserved cd epitopes showed milder illness after infection with pandemic h n influenza virus [ ] . furthermore, wilkinson et al. [ ] showed that, in the absence of antibody responses, pre-existing cd + t-cells respond to influenza internal proteins, reduce the severity of illness, and demonstrate lower shedding of virus [ ] . recently, zhang et al. [ ] developed a recombinant baculovirus-based vaccine containing three tandem copies of the highly conserved extracellular domain of influenza m protein (m e) (bv-dual- m e); a second vaccine had an additional mucosal adjuvant heat-liable enterotoxin b (bv-dual- m e-ltb). mice inoculated with bv-dual- m e-ltb vaccine induced higher levels of mucosal antibodies and more efficient cellular immunity against different h n clades (clade , . . . , . . , and ) compared to non-adjuvanted bv-dual- m e vaccine. interestingly, it was discovered that mucosal immunity alone was insufficient for protection from lethal h n challenge, whereas adjuvanted vaccine provided enhanced protection against similar challenge through cd + t-cell response. to validate the role of cd + t-cells in enhancing protective immunity, mice with depleted cd + or cd + t-cells were inoculated with bv-dual- m e-ltb. mice with depleted cd + t-cells displayed reduced lung viral titers, but no significant effect on survival rate was observed. comparatively, depletion of cd + t-cells or both cd + t-cells and cd + t-cells markedly increased lung viral titers and decreased survival rate. further research on enhancing ha-specific immune responses utilized a baculovirus vaccine vector with additional elements to improve immunogenicity. these elements included cag promoter, mhc class i signal sequence (mhciss), mhc class i trafficking domain (mitd), and woodchuck hepatitis virus posttranscriptional regulatory element (wpre). cag promoter enables robust expression of ha, while mhciss and mitd enhance mhc class i and ii antigen presentation, which stimulates the epitope-specific cd + and cd + t-cells. wpre, on the other hand, elevates the ha gene expression and increases immunogenicity. as shown in the study, mice immunized with baculovirus possessing the additional elements induced higher levels of ha-specific igg and igg a, higher hemagglutination inhibition titers, and better th and ifn-γ + /cd + t-cell responses compared to baculoviruses without the additional elements [ ] . furthermore, the role of wpre and itrs (inverted terminal repeats) in enhancement of ha expression and their effect on induction of humoral and cellular immune responses was studied. two series of baculovirus that use different promoters to drive the expression of ha were generated: bv-s series (bv-s-ha, bv-s-itrs-ha, bv-s-con-ha), which utilizes the cmv promoter for the expression of ha, and bv-a series (bv-a-ha, bv-a-itrs-ha, bv-a-con-ha), which utilizes white spot syndrome virus (wssv) immediate-early promoter one (ie ) for the expression of ha. results from immune studies indicate that baculoviral vaccine with wssv ie promoter induces a higher level of humoral and cellular immunity compared to similar baculoviral vaccine utilizing the cmv promoter [ ] . in another study, recombinant baculovirus vectors with an egfp expression cassette under p and cmv promoters were examined. the baculoviral vectors were modified to express ha using baculovirus polyhedrin (polh) promoter (vac-ha) or using dual-promoter with polh and cmv promoter (vac-ha-dual). while ha was expressed efficiently in sf insect cells for both constructs, mice vaccinated with vac-ha induced higher levels of igg and iga compared to mice vaccinated with vac-ha-dual. in addition, stimulation of splenocytes in immunized mice with ha-specific peptide triggered th immune response for vac-ha-immunized mice, whereas vac-ha-dual-immunized mice showed th -biased immune response. moreover, vac-ha-dual vaccine was more effective in inducing ha-specific cd + and cd + t-cell response in vitro [ ] . it has been reported that the signal peptide of membrane proteins is important in directing the protein to the endoplasmic reticulum membrane and in protein trafficking [ ] . the transmembrane (tm) domain plays a role in protein trafficking, membrane anchoring, membrane fusion, and viral budding [ , ] , whereas the ct domain is crucial for envelope incorporation, virus budding, interaction with the components of viral core [ , ] , and incorporation of influenza ha into the baculovirus envelope [ ] . supporting these observations was the research by tang et al. [ ] on the functional role of signal peptide (sp), ctd regions of gp , and tm of ha in the incorporation of ha into the baculovirus envelope. the study demonstrated enhanced ha incorporation and increased hi titers for baculovirus with gp sp, ctd, and ha tm. in stark contrast, recombinant baculoviruses containing different combinations of gp or ha domains (gp sp or ha-sp with ha-tm and ha-ctd; gp -tm or ha-tm with ha-sp and gp ctd; gp sp or ha-sp with gp -tm and gp -ctd) showed the opposite effect on ha incorporation and hi titers. based on the results, the second generation of tetravalent baculoviral vaccine was developed using gp -sp, ha-tm, and gp -ctd. the vaccine displays has from four subclades of h n influenza viruses and induces strong humoral and cellular immunity against homologous and heterologous h n strains [ ] . in another study, efficiency of wssv ie promoter-mediated h ha expression by recombinant baculovirus was examined [ ] . also, they confirmed that ha expressed by the baculovirus was able to successfully translocate to the plasma membrane of the infected cells and observed that surface-displayed ha was able to sustain its antigenic conformation and authentic cleavage [ ] . it has been reported that baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (vsvg) from vesicular stomatitis virus (vsv) effectively increased transduction levels in mammalian cells [ ] and has the capability to increase the amount of expressed protein displayed on the surface of the entire viral envelope [ , ] . in addition, recombinant baculovirus which displays ha and co-expresses vsvg under wssv ie showed ha displayed on the baculovirus surface has high ha activity. intranasal or intramuscular immunization of chickens with baculovirus containing wssv ie induced strong ha-specific antibodies and hi titer compared to baculovirus with cmv promoter. nevertheless, co-expression of vsvg by both viruses contributed to increased anti-ha antibody level and hi activity [ ] . similarly, recombinant baculovirus pseudotyped with vsvg (bv-g-ha) was shown to successfully transduce into mammalian cell, mediate gene delivery, and enable efficient expression of h ha. an immunization study revealed that the vaccine candidate induced higher levels of h ha-specific antibodies and cellular immunity compared to the mice vaccinated with µg of dna vaccine. furthermore, immunized chickens, administered with varying doses, demonstrated complete protection from a lethal dose of highly pathogenic h n avian influenza virus [ ] . however, the vaccine did not efficiently protect mice against an evolutionarily distant strain [ ] , possibly due to a high degree of genetic divergence of the influenza virus [ ] . for example, current monovalent inactivated whole virus vaccine can induce neutralizing antibodies against the homologous strain but shows lower magnitude of response against heterologous strains [ ] . hence, it is necessary to develop a vaccine that expresses antigens covering the major circulating viruses [ ] . therefore, to broaden the protective efficacy of monovalent bv-g-ha vaccine, a pseudotyped baculovirus-based bivalent vaccine that encodes ha from clade . . and clade influenza viruses was developed. mice immunization with pseudotyped baculovirus-based bivalent vaccine induced significantly higher humoral and cellular immune responses and provided complete protection against h n viruses from clade . . and clade compared to other candidates used in the study [ ] . similarly, the cross-protective efficacy of wssv ie promoter-controlled bivalent baculovirus surface-displayed ha of a/indonesia/ / and a/anhui/ / (bivalent-bacha) was tested in another study. mice orally vaccinated using live bivalent-bacha vaccine showed both systemic and mucosal immunity, unlike mice immunized subcutaneously with live or inactivated bivalent vaccine, which induced only robust systemic immunity. the level of systemic immunity induced by oral bivalent vaccine in immunized mice was lower compared to subcutaneous immunization. however, mice orally immunized with bivalent-bac-ha vaccine demonstrated strong ha-specific mucosal iga response. in addition, oral vaccination induced potent cross-clade neutralizing antibodies against distinct clades ( , . . , . . . , . . , . . , , , and ) of h n viruses which are absent in monovalent inactive whole h n vaccination, whereas mice subcutaneously vaccinated with live or inactivated bivalent-bacha vaccine showed low neutralization titer against clade h n compared to other clade of viruses. enzyme-linked immunospot assay revealed that mice immunized orally or subcutaneously with live bivalent vaccine triggered both ifn-γ-secreting th cells and il- -secreting th cells, while mice immunized subcutaneously with inactive adjuvanted bivalent vaccine stimulated only the il- response. moreover, mice vaccinated orally or subcutaneously with live bivalent bac-ha vaccine showed complete cross-protection against clade and clade . . . h n viruses, suggesting that this bivalent vaccine can, potentially, protect against cross-clade h viruses during pandemic situations [ ] . since baculovirus has been proved to efficiently express foreign genes and is capable of transduction in a variety of cell lines, researchers utilized this virus as a potential vaccine vector against various infectious agents. furthermore, additional elements, like vsvg protein, were included by researchers to increase the efficiency of transduction, gene delivery, and expression in the recipient host [ ] . while recombinant baculoviral vector expressing both vsv-g and influenza ha was shown to evoke both humoral and cellular immune responses and provided effective protection against lethal virus challenge in mouse and chicken hosts [ ] , the high cytotoxicity of vsv-g protein [ ] and its immediate inactivation by serum complement systems impedes the use of the element in a vaccine delivery vehicle [ ] . recent reports have shown that human endogenous retrovirus (herv) envelope-coated recombinant baculovirus-based human papillomavirus l vaccine significantly improved the expression of l in mammalian cells and induced humoral immune responses which are comparable with the level obtained using commercial cervical cancer virus-like particles vaccine [ ] . further, it has been reported that herv envelope-coated baculovirus-based ha of the h n pandemic influenza vaccine induced a strong cellular immune response compared to the commercial vaccine [ ] . in another study, prabakaran et al. [ ] developed a recombinant baculovirus displayed ha vaccine under the control of wssv ie promoter (bacha) against the pandemic h n virus. the vaccine showed successful ha expression on its envelope, and mice vaccination studies showed that both the live and adjuvanted with inactive form of recombinant baculovirus induced ha-specific antibody responses and offered complete protection against lethal viral infection [ ] . studies have shown that high neutralizing antibodies generated against influenza virus target specific regions in the globular head of influenza ha. moreover, the ha stalk domain is well conserved and antibodies targeting this region are capable of neutralizing the heterologous influenza viral subtypes [ ] [ ] [ ] [ ] [ ] [ ] . for example, chimeric ha constructs expressing the globular head and stalk regions from different subtypes induced antibodies that showed broad protection [ , , ] . similarly, mice immunized with recombinant baculovirus displayed ha of the pandemic h n influenza virus induced ha-stalk-specific antibodies and demonstrated protective immunity against homologous and heterologous h n subtype viruses [ ] . in recent decades, avian h influenza virus was responsible for numerous influenza outbreaks among poultry industries in europe and north america. since , none of these poultry-adapted viruses has evolved to widely infect humans or cause a pandemic. however, there are cases where the virus acquired the ability to infect mammals, as evidenced by the largest outbreak of highly pathogenic h n (nl/ . ) in the poultry industry of the netherlands, where human infections were reported [ ] . h n viruses isolated from the patients were shown to possess replicative potential in human conjunctiva and ability for human-to-human transmission. interestingly, the virulence of the virus in chickens and turkeys increased with acquisition of additional amino acids at the hemagglutinin (ha) cleavage site [ ] . in , human infections with avian influenza a h n subtype were first reported in china and caused several waves of human infection. it was speculated that the virus possesses high mutagenicity, which contributed to its ability to infect humans. higher mutagenicity may be the cause of enhanced pathogenicity, as supported by recent human cases, where the isolated virus was found to have mutations associated with reduced susceptibility to neuraminidase inhibitors used in antiviral treatment [ ] . since h subtype viruses pose a major challenge for public health and the poultry industry, numerous vaccines have been generated against these subtype viruses using various platforms. recently, baculovirus displayed ha vaccine against h n (nl ) was developed and its efficacy was tested via intranasal (i.n.) or subcutaneous (s.c.) administration in mice. it was observed that intranasally immunized mice with live baculovirus displayed ha vaccine induced higher or comparable humoral, mucosal, and cellular immune responses compared to other vaccine candidates, irrespective of immunization route. additionally, the immunization provided % complete protection, whereas no protection was observed in mice vaccinated with other vaccine candidates [ ] . in another study, recombinant baculovirus-based vaccine (bacha) against avian h n virus with high pandemic potential was generated, and its immunogenicity and cross-protective efficacy was evaluated in mice. they found that live bacha i.n. immunized mice elicited high levels of ha-specific iga mucosal immune response and humoral immune response, comparable to mice administered subcutaneously with live bacha. on the other hand, subcutaneous immunization with adjuvanted inactive bacha stimulated robust humoral immune response and induced higher neutralizing antibodies against h n viruses and other h subtype (h n and h n ) viruses compared to other vaccine candidates. in addition, subcutaneous immunization with inactive whole h n vaccine protected % against rg-h n challenge but demonstrated insufficient protection against rg-h n (nl ) subtype, whereas mice vaccinated subcutaneously with live or inactive recombinant baculovirus against h n (nl ) protected only - % of mice against h n challenge. furthermore, mice immunized i.n. with live bacha of h n or h n showed complete protection against both h n and h n infection in a challenge study [ ] . it has been reported that the differences between the immunogenicity of ha of h n (nl ) and ha of h n might be due to the amino acid changes at a t, which introduced a potential glycosylation site at position n, masking the epitopes of h ha (nl ) [ , ] . to account for this difference in immunogenicity, kumar et al. [ ] compared the ha of h n and h n viruses and selected three positions in their study: (a) , (b) , and (c) , which are located within or near the receptor-binding site of h n ha protein. by site-directed mutagenesis, they generated six mutant constructs by single or double amino acid substitution at these three positions (t a, t a, i v, t a- a, t a-i v, and i v-t a) . among the generated mutant constructs (bac-ha m ), mice vaccinated with constructs t a, t a-i v, and i v-t a induced higher hemagglutination inhibition and neutralization titer that neutralizes both h n and h n viruses compared to mice immunized with bacha or other mutant vaccines. the result shows that the substitution of amino acids responsible for forming potential glycan motif-epitope masks would improve immunogenicity of the vaccine candidates. in another study, mice i.n. immunized with baculovirus-displaying ha of low pathogenic influenza h subtype triggered higher ha-specific humoral, mucosal, and cellular immunity compared to mice immunized with inactivated influenza virus. moreover, regardless of immunization route, it conferred % protection against lethal homologous mouse-adapted h n virus [ ] . moreover, recombinant baculovirus with cmv-polyhedrin dual promoter for expressing chimeric ha of h n was shown to efficiently express ha in both mammalian and insect cells, induce strong immune response, and provide % protection against lethal h n viral challenge in mice, unlike other vaccine candidates observed [ ] . shrimp immunity relies on an innate immune mechanism, and their lack of adaptive immunity hinders vaccine development against shrimp pathogens. though limitations exist in developing shrimp vaccines, the presence of quasi-immune responses after natural or experimental infection with white spot syndrome virus (wssv) in p. japonicas and the resistance against experimental rechallenge with wssv due to neutralizing factors in the survivors' plasma [ , ] encouraged the researchers to continue the development of vaccines against the wssv virus. several attempts have been made to develop vaccines by targeting the immunogenic vp protein of wssv virus [ ] [ ] [ ] [ ] syed musthaq et al. [ ] , developed a recombinant-based vaccine (bac-vp ) that displays recombinant vp (rvp ) on its envelope. confocal and transmission electron microscopy analysis confirmed that rvp was able to localize on the plasma membrane of infected insect cells, and the virus also successfully acquired rvp on its envelope from the insect cell membrane during the budding process. approximately . µg/ml of vp protein was anchored on the bac-vp envelope ( viral particles). furthermore, its protective efficacy against wssv infection was tested via injection, oral, or immersion methods in shrimp. the bac-vp -injected shrimp successfully transcribed and translated vp and showed survival rates of . % and . % against wssv virus challenge at and days postvaccination, respectively [ ] . on the other hand, the transcriptional levels of lipopolysaccharide and β- , -glucan binding protein (lgbp) and signal transducer and activator of transcription (stat) genes (which are crucial for triggering innate immune responses) [ ] were altered at different days postinfection (dpi) in shrimp vaccinated with bac-vp by oral or immersion routes. the expression level of stat gene in the control group was downregulated at various dpi as wssv infection progressed, but in the vaccinated group, stat gene level decreased until dpi and started to increase at dpi and dpi. moreover, the levels of lgbp gene in the control group was upregulated as infection progressed, whereas the vaccinated group showed an increasing level at early infections, which later decreased as wssv infection cleared. these results confirm that oral vaccination of bac-vp triggers the innate immune responses and, possibly, was responsible for higher survival rates of . % and . % upon wssv challenge at and days postvaccination. also, shrimp vaccinated with bac-vp immersion vaccine showed % and . % survival rates compared to % mortality obtained with the control group in a challenge study [ ] . porcine reproductive and respiratory syndrome virus (prrsv), classical swine fever virus (csfv), and porcine circovirus type (pcv ) are the major pathogens continuously affecting the pig industry and cause significant economic losses worldwide. to date, researchers all over the world have contributed various forms of vaccines utilizing different strategies targeting these pathogens. virus-based vectored vaccines were developed using modified vaccinia virus ankara, canine adenovirus- , pseudorabies virus, and semliki forest virus [ ] [ ] [ ] [ ] . however, these vectors are of mammalian origin and pose a biohazard threat. in contrast, recombinant baculoviruses possess good safety profiles and have been utilized to develop vaccines against prrsv, csfv, and pcv viruses. using gp sp, tm, and ct domains, xu et al. [ ] developed recombinant baculovirus-based bivalent vaccine vector (bacsc-dual-gp -cap) that surface-displayed gp (major immunogenic inducer of neutralizing antibodies against prrsv infection) and pcv (major immunogenic capsid (cap) protein). a swine vaccination study demonstrated that this vaccine candidate stimulated potent gp and cap-specific neutralizing antibodies and induced higher lymphocyte proliferation responses compared to control groups, which indicates that the bivalent vaccine can potentially be used as a vaccine against a mixed prrsv and pcv infections. in another study, the orfs of prrsv were successfully displayed on the baculovirus envelope using the gp sp, h n tm, and gp ctd regions. the antibody responses elicited by the vectored vaccines were sustained for up to days after the booster, whereas the titers in the inactivated prrsv-vaccinated group were reduced dramatically at this time point. the enhanced immune responses could be due to the in vivo transduction of the prrsv genes by baculovirus vaccine, which activates the endogenous antigen-processing and -presenting pathways. amongst the vaccines developed in this study, baculoviruses that displayed orf a, orf , and orf induced higher antibody responses in mice compared to other vaccine candidates [ ] . a recombinant baculovirus-based vaccine that contains the pcv cap and vsvg protein (bv-gd-orf ) was developed. the expression of pcv was placed under the control of cmv and polh promoters, whereas expression of vsvg was placed under the p promoter. due to vsvg-mediated transduction and the successful expression of the pcv cap protein in transduced cells, bv-gd-orf behaved as a subunit/dna vaccine and induced robust cap protein-specific humoral and cellular immune responses [ ] . studies reported that e or e rns glycoprotein are the main immunogenic proteins of csfv, which can induce neutralizing antibodies and provide protective immunity against csfv. xu et al. generated recombinant baculoviruses that surface-displayed either e (bacsc-e ) [ ] or e rns (bacsc-e rns ) [ ] as fusion forms with gp domains. the presence of the gp domains did not alter the viral titers of the recombinant viruses. moreover, a mice vaccination study revealed that bacsc-e or bacsc-e rns induced stronger e -or e rns -specific antibodies, respectively, compared to other vaccine candidates, suggesting that the vaccine candidates could potentially be applied in the treatment of csfv infection. respiratory syncytial virus (rsv) is a common contagious virus that causes acute lower respiratory tract infection in infants, young children, elderlies, and immunocompromised individuals [ ] . currently, there is no approved vaccine available to treat this infection in humans. studies have reported that immunization with recombinant viral vector expressing rsvg protein showed immune pathological issues by enhancing th skewing of the cd + t-cell response [ ] [ ] [ ] . countering the issue, zhang et al. [ ] developed recombinant baculovirus vaccines that showed reduced immune pathological conditions in mice. four vaccine constructs were developed: bac-tf (displays the f ectodomain (tf) on the envelope), bac-cf (expresses full-length f protein in transduced mammalian cells), bac-cf/tf (displays tf on the envelope and expresses f in cells), and bac-cf/tf -visa (contains additional virus-induced signaling adaptor (visa gene). bac-cf and bac-cf/tf showed an increased mixed th /th cytokine response, higher levels of igg a/igg antibody responses, and reduced immunopathology upon rsv challenge compared to bac-tf . moreover, co-expression of visa reduced the th immune responses induced by bac-cf/tf and protected lungs from inflammatory immune response upon a subsequent rsv challenge. therefore, the inclusion of visa elements could be an effective alternate vaccine strategy against rsv infection [ ] . rabies virus (rabv) is highly infectious and poses a constant threat to humans and animals. traditionally, vaccine development against rabv mainly focused on inducing neutralizing antibodies [ ] [ ] [ ] . however, studies have shown that, in addition to humoral immunity, cellular immune responses are important for providing protective immunity against rabv [ , , ] . previously, a recombinant baculovirus viral vector vaccine against rabv glycoprotein (rvg) was constructed (bv-rvg/rvg). the vector expresses two types of rvg antigen (baculovirus-expressed rvg and in vivo expressed rvg) to enhance specific immune responses against the rvg antigen. based on immunization study, bv-rvg/rvg-immunized mice displayed higher humoral and cellular immune responses and offered complete protection against rabv compared to mice immunized with other vaccine candidates. the enhanced immunogenicity could be due to immediate recognition of rvg incorporated into bv-rvg/rvg by immunocompetent cells, resulting in presentation to dendritic cells (dcs) and, subsequently, activation of b cells to secrete viral-specific neutralizing antibodies. furthermore, cmv-mediated in vivo rvg expression by bv-rvg/rvg activated the endogenous antigen presentation pathway, inducing persistent and specific immune responses [ ] . in another study, feng et al. [ ] displayed the entire ectodomain of sars-cov spike protein (s) on the baculovirus envelope using gp signal peptide and vsv-g membrane anchor. in mice, the vaccine induced specific neutralizing antibodies against s protein in the absence of adjuvants [ ] . in a subsequent study, researchers developed recombinant baculovirus vaccines against avian infectious bronchitis virus (ibv), japanese encephalitis virus (jev), and bovine herpesvirus- (bhv- ) with the gp domains (sp, tm, and ctd). chickens immunized with baculovirus displayed s protein of ibv vaccine induced more humoral and cellular immune responses and showed better protection ( %) compared to other vaccines studied [ ] . on the other hand, glycoprotein e of jev displayed on the baculovirus envelope induced strong neutralizing antibodies in mice and swine and offered % protection in vaccinated mice against a lethal jev challenge [ ] . similarly, bhv- envelope glycoprotein (gd) displayed on the baculovirus envelope elicited strong neutralizing antibody against bhv- in mice [ ] . enterovirus (ev ) immunogenic capsid protein (vp ) is a non-membrane protein which requires additional regions, like sp, tm, and ctd of gp , ha, or other domains for successful incorporation into envelope, while membrane proteins like ha or na (neuraminidase) can be translocated to the plasma membrane and incorporated onto the envelope during viral budding. meng et al. [ ] generated recombinant baculoviruses, with wssv ie (bac-pie -gp -vp ) or polyhedrin promoter (bac-pph-gp -vp ), which surface-displayed chimeric vp (vp was fused in between gp sp and mature domain). transmission electron microscopy analysis confirmed that vp was successfully anchored on the recombinant baculoviral envelope. bac-pie -gp -vp -immunized mice were found to induce slightly higher vp -specific antibodies with potent neutralizing activity (can neutralize c subgenogroups and heterologous subgenogroups) than bac-pph-gp -vp and to show neutralizing activity comparable with inactivated ev vaccine. subsequently, premanand et al. [ ] generated the recombinant baculovirus (bac-vp ) that surface-displayed the chimeric vp using minimal fusion partners, like gp sp ( aa), h n -ha transmembrane domain ( aa), and gp ctd ( aa) to facilitate better incorporation of vp and higher baculovirus titers. expectedly, bac-vp contains -fold higher vp ( ng/µg) on its envelope compared to vp on bac-pie -gp -vp envelope ( ng/µg). bac-vp -immunized mice elicited stronger vp -specific antibody responses and cross-neutralization activity against homologous or heterologous strains compared to bac-pie -gp -vp and bac-pph-gp -vp vaccines. moreover, bac-vp provided % protection against a mouse-adapted lethal strain of ev [ ] . in both studies mentioned above, vp was anchored on the viral envelope similar to type transmembrane protein with unrestricted n-terminus exposure. mimicking other types of transmembrane protein, kolpe et al. generated recombinant baculovirus (bac-na-vp ) which surface-displayed vp using influenza a neuraminidase (na), resulting in a type ii transmembrane-anchored pattern with a distal c-terminus. mice immunization studies revealed that bac-na-vp induced both humoral and cellular immune responses and provided % protection of suckling balb/c mice against mouse-adapted ev -b strain challenge [ ] . infectious bursal disease virus (ibdv) is the causative agent of infectious bursal disease, which affects chickens and causes considerable economic loss for the poultry industry [ , ] . it has been reported that live attenuated vaccines elicited lower level of antibodies and failed to protect the chickens against ibdv [ ] . to target this issue, xu et al. [ ] generated a recombinant baculovirus that surface-displayed his -tagged vp protein using the gp tm and ct domains (bacsc-vp ). bacsc-vp -immunized chickens demonstrated higher levels of neutralizing antibodies and better protective immunity against a very virulent ibdv strain than control groups. furthermore, ibdv-specific lymphocytes were observed in bacsc-vp -vaccinated chickens. also, the major immunogenic capsid protein vp of canine parvovirus type (cpv- ) and immunogenic proteins σc and σb of avian reovirus (arv) have been utilized to develop a baculovirus-based vaccine against cpv- and arv, respectively. fusion of gp domains (sp, tm, and ctd) with the capsid proteins allows their incorporation into the baculoviral envelope. an animal vaccination study revealed that baculovirus that surface displayed vp of cpv- induced higher neutralizing antibodies in mice compared to other vaccines tested [ ] . moreover, recombinant baculoviruses that displayed immunogenic proteins of either σc or σb of arv elicited strong antibody titers with higher neutralizing activities compared to antibody responses by other vaccine candidates [ ] . malaria, a global disease caused by plasmodium parasites, is endemic in tropical and subtropical regions and results in up to million deaths annually. a recombinant protein-based subunit vaccine containing a part of a major surface protein from plasmodium falciparum circumsporozoite protein (pfcsp) provided short-lived protective immunity, and the effective period was affected by age group and malaria transmission intensity [ , ] . another vaccine plasmodium falciparum sporozoite (pfspz) was reported to provide protection against homologous and heterologous human malaria infection [ ] [ ] [ ] . aiming to curb the spread of malaria, the who has set to a goal of % vaccine efficiency by the year . novel and alternate strategies will be required to ensure the development of more effective vaccines against malaria infection. to develop a vaccine with dual functions (such as a malaria vaccine that can act as a liver-directed gene delivery vehicle), yoshida et al. [ ] developed the recombinant baculovirus-based vaccine that displayed rodent malaria parasite plasmodium berghei circumsporozoite protein (pbcsp). in the immunization study, the vaccine induced both humoral and cellular immune responsea and provided % protection against sporozoite challenge [ ] . subsequently, a series of baculovirus-based vaccines expressing pfcsp (with deleted glycosylphosphatidylinositol gpi anchor region) under the control of polh or cmv promoter was developed. acnpv.cs vector expresses the cs protein in apcs under the control of the cmv promoter to enable (preferentially) induction of cd + t-cells upon mhc class i presentation. acnpv.cs vector expresses a cs protein with gp as a fusion form using the polyhedrin promoter in insect cells, which allows the incorporation of cs on the baculovirus envelope during the budding process (mimicking the p. falciparum sporozoite). uptake of acnpv.cs into apcs triggers cd + cells via mhc class ii presentation and induces a potent humoral immune response. acnpv.cs-cs combines expression and presentation of recombinant cs antigens in mammalian cells to induce both cs-specific cd + and cd + t-cells. a mouse vaccination study showed that the acnpv.cs-cs vaccine induced efficient humoral and cellular immune responses with intramuscular immunization compared to other vaccine candidates [ ] . recombinant baculovirus dual expression-based malaria vaccine, which drives pbcsp expression by polh and cmv promoters, was developed. mice immunized with the vaccine induced both th and th responses after intramuscular inoculation and provided complete protection against sporozoite challenge [ ] . another baculovirus-based vaccine candidate which displays the major surface antigen of p. falciparum, ppfs , was developed as a transmission-blocking vaccine against human malaria. both i.m. and i.n. immunized mice showed high levels of pfs -specific antibodies and effective transmission-blocking response, with an % (intranasal) and % (intramuscular) reduction in oocyst intensity [ ] . in another case study, a recombinant baculovirus-based plasmodium vivax transmission-blocking vaccine using autographa california nuclear polyhedrosis virus (acnpv-dual-pvs ) was developed. acnpv-dual-pvs not only displayed pvs on the acnpv envelope in native conformation, but also expressed pvs upon transduction of mammalian cells. acnpv-dual-pvs induced pvs -specific antibodies in intranasally or intramuscularly immunized mice. moreover, in a rabbit immunization study, the vaccine induced significant transmission-blocking response, regardless of immunization route (subcutaneous, intramuscular, and intranasal) [ ] . yoshida et al. [ ] developed a baculovirus-based nasal drop vaccine (acnpv-pymsp surf ) that surface-displayed the carboxyl terminus of merozoite surface protein (pymsp ) from plasmodium yoelii. intranasal and intramuscular immunization of mice with acnpv-pymsp surf induced mixed th /th immune responses and provided % protective efficacy. however, intranasal immunization elicited higher pymsp -specific antibody titers and stronger natural boosting after a challenge study [ ] . in another study, a baculovirus dual-expression system-based pfcsp protein (bdes-pfcsp) vaccine was generated. based on an immunization study, the vaccine was found to induce mixed th /th immune responses in mice, generate a higher level of pfcsp-specific antibodies, and confer significant protection against malaria. in addition, sera from immunized monkeys prevented sporozoite invasion in hepg cells [ ] . a multistage malaria vaccine was developed using the baculovirus platform to target and inhibit the plasmodium parasite in its pre-erythrocytic and sexual stages. the generated baculovirus displayed correct folding of both p. vivax circumsporozoite protein (pvcsp) and p (pvs ) protein in fusion form (bdes-pvs -pvcsp) and acted as a pre-erythrocytic and a transmission-blocking activity vaccine. the bdes-pvs -pvcsp vaccine induced higher antibody levels against pvs and pvcsp while providing % protection against malaria and % transmission-blocking activity [ ] . in a recent study, human decay-accelerating factor (hdaf) was incorporated into a baculovirus vector, which effectively displayed hdaf and pfcsp on the surface of the baculoviral envelope to prevent serum complement inactivation. following intramuscular immunization, the modified vaccine conferred higher protective efficacy ( %) compared to control group ( %) in a challenge study [ ] . mucosal surfaces (respiratory, gastrointestinal, and urogenital tracts) provide the major route of entry for various microorganisms, and the mucosal immune system acts as first line of defense against microbial infection [ ] . therefore, triggering of the mucosal immune system is important in preventing infection, and this can be achieved by mucosal vaccinations via intranasal, oral, pulmonary, rectal, or vaginal routes of administration. among these routes, oral or intranasal immunizations are the two most viable options for vaccine delivery. the nasal route, in particular, offers several advantages, like the possibility of self-administration, the induction of both mucosal and systemic immunity in the gastric mucosa, respiratory tracts and genital tracts, as well as the lower doses of antigen and adjuvant required compared with oral vaccination. several nonhuman viral vectors have been developed in recent years, among which is the baculoviral vector. baculovirus has been reported to possess strong adjuvant properties in mammals, promoting enhanced humoral, mucosal, and cellular immune responses against co-administered antigens. supporting this are previous reports which indicated that mice immunized with baculovirus genomic dna and ovalbumin showed humoral and cytotoxic t-lymphocyte responses against co-administered antigens. the abundance of cpg motifs in the baculovirus improves its ability to induce innate immune responses through the tlr /myd -dependent endosomal recognition pathway and dna-dependent activator of interferon regulatory factor-mediated pathway [ ] . other studies have shown that intranasal inoculation of mice with wild-type baculovirus provided protection against h n , h n , and other influenza b strains [ ] , which highlighted the potential of the baculoviral vector in the development of vaccines against mucosal acquired pathogens. intranasal immunization of mice with live recombinant baculovirus (bac-ha) vaccine induced significantly higher mucosal iga immune response against h n -ha compared to other intranasally delivered vaccines. the higher immune response could be attributed to the efficient transduction of baculoviral vector or the strong adjuvant property of baculovirus due to the presence of unmethylated cpg motifs. in addition, i.n delivery of bac-ha stimulated higher amounts of ifn-γ and il- compared to s.c immunization of live bac-ha, which is similarly reported in other studies [ , ] . the enhancement of cytokine levels could be due to recall response in splenocytes or due to the presence of nasopharynx-associated lymphoid follicles in the nasal mucosa that trigger antigen-specific th lymphocytes, ctls, and iga immune responses. a challenge study in mice revealed that i.n immunized mice with live bac-ha showed % protection against lethal viral challenge compared to other vaccines, irrespective of route of administration. even though s.c immunized mice achieved a higher level of igg antibodies, the incapability of bac-ha in protecting against lethal viral challenge could be due to lack of mucosal iga, which is more crucial in clearing upper respiratory tract infection than igg [ ] . in another study, a recombinant baculovirus-based vaccine against another pandemic potential avian h n virus was generated (bac-ha). they found that mice intranasally immunized with live bac-ha elicited higher levels of ha-specific mucosal and humoral immune responses. mice vaccinated subcutaneously with live or inactive baculovirus displayed ha of h n (nl ) only protected - % of mice against h n challenge, but mice immunized intranasally with live bac-ha of h n or h n showed complete protection against both h n and h n infection in a challenge study. the result indicates the importance of additional mucosal immunity in recovery and protection during h subtype infection [ ] . the inclusion of adjuvant improves the immune response induced by the vaccine, and the cause of the better immune response in mucosal adjuvants, in particular, is due to the induction of protective local and systemic immune responses against various infectious diseases [ ] . also, intranasal immunization of recombinant baculovirus-displaying ha of h n virus induced high levels of mucosal iga and serum igg immune responses in mice. combination of recombinant mucosal adjuvant ctb further enhanced serum igg and iga responses compared to unadjuvanted log or log ha titer bac-ha alone. log ha titer live bacha with µg of rctb conferred % protection against homologous and heterologous h n strains [ ] , suggesting that the combination of safe adjuvants with vaccines would be ideal for optimal efficacy of intranasal vaccines. oral administration is considered as another efficient route for delivering the mucosal vaccines. it is non-invasive, safe, and shows good patient compliance. for example, oral polio vaccine against highly contagious poliovirus helped to eradicate poliovirus infection in most parts of the world [ ] . moreover, studies have shown that oral vaccination can prevent infection of lungs and is capable of inducing mucosal immune responses (iga) in the respiratory tract, providing protection against influenza viruses. prabakaran et al. [ ] reported that live recombinant baculovirus displaying ha of h n virus (bac-ha) was able to express ha in epithelial cells of intestines of orally vaccinated mice and induced strong systemic and mucosal immune responses. mice immunized with live bac-ha of h n without any inclusion of adjuvants conferred % protection against homologous and heterologous h n strains, while immunization with inactivated bac-ha resulted in a lower survival rate of . %. the reduced immune responses of the inactivated bac-ha could be due to loss of transduction ability, whereas the live bac-ha was able to bind to the receptors that are expressed in the intestinal cells, resulting in gene delivery and stimulation of cell-mediated immune responses [ ] . moreover, the formulation of inactivated vaccine can have a huge impact on its immunogenicity. in one study, the immunogenicity lost by inactivated baculovirus displayed ha (bac-ha) vaccine was recovered by encapsulation using reverse micelle of phosphatidylcholine adjuvant. mice orally immunized with encapsulated inactive bac-ha induced systemic and mucosal immune response comparable to live bac-ha [ ] . however, there are limitations in the use of mucosal adjuvants in humans. aside from effectiveness of the adjuvant, the major concern in using mucosal adjuvants is its possible detrimental impact on humans. therefore, adjuvant used in mucosal immunization has to be proven safe for use. premanand et al. [ ] utilized naturally occurring lipid-based vesicles (bilosomes) as a mucosal adjuvant to coat the recombinant baculovirus (bac-vp ). an oral immunization study revealed that bac-vp , associated with bilosomes, induced higher vp -specific immune responses and conferred complete protection in a challenge study [ ] . to overcome the hurdles of selecting safe mucosal adjuvants, data analysis can be applied to search for suitable mucosal adjuvants and to understand their mechanism of action. in addition to new technologies, novel concepts are welcomed in the development of suitable mucosal adjuvants. finally, considerations on the delivery route and the appropriate combination of vaccine and adjuvant are essential in ensuring optimal effectiveness of vaccination in humans. baculovirus surface-displayed vaccines have been proven effective in inducing protective immune responses in animal models. however, further advancement in the context of immunogenicity will be made by utilizing hybrid promoters for the enhanced expression and anchoring of complex protein on recombinant baculoviral envelopes. in addition, inclusion of novel regulatory elements and molecular adjuvant property-carrying motifs in the vector will certainly boost the immunogenicity of antigen displayed on the baculovirus envelope. the main obstacle in implementing baculovirus surface-displayed vaccine as a human vaccine candidate is the lack of safety guidelines for human immunization. however, international organizations, like organization of economic co-operation and development in [ ] directorate-general in [ ] , concluded that no adverse effects of baculovirus on human health have been observed in safety tests, and they are not pathogenic, carcinogenic, or genotoxic in mammalian cells. however, environmental and ecological impacts of the baculovirus in invertebrates will be reduced by modification of baculovirus essential genes required for replication and budding, allowing the development of a baculovirus surface-displayed vaccine with an optimal safety profile in the future. funding: this research received no external funding. active and passive immunity, vaccine types, excipients and licensing wanted, dead or alive: new viral vaccines patients with diabetes mellitus undergoing cardiac surgery are at greater risk for developing intraoperative myocardial acidosis development of th and th populations and the nature of immune responses to hepatitis b virus dna vaccines can be modulated by codelivery of various cytokine genes heterologous protection against influenza by injection of dna encoding a viral protein dna vaccines: roles against diseases effective induction of high-titer antibodies by viral vector vaccines immune control of an siv challenge by a t-cell-based vaccine in rhesus monkeys the complete dna sequence of autographa californica nuclear polyhedrosis virus autographa californica nuclear polyhedrosis virus: comparative infectivity of the occluded, alkali-liberated, and nonoccluded forms nucleotide sequence of the p polypeptide gene of autographa californica nuclear polyhedrosis virus characterization of a trichoplusia ni hexamerin-derived promoter in the acmnpv baculovirus vector promoter influence on baculovirus-mediated gene expression in permissive and nonpermissive insect cell lines differential gene expression mediated by late, very late and hybrid baculovirus promoters high-level expression and improved folding of proteins by using the vp late promoter enhanced with homologous dna regions utility of temporally distinct baculovirus promoters for constitutive and baculovirus-inducible transgene expression in transformed insect cells baculovirus as a gene delivery vector: recent understandings of molecular alterations in transduced cells and latest applications in vivo application and tracking of baculovirus baculovirus as vaccine vectors baculovirus as an avian influenza vaccine vector: differential immune responses elicited by different vector forms memory t cells established by seasonal human influenza a infection cross-react with avian influenza a (h n ) in healthy individuals intranasal immunization with synthetic recombinant vaccine containing multiple epitopes of influenza virus a universal epitope-based influenza vaccine and its efficacy against h n characterization of the immune responses elicited by baculovirus-based vector vaccines against influenza virus hemagglutinin hemagglutinin displayed baculovirus protects against highly pathogenic influenza a pseudotype baculovirus-mediated vaccine confers protective immunity against lethal challenge with h n avian influenza virus in mice and chickens wssv ie promoter is more efficient than cmv promoter to express h hemagglutinin from influenza virus in baculovirus as a chicken vaccine cross-protective efficacy of bivalent recombinant baculoviral vaccine against heterologous influenza h n challenge protective efficacy of a human endogenous retrovirus envelope-coated, nonreplicable, baculovirus-based hemagglutin vaccine against pandemic influenza h n baculovirus displaying hemagglutinin elicits broad cross-protection against influenza in mice intranasal immunization of baculovirus displayed hemagglutinin confers complete protection against mouse adapted highly pathogenic h n reassortant influenza virus cross-protective efficacy of baculovirus displayed hemagglutinin against highly pathogenic influenza h subtypes immunization with baculovirus displayed h hemagglutinin vaccine protects mice against lethal h influenza virus challenge a baculovirus dual expression system-based vaccine confers complete protection against lethal challenge with h n avian influenza virus in mice oral vaccination of baculovirus-expressed vp displays enhanced protection against white spot syndrome virus in penaeus monodon baculovirus as a prrsv and pcv bivalent vaccine vector: baculovirus virions displaying simultaneously gp glycoprotein of prrsv and capsid protein of pcv a novel baculovirus vector shows efficient gene delivery of modified porcine reproductive and respiratory syndrome virus antigens and elicits specific immune response induction of robust immunity response in mice by dual-expression-system-based recombinant baculovirus expressing the capsid protein of porcine circovirus type baculovirus surface display of e envelope glycoprotein of classical swine fever virus and immunogenicity of the displayed proteins in a mouse model baculovirus surface display of e rns envelope glycoprotein of classical swine fever virus baculovirus vectors expressing f proteins in combination with virus-induced signaling adaptor (visa) molecules confer protection against respiratory syncytial virus infection rabies-virus-glycoprotein-pseudotyped recombinant baculovirus vaccine confers complete protection against lethal rabies virus challenge in a mouse model baculovirus surface display of sars coronavirus (sars-cov) spike protein and immunogenicity of the displayed protein in mice models bacmam virus-based surface display of the infectious bronchitis virus (ibv) s glycoprotein confers strong protection against virulent ibv challenge in chickens baculovirus surface display of e envelope glycoprotein of japanese encephalitis virus and its immunogenicity of the displayed proteins in mouse and swine models a chimeric baculovirus displaying bovine herpesvirus- (bhv- ) glycoprotein d on its surface and their immunological properties display of vp on the surface of baculovirus and its immunogenicity against heterologous human enterovirus strains in mice display of enterovirus vp on baculovirus as a type ii transmembrane protein elicits protective b and t cell responses in immunized mice baculovirus virions displaying infectious bursal disease virus vp protein protect chickens against infectious bursal disease virus infection localization of the vp protein of canine parvovirus type on the baculovirus envelop and its immunogenicity in a mouse model baculovirus surface display of sigmac and sigmab proteins of avian reovirus and immunogenicity of the displayed proteins in a mouse model daf-shielded baculovirus-vectored vaccine enhances protection against malaria sporozoite challenge in mice protective efficacy of baculovirus dual expression system vaccine expressing plasmodium falciparum circumsporozoite protein baculovirus-vectored multistage plasmodium vivax vaccine induces both protective and transmission-blocking immunities against transgenic rodent malaria parasites functional immunogenicity of baculovirus expressing pfs , a human malaria transmission-blocking vaccine candidate antigen intranasal and intramuscular immunization with baculovirus dual expression system-based pvs vaccine substantially blocks plasmodium vivax transmission baculovirus-based nasal drop vaccine confers complete protection against malaria by natural boosting of vaccine-induced antibodies in mice a baculovirus dual expression system-based malaria vaccine induces strong protection against plasmodium berghei sporozoite challenge in mice baculovirus virions displaying plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection baculovirus-based vaccination vectors allow for efficient induction of immune responses against plasmodium falciparum circumsporozoite protein recombinant baculovirus associated with bilosomes as an oral vaccine candidate against hev infection in mice influenza (seasonal) . available online: www influenza vaccines: challenges and solutions the genesis of a pandemic influenza virus characterization of the influenza virus polymerase genes on the origin of the human influenza virus subtypes h n and h n avian-to-human transmission of the pb gene of influenza a viruses in the and pandemics n-glycosylation of recombinant human interferon-γ produced in different animal expression systems jupin, i. evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus rna-dependent rna polymerase domain expressed in a baculovirus-insect cell system the disulfide bond structure of plasmodium apical membrane antigen- insect baculoviruses strongly potentiate adaptive immune responses by inducing type i ifn baculovirus display strategies: emerging tools for eukaryotic libraries and gene delivery expression of foreign proteins on the surface of autographa californica nuclear polyhedrosis virus ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses truncated vesicular stomatitis virus g protein improves baculovirus transduction efficiency in vitro and in vivo avian influenza virus hemagglutinin display on baculovirus envelope: cytoplasmic domain affects virus properties and vaccine potential eukaryotic virus display: engineeringthe major surface glycoprotein of the autographa californica nuclear polyhedrosis virus (acnpv) for the presentation of foreign proteins on the virus surface cell-mediated protection in influenza infection evolutionarily conserved protein sequences of influenza a viruses, avian and human, as vaccine targets cellular immune correlates of protection against symptomatic pandemic influenza preexisting influenza-specific cd + t cells correlate with disease protection against influenza challenge in humans recombinant baculovirus vaccine containing multiple m e and adjuvant ltb induces t cell dependent, cross-clade protection against h n influenza virus in mice baculovirus vector as an avian influenza vaccine: hemagglutinin expression and presentation augment the vaccine immunogenicity construction of recombinant baculoviruses expressing hemagglutinin of h n avian influenza and research on the immunogenicity transport of proteins across the endoplasmic reticulum membrane functional analysis of the transmembrane (tm) domain of the autographa californica multicapsid nucleopolyhedrovirus gp protein: substitution of heterologous tm domains the effects of foreign transmembrane domains on the biosynthesis of the influenza virus hemagglutinin requirement for a non-specific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus spike protein-nucleocapsid interactions drive the budding of alphaviruses protective immunity against influenza h n virus challenge in mice by intranasal co-administration of baculovirus surface-displayed ha and recombinant ctb as an adjuvant gastrointestinal delivery of baculovirus displaying influenza virus hemagglutinin protects mice against heterologous h n infection in vivo gene transfer in mouse skeletal muscle mediated by baculovirus vectors construction and immunogenicity of pseudotype baculovirus expressing toxoplasma gondii sag protein in balb/c mice model a consensushemagglutinin-based dna vaccine that protects mice against divergent h n influenza viruses a clinical trial of a whole-virus h n vaccine derived from cell culture neutralizing epitopes of influenza virus hemagglutinin: target for the development of a universal vaccine against h n lineages protective immunity elicited by a pseudotyped baculovirus-mediated bivalent h n influenza vaccine a stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus g pseudotypes vsv-g pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum baculovirus as versatile vectors for protein expression in insect and mammalian cells subcutaneous immunization with baculovirus surface-displayed hemagglutinin of pandemic h n influenza a virus induces protective immunity in mice design of an ha -based escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge intranasal adenovirus-vectored vaccine for induction of long-lasting humoral immunity-mediated broad protection against influenza in mice chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies hemagglutinin stalk antibodies elicited by the pandemic influenza virus as a mechanism for the extinction of seasonal h n viruses induction of broadly neutralizing h n influenza antibodies by vaccination influenza virus vaccine based on the conserved hemagglutinin stalk domain influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes h n influenza virus infection induces broadly reactive hemagglutinin stalk antibodies in humans and mice avian influenza a virus (h n ) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome role of hemagglutinin cleavage for the pathogenicity of influenza virus european centre for disease prevention and control. influenza a(h n ) virus in china-implications for public health- th update the molecular basis of the pathogenicity of the dutch highly pathogenic human influenza a h n viruses vaccination with a synthetic peptide from the influenza virus hemagglutinin provides protection against distinct viral subtypes amino acid substitutions improve the immunogenicity of h n ha protein and protect mice against lethal h n viral challenge quasi-immune response of penaeus japonicus to penaeid rod-shaped dna virus (prdv) a time-course study on the resistance of penaeus japonicus induced by artificial infection with white spot syndrome virus vaccination trials with penaeus japonicus to induce resistance to white spot syndrome virus effect of white spot syndrome virus envelope protein vp expressed in silkworm (bombyx mori) pupae on disease resistence in procambarus clarkii sahul hameed, a.s. oral delivery of dna construct using chitosan nanoparticles to protect the shrimp from white spot syndrome virus (wssv) oral delivery of dna vaccine encoding vp against white spot syndrome virus in crayfish by attenuated salmonella typhimurium localization of vp on the baculovirus envelope and its immunogenicity against white spot syndrome virus in penaeus monodon innate immunity in higher insects construction and characterization of a recombinant canine adenovirus expressing gp and m proteins of porcine reproductive and respiratory syndrome virus expression of open reading frame protein of porcine reproductive and respiratory syndrome virus using semliki forest virus expression system protective immunity induced by a recombinant pseudorabies virus expressing the gp of porcine reproductive and respiratory syndrome virus in piglets co-expressing gp and m proteins under different promoters in recombinant modified vaccinia virus ankara (rmva)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (prrsv) respiratory syncytial virus infection in elderly and high-risk adults an epidemiologic study of altered clinical reactivity to respiratory syncytial (rs) virus infection in children previously vaccinated with an inactivated rs virus vaccine respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine understanding respiratory syncytial virus (rsv) vaccine-enhanced disease immunogenic and antigenic properties of recombinant soluble glycoprotein of rabies virus the immune response to rabies virus infection and vaccination the type i interferon response bridles rabies virus infection and reduces pathogenicity collaboration of antibody and inflammation in clearance of rabies virus from the central nervous system rabies virus expressing dendritic cell-activating molecules enhances the innate and adaptive immune response to vaccination induction of protective immune responses against ev in mice by baculovirus encoding a novel expression cassette for capsid protein vp research on infectious bursal disease-the past, the present and the future a recombinant newcastle disease virus (ndv) expressing vp protein of infectious bursal disease virus (ibdv) protects against ndv and ibdv avian adenovirus celo recombinants expressing vp of infectious bursal disease virus induce protection against bursal disease in chickens first results of phase trial of rts,s/as malaria vaccine in african children seven-year efficacy of rts,s/as malaria vaccine among young african children attenuated pfspz vaccine induces strain-transcending t cells and durable protection against heterologous controlled human malaria infection safety and efficacy of pfspz vaccine against plasmodium falciparum via direct venous inoculation in healthy malaria-exposed adults in mali: a randomised, double-blind phase trial protection against plasmodium falciparum malaria by pfspz vaccine inside the mucosal immune system involvement of the toll-like receptor signaling pathway in the induction of innate immunity by baculovirus baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice single intranasal mucosal mycobacterium bovis bcg vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis comparative evaluation of intranasal and subcutaneous route of immunization for development of mucosal vaccine against experimental tuberculosis mucosal adjuvants: opportunities and challenges oral vaccines: directed safe passage to the front line of defense reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against h n infection in mice consensus document on information used in the assessment of environmental applications involving baculovirus; series on harmonization of regulatory oversight in biotechnology number european commission's health & consumer protection directorate-general we are grateful for the financial support received from temasek life sciences laboratory, singapore. the authors declare no conflict of interest. key: cord- -smlq y authors: dhakal, santosh; renukaradhya, gourapura j. title: nanoparticle-based vaccine development and evaluation against viral infections in pigs date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: smlq y virus infections possess persistent health challenges in swine industry leading to severe economic losses worldwide. the economic burden caused by virus infections such as porcine reproductive and respiratory syndrome virus, swine influenza virus, porcine epidemic diarrhea virus, porcine circovirus , foot and mouth disease virus and many others are associated with severe morbidity, mortality, loss of production, trade restrictions and investments in control and prevention practices. pigs can also have a role in zoonotic transmission of some viral infections to humans. inactivated and modified-live virus vaccines are available against porcine viral infections with variable efficacy under field conditions. thus, improvements over existing vaccines are necessary to: ( ) increase the breadth of protection against evolving viral strains and subtypes; ( ) control of emerging and re-emerging viruses; ( ) eradicate viruses localized in different geographic areas; and ( ) differentiate infected from vaccinated animals to improve disease control programs. nanoparticles (nps) generated from virus-like particles, biodegradable and biocompatible polymers and liposomes offer many advantages as vaccine delivery platform due to their unique physicochemical properties. nps help in efficient antigen internalization and processing by antigen presenting cells and activate them to elicit innate and adaptive immunity. some of the nps-based vaccines could be delivered through both parenteral and mucosal routes to trigger efficient mucosal and systemic immune responses and could be used to target specific immune cells such as mucosal microfold (m) cells and dendritic cells (dcs). in conclusion, nps-based vaccines can serve as novel candidate vaccines against several porcine viral infections with the potential to enhance the broader protective efficacy under field conditions. this review highlights the recent developments in nps-based vaccines against porcine viral pathogens and how the nps-based vaccine delivery system induces innate and adaptive immune responses resulting in varied level of protective efficacy. viruses are the obligate intracellular nano-sized particles, which depend on host cell machinery for propagation and survival. they carry deoxyribonucleic acid (dna) or ribonucleic acid (rna) as their genomic material. there are several viruses from both dna and rna virus families that infect and produce disease in pigs [ ] . there are many economically important swine viral infections which cause considerable morbidity and mortality, and responsible for significant economic losses to the pork industry (table ). depending on their cellular and tissue tropisms, viruses cause pathological changes and clinical signs associated with respiratory system, reproductive and gastrointestinal tracts, nervous system, skin and extremities, alone or in combinations [ , ] . porcine reproductive and respiratory syndrome virus (prrsv), an enveloped and positive-stranded rna virus of arteriviridae family, causes porcine reproductive and respiratory syndrome (prrs) [ ] . prrs is responsible for over one billion dollar loss per year through direct and indirect costs in the us swine industry [ ] . two entirely distinct genotypes of prrsv circulate in european (genotype /prrsv ) and north american countries (genotype /prrsv ) and cause tremendous economic loss. prrsv is transmitted through oral-nasal secretions and semen. the clinical signs include fever, anorexia, mild to severe respiratory problems, abortion and reproductive failures. it is the most common pathogen associated with porcine respiratory disease complex (prdc) [ ] . swine influenza (flu) constitutes another persistent health challenge to the global pig industry. flu infection is caused by influenza a virus of orthomyxoviridae family which has negative-sense, single-stranded, segmented rna genome. influenza virus is transmitted through direct contact with infected animals or contaminated fomites, aerosols and large droplets [ ] . the clinical signs of influenza infection include fever, anorexia, loss of weight gain and respiratory problems. influenza associated economic losses are due to morbidity, loss of body weight gain, increased time to market, secondary infections, medication and veterinary expenses [ ] . influenza of swine origin occasionally infect humans and can even lead to pandemics as of [ ] . porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) are enteric pathogens of young pigs [ ] . these viruses belong to coronaviridae family and have positive-sense, single-stranded rna genome. tgev did serious economic damage to the swine industry in s but with the advent of vaccines it has been largely controlled [ ] . pedv still results in high morbidity and mortality in neonatal piglets with clinical signs like severe diarrhea, vomiting, dehydration and death. in / , pedv outbreak in the us led to over a billion-dollar loss [ ] . rotaviruses are double-stranded rna viruses of reoviridae family, cause enteric infections in pigs. rotavirus of groups a, b, c, e and h are involved in porcine enteric infections. some of these porcine rotaviruses also have zoonotic potential [ ] . foot and mouth disease (fmd) is another highly contagious, acute viral disease in pigs. the etiologic agent, fmd virus (fmdv), is a positive-sense, single-stranded rna virus of picornaviridae family [ ] . fmdv is transmitted through direct contact with infected animals or contaminated sources. clinical signs include high fever, appearance of vesicular lesions on the extremities, salivation, lameness and death. fmdv causes frequent epizootics in many parts of the world resulting in severe economic loss, food insecurity and trade restrictions [ ] . classical swine fever (csf) or hog cholera can result in high morbidity and mortality in pigs. it is caused by csf virus (csfv), an enveloped, positive-sense, singlestranded virus of flaviviridae family. transmission of csfv occurs through oral-nasal routes after contact with infected pigs or contaminated resources and even vertically from infected sows to piglets [ ] . clinical signs include fever, anorexia, respiratory problems, neurological disorders, reproductive failures and death. csf is a notifiable disease to world organization for animal health (oie). the economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs [ ] . for example, the / outbreak of csfv in the netherland resulted in death of million pigs and economic losses of . billion dollars [ ] . united states is free of csfv; however, this virus is endemic in many parts of the world including central and south america, africa and asia. porcine circovirus (pcv ), a single-stranded dna virus of circoviridae family, causes multi-systemic disease referred as porcine circovirus-associated disease (pcvad). pcv is transmitted horizontally as well as vertically. direct contact is the most efficient way of horizontal transmission of this virus. the clinical signs of pcv infection include poor weight gain, respiratory problems, dermatitis, enteritis, nephropathy and reproductive failures [ ] . five genotypes of pcv (pcv a to pcv e) are identified and circulate with high prevalence in swine herds causing significant economic losses worldwide [ ] . porcine parvovirus (ppv) is the common cause of reproductive failure in swine herds. this single-stranded dna virus of parvoviridae family is transmitted through oral-nasal routes. stillbirths, mummification, embryonic death, and infertility (smedi syndrome) are linked to ppv infection. conventionally, ppv was considered genetically conserved but recent evidences suggest that several virulent strains have emerged due to its high mutation rate [ ] . aujeszky's disease or pseudorabies in pigs is caused by suid herpesvirus , a double stranded dna virus belonging to herpesviridae family. the causative agent is spread primarily through direct animal-to-animal (nose-to-nose or sexual) contact. pseudorabies is characterized by nervous disorders, respiratory problems, weight loss, deaths in younger piglets and reproductive failures; and is one of the most devastating infectious diseases in pig industry [ , ] . african swine fever (asf) causes hemorrhagic infection with high morbidity and mortality. the etiologic agent, asf virus (asfv), is a double stranded dna virus of asfarviridae family [ ] . virus transmission occurs through direct contact with infected animals, indirect contacts with fomites or through soft tick species of the genus ornithodoros. clinical disease may range from asymptomatic infection to death with no signs. acute infections are characterized by high fever, anorexia, erythema, respiratory distress, reproductive failure in pregnant females and death [ ] . asf is oie notifiable disease. united states is free of asfv, however, this virus is endemic in domestic and wild pig population in many parts of the world with possibility of transmission to the us and other nonendemic regions through animal trades [ ] . the economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs [ ] . besides the rna and dna viruses described above, many other emerging and re-emerging viruses such as porcine hepatitis e virus, porcine endogenous retrovirus, porcine sapovirus, japanese encephalitis virus, encephalomyocarditis virus and others cause variable degree of impact in swine health and economic losses in pig industry globally [ , , ]. different types of vaccines that are available against economically important swine viruses are listed in table . vaccines against prrsv are being used in the us since s [ ] . both inactivated and modified-live virus vaccines are available and used globally. these vaccines are effective in reducing clinical disease and viremia mainly against homologous but not against heterologous infections [ ] . therefore, different strategies are ongoing to develop live, inactivated, subunit and mucosal prrsv vaccines to induce better immunity and broader protection [ , [ ] [ ] [ ] . swine influenza vaccines are also most effective when the vaccine strains closely match with the circulating strains [ , ] . to increase the immunity and protection, vaccines containing multiple strains of influenza a virus (iav) and autogenous vaccines are widely used [ , ] . cocirculation of multiple lineages of iav and frequent antigenic drift are responsible for reduced field efficacy of current swine influenza vaccines. moreover, the most commonly used whole inactivated iav vaccines administered via intramuscular route do not induce adequate mucosal antibody and cellular immune responses, suffer maternal antibody interference in young piglets and even can cause enhanced respiratory disease [ , ] . the emergence of highly virulent strains of pedv in recent years has highlighted the need of safe and effective vaccines against porcine enteric coronaviruses that prevents clinical disease, mortality and virus shedding in neonates [ ] . modified live vaccines against rotavirus are available for use in pigs against rotavirus a but their efficacy under field conditions is questionable indicating the need of alternatives for porcine rotavirus management [ ] . the available inactivated vaccines provided great help in prevention and control of fmd outbreaks in many countries. however, the development of these vaccines needs high level biocontainment facilities. further, the fmdv serotypes undergo continuous antigenic drift and escape the vaccine-induced immunity [ ] . thus, fmd vaccines with less stringent regulatory procedures and multi-serotype protective efficacy are needed in the future. safe and highly efficacious live-attenuated vaccines are available against csfv but differentiation of infected from vaccinated animals (diva) is not possible with these vaccines, which limit their use during outbreak control or disease eradication programs [ ] . inactivated whole virus or subunit vaccines based on pcv a are highly adopted in pig farms and are efficacious in reducing clinical signs and improving the production parameters. however, infections are still widespread in vaccinated farms [ , ] . further, the replacement of pcv a to pcv b and recently to pcv d is in part contributed by the selection pressure exhibited by pcv a-based vaccines [ ] which highlights the need of vaccines that protect against multiple genotypes. the currently used inactivated vaccines of porcine parvo virus protect against old ppv strains but not against the newly emerging strains demanding for more efficacious vaccines [ , ] . fortunately, pseudorabies has been eradicated in many countries including the us by using inactivated and attenuated vaccines together with stringent biosecurity measures. however, it is still a problematic disease in many countries including china and is also maintained in feral swine populations in other countries [ , ] . the frequent emergence of virulent strains even in the vaccinated herds demands updated vaccine technology to achieve efficient control and ultimate global eradication of pseudorabies [ , ] . vaccine is not available so far against asfv, and the control measures depend entirely on early identification and culling of infected herds and adoption of strict sanitary measures [ ] . vaccine development is hindered by the antigenic diversity and multitude of immune-evasion strategies used by the virus. an effective vaccine will definitely help in control and eradication of asfv from endemic countries and prevent its geographical expansion [ ] . [ , ] porcine epidemic diarrhea (ped) rna particle, inactivated and live-attenuated virus (in asia) protective immune response in sows better mucosal immunity [ , ] foot and mouth disease (fmd) inactivated virus less stringent requirements in vaccine production protection against multiple serotypes [ ] classical swine fever (csf) live-attenuated virus diva potential [ ] porcine circovirus associated disease (pcvad) inactivated, recombinant subunit multi-genotype protection [ , ] porcine parvovirus infection inactivated virus protection against novel strains [ , ] pseudorabies inactivated, live-attenuated virus protection against novel emerging strains [ , ] african swine fever (asf) none novel cross-protective vaccine [ ] importance of nanoparticle-based vaccine delivery platforms development of vaccines has made significant impact on reducing the viral infectious disease burden in both humans and animals. however, there are still many diseases for which either we do not have vaccines or need substantial improvements over existing ones [ , ] . in the past few decades, nanoparticles (nps)-based technologies have elicited significant interests in the development of novel vaccine candidates as they offer multiple benefits over inactivated virus or subunit soluble antigens. nps-based vaccines (nanovaccines) are prepared either by encapsulating vaccine components within the nps or by decorating the particle surface with viral antigens. nps protect antigens from proteolytic degradation, prolong their bioavailability and maintain slow and sustained antigen release. all of these properties help in induction of better immune responses compared to soluble antigen vaccines [ ] . the different mechanisms used by various nps to facilitate immune modulation of antigen presenting cells (apcs) are depicted graphically in figure . briefly, nps can enhance antigen adsorption and uptake by apcs; they can also facilitate antigen processing mechanisms; nps can induce maturation of dcs and promote antigen cross-presentation through major histocompatibility complex (mhc) class i to cd + t cells; and induce production of different innate cytokines that regulate humoral and cellular immune responses. nps-loaded antigens are readily phagocytosed by apcs; soluble antigens are not [ ] . moreover, dendritic cells (dcs), the key player involved in bridging innate and adaptive immunity, preferentially internalize nps compared to microparticles (> nm). for example, when poly(lactic-co-glycolic acid) (plga) particles of size nm to µm encapsulating ovalbumin were tested on mouse bone-marrow derived dendritic cells, nm sized particles were taken up efficiently compared to larger ones [ ] . the nm sized plga nps resulted in greater activation of dcs and stronger antigen-specific t cells responses in immunized mice compared to soluble antigens and larger particles [ ] . besides controlled delivery of antigens, nps also provide adjuvant-like functions. vaccine adjuvants either work as antigen delivery systems facilitating antigen uptake and presentation by apcs or they activate innate immune receptors for cytokine production and maturation/migration of dcs [ ] . adjuvant-induced innate immune responses determine the type of adaptive immune responses generated such as t helper (th ) versus t helper (th )-biased immunity [ ] . alum, the most widely used adjuvant in humans, is safe and inexpensive. its compatibility has been proved favorable with different vaccine antigens. however, despite inducing potent antibody responses, alum is a weak-inducer of cell-mediated immunity. adverse reactions are observed at injection site with alum-based adjuvants [ , ] . in veterinary vaccines, oil-in-water emulsions or saponins are the most common adjuvants. these can also cause adverse reactions at the injection sites [ , ] . while number of adjuvants are available for parenteral vaccinations, very limited options are available for intranasal (in) or other alternative routes of immunization [ , , ] . nps can serve as an alternative adjuvant for human and animal use as they act both as antigen delivery system and activate the innate immune responses [ ] [ ] [ ] . further, the modern vaccination approach has shifted from traditional whole pathogen-based antigens to small fraction (subunit) of the pathogen. however, purified whole inactivated pathogen and subunit or recombinant antigens by themselves are poorly immunogenic and require a potent immunostimulatory platform to augment the immune response. this can be achieved through nps-based technologies [ , ] . nps-based platforms can be used to deliver multiple antigens or antigen/adjuvant combinations, which improves antigen uptake and concurrent activation of apcs leading to innate immune programming [ , ] . co-delivery of cpg oligodeoxynucleotide and tetanus toxoid in nanospheres induced significantly greater t cell proliferative response and to times greater igg antibody isotypes in mice after subcutaneous immunization compared with the group that received tetanus toxoid and cpg oligodeoxynucleotide in soluble form [ ] . likewise, co-delivery of melanoma antigen and toll-like receptor (tlr) agonist in plga nps induced therapeutic anti-tumor effects that are mediated through potent cd + t cell activation [ ] . nps can be surface modified to target microfold (m) cells, macrophages or dcs, and could be used for mucosal vaccination through oral, nasal or other mucosal routes of immunization. in mice, surface coating of plga nps encapsulating hepatitis b virus vaccine antigens with lectin resulted in efficient targeting of oral delivered nps to mucosal m cells and induced secretary iga antibody response in mucosal surfaces [ ] . likewise, dcs targeted chitosan nps loading plasmid dna encoding nucleocapsid protein of severe acute respiratory syndrome coronavirus (sars-cov) induced better nucleocapsid protein-specific mucosal iga antibody response compared to soluble unentrapped antigens after nasal immunization in mice [ ] . a targeted t-cell mediated immune response is critical in protection against intracellular pathogens such as viruses. beneficially, nps-delivered antigens are useful in antigen cross-presentation to cytotoxic t lymphocytes (ctls) and development of robust cell-mediated immune response [ , ] . plga-based particulate vaccines are shown to induce efficient t-cell immunity in mice and pigs [ ] [ ] [ ] [ ] . similarly, rodent and pig studies have shown that polyanhydride nps-based vaccines also enhance cellular immunity [ , ] . thus, immunogenic properties of different polymer-based nps could be exploited to improve the efficacy of vaccines for use against porcine viral infections. in this review, only studies conducted in pigs related to the development and evaluation of nps-based vaccine candidates by using virus-like particles (vlps), biodegradable polymers, polysaccharides and liposomes against porcine viral infections are included (table ) . vlps are constructed using viral structural proteins, which can self-assemble but are non-infectious as they lack the viral genomic material. vlps mimic the virion and can effectively induce innate and adaptive immune responses [ ] . vlps are produced using different bacterial, insect, yeast or mammalian expression systems [ ] . due to their smaller size and particulate nature, vlps-based vaccines are processed and presented not only through mhc class ii but also through mhc class i pathway leading to the generation of antibodies as well as ctl responses [ , ] . the potential use of vlps in porcine viral vaccine development is evident through the success in commercialization of human papilloma virus (hpv), hepatitis b virus and malaria vaccines by adapting this technology [ ] . in one study, prrsv vlps containing five (gp , gp , gp , gp a and m) and two (gp and m) viral surface proteins were generated using the baculovirus expression system. prrsv vlps vaccine was mixed at : ratio with mycobacterium tuberculosis whole cell lysate (m. tuberculosis wcl) adjuvant and administered into pigs. vlps-vaccinated pigs were partially protected with -log reduction of virus titers in lungs. vlps-vaccinated pigs also had enhanced ifn-γ response compared to mock challenge pigs [ ] . however, in another study, when pigs were vaccinated in with prrsv vlps expressing n, m, gp and e proteins, enhanced viremia accompanied with higher level of ifn-α cytokine response was observed [ ] . the contrasting results in prrsv vlps study suggest the need for further research to fully evaluate the potential of vlps-based prrsv vaccines for swine. influenza-associated vlps expressing ha, na and m proteins of pandemic (h n ) virus were inoculated twice intramuscularly with or without emulsigen (mvp lab, usa) adjuvant to pigs. this vaccine induced robust serum igg, mucosal iga and virus neutralizing antibody responses in pigs. after homologous virus challenge, vlps-vaccinated pigs had significantly reduced pneumonic lesions and virus titers were substantially lowered in upper and lower respiratory tracts compared to mock vaccinated animals [ ] . many studies have been conducted with the goal to develop vlps-based fmdv vaccine using various expression systems encoding different viral antigens. rabbit hemorrhagic disease virus (rhdv) vlps expressing t-cell epitope of a protein of fmdv (rhdv- a-vlps) was generated. this vlps vaccine induced maturation of bone marrow derived dendritic cells in vitro [ ] . pigs immunized im with rhdv- a-vlps together with montanide isa adjuvant (seppic, france) induced higher serum igg and iga antibody responses. this vaccine also increased number of ifn-γ secreting cells and lymphoproliferative responses in pbmcs compared to vaccine delivered without adjuvant and in rhdv- a-vlps inoculated pigs; however, challenge experiments were not performed [ ] . guo et al. constructed fmdv vlps expressing capsid proteins vp , vp and vp and immunized pigs by im route [ ] . vlps-vaccinated pigs produced virus-specific neutralizing antibodies and ifn-γ response in peripheral blood mononuclear cells (pbmcs) as good as the inactivated fmdv vaccine control. after challenge with homologous virus, vlps-vaccinated pigs did not show specific clinical signs [ ] . in another study, vp epitope peptides (ep - ) of fmdv were inserted into the coat protein genes of male-specific coliphage (ms ) (cp-ep - vlps) and injected im to pigs. this formulation resulted in induction of virus neutralizing antibodies and protected % of the immunized pigs compared to only % protection in peptide alone vaccinated animals. however, the protection was lower than inactivated vaccine ( %) indicating the need : of further improvement in this vlps either by using longer sequence of epitope or addition of other adjuvants [ ] . vlps generated by insertion of vp epitopes of fmdv into porcine parvovirus vp were administered im to pigs. this vlps-vaccine induced higher virus neutralizing antibodies compared to synthetic peptide vaccine and resulted in better protection to challenge fmdv infection [ ] . vlps have also been developed and tested against porcine neurotropic viruses [ , ] . porcine encephalomyocarditis virus (emcv) vlps containing structural protein p , nonstructural protein a and protease c were generated. after im administration together with montanide ims n vg adjuvant (seppic), vlps-vaccine induced sustained production of virus neutralizing antibodies comparable to commercial vaccine control. there was absence of any severe injection site reactions in vlps-vaccinated pigs [ ] . this suggests the potential of developing vlps-based vaccine against emcv disease in pigs. likewise, in a recent study, japanese encephalitis virus genotype i (gi) vlps encoding premembrane (prm) and envelope (e) proteins were constructed. after subcutaneous immunization, this vaccine formulation induced robust neutralizing antibody response and protection against both homologous gi and heterologous giii jevs viruses. this finding indicates the cross-protection potential of vlps-based jev vaccine in pigs [ ] . early study on pcv vlps used full length cap protein in escherichia coli expression system [ ] . pigs vaccinated against pcv using cap vlps and isa adjuvant (seppic) by im route induced cap-specific igg antibodies. vaccinated animals were apparently healthy with normal body weight gain and absence of any clinical signs of disease [ ] . li et al. [ ] showed induction of cap-specific igg antibodies in pigs vaccinated by subcutaneous (sc) injection of cap vlps. vaccinated pigs demonstrated reduced fever, viremia and mild pathological changes in lungs and lymph nodes compared to unvaccinated challenge animals. in another study, vlps co-expressed with cap protein and porcine gm-csf were administered im to pigs. this vaccine formulation induced significantly higher virus neutralizing antibodies in pigs. after virus challenge, vlps-vaccinated pigs had normal body weight gain compared to cap protein alone and commercial pcv vaccine groups. virus clearance, however, was observed in equally in vlps as well as other control vaccine groups [ ] . only a single vlps-based vaccine study for porcine parvovirus was found [ ] . ppv-vlps expressing major structural protein vp were administered im with double oil emulsion (doe) mineral oil adjuvant to weaned pigs. there was an induction of significantly higher neutralizing antibodies in vlps-vaccinated animals compared to inactivated vaccine group. further, when gilts immunized with this formulation were challenged with virulent ppv, virus was not detected in any of the fetuses. thus, ppv-vlps can be a potential vaccine candidate to prevent ppv-induced reproductive failure [ ] . in summary, vlps of various origin can be used to develop more efficient vaccines against porcine viral infections. further studies are needed to evaluate their immunogenicity and protective efficacy under field conditions. plga is a co-polymer of lactic acid and glycolic acid. it is the most widely explored synthetic polymer in vaccine studies. it is a safe and non-toxic compound, and its hydrolysis products are readily assimilated into existing metabolic pathways [ ] . plga nanoparticles are prepared either by oil in water emulsification or nanoprecipitation methods [ , ] . plga nps bear a net negative charge. they enter apcs through pinocytosis and endocytosis, undergo reversal of charge and endolysosomal escape of entrapped vaccine cargo leading to antigen processing in cytoplasm, resulting in cross-presentation of antigen to cd + t cells through mhc class i pathway [ , ] . plga nps are involved in maturation of dcs of mice and human origin, and controlled release of entrapped antigens leading to efficient expansion and differentiation of memory t-cells [ , ] . in rodent studies, induction of robust t-cell immunity is observed with plga nps-based vaccines containing various vaccine antigens [ , ] . further, plga is approved for drug deliveries in humans by the us food and drug administration (fda) and european medicine agency (ema) [ ] . plga nps enhance antigen uptake and induce maturation of porcine apcs [ , , ] . single dose of in immunization with plga nps-encapsulated inactivated/ killed prrsv antigen (nps-kag) induced activation of innate natural killer (nk) cells, γδ t-cells and secretion of innate cytokine ifnα [ ] . nps-kag vaccine also induced greater frequency of cd + t cells; increased secretion of ifn-γ; lowered frequency of t-regulatory cells; and reduced secretion of inflammatory cytokines compared to control kag-vaccinated animals [ , ] . in a subsequent study, when nps-kag was co-administered in with m. tuberculosis wcl adjuvant, a balanced th / th immune response and augmentation of mucosal iga antibody response was observed. after heterologous prrsv challenge, pigs that received nps-based vaccine showed no clinical signs and also had significant reduction in lung virus load [ , ] . plga nps were also used to encapsulate highly conserved influenza peptides and evaluated for efficacy in pigs after in administration. plga nps-based subunit vaccine resulted in induction of epitope-specific t-cell response but not the antibody response [ ] . the t-cell biased immune response was also observed in pigs after in immunization with plga nps-encapsulated inactivated/killed influenza virus (plga-kag) vaccine in pigs [ ] . in plga-kag vaccine administered animals observed reduced fever; lowered pneumonic lesions; and increased virus clearance from lungs after heterologous virus challenge compared to kag vaccine controls [ ] . in another study, pedv kag was encapsulated in plga nps and used to immunize pregnant sows by in route. this nanovaccine induced higher virus-specific igg and neutralizing antibodies in serum and greater igg, iga and neutralizing antibody responses in colostrum. it also induced greater cell proliferation and ifn-γ responses in restimulated pbmcs compared to kag vaccine controls. importantly, piglets born to nps-vaccinated sows had higher virus neutralizing antibodies and were better protected against homologous virus challenge than kag controls [ ] . these studies suggest that plga nps can be used as an efficient means of enhancing virus-specific cell-mediated immune responses in pigs. polyanhydrides are another type of synthetic polymer widely studied for vaccine deliveries [ ] . polyanhydride nps are synthesized by polycondensation or emulsification processes and are biodegradable, biocompatible and safe for vaccine delivery [ , ] . they activate innate immune responses in a manner similar to lipopolysaccharides (lps) [ ] . the surface-eroding nature of polyanhydride nps provides safe microenvironment for the encapsulated antigens and facilitates slow and sustained antigen release [ , ] . induction of better antibody and cell-mediated immune responses by polyanhydride npsbased vaccines has been reported against viral, bacterial and parasitic infections [ , ] . inoculation of polyanhydride nps-based siv kag vaccine (kag-nanovaccine) by in route enhanced cell-mediated but not the antibody responses in pigs [ ] . after heterologous virus challenge, kag-nanovaccine group had six to eightfold reduction of nasal virus shedding compared to kag vaccine controls [ ] . in a subsequent study, when kag-nanovaccine formulation was supplemented with cpg-odn adjuvant, both cell-mediated as well as mucosal iga antibody responses were improved [ ] . after heterologous virus challenge, cpg-odn-adjuvanted kag-nanovaccine provided better protection through a significant reduction in influenza-induced fever, -fold reduction of nasal virus shedding and -fold reduction in lung virus titers compared to pigs immunized with five-times greater quantity of soluble killed antigen (kag) vaccine [ ] . this study also indicates the dose-sparing ability of polyanhydride nps. thus, polyanhydride nps can also be used to induce better cellular as well as humoral immune responses in pigs. chitosan, alginate and other polysaccharides have also attracted attention as materials for nps formulation and drug delivery studies. chitosan is a natural polymer derived from deacetylation of chitin and is composed of glucosamine and n-acetylglucosamine residues [ ] . due to the availability of amino and carboxyl groups in an acidic microenvironment, chitosan nps have net positive surface charge which makes them highly mucoadhesive and increases their half-time of antigen retention on mucosal surfaces [ , ] . further, chitosan nps can reversibly open the epithelial cell tight junctions thereby improving paracellular and intracellular antigen transport across mucosal epithelial surfaces [ , ] . chitosan nps also enhance antigen uptake by apcs, induce apc maturation and active secretion of innate cytokines [ , ] . thus, chitosan nps form an attractive mucosal vaccine delivery vehicle. chitosan-based nps are used in pigs to deliver adjuvants such as bee venom and plasmid encoding porcine il- and il- /il- genes, which improved induction of better virus-specific immune responses of respective vaccines against prrsv and pcv [ , ] . chitosan nps enhance antigen uptake by porcine apcs and activate them to produce innate cytokines including ifn-alpha, tnf-alpha and il- β [ ] . chitosan nps encapsulated siv kag (cnps-kag) vaccine administered twice through in route without any additional adjuvant in pigs induced the cross-reactive mucosal iga antibodies. chitosan nps-based vaccine also induced ifn-γ response in pbmcs and tracheobronchial lymph nodes (tbln) better than kag vaccine controls. this vaccine formulation substantially reduced the challenge heterologous virus titers by up to -fold in both the upper and lower respiratory tracts compared to soluble kag vaccine. this finding emphasizes the potential benefits of using chitosan nps in future development of mucosal swine influenza vaccine for pigs [ ] . recently, dendrimer-like-alpha-d-glucan (nano- ) nps derived from sweet corn variety sugary- was examined as an alternative, safe, cost-effective and potent adjuvant [ , ] . nano- are positively charged nps which efficiently adsorb negatively charged antigens through electrostatic interactions. rodent studies have shown that nano- nps enhance antigen uptake by dcs, induce their maturation, activate them to produce pro-inflammatory cytokines and help in induction of antigen-specific antibodies [ , ] . in a recent study, we observed that nano- nps with or without addition of siv killed antigen (kag) can stimulate porcine apcs and produce cytokines such as ifn-α, tnf-α and il- β [ ] . pigs immunized via in route with nano- nps adsorbed siv kag at two-to-one ratio (nano- + kag) resulted in cross-reactive mucosal iga responses better than kag controls. moreover, pigs immunized im with nano- adsorbed ovalbumin (nano- + ova) had significantly greater igg and igg antibodies in serum compared with pigs vaccinated with ova alone [ ] . these findings highlight the possibility of using cornderived nano- nps as a potential adjuvant in porcine viral vaccine development. liposomes can encapsulate both hydrophilic and hydrophobic molecules in aqueous and non-aqueous phases of their vesicles [ ] . liposome vesicles protect antigens from enzymatic degradation, enhance antigen internalization by apcs and maintain controlled release of antigens [ ] . liposome-encapsulated antigens can enhance both cellular and humoral immune responses [ , ] . in a pig study, liposome nps were used as an im adjuvant for a pcv dna vaccine [ ] . liposome nps-adjuvant induced higher neutralizing antibodies and ifnγ response in pigs and reduced viremia of a challenge virus compared to alum-adjuvanted vaccine, providing the evidence that liposome nps can be a potent adjuvant in pigs [ ] . in our recent study, we used liposome nps to encapsulate ten highly conserved peptides of different influenza viruses of human and pig origin and immunized pigs through in route co-administered with monosodium urate (msu) crystal adjuvant [ ] . the liposome-adjuvant based vaccine enhanced the mucosal iga antibody response and induced peptide and virusspecific t-helper/memory cells and ifnγ responses resulting in reduced fever and modest reduction in virus titers in the respiratory tract of pigs [ ] . these studies highlight the fact that liposome-based nps can be used as an attractive vaccine delivery platform against porcine viral infections. virus infections have significant impact on pig industry worldwide. use of available vaccines have definitely helped in achieving strong control over some of the porcine viral infections such as food and mouth disease, transmissible gastroenteritis, classical swine fever and pseudorabies. vaccination also helped in reducing the clinical signs and increasing the production parameters in pcv -associated disease. however, for many other porcine viruses, further improvements in existing vaccine platforms and development of novel vaccine delivery systems are necessary to: ( ) induce better mucosal and cell-mediated immunity; ( ) protect against emerging and re-emerging strains; ( ) enhance the breadth (heterologous, cross-genotype and heterosubtypic) of immunity; and ( ) differentiate between infected and vaccinated animals. nps-based vaccine delivery platforms such as vlps, biodegradable polymers and liposomes have great potential as they-( ) protect vaccine antigens from degradation; ( ) facilitate antigen uptake and processing by apcs; ( ) impart adjuvant potential; ( ) can be used in mucosal and other alternate routes of immunizations; and ( ) induce effective mucosal and cellular cross-protective (broader) immunity. research efforts are ongoing to develop porcine viral vaccines using nps-based technologies. however, more collaboration(s) and in-depth studies are warranted to make this innovative vaccine antigen delivery technology successful and practical for application in food animal industry. to date, almost all of the immunomodulatory mechanisms of nps-based vaccine delivery platforms have been studied in rodent disease models, which may or may not reflect the situation in pigs or other domestic animal species [ ] . likewise, proper understanding of effect of size, charge and other physicochemical properties of nps after delivery through different routes of immunization in pigs is necessary to make efficient translation of this robust nps-based vaccine technology. similarly, studies should also focus on nps stability at different storage conditions and immunogenicity over a long period of time as they will directly associate with commercial aspect of the vaccine product. recent advances in nps-based adjuvant and vaccine delivery platforms in pigs demonstrate great promise to yield better candidate vaccines against many porcine viral infections with enhanced efficacy in the field. these nanovaccine technologies can also be adopted to develop effective vaccines against viral infections in other animal species, and knowledge gained could be exploited for improving the efficacy of existing human viral vaccines. the important viral infections of pigs emerging and re-emerging swine viruses porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system assessment of the economic impact porcine reproductive and respiratory syndrome virus on united states pork producers optimal use of vaccines for control of influenza a virus in swine assessing production parameters and economic impact of swine influenza, prrs and mycoplasma hyopneimoniae on finishing pigs in a large production system the role of swine as "mixing vessel" for interspecies transmission of the influenza a subtype h n : a simultaneous bayesian inference of phylogeny and ancestral hosts vaccines for porcine epidemic diarrhea virus and other swine coronaviruses lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts porcine rotaviruses: epidemiology, immune responses and control strategies the pathogenesis of foot-and-mouth disease in pigs a review of classical swine fever virus and routes of introduction into the united states and the potential for virus establishment african and classical swine fever: similarities, differences and epidemiological consequences the classical swine fever epidemic - in the netherlands: descriptive epidemiology epidemiology and transmission of porcine circovirus type (pcv ) porcine circovirus type (pcv ) vaccines in the context of current molecular epidemiology molecular epidemiology and evolution of porcine parvoviruses pseudorabies virus in wild swine: a global perspective vaccines against pseudorabies virus (prv) a review of african swine fever and the potential for introduction into the united states and the possibility of subsequent establishment in feral swine and native ticks immunogenicity and safety of virus-like particle of the porcine encephalomyocarditis virus in pig genotype i of japanese encephalitis virus virus-like particles elicit sterilizing immunity against genotype i and iii viral challenge in swine improved vaccine against prrsv: current progress and future perspective challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective influenza a virus vaccines for swine foot-and-mouth disease vaccines classical swine fever vaccines-state-of-the-art ten years of pcv vaccines and vaccination: is eradication a possibility? pcv d- is the predominant type of pcv dna in pig samples collected in the u.s. during biology of porcine parvovirus (ungulate parvovirus ) control of swine pseudorabies in china: opportunities and limitations african swine fever: a re-emerging viral disease threatening the global pig industry vaccination greatly reduces disease, disability, death and inequity worldwide veterinary vaccines and their importance to animal health and public health role of sustained antigen release from nanoparticle vaccines in shaping the t cell memory phenotype biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine biodegradable particles as vaccine delivery systems: size matters modes of action for mucosal vaccine adjuvants vaccine adjuvants: putting innate immunity to work alum adjuvant: some of the tricks of the oldest adjuvant mechanism of immunopotentiation and safety of aluminum adjuvants adjuvants in veterinary vaccines: modes of action and adverse effects adjuvants for veterinary vaccines-types and modes of action recent progress in mucosal vaccine development: potential and limitations respiratory nanoparticle-based vaccines and challenges associated with animal models and translation chitosan nanoparticles act as an adjuvant to promote both th and th immune responses induced by ovalbumin in mice poly(anhydride) nanoparticles act as active th adjuvants through toll-like receptor exploitation liposome-based adjuvants for subunit vaccines: formulation strategies for subunit antigens and immunostimulators vaccine delivery using nanoparticles potent antigen-specific immune responses stimulated by codelivery of cpg odn and antigens in degradable microparticles enhancement of immune responses by co-delivery of a cpg oligodeoxynucleotide and tetanus toxoid in biodegradable nanospheres co-delivery of cancer-associated antigen and toll-like receptor ligand in plga nanoparticles induces potent cd + t cell-mediated anti-tumor immunity m-cell targeted biodegradable plga nanoparticles for oral immunization against hepatitis b dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein rapid endo-lysosomal escape of poly(dl-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery enhanced and prolonged crosspresentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles cytotoxic t cell vaccination with plga microspheres interferes with influenza a virus replication in the lung and suppresses the infectious disease entrapment of h n influenza virus derived conserved peptides in plga nanoparticles enhances t cell response and vaccine efficacy in pigs biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs induction of potent antigen-specific cytotoxic t cell response by plga-nanoparticles containing antigen and tlr agonist polyanhydride nanovaccine against swine influenza virus in pigs virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development virus-like particle engineering: from rational design to versatile applications phagocytic processing of exogenous particulate antigens by macrophages for presentation by class i mhc molecules efficient major histocompatibility complex class i presentation of exogenous antigen upon phagocytosis by macrophages major findings and recent advances in virus-like particle (vlp)-based vaccines development of a porcine reproductive and respiratory syndrome virus-like-particlebased vaccine and evaluation of its immunogenicity in pigs intranasal immunization of pigs with porcine reproductive and respiratory syndrome virus-like particles plus ′, ′-cgamp vaccigrade adjuvant exacerbates viremia after virus challenge pandemic h n influenza virus-like particles are immunogenic and provide protective immunity to pigs chimeric calicivirus-like particles elicit specific immune responses in pigs foot-and-mouth disease virus-like particles produced by a sumo fusion protein system in escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle promising ms mediated virus-like particle vaccine against foot-and-mouth disease immunogenicity of adenovirus-derived porcine parvovirus-like particles displaying b and t cell epitopes of foot-and-mouth disease characterization of porcine circovirus type (pcv ) capsid particle assembly and its application to virus-like particle vaccine development construction and immunogenicity of recombinant porcine circovirus-like particles displaying somatostatin a novel subunit vaccine co-expressing gm-csf and pcv b cap protein enhances protective immunity against porcine circovirus type in piglets a novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure poly lactic-co-glycolic acid (plga) as biodegradable controlled drug delivery carrier plga-based nanoparticles: an overview of biomedical applications the preparation of sub- nm poly(lactide-co-glycolide) microspheres for site-specific drug delivery delivery of a peptide via poly(d, l-lactic-co-glycolic) acid nanoparticles enhances its dendritic cell-stimulatory capacity duration of antigen availability influences the expansion and memory differentiation of t cells biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs plga nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination poly (d, l-lactide-co-glycolide) nanoparticle-entrapped vaccine induces a protective immune response against porcine epidemic diarrhea virus infection in piglets recent advances in polyanhydride based biomaterials amphiphilic polyanhydrides for protein stabilization and release activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants structural and antigenic stability of h n hemagglutinin trimer upon release from polyanhydride nanoparticles evaluation of cpg-odn-adjuvanted polyanhydride-based intranasal influenza nanovaccine in pigs chitosan-based gastrointestinal delivery systems strong adhesion and cohesion of chitosan in aqueous solutions contact timeand ph-dependent adhesion and cohesion of low molecular weight chitosan coated surfaces effect of chitosan on the permeability of monolayers of intestinal epithelial cells (caco- ) effect of chitosan on epithelial permeability and structure the effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells immunogenic properties of a bcg adjuvanted chitosan nanoparticle-based dengue vaccine in human dendritic cells nasal delivery of chitosan/alginate nanoparticle encapsulated bee (apis mellifera) venom promotes antibody production and viral clearance during porcine reproductive and respiratory syndrome virus infection by modulating t cell related responses enhancement of immune response of piglets to pcv- vaccine by porcine il- and fusion il- / gene entrapped in chitosan nanoparticles mucosal immunity and protective efficacy of intranasal inactivated influenza vaccine is improved by chitosan nanoparticle delivery in pigs alpha-d-glucan nanoparticulate adjuvant induces a transient inflammatory response at the injection site and targets antigen to migratory dendritic cells dendrimer-like alpha-d-glucan nanoparticles activate dendritic cells and are effective vaccine adjuvants corn-derived alphad-glucan nanoparticles as adjuvant for intramuscular and intranasal immunization in pigs liposomes as vaccine delivery systems: a review of the recent advances mucosal vaccine development based on liposome technology liposomeencapsulated antigens are processed in lysosomes, recycled, and presented to t cells development of porcine circovirus (pcv ) open reading frame dna vaccine with different adjuvants and comparison with commercial pcv subunit vaccine in an experimental challenge liposomal nanoparticle-based conserved peptide influenza vaccine and monosodium urate crystal adjuvant elicit protective immune response in pigs intranasal delivery of influenza antigen by nanoparticles, but not nkt-cell adjuvant differentially induces the expression of b-cell activation factors in mice and swine porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the research reviewed in this article was supported by agriculture and food research initiative competitive grant no. - - from the usda-nifa and nanovaccine institute ( - ), iowa state university to rgj. salaries and research supports were provided by the state and federal funds appropriated to oardc. we thank dr. steven krakowka for scientific editing of the manuscript. authors' contributions sd wrote the article; gjr edited and revised the article. both authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -z oztgi authors: palatnik-de-sousa, clarisa b. title: what would jenner and pasteur have done about covid- coronavirus? the urges of a vaccinologist date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: z oztgi nan vaccines are the best cost-benefit tools to control and eradicate infectious diseases. the live smallpox vaccination, called variolation, was the injection of the homologous virus and this promoted self-healing local lesions that guaranteed strong and long-lasting protection. however, since % of these variolations caused cases of smallpox in the vaccinated individuals, it was considered unsafe and was discontinued ( ) ( ) ( ) ( ) ( ) . in , edward jenner, who had been variolated, discovered the vaccination principle when he used the cowpox virus live-vaccine (vaccinia virus) to induce cross-immunity and prevent human smallpox in a child. his strong merit was to initiate the campaign that turned vaccination against smallpox obligatory and universal, and to discover that cross-protection promoted by a heterologous, although related, organism was sufficient to guarantee efficacy and reduce the safety issues of the homologous live-vaccine ( ) . in , due to the world health campaigns, smallpox was considered the first and only human viral infection ever eradicated ( , ) . ironically, jenner never knew that smallpox was induced by a virus ( , ) which suggests that, what is needed for the eradication of a disease is the systematic worldwide use of a potent and efficient vaccine. after the sabin anti-polio vaccine, which was launched in the 's, many other vaccines have been developed based on whole attenuated viruses. however, poliomyelitis was also induced by the sabin vaccine poliovirus in healthy subjects ( ) ( ) ( ) . this means that, nowadays, live-vaccination with whole wild or attenuated virus is no longer ethically possible, mainly because of the large population of immunocompromised subjects, in which a live-vaccine could cause the disease. due to these safety issues, whole virus and bacterial dead or inactivated vaccines have progressively substituted live-vaccines. we could guess that this is precisely what louis pasteur, the father of microbiology, would have done to control and eradicate covid . although he worked initially with the attenuation of viruses and bacteria, after his successful work with rabies, fowl cholera and anthrax ( ) , it became clear three steps were needed to develop a protective vaccine against infection. first, the organism should be isolated, then inactivated, and finally injected ( ) . in , pasteur's rabies vaccine employed an air dried fixed virus. semple improved the fixation by adding phenol ( ) . currently β-propiolactone is considered to be better than phenol or formaldehyde. however, it is carcinogenic ( ) . therefore, other methods like ultraviolet or gamma-irradiation, high pressure ( ) , visible ultrashort pulsed laser, and low-energy electron irradiation have been suggested ( ) . the most important advantage of whole inactivated vaccines is that, unlike the live or attenuated vaccines, they do not cause the disease ( table ). in fact, inactivated vaccines preserve the intact structure of the antigens and their b-cell epitopes that enable them to interact with the antibody paratopes, and promote the synthesis of neutralizing antibodies. they can not only stimulate the humoral, but the cellular immune responses as well, in a manner similar to live viruses, since they preserve the virus structures during inactivation ( ) . cross-presentation of conserved epitopes to the cytotoxic t cells (ctl), through the major hla class histocompatibility system, in addition to the viral pathogen associated patterns (pamps), which use innate immune receptors such as toll like receptor , can induce t cell mediated responses. in fact, in the late endosome of the infected apc three different events can occur: ( ) viral degradation following the exogenous pattern and presentation to cd t cells via the mhc class ii molecules, ( ) cross presentation pathway to cd t cells via the mhc class i molecules, and ( ) viral membrane fusion following the endogenous pathway. the above mentioned pathways along with the recognition of viral pamps, using prrs such as tlr , as well as production of cytokines such as ifn- can promote potent cellular mediated immune responses ( ) . however, in the case of influenza vaccines for instance, the inactivated formulations may not always induce t cell responses as potent as the live-vaccines. in fact, the inactivated vaccine may even prevent or suppress the induction of cross-reactive cd + t-cells ( ) . pamps are constitutive components of the virus and bacteria (viral or bacterial nucleic acids, polysaccharides, lipopolysaccharides, lipid a, monophosphoryl lipid a, bacterial peptidoglycans, etc.). they are compounds universally recognized by the innate immune system of healthy subjects, who build a natural protective response against them. in contrast, modern purified, fractionated, recombinant, or synthetic vaccines gained in safety but lost potency because they lack the pamps (table ) . while, vaccines using inactivated organisms with pamps have shown great success against polio, whooping cough, and tetanus ( , ) . if we use fixation of the structures of some of the isolates that cause the disease, then inactivate them and preserve their whole structures, the possible deleterious effect of the high mutagenicity detected in a few of the proteins of the virus ( ) would be overcome by the strong immune response generated against the whole virus structure. therefore, the mutagenicity should not be critical for the generation of protection, and would not damage the efficacy of the whole vaccine. furthermore, it might be that the whole virus inactivated vaccine could even induce some cross protection against other coronovirus agents of sars, which hold conservative structures ( ) . furthermore, whole inactivated vaccines are considered good candidates for designing universal vaccines capable of giving protection against multiple strains of influenza virus ( ) . it is true that an impressive amount of data about the dna sequencing of the virus has been gathered in a relatively short period of time and with that, the knowledge of its biological properties increased enormously ( , sequences in pubmed) ( , ( ) ( ) ( ) ( ) ( ) ( ) . however, for an urgent strategy, we could also take advantage of the lessons taught by the history of vaccinology in order to prevent the disease and save lives. furthermore, if we want to enhance the efficacy of the vaccine we should combine the inactivated virus with a good adjuvant. the adjuvant might contain saponins of quillaja saponaria molina, which induce antibodies of desired subtypes, promote both the cytotoxic antiviral cd + and cd + th cell responses against the infection and that been used with success in vaccines against leishmaniasis ( , , ) , cancer ( ) , malaria ( ), herpes zoster ( ) , and hiv ( ) . in spite of the valuable guidelines from the work of jenner and pasteur, who with much fewer resources, developed vaccines that controlled and eradicated smallpox that showed a % mortality rate ( ) and rabies with % mortality rate ( ) ; and even with all the knowledge acquired since then, there is no urgent combined international effort to produce one unique vaccine. instead, months after the description of the first covid cases in china, vaccines are reported to be in development all over the world and, only two of them contain the inactivated virus ( , ) . all the other formulations include live attenuated, non-replicating or replicating viral vector, recombinant protein, peptide base, virus-like particle, and virus dna and rna. most of these vaccines are focused on only one antigen of the coronavirus, therefore, these formulations will certainly be less potent than a vaccine made up of multiple antigens contained in the whole pathogen. furthermore, many of these formulations do not use the technology involved in any previously licensed vaccines ( ) . cepi (coalition for epidemic preparedness innovation) estimated the development of phase i clinical trials of vaccines, phase and trials for up to vaccines and progression to regulatory approval and production of up to candidates ( ) . in fact, by may th, seven vaccines had already entered phase i clinical trials: ( ) encapsulated mrna encoding protein s (moderna and niaid, usa); ( ) adenovirus expressing protein s (cansino biologics, china); ( ) dcs modified with lentivirus expressing several proteins and ctls (shenzen geno-immune medical, china); ( ) an apc modified with lentivirus expressing several viral proteins ( ); ( ) inno , sars cov dna injection (innovio, usa); ( ) chadox vaccine from the jenner institute, oxford university, (uk) which is a genetically modified adenovirus expressing coronavirus proteins ( ) , and is also being tested in a phase ii trial; and finally ( ) the whole inactivated coronavirus with alum by sinovac, china ( ) . furthermore, on july nd, the who communicates that there are covid candidate vaccines in clinical evaluation and more under pre-clinical assays ( ) . only one of the vaccines under clinical trials is currently supported by a peer reviewed scientific publication in science ( ). the results of its pre-clinical assays in the mouse, rat and non-human primate model were published before, without peer review on april th in the biorxiv. later on, on may th, the results of the chadox adenovirus vaccine of the jenner institute of oxford university were published with no peer review in the biorxiv ( ) . until june th, there has been no peer reviewed publication of this vaccine. regarding the formulations, the inactivated whole virus sinovac vaccine is composed of one isolate of sars-cov (cn ) obtained from a patient of china and alum adjuvant ( ) , while the chadox ncov vaccine of oxford is composed of a chimpanzee recombinant adenovirus, which expresses the s protein of sars-cov ( ) . sinovac biotech (china) in collaboration with several universities, public health institutions and the medical academy of the army of china have been able to produce a whole virus inactivated vaccine adjuvanted by alum that was stable and showed . to % sequence identity to other isolates also obtained from broncheoalveolar fluid (balf) of hospitalized patients (five in intensive care), from china, italy, united kingdom, switzerland and spain ( ) . the virus was propagated in cultures of vero cells in vitro and inactivated with β-propiolactone ( ) . the use of alum adjuvant is approved for human vaccines because it induces strong antibody responses, mainly of the igg and ige types that show efficacy against virus or bacterial diseases, which need neutralizing antibodies to be controlled. however, alum is a poor promoter of the cellular immune responses against pathogens ( ) . in contrast, the chadox ncov- vaccine developed by oxford university and astrazeneca is composed of a recombinant non-replicant chimpanzee adenovirus, which expresses the s protein of sars-cov- ( ) . different from the technology used for inactivated vaccines since the 's, this adenovirus platform was developed in ( ) . the authors aimed to include an adenovirus in the vaccine that would not infect humans, in order to avoid its potential rejection by human antibodies. the chosen chimpanzee adenovirus was phylogenetically related to the human adenovirus. the inventors deleted the region e of the chimpanzee adenovirus genome in order to render the virus defective and non-replicant, while the e region was excluded to increase the insert capacity. in addition, a bacterial artificial chromosome (bac) containing a codon-optimized full-length spike protein of sars-cov- with a human tpa leader sequence ( ) was added between the deleted e region and e to facilitate the genetic modifications. this approach has been reported to improve genetic stability ( ) . however, additional modifications were needed to guarantee that the e region of the virus would express a human, instead of a simian protein, that would enable the virus recognition and propagation inside human cells in in vitro culture, for large-scale virus production ( ) . regarding the number of samples, the sinovac inactivated vaccine was tested in groups of mice and rats and in cohorts of monkeys (macaca mulatta) ( ), while the chadox ncov- vaccine was only tested in groups of - mice and in rhesus monkeys using only monkeys as controls ( table ) ( ) . regarding the antibody response in mice and rats, the sinovac vaccine promoted high igg antibody titers against protein s, against its receptor binding domain, and to a lower extent against protein n ( table ) and also high titers of virus neutralizing antibodies. the cytokine expression induced by the inactivated vaccine in mice was not analyzed ( ) . in contrast, the chadox vaccine induced anti-s and s protein igg antibody titers (igg a, igg b, and igg ) and neutralizing antibodies in only three of the five balb/c mice, but showed ifn-γ, tnf-α, and il- expressed by cd + and cd + t cells and ifn-γ, tnfα, il- , and remarkably il- secreted to the supernatants ( ) ( table ) . furthermore, in vaccinated monkeys, seven days after infection, the sinovac inactivated vaccine at µg/dose induced high titers of igg antibodies directed against the s, rbd and lower levels of anti-n protein antibodies, high titers of virus neutralizing antibodies with no detected antibodydependent enhancement of disease (ade) ( ) . in contrast, anti-s protein igg and neutralizing antibodies were detected in the rhesus monkeys vaccinated with chadox ( table ) ( ) . moreover, regarding the concern of the increased proinflammatory events and cytokine storm related to the severity of covid , the sinovac vaccine was safe and did not promote any alteration in the frequencies of cd + , cd + , or cd t cells nor of secretion of ifn-γ, tnf-α, il- , il- , il- , or il- ( table ) ( ) . in contrast, increased levels of ifn-γ, tnf-α, il- , and il- were observed in monkeys vaccinated with the chadox vaccine ( ) ( table ) in which the frequencies of cytokine secreting t lymphocytes was not studied ( table ) . regarding cross-protection to other sars cov isolates, the sinovac inactivated vaccine protected mice and rats against the challenge with different virus isolates ( table ) suggesting its potential use all over the world ( ) . there is no available data concerning cross-protection for the chadox vaccine ( ) . besides, for a fair comparison of vaccine efficacies, the two sars-cov vaccines should be assayed in the same field trial, and the efficacy end-points should be determined prior to the assay. due to the urgency in saving lives, this might be not be feasible during the pandemic. however, for comparative purposes, early infection, disease, severe disease, and death due to covid or other causes should be recorded as vaccine efficacy end-points. for instance, reduction of the virus load in the nasal and pharynx mucosa indicates not only protection against early infection, but also the blockade of the transmission of infection by respiratory droplets. this means that this end point is particularly important when seeking a vaccine to interrupt the epidemic. in addition, clinical symptoms indicate disease, while pulmonary distress, cytokine storm, need of intensive care, intubation indicate severe disease. the number of deaths due to covid or other causes should also be recorded and compared in order to evaluate the reduction of mortality. notable, the sinovac inactivated vaccine reduced to zero the viral load in throat swabs (pharynx and crissum), anal swabs and all regions of both lungs of vaccinated and challenged monkeys ( ) indicating that the inactivated vaccine prevents not only the early infection but also blocks the transmission of the disease by droplets curtailing the epidemics. in contrast, no viral sgrna indicative of viral replication, could be detected in bal fluids, and in the lungs of two of six monkeys vaccinated with chadox and challenged ( ) ( table ). in fact, the lung viral load decreased by ∼ % in monkeys vaccinated with chadox . however, viral grna was detected in nose swabs of all vaccinated and challenged animals ( table ) ( ) , indicating that the chadox vaccine would not prevent the sars cov human infection nor block its transmission and interrupt the epidemics. vaccinated and infected subjects will continue to be infectious and spread sars cov- . however, the vaccine will probably, in most cases, reduce the pulmonary symptoms, and make the disease less severe. accordingly, the inventors of the chadox vaccine seem to be aware of the limitations of its efficacy when they describe it as a vaccine that prevents pneumonia in monkeys ( ) . unfortunately, neither the investigations of the sinovac nor the chadox vaccine have disclosed if any of their formulations prevent or reduces mortality. furthermore, in a report that analyzes the first results of the vaccine trials, published in nature, peter hotez considered that the oxford vaccine induced very modest titers of neutralizing antibodies and that considerable higher titers would be needed to afford protection ( ) . at the same time, hotez also says that the sinovac vaccine elicited a more promising antibody response in macaques monkeys ( ) . in spite of that, who disclosed that this vaccine is in fact being tested in uk in phase i, ii and iii trials ( ) and will be tested in a phase iii trial in brazil on , volunteers. consequently, contracts for large-scale fabrication have already been signed with the public laboratory of the brazilian ministry of health bio-manguinhos. in brazil, million doses are intended to be produced by bio-manguinhos and another million after the proven efficacy of the vaccine. at this point it is important to know which end-points of vaccine efficacy will be taken into consideration for such an important decision. on the other hand, the sinovac whole virus inactivated vaccine was also reported to have been successful in phase i and ii trials in - year olds (n = ) and in healthy elderly adults > years old (n = ) in china ( ) although these results have not yet been published in detail. more than % of the volunteers showed neutralizing antibodies ( ) . a recent contract has been signed between sinovac and the public laboratory instituto butantan of são paulo, brazil, in order to produce doses of the vaccine to immunize , healthcare professionals for a double-blind randomized phase iii trial in brazil, where the incidence of cases and deaths due to covid is still high ( ) . the results of the efficacy are expected in october . in return, the instituto butantan will gain the transfer of the technology and the license to manufacture , , doses for brazil. testing anti-covid vaccines in brazil became interesting because of the high morbidity and mortality and active expansion of the epidemics. an important warning is given by ewen callaway in his article published in nature ( ) , in which he asks for caution about the potential success of vaccines that arise from small animal or human studies. this might be the case of the moderna-niaid vaccine composed of lipid nano-particle encapsulated synthetic mrna, which encodes the spike s protein and already underwent phase i and ii clinical trials in usa ( ) . moderna company announced that phase iii trials are predicted to start in july and that studies in monkeys are underway in parallel. none of these mrna based vaccine has ever been licensed before ( ) . we conclude that the first results of anti-covid vaccine candidates confirm that the whole virus inactivated vaccine, which preserves the immunogenicity of all the antigens of the virus and contains pamps ( ) is more potent than the recombinant vaccines that have only the important s spicula protein, either expressed by an engineered adenovirus ( ) or by lnp encapsulated mrna ( ) . furthermore, the inactivated vaccine also contains the alum adjuvant. there are other examples support the superiority of whole inactivated vaccines above those expressing recombinants single antigens. for instance, . µg/dose of the trivalent inactivated influenza vaccine is as safe as, but more immunogenic than the . µg/dose of the recombinant baculovirus-expressed hemagglutinin flubok vaccine, in young children ( ) . this is a fast moving scenario and several phase clinical trials of covid vaccines have been published, either with or without peer reviews. two recombinant adenovirus vaccines expressing the s spike protein of sars-cov- , the chadox and the cansino vaccines ( , ) , and two other vaccines composed of mrna codifying for the s-protein (mrna , moderna vaccine) ( ) or its rdb domain (mrna bnt b pfizer-biontech vaccine) ( , ) have been assayed for safety and immunogenicity in phase i-ii clinical trials in humans. there were no serious adverse events related to any of the four vaccines ( ) ( ) ( ) ( ) ( ) . local and systemic reactions commonly including pain, feeling feverish, chills, muscle ache, headache, and malaise were recorded for all formulations ( ) ( ) ( ) ( ) ( ) and were reduced, in the case of the chadox vaccine, with use of prophylactic paracetamol ( ) . only the cansino vaccine was given as a single dose ( ) while chadox , moderna, and pfizer biontech vaccines were assayed in two-dose protocols ( , ( ) ( ) ( ) . anti-s protein igg responses rose by day ( ) and peaked or increased by day - , after the first ( ) or second immunization dose, respectively ( , ( ) ( ) ( ) . in addition, spike-specific t-cell responses detected by an ex-vivo interferon-γ enzyme-linked immunospot assay, peaked on day for the chadox ( ) and on day for the cansino adenovirus vaccine ( ) . moreover, the moderna mrna- , vaccine induced a th response against the s-protein peptide pools (tnf-α >il- >ifn-γ), with a minimal th cytokine expression (il- and il- ) and with cd t-cell responses, only detected at low levels, after the second vaccination ( ) . in agreement, most participants vaccinated with the pfizer-biontech mrna-rbd vaccine (bnt b ) also had th skewed t cell immune responses with rbd-specific cd + and cd + t cell expansion and ifn-γ produced by a high fraction of rbdspecific cd + and cd + t cells ( ) . furthermore, the levels of neutralizing antibodies raised by each one of the vaccines could be considered as correlates of their potential efficacy. while the mrna- of moderna disclosed % ec values ranging between and ( ), the maximal titer for the mrna rbd vaccine of pfizer-biontech was ( ) and for the chadox vaccine, from to ( ) . the cansino vaccine expresses its results as gmt ( - , ) impeding an accurate comparison ( ) . unfortunately, the results of phase i-ii clinical trial of the whole virus inactivated vaccine of sinovac have not yet been published in detail, therefore although the vaccine was tested in the largest number of individuals (n = , ) ( ), a fair comparison of the safety and immunogenicity results is not yet possible. ultimately, only the results of the phase iii trials will disclose the potential impact of the vaccines on reduction of deaths, clinical cases, and virus particles or viral rna in nasopharynx and will allow their efficacy and capability to interrupt the epidemic to be evaluated. in the imminence of a pandemic involving high mortality and economic distress, several factors could speed up the development of vaccines. one of them would be the use of an already standardized methodology. it is worth noting that most of the molecularly defined vaccines now in development would meet severe restrictions for large-scale production and this could led to an enormous delay to deliver vaccines for mass vaccination of the public. in contrast to this, nowadays large industries and public laboratories are authorized to produce inactivated vaccines against influenza. it is also reasonable to hypothesize that generation of protection and immunological memory against a group of antigens will be more efficient than that generated against a single antigen, no matter, how important it is. two concerns could be considered as the downside of the inactivated vaccines for sars diseases. the first would be the fear of an incomplete inactivation of the virus that could cause outbreaks among the vaccine production workers or in vaccinated populations ( ) . this concern is common to all vaccines produced with native antigens, which demand the production of large mass of pathogens. however, to guarantee safety, each batch of vaccine is submitted to validation of inactivation controls that include sequential passages assays of residual virus infectivity in embryonated eggs or tissue culture, and detection of live virus by tcid assays ( ) . the whole virus sars-cov- inactivated vaccine of sinovac includes validation of inactivation controls ( ) . the second concern would be the promotion of an antibody disease enhancement syndrome (ade) by the vaccine. this is usually related to non-neutralizing antibodies, which determine an increased lung pathology and it was observed before in vaccines against rsv and measles in the 's ( ) . since sars-cov- , mers-cov, and sars-cov- are phylogenetically related viruses that have caused epidemics over the last years and ade pathology was present for some sarscov- and mers vaccine candidates in animal models, there is also a concern about the induction of ade syndrome in humans vaccinated with sars-cov- vaccine candidates ( ) . however, ade pathology is not exclusive for inactivated vaccines and has been also demonstrated for the vectored vaccine expressing n protein, a replicon particle platform expressing s protein ( ), the recombinant protein s with or without gold nanoparticles ( ) and a mva vectored vaccine expressing s proteins ( , ) . with the aim of preventing these safety issues in sars-cov- vaccines cepi and the brighton collaboration safety platform for emergency vaccines (speac) convoked an expert scientific meeting on march and , in order to establish the assessment of the risk of ade during sars-cov- vaccine development ( ) . in murine models, ade was observed for an inactivated whole virus vaccine against mers ( ) and against sars-cov- ( , , ) . in fact an inactivated mers-cov vaccine, with and without adjuvant, induced in mice neutralizing antibody, reduced the viral load in lungs but showed mononuclear infiltrates containing eosinophils and eosinophils secreting il- and il- cytokines ( ) . a formalin double-inactivated sars-cov- (div) vaccine, adjuvanted or not, induced also immunopathology involving eosinophils in aged mice ( , ) . in addition, a formalininactivated sars-cov- vaccine promoted ade in nhp with macrophage and lymphocyte infiltration in the lungs and fibrin and protein-rich edema in the alveolar cavity ( ) . on the other hand, other inactivated vaccines against sars were reported as non-inducers of ade ( ) . fortunately, other studies disclosed the absence of ade in hamsters and monkeys immunized with whole inactivated vaccine against sars-cov- . these studies differed from the previous one in the use of β-propiolactone instead of formalin to inactivate the virus ( , ) . the conclusion was that, nhps could be used to evaluate the anti-covid vaccines with or without adjuvants to select the formulations with desired efficacy and reduced risk of ade ( ) . in addition, transgenic mice expressing the human ace receptor will be needed to evaluate the vaccine induced ade. the immunopathology was a consequence to a th type of response to the antigen and it was avoided in vaccines that drive the response to a th immunity, with or without adjuvants. also, it is known that the presence of fetal calf serum in the preclinical vaccine preparation may induce eosinophil influx to lungs ( ) . for instance, the passive transfer in nhps of human antibodies generated during phase trials, followed by viral challenge could be considered to assess the risk of disease enhancement ( ) . it was recommended to challenge the vaccinated animals with close related species in order to evaluate cross protection for future epidemic ( , ) . this has been done for the whole inactivated sinovac vaccine with other isolates of the sars-cov- ( ) . experts also recommended including animals vaccinated with formalin inactivated, alum-adjuvanted whole virus sars-cov- or sars-cov- , for immunopathology studies, as positive controls. this will help in establish accepted end-points to allow comparison ( ) . the group of experts considers that continuous monitoring of this risk will be needed during clinical trials. each effect observed should be discussed by the developers with their regulators who will ultimately define the actual requirements for clinical studies ( ) . if we consider that a whole virus inactivated vaccine with a potent qs saponin adjuvant is the ideal formulation for an anti-covid urgent first vaccine, the sinovac vaccine is not only the closest formulation to the ideal, only differing in the adjuvant, but also the one that can be developed the fastest. the potential use of alum, mf , as , as , or as , which contain qs saponin has been discussed ( , ) . it was concluded that, the immunopathology of sars vaccines was a consequence to a th type of response to the antigen and it was avoided in vaccines that drive the response to a th immunity, with or without adjuvants ( ) . time matters and is an extremely important factor considering the high daily rate of deaths worldwide. in fact, the assays of the sinovac vaccine in the mouse, rat, and macaque models seems to have been performed simultaneously from january to march, . in addition, this is the only vaccine with results already published in a peer reviewed journal ( ) . on the other hand, eradication of the pandemic or at least its control, as jenner knew in , will only be possible by a universal and simultaneous use of the same vaccine. this is what took place with smallpox, rabies, yellow fever, influenza, h n , etc. in spite of that, we do not see the united international effort to gather together resources for the production of enough doses of one vaccine to vaccinate the world. the support given to different research projects ( ) and the deposit of hundreds of patents confirms that. again, the urgent formulation might already be known and waiting to be rediscovered from the history of vaccinology. if different vaccines with diverse degrees of efficacy values are used, even the countries that have low incidence of covid will not be safe and will not be able to open their frontiers. to support the production of one ideal vaccine should be the common focus worldwide. maybe the observed multiple individualistic efforts that have arisen are due to the lack of leadership from the developed nations, which have the highest capacity to produce vaccines. in the usa, for instance, the economic interest of the large vaccine industries in preventive vaccination has recently decreased. conversely, they have started to invest in immunotherapies or drug treatments. in addition, in the usa there are not large public laboratories for production of the vaccines under a governmental request. consequently, the governmental public health decisions are restricted by the interests of the private vaccine companies. in contrast, in some developing countries, where infectious diseases are often the most important causes of mortality, public laboratories can produce large amounts of vaccine doses without the need to make a profit, under the auspices of their ministries of health. this is the case of instituto butantan and bio-manguinhos in brazil, instituto biológico de la plata and the administración nacional de laboratorios e institutos de la salud (anlis-malbrán) in buenos aires, argentina, and of the serum institute of india. fortunately, instituto butantan will produce the sinovac inactivated vaccine and bio-manguinhos the adenovirus chadox vaccine of oxford in brazil. the serum institute of india will also produce the chadox vaccine of oxford. finally, the modern policies for vaccine regulations should be taken into consideration. these regulations demand that phase i, phase ii, and phase iii trials should be developed with success before a government licenses a vaccine and uses it on phase iv trials and for industrialization. this usually takes at least a decade. although these tests enhance the confidence of a product, one might think that if we are dealing with a vaccine produced by a technology that is already well-established in many licensed vaccines, such as a whole inactivated virus, more rapid or simultaneous tests would be accepted as proofs of concepts ( ) . this would be another increased cost-benefit value of the vaccine, which takes into account the high mortality, worldwide incidence and impressive impact on the economy promoted by the quarantines. probably, this is what jenner and pasteur would have done. cp-s designed and wrote this article. smallpox vaccines: new formulations and revised strategies for vaccination smallpox: variolation anti-infectious human vaccination in historical perspective ann illustrated history of smallpox and its eradication from empiricism to rational design: a personal perspective of the evolution of vaccine development livro-introdução À virologia humana the delay in the licensing of protozoal vaccines: a comparative history the safety and immunogenicity of two novel live attenuated monovalent (serotype ) oral poliovirus vaccines in healthy adults: a double-blind, single-centre phase study development of thermostable lyophilized sabin inactivated poliovirus vaccine implications of a circulating vaccine-derived poliovirus in nigeria microbes infect rabies control and treatment: from prophylaxis to strategies with curative potential evaluation of different stabilizers and inactivating compounds for the enhancement of vero cell rabies vaccine stability and immunogenicity: in vitro study intranasal immunization with pressure inactivated avian influenza elicits cellular and humoral responses in mice inactivation methods for whole influenza vaccine production vaccination against human influenza a/h n virus prevents the induction of heterosubtypic immunity against lethal infection with avian influenza a/h n virus toward a modern synthesis of immunity: charles a. janeway jr. and the immunologist's dirty little secret recent advances in the discovery and delivery of vaccine adjuvants genetic diversity and evolution of sars-cov- human monoclonal antibodies against highly conserved hr and hr domains of the sars-cov spike protein are more broadly neutralizing genomic analysis of sars-cov- strains among indians returning from italy, iran & china, & italian tourists in india sars-cov- : structural diversity, phylogeny, and potential animal host identification of spike glycoprotein two linear epitopes on the sars-cov- spike protein that elicit neutralising antibodies in covid- patients attenuated sars-cov- variants with deletions at the s /s junction simulation of the clinical and pathological manifestations of coronavirus disease (covid- ) in golden syrian hamster model: implications for disease pathogenesis and transmissibility the proximal origin of sars-cov- immunogenicity assay of the leishmune vaccine against canine visceral leishmaniasis in brazil from mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine leish-f +gla-se thomsen-friedenreich (tf) antigen as a target for prostate cancer vaccine: clinical trial results with tf cluster (c)-klh plus qs conjugate vaccine in patients with biochemically relapsed prostate cancer vaccines against malaria immune responses to a recombinant glycoprotein e herpes zoster vaccine in adults aged years or older qs- promotes an adjuvant effect allowing for reduced antigen dose during hiv- envelope subunit immunization in humans smallpox virus plaque phenotypes: genetic, geographical and case fatality relationships rabies: a medical perspective the covid- vaccine development landscape draft landscape of covid- candidate vaccines rapid covid- vaccine development ensuring global access to covid- vaccines chadox ncov- vaccination prevents sars-cov- pneumonia in rhesus macaques rapid development of an inactivated vaccine candidate for sars-cov- on vaccine's adjuvants and autoimmunity: current evidence and future perspectives a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity coronavirus vaccine trials have delivered their first results-but their promise is still unclear china's sinovac plots pivotal covid- vaccine trial in brazil after positive phase clinical trial of efficacy and safety of sinovac's adsorbed covid- (inactivated) vaccine in healthcare professionals evaluation of the safety, reactogenicity and immunogenicity of flublok trivalent recombinant baculovirus-expressed hemagglutinin influenza vaccine administered intramuscularly to healthy children aged - months safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind, randomised controlled trial safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial an mrna vaccine against sars-cov- -preliminary report phase / study to describe the safety and immunogenicity of a covid- rna vaccine candidate (bnt b ) in adults to years of age: interim report. medrxiv concurrent human antibody and t h type t-cell responses elicited by a covid- rna vaccine. medrxiv sars vaccine development consensus summary report for cepi/bc march - , meeting: assessment of risk of disease enhancement with covid- vaccines gold nanoparticle-adjuvanted s protein induces a strong antigenspecific igg response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs antispike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine immunogenicity and protective efficacy in mice and hamsters of a βpropiolactone inactivated whole virus sars-cov vaccine updated insights into the mechanism of action and clinical profile of the immunoadjuvant qs- : a review thanks to prof. charles greenblatt of the hebrew university of jerusalem, for helpful comments and discussion. david straker was acknowledged for the english review of this manuscript. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © palatnik-de-sousa. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -ipa wz authors: poland, g. a.; ovsyannikova, i. g.; kennedy, r. b. title: personalized vaccinology: a review date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ipa wz abstract at the current time, the field of vaccinology remains empirical in many respects. vaccine development, vaccine immunogenicity, and vaccine efficacy have, for the most part, historically been driven by an empiric “isolate-inactivate-inject” paradigm. in turn, a population-level public health paradigm of “the same dose for everyone for every disease” model has been the normative thinking in regard to prevention of vaccine-preventable infectious diseases. in addition, up until recently, no vaccines had been designed specifically to overcome the immunosenescence of aging, consistent with a post-wwii mentality of developing vaccines and vaccine programs for children. it is now recognized that the current lack of knowledge concerning how immune responses to vaccines are generated is a critical barrier to understanding poor vaccine responses in the elderly and in immunoimmaturity, discovery of new correlates of vaccine immunogenicity (vaccine response biomarkers), and a directed approach to new vaccine development. the new fields of vaccinomics and adversomics provide models that permit global profiling of the innate, humoral, and cellular immune responses integrated at a systems biology level. this has advanced the science beyond that of reductionist scientific approaches by revealing novel interactions between and within the immune system and other biological systems (beyond transcriptional level), which are critical to developing “downstream” adaptive humoral and cellular responses to infectious pathogens and vaccines. others have applied systems level approaches to the study of antibody responses (a.k.a. “systems serology”), [ ] high-dimensional cell subset immunophenotyping through cytof, [ , ] and vaccine induced metabolic changes [ ]. in turn, this knowledge is being utilized to better understand the following: identifying who is at risk for which infections; the level of risk that exists regarding poor immunogenicity and/or serious adverse events; and the type or dose of vaccine needed to fully protect an individual. in toto, such approaches allow for a personalized approach to the practice of vaccinology, analogous to the substantial inroads that individualized medicine is playing in other fields of human health and medicine. herein we briefly review the field of vaccinomics, adversomics, and personalized vaccinology. vaccines have been one of the most effective public health strategies in preventing infectious diseases. a decade ago, we described the idea of vaccinomics and adversomics, based on the immune response network theory [ , ] , which utilizes immunogenetics/imunogenomics and systems biology approaches to understand the basis for inter-individual variations in vaccineinduced immune responses in humans, as well as the basis for adverse side effects from vaccines [ ] . vaccinomics and adversomics explore the influence of genetic and non-genetic regulation on the heterogeneity of vaccine-induced immune responses at both the personal and population levels [ ] . in particular, vaccinomics and adversomics utilize high-throughput, high-dimensional systems biology approaches, which aim to predict variations in protective and maladaptive innate and adaptive immune responses to vaccines [ ] [ ] [ ] [ ] , ] . in this regard, the basis of personalized (and predictive) vaccinology is the assessment of an individual's genetic background, sex, as well as other factors that may impact vaccine immunogenicity, efficacy, and safety [ ] [ ] [ ] [ ] . we and others have widely published on the applicability of the tools and concepts of vaccinomics, including immunogenetics and immunogenomics, to the knowledge-based directed development of new and improved vaccine candidates [ ] [ ] [ ] [ ] . the application of these concepts is likely to allow for explanation, quantification, and prediction of vaccine-induced protective immune responses-including the http://dx.doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. development of predictive immune signatures in response to vaccines. indeed, we have previously published what we believe is the first draft of a mathematical model and predictive equation describing the non-random events that lead to a pre-determined immune response [ ] : b i x iþe y = measure of immune response b o = intercept b i = coefficient for the ith variable x i and indicates the amount of change in y for a unit change in x i Ε = random deviations from the model we recognize that such an equation, given the current state of the science, is incomplete and cannot yet predict immune responses. but we present it as an early directional attempt to quantify such an equation. such an approach begins to move us into a st-century model of directed vaccine development and an advanced understanding of how, and by what mechanisms, vaccines and vaccine adjuvants trigger both useful and maladaptive innate and adaptive immune responses. we believe that vaccinomics and adversomics represent approaches counter to the standard methods of vaccine development until recently. historically, vaccine development has been empirical, despite many emerging and re-emerging complex, hyper-variable pathogens-many with elaborate immune escape mechanisms. in addition, vaccine coverage rates continue to suffer as society is risk-averse toward vaccines and demands levels of safety that may not be achievable. finally, the ''one-size-fits-all" approach to the practice of vaccinology ignores the complexity and diversity of the human immune system and host genome. thus, the promise of vaccinomics and related paradigms is to identify specific immune response profiles, immunosignatures, and biomarkers that predict vaccine safety and/or efficacy, and which may lead to new vaccine candidates. vaccinomics provides the opportunity to examine not only immune response genes likely to be involved in vaccine response, but also the possibility of identifying the influence of new (uncharacterized) genes on vaccine-induced immunity. in turn, the identification and directed study of such genetic variants allows recognition, often at the molecular level, of the effects of differential binding, processing, and expression/presentation of antigenic viral peptides used in vaccine development, identification of the differential range of presented peptides (genetic restriction), altered secretion patterns (cytokines) in response to vaccines or vaccine adjuvants, altered transcription of important genes (signaling molecules) and gene products, altered binding of virus/antigens by membrane-based receptors (tlrs, other), differential receptor function, expression, and affinities, and the impact of epigenetics on vaccine-induced immune responses. we have utilized this knowledge in our own laboratory to create a research-oriented paradigm of ''discover-validate-characterize-apply," which may be used in new candidate vaccine development ( fig. ) [ ] . in this paradigm, we have been able to utilize vaccinomics approaches to discover genetic variants that are significantly associated with subsequent downstream immune responses, validate that such variants are indeed associated, then seek to characterize the mechanism whereby such effects occur and, finally, apply this knowledge-often in functional studies that confirm the effect on immunity. such knowledge can be exploited in developing immune strategies to enhance or circumvent genetic restrictions, for example, in triggering vaccine-associated immune responses, by ''reverse engineering" around a given genetic or other obstacle to generating protective immune responses. there are a growing number of studies reporting unbiased genome-wide assessments of genetic variation and its influence on adaptive (humoral and cellular) vaccine-induced immune responses across multiple viral and bacterial vaccines. for example, candidate and gwas immunogenetic and phamacogenetic studies have identified polymorphisms in hla, kir, mica, and btn genes associated with immune responses to pathogens causing disease in humans, such as hepatitis c [ ] , mycobacterium leprae [ , ] , human immunodeficiency virus [ ] , and measles [ ] [ ] [ ] . similar studies have identified novel genes impacting immune responses to vaccines, including hepatitis b, rubella, influenza a, smallpox, anthrax, and mumps [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our gene association studies of measles-mumps-rubella (mmr) vaccines have demonstrated that inter-individual variations in measles vaccine virus-induced humoral and cellular responses are significantly associated with polymorphisms in immune response genes and, together with hla alleles, explain % of the inter-individual variability in humoral response [ , [ ] [ ] [ ] . these findings, which illustrated the importance of key hla alleles in the adaptive humoral immune response to measles vaccine, led to the identification of naturally processed and presented measles-derived peptides isolated from specific hla polymorphisms associated with vaccine non-and hyper-response [ , ] . these peptides containing specific components (adjuvants and biodegradable nanoparticles) are now being utilized in a reverse-engineering strategy to develop peptide-based candidate measles vaccines. likewise, homan et al. have attributed diminished protection to differential hla presentation of t and b cell epitopes between vaccine and wild type strains of mumps virus [ ] . this diminished efficacy could theoretically be overcome by incorporating defined critical immunogenic peptides into an improved vaccine. tlr genes represent an important link between the innate and the adaptive immune system [ , ] . as an example, we have demonstrated that measles vaccine-induced humoral responses are significantly associated with coding polymorphisms in the tlr (rs ) and tlr (rs ) genes [ ] . for the rubella vaccine and tlr gene, a tlr gene snp rs was associated with rubella-specific gm-csf production [ ] . our recent mumps vaccine study has identified and replicated tlr snps associated with a % decrease in antibody titer, and a tlr snp associated with a % increase in t cell response (unpublished data). these data strongly suggest that robust tlr activation by measles, mumps, and rubella viruses is crucial for optimal vaccine response. supporting these findings is a study demonstrating that an inactivated mumps vaccine containing a protollin-based tlr / adjuvant is highly immunogenic in a mouse model; it led to superior total igg levels, higher neutralizing antibody titers, greater mucosal iga production, and enhanced th /th cytokine secretion [ ] . one potential application of this finding is to identify the specific and critical interactions between tlrs (and other genes) and virus, leading to advances in our knowledge of the precise mechanisms driving immunity to mmr vaccine. significant sex differences in humoral and cellular immune responses to vaccines are apparent [ , ] . additionally, local and systemic adverse rates are generally higher in females versus males. protective antibody responses are significantly higher in females than males after vaccination against influenza, yellow fever, measles, mumps, rubella, hepatitis a and b, herpes simplex (hsv) , rabies, smallpox, and dengue viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . sex-based differences in humoral immune responses are observed through various age groups [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , suggesting that sex steroid hormones are not the singular mediators of sex differences in humoral immune responses to vaccines [ , ] . this suggests that genetic, or other, factors may be an important driver of sex-related differences in humoral immune response [ ] . despite significant evidence of immune response differences between the sexes, for the most part, vaccine studies have not examined and analyzed immune response outcomes by sex [ , ] . in fact, little information is known about potential mechanisms for sex-based effects, which should be a priority for vaccine research studies. discovery of specific factors involved in sex-based differences in immune response may allow the identification of new correlates of vaccine immunogenicity. in a cohort of older (ages - ) and younger (ages - ) previously vaccinated individuals, the seasonal trivalent influenza vaccine induced > . -fold higher a/h n -specific hai antibody titers in women than men across both age groups [ ] . similarly, a study of standard seasonal influenza vaccine and high-dose influenza vaccine responses in a sex-balanced cohort of elderly subjects (ages - ) demonstrated significantly higher rates of seroconversion in females than in males [ ] ; however, no significant differences in antibody measures were found between males and females after seasonal influenza vaccination in another cohort of older adults (ages - ) [ ] . a study by furman et al. examining gene expression, serum cytokines/ chemokines, cell subsets, and phosphorylation events found several serum markers (lept, il- ra, crp, gm-csf, and il- ) to be more highly expressed in females than males after influenza vaccine [ ] . this same report used a systems biology approach to identify a gene cluster involved in lipid biosynthesis that is regulated by testosterone and significantly correlated with poor humoral responses following influenza vaccination in men [ ] . these data suggest that this gene cluster (e.g., genes involved in lipid metabolism) could be an important driver of sex-related differences in humoral immune response. this collective knowledge could substantially assist future personalized vaccine development efforts through the generation of new knowledge and the identification of targets and biomarkers that predict vaccine responses in specific populations (e.g., females vs. males; young vs. old; obese vs. lean). further research is needed to clarify the effects of sex on immune response. identification of molecular immune signatures of sex differences in innate and adaptive immune responses to vaccines may provide evidence necessary for additional efforts in designing personalized vaccination and vaccinomics approaches (i.e., in which males and females might be vaccinated differently using different doses or different vaccines) to provide equal protection while reducing side effects [ , , ] . a significant global public health issue is the aging of the population. as individuals age, immunosenescence develops, leading to poorer immune responses to vaccines. immunosenescence is an age-related dysregulation of the immune system due to ageassociated changes in innate and adaptive immune system components, which leads to impaired immunity and protection following immunization or infection [ ] [ ] [ ] . published data reveal that innate and adaptive immunity is decreased with age, but the systems-level mechanisms for these findings are unclear [ , ] , particularly in regard to influenza and other viral vaccine responses where the morbidity, mortality, and associated healthcare costs are greater in older individuals [ ] . major signs of innate immune dysfunction commonly observed in the elderly include, but are not limited to, altered cytokine secretion; decreased nk cell activity; reduced tlr expression; and a chronic inflammatory state (elevated levels of il- b, mcp- , tnf-a, and serum il- ) known as ''inflamm-aging" [ , [ ] [ ] [ ] . age-related humoral immune dysfunction, for example, might be overcome through optimal stimulation of innate and/or th cell-specific genes, which may be different in males and females. for example, adjuvanted zoster subunit vaccine (hz/su) reduced the risks of herpes zoster, and postherpetic neuralgia in immunocompetent persons years of age and older [ ] . this hz/su vaccine contains varicella zoster virus glycoprotein e and a novel as b adjuvant system aimed to improve and preserve with age zoster-specific cd + t cell responses [ ] . a tlr agonist gla-se (glucopyranosyl lipid adjuvant formulated in a stable emulsion) has been shown to enhance th responses to influenza vaccine in older adults [ ] , suggesting a potential mechanism for targeting innate receptor agonists (e.g., tlrs) that enhance innate immune responses against influenza. given the substantially diminished efficacy of influenza and other vaccines with age and the importance of developing improved vaccines [ ] , data from vaccinomics studies could be used to inform directed and rational development of next-generation influenza vaccines-potentially circumventing immunosenescence-related factors. systems biology approaches provide a unique opportunity to identify biomarkers likely to be involved in immune responses to vaccination [ ] [ ] [ ] [ ] , , ] . fourati et al. applied a systems vaccinology approach to examine gene signatures and molecular pathways of age-related hyporesponse to hepatitis b vaccine (hbv) in naïve older adults [ ] . they observed the b cell signaling pathway (and higher memory b cell frequencies) and inflammatory pathway (and increased frequencies of activated pro-inflammatory innate cells) were strongly correlated with higher and low antibody responses to hbv, respectively. this signature, including serum cytokine profiling and flow cytometric correlates of response, predicted the antibody response to hbv with up to % accuracy [ ] . this study demonstrates that a systems biology approach can be used to predict age-related immune response to vaccination. obesity is another major public global health concern. in the us, % of adults and nearly % of children and adolescents are now overweight or obese [ ] . weight gains across all countries have been demonstrated to be associated with increasing socioeconomic status. obesity has been shown to be a predictor of impaired immunogenicity (e.g., decreased antibody response) to hepatitis b, tetanus toxoid, rabies, and influenza vaccines [ ] [ ] [ ] [ ] , and as such can be considered a marker, or state, of immunosuppression at its extremes. these data suggest that obesity is correlated with poorer vaccine-induced immune responses in humans, and further research is required to understand the immune mechanisms that are altered in obesity. as individuals age, their circulating leptin levels rise with a concomitant reduction in leptin signaling; this results in leptin resistance, which is a finding associated with obesity [ ] . leptin resistance has been shown to adversely affect the immune response in obese subjects, including responses to influenza virus [ , ] . for example, obese individuals demonstrate decreased activation of influenza-specific cd + t cells compared to healthyweight persons, including decreased production of ifn-c and granzyme b, suggesting that influenza vaccination may not be as effective in the obese population as in healthy-weight individuals [ ] . given only moderate seroprotection of influenza and other vaccines in obese older adults [ ] , and the importance of developing improved influenza vaccines [ ] , systems biology studies designed to identify the mechanisms for improved immune response are needed. in fact, data from vaccine studies could be used to inform directed and rational development of personalized vaccines that optimally stimulate innate and adaptive immune responses in males and females and overcome immune deficiencies induced by obesity [ ] . careful vaccine studies comparing lean and obese persons could provide foundational data used to improve vaccine-induced protection in the obese, a subpopulation with an elevated risk for serious vaccine-preventable illnesses and suboptimal vaccine-induced protective responses [ ] . adversomics utilizes tools-much like those used in vaccinomics-to identify, characterize, and predict adverse, or maladaptive, immune responses to vaccines [ , , ] . the promise of adversomics would be to develop or identify either predictors or immune signatures of maladaptive immune responses that lead to harm rather than benefit, and to better understand the generation and mechanisms of such maladaptive immune responses. we have asked the question, as have other scientists, ''does it make sense in the st century to give the same vaccine, dose, and at the same frequency to everyone, regardless of age, weight, gender, race, genotype, and medical condition?" for example, we give adult males and females the same dose, and the same number of doses of vaccines, ignoring the findings that females nearly always have superior humoral immune responses to males for all vaccines studied, and yet experience significantly more side effects-more adverse events, of greater duration, and of higher intensity [ , , ] . while the field is young in implementation, research has already revealed associations between specific genes or snps and adverse immune outcomes. for example, associations between cytokine gene expression and fever after smallpox vaccine have been identified [ ] . other studies have demonstrated correlations between smallpox vaccine-induced fevers and il a and il snps [ ] . other smallpox vaccine-induced adverse events such as fever, rash, and enlarged lymph nodes have been significantly associated with mthfr, irf , and il snps haplotypes [ ] . while smallpox vaccine is not used in the general population, such studies stand as examples of the usefulness of vaccinomic approaches. finally, other recent studies have identified generic fever gene networks (tnfa) after vaccine administration [ ] , and relationships between mmr vaccine administration and snps in ifi l, cd , scn a, a, and tmem (ano ) genes [ ] . despite the tremendous success of vaccines, vaccinologists face several current challenges, including difficulty in developing vaccines for hypervariable viruses (hiv, rhinovirus, hepatitis c virus, coronavirus) and complex pathogens (malaria, mycobacterium tuberculosis); newly emerging pathogens, such as zika virus (zikv); complications imposed by aging and immunosenescent populations; inadequate understanding of the neonatal and newborn immune systems; increasingly immune deficient or immunocompromised populations due to hiv, cancer, or medications; sexbased differences in vaccine response and adverse-event rates; enhanced scrutiny of vaccine safety; and as noted global increases in age and weight. in addition, vocal and active anti-vaccine groups whose messages are not easily countered by facts or scientific studies have materially and detrimentally affected vaccine coverage rates [ ] [ ] [ ] . vaccinomic approaches can be utilized to better understand these issues; this information can then be used to inform new approaches, new understandings, and new vaccine candidates. just as new technologies have created exciting new opportunities in personalized medicine, they have brought with them novel challenges in addition to those mentioned above. in order for the full potential of personalized vaccines to be achieved, we must overcome additional challenges, such as the need for the following: larger genotype:phenotype datasets (often in the many thousands to ten thousands) integrating increasingly diverse high-throughput, highdimensional data types biomarkers that can reliably distinguish which product patients receive based on the likelihood of their response or an adverse side effect vaccines with different mechanisms of action may require a move away from humoral correlates of protection for licensure; in this regard, correlates of protection based on cellular immune outcomes are likely to play an important role in future vaccines more sophisticated biostatistical and bioinformatics approaches that can identify patterns and causative networks within terabyte levels of extremely high dimensional data types from the economic side: methods of technology transfer and funding mechanisms to move novel vaccines developed through vaccinomic approaches into low and middle-income countries who often most need specific vaccines (malaria, others) we have seen the shift from ''vaccinology . ," which is the empirical ''isolate-inactivate-inject" paradigm, to ''vaccinology . "-the use of recombinant technology and novel adjuvants. however, even this paradigm is limited by our incomplete mechanistic understanding of adjuvants and innate immunity. as we adopt approaches such as those listed above, we envision a movement of the field into an era of ''vaccinology . ," during which we expect to see the use of vaccinomics and systems-level approaches to develop new vaccines; innovative vaccine-antigen packaging methods; and adjuvant development targeted at the innate response pathways best suited for a given pathogen. a common reaction to this paradigm of personalized vaccinology is questioning cost and economics. at one level, such considerations are simply ''too soon" in the development of the science to effectively answer. however, like progress being made in individualized medicine, it is likely that being able to provide the right vaccine to the right patient-for the right reasons and at the right dose-will lead to improved medical outcomes and reduced costs at the population level. personalized vaccinology is the goal of applying the concept of personalized medicine to vaccines. rapid strides in omics technologies and foundational work applying systems biology, computational immunology and reverse vaccinology have facilitated modern approaches to vaccine design and development enabling us to create vaccine formulations for new and re-emerging pathogens. egg-based influenza vaccines take > months to create. the recent licensure of cell culture-based influenza vaccines demonstrate that rapid, scalable processes can now be implemented in order to create vaccine against emerging influenza strains (e.g., h n , h n , h n , h n , h n ) within weeks [ ] and can be safely administered to individuals with egg allergies [ ] . the ebola outbreak in liberia, sierra leone, and guinea in provides an example of the need to rapidly develop vaccine candidates [ ] . dna vaccines, virus-like particle vaccines, and replicating/nonreplicating viral vector vaccines have all been created and tested. among the most promising are a replication-competent, recombinant vesicular stomatitis virus vector expressing the glycoprotein of ebola zaire (rvsv-zebov), [ ] a variety of adenovirusvectored vaccines expressing ebola glycoprotein, [ , ] a modified vaccinia virus ankara-based vaccine encoding the ebola zaire glycoprotein (mva-bn-filo), [ , ] and dna-based vaccinesone expressing glycoproteins from both zaire and sudan, and the other expressing the marburg glycoprotein [ ] . although the rvsv-based vaccine elicits high titers of neutralizing ab, it is contraindicated in children and those with compromised immune systems. viral vector vaccines present the problem of developing robust immunity to the vector as well as the target immunogen, limiting their usefulness to a single vaccination. the availability of vaccines in multiple vector backbones opens up the possibilities for prime-boost vaccination strategies for ebola, similar to those that have been applied to hiv, malaria, and tuberculosis [ ] [ ] [ ] [ ] . in this regard, a prime-boost regimen using the mva-based vaccine as the booster vaccination has shown considerable promise [ ] . another example of modern vaccine development being applied to a new pathogen can be seen with the response to zika virus. a purified, formalin-inactivated vaccine (zikv piv) has been developed by the walter reed army institute of research (wrair) [ ] and is being evaluated in several clinical trials (nct , nct , nct ), while other inactivated vaccines are in preclinical development [ ] . two variants of a plasmid dna vaccine containing the prm-env proteins have been developed by niaid and one of the formulations is currently in a phase i clinical trial (nct ) [ ] . inovio pharmaceuticals developed their own plasmid dna vaccine (also expressing prm-env), which is currently in two clinical trials (nct , nct ). rna-based vaccines [ ] and a variety of subunit and viral vector-based vaccines are also in development [ , , ] . dna and rna-based vaccines can be rapidly made at minimal costs compared to other formulations and are fairly stable, without the cold-chain requirements of live virus-based vaccines. subunit vaccines are typically safer than whole virus-based products, which represents an active area of investigation not only for pathogens with no existing vaccines, but also for improving on established vaccines. our group and others have identified pathogen-derived epitopes as preliminary steps in the development of safe, stable, and effective peptide-and protein-based vaccines for smallpox, influenza, measles, tuberculosis, staphylococcus, and myriad other viral and bacterial pathogens [ , [ ] [ ] [ ] [ ] [ ] . parallel efforts by different groups to create new vaccines result in a spectrum of potential products that can be uniquely tailored to specific population groups. live viral vaccines rapidly inducing robust immunity can be used in healthy individuals where time is of the essence (e.g., in outbreak scenarios), while inactivated or subunit vaccines can be used in vulnerable populations such as pregnant women or those with immunocompromising conditions, or in young children where the presence of maternal antibody interferes with whole virus vaccines. vaccines based on different viral vector backbones can be combined into effective primeboost regimens. vaccines with specific adjuvants may be most appropriate for the elderly in order to overcome immunosenescence, or in the very young in order to compensate for immune system immaturity. we, along with increasing numbers of other scientists, believe that personalized vaccinology will revolutionize the practice of vaccinology to the benefit of human health. as part of the development of this field of science, vaccinomics and adversomics will allow us to develop molecular immune signatures of adaptive and maladaptive immune responses to vaccines, develop early biomarkers of vaccine response in vaccine trials, identify who should get what vaccine and at what dose, and increase safety and public confidence in vaccines by reducing the likelihood of serious adverse events related to vaccines. in many ways, however, personalized vaccinology is most challenged by the difficulty in moving the field away from the post-wwii population-level paradigm of ''one dose of every vaccine for everyone," toward an individualized or personalized approach based on the unique factors relevant to a given individual. in his book, the structure of scientific revolutions [ ] , thomas kuhn recognized that ''we wrongly believe scientific progress is a process of linear accretion of knowledge, that science is predicated on the belief that the scientific community understands what the world is like, and that we suppress or resist 'fundamental novelties' because they are seen as subversive to our firmly held beliefs of what the world is like." later in his book, he suggests that ''new advances always have and always will reveal that science and medicine includes bodies of belief incompatible with beliefs we hold today, and that advancements come when we reject a time-honored scientific theory in favor of another incompatible with it." these cognitive biases have, in our opinion, been manifest in our discussions with scientific colleagues as we developed this field of science. schopenhaur, the german philosopher, suggested that new discoveries are at first ridiculed, then opposed, and finally accepted as self-evident. vaccinomics and adversomics appear to be moving from the ridiculed and opposed steps, and into the not-yet quite self-evident phase of the continuum. part of the challenge is that often the concept of personalized vaccinology suggests to the reader that a unique vaccine will be developed for each individual. while that is one tactic being used in the cancer-vaccine field, it is neither necessary nor practical for the prevention of infectious diseases. rather, the personalized vaccinology approach would suggest the development of specific vaccines based on factors that relate to overcoming the potential for poor immunogenicity and the potential for adverse events. an excellent example is influenza vaccines. a mere decade or so ago, only a trivalent injectable influenza vaccine was available. quadrivalent vaccines were unavailable. for with one exception, everyone received the same vaccine and dose, regardless of age, weight, immunosuppression state, etc. at the current time in the us, multiple influenza vaccines are available so that the right vaccine, for the right patient, can be given at the right time. for example, laiv (live attenuated influenza vaccine) can be used in younger subjects or the needle-phobic. high-dose or mf -adjuvanted vaccines can be chosen for the elderly. recombinant vaccines can be chosen for those with egg allergy, and so on. this is the approach that should be taken with all vaccines. in some cases it may mean merely adjusting the dose based on weight, gender, or age. in other cases it may mean utilizing an adjuvanted or non-adjuvanted vaccine based on immune status. other examples include the recently licensed mf adjuvanted influenza vaccine (fluad Ò ), which has demonstrably higher immunogenicity and efficacy than its nonadjuvanted counterparts, [ ] [ ] [ ] or the highly effective as adjuvanted zoster glycoprotein e vaccine, which does not contain live virus and may be more broadly suitable for administration to older individuals [ , ] . thus, the movement toward a new paradigm of vaccine practice, based on a personalized approach, is occurring in the st century based on new scientific knowledge, market demand, safety considerations, immunogenicity concerns, public health trends (age, obesity, other), and the simultaneous pull of individualized medicine in other medical arenas. the net result is likely to be higher vaccine coverage rates, increased public confidence in vaccines, improved immunogenicity and adverse event rates, and a reduction or elimination in the morbidity and mortality related to vaccine-preventable diseases. as a result, we anticipate a new era of personalized ''predictive vaccinology," whereby we abandon a ''one size and dose fits all vaccine approach" in order to design and develop new vaccines, and acquire the ability to make the following predictions for each individual: whether to give a vaccine based on likelihood of response (and perhaps need); the likelihood of a significant adverse event to a vaccine; and the number of doses likely to be needed to induce a protective response to a vaccine [ ] . current vaccine development is largely empirical. vaccines are tested by trial and error, are mass produced, and given to the entire population using the same antigen dose, route of administration, number of vaccinations, and at the same age. in contrast, the new vaccine-development paradigm begins with the ''discovery" of new knowledge by integrating unbiased, comprehensive analysis of the genome, transcriptome, proteome, metabolome, microbiome, and immunome-along with the assessment of multiple measures of immune function-in order to under-stand and evaluate perturbations of the immune system. findings are then ''validated" in replication cohorts or additional model systems. the new knowledge is then ''applied" to the creation of new vaccine formulations that can undergo additional testing to start a new round of ''discovery," or can move into clinical trials in order to develop vaccine products engineered to elicit (or avoid) specific effects on the immune system. each product is tailored to specific subgroups such that robust, protective immunity can be elicited in the old and young, lean and obese, or male and female, while avoiding inappropriate immune responses due to genetics, metabolism, race, gender, malnutrition, immunosuppression, and other host factors or underlying conditions. r ai , and contract no. hhsn c (n ai ). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. the authors would like to acknowledge the contributions of the centers for disease control and prevention (cdc), which provides financial support to the world health organization initiative for vaccine research (u ck ). dr. poland is the chair of a safety evaluation committee for novel investigational vaccine trials being conducted by merck research laboratories. dr. poland offers consultative advice on vaccine development to merck & co. inc., avianax, dynavax, novartis vaccines and therapeutics, emergent biosolutions, adjuvance, seqirus, and protein sciences. drs. poland and ovsyannikova hold three patents related to vaccinia and measles peptide research. dr. kennedy has received funding from merck research laboratories to study waning immunity to mumps vaccine. these activities have been reviewed by the mayo clinic conflict of interest review board and are conducted in compliance with mayo clinic conflict of interest policies. dissecting polyclonal vaccine-induced humoral immunity against hiv using systems serology cytometry by time-offlight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of cd + t cell phenotypes highresolution myogenic lineage mapping by single-cell mass cytometry metabolic phenotypes of response to vaccination in humans heterogeneity in vaccine immune response: the role of immunogenetics and the emerging field of vaccinomics vaccinomics, adversomics, and the immune response network theory: individualized vaccinology in the st century pharmacology, vaccinomics, and the second golden age of vaccinology a systems biology approach to the effect of aging, immunosenescence and vaccine response genetics and vaccines in the era of personalized medicine the weight of obesity on the human immune response to vaccination understanding immunosenescence to improve responses to vaccines vaccine immunogenetics: bedside to bench to population learning immunology from the yellow fever vaccine: innate immunity to systems vaccinology vaccine discovery and translation of new vaccine technology systems vaccinology: learning to compute the behavior of vaccine induced immunity additive effects of hla alleles and innate immune genes determine viral outcome in hcv infection role of hla, kir, mica, and cytokines genes in leprosy association of variants in bat -lta-tnf-btnl genes within p . region show graded risk to leprosy in unrelated cohorts of indian population immunogenetics of hiv disease the association of cd , slam, and cd cellular receptor gene snps with variations in measles vaccine-induced immune responses-a replication study and examination of novel polymorphisms associations between polymorphisms in the antiviral trim genes and measles vaccine immunity effects of vitamin a and d receptor gene polymorphisms/ haplotypes on immune responses to measles vaccine snp rs in hla-dpb loci as a major genetic determinant of response to booster hepatitis b vaccination: results of a genome-wide association study new genetic associations detected in a host response study to hepatitis b vaccine a genome-wide association study identifies polymorphisms in the hla-dr region associated with nonresponse to hepatitis b vaccination in chinese han populations polymorphisms in hla-dpb are associated with differences in rubella-specific humoral immunity after vaccination heme oxygenase- regulates the immune response to influenza virus infection and vaccination in aged mice genome-wide association study of antibody response to smallpox vaccine genome-wide genetic associations with ifngamma response to smallpox vaccine immunogenetics of seasonal influenza vaccine response human leukocyte antigens and cellular immune responses to anthrax vaccine adsorbed human leukocyte antigen and cytokine receptor gene polymorphisms associated with heterogeneous immune responses to mumps viral vaccine genetic polymorphisms associated with rubella virus-specific cellular immunity following mmr vaccination vaccinomics and personalized vaccinology consistency of hla associations between two independent measles vaccine cohorts: a replication study variability in humoral immunity to measles vaccine: new developments identification and characterization of novel, naturally processed measles virus class ii hla-drb peptides identification of class ii hla-drb * -bound measles virus peptides by d-liquid chromatography tandem mass spectrometry are cases of mumps in vaccinated patients attributable to mismatches in both vaccine t-cell and b-cell epitopes?: an immunoinformatic analysis toll-like receptors: critical proteins linking innate and acquired immunity toll-like receptors control activation of adaptive immune responses the role of polymorphisms in toll-like receptors and their associated intracellular signaling genes in measles vaccine immunity rubella vaccine-induced cellular immunity: evidence of associations with polymorphisms in the toll-like, vitamin a and d receptors, and innate immune response genes immunologic characterization of a novel inactivated nasal mumps virus vaccine adjuvanted with protollin the xs and y of immune responses to viral vaccines personalized vaccinology: one size and dose might not fit both sexes halfvs full-dose trivalent inactivated influenza vaccine safety and immunogenicity of a high dosage trivalent influenza vaccine among elderly subjects glycoprotein-d-adjuvant vaccine to prevent genital herpes gender effects on humoral immune responses to smallpox vaccine systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination immunogenicity and safety of yellow fever vaccination for hiv-infected patients atypical antibody responses in dengue vaccine recipients antibody responses and cross protection against lethal influenza a viruses differ between the sexes in c bl/ mice mechanisms of sex disparities in influenza pathogenesis reactogenicity and immunogenicity of an inactivated influenza vaccine administered by intramuscular or subcutaneous injection in elderly adults female children respond to recombinant hepatitis b vaccine with a higher titre than male sex-based biology and the rational design of influenza vaccination strategies sex-based differences in immune function and responses to vaccination sex bias in neuroscience and biomedical research immune cells have sex and so should journal articles gene signatures associated with adaptive humoral immunity following seasonal influenza a/h n vaccination vaccinomics and personalized vaccinology: is science leading us toward a new path of directed vaccine development and discovery? personalized vaccines: the emerging field of vaccinomics aging of the immune system: how much can the adaptive immune system adapt? the aging innate immune system understanding the immune response to seasonal influenza vaccination in older adults: a systems biology approach the aging of early b-cell precursors inflamm-aging. an evolutionary perspective on immunosenescence in vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection age-associated elevation in tlr leads to increased inflammatory responses in the elderly efficacy of the herpes zoster subunit vaccine in adults years of age or older efficacy of an adjuvanted herpes zoster subunit vaccine in older adults gla-se, a synthetic toll-like receptor agonist, enhances t-cell responses to influenza vaccine in older adults towards a sane and rational approach to management of influenza h n systems biology approach predicts immunogenicity of the yellow fever vaccine in humans systems biology of seasonal influenza vaccination in humans prevaccination inflammation and b-cell signalling predict age-related hyporesponse to hepatitis b vaccination prevalence of childhood and adult obesity in the united states obesity as a predictor of poor antibody response to hepatitis b plasma vaccine reduced tetanus antibody titers in overweight children incidence and variables associated with inadequate antibody titers after pre-exposure rabies vaccination among veterinary medical students association between obesity and vulnerability and serologic response to influenza vaccination in older adults the role of leptin in leptin resistance and obesity diet-induced obesity impairs the t cell memory response to influenza virus infection leptin and leptin-related gene polymorphisms, obesity, and influenza a/ h n vaccine-induced immune responses in older individuals obesity is associated with impaired immune response to influenza vaccination in humans leptin-based adjuvants: an innovative approach to improve vaccine response adversomics: the emerging field of vaccine adverse event immunogenetics adversomics: a new paradigm for vaccine safety and design cytokine expression patterns associated with systemic adverse events following smallpox immunization the immunogenetics of smallpox vaccination genetic basis for adverse events after smallpox vaccination identification of fever and vaccine-associated gene interaction networks using ontology-based literature mining common variants associated with general and mmr vaccine-related febrile seizures the clinician's guide to the anti-vaccinationists' galaxy trends affecting the future of vaccine development and delivery: the role of demographics, regulatory science, the anti-vaccine and consumer culture and vaccinomics understanding those who do not understand: a brief review of the anti-vaccine movement accelerated mass production of influenza virus seed stocks in hek- suspension cell cultures by reverse genetics cell culture-based influenza vaccines: a necessary and indispensable investment for the future ebola virus vaccines: where do we stand? a recombinant vesicular stomatitis virus ebola vaccine safety and immunogenicity of a chimpanzee adenovirus-vectored ebola vaccine in healthy adults: a randomised, double-blind, placebo-controlled, dose-finding, phase / a study safety and immunogenicity of a novel recombinant adenovirus type- vector-based ebola vaccine in healthy adults in china: preliminary report of a randomised, double-blind, placebo-controlled, phase trial safety and immunogenicity of novel adenovirus type -and modified vaccinia ankara-vectored ebola vaccines: a randomized clinical trial immunology and evolvement of the adenovirus prime, mva boost ebola virus vaccine safety and immunogenicity of ebola virus and marburg virus glycoprotein dna vaccines assessed separately and concomitantly in healthy ugandan adults: a phase b, randomised, double-blind, placebo-controlled clinical trial the prime-boost concept applied to hiv preventive vaccines control of a mucosal challenge and prevention of aids by a multiprotein dna/mva vaccine enhanced immunogenicity for cd + t cell induction and complete protective efficacy of malaria dna vaccination by boosting with modified vaccinia virus ankara enhanced immunogenicity of cd + t-cell responses and protective efficacy of a dna-modified vaccinia virus ankara prime-boost vaccination regimen for murine tuberculosis first inactivated zika vaccine trial vaccine development for zika virus-timelines and strategies prospects for a zika virus vaccine zika virus protection by a single low-dose nucleoside-modified mrna vaccination protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys preventative vaccines for zika virus outbreak: preliminary evaluation the identification of hla class ii-restricted t cell epitopes to vaccinia virus membrane proteins discovery of naturally processed and hla-presented class i peptides from vaccinia virus infection using mass spectrometry for vaccine development identification of mycobacterial rplj/l and rpsa/s proteins as novel targets for cd + t cells immunoinformatics analysis and in silico designing of a novel multi-epitope peptide vaccine against staphylococcus aureus high-density peptide arrays for malaria vaccine development the structure of scientific revolutions immunogenicity of intramuscular mf -adjuvanted and intradermal administered influenza enhanced vaccines in subjects aged over : a literature review enhanced and persistent antibody response against homologous and heterologous strains elicited by a mf -adjuvanted influenza vaccine in infants and young children the comparative effectiveness of adjuvanted and unadjuvanted trivalent inactivated influenza vaccine (tiv) in the elderly research reported in this publication was supported by the national institute of allergy and infectious diseases of the national institutes of health under award number u ai , r ai , key: cord- - o mryu authors: dhama, k.; mahendran, mahesh; gupta, p. k.; rai, a. title: dna vaccines and their applications in veterinary practice: current perspectives date: - - journal: vet res commun doi: . /s - - - sha: doc_id: cord_uid: o mryu inoculation of plasmid dna, encoding an immunogenic protein gene of an infectious agent, stands out as a novel approach for developing new generation vaccines for prevention of infectious diseases of animals. the potential of dna vaccines to act in presence of maternal antibodies, its stability and cost effectiveness and the non-requirement of cold chain have heightened the prospects. even though great strides have been made in nucleic acid vaccination, still there are many areas that need further research for its wholesome practical implementation. major areas of concern are vaccine delivery, designing of suitable vectors and cytotoxic t cell responses. also, the induction of immune responses by dna vaccines is inconclusive due to the lack of knowledge regarding the concentration of the protein expressed in vivo. alternative delivery systems having higher transfection efficiency and the use of cytokines, as immunomodulators, needs to be further explored. recently, efforts are being made to modulate and prolong the active life of dendritic cells, in order to make antigen presentation a more efficacious one. for combating diseases like acquired immunodeficiency syndrome (aids), influenza, malaria and tuberculosis in humans; and foot and mouth disease, aujesky’s disease, swine fever, rabies, canine distemper and brucellosis in animals, dna vaccine clinical trials are underway. this review highlights the salient features of dna vaccines, and measures to enhance their efficacy so as to devise an effective and novel vaccination strategy against animal diseases. the property of naked dna to get transfected to mammalian cells in vivo was first reported by ito ( ) ; and three decades later, the concept of dna vaccine was evolved by wolff et al. ( ) , when they administered a recombinant bacterial plasmid dna to obtain the expression of β-galactosidase gene in mice. this paved way for the development of nucleic acid based vaccination, an effective way for the in vivo expression of desired protein to initiate immune response (oshop et al. ; liu ) . the application of dna immunization as a new generation vaccine has been well studied since its invention, and a variety of such vaccines have undergone clinical trials, in veterinary practice (dunham ; oshop et al. ; babiuk et al. ; babiuk et al. ). the dna vaccines elicit desired immune responses viz. cell mediated immunity (cmi) and humoral immune response (hir); and it is much easier for their manipulation using recombinant dna techniques and production in bacteria using fed-batch fermentation (liu ; liu et al. ) . as an effective vaccine, plasmid dna have a gene encoding a protective antigen of a pathogen, which when injected into host, is transcribed and translated, to induce a specific immune response. the dna vaccines, described as genetic immunization to elicit a protective immune response, have been further improved by exploiting various gene delivery methods, cytokine adjuvants and prime-boost (dna vaccine priming and recombinant protein boosting) approaches (sharma and khuller ; jiang et al. ). dna vaccines have several advantages, which include simplicity of manufacture, biological stability, cost effectiveness and safety, ease of transport in lyophilized form and the ability to act in presence of maternal immunity. besides, different genes can be combined simultaneously, making it possible to develop multivalent vaccines. the demerits of dna vaccines, of theoretical levels and not yet proven are; integration into host genome, activation of proto-oncogenes, inactivation of tumor suppressor genes and the possibility of generating anti-nuclear antibodies (sharma and khuller ; dunham ) . however, as the merits of dna vaccines outnumber the hypothesized demerits, presently they have moved towards second stage clinical trials with promising results, for human diseases like acquired immunodeficiency syndrome (aids), herpes infections, rabies, ebola, tuberculosis, malaria, and leishmaniosis. however, a commercial product has not reached the market yet, due to the safety concerns raised by the international regulatory organizations. regarding veterinary practice, the last few years have seen numerous trials of dna vaccines against various animal diseases like foot and mouth disease (fmd) and herpes virus infection in cattle, aujeszky's disease and classical swine fever in swine, rabies and canine distemper in canines, and avian influenza, infectious bronchitis, infectious bursal disease and coccidiosis in birds (oshop et al. ; dunham ; ding et al. ; gupta et al. ; patial et al. ). one of the distinct advantages of the dna vaccines is the possibility of differentiating infected from the vaccinated animals (diva), for effective disease eradication programs. the utility of 'marker' dna vaccines has been reported for diseases like fmd and avian influenza (lee et al. ; grubman ) . even though, dna vaccines has ushered a new era in veterinary vaccinology, the potential of the vaccine to develop higher levels of immune response, has to be further improved. keeping in view of the appealing features, and ease of generation of dna vaccines, in the present review, authors have meticulously portrayed the salient features of dna vaccines, ways to improve its efficacy, and their potential applications in veterinary practice. salient features of dna vaccines and strategies to improve vaccine efficacy dna vaccines, generated using plasmids, include a gene encoding target antigen under the transcriptional control of an effective viral/eukaryotic promoter, along with a polyadenylation signal sequence (poly-a) and a bacterial origin of replication ( fig. ) (gurunathan et al. ) . the commonly used promoter has been derived from cytomegalovirus (cmv). the poly-a provides stability and effective translation; and the antibiotic resistance gene facilitates selection of bacteria (gurunathan et al. ; sharma and khuller ; liu ; brandsma ) . for complete optimization, the plasmid should have kozak sequence (gcca/gcc) upstream of initiator codon, and an enhancer, down stream of the poly-a signal (gurunathan et al. ) . as a vaccine, the amount of plasmid dna required for im administration is - μg in mice, - μg in small animals and . - . mg in large animals and humans; but, while using 'gene gun', only one hundredth of this amount is required (dunham ) . if properly optimized, the recombinant plasmids are capable of expressing the desired antigen in vivo (dunham ; babiuk et al. ; babiuk et al. ) . but, to elicit an effective immune response, the protein should undergo post-translational modification and retain their tertiary structure. after in vivo generation, the antigenic peptides are processed and presented by professional antigen presenting cells (apcs), like dendritic cells, by getting primed through direct transfection or by obtaining proteins from myocytes via cross presentation (fig. ) (fu et al. ) , to further allow presentation via both mhc i and ii, to induce cellular and humoral showing essential features of a dna vaccine construct. transcription unit comprises of a promoter, desired immunogenic or protective gene and a polyadenylation signal sequence. a bacterial origin of replication (ori) and an antibiotic resistance gene are also incorporated in the vector back bone to permit growth and selection of the plasmid in bacteria immunity. however, in some cases, dna vaccines have failed to produce measurable antibodies even when the host got protected, suggesting a major role for cellular immune responses (seo et al. ; kodihalli et al. ) . in addition, the potential of dna vaccines to act in presence of maternal antibodies is remarkable, due to the ability to provide a durable source of the antigen by dendritic cells. similarly, when administered with cationic liposomes, they enhance mucosal immunity, to protect the host from respiratory or enteric infections. besides, researchers have suggested the immunogenic properties of the plasmid itself, by virtue of having cpg motifs, functioning as adjuvant and up-regulator of natural killer (nk) cells (krieg ) . for improving dna vaccines, efforts have been made to enhance their efficacy, through efficient vectors, and vaccine delivery systems that combines delivery route and formulation. in animals, dna vaccines are found less effective due to host impedance, improper transfection and low level of expression. but, various delivery formulations and route could enhance the immune response. delivery methods like suppositories (loehr et al. ) , needle free injector system (van rooij et al. ) , mucosal delivery (barnes et al. ) and topical application (oshop et al. ) , have been found useful. vaccination professional apcs like dendritic cells and macrophages receives the secreted antigens (cross presentation) from transfected somatic cells or directly get transfected. they process and present the antigenic peptides to major histocompatibility complex (mhc) class ii molecules for helper t cells, which releases a variety of cytokines to augment activation of various cells of immune system. activation of cytotoxic t lymphocytes occurs by degraded antigenic peptides that are associated with mhc class i molecules. these two mechanisms help in the generation of cellular immune responses. for humoral or antibody responses, b lymphocytes recognize and respond to antigens that are present extracellularly, or as secreted antigens using gene gun, help in direct transfection of dendritic cells, favoring effective antigen presentation (dunham ; ulmer et al. ) . also, by using cationic lipid complexes and cytokine adjuvants, the efficacy could be significantly improved (min et al. ; stevenson ; manoj et al. a; dhama et al. ). however, the major lacuna that still persists is the lack of simultaneous generation of cmi and hir. hence, researchers have evolved various options to improvise dna vaccines for increasing cmi and hir together. addition of a eukaryotic secretory signal sequence have been found to improve the activity of cytotoxic and helper t cells. also, linking of ubiquitin molecule to enhance the proteasome-based degradation, and the use of toll like receptor (tlr) adapter molecule (myd ) along with the desired gene, may elicit a strong cmi and hir. further, the use of l-selectin or cytotoxic t lymphocyte antigen (ctla- ) ligands help better antigen targeting to the immune cells (chaplin et al. ) . similarly, targeting of the dendritic cells by heat shock proteins (hsp); microparticulate formulations, and complexing with nonionic block co-polymers, polycations or cochleates, may also increase vaccine efficacy (manoj et al. a) . targeted intra-cellular delivery of plasmid dna can be further achieved using intracellular bacteria and viruses (liu ; daudel et al. ). recently, prime-boost regimens and ways to increase the functional life of dendritic cells have been explored (ulmer et al. ; tsen et al. ). also, 'electroporation', few days prior to vaccination, has been found to force plasmid into cells or promoting influx of inflammatory cells like macrophages in myocytes (babiuk et al. ; peng et al. ). further, nanoparticle-mediated plasmid delivery has been suggested for augmenting the immune responses (jiang et al. ). veterinary vaccinology is a rapidly developing field and currently, vaccines are not only used for the prevention of diseases in animals, but also to help solve public health crisis, by effectively checking emerging or re-emerging pathogens of zoonotic significance. advancement in science and technology, together with improved knowledge in immunology, microbiology and recombinant technology has played pivotal roles in introducing novel ideas in vaccinology (babiuk et al. ; liu et al. ) . shams ( ) , has pointed out that subunit vaccines, dna vaccines and vectored vaccines are rapidly gaining acceptance as new generation animal vaccines. the last few years have seen the development of nucleic acid vaccines against many diseases like classical swine fever, aujeszky's disease, rabies, foot and mouth disease, brucellosis, bovine tuberculosis, equine herpes infections, avian infectious bronchitis, avian influenza, infectious bursal disease, chlamydiosis and avian coccidiosis (table ). dna vaccines have promoted a revolution in the concept of vaccination, which has been previously dominated by inactivated or live vaccines. among many advantages, dna vaccines also provide diva strategy, and hence vaccine-induced herd/flock immunity can be differentiated for effective sero-surveillance (lee et al. ) . hence, the latest research development in the field of nucleic acid-based vaccination strategy for preventing various bacterial and viral diseases of domestic animals as well as poultry has been discussed further in detail. bovines genetic immunization approach against bacterial diseases of bovines offers attractive possibilities for rapid and effective vaccine development. against brucellosis, an im administered l /l gene has been reported to result in intracellular expression of the immunodominant l /l protein (kurar and splitter ) . brucella abortus glyceraldehyde- -phosphate-dehydrogenase (gdph), a t and b cell reactive protein, which could induce partial protection when co-administered with il- ; and the copper-zinc super oxide dismutase (sod) antigen of b. abortus has also been utilized for the generation of an effective dna vaccine (rivers et al. ) . against mycobacterial infections in cattle, huygen ( ) has explained the utility and feasibility of the dna vaccine approaches. dna vaccine based on myobacterium bovis protein mpb- when tested in mice has shown to elicit protective immune responses (chambers et al. ) . the use of costimulatory molecule cd along with esat- gene has been suggested to enhance the immunogenic properties of mycobacterial dna vaccines. further, m. bovis ag b gene has been found suitable while incorporating in plasmid vector due to its ability to induce a th type of immune response (teixeira et al. ). aside to this, recently, dna vaccines used in combination with bacillus calmette guerin (bcg) elicited superior protection during challenge studies (cai et al. dhama et al. ) . against viral diseases of bovines, the earliest reports suggest the use of a gene encoding the vp protein of bovine rotavirus (brv), found effective in stimulating a th -like immune response (suradhat et al. ) . later, brillowska et al. ( ) reported the generation of an effective cellular immune response with the plasmid encoding envelope glycoprotein gp and transmembrane glycoprotein gp of the bovine leukemia virus (blv). also, dna vaccine encoding the fusion (f) gene of bovine respiratory syncytial virus (brsv) has been found to induce protection against the infection in calves. besides, several workers have developed successful dna vaccine strategies against bovine herpes virus- (bhv- ) infection (loehr et al. ; gupta et al. ; castrucci et al. ) . gupta et al. ( ) reported that dna immunization with gc gene of bhv- could induce neutralizing antibody and lympho-proliferative responses in bovines. also, bhv- gb gene along with il- has been suggested to enhance the ctl responses. further, suppositories containing plasmid coding for gd gene of bhv- induced mucosal immunity (loehr et al. ) ; and bovine cd co-stimulatory molecule linked to gd gene enhanced the immune responses (manoj et al. b ). it has also been suggested that gd gene confers higher protection when compared to gc gene. further, the intercellular trafficking ability of bhv- vp protein has been utilized to improve the efficacy of a dna vaccine encoding gd gene (zheng et al. ) . nucleic acid vaccine that could protect the cattle against bovine viral diarrhea virus (bvdv) infection has also been developed. plasmid dna, expressing the bvdv type glycoprotein e , induced virus-specific neutralizing antibodies (nobiron et al. ) . non-structural protein ns could also be used for inducing humoral immunity against bvd. further, liang et al. ( ) found that the dna prime boost regimens were effective for preventing bvd in cattle when compared to the administration of dna vaccine or protein vaccine alone. vp based dna vaccines are being utilized for developing effective vaccines against foot and mouth disease (fmd) (dong et al. ) . plasmid dna encoding the fmdv vp protein followed by boosting with a vp peptide conjugate resulted in production of high titers of neutralizing antibodies, suggesting that prime-boost strategy could be a key factor for the success of dna vaccine against fmd (jin et al. ) . also, the use of il- along with vp gene may provide enhanced immune response (shao et al. ) . recently, a microparticulate based dna vaccine has been developed that codes for the t and b cell epitopes of vp of the fmdv (wang et al. ) . dna vaccines are also being reported against some rickettsial diseases and ectoparasites of bovines. vaccine containing the gene of a major surface protein, msp b, of anaplasma marginale, offered partial protection against challenge infection (de andrade et al. ) . dna constructs involving orf of genes, cpg and groel and groes of ehrlichia ruminantium could partially protect cattle against "heart water" disease. the potential of dna immunization with plasmid encoding antigen bm to induce humoral and cellular immune responses against the tick, boophilus microplus, has also been studied (ruiz et al. ). ovines and caprines nucleic acid vaccines have been developed that could confer protection to the common bacterial diseases of sheep and goats. dna vaccination with genetically detoxified phospholipase d of corynebacterium pseudotuberculosis, linked with ctla- , protected sheep against caseous lymphadenitis (chaplin et al. ) . in case of anthrax in sheep, the protective antigen (pa ) gene of bacillus anthracis has been employed for developing highly promising dna vaccine (hahn et al. ). paratuberculosis or johne's disease, caused by mycobacterium avium subsp. paratuberculosis, has been successfully controlled by using plasmids coding mycobacterial heat shock protein antigen (hsp- ) (sechi et al. ) . against brucellosis, administering dna vaccine that encode brucella melitensis outer membrane proteins (omp), invasion protein b (ialb), periplasmic protein (bp ) and trigger factor (tf) have been found to induce significant immune responses, which could pave way for the effective control of brucellosis in goats gupta et al. ) . for preventing viral diseases of the small ruminants, dna vaccines have been developed that could protect against diseases like caprine arthritis-encephalitis (cae), foot and mouth disease (fmd), visna-maedi and rift valley fever. a plasmid expressing cae viral envelope gene with prime-boost vaccination strategy has shown to induce protective responses in goats (cheevers et al. ) . if using gene gun-based mucosal dna immunization against visna-maedi in sheep, the plasmid expressing the envelope gene of the virus in combination with ifnγ gene, is expected to give significant protection and also could restrict virus replication. for preventing fmd infection, niborski et al. ( ) reported the development of a vp -based nucleic acid vaccine and found that it was more effective when used along with poly lactide co-galactide (plg) formulation. against rift valley fever, the viral glycoprotein gene of rift valley fever virus (rvfv) when used as dna vaccine have been found to induce neutralizing antibodies during experimental studies. recently, babiuk et al. ( ) reported that a single dna vaccination with the hepatitis b virus surface antigen gene (hbsag), in combination with electroporation, approached the efficacy of the commercial subunit vaccine in maintaining long-term protective serum antibody titers against hepatitis b virus in sheep. dna vaccines are also being developed against many ecto-and endo-parasites of small ruminants. vaccination of sheep with a plasmid for the boophilus microplus antigen bm co-administered with plasmid encoding for ovine granulocyte monocyte-colony stimulating factor (gm-csf) provided significant levels of protection against b. microplus infestation ). an effective recombinant plasmid, encoding the kda sporozoite surface protein of cryptosporidium parvum has been developed for goats, which provided protective maternal immunity to offsprings (sagodira et al. ). also, schistosoma japonicum genes (sj gst and sj ) in dna vaccine formulation offered partial protection as evidenced by a reduction in parasite counts (shi et al. ) . similarly, a nucleic acid vaccine against tapeworms, using the w gene of taenia ovis, a protective membrane bound antigen, showed optimum humoral response (drew et al. ) . swine regarding the dna vaccines developed against bacterial diseases in swine, very few research works have been reported. against swine enzootic pneumonia (sep), caused by mycoplasma hyopneumoniae, plasmid dna coding the heat shock protein gene (p ) should be a suitable candidate as it is capable of inducing both th and th immune responses. against sep, the capability of p adhesin repeat region of m. hyopneumoniae to produce immunogenicity in mice in dna vaccine formulation has also been described (chen et al. ) . however, considerable research has been directed towards developing dna vaccines to prevent swine fever, fmd and psuedorabies infection in pigs. plasmid constructs have been developed that could protect swine against the classical swine fever (csf), which causes significant losses to the pig industry in many asian and european countries (wienhold et al. ; andrew et al. ) . immunization of pigs with a plasmid expressing the complete e protein of classical swine fever virus (csfv), conferred protection against viral challenge; and the co-delivery of il- , il- and cd further enhanced the protective responses (wienhold et al. ; andrew et al. ; li et al. ) . regarding fmd in swine, dna vaccines encoding vp gene of o, a and c strains of fmd virus showed protection against the disease when administered using 'gene gun' (benvenisti et al. ) . two vp epitopes (amino acid residues - and - ) have also been found suitable to elicit both fmdv-specific t cell proliferation and neutralizing antibodies (wong et al. ) . researchers have also developed successful dna vaccines using prm and envelope (e) genes for japanese encephalitis virus and nucleoprotein (n) gene for transmissible gastroenteritis virus. for constructing dna vaccines against psuedorabies (aujesky's disease), the immunogenic viral protein genes such as gb, gc and gd, are often considered (van rooij et al. ; gerdts et al. ; dory et al. ) . plasmids encoding these glycoproteins, when co-injected with cpg motifs, improved the humoral immune response and provided better clinical protection against lethal psuedorabies infection (dory et al. ) . also, it has been suggested that gb and gd genes primes the immune system efficiently even in the presence of maternal antibodies. the utility of dimethyl-dioctadecylammonium (dda), as an adjuvant to psuedorabies dna vaccine, has also been reported. for the prevention of porcine reproductive and respiratory syndrome (prrs) in swine, dna immunization strategies could be formulated utilizing the viral orf region that codes for a major envelope glycoprotein gp . for endo-parasitic infestations like taeniasis (cysticercosis), recently a dna vaccine using taenia solium b antigen has been developed (guo et al. ) . canines during the last couple of decades, immunoprophylactic agents have been developed that have greatly reduced the incidence of infectious diseases of pet animals. in canines, even though live vaccines have been found superior in efficacy, it is expected that new generation vaccines may dominate the market in the near future. focus has been directed for developing dna vaccines to eliminate the bacterial diseases like leptospirosis and lyme disease. leptospirosis, being a zoonotic disease is being given due attention, and many works has suggested the generation of successful vaccines based on its endoflagellar (flab ) gene. there exist cpg motifs within the flab gene, which could give the dna vaccine an additional immunostimulatory property, with out the use of adjuvants (dai et al. ) . also, hemolysis associated protein (hap ) gene has been found to elicit protection against leptospirosis when encoded in plasmids. against borrelia burgdorferi, a spirochete causing lyme's disease, dna vaccine experiments are under trial stages. the role of outer surface protein genes (ospa and ospc) of b. burgdorferi to elicit protective immune responses when administered as dna vaccines has also been explored. among the viral diseases affecting dogs, the important ones are rabies, canine distemper and parvoviral infections, many studies have been conducted to analyze the utility of dna vaccines. initially, it was against the deadly rabies, the first nucleic acid vaccine was successfully developed (xiang et al. ) . after this, it was jiang et al. ( ) who reported a plasmid dna vaccine to protect the canine population from parvovirus infections, utilizing vp gene of canine parvovirus (cpv). later, by using vp gene of cpv, a successful dna vaccine was developed (gupta et al. a) . now, most of the research has been centered on for developing dna vaccines against rabies and canine distemper (cd). the advantage of plasmid-based vaccine against rabies is that it is a valuable alternative for the mass production of cheaper rabies vaccine when compared to the cell culture based ones. a dna vaccine encoding the rabies virus glycoprotein (g) has been found to yield stronger and more durable virus neutralizing antibody titers in dogs, which has been proven beyond doubt by many researchers (rai et al. ; gupta et al. b) ; and the role of trans-membrane domain of the rabies virus glycoprotein in assisting humoral immune response has been experimentally demonstrated (gupta et al. ). further, the utility of a chimeric glycoprotein gene, constructed from different lyssa viruses, can be used as a multivalent vaccine. also, some works have suggested the advantage of dna vaccine administered intra-dermally in ear in inducing long lasting protective titer against rabies. recently, a bicistronic multivalent dna vaccine has been developed by patial et al. ( ) , which comprised of rabies virus (g) and parvovirus (vp ) genes for inducing neutralizing antibodies against both viral pathogens. it was sixt et al. ( ) who first developed a plasmid vaccine against canine distemper (cd) that encoded the hemagglutinin (ha) and fusion protein (f) gene against this highly infectious and lethal disease of pups. also, plasmids containing the nucleocapsid (n), f and ha genes of a virulent cd virus strain can be developed which could elicit strong humoral and cellular immune responses (jain et al. ) . further, immunization with n protein-based dna vaccine against cd to elicit serum n-specific igg responses and thereby generating satisfactory protection should also be considered as a viable option. similarly, the use of ha and f protein genes in a cationic lipid formulation should work well for protecting the pups from clinical disease even in the presence of high titers of maternally derived antibodies. dna vaccines are also being attempted for canines, targeting protozoan diseases (fukumoto et al. ) . the gene coding the babesia canis protein p has been found to induce protective immunity against canine babesiosis, while the p /lack antigen gene has been used against leishmaniosis. likewise, plasmid dna encoding a xenogenic tyrosinase can also function as tumor vaccine for prolonging the survival of dogs in cases of malignant melanoma. equines: the increasing international movement of horses and the relaxation of regulations have resulted in an increased incidence of equine infectious diseases. vaccination, along with management measures has become the primary method for the effective control rhodococcal infections, equine influenza, african horse sickness and herpes infections. the advent of recombinant technology has encouraged the development of new generation vaccines such as live-vectored vaccines and dna vaccines (minke et al. ). among bacterial diseases, dna vaccines have been generated to protect the foals from rhodococcus equi, which causes pyogranulomatous broncho-pneumonia. nucleic acid vaccine, expressing the virulence associated protein (vapa) gene of r. equi, induced an anamnestic response and has been found capable in generating specific igg antibodies (vanniasinkam et al. ) . against the equine herpesvirus- (ehv- ), a viral pathogen of horses causing respiratory, reproductive and neurological problems, the role of plasmid dna encoding the envelope glycoprotein d (gd) to induce humoral response, has been suggested; and the administration of gm-csf along with such vaccines significantly enhanced virus neutralizing antibody responses to ehv- (minke et al. ) . for equine influenza (ei), lunn et al. ( ) recorded complete protection of the experimental ponies during challenge after administering dna vaccine encoding hemagglutinin (ha) gene of ei virus. aside to this, administration of il- along with ha gene enhances vaccine efficacy and protection (olsen ) . to prevent the west nile virus infection in horses, the envelope protein genes (prm and e) have been incorporated in dna vaccine formulation to elicit satisfactory protection (hall and khromykh ) . similarly, giese et al. ( ) developed effective dna vaccines encoding the orf's ( and ) of equine arteritis virus (eav). development of nucleic acid vaccines has also been attempted for african horse sickness (vp gene) as well as against vesicular stomatitis (envelope gene). poultry historically, inactivated whole viruses with various adjuvant systems or live vaccines have been used for the successful prevention of various bacterial and viral diseases of poultry. however, the development of naked dna immunization as third generation vaccines has been well studied and recently a variety of such vaccines are in clinical trials for their use in poultry (oshop et al. ; dhama et al. ) . a plasmid dna vaccine encoding the enterotoxigenic escherichia coli k fimbrial protein elicited satisfactory protection during e. coli challenge (cho et al. ) . similarly, plasmids expressing the major outer membrane protein (momp) of chlamydophila psittaci serovar a and d strains have been found to induce protective immunity with significant reduction in clinical symptoms (vanrompay et al. ) . also, higher levels of protection against c. psittaci could be obtained while administering interferon gamma (ifn-γ) or vitamin d, along with dna vaccines. besides, dna vaccines have been developed against major viral infections of poultry like avian influenza, utilizing the ha gene of the virus (kodihalli et al. ; lee et al. ) . similarly, a vaccine encoding fusion (f) and haemagglutinin (hn) gene induced higher level of antibodies against newcastle disease (ndv) in chickens (loke et al. ) . for marek's disease, dna vaccine containing the clone of virulent serotype- mdv has been found useful during challenge infection in birds. vaccination with a mutated, nononcogenic v-src gene construct, derived from avian leucosis virus (alv), induced cytotoxic t-lymphocytes (ctl) to protect birds from tumors. protective utility of plasmid coded n protein gene of infectious bronchitis virus (ibv) has also been reported for which dna vaccines expressing the s glycoprotein of ibv has been suggested (seo et al. ) . against infectious bursal disease (ibd), vp gene or vp / / poly-protein gene of ibd virus-based dna vaccination has been found effective in protection . dna vaccination against duck hepatitis b virus has been shown to reduce viremia with rapid removal of the virus from the blood after the challenge. against chicken infectious anemia (cia), for the first time, the simultaneous in vitro and in vivo expression of viral proteins vp and vp has been studied for generating protective antibodies against the infection (senthil kumar et al. ) . also, dna vaccines have been developed against avian reovirus (σc protein gene), and egg drop syndrome (eds- ) virus (penton fiber gene fragment), and both these vaccines were found effective during their respective challenge studies. likewise, against protozoan infections, especially coccidiosis, successful dna vaccines have been developed, utilizing the - e and etmic genes in combination with cytokines to provide protection from this economically important infection of birds (min et al. ; ding et al. ) . vaccination with dna is one of the most promising novel immunization techniques against pathogens, for which conventional vaccination regimens have been less effective. after about years of experimentation, dna vaccines, nick named 'immunological silver bullet', have become well established in clinical trials. however, they have yet to proceed past the second phase trials primarily due to the inability to induce more potent immune responses in higher mammals. in small experimental animals, the milder host impedance has permitted the dna vaccines to induce lasting protective effects in contrast to much tougher host barriers in large animals. significant efforts have been put forward to identify methods of enhancing the immune response of plasmid dna to enable its practical implementation. prime importance has been given to develop vaccines to elicit both humoral and cellular immune responses. researchers have tried a variety of immune modulators, cytokines and co-stimulatory molecules, in this regard. if the potency is improved, plasmid dna vaccines, having numerous advantages, can be useful for the active immunization against infectious diseases of animals. considering the current trends and myriad possibilities, efforts should be targeted towards improving their delivery or to increase their immunogenic potential. poor cellular uptake and rapid in vivo degradation of plasmid dna has to be taken into account and novel delivery systems has to be developed along with the optimization of the plasmid vector. the major challenge in future is the improvement of the transfection efficiency of the dna vaccines. gene gun and electroporation can increase transfection and improve immune responses significantly, but these technologies have not yet advanced to routine use in animals. another promising approach is the development of microparticles as delivery systems or the non-invasive plasmid dna immunization. although the potency of the immune response has been weak while using topical application methods, stratum corneum disrupting agents and novel adjuvants may significantly improve them. further, the properties of dna vaccines have to be modulated via using cationic liposomes for promoting mucosal and systemic immunity, simultaneously. the current scenario of incorporating such novel methodologies unveils much promise regarding the development of effective, safe and economically viable nucleic acid vaccines. in this context, one should be optimistic regarding the continual research efforts for global implementation of dna vaccines as an effective immunological arsenal, which could ably address the threats posed by emerging and highly threatening infectious agents of animals. porcine interleukin- enhances dna vaccination against classical swine fever induction of immune responses by dna vaccines in large animals a single hbsag dna vaccination in combination with electroporation elicits long-term antibody responses in sheep recent developments in mucosal delivery of plasmid dna vaccines. current opinions in molecular therapy gene gun-mediate dna vaccination against foot-and-mouth disease virus dna vaccine design protection of cattle against bovine leukemia virus (blv) infection could be attained by dna vaccination a combined dna vaccine-prime, bcg-boost strategy results in better protection against mycobacterium bovis challenge vaccination trials against bovine herpesvirus- vaccination of mice and cattle with plasmid dna encoding the mycobacterium bovis antigen mpb targeting improves the efficacy of a dna vaccine against corynebacterium pseudotuberculosis in sheep prime-boost vaccination with plasmid dna encoding caprine-arthritis encephalitis lentivirus env and viral su suppresses challenge virus and development of arthritis evaluation of the immunogenicity of the p r adhesin of mycoplasma hyopneumoniae as a mucosal vaccine in mice a plasmid dna encoding chicken interleukin- and escherichia coli k fimbrial protein faeg stimulates the production of anti-k fimbrial antibodies in chickens analysis of cpg motifs in endoflagellar gene (flab ) and expression vector (vr ) of leptospiral dna vaccine. sichuan da xue xue bao yi xue ban use of attenuated bacteria as delivery vectors for dna vaccines immunization of bovines using a dna vaccine prepared from the jaboticabal strain of anaplasma marginale bm antigen induces a protective immune response against boophilus microplus following dna and protein vaccination in sheep immunity and disease resistance strategies in poultry: current and future prospects dna vaccines and prevention of infectious diseases in bovines: a review in ovo vaccination with the eimeria tenella etmic gene induces protective immunity against coccidiosis construction of recombinant plasmid with vp genes against asia i fmdv and elementary analysis of its immunological activity cpg motif in atcgat hexamer improves dna-vaccine efficiency against lethal pseudorabies virus infection in pigs humoral immune responses to dna vaccines expressing secreted, membrane bound and non-secreted forms of the taenia ovis w antigen the application of nucleic acid vaccines in veterinary medicine priming of cytotoxic t lymphocytes by dna vaccines: requirement for professional antigen-presenting cells and evidence for antigen transfer from myocytes prime-boost immunization with dna followed by a recombinant vaccinia virus expressing p induced protective immunity against babesia gibsoni infection in dogs potency of an experimental dna vaccine against aujeszky's disease in pigs stable and long-lasting immune response in horses after dna vaccination against equine arteritis virus development of novel strategies to control foot-and-mouth disease: marker vaccines and antivirals induction of protection against porcine cysticercosis in growing pigs by dna vaccination induction of immune responses in cattle with a dna vaccine encoding glycoprotein c of bovine herpesvirus- cloning of canine parvovirus vp gene and its use as dna vaccine in dogs immunogenicity of a recombinant plasmid dna containing glycoprotein gene of rabies virus cvs a dna vaccine that encodes rabies virus glycoprotein lacking transmembrane domain enhances antibody response but not protection induction of immune response in mice with a dna vaccine encoding outer membrane protein (omp ) of brucella melitensis m dna vaccines: immunology, application, and optimization comparison of the immunological memory after dna vaccination and protein vaccination against anthrax in sheep west nile virus vaccines on the use of dna vaccines for the prophylaxis of mycobacterial diseases a tumor reducing factor extracted by phenol from papillomatous tissue of cotton tail rabbits hemagglutinin (h) gene of canine distemper virus cloned in ptarget mammalian expression vector induces neutralizing antibody response in dogs nucleic acid immunization protects dogs against challenge with virulent canine parvovirus novel chitosan derivative nanoparticles enhance the immunogenicity of a dna vaccine encoding hepatitis b virus core antigen in mice dna prime followed by protein boost enhances neutralization and th type immunity against fmdv strategies for inducing protection against avian influenza a virus subtypes with dna vaccines cpg motifs in bacterial dna and their immune effects nucleic acid vaccination of brucella abortus ribosomal l /l gene elicits immune response generation of reassortant influenza vaccines by reverse genetics that allows utilization of a diva (differentiating infected from vaccinated animals) strategy for the control of avian influenza oral dna vaccination with polyprotein gene of ibdv delivered by attenuated salmonella elicits protective immune response in chickens a semliki forest virus replicon vectored dna vaccine expressing the e glycoprotein of classical swine fever virus protects pigs from lethal challenge priming with dna encoding e and boosting with e protein formulated with cpg oligodeoxynucleotides induces strong immune responses and protection from bovine viral diarrhea virus in cattle dna vaccines: a review dna vaccines: recent developments and future possibilities suppository-mediated dna immunization induces mucosal immunity against bovine herpesvirus- in cattle improved protection from velogenic newcastle disease virus challenge following multiple immunizations with plasmid dna encoding for f and hn genes antibody responses to dna vaccination of horses using the influenza virus hemagglutinin gene approaches to enhance the efficacy of dna vaccines modulation of immune responses to bovine herpesvirus- in cattle by immunization with a dna vaccine encoding glycoprotein d as a fusion protein with bovine cd adjuvant effects of ill beta, il- , il- , l- , ifn-alpha, ifn-gamma tgf-beta and lymphotactin in dna vaccination against eimeria acervulina use of dna and recombinant canarypox viral vectors for equine herpes virus vaccination efficacy of particle-based dna delivery for vaccination of sheep against fmdv dna vaccination against bovine viral diarrhoea virus induces humoral and cellular responses in cattle with evidence for protection against viral challenge dna immunization of dairy cows with the clumping factor a of staphylococcus aureus dna vaccination against influenza viruses: a review with emphasis on equine and swine influenza dna vaccination in avian virus neutralizing antibody response in mice and dogs with a bicistronic dna vaccine encoding rabies virus glycoprotein and canine parvovirus vp electric pulses applied prior to intramuscular dna vaccination greatly improve the vaccine immunogenicity development of rabies dna vaccine using a recombinant plasmid brucella abortus: immunity, vaccines and prevention strategies based on nucleic acids immune response in mice and cattle after immunization with a b. microplus dna vaccine containing bm gene protection of kids against c. parvum infection after immunization of dams with cp -dna immunization with dna vaccines encoding different mycobacterial antigens elicits a th type immune response in lambs and protects against mycobacterium avium subspecies paratuberculosis infection development of dna vaccine against chicken anemia virus simultaneously using it's vp and vp proteins the carboxyl-terminal -residue polypeptide of ib virus nucleocapsid induces ctls and protects chickens from acute infection recent developments in veterinary vaccinology dna fragment encoding human il- β - peptide enhances the immune responses elicited in mice by dna vaccine against foot-and-mouth disease dna vaccines: future strategies and relevance to intracellular pathogens laboratory and field evaluation of schistosoma japonicum dna vaccines in sheep and water buffalo in china cd virus dna vaccination induces humoral and cellular immunity and protects against a lethal intracerebral challenge dna vaccines and adjuvants dna immunization with a bovine rotavirus vp gene induces a th -like immune response in mice dna vaccine using m. bovis ag b antigen induces partial protection against experimental infection in balb/c mice enhancing dna vaccine potency by modifying the properties of antigen-presenting cells dna vaccines: recent technological and clinical advances effect of vaccination route and composition of dna vaccine on the induction of protective immunity against psuedorabies infection in pigs immune response to vaccines based upon the vapa protein of the horse pathogen, rhodococcus equi, in a murine model protection of turkeys against c. psittaci challenge by parenteral and mucosal inoculations and the effect of turkey interferon-gamma on genetic immunization enhanced immunogenicity of microparticulated multiepitope dna vaccine encoding t and b cell epitopes of foot and mouth disease virus in mice immunomodulatory effect of plasmids co-expressing cytokines in classical swine fever virus subunit gp /e -dna vaccination direct gene transfer into mouse muscle in vivo a dna vaccine against fmd elicits an immune response in swine which is enhanced by co-administration with interleukin- . vaccine vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus selection of protective epitopes for brucella melitensis by dna vaccination bovine herpesvirus vp enhances the efficacy of a dna vaccine in cattle key: cord- - fwz z n authors: kumar, amit; kumar, prateek; saumya, kumar udit; kapuganti, shivani k.; bhardwaj, taniya; giri, rajanish title: exploring the sars-cov- structural proteins for multi-epitope vaccine development: an in-silico approach date: - - journal: expert review of vaccines doi: . / . . sha: doc_id: cord_uid: fwz z n introduction: the ongoing life-threatening pandemic of coronavirus disease (covid- ) has extensively affected the world. during this global health crisis, it is fundamentally crucial to find strategies to combat sars-cov- . despite several efforts in this direction and continuing clinical trials, no vaccine has been approved for it yet. methods: to find a preventive measure, we have computationally designed a multi-epitopic subunit vaccine using immuno-informatic approaches. results: the structural proteins of sars-cov- involved in its survival and pathogenicity were used to predict antigenic epitopes. the antigenic epitopes were capable of eliciting a strong humoral as well as cell-mediated immune response, our predictions suggest. the final vaccine was constructed by joining the all epitopes with specific linkers and to enhance their stability and immunogenicity. the physicochemical property of the vaccine was assessed. the vaccine d structure prediction and validation were done and docked with the human tlr- receptor. furthermore, molecular dynamics simulations of the vaccine-tlr- receptor complex are employed to assess its dynamic motions and binding stability in-silico. conclusion: based on this study, we strongly suggest synthesizing this vaccine, which further can be tested in-vitro and in-vivo to check its potency in a cure for covid- . severe acute respiratory syndrome coronavirus (sars-cov - ) is the causative agent for the associated disease named as covid- , which had spread throughout the world and declared as a pandemic [ , ] . sars-cov- has been demonstrated to have a strong affinity for human respiratory receptors (hace ), which makes it more vulnerable to humans globally [ ] . sars-coronavirus infections are commonly found to be associated with multiple disorders, such as respiratory, enteric, hepatic, and neurological complications [ ] . in general, coronaviruses (cov) belong to a family coronaviridae, which consists of enveloped positive-sense, single-stranded rna viruses [ , ] . among the four genera of covs, sars-cov, mers-cov, and sars-cov- belong to betacoronaviruses [ ] . inside host cells, sars-cov- genome is translated into two large polyproteins: replicase polyprotein a (pp a) and ab (pp ab) [ ] . the larger pp ab comprises non-structural proteins (nsp -nsp ), which are responsible for constructing a viral replication-transcription complex [ ] . alongside the viral rna transcribes subgenomic rnas, which encode four structural proteins; spike (s), envelope (e), membrane (m), and nucleocapsid (n) and several accessory proteins [ ] . while accessory proteins are engaged in host immune suppression, including downregulation of the interferon pathway, structural proteins shape the virion, facilitating genome encapsulation, viral particle assembly, and release [ , ] . recently, one health concept is used and hypothesized that the prior exposure of humans with pet animals lowers the adverse effect of sars-cov- . the n and s protein epitopes in comparison with taxonomically related coronaviruses' epitopes share high sequence homology, which has high implications for vaccine designing against sars-cov- [ , ] . additionally, there are repurposing of drugs are also important to cobat this disease [ , ] . since the beginning of the st century, two outbreaks of sars-cov and mers-cov have led the researchers into designing vaccines against sars coronaviruses. the third outbreak, wherein sars-cov- infects promiscuously to humans, has given the need for speedy research into developing a broad-range vaccine against these coronaviruses. evidence shows that humoral and cell-mediated immune response plays a protective role against coronavirus infections, specifically against s and n proteins [ ] [ ] [ ] . although the effective antibody response is short-lived, the t cell responses are found to provide long-term protection [ , ] . similarly, t c cell response against the structural proteins s and n are long-lasting [ ] . previously, immunoinformatics approaches have been used to construct the vaccines against the zika virus nipah virus, and human papillomavirus, where in-vivo studies of the human papillomavirus showed promising results [ ] [ ] [ ] . hence, in this study, we have used immunoinformatic approaches to predict highly antigenic epitopes from sars-cov- structural proteins that would evoke a strong immune response in humans. for this purpose, we have used the structural proteins: spike, envelope, and nucleocapsid to predict b-cell, cytotoxic t lymphocyte (ctl) and helper t lymphocyte (htl) epitopes for construction of vaccine. thus, the multi-epitopic subunit vaccine would be capable of eliciting humoral as well as cellmediated immune responses. we have also performed the docking and molecular dynamic simulations (mds) between the vaccine and human toll-like receptor- (tlr- ) to study their binding stability. the outcome of the present study will result in a novel and immunogenic vaccine, which may be further accessed against sars-cov- . the iedb tools (http://tools.iedb.org/bcell/) were used to identify the b cell epitope and for verifying antigenicity, bepipred linear epitope analysis was performed. according to the bepipred linear epitope prediction method, residues with scores above the threshold (default value . ) are predicted to be part of an epitope having % sensitivity and % specificity [ ] . the iedb server (http://tools.iedb.org/mhcii/) was used for the prediction of mhc ii antigenic binders in translated sequences. top htl epitopes were sorted based on their percentile rank and ic value. epitopes with the lowest percentile rank were considered an excellent binding affinity, whereas, peptides with ic values in lower (< nm), middle (< nm), and higher (< nm) range corresponds to the highest, intermediate, and lowest binding affinity, for the t-cell, respectively [ ] . mhc class i binding/ctl epitopes for supertype a were predicted for all three proteins by using an online server netctl . (http://www.cbs.dtu.dk/services/netctl/) [ ] . default threshold of . was chosen for vaccine construction in this study (> . corresponds to % sensitivity and . % specificity; > . shows % sensitivity and . % specificity; > . shows % sensitivity and % specificity). mhc class i binding and proteasomal cleavage prediction was performed using artificial neural networks. accordingly, tap transport efficiency was predicted using a weight matrix. mhc class ii binder epitopes/htl, with the ability to induce cell-mediated immunity, were identified using the ifnepitope server (http://crdd.osdd.net/raghava/ifnepitope/). the prediction was performed by a motif and support vector machine (svm) hybrid approach [ ] . all the positive epitopes able to induce interferon-γ (ifn-γ) were selected for vaccine construct. selected high-scoring ctls, high-affinity htls, and b-cell epitopes were used to generate the vaccine sequence. the different epitopes were linked together using linkers: ctl linker (aay), htl linker (gpgpg), and b epitope linker (kk). to improve the immunogenicity of the vaccine, β-defensin (uniprot id: p ) as an adjuvant was added through an eaaak linker at the n-terminal of the construct. the servers antigenpro (http://scratch.proteomics.ics.uci.edu/ ) and vaxijen v . (http://www.ddg-pharmfac.net/vaxijen/ vaxijen/vaxijen.html) were used for the antigenicity evaluation of the final vaccine construct [ , ] . the vaxijen . server predicts the antigenicity of the multi-epitope vaccine peptide based on the physicochemical properties of the input protein. whereas, antigenpro server predicts the antigenicity of the multi-epitopic vaccine based on the protein microarray data analysis of the target organism. the server allertop v . (http://www.ddg-pharmfac.net/ allertop) and allergenfp were used to predict the allergenicity of multi-epitopic vaccine [ , ] . allertop v . classifies the vaccine protein sequence by machine learning methods for the classification of allergens. allergenfp is an alignmentfree online server that uses svm module to predict the allergenic and non-allergenic nature of proteins. vaccine construct was accessed for its physicochemical properties using the protparam server (http://web.expasy.org/prot param/) that predicts the theoretical pi, instability index, halflife, stability profiling, aliphatic index, and grand average of hydropathy of the sequence [ ] . protein secondary structure was determined using psipred, a web-based freely accessible online server (http://bioinf.cs. ucl.ac.uk/psipred/) [ ] which also gives information of contact analysis, fold recognition, structure modeling, function prediction, protein intrinsic disorder prediction and domain prediction of a query sequence [ ] . the disorder profile of the vaccine construct was generated using pondr pool of four predictors (http://original.disprot. org/metapredictor.php) [ ] [ ] [ ] and iupred a predictor [ ] as described in our previous reports [ ] [ ] [ ] . the tertiary structure of the vaccine construct was generated using the i-tasser web server [ ] . it provides an energy minimized model through iterative template-based fragment assembly simulations. the structure was further optimized upon adding hydrogen and missing side-chain atoms followed by minimization in schrodinger's protein preparation wizard using opls forcefield [ ] . this produced a high-quality model for protein-protein docking and molecular simulations. rampage [mordred.bioc.cam.ac.uk/~rapper/rampage.php] and procheck [ ] web servers were used for evaluation of the vaccine model based on the distribution of amino acids in the ramachandran plot. piper program embedded in the bioluminate module of schrodinger for protein-protein docking was implemented for docking of vaccine model and tlr- (pdb id: az) and tlr- (pdb id: j a) receptors [ , ] . it is an efficient tool that reduces false positive poses and performs a global search with fast-fourier transform (fft) approach. using conformations of input structures, the top clusters were selected with cluster radius Å, which then centralized, and the outcomes of the docking based on cluster size were evaluated. with the largest cluster size, the docked complex out of complexes was selected for molecular dynamics simulation. the ~ kda vaccine model in complex with tlr- receptor was evaluated for its binding stability in an aqueous environment for ns. with a total of , , molecules of tip p water model, neutralizing cl − ions, and . m concentration of salt, the complex was incorporated in a cubic box. recently updated forcefield charmm was implemented to generate the topologies of the protein-protein complex. the generated system was minimized for , steps of the steepest descent algorithm. further, the equilibration process under npt and nvt conditions for ns was done with parrinello-rahman and v-rescale methods for coupling of pressure and temperature, respectively. lastly, production md run for all three systems was performed in periodic boundary conditions for ns. lincs algorithm was used for calculating bond parameters. particle mesh ewald (pme) for long-range electrostatics with fourier spacing of . was used for production md. an analysis of md trajectory, root mean square deviation (rmsd), and root mean square fluctuation (rmsf) was calculated with gmx rms, and rmsf commands. for the production of final vaccine in large quantity, java codon adaptation tool (http://www.jcat.de/) was used for codon optimization and attached with expression vector. the optimized codon sequence includes codon adaption index (value . ) and %gc content ( - %), which denotes the high level of protein expression and dna stability. finally, restriction cloning was performed using tool snapgene v . . . the restriction sites ndei and xhoi were added to the n and c terminals of the optimized complementary dna sequences followed by their insertion within the pet- a(+) vector to ensure the polyprotein synthesis within the e coli expression system. htl epitopes for all structural proteins were predicted using iedb server for human mhc-ii alleles. among these, top epitopes based on their least percentile rank depicting their high affinity were selected. the final selection of epitopes was made on the basis of overlapping sequences, which were further assessed for their ifn inducing potential. epitopes with positive ifn inducing potential were finally selected for vaccine construction (table ) . for each structural protein, ctl epitopes for supertype a were predicted using an online server netctl . . a total of , , and epitopes were predicted for s, n, and e proteins, respectively (table ) . linear b-cell epitopes in all three structural proteins were predicted using another online server bcpreds. epitopes selected on the basis of prediction scores were further screened for their non-allergic and antigenicity potential. two epitopes each for s and n with high antigenicity scores were selected for final vaccine construction are tabulated in table . however, no b cell epitopes were found for the e protein. the designed construct comprising of ctl epitopes, htl epitopes, and b-cell epitopes were then joined using linkers. additionally, in order to improve the immunogenicity of multi-epitope vaccine, the construct was tagged with an adjuvant (β-defensin-tlr- agonist) at n terminal region, which enhances the natural immune response. the vaccine must be antigenic and non-allergic in nature and also induce humoral as well as cell-mediated immune responses against the targeted virus. our vaccine was observed to be antigenic with a respective probability score of . and . predicted by vaxijen v . and antigenpro servers. the construct was also detected as a non-allergen, checked using the allertop v . server. the vaccine construct is composed of amino acids and has a molecular weight of . kda. it is basic in nature (theoretical pi . ) and has a half-life of hrs in mammalian reticulocytes (in vitro). the instability index is estimated to be . , which denotes the stability of the protein. the aliphatic index ( . ) showed that the protein is thermostable, whereas grand average of hydropathicity is observed to be negative (− . ), indicating the hydrophilic nature of vaccine. the secondary structure was predicted using pesipred . server. as shown in figure , the vaccine contains several αhelices together with many the β-strands. six long helices from residues - , - , - , - , - , and - with a long β-strands of amino acids from residues - can be observed. the -residue long vaccine construct was modeled using i-tasser. the lowest energy predicted model was selected for further refinement and docking studies. however, the validation on ramachandran plot by procheck server suggested that % residues of the constructed vaccine model lies in the favored and allowed regions while the rest % are a part of generously allowed and disallowed regions (figure (b) ). similarly, rampage server also mapped nearly . % residues in favored and allowed regions, while . % residues were present in coordinates portraying the outlier region (figure (a) ). as predicted by secondary structure and disorder-based structure analysis, the tertiary structure of the construct has a high number of unstructured regions. based on disorder prediction data, it is largely disordered at its terminals. from the piper program, with highest cluster size , out of complexes ranging till , the complex , also with greatest minimal local energy, was chosen for further analysis. the vaccine model interacts with the tlr- receptor (pdb: az) through its n-terminal and the middle regions (figure ). the resultant interacting residues of vaccine-tlr- complex are shown in table . at the interface of docked complex, asn of vaccine is observed to form a h-bond with asp residue of the receptor. additionally, a few more aromatic h-bonds between val , leu residues of vaccine, and his of tlr- are identified. the complex if further stabilized by a pi-cation interaction between lys of vaccine and tyr of tlr- (table ). figure depicts a close view of vaccine-tlr- interface. several amino acids (red) of the receptor (green) are found engaged with vaccine residues (blue). additionally, we have also performed docking with another toll-like receptor (tlr- ) which is known to recognize the flagellin of bacteria. similar to tlr- , the vaccine model has shown significant interactions with tlr- through its n-terminal as well as residues of middle region where the n-terminal folds in the constructed . psipred graphical result of secondary structure prediction of vaccine is represented by different colors: α-helix (pink), β-sheet (yellow), and coil (gray). expert review of vaccines model, as shown in figure . residues such as tyr , glu from n-terminal, and thr , lys from middle regions of vaccine construct interacts with residues asp , ala , cys , and glu of tlr- , respectively, through aromatic hydrogen bonds, salt bridge, and hydrogen bonds. to analyze the stability and dynamic motions of docked complex, we performed a ns long md simulation using gromacs v . . the simulation system comprised ~ lacs atoms in a cubic box with proteins, solvent, and ions. based on the rmsd data, the complex was found to have an upward trend ranging from ~ to . nm till ns with small fractions of stability in between ( figure (a) ). however, according to rmsf values depicted in the graph of figure comparison with the other residues. overall, the average fluctuation in the residues of the vaccine model was near ~ . nm. moreover, the regions interacting with tlr- receptor are not disordered as per our analysis in figure (b) and had stability throughout the simulation. as calculated in visual molecular dynamics, the average h-bonds were found to be in vaccine-tlr- complex during md simulations (figure (c) ). additionally, disordered regions based on the sequence of vaccine were also predicted. the graph in figure (d) represents the moderate amount of disorder (~ % calculated as the mean of all predictors) within residues - and - near n-and c-terminal regions. the regions interacting with tlr- receptor are not disordered as per our analysis in figure (d) and had stability throughout the simulation. the cai improved value for codon optimized sequence was . , while gc content obtained for our vaccine construct was . %. overall, the vaccine sequence lies in a suitable category for its expression in e coli. finally, the vaccine construct was cloned with restriction sites ndei and xhoi in the e coli pet- a(+) vectors. finally, a cloned construct was generated, having a sequence length of base pairs (figure ). sars-cov- is highly pathogenic, which has caused more than lakh deaths globally until the writing of this manuscript. particularly among all, coronaviridae structural proteins s, m, e, and n are the main structural proteins and can be employed for protein/peptide-based vaccines. different human infecting coronaviruses like oc , hku , and mers-cov use a diverse set of lipids and receptors to enter into target cells [ , ] . in sars-cov, the spike glycoprotein interacts with ace- receptor and facilitates the fusion of viral membrane with the lungs membrane [ , , ] . e is an integral membrane protein with n-terminal region, a transmembrane domain (tmd), and a flexible hydrophilic c-terminal tail containing a pzdbinding motif in the last four amino acids [ , ] . envelope protein forms pentamer to lead viroporin like structure that exhibits a membrane-destabilizing ion-channel activity on the host membrane [ ] . cov envelope, which plays a vital role during virion assembly, consists mainly of m protein and only a small fraction of e protein [ ] . whereas n protein shows interaction with m protein in lipid membrane of infected cells forming a double-layered vesicular structure in a secreted form that further interacts with host proteins [ , ] . multiple vaccines are in clinical trial, but till date, no vaccine is approved against the sars-cov- . in a recent in-silico report, the vaccine model using spike glycoprotein has been constructed but limited to the docking with tlr receptor [ , ] . it was reported that human coronaviruses oc recognize the mhc class i supertype a [ ] . based on these reports, we have chosen the a super type for epitope prediction, which will help the vaccine construct to act significantly. according to information obtained from the world health organization (who), one vaccine is in phase , and two vaccines (dna and rna based) are in phase clinical trial. moreover, vaccines are in pre-clinical trials [ ] . previously, studies showed that the whole virus vaccine and virus-like particle vaccine against sars-cov are able to cure infection but leads to the pulmonary immunopathologic type lung disease [ ] . therefore, it is needed that the vaccine is selected on the basis of multi factors, i.e., antigenicity, immunogenicity, toxicity, allergenicity. in the present study, we have designed the vaccine construct by using immune-informatics approaches. three structural proteins (spike glycoprotein, nucleocapsid, and envelope) were selected to construct a multi-epitope vaccine, which is capable of eliciting the humoral and cell-mediated immune response. furthermore, the vaccine is antigenic, non-allergen, nontoxic, immunogenic in nature, and capable of ifn-γ production. the vaccine epitopes were joined with the help of specific linkers to enhance stability and immunogenicity. the vaccine has a molecular weight of . kda, basic in nature, and half-life of hrs in mammalian reticulocytes (in-vitro). moreover, vaccine was found to be highly stable and hydrophilic in nature. finally, the build vaccine construct was found to be moderately disordered, containing few disordered residues at n-terminal and relatively a vast region at c-terminus. as previously known, the vaccine candidates often have an ample amount of protein intrinsic disorder as they feature in the immune system [ ] . further, we also checked the interaction of vaccine construct through a modeled structure docked with two toll-like receptors (tlr- and tlr- ) structure. the adjuvant βdefensin showed interaction with tlr- receptor and few residues from the same region also showed interaction with tlr- . further, the stability of the vaccine-tlr- complex was monitored by implementing molecular dynamics simulations till ns. several residues at the n-terminal and middle region of vaccine construct were found to form stable interactions. finally, the cloned sequence vector was generated in-silico for the expression of the vaccine. newly emerged sars-cov- is declared as pandemic and scientists all over the world running a race against the time to find vaccine/drugs against covid- . despite that, many obstacles like allergenicity, immunogenicity, toxicity, etc. limit the progress of vaccine development. in recent times, advancement in immunoinformatics approaches led researchers to speed up vaccine development. in this context, by precise prediction and selection of multi-epitope, we strongly suggest to experimentaly validate this multi-subunit vaccine, in-vitro and in-vivo. ak, pk, kus, sk, tb: acquisition and interpretation of data, writing, and editing of the manuscript. features, evaluation and treatment coronavirus (covid- ). statpearls understanding covid- via comparative analysis of dark proteomes of sars-cov- , human sars and bat sars-like coronaviruses clinical findings in a group of patients infected with the novel coronavirus (sars-cov- ) outside of wuhan, china: retrospective case series sars-cov- causing pneumonia-associated respiratory disorder (covid- ): diagnostic and proposed therapeutic options is the discovery of the novel human betacoronavirus c emc/ (hcov-emc) the beginning of another sars-like pandemic? coronaviruses -drug discovery and therapeutic options this important review on coronavirus details about drug discovery coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- sars and mers: recent insights into emerging coronaviruses coronaviruses: an overview of their replication and pathogenesis emerging coronaviruses: genome structure, replication, and pathogenesis structure, function, and evolution of coronavirus spike proteins a new coronavirus associated with human respiratory disease in china comparative computational analysis of sars-cov- nucleocapsid protein epitopes in taxonomically related coronaviruses. microbes infect molecular basis of covid- relationships in different species: a one health perspective structural and evolutionary analysis indicate that the sars-cov- mpro is a challenging target for small-molecule inhibitor design probable molecular mechanism of remdesivir for the treatment of covid- : need to know more preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies a dna vaccine induces sars coronavirus neutralization and protective immunity in mice this important paper detail about vaccine development identification of an epitope of sars-coronavirus nucleocapsid protein long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study virus-specific memory cd t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection designing b-and t-cell multi-epitope based subunit vaccine using immunoinformatics approach to control zika virus infection strategic development of a next-generation multi-epitope vaccine to prevent nipah virus zoonotic infection in silico/in vivo analysis of high-risk papillomavirus l and l conserved sequences for development of cross-subtype prophylactic vaccine this important paper detail about insilico vaccine development followed by in vivo studies improved method for predicting linear b-cell epitopes nn-align. an artificial neural network-based alignment algorithm for mhc class ii peptide binding prediction large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction designing of interferon-gamma inducing mhc class-ii binders vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines high-throughput prediction of protein antigenicity using protein microarray data allertop -a server for in silico prediction of allergens allergenfp: allergenicity prediction by descriptor fingerprints protein identification and analysis tools on the expasy server protein secondary structure prediction based on position-specific scoring matrices the psipred protein analysis workbench: years on exploiting heterogeneous sequence properties improves prediction of protein disorder predicting intrinsic disorder from amino acid sequence pondr-fit: a meta-predictor of intrinsically disordered amino acids iupred a: context-dependent prediction of protein disorder as a function of redox state and protein binding the dark side of alzheimer's disease: unstructured biology of proteins from the amyloid cascade signaling pathway folding and structural polymorphism of p c-terminal domain: one peptide with many conformations understanding the penetrance of intrinsic protein disorder in rotavirus proteome the i-tasser suite: protein structure and function prediction protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments procheck: a program to check the stereochemical quality of protein structures decoys as the reference state) potentials for protein-protein docking piper: an fft-based protein docking program with pairwise potentials human coronaviruses oc and hku bind to -o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein mechanisms of coronavirus cell entry mediated by the viral spike protein cooperative involvement of the s and s subunits of the murine coronavirus spike protein in receptor binding and extended host range biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein the sars coronavirus e protein interacts with pals and alters tight junction formation and epithelial morphogenesis this imporatant paper detail about envelope protein and their role in pathogenesis sars coronavirus e protein forms cation-selective ion channels nucleocapsidindependent assembly of coronavirus-like particles by co-expression of viral envelope protein genes analysis of multimerization of the sars coronavirus nucleocapsid protein analyses of coronavirus assembly interactions with interspecies membrane and nucleocapsid protein chimeras development of epitope-based peptide vaccine against novel coronavirus (sars-cov- ): immunoinformatics approach consider tlr for new therapeutic development against covid- human coronavirus oc interacts with major histocompatibility complex class i molecules at the cell surface to establish infection who. who | coronavirus disease world health organization immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus insights into the immunological properties of intrinsically disordered malaria proteins using proteome scale predictions the authors would like to thank iit mandi for research facilities. rg is thankful to dbt, govt. of india (bt/ /iyba/ / ) to rg. ak is supported from dbt, govt. of india grant (bt/ /iyba/ / ). pk and sk were supported by mhrd-india for their funding. kus and tb are grateful to the icmr srf and dst inspire phd fellowships, respectively. key: cord- -w pbq lb authors: lindblad, e. b.; duroux, l. title: chapter mineral adjuvants ∗ ∗ the present chapter is an updated version of the chapter “mineral adjuvants,” published in immunopotentiators in modern vaccines, p. – . ed. virgil schijns & derek o'hagan, elsevier science publishers ( ). date: - - journal: immunopotentiators in modern vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: w pbq lb abstract mineral adjuvants comprise aluminum hydroxide and phosphate adjuvants as well as calcium phosphate adjuvants. in particular, the aluminum salts have achieved an undisputed status as the most commonly used adjuvants in human and veterinary vaccines. calcium phosphate adjuvant, later discovered by edgar relyveld, constitutes a very interesting alternative and has also been applied both in human and veterinary vaccines. new analytical tools applied in adjuvant research are about to take us to the next level of understanding mineral adjuvants. these tools have been used to characterize mineral adjuvants, but so far, in particular, aluminum-based adjuvants in terms of surface marker expression profiles, isotypic profiles, and cytokine profiles. in the past years, the discovery of adjuvant-mediated induction of the nalp inflammasome and its impact on the secretion of interleukin (il)- β and il- as proinflammatory mediators in the early phases of immune response has been described as an important mechanism for the function of these adjuvants. two categories of inorganic mineral compounds have been applied as immunological adjuvants in vaccine formulation, aluminum compounds and calcium phosphate. of these two, the aluminum compounds have the longest history and by far the most comprehensive record of use. both adjuvants are generally regarded as safe to use in human vaccines when used in accordance with the current vaccination schedules. , a.t. glenny and coworkers were the first to demonstrate the adjuvant effect of aluminum compounds in . glenny prepared a variety of diphtheria toxoid precipitates and investigated their immunogenicity. among these were toxoids precipitated by the addition of potassium alum [kal(so ) : h o]. glenny observed that injecting the diphtheria toxoid as an alum precipitate led to a significant increase in the immune response against the toxoid. , vaccines prepared in accordance with this principle have been used in practical vaccination and are referred to as alum-precipitated vaccines. this approach, however, has a number of drawbacks. it was found that such preparations could be highly heterogeneous, depending on which anions, such as bicarbonate, sulfate, or phosphate, were present at the time of precipitation, e.g., as buffer constituents or growth media residues in the antigen solution. in addition to this, al precipitation with the antigen takes place under alkaline conditions, and this may in some cases introduce alterations to the antigen. in contrast, preformed aluminum hydroxide, in the form of hydrated colloid "gels," has the ability to adsorb protein antigens from an aqueous solution and such gels can be preformed in a well-defined and standardized way. vaccine preparations based on adsorption of the antigen onto a preformed aluminum hydroxide adjuvant are referred to as aluminum-adsorbed vaccines, in contrast to the alum-precipitated vaccines mentioned earlier. data on the use of alum-precipitated vaccines can be found in the older literature, but in practical vaccination, the adsorption onto preformed aluminum hydroxide and aluminum phosphate gels has now almost completely substituted the alum precipitation in vaccine formulation. aluminum phosphate was introduced as an alternative adjuvant two decades after glenny's work. in , hans ericsson from sweden devised a method in which diphtheria toxoid was coprecipitated into a matrix of aluminum phosphate, corresponding to the alum precipitation method described earlier. lewis b. holt demonstrated the following year that preformed aluminum phosphate (prepared from aluminum chloride and trisodium phosphate) acted as an adsorbant and was adjuvant-active with diphtheria toxoid. aluminum hydroxide has been included into composite adjuvant formulations, such as the as adjuvant formulation proprietary to glaxo smithkline, which consists of aluminum hydroxide in combination with monophosphoryl lipid a (mpl). occasionally, the word "alum" is seen in the adjuvant literature to describe both aluminum hydroxide and aluminum phosphate gels, but this is incorrect use of terminology. potassium alum, kal(so ) : h o, is in accordance with the chemical definition of an alum, whereas neither aluminum hydroxide nor aluminum phosphate is. calcium phosphate was developed as an adjuvant by edgar h. relyveld in (relyveld, personal communication) . also in the case of calcium phosphate the adjuvant can be coprecipitated in the presence of the antigen, or it can be preformed in a carefully controlled chemical environment and subsequently used for adsorption of the antigen in question. aluminum hydroxide and aluminum phosphate adjuvants are generally prepared by exposing aqueous solutions of aluminum salts, typically as sulfates or chlorides, to alkaline conditions in a well-defined and controlled chemical environment. various soluble aluminum salts can be used for the production of aluminum hydroxide, but the experimental conditions (temperature, concentration, and even the rate of addition of reagents) strongly influence the results. , anions present at the time of preparation may coprecipitate and change the characteristics from those of "pure" aluminum hydroxide. aluminum phosphate gel can be seen as an example of such a preparation where the soluble aluminum salts are precipitated in the presence of sufficient amounts of phosphate ions. x-ray microanalysis ( fig. . ) is a way to obtain a ground element "fingerprinting" of mineral adjuvant preparations giving an indication of which salts were used as starting material in the preparation. stanley hem's group at purdue university studied the physicochemical nature of inorganic mineral gel preparations commonly used as vaccine adjuvants for more than years. using x-ray crystallography and infrared spectroscopy they demonstrated a boehmite-like (aluminum oxyhydroxide, alooh) pattern in preparations traditionally known as aluminum hydroxide, whereas commercialized aluminum phosphate gel adjuvant was identified as amorphous aluminum hydroxyphosphate. it was possible to calculate an average primary crystallite size of . Â . Â nm for the boehmite preparations. commercially available calcium phosphate (from reheis inc. nj, usa) was studied by x-ray diffraction, fourier transform infrared spectroscopy, and thermal analysis. this indicated that calcium phosphate adjuvant with the suggested formula of ca (po ) could be described as nonstoichiometric hydroxyapatite, ca Àx (hpo ) x (po ) Àx (oh) Àx , where x varies from to . in the original work, relyveld described the calcium phosphate adjuvant, prepared at the institut pasteur, as non-hydroxyapatite (relyveld, personal communication morphological studies using scanning electron microscopy (sem) have been performed ( fig. . ). it should, however, be remembered that dehydration of the adjuvant particles in the preparation for sem may lead to structures not completely identical to those presented to the immune system as vaccine adjuvants. in human vaccination, aluminum adjuvants have been primarily used in tetanus, diphtheria, pertussis, and poliomyelitis vaccines as part of standard childhood vaccination programs for more than years in many countries. later, aluminum adjuvants were also introduced in hepatitis a and hepatitis b virus vaccines, as well as in vaccines against human papillomavirus (causing genital warts and cervical cancers), and vaccines against lyme disease/borreliose and japanese encephalitis. other aluminum-adsorbed vaccines, against, e.g., anthrax, are available for special risk groups (table . ). in veterinary medicine, aluminum adjuvants have been used in a large number of vaccine formulations against viral e and bacterial diseases e (table . ), as well as in attempts to make antiparasite vaccines. e calcium phosphate was used as an adjuvant in vaccines against diphtheria, tetanus, bordetella pertussis, and poliomyelitis, , commercialized by institut pasteur. calcium phosphate was used as an adjuvant in the ipad series of vaccines by institut pasteur for approximately years. furthermore, calcium phosphate was tested as an adjuvant in experimental vaccine formulations with the gp antigen from human immunodeficiency virus. calcium phosphate has so far not been used as an adjuvant in commercial veterinary prophylactic vaccines. both aluminum hydroxide and calcium phosphate have been used as adjuvants in commercialized adsorbed allergen preparations for hyposensitization of allergic patients. limitations to the applicability of mineral adjuvants one obvious limitation for the application of aluminum adjuvants lies in the apparent th -like profile of these adjuvants. a th -biased immune response is not likely to protect against diseases for which th immunity and major histocompatibility complex (mhc) class ierestricted ctls are essential for protection, such as with, e.g., intracellular parasites or tuberculosis. another limitation lies in the fact that traditional aluminumand calcium-adsorbed vaccines are sensitive to freezing and therefore not lyophilizable. assays for the detection of damages to vaccine formulations induced by freezing have been published. , attempts have been made to overcome the sensitivity to freezing by adding lyoprotectants, such as trehalose, to the preparations. , aluminum adjuvants failed to provide satisfactory augmentation of the immune response against a number of infectious diseases, such as with influenza and typhoid fever vaccines. , in some approaches to vaccine preparation, aluminum adjuvants have shown limitations in their applicability in vaccines based on small-sized peptides. in some cases, e.g., with foot and mouth disease (fmd) virus peptides, the problem could be overcome by conjugating the peptide to a larger carrier molecule. in others, it could not. , aluminum adjuvants have been tested in a few dna vaccine formulations. here it was shown , that aluminum hydroxide had an inhibiting effect, whereas aluminum phosphate adjuvant augmented the immune response against the antigen encoded by the dna nucleotide. the content of phosphate in the dna molecule apparently gives it a high binding affinity to the aluminum hydroxide, which in turn prevents efficient transcription and translation into protein. it has recently been shown that certain metal ions potentially present in mineral adjuvant formulations may have a destabilizing effect on vaccine stability. schlegl et al. demonstrated that high amounts of residual cu þþ ions could interact with sodium metabisulfite, which is added to vaccine formulations to neutralize formaldehyde, leading to the formation of free radicals. these would in turn react with antigen integrity resulting in a significantly reduced shelf life of vaccines, as exemplified by a commercial vaccine against japanese encephalitis virus (ixiaro). there are limitations for the content of aluminum and calcium allowed in vaccines for humans, when administered as adjuvants. these limits are . mg aluminum per dose in europe, and in the united states, the limit is . mg aluminum per dose if determined by assay, . mg if determined by calculation, and . mg if safety and efficacy data justify it. in europe, the maximum allowed amount of calcium delivered by calcium phosphateeadjuvanted vaccines is . mg ca. there is, however, no obvious toxicological rationale behind limiting the amount of calcium in vaccines to . mg/dose. calcium phosphate is a natural constituent of mammals, and it is a component of bone replacement transplants in much higher amounts with no toxicological problems. the optimum dose of adjuvant is normally determined empirically in a pilot trial, but helpful guidelines are available in the literature. in veterinary vaccines, there is no defined maximum limit for the allowed content of aluminum adjuvants. here the dose is normally set from a balance between efficacy and local reactogenicity. for doseeresponse relations of both types of mineral adjuvants in combination with bacterial antigens the immunomodulation observed may reflect a composite effect between the mineral adjuvant itself and the immunomodulatory and adjuvantactive bacterial substances, known as pathogen-associated molecular patterns (pamps) or toll-like receptor (tlr) agonists such as muramyl peptides from peptidoglycans, lipopolysaccharides (lps), trehalose dimycolate ("cord factor"), or cpg motifs from bacterial dna. the immunostimulating effect of the traditional aluminum adjuvants is highly complex and must be attributed to several different mechanisms. in the older literature, the function of a repository adjuvant was originally described as to delay clearing from the injection site and sustain a gradual release of adsorbed antigen from the inoculated depot. although gradual release and delayed clearing may indeed play a role, it quickly became obvious that the gradual release was insufficient in explaining the mechanisms of adjuvant activity. however, the physical adsorption characteristics of antigen onto the adjuvant is still considered to be a very important mechanism for the function of mineral adjuvants. the literature holds examples of publications in which injection of adjuvant and unadsorbed antigen at distant sites have led to immunostimulation toward the antigen ; however, this is not the consistent picture, and the nature of the antigen chosen for the work may provide part of the explanation for deviating conclusions. as a general rule, the antigen should be adsorbed onto the adjuvant prior to immunization and the adsorption should be carefully monitored. a consequence of the physical attachment of the antigen onto the adjuvant is, that a soluble antigen upon adsorption may be presented to the immunocompetent cells in a "particulate" manner, which could facilitate antigen targeting, i.e., favor uptake by antigen-presenting cells (apcs). a likely explanation is that apcs may be more efficient in antigen uptake by phagocytosis than by pinocytosis. mannhalter and coworkers convincingly demonstrated enhanced uptake as well as antigen presentation as measured by t-cell proliferation of aluminum-adsorbed tetanus toxoid compared with the soluble toxoid, by human apcs in vitro. the physicochemical mechanisms behind the antigen adsorption itself is complex, and, depending upon the nature of the individual antigen and the characteristics of the adjuvant particles, some mechanisms may predominate over others. the primary mechanisms responsible for the adsorption have been explained partly by electrostatic attraction and partly by anionic ligand exchange. in addition, other intermolecular binding forces, like hydrophilicehydrophobic interactions and van der waals forces, may play a role in protein adsorption. each binding force plays its role in a given antigeneadjuvant combination, depending upon the nature of the antigen and the chemical environment: ph, ionic strength, presence of surfactants, etc. e as a general guideline, adsorption by electrostatic attraction is accomplished in the ph interval between the isoelectric point (iep) of the protein antigen and the point of zero charge (pzc) of the adjuvant, which is the equivalence of the iep, but for the adjuvant. this applies for both aluminum hydroxide and aluminum phosphate adjuvants. in this interval the adjuvant and the antigen will have opposite electrical charges, facilitating electrostatic attraction and adsorption ( fig. . ). the surface charge (sch) in millivolts of the adjuvant particle at c was described by the formula (stanley l. hem, personal communication): in this formula, which is derived from the nernst equation, "pzc" is the ph value at which the net charge of the adjuvant is zero and "ph" is the actual ph value of the chemical environment. sally seeber and coworkers concluded that aluminum hydroxide should be superior to aluminum phosphate in adsorbing proteins with an acid iep, and vice versa for proteins with an alkaline iep. however, antigens with a distinct polarity in terms of one part of the molecule having a clearly acidic iep and a distant part of the same molecule having a clearly alkaline iep may bind well to both, e.g., al(oh) and alpo , by electrostatic attraction. however, in such cases there may be a difference in the orientation of the adsorbed molecule in relation to the adjuvant. ligand exchange if the antigen contains phosphorylated groups (e.g., phosphorylated amino acids), ligand exchange between the antigen-associated phosphate and hydroxyl groups of the adjuvant may account for high-affinity binding to the adjuvant. this is the case, e.g., with hepatitis b virus surface antigen (hbsag) particles and has been shown in experiments using phosphorylated alpha-casein as model antigen. ligand exchange involves substitution of surface-associated hydroxyl groups with phosphate groups and leads to a decrease of the pzc of the adjuvant. adsorption due to ligand exchange may take place even in systems in which the adjuvant and the antigen have the same electrical charge and electrostatic repulsion would be expected. determination of the protein adsorption capacity of the adjuvant is highly recommended and can be measured by a variety of analytical methods. it is normally done by comparing the protein content in the aqueous phase of the antigen solution before and after adsorption onto the adjuvant. if an antibody specific for the antigen one wishes to adsorb is available, adsorption can be measured by immunoprecipitation techniques. it can be done by quantitative immunoelectrophoresis or by single radial immunodiffusion. without the use of an antibody it can be tested spectrophotometrically, e.g., by the bicinchoninic acid or bca method. however, one should be aware that contamination with the fine fraction of mineral gel particles can disturb the spectrophotometrical readings by light-scatter effects. enzyme-linked immunosorbent assay (elisa) methods have been designed in which aluminum-adsorbed antigens could be used directly as antigens in elisa assays. elisa methods were also applied for in vitro assessment of various viral antigens, i.e., pseudorabies, porcine parvovirus, and infectious bovine rhinotracheitis vaccines adsorbed onto aluminum hydroxide adjuvant. differential adsorption of complex mixtures of antigens can be measured by either immunoelectrophoresis or by high-performance liquid chromatography (hplc). if an antiserum raised against the complex antigen mixture is available, a crossed, twodimensional immunoelectrophoresis may reveal if single components from the complex solution of proteins remain unadsorbed. to use this approach, the precipitation band pattern from an electrophoresis run on the complex antigen mixture prior to adsorption is compared with the bands of an electrophoresis run on the supernatant of the same mixture after adsorption. unadsorbed components will retain their immunoprecipitation band pattern, whereas missing bands or reduced height of bands are indicative of complete or partial protein adsorption. an hplc chromatogram of the antigen mixture liquid phase before and after adsorption may provide a similar type of information. for the testing of adsorptive power of mineral adjuvants when used in diphtheria and tetanus vaccines, the old ramon flocculation test is still frequently used. in this test the results are given as lf (limits of flocculation). it is of relevance to distinguish between the adsorption capacity, which is the amount of antigen that is adsorbed at monolayer coverage of the adjuvant, and the adsorption coefficient, which is a measure of the strength of the adsorption force, not the least since there is evidence that very high adsorption coefficients may lead to reduction of the immune response. e various mathematical models can be used to describe protein adsorption quantitatively. adsorption of molecules to surfaces, like antigens to adjuvant particles, is governed by equilibrium thermodynamics and kinetic principles, where the proportion of surface covered by the adsorbate at equilibrium, in principle depends on the adsorbate concentration and the rate constants for adsorption and desorption, at a given temperature and pressure. in its simplest form, this process is described by the langmuir isotherm, which was originally derived to describe the adsorption of gas molecules to simple planar surfaces. when applied to the adsorption of molecules in solution by solid interfaces, the relation between the quantity of a solute molecule, q, which is bound to the surface of the adsorbent, and its concentration [a] is given by the following function, which is a rectangular hyperbola: with q max being the maximal quantity of adsorbed molecule and k eq being the equilibrium constant. this equation can also be written as the proportion of sites occupied on the surface, q (with q ¼ q/q max ), as a function of the concentration of adsorbate [a]: the strength of this model, which makes it popular and attractive, is that it is based on a physical theory and it allows with a simple data fitting procedure (e.g., nonlinear leastsquares method) to extract the equilibrium constant, which is a measure of the binding strength of the adsorbate to the surface. however, despite its popularity, the langmuir isotherm very rarely corresponds to the physical reality of macromolecule adsorption to adjuvant surfaces, as most of the underlying assumptions are generally violated (homogeneous and : binding sites, dynamic equilibrium at time of measurement. and no interactions between adsorbates). this is due to the complex nature of macromolecule (protein antigen) interactions with surfaces, which are typically rough and inhomogeneous; the equilibria conditions, which might not be achieved at the time of recording; the disparity and multivalent nature of protein-binding sites at their own surface; structure remodeling upon adsorption; proteineprotein interactions at high concentrations; etc. this topic is not discussed further here, and the reader is referred to the work by latour. when langmuir isotherms are fitted to "langmuir-looking" data plots, there is often the risk of underevaluating the k eq parameter (and associated free gibbs energy value) with the potential for drawing erroneous conclusions. in addition, when k eq values are very high and because of the shape of rectangular hyperbola, the dynamic range for data point collection can become compressed, making the effective concentration range very limited and experimentally impractical to handle. in order to correct for the limitations of the langmuir isotherm, other models were later developed, which are better suited to fit data from heterogeneous systems. hybrid models combining the langmuir and the freundlich isotherms, such as the toth isotherm, better satisfy the lower and upper ranges of adsorbate concentrations than any the two models that it is derived from. for a short and exhaustive review of adsorption isotherms, see foo and hameed. the toth equation takes the following form: as seen, the equation includes an additional parameter, t, which when t ¼ reduces to the langmuir equation. this parameter t quantifies the deviation from the ideal system formulated by langmuir and can indicate the level of heterogeneity in the adsorption process. a typical experiment would consist in measuring the amount of molecules bound to the surface of the adsorbent as a function of adsorbate concentration using various techniques, and until the saturation of the surface is achieved. in the case of insoluble adjuvant particles, like aluminum hydroxide, aluminum phosphate, or calcium phosphate, the amount of adsorbed antigen can be measured indirectly by the difference between the initial concentration in the solution and the final concentration (at equilibrium) after physical separation of the insoluble adjuvant (e.g., after sedimentation or filtration). the amount of bound adsorbate is plotted as a function of adsorbate concentration. a typical example of such an equilibrium fractional saturation is given in fig. . , where lysozyme was adsorbed to adju-phos particles. the data are fitted with the model isotherm, here langmuir and toth (fig. . ) . one can observe a linear increase of the bound fraction at low concentrations of lysozyme, followed by a gradual inflection of the plot as the concentration increases to reach an asymptotic region where all sites become occupied (fig. . ) . besides the apparent k eq , relevant information for the experimentalist is the maximal amount of bound adsorbate q max per surface area or weight of particulate material (value at asymptote), which can be used to determine the maximal vaccine dose. finally, the fitted q values give the fraction of sites occupied, which is also valuable information to determine the working concentration of antigen needed to achieve a desired coverage of the adjuvant particles. reducing the concentration (i.e., the amount) of antigen to achieve similar site occupancy can also prove economical with expensive antigens. figure . hen-egg lysozyme adsorption isotherm to adju-phos. the raw data was fitted with two model isotherms, langmuir (solid line) and toth (dotted line). data points were fitted using nonlinear least squares regression. the parameters extracted from the fitting are r , the adjusted coefficient of determination; b max , the maximal amount of adsorbed molecules (in nmoles); k eq , the apparent equilibrium constant (in liter/mole); and t, the exponent in the toth equation. increasing concentrations of lysozyme were admixed with a fixed amount of buffered adju-phos particles ( . ae . mm diameter, À mv z-potential) suspended in imidazole mm at ph . and incubated at c for min (apparent equilibria were reached within min). adsorbed lysozyme was deduced from the difference in ultraviolet absorbance at nm between the initial solution and the admixture supernatant after centrifugal sedimentation of the particles. finally, one should emphasize that the variables extracted by fitting with the two isotherms differ, as expected. it is noticeable that the goodness of fit, measured by the adjusted r , with the toth isotherm is slightly improved over the langmuir isotherm fig. . , suggesting that in this particular system the mechanism of adsorption does appear to deviate from the langmuir model. this situation may be very different in other systems in which significant deviation from the langmuir isotherm are encountered. it is also noticeable that somewhat different values for q max (maximal quantity of adsorbed molecules) and k eq are obtained from the two models (fig. . ) , as a reminder that critical judgment should be exerted upon their significance. in the older literature, aluminum adjuvants were often claimed not to be "t-cell adjuvants." however, this is mostly based on work from a period in which a "t-cell adjuvant" was primarily an adjuvant capable of inducing a delayed-type hypersensitivity (dth) response. such generalizations are now considered too simple and obsolete. it is correct that aluminum adjuvants are not efficient dth inducers in rodents, as pointed out by robert bomford. there is also very little evidence that aluminum adjuvants should be able to generate mhc class ierestricted cytotoxic t cells; so far there is only a single report from dillon and coworkers using a recombinant influenza vaccine in mice. however, as early as the s', the ability of aluminum adjuvants to induce eosinophilia was shown to require the presence of t cells and the reaction profile of aluminum adjuvants was shown to comprise stimulation of cd þ t cells. among the early observations in classical animal models was the demonstration of mannhalter that aluminum-adsorbed tetanus toxoid led to an increase in antigeninduced t-cell proliferation, apparently due to increased release of il- . in contrast, there was a lack of importance of il- in the augmentation of the primary antibody response in rabbits immunized with aluminum adjuvant. grun and maurer demonstrated that anti-il- a or anti-il- was able to inhibit an antigen-specific t-cell proliferative response after immunization with aluminum adjuvant. this was not the case if the mice were immunized with freund complete adjuvant. however, as the proliferative responses were inhibited by anti-cd antibody, regardless of the adjuvant used, it indicated that the proliferating cd þ t cells from mice immunized using aluminum adjuvant were of the th subset. lindblad and coworkers found a corresponding profile in c bl/ j mice while performing reverse transcriptasepolymerase chain reaction for il- -and il- -specific messenger rna (mrna) in the regional draining lymph nodes at day following vaccination with aluminumadjuvanted vaccine. it is interesting that a complex between al(oh) and il- (al(oh) /il- ) induced a th response, rather than a th response, when used as an adjuvant and the th -promoting effect of the al(oh) /il- complex was greatly augmented by the coadministration of exogenous il- . a new line of research was initiated with the introduction of gene knockout mice. this has since facilitated the study of the significance of interleukins in adjuvantmediated immunostimulation. in il- gene knockout mice, immunization with ovalbumin (ova) þ al(oh) elicited igg a titers of a similar magnitude as when ova was injected together with freund's complete adjuvant (fca). interestingly, the group immunized with ovaþ al(oh) continued to produce il- (a cytokine normally associated with the th profile). in contrast, when the il- À/À mice had been immunized using fca a similar stimulation of il- was not seen. this is in support of the idea that the major role of aluminum-induced il- in th-subset stimulation is to downregulate the th response. in a later study, jim brewer's group showed, using either stat -or il- ra-deficient mice, that although these mice were unable to further process an il- -mediated signal, the ability of aluminum hydroxide to induce il- was not abrogated. higher levels of il- were found in il- ra À/À mice than in the wild-type mice. it has been suggested that the th stimulation in il- À/À mice could be due to overlapping responses of il- and il- , since they both utilize a common signaling pathway via the il- receptor. however, they concluded that the th response could not be due to il- , since the il- response too is impaired in stat -or il- ra-deficient mice. the role of il- in the adjuvant activity of aluminum hydroxide and its effect on th induction was studied by brewers group. they demonstrated that il- deficient mice immunized with ova þ al(oh) had reduced il- production in lymph node cells compared with wild-type mice. however, if they added exogenous il- , it did not further enhance the aluminum-induced th response. although the aluminum adjuvant led to reduced il- production in il- À/À mice, this was not accompanied by a reduced level of serum igg . apparently, there is poor correlation between this particular antibody subclass and il- production. with calcium phosphate no cytokine data are yet found in the literature. it is not yet possible to investigate surface marker differentiation over time of cell populations in vivo due to the biological complexity of living organisms. however, in vitro models may lead to observations the validity of which may later be challengedd with all due care takendby in vivo control experiments. ulanova et al. were among the first to carry out systematic studies on the direct effect of aluminum hydroxide in cultures of human peripheral blood mononuclear cells (pbmcs). they found an increase in the expression of costimulatory and adhesion molecules: mhc class ii, cd , cd (formerly known as intercellular adhesion molecule , or icam- ), cd (formerly known as lymphocyte function-associated antigen or lfa- ), cd (maturation marker), and cd (formerly known as b - ) on the monocytes as well as an increase of mrna for il- . however, in the presence of anti-il- antibody or in highly purified monocyte cultures (i.e., depleted of cd þ t cells) there was no increase in mhc class ii expression. so, apparently, aluminum adjuvanteinduced monocyte-derived cytokines stimulated cd þ t cells to secrete il- , which in turn stimulated mhc class ii expression on the monocyte surface. rimaniol and coworkers cultivated human monocytes (pbmc) in medium alone or medium containing aluminum hydroxide adjuvant and observed the phenotypic macrophage changes. the changes encountered involved significant upregulation of hla-dr, as well as cd and cd . almost % of the macrophages obtained were positive for the scavenger receptor cd . however, incubation with aluminum hydroxide downregulated both fc g r and cd . macrophages, as they expressed a dendritic cell (dc)elike phenotype after incubation with aluminum hydroxide (hla-dr high , cd high , and cd À ), were further investigated for the expression of dce specific markers. the expression of cd increased after h of incubation with aluminum hydroxide, compared with noneal(oh) -stimulated cells. it turned out that adjuvant-stimulated macrophages were also superior in antigen presentation. based on these findings, rimaniol et al. concluded that stimulation with aluminum adjuvant led to differentiation of the macrophages into a form sharing some features with, but still distinctly different from, dcs. no similar data are at the moment available for the calcium phosphate adjuvant. in , a group at university of lausanne, headed by j€ urg tschopp, defined the inflammasome as "a molecular platform triggering activation of inflammatory caspases and processing of pro-il-b." this initiated a new line of research leading to a possible explanation for the mechanisms of action of aluminum adjuvants in the early phases of the immune response with the stimulation and excretion of proinflammatory cytokines. according to this approach uptake of al-adjuvanted vaccines by dcs is accompanied by k þ efflux and three intracellular proteins, known as nalp (also known as cryopyrin), cardinal, and asc, then join to form the so-called nalp inflammasome (fig. . ) , possibly through phagosomal destabilization. upon assembly the nalp inflammasome induces cleavage of the -kda procaspase- turning it into the active caspase- enzyme, which is able to cleave pro-il- b and pro-il- into their active counterparts: il- b and il- . these can then leave the dc as active, proinflammatory cytokines. e in nalp À/À mice no significant increase in il- b was seen when compared with the level seen in mice receiving saline or antigen alone, and since the process could take place in myd -deficient mice, it was not considered myd dependent. the synthesis of pro-il- b and pro-il- is affected by tlr agonists reacting with tlrs on the surface of the dc. upon reaction with surface-associated tlrs, it is believed that the nuclear factor (nf)-kb pathway is activated and the genes for pro-il- b and pro-il- are transcribed in the nucleus of the dc. additional mechanisms may hypothetically contribute to the availability of pro-il- b and pro-il- in vivo. for example, the antigen itself may contain pamps and thereby fulfill the function as tlr agonists. this draws a line back to the original sustained-release theory, as antigen released from an al-adjuvanted depot, as a consequence of interaction with interstitial fluid, may expose antigen-associated pamps to surface tlrs on nearby dcs attracted to the inoculum at the injection site. apparently, the activation of the nalp inflammasome in dc's is not limited to being a consequence of uptake of aluminum adjuvants. the nalp inflammasome can also be activated by uptake of, e.g., uric acid crystals. uric acid crystals are very powerful danger signals released from dying cells as breakdown products of nucleic acids and, in what appears to be a combination of the two views, it has been suggested that phagocytic cells taking up aluminum adjuvants may release uric acid crystals as danger signals, stimulating the formation of the nalp inflammasome in dcs. it remains to be established if other danger signals, such as hsp (a kda heat shock protein) also lead to the stimulation of the nalp inflammasome, but there are some indications that it may be the case. one observation considered supportive was reported by alexzander asea's group from the harvard medical school. they demonstrated that extracellular hsp added to human monocyte cultures elicited a rapid intracellular ca þþ flux, activated nuclear factor nf-kb, and upregulated the expression of proinflammatory cytokines, tnf-a, il- b, and il- . in a follow-up study, they demonstrated that hsp utilized both the tlr and tlr receptors for proinflammatory signal transduction in a cd -dependent fashion. a study of the literature suggests a difference in the profile related to stimulation of ige between the aluminum-and calcium-based adjuvants. the ability of aluminum adjuvants to stimulate the production of ige as part of the overall th profile is well-established. , although this has often been mentioned as a disadvantage, it has been difficult to demonstrate cases in which vaccination with aluminum adjuvants has led to ige-mediated allergy toward the vaccine antigen in practical vaccination. in contrast, aluminum adjuvants have been used to hyposensitize allergic patients for many years with good results, e.g., with the alutard series of vaccines (table . ). much of the work on the ige stimulation by aluminum adjuvants in animal models has been carried out using a dual setup model in which rodents were immunized using either aluminum adjuvant or fca, respectively, and antibody and cytokine profiles were compared. uede and coworkers in japan , pioneered this line of research three decades ago using keyhole limpet hemocyanin as antigen and demonstrated the involvement glycosylation-enhancing factors and fc g r þ t cells in a dichotomous regulatory pathway where aluminum adjuvant stimulated the synthesis of ige, whereas fca suppressed it. brewer and coworkers used the same approach of comparing the adjuvant profiles of aluminum hydroxide vs fca using gene-disrupted mice. they found that there was no ige production in il- gene-disrupted mice (il- À/À ) regardless of whether aluminum adjuvant or fca was used as adjuvant. this suggests that il- is an essential prerequisite for the induction of ige by aluminum adjuvants. the literature data suggest that the calcium phosphate adjuvant does not lead to significant stimulation of ige antibodies. vassilev compared passive cutaneous anaphylaxia in guinea pigs after two immunizations with either aluminum or calcium phosphate adjuvant using tetanus toxoid as antigen. he found that calcium phosphateeadjuvanted guinea pigs only had insignificant ige titers compared with the group that had received al-adjuvanted vaccines. in general, the research in this field is sparse and there are at present no data on the interleukin profile after immunization with calcium phosphate to illustrate possible underlying differences in the mechanisms behind such a difference. there are some interesting similarities between the immune response (e.g., stimulation of ige and eosinophilia) elicited by some helminthic parasites and the immune response following immunization with aluminum adjuvants that makes these adjuvants interesting candidates for antiparasitic vaccines. early experimental data suggested a protective superiority of specific ige after aluminum-adjuvanted vaccination in animal models against schistosomiasis infections. , aluminum is normally found in the blood and serum of humans and animals whether or not they have been vaccinated using aluminum adjuvants. the major source of this aluminum is apparently oral intake with the food and drinking water. persons with normal kidney function are known to excrete aluminum with the urine, whereas persons with impaired renal function may to some extent accumulate it and may over a life-long exposure reach al levels associated with systemic adverse reactions. the exposure to aluminum from vaccination, seen over a lifetime, is minimal compared with the daily intake of aluminum by drinking water, antiperspirants, and food additives in convenience food. for example, bread made with aluminum-based baking powder may contain up to mg aluminum per slice, and processed american cheese contains as much as mg aluminum per slice. even if it is taken into consideration that only as little as . % of the ingested aluminum may be taken up from the gastrointestinal tract, exposure to aluminum from the use of adsorbed vaccines in normal vaccination schedules will still be minimal in comparison. martyn and coworkers, based on a study in britain, reported the average daily intake of aluminum by humans from drinking water to be e mg. a major difference between aluminum-and calcium-based adjuvants lies in the in vivo clearing of the adjuvant inoculum and the metabolic fate of the degradation products. upon degradation of calcium phosphate, the two constituents can be reutilized in the normal physiological pathways for ca þþ and po À respectively, whereas in contrast to other metallic ions, like zn þþ and mg þþ , aluminum apparently does not act as essential trace element or coenzyme in the normal metabolism. however, previous claims that aluminum adjuvants are not broken down in situ and excreted have been shown to be incorrect. the in vivo clearing of parentally administered aluminum adjuvants has been investigated in rabbits by flarend et al. using adjuvants prepared from the isotope al. blood-and urine-excreted al was followed using accelerator mass spectroscopy for a period of days. as early as h following intramuscular (im) injection, radioactive labeled al could be detected in the blood and it was found that approximately three times more al was excreted from animals vaccinated with aluminum phosphate than those vaccinated with aluminum hydroxide. assumingly, interstitial fluid containing organic acids with an a-hydroxy carboxylic acid, able to chelate al, reacted more readily with aluminum phosphate than with aluminum hydroxide. at day , the rabbits were euthanized, the main organs were digested using nitric acid, and the radioactivity measured. the relative tissue distribution of radiolabeled al was: kidney > spleen > liver > heart > lymph node > brain. it is likely that the excretion through blood and urine described earlier primarily involves al dissolved under the influence of interstitial fluid, whereas the radioactivity detected in lymph nodes and spleen also involved al adjuvant taken up by apcs. following injection of aluminum hydroxide adjuvant containing . mg al the normal plasma concentration of ng al/ml only rose by approximately ng al/ml in flarend's rabbits. according to the calculation of flarend, a similar al dose injected into humans, provided similar clearing kinetics existed, would lead to an estimated increase of serum al of only . ng al/ml, equaling . % above the normal level of approximately ng al/ml. as the applied dose of . mg al corresponds to what is normally used in human vaccines, it seems that the amount of aluminum administered via vaccination does not contribute significantly to the normal exposure to aluminum in humans and serum levels of aluminum. aluminum hydroxide and aluminum phosphate adjuvants have been used for more than half a century now and are generally regarded as safe when used according to the current immunization schedules. in , the us ncvdg working group on safety evaluation of vaccine adjuvants with the participation of the us food and drug administration representatives concluded that "the extensive experience with this class of adjuvant for vaccine use has indicated that it is safe." this issue has been extensively reviewed previously. there is no evidence that aluminum adjuvants themselves should be immunogenic and act as haptens; accordingly they are not likely to cause harmful immune complex reactions and observations of contact hypersensitivity reactions are not commonly seen. , the aluminum adjuvants are not in themselves pyrogenic, and there is no evidence of carcinogenicity or teratogenicity attributed to their use. cases of local reactions have been reported. these may include swellings, indurations, erythemas, and cutaneous nodules, which can persist for up to weeks or sometimes longer. these reports often describe cases of hyposensitization of allergic patients who receive a large number of injections of adsorbed allergenic extracts over a limited period. in a vaccination program in sweden, elisabeth bergfors and her colleagues found itching local reactions in . % out of , vaccinees. a number of side effects observed after vaccination with adjuvanted vaccines must, however, be attributed to the vaccine preservatives, like thiomersal, betapropriolactone, or formaldehyde or, as mentioned, to bacterial toxins from the antigen preparation. significant resources have been spent on throwing light on a possible link between aluminum exposure and the prevalence of alzheimer disease. some researchers found aluminum deposits in ad brain tissue biopsies, , whereas others have not. , in a later report, it was suggested that the aluminum detection was an artifact caused by the staining reagents used in the preparation of the specimen. the canadian alzheimer society (http://www.alzheimer.ca/en/research/ alzheimer-s-disease-research/aluminum) concluded on their webpage: "at this point, there is no convincing evidence that aluminum increases a person's risk of developing alzheimer's disease." aluminum and calcium adjuvants should, along with water-in-oil emulsions, be regarded as depot-forming or repository adjuvants. with these adjuvants the formation of a temporary inflammatory focus attracting immunocompetent cells shortly after injection must be expected. , upon injection macrophages are attracted to the site to phagocytize and clear the inoculum. the local reaction may be negligible if the inoculum is rapidly dispersed from the injection site. however, if the inoculum resides for a prolonged period at the injection site (as is the case with repository adjuvants like mineral adjuvants or water-in-oil emulsions), then in situ accumulation of phagocytic and immunocompetent cells may in some cases manifest itself as an inflammatory focus accompanied by a transient swelling, local irritation, and redness. some observations of aluminum-adsorbed vaccines giving rise to more local reactions than unadsorbed vaccines with plain toxoid could in part be explained by the plain toxoid vaccine being dispersed from the injection site before a local reaction was established. any visible or palpable reaction at the injection site is in principle non grata, as it hinders the obtaining of a hypothetical and nonreactogenic "ideal adjuvant." however, it is important to realize that the mechanisms described are part of a normally functioning immune system. hence, use of repository adjuvants without temporarily also inducing an inflammatory focus around the inoculum may not be achievable. there are inconsistent observations regarding whether adsorption onto aluminum adjuvants leads to increased or decreased vaccine reactogenicity. , however, butler et al. found that adsorption onto aluminum hydroxide (alhydrogel) significantly reduced the side effects with combined diphteria-tetanus-pertussis (dtp) vaccines. the binding affinity of lipopolysaccharide (lps) to aluminum hydroxide is well established and was much higher, than to aluminum phosphate, approximately mg/mg al versus approximately mg/mg al. this difference is ascribed to the phosphate groups of lps giving a higher degree of ligand exchange with aluminum hydroxide than with aluminum phosphate. it is conceivable that the acute toxicity is reduced in adsorbed vaccines simply by a delayed release of toxic vaccine constituents, like pertussis toxin, peptidoglycans from gramnegative cell walls, or lps from the injection site. norimatsu found that adsorption of lps onto aluminum hydroxide prior to injection inhibited or mitigated systemic effects like the trembling, transient leucopenia and elevated serum tnf-a otherwise observed following im injection of lps in saline. also, the level of il- after administration of lps was reduced if the lps was adsorbed to aluminum hydroxide prior to injection. attempts have been made to link the presence of a local inflammatory focus in the myofascii (macrophagic myofasciitis, mmf) after im injections of al-adjuvanted vaccines to conditions, like myalgia and muscle fatigue. such manifestations can partly be explained by the formation of adjuvant granulomas in the muscle tissue. however, mmf was also claimed to be associated with neurological disorders having no obvious etiologic relation to the vaccination. however, such correlations are associated with statistical challenges. due to the very high vaccination coverage in the western countries, it is expected statistically that patients suffering from a wide range of unrelated diseases would all have been vaccinated with al-containing vaccines at some point in their medical history. in a controlled study in cynomolgus monkeys, it was not possible to detect any histological changes besides the local inflammatory focus itself and no abnormal clinical signs were associated with it. vaccinations may be given subcutaneously (sc) or im and the injection modus is not without importance in relation to local reactogenicity. when immunizing by the sc route the vaccine inoculum is introduced into a compartment with numerous sensory neurons (in contrast to the im compartment). the introduction of a local inflammatory response here may more easily lead to irritation and itching reactions. besides, a transient swelling, as a consequence of the inflammatory focus formed, may more easily be palpable through the skin. when immunizing by the im route, even a similar size swelling may be less easily visible and palpable as it is located in deeper lying tissue. when evaluating the profile of an adjuvant for possible new applications, very few adjuvants can match the aluminum adjuvants in terms of records of efficacy and safety profiles from a period of use reaching practically over an entire life span of humans. the aluminum adjuvants have their limitations, due to their sensitivity to freezing and to their apparent th -biased profile. however, it should be borne in mind that most of the pioneering work that led to the conclusion that aluminum adjuvants gave a fairly clear th stimulation was carried out at a time when only the th and th subsets were recognized. since then another three effector t-cell subsets have been identified: th , regulatory t cells, and follicular t-helper cells (t fh ). additional research is required to see to what extent, if any, aluminum-or calcium-based adjuvants may encompass also the stimulation of these t-cell subsets. over the past years, there has been an increasing interest in calcium phosphate as adjuvant, not only for conventional vaccines but also for the preparation of adsorbed allergens. calcium phosphate, being a natural constituent of the body and hence fully physiologically compatible, constitutes an interesting alternative to the aluminum adjuvants not yet fully explored. immunological adjuvants; . technical report series . world health organization vaccine adjuvants immunological notes xvii to xxiv rate of disappearance of diphtheria toxoid injected into rabbits and guinea-pigs toxoid precipitated with alum developments in diphtheria prophylaxis uber die f€ ahigkeit des tonerde-pr€ aparates b, diphtherie-toxin zu adsorbieren diphtheria immunization with fluid toxoid and alum-precipitated toxoid purification and adsorption of diphtheria toxoid purified precipitated diphtheria toxoid of constant composition uber ein tonerde-gel von der formel al (oh) . ii. mitteilung € uber hydrate und hydrogele uber die hydroxide und ihre hydrate in den verschiedenen tonerde-gelen aluminium compounds used as adjuvants in vaccines measuring the surface area of aluminium hydroxide adjuvant structure and adsorption properties of commercial calcium phosphate adjuvant avian infectious bronchitis: the protection afforded by an inactivated virus vaccine experiments with an inactivated hepatitis leptospirosis vaccine in vaccination programmes for dogs early protection of pigs against foot-and-mouth disease the influence of aluminium hydroxide content, dose volume and the inclusion of saponin on the efficacy of inactivated foot-and-mouth disease vaccines comparative evaluation of the potency of beta-propiolactone inactivated newcastle disease vaccines prepared from a lentogenic and a velogenic strain comparison of alum-adsorbed or non-alum-adsorbed oil emulsion vaccines containing, either pilate or non-pilate bacteroides nodosus cells in inducing and maintaining resistance of sheep to experimental foot rot vaccination against canine bordetellosis using an aluminium hydroxide adjuvant vaccine protective antigens in bovine pasteurellosis leptospira interrogans serovar. pomona vaccines with different adjuvants in cattle immunogenic effects of culture-derived exoantigens of cooperia punctata on calves before and after challenge exposure with infective larvae inocu ation of pigs against trichinella spiralis using larval excretory-secretory antigens resistance of onchocerca lienalis microfilariae in mice conferred by egg antigens of homologous and heterologous onchocerca species preparation and use of calcium phosphate adsorbed vaccines simultaneous administration of diphtheria-tetanus-pertussis-polio and hepatitis b vaccines in a simplified immunization program: immune response to diphtheria toxoid, tetanus toxoid, pertussis, and hepatitis b surface antigen humoral response in rabbits immunized with calcium phosphate adjuvanted hiv- gp antigen calcium phosphate adjuvanted allergens adjuvant modulation of immune responses to tuberculosis subunit vaccines validation of the shake test for detecting freeze damage to adsorbed vaccines structural damages in adsorbed vaccines affected by freezing inhibition of aggregation of aluminum hydroxide adjuvant during freezing and drying stabilization of a recombinant ricin toxin a subunit vaccine through lyophilization lack of adjuvant effect of alpo on purified influenza virus hemagglutinins in man the present status of field and laboratory studies of typhoid and paratyphoid vaccines immune response to uncoupled peptides of foot-and-mouth disease virus immunological priming with synthetic peptides of foot-and-mouth disease virus comparison of antibody avidity and titre elicited by peptide as a protein conjugate or as expressed in vaccinia the influence of different adjuvants on the immune response to a synthetic peptide comprising amino acid residues - of herpes simplex virus type enhancement of dna vaccine potency using conventional aluminum adjuvants co-delivery of a dna vaccine and a protein vaccine with aluminium phosphate stimulates a potent and multivalent immune response influence of elemental impurities in aluminum hydroxide adjuvant on the stability of inactivated japanese encephalitis vaccine, ixiaroÒ vaccines for human use code of federal regulations the effect of adjuvant calcium phosphate coating on a porous-coated femoral stem cpg motifs in bacterial dna and their immune effects immunopotentiating effects of the adjuvants sgp and quil-a. i. antibody responses to t-dependent and t-independent antigens adjuvant properties of aluminum and calcium modulation of the human immune response by the non-toxic and non-pyrogenic adjuvant aluminium hydroxide: effect on antigen uptake and antigen presentation characterization of aluminium hydroxide for use as an adjuvant in parenteral vaccines contribution of electrostatic and hydrophobic interactions to the adsorption of proteins by aluminium-containing adjuvants effect of anions on model aluminium-adjuvant-containing vaccines elutability of proteins from aluminium-containing vaccine adjuvants by treatment with surfactants effect of ph on the elution of model antigens from aluminiumcontaining adjuvants predicting the adsorption of proteins by aluminium-containing adjuvants a novel bipolar mode of attachment to aluminium-containing adjuvants by bbg na, a recombinant subunit hrsv vaccine mechanism of adsorption of hepatitis b surface antigen by aluminium hydroxide adjuvant effect of the degree of phosphate substitution in aluminium hydroxide adjuvant on the adsorption of phosphorylated proteins the adsorption of serum proteins to aluminium hydroxide gel examined by means of quantitative immunoelectrophoresis the theory and practical application of adjuvants measurement of protein using bicinchoninic acid an enzyme-linked immunosorbent assay for the detection of antitetanus toxoid antibody using aluminium-adsorbed coating antigen in vitro assessment of viral antigen content in inactivated aluminium hydroxide adjuvanted vaccines relationship between the strength of antigen adsorption to an aluminum-containing adjuvant and the immune response effect of the strength of adsorption of hepatitis b surface antigen to aluminum hydroxide adjuvant on the immune response effect of the strength of adsorption of hiv sf dv gp to aluminum-containing adjuvants on the immune response the constitution and fundamental properties of solids and liquids the langmuir isotherm: a commonly applied but misleading approach for the analysis of protein adsorption behavior over the adsorption in solution state equations of the solid gas interface layer insights into the modeling of adsorption isotherm systems the comparative selectivity of adjuvants for humoral and cell-mediated immunity. ii. effect on delayed-type hypersensitivity in the mouse and guinea pig, and cell-mediated immunity to tumour antigens in the mouse of freund's incomplete and complete adjuvants, alhydrogel, corynebacterium parvum, bordetella pertussis, muramyl dipeptide and saponin induction of protective class i mhc-restricted ctl in mice by a recombinant influenza vaccine in aluminium hydroxide adjuvant eosinophil response to alum adjuvants: involvement of t cells in non-antigen-dependent mechanisms different t helper cell subsets elicited in mice utilizing two different adjuvant vehicles. the role of endogenous interleukin-l in proliferative responses a limited role of il- in immuneenhancement by adjuvants adsorption to aluminium hydroxide promotes the activity of il- as an adjuvant for antibody as well as type cytokine responses to hiv- gp interleukin- plays a role in both the alum-induced t helper response and the t helper response induced by alum-adsorbed interleukin- in interleukin- -deficient mice, alum not only generates t helper responses equivalent to freund's complete adjuvant, but continues to induce t helper cytokine production aluminium hydroxide adjuvant initiates strong antigen-specific th responses in the absence of il- -or il- -mediated signaling the common vaccine adjuvant aluminum hgydroxide up-regulates accessory properties of human monocytes via an interleukin- -dependent mechanism aluminium hydroxide adjuvant induces macrophage differentiation towards a specialized antigen-presenting cell type the inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of pro-il-b silica crystals and aluminum salts activate the nalp inflammasome via phagosomal destabilization cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the nalp inflammasome aluminum hydroxide adjuvants activate caspase- and induce il- beta and il- release cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by nlrp crucial role for the nalp inflammasome in the immunostimulatory properties of aluminium adjuvants molecular identification of a danger signal that alerts the immune system to dying cells uric acid: a possible mediator of the adjuvant effect of alum in mice immunized with ovalbumin hsp stimulates cytokine production through a cd -dependent pathway, demonstrating its dual role as a chaperone and a cytokine novel signal transduction pathway utilized by extracellular hsp influence of adjuvants on the quantity, affinity, isotype and epitope specificity of murine antibodies hapten-specific ige antibody responses in mice. i. secondary ige response in irradiated recipients of syngeneic primed spleen cells formation of ige binding factors by rat t lymphocytes. v. effect of adjuvant for the priming immunization on the nature of ig e binding factors formed by antigenic stimulation formation of ige binding factors by rat t lymphocytes. vi. cellular mechanisms for the formation of ige-potentiating factor and ige-suppressive factor by antigenic stimulation of antigen primed spleen cells aluminium phosphate but not calcium phosphate stimulates the specific ige response in guinea pigs to tetanus toxoid induction of resistance to schistosoma mansoni by immunization with subfractions of worms protection against schistosoma mansoni achieved by immunization with sonicated parasite updated aluminum pharmacokinetics following infant exposures through diet and vaccination aluminum and alzheimer's disease: after a century of controversy, is there a plausible link ? geographical relation between alzheimers disease and aluminium in drinking water in vivo absorption of aluminium containing vaccines using al safety evaluation of vaccine adjuvants safety evaluation of vaccine adjuvants contact sensitivity to aluminium adjuvants e a balance between toxicity and adjuvanticity persistent subcutaneous nodules in children hyposensitized with aluminium-containing allergen extracts unexpectedly high incidence of persistent itching nodules and delayed hypersensitivity to aluminium in children after use of adsorbed vaccines from a single manufacturer lipopolysaccharides of bordetella pertussis endotoxin alzheimer's disease: x-ray spectrometric evidence of aluminum accumulation in neurofibrillary tangle-bearing neurons brain aluminum, magnesium and phosphorus contents of control and alzheimer-diseased patients absence of aluminium in alzheimer's disease brain tissue: electron microprobe and ion microprobe studies absence of aluminium in neuritic plaque cores in alzheimer's disease microanalysis of senile plaques using nuclear microscopy local tissue irritating effects and adjuvant activities of calcium phosphate and aluminium hydroxide with different physical properties reactions and antibody responses to reinforcing doses of adsorbed and plain tetanus vaccines aluminium compounds as adjuvants for vaccines and toxoids in man: a review advantages of aluminium hydroxide adsorbed combined diphtheria, tetanus, and pertussis vaccines for the immunization of infants detoxification of endotoxin by aluminium hydroxide adjuvant effects of aluminium adjuvant on systemic reactions of lipopolysaccharides in swine central nervous system disease in patients with macrophagic myofasciitis the artwork in fig. key: cord- - adloi o authors: cunha, rafes d. s.; da silva junior, camilo l.; costa, camilla a.; de aguiar, hulliana m.; junqueira júnior, danilo g. title: comparison of immunity against canine distemper, adenovirus and parvovirus after vaccination with two multivalent canine vaccines date: - - journal: vet med sci doi: . /vms . sha: doc_id: cord_uid: adloi o background: viral diseases are a major cause of morbidity and mortality in puppies. there is a belief among veterinary practitioners and even educational institutions that the vaccines made in brazil against canine distemper virus (cdv), canine parvovirus (cpv) and canine adenovirus (cav) are ineffective or only partially effective. objectives: this study aimed at comparing the immunity of two multivalent vaccines in adult dogs in the city of uberlândia, minas gerais state, brazil. methods: the study was carried out at the animal protection association and a total of adult mongrel dogs were selected and divided into two groups. group a was immunized with two doses of elevencell(®) vaccine and group b received two doses of imported vaccine from the united states; each group was made up of females and males. results: in group a, the elevencell vaccine generated a protective antibody titre against cdv in out of subjects ( . %), cpv in out of subjects ( . %) and cav in out of subjects ( . %). in group b, the imported us vaccine generated a protective antibody titre against cdv in out of subjects ( . ), cpv in out of subjects ( %) and cav in out of subjects ( . %). there was no statistical difference between titres generated between vaccine types for any of the three diseases tested. conclusion: elevencell vaccine titres were not inferior to the imported us vaccine in conferring protective titres against cdv, cpv and cah, which confirms the efficacy of this product. viral diseases are a major cause of morbidity and mortality in puppies. in this canine population, there is a higher prevalence of canine distemper, parvovirosis and canine infectious hepatitis (vila nova et al., ) . these three diseases are aetiologically different, but they can be prevented by vaccination with recombinant or live-attenuated vaccines (day, horzinek, schultz, & squires, ) . canine distemper virus (cdv) induces several clinical signs, including fever, dyspnoea, diarrhoea and neurological disorders. these signs may vary according to the host immune status and virus strain. puppies are the most susceptible group to this infection and present the highest fatality rate (martella, elia, & buonavoglia, ) . parvoviruses is caused by canine parvovirus type (cpv- ), characterized by tropism through rapidly dividing cell lines and affecting dogs at different ages. this disease causes a severe enteric infection with bloody diarrhoea, immune suppression and also high fatality rates. the continuous incidence of enteritis is due to the ability of the virus to mutate, which gives rise to new, more resistant and virulent subspecies (goddard & leisewitz, ) . caused by canine adenovirus type (cav- ). this virus has tropism for hepatocytes and endothelial cells, which can cause hepatocellular necrosis and systemic bleeding. unvaccinated puppies are the most susceptible to this infection and present non-specific clinical signs, which requires differential diagnosis of other diseases such as canine distemper (decaro, martella, & buonavoglia, day et al., ) . therefore, to choose an appropriate vaccine and the right age for vaccination, it is crucial to seek veterinary advice. there is a belief among veterinary practitioners or even educational institutions that the vaccines made in brazil against cdv, cpv and cav are ineffective or only partially effective. however, there are no published scientific data to support this. a study carried out in viçosa, minas gerais (brazil), showed that the facility where vaccination is performed (veterinary clinics or agricultural stores) is not a determining factor for successful immunization, but rather adherence to the schedule recommended (monti, viana, dias, moraes, & salcedo, ) . the lack of research providing a better understanding of the effectiveness of vaccines made in brazil may influence the opinion of clinicians and pet owners when choosing the best immunogen. thus, this study aimed to compare two commercial vaccines, one made in brazil and another coming from abroad, for efficacy against three diseases, namely: canine distemper, parvovirosis and canine infectious hepatitis. this study consisted of a randomized double-blind comparative trial. all procedures were evaluated and approved by the ethics committee on the use of animals at the centro universitário do triângulo (unitri) under the protocol / - . the data that support the findings of this study are available on request from the corresponding author. the data are not publicly available due to ethical restrictions. this trial was performed at associação de proteção animal (animal protection association, apa in short) in uberlândia, minas gerais state, brazil. apa, an institution founded in ,which has a total of housing units divided into three sectors for dogs, as well as a nursery with housing units for cats and dogs plus two catteries, totalling dogs and about cats. these animals were rescued from the streets, where they had been abandoned, abused or injured. for this study, the criteria for inclusion were animals that had no clinical signs of distemper, parvoviruses and infectious hepatitis, they were dewormed, presented with a medical history inside the shelter (more than a sheltered year) and had negative results in the colorimetric test for the studied antigens. animals with a change in the physical examination, under the age of or over years, less than a year housed or had positive results in the colorimetric test were excluded. a total of dogs were selected (sampling error %), half of them males and half females. the animals studied were mongrel adult dogs aged between and years that received the same diet plus water (ad libitum) and were housed in the same housing unit. all animals underwent a thorough physical examination by a veterinarian in order to check for the presence of petechiae, ectoparasites, overt organomegaly and any other abnormalities that could be identified in the examination and interfere with the results. randomization was adopted first stratified by sex, by selecting males and females. then they were separated into blocks of two animals with two sequences of intervention. to guarantee the blinding of the study, the researchers had no contact with vaccines and animals until the moment of the vaccination. the vaccines were stored, prepared and coded by a guest veterinarian who was unaware of the purpose of the experiment. thus, both animals and researchers were blinded for the protocol used in the vaccination. at the end, each group was composed of males and females. group a was given v elevencell vac (made in brazil at labovet ® ) and group b received immunization with a vaccine imported from the united states (vangard ® plus, zoetis inc.). one of the vaccines used in this study, brand name v elevencell vac, contains live-attenuated virus antigens of distemper, canine parvovirus, infectious hepatitis, adenovirus type , canine parainfluenza virus, coronavirus-inactivated antigen and five leptospira serovars (l. samples were collected on two occasions: day (also known as d blood samples were collected from the cephalic or saphenous vein and refrigerated for clot retraction, followed by centrifugation and serum separation. serum was stored at a temperature of − °c until the tests were performed. all analyses were performed in a clinical laboratory at unitri. the pre-and post-vaccination responses were evaluated using the commercially available kit immunocomb ® (biogal galed labs) based on solid-phase 'dot'-elisa technology and designed for detecting seru-migg or igm levels, validated against gold standard tests: virus neutralization assay(vn) and haemagglutination inhibition assay (hi). in addition, this test kit is a qualitative and quantitative method that provides a diagnosis within min at room temperature (the best results are obtained at a temperature of - ºc) and consists of: (a)a developing plate with wells containing elisa test solutions; (b) an immunocomb card that is inserted in these wells and im- interpretation of the test results according to the manufacturer uses a colour scale from s to s . there are four levels of interpretation:s , negative; s - ,inappropriate immunity; ≥s ,positive; ≥s ,strongly positive. all dogs with a reading equal to or higher than s were regarded as immunized or protected. the same titre was used for all three diseases. the test presented the following values for specificity (sp) and sensitivity (se): cav, sp % and se %; cpv, sp % and se %; cdv, sp % and se % (biogal galed labs acs ltd., ). the cut-point s indicates a significant response of anti-cav antibodies ( : titre in vn), anti-cpv antibodies ( : titre in hi) and anti-cdv antibodies ( : titre in vn). the data for the animals were entered individually into excel spreadsheets (version ; microsoft corp.). as the procedure is a scale test with a non-normal distribution, the median post-vaccination titre response was obtained, as well as its comparison using the mann-whitney non-parametric test at a significance level of %. descriptive statistics were used to calculate the frequencies of animals immunized, and proportions were compared using the binomial test for two proportions at a significance level of %. all analyses were carried out using bioestat . software (ayres, ayres junior, ayres, & santos, ). of the animals selected and randomly distributed into two groups, only were analysed because three were adopted during the trial and one died as a result of trauma unrelated to enrolment in the study. thus, each group consisted of dogs. before immunization, both groups of animals presented results of ≤ on the colorimetric scale, which means that all of them were eligible to take part in the vaccination protocol. when analysing antibody titres against canine distemper, . % ( / ) of the animals of group a were protected (i.e. with a titre of ≥ )and . % ( / ) of group b were protected; thus, there was no significant difference between the groups (p = . ). both groups had a median response of . on the colorimetric scale and again there was no difference between the groups. a was shown to have a protective titre of ≥ in . % ( / ) and group b in % ( / ). there was no statistical difference between the groups (p = . ). both groups had a median response of on the colorimetric scale and again there was no difference between the groups. for analysis of antibody titres against adenovirus, group a was shown to have a protective titre of ≥ in . % ( / ) and group b in . % ( / ); thus, there was no statistical difference (p = . ). group a had a median response of . for colorimetric titration and group b showed a median response of . . however, these differences were not statistically significant. table shows the frequency of test results for both groups distributed according to the colorimetric scale of the immunocomb ® kit. randomized trials are a powerful tool for reducing bias. by distributing the animals randomly into groups, this ensures uniformity between them. coupled with a double-blind strategy, this helps to avoid any bias that could favour a particular treatment or control (oliveira & parente, ) . although no statistical difference between the two vaccines has been shown, a comparable proportion of animals was protected using vaccine v made in brazil, which reinforces the quality of the product in comparison to the vaccine imported from the united states. several different factors can affect vaccine induction of a protective titre and may account for the lack of an appropriate response in some animals: factors such as storage conditions, nutritional status of the animal, maternal antibody titres and vaccine immunogenicity (day et al., ; monti et al., ) . in relation to storage conditions, the vaccines used in this study were stored according to both manufacturers' guidelines and normative instructions (ima, ) at a temperature between °c and °c in a cold chamber, which ensures the quality of the products. another common cause of vaccination failure involves high levels of maternal antibodies, which can inhibit or neutralize the action of the vaccine (nandi, kumar, mohapatra, & ravishankar, ) . however, all animals immunized in this study were adults and therefore there was no correlation between vaccination failure and maternal antibody presence. ecto-and endoparasites can also influence the effect of the vaccine because these parasites extract nutrients from the host, causing weakness, anaemia, increased stress and secondary bacterial infections (bowman, lynn, eberhard, & alcarez, ) . thirty days before the beginning of vaccination, all animals were given fenbendazole, a broad-spectrum benzimidazole anthelmintic drug used against endoparasites, and also fipronil for the control of ectoparasites. additionally, when selecting the animals for this trial, those that presented with apathy, weight loss, pale mucous membranes, petechiae and ectoparasites were excluded from the study. every effort was made to control any variables that could interfere with the immune response of each animal individually. there were limitations to this study that can be addressed in the future. the absence of public and private funding for execution of the project limited the tests that could be carried out, such as complete blood count, imaging tests to evaluate the spleen and liver and also individual quantification of antibodies by spectrophotometry. both vaccines are effective in the protection of dogs and the v elevencell vac made in brazil has been shown to be an appropriate immunogen to induce a strong immune response in a highly challenging environment such as the apa shelter. on behalf of all authors, the corresponding author states that there is no conflict of interest. note: scale: - , inappropriate immunity; - , positive; - , strongly positive. all dogs with a reading equal to or higher than were regarded as immunized or protected. all dogs with a reading of or show the presence of some immune memory cells against the virus tested. manual of infectious hepatitis, parvovirus and distemper igg antibody test kit georgis' parasitology for veterinarians estratégias para vacinação de animais de companhia: cães e gatos wsava guidelines for the vaccination of dogs and cats canine adenoviruses and herpesvirus canine parvovirus. veterinary clinics of north america: small animal practice portaria n° de de outubro de canine distemper virus anticorpos contra o vírus da cinomose de cães vacinados em diferentes estabelecimentos emergence of canine parvovirus- variants and its impact on vaccination understanding randomized controlled trials evaluation of the humoral immune response induced by vaccination for canine distemper and parvovirus: a pilot study key: cord- -ah dvnxv authors: cao, weiping; kim, jin hyang; reber, adrian j.; hoelscher, mary; belser, jessica a.; lu, xiuhua; katz, jacqueline m.; gangappa, shivaprakash; plante, martin; burt, david s.; sambhara, suryaprakash title: nasal delivery of protollin-adjuvanted h n vaccine induces enhanced systemic as well as mucosal immunity in mice date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ah dvnxv sporadic, yet frequent human infections with avian h n influenza a viruses continue to pose a potential pandemic threat. poor immunogenicity of unadjuvanted h n vaccines warrants developing novel adjuvants and formulations as well as alternate delivery systems to improve their immunogenicity and efficacy. here, we show that protollin, a nasal adjuvant composed of neisseria meningitides outer membrane proteins non-covalently linked to shigella flexneri a lipopolysaccharide, is a potent nasal adjuvant for an inactivated split virion h n clade a/viet nam / (a/vn/ / ) vaccine in a mouse model. protollin-adjuvanted vaccines elicited enhanced serum protective hemagglutination inhibition titers, mucosal iga responses, and h n -specific cell-mediated immunity that resulted in complete protection against a lethal challenge with a homologous virus as well as a heterologous clade virus a/indonesia/ / (a/in/ / ). detailed analysis of adaptive immunity revealed that protollin increased the frequency of lymphoid- as well as local tissue-resident antibody-secreting cells, local germinal center reaction of b cells, broad-spectrum of cd t cell response. our findings suggest that nasal delivery of h n vaccine with protollin adjuvant can overcome the poor immunogenicity of h n vaccines, induce both cellular and humoral immune responses, enhance protection against challenge with clade and clade h n viruses and achieve significant antigen dose-sparing. the world has already experienced three influenza pandemics in the th century in addition to the recent pandemic caused by swine-origin h n virus, which has taught us how newly emerging pathogens can be a risk to global populations [ ] . highly pathogenic h n avian influenza viruses continue to be prime candidates for the next influenza pandemic, as they have steadily caused fatal infections in the human population [ ] . so far, direct human-to-human transmission appears to be infrequent; however, the accumulation of mutations may break this genetic barrier and generate an h n virus that is transmissible among humans potentially causing pandemic. the inherently poor immunogenicity of unadjuvanted h n influenza vaccines warranted efforts to explore novel adjuvants and alternate delivery systems to improve immunogenicity and protective efficacy of h n vaccines. intradermal (i.d.), oral and nasal delivery of vaccines represent alternative routes to conventional intramuscular (i.m.) delivery [ ] . unlike i.m. and i.d. routes, oral and nasal deliveries are noninvasive and needle-free that are known to induce mucosal and systemic immune responses [ ] . since degradation of antigens by proteolytic enzymes, poor absorption, need for large doses of antigen, and concerns about tolerance still remain as challenges for oral delivery of non-replicating vaccine antigens [ ] , the nasal route has become an attractive option for vaccination, particularly against respiratory pathogens as the respiratory tract is equipped with pathogen sensing defense mechanisms, has a large surface area for absorption, and is highly vascularized [ , ] . a live attenuated seasonal influenza vaccine, which is delivered intranasally (laiv, flumist Ò , fluenz tm ) (medimmune astrazenica) is approved for healthy individuals of - years of age in the us. however, the absence of correlates of immunity, cold-storage requirement, and potential of genetic recombination with seasonal influenza viruses may limit the use of laiv as a pre-pandemic influenza http vaccine [ ] . therefore, an ideal h n vaccine formulated/adjuvanted for nasal delivery should induce mucosal as well as systemic serological and cellular immune responses that can correlate with protection. protollin, a nasal adjuvant composed of neisseria meningitidis outer membrane proteins (omps) noncovalently complexed to shigella flexneri a lipopolysaccharide (lps) activates the innate immune system via activation of tlr and tlr by omp and lps respectively. protollin has been shown to induce effective systemic as well as mucosal antibody responses in preclinical studies when administered with several viral antigens such as measles, sars corona virus, rsv, and recombinant plague antigen f -v [ ] [ ] [ ] [ ] [ ] . therefore, protollin represents an attractive adjuvant suitable for nasal delivery of pre-pandemic h n vaccines. in this study, we evaluated the immunogenicity and protection against challenge conferred by protollinadjuvanted h n inactivated split vaccine. h n monovalent influenza split vaccines (a/vn/ / and a/in/ / ) were provided by glaxosmithkline vaccines (ste-foy, quebec, canada). protollin from gsk vaccines consists of proteosomes (outer membrane proteins derived from wild-type neisseria meningitidis group b) noncovalently complexed in approximately a : ratio with lipopolysaccharide (lps) isolated from shigella flexneri serotype a. the amounts of protollin were expressed as lg of lps and the amount of h n vaccine was expressed as lg of ha. two virus strains were made by reverse genetic engineering, ha and na from clade a/viet nam/ / (a/vn/ / ) or clade a/indonesia/ / (a/in/ / ) and the remaining six gene segments from a/puerto rico/ / -(pr ). viruses were propagated in -day old embryonated chicken eggs for h. pooled allantoic fluid was clarified by centrifugation, aliquoted, and stored at À °c until use. female balb/c mice (jackson laboratory, bar harbor, maine) weeks of age were anesthetized with intraperitoneal injection (i.p.) of , , -tribromoethanol in tert-amyl alcohol (avertin; sigma-aldrich, st. louis, mo) before nasal inoculation with , , or . mg of h n vaccine, either alone or with mg of protollin [ , ] in a final volume of ml administered slowly drop by drop at ml each per nostril. both the vaccine antigens and adjuvants were mixed prior to immunization. mice nasally administered with adjuvant alone were used as negative controls. four weeks later, mice received a second dose of the same vaccine preparation. one week after boost, mice were euthanized for collection of spleen and bone marrow tissues to assess adaptive immunity. bronchoalveolar lavages (bal) and nasal lavages were also performed at the same time by injecting . ml pbs with protease inhibitors and re-collecting the fluid to measure mucosal antibody response by elisa. mice were bled weeks post-primary and again weeks post-boost to measure hemagglutination inhibition (hi) titers using horse red blood cells. four weeks after boost, mice from each group were challenged with ld of a/vn/ / or a/in/ / viruses. animal were monitored daily for morbidity by changes in body weight and any mouse that lost > % of preinfection body weight was euthanized. animal research was con-ducted under the guidance of the cdc's institutional animal care and use committee. statistical analysis was performed using graphpad prism . software (graphpad software, la jolla, ca). the student's t test was used to analyze differences in antibody isotype between two groups. one-way analysis of variance with bonferroni post analysis was used to analyze differences among treatments. the mann-whitney test was used to determine significance among hi titers. finally, the logrank (mantel-cox) test was used to compare percent survival among groups of mice. all differences were considered statistically significant when the p-value was . . to investigate the adjuvant effect of protollin, mice were nasally administered with , , or . mg of h n vaccine with or without mg of protollin using a prime-booster regime and then challenged with homologous a/vn/ / viruses. all mice immunized with or . mg of h n vaccine alone succumbed to challenge between day and post-challenge, while % of mice that received the highest dose of vaccine ( mg) initially showed significant weight loss but eventually survived challenge ( fig. a and b ). all mice immunized with vaccine plus protollin including mice that received the lowest vaccine antigen dose ( . mg), survived and no significant weight loss was observed ( fig. a and b) . these data demonstrate that protollin significantly enhanced the protective efficacy of h n vaccine. to test whether the protollin-mediated protection extended against cross-clade h n virus, h n vaccine-immunized mice were challenged with the clade virus, a/in/ / . mice immunized with either protollin or vaccine alone ( . or mg) succumbed to challenge ( fig. c and d) . among the mice immunized with mg vaccine alone, % of mice showed % weight loss and eventually recovered and survived. however, protollinadjuvanted vaccine, even at the lowest vaccine dose ( . mg) significantly reduced weight loss and completely protected mice against the lethal challenge ( fig. c and d) . therefore, protollin also enhanced cross-clade protection with significant antigen dosesparing. next, we assessed whether the improved protection conferred by protollin-adjuvanted h n vaccine was due to enhanced antibody titers. mice were immunized as described in fig. and sera were collected at weeks following primary immunization (week ) and booster immunization (week ) to measured hi titers against a homologous a/vn/ / virus as well as heterologous a/in/ / virus. mice immunized with unadjuvanted h n vaccine did not induce detectable antibody titers following primary immunization. however, mice immunized with protollin -adjuvanted h n vaccine ( mg ha) developed low level of antibody titers even after a single vaccination (data not shown). at week , all mice immunized with vaccine plus protollin developed significantly higher levels of hi titers against both homologues and heterologous viruses ( fig. a and c) . however, none of the mice immunized with vaccine alone had detectable hi titers against a/ vn/ / virus ( fig. a) and only a few mice immunized with either or mg ha of unadjuvanted vaccine developed detectable hi titers against a/in/ / virus (fig. c) . furthermore, vaccine alone elicited more igg production than igg a, whereas protollin adjuvantation induced a mixed igg and igg a response with a clear trend towards a t h response (igg a-dominant) ( fig. b and d) . these results indicate that protollin significantly enhanced the immunogenicity of h n vaccine and skewed the immune response towards a t h response. to determine whether protollin increased the frequency of agspecific memory b cells, mice were immunized as described in fig. and the frequency of h n -specific antibody-secreting cells (ascs) in spleen and bone marrow was examined by elispot assay. as shown in fig. a and b, the frequency of h n -specific igg and igm ascs in the spleen increased in mice immunized with unadjuvanted h n vaccine; protollin significantly increased the frequency of h n -specific igg and igm ascs even at the lowest dose ( . mg), although ag-specific igm ascs frequency is much lower than that of ag-specific igg ascs. in bone marrow, the frequency of h n -specific igg ascs was similarly enhanced by the use of protollin as an adjuvant, while the frequency of igm ascs was minimally affected by protollin ( fig. c and d) . together, these data suggest that intranasal administration of h n vaccine with protollin enhanced the overall frequency of ascs with significant dose-sparing effect. next, the activation of vaccine-specific t cells in spleen was assessed in mice immunized with mg h n vaccine with or without protollin. for cd t cells, while the production of il- and ifnc was detectable in mice immunized with unadjuvanted vaccine as compared to the protollin control group, protollin adjuvantation further increased the il- and ifnc production significantly (fig. e ). on the other hand, il- , il- , tnfa, and il- production by activated cd t cells was comparable between protollin alone vs. vaccine group; however, co-administration with protollin and vaccine significantly increased il- , tnfa and il- production from cd t cells. vaccine-specific cd t cells responses were slightly increased upon protollin adjuvantation but this increase was not statistically significant (data not shown). in summary, our data suggest that protollin-adjuvanted h n vaccine enhanced a broad spectrum of the cd t cell activation pathways: t h (ifnc and tnfa), t h (il- and il- ) and t h (il- ). systemic increase in asc frequency by protollin prompted us to assess the extent of homing of ascs back to the local immune induction site. thus, we measured the h n -binding iga by elisa week following booster immunization in the fluids from bronchoalveolar (bal) and nasal lavage from mice that had been immunized as previously described in fig. . the iga secretion from unadjuvanted h n vaccine antigen-immunized mice was barely detectable both in nasal lavage (fig. a) and bal (fig. b ). in contrast, the mice immunized with h n vaccine antigen plus protollin produced a/vn/ / -specific iga at a minimum of fold higher than that of mice immunized with the unadjuvanted vaccine. in addition, protollin significantly enhanced clade a/ in/ / -specific iga secretion both in nasal and bal as compared to vaccine alone ( fig. c and d) ; secretion of igg antibodies was also greatly enhanced by protollin and the reactivity was extended against clade , a/in/ / virus (fig. a-d) . however, the frequency of igm ascs was much lower and minimally affected by protollin (fig. e-h) . these data indicate that nasal administration of protollin-adjuvanted h n vaccine enhanced local mucosal immunity throughout the respiratory tract. next, the activation status of b cells at peak of the primary response (day ) was assessed by flow cytometric analysis. com- pared to mice immunized with vaccine alone, mice immunized with vaccine plus protollin had a higher frequency of b cells participating in gc reaction (fig. a) , higher overall mean fluorescence intensity of cd among gc-participating b cells, and more gc-b cells expressing high level of cd ( fig. c and d) . the plasma cell differentiation was at low level and did not reveal differences among groups (fig. b) . interestingly, protollin alone recruited b cells into gc reaction and induced activation (fig. a) , emphasizing the significant role of innate signaling through tlr and tlr in b cell activation. nonetheless, their recruitment to gc reaction and activation did not result in the production of ag-specific antibodies (fig. ) . we also measured the frequency of the splenic ag-specific igg and igm during the primary response and found that protollin adjuvantation significantly increased the early igg secretion and marginally igm secretion ( fig. e and f) . overall, these data suggest that protollin recruited more naïve b cells at the early phase of the vaccine response. the spread and evolution of highly pathogenic influenza h n virus in birds worldwide and the increasing number of cases of direct transmission to humans leading to fatalities, have raised concern about an imminent h n influenza pandemic [ ] . vacci-nation is unquestionably one of the most cost-effective public health interventions available to protect against such pandemics. however, currently available unadjuvanted h n vaccines administered by i.m. route are poorly immunogenic. new strategies to improve the immunogenicity of vaccines with adjuvants, novel formulations and alternate delivery methodologies to meet the demands of global populations are still urgently needed. in the current study, we evaluated the use of protollin-adjuvanted h n vaccine as a nasally delivered, pre-pandemic vaccine in a mouse model. the mucosal immune system in the respiratory track plays an important role in prevention of influenza virus infection. the upper respiratory tract is equipped with not only the physical and dynamic barrier of mucus as well as both soluble and membrane bound pathogen sensors but also contains nasopharyngealassociated lymphoid tissues (nalt) that are enriched in agspecific mucosal effector cells as well as innate immune cells [ ] . hence, nasal delivery of antigens triggers local mucosal as well as a systemic immune response, which is crucial against respiratory pathogens including influenza [ ] . nasal delivery of split, inactivated influenza vaccine generally requires a mucosal adjuvant to induce strong protective immune responses [ ] . some mucosal adjuvants containing bacterial toxin derivatives, including esherichia coli heat labile enterotoxin (lt) have been clinically eval- . the data is representative of independent experiments ( - mice per group) and error bars represent standard error of the mean (sem). oneway analysis of variance with bonferroni post analysis was used to analyze differences among treatments. the student's t test was used to analyze differences in antibody isotype between two groups. uated with subunit influenza vaccines and ultimately licensed in europe, but soon withdrawn from the market due to an increased incidence of bell's palsy post-vaccination [ ] . clinical trials with proteosome-adjuvanted either monovalent h n or trivalent inactivated vaccines have shown that proteosome-adjuvanted vaccines are well-tolerated, while inducing significantly higher serum hi and mucosal secretory iga titers as compared to unadjuvanted vaccines [ ] [ ] [ ] . preclinical studies using protollin-adjuvanted h n vaccines also showed the similar results [ , ] consistent with this, our data showed that protollin-adjuvanted h n vaccine induced significantly higher levels of mucosal iga antibodies against a/vn/ / virus both in nasal washes and lung, with considerable dose-sparing effect. the breadth of the mucosal iga response was extended against clade virus, a/in / , as a pre-pandemic vaccine formulation, cross-clade immunity is an important and desirable feature, because the current endemic h n viruses continuously evolve in ha antigenicity through antigenic drift and reassortment, thus making it hard to predict which strain will cause a pandemic. protollin also induced cross-reactive igg as well as low, yet detectable levels of igm antibodies. lung-resident memory b cells as well as the mucosal, secretory iga and/or igg antibodies play a crucial role in conferring protection against influenza virus challenge [ , ] . the mice immunized with protollin-adjuvanted h n vaccine were completely protected against lethal challenge with clade and clade viruses. therefore, the protollin-adjuvanted h n represents a novel pre-pandemic vaccine candidate that is efficient in inducing protective mucosal and systemic immunity. the adjuvant properties of protollin have been documented in conjunction with antigens from various infectious agents including measles [ ] , recombinant sars-spike glycoprotein [ ] , respiratory syncytial virus (rsv) [ , ] , and recombinant plague antigen f -v [ ] . the level of serum igg response induced by protollin is generally comparable to that by alum-adjuvanted vaccines and shows a mixed t h /t h response with a t h trend (higher igg a/igg ratio) as compared to unadjuvanted vaccines [ , , ] . consistent with these findings, our data demonstrated that protollin potentiated the immunogenicity of h n vaccine. after a single vaccination, mice immunized with protollin -adjuvanted h n vaccine ( mg) developed detectable levels of serum hi titers. following booster immunization, protollin -adjuvanted h n vaccine significantly increased serum hi titers compared to unadjuvanted vaccine, which coincided with complete protection against homologous virus challenge. the breadth of antibody response was also broadened by protollin-adjuvanted h n vaccine, as they significantly increased serum hi titers against a/in/ / virus compared to the vaccine alone group and fully protected mice against a/in/ / virus challenge. protection against influenza virus infection includes antibodymediated neutralization/blocking of virus and cell-mediated clearance of virus-infected cells. although the antigen-specific cd t cell responses was not significantly enhanced by protollin adjuvantation (data not shown), protollin-adjuvanted vaccines significantly increased the production of cytokines by antigen-specific cd t cells: t h (ifn-c, tnfa), t h (il- ) and t h (il- ). ifnc, as a representative cytokine of t h response promotes overall microbial killing through enhancing phagocytosis and recruiting mononuclear cells into the site of infection, while favoring the production of igg a from b cells [ ] [ ] [ ] . on the other hand, il- along with il- , shapes the t h response and promotes the production of igg isotype [ ] . consistent with the cytokine profiles, the mice immunized with protollin-adjuvanted h n vaccine showed a mixed igg /igg a response with higher production of igg a than igg . il- is typically associated with pathogenesis of inflammatory diseases [ ] . however, the protective role of il- for survival against high dose challenge and severe cases has been recently demonstrated [ , ] . of interesting note, protollin-adjuvanted h n vaccine elicited a sharp increase in il- production as compared to vaccine alone. il- is an anti-inflammatory cytokine produced by natural cd + cd + foxp + regulatory t cells (tregs) as well as antigen-specific cd + and cd + effector t cells [ ] [ ] [ ] . it remains unclear the source of il- in our study, but regardless, the enhanced il- production indicates the fine balance between effector vs. control mechanisms was achieved by protollin. using gene knock-out mice, tlr has been shown to be critical for antigen-specific antibody responses in protollin-adjuvanted rsv vaccine, while myd was required to elicit a balanced t h /t h immune responses. although tlr is required for nanoparticle formation, it is not playing a role in the adjuvanticity of protollin [ ] . in summary, protollin demonstrated a significant antigen dosesparing effect. with protollin-adjuvantation, h n vaccine antigen at the -fold lower dose elicited higher serum hi titers, mucosal antibody responses than the highest vaccine antigen dose ( mg ha/mouse) against both clade and clade h n viruses. these enhanced immune responses culminated in enhanced protection against challenge with lethal doses of h n viruses of either clade or clade . therefore, nasal delivery of protollin-adjuvanted h n vaccines is a promising approach that merits further development to prepare for a h n pandemic. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention or the funding agencies. w.c., j.k., s.s. designed the experiments and interpreted the data; w.c., j.k., a.r. and m.h. did the in vivo experiments; j.b. did the challenge experiment; x.l did the hi assay; w.c., j.k., and s.s. wrote the manuscript. s.g., j.k., m.p. and d.b. edited the manuscript. dsb and mp were employees of the gsk group of companies at the time of the study, own stock options in gsk and are listed as inventors on patents owned by the gsk group of companies. the remaining authors declare no commercial or financial conflict of interest. work was supported by the influenza division, centers for disease control and prevention. . protollin increased the early b cell response at the local lymph nodes and spleen. balb/c mice ( - mice/group) were nasally administered with mg ha h n split virion vaccine antigen with or without mg protollin or protollin alone as a control. a week later, mediastinal lymph nodes and/or spleens were collected and b cells (b + cd À ) were stained. the lymph nodes were analyzed for percentage of gc-participating b cells (b + cd À gl + cd À ) (a), plasma cells (b À cd + ) (b), the mean fluorescence intensity (mfi) of cd (c) as well as % b cells expressing high cd (d) among gc-participating b cells. spleen were harvested and the frequency of a/vn/ / -specific igg + ascs (e) or igm + ascs (f) were measured by elispot assay. the number of a/vn/ / -specific igg + (or igm + ) ascs were normalized against the number of total igg + (igm + ) secreting ascs and presented as % ag-specific igg + (igm + ) b cells. the data is representative of at least independent experiments ( - mice each group) and the error bars represent standard error of the mean (sem). one-way analysis of variance with bonferroni post analysis was used to analyze differences among treatments. two years after pandemic influenza a/ /h n : what have we learned? human influenza a h n virus related to a highly pathogenic avian influenza virus microneedle and mucosal delivery of influenza vaccines mucosal immunity and nasal influenza vaccination development of oral vaccines to stimulate mucosal and systemic immunity: barriers and novel strategies inside the mucosal immune system nasal vaccines live attenuated influenza vaccine a novel intranasal protollin-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice intranasal protollin-formulated recombinant sars s-protein elicits respiratory and serum neutralizing antibodies and protection in mice c bl/ mice are protected from respiratory syncytial virus (rsv) challenge and il- associated pulmonary eosinophilic infiltrates following intranasal immunization with protollin-ersv vaccine murine host responses to respiratory syncytial virus (rsv) following intranasal administration of a protollinadjuvanted, epitope-enhanced recombinant g protein vaccine intranasal protollin/f -v vaccine elicits respiratory and serum antibody responses and protects mice against lethal aerosolized plague infection effects of different adjuvants in the context of intramuscular and intranasal routes on humoral and cellular immune responses induced by detergent-split a/h n influenza vaccines in mice research funding. a framework for decisions about research with hpai h n viruses proteosomeadjuvanted intranasal influenza vaccines: advantages, progress and future considerations use of the inactivated intranasal influenza vaccine and the risk of bell's palsy in switzerland intranasal administration of a proteosome-influenza vaccine is well-tolerated and induces serum and nasal secretion influenza antibodies in healthy human subjects safety and immunogenicity of a proteosome -trivalent inactivated influenza vaccine, given nasally to healthy adults a nasally administered trivalent inactivated influenza vaccine is well tolerated, stimulates both mucosal and systemic immunity, and potentially protects against influenza illness adjuvanted inactivated influenza a(h n ) vaccines induce stronger immunogenicity in mice and confer higher protection in ferrets than unadjuvanted inactivated vaccines memory b cells in the lung participate in protective humoral immune responses to pulmonary influenza virus reinfection cross-protection in mice infected with influenza a virus by the respiratory route is correlated with local iga antibody rather than serum antibody or cytotoxic t cell reactivity interleukin- and interferon-gamma: the quintessence of a mutual antagonistic relationship functional diversity of helper t lymphocytes cellular responses to interferongamma th and th hypercytokinemia as early host response signature in severe pandemic influenza il- deficiency unleashes an influenza-specific th response and enhances survival against high-dose challenge imbalanced pro-and anti-th responses (il- /granulocyte colonystimulating factor) predict fatal outcome in pandemic influenza the development and function of memory regulatory t cells after acute viral infections the regulatory t cells in antiinfluenza antibody response post influenza vaccination effector t cells control lung inflammation during acute influenza virus infection by producing il- tlr and myd control protection and pulmonary granulocytic recruitment in a murine intranasal rsv immunization and challenge model we thank the members of influenza division, centers for disease control and prevention (cdc) for providing reagents and constructive comments during the course of this investigation. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.vaccine. . . . key: cord- -tdvb fhv authors: lv, huibin; wu, nicholas c.; mok, chris k. p. title: covid‐ vaccines: knowing the unknown date: - - journal: eur j immunol doi: . /eji. sha: doc_id: cord_uid: tdvb fhv vaccine development against sars‐cov‐ has drawn attention around the globe due to the exploding pandemic. although covid‐ is caused by a new coronavirus, sars‐cov‐ , previous research on other coronavirus vaccines, such as fipv, sars and mers, has provided valuable information for the rapid development of covid‐ vaccine. however, important knowledge gaps remain – some are specific to sars‐cov‐ , others are fundamental to immunology and vaccinology. here we discuss areas that need to be addressed for covid‐ vaccine development, and what can be learned from examples of vaccine development in the past. since the beginning of the outbreak, the research progress on covid‐ has been remarkable. we are therefore optimistic about the rapid development of covid‐ vaccine. this article is protected by copyright. all rights reserved namely inactivated virus, lipid nanoparticle (lnp)-encapsulated mrna, and plasmid dna, are in phase i clinical trials (https://www.who.int/blueprint/priority-diseases/key-action/novel-coronavirus-landscape-ncov.pdf). other platforms, such as live attenuated virus, protein subunit, are also being evaluated at the pre-clinical stage. although there is an urgent need for these vaccines to become available, several critical features of the covid- vaccine will need to be assessed, based on the experience from previous vaccine development for feline-covs, mers-cov, sars-cov and other viruses ( figure ). it is highly desirable that a potential vaccine will induce a potent antibody response as well as long-term . identifying the epitopes that are targeted by these antibodies will be crucial to determine the antigenic sites on the spike of sars-cov- . although traditionally an effective vaccine should mainly aim to elicit a high titer of neutralizing antibodies, it was found that some non-neutralizing antibodies could provide in vivo protection through antibody dependent cell-mediated cytotoxicity (adcc) [ ] . thus, it will be interesting to distinguish the functions of the antibodies isolated from volunteers in clinical trials in order to understand in molecular terms vaccines' effectiveness. it is also important to note that t-cell immunity was found to be elicited by sars-cov or mers-cov dna vaccines (both express trimeric spike protein) [ , ] . these results imply that using sars-cov- spike protein as immunogen may also elicit cellular immune response, in addition to humoral immune response. there is a concern that some antibodies elicited by the sars-cov- vaccine may cause an adverse effect such as antibody-dependent enhancement (ade) or enhanced respiratory disease (erd) [ suggesting such a potential complication may not be a concern, at least for rbd-based sars-cov- vaccine development [ ]. to enhance the magnitude and quality of the adaptive response, adjuvants are included in some vaccination formulations [ ] . different adjuvants drive different immunological signatures and, hence, are optimal for protection against different pathogens [ ] . for example, adjuvants can modulate the ratio of t helper (th ) and t helper (th ) responses, increase the generation of memory cells, and alter the breadth and affinity of the response [ ] . determining which adjuvants can enhance protective vaccine response to sars-cov- will be important. because immune responses in different age groups are not the same, optimal vaccination strategy may be varied for different age groups [ ] . for example, the mf -adjuvanted influenza vaccine, fluad, is only licensed and approved for adults aged years and older, to elicit a higher protective immune response in the elderly this article is protected by copyright. all rights reserved. (https://www.cdc.gov/flu/prevent/adjuvant.htm). as it is clear that the mortality rate in the elderly is the highest among the covid- patients, a specific vaccination strategy for this group will need to be considered during vaccine development. to accelerate vaccine development, animal infection models for sars-cov- are needed. although macaques show covid- -like disease upon sars-cov- infection, the non-human primate model is usually not readily accessible to most laboratories [ ] . ferrets and golden syrian hamster are presented as alternatives, as all show mild disease signs and virus shedding after challenge with sars-cov- [ , ] , and are usually more widely available than non-human primates. nonetheless, mouse is the most commonly used animal model during vaccine development in general. although wild type mouse is not susceptible to the infection of sars-cov- , transgenic mice that express the human angiotensin-converting enzyme (hace ) receptor, which was previously established for sars-cov study [ ] , showed significant pathogenicity upon the infections of sars-cov- [ ] . expressing hace through adenoviral transduction may provide another possible approach to generate a mouse model for sars-cov- infection. the feasibility of this approach is suggested by a previous study that used adenoviral transduction to generate a mouse model for mers-cov infection [ ] . the advantage of this approach is that it can be applied to multiple genetic backgrounds including outbred mice. these animal models are thus suitable to evaluate vaccine candidates in preclinical settings. there is also a pressing need to define correlates of protection, which have the additional benefit of serving as a benchmark for vaccine evaluation without the need for challenge studies, an approach that has been used for many different pathogens [ ] . a typical example is influenza vaccine, where a hemagglutination inhibition (hai) titer of ≥ : is a surrogate of protection [ ] . however, the exact serological parameters that provide best correlation with protection against sars-cov- infection will need to be investigated. on a related note, evaluating the neutralizing antibody titer against this article is protected by copyright. all rights reserved. therefore, pseudovirus assays that can be performed in a bsl setting are also valuable for vaccine development [ , ] . zoonotic coronaviruses are likely to be a continuous threat [ ] . although generation of a pan-coronaviruses vaccine seems unlikely, owing to the high genetic diversity among different genera, it might be possible to develop a pan-betacoronavirus vaccine to prevent the potential risk from new subtypes identified in bats, pangolins or other species [ ] . indeed, cross-reactivity between different betacoronaviruses has been found in human samples. for example, a conserved cd t-cell epitope can mediate cross-reactive protection between sars-cov and mers-cov [ ] . in addition, our recent study has revealed a conserved antibody epitope shared by sars-cov- and sars-cov [ ] . these results indicate a potential roadmap for the development of universal vaccine against coronaviruses. although it is not possible to predict, at this moment, the strain of the next coronavirus that will jump species, continuing exploration of the b-cell repertoire to identify cross-reactive epitopes will be important for the development of a coronavirus vaccine with high breadth of coverage. taken together, while therapeutic approaches for covid- are urgently needed (reviewed in [ ] ) given the increasing number of active cases, the ultimate goal of establishing sterilizing immunity in this article is protected by copyright. all rights reserved. uninfected individuals will require an sars-cov- vaccine. we are confident that the rapid and collaborative efforts among researchers around the globe will offer an effective countermeasure to covid- in the near future. the sars-cov- receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement vaccine adjuvants: putting innate immunity to work different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens evolution of the immune system in humans from infancy to old age comparative pathogenesis of covid- , mers and sars in a nonhuman primate model simulation of the clinical and pathological manifestations of coronavirus disease (covid- ) in golden syrian hamster model: implications for disease pathogenesis and transmissibility infection and rapid transmission of sars-cov- in ferrets lethal infection of k -hace mice infected with severe acute respiratory syndrome coronavirus the pathogenicity of sars-cov- in hace transgenic mice rapid generation of a mouse model for middle east respiratory syndrome correlates of protection induced by vaccination correlates of protection to influenza virus, where do we go from here? characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov establishment this article is protected by copyright. all rights reserved. and validation of a pseudovirus neutralization assay for sars-cov- a sars-like cluster of circulating bat coronaviruses shows potential for human emergence cells mediate protective immunity against emerging respiratory coronaviruses. immunity a highly conserved cryptic epitope in the receptor-binding domains of sars-cov- and sars-cov controlled modulation of innate immunity to prevent and treat covid- disease we thank profs roberto bruzzone and stanley perlman for helpful discussions. the authors declare no financial or commercial conflict of interest.this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. an ideal covid- vaccine should offer long-term protection with no adverse effect. the current status of covid- vaccine development and factors that need to be considered are discussed. key: cord- - h c yq authors: locht, camille title: vaccines against covid- date: - - journal: anaesth crit care pain med doi: . /j.accpm. . . sha: doc_id: cord_uid: h c yq nan for sars-cov- , it has been demonstrated for dengue . one way to overcome this potential risk is to include antigens/epitopes that generate cell-mediated immunity, particularly via cd + t cells. this has been shown protective against dengue, even in the presence of diseaseenhancing antibodies . especially tissue-resident memory cd + t cells generated in the upper airways may be important for long-lasting protection, as has been shown for influenza . several hundred covid- -specific vaccines are at various stages of development in academia and industry and make use of a variety of different generic platforms, such as inactivated virus, purified recombinant viral proteins with or without adjuvant, replicating and non-replicating viral vectored antigens, antigen-encoding dna or mrna. some of them build on technologies approved for other vaccines, others are novel and have not yet been used for large-scale vaccination. this editorial will focus on vaccines in clinical development with data published in peer-reviewed articles. the first clinical trial data were published in june . the trial was a dose-escalation study of recombinant adenovirus type- vectored s. the vaccine was shown tolerable, although - % of participants reported adverse events, mostly mild or moderate. it induced neutralising antibody and t cell responses with seroconversion in - % of the vaccine recipients. however, pre-existing vector-neutralising antibodies diminished the immune responses. furthermore, immunogenicity was sub-optimal in older participants. this study was followed by a phase , randomised, double-blind trial , including participants. sero-conversion was seen in more than % and neutralising antibodies were generated in % of vaccine recipients. ifn- responses were also seen in roughly % of the vaccinees. again, the vaccine induced lower antibody responses in older participants and subjects with pre-existing antivector immunity. the vaccine at a x viral particles/ml dose is now in a phase trial in brazil. to overcome the immune-interference by pre-existing immunity to the vector, a replicationdeficient simian adenovirus-vectored vaccine was engineered to encode s. a phase / , singleblind, randomised controlled study with this vaccine at x viral particles/ml in healthy adults showed acceptable safety . local and systemic reactions were frequent but could be reduced by paracetamol. the vaccine also induced t cell and antibody responses, including virus-neutralising antibodies. after a booster given to some participants, antibody responses increased neutralising antibodies were generated in all participants. this vaccine is currently in phase in several countries. a combination of two adenovirus-vectored vaccines (types and ), both carrying s, was tested in a non-randomised, non-placebo-controlled phase / trial in a total of volunteers in russia . in phase , each vaccine was tested individually, followed by phase , in which the participants were primed with rad and boosted with rad . the two studies combined included volunteers. adverse events included injection-site pain in % of the participants, as well as hyperthermia, headache, asthenia, and muscle and joint pain. all participants generated high titres of igg recognising the receptor-binding domain of s. neutralising titres were also induced, as well as cd + and cd + t cell responses. although safety and immunogenicity reported in this study were in line with the previous studies on adenovirusvectored vaccines, this study has raised concerns about the validity of the reported results, especially in the context of potential accelerated distribution of the vaccine in the population in russia . an inactivated, alum-adjuvanted whole-virus vaccine has also undergone a phase / trial . in phase with participants, three intramuscular injections of . , and µg/dose were compared to alum, followed by phase with participants comparing two injections of µg/dose with alum. adverse reactions, mild and self-limiting injection site pain and fever occurred in to . % of vaccine recipients and were not more frequent than in the alum control group. the geometric mean titres of neutralising antibodies ranged from to . two rna-based vaccines are currently in phase , mrna- and bnt b , with the aim of showing at least % efficacy. bnt b is a lipid-nanoparticle-formulated, nucleosidemodified mrna encoding the receptor-binding domain of s. a phase / placebo-controlled, dose-escalation study on adults, randomised to receive doses of , or µg showed dose-dependent reactogenicity . antibodies recognising the receptor-binding domain and neutralising antibodies were also elicited in a dose-dependent manner and increased after a second dose. this vaccine was subsequently compared to a second version, bnt b , encoding stabilised, membrane-anchored full-length prefusion s . interestingly, both vaccines elicited similar dose-responses in younger and older adults, and bnt b was selected for further clinical development because of improved safety. similarly, mrna- also codes for stabilised prefusion s. a phase , open-label trial in young adults showed acceptable safety and reactogenicity and the induction of neutralising antibodies after two injections . this vaccine was also tested for safety in older adults, age to and above , using two doses of µg or µg. in this population, adverse events were moderate for both dosages, but the -µg dose was chosen because it induced stronger neutralising antibody titres. the vaccine also induced cd + t cell responses, including ifn-, il- and tnf- production. in addition to these studies, more than trials have been registered so far on clinicaltrials.gov (https://clinicaltrials.gov/ct /results?term=covid- +vaccine&search=search), but safety, efficacy or immunogenicity data are lacking for most of them. besides sars-cov- -specific vaccines, these trials also include studies on heterologous vaccines, in particular the bacillus calmette-guérin (bcg). bcg is known to induce heterologous protection, especially against respiratory infections, by the generation of trained innate immunity, and may therefore potentially protect against covid- . vaccines take usually at least to years to be developed, due to the phasing of vaccine development, from pre-clinical to clinical phases , and , the latter being the conclusive efficacy trial. these phases are usually conducted sequentially, as they become increasingly costly. therefore, before engaging resources to the following phase, it is important to ensure that the data of the previous phase are convincing enough to warrant further development. anti-covid- vaccine development has proceeded at an unprecedented pace, as several phases are conducted simultaneously because of massive financial resources poured into vaccine development. within months rather than years, more than vaccines have entered the clinical development pipeline, including a dozen in phase / trials. simultaneously, largescale manufacturing was launched before data on safety and efficacy were gathered to make safe and effective vaccines readily available. however, considering the width of the covid- pandemic, mounting to close to , , global deaths at the time this article was prepared, it remains uncertain whether enough vaccines will be available. vaccine roll out will take time and is accompanied by difficult decisions about priorities as to who should receive the vaccines first . furthermore, accelerated vaccine development may also lead to confusion in the public perception and concern about safety and efficacy of anti-covid- vaccines once approved, and about vaccines in general. furthermore, there is a risk that anti-vaccine movements will use this opportunity to strengthen their position, which may have disastrous consequences, as vaccine hesitancy is regarded as one of the most important global health threats today. yet, the widespread use of safe and efficacious anti-covid- vaccine is the only hope for us to return to a "normal" life. j o u r n a l p r e -p r o o f resident memory cd + t cells in the upper respiratory tract prevent pulmonary influenza virus infection safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, nonrandomized, first-in-human trial immunogenicity and safety of a recombinant adenovirus type- -vectored covid- vaccine in healthy adults aged years or older: a randomized, double-blind, placebo-controlled, phase trial safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind, randomized controlled trial safety and immunogenicity of an rad and rad vectorbased heterologous prime-boost covd- vaccine in two formulations: two open, non-randomised phase / studies from russia russian sars-cov- vaccine effect of an inactivated vaccine against sars-cov- on safety and immunogenicity outcomes, interim analysis of randomized clinical trials phase / study of covid- rna vaccine bnt b in adults rna-based covid- vaccine bnt b selected for a pivotal efficacy study. medrxiv an mrna vaccine against sars-cov- -preliminary report safety and immunogenicity of sars-cov- mrna- vaccine in older adults covid- : vaccine roll out could take a year and will require difficult priority decisions reassuring the public and clinical community about the scientific review and approval of a covid- vaccine key: cord- -s tw x authors: shen, m.; zu, j.; fairley, c. k.; pagan, j. a.; an, l.; du, z.; guo, y.; rong, l.; xiao, y.; zhuang, g.; li, y.; zhang, l. title: projected covid- epidemic in the united states in the context of the effectiveness of a potential vaccine and implications for social distancing and face mask use date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: s tw x background: multiple candidates of covid- vaccines have entered phase iii clinical trials in the united states (us). there is growing optimism that social distancing restrictions and face mask requirements could be eased with widespread vaccine adoption soon. methods: we developed a dynamic compartmental model of covid- transmission for the four most severely affected states (new york, texas, florida, and california). we evaluated the vaccine effectiveness and coverage required to suppress the covid- epidemic in scenarios when social contact was to return to pre-pandemic levels and face mask use was reduced. daily and cumulative covid- infection and death cases were obtained from the johns hopkins university coronavirus resource center and used for model calibration. results: without a vaccine, the spread of covid- could be suppressed in these states by maintaining strict social distancing measures and face mask use levels. but relaxing social distancing restrictions to the pre-pandemic level without changing the current face mask use would lead to a new covid- outbreak, resulting in . - million infections and , - , deaths across these four states over the next months. in this scenario, introducing a vaccine would partially offset this negative impact even if the vaccine effectiveness and coverage are relatively low. however, if face mask use is reduced by %, a vaccine that is only % effective (weak vaccine) would require coverage of - % to suppress the epidemic in these states. a vaccine that is % effective (moderate vaccine) would only require - % coverage to suppress the epidemic. in contrast, if face mask usage stops completely, a weak vaccine would not suppress the epidemic, and further major outbreaks would occur. a moderate vaccine with coverage of - % or a strong vaccine ( % effective) with coverage of - % would be required to suppress the epidemic. delaying vaccination rollout for - months would not substantially alter the epidemic trend if the current interventions are maintained. conclusions: the degree to which the us population can relax social distancing restrictions and face mask use will depend greatly on the effectiveness and coverage of a potential covid- vaccine if future epidemics are to be prevented. only a highly effective vaccine will enable the us population to return to life as it was before the pandemic. the coronavirus disease is resulting in enormous health and economic losses in the united states (us). [ ] [ ] [ ] [ ] as of th october , there are more than million cases of covid- and more than , deaths in the us. the cooler weather in the us is seeing evidence of second waves of infection occurring in many us states. some us politicians are suggesting that an effective vaccine would mean that americans could return to normal life so that citizens would no longer need to socially distance or wear face masks, and the economy can be fully revived. however, the degree to which these restrictions could be eased will be dependent on the effectiveness of the potential covid- vaccines which is currently unknown. [ ] [ ] [ ] to allow careful planning about what restrictions may need to be continued, research is urgently needed to project how the effectiveness of a potential vaccine may affect the trajectory of the covid- pandemic in the us. it is also important to determine how the current nonpharmaceutical interventions can be integrated into an overall covid- control strategy that includes vaccines of different effectiveness. there are three key questions that need to be addressed to plan an effective control strategy once an effective vaccine becomes available. these are: ( ) how effective would the vaccine need to be to suppress the pandemic? ( ) what proportion of the population would need to be vaccinated? and ( ) what levels of social distancing measures and face mask use would need to be maintained in the context of different values of vaccine effectiveness and coverage? to address these questions, we developed dynamic simulation models of covid- for the four hardest-hit states in the us (new york, texas, florida, and california). the models were developed for each state because each state has different covid- policies for social distancing measures and mask use. each of the state-level models was calibrated based on the most recent covid- data of that state. the models were then used to project the averted covid- cases and deaths at different levels of vaccine effectiveness and coverage for the four states. given that the proportion of people who wear face masks is different across the four states, we further examined how face mask use would influence the effect of a potential vaccine in controlling the pandemic. findings from this study provide timely information that can be used by policymakers to plan for the potential release of a covid- vaccine and understand its effect across different regions in the us under different social distancing and face mask use scenarios. we obtained covid- data for new york, texas, florida, and california from the johns hopkins university coronavirus resource center. the data included the number of daily and cumulative confirmed cases and deaths from th january to th september . these data were used to calibrate the model for each state. we developed a dynamic compartmental model to capture the transmission of covid- in each state. figure shows the structure of our model. the population in each state was divided into ten compartments (susceptible individuals (s), vaccinated individuals (v), latent infections (e), asymptomatic infections (a, infected individuals without symptoms), undiagnosed infections with mild/moderate (i ) and severe/critical symptoms (i ), diagnosed infections with mild/moderate (t ) or severe/critical symptoms (t ), and recovered (r) and deceased (d) cases). susceptible and vaccinated individuals could be infected by contacts with latent, asymptomatic, and undiagnosed infections with mild/moderate and severe/critical symptoms in public settings (e.g., public transportation, supermarkets, . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint workplaces, etc.) and households (home or other private settings) with a probability ߉ and Λ (the force of infection), respectively (detailed descriptions of ߉ and Λ are provided in the supplementary appendix). our model did not consider population mobility in these states. latent individuals could progress to the infectious compartments with mild/moderate symptoms or asymptomatic compartments at a rate k . the probability that an individual is asymptomatic after an infection is ߩ . infectious individuals with mild/moderate and severe/critical symptoms are diagnosed and treated at the rates ߙ ଵ and ߙ ଶ , respectively. we assumed that diagnosed individuals were isolated and could not infect others. undiagnosed and diagnosed mild/moderate cases could progress to the severe/critical stages at the rates ݇ ଶ and ݇ ଷ , respectively. asymptomatic infections and undiagnosed mild/moderate cases were assumed to recover naturally at the rate ߛ . diagnosed mild/moderate and severe/critical cases could recover at the rates ߛ ଵ and ߛ ଶ , respectively. undiagnosed and diagnosed severe/critical cases could die due to the disease at the rates ߤ ଵ and ߤ ଶ , respectively. we assumed that the face mask effectiveness to prevent infection is % ( % ci, %- %) based on a meta-analysis on the effectiveness of face masks for covid- . we obtained data on the proportion of people who always wear a face mask at the county level from the new york times (based on about , interviews conducted by dynata from nd july to th july . we then estimated state-level face mask use by combining county-level data. the proportions of people who always wear a face mask in new york, texas, florida, and california were estimated to be . %, . %, . %, and . %, respectively. we denoted the vaccination rate as w and the effectiveness of the vaccine against the acquisition of infection as ε . that is, the probability of being infected for a vaccinated individual was െ ε of that for an unvaccinated individual when the vaccine is available. there is no covid- vaccine data publicly available in the us right now; as such, we varied the vaccine effectiveness ε from to % and assumed the vaccine coverage rate (v/(v+s)) changed from to % by varying the vaccination rate w. we estimated some of the model parameters using a nonlinear least-squares method (see supplementary appendix) and calibrated the model using data on daily and cumulative confirmed infections and deaths. figure shows the model calibration results for all the four states. the other model parameters were estimated from the literature (see tables s -s in the supplementary appendix). [ ] [ ] [ ] [ ] [ ] in each simulation, we calculated the sum of square errors between the model output and data and selected the top % with the least square errors to generate % confidence intervals. the detailed calibration procedure is described in the supplementary appendix. all analyses and simulations were performed in matlab r b. we projected the number of cases and deaths under the following four scenarios: ( ) the no vaccine scenario in which social distancing restrictions are relaxed (public person-toperson contact rates recovered to % of the pre-pandemic level), but the vaccine has not been developed; ( ) the vaccine and face mask scenario in which people are vaccinated (with different effectiveness and coverage) while social distancing restrictions are relaxed, and the baseline face mask use rate is maintained; ( ) the vaccine and reduced face mask scenario in which people are vaccinated while social distancing restrictions are relaxed, and half of the baseline face mask use rate is maintained; ( ) the vaccine and no face mask scenario in which . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint people are vaccinated while social distancing restrictions are relaxed and face masks are no longer used. we assume that natural and vaccine-induced immunity would last for at least one year. we then calculated the number of averted infections and deaths after one year for scenarios ( )-( ), compared to scenario ( ), and plotted them as a function of vaccine effectiveness and coverage. the threshold of vaccination curve was defined as the combination of vaccine effectiveness and coverage such that social distancing restrictions may be relaxed while the covid- epidemic can be retained at a sustainably low endemic level or eliminated. our results (figures - ) show that, in the absence of a vaccine, if social distancing restrictions were relaxed while the current face mask use rate was maintained, there would be . - million new infections and , - , new deaths across the four states within one year. if the current face mask use rate was maintained, introducing an effective vaccine would always decrease the number of infections and deaths. greater vaccine effectiveness and/or coverage rate would lead to more averted infections and deaths. however, if the face mask use rate decreased by %, a low vaccine effectiveness and coverage rate may not be enough to eliminate or suppress the pandemic to a low level without further major outbreaks. if people no longer wore face masks, a greater vaccine effectiveness and coverage rate would be needed to suppress the pandemic. we define vaccine effectivenesses of %, %, and % as weak, moderate, and strong, respectively, and present state-specific results next. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . if the face mask use decreased by % (figures e and e) or there was no face mask use (figures f and f) , greater vaccine effectiveness and coverage would be needed to suppress the pandemic. for example, if the vaccine effectiveness was weak, the vaccine coverage should be greater than . % to suppress the pandemic under % face mask use. deferring the rollout of vaccine by two months would require a . % coverage to suppress the pandemic (figures s -s in the supplementary appendix). when no face mask was used, and the vaccine was weak, even % coverage would not suppress the pandemic in texas. in if the face mask use decreased by % and the vaccine effectiveness was weak (figures h and h) , the threshold of vaccination curve showed that the coverage should be greater than . % to suppress the pandemic. if the vaccine effectiveness was moderate or strong, the vaccine coverage should be greater than . % and . %, respectively, to suppress the pandemic. deferring the rollout of vaccine by two months with % of the current face mask use would require . % and . %, respectively, to suppress the pandemic ( figures s -s in the supplementary appendix). if no face mask was used, the required vaccine coverage rates would be . % and . % under the moderate and strong effectiveness, respectively, to suppress the pandemic (figures i and i) . in the state of california, if the current face mask use rate was maintained and the vaccine was weak, % coverage could avert cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . if the face mask use decreased by %, and the vaccine was weak, the vaccine coverage should be greater than . % to suppress the pandemic (figures k and k ). if the vaccine was moderate or strong, the vaccine coverage should be greater than . % and . %, respectively, to suppress the pandemic. if no face mask was used, and the vaccine effectiveness was weak, even % coverage would not decrease the number of infections or deaths (figures l and l) . if the vaccine effectiveness was moderate, the vaccine coverage of great than . % would be required to suppress the pandemic based on the threshold of vaccination curve. if the vaccine effectiveness was strong, less vaccine coverage ( . %) would be required. similar to the other states, deferring the rollout of vaccine would only moderately decrease the vaccine coverage required to suppress the pandemic in california. our study addressed important questions related to what would be needed to suppress covid- in new york, texas, florida, and california under different scenarios of vaccine effectiveness, uptake, and face mask use. the results suggest that the number of covid- cases would decrease in the four most severely affected states in the us under the current approach of relying primarily on social distancing and mask use. however, if these measures are relaxed before an effective vaccine becomes available, the epidemic will likely rebound with further major outbreaks. so far, all four states have partially or fully reopened their economies, but face mask use in public space remains mandatory or recommended. our study suggests that if face mask use was maintained at the current level, vaccination if it were only moderately effective would result in lower numbers of new infections and deaths even when social distancing returned to normal. if face mask usage was halved in these states, then a weak vaccine ( % effectiveness) would require - % population coverage to suppress the epidemic, whereas a moderate vaccine ( % effectiveness) would require - % population coverage and a strong vaccine ( % effectiveness) would require only - % population coverage to suppress covid- . in all scenarios social distancing was assumed to return to pre-epidemic levels. in contrast, if face mask usage is reduced to zero then a weak vaccine would not provide substantial protection, and further outbreaks are anticipated, but a moderate vaccine may suppress the epidemic with vaccination coverage of - %, and a strong vaccine would require - % coverage. for social distancing to be returned to the pre-pandemic level in the four most severely covid- affected states in the us, at least half of its population needs to receive a vaccine with moderate effectiveness. however, the state of california, in particular, will need a vaccine coverage of close to % to suppress the covid- epidemic, such that both social distancing restrictions and the requirement for face mask use can be relaxed. the requirement of higher vaccination coverage in california is likely due to a higher proportion of susceptible individuals compared to the other three states. in other words, the prevalence of infected individuals with natural immunity is lower in california. a recent study indicated that vaccinating % of the us population with a vaccine of % effectiveness is necessary to achieve herd immunity and eliminate covid- . a separate study also also confirmed this estimate and demonstrated that with a moderately effective vaccine ( % effectiveness), the threshold coverage for the us population would be at least - % when the virus reproduction number varied between . - . . given that the willingness to take a covid- vaccine in the us has been estimated at only %, , only a strong vaccine with high effectiveness of nearly % would be sufficient to suppress the epidemic alone and enable relaxation of social distancing and face mask requirement. if a strong vaccine is not attainable, a moderate vaccine and maintaining a relatively low face . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint mask usage of - % would also be a plausible alternative to achieve the same target. that is, vaccination combined with a modest level of non-pharmaceutical measures, such as face mask use in common public spaces (shopping malls and transportation), may be a viable option to continue suppressing the epidemic in the long term. our findings demonstrate that the timing of a vaccination rollout may only have a small impact on the threshold vaccination coverage. deferring vaccination rollout by one or two months did not substantially change the required threshold coverage. however, if a safe and effective vaccine becomes available, it should be delivered to the population as early as possible to support the economic recovery of the country. [ ] [ ] [ ] despite reopening the economy in these states, the restrictions related to interstate and international travel have significantly disrupted the recovery of the us economy. only an effective vaccination program is able to counteract these restrictions. [ ] [ ] [ ] our study has a number of limitations. first, our model did not take into consideration the age structure of the population because data are currently not available on the different effects of a potential vaccine across age groups. second, the model did not distinguish between vaccine types (e.g., inactivated, live attenuated, recombinant protein) and the doses of vaccine. we used the average vaccine effectiveness to address the difference of vaccine types and doses in the model. third, the model did not consider the lag time required for the vaccine to become effective and assumed an immediate immune response and protection after vaccination. our sensisivity analyses showed that one-or two-month delay of immune response would have little impact on the results. fourth, we assumed that the vaccine protection lasts for at least one year and, thus, did not project the epidemic beyond one year. if the vaccine protection duration was shorter than one year, it would need larger vaccine coverage to suppress the epidemic. finally, the model did not consider issues related to vaccine availability, distribution, and the cost-effectiveness of vaccination, , , which would be important future research directions when more data (e.g., vaccine cost, quality of life for covid- patients) become available. our study indicates that for people to return to their pre-pandemic normal life, a potential vaccine needs to have moderate effectiveness of % and cover at least - % for the four most severely affected states in the us. maintaining a - % face mask use would reduce the required vaccine coverage below the willingness level of vaccination of the us population. delaying vaccination rollout for - months would not substantially alter the epidemic trend if the current non-pharmaceutical interventions were maintained. the findings from this study can inform the planned rollout of covid- vaccines and the continued implementation of non-pharmaceutical interventions such as social distancing and mask use mandates. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint . the blue areas denote % confidence intervals. dashed lines, dash-dot lines, and dotted lines denote the social distancing order (public person-to-person contact rates decreased), face mask order, and reopening (public person-to-person contact rates recovered to no more than % of the pre-pandemic level) policies that were implemented in each state, respectively. . it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . disease and healthcare burden of covid- in the united states revealing the unequal burden of covid- by income, race/ethnicity, and household crowding: us county versus zip code analyses estimation of excess deaths associated with the covid- pandemic in the united states the potential health care costs and resource use associated with covid- in the united states: a simulation estimate of the direct medical costs and health care resource use associated with covid- infections in the united states. health aff (millwood) sars-cov- immunity: review and applications to phase vaccine candidates. the lancet an mrna vaccine against sars-cov- -preliminary report immunogenicity and safety of a recombinant adenovirus type- -vectored covid- vaccine in healthy adults aged years or older: a randomised, double-blind, placebo-controlled, phase trial. the lancet community use of face masks and covid- : evidence from a natural experiment of state mandates in the us: study examines impact on covid- growth rates associated with state government mandates requiring face mask use in public. health aff (millwood) physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov- and covid- : a systematic review and meta-analysis. the lancet what is required to prevent a second major outbreak of sars-cov- upon lifting quarantine in wuhan city assessing the effects of metropolitan-wide quarantine on the spread of covid- in public space and households modelling the epidemic trend of the novel coronavirus outbreak in china can self-imposed prevention measures mitigate the covid- epidemic will an imperfect vaccine curtail the covid- pandemic in the us? medrxiv vaccine efficacy needed for a covid- coronavirus vaccine to prevent or stop an epidemic as the sole intervention acceptability of a covid- vaccine among adults in the united states: how many people would get vaccinated? vaccine attitudes toward a potential sars-cov- vaccine: a survey of us adults the equitable distribution of covid- therapeutics and vaccines priorities for the us health community responding to covid- fairly prioritizing groups for access to covid- vaccines partitioning the curve-interstate travel restrictions during the covid- pandemic planning for a covid- vaccination program a strategic approach to covid- vaccine r&d covid- -navigating the uncharted key: cord- -w wxsg p authors: dimitrov, kiril m.; afonso, claudio l.; yu, qingzhong; miller, patti j. title: newcastle disease vaccines—a solved problem or a continuous challenge? date: - - journal: veterinary microbiology doi: . /j.vetmic. . . sha: doc_id: cord_uid: w wxsg p abstract newcastle disease (nd) has been defined by the world organisation for animal health as infection of poultry with virulent strains of newcastle disease virus (ndv). lesions affecting the neurological, gastrointestinal, respiratory, and reproductive systems are most often observed. the control of nd must include strict biosecurity that prevents virulent ndv from contacting poultry, and also proper administration of efficacious vaccines. when administered correctly to healthy birds, nd vaccines formulated with ndv of low virulence or viral-vectored vaccines that express the ndv fusion protein are able to prevent clinical disease and mortality in chickens upon infection with virulent ndv. live and inactivated vaccines have been widely used since the ’s. recombinant and antigenically matched vaccines have been adopted recently in some countries, and many other vaccine approaches have been only evaluated experimentally. despite decades of research and development towards formulation of an optimal nd vaccine, improvements are still needed. impediments to prevent outbreaks include uneven vaccine application when using mass administration techniques in larger commercial settings, the difficulties associated with vaccinating free-roaming, multi-age birds of village flocks, and difficulties maintaining the cold chain to preserve the thermo-labile antigens in the vaccines. incomplete or improper immunization often results in the disease and death of poultry after infection with virulent ndv. another cause of decreased vaccine efficacy is the existence of antibodies (including maternal) in birds, which can neutralize the vaccine and thereby reduce the effectiveness of nd vaccines. in this review, a historical perspective, summary of the current situation for nd and ndv strains, and a review of traditional and experimental nd vaccines are presented. newcastle disease (nd) was first recognized ninety years ago and continues to be a problem for poultry producers. at least four defined panzootics have been recognized (miller and koch, ) ; negatively impacting not only economic livelihoods, but also human welfare by decreasing food supplies (alders, ) . after the initial, almost simultaneous, identification of newcastle disease in in indonesia, england, and possibly korea, nd was identified to the philippines, india, japan, australia, and kenya. by it was also reported in palestine, syria, french congo (present day gabon, republic of congo, and central african republic), the island of sicily, europe, and the united states. in the 's as part of the nd and rd panzootics, nd was reported in hawaii, canada, mexico, central and south america, china and throughout europe. these panzootics were aided both by the trade and movement of exotic psittacine birds without strict quarantine guidelines, the ubiquitous and synanthropic nature of pigeons, and the industrialization of the poultry industry (alexander, ) . newcastle disease viruses are single stranded, non-segmented, negative sense rna viruses encoding for at least six structural proteins and comprising one of three genome sizes: , , , , and , nucleotides (miller and koch, ) . the six proteins are the nucleocapsid, phosphoprotein, matrix, fusion, hemagglutininneuraminidase and the polymerase. since the nucleotide sequences for the fusion (f) and hemagglutinin-neuraminidase (hn) genes of newcastle disease viruses (ndv) were published in the 's, fusion (f), and hemagglutinin-neuraminidase (hn) full gene sequences have been placed into genbank ( th august ). the sequence motifs of the f protein cleavage sites have been proven to be reliable indicators of virulence (toyoda et al., ) , which has helped to develop more efficient molecular diagnostic assays (rue et al., ) . clinical disease affecting the neurological, gastrointestinal, reproductive, and respiratory systems (miller and koch, ) are most often observed in naïve, unvaccinated, or poorly vaccinated birds. clinical signs vary depending on the species of bird, the strain and challenge dose of the virus, and the immunity of the host (miller and koch, ) . virus isolation, either in specific pathogen free (spf) or ndv antibody-free embryonating chicken eggs or cell cultures, coupled with hemagglutination inhibition (hi) with ndv specific antiserum remain the definitive diagnostic assay for nd (oie, ) . the mean death time from the minimum lethal dose (mdt/mld) assay performed on spf embryonating chicken eggs continues to provide information on virulence for experimental purposes (miller and koch, ) and the intracerebral pathogenicity index (icpi) is required by the world organisation for animal health (oie) due to the assay's ability to discern mixed infections that may be otherwise missed using molecular techniques (oie, ) . like all other rna viruses, ndv is constantly evolving. as of , newcastle disease viruses have been grouped genetically into two classes with only one genotype of class i and genotypes of class ii. historically, grouping ndv strains into genotypes based on the similarities of the genomes began as a way to provide epidemiological information (lomniczi et al., ) . the ability to explore the virus repositories of laboratories that had collected ndv strains over the years and to use new sequencing technologies provided previously unknown information as to how the various strains from different outbreaks related to one another. a better understanding on the epidemiological relations among the circulating ndv, their genetic diversity and characteristics, and global distribution is crucial for developing new vaccines and vaccination strategies. genetic evaluation of the oldest ndv strains grouped them into what is now known as genotypes i, ii, iii, iv, and ix (dimitrov et al., b) . while genotype i includes predominantly ndv of low virulence, genotype ii strains may be virulent or of low virulence, and all of the characterized strains from iii, iv, and ix contain cleavage site motifs of virulent strains. excluding one virus from the s, most genotype ix ndv strains were isolated after the 's in china (dimitrov et al., a) . genotypes v, vi and viii were regularly isolated prior to (dimitrov et al., b) . genotype vii were first isolated in the early 's in italy, spain, the netherlands, belgium, and germany, and those strains were genetically most similar to ndv strains from indonesia in the late 's (lomniczi et al., ) . the newly classified genotype x strains consisting of strains of low virulence were identified as early as in the usa (diel et al., a) . along with the f and hn sequences, the addition of ndv complete genome sequences into genbank has assisted in the phylogenetic characterization of additional virulent genotypes from through (dimitrov et al., b) . viruses of genotype xi have been exclusively isolated from chickens in madagascar between and and seem to have common ancestry with viruses of genotype iv. viruses of genotypes xiv, xvii and xviii appear to have limited geographic distribution and have been isolated predominantly from domestic gallinaceous birds in west and central africa during - (dimitrov et al., b; samuel et al., ) . while genotype xii was first reported in peru in and china in , it has been also found in colombia in (dr. claudio afonso, unpublished data) . in recent years the number of reported viruses has increased (dimitrov et al., b ). an average of countries reporting nd outbreaks yearly from to and the increasing number of genotypes demonstrate the broadening of virulent ndv genetic diversity, suggesting that perhaps vaccination may have contributed to this effect. furthermore, the currently used vaccine strains (mainly genotypes i and ii) are three to seven decades old and are genetically distant ( . - . % nucleotide distance) from the currently circulating virulent ndv (dimitrov et al., b) . such high genetic distance between the vaccine and the contemporary ndv strains prevents effective reduction of shedding of the virulent virus from vaccinated birds, as discussed later in this review (miller et al., (miller et al., , . to complement vaccination, control of nd has been facilitated by strict biosecurity, which prevents the virus from contacting poultry (miller and koch, ) . this included implementing quarantine stations for imported birds, controlling the movement of birds and eggs inside the areas of concern, and the stringent and proper administration of vaccines. currently, the effective containment of nd outbreaks is normally achieved with the utilization of a combination of vaccinations, rapid diagnostic assays, and culling of infected flocks. from the early 's to the late 's live and inactivated nd vaccines were the only vaccine platforms available and were used to decrease economic losses resulting from morbidity and mortality (gallili and ben-nathan, ) . inactivated vaccines became commercially available in the usa in , but were not adopted by the poultry industry at that time as they were comparatively expensive, and were unable to prevent clinical disease to a sufficient level to merit wide spread use. the first live vaccines licensed in were formulated with strains now designated as virulent that produced disease in younger birds and were only applicable for use in chickens at least four-week old and needed to be applied with a wing-web application (goldhaft, ) . during this time several laboratories were investigating ndv strains that could be used as a live vaccine with acceptable levels of post-vaccinal clinical disease symptoms. within two years, two ndv strains of low virulence (b and lasota) isolated from chickens from the usa were also licensed for use (goldhaft, ; hitchner, ) . shortly after these products were available for individual bird administration, mass application techniques deemed necessary for larger commercial settings were investigated and implemented despite varying responses, because of uneven coverage in flocks and less than an optimal percentage of seroconverting birds (lancaster, ) . since the earliest implementation of live nd vaccination, the transfer of antibodies to offspring that can even partially neutralize the live nd vaccines, was known to be a complication. it was also evident that even the vaccines formulated with the more virulent vaccine strains would not provide lifelong immunity and that additional vaccinations would be necessary in layers and breeders. unfortunately, many of the problems in controlling nd that were clear from the first few decades of nd vaccine use, continue in . when an outbreak of nd occurs from infections of poultry with the virulent forms of the virus, referred to as mesogenic or velogenic ndv, the country is obligated to report it to the oie, and trading partners may suspend imports of poultry or poultry products from that country. the world livestock disease atlas surveyed countries included in the oie animal health yearbooks from to and concluded that newcastle disease is the fourth most problematic disease of poultry, behind highly pathogenic avian influenza, avian infectious bronchitis, and low pathogenic influenza (anonymous, ) . when evaluating the number of wild animals lost through destruction, disease or slaughter, nd ranked th out of the diseases evaluated (anonymous, ) . the widespread distribution of nd and the high number of annual outbreaks demonstrate that although globally used, current nd vaccines and vaccination practices alone cannot control the disease. countries most affected during - in descending order were iran, south africa, israel, china, vietnam, columbia, romania, south korea, kuwait, and sweden (anonymous, ) . with countries reporting nd outbreaks on average per year from to , nd ranked nd only behind rabies in the reported disease outbreaks (anonymous, ) . from to , countries confirmed nd outbreaks in domestic poultry with , and countries reporting nd outbreaks in , , and , respectively (www.oie.int/wahis_ /public/wahid.php/ diseaseinformation/statuslist). underreporting of nd, especially in areas where virulent ndv is endemic in poultry, may mean that these numbers are underestimating an already bad situation. many of the countries affected by nd also lack good biosecurity practices. thus, nd will likely persist in these areas of the world until both vaccines and biosecurity practices are improved. vaccination efforts against nd are focused in the poultry sector. however, it is likely that all bird species are susceptible to infection with ndv strains, and to date, more than avian species have been documented with ndv infections (kaleta and baldauf, ) . besides chickens, other birds (pigeons, cormorants, psittacines, pheasants, peafowl, wild waterfowl and shorebirds) are often reported with infections of virulent ndv (cardenas diel et al., a; pearson and mccann, ) and ndv of low virulence (kim et al., ) . in general, turkeys are slightly more resistant to ndv than chickens; however, some wild-bird ndv strains that have adapted to turkeys are able to infect this species more readily (dr. patti miller, unpublished data) . geese and ducks also show some resistance to infection, with ducks rarely presenting signs of clinical disease and geese showing slightly higher susceptibility, depending on the strain of ndv. when genotype v ndv strains began to be isolated in the usa, in addition to chickens, peafowl were among the birds noted to present clinical disease (pearson and mccann, ) . during - virulent ndv strains from captive, non-poultry species (peafowl, pigeons, pheasants, and parakeets) in pakistan were nearly identical to those isolated from chickens in the same geographical locations, suggesting the existence of an epidemiological link between the two groups of birds (submitted for publication). the roles of the different bird species reported with infections of ndv in the maintenance of the disease are still unclear. nevertheless, their importance should not be underestimated as sometimes they are reared in large flocks in close proximity to poultry, and their vaccination (when appropriate and feasible) could contribute to the control of nd. vaccination protocols for these species are dependent on national and international regulations and the specific epidemiological situation (oie, ) . the small size of the ndv genome, along with the low likelihood of genetic recombination, facilitates the use of ndv as a vaccine vector. information can be found in other reviews that summarize the body of knowledge for each gene individually, which has facilitated the genetic manipulation that has furthered recombinant ndv (rndv) vaccine development (ganar et al., ) . the virus can also replicate in many mammalian species, allowing ndv to be used as a vector for development of veterinary and human vaccines against other diseases such as rabies, west nile disease, infectious bursal disease, canine distemper, influenza, ebola, severe acute respiratory syndrome, human immunodeficiency syndrome, respiratory syncytial virus syndrome, among others (kim and samal, ) . however, there is no evidence of mammals serving as a biological vector for the spread of nd to poultry, although mammals, including humans, often serve as mechanical vectors for the virus. there are three main goals when using vaccination to help control nd: i) decrease or eliminate clinical disease; ii) decrease the amount of virulent virus shed; and iii) increase the infectious dose of the challenge virus . unfortunately, only the first goal is considered to be an objective of current control strategies, as field veterinarians do not have the tools to assess the effectiveness of vaccination on the accomplishment of the second and third objectives. biosecurity is a critical component of keeping the challenge virus away from the flock before they achieve a protective level of immunity, or ideally preventing any exposure. the success of any nd vaccination program also may depend on a minimum of % of the flock receiving a proper dose and responding to vaccination to achieve herd immunity (van boven et al., ) . those studies were performed under optimal conditions and remain to be validated in the field, under suboptimal conditions that may include deficient nutrition, stress, immune suppression, and repeated challenges. exemplary current vaccination strategies for different poultry sectors have been provided in the merck veterinary manual (www.merckvetmanual. com), however, these vary depending on the specific nd epidemiologic situation. table summarizes the main properties of live, inactivated and vectored vaccines, the most widely used nd vaccines. worldwide, the most commonly used nd vaccines are live vaccine viruses formulated with strains isolated in the 's and 's. viruses circulating in poultry were the source of the lasota, b , and vg/ga vaccines. all of those viruses belong to genotype ii and are genetically and antigenically highly related among themselves (> % nucleotide identity). the main differences among those vaccines are the tropism and the capacity to replicate in naïve chickens, which is highest in lasota and results in higher levels of neutralizing antibodies compared to other strains (meulemans, ) . thus, the lasota strain is nearly always used in countries where virulent ndv is endemic (diel et al., b) . the vg/ga strain is normally sold as an enterotropic vaccine, and the b strain as the most attenuated vaccine to be used in cases of low challenges or in very young birds. while live vaccines provide both mucosal and humoral immunity and can be administered using mass application techniques, they may cause clinical respiratory disease, drop in egg production, and are easily inactivated when not kept at the required temperature (commonly c) (winterfield and dhillon, ) . the effectiveness of live nd vaccines is directly correlated with the dose of the administered vaccine, and under experimental conditions the mean embryo infectious vaccine dose (eid ) of - reliably achieves % protection against mortality in adult spf chickens, but did not prevent challenge virus infection and replication (cardenas garcia et al., ; cornax et al., ; . doses of the lasota vaccine of eid or higher produced strong humoral immune responses and no increase in titers was observed after challenge, suggesting little to no replication of the challenge virus (cornax et al., ) . similar survival rates and viral shedding amounts after challenge with virulent ndv strains from genotype vii have been observed with the lasota vaccines in spf chickens (dr. cardenas garcia, university of georgia, usa, personal communication) (samuel et al., ) . cornax et al. results suggested that it may be possible to achieve the three objectives of vaccination when a very high dose of vaccine is used (cornax et al., ) . those results appear to be confirmed by results in the field in which a more aggressive vaccination strategy often results in improved nd control. unfortunately, the administration of high doses of classical nd vaccines significantly increases the cost of vaccination, thus this practice is not widely utilized. a second group of traditional vaccines that is widely used are vaccine strains from class ii genotype i (i.e. i , v , and phy-lmv ), which are avirulent and safely used in chickens of all ages (cardenas . strains of ndv that have increased stability to heat are especially advantageous in rural areas of the world with limited refrigeration. the i- strain has improved thermostability in comparison to the v nd vaccine strain it was derived from, and is mainly used in areas with higher ambient temperatures (alders, ) . the i- seed strain is produced by the australian centre for international agricultural research and provided to countries for the production of nd vaccines for village poultry flocks (copland and alders, ) . progress in identifying and characterizing other thermostable and immunogenic ndv strains continues (jeong et al., ) . these vaccines are also effective in preventing clinical signs upon infection with virulent ndv, but like the others, do not prevent viral replication (susta et al., b ). an additional consideration for village poultry is the number of doses able to be purchased and the ease at which the vaccine can be reconstituted. using a freeze-dried pelleted lasota ndv strain, researchers were able to provide a consumer friendly and costeffective vaccine for smaller flocks ( doses) that had more heat resistance than the usual lyophilized product (lal et al., ) . a similar pelleted commercial product formulated with the vg/ga strain, in an effervescent tablet can be reconstituted in drinking water and administered within two hours by mass application methods. inactivated nd vaccines have the disadvantage of requiring a withdrawal period before vaccinated birds can be processed for human consumption, and each vaccine requires individual administration by a subcutaneous or intramuscular injection. even though birds vaccinated with inactivated vaccines tend to have higher humoral antibody levels, they do not develop a strong cell mediated response (schijns et al., ) , and shed larger amounts of virulent challenge virus compared to birds vaccinated with live nd vaccines (miller et al., , . although live and inactivated vaccines protect against clinical disease in spf chickens, there are continuous reports of vaccine failures under field conditions (perozo et al., ; rehmani et al., ) . one of the possible reasons for these failures may be poor vaccination response that is also dependent on field-associated factors unrelated to the vaccines, such as immunosuppression (meulemans, ) from infections prior to nd vaccination. different avian pathogens such as infectious bronchitis virus, gallid alphaherpesvirus , infectious bursal disease virus, and mycoplasma spp. have been associated with immunosuppressive effects. understanding the role of field factors and field immunosuppressing agents during and after nd vaccination may lead to the development of more effective vaccines or vaccination strategies. vaccines that are co-expressing antigens of different pathogens and are simultaneously inducing immunity against several avian diseases would be of great value. presence of maternal antibodies interferes with the development of active immunity when live vaccines are administered via intramuscular, subcutaneous, intranasal route, in drinking water, and through aerosol. in chickens with maternal immunity, the best response to live nd vaccine is achieved through conjunctival and intranasal routes of administration, perhaps due to the development of local immunity induced by these vaccines. however, immunity induced by inactivated vaccines was less affected by the presence of maternal antibodies (lancaster, ) . for more than years, efforts have been directed towards the development of recombinant vaccines against nd, using other avian viruses as vectors. in , the fowlpox virus (fpv) vectorbased vaccines expressing the ndv f or hn protein were proven to protect chickens from a challenge with virulent ndv (boursnell et al., ) . later, multiple studies were conducted, employing both genes (alone or in combination, also with other viral genes), to investigate the protective efficacy of the recombinant vaccines (karaca et al., ; taylor et al., ) . while some have shown that maternal antibodies to the influenza a virus hemagglutinin (ha) protein can interfere with recombinant fpv (rfpv) vaccines expressing ha (faulkner et al., ) , others have shown that immunity to fpv from previous fpv vaccinations, not maternal antibodies, are the problem (bublot et al., ) . at least two commercial rfpv-nd vaccines have been registered and are sold commercially. however, the rfpv-nd vaccines are not widely used because they cannot be applied through mass methods. furthermore, previous exposure to fpv, which is commonly present in the environment, decreases efficacy of the rfpv vaccines. the meleagrid alphaherpesvirus , commonly known as herpesvirus of turkeys (hvt) or a serotype marek's disease virus, is one of the most widely used vectors in recombinant vaccine production. in the early 's, morgan et al. and reynolds et al. first showed the protective efficacy of hvt vector-based vaccines to protect chickens from nd and marek's disease (morgan et al., ; reynolds et al., ) . these vaccines are made by inserting the coding region of the fusion protein of ndv into the thymidine kinase site of the viral genome, and are capable of expressing the protein encoded by the gene during replication. currently, two bivalent commercial recombinant hvt (rhvt) vaccines have been registered and are used internationally. while the replication of rhvt-nd vaccines appears to be mildly hindered by the presence of maternal antibodies (le gros et al., ) , they are able to prevent clinical disease and mortality when challenged with a virulent ndv six weeks after vaccination (sonoda et al., ) . the antibodies induced after the administration of the rhvt-nd vaccines occur at the same time in which maternal antibodies are waning. the rhvt-nd vaccines have many benefits, among these is that they can be administered in ovo at the hatchery or subcutaneously after hatch, and produce long-term immunity (armour and garcía, ; esaki et al., ) . however, the rhvt-nd vaccines are cell associated, so like marek's disease vaccines, they are required to be kept in liquid nitrogen, and to be administered within an hour of being thawed. unfortunately, rhvt-nd require four weeks before full immunity will be reached (palya et al., ) , which would require the strictest level of biosecurity to prevent infection during that period. this may be impossible in countries where nd is endemic. recombinant hvt vaccines have been widely used in countries where minimum viral challenges exist; however, in endemic countries, these vaccines may need to be used in combination with other nd vaccines to confer acceptable protection. after hatch, the administration of a killed or live nd vaccine to birds that were vaccinated in ovo with rhvt-nd vaccine, increases the level of immunity to facilitate more complete protection and helps decrease the amount of virulent ndv shed after challenge (palya et al., ) . this approach is commonly referred to as a prime-boost strategy. because of the lack of simple serologic tests to measure the immune response to rhvt-nd vaccines expressing the ndv fusion protein, a quantitative real-time polymerase chain reaction assay to evaluate the rhvt-nd vaccine load from feather follicles has been developed (rauw et al., ) . this approach may be helpful in evaluating if rhvt vaccine administration was successful. a commercial elisa kit has been recently advertised that can detect anti-ndv antibodies induced by rhvt-nd vaccines. it is important to note that the use of one rhvt vaccine in ovo prevents the use of other rhvt-vectored vaccines subcutaneously in the same birds after hatch, as the immunity that is induced from the first vaccine will neutralize the viruses from the second application after it is administered (schat, ) . however, the simultaneous subcutaneous administration of a recombinant marek's disease virus vaccine of serotype (rispens strain) expressing the protein encoded by the vp gene of ibdv with a rhvt-nd vaccine resulted in %, %, and % survival after challenge (five weeks after vaccination) with marek's disease virus, ibdv, and ndv, respectively (ishihara et al., ) . a recombinant infectious bursal disease virus (ibdv) containing the hn of ndv has also been created, but it only provided - % protection to spf birds following a virulent ndv challenge (li et al., b) . during the late 's, reverse genetics technology was developed to rescue infectious ndv from assembled sub-genomic overlapping cdna fragments under control of a t rna polymerase promoter (peeters et al., ; romer-oberdorfer et al., ) . this technology allows researchers to genetically manipulate the genome of ndv and insert non-ndv genes in it. this lead to the development of genetically engineered vaccines while retaining the replication competency of the original virus. since then, using the reverse genetics technology, many strains of ndv have been developed as vectors to express proteins encoded by the inserted foreign gene for the purposes of developing avian vaccines and for human cancer therapy. a comprehensive review lists the main features of ndvs that allows them to be promising vaccine vector: they replicate well in vivo, induce a robust mucosal and systemic immune response, allow easy genetic manipulation, and avirulent strains usually used for vaccine vectors are safe, do not recombine, and do not incorporate into the dna genome during replication (kim and samal, ) . many recombinant nd vaccines have been created by the insertion of a foreign gene into an intergenic region of the ndv genome for the expression and dual use as a vaccine against both the ndv and the second agent. the evaluation of these vaccine candidates in clinical trials revealed different levels of protection against targeted pathogen challenge (dinapoli et al., ; huang et al., ; nakaya et al., ; park et al., ; yu et al., ) . although most of them are not yet commercially available, numerous experimental vaccines using ndv of low virulence as vectors have been developed and investigated for protection against different avian diseases. huang et al. reported the generation of lasota vector-based vaccine expressing the vp protein of a variant ibdv in (huang et al., ) . a decade later another experimental recombinant ndv (rndv)-ibdv (vp ) vaccine was evaluated in spf and commercial broilers after in ovo administration and resulted in - % hatchability and - % survivability after intramuscular challenge with virulent ndv (ge et al., ) . in , two teams reported the successful formulation of ndv (lasota) vector-based vaccines expressing, the gallid alphaherpesvirus (commonly known as infectious laryngotracheitis virus) surface glycoproteins gb, gc and gd, together or separately (kanabagatte basavarajappa et al., ; zhao et al., ) . with a prime-boost application, -day-old spf birds survived a virulent ndv challenge two weeks after the last vaccine was administered (kanabagatte basavarajappa et al., ) . during the same year, recombinant lasota virus containing the ibv s gene was also generated as a priming vaccine (toro et al., ) . following the emergence and worldwide spread of the high pathogenicity avian influenza virus (hpaiv) h n in the 's, and the increasing need for better protection of the poultry industry against hpai and low pathogenicity avian influenza, multiple ndv vector-based vaccines containing different avian influenza virus (aiv) hemagglutinin (ha) genes were created after replacing the polybasic cleavage site of hpaiv with a low pathogenicity cleavage site: e.g. h , h , h , h (goff et al., ; lardinois et al., ; park et al., ; romer-oberdorfer et al., ; schroer et al., ) . recently, the experimental use of a modified virulent ndv vector-based vaccine expressing aiv h protein was reported and showed the potential of virulent ndv to be used as vectors (kim et al., ) . a ndv vector-based vaccine expressing h protein was recently commercialized (sarfati-mizrahi et al., ) . these rndv-aiv vaccines are heavily used in the field in china with roughly . billion doses having been applied from through (li et al., a) . limited data from mexico is available, but during a three-month period in , million doses of rndv-aiv were administered (villarreal, ). in addition to the "conventional model" for foreign gene expression through an additional independent transcription unit (itu), different approaches for expression of a foreign gene by ndv have been explored (wen et al., ) (gao et al., ) . some of them increased the capacity of expressing a larger gene or more than one foreign gene. expression of a foreign protein through an internal ribosomal entry site (ires) from a second open reading frame in a ndv vector has also been investigated , and results suggest that the np gene downstream noncoding region is the optimal insertion site for a high level of foreign gene protein expression. not all rndv vaccines are equal in their levels of immunogenicity or their ability to replicate in chickens, therefore, each vaccine that is created should be evaluated for its ability to replicate in chickens, and to induce a protective immune response against a virulent ndv challenge. the immune response to vaccination with recombinant vaccines is influenced by many factors, and the expression of the desired level of foreign genes is undoubtedly one of the most important and critical factors for the success of the vaccine. the level of foreign gene expression from a ndv vector can be affected by the rate of replication, the tissue tropism of the viral vector, the size and the sequence of the foreign gene insert, and the genomic location of the foreign gene in the vector. among these, the genomic location of the foreign gene has been shown to be crucial. to date, most of the foreign genes have been inserted into a non-coding region in the ndv genome as an additional independent transcription unit that consists of ndv gene start (gs), the foreign gene, and ndv gene end (ge) sequences (dinapoli et al., ; huang et al., ; nakaya et al., ; park et al., ; yu et al., ; zhao et al., ) . based on the sequential transcription of negative stranded rna viruses (lamb and parks, ) , the best position for foreign gene expression is hypothesized to be the closest to the 'end of ndv genome. however, the insertion of a foreign gene as an itu into a promoter-proximal position may interfere with ndv replication more seriously than a promoter-distal position, resulting in lower levels of foreign gene expression (carnero et al., ; zhao and peeters, ; zhao et al., b) . therefore, a balance in virus replication and the abundance of foreign gene expression must be considered for selection of a foreign gene insertion site. the insertion of a foreign gene more proximal to the end, between the np and p gene, expressed a low level of the foreign protein (carnero et al., ) . further studies supported the conclusion that the p and m junction region is the optimal insertion site for an optimal level of foreign gene expression by a ndv vector (nakaya et al., ; zhao et al., b) . an important aspect that has to be considered when using ndv as a vaccine vector is the efficacy of the virus in the presence of both vector-and insert-specific maternal antibodies (armour and garcía, ) . it has been previously demonstrated that the efficacy of recombinant ndv vector-based vaccines expressing avian influenza proteins was reduced when the vaccines were administered in birds with pre-existing anti-nd and anti-aiv antibodies (faulkner et al., ; sarfati-mizrahi et al., ; schroer et al., ) . notably, not all rndv vector-based vaccines have been evaluated in ndv-challenge experiments. autogenous vaccines were the first true antigenically matched vaccines used in poultry (smith, ) . however, autogenous vaccines are not defined and are difficult to use, especially in food animals, as they are usually inactivated and have long withdraw periods. it is not uncommon for the virus strains used in vaccines for respiratory diseases caused by paramyxoviruses, such as measles and canine distemper, to be changed over time to improve the efficacy of the vaccine and the achieved immunity (griffin and pan, ; martella et al., ) . these changes are made when the virulent challenge strains accumulate too many genomic mutations over time, and the vaccine virus strains are no longer similar antigenically to the challenge strain. avian influenza viruses with their multiple serotypes require serotype specific vaccines to prevent morbidity and mortality. in addition, the shedding of virulent hpai challenge virus after infection was decreased with smaller amounts hpai shed when the vaccine and challenge virus were more similar (swayne, ) . the principle of utilizing a ndv strain closely related or homologous to the challenge strain was tested by our laboratory and produced similar findings (miller et al., , (miller et al., , . live and inactivated antigenically matched nd vaccines have been developed using reverse genetics. one type of such vaccines contain viruses that are identical to the circulating virulent ndv with the exception of the fusion protein cleavage site, which is modified to decrease virulence. another type of vaccines, developed by our laboratory, is based on the use of a vaccine backbone (e.g. lasota) with the replacement of the fusion and hemagglutinin neuraminidase genes. these two genes are replaced with ones homologous to currently circulating viruses, with the modification of the cleavage site of the fusion protein, which is normally engineered to be identical to the cleavage site of the lasota vaccine strain. when live and inactivated nd vaccine strains were antigenically matched they produced a higher humoral immune response to the challenge viruses than the heterologous vaccine, and the amount of virulent nd challenge virus shed from vaccinated birds was lower than the amount secreted by the heterologous vaccine (miller et al., (miller et al., , . the efficacy of inactivated vaccines from genotypes i, ii, v, vi, vii, and xii against challenge viruses of different genotypes were evaluated and found, under optimal conditions, to prevent to % of the birds from having morbidity and mortality against all the virulent ndv strains used no matter their genotype (miller et al., , (miller et al., , . however, when heterologous (non-matching genotypes) live or inactivated nd vaccines were administered properly and chickens were given enough time to develop a proper immune response, all the birds lived and showed no sign of disease , supporting the claim that the administration of the proper amount of vaccine is also crucial to nd control (dortmans et al., ) . more recently, recombinant nd vaccines with the f and hn protein genes homologous to the challenge virus in a lasota backbone have been shown to induce higher levels of antibodies, and reduced viral shedding after challenge in comparison to the commercial lasota vaccine. furthermore, when birds were suboptimally vaccinated with low doses of vaccines given only seven days before challenge with a virulent ndv, a decrease in morbidity and mortality rates was observed with one homologous vaccine compared to a traditional heterologous nd vaccine (cardenas garcia et al., ) . teams from korea (cho et al., ) , china , and indonesia (xiao et al., ) have had similar findings in terms of reduction of viral shedding, while others have reported no improvement with their homologous rndv vaccines (dortmans et al., ) . the primary benefit of antigenically matched vaccines compared to traditional vaccines is a possible decrease in the amount of challenge virus shed from vaccinated chickens, but this parameter is not one that has been routinely part of evaluating nd vaccine efficacy for commercial production. our recent finding of improvements in clinical protection in sub-optimally vaccinated chickens suggests that the advantages of antigenically matched vaccines may be even clearer in the field than under laboratory conditions. vaccines homologous to ndv genotype v are heavily used in mexico with . billion doses applied from january through july (dr. arnulfo toscano, investigación aplicada s.a.de c.v., mexico, personal communication), suggesting that the market is starting to appreciate the benefits of a reduction in viral shedding. a disadvantage of homologous vaccines is the difficulty of the vaccine company to work with virulent ndv in the laboratory because of the need for higher biosecurity. most countries do not have vaccine companies with the level of biosecurity to safely make antigenically matched vaccines. chickens are vaccinated conveniently in ovo at or days of embryonation when they are moved into hatching trays. this system presents the ndv antigen to both the respiratory and gastrointestinal tracts and allows newly hatched chickens to develop an early immune response (kapczynski et al., ) . however, live ndv given in ovo can cause decreased hatchability and weak chicks. an innovative approach was developed to use an antigen-antibody complex live nd vaccine for in ovo vaccination to slow the replication without adversely affecting the hatchability. the ndv specific antibodies were able to dissociate from the vaccine virus after the birds hatched, resulting in acceptable hatchability, otherwise not achieved with even the least virulent, asymptomatic wild type or recombinant ndv strains administered in ovo (kapczynski et al., ) . over the last few decades understanding of the mechanisms of innate immunity increased significantly. innate immune system specifically recognizes foreign pathogens (or their pathogenassociated molecular patterns) with the help of pattern recognition receptors (prr). toll-like receptors (tlr) are a type of prr and efforts have been made to enhance vaccine potency and to stimulate immune responses by using tlr ligands as adjuvants (gupta et al., ) . in a recent review by gupta et al., chicken tlrs agonists and their use as adjuvants in vaccines against poultry infectious diseases (including nd) have been extensively described (gupta et al., ) . ramakrishnan and colleagues demonstrated significant up-regulation of the transcriptional expression of interferon (ifn)-b, ifn-g, interleukin (il)- b, il- , and tlr- genes in chicken peripheral blood mononuclear cells after administration of resiquimod (r- , a tlr- agonist) and the synergetic effect of r- and lipopolysaccharide, a tlr- agonist . the use of r- as an adjuvant for inactivated nd vaccine administered intramuscularly in chickens showed significantly upregulated expression of ifn-a, ifn-b, ifn-g, il- b, il- , inducible nitric oxide synthase and mhc-ii genes and increased protection after virulent ndv challenge when compared to two inactivated nd vaccines used alone . lipopolysaccharide used in combination with liposome encapsulated ndv administered to chickens via the intranasal route induced significant tracheal iga and serum igg levels, with increased levels of cd + and cd + cells, and % survival after virulent ndv challenge (tseng et al., ) . when polyinosinic:polycytidylic acid (poly i: c, tlr- ligand) was used in chicken embryo cells and exposed to ndv, it induced an antiviral state and reduced the plaque-forming capacity of the ndv (gupta et al., ) . another approach utilizing the tlr agonist effects is the use of oligodeoxynucleotides containing unmethylated cpg motifs (cpg odn), which has also been proven to have immunostimulatory effects. cpg motifs are recognized as pathogen patterns by the innate immune system to activate defensive mechanisms and induce the immune response of immunized chickens (vleugels et al., ) . after internalization by target cells, cpg odns reach the late endosomal/lysosomal compartment where they signal by interacting with tlr- (chicken ortholog of tlr- ). after intramuscular and intranasal administration, cpg odn enhanced the activity of a nd vaccine in chickens; increasing the systemic antigen-specific igg levels in serum (t-cell proliferation), and mucosal (iga) levels when administered intranasally. specific-pathogen-free chickens covaccinated with nd vaccines and at least mg of cpg odn by either route were protected from challenge with an otherwise lethal dose of virulent ndv (zhang et al., (zhang et al., , . vaccines containing cpg odns can be applied either systemically or via the mucosa, have good safety profiles, increase the immunogenicity of co-administered vaccines by improving the function of professional antigenpresenting cells, and boost the humoral and cellular vaccine-specific immune responses, highlighting their potential to be used as effective adjuvants for ndv and other poultry vaccines (vleugels et al., ) . the co-expression of immunostimulatory cytokines by virus vectors has been suggested to improve protective immunity induced by several avian vaccines (armour and garcía, ) . chicken ifn-g (chiinf-g) is a macrophage activation modulator, inhibits viral replication, promotes development of the th response by inhibiting th cytokine production, and enhances antigen presentation and antigen processing and destruction of intracellular pathogens. this cytokine has been shown to improve protection and enhance immune responses in avian species against different avian pathogens; however, no commercial product has ever reached the market, and controversial results highlight the need for further evaluation of the potential of this cytokine (cardenas garcia et al., ) . cardenas garcia et al. demonstrated that co-delivering chicken ifn-g with a nd vaccine using three vaccination systems (dna-vaccine administered in ovo, recombinant vaccine expressing chiinf-g used in ovo, and inactivated recombinant vaccine expressing chiinf-g administered subcutaneously in two-week-old spf chickens) did not improve the immunogenicity or the protective efficacy of the evaluated vaccine candidates (cardenas garcia et al., ) . expression of chicken il- by a highly virulent strain of ndv led to decreased systemic viral load, but did not significantly affect mortality in chickens (susta et al., a) , while expression of ifng by the same virulent ndv attenuated the virus and decreased morbidity and mortality in spf chickens (susta et al., ) . activation of tlrs or over-expressing certain cytokines result in the up-or down-regulation of various interleukins, chemokines and interferons, which in turn directs the immune response towards either a th or th , or a mixed immune response. some tlr combinations can produce a stronger and selective immune response, and others can down-regulate cytokine expression (gupta et al., ) . cytokines are components of a fine-balanced network of immune responses with multiple feedback loops (schat, ) ; thus, it is possible that inserting cytokine genes or using tlr ligands may deregulate the fine-tuning that exists in the naturally perfectly-timed system that leads to an enhanced immune response (cardenas garcia et al., ) . in addition, the immunomodulatory effect of cytokines and tlr ligands may depend on various factors, such as the type of the pathogen, the amount and type of the co-delivered antigen, the relative time of administration, and the amount of delivered cytokine/ligand. further studies to evaluate these approaches and the level of protection they induce in comparison to the existing traditional vaccines are necessary. the use of other adjuvants to enhance the immune responses induced by nd vaccines has also been investigated. chitosan is a nontoxic, biocompatible, biodegradable polysaccharide derived from the exoskeleton of crustaceans and insects. it has been shown to improve the th pathway of immunity by inducing a stronger and earlier peripheral cellular immune response with no effect on the systemic, lachrymal and digestive antibody-mediated immunity after oculonasal co-delivery with live nd vaccine in day-old chickens (rauw et al., a) . the same scheme was also evaluated as a second vaccination after an in ovo vaccination with a rhvt-nd vaccine at day of embryonation. the combination of rhvt-nd with mucosal co-delivery live ndv and chitosan demonstrated the best protection against mortality and morbidity, as well as the strongest reduction of virus shedding correlated to higher levels of cellular immunity and gastrointestinal antibody-mediated immunity in comparison to each of the vaccines used alone (rauw et al., b) . the rapid development of nanotechnology has provided various biodegradable nanomaterials that have become useful in vaccine research. in particular, nanoparticle systems have long been developed as vaccine delivery vehicles in human vaccines by providing protection from maternal antibodies and nucleases, leading to an interest in their use against animal pathogens (chahal et al., ; dai et al., ; zhao et al., b) . the nanoparticle delivery systems protect the delivered antigen from disruption and have advantages such as higher antigen uptake, controlled release, and increased duration of responses (dai et al., ; zhao et al., b) . many types of materials, such as polylactic acid, poly (lactide-co-glycolide), calcium phosphate, carboxymethylcellulose, chitosan, and magnesium phosphate among others, can be used as nanoparticle carriers and administered by multiple routes including oral, mucosal, and parenteral (dai et al., ; zhao et al., b) . two chitosan derivatives, o- -hydroxypropyltrimethyl ammonium chloride chitosan and n- -hydroxypropyl trimethyl ammoniumchloride chitosan, have been utilized to make nanoparticles as a mucosal delivery vehicle for live attenuated nd vaccines (dai et al., ; zhao et al., b) . in both systems the release of ndv was effective and sustainable and resulted in stronger cellular, humoral, and mucosal immune responses as measured by levels of specific humoral and local antibodies, spleen lymphocyte proliferation, and levels of cytokines. both systems also conferred protection upon challenge with virulent ndv and no clinical signs or microscopic lesions were observed in vaccinated birds, while % mortality and some hyperplastic changes were observed in the chickens vaccinated with a traditional commercial nd vaccine. silver @sio and double hydroxide @sio nanoparticles have been developed for intranasal delivery of dna nd vaccines (zhao et al., a (zhao et al., , a . in experiments with spf chickens these sio nanoparticles showed very low toxicity, sustainable release after initial burst, and induced stronger cellular, humoral, and mucosal immune responses compared to the intramuscular administration of the same vaccine or the naked dna plasmid. both systems demonstrated % protection in chickens after challenge with the virulent f ndv strain. great potential lies in nonretroviral mrna-based vaccines. recently, a delivery system required to deploy conventional unmodified replicon mrnas based on the genomes of venezuelan equine encephalitis virus (veev) as a vaccine was developed (chahal et al., ) . this system supports self-amplification via a double-stranded rna intermediate in the cytoplasm to drive efficient expression of the immunogenic antigen. single dose modified dendrimer nanoparticle (mdnp)-delivered veev replicon rnas encoding single or multiple pathogen proteins were shown to protect mice against lethal infection of h n influenza virus (a/wsn/ ), ebola virus or toxoplasma gondii. this synthetic highly innovative, adjuvant-free system is flexible and can multiplex (co-formulate and co-express) different antigenexpressing replicons (i.e. simultaneously raise immunity against multiple antigens from a single disease and/or multiple antigens from multiple diseases). moreover, it induces vital antigen-specific cd + t-cell and antibody responses without additional adjuvants (chahal et al., ) . while the system has not been tested in chickens, some of its main advantages are that the design and production of the vaccines take only weeks, do not require high biosecurity level facilities, and allow for a rapid and on-demand response to currently circulating pathogens, including highly diverse ndv strains. virus-like particles (vlp), although available for a long time, are increasingly being considered as viral vaccines. vlps are formed by the assembly of viral structural proteins and lipids, but without the incorporation of the viral genome and may offer significant advantages over many currently used or developing vaccine technologies: i) they are not infectious and cannot spread infection; ii) there is no chance of reversion of virulence or recombination; iii) they mimic the structure of the infectious virus; iv) they are very immunogenic; and v) stimulate both humoral and cellular immune responses (mcginnes et al., ; morrison, ) . many different paramyxovirus vlps can be produced upon expression of the m protein or m protein with various combinations of the glycoproteins and np after the transfection of cells with vectors containing cdnas encoding for these proteins, with nd vlps having high efficiency of release (morrison, ) . newcastle disease vlps, although not being commercialized, have a potential to incorporate glycoproteins of different strains of ndv, could potentially be used against different nd viruses (mcginnes et al., ) , and have significant potential applications as they can be designed to express protein sequences from many pathogens (morrison, ) . one aspect of vaccine production that has been addressed in many, but not all areas of the world, is the quality assurance standards for the production and testing of nd vaccines (gallili and ben-nathan, ) . the code of federal registration, title , from the animal and plant health inspection service (aphis) of the united states department of agriculture (usda) (sections . and . ), the manual of diagnostic tests and vaccines for terrestrial animals (sections . . , and . . ) and principles of vaccine production from the oie (oie, (oie, , , and council directives / /eec, and / /eec from the commission of european communities regulate the development and testing of nd vaccines to ensure that only reliable, safe, and effective products are available. however, as recent as , nd vaccines illegally transported from mexico, intended for use in fighting gamecocks in california, usa, were confiscated at the border between the countries and shown to be contaminated with virulent ndv, in addition to the vaccine ndv strain (pedersen et al., ) . extensive use of currently available vaccines, strict quarantine combined with rapid diagnostics and biosecurity, and stamping out and other containment measures seem to keep nd under control in developed countries. however, as evident from the multiple outbreaks occurring worldwide, current vaccination strategies are not fully efficacious under different environmental conditions and the development of new concepts for vaccine generation are needed. to enhance the efficacy of vaccines and to improve the immune responses induced by them, investigation of innovative approaches together with the development of safe and novel strong adjuvants are necessary. future nd vaccine systems that allow rapid development to target emerging ndv strains, and enable design of multiplexed vaccines, will have advantage over currently existing vaccines. none. making newcastle disease vaccines available at village level newcastle disease virus-an avian paramyxovirus administration of tlr agonist, resiquimod, in different types of chicken induces a mixed th and th response in the peripheral blood mononuclear cells world livestock disease atlas. a quantitative analysis of global animal health dataa current and future applications of viral-vectored recombinant vaccines in poultry. the poultry informed professional. department of population health insertion of the fusion gene from newcastle disease virus into a non-essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity induced by the recombinant development and use of fowlpox vectored vaccines for avian influenza molecular epidemiology of newcastle disease in mexico and the potential spillover of viruses from poultry into wild bird species development of an improved vaccine evaluation protocol to compare the efficacy of newcastle disease vaccines effects of chicken interferon gamma on newcastle disease virus vaccine immunogenicity optimization of human immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h n influenza, and toxoplasma gondii challenges with a single dose characterization of a recombinant newcastle disease vaccine strain the australian village poultry development programme in asia and africa characterization of live lasota vaccine strain-induced protection in chickens upon early challenge with a virulent newcastle disease virus of heterologous genotype newcastle disease virus fusion and haemagglutinin-neuraminidase proteins contribute to its macrophage host range o- -hydroxypropyltrimethyl ammonium chloride chitosan nanoparticles for the delivery of live newcastle disease vaccine newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens genetic diversity of avian paramyxovirus type : proposal for a unified nomenclature and classification system of newcastle disease virus genotypes complete genome and clinicopathological characterization of a virulent newcastle disease virus isolate from south america newcastle disease viruses causing recent outbreaks worldwide show unexpectedly high genetic similarity to historical virulent isolates from the temporal, geographic, and host distribution of avian paramyxovirus (newcastle disease virus) newcastle disease virus outbreaks: vaccine mismatch or inadequate application? protection and antibody response caused by turkey herpesvirus vector newcastle disease vaccine passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination newcastle disease vaccines newcastle disease virus: current status and our understanding expression of transgenes from newcastle disease virus with a segmented genome novel in-ovo chimeric recombinant newcastle disease vaccine protects against both newcastle disease and infectious bursal disease induction of cross-reactive antibodies to novel h n influenza virus by recombinant newcastle disease virus expressing a north american lineage h subtype hemagglutinin historical note on the origin of the la sota strain if newcastle disease virus measles: old vaccines, new vaccines toll-like receptor-based adjuvants: enhancing the immune response to vaccines against infectious diseases of chicken serendipity in science-discovery of the b- strain of newcastle disease virus a recombinant newcastle disease virus (ndv) expressing vp protein of infectious bursal disease virus (ibdv) protects against ndv and ibdv combination of two marek's disease virus vectors shows effective vaccination against marek's disease, infectious bursal disease, and newcastle disease immunization with a thermostable newcastle disease virus k / strain originated from wild mallard duck confers protection against lethal viscerotropic velogenic newcastle disease virus infection in chickens newcastle disease in free-living and pet birds a recombinant newcastle disease virus (ndv) expressing infectious laryngotracheitis virus (iltv) surface glycoprotein d protects against highly virulent iltv and ndv challenges in chickens protection from clinical disease against three highly virulent strains of newcastle disease virus after in ovo application of an antibody-antigen complex vaccine in maternal antibodypositive chickens immune responses of poultry to newcastle disease virus recombinant fowlpox viruses coexpressing chicken type i ifn and newcastle disease virus hn and f genes: influence of ifn on protective efficacy and humoral responses of chickens following in ovo or post-hatch administration of recombinant viruses newcastle disease virus as a vaccine vector for development of human and veterinary vaccines phylogenetic diversity among low virulence newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to poultry-origin isolates modified newcastle disease virus vectors expressing the h hemagglutinin induce enhanced protection against highly pathogenic h n avian influenza virus in chickens development of a low-dose fast-dissolving tablet formulation of newcastle disease vaccine for low-cost backyard poultry immunisation paramyxoviridae iii. control measures. live and inactivated vaccines compared potency of a recombinant ndv-h vaccine against various hpai h n virus challenges in spf chickens field efficacy trial of a novel hvt-ibd vector vaccine for -day-old broilers avian influenza vaccines against h n 'bird flu recombinant infectious bursal disease virus expressing newcastle disease virus (ndv) neutralizing epitope confers partial protection against virulent ndv challenge in chickens generation by reverse genetics of an effective attenuated newcastle disease virus vaccine based on a prevalent highly virulent chinese strain newcastle disease outbreaks in recent years in western europe were caused by an old (vi) and a novel genotype (vii) lights and shades on an historical vaccine canine distemper virus, the rockborn strain assembly and biological and immunological properties of newcastle disease virus-like particles control by vaccination newcastle disease antigenic differences among newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge comparison of viral shedding following vaccination with inactivated and live newcastle disease vaccines formulated with wild-type and recombinant viruses effects of newcastle disease virus vaccine antibodies on the shedding and transmission of challenge viruses protection of chickens from newcastle and marek's diseases with a recombinant herpesvirus of turkeys vaccine expressing the newcastle disease virus fusion protein newcastle disease virus-like particles as a platform for the development of vaccines for human and agricultural pathogens recombinant newcastle disease virus as a vaccine vector newcastle disease. manual of diagnostic tests and vaccines for terrestrial animals: mammals, birds and bees. biological standards commission. world organisation for animal health principles of veterinary vaccine production. manual of diagnostic tests and vaccines for terresrial animals. world organisation for animal health advancement in vaccination against newcastle disease: recombinant hvt ndv provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions onset and long-term duration of immunity provided by a single vaccination with a turkey herpesvirus vector nd vaccine in commercial layers engineered viral vaccine constructs with dual specificity: avian influenza and newcastle disease the role of indigenous wild, semidomestic, and exotic birds in the epizootiology of velogenic viscerotropic newcastle disease in southern california, - isolation of a virulent newcastle disease virus from confiscated lasota vaccine rescue of newcastle disease virus from cloned cdna: evidence that cleavability of the fusion protein is a major determinant for virulence biological and phylogenetic characterization of a genotype vii newcastle disease virus from venezuela: efficacy of field vaccination synergy of lipopolysaccharide and resiquimod on type i interferon, pro-inflammatory cytokine, th and th response in chicken peripheral blood mononuclear cells the positive adjuvant effect of chitosan on antigenspecific cell-mediated immunity after chickens vaccination with live newcastle disease vaccine improved vaccination against newcastle disease by an in ovo recombinant hvt-nd combined with an adjuvanted live vaccine at day-old quantification of rhvt-f genome load in feather follicles by specific realtime qpcr as an indicator of ndv-specific humoral immunity induced by dayold vaccination in spf chickens presence of virulent newcastle disease virus in vaccinated chickens in farms in pakistan a recombinant hvt vaccine expressing newcastle disease virus antigens protects chicks against a lethal newcastle disease challenge. forty-second western poultry disease conference generation of recombinant lentogenic newcastle disease virus from cdna level of protection of chickens against highly pathogenic h avian influenza virus with newcastle disease virus based live attenuated vector vaccine depends on homology of h sequence between vaccine and challenge virus evolutionary changes affecting rapid diagnostic of newcastle disease viruses isolated from double-crested cormorants adjuvant potential of resiquimod with inactivated newcastle disease vaccine and its mechanism of action in chicken phylogenetic and pathotypic characterization of newcastle disease viruses circulating in west africa and efficacy of a current vaccine protective dose of a recombinant newcastle disease lasota-avian influenza virus h vaccine against h n highly pathogenic avian influenza virus and velogenic viscerotropic newcastle disease virus in broilers with high maternal antibody levels back to the past: do vector vaccines represent the future? practical aspects of poultry vaccination efficacy of newcastle disease virus recombinant expressing avian influenza virus h hemagglutinin against newcastle disease and low pathogenic avian influenza in chickens and turkeys autogenous vaccines: current use patterns and end users' needs in the integrated broiler industry development of an effective polyvalent vaccine against both marek's and newcastle diseases based on recombinant marek's disease virus type in commercial chickens with maternal antibodies expression of interferon gamma by a highly virulent strain of newcastle disease virus decreases its pathogenicity in chickens expression of chicken interleukin- by a highly virulent strain of newcastle disease virus leads to decreased systemic viral load but does not significantly affect mortality in chickens pathologic characterization of genotypes xiv and xvii newcastle disease viruses and efficacy of classical vaccination on specific pathogen-free birds vaccines for list a poultry diseases: emphasis on avian influenza efficacy of a recombinant fowl pox-based newcastle disease virus vaccine candidate against velogenic and respiratory challenge infectious bronchitis virus s expressed from recombinant virus confers broad protection against challenge structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of newcasltle disease virus effect of lipopolysaccharide on intranasal administration of liposomal newcastle disease virus vaccine to spf chickens avian influenza in mexico stimulatory effect of cpg sequences on humoral response in chickens development of a novel thermostable newcastle disease virus vaccine vector for expression of a heterologous gene comparative immune response from vaccinating chickens with lentogenic newcastle disease virus strains generation by reverse genetics of an effective, stable, live-attenuated newcastle disease virus vaccine based on a currently circulating, highly virulent indonesian strain protection by recombinant newcastle disease viruses (ndv) expressing the glycoprotein (g) of avian metapneumovirus (ampv) subtype a or b against challenge with virulent ndv and ampv vaccination with newcastle disease vaccine and cpg oligodeoxynucleotides induces specific immunity and protection against newcastle disease virus in spf chicken enhancement of mucosal immune responses by intranasal co-delivery of newcastle disease vaccine plus cpg oligonucleotide in spf chickens in vivo development of a newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism recombinant newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication newcastle disease virus (ndv) recombinants expressing infectious laryngotracheitis virus (iltv) glycoproteins gb and gd protect chickens against iltv and ndv challenges synthesis, characterization, and immune efficacy of layered double hydroxide@sio nanoparticles with shell-core structure as a delivery carrier for newcastle disease virus dna vaccine p and m gene junction is the optimal insertion site in newcastle disease virus vaccine vector for foreign gene expression iga response and protection following nasal vaccination of chickens with newcastle disease virus dna vaccine nanoencapsulated with ag@sio hollow nanoparticles biological evaluation of n- -hydroxypropyl trimethyl ammonium chloride chitosan as a carrier for the delivery of live newcastle disease vaccine the authors gratefully acknowledge david suarez for his useful comments on the manuscript. this work was supported by usda/ ars cris - - . the mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. the usda is an equal opportunity provider and employer. key: cord- - o gqe v authors: sanami, samira; zandi, milad; pourhossein, behzad; mobini, gholam-reza; safaei, mohsen; abed, atena; arvejeh, pooria mohammadi; chermahini, fatemeh amini; alizadeh, morteza title: design of a multi-epitope vaccine against sars-cov- using immunoinformatics approach date: - - journal: int j biol macromol doi: . /j.ijbiomac. . . sha: doc_id: cord_uid: o gqe v severe acute respiratory syndrome coronavirus (sars-cov- ) caused covid- disease in china. so far, no vaccine has licensed to protect against infection with covid- , therefore an effective covid- vaccine needed. the aim of this study was to predict antigenic peptides of sars-cov- for designing the covid- vaccine using immunoinformatic analysis. in this study, t and b-cell epitopes of s protein were predicted and screened based on the antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. the epitopes were joined by the appropriate linker. lt-iic as an adjuvant was attached to the end of the structure. the secondary and d structure of the vaccine was predicted. the refinement process was performed to improve the quality of the d model structure; the validation process is performed using the ramachandran plot and prosa z-score. the proposed vaccine's binding affinity to the hla-a : and hla-drb _ : molecule was evaluated by molecular docking. using molecular dynamics, the stability of vaccine-hla complexes was also evaluated. finally, in silico gene cloning was performed in the pet a (+) vector. the findings suggest that the current vaccine may be a promising vaccine to prevent sars-cov- infection. in december , severe acute respiratory syndrome coronavirus (sars-cov- ) caused covid- disease in china, which was associated with a cluster of respiratory infections [ ] . infection with sars-cov- has spread to various countries and created a significant threat to public health. following the outbreak of sars-cov in and and mers-cov outbreak in , the sars-cov- outbreak is a recurrence of the previous occurrence with new pressure, and the bat is highly probable to be the origin of all types of cov [ ] . researchers have confirmed the transmission of this virus from human to human, which has led to a further outbreak of the disease [ ] . coronaviruses are classified into four genera: alpha, beta, gamma, and delta coronavirus. alpha and bata coronaviruses infect bats and human [ , ] , sars-cov- is a member of β coronavirus genera [ ] . sars-cov- is a single strand, positive-sense rna virus with a genome of around kb in length [ ] . in all coronaviruses, the structural proteins comprise spike (s), envelope (e), membrane protein (m), and nucleoprotein (n) [ , ] . sars-cov- uses the s protein as the key to binding and entering host cells therefore understanding its structure and function is important. the s protein is a type i transmembrane protein and highly glycosylated. spike glycoproteins assemble into trimers on the viral particle surface. s glycoprotein has three domains including ectodomain, transmembrane, and endodomain. ectodomain subdivided into s and s subunits, s is important for binding to host cell receptors, the amino acid sequences of s are more variable than s [ ] . s protein is an attractive target for vaccine development for the following reasons: s protein is located at the virus surface and uses angiotensin-converting enzyme receptor in respiratory epithelial cells to enter the host cells [ , ] , it also can induce neutralizing antibodies [ ] . the development and production of a vaccine are costly and will take many years to achieve this goal, the various strategies were used to reduce the time and costs of this process [ ] . reverse vaccinology or vaccinomics is a new method that combines immunogenomics and bioinformatics to develop novel vaccines [ ] . this method has many benefits over conventional vaccinology, it decreases the vaccine development time and costs and it also makes possible to study non-cultivable or risky microorganisms [ ] . scientists are working very hard to combat this virus, but still, there is no drug or vaccine available to protect against infection with covid- [ ] . thus, in this regard, we decided to design a new multiepitope vaccine against sars-cov- infection using reverse vaccinology approaches. in this research, first, the ctl, htl, and b-cell epitopes of the s protein were predicted using propred- , propred, and abcpred servers, respectively, and then were selected base on antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. the selected epitopes were joined together using the appropriate linker to construct the main core of the multi-epitope vaccine. since the multi-epitope vaccine has poor antigenicity, lt-iic as an adjuvant to overcome this weakness was attached to the end of the structure. lt-iic is a type ii subfamily of ltbs consisting of amino acids. moreover, when coadministered with a multi-epitope vaccine it shows an increase in cd + t cell immune responses [ ] . next, the physicochemical properties of the construct were investigated, the d structure of the protein was predicted, and finally, its affinity to the mhc i and ii molecules was investigated through docking, following that, was performed the molecular dynamics (md) simulation of docking complexes. finally, the vaccine coding gene expression in escherichia coli (e. coli) was evaluated, and in silico gene cloning was performed in the pet a (+) vector. the amino acid sequences of sara-cov- s protein (qii . ) and lt-iic (h w f ) were obtained in fasta format from ncbi (https://www.ncbi.nlm.nih.gov/) and uniprot (https://www.uniprot.org/) databases, respectively. the binding epitopes to mhc i alleles were predicted using the propred- server (http://crdd.osdd.net/raghava/propred /). propred- server is based on a method of predicting epitopes that bind to different alleles of mhc i [ ] . to predict htl epitopes was used the propred server (http://crdd.osdd.net/raghava/propred/) which uses quantitative matrices to predict t-cell epitopes binding to different alleles of mhc ii [ ] . in this study, peptide fragments with default parameters for mhc i and ii alleles with the highest coverage of iran's population were predicted. gamma interferon (ifn-γ) is a cytokine that plays a key role in both innate and adaptive immune response; ifnepitope server (https://webs.iiitd.edu.in/raghava/ifnepitope/predict.php) determined the ifn-γ-inducing htl epitopes. abcpred server (http://crdd.osdd.net/raghava/abcpred/) using an artificial neural network predicted the b-cell epitopes. this is the first server developed using fixed-length patterns based on a recurrent neural network. the server predicts peptides - mer in length and the threshold is among . to . in this study, the server was predicted peptide fragments of mer length, and the threshold was set at . [ ] . antigenicity, toxicity, and allergenicity were evaluated for predicted epitopes. the antigenicity of the epitopes were calculated by vaxijen v . server (http://www.ddgpharmfac.net/vaxijen/vaxijen/vaxijen.html), which is based on the transformation of the protein sequences auto cross-covariance (acc) into uniform vectors of main amino acid properties. the performance accuracy of the vaxijen server varied from to %, based on the selected model organism. in this study, the vaxijen v . server determined the epitopes antigenicity based on a threshold of . and a virus model [ ] [ ] [ ] . the epitopes toxicity were determined using the toxinpred server (http://crdd.osdd.net/raghava/toxinpred/). this server predicts of toxic or non-toxic peptides from a wide variety of peptides, along with their important physical and chemical properties such as hydrophobicity, hydropathicity, amphipathicity, molecular weight, and pi charge [ ] . the allertop v. . server (https://www.ddg-pharmfac.net/allertop/index.html) predicted epitopic allergenicity. the approach used in this server is based on the automatic cross-covariance (acc) transformation of protein sequences into uniform vectors of equal length [ ] . the pir peptide matching program (https://research.bioinformatics.udel.edu/peptidematch/index.jsp) was used to investigate the presence or absence of similarity with the human proteome in predicted epitope sequences [ ] . the selected t and b-cell epitopes from the previous step were placed in the vaccine structure. kk and gpgpg linkers joined the screened t and b-cell epitopes in a structure of multi-epitope vaccine, respectively. the lt-iic amino acid sequence was attached to the merged epitopes at the n-terminus using the eaaak linker to boost the immunogenicity of the vaccine. an effective vaccine should not only cause strong immune responses but should also have appropriate physicochemical properties during the j o u r n a l p r e -p r o o f development cycle. protparam server (https://web.expasy.org/protparam/) calculated the amino acid content and some of the designed vaccine's physicochemical properties such as molecular weight, theoretical pi, half-life in the mammalian reticulocytes, instability index, aliphatic index, and grand average of hydropathy (gravy) [ ] . a standard method for determining the molecular weight of a protein, given the number of amino acids that make it up, is to multiply that number by the average weight of an amino acid, which is da [ ] . the isoelectric point is the ph at which amino acid or protein loses its charge and is, therefore, unable to move in a direct current electric field. knowing the theoretical pi of a protein can be very useful for selecting and optimizing the methods used for protein purification, including ion-exchange chromatography and isoelectric focusing electrophoresis [ ] . the half-life is the time it takes for half of the protein to disappear inside the cell after synthesis [ ] . the instability index of proteins indicates their stability in the test tube so that proteins with an instability index of less than are predicted as stable proteins [ ] . the aliphatic index of a protein is the relative volume of protein occupied by aliphatic side chains, this factor indicates the stability of the protein against heat [ ] . the gravy is computed by dividing the sum of the hydropathic values of all amino acids by the number of residues in the sequence [ ] . the gravy demonstrates the amphipathic nature of the proteins, the negative and positive values suggest the hydrophilic and hydrophobic nature of the designed vaccine, respectively [ ] . solpro server (http://scratch.proteomics.ics.uci.edu/) predicted the protein tends to be soluble when overexpression in e. coli [ ] . antigenpro server (http://scratch.proteomics.ics.uci.edu/) was used to estimate the antigenicity of the vaccine, the accuracy of the server was determined to be % based on cross-validation experiments using the combined dataset [ ] . allergenicity and toxicity of the vaccine were determined by allertop v. . and toxinpred servers, respectively. prabi server (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/npsa/npsa_gor .html) used gor method to predict the secondary structure of the engineered vaccine. gor iv with a mean accuracy of . % uses all possible pair frequencies within a amino acid residues window [ ] . dpro server (http://scratch.proteomics.ics.uci.edu/) predicted the tertiary structure of the multiepitope vaccine [ ] . drefine server (http://sysbio.rnet.missouri.edu/ drefine/) improved the quality of the d model from the previous stage. drefine refining protocol includes a two-step process as follows: (i) optimization of the hydrogen bonding network and (ii) minimization of atomic energy by integrating physics with knowledge-based force fields [ ] . model validation is an important step to detect possible errors in crude structures and to compare the quality of models before and after the refinement process [ ] , for this purpose, rampage (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) and prosa (https://prosa.services.came.sbg.ac.at/prosa.php) servers were used in this study. ramachandran plot was generated using the rampage server. the most important application of the ramachandran plot is to predict the quality of different protein structures regarding experimental methods (x-ray crystallography, nmr, and cryo-em). acceptable and nonacceptable quality protein structures consisted of a collection of torsional angles in the allowed and disallowed regions, respectively [ ] . the prosa j o u r n a l p r e -p r o o f server evaluated the overall quality of the protein models in the form of a z-score. when the predicted model's z-scores are outside the normal range of native proteins, it indicates an incorrect structure [ ] . cluspro . server (https://cluspro.org) performed molecular docking between vaccine candidate and two of the most common binding alleles in the population of iran including hla-a : with pdb entry of x q, and hla-drb _ : with pdb entry of aqd. cluspro can discriminate the hypothetical usercreated structures and run any server-compatible docking algorithms. additional functions allow the users to modify the structure, determine the attraction and repulsion, or identify pairwise distance restrictions [ ] [ ] [ ] . md simulation was performed using gromacs . . (groningen machine for chemical simulations) software to investigate the behavior of vaccine, hla-a : , and hla-drb _ : molecules in docking structure [ ] . the input structures were prepared using the amber ff sb force field. the correct hydrogen status of histidines was established for all the histidines presented in the structure, and the disulfide bonds were defined for the protein. then, the surface charge of the structure was neutralized by adding several sodium and chlorine ions. the protein was inserted into a layer of tip p water molecules at angstroms using gmx solvate software. energy minimization was performed on structures with the -step by steepest descent method to eliminate van der waals interactions and to form the hydrogen bonds between water and complex molecules. in the next step, the system temperature gradually increased from up to k for ps in constant volume and was then equilibrated in constant pressure at ps. md simulation was performed at °c ( k) for ns. non-bonding interactions with a distance of angstroms were calculated using the pme method. to increase the computational speed, the shake algorithm was used to limit the bonds involved in the hydrogen atom. backtranslation of the amino acid sequence of the multi-epitope vaccine was conducted using the gene infinity server (http://www.geneinfinity.org/sms/sms_backtranslation.html). the codon adaptation index (cai) and gc content of optimized dna were assessed by the genscript server (https://www.genscript.com/tools/rare-codon-analysis). finally, the multi-epitope vaccine sequence was inserted into the pet- a (+) vector by the snapgene tool. using the propred- server, which was set to default parameters, were predicted ctl epitopes ( mer) for human mhc-i alleles including hla-a* , hla-a* , hla-b* , hla-b* and hla-b* ( table ). the propred server predicted htl epitopes ( mer) for the alleles of mhc-ii, namely, drb * : , drb * : , drb * : , drb * : , drb * : and drb * : . next, epitopes capable of binding to at least four types of mhc alleles were assessed using the ifnepitope server for their ability to induce ifn-γ, the result of this step suggested that htl epitopes were capable of inducing ifn-γ (table ). using the default parameters of the abcpred server were predicted j o u r n a l p r e -p r o o f linear b-cell epitopes ( table ) . the predicted ctl, htl, and b-cell epitopes were evaluated for overlapping, antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. it is noteworthy that among the overlapped epitopes were selected epitopes that had a higher antigenicity and improved quality of the second and three-dimensional structure of the vaccine by being in the chimeric structure. finally, , , and epitopes were selected from ctl, htl, and b-cell epitopes, respectively. the final vaccine structure consists of four domains: lt-iic, linear b-cell, ctl, and htl epitopes, which were placed in the vaccine construct by different linkers (fig. ) . the protparam results revealed that the multi-epitope vaccine comprised of amino acids. the vaccine's molecular weight was . kda and its theoretical pi value was . . the half-life of the vaccine was h in mammalian reticulocytes, more than h in yeast, and more than h in e. coli. the instability index, aliphatic index, and gravy were predicted . , . , and - . , respectively. the solpro server result showed the vaccine to be soluble. the antigenicity of the final sequence (including the adjuvant ) and the main sequence (without adjuvant) of vaccine were predicted using the antigenpro server to be . and . , respectively. the vaccine sequence was predicted to be non-allergenic and non-toxic using allertop v. . and toxinpred servers, respectively. the prabi server predicted that . , . and . % of the total amino acids of vaccine construct were alpha helix, extended strand, and random coil, respectively. vaccine primary d model was predicted using the dpro server. the drefine server performed the process of refining the protein model. five parameters, including drefine, gdt-ts, gdt-ha, rmsd, rwplus, and molprobity, ranked the optimized models. the higher gdt-ts, rmsd and gdt-ha scores, and the low scores of drefine, molprobity, and rwplus showed a higher quality model. based on these factors, refined model no. was selected, which is indicated in the first row of table , the raw d structure and refined model of the vaccine are depicted in fig. a and b . the validation process was performed using the ramachandran plot and the prosa z-score. ramachandran plot result revealed that in the primary model, . %, . %, and . % of residues were located in favored, allowed, and outlier regions, respectively (fig. a) , while in the refined model, . %, %, and . % of residues were found j o u r n a l p r e -p r o o f in favored, allowed, and outlier regions, respectively (fig. b) . z-score of the initial model was calculated - . (fig. a) , while the z-score of the refined model was estimated - . (fig. b ). the interaction of the refined model of vaccine with hla-i and hla-ii molecules was investigated using the cluspro . server. proteins with pdb entry of x q and aqd were identified separately as receptor and vaccine as a ligand to do molecular docking. the server provided clusters from each complex and ranked the clusters based on the amount of energy. cluster no. . in the results of the interaction between the hla-a : and hla-drb _ : molecules with the multi-epitope vaccine showed the lowest energy weighted score of - . and- . kcal/mol, respectively ( fig. a and a ). the pdbsum server was used to obtain accurate information on the interaction between residues in docking complexes. a total of vaccine residues coupled with residues of chain a from hla-a : molecule. a number of hydrogen bonds were formed between the residues of the chain a from the hla-a : molecule and the residues of the vaccine, which included pro -his , glu -lys , glu -his , asn -gln , glu -tyr , arg -tyr , lys -tyr , asp -lys , glu -lys , lys -gly , lys -asn , lys -ala , lys -leu , arg -ser , asp -asn , arg -ile , arg -ala and thr -trp (fig. b ). in the vaccine-hla-drb _ : complex and residues of the vaccine associated with residues of the chain a and residues of the chain b from the hla-drb _ : molecule, respectively. hydrogen bond of thr -ser was established between the vaccine and chain a from hla-drb : molecule and hydrogen bonds, including glu -lys , glu -lys , gln -ala , gly -arg , his -arg , glu -arg , glu -lys , thr -lys , glu -gln , asn -val , asn -gly , asn -thr , thr -thr and gln -arg were formed between the chain b from hla-drb : molecule and vaccine (fig. b ). the best molecular docking complexes were used as inputs to md simulation. at this stage, md simulation of both complexes was performed for ns and the results were evaluated as root mean square deviation (rmsd), root mean square fluctuation (rmsf), and radius of gyration (rg). rmsd between the created structures during md simulation is an appropriate parameter to ensure the stability of the vaccine-hla complexes. rmsd of the hla-a : molecule in the vaccine -hla-a : complex reached . nm after approximately ps and showed a slight variation around this amount until the end of the md simulation. the rmsd of the vaccine showed a significant increase from the beginning of the md simulation until ps, after that, there was a downward trend up to , , from this point to until ps the trend was approximately increasing (fig. a) . the profile of rmsd changes of hla-drb _ : molecule in the vaccine -hla-drb _ : complex was similar to hla-a : , while the rate of rmsd changes in the vaccine was relatively different. at the beginning of the md simulation, the value of rmsd showed a sharp increase until ps which reached nm, of this point until the end of the md simulation was observed no significant changes. (fig. b) . rmsf determined the flexibility of any residue in the vaccine-hla complexes. rmsf result showed that the rmsf of most amino acids in the chain a of the hla-a : molecule was high and the maximum and minimum rmsf values were . and . , respectively (fig. a) . two regions on the chain b of the molecule showed relatively high fluctuations, which included regions - and - . in the range of j o u r n a l p r e -p r o o f - , the rmsf reached . nm (fig. b) . the study of vaccine structure in this complex showed high rmsf in most of its amino acids. the two regions of the vaccine showed very high levels of rmsf of . and . nm, which included amino acids in regions - and - , respectively (fig. c ). most parts in chain a of hla-drb _ : molecule in the vaccine -hla-drb _ : complex including amino acids in areas - , - , - , - , - and - showed high fluctuations (fig. a) . in chain b, the lowest and highest value of rmsf was . and . nm, respectively (fig. b) . the vaccine showed very high fluctuations in most areas and had a maximum rmsf of . nm (fig. c) . rg for the hla-a : molecule in the vaccine -hla-a : complex was . nm at the beginning of the simulation and showed no change from the beginning of the simulation to the end (fig. a) . while rg of the hla-drb _ : molecule in the vaccine -hla-drb _ : complex was . nm at the beginning of the md simulation and had a slight increase up to ps, then remained constant until the end of the md simulation (fig. b) . in the presence of the hla-a : molecule, the rg value of the vaccine was . at the beginning of the simulation, and during the simulation period, a slight increase and decrease were observed in this parameter, and at the end of the simulation time, the rg value reached . (fig. a) . the rg level of the vaccine in the presence of the hla-drb _ : molecule showed a relatively significant decrease from the beginning of the simulation to ps then remained constant from that point until the end of the simulation period (fig. b ). the gene infinity server conducted backtranslation of the vaccine construct. genscript tool was used to evaluating the key properties of the gene sequence including the codon adaptation index (cai) and gc content. the optimized nucleotide sequence cai value was and the gc content of the optimized sequence was . % in e. coli. recognition sites for bamhi and xhoi restriction enzymes were added to ′ and ′ end of the optimized gene, respectively. the adapted codon sequence was inserted into the pet a (+) vector using snapgene software (fig. ). the covid- outbreak demonstrates a pandemic threat to global public health [ ] . the sars-cov- can be transmitted from person to person, even when the infected individual has no clinical symptoms, and sometimes cannot be detected in the infected individual for several days or even weeks. there are cases where a person has clinical symptoms of covid- , but the diagnosis test is negative, so the best way to prevent coronavirus from occurring is to get a vaccine [ ] , but unfortunately, because of the novelty of the virus, there is still no licensed vaccine for the disease. however, researchers and pharmacists around the world are working at an unprecedented pace to develop an effective virus vaccine, and clinical trials of several candidate vaccines have begun, including mrna- , ino- , and chadox ncov- [ ] . reverse vaccinology technology has been widely used to determine the epitopes for the development of multi-epitope vaccines in various organisms. in recent months, several reverse vaccinology studies have also been conducted to design the covid- vaccine. in a study conducted by enayatkhani et al. the b-cell and t epitopes of n, orf a, and m proteins were predicted, a multi-epitope vaccine candidate was constructed, then were performed bioinformatics evaluations [ ] . peele et al. designed a vaccine candidate using b-cell and t epitopes of s protein by in silico methods [ ] . kalita et al. selected n, m, and s proteins as the target antigen for the prediction of t and b-cell epitopes and designed a multi-epitope vaccine against sars-cov- [ ] . guglielmo lucchese predicted s j o u r n a l p r e -p r o o f protein antigenic peptides from sars-cov- using immunoinformatics methods [ ] . ahmed et al. predicted a collection of b cell and t cell epitopes derived from the s and n proteins of sars-cov- [ ] . in each of the studies mentioned above, different proteins have been used to predict epitopes, also, the type of bioinformatics softwares, linkers, and many other factors are different in each study, therefore the laboratory evaluations of each of these designed vaccines show different results. this work focussed on the in silico design of a potential multi-epitope vaccine for covid- using s protein. it is important to identify b-cell and t epitopes of the target antigens in order to develop a multi-epitope vaccine. in the current study, we predicted a large number of ctl, ifn-γ-inducing htl, and b-cell epitopes for s protein then selected high-ranked epitopes based on antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. linkers play a dual role in the multi-epitope vaccine structure : (i) the prevention of the generation of junctional epitopes (neoepitopes), which is the major concern in designing multi-epitope vaccines, and (ii) promotion of the immune processing and presentation of hla-ii binding epitopes [ , ] . to design the vaccine construct, the selected t and b-cell epitopes were fused using the kk and gpgpg linkers, respectively. lt-iic was attached to the n-terminal of the multiepitope sequence by an eaaak linker. the designed vaccine was evaluated for its physicochemical features, antigenicity, allergenicity, and toxicity .the vaccine's molecular weight was . kda, which makes it an acceptable vaccine since proteins with a molecular weight of less than kda are considered to be more appropriate targets for the development of vaccines, as proteins with a lower molecular weight can be easily purified [ ] , the vaccine construct was basic in nature and the total numbers of negatively and positively charged residues were and , respectively. the vaccine structure has a half-life of , , and h in mammalian, yeast, and e. coli, respectively, therefore the vaccine is exposed to the immune system for a relatively long time and causes more immune responses [ ] . the recombinant vaccine's instability index was calculated at . , and as it was less than , it was considered to be a stable protein [ ] . the calculated aliphatic index and the gravy were . and - . , respectively. the higher aliphatic index value suggests greater thermostability and the negative gravy value shows the vaccine's hydrophilic nature therefore it can show strong interaction with water molecules [ ] . the sequences of vaccine (with and without adjuvant) were both antigenic, and adding the adjuvant to the n-terminal of the vaccine sequence increased the vaccine's antigenicity. results also suggested that the vaccine was soluble, non-allergenic, and non-toxic. to obtain a high-quality d structure, the initial d structure was subjected to the refinement process. the quality of the initial and refined models were assigned using ramachandran plot and prosa z-score. ramachandran plot data showed that . % of residues of the initial model was located in the outlier region while this value was reduced to . % after the refinement process. in addition, the z-score of primary model was calculated - . , while this parameter was determined - . after refining. these results suggest that the three-dimensional structure of the vaccine has improved after refinement. cluspro server conducted the docking process between the multi-epitope vaccine with hla-a : and hla-drb _ : molecules, the value of lowest energy in the selected complexes of vaccine-hla-a : and vaccine-hla-drb _ : were - . and - . , respectively, which indicated that the interaction of the vaccine with the hla-drb _ : to be stronger than its interaction with the hla-a : . the results of the rmsd evaluation showed that the vaccine -hla-drb _ : complex reached stability at ns times, while the vaccine -hla-a : complex required more time to stabilize. the rmsf result suggested that the flexibility in the regions between vaccine-hla in the both complex and the results of rg showed that the vaccine structure in both complexes was densified during simulation, but gained more density in the vaccine-hla-drb _ : complex. in practice, some essential factors including cai and gc content of the gene need to be optimized to achieve an optimal j o u r n a l p r e -p r o o f level protein expression in e. coli. according to the results, the value of the cai was , the cai value was greater than . , which suggests a higher probability of protein expression at a high level [ ] . gc content was . %, and the ideal percentage of gc content ranges from to %. the results of bioinformatics evaluation of the vaccine showed that this vaccine candidate may have a high potency against sars-cov- but the in vitro and in vivo studies are needed to confirm these results. the purpose of this research was to develop a covid- vaccine using bioinformatics approaches. to achieve this aim were predicted t and b-cell epitopes of s protein and then screened for antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. the eighteen high-ranked epitopes and lt-iic were incorporated into the final construct. after evaluating the physicochemical properties of the vaccine construct, its secondary and three-dimensional structure was predicted. structural quality, binding affinity to mhc-i and ii, and stability of the docked complexes were assessed. finally, the expression level of the vaccine in e. coli strain k was evaluated. significant results were reported in this research, experimental confirmation of the engineered vaccine required to further assess the efficacy of this vaccine candidate. j o u r n a l p r e -p r o o f clinical features of patients infected with novel coronavirus in the novel coronavirus disease (covid- ) pandemic: a zoonotic prospective sars-cov- causing pneumoniaassociated respiratory disorder (covid- ): diagnostic and proposed therapeutic options host factors in coronavirus replication, roles of host gene and non-coding rna expression in virus infection discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus a novel coronavirus from patients with pneumonia in china covid- : a new challenge for human beings the genome sequence of the sars-associated coronavirus unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage mechanisms of coronavirus cell entry mediated by the viral spike protein cryo-em structure of the -ncov spike in the prefusion conformation consider tlr for new therapeutic development against covid- identification of an antigenic determinant on the s domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies history of vaccination application of pharmacogenomics to vaccines reverse vaccinology probable molecular mechanism of remdesivir for the treatment of covid- : need to know more lt-iic, a new member of the type ii heat-labile enterotoxin family encoded by an escherichia coli strain obtained from a nonmammalian host propred : prediction of promiscuous mhc class-i binding sites propred: prediction of hla-dr binding sites prediction of continuous b-cell epitopes in an antigen using recurrent neural network vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines identifying candidate subunit vaccines using an alignmentindependent method based on principal amino acid properties bioinformatic approach for identifying parasite and fungal candidate subunit vaccines in silico approach for predicting toxicity of peptides and proteins dna and peptide sequences and chemical processes multivariately modelled by principal component analysis and partial least-squares projections to latent structures a fast peptide match service for uniprot knowledgebase protein identification and analysis tools on the expasy server exoproteome and secretome derived broad spectrum novel drug and vaccine candidates in vibrio cholerae targeted by piper betel derived compounds isoelectric point separations of peptides and proteins how are substrates recognized by the ubiquitin-mediated proteolytic system correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence thermostability and aliphatic index of globular proteins a simple method for displaying the hydropathic character of a protein development of multi-epitope driven subunit vaccine against fasciola gigantica using immunoinformatics approach scratch: a protein structure and structural feature prediction server high-throughput prediction of protein antigenicity using protein microarray data gor secondary structure prediction method version iv meshi: a new library of java classes for molecular modeling designing an efficient multiepitope peptide vaccine against vibrio cholerae via combined immunoinformatics and protein interaction based approaches prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins the cluspro web server for protein-protein docking new additions to the c lus p ro server motivated by capri how good is automated protein docking? gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers sars-cov- : an emerging coronavirus that causes a global threat design of a peptide-based subunit vaccine against novel coronavirus sars-cov- a review of sars-cov- and the ongoing clinical trials reverse vaccinology approach to design a novel multi-epitope vaccine candidate against covid- : an in silico study design of multi-epitope vaccine candidate against sars-cov- : a in-silico study epitopes for a -ncov vaccine preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies a novel design of a multi-antigenic, multistage and multi-epitope vaccine against helicobacter pylori: an in silico approach exploring dengue genome to construct a multi-epitope based subunit vaccine by utilizing immunoinformatics approach to battle against dengue infection codon adaptation index as a measure of dominating codon bias we would like to thank dr. shahram tahmasebian and dr. sorayya ghasemi for their valuable feedback and suggestions. the authors have declared no competing interest. none table . results of ctl epitope prediction. four ctl epitopes were selected (labeled with *) for incorporation in the proposed vaccine amino acid sequence. key: cord- -ywb cq authors: sarkar, indranil; garg, ravendra; van drunen littel-van den hurk, sylvia title: selection of adjuvants for vaccines targeting specific pathogens date: - - journal: expert rev vaccines doi: . / . . sha: doc_id: cord_uid: ywb cq introduction: adjuvants form an integral component in most of the inactivated and subunit vaccine formulations. careful and proper selection of adjuvants helps in promoting appropriate immune responses against target pathogens at both innate and adaptive levels such that protective immunity can be elicited. areas covered: herein, we describe the recent progress in our understanding of the mode of action of adjuvants that are licensed for use in human vaccines or in clinical or pre-clinical stages at both innate and adaptive levels. different pathogens have distinct characteristics, which require the host to mount an appropriate immune response against them. adjuvants can be selected to elicit a tailor-made immune response to specific pathogens based on their unique properties. identification of biomarkers of adjuvanticity for several candidate vaccines using omics-based technologies can unravel the mechanism of action of modern and experimental adjuvants. expert opinion: adjuvant technology has been revolutionized over the last two decades. in-depth understanding of the role of adjuvants in activating the innate immune system, combined with systems vaccinology approaches, have led to the development of next-generation, novel adjuvants that can be used in vaccines against challenging pathogens and in specific target populations. development of vaccines against infectious diseases is one of the most remarkable accomplishments in the history of mankind [ ] . smallpox has been eradicated from the world, and other diseases like diphtheria, poliomyelitis, pertussis, measles, and neonatal tetanus are significantly controlled by vaccination [ , ] . while live attenuated vaccines are immunogenic, there is a chance of live virus-induced disease progression in populations with underdeveloped or compromised immune systems [ ] . in contrast, inactivated virus vaccines are safe, but unsuitable when natural infection by the pathogen itself fails to induce any long-term immunity. recombinant subunit vaccines are considered as one of the most attractive modern vaccine types due to their high safety profiles. however, subunit vaccines are not inherently immunogenic [ , ] . to overcome this limitation, adjuvants are incorporated in subunit vaccines to enhance immunogenicity of the vaccine antigen. adjuvants facilitate the development of vaccines targeting pathogens against which live attenuated or inactivated vaccines are ineffective or undesirable. identification and selection of new adjuvants is thus critical, but also challenging, for successful subunit vaccine development. the fact that only a few adjuvants were approved for use in human vaccines till a few years ago can be at least partially attributed to the dearth of knowledge of the mechanism of action of adjuvants. however, presently six adjuvants (alum, as , mf , as , as and cpg odn) are approved for use in human vaccines. this was possible because structural characterization of several adjuvants and identification of various pattern recognition receptors (prrs) and co-stimulatory ligand receptors have enabled us to better understand the mode of action of adjuvants at a molecular level. understanding the mode of action of adjuvants is critical in designing vaccines that elicit pathogen-specific effector and long-term memory responses and in assessing the adjuvant safety at developmental and regulatory stages. the possible mechanisms of action by which adjuvants exert their adjuvanticity are discussed in the subsequent sections and represented schematically in figure . adjuvants as a delivery system in subunit vaccines [such as liposomes, immune stimulating complexes (iscoms) and nanoparticles] are considered effective in stimulating protective immunity [ ] . such adjuvants prevent rapid degradation of proteins and peptides in vivo, thereby enhancing the dose effectiveness of the vaccine antigen. liposomes are used in vaccine formulations against influenza, chlamydia, malaria, and tuberculosis (tb). coadministration of antigen with cationic liposomes leads to the induction of stronger antigen-specific immune responses than neutral or anionic liposomes [ ] . liposomes are effective vaccine delivery systems and act as carriers in adjuvants such as as , a liposome-based formulation consisting of monophosphoryl lipid a (mpla) and qs- [ ] . improved saponin-based tensoactive adjuvants (iscom, iscomatrix, and matrix-m tm ) are particulate antigen delivery systems with powerful immunostimulating activity [ ] . in iscoms, saponin, cholesterol and phospholipid form cage-like structures ( - nm in diameter). the adjuvant iscomatrix has a structurally similar structure but without the incorporated antigen (the antigen can be formulated with iscomatrix to prepare an iscomatrix vaccine) [ ] . both antigen delivery and immunostimulatory properties are present in iscomatrix [ ] . within the first few hours of injection, the antigen-iscomatrix complex traffics into draining lymph nodes (dlns) where antigen delivery takes place for uptake by the resident dendritic cells (dcs) and other antigen presenting cells (apcs) [ ] . iscoms are currently used in the development of influenza vaccines and iscomatrix in hepatitis c virus (hcv), influenza and candidate cancer vaccines in humans. the matrix-m tm adjuvant is being evaluated in vaccines against influenza, herpes simplex virus (hsv) type and malaria [ ] . nanoparticles are polymeric colloidal carriers of antigens, which enable site-directed delivery and prolonged release of antigen and facilitate alternative modes of vaccine administration (such as inhalation, optical or topical delivery). examples of polymeric nanoparticles are poly(lactic-co-glycolic acid) (plga), poly(lactic acid) (pla), poly(glycolic acid) (pga), poly(hydroxybutyrate) (phb) and chitosan [ ] aluminum salts are also used as delivery systems. crystals of alum bind to and alter the lipids of the dc plasma membrane to trigger cell activation that facilitates delivery of antigen, without alum itself being internalized by the dcs [ ] . aluminum salts are used as adjuvants in human vaccines against diphtheria, tetanus, pertussis, rabies, anthrax, and hepatitis a and b [ ] . in vitro studies revealed that the oil-inwater emulsion adjuvant, mf increases both phagocytosis and pinocytosis indirectly to promote better antigen uptake by apcs. instead of directly targeting dcs for antigen uptake, mf acts upstream by promoting influx of dc precursor cells and augmenting their differentiation into dcs [ ] . the safety of mf was demonstrated in various clinical studies with antigens from hepatitis b virus (hbv), hcv, cytomegalovirus (cmv), hsv and human immunodeficiency virus (hiv) [ ] . similar to mf , as does not directly activate dcs in vitro. intramuscular injection of as in mice promotes influx of monocytes, dcs, and granulocytes into the dlns. as is article highlights • identification of new prrs and their agonists are expected to lead to the identification of more adjuvants; in particular, prr agonists in combination adjuvants hold great promise. • systems vaccinology will provide a better understanding of the mode of action of adjuvants and allow identification of unique biomarkers of adjuvanticity. • there are many pathogens for which host-pathogen interactions have not been characterized in detail. such knowledge on hostpathogen interaction combined with the mechanism of action of adjuvants will lead to the use of specific adjuvants in vaccines against distinct pathogens. figure . schematic representation to highlight the possible mechanism of action by which adjuvants exert their adjuvanticity. adjuvants can serve as a depot that mediates recruitment of apcs or act as a delivery system to facilitate uptake of antigen by the apcs. adjuvants may activate innate immune responses by signaling through cell surface clrs (such as dectin- , dectin- , mincle), cytosolic nlrs, cell surface tlrs, endosomal tlrs or cytosolic rig-i and mda . signaling via prrs may lead to the activation of several transcription factors, which results in the production of pro-inflammatory cytokines, chemokines and type i ifns. secretion of chemokines due to adjuvants may also result in the recruitment and infiltration of more immune cells. adjuvants can activate c-gas that participates in the stingmediated irf -type i ifn pathway. adjuvants can enhance the expression of mhc and co-stimulatory molecules to mediate efficient presentation of antigen to naïve cd + t cells. depending upon the class of adjuvant, cellular (th ) and/or humoral (th ) immune responses may be induced. adjuvants also play important roles in gc reaction, affinity maturation and long-lived memory responses as a part of humoral immunity. also responsible for enhanced antigen uptake, by monocytes in particular, and antigen presentation in the dlns; this process is mediated by the presence of α-tocopherol in as [ , ] . the safety of squalene-based adjuvants (such as mf and as ) has been demonstrated by toxicological studies in animal models as well as in phase i-iii studies in humans [ ] [ ] [ ] depot effect refers to slow and prolonged antigen release at the site of injection providing continuous stimulation of the immune system. this facilitates enhanced antigen uptake by the apcs, which correlates to induction of high antibody titers. adjuvanticity of alum was originally thought to be due to depot effect; however, according to recent evidence, a depot effect is not the only mechanism by which it exerts its adjuvant activity [ ] . in a mouse model, alum was found to rapidly induce an inflammatory environment (via induction of inflammatory chemokines) that in turn triggers neutrophil recruitment and swarming at the injection site. furthermore, alum induces neutrophil death, resulting in the release of extracellular dna strands (neutrophil extracellular traps or nets) that play a significant role in the adjuvant action of alum [ ] . oil-in -water emulsions such as emulsigen®, water-in-oil emulsions such as cationic adjuvant formulation (caf) (a cationic liposome consisting of a combination of dimethyldioctadecylammonium/α,α'-trehalose , '-dibehenate or dda/tdb), as well as biodegradable micro-and nano-particles exhibit adjuvant activity via a depot effect in mice [ , ] . cationic liposomes exhibit long depot effects at the site of injection and strong electrostatic interactions with apcs. in contrast, adjuvants such as mf or iscoms do not require depot formation to exert their adjuvant activities; rather, antigen and adjuvant are cleared rapidly from the site of administration. a biodistribution study of as in mice conducted by radiolabelling each component of as revealed that all constituents of as infiltrated into the dlns within min of injection [ ] and that - % of each constituent of as was cleared from the injection site days post injection, suggesting dissociation of as [ ] . similarly, intramuscular injection of an as -adjuvanted vaccine in mice indicated rapid clearance of antigen and qs- from the injection site and into the dlns. differential biodistribution dynamics and pharmacokinetics of antigen and qs- suggested that the as and the vaccine antigen were not physically associated with each other [ ] . similarly, in matrix adjuvant formulations (consisting of nanoparticles comprised of saponin, cholesterol, and phospholipid), a physical association between matrix and antigen is not required for potent immune stimulation [ ] . in contrast, electrostatic interaction of antigen with adjuvant is required for optimal adjuvant activity of caf . similarly, optimal adjuvanticity of virosomes is achieved when the antigen of interest is associated with the virosome either through encapsulation or attachment to the bilayer via hydrophobic interaction [ ] . the success of the yellow fever vaccine yf- d, a live attenuated virus vaccine, can be attributed to its ability to activate multiple tlrs including tlr , , and , on or in dcs in mice [ ] . yf- d also activates human monocyte-derived dcs and plasmacytoid dcs (pdcs) [ ] . tlr -tlr complexes are activated by the lipopeptide analog pam csk , while tlr -tlr complexes are activated by the macrophage activating lipopeptide- (malp- ) from mycoplasma [ ] . poly(i:c) is a ligand for endosomal tlr , cytosolic retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated protein (mda ). poly(i:c) and its two derivatives, polyi:c u (ampligen) and poly(ic:lc) (hiltonol) are used in clinical trials against both tumors and infectious diseases such as hiv [ ] . tlr is targeted by monophosphoryl lipid (mpl)a. as (containing mpl) is approved for use in hbv (fendrix) and human papilloma virus (hpv) vaccines (cervarix) [ , ] . as (containing mpl) is also used in a malaria vaccine (rts,s) and the varicella zoster virus vaccine (hz/su) that have been found to be efficacious in phase iii trials [ ] . tlr recognizes bacterial flagellin. tlr signaling in cd + cd b + dcs plays an important role in intestinal iga production and th differentiation, and leads to myd -dependent, strong nuclear factor (nf)-κb activation and th- type immune responses in mice [ , ] . in contrast, flagellin acts as a th -polarizing factor for cd + t cells from human neonates and adults [ ] . tlr and tlr recognizing single-stranded rna (ssrna) molecules are targeted by small-molecule immune potentiator (smip)-based adjuvants such as imiquimod and resiquimod used in hpv virus-like particle (vlp) vaccines [ ] . tlr signaling induces b cell-mediated production of immunoglobulins (ig), interleukin (il)- and tnf-α, and natural killer (nk) cellmediated production of ifn-γ, while tlr signaling induces t cell proliferation, production of ifn-γ, il- , and il- , and memory t cell activation [ ] . tlr recognizes unmethylated cpg motif-containing microbial dna or immunostimulatory sequences (iss). tlr agonists are used in hbv vaccines to promote higher levels of protective antibodies. consequently, fewer immunizations and lower antigen doses are needed. in mice, tlr signaling leads to th type pro-inflammatory responses (il- , il- , il- , il- , tnf-α and ifn-γ), upregulation of major histocompatibility complex (mhc) and costimulatory molecules, and increased cd + t cell responses, while tlr -mediated b cell activation is responsible for induction of humoral immunity and antibody class switching [ ] . intracellular nlrs such as nod and nod receptors recognize diaminopimelatic acid (dap)-containing muropeptide from gram-negative bacteria, while nod detects the muramyl dipeptide (mdp) component present in all bacterial peptidoglycans. the adjuvanticity of the mucosal adjuvant cholera toxin (ct) is mediated through the nod receptor [ ] . adjuvants that are inducers of damage-associated molecular patterns (damps) trigger innate immune responses in vivo by damaging the host cells, thereby resulting in the release of damp factors (ex. rna, dna) for subsequent activation of the innate immune receptors [ ] . the cytosolic receptor nlrp is recognized by adjuvants such as quil-a and chitosan, as well as atp, mdp, uric acid crystals and silica. these compounds generate damp signals, such as reactive oxygen species (ros) or induce potassium efflux to activate nlrp . alum's adjuvanticity in mice is also attributed to the activation of nlrp / nacht (via swelling and rupture of the phagolysosome, release of cathepsin b into cytosol, subsequent activation of caspase and release of il- β), leucine-rich repeat (lrr), and pyrin-paad-dapin domain (pyd) domains-containing protein (nalp ) inflammasome, release of uric acid or activation of the stimulator of interferon (ifn) genes (sting)-ifn regulatory factor (irf) pathway due to the release of dna [ , ] . however, validation of these hypotheses for humans is warranted (ex. a direct role of il- β in adjuvanticity of alum in humans is debatable) [ ] . for instance, it is generally concluded that nlrp inflammasome and caspase are sometimes, but not always, required for induction of th cellassociated antibody responses in response to aluminum salts in vivo [ ] activation of caspase- is nlrp -dependent in vitro. however, no such role of nlrp is observed for qs- in vivo. qs- when formulated in liposomes activates human dcs by promoting cholesterol-dependent endocytosis with subsequent lysosomal destabilization and syk activation similar to alum [ ] . in mice, endocine, a lipid adjuvant, causes cellular damage to generate damps such as lactate dehydrogenase (ldh), dna and rna [ ] . chitosan induces release of mitochondrial dna into the cytoplasm to activate the nlrp inflammasome in mice [ ] . mf (but not aluminum hydroxide or calcium phosphate adjuvants) induces release of extracellular atp from the muscle in mice that acts as a danger signal [ ] . other damp adjuvants such as hydroxypropyl-βcyclodextrin (bcd) induce local cellular stress and death resulting in the release of the host cellular dna that serves as a damp to induce th -type immune responses [ ] . cytosolic dsdnas are sensed by several other prrs such as absent in melanoma (aim ), as well as by the protein cyclic guanosine monophosphate-adenosine monophosphate (cgamp) synthase (cgas), which simultaneously leads to the activation of sting-dependent tbk -irf -ifn- pathways and rela-tnf-α pathways [ ] . sting can bind cyclic dinucleotides (cdn), cyclic di-gmp (cdg) and cyclic di-amp (cda). in mice, cdg appears to be a safer mucosal adjuvant than cholera toxin [ ] and to promote protective immunity against h n influenza, staphylococcus, streptococcus and klebsiella infections. in mice, chitosan triggers release of intracellular dna that results in the engagement of the cgas-sting pathway in dcs to induce type ifn production and isgs, thereby promoting robust th immunity. this leads to the upregulation of costimulatory immune markers and the subsequent activation of dcs, as well as induction of igg c and cell-mediated immunity (cmi) [ ] . carbohydrate-based adjuvants include glucans, fructans, mannans, chitin/chitosan and other carbohydrate compounds derived from mycobacterium spp. (including lipoarabinomannan, muramyldipeptide/mdp, trehalose- - -dimycolate/tdm), as well as lps and saponin compounds (including qs- , a saponin in an oil-in-water emulsion) [ ] . in human monocyte-derived dcs (modc), qs- (also a component of as ) is endocytosed via the action of membrane cholesterol, with subsequent lysosomal destabilization and syk activation to promote a pro-inflammatory transcription program. in addition, cathepsin b (a lysosomal cysteine protease) is required for activation of modcs in vitro and also required for adjuvant activity of qs- in vivo [ ] . in another study, ln-resident cd b + cd + macrophages played a key role in the adjuvant activity of qs- . upon intramuscular injection in mice, qs- leads to rapid induction of local innate immune responses in the dlns and co-localises with ln-resident macrophages that are crucial for innate and effector responses to antigens formulated with qs- (via caspase / and inflammasome activation) [ ] . the primary mechanism of action of carbohydrate-based adjuvants involves interaction with prrs such as tlrs, nod and c-type lectin receptors (clrs) dectin- , dectin- and mincle on monocytes and apcs. this interaction activates nf-κb to induce inflammatory chemokine and cytokine responses [ ] . carbohydrate adjuvants also activate complement pathways to generate complement components acting as opsonins and chemokines. other important mechanisms of action of carbohydrate-based adjuvants include chemotaxis of lymphocytes, inflammasome activation (e.g. zymosan and mannans), and pore-formation, facilitating antigen entry into apcs (via interaction with cholesterol in the plasma membrane, e.g. qs- ) [ ]. adjuvants induce a series of signal transduction pathways to exert their action at both innate and adaptive levels. intramuscular injection of mpl or aso in mice is responsible for nf-κb activation in the muscles and local dlns [ ] . synthetic derivatives of mpl induce activation of tlr and selectively activate the p mapk pathway, which is strongly associated with optimal induction of ifn-γ-induced protein (ip- ), tnf-α and il- in mice [ ] . injection of as adjuvanted vaccine promotes release of ifn-γ by ln-resident nk cells and cd + t cells. pathway enrichment analysis of differentially expressed genes in injection site-dlns revealed that the ifn-signaling pathway was most enriched at and h, while the il- -driven anti-inflammatory pathway was also triggered by as at h post-injection. increased levels of ifnγ were detected early in the serum and dlns of humans and macaques vaccinated with as -adjuvanted vaccine, respectively [ ] . cellular and molecular synergy between mpl and qs- used in as in combination was responsible for this early ifn-γ response, which in turn, enhances the immunogenicity of as -adjuvanted vaccines [ ] . il- as an adjuvant activates janus kinase (jak)-signal transducer and activator of transcription (stat), phosphoinositide -kinase (pi k) and mapk pathways, thereby promoting b-cell and t-cell differentiation via sustained activation of stat and th differentiation through irf [ ] . subtle chemical alterations to mpla were made to develop a designer smip-based tlr -agonist known as sla, which induces trif signaling to produce th biased cytokines and chemokines like ifn and ip- , and less il- β. sla in oil-in-water emulsion (sla-se) was produced capitalizing on the knowledge that a combination of ifn and caspase-dependent inflammasome signaling leads to powerful adjuvant action [ ] . activation of the nf-κb pathway, as well as the p and jnk mapk pathways, programs dcs to produce il- p and to induce th responses. the extracellular signal-regulated kinase (erk)-c-fos mapk pathways favor th -type responses, while erk-retinaldehyde dehydrogenase (raldh) enzymes or β-catenin program dcs to induce t regulatory (treg) responses. similarly, complete freund's adjuvant (cfa) induces transcription of mhc-ii and b cell activation markers via the lyn-syk-pi k, the calcineurin-nuclear factor of activated t-cells (nfat) and the ras-mek-erk signaling pathways [ ] . saponin adjuvanticity relies on myd -mediated and il- receptor-signaling pathways [ ] . chitosan engages cgas-sting pathways to induce igg c and th responses in mice [ ] . intact myd signaling in each of the three types of apcs (dcs, macrophages and b cells) is essential for robust activity of tlr ligand-based vaccine adjuvants (porb, a tlr ligand and cpg, a tlr ligand) such as induction of in vivo cytokine responses, germinal center (gc) formation and antibody production [ ] . based on microarray analysis, mosca et al. demonstrated that three potent human vaccine adjuvants, mf , cpg odn, and alum, modulate a common set of genes ['adjuvant core response genes'] encoding cytokines, chemokines, innate immune receptors, ifn-induced proteins and adhesion molecules in mouse muscles [ ] . the establishment of a local immunocompetent environment due to such nonpathogenic inflammatory responses is associated with vaccine adjuvanticity. when compared to cpg odn and alum, mf was found to be the stronger inducer of adjuvant core response genes, which was reflected in enhanced and more rapid influx of mhc-ii + and cd b + cells into the injection site and more efficient transport of antigen to the dlns [ ] . both alum and mf induced chemokines involved in cellular influx such as ccl , ccl , ccl , and cxcl- [ ] . the presence of α-tocopherol in as promotes induction of leukocyte-recruiting chemokines (ccl , ccl and ccl ), neutrophil-mobilising cytokine (granulocyte colonystimulating factor (csf )) and pro-inflammatory cytokine/ chemokines (il- and cxcl- ), in mice which is in agreement with increased recruitment of granulocytes and antigenloaded monocytes into the dlns [ ] . aluminum adjuvants facilitate recruitment and differentiation of inflammatory monocytes (f / int cd b + lyg − ly c + ) into inflammatory dcs, thereby enhancing both humoral and cellular immunity [ ] . many of the above results with alum and mf have been confirmed in non-human primates [ ] . for example, vaccination with hiv vaccine adjuvanted with alum or mf triggered recruitment of monocytes, dcs, and neutrophils in the muscle [ ] . subcutaneous administration of iscomatrix in sheep induces a rapid and transient production of cytokines (il- , il- , ifn-γ) and influx of innate cells such as nk cells, nkt cells, neutrophils, migratory dcs (cd + cd − ) and cd α + dcs to the dlns [ ] . a combination adjuvant consisting of poly(i:c), a host defense peptide and pcep when delivered intranasally transiently induces production of chemokines and cytokines in murine respiratory tissues, which promotes infiltration and activation of dcs, macrophages, and neutrophils to generate improved mucosal and systemic immune responses [ ] . (a) improvement of the quality of antibody responses: innate immune responses play a profound role in regulating the magnitude, quality and persistence of antibody responses. the magnitude of the antibody response is critical in conferring protection against diphtheria, hepatitis a, lyme disease, tetanus, yellow fever, polio, rabies, and pneumococcal infections [ ] , while for rsv and meningococcal infections, the magnitude and quality of the antibody and cell-mediated response are important. adjuvant systems such as as are used in malaria (rts,s), herpes zoster (hz/su), tb and hiv vaccines, while as is used in several influenza vaccines such as trivalent inactivated h n influenza, h n prepandemic influenza, and candidate h n and h n pandemic influenza vaccines. as is used in licensed hpv- / and hbv vaccines. such adjuvant systems are known to augment antigen-specific t cell and antibody responses [ ] . in a murine study, the er stress-related pathway was found to potentially contribute to the adjuvanticity of as in vivo [ ] . furthermore, the expression of the er stress sensor kinase ire α by myeloid cells was involved in adjuvant activity of as . the er stress-related pathway was required for as mediated induction of il- , robust tfh responses, and antigenspecific antibody affinity maturation. il- plays a crucial role in differentiation of tfh cells as well as in the expression of il- by tfh cells and in production of antibodies [ ] . alum-adjuvanted vaccines act on il- producing gr- + cells to facilitate optimal priming, clonal expansion and antibody production by antigen-specific b cells in mice [ ] . in b cells, tlr ligands as adjuvants induce upregulation of surface markers involved in antigen uptake (mhc-i and mhc-ii) and surface markers involved in cross-talk with the t cells (cd , cd , and cd ), which ultimately leads to increased antigenspecific antibody production [ ] . however, when as is used as an adjuvant in the same hbv and hpv vaccine, instead of aluminum salts, higher levels of antibodies are induced in humans, indicating the added benefit of mpl (a tlr agonist) in humans [ ] . emulsigen®, an oil-in-water adjuvant, similar to mf and as , boosts innate responses and increases the number of cd + t cells required for robust antibody responses [ ] . mf supports induction of t follicular helper (tfh) cells and gc responses to vaccination by an unknown mechanism [ ] . in humans, mf and as promote early inflammation similar to results from animal studies [ ] . type ifns responses are induced in children as early as day postimmunization with mf -adjuvanted influenza vaccine, but in children immunized with non-adjuvanted vaccine, type i ifn responses are weaker and delayed. such early innate immune responses are reflected in induction of antigenspecific tfh cells days post-vaccination, and enhanced antibody responses in humans immunized with mf -adjuvanted influenza vaccine [ ] . (b) induction of gc reactions to promote memory b cell development: immunological memory is a distinctive hallmark of the adaptive immune system that contributes to protective immunity against infectious diseases. the gc reaction is central to memory development. induction of certain key molecules such as cd , inducible t-cell costimulator (icos), il- , programmed death-ligand (pd- ), cd , irf , and b-cell lymphoma protein (bcl- ) play a critical role in regulation of gc differentiation, affinity maturation and long-lived memory responses [ ] . tlrs expressed on gc b cells, follicular dcs (fdcs) and t cells have a profound effect on induction of antibody responses. nanoparticles resembling virions in size and containing tlr ligands, mpl and r , in combination with h n hemagglutinin mediate increased persistence of gcs, which significantly influence the differentiation of memory b cells critical for long-lived antibody responses in mice [ ] . a subset of cd + t cells, icos + cxcr + cxcr + t cells, was associated with protective antibody responses conferred by a trivalent split-virus influenza vaccine and efficiently induced memory b cells to differentiate into plasma cells [ ] . novel adjuvants may enhance b-cell activation in gcs and bonemarrow plasma cell survival. for example, the heat-labile enterotoxin (lt) of escherichia coli, ltk , when administered parenterally to neonatal mice, facilitates maturation of follicular dcs and generation of gcs [ ] . . . induction of cellular immunity: effector th /th / th and memory t cell responses signaling via tlr , tlr , tlr , tlr , and tlr promotes th biased immunity, while signaling via tlr /tlr , tlr /tlr and tlr promotes th -biased immunity. cd c + cd b − cd α + dcs localized in the marginal zones of lns induce th responses, as well as exhibit cross-presentation of antigens in vivo and ex vivo in mice [ ] . in humans, bdca + (cd c + ) and bdca + (cd + ) dcs (equivalent to murine cd α − and cd α + , respectively) are involved in cross-presentation of extracellular antigens [ ] . as a result of tlr -mediated enhanced mhc-i expression and type i ifn production, poly(i:c) promotes antigen cross-presentation to cd + t cells and antigen-specific ctls. in contrast, alum promotes th responses (strong antigen-specific igg and ige) and does not induce cd + t cell immunity, and even inhibits th immune responses in mice [ ] . however, when alum is present in combination with mpla, th responses can be generated as is found for aso [ ] . it is unclear whether such an aluminum salt-induced th bias exists in humans [ ] . rather, poor t cell responses are induced by aluminum salts in humans, possibly due to poor stimulation of the innate immune system [ ] . squalene-based oil emulsion is a potent inducer of both th -and th -mediated immunity and is well tolerated [ ] . adjuvants such as qs- , mf or cfa preferentially induce th -biased or a mixed th / th and th /th immune response. experimental cafs combined with immunostimulators such as tdb in tb vaccines stimulate both cellular and humoral immune responses, as well as promote efficient polyfunctional memory t cells, and th -and th -biased immune responses in mice [ ] . a sting-activating adjuvant, cdn, when formulated in a subunit vaccine and delivered intranasally, promoted a robust th immune response that correlated with long-lasting enhanced protection against mycobacterium tuberculosis in mice. adjuvanted vaccines promoting th responses may protect against intracellular pathogens by recruiting protective t cells earlier during infection [ ] . in neonates, cd + t cells are polarized towards th responses and reduced th responses. however, novel adjuvants such as ic and caf can induce adult-like th responses in newborn mice [ ] . cdg as a mucosal adjuvant induces th and th immune responses in mice [ ] . caf predominantly induces cd + t cell responses, while caf (consisting of dda, tdb and poly(i:c)) induces both cd + and cd + t cell responses [ ] . replacing as in an rts,s/ as candidate malaria vaccine with as improved antibody and cd + t cell responses, as well as protective efficacy as demonstrated by a randomized, double-blind, phase a trial [ ] . activated murine sub-capsular macrophages produce il- , which promotes generation of cd + t cells, while activated apcs also produce il- or il- , promoting generation of tfh cells, which in turn favors production of high-avidity antibodies by b cells in both mice and humans [ ] . intramuscular injection of mice with as induces early ifn-γ production by ln-resident nk cells, which is mediated by synergistic action of il- and il- in promoting ifn-γ responses. early ifn-γ production by nk cells is a prerequisite for optimal activation of dcs and induction of antigen-specific cd + t responses to as -adjuvanted antigens [ , ] . such ifn-γ responses were also observed in lns of macaques when they were injected with as [ ] . elevated levels of ifn-γ in the serum at day post-immunization and an increase in the number of cytokineproducing antigen-specific cd + t cells was also observed in humans immunized with rts,s malaria vaccine [ ] . the mechanisms of action of different adjuvants are summarized in table . the adjuvants that are licensed for use in human vaccines are listed in table , while the adjuvants in clinical trials are listed in table . . selection of adjuvants based on their mechanism of action against distinct types of pathogens mucosal surfaces are an attractive target for pathogens whose port of entry are gastrointestinal (e.g. polio virus, escherichia, salmonella, shigella, vibrio and helicobacter), respiratory (influenza virus, m. tuberculosis or mtb, adenovirus, coronavirus, rhinovirus and rsv) or urogenital tract (hsv, hpv, hiv- , chlamydia and neisseria) [ ] . mucosal adjuvants can be categorized as toxin-based (lt and ct), immunostimulatory (mpl, cpg, and qs ) and delivery system (emulsigen® and iscoms). two commonly used oral toxin-based adjuvants are [ ] . both are potent, but also toxic, when used as mucosal adjuvants. protective efficacy was attained when intranasal vaccines containing mutant lt adjuvants were used against hsv, bordetella pertussis and streptococcus pneumoniae [ ] . natural infection with rsv induces poor antibody responses with impaired effector functions, and perturbs localization and persistence of effector and memory t cells [ ] . induction of a potent, local mucosal immune response is required to prevent infection and a high systemic antibody response is also required to interrupt disease progression. nanoemulsions are safe for intranasal delivery and induce th and th responses in mice with no significant inflammation [ ] . sastry et al. tested multiple adjuvants such as sigma adjuvant system (sas, an oil-in-water adjuvant), carbopol, alum, adjuplex, poly(i:c), poly(ic:lc), mpla, addavax and montanide isa, and found that the adjuvantmediated increase in rsv-specific neutralizing antibody responses was context-dependent (i.e. whether pre-existing immunity was present or not) and species-specific (i.e. mice vs. calves) [ ] . formulation of the rsv fusion (f) protein with a combination adjuvant, triadj, elicited protective, mucosal and systemic, immune responses against rsv in mice and cotton rats [ ] . the mucosal epithelial barrier limits the bioavailability of vaccine antigens for sampling by apcs. adjuvants such as polyethyleneimine and chitosan are used as penetration enhancers and immunostimulants to augment binding to the mucosal surfaces and activate innate immunity in a mouse model [ ] . chitosan polymeric nanoparticles stimulate the nasal-associated lymphoid tissues (nalt) to produce mucosal secretory iga, igg, tnf-α, il- , and ifn-γ. pro-inflammatory cytokines and chemokines also act as mucosal adjuvants. proinflammatory cytokines such as il- , il- , il- , il- , and il- induce mucosal cd + ctls and antigen-specific iga. similarly, chemokines such as ccl (or mcp- ) enhance mucosal iga and ctl responses [ ] . neutralizing antibodies may protect against some acute self-limiting mucosal pathogens, but for highly invasive pathogens causing chronic infections (such as hiv, hcv, herpesviruses, and mycobacteria), mucosal innate and adaptive immune responses including cd + , th , and cd + ctls, as well as secretory iga and igg neutralizing antibodies at the port of pathogen entry, are required for effective and optimal protection [ ] . mucosal adjuvant-containing vaccines elicit both local and systemic immune responses, effective at local as well as distant sites [ ] . to control enteropathogens, orally administered vaccines must overcome challenges of antigen degradation and immune tolerance [ ] . biodegradable micro-or nanoparticles are required that are resistant to low ph and can target antigen to m cells. u-omp , a bacterial protease inhibitor from brucella abortus, is an oral adjuvant suitable for subunit vaccine formulation, which can inhibit stomach and gut proteases and delay antigen digestion at the lysosome to enhance antigen presentation and recruitment of immune cells to gastrointestinal mucosa [ ] . intranasal immunization of mice with poly-i:c u (ampligen) in an h n influenza vaccine promoted increased levels of protective, mucosal iga and systemic igg [ ] . however, only a few mucosal vaccines are licensed for humans, primarily due to a dearth of safe and effective mucosal adjuvants [ ] . although during the last few decades there has been a constant development of new and effective mucosal adjuvants, most of them are toxic. for example, ltk when intranasally injected can trigger transient facial nerve paralysis or bell's palsy [ ] . thus, there is an urgent need to address safety issues of mucosal adjuvants. in a phase iii trial, poly-i:c u (ampligen) was demonstrated to be safe [ ] . a randomized phase i/ii trial was conducted to determine the safety and efficacy of ampligen in patients with stage ii-iv human epidermal growth factor receptor (her )-positive breast cancer. the result from this trial will give important insight into the application of ampligen in therapeutic cancer vaccines [ ] . pathogenic fungi and protozoan parasites have complex life cycles and switch among several different forms during their life. histoplasma capsulatum grows as a mold in the soil at low temperature, but upon inhalation into the lungs, it switches to yeast form and causes histoplasmosis. interaction of infected macrophages with cd + and cd + t cells leads to increased production of th cytokines, il- , ifn-γ, and tnf-α, that are critically important in generating protective immunity against h. capsulatum infection in mice. leukotrienes, lipid mediators derived from arachidonic acid metabolism, are found to be potent adjuvants against such fungal infections [ ] . malaria vaccine development is impeded by the complex life cycle of plasmodium spp., the intracellular stage in its life cycle, large physical size, surface antigenic diversity and enormous genetic and genomic plasticity [ ] . the parasite replicates intracellularly (and thus is partially protected from immune recognition) and also sequesters any innate immune ligand away from prrs in the sporozoite and gametocyte stages of their life cycle. a malaria vaccine needs to establish humoral immunity to prevent merozoites from entering the erythrocytes and the liver or destroy the merozoites through opsonization and cmi. rts,s/as (mosquirix) is a malaria candidate vaccine targeted against the infectious sporozoite stage and designed to enhance both antigen-specific humoral and cellular immunity. th effector cells are essential to target asexual blood stages, while eventual control and/or clearance of the parasites requires antibody-mediated responses [ ] . mpl and qs- , the two components used in as have important functions. mpl is a tlr agonist that induces production of ifn-γ by t cells and antibody isotype switching to igg a/c in mice, while qs- induces neutralizing antibodies and cytotoxic t cell responses [ ] . as requires synergistic activities of both mpl and qs- for optimal adjuvant activity. as in combination with plasmodium antigens induces rapid and transient innate immune responses in the injection site and dlns, activates immune cells (including apcs), as well as generates -fold higher antibody titers when compared to natural exposure [ ] . however, in a large phase iii trial in children and young infants in seven sub-saharan african countries, although rts,s/as prevented a considerable number of cases of clinical malaria in infants and young children over - years, the vaccine efficacy declined with subsequent follow-ups in the infants, and did not provide significant protection against severe malaria [ ] . nonetheless, rts,s/as plays a significant role in the control of malaria in areas of high transmission when used in conjunction with other effective preventive measures (rts,s clinical trials partnership). poly(i:c) and its derivatives are of great importance for vaccines that need to induce a th /ctl immune response against various viruses and pathogens including p. falciparum [ ] . pam csk was used in a malaria vaccine containing p. falciparum circumsporozoite protein b cell epitopes and universal t cell epitopes, which resulted in the induction of high titers of antigenspecific igg , igg and igg in immunized volunteers [ ] . herpesviruses are large viruses with a complex genome. primary infection with varicella zoster virus (vzv) causes varicella (chickenpox) and may go into latent phase in human cranial and dorsal root ganglia. aging or immune dampening results in decline of vzv-specific cmi, which may induce reactivation of the virus and cause shingles. hence, cmi is necessary to prevent reactivation of the latent virus. the vzv vaccine hz/su (shingrix) composed of the vzv glycoprotein e subunit (ge) antigen and as b was recently approved for the prevention of herpes zoster in adults aged years or older [ ] . as was selected as the adjuvant for the vzv vaccine, because compared to other adjuvant systems, as induced higher numbers of ifn-γ secreting cd + t cells, and thus improved t cell as well as antibody responses, with acceptable clinical safety profiles [ ] . hpv effectively evades innate immunity by inhibiting the ifn receptor signaling pathways and activation of isgs via the e and e proteins. hpv also downregulates tlr and does not induce any danger signal to alert the immune system [ ] . this prolongs the duration of infection and delays the onset of adaptive immunity. thus, an effective cmi is required to clear and control hpv infection. effective vaccine-induced immunity against hpv should consist of cmi to the early proteins, e and e , and neutralizing antibodies against the virus coat protein l . two currently approved hpv vaccines, cervarix (a bivalent hpv / vaccine, gsk) and gardasil (a quadrivalent hpv / / / vaccine, merck) are highly protective against hpv , , and [ ] . both are li vlps; however, cervarix is as adjuvanted, while gardasil is aahs (amorphous aluminum hydroxyphosphate sulfate)-adjuvanted. vlps strongly activate the stromal dcs in the injection site that migrate to the dlns, or may directly bind to the surface of apcs or other immune cells and migrate to the lns, where they prime naïve b cells [ ] . according to a recent study in girls aged - years, two doses of cervarix elicited superior hpv- / antibody responses compared to two or three doses of gardasil. the differences in immunogenicity between the two vaccines may be due to the different types of adjuvants used. as enhances humoral immune responses and cmi by triggering local and transient cytokine responses that promote enhanced activation and presentation ability of apcs [ ] . significantly higher antibody titers are induced in mice immunized with hpv- l vlps adsorbed onto aahs as compared to vlps adsorbed onto aluminum hydroxide along with induction of an improved l -specific ifn-γ secreting t cell response [ ] . mtb causing tb is an intracellular pathogen that has the ability to survive within the hostile environment of the alveolar macrophages after being phagocytosed and to multiply unchecked. cd + t cells, cd + t cells, ctls, th cells, nk cells, and activated macrophages are critical in controlling mtb infections. bacillus calmette-guérin (bcg) vaccine fails to protect adults from pulmonary tb and prevent transmission of mtb in adolescents and adults [ ] . thus, there is an urgent need for improved vaccines against tb. one of the potential vaccine strategies against mtb is to eliminate or control latent infection and prevent reactivation or progression to clinical tb in latently infected patients. this may be accomplished by incorporating adjuvants that are capable of inducing both cd + and cd + t cell responses in both immunocompetent and immunocompromised individuals. mechanisms of antibody-mediated protection against tb include opsonization, complement activation, and fc receptor engagement. current research is focused on adjuvants that act on innate lymphoid cells (ilcs), nk cells and non-classical t cells such as cd , mr , hla-e and γδ t cells present in large numbers in the circulation and mucosa [ ] . although the immune correlates of protection from tb disease are not validated yet, vaccines currently in clinical development predominantly focus on generating cd + and cd + th -type immune responses. adjuvants such as mineral salts, saponin, emulsigen®, micro-or nanoparticles, toxin derivatives, cationic lipids, cpg dna, adjuvant systems and cytokines have been tested in subunit vaccine preparations, either alone or in combination with bcg in a prime-boost strategy [ ] . the strongest th -inducing adjuvants for tb are unmethylated mycobacterial dna and cpg odn, which promote ctl activation and ifn-γ production [ ] . tlr / and tlr / ligands are presented on the surface of mtb (triacylated and diacylated forms of mycobacterial p lipoprotein) or secreted by the bacterium, while nlrs such as nod are responsible for intracellular recognition of mycobacteria [ ] . novel adjuvants, including dda, tdb, ic , poly(i:c), gelatin, cpg odn, mpla, glycopyranosyl lipid adjuvant (gla) in combination with squalene (se) known as gla-se, mf , caf , and as b are also being clinically tested. dda promotes generating humoral, cell-mediated and ifn-γ responses against mtb, while as and mf induce strong th immunity against mtb. all these adjuvanted subunit vaccines induce protective immunity and enhance bcg-primed immunity in animal models [ ] . in a randomized, double-blind, phase b trial, a candidate tuberculosis vaccine, m /as e , demonstrated a clinically acceptable safety profile and conferred % protection against active pulmonary tuberculosis in adults with latent mtb infection [ ] . nanoparticle-based vaccines are critical for the induction of protective th -type immune responses to intracellular pathogens. the liposomal caf adjuvant promoted th and long-lasting memory t cell response in human tb vaccination trials. caf -adjuvanted tb vaccine stimulates the clr, and mincle, and triggers the syk/card signaling cascade to activate the th signaling pathway [ ] . one of the biggest challenges in vaccine development is the fact that the immunological mechanisms that govern vaccine safety and efficacy are still largely unknown. in recent years, systems vaccinology has emerged as an interdisciplinary approach that relies on high-throughput omics-based techniques to study vaccine-induced changes in the entire genome, set of transcripts, proteins, and metabolites in various tissues. a systems vaccinology approach has been used to elucidate immune responses to vaccines against yellow fever [ ] , influenza [ ] , malaria [ ] , smallpox [ ] and hiv [ ] . in addition, a systems vaccinology approach identified molecular and cellular immune signatures of a vaccine against bordetella pertussis [ ] . ip- was identified as an early innate immune signature that correlated with antibody responses to an ebola vaccine (rvsv-zebov) [ ] . computational analysis of the transcriptomic profile in human peripheral blood mononuclear cells (pbmcs) induced by yellow fever vaccine yf- d identified two molecular signatures: eukaryotic translation initiation factor alpha kinase (eif ak ) and tnfrsf , encoding the receptor for the b-cell growth factor blys-baff [ ] . eif ak correlated with the magnitude of the cd + t cell responses, while tnfrsf correlated with the magnitude of neutralizing antibody responses. other genes such as calreticulin, c-jun, and glucocorticoid receptor were also induced by yf- d, and this induction correlated with cd + t cell responses [ ] . in another study with young healthy adults, intramuscularly administered tiv induced higher antibody levels and plasmablasts when compared to intranasally delivered live attenuated influenza vaccine (laiv) with induction of distinct transcriptional signatures such as enhanced expression of type ifn genes in laiv recipients, but not in tiv recipients [ ] . based on a systems vaccinology approach, tlr agonists as adjuvants were found to potently enhance the immunogenicity of influenza vaccine, resulting in an improved antibody response in humans [ ] . the longevity of the immunoglobulin response post vaccination could be predicted from the ability of the adjuvanted vaccine to induce proliferation of antigen-specific il- + icos + cxcr − cd + t cells in the peripheral blood. systems vaccinology also identified two biomarkers (junb and ptx ) of mf and the skeletal muscle tissue cells (in addition to apcs) as direct target of mf for its adjuvant action in mice [ ] . caproni et al. investigated molecular signatures induced by different tlr-dependent (cpg odn, resiquimod and pam csk ) and tlr-independent (mf and alum) adjuvants in influenza subunit vaccines to establish the innate immune correlates of adjuvanticity by using dna microarrays in a mouse model [ ] . two adjuvants, mf and pam csk increased overall antibody and hai titers, and induced active infiltration of cd b + cells, especially neutrophils, to the injection site. this suggests early induction of cd b + cells due to an emulsion-based adjuvant to be predictive of subsequent robust humoral immunity. systems vaccinology has also been applied to identify novel mechanisms of induction of th responses by an adjuvant. for instance, the th -promoting adjuvant activity of cysteine protease allergen is dependent on the production of ros by dcs. as a result of induction of ros, oxidized lipids are induced that in turn promote epithelial cell-mediated production of thymic stromal lymphopoietin (tslp), resulting in the recruitment of il- + basophils to the lns for induction of th -type immune responses in mice [ ] . genes associated with memory b cell formation and productive antibody responses such as bcl , bcl a, tank, plcg , and cd are induced when mice are immunized with ovalbumin (ova) adjuvanted with tlr and tlr agonists [ ] . in a study with the candidate malaria vaccine rts,s/as b in human subjects, enhanced expression of genes involved in immunoproteasome formation, psme in particular, was found to be responsible for conferring protection from parasitemia. induction of the immunoproteasome enhances mhc antigen presentation, which in turn, indirectly enhances antibody responses and directly augments cd + t cell development and production of ifn-γ, tnf-α, il- , and cd l. the above immune signatures may contribute to the protective efficacy of the candidate malaria vaccine [ , ] . a comparative systems analysis of four vaccine adjuvants, gla-se, ic , caf , and alum, in mice revealed distinct molecular signatures. gla-se induced massive changes in the transcriptomic profile in the whole blood and dlns that correlated with increased cellular influx (such as cd c + gr + mdcs) in the dlns, in contrast to limited transcriptomic changes induced by other adjuvants. co-expression analysis of differentially expressed genes in whole blood revealed that caf and gla-se (but not ic ) induced transcriptional signatures related to innate immune responses. the analysis also revealed modules enriched for genes associated with tfh and gc-mediated b cell responses; for example, gla-se induced nfatc , nfatc , and il r; caf induced batf and ic induced pou af . a systemic analysis of protective immune responses to three rts,s vaccinations with a subsequent controlled human malaria challenge of the vaccine recipients with plasmodium-infected mosquitoes was carried out. molecular signatures of b cell and plasma cells in human pbmcs were found to be positively correlated to protection, while the nk cell signatures correlated negatively with protection, indicating multiple mechanisms of protective immunity against p. falciparum [ ] . in a study by burny et al., different adjuvants [as b , as e , as , as , and alum/al(oh) ] induced common innate pathways and were responsible for improved adaptive responses when used with a model antigen (hbv surface antigen or hbsag) in humans. as b , as e , and as induced comparable innate profiles and so did as and alum. furthermore, the ability to activate innate immunity (ifn-signaling pathway, in particular) was linked to enhanced adaptive responses elicited by as -and as -adjuvanted vaccine. early changes in immune markers, such as crp, il- , ifn-γ, and ip- , correlated with the magnitude of the adaptive responses [ ] . systems vaccinology also identifies signatures of vaccine safety and efficacy. non-specific adverse side effects observed for vaccines that fail in human clinical trials are frequently associated with over-stimulation of certain components of the innate immune system. systems vaccinology can be applied to screen adjuvants to help design protective and safe vaccines [ ] . correlates of protection have been established for a number of licensed vaccines as reviewed by tomaras et al. [ ] . however, attempts to identify correlates of protection are still ongoing for tb, while the commonly assumed immune correlates often fail to correctly predict an individual's risk of developing malaria [ ] . for hiv, complex immune correlates of protection characterized by multiple types of immune responses are found to be involved in controlling hiv- transmission [ ] . for vaccines for which the immune correlates of protection are unknown, systems vaccinology approaches can be used to identify signatures induced rapidly after vaccination that will help to predict the later immune outcome. a systems vaccinology approach can also help in identifying vaccine non-responders as well as vaccine high-and low-responders [ ] . innate and adaptive immune responses are profoundly influenced by any significant changes in metabolic activity. inflammation triggered by vaccine adjuvants results in a shift in energy supply leading to metabolic acidosis and impaired oxygen supply, which in turn results in phenotypic shifts. these phenotypic shifts heavily affect the metabolic state of an individual. lipid metabolism plays an important role in inflammation. liquid chromatography-mass spectrometry (lc-ms) is employed to identify and quantify cell-or tissue-specific metabolites [ ] . metabolite immune-correlates such as nucleotides, amino acids, lipids, fatty acids, and anti-oxidants may represent inflammatory mediators and/or biomarkers that profoundly influence several inflammatory processes such as cellular infiltration, activation of signaling pathways and oxidative stress [ ] . thus, a comprehensive understanding of the molecular signatures induced by adjuvants early after vaccination will help to predict the later adaptive immune responses in humans. furthermore, such knowledge will also improve or help in (re-)designing next-generation adjuvants and drive the development of next-generation vaccines with the concerted effort of vaccinologists, clinicians, systems biologists, statisticians, as well as industrial and regulatory authorities. the relationship between adjuvants, innate pathways/ receptors activated, immune responses triggered, and the type of pathogens ideal for such adjuvant-mediated immune responses are summarized in table . in this review article, we have summarized the mechanism of action of different classes of adjuvants. we also discussed why this knowledge is important in context of distinct disease targets and how this knowledge can be utilized to improve the development of adjuvanted vaccines against challenging pathogens. we also briefly highlighted the important role of the new-age systems vaccinology approaches in better understanding an adjuvant's mode of action, and identification of unique cellular and molecular biomarkers of adjuvanticity. it is important to note here that mouse models offer flexibility and accessibility to study intricate facets of the mechanism of action of adjuvants and responses to immunization with adjuvanted vaccines. while the results from animal studies often overlap with the results from human studies, there are several dissimilarities as well. for instance, mf -adjuvanted vaccine in mice induces both cell-mediated and humoral immune responses, which is not observed in humans at any age, rather they tend to develop a th -th response. even in humans, immune responses need to be investigated not only in the serum but also in the dlns and other lymphoid organs; not only at the priming site but also in the distant effector sites such that a holistic and reliable assessment of mechanism of action of adjuvant and/or vaccine can be made encompassing all possible immune parameters. even the immunological correlates that have recently been identified by gene profiling or systems vaccinology for different adjuvants/vaccines are defined more at the population level and much less at the individual level [ ] . all these considerations must be taken into account while designing effective and safe vaccines/adjuvants. recent advancements have allowed researchers to conclude that clinical-grade adjuvants have distinct immunological profiles and signatures, which can be used to target different pathogens. based on pathogen-specific immune response requirements (i.e. th , th or th responses, or mixed th / th or th /th responses, etc.), next-generation adjuvants can be rationally developed and incorporated into human vaccines. currently, all approved human adjuvants mostly induce only antibody responses. however, recent adjuvant research has led to the development of novel adjuvants capable of inducing cmi (especially required for malaria, tb and hiv), as well as antibody responses. new immunostimulatory adjuvants or immunomodulatory compounds are under investigation to induce cmi and high antibody titers. novel combination adjuvants are being tested in candidate human vaccines with promising results that have strong implications for use in vaccines against challenging infectious pathogens and different target populations. this is potentially due to activation of multiple innate immune sensing signal transduction pathways by combination adjuvants. novel adjuvants are required that can target emerging new pathogens or reemerging old pathogens. such pathogens often have a more complex host-pathogen interaction, which needs better understanding and further characterization. among these new-generation adjuvants, several are proprietary, which may make it difficult to purchase them and conduct independent parallel trials. factors such as genetic background, preexposure to pathogens or vaccine antigens, age, nutritional and immunological status of vaccine recipients, all dictate the final effectiveness of adjuvanted vaccines. nevertheless, with the aid of structural, systems and reverse vaccinology, epitope prediction and other technological advancements, adjuvant technology is now gradually progressing towards a more personalized approach. there has been an exponential growth in the field of adjuvant research. while alum was historically used as the only licensed adjuvant for more than years, six new adjuvants were approved in the last years. the next five years will see substantial progress in obtaining licensure for more varied types of adjuvants. understanding innate immunity and the role of prr agonists as adjuvants in stimulating the innate immune system has revolutionized adjuvant technology. systems biology has immense contribution in the development of effective and potent adjuvants, and will continue to do so in the coming years. correlates of adjuvanticity or immune signatures, and biomarkers of adjuvant safety and protective efficacy will further streamline adjuvant research. tailor-made adjuvants will find their use against distinct pathogens and in specific target populations. better characterization of adjuvants by new omics-based technologies will facilitate licensing of new adjuvants. since there is a pressing need for developing vaccines against a multitude of very virulent/emerging pathogens, we need to develop subunit vaccines and not live vaccines, and hence adjuvant selection is critical. the mucosal surface is the preferred route of entry for most pathogens. therefore, mucosal immunization is considered to be most effective in preventing mucosally transmitted infections. however, the major hurdle in the development of mucosal vaccines is the lack of safe and effective mucosal adjuvants due to toxicity issues. there is specific need for standardized, more comprehensive and pertinent methodologies for safety evaluation to enable development of safe mucosal adjuvants [ ] . mucosal adjuvants are also required to promote bioavailability of vaccine antigen. another requirement is the development of mucosal adjuvants with an optimal targeting ability so as to reduce undesirable adverse side effects. since efficacy and toxicity of most mucosal adjuvants appear to be intrinsically linked, a risk-benefit ratio needs to be ascertained for these adjuvants. attention must also be directed to studying antigen-adjuvant interactions instead of irrational mixing of an adjuvant in a vaccine formulation [ ] . oil-in-water emulsions are very promising adjuvants and characterization/analysis of components added in emulsion preparations in more detail will facilitate improvement of such adjuvants [ ] . papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers vaccine-preventable disease table working group. historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states modes of action for mucosal vaccine adjuvants vaccine adjuvants: from to and beyond. vaccines (basel) an overview of adjuvant formulations and delivery systems liposomal vaccine delivery systems liposomes as vaccine delivery systems: a review of the recent advances iscom-based vaccines: the second decade iscomatrix adjuvant for antigen delivery iscomatrix: a novel adjuvant for use in prophylactic and therapeutic vaccines against infectious diseases meta-analysis on randomized controlled trials of vaccines with qs- or iscomatrix adjuvant: safety and tolerability vaccine delivery using nanoparticles alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity vaccine adjuvants: mode of action vaccines with the mf adjuvant do not stimulate antibody responses against squalene from discovery to licensure, the adjuvant system story correlates of adjuvanticity: a review on adjuvants in licensed vaccines design and evaluation of a safe and potent adjuvant for human vaccines novel respiratory syncytial virus-like particle vaccine composed of the postfusion and prefusion conformations of the f glycoprotein adjuvants: classification, modus operandi, and licensing neutrophil swarming and extracellular trap formation play a significant role in alum adjuvant activity comparison of multiple adjuvants on the stability and immunogenicity of a clade c hiv- gp trimer non-clinical safety and biodistribution of as -adjuvanted inactivated pandemic influenza vaccines enhancement of adaptive immunity by the human vaccine adjuvant as depends on activated dendritic cells iscom technology-based matrix m adjuvant: success in future vaccines relies on formulation working together: interactions between vaccine antigens and adjuvants yellow fever vaccine yf- d activates multiple dendritic cell subsets via tlr , , , and to stimulate polyvalent immunity identification of novel synthetic toll-like receptor agonists by high throughput screening immune adjuvant effect of molecularly-defined toll-like receptor ligands. vaccines (basel) • in-depth review of the use of tlr-ligands as adjuvants and discussion of the mechanism of action of such ligands efficacy of a prophylactic adjuvanted bivalent l virus-like-particle vaccine against infection with human papillomavirus types and in young women: an interim analysis of a phase iii double-blind, randomised controlled trial new hepatitis b vaccine formulated with an improved adjuvant system rectal and vaginal immunization of mice with human papillomavirus l virus-like particles microbiota influences vaccine and mucosal adjuvant efficacy towards an understanding of the adjuvant action of aluminium rna is an adjuvanticity mediator for the lipid-based mucosal adjuvant the vaccine adjuvant chitosan promotes cellular immunity via dna sensor cgas-stingdependent induction of type i interferons viral evasion of dna-stimulated innate immune responses the mucosal adjuvant cyclic di-gmp enhances antigen uptake and selectively activates pinocytosis-efficient cells in vivo carbohydrate-based immune adjuvants lysosome-dependent activation of human dendritic cells by the vaccine adjuvant qs- central role of cd (+) lymph node resident macrophages in the adjuvanticity of the qs- component of as adjuvants in the driver's seat: how magnitude, type, fine specificity and longevity of immune responses are driven by distinct classes of immune potentiators. vaccines (basel) excellent insight into the correlates of adjuvanticity of wellknown and specialized adjuvants, roles on induction of adaptive immune responses as well as targeted and rational use of adjuvants in vaccines as , an aluminum salt-and tlr agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity selective activation of the p mapk pathway by synthetic monophosphoryl lipid a cellular and molecular synergy in as -adjuvanted vaccines results in an early ifngamma response promoting vaccine immunogenicity il- signaling in immunity a structure-function approach to optimizing tlr ligands for human vaccines molecular characterization of in vivo adjuvant activity in ferrets vaccinated against influenza virus mode of action of adjuvants: implications for vaccine safety and design toll-like receptor ligand-based vaccine adjuvants require intact myd signaling in antigen-presenting cells for germinal center formation and antibody production molecular and cellular signatures of human vaccine adjuvants identification of molecular and cellular biomarkers induced in mouse muscle upon injection with mf oil-in-water emulsion the adjuvants aluminum hydroxide and mf induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells adjuvant system as containing alpha-tocopherol modulates innate immune response and leads to improved adaptive immunity alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells induction of lymphocyte recruitment in the absence of a detectable immune response littel-van den hurk s. formulation of the respiratory syncytial virus fusion protein with a polymer-based combination adjuvant promotes transient and local innate immune responses and leads to improved adaptive immunity immunological mechanisms of vaccination different adjuvants induce common innate pathways that are associated with enhanced adaptive responses against a model antigen in humans activation of the endoplasmic reticulum stress sensor ire alpha by the vaccine adjuvant as contributes to its immunostimulatory properties promotion of b cell immune responses via an alum-induced myeloid cell population preclinical evaluation of bacterially produced rsv-g protein vaccine: strong protection against rsv challenge in cotton rat model towards an evidence based approach for the development of adjuvanted vaccines highlights the role of immunepotentiators in activation of the innate immune system, which is implicated in the development of next-generation adjuvants as well as clinical adjuvanted-vaccines immunity to viruses: learning from successful human vaccines induction of icos +cxcr +cxcr + th cells correlates with antibody responses to influenza vaccination the adjuvant lt-k can restore delayed maturation of follicular dendritic cells and poor persistence of both protein-and polysaccharide-specific antibody-secreting cells in neonatal mice adjuvants enhancing cross-presentation by dendritic cells: the key to more effective vaccines? front immunol sting-activating adjuvants elicit a th immune response and protect against mycobacterium tuberculosis infection a liposome-based mycobacterial vaccine induces potent adult and neonatal multifunctional t cells through the exquisite targeting of dendritic cells intranasal c-di-gmpadjuvanted plant-derived h influenza vaccine induces multifunctional th cd + cells and strong mucosal and systemic antibody responses in mice randomized, double-blind, phase a trial of falciparum malaria vaccines rts,s/ as b and rts,s/as a in malaria-naive adults: safety, efficacy, and immunologic associates of protection mechanisms of action of adjuvants synthetic cationic peptide idr- provides protection against bacterial infections through chemokine induction and enhanced leukocyte recruitment (tlr ) interacts with flagellin and profilin through disparate mechanisms cpg oligonucleotides as cancer vaccine adjuvants. vaccines (basel) flagellin as a vaccine adjuvant the development of mucosal vaccines for both mucosal and systemic immune induction and the roles played by adjuvants discusses the role of mucosal adjuvants in stimulating both mucosal and systemic immunity against pathogens infecting via mucosal surfaces u-omp from brucella abortus is a useful adjuvant for vaccine formulations against salmonella infection in mice choice and design of adjuvants for parenteral and mucosal vaccines. vaccines (basel) vaccine development for respiratory syncytial virus adjuvants and the vaccine response to the ds-cav -stabilized fusion glycoprotein of respiratory syncytial virus van den hurk s. induction of mucosal immunity and protection by intranasal immunization with a respiratory syncytial virus subunit vaccine formulation mucosal adjuvants: opportunities and challenges what role does the route of immunization play in the generation of protective immunity against mucosal pathogens? intranasal immunization with h n vaccine plus poly i:poly c u, a toll-like receptor agonist, protects mice against homologous and heterologous virus challenge tlr agonists and proinflammatory antitumor activities leukotrienes are potent adjuvant during fungal infection: effects on memory t cells antigenic diversity and immune evasion by malaria parasites immune mechanisms in malaria: new insights in vaccine development induction of cross-reactive cytotoxic t-lymphocyte responses specific for hiv- gp using saponin adjuvant (qs- ) supplemented subunit vaccine formulations the future of the rts,s/as malaria vaccine: an alternative development plan vaccine adjuvant uses of poly-ic and derivatives recommendations of the advisory committee on immunization practices for use of herpes zoster vaccines cell-mediated immune responses to a varicella-zoster virus glycoprotein e vaccine using both a tlr agonist and qs in mice hpv -immune response to infection and vaccination efficacy of human papillomavirus l protein vaccines (cervarix and gardasil) in reducing the risk of cervical intraepithelial neoplasia: a meta-analysis immunobiology of hpv and hpv vaccines comparative immunogenicity and safety of human papillomavirus (hpv)- / as -adjuvanted vaccine and hpv- / / / vaccine administered according to -and -dose schedules in girls aged - years: results to month from a randomized trial adjuvants in tuberculosis vaccine development tb vaccine development and the end tb strategy: importance and current status nod and toll-like receptors are nonredundant recognition systems of mycobacterium tuberculosis multi-stage subunit vaccines against mycobacterium tuberculosis: an alternative to the bcg vaccine or a bcg-prime boost? phase b controlled trial of m /as e vaccine to prevent tuberculosis systems biology approach predicts immunogenicity of the yellow fever vaccine in humans systems biology of vaccination for seasonal influenza in humans expression of genes associated with immunoproteasome processing of major histocompatibility complex peptides is indicative of protection with adjuvanted rts, s malaria vaccine integrated analysis of genetic and proteomic data identifies biomarkers associated with adverse events following smallpox vaccination molecular signatures of a tlr agonist-adjuvanted hiv- vaccine candidate in humans molecular and cellular signatures underlying superior immunity against bordetella pertussis upon pulmonary vaccination systems vaccinology identifies an early innate immune signature as a correlate of antibody responses to the ebola vaccine rvsv-zebov predictive markers of safety and immunogenicity of adjuvanted vaccines mf and pam csk boost adaptive responses to influenza subunit vaccine through an ifn type i-independent mechanism of action the t helper type response to cysteine proteases requires dendritic cell-basophil cooperation via ros-mediated signaling key roles of adjuvants in modern vaccines systems analysis of protective immune responses to rts,s malaria vaccination in humans complex immune correlates of protection in hiv- vaccine efficacy trials identification of immune signatures predictive of clinical protection from malaria systems vaccinology: enabling rational vaccine design with systems biological approaches new approaches to understanding the immune response to vaccination and infection vaccine adjuvants: putting innate immunity to work the importance of adjuvant formulation in the development of a tuberculosis vaccine recent advances of vaccine adjuvants for infectious diseases the authors like to thank all the current and previous members from the laboratory as well as the animal care facility at vido-intervac, university of saskatchewan, canada for their contribution. this is vido-intervac manuscript no . publication of this manuscript is supported by grant mop from the canadian institutes of health research (cihr). the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. key: cord- -c gmu authors: davis-wurzler, gina m. title: current vaccination strategies in puppies and kittens date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: c gmu motivation in writing this article stems from many things: a lack of time spent in the veterinary curriculum discussing vaccines, a growing concern(by the general public and the veterinary community) regarding adverse reactions associated with vaccines, and a desire to prevent a recurrence of preventable infectious diseases resulting from a fear-driven cessation of vaccine administration. the objectives of this article are to present a basic review of immunology as related to vaccines, to discuss general guidelines for pediatric vaccines in canine and feline patients,and to offer suggestions as to how we can most positively influence our patients' health from the first visit. has a minimal effect in these species. it also occurs during initial suckling through the ingestion of colostrum and lactation, which have more significant effects in these species [ ] . this maternal immunity does provide initial protection against many pathogens but, of course, is dependent on the health and immune status of the mother as well as the health of the fetus and neonate. although this may result in temporary protection for the infant, in the long term, it may be deleterious to that individual's health by essentially keeping that animal naive to different antigens (eg, maternal antibody interference with vaccination of the neonate). maternal or passive immunization is effective in protecting neonates for the first several weeks of life but begins to decline and lose the ability to protect against diseases rapidly as the maternal antibodies are degraded through natural catabolic processes. between the ages of and weeks, depending on multiple factors (including the species; amount of maternal antibody produced, transferred, and absorbed; and individual health status of the neonate), most puppies and kittens have maternal antibody levels below protective levels. if present at high enough levels, however, maternal antibodies can interfere with the neonate's ability to respond to vaccination, because the circulating maternal antibody within the puppy or kitten may effectively respond to and neutralize the vaccine antigen or render it ineffective by preventing recognition of the antigen by the immune system [ ] . this is one reason for multiple sequential vaccines in kittens less than weeks of age and puppies less than weeks of age. maternal antibodies can interfere with immunization, although the level of maternal antibody present may not be protective against pathogens. a functioning immune system is composed of multiple parts. innate immunity is the oldest (evolutionarily), least specific, and most immediate (in terms of response to potential invaders and/or pathogens) form of immunity. macrophages, neutrophils, dendritic cells, and natural killer (nk) cells, combined with numerous products produced by these cells, comprise the innate immune system. examples of some of the chemical components produced and released by these cells in response to microbial invasion include lysozyme, complement, and various cytokines, such as tumor necrosis factor-a and interleukins, as well as various vasoactive molecules, such as histamine [ ] . active immunization is the process of the individual responding to an antigenic stimulus appropriately by natural infection or vaccination. active immunization is processed through the acquired immune system. the two main types of acquired immunity are cell-mediated immunity and antibody, or humoral, immunity. cell-mediated immunity is predominantly directed against pathogens that typically are obligate intracellular organisms. examples include viruses, some obligate intracellular bacteria, some fungi, and protozoa. t lymphocytes are the predominant effector cells and are dependent on foreign protein (antigen) being presented to them before they can take effect against the pathogens; thus, multiple cell types are involved in forming cell-mediated immunity. antibody or humoral immunity is predominantly directed against pathogens that can survive outside the host or at least survive extracellularly. examples include most bacteria, fungi, protozoa, and helminths. multiple cells act in concert to confer humoral immunity as well, but the primary effector cell is the b lymphocyte [ ] . kittens and puppies have varying degrees of ability to respond to antigens, whether attributable to natural or vaccine exposure, based on antigen load, route of exposure, antigenic virulence, genetics of the individual animal, and levels of persistent maternal immunity. in naive animals whose maternal immunity has declined sufficiently so as not to interfere with an immune response, the first vaccine should stimulate a primary immune response. this initial exposure and recognition process and the ability to produce antibody to respond to the antigen typically take to days. subsequent exposures to the same antigen elicit a stronger response; a greater amount of antibody is produced, and the subsequent response is faster. this is known as the secondary, or anamnestic, immune response. although multiple cell lines are involved in this response, subsets of t and b lymphocytes known as memory cells preserve the host's ability to recognize and respond to antigens to which the animal has previously been exposed [ ] . to design, recommend, and actuate an effective plan for each patient, a practitioner must have familiarity with multiple variables. those variables include the duration of protection conferred on the neonate by the mother, the typical length of time maternal antibody may persist and pose interference with the young animal's ability to respond fully to a vaccine, and the length of time needed for an appropriate response. in addition, knowledge of the various diseases that pose risks to pediatric patients and of safe efficacious vaccines available is critical. in essence, we must assess each patient as an individual within the population to provide optimal wellness over the lifetime of each individual as well as the population. this rationale has led to the concept of core and noncore vaccines, two terms commonly used when discussing vaccination within the veterinary field. criteria for assigning vaccines into these categories as well as a third category, ''generally not recommended,'' are based on the following factors [ ] [ ] [ ] [ ] [ ] : . morbidity and mortality associated with the specific disease (does the organism cause serious illness, or does it cause a mild transient disease that may pose only minimal risk to the individual or population?) . prevalence or incidence rate of the disease (although a specific disease may not commonly be seen, the organism is ubiquitous in the environment and therefore poses a risk to the individual or population) . risk of the individual for exposure to the disease (eg, indoor-only animal versus free-roaming individual, regional variations of occurrence) . efficacy of the vaccine (does the vaccine prevent infection or simply ameliorate some signs or length of the disease?) . risks associated with administering the vaccine (are the risks associated with that vaccine greater than the risk of the disease?) . potential for zoonotic disease . route of infection or transmissibility when these criteria are assessed, general guidelines may be generated for the individual practitioner as well as for the veterinary community at large. again, guidelines are not to be thought of as absolutes, nor are they to be used to establish standard of care. they are, simply stated, tools for each of us to use to promote optimal wellness for our patients when considering all factors affecting the individual's health (environmental, organismal [pathogen and host], owner concerns, and current vaccine technologies) [ ] [ ] [ ] [ ] [ ] . there are multiple vaccines available for our canine and feline patients; yet, most vaccines fall within three basic categories. assignment of vaccine products (which are considered biologic agents rather than drugs and are therefore assessed and approved by the us department of agriculture [usda] rather than the us food and drug administration [fda]) into these categories is based on how the product is created. simply stated, modified-live vaccines (ml) are vaccines created by altering (attenuating) the pathogen in some way so that it is no longer able to cause serious or clinical disease in the targeted species. modified-live virus vaccines (mlv) are therefore vaccines containing live but avirulent virus. killed vaccines are vaccines produced by inactivating the pathogen completely, rendering it incapable of reproducing and thereby unable to cause disease. the third category of vaccines consists of recombinant vaccines. there are multiple types of recombinant vaccines, and this category itself has three subcategories. these vaccines use genetic technologies to introduce genetic material directly into the host (no vector is used [eg, purified subunit vaccines, type i recombinant]), alter the genetic material to change its virulence (gene deletion, type ii recombinant), or incorporate genetic material from the desired pathogen into an attenuated vector organism (eg, feline recombinant rabies, type iii recombinant) [ , ] . within the near future, multiple new technologies are likely to provide us with even more choices, hopefully providing our patients with better protection against disease with minimal vaccine-associated risks. for a comparison between vaccine types, the reader is referred to table . vaccines are available in single-dose and multiple-dose (tank) vials. the use of single-dose vial vaccines is highly recommended in these species. conversely, the use of multiple-dose vials is discouraged because of the increased risk of contamination and the inability to ensure consistent levels of antigen and adjuvant in individual doses from a single vial [ , ] . multivalent vaccines are not recommended in cats other than the core feline vaccine designed to protect against feline panleukopenia, feline herpesvirus i (fhv-i), and feline calicivirus (fcv). because of increased inflammation at the site of multivalent vaccines, all other vaccines should be given as a separate vaccine at the indicated site (see discussion on feline core and noncore vaccines) [ , ] . allowing vaccines to acclimate to room temperature before administration, particularly in cats, is recommended, because the administration of cold vaccines was found to have an increased association with tumorigenesis in cats [ ] . the practitioner is advised always to follow manufacturer's directions for dose and route of administration. using a topical product parenterally or splitting doses should never be done. administration of a modified-live bacterin vaccine designed for topical administration yet administered parenterally may have serious and potentially fatal consequences (fig. ) . a full dose is required to stimulate the immune system; there is no medical basis for giving a smaller dose to a toy-breed dog, and this practice could lead to vaccine failure in that animal. if done with a rabies vaccine, the practitioner is not following federal requirements, which carries potential legal implications [ , ] . the interval between various vaccines, whether using the same product serially in the initial series or using different products in an adult animal, should never be less than to weeks. interference between the first product administered and a second vaccine product may lead to failure to respond to that second vaccine optimally. the exact mechanism of this interference is unknown but may be associated with interferon produced by cells processing an ml agent or by transient immunosuppression by an ml agent. multiple vaccines administered at the same time do not seem to elicit this interference and is therefore an acceptable practice [ , ] . the reader is referred to tables and for a comparison between pediatric canine and feline core, noncore, and generally not recommended vaccines. the diseases that fall within this category carry high rates of morbidity or mortality; they are of public health concern, are readily transmissible, or may be ubiquitous in the environment. in addition, safe efficacious vaccines are available and provide sterile immunity (prevent infection) or confer a high degree of protection (do not prevent infection but may confer protection, such that the animal does not develop clinical signs of disease) [ , ] . essentially, the vaccines that fall within this category are recommended for each individual within the population regardless of that animal's lifestyle or locale. canine distemper virus (cdv), an enveloped morbillivirus, has been well controlled because of widespread vaccination programs over the past several decades. the disease still persists, however, and in addition to high virulence, it is readily transmissible. infection with the virus causes respiratory, gastrointestinal, and neurologic signs and is often fatal [ ] . the distemper vaccine is commonly administered as part of a multivalent product. the general recommendation is to use a modified-live or recombinant, multivalent product beginning at to weeks of age and to give serial vaccines every to weeks until the puppy has reached to weeks of age [ , , ] . currently, there is one product that contains modified-live distemper virus, canine adenovirus type ii (cav-ii), and canine parvovirus (cpv) (continuum; intervet, millsboro, delaware), but there are numerous vaccines with high safety and efficacy records that also include canine parainfluenza virus. there is also a vaccine that combines a recombinant canarypox-vectored cdv with modified-live adenovirus ii, parainfluenza, and parvovirus (recombitek; merial ltd, duluth, georgia), and this vaccine seems to be less affected by maternal antibody interference than mlv vaccines [ ] . other studies support the improved ability of recombinant vaccines to overcome maternal antibody interference as compared with mlv vaccines [ , ] . most puppies receive two or three distemper vaccinations, depending on the age when they are first presented to the veterinarian. it is, however, the interval between or the ''timing'' of the vaccinations rather than the ''number'' that is important. serial vaccinations help to increase the likelihood of a complete response of the patient and thereby decrease the risk of vaccine failure that may occur when only one vaccine is administered. in addition, by eliciting a secondary immune response, they may help to increase the level of circulating antibody and decrease the lag time between exposure to an antigen and achievement of maximal antibody level [ ] . potential causes for vaccine failure include an mlv vaccine that was improperly stored and therefore lost its efficacy, the vaccine was improperly administered (wrong route or accidental loss of vaccine onto the skin of the patient), the patient's immune system did not respond (the immune system may have been responding to another antigenic challenge, or the vaccine may have been given too soon after a previous vaccine), and maternal interference [ ] . in theory, if a puppy were kept ''sequestered'' from exposure to this virus, one mlv distemper vaccine administered after weeks of age would confer protection for at least year [ , ] . in reality, however, most pet owners are not inclined to isolate their puppies for the first months of life, nor should they. early socialization is an important part of families bonding with their puppies. exposure to various people, other dogs, and new places helps to decrease behavior problems in young adult and mature dogs [ ] . as long as the last distemper vaccine is administered after weeks of age, the puppy should be able to mount a strong active response and fully overcome any residual maternal antibody. the current recommendation is to have the puppy return year later (when approximately months old) for administration of another dose of distemper vaccine. after the first ''annual'' vaccination, triennial immunization is recommended for mlv vaccines [ , ] . annual revaccination is currently recommended if the recombinant product is used [ , ] . there are two types of adenovirus that cause disease in our canine patients. canine adenovirus type i (cav-i), a nonenveloped virus in the family adenoviridae, causes the potentially fatal disease infectious canine hepatitis. clinical signs include fever, depression, vomiting and diarrhea, and potential abbreviations: ab, antibody; felv, feline leukemia virus; fvr, feline viral rhinotracheitis; mlv, modified-live virus; pma, persistent maternal antibodies. a because of increased susceptibility for infection in kittens, vaccination against felv is strongly recommended for all kittens. in single-cat households, households with known negative viral status of all cats, and households with indoor only cats, the practitioner may elect to consider this a noncore vaccine. petechiation and ecchymotic hemorrhage secondary to hepatic dysfunction. in addition, uveitis and renal disease are associated with infection with this virus. cav-ii causes respiratory tract disease. cav-i is associated with severe potentially fatal disease, and protection against this disease is recommended. transmission is via the oronasal route and exposure to infected secretions. cav-ii infection typically results in mild self-limiting disease and is therefore considered to be a noncore disease; however, the mlv product designed for prevention of cav-i has been associated with adverse effects, such as uveitis and corneal edema (an arthus reaction, similar to effects caused by natural infection) [ , ] . the current recommendation is to use the cav-ii mlv because it stimulates the immune system to protect against cav-i and cav-ii without the associated adverse reaction caused by the type i vaccine [ , , ] . a modified-live adeno-type ii virus is typically included in a multivalent injection (as mentioned previously) and is therefore usually administered at intervals of to weeks, beginning between and weeks of age and ending between and weeks of age. a vaccination year later is recommended before instituting triennial vaccinations. canine parvovirus cpv is a nonenveloped type parvovirus. the predominant form currently causing infection in the united states is type b, but other subtypes exist and cause disease elsewhere [ ] . because the virus is nonenveloped, it may exist (outside a host) under certain environmental conditions and is somewhat resistant to many disinfectants. transmission is via the fecal-oral route, and clinical signs include lethargy, anorexia, pyrexia, vomiting, and diarrhea (typically hemorrhagic). young animals seem to be at highest risk for developing severe life-threatening disease. the current recommendation for vaccination is to use a multivalent mlv beginning at to weeks of age and to repeat the vaccine at intervals as stated previously (every to weeks until the puppy is to weeks of age). there has been some concern that certain breeds may be at increased risk for contracting and developing severe parvoviral disease (eg, doberman pinschers, rottweilers), but it is generally agreed that these breeds mount an appropriate response to a quality product if the last vaccine is given between and weeks of age [ , ] . recent studies using mlv cpv b strains showed a higher antibody response to cpv and cpv b and that the cpv b strain vaccines were better able to overcome maternal antibody interference than the cpv strain vaccines used [ , ] . an alternative would be to use serial vaccinations of killed virus if the practitioner were concerned about the potential for vaccine-induced clinical disease in one of the breeds believed to be more susceptible to this virus; however, these vaccines are less immunogenic [ ] . immunization year after completing the initial puppy series is recommended, with subsequent triennial vaccinations [ , , ] . rabies virus, an enveloped virus in the rhabdoviridae family, is capable of infecting all mammals [ ] . because it is an enveloped virus, it is not stable in the environment and is readily inactivated by most common disinfectants. the virus is transmitted through infected saliva, most commonly from a bite by an infected animal. clinical signs range from anxiety or other vague behavior changes to pica, dysphagia, photophobia, and paralysis. because of the zoonotic potential and implications regarding public health, canine vaccination programs are strongly regulated and enforced. the current recommendation is to vaccinate puppies using a killed virus vaccine at a minimum of or weeks of age. state regulations vary as to the minimum age for canine rabies vaccination; in california, the legal minimum age of canine vaccination against rabies is weeks. a second rabies vaccine (killed product) is administered year later and then annually or triennially thereafter depending on local regulations [ , ] . it is the practitioner's professional responsibility to acquire knowledge of and maintain adherence to regional laws regarding rabies vaccination frequency [ ] . vaccines in the noncore category have limited efficacy, or the organism causing disease is not readily transmissible or may have limited geographic distribution or prevalence. additionally, the diseases these vaccines are designed to prevent may be so mild or self-limiting that the risk associated with administering the vaccines may be greater than that of the actual disease. finally, some vaccines may interfere with common screening methods for disease detection and are therefore not recommended unless absolutely warranted for a specific individual. it is the burden of the practitioner, along with the pet owner, to make decisions regarding which, if any, of the noncore vaccines should be administered to a puppy [ ] [ ] [ ] [ ] . a bacterial pathogen that causes acute hepatic and renal disease, leptospirosis is typically transmitted through urine of infected animals (reservoir hosts include dogs, rats, wildlife, and livestock) and in contaminated water. there are at least two different species (leptospira interrogans and leptospira kirschneri) that can infect dogs, with multiple serovars (variants of the same species) of l interrogans causing disease in dogs [ ] . although these organisms have the potential to cause serious disease, dogs are not likely to be at risk in a mostly urban and controlled environment (eg, housed in a fenced yard with no exposure to wildlife or livestock). a dog that frequents rural environments or has exposure to waterways or livestock is definitely at risk of infection, however, and should therefore be protected against the disease. again, the initial puppy appointments should involve a thorough history and include the owner's plans for that dog's future use. if an owner brings a labrador retriever puppy to the veterinarian for ''whatever vaccines it needs,'' it is up to the practitioner to ask: ''will it be a hunting dog, will it be used in field trials, or will it be exposed to wildlife and waterways?'' the border collie that lives on a working sheep ranch surely should be vaccinated as well. conversely, a long-haired miniature dachshund that spends its days on its owner's lap is at minimal risk of exposure; therefore, vaccination is most likely not warranted. in essence, the regional distribution, seasonality (increased prevalence during and immediately after the rainy season), and lifestyle of the puppy should factor into the decision as to whether the puppy should be vaccinated. if the decision is made to vaccinate against leptospirosis, the general recommendation is to wait until the puppy is at least weeks of age; at that time, a killed or purified subunit vaccine is administered. infection is serovar specific, and no cross-protection is seen between different serovars; therefore, vaccination with those serovars known to cause disease in a given region is recommended. currently, there is one killed purified subunit vaccine (leptovax; fort dodge animal health, overland park, kansas) that contains four serovars (icterohaemorrhagiae, canicola, pomona, and grippotyphosa); however, the duration of immunity against grippotyphosa and pomona is unknown, and there are no vaccines available for autumnalis or bratislava. an initial series of two to three vaccinations should be administered monthly and repeated at least annually thereafter as long as exposure to the agent exists [ ] . the recommendation to wait until the puppy is at least weeks old before administering the leptospirosis vaccine is based on the increased potential for adverse events associated with this vaccine and to increase the likelihood of a complete immune response minimizing ineffective vaccinations [ , ] . bordetella bronchiseptica is a bacterial agent that causes infectious tracheobronchitis. infection with this agent may occur in concert with other agents infecting the respiratory tract (eg, canine parainfluenza virus, cav-ii). transmission occurs via direct contact or through aerosolized microdroplets from infected dogs and is most likely to occur under crowded conditions, such as boarding and grooming facilities and dog show venues. the current recommendation is to vaccinate puppies at risk a minimum of week before potential exposure with a combination vaccine containing an avirulent live bacterin for b bronchiseptica and a modified-live canine parainfluenza virus. although the vaccine can be administered to puppies as young as to weeks of age, it is generally not indicated unless the puppy is in a kennel environment [ ] . many organized obedience classes commonly require proof of vaccination against bordetella at the time of enrollment or before beginning the course. the general consensus is that intranasal vaccines are superior to parenteral vaccines because they stimulate rapid local immunity [ , ] . if the puppy is intermittently exposed throughout the year (eg, traveling to shows or boarding or grooming facilities), the vaccine should be administered every months. as stated previously, parainfluenza may occur in concert with other respiratory tract agents. the vaccine recommendations are as stated for b bronchiseptica if indicated. there are multiple products available, but the product currently recommended is the combination intranasal vaccine containing a modified-live parainfluenza virus with an attenuated b bronchiseptica bacterin. for optimal protection, the vaccine should be administered every months to annually if indicated. alternatively, many multivalent products containing modified-live cdv, cav ii, cpv, and parainfluenza are available and appropriate for use [ ] . borrelia burgdorferi is a vector-borne spirochete bacterium responsible for lyme disease (borreliosis). transmission occurs when an infected tick (various species within the ixodes genera, also referred to as ''hard ticks'') bites and remains attached to a host, in this case, a puppy. direct horizontal transmission is not likely to occur; therefore, the risk to human beings and other pets is thought to be minimal. if a puppy has a significant burden with infected ticks, this, of course, increases the exposure to others in the household; however, because ticks typically do not reattach once they have taken a complete meal, the risk is thought to be quite small unless appropriate tick control is not instituted [ ] . vaccination to protect against lyme disease is controversial, because the duration of immunity and degree of protection provided by vaccination are unknown and vaccination with some vaccines interferes with standard screening diagnostics [ ] . therefore, vaccination against lyme disease is warranted only if a puppy is at high risk for tick exposure and only if it lives in a borrelia endemic area. there are killed and recombinant (outer surface protein a [ospa] subunit) vaccines available for use against b burgdorferi, and if vaccination is deemed warranted, the current recommendation is to use one of the subunit vaccines before exposure to ticks. the vaccine can be given as early as weeks of age and should be repeated to weeks later [ ] . the best prophylaxis is likely achieved by using appropriate tick prevention, such as fipronil with methoprene spray or spot-on products (frontline top spot; merial ltd, iselin, new jersey), amitraz collars (preventic collar; virbac, fort worth, texas), or an imidacloprid/permethrin topical product (canine advantix; bayer animal health, shawnee mission, kansas) [ , ] . these products should be chosen and recommended carefully by the veterinarian based on household situations, owner concerns, and age of the puppy. this virus, also a morbillivirus, can stimulate an immune response that is crossprotective against cdv. the indication for using this vaccine is for puppies that may have maternal antibody to distemper virus sufficient to cause interference with distemper vaccination but not adequate to protect against infection. if indicated (see discussion of special circumstances), a single vaccination with an mlv vaccine should be given intramuscularly as early as weeks of age. subsequent immunizations with mlv cdv vaccines should be given serially as recommended previously (see section on cdv) [ , , ] . canine measles vaccines should never be administered to female puppies older than weeks of age because they may develop an acquired immune response to the virus. this could be problematic if a female puppy vaccinated against measles at weeks of age, for example, later became pregnant. if the dog developed antibodies to the measles virus and maintained immunologic memory, it would confer measles antibody to puppies via passive transfer, thus rendering measles vaccination in those puppies ineffective. a more appropriate alternative to administering a measles vaccine to a young puppy thought to be at risk for infection but too young to receive an mlv cdv vaccine would be to use a recombinant cdv vaccine, thereby decreasing the likelihood of maternal interference [ ] an enveloped virus belonging to the family coronaviridae, this virus is transmitted via the fecal-oral route. vaccination against this disease is generally not recommended, because the vaccines provide questionable protection and the actual prevalence of the disease is unknown. those most likely to be infected and develop clinical disease are neonates less than weeks of age. clinical signs may include diarrhea, possibly hemorrhagic but typically self-limiting. the general recommendation is to vaccinate puppies against cpv (as recommended previously), because this practice seems to confer protection against coronavirus in addition to preventing infection with cpv- [ , ] . this protozoal parasite causes diarrhea in canine and feline patients as well as in many other mammalian species, including human beings. transmission is via the fecal-oral route, with animals contracting the agent from contaminated feces or water. there is a killed vaccine available; however, vaccination against this agent is typically not recommended, because most animals are not at risk to contract the parasite, the vaccine does not prevent infection (it may ameliorate clinical signs and decrease cyst shedding), and the disease is readily amenable to therapy (fenbendazole, albendazole, and metronidazole are off-label uses but commonly accepted as standard of care). because puppies should be prophylactically dewormed at regular intervals, it is unwarranted to use this vaccine even if the disease is suspected, because a standard anthelmintic dose of fenbendazole given for several days should resolve the infection [ , , , ] . a vaccine designed to protect against envenomation by crotalus atrox, the western diamondback rattlesnake, was released onto the market recently. the original provisional licensure was granted to provide possible protection against that single species of snake and was granted for use only in california. recently, the company was granted extended provisional licensure for multiple states and has extended their claim for potential protection against multiple species of members of the crotalidae (pit vipers). at this time, no challenge studies have been performed in the canine species to validate efficacy claims; all claims are based on antibody titer to the venom component included in the vaccine, to murid challenge studies, and to anecdotal reports of protection of naturally occurring envenomation [ ] . the manufacturer does not claim that vaccination with this product completely protects against the effects of envenomation; rather, the manufacturer claims that it may slow the onset of clinical signs and decrease the severity of signs. immediate veterinary care is still the ''gold standard'' for any snakebite. because of the great potential for variability in envenomation (site of bite on an animal, size of the snake, amount of venom injected into an animal, and species of snake), field observations and anecdotal reports of protection are difficult to substantiate. challenge studies done under controlled conditions are likely necessary to validate the efficacy of this product. at present, because of the preceding statements, this vaccine is not recommended for general use. aversion training and keeping dogs out of areas known to favor rattlesnake habitation as well as immediate veterinary evaluation and care are still the standard recommendations for preventing and treating disease associated with rattlesnake envenomation. canine adenovirus type i as stated in the canine core vaccine section, cav-i causes serious disease in dogs; however, the use of the cav-i vaccine is associated with a high incidence of adverse events. vaccination with the cav-ii vaccine induces an immune response that is protective against cav-i and cav-ii without the adverse effects. the recommendation is to use the cav-ii vaccine as part of the canine core vaccination program; the cav-i vaccine should not be used [ ] . feline panleukopenia, a nonenveloped parvovirus closely related to cpv, causes serious and often fatal disease in kittens. transmission typically occurs from direct contact with infected animals, although in utero infection and fomite transmission also occur. clinical signs typically include pyrexia, anorexia, lethargy, and vomiting and diarrhea. kittens may be immunosuppressed subsequent to pancytopenia associated with this viral infection. kittens infected in utero may exhibit cerebellar disease. prevention is achieved using mlv vaccines beginning between and weeks of age. the standard recommendation is to use a parenteral product (as opposed to intranasal products, which have a higher incidence of postvaccinal viral shedding and potential for clinical disease induced by the more virulent viruses in these vaccines) [ , , , ] . as is the case for canine cdv, cav, and cpv, the core feline diseases, with the exception of rabies, are typically administered in a multivalent product in a series. there are numerous vaccine products containing feline panleukopenia virus, herpesvirus i, and calicivirus (see additional discussion). the current recommendation is to choose an mlv nonadjuvanted product from a reputable manufacturer. vaccines are administered subcutaneously in the right thoracic limb and given every to weeks until the kitten is to weeks of age. repeat administration is recommended year later before instituting a triennial schedule [ , , ] . feline herpesvirus i fhv-i, also known as feline viral rhinotracheitis virus, is an enveloped virus causing respiratory tract disease in cats. clinical signs include sneezing, nasal congestion and discharge, conjunctivitis, and ocular discharge. in addition, kittens may exhibit pyrexia, anorexia, and lethargy along with oral and/or lingual ulcerations and associated hypersalivation. in some cases, ulcerative crusting dermatitis that may mimic other dermatologic disease occurs [ ] . the virus typically causes upper respiratory disease, but the lower respiratory tract may become involved, especially in neonates or debilitated animals. infection with this virus is lifelong, although many cats ''recover'' and do not show clinical signs. cats infected with fhv-i may have recurrent ''outbreaks,'' especially in times of stress or if their immunity is otherwise compromised. cats may persistently shed the virus and act as a source of infection in shelters, catteries, and multiple-cat households. therefore, prevention before exposure is key in controlling this disease [ , ] . vaccination with an mlv or recombinant vaccine beginning as early as to weeks of age is recommended. this is commonly administered as part of a multivalent product and is given subcutaneously in the right thoracic limb. the current recommendation is for kittens to receive a second vaccination weeks later. the last vaccine in the series should be given when kittens are at least weeks old. the vaccine should be given year later before beginning the triennial schedule [ , ] . feline calicivirus fcv causes respiratory tract disease in kittens and cats. because it is a nonenveloped virus, it is more resistant to disinfectants and may therefore persist in the environment. signs are similar to those associated with fhv-i, but lameness and stomatitis are also commonly seen. transmission of fhv-i and fcv is through direct contact, exposure to contaminated secretions, aerosolization, and fomites [ , ] . a newer highly virulent strain of fcv was recently identified and carries a high incidence of mortality. transmission is through direct contact or via fomites. prior vaccination against fcv does not seem to be protective against this strain, and adult cats seem to be more severely affected than kittens [ , ] . the current recommendation is as previously discussed for panleukopenia and fhv-i: administering an mlv or recombinant virus parenteral vaccine beginning at or weeks of age, with a subsequent dose of vaccine weeks later (last vaccination should be when the kitten is at least weeks old). a booster vaccination should be administered year later and then every years [ , ] . as stated previously, rabies virus affects all mammals; in this country, most documented rabies cases in pet animals occur in cats [ ] . because of the significant risk to pets, wildlife, and human beings, vaccination against rabies virus is highly recommended for all kittens and cats, even those kept inside [ , ] . local requirements vary, but the general recommendation is that all kittens should be vaccinated beginning at weeks of age with the recombinant rabies vaccine designed for use in cats [ ] . this product uses gene-splicing technology: reverse transcriptase is applied to rabies viral rna to create complementary dna. the segment of rabies virus dna that codes (a codon) for the immunogenic protein associated with the virus (glycoprotein g) is then spliced from the rabies dna and inserted into a canarypox virus. the canarypox virus is attenuated and nonpathogenic to mammalian cells and therefore carries no potential to cause disease in this species. because the vaccine is essentially a modified-live product, the canarypox virus can enter cells, delivering the codon for rabies virus glycoprotein g to its targeted site. once inside the cell, the canarypox virus is unable to replicate, but the rabies glycoprotein g codon is preserved, leading the host cell to express the glycoprotein on its surface. this stimulates cell-mediated and humoral immune responses. in addition to the benefit of stimulating both types of immunity, the fact that no adjuvant is needed is beneficial [ ] . rabies vaccines should be administered subcutaneously in the right pelvic limb as distally as is reasonably possible; the level of the stifle is acceptable, and areas distal to the tarsus are not appropriate. currently, there is only a recombinant rabies vaccine approved for use in cats (purevax feline rabies vaccine; merial ltd, duluth, georgia). the current usda approval and label state that this product should be administered annually. there are multiple killed virus rabies vaccines approved for use in cats, with initial vaccination occurring at weeks of age and a subsequent vaccination year later. these products are highly efficacious but may carry an increased association with the development of fibrosarcoma formation because they contain adjuvants [ , , , , ] . because regulations vary depending on the state or region, the veterinary practitioner must be familiar with local laws regarding rabies vaccination in this species [ ] . feline leukemia virus (felv) is a retrovirus affecting cats of any age, but kittens and juvenile cats seem to be most susceptible to infection [ ] . clinical signs are numerous and nonspecific, and they include pyrexia, failure to thrive, and chronic or recurrent respiratory tract and gastrointestinal disease. infection in kittens occurs via vertical transmission from the queen to the fetus but may also spread horizontally from the queen to the kitten during lactation and grooming. transmission also occurs through direct and usually prolonged contact with other infected cats from behaviors like grooming and sharing food and water bowls as well as litter boxes. viral screening using an elisa test designed to detect antigenemia should be performed on all kittens, even if their owners plan to house them strictly indoors. because the elisa test detects antigen, maternal antibody and vaccination do not interfere with test results. therefore, kittens of any age may be tested [ ] . if a kitten is antigen-negative, the current recommendation is to administer a recombinant vaccine on the second visit. a second dose of vaccine should be administered weeks later, followed by vaccination year after administration of the last felv vaccine in the kitten [ , ] . the recommended site for administration of any felv vaccine is the left pelvic limb as distally as is reasonably possible [ ] . currently, there is only one recombinant felv vaccine available (purevax recombinant leukemia vaccine; merial ltd, duluth, georgia). this vaccine is administered with a needle-free high-pressure device that deposits the vaccine in skin, subcutaneous, and muscle tissues (vetjet delivery system, merial ltd, duluth, georgia, which is manufactured by bioject, tualatin, oregon) [ ] . there are killed virus vaccines that are efficacious; however, because they contain killed virus, they require an adjuvant to maximize the host's immune response. because of documented associations between adjuvants and the formation of fibrosarcomas, the use of adjuvants in cats should be avoided when adjuvantfree products with comparable efficacy are available. the most recent findings linking injections of any type with fibrosarcoma formation in this species further support the use of a needle-free system as a viable means of vaccine delivery [ , , ] . under the current vaccine guidelines released in in the american veterinary medical association council on biologic and therapeutic agents' report on cat and dog vaccines, vaccination against felv is only indicated if a kitten or cat is allowed to go outside or if the kitten or cat lives with an felv-positive cat. because kittens are most vulnerable to infection and may have exposure if outdoors and because immunity increases with age, it is rational to vaccinate all kittens against this disease with a repeat vaccination year later. subsequent to that, if the cat is housed strictly indoors and does not live with an infected (felv-positive) cat, additional vaccinations are not indicated [ ] . chlamydophila felis, formerly known as chlamydia psittaci, is a bacterium that causes upper respiratory tract disease in kittens and cats. the most common sign is conjunctivitis, but sneezing and nasal discharge may also be present. transmission is typically through direct contact with infected cats. kittens are most commonly affected but usually recover fully with appropriate antibiotic therapy-topical oxytetracycline (ophthalmic ointment) or systemic tetracycline or doxycycline. vaccination against this agent typically does not prevent infection but may prevent clinical signs of disease. because the vaccine does not fully prevent infection and carries an association with adverse events that may be greater than the actual disease, routine vaccination of household pets with this product is generally not recommended. it may be of use in some environments in which the risk of infection is high, however, such as shelters or catteries [ , ] . if vaccination is deemed appropriate by the practitioner, an attenuated parenteral vaccine can be given to kittens beginning at weeks of age, with a second dose given to weeks later [ ] . this bacterial agent causes respiratory tract disease in cats, and cats affected by stress, poor nutrition, or overcrowding seem to be more susceptible. although many infected kittens show mild self-limiting disease with signs that include pyrexia, sneezing, and nasal and ocular discharge, bronchopneumonia has been documented. there is a topical modified-live bacterin vaccine designed for use in this species, but it is generally not recommended for routine use. if the practitioner thinks protection against b bronchiseptica is warranted based on the kitten's risk of exposure (eg, attends cat shows, goes to a boarding facility), administration of the vaccine designed for use in cats may be considered [ ] . a single dose of the ml intranasal vaccine can be given to kittens as young as weeks of age [ ] . the product designed for use in dogs should not be used in cats. there are multiple vaccines in addition to those described and recommended previously; however, many of these diseases pose a minimal risk to most of the feline population or the vaccines are minimally efficacious at preventing infection or disease and therefore are generally not recommended. additional reasons not to use some of these products are vaccine interference with screening tests and adverse events associated with some vaccines. a retrovirus, feline immunodeficiency virus (fiv) primarily affects cats by compromising their immune system, leaving them vulnerable to opportunistic infections. in addition to immunosuppression, with most of the effect targeted against the cell-mediated (t-cell) immune response, infection with fiv carries an increased risk for development of certain types of neoplasia, with b-cell lymphoma being the most common. transmission occurs most commonly from breeding and fighting. the virus is not spread through casual contact between housemates not engaging in the behaviors stated previously, nor is it spread through casual encounters between nonbreeding and nonfighting cats outside. naturally occurring infection of kittens from queens is rare; however, kittens can become fiv antibody-positive via passive transfer from ingestion of colostrum of fiv-positive queens or queens previously vaccinated against fiv [ ] . fiv antibody levels acquired from maternal transfer in kittens that are actually negative for fiv virus decline over the first several months of life. the standard screening test for fiv is an elisa test designed to detect fiv antibody. the elisa was designed to detect antibody rather than antigen, because infected cats produce high levels of circulating antibody in contrast to low levels of circulating virus [ ] . because kittens may have circulating fiv antibody, although actually being negative for fiv antigen, it is generally not recommended to test kittens less than months of age. if a kitten is tested and a positive result is obtained, the test result should be repeated with a different methodology (western blot or polymerase chain reaction [pcr] ) and should be repeated once the kitten is older than months of age [ ] . if a kitten is truly not infected, the maternal antibody wanes by months of age, leading to seroconversion. if, however, a kitten or cat remains seropositive, the recommendation is made to keep the cat indoors only from that point on so as to prevent infection of other cats and to decrease exposure to potential environmental pathogens. fiv-infected cats can live for years; unless otherwise indicated by concurrent disease, euthanasia is generally not indicated for most owned pets. there is a killed fiv vaccine available, but the efficacy of this product is still unknown. there are five known subtypes of fiv virus, and the vaccine has been formulated to protect against subtypes a and d; however, the predominant subtype infecting cats in north america and europe seems to be subtype b. it is unknown if cross-protection exists between the different subtypes [ ] . because the vaccine elicits a strong antibody response, vaccinated kittens and cats become seropositive on elisa and western blot tests, because both tests detect antibody. a pcr test is available but is currently only performed at certain laboratories. because of the increased technologic needs and increased costs of this test, it is not considered the standard screening test. if done under specific conditions, it can detect virus and therefore may be of benefit in differentiating between cats with viremia (truly infected cats) and kittens or cats with circulating antibody attributable to maternal transfer or vaccination. because of the nature of transmission of the virus and interference with the standard screening methods for infection, vaccination against fiv is not currently recommended. keeping cats indoors if possible, neutering all cats going outside, and preventing exposure to stray or feral cats that may be more likely to engage in fighting behaviors remain the gold standards for preventing this disease [ , ] . the disease feline infectious peritonitis (fip) is caused by a member of the coronaviridae. feline enteric coronavirus (fecv) and fip virus are two phenotypes of the same virus. fecv transmission occurs through the fecal-oral route, where it typically infects the intestinal epithelium, but the organism can be transmitted via fomites and persists for long periods in the environment. most cats infected with fecv do not show clinical signs of disease or may have transient diarrhea; some persistently shed the virus in their feces [ ] . fecv can, however, undergo random mutations within a host, creating fip virus, although the virus does not mutate to this form in most cats and most cats do not develop fip. the fip virus enters and replicates within macrophages, where it can then be disseminated throughout the body. clinical signs are numerous but commonly include weight loss, failure to thrive, diarrhea, pyrexia, and chronic respiratory tract disease. two main types of the disease exist, the dry (noneffusive) and the wet (effusive) forms. both are ultimately fatal diseases [ ] . although there is a vaccine available, efficacy and indication for use are believed to be minimal, if at all [ ] . the current recommendation is not to use this vaccine based on efficacy concerns and the minimal risk of infection in most kittens and cats. infection with fecv and mutation with subsequent development of disease occur most commonly in multiple-cat households (five or more), catteries, and shelters. the standard screening test for fip is a serologic indirect immunofluorescent antibody (ifa) test designed to detect antibody. this test may be of some value, but results need to be interpreted with caution and concomitantly with signalment, clinical signs, and other laboratory data. prior vaccination against fip yields positive ifa results, further posing potential complications in routine screening of this disease. in general, kittens are most vulnerable to this disease, with greater than % of cats with fip being less than years of age [ ] . prevention is directed toward decreasing stress in kittens and cats in multiple-cat households and preventing exposure of naive kittens and cats in environments known to have high endemic levels of fecv and at depopulating catteries known to have high prevalence rates of fecv and fip [ , ] . because of the complexity of this disease and the limited space and objectives of this discussion, readers are encouraged to review the texts by greene [ ] and ettinger and feldman [ ] for a more comprehensive review of this disease. as discussed in the canine section, vaccination with this product does not prevent infection but may decrease fecal shedding of infective cysts. because vaccination does not prevent infection and the organism is readily treated with fenbendazole or metronidazole, routine use of this product in cats is generally not recommended [ ] . vaccines are potent biologic agents designed to prevent disease. any foreign product administered to an animal has the potential to be associated with an unexpected response by that animal. although vaccines must meet usda requirements for safety, efficacy, potency, and purity, there still exists the potential for adverse events with products that have met those standards. veterinarians should always report adverse events related to vaccination to the vaccine manufacturer. some adverse events are more likely to occur with certain agents, whereas others seem to have an increased rate of occurrence in certain breeds. still others may be idiosyncratic and are not predictable. the following is offered as a brief overview of some types of adverse events associated with vaccination and to offer suggestions as to how a practitioner might best respond to and prevent those events from recurring. the reactions seen most commonly are local inflammation at the site of the injection or general malaise, pyrexia, and anorexia for to days after vaccination [ ] . most of these reactions are self-limiting and require nothing more than monitoring by the animal owner. it is appropriate for the practitioner to note any reaction, along with a description of signs exhibited, in the medical record and to offer supportive care if indicated. in some instances, administration of an ml causes transient mild clinical disease. supportive care and isolation from unvaccinated animals are recommended, because the vaccinated animal showing clinical disease sheds the vaccinal organism and is potentially infectious to other animals [ ] . contact information for vaccine manufacturers, support agencies, and disease-reporting organizations is included in table . feline injection site sarcomas, also known as feline vaccine-associated sarcomas or fibrosarcomas, develop secondary to local inflammation of injection sites. there is an increased risk for development of these tumors associated with adjuvants and certain repositol agents, such as long-acting penicillin and corticosteroid injections [ ] . measures to prevent these tumors are aimed at decreasing the local inflammatory response by avoiding the use of adjuvants in this species and administering only those vaccines indicated for the individual animal. multiple vaccines should not be administered in one site because this may increase the amount of inflammation in that site. following the recommended sites for injection is strongly recommended (see individual vaccine sections for specific sites) [ , ] . there are specific guidelines as to how a practitioner should proceed if a cat develops a swelling at the site of a vaccination or injection. the practitioner is advised to monitor the patient closely, documenting three-dimensional measurements and temporal association if a mass or swelling develops at the site of a vaccination. the ''three-two-one rule'' developed by the feline vaccine-associated sarcoma task force should be closely applied. ''three'' refers to persistence of the mass for months or longer, ''two'' refers to a size of cm or greater, and ''one'' applies if the mass increases in size after month. if any of these criteria are met, the mass should be biopsied using wedge technique or needle biopsy, allowing for complete resection of the biopsy margins in the future and subsequent referral to an oncologist or surgical oncologist if fibrosarcoma is confirmed. fine needle aspiration is not recommended for evaluation of potential injection site sarcomas [ , ] . most vaccine manufacturers have programs established to help defray the medical and surgical costs associated with these tumors, and the practitioner is advised always to notify the vaccine manufacturer whenever an adverse event is seen. type i hypersensitivity type i hypersensitivity, also known as immediate hypersensitivity and, in some cases, anaphylaxis, is mediated by ige antibody. the host's immune system may react to anything contained within the vaccine product, including cellular products used for culture, adjuvant, preservative, and the antigen itself, and such a reaction typically occurs within to hours after the administration of a vaccine. in the dog, the most common signs are angioedema (fig. ) , urticaria, and pruritus, but symptoms may progress to respiratory distress and fulminant vascular collapse (anaphylaxis). in the cat, the acute onset of vomiting and diarrhea with associated hypovolemia and respiratory and vascular shock may be seen [ ] . if an animal develops any of these signs within the first several hours after vaccination, it should be presented to the veterinarian immediately for emergency medical care and support. it is not the goal of this review to offer therapies for shock; thus, the reader is referred to emergency veterinary literature for recommended therapies. the point here is to advise the practitioner to proceed with caution when using vaccines that may have a higher incidence of these reactions or in breeds that may be at increased risk for immediate hypersensitivity. the increased association between leptospirosis vaccines and type i reactions is well documented, and there are reports that toy breeds, particularly miniature dachshunds, may be at increased risk for type i reactions associated with leptospirosis vaccination [ ] . if an animal does have a type i reaction to a vaccine, the signs exhibited by the patient, interval between vaccine administration and onset of signs, and therapeutics administered should be well documented in the medical record as well as plans for future vaccination of that patient. ideally, once an animal has this type of reaction to a vaccine, that product should not be used again in that patient. all subsequent vaccines should be administered after a complete physical examination, and the vaccine should be given early in the day to allow monitoring of the patient in the hospital for several hours; however, if this is not possible, the patient should remain in the veterinary hospital for monitoring for at least minutes, followed by subsequent monitoring by the owner at home for several hours. pretreatment with diphenhydramine is an option; it is given parenterally (subcutaneous or intramuscular route) at a dose of . - . mg/kg to minutes before vaccination if hypersensitivity is a concern. administration of corticosteroids concurrently with vaccination to prevent a hypersensitivity reaction is neither appropriate nor recommended because of potential immunosuppression and vaccine interference, however [ , ] . the patient's medical record should be identified, outside and inside, to prevent future accidental readministration of that product. advising the owner that the patient should never receive that product again is important. type ii hypersensitivity reactions (autoimmune reactions) are suspected to occur in dogs secondary to vaccine administration. although this theory is yet unproven, there are reports of dogs developing immune-mediated thrombocytopenia and immune-mediated hemolytic anemia temporally associated with recent vaccination. if a dog develops either of these conditions within to months after vaccine administration, the practitioner would be advised to consider the risk/ benefit ratio of subsequent use of that product in that patient [ , ] . type iii hypersensitivity reactions are immune complex reactions. examples include the anterior uveitis associated with the use of the cav-i vaccine and table the complement-mediated rabies vaccine-induced vasculitis-dermatitis seen in dogs. other examples include glomerulonephritis and polyarthritis. antihistamine administered at the time of vaccine does nothing to prevent the reaction, nor is it recommended to administer corticosteroids concurrently with vaccination. once an animal has had this type of reaction, subsequent use of that product should be avoided in that patient [ , ] . type iv hypersensitivity reactions are cell-mediated responses occurring locally or systemically. examples include sterile granulomas at the sites of vaccine administration or polyradiculoneuritis. many sterile granulomas resolve without any intervention; however, for more severe reactions, the practitioner is referred to various medical texts for recommendations [ , ] . the previous discussion applies mainly to puppies and kittens owned by individuals. puppies and kittens housed in shelters face unique challenges, as do orphaned animals. these animals may not have received colostrum, and it is more likely that their mothers were not adequately vaccinated. the implications are that these animals are less likely to have received maternal antibodies, leaving them more vulnerable in the earliest stages of life. in addition, they frequently are malnourished, have an increased parasite burden, and are placed in crowded environments, possibly with high numbers of endemic pathogens. the american animal hospital association task force is currently developing recommendations specifically designed for puppies in these environments. in general, neonates that may not have received colostrum or are housed under these conditions may be vaccinated at an earlier age and ideally should be vaccinated before or at the time of entry into the shelter. the recombinant distemper vaccine could be given in this circumstance and should be administered . vaccination against additional diseases (canine and feline upper respiratory diseases) is indicated as well (see previous vaccine sections). husbandry is extremely important in these animals: providing proper nutrition, anthelmintics, and clean and dry housing is paramount. in general, these animals are special subsets of the general population facing challenges that most young animals do not experience. fiscal considerations and overall population health applies in these cases much more so than to individual client-owned pets. vaccines are perhaps one of the practitioner's greatest tools in preventing disease and maintaining individual and population health. they are to be used with forethought based on the risk of disease to the population and the individual balanced with assessment of the risks associated with individual vaccines. it is the practitioner's role to educate pet owners regarding actual risks associated with undervaccination and overvaccination. the goal is to reach the highest level of overall animal health with the minimum number of adverse events based on scientific and epidemiologic merit. immunity in the fetus and newborn the defense of the body b cells and their response to antigen report of the american animal hospital association (aaha) canine vaccine task force: executive summary and canine vaccine guidelines and recommendations avma council on biologic and therapeutic agents' report on cat and dog vaccines vaccines and vaccinations: guidelines vs. reality vaccines and vaccinations: the strategic issues report of the american association of feline practitioners and academy of feline medicine advisory panel on feline vaccines infectious diseases of the dog and cat in: veterinary immunology an introduction. th edition. philadelphia: saunders elsevier multicenter case-control study of risk factors associated with development of vaccine-associated sarcomas in cats philadelphia: saunders elsevier canine viral disease canine vaccination protection of dogs against canine distemper by vaccination with a canarypox virus recombinant expressing canine distemper virus fusion and hemagglutinin glycoproteins vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection using recombinant attenuated poxvirus vaccines bsava manual of canine and feline behavioural medicine. quedgeley, gloucester (united kingdom): british small animal veterinary association evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus, infectious canine hepatitis virus, and distemper virus experimental challenges infectious canine hepatitis and canine acidophil cell hepatitis infectious diseases of the dog and cat seroconversion of puppies to canine parvovirus and canine distemper virus: a comparison of two combination vaccines canine parvovirus (cpv) vaccination: comparison of neutralizing antibody responses in pups after inoculation with cpv or cpv b modified live virus vaccine duration of serologic response to five viral antigens in dogs national association of state public health veterinarians. compendium of animal rabies prevention and control prevalence of and risk factors for leptospirosis among dogs in the united states and canada: cases ( - ) leptospirosis: a re-emerging zoonotic disease canine vaccination infectious diseases of the dog and cat canine borreliosis diseases caused by systemic bacterial infections infectious diseases of the dog and cat impact of giardia vaccination on asymptomatic giardia infections in dogs at a research facility enteric protozoal infections, giardiasis presented at the dr. ross o. mosier th annual western veterinary conference use of serologic tests to predict resistance to feline herpesvirus , feline calicivirus, and feline parvovirus infection in cats other feline viral diseases infectious diseases of the dog and cat update on feline calicivirus: new trends an outbreak of virulent systemic feline calicivirus disease rabies surveillance in the united states during epidemiologic evidence for a causal relation between vaccination and fibrosarcoma tumorigenesis in cats feline leukemia virus report of the american association of feline practitioners and academy of feline medicine advisory panel on feline retrovirus testing and management comparison of the safety and efficacy of a recombinant feline leukemia virus (felv) vaccine delivered transdermally and an inactivated felv vaccine delivered subcutaneously chlamydial infections textbook of veterinary internal medicine feline immunodeficiency virus infection feline immunodeficiency virus infection and related diseases infectious diseases of the dog and cat feline infectious peritonitis and feline enteric coronavirus infectious diseases of the dog and cat textbook of veterinary internal medicine vaccine-associated feline sarcoma task force. vaccine-associated feline sarcomas vaccine-associated feline sarcoma task force. the current understanding and management of vaccine-associated sarcomas in cats plumb's veterinary drug handbook vaccine-associated adverse events immune complexes and type iii hypersensitivity key: cord- -ty wbtkv authors: chugh, tulsi title: timelines of covid- vaccines date: - - journal: curr med res pract doi: . /j.cmrp. . . sha: doc_id: cord_uid: ty wbtkv nan potential conflicts of interest: the author has nothing to disclose keywords: coronavirus, covid- , vaccine, sars-cov- world health organisation discussed the "top threats to human health in ," and developed a strategic plan to meet the challenges. among the communicable diseases. emerging and re-emerging viral pathogens causing global pandemic with devastating results were emphasised. severe acute respiratory syndrome coronavirus- (sars-cov- ) has caused a pandemic of coronavirus disease - (covid- ) with global public health and economic crisis. there is an urgent need for diagnostic and therapeutic countermeasures and rapid development of a vaccine for prevention and control of this formidable disease. since the who notification of first case of this disease on st dec, and a complete genome sequence of the virus on jan , , global attempts to produce a suitable vaccine are ongoing in scores of laboratories. most of the coroviruses in humans including covid- are derived from bats and there is a variable similarity between them. the whole genome sequence of covid - has about % similarity to that of mers-cov. the novel virus is a single strand rna which attaches to the human cell using through angiotensin converting enzyme (ace- ) receptors like middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars). there is a continuing evolution of this virus globally including the ones seen in eastern india. the geographic distribution, and severity of disease and risk of subsequent waves caused by these genomic variants is currently unknown. phases of vaccine development. , various phases of vaccine development are: preclinical trials are done in suitable animals for safely and efficacy by challenge studies. clinical studies are made in phase: phase i: vaccines are given to a limited number of human volunteers with emphasis on safety and also to monitor the immune response. phase ii: this is done by vaccine administration to a few hundred volunteers of various age groups to further study safety and efficacy. phase iii: it is done by giving vaccine to thousands of volunteers to monitor protection and safety. phase iv: it is post-marketing surveillance for protection and any adverse events. finally the data is analysed and evaluated for submission to regulatory authorities for approval. the whole process may take several years. however, in an emergency situation as now, it can be compressed by simultaneous phase to trials and scientific collaboration in various institutions. around vaccine candidates are under active development and are in human clinical trials. virus vaccine candidates: . inactivated virus vaccines: virus may be inactivated by treatment with heat or a chemical but viral surface proteins a left actue. however, it may not be potent immunogenic and repeat doses may be required. example: salk vaccine. . live -attenuated vaccine: virus is modified so that it is immunogenic but a virulent (sabin poleo vaccine). . non-replicating viral vector vaccines: genes of viral proteins are identified and put into a harmless carrier virus to be delivered into the host cell which make the viral protein and stimulate immune system of the host. university of oxford with astrazeneca are using this approach. . replicating viral vector vaccines: the harmless viral vector may multiply in the host, produce copies of vaccine proteins and produce protective antibodies. used for ebola vaccine. . rna vaccine: rna codes for the spike protein on the surface of covid- . rna vaccine stimulates immune system to produce protective antibodies against viral s protein. carries genetic instruction to host cells to produce rna which in true stimulates immune system to produce antibodies. the actual viral protein is injected and immune system produces corresponding protective antibodies. novavax is using this approach. repurposed vaccine , , , bacillus calmette-guerin (bcg) vaccine is a live attenuated vaccine against tuberculosis. it is the most used vaccine used in the world and ~ %. children in india are vaccinated at birth. various epidemiological studies show that it significantly reduces disseminated tuberculosis and its mortality in infants. this is due to protection by bcg against unrelated respiratory pathogens and neonatal sepsis. bcg activates innate cells which engulf and kill virus. (trained immunity). another related organism, mycobacterium vaccae also has protective action in mouse model and humans against tuberculosis when used for immunotherapy. bcg enhances innate immunity and may reduce viral load of covid- and may reduce cytokine storm. bcg has a general boosting effect. some ecological studies do suggest a "link between prior bcg vaccine and recorded cases of covid- ". however, more studies are required. professor david levine from university of california, berkeley states that there is a "shred of evidence" from studies in spain and italy that bcg may protect against covid- . timeline of vaccines , , as frantic efforts are being made to have the vaccines at the earliest. it is expected that we may have these in place by early next year. however, there are several logistical and policy dilemmas: affordability, fair distribution in various countries, priority of professional individuals, dosage, vaccine hesitancy, repeat doses, and prohibitive cost. the author has nothing to disclose covid- : a comprehensive review of a formidable foe and the road ahead pandemic preparedness: developing vaccines and therapeutic antibodies for covid- the early landscape of covid- vaccine development in the uk and rest of the world bcg-induced trained immunity: can it offer protection against covid- ? bcg vaccination enhances the immunogenicity of subsequent influenza vaccination in healthy volunteers: a randomized, placebocontrolled pilot study improved immunotherapy for pulmonary tuberculosis with mycobacterium vaccae effects of mycobacterial vaccae vaccine in a mouse model of tuberculosis: protective action and differentially expressed genes mutations in sars-cov- viral rna identified in eastern india: possible implications for the ongoing outbreak in india and impact on viral structure and host susceptibility spike mutation pipeline reveals the emergence of a more transmissible form of sars-cov- . biorxiv sars-cov- rates in bcg-vaccinated and unvaccinated young adults key: cord- -hbd euq authors: søborg, christian; mølbak, kåre; doherty, t. mark; ulleryd, peter; brooks, tim; coenen, claudine; van der zeijst, ben title: vaccines in a hurry date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: hbd euq preparing populations for health threats, including threats from new or re-emerging infectious diseases is recognised as an important public health priority. the development, production and application of emergency vaccinations are the important measures against such threats. vaccines are cost-effective tools to prevent disease, and emergency vaccines may be the only means to prevent a true disaster for global society in the event of a new pandemic with potential to cause morbidity and mortality comparable to the spanish flu, the polio epidemics in the s, or the sars outbreak in if its spread had not been contained in time. given the early recognition of a new threat, and given the advances of biotechnology, vaccinology and information systems, it is not an unrealistic goal to have promising prototype vaccine candidates available in a short time span following the identification of a new infectious agent; this is based on the assumption that the emerging infection is followed by natural immunity. however, major bottlenecks for the deployment of emergency vaccine are lack of established systems for fast-track regulatory approval of such candidates and limited international vaccine production capacity. in the present discussion paper, we propose mechanisms to facilitate development of emergency vaccines in europe by focusing on public–private scientific partnerships, fast-track approval of emergency vaccine by regulatory agencies and proposing incentives for emergency vaccine production in private vaccine companies. although progress in medical science has eradicated one infectious disease (smallpox) and threats from other infections such as polio have been reduced by widespread vaccination, new infectious diseases emerge at historically surprisingly high rates-more than one disease per year. there are several explanations. globalisation with its correspondingly increased transport of persons, products and animals can rapidly spread new infectious diseases around the world. furthermore, the condensing of populations with worldwide urbanization and encroachment of humans into new habitats, facilitating close contact with wild animals creates new hazards for transmission of zoonotic infectious agents from animals to man and possibly in the reverse direction, transmitting human pathogens to animals [ , ] . it has been suggested that more than % ( / ) of all new human pathogens in the last century originated from an animal reservoir [ ] . there is an international recognition of the importance of emerging infectious disease in an age of changes, as recently under-lined by the world health organisation: "it would be extremely naïve and complacent to assume that there will not be another disease like aids, another ebola or sars, sooner or later" [ ] . the recent experience with emergence of chikungunya virus in italy underlines these issues. the spread of chikungunya exemplifies how easily a well-known virus from a subtropical region in africa is able to shift vector from one mosquito vector (aedes aegypti) to another (aedes albopictus), disseminate to other climatic zones -including europe -and cause disease in a susceptible population [ ] . the adaptation to a new vector can probably be ascribed to a point mutation in the virus, whereas international travel served as the means of introduction of the virus to the competent vector. a. albopictus have recently become prominent around the mediterranean basin from greece to spain and other arboviral diseases including dengue and west nile virus may use the same vector-possibly causing the next outbreak in europe. climate change may boost this development further by expanding the range of vectors and their capacity to spread disease, together with other activities that transfer potential vectors to new areas. a. albopictus has extended its distribution to europe and the americas as its larvae can be transported in used automobile tyres [ ] . emerging infections have impact not only on the health but also on the economics of the afflicted region. the sars epidemic was estimated to have a direct cost of billion us$ in asia, and the international community was on the verge of a true disaster of even larger dimensions. this blow could have disrupted health care services, and affected societies and economies for years. it was a fortunate coincidence that sars was not transmissible before the onset of the patient's signs and symptoms of disease. hence, the epidemic was contained by traditional measures of disease control such as early case finding, isolation and quarantine. if this had not been the case, the rapid development and use of an emergency vaccine might have been the only feasible measure to prevent further spread. over the last century, vaccines have been shown to be one of the most cost-effective ways to prevent and control diseases. in some situations, such as the re-emergence of smallpox or a new influenza pandemic with a severity comparable to the spanish flu, emergency vaccinations may be the only way to prevent a true disaster for europe and the global society. following the early recognition of a new threat, the current advances in biotechnology, vaccinology (including reverse genetics) and information systems offer us the possibility of developing promising vaccine candidate shortly after the identification of a new infectious agent, under the assumption that this emerging infection is followed by natural immunity. however, additional major bottlenecks for the deployment of emergency vaccines include the lack of established systems for fast-track regulatory approval of such candidates and limited international vaccine production capacity. in the present discussion paper, we address mechanisms to facilitate development of emergency vaccines in europe by focusing on regulatory aspects and proposing incentives for emergency vaccine production in private vaccine companies. the control of poliomyelitis by rapid development of a vaccine shows that it can be done. it exemplifies what is possible with a strong governmental commitment, public demand and dedication. it also underlines the importance of government institutions taking the lead and responsibility in vaccine development. within only one year -from to -double blinded placebo controlled studies were conducted involving , vaccines and , controls receiving placebo. these trials proved safety and efficacy, leading to licensing of the vaccine shortly after [ ] . the fruit of basic immunology and vaccinology research led to the success of the vaccine and can be condensed into four major discoveries. firstly, characterization of the poliovirus and the definition of three serotypes leading to a trivalent vaccine [ ] . secondly, pathogenicity: the discovery that poliovirus causes paralysis [ ] . thirdly, proof of principle: confirmation that neutralizing antibodies protect against disease [ ] and finally, that virus could be grown in cell cultures for mass vaccine production [ ] . the vaccine campaign was a huge success, and was accepted very well by the population, leading to a steep decrease in polio cases in the years immediately following the vaccine's deployment in europe and usa. today, polio is eradicated in most parts of the world and remains present only in a few of the poorest countries. on the other hand, diseases selected for mass vaccination have to be chosen carefully. the attempt to prevent the spread of swine influenza by vaccination, in usa , is a good example of why to think twice before initiating mass vaccination. influenza specialists were worried that an influenza strain isolated from swine might cross the species barrier and cause a repeat of the spanish flu pandemic from . although no human cases were detected, the decision to start mass vaccination was made and more than million people were vaccinated within a few months. however, suspicion that vaccination was increasing the risk of guillain-barré syndrome as a side effect soon stopped the vaccination campaign [ ] . thus, with a strong public commitment and vital basic knowledge about the pathogen, a successful vaccine can be developed within a short timeframe. however, the decision whether to implement a new vaccine needs to be based on solid risk assessment. potential and unknown hazards associated with the early mass deployment of a new vaccine must be weighed against the risk and nature of the disease. vaccine development -in particular for emergency vaccinesneeds a different business plan than the market-driven approach that underlies the pharmaceutical industry [ ] . private vaccine companies cannot be expected to use resources on improving existing vaccines or developing new vaccine candidates for emerging infectious diseases when there is no current market or if the market is too small or diffuse to be economically feasible. hence, it is important that governments find mechanisms and funding to ensure the fundamentals for the success of a vaccine, namely basic and applied research in public health institutions and academia. furthermore, clear communication is necessary between governments and the vaccine industry on which vaccines need to be developed from a public health perspective. the challenge is to find incentives for the vaccine industry to take part in the development of products that currently do not have a clear market. one solution might be public support to public-private research and development of vaccine candidates in their early preclinical/clinical phases or advance assurances of confirmed purchases of certain volumes of vaccines if they make it to licensure. the european union has promoted the concept of public-private partnerships, but this concept has not resulted in important changes so far [ ] . it is important to find new ways to achieve these aims: it will be too late by the time we suddenly need new vaccines against an emerging epidemic. public-private partnerships in particular are necessary to secure vaccine production from laboratory bench through pilot plant to mass scale industrial production. specific contracts between governments and vaccine companies must be in place to secure that private production capacity is available for emergency vaccination production if needed. as mentioned, modern biotechnology has opened novel approaches for the development of new vaccines allowing production to be carried out in only a few months under the best circumstances (see table ). but obtaining data on clinical efficacy for licensure and regulatory approval will be a major bottleneck for making use of the current technology. without some indication of the immune response required for protection, basic efficacy studies will be difficult. without an animal model, they will be all but impossible. that leaves only the possibility of going to human studies without an indication that the vaccine is effective-a step that is highly unlikely, even in a dire situation. this leads us back to the responsibility of governments and international agencies -including the european union -for laying the groundwork. the first vaccines for sars were developed this way, leveraging preclinical work that had already been done on other coronaviruses [ ] . seasonal influenza vaccines are also made this way; working on the assumption that what has proved efficacious in the past against a related virus will prove efficacious in the future table a toolbox for the rapid development, production and deployment of an emergency vaccine. early recognition of an emerging microbial threat identification and characterization of the causative agent rapid understanding of natural history, pathogenesis, molecular biology and epidemiology; building on work in related pathogens as well as ongoing clinical, laboratory and epidemiological studies identification of potential vaccine candidates identification of potential delivery systems and suitable adjuvant to improve immunogenicity and sparing of antigen and dosages production at pilot plant level development and acceptance of correlates of immunity development and acceptance of correlates of safety limited trials in animals and humans based on these correlates as outcome measures fast-track approval of the vaccines enhancing production capacity by public-private partnerships based on risk assessment and defined objectives: implementation of emergency vaccination post-licensure follow-up of emergency vaccination with data accessible in real-time to medicine-and public health agencies as a surrogate for phase iii trials and ensuring development with advance purchase agreements to establish a market. testing novel vaccine candidates also becomes a bottleneck: just any combination of antigen and delivery system may not be effective. toxicity testing -which is designed to catch only acute problems with a vaccine -can be generally applied to most vaccines. in theory, it may be possible to produce basic safety data even before a complete product is available by testing "mock-up vaccines" in which a placeholder antigen or antigens are used [ ] . this would provide information on the safety of the delivery system including adjuvant, which is generally the most reactogenic part of a vaccine. safety testing of a specific product would still be required, but the reactogenicity of the generic components would already be defined and be one less variable to consider in designing the vaccine. however, efficacy studies require animal models of the infection or the disease and this is both time-and money-intensive. in the absence of a defined animal model, the obvious choice is nonhuman primates, as the closest match to humans, but even if this is possible (i.e. the pathogen will infect primates and produce disease) it may not be practical. both primate and non-primate animal research facilities are in short supply. support of animal facilities is likely to pay dividends when a need arises for rapid assessment of new vaccines. this should be relatively easily put into place, by making it an explicit goal, since it is really only an expansion of existing activities by research supporting agencies. certainly, in the united states, the biodefense initiative has led to a significant expansion in capacity. but it cannot be put into place on an ad hoc basis, nor is it likely that the private sector will become involved-the return on investment in possible emerging diseases is highly uncertain. such facilities take years to establish and their benefits are primarily in research and public health: therefore bodies involved in research and public health will need to take the initiative. once a potential vaccine has been produced and some evidence for efficacy and safety produced in animals through accepted correlates, the same data needs to be reproduced in humans. the current procedures were designed with safety as the foremost consideration and rapidity is not a characteristic of the process: it can take - years (occasionally longer) for a new vaccine to pass through clinical trials. under normal conditions, this is a sensible application of risk/benefit analysis with the emphasis on "first, do no harm". by definition, emerging diseases are not a major health risk-until a significant outbreak occurs. thus, clinical assessment is built around gradual steps-first screening for major risks (phase i, trials generally conducted in very small groups) then subsequently screening for less frequent risks (phase ii, in larger, but still small groups). it is not until phase iii studies, which are often large (thousands to tens of thousands), that efficacy data are expected to be produced. and while phase iii studies are large, they still occasionally fail to uncover rare risks, which only emerge after hundreds of thousands of people have been vaccinated, for example, intussusception of the bowel after administration of rotashield, a vaccine to prevent diarrhoea caused by rotavirus infection [ ] . such events are only uncovered during post-licensure pharmacovigilance. in an epidemic situation, however, the risk/benefit balance changes: if morbidity and mortality due to the pathogen is high, then even a vaccine with significant side effects becomes much more acceptable. it is therefore important to develop procedures for alternative pathways of approval. this should be done in close collaboration with regulatory agencies, and be based on accepted correlates of immunity and safety plus [probably] limited human data. while it will ultimately be up to governments with who guidance under international health regulations to decide what constitutes an "emergency" in which it could be invoked, the bare bones of a "fast-track approval" system for new treatments already exists (influenza mock-up vaccines emea) [ ] , and this procedure could be expanded to accelerate vaccine-testing in clinical trials during a public health emergency. in most cases, the greatest amount of time in a clinical trial is devoted to paperwork, to ensure that the trial complies with regulations designed to minimize risk to participants, ensure transparency and provide a paper trail as a shield against future litigation, should things go awry. in diseases with a poor survival chance -aggressive cancers, for example, or anti-retroviral therapy in the early days of the hiv epidemicregulatory agencies tended to be more forgiving. in such a situation, while safety remains a major issue (particularly for preventive vaccines administered to healthy individuals), testing for efficacy assumes greater importance.the demands for faster processing of vaccines can be addressed by the following steps: . an already-defined regulatory framework within which fasttrack clinical trials can be conducted. this should contain rules for priority review and approval, some (limited) protection against liability to open the process to commercial entities (as they are best equipped for large scale production and distribution) and rules for invoking such a process. they may not (perhaps should not) lead to open-ended approval of a product, being instead intended to allow limited release. . rapid access for vaccine developers to the appropriate regulatory authorities within the emea and authorization for regulators to draw on necessary expertise (perhaps in the form of expert panels) to enable assessments to be made quickly. regulations on relaxing approval (perhaps in the form of approval for a limited time) for import of vaccines not currently approved for use in europe. . a process whereby approval can be granted under the understanding that the complete necessary paperwork can be submitted retrospectively-enabling a rapid progress of efficacy trials, as soon as initial data suggests a vaccine is safe, without waiting for collation, submission and approval before progressing to the paperwork for the next step. . a broader acceptance of surrogate data, e.g., correlates of protection and safety, in the early steps. if (for example) animal toxicity studies raise no concerns, it may be possible to proceed directly to combine phase i/ii studies. this would provide human safety data, but at the cost of placing slightly more participants at risk. the payoff would be earlier access to data indicating whether the vaccine is immunogenic and stimulates the desired type of immune response, plus a more rapid assessment of vaccine safety. acceptance of immunogenicity data as a surrogate for efficacy, based on animal models (and it is here that expert panels will be crucial) might allow rapid release of a product from phase iii trials, subject to the study continuing to collect efficacy data. such an approach could shorten vaccine-testing time by months to years. . finally, while it is possible to enter phase i trials with an experimental product, phase ii has stricter requirements and the product tested needs to be closer to, if not identical with, that which will be taken into phase iii trials-which itself will be the final product. some flexibility on vaccine composition would allow a more rapid progress to phase iii. phase iii trials are intended to demonstrate that a product is efficacious. this is never an easy task and for emerging diseases is complicated by the fact that such diseases are, by definition, not endemic. that means that without reliable surrogate markers, efficacy studies can only be done in endemic countries or selected high-risk populations. it can be expected that even in countries where the disease is endemic, people will be reluctant to accept testing of a vaccine for which safety data and approval has been fast-tracked: some form of compensation mechanism is almost certain to be required to encourage manufacturers and the public to participate in a clinical trial. this passes out of the remit of organizations such as emea and into that of international cooperation, which needs to be arranged at the governmental level. the risks of rapidly proceeding into phase iii can be ameliorated by compromising (to some extent) impartiality. to avoid bias, such large studies are normally blinded and results assessed at the end of the study. however, in a situation where large numbers of people are being vaccinated with a "fast-tracked" vaccine safety concerns will be higher than normal. by putting enhanced surveillance into place and assessing data from cohorts within the main phase iii study, data on adverse events possibly associated with vaccination and efficacy of the vaccine could be collected much faster. objectivity could be maintained by maintaining blinding with regard to the study monitors. this approach essentially expands the role of the data safety management board, whose role is normally to oversee the safety of the study and who do review results on an ongoing basis, to cover decision-making on vaccine efficacy. in such a case, they would need to involve the study designers, which may raise issues of conflict of interest. this can be addressed by again involving expert review panels, but that will almost certainly face resistance from commercial developers who would face exposure of their operating procedures. but where there is an overwhelming public interest in rapid assessment of vaccine efficacy, the conventional rules may need to be relaxed and increased transparency is the safest counter to decreased regulatory oversight. in conclusion, new infectious diseases are emerging at a historically high rate. to secure both public health and economic stability in the future effective countermeasures have to be instituted in advance at governmental levels. implementing fast-track approval systems for emergency vaccines by the regulatory agencies, and underpinning public private partnerships to enable production in the absence of a market would be an important step in order to be prepared for a new pandemic. finally, innovative research towards the understanding of vaccine safety and efficacy and leading to shorter development times should be promoted. american association of physical anthropologists meeting perceived vaccination status in ecotourists and risks of anthropozoonoses surveillance and response to disease emergence a safer future, global health in the century. the annual health report who infectious diseases chikungunya: no longer a third world disease the used tyre trade: a mechanism for the worldwide dispersal of container breeding mosquitoes making history: thomas francis m.d. jr. and the salk poliomyelitis vaccine field trial differentiation of types of poliomyelitis viruses; the grouping of strains into three basic immunological types viremia in human poliomyelitis evaluation of red cross gamma globulin as a prophylactic agent for poliomyelitis iv. final report of results based on clinical diagnoses cultivation of the lansing strain of poliomyelitis virus in cultures of various human embryonic tissues guillain-barre syndrome following vaccination in the national influenza immunization program, united states - why certain vaccines have been delayed or not developed at all european union dg interternal policies of the union a dna vaccine induces sars coronavirus neutralization and protective immunity in mice pandemic vaccines: promises and pitfalls rotavirus vaccines: an overview key: cord- - jikrn authors: borja-cabrera, g.p.; santos, f.n.; bauer, f.s.; parra, l.e.; menz, i.; morgado, a.a.; soares, i.s.; batista, l.m.m.; palatnik-de-sousa, c.b. title: immunogenicity assay of the leishmune(®) vaccine against canine visceral leishmaniasis in brazil date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: jikrn leishmune(®) is the industrialized version of the fml-saponin vaccine which has been shown to develop – % protection in vaccinated dogs and – % vaccine efficacy against field canine visceral leishmaniasis (cvl) in brazil. leishmune(®) has been proven to be safe and tolerable and a transmission-blocking vaccine which renders vaccinated dogs non-infectious to sand fly vectors. in the present investigation, healthy seronegative dogs of endemic and epidemic areas of brazil were monitored for leishmune(®)-induced immunogenicity during a -year trial. another group of untreated exposed dogs was also studied in parallel. both groups were seronegative on day . the strong immunogenicity induced by leishmune(®) vaccine was demonstrated by the % of fml-seroconversion, increase in absorbencies, the . % dth positive reactions and increase in skin test size diameters, the average increase in cd + total lymphocytes population in blood ( . %), expected for qs saponin-containing vaccine, the sustained proportions of cd + t cells, and the average increased proportions of cd + b lymphocytes ( . %). the leishmune(®)-induced protection against cvl is demonstrated by the results: . % asymptomatic dogs (at the end of first year) and % healthy survivors (at the end of the second year) among vaccinated dogs, compared to the . % asymptomatic and % survivor dogs (p < . ) monitored in the untreated exposed cohort. in spite of the low vaccine coverage, it was possible to detect a . % (p < . ) reduction in belo horizonte and an . % (p < . ) reduction in araçatuba of the incidence of cvl among vaccinated dogs, when compared to the global incidence of cvl of each town, respectively. our preliminary results support the potential use of leishmune(®) to prevent cvl epidemics. visceral leishmaniasis (vl), a chronic and severe protozoa infection, is fatal if untreated after the beginning of symptoms. the disease is a canid zoonosis (zvl) caused by leishmania chagasi in america and by leishmania infantum in the mediterranean basin and middle east, and an anthroponosis caused by leishmania donovani in africa, india and asia. nowadays there are , new human cases registered annually worldwide [ , ] . the drug resistance and toxicity of chemotherapy, the increase of the disease incidence of immunocompromised subjects, and the difficulties of the epidemiological control, which is based upon sacrificing of seropositive dogs, emphasizes the need for safe pro- [ ] [ ] [ ] [ ] achieving different levels of protection, but no data from field trials of any dna formulation is so far available [ ] . despite the recent intensification in research for a canine vaccine only two second-generation vaccines with native antigens have progressed to phase iii field trials: the fml-saponin [ , ] and the liesap vaccines [ ] . the fml (fucose-mannose ligand) glycoproteic complex [ ] , antigenic for both humans [ ] and dogs [ ] , was formulated, as a second-generation vaccine, with quillaja saponaria saponin and underwent phase i-iii trials becoming the leishmune ® licensed vaccine in brazil [ ] . the fml was immunogenic, immunoprophylactic and immunotherapeutic, in mice and hamsters and canine field trials [ , , [ ] [ ] [ ] [ ] . in the first phase iii dog assay [ ] , deaths and symptomatic cases among placebo-treated dogs ( %) were detected and confirmed by parasite analysis and pcr. no deaths were detected among vaccines and infection was confirmed in / oligosymptomatic dogs ( . %), resulting in % protection and % vaccine efficacy [ ] . in the second field assay [ ] , the infective pressure was higher and years after vaccination, deaths were detected in / ( %) placebotreated and / ( %) vaccinated dogs, resulting in % of protection and % vaccine efficacy. this protection lasted for at least . years and was concomitant with the reduction of the human incidence of the disease in the area [ ] . the fml-vaccine also produced an immunotherapeutic effect when administered to l. donovanior l. chagasi-infected dogs while they were still asymptomatic [ ] . the decrease in the canine and human incidence of visceral leishmaniasis in the vaccinated area [ ] and the maintenance of normal proportions of cd and cd lymphocyte levels in the blood of vaccinated dogs [ ] indicate that dog vaccination with the fmlvaccine reduces dog infectivity to sand flies [ ] . liesap + mdp vaccine, the other second-generation vaccine with native antigens, was recently used in a field assay with naturally exposed dogs of south france [ ] . in this trial any dog showing clinical and/or serological evidence, infection was confirmed (or not) by the presence of parasites in bone marrow cultures and by pcr analysis. after years, the incidence of infection was . % ( / ) in vaccines versus . % ( / ) in placebo-treated dogs ( % vaccine efficacy) [ ] . the authors claimed a vaccine efficacy of % based on the confirmation of infection by very sensitive methods such as pcr or culture, instead of cvl deaths [ ] which did not occur in this area of lower incidence. the fml-vaccine unlike the liesap vaccine revealed protection not only against infection, but also against severe disease and deaths due to cvl [ , ] reducing morbidity and mortality [ , ] which are much stronger criteria of protection [ ] . the fml-vaccine was licensed in brazil, for dog prophylaxis against zvl, under the brand leishmune ® [ ] . dogs vaccinated with leishmune ® (fml-licensed vaccine) are not infectious for sand flies [ ] , as indicated by a complete absence of clinical signs and of parasites in the skin, lymph node and blood pcr-amplified samples. exposed untreated controls on the other hand, were symptomatic ( %) and showed parasites in their lymph nodes ( . %), leishmania dna detected by pcr in blood ( . %) and immunohistochemical reactions in skin ( %) [ ] . leishmune ® is a transmission-blocking vaccine [ ] and when used with increased adjuvant concentration was also effective in immunotherapy on experimental cvl [ ] . recently we described the safety analysis of leishmune ® vaccine performed in a cohort of dogs from brazilian endemic and epidemic areas of canine and human visceral leishmaniasis. the vaccine proved to be tolerable and safe [ ] . in the present investigation we report the immunogenicity assay of leishmune ® , monitored in the same dog cohort, confirming the immuno protective potential previously described for the fml-saponin vaccine [ , ] and disclosing the potential use of leishmune ® vaccine to interrupt epidemics. six hundred healthy dogs from the canine visceral leishmaniasis endemic towns of araç atuba, andradina, valparaíso, guararapes, bauru (são paulo state) and belo horizonte, nova lima, sete lagoas (minas gerais state), brazil, showing previous negative results in leishmania-serology by the immunofluorescent assay [ ] were selected for vaccination with three doses of leishmune ® (fort dodge animal health, campinas, sp, brazil), in a -day interval, through the subcutaneous (sc) route [ ] and a booster in month . on day , before vaccination, from the dogs were excluded due to their positive reaction to the more sensitive fmlelisa assay [ ] . the remaining dogs, seronegative to the fml antigen, asymptomatic and showing good physical condition, became the trial group of this investigation. each of the veterinarians participating in this trial vaccinated dogs with three doses of leishmune ® , making a total of doses. the animals were monitored for their anti-fml igg serum antibody titters by the fml-elisa assay [ ] at days and and in months and , and by their intradermal response to the l. donovani promastigote lysate [ , ] antigen in months and . the serum was collected and intradermal test was carried out before injection of the vaccine booster in month . also, clinical evaluations were performed every months, during the -year period ( ) ( ) ( ) . alopecia, onychogryphosis, cachexia, anorexia, apathy, disseminated ulcers, skin lesions, keratitis, renal failure, loss of weight, lymph node enlargement or diarrhoea were recorded as visceral leishmaniasis symptoms. in vaccinated symptomatic animals, leishmania infection was confirmed either by pcr analysis of lymph node aspirates and/or blood samples [ ] or by direct microscopical observation of leishmania amastigotes in giemsa stained lymph node smears [ ] . the leishmune ® -vaccinated dog cohort included animals ( %) from different breeds and of ( %) mongrel dogs [ ] . all animals were previously vaccinated against distemper, parvovirosis, parainfluenza virus, leptospirosis, coronaviruses, type adenoviruses and rabies. for ethical reasons, veterinarians were not able to keep an untreated and exposed control dog population. for the purpose of comparison, asymptomatic fml-seronegative dogs from another endemic area (jardim progresso, natal, rn, brazil), with similar canine incidence [ ] were included in this study as the exposed untreated group. in this investigation, all manipulations performed on the animals were conducted to ensure minimal animal suffering, as recommended by the nih regulation. each leishmune ® prophylactic vaccine dose [ ] was composed of lyophilized fml antigen adjuvanted with saponin, reconstituted in ml nacl . % sterile saline solution at the moment of vaccination and administered subcutaneously. the fml-vaccine, leishmune ® , is patented: inpi number: pi - ( march ) assigned to universidade federal do rio de janeiro, brazil and is the first second-generation vaccine licensed against leishmaniasis, since th june [ ] . this was determined by injecting dogs intradermally, in the inner aspect of the right hind leg, with . ml of l. donovani freezethawed antigen containing g protein in nacl . % sterile saline solution ( stationary phase promastigotes/ml). the left hind leg received only . ml saline. measure of the increase of intradermal reaction was performed h after antigen injection. indurate areas were marked and each time the values of the saline control were subtracted from the reaction due to the leishmania antigen. reactions showing diameters ≥ mm were considered positive [ , ] . in month after vaccination, pbmc of randomly chosen leishmune ® -vaccinated dogs from araç atuba and andradina were analysed by flow cytometry. three milliliters of blood from the cephalic vein was collected from each dog in heparin-tubes, transported at room temperature and processed h after collection [ ] . for the ex vivo analysis, l of blood was incubated for min at room temperature, with l of each one of the following monoclonal antibodies diluted in facs dil solution ( % fcs-supplemented pbs buffer): anti-thy- (rat-igg bclone ykix . ) ( : ), anti-cd (rat-igg a-clone ykix . ) ( : ), anti-cd (rat-igg a-clone ykix . ) ( : ), and anti-cd (rat-igg -clone ycate . ) ( : ). facs dil solution was used as negative control. after this period, ml of pbs-w (pbs buffer with . % bovine serum albumin and . % sodium azide) were added to each tube and the mixture was homogenised, and centrifuged at rpm, at room temperature, for min. the supernatants were aspirated and pellets homogenised and added to l of anti-rat fitc conjugate ( : ) (serotec, uk) except for the facs dil cell control. at this time, l of the fitc-labelled mouse anti-human-cd (mouse-igg -clone iob a) monoclonal antibody (immunotech co., marseille, france) was used in a direct immunofluorescence procedure. all suspensions were homogenised, incubated for min at room temperature in the dark and treated with ml of the / diluted lysis solution during vortex homogenisation (becton & dickinson, usa). the mixtures were further incubated for min at room temperature in the dark and further centrifuged at rpm for min. supernatants were discarded and the pellet-containing tubes were inverted on to absorbent paper. all these procedures were repeated twice after the addition of ml pbs. the pellets were homogenised carefully and finally fixed with l of . % formaldehyde-pbs. the relative immunofluorescence of cells was counted in a total of , events measured in a becton dickinson facscalibur apparatus and further analysed using windows multiple document interface flow cytometry application (winmdi) version . software [ ] . as control, facs analysis of pbmc of nine normal healthy untreated dogs was also performed. comparison of proportions was carried out using the -test. to test the significance of the differences between groups we used the % confidence interval of the averages. five hundred and fifty dogs previously assayed for leishmune ® safety analysis [ ] were also monitored for the vaccine-induced immunogenicity during a -year trial ( ) ( ) ( ) . another group of untreated exposed dogs were also studied. the differences between the two groups after vaccination were highly significant in all variables (p < . ) ( table ) . both the vaccinated and the untreated groups were seronegative at day , but a strong fmlseroconversion ( %) was detected after complete vaccination on day . by this time, % of the untreated controls developed anti-fml antibodies due to their exposure to natural infection. the vaccine increased, not only the number of dogs with positive table two-year evolution of immunogenicity and incidence of zvl in cohorts of leishmune ® the delayed type of hypersensitivity response (dth) against the leishmanial lysate was positive in % of the vaccinated dogs, in month , and increased along time (table ) disclosing that, as desired for a protective vaccine against cvl, leishmune ® prophylactic vaccine enhances not only the humoral response but also triggers the cellular immune response against the parasite as well. the size of skin test reactions can be used as a measure of potency of a vaccine. the mean ± s.d. diameter of the skin tests at month was . ± . mm (n = ). the skin tests diameters significantly increased until month to . ± . (n = , p < . ), when dth reactions were positive in . % of the vaccines. these results confirm that the natural booster in an endemic area, while sustaining the humoral response of leishmune ® vaccines, contributes to enhance the specific anti-l. chagasi cellular immune response which is known to be responsible for protection against cvl. while both vaccinated and untreated dogs were both healthy at the beginning of the study, the untreated dog group developed a greater number of zvl symptoms along time reaching . % of the cohort by the end of the first year. meanwhile, clinical signs were detected in only . % of the vaccines during the same period (table ) . accordingly, while the cumulative proportions of deaths due to confirmed zvl in the untreated dogs reached % of the cohort, only % of the vaccinated dogs died of zvl during the trial. our results indicate the strong protective prophylactic effect of leishmune ® in seronegative dogs of endemic areas. table summarizes the results of the immunophenotype analysis of pbmc of a randomly selected sample of dogs, collected the great difference in the incidence of zvl disclosed in vaccines and controls (table ) could be partially due to the different infective pressure of the towns where leishmune ® -vaccinated dogs and untreated controls were located. however, when we compared the incidence of zvl in leishmune ® -vaccinated dogs to the incidence of the total dog population of the same town, the vaccine-induced protection was also evident. table shows the official data mean values of canine incidence of zvl during the period - . we show that the zvl incidence in belo horizonte, decreased from . % in the total population [ ] to . % (p < . ) in the leishmune ® -vaccinated dogs, in the first years of the vaccine use. the more striking effect of the prophylactic vaccination was seen in araç atuba, where the incidence of zvl decreased from . % of the total population to . % in vaccines (p < . ), leading to an . % reduction in the zvl incidence and thus confirming the strong protective effect of leishmune ® . the cohort of vaccinated dogs analysed in this investigation was the same previously used for the safety analysis of the leishmune ® vaccine [ ] , which confirmed that the formulation was tolerable and safe. in this investigation, we confirmed the strong immunogenicity of leishmune ® vaccine, previously shown for the fml-saponin vaccine in the field [ , ] indicating that the commercial formulation maintains the characteristics of the laboratory-prepared vaccine. soon after complete vaccination, the percent of seropositivity to fml in dogs treated with the fml-saponin vaccine was % [ ] , while it reached % in leishmune ® vaccines. this indicates the earlier achievement of humoral response induced by the commercial formulation, which is an important indicator of the vaccine potency. the humoral response was also sustained at high levels for months after vaccination, probably due to the natural booster effect of l. chagasiinfected sand flies of the endemic area. conversely, a previous kennel experiment in a non-endemic area, showed in leishmune ®vaccinated unexposed dogs, a decrease in absorbencies from day ( . ± . ) to month after vaccination ( . ± . ) (unpublished results). twelve months after vaccination, a positive dth response to leishmanial antigen was present in % of the fml-saponinvaccinated dogs ( . mm average skin test diameter) [ ] , and in . % of leishmune ® -vaccinated dogs which showed slightly larger diameters of skin tests ( . mm), indicating that beside the antibody response, the induction of the cellular immune response against leishmania lysate was also preserved in the commercial formulation. leishmune ® vaccination induced an increase in cd + total lymphocytes population in blood, also observed after dog immunotherapy with the fml-saponin vaccine [ ] . this was expected for a vaccine containing the qs saponin adjuvant of q. saponaria molina and it was related to its hydrophobic normonoterpene moiety [ , ] but also present in the deacylated saponins of q. saponaria molina [ ] and the deacylated saponins of calliandra pulcherrima [ ] which lack the hydrophobic moieties. evidence of the involvement of cd + lymphocytes in protection against intracellular parasitic infection has increased recently [ ] . cd -defficient mice failed to control leishmania parasite growth [ ] . also, cd specific cells are primed during natural infection or human vaccination and secrete ifn-␥, when re-stimulated in vitro with leishmania antigens [ ] [ ] [ ] . in naturally infected asymptomatic dogs, increased levels of cd + lymphocytes appeared as the major phenotypic feature, as well as in dogs bearing a lower parasite load [ ] , indicating a correlation with natural protection. while no effect was observed after l. infantum lysate vaccination [ ] , an increase in cd + t cells was observed after vaccination with the q. saponaria saponin-containing vaccines: fml-quila ( . %), fml-saponin r ( . %) [ ] , leishmune ® ( . %) [ ] and the l. brasilensis lysate-saponin vaccine ( . %) [ ] . the intense cell proliferation and increased nitric oxide production during in vitro stimulation by l. chagasi soluble antigens, suggests the induction of a potential resistant profile [ ] . the total levels of cd + and cd + lymphocytes are expected to decrease in advanced canine visceral leishmaniasis [ , , , ] with the specific decrease of cd + cells being correlated with dog infectivity to sand flies [ ] . indeed, the average percent value of cd lymphocytes in naturally infected dogs with visceral leishmaniasis was . % [ ] . on the other hand, vaccination against zvl is expected to expand or sustain the cd + t cell levels. the expansion or sustention of cd + t cell levels of dogs treated with l. infantum vaccine immunochemotherapy was % [ ] , with the fml-quila vaccine it was . %, with fml-saponin r . % [ ] and with the leishmune ® vaccine was . % [ ] . sustained cd + t cell were found also in this investigation in dogs vaccinated with leishmune ® , and sustained l. chagasi-specific cd + t cell proportions were found before in dogs treated with leishmune ® after infection [ ] indicating that the commercial formulation maintains the immunogenicity and potency demonstrated by the fml-saponin vaccine in the prophylaxis [ ] and immunotherapy [ ] against zvl. both the leishmune ® prophylactic vaccine, which contains . mg of [ ] and the leishmune ® immunotherapeutic [ ] or the l. brasiliensis [ ] vaccines, that contain mg of the riedel de haen saponin, maintain the normal levels of cd + lymphocytes in vaccinated unexposed [ ] , exposed [ ] or challenged [ ] dogs. higher proportions of cd cells were detected in this investigation in the leishmune ® -vaccinated dogs. the levels of cd + cells were higher in vaccinees, with a mean average that fell outside the ci % interval of the normal dogs. increased cd + b cell levels were also found in dogs vaccinated with the l. brasiliensis-saponin vaccine that uses the same adjuvant included in leishmune ® [ ] and in unexposed dogs vaccinated with leishmune ® [ ] . the increase in total and in leishmania-specific cd + b circulating lymphocytes was synchronous with the induction of an intense humoral response [ ] . also, a positive correlation was found between pbmc proliferation in response to l. chagasi antigen and cd + b cells. this was considered an indication of the major apc function of the cd + cells [ ] . on the other hand, decreased cd + b cell proportions are expected to occur in untreated infected dogs with advanced zvl [ , ] , which also exhibit hypergammaglobulinemia, a hallmark of human and canine visceral leishmaniasis [ ] . these facts suggest that the cd + b lymphocyte population, which increased after vaccination with leishmune ® is involved not merely in the expansion of the total humoral response but in the increase of the specific synthesis of the igg anti-fml antibodies that are related to protection [ ] [ ] [ ] [ ] [ ] and blockage of the transmission of vl in the field [ ] . in this investigation, we have demonstrated the strong immunogenicity of leishmune ® in healthy exposed dogs in epidemic areas, which consequently exhibit a strong and sustained humoral and cellular immune response against the parasite. a classic phase iii trial, with random double-blind selected controls could not be performed because of the ethical restrictions of the veterinarians from these epidemic areas, who refused to include untreated healthy dogs as exposed controls. leishmune ® -induced protection against zvl, is however suggested by the results of . % of asymptomatic dogs (at the end of first year) and % healthy survivors (at the end of the second year) among vaccinated dogs, compared to the . % of asymptomatic and % survivor dogs monitored in an untreated exposed cohort in another endemic area. although a vaccine against zvl is considered an efficient tool for eradication of human and canine visceral leishmaniasis [ ] and leishmune ® is the first vaccine in the world to be licensed against zvl [ ] , its vaccine coverage in brazil is still very low. in spite of this the incidence of zvl among vaccinated dogs in belo horizonte suffered a significant reduction of . % (p < . ), and there was an . % (p < . ) significant decline in araç atuba, when compared to the global incidence of zvl of both towns, respectively. thus our preliminary results then support the potential use of leishmune ® for the prevention of zvl epidemics. the tdr fifteenth programme report. research progress - . new and improved tools control of zoonotic visceral leishmaniasis. is it time to change strategies? the logic of visceral leishmaniasis control autoclaved leishmania major vaccine for prevention of visceral leishmaniasis: a randomised, doubled-blind, bcg-controlled trial in sudan double-blind randomized efficacy field trial of alum precipitated autoclaved leishmania major vaccine mixed with bcg against canine visceral leishmaniasis in meshkin-shahr district phase iii randomized double blind clinical trial on the efficacy of a vaccine against canine visceral leishmaniasis in urban area of montes claros, mg, brazil protection against experimental visceral leishmaniasis infection in dogs immunized with purified excreted secreted antigens of leishmania infantum promastigotes immunogenicity in dogs of three recombinant antigens (tsa, leif and lmsti ) potential vaccine candidates for canine visceral leishmaniasis immunization with h , haspb and mml leishmania proteins in a vaccine trial against experimental canine leishmaniasis failure of a multi-subunit recombinant leishmanial vaccine (mml) to protect dogs from leishmania infantum infection and to prevent disease progression in infected animals protective vaccination against canine visceral leishmaniasis using a combination of dna and protein immunization with cysteine proteinases type i and type ii of l. infantum vaccination with plasmid dna encoding kmp , tryp, lack, and gp does not protect dogs against leishmania infantum experimental challenge protection in dogs against visceral leishmaniasis caused by leishmania infantum is achieved by immunization with a heterologous prime-boost regime using dna vaccine and vaccinia recombinant vectors expressing lack vaccine congress on nucleoside hydrolase dna vaccine against visceral leishmaniasis vaccines for leishmaniasis in the fore coming years a phase iii trial of efficacy of the fml-vaccine against canine kala-azar in an endemic area of brazil (são gonç alo do amarante, rn) long lasting protection against canine kala-azar using the fml-quila saponin vaccine in an endemic area of brazil (são gonç alo do amarante) long-lasting protection against canine visceral leishmaniasis using the liesap-mdp vaccine in endemic areas of france: double-blind randomised efficacy field trial mendonç a-previato l. inhibition of leishmania donovani promastigote internalization into murine macrophages by chemically defined parasite glycoconjugate leishmania donovani: titration of antibodies to the fucose mannose ligand as an aid in diagnosis and prognosis of visceral leishmaniasis the fml-elisa assay in diagnosis and prognosis of canine visceral leishmaniasis leishmune ® vaccine blocks the transmission of canine visceral leishmaniasis. absence of leishmania parasites in blood, skin and lymph nodes of vaccinated exposed dogs análise do potencial diagnóstico, prognóstico e imunoprotetor do antígeno fml (ligante de fucose manose) de leishmania (l.) donovani, no calazar canino experimental e de área endêmica saponins, il and bcg adjuvant in the fml-vaccine formulation against murine visceral leishmaniasis the fml vaccine (fucose-manosse ligand) protects hamsters from experimental kalaazar. ciência e cultura immunotherapy against murine experimental visceral leishmaniasis with the fml-vaccine effective immunotherapy against canine visceral leishmaniasis with the fml-vaccine guidelines for the evaluation of plasmodium falciparum vaccines in populations exposed to natural infections. tdr/mal/vac/ . world health organization the fml-vaccine (leishmune ® ) against canine visceral leishmaniasis: a transmission blocking vaccine immunotherapy against experimental canine visceral leishmaniasis with the saponin enriched-leishmune® vaccine safety trial using the leishmune ® vaccine against canine visceral leishmaniasis in brazil improving methods for epidemiological control of canine visceral leishmaniasis based on a mathematical model. impact on the incidence of the canine and human disease acylated and deacylated saponins of quillaja saponaria mixture as adjuvants for the fml-vaccine against visceral leishmaniasis vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity pulcherrima saponin, from the leaves of calliandra pulcherrima, as adjuvant for visceral leishmaniasis importance of cd t cell-mediated immune response during intracellular parasitic infections and its implications for the development of effective vaccines cd + t cells are required for primary immunity in c bl/ mice following lowdose, intradermal challenge with leishmania major evaluation of the stability and immunogenicity of autoclaved and nonautoclaved preparations of a vaccine against american tegumentary leishmaniasis flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant lack and soluble leishmania antigen in human cutaneous leishmaniasis differences in gamma interferon production in vitro predict the pace of the in vivo response to leishmania amazonensis in healthy volunteers phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by leishmania chagasi canine leishmaniasis transmission: higher infectivity amongst naturally infected dogs to sand flies is associated with lower proportions of t helper cells despite leishvaccine and leishmune trigger distinct immune profiles, their ability to activate phagocytes and cd + t-cells support their highquality immunogenic potential against canine visceral leishmaniasis immunogenicity of a killed leishmania vaccine with saponin adjuvant in dogs lymphocyte subset abnormalities in canine leishmaniasis the immune response and pbmc subsets in canine visceral leishmaniasis before, and after, chemotherapy evaluation of a specific immunochemotherapy for the treatment of canine visceral leishmaniasis visceral leishmaniasis in the german shepherd dog. i. infection, clinical disease, and clinical pathology igg /igg antibody dichotomy in sera of vaccinated or naturally infected dogs with visceral leishmaniasis specific igg and igg antibody responses of dogs to leishmania infantum and other parasites analysis of the humoral response against total and recombinant antigens of leishmania infantum: correlation with disease progression in canine experimental leishmaniasis leishmania infantum-specific igg, igg and igg antibody responses in healthy and ill dogs from endemic areas. evolution in the course of infection and after treatment infectiousness in a cohort of brazilian dogs: why culling fails to control visceral leishmaniasis in areas of high transmission the most studied first-generation vaccine, composed of total leishmania lysate and bcg, protected against vl in sudan [ ] and against canine visceral leishmaniasis (cvl) in iran [ ] but not in brazil [ ] . lemesre et al. [ ] , using a second-generation vaccine with the culture media of l. infantum containing a -kda excreted protein in formulation with mdp (liesap) obtained protection in beagles in a kennel assay [ ] . regarding the recombinant vaccines, the multicomponent leish- f fusion protein in combination with mpl-se or adjuprime was only immunogenic in dogs challenged with l. chagasi [ ] and l. infantum (mml) [ ] and failed to prevent l. infantum natural infection or the progression of disease in dogs in an open kennel trial [ ] . a few third-generation dna vaccines have been tested in dogs against experimentally induced cvl key: cord- -rlliacex authors: kremer, eric j. title: pros and cons of adenovirus-based sars-cov- vaccines date: - - journal: mol ther doi: . /j.ymthe. . . sha: doc_id: cord_uid: rlliacex nan with the rise of molecular biology, vaccine designs became more nuanced and the use of viral vectors emerged. an example is the evolution and checkered history of vaccines based on adenoviruses (ads). live ad types (ad ) and (ad ) have been used in north american military recruits since the s to prevent severe respiratory illness. similarly, dogs in western countries are vaccinated with an attenuated canine type to prevent infection of the more virulent type . many of the first replication-defective ad "vectors" in the early s were vaccines. the original ad vaccine design was relatively simple: delete a region of the viral genome that the virus needs to propagate, provide these functions via transcomplementing cells (e.g., frank graham's cells) so that one could grow the vaccine, and then insert into the virus genome an expression cassette coding for the targeted epitopes. fast forward to . the sars-cov- pandemic may be headed toward historic proportions-although still far from the spanish flu ( million deaths) and aids ( million deaths)-inflicting havoc on families, communities, and economies and overwhelming health care facilities. clearly, we need a vaccine. are ad-based vaccines targeting the spike and capsid proteins our best bet? after almost years of working with ads, their biochemical properties are well characterized: ads are simple to make (in weeks a graduate student could generate enough of a novel ad vaccine to treat a thousand mice and dozens of monkeys), easy to purify to high titer, genetically stable, easily stockpiled, relatively inexpensive, and can be delivered via aerosol, oral, intradermal, and intramuscular routes. the aerosol route is particularly relevant when targeting a respiratory virus. it is also important that ad-based vaccines tend to induce b cell and t cell responses. hundreds of millions of euros, dollars, and yen have been invested in advancing ad-based vaccines. these advances include production and purification methods, genetic incorporation of epitopes into the capsid so that mononuclear phagocytes present these antigens via major histocompatibility complex (mhc) class i and ii pathways, cloaking the capsid with polymers/shields to prevent neutralization by antibodies (nabs), retargeting the vector to professional antigen-presenting cells, using helper-dependent vectors (so that the vector-infected cell only expresses the target epitopes and not ad antigens), using ad types with a lower level of seroprevalence in some populations, and single-cycle replication of vaccines to produce massive amounts of antigens. each tweak, alone or in combination with others, has improved vaccine efficacy in preclinical trials. as sars-cov- became a pandemic, it is astonishing that, in the case of the ad-based vaccine frontrunners, little has changed from the basic design of years ago. some used the well-trodden path of an ad -based vaccine, while others switched to human or simian ads that have low seroprevalence in europe and north america (but not necessarily in africa or asia). conceptually, ad serotype switching to avoid nabs is at least years old. the advent of simian ad vaccines was not developed following a rigorous testing of all > different ad types but was most likely the result of intellectual property issues and the ability to produce simian ads in good manufacturing practice (gmp)-compliant cells. one presumes that the subsequent rounds of ad-based coronavirus disease (covid- ) vaccine candidates will be more sophisticated. should we go "all in" on an ad-based vaccine against sars-cov- ? the first question is safety. there are few drugs or biologicals that do not have side effects or cause adverse reactions. weighing the advantages versus disadvantages during the current pandemic can be idiosyncratic, and the strength of the reasoning varies by population, culture, religious beliefs, and bizarrely (for those of us outside the usa) even political affiliation. current criteria limit the window to identify adverse reactions to months. in addition to swelling and pain at the injection site, common to some vaccines, ad-based vaccine adverse effects include fever, pneumonia, diarrhea, transient neutropenia and lymphopenia, fatigue, labored breathing, headaches, liver damage, and fasting hyperglycaemia. rare but grave adverse reactions include neuropathies such as bell's palsy, guillain-barré syndrome, gait disturbance, and transverse myelitis, an inflammatory condition in the spinal cord. the origin of these effects lies in pre-existing anti-ad immunity. most adults have been infected by multiple ad types and have persistent ad infections. together, this promotes long-lived anti-ad b cell and t cell responses, include regulatory t cells (t reg s) that can dampen the t cell responses. when injected with a bolus of ad antigens (the vaccine), the response includes re-activation of anti-ad effector memory t cells (t em s), which return via homing receptors to the mucosal environments-where most ad infections occur-and increased production of antibodies. why are mucosal homing anti-ad t em s important? some pathogens, like hiv, infect activated t cells in the mucosa. if you live in an environment where hiv acquisition is a risk, then the last thing you want to do is provide hiv with an easy target. increased hiv spread will fall below the radar of vaccine safety criteria because it is typically not included as an endpoint measurement. an ideal vaccine should provide rapid, multifaceted, long-term protection. few can argue with the preclinical data that demonstrate that ad-based vaccines generate rapid, antigen-targeted immune response in mice, rabbits, hamsters, and monkeys. assays in mice raised in a pathogen-free environment have suggested that ad-based vaccines are fabulously efficient at generating rapid b cell and t cell responses. while most readily admit that data in mice are suggestive and vaccines need to be trialed in hosts with a complex immunological history, clinical trials are being launched with mouse data. of course, preclinical trials in monkeys have more credibility because each subject has its own genetic differences and immunological history. many monkeys will host their own ad types. however, the number of monkeys used in preclinical studies is too small to provide robust safety evaluation. moreover, i cannot recall preclinical vaccine studies that included a challenge step months post-vaccination. yet, such data would be critical because sars-cov- will not disappear within the foreseeable future. hence, efficacy is not about whether a vaccine can prevent infections for months but whether it will protect us for - months (although this caveat is not limited to ad-based vaccines). most data suggest that the immune response to coronavirus is transient (< months). with regard to covid- , the more severe the clinical complications, the greater the immune response against sar-cov- , as is the case for most viral infections. therefore, asymptomatic/mild covid- would induce a lower immune response that would disappear within weeks. fold this onto the kinetics and efficacy of an ad-based vaccine, and the law of parsimony suggests that we need long-term antigen expression in a favorable environment to generate long-term protection. yet, in % our anti-ad t em s will target vaccine-infected cells. is it possible that ad-specific t reg s could prevent lysis of vaccine-infected cells and allow long-term expression of sars-cov- antigens? this would be a welcome surprise. follow-up studies from hiv and ebola ad-based vaccines suggest that a -dose vaccine regimen can induce immune hallmarks of protection for up years. but the idea of doses/person for billions of people during the first round of vaccinations-perhaps followed by booster shots every year-is difficult to imagine. while at least one phase trial suggested that a -shot ad-based vaccine could be sufficient, it is my bet that this will be transient protection in most adults and particularly ephemeral protection in seniors. although ad-based vaccines could be used alone by including type switching, the inclusion of other vaccine platforms (other viral vectors, proteins/peptides, nucleic acid-mediated expression, virus-like particles, inactivated or attenuated coronaviruses, etc.) may reduce the limitations of each platform and increase the breadth of the immune response. looking forward, another key question is whether we must look beyond the th century mentality with the aim of generating vaccines that prevent covid- as opposed to sars-cov- spread, but that is a subject for a future editorial. eric j. kremer emergence and re-emergence of respiratory adenoviruses in the united states a review of years of human adenovirus seroprevalence recombinant adenovirus type hiv gag/pol/nef vaccine in south africa: unblinded, long-term follow-up of the phase b hvtn /phambili study lessons for covid- immunity from other coronavirus infections key: cord- -b tj x authors: giersing, birgitte k.; vekemans, johan; nava, samantha; kaslow, david c.; moorthy, vasee title: report from the world health organization’s third product development for vaccines advisory committee (pdvac) meeting, geneva, – th june date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: b tj x abstract the third meeting of who’s product development for vaccines advisory committee (pdvac) was held in june , with a remit to revisit the pathogen areas for which significant progress has occurred since recommendations from the meeting, as well as to consider new advances in the development of vaccines against other pathogens. since the previous meeting, significant progress has been made with regulatory approvals of the first malaria and dengue vaccines, and the first phase iii trials of a respiratory syncytial virus (rsv) vaccine candidate has started in the elderly and pregnant women. in addition, pdvac has also supported vaccine development efforts against important emerging pathogens, including middle eastern coronavirus (mers cov) and zika virus. trials of hiv and tuberculosis vaccine candidates are steadily progressing towards pivotal data points, and the leading norovirus vaccine candidate has entered a phase iib efficacy study. who’s immunization, vaccine and biologicals (ivb) department is actively working in several pathogen areas on the recommendation of pdvac, as well as continuing horizon scanning for advances in the development of vaccines that may benefit low and middle income countries (lmics), such as the recent licensure of the enterovirus (ev ) vaccine in china. following on from discussions with who’s strategic advisory group of experts (sage) on immunization, pdvac will also look beyond licensure and consider data needs for vaccine recommendation and implementation to reduce the delay between vaccine approval and vaccine impact. who's pdvac was established by the department of immunization, vaccines and biologicals (ivb) in , following a review of who's process for strategic priority setting for vaccines. the need for a group to advise who specifically on vaccine product development was highlighted, to accelerate vaccine availability and ensure accessibility of vaccines to low and middle income countries (lmics). pdvac's remit is to advise on the product development strategy of vaccine candidates at phase ii of clinical evaluation or earlier, and to report its proceedings to the who's principal committee on immunization policy recommendations: the strategic advisory group of experts on immunization (sage). the pdvac committee has a critical role in assessing the evolving vaccine development landscape and in helping to define where and how who can be most impactful, according to three criteria: likelihood of a product emerging from the pipeline, as defined by probability of technical and regulatory success, and the extent of awareness, activity and investment in a given area, a clear role for who with perceived added value for engagement in the pathogen area. typically, who engages in a pathogen area by working with a broad set of key vaccine development stakeholders to develop consensus on pivotal clinical trial design, vaccine roadmaps, or guidance documents on desired vaccine properties, referred to as preferred product characteristics (ppcs). ppcs define who preferences for the properties of vaccines to be used in lmics that are - years from licensure, and inform target product profiles in use by manufacturers and funders for vaccines. pdvac also encourages developers to be aware of the process and requirements for who prequalification (pq). who prequalification is a service to unicef and other un agencies that purchase vaccines once they have been licensed, to determine the acceptability, in principle, of vaccines from different sources for supply to these agencies. it aims to ensure that diagnostics, medicines, vaccines and immunizationrelated equipment and devices for high burden diseases meet global standards of quality, safety and efficacy, and are appropriate for use in lmics contexts in order to optimize the potential benefit of these interventions [ ] . the third pdvac meeting was held in geneva from - th june . dr. jean-marie okwo-bele, director of ivb, opened proceedings with a synopsis of the significant milestones in vaccine development in the nine months since the previous meeting in september : the first dengue and malaria vaccines have been licensed or achieved the equivalent of licensure, respectively, the first rsv vaccine candidate has entered phase iii studies in the elderly and pregnant women, the most advanced hiv vaccine candidate has met its endpoints in the interim analysis of a phase ii study, and preparations to commence an efficacy study are underway, who convened the mers-coronavirus r&d community, and a phase i clinical study is now underway (nct ), ebola virus vaccines are under review and have progressed to the point of consideration for licensure in record time, there are co-ordinated efforts to develop a zika virus vaccine as expeditiously as possible. a pdvac working group has overseen the development of a zika virus vaccine target product profile (tpp), and developed regulatory considerations towards phase i and emergency use authorization. in addition to these significant advances in vaccine development, the uk government published in may the report on 'tackling drug-resistant infections globally' that it commissioned in collaboration with the welcome trust [ ] . the report highlights the urgent need to reduce reliance on currently available antimicrobials, without which today's , deaths per year from drug resistant microbes is forecasted to increase to million, by . the cost in terms of lost global production due to infections that are not controllable due to antimicrobial resistance (amr) is estimated to be $ trillion by if no action is taken [ ] . the development of vaccines against pathogens that are currently controlled by antimicrobials has become an imperative, as they have the potential to reduce the prevalence and spread of drug resis-tance, as well as to reduce the use of antimicrobials more broadly [ ] . the decade for vaccines' global vaccine action plan (gvap) mid-term review, required an assessment of progress against objectives since its inception in , and strategic planning to achieve the stated targets within the remaining years. part of pdvac's remit is to review the vaccine development pipeline and consider the priority activities for ivb, within this context. during the remaining timeframe of gvap, a number of vaccines could reach licensure, and who needs to ensure early engagement with policy makers regarding potential vaccine implementation, as well as alignment with gavi's vaccine investment strategy. to facilitate information sharing, and tracking of progress within the global vaccine development community, the who has established and maintains an online 'vaccine pipeline tracker' in which information regarding all current clinical studies in several different pathogen areas can be found [ ] . in addition, landscape analyses for pathogens from the meeting have been collated within a special issue of the journal 'vaccine' and all are available through open access [ ] . these documents are authored by independent subject matter experts and review the status of vaccine candidate development, as well as assessing possible pathways to regulatory approval. pdvac reports progress on the global vaccine development pipeline to who's strategic advisory group of experts (sage) on immunization. at the meeting in april , advances in the development of interventions (vaccines and monoclonal antibodies) for respiratory syncytial virus (rsv) were presented for information. the reports from the october and april sage meetings are available online [ , ] . much of the discussion focused on the need to better understand the key factors for early for implementation, as well as safety and efficacy data to support the assessment of a vaccine for policy recommendation. as emerging vaccines are likely to require new vaccination platforms, such as maternal immunization, or visits outside of the current vaccination schedule, such as for the recently licensed malaria vaccine rts,s, cost-effectiveness data informing their optimal use and potential impact must be generated in line with conventional clinical data required for regulatory approval, to minimise the delay between vaccine licensure and uptake [ ] . the goals of this third pdvac meeting were to revisit the pathogen areas where there has been significant progress to report since recommendations from the meeting, as well as to: review status of vaccine development in new pathogen areas where there has been significant vaccine development progress, or where there is significant disease burden but r&d has stalled, refine the workplan and strategic directions for ivb in specific pathogen areas, identify cross-cutting issues that accelerate vaccine development or prepare for policy decisions, where appropriate, consider how to better align pdvac's vaccine development activities and strategies with other areas of research, to inform the vaccine development community regarding steps to be considered beyond vaccine licensure, and who processes for vaccine policy recommendation. the gvap is a -year strategic framework derived from the decade of vaccines collaboration [ ] to prevent millions of deaths by through more equitable access to existing vaccines for people in all communities. within this framework is a specific objective that supports research and development of innovations that will maximise the benefits of immunization, with indicators for progress towards development of hiv, malaria, tuberculosis and influenza vaccines. the gvap has just completed its midterm review stage, and following the recommendation from sage [ ] , the gvap assessment will highlight advances made in these areas. these four pathogen areas are standing agenda items for discussion at pdvac. in , mycobacterium tuberculosis (mtb) killed . million people ( . million of whom were co-infected with hiv) and is now the world's most deadly infectious disease [ ] . approximately , cases/annum are multi-drug resistant (mdr) or extensively drug resistant (xdr) and some strains are untreatable. in , six million new cases of mtb were reported to who, fewer than two-thirds ( %) of the . million people estimated to have contracted the disease. this means that % of new cases were not detected or reported. a vaccine is imperative to achieving the end tb goals [ ] , particularly through reaching the population who are undiagnosed and continue to transmit disease. as such, the tb vaccine development community has turned its focus to the development of vaccines targeted to adolescents and adults as the age-groups with highest burden of active disease and the source of mtb transmission. modelling studies suggest that prevention of pulmonary disease in this population from primary infection and from reinfection or reactivation of existing infections is the most effective strategy to prevent mtb infection and disease in infants and children [ ] . the most advanced vaccine candidates are targeting this indication, including current neonatal bcg replacement candidate vaccines that are also undergoing evaluation as a booster in later life. several of these candidates are in proof-of-concept clinical studies and are approaching key endpoints through prevention of infection or disease, or prevention of disease due to reinfection in this these target populations in the next - months [ ] . with this in mind, pdvac recommended that who prioritize and facilitate consensus building with respect to the development of strategic goal(s) and ppc(s) for vaccines targeted to adolescents and adults, in the first instance. there are several candidates and platforms in the pipeline that target this goal in this population, as well as other important target populations [ ] . pdvac acknowledged the significant need for development for these vaccines in parallel, as well as continued efforts to understand the biological mechanism of disease to support the immunological rationalization of candidates. the pox-protein public private partnership (p ) consisting of sanofi, glaxosmithkline (gsk), bill & melinda gates foundation, the us military hiv research program (mhrp), and the hiv vaccine trials network (hvtn) have been collaborating with the us national institutes of allergy and infectious disease (niaid) to optimize and assess the efficacy of the alvac/heterologous prime boost approach, following the demonstration of partial efficacy in the rv trial in thailand [ ] . the interim data from a phase i/ ii study (hvtn ) met its humoral and cellular immunological 'go' criteria, exceeding the rv responses against sub-saharan clade c antigens. extrapolation of these responses to those observed with rv , suggest that the optimised vaccine could offer at least % protection following a month booster. based on these data, a randomised placebo controlled phase iib/iii efficacy trial (hvtn ) enrolling subjects was initiated in late in south africa, and will evaluate alvac (clade c) prime/ bivalent recombinant gp protein with mf adjuvant as a heterologous boost, as well as the effect of a booster at months [ ] . futility analyses will be undertaken early in the year followup period. correlate of protection studies and assessment of crossreactivity to other regional clades are included in the study design. discussions with the south african medicines control council (mcc) are ongoing, and licensure in south africa could be as early as . other vaccine candidates are in development, including janssen's heterologous prime boost approach with ad /gp , currently undergoing dose regimen selection in phase i/iia trials. antibody-mediated prevention using broadly neutralizing, potent monoclonal antibody (bnmabs) approaches are also undergoing phase i/iia clinical evaluation. the niaid/vaccine research centre's vrc broadly neutralising mab is the most advanced candidate which has been shown to neutralise cd binding of % of viral isolates. hvtn /hptn and hvtn /hptn are phase iib studies to evaluate the efficacy of vrc in reducing acquisition of hiv- infection in high risk populations in the americas and sub-saharan africa, and started enrolment in . if shown to be effective, administration of vrc could be positioned as a long-acting supplement to increase effectiveness of anti-retrovirals. pdvac commended the advances in hiv vaccine development, and requested to be kept informed about progress with hvtn . currently, there are no known intentions for global studies with the p candidate vaccine, or to seek who prequalification. pdvac encourages the p partners and the south african hiv vaccine development community to keep who fully informed about progress with the trial. concerns were expressed regarding the lack of follow-on studies in thailand, given that the initial landmark rv trial was performed there. despite the substantial reduction over the last years (over % for global malaria mortality in children aged < years), mainly due to greater investments in malaria control, the who estimates there were million malaria cases in , % of which were in africa. of the , people who died from the disease in , % reside in africa [ ] . given the increase in multi-drug and insecticide resistance, there remains an urgent need for a vaccine to combat malaria. as reported in the pdvac meeting summary [ ] , the european medicine's agency (ema) provided a positive scientific opinion, indicating a favorable assessment of the risk-benefit balance of rts,s/as from a regulatory perspective. in october , two advisory bodies to who, namely sage and the malaria policy advisory committee (mpac), recommended pilot implementation studies of the -dose schedule of the rts,s/as vaccine in - distinct epidemiological settings in sub-saharan africa, at sub-national level, covering moderate-to-high transmission settings, with three doses administered to children between and months of age, followed by a fourth dose - months later. the intent of these pilot studies is to assess: the feasibility of providing all four doses of rts,s to the target age group through existing health services; the impact of rts,s on child mortality; whether there are any safety issues, particularly evidence of any causal relationships between rts,s administration and either meningitis or cerebral malaria (both signalled in the phase iii trials), whether introduction of the vaccine impacts positively or negatively on existing country immunization programs and on the use of currently recommended malaria control measures. in , the malaria vaccine technology roadmap was updated to include licensure of vaccines targeting plasmodium falciparum and plasmodium vivax by , with protective efficacy of at least % against clinical malaria, and that reduce transmission of the parasite and thereby substantially reduce the incidence of human malaria parasite infection [ ] . the vaccine candidate pipeline is robust, and includes novel antigens and platforms [ ] . second generation vaccines are expected to provide higher protection than rts,s in the longer term. optimised tools are needed to measure incremental improvements and predict potential cost effectiveness of new candidates. the development of controlled human malaria infection (chmi) models, efforts to harmonize elements of clinical trial design and standardization of various assays continue. pdvac stressed the importance of the development of nd generation malaria vaccines in parallel to the pilot implementation program for rts,s, and proposed that the current version of the vaccine roadmap be updated, potentially in , in light of the rts,s pilot implementation. in , pdvac noted that development of universal influenza vaccines will be challenging and protracted, particularly due to the lack of a regulatory pathway for novel antigens that operate through induction of t-cell immunity. rather, pdvac recommended that there be a focus on the definition of, and the collection of data to support implementation of 'improved' seasonal flu vaccines that would offer more immediate impact in lmics. pdvac advised who to develop strategic public health goals and ppcs for improved seasonal influenza vaccines, and to provide guidance on data requirements that would be needed to establish improved performance of such vaccines. a working group has been established, and has proposed a draft statement of unmet public health need: 'safe and well-tolerated influenza vaccines that are effective at preventing severe influenza illness, that provide protection beyond a single year, and that are programmatically suitable for use, are needed for low-and middleincome countries.' draft -and -year strategic goals for development of influenza vaccines that induce broader and more durable protection against severe illness caused by influenza a strains have been developed. these strategic goals and the draft ppc for nextgeneration influenza vaccines were presented at the upcoming eighth who meeting on development of influenza vaccines [ ] . pdvac reaffirmed the value of ppcs based on the two different approaches. there is a public health need to develop improved performance of currently available seasonal vaccines to offer protection over multiple seasons, and against drifted strains, with a view to generating shorter timelines to achieving availability and access in lmics. as part of this effort, it will be necessary to define the criteria needed to demonstrate clinical benefit, and additional data requirements to support policy recommendations. efforts to develop 'universal' vaccines that target conserved antigens, or conserved components of antigens, should continue in parallel, with a focus on identifying correlates of protection to support a regulatory pathway for this novel class of vaccines. diarrheal disease remains the second leading cause of death in children under years of age. although mortality has declined over the past four decades, morbidity has not declined significantly, despite improvements in water and sanitation and benefits from oral rehydration therapy. there are nearly . billion cases of diarrheal disease every year, many with acute and chronic effects such as growth stunting and cognitive impairment. these long term sequelae significantly impact quality of life and economic potential, and are estimated to affect one-fifth of children globally. in , pdvac recommended that who expand its remit to include support for enteric vaccine development, particularly against enterotoxigenic escherichia coli (etec) and shigella. one of the main objectives of the planned who engagement in this area will be to ensure that the design of the phase iii efficacy study, including definition of primary/secondary endpoints and long-term follow up, and the data generated, will be relevant to support a policy recommendation from sage. another key objective is to develop a who preferred product characteristics document which outlines who preferences, including considerations for development towards a potential combined vaccine. several vaccines are in development, with two etec candidates and seven shigella candidates currently in clinical studies. for etec, the most advanced vaccine is etvax adjuvanted with dmlt, which is being developed for both a pediatric and traveller's indication. a phase i/ii dose escalation, age de-escalation study in children is currently ongoing in bangladesh, with intent to further age deescalate into week-old infants in late . in parallel, a phase iib study in travellers is planned to begin in . based on an encouraging phase iib immunization and challenge study and additional positive protection studies in non-human primates (nhps), an adhesin-based subunit etec vaccine (fta) is moving forward with an accelerated clinical program designed to move a complete multi-valent vaccine into descending age field trials in . the most advanced shigella candidate is trivalent shigella killed whole cell (tswc) composed of formalin-inactivated s. flexneri a, s. flexneri a, and s. sonnei, expected to offer coverage across about % of isolates. a phase i study has been completed and a challenge trial with s. flexneri a prototype will begin in , followed by a study that will assess co-administration with etvax. both etvax and tswc are being developed for oral administration. other promising shigella vaccines in early stage clinical testing include two live attenuated vaccines, wrrs and shigetec. wrrs is in a descending age study in bangladesh, while shigetec, which is a combination shigella-etec combination vaccine, will begin a phase i study in early . three subunit approaches for shigella are also in phase i/ii studies; the prototype s. flexneri a bioconjugate vaccine (flexyn a), invaplex and the generalized module for membrane antigens (gmma). one of the critical strategic issues is whether to prioritize the licensure and approval of an etec vaccine, or to focus on the development of a combination with shigella that will likely delay the timeline to vaccine availability. epidemiologic data suggest that both intra-and inter-country disease heterogeneity is likely to exist and this may drive vaccine preferences, and presentation optimization. these data are critical to inform decision-making by country policymakers. for this reason, development of who derived preferred product characteristics for etec and shigella vaccines, alone and in combination is needed. in april , plos released a collection on 'the global burden of norovirus & prospects for vaccine development' [ ] , which includes the most current estimates on global norovirus disease burden of over , deaths in low resource countries, and a global economic burden of more than $ billion [ ] . recent molecular analyses of samples from the community based longitudinal birth cohort mal-ed study suggest that norovirus is the most common diarrheal pathogen in the first year of life, and the second most common in the second year of life. there are vaccine candidates in development, including three strategies to develop a combination vaccine against other enteric pathogens. however, only one candidate, which is composed of two vlps based on the gi.i and gii. norovirus genotypes, has entered clinical studies, a phase iib study began recently [ ] . the advent of cell culture methods for norovirus will facilitate many advancements, including the optimization of a neutralization assay and enable the assessment of antisera against this vaccine to block binding of a diverse genotypes. in addition, in response to the pdvac recommendation to consider incorporating norovirus surveillance within the who global rotavirus surveillance network, a survey of the capability and capacity at representative global sites has been performed to support a pilot study proposal. the recently published epidemiology and burden of disease data indicate that norovirus fulfils the pdvac criterion of unmet public health for a vaccine in lmics. however, the ability of the candidates in the pipeline to offer protection over the range of circulating and emerging viral genotypes, and therefore the duration of protection of these vaccines, is currently unknown. it is conceivable that the vaccine will need to be periodically re-formulated, to include emerging genotypes. in addition to infants as a priority target population, adults and particularly the elderly are at risk, requiring the potential need for two vaccine formulations and/or presentations. fortunately, at the current time, development of a norovirus vaccine that may offer efficacy in the context of low and middle income countries is proceeding with investment from the private sector, however an assessment of vaccine programmatic suitability and applicability to prequalification is needed, prior to phase iii trials to ensure the vaccine is appropriate for use in lmics, assuming it is demonstrated to offer coverage over circulating genotypes within lmics. rotavirus is the leading cause of severe diarrhea among all children below years of age worldwide, causing - % of severe diarrheal hospitalisations, and is associated with significant mortality, with the latest mortality estimates at , deaths in [ ] . the introduction of the live-attenuated oral rotavirus vaccines, rotateq and rotarix, in has had significant direct and indirect impact in countries where they are in use, including saving lives and reducing hospitalizations. however, in gavieligible and lmic countries in asia and africa the vaccine effectiveness is lower, with protective efficacy observed from to % against severe rotavirus diarrhea over the first year of life. waning of protection has also been observed in these settings, with lower protection rates ( - %) in the second year of life. in comparison, in high-income countries protection is higher ( - %) and persists into the second year of life. thus, despite the enormous success of the live oral rotavirus vaccines, several challenges and issues remain such as the lower protection in gavi-eligible and lmic countries in africa and asia, together with the high cost of available vaccines. despite an overall acceptable safety profile, the intussusception rate seems to be slightly increased by vaccination (occurrence - / oral rotavirus vaccine recipients) in high income countries. several new oral, live-attenuated vaccines, composed of alternative strains, are in mid-to late-stage clinical development. the current who guidance document for the quality, safety and efficacy of oral live attenuated rotavirus vaccines [ ] would be applicable for these next generation oral, live-attenuated vaccines. of these new oral rotavirus vaccines, rotavac c (developed by bbil) is the only vaccine currently licensed for use in children, having been approved for use in india in . this vaccine is available on the private market in india and staged roll out in public health system is planned in four states in india. another live rotavirus vaccine is being evaluated in a randomised placebo controlled trials in india (nct ) and in niger (nct ). efforts are underway to develop non-replicating rotavirus vaccines (nrrv) as second generation rotavirus vaccines, which may avoid the risk of intusseseption. the most advanced candidate is p -vp ⁄ , a trivalent truncated vp ⁄ of rotavirus genotypes p [ ] , p [ ] and p [ ] , currently in phase ii clinical testing with a parenteral route of administration (nct ). for both nrrvs and additional oral, live-attenuated vaccines in development, pdvac encouraged the rationalization of target product profiles for these new candidates, to clearly articulate the distinguishing/advantageous features over the existing vaccines, i.e. cost, safety, efficacy in lmic, stability, breath of protection, etc. the potential for any of these vaccine candidates to be included in combination with other emerging enteric vaccines will clearly be advantageous and should be encouraged and explorations of combination with ipv could be considered. clostridium difficile is the leading cause of healthcare-associated diarrhoeal disease in the high-income countries, and is strongly associated with increasing age and frailty, immunodeficiency and in particular, modification of the normal flora through antibiotic use. the results of infection range from asymptomatic carriage through mild infection to severe diarrheal disease, with complications including pseudomembraneous colitis and toxic megacolon. in the us alone, it is believed to have caused approximately . million infections and , deaths in [ ] . current interventions include antibiotic treatments, but their use can trigger relapse on withdrawal. data on the burden of disease in lmics is lacking, however hospital based studies in india, thailand and south korea suggest that the c. difficile infection is widespread, and global (douce, manuscript in preparation). there is a correlation between toxin neutralising antibody in human serum and disease protection; antibodies against toxin a are associated with protection against acute diarrhea, whilst immune responses to toxin b appear to be effective against severe disease and relapse. toxin-mediated disease is recapitulated in the syrian golden hamster, which is the standard preclinical model for demonstration of proof of concept. currently there are three vaccines in clinical development. a toxoid vaccine candidate (containing toxins a and b) recently completed a phase ii study in healthy adults and demonstrated induction of high levels of neutralizing antibodies [ ] and a phase iii study has been initiated. a genetically modified, detoxified whole cell vaccine has also completed phase ii, alhough results have not yet been reported. in phase i, the vaccine was shown to be safe and induced toxin-specific neutralizing antibodies that were sustained for months [ ] . the third candidate is an adjuvanted recombinant protein encoding binding domains of both toxins, and the results of a phase i trial has been reported [ ] , and a phase ii study has been completed. passive immunity by administration of a monoclonal antibody is also in phase iii evaluation (nct and nct ). pdvac agreed that the role for who in facilitating c. difficile vaccine development is not clear given the lack of data regarding the disease burden in lmics. however, it would be useful to understand the potential effectiveness of a vaccine in low resource contexts, and pdvac raised the possibility of testing existing samples from the gems and mal-ed studies for the presence of c. difficile. in addition, it would be helpful to assess the impact that these vaccines many have on reducing the use, and cost of antibiotics, and to consider this in the value proposition for vaccine decision-making. h. pylori is a highly motile, gram-negative bacterium that infects the mucus layer lining the stomach. infection typically occurs in childhood, although symptoms and clinical disease develop in only a minority of infected individuals during their lifetime. h. pylori is associated with gastritis, which causes several pathologies including gastric peptic and duodenal ulcer disease. most significantly, long term infection can result in gastric adenocarcinoma (ga) in later years of life; - % of ga cases are due to h. pylori infection. ga is the rd leading cause of death due to cancer, globally ( , deaths in , . % of all cancers) [ ] . the global prevalence of h. pylori is believed to be approximately % with the highest mortality rates in east asia and eastern europe. the route of transmission is poorly characterised but the oraloral route appears to be a common mechanism, as well as vertical transmission from mother to child. if untreated, most h. pylori infections are sustained for life, and % of those infected are thought to develop an associated pathology. if diagnosed, h. pylori infections are currently treatable with combination antimicrobial therapies. however antibiotic resistance is increasing, with % of patients in some countries currently failing first treatment and % failing two rounds of therapy. antimicrobial treatment offers no protection against reinfection. the choice of indication for an h. pylori vaccine is challenging: a prophylactic vaccine would likely need to be given to children in the first few years of life (to reach the maximum number of the target group while uninfected) but would need to offer long term protection to demonstrate clinical benefit against ga. an effective therapeutic vaccine however could be given at almost any age and would ideally be given by the th decade of life, prior to the peak of ga development which typically occurs from years of age. the most advanced candidate is a urease toxin fusion approach and has completed phase iii trials in children, in china, and demonstrated . % efficacy against natural acquisition of infection [ ] . however, protection appeared to wane to % over - years and next steps for this vaccine are not clear. several other candidates are in preclinical development with one close to phase i studies. pdvac concluded that the burden of h. pylori is significant, and that a vaccine that is able to protect against infection, with sufficiently long duration of protection, would be of public health benefit. therapeutic candidates are currently too upstream in development for there to be a role for pdvac. maternal immunization is increasingly considered as a strategy to prevent maternal and/or neonatal disease. this approach has been proven to protect against maternal and neonatal tetanus and has been in place for decades. who recommends influenza and pertussis vaccination of pregnant women to prevent disease in mothers and newborns, respectively. however, for the first time there are now vaccines in development, specifically indicated for immunization of pregnant women as the target population. respiratory syncytial virus vaccines are most advanced in this area followed by group b streptococcal vaccines. since the pdvac meeting, a special journal issue dedicated to the issues regarding the maternal immunization vaccination strategy has been published and a great deal of work is underway to strengthen the maternal immunization platform [ ] . due to the advanced stage of rsv vaccine and monoclonal antibody development, rsv was presented to sage for information in april . rsv causes . million episodes of lower respiratory infection (lri) annually in children and approximately , deaths, % of which are in lmics [ ] . recently updated estimates for rsv acute and severe lri (community based and hospitalized) disease and deaths will be published by the rsv global epidemiology network (rsv-gen) in early . in addition, the pneumonia etiology research for child health (perch) study will present and publish results on the etiology of severe and very severe pneumonia in hospitalized infants and children in sites in africa and asia. preliminary data analyses indicate that rsv was the leading pathogen in infants with severe pneumonia in this study. there are four rsv intervention strategies currently in development: ( ) maternal immunization to enable passive transfer of maternal antibodies to the foetus in utero, ( ) birth or early infant passive immunization with a long-acting monoclonal antibody, ( ) active pediatric immunization and ( ) vaccination of the elderly. the most advanced maternal immunization candidate begun phase iii efficacy testing in late following the demonstration of induction of palivizumab-competing antibodies (measured by elisa) in women of childbearing age (pmid: ) and pregnant women. this efficacy trial has a group sequential design and will enroll - participants in a randomised placebo controlled trial across multiple sites in both the northern and southern hemispheres, and is expected to take - years to complete. monoclonal antibody development for the prevention of rsv in pediatrics is the next most advanced, with an extended half-life candidate (medi ) that has been shown to be more potent in vitro than the currently licensed palivizumab. one dose may offer protection for up to months. a phase iib clinical study in infants born at - week gestation is planned, and the fda recently granted fast-track designation for this product. since the palivizumab patent recently expired, who in collaboration with the university of utrecht will develop a 'biosimilar' of palivizumab and reduce costs for lmic markets through high yield production and a novel financing plan [ ] . the estimated price is $us per child for the full month dose series and the first market authorization is expected in late . pediatric rsv vaccine candidates are the least advanced, however two adenovirus-based approaches have entered the clinic since the last pdvac meeting. a chimp adenovirus (chad) candidate is currently in phase i testing in adults, to be followed by age de-escalation into seropositive, and ultimately seronegative infants. ad is also being evaluated as a heterologous primeboost regimen, currently in phase i testing in adults. a number of pediatric vaccine candidates developed by the laboratory of infectious diseases, nih are in phase i trials in infants and children. of note, a vaccine containing a deletion of the m - gene showed evidence of diminished replication, enhanced immunogenicity, and asymptomatic 'boosting' (anamnestic response) following naturally acquired rsv infection (pmid: ). two vaccine candidates are in clinical development for the elderly with a post-fusion f-based adjuvanted nanoparticle in phase iii efficacy testing, with data expected in early . pdvac fully supported the following sage recommendations and called for who and partners to develop plans to support global policy-making for rsv maternal immunization as well as passive immunization with long-acting mab, following licensure. particular areas of emphasis include: ( ) rsv surveillance to determine seasonality and age-stratified rsv disease burden and community morbidity and mortality, especially in africa and south-east asia ( ) assessment of the long term effects of rsv interventions and the potential impact of vaccination on reducing recurrent wheeze, which, if demonstrated, would substantially increase the costeffectiveness and impact of rsv preventive interventions ( ) generation of cost-effectiveness and impact data. sage also emphasized the need for strengthening of the maternal immunization platform in collaboration with the influenza, tetanus and pertussis vaccine communities, along with preparations for potential country introductions of rsv vaccine. there is an urgent need to establish a who prequalification pathway for monoclonal antibodies, which does not currently exist. as a rsv vaccine or extended half-life monoclonal ab may become available in the next years, it will also be imperative to initiate early discussions with financing bodies, and to align with the gavi vaccine investment strategy (vis) to avoid delay in achieving the potential major public health impact of rsv immunization if recommended for use by who. globally, gbs remains the leading cause of sepsis and meningitis in young infants, with its greatest burden in the first days of life. intrapartum antibiotic prophylaxis (iap) for women at risk of transmitting gbs to their newborns has been effective in reducing the young infant gbs disease burden in many high income countries, but iap uptake is limited and difficult to implement in lmics. immunization of pregnant women with a gbs vaccine represents an alternative pathway to protecting newborns and young infants from gbs disease, through prevention of gbs colonization and transplacental antibody transfer to the fetus in utero. pdvac prioritized gbs in and encouraged who to engage on developing guidance on the development pathway for gbs vaccines, including development of a ppc guidance document and a vaccine roadmap. in april , who convened its first consultation on gbs vaccine development [ ] . the focus was on gbs maternal immunization development programs targeting lmic with the ultimate goal of reducing global newborn and young infant deaths. the major knowledge gaps about the disease burden characterization were identified. recent data suggesting that gbs is an under-reported cause of stillbirth may have profound implications on the estimate of the global public health impact of a future gbs vaccine. the relationship between gbs colonization and prematurity should also be clarified. disease surveillance in hic also suggest an important residual unmet medical need, despite implementation of iap. two major pharmaceutical companies are currently developing a multivalent polysaccharide conjugate vaccine, based on the available evidence of an association between trans-placental maternalfoetal transfer of antibodies targeting polysaccharides of the gbs envelope, acquired as a consequence of natural exposure, and a reduced risk of invasive infant disease. a vaccine incorporating five of the eleven described gbs serotypes is predicted to cover over % of the global circulating serotypes, but the risk of serotype replacement is unknown. an alternative approach is targeting surface expressed proteins, in an attempt to confer broad protection across all serotypes. epidemiological studies evaluating the role of maternal antibodies acquired following natural exposure will determine whether a protective threshold at birth can serve as an acceptable vaccine-induced correlate of protection. until additional epidemiological and immunological data are available, estimating vaccine efficacy against invasive gbs disease in neonates and young infants in a double blind placebo-controlled vaccine trial remains the gold standard for generating the evidence required to determine potential public health impact and inform policy decision-making. pdvac endorsed the consensus-based prioritization of future activities including the development of a ppc and vaccine development technology roadmap. efforts should be made to raise awareness of the burden of gbs disease and potential public health value of a gbs vaccine, particularly in countries that lack local epidemiological data. as with rsv, efforts must be made to leverage and strengthen the maternal immunization platform by alignment with other vaccines that are administered in pregnancy, including the brighton collaboration's considerations for safety monitoring through the global alignment of immunization safety assessment in pregnancy (gaia) [ ] . antimicrobial-resistant infections currently claim at least , lives each year across europe and the us alone, but amr affects many hundreds of thousands in other areas of the world [ ] . in european countries, more than % of bloodstream staphylococcus aureus infections are caused by methicillin-resistant strains (mrsa), with several of these countries seeing resistance rates closer to %. emerging resistance to treatments for other diseases, such as tb, malaria and hiv, have enormous impacts in lower-income settings, and by , the death toll due to amr infections in africa is predicted to be approx. , , per year. as mentioned above, each year almost . million cases of drugresistant tb are reported, and these are extremely costly to treat; an mdr case costs - -fold more to treat than drug a sensitive case, while an xdr case is - -fold more expensive [ ] . the who estimates that approximately $ billion per year is required to support tb care and control efforts in lmics. this is significantly more than the current investment in tb vaccine development programs. the o'neill review on antimicrobial resistance estimated that by drug-resistant infections could be claiming million lives per year and at an economic cost to the global gdp in excess of $ trillion. at the sixty-eighth world health assembly in may , a global action plan to tackle antimicrobial resistance, including antibiotic resistance, was adopted [ ] . its goal is to ensure continuity of successful treatment and prevention of infectious diseases with effective and safe medicines -including vaccines -that are quality-assured, used in a responsible way, and accessible to all who need them. the amr global action plan (gap) is based on work streams ranging from national plans, stewardship of antibiotics, encouraging r&d through developing new business plans and assessing environmental drivers. one work stream focuses on vaccines to prevent amr. the who gap workstream on vaccines to prevent amr is based on three complementary approaches: increasing the use of existing vaccines; developing vaccines against high burden diseases currently treated systematically with antibiotics; and prioritizing the development of vaccines for diseases where antibiotic resistance is significant. these three approaches, and the challenges associated with implementing them are summarised below: a). increasing use of existing vaccines: while it is logical that increasing the use of existing vaccines would reduce infections and result in reduced use of antibiotics, it is not always clear which vaccines, in which populations, would have the greatest impact on reducing antibiotic use and potentially amr, and should therefore be prioritized. for example, it has been shown that the use of pcv- in children results in a roughly % reduction in antibiotic-resistant strains of s. pneumonia, and the use of pcv- reduces outpatient antibiotic purchase, leading to the suggestion that global pediatric coverage with pcv could prevent million days of antibiotic use in children annually. however, it is thought that the bulk of pneumonia, and antibiotic use for s. pneumonia infections, is in older adults. this would suggest that demonstrating efficacy of pneumonia vaccines to reduce antibiotic use in older adults and an expanded use of these vaccines in that population group where very few countries have a vaccination policy may have a substantial impact on reducing amr. other existing vaccines which could impact antibiotic use include pertussis, haemophilus influenza, neisseria meningitides, typhoid, as well as influenza which, although not directly susceptible to antibiotic treatment, does result in bacterial super-infections and accounts for up to % of excess (winter-related) antibiotic prescriptions in some countries. in order for a rational evidence-based policy on expanding the use of existing vaccines a prioritization exercise needs to be performed, taking into account the disease burden in different populations, the antibiotic use associated with that disease burden, and an evaluation of how many days of antibiotic use would be avoided with each dose of vaccine administered. this exercise is particularly challenging since in most of the world antibiotics are taken in response to a symptom, rather than an identified infection. this means that, for example, preventing salmonella typhi-induced infection with vaccines may have minimal impact on antibiotic use for severe diarrhea. the prioritization exercise therefore needs to consider not only the disease burden, but the symptom burden and the proportion of that burden due to the vaccine-preventable infection. b). developing vaccines for diseases that are consistently treated with antibiotics, where amr is not currently an issue, but where the vaccine could reduce antibiotic use. one such example is group a streptococcus (gas). while gas is not directly associated with antibiotic resistance (there is little evidence of resistance to date) it has a high disease burden and is a source of extensive antibiotic use. in addition, it is thought that vaccine development is feasible. however to date there has been no significant effort from industry, possibly because of the weak market assessment since it can be treated with antibiotics, and such treatment is cheap. however the indirect costs from such antibiotic use, increased environmental exposure to antibiotics and expansion of amr have not been considered. taking these costs into account may contribute to the value proposition for developing and using such a vaccine. other such candidates could include group b streptococcus, and m. catarhalis and non-typeable haemophilus influenza, both responsible for otitis media which is another source of significant antibiotic prescription. while including potential impact on reduction of antibiotic use, prioritization of these candidate vaccines also needs to consider technical feasibility, whether the antibiotic use is appropriate, and whether alternative non-antibiotic approaches may make vaccine use less attractive. for example: while there are over million cases of otitis media per year in under - year olds, for which antibiotic treatment is usually prescribed which could justify development of a vaccine, most otitis media resolves and new guidelines recommend limiting antibiotic treatment. another example is urinary tract infections which are frequent in elderly patients and a cause of significant antibiotic use, yet there is little supporting evidence that these infections could be effectively reduced by vaccination. a prioritization exercise is therefore required for vaccines that are considered technically feasible, takes into account the potential amr impact, and therefore could contribute to the cost effectiveness of the vaccine if they were developed. to achieve this, the evaluation of impact on amr is recommended to be included in the review of vaccine conducted by pdvac. c). the third and most challenging approach is developing vaccines against pathogens that are frequently antibiotic resistant and becoming increasingly difficult to treat, the so-called eskape pathogens [ ] . this list includes staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumonia, as well as clostridium difficile and tuberculosis. there are numerous challenges in this approach. the first is that although infection with these may result in significant morbidity, the current global disease burden of many of these infections remains relatively low so prophylactic vaccination of the entire population would not be cost-effective. tuberculosis is however an example with significant disease burden and rapidly expanding multi-drug resistance. secondly many of these infections are associated with ageing, where immune decline may make immune interventions poorly effective, or are associated with penetrative medical interventions where the time available to induce a protective immune response may be insufficient. and finally, despite significant efforts to make effective vaccines against some of these has so far proven to be difficult. of these, tuberculosis appears to have the greatest global burden and public health impact, and tb vaccines are also the subject of extensive research. the amr gap activity in this area, to be conducted by ivr is firstly to promote tb vaccine research and development through facilitation of preferred product characteristics and the establishment of a roadmap for vaccine use, and highlighting the impact that the vaccine will have on antibiotic use and antibiotic resistance. additional activities involve monitoring the state of development of vaccines against the pathogens that are becoming antibiotic resistance, and facilitating their development. gas is a ubiquitous human pathogen that causes a broad disease spectrum, from mild to severe, the most serious of which is rheumatic heart disease (rhd). rhd affects approximately million people globally, of whom million experience heart failure and an estimated , die. gas is also a major cause of invasive disease, with a case fatality rate of - % in high income countries, and as high as % in lmics [ ] . on the milder end of the spectrum, gas causes approx. million cases of pharyngitis per year, resulting in - % of cases being treated with broad spectrum antibiotics, rather than penicillin ( % of cases), to which gas is universally susceptible. this extensive use of unnecessary and inappropriate antibiotics increases the likelihood of amr emergence against antibiotics that are used to treat a range of pathogens. previous human challenge studies, as well as preclinical animal models suggest that it is feasible to develop a vaccine against gas, and since the previous pdvac meeting, phase i studies for one candidate has been initiated in adults, and two additional candidates are expected to enter phase i studies in the next months. despite this encouraging progress, significant debate remains as to the appropriate indication and optimal clinical endpoints, and the regulatory pathway for a vaccine to prevent or reduce rhd is unclear. in addition, there is a perception that increased prescription of penicillin would be an equally as effective and a significantly more cost effective method of reducing conditions that result from gas infection. these issues are likely major stumbling blocks in incentivising investment in gas vaccine development. gas has been prioritized by pdvac previously, with a recommendation to develop a business case for both a global market, and also specifically for lmics which would focus on prevention of severe outcomes in resource poor settings. despite significant effort, it has been very difficult to engage stakeholders in this activity. on the recommendation of pdvac, who convened a consultation in december to examine the value proposition for gas vaccines, considering its potential impact across both high income and lower income settings -including the consideration of how current antibiotic treatment practices may increase amr, as well as to investigate the perceived regulatory obstacles. s. aureus is a bacterium that is found as both an asymptomatic colonizer of the skin and nares of human hosts, as well as a frequent cause of human disease. it causes a spectrum of clinical manifestations of varying severity, and is the most commonly isolated pathogen from skin and soft-tissue infections, septic arthritis, pneumonia, endovascular infections, osteomyelitis, catheter/other foreign-body infections, septicaemia, and toxic shock syndrome. methicillin-resistant s. aureus (mrsa) has been documented to be emerging at a rapid and increasing rate since the antibiotic was first introduced in , and hospital-associated mrsa (ha-mrsa) clones are now recognized to be the leading cause of nosocomial infections both in the united states and around the world, in high income as well as lmics. the emergence of communityassociated mrsa (ca-mrsa) in the past several decades is of concern, as is the emergence of highly resistant vancomycin-resistant s. aureus (vrsa). to date, active and passive immunization approaches have been based on increasing the concentration of opsonic antibodies to single surface antigens, and all have failed to demonstrate protection. antigenic variation, the multiple invasion pathways and lack of a surrogate of protection all present significant obstacles to vaccine development. following the failure of single antigen vaccine approaches, most development efforts are now focused on multiple antigens, and a number of candidates are in preclinical development. one multi antigen approach, comprised of antigens including two capsule polysaccharides, clumping factor a and a manganese transport protein, is the most advanced [ ] . current efforts are also focused on further characterizing the immunopathology and immunity of s. aureus infections to identify new antigenic targets, and developing more representative preclinical models in which opsonising and/or neutralising immune responses are measured. to date, none of the vaccine candidates in development have contemplated target populations or indications that are prevalent in lmics. focus has been on development of a vaccine that will protect against life-threatening s. aureus infections in high income countries, but it is hoped that such a vaccine would also protect against all s. aureus infections including more commonly encountered skin and soft tissue infections, and therefore be applicable in lmic contexts. since the pdvac meeting, a new global health sector strategy on sexually transmitted infections has been developed for - and adopted by who member states at the th world health assembly. within this strategic framework, sti vaccine development was highlighted as key need for future sti control [ ] . in addition, the global roadmap for vaccines against stis has been updated and included in the who special issue on pipeline vaccines published in vaccine [ ] . currently, the only sti vaccine candidates that are undergoing or approaching clinical development are against herpes simplex virus (hsv) and chlamydia trachomatis, and as such discussion was limited to these pathogens. hsv is the leading cause of genital ulcer disease, and a particular concern for lmics as it increases both acquisition and transmission of hiv infection. hsv type and type disease burden estimates were recently updated [ , ] , and it is estimated more than half a billion people live with genital hsv infection, worldwide. pdvac previously recommended that improved global estimates of neonatal herpes burden be generated, and assessment of available data has recently been completed with preliminary estimates of > , new cases globally, an incidence rate of approx. / , births, which is concerning because of a case fatality rate of % (looker, submitted) . the incidence is likely to be under-estimated in lmics where hsv infection rates are highest and poor healthcare infrastructure means that neonatal herpes cases are likely to be undetected, but primary data are lacking. ongoing evaluation of hsv infection as part of the child health and mortality prevention surveillance (champs) network will help to address this burden gap. at the meeting, the advance of therapeutic vaccine candidates for hsv- was highlighted, and the role of these types of vaccines in modulating the interaction between hsv and hiv acquisition was discussed as an important consideration for these vaccines in lmics. in consideration of this, and with who support, a systematic review/meta-analysis of hsv- and risk of hiv acquisition including studies will inform modelling of the potential impact of an hsv- vaccine on hiv incidence, and is expected to be published in late . a review of biological mechanisms of hsv-hiv interaction and implications for vaccine development has also been drafted. the pipeline for therapeutic vaccines remains robust, with candidates in clinical development, the most advanced of which now has data demonstrating significant reductions in hsv shedding ( %) and days with genital lesions ( %) over months [ ] . in response to these positive data, niaid has formed an hsv working group to propose desired characteristics for therapeutic and prophylactic vaccines for hsv, including indication, priority target populations, clinical trial endpoints, and safety and efficacy criteria. this document could form the foundation for a who consultative process to generate a guidance document on preferred product characteristics (ppc). pdvac encouraged who to actively collaborate and support development of ppcs for hsv vaccines. chlamydia trachomatis is a gram-negative bacterium that can infect genital, ocular and lung epithelium. it includes three sets of serovars: -serovars ab, b, ba, or c -cause ocular trachoma, which can lead to blindness -serovars d-k -cause sexually transmitted infection resulting in urethritis, cervicitis, pelvic inflammatory disease (pid) (and associated infertility, ectopic pregnancy, and chronic pelvic pain), neonatal pneumonia, and neonatal conjunctivitis -serovars l , l and l -cause lymphogranuloma venereum c. trachomatis can ascend to the upper genital tract and cause pelvic inflammatory disease (pid), which can in turn lead to long-term sequelae including tubal factor infertility, ectopic pregnancy, and chronic pelvic pain. other adverse outcomes of chlamydia include preterm birth, neonatal conjunctivitis and pneumonia, and increased hiv risk. currently management is through screening programs in some high income countries that are not feasible in resource constrained settings, where most cases are likely never diagnosed. who estimates that there were million new cases of chlamydia in [ ] with most cases among adolescents and young adults. the global burden of chlamydia-associated pid, infertility and other sequelae has not been well characterised and estimates of the proportion of infertility presumed to be associated with genital infection (e.g., have a fallopian tube etiology) in africa are outdated [ ] , but are thought to be approx. - % in women seeking fertility care. there are several vaccine candidates currently in preclinical development, with a subunit vaccine based on the chlamydial major outer membrane protein (momp) and live-attenuated (plasmid-deficient) approaches being the most advanced. the momp candidate entered phase i clinical testing in late , and a phase i study with the live attenuated candidate will commence in . the intended goal of a chlamydia vaccine is to decrease upper genital tract sequelae, however pid is challenging to use as clinical endpoint as it is difficult to definitively diagnose and the causes of pid are multi-factorial (typically the result of c. trachomatis in / of cases). the chlamydia vaccine community is seeking guidance and consensus building on clinical endpoints for clinical studies, including evaluating the potential role of biomarkers, radiologic, and other measures of upper tract ascension, infection, inflammation and damage. improved global burden of disease data and vaccine impact modelling on long term sequelae are also needed to define the investment case for these vaccines. pdvac commended the progress towards the first vaccine study against chlamydia since the s and look forward to discussing the path ahead once the early clinical data are available. this section refers to vaccines that have been licensed, or are approaching licensure in some areas of the world, but are currently limited in their use outside any single who region. in some instances, the vaccines may have the potential of offering broader public health impact by expanding approval and use in other geographical regions, and pdvac is seeking to understand the perspective in this regard. ev is one of the most common causes of hand-foot-andmouth disease (hfmd). sporadic ev outbreaks have occurred globally since it was first isolated in , but from the late s a series of large hfmd epidemics caused by ev have been reported in the asia-pacific region. in china alone, . million cases were reported between and , of which ( . %) were fatal [ ] . children less than years of age have the highest risk of disease, and although infection is unusually mild and selflimiting, severe infections can result in neurological and cardiopulmonary complications, and death. several ev vaccine candidates are in development, and it was stated at the meeting that the chinese national regulatory authority has licensed two ev vaccines, with another in progress. the first licensed vaccine was developed by the institute of medical biology, chinese academy of medical science, and has been approved for use to prevent ev disease in - month olds, based on a phase iii study that demonstrated % efficacy over month [ ] . sinovac has also licensed in activated vaccine, with supportive phase iii data in - month olds [ ] . beijing vigoo is in the process of licensing its inactivated ev vaccines in china (nct ). all three vaccines are adjuvanted with aluminum hydroxide. given that ev outbreaks occur in other areas of the world (recently reported in spain [ ]), further discussions are warranted in the international health community about how to assess the role of the chinese vaccines during outbreaks outside china. in the wake of the - ebola outbreak, various strategies were proposed to avoid such crises from reoccurring. key to improving r&d preparedness and response is determining which pathogens are likely to be the greatest threat, creating consensus with respect to product development strategies and coordinating global funding for complementary r&d efforts going forward. to tackle these questions, and at the request of its member states, who convened a broad global coalition to develop the r&d blueprint [ ] as a sustainable platform for accelerated r&d, with two complementary objectives: to develop (and implement) a roadmap for r&d preparedness for known priority pathogens, and to enable roll-out of an emergency r&d response as early and as efficiently as possible the main approaches underpinning the improvement of preparedness within the r&d blueprint include: as reported in the meeting summary, a consultation to initiate work towards a mers cov roadmap was held in december with aims of defining the key basic and applied research activities, identifying the priority technologies and capacities to support vaccine development, and finally understanding the financing/procurement opportunities. following this meeting, a draft roadmap was developed and underwent public consultation prior to finalisation and publication [ ] . it is well accepted that the extraordinary rate of ebola virus vaccine development was as a result of unprecedented collaboration and co-ordination of global vaccine r&d activities, and the availability of a number of candidate vaccines that could enter clinical phase evaluation [ ] . in the face of another pheic so soon following ebola virus disease (evd) outbreak, the global vaccine community is rallying, and reflecting on lessons learned from the experience in west africa only years ago. at the time of responding to the evd emergency, the availability of well-characterised pre-clinical models and robust data was essential for the comparative evaluation and selection of candidates to move into clinical studies. novel recombinant viral vector platforms, in combination with recombination proteins have been validated by evd experience and it could be argued are now less risky for development of vaccine against future pathogens, but manufacturing feasibility and scale-up capabilities still need to be confirmed for the most novel platforms. critically, sustainable public sector push and pull investment mechanisms beyond the initial emergency response phase need to be created, to incentivise manufacturers to engage in the long term commitment to developing and licensing vaccines that may only be used in outbreak or emergency scenarios. pdvac noted that a target product profile for a second generation ebola virus vaccine is under development that will likely cover ebola zaire, ebola sudan, and marburg filoviruses, and will need to demonstrate longer duration of protection. this tpp will provide guidance about whos preferences and minimally acceptable criteria for vaccines in this area. during the discussion it was clarified that who tpps include minimally acceptable criteria, whereas preferred product characteristics specify only preferences. the status of zika virus epidemiology and the understanding of its pathogenesis and associated sequelae are evolving so rapidly that publications on these issues are almost immediately out of date. pdvac's role has been to oversee a working group that has developed a target product profile (tpp) for use in an emergency, or future outbreak scenario. the tpp was made available for public consultation, after which subject matter experts, global regulators, developers and manufacturers were convened to discuss the regulatory considerations for developing a vaccine with the characteristics described in the tpp. the finalised tpp and position paper are publically available [ ] . in addition to reviewing the status of vaccine development against pathogens, pdvac considered a number of cross-cutting issues that could better integrate and therefore facilitate product development efforts for vaccines and other interventions. in addition to the significant morbidity and mortality that drives the development of vaccines against pathogens for which vaccines are currently not available, the who estimates that there are approximately . million deaths per year in children under from vaccine preventable diseases [ , ] . one of the reasons for this striking immunization gap is the cost and logistical challenges of delivery of these vaccines, over and above the cost of their manufacture. the remit of who's immunization practices advisory committee (ipac) is to provide strategic advice on immunization practices, tools, and technologies intended to improve the delivery of immunization programs at the country level. it oversees the recently formed delivery technologies working group (dtwg) composed of public health organizations, funders and procurement agencies as well as vaccine developers to evaluate r&d in novel delivery technologies and devices, for example the microarray patch, and compact, pre-filled auto-disable injection technologies (cpad). of particular focus for this group is the development and evaluation of a framework to analyze high-level trade-offs between important variables such as development, procurement and supply chain costs, coverage, efficacy, and safety in order to facilitate investment decisions by product developers, vaccine manufacturers, global policy makers, in-country decision makers and procurement agencies. this framework is referred to as total systems effectiveness (tse). the intent of this delivery technology working group is to offer a platform for discussion and guidance regarding vaccine preferences for lmics, early on in development, so that ultimately the vaccine is suitable for programmatic use. the dtwg reports directly to ipac, but has potential overlap with activities that are overseen by pdvac, particularly in consideration of second generation vaccines or new vaccines that may be developed with an alternative presentation to that of a needle and syringe. pdvac was supportive of the dtwg and encouraged continued communication between vaccine development and device/delivery technology development to identify potential opportunities for novel combination product development. there are several pathogen areas where mab are being developed as vaccine-like interventions, as their single dose regimen and long half-life render them amenable for lmics contexts, where they could offer significant public health benefit. candidates for rsv and rabies are approaching licensure within the next years, and a who procedure for who prequalification is urgently needed to avoid delay implementation. this gap has been recognized and will be addressed. the scope of pdvac overlaps with several other research agendas such as gvap, amr, new delivery technologies and development and consolidation of maternal immunization platforms. the pdvac research agenda needs to be clearly communicated, and pdvac and ivb will strive to be well-informed of efforts in other research areas, to help shape and align strategy where appropriate. future pdvac meetings will consider these potential overlaps in more detail, as well as how pdvac and ivb can facilitate development of integrated product development approaches. since its inception in , pdvac has reviewed the pipeline and vaccine development status of different pathogens. pdvac will continue to review new pathogen areas as candidates progress into clinical studies, providing that who engagement will likely facilitate the use of vaccines to reduce disease burden in lmics. one such pathogen is cytomegalovirus (cmv), which is a leading cause of congenital infections worldwide, resulting in - % of infants developing permanent sequelae including hearing loss and neurodevelopmental disabilities. there are little data on cmv infection in lmics, but a recent systemic review suggests that birth prevalence ranges are higher in lmics than in europe and north america [ ] , and several clinical trials of vaccine candidates are ongoing. as such, an assessment of cmv vaccine development will be undertaken by pdvac . with rsv, tb, hiv and enteric candidates approaching pivotal data points, understanding what data are needed to support earlier policy implementation and outcomes will be key, as well as understanding the potential impact of vaccines within the broader control strategy -including diagnostics and other preventatives -for these pathogens. vaccine impact modelling, and understanding the composite set of cost drivers through to vaccine delivery will be important. interaction with who's ipac and pq teams will increase going forward, to strengthen the link between product development and programmatic requirements. under the recommendation of pdvac, who will seek to broaden its role to support development of value propositions for vaccines against pathogens for which there is a poorly defined business case, for example gas and hsv. raising awareness of lmic disease burden and requirements/procedures for access to lmics markets may help to incentivise financing development of these vaccines. of key consideration may be the potential for these vaccines to reduce the emergence of amr, and pdvac recommended that this be considered as a criterion in future landscape analyses and ppc guidance documents. pdvac and who will continue to align activities with the priorities within the who r&d blueprint. pdvac is aware that several other organizations which are responsible for emergency preparedness have been through a process to prioritize their r&d agendas, with some commonality and some complementarity to the pathogens listed in the who blueprint. in this arena, pdvac will continue its horizon scanning role, and will advocate for commitment to product development of vaccines for emerging diseases to progress through robust preclinical proof of concept to generation of phase i data, as a minimum. pdvac strongly recommends the collaboration with other groups to co-ordinate advocacy and funding for vaccine development to prepare for the inevitable future emergencies. procedure for assessing the acceptability, in principle, of vaccines for purchase by united nations agencies tackling drug resistant infections globally: final report and recommendation how can vaccines contribute to solving the antimicrobial resistance problem? who vaccine pipeline tracker moorthy vasee s, who product development for vaccines advisory committee (pdvac) pipeline analyses for pathogens mind the gap: jumping from vaccine licensure to routine use global vaccine action plan (gvap) impact and cost-effectiveness of new tuberculosis vaccines in low-and middle-income countries status of vaccine research and development of vaccines for tuberculosis vaccination with alvac and aidsvax to prevent hiv- infection in thailand report from the world health organization's product development for vaccines advisory committee (pdvac) meeting who global consultation on universal influenza vaccines the global burden of norovirus & prospects for vaccine development the vast and varied global burden of norovirus: prospects for prevention and control global, regional, and national estimates of rotavirus mortality in children < years of age who guidance document for the quality, safety and efficacy of oral live attenuated rotavirus vaccines current status of clostridium difficile infection epidemiology defining the optimal formulation and schedule of a candidate toxoid vaccine against clostridium difficile infection: a randomized phase clinical trial a phase , placebo-controlled, randomized study of the safety, tolerability, and immunogenicity of a clostridium difficile vaccine administered with or without aluminum hydroxide in healthy adults safety, immunogenicity and dose response of vla , a new vaccine candidate against clostridium difficile, in healthy volunteers efficacy, safety, and immunogenicity of an oral recombinant helicobacter pylori vaccine in children in china: a randomised, double-blind, placebo-controlled, phase trial brewinski isaacs and a. sobanjo-ter meulen advancing maternal immunization programs through research in low and medium income countries global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis moorthy vasee s. who consultation on group b streptococcus vaccine development: report from a meeting tb vaccine research & development: a business case for investment clinical relevance of the eskape pathogens invasive group a streptococcus infection among children development of a multicomponent staphylococcus aureus vaccine designed to counter multiple bacterial virulence factors the global roadmap for advancing development of vaccines against sexually transmitted infections: update and next steps global estimates of prevalent and incident herpes simplex virus type infections in global and regional estimates of prevalent and incident herpes simplex virus type infections in global estimates of the prevalence and incidence of four curable sexually transmitted infections in based on systematic review and global reporting the pill, chlamydia and pid hand, foot, and mouth disease in china, - : an epidemiological study an inactivated enterovirus vaccine in healthy children efficacy, safety, and immunogenicity of an enterovirus vaccine in china a roadmap for mers-cov research and product development: report from a world health organization consultation on a path to accelerate access to ebola vaccines: the who's research and development efforts during the - ebola epidemic in west africa meeting report: who consultation on considerations for regulatory expectations of zika virus vaccines for use during an emergency global, regional, and national causes of child mortality in : a systematic analysis systematic review of the birth prevalence of congenital cytomegalovirus infection in developing countries we gratefully acknowledge the pathogen specific landscape analyses that were prepared by the following individuals: this report contains the collective views of an international group of experts, and does not necessarily represent the decisions or the stated policy of the world health organization, nor of the u. s. government.bsg is an employee of the u.s. government. this work was prepared as part of his official duties. title u.s.c. § provides that 'copyright protection under this title is not available for any work of the united states government'. key: cord- -h wvx gw authors: imperiale, michael j.; casadevall, arturo title: the importance of virology at a time of great need and great jeopardy date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: h wvx gw nan a s we enter , one needs to look no farther than the daily news reports to appreciate the ongoing burden of viral diseases. last year, ebola reemerged in west africa, claiming thousands of lives and affecting many thousands more. cases of middle east respiratory syndrome (mers) continue to be reported, with the possibility of a severe acute respiratory syndrome (sars)-like epidemic ever present. chikungunya virus has spread to the western hemisphere, and the infection is now epidemic in the caribbean and southern united states. we are also living in a world in which hundreds of millions of people are chronically infected with hepatitis b and c viruses (hbv and hcv, respectively). the rate of new hiv infections has declined, but millions remain infected, and it, too, has already cut short far too many lives. viruses account for up to % of all human cancers, and although a large percentage of new human papillomavirus (hpv) and hbv infections can now be prevented by vaccination, many are already infected, and the vaccines are not being used to their full potential. we are in the middle of our annual encounter with influenza virus, never knowing when the next strain to which there is little or no preexisting immunity will arise. in - , a mismatch between the h n strain in the influenza vaccine and the circulating virus has led to a poorly protective vaccine, which highlights the need for new vaccines. in recent months, there has been a recurrence of measles in the united states associated with a refusal by some parents to vaccinate their children, and the outbreak continues at the time of this writing ( ). this accounting is of only some viruses and is limited to those that infect humans! there can be no argument that humankind's best hope of preventing and treating these diseases comes from a vigorous research enterprise. vannevar bush recognized this after world war ii in the landmark report "science, the endless frontier," in which he convinced the u.s. government that investment in basic research at universities would yield tremendous dividends (https:// www.nsf.gov/od/lpa/nsf /vbush .htm). indeed, we have now almost eliminated polio due to the development of vaccines and have converted hiv infection from a certain death sentence to a largely manageable state, and, as mentioned above, we have our first anticancer vaccines by preventing some viral infections. the tremendous reduction in mortality from such diseases as variola, measles, and rubella came about only because the causative viruses were identified, cultivated, attenuated, and made into effective vaccines by biomedical research. in addition to having applied these practical findings, we have gained important fundamental insights into the biology not only of viruses but also of the cells that they infect, and that information is being applied to find cures against other diseases, such as cancer. all these advances, and more, have come about because of public trust in science and investment in scientific research. despite all this good news, much remains to be done. it was recently estimated that there are , mammalian viruses ( ), many of which may have the potential for human transmission. even if only a small fraction of these viruses can jump into humans and cause disease, humanity is living under a tremendous threat from viral zoonoses. less expensive drugs are needed for treating those with viral infections such as hepatitis c. while the hpv vaccine can prevent many infections, there are viral types that are not covered by the vaccine, and many millions of people were infected before the vaccine came on the market. infections with numerous other viruses are not treatable due to the lack of effective antivirals. clearly, vaccines against hiv, hepatitis c, and ebola, to name a few, would save countless lives. new pathogens continue to emerge, and existing nonviral pathogens become resistant to common antibiotics. hence, we are living at a time of great need for the discipline of virology. we think that the field of virology and, by extension, the field of microbiology are at a critical crossroads. funding for research in the united states and elsewhere is stagnant, if not losing pace with inflation. working with the most pathogenic organisms requires even higher costs and is heavily regulated. some senior scientists are rethinking their career choices, and there is growing concern that young scientists will be discouraged from entering the field, especially in areas of controversial research, such as studies of the transmissibility of highly pathogenic influenza viruses ( ). while some have argued that virology is a dying field, that assertion has been elegantly refuted by dan dimaio ( ). adding to these stresses, scientists and society are struggling with a new antiintellectual movement that challenges scientific conclusions, from anthropomorphically induced climate change to the absence of any link between vaccines and autism. the rise of antivaccine movements is of particular concern to society, for reduced vaccination rates threaten to undermine some of the greatest accomplishments of virology and public health in the th century. the combination of reduced funding, increased regulation, experimental controversies, and the emerging antiscience intellectual milieu is a toxic blend that makes this time one of great jeopardy for virology. while we scientists cannot directly control funding or regulations, we can take charge of some aspects of the research enterprise in a way to ensure that it continues to benefit society. first, we can continue to advocate for better funding by the federal government. this requires engaging our elected officials both directly and indirectly by continuing to educate them and the public at large about the importance of fundamental research in infectious diseases. the advocacy group research!america has a number of helpful tips on its website (http://www.researchamerica.org). second, we need to demonstrate to the public that we are being good stewards of their investment by working safely in the laboratory. there have been a number of high-profile biosafety lapses over the past year, and the negative publicity surrounding these events may lead to more regulation and less funding support for exactly the types of research that we most critically need. we therefore argue that each of us needs to pay special attention to biosafety in and the longer term. third, in controversial areas, such as studies of transmissibility involving pathogens with pandemic potential, it needs to be clearly articulated why some types of experiments need to be done by vigorously engaging in scientific debate using the tools of science, all the while acknowledging that there are risks and taking every step to mitigate those risks. third, every scientist needs to become a foot soldier in confronting the pervasive spread of antiscientific attitudes, such as the antivaccination movement, which threaten to undermine the great advances society has made in so many aspects of everyday life, including reducing mortality from many infectious diseases. although virology is currently at the epicenter of these converging storms, the issues that it faces are relevant to all of microbiology and, by extension, to all of science and the society that it serves. a little added effort on our parts will go a long way to ensuring continued public confidence in what we do to make their lives healthier. measles cases exceed in us outbreak a strategy to estimate unknown viral diversity in mammals is the debate and "pause" on experiments that alter pathogens with pandemic potential influencing future plans of graduate students and postdoctoral fellows? mbio is virology dead? mbio the views expressed in this article do not necessarily reflect the views of the journal or of asm key: cord- - u x z authors: yang, william h.; dionne, marc; kyle, michael; aggarwal, naresh; li, ping; madariaga, miguel; godeaux, olivier; vaughn, david w. title: long-term immunogenicity of an as -adjuvanted influenza a(h n )pdm vaccine in young and elderly adults: an observer-blind, randomized trial() date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: u x z background: this study (nct ) evaluated the immunogenicity and relative protective efficacy of one dose of influenza a(h n )pdm vaccine with or without as (an α-tocopherol oil-in-water emulsion based adjuvant system). methods: four thousands and forty-eight healthy adults aged ≥ years were randomized ( : ) to receive one dose of either the adjuvanted split virion ( . μg hemagglutinin antigen [ha]/as ) or non-adjuvanted ( μg ha) vaccine. hemagglutination inhibition [hi] antibody response was evaluated before vaccination and at days , and (month ). safety of the study vaccines was evaluated during the entire study duration. results: at day , both study vaccines induced hi immune responses meeting the us regulatory criteria in subjects – years (seroprotection rate [spr]: . % [ . – . ]; seroconversion rate [scr]: . % [ . – . ] in the as -adjuvanted group; spr: . % [ . – . ]; scr: . % [ . – . ] in the non-adjuvanted group) and > years of age (spr: . % [ . – . ]; scr: . % [ . – . ] in the as -adjuvanted group; spr: . % [ . – . ]; scr: . % [ . – . ] in the non-adjuvanted group). the as -adjuvanted vaccine induced higher hi geometric mean titers than the non-adjuvanted vaccine at all time points. at month , only subjects – years of age from both vaccine groups still met the us regulatory criteria (spr: . % [ . – . ]; scr: . % [ . – . ] in the as -adjuvanted group; spr: . % [ . – . ]; scr: . % [ . – . ] in the non-adjuvanted group). protective efficacy was not evaluated due to low number of rt-qpcr-confirmed a(h n )pdm influenza cases. through month , serious adverse events (in subjects: in the as -adjuvanted and in the non-adjuvanted group) and potentially immune mediated diseases ( in the as -adjuvanted and in the non-adjuvanted group) were reported. conclusion: a single dose of either adjuvanted or non-adjuvanted influenza a(h n )pdm vaccine induced protective hi antibody levels against the a/california/ / strain that persisted through month in the – years population. mass immunization is considered to be an effective prophylactic method of mitigating influenza pandemic-associated morbidity and mortality [ ] [ ] [ ] [ ] . due to the novel antigenic characteristics of the swine-origin influenza a(h n ) pandemic virus [influenza (a(h n )pdm ] [ , ] , the seasonal influenza vaccines available at the time of the - h n pandemic were unlikely to confer protection against the novel virus [ , , ] . the world health organization (who) encouraged the development and use of adjuvanted influenza a(h n )pdm vaccines [ , ] , with the aim of dose-reduction, antigen-sparing and to potentially provide broader vaccine efficacy against drifted strains through cross-reactive immunity [ ] . based on the experience of developing a pre-pandemic a/h n influenza vaccine utilizing as (an ␣-tocopherol oil-in-water emulsion based adjuvant system) [ , ] that was well-tolerated and highly immunogenic in adults [ ] [ ] [ ] , an as -adjuvanted influenza a(h n )pdm vaccine with . g hemagglutinin (ha) content was developed [ ] [ ] [ ] . this large-scale, randomized study in subjects ≥ years of age assessed whether one dose of as -adjuvanted . g ha influenza a(h n )pdm vaccine elicited immune response that met the us and european regulatory criteria. additionally, noninferiority and superiority of this vaccine protective efficacy versus a non-adjuvanted g ha influenza a(h n )pdm vaccine were evaluated. in this phase iii, observer-blind, randomized study (nct ), adults ≥ years of age were enrolled across centers in the us and in canada between november and december , . they were randomized (allocation ratio : ) to receive one dose of either a monovalent as -adjuvanted . g ha a/california/ / pandemic influenza vaccine or a non-adjuvanted g ha a/california/ / pandemic influenza vaccine. the enrolment stratification was by age ( : : : ; - years, - years, - years, ≥ years). the subjects and study personnel involved in evaluating end points were blinded to the intervention administered. double blinding was not possible because the vaccine preparation required mixing of as and a(h n )pdm antigen from two vials. randomization was performed using a central, internet-based system that balanced groups with respect to center, age strata and previous seasonal influenza vaccination. adults were excluded from enrolment: if they had a history of physician-confirmed a(h n )pdm influenza infection or vaccination, those who received any vaccination other than a seasonal influenza vaccine within days preceding study start, those with confirmed or suspected immunosuppressive or immunodeficient conditions, diagnosed with or undergoing treatment for cancer, and/or with a history of allergic/anaphylactic reactions following previous influenza vaccination. in addition, laboratory screening was performed to exclude those with results outside of protocol-specified normal ranges. the following safety laboratory parameters were tested to evaluate the participants' eligibility: hepatic aminotransferases, total and direct bilirubin, alkaline phosphatase, creatinine, serum urea nitrogen, hemoglobin, hematocrit, white blood cell count and platelet count. active surveillance of influenza-like infections (ilis: defined as fever ≥ . • c/ . • f or new or worsening myalgia accompanied by new or worsening cough or sore throat) was done during study visits and through bi-weekly telephonic contact through day ( months after the initially planned administration of the second study vaccine dose). additionally, the subjects were instructed to contact the study sites if they develop any ili symptoms. once the study site had been notified of a possible ili episode, a visit for nasal and throat swab sample collection was scheduled within days of symptom onset and before initiating any antimicrobial/influenza antiviral therapy. if an ili episode was reported more than days after onset, no swab specimen was collected. written informed consent was obtained from all subjects prior to conducting any study-related procedures. the study was conducted in accordance with the good clinical practice guidelines, the declaration of helsinki and local regulations. all study-related documents were approved by institutional review boards. the influenza a(h n )pdm vaccine was a monovalent, inactivated, split-virion antigen suspension (a/california/ / strain) adjuvanted with as (arepanrix tm , a trademark of glaxosmithkline vaccines) or administered as plain antigen. the h n viral seed for the vaccine was prepared as per who recommendations [ ] . as is an oil-in-water emulsion based adjuvant system containing squalene ( . mg per dose), dl-␣-tocopherol ( . mg) and polysorbate ( . mg). the as -adjuvanted influenza a(h n )pdm vaccine doses were prepared by mixing the a(h n )pdm antigen and as ( : ) from separate multidose vials. . ml of the assigned study vaccine was administered into the deltoid muscle within min after mixing the antigen and the adjuvant. the first co-primary objective of the study was to evaluate hi antibody responses days after vaccination in the as adjuvanted vaccine group based on the center for biologics evaluation and research (cber) and committee for medicinal products for human use (chmp) criteria for pandemic influenza vaccines in adults [ , ] . at least rt-qpcr-confirmed a/california influenza cases were required to evaluate the second co-primary objective on noninferior protective efficacy followed by superiority. as only three rt-qpcr-confirmed a/california influenza cases were diagnosed during the study, descriptive analyses of the influenza attack rate and vaccine efficacy improvement (vei) were computed only for ili and pneumonia cases. the study also assessed whether the non-adjuvanted g ha influenza a(h n )pdm vaccine elicited immune responses that met the us and european regulatory criteria, days after vaccination and whether these criteria were met for either study vaccines at day (in a small subset of subjects) and at day (month ). hemagglutination inhibition (hi) antibody levels in serum samples were assessed at glaxosmithkline vaccines central laboratory using a validated in-house assay [cut-off: ≥ : ] that used chicken erythrocytes, as previously described [ ] . the a/california/ / strain was used as the antigen strain. rt-qpcr was performed on viral rna from the clinical samples as described previously [ ] . viral load values were quantified and the sample was considered positive when the measured viral load was equal to or above the assay cut-off [ ] . serum samples were collected before vaccination (day ), at days , (in a subset of subjects) and (month ) for assessment of humoral immune response and for clinical chemistry and hematology assessments at days , and . the immunological assessment was based on hi antibody seroconversion rates (scr), seroprotection rate (spr) and geometric mean fold rise (gmfr), against the vaccine homologous strain. post hoc exploratory analyses included the assessment of possible correlation of hi antibody response with body mass index (bmi) and with previous influenza vaccination history. further assessments were performed to identify the respiratory viruses isolated from swab samples from ili cases using xtag respiratory viral panel (rvp) fast assay (luminex molecular diagnostics inc., toronto, canada) [ , ] . subjects used diary cards to record the solicited local and general symptoms occurring within days following vaccination and the unsolicited adverse events occurring within days following vaccination. potential immune-mediated diseases (pimds: subset of aes that include both autoimmune diseases and other inflammatory and/or neurologic disorders which may/may not have an autoimmune etiology) and serious adverse events (saes) were recorded throughout the study period. the intensity of all solicited adverse events except fever was graded on a scale of ( - ), grade being those that did not interfere with normal activities and grade being those that prevented normal activities (grade redness and swelling: diameter > mm; grade fever: temperatures ≥ . -≤ . • c. fever was graded on a scale of ( - ), grade being temperatures > . • c. based on clinical judgment, the investigators assessed whether the aes/saes were potentially related/not related to the study vaccine. serum samples for the analysis of clinical safety laboratory parameters were collected at days and . the following laboratory parameters were tested: hepatic aminotransferases, total and direct bilirubin, alkaline phosphatase, creatinine, serum urea nitrogen, hemoglobin, hematocrit, white blood cell count and platelet count. the sample size was calculated taking into consideration the co-primary objectives. overall, evaluable subjects ( for vei evaluation) in each of the two treatment groups (accounting for % and % drop-out rates for the co-primary objectives) was estimated to provide a power of . % to meet the co-primary objectives, assuming %/ % as reference for spr/scr in subjects - years and > years of age, respectively, % vaccine efficacy for the non-adjuvanted influenza a(h n )pdm vaccine, and an attack rate of % in subjects who do not receive any h n vaccine (pass ; one-sided test, one-sided alpha = . %). the scr, spr and gmfr and incidence rates of solicited and unsolicited adverse events were calculated with % confidence interval (ci). the analyses of immunogenicity were performed on the according to protocol (atp) cohort which included evaluable subjects meeting eligibility criteria and adhering to protocoldefined procedures. a cox regression model, including the vaccine group as a fixed effect, age and baseline antibody titer as covariates was used to estimate the vei for the any ili cases and any pneumonia cases (the first event was considered if multiple events were reported by a subject). all statistical analyses were performed using statistical analysis software (sas) version . . a total of subjects were screened, received vaccine, and completed the study through day . the reasons for withdrawals and elimination of subjects from the analyses at different time points are presented in fig. . the mean age of subjects in the tvc at the time of vaccination in the - years age group was . years (range: - years); > years age group was . years (range: - years). overall, . % and . % of subjects in the respective two age groups were female and the majority of subjects were caucasians ( . % and . %, respectively). co-primary objectives: the first co-primary objective was met. a single dose of the as -adjuvanted . g ha influenza a(h n )pdm vaccine elicited hi immune responses in the - years and > years age groups that met the cber regulatory criteria at day ( table ). the chmp criteria were met in the - years and > years age groups (data not presented). the second co-primary objective was not evaluated as only three rt-qpcr-confirmed a/california influenza cases were identified (as -adjuvanted: ; non-adjuvanted: ). secondary objectives: in the day subset (n = ) which received the as -adjuvanted . g ha influenza a(h n )pdm vaccine, the cber criteria were met in the - years age group and > years age group (table ) . at day (month ), the cber criteria were met only for subjects - years of age (table ). subjects > years of age had a ll of the % ci for spr of . %, thus not fulfilling the cber criteria at this time point. at day , a single dose of the non-adjuvanted g ha influenza a(h n )pdm vaccine elicited hi immune responses in subjects - years and > years of age that met the cber regulatory criteria (table ) . only those in the - years age group met the cber criteria at day and at day (month ). at this time points subjects > years of age had a lls of the % ci for spr of . and . %, respectively and lls of the % ci for scr of . % and . %, respectively, thus not fulfilling the cber criteria. the chmp criteria were met at day and day in the - years and > years age groups for both study vaccines. at day , the chmp criteria were met in the - years age group but not in the > years age group for both study vaccines (data not presented). hi antibody gmts in both age groups were higher at all post-vaccination time points for those who received the as adjuvanted influenza a(h n )pdm vaccine compared to those who received the non-adjuvanted vaccine; gmts were generally lower in the > years compared to the - years age group at all time points (table ) . persistence of hi antibody response at day (month ) was observed for both study vaccines, although at lower levels compared to that observed at day (table ) . overall, the immune response against the vaccine homologous strain appeared to decrease with advancing age (fig. /web-appendix table ). post hoc exploratory analyses showed that hi antibody responses were mostly comparable across healthy weight, overweight and obese subjects. no clear patterns emerged due to the modest number of subjects in the underweight category (web-appendix table ). a higher hi antibody was observed among influenza vaccine-naïve subjects, compared with those with previous seasonal influenza vaccination, in terms of hi antibody gmts and gmfrs (web-appendix table ). [ . %] in the as -adjuvanted and non-adjuvanted treatment groups) were reported and were sampled during the study. of these, samples were tested and only three cases ( . %) of a(h n )pdm were confirmed by rt-qpcr (one and two cases respectively). the incidence of ili cases was comparable between the two treatment groups, except through day ( respiratory viruses: rhinovirus, identified from ( . %) nasopharyngeal swabs, was the most frequently determined respiratory virus (table ). solicited adverse events: pain at the injection site was the most frequently reported solicited local adverse event. it was reported for . % and . % of subjects in the - years age group who received the as -adjuvanted and the non-adjuvanted influenza a(h n )pdm vaccine, respectively (p < . ) and for . % and . % of subjects in the > years group who received the adjuvanted and the non-adjuvanted influenza a(h n )pdm vaccine, respectively (p < . ) (fig. /web-appendix table ). additionally, in both age groups, a statistically significant higher percentage of subjects receiving the adjuvanted vaccine reported redness and swelling compared with non-adjuvanted vaccine group (p < . for both). muscle ache (as -adjuvanted/nonadjuvanted: - years: . %/ . %, p < . ; > years: . %/ . %, p < . ), fatigue (as -adjuvanted/non-adjuvanted: - years: . %/ . %, p < . ; > years: . %/ . %, p = . ) and headache (as -adjuvanted/non-adjuvanted: - years: . %/ . %, p = . ; > years: . %/ . %, p = . ) were the most frequently reported solicited general adverse events (fig. /web-appendix table ). in the - years age group, a higher percentage on subjects receiving the adjuvanted vaccine reported joint pain, shivering and sweating compared with nonadjuvanted group (p < . for all). in the > years group joint pain was reported by a higher percentage of subjects receiving adjuvanted vaccine compared with the subjects receiving the nonadjuvanted vaccine (p = . ). in this age group, no statistically significant differences were observed between vaccine groups in terms of shivering, sweating and fever (p > . ). solicited local and general adverse events of grade intensity were reported for ≤ . % of subjects. in the - years age group, the incidence of pain at the injection site, joint pain and muscle aches of grade intensity was significantly higher in the adjuvanted vaccine group compared with the non-adjuvanted group (p < . for pain at the injection site; p = . for joint pain; p = . for muscle aches). the incidence of other solicited symptoms of grade intensity in this age group, as well as in the > years age group, was not statistically significant different between the adjuvanted and the non-adjuvanted vaccine groups (p > . ). reporting of solicited adverse events was higher in the - years age group. unsolicited adverse events: a total of subjects ( . %; as -adjuvanted: table ); subjects in the as -adjuvanted treatment group, - years: . %, > years: . %, and subjects in the non-adjuvanted treatment group, - years: . %, > years: . %. two of these events, intestinal obstruction (as -adjuvanted treatment group) and multiple sclerosis (non-adjuvanted treatment group) were considered by the investigator to be possibly related to study vaccine and were also considered pimds. through day (month ), pimds according to the predefined list of pimd preferred terms were reported, with and in as -adjuvanted and non-adjuvanted influenza treatment groups, respectively. seven fatal saes were reported, and in as -adjuvanted and non-adjuvanted treatment groups, respectively. all were assessed by investigators as not related to vaccination. a detailed description of all fatal saes is provided in web appendix table . overall, samples had laboratory values for the hematological and biochemical parameters outside the normal laboratory reference range at days and . of these, were from subjects in the adjuvanted vaccine group and were from subjects in the non-adjuvanted vaccine group. data from this large, controlled study in adults years of age and older demonstrated that a single dose of as -adjuvanted or non-adjuvanted influenza a(h n )pdm vaccine elicited strong hi immune responses days later that met the chmp and the more stringent cber criteria for pandemic influenza vaccines. the hi antibody response persisted through six months after vaccination for both vaccines, although the cber criteria were met only in the - years age group and chmp criteria in the - years age group. the co-primary objective concerning relative vaccine efficacy against influenza was not evaluated due to the small number of rt-qpcr-confirmed h n / influenza cases. the low number of cases observed may be partially due to the timing of the study; the start of study vaccination followed the peak of a(h n )pdm virus transmission in the us and canada by a week or more (last week of october, ), by which time a(h n )pdm circulation had diminished considerably. published estimates of as -adjuvanted influenza a(h n )pdm vaccine effectiveness against influenza range from . % to . % [ ] [ ] [ ] [ ] . overall, the incidence of ili cases was comparable between the two groups, except in the first days after vaccination ( versus ili cases in the as -adjuvanted and non-adjuvanted treatment groups, respectively). this study was not sufficiently powered to detect statistical significance in this analysis. the data for elderly subjects from the present study are in agreement with observations made in previous studies that one dose of the as -adjuvanted . g ha influenza a(h n )pdm vaccine may be insufficient to meet cber criteria at months in elderly [ ] and two doses of vaccine administered days apart induce longterm persistence of hi antibodies at putatively protective levels [ ] [ ] [ ] . nicholson et al. demonstrated that two doses of a different as -adjuvanted influenza a(h n )pdm vaccine elicited hi immune responses that persisted at seroprotective levels in > % of subjects ≥ years of age, up to six months after vaccination, although at lower levels compared to younger adults (p < . ) [ ] . similar to other observations [ , , , [ ] [ ] [ ] [ ] [ ] [ ] , our results showed that previous seasonal vaccination appeared to negatively influence the strength of the immune response elicited by the influenza a(h n )pdm vaccines, especially in terms of longterm immunogenicity. there are conflicting reports on whether previous seasonal influenza vaccination increases the risk of subsequently contracting a(h n )pdm infection requiring medical attention [ , ] . the effect of bmi on immune response was also studied. consistent with previous trials [ , ] , in the present study, high bmi did not appear to impair hi antibody response shortly after vaccination. however, sheridan et al. reported a decrease in hi antibody titers in obese subjects months after vaccination [ ] , an observation also made in the present study. the reactogenicity and safety profile was in agreement with available data in adults and children [ , , ] . the frequency of solicited local adverse events in this study was higher in the as -adjuvanted versus the non-adjuvanted treatment group and the frequency of solicited adverse events were comparatively lower in the > years age group. previous clinical trials of influenza a(h n /)pdm vaccines [ , , , ] comparing safety outcomes between adjuvanted and non-adjuvanted vaccines reported similar observations, with higher frequency of both local and general adverse events with adjuvanted vaccines compared with non-adjuvanted vaccines. in our study, we did not observe any differences between the two vaccine groups in terms of saes considered as possibly related to vaccination ( in each group). although an imbalance in the number of fatal saes was observed between the adjuvanted and non-adjuvanted group ( versus ), none were considered to be related to vaccination and they all occurred in subjects with a relevant medical history. a gradual decrement in the hi antibody gmts elicited by both study vaccines against the a(h n )pdm vaccine strain in older subjects was observed and this could be attributed to "immunosenescence" [ , ] . a decreasing trend with advancing age was also observed in the frequency of solicited adverse events. a possible limitation of this study was the absence of blood samples collection for assessment of the immune response after day (month ). this period of six months was anticipated to cover the period of transmission of influenza virus during one season. a recently published study enrolling subjects randomized to receive one or two doses of the same adjuvanted vaccine and followed up to months, showed that regulatory criteria were met months after the administration of the last vaccine dose in subjects aged - years receiving either one or two vaccine doses and in subjects aged > years receiving two vaccine doses [ ] . at day (month ) the regulatory criteria were still met only in subjects aged - years who received two vaccine doses. in conclusion, a single dose of either adjuvanted or nonadjuvanted influenza a(h n )pdm vaccines elicited protective levels of hi antibodies against the vaccine homologous a/california/ / strain that persisted up to day (month ) in the - years population. adjuvantation potentially offers the opportunity for antigen-sparing, making this as -adjuvanted influenza a(h n )pdm vaccine a candidate to help meet the demands for the large number of vaccine doses required to mitigate pandemic influenza. caroline gesualdi for clinical study management, janine linden for preparation of the study protocol and related study documentation, dorothy slavin, clinical safety representative, rosalia calamera, clinical data coordinator, karl walravens lab manager and clinical readout. finally, we thank avishek pal (glaxosmithkline vaccines) and adriana rusu (xpe pharma and science) who provided medical writing services and dr. santosh mysore (xpe pharma and science, c/o glaxosmithkline vaccines) for editorial assistance and manuscript coordination. financial disclosure: the study was funded by the us department of health and human services (hhs), assistant secretary of preparedness and response (aspr), biomedical advanced research and development authority (barda) and glaxosmithkline biologicals sa. glaxosmithkline biologicals sa was involved in all stages of the study conduct and analysis. glaxosmithkline biologicals sa also took in charge all costs associated with the development and the publishing of the present manuscript. all authors had full access to the data. the corresponding author had final responsibility to submit for publication. conflict of interest: all investigators received compensation for study involvement and travel related to this study. ping li, miguel madariaga, olivier godeaux and david vaughn are/were employees of glaxosmithkline group of companies and report receiving restricted shares of the company. contributorship: w.y., m.d., m.k. and n.a. contributed to the data collection, data interpretation and critical review of the manuscript drafts. p.l., m.m., o.g. and d.w.v. contributed to the study design, data analysis and interpretation as well as to the critical review of all drafts of the manuscript. trade mark statement: arepanrix is a trade mark of glaxosmithkline group of companies. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.vaccine. . . . trial of influenza a(h n ) monovalent mf -adjuvanted vaccine safety and immunogenicity of pandemic influenza a h n vaccines in china: a multicentre, double-blind, randomised, placebo-controlled trial safety and immunogenicity of a pandemic influenza a h n vaccine when administered alone or simultaneously with the seasonal influenza vaccine for the - influenza season: a multicentre, randomised controlled trial an adjuvanted pandemic influenza h n vaccine provides early and long term protection in health care workers emergence of a novel swine-origin influenza a(h n ) virus in humans antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans serum cross-reactive antibody response to a novel influenza a(h n ) virus after vaccination with seasonal influenza vaccine cross-reactive antibody responses to the pandemic h n influenza virus pandemic influenza a(h n ) virus vaccine -conclusions and recommendations from the who recommendations on pandemic (h n ) vaccines. pandemic (h n ) : briefing note immunogenicity and safety of varying dosages of a monovalent h n influenza vaccine given with and without as adjuvant system in healthy adults and older persons adjuvant system as containing ␣-tocopherol modulates innate immune response and leads to improved adaptive immunity development and evaluation of as , an adjuvant system containing ␣-tocopherol and squalene in an oil-in-water emulsion immunogenicity and tolerability of an as a -adjuvanted prepandemic influenza vaccine: a phase iii study in a large population of asian adults a phase ii, open-label, multicentre study to evaluate the immunogenicity and safety of an adjuvanted prepandemic (h n ) influenza vaccine in healthy japanese adults broad clade cross-reactive immunity induced by an adjuvanted clade rh n pandemic influenza vaccine immunogenicity and safety in adults of one dose of influenza a h n v vaccine formulated with and without as (a)-trial. adjuvant: preliminary report of an observer-blind, randomized controlled trial effect on cellular and humoral immune responses of the as adjuvant system in an a/h n / influenza virus vaccine administered to adults during two randomized controlled trials immunogenicity and safety of a novel as a -adjuvanted h n pandemic influenza vaccine in adults in japan who biosafety risk assessment and guidelines for the production and quality control of human influenza pandemic vaccines us food and drug administration (fda) for industry, clinical data needed to support the licensure of pandemic influenza vaccines guideline on influenza vaccine prepared from viruses with the potential to cause a pandemic and intended for use outside of the core dossier context (emea/chmp/vwp/ / ). european agency for the evaluation of medicinal products ten years of experience with the trivalent split-influenza vaccine use of real-time polymerase chain reaction (rtpcr) as a diagnostic tool for influenza infection in a vaccine efficacy trial comparison of the luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections comparison of the luminex respiratory virus panel fast assay with in-house real-time pcr for respiratory viral infection diagnosis effectiveness of the pandemic h n influenza vaccines against laboratoryconfirmed h n infections: population-based case-control study association between the and seasonal influenza vaccine and pandemic h n illness during spring-summer : four observational studies from canada protective effect of single-dose adjuvanted pandemic influenza vaccine in children age-specific effectiveness of an oil-inwater adjuvanted pandemic (h n ) vaccine against confirmed infection in high risk groups in england evaluation of immune response following one dose of an as a -adjuvanted h n pandemic influenza vaccine in japanese adults years of age or older safety and longterm humoral immune response in adults after vaccination with an h n pandemic influenza vaccine with or without as adjuvant characterization and long-term persistence of immune response following two doses of an as a -adjuvanted h n influenza vaccine in healthy japanese adults immunogenicity and safety of a two-dose schedule of whole-virion and as a -adjuvanted influenza a(h n ) vaccines: a randomised, multicentre, age-stratified, head-to-head trial immunogenicity of a monovalent influenza a(h n ) vaccine in infants and children: a randomized trial safety and immunogenicity of a prototype adjuvanted inactivated split-virus infl uenza a (h n ) vaccine in infants and children predictors of immune response and reactogenicity to as b-adjuvanted split virion and nonadjuvanted whole virion h n ( ) pandemic influenza vaccines immunogenicity of a monovalent influenza a(h n ) vaccine among pregnant women: lowered antibody response by prior seasonal vaccination influenza vaccine effectiveness in the community and the household as -adjuvanted versus non-adjuvanted inactivated trivalent influenza vaccine against seasonal influenza in elderly people: a phase randomised trial no association between and influenza vaccine and influenza a(h n )pdm virus infection association between obesity and vulnerability and serologic response to influenza vaccination in older adults obesity is associated with impaired immune response to influenza vaccination in humans clinical evaluation of an as -adjuvanted pandemic influenza h n vaccine in children (preliminary report) trial of influenza a(h n ) monovalent mf -adjuvanted vaccine a rapid immune response to influenza a(h n ) vaccines in adults: a randomized, double-blind, controlled trial the unmet need in the elderly: how immunosenescence, cmv infection, comorbidities and frailty are a challenge for the development of more effective influenza vaccines influenza vaccination for older adults longterm persistence of humoral and cellular immune responses induced by an as a-adjuvanted h n influenza vaccine: an open-label, randomized study in adults aged - years and older we are grateful to the new york medical college, new york for providing the vaccine virus strain. the authors are indebted to the participating study volunteers, clinicians, nurses and laboratory technicians at the study sites. we are grateful to the principal investigators, drs. key: cord- -zkk d gd authors: muzumdar, jagannath m.; cline, richard r. title: vaccine supply, demand, and policy: a primer date: - - journal: j am pharm assoc ( ) doi: . /japha. . sha: doc_id: cord_uid: zkk d gd objective: to provide an overview of supply and demand issues in the vaccine industry and the policy options that have been implemented to resolve these issues. data sources: medline, policy file, and international pharmaceutical abstracts were searched to locate academic journal articles. other sources reviewed included texts on the topics of vaccine history and policy, government agency reports, and reports from independent think tanks. keywords included vaccines, immunizations, supply, demand, and policy. study selection: search criteria were limited to english language and human studies. articles pertaining to vaccine demand, supply, and public policy were selected and reviewed for inclusion. data extraction: by the authors. data synthesis: vaccines are biologic medications, therefore making their development and production more difficult and costly compared with “small-molecule” drugs. research and development costs for vaccines can exceed $ million, and development may require years or more. strict manufacturing regulations and facility upgrades add to these costs. policy options to increase and stabilize the supply of vaccines include those aimed at increasing supply, such as government subsidies for basic vaccine research, liability protection for manufacturers, and fast-track approval for new vaccines. options to increase vaccine demand include advance purchase commitments, government stockpiles, and government financing for select populations. conclusion: high development costs and multiple barriers to entry have led to a decline in the number of vaccine manufacturers. although a number of vaccine policies have met with mixed success in increasing the supply of and demand for vaccines, a variety of concerns remain, including developing vaccines for complex pathogens and increasing immunization rates with available vaccines. new policy innovations such as advance market commitments and medicare part d vaccine coverage have been implemented and may aid in resolving some of the problems in the vaccine industry. c urrently, vaccine-preventable disease levels are at near record lows. this was not the case at the beginning of the th century, when infectious diseases were the greatest threat to public health and the leading cause of death in the united states and elsewhere. during that period, few effective treatments or measures were available for preventing large numbers of deaths from these diseases, despite the fact that in , edward jenner performed the western world's first vaccination. during the th century, the "golden age" of vaccines witnessed the development and acceptance of vaccines for diphtheria (discovered in , but not used widely until the s), tetanus toxoids ( ), influenza vaccine (first used in ), polio vaccine with inactive virus ( ) and live attenuated virus ( ), measles ( ) , and combination measles-mumps-rubella (mmr) vaccine ( ) . estimates indicate that these vaccines have prevented more than million deaths per year worldwide from infectious diseases. vaccines have been well documented as one of the greatest achievements of medicine and are among the most cost-effective interventions in public health. for example, zhou et al. evaluated the economic impact of the routine u.s. childhood immunization schedule. they reported that for every dollar invested in childhood vaccination against nine vaccine-preventable diseases, $ . was saved in direct medical care costs. further, when indirect benefits were taken into account, such as parental absenteeism costs incurred in caring for ill chil- synopsis: supply and demand issues in the vaccine industry and the policy options that have been implemented to resolve these issues are reviewed in the current work. although vaccines have been responsible for some of the greatest successes in public health, the vaccine market is fragile and requires both supply-and demand-side interventions. vaccine availability has been limited by the number of suppliers, high research and development and production costs, and safety problems leading to increased regulatory requirements. demand for vaccines has been constrained by rapidly increasing vaccine costs, financing issues that have hindered efforts to achieve targets set for population immunization rates, and parental attitudes regarding the safety and efficacy of vaccine products. analysis: to date, a patchwork of policies to make the vaccine market more attractive for private firms and to increase patient access to these products has been implemented by the u.s. government and private philanthropies. according to the authors, an integrated policy approach that preserves incentives for market entry and innovation in the vaccine industry while addressing parental vaccine concerns and increasing immunization funding and reimbursement for both providers and patients is needed. dren, the amount saved rose to $ . . salo et al. assessed the cost effectiveness of influenza vaccination of children aged months to years and found that influenza vaccination for healthy children (all age groups) was more effective and less costly than not vaccinating children against influenza. these findings are due in part to the fact that many vaccines result in long-term or lifelong protection of the recipient and people they contact. through the process of "herd immunity" and "herd effect," a vaccines protect not only those who receive them but also those who cannot or do not receive the vaccine because of medical conditions, parental indifference, or religious or philosophical objections to vaccinations. , ( a the probability of unvaccinated individuals contracting a disease when part of a larger group with a certain seroprevalence [herd immunity] is called the herd effect.) however, of note, if a susceptible person strays outside the herd or if the herd changes, that person is still susceptible. compared with pharmaceutical products, the number of lives saved per invested dollar is substantial. economists have reported that this increased life expectancy has made a considerable contribution to economic growth. , in fact, it has been argued that more than one-half of the growth in real income in the first half of the th century is attributable to the declining mortality associated with the discovery of vaccines. , mass immunization programs have resulted in % eradication of smallpox from the world, elimination of diphtheria and polio, and % eradication of measles, mumps, and rubella in the united states. haemophilus influenzae type b (hib) vaccines have been successful in reducing childhood mortality. in addition, vaccination of healthy adults has resulted in decreased work absenteeism and decreased use of health care resources, including less use of antibiotics. , nevertheless, this success of vaccines is threatened because of several factors. problems related to vaccine research and development (r&d), manufacturing complexities, supply and distribution, safety issues, and financing have become areas of major concern. symptoms of this crisis include a decline in the number of vaccine producers from in to in and a decline in the number of licensed vaccine products from in to in . (some of these are combination products.) , eight of these vaccine products are currently produced by five major companies: sanofi pasteur, chiron (a business unit of novartis vaccines and diagnostics), glaxosmithkline, merck vaccines & infectious diseases, and wyeth vaccines. should any one of these suppliers cease production, it could take years for a replacement vaccine to be licensed and become available publicly. beginning in late , the united states faced shortages of of the recommended childhood vaccines. affected vaccines included diphtheria-tetanus-acellular pertussis (dtap), mmr, varicella, and pneumococcal conjugate vaccines. suspension of production of pedvaxhib and comvax by merck and a subsequent voluntary recall of certain lots of both vaccines on december , , led to a considerable disruption in the supply of hib-containing vaccines. thus, the dearth of suppliers appears to have affected the stability of vaccine supply. compounding these problems, the epidemiology of several diseases is changing. west nile virus killed at least people in the united states in , and cases of dengue fever, formerly known only in tropical areas, have been reported in texas. the centers for disease control and prevention (cdc) received reports of , cases of malaria in among individuals in the united states or its territories. this total represents an increase of . % from the , cases reported for . with rapid intercontinental transportation and a larger global population, diseases can travel and spread to many countries in little time. cdc issued a health advisory on april , , regarding a measles outbreak in arizona that was linked to importation of the measles virus from switzerland. the first case, with rash onset on february , , occurred in an adult visitor from switzerland who was hospitalized with measles and pneumonia. in another dramatic example, severe acute respiratory syndrome spread from asia to north america quickly, eventually infecting , people worldwide, of whom died, when a -year-old woman carried the infection from hong kong to toronto, where it eventually caused deaths. , objectives the current report seeks to provide an overview of the vaccine industry and public policy affecting it. specifically, we sought to ( ) highlight issues faced by vaccine manufacturers that make the vaccine industry a unique segment of the prescription drug industry, ( ) provide an overview of the vaccine market with regards to vaccine supply and demand, and ( ) provide an overview and critical evaluation of policy options proposed and implemented by various parties to address vaccine supply and demand problems. this research consists of a narrative literature review and critical analysis of the information retrieved. search criteria were limited to english language and human studies. keywords used for the search included vaccines, immunizations, supply, manufacturing, demand, policies, and push-pull solutions. indices such as medline, policy file, and international pharmaceutical abstracts were searched and the results augmented with reports produced by government agencies (e.g., government accountability office) and independent think tanks. a variety of sources were reviewed, including reports from academic journals and current texts on vaccine history and policy. articles pertaining to vaccine demand, supply, and public policy were selected and reviewed for inclusion in the current work. vaccines are biologics that introduce "weakened or killed disease-causing bacteria, viruses, and/or their components" or toxoids into a person or animal to stimulate an immune reaction that the body will remember if exposed to the same pathogen in the future. this unique property sets them apart from other segments of the pharmaceutical industry, such as "small-molecule" or products derived from traditional organic chemistry methods and from other biologically derived products used in a therapeutic capacity. as such, when a private firm considers entering the vaccine market, they face several important barriers to entry, some of which are shared with these product segments and others that are unique to vaccines. these are discussed in detail below. new vaccines begin with the recognition of an infectious disease burden worth preventing. basic research regarding pathogens and immune responses, often funded by the national institutes of health (nih), vaccine manufacturers, and nonprofit organizations such as the bill & melinda gates foundation, is performed mainly at universities. certain vaccines for yellow fever, typhoid, and anthrax are funded and developed in the department of defense. before entering clinical trials, prototype vaccines undergo toxicology testing that is conducted in a good laboratory practice-compliant laboratory. private firms then build on this knowledge to develop clinically feasible vaccine products and shepherd them through clinical testing. the vaccine's manufacturer then must submit a biological license application (bla) to the food and drug administration (fda) for evaluation and approval before marketing. the approval process tests extensively for safety and efficacy, along with purity and absence of contaminants. if data raise serious concerns about product safety or efficacy during any phase, fda may request additional information or studies or may halt ongoing clinical studies. the entire research, development, and approval process may require years or more. estimates of the cost of this process range from $ million to $ million ($us ). , table summarizes information on the different phases in vaccine research. manufacturing complexities. although vaccine manufacturing regulation originally was controlled by the u.s. public health service under the biologics control act of , this authority now rests with fda. the majority of vaccines approved by fda are manufactured from live (attenuated) or killed (inactivated) organisms. some are based on partially purified components of an organism such as diphtheria and tetanus, and a handful are recombinantly produced, such as the hepatitis b vaccine. vaccines are manufactured by at least three methods: egg-based (e.g., influenza vaccine), cell-derived (e.g., polio vaccine), or recombinant (e.g., hepatitis b vaccine). for bacterial vaccines, the bacterial pathogens are grown in bioreactors using media developed for optimizing the yield of the antigen (e.g., hib) ( figure ). as such, small deviations in the manufacturing process can have a major impact on the potency and/or purity of these products. thus, fda production facility requirements are rigorous, and these stringent regulatory hurdles add to the production costs of vaccines. because new vaccines generally are more complex than older products, vaccine suppliers face increasingly stringent regulation of manufacturing facilities even after a vaccine is approved. suppliers undergo frequent inspections of their production facilities by each country in which the vaccine product is licensed and by fda. individual product batches require separate approval for release, and slight modifications reviews vaccine policy to production processes or the packaging of products may trigger expensive and time-consuming product reviews. fda also requires frequent upgrades of vaccine production facilities to reflect state-of-the-art manufacturing processes. recently, fda quality control inspections led to merck's recall of . million doses of childhood vaccines to protect against meningitis, pneumonia, and hepatitis b because of contaminated manufacturing equipment. , the recall involved lots of the hib vaccine pedvaxhib and two lots of a combination vaccine for both hib and hepatitis b sold under the brand name comvax. most vaccine manufacturers are profit-seeking firms, not public health agencies. as such, they are not obligated to develop vaccines. these manufacturers face the decision of whether to invest large amounts of capital in vaccine r&d for a small portion of the global pharmaceutical market representing approximately . % of all pharmaceutical revenues. most vaccines are not "blockbuster" pharmaceuticals that yield large profits or returns on investment. although pharmaceuticals in the aggregate are a large market representing approximately $ billion annually ($us ) worldwide, sales of vaccines are estimated at just $ . billion to $ billion per year, with about one-quarter of total sales in the united states. this $ -billion market is controlled primarily by the five major manufacturers. moreover, most vaccines are used at most several times in a lifetime, whereas therapeutic biologics and small-molecule drugs often are used every day. thus, markets for small-molecule and biotechnology drugs treating chronic diseases are considerably more attractive to investors than vaccines. safety concerns, both real and unsubstantiated, continue to be a threat to the present vaccine market. vaccines are biologics and therefore are more difficult to produce with consistent precision than small-molecule drugs. they are subject to variability in the manufacturing process and require careful handling. despite intensive quality regulation, the biologic nature of vaccines, inherent uncertainties in manufacturing, and safety concerns make vaccine manufacturers targets for tort litigation for patients suffering an illness after vaccination. a surge of lawsuits in the s resulted in serious concerns regarding the supply of the dtap combination vaccine, as well as other vaccines. concerns have been raised in the united states regarding the safety of thimerosal, which is a mercury-containing preservative used in some vaccines. another concern is with the false association of mmr combination vaccine and autism in children. however, to date, studies have not shown an association between neurodevelopmental disorders and thimerosal. , in addition, no evidence has been found demonstrating a link between vaccination with the mmr vaccine and autism in children. rotashield, a rotavirus vaccine licensed in , was permanently withdrawn in when it was found to cause a rare but serious intestinal obstruction in some recipients. these safety concerns have been a reason for a change in the attitudes of some parents regarding having their children immunized. taken together, liability issues and safety concerns provide important disincentives to manufacturers considering developing and manufacturing vaccines. immunization rates for recommended vaccines among children in the united states have been consistently high. the immunization regimen was simple, costs incurred were small, and many (if not most) public schools required proof of immunization as a condition of attendance. , most children received vaccines from private practitioners with their parents paying for this service through third-party insurance or out of pocket. underprivileged children often received free immunizations from local health departments, with costs paid from general revenue funds at the local and state level. in the s, the cost of recommended immunizations began to increase, primarily as a result of the introduction of table displays a comparison of the costs for recommended vaccines for children to years of age for and . according to the national immunization survey (nis) for children, although the number of children vaccinated had reached record highs, for some vaccines, coverage among children was lower and varied with poverty level. also, according to recent cdc data, substantial gaps continue to remain in vaccination coverage for adults. these shortcomings may be due in part to the increasing costs of vaccines. given the increasing costs associated with vaccination and the increasing number of vaccine doses, financing for this service has taken on greater importance. currently, u.s. vaccine financing is a joint responsibility shared by the private and public sectors. as of , more than one-half of the vaccines recommended for children were purchased through federal contract, whereas vaccines for adults typically are covered by private insurance. private health plans often have insurance coverage for vaccines. however, some children enrolled in private health plans do not have coverage for vaccines and are considered underinsured for immunization. finally, some studies have shown that health care provid-ers have concerns regarding the costs of purchasing and administering vaccines and their level of reimbursement from public and private insurers. providers must order and purchase many vaccines (e.g., influenza) months before they are administered, resulting in substantial capital outlay coupled with delayed reimbursement. recently, freed et al. conducted a survey exploring physicians' perspectives on reimbursement for childhood immunizations. approximately one-half of the study respondents reported financial reasons and low profit margins from immunizations as factors affecting their purchase and administration of vaccines. these authors concluded that physicians who provide vaccines to children and adolescents are dissatisfied with third-party reimbursement levels and the increasing financial strain on their practices from immunizations. thus, increasing vaccine prices, a greater number of vaccine doses, and declining provider reimbursement for these products appear to be factors constraining both patient and provider demand for these products. parental beliefs regarding vaccine safety and efficacy have led to a decrease in the demand for recommended vaccines. , - for example, kennedy et al. reported that % of parents with a child still living at home in were opposed to compulsory vaccination laws and that this opposition was associated significantly with beliefs in the safety and efficacy of vaccines. in an analysis of the - nis, gust et al. found that more than % of parents had delayed their child's for recombinant vaccines, this involves many unit operations of column chromatography and ultrafiltration. formulated vaccine may include an adjuvant to enhance the immune response, stabilizers to prolong shelf life, and/or preservatives to allow multidose vials to be delivered. reviews vaccine policy first vaccination and that % had refused vaccination. concerns about vaccine safety were associated significantly with both of these behaviors. as described above, at least two important positive externalities (i.e., benefits accruing to individuals other than the original supplier and patient) can be attributed to vaccines: ( ) vaccination helps protect even those individuals not receiving the vaccine by reducing the transmission of a given disease and ( ) reductions in the burden of infectious disease in the th century have been linked to considerable economic expansion during that period. however, vaccine manufacturers cannot capture these third-party benefits. this problem, together with other supply-side (e.g., barriers to entry) and demand-side (e.g., vaccine financing) issues have resulted in market failure (i.e., a quantity and variety of vaccine products supplied that is below the social optimum) in the vaccine market. thus, both government policy makers and various health philanthropies have implemented a number of proposals aimed at overcoming these issues. these policies can be described as either "push" or "pull" strategies. push strategies seek to address supply-side issues in the vaccine market by providing direct assistance to ease the burden of research, development, and production costs, whereas pull strategies are designed to manipulate demand for vaccines, thereby improving the likelihood of a return on investment by increasing the number of immunizations administered. thus, push mechanisms can be thought of as funding inputs, while pull mechanisms can be thought of as paying for outputs. financial incentives. large, government-funded research and academic institutions play a vital role in basic vaccine research. public funding of vaccine discovery and early developmental efforts coupled with tax subsidies to private firms can reduce manufacturers' upfront financial outlays substantially and alter return on investment calculations for vaccine research favorably. , for example, nih sponsors approximately one-third of all vaccine-related basic research. most of this funding is in the form of grants to academic institutions and health-related agencies. the bioshield act of (p.l. - ) conferred more authority and leadership in the vaccine development effort on the national institutes of allergy and infectious diseases (niaid). the law increased the federal share of bioterrorism projects and allowed niaid to hire technical experts and to award grants and contracts for advancing r&d efforts for specific vaccines. to date, funds from the act have provided support for the r&d of new smallpox and anthrax vaccines. after the bioshield act, in , congress enacted the pandemic and all-hazards preparedness act (p.l. - ). this act gave authority for the advanced development and acquisitions of medical countermeasures to the biomedical advanced research and development authority. fda fast-track mechanism. the fda modernization act of (p.l. - ) directed fda to issue guidance describing its policies and procedures pertaining to fast-track products. fda's fast-track mechanism is designed to facilitate the development and expedite the review of new vaccines intended to treat serious or life-threatening conditions. the mechanism emphasizes early communication between the manufacturer and fda. this allows the manufacturer and fda to discuss development plans and strategies that can improve the efficiency of preclinical studies of the drug and focus efforts on the design of the major clinical efficacy studies before a formal submission of a bla. this early interaction can help clarify goals and plan early for obstacles that might delay approval decisions for a new vaccine. biovest international inc.'s biovaxid (a therapeutic vaccine focused on follicular non-hodgkin's lymphoma) and intracel's oncovax vaccine (designed to prevent recurrence in stage colon cancer) are recent examples of vaccines that have been granted fast-track status. fda accelerated approval. for certain biological products that are being tested for treatment of a serious or life-threatening illness, fda regulations allow "accelerated approval" of the biologic product based on the biologic products' "meaningful therapeutic benefit over existing treatments." , fda grants this approval on the basis of adequate and well-controlled clini- cal trials establishing that the biological product has an effect on a surrogate endpoint that is reasonably likely to predict clinical benefit. for example, in an effort to meet the increasing need for a flu vaccine, the fda approved fluarix, an influenza vaccine for adults that contains inactivated virus through this mechanism. the manufacturer demonstrated that after vaccination with fluarix, adults made levels of protective antibodies in the blood that fda believes are likely to be effective in preventing influenza. fluarix was the first vaccine approved using the accelerated approval process. fda priority review. under the fda modernization act, reviews for new drug applications (ndas) or blas are designated as either standard or priority. the review period changes depending on the designation given to the drug. drugs given a standard designation usually require months to more than year for review. the priority designation can, however, shorten the anticipated amount of time until approval decision from months to months for some products. the priority review process begins only when a manufacturer officially submits a bla (or an nda). priority review, therefore, does not alter the steps taken in a vaccine's development or testing for safety and effectiveness. merck's human papillomavirus vaccine-the first developed to prevent cervical cancer-was evaluated and approved in months under the priority review process. liability protections and safety solutions. because pharmaceutical manufacturers have expressed liability concerns as an important reason for abstaining from vaccine development, proposals addressing these concerns have been seen as necessary incentives to participation in vaccine development. in response to the safety concerns and the lawsuits against vaccine manufacturers, congress enacted the national childhood vaccine injury act of (p.l. - ). this legislation established the vaccine injury compensation program in , which ensures that individuals or families of individuals who may have been injured as a result of a routinely recommended vaccine are quickly, easily, and appropriately compensated. an individual claiming injury or death from a vaccine files a petition for compensation with the court. the petition is reviewed to determine whether it meets the criteria for compensation. a vaccine injury table lists and explains injuries/conditions that are presumed to be caused by vaccines. it also lists time periods in which the first symptom of these injuries/conditions must occur after receiving the vaccine. to qualify for compensation, a petitioner must show that an injury found in the vaccine injury table occurred or must prove that the vaccine caused the condition. a case found eligible for compensation is scheduled for a hearing to assess the amount of compensation. most noncompensable claims receive awards for attorney fees and costs. congressional approval of this act also set in motion the vaccine adverse event reporting system for monitoring vaccine adverse events. the homeland security act of (p.l. - ) protects manufacturers and health care workers who administer the smallpox vaccine from tort liability and restricts the liability assumed by the united states to negligence of those parties. the smallpox emergency personnel protection act of (p.l. - ) created a mechanism to compensate individuals who, in response to a secretarial request for smallpox vaccine preparedness, are injured by the vaccinia virus used in the smallpox vaccine. vaccine recipients and individuals contacted by them are eligible for medical care expense reimbursement, lost income benefits, and death benefits, administered through the health resources and services administration. the public readiness and emergency preparedness act of , (p.l. - ) is a tort liability shield that immunizes vaccine manufacturers, distributors, program planners, and administrators. the act protects these entities from financial risk in the event of any loss related to the manufacture, testing, development, distribution, administration, and use of countermeasures against chemical, biological, radiological, and nuclear agents of terrorism, epidemics, and pandemics. public-private partnerships. donors, foundations, and other partners have created a public-private partnership known as the global alliance for vaccines and immunization (gavi), the mission of which is to save children's lives and protect people's health through the widespread use of vaccines. as a gavi partner, the bill and melinda gates foundation has invested millions of dollars in r&d for vaccines for diseases such as malaria and human immunodeficiency virus, currently the leading killers of children and adults around the world. gavi has established public-private partnerships to accelerate late-stage development and introduction of priority vaccines against disease such as rotavirus and pneumococcus. stockpiles. stockpiles are, put simply, an artificial enhancement to current market demand levels in anticipation of periods when supply will be insufficient to meet demand. government funding of vendor-managed stockpiles of childhood vaccines ensures that some excess vaccine supply is always available to buffer supply problems when they occur. currently, the united states has a large enough stockpile of smallpox vaccine to vaccinate every person in the country who might need it in the event of an emergency. the government also expects to stockpile nearly million doses of an investigational vaccine against pandemic influenza, and studies are under way to develop mechanisms that could stretch that supply to cover more than one-third of the population. cdc also maintains a large anthrax vaccine stockpile. advance market commitments. advance market commitments involve donors who commit to buying yet-to-be-developed vaccines in bulk for poor nations if drug makers are able to deliver a vaccine that meets specifications and a price can be settled on in advance. supporters of advance market commitments range from the gavi partners to pope benedict xvi. donors have agreed to test this mechanism for a vaccine for pneumococcal disease. to date, the gates foundation, the united kingdom, italy, canada, norway, and russia have committed a total of $ . billion for the project. vaccine bonds. the united kingdom has taken a lead in promoting an international financing facility for immunization (iffim) iffim has raised more than $ billion in capital markets to immunize poor children in developing nations against reviews vaccine policy vaccine-preventable diseases. iffim plans to invest $ billion over the next decade to immunize million people who would not otherwise be protected from diseases that no longer represent public health threats in developed countries. the iffim mechanism concentrates on the funding for vaccine research by using long-term government commitments as security bonds issued in the capital markets. the cash received for the bonds then can be used for research and future purchase of vaccines. whenever the bonds are issued, iffim pays bondholders a modest rate of interest. as money pledged by donor governments becomes available gradually over years, these funds will be used to repay the capital value of the bonds. iffim was able to double the resources gavi has been able to allocate-$ . million in compared with $ . million in . vaccine financing programs. historically, the u.s. immunization system has been financed through public-private sector partnerships. the public sector purchases vaccines for approximately % of the birth cohort. section (a federal discretionary grant program to all states), the vaccines for children (vfc) act of (p.l. - ), and state funds are major public sector sources for vaccine financing. private sector vaccine purchases are covered through private health insurance and account for % to % of the pediatric vaccines sold annually in the united states. the federal government has played an evolving role in building the immunization structure in the united states. the earliest legislation pertaining to vaccine financing is the social security act of . title v of this act pertains to immunization services for children and their mothers. in , congress enacted the vaccine assistance act (section of the public health service act). this legislation provided grants to social service agencies and local health departments for immunization infrastructure and vaccine purchases. however, barriers to immunization access still remained in some areas as a result of considerable variability in immunization efforts by state and local governments. the deficiencies in this legislation were highlighted by the measles epidemic of - , which involved more than , cases and led to deaths. , substantial numbers of unimmunized preschool children, particularly in inner-city areas, contributed to this event. to ensure that vulnerable children had more reliable access to vaccines, the government refocused their funding resources on helping individual states in building immunization infrastructure. vfc is a state-operated federal entitlement program that provides free advisory committee on immunization practices-recommended vaccines to children years of age or younger who are uninsured, alaska native or native american, eligible for medicaid, or receive their vaccines in a federally qualified health center. at the state level, funds are earmarked for vaccine purchase and immunization programs. state funds also have been used to purchase vaccines for children and adolescents not eligible for vfc. a combination of vfc, state/local, and section program funds (i.e., vfc only, vfc and underinsured, vfc and underinsured select, universal, and universal select) has been used by a number of states to purchase all recommended vaccines for children in the state, including the privately insured. many states use universal programs that expand the eligibility for vfc vaccines by supplementing vfc purchases at federally discounted prices. the universal purchase states have been successful in raising vaccination rates among the underinsured and increasing access to newer and more expensive vaccines for children without insurance. however, criticisms of the universal purchase programs have been raised, including ( ) vaccine manufacturers' claims that universal purchase programs unfairly provide for the purchase of all vaccines at lower government contract prices, thus eliminating the private market for vaccines and decreasing revenue; ( ) although immunization charges are reduced under this program, patients still pay for the vaccine administration fee; and ( ) some contend that taxpayer money should not be spent to provide free vaccines for children whose insurance would otherwise pay for it. state medicaid and state children's health insurance program funds also are provided for vaccine purchase, although the level of medicaid funding varies from state to state. in contrast to vaccine coverage for children, adults are far less likely to be covered for immunization services and frequently face a problem of underinsurance. the federal medicare program covers some immunizations for all eligible beneficiaries through the medicare part b program. the selected immunizations include influenza, pneumococcal, and hepatitis b vaccinations. certain other vaccines (e.g., tetanus toxoid) also are covered if their administration is considered necessary in the treatment of another covered illness. the part d program generally covers those vaccines not available for reimbursement under medicare parts a or b when administration is reasonable and necessary for the prevention of illness. private insurance coverage of immunizations for working-age adults varies widely by the type of health plan. for example, health maintenance organizations typically have the highest coverage levels, while preferred provider organizations and indemnity plans historically have covered immunization services less frequently. by saving millions of lives and millions of dollars, vaccines have been responsible for some of the greatest successes in public health. however, the struggle against infectious disease is a continual process requiring new vaccines for the challenges that may confront human health in the future. the vaccine market is fragile and requires both supply-and demandside interventions. vaccine availability has been limited by the number of suppliers, high r&d and production costs, and safety problems leading to increased regulatory requirements. demand has been constrained by rapidly increasing vaccine costs, financing issues that have constrained efforts to achieve targets set for population immunization rates, and parental attitudes regarding the safety and efficacy of vaccine products. to date, the u.s. government, in concert with private philanthro-vaccine policy reviews pies, has implemented a patchwork of policies to make the vaccine market more attractive for private firms and to increase access to these products for individuals. we would argue that what is needed is an integrated policy approach that preserves incentives for market entry and innovation in the vaccine industry while simultaneously addressing parental vaccine concerns and increasing immunization funding and reimbursement for both providers and patients. year legislation description the vaccine act the first federal law dealing with patient protection and therapeutic substances. an agent to be appointed for preserving the genuine vaccine matter and to furnish the same to any citizen of the united states, whenever it may be applied for, through the medium of the post office. packets not exceeding half an ounce and relating to vaccination to go free of postage to and from the agent. biologics control act in addition to the testing of the final product, this act also mandated the testing and control of manufacturing materials and establishments. vaccine assistance act (section of the public health service act) infrastructure support for preventive health services such as immunization activities, including vaccine purchase assistance, is provided under section of the public health service act. national childhood vaccine injury act ensures that children who might be injured as a result of a routinely recommended vaccine are quickly, easily, and appropriately compensated. this act set into motion vaers for monitoring vaccine adverse events. vaccines for children act state-operated federal entitlement program that provides free acip-recommended vaccines to eligible children through age years. fda modernization act: fast-track mechanism the mechanism is designed to facilitate the development and expedite the review of new potential vaccines intended to treat serious or life-threatening conditions. fda modernization act: priority review reviews for ndas or blas are designated as either standard or priority. the review period changes depending on the designation given to the drug. homeland security act protects manufacturers and health care workers who administer the smallpox vaccine from tort liability and restricts that liability assumed by the united states to negligence of those parties. smallpox emergency personnel protection act mechanism to compensate individuals who, in response to a secretarial request for smallpox vaccine preparedness, are injured by the vaccinia virus used in smallpox vaccines. vaccine recipients and their contacts are eligible for medical care expense reimbursement, lost income benefit, and death benefits, administered through hrsa. project bioshield act increased the federal share of bioterrorism projects and allowed niaid to hire technical experts and to award grants and contracts for advancing the research and development efforts in vaccine areas. pandemic and all-hazards preparedness act intended to improve u.s. public health and medical preparedness and response capabilities for emergencies, whether deliberate, accidental, or natural. public readiness and emergency preparedness act tort liability shield intended to protect vaccine manufacturers, distributors, program planners, and administrators of vaccines from financial risk in the event of a loss from vaccines. abbreviations used: acip, advisory committee on immunization practices; bla, biological license application; hrsa, health resources and services administration; nda, new drug application; niaid, national institutes of allergy and infectious diseases; vaers, vaccine adverse event reporting system. immunizations in the united states: success, structure, and stress the vaccine industry: does it need a shot in the arm? national health policy forum vaccines: the controversial story of medicine's greatest lifesaver. part ii vaccine manufacturing: challenges and solutions economic evaluation of the -vaccine routine childhood immunization schedule in the united states cost-effectiveness of influenza vaccination of healthy children herd immunity and herd protection vaccines in the public eye demographic change and economic growth in east asia the health and wealth of nations vaccination greatly reduces disease, disability, death and inequity worldwide the health of nations: the contribution of improved health to living standards impact of vaccines universally recommended for children: united states, - . mmwr morb mortal wkly rep effectiveness and cost-benefit of influenza vaccination of healthy working adults: a randomized control trial vaccines for preventing influenza in healthy adults financing vaccines: in search of solutions that work putting markets to work in vaccine manufacturing why are pharmaceutical companies gradually abandoning vaccines? centers for disease control and prevention. vaccines & immunizations: immunization news strengthening the supply of routinely recommended vaccines in the united states: recommendations from the national vaccine advisory committee red book online: special alert: hib shortage vaccine shortages: history, impact, and prospects for the future reuters health information. tropical dengue fever may threaten u.s.: report. accessed at malaria surveillance: united states epidemiological and genetic analysis of severe acute respiratory syndrome just the facts: vaccines provide effective protection and fda makes sure they are safe crs report for congress: vaccine policy issues the epidemiology of rotavirus diarrhea in the united states: surveillance and estimates of disease burden vaccine product approval process the price of innovation: new estimates of drug development costs the fragility of the us vaccine supply merck recalls childhood vaccine financing vaccines in the st century lessons learned: new procurement strategies for vaccines: final report to the gavi board early thimerosal exposure and neuropsychological outcomes at to years neuropsychological performance years after immunization in infancy with thimerosal-containing vaccines autism's false prophets: bad science, risky medicine, and the search for a cure withdrawal of rotavirus vaccine recommendation financing immunizations in the united states vaccine shortages: history, impact, and prospects for the future national, state, and local area vaccination coverage among children aged - months: united states new data show unacceptably low adult immunization rates and that adults unaware of infectious disease threat. accessed at www.reuters.com/article/pressrelease/ idus + epidemiology: infectious diseases: preparing for the future gaps in vaccine financing for underinsured children in the united states estimating medical practice expenses from administering adult influenza vaccinations primary care physician perspectives on reimbursement for childhood immunizations qualitative analysis of mothers' decision-making about vaccines for infants: the importance of trust vaccine beliefs of parents who oppose compulsory vaccination parents with doubts about vaccines: which vaccines and reasons why cost-benefit analysis: concepts and practice crs report for congress: rs project bioshield project bioshield: protecting americans from terrorism department of health and human services. pandemic and all-hazard preparedness act guidance for industry, fast track drug development programs: designation, development, and application review biovest moves to fast track with cancer vaccine fda grants "fast track" designation for the development of oncovax government printing office. cfr subpart e: accelerated approval of biological products for serious or life-threatening illnesses cfr -code of federal regulations title fda approves new influenza vaccine for upcoming flu season fast track, priority review and accelerated approval fda licenses new vaccine for prevention of cervical cancer and other diseases in females caused by human papillomavirus department of health and human services, health resources and services administration. national vaccine injury compensation program (vicp). accessed at www.hrsa.gov/vaccinecompensation crs report for congress: rl , homeland security act of : tort liability provisions crs report for congress: rl , smallpox vaccine injury compensation public readiness and emergency preparedness act questions and answers. accessed at www.hhs.gov/disasters/emergency/manmadedisasters/bioterorism/medication-vaccine-qa.html accessed at www.gavialliance.org the problems and promise of vaccine markets in developing countries department of health and human services. cdc efforts in implementing a smallpox vaccination program builds stockpile of vaccine for flu pandemic why a market-driven vaccine plan faces big obstacles vaccine bonds provide model for other aid projects. accessed at www.medscape.com/viewarticle/ gavi alliance announces dramatic funding boost for hib vaccine assuring vaccination of children and adolescents without financial barriers: recommendations from the national vaccine advisory committee (nvac) measles surveillance: united states accessed at www. cdc.gov/vaccines/programs/vfc childhood vaccine supply policy impact of north carolina's universal vaccine purchase program by children insurance status adult immunization. accessed at www.cms.hhs.gov/adultimmunizations instructions: the assessment test for this activity must be taken online; please see "cpe processing" below for further instructions. there is only one correct answer to each question. this cpe will be available at www.pharmacist.com no later than july , . . which of the following is the process by which vaccines protect not only those who receive them but also those who cannot or do not receive the vaccine? a. passive immunization b. active immunization c. herd to obtain . contact hour of continuing pharmacy education credit ( . ceus) for "vaccine supply, demand, and policy: a primer," go to www.pharmacist.com and take your test online for instant credit. cpe processing is free for apha members and $ for nonmembers. a statement of credit will be awarded for a passing grade of % or better. you have two opportunities to successfully complete the posttest. pharmacists who complete this exercise successfully before july , , can receive credit.the american pharmacists association is accredited by the accreditation council for pharmacy education as a provider of continuing pharmacy education. the acpe universal activity number assigned to the program by the accredited provider is - - - -h -p."vaccine supply, demand, and policy: a primer" is a home-study continuing education activity for pharmacists developed by the american pharmacists association. key: cord- -jahnjvyk authors: hasford, joerg title: large simple double-blind randomized trials for the rapid assessment of the effectiveness of covid- vaccines date: - - journal: j infect dis doi: . /infdis/jiaa sha: doc_id: cord_uid: jahnjvyk nan large simple double-blind randomized trials for the rapid assessment of the effectiveness of covid- vaccines to the editor-the coronavirus disease (covid- ) pandemic has brought not only far too many losses of human lives but an economic crisis as well. thus, effective treatments and vaccines for severe acute respiratory syndrome coronavirus (sars-cov- ) are urgently needed. eyal et al have discussed challenge studies [ ] to accelerate the assessment of vaccine effectiveness. in a challenge study, all participants are vaccinated with placebo or with the test vaccine and are then intentionally exposed to doses of sars-cov- . there is already a worldwide initiative to register volunteers for such studies [ ] . as all participants have been exposed, the effectiveness of a vaccine can be assessed with smaller sample sizes and possibly more quickly compared to the conventional trial with community participants; however, challenge studies are accompanied by serious ethical issues [ ] . first, the characteristics of a distinct group of volunteers without risks for fatal progression or serious late complications of covid- need to be reliably known. second, a highly effective and safe treatment should be available for patients with covid- , so that fatalities and persistent adverse consequences can be avoided. both these problems are not yet solved. third, the consent of the volunteer must be with their full understanding of comprehensive information, including appreciation of potential long-term consequences. it may be questioned, however, whether someone at age years or so can imagine the consequences of a scarred lung occurring many years later. finally, it cannot be taken for granted that a vaccine that works in a challenge study with young, healthy volunteers will work in elderly patients with possible comorbid conditions [ ] . thus, even the social value of challenge trials can be questioned. fortunately, there is an ethically more acceptable alternative for an accelerated evaluation of sars-cov- vaccines. the large, simple, randomized trial (lsrt), as proposed by yusuf et al in , is a reliable, methodologically and ethically sound alternative [ ] . characteristics of this design include: wide, simple eligibility criteria; central randomization; recording of only few baseline data; simple and short-term treatment; reduced or no follow-up visits; and outcome assessments of hard endpoints, preferably by registries. using this design, truly large randomized trials with sample sizes beyond patients have been carried out and answered important questions [ ] . probably due to the increasing bureaucratization of clinical research activities, including very costly monitoring, in the last years there has been an almost complete disappearance of this trial design. at the current stage of evaluation of the effectiveness of sars-cov- vaccines, this design should be revived as it is ideally suited for this task. there would be wide eligibility criteria with very few exclusion criteria as the vaccine should become available for almost everybody. the investigational vaccine is a or -time treatment only and no follow-up visits are needed. the outcomes, covid- or death, can be collected either by registries available in many countries (eg, in the united states, united kingdom, or scandinavia) or by patient reporting. vaccine safety information can be collected by established systems like the vaccine safety datalink in the united states [ ] , prescription event monitoring programs (eg, the drug surveillance research unit in the united kingdom [ ] ), or by direct patient safety reporting on websites, including those accessible with smartphones [ ] , which can be specifically designed for vaccine trials. among the advantages of using the lsrt design are that it allows central randomization of large numbers of volunteers within a short time and rapid collection of the relevant outcomes at a low cost compared to the conventional phase trials with many follow-up visits and extensive monitoring. adaptive design features (eg, modification of the eligibility criteria considering the accruing safety information) are feasible as well. given the wide entry criteria, the results provide external validity for large parts of the population compared to any challenge trial, which would need to focus on participants with extremely low risks for developing serious covid- . as there will be very many people who would like to participate in such a vaccination trial, the sample sizes needed should be achieved within a very short time. when the lsrt double-blind design is used, the validity of the results is assured and it does not generate the serious ethical issues inherent in challenge trials. regarding the salk vaccine, large randomized trials with sample sizes of more than were done in the early s [ ] and such lsrts should be feasible in . thus, the sponsors of vaccine trials and the drug regulatory agencies should start the preparatory work now to be ready once an investigational vaccine is ready to be administered on a large scale. disclaimer. the opinions expressed do not necessarily represent those of the association of medical ethics committees in germany. potential conflicts of interest. author certifies no potential conflicts of interest. the author has submitted the icmje form for disclosure of potential human challenge studies to accelerate coronavirus vaccine licensure covid- human challenge trials experimental infections in humans-historical and ethical reflections vaccines to prevent infectious diseases in the older population: immunological challenges and future perspectives why do we need some large, simple randomized trials isis- : a randomised comparison of streptokinase vs tissue plasminogen activator vs anistreplase and of aspirin plus heparin vs aspirin alone among , cases of suspected acute myocardial infarction vaccine safety datalink patent safety making history: thomas francis, jr, md, and the salk poliomyelitis vaccine field trial key: cord- -ruf vvz authors: sohrab, sayed sartaj title: an edible vaccine development for coronavirus disease : the concept date: - - journal: clin exp vaccine res doi: . /cevr. . . . sha: doc_id: cord_uid: ruf vvz a novel coronavirus was emerged in december from wuhan city, china and has now become a global threat to human health. currently, the coronavirus disease (covid- ) has spread to more than countries with , deaths and , confirmed cases. currently, there is no vaccine available against covid- . the traditional vaccines development requires more time and high cost and due to this, the disease outbreaks becomes more challenging. now a days, plants have become more attractive platform for edible vaccine production than the other system. the development of an edible vaccine in a selected plant system has many significant advantages such as; easy and efficient oral delivery, low cost with higher scale production, avoidance of any trained medical personnel for delivery, lack of any pathogenic infection, multicomponent expression in a single plant, and so forth. in this manuscript, the concept, development, and importance of an edible vaccine have been discussed. by using this plant-based platform, an edible vaccines can be produced in many crops like banana, cucumber, carrot, lettuce, and tomato against various diseases. due to increasing cases globally with covid- , there is an urgent requirement to develop an ideal vaccine and antiviral therapy against this virus to control the disease worldwide. china with , deaths and , laboratory confirmed cases [ , ] . based on medical and healthcare workers, the medical supplies are getting low due to the high number of cases in wuhan city. to manage the cases, china has announced to build two new hospitals with , beds within days which is functional now. the outbreak caused by covid- in wuhan is an epidemic threat to global health [ , ] . currently, there are many systems available such as bacteria, yeast, and mammalian cell lines to express the recombinant subunit vaccines and therapeutic proteins but all the above systems have some disadvantages like high cost, safety, and target integrity. while, by using plant-based system is very useful and avoids all the issue as compared to other system and provides low cost and better safety with high scalability without harm with any pathogenic infections. this is the most recent and alternative technology for vaccine development. the expected protein folding and post-translational modifications of the proteins can be produced in the correct form in plant-based systems with the desired biological functions [ ] . plant-based edible vaccines have been recently introduced for vaccine production. the main goals of plant-based edible vaccines are the transformation and production of antigens into plants and the oral consumption of such vaccine to induce antigen specific immune responses. currently, the use of plant-based expression system platform have been extensively utilized for the expression and purification of vaccines, recombinant proteins, enzymes, and many bio-pharmaceuticals in a variety of plant species, including potato, corn, tomato, carrot, lettuce, and spinach and have reached at advanced stage of pre-clinical and clinical evaluation. the oral administration of edible vaccines is a preferable route of vaccination for being a simple and safe route of administration; the low production cost allows for local production and minimal plant material processing; natural bio-encapsulation and hence, stability in the gastrointestinal (gi) tract; and protective immunogenicity at the gi mucosa. the current status of plant-based vaccine and their clinical evaluation are summarized in table [ , ] . the plant-based vaccine development idea was started years ago and since then many vaccine proteins have been produced in plants for human and animal diseases [ ] [ ] [ ] [ ] [ ] . since more than years, plant science has grown tremendously and shown great importance towards molecular pharming and have significant achievements in large scale with low cost production of recombinant proteins, enzymes, and pharmaceuticals compounds. currently, various pharmaceutical compounds, antigen, antibodies, hormones, enzymes, and growth regulators have been developed into multiple plants system [ ] [ ] [ ] [ ] . the main objective of this review is to provide the latest information about the use of plant-based platform for the development of an edible vaccine. for plant-based vaccine development, the desired plant with efficient and quick regeneration and transformation properties should be selected. the desired gene can be delivered into plant cells to express desired proteins/pharmaceuticals compounds with low cost and high scale [ , , ] . this technology has attracted the researchers because of elimination of a specific animal requirement to conduct the experiment. the edible vaccines can be designed in such a way that, the expressed and produced proteins should not be pathogenic. the production of conventional vaccines is very expensive, and they require purification and refrigeration. apart from that, the plant-based vaccines are the most suitable for children as an oral delivery. currently, world health organization has suggested new technologies for the vaccine development. the specific proteins can be expressed into desired plants with very less cost and can be grown to the required locations so that, an edible vaccine can be available to the needy population globally, especially in the developing countries. due to high cost, storage, refrigeration, transportation, and requirements of trained medical personnel, an injectable vaccine cannot be easily taken in developing countries. additionally, various pathogenic organism, bacterial and viral diseases can be easily transmitted by re-use of needles. mono-clonal antibodies are being used in the treatment of arthritis and cancer. the monoclonal antibodies can be easily produced in desired transgenic plants with very less cost and time duration. it has been reported that the developed transgenic rice was stable at room temperature for months. because these vaccines are needle-free [ ] , they have the added advantage of eliminating the associated waste and potential for dissemination of blood borne-infections. antigens are released in the form of vaccine through bio-encapsulation which protects them from gastric enzymes. the released proteins were absorbed by m cells in the intestinal wall and passed on to the macrophages and antigen-presenting cells and local lymphocyte populations generating serum immunoglobulin (ig)g, ige, and local iga responses and memory cells which neutralize the attack by a real pathogen. the development and stability of edible vaccine as an antigen has been investigated in many recent reports. the current status of plant-based vaccines has been reviewed and presented [ ] . the concept of edible vaccine development has been presented in fig. . currently, the plant-based vaccine has many valuable advantages as compared to traditional vaccines which include. adjuvants require to enhance immune responses. ( ) orallyintroduced antigens elicit mucosal immunity. ( ) it is easy to bulk produce onsite, transport and stored with less cost and without refrigeration. ( ) no injection and need for trained medical person. ( ) easy to express, separate and purify the protein. ( ) they can be stored as seeds and oils and dried tissue without any refrigeration. ( ) they do not have any risk of contamination and disease spread. ( ) there is the possibility of enhanced compliance, especially in children [ ] . edible vaccines have received considerable attention from researchers in both academia and industry. the first plant-derived rabies vaccine was produced in tomatoes and offered the advantages of high biomass yields and the increased containment by growth in greenhouses. lettuce and bananas have also been utilized for the production of plant-based vaccines [ ] [ ] [ ] . the edible can be developed in many desired plants. recently, there are different crops like tomato, carrot, corn, cucumber, lettuce, and spinach plants that have been utilized as a green factory to express, and purify the desired protein, enzymes, antigens, and biopharmaceuticals compounds [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the most important advantage of plant expression systems is the low cost. tomato can be used as for vaccine production and the used as salad which facilitates the easy oral delivery of a particular vaccine. as an example, tomato has excellent biomass and easily transformed and generate the whole plant within short period and this provides an ideal choice for vaccine production. therefore, tomato is no doubt a green vaccine factory. currently, there is no approved vaccine or treatment with proven efficacy against newly emerged cov covid- . while previous work on sars-cov vaccines have shown that the spike glycoprotein (s) is the main inducer of neutralizing antibodies. the covs infection causes severe respiratory disease with clinical symptoms; specifically, patients develop acute respiratory symptoms. the spike protein can be used to develop a vaccine against the covid- . the spike (s) protein gene or subunit of spike like s subunit can be cloned into a plant expression vector and the desired plant like tomato, cu-cumber, or lettuce can be transformed. the developed transgenic plants can be used as salad and easily delivered orally and immunized the human against the newly emerged virus. several teams are working on a vaccine to protect against the new cov. recently, the therapeutic options for covid- has been discussed and suggested in a published paper [ ] . however, the development of a protective vaccine is of great importance to prevent and control the spread of the virus as well as any future outbreaks. the newly emerged cov has now become a global threat to human health. as on february , , this virus has been spread to more than countries including china with , deaths and , confirmed cases [ , ] . the fast spread of this virus has attracted the researchers towards the development of an urgent vaccine to control the virus infection and disease spread. the full-genome of covid- has provided the valuable information which can be used to design and develop an effective vaccine [ ] . the plant-based edible vaccine has now attracted many researchers and pharmaceutical companies to develop fast and cheaper vaccines against many pathogens. this technology has got more publicity in the last years and have proven the efficiency to express the desired protein either as antigens or pharmaceuticals therapeutic compounds in the desired plant cells. this novel technology provides the high and fast expression, purification, and better stability of desired proteins in to plant cells as well as their removal of refrigeration requirement and trained medical personnel for delivery. the edible vaccine also removes the risk factors and safety and regulatory issues related to living organisms. the edible vaccine can be grown at the site of requirement and orally delivered in the form of salad. therefore, this technology is emerging as a novel and alternative methods for large scale edible vaccine production, manufacturing, and processing as well as commercialization and easily availability to the needy persons globally. this technology will provide an effective vaccination against many diseases in human and animal globally. based on current information, it is concluded that there is an urgent need to develop a vaccine against this fast spreading novel cov by using various vaccine development technology. the edible vaccine technology will provide a fast and cheaper vaccine not only this new virus but also against many other pathogens. disease outbreak news (dons) [internet]. geneva: world health organization novel coronavirus ( -ncov) situation summary recent progress in the development of plant derived vaccines clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond recent development and future prospects of plant-based vaccines plant-based vaccines against viruses plants as an alternative source of therapeutic proteins new strategies toward edible vaccines: an overview plant-made vaccines and reagents for the one health initiative production of tetravalent dengue virus envelope protein domain iii based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development process development strategies in plant molecular farming farming of plant-based veterinary vaccines and their applications for disease prevention in animals hussein s. plant-based vaccines: production and challenges needle-free vaccine delivery therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in wuhan, china: version preliminary phylogenetic analysis of ncov- genomes key: cord- - y d brz authors: häfner, sophia title: contagion . () date: - - journal: microbes infect doi: . /j.micinf. . . sha: doc_id: cord_uid: y d brz nan contagion . * vaccination is probably medicine's greatest success story up to date [ ] . smallpox has definitively been eradicated in , poliomyelitis is close to extirpation, measles, pertussis or tetanus are widely controlled [ ] . vaccines comprise an arsenal of dead and mutated specimens, pieces and secretions, synthetic peptides and chimaeras, all of them dispensing acquired immunity to a specific disease. live attenuated vaccines, in particular, miming best the "natural" infection, hold the advantage of eliciting the most complete and long-lasting immunization, are relatively inexpensive to produce, suitable to be distributed to large populations and do not require adjuvants. random chemical mutagenesis has given way to site-directed genetic engineering and multiple licensed vaccines for bacterial and viral pathogens [ ] . vaccination is a fascinating performance. it is the interplay between the immune system and the invading pathogens, mobilizing in turns hordes of specialized cell populations, shaped and tuned by eras of co-evolution. but as expected from any highly complex, multi-leveled system, it is not entirely predictable. the work of yanfen ma and colleagues in this issue prove how unforeseeable the genetic manipulation of the pathogen can be, where the simple insertion of a transgene produces an attenuated strain even in the absence of triggering the intended suicide mechanism [ ] . vaccination started long before the molecular details behind infection, contagion and immunity were elucidated, and vaccine production has partially stayed a carefully supervised empirical science. several attenuated strains used for vaccination arose by chance over passages, while the very necessity to maintain and amplify the strains requires further passaging, rendering vaccine production also a major logistic challenge, in order to assure the safety, quality and homogeneity of products over the world, including the panoply of issues linked to the preservation, transport and administration of the vaccines [ ] . vaccination is an economic and political issue. currently, south korea and china are in the grip of the largest outbreak of mers (middle east respiratory syndrome) since the first reports of the virus in , a virus with high structural similarity to the sars coronavirus (severe acute respiratory syndrome), which infected and killed people in / . mers originated in bats, uses camels and dromedaries as an animal reservoir, can be transmitted from human to human and elicits respiratory distress plus multiorgan failure in its victims, with a fatality rate of about % [ ] . however, despite the repeated call from scientists and several valuable discoveries which could probably be transformed into treatments and vaccines [ ] , the pharmaceutical companies showed limited interest in investing into mers, as the disease kept a relatively low profile over the past three years and numerous other catastrophes fought simultaneously for economic and media attention. now, at the time of mid-june, mers accounts for cases of infection, casualties and over quarantined people and the need for efficient countermeasures urges [ ] . yet the epidemics market is unpredictable and the career of an average health minister can be endangered as much by prudence and vaccine stockpiling as by negligence. vaccination is a sociological issue. individual freedom and rights come across social responsibility and civic duty [ ] . as vaccination works by herd immunity to dyke up the spread of infectious pathogens, it requires a vast majority of individuals to conform to mandatory vaccination, notably members of specific professions such as health care workers [ ] . however, the mentality of the modern western world has shifted to the worship of the individual, asserting round the clock that it is the duty of society to insure its unconditional well-being, and not that it might be the individual's duty to ensure the society's wellbeing. accepting a minor risk at the individual level in order to decrease the risk at the group level has become way less self-evident for a generation that has never witnessed the real ravages of vaccine-preventable diseases but is rather obsessed with the precise caloric content of % organic quinoa instead. moreover, personal exemptions from mandatory vaccination are often allowed e states of the usa give leave to religious and to philosophical exemptions, whatsoever the precise definition of the latter might be [h] . yet, * article highlight based on "inducible suicide vector systems for trypanosoma cruzi" by yanfen ma et al. [ ] . studies show that outbreaks of measles and pertussis correlate with higher numbers of exemptors and anti-vaccine movements [ ] . and finally, vaccination is the sad occasion to study the spread of a novel, extremely virulent and contagious disease [ ] . the one of misinformation, fear and doubt, via a medium a thousand times more efficient then the release of an influenza virus in the middle of woodstock e the internet. the global reach and instantaneous nature of virtual information has rendered harmless neighborhood rumor spreading a dangerous and powerful tool with a major sociological and political impact [ ] . scientists might roll their eyes over ten thousands of "likes" of a webpage claiming that vaccines cause homosexuality and tear one's hair because of andrew wakenfields fraudulent research linking mmr vaccine to autism and the ensuing "overnight autism" stories. once anti-vaccine forums appear in the vicinity of the centers for disease control (cdc) upon online research for "vaccines", leading to noticeable vaccine refusal, delayed vaccine schedules, measles being declared endemic in the uk and diphtheria cases in spain however, the alarm bell should ring. moreover, while the veracity of the content of any account, preferentially emphasized by horrifying pictures, is not put to the test, the tool that is meant to democratically make information available to everyone is ironically a hotspot of censorship -the content of many fervid anti-vaccine sites is tightly controlled and any comment relating a deviant view is immediately removed [ ] . partially due to a hint of snobbism, the scientific and medical community is running behind in the world of web . . neither do sober statistics and evidence-based information written in technical jargon have the same appeal as emotion-driven horror stories, nor does an equivalent in form of pro-vaccine narratives and listings of uneventful vaccinations exist [ ] . studies have shown that the tone and nature of comments published alongside a neutral article presenting a new technology significantly skew the reader's interpretation of risks associated with the latter, way more than the effective article content, demonstrating how easily the average internet user is manipulated by his peers [ ] . as long as no vaccine has been developed against the propagation of myths and conspiracies, the scientific world should better speed up its domestication of social media in order to re-establish a correct perception of the risk-benefit output of vaccines. as for the alternative solutions, web cartoonist and ex-nasa roboticist randall munroe, furnishing "serious scientific answers to absurd hypothetical questions" via his "what if?" blog [ ], has addressed the issue if common cold (i.e. rhinoviruses) could be eradicated by all individuals of the planet keeping the maximal possible distance from each other (i.e. about m) for a couple of weeks. apart from not working, due to reservoirs in for example immunocompromised persons, this would probably cause the breakdown of global economics [ ] . currently, no suitable vaccine is available for protecting against t. cruzi infection, despite considerable research in this area. although several experimental vaccines provide some protections against t. cruzi infection, as compared to live attenuated t. cruzi vaccines, these approaches do not provide a strong and long-lasting immunity against t. cruzi infection. in t. cruzi, attenuated parasites still pose health risks, since it is has been impossible to eliminate the parasitism in the immunized hosts, and attenuated parasites may revert to virulence particularly when immunized hosts develop immune compromise. to overcome this obstacle, we sought to develop a new method to prevent persistence, based on the introduction of an inducible detrimental/toxic gene into t. cruzi, which results in killing the organism upon the induction of the detrimental/toxic gene. such an inducible strain could be useful as a vaccine to induce protective immunity. once a protective immune response is present, any latent parasites could be eliminated by inducing the detrimental/toxic gene, producing a "sterile" immunity. the system we have developed can be used in any strain of t. cruzi (including field isolates) allowing this approach to create multiple vaccine strains. . what was your first reaction when you faced the results? did you expect them? we expected the toxin-dddha to kill the organism upon induction. however, we also unexpectedly made a discovery when we tested whether the ddd system could be regulated to stabilize fusion proteins in intracellular amastigotes. human foreskin fibroblasts were infected with ptrex-gfp-dddha strain and in absence of induction, amastigotes were replicating inside the host cells normally. then, nm tmplactate was added to the culture medium to induce gfp-dddha stabilization, which resulted in intracellular amastigote death. the dddha peptide is detrimental to intracellular amastigotes. . how will the project go on? this system has significant potential as a bio-safety device in t. cruzi which can be used in combination with other techniques such as gene deletion or radiation to develop safe animal and human vaccines. . what is the take-home message of the article? we established effective inducible systems for t. cruzi employing the degradation domain based on the escherichia coli dihydrofolate reductase (ecdhfr). the dhfr degradation domain (ddd) can be stabilized by trimethoprim-lactate and can be used to express detrimental or toxic proteins to induce intracellular amastigote death. the transgenic strains were attenuated in mouse experiments producing no pathological changes and inoculation with these dddha strains in mice provided strong protection against lethal wild type infection. the technique may lead to a breakthrough in t. cruzi vaccine design. . do you have a personal motto, quote or leading sentence? "imagination is more important than knowledge" . what advice would you give to the young next-generation scientists? ask fundamental questions and do good science. i would like to meet carlos chagas in order to define the disease mechanism and invent methods to prevent the disease. i would like to check out if there will be a safe and reliable vaccine to prevent t. cruzi infection and effective and safe drugs for treatment of chagas disease. vaccines are antigenic substances that provoke an immune response, leading to long-lasting protective immunity against one or several related pathogens vaccines exist against numerous bacterial, viral and parasitic pathogens they can consist in inactivated or attenuated microorganisms, fragments or products of the pathogen as well as chimeric constructs stable integration of constructs carrying the dhfr degradation domain (ddd) alone or coupled to either gpf, a-toxin or cecropin a was achieved in trypansoma cruzi sufficient degradation of gfp, a-toxin and cecropin a by the ddd domain was observed in the absence of stabilization upon addition of trimethoprim (tmp)-lactate, stabilization of the gfp and a-toxin proteins was reached, killing the intracellular amastigotes in mice, the transgenic strains are highly attenuated with and without tmp-lactate addition and confer protection against wild-type t. cruzi inducible suicide vector systems for trypanosoma cruzi live attenuated vaccines: historical successes and current challenges live attenuated vaccines for invasive salmonella infections middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor calling for rapid development of a safe and effective mers vaccine individual and community risks of measles and pertussis associated with personal exemptions to immunization ethics of mandatory vaccination for healthcare workers social science. science, new media, and the public story and science: how providers and parents can utilize storytelling to combat anti-vaccine misinformation what if? serious scientific answers to absurd hypothetical questions key: cord- -swxs pr authors: lacaille-dubois, marie-aleth title: updated insights into the mechanism of action and clinical profile of the immunoadjuvant qs- : a review date: - - journal: phytomedicine doi: . /j.phymed. . sha: doc_id: cord_uid: swxs pr abstract background vaccine adjuvants are compounds that significantly enhance/prolong the immune response to a co-administered antigen. the limitations of the use of aluminium salts that are unable to elicite cell responses against intracellular pathogens such as those causing malaria, tuberculosis, or aids, have driven the development of new alternative adjuvants such as qs- , a triterpene saponin purified from quillaja saponaria. purpose the aim of this review is to attempt to clarify the mechanism of action of qs- through either receptors or signaling pathways in vitro and in vivo with special emphasis on the co-administration with other immunostimulants in new adjuvant formulations, called adjuvant systems (as). furthermore, the most relevant clinical applications will be presented. methods a literature search covering the period – was performed using electronic databases from sci finder, science direct, medline/pubmed, scopus, google scholar. results insights into the mechanism of action of qs- can be summarized as follows: ) in vivo stimulation of th humoral and th cell-mediated immune responses through action on antigen presenting cells (apcs) and t cells, leading to release of th cytokines participating in the elimination of intracellular pathogens. ) activation of the nlrp inflammasome in mouse apcs with subsequent release of caspase- dependent cytokines, il- β and il- , important for th responses. ) synthesis of nearly qs- analogs, allowing structure/activity relationships and mechanistic studies. ) unique synergy mechanism between monophosphoryl lipid a (mpl a) and qs- , formulated in a liposome (as ) in the early ifn-γ response, promoting vaccine immunogenicity. the second part of the review is related to phase i-iii clinical trials of qs- , mostly formulated in ass, to evaluate efficacy, immunogenicity and safety of adjuvanted prophylactic vaccines against infectious diseases, e.g. malaria, herpes zoster, tuberculosis, aids and therapeutic vaccines against cancer and alzheimer's disease. conclusion the most advanced phase iii clinical applications led to the development of two vaccines containing qs- as part of the as, the herpes zoster vaccine (hz/su) (shingrix™) which received a license in from the fda and a marketing authorization in the eu in and the rts,s/as vaccine (mosquirix™) against malaria, which was approved by the ema in for further implementation in sub-saharan countries for routine use. vaccines are the most effective and least expensive methodology for preventing diseases caused by infectious pathogens. recently emerging or re-emerging diseases such as the severe acute respiratory syndrome (sars) in , h n influenza pandemic in or ebola virus in (meaning to help) are substances used in combination with a specific antigen (ag) to produce a more potent and persistent immune response against a specific disease than the ag alone. the aim is to confer longterm protection, and with the additional benefit that less antigen and fewer injections are needed (lee and nguyen, ) . adjuvants are used in many vaccines, but their mechanisms of action have not been fully elucidated. they include diverse classes of compounds such as mineral salts, microbial products, emulsions, saponins, cytokines, polymers, microparticules and liposomes (awate et al., ) . up to now, few adjuvants have been approved for use in humans (del giudice et al., ) . aluminium salts, which have been extensively used as adjuvants in vaccines for more than years, induced robust antibody responses but weak th cell type responses, which are instrumental for protection against many pathogens. the new generation of vaccines using purer components for safer vaccines (e.g.,subunits vaccines with highly purified recombinant antigens) are less immunogenic in contrast to live attenuated or inactivated whole-cell vaccines. therefore, they required the induction of strong cellular responses including cd + t-helper cells (th) and cd + cytotoxic t lymphocytes (ctl) in addition to antibody (ab) responses. thus, the development of new adjuvants is necessary for vaccines that require a cell-mediated immune response (cmi). among them, the triterpene glycosides represent an interesting class of clinically relevant adjuvants, particularly those of quillaja saponaria (qs). these amphiphatic plant glycosides possess a variety of pharmacological activities including immunoadjuvant, antitumor, anti-inflammatory, and antimicrobial properties which have been extensively studied in many in vitro and in vivo bioassays (lacaille-dubois and wagner, , , lacaille-dubois, , ). despite the apparent success of qs- as vaccine-adjuvant in the last years, there is an urgent need for a deeper understanding of its mode of action which could accelerate the development of new vaccine strategies. this requires an interdisciplinary approach between chemists, biochemists, molecular biologists and immunologists. the following review will summarize updated insights into the modes of action, the structure/activity relationships allowing detailed mechanistic studies, and the clinical status of the vaccine adjuvant qs- . with a growing understanding of the innate immune system (response first mediated by antigen presenting cells (apcs)) and its role in activation and modulation of adaptive immune responses (ag-specific b and t cell responses) di pasquale et al., ) , the mechanisms of action of adjuvants are being elucidated. they can act on one or more of the following targets to increase response to ags: ( ) sustaining release at the injection site (depot effect), ( ) transient secretion of cytokines and chemokines, ( ) recruitement of various immune cells (neutrophils, monocytes, eosinophils, macrophages and dendritic cells (dcs) at the injection site leading to a local immune-competent environment, ( ) expression by the recruited apcs of various pathogen recognition receptors (prrs) both on their surface (toll-like receptors, tlrs, c-type lectin receptors, clrs), and intracellularly (nucleotide oligomerization domain (nod)-like receptors (nlrs) and retinoic inducible gene- (rig)-like receptors (rlrs)), which are recognized and/or activated by adjuvants, ( ) maturation and activation of recruited apcs which up-regulate the expression of major histocompatibility complex (mhc)-i and/or mhc-ii and activation of co-stimulatory signals cd , cd / , ( ) increased capacity of apcs for ag processing and presentation by mhc, ( ) migration of the mature apcs to the draining lymph nodes (dlns) to interact with ag-specific b or t lymphocytes (through receptor-ligand interactions, mhc-t cell receptor (mhc-tcr), cd -cd l, cd / -cd ) which are activated to produce potent ab-secreting b cells and/or effector cd + t cell responses (awate et al., ) . researchers continue to characterize the adaptive immune response and to clarify the respective roles of vaccine-induced immune effectors ( fig. ) . they include abs produced by b lymphocytes that bind specifically to a toxin or a pathogen, memory b cells produced only when b cells received t cell help having the ability to respond rapidely on reexposure of the same antigen, cytotoxic cd + t lymphocytes that limit the spread of infectious agents by killing infected cells or secreting specific cytokines, and cd + t helper (th) lymphocytes. various th subsets have been defined through their cytokine profiles and ability to induce b cell and cd + t cell responses (reed et al., ; siegrist ). the th -type cd + t cells essentially release the awate et al., ; reed et al., ; lacaille-dubois and wagner, ) . phytomedicine ( ) proinflammatory cytokines interleukin- (il- ), interferon-γ (ifn-γ), tumor necrosis factor-α (tnf-α), which participate in the elimination of intracellular pathogens both directly (cytokine response) or indirectly via cd + t cell activation and differentiation in ctls (cellular immune response) and the generation in mice of the complement-fixing abs igg a and igg . the th -type cd + t cell response is characterized by secretion of il- , il- and il- providing b cell help and the preferential induction of igg , ige, and iga in mice, and ige and igg in humans (humoral response) (guy, ) . the saponin qs- has been isolated from quillaja saponaria tree bark (kensil et al., ) and is one of the most potent adjuvants known which is currently used in exploratory, and a few licensed, vaccines. qs- is a mixture of two isomeric molecules, qs- apiose (qs- api), and qs- xylose (qs- -xyl) ( : ), each having four domains: the triterpene quillaic acid, a branched trisaccharide linked at c- through an o-heterosidic bound, a linear tetrasaccharide linked at c- through an ester bound, and a glycosylated pseudodimeric acyl chain attached through a labile ester linkage at c- of the fucose unit of the tetrasaccharide (fig. ) . this compound showed a potent adjuvant activity against a wide variety of ags with low toxicity in preclinical studies in mice, guinea pigs, monkeys and baboons. it was shown to stimulate th and th immune responses, when added to vaccine (kensil, ) . this adjuvant is a candidate for many clinical trials of vaccines directed against infectious diseases, cancer, and others. however, the exact role of qs- , either through activation of receptors or signaling pathways, is poorly characterized. some hypotheses advanced that qs- may facilitate ag uptake into apcs by binding to cell surface lectins through its carbohydrate domains, leading to specific cytokine profiles that enhance the t and/or b cells' response (marciani, ) . an effective mechanism of action in which qs- apparently acts on both dcs and t cells was proposed (marciani, ) (fig. ) . exogenous ags and qs- enter dcs by cholesterol-dependent endocytosis. the high affinity of qs- for endosomal membrane cholesterol resulted in the destabilization of the membrane (lorent et al., ) leading to pore formation, thus facilitating the escape of the antigen to the cytosol for further processing inside the cell into peptides. they are then loaded to mhc-i and presented on the dc surface to naïve cd + t cells to yield ctls. the role of aldehyde in qs- was highlighted in the t cell by forming an imine with the amino group of the t cell surface receptor such as cd , providing a costimulatory signal to the t cell. this signal together with the mitogen-activated protein kinase (mapk) erk resulting from the activation of the tcr-mhc interaction, and the changes of the ionic balance na + /k + , stimulates t cell activation resulting in the secretion of th cytokines (marciani, ) (fig. ) . recently, studies in mouse apcs (dcs and macrophages) identified qs- as an activator of the nlrp inflammasome. qs- in combination with the tlr -agonist -o-deacyl- ′-monophosphoryl lipid a (mpl a), was shown to activate the acs-nlrp inflammasome, a multiprotein complex and cause subsequent release of caspase- dependent proinflammatory cytokines il- β/il- that can promote th cell maturation or drive inf-γ-mediated th responses, respectively (marty-roix et al., ) . thus, inflammasome signaling has the potential to direct the development of t helper subsets. however, the role of this signaling pathway in vivo remained to be clarified. although qs- has been used in many clinical investigations of vaccines, several drawbacks inherent to the natural product, such as its chemical instability, scarcity, heterogeneity, dose-limiting toxicity and poorly understood mechanism of action have limited its widespread clinical advancement, except for the recent malaria (mosquirix™) and shingles (shingrix™) vaccines developed by glaxosmithkline (gsk). to overcome these limitations, structural modifications of the natural product through chemical synthesis have been undertaken during the last decade, leading to the preparation of nearly saponin analogs, which have provided key insights into structure/activity relationships (sar) which are summarized in fig. . these achievements allowed the identification of new novel saponins probes with potent adjuvant activities, increased stability, and decreased toxicity, and the investigation into their mechanisms of action (fernández-tajeda et al., ; fernández-tejada, ) . firstly, the total synthesis of each isomeric qs- produced this potent adjuvant in high purity and homogeneous composition. then, some qs- variants (sqs- , sqs- , sqs- ) were chemically synthesized with more stable amide linkages in the acyl chain instead of the unstable native esters (fig. ). their immunological evaluation in mice demonstrated adjuvant activities comparable to qs- , as assessed by measuring ab responses by elisa one week after booster injection (day ) and significantly lower toxicity than the natural product, except for sqs- . further modifications in the acyl chain with a dodecanedioic acid and truncation of the tetrasaccharide led to a trisaccharide variant with potent adjuvant activity and whose toxicity issue is not completely resolved . this simplification led to further structural variations at the linker (ester linkage at c- ) between the triterpene and the linear oligosaccharide in order to modulate the distance and orientation of these two domains. the variants exhibit striking differences in adjuvant activity and toxicity in mouse vaccination models, yielding new insights into the structural requirements for adjuvant activity. molecular dynamics simulations of these compounds and qs- api have revealed distinctive conformational features correlating with adjuvant activity, which may help in the design of new analogs (walkowicz et al., ) . the modifications of the tetrasaccharide into a trisaccharide variant, and the acyl chain into a terminal amine variant enables the synthesis of novel acyl chain variants bearing fluorescent and iodinated substituents for subsequent imaging and in vivo biodistribution studies (fernández-tejada et al., ) . they were adjuvant-active in several mouse-vaccination models and less toxic than qs- . early studies of biodistribution and fluorescence imaging allowed investigations into the enigmatic mechanism of action of these saponins. preliminary results in vaccinated mice suggested the potential role of these fluorescent probes in ag ovalbumin (ova) trafficking by apcs from the site of injection to the lns for its presentation to the immune system and yielded some mechanistic understanding of the potentiation of the immune response (fernández-tejada et al., ) . extensive structure/function studies have shown that aryl iodide derivatives with a free c- oh display a good efficacy/ toxicity profile. it was concluded that the trisaccharide at c- was not required for adjuvant activity. modifications at c- aldehyde and c- hydroxyl have shown that echinocystic acid aryl iodide derivatives lacking the c- aldehyde but retaining the c- alcoholic function ( fig. ) exhibited potent adjuvant activity and no toxicity in a preclinical vaccination model using a four-component vaccine (muc -klh, ova, gd -klh) when tested at doses of and μg. these data indicated that the c- aldehyde previously suggested to participate in schiff's base formation with a presumed t cell surface receptor target (marciani, ) , is not required for adjuvant activity and revealed the previously unknown role of the c- alcohol in enhancing activity. this opens the door to semi-synthesis from easily available alternative precursors and allowed detailed mechanistic studies (fernández-tejada et al., ) . one of the promising approaches to improving efficiency of newly developed vaccines is given by the combination of several adjuvants into single formulations, such as the adjuvant systems as , as , as (garçon et al., ) . as , developed more than years ago, is a liposome-based adjuvant comprising two immunostimulants, mpl and qs- . liposomes are synthetic nanospheres consisting of phospholipid bilayers that can encapsulate antigens and act as antigen delivery vehicles (garçon et al., ) . mpl is a detoxified synthetic derivative of the lipopolysaccharide (lps) from the bacteria salmonella minnesota and induces activation of innate immunity via tlr- , stimulating nf-kb transcriptional activity. it stimulates the induction of ag-specific t cells producing interferon-γ (ifn-γ) and igg a antibodies. qs- from q. saponaria (licensed by gsk from antigenics inc., a wholly owned subsidiary of agenus inc., lexington, ma, usa) in the early evaluations as an adjuvant was suggested to promote high agspecific ab responses and cd + t cell responses in mice and high agspecific ab responses in humans (didierlaurent et al., ) . qs- , as an amphiphilic compound is known for its hemolytic properties, which can be abrogated through formulation in liposomes in the presence of cholesterol such as in as (beck et al., ; garçon and di pasquale, ) . this resulted in an acceptable tolerability profile for its use in human vaccines. as contains mpl/qs- in an oil-in-water emulsion. as , combining qs- , mpl, and cpg (an oligodesoxynucleotide and tlr- agonist), in a liposome is the most complex combination of adjuvants which has been used for applications in cancer immunotherapy. the mechanism of action of qs- in a liposome is poorly understood despite the clinical efficacy of as . therefore, some studies were carried out in order to tentatively elucidate the adjuvant effect of qs- in such a formulation. it was observed after an intramuscular (im) immunization in mice against two model antigens, hepatitis b surface antigen (hbsag) and ova that qs- , rapidly accumulated in cd + resident macrophages of the dln, where it induces caspase- activation. these early events then organized the recruitment of innate immune cells and activation of dcs and subsequent t cell priming. further analysis showed that the adjuvant effect of qs- depended on the integration of caspase- and myd pathways at least in part through local release of hmgb (high-mobility-group protein b ) (detienne et al., ) . a recent study reported that qs- directly activated human monocyte-derived dendritic cells (mo-dcs) and promoted a pro-inflammatory transcriptional program . in this model, qs- was endocytosed via a cholesterol-dependent mechanism and accumulated in lysosomes where it induces their destabilization through pore formation and potential release of their contents. this resulted in inflammasome activation, syk-(a tyrosine kinase) and cathepsin-b (a cysteine protease) dependent cell activation and cytokine production in mo-dcs. lysosomal disruption could also affect ag processing and translocation to the cytosol for presentation on mhcs. finally it was shown that cathepsin b contributes to the adjuvant properties of qs- on both cd + and cd + ag-specific t-cell responses in vivo. the adjuvant systems containing mpl/qs in combination with hbsag were shown to induce very strong and persistent humoral and cellular immune responses in healthy adults, as b inducing the strongest and most durable specific cmi after two doses, without serious adverse events. the cd + response was characterized in vitro by high lymphoproliferation, high ifn-γ and moderate il- production. this response was confirmed ex vivo by detection of il- and ifn-γ producing cd + t cells and in vivo by measuring increased levels of ifn-γ in the serum (vandepapelière et al., ) . therefore as b has been selected as lead as for the development of vaccines. experiments were conducted in mouse models with a recombinant varicella-zoster virus (vzv) glycoprotein e (ge) and the fluorescently labeled qs- incorporated in as (hz (shingle) vaccine formulation). as induces a rapid and transient activation of the innate immune system, both at the injection site and in the dln, leading to the generation of high number of efficient ag-loaded dcs in the dln to promote ag-specific adaptive immune responses (didierlaurent et al., ) . a comparative study of the immunogenicity of different formulations containing the subunit ge was carried out in a vzv-primed mouse model (dendouga et al., ) . this model was chosen in order to mimic the clinical setting, in which the large majority of persons at elevated risk for hz are vzv sero-positive. an hz candidate vaccine comprising ge ( μg) and as b ( μg each of mpl and qs- ) was shown to be highly immunogenic in mice, inducing higher responses of cd + t cells expressing il- and/or inf-γ and higher humoral responses compared to other ge/as formulations with different ag doses ( . , . , μg) or with lower doses of adjuvant ( . and . μg each of mpl and qs- , corresponding to as e and as f , respectively) (dendouga et al., ) . furthermore, the contribution of each component of as b to the induction of cellular and humoral response was assessed after immunization of mice with ge either unadjuvanted, or adjuvanted with qs- ( μg), mpl ( μg) (both in liposomes), and as . the results showed that the ge-specific cytokine (ifn-γ, il- ) producing cd + t cell responses induced by ge/liposome+qs and ge/liposome+mpl were of similar and low magnitude, whereas the response induced by ge/as b was significantly higher than ge/liposome+qs ( . fold difference) and ge/liposome+mpl ( . fold difference). this clearly showed that qs- and mpl acted synergistically to induce a high frequency of ge-specific cd + t cells. for the anti-ge antibody responses, an additive effect, rather than a synergistic effect was observed (dendouga et al., ; didierlaurent et al., ) . these results suggested that the ge/as b vaccine candidate was appropriate for further clinical assessment. an investigation of the molecular and cellular mechanism of as adjuvanted vaccines was undertaken in order to clarify how immunostimulants function in combination. . results from in vivo and clinical studies showed that the combination of mpl/ qs- in as resulted in the stimulation of de novo pathways that were not triggered by the individual components. mice were immunized twice, weeks apart, with the rts,s antigen of the malaria vaccine, formulated either without adjuvant, with mpl, with qs- (both included in liposomes) or with as . rts,s is a recombinant protein consisting of repeated sequences of the plasmodium falciparum circumsporozoite protein (csp-repeat region, (r)) and t-cell epitope domain (t) linked to the hepatitis b surface antigen (hbsag) (s), and hbsag alone (s) (moris et al., ) . the csp-specific immune response was measured after the second dose. the results clearly showed that mpl and qs- act synergistically to induce strong antibody and agspecific cd + t cell responses. in contrast, very low levels of cd + t cell responses were generated against csp. further investigations highlighted the key role of the early release of ifn-γ by innate resident cells (nk and cd + t cells) in the ln, draining the injection site, which is essential for the development of the cd + t cell response (th immunity). this was confirmed by measuring the levels of the cytokine after immunization of mice with hbs adjuvanted with as or with its components. as induced the early production of ifn-γ with a peak in concentration at h post immunization ( pg/ml), whereas mpl or qs- alone failed to significantly induce ifn-γ. this effect results from the mpl/qs- synergy and is controlled by macrophages, il- and il- . qs- containing adjuvants have been assessed in more than human clinical trials. as is a key component of the recently developed malaria and hz vaccines and other candidate vaccines under development against infectious diseases, and cancer didierlaurent et al., ; garçon and di pasquale, ) . recent clinical studies of prophylactic vaccines against malaria, hz, hiv, tuberculosis from an efficacy, immunogenicity and safety points of view (table ) and of therapeutic vaccines against melanoma, nsclc and alzheimer's disease, will be reported below. despite effective medicines, insecticide-treated nets and indoor insecticide spraying, malaria killed , people in , particularly sub-saharan african children (world malaria day, ) . the ag of the candidate malaria vaccine rts,s targets the csp which is expressed on the plasmodium sporozoite during the pre-erythrocytic stage of its lifecycle, the stage between mosquito bite and liver infection. rts,s /as , was the first malaria vaccine candidate, in which an adjuvant system was used (garçon et al., ) . however, rts,s/as was shown in a phase ii trial to be well tolerated, to induce strong humoral and cellular immune response and to improve protection against plasmodium falciparum challenge in comparison to rts,s/as (kester et al., ) . namely, it induced high levels of anti-csp igg titers and csp-specific polyfunctional cd + t cells expressing il- , ifnγ, tnfα or cd l which have been associated with protection in the experimental malaria challenge model in adults. other clinical trials in children and adults confirmed these observations and no serious adverse events were reported (agnandji et al., ; olotu et al., ; ansong et al., ; white et al., ; levast et al., ; leroux-roels et al., a) . therefore the development of rts,s/as was stopped and rts,s/s /as was consequently selected for phase iii development . analyses of data in term of efficacy, immunogenicity and safety of phase i-iii trials including adults, infants, and children from sub-saharan countries were reviewed (agnandji et al., ) . clinical trials in children with newly adjuvanted vaccines led to the conclusion of clinical safety except for some unexplained cases of meningitis in one phase iii as adjuvanted malaria vaccine trial, which warrant further safety evaluations (stassijns et al., ) . important insights into the molecular mechanism of protective immunity against malaria were achieved involving additional immunogenicity analyses into the potential contributive roles of csp-specific abs and csp-specific cmi to protection (moris et al., ; kazmin et al., ) leading to the identification of the nf-kb and ifn-γ pathways (van den berg et al., ). the first clinical large-scale phase iii trial (the rts,s clinical trials partnership, ) evaluating a malaria vaccine involving , infants and young children was completed in december at sites from african countries (burkina faso, gabon, ghana, kenya, malawi, mozambique, and tanzania). safety and efficacy studies of the rts,s/ as vaccine (mosquirix™) showed that it prevented many cases of clinical and severe malaria over the months after dose . vaccine efficacy was higher in children ( - months) than in infants ( - weeks), but even at modest levels ( . %). the final results in (the rts,s clinical trials partnership, ) from the same trial, showed the enhanced efficacy by administration of a booster dose in both age categories, months after the first three injections. with regards to risks, the safety of mosquirix is similar to that of other vaccines, the most common side effects being fever, pain and swelling at the injection sites. thus, the vaccine has the potential to make a substantial contribution to malaria control when used in combination with other effective control measures, especially in areas of high transmission. the ratio benefit/risk of mosquirix was found to be acceptable by the european medicines agency's committee for medicinal products for human use (ema/chmp) which delivered a positive scientific opinion in for use outside the european union (eu) (ema press release, ). furthermore, the world health organization recommended a large-scale pilot implementation of the vaccine in young children with a four-dose schedule in african regions of moderate to high parasite transmission (who, ) . three countries, ghana, kenya, and malawi were selected by the who in for this largescale pilot program which started in . it will be the first malaria table clinical trials involving newly adjuvanted prophylactic vacccines. vaccine provided to young children through routine immunization programmes (malaria vaccine implementation programme, ). the results of two phases ( - , and - ) providing insights on the feasibility of delivering the vaccine in real-life settings and on its safety profile in the context of routine use, will contribute to the future decisions on the wider-scale use of the vaccine. herpes zoster (hz), also known as shingles is a common, painful disease occurring principally in adults aged > years and immunocompromised individuals. hz is caused by the reactivation of the latent varicella-zoster virus (vzv) in the dorsal root or cranial ganglia usually many years after a primary vzv infection (chickenpox) that occurs during childhood . hz can lead to serious complications including post herpetic neuralgia (phn). a phase i/ii trial showed that a subunit vaccine containing the recombinant vzv glycoprotein e (ge) as ag and as b (called hz/su) was well tolerated and highly immunogenic with cd + t cell and humoral immune responses, being more immunogenic than a live attenuated vaccine (leroux-roels et al., ) . a phase ii trial in adults > years of age evaluating different formulations (containing , , or μg ge combined with as ) and different schedules showed that all formulations elicited robust humoral and cd + t cell immune responses that persisted for at least years after vaccination. two doses of ge/ as b were more immunogenic than one (chlibek et al., ) . in a follow-up study conducted on healthy adults, it was shown that the ge specific cd + t cell and anti-ge antibody responses persisted for years after two doses of μg ge/as b , without any safety concerns . therefore, the dose of μg ge was selected for other clinical developments. a two randomized placebo-controlled phase iii efficacy trials in countries evaluated the efficacy and safety of hz/su as compared to placebo in older adults (> and > years of age). participants received two im doses of the vaccine or placebo, months apart. hz/su has demonstrated high efficacy ( . % and . % for adults > years and > years, respectively) in preventing hz and phn in all age groups with an acceptable safety profile (lal et al., ; cunningham et al., ) . the robust immune responses remained for years after vaccination as assessed in subsets of participants of the phase iii trials. persistent anti-ge ab and ge-specific polyfunctional cd + t cell responses are likely important mechanisms by which hz/su confers the high efficacy against hz (cunningham et al., ) . in an extension study of the original phase ii trials reported by chlibek et al. ( chlibek et al. ( , , it was observed that hz/ su induces cell mediated and humoral immunity persisting up to years post initial vaccination in adults > years old, confirming statistical prediction models based on immune responses measured at earlier time points. immune responses are predicted to persist up to years after the initial vaccination (schwarz et al., ) . the acceptable benefit/ risks profile led to the licensing by the u.s. food and drug administration (fda) in of the hz/su vaccine (shingrix™) and a marketing authorization valid throughout the eu on march (shingrix, inn-herpes zoster vaccine. ema/ / ). shingrix is preferred to zostavax (a live attenuated vaccine), providing strong protection against shingles and phn. first introduced in , the bacillus calmette-guérin (bcg) vaccine is the only currently licensed vaccine available to protect against tuberculosis (tb), a disease caused by the bacteria mycobacterium tuberculosis (m.tb.). it protects children from meningeal and severe forms disseminated of tb and death, but offers limited protection against pulmonary tb in adolescents and adults, which is the form of the disease responsible for the vast majority of transmission and tb-related morbidity and mortality. therefore, there is an urgent need for a new and improved vaccine against tb for controlling this disease that continues to pose a global health threat with . million deaths in (van der meeren et al., ) . the m /as candidate vaccine contains the recombinant fusion protein derived from the two immunogenic m. tuberculosis ags (mtb a and mtb a) in as . a first phase i/ii randomized, controlled clinical study with m /as was carried out in belgium in healthy adults volunteers. the results showed that the vaccine was clinically well tolerated and induced high and persistent (up to three years) specific th cd + t-cell (co-expressing cd l, il- , tnf-α and ifn-γ) and ab responses, supporting its further clinical evaluation in tb-endemic settings (leroux-roels et al., ) . similar conclusions were drawn in a phase ii controlled trial conducted on healthy, hiv uninfected adolescents living in an area with high tb burden in south africa (penn-nicholson et al., ) . in a phase ii study m /as given to infants after or concomitantly with expanded-programme-on-immunization (epi) vaccines had an acceptable safety profile. these results suggested no interference of immunogenicity profiles occurred following co-administration of m /as and epi vaccines. two m /as doses elicited higher immune responses than one dose (ikodo et al., ) . in a large phase iib trial conducted on hiv-uninfected adults latently infected with mtb in three african countries, m /as e was shown to provide % protection for mtbinfected adults against active pulmonary tb, without evident safety concerns (van der meeren et al., ) . these results suggest further evaluations of m /as as a possible vaccination strategy against tb. there were approximately . million people living with hiv at the end of with . million people becoming newly infected in (hiv/aids, key facts, july ). while treatments are available to treat or to prevent hiv infections, there is no vaccine currently licensed. despite ongoing prevention efforts, there is an urgent need for a safe and effective prophylactic vaccine. prevention of infection through induction of neutralizing abs, induction of strong cellular immune responses in order to delay progression and reduce the transmission rate in high risk population, are the major aims for an hiv vaccine. although clinical attempts were disappointing, they contributed valuable insights into immune protective immunity. a randomized double-blind study conducted on healthy hiv-seronegative adults showed that a vaccine formulation containing gp , nef and tat ags and as b induced strong ab response and cd + t cell responses characterized by high lymphoproliferative capacity and il- production, that were maintained up to months after the last vaccine dose. a cd + t cell response was not observed (leroux-roels et al., ) . this study conducted with different adjuvants underlined the superiority of responses with as b in comparison with other adjuvants, confirming previous findings. another candidate hiv- vaccine consisting of a recombinant fusion protein (f ) encoding four hiv- clade b ags (p , p , reverse transcriptase (rt) and nef) adjuvanted with as was studied in a phase i/ii trials with healthy hiv-seronegative volunteers (van braeckel et al., ) . this vaccine was shown to be immunogenic with an acceptable safety profile. after two doses of μg, strong polyfunctional (cd l, il- , tnf-α, ifn-γ phenotype) and persistent cd + t cell responses were induced, suggesting that this vaccine merits further evaluation both in a prophylactic setting and as a potentially disease-modifying therapeutic vaccine in hiv- infected subjects. a phase i clinical study in such subjects has been initiated (harrer et al., ) . a phase ii study was conducted with the f /as b candidate vaccine on healthy adults in order to evaluate the effect of chloroquine on the specific cd + t cell responses and the safety profile of a booster dose (leroux-roels et al., b) . the results showed that a booster dose, administered alone or two days after chloroquine induced a strong ab immune response and robust f -specific cd + t cell response, but no significant cd + t cell response. this suggest that two primary doses of the f /as b vaccine induce long-lasting memory b cell and cd + t cell responses in healthy adults, confirming previous findings of the phase i/ii study (leroux-roels et al., b) . this vaccine was shown to be immunogenic with a clinically acceptable safety profile, but doesn't show significant viral efficacy in antiretroviral therapy-naïve hiv- -infected adults, results confirmed in a long term study (dinges et al., ; harrer et al., ) . the use of immunotherapeutic vaccines in cancer patients is aimed at inducing immune responses against existing tumors rather than protecting healthy individuals. they have been used in phase ii and iii trials against melanoma and lung cancer after surgical resection, and the disappointing results of the most recent studies will be shortly presented here. enhanced production of ganglioside gm is observed in different human tumor types including melanoma. anti-gm vaccines such as gm conjugated to a carrier keyhole limpet hemocyanin (klh) and administered with qs- induced high igm and igg ab responses but failed to show a beneficial effect in melanoma patients as it was shown in a phase iii clinical trial in term of relapse free survival, distant metastasis-free survival and overall survival (eggermont et al., ) . a five-years survival occurs for less than % of the patients with completely resected non-small-cell-lung cancer (nsclc). therapeutic cancer vaccination has the potential for eliminating remaining cancer cells after complete resection, thereby decreasing relapse rates and improving survival (vansteenkiste et al., ) . ag-specific immunotherapies enhance t-cell responses against specifically expressed tumor ags. in this context, the use of mage-a (melanoma antigen family a, ) as ag, which is expressed in a wide array of malignancies can be an application in cancer immunotherapy (esfandiary and ghafouri-fard, ) . the mage-a /as immunotheraputic vaccine was assessed for clinical efficacy in an international, multicenter phase iii study in surgically resected nsclcs. this study confirmed the acceptable clinical safety profile of the vaccine, but did not improve overall survival. therefore, its further development for use in nslcs has been stopped (vansteenkiste et al., ) . similar conclusions were drawn from a phase iii trial of a mage-a /as vaccine in patients with resected, mage-a -positive, stage iii melanoma, which led to the arrest of the clinical development of this vaccine for use in melanoma (dreno et al., ) . the pathogenesis of alzheimer disease (ad) is associated with the accumulation of amyloid β (aβ) and/or hyperphosphorylated tau proteins in the brain. active immunotherapy of ad is the most extensively studied approach, in order to evaluate its ability to elicite anti-aβ antibodies, to induce aβ clearance, and reduce aβ accumulation in the brain. vaccination with a full length aβ peptide (aβ - ) successfully elicited anti-aβ-ab in human subjects with ad, but adverse effects such as meningoencephalitis were observed, attributed to t cell activation (winblad et al., ) . to avoid this safety issue, a n-terminal (aβ − ) peptide conjugated to a carrier protein called vanutide cridificar (acc- ) was produced. in preclinical studies, it was shown to generate nterminal aβ abs in adult nonhuman primates without inducing aβ-directed t cell response, supporting further clinical studies. several phase ii trials in patients with mild to moderate ad were conducted in usa, europe, and japan by using doses of acc- ( , and μg) with and without the adjuvant qs- ( μg) or placebo to investigate doserange, safety, immunogenicity and long term treatment (arai et al., ; pasquier et al., ) . acc- +qs- elicited high, sustained anti-aβ igg ab titers compared with acc- alone, highlighting the role of qs- in this effect. no difference was observed among the three doses tested. furthermore, acc- at all dose levels with or without qs- was generally safe and well tolerated, but exploratory cognitive evaluations did not show differences between treatment groups and placebo. similar safety, tolerability and anti-aβ-igg immunogenicity profiles were observed in long term phase ii extensions studies, suggesting that side effects do not limit in principle anti-amyloid active immunotherapy (hüll et al., ) . however, larger trials of adequate duration, with optimized dosing may be needed to attest efficacy of anti-aβ vaccine therapy for ad. this overview provides a summary of recent reports on the mechanism of action of qs- , a promising triterpene glycoside vaccine adjuvant isolated from quillaja saponaria and the most relevant clinical applications. this compound showed a potent adjuvant activity stimulating humoral and cell-mediated immunity against a wide variety of ags and presented advantages over aluminium salts by inducing a th type immune response, necessary for controlling most intracellular pathogens. it is unlikely that a single mechanism of action could explain its effects as an adjuvant. this contribution highlights: ) the role of qs- acting on both apcs and t cells. the interaction between the aldehyde function and t-cell receptors such as cd , delivering co-stimulatory signals together with the presentation of the ag peptides by apcs to the t-cell resulted in the activation of t lymphocytes and secretions of th cytokines, an important immune effector mechanism for the elimination of infected cells. ) a novel mechanism of action based on the activation of caspase dependent nlrp inflammasome in mouse apcs, leading to the secretion of proinflammatory cytokine il- β/il- , which are important for th responses. ) the synthesis of nearly structural analogs of qs- and detailed sar studies leading to more easily available, and stable molecules with an improved activity/toxicity profile, among them a novel aryl iodinated simplified echinocystic derivative showing a potent adjuvant activity and considerably attenuated toxicity. ) the signaling pathways of qs- when formulated in a liposome, such as the rapid accumulation in dln resident cd + macrophages, where it induces caspase- activation and hmgb release. these events orchestrate the recruitment of innate immune cells and activation of dcs leading to t cell priming. a direct activation of dcs was also reported, resulting in release of cathepsin b which contribute to specific t cell responses in vivo. ) the development of adjuvant systems (as) including a combination of immunostimulants in a formulation, such as as (qs- / mpl in a liposome), in which they were shown to act synergistically in enhancing cmi responses. ) the high number of clinical applications in term of immunogenicity, efficacy, safety of qs- alone or in adjuvant systems in many prophylactic vaccines against complex pathogens, e.g. malaria, herpes zoster, tuberculosis, hiv, and therapeutic vaccines, e.g. cancer, ad. ) the licensing by the us fda in of a vaccine against shingles called hz/su (shingrix™) containing ge/as and its commercialization in the eu since march . ) the first malaria vaccine (mosquirix™) containing rts,s/as receiving a regulatory approval by the ema in for use outside of europe and being currently under exploration in sub-saharan regions for its use in routine. ) the disappointing phase iii clinical results in term of disease-free survival of patient with immunotherapeutic vaccines adjuvanted with qs- /as against nsclc and melanoma despite promising results of phase ii trials. ) the encouraging results of a phase ii trial confirming that the long term treatment with the immunotherapeutic vaccine vanutide cridificar (acc- ) adjuvanted with qs- against alzheimer's disease was well tolerated with an acceptable safety profile. the development of adjuvants in vaccines represents an area in constant evolution with substantial advances over the past two decades. qs- in ass is a key component of multiple vaccines targeting infections and endemic diseases in developing countries. recent research has led to the commercialization of shingrix™ and the approval of mosquirix™, two vaccines against hz in adults and malaria in children, respectively, which contain qs- as a part of their formulation. to ensure a continuous supply of this exciting molecule, the researchers aimed in the near future to find an alternative innovative process of producing this compound on a large scale based on plant cell-culture. this might overcome some limitations of the current traditional techniques of extraction with tedious purification processes and chemical synthesis involving many complex reactions. however, the synthetic technology initially used for the first synthesis of qs- , was subsequently applied to preparing novel structural analogs with potent activity and low toxicity, leading to sar studies and a better understanding of the mode of action. further work in this area could facilitate novel saponin design that will lead to the discovery of new adjuvants and improved adjuvant-antigen combinations for future vaccines, particularly against infectious tropical diseases such as dengue, leishmaniasis, and chagas diseases, which continue to cause significant morbidity and mortality in developing countries. evaluation of the safety and immunogenicity of the rts,s/as e malaria candidate vaccine when integrated in the expanded program of immunization clinical development of rts,s/as malaria vaccine, a systematic review of clinical phase i-iii trials t cell responses to the rts,s/as e and rts,s/as d malaria candidate vaccines administered according to different schedules to ghanaian children vanutide cridificar and the qs- adjuvant in japanese subjects with mild to moderate alzheimer disease: results from two phase studies mechanisms of action of adjuvants detection of liposomal cholesterol and monophosphoryl lipid a by qs- saponin and limulus polyphemus amebocyte lysate safety and immunogenicity of three different formulations of an adjuvanted varicella-zoster virus subunit vaccine in older adults: a phase ii, randomized controlled study long-term immunogenicity and safety of an investigational herpes zoster subunit vaccine in older adults cellular and molecular synergy in as -adjuvanted vaccines results in an early ifnγ response promoting vaccine immunogenicity efficacy of herpes-zoster subunit vaccine in adults years of age or older immune responses to a recombinant glycoprotein e herpes zoster vaccine in adults aged years or older correlates of adjuvanticity: a review on adjuvants in licenced vaccines cellmediated immune responses to a varicella-zoster virus glycoprotein e vaccine using both a tlr agonist and qs- in mice central role of cd + lymph node resident macrophages in the adjuvanticity of the qs- component of as enhancement of adaptive immunity by the human vaccine adjuvant as depends on activated dendritic cells adjuvant system as : helping to overcome the challenges of modern vaccines the f /as b hiv- vaccine candidate is safe and immunogenic, but doesn't show viral efficacy in antiretroviral therapy-naïve, hiv- infected adults: a randomized controlled trial vaccine adjuvants: from to and beyond. vaccines mage-a immunotherapeutic as ajuvant therapy for patients with resected, mage-a -positive, stage iii melanoma (derma): a double blind, randomized, placebo-controlled adjuvant ganglioside gm -klh/qs- vaccination versus observation after resection of primary tumor > . mm in patients with stage ii melanoma: results of the eortc randomized phase iii trial mage-a : an immunogenic target used in clinical practice design, synthesis and evaluation of optimized saponin variants derived from the vaccine adjuvant qs- development of a minimal saponin vaccine adjuvant bsed on qs- development of improved vaccine adjuvants based on the saponin natural product qs- through chemical synthesis from discovery to licensure, the adjuvant system story glaxosmithkline adjuvant systems in vaccines: concepts, achievements and perspectives understanding modern vaccines: perspectives in vaccinology the perfect mix: recent progress in adjuvant research safetey and immunogenicity of an adjuvanted protein therapeutic hiv- vaccine in subjects with hiv- infection: a randomized placebo-controlled study long-term follow-up of hiv- -infected adults who received the f /as b hiv- vaccine candidate in two randomized controlled trials long term extensions of randomized vaccination trials of acc- and qs- in mild to moderate alzheimer's disease safety and immunogenicity of the m /as candidate tuberculosis vaccine when given as a booster to bcg in gambian infants: an open-label randomized controlled trial system analysis of protective immune responses to rts,s malaria vaccination in humans saponins as vaccine adjuvants separation and characterization of saponins with adjuvant activity from quillaja saponaria molina cortex randomized, doubleblind, phase a trial of falciparum malaria vaccines rts,s/as b and rts,s/as a in malaria-naïve adults: safety, efficacy, and immunologic associates of protection a review of the biological and pharmacological activities of saponins bioactive saponins from plants: an update new perspectives for natural triterpene glycosides as potential adjuvants saponins as immunoadjuvants and immunostimulants bioactive saponins with cancer related and immunomodulatory activity: recent developments efficacy of an adjuvanted herpes zoster subunit vaccine in older adults recent advances of vaccine adjuvants for infectious diseases evaluation of the immune response to rts,s/as and rts,s/as adjuvanted vaccines immunogenicity and safety of a booster dose of an investigantional adjuvanted polyprotein hiv- vaccine in healthy adults and effect of administered chloroquine improved cd + t cell responses to mycobacterium tuberculosis in ppd negative adults by m /as as compared to the m /as and mtb f/as tuberculosis candidate vaccine formulations: a randomized trial strong and persistent cd + t-cell response in healthy adults immunized with a candidate hiv- vaccine containing gp , nef and tat antigens formulated in three adjuvant systems a phase / clinical trial evaluating safety and immunogenicity of a varicella zoster glycoprotein e subunit vaccine candidate in young and older adults vaccine potentiation by combination adjuvants amphiphilic nature of saponins and their effects on artificial and biological membranes and potential consequences for red blood and cancer cells vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity elucidating the mechanisms of action of saponin-derived adjuvants identification of qs- as an inflammasome-activating molecular component of saponin adjuvants characterization of t-cell immune responses in clinical trials of the candidate rts,s malaria vaccine efficacy of rts,s/as e malaria vaccine and exploratory analysis on anti-circumsporozoite antibody titres and protection in children aged - months in kenya and tanzania: a randomized controlled trial two phase multiple ascending dose studies of vanutide cridificar (acc- ) and qs- adjuvant in mild-to-moderate alzheimer's disease the vaccine study team, . safety and immunogenicity of vaccine m /as e in adolescents in a tb endemic setting key roles of adjuvants in modern vaccines efficacy and safety of the rts,s/as malaria vaccine during months after vaccination: a phase randomized, controlled trial in children and young infants at african sites efficacy and safety of rts,s/as malaria vaccine with or without a booster dose in infants and children in africa: final results of a phase , individually randomised, controlled trial vaccines", section , general aspects of vaccination a systematic review and metaanalysis on the safety of newly adjuvanted vaccines amound children persistence of immune response to an adjuvanted varicella-zoster virus subunit vaccine for up to year nine in older adults an adjuvanted polyprotein hiv- vaccine induces polyfunctional cross-reactive cd + t cell responses in seronegative volunteers predicting rts,s vaccine-mediated protection from transcriptomes in a malaria-challenge clinical trial phase b controlled trial of m /as e vaccine to prevent tuberculosis vaccine adjuvant systems containing monophosphoryl lipid a and qs- induce strong and persistent humoral and t cell responses against hepatitis b surface antigen in healthy adult volonteers efficacy of the mage-a immunotherapeutic as adjuvant therapy in patients with resected mage-a -positive non-small-cell lung cancer (magrit): a randomized, double-blind, placebo-controlled quillaja saponin variants with central glycosidic linkage modifications exhibit distinct conformations and adjuvant activities lysosome-dependent activation of human dendritic cells by the vaccine adjuvant qs- the relationship between rts,s vaccine-induced antibodies, cd + t cell responses and protection against plasmodium falciparum infection active immunotherapy options for alzheimer's disease. alzheimer's res. ther. , . world health organisation, . malaria vaccine: who position paper first-malaria-vaccine-receivespositive-scientific-opinion-ema_en.pdf. ema press release . first malaria vaccine receives positive scientific opinion from ema there are no conflicts of interest associated with this publication. key: cord- - xkt u authors: petrini, stefano; righi, cecilia; iscaro, carmen; viola, giulio; gobbi, paola; scoccia, eleonora; rossi, elisabetta; pellegrini, claudia; de mia, gian mario title: evaluation of passive immunity induced by immunisation using two inactivated ge-deleted marker vaccines against infectious bovine rhinotracheitis (ibr) in calves date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: xkt u different types of vaccines against infectious bovine rhinotracheitis (ibr) are commercially available. among these, inactivated glycoprotein e (ge)-deleted marker vaccines are commonly used, but their ability to induce passive immunity is poorly known. here, we evaluated the passive immunity transferred from dams immunised with commercial inactivated ge-deleted marker vaccines to calves. we vaccinated pregnant cattle devoid of neutralising antibodies against bovine alphaherpesvirus (bohv- ) and divided them into two groups with animals each. both groups were injected with a different inactivated ge-deleted marker vaccine administrated via intranasal or intramuscular routes. an additional pregnant cattle served as the unvaccinated control group. after calving, the number of animals in each group was increased by the newborn calves. in the dams, the humoral immune response was evaluated before calving and, subsequently, at different times until post-calving day (pcd ). in addition, the antibodies in colostrum, milk, and in serum samples from newborn calves were evaluated at different times until pcd . the results indicated that inactivated glycoprotein e (ge)-deleted marker vaccines are safe and produce a good humoral immune response in pregnant cattle until calving and pcd . moreover, results showed that, in calf serum, passive immunity persists until pcd . bovine alphaherpesvirus (bohv- ) causes various clinical syndromes among cattle populations, including respiratory disease, vaginitis, balanoposthitis, abortions, enteritis, and encephalitis, which may be observed after acute infection or subsequent to viral recrudescence, following periods of stress [ , ] . to date, finland, sweden, norway, denmark, austria, switzerland, as well as the province of bolzano in italy, have successfully employed a "test-and-slaughter" strategy. other european union states have implemented compulsory eradication programmes combining "test-and-removal" with "vaccination" using marker vaccines, conversely to vaccination strategies used in the usa, where non-marker vaccines are used [ ] . marker vaccines are derived from the deletion of one or more genes responsible for the synthesis of glycoproteins or enzymes [ , ] . in europe, glycoprotein e (ge) of bohv- is the gene marker that is most commonly deleted in modified live or inactivated bohv- diva (differentiating infected from vaccinated animals) vaccines [ , ] . this type of immunisation makes it possible to differentiate animals immunised with marker vaccines (ge negative) from those infected or inoculated with traditional non-deleted vaccines (ge positive) through diagnostic tests specific for bohv- ge [ , , ] . however, a disadvantage of using live marker vaccines is that the virus can remain in a latent state in the immunised animals such that it can be reactivated and excreted following exposure to an immunosuppressive stimulus [ ] . further, marker vaccines administered both intramuscularly and intranasally induce a marked humoral and cell-mediated immune responses [ ] , however, little information is available regarding the induction of passive immunity. regarding traditional vaccines, several reports have shown that cattle vaccinated with non-deleted modified-live vaccines transfer neutralising antibodies (na) to the newborn calves that can protect them from experimental infection [ , ] , and other studies have demonstrated that a poor titre of colostral antibodies increases the risk of contracting bohv- infection in the calf [ ] . it has also been reported that the concentration of maternal antibodies against bohv- in calves after birth and after colostrum ingestion is directly proportional to that present in the mother's serum [ , ] . though extensive studies have reported on marker vaccines, there is a need to assess their ability to induce passive immunity. here, we have made a concerted effort to evaluate passive immunity transmitted from cattle to newborn calves following immunisation with different inactivated marker vaccines. two commercial vaccines were selected for the study ( table ) . the products were identified as follows: vaccine a, a ge-negative, inactivated strain of bohv- containing light paraffin oil; and vaccine b, a ge-negative, inactivated strain of bohv- containing aluminium hydroxide, saponin, and thiomersal. the vaccines were administered as follows: doses of ml each were given to each animal (pregnant females) days apart. vaccine a was injected to the animals intranasally (i.n.), whereas vaccine b was injected intramuscularly (i.m.) into the neck muscle. the cattle received their first dose of vaccine injection in the th month of pregnancy. all animals in the study were from a single dairy herd, located in central italy and owned and run by a local veterinarian. according to the farm records, no vaccine against bohv- had been used before and no recent history of respiratory disease was registered. eighteen pregnant friesian cattle of approximately - years of age and devoid of na and elisa antibodies against glycoprotein e (ge-elisa) of bohv- were selected. the animals had previously had two pregnancies. the status of the animals was confirmed throughout the experimental period based on the absence of clinical symptoms and serological and virological investigation against ibr. the animals were fed twice per day with unifeed mixtures and water ad libitum. moreover, after calving, milking was performed three times per day. the maintenance and experimental protocols were established according to the european legislation on the protection of animals used for scientific purpose [ ] . the experiments were carried out with the approval of the italian ministry of health under the authorisation number / -pr. the number of animals in each group was determined through the sampling procedure envisaged for an experimental clinical study that was used to compare the proportions in terms of superiority. a percentage of was considered for the proportion of the appearance of the study event in the control group and a percentage of was used as the proportion of the appearance of the study event in the experimental group, with an alpha error of % and a study power of %. the cattle were divided into three groups with six pregnant animals each. the first and second group were injected with vaccine a and b, respectively (table ) , whereas the third group served as an unvaccinated control group. the animals in each group were housed in separate pens on the farm. after calving, the number of animals in each cattle group increased by six newborn calves, and these were fed with l of colostrum or milk administered twice per day throughout the experimental period. subsequently, days post-birth, the newborn calves were fed with concentrated feed and hay in addition to milk from approximately days after birth. data on the following possible adverse reactions after vaccination were obtained from the local veterinarian: (i) localised swelling at the injection site; (ii) slight transient slight increase in body temperature; (iii) anaphylactic shock; (iv) abortion; (v) stillbirth; or (vi) birth of weak, unthrifty calves. in the case of abortion, the foetus and placenta with serum samples were submitted for laboratory investigations. clinical conditions were monitored throughout the experiment. on the day of vaccination (time ), day , and day post-vaccination days (pvds), serum samples were collected from the cattle and tested for the presence of bohv- ge-elisa antibodies and nas. in addition, serum samples were taken from all animals on , , , , , , , , , , and post-calving days (pcds) to assess bohv- antibodies by gb-elisa, ge-elisa, and virus neutralisation test (vnt). simultaneously, colostrum or milk samples were collected from lactating cattle to test for gb-elisa, ge-elisa, or indirect elisa of bohv- . blood samples of approximately ml were collected from the coccygeal or jugular vein of pregnant cattle and calves, respectively. for each animal, disposable needles and vacutainer tubes were used. subsequently, the samples were centrifuged at × g for min at • c to extract the serum. in addition, colostrum and milk samples were uniformly collected from the teats of each animal to obtain ml of each using conical tubes. later, they were centrifuged at × g for min at • c to obtain skimmed samples. all samples were transported with refrigeration to the laboratory within h of collection before testing. afterwards, all samples were stored at − • c for further serological studies. two commercial elisa tests (idexx ibr ge ab test, maine, usa; idexx ibr gb x ab, maine, usa) were used in parallel to examine the collected sera, colostrum, or milk samples. in addition, indirect elisa (idexx bhv bulk milk ab, maine, usa) was used only for colostrum and milk samples. the protocols described by the kit manufacturer were followed and the results were also expressed according to the instructions of the manufacturer. the microplates were read using an automated plate reader and the data were analysed using the magellan software (tecan ag, switzerland). the serum samples were tested using the protocol described by the oie manual of diagnostic tests and vaccines for terrestrial animals [ ] . briefly, µl of undiluted serum samples and two-fold dilutions of each were mixed with µl of tcid of bohv- (los angeles strain / ) in -well microtitre plates. the samples were incubated at • c for h and then , madin-darby bovine kidney cells in µl were added to each well. the cells were provided by biobanking of veterinary resources (bvr, brescia, italy) and identified with the code bs cl . after days of incubation at • c, the plates were read using the inverted tissue culture microscope to determine cytopathic effects. neutralisation titres were expressed as the highest dilution inhibiting cytopathology. overall, animals were used in this study, which included the pregnant cattle and their newborn calves. the titres of antibodies were measured on a logarithmic scale with base . means of the titres were calculated for each animal group and for all sampling times. the nonparametric wilcoxon mann-whitney test was used to evaluate the presence of any statistically significant differences in immunity induced by vaccination between the two ge-deleted marker vaccines and the unvaccinated controls. the differences between group a and group b with respect to the control group at each sampling time were studied considering a significance level at p-value ≤ . . all statistical analyses were performed using stata software v. . (statacorp lcc, texas, usa). after immunisation, no clinical signs or adverse reactions were observed in any of the pregnant cattle or animals immunised i.n. with vaccine a or i.m. with vaccine b. moreover, throughout the experimental period, no clinical signs of ibr infection were seen in the calves born from cattle immunised with vaccine a or b, except for two calves. these two animals, born to vaccine a-immunised cattle showed mono-lateral discharge at two months of age, and, following a nasal swab for virologic and bacteriologic investigations, were found to be infected with only one staphylococcus spp. consequently, they were treated with antibiotics (ceftiofur hydrochloride). after days post-vaccination, all pregnant cattle had nas to bohv- at a mean na titre of . log . titres in cattle vaccinated with both vaccine a and vaccine b showed a significant difference compared to the control (p = . ); the mean titre was . log . this value was increased to . log (p = . ; vaccine a) and . log (p = . ; vaccine b) at pvd . no seroconversion was detected in the unvaccinated controls (table ) . after days post-calving, the mean na titre of cattle immunised with vaccine a did not decrease compared to that on the day of calving ( . log ; p = . ). however, pcd onwards, the na titres started declining and continued to do so till pcd (antibody titres reached . log ; p = . ), after which it remained constant until pcd (table ) . conversely, after days post-calving, the mean na titres of cattle immunised with vaccine b decreased to . log (p = . ) and, then, remained constant up to pcd . subsequently, they decreased up to pcd to an antibody titre of . log (p = . ). in addition, after vaccination, all pregnant cattle remained seropositive for gb and seronegative for ge. conversely, no seroconversion was detected in the unvaccinated controls (table ) . on the day of calving (approximately h after birth), calves born to vaccine a-immunised cattle had nas with a mean titre of . log (p = . ), which remained constant up to pcd . this titre decreased to . log (p = . ) on pcd . subsequently, it declined up to pcd , reaching . log (p = . ). conversely, on the day of calving, the calves of cattle immunised with vaccine b had nas titre of . log (p = . ), which persisted till pcd . it, then, started declining up to pcd , when antibody titres reached . log (p = . ). after calving, all calves remained seropositive for bohv- gb and seronegative for bohv- ge. in contrast, no seroconversion was detected in the control group (table ). the colostrum and milk samples collected from cattle immunised with vaccine a or b were seronegative for ge and seropositive for gb or indirect elisa antibodies throughout the experimental period. in contrast, the colostrum or milk samples collected from unvaccinated cattle were seronegative for gb, ge, and indirect elisa antibodies (table ). currently, in european countries, the diva strategy is considered to be one of the first-line interventions in bohv- eradication programs in areas with a high prevalence of the disease [ , ] . in italy, a national surveillance plan for ibr is only active for autochthonous cattle breeds and recommends the use of marker vaccines to decrease ibr seroprevalence [ ] . it is widely known that marker vaccines induce a marked humoral and cell-mediated immune response [ , ] ; however, only little information is available regarding passive immunity induced by these vaccines. previous studies have suggested that passive immunity would be induced in the calves after maternal vaccination. however, this study is important to provide this. we performed several experiments using two ge-deleted marker vaccines, administered via an intranasal (vaccine a) or intramuscular (vaccine b) route. both inactivated ge-deleted marker vaccines did not induce any clinical signs or adverse reactions. such effects, especially abortion and anaphylactic shock, have been reported when modified-live attenuated vaccines are administered to pregnant cattle in the pre-partum period [ , ] . the results of this study, in accordance with those reported previously [ , ] , suggested no risk of adverse reactions following the administration of either inactivated ge-deleted marker vaccine. in addition, the outcomes of the present study are in accordance with those of other studies [ ] , showing that the vaccination of pregnant cattle can prevent ibr-induced abortion. it is currently known [ , ] that other herpesviruses are involved in cattle abortion, but little information is available on the ability of vaccines prepared against bohv- to protect against abortion induced by bovine gammaherpesvirus (bohv- ) and bovine alphaherpesvirus (bohv- ). the response to vaccination was determined by the assessment of na titres against bohv- or by ge-elisa of serum bohv- in pregnant cattle. results indicated that na titres against bohv- were increased at pvd , compared to control levels. significantly (p = . ; p = . ) elevated titres were also observed at pvd . these findings are in agreement with those of other studies in which an increased colostral antibody production against bohv- and various pathogens were detected after the vaccination of pregnant cattle during the pre-partum period [ , , , ] . in contrast, in a study conducted by lemaire et al., a low level of antibodies against bohv- was observed in two cows after two vaccinations with inactivated ge-deleted marker vaccines [ ] . moreover, in calves, a decline in maternal antibodies against bohv- and bovine viral diarrhea virus was shown at or days of age, respectively [ , ] . similar results were also seen in another study, in which a vaccine containing rotavirus, coronavirus, and escherichia coli was used to immunise pregnant cattle and led to high titres of specific antibodies in their colostrum and milk [ ] . further, agianniotaki et al. demonstrated the presence of nas against lumpy skin disease virus until days post-calving [ ] . in the present study, the pregnant cattle showed negative results for ge-elisa during the vaccination period. these results are in agreement with those of reports demonstrating that serum samples collected from animals immunised with ge-deleted marker vaccines were also negative for ge-elisa [ , , ] . moreover, this result showed that, during the experimental period, the field virus did not circulate in the experimental groups. cattle naturally or experimentally exposed to bohv- can be positive for ge-elisa antibodies [ ] . the experiments performed herein showed that pregnant cattle immunised with the two ge-deleted marker vaccines could transfer passive immunity to calves. in fact, on the day of calving, both groups injected with vaccine a or b showed high na serum levels against bohv- . after birth, calves are born in an agammaglobulinemia state and need colostrum with a high level of immunoglobulins (igs). in the first h, these igs can penetrate the bloodstream by binding to the fc receptors located in the intestinal brush border [ , ] . produced by b cells or plasma cells in the blood of lactating cows [ ] , these igs are responsible for the early protection of newborns against different infections. furthermore, colostrum contains leukocytes and several antimicrobial proteins, such as complement c , lactoferrin, lactoperoxidase, and lysozyme. these leukocytes, together with maternal igs, are involved in conferring orogastric protection [ ] ; specifically, they can enter the circulation through intestinal adsorption to promote neonatal cellular immunity [ ] . in our research, on the day of calving, the cattle immunised with vaccines showed a good level of nas, which decreased progressively up to pcd . in addition, calves in both the groups showed na titres similar to those of their mothers approximatively h after birth. subsequently, the antibody titres decreased until the end of the experiment, when they reached negative values. in both of the vaccinated groups, the difference in antibody titres between cattle and newborn calves could be due to the passage of igs from mothers to newborns through the colostrum. these results are similar to those obtained by cervenak et al., showing that when the time of calving approaches, igg massively decreases in maternal serum samples as it binds to mammary epithelial cells. then, igg is released with colostrum and milk during lactation. calves undergo so-called gut closure by approximately - h after birth and the adsorbed colostral igg (igg , igg ) levels fall below % [ ] . after pcd , the antibody titres in milk gradually decreased up to pcd . the data obtained in this study supported the results of mechor et al., who reported a correlation in serum na titres between cattle and newborn calves [ ] . in addition, the antibody titres observed in both the groups of newborn calves, born to the cattle immunised with vaccine a or b ( . log and . log , respectively), on pcd were lower than those required to protect them against infection by bohv- . several studies have shown that nas higher than a value of . log can protect calves against experimental infection [ , , ] . moreover, it has been shown that these antibodies appear in nasal secretions from calves as early as the first day after the ingestion of colostrum [ ] . the nas secreted in the respiratory tract mucosa, primarily of the igg class, persist for to days after birth and serum antibodies can be detected until calves reach several months of age [ ] . these results are similar to those of other studies [ ] showing the persistence of nas up to pcd when using non-marker vaccines. this study was carried out based on current field conditions with different variables, including the geographical position of the farm, weather, nutrition, and health status of the herd. generally, dairy cattle are subjected to stress that can negatively affect their serological responses to vaccination. in this study, there was no evidence of stressors as both intramuscularly-and intranasally-vaccinated animals produced humoral immunity with high antibody titres. conversely, other authors, using modified-live vaccines, found that the aforementioned factors can negatively affect the antibody response after vaccination [ ] . in this study, no challenge with virulent bohv- virus was given because this research aimed to evaluate the ability of the two ge-deleted marker vaccines to induce passive immunity. moreover, this study was intended to form the basis of additional research evaluating the effect of different vaccines against ibr. thus, future studies will be conducted to assess whether passive immunity in calves can protect them against experimental infection using virulent bohv- virus. overall, the two inactivated ge-deleted marker vaccines against bohv- were shown to (i) be innocuous for pregnant cattle, (ii) effectively transfer passive immunity from dams to calves up to pcd , and (iii) be suitable for immunisation in ibr eradication programs. further studies will be conducted to evaluate if passive immunity induced by these vaccines can protect calves from challenge with a virulent (wt) strain of bohv- . author contributions: conceptualization, s.p., c.r., and c.i.; stata software v. . , e.s.; methodology and investigations, e.r., c.p., p.g. and g.v.; data curation, c.r. and c.p.; manuscript writing, review, and editing, s.p. and g.m.d.m. all authors have read and agreed to the published version of the manuscript. bhv- infection in cattle: an update bovine herpes virus infections in cattle bovine herpesvirus- : evaluation of genetic diversity of subtypes derived from fields strains of varied clinical syndromes and their relationship to vaccine strains the use of marker vaccines in eradication of herpesviruses vaccination of calves against bovine herpesvirus- : assessment of the protective value of eight vaccines epidemiology and control of bovine herpesvirus infection in europe cell-mediated immune responses induced by bhv- : rational vaccine design surveillance of infectious bovine rhinotracheitis in marker-vaccinated dairy herds: application of a recombinant ge elisa on bulk milk samples antibody response to bovine alphaherpesvirus (bohv- ) in passively immunized calves a study on latency in calves by five vaccines against bovine herpes virus- infection enhancement of the immune response and virological protection of calves against bovine herpesvirus type with an inactivated ge-deleted vaccine effect of maternally antibody upon vaccination with infectious bovine rhinotracheitis and bovine virus diarrhea vaccines protection of newborn calves against fatal multisystemic infectious bovine rhinotracheitis by feeding colostrums from vaccinated cows maternally derived humoral immunity to bovine viral diarrhea virus (bvdv) a, bvdv b, bvdv , bovine herpes virus- , parainfluenza- virus bovine respiratory syncyntial virus, mannheimia haemolytica and pasteurella multocida in beef calves, antibody decline by half-life studies and effect on response to vaccination predicted ages of dairy calves when colostrum-derived bovine viral diarrhea virus antibodies would no longer offer protection against diseases or interfere with vaccination effect of age at time of vaccination on antibody titres and feedlot performance in beef calves on the protection of the animals used for scientific purposes manual of diagnostic tests and vaccines for terrestial animals national surveillance plan for infectious bovine rhinotracheitis (ibr) in autochthonous italian cattle breeds: results of the first year of activity inactivated bovine herpesvirus marker vaccines are more efficacious in reducing virus excretion after reactivation than a live marker vaccine anti-bovine herpesvirus and anti-bovine viral diarrhea virus antibody responses in pregnant holstein dairy cattle following administration of a multivalent killed virus vaccine safety of a modified-live combination vaccine against respiratory and reproductive diseases in pregnant cows bovine herpesvirus modified live virus vaccines for cattle reproduction: balancing protection with undesired effects prevention of abortion in cattle following vaccination against bovine herpesvirus : a meta-analysis partial protection induced by a bhv- recombinant vaccine against challenge with bhv- a recombinant bovine herpesvirus defective in thymidine kinase and glycoprotein e is immunogenic for calves and confers protection upon homologous challenge and bohv- challenge stimulation and analysis of the immune response in calves from vaccinated pregnant cows antibody response to glycoprotein e after bovine herpesvirus type infection in passively immunized, glycoprotein e-negative calves colostral and milk responses of cows vaccinated with a single dose of a combined vaccine against rotavirus, coronavirus and escherichia coli f (k ) colostrum transfer of neutralizing antibodies against lumpy skin disease virus from vaccinated cows to their calves passive immunization, an old idea revisited: basic principles and application to modern animal production system the neonatal fc receptor plays a crucial role in the metabolism of igg in livestock animals passive immunity in newborn calves cytotoxicity of human peripheral blood and colostral leukocytes against shigella species humoral and cellular factors of maternal immunity in swine evaluation of safety and efficacy of dna vaccines against bovine herpesvirus- (bohv- ) in calves demonstration of colostral antibodies in the nasal secretion of calves and their protective effect against infection evaluation of safety and efficacy of an intranasal vaccine containing a temperature-sensitive strain of infectious bovine rhinotracheitis virus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors are grateful to professor fernando a. osorio, school of veterinary medicine & biomedical sciences, university of nebraska-lincoln (usa) for providing a critical review of this manuscript and to professor gigliola canepa, university of milan (i), for revising the language of the manuscript. the authors also thank dr. roberto sabato for kindly taking part in sample collection. the authors declare no conflict of interest. key: cord- -y tbvs t authors: ramvikas, m.; arumugam, m.; chakrabarti, s.r.; jaganathan, k.s. title: nasal vaccine delivery date: - - journal: micro and nanotechnology in vaccine development doi: . /b - - - - . - sha: doc_id: cord_uid: y tbvs t the mucosal surfaces represent the major site of entry of many pathogens, and major challenges in vaccine development include safety and stability in a suitable dosage form. micro- and nanocarrier-based delivery systems as nasal vaccines induce humoral, cellular, and mucosal immunity. the nasal route of vaccination could also offer immunity at several distant mucosal sites (oral, rectal, vaginal, and pulmonary), which is considered a simplified and cost-effective mode of vaccination with enhanced patient compliance. most of the nasal vaccine delivery systems in the form of microparticulates, nanoparticulates, and liposomes are currently under development and prove to offer immunity in animal models. the importance and potential of the nasal route of administration for vaccines is unexplored, and this chapter outlines the opportunities, challenges, and potential delivery solutions to facilitate the development of improved nasal vaccines for infectious diseases. mucosal infections are caused by emerging pathogens such as bacteria and viruses, which are mucosally transmitted and replicate in the mucosal tissues. these pathogens enter the human through single or multiple mucosal sites such as oral, nasal, conjunctival, respiratory, gastrointestinal, and genitourinary tracts, invade the bloodstream, and finally lead to infection. approximately % of deaths in children younger than years are due to mucosal infections. bacteria such as streptococcus pneumoniae, staphylococcus aureus, anaerobic bacteria, streptococcus viridans, α-streptococci, enterobacteriaceae, and viruses such as haemophilus influenzae, corona virus, and human adenovirus cause mucosal infections. the mucosal infections are treated by antibiotics, and vaccines are administered through oral, parenteral, and nasal routes. a nasal vaccine induces mucosal immune responses and systemic immunity, which provides better protection against infectious agents. yet developing vaccine delivery systems that induce humoral and cell-mediated response with mucosal immunity has been challenging to date. different mucosal routes are being explored using different delivery systems, and the nasal route is preferred as compared to other mucosal routes. the mucosal route of vaccine delivery is one of the better alternatives to conventional multiple injection vaccines, which employ traumatic procedures and may also lead to the spread of infectious agents via contaminated syringes. mucosal vaccine development has grown extensively, and a multitude of vaccine delivery systems have been developed for application via ocular, nasal, oral, rectal, and vaginal routes. of all the different routes of mucosal vaccination, the oral and nasal routes are most accepted and easily accessible. for many decades, intranasal applications of tobacco snuff, cocaine, and various hallucinogenic and psychotropic agents have been in practice. similarly, for many years synthetic drugs have been administered intranasally for their local effect on the mucosa (eg, antihistamines, antibiotics, and nonpeptide drugs). the first nasal influenza vaccine was introduced in , but it was later withdrawn from the market due to potential toxicity problems. another intranasal vaccine (flumist) was launched in and was administered using a syringe sprayer. success of a vaccine depends not only on the delivery but also on the route of administration; for example, nasal immunization elicits potent immunoglobulin a (iga) secretion in the respiratory tract. nasal administrations of vaccines have been shown to achieve a better systemic bioavailability and protection from gastric enzymes compared with parenteral and oral administration. nasal delivery of vaccines acts as a "first entry block," that is, blocks the pathogen entry, while invading to the mucosal surface by inducing local microbial-specific immune responses, thus increasing the general efficacy of the vaccine. in addition, vaccine uptake into the blood circulatory system by absorption through mucosa can be relatively fast. it is the most appropriate method of immunization because it is rich in t cells, b cells, and plasma cells and stimulates both antigen-specific systemic and mucosal adaptive immune responses. it provides better patient compliance due to the needle-free delivery. the nasal route is considered an attractive route for vaccine administration with the following advantages: • better patient compliance • numerous microvilli present in the nasal epithelium provide a better absorption surface • mucosal and systemic immune response can be induced • easy immunization of large population groups • nasal immunization does not require needles and syringes many challenges stand in the way of developing nasal vaccines. when nasal vaccines are administered directly on mucosal surface, the antigens may be diluted by mucosal secretions, seized in mucus gels, attacked by proteases and nucleases, and obstructed by epithelial barriers. major challenges in the development of nasal vaccines include: • a relatively large dose of vaccine is required, and it is difficult to administer through the nasal route and also difficult to monitor the actual dose that crosses the mucosa. • costly innovative vaccination strategy. • efficacy of nasal vaccines may be limited due to the including mucociliary clearance and the inefficient uptake of soluble antigens by nasal epithelial cells in the nasal cavity. • nasal delivery may require adjuvants to enhance their immunogenicity and delivery to the mucosal tissues. • lack of multiple human-compatible mucosal adjuvants. • rapid nasal clearance may not allow sufficient retention for antigen to be taken up by antigen-presenting cells (apcs) in the nasal-associated lymphoid tissue (nalt). • several enzymes that are present in the nasal mucosa might affect the stability of drugs. for example, proteins and peptides are subjected to degradation by proteases and amino-peptidase at the mucosal membrane. • delivery volume in the nasal cavity is restricted to - µl. • high molecular weight compounds cannot be delivered through this route (mass cutoff ∼ kda). • low antigen entrapment efficiency mode. • normal defense mechanisms like mucociliary clearance and ciliary beating affect the permeability of the drug. the nose is a vital organ in the human body for breathing. the nose has a more complex role as a complete system of defense against inhaled air and air conditioning. the nasal anatomical and physiological structure provides support for nasal immunization against upper respiratory mucosal diseases. nasal anatomy, nasal morphology and physiology, nasal secretions, nasal mucosa, olfactory region, and blood supply to the nasal cavity are described with respect to their link with the vaccine administration route. the nasal cavity is protected by the viscerocranium in the human head. the human nose is divided by the median septum into two symmetrical halves; each half opens to the face through the nostrils and extends posteriorly to the nasopharynx. the nasal cavity and the nasal vestibule are the anterior part, which opens to the face through the nostrils. the atrium is placed in an intermediate region between the vestibule and the respiratory region. the respiratory region, the nasal turbinates, occupies more area of the nasal cavity. it possesses lateral walls that divide it into three sections composed of the superior nasal turbinate (superior region), nasal turbinate (middle region), and inferior turbinate (inferior region). the attachment of the turbinate to the lateral wall is more complex in animals than in humans. these folds provide the nasal cavity with a very high surface area compared to its small volume. the basic functions of the nose are filtration of particles and heating and humidification of inspired air before it reaches the lungs. the olfactory region situated above the superior nasal turbinate possesses specialized ciliated olfactory nerve cells for smell perception. the human nasal cavity has a total volume of - ml and a total surface area of approximately cm , of which the respiratory region covers about %. each nasal cavity can be subdivided into different regions such as nasal vestibule, turbinate (inferior, middle, and superior), olfactory region, frontal sinus, sphenoidal sinus, and cribriform plate of ethmoid bone. nalt is present in the nasal cavity, which is situated in the nasopharynx. the central axon of these nerve cells passes through the cribriform plate of the ethmoid bone and into the olfactory bulb. the nasal vestibule has numerous nasal hair (vibrissae) that filter large airborne particles ( fig. . ) . the anterior section of the nasal portion is constituted by a stratified squamous epithelium with sebaceous glands and the posterior section of the nasal portion by pseudostratified columnar cells presenting microvilli. the nasal respiratory mucosa is considered the most important area for delivering drugs systemically, as its made of epithelium, basement membrane, and lamina propria. the nasal respiratory epithelium consists of pseudostratified columnar epithelial cells, goblet cells, basal cells, and mucous and serous glands. , most of the epithelial cells on their apical surface with microvilli and the major part have fine projections, called cilia. the nasal secretion, nasal mucosa, olfactory region, and blood supply to the nasal cavity play a major role in nasal physiology. nasal secretions originate mostly from submucosal glands and goblet cells. mucus is composed of water ( %); glycoproteins ( %); albumin, immunogobulins, lysozyme, lactoferrin and other proteins ( %); inorganic salts ( %); and lipids (< %). glycoproteins are in low proportion, which provides mucus with its characteristic viscoelastic properties. the mucus layer is divided into the low viscosity lower layer of about - µm thick and the more viscous upper layer of about . - µm thick. the human nasal ph is approximately - with an average baseline of . . the nasal respiratory mucosa consists of epithelium, basement membrane, and lamina propria. the nasal respiratory epithelium contains pseudostratified columnar epithelial cells, goblet cells, basal cells, mucous, and serous glands. many of the epithelial cells are covered on their apical surface with fine projections of microvilli (called cilia), which enhance the respiratory surface area. the nasal epithelium is covered with a thin mucus layer, which is produced by secretory glands and goblet cells. nasal mucus is responsible for several physiological functions, such as humidification and warming of the inhaled air, and offers physical and enzymatic protection. the olfactory region is located in the top of the nasal cavity about - cm and extends down the septum and lateral wall. neuroepithelium, which is the only part of the central nervous system (cns), is directly exposed to the external environment. the olfactory area is made of pseudostratified columnar epithelium and is composed of supporting cells, basal cells, microvillar cells, and the typical receptor or olfactory cells. olfactory regions also contain olfactory receptors (receptors for smell sensations) and small serous glands (which produce secretions that act as a solvent). the total surface area of the olfactory epithelium is - mm. the nasal cavity vasculature requires rich blood supply to fulfill the basic physiological functions such as heating, humidification, olfaction, mucociliary clearance, and immunological roles. the nasal vascular bed is intended as such for rapid and easy exchange of fluid and dissolved excipients between blood vessels and nasal tissue. the capillary flow in the nasal mucosa was reported to be . ml/g/min. antigen uptake in the nose is mostly by two mechanisms: paracellular (aqueous pathway) and transcellular (lipoidal) processes ( fig. . ). the most efficient area for drug absorption is the highly vascularized lateral wall of the nasal cavity: the mucosa lined over the turbinates or conchae. • in the paracellular mechanism, the antigen uptake nasally is through the aqueous route of transport, and this route is slow and passive. the process mainly depends on an inverse log-log correlation between intranasal absorption and the molecular weight of water-soluble compounds/antigens. poor bioavailability is observed for antigens with a molecular weight greater than kda in the paracellular process. an example is chitosan widening the tight junctions between epithelial cells. • in the transcellular mechanism, the antigen uptake nasally is through a lipoidal route and is responsible for the transport of lipophilic antigen based on lipophilicity. other than transcellular and paracellular processes, antigens may also cross cell membranes by an active transport route via passive diffusion (depends on ph of environment and pka of the drug), carrier-mediated means, or transport through the opening of tight junctions in mucosa (ie, through organic cation or amino acid transport), and endocytic process [uptake of antigen mediated by microfold (m) cells]. there are various factors that affect the antigen uptake in the nasal cavity after nasal immunization, but the bioavailability of a nasal delivery vaccine depends on two major factors: nasal physiological factor and physiochemical properties of nasal vaccine formulation. there are different physiological factors that regulate the absorption of antigen. • the permeability of a nasal vaccine depends on the type of cells and number of cells in the nasal cavity. • secretions of nasal mucosa enzymes such as lactate dehydrogenase, oxidative, conjugative enzymes, peptidases, and proteases also act as a barrier and degrade the nasal vaccine. • stimulation of nasal mucosa plays a role in mucosal absorption. parasympathetic stimulates increase permeability of a nasal vaccine. • viscous nasal mucus secretion may retard the uptake of antigen. • permeation of antigen is altered at night (chronokinetics) due to clearance rates and mucosal secretions. • generally, ph of the nasal cavity for adults is . - . and for infants is . - . ; for better antigenic absorption, the developed nasal formulation ph should be within . - . . • mucociliary clearance (mcc) takes about min in the nasal cavity, and increased mcc decreases antigen uptake. • mucociliary functioning may affect nasal mucosa due to diseases such as the common cold, rhinitis, etc. • environmental conditions (ie, increases in temperature and increases in mucus secretion) also affect the mucociliary clearance. • antigen concentration, dose, and volume of administration are three interrelated parameters that affect the performance of the nasal antigen delivery. • ph of nasal formulation should be in the range between . and . because lysozymes found in nasal secretions are responsible for destruction of certain bacteria at acidic ph. the lysosomes will be inactivated at ph above and susceptible to microbial infection in nasal tissue. • pharmaceutical dosage form is another important factor to consider while developing a nasal formulation. nasal drops are simpler and more convenient than powder, gel, and suspension sprays. • pharmaceutical excipients used in the formulation such as buffer components, antioxidants, preservatives, surfactants, humactants, solubilizers, and gelling/viscosifying agents also play a crucial role in the delivery of the antigen. nasal clearance is one of the major challenges in the development of a nasal vaccine. in nasal mucosa, mucus acts as a sticky fluid and cilia act as a motivator, which prevents foreign substances, pathogens, and particles from being carried by inhaled air into the lungs. in physiological conditions, mucus is transported at a rate of mm/min, and its transit time in the human nasal cavity is - min. it depends on the length, density, and beat frequency of cilia as well as the amount and viscoelastic properties of the mucus. the absorption of antigen is influenced by the residence (contact) time between the antigen and the epithelial tissue in mucosa. if the mucociliary clearance increases, the antigen absorption decreases (ie, inversely proportional to each other). nasal mucociliary clearance can also be stimulated or inhibited by the active ingredient (ie, antigen and/or inactive ingredients such as preservatives, absorption enhancers, and/or other excipients), thus affecting the delivery of antigen to the absorption site. a prolonged residence time in the nasal cavity may also be achieved by using bioadhesive polymers or a particulate delivery system or by increasing the viscosity of the formulation. deposition of antigen in an anterior and posterior region of the nose also affects the antigen absorption of nasal formulation. the anterior portion provides a longer nasal residence time for antigen with low permeability, but antigen permeability is higher in the posterior portion of the nose with shorter nasal residence time. an example is that nasal sprays are deposited anteriorly and cleared slowly into the nasal pharynx by mucociliary clearance. nasal drops are deposited posteriorly and removed rapidly into the nasal pharynx. nalt is the primary target site for nasally administered vaccines, and it consists of lymphoid follicles that occur directly beneath the mucosal follicle-associated epithelial (fae) cells. nalt is in the waldeyer's ring, which includes the pharynx and tonsils. the inductive site for nasal immunity is nalt, m cells, b cells, t cells, dendritic cells (dcs), and regional and cervical lymph nodes. nasally immunized vaccine is transported to nalt through the fae that contains specialized villous m cells for process and presentation. villous m cells lack a brush border, which facilitates the binding and delivery of antigens and also does not secrete mucus or any enzymes. nalt is involved in the induction of dc, b cell, and t cell responses following nasal vaccination and also produces local defense against invading pathogens. part of the soluble antigens may migrate to regional draining lymph nodes through the major histocompatibility complex (mhc) to th cells. activation of antigen-specific cd + t helper cells (th cells) interacts with b cells, which develop into iga committed (iga+). the iga+ b cells rapidly migrate from the nalt to the draining cervical lymph nodes in the nasal passage where they differentiate into iga-producing plasma in the presence of cytokines such as il- and il- that are produced by th cells and secrete iga in dimers. dimeric iga then becomes s-iga by binding to the polymeric ig receptor, which transports iga to effector sites. s-iga is able to bind toxins, bacteria, or viruses and neutralize their activity, thus preventing entry into the body or reaching the internal organs, and forms a first barrier of defense against invading antigens. intranasally administered vaccines elicit neutralizing iga, preventing colonization of the throat and systemic igg antibodies and facilitating clearance from systemic sites (fig. . ) . immune-competent cells in tonsillar lymphoid tissue and ciliated and nonciliated cells also play a role in immunity generation. the production of iga by both the adenoid tissue and the nasal mucosa contributes significantly to immune protection against inhaled bacteria and viruses. mucosa defends against invading pathogens through two types of immune systems: the innate immunity system and adaptive immune system. the key role of innate immunity and adaptive immunity at the mucosal surface are discussed in the following sections. mucosal surfaces of the nasal tract and respiratory tract are adorned with a potential barrier known as epithelial cell lines. epithelial cells in nasal mucosa are active participants in mucosal defense. epithelia and their associated gland produce innate defenses including mucins and antimicrobial proteins. epithelial cells detect the dangerous/foreign microbial components through pattern recognition receptors such as toll-like receptors (tlrs) and send the cytokine and chemokine signals to mucous membrane-associated apcs, such as dcs and macrophages, to trigger nonspecific/innate defenses and stimulate adaptive immune responses. an important characteristic of mucosa is the production and secretion of dimeric iga that is resistant to degradation in the protease-rich surroundings of mucosal surfaces. in humans, production of iga is more comparable to other immunoglobulin isotypes, and high concentrations of iga antibodies (more than mg/ml) are present in the mucosal surface-associated secretions. iga facilitates the entrapment of microbes and foreign bodies into the mucus by avoiding direct contact of pathogens with the mucosal surface, which is known as "immune exclusion." there are numerous mucosal routes of immunization available as an alternative to current parenteral immunization, namely oral, nasal, pulmonary, vaginal, and rectal. the nasal route is more attractive for several reasons. it is a practical site for easy selfadministration, with the use of commercially available wide delivery devices. nasal immunization generally requires much lower doses of antigen compared with the oral or sometimes parenteral route. nasal immunization does not expose antigens to low ph and digestive enzymes like protease and nuclease, and a protective coating of mucus limits access of antigen into the mucosal epithelium. administration of vaccines through the nasal route may predominantly induce potent immune responses. different types of delivery vehicles suitable for the nasal drug delivery systems are being explored; some are being researched, and a few are in clinical phases. they are classified into two major categories: replicating delivery systems and nonreplicating delivery systems. in a replicating delivery system genetically altered live virus acts as a vector; it proliferates in the host tissues after immunization. it is one of the more prevalent and effective presentations of the antigen and more likely to be effective at stimulating an immune response following oral and nasal delivery. there are different virus vector (rhabdo virus, polio virus, influenza virus, cowpea mosaic virus) [ ] [ ] [ ] and bacterial vector (shigella, salmonella, listeria) replicating delivery systems able to induce a ctl response that produces longer-lasting immunity. introduction of multiple foreign genes can replace the nonessential regions of the viral genome, resulting in an immune response against multiple pathogens. this tactic enables the recombinant virus to be used as a vaccine for two or more infectious agents. vaccine priming with hiv-env-expressing influenza virus induced systemic cellular response in mice. a single intranasal dose of shigella vector based hiv- vaccine produced cd + t cell response comparable to systemic immunization. replicating antigen delivery systems are not preferred due to their complexity of large exploration. nonreplicating delivery systems are those which mimic the antigens to the immune system and cause similar uptake by apcs. different kinds of nonreplicating delivery systems include: • liposomes • micro-and nanoparticulate systems • immune-stimulating complexes (iscoms) • virus-like particles (vlps) • emulsions • bioadhesive delivery systems liposomes are small artificial vesicles made of bilayer lipid molecules such as phospholipid and cholesterol which encapsulate antigens. liposomes are a promising delivery system because of their size, ampiphilic nature, and biocompatibility. these vesicles form spontaneously when an aqueous solution is added to a dried film of the lipid components. the hydrophilic (water-soluble or polar head) portion of the lipid molecule is present toward the aqueous phase, and the hydrophobic (water-insoluble) portion is allied inside the membrane. many of the phospholipids used to make liposomes are from food products (eg, egg yolk or soybeans), so they are nontoxic and safe. the potency of liposomes depends on different factors such as the surface of the lipid layers, electric charge, composition, and method of preparation. liposomes can also act as an adjuvant, which leads to the induction of potential immune response with low antigen payload. they can also convert nonimmunogenic substances into immunogenic forms (eg, by rendering soluble substances particulate in nature). liposomes are taken up by macrophages and by m cells for antigen processing and/or presentation to other lymphoid cells for the induction of immune responses. they can also directly present antigens to lymphoid cells for the induction of immune responses. nasal application of liposomes containing bacterial polysaccharide antigens has been tested in balb/c mice and revealed that enhanced immune responses in pulmonary secretions was observed in mice after immunization of the liposomal antigen in comparison with antigen alone and as an oral immunization. apart from this, many investigations carried out on liposomes using different antigens are described in table . . a nasal influenza vaccine, based on a liposome (virosomal) formulation of influenza virus subunits, has recently been marketed in europe by the swiss serum institute (berne, switzerland). liposomes could be a promising delivery system for nasal vaccines. particulate carriers have attracted considerable interest as antigen carriers for achieving delivery of antigens at a specific site in the body. nanoparticles range in size from to iga antibody and ctl responses [ ] nm ( µm), while microparticles range from to µm; the smaller particle size promotes faster adsorption. other added advantages include a more stable system protecting from the hostile environment of nasal mucosa, prolonged release, bioadhesion properties, and induction of a significant immune response with reduced dose. depending on the method of preparation, microparticles or nanoparticles can be prepared with different properties and release characteristics. the polymers used for the particulate system are biomolecules such as proteins, peptides, polynucleotides, and polysaccharides or synthetic polymers such as polylactide-polyglycolide copolymers, polyacrylates, polyε-caprolacton, and n-trimethyl chitosan-poly(γ-glutamic acid). nano-and microsized particulate carriers are prepared with the antigenic molecule and deliver it to the desired site of action to induce potent and long-lasting immune responses. the mechanisms by which these particulate systems alter the induction of immune responses are size-dependent penetration, depot effect, repetitive antigen display, crosspresentation, and release of soluble mediators such as cytokines that regulate the immune response. many studies carried out on micro-and nanoparticulate-based nasal vaccine delivery systems using different antigens are shown in table . . hence a polymer-based micro-/nanoparticulate system can be exploited as a viable nasal vaccine delivery system that is capable of delivering a multitude of antigens at the targeted sites and inducing desired immune response. an iscom is a highly versatile and effective antigen presentation system, which is a spherical open cage-like structure (typically - nm in diameter) that forms spontaneously on mixing cholesterol, a lipid such as phosphatidyl choline, and the mixture of saponins that comprise quillaja saponins a. the antigen-enveloped quillaja saponin acts as a strong inherent adjuvant as well. in morein and colleagues showed the potential induction of immune responses upon immunization with iscoms containing viral and bacterial membrane glycoproteins. iscoms are the potent inducers of not only humoral (antibody-mediated) immune responses but also cellular (t cell-mediated) immune responses. presentation of antigens unified with the iscom matrix allows their processing via the endogenous and exogenous pathways, resulting in the stimulation of both cd + and cd + t cells. moreover, adjuvant activity of the quil a moiety induces long-lasting immune responses. the antibody levels stimulated by iscoms are found to be equivalent to those after immunization with conventional adjuvants such as complete freund's adjuvant or alum. some studies revealed that immunization with iscoms induces a wide range of immune responses including all subclasses of igg and cell-mediated immune responses such as delayed-type hypersensitivity (dth) in vivo, antigen-specific proliferative responses and cytokine production in vitro. [ ] [ ] [ ] an additional property of iscoms is their capability to enter the endogenous antigenprocessing pathway and then mhc class i-restricted cytotoxic t cells (ctl). intranasal immunization of echinococcus granulosus surface antigen iscoms evokes higher serum iga titer in relation to igg than the subcutaneous route in mice. several studies on different antigens using iscom-based delivery systems are shown in table . . hence iscom-based delivery systems may be suitable for nasal vaccine administration. vlps are a promising option for vaccine delivery to the nasal site because they are easy to access, highly vascularized, self adjuvanting, and highly immunostimulatory and have a relatively large surface area and low proteolytic activity. vlps are inert with a - nm self-assembled empty capsid protein, which contains no dna/rna and shows a similar size and shape as viruses. vlps are efficiently taken up by dcs and induce potent immune responses after nasal immunization, which induces systemic immunity as well as both local and distal mucosal immunity via the common mucosal immune system (cmis). it is even more superior to parenteral administration at eliciting iga at distal mucosal sites. the vlps can be produced within mammalian cells, insect cells, yeast, bacteria, and even plants through recombinant dna techniques. , due to these properties, vlps are exploited as the delivery system for different protein/peptide antigens. despite successful usage and potent immunity, vlps as a delivery system for protein antigens are still limited by the relatively complicated genetic modification on protein fusion and the subsequently required structural integrity characterization. vlp technology has been used for many years. an examples includes recombinant hepatitis b surface antigen (hbsag), produced in saccharomyces cerevisiae or pichia pastoris yeasts. the entire recombinant core antigen (hbcag) of the hepatitis b virus (hbv) promotes th immunomodulation of the immune response to coadministered antigens, including the hbv surface antigen (hbsag) after nasal administration. this concept also applies to hiv, dengue, and chronic hepatitis c immunotheraphy; details are discussed in table . . vlps elicit a potent immune response and increase the immunogenicity of poorly immunogenic antigens, including self-proteins. the potential of micro-/nanoemulsion for various routes of administration has continuously been explored for the past two decades. due to low globule size and lipophilic nature, emulsions are widely chosen as a delivery system to enhance uptake across nasal mucosa. emulsions are of various types including oil-in-water (o/w) and water-in-oil (w/o). most of the novel adjuvants are made up of emulsions such as incomplete freund's adjuvant, adjuvant , montanide, and mf . emulsions are isotropic, transparent, thermodynamically stable, and low viscosity colloidal dispersions, which are stabilized by an interfacial film of alternating surfactants and cosurfactant molecules. ease of administration and scale-up is one of the important advantages over other drug delivery systems such as solid dispersion, liposomes, and particulate delivery systems. emulsion formulation contains different inactive ingredients such as oil (soybean oil, sesame oil, isopropyl myristate, etc.), surfactant (tweens, span , chromophores, peg, lecithin, cetrimide, etc.), cosurfactant (ethanol, propanol, peg, etc.), and aqueous phase. mucoadhesive polymer such as different grades of carbopol (eg, p/ p/ p, sodium alginate, pluronics, etc.) is generally incorporated in the composition of an emulsion to prolong the release of antigens. micro-/nanoemulsion provides good sprayability for nasal vaccine delivery compared with other particulate delivery systems. factors to consider during the preparation of a formulation include nature and concentration of oil, surfactant, cosurfactant, and aqueous phase; ratio of oil/ surfactant/cosurfactant; and temperature and ph of the environment. dilutability, particle size, ph, zeta potential, viscosity, and freeze/thaw cycling are critical parameters in the development of emulsion vaccines. nanoemulsion nasal vaccines have been studied for several diseases like hepatitis b, hiv, and influenza. some studies reported that the nasal vaccine immunization produced strong igg and iga antibody levels, which is similar to alum adjuvant-based vaccines. the emulsion-based delivery approach might be promising for low-cost nasal vaccine immunization for the populations of developing and underdeveloped countries. in a bioadhesive delivery system the antigen carrier system adheres to a biological tissue for an extended period. nasal mucosa bioadhesion protects the antigen from mucosal enzymes and increases retention time. bioadhesive-based nasal vaccines are preferred to overcome nasal clearance issue, to facilitate absorption, and for extended antigen delivery. bioadhesive polymers can be used to increase the nasal residence time by slowing mucocilliary clearance, which allows absorption for a longer time with the nasal mucosa and results in a subsequent increased in absorption. different bioadhesive polymers such as chitosan, carrageenan, carbopol, hydroxypropyl methylcellulose (hpmc), k m and e , sodium alginate, sodium carboxy methylcellulose, polyvinyl pyrrolidone (pvp) , and xanthan gum are used in the formulations. bioadhesion occurs between polymermucin chains through van der waals, hydrogen, hydrophobic, and electrostatic forces. nasal immunization with different antigens such as tetanus toxoid, influenza, pertussis, and diphtheria following encapsulation and administration using different bioadhesive polymers such as pluronic f , chitosan and its combination, , and peg-coated polylactic acid shows enhanced immune response via induction of igg and iga responses. chitosan-based influenza nasal vaccine produced effective immune response in human clinical trials. bioadhesive drug delivery systems may be a potential tool for extended antigen delivery through nasal immunization. intranasal immunizations are simple, easy, convenient, and safer than other routes of administration. the delivery system selection depends upon the antigen being used for the proposed indication, patient type, and marketing preferences. there are different options available to deliver nasal vaccine such as drops, powder, aerosol sprays, and the application of nasal gel. nasal drops are convenient and the most simple method for delivery of nasal vaccines. the nasal drops are administered using a nasal dropper or syringes. the major disadvantage of this system is the lack of dose precision and difficulty for the pediatric population. some studies reported that nasal drops deposit human serum albumin in the nostrils more efficiently than nasal sprays. nasal powder formulations are highly stable compared to liquid formulations. nasal powders can extend the residence time for powder formulations on the nasal mucosa, potentially increasing the local and systemic immune response. however, the production of nasal dry powders is quite complicated with required particle size, particle distribution, and performance characteristics when compared with other dosage forms. the aerosol route of delivery of vaccines is one of the most preferred for nasal administration compared with other nasal dosage forms and also less reactogenic than the subcutaneous route of administration. aerosol vaccination via the lungs targets an epithelium critical to host defense against inhaled pathogens and provides an exciting opportunity in the development of newer and more effective tuberculosis (tb), measles, and influenza vaccines. an aerosol vaccination usually depends on the target pathogen and the sites of the inductive immunity. the aerosols are available in liquid (solution, suspension, and emulsion) and solid forms. in addition to vaccine antigens, carrier/ solvent, emulsifier/surfactants, corrosion inhibitors, and propellant selection and compatability play a critical role in achieving required immunogenicity in infants. aerosol vaccine immunogenecity achievement is based on antigen particle size for prevention of upper respiratory (eg, boredetella pertussis, chlamydia pneumonia) and lower respiratory (eg, streptococcus pneumoniae, bacillus anthracis) bacteria and virus disease. larger particles (∼ µm) are needed for the aerosol vaccination to prevent upper respiratory tract infection and smaller particles (≤ µm) for lower respiratory tract infection. the dried forms of vaccines with optimum particle size are traditionally prepared by freeze drying or spray drying. the aerosol form of vaccines was administered to many human subjects for a longer period and found to provide excellent protection for diseases such as influenza a and measles. , therefore aerosol immunization may be a promising method of vaccination. nasal gels are generally used for colds, allergies, low humidity, or overuse of decongestant. nasal vaccine gels are a high-viscosity solution or suspension in which antigenic molecules are dispersed. the advantages of a nasal gel include the reduction of nasal clearance and anterior leakage due to highly viscous formulation, reduction of irritation by using soothing/emollient excipients, and target delivery to mucosa for better absorption. in addition, it may potentially enhance the immune response, reduce the antigen and/or adjuvant dose, sustain antigen release, and improve antigen uptake with enhanced antigen stability. special application techniques are required for the administration of nasal gel vaccines because of their highly viscous formulation and poor spreading abilities. a wide variety of gelling polymers are available for formulation such as pullulan, deaceylated gellan gum, xantham gum, chitosan, and polyethylene glycol, used to encapsulate vaccine/adjuvant formulation as gel particles. viscosity, sol-gel transition temperature and gelling time, and gel strength and its texture are critical parameters in the development of nasal gel vaccines. recently, pneumococcal surface protein-a nasal gel vaccine, clostridium botulinum type-a neurotoxin bohc/a, and tetanus toxoid were studied in animal models and enhanced both humoral and cellular immunity. nasal gel is an alternative and promising novel dosage delivery system to achieve the immune response. nasal vaccine administration has been considered as an alternative method and found equivalent or superior to parenteral and other mucosal administration. it avoids the risk of transmitting diseases like hepatitis b, hiv, and other agents through improper injection practices and improves patient compliance. the intranasal vaccine delivery system induces both mucosal and systemic immune response, which avoids entry of pathogen in all mucosal routes. multiple delivery systems are being explored (both developmental and clinical phases) and can open the door for practical development of nasal vaccine delivery systems. global regional and national causes of child mortality in : a systematic analysis human nasal challenge with streptococcus pneumoniae is immunising in the absence of carriage staphylococcus aureus binding to human nasal mucin effects of viral and bacterial infection on nasal and sinus mucosa bacterial adherence to nasal mucosal cells diseases of the sinuses: comprehensive textbook of diagnosis and treatment nasal drug delivery in pharmaceutical and biotechnology: present and future. e mucosal penetration enhancers for facilitation of peptide and protein drug absorption design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens nasal drug delivery nasal delivery of high molecular weight drugs current challenges in non-invasive insulin delivery systems: a comparative review nasal route: a novelistic approach for targeted drug delivery to cns nasal delivery of insulin using chitosan microspheres nasal mucociliary clearance as a factor in nasal drug delivery anatomy and physiology of the nose anatomy, physiology and function of the nasal cavities in health and disease intranasal drug delivery: how, why and what for? intranasal drug delivery for systemic medications usa: mosby anatomical and histological factors affecting intranasal drug and vaccine delivery morphology of human olfactory epithelium advantagious nasal drug delivery system: a review review on nasal drug delivery system with recent advancement drug absorption studies: in situ, in vitro and in silico models. in: kom d, editor. invitro cellular models for nasal drug absorption studies the isoenzyme pattern of lactate-dehydrogenase in nasal secretions review on nasal drug delivery system the nose and paranasal sinuses physiology and anatomy enhanced bioavailability of drugs via intranasal drug delivery system advances in nasal trans-mucosal drug delivery nasal drug delivery: an approach of drug delivery through nasal route nasal drug delivery: possibilities, problems & solutions review: clinical opportunities provided by the nasal administration of peptides mucosal vaccines: the promise and the challenge nalt-versus peyer's-patch-mediated mucosal immunity recent progress in mucosal vaccine development: potential and limitations the microbial ecology and immunology of the adenoid: implications for otitis media collaboration of epithelial cells with organized mucosal lymphoid tissues advantages of intranasal vaccination and considerations on device selection tracking human antigen-specific memory b cells: a sensitive and generalized elispot system strong cellular and humoral anti-hiv env immune responses induced by a heterologous rhabdoviral prime-boost approach intranasal immunization with a plant virus expressing a peptide from hiv- gp stimulates better mucosal and systemic hiv- specific iga and igg than oral immunization immunogenicity of an hiv- gag dna vaccine carried by attenuated shigella prime-boost immunization schedules based on influenza virus and vaccinia virus vectors potentiate cellular immune responses against human immunodeficiency virus env protein systemically and in the genitorectal draining lymph nodes vaccination with a shigella dna vaccine vector induces antigen-specific cd + t cells and antiviral protective immunity the use of natural and synthetic phospholipids as pharmaceutical excipients liposomes as vaccine delivery systems: a review of the recent advances liposomes act as stronger sub-unit vaccine adjuvants when compared to microspheres adjuvant activity of monophosphoryl lipid a for nasal and oral immunization with soluble or liposome-associated antigen adjuvant activity of monophosphoryl lipid a for nasal and oral immunization with soluble or liposome-associated antigen humans immunized with streptococcus mutans antigens by mucosal routes intranasal immunization with proteoliposomes protects against influenza mucosal immunoadjuvant activity of liposomes: role of alveolar macrophages surface modified liposomes for nasal delivery of dna vaccine intranasal delivery of nanoparticles encapsulating bpi v proteins induces an early humoral immune response in mice adjuvanted poly(lactic-coglycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs immunization with biodegradable nanoparticles efficiently induces cellular immunity and protects against influenza virus infection preventative vaccine-loaded mannosylated chitosan nanoparticles intended for nasal mucosal delivery enhance immune responses and potent tumor immunity development and characterization of bipolymer based nanoparticulate carrier system as vaccine adjuvant for effective immunization strong systemic and mucosal immune responses to surface-modified plga microspheres containing recombinant hepatitis b antigen administered intranasally evaluation of mucoadhesive nanoparticle based nasal vaccine chitosan nanoparticle encapsulated hemagglutinin-split influenza virus mucosal vaccine induction of protective immunity against chlamydia trachomatis genital infection by a vaccine based on major outer membrane protein-lipophilic immune response-stimulating complexes intranasal immunisation with influenza-iscom induces strong mucosal as well as systemic antibody and cytotoxic t-lymphocyte responses iscom is an efficient mucosal delivery system for mycoplasma mycoides subsp. mycoides (mmmsc) antigens inducing high mucosal and systemic antibody responses the immunostimulating complex (iscom) is an efficient mucosal delivery system for respiratory syncytial virus (rsv) envelope antigens inducing high local and systemic antibody responses intranasal delivery of norwalk virus-like particles formulated in an in situ gelling, dry powder vaccine a novel intranasal virus-like particle (vlp) vaccine designed to protect against the pandemic influenza a virus (h n ) intranasal vaccination with an adjuvanted norwalk virus-like particle vaccine elicits antigen-specific b memory responses in human adult volunteers intranasal immunization with siv virus-like-particles (vlps) elicits systemic and mucosal immunity efficacy, immunogenicity and stability of a novel intranasal nanoemulsion-adjuvanted influenza vaccine in a murine model formulation and characterization of nanoemulsion intranasal adjuvants: effects of surfactant composition on mucoadhesion and immunogenicity nasal immunization with a recombinant hiv gp and nanoemulsion adjuvant produces th polarized responses and neutralizing antibodies to primary hiv type isolates pre-clinical evaluation of a novel nanoemulsion-based hepatitis b mucosal vaccine a novel bioadhesive intranasal delivery system for inactivated influenza vaccines proteosomes, emulsomes, and cholera toxin b improve nasal immunogenicity of human immunodeficiency virus gp in mice: induction of serum, intestinal, vaginal, and lung iga and igg capric acid and hydroxyl propyl methylcellulose increase the immunogenicity of nasally administered peptide vaccines the mucosal adjuvant potential of cross-linked dextran microspheres as dry powder nanoparticles and microparticles for drug and vaccine delivery peg-pla nanoparticles as carriers for nasal vaccine delivery nasal delivery of high molecular weight drugs novel n-trimethyl chitosan-poly(γ-glutamic acid) nanoparticles for mucosal delivery of vaccines applications of nanotechnology for immunology iscom-based vaccines: the second decade iscoms: an adjuvant with multiple functions iscomtm-based vaccines: the second decade immune stimulating complexes as mucosal vaccines the serum antibody response distributed in subclasses and isotypes after intranasal and subcutaneous immunisation with influenza virus immunostimulating complexes immune stimulating complexes containing quil a and protein antigen prime class i mhc-restricted t lymphocytes in vivo and are immunogenic by the oral route effect of iscoms and their adjuvant moiety (matrix) on the initial proliferation and il- responses: comparison of spleen cells from mice inoculated with iscoms and/or matrix intranasal immunization of mice with echinococcus granulosus surface antigens iscoms evokes a strong immune response, biased towards glucidic epitopes virus-like particles as immunogens the coming of age of virus-like particles an efficient plant viral expression system generating orally immunogenic norwalk virus-like particles virus-like particle-based human vaccines: quality assessment based on structural and functional properties virus-like particles as vaccine antigens and adjuvants: application to chronic disease, cancer immunotherapy and infectious disease preventive strategies microemulsions for nasal drug delivery system: an overview novel adjuvants & delivery vehicles for vaccines development: a road ahead characterization of stability and nasal delivery systems for immunization with nanoemulsion-based vaccines bioadhesive and formulation parameters affecting nasal absorption in situ gelling, bioadhesive nasal inserts for extended drug delivery: in vitro characterization of a new nasal dosage form bioadhesive polymers for controlled drug delivery projuvant (pluronic f /chitosan) enhance the immune response to intranasally administered tetanus toxoid chitosan as a novel nasal delivery system for vaccines almeida aj, alpar ho. nasal delivery of vaccines a review on intranasal drug delivery system development of nasal drug delivery: a review dry powder nasal vaccines as an alternative to needle-based delivery a meta-analysis of studies comparing the respiratory route with the subcutaneous route of measles vaccine administration a fast-rotating spectral shadow band radiometer for marine applications feasibility of aerosol vaccination in humans measles aerosol vaccination mucoadhesive hydrogels nanogel-based pneumococcal surface protein a nasal vaccine induces microrna-associated th cell responses with neutralizing antibodies against streptococcus pneumoniae in macaques nanogel antigenic proteindelivery system for adjuvant-free intranasal vaccines key: cord- -byqhzyzi authors: zhang, dingmei; lu, jiayuan; lu, jiahai title: enterovirus vaccine: close but still far date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: byqhzyzi background: enterovirus (ev ), a member of the enterovirus genus of the picornaviridae family, is one of the causative pathogens of hand-foot-and-mouth disease (hfmd) and the most common etiological agent isolated from hfmd patients complicated with neurological disorders. ev has become an increasingly important neurotropic enterovirus in the post-poliomyelitis eradication era. effective antiviral agents and vaccines against this virus are currently still under development. we reviewed publications on the development of ev vaccines in order to provide an overview of the field. methods: fifty-five articles on ev vaccine development, published from to , were collected from sun yat-sen university library and reviewed. results: various types of vaccine have been developed for ev . in results published to date, all vaccines for ev under development appear to elicit an immune response in rodents or in monkeys. according to the established regulatory standards, it may be relatively easy to acquire a license to use the inactivated virus in order to meet the immediate demands for ev control . with regard to the attenuated vaccine, it is critical to increase the genetic stability before clinical use, due to the risk of virulent revertants. the virus-like particle (vlp) vaccine, not only conserving the conformational epitopes, but also having no risk of virulent revertants, is another promising vaccine candidate for ev , but needs further development. the vp capsid protein is the backbone antigen protein for developing subunit vaccine and epitope vaccine; these remain viable potential vaccine strategies worthy of further study and development. conclusions: the conservation of the three-dimensional structure is important for the ev inactivated vaccine and vlp vaccine to induce a strong immune response. to develop ev vaccines with a high protection efficacy, strategies such as the use of adjuvant, strong promoters, tissue-specific promoters, and addition of mucosal immune adjuvant should be considered. enterovirus (ev ), a member of the enterovirus genus of the picornaviridae family, is the most frequently detected pathogen in hand-foot-and-mouth disease (hfmd) patients complicated with neurological dysfunction. ev was first isolated in california in , and its association with hfmd was verified in . , it was later confirmed as the causative agent responsible for hfmd outbreaks in hungary, australia, hong kong, taiwan, japan, and singapore. moreover, in and , a large outbreak occurred in mainland china. [ ] [ ] [ ] children under years of age have been found to be particularly susceptible to the severest form of ev -associated neurological disease. this is an important public health problem causing serious clinical illness and, potentially, death in young children. ev possesses a single-stranded rna genome of approximately nucleotides, consisting of a single open reading frame (orf) flanked by -untranslated regions ( utr) and -untranslated regions ( utr). the orf is expressed as a large polyprotein that can be cleaved into p , p , and p regions. the p region encodes four structural proteins vp , vp , vp , and vp . the p and p regions encode nonstructural proteins, such as proteases a, b, and cd, responsible for virus replication and virulence. protease a autocatalytically cleaves p at its n-terminus and liberates p from the nascent polyprotein, while protease cd cleaves the p precursor into vp , vp and vp (vp and vp ). these three structural proteins spontaneously assemble and form the crystalline virus-like particles. though there has been a significant increase in ev epidemic activity throughout the asia-pacific region, effective antiviral therapies and vaccines have, to-date, not been available. the development of effective vaccines is a top priority in terms of control strategies. below is an overview of the field of ev vaccine preparation to date. background: enterovirus (ev ), a member of the enterovirus genus of the picornaviridae family, is one of the causative pathogens of hand-foot-and-mouth disease (hfmd) and the most common etiological agent isolated from hfmd patients complicated with neurological disorders. ev has become an increasingly important neurotropic enterovirus in the post-poliomyelitis eradication era. effective antiviral agents and vaccines against this virus are currently still under development. we reviewed publications on the development of ev vaccines in order to provide an overview of the field. methods: fifty-five articles on ev vaccine development, published from to , were collected from sun yat-sen university library and reviewed. results: various types of vaccine have been developed for ev . in results published to date, all vaccines for ev under development appear to elicit an immune response in rodents or in monkeys. according to the established regulatory standards, it may be relatively easy to acquire a license to use the inactivated virus in order to meet the immediate demands for ev control . with regard to the attenuated vaccine, it is critical to increase the genetic stability before clinical use, due to the risk of virulent revertants. the virus-like particle (vlp) vaccine, not only conserving the conformational epitopes, but also having no risk of virulent revertants, is another promising vaccine candidate for ev , but needs further development. the vp capsid protein is the backbone antigen protein for developing subunit vaccine and epitope vaccine; these remain viable potential vaccine strategies worthy of further study and development. conclusions: the conservation of the three-dimensional structure is important for the ev inactivated vaccine and vlp vaccine to induce a strong immune response. to develop ev vaccines with a high protection efficacy, strategies such as the use of adjuvant, strong promoters, tissue-specific promoters, and addition of mucosal immune adjuvant should be considered. ß international society for infectious diseases. published by elsevier ltd. all rights reserved. as conventional vaccines, inactivated virus vaccines, such as inactivated influenza vaccine and inactivated hepatitis a vaccine, have been successfully used in the human. seroepidemiologic studies have indicated that the preexisting neutralizing antibody to ev is protective against the severe outcomes of infection. , yu et al. and wu et al. showed that passive transfer of serum from formalin-inactivated and heatinactivated virus vaccine immunized adult mice, could provide protection against ev challenge in neonatal mice; meanwhile, maternal immunization with inactivated ev vaccine was able to prolong the survival of suckling mice after ev lethal challenge. these results show the value of the inactivated virus vaccine for the effective control of ev . however, the conservation of the threedimensional structure is important in order to induce a strong immune response. therefore, for the heat-inactivated virus, a much higher dose of viral antigen and adjuvant are required to achieve an acceptable level of immunogenicity and protection. obviously, an ideal vaccine strain is required for the large-scale preparation of the inactivated ev vaccine, as has been the case for the sabin oral polio vaccine (opv) strain. lin et al. developed an ev strain, yn - a, exhibiting a rapid growth rate in vero cells with a larger plaque size and a lower lethal dose (ld) in newborn mice. lin and coworkers showed that mouse antiserum raised against yn - a was able to neutralize a broad range of ev strains isolated from patients of a variety of geographic origins at different points in time. yn - a possesses desirable features, such as a high viral yield, the ability to propagate in serum-free medium, and strong immunogenicity, as well as broad-based antigenic coverage and passage stability, indicating its potential for development as an inactivated vaccine strain. a powerful cell system is also important in the development of an inactivated vaccine. as shown by wu and coworkers, a serumfree vero cell culture with a g/l cytodex microcarrier concentration in a -l bioreactor has also been established, yielding a high titer of .  tcid /ml ev production. on the basis of the study of wu et al., liu et al. showed that the serum-free culture increased post-infection cell death and reduced the virus productivity, but elicited a higher neutralizing antibody titer in immunized mice as compared to the serum-containing cultures. therefore, the serum-free microcarrier culture is a valuable technique for developing inactivated ev vaccines on a large scale. an example of a successful attenuated strain vaccine is sabin opv. this was introduced in the early s due to its easier administration, lower cost, and higher intestinal muscosal immunity than the inactivated polio vaccine (ipv), and has since been approved for worldwide application for poliovirus eradication. , because of the similarities between poliovirus and ev , arita et al. , have developed an ev attenuated strain, ev (s - ), carrying mutations in the utr, d polymerase ( d pol ) and utr non-translated based on the attenuation determinants of poliovirus. this ev (s - ) strain is characterized by attenuated neurovirulence and limited spread of virus. in a subsequent study by arita et al., three cynomolgus monkeys were inoculated with ev (s - ), followed by a lethal challenge with the parental virulent strain ev (brcr-tr); they suffered mild neurological symptoms (tremor), but survived the lethal challenge without exacerbation of the symptoms. moreover, the sera from the immunized monkeys showed a broad spectrum of neutralizing activities against different genotypes of ev . these findings indicate that ev (s - ) acts as an effective antigen. however, it does cause mild neurological symptoms when inoculated via the intravenous route. additional studies are required to ensure that further attenuation produces effective attenuated vaccine strains. ev infection via the oral route did not efficiently cause neurological disorders in the inoculated monkeys. therefore the cynomolgus monkeys were inoculated by intravenous route instead of the oral route to evaluate the antigenicity of the attenuated ev vaccine. thus, to develop an oral ev attenuated vaccine, a valid animal model of ev infection by the oral route is urgently needed. meanwhile, it is well known that in a small number of opv recipients and their close contacts, especially those with primary humoral immunodeficiencies, the vaccine strain can mutate to a neurovirulent strain during opv replication and cause vaccineassociated paralytic poliomyelitis (vapp), which is an adverse side effect of opv. - a world health organization collaborative study found that the vapp rate was one in every . million doses administered for vaccine recipients and one in every . million doses administered for contacts. therefore, the genetic stability of the attenuated opv is a major concern, and efforts should be made to further attenuate the neurotoxic effects and increase the genetic stability of the attenuated ev vaccine before clinical use. to overcome the potential problem of reversion to virulence of attenuated strain vaccine, subunit vaccines consisting of only one or a few 'subunit' proteins of the pathogen that can stimulate immune responses directed at the intact virus have been developed using recombinant dna technology. in common with other enteroviruses, the vp , vp , and vp of ev are responsible for the antigenic diversity of enteroviruses, but the vp , the major capsid protein of ev , is clustered with neutralization epitopes and has the potential to act as an antiviral subunit vaccine. wu et al. have described a recombinant vp protein expressed in escherichia coli bl , showing that the vp protein with a complete adjuvant is able to elicit a neutralizing antibody response, enhance t helper cell proliferation, and induce high levels of interleukin (il)- and interferon (ifn)-g in mice, providing direct evidence that the vp protein contains neutralizing epitopes independent of other viral capsid proteins; this paves the way for the use of vp as a backbone antigen for developing subunit vaccines against ev . transgenic edible plants and mammalian glands are possible alternatives to prokaryotic and eukaryotic cell culture systems, offering a palatable oral delivery system, which can elicit a good mucosal immune response as well as systemic humoral and cellular immune responses, making it particularly suitable for protecting against infectious agents intruding via the mucosal surface. , ev initiates disease following implantation in the gut mucosa, showing the potential of an oral vaccine for immunization against ev infection. chen and colleagues have developed vp -expressing transgenic tomato fruits. these were used as a mouse free-feeding oral vaccine. the vp -specific fecal iga and serum igg were then observed in mice, and both humoral and cellular immunity against ev were established, showing the potential use of the transgenic tomato as an oral vaccine. meanwhile, other ev oral vp vaccine delivery systems, such as milk of transgenic mice described by chen et al. and the salmonella-based method by chiu et al. have been extensively explored. for oral vaccines, gastric acid and enzymatic digestion are major concerns, since they may interfere with vaccine absorption. the enterovirus genus can withstand human gastric acid and remain infectious below ph . . however, vp is a capsid protein on the surface of the ev particle, and whether it can resist human gastric acid has not been fully addressed. meanwhile, digestive enzymes may also degrade the antigens. in the experiment of chen et al., mice gavaged with vp protein produced more vp -specific antibodies than mice fed transgenic tomato containing more vp protein, in both sera and feces, indicating that chewing and digestion cause degradation of vp antigen. also, the oral rotavirus vp protein vaccine has been shown to provide lower levels of antibodies and less protection than immunization by injection of the rotavirus vp protein, indicating the possible interference of digestive enzymes. moreover, it has been difficult to determine the precise dose of antigens for immunization, since competition with food and microbial antigens interferes with the absorption rate of vaccine components. therefore, to improve oral vaccine delivery, many strategies have been developed, such as using tissue-specific promoters, the addition of mucosal immune adjuvant, using liposomes to protect the fusion peptides in the phospholipid bilayer vesicles from the gastric enzymes, and n-trimethyl chitosan nanoparticles. , for the oral ev vp subunit vaccine, exploring strategies to protect antigens from enzyme degradation in the gut is necessary. dna vaccines are expressed intracellularly in the same manner as during natural viral infection and can stimulate either humoral immunity, cellular immunity, or both. also, dna vaccines only deliver the target subunit antigen and thus cause fewer adverse effects, making them another valuable vaccine choice for most viral infections. tung and co-workers developed an ev dna vaccine by inserting the vp gene into a eukaryotic expression vector and evaluated the immune response in mice. their study results showed that the anti-vp igg level increased in mice immunized with dna vaccine; in contrast, this level declined after boosting immunization. furthermore, although the anti-vp igg exhibited neutralizing activity against ev , the neutralizing effect of the sera of mice immunized with the vp dna vaccine was much lower than that of ev -infected human serum. another dna vaccine developed by wu et al., elicited a high neutralization titer and stable titer level, which could be detected even at a late postimmunization time. however, because the dna vaccine contains fewer antigenic epitopes, it induces a weaker immune stimulation than the whole virus particles. therefore, strategies to increase the immune stimulation ability of dna vaccines have been developed including: incorporation of immunostimulatory sequences in the backbone of the plasmid, co-expression of stimulatory molecules, use of localization/secretory signals, and an appropriate delivery system, as well as adjuvants and optimization of transgene expression. , [ ] [ ] [ ] all these techniques can help to prepare a better ev dna vaccine. an epitope peptide vaccine consisting of a well-defined immunogenic epitope stimulates an effective and specific protective immune response while avoiding potential undesirable effects. the host immune response developed upon any viral infection is primarily cd + t cell-dependent, including the induction of a cytotoxic cellular response and efficient antibody response. thus, identification of cd + t cell epitopes and b cell epitopes is of great importance in the design of effective epitope peptide vaccines. foo and colleagues [ ] [ ] [ ] have published several studies aimed at identifying the t-cell and b-cell epitopes of ev . in these studies, they identified three regions, - , - , and - , spanning amino acids of the vp protein; they showed that these three regions could induce proliferation of cd + t cells, then producing abundant il- and ifn-g upon stimulation. additionally, among the three peptides, amino acids - induced the strongest proliferative response and highest cytokine production. furthermore, in order to identify the neutralizing linear epitopes, overlapping synthetic peptides spanning the vp capsid protein of ev were used to immunize mice. peptides containing amino acids - and - of vp protein were capable of eliciting neutralizing antibodies against ev , and the neutralizing antibodies elicited by the synthetic peptide - were able to confer good in vivo passive protection against homologous and heterologous ev strains in suckling balb/c mice. moreover, the monoclonal antibody generated by immunizing mice with amino acids - of vp showed strong neutralizing activity against ev in an in vitro neutralization assay. therefore, the epitope peptide vaccine represents a promising candidate for ev . the identification of more t-cell and b-cell epitopes of the vp protein, as well as a combination epitope peptide vaccine, should be considered in the search for a more effective epitope peptide vaccine. vlps are empty particles composed of all major structural proteins, mimicking the organizations and conformations of the native particles. to date, it has been shown that a wide range of vlps of clinically important viruses (e.g., hiv and severe acute respiratory syndrome coronavirus) induce effective neutralizing antibodies and cytotoxic t cell responses. , the vlp vaccine, not only conserving the conformational epitopes, but also having no risk of virulent revertants, is also a promising vaccine strategy for ev . hu et al. and chung et al. used a recombinant baculovirus expression system to express the cd and p proteins of ev ; the cd protein cleaves p precursor into vp , vp , and vp , which spontaneously assemble to form vlps, inducing both th and th immune responses. more importantly, the vlp immunization of mother mice conferred protection to neonatal mice against the lethal viral challenge, indicating ev vlp to be a promising vaccine. chung et al. also found that compared with the intact vlps, the denatured vlps elicited significantly lower levels of neutralizing antibodies and conferred lower degrees of protection against virus challenge, which highlights the importance of preserving the conformation-dependent epitopes in preventing ev infection. at present, the vlps are mostly developed using insect cells and the strict culture conditions limit the required large scale of vaccine production. thus, transgenic plants or yeast that can produce vlps to be delivered by either oral administration or injection, might be promising expression systems. ev is one of the causative pathogens of hfmd, often complicated with neurological disorders. due to the similarities between poliovirus and ev in many virological and clinical aspects, the success of the oral polio vaccine and inactivated-virus preparation in controlling poliomyelitis and eradicating the poliovirus, highlight the potential for controlling ev by vaccination. poliovirus vaccine technology, both live attenuated and inactivated virus vaccines, can be adapted to control ev infection. in recent years, various types of vaccine against ev have been developed, but these have as yet remained at the preclinical stage. outbreaks of ev have been reported around the world since . in economically developed nations, it typically causes a mild illness, and most patients usually recover quickly. however, since the late s, there has been a significant increase in ev epidemics, and it has emerged as a serious threat to public health throughout the asia-pacific region. developed countries with the resources for vaccine research and development do not view ev as a priority, and the vaccine industry in developed countries has little incentive to develop a vaccine to ev . at present, there are only a few vaccine industries in the asia-pacific region undertaking ev vaccine preparation. therefore, to effectively control ev , more effort and cooperation worldwide is needed. because ev mainly threatens the children in developing countries, an ideal ev vaccine would have to be inexpensive, safe, convenient to administer, and acceptable to parents. for the inactivated virus vaccine, the established regulatory standards may allow a license to be obtained to meet the immediate demands for ev control. due to the need to conserve the threedimensional structure, the formalin-inactivated virus vaccine is a potential candidate vaccine for ev . an oral ev attenuated vaccine has the potential to control ev in the same way as opv controlling poliovirus, though further attenuation procedures are needed. at present, there are five genotypes of ev . crossprotection to the different genotypes for all the ev vaccines under current development is unclear. hence, the preparation of a vaccine strain providing wide cross-protection is another important issue for ev vaccine development. there are no conflicts of interest with regard to employment, consultancy, stock ownership, honoraria, paid expert testimony, patent applications/registrations, or grants. we declare that we have no funding source. outbreaks of hand, foot, and mouth disease by enterovirus . high incidence of complication disorders of central nervous system an apparently new enterovirus isolated from patients with disease of the central nervous system new enterovirus type associated with epidemic of aseptic meningitis and-or hand, foot, and mouth disease a large-scale epidemic of hand, foot and mouth disease associated with enterovirus infection in japan in virological diagnosis of enterovirus type infections: experiences gained during an epidemic of acute cns diseases in hungary in outbreak of enterovirus infection in victoria, australia, with a high incidence of neurologic involvement monoplegia caused by enterovirus : an outbreak in hong kong an epidemic of enterovirus infection in taiwan. taiwan enterovirus epidemic working group outbreak of central nervous system disease associated with hand, foot, and mouth disease in japan during the summer of : detection and molecular epidemiology of enterovirus direct detection of enterovirus (ev ) in clinical specimens from a hand, foot, and mouth disease outbreak in singapore by reverse transcription-pcr with universal enterovirus and ev -specific primers enterovirus outbreak in the people's republic of china in ministry of health of the people's republic of china epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus in taiwan poliovirus polypeptide precursors: expression in vitro and processing by exogenous c and a proteinases formation of enterovirus-like particle aggregates by recombinant baculoviruses co-expressing p and cd in insect cells the efficacy, effectiveness and cost-effectiveness of inactivated influenza virus vaccines a randomised comparison of two inactivated hepatitis a vaccines, avaxim and vaqta, given as a booster to subjects primed with avaxim risk factors of enterovirus infection and associated hand, foot, and mouth disease/herpangina in children during an epidemic in taiwan neutralizing antibody provided protection against enterovirus type lethal challenge in neonatal mice protection against lethal enterovirus infection in newborn mice by passive immunization with subunit vp vaccines and inactivated virus characterization of a vero cell-adapted virulent strain of enterovirus suitable for use as a vaccine candidate optimization of microcarrier cell culture process for the inactivated enterovirus type vaccine development high immunogenic enterovirus strain and its production using serum-free microcarrier vero cell culture oral polio vaccines and their role in polio eradication in india role of injectable and oral polio vaccines in polio eradication temperaturesensitive mutants of enterovirus show attenuation in cynomolgus monkeys cooperative effect of the attenuation determinants derived from poliovirus sabin strain is essential for attenuation of enterovirus in the nod/scid mouse infection model an attenuated strain of enterovirus belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys vaccinederived polioviruses and the endgame strategy for global polio eradication novel btk mutation presenting with vaccine-associated paralytic poliomyelitis a case of vaccine-associated paralytic poliomyelitis adverse events following poliomyelitis vaccine enterovirus : the virus, its infections and outbreaks oral immunization using live attenuated salmonella spp. as carriers of foreign antigens oral vaccine delivery: can it protect against non-mucosal pathogens? oral immunization of mice using transgenic tomato fruit expressing vp protein from enterovirus expression of vp protein in the milk of transgenic mice: a potential oral vaccine protects against enterovirus infection protection of neonatal mice from lethal enterovirus infection by maternal immunization with attenuated salmonella enterica serovar typhimurium expressing vp of enterovirus efficient intranasal immunization of newborn mice with recombinant adenovirus expressing rotavirus protein vp against oral rotavirus infection oral vaccination with liposome-encapsulated recombinant fusion peptide of urease b epitope and cholera toxin b subunit affords prophylactic and therapeutic effects against h. pylori infection in balb/c mice in vitro and in vivo study of n-trimethyl chitosan nanoparticles for oral protein delivery dna vaccines: immunology, application, and optimization dna vaccine constructs against enterovirus elicit immune response in mice intradermal immunization with novel plasmid dnacoated nanoparticles via a needle-free injection device chemical adjuvants for plasmid dna vaccines dna vaccines: improving expression of antigens identification of human cd t-cell epitopes on the vp capsid protein of enterovirus identification of neutralizing linear epitopes from the vp capsid protein of enterovirus using synthetic peptides passive protection against lethal enterovirus infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide generation of neutralizing monoclonal antibodies against enterovirus using synthetic peptides chimeric gag-v virus-like particles of human immunodeficiency virus induce virus-neutralizing antibodies assembly of human severe acute respiratory syndrome coronavirus-like particles expression, purification and characterization of enterovirus- virus-like particles immunization with virus-like particles of enterovirus elicits potent immune responses and protects mice against lethal challenge we would like to express our thanks to associate prof. xia guo (the hong kong polytechnic university), dr. zhiyong guo (sun yatsen university) and dr. e. mengue mengue (sun yat-sen university) for their help in manuscript modification. key: cord- -y z l ji authors: carter-pokras, o.; hutchins, s.; gaudino, j.a.; veeranki, s.p.; lurie, p.; weiser, t.; demarco, m.; khan, n.f.; cordero, j.f. title: the role of epidemiology in informing united states childhood immunization policy and practice date: - - journal: ann epidemiol doi: . /j.annepidem. . . sha: doc_id: cord_uid: y z l ji one of the ten greatest public health achievements is childhood vaccination because of its impact controlling and eliminating vaccine-preventable diseases (vpds). evidence-based immunization policies and practices are responsible for this success and are supported by epidemiology that has generated scientific evidence for informing policy and practice. the purpose of this report is to highlight the role of epidemiology in the development of immunization policy and successful intervention in public health practice that has resulted in a measurable public health impact: the control and elimination of vpds in the united states. examples in which epidemiology informed immunization policy were collected from a literature review and consultation with experts who have been working in this field for the past years. epidemiologic examples (e.g., thimerosal-containing vaccines and the alleged association between the measles, mumps, and rubella (mmr) vaccine and autism) are presented to describe challenges that epidemiologists have addressed. finally, we describe ongoing challenges to the nation’s ability to sustain high vaccination coverage, particularly with concerns about vaccine safety and effectiveness, increasing use of religious and philosophical belief exemptions to vaccination, and vaccine hesitancy. learning from past and current experiences may help epidemiologists anticipate and address current and future challenges to respond to emerging infectious diseases, such as covid- , with new vaccines and enhance public health impact of immunization programs for years to come. epidemiology is the foundation of effective immunization policy and practice in the united states . epidemiologic methods, such as surveillance of vaccine-preventable diseases (vpds) and vaccination coverage, risk factor identification for both disease and lack of vaccination, community intervention and effectiveness studies, and assessment of access to and quality of vaccination services have contributed to the historic reduction or elimination of many vpds in the united states and the americas. epidemiology has contributed to immunization policy and practice at most levels of the immunization field--from vaccine development to ensuring that vaccines reach those who need them and result in the desired public health impact, disease control, and when feasible, disease elimination. for example, surveillance and studies of childhood infectious diseases provide the basis of morbidity and mortality data used to make j o u r n a l p r e -p r o o f immunization was selected as an example for examination of epidemiology in informing public health policy and practice because childhood immunization is one of the ten greatest public health achievements in the united states--it saves lives and is cost-effective. , - a study of . million children years of age or younger born during - found that routine childhood vaccination prevented million cases of illnesses and , premature deaths from vpds, resulting in a net savings of an estimated $ billion in direct medical costs and $ . trillion in societal costs to the united states. , this paper highlights the role of epidemiology in immunization policy development and public health practice that have led to major reductions in vpds. the success of childhood immunization programs has resulted from coordinated efforts that began with a rigorous science base--including epidemiologic methods and studies--that informed decision-making, led to public health policy, and continues to guide immunization services delivery. the working definition for policy in this paper is one generally used in public health: "a law, regulation, procedure, administrative action, incentive, or voluntary practice of governments and other institutions." this definition can be further summarized as described by torjman: "those decisions that seek to achieve a desired goal that is considered to be in the best interest of all members of society." j o u r n a l p r e -p r o o f through this examination of how epidemiology contributed to the successes, we also highlight lessons learned from immunization policy and practice that may be applicable to other public health programs, particularly those priorities delineated in healthy people . the united states has a robust policy-making apparatus for immunization policy development that supports all stages, from vaccine development to immunization practice. many stakeholders in the public and private sector are engaged at each step--from the consideration of candidate vaccines to vaccination of children once the food and drug administration (fda) (figure ) license new vaccines. many groups share responsibility in program implementation at the state, local, and even the health care office level in order to ensure high vaccination coverage, and reduction and control of vpds. vaccine development requires a large and diverse research infrastructure with funding from public and private sectors that begins by identifying diseases suitable for vaccine development ( figures and ) . once a candidate vaccine is developed, rigorous testing for safety, tolerability, immunogenicity, and efficacy follows with phase i, ii and iii clinical vaccine trials ( figure ). the private sector funds most clinical trials to demonstrate the safety, tolerability, immunogenicity, and efficacy of a candidate vaccine while the public sector funds vaccine development for selected vaccines and establishes priorities for vaccine development. developing new vaccines is an expensive and high-risk proposition, estimated to cost up to $ million dollars per vaccine and is a lengthy process, often taking more than a decade to bring a vaccine from development to market. the fda in the united states plays a key role in j o u r n a l p r e -p r o o f examining a candidate vaccine for its composition and source and the methods used for, and findings from,testing the vaccine's safety, purity and potency. only after the fda reviews and accepts the evidence from these initial steps, will it further examine evidence from human clinical trials about safety, tolerability, immunogenicity, and efficacy for the candidate vaccine in humans after finding a candidate vaccine to be safe and efficacious in humans, fda can then proceed to issue a license for the manufacture and commercial distribution for the vaccine ( figure ). , once the fda approves a vaccine, advisory committees such as the advisory committee on immunization practices (acip) recommend whether a new vaccine targeted for children and adults should be included in its recommended schedules for routine immunization ( figure ). , state and local immunization programs and health care providers play major roles in ensuring that vaccine coverage of a new vaccine quickly reaches high levels, and that established vaccines maintain a high coverage level needed to reduce or control vpds. professional organizations, such as the american academy of pediatrics (aap), the american academy of family physicians (aafp), and the american college of physicians (acp), make recommendations to their members on best practices to ensure high vaccination coverage and, in collaboration with the acip, recommend a schedule of routine immunization. government programs and insurance companies have a major role in the financing of vaccine purchase, and access to those vaccines. insurance companies cover many immunizations through their health care coverage plans. government programs, such as the vaccines for children program (vfc), provide targeted funding to cover costs for all acip-recommended vaccines for uninsured and underinsured children ages years and younger. many stakeholders from federal, state and local agencies, health plans, hospitals, clinics, employers, health care providers and philanthropic organizations play key roles in the implementation and day-to-day operation of the united states j o u r n a l p r e -p r o o f immunization system. the complex infrastructure of laws, regulations, funding streams, and programs continues to be informed by a spectrum of diverse epidemiologic surveillance and studies. we now describe some key elements of the federal agencies and respective advisory committees that inform immunization policy development. the national vaccine program office (nvpo), provides strategic leadership and coordination among federal agencies and other stakeholders to help reduce the burden of preventable infectious diseases. nvpo and national vaccine advisory committee (nvac) were established to comply with section of the public health service act. , nvpo obtains advice from the national vaccine advisory committee (nvac), which recommends approaches to control and prevent human infectious diseases through vaccine development, and provides advice on prevention of adverse reactions to vaccines ( figure ). , one example of nvac's key role was during and after the time of the major measles resurgence of the s was when it issued a call for action to eliminate endemic measles in the united states by using epidemiological evidence to improve childhood vaccination along with simultaneous monitoring of burden of measles. use of scientific evidence by nvac and the advisory committee for immunization practices' (acips') is a strong example of how epidemiology has contributed to the development of evidence-based national policy and has strengthened the immunization system in the united states. , , this example is discussed later in the article. j o u r n a l p r e -p r o o f as mentioned earlier, in the united states, vaccine development is supported by a combination of public and private sector research. in the public sector, the federal government through the united states department of defense, the national institutes of health, and other agencies within the department of health and human services (hhs) funds vaccine development. vaccine manufacturers invest significantly in all phases of vaccine development. the fda is the government regulatory agency that approves vaccines for commercial use. the sponsor of a vaccine submits the required documentation on safety, efficacy, and other aspects of the candidate vaccine to the fda. following internal reviews, the proposal is presented to the vaccines and related biological products advisory committee (vrbpac) (figure ), which then makes recommendations for licensing and additional data requests based on this evidence. the fda administrator makes the decision to approve the candidate vaccine based on the recommendation of the advisory committee. if approved, a vaccine license is issued with specific indications, precautions and contraindications. the advisory committee on immunization practices (acip), provides advice and guidance to the director of the centers for disease control and prevention (cdc) regarding use of vaccines and related agents for control of vaccine-preventable diseases in the civilian population of the united states ( figure ). , once a vaccine is licensed, and following a comprehensive review of the scientific evidence, the acip recommends vaccines for routine use and provides guidance on vaccine administration schedules likely to achieve the best levels of disease protection. years. , [ ] [ ] [ ] the increased availability and recommendations for more childhood vaccines represent remarkable achievements of the maturing immunization system of the united states to prevent vaccine preventable diseases, but have contributed to growing concerns about vaccine safety acceptability. [ ] [ ] [ ] [ ] [ ] the community preventive services task force, established by hhs in , develops guidance on community-based approaches to increase vaccination coverage based on available scientific evidence. , , this taskforce has provided evidence-based guidance for effective community-based approaches to reach and sustain high vaccination coverage ( figure ). effective strategies recommended include "multi-component" efforts such as combining health care system and community interventions together, use of client reminder/recall and provider reminder systems, use of client incentives, use home visits, and implementing state or local school immunization requirements for attendance. from this point on, we use the terms, "surveillance" and "monitoring" interchangeably to refer to the ongoing, systematic collection, analysis, interpretation, and dissemination of data regarding a health-related event for use in public health action to reduce morbidity and mortality and to improve health in contrast to "point in time" epidemiologic studies and outbreak investigation data use. vpds and reports national summaries of notifiable diseases, a regular feature in the mmwr. , cdc also monitors sporadic, endemic, epidemic and pandemic disease incidence overall and among population sub-groups to target and improve disease prevention and control efforts, including national elimination and global eradication initiatives. the recognition that hpv and hepatitis b vaccines can prevent cancer, has led to the inclusion of cancer and more recently precancerous disease surveillance and registries as data sources for monitoring the impact of vaccines in reducing cervical and liver cancer, respectively. [ ] [ ] [ ] [ ] since the s, after the resurgence of measles, the national immunization survey (nis) has been measuring immunization coverage at national and state levels using standardized methods. the nis originally targeted children - months of age, but now includes adolescents in a module designated as nis-teen. , the nis (preschool child) and nis-teen are multi-modal telephone-based surveys of parents with provider verification of immunization records. the nis has been essential in monitoring coverage for new vaccines as they are incorporated into the recommended immunization schedule. ensuring the safety of vaccines is a key component of table, those affected can apply for compensation through a streamlined process that avoids lengthy litigation. , immunization policy, practice, and epidemiology are necessarily intertwined. epidemiology informs policy and strategies to be incorporated into immunization practice through a process j o u r n a l p r e -p r o o f that begins with the consideration of what diseases may be preventable by a vaccine and continues with the identification of evidence-based strategies to effectively ensure high immunization coverage and optimally control or eliminate vpds. the development of childhood vaccines is preceded by collection and analysis of epidemiological data on the incidence of vpd-related conditions, disease morbidity and mortality, and evidence that infection confers protection against recurrence of the disease ( figure ). a recent example of this process related to the severity of varicella disease including mortality among adults in the united states prior to development of the varicella vaccine. also, as we write during the current pandemic, we are seeing unprecedented international scientific efforts to respond to the widespread community transmission of the novel coronavirus, sars-cov- , and the resulting waves of suffering and death related to covid- . these field clinical trials provide efficacy data and additional safety data about candidate vaccines. , these clinical trials are developed using rigorous epidemiologic methods, which include identifying the targeted trial population, randomization of participants to vaccination or placebo/alternative comparator groups, surveillance of the disease targeted by the vaccine, and monitoring of adverse events following vaccine administration. there are many examples of how epidemiologic evidence from vpd surveillance systems and outbreak investigation have contributed to better understanding of vaccine effectiveness and have led to changes in recommendations of vaccine administration. following introduction of a new vaccine, it is necessary to measure its population effectiveness in reducing the incidence of the targeted condition. results from ongoing surveillance of vpds and studies of reported outbreaks also provide opportunities to investigate waning vaccine immunity, reduced vaccine effectiveness, and gaps in vaccination due to missed opportunities to vaccinate during clinical encounters and/or vaccine hesitancy. the contribution of epidemiologic studies is evident, for example, in the development of recommendations for pertussis vaccines. studies of several pertussis outbreaks provided evidence that adults and adolescents were sources of disease transmission to young children, and that previously vaccinated adolescents were responsible for school outbreaks because of waning immunity. these findings led to additional child dose recommendations and the development of a new acellular vaccine booster recommended for adolescents and adults. [ ] [ ] [ ] the evidence of both waning immunity and that vaccinated pregnant women were able to provide passive immunity to their developing fetuses, also led to recommendations for routine tetanus and influenza vaccination for pregnant women. , epidemiologic studies of measles outbreaks led to the recognition that measles vaccination before months of age was associated with lower vaccine effectiveness. this was the basis for the acip recommendation that the first measles dose be administered on or after months of age. similarly, evidence from outbreaks among college students and school children showed insufficient effectiveness of a single measles dose to provide herd immunity. this led to recommendations for two doses of measles vaccines, one at - months and a second at to years of age. , other examples include a study of pertussis risk relative to the receipt and time since vaccination of the fifth dose of diphtheria and tetanus toxoids, and acellular pertussis vaccine (dtap) during an outbreak, and the role of varicella surveillance leading to change in immunization schedule from a single varicella dose to a two-dose schedule. epidemiologic studies have been used to evaluate new, and untested outbreak control interventions, such as evaluating recommendations to health care providers to vaccinate children using cdc's minimum immunization intervals during pertussis outbreaks and to use a third vaccination, during recent upsurges in mumps outbreaks. from to , the united states experienced a major nationwide resurgence of measles, which was detected by cdc's measles surveillance. the response to these events perhaps provides the best case-study of how epidemiologic evidence has informed, refined, and redirected united states immunization policy and practice. examination of reasons for the resurgence identified two kinds of outbreaks: ( ) large outbreaks among unvaccinated preschoolaged children, mainly in large urban centers, and ( ) smaller outbreaks among vaccinated children who, we know retrospectively, needed a second dose of a measles-containing vaccine. , additional analyses showed that unvaccinated preschool-aged outbreaks affected mostly young minority children in urban areas, with african american, latino, and american indian/alaska native children who contracted measles at rates three to times higher than white children did. the nvac examined evidence that pointed to challenges in the united states immunization system that likely contributed to the measles resurgence and to low immunization coverage rates that were well below healthy people objectives for preschool children. low vaccination coverage was primarily attributed to barriers in access to vaccination services or to missed opportunities to vaccinate by health care providers. , cost of vaccine was a key risk factor for uninsured or underinsured children. studies indicated that children visiting health care providers did not always receive all the recommended vaccines they were due, suggesting that missed opportunities to vaccinate were also important risk factors. , , - nvac's report concluded that immunization services needed to be enhanced and expanded. to guide efforts to increase vaccination rates, the report recommended that a national, standardized surveillance system to track age-appropriate immunization coverage across jurisdictions was necessary. this led to the creation of the national immunization survey to track the uptake of new childhood vaccines and monitor vaccination rates among young children - months of j o u r n a l p r e -p r o o f age to guide initiatives to more completely vaccinate these children with all recommended vaccines. , [ ] [ ] [ ] [ ] [ ] the key nvac findings and recommendations were published in , in what is now considered a report of historic significance. the nvac recommendations were embraced by policy makers and resulted in the launch of the childhood immunization initiative (cii). the cii, a presidential initiative, included several key elements: ( ) improving access to immunization services, ( ) developing immunization information systems, ( ) providing free vaccines to uninsured children (the vaccines for children program), and ( ) creating the national immunization program at cdc, now within the national center for immunization and respiratory diseases. improved access to immunization services improving access required addressing missed opportunities for immunizations. at the time, there were differences in recommendations between the acip, the american academy of pediatrics (aap) and the american academy of family practice (aafp). a major accomplishment of the cii was harmonizing the childhood immunization schedule jointly endorsed by acip, aap, and aafp, revisions of which have become a well-established convention and practice standard since . [ ] [ ] [ ] to address missed opportunities, programs targeted health care providers to remind them to make every child's medical visit, including acute and chronic care visits, a vaccination visit. tools are now available to health care providers to help them assess and improve immunization practices and identify ways to eliminate missed opportunities for vaccination at their offices. immunization registries or immunization information systems (iis) before the cii, most parents did not know the immunization status of their child. use of completed immunization cards and access to scattered immunization records among child providers were very limited and there were no electronic medical records that would allow clinicians to accurately assess immunization status at every visit (something particularly difficult at emergency room visits). immunization registries were developed to assist in the immunization assessment at each health j o u r n a l p r e -p r o o f care visit. by the mid- s, provider-based and population-or community-based immunization registries, now called immunization information systems (iis), were created for use by health providers to address immunization record scatter across clinics. iis are powerful and effective tools that provide timely access to immunization status at the point of care and have reduced missed opportunities by targeting under-vaccinated children for vaccination reminders and recalls, even before the introduction of electronic health record (ehr) systems. , [ ] [ ] [ ] as new vaccines were added to the immunization schedule, the combined series have been expanded. table includes a glossary of selected measures of vaccine completeness. , the acip expansion of recommended vaccines to adolescents and adults led to upgrades of the nis to specifically measure vaccination coverage for adolescents, including tetanus-diphtheriaacellular pertussis (tdap) and meningococcal conjugate vaccine (menacwy), by creating the nis -teen module in . , , in , monitoring for human papillomavirus (hpv) vaccination was added. like the original preschool child nis, this nis adolescent module includes provider-verified receipt of vaccines rather than relying on self-reported vaccination and provides data at state and selected local levels. vaccination coverage among young children and adolescents is found in figure and tables and . the end of the th century and the subsequent decades of the st century have witnessed further declines and the control of many vpds. polio has been eliminated from the americas and most of the world and it is near eradication worldwide. diseases like diphtheria, tetanus, measles, in spite of the retraction, this article created major concerns among parents considering vaccinating their children and continues to affect vaccination coverage of the mmr vaccine. a large epidemiologic study in denmark provided strong evidence of a lack of association between mmr and autism. similarly, a study in the united kingdom did not find any association between mmr and autism. the institute of medicine in the united states examined all available evidence and concluded that there was no evidence to link mmr vaccination and autism. the consequences of the subsequently retracted wakefield article include dramatic initial declines in mmr vaccination coverage in some countries. there were numerous resulting outbreaks of measles and mumps in the united kingdom, france, and elsewhere. [ ] [ ] [ ] [ ] surveillance documented that, in , the united states experienced cases in states, the largest number of measles cases since endemic measles was eliminated in . nis has also confirmed other study findings that suggest that those who intentionally delayed vaccination are significantly more likely to have heard or read unfavorable information about vaccines than parents who did not intentionally delay. additionally, parents who intentionally delayed due to vaccine safety or efficacy concerns were significantly more likely to seek information from the internet rather than from a health care provider compared with parents who delayed because of child illness. differences by race have been documented in j o u r n a l p r e -p r o o f these analyses--the percentage of parents who intentionally delayed immunizations was highest among white, non-hispanics ( . %), american indian/alaska natives ( . %), followed by asians ( . %), hispanics ( . %), and blacks ( . %). further analyses are needed to evaluate which parental, community and other characteristics and risk factors underlie these notable differences by racial/ethnic groups in childhood vaccine delays, for example examining how differences in historical experiences with vpds and trust may influence vaccine decision making among different groups. findings about intentional delays in immunization among some -year-old children and the ability of parents to claim religious or philosophical exemptions raise questions about the influence of the ease of parent claims in some states and higher state vaccination exemption rates. one study found that states enacting stricter exemption policies tend to have lower rates of exemptions. in recent years, congress and states, such as california, vermont, utah, washington and oregon, have passed or attempted to pass laws to modify or eliminate the use of non-medical exemptions. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] these policy initiatives are being met with public controversy and opposition by nationally-organized and grassroots groups communicating vaccine safety, civil liberty, other concerns, and also anti-vaccine sentiments. [ ] [ ] [ ] [ ] [ ] the legal viability and public health effectiveness of these more restrictive strategies remain to be determined. early studies of california's non-medical exemption elimination show that, while non-medical exemptions declined, geographic clustering of these exemptions remained leaving populations of students at-risk for vpds in a number of communities. [ ] [ ] [ ] epidemiological studies clearly play a key role in monitoring changing child immunization coverage, non-medical exemptions to school immunization requirements and other measures of vaccine hesitancy trends and the impact of policy changes and the interventions to address them. another important immunization practice issue is addressing differences in vpd morbidity and disparities in vaccination coverage among special populations. epidemiological studies proved to be particularly relevant when examining the impact of haemophilus influenzae type b (hib) and hepatitis a (hepa) vaccines on the american indian/alaska native (ai/an) population. the introduction of the hib vaccine significantly reduced hib incidence in ai/an children. surveillance proved to be critical in demonstrating a greater response with the first dose of the polyribosylribitol phosphate conjugated to the meningococcal outer membrane protein (prp-omp)-containing hib vaccines for ai/an infants providing earlier protection. in fact, when alaska switched from prp-omp to non-omp vaccine during a vaccine shortage, ai/an hib incidence increased. , again, epidemiological evidence was important to guide immunization practice. besides experiencing higher hib disease incidence, ai/an children historically had more than a five-fold higher incidence of hepa virus infection and were experiencing frequent large-scale outbreaks every - years. with the implementation of routine hepa vaccination in among high-risk populations (e.g., ai/ans), disease incidence and outbreak disparities were completely eliminated. as another special population, the amish were the last group to experience a polio outbreak in the united states. in , pennsylvania noted an increase in hib disease among amish preschool children. an epidemiologic study of hib carriage showed high levels of hib carriage and low vaccination coverage among amish households. a study among amish parents who did j o u r n a l p r e -p r o o f not vaccinate their children found that only % identified personal-belief objections as a factor, % reported that vaccination was not a priority compared with other daily activities, and % would vaccinate children if offered locally. these findings encouraged the state to target hib vaccination programs to amish communities and craft specific educational messages to amish parents leading to a reduction in hib disease in this special population. these examples show how public health used epidemiologic surveillance to document increases in disease incidence and disparities in vaccination coverage in special populations in order to respond with targeted interventions to address these problems and achieve disease prevention successes. epidemiologists are improving their methods to track new vaccine uptake, especially for newer vaccines including the multi-dose human papillomavirus (hpv) vaccine to prevent cervical cancer and the tdap booster for adolescents and adults. the hpv vaccine experience epidemiologists have looked closely at the factors associated with rates of hpv vaccine initiation and completion to examine vaccine uptake and acceptance. observed differences pointed out that further research was needed to better understand population-specific barriers to completion of the hpv series. monitoring hpv uptake, first among adolescent girls and later among adolescent boys, epidemiologists focused on identifying risk factors associated with low hpv vaccination.a telephone survey of mothers of - -year-old girls found that the predominant perception was that their daughters were at low risk for hpv infections and hpv-related diseases. findings also showed that mothers and their health care providers lacked sufficient knowledge about hpv disease and hpv vaccines. many mothers also reported that they believed that their daughters were currently too young to receive the hpv vaccine although receipt might be more acceptable at later ages. also, mothers reported significant concerns about the long-term safety of these vaccines. the most commonly identified reasons for mothers accepting these vaccines for their daughters included: their perceptions that their daughters were at high risk for acquiring hpv; their beliefs that the vaccine had a favorable safety profile; their intentions to prevent cervical j o u r n a l p r e -p r o o f cancer among their daughters and protect them against cancer; their own personal experience with hpv infection or hpv-related diseases; and their recalling strong physician recommendations to vaccinate their daughters. these findings have been shaping the messages and strategies to promote hpv vaccination with a stronger focus on the cancer prevention benefit of this vaccine. as in other countries, the impact of the covid- pandemic on the united states immunization system and policies is starting to become apparent as covid- continues to rapidly spread across communities. since public health authorities across the united states have needed to urgently implement non-pharmaceutical public health disease containment measures (e.g., shelter-in-place, postponements of noncritical health care visits), early epidemiological studies are already documenting a dramatic decline in ordering and administration of childhood vaccines, vfc clinic capacity to vaccinate children, and immunization coverage rates for vpds. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] rapid development of new covid- vaccines is an imperative because of the severe consequences of covid- disease, which is disproportionately impacting people over years of age, people with heart disease, diabetes, other chronic diseases, essential service workers and populations of color. [ ] [ ] [ ] [ ] however, as new vaccines for covid- are being developed and tested, new reports also suggest the emergence of major challenges for new covid- vaccination uptake. [ ] [ ] [ ] several reports state that that up to % percent of polled respondents were hesitant about accepting new covid- vaccines when they become available. , j o u r n a l p r e -p r o o f previous epidemiological studies have shown that after vaccine supply chain disruptions and shortages have occurred, uptake of vaccine may slowly recover and could remain persistently lower than prior uptake well behind recommended target coverage rates when supplies become available. re-engaging patients for clinical preventive services and increasing vaccination among people who have previously declined or fallen behind schedule during and after the covid- crisis are critical strategies to prevent other vpd outbreaks, which could further strain our health care system, emergency response systems, and economy and, thus, slow economic and societal recovery from the pandemic. [ ] [ ] [ ] with delays in vaccinations, vaccine hesitancy and upcoming seasonal influenza transmission, during the pandemic, we face new challenges that risk losing historical achievements in individual and community health and new unknown risks of further preventable illnesses, disabilities and death. , [ ] [ ] [ ] [ ] previous epidemiologic evidence suggests that by reducing the incidence of vpds such as influenza and pneumococcal disease, we also would reduce burden on the health care facilities that are already under pressure in communities responding to the waves of covid- outbreaks and community-wide transmission. immunization policy makers, public health practitioners and health care providers must plan new immunization initiatives that include proactively and transparently gaining back the trust of an already skeptical public whose trust in public health and health care advice during this pandemic have been sorely tested. , [ ] [ ] [ ] epidemiologic surveillance, research and program evaluation will be essential nationally, regionally and within communities to guide needed interventions that successfully respond to these new public health challenges. more challenging is the ongoing need to develop new, specific vaccines for emerging diseases with high morbidity and mortality and rapid spread as real-time countermeasures, notable at the time of this writing during the covid- pandemic. [ ] [ ] [ ] [ ] , especially challenging is that currently governments are usually the sole funding source for vaccine development unless commercial manufacturers offer to help and see financial and other incentives including the potential for more routine population-wide use. [ ] [ ] [ ] [ ] [ ] [ ] [ ] to be ready to respond effectively to the eventuality of new, emergent vaccine-preventable outbreaks and community-wide biological attacks, policy makers, health officials, legislators and other stakeholders can work together to ensure that policies are in place to expedite development of new vaccines, ensure vaccine safety and efficacy, and determine appropriate resources in a timely fashion. public/private partnerships can be developed to meet the demands for research and development of new vaccines and to establish capacity for production. additional public health system capacity across all levels and communities could be enhanced and sustained in order to address mass vaccine distribution and administration by health care providers, vaccination monitoring, disease surveillance, and program and policy evaluations to meaningfully inform policy and program decisions in realtime. j o u r n a l p r e -p r o o f uniform, quick, appropriate and timely reporting of disease cases and adverse events by physician offices, hospitals, laboratories, schools or other institutions such as child-care and correctional facilities can be more firmly established. enhanced electronic reporting from electronic laboratory and health record systems, data analyses and information dissemination can be enhanced to function more rapidly in real-time. rapid surveillance using electronic data is needed to provide more timely and accurate situational status assessments, target services and improve response time to public health emergencies. epidemiologists can expand their use of methods from other public health disciplines, such as community-based participatory research, qualitative research, rapid-cycle quality improvement work and evaluation methods to better identify vaccine acceptance disparities and differences in perceptions, knowledge, attitudes, and beliefs among specific populations, including providers. interventions that overcome the barriers and address the needs of special populations can be developed, implemented, evaluated and disseminated. epidemiology remains essential for informing policy and programmatic practice decision making to prevent and respond to vpds. epidemiologic studies of the large united states measles resurgence identified major factors by further identifying determinants of low vaccination coverage. these efforts were crucial for focusing policies and programmatic strategies at national, state and local levels. surveillance and epidemiologic research have also been essential in monitoring the impact of vaccinations on infectious disease incidence and vaccine acceptance j o u r n a l p r e -p r o o f by clinicians, parents and patients. while epidemiology has positively influenced changes in immunization policy and led to historic reductions in vpds, the reduction of vpd incidence has created new challenges in our ability to help parents and providers understand why vaccines remain essential. recent developments have led to public questioning of the value and risks of vaccinations while vaccine acceptance is high. , [ ] [ ] [ ] , , , , however, the nation must be vigilant in continuously measuring vaccine use, vaccine-preventable diseases, and vaccine safety, to avoid the trap of being victims to our own success. j o u r n a l p r e -p r o o f table . vaccination coverage among adolescents ages - years, by race/ethnicity and selected vaccines and doses* -national immunization survey -teen, (nis -teen), united states, † vaccine-preventable disease table working g. historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states elimination of measles and of disparities in measles childhood vaccine coverage among racial and ethnic minority populations in the united states. the journal of infectious diseases centers for disease control and prevention. benefits from immunization during the vaccines for children program era -united states ten great public health achievements--united states ten great public health achievements--united states economic evaluation of the routine childhood immunization program in the united states office of the associate director for policy and strategy. definition of policy what is policy? ottawa, ca: caledon institute of social policy office of disease prevention and health promotion assembling a global vaccine development pipeline for infectious diseases in the developing world united states food & drug administration. blood, vaccines and other biologics the history of the united states advisory committee on immunization practices (acip) united states department of health and human services united states department of health and human services. vaccines & immunizations. national vaccine advisory committee (nvac) centers for disease control and prevention. recommendations of the advisory committee on immunization practices: programmatic strategies to increase vaccination coverage by age years--linkage of vaccination and wic services. mmwr morbidity and mortality weekly report recommendations of the advisory committee on immunization practices: programmatic strategies to increase vaccination rates--assessment and feedback of provider-based vaccination coverage information. mmwr morbidity and mortality weekly report children's hospital of philadelphia. vaccine history: developments by year vaccination and risk communication: summary of a workshop reviews of evidence regarding interventions to improve vaccination coverage in children, adolescents, and adults. the task force on community preventive services institute of medicine (us) committee on immunization finance policies and practices calling the shots: immunization finance policies and practices nonmedical exemptions from school immunization requirements: a systematic review increasing appropriate vaccination: vaccination requirements for child care updated guidelines for evaluating public health surveillance systems: recommendations from the guidelines working group national notifiable disease surveillance system (nndss). national notifiable conditions using population-based cancer registry data to assess the burden of human papillomavirus-associated cancers in the united states: overview of methods annual report to the nation on the status of cancer, - , featuring the increasing incidence of liver cancer the hpv vaccine impact monitoring project (hpv-impact): assessing early evidence of vaccination impact on hpv-associated cervical cancer precursor lesions monitoring effect of human papillomavirus vaccines in us population, emerging infections program national immunization survey: the methodology of a vaccination surveillance system national vaccination coverage among adolescents aged - years--united states, . mmwr morbidity and mortality weekly report safety monitoring in the vaccine adverse event reporting system (vaers) the vaccine safety datalink: successes and challenges monitoring vaccine safety the food and drug administration's post-licensure rapid immunization safety monitoring program: strengthening the federal vaccine safety enterprise national vaccine injury compensation program. health resources and services administration vaccine development: from concept to early clinical testing trends in varicella mortality in the united states: data from vital statistics and the national surveillance system a strategic approach to covid- vaccine r&d the road to immunity during covid- : developing & distributing a vaccine. special presentation and panel discussion from the covid- conversations webinar series sponsored by the special presentation and panel discussion webinar sponsored by the fred hutchinson cancer research center rapid covid- vaccine development tetravalent dengue vaccine reduces symptomatic and asymptomatic dengue virus infections in healthy children and adolescents aged - years in asia and latin america. the journal of infectious diseases safety overview of a recombinant live-attenuated tetravalent dengue vaccine: pooled analysis of data from clinical trials preventing tetanus, diphtheria, and pertussis among adolescents: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccines recommendations of the advisory committee on immunization practices (acip) preventing tetanus, diphtheria, and pertussis among adults: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine recommendations of the advisory committee on immunization practices (acip) and recommendation of acip, supported by the health care infection control practices advisory committee (hicpac), for use of tdap among health-care personnel prevention and control of seasonal influenza with vaccines measles vaccine efficacy in children previously vaccinated at months of age mmwr morbidity and mortality weekly report acip releases supplementary report on measles prevention association of childhood pertussis with receipt of doses of pertussis vaccine by time since last vaccine dose, california prevention of varicella: recommendations of the advisory committee on immunization practices (acip) did children receive dtap vaccinations earlier after accelerated dtap immunization recommendations by local public health during pertussis outbreaks in oregon?: an evaluation of immunization practice changes among community clinicians. paper presented at: annual meeting of the council of state and territorial epidemiologists catch-up immunization schedule for persons aged months- years who start late or who are more than month behind, united states impact of a third dose of measles-mumpsrubella vaccine on a mumps outbreak adverse events following a third dose of measles, mumps, and rubella vaccine in a mumps outbreak patterns of transmission in measles outbreaks in the united states, - . the new england journal of medicine measles outbreak among unvaccinated preschool-aged children: opportunities missed by health care providers to administer measles vaccine missed opportunities for immunizations: a review of the evidence causes of low preschool immunization coverage in the united states recommended by the national vaccine advisory committee centers for disease c, prevention. county-level trends in vaccination coverage among children aged - months -united states national, state, and local area vaccination coverage among children aged - months--united states vaccination coverage among children aged - months -united states coverage among children - months by state, hhs region, and the united states vaccination coverage among u.s. children aged - months entitled by the vaccines for children program the measles epidemic. the problems, barriers, and recommendations. the national vaccine advisory committee the president's child immunization initiative--a summary of the problem and the response effectiveness of a practice-based intervention to increase vaccination rates and reduce missed opportunities ?cdc_aa_refval=https% a% f% fwww.cdc.gov% fpertussis% fout breaks% ftrends.html. accessed assessing immunization coverage in private practice infants and children (wic childhood immunization registries designed to expedite: immunization registry information systems immunization information systems to increase vaccination rates: a community guide systematic review comunity preventive services task force. recommendation for use of immunization information systems to increase vaccination rates immunization information systems use during a public health emergency in the united states vaccines for children program overview of the national health interview survey and its sample design. vital health stat vaccination coverage of -year-old children--united states vaccination coverage among children in kindergarten -united states, - school year. mmwr morbidity and mortality weekly report centers for disease control and prevention. human papillomavirus vaccination coverage among adolescent girls national, regional, state, and selected local area vaccination coverage among adolescents aged - years -united states ?cdc_aa_refval=https% a% f% fwww.cdc.gov% fvaccines% fpare nts% fdiseases% fchild% f -diseases.html. accessed changes in childhood immunization decisions in the united states: results from & national parental surveys risk factors associated with parents claiming personal-belief exemptions to school immunization requirements: community and other influences on more skeptical parents in oregon parental hesitancy about routine childhood and influenza vaccinations: a national survey understanding thimerosal, mercury, and vaccine safety ileal-lymphoid-nodular hyperplasia, nonspecific colitis, and pervasive developmental disorder in children ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children world health organization (who) an assessment of thimerosal use in childhood vaccines thimerosal in vaccines: a joint statement of the american academy of pediatrics and the public health service. mmwr morbidity and mortality weekly report immunization safety review: thimerosal-containing vaccines and neurodevelopmental disorders prevalence and characteristics of autism spectrum disorder among children aged years--autism and developmental disabilities monitoring network, sites, united states a population-based study of measles, mumps, and rubella vaccination and autism. the new england journal of medicine autism and measles, mumps, and rubella vaccine: no epidemiological evidence for a causal association institute of medicine (us) immunization safety review committee an outbreak of mumps in the metropolitan area of walsall, uk. international journal of infectious diseases : ijid : official publication of the international society for infectious diseases measles outbreak--california measles elimination efforts and - outbreak, france. emerging infectious diseases crucial lessons from the recent measles outbreak. the huntington post vaccine-preventable outbreaks: still with us after all these years the timing of pertussis cases in unvaccinated children in an outbreak year: oregon increase in measles cases -united states the impact of epidemics of vaccine-preventable disease on vaccine uptake: lessons from the - us pertussis epidemic association between vaccine refusal and vaccine-preventable diseases in the united states: a review of measles and pertussis notes from the field: community outbreak of measles cost of washington measles outbreak tops $ m. the oregonian processes for obtaining nonmedical exemptions to state immunization laws religious healing in the courts: the liberties and liabilities of patients, parents, and healers paper presented at: webinar at northwest center for public health practice (nwcphp) ; school of public health identifying communities in patterns of exemptions to school immunizations. paper presented at: national immunization conference national, regional, state, and selected local area vaccination coverage among adolescents aged - years--united states the association between intentional delay of vaccine administration and timely childhood vaccination coverage improving nonmedical vaccine exemption policies: three case studies vaccine controversy: oregon senator's school bill renews exemption fight hundreds rally outside capitol to oppose bill that would limit vaccine exemptions in oregon over , descend on wa state capitol to challenge vaccine mandate bills to oregon lawmakers: reject vaccine bill mandatory health care provider counseling for parents led to a decline in vaccine exemptions in california conditional admission, religious exemption type, and nonmedical vaccine exemptions in california before and after a state policy change elimination of nonmedical immunization exemptions in california and school-entry vaccine status impact of immunizations on the disease burden of american indian and alaska native children haemophilus influenzae type b disease and vaccine booster dose deferral, united states prevention and control of haemophilus influenzae type b disease: recommendations of the advisory committee on immunization practices (acip) haemophilus influenzae type b disease among amish children in pennsylvania: reasons for persistent disease human papillomavirus vaccination series initiation and completion understanding the reasons why mothers do or do not have their adolescent daughters vaccinated against human papillomavirus effects of the covid- pandemic on routine pediatric vaccine ordering and administration -united states, . mmwr morbidity and mortality weekly report provision of pediatric immunization services during the covid- pandemic: an assessment of capacity among pediatric immunization providers participating in the vaccines for children program -united states please continue vaccinating patients during covid- maintaining childhood immunizations and well-child care during covid- pandemic routine vaccination during the covid- outbreak decline in child vaccination coverage during the covid- pandemic -michigan care improvement registry vaccinations have sharply declined nationwide during the covid- pandemic: rates of childhood immunization have fallen across the u.s., raising the risk of vaccine-preventable disease outbreaks the dual epidemics of covid- and influenza: vaccine acceptance, coverage, and mandates exclusive: a quarter of americans are hesitant about a coronavirus vaccine. -reuters/ipsos poll one in three americans would not get covid- vaccine american academy of family physicians. inside look at using telemedicine during covid- pandemic - estimated influenza illnesses, medical visits, hospitalizations, and deaths averted by vaccination flu vaccine coverage, united states - influenza season estimated influenza illnesses, medical visits, hospitalizations, and deaths in the united states- - influenza season planning for a covid- vaccination program the reemergence of vaccine-preventable diseases: exploring the public health successes and challenges. testimony before the committee on health, education, labor and pensions, united states senate group shape vaccine delivery working group. from refrigerator to arm: issues in vaccination delivery vaccine refusal, mandatory immunization, and the risks of vaccine-preventable diseases. the new england journal of medicine > polio: > mmr: > hib: > hepb: > varicella abbreviations: dtp/dtap = diphtheria and tetanus toxoids and whole cell pertussis vaccine or diphtheria and tetanus toxoids and acellular pertussis vaccine mmr = measles-mumps-rubella vaccine hib = haemophilus influenzae type b vaccine hep b = hepatitis b vaccine; varicella= varicella vaccine; and pcv = pneumococcal conjugate vaccine abbreviations: dtp/dtap = diphtheria and tetanus toxoids and whole cell pertussis vaccine or diphtheria and tetanus toxoids and acellular pertussis vaccine mmr = measles-mumps-rubella vaccine hib = haemophilus influenzae type b vaccine hepb = hepatitis b vaccine varicella=varicella vaccine pcv = pneumococcal conjugate vaccine hepa = hepatitis a vaccine *selected vaccines and dosages are in accordance with immunization objectives from healthy people and follow the cdc's recommended immunization schedule for children and adolescents ages years or younger : : *: : : ) includes ≥ doses of dtap, ≥ doses of poliovirus vaccine, ≥ dose of measles-containing vaccine, the full series of hib (≥ or ≥ doses, depending on product type figure . vaccine coverage among preschool-aged children -united states abbreviations: dtp/dtap = diphtheria and tetanus toxoids and whole cell pertussis vaccine or diphtheria and tetanus toxoids and acellular pertussis vaccine mmr = measles-mumps-rubella vaccine hib = haemophilus influenzae type b vaccine hep b = hepatitis b vaccine; varicella= varicella vaccine pcv = pneumococcal conjugate vaccine rv = rotavirus vaccine hep a = hepatitis a vaccine + from the united states immunization survey note: no data are available for - . children in the united states immunization survey and national health interview survey were ages - months centers for disease control and prevention. coverage among children - months by state, hhs region, and the united states national, regional, state, and selected local area vaccination coverage among adolescents aged - years -united states centers for disease c, prevention. benefits from immunization during the vaccines for children program era -united states race/ethnicity non-hispanic white *selected vaccines and dosages are in accordance with immunization objectives from healthy people and follow the cdc's recommended immunization schedule for children and adolescents aged years or younger. , **includes those with >= doses, and those with doses when the first hpv vaccine was initiated prior to age years and there was at least five months minus four days between the first and second dose. ***among adolescents with no history of varicella. † numbers in parentheses refer to the number of doses of that vaccine being reported in this figure. key: cord- - vsynfgn authors: kostoff, ronald n.; briggs, michael b.; porter, alan l.; spandidos, demetrios a.; tsatsakis, aristidis title: covid- vaccine safety date: - - journal: int j mol med doi: . /ijmm. . sha: doc_id: cord_uid: vsynfgn in response to the sars-cov- outbreak, and the resulting covid- pandemic, a global competition to develop an anti-covid- vaccine has ensued. the targeted time frame for initial vaccine deployment is late . the present article examines whether short-term, mid-term, and long-term vaccine safety can be achieved under such an accelerated schedule, given the myriad vaccine-induced mechanisms that have demonstrated adverse effects based on previous clinical trials and laboratory research. it presents scientific evidence of potential pitfalls associated with eliminating critical phase ii and iii clinical trials, and concludes that there is no substitute currently available for long-term human clinical trials to ensure long-term human safety. the new outbreak of sars-cov- from december precipitated a world-wide crisis. globally, lockdowns of different severity levels were imposed ( ) . while the number of daily deaths attributable to covid- appears to have decreased substantially by june , the increasing numbers of 'cases' (positive test results for viral exposure) have raised some concerns regarding the ability of governments and decision-making authorities to reduce viral transmission and subsequent consequences ( ) ( ) ( ) . currently, at months following the outbreak, no specific treatment for severe forms of covid- has achieved consensus within the medical community, although several potential therapies appear to have produced more or less encouraging results ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the methods used to contain the spread of the virus have been the traditional social distancing, quarantine, use of disinfectant substances, and wearing of protective face masks ( ) ( ) ( ) . these measures have adverse consequences, both psychological and economic, and have resulted in substantial disagreement among the medical community and political decision-makers regarding their efficacy ( , , ) . in parallel with the imposed restrictions to prevent viral spread and the testing of (mainly) repurposed anti-viral treatments is the accelerated development of vaccines to prevent/restrict potential viral damage. questions have been raised as to whether an accelerated vaccine development can be accomplished safely, preventing potential adverse vaccine effects not only in the short-term, but also in the mid-and long-term (https://smartech.gatech.edu/handle/ / ). currently (mid-september, ), there is avid competition regarding the development and commercialization of a vaccine by early ( , ) . one candidate vaccine, sputnik- , was approved by the ministry of health of the russian federation on august , ( ) . these accelerated vaccine development efforts suggest that safety testing was performed in ≤ year, a time frame significantly shorter than that of - years typically associated with the commercialization of a vaccine ( ) . it is difficult to see how mid-and long-term safety testing for the proposed vaccine (or any vaccine or drug) can be performed credibly in such a compressed time frame (https://smartech.gatech.edu/handle/ / ) for reasons described below. the underlying models may be lacking. this could impact the credibility of any conclusions on safety or toxicity ( ) . thus, while these models may provide interesting insight, they cannot substitute for human trials, at least at this point in their development. animal experimentation. there are several examples where whole animal experiments have been poor predictors of human responses to environmental exposures or drugs. isotretinoin (acutane), for example, has been demonstrated to cause birth defects in rabbits, monkeys and humans, but not in mice or rats. as another example, corticosteroids are teratogenic in experimental animals, but not in humans. in addition, there is the well-known example of thalidomide, 'a teratogen in humans, but not in many experimental animal species' ( ) . why are some animal experiments poor predictors of human outcomes and responses? the studies may be designed poorly and may be inadequate methodologically; the studies may not be replicated or subject to meta-analyses; the metabolic pathways or drug metabolism of humans differ from those of the tested species or strains and 'disease manifestations in the animals are distinct from those encountered in humans' ( ) . laboratory animal experiments allow for the selection of test animals with short lifetimes, and can identify adverse health effects over the animals' lifetimes (the analog of long-term human effects), and perhaps one or two generations beyond. as previously stated ( ) , how well the response of the species selected for the experiments reflects the response of humans remains to be determined. additionally, laboratory animals are typically exposed to one toxic stressor (vaccines, in the present case), whereas human beings are exposed to myriad toxic stressors daily and over their lifetimes ( ) ( ) ( ) ( ) . these toxic stressor exposures may substantially alter the effects of a vaccine ( ) . to simulate the human real-life experience, a number of animal experiments would have to be performed to reflect the effects of various combinations of the thousands of toxic stressors (in conjunction with vaccines) to which human beings could be exposed (and other exposures that, by themselves are not toxic, but in combination are toxic) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . these experiments would require vast amounts of resources, particularly money and time. human clinical trials. human trials have at least two advantages over laboratory animal experiments. first, there are no concerns regarding species differences that occur when extrapolating from laboratory animal testing results to potential human impacts. second, humans are exposed to myriad toxic stressors before, during and after the trial period, providing results that mirror the real-life experience. in all cases, human trials will be most relevant if the characteristics of the trial population reflect those of the target/user population. the disadvantages of human clinical trials are as follows: i) the exposures to toxic stimuli are either not known, or, if they are known, have not been estimated accurately; and ii) the identification of the long-term effects requires long periods of time (https://smartech.gatech.edu/handle/ / ). how much time is required? in a previous study of vaccines and autoimmunity ( ), the authors concluded that 'latency periods can range from days to years for postinfection and postvaccination autoimmunity'. mid-term adverse effects of vaccines, such as central nervous system (cns) inflammatory demyelination ( ) and diabetes ( ) have been shown to emerge after approximately years. longer-term effects, such as cancer, alzheimer's disease, parkinson's disease, etc., have not been studied. in fact, vaccine inserts typically state that carcinogenic effects (and mutagenic and fertility effects) have not been studied ( ) [e.g., for the mmr vaccine it is stated that 'm-m-r ii has not been evaluated for carcinogenic or mutagenic potential, or potential to impair fertility ... animal reproduction studies have not been conducted with m-m-r ii'; and for the hpv vaccine it is stated that 'gardasil has not been evaluated for the potential to cause carcinogenicity, genotoxicity or impairment of male fertility' ( ) ]. several decades of close tracking would be required to identify such adverse effects. an overlooked issue associated with the vaccine discussions is potential transgenerational effects. transgenerational studies of adverse substance effects tend to be focused on environmental causes; however, there are some examples of such studies for drugs. a previous study on chemotherapy-induced late transgenerational effects ( ) has raised some concerns, both due to the scarcity of such studies in the literature and the transmission of adverse effects deep in the generational chain. due to the inadequate safety testing of several toxic stimuli in the past (including vaccines), it remains uncertain as to whether a number of diseases currently affecting humanity may be due in part to the actions of our predecessors passed on to us through transgenerational effects. it is uncertain as to whether any of the drugs, vaccines, foods or radiation exposures of our predecessors, which were not tested for transgenerational effects, are adversely affecting human life at present. of note, the question remains whether humanity is currently willing to pass on potential devastating diseases to future generations due to the present need for the speedy development of a vaccine, bypassing adequate long-term and transgenerational safety testing. there are also ethical issues of concern associated with accelerated vaccine development, particularly with the drastic reduction in time devoted to clinical trial phases ii and iii ( ) . the main target population for a vaccine is the most vulnerable demographically: the elderly with high comorbidities and dysfunctional immune systems. yet, the test demographic population being used for the initial clinical trials is the relatively young and healthy population (as discussed below). this leads to uncertainty regarding the efficacy of the trial, raising issues as to how the results from a young healthy population can be extrapolated to an elderly and vulnerable population. additionally, in myriad cultures, it is the elderly who sacrifice for the benefit of the young. this tradition is being inverted in the present accelerated testing regimen. for any new product, the decision to implement (whether for commercial or non-commercial purposes) typically involves a tradeoff between costs and benefits. in the ideal case, the projected benefits would far outweigh the projected costs. the potential costs and/or benefits may be known to high, modest, or low degrees of certainty. thus, a risk factor must be applied to the costs and benefits, reflecting the level of uncertainty about the projections. the vaccine costs in this discussion are the potential adverse health effects from a covid- vaccine, particularly for the mid-and long-term. for a vaccine with high levels of uncertainty as to the projected costs, a high risk factor is required. for the tradeoff to justify moving forward, a very high level of benefits would be required. the cost-benefit tradeoff for a covid- vaccine would be different for groups with different vulnerabilities to the disease. for simplicity, the target population for vaccination could be divided into groups: the highly vulnerable, and the remainder of the population. the demographic population most vulnerable to the more severe consequences of covid- tends to be the elderly with high comorbidities and others with compromised immune systems ( ) . it is a small fraction of the total population, although a somewhat greater fraction of the senior population. the remainder of the population, when infected with the sars-cov- virus, usually displays no symptoms or minimal symptoms. this demographic sub-division is similar to that for influenza and for the sars pandemic ( ) . the vaccine tradeoff analysis will differ for each of these two groups. for the most vulnerable, the main consideration is to survive the season. the mid-and long-term effects may be of lesser importance (although for the few younger members of this demographic population with highly compromised immune systems, the mid-and long-term adverse effects would not be negligible). for the least vulnerable (the vast majority of the population), the need for a vaccine is unclear, since the adverse effects of the virus appear to be minimal for most. this least vulnerable demographic population would have to bear the brunt of any potential mid-and long-term adverse health impacts that may result from a vaccine inadequately tested for these effects. thus, a vaccine that proved efficacious for the very short-term for all demographics may potentially be justifiable (albeit high-risk) for the most vulnerable demographic population. however, it is difficult to ascertain how such a vaccine could be justified for the remaining demographics. furthermore, the question remains of what are the present prospects for a vaccine efficacious even in the short-term. trial results for a highly-promoted covid- vaccine reported publicly have exhibited adverse effects of varying severity, where the test group was relatively young and very healthy ( , ) , unlike the highly vulnerable elderly target group with comorbidities. in other words, even short-term efficacy has not yet been demonstrated for the least vulnerable demographic population, much less the most vulnerable demographic population who would be the most justifiable target of the vaccine. in the present political environment, there is the potential that the majority of the population could be required to be vaccinated, even those demographics that were not vulnerable to the severe effects of covid- , and particularly those in the youngest demographic. the potential adverse consequences of such a mass inoculation with a vaccine not adequately tested for mid-and long-term adverse effects could be substantial. not applicable. no funding was received. not applicable. all authors substantially contributed to the conception, writing and revision of the work and approved the final content of the manuscript. not applicable. not applicable. das is the editor-in-chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article. the other authors declare that they have no competing interests. a new threat from an old enemy: re-emergence of coronavirus (review) post-lockdown guidelines covid- in northern italy: an integrative overview of factors possibly influencing the sharp increase of the outbreak (review) the under-reported role of toxic substance exposures in the covid- pandemic editorial: nicotine and sars-cov- : covid- may be a disease of the nicotinic cholinergic system zinc and respiratory tract infections: perspectives for covid- (review) spandidos da, nikolouzakis tk, drakoulis n and tsatsakis a: comprehensive analysis of drugs to treat sars-cov- infection: mechanistic insights into current covid- therapies (review) spandidos da and tsatsakis a: a dissection of sars-cov with clinical implications (review) a perspective on emerging therapeutic interventions for covid- sars-cov- : repurposed drugs and novel therapeutic approaches -insights into chemical structure-biological activity and toxicological screening impact of self-imposed prevention measures and short-term government-imposed social distancing on mitigating and delaying a covid- epidemic: a modelling study nazarenko y: air filtration and sars-cov- protection and disinfection policies against sars-cov- (covid- ) effects of wearing n and surgical facemasks on heart rate, thermal stress and subjective sensations covid- : important potential side effects of wearing face masks that we should bear in mind fact sheet: explaining operation warp speed covid- vaccine tracker. regulatory affairs professionals society towards effective covid- vaccines: updates, perspectives and challenges (review) are we rushing too much? the role of toxic stimuli combinations in determining safe exposure limits covid- , an opportunity to reevaluate the correlation between long-term effects of anthropogenic pollutants on viral epidemic/pandemic events and prevalence human exposure to chemical mixtures: challenges for the integration of toxicology with epidemiology data in risk assessment simulating real-life exposures to uncover possible risks to human health: a proposed consensus for a novel methodological approach new challenges in risk assessment of chemicals when simulating real exposure scenarios; simultaneous multi-chemicals' low dose exposure a small jab -a big effect: nonspecific immunomodulation by vaccines setting safer exposure limits for toxic substance combinations behavioral impacts of a mixture of six pesticides on rats a mixture of routinely encountered xenobiotics induces both redox adaptations and perturbations in blood and tissues of rats after a long-term low-dose exposure regimen: the time and dose issue genotoxic, cytotoxic, and cytopathological effects in rats exposed for months to a mixture of chemicals in doses below noael levels the effect of chronic vitamin deficiency and long term very low dose exposure to pesticides mixture on neurological outcomes -a real-life risk simulation approach six months exposure to a real life mixture of chemicals' below individual noaels induced non monotonic sex-dependent biochemical and redox status changes in rats adverse and hormetic effects in rats exposed for months to low dose mixture of chemicals: rlrs part iii hormetic neurobehavioral effects of low dose toxic chemical mixtures in real-life risk simulation (rlrs) in rats vaccines and autoimmunity hepatitis b vaccine and the risk of cns inflammatory demyelination in childhood clustering of cases of insulin dependent diabetes (iddm) occurring three years after hemophilus influenza b (hib) immunization support causal relationship between immunization and iddm johns hopkins bloomberg school of public health: package inserts and manufacturers for some us licensed vaccines and immunoglobulins under-reporting of adverse events in the biomedical literature covid- vaccines: ethical framework concerning human challenge studies coronavirus disease: vs. the flu. johns hopkins medicine an mrna vaccine against sars-cov- -preliminary report key: cord- -s yx u authors: tizard, ian r. title: adverse consequences of vaccination date: - - journal: vaccines for veterinarians doi: . /b - - - - . - sha: doc_id: cord_uid: s yx u the importance of adverse effects from vaccination must not be overstated. vaccine benefits greatly exceed any risks from the procedure. neither must they be minimized. unnecessary vaccination must be discouraged. hypersensitivity reactions to vaccine components are real and must be guarded against. residual virulence, although a concern tends to be more a hypothetical than a real problem. progressive improvements in animal vaccines have significantly reduced the chances of adverse effects occurring, although some issues persist. one such example is injection-site sarcomas in cats. another issue is the influence of animal size on the prevalence of adverse events in dogs. vaccination is the only safe, reliable, and effective way of protecting animals against the major infectious diseases. society does not remember the devastating toll taken by infectious diseases before the development of modern vaccines. exaggerated fear of negative side effects has discouraged owners from having their pets (and themselves) from being vaccinated. the rise of the internet and the development of social media have enabled those who oppose vaccination to spread their opinions. those who resist vaccination for themselves or their children are unlikely to be enthusiastic about vaccinating their pets. much of this resistance is a result of adverse events and controversy regarding effectiveness associated with the earliest vaccines. in spite of the fact that these problems have long been solved, it takes a considerable time before confidence is restored. there is a lack of awareness of the rigorous safety tests that modern vaccines must undergo before they are marketed. good manufacturing practices and the quality control procedures used by the biologics industry, together with rigorous regulatory controls, serve to minimize the occurrence of these events. past issues have been corrected and vaccine safety has steadily improved. modern vaccines are safe to use and overwhelmingly beneficial. adverse events associated with vaccination that might compromise the health of an animal are usually rare, mild, and transient. hypothetical, speculative, or historical adverse effects sometimes dominate perceptions. nevertheless, it has been truly said, "the most dangerous vaccine is the one not given." in reading this chapter the reader should be aware that the events described here are rare, somewhat historical, and relatively unimportant when compared with the benefits of vaccination. drivers of vaccine usage differ significantly between companion animals and commercial livestock. owners of companion animals are concerned for the health and well-being of their pets and are intolerant of any adverse events that cause discomfort, pain, or sickness. livestock producers in contrast vaccinate to maintain livestock health, prevent disease spread, maximize economic return, and to minimize zoonotic disease risks. vaccines that cause a drop in milk production, decreased feed conversion, increased time to market, or a decline in carcass quality may have significant economic consequences and will not be used. in determining whether a vaccine causes an adverse effect, the following three principles should apply. first, is the effect consistent? the clinical responses should be the same if the vaccine is given to a different group of animals, by different investigators, and irrespective of the method of investigation. second, is the effect specific? the association should be distinctive and the adverse event linked specifically to the vaccine concerned. it is important to remember that an adverse event may be caused by vaccine adjuvants and components other than the major antigens. finally, there must be a temporal relationship. administration of the vaccine should precede the earliest manifestations of the event or a clear exacerbation of a continuing condition. the us centers for disease control and prevention (cdc) has classified adverse events as follows: . vaccine-induced events: these are events that would not occur in the absence of vaccination and are therefore attributed to the vaccine. an example would be an allergic response to a vaccine component such as egg protein. vaccine potentiated reactions: these are events that might have occurred anyway but may have been precipitated by the vaccine. one possible example is purpura hemorrhagica in horses. . programmatic error: events that occur in response to technical errors in vaccine storage, preparation, handling, and administration. . coincidental events: these are simply events that happen by chance or result from some underlying illness. the use of vaccines is not free of risk, and an owner has reason to be upset if their healthy animal is sickened by the administration of a vaccine. residual virulence and toxicity, allergic responses, disease in immunodeficient hosts, neurological complications, and harmful effects on the fetus are potential risks associated with the use of vaccines (table . ). veterinarians should use only licensed vaccines, and the manufacturer's recommendations must be carefully followed. before using a vaccine, the veterinarian should consider the likelihood that an adverse event will happen, and also the possible consequences or severity of this event. these factors must be weighed against the benefits to the animal. a common but mild complication requires a very different consideration than a rare, severe complication (table . ). the issue of the risk associated with vaccination remains in large part a philosophical one because the advantages of vaccination are well documented and extensive, whereas the risk for adverse effects is poorly documented, and in many cases, largely speculative. nevertheless, established facts should be recognized, unsubstantiated allegations rebutted by sound data, and uncertainties acknowledged. for example, there is absolutely no evidence that vaccination itself leads to ill health. although difficult to prove, a negative, competent statistical analysis has consistently failed to demonstrate any general adverse effect of vaccination. identification of an adverse event is based on the clinical judgment of the attending veterinarian and is therefore subject to bias. standard case definitions of a vaccine-associated adverse event are not yet available. it still is often difficult to distinguish association from causality (box . ). traditionally, adverse events resulting from vaccine administration have been reported by veterinarians to manufacturers or government agencies. the resulting numbers have been difficult to analyze satisfactorily for two major reasons. first, reporting is voluntary, so significant underreporting occurs. adverse events are often regarded as insignificant, or it may be inconvenient to report them. second, very little data has been available on the number of animals vaccinated. although manufacturers know the number of doses of vaccine sold, they are unable to measure the number of animals vaccinated. it has, however, proved possible by examining the electronic medical records of a very large small animal general practice, to determine the prevalence of vaccine-associated adverse events in over a million dogs. the use of a standardized reporting system within a very large population has permitted objective analysis of the prevalence of adverse events occurring within three days of vaccine administration. out of , , dogs receiving , , vaccine doses, adverse events were recorded ( . / , dogs); . % of these events occurred on the same day the vaccine was administered, . % were considered to be allergic reactions, . % were classified as anaphylaxis, and . % were considered "vaccine reactions" and were likely caused by innate immune responses. three dogs died. the lowest rate of such events was associated with bordetella more than in animals showing adverse reactions (. %) common greater than but less than animals per animals vaccinated ( %- %) uncommon more than but less than animals per animals vaccinated ( . %- %) rare more than but less than animals per , animals vaccinated ( . %- . %) very rare less than animal in , reported (, . %) autism spectrum disorder is a chronic developmental disorder in children. its causes are largely unknown. it usually becomes apparent in young children over one year of age at around the same time they receive their initial vaccinations. in a paper published in , a physician studied children with autism. he asked the parents if the children had been vaccinated, with the measles, mumps, and rubella vaccine, within the previous two weeks. eight said yes, so the author went on to assert in his paper that this vaccine caused autism. he postulated that autism resulted from measles infection. the paper was eventually retracted and the author lost his medical license. subsequent population-based studies have failed to demonstrate any link between vaccination and autism. thousands of children are vaccinated every year and large amounts of data are available for analysis. all these show the same thing. there is no link between vaccination and autism risk. however, the word was out. the internet and twitter spread the word. additionally, pet owners began to claim that their dog's behavior had changed after vaccinationcanine autism. the british veterinary association felt obliged to issue a statement regarding these claims. "there is currently no reliable scientific evidence to indicate autism in dogs or a link between vaccination and autism. vaccinations save lives and are an important tool in keeping our pets healthy. all medicines have potential side-effects but in the case of vaccines, these are rare and the benefits of vaccination in protecting against disease far outweigh the potential for an adverse reaction." vaccination and the highest rate with lyme disease vaccine. additional analysis indicated that the risk of adverse events was significantly greater for small dogs than for large dogs ( fig. . ); for neutered than for sexually intact dogs; and for dogs that received multiple vaccines on one occasion. each additional vaccine dose administered increased the risk of an adverse event occurring by % in dogs under kg and by % in dogs heavier than kg ( fig. . ). highrisk breeds included dachshunds, pugs, boston terriers, miniature pinschers, and chihuahuas. overall, the increased prevalence of adverse events in young adult, small-breed, neutered dogs and their relationship to multiple dosing suggests that veterinarians should look carefully at the practice of giving the same vaccine dose to all dogs irrespective of their size. in another report, from japan dogs showed an adverse event out of , vaccinated ( . / , doses). (vaccines used included canine parvovirus, canine distemper, canine adenovirus , canine coronavirus, and leptospirosis.) of these dogs, died, had anaphylaxis, developed dermatological signs, and showed gastrointestinal signs. about half the anaphylaxis events occurred within minutes of vaccination. additional analysis of these anaphylaxis cases reported % collapse, % cyanosis, and both collapse and cyanosis in % of affected dogs. breeds affected included miniature dachshunds ( %; these accounted for about % of all the anaphylaxis cases), chihuahuas ( %), mixed breeds ( %), and toy poodles ( %). miniature schnauzers also appeared to be unusually prone to anaphylaxis. the highest frequency of adverse reactions occurred in dogs under kg. most adverse events were observed within hours after vaccination. the adverse event rate in japan as reported here ( . / , doses) is much higher than in the united kingdom ( . / , doses), or in the united states ( . / , dogs). vaccines may elicit mild transient injection site reactions as a result of inflammation. these inflammatory responses may manifest themselves within two to three days. as pointed out in chapter , some degree of inflammation is required for the efficient induction of protection. this may cause pain or pruritus. the sting produced by some vaccines may present problems, not only to the animal being vaccinated, but also to the vaccinator, if the animal reacts violently. lethargy, anorexia, soreness, minor behavioral changes, and tenderness at the vaccine site are normal postvaccinal responses and should resolve within to hours. swellings may develop at the reaction site less commonly. these may be firm or edematous and may be warm to the touch. they appear within hours and can last for about a week. unless an injection-site abscess develops, these swellings leave little trace. vaccines containing killed gram-negative bacteria may be intrinsically toxic owing to the presence of pathogen-associated molecular patterns such as endotoxins, lipids, muramyl peptides, and porins that can bind to pattern recognition receptors and provoke cytokine release. in extreme cases this may lead to anorexia, and fever. although such reactions are usually only a temporary inconvenience to male animals, they may be sufficient to provoke early embryonic deaths in pregnant females. it may be prudent to avoid vaccinating pregnant animals unless the risks of not giving the vaccine are considered to be too great. vaccination with either immunestimulating complex (iscom) vaccines or live recombinant vectored vaccines against influenza and tetanus may induce an acute-phase response in horses. innate immune responses may reduce an animal's growth rate and diminish its feed efficiency. this growth suppression can be mimicked by injection of interleukin (il)- and tumor necrosis factor (tnf)-a. these cytokines act on the brain to reduce appetite while at the same time, causing degradation of skeletal muscle. intranasal vaccines such as those containing bordetella bronchiseptica and some viruses may cause transient cough or sneezing. this simply reflects the mild innate response triggered as the vaccine organisms invade the upper respiratory tract. vaccines have the potential to cause rare but serious allergic reactions (type i hypersensitivity). for example, allergic responses may occur when an animal produces immunoglobulin (ig)e in response, not only to the immunizing antigen, but also to other components in vaccines. the most significant allergens are often vaccine excipients. for example, reactions are most likely to occur after injection of vaccines that contain trace amounts of fetal calf serum (specifically bovine serum albumin), egg proteins (ovalbumin), or gelatin. (gelatin and serum albumin are added to vaccines as stabilizers to protect the vaccine antigens during the freeze-drying process.) some vaccines may also contain antibiotics such as neomycin to which an animal may be sensitized. severe allergic responses have been associated with the use of killed foot-and-mouth disease, rabies, and contagious bovine pleuropneumonia vaccines in cattle. signs include angioedema, affecting mainly the head and ears, urticaria, pruritus, acute-onset diarrhea, vomiting, dyspnea, and collapse. all forms of hypersensitivity are more commonly associated with multiple injections of antigens and therefore tend to be associated with the use of killed vaccines. it is important to emphasize that a type i hypersensitivity reaction is an immediate response to an antigen and occurs within a few minutes after exposure to an antigen ( fig. . ). it is good practice to keep an animal in the clinic for to minutes after vaccination to ensure that any immediate problems can be promptly recognized and treated (box . ). reactions occurring more than two or three hours after administration of a vaccine are likely not type i hypersensitivity reactions. in type ii hypersensitivity reactions, antibodies directed against an animal's own cells act together with complement to cause cell lysis. these antibodies are usually induced by the presence of animal cells in the vaccine. natural hemolytic disease of the newborn (hdn) in calves is very rare, but it has resulted from vaccination against anaplasmosis or babesiosis. these vaccines contain pooled red cells from infected calves. in the case of anaplasma vaccines, for example, the blood from infected donors is pooled, freeze-dried, and mixed with adjuvant before being administered to cattle. the vaccine against babesiosis consists of fresh, infected calf blood. both vaccines cause infection, and consequently, the development of immunity in recipients. they also stimulate the production of antibodies against the injected red cells. if cows sensitized by these vaccines are then mated with bulls carrying the same blood groups, they can transmit these antibodies to their calves through colostrum. the calves that drink this colostrum may then develop hemolytic disease. hdn in piglets had a similar pathogenesis when sows were immunized with a hog cholera vaccine containing pig blood. beginning in , multiple outbreaks of an unexplained hemorrhagic disease in newborn beef calves were reported from many countries in western europe. affected calves showed sudden onset bleeding including nasal hemorrhage, petechiation on mucus membranes, and excessive bleeding from minor wounds such as injection, or ear-tag sites. the disease appeared to days after birth and affected calves could die within hours. it is now called bovine neonatal pancytopenia (bnp). investigation showed an early drop in platelets, monocytes, and neutrophils was followed by drops in erythrocyte and lymphocyte numbers. the net result was a profound pancytopenia. the bone marrow could be completely aplastic. mortality was as high as % in severely affected calves, but there were also many subclinical cases. because this disease only occurred in suckled calves and developed within hours of first suckling, it appeared to result from the consumption of colostrum. further investigations showed that the colostrum from these cows contained antibodies directed against the major histocompatibility complex (mhc) class i molecules expressed on neonatal leukocytes and bone marrow stem cells. cells of the thrombocyte, lymphocyte and monocyte lineages, and precursors of neutrophil, erythrocyte, and eosinophil lineages were affected. further investigations showed that the disease was triggered by administration of a specific vaccine against bovine virus diarrhea (bvd). this vaccine-pregshure-contained inactivated bovine viral diarrhea virus (bvdv) grown in bovine kidney cells. a potent, oil-in-water emulsion adjuvant containing quil a, cholesterol, and mineral oil was then added. immunization with this anaphylaxis is a life-threatening medical emergency. deterioration can occur very rapidly and time is of the essence. initiate treatment immediately. if an animal is undergoing acute anaphylaxis take the following steps: . stop administering the vaccine. in the case of dogs and cats, administer epinephrine : at . mg/kg intramuscularly. repeat every - minutes if necessary. if very severe and shock has developed, place an intravenous catheter and administer . mg/kg of : , epinephrine by slow intravenous infusion and monitor blood pressure and perfusion. alternative routes of administration include intracardiac or intratracheal. avoid subcutaneous administration because epinephrine is a potent vasoconstrictor and absorption is delayed. in the case of foals administer epinephrine : at . to . mg/kg ( . - ml for a kg foal) given slowly intravenously or intramuscularly. in adult horses administer epinephrine at . mg/kg ( - ml for a kg horse) slowly intravenously. if the condition is mild, this dose may be administered intramuscularly. repeat every - minutes if necessary. . secure the airway, intubate if necessary, and administer oxygen to animals showing respiratory symptoms. . provide isotonic shock crystalloid fluids (normal saline or lactated ringer solution) intravenously to help restore adequate blood pressure in hypotensive animals. the volume required depends on the animal's response, but may be as high as ml/kg for dogs and ml/kg for cats. administer an h -antihistamine such as diphenhydramine every - hours if necessary. . once the animal is stabilized consider administering a fast acting glucocorticosteroid by the slow intravenous route to prevent late-phase responses. vaccine induced antibodies against the bovine kidney cells in some cows. these antibodies, when transferred to calves via colostrum, bound to their leukocytes and bone marrow stem cells, killed them, and so induced pancytopenia (fig. . ) . not all calves born from cows that received this specific vaccine developed clinical disease. the quantity and specificity of their antibody response determined the risk to their calves. antibody levels remained high in some cows for many years and were boosted by each pregnancy. as a result, bnp cases occurred for many years after pregshure was removed from the market in . type iii hypersensitivity reactions (immune-complex-mediated) may be induced by vaccination. the deposition of immune-complexes in tissues may cause local inflammation or cause a generalized vasculitis such as purpura. some rabies vaccines may also induce a local complementmediated vasculitis in the skin resulting in ischemic dermatitis and local alopecia. this may occur at the injection site or at remote locations such as the ear tips, footpad, tail, or scrotum. this vasculitis is most often seen in small dogs such as dachshunds, miniature poodles, bichon frises, and terriers. in dogs infected with canine adenovirus- (cav- , infectious canine hepatitis), an immunecomplex-mediated uveitis and a focal glomerulonephritis both develop. the uveitis, commonly called "blue-eye," is seen both in dogs with natural infections and in those vaccinated with live attenuated cav- vaccine (fig. . ). the uveitis results from the formation of virus-antibody complexes in the anterior chamber of the eye and in the cornea with complement activation and consequent neutrophil accumulation. the neutrophils release enzymes and oxidants that damage corneal epithelial cells, leading to edema and opacity. the condition resolves spontaneously in about % of affected dogs. replacing cav- with cav- in vaccines has largely eliminated this problem. see chapter . type iv hypersensitivity (delayed) reactions are t-cell-mediated inflammatory responses. they may occur at the injection site in response to vaccination, but a more common reaction is local granuloma formation. this may be in response to persistent adjuvants containing alum or oil. vaccines containing a water-in-oil adjuvant produce larger and more persistent lesions at injection sites than vaccines containing alum or aluminum hydroxide. these lesions may develop into sterile abscesses and if the injection site is dirty, these abscesses may become infected. injection site lesions are of major concern in the meat industries. modified live vaccines must be able to establish themselves transiently in a vaccinated animal yet at the same time not cause disease. they must be safe in animals and their human companions. they must be as stable as possible to enable long-term storage. they must be environmentally safe. it may be possible to achieve minimal virulence with maximal immunogenicity, but this may be unattainable in animals with any defects in their immune function. the normal distribution of immunological competence in an outbred population is such that some animals will inevitably be susceptible to an otherwise avirulent organism. this immunosuppression may result from minor stresses, but equally important some common viral infections such as canine distemper, feline pancytopenia, or feline leukemia also cause immunosuppression to a degree that an animal may become susceptible to otherwise avirulent vaccinal agents. it is also appropriate to point out that modified live vaccines are attenuated for a specific target species for administration by a specified route. if administered to the wrong species or in the wrong way residual virulence may cause disease. thus some modified live vaccines may retain the ability to cause disease. a good example is brucella abortus strain . although highly immunogenic in cattle, s can cause severe reactions in vaccinated cows. swelling, fever, anorexia, depression, and a drop in milk yield have been reported. s can also cause abortion in pregnant cows and orchitis in bulls and humans. safer attenuated brucella vaccines are now available. similar residual virulence hazards are associated with the soremouth vaccine and the sheep toxoplasmosis vaccine. some modified live herpes vaccines or calicivirus vaccines given intranasally may spread to the oropharynx and result in persistent infection. in these cases, the vaccine virus may infect (and protect) other animals in contact. because live vaccine strains may be released into the environment, safety issues involving not only the animal but also its environment must be addressed. are there changes in the tissue tropism of the virus? are there changes in the carrier through the incorporation of new foreign genes? is there reversion to virulence through the incorporation of complementation genes? is there exchange of genetic information with other wild type or vaccine strains of the carrier? will the carrier spread unwanted genes such as antibiotic resistance into the environment? these questions are highly relevant in the aquaculture industry where modified live vaccine viruses may escape into the aquatic environment (chapter ). postvaccinal canine distemper encephalitis is a rare complication that may develop in dogs and ferrets after administration of modified live canine distemper vaccines. affected animals may show neurologic signs such as aggression, incoordination, and seizures, or die suddenly. the pathogenesis of this condition is unclear. it may be the result of residual virulence, increased susceptibility, or triggering of a latent paramyxovirus by the vaccine. vaccination during pregnancy carries uncertain risks, especially when live vaccines are used. the fetal immune system may not have developed sufficiently to defend itself against the vaccine strain of the virus. mlv bluetongue virus vaccine has been reported to cause malformations in the offspring of ewes vaccinated while pregnant. the severity of the lesions depends upon the stage of pregnancy at vaccination. for example, mlv bluetongue administered to ewes between and days of gestation has caused hydranencephaly and retinal dysplasia in lambs. live erysipelothrix rhusiopathiae vaccines have been reported to cause abortions in sows. the stress from this type of vaccination may also be sufficient to reactivate latent infections; for example, reactivation of equine herpesviruses has been triggered by vaccination against african horse sickness. a modified live virus (mlv) parvovirus vaccine administered during pregnancy has been reported to cause hydranencephaly and cerebellar hypoplasia in kittens. many viruses promote their own survival by suppressing their host's immune system. although immunosuppression is greatest in virulent strains, some mlvs may remain somewhat immunosuppressive. for example, some mlv canine parvovirus strains may depress t cell responses to mitogens in puppies for two to five weeks following administration, or even cause a lymphopenia. similarly, mlv canine distemper may cause immunosuppression and thrombocytopenia. in view of this it may be best to avoid performing elective surgery on dogs for at least one week postvaccination. mlv bovine viral diarrhea (mlv-bcd) vaccines may suppress neutrophil functions and lymphocyte blastogenesis in vaccinated calves. as a result, they may potentiate intercurrent infections. mlv-bvd may also induce mucosal disease to days after vaccination. vaccination with an mlv-bhv vaccine has been shown to exacerbate the lesions of experimental moraxella-induced pinkeye (chapter ). several vaccine combinations may also result in transient immunosuppression. for example, a combination of distemper and adenovirus vaccines can reduce canine lymphocyte counts and their responsiveness to mitogens, although the individual components are not detectably immunosuppressive. this t cell suppression may be accompanied by simultaneous enhancement of b cell responses and raised immunoglobulin levels. many of these cases of "immunosuppression" attributed to vaccines may however simply reflect alterations in the th /th balance or transient alterations in lymphocyte recirculation patterns. they are rarely of clinical significance. as pointed out in an earlier chapter, older vaccine viruses were attenuated by prolonged passage in tissue culture or eggs. in some cases, it is possible to reverse the attenuation process by backpassage through their natural hosts. for example, attenuated distemper strains cannot grow in canine lung macrophages. back-passage of the canine distemper virus (cdv) rockborn strain for as few as three passages in puppies resulted in the virus regaining this ability. by four passages the virus could cause weight loss. by five passages, immunosuppression returned. the virus that had been back-passaged six to seven times had regained its virulence. the use of genetically defined, gene deleted attenuated vaccines has largely eliminated this type of problem. louis pasteur's first rabies vaccine contained dried rabbit brain tissue. when injected into patients it induced antibodies against myelin basic protein and an acute demyelinating encephalomyelitis developed in about . % of recipients. rabies vaccines have had an undeserved bad reputation ever since. in , it was proposed that a new syndrome existed that linked diverse human autoimmune diseases with the use of adjuvanted vaccines. it was called autoimmune/ autoinflammatory syndrome induced by adjuvants (asia). this syndrome has been investigated to determine whether it is an insignificant clinical term or whether there is an underlying mechanism that links adjuvants to autoimmunity. aluminum-containing adjuvants were claimed to be the "cause" of asia. however, patients receiving allergen-specific immunotherapy receive up to times more injected aluminum than regular vaccine recipients and have a lower incidence of autoimmune disease. current data does not support the causation of asia by vaccine adjuvants. there is a lack of any reproducible evidence for any link between adjuvants and autoimmunity. one obvious problem with this proposed syndrome is that vaccination is so commonplace whereas autoimmunity remains uncommon. after all, huge numbers of people receive influenza vaccines annually without untoward effect. there is a single animal study that appears to show that a link might exist between vaccination and the development of autoimmunity. a retrospective analysis of the history of dogs presenting with immune-mediated hemolytic anemia (imha) showed that of ( %) dogs with imha had been vaccinated within the previous month, compared with a randomly selected control group of dogs in which % had been vaccinated. dogs with imha that developed within a month of vaccination differed in some clinical features from dogs with imha unassociated with prior vaccination. some studies using very large databases have tended to confirm this effect, in that they showed an approximately three-fold increase in diagnoses of autoimmune thrombocytopenia, and a two-fold increase in diagnoses of imha in dogs in the days following vaccination, compared with other time periods. other studies have failed to show any association between vaccination and imha. the overall prevalence of these diseases remains low, and they can be diagnosed at times not temporally associated with vaccination. vaccination may therefore serve as a trigger for these diseases in some dogs-a vaccine potentiated reaction. contaminating thyroglobulin found in some vaccines (usually from the presence of fetal bovine serum) may lead to the production of antithyroid antibodies in vaccinated dogs. lymphocytic thyroiditis has been found in % of beagles on necropsy, but there was no association detected between vaccination and the development of this thyroiditis. in the s, a swine influenza vaccine induced guillain-barré syndrome (an autoimmune polyradiculoneuritis) in about case per , human recipients. (current influenza vaccines have a risk of about : million. it appears that the older influenza vaccine was unique in this respect.) cases of this syndrome in dogs have been rarely reported. in some animals, the administration of potent, adjuvanted vaccines may stimulate the transient production of autoantibodies to connective tissue components such as fibronectin and laminin. vaccination of some weimaraner puppies may lead to the development of a severe hypertrophic osteodystrophy. the disease appears within days of administration of mlv canine distemper vaccine. systemic signs include anorexia, depression, fever, and gastrointestinal, nervous, and respiratory symptoms, in addition to symmetrical metaphyseal lesions with painful swollen metaphyses. radiological examination shows radiolucent zones in the metaphyses, flared diaphyses, and formation of new periosteal bone. it is possible that the condition is triggered by the vaccine in genetically susceptible animals. these dogs may have a preexisting immune dysfunction with low concentrations of one or more immunoglobulin classes, recurrent infections, and inflammatory disease. it has been suggested that weimaraners are especially susceptible to this condition and that they therefore receive only killed virus vaccines. a mild transient polyarthritis has been reported in some dogs following vaccination. the dogs show a sudden onset of lameness with swollen and painful joints within two weeks of vaccination. the dogs recover within two days. no specific breed or vaccine has been associated with this problem. vaccination against calicivirus has been associated with polyarthritis and a postvaccination limping syndrome in cats. a search of web sites regarding vaccination of pets reveals that a large number express great concern regarding the practice of overvaccination. by this is meant the use of unnecessary vaccines and by implication a significant threat to the health of pets. conversely a search of pubmed, the ncbi web site, reveals only a single scientific paper regarding this subject. the paper describes renal disease in a spaniel that received seven doses of vaccine from its owner, one vaccine per month, in the absence of any veterinary supervision. as a result, the dog developed immune-complex lesions in its kidney glomeruli. this was very likely a type iii hypersensitivity nephropathy. clearly administration of excessive and unneeded vaccines is inappropriate. there are no health benefits and each additional dose of vaccine carries with it the chances of an untoward event. as pointed out throughout this text, the risk/benefit assessment of any vaccination procedure must be a subject for discussion between a veterinarian and the pet owner. there are many reasons why a veterinarian may suggest that it may be beneficial to vaccinate an animal and it is inappropriate to blame those vets who choose to vaccinate animals more frequently than currently recommended without a full knowledge of each specific case. this is called clinical judgment. these are discussed in chapter . errors in manufacture and administration modified live vaccines cannot contain preservatives (except antibiotics in viral vaccines). as a result, occasional cases of vaccine contamination have occurred. these have been a major issue in the past when viral identification required culturing. modern identification techniques such as the polymerase chain reaction have made such contamination a thing of the past. there are numerous examples of such contamination. for example, mycoplasma contamination was a feature of many live virus veterinary vaccines. the pestivirus of border disease contaminated some soremouth and pseudorabies vaccines; bovine leukemia virus has contaminated bovine blood vaccines such as those against babesiosis and anaplasmosis. bluetongue virus has contaminated some canine vaccines. injection site selection should include consideration of potential adverse reactions in addition to the hypersensitivity reactions described earlier. for example, injection in the gluteal muscles/hip region of cattle should be discouraged because gravitational drainage along fascial planes can occur. should an abscess develop, considerable tissue damage may occur and result in eruptions in undesirable locations with lesions that require prolonged time to heal. they may result in unacceptable blemishes in meat destined for human consumption (chapter ). veterinarians and other vaccine users may be inadvertently exposed to animal vaccines as a result of unintended inoculation or spraying. some of these vaccines may cause sickness. veterinarians, their assistants, and other animal handlers should be especially careful when administering injectable vaccines to avoid needle-stick and eye injuries. if an individual is accidentally self-injected with a mineral oil-adjuvanted vaccine, seek immediate medical treatment regardless of the dose injected. with the notable exception of brucellosis, these events are rarely reported. nevertheless, accidents do occur and veterinarians should be fully aware of these risks. brucellosis is an existential hazard to veterinarians. the cdc has established a passive surveillance registry. in the two years to , individuals reported needlestick injury related exposure to the brucella vaccine strain rb , five were splashed in the eye, and one was splashed into an open wound. although most received antibiotics, reported clinical disease. approximately to million doses of brucella vaccines were administered annually in to . it is estimated these would have resulted in at least needle-stick injuries, suggesting that exposure to rb is substantially under-reported. a vaccinia recombinant rabies vaccine bait has been air-dropped across many states in the united states to vaccinate wildlife. several instances of human exposure to these baits have been reported. (the vaccine baits have toll-free numbers printed on them.) in ohio, there were reports of bait contact and of these involved contacts with the vaccine. one individual developed a severe vaccinia infection and had to be hospitalized. bordetella bronchiseptica causes respiratory disease in dogs and atrophic rhinitis in pigs. infection of humans is rare but has been documented. in at least one case a young boy was inadvertently sprayed in the face with a "kennel cough vaccine." he had been holding his dog but the dog moved. he developed a pertussis-like respiratory disease that lasted several months despite antibiotic treatment. there have been reports of clients experiencing respiratory difficulty following administration of an intranasal vaccine to their dogs. needle-stick injuries are not uncommon and many involve vaccines. a woman was inadvertently inoculated with the sterne anthrax vaccine while vaccinating her horse. she did not develop anthrax but did develop a local reaction within hours. serious inflammatory reactions are associated with injected mycobacterium paratuberculosis vaccine. self-injections appear to be a major issue in the aquaculture industry where workers have to work fast to vaccinate slippery fish. veterinarians are encouraged to report all adverse reactions to the vaccine's manufacturer and the regulatory authorities. this provides both with the critical information that is used to evaluate and monitor vaccine safety in the field. in this way vaccine safety can be progressively improved. adverse reactions should be reported to the vaccine manufacturer first. after that, they should be reported to the appropriate regulatory authorities. in the united states, adverse vaccine events should also be reported to the us department of agriculture aphis center for veterinary biologics at - - - . they have an online electronic report form. reports can also be made by fax or mail. vaccine lot and serial numbers should be noted in vaccination records because this will facilitate an investigation. the use of standardized reporting systems is encouraged. web: http://www.aphis.usda.gov/animal_health/vet_biologics/vb_adverse_event.shtml fax or mail: download the pdf form at http://www.aphis.usda.gov/animal_health/vet_ biologics/publications/adverseeventreportform.pdf and fax to ( ) - or mail to the center for veterinary biologics (cvb), dayton avenue, po box , ames, iowa , usa. telephone: ( ) - in canada, suspected adverse events (sae) should be reported to the canadian center for veterinary biologics (ccvb) in ottawa at - - - . as stipulated by the health of animals regulations, all reports that indicate "serious expected" or "serious unexpected" adverse events related to the use of a veterinary biologic, including lack of efficacy, must be reported to ccvb within days of that information becoming known to the permit or license holder. follow-up reports, including case conclusions, must be submitted to ccvb in a timely manner. all other reports should be investigated by the license/permit holder, summarized in a summary update report, and submitted to ccvb every six months. summary update reports should be submitted within days of the end of the reporting period. sae related to veterinary biologics are categorized as one of the following: adverse event (ae), serious ae, unexpected ae, and lack of efficacy. a causality assessment should also be assigned to each sae. each case should be classified as probable, possible, unlikely, or unknown. form cfia/acia , notification of suspected adverse events to veterinary biologics, may be found at http://inspection.gc.ca/english/for/pdf/c e.pdf. in the united kingdom adverse events should be reported to the veterinary medicines directorate. forms can be obtained at their website at www.vmd.defra.gov.uk or by calling their pharmacovigilance team at . the veterinary medicines directorate (vmd), an agency of the department for environment, food, and rural affairs, is responsible for the suspected adverse reaction surveillance scheme (sarss) for veterinary medicines. adverse reactions in animals in the united kingdom should be reported at http://www.vmd.defra.gov.uk/ adversereactionreporting/default.aspx. human reactions to veterinary medicines in the united kingdom should be re new zealand in new zealand adverse event reports should be made to the ministry for primary industries adverse events in humans associated with accidental exposure to the livestock brucellosis vaccine rb human illness associated with use of veterinary vaccines evaluation of the safety of vaccinating mares against equine viral arteritis during mid or late gestation or during the immediate postpartum period pharmacovigilance: suspected adverse events vaccination and ill-health in dogs: a lack of temporal association and evidence of equivalence vaccine hypersensitivity-update and overview adverse reactions to vaccination: from anaphylaxis to autoimmunity revisiting adverse reactions to vaccines: a critical appraisal of autoimmune syndrome induced by adjuvants (asia) vaccine-induced enhancement of viral infections rabies vaccine is associated with decreased all-cause mortality in dogs vaccine-associated adverse events large-scale survey of adverse reactions to canine non-rabies combined vaccines in japan adverse events after vaccine administration in cats: , cases adverse events diagnosed within three days of vaccine administration in dogs adverse vaccinal reactions in dogs and cats a space-time cluster of adverse events associated with canine rabies vaccine aa amyloidosis in vaccinated growing chickens ige reactivity to vaccine components in dogs that developed immediate-type allergic reactions after vaccination membranoproliferative glomerulonephritis possibly associated with over-vaccination in a cocker spaniel fatal adverse pulmonary reaction in calves after inadvertent intravenous vaccination anaphylaxis in dogs and cats comparative vaccine-specific and other injectable-specific risks of injection-site sarcomas in cats immune modulation following immunization with polyvalent vaccines in dogs key: cord- -baqaqsli authors: dreesen, david w. title: animal vaccines date: - - journal: rabies doi: . /b - - / - sha: doc_id: cord_uid: baqaqsli rabies in terrestrial animals, primarily carnivores, is caused by the classic genotype rabies virus. even though the widespread vaccination of domestic dogs has been the one most effective factor in the reduction of human rabies, the number of human deaths worldwide is greater than that of the combined deaths from polio, meningococcal meningitis, japanese encephalitis, yellow fever, severe acute respiratory syndrome and avian influenze (bird flu).tools are available in highly efficacious and safe animal and human vaccines. multiple factors can, however, prevent their use effectively in many areas of the world. for several decades, virtually all rabies nerve tissue origin (nto) vaccines were inactivated with phenol using the method described by semple. the nto vaccines currently in use for mass vaccination campaigns in africa, latin america, and the caribbean are primarily produced from rabies virus-infected suckling mouse brains or lamb brains. these vaccines are shown to be effective in campaigns. however, nto-killed vaccines for dogs and other animals have often, in the past, resulted in post-vaccinal nervous system reactions that could result in the death of the vaccinated animals. in his quest for a means to prevent rabies in humans, louis pasteur initiated research into animal rabies vaccine in france in the early s (bunn, ) . virus obtained from a rabid dog was first serially passed in rabbits by intracerebral inoculation at specified time intervals. dogs were then vaccinated at various time intervals and challenged with rabies virus. although this method produced acceptable results, pasteur found that by serial intracerebral inoculation of monkeys with the dog origin virus, the incubation period increased while the virulence of the virus decreased. by using this regimen, pasteur demonstrated that dogs vaccinated were resistant to subsequent challenge with virulent street (non-laboratory propagated) rabies virus. in , pasteur attenuated, or weakened, the virus by desiccation (bunn, ) to improve on the safety of these early attempts to produce a rabies vaccine. in a review by friedberger and frohner ( ) , it was reported that hogyes and protopopoff and others conducted further studies to improve on the safety and efficacy of vaccines for dogs and to reduce the number of doses needed. in , the first international rabies conference recommended that fixed virus for canine rabies vaccines be completely inactivated or attenuated so that they caused no disease in dogs vaccinated either subcutaneously (sc) or intramuscularly (im) (schoenig, ) . for the next several decades, virtually all rabies nerve tissue origin (nto) vaccines were inactivated with phenol using the method described by semple (bunn, ) . the nto vaccines currently in use for mass vaccination campaigns in africa, latin america and the caribbean are primarily produced from rabies virus-infected suckling mouse brains or lamb brains. these vaccines have been shown to be effective in campaigns (who, ) . however, nto killed vaccines for dogs and other animals have often, in the past, resulted in post-vaccinal nervous system reactions that could result in the death of the vaccinated animals (bunn, ) . better vaccines were needed. embryonated chicken eggs were used by koprowski and cox ( ) for serial passage of the flury strain (a human rabies virus isolate). the virus was initially passed times in -day-old chicks. vaccine produced from the th to the th chicken embryo passage lost its viscerotropic properties but retained some neurotropic properties. this was designated as flury low-egg passage (lep). while effective in dogs, the vaccine occasionally caused rabies in young pups, cats and cattle (bunn, ) . to increase the safety of the vaccines in these species, koprowski et al. ( ) increased the passages of the flury strain in embryonated eggs until the virus was found to be non-pathogenic for dogs when inoculated intracerebrally following the th passage. this flury high-egg passage (hep) vaccine was declared safe for im use in cats and cattle as well as puppies months of age. however, since cases of vaccine-induced rabies occurred in cats administered im with the flury-hep vaccine, it was later withdrawn from the market (cabasso etal., ; dean and guevin, ) . the flury and kelev strains of the rabies virus are used to produce chick embryo origin (ceo) modified live virus (mlv) vaccines. tissue culture (tc) vaccines, such as those derived with the street alabama dufferin (sad) strain, which was adapted to hamster kidney cells (fenji, ) and the evelyn-rokitnicki-abelseth (era) strain, are grown on porcine kidney cells (abelseth, ) and are commonly used to produce mlv vaccines (reculard, ) . several other mlv vaccines have been produced over the years. these mlv vaccines, especially those using the ceo, sad and era strains are still used extensively in asia and africa and parts of europe and have been adapted for oral immunization of carnivores, including domestic dogs and cats (blancou and meslin, ) . the tc mlv vaccines produce fewer allergic reactions than the ceo vaccines. potency tests for mlv vaccines for animal use consist of measuring the titer of infectious virus in a animal vaccines sample from each filling lot (see section . . ). if the titer is as high as that proved efficacious in the species of animal for which the vaccine is intended, the vaccine is released for use (sizaret, ) . even though mlv vaccines have been trustworthy over the years, the use of inactivated (killed) cell culture vaccines is increasing in areas of the world where mlv vaccines are still in use. the who does not recommend mlv vaccines for parenteral use in animals (who, ) and no mlv rabies vaccines are currently licensed for use in the usa. the concept of oral rabies vaccines (orv) was first proven to be successful in (baer et al., ) . using the sad berne strain of virus adapted from the era strain, several types of mlv orv vaccines have been produced for use in baits for free-ranging animals that serve as vectors for the maintenance and transmission of the disease in wildlife species . orv have been used extensively in europe since and in canada from with considerable success (isara et al., ; aubert et al., ) . unfortunately, the live-virus sad vaccines contained some degree of residual pathogenicity for wild rodents (artois et al., ) and resulted in partially impaired immune responses in fox cubs < weeks old born from sad b -vaccinated vixens, resulting in insufficient protection against rabies (muller et al., ) . since the early to mid- s, the sad strain used in vaccine has been replaced by the sag- and sag- (sad-avirulent-gif) strains in the development of vaccines. the sag- strain, the strain of choice, is a double mutant isolated from the sad berne strain after two successive selection steps utilizing anti-glycoprotein monoclonal antibodies. this strain is avirulent following intracerebral inoculation of immunocompetent mice and protects the mice against challenge with challenge virus standard (cvs) (lafay et al., ) . the sag- strain of rabies virus, packaged in chicken-head baits, has successfully protected captive african wild dogs against rabies challenge (knobel et al., ) . in studies conducted at the centers for disease control and prevention in atlanta, georgia, usa, the sag- vaccine produced no clinical illness in laboratory vaccinates (beagles) and residual sag- virus was isolated from only one of oral swabs from the dogs (fekadu, et al., ; orciari et al., ) . no orv derived from sad/sag origin vaccines are currently licensed for use in the usa (compendium, ) . a recombinant vaccinia virus expressing the rabies virus glycoprotein gene (v-rg) was developed by inserting the cdna of the glycoprotein gene of the era strain into the thymidine kinase gene of the copenhagen strain of vaccinia virus. initial studies of this new orv were conducted to determine whether the v-rg recombinant virus vaccine satisfied the various criteria that had to be met for such a vaccine to be distributed in the wild (kieny et al., ; wiktor et al., ) . criteria included: the vaccine would be effective when delivered by an oral bait; the baits would be readily accepted by target species but would be rabies virusfree; the vaccine in the baits would have reasonably long-term genetic and thermal stability; the vaccine would be biologically contained in the host; oral exposure to baits with the vaccine would produce full protection against rabies virus challenge; that no non-target species would develop rabies if they ingested the baits; and that the baits were clearly identified and safe for contact with humans. in an extensive series of trials carried out in the usa and in france, these criteria were met and the vaccine was licensed in for raccoons to prevent spread of raccoon rabies (and later to prevent the spread of mexican dog rabies to texas coyotes along the south texas border with mexico) by the us department of agriculture (usda) (wiktor et al., ; rupprecht et al., ; brochier et al., brochier et al., , brochier et al., , desmettre et al., ) . the single licensed product is produced only by merial, inc., athens, ga, usa as raboral v-rg tm for use by governmental (state public health) agencies as an orv for raccoons and coyotes. in the usa, raboral v-rg tm is currently delivered to raccoons and coyotes in an extended fishmeal polymer bait, which contains mg of tetracycline hydrochloride as a bone biomarker and a plastic sachet containing . ml of the vaccine. an extruded poultry-based bait with identical vacdne content has been shown to be more effective for targeting gray foxes (merial, inc., athens, ga, usa). the successful use of the orv to achieve containment or elimination of rabies in some terrestrial wildlife animals in the usa and canada is indicated by the effective containment to near elimination of red fox rabies in southern ontario (macinnes et al., ) , canine rabies in south texas (fearneyhough et al., ) and raccoon rabies in ohio (krebs et al., ) , southern ontario (rosatte et al., ) and eastern new brunswick (slate et al., ) . in , over million baits were distributed in states in the usa (slate et al., ) . new and potentially more effective oral vectored vaccines and more effective baits, including a fishmeal coated sachet bait, are being developed for orv (slate et al., ) . a canarypox-rabies glycoprotein recombinant vaccine was developed and found to be as effective as other poxvirus-rabies glycoprotein recombinants (taylor et al., (taylor et al., , . live canarypox virus that expresses the rabies virus glycoprotein has been licensed in the usa as a parenteral monovalent vaccine for cats and as a combination rabies vaccine for cats with feline panleukopenia virus, feline parvovirus and feline calicivirus vaccines included in the product. a combination canarypox-rabies vaccine with the whole-cell bacterin of neorickettsia risticii included is also licensed for use in the prevention of potomac fever in horses. these are the only rabies virus glycoprotein vaccines currently licensed in the usa (compendium, ) . a recombinant adenovirus-vectored vaccine expressing rabies virus glycoprotein (adrab.gp) was shown to be capable of inducing antibody immune responses in greyhound dogs immunized either subcutaneously or intramuscularly. the dogs had been previously vaccinated for rabies but had low or no rabies antibody titers (tims et al., ) . this vaccine holds promise as a rabies virus vaccine for dogs. the inactivated vaccines require that the rabies virus be produced in high concentrations. this is initially done by growing the virus strain (primarily cvs- , pittman-moore (pm)-nil and pasteur virus (pv)-bhk strains) in the brain tissue of rabbits, baby hamster kidney (bhk) cells, suckling mouse brains (smb), guinea pig brain cells, chick embryo cells (ceo), vero cells or other substrates (precausta and soulebot, ; reculard, ) . neonatal mice can be used as they lack the immunogenic (or allergenic) myelin that caused encephalomyelitis occasionally noted in animals vaccinated with earlier smb nto killed vaccines. the production methods used for the tco rabies vaccines have allowed less allergenic but more immunologic products (greene and rupprecht, ) . various methods, which are still valid, have been used to render the virus non-pathogenic or essentially inactivated (killed) as vaccines. these include, but are not limited to, beta propiolactone (bpl), uv light, and acetylethylamine as well as other amines. phenol and formaldehyde are no longer recommended for virus inactivation (reculard, ) . the most commonly used inactivating agent is bpl. once inactivated, adjuvants are added in order to increase the immune response to the antigen. the most common adjuvants are aluminum hydroxide, aluminum phosphate, saponin (in cattle vaccines) and, rarely, oil adjuvants (precausta and soulebot, ) . much of the information on cell lines, inactivating methods and adjuvants is proprietary and cannot be reported specifically for any one vaccine. the stability of these inactivated cell culture vaccines has allowed the rabies vaccine to be combined with other vaccines and bacterins such as canine distemper, canine adenovirus type , leptospira and parvovirus for canines. for cats, the combination vaccines include feline panleukopenia-virus, feline parvovirus and feline calicivirus. a combined rabies and foot-and-mouth disease vaccine is available for cattle, sheep and goats (who, ) . the potency and safety of the inactivated rabies vaccines have proven to be quite good. in , the national institutes of health (nih) of the us department of health and human services (dhhs) adopted a mouse inoculation test to measure the potency of inactivated vaccines (seligmann, ) . this was necessitated because of the poor performance of the initial manufactured tissue culture origin vaccines (bunn, ) . although a number of other tests to measure vaccine potency are used throughout the world, the nih test is considered the 'gold standard' for measuring the ability of an inactivated vaccine to protect a mouse against virus challenge. the nih test relies on challenge exposure of immunized mice to one virus strain (cvs), a strain thought to be derived from the original pasteur isolate (baer, ) . this test has some inherent bias towards vaccine from the same virus strain origin when comparing vaccine efficacy across the variety of strains (i.e. sad, flury strain vaccines) used to prepare vaccines (barth et al., ) , but this bias does not occur when non-pasteur stain vaccines are tested for protective potential against wild virus strains (baer, ; wunderli et al., a) . in addition, the nih uses two doses of vaccine administered at a one-week interval by an intraperitoneal challenge two weeks later. this vaccination route is quite different from that used for routine administration of rabies vaccine. the second dose prevents an evaluation of the vaccine's primary immunologic potential and the challenge results in a disruption of the blood-brain barrier, allowing neutralizing antibodies in the serum to prevent infection. as a result of these limitations, the who has acknowledged that the nih test needs some improvements or further suggesting that a new rabies potency test may be needed (who, , i ). two recent reports have proposed an alternative method that avoids these shortcomings (wunderli et al., a (wunderli et al., , b . due to the higher antigenic mass and the use of adjuvants, inactivated rabies vaccines have produced post-vaccinal local and systemic reactions. the most common non-neurologic reactions include soreness, lameness and regional lymphadenopathy in the injected limb. fever and anaphylaxis have also been reported (dreesen, ; greene and rupprecht, ) . focal vasculitis and granulomas have been seen - months after vacdnation (greene and rupprecht, ) . post-vaccinal sarcomas may develop as a result of sustained inflammatory reactions at the site of the vaccination that involve the underlying dermas. such postvaccinal sarcomas are often aggressive and invasive, espedally in cats, months to years following vaccination (dubielzig et al., ; kass et al., ; greene and rupprecht, ) . a review of cases of fibrosarcomas in cats following single vacdnation showed that % of the cats with vaccination-site tumors had received rabies vaccine, % were administered a non-rabies combination vaccine and % received a feline leukemia vaccine (hendrick et al., ) . it is not unusual for palpable lesions to occur in cats administered killed vaccine subcutaneously (schulze et al., ) . adverse incidence rates for reactions to rabies vaccination in a retrospective study of ferrets was % when the rabies vaccine was given alone and . % when given in combination with distemper vaccine. the most common adverse events were vomiting and diarrhea (moore et al., ) . the new generation of vectored recombinant vaccines now appearing on the market, such as the avipoxvirus vaccine recently licensed for use for cats in the usa (a rabies glycoprotein, live canarypox vectored vaccine) appears to produce few, if any, allergic or neoplastic reactions (greene and dreesen, ; greene and rupprecht, ) . the who's world survey of rabies reported that there are at least countries or territories that reported producing animal rabies vaccines during . for the production of animal rabies vaccines, countries use cell culture, seven use neural tissue and six countries use embryonated eggs (who, ) . four countries produced more than one type of vaccine. both mlv and inactivated vaccines are produced worldwide. the who world survey of rabies reported that brazil is the major producer of nto rabies vaccines for animal use followed by bangladesh, romania, tunisia and e salvador (who, ) . these five countries account for . % of the . million doses of nto vaccine, primarily smb origin (fuenzalida strain), reported produced for the year. this same survey reported that the usa produced approximately million doses of tco rabies vaccines, % of all tco animal vaccines produced. vietnam is reportedly the primary source of embryonated egg-origin animal vaccine, producing % of this vaccine produced worldwide. it should be noted here that argentina, france, germany, india and a number of other countries that presumably produce animal rabies vaccines did not contribute to the who report. during the -year period - , the availability of rabies vaccines for dogs and cats in latin america grew by . % and the total doses of vaccine administered for these species rose by . % (redipra, ) . vaccine coverage increased from . % in brazil to . % in the southern cone (argentina, chile, paraguay, uruguay). however, there was a . % decline in the andean area (bolivia, columbia, ecuador, peru, venezuela) and a . % decline in central america. this same report denotes that, in the andean area, % of the canine population was vaccinated, in the southern cone . %, brazil %, central america %, mexico % and latin caribbean %. the who recommends that % of dogs in a population should be effectively immunized to prevent an epidemic of canine rabies (coleman and dye, ) . there were laboratory confirmed canine rabies cases in all of latin america during and during . during the same periods, cattle accounted for and cases and other domestic animals accounted for and cases respectively. many types of rabies vaccines are currently marketed in the usa for use in domestic animals. there are inactivated monovalent rabies vaccines licensed for dogs and cats, two for ferrets, four for horses, four for cattle and five for sheep. two inactivated vaccines are combined with other biologics for use in horses. in , a new generation of vaccines was licensed for use in cats. these are the live canarypox-rabies virus glycoprotein recombinant vectored vaccines, either monovalent (one licensed vaccine) or in combination with feline panleukopenia virus, feline parvovirus and feline calicivirus vaccines (compendium, ) . a live vaccinia-rabies virus glycoprotein recombinant vectored vaccine is licensed for restricted use in wildlife raccoons and coyotes. as stated earlier, there are no mlv (attenuated) rabies vaccines licensed for use in the usa. all currently licensed killed rabies vaccines intended for use in carnivores must protect of or of (or a statistically equivalent number) animals from an im challenge with a rabies virus for days post challenge and % of controls must die from the challenge (code of federal regulations, ). alternative challenge requirements have been outlined when the test animals are of a species other than carnivores (code of federal regulations, ). the us department of agriculture (usda), animal and plant health inspection service (aphis), center for veterinary biologics has jurisdiction over licensure of rabies vaccines in the usa. the national association of state public health veterinarians (nasphv) publishes annually the compendium of animal rabies prevention and control (compendium, ) this compendium is a basis for animal rabies programs and the nasphv issues it as recommendations. some states (e.g. georgia) and various cities and counties adopt the recommendations in the compendium as regulations for animal rabies control and prevention. the inactivated tco vaccines should be used in animals at months of age or older and then again one year later. this minimum age precludes maternal antibody blockage and recognizes the immature immune system's often poor response (greene and dreesen, ) . depending on the vaccine, the animal species and, at times, local regulations, the animals should be vaccinated annually or triennially thereafter (compendium, ) . depending on the vaccine type and the species, the vaccine is administered either im or sc, while some vaccines can be administered either way. the minimum age for animal vaccination is weeks of age for the licensed vectored vaccines. regardless of the rabies vaccine type, only when the antibody response peaks, at approximately days after primary vaccination, is the animal considered fully immunized, if vaccination has been administered in accordance with the manufacturer's recommendations. from an epidemiologic viewpoint, the effectiveness of canine rabies prevention and control programs can be measured by comparing reports of rabies in dogs with reports of increases in cat rabies. this was apparent during the recent raccoon rabies epidemic in the middle atlantic and northeastern usa (krebs et al., ; hanlon and rupprecht, ) . the increase in rabies cases in cats, while dog rabies cases remained substantially unchanged, reflects the vaccine status of the two populations as well as the number of feral animals in the two populations (eng et al., ; petronek, ; dreesen, ) . of respondents in a survey of state and community health officials by johnson and walden ( ) , % stated that canine rabies vaccination was required by state law while only % stated that cat vaccination was state law. the need for cat vaccination and feral population control cannot be overemphasized (dreesen, ) . johnson and walden's survey ( ) also noted that over-the-counter sales of rabies vaccines was permitted in states and that, at that time, vaccination of wolfhybrids was permitted in states; however, in all but two of these states the owner must sign a liability statement. fourteen other states did not address the wolf-hybrid issue at all. in , after extensive studies at the centers for disease control and prevention (niezgoda et al., ) , a rabies vaccine for ferrets was approved by the usda, aphis. the ferret should be treated in a similar manner as a dog or cat in regard to vaccination and post-exposure management (compendium, ) . vaccination of wolf-hybrids with canine rabies vaccine is still a matter of considerable debate. in a meeting of taxonomists in , it was concluded that rabies vaccines for dogs would probably protect wolves and their hybrids as they are genetically virtually indistinct from the domestic dog (dreesen, ) . at least one well-documented case of rabies has occurred in a properly vaccinated wolf-hybrid (jay et al., ) . this animal was vaccinated with a -year vaccine at months of age and received other vaccines and bacterins and an antihelminthic on the same day. six months later the animal was found with a dead skunk in its mouth. within weeks the animal developed signs suggestive of rabies, was euthanized and rabies was confirmed in the laboratory. currently, there are no licensed rabies vaccines for wolf-hybrids and the compendium states that wild animals and hybrids (offspring of wild animals crossbred to domestic animals) should not be kept as pets. an animal can be considered to be immunized against rabies virus exposure approximately days after the primary rabies vaccination, which is consistent with a peak antibody response (compendium, ) . thus, an animal is considered immunized if the primary vaccination was administered at least days previously and the follow-up vaccinations have been administered as recommended by the package insert and/or the compendium ( ). the nasphv (compendium, ) recommends that unvaccinated dogs, cats and ferrets exposed to a known or suspected rabid animal should be euthanized immediately. if not euthanized, the animal should be placed in strict quarantine for months and vaccinated either upon entry into isolation or one month prior to release. animals with expired vaccinations should be evaluated on a case-by-case basis. currently, vaccinated dogs, cats and ferrets should be revaccinated immediately following exposure and kept under control and observation for days. it has been shown that there is some evidence that the use of vaccine alone will not reliably prevent rabies from occurring in an unvaccinated domestic animal (hanlon et al., ) . vacdnated livestock exposed to rabies should be revaccinated and observed for days (compendium, ) . if not previously vaccinated, food animals should be slaughtered within days with disposal of tissues in the exposed area. if not slaughtered within this time period, the animal should be closely observed for months. as previously mentioned, the compendium ( ) is issued as recommendations only. some states do not strictly adhere to the recommendations. for example, the texas health and safety code originally followed the previously noted recommendations for animals exposed to rabies (clark and wilson, ) . however, in , the code was amended; unvaccinated domestic animals exposed to a rabid animal were to be euthanized or vaccinated immediately after exposure, kept in isolation for days and given booster vaccinations in the third and eighth week of isolation. this regimen was based loosely on recommendations for humans exposed to rabies virus. a retrospective study conducted by clark and wilson ( ) found that . % of unvaccinated animals did not develop rabies during the - period during which the recommendations of the nasphv were followed. two pep failures did occur ( . %). for the period - , after the texas code was amended to allow pep for unvaccinated animals exposed to rabies, of animals ( . %) that received the pep booster vaccinations did not die of rabies. there was no statistical difference between the two regimens under conditions followed in texas. in a follow-up study for the years - , wilson and clark ( ) found only four of ( . %) domestic animals that received the pep protocol, as recommended, during the previous -year period developed clinical rabies. they concluded that this is an effective pep protocol and 'has been proven to be effective for the control of rabies in animals'. this alternative method of pep for unvaccinated domestic animals exposed to rabies, as practiced in texas, has not been endorsed in the compendium ( ) . propagation of rabies virus in pig kidney cell culture partial pathogenicity for rodents of vaccines intended for oral vaccination against rabies: a comparison oral wildlife rabies vaccination field trials in europe, with recent emphasis on france evaluation of an animal rabies vaccine by use of two types of potency tests oral vaccination of foxes against rabies nih test, a problematic method for testing potency of inactivated rabies vaccine modified live-virus rabies vaccination for oral immunizations of carnivores elimination of fox rabies from belgium using a recombinant vaccinia-rabies vaccine: an update large-scale eradication of rabies using recombinant vaccinia-rabies vaccine (letter) use of a vaccinia-rabies recombinant virus for the oral vaccination of foxes against rabies canine and feline rabies vaccines, past and present vaccination of cats against rabies postexposure rabies prophylaxis and preexposure vaccination failure in domestic animals title immunization coverage required to prevent outbreaks of dog rabies compendium of animal rabies prevention and control rabies vaccination of cats use of vaccinia rabies recombinant for oral vaccination of wildlife preexposure rabies immunization mycoplastic sarcoma originating at the site of rabies vaccination in a cat rabies surveillance, united states results of an oral vaccination program for coyotes efficacy and safety of an oral rabies vaccine (sag- ) in dogs propagation of rabies virus in a culture of hamster kidney cells friedberger and frohner's veterinary pathology rabies. in: infectious diseases of the dog and cat rabies and other lyssavirus infections the reemergence of rabies postexposure prophylaxis for prevention of rabies in dogs comparison of fibrosarcomas that developed at vaccination sites and at non-vaccination sites in cats: cases ( - ) sylvatic rabies in italy: epidemiology studies on chick-embryo-adapted rabies virus. vi. further changes in pathogenic properties following prolonged cultivation in the developing chick embryo rabies surveillance in the united states during rabies surveillance in the united states during vaccination against rabies: construction and characterization of sag , a double avirulent derivative of sadbern elimination of rabies from red foxes in eastern ontario incidence and risk factors for adverse events associated with distemper and rabies vaccine administration in ferrets effect of maternal immunity on the immature response to oral vaccination against rabies in young foxes lyssavirus p gene characterization provides insights into the phylogeny of the genus and identifies structural similarities and diversity within the encoded phosphoprotein pathogenesis of experimentally induced rabies in the domestic ferret rapid clearance of sag- rabies virus from dogs after oral vaccination free roaming and feral cats -their impact on wildlife and human beings vaccines for domestic animals cell-culture vaccines for veterinary use report of the viii meeting of directors of national rabies control programs in latin america emergency response to raccoon rabies introduction into ontario oral vaccination of wildlife against rabies: opportunities and challenges in prevention and control oral immunization and protection of raccoons (procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine experimental studies with killed canine rabies vaccine repeated physical and cytologic characterizations of subcutaneous postvaccinal reactions in cats potency tests requirements of the united states national institutes of health (nih) general considerations in testing the safety and potency of rabies vaccines status of oral rabies vaccination in wild carnivores in the united states biological and immunogenic properties of a canarypoxrabies recombinant, alvac-rg (vcp ) in non-avian species efficacy studies on a canarypox-rabies recombinant virus adult dogs receiving a rabies booster dose with a recombinant adenovirus expressing rabies virus glycoprotein develop high titers of neutralizing antibodies world health organization expert consultation on rabies world health organization. requirements for rabies vaccine for veterinary uses (requirements for biological substances no world survey of rabies no. for the year . p. i. geneva: world health organization world survey for rabies no. for the year . whoicdsicsriephi . p. . geneva: world health organization world health organization expert consultation on rabies immunogenic properties of vaccinia recombinant virus expressing the rabies glycoprotein protection from rabies by a vaccinia recombinant containing the rabies virus glycoprotein gene rabies control in south and southeast asia postexposure rabies prophylaxis protocol for domestic animals and epidemiologic characteristics of rabies vaccination failures in texas: - effects of vaccine route and dosage on protection from rabies after intracerebral challenge in mice effect of heterogeneity of rabies virus strain and challenge route and efficacy of inactivated rabies vaccines in mice rabies virus key: cord- -wyanwvxa authors: sen, adrish; ding, siyuan; greenberg, harry b. title: chapter the role of innate immunity in regulating rotavirus replication, pathogenesis, and host range restriction and the implications for live rotaviral vaccine development date: - - journal: mucosal vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: wyanwvxa abstract rotaviruses (rvs) are important causative agents of viral gastroenteritis in the young of most mammalian species studied, including humans, in which they are the most important cause of severe gastroenteritis worldwide despite the availability of several safe and effective vaccines. replication of rvs is restricted in a host species-specific manner, and this barrier is determined predominantly by the host interferon (ifn) signaling and the ability of different rv strains to successfully negate ifn activation and amplification pathways. in addition, viral attachment to the target intestinal epithelial cells also regulates host range restriction. several studies have focused on the role of the innate immune response in regulating rv replication and pathogenesis. the knowledge accrued from these efforts is likely to result in rational attenuation of rv vaccines to closely match circulating (and host species-matched) virus strains. in this chapter, we review prevalent models of rv interactions with innate immune factors, viral strategies employed to regulate their function, and the implications of these findings for improved rv vaccine development. rotaviruses (rvs) remain one of the two most important viral causes of gastroenteritis despite the availability of several safe and effective live attenuated vaccines [ , ] . rotavirus infection has its biggest health impact on children under the age of years, in whom it still accounts for approximately , deaths annually, almost entirely in less-developed countries [ ] . rvs can infect many cells of the nonimmune host, but the overwhelming bulk of viral replication occurs in the mature villus tip cells of the small intestine [ ] . in this review, we focus on the regulation of rotavirus replication by the host innate immune system, the host-restricted nature of the innate immune response to specific rotavirus strains, and the practical utility of these host range barriers in the development of safe and effective rv vaccines. infection with rv results in the immediate activation of a conserved cellular innate immune signaling pathway that involves multiple pattern recognition receptors (prrs) recognizing discrete rv-encoded pathogen-associated molecular patterns (pamps). a primary purpose of this diverse host-signaling system is to induce different types of interferons (ifns) and a set of virus-induced stress genes (visgs) through two principal transcriptional factors: nuclear factor-κb (nf-κb) and ifn regulatory factor (irf ) [ , ] . the induced ifns and visgs then function to restrict rv replication and pathogen-induced cell injury [ ] . of note, rvs, like virtually all other viral pathogens, have evolved a set of countermeasures to inhibit the host innate immune response, and these countermeasures are most pronounced during homologous rv infection (rv infection with a strain routinely isolated from that specific host species) [ ] . interestingly, rv strains that differ in their ability to regulate the secretion of ifns similarly induce this early recognition pathway, as indicated by the transcriptional upregulation of ifns and several visgs [ ] . based on the collective evidence, initial rv transcription engages the two related prrs rig-i and mda- (members of the family of rig-i-like receptors, or rlrs) [ , ] , which then trigger activation of the mitochondrial antiviral-signaling protein (mavs). these receptors are likely to be stimulated by early rv transcriptional by-products such as exposed -phosphate groups, incompletely methylated -cap structures, and local dsrna structures such as panhandle loops in viral transcripts [ ] . in addition to inducing the secretion of different ifns, rlr responses to rv are likely to orchestrate other host responses. rotavirus activation of mda- results in apoptosis, which occurs mostly in the pancreas of rv-infected mice, indicating that such prrdependent consequences can occur in a cell or organ type-specific fashion [ ] (chapter : innate immunity at mucosal surfaces). in addition to rig-i-and mda- -dependent host responses to rv rna, other sensors are also recruited by the innate immune machinery to trigger early anti-rv responses. among these is a third member of the rlr family: lgp , which seems to exert a proviral effect on rv replication [ ] and whose activation during rv infection may represent a viral strategy to dampen this pathway. yet another player in the innate recognition of rv is the dsrnadependent protein kinase pkr, which is essential for rv-infected cells to secrete ifn [ ] . the molecular basis for pkr's role during rv infection is not well understood, but given the importance of pkr in antiviral signaling in general and its inhibition by a majority of viruses, pkr is likely to be important for rv pathogenicity. distinct from the cytosolic receptors discussed above, rv recognition also involves the toll-like receptors (tlrs), a class of viral receptors that function in the context of cellular membranes, including surface and endosomal membranous components. this aspect of innate rv recognition possibly reflects the rv entry process that exploits endosomal vesicle transport to gain access into host cells. so far, tlr , tlr , tlr , and tlr have all been implicated as potential players in the innate rv detection cascade [ À ] . the ability of tlr to recognize and regulate rv and thus perpetuate an antiviral effect has been tied to tlr 's agedependent expression in the intestine [ ] . since rv typically causes severe disease and replicates in the intestine of infants and young children (in many mammalian species), coincidental with lower levels of tlr expression in infants [ ] , one possible implication is that age-restricted rv intestinal replication is partly due to enhanced tlr signaling with age in this mucosal compartment. other tlrs play specialized roles in discrete types of cells during rv infection. the rvencoded enterotoxin nsp may function as a pamp and in macrophages triggers inflammatory cytokine secretion by a tlr -dependent pathway [ ] . during rv infection in human enteroid cultures [ ] and in different species of mammals [ À ], different types of ifns are secreted, and as will be discuss below, antiviral actions of these ifns are actively countered in a host-range-specific manner by pathogenic rvs. of the ifns, type i ifn is mostly expressed in the intestinal hematopoietic cell compartment rather than in the epithelium where rv primarily replicates [ ] . studies to date have implicated tlrdependent signaling in dendritic cells in the type i ifn secretion process during rv infection [ , , ] . infection of plasmacytoid dendritic cells with rv, which are a major source of type i ifn secretion during viral infections, leads to endosome-dependent (and possibly tlr -mediated) type i ifn secretion that is triggered by viral genomic dsrna (or, potentially, a rv structural protein) [ , , , ] . a central role for tlr-dependent defense against rv is also indicated by the finding that the absence of myd , an essential convergent adaptor in signaling from the different tlrs, results in increased rv infectivity, severity of diarrhea, and impaired humoral immunity [ ] . in addition, rv is susceptible to the antiviral effects of tlr , since activation of this receptor by bacterial flagellin prevents or cures rv infection by a process that involves the secretion of il [ , , ] . inflammasomes are cytosolic multiprotein complexes that remain quiescent at resting state [ ] . upon activation, apoptosis-associated speck-like protein containing card protein, named asc (encoded by pycard) and caspase- (encoded by casp ), oligomerize and mediate the proteolytic processing of proinflammatory cytokines such as pro-il- β and pro-il- and the pore-forming protein gasdermin d, ultimately leading to a lytic form of cell death known as pyroptosis [ ] . these events not only contribute to the host defense against bacterial and other microbial infections, but also regulate the homeostasis of the immune system and the development of various inflammatory diseases and cancer [ ] . although it is known that aim and ifi inflammasomes recognize dna viruses and that nlrp inflammasome responds to general stress and breach of plasma/endosomal membrane integrity [ , ] , how inflammasomes control rna virus replication is less well understood. in addition, whether noncanonical inflammasomes operate in cell types other than myeloid cells is largely unknown. recently, we found that oral infection of suckling mice with murine rv-induced robust activation of casp in the small intestinal tissue, indicating a potential role for inflammasomes in rv pathogenesis [ ] . of note, in contrast to other nod-like receptors, including nlrp , nlrp , nlrc , and naips, targeted deletion of nlrp b in intestinal epithelial cells (iecs) of suckling mice led to an increase in diarrhea, rv shedding in the feces, and intraintestinal viral replication compared to wild-type pups, highlighting a crucial role of nlrp b in controlling rv replication. mechanistically, we found that during rv infection, dexh-box helicase (dhx ) binds to viral rna pamp and interacts with nlrp to activate the downstream signaling pathway ( fig. . ) . furthermore, primary mouse intestinal enteroids generated from dhx -or nlrp deficient mice produced less il- and underwent less pyroptosis compared to wildtype enteroids upon rv infection, confirming a role for dhx in the activation of the nlrp b inflammasome during rv infection [ ] . identification of the dhx -nlrp b-asc-casp cascade as a novel rv-sensing pathway opened up new research directions. are there other inflammasome sensors of rv or other enteric viruses? how do different rnabinding proteins (dhx , rig-i, mda- , etc.) coordinate in the cytoplasm? what is the physiological relevance of theses sensors in the human intestine? addressing these fundamental issues will provide new insights into the biological functions of host innate immune recognition during acute rv infection and, more generally, in overall human enteric health and disease. in addition, answering these basic questions is likely to inform more practical considerations, such as the development of better therapeutics and preventive strategies for enteric infectious diseases. the concept of host range restriction (hrr) is central to many aspects of rv replication and disease, including the development of several of the currently available safe and effective rv vaccines [ ] . rvs are distinguished by being highly pathogenic and infectious to their homologous host species (i.e., the species of host normally infected by the particular rv strain and the species in which that rv strain spreads efficiently) [ ] . rvs are also subject to very severe species-specific restriction of replication and transmission in heterologous host species [ ] . these fundamental properties of rvs are not only of great importance for viral pathogenicity. they also form the basis for several licensed live attenuated orally administered rv vaccines (which are attenuated by virtue of their hrr). in traditional continuous cell line culture systems, most rv strains efficiently block the induction of type i ifn and have evolved to target several different host factors that regulate the ifn pathway [ ] . this multipronged subversion of the ifn response is accomplished primarily by the versatile rv nonstructural nsp protein, the product of rv gene (see below). most ifn-sensitive rv strains encode forms of nsp that exhibit defective ifn inhibition and therefore elicit enhanced ifn secretion [ À ] . although such strains are still viable infectious agents, their ability to replicate is substantially hampered. in addition, ifn sensitivity of rvs encoding full-length "functional" nsp proteins also occurs in specific cell lines, possibly reflecting nsp 's inability to target host innate factors across different species [ ] . enteric infection of suckling (i.e., -to -dayold) mice with a homologous murine rv compared to a heterologous simian rv strain reveals a substantial (b À log) host restriction of the simian rv replication in the intestine [ , , , ] . this host restriction phenotype is substantially reduced (down to log or less) in mice lacking and three ifn receptors (ifnrs) or stat , a key transcription factor relaying antiviral signals from different ifnrs [ , , , ] . the replication phenotype strongly cosegregates with the genetic origin of the vi. mucosal vaccines for viral diseases murine or simian rv nsp encoding gene segments. the suckling mouse thus presents a highly tractable model in which ifn-specific effects on rv replication can be studied within the biologically relevant framework of intestinal rv replication in a natural host and in a host-range-restricted manner. ectopic parenteral injection of purified ifn types i, ii, or iii in many species, including mice, results in the activation of the key downstream transcription factor stat in small iecs (the predominant site of rv replication) [ ] . multiple lines of evidence indicate important roles for ifn types i, ii, and iii in restricting rv replication in the gut and in cell culture [ , , , , ] . in mouse embryonic fibroblasts lacking both types i and ii ifnrs, the replication of several nonmurine rv strains is substantially enhanced (by four to five orders of magnitude). in suckling mice lacking the types i, ii, and iii ifnrs (either singly or in combination) significant enhancement of simian rv intestinal replication occurs demonstrating the sensitivity of heterologous nonmurine rvs to different ifn types in the mouse gut [ , , , ] . in contrast, replication of the homologous murine rv is quite resistant to these ifns (b log or less replication gain). the collective evidence thus highlights the important role of different types of ifns (and their inhibition by the rv nsp protein) in rv pathogenicity and attenuation [ , , , , ] . deciphering the mechanisms underlying these interactions is key for rational rv strain attenuation and designing improved third-generation live attenuated rv vaccines. in a manner analogous to that of other rna viruses, rv-induced ifn activation is dependent on an intact rna sensing pathway [ ] . postrecognition of viral rna by the cytoplasmic sensors rig-i and/or mda- , epithelial cells activate the mavs, a mitochondriaresident adaptor protein that is alternatively known as ifn-β promoter stimulator (ips- ), card adapter inducing ifn beta (cardif), or virus-induced signaling adapter (visa) [ À ] . mavs serves as a central hub in the ifn induction pathway by activating further downstream kinases including tank-binding kinase (tbk ) and inhibitor of kappa light polypeptide gene enhancer in b cells, kinase epsilon (ikk-ε) that phosphorylate irf and nf-κb, respectively [ ] . these molecules translocate into the nucleus upon phosphorylation and function as transcription factors, which ultimately leads to the expression of different ifns and the activation of ifnstimulated response elements (isres). in addition to irf , irf has been characterized as an important transcription factor for type i ifn induction in immune cells, in particular dendritic cells [ ] . similar to irf , irf undergoes phosphorylation and subsequent translocation into the nucleus in response to rv infection and activates ifn expression by functioning as transcription factors. to block such an important pathway, the rv-encoded nsp protein efficiently degrades both irf and irf in a virus-strain-dependent manner [ , ] . this process takes place first through the recognition of irf using an ellis motif localized at the c-terminal end of nsp present on the nsp molecule derived from simian, murine, and some other nonhuman rv strains [ ] . the nsp Àirf interaction subsequently results in a rapid and efficient degradation of irf at the proteasome and suppression of ifn production in rv-infected cells (fig. . ) . besides the irf family members, nf-κb has been shown to be another key arm of the host innate immune response downstream of mavs in many virus-infected cells [ ] . nf-κb signaling is robustly activated by virus infection as well as proinflammatory cytokines, including il- β and tnf-α, the latter of which has recently been shown to be directly antiviral against rv [ ] . for rv infection of ht- cells, the secretion of il- is dependent on the nf-κb activation [ ] . in a suckling mouse model, other chemokines such as ccl , ccl , cxcl , and gm-csf were also upregulated following rv infection [ ] , although whether these canonical nf-κb cytokines are activated through mavs or tlrs remains unknown. similar to irf , β-trcp, a critical protein essential for degrading cellular nf-κb inhibitor iκb, is also targeted by nsp for proteasomal degradation [ ] . in the case of β-trcp, the binding domain within nsp maps to a c-terminal dsgis motif in human and porcine rv strains [ ] . importantly, this is the same region as the ellis motif responsible for irf binding mentioned above. this interesting dichotomy of nsp Àsubstrate interaction may stem from the distinct contribution of irf versus β-trcp in ifn induction in different human and animal rv species [ ] . in contrast to the previous speculation of nsp as a viral e ligase due to the presence of an n-terminal ring finger domain, we recently discovered an interesting codestruction mechanism, in which nsp localizes to the golgi apparatus and hijacks the host cullin Àring box protein e ligase complex to induce the proteasomal degradation of both β-trcp and nsp itself [ ] . chemical blockade or sirna knockdown of cullin- components impaired nsp 's ability to degrade β-trcp, leading to a significant increase in the levels of β-trcp and reduced rv replication (fig. . ) . interestingly, the cullin complex did not appear to be required for nsp -mediated irf degradation, suggesting an alternative mechanism of action at work. more recent unpublished data from our lab indicate that in addition to irfs and β-trcp, mavs itself is also subject to rv inhibition. mavs levels were significantly reduced during rv infection, and this process is mediated, surprisingly, by the rna methyl-and guanylyltransferase vp protein (fig. . ) . by localizing to the mitochondria and binding to mavs through an n-terminal domain, vp induced efficiently proteasomal degradation of mavs in a host-species-and virus-strain-specific manner. mavs inhibition by vp is another striking manifestation of rv's ability to subvert host innate immune signaling. this is the first example of mavs degradation by an enteric virus, and it would be of interest to further test other enteric rna viruses such as norovirus. the antiviral ifn response to rv infection follows a biphasic pattern consisting of an pathway. rv rna pamp activates the rna sensing pathway, which is antagonized by rv-encoded proteins at multiple steps. in unpublished studies, vp has been shown to directly induce the proteasomal degradation of mavs. in addition, nsp from different rv strains degrades either irf or β-trcp, the latter of which is mediated by the host cullin Àe ligase complex. together, these rv proteins work in concert to dampen ifn induction in rv-infected cells. vi. mucosal vaccines for viral diseases initial ifn induction phase followed by a ligand-mediated (and ifn receptor-relayed) amplification phase [ , ] . as was discussed above, rvs are adept at inhibiting ifn induction and the rv nsp protein functions to degrade the essential factors β-trcp and/or irf during ifn induction in a rv strainspecific manner [ ] . interestingly, in spite of viral antagonism of ifn induction, infection with rv leads to the transcriptional induction and secretion of different ifn types in both cell culture and in vivo [ , , , , , ] . at least two factors contribute to the failure of rv to completely suppress the induction of ifn secretion. first, synthesis of the ifn antagonist nsp occurs only after viral entry, uncoating of the virion, rv transcription, and translation. during this initial infection process, several byproducts of viral transcription are generated that act as potent triggers of the ifn induction pathway [ , ] . second, rv entry into different types of cells may not always result in productive infection. for example, rv exposure to primary human pdcs results in two distinct populations of cells that differ in their level of viral infectivity [ ] . dendritic cells that are not productively infected nevertheless exhibit robust activation of the ifn induction response [ , ] . given the remarkable efficiency of ifn secretion in this cell type, they are a likely source of substantial ifn secretion from a nonepithelial cellular compartment where rv does not replicate efficiently. in suckling mice, infection with rv leads to significant induction of different types of ifns, of which the type i ifns are induced primarily in intestinal immune cells rather than being derived from iecs, where viral infectivity is maximal [ ] . the secretion of ifns from different cell types poses a unique challenge to successful viral propagation and spread to uninfected bystander iecs. this is because ifn binding to its cognate cell surface receptor activates a positive feedback loop that amplifies the expression of ifns as well as more than different ifn-stimulated genes [ ] . this ifn release then efficiently amplifies the expression of antiviral proteins targeting a variety of viral replication steps in uninfected bystander cells. each of the three major ifn types (i, ii, and iii) that are found to be induced in the intestine following rv infection is capable of mediating phosphorylation of the key convergent transcription factor stat (at y , an event that is critical for unlocking the transcriptional program resulting in an antiviral state) (fig. . ) . each of these ifn types is biologically relevant in the context of modulating rv infection and spread [ , ] . several lines of evidence indicate that rv employs potent countermeasures to subvert the antiviral state mediated by secreted ifns during initial infection [ , , , , ] . in cell culture, addition of purified exogenous ifns after rv adsorption does not significantly hamper viral replication; instead, ifn treatment of cells prior to rv infection is required to achieve efficient rv replication restriction [ ] . in the rv suckling mouse model, infection with a homologous murine rv and infection with a heterologous simian rv strain result in comparable levels of induction of different ifns from the intestine [ , ] . however, as was noted above, the presence of ifns during infection has a negligible effect on murine rv replication (b -log restriction in titer) but has a potent effect on heterologous simian rv replication ( -to -log restriction in titer) [ , ] . these observations suggest that in order to replicate successfully, homologous rvs have evolved strategies to induce resistance to the actions of different secreted ifn types in cells prior to their actual infection (bystander cells). classical reassortment genetic studies of the ifn-mediated replication phenotype of murine and simian rvs implicated a constellation of rv genes (encoding the vp , nsp , nsp , and nsp viral proteins) in determining the resistance to ifn signaling [ ] . of these, nsp is a necessary and the major determinant of efficient intestinal rv replication in an ifn-dependent fashion. direct evidence for rv subversion of the antiviral state mediated by exogenous ifns comes from the finding that rv-infected ht- cells (a human iec colonic cancer cell derived line) are able to efficiently block stat -y phosphorylation in response to exogenously added purified ifns i and ii [ , ] . using single-cell analytic techniques, ifn-mediated stat inhibition is found to occur within rvinfected cells. remarkably, stat responses to exogenous ifn ligand are also potently inhibited in rv uninfected bystander cells, which do not express any detectable viral antigen [ ] (fig. . ) . although initially described for a porcine rv strain sb -a, this bystander inhibitory effect has now been observed in vitro with several other rv strains, albeit with lower efficiency (sen and greenberg, unpublished observations). the ability of rv to block ifndependent signaling has also been observed in vivo. suckling mice infected with murine rv are able to significantly suppress iec stat -y phosphorylation and subsequent transcription that occurs in response to parenterally figure . rv regulation of the ifn amplification pathway. following infection, rv nsp efficiently blocks exogenous ifn-directed phosphorylation of stat at y , shown here for the ifnar ( ). in the virus-infected cell, rv also mediates the lysosomal degradation of receptors for types i, ii, and iii ifn by an unknown process ( ) . along with irf and irf , the viral nsp protein also proteasomally degrades irf and irf in the infected cells, which are required for optimal ifn amplification responses ( ). in addition, rv can inhibit the nuclear translocation of stat -py by an unknown mechanism ( ) . remarkably, in addition to these viral effects in infected cells, rv also potently inhibits stat phosphorylation in uninfected bystander cells in response to different types of ifns ( ) . the viral and host factors underlying this bystander inhibition of ifns are unknown. vi. mucosal vaccines for viral diseases administered purified ifns i or iii [ ] . although ifna , ifna , and ifna transcripts are induced in the intestines of murine rv-infected mice, transcriptional analysis of isolated mature villous enterocytes revealed that within this compartment (where rv replication predominantly occurs), both infected and bystander cells fail to amplify the type i ifn genes [ ] . in the villous epithelium, rv bystander cells also do not express elevated levels of transcripts encoding irf , which is upregulated in response to stimulation of cells with secreted ifns and is critical for the optimal expression of several antiviral genes. the effectors in rv-infected cells that mediate stat inhibition in bystander cells and the rotaviral factors responsible have not been identified. in contrast, rv inhibition of ifndirected stat activation in rv-infected cells is well characterized [ , , ] . recent findings demonstrate that at the single-cell level, rv infection results in the efficient depletion of type i, ii, and iii ifnrs within rv (vp antigen ) infected cells [ ] . such rv-mediated ifnr degradation is unlikely to be directed by secreted ifns (i.e., by a ligand-dependent pathway) and despite prolonged infection of cells is restricted exclusively to the subset of cells expressing vp . the depletion of ifnrs in rv-infected cells occurs from to hours postinfection (hpi) onwards by a lysosomalÀproteasomal pathway of protein degradation and is not observed in the rv (vp antigen) uninfected bystander cells, which are nevertheless highly refractory to ifn-directed stat activation (fig. . ) . the relevance of rv-mediated ifnr degradation is shown in vivo by the significant decrease in intestinal type i and ii ifnr protein expression in the small intestine following murine rv infection [ ] . degradation of different types of ifnrs by rv represents an ingenious strategy to ensure that any autocrine ifn antiviral amplification is inhibited, thus allowing viral replication and cell to cell spread to proceed efficiently [ ] . interestingly, these findings indicate the likelihood that rv targets a common hostsignaling pathway that is responsible for the expression of all three ifnrs. continuing to unravel the mechanisms by which rv also inhibits the response to different ifns in bystander cells is important for several reasons. first, since the level of rv replication substantially determines host pathogenicity, such information will enable more rational attenuation of homologous and heterologous rv strains and their use as candidate thirdgeneration live vaccines. second, for several diseases (including sepsis and systemic lupus erythematosus), an excessive ifn response is undesirable and implicated as a causative and/or exacerbation trigger of disease. in these situations, discovering novel therapeutic modalities that can dampen ifn signaling is potentially valuable. other rv strategies have also been identified that are directed at disrupting stat signaling during infection. the ability of rv to perturb stat signaling was first reported by holloway and colleagues [ , ] , who observed that as early as hpi, several rv strains inhibited the nuclear translocation of phosphorylated stat -y in response to exogenous ifn stimulation (fig. . ) . although viral factors responsible for this inhibitory effect downstream of stat activation have not yet been identified, it is possible that redundancy in rv inhibition of the stat pathway exists, perhaps indicative of the vital role of inhibiting ifn signaling in enabling rv replication. the ability of the rv nsp protein to target irfs for proteasomal degradation extends to irf and irf , two additional factors that are critical for the optimal amplification of ifndependent antiviral responses [ , ] . whereas early induction of different ifns and antiviral transcripts is mediated primarily by irf , ifnmediated signaling results in an increase in irf expression, which subsequently orchestrates the amplification of ifns and of isgs. the ifn-mediated effects on transcription of several genes (including irf ) require the assembly of a transcriptional complex isgf , which includes stat and irf . the role of irf and irf degradation in ifn-dependent responses during rv infection has not yet been well studied. nevertheless, the degradation of irf and irf by nsp is likely to be an additional weapon in the rv arsenal that can be used to halt an efficient ifn amplification response (fig. . ). in addition to the ifn induction and amplification pathway, rv is equipped with the ability to block further downstream effector antiviral proteins. one such example is ribonuclease l (rnasel), a key enzyme in the ifn-inducible - -oligoadenylate synthetase (oas)-rnasel pathway responsible for potent rna degradation, including both host and viral rna molecules [ ] . the rv rna capping enzyme vp encodes a c-terminal phosphodiesterase (pde) domain that was recently shown to induce the degradation of , -oligoadenylate, the second messenger responsible for rnasel activation and dimerization [ ] . the rv vp pde domain functionally replaced the comparable domain in the murine coronavirus ns protein and inhibited rnasel activity. a more recent study suggests that another vp -independent, yet-to-be identified, mechanism also exists and contributes to rv inhibition of rnasel [ ] . however, the actual physiological roles of rnasel in modulating rv replication and of vp in antagonizing rnasel in vivo remain to be demonstrated. there are currently two time-honored and demonstrably successful approaches to developing safe and effective human viral vaccines [ ] . in the first case, a wide variety of current viral vaccines rely on the parenteral administration of replication-incompetent inactivated whole virus, the parenteral administration of a viral protein(s) component, or the administration a molecularly produced virus-like particle (vlp). all of these entities are selected because immunity to the individual protein, inactivated whole virus, or vlp induces protective immunity to the host and, at the same time, is both safe and well tolerated. there are numerous highly successful examples of this category of viral vaccine (e.g., the inactivated polio vaccine, the various preparations of inactivated influenza hemagglutinin-based vaccines, the human papilloma vlp vaccine, the hepatitis b virus vaccine, and the recently licensed herpes zoster ge protein-based vaccine). in all cases, these vaccines are administered parenterally. several are administered with adjuvants of one kind or another to enhance immune responses, and in all cases, they appear to function primarily by stimulating systemic immunity, with the primary effector function generally mediated by systemic antibodies. of note, none of these types of vaccines are directed at a predominantly enteric pathogen, although an investigation of a potential parenterally administered norovirus vlp vaccine is currently under way [ ] , and there are plans to study the utility of a heat-inactivated rv virion-based vaccine in humans [ ] . the greatest advantages of the inactivated/recombinant protein-based vaccines are their general safety and the fact that they can be produced even when the actual pathogen cannot be readily propagated. the general disadvantage of such vaccines is that they are almost always less effective at stimulating t-cell-based immune responses, they are not very efficient at stimulating mucosal immune responses, that highly effective mucosal immune adjuvants are not yet readily available, and in some cases, immune responses to these inactivated vaccine preparations tend to diminish over time more rapidly than do responses to several live viral vaccines. other potential disadvantages of inactivated vaccines become apparent when the antigen or antigens required to induce protective immunity are difficult to synthesize artificially or when immunity is most potent when it is directed at multiple antigens that are correctly folded only on the actual or recombinant multiprotein viral particle. of note, rvs have at least two separate proteins (vp and vp ) that are targets of protective antibodies, and vp is cleaved by enteric trypsin into two separate protein components: vp * and vp * [ ] . both vp * and vp * are individually targeted by protective antibody responses. vp is correctly folded into its proper antigenic trimeric form only within the context of the rv virion, and a similar issue likely is true for vp *. on the other hand, the rv vp * protein can be relatively simply and accurately synthesized in several prokaryote and eukaryote systems, and because of this feature, it is currently being examined as a potential inactivated vaccine to be administered parenterally [ ] (chapter : development of oral rotavirus & norovirus vaccines). the second highly successful approach to human viral vaccine development has been the production of live attenuated viral vaccines that actually infect susceptible people but are attenuated to such a degree that their level of reactogenicity and pathogenicity is acceptable, while they reliably generate protective immunity. as with inactivated vaccines, a number of highly effective replication-competent viral vaccines are currently available (e.g., oral sabin polio vaccine, measles vaccine, rubella vaccine, yellow fever vaccine, smallpox vaccine, live attenuated influenza vaccine, and, of course, several licensed rv vaccines, such as rotateq, rotarix, and rotavac) [ ] . most but not all of the live attenuated viral vaccines are administered by parenteral injection; however, oral polio, rotavirus, and live attenuated influenza vaccine are all administered to a mucosal surface (the gi tract and the respiratory tract, respectively). the general thinking is that live attenuated viral vaccines more closely mimic the type and level of immunity induced by natural infection than parenterally administered inactivated vaccines do. if natural infection is an effective preventative of severe secondary infection, then reproducing it without undue reactogenicity can be desirable. this feature is present when natural immunity is operative primarily at a mucosal surface, as is the case for rvs. natural rv infection efficiently protects against severe reinfection, and for this reason, a variety of experimental and licensed live attenuated rv vaccines have been developed or proposed. the key issue to overcome in developing a live viral vaccine is to discover a method that reliably attenuates viral pathogenicity while retaining the ability of the viral infection to induce protective immunity. traditionally, such modification has been accomplished by multiple passage of virulent viral strains in cell culture with the hope that such multiple passages will lead to the acquisition of sufficient mutations in the viral genome (acquired to enhance cell culture replication) and that these cell culture adaptations will attenuate viral pathogenicity in the target host. this strategy is vi. mucosal vaccines for viral diseases time honored and has been used to develop multiple vaccines (e.g., live polio, measles, mumps, rubella, yellow fever, and some rv vaccines). while this approach is frequently successful, there is no way to determine how many passages are needed to eliminate residual virulence while retaining immunogenicity, so it is often tedious and always an inexact approach. in addition, concerns about reversion to virulence are always present. that said, this approach was used successful to develop a highly effective human rv vaccine (r , rotarix), which consist of a single human rv strain that was repeated passed in several cell culture systems and, over the passage history, became attenuated in people [ ] . while the genomic sequence of both the virulent wildtype parental rotarix strain and the eventual vaccine strain are known, the exact genomic mutations responsible for attenuation are unclear. what has been established, however, is that, given the very substantial and decade-long safety record, this human rv vaccine strain has sufficient attenuating mutations to ensure a high degree of genetic stability in humans. of note, the r rotarix vaccine represents a single g and [p] serotype yet reliably induces protective immunity to virtually all frequently circulating human rv strains. this finding strongly supports the conclusion that serotype-specific immunity is not a major determinant of immunity to severe rv disease in humans [ ] . the other strategy that has proven highly successful for the reproducible attenuation of the rvs used in live attenuated rv vaccines has been to take advantage of the hrr (see above) of heterologous (nonhuman origin) rvs as vaccine candidates for humans [ ] . several currently licensed rv vaccines (e.g., the r vaccines rotateq and rotasil and the r vaccine rotavac) consist of either natural or experimentally selected reassortants between animal rvs (in these cases, all bovine rvs) and human rvs. both r vaccines are pentavalent constructs derived experimentally on the basis of bovine rv genomes but containing individual vp -or vp s-encoding genes derived from various human serotypes. rotateq is broadly licensed around the world, while rotasil is currently licensed only in india [ ] . rotavac is, interestingly, a naturally occurring reassortant rv derived from a human rv and a bovine rv. it contains only a bovine vp , with all other ten rv gene segments derived from a human rv strain. this virus was originally isolated from a neonatal nursery where asymptomatic rv infection was endemic [ ] . finally, of relevance to this review, an entirely lamb origin rv strain is currently licensed for rv prevention in china. this vaccine is also highly attenuated, presumably because of the hrr of a lamb origin rv in humans. while this vaccine seems safe, the data on its efficacy are limited [ ] . the key point here is that all these animal virus origin based rv vaccines are highly attenuated in humans, and this attenuation, as was discussed above, is most likely based on their host range replication restriction in humans. this hrr is primarily due to the inability of heterologous rv to efficiently suppress the human intestinal innate immune response, primarily the human ifn response, owing to the presence of heterologous nsp s in the vaccine candidates. the active human ifn response to these heterologous rv vaccines suppresses their replication sufficiently to restrict pathogenicity and reactogenicity but not so much that the generation of effective rv immunity is suppressed. however, in the case of the indian rotavac vaccine, attenuation might be based on the heterologous bovine origin vp , which might be expected to reduce rv binding to human iecs. the big question for the future is whether, with the advent of a tractable reverse genetic system and our improved understanding of the genetic determinants of hrr, we will be able to better fine-tune the replication competence and immunogenicity potential of rv vaccine candidates to further enhance their efficacy. pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (mal-ed) global, regional, and national estimates of rotavirus mortality in children , years of age fields virology the battle between rotavirus and its host for control of the interferon signaling pathway activation of host pattern recognition receptors by viruses or not totranslate: viral and host mrna regulation by interferon-stimulated genes distinct roles of type i and type iii interferons in intestinal immunity to homologous and heterologous rotavirus infections the early interferon response to rotavirus is regulated by pkr and depends on mavs/ips- , rig-i, mda- , and irf rig-i/mda /mavs are required to signal a protective ifn response in rotavirusinfected intestinal epithelium characterization of rotavirus rnas that activate innate immune signaling through the rig-i-like receptors the innate immune receptor mda limits rotavirus infection but promotes cell death and pancreatic inflammation myd -mediated tlr signaling protects against acute rotavirus infection while inflammasome cytokines direct ab response rotavirus activates lymphocytes from non-obese diabetic mice by triggering toll-like receptor signaling and interferon production in plasmacytoid dendritic cells rotavirus nsp triggers secretion of proinflammatory cytokines from macrophages via toll-like receptor porcine small intestinal epithelial cell line (ipec-j ) of rotavirus infection as a new model for the study of innate immune responses to rotaviruses and probiotics rotavirus structural proteins and dsrna are required for the human primary plasmacytoid dendritic cell ifnalpha response viral infection. prevention and cure of rotavirus infection via tlr /nlrc -mediated production of il- and il- age-dependent tlr expression of the intestinal epithelium contributes to rotavirus susceptibility a paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection frequencies of virus-specific cd ( ) and cd ( ) t lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults the cloning of cattle interferon-a subtypes isolated from the gut epithelium of rotavirusinfected calves rotavirus induces alphainterferon release in children with gastroenteritis treatment of rotavirus infection in neonate and weanling pigs using natural human interferon alpha interferon response in colostrum-deprived newborn calves infected with bovine rotavirus: its possible role in the control of the pathogenicity interferon activity in rotavirus infected newborn calves innate immune response to homologous rotavirus infection in the small intestinal villous epithelium at single-cell resolution plasmacytoid dendritic cells promote rotavirus-induced human and murine b cell responses role of interferon regulatory factor in type i interferon responses in rotavirus-infected dendritic cells and fibroblasts flagellin treatment protects against chemicals, bacteria, viruses, and radiation interferon-lambda and interleukin act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection the inflammasomes inflammasome-activated gasdermin d causes pyroptosis by forming membrane pores inflammatory bowel disease and the nlrp inflammasome the aim inflammasome is essential for host defense against cytosolic bacteria and dna viruses k( ) efflux is the common trigger of nlrp inflammasome activation by bacterial toxins and particulate matter nlrp b inflammasome restricts rotavirus infection in intestinal epithelial cells global impact of rotavirus vaccination on childhood hospitalizations and mortality from diarrhea roles of vp and nsp in determining the distinctive replication capacities of simian rotavirus rrv and bovine rotavirus uk in the mouse biliary tract rotavirus infection diversity of interferon antagonist activities mediated by nsp proteins of different rotavirus strains the rotavirus interferon antagonist nsp : many targets, many questions rotavirus nsp mediates degradation of interferon regulatory factors through targeting of the dimerization domain rotavirus nsp inhibits expression of type i interferon by antagonizing the function of interferon regulatory factors irf , irf , and irf comparative proteomics reveals strainspecific beta-trcp degradation via rotavirus nsp hijacking a host cullin- -rbx complex rotavirus nsp inhibits nfkappab activation by inducing proteasome-dependent degradation of beta-trcp: a novel mechanism of ifn antagonism zinc-binding domain of rotavirus nsp is required for proteasome-dependent degradation of irf and autoregulatory nsp stability interferon regulatory factor is a cellular partner of rotavirus nsp nsp of human rotaviruses commonly inhibits nf-kappab signalling by inducing beta-trcp degradation irf inhibition by rotavirus nsp is host cell and virus strain dependent but independent of nsp proteasomal degradation role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice permissive replication of homologous murine rotavirus in the mouse intestine is primarily regulated by vp and nsp rotavirus degrades multiple type interferon receptors to inhibit ifn signaling and protects against mortality from endotoxin in suckling mice variation in antagonism of the interferon response to rotavirus nsp results in differential infectivity in mouse embryonic fibroblasts ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf visa is an adapter protein required for virus-triggered ifn-beta signaling phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf activation irf- is the master regulator of type-i interferon-dependent immune responses rotavirus nonstructural protein subverts innate immune response by inducing degradation of ifn regulatory factor structural basis for concerted recruitment and activation of irf- by innate immune adaptor proteins tnf-alpha exerts potent antirotavirus effects via the activation of classical nf-kappab pathway rotavirus stimulates il- secretion from cultured epithelial cells rotavirus and coxsackievirus infection activated different profiles of toll-like receptors and chemokines in intestinal epithelial cells putative e ubiquitin ligase of human rotavirus inhibits nf-kappab activation by using molecular mimicry to target beta-trcp silencing the alarms: innate immune antagonism by rotavirus nsp and vp rotavirus nsp protein inhibits interferon-mediated stat activation interferons and viruses: an evolutionary arms race of molecular interactions interferon gamma and interleukin , but not interferon alfa, inhibit rotavirus entry into human intestinal cell lines rotavirus inhibits ifn-induced stat nuclear translocation by a mechanism that acts after stat binding to importin-alpha rotavirus antagonizes cellular antiviral responses by inhibiting the nuclear accumulation of stat , stat , and nf-kappab viral encounters with ', '-oligoadenylate synthetase and rnase l during the interferon antiviral response homologous ', '-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity rotavirus controls activation of the - -oligoadenylate synthetase/rnase l pathway using at least two distinct mechanisms plotkin's vaccines status of vaccine research and development for norovirus does a monovalent inactivated human rotavirus vaccine induce heterotypic immunity? evidence from animal studies vp -and vp -specific antibodies mediate heterotypic immunity to rotavirus in humans safety and immunogenicity of a parenterally administered rotavirus vp subunit vaccine in healthy adults efficacy of human rotavirus vaccine against rotavirus gastroenteritis during the first years of life in european infants: randomised, double-blind controlled study rotavirus vaccines -a new hope jennerian and modified jennerian approach to vaccination against rotavirus diarrhea using a quadrivalent rhesus rotavirus (rrv) and human-rrv reassortant vaccine safety and efficacy of a pentavalent human-bovine (wc ) reassortant rotavirus vaccine efficacy of a monovalent humanbovine ( e) rotavirus vaccine in indian infants: a randomised, double-blind, placebo-controlled trial effectiveness of the live attenuated rotavirus vaccine produced by a domestic manufacturer in china studied using a population-based case-control design key: cord- -gevrlcvy authors: shewen, p.e.; carrasco-medina, l.; mcbey, b.a.; hodgins, d.c. title: challenges in mucosal vaccination of cattle date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: gevrlcvy recognition of the mucosal portal of entry for many infectious diseases and of the relevance of mucosal immune response to protection has encouraged the development of vaccines administered by mucosal routes, principally oral and intranasal, for stimulation of intestinal and nasopharyngeal lymphoid tissues respectively. the oral route is problematic in cattle and other ruminants where antigen degradation in the rumen is likely, prior to transit to the intestine. on the other hand, rumination can be exploited for exposure of nasopharyngeal tissues during cudding if vaccine antigen is expressed by a fibrous feed like alfalfa. an increase in anti-leukotoxin (lkt) iga was demonstrated in nasal secretions of calves following feeding of alfalfa expressing a truncated lkt from mannheimia haemolytica, and there is evidence suggesting that such vaccination may protect against experimentally induced pneumonia. intranasal vaccination is an alternative approach for use in pre-ruminating calves. intranasal administration of iscoms carrying soluble antigens of m. haemolytica, including native lkt, induced lkt specific iga in nasal secretions after vaccination at and weeks of age. subcutaneous (s.c.) administration of the same vaccine induced lkt specific igg in both serum and nasal secretions, whereas s.c. administration of a commercial m. haemolytica vaccine did not. regardless of the vaccination strategy employed it is difficult to assess the immunogenicity of mucosally administered vaccines because production of secreted antibodies tends to be transient, and they do not persist on the mucosal surface in the absence of ongoing antigenic stimulation. an additional challenge is demonstration of vaccine efficacy in response to experimental infection. protection of the mucosally vaccinated animal will most probably result from recall response, which may not amplify sufficiently to counter the effects of experimental pulmonary delivery of a large bolus of virulent bacteria, even though the response would suffice over the more prolonged and gradual infection that occurs in natural induction of pneumonia. the vast majority of infectious diseases in all species are initiated by colonization of, or entry across, mucosal surfaces of the respiratory, intestinal or urogenital tracts. there has, therefore, been a great deal of interest in immune response at these sites and in development of vaccines that target these portals of entry (hodgins et al., ) . the reality is that most current vaccines for such infections are delivered parenterally and act thorough induction of systemic rather than mucosal immunity. protection is typically mediated by ''spill-over'' of mediators onto mucosal surfaces or by blocking of infection once the mucosal surface is breached; examples include vaccines for influenza viruses, vibrio, salmonella, and veterinary immunology and immunopathology ( ) - a r t i c l e i n f o mucosal immunity vaccination mannheimia haemolytica cattle a b s t r a c t recognition of the mucosal portal of entry for many infectious diseases and of the relevance of mucosal immune response to protection has encouraged the development of vaccines administered by mucosal routes, principally oral and intranasal, for stimulation of intestinal and nasopharyngeal lymphoid tissues respectively. the oral route is problematic in cattle and other ruminants where antigen degradation in the rumen is likely, prior to transit to the intestine. on the other hand, rumination can be exploited for exposure of nasopharyngeal tissues during cudding if vaccine antigen is expressed by a fibrous feed like alfalfa. an increase in anti-leukotoxin (lkt) iga was demonstrated in nasal secretions of calves following feeding of alfalfa expressing a truncated lkt from mannheimia haemolytica, and there is evidence suggesting that such vaccination may protect against experimentally induced pneumonia. intranasal vaccination is an alternative approach for use in pre-ruminating calves. intranasal administration of iscoms carrying soluble antigens of m. haemolytica, including native lkt, induced lkt specific iga in nasal secretions after vaccination at and weeks of age. subcutaneous (s.c.) administration of the same vaccine induced lkt specific igg in both serum and nasal secretions, whereas s.c. administration of a commercial m. haemolytica vaccine did not. regardless of the vaccination strategy employed it is difficult to assess the immunogenicity of mucosally administered vaccines because production of secreted antibodies tends to be transient, and they do not persist on the mucosal surface in the absence of ongoing antigenic stimulation. an additional challenge is demonstration of vaccine efficacy in response to experimental infection. protection of the mucosally vaccinated animal will most probably result from recall response, which may not amplify sufficiently to counter the effects of experimental pulmonary delivery of a large bolus of virulent bacteria, even though the response would suffice over the more prolonged and gradual infection that occurs in natural induction of pneumonia. ß elsevier b.v. all rights reserved. (corbeil et al., ; wilkie and markham, ) . despite these successes, development of mucosally delivered vaccines remains an area of active investigation in many laboratories, for both human and veterinary pathogens. why vaccinate mucosally? immune mediators, both immunoglobulins and effector t cells generated by mucosal exposure to antigens differ from those generated by systemic immunization (boyaka et al., ) . certainly, where the goal is prevention of infection, the presence of mediators on the mucosal surface is needed. memory cells generated at mucosal sites and in draining lymph nodes, home preferentially to other mucosal locations providing a primed response at all potential portals of exposure (youngman et al., ) . there are also non-immunologlical reasons for seeking vaccines that are delivered without injection, including ease of delivery and the absence of injection site reactions. vaccination of food producing animals would be facilitated by mass delivery of vaccine in feed, water or by aerosol, meaning less labor cost for producers and reduced stress on the animals. additionally, carcass condemnation due to needle breakage or injection site reactions would be avoided (roeber et al., ) . increasing consumer pressure for organically produced food and a natural approach to disease management is more compatible with disease prevention using noninvasive methods of vaccine delivery. mucosal delivery of antigens triggers immune response in mucosa-associated lymphoid tissues including the peyer's patches of the small intestine, the tonsil and associated pharyngeal lymphoid tissues, the bronchus associated lymphoid tissues of the lung and diffuse lymphoid aggregates lining the urogential tract. induction sites for mucosal immunity have been most thoroughly described for the intestinal tract where antigens are delivered to underlying peyer's patches by specialized membranous non-ciliated epithelial cells, m cells, located in villous crypts, and by dendritic cells (dcs) that send processes to the surface between ciliated columnar epithelial cells of the villi. these dcs deliver antigen to peyer's patches and to draining mesenteric nodes (meeusen et al., ) . similar mechanisms for antigen acquisition exist at other mucosal sites, including in tonsilar crypts and in balt where both m cells and epithelial dcs have been identified (gebert and pabst, ; stanley et al., ) , and presumably also at genital sites (hodgins et al., ) . experimentally, calves have been immunized by the vulvovaginal and rectal routes (loehr et al., (loehr et al., , , but most mucosal vaccines deliver antigens by oral administration, which targets intestinal induction, or intranasally which targets pharyngeal and, depending on particle size, deeper respiratory tissues. there are many challenges inherent in mucosal antigen delivery. intranasal vaccines must be delivered in dosages sufficient to overcome innate clearance mechanisms and facilitate uptake in the pharynx. oral vaccines must be protected against degradation by digestive processes, mechanisms are needed to facilitate antigen adherence to the mucosal epithelium and avoid clearance with the mucociliary blanket coating the gut, and the potential for induction of tolerance rather than active immunity must be considered . in mouse models, oral delivery of auto-antigens may lead to oral tolerance and reversal or reduction of autoimmunity. however, oral tolerance has not yet been reported in studies where plant expressed antigens have been delivered as vaccines in mice or other monogastric animals (arntzen et al., ; rice et al., ) . successful oral vaccines exist; vaccines for polio and rabies are excellent examples. vaccines using avirulent live bacteria (e.g. salmonella, bordetella) or viruses (e.g. adenovirus, coronavirus, reovirus) have been shown to be quite effective in stimulating mucosal immune responses and in cases where the vaccine organism is at least minimally invasive, systemic responses as well. perhaps the greatest challenge for mucosal immunization lies in vaccination with non-replicating antigens. such preparations are difficult to protect against digestion, tend not to adhere to mucosal surfaces, and generally fail to trigger the danger signals needed to initiate appropriate cellular stimulation for active immune response. experimentally several strategies have been employed to overcome these inherent challenges. vaccine antigens have been enclosed within microspheres, linked to bacterial toxin subunits such as ct or lt, or administered with molecules that induce danger signals, like cpg motifs, or with cytokines to stimulate lymphocyte activation (holmgren et al., ) . ruminating animals pose a particular challenge for development of orally administered vaccines. encapsulation of antigens in polymer microparticles or microspheres that resist digestion in the rumen is one approach that shows promise for stimulation of mucosal immune response in peyer's patches by oral delivery (bowersock et al., ) . an alternative approach is to exploit the process of rumination for exposure of the pharyngeal lymphoid tissues including the tonsil during cudding. this could be a particularly valuable approach for vaccination against both respiratory and intestinal diseases, since it is recognized that memory cells produced in tonsilar lymphoid tissue migrate preferentially to the lung and intestine, priming these sites for subsequent natural exposure (brandtzaeg and johansen, ) . with this in mind, we hypothesized that delivery of protective antigens in a palatable fibrous feed, such as alfalfa, could lead to repeated exposure of pharyngeal lymphoid tissue by cudding during rumination. because of its relevance as an economically important disease of cattle, we selected bovine pneumonic pasteurellosis as the target disease for study. our extensive experience with immunity to the principal causative bacterium mannheimia haemolytica and its protective antigens made this both an appropriate and convenient model system to test both the immunogenicity and efficacy of edible vaccines comprised of transgenic alfalfa. the first antigen targeted in this research was the leukotoxin (lkt). many studies have demonstrated lkt's importance in pathogenesis and the correlation between the presence of anti-lkt antibodies in serum and protection against pneumonia; however, most suggest that an anti-lkt response is essential but not sufficient alone to provide immunity (shewen and wilkie, ; jeyaseelan et al., ) . the transgenic alfalfa used in these experiments expressed a truncated form of leukotoxin (lkt ) that contained the neutralizing epitope (lee et al., ) . the concentration of lkt was at least mg/g of dried plant material, estimated as a percentage of total soluble protein. posttranslational modification of proteins occurs in plants ( gomord and faye, ) , including both n-linked and olinked glycosylation, but plant glycosylation patterns can differ from those found in bacteria. since the extent of glycosylation and type of glycan added to proteins can alter their immunogenicity, an early step in this work was demonstration of the immunogenicity of plant expressed lkt following intramuscular inoculation of rabbits, and verification that antisera from immunized rabbits recognized both recombinant and native lkt (lee et al., ) . use of transgenic alfalfa as a vehicle provides an efficient means for delivery of antigen that also furnishes protection against immediate dilution and destruction in the rumen. the natural process of cudding means that the fibrous feed is regurgitated, chewed slowly and held as a cud in the posterior oral cavity. typically this activity occurs - times over a period of several days, spraying the pharyngeal lymphoid tissues with antigen during each cycle, before it is finally digested and passed on. with this system, concerns about delivery, avoidance of innate clearance mechanisms and antigen destruction are addressed, but at least two major challenges remain as barriers to vaccine development. the first is demonstration of immunogenicity when the anticipated response is predominately mucosal and therefore inherently both difficult to sample and transient in the absence of continual antigen stimulation. it is also quite possible that pharyngeal exposure will merely prime the lung for an anamnestic response on infection, rather than lead to production of mucosal antibodies in response to the levels of antigen delivered by vaccination. the second related challenge is demonstration of efficacy, given that protection is most likely derived from a recall response. such response should suffice during natural exposure since this is gradual and continual over a period of hours or days, but can easily be overwhelmed in experimental challenge where the successful challenge model uses intrabronchial delivery of a large number of organisms, sufficient to cause pneumonia, as a single bolus. to determine the extent of these challenges and to address related questions of dose and duration of feeding needed for immunization, we have conducted a series of pilot studies feeding transgenic alfalfa to small groups of calves, typically two vaccinates and two controls that receive an equal amount of wild type alfalfa in lieu of the transgenic feed. for initial studies we opted to use colostrum deprived animals, reared in an isolation facility, to avoid interference by passive maternal antibodies and to minimize commensal colonization by m. haemolytica that might confound demonstration of response to vaccination. the animals were not germfree, nor were they caesarian derived, thus there was a low level of colonization and a low baseline antibody titer in serum at the time of first feeding, typically about months of age. all experiments were conducted under approval of the university of guelph animal care committee and adhered to the canadian council of animal care guidelines for use of animals in research. growth, processing, storage and feeding of the transgenic alfalfa as well as disposal of animals and animal waste were as specified in letters of permission from the canadian food inspection agency, which is responsible for regulation of gmos and use of experimental vaccines. in early trials, calves were fed in two rounds, g of dried alfalfa, each day for days, at a -week interval. they were then challenged approximately weeks after the second feeding by intrabronchial administration of ml of m. haemolytica (atcc ) at approximately cfu/ ml. this dose was estimated to cause infection sufficient to elicit recall response, but not produce pneumonia in controls. calves were euthanized or days postchallenge and mononuclear cells were harvested from blood, tonsil, spleen, and retropharyngeal, bronchial and mesenteric lymph nodes. serum and nasal swabs were collected at various points throughout the trial, and the presence of antibodies was determined by an lkt specific elisa using either alfalfa expressed lkt or native leukotoxin, in log phase serum-free culture supernatant, as the antigen. the latter elisa was further adapted as an elispot assay for detection of antibody secreting cells in mononuclear cell preparations following incubation with native leukotoxin. mononuclear cell culture supernatants were also assayed for production of interferon gamma using a commercial kit (bovigam, pfizer). during these studies it was noted that feeding of transgenic lkt alfalfa led to an increase in lkt specific iga in nasal secretions week following the second feeding ( fig. ) . this increase was transient and by the time of challenge levels of specific nasal iga were similar in vaccinates and controls. no changes in serum antibodies were observed prior to challenge. low level intrabronchial challenge with m. haemolytica, < cfu/ml, resulted in an increase in both serum and nasal antibodies in all calves at days post-challenge. these observations illustrate the difficulty in demonstrating humoral immune response to mucosal vaccination. the response is likely to be local, not systemic, and transient. thus sampling site and timing become critical. a similar response to vaccination was recently observed in a larger scale feeding trial in colostrum sufficient calves ( vaccinates and controls) (fig. ) . baseline serum antibodies to lkt were higher in all calves than previously observed in colostrum deprived animals, and feeding did not result in a noticeable change in serum antibodies. several calves fed recombinant alfalfa maintained elevated nasal iga to lkt from week after the second feeding up to the day of challenge. that the response was sustained in these animals may be a characteristic of conventional calves, since continuous antigen stimulation due to natural exposure could help maintain the response that was enhanced by vaccination. in this experiment where the challenge, ml of m. haemolytica at . cfu/ml, was intended to induce pneumonia, a correlation between the level of anti-lkt nasal iga and protection against pneumonia was observed. however, there was also a correlation between the level of anti-lkt igg in serum and protection, even though we could not demonstrate an effect of vaccination on the circulating level of those antibodies. thus, despite the encouraging nasal antibody results, we cannot confidently associate protection with vaccination. during one pilot study the challenge dose, at cfu/ ml, was sufficient to induce pneumonia in the two control calves (lesion scores / for both, % and % pneumonic tissue). both calves fed alfalfa expressing lkt had no clinical signs of pneumonia and no lesions at necropsy. although caution must be exercised in interpreting this as evidence of vaccine efficacy, since calf numbers are small, several interesting observations were made with respect to the response of mononuclear cells in susceptible versus resistant calves. vaccinated calves had lkt specific antibody producing cells in and day old cultures of cells derived from blood and bronchial nodes harvested at necropsy and there was evidence of class switching since igg and iga producing cells were present as well as cells producing igm. each of the control calves had only a single blood cell ( in ) producing antibodies, one igm and one iga. there was no interferon gamma production by blood mononuclear cells of any calves, response in tonsil cells was very weak, and there was no differential between vaccinated and control calves in ifng response in bronchial node cells. spleen and retropharyngeal node cells from vaccinates produced ifng rapidly within h of incubation, whereas cells from control calves responded later or not at all. the most striking difference was observed on culture of cells from mesenteric nodes with lkt. cells from vaccinates responded by producing ifng, controls did not (fig. ) . this was especially interesting since the challenge was pulmonary, not intestinal; thus, one would not expect activation of lymphocytes in these nodes. does this confirm trafficking of antigen-specific memory cells following exposure of the nasopharynx to transgenic alfalfa or does this suggest that vaccine antigen survived rumination and sensitized galt by transport across the intestinal epithelium? these questions remain to be addressed but at least we have identified a tissue to target in ongoing investigations. is the edible format the future for veterinary vaccines in ruminanting animals? perhaps, but even if that approach succeeds it will not address the question of pneumonia in younger calves that are not yet ruminating. bacteria in the pasteurellaceae family are major contributors to enzootic pneumonia that occurs at - weeks of age in both veal calves and replacement dairy heifers (van donkersgoed et al., ) . mucosal delivery of vaccine is also a relevant goal in these neonates, in particular intranasal delivery has appeal for both logistical (ease of delivery) and immunological (targets nasopharyngeal lymphoid tissues) per- fig. . iga antibodies to leukotoxin (lkt) in nasal secretions, determined in an elisa assay using alfalfa expressed lkt as antigen. od = optical density against lkt alfalfa minus od using a mock antigen preparation derived from alfalfa expressing gfp. grids span feeding days, solid vertical bar indicates day of challenge, oval indicates increase in specific iga days after feeding in vaccinated calves ( vaccinates, control). group means for iga antibodies to leukotoxin in nasal swabs obtained from calves fed alfalfa expressing lkt and control calves fed wild-type. median sample to positive (s/p) ratios determined in an elisa assay using native lkt as antigen. grids span feeding days, solid vertical bar indicates day of challenge. spectives. the added difficulty in calves of this age is the potential for interference in active immune response by passive maternal antibodies. in fact, pneumonia, at - weeks of age, occurs precisely during the period when passive immunity has waned to the extent that it is no longer protective, but the effects of interference with active immune response have delayed generation of protective immunity (prado et al., ) . previously we demonstrated that calves do not produce leukotoxin neutralizing antibodies in response to parenteral vaccination with a commercial m. haemolytica culture supernatant vaccine (presponse, wyeth/fort dodge) prior to weeks of age (hodgins and shewen, ) . earlier induction of active immunity may be possible through selection of an appropriate adjuvant or delivery by a mucosal immune route. immune stimulating complexes (iscoms) are an antigen delivery and adjuvant system (morein et al., ) wherein many antigens can be incorporated within or on the surface of small ( - nm) cage like structures formed of cholesterol, saponin and phosphatidylcholine. antigens contained in multivalent subunit iscom vaccines are found both within the cytosol and endosomic vesicles of antigen presenting cells (villacres et al., ) . iscom vaccines have been shown to stimulate both humoral and cell mediated immune responses and are claimed to override the down-regulatory effects of passively acquired maternal antibodies (nordengrahn et al., ; hagglund et al., ) . iscoms, prepared using supernatants from log-phase cultures, were shown to contain native lkt, as the target antigen, as well as other soluble antigens of m. haemolytica. iscom vaccines were used to vaccinate groups (n = or per group) of colostrum fed dairy heifers at and weeks of age, by either the intranasal or subcutaneous routes. response to vaccination was compared to that in unvaccinated controls and a group of calves that received a commercial vaccine, presponse sq (wyeth/fort dodge) subcutaneously at and weeks. all vaccinated calves were challenged to assess recall response by subcutaneous vaccination with the commercial vaccine at weeks of age, an age where response to vaccination might normally be expected. all three vaccines were standardized by antigen capture elisa to contain concentrations of lkt equivalent to that present in the commercial vaccine. sera and nasal swabs were collected weekly from to weeks of age and antibody responses were determined by direct and indirect agglutination, for antibodies to bacterial surface antigens, and by elisa for isotypic response to lkt. antibody responses were expressed as the change in titer from that at week , the time of initial vaccination, to adjust for antibodies present due to passive transfer. subcutaneous vaccination with iscoms induced an increase in the direct agglutination titer in serum at week , one week after the second vaccination and the titer remained elevated for the duration of the study (data not shown). vaccination with the commercial s.c. vaccine had no effect on titer by any assay before weeks of age. the earlier i.m. formulation of the same vaccine had been shown previously to induce agglutinating antibodies and igm to capsular polysaccharide, but not lkt neutralizing responses, in calves vaccinated at and weeks of age (hodgins and shewen, ) . both iscom vaccines induced an increase in serum igg to lkt week following the second dose (fig. , top panel) . the rate of decline in titer, from week to , was less in calves receiving presponse compared to controls. titers in control calves rose by week , consistent with a naturally induced active immune response to commensal colonization. as hoped, intranasal vaccination induced a significant change in lkt specific iga in nasal secretions week following first vaccination at week , the earliest age at which we have succeeded in inducing active immunity to lkt to date. this may be particularly important given the correlation between nasal iga to lkt and protection, recently demonstrated in our edible vaccine trial (above). by weeks of age nasal iga titers had begun to increase in all groups (fig. , middle panel) . interestingly, subcutaneous vaccination with iscom vaccine induced an increase in lkt specific igg in nasal secretion, which could relate to spillover from systemic response, but may reflect homing of memory cells to the nasopharynx permitting enhanced response to natural exposure locally (fig. , lower panel) . thus iscom vaccines, but not the commercial vaccine, induced immune response to lkt in serum and nasal secretions following vaccination at and weeks of age. this preceded responses arising from natural exposure that were evident at weeks of age. the hope would be that this earlier response would protect should the calves receive a challenge sufficient to induce pneumonia in the - week old period, but that was not assessed in this trial. these studies demonstrate that it is possible to detect immune response to mucosal vaccination targeted at the nasopharyngeal lymphoid tissue by examining antibodies in nasal secretions. we have also collected saliva and feces from many of these animals and will analyze those to determine their utility as alternative indicators of mucosal response. we could demonstrate enhanced response, compared to controls, in sera from some calves vaccinated mucosally using an edible vaccine, but only post-challenge. this would be important as a protective response to natural exposure, but is not useful for demonstration of immunogenicity in response to vaccination per se. additionally, though it may be adequate to stave off pneumonia during the course of natural infections, the recall response was not adequate to provide protection against an experimental challenge sufficient to induce pneumonia using a single bolus exposure. therefore, it may be necessary to further refine challenge protocol to enable differentiation, for example by adjusting challenge dose to animal weight, prechallenge serum titer or other criteria. additionally it is important to continue to examine immune response to mucosal antigen exposure, to improve our understanding of factors that lead to its stimulation and those parameters that reflect stimulation. this will assist in finding new and innovative means to enhance responsiveness, including adjuvants and delivery systems, and improved methods for detection of immunogenicity. author p. shewen is co-author on patents that protect the commercial vaccine presponse and receives loyalty revenue from wyet/fort dodge related to this vaccine. she currently holds a contract with dow agrosciences that funds an unrelated project. with these exceptions, she and her co-authors have no financial or personal relationship with other people or organizations that could inappropriately influence or bias the paper entitled ''challenges in mucosal vaccination of cattle''. isolation unit for excellent care of calves. the natural sciences and engineering research council of canada; the ontario ministry of agriculture, food and rural affairs; the ontario cattlemen's association and the agricultural adaptation council, and dow agrosciences funded various portions of this research. plant-derived vaccines and antibodies: potential and limitations oral vaccination of animals with antigens encapsulated in alginate microspheres mucosal vaccines: an overview mucosal b cells: phenotypic characteristics, transcriptional regulation, and homing properties immunity in the female bovine reproductive tract based on response to campylobacter fetus m cells at locations outside the gut posttranslational modification of therapeutic proteins in plants bovine respiratory syncytial virus iscoms-protection in the presence of maternal antibodies mucosal veterinary vaccines: comparative vaccinology serologic responses of young colostrum fed dairy calves to antigens of pasteurella haemolytica a mucosal immunization and adjuvants: a brief overview of recent advances and challenges role of mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis towards development of an edible vaccine against bovine pneumonic pasteurellosis using transgenic white clover expressing a mannheimia haemolytica a leukotoxin fusion protein gene gun-mediated dna immunization primes development of mucosal immunity against bovine herpesvirus- in cattle suppository-mediated dna immunization induces mucosal immunity against bovine herpesvirus- in cattle advances in mucosal vaccination immune response versus mucosal tolerance to mucosally administered antigens a novel structure for antigenic presentation of membrane protein from enveloped viruses equine herpesvirus type (ehv- ) as a predisposing factor for rhodococcus equi pneumonia in foals: prevention of the bifactorial disease with ehv- immunostimulating complexes maternally and naturally acquired antibodies to mannheimia haemolytica and pasteurella multocida in beef calves plant-made vaccines: biotechnology and immunology in animal health frequencies of injection-site lesions in muscles from rounds of dairy and beef cow carcasses vaccination of calves with leukotoxic culture supernatant from pasteurella haemolytica characterization of ovine nasal associated lymphoid tissue and identification of m cells in the overlying follicle-associated epithelium epidemiological study of enzootic pneumonia in dairy calves in saskatchewan internalization of iscom-borne antigens and presentation under mhc class i or class ii restriction bronchoalveolar washing cells and immunoglobulins of clinically normal calves lymphocyte homing: chemokines and adhesion molecules in t cell and iga plasma cell localization in the mucosal immune system key: cord- -pceenwb authors: falo, louis d. title: advances in skin science enable the development of a covid- vaccine date: - - journal: j am acad dermatol doi: . /j.jaad. . . sha: doc_id: cord_uid: pceenwb nan expressing adenovectors and adjuvant in the same mnas resulting in a vaccine that induced both antibody responses and enhanced cytotoxic cellular immunity that is likely important for "universal" vaccines and cancer immunotherapies. taken together, these and studies by others demonstrate the potential for the development of cutaneous immune engineering strategies to control systemic immune responses including the potential for developing novel vaccine strategies and immunotherapies, and even negative immunization strategies to treat systemic allergy and autoimmune diseases. advances in skin biology are making important contributions to the fight against the covid- pandemic demonstrating once again that dermatology is more than skin deep. the immunological anatomy of the skin antigen-presenting cells in the skin microneedles for drug and vaccine delivery microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development improved cutaneous genetic immunization by microneedle array delivery of an adjuvanted adenovirus vaccine key: cord- - g gp authors: poland, gregory a. title: tortoises, hares, and vaccines: a cautionary note for sars-cov- vaccine development date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: g gp nan tortoises, hares, and vaccines: a cautionary note for sars-cov- vaccine development in the aesop fable, ''the hare and the tortoise," the tortoise unexpectedly beats the hare in a race. the moral of the story is that the race is not always to the swift. this same moral also appears in the wisdom literature of the old testament book of ecclesiastes : , which more generally concerns the limitations of human wisdom-which is nearly always disregarded by humans themselves. in late december , the world was notified of an unusual cluster of severe respiratory disease occurring in wuhan, china. very soon thereafter, the causative agent was identified as the now-named sars-cov- virus-a betacoronavirus that had crossed the species barrier to infect humans. in the last few months, this virus has circulated worldwide and caused over million identified cases and , deaths as of this writing, and those numbers are certainly an under-estimate. almost immediately, the call went forth that a vaccine was needed. i agree and so does every serious scientist knowledgeable about the issue. there is no question that a vaccine against this virus, and other as-yet-to-come coronaviruses, is imperative to protect human health and to quickly respond to future viral introductions, epidemics, and pandemics. but, alarmingly, scientists began to speak of the promise of a vaccine being available in ''months"-promises that began to circulate in the media almost as quickly as the virus. vaccine development has a long and documented history. in the us, as is true to greater and lesser degrees around the world, vaccines go through both scientific and regulatory pathways. these pathways, informed by science and the past history of successes and failures, are designed to maximize the chances of efficacy and safety. further, these pathways are designed to be deliberate, reflective, evidence-based, and peer-reviewed . . . in short, to maximize the chance that the data generated are robust, interpreted correctly, and lead to safe and effective vaccines when used in the population-at-large. perhaps the fastest a vaccine has been licensed in response to a new human pathogen of public health concern is the example of ebola virus. from the first cases to licensure in the us took some years, although work on a vaccine had started in the s. even the pandemic influenza a/h n vaccine in took over months to produce and distribute, and this was for a vaccine we had decades of experience in producing and testing with annual strain changes. even then, many concerns were raised by the public of an ''experimental and untested" vaccine being foisted on the public. it turns out that perception is important (at least in terms of vaccine uptake), and that human decision-making under conditions of uncertainty is both biased and flawed, particularly under distorting influences such as economic incentives or perceived losses, peer pressure, and wide-spread fear. what does history teach us in regard to vaccine development? first, expect the unexpected. research is non-linear and often presents problems and barriers that are unanticipated. from these we learn (supposedly) and build on both successes and failures for the future. in vaccine development, we need only look back a handful of decades to recall failed vaccines against measles and rsv that used inactivated virus approaches. these vaccines led to antibody enhanced disease (aed) in people who were immunized and later infected with wild virus [ , ] . more recently, despite careful studies through years of preclinical and phase - clinical trials, aed was detected in post-licensure studies of dengue vaccine [ ] . second, rna viruses accumulate mutations that can sometimes circumvent vaccine-induced immunity. for example, influenza viruses mutate so fast that nearly annual strain changes are necessary for influenza vaccines. this occurs despite vaccines containing both h and n protein antigens, rather than depending upon single protein/antigen preparations. third, issues of broad immunogenicity exist. given that this is an rna virus, i believe it is critical that more than one viral antigen be included in the vaccine. while the significance remains unknown to date, researchers have already identified at least one mutation in the receptor binding domain of the s gene [ ] . further mutations could conceivably lead to issues of original antigenic sin with resultant disease enhancement after exposure or to vaccines that simply are not effective into the future. ''s only" vaccines risk these issues, whereas vaccines that include other relevant sars-cov- viral antigens considerably reduce this risk. fourth, decisions must be made regarding how much safety data is needed before initiating first-in-man clinical trials. of concern is the push for starting clinical trials in the absence of completed animal studies. novel phase i vaccines should not be administered to humans prior to completion and evaluation of appropriate animal studies for safety, toxicity, and immunogenicity. rushing through animal studies, using irrelevant or single animal species models, and avoiding non-human primate studies is simply transferring risk from animals to humans in an attempt to rush vaccine development. this may be even more important in studies of novel vaccine antigens, vaccine approaches, and concomitant adjuvants or immunostimulants. fifth, some are beginning to call for human challenge models as a method for quickly moving through vaccine development. this would require extensive discussion and ethical consultation to consider factors such as the lack of known effective treatment, the balance between https://doi.org/ . /j.vaccine. . . - x/Ó elsevier ltd. all rights reserved. vaccine ( ) - rushed studies to get quickly to licensure presuppose evidences of safety, efficacy, and benefit. these should not be supposed; rather, the burden of proof lies upon the vaccine developer to demonstrate that those presuppositions are justified. for example, what level of risk are we willing to tolerate to immunize against an infection that may disappear in the next year or two? or that could diminish in severity in the short-to mid-term? or to administer to young children whose risk of both serious illness or death is quantifiably very, very low? this begs the question of how to license a vaccine in the midst of an ongoing pandemic like sars-cov- . might reasonable ''accommodations" be made for such a scenario? several seem worth immediate discussion: -could a vaccine be provided through an eua mechanism for mentally competent adults who meet certain risk guidelines, and in the context of study enrollment and data collection, and enhanced informed consent? -could a vaccine be provided through a revised definition of a compassionate use mechanism in the highest risk subjects after signing waivers of responsibility and enhanced informed-consent procedures? who should be included-perhaps healthcare providers and first responders who share the highest risk of infection as a starting point? -what, if any, animal models might be developed that allow the ''animal rule" to be utilized in an effort to accelerate research and licensure? -if phase i and ii trials are conducted earlier than normal procedure, could a phased initiation of studies from highest risk to lowest risk subjects be utilized? -might one conceive of differential regulatory pathways for vaccine candidates using well-understood antigens, vaccine methodology, adjuvants, manufacture, and routes of administration (tbd) versus those using novel delivery technology and novel antigens or adjuvants? -as mentioned above, human challenge studies have been advanced as a method to rapidly determine efficacy in discussions i have had with other vaccinologists. could this be a viable strategy in accelerating licensure? to date, no ethical framework has been advanced to support such an idea. -what will be the endpoint for determining vaccine efficacyprevention of infection? prevention of severe disease? prevention of viral shedding? other? -will different vaccines and different regulatory pathways be feasible for different members of the population with differing risk:benefit ratios? for example, administering a vaccine to a healthy and robust -year-old with no underlying comorbidities should require an exceptionally high safety and efficacy threshold. might that safety profile be somewhat different (to be defined) in an exceptionally high risk -yearold with multiple co-morbidities? what about for pregnant women or younger but immunocompromised persons? these and other such questions are raised to consider more carefully and thoughtfully how best to approach the development and distribution of a covid- vaccine. under current knowledge and disease severity, a vaccine is urgently needed. but such vaccine development must begin and progress cognizant of the many lessons learned from the past. in addition to safety issues, i raise concern over ''s-only" vaccine approaches for the mid-to long-term control of this rna virus. we need a vaccine-and we need it as quickly as one can be developed-that demonstrates safety and efficacy in adequately powered studies. such an extraordinary event as covid- is an argument for carefully developing a new playbook for how to develop novel vaccines against emerging pathogens in the context of epidemics and pandemics. modern science has the ability to rapidly develop vaccine candidates, but wisdom lies in attending to the many lessons of the past . . . including that of the tortoise and the hare. atypical measles and enhanced respiratory syncytial virus disease (erd) made simple brief history and characterization of enhanced respiratory syncytial virus disease dengvaxia sensitizes seronegatives to vaccine enhanced disease regardless of age analysis of the mutation dynamics of sars-cov- reveals the spread history and emergence of rbd mutant with lower ace binding affinity vaccine research group, c guggenheim building key: cord- -boxxlopy authors: devi, arpita; chaitanya, nyshadham s. n. title: in silico designing of multi-epitope vaccine construct against human coronavirus infections date: - - journal: journal of biomolecular structure & dynamics doi: . / . . sha: doc_id: cord_uid: boxxlopy single stranded rna viruses were known to cause variety of diseases since many years and are gaining much importance due to pandemic after the identification of a novel corona virus (severe acute respiratory syndrome-coronavirus (sars-cov- )). seven coronaviruses (covs) are known to infect humans and they are oc cov, nl cov, hku cov, middle east respiratory syndrome, sars cov, and sars cov- . virus replication weakens the immune system of host thereby altering t-cell count and much of interferon response. although no vaccine or therapeutic treatment has been approved till now for cov infection, trials of vaccine against sars cov- are in progress. one of the epitopes used for vaccine production is of the spike protein on the surface of virus. the work focuses on designing of multi-epitope vaccine construct for treatment of seven human cov infections using the epitopes present on the spike protein of human covs. to address this, immuno-informatics techniques have been employed to design multi-epitope vaccine construct. b- and t-cell epitopes of the spike proteins have been predicted and designed into a multi-epitope vaccine construct. the tertiary structure of the vaccine construct along with the adjuvant has been modelled and the physiochemical properties have been predicted. the multi-epitope vaccine construct has antigenic and non-allergenic property. after validation, refinement and disulphide engineering of the vaccine construct, molecular docking with toll-like receptors (tlrs) have been performed. molecular dynamics simulation in aqueous environment predicted that the vaccine-tlrs complexes were stable. the vaccine construct is predicted to be able to trigger primary immune response in silico. communicated by ramaswamy h. sarma coronavirus (cov) belong to large and enveloped subfamily viruses contain sense single strand rna that are categorized into four genera namely alpha, beta, gamma, and delta, among them alpha-and beta-covs known to infect humans (yi et al., ) . seven different types of corona virus that infect humans of alpha covs includes e and nl ; and beta-covs includes oc , hku , mers-cov, and severe acute respiratory syndrome (sars-cov) and sars-cov- (waleed, ) . s, m, n, structural proteins are encoded by genome of cov and are common for all types of virus whereas e proteins are encoded in human coronavirus e (hcov- e) and hcov-nl genome, while he protein is encoded by hcov-oc and hcov-hku genome apart from structural proteins. the spike (s) protein mediates receptor-binding and fusion with the host cell membrane. the membrane (m) protein plays an important role in viral assembly. the nucleocapsid protein (n) regulates viral rna synthesis, and may interact with m protein during virus budding. the hemagglutinin-esterase (he) glycoprotein found only in the beta-covs and hemagglutinin moiety binds to neuraminic acid on the host cell surface thereby permitting initial adsorption of virus to the membrane. hcov- e genome consists of , bases that infects humans and bats (lim et al., ) . along with hcov-oc , it causes common cold (gaunt et al., ) . hcov- e is associated with a range of respiratory symptoms, ranging from the common cold to pneumonia, hcov- e encodes four structural proteins, spike (s), envelope i, membrane (m), and nucleocapsid (n). hcov-nl , a member of coronaviridae is large-envelope virus of positive-sense single-stranded rna genome of $ , bases in length that contains a cap structure at its end and a poly (a) tail at its end. the genome consists of and noncoding regions (ncrs), and the genes encodes for four structural proteins namely spike (s), envelope i, membrane (m), and nucleocapsid (n) (kim et al., ) . the hcov-oc genome encompasses , nucleotides of which two-thirds of the genome consists of two large replicase open reading frames (orfs) and end includes several structural and accessory protein genes: an envelope-associated he glycoprotein gene, a spike (s) glycoprotein gene; an envelope i protein gene; a matrix (m) glycoprotein gene; and a nucleocapsid (n) phosphoprotein gene (vijgen et al., ) . the genome of cov-hku is , nucleotides; polyadenylated rna with gc content is %, which is lowest among all known coronaviruses. the genome organization is with the characteristic gene order -replicase, spike (s), envelope i, and membrane (m), nucleocapsid (n)- . both and ends contain short untranslated regions (utrs). the end of the genome consists of a putative leader sequence (woo et al., ) . the first imported mers-cov strain was named chinagd and , nucleotide long, including the and utrs (lu, ) . the genome structure is a single-stranded rna (ssrna) encoding replicase polyproteins (orfs ab and a), structural proteins (e, n, and m), a surface (spike) glycoprotein (s), and sulphidral proteins (orfs , a, b, and ) (mackay & arden, ) . mers-cov has been assumed to be transmitted from bats and spread to humans through intermediate hosts (coleman & frieman, ) . sars genome contains , bases with cap structure and polyadenylation tract. this complex then associates with the m protein in the membranes of the er, and virus particles form as the nucleocapsid complex buds into the lumen of the er (marra et al., ) . the single-stranded rna genome of the sars cov- was , nucleotides in size, contains two flanking utrs and a single long orf encoding a polyprotein, it is arranged in order of -replicase (orf /ab)-structural proteins (spike (s)-envelope i-membrane (m)-nucleocapsid (n))- and lacks the he gene (chan et al., ) . no drugs or vaccines are available to treat any of the hcov infections. therapies to treat covid- caused by sars cov- include antiviral drugs (lu, ) , which includes remdesivir (warren et al., ) , baricitinb, interferon-a, lopinavir/ritonavir, and ribavirin (zumla et al., ) ; immunosuppressant, steroids, and antibodies from plasma of recovered patients (kreil & farcet, ) . according to who, hydroxychloroquine is a promising candidate to reduce the pathogenicity of sars-cov- until date. studies revealed that several viral structural proteins such as small envelope i, membrane (m), nucleocapsid (n), and spike (s) play important roles in sars infection (simmons et al., ) . the -kda spike protein plays a central role in the host cell attachment and entry processes and comprised of s and s subunits. the s subunit contains a signal peptide, n-terminal domain, and receptor-binding domain, while the s subunit contains a fusion peptide, heptad repeat (hr) and , transmembrane domain i, and cytoplasmic domain (cp) (chan et al., ) . coronavirus s proteins contain short amino-terminal hydrophobic signal sequence motifs (von heijne, ) . although some coronavirus spike proteins cleaved between the s and s regions as part of activation process, unlike sars-cov spike not cleaved before, internalized inside the host (xiao et al., ) . the more variable amino-terminal region of the spike protein (s ) seems to contain the receptor-binding activity (wong et al., ) . the more conserved s region contains the transmembrane anchor, palmitic acid acylation site (thorp et al., ) that is important for membrane fusion (mcbride & machamer, ) , and the coiled-coil fusion motor domain (bosch et al., ) . high-resolution x-ray crystallography structures obtained for coronavirus spike protein reveals sars-cov in conjunction with angiotensin-i-converting enzyme (ace ) (pdb id: d g, d h, d i, acc, acd) a cellular receptor for sars-cov (li et al., ) and hcov-nl (hofmann et al., ) (pdb id: kbh). image analysis of cryoelectron micrographs of sars-cov (beniac et al., ) and other coronaviruses (neuman et al., ) confirms that spikes exist as a homotrimer in the native perfusion state on virion-and protein-mediated fusion to its receptor-binding triggers conformational changes including disulphide reshuffling (lavillette et al., ) . spike protein is incorporated into virion through interactions with the membrane protein m (godeke et al., ) . there are different kinds of receptors for different virus for effective binding to its host. e virus enters the host cell by binding to the aminopeptidase n receptor (fehr & perlman, ) . nl virus enters its host cell by the ace receptor (brielle et al., ) . oc virus enters its host cell by binding to the n-acetyl- -o-acetylneuraminic acid receptor (li, ) . hku virus enters its host cell by binding to the n-acetyl- -o-acetylneuraminic acid receptor (lim et al., ) . mers-cov virus enters host cell by binding to the dpp receptor (fehr & perlman, ) . sars cov virus infects the epithelial cells within the lungs and enters the host cell by binding to ace (ge et al., ) . sars cov- virus enters the human cells by binding to ace receptor (letko et al., ) . three hcovs, mers cov, sars cov, and sars cov- causes severe symptoms in humans. middle east respiratory syndrome (mers) first reported in saudi arabia in september and sars was identified in southern china in . recently, sars cov- became a pandemic spreading to countries and territories. many researchers are working towards development of vaccine for sars cov- . thus, our work focuses on designing of multi-epitope vaccine against all the hcovs (known so far) to avoid any repeated outbreak in near future. s-protein of coronaviruses is the main agent for attachment and entry of the viruses into the human cell. the epitopes from the s-protein of seven hcovs used to design a multi-epitope vaccine construct. sequences of full-length proteins of hcov-oc , ku , e, nlc , mers, sars, and novel sars were retrieved from uniprot (http://uniprot.org) and ncbi (http://ncbi.nlm.nih.gov) databases. sequence of oc , ku , e, nlc , and sars coronavirus was deposited with uniprot id p , q u , p , q q s , and p , respectively. sequence of mers cov and novel sars cov- was deposited in ncbi database with accession nos. akj and qih , respectively. clustal omega was used for multiple sequence analysis and phylogenetic analysis (madeira et al., ) . full length sequences of hcov-oc , ku , e, nlc , mers, sars, and novel sars were provided in fasta format to perform the analysis. clustal omega uses seeded guide trees and hidden markov model profile-profile technique to align two or more sequences. the phylogenetic analysis was performed with a neighbour-joining tree. percent identity matrix was generated after phylogenetic analysis. multiple sequence alignment was done to check the sequence similarity of the s-protein of the coronaviruses. . . b-cell, ctl, and htl epitope prediction b-cell epitopes were predicted using the modelled tertiary structures of s-protein of hcovs in ellipro webserver (ponomarenko et al., ) . each predicted epitope is given a score known as protrusion index value by the server. maximum score is and higher the score better is the predicted epitope. for each protein, a number of epitope has been predicted. for cytotoxic t-cell lymphocyte (ctl) epitope prediction netctl . webserver was used (larsen et al., ) . this server predicts the mhc-i-binding epitopes by using neural network algorithm. the server gives a score based on predicted mhc class i-binding affinity, proteosomal c terminal cleavage and tap transport efficiency. higher score indicates high specificity of the epitopes towards mhc-i. the epitope with score > was further analysed. helper t-cell lymphocyte (htl) epitopes were predicted in iedb webserver (wang et al., ) . the server predicted the mhc-ii-binding epitopes using iedb recommended consensus approach. prediction of mhc-iibinding epitopes was done using hla-drb  : , hla-dpa  /dpb  : , hla-dqa  : /dqb  : alleles. the server gives a percentile rank to the predicted epitopes. lower rank indicates good binders. hence, the epitopes with percentile rank of < were further analysed for their ability to induce ifn-c production by using the ifnepitope server (http://crdd.osdd.net/raghava/ifnepitope/). the b-cell, ctl and ifn-c producing htl epitopes were further screened for antigenicity, allergenicity, and toxicity properties. the antigenicity of the epitopes and vaccine constructs was predicted using vaxigen v . (doytchinova & flower, ) webserver. vaxigen v . uses alignment-free approach for antigen prediction. the allergenicity of the epitopes and vaccine construct was predicted using allertop v . (dimitrov et al., ) webserver. allertop uses amino acid e-descriptors, auto-and cross-covariance transformation, and several machine learning methods for classification of allergens. the toxicity of the epitopes was predicted using toxinpred webserver (gupta et al., ) . toxinpred can predict toxicity of peptides with < residues. hence, the epitopes with more than residues were chopped into fragments of residues and checked for toxicity. the high scoring b-cell and ctl epitopes and low scoring htl epitopes with non-antigenic, non-allergenic and nontoxic properties were linked by appropriate linkers to design the multi-epitope construct. also, s ribosomal protein l / l (ncbi accession no. p whe and uniprot id p a k ) was used as an adjuvant and was attached to the n-terminal through eaaak linker (kar et al., ; naz et al., ; samad et al., ) . the adjuvant is believed to improve the antigenicity of the vaccine. the b-cell and htl epitopes were linked by gpgpg linkers and ctl epitopes were linked by aay linkers (kar et al., ; samad et al., ) . a xhis tag was incorporated at the c-terminal end to aid in protein purification and identification. secondary structure of the vaccine construct was predicted in raptorx webserver (wang et al., ) . the secondary structures are predicted in the form of helix, b-sheet and loop. tertiary structures were predicted in i-tasser webserver (roy et al., ) . i-tasser server stands for iterative threading assembly refinement server and is an integrated platform for automated protein structure and function prediction. i-tasser first generates fragments of three-dimensional ( d) atomic models from the primary sequence by multiple threading alignments. then the fragments are reassembled into full-length models by replica-exchange monte carlo simulations. the d model obtained for the vaccine construct was refined in the drefine server (http://sysbio.rnet.missouri.edu/ drefine/). the server combines iterative optimization of hydrogen bonding network and atomic level energy minimization using composite physics and knowledge-based force fields for tertiary structure refinement (bhattacharya et al., ) . disulphide engineering was done to rationally incorporate of disulphide bonds in the modelled structure of the vaccine construct. disulphide engineering was done using disulphide by design v . webserver (craig & dombkowski, ) . the server rapidly assessed for proximity and geometry of the amino acid residues consistent with disulphide formation. tertiary structure model validation detects potential errors in predicted d models. the structure validation was performed in saves v . server (https://servicesn.mbi.ucla.edu/ saves/). a ramachandran plot was obtained in this server to visualize energetically allowed and disallowed dihedral angles psi (w) and phi (/) of an amino acid. the server also gave errat score which is used to analyse non-bonded atom-atom interactions compared to reliable high-resolution crystallography structures. verify d score is also obtained from this server. this score determines the compatibility of an atomic model ( d) with its primary sequence ( d). the overall model quality was assessed in prosa webserver (https://prosa.services.came.sbg.ac.at/prosa.php). protparam webserver (https://web.expasy.org/protparam/) was used to predict various physiochemical properties of the constructs such as amino acid composition, theoretical isoelectric point (pi), instability index, in vitro and in vivo halflife, aliphatic index, and molecular weight. grand average of hydropathicity (gravy) was calculated in gravy calculator webserver (http://www.gravy-calculator.de/). toll-like receptors (tlrs) are immune receptors whose activation leads to intracellular signalling pathway. tlr and tlr induce the production of nf-jb. tlr induces the activation of irf and nf-jb. tlr activation leads to an intracellular signalling pathway nf-jb and inflammatory cytokine production. tlr induces the activation of activation leads to an intracellular signalling pathway nf-jb and il . all the tlrs are located at the cell membrane thus; they are the first molecules to come in contact with pathogens. adjuvant, s ribosomal protein attached to the n-terminal of the vaccine construct has the ability to activate tlr . however, other tlrs may also come in contact with the adjuvant. thus, molecular docking of the multi-epitope vaccine construct with tlr (pdb id: nig), tlr (pdb id: a z), tlr (pdb id: g a), tlr (pdb id: j a), and tlr (pdb id: r a) receptor was performed using the cluspro . (kozakov et al., ) and hdock (yan et al., ) webservers in order to evaluate the interaction between ligand and receptor and consequently the activation of an immune response. before docking, the x-ray crystallized structures of tlrs were prepared by removing solvent molecules and other ligands followed by addition of hydrogen atoms and energy minimization in swiss pdbviewer deep view software package. binding pockets of tlrs were searched using castp . webserver (http://sts.bioe.uic.edu/castp/calculation.html). the vaccine construct and vaccine construct-tlr complexes were subjected to ns molecular dynamics simulation using gromacs . . software package (van der spoel et al., ) . for the simulation, charmm forcefield and spc/e water model was used. the simulation system was neutralized by adding the suitable number of na þ /clions. the solvated system was energy minimized in steps using steepest descent method iterations. after setting the temperature at k and pressure at bar, production simulation was run for ns to evaluate dynamics of vaccine construct and vaccine construct-tlr complexes. the rmsd, rmsf, and radius of gyration profiles of the vaccine construct and vaccine construct-tlr complexes were generated. codon biasness occurs between prokaryotic and eukaryotic genes. codon optimization therefore becomes necessary for protein expression in a prokaryotic host. codon optimization of the designed multi-epitope vaccine construct was done in java codon adaptation tool (jcat) server (http://www. prodoric.de/jcat) for gene expression in the e. coli (strain k ) host. rho-independent transcription termination, prokaryote ribosome-binding site, and restriction enzymes cleavage sites were avoided during codon optimization. codon adaptation index (cai) and percentage gc content was also received as output along with the optimized nucleotide sequence. the optimized nucleotide sequence of the final vaccine construct was cloned into the e. coli pet- a (þ) vector using snapgene tool and ndei and bglii restriction sites were introduced to the n and c-terminals of the sequence, respectively. to predict the probable immune response of the designed multi-epitope vaccine construct in human immune system, in silico immune simulations were conducted using the c-immsim server (http:// . . . /c-immsim/index.php) (rapin et al., ) . c-immsim is a novel in silico approach for the study of the mammalian immune system the tool is a combination of a mesoscopic scale simulator of the immune system with machine learning techniques for molecular-level predictions of major histocompatibility complex (mhc)-peptide-binding interactions, linear b-cell epitope discovery, and protein-protein potential estimation. all simulation parameters were kept as default and a three dose of injection ( antigens) at weeks apart was given. the response after the injections was analysed. s-proteins of seven hcovs causing mild and severe disorders in human have been studied. primary structure comparison of the s-proteins revealed a sequence similarity towards the c-terminal of the proteins (supplementary figure ) . based on similarity, it was found that s-protein of sars cov- and sars cov share the highest identity ( . %) among all the hcovs. s-protein of oc cov and ku cov share . % identity and e cov, and nl cov share . % identity. s-protein of mers cov shares highest identity with oc cov ( . %). the percent identity matrix of the s-proteins of hcovs is shown in table . tertiary structure of full-length s-proteins of hcovs shows a similar y-shaped structure. arm a is the receptor-binding domain. arm c contains the transmembrane domain and the cytoplasmic domain. from figure , it is clearly seen that the arms a and b of s-protein of e cov and nl cov is in closed conformation. the arms a and b of s-proteins of all other hcovs are in open conformation. another difference in the tertiary structure is seen in the c arm, which is the c-terminal tail of s-proteins. the c arm of s-protein of e cov and nl cov is in an elongated form, whereas the c arm is in a loop like structure in the other hcovs. b-cell epitopes of different lengths were predicted for each s-protein of hcovs (supplementary tables - ) . ctl epitopes of residues along with their mhc-binding affinity, c-terminal cleavage affinity and transport affinity were predicted (supplementary table ). htl epitopes of residue length interacting with hla-drb  : and hla-dqa  : / dqb  : alleles were predicted (supplementary tables - ). the epitopes were subjected to allergenicity, antigenicity and toxicity prediction. the epitopes with best scores and non-allergenic, antigenic, and non-toxic properties were selected for the vaccine construct. eighteen epitopes from the s-protein of hcovs were linked by linkers to form the final vaccine construct ( table ) . the b-cell and htl epitopes were linked by gpgpg linkers whereas the ctl epitopes were linked by aay linkers. the tlr agonist, s ribosomal l /l protein was added to the amino terminus of the vaccine construct using an eaaak linker and xhis tag was added at the c-terminal to help in protein purification and identification. the final vaccine construct designed consisted of amino acid residues (figure ) . the antigenicity of the vaccine construct including the adjuvant sequence and his-tag was predicted by the vaxijen . server to be . with a bacteria model at a threshold of . . the main vaccine sequence without adjuvant and histag gave scores of . with bacteria model at a threshold of . on vaxijen . server. thus, the results indicate that the designed vaccine is antigenic in nature. the presence of adjuvant and his-tag doesn't change the antigenic property of the construct. the vaccine construct with and without the adjuvant and his-tag is predicted to be non-allergenic by allertop v. and allergenfp servers. the calculated molecular weight of the vaccine construct is . kda. theoretical pi value of the construct is . , indicating the protein is slightly acidic in nature. the estimated half-life of the vaccine construct is h mammalian reticulocytes in vitro, and > h in yeast in vivo and > h in e. coli in vivo. the instability index (ii) is computed to be . , which classify the protein as stable. the aliphatic index is calculated to be . and grand average of hydropathicity (gravy) score is predicted to be À . , indicating that protein is hydrophilic in nature. the secondary structure of the multi-epitope vaccine construct with adjuvant and his-tag is predicted to be composed of % a-helix, % b-sheet, and % coils. of the total residues % are predicted to be exposed and % residues are predicted to be buried. tertiary structure prediction by i-tasser server predicted five tertiary structure models of the peptide aptamer. ten threading templates were used for the prediction, of which jx a, rqua, nzka, and ijoj were the best templates. all the templates showed good alignment as per their z-score values, ranging from . to . . the calculated c-score of the five predicted models ranges from À . to À . . the c-score range is generally between À and and higher value indicates higher confidence (lee et al., ) . thus, the model with the highest c-score was selected for further studies. this model had a tm-score of . ± . with an estimated rmsd of . ± . Å. the tmscore is a measure of structural similarity between two structures (shaik, ) . a tm-score of > . indicates a model of correct topology and a tm score < . means random similarity (shaik, ) . these cut-off values are independent of the length of protein. the tertiary structure of the vaccine construct was refined on drefine server and it yielded five models. model was found to be the best model based on model quality scores for all refined models (figure (a) ). the model quality scores included high accuracy global distance test (gdt-ha) score of . , global distance test total score of . , rmsd score of . Å, molprobity score of . , rwplus score of À , . , and drefine score of , . . this model was chosen as the final vaccine construct model for further analysis. disulphide engineering was performed with selected pair of residues (pro -asn , gly -ala , gly -gly , pro -leu , glu -phe , glu -ala , gln -cys , phe -try , leu -asn , and ala -phe ). the selection was done on the basis of energy, v value, and b-factor. the residue pairs with energy of kcal/mol to . kcal/mol (mean value is . kcal/mol, and the th percentile is . kcal/mol), v value of À to À and þ to þ (mean value is À and þ ) and b-factor of - (mean b-factor for residues involved in stabilizing disulphide bonds is . ) (craig & dombkowski, ) . after refinement and disulphide engineering of the modelled structure, validation was done. the overall model quality was À as calculated by prosa. the ramachandran plot generated showed that . % of the residues are in favourable region, . % residues are in the allowed region, and . % residues are in outline region. the quality and potential errors in the tertiary structure were verified by errat. the chosen model after refinement had an overall quality factor of . % with errat. verify d also passed the modelled structure as > % of the residues have averaged d- d score ! . . verify d determines the compatibility of an atomic model ( d) with its own amino acid sequence ( d). binding pocket prediction of the studied tlrs revealed one pocket. however, the solvent accessible surface area and volume of the binding pocket of all the tlrs are not similar (table ) . molecular docking was performed in hdock server and cluspro . server. hdock server predicted complexes in each case from which the lowest scoring complex was studied. cluspro . predicted complexes from which the lowest energy complex was studied. from figure , it can be seen that the vaccine construct has almost similar binding pattern to tlr , tlr , and tlr . the adjuvant containing arm of the vaccine could interact with the predicted binding pocket of the tlrs. based on binding energy, tlr -vaccine construct complex is predicted to be the best complex in both the servers (table ) . thus, although the adjuvant used is a known tlr agonist, the vaccine construct may have affinity towards tlr and tlr as well. molecular dynamics simulation of the vaccine construct revealed its stability throughout the simulation. the average rmsd of the vaccine construct alone was . Å. upon binding to tlrs, the average rmsd of the vaccine construct increased. the vaccine construct-tlr complexes were found to be stable with no major fluctuation ( figure (a) ). the vaccine construct has shown a fluctuation at ns but was stable after that. one major fluctuation in the vaccine construct-tlr complex was seen at ns. the complex was found to be stable after that. the average rmsd of this complex was . Å which is higher than all other complexes. the rmsf of vaccine construct showed that the n-terminal residues and the residues between and positions have highest movement ( figure (b) ). the n-terminal of the vaccine construct (residue - ) consists of the adjuvant and is involved in interaction with tlrs. however, when the vaccine construct is bound to the tlrs, the rmsf of the highest fluctuating residues of the construct has decreased. this can be due to interaction with tlrs. radius of gyration of vaccine construct after ns simulation was calculated to be . nm. however, the radius of gyration of vaccine-construct-tlr complexes ranged between . and . nm. the radius of gyration profile against time (in ns) reveals that the vaccine construct alone and vaccine construct-tlr complexes were in a compact state till the end of molecular dynamics run (figure (c) ). codon optimization of the vaccine construct in e. coli (strain k ) was done for expression of protein. the optimized codon sequence was nucleotides long. the cai of the optimized nucleotide sequence was . which means that the nucleotide sequence contains the most frequently occurring codons (rapin et al., ) . the average gc content of the optimized sequence is . %. higher the gc content, better is the expression of a protein in prokaryotes (zhou et al., ; du, ) . the optimized nucleotide sequence was inserted into the pet a (þ) vector using snapgene software ( figure ). immune simulation by c-immsim server showed the immune responses of three injections. the antigen is calculated to be present for about days after each dose. after first dose, very low level of igm is seen to be present. but after second and third dose, the level of igm and igg subsequently increased. after third dose, the total level of igm and igg antibodies is found to be higher than the level of antigen. similarly, level of memory b-cell is seen to be increasing after each dose. the level of memory t-cells also increased after second dose. however, after third dose, there was no further increase in the level of memory t-cells. the level of ifn-c was found to be increasing at the same level after each dose. the level of ifn-c dropped to basal level at the end of fourth week and subsequent injection of the antigen again triggered the expression of ifn-c. another cytokine il- was found to reach its maximum expression after the second dose. the level of igg, igm, and cytokines dropped gradually after weeks but the level of memory b-cells and memory t-cells were found to be constant. thus, the vaccine construct is predicted to trigger mostly igg, igm, b-cell, t-cell, and cytokines (ifn-c and il ) (figure ). three hcov s are known to cause severe respiratory symptoms. mers cov outbreak was seen in , sars cov other hcovs, e, nlc , oc , and ku causes mild respiratory symptoms but e and ku is also known to cause pneumonia. until the outbreak of sars cov- , there is no approved treatment for any of the hcov infection. after the outbreak of sars cov- , researchers are trying to develop vaccine against sars cov- . b-cell, ctl, and htl epitopes are used as antigenic determinants for efficient vaccine production had much focused on spike (s) protein, which is gaining much attention in research. here in this paper we had focused on antigenic determinants on the sprotein of hcovs and their use as effective vaccine candidates through in silico approach. sequence comparison of s- proteins of human corona viruses shows similarity towards the c-terminal end. however, tertiary structure shows a similar y-shaped conformation of the s-proteins. to get insights and details about various antigenic determinants of s-proteins of virus different lengths of b-cell epitopes were predicted using ellipro server. ctl epitopes were predicted using netctl . server and htl epitopes were predicted using iedb server. the htl epitopes were checked for their ability to produce ifn-c using ifn epitope server. htl epitopes predicted for hcovs e and ku didn't have the ability to produce ifn-c. all the predicted epitopes were further checked for allergenic, immunogenic, and toxic properties. the high-scoring epitopes (as per the servers) with antigenic, non-allergenic, and non-toxic properties were selected for vaccine construct designing. multi-epitope vaccine construct was designed by linking the selected b-cell and htl epitopes with gpgpg linkage. the ctl epitopes were linked by aay linkers. s ribosomal protein, which is a tlr agonist is used as an adjuvant and linked to the n-terminus with eaaak linkage .also, for ease of protein purification,  his tag is added at c-terminus, thus yielding a final construct of amino acids. immunogenic properties such as antigenic and allergic predictions performed using vaxijen v . and allertop v. server indicates designed vaccine construct is antigenic and non-allergic in nature. the molecular weight of the designed multi-epitope vaccine construct is found to be . kda with pi of . and a half-life of h in mammalian reticulocytes. the vaccine construct is also predicted to be stable and hydrophilic in nature. secondary structure prediction of multi-epitope vaccine construct as found to have % a-helix, % b-sheet, and % coils. of the total residues, % are predicted to be exposed and % residues are predicted to be buried. the tertiary structure of the vaccine construct was refined on drefine server. the refined model gdt-ha score of . , global distance test-total score of . , rmsd score of . Ð, molprobity score of . , rwplus score of À , . , and drefine score of , . . disulphide engineering was performed with ten selected pair of residues (pro -asn , gly -ala , gly -gly , pro -leu , glu -phe , glu -ala , gln -cys , phe -try , leu -asn , and ala -phe ). the selection was done on the basis of energy, ꭓ value, and b-factor. after refinement and disulphide engineering of the modelled structure, validation was done. the overall model quality was À as calculated by prosa. the ramachandran plot generated showed that . % of the residues are in favourable region, . % residues are in the allowed region and . % residues are in outline region. the model had an overall quality factor of . % with errat. verify d also passed the modelled structure as more than % of the residues have averaged d- d score ! . . molecular docking was performed in hdock server and cluspro . server. the vaccine construct has almost similar binding pattern to tlr , tlr , and tlr . the adjuvant containing arm of the vaccine could interact with the predicted binding pocket of the tlrs. based on binding energy, tlr vaccine construct complex is predicted to be the best complex in both the servers. the stability of the tlr-vaccine construct complexes were checked by performing a ns molecular dynamics simulation in gromacs . . software package. charmm force field and spc/e water model was chosen for molecular dynamics simulation. the average rmsd of the vaccine construct alone was . Å. upon binding to tlrs, the average rmsd of the vaccine construct increased. the vaccine construct-tlr complexes were found to be stable. tlr -vaccine construct complex showed fluctuation at ns. after ns, there was no major fluctuation in the rmsd profile of tlr -vaccine construct complex. rmsf profile of the vaccine construct showed the presence of two highly fluctuating regions. the regions are the n-terminal region and the region between and positions. the nterminal of the vaccine construct (residues - ) consists of the adjuvant and is involved in interaction with tlrs. interaction of the vaccine construct with tlrs have decreased the fluctuations of the two regions. radius of gyration of vaccine construct after ns simulation was calculated to be . nm. however, the radius of gyration of vaccine-construct-tlr complexes ranged between . and . nm. this change in radius of gyration reveals the compactness of the vaccine construct-tlr complexes. the vaccine construct must be cloned into a suitable vector for expression and purification. pet a(þ) is a suitable vector for protein expression. prokaryotic vectors often have codon biasness while expressing eukaryotic proteins. thus, codon optimization was done to avoid codon biasness. the cai of the optimized nucleotide sequence was . and average gc content of the optimized sequence is . %. these two parameters show that the vaccine construct will be highly expressed in the vector. in silico prediction of primary immune response after vaccine injection was also done in c-immsim server. three injections of antigen molecules without lps were given weeks apart and immune response was recorded for days. the antigen was seen to be present for about days after each dose. after first dose, very low level of igm was generated. but after second and third dose, the level of igm and igg subsequently increased. after third dose, the total level of igm and igg antibodies is found to be higher than the level of antigen. similarly, level of memory b-cell increased after each dose. the level of memory t-cells also increased after second dose. however, after third dose, there was no further increase in the level of memory t-cells. the level of ifn-c was found to be increasing at the same level after each dose. the level of ifn-c dropped to basal level at the end of fourth week and subsequent injection of the antigen again triggered the expression of ifn-c. another cytokine il- was found to reach its maximum expression after the second dose. the level of their cytokines such as il , il , il , il , il , ifn-a, tgf-b, and ifn-b did not undergo much change even after three doses of antigen injection. the level of igg, igm, and cytokines dropped gradually after weeks but the level of memory b-cells and memory t-cells were found to be constant. thus, the vaccine construct is predicted to trigger mostly igg, igm, b-cell, t-cell, and cytokines (ifn-c and il ). complete eradication of a disease or infection is not possible without a specific treatment. there is always a possibility that previous epidemics can re-emerge and cause severe damage to coming generations. the outbreak of mers and sars cov caused severe damage to some countries but till date no vaccine or treatment has been approved. the present outbreak of sars cov- has triggered the importance of vaccine. thus, in this work, we have employed immuneinformatics tools to design multi-epitope vaccine construct against the s-protein of seven hcovs. the s-protein is the medium by which the viral genome transfers to human cells. thus, the vaccine construct designed is expected to trigger host immune system to generate antibodies against the sprotein epitope and restrict the entry of viral genome to host cells. this multi-epitope vaccine may help in boosting the host immune system against seven hcovs. architecture of the sars coronavirus prefusion spike drefine: an interactive web server for efficient protein structure refinement the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the sars-cov- exerts a distinctive strategy for interacting with the ace human receptor genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan. emerging microbes and infections coronaviruses: important emerging human pathogens disulfide by design . : a webbased tool for disulfide engineering in proteins allertop v. -a server for in silico prediction of allergens vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines the gc content as a main factor shaping the amino acid usage during bacterial evolution process coronaviruses: an overview of their replication and pathogenesis epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method isolation and characterization of a bat sars-like coronavirus that uses the ace receptor assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein in silico approach for predicting toxicity of peptides and proteins human coronavirus nl employs the severe acute respiratory syndrome coronavirus receptor for cellular entry a candidate multi-epitope vaccine against sars-cov- complete genome sequence of human coronavirus nl cn / , first isolated in south korea the cluspro web server for protein-protein docking immunoglobulins and virus antibody titers: of past needs, current requirements, and future options large-scale validation of methods for cytotoxic tlymphocyte epitope prediction significant redox insensitivity of the functions of the sars-cov spike glycoprotein: comparison with hiv envelope relative codon adaptation index, a sensitive measure of codon usage bias functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses structure, function, and evolution of coronavirus spike proteins angiotensin-converting enzyme is a functional receptor for the sars coronavirus human coronaviruses: a review of virus-host interactions drug treatment options for the -new coronavirus ( -ncov) complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd the embl-ebi search and sequence analysis tools apis in the genome sequence of the sars-associated coronavirus palmitoylation of sars-covs protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with m protein designing multi-epitope vaccines to combat emerging coronavirus disease (covid- ) by employing immuno-informatics approach supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy ellipro: a new structure-based tool for the prediction of antibody epitopes computational immunology meets bioinformatics: the use of prediction tools for molecular binding in the simulation of the immune system i-tasser: a unified platform for automated protein structure and function prediction designing a multi-epitope vaccine against sars-cov- : an immunoinformatics approach essentials of bioinformatics, volume i: understanding bioinformatics: genes to proteins characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry palmitoylations on murine coronavirus spike proteins are essential for virion assembly and infectivity gromacs: fast, flexible, and free complete genomic sequence of human coronavirus oc : molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event analysis of the distribution of charged residues in the n-terminal region of signal sequences: implications for protein export in prokaryotic and eukaryotic cells understanding the mosaic of covid- : a review of the ongoing crisis peptide binding predictions for hla dr, dp and dq molecules protein -class secondary structure prediction using conditional neural fields therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys a -amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia the sars-cov s glycoprotein: expression and functional characterization the hdock server for integrated protein-protein docking covid- : what has been learned and to be learned about the novel coronavirus disease analysis of the relationship between genomic gc content and patterns of base usage, codon usage and amino acid usage in prokaryotes: similar gc content adopts similar compositional frequencies regardless of the phylogenetic lineages coronaviruses-drug discovery and therapeutic options a.d. acknowledges csir-srf for providing financial assistance. n.s.n.c. acknowledges icmr-srf for proving financial assistance. the authors acknowledge the anonymous reviewers for helping to improve the manuscript by providing critical suggestions. no potential conflict of interest was reported by the authors. key: cord- - q wkwrf authors: chattopadhyay, saborni; chen, jui-yi; chen, hui-wen; hu, che-ming jack title: nanoparticle vaccines adopting virus-like features for enhanced immune potentiation date: - - journal: nanotheranostics doi: . /ntno. sha: doc_id: cord_uid: q wkwrf synthetic nanoparticles play an increasingly significant role in vaccine design and development as many nanoparticle vaccines show improved safety and efficacy over conventional formulations. these nanoformulations are structurally similar to viruses, which are nanoscale pathogenic organisms that have served as a key selective pressure driving the evolution of our immune system. as a result, mechanisms behind the benefits of nanoparticle vaccines can often find analogue to the interaction dynamics between the immune system and viruses. this review covers the advances in vaccine nanotechnology with a perspective on the advantages of virus mimicry towards immune potentiation. it provides an overview to the different types of nanomaterials utilized for nanoparticle vaccine development, including functionalization strategies that bestow nanoparticles with virus-like features. as understanding of human immunity and vaccine mechanisms continue to evolve, recognizing the fundamental semblance between synthetic nanoparticles and viruses may offer an explanation for the superiority of nanoparticle vaccines over conventional vaccines and may spur new design rationales for future vaccine research. these nanoformulations are poised to provide solutions towards pressing and emerging human diseases. vaccination is a process of introducing antigenic material to activate an individual's immune system to develop adaptive immunity to a pathogen. it has proven to be the most successful and cost-effective prophylactic measure against infectious diseases. vaccines have been responsible for eradicating or effectively managing many major diseases, including smallpox, measles, mumps, rubella, diphtheria, tetanus, pertussis, polio, and yellow fever [ ] . despite the many examples of successful vaccines, many disease threats, such as human immunodeficiency virus (hiv), tuberculosis, dengue, and malaria, lack an effective prophylactic measure. thus, development of new vaccine formulations and technology remains an ongoing quest [ ] . conventionally, vaccine formulations are comprised of biological materials in the form of attenuated viruses, killed pathogens, or subunit protein antigens. each platform has its distinct advantages and shortcomings, frequently presenting a trade-off between safety and efficacy. for example, live attenuated vaccines are excellent at inducing long lasting protective immunity and strong immune response, but their "live" nature poses safety concerns, especially to individuals who may be immunocompromised. on the other hand, subunit vaccines are safer to administer, but they are less immunogenic and less effective at eliciting cellular immunity for disease protection (fig. ) . emerging technology and formulations to combine the advantages of live attenuated and subunit vaccines thus continue to be developed with the aim of maximizing vaccine safety and potency. in the last decade, advances in materials engineering have opened up new avenues for ivyspring international publisher innovative vaccine designs. in particular, synthetic nanoparticles have been widely adopted for vaccine development [ , ] . these particles, typically to nm in diameter, have shown effective immune potentiation in vivo, capable of inducing strong humoral and cellular immune responses against antigen targets. compared to live attenuated viral vaccines, synthetic nanoparticles promise better safety profiles because of their non-replicating nature. they are also readily amenable to different infectious pathogens [ ] . from a holistic view, many advantages of nanoparticle vaccines may be attributed to their intrinsic semblance to natural viruses (fig. ) . many viral features, such as nanoscale morphology, repetitive multivalent antigen display and controlled antigen/adjuvant delivery are conducive to immune processing on both physiological and cellular levels. as our immune system has evolved to effectively respond to infectious viral nanoparticles, it should not come as a surprise that nanoformulations adopting virus-like features can be more potent than conventional subunit formulations. to this date, numerous nanoparticle-based platforms have been examined for vaccine applications, and many have demonstrated encouraging efficacy against many pressing infectious threats, including malaria, influenza, ebola, and hiv [ ] [ ] [ ] [ ] . also worth noting is the rapidly expanding field of nanoparticle-based anticancer vaccines, which exploits nanotechnology for enhanced induction of antigen-specific cellular immune responses against oncologic malignancies [ ] [ ] [ ] [ ] . many design considerations in nanoparticle vaccines can be traced to the fundamental interaction dynamics between viruses and the immune system [ ] . upon close scrutiny, virus-like mimicry in terms of size, geometry, antigen display, and adjuvanticity can be commonly observed among vaccine nanoformulations. in light of the emerging landscape of nanoparticle vaccine research, this review highlights some of the underlying principles behind the advantages of nanoparticle vaccines. in the first section, commonly used biomaterials such as lipids, polymers and inorganic compounds are reviewed in the context of vaccine applications. in the sections that ensue, various nanoparticle design aspects are explored from a virus-mimetic perspective including lymphatic delivery, antigen display, and interaction with immune cells. lastly, we highlight conjugation strategies that couple subunit protein antigens with nanocarriers. this review also summarizes the various strategies for conjugating antigens/adjuvants with nanocarriers. the review aims to provide a holistic view of the recent advances for the next generation of immunomodulatory vaccines. towards mimicking viral features, preparing nanoparticulates in the range of to nm in size is the first step prior to subsequent vaccine development. it is therefore imperative to have an understanding of the various materials and their properties for nanoparticle preparations. the diverse range of nanomaterials for biomedical research encompasses a wide range of organic and inorganic matters, such as phospholipids, polymers, metal, silica, and carbon (fig. ) . these nanomaterials can be modified to provide a functionalized and stable interface for different biomedical applications. in the context of vaccination, many nanocarriers have been modified to trigger specific immune responses analogous to natural defense mechanisms against viral invasion. the following section summarizes the different synthetic nanoparticles used in vaccine applications with an overview on their functionalizability and immunological adjuvanticity. liposomes are self-assembled, spherical vesicles consisting of a phospholipid bilayer and an aqueous inner core [ , ] . they are prepared from phospholipids with fatty-acid chains of defined length and saturation. the choice of phospholipids and the addition of cholesterol influence liposomal stability and performance [ ] [ ] [ ] . the vesicles can have a unilamellar structure with one layer of phospholipids or a multi-lamellar structure with several concentric phospholipid shells. this platform is highly versatile for cargoes delivery and allows for incorporation of hydrophilic molecules in the inner core and hydrophobic molecules within the phospholipid bilayers. antigens and adjuvants may thus be incorporated into liposomes based on their lipophilicity [ , ] . the lipid bilayered structure of liposomes is structurally analogous to enveloped viruses, which are formed from budding of infected cells and are wrapped in pieces of cell's plasma membrane. the inherent semblance to viral particles may help explain the innate adjuvanticity of liposomes upon incorporation with protein and peptide antigens [ ] . numerous antigen targets, ranging from toxoid, viral antigens, and bacterial antigens, have been observed to elicit enhanced humoral responses following liposomal incorporations [ ] . mechanistic studies have shed light on the liposomes' adjuvanticity. for instance, liposomes have been shown to be capable of modulating cd + t cell mediated immune responses [ ] , reflecting facilitation of antigen processing via the classical mhc i and mhc ii pathways. liposomes have also been reported to promote the development of t cell-independent b cell immune responses [ ] and have potential to promote long-term immunity through the development of t-cell memory [ ] . in addition to their innate adjuvanticity, liposomes have been incorporated with a plethora of adjuvants ranging from small molecules [ , ] , glycolipids [ ] [ ] [ ] [ ] , oligodeoxynucleotides [ ] [ ] [ ] [ ] , to cyclic dinucleotides [ , ] . the structural fluidity of liposomes offers flexibility in platform modification towards virus mimicry. for example, the development of virosomes, which are fusion vesicles prepared from viral particles and synthetic liposomes, is an adaption that highlights the fundamental similarity between liposomes and viruses. derived from the membrane vesicles of viruses, virosomes consist of liposome-like lipid vesicles and viral envelope glycoproteins. the fusion vesicles retain some viral characteristics (i.e. epitope presentation) and have been applied for both drug delivery and vaccination purposes [ , ] . the applicability of this platform as a vaccine candidate has been demonstrated extensively using influenza virus-derived virosomes [ ] [ ] [ ] [ ] . in studies that apply virosomes for anti-influenza vaccination, derivatized influenza virosomes were shown to be non-infective as their genetic materials were removed. upon in vivo administration, these virosomes were rapidly uptaken by antigen presenting cells and in turn activated numerous other immune cells [ ] [ ] [ ] [ ] . immunization with virosomes was reported to reactivate influenza-specific memory cd + t cells that subsequently supported the proliferation of antigen-specific effector cells [ ] , leading to enhanced anti-influenza immune responses [ ] . besides influenza-based virosomes, induction of cytotoxic t lymphocyte responses has been demonstrated with a sendai virus-based carrier system loaded with ovalbumin (ova). it was demonstrated that sendai virosomes fused with ova elicited stronger ctl responses against the model antigen [ ] . liposomes may also be modified to enhance the stability of carriers in a manner analogous to how viruses employ viral matrix proteins to stabilize their lipid envelope [ ] . in a study by moon et. al., interbilayer-corsslinked multilamellar vesicles have been prepared by covalently crosslinking multiple layers of liposomes via thiol chemistry [ ] . these multi-lamellar liposomes showed added stability owing to short covalent bonds that crosslinked adjacent lipid layers within the vesicle walls. this modification served to address some of the shortcomings of liposomes, facilitating enhanced antigen encapsulation and increased particle stability. also belonging to the class of lipid-based nanoparticles are an emerging class of lipoplexes, which consist of cationic lipid derivatives for the complexation with nucleic acids [ , , ] . immunostimulatory rnas or mrnas encoding specific antigen targets have been formulated into lipoplexes to trigger immune responses. the function of these lipoplexes can be likened to the immune response induction by rna viruses [ ] . as rnas are delivered intracellularly by lipoplexes, they activate innate immune receptors, leading to an upregulation of type i interferons, which may further trigger a multitude of downstream immunological pathways [ ] . concurrently, these rnas are translated into antigens of interest, thereby promoting an antigen-specific immune response. lipoplexes carrying the mrna of target antigens have recently been shown in a phase i clinical trial to induce strong cellular responses against tumor antigens in humans [ ] . a wide variety of polymers have been applied to the development of nanoparticle vaccines. synthetic polymeric nanoparticles are typically solid particles between nm to nm. they have been an attractive platform for vaccine delivery as antigens and adjuvants can be either surface attached to or interior loaded inside these nanoparticles [ ] [ ] [ ] [ ] [ ] . in particular, controlled release of biomolecules is one of the strongest advantage of polymeric nanoparticles, the release kinetics of which can be regulated by tuning of the copolymer composition and molecular weight [ ] . typically, polymeric nanoparticles are formed via self-assembly of amphiphilic copolymers under an emulsion or nanoprecipitation process [ ] [ ] [ ] . notable polymeric nanoparticles in vaccine development are as follows. poly(lactic-co-glycolic acid)(plga) is one of the most commonly used polymers for biomedical applications [ ] . plga-based nanoparticles are known to be biodegradable, non-toxic and non-immunogenic. upon administration, the polymer is degraded into lactic acids and glycolic acids in vivo to be safely metabolised in the body. in vaccine applications, plga nanoparticles provide a robust platform for antigen functionalization and have been used to carry antigen derived from various pathogens. through surface conjugation or interior encapsulation, antigens including those derived from plasmodium vivax [ ] , hepatitis b virus (hbv) [ ] , bacillus anthracis [ ] , tetanus toxin [ ] , model antigens such as ovalbumin [ ] have been associated with plga-based delivery systems for enhanced immune potentiation. adjuvants have also been encapsulated or chemically attached to the plga polymer backbone for controlled delivery to enhance immune responses [ ] [ ] [ ] [ ] . natural polymers based on polysaccharide, such as pullulan [ ] [ ] [ ] , alginate [ ] , and chitosan [ ] [ ] [ ] [ ] [ ] [ ] , have been explored for nanoparticle vaccine preparations. in particular, chitosan-based nanoparticles have been widely studied due to their biocompatibility, biodegradability, non-toxic nature and their ability to be easily modified into desired shapes and sizes [ ] [ ] [ ] . more intriguing is the recent discovery of chitosan's immune potentiating mechanism via the dna sensing cgas-sting pathway [ ] . the sting pathway is triggered in the host cells by many viral pathogens and plays a major role in both the innate and adaptive immunity [ ] . upon interaction with dendritic cells, chitosan induces type i interferons in a cgas and sting-dependent fashion, mediating cellular maturation and the promotion of th responses. chitosan-based nanoformulations have been widely adopted for vaccine development, examples of which include vaccines against clostridium botulinum type-a neurotoxin [ ] , neospora caninum [ ] , hbv [ ] , and newcastle disease [ ] . the polysaccharide polymer has also been applied to enhance the potency of dna vaccines against viral, bacterial, and parasite infections [ , , ] . other polymeric nanoparticle platforms include poly(γ-glutamic acids)(γ-pga) nanoparticles [ , ] , polystyrene nanoparticles [ , ] , and poly-alkyl acrylate based nanoparticles [ , ] . γ-pga are comprised of amphiphilic poly(amino acid)s, which self-assemble into nano-micelles with a hydrophilic outer shell and a hydrophobic inner core. they are generally used to encapsulate hydrophobic antigens. polystyrene nanoparticles are solid particles consisting of polymerized styrene monomers that can be conjugated to a variety of antigens. poly-alkyl acrylate based nanoformulations have been prepared with poly (methylmethacrylate) (pmma), poly (ethylacrylic acid) (peaa), poly (propylacrylic acid) (ppaa) and poly (butylacrylic acid) (pbaa). studies have shown that polyacrylate-based nanoparticles show an inherent adjuvanticity with several model antigens [ , , ] . the type of polymer used in the nano-formulation strongly affects the structure, properties and applications of the particles. as gold is chemically inert, gold nanoparticles (aunps) have been studied extensively for biomedical applications. aunps can be synthesized reproducibly with a high level of precision, offering an ultrastable metallic core for further modifications. capable of being modified with a plethora of chemical functional groups such as thiols, phosphines, amines, and by extension protein antigens, aunps have been used broadly in many vaccination studies. a multitude of biomolecules, ranging from toxin, viral antigens, bacterial antigens, parasite proteins, and tumor antigens, have been coupled with gold nanoparticles to enhance immune responses. aunps vaccines have been explored in clinical trials for hepatitis b and malaria vaccinations [ , ] , and they also allow anchoring of nucleic acids for dna vaccine applications [ ] . aunps have been modified with different adjuvants as well, such as chitosan [ ] and cpg oligodeoxynucleotides [ , ] , and many alternative surface functionalization strategies have been explored to further enhance immune potentiation. aunps surface modified with cetyltrimethylammonium bromide (ctab), poly(diallydimethylammonium chloride) (pddac), and polyethyleneimine (pei), have been used as a dna vaccine adjuvant for human immunodeficiency virus (hiv) [ ] . these modifications were found to further boost the adjuvanticity of the aunp carrier. gold's unique malleability makes aunps an attractive platform for vaccinology studies [ ] . aunps can be fabricated into myriad of shapes (i.e. sphere, rod, cube, etc.) [ ] with tunable yet sharply distributed size range between - nm [ ] . this morphological tunability adds an additional dimension of virus mimicry with the introduction of non-spherical particles. gold nanorods, for instance, have been used as a vaccine vector for the delivery respiratory syncytial virus (rsv) f protein [ ] . these non-spherical particles bear resemblance to rod-like viruses that can also be frequently observed among different virus genera [ ] . the size modularity of aunps also allows for examining the impact of particle size on vaccine delivery, which will be discussed in details in later sections of this review. also worth noting is the more recent discovery that aunp size and shape can modulate the inflammatory responses at the cellular level [ ] . in a study that compares gold nanospheres, gold nanorods, and gold nanocubes, niikura et al. showed that whereas the nanospheres and the nanocubes induced tumor necrosis factor-α (tnf-α), il- , il- , and granulocyte macrophage colony-stimulating factor (gm-csf), the nanorods induced interleukin- β and interleukin- via an inflammasome-dependent mechanism [ ] . this shape-dependent immunological property may be due to the differing surface energies associated with different nanoscale features, which may promote varying levels of stress upon cellular uptake [ ] . silica nanoparticles offer a range of particle sizes and shapes via controlled synthesis using sol-gel chemistry. an abundance of silanol groups on silica nanoparticle surface allow for functional modifications for increasing specific cellular recognition, facilitating attachment of specific biomolecules, and modulating cellular uptake [ ] [ ] [ ] . nanoscale pores can be integrated into silica nanoparticles, yielding mesoporous silica nanoparticles (msns) with more versatile cargo-carrying capacity for vaccination purposes [ , ] . the pore size and surface functionalization of msns can be modified to control the encapsulation and release of antigens or adjuvants of choice [ ] [ ] [ ] [ ] . use of silica nanoparticles in vaccine applications include formulations against snake venom, e. coli [ ] , porcine circovirus [ ] , hiv [ ] , and other model antigens [ ] . carbon-based nanoparticles, such as carbon nanotubes and graphene, have also been studied as vaccine carriers [ , ] . owing to their high aspect ratio and large surface area, these carbon-based nanoparticles may carry a high proportion of antigens for immune activation. in a study on anticancer vaccination, villa et al. conjugated single-wall carbon nanotubes (swcts) with wilm's tumour protein, an antigen upregulated in many cancers. antigen conjugated swcts showed good uptake by dendritic cells and macrophages in vitro, and subcutaneous immunization with these swcts promoted induction of antigen-specific igg. in contrast, the free peptide formulation failed to induce an antibody response against the tumor antigen [ ] . in another study, graphene nanosheets were used to deliver antigens to facilitate antigen cross-presentation to cd + t cells [ ] . xu et al. also demonstrated the use of a dual polymer-modified graphene formulation as an effective adjuvant to enhance the immunogenicity of h. pylori derived antigen (alum-ure b) [ ] . semiconductor quantum dots have been applied for vaccine applications, offering a versatile platform for nanoparticulates antigen delivery with the added benefit of particle tracking. in a study by cambi et al., antigen-conjugated quantum dots of virus-like dimension were shown to combine antigen delivery and bioimaging functionalities, enabling immune cell tracking following antigen uptake. it presented the possibility of tracking antigen-presenting cells in vivo [ ] . along similar lines, sen et al. showed that quantum dots can induce t cell proliferation and ifn-γ production in vivo while being traceable within lymph nodes [ ] . other nanoparticle platforms with imaging functionality, including polymeric upconversion nanoparticles [ ] , and iron oxide nanoparticles [ ] , have also been studied as antigen carriers for vaccine applications. these platforms offer the capability for examining the function mechanisms behind the benefits of nanoparticle vaccines. one of the biggest advantages of nanoparticle vaccines is their ability to efficiently drain and accumulate to lymph nodes for enhanced immune processing. the lymphatic system is a subset of the circulatory system that consists of a complex network of vessels, tissues and organs. the lymphatic vessels conduct lymph between different parts of the body. as blood exits the blood vasculature to become interstitial fluid, the lymphatic system provides a return route for this interstitial fluid to the blood vessels in the form of lymph. in the process, it regulates fluid balance within tissues [ ] . underlying the lymphatic system are numerous lymph nodes that scatter throughout the body. these lymph nodes are the homing sites of migratory dendritic cells that present engulfed antigens. resident in these peripheral lymphoid organs are an abundance of specialized macrophages and other lymphocytes which play a major role in antigen capture and processing for adaptive immune responses. as lymph passes through lymph nodes, resident macrophages further capture passing antigens [ ] . in other words, the lymphatic system functions as a filter system of bodily fluids, trapping antigens in the lymph nodes for immune processing. viruses and virus-like nanoparticulates can accumulate in lymph nodes via both cell-mediated lymphatic delivery and convective lymphatic transport. the cell-mediated transport is mediated primarily by migratory dendritic cells, which take up antigens outside of the lymphatic system (i.e. skin and lung) and enter lymph nodes via either high endothelial venules or lymph vessels [ , ] . in comparison to small protein antigens, viruses and nanoparticle vaccines are more favorable to this hitchhiking mechanism. their particulate nature promotes receptor-mediated, complement-mediated, or other intracellular uptake mechanisms [ , ] . antigens associated with nanocarriers are routinely observed to be more efficiently uptaken by dendritic cells compared to soluble antigens, thereby enabling more effective lymph node delivery and cross presentation [ , [ ] [ ] [ ] [ ] . on the other hand, the nanoscale morphology of viruses and virus-like particulates allows them to move freely in lymphatic vessels to draining lymph nodes. upon lymph node entry, a special subset of macrophages is responsible for the capture of these nanoparticulates. in a study on lymphatic tracking of viruses by junt et al., macrophages in the subcapsular sinus and in the medulla of lymph nodes were shown to be responsible for the lymph node accumulation of subcutaneously administered inactivated vesicular stomatitis virus, adenovirus, and vaccinia virus [ ] . depletion of these macrophages resulted in an enhanced virus level that circulated back to the blood, highlighting both viruses' convective transport in the lymphatic system and the macrophages' role in virus filtration. this gatekeeper function by the lymph node-resident macrophages serves to limit blood-borne infection and promote immune processing. exploiting the same transport mechanisms aimed at detaining viruses, nanoparticle vaccines can effectively target immune cells in lymph nodes, delivering antigens or adjuvants following administration in peripheral tissues [ , , ] (fig. a) . the benefit and focus on lymph node targeting also explain why particulate vaccines are most commonly administered subcutaneously as opposed to the conventional intramuscular route for subunit vaccines; free lymphatic drainage and access to immune cells in lymph nodes following injection into the interstitium likely outweigh the "depot effect" afforded by the intramuscular route [ , ] . lymphatic targeting by nanoparticles have also been observed following administration via different delivery routes, including pulmonary [ ] , oral [ ] , intra-peritoneal [ ] routes. such favorable distribution profile allows tailoring of nanoparticle vaccines towards targeting specific immunological compartments against different infectious threats. studies on the influence of nanoparticle size on lymph node targeting began in the s as scientists aimed to maximize lymphatic delivery of drugs for treating metastatic cancer. it was generally observed that following subcutaneous injections liposomes smaller than nm were able to enter the lymphatic capillaries whereas larger liposomes remained at the injection sites [ ] [ ] [ ] . in a study by oussoren et al. using isotope labelled liposomes between to nm in diameter in lymphatic tracking, lymphatic uptake was found to be inversely proportional to the liposome size. nm, nm, nm and nm liposomes had approximately %, %, %, and % of the injected dose entering the lymphatic system, respectively. curiously, despite higher lymphatic entry by smaller liposomes in the study, liposome accumulation in the draining lymph node was similar across the differently sized formulations. it was found that the majority of the small liposomes, which consisted of egg-phosphatidylcholine and egg-phosphatidylglycerol, passed through the lymph node and were ultimately captured by the liver and the spleen [ ] . the result highlighted the dynamic relationship between particle size and lymph node accumulation; as smaller particles are more likely to enter the lymphatic system, they are also more likely to evade the filtering mechanism of lymph nodes. the authors showed that incorporating phosphotidylserine, a lipid more susceptible to macrophage recognition and capture, increased the lymph node accumulation by -fold. other lipid modification strategies, such as steric stabilization and ligand functionalization, have also been reported to influence the lymph node accumulation of liposomes following lymphatic uptake [ ] . later studies using solid nanoparticles on examining the effect of particle size on lymph node delivery echo earlier findings based on liposomes. using polystyrene beads, manolova et al. confirmed the size-dependent particle transport in the lymphatic system. upon subcutaneous delivery, large particles between to nm were found to be associated with dendritic cells from the site of injection, and small nanoparticles between to nm could drain freely in the lymphatic system, effectively targeting lymph node-resident dendritic cells and macrophages [ ] . in a study by reddy et al. on examining nanocarriers as a vaccine delivery platform, pluronic-stabilized polypropylene sulfide nanoparticles of well-defined sizes were investigated. using fluorescence microlymphangiography, the investigators showed a clear distinction between nm and nm particles regarding their lymphatic uptake. following injection into mouse tails, nm particles were efficiently drained to the lymphatic vessels, whereas the interstitial transport of nm particles was less efficient. in contrast to prior studies with liposomes, the nm particles also showed higher lymph node accumulation, resulting in a -fold enhancement in lymph node delivery as compared to the nm particles (fig. b, c) [ ] . such enhanced lymph node delivery, which was absent in earlier liposomal studies, could be attributed to both increased colloidal stability of the polymeric nanoparticles and the particles' ability to elicit complement activation. as the polypropylene sulfide particles were functionalized with surface hydroxyl groups to trigger the proteolytic cleavage of c complement protein, the danger signal associated with the complement activation could facilitate macrophage uptake upon lymph node entry [ ] , thereby reducing particle escape from the lymph node's filtering mechanism. the size-dependent lymph node targeting was also observed in other solid particle platforms. in a study by gao et al. that compared nm and nm gold nanoparticles as antigen carriers, nm gold nanoparticles yielded . -fold higher lymph node accumulation in terms of total gold delivery. upon conversion to particle number and total particle surface area, nm gold nanoparticles had an enhancement of -fold and . -fold as compared to nm particles, respectively [ ] . in general, nanoparticles between and nm, a length scale that coincides with viral particles, can exploit interstitial flow for lymphatic delivery. within this length scale, smaller particle size tends to favor lymph node accumulation. given the privilege of nanocarriers in lymphatic transport, nanoparticles have been shown to enhance the delivery of target antigens to lymph nodes and resident immune cells for processing and immune activation. in the aforementioned study on polypropylene sulfide particles, for instance, reddy et al. demonstrated increased resident dendritic cell activation in the lymph node by ovalbumin-conjugated nanoparticles. in their animal study, strong anti-ovalbumin humoral response was observed [ ] , highlighting the benefit of nanoparticle-mediated lymph node delivery on enhancing antigen processing. moon et al. also showed nanoparticle vaccines can promote preferential accumulation of antigens in the draining lymph nodes and enhance expansion of antigen-specific t cells. using interbilayer-crosslinked multilamellar vesicles (icmvs), a lipid-based nanoformulation consisting of multiple layers of lipid vesicles interconnected via thiol chemistry, the investigators demonstrated enhanced antigen delivery to total dcs, macrophages and plasmacytoid dcs in the lymph nodes [ ] . interestingly, liposomes of comparable sizes were much less effective in shuttling antigens to draining lymph nodes in the same study. this observation highlighted the importance of nanoparticle stability in vaccine design as the icmvs were more colloidally stable than liposomes. icmv-mediated antigen delivery resulted in significantly higher humoral and cellular responses as compared to the free antigens and the liposomal formulations. the effect of nanoparticle carrier on antigen transport was also shown in a study by chen et al., who demonstrated effective vaccination against coronaviruses using gold nanoparticle-adsorbed viral antigens. these antigen-coated nanoparticles were structurally analogous to coronaviruses in terms of size and antigen display. immunofluorescence quantification showed that viral spike proteins delivered with nm gold nanoparticles increased lymph node delivery by approximately -fold compared to free spike proteins. these virus-like particles showed high immunogenicity in both murine and avian models and enhanced anti-viral iga and igg titers and cellular immune responses in comparison to free protein antigens and a commercial wiv vaccine [ ] . in addition to delivering antigens for more effective immune processing, nanoparticles have been extensively applied to localize immunological adjuvants to lymph nodes for improved safety and potency. while conventional adjuvants such as alum have been widely employed clinically to promote humoral responses [ ] , more recent development in adjuvant research has identified many pathogen-associated molecular patterns (pamps) as promising adjuvant candidates towards promoting both humoral and cellular responses [ ] . these molecular danger signals are often similar to viral pathogens regarding their immune potentiating mechanisms, triggering innate immunity and in turn facilitating adaptive immune responses. many pamps (i.e. cpg-odn, poly(i:c), and cyclic dinucleotides) as well as other molecular agonists of toll-like receptors (tlrs) (i.e. imiquimod and resiquimod) are known to induce strong immune responses. however, their potency poses safety concerns over the likely induction of systemic inflammation. nanoparticle-based delivery thus offers a desirable strategy in guiding these immunological modulators to lymph nodes, increasing their effective concentration and reducing their systemic reactogenicity. in one example, nunh et al., constructed a ph-degradable nanogel platform ligated with imidazoquinoline (imdq), a tlr / agonist, and showed retention of the adjuvant at the injection site and the draining lymph node. the adjuvant in its free form elicited systemic inflammatory responses, but this side effect was largely obviated with the nanogel formulation. the targeting effect of the nanoformulations also resulted in recruitment of monocytes to the draining lymph node. a large number of immune cells, including b cells, dcs, and macrophages were shown to readily take up these adjuvant-loaded nanogels [ ] . in another study by ilyinskii et. al., synthetic vaccine particles encapsulating resiquimod (r , a tlr / and tlr ligand) augmented humoral and cellular immune responses to both soluble and nanoparticle-delivered proteins compared to that observed with free adjuvants. the adjuvant-loaded nanoparticles promoted local cytokine induction in the lymph nodes and reduced systemic cytokine production observed with free r . moreover, while injection of the nanoformulation led to sustained expressions of ifn-γ, il- , and il- β in lymph nodes after hours, free r induced only modest levels of il- and ifn-β [ ] . cpg-odn, an agonist of tlr- , is another adjuvant that's frequently coupled with nanocarriers for vaccination studies [ , , , , , ] . some of the primary advantages of nanoparticle-based cpg formulations include strong t cell responses, dosage sparing, and reduced systemic side effects. such formulations have been commonly applied in anticancer vaccination efforts owing to the need for high cell-based immune responses for effective tumor containment. transport of viral antigen and immune-potentiating adjuvants by viruses to immune cells is a highly coordinated event as viral particles shuttle both antigen targets and adjuvanting nucleic acids simultaneously. such antigen/adjuvant coordination, or its absence, has been shown to strongly influence immune cell activation [ ] . in addition, the immune system can respond to viruses through multiple pamps, including glycoprotein, dna, dsrna, and ssrna, activating a broad spectrum of signalling pathways for heightened antiviral immunity [ ] . the synthetic flexibility of nanoparticles has thus been exploited to emulate the co-delivery capacity of viral pathogens to boost immune responses. in some cases, antigen and adjuvant are localized on the same nanoparticles for synchronized delivery. ma et al. incorporated a hepatitis b surface antigen (hbsag) and cpg adjuvant onto plga nanoparticles via conjugation with dopamine, allowing the particles to display both the viral antigen and the immune activator. the study showed that the pathogen-mimicking particle enhanced the recruitment of immune cells to the injected site, activated bone marrow derived dendritic cells, and induced strong humoral and cellular immune responses [ ] . in another study conducted by kuai et al., a disc-like synthetic high-density lipoprotein (shdl) was applied to integrate both target antigens of cd + t cells and cpg-odn for anticancer vaccination. the nanodisc drastically improved the co-delivery of antigens and adjuvants to lymph nodes compared to soluble vaccine and induced a -fold enhancement in antigen-specific cd + cytotoxic t-lymphocyte response as compared to the free peptide and adjuvant control. besides incorporating antigens and adjuvants on the same particle for co-delivery, a number of studies demonstrated that delivering antigen and adjuvant in separate but similar nanocarriers can also elevate antigen-specific immune responses. in one example, an hiv antigen (hivgp ) was anchored on the surface of liposomes and co-delivered with a liposomal formulation of cd-gmp, a potent agonist of the sting pathway. the study showed a substantial accumulation of the sting agonist in draining lymph nodes [ ] , and it is expected that the liposome-bound peptide antigen was delivered in a similar fashion. as a result, enhanced activation of antigen presenting cells and increased levels of antibodies against the hiv antigen were observed. immune stimulation by formulations containing separate antigen-and adjuvant-loaded nanoparticles was also reported in a study exploring the benefit of multi-adjuvant loaded particles. by incorporating multiple distinctive activators of tlrs, including monophosphoryl lipid a (a tlr agonist) and r (a tlr agonist), kasturi et al. demonstrated adjuvant synergism that triggered elevated antigen-specific humoral responses [ ] . using ovalbumin and hemagglutinin of influenza viruses, the investigators demonstrated long-lasting humoral responses and evidence of memory b cell formation following immunization with the nanoparticle vaccine. these examples highlight the functional versatility of synthetic nanoparticles, which can facilitate different modes of virus mimicry for immune activation. in addition to the role of lymph nodes in trapping nanoscale particles for immune processing, the immune system has also adapted to the repetitive antigen display on viruses for effective potentiation. virus surfaces display antigenic epitopes in an ordered and highly repetitive fashion, and the presentation of repetitively arranged and appropriately spaced antigens on the surface of virus or virus-like particles has been linked to enhanced immune responses [ ] . many reports have shown that numerous components of the mammalian immune system have evolved to respond strongly to the repetitive antigen patterns frequently found on pathogens [ ] [ ] [ ] . in comparison, non-repetitive antigens are usually less effective in inducing immune responses [ ] [ ] [ ] [ ] . repetitive motifs on viral surfaces are also found to activate the complement system [ ] engaging the cd -cd complex, which further facilitates b cell activation and amplify other immune processing pathways [ ] . understanding the link between structural features of antigen display and immunological induction is vital in designing nanoparticulate vaccines. multivalent interactions promote b-cell receptor (bcr) clustering and signaling and facilitate receptor-mediated internalization of antigen. antigen features, such as epitope affinity, valency, or co-receptor recruitment can impact b and t cell signaling. in a study that used antigen-conjugated polymer to assay the impact of antigen valency on b cell activation, puffer et al. showed that clustering of bcrs by multivalent antigens is crucial for antigen-dependent signaling. the antigen-conjugated polymers clustered unbound bcrs and contributed to enhanced intracellular signalling [ ] . whereas the multivalent antigen-polymer conjugates elicited antibody production, free antigens failed to trigger humoral responses (fig. a) . highly repetitive surfaces are also known to bind strongly to natural igm antibodies through multivalent, high-avidity interactions [ ] . such antibody binding also facilitates cellular uptake of particles by macrophages and dendritic cells, which can in turn enhance immune processing through increased antigen presentation. it is also worth noting that many important components in the humoral arm of innate immunity, such as complement c q, pentraxins, ficolins and collectins, are multimeric structures that favor high-avidity interactions with repetitive pathogen surfaces [ ] (fig. b) . these observations highlight how the immune system has been primed to respond to repetitive motifs frequently found on viral particles. unlike free subunit antigens, nanoparticle vaccines present a high concentration of antigens on their surfaces. a nanoparticle can be surface functionalized with up to hundreds of antigens, effectively emulating the antigen display on viral surfaces [ , ] . the multivalent antigen display on nanoparticles enhances antibody responses by efficiently cross-linking bcrs, activating complement, and facilitating antibody binding. these factors play synergistic roles in promoting b cell differentiation and stimulating dc-mediated t cell priming. although synthetically prepared nanoparticles have yet to show the level of ordered antigen arrangement found on viruses and virus-like particles derived from cell culture systems [ , ] , ongoing studies continue to demonstrate emerging techniques to couple antigens of interest to nanocarriers. in the last section of the review, we review commonly used methods for coupling antigens with nanoparticles. these strategies may spur novel approaches for preparing nanoparticle vaccines. association of protein antigens with nanoparticles can be divided into particle encapsulation and surface association. while many studies show excellent nanoparticle vaccine potency with encapsulated antigens, surface associated antigen may benefit from the innate immune factors and immune processing mechanisms described in the previous section. techniques to associate protein or peptide antigens can be categorized into multiple categories. the different modes of antigen association are highlighted as follows. thiol and amine groups on protein or peptide antigens are frequently exploited for bioconjugation with nanoparticles. the thiol group on a cysteine amino acid is a powerful nucleophile with the capacity to form covalent linkage. several linker groups, such as maleimide and succinimidyl -( -pyridyldithio)propionate (spdp), facilitate the conjugation between thiol-containing antigens with nanoparticles [ ] . in a maleimide-thiol reaction, the nucleophilic thiolate anion attacks the π-bond of maleimide, forming the enolate intermediate and yielding the desired conjugate. this technique has been widely used to assemble nanoparticle vaccines [ ] [ ] [ ] . in a study on refining a liposomal formulation of hiv vaccine, thiol chemistry was exploited to control the physiological conformation and density of the target antigen to modulate immune responses [ ] . thiols groups also readily associate with gold surfaces. the strong interaction between sulfur and gold drives the sulfur atom to fill the free orbitals of a gold atom, creating a coordinate covalent bond [ , ] . such approach has been extensively applied to associate nucleic acids [ ] [ ] [ ] [ ] [ ] and antigens [ , ] with gold nanoparticles. amine groups, on the other hand, are present on all protein and peptide antigens, which can be linked to nanocarriers via amide bond formation, typically through carbodiimide crosslinker chemistry. bioconjugations with amine-containing antigens are commonly performed using -ethyl- -( -(dimethylamino)propyl)carbodi-imide (edc) and n-hydroxysuccinimide (nhs). nhs or its sulfonated from (sulfo-nhs) is efficiently coupled to carboxyl groups with the aid of edc to form nhs esters. the nhs esters then covalently conjugate to primary amines to form an intermediate compound that is subsequently hydrolysed to the desired conjugate [ ] . many nanoparticle vaccines have been prepared with the edc/nhs conjugation method with a high coupling yield [ , ] . polydopamine functionalization is another conjugation approach that is increasingly applied to associate proteins and peptides with nanoparticles. in alkaline ph, dopamine undergoes oxidative self-polymerization to form a layer of polydopamine that can coat almost any type of material [ ] [ ] [ ] . this mussel-like adhesive layer enables a secondary reaction with biomolecules containing thiol or amine groups [ , ] . the technique has been demonstrated on both organic and inorganic nanoparticles [ , ] . dopamine-incorporated polymers have also been employed to adsorb proteins for macrophage-targeted delivery [ ] . the versatile technique can be applied to conjugate multiple cargoes for vaccine applications [ ] . electrostatic attraction between oppositely charged antigens and nanoparticles have been exploited to prepare nanoparticle vaccines. in general, cationic nanocarriers are prepared for the association of anionic protein antigens. in the case of liposomes, cationic lipid dotap is frequently applied to render the nanocarrier positively charged. dotap-based liposomes have been shown to absorb hpv e peptides [ ] , enhancing antigen-specific cd t cell response and increasing antitumor responses. strategies have also been employed to prepare antigens with added anionic moieties via recombinant protein engineering. by expressing hpv e protein and ovalbumin with an anionic lipoprotein, shen et al. demonstrated enhanced antigen association efficiency and retention to dotap liposomes. the vaccine formulation elevated antigen-specific cellular responses and inhibited tumor growth in a mouse model [ , ] . in another work based on print (particle replication in non-wetting templates) technology, electrostatic interaction was exploited to generate nanoparticle vaccines of cylindrical shape. cationic particles were prepared by blending positively charged polymers with plga prior to the imprint lithography process. upon mixing with anionic hemagglutinin proteins of influenza viruses, a high level of antigen binding to the particle surface was achieved [ ] . immunization with the nanoparticle vaccine elicited a more potent anti-influenza antibody response compared to a commercial vaccine based on inactivated subunit influenza viruses. unlike other spherical nanoparticle vaccines, the platform offers the ability to mimic the filamentous shape that can be found among many virus species. synthetic nanoparticles have high surface energies owing to their large radii of curvature. as a result, adsorption of protein can occur spontaneously owing to a combination of weak interaction forces, leading to the formation of protein corona formation [ ] . this phenomenon was demonstrated to facilitate the assembly of synthetic virus-like nanoparticles [ ] . upon mixing gold nanoparticles with viral antigens, rapid protein adsorption took place on the nanoparticle surface. this adsorption led to colloidal stabilization of gold nanoparticles, preventing their aggregation in biological buffers. biochemical analysis showed the facile assembly method surrounded each nanoparticle with approximately coronavirus spike proteins, inducing enhanced anti-viral immune responses. the adsorption approach has been used extensively to associated antigens with nanocarriers, including nanoparticles made of gold [ ] , calcium phosphate [ , ] , aluminium hydroxide [ ] , carbon [ , ] , silica [ , ] , and organic polymers [ , ] . the range of adsorbed biomolecules vary from dna [ , , ] , adjuvant [ ] , small peptides, and protein antigens [ , , , ] . it is important to note that formation of protein corona is a dynamic process that can be strongly influenced by both nanomaterials and antigen of interest [ ] . studies have also shown that antigens can undergo conformational changes upon nanoparticle adsorption [ ] , which could have both positive and negative effects on the resulting vaccine formulations. coating nanoparticles with cellular membranes is an emerging functionalization approach that can pave ways to novel vaccine formulations with virus-mimetic features. it has been shown that following dispersion of cell membrane vesicles with nanoparticles, highly controlled membrane coating can be achieved, yielding unilamellar membrane cloaked nanoparticles retaining membrane proteins in their right-side-out orientation [ ] [ ] [ ] . these nanoparticles are structurally analogous to enveloped viruses consisting of cell-derived lipid membranes stabilized via viral capsids or matrix proteins [ ] . the membrane coating approach has been adapted for vaccine preparations against cancer and bacteria [ , [ ] [ ] [ ] . enhanced dendritic cell activation and presentation of a melanoma-associated tumor antigen (gp ) was demonstrated by fang et al. using plga nanoparticles cloaked in b -f membranes [ ] . the enhanced immune response to the membrane antigen was attributed to increased cellular delivery and colocalization with immunological adjuvant facilitated by the nanoparticles. gao et al. also showed that bacterial outer membrane vesicles (omvs) can be rendered more immunogenic following coating on gold nanoparticles. the omv-coated nanoparticles significantly increased antigen delivery to lymph nodes and elevated production of cytokines associated with bacterial containment [ ] . the membrane cloaking approach offers a titillating strategy towards future vaccine designs as the method enables coupling of membrane-anchored antigens in their native conformation with immunogenic nanocarriers. advances in nanotechnology and its adoption in vaccinology have helped push the boundaries of non-live, subunit vaccines, resulting in many exciting demonstrations of effective immune potentiation by nanoformulations. not only can nanoparticle vaccines enhance humoral responses against target antigens, they have been shown to promote cell-based immunity as well as immunological memory. these are hallmarks of good vaccine formulations that are often inherent to live attenuated viruses. an increasing number of studies have shed light on the mechanisms behind the benefits of nanoparticle vaccines, including lymph node targeting, multivalent antigen display, and coordinated delivery of antigens and adjuvants. these features can find their mechanistic analogues in the immunological processing of viruses. given the frequent semblance between nanoparticle vaccines and viruses regarding their size, morphology, antigen display, and adjuvanticity effect, nanoparticles present a compelling platform in bridging the gap between live and non-live vaccines. emerging techniques in nanoparticle functionalization also pave ways to novel formulation designs that promise controlled immune modulation. going forward, understanding of virology may assist scientists and engineering in preparing emerging nanoformulations with advanced virus-like features. such vaccine nanotechnology is envisioned to improve vaccine safety, potency, and availability, offering compelling platforms towards addressing the many public health threats yet to have effective prophylaxis and treatment options. smallpox and its eradication. world health organization geneva translating innate immunity into immunological memory: implications for vaccine development nanoparticle vaccines biomaterials for nanoparticle vaccine delivery systems dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h n influenza, and toxoplasma gondii challenges with a single dose nanoparticulate sting agonists are potent lymph node-targeted vaccine adjuvants enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand t-fh cells and promote germinal center induction dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h n influenza, and toxoplasma gondii challenges with a single dose designer vaccine nanodiscs for personalized cancer immunotherapy systemic rna delivery to dendritic cells exploits antiviral defence for cancer immunotherapy enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes generation of effector memory t cell-based mucosal and systemic immunity with pulmonary nanoparticle vaccination exploiting lymphatic transport and complement activation in nanoparticle vaccines liposomal vaccine delivery systems liposomes as delivery systems for nasal vaccination: strategies and outcomes targeting of drugs: implications in medicine. the lancet liposomes--bags of potential clinical prospects for liposomes endothelial endocytic pathways: gates for vascular drug delivery. current vascular pharmacology lipid carriers for gene therapy liposomes as immunological adjuvants immunological adjuvants: a role for liposomes highly efficient antiviral cd + t-cell induction by peptides coupled to the surfaces of liposomes t cell-independent b cell response is responsible for abc phenomenon induced by repeated injection of pegylated liposomes effect of liposomal antigens on the priming and activation of the immune system by dendritic cells liposomal resiquimod for the treatment of leishmania donovani infection adjuvant effect of cationic liposomes for subunit influenza vaccine: influence of antigen loading method, cholesterol and immune modulators liposomes containing monophosphoryl lipid a: a potent adjuvant system for inducing antibodies to heroin hapten analogs liposomes containing glucosyl ceramide and the adjuvant monophosphoryl lipid a specifically bind t bacteriophage: a self-assembling nanocarrier inhibition of cd l(+) t-cell response in vitro via a novel sulfo-glycolipid, beta-sqag liposome that binds to cd l molecule on the cell surface growth-inhibition of tumor-cells by a liposome-encapsulated, mycolic acid-containing glycolipid, trehalose , , '-trimycolate coencapsulation of cpg oligodeoxynucleotides with recombinant leishmania major stress-inducible protein in liposome enhances immune response and protection against leishmaniasis in immunized balb/c mice. clinical and vaccine immunology adjuvant effect of liposome-encapsulated natural phosphodiester cpg-dna enhancement of immunomodulatory activity by liposome-encapsulated natural phosphodiester bond cpg-dna in a human b cell line induction of immunological memory response by vaccination with tm sf epitope-cpg-dna-liposome complex in a mouse hepatocellular carcinoma model a new adjuvant delivery system 'cyclic di-gmp/ysk liposome' for cancer immunotherapy the virosome concept for influenza vaccines virosomes: evolution of the liposome as a targeted drug delivery system induction of cytotoxic t lymphocyte activity by fusion-active peptide-containing virosomes epaxal®: a virosomal vaccine to prevent hepatitis a infection. expert review of vaccines influenza virosomes as a combined vaccine carrier and adjuvant system for prophylactic and therapeutic immunizations. expert review of vaccines influenza virosomes as vaccine adjuvant and carrier system. expert review of vaccines vaccine delivery: a matter of size, geometry, kinetics and molecular patterns virosome-mediated delivery of protein antigens to dendritic cells virosome-mediated delivery of tumor antigen to plasmacytoid dendritic cells influenza virosomes enhance class i restricted ctl induction through cd + t cell activation influenza virosomes as a vaccine adjuvant and carrier system. expert review of vaccines sendai virus fusion protein-mediates simultaneous induction of mhc class i/ii-dependent mucosal and systemic immune responses via the nasopharyngeal-associated lymphoreticular tissue immune system subunit interactions of vesicular stomatitis-virus envelope glycoprotein stabilized by binding to viral matrix protein interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses lipid-derived nanoparticles for immunostimulatory rna adjuvant delivery enhancement of dendritic cells transfection in vivo and of vaccination against b f melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger rna sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion type i interferons in host defense in vitro degradation and release profiles of poly-dl-lactide-poly(ethylene glycol) microspheres with entrapped proteins nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines mucosal delivery of vaccines: role of mucoadhesive/biodegradable polymers nanoparticles for nasal vaccination biodegradable polymer microspheres as vaccine adjuvants and delivery systems a review of the formation and classification of amphiphilic block copolymer nanoparticulate structures: micelles, nanospheres, nanocapsules and polymersomes current advances in research and clinical applications of plga-based nanotechnology biodegradable nanoparticles for drug delivery and targeting. current opinion in solid state and materials science plga-based nanoparticles: an overview of biomedical applications enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand tfh cells and promote germinal center induction enhancement of t helper type immune responses against hepatitis b virus core antigen by plga nanoparticle vaccine delivery a single-dose plga encapsulated protective antigen domain nanoformulation protects mice against bacillus anthracis spore challenge dose sparing of cpg oligodeoxynucleotide vaccine adjuvants by nanoparticle delivery role of sustained antigen release from nanoparticle vaccines in shaping the t cell memory phenotype adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release programming the magnitude and persistence of antibody responses with innate immunity a mucosal vaccine against chlamydia trachomatis generates two waves of protective memory t cells design and development of immunomodulatory antigen delivery systems based on peptide/peg-pla conjugate for tuning immunity in vitro stimulation of cd and cd t cells by dendritic cells loaded with a complex of cholesterol-bearing hydrophobized pullulan and ny-eso- protein: identification of a new hla-dr -binding cd t-cell epitope t cell immunomonitoring and tumor responses in patients immunized with a complex of cholesterol-bearing hydrophobized pullulan (chp) and ny-eso- protein nanogel-based immunologically stealth vaccine targets macrophages in the medulla of lymph node and induces potent antitumor immunity bioreducible alginate-poly(ethylenimine) nanogels as an antigen-delivery system robustly enhance vaccine-elicited humoral and cellular immune responses immune response by nasal delivery of hepatitis b surface antigen and codelivery of a cpg odn in alginate coated chitosan nanoparticles enhanced immune response and protective effects of nano-chitosan-based dna vaccine encoding t cell epitopes of esat- and fl against mycobacterium tuberculosis infection nanogel vaccines targeting dendritic cells: contributions of the surface decoration and vaccine cargo on cell targeting and activation assessment of the immune responses to treponema pallidum gpd dna vaccine adjuvanted with il- and chitosan nanoparticles before and after treponema pallidum challenge in rabbits preparation and efficacy of a live newcastle disease virus vaccine encapsulated in chitosan nanoparticles an effective mannosylated chitosan nanoparticle dna vaccine for fmd virus biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine chitosan-based systems for the delivery of vaccine antigens. expert review of vaccines chitosan microparticles and nanoparticles as biocompatible delivery vehicles for peptide and protein-based immunocontraceptive vaccines chitosan: an adjuvant with an unanticipated sting structural mechanism of cytosolic dna sensing by cgas vaccination of mice with chitosan nanogel-associated recombinant ncpdi against challenge infection with neospora caninum tachyzoites preparation and characterization of biodegradable nanoparticles based on poly(gamma-glutamic acid) with l-phenylalanine as a protein carrier methods for nano-particle based vaccine formulation and evaluation of their immunogenicity systemic immune responses in sheep, induced by a novel nano-bead adjuvant synthesis and characterization of poly(ethyl- -cyanoacrylate) nanoparticles with a magnetic core san biagio pl. preparation, characterization and in vitro antimicrobial activity of ampicillin-loaded polyethylcyanoacrylate nanoparticles new adjuvants on a polymethylmethacrylate base the use of new polymethylmethacrylate adjuvants for split influenza vaccines potential of nanocarriers in antigen delivery: the path to successful vaccine delivery high-density sub- -nm peptide-gold nanoparticle complexes improve vaccine presentation by dendritic cells in vitro preclinical and clinical progress of particle-mediated dna vaccines for infectious diseases gold nanoparticles as a potential carrier for transmucosal vaccine delivery gold nanoparticle delivery of modified cpg stimulates macrophages and inhibits tumor growth for enhanced immunotherapy immunomodulatory spherical nucleic acids surface-engineered gold nanorods: promising dna vaccine adjuvant for hiv- treatment nanotechnology in vaccine delivery gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo vaccine delivery using nanoparticles gold nanorod vaccine for respiratory syncytial virus phylogeny of capsid proteins of rod-shaped and filamentous rna plant viruses: two families with distinct patterns of sequence and probably structure conservation understanding biophysicochemical interactions at the nano-bio interface bioconjugated nanoparticles for dna protection from cleavage polyethyleneimine coating enhances the cellular uptake of mesoporous silica nanoparticles and allows safe delivery of sirna and dna constructs hyaluronic acid modified mesoporous silica nanoparticles for targeted drug delivery to cd -overexpressing cancer cells ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism recent advances in the rational design of silica-based nanoparticles for gene therapy studies on mcm- mesoporous silica for drug delivery: effect of particle morphology and amine functionalization mesoporous silica nanoparticles as antigen carriers and adjuvants for vaccine delivery immunological parameters related to the adjuvant effect of the ordered mesoporous silica sba- mesoporous silica nanoparticles act as a self-adjuvant for ovalbumin model antigen in mice ordered mesoporous silica sba- : a new effective adjuvant to induce antibody response immunization of mice by hollow mesoporous silica nanoparticles as carriers of porcine circovirus type orf protein binding of hiv- gp glycoprotein to silica nanoparticles modified with cd glycoprotein and cd peptide fragments enhanced mucosal and systemic immune responses obtained by porous silica nanoparticles used as an oral vaccine adjuvant: effect of silica architecture on immunological properties applications of carbon nanotubes in drug delivery spontaneous protein adsorption on graphene oxide nanosheets allowing efficient intracellular vaccine protein delivery single-walled carbon nanotubes deliver peptide antigen into dendritic cells and enhance igg responses to tumor-associated antigens functionalized graphene oxide serves as a novel vaccine nano-adjuvant for robust stimulation of cellular immunity ligand-conjugated quantum dots monitor antigen uptake and processing by dendritic cells quantum dots for tracking dendritic cells and priming an immune response in vitro and in vivo antigen-loaded upconversion nanoparticles for dendritic cell stimulation, tracking, and vaccination in dendritic cell-based immunotherapy lipopeptide-coated iron oxide nanoparticles as potential glycoconjugate-based synthetic anticancer vaccines interstitial flow and its effects in soft tissues trafficking of b cell antigen in lymph nodes dendritic cells and the control of immunity the mechanism of uptake of biodegradable microparticles in caco- cells is size dependent size-dependent endocytosis of nanoparticles particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model characterization of poly(d,l-lactic-co-glycolic acid) based nanoparticulate system for enhanced delivery of antigens to dendritic cells targeting nanoparticles to dendritic cells for immunotherapy a multifunctional core-shell nanoparticle for dendritic cell-based cancer immunotherapy nanoparticles target distinct dendritic cell populations according to their size subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral b cells lymphatic targeting with nanoparticulate system in vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles role of intramuscular administration of water-in-oil emulsions as a method for increasing the delivery of anticancer to regional lymphatics studies on pharmaceutical modification of anticancer agents. ii. enhanced delivery of bleomycin into lymph by emulsions and drying emulsions lymphatic uptake of pulmonary delivered radiolabelled solid lipid nanoparticles effect of lipid core material on characteristics of solid lipid nanoparticles designed for oral lymphatic delivery lymphatic targeting of polymeric nanoparticles after intraperitoneal administration in rats fate of cholesterol-rich liposomes after subcutaneous injection into rats distribution of radiolabeled multilamellar liposomes injected intralymphatically and subcutaneously subcutaneous administration of liposomesa comparison with the intravenous and intraperitoneal routes of injection lymphatic uptake and biodistribution of liposomes after subcutaneous injection. ii. influence of liposomal size, lipid compostion and lipid dose liposomes to target the lymphatics by subcutaneous administration. advanced drug delivery reviews mechanisms of phagocytosis in macrophages. annual review of immunology modulating antibacterial immunity via bacterial membrane-coated nanoparticles synthetic virus-like particles prepared via protein corona formation enable effective vaccination in an avian model of coronavirus infection alum's adjuvant action: grease is the word key roles of adjuvants in modern vaccines ph-degradable imidazoquinoline-ligated nanogels for lymph node-focused immune activation adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release activation of toll-like receptor and production of epitope specific antibody by liposome-encapsulated cpg-dna targeting the tumor-draining lymph node with adjuvanted nanoparticles reshapes the anti-tumor immune response regulation of antiviral t cell responses by type i interferons pathogen recognition and inflammatory signaling in innate immune defenses pathogen-mimicking polymeric nanoparticles based on dopamine polymerization as vaccines adjuvants induce robust humoral and cellular immune responses antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity regulation of igg antibody responses by epitope density and cd -mediated costimulation the role of antibody concentration and avidity in antiviral protection the influence of virus structure on antibody responses and virus serotype formation neutralizing antiviral b cell responses. annual review of immunology determinants of autoantibody induction by conjugated papillomavirus virus-like particles the influence of antigen organization on b cell responsiveness conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies induction of autoantibodies to mouse ccr with recombinant papillomavirus particles the role of complement in inflammation and adaptive immunity structure and function: lessons form cd activating b cell signaling with defined multivalent ligands on natural and artificial vaccinations an integrated view of humoral innate immunity: pentraxins as a paradigm applications of nanotechnology for immunology reorienting our view of particle-based adjuvants for subunit vaccines multi-antigen avian influenza a (h n ) virus-like particles: particulate characterizations and immunogenicity evaluation in murine and avian models virus like particles as immunogens and universal nanocarriers conjugation of ovalbumin to trimethyl chitosan improves immunogenicity of the antigen a method for producing protein nanoparticles with applications in vaccines optimization, production, and characterization of a cpg-oligonucleotide-ficoll conjugate nanoparticle adjuvant for enhanced immunogenicity of anthrax protective antigen liposomal vaccines incorporating molecular adjuvants and intrastructural t-cell help promote the immunogenicity of hiv membrane-proximal external region peptides gold nanoparticles and vaccine development gold nanoparticles for nucleic acid delivery what controls the melting properties of dna-linked gold nanoparticle assemblies? a dna-based method for rationally assembling nanoparticles into macroscopic materials oligonucleotide-modified gold nanoparticles for intracellular gene regulation regulating immune response using polyvalent nucleic acid-gold nanoparticle conjugates m e-immobilized gold nanoparticles as influenza a vaccine: role of soluble m e and longevity of protection gold nanoparticle-m e conjugate coformulated with cpg induces protective immunity against influenza a virus systematic investigation of edc/snhs-mediated bioconjugation reactions for carboxylated peptide substrates lysine-tagged peptide coupling onto polylactide nanoparticles coated with activated ester-based amphiphilic copolymer: a route to highly peptide-functionalized biodegradable carriers toll-like receptor- agonist functionalized biopolymer for mucosal vaccination substrate-independent layer-by-layer assembly by using mussel-adhesive-inspired polymers mussel-inspired surface chemistry for multifunctional coatings single-molecule mechanics of mussel adhesion materials from mussel-inspired chemistry for cell and tissue engineering applications mussel-inspired adhesives and coatings gold nanoparticles conjugated dopamine as sensing platform for sers detection engineering nanoparticle-coated bacteria as oral dna vaccines for cancer immunotherapy dopamine-conjugated poly(lactic-co-glycolic acid) nanoparticles for protein delivery to macrophages induction of cytotoxic t-lymphocytes and antitumor activity by a liposomal lipopeptide vaccine filamentous influenza virus enters cells via macropinocytosis respiratory syncytial virus assembles into structured filamentous virion particles independently of host cytoskeleton and related proteins development of a nanoparticle-based influenza vaccine using the print technology immunogenic properties of colloidal gold antigen adsorbed calcium phosphate nanoparticles stimulate both innate and adaptive immune response in fish tb trifusion antigen adsorbed on calcium phosphate nanoparticles stimulates strong cellular immunity in mice aluminum hydroxide nanoparticles show a stronger vaccine adjuvant activity than traditional aluminum hydroxide microparticles complement activation and protein adsorption by carbon nanotubes spontaneous protein adsorption on graphene oxide nanosheets allowing efficient intracellular vaccine protein delivery mesoporous silica nanoparticles as antigen carriers and adjuvants for vaccine delivery dna prime and protein boost immunization with innovative polymeric cationic core-shell nanoparticles elicits broad immune responses and strongly enhance cellular responses of hiv- tat dna vaccination novel chitosan particles and chitosan-coated emulsions inducing immune response via intranasal vaccine delivery cationic microparticles: a potent delivery system for dna vaccines induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus dna vaccines cationic microparticles are an effective delivery system for immune stimulatory cpg dna anionic microparticles are a potent delivery system for recombinant antigens from neisseria meningitidis serotype b sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide)s facilitate the preparation of small negatively charged biodegradable nanospheres biomolecular coronas provide the biological identity of nanosized materials investigation of the effects of surface chemistry and solution concentration on the conformation of adsorbed proteins using an improved circular dichroism method erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform nanoparticle biointerfacing by platelet membrane cloaking marker-of-self' functionalization of nanoscale particles through a top-down cellular membrane coating approach structural basis for membrane fusion by enveloped viruses nanoparticle-detained toxins for safe and effective vaccination nanoparticle-based antivirulence vaccine for the management of methicillin-resistant staphylococcus aureus skin infection cancer cell membrane-coated nanoparticles for anticancer vaccination and drug delivery bacterial protein toxins targeting rho gtpases the authors acknowledge support from the ministry of science and technology, r.o.c. the authors have declared that no competing interest exists. saborni chattopadhyay is a phd candidate in the institute of biomedical sciences at academia sinica, taiwan. she received her m. tech in biological sciences and bioengineering from indian institute of technology kanpur. her research interests include cancer, therapeutics and drug delivery. saborni is an avid reader and enjoys travelling.jui-yi chen is a research assistant in the institute of biomedical sciences at academia sinica, taiwan. she received her master's degree in material science and engineering from the national tsing-hua university in taiwan. her research interests include biomimetic materials and drug delivery. key: cord- -t xjy y authors: nazneen akhand, mst rubaiat; azim, kazi faizul; hoque, syeda farjana; moli, mahmuda akther; joy, bijit das; akter, hafsa; afif, ibrahim khalil; ahmed, nadim; hasan, mahmudul title: genome based evolutionary study of sars-cov- towards the prediction of epitope based chimeric vaccine date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: t xjy y sars-cov- is known to infect the neurological, respiratory, enteric, and hepatic systems of human and has already become an unprecedented threat to global healthcare system. covid- , the most serious public condition caused by sars-cov- leads the world to an uncertainty alongside thousands of regular death scenes. unavailability of specific therapeutics or approved vaccine has made the recovery of covi- more troublesome and challenging. the present in silico study aimed to predict a novel chimeric vaccines by simultaneously targeting four major structural proteins via the establishment of ancestral relationship among different strains of coronaviruses. conserved regions from the homologous protein sets of spike glycoprotein (s), membrane protein (m), envelope protein and nucleocapsid protein (n) were identified through multiple sequence alignment. the phylogeny analyses of whole genome stated that four proteins (s, e, m and n) reflected the close ancestral relation of sars-cov- to sars-cov- and bat coronavirus. numerous immunogenic epitopes (both t cell and b cell) were generated from the common fragments which were further ranked on the basis of antigenicity, transmembrane topology, conservancy level, toxicity and allergenicity pattern and population coverage analysis. top putative epitopes were combined with appropriate adjuvants and linkers to construct a novel multiepitope subunit vaccine against covid- . the designed constructs were characterized based on physicochemical properties, allergenicity, antigenicity and solubility which revealed the superiority of construct v in terms safety and efficacy. essential molecular dynamics and normal mode analysis confirmed minimal deformability of the refined model at molecular level. in addition, disulfide engineering was investigated to accelerate the stability of the protein. molecular docking study ensured high binding affinity between construct v and hla cells, as well as with different host receptors. microbial expression and translational efficacy of the constructs were checked using pet a(+) vector of e. coli strain k . the development of preventive measures to combat covid- infections might be aided the present study. however, the in vivo and in vitro validation might be ensured with wet lab trials using model animals for the implementation of the presented data. novel coronavirus named sars-cov- / -ncov was identified at the end of in wuhan, a city in the hubei province of china, causing severe pneumonia that leads to huge death cases . gradually this virus emerged as a new threat to the whole world and affecting almost all parts of the world. to date, the pathogen has affected countries, and thus becoming a global public health emergency. global public health concern with pandemic notion of covid- was declared on january th, by the world health organization (who, ) . agin, an adverse situation has also been announced on march for increasing the infections of covid- (kunz and minder, ) . till april , , total virus affected people around the world exceeded , , and more than , committed death, while , people fully recovered from the infection (who, ) . the alarming situation is that the number of confirmed cases worldwide has exceeded one million by this time. it took more than three months to reach the first confirmed cases, while required only days to detect the next cases. the situation is getting worse in european region. total death cases in italy, spain, usa, france, united kingdom was , , , and respectively (till april , ) and this number is exacerbating day by day (who, ) . some common clinical manifestations of covid- is fever, sputum production, shortness of breath, cough, fatigue, sore throat and headache which leads to severe cases of pneumonia. a few patients also have gastrointestinal symptoms with diarrhea and vomiting (guan et al., ) . though several early studies showed that the mortality rate for sars-cov- is not as high ( - %), the latest global death rate for covid- is . % which indicates the increasing trends . the investigation of chinese center for diseases control and prevention ( ) revealed that the prevalence of covid- is more apparent in the people ages years rather than the lower age groups (jeong-ho et al., ) . high fever and lymphocytopenia were found more common in covid- , though the frequency of the patient without fever condition is also higher than in the earlier outbreaks caused by sars-cov ( %) and mers-cov ( %) chen et al., ) . sars-cov- is a betacoronavirus that has a positive sense, - kb in length, single stranded rna molecule as its genetic material and belongs to the family coronaviridae, order nidovirales (hui et al., ) . it shares genome similarity with sars-cov ( . %) and bat coronavirus ( %) zhu et al., ) . however, there are still obscured hypothesis regarding the vector or carrier of sars-cov- , though its detection was primarily linked to wuhan's huanan seafood wholesale market (lu et al., ; who, ) . though the species of sars-cov- and bat coronavirus shares sufficient sequence similarities with the covid- , the known way mechanism of infection to the host, and the death rate is quite different in case of the novel coronavirus. in addition, there is an evolutionary distance between sars-cov- and bat coronavirus as well as the covid- (hu et al., ; wu, b) . because of high sequence variability of the pathogen, many of the efforts that have been undertaken to develop vaccine against sars-cov , remain unsuccessful . therefore, there is an urgent need to develop vaccines for treatment of sars-cov- based on the understanding of actual evolutionary ancestral relationship. while some natural metabolites and traditional medication may come up with comfort and take the edge off few symptoms of covid- , there is no proof that existing treatment procedures can effectively combat against the diseased condition (who, ). however, inactivated or live-attenuated forms of pathogenic organisms are usually recommended for the initiation of antigen-specific responses that alleviate or reduce the possibility of host experience with secondary infections (thompson & staats, ) . moreover, all of the proteins are not usually targeted for protective immunity, whereas only a few numbers of proteins are necessary depending on the microbes (tesh et al., , li et al., ) . depending on sufficient antigen expression from experimental assays, traditional vaccine could take years to develop, while sometimes can lead to undesirable consequences (purcell et al., , petrovsky & aguilar, . reverse vaccinology approach, on the other hand, is an effective way to develop vaccine against covid- . in this method, computation analysis towards genomic architecture of pathogenic candidate could predict the antigens of pathogens without the prerequisite to culture the pathogens in lab condition. although, few pathogens that challenge to develop effective vaccines so far may become possible through such approach (rappuoli, ) which initiates a huge move in the development of vaccine against the deadly pathogens. the strategy included the comprehensive utilization of bioinformatics algorithm or tools to develop epitope based vaccine molecules, though further validation and experimental procedures are also needed (moxon et al., ) . in addition, peptide based subunit vaccines are biologically safer due to the absence of continuous in vitro culture during the production period, and also implies an appropriate activation of immune responses (purcell et al., ; dudek et al., ) . such immunoinformatic approaches have already been employed by the researchers to design vaccines against a number of deadly pathogens including ebola virus (khan et al., ) , hiv (pandey et al., ) , areanaviruses , marburgvirus , norwalk virus (azim et al., b) , nipah virus (saha et al., ) , influenza virus (hasan et al., b) and so on. at present, a suitable peptide vaccine against sars-cov- is urgently necessary that could efficiently generate enough immune response to destroy the virus. hence, the study was designed to develop a chimeric recombinant vaccine against covid- by targeting four major structural proteins of the pathogen, while revealing the evolutionary history of different species of coronavirus based on whole genome and protein domain-based phylogeny. complete genomes of the covid- and other coronaviruses were retrieved from the ncbi (https://www.ncbi.nlm.nih.gov/), using the keyword 'coronavirus' and the search option 'nucleotide'. a total complete genomes were retrieved, with unique identity (supplementary file ). protein sequence of the spike, envelope, membrane and nucleocapsid were also retrieved from the corresponding genome sequences found in ncbi (supplementary file ). tthe complete genome sequences of coronaviruses and the proteins of envelope, envelope, membrane and nucleocapsid were employed to construct different phylogenetic trees. multiple sequence alignment (msa) of the complete genome and protein sequences were performed using mafft v . (katoh & standley, ) tool. for the whole genome alignment, we used mafft auto algorithm, while for the protein sequences alignment, mafft g-ins-i algorithm was used using default parameters. next, alignment was visualized using the jalview- . (waterhouse et al., ) . alignment position with more than % gaps was pruned from coronavirus genome using phyutility . . program (smith & dunn, ) . again, more than % gaps from the spike protein alignment was removed. partitionfinder- . . (lanfear et al., ) indicated the best fit substitution model of the completed genome sequences and the protein sequences. the phylogeny of the whole genome sequences of coronavirus was constructed using both the maximum likelihood method and bayesian method. raxml version . . (stamatakis, ) with the substitution model gtrgammai was used using rapid bootstrap replicates. mrbayes version . . (ronquist et al., ) with invgamma model was used for the corona virus genomes. phylogenetic analyses of four different protein sequences were performed by using raxml- . . tool. for spike and nucleocapsid proteins, we found protgammaiwag and protgammaiwag as the best fil model, respectively. again, protgammawag was the best fit model of evolution for both the membrane and envelope proteins. for the retrieval of the domain sequences of the stated protein sequences, interpro database (https://www.ebi.ac.uk/interpro/) was utilized. finally, the interactive tree of life (itol; embl, heidelberg, germany) was used for the visualization of the phylogenetic trees. all the trees were rooted in the midpoint. in the present study, reverse vaccinology technique was utilized to model a novel multiepitope subunit vaccine against -ncov. the scheme in figure represents the complete methodology that has been adopted to develop the final vaccine construct. among proteins (available in the ncbi database) from different strains of novel corona virus, four structural proteins, i.e. spike glycoprotein, membrane glycoprotein, envelope protein and nucleocapsid protein, were prioritized for further investigation (supplementary file ). after sequence retrieval from ncbi, the sequences were subjected to blastp analysis to find out the homologous protein sequences. multiple sequence alignment was done by using clustal omega to identify the conserved regions (sievers and higgins, ) . the topology of each conserved regions were predicted by tmhmm server v. . (http://www.cbs.dtu.dk/services/tmhmm/), while the antigenicity of the conserved regions was determined by vaxijen v . (doytchinova and flower, a) . only the common fragments were used for t-cell epitopes enumeration via t-cell epitope prediction server of iedb (http://tools.iedb.org/main/tcell/) (vita et al., ) . again, tmhmm server was utilized for the prediction of transmembrane topology of predicted mhc-i and mhc-ii binding peptides followed by antigenicity scoring via vaxijen v . server (krogh et al., ; doytchinova and flower, b) . the epitopes which have antigenic potency were picked and used for preceding analysis. the level of conservancy scrutinizes the ability of epitope candidates to impart capacious spectrum immunity. homologous sequence sets of the chosen antigenic proteins were retrieved form the ncbi database by utilizing blastp tool. later, conservancy analysis tool (http://tools.iedb.org/conservancy/) in iedb was used to demonstrate the conservancy level of the predicted epitopes among different viral starins. the toxicity of non-allergenic epitopes was enumerated by using toxinpred server (gupta et al., ) . among different ethnic societies and geographic spaces, the hla distribution varies around the world. population coverage study was conducted by using iedb population coverage calculation server (vita et al., ) . to check the allergenicity of the proposed epitopes, four distinct servers i.e. allergenfp , allertop (dimitrov et al., ) , allermatch (fiers et al., and allergen online (http://www.allergenonline.org/) servers were utilized. three different algorithms i.e. bepipred linear epitope prediction . (jespersen et al., ) , emini surface accessibility prediction (emini et al., ) and kolaskar and tongaonkar antigenicity scale (kolaskar and tongaonkar, ) from iedb predicted the potential b-cell epitopes within conserved fragments of the chosen viral proteins. top ctl, htl and b cell epitopes were compiled to design the final vaccine constructs in the study. each vaccine constructs commenced with an adjuvant followed by top ctl epitopes, htl epitopes and bcl epitopes respectively. for construction of novel corona vaccine, the chosen adjuvants i.e. l /l ribosomal protein, beta defensin (a mer peptide) and haba protein (m. tuberculosis, accession number: agv . ) were used (rana and akhter, ) . several linkers such as eaaak, gggs, gpgpg and kk in association with padre sequence were incorporated to construct fruitful vaccine sequences against covid- . the constructed vaccines were then analyzed whether they are non-allergenic by utilizing the following tool named algpred (azim et al., ) . the most potential vaccine among the three constructs was then determined by assessing the antigenicity and solubility of the vaccines via vaxijen v . (doytchinova and flower, b) and proso ii server (smialowski et al., ) , respectively. protparam tool (https://web.expasy.org/protparam/), provided by expasy server (hasan et al., c ) was used to functionally characterize (gasteiger et al., ) the vaccine constructs. the studied functional properties were isoelectric ph, molecular weight, aliphatic index, instability index, hydropathicity, estimated half-life, gravy values and other physicochemical characteristics. alpha helix, beta sheet and coil structures of the vaccine constructs were analyzed through gor secondary structure prediction method using prabi (https://npsaprabi.ibcp.fr/). in addition, espript . (robert & gouet, ) was also used to predict the secondary structure of the stated protein sequences. vaccine d model was generated on the basis of percentage similarity between target protein and available template structures from pdb by using i-tasser (peng and xu, ) . the modeled structures were further refined via fg-md refinement server. structure validation was performed by ramachandran plot assessment in rampage (hasan et al., b) . by utilizing dbd server, probable disulfide bonds were designed for the anticipated vaccine constructs (craig and dombkowski, ) . the value of energy was considered < . , while the chi value for the residue screening was chosen between - to + for the operation (hasan et al., b) . the b-cell epitopes of putative vaccine molecules were predicted via ellipro server (http://tools.iedb.org/ellipro/) with minimum score . and maximum distance of Å (ponomarenko et al., ) . moreover, ifn-inducing epitopes within the vaccine were predicted using ifnepitope with motif and svm hybrid detection strategy (hajighahramani et al., ). normal mode analysis (nma) was performed to predict the stability and large scale mobility of the vaccine protein. the imod server determined the stability of construct v by comparing the essential dynamics to the normal modes of protein (aalten et al, ; wuthrich et al., ) . it is a recommended alternative to costly atomistic simulation (tama and brooks, ; cui and bahar, ) and shows much quicker and efficient assessments than the typical molecular dynamics (md) simulations tools (prabhakar et al., ; awan et al., ) . the main-chain deformability was also predicted by measuring the efficacy of target molecule to deform at each of its residues. the motion stiffness was represented via eigenvalue, while the covariance matrix and elastic network model was also analyzed. patchdock server was prioritized for docking between different hla alleles and the putative vaccine molecules. in addition, the superior construct was also docked with different human immune receptors such as, ace , apn, dpp and tlr- .the d structure of these receptors were retrieved from rcsb protein data bank. detection of highest binding affinity between the putative vaccine molecules and the receptor was experimented based on the lowest interaction energy of the docked structure. jcat tool was utilized for codon adaptation in order to fasten the expression of vaccine construct v in e. coli strain k . for this, some restriction enzymes (i.e. bgli and bglii), rho independent transcription termination and prokaryote ribosome-binding site were put away from the work (grote et al., ) . after that, the mrna sequence of constructed v vaccine was ligated within bgli ( ) and bglii ( ) restriction site at the c-terminal and n-terminal sites respectively. snapgene tool was utilized for in silico restriction cloning (solanki and tiwari, ). in the phylogenetic analysis, we introduced different coronavirus from three different genera: (forni et al., ; zhou et al., ; zumla et al., ) . among these, the first five species belong to the beta coronavirus genera, while the last two belongs to the alpha genera. apart from the human coronaviruses, we introduced other coronaviruses which choose different species of bats, whale, turkey, rat, mink, ferret, swine, camel, rabbit, cow and others as host (supplementary table- domain analysis of spike protein of coronaviruses reveals that they contain mainly one signature domains namely, coronavirus s glycoprotein (ipr ), which is present in all the candidates. all other betacoronavirus contains spike receptor binding protein (ipr ), coronavirus spike glycoprotein hapted receptor domain (ipr ) and spike receptor binding domain superfamily (ipr ). sars-cov- contains an extra domain, namely spike glycoprotein n-terminal domain (ipr ), which is also present in some the sub-genera (embecovirus) of betacoronavirus, but not in covid- . one important finding in our study is that the covid- candidates do not contain the domain spike glycoprotein (ipr ), which is present in the sars-cov- (figure ) . the secondary structure prediction study shows a large numbers of cysteine residues which contribute to the formation of disulfide bonds within the spike protein. most of them fall within the s spike protein, which is amino acid long in sars-cov- , while amino acids long in covid- . the rgd motif which is conserved within the covid- is present in the vicinity of the s protein. it exists as kgd that clearly demonstrates the mutation over the short time period. again, the receptor binding domain and receptor binding motif analyses disclose variations within several region between the covid and sars-cov- (supplementary file ). the domain-based phylogenetic analysis reflects two main divisions, where the all the novel betacoronavirus i.e., covid form clade with the sars-cov- ; while other betacoronavirus fall in another clade which further divide to give rise different sub-genera. this clearly shows that the covid- exerts specific ancestral connection to the sars-cov- in terms of spike glycoproteins. interestingly, our study also revealed close relatedness of both the sars-cov- and covid- to the bat betacoronavirus that belongs to the hibecovirus sub-genus. however, in our study, the bat coronaviruses of nobecovirus subgenus did not fall into the same clade of novel coronaviruses. the phylogenetic study and msa also revealed that, the functional portion of the spike glycoprotein domain and spike glycoprotein n-terminal domain might be lost from the covid- during the course of evolution. the envelope proteins of both betacoronavirus and alphacoronavirus contain only one protein domain (ipr ) namely, nonstructural protein ns or small envelope protein e (ns /e). this domain is well conserved in coronavirus and also found in murine hepatitis virus. on the other hands, the gamma coronavirus shows the exception, which possess (ipr ) ibv c protein domain, which thought to be expressed from the orf c gene of infectious bronchitis virus (jia & naqi, (figure ) . in spite to the previous findings, where it was found that the envelope proteins of the mers virus and sars-cov- exerted close proximity in terms of secondary structure and functions (surya et al., ) . unlike to earlier finding, we got that gamma corona virus candidate in our study shows close connection with both sars-cov- and covid- in terms of envelope proteins. membrane the length of nucleocapsid proteins of betacoronavirus genus ranges from to amino acids. three signature domains are mainly present in the nucleocapsid proteins, which are: coronavirus nucleocapsid protein (ipr ), nucleocapsid proteins c-terminal (ipr ) and nucleocapsid proteins n-terminal (ipr ). however, in our experiment, we didn't find these domains in hcov-hku ( figure ) showed that among the immunogenic conserved sequences from the corresponding proteins except spike glycoprotein met the criteria of desired exomembrane characteristics (table ) . a plethora of immunogenic epitopes were generated from the conserved sequences that were able to bind with most noteworthy number of hla cells (supplementary table , supplementary table , supplementary table and supplementary table ). top epitopes with exomembrane characteristics were ranked for each individual protein after investigating their antigenicity score and transmembrane topology (table ) . epitopes from each protein showed high level of conservancy up to % (table ). toxinpred server predicted the relative toxicity of each epitope which indicated that the top epitopes were non-toxin in nature (supplementary table ). population coverage of four structures proteins were also done for the predicted ctl and htl epitopes. from the screening, results showed that population of the various geographic regions could be covered by the predicted t-cell epitopes ( figure ) . finally, the allergenic epitopes were excluded from the list based on the evaluation of four allergenicity prediction server (supplementary table ). top b-cell epitopes were predicted for spike glycoprotein, membrane glycoprotein, envelope protein and nucleocapsid protein using distinct algorithms (i.e. bepipred linear epitope prediction, emini surface accessibility, kolaskar & tongaonkar antigenicity prediction) from iedb. epitopes were also allowed to analyze their vaxijen scoring and allergenicity (table ) . three putative vaccine molecules (i.e. v , v and v ) were constructed, each comprising a protein adjuvant, eight t-cell epitopes, twelve b-cell epitopes and respective linkers (supplementary table ). padre sequence was included to extend the efficacy and potency of the constructed vaccine. the putative vaccine constructs, v , v and v were , and residues long respectively. however, allergenicity score of v (- . ) revealed that it was superior among the three constructs in terms safety and efficacy. v also had a solubility score ( . ) ( figure e ) and antigenicity ( . ) over threshold value (table ) . protparam tool was employed to analyze the physicochemical properties of v . figure ) . tertiary structure of the putative vaccine construct v was generated using i-tasser server ( figure a and b). the server used best templates with highest significant (measured via zscore) from the lomets threading program to model the d structure. after refinement, ramachandran plot analysis revealed that . % and . % residues were in the favored and allowed regions respectively, while only residues ( . %) occupied in the outlier region ( figure c ). the overall quality factor determined by errat server was . % ( figure d ) ellipro server predicted a total conformational b-cell epitopes from the d structure of the construct v . epitopes no. were considered as the broadest conformational b cell epitopes with amino acid residues (figure and supplementary table ). stability of the vaccine construct v was investigated through mobility analysis ( figure a and b), b-factor, eigenvalue & deformability analysis, covariance map and recommended elastic network model. results revealed that the placements of hinges in the chain was insignificant ( figure c ) and the b-factor column gave an averaged rms ( figure d ). the estimated higher eigenvalue . e - ( figure e ) indicated low chance of deformation of vaccine protein v . the correlation matrix and elasticity of the construct have been shown in figure g and figure h , respectively. the structural interaction between hla alleles and the designed vaccines were investigated by molecular docking approach. the server detected the complexed structure by focusing on complementarity score, ace (atomic contact energy) and estimated interface area of the compound ( table ). the molecular affinity between the putative vaccine molecules v and several immune receptors were also experimented. the result showed that construct v interacted with each receptor with significantly lower binding energy (figure ). the codon adaptation index (cai) and gc content for the predicted codons of the putative vaccine constructs v were demonstrated as . and . % respectively. an insert of bp was found which lacked the restriction sites for bgli and bglii, thus providing comfort zone for cloning. the codons were inserted into pet a(+) vector alongside two restriction sites (bgli and bglii) and a clone of base pair was generated ( figure ). in december , a new coronavirus prevalence flourished in wuhan, china, causing clutter among the medical community, as well as to the rest of the world . the new species has been renamed as -ncov or, sars-cov- , already causing considerable number infections and deaths in china, italy, spain, iran, usa and to a growing degree throughout the world. the major outbreak and spread of sars-cov- in forced the scientific community to make considerable investment and research activity for developing a vaccine against the pathogen. however, owing to high infectivity and pathogenicity, the culture of sars-cov- needs biosafety level conditions, which may obstructed the rapid development of any vaccine or therapeutics. it had been found that about companies and academic institutions are engaged in such works (spinney et al., , ziady et al., . among the potential sars-cov- vaccines in the pipeline, four have nucleic acid based designs, four involve non-replicating viruses or protein constructs, two contain live attenuated virus and one involves a viral vector (pang et al., ) , while only one, called mrna- (developed by niaid collaboration with moderna, inc.), has confirmed to start phase- trial (nih, ) . however, in this study we emphasized on a different approaches by prioritizing the advantages of different genome and proteome database using the immunoinformatic approach. computational vaccine predictions were adopted by the researchers to design vaccines against both mers-cov (sudhakar et al., ; fernando et al., ) and sars-cov- (yang et al., ; oany et al., ) , targeting the outer membrane or functional proteins (sharmin and islam, ) . several in silico strategies have also been employed to predict potential t cell and b cell epitopes against sars-cov- , either emphasizing on spike glycoprotein or envelope proteins (behbahani, ; rasheed et al., ) . none of the studies, however, focused on other structural proteins. moreover, random genetic changes and mutations in the protein sequences (yin, ) may obstruct the development of effective vaccines and therapeutics against human coronavirus in the future. hence, the present study was employed to identify the similarity and divergence among the close relatives of the target pathogen and develop a novel chimeric recombinant vaccine considering all major structural proteins i.e. spike glycoprotein, membrane glycoprotein, envelope protein and nucleocapsid protein simultaneously. the topology of the phylogenetic trees of the whole genome and the stated four proteins sequences from different species of coronaviruses reveal that sars-cov- and bat coronaviruses are the closest homologs of the novel coronaviruses. our results infer a significant level of similarities within the covid- and sars-cov- which was also aligned with the previous findings (jaimes et al., ; wu, a) . the sequence similarities between the sars-cov, bat coronaviruses and the covid- from the reported studies (hu et al., ; wu, b) suggests that those are distantly related, in spite those are capable of infecting the humans and therefore possess the adaptive convergent evolution. interestingly, the covid- envelope proteins form clade with the turkey coronavirus which belongs to gamma coronavirus genus. so, in terms of envelope proteins, the envelope gene of turkey coronavirus might contribute to the convergence process, which need further analysis. in addition, from the domain-based phylogeny of nucleocapsid proteins, it can be deduced that this protein might have originated in bats and was transmitted to camels and then later on choose human as the potential host. overall, the covid- might go through complex adaptation strategies in order to be transmitted into the human via different animals. the homologous protein sets for four structural proteins of coronavirus were sorted to identify conserved regions through blastp analysis and msa. only the conserved sequences were utilized to identify potential b-cell and t-cell epitopes for each individual protein (table ) . thus, our constructs are expected to stimulate a broad-spectrum immunity in host upon administration. cytotoxic cd +t lymphocytes (ctl) play a crucial role to control the spread of pathogens by recognizing and killing diseased cells or by means of antiviral cytokine secretion (garcia et al., ) . thus, t cell epitope-based vaccination is a unique process to confer defensive response against pathogenic candidates (shrestha, ) . approximately mhc-i peptides (ctl epitopes) and mhc-ii peptides (htl epitopes) were predicted via iedb server, from which we screened the top ones through analyzing the antigenicity score, transmembrane topology, conservancy level and other important physiochemical parameters employing a number of bioinformatics tools ( table ). the top epitopes from each protein was further assessed by investigating the toxicity profile and allergenicity pattern. different servers rely on different parameters to predict the allergenic nature of small peptides. therefore, we used distinct servers for such assessment and the epitopes predicted as non-allergen at least via servers were retained for further analysis (supplementary table ). vaccine initiates the generation of effective antibodies that are usually produced by b cells and plays effector functions by targeting specifically to a foreign particles (cooper & nemerow, ) . the potential b cell epitopes were generated by three different algorithms (bepipred linear epitope prediction . , kolaskar and tongaonkar antigenicity prediction and emini surface accessibility prediction) from iedb database (table ) . suitable linkers and adjuvants were used to combine top finalized epitopes from each protein that led to develop a multi epitope vaccine molecules (supplementary table ). as padre sequence was usually recommended to lessen the polymorphism of hla molecules in the population (ghaffari-nazari et al., ) , it was also considered to construct the final vaccine molecule. here, adjuvants would enhance the immunogenicity of the vaccine constructs and appropriate separation of epitopes in the host environment would be ensured by the linker (yang et al., ) . allergenicity, physiochemical properties, antigenicity and three-dimensional structure of vaccine constructs were characterized, and it had been concluded that v was superior to v and v vaccine constr. the final construct also occupied by several interferon-α producing epitopes (supplementary table ). the vaccine protein (v ) was subjected to disulfide engineering to enhance its stability. analysis of the normal modes in internal coordinates by imods was employed to investigate the collective motion of vaccine molecules (lopez-blanco et al., ) . negligible chance of deformability at molecular level was analyzed for the putative vaccine construct v , thereby strengthening our prediction. moreover, molecular docking was investigated to analyze the molecular affinity of the vaccine with different hla molecules i.e. drb * , drb * , drb * , drb * , drb * and drb * (table ) . it had been reported that a specific receptor-binding domain of cov spike protein usually recognizes its host receptor ace (angiotensin-converting enzyme ) (li. et al., ; li, ) . previous studies also identified dipeptidyl peptidase (dpp ) as a functional receptor for human coronavirus (raj et al., ) . therefore, we performed another docking study prioritizing these immune receptors to strengthen our prediction ( figure ). results showed that the designed construct bound with the selected receptors with minimum binding energy which was biologically significant. finally, in-silico restriction cloning was adopted to check the suitability of construct v for entry into pet a (+) vector and expression in e. coli strain k (figure ). traditional ways to vaccine development are time consuming and laborious. moreover, the result may not be always as expected or fruitful (stratton et al., ; hasan et al., ) . in silico prediction and prescreening methods, on the contrary, offer some advantages while saving time and cost for production. therefore, the present study may aid in the development of preventive strategies and novel vaccines to combat infections caused by -ncov. however, further wet lab trials involving model organism needs to be experimented for validating our findings. the darker the greys, the stiffer the springs (h). a comparison of techniques for calculating protein essential dynamics mutation-structure function relationship based integrated strategy reveals the potential impact of deleterious missense mutations in autophagy related proteins on hepatocellular carcinoma (hcc): a comprehensive informatics approach conglomeration of highly antigenic nucleoproteins to inaugurate a heterosubtypic next generation vaccine candidate against arenaviridae family. biorxiv immunoinformatics approaches for designing a novel multi epitope peptide vaccine against human norovirus in silico design of novel multi-epitope recombinant vaccine based on coronavirus spike glycoprotein genomic variance of the -ncov coronavirus epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study pathogenicity and transmissibility of -ncov-a quick overview and comparison with other emerging viruses the role of antibody and complement in the control of viral infections disulfide by design . : a web-based tool for disulfide engineering in proteins normal mode analysis theoretical and applications to biological and chemical systems allertop v. -a server for in silico prediction of allergens allergenfp: allergenicity prediction by descriptor fingerprints identifying candidate subunit vaccines using an alignmentindependent method based on principal amino acid properties a server for prediction of protective antigens, tumour antigens and subunit vaccines epitope discovery and their use in peptide based vaccines induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide engineering a replication-competent, propagation defective middle east respiratory syndrome coronavirus as a vaccine candidate allermatch™, a webtool for the prediction of potential allergenicity according to current fao/who codex alimentarius guidelines molecular evolution of human coronavirus genomes structural basis of t cell recognition improving multi-epitope long peptide vaccine potency by using a strategy that enhances cd +t help in balb/c mice a decade after sars: strategies for controlling emerging coronaviruses jcat: a novel tool to adapt codon usage of a target gene to its potential expression host clinical characteristics of coronavirus disease in china silico approach for predicting toxicity of peptides and proteins vaccinomics strategy for developing a unique multi-epitope monovalent vaccine against marburg marburgvirus. infection, genetics and evolution contriving a chimeric polyvalent vaccine to prevent infections caused by herpes simplex virus (type- and type- ): an exploratory immunoinformatic approach reverse vaccinology approach to design a novel multi-epitope subunit vaccine against avian influenza a (h n ) virus, microbial pathogenesis genomic characterization and infectivity of a novel sars-like coronavirus in chinese bats. emerging microbes and infections clinical features of patients infected with novel coronavirus in wuhan, china the continuing -ncov epidemic threat of novel coronaviruses to global health-the latest novel coronavirusoutbreak in wuhan structural modeling of -novel coronavirus (ncov) spike protein reveals a proteolytically-sensitive activation loop as a distinguishing feature compared to sars-cov and related sars-like coronaviruses chinese scientists race to develop vaccine as coronavirus death toll jumps". south china morning post bepipred- . : improving sequence-based bcell epitope prediction using conformational epitopes sequence analysis of gene , gene and gene of avian infectious bronchitis virus strain cu-t mafft multiple sequence alignment software version : improvements in performance and usability epitope-based peptide vaccine design and target site depiction against ebola viruses: an immunoinformatics study a semi-empirical method for prediction of antigenic determinants on protein antigens predicting transmembrane protein topology with a hidden markov model: application to complete genomes covid- pandemic: palliative care for elderly and frail patients at home and in residential and nursing homes partitionfinder : new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses receptor recognition mechanisms of coronaviruses: a decade of structural studies peptide vaccine: progress and challenges angiotensin-converting enzyme is a functional receptor for the sars coronavirus imods: internal coordinates normal mode analysis server outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein nih clinical trial of investigational vaccine for covid- begins. study enrolling seattle-based healthy adult volunteers. accessed on design of an epitope-based peptide vaccine against spike protein of human coronavirus: an in silico approach. drug design, development and therapy immunoinformatics approaches to design a novel multi-epitope subunit vaccine against hiv infection. vaccine potential rapid diagnostics, vaccine and therapeutics for novel coronavirus ( -ncov): a systematic review exploiting structure information for protein alignment by statistical inference vaccine adjuvants: current state and future trends. immunology and cell biology monomerization alters the dynamics of the lid region in campylobacter jejuni cstii: an md simulation study more than one reason to rethink the use of peptides in vaccine design dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc reverse vaccinology silico identification of novel b cell and t cell epitopes of wuhan coronavirus ( -ncov) for effective multi epitope-based peptide vaccine production deciphering key features in protein structures with the new endscript server mrbayes . : efficient bayesian phylogenetic inference and model choice across a large model space in silico identification and characterization of common epitope-based peptide vaccine for nipah and hendra viruses. asian pacific journal of tropical medicine a highly conserved wdypkcdra epitope in the rna directed rna polymerase of human coronaviruses can be used as epitope-based universal vaccine design role of cd + t cells in control of west nile virus infection clustal omega, accurate alignment of very large numbers of sequences protein solubility: sequence based prediction and experimental verification phyutility: a phyloinformatics tool for trees, alignments and molecular data subtractive proteomics to identify novel drug targets and reverse vaccinology for the development of chimeric vaccine against acinetobacter baumannii when will a coronavirus vaccine be ready?". the guardian. retrieved raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies immunization safety review: vaccinations and sudden unexpected death in infancy platform strategies for rapid response against emerging coronaviruses: mers-cov serologic and antigenic relationships in vaccine design potential factors influencing repeated sars outbreaks in china covid- : epidemiology, evolution, and cross-disciplinary perspectives mers coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels symmetry, form, and shape: guiding principles for robustness in macromolecular machines efficacy of killed virus vaccine, live attenuated chimeric virus vaccine, and passive immunization for prevention of west nile virus encephalitis in hamster model. emerging infectious diseases cytokines: the future of intranasal vaccine adjuvants. clinical and developmental immunology the immune epitope database (iedb) . a novel coronavirus outbreak of global health concern. the lancet jalview version -a multiple sequence alignment editor and analysis workbench coronavirus disease (covid- ) outbreak infection prevention and control during health care when covid- is suspected: interim guidance characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention a new coronavirus associated with human respiratory disease in china strong evolutionary convergence of receptor-binding protein spike between covid- and sars-related coronaviruses strong evolutionary convergence of receptor-binding protein spike between covid- and sars-related coronaviruses correlations between internal mobility and stability of globular proteins an evolutionary rgd motif in the spike protein of sars-cov- may serve as a potential high risk factor for virus infection ? in silico design of a dna-based hiv- multiepitope vaccine for chinese populations a dna vaccine induces sars coronavirus neutralization and protective immunity in mice genotyping coronavirus sars-cov- : methods and implications atomic-level protein structure refinement using fragment-guided molecular dynamics conformation sampling a pneumonia outbreak associated with a new coronavirus of probable bat origin network-based drug repurposing for novel coronavirus -ncov/sars-cov- a novel coronavirus from patients with pneumonia in china biotech company moderna says its coronavirus vaccine is ready for first tests coronaviruses-drug discovery and therapeutic options authors would like to acknowledge the department of biochemistry and chemistry, department of microbial biotechnology and department of pharmaceuticals and industrial biotechnology of sylhet agricultural university for the technical support of the project. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. authors declare that they have no conflict of interests. supplementary table : predicted ctl and htl epitopes of spike glycoprotein. key: cord- -jek pd authors: fisher, kimberly a.; bloomstone, sarah j.; walder, jeremy; crawford, sybil; fouayzi, hassan; mazor, kathleen m. title: attitudes toward a potential sars-cov- vaccine: a survey of u.s. adults date: - - journal: ann intern med doi: . /m - sha: doc_id: cord_uid: jek pd background: coronavirus disease (covid- ) has rapidly instigated a global pandemic. vaccine development is proceeding at an unprecedented pace. once available, it will be important to maximize vaccine uptake and coverage. objective: to assess intent to be vaccinated against covid- among a representative sample of adults in the united states and identify predictors of and reasons for vaccine hesitancy. design: cross-sectional survey, fielded from through april . setting: representative sample of adults residing in the united states. participants: approximately adults drawn from the amerispeak probability-based research panel, covering approximately % of the u.s. household population. measurements: intent to be vaccinated against covid- was measured with the question, “when a vaccine for the coronavirus becomes available, will you get vaccinated?” response options were “yes,” “no,” and “not sure.” participants who responded “no” or “not sure” were asked to provide a reason. results: a total of amerispeak panel members responded. overall, . % of participants (n = ) intended to be vaccinated, . % (n = ) were not sure, and . % (n = ) did not intend to be vaccinated. factors independently associated with vaccine hesitancy (a response of “no” or “not sure”) included younger age, black race, lower educational attainment, and not having received the influenza vaccine in the prior year. reasons for vaccine hesitancy included vaccine-specific concerns, a need for more information, antivaccine attitudes or beliefs, and a lack of trust. limitations: participants' intent to be vaccinated was explored before a vaccine was available and when the pandemic was affecting a narrower swath of the united states. questions about specific information or factors that might increase vaccination acceptance were not included. the survey response rate was . %. conclusion: this national survey, conducted during the coronavirus pandemic, revealed that approximately in adults were not sure they would accept vaccination and in did not intend to be vaccinated against covid- . targeted and multipronged efforts will be needed to increase acceptance of a covid- vaccine when one becomes available. primary funding source: agency for healthcare research and quality. c oronavirus disease is caused by the ␤-coronavirus severe acute respiratory syndrome coronavirus (sars-cov- ). this virus has rapidly become a major global threat, instigating a pandemic affecting more than countries and people and leading to nearly deaths worldwide ( ) . the pandemic has overwhelmed hospital systems, undermined economic activity worldwide, and instilled fear into the general populace ( , ) . an international poll conducted in april found that % of those surveyed identified covid- as the most concerning national issue, overtaking unemployment, health care, and poverty ( ) . in a separate survey conducted at the same time in the united states, more than % of participants were very or somewhat concerned about being infected with coronavirus ( ) . in response to the massive global effects of covid- , multiple laboratories worldwide are working to create an effective vaccine. the possibility that one will be available in to months is seen by many as the most promising means of controlling the covid- pandemic. over the past century, vaccinations have become a routine and effective preventive measure in reducing the rate of and eradicating or nearly eradicating certain viral illnesses ( ) . besides providing direct immunity and preventing disease among vaccinated individuals, vaccines have been shown to reduce infections even among individuals who are not vaccinated, through herd immunity, if a sufficient proportion of the population is immune ( ) . many pharmaceutical companies and research labs are currently working with messenger rna, dna, subunit, virus-like particles, and viral vectors to discover an effective vaccine for the covid- pandemic ( , ) . on an unprecedented timeline, multiple vaccines have been developed and are currently being tested in large-scale phase trials ( ) , suggesting that a vaccine may be available in the foreseeable future. the great potential of a vaccine against covid- is tempered by rising vaccine skepticism in the united states and worldwide, which may present challenges to widespread vaccine uptake when a vaccine becomes available ( ) ( ) ( ) ( ) . it is unknown whether the unprecedented and severe effects of covid- in the united states will overcome vaccine skepticism and foster widespread acceptance of and demand for vaccination. we assessed intent to be vaccinated for the novel coronavirus with the question, "when a vaccine for the coronavirus becomes available, will you get vaccinated?" followed by the response options "yes," "no," and "not sure." participants who responded "no" or "not sure" were asked one of the following open-ended questions, respectively: "what makes you unwilling to get the vaccine?" or "what makes you unsure whether you will get the vaccine?." to assess perceived risks of infection, we asked, "what is your best guess as to whether you will get the coronavirus within the next months?"; response options were "i don't think i will get the coronavirus," "i think i will get a mild case of the coronavirus," "i think i will get seriously ill from the coronavirus," or "i have already had the coronavirus." survey items are shown in appendix table (available at annals.org). we conducted rounds of pilot testing of the main question assessing intent to be vaccinated among a convenience sample of over individuals and did not detect any problems. data on participant characteristics were provided by norc and included age, sex, race/ethnicity, educational attainment, household income, household size, marital status, employment status, geographic location, urban or rural location (addresses within a metropolitan statistical area were categorized as urban), receipt of influenza vaccination in the prior year, and self-rated overall health status. norc collects data on healthrelated variables (such as receipt of influenza vaccination and self-rated overall health status) upon enrollment or soon after for most panel members; if a panel member has not responded to a specific item, that item may be included on subsequent surveys. all data provided to the investigators were fully deidentified. participant characteristics were summarized by using frequencies and percentages. we used crosstabulations and tests to estimate unadjusted associations of participant characteristics and perceived personal risk for coronavirus with the -category outcome intent to get vaccinated. to better distinguish characteristics associated with responses of "not sure" versus "yes" and characteristics associated with responses of "no" versus "yes," we also calculated separate tests and associated p values for these sets of comparisons. to estimate corresponding adjusted (multivariate) associations, we used multinomial logistic regression, an extension of binomial logistic regression that compares each of or more nonordered outcome categories to the reference category. in particular, we modeled both natural log [pr (not sure)/pr (yes)] and natural log [pr (no)/pr (yes)] as a function of participant characteristics. this approach allows different associations with covariates for the comparisons while providing overall p values for covariates. whereas coefficients from a binomial logistic regression model are typically exponentiated to obtain odds ratios, exponentiated coefficients from a multinomial logistic regression model are interpreted as relative risk ratios (rrrs). an illustrative calculation is provided in the footnote to table . characteristics that were not statistically significant (p < . ) in the multivariate multinomial modeling were omitted in the final model; these characteristics were found to be correlated with predictors retained in the final model (for example, household income was related to education). we considered the possibility that inclusion of prior receipt of influenza vaccine in the model may obscure other predictors of covid- vaccine hesitancy owing to overlap in the reasons for reluctance to get an influenza or covid- vaccine. we therefore repeated the primary analysis after removing receipt of influenza vaccine from the model. adjusted percentages were calculated for each predictor category by fixing all other predictors at their observed dis- attitudes toward a potential sars cov- vaccine among u.s. adults tributions. to assess model performance, we calculated c-statistics and hosmer-lemeshow statistics separately for binomial logistic regressions for "not sure" versus "yes" and "no" versus "yes." all analyses incorporated survey sampling weights based on gender, age, education, race/ethnicity, and region. analyses were conducted by using sas, version . . we used thematic analysis to inductively generate codes and identify themes in the responses to the open-ended query soliciting reasons for vaccine hesitancy ( ) . the coding team included investigators with backgrounds in health communication, health literacy, patient-provider communication, clinical medicine, and clinical social work; all coding team members had prior experience in qualitative analysis. a coding framework was created on the basis of initial review of all responses. codes and associated definitions were revised and refined through iterative application and discussion. two analysts (k.f., s.b.) then independently coded all responses. more than code could be assigned to a response if applicable. coding discrepancies were discussed until agreement was reached; the third member of the coding team was available to adjudicate but was not needed. codes were assigned in excel; final codes were merged into spss, version , to facilitate data manipulation and summarization. our study was determined to be exempt by the university of massachusetts medical school institutional review board. dr. fisher is supported by agency for healthcare research and quality grant k hs . the funder had no role in the design, conduct, or analysis of this study. the amerispeak omnibus survey was released to panel members, and a total of ( . %) responded. most participants ( . %) completed the survey via the web; the remainder ( . %) completed it via telephone interview. twelve participants did not respond to the question on intent to be vaccinated; all results presented here are based on the participants who responded to this question. a majority of participants ( . %) were white, approximately one third ( . %) were years of age or older, and . % were female. participants had varied levels of educational attainment, with more than one third ( . %) having a high-school diploma or less. most participants perceived their risk for coronavirus to be low, predicting that they will either not get the coronavirus ( . %) or that they will get a mild case of the coronavirus ( . %) in the next months. only participants ( . %) predicted they will get seriously ill from the coronavirus. approximately one half ( . %) of participants reported having received the influenza vaccine previously. additional participant characteristics are shown in table . overall, . % of participants (n = ) intended to be vaccinated, . % (n = ) were not sure whether they would be vaccinated, and . % (n = ) did not intend to be vaccinated. participant characteristics associated with a higher chance of responding "no" or "not sure" versus "yes" were being younger (< years), female, or black or hispanic; having lower educational attainment, lower household income, or larger household size, and being less likely to report having received an influenza vaccine. in addition to these differences, participants who responded "not sure" were more likely to live in the south or west and to believe they were at less personal risk for coronavirus despite providing lower ratings of their overall health. participants who responded "no" were more likely to live in a rural setting ( table ) . after adjustment for differences in participant characteristics ( table ) , factors that were independently associated with vaccine hesitancy (response of "no" or "not sure") include younger age (< years), black race, educational attainment of less than a college degree, and not receiving an influenza vaccine in the prior year. participants who did not have a high school diploma had a nearly -fold higher relative likelihood of responding "no" versus "yes" compared with those who had a college degree or higher (rrr, . [ % ci, . to . ]). black race was associated with a more than -fold higher chance (rrr, . [ci, . to . ]) of not intending to be vaccinated versus intending to be vaccinated compared with white race. participants who had previously received an influenza vaccine had a % lower relative likelihood of responding "no" versus "yes" (rrr, . [ci, . to . ]) compared with those who had not received an influenza vaccine. other characteristics, such as female sex, some age strata, hispanic ethnicity, and perceived personal risk for coronavirus, were associated with vaccination intent but did not consistently achieve statistical significance for both response categories ("not sure" and "no"). living in a rural area was strongly associated with responding "no" when asked about intent to be vaccinated, but not with responding "not sure." household income, household size, region, and self-reported health were not significantly associated with vaccination intent after adjustment for the characteristics in table . results including these as model predictors were similar (data not shown). removal of prior receipt of influenza vaccine from the multinomial model resulted in an increase in the relative risk ratios comparing "no" versus "yes" for age groups ( to years and to years), such that the ci no longer included while other results remained similar (appendix table , available at annals .org). because one of the main goals of our study was to predict who may be hesitant to be vaccinated against covid- and prior receipt of influenza vaccine offers a pragmatic way to identify these individuals, we report the findings from the model that included prior receipt of influenza vaccine. hosmer-lemeshow statistics for "not sure" versus "yes" and for "no" versus "yes" were not statistically significant (p = . and . , respectively), and corresponding c-statistics were . and . , indicating excellent model fit and performance. of the participants who were unsure or did not intend to be vaccinated, ( . %) provided a reason for their response and constitute the sample for the qualitative analysis. the remaining participants who answered "not sure" or "no" ( . %) did not provide a reason for their hesitancy (for example, they did not respond, responded simply "don't know," or provided an uninterpretable response). participants' reasons for being unsure or not intending to be vaccinated are broadly categorized as having specific concerns about the vaccine; needing additional information; holding antivaccine attitudes, beliefs, or emotions; and not trusting entities involved in vaccine development, testing, or dissemination ( table ). the most common reasons cited by participants who were not sure whether they will be vaccinated included specific concerns about the vaccine (such as safety or effectiveness) or a need for more information. in contrast, the most common reasons provided by participants who did not intend to be vaccinated included antivaccine attitudes, beliefs, or emotions, and lack of trust. illustrative quotes are provided in appendix table (available at annals .org). in this large, nationally representative sample, nearly one half ( . %) of participants indicated hesitancy to be vaccinated against covid- when a vaccine becomes available. this finding is especially striking considering that the survey was conducted during mid-april , when the number of deaths per day due to covid- were at or near peak levels of the initial surge in the united states ( ). the percentage of individuals who intend to be vaccinated ( %) is only slightly higher than the percentage of adults who received the influenza vaccination ( %) during the - influenza season ( ); this is surprising, attitudes toward a potential sars cov- vaccine among u.s. adults given the increased severity, death rate, societal disruption, and resultant media coverage associated with the covid- pandemic. increasing vaccination rates are expected to confer substantial benefits, including reductions in covid- related hospitalizations, strain on hospital capacity, and deaths. for example, it has been estimated that increasing influenza vaccination coverage by percentage points could have prevented to hospitalizations in the - influenza season ( ) . the increased severity of covid- compared with influenza suggests that the magnitude of benefit of increased coronavirus vaccination coverage could be even greater. the percentage of individuals who will need to be vaccinated to achieve herd protection is not yet defined for covid- because it depends on vaccine effectiveness, patterns of population mixing, vaccination patterns, and the basic reproduction number (r ) ( ) of the novel coronavirus. using a pooled estimate of the r of . ( ) and assuming a best-case scenario in which a vaccine has perfect effectiveness yields a projection that at least % of the population will need to be vaccinated to achieve herd protection. in fact, a newly developed coronavirus vaccine is unlikely to be perfectly effective, so the coverage required to achieve herd immunity will almost certainly be higher than %. considering that intent as assessed in our study does not account for incomplete followthrough and barriers to vaccine access, it is likely that a substantial gap will exist in the number needed to be vac- attitudes toward a potential sars cov- vaccine among u.s. adults cinated to achieve herd protection and the number who receive vaccination. concerted efforts will be needed to persuade the large percentage of individuals who are unsure about or opposed to being vaccinated against covid- if we are to realize the substantial benefits afforded by high immunization coverage rates. we found several independent predictors of being hesitant to be vaccinated against covid- ; the strongest were lower educational attainment, black race, not having had a recent influenza vaccination, and perceived personal risk for coronavirus, consistent with the findings of a national survey conducted by rti ( ) . evidence that these characteristics are predictive of vaccine hesitancy could be useful in targeting vaccine messaging and outreach to populations at risk for not being vaccinated. our findings highlight the importance of social determinants of health, such as educational status (a close proxy for health literacy [ ] ) and race/ethnicity, and their influence on preventive health behaviors ( ) . racial disparities in vaccination rates have been described for other vaccinations. for example, rates of influenza vaccination among african american persons ( . %) and hispanic persons ( . %) were substantially lower than among white persons ( . ) during - ( ). these differences are particularly concerning given the disproportionately high toll of covid- among african american communities ( - ). the confluence of increased covid- disease burden and potential for decreased receipt of vaccination has the potential to substantially magnify health-related disparities experienced by african american persons. our findings highlight the need for vaccine implementation strategies that anticipate racial gaps in covid- vaccination. these strategies could draw on the approaches used to successfully close racial disparities in measles vaccination while being mindful of persistently lower rates of influenza vaccination rates among minority adults stemming from lack of trust in health care ( ) . prior research has demonstrated the importance of social norms and perceived disease risk in influencing vaccination decisions among african american persons and could be explored as a means of fostering coronavirus vaccine acceptance among this population ( , ) . the association between intent to be vaccinated and perceived risk for coronavirus suggests this may be a particularly important lever for promoting vaccination. in addition to being targeted for populations least likely to be vaccinated, such as members of racial and attitudes toward a potential sars cov- vaccine among u.s. adults annals.org annals of internal medicine ethnic minority groups and individuals of low health literacy, successful vaccination campaigns will need to leverage an understanding of why individuals may be hesitant to be vaccinated in order to tailor messaging to mitigate these concerns. concern about vaccine safety was one of the most commonly cited reasons for being unsure about accepting vaccination in the present study, consistent with studies of other vaccines ( ). a reuters poll found that approximately % of americans would agree to be vaccinated against covid- if they received assurances about the safety of the vaccine ( ). collectively, these findings suggest that transparent reporting of vaccine safety in a way that people of all educational levels can understand is likely to be an effective strategy to increase public uptake of vaccination. however, many participants in our study and the reuters poll indicated hesitancy to be among the first to be vaccinated, which will probably delay achievement of high vaccination coverage rates for covid- . over one half ( . %) of respondents who provided a reason for not intending to be vaccinated referred to antivaccine attitudes, beliefs, or emotions. of these, many indicated only that they did not like, want, or believe in vaccines, whereas others made explicit reference to scientifically inaccurate information, such as the association between vaccines and autism and that it is not possible to vaccinate against a virus. these beliefs and essentially emotional responses to vaccination are likely to be among the hardest to overcome, because information alone is unlikely to have an effect. it may be that messages designed to engage and influence emotions, such as narratives or stories, will be more effective than expository or informational health messages ( ). lack of trust was the second most common reason for responding "no" to intent to be vaccinated. trust has been shown to be a determinant of vaccine uptake ( ), suggesting this finding is likely to be of consequence and indicating a need for strategies aimed at increasing trust among individuals with greater degrees of vaccine skepticism. we found that circulating conspiracy theories about the coronavirus vaccination have taken hold among a small percentage of participants, in addition to more common misconceptions about vaccines. further research is needed to develop effective strategies to combat conspiracy theories and misinformation ( ). some participants in our study also cited prior experience with the influenza vaccine "not working" as a reason to believe a vaccine against the coronavirus will not be effective, demonstrating the negative effects of perceived ineffective vaccines on overall vaccine acceptance. given the real possibility for variable rates of effectiveness among the covid- vaccines currently in development and the possible need for revaccination, public health officials might consider proactively acknowledging this possibility to avoid further loss of trust if or when this happens. surprisingly, very few vaccine-hesitant participants indicated a need or desire for a recommendation from a physician. however, there is evidence that patients whose physicians recommend a vaccine are more likely to be vaccinated than patients who do not ( ). it has been argued that physicians are well-positioned to address misinformation, discuss risk, and convey the seriousness of covid- in a way that is tailored to the unique needs of the individual patient during an encounter ( ). such conversations may be the ideal but may be difficult to implement in time-limited primary care encounters, where there are typically many competing priorities. in addition, the effectiveness of such conversations will almost certainly depend on the patient having trust in the physician and the physician having the requisite time, skills, and comfort to address the emotion-laden topic of vaccine hesitancy. given the time constraints of primary care and the potential need for physicians to receive additional training to enable them to successfully address vaccine-related concerns, health systems might consider an alternative strategy in which trained vaccine counselors use motivational interviewing to engage vaccine-hesitant individuals. this approach has been effective at increasing rates of infant vaccine coverage and adolescent human papillomavirus vaccination ( , ). we have identified characteristics, such as not previously receiving an influenza vaccine, that are readily available in the electronic health record and could easily be used to identify covid- vaccine-hesitant individuals who might especially benefit from the motivational interviewing approach. our findings suggest that a multipronged approach may be needed in which trusted physicians promote vaccine uptake against a backdrop of innovative approaches and channels to combat vaccine misinformation, consistent with the body of literature of strategies to address vaccine hesitancy ( ). a strength of our study is that the large, nationally representative sample allows generalization of our findings. in addition, the timing of the survey administration coincided with a peak time of the pandemic in many parts of the united states, making the findings particularly timely and salient. our study also has limitations. first, we queried individuals about their intent to be vaccinated at a time when a vaccination is not yet available. it is possible that as more details regarding a potential vaccine are known, some participants who indicated their response depended on additional information may change their response. in addition, our study was not designed to determine what additional information is needed, or how best to deliver it. future research is needed to better delineate the types of assurances needed and the messengers most likely to be trusted (for example, community leaders and religious leaders). in conclusion, we found that a substantial proportion ( . %) of participants in a national survey conducted during the coronavirus pandemic would be hesitant to accept vaccination against covid- . black race was one of the strongest independent predictors of not accepting vaccination; this is especially alarming, given the outsized impact of covid- among african-americans. our findings suggest that many of the individuals who responded "not sure" may accept vaccination if given credible information that the vaccine is safe original research attitudes toward a potential sars cov- vaccine among u.s. adults and effective. as vaccine development proceeds at an unprecedented pace, parallel efforts to proactively develop messages to foster vaccine acceptance are needed to achieve control of the covid- pandemic. ) "there is no way i trust big pharma companies." vaccine development or testing processes ( . ) ( . ) "i'm thinking a vaccine now might be approved too quickly because of political pressure." "rushing to get a vaccine out will be a danger." continued on following page world health organization. coronavirus disease (covid- ): situation report . world health organization; . accessed at www.who.int/docs/default-source/coronaviruse/situation-reports / covid- -sitrep- .pdf?sfvrsn= f _ on the socio-economic implications of the coronavirus pandemic (covid- ): a review asian critical care clinical trials group. intensive care management of coronavirus disease (covid- ): challenges and recommendations what worries the world topline & methodology. . accessed at www.ipsos.com /sites/default/files/ipsos-coronavirus-us-aggregate-topline- vaccines through centuries: major cornerstones of global health. front public health herd immunity": a rough guide current status of potential therapeutic candidates for the covid- crisis microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development accessed at www.nytimes.com/interactive/ /science/coronavirus-vaccine-tracker national update on measles cases and outbreaks-united states who releases list of threats to global health institute for health metrics and evaluation projected population benefit of increased effectiveness and coverage of influenza vaccination on influenza burden in the united states estimate of the basic reproduction number for covid- : a systematic review and meta-analysis predicting willingness to vaccinate for covid- in the us the prevalence of limited health literacy health literacy and preventive health care use among medicare enrollees in a managed care organization hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease -covid-net, states racial and ethnic disparities in sars-cov- pandemic: analysis of a covid- observational registry for a diverse u.s. metropolitan population. medrxiv. preprint posted online the influence of social norms on flu vaccination among african american and white adults open ended if not sure: what makes you unsure whether you will get the vaccine?open ended what is your best guess as to whether you will get the coronavirus within the next months? i don't think i will get the coronavirus i think i will get a mild case of the coronavirus i think i will get seriously ill from the coronavirus i have already had the coronavirus appendix reference to specific conspiracy theories ( . ) ( . ) "[. . .] i personally do not believe that the virus was fully caused by infected animals in wuhan.[. . .] i believe that the vaccine is a governmental covert method to kill off more people, and then some." "as long as bill gates is involved with any of this, there's no way in hell i or anyone in my family would do this." "because i heard the government was to put a chip in you when you get the vaccination and i do not want a chip inside of me." distrust unspecified ( . ) ( . ) "i don't trust them." key: cord- -ob l bcf authors: bar-zeev, naor; inglesby, tom title: covid- vaccines: early success and remaining challenges date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: ob l bcf nan in the lancet, denis y logunov and colleagues from the n f gamaleya research institute of epidemiology and microbiology in russia present findings from two phase / , non-randomised, open-label studies of a heterologous, replication-deficient, recombinant adenovirus vector-based vaccine in both frozen and lyophilised formulations. the researchers enrolled healthy adult volunteers (aged - years) into the two studies ( people in each study); ( %) participants were men and ( %) were women. the primary outcome measures of the studies were safety and immunogenicity (antigen-specific humoral immunity). in phase of each three-arm study, two groups of nine volunteers received one dose of either recombinant adenovirus type (rad ) vector or recombinant adenovirus type (rad ) vector, both carrying the gene for severe acute respiratory syndrome coronavirus (sars-cov- ) spike glycoprotein (rad -s and rad -s), in lyophilised or frozen form. in phase , another group of healthy adult volunteers in each study received sequential doses of rad -s followed by rad -s of one of the two formulations. adverse events were mostly mild, with the most common adverse events being pain at injection site ( [ %]), hyperthermia ( [ %]), headache ( [ %]), asthenia ( [ %]), and muscle and joint pain ( [ %]). adverse events occurred at similar frequency for each vaccine vector, with each formulation, and after each dose. serious adverse events did not arise in this small cohort. both formulations of the vaccine were immunogenic in all participants, inducing neutralising humoral and cell-mediated responses. in phase , % of participants had detectable antibodies at days after the priming dose, rising to % by day , with substantial titre rises after the boosting dose. the two studies by logunov and colleagues have several strengths. first, adenoviruses are ubiquitous, so humans might not be immunologically naive. previous immunity to the vector might interfere with adenovirusvectored covid- vaccine efficacy. indeed, findings of an adenovirus type -vectored covid- vaccine trial suggested such immunity could affect covid- responses. in another study, a chimpanzee adenovirus vector was used, since humans will presumably be naive to at least the first dose. logunov and colleagues used immunologically distinct (heterologous) vectors in their two-dose (prime-boost) regimen. they investigated cross-vector heterologous immunity and previous antivector adenovirus immunity, and neither affected covid- immunogenicity. a second strength is the threshold for neutralisation used in the two studies. neutralisation assays vary from study to study. neutralisation is tested by examining whether plasma from a recently vaccinated individual can prevent cellular damage on in-vitro exposure of cells to sars-cov- . both the degree of such protection (how many damaged cells are allowed) and the dose of the infecting virus vary across studies. expecting a vaccine to result in less than complete neutralisation is not inherently wrong but sets the bar low and makes it easier to claim neutralising activity. in logunov and colleagues' studies, however, the threshold for neutralisation was set high in two regards: the inoculating viral dose was large, and no arising cellular damage was allowable. essentially, the assay was set at full neutralisation. this high bar implies these researchers took an a-priori risk that their vaccine might fail the test. it did not. it remains to be seen if other manufacturers will set a similar high standard. a third strength is that the vaccine, similar to other adenovirus-vectored and mrna covid- vaccines before it, - induced broad immune responses. although not specifically discussed, the results imply a t-helper- -cell-weighted response that might be important for vaccine safety, potentially reducing the risk of antibodydependent enhanced disease. a fourth strength was development of two vaccine formulations, frozen and lyophilised. a lyophilised formulation could mean stability within the existing global vaccine refrigerated cold chain that is needed to maintain vaccine efficacy from factory to recipient, a hurdle other vaccines are yet to address. although more costly to produce at scale, product stability will maximise reach in remote terrain, a must if universal and equitable coverage is to be achieved. some limitations of the studies by logunov and colleagues are notable. in the study of the frozen vaccine formulation, the population included young military personnel. soldiers are likely to be fitter and healthier than the general population. moreover, in older adults, immune senescence might make vaccines less immunogenic, and this age group was absent from this study. sex imbalance occurred in the study arms because there was no random allocation. a control arm was conspicuously absent. two participants were of asian descent, with the rest of the participants of white european ethnic origin. clearly, much more remains to be learned from the phase randomised trial planned to include civilian volunteers and, hopefully, broadly inclusive of groups at risk. this covid- vaccine candidate from russia joins two other adenovirus-vectored covid- vaccine candidates, which have been reported in randomised trials in the lancet, , and an mrna vaccine candidate reported in a non-randomised trial. similar to these studies before it, logunov and colleagues' studies are encouraging but small. the immunogenicity bodes well, although nothing can be inferred on immunogenicity in older age groups, and clinical efficacy for any covid- vaccine has not yet been shown. a wide portfolio of early covid- vaccine candidates will hopefully provide more successful vaccines that are broadly protective across risk groups and that increase the global availability of what will be a precious limited commodity. showing safety will be crucial with covid- vaccines, not only for vaccine acceptance but also for trust in vaccination broadly. safety outcomes up to now are reassuring, but studies to date are too small to address less common or rare serious adverse events. unlike clinical trials of therapeutics, in which safety is balanced against benefit in patients, vaccine trials have to balance safety against infection risk, not against disease outcome. since vaccines are given to healthy people and, during the covid- pandemic, potentially to everyone after approval following phase trials, safety is paramount. licensure in most settings should depend on proven short-term and long-term efficacy against disease (not just immunogenicity) and more complete safety data, including rates of vaccine failure to prevent infection (not merely disease) and rates of occurrence of antibody-dependent enhanced disease, ideally from very large scale, flexible multivaccine trials with prolonged, blinded safety and efficacy follow-up. , safety assurance will then require further large-scale surveillance after licensure. such surveillance is not well established in many settings, and rapid efforts need to be made by governments, regulators, and global research funders to get those systems in place. surveillance will also be vital for showing transmission reduction, which is unlikely to come from phase trials since these are powered to detect covid- disease outcomes and not asymptomatic sars-cov- infection. to be sure, most past vaccines were designed to target disease and not infection as such, but with covid- , the general public could be expecting striking reductions in disease transmission after widespread vaccine introduction. such effects would be very welcome if they occur, but they are far from certain. a vaccine that reduces disease but does not prevent infection might paradoxically make things worse. it could falsely reassure recipients of personal invulnerability, thus reducing transmissionmitigating behaviours. in turn, this could lead to increased exposure among older adults in whom efficacy is likely to be lower, or among other higher-risk groups who might have lower vaccine acceptance and uptake. in view of the ongoing painful toll of the covid- pandemic and its magnitude, the more vaccine candidates that have successful early results the better. ultimately, all vaccine candidates will need to show safety and prove durable clinical efficacy (including in groups at greater risk) in large randomised trials before they can be put into widespread use. equitable access will require multiple vaccine producers and providers in a range of settings. each of their successes will together lead us towards our collective, longed for, new day. we declare no competing interests. *naor bar-zeev, tom inglesby nbarzee @jhu.edu safety and immunogenicity of an rad and rad vector-based heterologous prime-boost covid- vaccine in two formulations: two open-label, non-randomised phase / studies from russia immunogenicity and safety of a recombinant adenovirus type- -vectored covid- vaccine in healthy adults aged years or older: a randomised, double-blind, placebo-controlled, phase trial safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind, randomised controlled trial an mrna vaccine against sars-cov- : preliminary report consensus summary report for cepi/bc march - , meeting: assessment of risk of disease enhancement with covid- vaccines vaccine confidence in the time of covid- covid- vaccine trials should seek worthwhile efficacy key: cord- -y s iv authors: logunov, denis y; dolzhikova, inna v; zubkova, olga v; tukhvatullin, amir i; shcheblyakov, dmitry v; dzharullaeva, alina s; grousova, daria m; erokhova, alina s; kovyrshina, anna v; botikov, andrei g; izhaeva, fatima m; popova, olga; ozharovskaya, tatiana a; esmagambetov, ilias b; favorskaya, irina a; zrelkin, denis i; voronina, daria v; shcherbinin, dmitry n; semikhin, alexander s; simakova, yana v; tokarskaya, elizaveta a; lubenets, nadezhda l; egorova, daria a; shmarov, maksim m; nikitenko, natalia a; morozova, lola f; smolyarchuk, elena a; kryukov, evgeny v; babira, vladimir f; borisevich, sergei v; naroditsky, boris s; gintsburg, alexander l title: safety and immunogenicity of an rad and rad vector-based heterologous prime-boost covid- vaccine in two formulations: two open, non-randomised phase / studies from russia date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: y s iv background: we developed a heterologous covid- vaccine consisting of two components, a recombinant adenovirus type (rad ) vector and a recombinant adenovirus type (rad ) vector, both carrying the gene for severe acute respiratory syndrome coronavirus (sars-cov- ) spike glycoprotein (rad -s and rad -s). we aimed to assess the safety and immunogenicity of two formulations (frozen and lyophilised) of this vaccine. methods: we did two open, non-randomised phase / studies at two hospitals in russia. we enrolled healthy adult volunteers (men and women) aged – years to both studies. in phase of each study, we administered intramuscularly on day either one dose of rad -s or one dose of rad -s and assessed the safety of the two components for days. in phase of the study, which began no earlier than days after phase vaccination, we administered intramuscularly a prime-boost vaccination, with rad -s given on day and rad -s on day . primary outcome measures were antigen-specific humoral immunity (sars-cov- -specific antibodies measured by elisa on days , , , , and ) and safety (number of participants with adverse events monitored throughout the study). secondary outcome measures were antigen-specific cellular immunity (t-cell responses and interferon-γ concentration) and change in neutralising antibodies (detected with a sars-cov- neutralisation assay). these trials are registered with clinicaltrials.gov, nct and nct . findings: between june and aug , , we enrolled participants to the two studies ( in each study). in each study, nine volunteers received rad -s in phase , nine received rad -s in phase , and received rad -s and rad -s in phase . both vaccine formulations were safe and well tolerated. the most common adverse events were pain at injection site ( [ %]), hyperthermia ( [ %]), headache ( [ %]), asthenia ( [ %]), and muscle and joint pain ( [ %]). most adverse events were mild and no serious adverse events were detected. all participants produced antibodies to sars-cov- glycoprotein. at day , receptor binding domain-specific igg titres were with the frozen formulation and with the lyophilised formulation, and neutralising antibodies were · with the frozen formulation and · with the lyophilised formulation, with a seroconversion rate of %. cell-mediated responses were detected in all participants at day , with median cell proliferation of · % cd (+) and · % cd (+) with the frozen formulation, and a median cell proliferation of · % cd (+) and · % cd (+) with the lyophilised formulation. interpretation: the heterologous rad and rad vector-based covid- vaccine has a good safety profile and induced strong humoral and cellular immune responses in participants. further investigation is needed of the effectiveness of this vaccine for prevention of covid- . funding: ministry of health of the russian federation. covid- was first reported in wuhan, china, at the end of december, . the disease is an acute respiratory illness ranging in severity from mild to severe, with death in some cases; many infected people are asymptomatic. since the end of january, , cases of covid- have been reported in more than coun tries around the world. on march , , who described the spread of covid- as a pandemic. the causative agent of covid- is the betacoronavirus severe acute respiratory syndrome coronavirus (sars-cov- ). sars-cov- can be transmitted in many ways, with the main route of transmission via contact with infected people (eg, by secretions, particularly droplets). as of aug , , there have been more than million laboratory-confirmed cases of sars-cov- infection, and more than deaths. because of the rapid global spread of sars-cov- infection and the high mortality rate, development of a vaccine is an urgent task. vaccination will restrict the spread of covid- and reduce mortality. intensive research and development of vaccines is currently underway in china, russia, the uk, the usa, and other countries. according to who, on aug , , candidate covid- vaccines based on different platforms (vectored, dna, mrna, inactivated, etc) were being tested in clinical trials. prevention of sars-cov- infection might be achieved by targeting the spike protein (glycoprotein s), which interacts with the ace receptor and enables entry of sars-cov- into the cell. blocking this interaction decreases viral internalis ation and replication. [ ] [ ] [ ] most vaccines that are currently in development target glycoprotein s as the main antigen. the structure and function of the sars-cov- glycoprotein s is similar to that of other highly pathogenic betacoronaviruses, such as middle east respiratory syndrome coronavirus (mers-cov) and severe acute respiratory syndrome coronavirus (sars-cov). glycoprotein s consists of two subunits: s contains a receptor-binding domain (rbd), which interacts with the ace receptor on the cell surface; s mediates the fusion of viral and cell membranes via formation of a six-helix bundle fusion core. , to protect against sars-cov- infection, it is important to form neutralising antibodies targeting s rbd, s n-terminal domain, or the s region; these antibodies block binding of the rbd to the ace receptor and prevent s -mediated membrane fusion or entry into the host cell, thus inhibiting viral infection. , when developing a vaccine (particularly during a pandemic), it is important to consider that a protective response must develop in a short time (eg, up to month). moreover, previous work on vaccines for mers-cov and sars-cov showed that both humoral and cellular (cytotoxic) immune responses are important to induce a protective immune response. to achieve these goals, one of the most attractive options is for vaccines to be based on recombinant viral vectors, which can induce humoral and cellular immune responses and form protective immunity after one or two doses. , recombinant adenovirus vectors have been used for a long time, with safety confirmed in many clinical studies of various preventive and therapeutic drugs. [ ] [ ] [ ] [ ] [ ] [ ] [ ] moreover, the long-term effects of vectors based on adenoviruses have been investigated, by contrast with newer methods that remain to be studied long term. for evidence before this study we searched clinicaltrials.gov and pubmed up to aug , , with the terms "covid- " or "sars-cov- " and "vaccine" and "clinical trial", with no date or language restrictions, to find information about adenovirus-based covid- vaccine candidates in active clinical trials. according to who, on aug , , candidate vaccines based on different platforms (vectored, dna, mrna, inactivated, etc) were being tested in clinical trials against severe acute respiratory syndrome coronavirus (sars-cov- ) proteins. recombinant viral vector-based vaccines are promising candidates for covid- prevention because they induce humoral and cellular immune responses and can provide protective immunity after one or two doses. several candidate covid- vaccines have been tested in clinical trials, including an adenovirus type (ad ) vector-based vaccine (cansino biological/beijing institute of biotechnology, china), an ad vector-based vaccine (johnson & johnson, usa), and a vaccine containing a simian adenoviral vector (astrazeneca/university of oxford, uk). since boosting vaccination is necessary for formation of a more powerful immune response, the effectiveness of such vaccination can be reduced when using a homologous vector (because of formation of an immune response not only to the target antigen but also to the vector components after priming vaccination). we designed a covid- vaccine with two different adenoviral vectors (recombinant ad [rad ] and recombinant ad [rad ]), both carrying the gene for sars-cov- spike glycoprotein (rad -s and rad -s), and we implemented a prime-boost regimen. we did two open, phase / non-randomised trials of two formulations (frozen and lyophilised) of the vaccine in healthy adult volunteers. safety of the two individual vaccine components (rad -s and rad -s) was confirmed in phase . both components were then administered as a prime-boost vaccination in phase , with testing for safety and immunogenicity. the vaccine was well tolerated and produced humoral and cellular immune responses in healthy adults. igg responses were elicited in all participants, with geometric mean titres significantly higher than those reported in people who have recovered from covid- . antibodies to sars-cov- glycoprotein and neutralising antibodies increased significantly at day and continued to increase throughout the observation period. specific t-cell responses peaked at day after vaccination. our findings indicate that a heterologous rad and rad vector-based covid- vaccine is safe and immunogenic in healthy adults. further investigation is needed of the effectiveness of this vaccine for prevention of covid- . formation of a robust long-lasting immune response, a prime-boost vaccination is advisable, which is widely used with registered vaccines for diseases including hepatitis b and ebola virus disease. when using vector-based vaccines, immune responses are formed not only to the target antigen but also to the vector component. as a result, the best vaccination scheme is heterologous vaccination, when different viral vectors are used to overcome any negative effects of immune response to vector components. [ ] [ ] [ ] such an approach was successfully used with an ebola virus disease vaccine developed in russia and licensed in . we designed a novel, heterologous adenoviral vectorbased vaccine against sars-cov- suitable for primeboost vaccination. the vaccine was designed with two recombinant adenovirus vectors and was developed as two formulations (frozen [gam-covid-vac] and lyophilised [gam-covid-vac-lyo]). we aimed to assess safety and immunogenicity of both vaccine formulations and to compare the humoral immune response with that recorded in people who have recovered from covid- . we did two open, phase / non-randomised studies at hospitals in russia (burdenko hospital and sechenov university, moscow, russia). for each study, healthy adult volunteers (aged - years) were preselected to be included in the volunteer register; all adults provided signed informed consent to be included in this database for study participation. volunteers were screened by demographic data, had a physical examination and bodyweight mea sured, were assessed for vital functions (eg, blood pressure, pulse, and temperature), had a blood test for clinical and biochemical testing, were screened for infections such as hiv, hepatitis, and syphilis, underwent pcr for sars-cov- and had a test for antibodies to sars-cov- , and had a urine test for drugs, alcohol, and pregnancy (in women). we included adult volunteers of both sexes with a body-mass index of · - · kg/m², who had a negative pcr and negative igg and igm to sars-cov- , and who had no history of covid- or contact with patients with covid- . volunteers had no infectious diseases at the time of vaccination and for days before vaccination, and they did not receive any other vaccinations within days of participation in the study. based on the results of the preliminary screening, volunteers were selected ( for each clinical trial) for inclusion in the register of volunteers planning to take part in the study of vaccines against covid- . as soon as the volunteers were included in the register they began self-isolation. all participants provided written informed consent. the two studies were reviewed and approved by appropriate national and local competent authorities, including the regulator (department of state regulation for medicine distribution, approval nos and ) and the ethics committee of the ministry of health of the russian federation. the vaccine comprises two vector components, recombinant adenovirus type (rad ) and recombinant adenovirus type (rad ), both of which carry the gene for sars-cov- full-length glycoprotein s (rad -s and rad -s). both components were developed, manufactured, and stored by n f gamaleya national research centre for epidemio logy and microbiology (moscow, russia) according to good manufacturing practices. a full dose of the vaccine was ¹¹ viral particles per dose for both recombinant adenoviruses and all participants received full doses. the dose was set based on findings of preclinical studies (unpublished data). the vaccine was manufactured as two formulations, frozen (gam-covid-vac) and lyophilised (gam-covid-vac-lyo). the frozen vaccine has a volume of · ml (per dose) and the lyophilised vaccine needs to be reconstituted in · ml of sterile water for injection (per dose). the study of gam-covid-vac was done at a branch of burdenko hospital, an agency of the ministry of defence. both civilian and military volunteers took part in that study. military personnel were contract employees (who received a salary for their work) and not individuals conscripted for compulsory military service. the study of gam-covid-vac-lyo took place at sechenov university and all volunteers in that study were civilians. in all cases, vaccines were administered intramuscularly into the deltoid muscle. during phase of both studies, participants received one dose intramus cularly of either rad -s or rad -s and were assessed for safety over days. phase of both studies began no earlier than days after phase vaccination, after an interim safety assessment had been done. during phase , participants received prime-boost vaccination, with one dose of rad -s administered intramuscularly on day and one dose of rad -s administered intramuscularly on day . injection-site reactions, systemic reactogenicity, and medication use to alleviate such symptoms were monitored for days after the first injection (in phases and ) and at day (phase only). no randomisation or special selection was done for phases and . participants were included as soon as informed consent was signed. participants underwent clinical and laboratory assessments on days , , and in phase and on days , , , and in phase . laboratory analyses included complete blood and urine counts, alanine aminotransferase, aspartate amino transferase, protein, bilirubin, total cho lesterol, lactate dehydro g enase, alkaline phosphatase, prothrombin index, glucose, urea, and creatinine. immune status was analysed on days and in phase and on days , , and in phase . volunteers were in hospital for days from the start of vaccination. information on adverse events was recorded daily. determination of immunogenicity is described in detail in the appendix (pp - ). in brief, antigen-specific humoral immune responses were analysed on days , , , and in phase and on days , , , , and in phase . the titre of glycoprotein-specific antibodies in serum was ascertained by elisa. to test anti-sars-cov- igg, we used an elisa that was developed at n f gamaleya national research centre for epidemiology and micro biology and registered for clinical use in russia (p h / - - ). the elisa measures iggs specific to the rbd of sars-cov- glycoprotein s. the titre of neutralising antibodies was measured on days , , and in phase and on days , , , and in phase and was ascertained by microneutralisation assay using sars-cov- (hcov- /russia/ moscow_pmvl- / ) in a -well plate and a % tissue culture infective dose (tcid ) of . cellmediated immune responses were measured on days , , and after the first injection by determination of antigen-specific proliferating cd + and cd + cells by flow cytometry and by quantification of interferon-γ release. to compare post-vaccination immunity with natural immunity that forms during infection with sars-cov- , we obtained convalescent plasma from blood samples of people from moscow who had recovered after covid- (between march and aug , ). convalescent plasma was obtained from people who had had a laboratory-confirmed covid- diagnosis, who had been recovered for at least weeks, and who had tested negative by pcr twice. the average time from recovery to convalescent plasma collection was about month. convalescent plasma was collected from people who had had mild (fever ≤ °c without pneumonia) and moderate (fever > °c with pneumonia) disease severity. humoral immune responses were ascertained as men tioned above. primary outcome measures were safety and immunogenicity of the covid- vaccine. the primary outcome measure for safety was the number of participants with adverse events from day to day after vaccination in phase and from day to day after vaccination in phase . the primary outcome measure for immunogenicity was change from baseline in antigen-specific antibody levels at days (from day to day ), measured by elisa. secondary immuno genicity outcome measures were virus neutra lising antibody titres (on days , , and after vaccination in phase and on days , , , and after vaccination in phase ) and determination of antigen-specific cellular immunity (specific t-cell immunity and interferon-γ production or lymphoproliferation) on days , , and after vaccination. the sample size for both studies was calculated from previous clinical trials of a mers vaccine based on the same recombinant viral vectors as used in our vaccine but carrying the mers-cov glycoprotein s gene. preliminary results of a study of a mers vaccine in which more than people participated showed a seroconversion rate of %. when calculating the sample size for our study, we expected % efficiency, which required inclusion of participants in each study. considering the possibility of early dropout of volunteers, we decided that volunteers should be recruited into the immunogenicity assessment group in phase of each study. a total sample size of ( in each study) was expected to produce reliable data on adverse events. all statistical calculations were done in graphpad prism . normality of the data distribution was assessed with the d'agostino-pearson test. paired samples were compared with the wilcoxon test and unpaired samples with the mann-whitney u test. correlation analysis was done with spearman's test; the correlation coefficient r shows interactions between two datasets and takes values either from to (in the case of a positive correlation) or from - to (in the case of a negative correlation). we used the mann-whitney u test to compare at various timepoints antibody titres, the level of proliferating cd and cd cells, and increases in concentrations of interferon-γ between volunteers receiving the two vaccines, and when comparing antibody titres in volunteers on days and after vaccination with antibody titres in convalescent plasma. we used the wilcoxon test to compare data within the same group of volunteers at different timepoints (eg, when comparing day to day ). these trials are registered with clinicaltrials.gov, nct and nct . the funder had no role in study design, data collection, data analysis, data interpretation, or writing of the report. all authors had full access to all data in the studies and had final responsibility for the decision to submit for publication. between june and aug , , healthy adults were enrolled to the two studies from the volunteer register (figure ). adults were selected at the beginning of each study from the volunteer registry; participants were included in each study and five people were kept as backup volunteers in case of dropouts (two for phase and three for phase ). nine participants in each study received rad -s in phase , nine received rad -s in phase , and received sequential injections of rad -s (on day ) and rad -s (on day ) in phase . all volunteers in the main group were analysed and additional volunteers from the backup groups were not needed. thus, in each study, volunteers were vaccinated. more men than women took part in the study (table ) . in both studies, systemic and local reactions (table ) (combined data for both the lyophilised and frozen vaccine formulations). comparing data for antibody responses to sars-cov- at days and with data for antibody responses in convalescent plasma showed that postvaccination elisa titres were signifi cantly higher than were titres after covid- (for both days and , p< · ), whereas significant differences in neutralising antibodies were not seen (p= · ; figure ). we also analysed the correlation between sars-cov- rbd elisa titres and neutralising antibody titres and noted a strong correlation between these variables (r= · , % ci · - · ; p< · ; appendix p ). when analysing antigen-specific iggs, the seroconversion rate was % for both vaccine formulations on days and of the study, and when analysing neutralising antibody responses, serocon version was % on day of the study for both vaccine formulations. seroconversion rates on days , , , and (in phase ) are presented in the appendix (pp - ). descriptive statistics for humoral immune responses are presented in the appendix (pp [ ] [ ] [ ] [ ] . cellular immune responses showed formation of antigen-specific cells of both t-helper (cd + ) and t-killer (cd + ) cells, and an increase in the concentration of interferon-γ secretion in peripheral blood mononuclear cells, in % of volunteers (figure ). cells from vaccinated participants proliferated significantly in response to glycoprotein s, particularly on day . the number of participants with cd + and cd + proliferative responses to antigen are shown in the appendix (p ). cell-mediated responses were detected in all participants at day , with median cell proliferation of · % cd + to investigate the effect of the pre-existing immune response to adenoviral vectors, neutralising antibodies to recombinant vectors were measured in all participants on days and in both studies (figure ). after one injection of vaccine components, not only is an immune response to target antigen formed but also an immune response is seen to components of the vaccine vector. further, a correlation analysis was done to compare the level of neutralising antibodies to recombinant vectors with the level of antigen-specific antibodies (appendix p ). no significant correlation was noted between the titre of neutralising antibodies to recombinant viral vectors on day and the titre of rbd-specific iggs in serum samples of participants on days , , and from the start of vaccination in participants in phase of each study and on days , , , and from the start of vaccination in participants in phase of each study. moreover, formation of cross-reactive neutralising antibodies to vectors rad and rad was analysed. administration of rad did not increase the titre of neutralising antibodies to rad on day , and vice versa, which indicates the absence of cross-reactivity with respect to vaccine components ( figure ) . thus, the presence of a pre-existing immune response to the components of vaccine vectors rad and rad does not affect the titre of rbd-specific antibodies in the serum of participants. these findings of two open, phase / non-randomised studies of a heterologous prime-boost covid- vaccine based on recombinant adenoviral vectors rad -s and rad -s show that the vaccine is safe, well tolerated, and induces strong humoral and cellular immune responses in % of healthy participants. all reported adverse events were mostly mild. the most common systemic and local reactions were pain at the injection site, hyperthermia (body temperature - °c), headache, asthenia, and muscle and joint pain, which are typical for vaccines based on recombinant viral vectors. no serious adverse events were reported during the study. in general, the adverse event profile did not differ from those reported in pub lished work for other vector-based vaccines. , [ ] [ ] [ ] the incidence of adverse events in our studies was slightly lower than in other work; a comparative clinical study with other vaccines is needed to confirm these findings. in preclinical studies of the vaccine (unpublished data), robust humoral and cellular immune responses were elicited in non-human primates, providing protection from sars-cov- infection. the vaccine showed % protectivity in a lethal model of sars-cov- challenge in immunosuppressed hamsters. no antibody-dependent enhancement of infection was seen in vaccinated and sars-cov- -challenged animals. in general, titres of neutralising antibodies to sars-cov- were lower than those reported in studies of vaccines based on mrna and chadox . , in our study, we used a high dose of virus ( tcid ) and a small amount of serum ( µl serum and µl of virus), whereas in studies of other vaccines, doses of - tcid and a larger amount of serum were used for analyses. , despite the fact that research results cannot be compared with each other in this case, we can make a comparison between titres of neutralising antibodies in vaccinated volunteers and in convalescent plasma. we showed that volunteers who received the heterologous rad and rad vaccine elicited the same titre of sars-cov- neutralising antibodies as did people who had recovered from covid- . according to our study protocols (nct and nct ), the t-cell response in healthy adult volunteers after vaccination was to be assessed using two methods. first, by measuring percentages of proliferating cd and cd t (cd + ) cells in response to antigenic re-stimulation in culture. second, by measuring interferon-γ in culture medium produced by peripheral blood mononuclear cells. interferon-γ is a marker cytokine of t-helper- biased cellular response towards vaccination, and high rates of antigen-specific cd + t cells generally correspond to potentiation of t-helper- polarisation. we understand that results obtained from both assays could indirectly characterise the t-helper- response. in the phase clinical trial, we will supplement our research methods with more focus on t-helper- and t-helper- polarisation. the main issue that can limit use of vectors based on recombinant adenoviruses is widespread pre-existing immunity in the human population. after vaccination with an adenoviral vector, immune responses form not only to the target antigen but also to the vector proteins (particularly in case of pre-existing immunity). in our study, despite formation of neutralising antibodies to recombinant adenoviruses after vaccination with rad and rad , formation of a humoral immune response to target antigen (sars-cov- glycoprotein s) in vaccinated volunteers was not affected. moreover, neutralising antibodies to rad did not neutralise rad when serum samples from vaccinated volunteers were obtained and analysed days after immunisation (and vice versa). thus, use of a heterologous prime-boost immunisation, when rad -s is used for priming and rad -s is used for boosting, is an effective approach to elicit a robust immune response and to overcome the immune response that is formed to the components of a viral vector. for more accurate estimation of the effect of preexisting immunity on vaccination, the number of observations should be increased and analysed during future research. limitations of our studies include the short duration of follow-up ( days), inclusion of only male volunteers in some parts of phase , the low number of participants (n= ), and no placebo or control vaccine. despite planning to recruit healthy volunteers aged - years, in general, our study included fairly young volunteers. further research is needed to evaluate the vaccine in different populations, including older age groups, individuals with underlying medical conditions, and people in at-risk groups. participants in these phase / trials will be followed up to days after initial immunisation. we designed the vaccine in two formulations, frozen (storage at - °c) and lyophilised (storage at - °c). the lyophilised form was developed for vaccine delivery to hard-to-reach regions of russia, and the frozen form was developed for large-scale use. production volumes in a pandemic will be strongly biased towards the frozen vaccine, since production of a lyophilised form takes much more time and resources. in conclusion, these data collectively show that the heterologous vaccine based on rad -s and rad -s is safe, well tolerated, and does not cause serious adverse events in healthy adult volunteers. the vaccine is highly immunogenic and induces strong humoral and cellular immune responses in % of healthy adult volunteers, with antibody titres in vaccinated participants higher than those in convalescent plasma. unprecedented measures have been taken to develop a covid- vaccine in russia. based on our own experience in developing vaccines against ebola virus disease and mers, the covid- vaccine has been developed in a short time. preclinical and clinical studies have been done, which has made it possible to provisionally approve the vaccine under the current decree of the government of the russian federation of april , , no on aug , (registration no lp- [gam-covid-vac]) and on aug , (registration no lp- [gam-covid-vac-lyo]). provisional licens ure requires a large-scale study, allows vaccination in a consented general population in the context of a phase trial, allows the vaccine to be brought into use in a population under strict pharmacovigilance, and to provide vaccination of risk groups. the phase clinical trial was approved by the appropriate national and local competent authorities, including the regulator (department of state regulation for medicine distribution) and the ethics committee of the ministry of health of the russian federation, on aug , (approval ). the phase clinical trial is planned with involvement of volunteers from different age and risk groups. the phase clinical trial will be undertaken with constant monitoring of the condition of volunteers through an online application, and each dose of vaccine will have its own qr code, which will be assigned to the volunteer. dyl is the principal investigator, did research, and coordinated the study. ivd and dvs wrote the draft report. ivd, nll, yvs, and eat coordinated the study. ivd, ovz, ait, asd, dmg, ase, avk, agb, fmi, op, tao, ibe, iaf, diz, dvv, dns, and ass collected data. ivd, ovz, ait, yvs, eat, nll, dae, nan, and mms contributed to data analysis and data interpretation. dyl, dvs, bsn, and alg edited the report. lfm, eas, evk, vfb, and svb did research. alg organised the research and had final responsibility for the decision to submit for publication. all authors critically reviewed the report and approved the final version. alg and dyl report funding from the ministry of health of the russian federation. ovz, tao, ivd, op, dvs, dmg, asd, ait, dns, ibe, eat, agb, ase, ass, svb, dyl, bsn, and alg report patent pending (patent for the use of vector constructs for the induction of immunity to sars-cov- ). all other authors declare no competing interests. individual participant data will be made available on request, directed to the corresponding author (dyl). after approval of a proposal, data can be shared through a secure online platform. covid- ) pandemic director-general's opening remarks at the media briefing on covid- transmission of sars-cov- : implications for infection prevention precautions: scientific brief draft landscape of covid- candidate vaccines structure analysis of the receptor binding of -ncov angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target sars-cov- pandemic and research gaps: understanding sars-cov- interaction with the ace receptor and implications for therapy severe acute respiratory syndrome coronavirus (sars-cov- ): an overview of viral structure and host response inhibition of sars-cov- (previously -ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion interaction of the spike protein rbd from sars-cov- with ace : similarity with sars-cov, hot-spot analysis and effect of the receptor polymorphism neutralizing antibodies against sars-cov- and other human coronaviruses immunoinformatic analysis of t-and b-cell epitopes for sars-cov- vaccine design middle east respiratory syndrome coronavirus (mers-cov): infection, immunological response, and vaccine development immunological responses against sars-coronavirus infection in humans new vaccine technologies to combat outbreak situations virus-vectored ebola vaccines adenovirus vectors for gene therapy, vaccination and cancer gene therapy adenoviral vectors for gene transfer and therapy adenoviruses as vaccine vectors gene therapy finds its niche methods and clinical development of adenovirus-vectored vaccines against mucosal pathogens adenoviral vector-based strategies against infectious disease and cancer the first approved gene therapy product for cancer ad-p (gendicine): years in the clinic us centers for disease control and prevention. hepatitis b vis safety and immunogenicity of gamevac-combi, a heterologous vsv-and ad -vectored ebola vaccine: an open phase i/ii trial in healthy adults in russia heterologous prime-boost vaccination a heterologous vectored vaccine for prevention of middle east respiratory syndrome induces long protective immune response against mers-cov an mrna vaccine against sars-cov- : preliminary report immunogenicity and safety of a recombinant adenovirus type- -vectored covid- vaccine in healthy adults aged years or older: a randomised, double-blind, placebo-controlled, phase trial safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind, randomised controlled trial th /th cytokine responses following hiv- immunization in seronegative volunteers memories that last forever: strategies for optimizing vaccine t-cell memory this research was supported by the ministry of health of the russian federation (state assignment no - - - , to alg and dyl). we thank the study participants, site research staff, and members of the trial management groups, trial steering committee, and independent data monitoring committee. key: cord- - pxdeva authors: nicholson, karl g; wood, john m; zambon, maria title: influenza date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: pxdeva although most influenza infections are self-limited, few other diseases exert such a huge toll of suffering and economic loss. despite the importance of influenza, there had been, until recently, little advance in its control since amantadine was licensed almost years ago. during the past decade, evidence has accrued on the protection afforded by inactivated vaccines and the safety and efficacy in children of live influenza-virus vaccines. there have been many new developments in vaccine technology. moreover, work on viral neuraminidase has led to the licensing of potent selective antiviral drugs, and economic decision modelling provides further justification for annual vaccination and a framework for the use of neuraminidase inhibitors. progress has also been made on developing near-patient testing for influenza that may assist individual diagnosis or the recognition of widespread virus circulation, and so optimise clinical management. despite these advances, the occurrence of avian h n , h n , and h n influenza in human beings and the rapid global spread of severe acute respiratory syndrome are reminders of our vulnerability to an emerging pandemic. the contrast between recent cases of h n infection, associated with high mortality, and the typically mild, self-limiting nature of human infections with avian h n and h n influenza shows the gaps in our understanding of molecular correlates of pathogenicity and underlines the need for continuing international research into pandemic influenza. improvements in animal and human surveillance, new approaches to vaccination, and increasing use of vaccines and antiviral drugs to combat annual influenza outbreaks are essential to reduce the global toll of pandemic and interpandemic influenza. influenza viruses have segmented genomes and show great antigenic diversity. of the three types of influenza viruses-a, b, and c-only types a and b cause widespread outbreaks. influenza a viruses are classified into subtypes based on antigenic differences between their two surface glycoproteins, haemagglutinin and neuraminidase. haemagglutinin subtypes (h -h ) and nine neuraminidase subtypes (n -n ) have been identified for influenza a viruses (figure ). viruses of all haemagglutinin and neuraminidase subtypes have been recovered from aquatic birds, but only three haemagglutinin subtypes (h , h , and h ) and two neuraminidase subtypes (n and n ) have established stable lineages in the human population since . only one subtype of haemagglutinin and one of neuraminidase are recognised for influenza b viruses. although most influenza infections are self-limited, few other diseases exert such a huge toll of suffering and economic loss. despite the importance of influenza, there had been, until recently, little advance in its control since amantadine was licensed almost years ago. during the past decade, evidence has accrued on the protection afforded by inactivated vaccines and the safety and efficacy in children of live influenza-virus vaccines. there have been many new developments in vaccine technology. moreover, work on viral neuraminidase has led to the licensing of potent selective antiviral drugs, and economic decision modelling provides further justification for annual vaccination and a framework for the use of neuraminidase inhibitors. progress has also been made on developing near-patient testing for influenza that may assist individual diagnosis or the recognition of widespread virus circulation, and so optimise clinical management. despite these advances, the occurrence of avian h n , h n , and h n influenza in human beings and the rapid global spread of severe acute respiratory syndrome are reminders of our vulnerability to an emerging pandemic. the contrast between recent cases of h n infection, associated with high mortality, and the typically mild, self-limiting nature of human infections with avian h n and h n influenza shows the gaps in our understanding of molecular correlates of pathogenicity and underlines the need for continuing international research into pandemic influenza. improvements in animal and human surveillance, new approaches to vaccination, and increasing use of vaccines and antiviral drugs to combat annual influenza outbreaks are essential to reduce the global toll of pandemic and interpandemic influenza. we reviewed international reports published in english before december, . the data for this non-systematic review of articles were identified by searches of medline, embase, integrated science citation index, pubmed, and the cochrane library electronic databases with relevant keywords. we also searched cited references in retrieved articles, reviewed articles we have collected over many years, referred to the textbook of influenza, and used knowledge of new data presented at international scientific meetings. because of the large number of articles that are published every year and limitations on the number of citations, we gave emphasis to clinically relevant issues, particularly disease burden, the emergence of new subtypes, vaccines, and antivirals, and diagnosis. we gave priority to randomised controlled trials when available, to larger studies, articles published in high-impact journals that have a wide readership, and the systematic review and economic decision modelling, for the prevention and treatment of influenza, commissioned by the health technology assessment programme on behalf of the national institute of clinical excellence. we also drew on our own knowledge when it seemed appropriate to fill in the gaps in the published work and included several recent pertinent articles. haemagglutinin facilitates entry of the virus into host cells through its attachment to sialic-acid receptors. it is the major antigenic determinant of type a and b viruses to which neutralising antibodies are directed and the crucial component of current influenza vaccines. an important function of neuraminidase, the second major antigenic determinant, is to catalyse the cleavage of glycosidic linkages to sialic acid, thereby assisting in the release of progeny virions from infected cells. accordingly, neuraminidase has become an important target for antiviral activity. the m ion channel of influenza a, which is blocked by the antiviral drug amantadine, regulates the internal ph of the virus, which is crucial during early viral replication. the epidemiological behaviour of influenza in people is related to the two types of antigenic variation of its envelope glycoproteins-antigenic drift and antigenic shift. during antigenic drift, new strains of virus evolve by accumulation of point mutations in the surface glycoproteins. the new strains are antigenic variants but are related to those circulating during preceding epidemics. this feature enables the virus to evade immune recognition, leading to repeated outbreaks during interpandemic years. antigenic shift occurs with the emergence of a "new", potentially pandemic, influenza a virus that possesses a novel haemagglutinin alone or with a novel neuraminidase. the new virus is antigenically distinct from earlier human viruses and could not have arisen from them by mutation (figure ). four or five pandemics of influenza occurred during the th century with intervals of - years. the h n pandemic of - was the most devastating, with - million deaths; an estimated · million excess deaths, representing % of the population, occurred in india alone. however, the cumulative mortality from influenza during the intervening years is generally many times greater than that associated with pandemics. although influenza a or b viruses circulate virtually every winter in temperate zones of the northern and southern hemispheres, quantification of the burden of influenza on consultations, emergency-department examinations, hospital admissions, and mortality has been difficult because influenza lacks pathognomonic features, it cocirculates with other respiratory pathogens, and it causes a range of nonspecific complications, such as exacerbations of chronic cardiopulmonary disease. nevertheless, there is much evidence that the h n subtype of influenza a virus causes more severe illness than h n or influenza b, - more hospital admissions for pneumonia and influenza, and higher numbers of excess deaths. during outbreaks, sentinel schemes, such as the royal college of general practitioners' network in england, report increased consultation rates for influenza-like illness and other respiratory syndromes that are strongly associated with excess mortality. in england and wales, an estimated - people died during each of the epidemics between - and - . these estimates are about ten times the number of death certifications for influenza, because the disease is the cause of many "hidden deaths". in the usa, during the about % of these influenzaassociated excess deaths are among people aged years and older. although there are age-related increases in deaths from influenzal illness in both at-risk and low-risk groups, most deaths and hospital admissions occur in elderly people with chronic cardiopulmonary disorders. among toddlers, rates of influenza-associated hospital admission in the usa have ranged from about per population for those with high-risk conditions to per for those without high-risk conditions. [ ] [ ] [ ] [ ] admission rates are highest among children younger than year and are similar to rates found among people aged years and older. , among children in hong kong, china, the numbers of excess hospital admissions attributed to influenza are very high in children younger than months ( and per in and , respectively) and decrease with age ( and per children aged - months; and per children aged - years; and per children aged - years; and and per children aged - years). in the tropics and subtropics, influenza occurs either throughout the year with no distinct seasonality or visible excess mortality, or twice a year, with the more intense activity during the rainy season. consequently, the morbidity and mortality from influenza are probably greatly underestimated in these regions. during summer, , an epidemic of respiratory illness with cases and % case-mortality affected madagascar; it was attributable to influenza a/panama/ / -like (h n ) virus. the loss of life was greatest in young children and was ascribed to malnutrition and poor access to health care. another outbreak attributable to influenza a/panama/ / -like (h n ) virus occurred during november and december, , in the district of bosobolo, democratic republic of congo. the casefatality rate was · % in children younger than years and · % in people over . these rates illustrate the seriousness of such outbreaks and are one of the reasons why improved linkage of morbidity and mortality analysis with virological surveillance is one of the key objectives of the who global agenda on influenza, formulated in . in southern china, influenza viruses circulate throughout the year. there is evidence for the origin in china of the viruses that caused the pandemics of h n influenza in , h n influenza in , and the re-emergence of h n influenza in . recent outbreaks of avian influenza a h n and h n in people in hong kong show the importance of virological surveillance in this region for the early detection of potentially pandemic viruses. there is also evidence that some drift variants circulate in china for up to years before causing epidemics in europe and north america. , this region is thought to provide an appropriate ecological niche for the emergence of new influenza viruses with pandemic potential, owing to the proximity of dense populations of people, pigs, and wild and domestic birds, thereby facilitating genetic reassortment of viruses from different species (figure ), or for the emergence of drift variants, given the high human population density and year-round virus circulation. these observations provided the impetus for improving the who global influenza surveillance programme in china that has provided many of the vaccine strains recommended by who in the past decade. the examples cited below indicate the unpredictability of influenza-virus variation and the great capacity for evolution, but they also show that novelty alone is insufficient for the emergence of pandemic influenza. adaptation to replication in human beings, the ability to spread from person to person, and a susceptible population are also prerequisites. thus, the emergence of new influenza-virus variants in the human population does not necessarily herald pandemic influenza. influenza a/hong kong/ (h n ) in may and november-december, , cases of influenza h n infection were identified in people in hong kong. this outbreak, which followed serious outbreaks of avian h n influenza in chicken farms, signalled the possibility of an incipient pandemic. the human influenza isolates were of avian origin and were not derived by reassortment. the high mortality (six of patients died from acute respiratory distress syndrome or multiple organ failure, most previously healthy young adults ) suggested an unusually aggressive clinical course. deterioration was rapid, with pneumonia necessitating ventilatory support developing within a few days of illness onset. striking features of severe cases were the early onset of lymphopenia and high concentrations of serum transaminases. fortunately, there were few if any secondary infections, and the h n outbreak ceased when all chickens in hong kong (about · million) were slaughtered. the territory's poultry stocks were again depopulated when highly pathogenic a/hong kong/ (h n ) virus reemerged in flocks in may, , and february and april, . however, no further human cases of h n influenza were identified until february, , when two cases were confirmed in a family of hong kong residents. the first patient, a -year-old boy who was admitted to hospital in hong kong and recovered, became unwell during travel to fujian province, mainland china. the boy's -year-old father died in a hong kong hospital and his -year-old sister died in a hospital while the family was in china; the cause of her death is not known. genetic analysis of the two h n isolates showed that the virus genes were purely avian in origin, but differed from the strains that infected human beings. after the h n outbreak in hong kong, heightened surveillance in the adjoining guandong province led to recovery of nine human isolates of h n virus during july-september, . in march, , influenza h n viruses were isolated from two children in hong kong. the illness in both was mild and self-limited. no serological evidence of h n infection was found in family members or health-care workers who had close contact with the children; thus, h n viruses, like h n viruses, seem not to be easily transmitted from person to person. three lineages of h virus have been defined, with the prototype viruses being g , g , and y . the g "avian" h n viruses isolated from human beings have some receptor properties similar to those of other human viruses-ie, binding to ␣ , sialic acid linkages, in contrast to the binding preference to the ␣ , linkages normally found with avian influenza viruses. in hong kong, antibody to h viruses was found in about % of blood donors, which suggests that human infection with h n may occur in this locality. surveillance of pigs in southern china has shown that h n viruses are cocirculating with human a/sydney/ -like h n viruses and other porcine h n and h n viruses. together, these observations indicate that all the precursors of potentially pandemic h human-avian reassortants are in place. during february, , a new influenza h n virus was isolated from patients with influenza-like illness in england and the middle east. in the uk it affected mainly young children. these h n viruses arose after reassortment of the segments of the currently circulating influenza a (h n ) and a (h n ) subtypes. although influenza a (h n ) viruses have been identified previously, during - , when influenza a (h n ) viruses were isolated in six cities in china, the virus did not spread further. , the limited effect of h n in and during the - and - seasons is attributable to the good pre-existing immunity in the population. in , four people contracted purulent conjunctivitis within days of post-mortem examination of harbour seals that died during an outbreak of influenza a/seal/mass/ / (h n ), an a/fowl plague/dutch (h n )-like virus, in cape cod, ma, usa. subsequently, a/seal/mass/ / (h n ) was recovered from the conjunctiva of an investigator who developed conjunctivitis when an infected animal sneezed into his face. in , avian h n virus was isolated in the uk from a woman with conjunctivitis who kept ducks. although none of these six patients had respiratory symptoms, an outbreak of highly pathogenic avian h n influenza in poultry farms in the netherlands, which began at the end of february, , was associated with fatal respiratory illness in one of human cases by april . the person who died was a previously healthy -year old veterinary surgeon who developed severe headache, renal impairment, interstitial pneumonia, and acute respiratory distress after visiting an affected poultry farm. most patients presented with conjunctivitis (n= ), and only seven (< %) had respiratory illness. transmission of h n influenza from poultry workers to family members was found on three occasions. most virus isolates obtained from human beings had not accumulated significant genetic changes, including those from cases of human-to-human transmission. however, the virus isolated from the person who died had aminoacid substitutions, which suggests a role in pathogenicity. rapid, near-patient tests for influenza can aid clinical management, but the usefulness of existing tests for decisions on whether to start antiviral drug treatment is limited because they are complex or have low sensitivities (table ) . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, rapid influenza tests can show whether virus is circulating in specific populations or localities, and they may become a useful adjunct to surveillance programmes. thus, treatment with anti-influenza drugs commonly depends on patients' symptoms. although influenza has no pathognomonic features, it was diagnosed correctly in clinical trials in about two-thirds of adults when the clinical entry criteria were met. cough and fever (temperature · °c or above) are the most predictive symptoms of influenza, but fever may not be present in elderly people. in primary care, about - % of patients with influenza-like illness have the disease during outbreaks. [ ] [ ] [ ] thus, optimum implementation of guidelines for antiviral treatment depends on continuous community-based clinical and virological surveillance and awareness by general practitioners that influenza is circulating. most of what we know about highly pathogenic influenza viruses derives from studies with avian influenza viruses in birds. this situation is potentially relevant to disease in human beings because some mechanisms of pathogenicity in birds may operate in mammals and new human influenza a strains may come ultimately from the avian reservoir. tissue tropism and the capacity for systemic spread are the most important determinants of pathogenicity in birds. the molecular correlates of these pathogenic properties reside in the viral haemagglutinin and have been well studied. [ ] [ ] [ ] however, in mammals, factors other than viral haemagglutinin are involved in determining pathogenicity, including viral non-structural protein , pb , and neuraminidase. recovery of nucleic acid of the pandemic virus from post-mortem tissue or preserved human remains has shown that this highly pathogenic virus did not have the molecular motifs in haemagglutinin associated with virulence in avian strains. , the use of reverse genetics techniques has allowed the direct manipulation of influenza-virus gene products and creation of new recombinant viruses. selective testing of specific mutations engineered into recombinant viruses in a mouse model has shown that greater virulence is obtained by specific molecular properties of haemagglutinin, but these do not fully explain pathogenicity. the balance between viral replication and host immune response determines the outcome of viral infection. highly virulent viruses, such as h n , have a remarkable capacity to resist the antiviral effects of host cytokines. infection of human macrophages also results in induction of high cytokine expression, suggesting that severe outcome of infection is due both to lack of inhibition of viral replication by cytokines and to excess induction of cytokines leading to tissue damage in the infected host. the key genetic component determining replication of highly pathogenic virus may be the nonstructural protein of the virus, which has been identified as the major immune modulator. factors contributing to the pathogenesis of influenza in people are incompletely understood. these include understanding of the nature of tissue restriction of proteases, differences in reactivity of the innate immune system at different stages of life, and varying susceptibilities in different human populations, leading to different ranges of disease in certain populations; for example, encephalopathy is well recognised in japan, but less so in other populations. , study of mechanisms of virulence should provide more than simply elucidation of pathogenesis, notably development of alternative means of attenuating influenza viruses (eg, by deletion of non-structural protein or m [matrix protein ] genes) to make safer vaccine strains. current vaccines are produced from virus grown in fertile hens' eggs and inactivated by either formaldehyde or ␤-propiolactone. they consist of whole virus, detergent-treated split product, or purified haemagglutinin and neuraminidase surface antigen formulations of the three virus strains currently recommended by who. about countries have government-funded national immunisation programmes, and influenza vaccine is available in many others. about million of the world's population of billion were vaccinated during . specific vaccine recommendations vary, but most involve annual vaccination of elderly people and those with certain chronic medical disorders. these recommendations were founded on proven morbidity and mortality in the at-risk groups, consistent demonstrations of vaccine efficacy in military recruits, recognition of the relation between antibody and protection, and proof of vaccine antigenicity. whole-virus vaccines are not widely available because they cause adverse reactions in young children, whereas split-product formulations and those containing purified surface antigen are well tolerated and extremely safe. the virtual absence of published reports suggests that hypersensitivity reactions are rare. recent randomised controlled trials efficacy and effectiveness of current vaccines studies on the efficacy and effectiveness of inactivated influenza vaccines reveal substantial benefits. in children, meta-analysis of double-blind - and singleblind , randomised controlled trials estimated the efficacy of vaccine in preventing symptomatic laboratory-confirmed influenza at % (table ) . other benefits include reductions in school absenteeism, otitis media, asthma exacerbations, and febrile respiratory illness in unvaccinated household contacts. , [ ] [ ] [ ] [ ] [ ] in adults of working age, meta-analysis of two randomised controlled trials of split influenza vaccines , estimated the efficacy in preventing laboratory-confirmed influenza at % (table ) . associated benefits include reductions in absenteeism, consultations, antibiotic use, and use of over-thecounter medication. [ ] [ ] [ ] the frequency of laboratory-confirmed influenza fell by % in vaccinees in a randomised controlled trial (table ) and by % in a prospective cohort study of elderly people living in the community. many cohort and case-control studies have shown lower rates of hospital admissions for pneumonia and influenza, - all respiratory disorders, , respiratory disorders and heart failure, deaths from pneumonia and influenza, , and all-cause mortality , , in vaccinees than in controls (table ) . a meta-analysis of reports published before showed that vaccination reduces numbers of cases of influenza-like illness by %, hospital admissions for pneumonia and influenza by %, and all-cause mortality by %. a prospective observational study of nursinghome residents in japan showed a % reduction in laboratory-confirmed influenzal illness among vaccinees. vaccination also reduced numbers of hospital admissions among vaccine failures and thus appears to ameliorate illness severity. gross and colleagues did a meta-analysis of cohort studies. the odds ratios for development of respiratory illness ( · ) or pneumonia ( · ), hospital admission ( · ), and mortality ( · ) indicate substantial protection. vaccination of elderly patients with chronic lung disease reduces hospital admissions for pneumonia and influenza by %, all-cause mortality by %, and complications (death, exacerbations of lung disease, pneumonia, heart failure, angina, and myocardial infarction) by %. influenza vaccination also prevents heart failure, brain infarction, recurrent myocardial infarction, and primary cardiac arrest, indicating important benefits in patients with cardiovascular disease. vaccination of patients with diabetes mellitus is associated with an estimated % reduction in hospital admissions, mostly ( %) for reasons of diabetic control. thus, influenza vaccine protects against several potentially fatal events that explain the many hidden deaths that accompany epidemics. staff have been implicated as the source of influenza in several outbreaks in nursing homes. accumulating that influenza vaccination of staff caring for elderly people in long-stay facilities provides benefits to residents. [ ] [ ] [ ] one study showed substantial herd immunity ( - % protection) among patients exposed to staff with high vaccine coverage. during the past decade, there have been many new developments in vaccine technology that have aided vaccine production or aimed to improve vaccine immunogenicity and acceptability. subunit influenza vaccine with adjuvant mf , an emulsion of squalene in water for parenteral use, is licensed in some european countries but not the uk. mf significantly increases haemagglutinationinhibition antibody responses to interpandemic influenza a h n and influenza b antigens, particularly in older people with chronic diseases, and is well tolerated despite slightly higher rates of transient mild local reactions than with other vaccines. virosomes consist of bilayers of phospholipids (liposomes) containing virus surface proteins embedded in the bilayer. virosomes have been extensively evaluated in various human populations. typically, virosomes induce higher concentrations of antibody after vaccination, higher rates of seroconversion, and a greater proportion of individuals with "protective" antibody titres than conventional inactivated vaccines. virosomal influenza vaccine for parenteral use became available in the uk during the - season. cell-culture vaccines offer the potential of being able to respond quickly to epidemics or a pandemic at any time of the year and avoid the risk of contaminated eggs, which could affect the bioburden and endotoxin content of vaccines. moreover, influenza viruses grown in mammalian cells more closely resemble those present in clinical samples than viruses isolated and grown in eggs, hence offering the potential for more effective vaccine. influenza vaccines prepared with madin darby canine kidney (mdck) and african green monkey (vero) cells as substrate have been licensed in the netherlands but are not yet available commercially. live attenuated influenza vaccines intranasal delivery of live influenza vaccines offers the advantage of mimicking natural infection, thereby providing a broader immunological response and more durable protection than with inactivated vaccines. strategies for use of live influenza vaccines based on the transfer of genes coding for cold adaptation (ca) and temperature sensitivity from an attenuated parental virus donor have been used in russia for many years and were approved in june, , by the us food and drug administration for use in healthy children and adolescents aged - years, and in healthy adults aged - years after three decades of clinical evaluation. vaccines based on ca replicate well at the temperatures found in the nasopharynx but not at temperatures in the lower airways. in young children, a recent study of us ca vaccine showed very high protection with benefits in terms of both influenzal illness and otitis media. during the second year of this study, ca vaccine afforded a high degree of protection against a variant not closely matched to the vaccine antigen. studies in nursing-home residents have suggested that a combination of live and inactivated influenza vaccines may improve protection in these communities. immunostimulating complexes are cage-like structures that were originally formed as a complex between cholesterol and saponin derived from the tree quillaia saponaria. vaccines containing a defined saponin called iscoprep stimulated an accelerated serum antibody response in human beings compared with conventional inactivated vaccines, an improved proliferative t-cell response, and a cytotoxic t-cell response. although nasally delivered influenza vaccine could greatly increase vaccine coverage and provide mucosal immunity, intranasal administration of conventional inactivated influenza vaccines has typically been unsuccessful. in animals, incorporation of a mucosal adjuvant derived from bacteria has been necessary to improve immunogenicity, and several such vaccines administered intranasally have been evaluated clinically, with promising results. an intranasal spray formulation containing trivalent subunit influenza vaccine prepared from virosomes and wild-type escherichia coli enterotoxin was licensed briefly in switzerland. although it met the immunogenicity criteria set for yearly relicensing of conventional influenza vaccines, it was withdrawn after a possible association with bell's palsy could not be discounted. various microparticles are being investigated as adjuvants and delivery systems, for parenteral delivery of influenza-virus antigens or delivery to mucosal sites including the gut. subunit influenza vaccines have been prepared from recombinant haemagglutinin and neuraminidase proteins expressed in insect cells by baculoviruses. the recombinant haemagglutinins are well tolerated by young adults and elderly people, and there are significant doseresponse effects for both h and h haemagglutinin vaccines. phase i and virus-challenge studies of baculovirus-expressed recombinant neuraminidase in healthy volunteers have had promising results. the development of reverse-genetics techniques for negative-sense rna viruses has allowed the direct manipulation of influenza-virus gene products and creation of new recombinant viruses. this approach offers enormous potential for preparing interpandemic vaccines. nucleic-acid vaccines dna vaccines present a promising new approach to vaccination, evoking a full range of immune responses, including antibody, cytotoxic, and helper-t-cell responses. dna vaccines with constructs encoding the nucleoprotein (np), haemagglutinin, neuraminidase, matrix protein (m ), and non-structural protein of influenza virus have been studied extensively, either singly, in combination with one another, or together with dna encoding various cytokines. currently, two drug classes are available to manage influenza: the inhibitors of m , amantadine and rimantadine, and the neuraminidase inhibitors, zanamivir seminar the lancet • vol • november , • www.thelancet.com and oseltamivir. rimantadine causes less neurotoxicity than amantadine but is not available in most parts of the world and is not discussed further. amantadine inhibits the m membrane protein ionchannel activity of the influenza a virus but has no effect on influenza b. amantadine has three important limitations: its range of activity excludes influenza b; it has adverse side-effects, including insomnia, lightheadedness, hallucinations, dizziness, headache, and falls, which are particularly troublesome in elderly people; and drug resistance emerges rapidly during treatment. the genetic basis of resistance is a single nucleotide change, resulting in an aminoacid substitution at position , , , , or in the membrane-spanning region of m . estimates of amantadine's therapeutic effectiveness are uncertain owing to the clinical and methodological heterogeneity of clinical trials, a paucity of data by dose, the small number of trials in children and elderly people, and low trial-quality scores. treatment of healthy adults with - mg daily of amantadine cuts the duration of fever compared with placebo by day. there are few data on use of the currently licensed dose in the uk, mg daily. at this dose, amantadine reduced the duration of fever compared with placebo by day, but in a meta-analysis of data from six trials involving a total of patients the effect did not attain statistical significance. there is no high-quality evidence from randomised controlled trials of the effectiveness of amantadine mg daily for the treatment of influenza in at-risk individuals, and illness was significantly shortened with treatment by · days in only one of two small randomised controlled trials in children. , no randomised trial has tested amantadine during outbreaks in nursing homes. moreover, its use in this setting is complicated by toxicity, treatment failures, and frequent recovery of drug-resistant virus (about %). amantadine prophylaxis of other populations during interpandemic outbreaks is precluded by the lack of high-quality evidence from randomised controlled trials at the licensed dose and the high incremental cost per quality-adjusted life-year gained. this second-generation neuraminidase inhibitor is a potent and specific inhibitor of a wide range of influenza virus types a and b. it has poor oral bioavailability and is delivered through an inhaler. zanamivir is licensed for the treatment of influenza a and b in people aged years and over. it is well tolerated; the number, type, and severity of adverse events in healthy adults or people with stable chronic underlying medical disorders differ little from those with placebo. the main safety concern is that inhaled zanamivir may cause bronchospasm. however, respiratory viruses including influenza regularly exacerbate asthma and chronic obstructive pulmonary disease, so the role of zanamivir in bronchospasm is unclear. zanamivir was administered with apparent safety in two studies involving patients with asthma or chronic obstructive pulmonary disease. , difficulty in using the inhaler may limit use of zanamivir. in one study, half of a very elderly group were unable to use the inhaler after training, and two-thirds zanamivir healthy individuals, aged - years · ( · to · ) · ( · to - · ) · (- · to · ) · ( · to · ) at-risk individuals, including older than years · (- · to · ) · ( · to · ) · (- · to · ) · (- · to · ) healthy children · ( · to · ) · ( · to · ) · (- · to · ) · (- · to · ) "all" individuals · ( · to · ) · ( · to · ) · ( · to · ) · ( · to · ) healthy individuals, aged - years · ( · to · ) · ( · to · ) · ( · to · ) · ( · to · ) at-risk individuals, including older than years Ϫ · (- · to · ) · (- · to · ) · ( · to · ) · ( · to · ) healthy children · ( · to · ) · ( · to · ) · ( · to · ) · ( · to · ) "all" individuals · ( · to · ) · ( · to · ) · ( · to · ) · ( · to · ) itt=intention-to-treat. were unable to use it the next day. however, in three other studies, it was used successfully by about % of more than elderly people. summary results, which draw from published [ ] [ ] [ ] [ ] [ ] [ ] and unpublished treatment studies with zanamivir, are shown in table . overall, symptoms were alleviated sooner with zanamivir than with placebo-a median of · days on an intention-to-treat basis and · days for the influenzapositive subgroup. with zanamivir, the median time to return to normal activities was · days shorter for the treatment group, for both the intention-to-treat and influenza-positive populations. in a pooled analysis of intention-to-treat data from trials including both otherwise healthy and at-risk individuals, antibiotics were given to a smaller proportion of patients receiving zanamivir than of those assigned placebo (table ). similar, but nonsignificant, reductions in need for antibiotics in high-risk individuals and in pneumonia were seen with treatment in published and unpublished marginal analyses. seasonal prophylaxis with zanamivir mg daily of mostly unvaccinated healthy adults provided an estimated % reduction in the incidence of laboratory-confirmed clinical influenza compared with placebo, and metaanalysis of two randomised controlled trials of postexposure prophylaxis, with treatment given for days or days, suggested % protection against symptomatic laboratory-confirmed influenza. oseltamivir this third-generation neuraminidase inhibitor is an orally active prodrug of oseltamivir carboxylate. it is licensed for the treatment of influenza a and b in people aged year or older and for the prophylaxis of influenza a and b in people aged years or older. the frequency of nausea is - % higher and of vomiting up to % higher than with placebo; , these gastrointestinal side-effects can be ameliorated if the drug is taken shortly after food. summary results, which draw from published [ ] [ ] [ ] and unpublished treatment studies with oseltamivir (table ) show that for all treatment groups combined, symptoms were alleviated sooner with oseltamivir than with placebo, by · days on the basis of intention to treat and · days for the influenza-positive subgroup. similarly, normal activities were resumed · days and · days sooner with oseltamivir for the intention-to-treat and influenzainfected group, respectively. treatment with oseltamivir reduces the frequencies of otitis media, antibiotic use, pneumonia, and hospital admissions. in children with influenza, the frequency of otitis media was % with placebo and % with oseltamivir. the rate of antibiotic use in the intention-totreat population in one study was · % (eight of ) with placebo and · % (one of ) with treatment. pooled marginal analyses showed lower rates of antibiotic use for lower-respiratory-tract complications in "healthy" and "high-risk" people with influenza with oseltamivir than with placebo; a lower frequency of pneumonia in the influenza-positive group of ten studies ( % among placebo recipients vs < % with oseltamivir; table ); and a significant reduction in the occurrence of hospital admissions in influenza-positive populations of ten trials ( · % vs · %). three different strategies in preventing laboratoryconfirmed symptomatic influenza with oseltamivir have been investigated in randomised controlled trials. metaanalysis of data from two trials of seasonal prophylaxis in non-vaccinated healthy adults with oseltamivir, mg once daily, gave an estimate of % protection. in households, postexposure prophylaxis with oseltamivir, mg once daily for days, gave % protection. similarly, seasonal prophylaxis of mostly vaccinated elderly people receiving residential care with oseltamivir, mg daily for weeks, provided % protection. resistance to neuraminidase inhibitors influenza viruses with low susceptibility to the neuraminidase inhibitors have been isolated in vitro and in vivo. resistance involves either a mutation in the active site of the neuraminidase, altering its sensitivity to inhibition, or a mutation in the haemagglutinin. mutations in haemagglutinin that confer drug resistance decrease the affinity of the protein for the cellular receptor, thus enabling virus to escape from infected cells without the need for viral neuraminidase. to date, few viruses with altered susceptibility to neuraminidase inhibitors have been recovered from patients. the first report of emergence of neuraminidaseinhibitor resistance (r k) during treatment with zanamivir involved a recipient of a bone-marrow transplant. during clinical trials with oseltamivir, · % (four of ) of post-treatment isolates from adults and adolescents and · % (nine of ) from children had low neuraminidase-inhibitor susceptibility, indicating that such viruses are likely to emerge in clinical practice. three resistant variants with neuraminidase mutations (e v, h y, and r k) that have emerged in clinical trials show low infectivity and virulence in animal models, thus the relevance of these mutations in clinical practice remains uncertain. in , an international neuraminidase susceptibility network was established to oversee global surveillance of neuraminidase-inhibitor resistance. the uk national institute for clinical excellence (nice) has recently issued new guidance on the interpandemic use of antivirals for the treatment of influenza. amantadine is not recommended. neither zanamivir nor oseltamivir is recommended for the treatment of influenza in children or adults unless they are at risk. within their licensed indications, zanamivir and oseltamivir are both recommended for the treatment of atrisk adults, and oseltamivir for the treatment of at-risk children, who present with influenza-like illness and can start therapy within h of the onset of symptoms, when it is known that influenza a or b is circulating in the community. nice guidance on the use of antiviral drugs for the prevention of influenza was also issued lately. oseltamivir is recommended for postexposure prophylaxis of influenza in at-risk people aged years and older, who can begin prophylaxis within h, if they live in a residential care establishment, whether or not they have been vaccinated, and a resident or staff member has influenza-like illness; or if they are not effectively protected by vaccination and can begin prophylaxis within h of exposure. oseltamivir is not recommended for postexposure prophylaxis of healthy people up to age years or for seasonal prophylaxis. amantadine is not recommended for either postexposure or seasonal prophylaxis. this guidance does not cover the circumstances of a pandemic. the hong kong "chicken flu" situation in and the rapid global spread of severe acute respiratory syndrome highlighted how ill prepared we are to introduce preventive measures for pandemic influenza. the problems encountered in were due mainly to the dangers of working with the chicken h n virus and the need to produce a safe vaccine strain. conventional technology was unable to produce a safe productive vaccine strain. however li and colleagues were able to modify the haemagglutinin gene of the a/hong kong/ virus. they deleted the series of basic aminoacid residues at the cleavage site associated with virulence, then by use of reverse genetics rescued the modified haemagglutinin gene and the neuraminidase gene from the wild-type a/hong kong/ virus into ca a/ann arbor/ / virus. the resultant ca virus was non-pathogenic in animal models of infection, grew well in eggs, and protected chickens from challenge with lethal virus; it could be a suitable candidate vaccine strain. these experiments show the potential for using reverse genetics technology to develop live and inactivated vaccines for both pandemic and interpandemic use. a second strategy for pandemic vaccine development is the use of recombinant haemagglutinin. however, the disappointing results from clinical trials of baculovirusexpressed haemagglutinin from the a/hong kong h n virus, even after two doses of up to µg, question the role of this strategy alone. clinical trials of conventional inactivated-surface-antigen vaccine produced from an h n virus showed that extremely poor antibody responses were stimulated, even after two doses, whereas an h n subunit vaccine with mf adjuvant was much more immunogenic. the benefit of adjuvants for use in naïve populations has also been shown with a whole-virus h n vaccine with aluminium salts adjuvant. thus, like mf , aluminium salts have promise in increasing vaccine coverage in response to pandemic influenza by allowing scarce antigen to be used more efficiently. from the limited information available, conventional influenza vaccines seem not to be sufficiently immunogenic in a pandemic situation and two doses in conjunction with an adjuvant may be needed. different dosing strategies with various influenza-virus subtypes should be investigated so a robust strategy can be developed. vaccines will be in very short supply during the first stages of a pandemic, and antiviral drugs could have an important role in prevention. who has recently prepared draft guidelines for use of both vaccines and antiviral drugs during a pandemic; they emphasise the need to stockpile drugs and to develop plans for their distribution and use. as with vaccines, there are gaps in our knowledge as to how antiviral drugs should be used. research is urgently required to ensure effective use of both vaccines and drugs in response to an emerging pandemic. kgn has received fees or honoraria from roche pharmaceuticals, wyeth, and berna biotech for speaking at meetings on influenza; research has been supported by chiron vaccines, aventis pasteur, wyeth, and berna biotech. solvay and berna biotech have provided h avian influenza viruses free of charge for a project on candidate pandemic vaccines. mz has received honoraria from berna biotech to speak at pharmaceutical-industry conferences on influenza and has been supported in attendance at an international who workshop on virus neutralisation sponsored by glaxosmithkline. the health protection agency has received funding from chiron vaccines, wyeth vaccines, aventis pasteur, roche, and glaxosmithkline, to carry out analytical work on a contractual basis in mz's laboratory. jmw has no conflicts of interest to declare in relation to this paper. none of these sources of funding had any role in the writing of this seminar. we received no funding in relation to this seminar other than a small payment from the lancet. learning from experience? uses of error reflecting on my medical errors over years of clinical experience, i was disturbed to note that most errors that came to my mind were from the early part of my career. is this a sign that i really did become a better doctor or is it a symptom of ageing? were my early errors more influential on my subsequent practice or are my current errors not recognised or not made known to me by sympathetic colleagues? during my first house job, a middle-aged man was under my care for several months with an infected pleural cavity following a plombage operation for tuberculosis some years previously. he was memorable not only because of his sickness, but also because he was the first patient to give me a present, which i have to this day. months later, i was a casualty officer at a teaching hospital. an intern showed me the chest radiograph of a man with a febrile illness and an opaque hemithorax. i lectured the intern on my patient with the infected plombage, not thinking for a moment that this story was directly relevant to the radiograph i was shown. the patient died of a cardiac arrest in the casualty department. the autopsy showed an infected plombage site and death from untreated septicaemia. years after qualifying, while a locum physician in the northern parts of canada, i was required to do a coroner's post-mortem on a middle-aged man found dead in his cabin. fortunately, the policeman knew what to do and this overcame my anxiety. hours later, i had no doubt that the man was dead, but knew neither why nor how. i dissected the heart and coronary vessels and decided they were atheromatous and that death was from natural causes, probably from myocardial ischaemia. in the coroner's court, i presented my conclusions and the case was closed. i received the pathologist's report as i was due to finish the locum . . . normal heart! being on-call for a unit and not just one's own patients can be an onerous task, particularly as the staff become more numerous and anonymous. in the past years, i have been required to be on-call for a unit of ten consultants at two hospitals, one of which i visit only when on call. i was telephoned in the middle of the night about a patient who had had a cardiac arrest. i did not recognise the patient's name. i was told the arrest team wanted me to advise on their "enthusiasm" for resuscitation. i said that if she was not readily resuscitated, they should not resort to extreme measures, presuming she was a cancer patient admitted under the care of one of my oncology colleagues. on visiting the ward the next morning, i learnt that the patient who had died was a young woman under my care. (i had been given her first name and not her surname). she had been successfully treated for ovarian cancer years previously and had been diagnosed with metastatic disease a couple of weeks earlier. she was neutropenic after the first chemotherapy treatment and a fatal outcome was not anticipated, particularly since she was being treated with curative intent. systematic review and economic decision modeling for the prevention and treatment of influenza a and b textbook of influenza the impact of influenza epidemics on mortality: introducing a severity index differing virulence of h n and h n influenza strains comparison of infection rates and severity of illness for influenza a subtypes h n and h n the tecumseh study of illness: xiii, influenza infection and disease, - the impact of influenza epidemics on hospitalizations impact of influenza and respiratory syncytial virus on mortality in england and wales from mortality associated with influenza and respiratory syncytial virus in the united states population-based study on incidence, risk factors, clinical complications and drug utilization associated with influenza in the united kingdom survey of underlying conditions of persons hospitalized with acute respiratory disease during influenza epidemics in houston burden of influenza illness in children with asthma and other chronic medical conditions influenza and the rates of hospitalization for respiratory disease among infants and young children effect of influenza on hospitalizations, outpatient visits, and courses of antibiotics in children influenzarelated hospitalisations among children in hong kong outbreak of influenza influenza: global surveillance for epidemic and pandemic variants global influenza surveillance: tracking a moving target in a rapidly changing world human influenza a h n virus related to a highly pathogenic avian influenza virus clinical features and rapid viral diagnosis of human disease associated with avian influenza a h n virus surveillance of influenza viruses in guandong province, china in : a preliminary report human infection with influenza h n lack of evidence for humanto-human transmission of avian influenza a h n viruses in hong kong, china molecular characterization of h n influenza viruses: were they the donors of the ''internal'' genes of h n viruses in hong kong? emergence of influenza a h n reassortant viruses during molecular diagnosis of influenza human influenza a (h n ) viruses isolated from china origin and evolutionary characteristics of an antigenic reassortant virus isolated from man in china conjunctivitis in human beings caused by influenza a virus of seals avian influenza virus isolated from a woman with conjunctivitis avian influenza, human-netherlands ( ): fatal case. reported in: promed-mail application of directigen flu-a for the detection of influenza a virus in human and nonhuman specimens comparison of rapid diagnostic techniques for respiratory syncytial and influenza a virus respiratory infections in young children performance of virus isolation and directigen flu a to detect influenza a virus in experimental human infection effect of rapid diagnosis on management of influenza a infections evaluation of directigen flu a and b for rapid diagnosis of influenza type a and b infections evaluation of a new dot blot enzyme immunoassay (directigen flu a+b) for simultaneous and differential detection of influenza a and b virus antigens from respiratory samples evalaution of diagnostic tests for influenza in a pediatric practice evaluation of a neuraminidase detection assay for the rapid detection of influenza a and b virus in children bedside diagnosis of influenzavirus infections in hospitalized children quickvue influenza test for rapid detection of influenza a and b viruses in a pediatric population enhancing public health surveillance for influenza virus by incorporating newly available rapid diagnostic tests clinical signs and symptoms predicting influenza infection influenza b presentation in vaccinated elderly patients: diagnostic considerations. options for the control of influenza iv medical practicebased influenza surveillance: viral prevalence and assessment of morbidity surveillance of respiratory pathogens and influenzalike illnesses in general practices-the netherlands, winter / contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study pathogenesis of influenza a and b in humans structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation role of hemagglutinin cleavage for the pathogenicity of influenza virus structural features of the avian influenza virus haemagglutinin that influence virulence initial genetic characterization of the "spanish" influenza virus origin and evolution of the "spanish" influenza virus hemagglutinin gene molecular basis for high virulence of hong kong h n influenza a viruses lethal h n influenza viruses escape host anti-viral cytokine responses induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? influenza a virus lacking the ns gene replicates in interferon deficient systems encephalitis and encephalopathy associated with an influenza epidemic in japan influenza-associated encephalopathy in japan randomised placebo-controlled crossover trial on effect of inactivated influenza vaccine on pulmonary function in asthma effects of inactivated influenza virus vaccination on bronchial reactivity symptom scores and peak expiratory flow variability in patients with asthma the safety of inactivated influenza vaccine in adults and children with asthma the guillain-barré syndrome and the and - influenza vaccines live attenuated and inactivated influenza vaccine in school-age children comparison of heterotypic protection against influenza a/taiwan/ (h n ) by attenuated and inactivated vaccines to a/chile/ -like viruses efficacy of inactivated and cold-adapted vaccines against influenza a infection, to : the pediatric experience comparison of us inactivated split virus and russian live attenuated, cold-adapted trivalent influenza vaccines in russian schoolchildren studies of the - inactivated influenza vaccine among children attending day care: immunologic response, protection against infection, and clinical effectiveness a randomized controlled trial of cold-adapted and inactivated vaccines for the prevention of influenza a disease effectiveness of influenza vaccine in health care professionals the efficacy of influenza vaccination in elderly individuals: a randomized double-blind placebo-controlled trial efficacy of influenza vaccine in the elderly in welfare nursing homes: reduction in risks of mortality and morbidity during an influenza a (h n ) epidemic clinical manifestations and incidence of oculo-respiratory syndrome following influenza vaccination-quebec ors during the - influenza vaccination season efficacy of attenuated and inactivated influenza vaccines in school children and their unvaccinated contacts in novgorod, russia influenza vaccination in the prevention of acute otitis media in children influenza a vaccine decreases the incidence of otitis media in to -month-old children in day care does influenza vaccination prevent asthma exacerbations in children? effectiveness of influenza vaccination of day care children in reducing influenza-related morbidity among household contacts the effectiveness of vaccination against influenza in healthy, working adults randomized, placebo-controlled double blind study on the efficacy of influenza immunization on absenteeism of health care workers effectiveness and costbenefit of influenza vaccination of healthy working adults: a randomized controlled trial influenza a among community-dwelling elderly persons in leicestershire during winter - ; cigarette smoking as a risk factor and efficacy of influenza vaccination influenza vaccination programs for elderly persons: cost effectiveness in a health maintenance organization benefits of influenza vaccination for low-, intermediate-, and high-risk senior citizens influenza vaccine effectiveness in preventing hospitalizations and deaths in persons years or older in minnesota clinical effectiveness of influenza vaccination in manitoba effectiveness of influenza vaccine in reducing hospital admissions during the - epidemic influenza vaccine effectiveness in preventing hospitalization for pneumonia in the elderly influenza vaccine effectiveness in preventing hospitalization among the elderly during influenza type a and type b seasons reduction in hospital admission for pneumonia in non-institutionalised elderly people as a result of influenza vaccination: a case-control study in spain effectiveness of influenza vaccination in the elderly in a community in italy influenza vaccination, hospitalisations, and costs among members of a medicare managed care plan study of the effectiveness of influenza vaccination in the elderly in the epidemic of - using a general practice database a meta-analysis of effectiveness of influenza vaccine in persons aged years and over living in the community the efficacy of influenza vaccine in elderly persons: a meta-analysis and review of the literature relation between influenza vaccination and outpatient visits, hospitalization, and mortality in elderly persons with chronic lung disease is immunizing all patients with chronic lung disease in the community against influenza cost-effective? evidence from a general practice based clinical prospective cohort study in utrecht, the netherlands association between influenza vaccination and reduced risk of brain infarction association of influenza vaccination and reduced risk of recurrent myocardial infarction influenza vaccination and the risk of primary cardiac arrest effectiveness of influenza vaccine in reducing hospital admissions in people with diabetes influenza vaccination of health care workers in long-term care hospitals reduces the mortality of elderly patients effects of influenza vaccination of health-care workers on mortality of elderly people in long-term care: a randomised controlled trial influenza vaccination levels and influenza-like illness in long-term-care facilities for elderly people in niigata, japan, during an influenza a (h n ) epidemic a new mf- -adjuvanted influenza vaccine enhances the immune response in the eldeerly with chronic diseases: results from an immunogenicity meta-analysis liposomal influenza vaccine the efficacy of live attenuated, cold-adapted, trivalent, intranasal influenzavirus vaccine in children efficacy of vaccination with live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine against a variant (a/sydney) not contained in the vaccine amantadine and rimantadine for preventing and treating influenza a in adults (cochrane review) guidance on the use of zanamivir, oseltamivir and amantadine for the treatment and prophylaxis of influenza zanamivir: a review of clinical safety us food and drug administration. relenza pulmonary function and airway responsiveness in mild to moderate asthmatics given repeated inhaled doses of zanamivir efficacy and safety of inhaled zanamivir for the treatment of influenza in patients with asthma or chronic obstructive pulmonary disease: a double-blind, randomised, placebo-controlled, multicentre study comparison of elderly people's technique in using two dry powder inhalers to deliver zanavivir: randomised controlled trial efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenza virus infections randomised trial of efficacy and safety of inhaled zanamivir in treatment of influenza a and b infections clinical efficacy and safety of the orally inhaled neuraminidase inhibitor zanamivir in the treatment of influenza: a randomised, double-blind, placebocontrolled european study inhaled zanamivir for the prevention of influenza in families efficacy and safety of inhaled zanamivir for the treatment of influenza in patients with asthma or chronic obstructive pulmonary disease: a double-blind, randomised, placebo-controlled, multicentre study zanamivir for treatment of symptomatic influenza a and b infection in children five to twelve years of age: a randomized controlled trial randomized, placebo-controlled studies of inhaled zanamivir in the treatment of influenza a and b: pooled efficacy analysis zanamivir for the treatment of influenza a and b infection in high-risk patients: a pooled analysis of randomized controlled trials zanamivir in the prevention of influenza among healthy adults: a randomized controlled trial short-term treatment with zanamivir to prevent influenza: results of a placebocontrolled study use of the selective oral neuraminidase inhibitor oseltamivir to prevent influenza effectiveness of oseltamivir in preventing influenza in household contacts: a randomized controlled trial efficacy and safety of oseltamivir in treatment of acute influenza: a randomised trial efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza oral oseltamivir in treatment of influenza in children long term use of oseltamivir for the prophylaxis of influenza in a vaccinated frail elderly population evidence for zanamivir resistance in an immunocompromised child infected with influenza b virus guidance on the use of oseltamivir and amantadine for the prophylaxis of influenza recombinant influenza a virus vaccines for the pathogenic human a hong kong (h n ) viruses safety and immunogenicity of a recombinant hemagglutinin vaccine for h influenza in humans safety and antigenicity of non-adjuvanted and mf- adjuvanted influenza a/duck/singapore/ (h n ) vaccine: a randomized trial of two potential vaccines against h n influenza pandemic preparedness: lessons learnt from h n and h n candidate vaccines key: cord- -nnm n a authors: varadé, jezabel; magadán, susana; gonzález-fernández, África title: human immunology and immunotherapy: main achievements and challenges date: - - journal: cell mol immunol doi: . /s - - - sha: doc_id: cord_uid: nnm n a the immune system is a fascinating world of cells, soluble factors, interacting cells, and tissues, all of which are interconnected. the highly complex nature of the immune system makes it difficult to view it as a whole, but researchers are now trying to put all the pieces of the puzzle together to obtain a more complete picture. the development of new specialized equipment and immunological techniques, genetic approaches, animal models, and a long list of monoclonal antibodies, among many other factors, are improving our knowledge of this sophisticated system. the different types of cell subsets, soluble factors, membrane molecules, and cell functionalities are some aspects that we are starting to understand, together with their roles in health, aging, and illness. this knowledge is filling many of the gaps, and in some cases, it has led to changes in our previous assumptions; e.g., adaptive immune cells were previously thought to be unique memory cells until trained innate immunity was observed, and several innate immune cells with features similar to those of cytokine-secreting t cells have been discovered. moreover, we have improved our knowledge not only regarding immune-mediated illnesses and how the immune system works and interacts with other systems and components (such as the microbiome) but also in terms of ways to manipulate this system through immunotherapy. the development of different types of immunotherapies, including vaccines (prophylactic and therapeutic), and the use of pathogens, monoclonal antibodies, recombinant proteins, cytokines, and cellular immunotherapies, are changing the way in which we approach many diseases, especially cancer. the knowledge of human immunology has improved exponentially in recent years, and more advances in the near future are certainly imminent. the immune system is extremely complex, but we are now developing new tools and skills to study it. several factors have been involved in these advancements, and the most important ones include the development of thousands of different monoclonal antibodies that allow the identification of a large variety of cell subpopulations and the functional analysis of immune cells. these tools, together with new and sophisticated technologies, such as single-cell analysis, imaging techniques, omics (including massive dna-rna sequencing, proteomics, and metabolomics data and new tools for processing these data, such as artificial intelligence and machine learning approaches, mathematical modeling, etc.), newly designed animal models (using conventional transgenic/knockout/knock-in mice or new technologies such as crispr-cas (clustered regularly interspaced short palindromic repeats-crispr-associated protein ), are increasing our knowledge about how our immune system functions. the study of the interaction between the immune system and other systems, such as the nervous and endocrine systems or the microbiome, in several illnesses has produced interesting results with important clinical applications. all of these advances can be applied to several immunemediated pathologies, but overall, the success achieved with some types of immunotherapies in recent years is revealing new ways to explore and manipulate the immune system for our benefit. writing a review about human immunology is a significant challenge, but we have attempted to bring together recent knowledge about the immune system, immune-mediated illnesses and types of immunotherapies. the last two decades have witnessed a major revolution in the field of immunology. the traditional classification of the immune system into two different arms, namely, innate and adaptive components that collaborate to respond to foreign antigens or to perform self-/nonself-discrimination, has become much more complex. the development and application of new technologies have provided new findings and created a new landscape in which the immune system establishes cross talk, not only between immune components but also with commensal microorganisms , and other important systems, such as the endocrine and nervous systems [ ] [ ] [ ] . these developments have forced immunologists to reformulate the immunological architecture that confers protection, which has made the study of the immune system especially attractive. moreover, these advances have led to an increased interest in better understanding, managing, and manipulating the immune response in both health and disease. the characterization of new immune cell subsets has been a constant feature in the immunology field. this evolution is clearly reflected in the discovery of an innate counterpart of t lymphocytes, collectively named innate lymphoid cells (ilcs) , and in the identification of different types of effector cd and regulatory t cells . innate lymphoid cells (ilcs). ilcs are lymphocytes, but in contrast to adaptive immune cells, they can colonize lymphoid and barrier tissue sites during fetal development, do not undergo somatic recombination and do not express antigen-specific receptors , . in addition to lymphoid organs, ilcs are enriched in barrier tissues, such as the gastrointestinal tract, airways, and skin , . these innate cells have been considered to be tissue-resident cells, but recent studies suggest that ilcs can migrate through the lymphatic system during homeostasis or enter into the circulation upon infection and inflammation , . currently, five different ilcs are defined on the basis of their transcription factor expression, different cytokine production and/or developmental patterns : natural killer (nk) cells (discussed below), lymphoid tissue inducer cells (ltis) and three subsets of helper-like ilcs (ilc s, ilc s, and ilc s), which are considered to be the innate counterparts of t helper (th) , th , and th cells, respectively. the main focus of this review is ilcs. ilc s are dependent on the t-box transcription factor t-bet and produce interferon gamma (inf-γ), but they differ in the expression of eomesodermin transcription factor . ilc s express cd in humans and cd r in mice, but the natural cytotoxicity receptor nkp (also known as ncr ) is expressed in both species , . ilc s constitute the most homogeneous class of ilcs; they are dependent on gata and rorα, and they produce type cytokines, mainly interleukin (il- ) and il- . ilc s are involved in immune responses to parasite infection, and in humans, they express chemoattractant receptor-homologous molecule expressed in t h cells (crth ) and high levels of cd , whereas most mouse ilc s express st (a member of the il- receptor family) , . the development and function of ilc s depend on the transcription factor rorγt. both human and mouse ilc s can produce granulocyte macrophage colony-stimulating factor (gm-csf), il- , and/or il- , . in humans, two major ilc subsets can be distinguished on the basis of the expression of the natural cytotoxicity receptor nkp (also known as ncr ) , . both types can produce il- , but the production of il- is mainly confined to nkp + ilc s. extensive research has focused on deciphering the role of ilcs to ensure the maintenance of tissue homeostasis and immune protection , . ilcs express particular sets of receptors in a tissuespecific manner, and these allow the detection of host-derived signals (including those from alarmins, neuronal mediators, microbia, and the diet) . the integration of these endogenous signals is essential for the maintenance of tissue homeostasis, but dysregulation of ilc responses leads to inflammation and disorder , . ilc are mainly involved in early protection against viruses and bacteria , , but their response to dysregulated local proinflammatory cytokine production in adipose tissues leads to the development of metabolic disorders and obesity . il- and il- produced by ilc s induce goblet cell differentiation and the recruitment of eosinophils, basophils, and mast cells , which are involved in protection against infection by helminths and viruses, but when uncontrolled, these cells drive allergic responses and metabolic disorders. moreover, the depletion of ilc s in animal models suggests a role for these cells in atopic dermatitis and asthma . ilc s are abundant in mucosal tissues, and ncr + ilc s have been proven to be essential for regulating the balance between commensal and pathogenic bacteria through the production of il- . in contrast, ncr − ilc s can promote colitis in a model of inflammatory bowel disease . the lack of immunodeficiency in ilc-deficient patients led to the proposal that ilcs are dispensable in the presence of functional t cells and b cells . however, recent studies support the idea that ilcs cannot be considered to have functions that only duplicate those of the adaptive immune system. in addition to those showing the essential role of lti cells in the formation of secondary lymphoid organs during embryogenesis and the postnatal development of intestinal lymphoid clusters, recent studies also provide evidence that subsets of ilcs express multiple factors that modulate the adaptive immune response in health and disease , . in particular, ilc s and ilc s modulate the t-cell response. studies in mice suggest that in healthy intestine, ilc s express major histocompatibility complex (mhc) class ii molecules but lack the expression of costimulatory molecules; therefore, they inhibit microbiota-specific t-cell responses, thus preventing intestinal inflammation . it seems that the interaction between ilc s and tfh cells limits il- secretion and the production of iga by mucosal b cells . studies with murine models have significantly contributed to the classification and understanding of the role of ilcs in the immune system, especially since similarities have been observed between ilcs identified in mice and humans . however, the differences between these two species present real challenges , because human ilcs have unique attributes that are only now being elucidated, with further work required in this exciting field. the roles of ilcs in immunity and their cross talk with other components of the immune response await further analysis. detailed coverage of this topic is beyond the scope of this review, and we refer the reader to recent reviews that provide more information on the biology of human and mouse , ilcs. t cells and plasticity. t cells are categorized as tα/β and tγ/δ cells, depending on the type of t-cell receptor (tcr) that they express . human tγ/δ cells, similar to their murine counterparts, are a minor population ( - % of nucleated cells) in peripheral blood, but are especially abundant in barrier tissues such as the epidermis [ ] [ ] [ ] . the three main subsets of t cells carrying α/β receptor are the cd +t helper cells and cd +cytotoxic and cd + cd + regulatory t cells . new effector cd + helper t-cell subsets (initially classified as th and th ) , have been recently described, and at least six human th cell subsets have been identified to date: th , th , th , tfh, th , and th cells , . all of these cells recognize foreign peptides presented by class ii mhc molecules on antigenpresenting cells (dendritic cells, macrophages, and b lymphocytes). th cells are required to activate macrophages and cellmediated immunity to kill intracellular pathogens , whereas th cells are important in facilitating eosinophils to fight against parasitic helminths and b cells for antibody production and antibody class-switching to generate iga or ige . th cells are required to mobilize neutrophils for the clearance of fungi and extracellular bacteria, and they are also involved in mucosal protection . th and th cells are also involved in mucosal immunity; th cells protect against parasites , , and th cells prevent microbial translocation across epithelial surfaces and promote wound healing , . as mentioned in the introduction to ilcs, studies on human th cells isolated from lymphoid organs and blood samples, along with recent observations on the developmental mechanism of distinct th cell subsets, have revealed both similarities and differences of human and mouse th cells , , . tfh cells are very important for germinal center reactions, antibody class switching, affinity maturation, and the development of high affinity antibodies and memory b cells , . at the surface marker level, tfh cells are generally characterized by the expression of cxcr , the chemokine receptor for cxcl , which is highly expressed on b-cell follicles for expressing inducible t-cell costimulator (icos) and programmed death protein (pd- ) , , which enable their involvement in the interaction of tfh cells and b cells . the definition of a given t cell lineage is based on its ability to sense different inductive cytokines, to produce particular cytokines or to express a lineage-specifying transcription factor. th cells produce ifn-γ and express t-bet ; th cells are characterized by il- , il- , and il- production and gata- expression , ; ptregs, which are induced in the periphery from naïve precursors, produce tgf-β and express foxp (tr cells are il- -secreting tregs that do not express foxp ) . th cells produce il- a, il- f, and il- and express rorγt , , and tfh cells produce il- and il- and express the bcl transcription factor. in addition, th cells, which produce il- and express the aryl hydrocarbon receptor (ahr) , , and th cells, are characterized by the expression of il- and the transcription factor pu. . additional levels of regulation, such as the differential expression of micrornas, long noncoding rnas (lncrnas), and protein stability and function, have been found to control various aspects of th cell differentiation and effector function , . cd + cytotoxic t cells express the dimeric cd marker and have specific lytic capacity to target cells through several mechanisms, including the release of cytotoxic granules, secretion of cytokine tumor necrosis factor alpha (tnfa) and interferon gamma, and the induction of cell death through the interactions of fas and the fas ligand , . their tcrs are restricted to interactions with peptides presented by class i mhcs. regulatory t cells (tregs) include thymically derived and peripherally induced regulatory t cells (ttregs and ptregs, respectively), and they produce either il , tgf-beta, il- or combinations of these proteins . ttregs express the transcription factor foxp and secrete il and tgf-β; ptregs, which are induced in the periphery from naïve precursors, can also be subdivided into il- -induced tregs [tr cells] (which secrete large amounts of il- and moderate levels of tgfβ), th cells (which produce il- and tgf-β), and tgfβ-induced tregs [itregs], which may or may not express foxp . moreover, new subsets of regulatory t cells have been described. they include follicular regulatory t cells (which express foxp and bcl- and cxcr ), which modulate the function of tfh cells and fine-tune the germinal center response [ ] [ ] [ ] , and a il- dependent regulatory population of cells (referred to as itr cells), which show potent suppressive potential in several mouse disease models . other regulatory populations have also been described, including bregs and cd + tregs, which are the analogous counterparts of tregs [ ] [ ] [ ] . recent studies have revealed the capacity of differentiated t cells, particularly th cell and ptreg subsets, to change their phenotype in response to changing contexts [ ] [ ] [ ] [ ] [ ] . becattini et al. found that human memory cd t cells primed in vivo by pathogens (e.g., candida albicans and mycobacterium tuberculosis) or vaccines (tetanus toxoid) are highly heterogeneous, both at the population and clonal levels. with respect to studies on human arthritis, nistala et al. proposed that th cells are recruited to the joint and converted to th / or th cells in response to local il- levels. this plasticity has also been observed with in vitro assays under conditions that mimic a disease site, namely, low tgf-β and high il- levels . these results are inconsistent with the original idea of th lineage stability and provide new possibilities for disease treatment aimed at inducing particular th subsets to modulate the immune response against pathogens or to control detrimental immunity , , . trained and adaptive immune memory other classical concepts in fundamental immunology, such as immune memory, are also changing. the specificity and the capacity to generate long-lived memory cells are two properties that have been classically used to distinguish innate immunity from adaptive immunity. adaptive immunity is clearly based on the specific recognition of antigenic determinants by somatically diversified receptors (b cell and t cell receptors (bcr and tcrs, respectively)) and on its capacity to respond more effectively to restimulation with the same antigen. in contrast, innate immune responses have traditionally been considered nonspecific and without the capacity to adapt . however, the discovery of germline-encoded pattern recognition receptors (prrs) and the "trained innate" immunity (or innate immune memory) have provoked a shift in our understanding of the immune response. in , medzhitov et al. demonstrated that pattern recognition receptors (prrs) expressed on innate cells recognize invariant molecular structures expressed by invading pathogens . after the interaction, prrs trigger the expression of costimulatory molecules and activate important signaling pathways to induce the activation of innate and adaptive immune cells. prrs mainly belong to four families: toll-like receptors (tlrs), nod-like receptors (nlrs), c-type lectin receptors (clrs), and peptidoglycan recognition proteins (pgrps) , . the profiles of prrs expressed by innate cells can lead to partially specific recognition of a type of microorganism; e.g., innate cells can distinguish between gramnegative and gram-positive bacteria and modulate the immune response based on this recognition, although they cannot differentiate between bacterial species . the idea that only jawed vertebrates developed immunological memory has also been challenged by the observation of resistance to reinfection in organisms that lack an adaptive immune response, such as plants and invertebrates , . recent studies have shown that monocytes and macrophages exposed to candida albicans or β-glucans exhibited an enhanced secondary response . in addition, immunization of mice with bacillus calmette-guérin (bcg, the tuberculosis vaccine) induces t cellindependent protection against secondary infections by candida albicans, schistosoma mansoni or influenza virus [ ] [ ] [ ] [ ] . thus, organisms are protected not only against the original microorganism but also to unrelated pathogens. the mechanisms underlying the establishment of this innate immune memory differ from those involved in adaptive immune memory . after infection or vaccination, innate immune cells (such as monocytes and macrophages) display long-term functional changes through epigenetic and metabolic reprogramming, including histone acetylation, methylation and modulation of noncoding rnas [ ] [ ] [ ] . in turn, the faster and more pronounced reactivity of adaptive immune cells (t and b lymphocytes) upon reinfection is characterized by permanent changes in the genome of cells, such as mutations, gene rearrangement, clonal expansions, as well as epigenetic modifications, all of which ensure a more persistent effect than is endowed by trained immunity , , . other cells for which immunological memory has been described include tγ/δ cells and innate lymphoid cells . recently, some authors have proposed that nk cells are also capable of immunological memory [ ] [ ] [ ] [ ] . antigen-specific recall responses by human nk cells were observed by nikzad et al. in humanized mice and in varicella zoster virus (vzv)-exposed adult human volunteers, in which cytotoxic nk cells were recruited to sites of an vzv test antigen challenge on the skin. sensitization with haptens using mice lacking t cells and b cells led to the generation of hapten-specific memory nk cells . the recall response persisted for more than four months after priming, and was adoptively transferred to naïve mice . interestingly, nk cells exhibit memory that is not only specific to a given virus, such as cytomegalovirus , , but that is also induced in the absence of a defined antigen , . furthermore, new studies suggest that trained immunity is not a phenomenon that is restricted to immune cells, because epithelial stem cells also retain memory of previous inflammatory challenges by displaying an enhanced wound-healing capacity upon skin damage . given the data outlined above, immunological memory is now recognized to be highly diverse and not restricted to b cell-or t cell-mediated adaptive immunity. much remains to be learned in this field, but the different manifestations of immunological memory described above offer an important basis for clinical applications, such as the development of novel vaccination strategies or new therapies for pathological situations in which immunological memory can be detrimental, such as allergies or autoimmune diseases , , . interaction of the immune system and the microbiome the immune system has evolved in the presence of commensal microorganisms that colonize barrier surfaces of vertebrates and invertebrates , . the cross talk between the natural host microbiome and immune system is particularly interesting in the gastrointestinal tract, where the density and diversity of indigenous bacteria, viruses and fungi are greatest compared to those of other anatomical sites . in the literature, reports of observed changes in microbial community composition during diseases are diverse and include those in inflammatory bowel disease (ibd), obesity, metabolic syndrome, and multiple sclerosis [ ] [ ] [ ] [ ] [ ] . however, the microbiome can be influenced by different factors, such as the specific niche that it occupies, diet, stress, environmental factors, and host genetics, and a specific correlation does not necessarily infer causation. the presence of these commensals in mucosal tissues has been known since before metchnikoff, but the current knowledge on the role of the microbiome in shaping the immune system throughout life came mostly from the development of next-generation sequencing (in particular, the reduction in the cost of s ribosomal rna gene sequencing) and the use of germ-free animal models, which can be colonized even with human microbiota . germ-free mice are characterized by atrophy of peyer's patches with few germinal centers and isolated lymphoid follicles, a lower number of b, t, and dendritic cells and a decreased level of immunoglobulins, particularly iga and igg . these effects are observed at the mucosal and systemic levels, and they can be reversed within weeks after the colonization of germ-free mice with commensal bacteria . moreover, colonization with commensal bacteroides fragilis revealed the immunomodulatory effect of bacterial polysaccharides in restoring systemic cells and the differentiation of cd + t cells into regulatory t cells (foxp + tregs), which in turn favor mucosal immunomodulation . the induction of th cell maturation by segmented filamentous bacteria has also been reported . these important examples emphasize the major roles of the commensal microbiome in the maturation of mucus-associated lymphoid tissue and the systemic immune system. the development of new technologies to better track the locations and activities of distinct microbial populations is essential to elucidate host-microbe interactions, through which other systems, such as the nervous system, seem to play important roles , - . the better characterization of some immune cell subsets, trained immunity, and host-microbiome interactions provides a few very good examples that prove the maturation of immunology in the last few decades. in this sense, studies with mouse models have significantly contributed to the increase in our fundamental knowledge; however, the differences between murine and human immunology are notable, and conclusions drawn from mouse studies are sometimes not fully translated to humans . if we want to fully exploit the power of the immune system for human health, greater effort is required for understanding human immunology. immunologists, in cooperation with experts from other fields, have developed a variety of protocols and tools to achieve greater selectivity in the identification and analysis of human cell subsets, types of cytokines and receptors, chemokines, etc. these tools range from biological approaches that rely on next-generation sequencing, mass spectrometry, and bioinformatics to immune monitoring technologies based on multiparameter flow cytometry and single-cell gene expression analysis. although not without limitations, these techniques provide a much better picture of the whole immune system than individual and independent approaches. immune-mediated illnesses comprise a wide variety of diseases characterized by the dysregulation of a normal immune response. most of these illnesses are complex disorders believed to arise from a combination of genetic and environmental factors . infectious diseases infectious diseases are caused by pathogens (viruses, bacteria, fungi or parasites that infect the host body), and they remain a leading cause of mortality worldwide. prominent examples include illnesses produced by mycobacterium tuberculosis, human immunodeficiency virus (hiv), plasmodium falciparum or the current coronavirus disease (covid- ) outbreak caused by the severe acute respiratory syndrome coronavirus (sars-cov- ), which has already infected millions of people and produced thousands of deaths in many countries. for a number of years, many people believed koch's postulates, which implied that virulence traits reside solely in the pathogen. however, recent advances in molecular biology have shown that host genes play major roles in infection, together with a wide range of environmental variables . to date, six gene products endowing infectious disease susceptibility have been validated in the literature: ( ) hemoglobin subunit beta; ( ) band -anion transport protein; ( ) duffy antigen/ receptor, which is associated with plasmodium spp. infections; ( ) the prion protein associated with creutzfeldt-jakob disease; ( ) fucosyltransferase and , which is associated with norwalk virus infections; and ( ) c-c motif chemokine receptor (ccr ) coreceptor, encoded by an immune-related gene and leads to the impairment of the entry of the human immunodeficiency virus (hiv) into helper t cells, thus avoiding/decreasing the progression to acquired immunodeficiency syndrome . another gene associated with infectious disease and the immune system is the natural-resistance-associated macrophage protein (nramp ), which encodes an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. it is a susceptibility locus for increased ratios of infection with leishmania spp. parasites and certain strains of salmonella spp., mycobacterium bovis and mycobacterium tuberculosis , . in addition, it has been suggested that functional variants of immunoglobulin fc gamma riia (cd ) are related to the development of invasive encapsulated bacterial infections . moreover, because of recently acquired genomic data, new human polymorphisms have been discovered, some of which play roles in changing immunoglobulin levels, seroconversion rates or the intensity of antigen-specific immune responses. in addition, they also contribute to human susceptibility to infection by viruses such as influenza, rhinovirus and respiratory syncytial virus . these polymorphisms are mapped within the mhc (hla-dqb * , hla-drβ , or hla-dpβ ), natural killer cell immunoglobulin-like receptors and (kir dl and kir ds ) and natural killer lectinlike receptor d (kldr- ) . several recent studies available as preprints have analyzed certain genes that may explain the differences in the variable expression of and susceptibility to covid- by patients, either by affecting the host receptor for the virus (angiotensin i converting enzyme (ace- )) , immune genes (tlr and others) or blood groups (group o seems to be the most protective) , and more extensive omics studies are now underway with larger numbers of patients. in , the physician paul ehrlich first used the term "horror autotoxicus" to describe the way autoimmunity contradicts the natural aversion to self-injury ("living with the enemy", reviewed in ). currently, according to the american autoimmune related disorders association, more than autoimmune diseases have been identified. historically, these diseases were considered to be rare, but current epidemiological data have shown that they affect approximately - % of the population worldwide. some of the most common autoimmune diseases include type diabetes, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease (https://www.aarda.org/diseaselist/). although significant progress has been made in understanding the mechanisms of autoimmune diseases and the nature of selftolerance, these disease remain major burdens on health systems around the world. autoimmune diseases arise when the immune system attacks normal components of the body . the concept of immune tolerance is defined as the ability of the immune system to prevent the targeting of self-molecules, self-cells or self-tissues. on the other hand, the failure to distinguish self from nonself is often termed a break of tolerance, and it is the basis for an autoimmune disease . what are the mechanisms that lead to a break in tolerance? autoimmune diseases are complex disorders that are believed to arise from a combination of genetic (mutations and higher inheritance frequency of some types of major histocompatibility complex alleles), epidemiological (age and sex) and environmental (infections, microbiota, tobacco, chemicals and pharmaceutical drugs). factors these factors trigger a break in self-tolerance with the activation of self-reactive lymphocytes through several mechanisms, such as molecular mimicry, the overexpression and abnormal expression of mhc class ii molecules in peripheral tissues, thymic aging, and immunodeficiencies (discussed below) and many others. some lymphocytes escape control due to polymorphisms in several genes that affect the routes of lymphocyte activation. other causes may include defective antigen presentation by some mhc variants with specific polymorphisms. therefore, the self-reactive lymphocytes that have escaped control and react against self-constituents initiate the autoimmune process . although a large number of genome-wide association studies (gwas) have led to the identification of hundreds of polymorphisms associated with the development of different autoimmune diseases, it has proven difficult to define the role of most of these polymorphisms in the breakdown of tolerance to a selfantigen [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is worth highlighting, however, that the mhc remains the main genetic factor associated with human autoimmunity , . other gene variants identified are common to many autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type i diabetes, ulcerative colitis, autoimmune hepatitis and numerous other autoimmune diseases. for example, the protein tyrosine phosphatase nonreceptor type (ptpn ) gene encodes a protein that inhibits t-cell activation in the adaptive immune system, whereas it promotes myeloid cell activation; interferon regulatory factor -transportin (irf -tnpo ) is involved in the accumulation of lymphocytes within lymphoid organs and failed elimination of autoreactive naïve t cells; btb domain and cnc homolog (bach ) has a critical role in immunoglobulin class-switching recombination, somatic hypermutation of immunoglobulin encoding genes and the activation of tissue macrophages. a more complete list of genes associated with autoimmunity can be found in the review by wang et al. researchers are currently looking for the missing heritability in autoimmune diseases by focusing on the study of methylome profiles, genetic cargos in extracellular vesicles, genetic alterations, and ways in which the microbiome may affect these diseases. immune-mediated rejection of tissue allografts was first described in by the british immunologist peter medawar , . only three years later, george snell described the mhc, which carries the histocompatibility genes, and one decade later, jean dausset described the human leukocyte antigen (hla); each of these scientists was recognized with the nobel prize in physiology and medicine . since its discovery, mhc has emerged as the most polymorphic gene locus in eukaryotes with hla and related alleles, more than nucleotide variants reported in the individual-participant data-international immunogenetics/ human leukocyte antigen (ipd-imgt/hla) work group database (https://www.ebi.ac.uk/ipd/imgt/hla/), release . . , / / . although the main barrier for long-term organ and tissue grafting is driven by hla incompatibilities, other important players play roles in transplant rejection. in particular, minor histocompatibility antigens, which are peptides derived from allelic variants of normal cellular proteins, presented by class i or ii mhc antigens induce cellular immune responses in hla-matched individuals who lack the same allelic variant . natural killer (nk) cells also play important roles in transplantation through their killer cell immunoglobulin-like receptors (kirs), which are receptors for hla class i molecules. nk cells expressing an inhibitory kir-binding self-hla can be activated when exposed to allografts that lack a ligand for the inhibitory receptor . the locus that codifies these receptors displays a considerable degree of polymorphism, with alleles reported in the individual-participant data-international/killer cell immunoglobulin-like receptors (ipd/kir) work group database, release . . , / / . more recently, we have begun to appreciate the importance of non-hla genetic factors in the development of transplant rejection; examples include polymorphisms in the genes encoding cytokines, such as tumor necrosis factors (tnf), interleukins (il- , il- and il- ), interferon gamma (ifn-γ), and transforming growth factor-β (tgf-β ). other genes encode pathogen recognition receptors, with nucleotide-binding oligomerization domaincontaining (nod (card )) being the most widely studied, although conclusive data have not been obtained to date . primary immunodeficiencies (pids) comprise a heterogeneous group of more than genetic disorders that result in defects in the immune response . pids are considered mendelian disorders because they are mainly autosomal recessive disorders that often display incomplete penetrance, which affects the severity and onset of the disease. with the exception of immunoglobulin a (iga) deficiency, pids are considered to be rare disorders, as their prevalence worldwide ranges from to among , people . unsurprisingly, these types of diseases are not uncommon in highly consanguineous populations such as those in the middle east/northern africa (mena) region. the incidence of consanguinity marriage in these areas ranges between and %, which leads to a unique population in which autosomal recessive diseases arise, with the prevalence of pid in these countries as high as in , people . although more than genes have been described for pids, approximately % of the causal genes remain unknown, and next-generation sequencing studies performed in mena populations are contributing to the search for currently unknown genes that cause pids . a complete and updated list of pid-causing genes and diseases can be found at the european society for immunodeficiencies (esid) webpage (https://esid.org) . clinical manifestations of pids are highly variable; many disorders involve an increased susceptibility to several types of infections, but some patients develop autoimmune diseases. patients usually present recurrent sinus or ear infections or pneumonia within a one-year period; other indicators are failure to thrive, poor response to prolonged use of antibiotics, and persistent thrush or skin abscesses . depending on the affected pathway, pids are associated with varying levels of severity, times of onset, and risks of infection by certain groups of microorganisms. according to the international union of immunological societies (iuis) (https://iuis.org/ committees/iei/), inborn errors of immunity can be classified as follows: (a) immunodeficiencies that affect cellular and humoral immunity; (b) combined immunodeficiency (cid) with associated or syndromic features; (c) predominant antibody deficiencies; (d) diseases of immune dysregulation; (e) congenital defects of phagocyte number, function, or both; (f) defects in intrinsic and innate immunity; (g) autoinflammatory disorders; (h) complement deficiencies; and (i) phenocopies of a pid , . however, pids are broadly classified as follows according to the component of the immune system affected: • t-cell immunodeficiency, e.g., defects in the ifn-γ/il- pathway and mutations in the autoimmune regulator (aire) gene. • b-cell (antibody-mediated) immunodeficiency: gamma-globulinemia, x-linked common variable immunodeficiency (cvid), selective iga deficiency, specific antibody deficiency, and igg subclass deficiency. • combined immunodeficiency: wiskott-aldrich syndrome, ataxia telangiectasia, digeorge syndrome and severe combined immunodeficiency (scid). • phagocyte defects: chronic granulomatous disease, hyperimmunoglobulin e (ige) syndrome and leukocyte adhesion deficiency. • complement defects (deficiency in early, late or regulatory complement components) . autoinflammatory diseases systemic autoinflammatory diseases (aids) are characterized by recurrent acute inflammatory episodes secondary to a dysregulated inflammatory process that typically develops during childhood, with recurrent episodes of fever, rashes, and disease-specific patterns of organ inflammation. genetically speaking, these are hereditary disorders, andto date, more than genes (table ) have been identified as causes of aids, which can be grouped according to the pathway that is altered . ( ) inflammasome. the inflammasome is a multiprotein intracellular complex that detects pathogenic microorganisms and stressors and activates the highly proinflammatory cytokines il- β and il- . genes affected in this group are mefv (mediterranean fever pyrin innate immunity regulator), which is related to familial mediterranean fever (fmf); nlrc (nlr family card domaincontaining ); nlrp (nlr family pyrin domain-containing ) and wdr (wd repeat domain ) . ( ) type-i interferon (ifn)-mediated disorders. these disorders are characterized by the upregulated expression of genes induced by ifn. the gain of function by variants of tmem (transmembrane protein ) is the core manifestation of this disorder group, but other genes have been identified, including ddx (dexd/h-box helicase ), dnase (lysosomal deoxyribonuclease ), pola (dna polymerase alpha subunit) and usp (ubiquitin-specific peptidase ) , . ( ) ubiquitination disorders. ubiquitination is a process that marks proteins for degradation via the proteasome, which is required for the processing of intracellular antigens (such as virus proteins or mutated tumor proteins) and their presentation by class i hla molecules. ubiquitination involves three main steps: activation, conjugation and ligation, which are performed by ubiquitin-activating enzymes (e s), ubiquitin-conjugating enzymes (e s), and ubiquitin ligases (e s). ubiquitination disorders are caused by variants of the psmb , psmb , psma and psm genes (proteasome s subunit beta , subunit beta , subunit alpha and subunit alpha , respectively), affecting the proteasome subunits, proteasome maturation protein gene (pomp) and/or proteasome assembly chaperone (psmg ), by encoding proteasome assembly molecules . in addition, other genes in this group, such as otulin (otu deubiquitinase with linear linkage specificity), encode ubiquitin peptidases, i.e., proteins involved in ubiquitination assembly complexes, such as hoil- (hemeoxidized irp ubiquitin ligase ) and hoip (nhp -like protein homolog). finally, the loss of function due to variants of the tnfaip (tnf-alpha-induced protein , also known as a ) gene, which encodes a protein with ubiquitin ligase and ubiquitinase activity, has also been described . ( ) inflammatory or innate immune regulators. a large number of genes have been found to affect the pathways/ mechanisms involved in macrophage and b-cell differentiation and lymph node development, among many functions. genes in this group include ada (adenosine deaminase ), tnfrsf a (tnf receptor superfamily member a), adgre (adhesion g protein-coupled receptor e ), trnt (trna nucleotidyltransferase ), lacc (laccase domain-containing ) and ap s (adaptor related protein complex subunit sigma ) . allergy allergic diseases can be termed complex diseases that involve both genetic and environmental factors, and they influence not only the development of ige-mediated sensitivity in the case of hypersensitivity type i allergies but also the subsequent development of clinical symptoms in a range of tissues, including skin, nose, and lung tissue . since the first report of a link between chromosome q and atopy in , knowledge about the common risk variants for allergic diseases has increased exponentially, mainly because of gwas. most allergic diseases have allergy-related traits such as asthma, with the strongest association mapped to chromosome q . however, the disease-associated gene at this locus remains unclear; one of the candidate genes is ormdl (sphingolipid biosynthesis regulator ) due to its role in sphingolipid synthesis and the regulation of eosinophils. other genes associated with asthma are interleukin (il ) and its receptor, il rl (interleukin receptor-like ), hla region, smad (sma-and mad-related protein ) and il rb (interleukin receptor subunit beta) . as asthma and other allergic-associated traits could be present in patients without allergies, some researchers performed gwas analysis on cohorts of patients who had high levels of allergenspecific immunoglobulin e (ige) or a positive skin prick test. as a were identified, and the strongest association was on chromosome q . this locus has been associated with two genes: c orf (emsy transcriptional repressor, brca interacting), a potential regulator of interferon-stimulated gene, and lrrc (leucine rich repeat-containing ), which is involved in transforming growth factor beta (tgfβ)-signaling in t regulatory cells. the rest of the associated loci involved in the pathogenesis of allergy highlight the importance of the th responses (stat (signal transducer and activator of transcription ), tslp (thymic stromal lymphopoietin), bcl (b-cell lymphoma protein), il rl (interleukin receptor-like ), il (interleukin ), gata (transacting t-cell-specific transcription factor binding protein )); innate immunity (tlr / / (toll-like receptor / / )); tgfβ-signaling (lrrc (leucine rich repeat-containing ), smad (mothers against decapentaplegic homolog )); t-cell (il (interleukin ), ptger (prostaglandin e receptor )) and t regulatory box (lrrc (leucine rich repeat-containing ), il- , nfatc (nuclear factor of activated t cells ), foxa (forkhead box a )) . in the last two years, researchers have focused on epigenomewide association study (ewas) of allergy processes. the epigenetic landscape is specific for a given cell; thus, ewas requires careful selection of the relevant cell type for a given biomedical condition. for allergies, ewas has mainly been performed on nasal mucosal cells and whole blood (although the result was later normalized by the number of circulating eosinophils). nasal mucosal cells comprise cd + t cells, cd + t cells, myeloid cells, innate lymphoid cells, b cells, double-negative t cells, granulocytes, cd + cells, and plasma cell populations . in all of these studies, cpg-associated regions were identified, from which the smad gene, coding for an important regulator of t-cell differentiation, was replicated in three independent cohorts . of all of the genes in whole blood identified using ewas, only the acot (acyl-coa thioesterase ), epx (eosinophil peroxidase), gja (gap junction protein alpha ) and mettl (methyltransferase-like ) genes were confirmed in the nasal cell populations . in , ehrlich proposed the idea that mutant cells arise continuously and that the immune system scans for and eradicates these mutant cells before they manifest clinically . however, immune surveillance remained a controversial topic until its acceptance in the s . immune surveillance is the recognition and elimination of cancerous cells by lymphocytes, which act as sentinels that recognize transformed cells. ultimately, during tumor progression, cancer cells show low immunogenicity and resistance to immune effector cells, thus expanding and escaping immune control. the way in which cancer cells modify the immune system has been called immune editing . the key of immunosurveillance is cancerous cell expression of tumor antigens that can activate various immune cell phenotypes; for simplicity, any overexpressed, mutated, dysregulated, or rearranged gene product expressed by a cancerous cell may be considered a tumor antigen. it is critical to consider that most of these proteins, except those derived from virus-infected cancer cells, are primarily self-proteins, but they are expressed with mutation(s) or minor changes in their antigenic structure . one mechanism by which cancer cells escape from immune recognition is antigenic modulation. for example, the loss of mhc class i molecule expression leads to aberrant antigen masking, which is one of the mechanisms described for tumor cells that escape specific antitumor t-cell immune responses . in addition, the mhc-peptide-t cell receptor complex elicited by a tumor antigen shows weak stability, since high-affinity t-cells tend to be rendered tolerant to these antigens . another mechanism is the direct inhibition induced by cancer cells due to their interaction with surface regulatory molecules, table . also called checkpoint molecules. these molecules include programmed cell death- (pd ) and cytotoxic t-lymphocyteassociated protein (ctla- ), which induce the inhibition of host t cells. although these checkpoints usually help conventional immune responses control immune activation, they can also be used by tumor cells to inhibit antitumoral t-cell responses . pd ) is a transmembrane protein expressed on t, b, and nk cells, and it binds to pd ligands (pd-l and pd-l ) on target cells. when it binds to its ligand on tumor cells, pd inhibits tumor cell apoptosis, causes peripheral effector t-cell exhaustion, and promotes the conversion of effector t cells into regulatory t cells , . ctla is also a physiological negative regulator of t-cell activation. the interaction with cd /cd in the tumor leads to the inhibition of t-cell function and suppressed effector activity . knowledge of these two checkpoint inhibitors has opened the door to new antitumoral therapeutic approaches, such as the use of monoclonal antibodies that block the aforementioned interactions (anti-pd , anti-pd-l , or anti-ctla- ), which are called checkpoint inhibitors . in addition, tumor cells create an inhibitory microenvironment around them. malignant cells can recruit other cells, such as immune cells and fibroblasts, which can be corrupted by tumor cells. the interaction between tumor and nontumor cells creates the tumor microenvironment, which is mostly driven by the dynamics of the tumor promoting the proliferation/expansion of cancer cells. for example, tumor and stromal cells release multiple factors, such as the chemokine ccl (c-c motif chemokine ligand ), which inhibits effector t-cell functions and attracts tregs to the microenvironment . tumor cells use different mechanisms to promote cancer progression and further metastasis. the complete immunological eradication of cancer is the goal of antitumoral immunotherapy and is discussed later in this review. immunosenescence and inflammaging aging is accompanied by the decline and dysregulation of immune efficacy, which results in an increased vulnerability to infectious diseases, diminished responses to vaccination, and reduced tumor clearance. immune alterations mainly manifest as a reduction in the number of naïve peripheral blood cells and a relative increase in some types of memory cells . natural aging causes progressive atrophy of the thymus, which is called thymic involution. the endpoint is a significant decrease in naïve t cells, which reduces the diversity of the t-cell antigen receptor (tcr) repertoire and culminates in disrupted t-cell homeostasis . the cellular and molecular hallmarks of aging have been described as genomic instability, telomere attrition, epigenetic alterations, sarcopenia, changes in intracellular communications, cellular senescence, immunosenescence and mitochondrial dysfunction . the process of aging alters the innate and adaptive immune systems. in terms of innate immunity, aging results in a decreased number of circulating monocytes and dendritic cells, reduced phagocytic properties of macrophages and neutrophils, and impaired antigen presentation by dendritic cells . as mentioned above, aging also generates a reduction in the t-cell and b-cell receptor repertoire due to the accumulation of senescent or exhausted lymphocytes, together with a decrease in the number of circulating naïve t and b cells , . on the other hand, nk cell cytotoxicity is maintained in centenarians, and an increase in the number of these cells is observed in healthy aging people . moreover, cd + t cells exhibit cytotoxic features in centenarians; this is an acquired characteristic for cd + t cells that usually have helper, but not cytotoxic functions under physiological conditions . in addition to these features, chronic inflammation is considered the key that underlies the phenomenon called 'inflammaging', which is related to elevated self-reactivity and results in the typical chronic low-grade, systemic inflammatory phenotype observed in the elderly in the absence of acute infection. currently, it is believed that self-reactive t cells are the main contributors to this process. it has been proposed that this basal inflammatory state contributes to the development of some diseases, such as type ii diabetes, alzheimer's disease and atherosclerosis . understanding the mechanisms of age-related disorders in immune regulation is important for identifying more efficient strategies of immune rejuvenation and for the effective induction of vaccination-mediated immunity in older individuals . immunotherapy includes the use of certain components of the immune system (antibodies, cells, cytokines, etc.) for the treatment of various cancers and autoimmune diseases and the manipulation of the immune system through vaccines for the prevention and treatment of infectious and allergic diseases (fig. ) . immunotherapy using microorganisms or their components in vaccines was first practiced centuries ago; soluble substances such as poly-and monoclonal antibodies, as well as cytokines, have been used for many years, but recently, cellular immunotherapy has emerged in clinical practice. although immunotherapy can be used for many diseases (infections, autoimmune diseases, macular degeneration, allergic diseases, etc.), it is being used most expansively in the cancer field. the main goal is to destroy the tumor, either directly or indirectly (by enhancing the patient's immune system), while offering greater specificity and fewer side effects than conferred by conventional therapies. pathogens and vaccines for infectious diseases immunotherapy associated with pathogens was first linked to the prevention of infectious diseases, starting from variolization (in the x century), followed by edward jenner's vaccination against smallpox (in the xviii century) and subsequently many other preventive vaccines for infectious diseases. the great advances in the knowledge about infectious diseases took place in the nineteenth century, but the xx and xxi centuries are clearly the vaccination centuries, as many new successful vaccines (with attenuated or dead pathogens, subunits, recombinant proteins, carbohydrates or dna) introduced against a variety of pathogens. more recently, and with the increased knowledge of the human microbiome, the use of microorganisms in therapy has seen a resurgence. some intestinal infections, such as those produced by clostridium difficile, can be cured with the transfer of intestinal bacteria from healthy people (feces transplantation) . numerous other attempts to use microorganisms to cure inflammatory illnesses (crohn's disease, ulcerative colitis, etc.) have met with limited success , which indicates that this type of therapy is much more complex than initially anticipated. as a consequence, many more studies are required to ensure that this approach can be used for curative immunotherapy. researchers are also working on genetically modified or artificial bacteria (e.g., based on salmonella enterica, listeria monocytogenes or lactobacillus lactis), but only limited effects have been observed to date . oncolytic viruses (ovs). although the use of bacteria in antitumoral therapy has been largely restricted, the use of therapeutic viruses is increasing. virus-based therapy was introduced in the s with the use of adenovirus, but only in recent years has it been used in practice in the clinic. oncologic viruses have the capacity to attack tumor cells in a preferential manner and induce immunogenic cell death (icd) and host antitumor immunity (fig. ) . the first virus approved for use in therapy was a recombinant oncolytic adenovirus named h , which was licensed in by the china food and drug administration (cfda) for treating head and neck carcinoma in combination with chemotherapy . ten years later, the oncolytic attenuated-modified virus herpes simplex i-talimogene laherparepvec (t-vec, imlygic®) was approved by both european (emea) and american (fda) agencies for the treatment of melanoma . the virus is modified by the insertion of human gm-csf and deletion of the icp gene. since the approval of t-vec, a new era has dawned on the use of ovs in cancer therapy , . currently, oncolytic viruses from the adenoviridae, herpesviridae, picornaviridae, reoviridae and poxviridae families are in different phases of clinical studies for several types of tumors , . for example, reovirus against brain tumors (alone or combined with other therapies) or maraba virus against triple-negative breast tumors , offer some hope to patients with these types of cancer. viral sequences can be modified by genetic engineering techniques, thus making the virus more prone to infect some cells and enhancing viral infiltration and tumor tropism. combinations with other components (immunomodulators, drugs, and cytokines) are also being explored to suppress antiviral immunity and enhance antitumoral cytotoxicity . vaccines for cancer prevention. it is clear that certain viruses and bacteria play roles in cancer development. viruses such as genital herpes, hepatitis b, epstein barr or human papilloma and bacteria such as helicobacter pylori have been associated with cancers of the uterus and liver, in burkitt's lymphoma, and oral/genital and stomach cancers, respectively . therefore, immunization against these pathogens offer protection not only from infection but also from cancer. therapeutic vaccines. once an illness has developed, the intention of a therapeutic vaccine is to eliminate or decrease its pathology. thus, vaccines are used for cases of allergies, cancers and autoimmune diseases. allergy (type ): allergen-specific immunotherapy (ait) aims to modulate the immune system against an allergen, thus modifying the natural course of the allergic disease and conferring longlasting benefits . the basic ait involves the introduction of repeated doses of allergen (either injectable or sublingual allergen extract tablets) and often in escalating doses in a controlled manner, followed by a maintenance phase. in cases for which long-lasting tolerance is acquired, therapy may be discontinued. allergen extracts can be obtained from different sources, such as cat hair and pelt, mites, different types of pollen, venom protein, foods, etc. allergy vaccines are currently the only effective therapy that can stop the progression of the illness because treatment with anti-inflammatory drugs, such as anti-histaminic drugs or corticoids, mitigates the symptoms of the allergic processes but does not modify the natural course of the disease , . ait has been shown to induce the activation of antigen-specific tregs and il- -producing bregs (br ) subtype cells, which is combined with anergy caused by th cells and the production of allergen-specific igg antibodies that can compete with ige for binding to allergens . in the past, most vaccines were developed using natural allergen extracts. however, significant progress has been made in recent years to correctly characterize the allergen at the molecular level, and some of these allergens are now being produced by recombinant technologies, nucleic acid-based strategies, or synthetic peptide chemistry . cancer: another therapeutic approach for vaccines is in the field of cancer. therapeutic cancer vaccines that contain self-or nonself-patient tumor lysates, viral vectors, mutated tumor proteins or peptides, among other types administered in the presence of adjuvants can activate the immune system to induce antitumoral responses . the goal is to activate the th and tc cell compartments to expand specific cytotoxic t and nk cells directed against tumor cells. some vaccines are more immunogenic than others, and this effect can be related to several factors, such as the types/numbers of genetic mutations in the tumor, expression of neoantigens, production of viral proteins, an immunosuppressive environment, lack of expression of histocompatibility complex molecules, etc., which together may explain the large variability in tumor elimination . therapeutic cancer vaccines are generally very safe, and major secondary effects have not been observed, although large differences in patient responses are detected. moreover, this strategy may be used in conjunction with other complementary therapies , such as monoclonal antibodies, chemotherapy or cellular therapy , . several patients are currently taking part in clinical trials and are receiving therapeutic cancer vaccines against different types of tumors, such as lung (clinicaltrials.gov identifier: nct ), prostate (clinicaltrials. gov identifier: nct ) or pancreas (clinicaltrials.gov identifier: nct ), using individual or combined therapies. autoimmunity: in the case of therapeutic vaccines for autoimmune diseases, such as multiple sclerosis, diabetes, myasthenia gravis or guillain barré syndrome, the intention is to induce tolerance to self-antigens through the activation of regulatory cells (tregs and bregs) and tolerogenic dendritic cells, thus avoiding the immune response to self-components . due to the large variety of autoimmune diseases, the different etiologies and extensive variability, even in the same type of disease, designing a vaccine that can be useful for a wide range of patients is very difficult. however, several researchers are obtaining good results in animal models with nanostructures and peptides that induce specific tolerance, and it is predicted that, in the near future, these types of therapies will be applied to patients suffering from autoimmune diseases (reviewed by serra and santamaria ). polyclonal antibodies (pabs)-serotherapy the discovery of antibodies by dr. e. von behring and kitasato at the end of the xix century highlighted the potential of antibodies to neutralize tetanus and diphtheria toxins. this discovery opened the way to exploring the potential clinical applications of conventional antiserum-containing polyclonal antibodies from immunized animals/humans . this "serotherapy" was initiated by dr. roux and dr. yersin, who used antidiphtheria serum to treat several children . after this initial success, the use of serotherapy was increased for use against diphtheria and other diseases but also led to the identification of problems, such as immunogenicity with the formation of immune complexes (arthus reaction), the variability and limitation of the antibody batches, the content of a mixture of classes and subclasses of antibodies with different biological activities, and their temporal effects. for all of these reasons, therapy with polyclonal antibodies was very much restricted to special cases, such as the use of gamma-globulins for the prevention of rhesus (rh) maternal-fetal incompatibility and tetanus or snake venom toxicity . with the identification of gamma-globulin-deficient patients by dr. bruton in , the use of immunoglobulins as therapeutic molecules for the treatment of humoral immunodeficiencies was initiated. however, some problems were encountered in the initial phases, mostly related to the serum preparation and aggregation/ fragmentation of antibodies. since their initial use, several efforts have been made to avoid impurities and to improve the purification process, and several commercial products are now available (as intravenous or subcutaneous preparations). currently, many patients with humoral immunodeficiencies are successfully being treated to prevent them from catching infectious diseases. more recently, the therapeutic applications of immunoglobulins have expanded to other diseases, such as against covid- caused by sars-cov- infection (see below), autoimmune disorders and kawasaki syndrome in children . the beneficial effects seem to be mediated by several immunological mechanisms, including viral neutralization, inhibition of inflammatory cells and activation of immune regulators . monoclonal antibodies (mabs) the development of monoclonal antibodies (mabs) by c. milstein and g. köhler in (nobel prize winners for physiology/ medicine in ) changed medicine and immunology completely, along with many other disciplines. monoclonal antibodies are produced from the fusion of two cells to generate a hybrid cell or hybridoma with two characteristics, i.e., the production of one specific antibody and immortality. dr. milstein is considered to be the father of modern immunology for his crucial contribution . the development of many different mabs has enabled the identification of new molecules and the development of more accurate diagnostic approaches; specific, fast and inexpensive technologies; processes for the purification/concentration of compounds; and better and more specific therapy. mabs can now be used against specific targets according to the concept of the "magic bullet", a term coined by dr. paul ehrlich at the beginning of the xx century (reviewed in ref. ). numerous different mouse and rat mabs were produced against several molecules, but due to their murine origin, patients treated with these mabs suffered from hypersensitivity and immune responses , . thus, most mabs currently used in clinical applications are linked to radioactive elements and used for diagnostic purposes (table ) . in an effort to avoid immunogenicity, mabs were subsequently modified by genetic engineering approaches to carry mostly sequences of human origin. several research groups and companies developed chimeric and humanized mabs ( table ) , and these mabs included additional modifications, such as changes in the carbohydrates (glycosylation) and/or antibody regions, with the aim of improving their therapeutic action [ ] [ ] [ ] [ ] [ ] . moreover, fragments of recombinant antibodies (fabs, single-chain fvs, different v regions, fusion proteins, smaller antibodies, etc.) increased the variability of these potential therapeutic agents. the generation of fully human mabs took more time due to technical difficulties and ethical issues; therefore, researchers sought alternative methods to conventional approaches, such as the development of transgenic animals carrying human immunoglobulin genes using minilocus vectors, artificial yeast/human chromosomes or p vectors. the generation of knockout mice (in which mice lack ig genes) and further crosses with transgenic mice carrying human antibody sequences led to the generation of mouse strains that were able to produce fully human mabs , . other initiatives, such as the generation of immunodeficient mice in which human bone marrow or libraries of recombinant phages carrying human variable genes were reconstituted, allowed the development of more fully human antibodies ( table ) . sir greg winter, nobel prize winner in chemistry in , , became the pioneer of mab humanization through the genetic engineering of an antibody (campath ), later developing a fully human antibody (antitumor necrosis factor alfa, tnf-a) using recombinant phage technology , , . several companies are currently developing human antibodies using these and new technologies (reviewed in , , , ) . since , the list of approved mabs for human therapy has continued to increase (table ) , and many more mabs are in clinical trials [ ] [ ] [ ] . the versatility of mabs is based on a different mechanism of action : human immunology and immunotherapy: main achievements and challenges j varadé et al. -neutralization/blocking of soluble elements. for example, the neutralization of cytokines (tnf-α) and growth factors (vascular endothelium growth factor) prevents the exhibition of their effects, i.e., inflammatory and angiogenic effects, respectively , . -complement activation. igg/igm antibodies activate the complement cascade by the classical route, which leads to the death of the target cell , . -cytotoxicity mediated by nk cells. nk cells can facilitate mab killing of target cells. the mab, after binding to a target cell, can attach to fc receptors on the surface of nk cells to trigger the release of granzymes and perforin, thus inducing cell target death , . -induction of cell death by apoptosis. certain mabs directed against some membrane molecules can directly activate apoptosis . -blocking activation signals. antibodies can block some membrane receptors and avoid cell activation/proliferation activation/proliferation , . -carriers of toxins, pro-drugs, enzymes, and radioactive elements. mabs are able to concentrate select compounds around target cells, providing a much more selective therapy than conventional chemo-or radiotherapy . -check point inhibitors. leading to a recent revolution in cancer therapy, the identification of several inhibitory molecules can be blocked by mabs, thus leading to the activation and proliferation of antitumoral t cells. molecules such as ctla- and pd and its ligand pdl- , maintain immune cells under controlled conditions. however, it is possible to reactivate the antitumoral immune responses by blocking some of these molecules with mabs, either directed to only one of them or by using various antibodies in combination (for example, against ctla- and pd ) . the results obtained with these therapeutic mabs against checkpoint inhibitors in some types of cancer have been amazing. for their contribution to the understanding of the roles of ctla- and pd- , the swedish academy gave the nobel prize in to dr. j.p. allison and dr. t. honjo, respectively . however, this therapy is not efficacious in all types of cancers for several reasons, such as the expression of these and other checkpoint inhibitors in immune cells, the number of antitumoral cells in each patient, an immunosuppressant microenvironment, the rate of cancer mutations, and the expression of histocompatibility molecules. there is a large list of recombinant proteins that are currently being used for human therapy, including interleukin (il- ), interferons (ifns) and gm-csf. il- was identified in as a growth factor for t lymphocytes, and soon after dr. rosenberg started to use it in antitumoral therapy , . years later, in , il- was approved by the fda for patients with metastatic renal cancer and in for the treatment of metastatic melanoma . interferon (ifn) was described in by isaacs and lindenmann . the interferon family is the largest family of cytokines and is classified into three different types (i, ii, and iii). type i ifns (including ifn-α and ifn-β) exhibit several molecular actions that may be very relevant for use in therapy for a range of pathologies (such as autoimmune diseases and cancers) . in , the fda approved human ifn-α a and ifn-α b for patients with hairy cell leukemia and later on for patients with multiple sclerosis. since their initial use, these interferon species have been approved for many other diseases, including chronic hepatitis b and c, lymphoma, advanced melanoma, and as adjuvants together with other therapies for several types of cancers , . another cytokine is gm-csf, which activates the production of granulocytes and monocytes from bone marrow myeloid progenitors and has shown adjuvant antitumoral effects , . other cytokines, such as il- , il- , il- , il- , il- , and il- , , are being tested in several clinical trials, and it is expected that some of them, either alone or in combination, can be used in future antitumoral therapy. other recombinant proteins are already on the market, some of which are derived from antibodies, with some advantages such as small size, low immunogenicity and general ease of production. examples are etanercept and abatacept (ctla- ig), which were approved by the european medicines agency in and , respectively. the former is a chimeric protein that carries the external portion of the tumor necrosis factor (tnf) receptor linked to the igg fc region, which captures soluble tnf to block its inflammatory effects . the latter example is a fusion protein that combines the extracellular portion of human ctla- and igg fc. abatacept is a competitive inhibitor that blocks t-cell activation and can be used in the treatment of inflammatory illnesses such as rheumatoid arthritis . natural killer (nk) and lymphokine-activated killer (lak) cells. natural killer (nk) cells were described in the s based on their capacity to eliminate tumor cells without prior sensitization, with differences observed compared with specific cytotoxic t cells (which are activated based on the recognition of the target cells) , . in , piontek et al. reported that nk cells have the ability to preferentially kill cells that had lost the expression of the major histocompatibility complex class i molecules , . lymphokine-activated killer (lak) cells are a heterogeneous population that includes not only nk but also nkt and t cells, which can be generated in an in vitro culture of peripheral blood mononuclear cells (pbmcs) in the presence of il- . dr. rosenberg and collaborators carried out studies using these cells in the presence of il- (reviewed by rosemberg ). these lak cells showed good antitumoral responses in % of the melanoma patients who received them as therapy . however, secondary effects such as liver toxicity and the expansion of the treg population limited their therapeutic effect. researchers started to design new recombinant il- with some mutations to avoid the activation of tregs , with linking it to polyethylene glycol (peg) to increase its half-life and efficacy . another cytokine described later, il- , showed similarities to il- in many respects , and it had some unique advantages, such as the capacity to activate nk and cytotoxic t cells (tc) but not tregs. il- is being used in different versions (alone, as a heterodimer with receptor il- /il ra or il rα igfc, or in an agonist complex with alt- ) and in combination with other therapies in several clinical trials (examples: nct , nct , and nct ). more recently, researchers have focused their attention on other cytokines and combinations (such as il- , il- , and il- ) , which are able to activate nk cells in vitro and induce a good responses in animal models. in some human clinical trials, remission has been observed for patients with acute myeloid leukemia , , which broadens the options for the use of nk cells in the treatment of this pathology. the properties of nk cells reveal their versatility as treatments against tumors. nk cells are able to kill tumors through several mechanisms, including receptor-mediated cytotoxicity, antibodydependent cell-mediated cytotoxicity (adcc) and death receptormediated apoptosis, but they also secrete cytokines such as interferon gamma that enhance the antitumoral adaptive immune response. nk cell adoptive transfer (either autologous or allogenic nks) is currently being tested in clinical trials for hematological diseases and solid tumors, and numerous research groups have recognized their potential in other situations, such as transplant rejection and pregnancy. nk cell lines, memory-like nk cells and stem cell-derived nk cells are additional types of cells that can be used for tumor immunotherapy . regarding other cellular therapies, nk cells as substitutes for t cells for use upon transformation with an chimeric antibody receptor (car) are being explored (see below). dendritic cells. paul langerhans identified dendritic cells in human skin in , but these cells were not named until by dr. ralph m. steinman (nobel prize in ) and dr. zanvil a. cohn, who chose the term because the cell morphology, with long extensions, resembles that of neuronal dendrites . in humans, dendritic cells are obtained from different sources that vary in origin, maturation state and tissue distribution (skin, lymphoid tissue, circulating cells). among the main types of dendritic cells, plasmocytoids are conventional myeloid dc and dc , pre-dc and monocyte-derived dendritic cells. in the epidermis, there are three types: langerhans cells (lc), monocyte-derived lc-like cells and inflammatory dendritic epidermal cells (idecs) . as indicated above, dcs are antigenpresenting cells and are the only cells that are able to activate naïve t lymphocytes. a subpopulation of dcs also carries out a process known as cross-presentation. in this way, they facilitate the activation of both helper and cytotoxic t lymphocytes . in addition to their participation in the immune response, they can be used in antitumoral therapeutic vaccines , . it is possible to generate a type of blood monocyte-derived dendritic cell in the presence of a mixture of cytokines in culture -a process that induces their subsequent maturation and activation in the presence of tumor antigens (cell lysates, recombinant or purified antigens, peptides, rna, dna, and viral vectors ). these cells can also be obtained from bone marrow hematopoietic cd + progenitor cells . other sources, such as circulating or skin dendritic cells, are relatively scarce and are therefore not usually used. after their differentiation and activation in vitro , , dcs are exposed to tumor antigens and infused back into the patient (either by blood infusion or injected into areas near the lymph nodes or even directly into them) to reach the secondary lymphoid organs as soon as possible, at which point they can present antigens to the t cells. this approach is a type of individualized therapy and is therefore expensive. the first approved vaccine in which autologous dendritic cells were used was sipuleucel-t (provenge) , which was a treatment for prostate cancer refractory to hormonal treatment. immunotherapy with dendritic cells is currently being tested in more than clinical trials for various tumors: brain, pancreas, mesothelioma, melanoma and many others (clinicaltrials.gov identifiers: nct , nct , nct , and nct , respectively). the data indicate that the therapy is well tolerated and has led to increased patient survival in some trials. furthermore, complete cure and partial remission outcomes have also been observed. the lack of efficacy on other tests was probably due to the presence of immunosuppressive factors in the tumor environment. another therapeutic use of dendritic cells involves their induction of immunosuppression both in transplants and in autoimmune diseases . in an autoimmune pathology such as multiple sclerosis, the intention is to achieve stable tolerogenic dendritic cells that can act against some autoantigens (such as myelin peptides) in the presence of vitamin d , dexamethasone, or other agents . phase i clinical trials have generally shown good tolerance to this therapy without serious adverse effects . however, greater control of this treatment is necessary in several respects to obtain the best therapeutic results ; e.g., the human immunology and immunotherapy: main achievements and challenges j varadé et al. type of dendritic cells and ex vivo differentiation, the antigens used, and the injection route are important considerations. gamma/delta t cells (tγ/δ). human t cells expressing γ/δ tcr cells have interesting properties, including the capacity to kill a broad range of tumor cells. the advantages of these cells in cancer therapy are based on their independence from mhc expression on tumor cells and that their relative insensitivity to some inhibitor molecules (such as pd- ). the initial clinical application, with the adoptive transfer of autologous vδ + cells after ex vivo expansion, showed only sporadic responses , and different exploratory studies are currently being carried out to increase their clinical therapeutic use. allogeneic vδ + cells are also being explored in cancer therapy; e.g., they are being used against refractory hematological malignancies and advanced cholangiocarcinoma . although the basis of immune regulation was suggested centuries ago, regulatory t cells were described by sakaguchi et al. as cd + cd + natural regulatory t cells that expressed the forkhead box p transcription factor (foxp ) . later, induced or adaptive regulatory t cells were also identified, including different subsets that carry several phenotypic markers and express various cytokine secretion profiles . all of these factors play crucial roles in the maintenance of immunological self-tolerance by suppressing autoreactive t cells. the manipulation of tregs to achieve therapeutic outcomes is a field of great interest, because of both their expansion and activation in diseases, such as allergic and autoimmune diseases, and as a potential targets for cancer immunotherapy . tumor-infiltrating lymphocytes (tils). lymphocytes that infiltrate solid tumors are called tumor-infiltrating lymphocytes (tils). in , thomas and burnet proposed that the immune system performs tumor immune vigilance, with lymphocytes as sentinel cells leading to the elimination of somatic cells transformed by spontaneous mutations , . since the end of the s, dr. rosenberg has been trying to prove and improve the effective use of tils. the process starts with surgery and the isolation of tils from a tumor, followed by til activation in culture in the presence of cytokines, cellular expansion and, finally reinfusion into the patient. since its inception, this therapy has been improved markedly, with an increase in optimal responses from less than % to the current - %, in some cases. these higher success rates are due, in particular, to the prior preparation of the patient, including the depletion of lymphoid tissues, to avoid an expansion of regulatory cells , myeloid suppressor cells and other cells that can compete with the transferred tils. currently, there are more than trials in which tils are being used alone or in combination with other immunotherapies on several tumors, such as melanoma, metastatic colorectal cancer, glioblastoma, pancreatic cancer, hepatobiliary cancer, ovarian cancer and breast cancer. this individualized therapy has limitations; it can only be used on solid tumors, and the number and specificity of the tils and the type of tumor and microenvironment make standardizing this therapy difficult. chimeric antigen receptor (car). since tils include a variety of t lymphocytes with different specificities, the next step was to obtain t cells of a single type (monoclonal cells) carrying a clonal receptor capable of recognizing tumor antigens. this effort was carried out for the first time in mice and subsequently, in , in humans with a transgenic tcr against the mart- melanoma antigen , . these types of receptors are known as ttcrs, but their ability to recognize antigens is restricted since they can only identify the peptides presented by antigen-presenting cells on self-histocompatibility molecules. this situation changed because of one of the latest revolutions in antitumor therapy, the development of t lymphocytes that carry a chimeric antigen receptor (car) based on a specific antibody directed to a target surface molecule , . these modified t cells can directly recognize tumor cells without required antigen processing or presentation by professional antigen-presenting cells. moreover, the car includes all of the necessary elements for intracellular signaling and activation of helper and cytotoxic t lymphocytes. car therapy was developed by one of its pioneers, dr. carl june at the university of pennsylvania in the united states , who used modified t lymphocytes that carried a chimeric antigen receptor to target cd + leukemic b cells. after interacting with cd + cells, these modified car t cells were activated and able to proliferate and exert cytotoxic functions against target cells. in this case, both tumor and healthy b cells were affected. although bone marrow continues to produce b lymphocytes, in cases of severe b lymphopenia, it is possible to provide exogenous immunoglobulins periodically. the whole process of the current car t-cell therapy begins with blood donation, from which lymphocytes are purified and genetically modified in vitro by a viral vector, which carries the genes coding for the chimeric antigen receptor. the cells are expanded in the presence of cytokines in culture and are subsequently reinfused into the patient. this type of cellular immunotherapy is individualized for each patient, with his/her car t cells ultimately destroying the tumor. since the first generation of cars appeared, namely, a chimeric receptor composed of an anti-cd -specific single-chain variable fragment linked to a transmembrane domain and intracellular signaling domain of the t cell receptor (cd ζ chain), researchers started to modify the original design. new generations of cars, including the cd ζ subunit together with other signaling domains, such as cd , cd , cd ( - bb), cd , and icos, or combinations (cd ζ, cd , and cd ) , have been developed in the second and third generations of cars to improve several aspects, such as the activation, proliferation and survival of car t cells. the fourth generation of cars show improved the antitumoral effects by carrying additional molecules (such as cytokines or drugs), improvements to the safety of car tcell therapy through the use of suicide genes and many new designs, such as dual cars or the so-called split universal and programmable (supra) car system . in addition to t cells, other types of cells, such as nk cells, are now being explored for use in antitumoral responses . in an effort to avoid using personalized treatment, researchers are now working on universal cars that may be used on many different patients without inducing the problem of rejection [ ] [ ] [ ] [ ] . the encouraging results obtained with this therapy have led to interest from companies, and some commercialized examples are available, although many more "in-house" or academia-produced cars are in clinical trials. car t-cell therapy was initially designed for use against hematological cancers (leukemia and lymphomas), but many new opportunities have been opened for its use against solid tumors , infectious diseases (such as hiv) , allotransplantation, autoimmune diseases and severe allergies . china and the usa are the leading countries in producing car t-cell therapy, and numerous clinical trials are underway. immunotherapy for covid- patients coronavirus disease (covid- ), which is produced by severe acute respiratory syndrome coronavirus (sars-cov- ), affects millions of people in many countries. most of the infected patients ( - %) are asymptomatic or have mild symptoms, but the disease in some patients progresses to a moderate or severe illness that requires hospitalization in intensive care units because of respiratory distress, multiorgan failure, and/or other pathologies, and more than one-half million fatal cases have been human immunology and immunotherapy: main achievements and challenges j varadé et al. reported worldwide. the most vulnerable population includes aging patients and those with comorbidities such as hypertension, diabetes and cardiovascular diseases. there are several aspects of the covid- pathogeny that suggest an overreaction of the immune system in severely ill patients, with increased levels of inflammatory cytokines such as il- , il- and others (creating the so-called "cytokine storm"), together with blood lymphopenia and cd t cell and nk cell exhaustion. special therapies have not yet been identified to cure these patients, and preventive vaccines are not yet available, but some immunotherapies have been proposed as adjunct therapies, and some of these are currently in different phases of clinical trials . the immunotherapeutic strategies include the following: future challenges in immunotherapy immunotherapy has been used for centuries, but only in recent years has this area expanded rapidly in several respects, mostly by the use of soluble elements (monoclonal antibodies and cytokines) and, more recently, with immune cells (cellular immunotherapy). there are many fields in which immunotherapy faces a range of challenges: vaccines. . researchers are working on reducing the number of injections by employing a combination of vaccines. several current vaccines contain components from - pathogens in a single injection, and these are able to provide adequate protection against all of these pathogens . . researchers are developing more stable and durable vaccines. improvements in the half-lives of vaccines, for example, by lyophilization, while maintaining immunogenicity is expected to reduce current problems, especially those involved in the transportation of vaccines to remote areas . in this respect, nanotechnology can help in the design of more stable vaccines that lead to slow antigen release and improved immunogenicity . . researchers are working on vaccines that confer protection against all serotypes of a specific pathogen (universal). this outcome is especially important for pathogens with high variability (such as the influenza virus). researchers are designing vaccines that can protect against several variants by using common regions that can induce protective immune responses to all or most of the variants . . researchers are developing alternative routes of administration (e.g., oral, inhaled, intranasal, skin, rectum, vagina) as substitutes for intramuscular or subcutaneous injections. one of the problems to be solved is the immune tolerance developed to elements delivered by the oral route, but some vaccines are already effectively administered by this route (such as the oral polio, cholera, typhoid fever and rotavirus vaccines). the intranasal route has also proven effective for some vaccines (nasal influenza vaccine), and vaccines administered through other routes are under investigation. . researchers are seeking the early protection of newborns . newborns are very susceptible to infections due to their immature immune system . moreover, the protection exerted by maternal antibodies transferred through the placenta during pregnancy against some pathogens interferes with the development of the newborn's own immune response. greater knowledge on ways to activate the immature immune system early will enable the development of vaccines for newborns. moreover, immunization of pregnant women may help to enhance neonatal protection against several pathogens . . researchers are developing new and more effective vaccines. this effort is crucial for very prevalent pathogens such as mycobacteria tuberculosis, hiv virus or plasmodium falciparum. although there are treatments against these pathogens, most are not curative-as in the case of hiv; prevention is the best way to stop their spread. . researchers are working to address emerging pandemics. in the case of new pathogens, such as sars-cov- , which has produced a recent global outbreak, effective vaccines are urgently required . new technologies for vaccine formulations and routes of administration, the identification of immune-related factors of protection and modifications to the governmental regulatory and approval process for vaccines for emerging pathogens are challenges that must be faced to achieve a rapid vaccination procedure for outbreaks. hundreds of vaccines against sars-cov- (using different strategies such as live attenuated or inactivated pathogens, viral vector-based, viral rna, dna, recombinant proteins, peptides, etc.) are now under development, and some are in clinical trials. however, the need to develop a new vaccine in a short period of time should not negate the main principles of vaccination use: safety and immune protection. . researchers are working on genetic (rna, dna) vaccines because they have great advantages, including no requirement for growing a pathogen. genetic vaccines can be obtained in a much shorter time, with much faster and safer production processes, and can be transported much more easily. however, the immunogenicity of these vaccines must be improved, and other problems need be avoided, such as the potential deleterious effects of integrating vaccine sequences into cells . . researchers are developing safer and more powerful adjuvants. many years ago, the only adjuvant authorized for vaccines was aluminum hydroxide (alum), but currently, several adjuvants are on the market . the use of ligands that activate the innate immune response, such as those linked to tlrs or nanostructures with adjuvant effects, is currently under study. . researchers are boosting trained immunity, a new concept related to the innate immune memory-like described for nk cells (expansion) and macrophages (epigenetic modifications). knowledge of how to handle trained immunity will enable better vaccine design and more effective secondary responses . . researchers are seeking to eradicate diseases from the earth. the greatest challenge, eradicating disease is possible in the short term for some pathogens, such as poliovirus. very few cases of polio have been recently reported, and these reports came from only three countries; therefore, it is feasible that this disease can be eradicated in the near future. human immunology and immunotherapy: main achievements and challenges j varadé et al. . advances are challenged by the anti-vaccine movement. paradoxically, there are people who doubt the beneficial effects of vaccines, and they are putting the health of their own children and society in general at risk . the effectiveness of community protection conferred through vaccinated people is disrupted by decreased numbers of immunized persons. this lesser coverage enables pathogens to infect the most susceptible people, such as small children, elderly patients and those who cannot be vaccinated due to certain pathologies or because they are undergoing immunosuppression treatment. thus, news about the return of illnesses that were nearly forgotten, such as tetanus in italy (the first case in years), the death of a child in catalonia from diphtheria, or the exponential increase of measles cases (already counted in the thousands) worldwide , should make parents think carefully about the risks of not protecting children by vaccines. the world health organization (https://www.who.int/topics/vaccines/en/) argues that anti-vaccine movements can roll back all the achievements thus far in this field and have cited this issue as one of the main challenges to be resolved. addressing the antivaccine movement requires a coordinated effort of professionals to inform parents adequately and perhaps other types of coercive measures that some countries are already applying (financial fines, denial of access to public assistance in childcare units, removal of authorization to travel/live in some countries, new laws, and so on). antibodies and cytokines. immunotherapy with monoclonal antibodies has been a true revolution for many pathologies, as has the use of certain cytokines and recombinant fusion proteins. it is therefore predicted that these approaches may have a bright future, and regulatory agencies are expected to authorize many more mab-based therapies in the coming years, especially given the good results obtained in clinical trials. complete antibodies or those modified to increase their functionality or decrease their immunogenicity, combinations of antibodies and cytokines, antibody fragments, etc., are only some of the many possibilities for this type of product, which will expand the range of therapeutic options. one of the main problems regarding the use of antibodies in therapy, especially in cancer, is based on their often unpredictable efficacy. large variability in terms of remission and durable clinical benefits between patients is observed (for example, in the antitumoral responses by antibodies directed to the checkpoint inhibitors). thus, the main challenge is to understand the situations in which an antibody will have the desired effect. it is crucial to find validated biomarkers (with predictive and/or prognostic value) that can help to stratify or select patients for the best immunotherapy. a better understanding is also required for tumor heterogeneity, resistance to some drugs and immunosuppressive microenvironments . an in-depth immunological study, together with a personalized approach, is certainly the way to improve the success of these types of therapies. in combination with conventional therapies (radiotherapy, chemotherapy, and surgery), other immunotherapeutic drugs or cellular immunotherapies can also help to maximize the efficacy of this immunotherapy, but increases in toxicity will be another challenge to face. pathogens. the use of oncolytic viruses (ovs), bacteriophages that selectively infect bacteria, modified pathogens for vaccines or for antitumor immuno-activation, and the manipulation/ modification of the microbiota are some of the therapies that are being considered. ovs are designed to kill tumor cells and to activate the immune system against those cells. however, many of ovs have shown limited therapeutic effects when applied in monotherapy; therefore, much more work is required to improve their systemic antitumor effects and avoid the attenuation of the virus, which limits the viral replication. several obstacles, such as low viral delivery and spread, resistance to therapy and antiviral immunity, have been observed . thus, the main challenges with oncolytic viruses are addressed by improving their antitumoral efficacy, including the optimization of viral delivery, the development of ovs engineered to activate the immune system (e.g., by releasing cytokines), and their use as adjuvant therapies or in combination with other immunotherapeutic agents, such as immunomodulators . regarding gut microbiota manipulation as a therapeutic approach, fecal microbiota transplantation is an effective therapy for recurrent clostridium difficile infection and is now being investigated for other indications, such as inflammatory bowel disease and cancer. some of the challenges facing microbiome transplantation are the lack of precise knowledge about the complete microbiome and the mechanisms of action involved in its therapeutic capacity, the large variability of its effectiveness and the external factors that affect it. more studies are centered on understanding how to manipulate bacterial colonies, the discovery of therapeutic molecules, nutrient competitions, etc., that are required for successful application. the best type of therapy (either individual or the combination of bacteria) is also under debate, along with how to reach the market by translating this individualized therapy into commercial scale products. the safety and potential adverse long-term effects are also being assessed. other components (nanomaterials and small molecules). nanomaterials. to obtain approval for the use of other elements from incipient fields, such as the use of different types of nanostructures, either alone or in combination with other immunotherapies, it is important to resolve certain issues. in the case of nanoparticle use, a better understanding of the interaction between nanomaterials and biological media; nanoparticle biodistribution, metabolism and biocompatibility; and the reproducibility of the synthesis and scaled up production of nanomaterials are among the issues to address. small molecules. a greater knowledge of several molecules involved in the immune system has led to the development of new therapeutic agents, which have been synthesized by traditional chemistry and block or activate intracellular signaling. the low cost of production of these molecules, along with the scaling and reproducibility of small-molecule batches, has attracted the attention of pharmaceutical companies interested in a whole set of new immunomodulatory drugs. a better understanding of the mechanism of action of small-moleculebased drugs and proof that they offer higher efficacy than existing therapies, either in monotherapy or in combination therapy, are challenges that face those seeking to engineer new types of targeting molecules. cellular immunotherapy. to date, cellular immunotherapy has been an individualized therapy with high production costs, and it requires the involvement of multidisciplinary groups in hospitals. a real challenge in the field of cellular immunotherapy is the acquisition of universal off-the-shelf cell therapies to replace those currently made to order in a very personalized manner. the development of universal cells, for example, in the case of car tcell therapy, would increase the number of patients who could benefit from this treatment at thus reduce the costs. other challenging aspects of cellular immunotherapy are the life-threatening toxicity of induced and their lack of effect on solid tumors, which is mostly due to the immunosuppressive tumor microenvironment. this approach requires new strategies to overcome these difficulties. in addition to cancer, cellular immunotherapy has a long history of use against autoimmunity, infectious diseases, allergies and transplantation rejection. it is also important to find biomarkers for prognosis/prediction that can help to optimize this method. other therapies that involve the use of activated nk cells, tumor-infiltrating lymphocytes, vaccination with dendritic cells, etc., are having partial clinical success. similar to other treatments, these approaches require further study, but it is feasible that they may become reality in the near future. greater knowledge of the immune system, especially concerning the variety of cellular and humoral components and the close regulation among them, the interaction with other systems or with elements such as the microbiota, will allow the development of new types of therapies that may be safer, more effective and specific but with much lower toxicity than found in current therapies. this long journey has been possible due to the efforts of numerous researchers (throughout the centuries), who have contributed with their work, creativity, successes and failures to advance our knowledge of the immune system, cellular components, membrane markers, interactions, signaling pathways and many more aspects. this great combined effort has paved the way for the achievements that are currently being realized. the microbiota in adaptive immune homeostasis and disease the enteric network: interactions between the immune and nervous systems of the gut chronobiology of the neuroimmunoendocrine system and aging neuroimmune interactions: from the brain to the immune system and vice versa neuroimmune interactions: how the nervous and immune systems influence each other innate lymphoid cells: years on heterogeneity of human cd + t cells against microbes the biology of innate lymphoid cells innate lymphoid cell development: a t cell perspective tissue residency of innate lymphoid cells in lymphoid and nonlymphoid organs tissue signals imprint ilc identity with anticipatory function innate lymphoid cells: major players in inflammatory diseases innate immune defenses mediated by two ilc subsets are critical for protection against acute clostridium difficile infection human innate lymphoid cell subsets possess tissue-type based heterogeneity in phenotype and frequency helper-like innate lymphoid cells in humans and mice functional and phenotypic heterogeneity of group innate lymphoid cells neuropilin- is expressed on lymphoid tissue residing lti-like group innate lymphoid cells and associated with ectopic lymphoid aggregates adipose type one innate lymphoid cells regulate macrophage homeostasis through targeted cytotoxicity physiological regulation of innate lymphoid cells adipose-resident group innate lymphoid cells promote obesity-associated insulin resistance ilc confer early host protection at initial sites of viral infection type innate lymphoid cells control eosinophil homeostasis type innate lymphoid cells: new players in asthma and allergy complementarity and redundancy of il- -producing innate lymphoid cells innate lymphoid cells sustain colon cancer through production of interleukin- in a mouse model evidence of innate lymphoid cell redundancy in humans functional interactions between innate lymphoid cells and adaptive immunity regulation of the adaptive immune system by innate lymphoid cells innate lymphoid cells regulate cd + t-cell responses to intestinal commensal bacteria antigen-presenting ilc regulate t cell-dependent iga responses to colonic mucosal bacteria of mice and not men: differences between mouse and human immunology human innate lymphoid cells innate lymphoid cells-how did we miss them? innate lymphoid cell interactions with microbiota: implications for intestinal health and disease the human γ/δ+ and α/β+ t cells: a branched pathway of differentiation human αβ and γδ t cells in skin immunity and disease human γδ t cells: candidates for the development of immunotherapeutic strategies the single-cell phenotypic identity of human cd + and cd + t cells two types of murine helper t cell clone. i. definition according to profiles of lymphokine activities and secreted proteins the expanding universe of t-cell subsets: th , th and more regulation of human helper t cell subset differentiation by cytokines the effector t helper cell triade regulation of the development of type t-helper cells in allergy il- and th cells transforming growth factor-β 'reprograms' the differentiation of t helper cells and promotes an interleukin -producing subset il- and th in parasite immunity identification of a human helper t cell population that has abundant production of interleukin and is distinct from th- , th and th cells human immunology and immunotherapy: main achievements and challenges production of interleukin but not interleukin by a subset of human skin-homing memory t cells pathophysiology of t follicular helper cells in humans and mice translational mini-review series on th cells: development of mouse and human t helper cells blockade of b -h (programmed death ligand ) enhances humoral immunity by positively regulating the generation of t follicular helper cells pd- regulates germinal center b cell survival and the formation and affinity of long-lived plasma cells bcl and maf cooperate to instruct human follicular helper cd t cell differentiation nonclassical mhc class i-dependent invariant t cells are evolutionarily conserved and prominent from early development in amphibians cxc chemokine receptor expression defines follicular homing t cells with b cell helper function a novel transcription factor, t-bet, directs th lineage commitment kinetics of gata- gene expression in early polarizing and committed human t cells the transcription factor gata- is necessary and sufficient for th cytokine gene expression in cd t cells il- -secreting regulatory t cells do not express foxp but have comparable regulatory function to naturally occurring cd + cd + regulatory t cells surface phenotype and antigenic specificity of human interleukin -producing t helper memory cells development, cytokine profile and function of human interleukin -producing helper t cells th cells represent a distinct human t cell subset involved in epidermal immunity and remodeling differentiation, regulation and function of th cells role of micrornas and long-non-coding rnas in cd + t-cell differentiation microrna-mediated regulation of t helper cell differentiation and plasticity heterogeneity of cd + and cd + t cells interleukin- : expanding its job profile foxp + follicular regulatory t cells control the germinal center response t follicular regulatory cells in mice and men regulatory t cells can migrate to follicles upon t cell activation and suppress gc-th cells and gc-th cell-driven b cell responses il- -mediated induction of a potent regulatory t cell population human cd + hla-dr+ regulatory t cells, similarly to classical cd + foxp + cells, suppress immune responses via pd- /pd-l axis new insights into the biology of cd regulatory t cells regulatory b cells: origin, phenotype, and function demethylation of the rorc and il a in human cd + t lymphocytes defines th origin of nonclassic th cells brief report: etanercept inhibits the tumor necrosis factor αdriven shift of th lymphocytes toward a nonclassic th phenotype in juvenile idiopathic arthritis il- β promotes the differentiation of polyfunctional human ccr + cxcr + th / cells that are specific for pathogenic and commensal microbes functional heterogeneity of human memory cd + t cell clones primed by pathogens or vaccines th plasticity in human autoimmune arthritis is driven by the inflammatory environment harnessing the plasticity of cd + t cells to treat immune-mediated disease immunological memory: what's in a name? a human homologue of the drosophila toll protein signals activation of adaptive immunity prr function of innate immune receptors in recognition of bacteria or bacterial ligands tlrs/nlrs: shaping the landscape of host immunity pattern recognition receptors and inflammation systemic acquired resistance a specific primed immune response in drosophila is dependent on phagocytes specific memory within innate immune systems candida albicans infection affords protection against reinfection via functional reprogramming of monocytes the rof bcg/ppd-activated macrophages in resistance against systemic candidiasis in mice nonspecific protection of mice against influenza virus infection by local or systemic immunization with bacille calmette-guérin bcg as a case study for precision vaccine development: lessons from vaccine heterogeneity, trained immunity, and immune ontogeny bcg-induced protection: effects on innate immune memory trained immunity: a program of innate immune memory in health and disease defining trained immunity and its role in health and disease epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity inflammation induces dermal vγ + γδt memory-like cells that travel to distant skin and accelerate secondary il- -driven responses innate lymphoid cell memory t cell-and b cellindependent adaptive immunity mediated by natural killer cells critical role for the chemokine receptor cxcr in nk cellmediated antigen-specific memory of haptens and viruses expansion of a unique cd + nkg chi natural killer cell subset during acute human cytomegalovirus infection cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated nkg c+ natural killer cells with potent function human natural killer cells mediate adaptive immunity to viral antigens cytokine-induced memory-like natural killer cells memory-like responses of natural killer cells epithelial cells: liaisons of immunity non-specific effects of vaccines: current evidence and potential implications live attenuated vaccines against pertussis β-glucan derived from aureobasidium pullulans is effective for the prevention of influenza in mice homeostatic immunity and the microbiota bacterial community variation in human body habitats across space and time low diversity of the gut microbiota in infants with atopic eczema low gut microbiota diversity in early infancy precedes asthma at school age multiple sclerosis patients have a distinct gut microbiota compared to healthy controls decreased bacterial diversity characterizes the altered gut microbiota in patients with psoriatic arthritis, resembling dysbiosis in inflammatory bowel disease causal effects of the microbiota on immune-mediated diseases human microbiota-associated mice: a model with challenges the gut microbiota shapes intestinal immune responses during health and disease reversible microbial colonization of germ-free mice reveals the dynamics of iga immune responses an immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system immune responses to gut microbiotacommensals and pathogens the gut microbiota influences blood-brain barrier permeability in mice regulation of prefrontal cortex myelination by the microbiota indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders genetics of immune-mediated inflammatory diseases host genetic regulation of immune-based and infectious diseases: introduction to mammalian genome special issue: genetics of infectious disease genetic predisposition to infectious disease variations in the nramp gene and susceptibility to tuberculosis in west africans natural resistance to infection with intracellular parasites: isolation of a candidate for bcg association of human fc gamma riia (cd ) polymorphism with susceptibility to and severity of meningococcal disease klrd -expressing natural killer cells predict influenza susceptibility human genetic variants and age are the strongest predictors of humoral immune responses to common pathogens and vaccines ace gene variants may underlie interindividual variability and susceptibility to covid- in the italian population relationship between the abo blood group and the covid- susceptibility autoimmunity versus horror autotoxicus: the struggle for recognition multistep pathogenesis of autoimmune disease human autoimmune diseases: a comprehensive update mechanisms of human autoimmunity analysis of the genetic component of systemic sclerosis in iranian and turkish populations through a genome-wide association study gwas for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways fine-mapping and functional studies highlight potential causal variants for rheumatoid arthritis and type diabetes approaches and advances in the genetic causes of autoimmune disease and their implications meta-analysis of immunochip data of four autoimmune diseases reveals novel single-disease and cross-phenotype associations the pathogenesis of systemic lupus erythematosus: harnessing big data to understand the molecular basis of lupus a second study of the behaviour and fate of skin homografts in rabbits: a report to the war wounds committee of the medical research council immunity to homologous grafted skin; the relationship between the antigens of blood and skin the ebmt handbook: hematopoietic stem cell transplantation and cellular therapies ipd-imgt/hla database effects of mismatching for minor histocompatibility antigens on clinical outcomes in hla-matched, unrelated hematopoietic stem cell transplants the impact of hla class i-specific killer cell immunoglobulin-like receptors on antibody-dependent natural killer cell-mediated cytotoxicity and organ allograft rejection incomplete penetrance in primary immunodeficiency: a skeleton in the closet primary immunodeficiency diseases in highly consanguineous populations from middle east and north africa: epidemiology, diagnosis, and care current genetic landscape in common variable immune deficiency human inborn errors of immunity: update on the classification from the international union of immunological societies expert committee the iuis phenotypic classification for primary immunodeficiencies update on the genetics of autoinflammatory disorders an update on autoinflammatory diseases: interferonopathies novel proteasome assembly chaperone mutations in psmg /pac cause the autoinflammatory interferonopathy candle/praas genetics of allergic disease linkage between immunoglobulin e responses underlying asthma and rhinitis and chromosome q genetics of allergy and allergic sensitization: common variants, rare mutations innate and adaptive nasal mucosal immune responses following experimental human pneumococcal colonization epigenome-wide analysis links smad methylation at birth to asthma in children of asthmatic mothers recent findings in the genetics and epigenetics of asthma and allergy ueber den jetzigen stand der karzinomforschung cancer immunoediting from immune surveillance to immune escape tumor immunology and tumor evolution: intertwined histories natural history of hla expression during tumour development cancer immunology and immunotherapy anti-pd- and anti-ctla- therapies in cancer: mechanisms of action, efficacy, and limitations. front t-cell exhaustion: characteristics, causes and conversion ctla- /cd pathway regulates t cell infiltration into pancreatic cancer molecular interactions of antibody drugs targeting pd- , pd-l , and ctla- in immuno-oncology immunosenescence and its hallmarks: how to oppose aging strategically? a review of potential options for therapeutic intervention contributions of age-related thymic involution to immunosenescence and inflammaging aging and the immune system: the impact of immunosenescence on viral infection, immunity and vaccine immunogenicity single-cell transcriptomics reveals expansion of cytotoxic cd t cells in supercentenarians vaccines, new opportunities for a new society smallpox in the post-eradication era the contribution of vaccination to global health: past, present and future bioimmunoadjuvants for the treatment of neoplastic and infectious disease: coley's legacy revisited effect of bacillus calmette-guerin infection on transplanted tumours in the mouse cost-effectiveness analysis of fecal microbiota transplantation for recurrent clostridium difficileinfection in patients with inflammatory bowel disease efficacy of fecal microbiota transplantation for recurrent c. difficile infection in inflammatory bowel disease therapeutic bacteria to combat cancer; current advances, challenges, and opportunities oncolytic viruses: how "lytic" must they be for therapeutic efficacy? oncolytic virotherapy for head and neck cancer: current research and future developments. oncolytic virother talimogene laherparepvec: first global approval the current status and future prospects of oncolytic viruses in clinical trials against melanoma, glioma, pancreatic, and breast cancers two roads for oncolytic immunotherapy development new viruses for cancer therapy: meeting clinical needs intravenous delivery of oncolytic reovirus to brain tumor patients immunologically primes for subsequent checkpoint blockade pre-surgical neoadjuvant oncolytic virotherapy confers protection against rechallenge in a murine model of breast cancer neoadjuvant oncolytic virotherapy before surgery sensitizes triple-negative breast cancer to immune checkpoint therapy oncolytic viruses for cancer therapy: barriers and recent advances the prevention of infection-associated cancers therapeutic vaccines for allergic disease immunotherapy-vaccines for allergic diseases vaccine development for allergen-specific immunotherapy based on recombinant allergens and synthetic allergen peptides: lessons from the past and novel mechanisms of action for the future immunological mechanisms of allergenspecific immunotherapy recombinant allergens for immunotherapy therapeutic cancer vaccines antigenic variability: obstacles on the road to vaccines against traditionally difficult targets towards personalized, tumour-specific, therapeutic vaccines for cancer human nk cells: surface receptors, inhibitory checkpoints and translational applications therapeutic vaccines for autoimmune diseases nanoparticle-based approaches to immune tolerance for the treatment of autoimmune diseases der diphtherie-immunitat and der tetanus-immunität bei thieren passive serum therapy to immunomodulation by ivig: a fascinating journey of antibodies contribution à l'étude de la diphthérie. e mémoire ivig-mediated effector functions in autoimmune and inflammatory diseases update on the use of immunoglobulin in human disease: a review of evidence continuous cultures of fused cells secreting antibody of predefined specificity césar milstein, the father of modern immunology ehrlich's magic bullet concept: years of progress human immune response to multiple injections of murine monoclonal igg will immunogenicity limit the use, efficacy, and future development of therapeutic monoclonal antibodies? the state-of-play and future of antibody therapeutics fully human antibodies from transgenic mouse and phage display platforms phage libraries for generation of clinically useful antibodies phage display-derived human antibodies in clinical development and therapy progress and challenges in the design and clinical development of antibodies for cancer therapy production of antigen-specific human monoclonal antibodies: comparison of mice carrying igh/κ or igh/κ/λ transloci antigen-specific human monoclonal antibodies from transgenic mice nobel prize in chemistry: phage display of peptides and antibodies harnessing evolution to make medicines (nobel lecture) phage display libraries for antibody therapeutic discovery and development making antibodies by phage display technology antibodies to watch in human immunology and immunotherapy: main achievements and challenges antibodies to watch in antibodies to watch advances in the assessment and control of the effector functions of therapeutic antibodies molecular mechanisms of action of anti-tnf-α agents -comparison among therapeutic tnf-α antagonists quantitative comparison of the neutralizing capacity, immunogenicity and cross-reactivity of anti-tnf-α biologicals and an infliximabbiosimilar cytotoxic mechanisms of immunotherapy: harnessing complement in the action of anti-tumor monoclonal antibodies complement in monoclonal antibody therapy of cancer monoclonal antibodies: a review strategies and challenges for the next generation of antibody-drug conjugates next generation antibody drugs: pursuit of the 'highhanging fruit' development of immune checkpoint therapy for cancer enhancement of antitumor immunity by ctla- blockade pd- and pd- ligands: from discovery to clinical application a nobel prize-worthy pursuit: cancer immunology and harnessing immunity to tumour neoantigens in vitro growth of murine t cells. v. the isolation and growth of lymphoid cells infiltrating syngeneic solid tumors il- : the first effective immunotherapy for human cancer the interferons: years after their discovery, there is much more to learn interferons α and β in cancer: therapeutic opportunities from new insights antitumour actions of interferons: implications for cancer therapy dual role of gm-csf as a pro-inflammatory and a regulatory cytokine: implications for immune therapy comparisons of therapeutic efficacy and safety of ipilimumab plus gm-csf versus ipilimumab alone in patients with cancer: a meta-analysis of outcomes cytokines in the treatment of cancer immunomodulatory cytokines as therapeutic agents for melanoma manufacturing history of etanercept (enbrel ® ): consistency of product quality through major process revisions the evolving clinical profile of abatacept (ctla -lg): a novel co-stimulatory modulator for the treatment of rheumatoid arthritis natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. ii. characterization of effector cells natural" killer cells in the mouse. ii. cytotoxic cells with specificity for mouse moloney leukemia cells. characteristics of the killer cell yac- mhc class i variants reveal an association between decreased nk sensitivity and increased h- expression after interferon treatment or in vivo passage selective rejection of h- -deficient lymphoma variants suggests alternative immune defence strategy lymphokineactivated killer cell phenomenon. lysis of natural killer-resistant fresh solid tumor cells by interleukin -activated autologous human peripheral blood lymphocytes a next-generation tumor-targeting il- preferentially promotes tumor-infiltrating cd + t-cell response and effective tumor control nktr- , an engineered cytokine with biased il receptor binding, increased tumor exposure, and marked efficacy in mouse tumor models the shared and contrasting roles of il and il in the life and death of normal and neoplastic lymphocytes: implications for cancer therapy natural killer cells in cancer immunotherapy immunotherapy in acute myeloid leukemia and myelodysplastic syndromes: the dawn of a new era? high-risk leukemia: past, present, and future role of nk cells natural killer cell adoptive transfer therapy ueber die nerven der menschlichen haut nobel prize in physiology or medicine uncovering the mysteries of langerhans cells, inflammatory dendritic epidermal cells, and monocyte-derived langerhans cell-like cells in the epidermis dendritic cell science: more than years of history dendritic cell-based immunotherapy generation of human monocytederived dendritic cells from whole blood dendritic cells in cancer immunotherapy proliferating dendritic cell progenitors in human blood dendritic cells and cancer immunity sipuleucel-t for the treatment of prostate cancer: novel insights and future directions clinical tolerogenic dendritic cells: exploring therapeutic impact on human autoimmune disease dendritic cells as gatekeepers of tolerance high-dose cholecalciferol supplementation significantly increases peripheral cd + tregs in healthy adults without negatively affecting the frequency of other immune cells gamma delta t cell therapy for cancer: it is good to be local successful adoptive transfer and in vivo expansion of haploidentical γδ t cells allogenic vγ vδ t cell as new potential immunotherapy drug for solid tumor: a case study for cholangiocarcinoma immunologic selftolerance maintained by activated t cells expressing il- receptor alpha-chains (cd ) control of regulatory t cell development by the transcription factor foxp cd + cd + regulatory t cells: i. phenotype and physiology regulatory t cells: a potential target in cancer immunotherapy cancer immunoediting: from immunosurveillance to tumor escape the immunobiology of cancer immunosurveillance and immunoediting depletion of cd +cd high regulatory t cells from tumor infiltrating lymphocytes predominantly induces th type immune response in vivo which inhibits tumor growth in adoptive immunotherapy human immunology and immunotherapy: main achievements and challenges adoptive transfer of mart- t-cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma tcr transgenes and transgene cassettes for tcr gene therapy: status in t cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia chimeric antigen receptor-modified t cells in chronic lymphoid leukemia suicide gene therapy to increase the safety of chimeric antigen receptor-redirected t lymphocytes universal chimeric antigen receptors for multiplexed and logical control of t cell responses use of car-transduced natural killer cells in cd -positive lymphoid tumors il- and ccl expression in car-t cells improves immune cell infiltration and car-t cell survival in the tumor cancer immunotherapy using car-t cells: from the research bench to the assembly line next-generation chimeric antigen receptor t-cell therapy: going off the shelf engineering natural killer cells for cancer immunotherapy car t cells in solid tumors: blueprints for building effective therapies chimeric antigen receptor t-cell approaches to hiv cure car t cells for infection, autoimmunity and allotransplantation chimeric antigen receptor-redirected regulatory t cells suppress experimental allergic airway inflammation, a model of asthma adjunct immunotherapies for the management of severely ill covid- patients use of convalescent plasma therapy in two covid- patients with acute respiratory distress syndrome in korea effectiveness of convalescent plasma therapy in severe covid- patients immunogenicity and safety following primary and booster vaccination with a hexavalent diphtheria, tetanus, acellular pertussis, hepatitis b, inactivated poliovirus and haemophilus influenzae type b vaccine: a randomized trial in the united states tools and approaches to ensure quality of vaccines throughout the cold chain polymeric nanocapsules for vaccine delivery: influence of the polymeric shell on the interaction with the immune system development of universal influenza vaccines targeting conserved viral proteins the national vaccine advisory committee at : impact and opportunity vaccine responses in newborns immunological and clinical benefits of maternal immunization against pertussis: a systematic review developing covid- vaccines at pandemic speed perspective sars-cov- vaccines: status report gene-based vaccines: recent technical and clinical advances comparative safety of vaccine adjuvants: a summary of current evidence and future needs trained immunity-based vaccines: a new paradigm for the development of broad-spectrum anti-infectious formulations misinformation lingers in memory: failure of three pro-vaccination strategies measles outbreak in unvaccinated and partially vaccinated children and adults in the united states and canada cancer immunotherapy, part : challenge and future trends oncolytic viruses: overcoming translational challenges oncolytic virus immunotherapy: future prospects for oncology the role of gut microbiota in clostridium difficile infection a.g-f conceptualized the study and conceived the project, and all the authors participated in writing the paper. competing interests: a.g-f is a copromoter of the spin-off nanoimmunotech. there are no competing financial interests in relation to the work described. key: cord- -srrfr es authors: tregoning, j. s.; brown, e. s.; cheeseman, h. m.; flight, k. e.; higham, s. l.; lemm, n.‐m.; pierce, b. f.; stirling, d. c.; wang, z.; pollock, k. m. title: vaccines for covid‐ date: - - journal: clin exp immunol doi: . /cei. sha: doc_id: cord_uid: srrfr es since the emergence of covid‐ , caused by the sars‐cov‐ virus at the end of , there has been an explosion of vaccine development. by september , a staggering number of vaccines (more than ) had started preclinical development, of which had entered clinical trials, including some approaches that have not previously been licensed for human vaccines. vaccines have been widely considered as part of the exit strategy to enable the return to previous patterns of working, schooling and socializing. importantly, to effectively control the covid‐ pandemic, production needs to be scaled‐up from a small number of preclinical doses to enough filled vials to immunize the world’s population, which requires close engagement with manufacturers and regulators. it will require a global effort to control the virus, necessitating equitable access for all countries to effective vaccines. this review explores the immune responses required to protect against sars‐cov‐ and the potential for vaccine‐induced immunopathology. we describe the profile of the different platforms and the advantages and disadvantages of each approach. the review also addresses the critical steps between promising preclinical leads and manufacturing at scale. the issues faced during this pandemic and the platforms being developed to address it will be invaluable for future outbreak control. nine months after the outbreak began we are at a point where preclinical and early clinical data are being generated for the vaccines; an overview of this important area will help our understanding of the next phases. in november , a cluster of pneumonia cases was detected in wuhan, china [ ] . these were the first cases of covid- caused by the novel beta-coronavirus sars-cov- . the genetic information was made publicly available on january , days after the first declared case. sixty-three days after the sars-cov- sequence was published, on march , the first doses of the first human vaccine were being tested. by september , the sars-cov- vaccine landscape included candidates being tested in clinical trials and more than candidates. as the results from the phase i trials and earliest phase ii/iii trials emerge, this review will cover the platforms under development, the type of immune response required and the path to a clinical product. coronaviruses are unusually large enveloped rna viruses, with a large positive-sense, single-stranded rna genome. the integrity of this lengthy genome is maintained by a proof-reading replicase. the sars-cov- genome encodes open reading frames (orf), many of which have unknown functions (fig. ). orf a and orf b both encode polyproteins, which are cleaved into multiple non-structural proteins. orf encodes the envelope protein, a viroporin [ ] , and orf encodes the membrane protein; together, they coordinate viral assembly and release. orf encodes the nucleocapsid (n) protein. orf encodes the spike (s) surface glycoprotein, the viral entry protein and key antigenic determinant, which binds the angiotensin converting enzyme (ace ) receptor on the host cells. ace is commonly found on type ii pneumocyte cells in the airways. sars-cov- has a - times higher affinity for ace than the related coronavirus sars-cov- [ ] , which was responsible for the - sars outbreak. sars-cov- is able to bind ace from a wide range of mammalian species [ ] . having bound ace , spike protein is cleaved by a host cell surface bound proteinase, either furin or tmprss , enabling entry of the viral capsid. there may be a relationship between the mechanism of viral entry via ace and the pathogenesis of disease. human coronaviruses can cause both mild (oc , hku , e and nl ) and severe (sars-cov- , sars-cov- and mers) disease. for most patients (approximately %), sars-cov- causes an asymptomatic infection or mild symptoms [ ] . the following signs are associated with a virus positive polymerase chain reaction (pcr) test: fatigue, fever, chills, loss of appetite and persistent cough [ ] . a striking feature of infection with sars-cov- is anosmia, a loss of smell and taste, reported in approximately % of cases in one study [ ] . whether viral spread to the lower respiratory tract is a precursor for severe disease is unclear; pneumonia with characteristic pulmonary ground glass opacity changes on chest ct scans is common, even in asymptomatic individuals [ ] . blood clotting, respiratory compromise, renal damage and cardiovascular collapse are all features of severe disease. the greatest risk factor for severe covid- disease is age: the remarkable relationship with age is consistently observed, despite geographic variability in reported case fatality rates [ ] . while sars-cov- is a new virus and, therefore, the exact correlates of protection are not completely defined, there are precedents from other respiratory infections in general and coronaviruses in particular [ ] . there has been discussion that natural immunity to sars-cov- declines quickly; whether this is the case is still unclear. it is our speculation that because vaccines aim to evoke an immune response they could be more immunogenic than the virus itself, which might have mechanisms to dampen immune response: whether this speculation is correct or not is yet to be determined. the t cell response is important in the control of other respiratory infections, and therefore likely to be the spike (s) protein is the major antigenic determinant, coat is made of spike (s), membrane (m) and envelope (e) proteins. the rna in encapsulated with the nucleocapsid (n) protein). created with biorender.com important in covid- [ ] . models of sars-cov- indicate that t cells can be protective. cd + t cell depletion in mouse models delayed viral clearance and enhanced disease [ ] ; similarly, t cell transfer resulted in rapid viral clearance and disease amelioration [ , ] . sars-cov- -specific cd + t resident memory were protective in a mouse model in the absence of antibody [ ] . t cell memory can be long-lived; sars-cov- t cells were detected years after infection [ , ] . for sars-cov- , t cell responses have been observed to a range of antigens, including s, m, n and other orfs [ ] . sars-cov- -specific t cells have been detected in individuals who had asymptomatic or mild covid- [ ] and sars-cov- -specific t cells have been observed in contacts of infected individuals [ ] . patients suffering from covid- had fewer t cells than healthy controls [ ] . t cells, especially cd + t cells, can influence the immune response through the production of cytokines, and elevated cytokines have been associated with exacerbated disease [ ] . the skewing of the cd + t cell response is likely to be important. t helper type (th ) responses are central to the successful control of sars-cov- and mers-cov [ ] . th responses have been speculated to be deleterious [ ] , and increased th cytokines were seen in severe disease [ ] . regulatory t cells are important in the resolution of infection, and were observed to be elevated in covid- patients [ ] . circulating follicular t helper cells, important in defining recall antibody response to infection, have been observed in a small number of individuals with covid- [ ] . it is not clear whether the 'cytokine storm' is a cause or effect of disease; understanding this relationship is critical in monitoring vaccine safety. antibody response. the humoral response is pivotal in later stages of infection and helps to inhibit subsequent reinfection. virus-specific antibodies were detectable in - % of sars-cov- and mers-cov patients weeks after onset of symptoms [ ] [ ] [ ] [ ] [ ] [ ] [ ] , with delayed antibody responses associated with more severe disease. a number of studies have been performed to try to more clearly understand the antibody response to sars-cov- ; a systematic review of studies on antibody to coronaviruses [ ] observed that antibody was rarely seen in the first days of infection, but rose in the second and third weeks postinfection. it is unclear whether antibodies correlate with covid- severity. antibodies are likely to be an important part of vaccine-induced protection. in sars-cov- , the antibody response is short-lived [immunoglobulin (ig)m and iga responses last less than months and igg lasts approximately year]; this is possibly the same for sars-cov- [ ] . human challenge studies using non-covid- coronavirus strains suggest that higher antibody levels correlate with protection [ ] . these challenge studies have also suggested that reinfection is possible [ ] , but the dose in challenge studies may be higher than experienced during natural infection. two recent studies have observed natural reinfections with sars-cov- , one asymptomatic [ ] and one symptomatic [ ] , although this is in the context of more than million recorded cases globally, suggesting that it is a rare event. because of the overlap between sars-cov- and sars-cov- spike proteins, antibodies could be cross-neutralizing [ ] . however, the most potent specific, neutralizing monoclonal antibodies against the receptor binding domain (rbd) of sars-cov- did not bind to the spike protein of sars-cov- [ ] . one promising observation is that isolated neutralizing antibodies have minimally mutated vdj genes, which make inducing them possible with fewer rounds of vaccination [ ] . most attention has focused upon neutralizing igg antibodies in the serum, but other antibody-mediated mechanisms may be important in disease pathogenesis. fragment crystallizable (fc) and fc receptor (fcr) interactions can regulate the inflammatory response [ ] and the sars-cov- virus-antibody complex could potentially trigger such fcr-mediated inflammatory responses, causing acute lung injury [ ] . the iga response may be important in determining disease severity of covid- patients, but remains relatively unexplored so far [ ] . one concern with vaccine development for sars-cov- is that the immune response can cause disease, often in the act of clearing the infection. understanding vaccineinduced immunopathology is critically important for all emerging infectious diseases. vaccines for emerging infections will, by necessity, require a shorter turn-around from discovery to deployment, and therefore predicting safety early in the process is critical. vaccine-induced immunopathology can either present as an acute response to the vaccine itself or as disease enhancement after viral infection. vaccines can occasionally induce an acute autoimmune disease. this was observed during the h n swine flu outbreak, where vaccination in the united states led to an increased risk of guillain-barré syndrome (gbs) [ ] . the mechanism has not been fully determined, but one suggestion is off-target antibodies against ganglioside gm . off-target autoimmune effects were also observed during the h n swine flu pandemic, with narcolepsy observed in a subset of children immunized with a vaccine adjuvanted with as [pandemrix; glaxosmithkline (gsk)]. there was a very tight association with hla-dqb * : [ ] . the proposed mechanism is inhibition of the hypocretin signalling pathway. curiously, another swine flu vaccine made by gsk (arepanrix; gsk) using the same adjuvant was not associated with narcolepsy [ ] , suggesting that the side effect was not caused by the adjuvant. the level of viral proteins, specifically nucleoprotein, may have been the problem [ ] ; anti-nucleoprotein antibodies have been seen to cross-react with hypocretin [ ] . these acute events are relatively rare; the rate of gbs was per million individuals vaccinated and narcolepsy at approximately per million individuals vaccinated (all in individuals aged less than years) [ ] . the delayed effects of vaccines are difficult to predict; post-licensure monitoring will be critical, especially as the vaccines will potentially have been tested in fewer people during the prelicensure phases than other licensed products. disease enhancement following infection of vaccinated individuals has been seen in other viral diseases; for example, measles, respiratory syncytial virus (rsv) and dengue virus. of children who received formalin-inactivated measles vaccine and were then subsequently exposed to the wild-type measles virus, - % developed a severe form of the disease [ ] , causing the vaccine to be withdrawn in . a similar situation was observed with formalin-inactivated rsv vaccination (fi-rsv) in a clinical trial in . the fi-rsv vaccine induced mainly non-protective antibodies, and children who were seronegative to the virus prevaccination had enhanced disease and hospitalization compared to the control groups [ ] . vaccine-enhanced disease has also been observed with the live attenuated tetravalent dengue vaccine (dengvaxia; sanofi pasteur inc., swiftwater, pa, usa), specifically in seronegative children [ ] . disease enhancement following vaccination can occur by two main mechanisms: priming for a detrimental t cell response and priming for antibodies that can increase the risk of infection or severe disease. the cellular response to vaccination, particularly t cells and eosinophils, and the inflammatory mediators these cells release has been suggested to promote vaccineenhanced disease [ ] [ ] [ ] . whether sars-cov- vaccine platforms will have negative outcomes on infection is currently speculative, and draws upon experience with other respiratory viruses. one important factor determining the t cell response is antigen selection. specific epitopes can affect t cell polarization and activation, therefore antigen selection for vaccine applications requires careful consideration [ ] . both the s and n proteins of sars-cov- have epitopes that are recognized by cd + and cd + t cells. some vaccines which used the n protein induced an eosinophilic response associated with vaccine-enhanced disease [ ] , and post-vaccination challenge of animals immunized with sars-cov- n protein induced severe pneumonia [ ] . mismatch of epitopes between vaccine and challenge strain can also lead to t cell enhanced disease due to original antigenic sin, as seen in dengue [ ] . the vaccine platform may be critical in determining disease outcome on infection. immunopathology in animal models has most commonly been linked to inactivated, alum-adjuvanted vaccines. for example, double inactivation [ultraviolet (uv) and formalin] of sars-cov- enhanced the eosinophilic response from the vaccine, eliciting a proinflammatory pulmonary response and failing to provide complete protection [ ] . enhanced disease was also observed following immunization with a gamma-irradiated mers-cov vaccine [ ] . the mode of inactivation can influence both the quality of antibodies and the polarization of the t cell response to the vaccine. formalin inactivation in particular has been associated with deleterious th skewing by the addition of carbonyl groups [ ] , and th skewing has been seen for a formalin-inactivated vaccine for sars-cov- [ ] . other methods of inactivation have been explored; for example, beta-propiolactone, uv or gamma radiation, which could prove to be a promising avenue forward for eliciting the correct t cell response [ ] . immunopathology is not restricted to inactivated vaccines. it can occur following immunization with a range of vaccine platforms; for example, it has been seen in animal models of rsv vaccination with both viral vectors and dna vaccines [ ] . similarly, a range of vaccines against sars-cov- induced th -directed pulmonary immunopathology in mouse models [ ] . age at vaccination may also be an important consideration in immunopathology: the fi-rsv vaccine was given to infants. infants have a different immune response to adults, and this may predispose towards a qualitatively different immune memory [ ] . the humoral arm of the adaptive immune response can also contribute to disease, called 'antibody-dependent enhancement' (ade). ade has been observed with flaviviruses, coronaviruses and some viruses of the paramyxoviridae family [ ] . ade can occur in two ways, either by causing immune complexes or by enhancing infection. antibodies are bispecific molecules; as such, they can form antigen-antibody complexes. these complexes can cause direct damage when complex deposition in the vasculature leads to complement deposition and vessel damage, as seen after the feline coronavirus infection [ ] . immune complexes can trigger macrophage activation leading to the release of proinflammatory cytokines. immune complexes have been proposed to have a role in the enhanced disease seen after fi-rsv immunization [ ] , and may have a role in sars-cov- [ ] . antibodies can also increase viral disease by enhancing infection; some viruses utilize antibodies to enter target cells. in the case of dengue virus, pre-existing antibodies for one serotype of the virus can cause enhancement of infection upon subsequent exposure to a new serotype [ ] . a number of mechanisms have been proposed: antibody bound to virus could facilitate entry into macrophages through their fcrs [ ] and antibody might stabilize viral surface antigen into a mature form [ ] . the avidity of the antibody has been suggested as an important factor, with low antibody avidity a risk factor [ ] . ade has been reported in sars-cov- after viral challenge in mice [ ] , ferrets [ ] and macaques [ ] using a range of different vaccine strategies. in mers-cov, a neutralizing monoclonal antibody targeting the spike protein promoted viral entry via the fc receptor [ ] . it is not yet known whether antibodies to sars-cov- will enhance disease, but it is something that is being closely monitored [ ] . animal models. as coronaviruses have previously been associated with immunopathogenesis, vaccine-enhanced disease is a potential concern for efficient vaccine design for sars-cov- . the use of models can improve understanding [ ] , potentially predicting correlates of protection or disease. the ideal animal model is permissive to infection with the virus and reproduces the pathology and clinical course observed in humans. since the sars-cov- outbreak in - a range of species, including hamsters, cats, ferrets and non-human primates, have all been used to study pathogenesis of coronaviruses [ , ] . despite productive infection in a wide range of laboratory species, few displayed overt clinical disease. several inbred mouse strains have been investigated to model sars-cov- , including balb/c, c bl/ , rag −/− and svev mice. although young adult mice infected with varying doses of sars-cov- showed evidence of infection, the inbred strains do not accurately reflect the alveolar damage seen in humans [ ] . however, aged mice show signs of clinical disease despite, in many cases, the absence of the lung lesions seen in humans [ ] , and therefore have been used more extensively than younger mice. transgenic mice expressing human ace (hace ) have also been generated; disease severity in transgenic mice largely correlated with the level of hace expression, and when challenged with sars-cov- they developed severe infection and % mortality was reached by day [ ] . mers-cov appears to be even more challenging to model, with most species resistant to infection, except for some primate species [ ] and camelids [ , ] . the same models are being used for sars-cov- . infection of human ace transgenic mice with sars-cov- led to weight loss and viral rna was detectable in the lungs, as well as lung pathology [ ] . symptomatic infection and transmission of sars-cov- between animals has been observed in hamsters [ ] , and asymptomatic infection and transmission of sars-cov- has been observed in ferrets [ ] . sars-cov- is also infectious in experimental settings using cats, but not dogs, pigs, chickens or ducks [ ] . as with sars-cov- , non-human primates, e.g. rhesus or cynomologous macaques, have been helpful for evaluating immune protection [ ] . human challenge. as animal models do not fully recapitulate human disease, alternative strategies may be required. controlled human infection models (chim) are studies in which participants (either vaccinated or not) are intentionally challenged with an infectious organism [ ] . chim trials of sars-cov- vaccine candidates could be particularly beneficial in vaccine and drug efficacy studies, especially if the community infection rate has declined due to epidemiological interventions [ ] . the deliberate exposure of healthy individuals to sars-cov- requires a tight ethical and regulatory framework [ ] . the major concerns are that we do not have complete understanding of the long-term sequelae of sars-cov- infection and there is a lack of rescue therapy to enable the resolution of severe infection, although recent findings suggest that dexamethasone may reduce mortality in severe disease [ ] and remdesivir (gilead sciences, foster city, ca, usa) may improve clinical status [ ] . the lack of rescue therapy is not unique to a sars-cov- chim. rhinovirus and rsv chim do not have a specific anti-viral treatment but are self-resolving, which may also be true for sars-cov- in healthy young adults. there are also challenges associated with the manufacture of a challenge virus stock, which requires a high-containment [biosafety level iii (bsliii)] laboratory. at the time of writing, no study had been established, although the world health organization (who) has published guidance [ ] and several academic and contract research organizations are investigating the approach [ ] . an alternative use of deliberate human infection has been proposed: to infect young, lowrisk individuals to build herd-immunity, and therefore safeguard the unvaccinated, immunocompromised and immunologically naive [ , ] . however, this strategy is unattractive because the risk factors for severe disease are not fully understood: ethically there are also questions about infecting groups of individuals for the greater benefit, especially if there is a financial incentive. a huge range of vaccine approaches against sars-cov- have been proposed (table ) . these include traditional approaches -inactivated, live attenuated and protein/adjuvant approaches and more novel, as yet, unlicensed approaches -viral vectors and nucleic acids. this has been a rapidly evolving field and some of the vaccines are more advanced than others. we are focusing upon those that are in clinical trials at the time of writing ( table ) . several factors need to be considered before any vaccine progresses to widespread usage. first and foremost is vaccine safety and efficacy. closely linked is the scope for global scale-up manufacture to produce enough doses to achieve herd immunity. the spike (s) protein. before looking at the platforms being developed, the antigen needs to be considered. based on experience with sars-cov- , most vaccines target the sars-cov- spike protein. within the spike, the receptor binding domain (rbd) responsible for binding to and entering host cells is the primary target of neutralizing antibodies [ ] , and some vaccines only include this region. however, a recent study that isolated monoclonal antibodies found that most of them targeted areas outside the rbd [ ] . an important consideration is the correct folding of the protein, both during production and when the vaccine is in storage prior to deployment. the coronavirus spike is a type fusion protein and is metastable, undergoing an irreversible conformational change to enable membrane fusion [ , , ] . this may affect the ability of the antigen to induce neutralizing antibodies. a similar effect has been seen with the rsv fusion (f) glycoprotein. antibodies specific to prefusion f (pre-f) have better neutralizing capacity than post-fusion f-specific antibodies [ ] [ ] [ ] [ ] : stabilization of the pre-f form can lead to better responses. based on this, prefusion sars-cov- spike protein could elicit a more potent immune response and stabilized sars-cov- spike proteins have been generated with stabilizing proline mutations in the s domain [ , , ] . coronavirus nucleocapsid (n) is also immunogenic: antibodies against the sars-cov- n protein are abundant and longer-lived than those against the s protein in recovered patients [ ] . interestingly, in model systems of sars-cov- , immunization with the n protein is associated with vaccine-enhanced disease [ , ] . it is not known whether the n protein is a potential protective immunogen for sars-cov- , although vaccine approaches that use whole virus -either inactivated virus or live attenuated approaches -will potentially include n protein. the n protein can be a useful diagnostic for infection during phase iii trials of s protein-based vaccines. while the emphasis has been on the generation of neutralizing antibodies, targeting t cell epitopes may provide additional protection [ ] . in other respiratory viruses, for example rsv, t cell only strategies can enhance disease [ ] and t cells can be deleterious in dengue [ ] , although less evidence of this has been seen with influenza vaccines [ ] . it is not yet clear whether sars-cov- behaves more like rsv or influenza. drawing on information about sars-cov- and mers-cov and using bioinformatics, potential immunogenic epitopes in the sars-cov- proteome have been predicted. a total of human leucocyte antigen (hla) class i and hla class ii epitopes common between sars-cov- and sars-cov- were found [ ]. t cell responses against the structural proteins of sars-cov- were found to be more immunogenic than non-structural proteins [ ] . a wide range of different platforms have been developed, which can be loosely grouped as proteins, inactivated virus, vectored vaccines, live attenuated and nucleic acid (fig. ) . this is clearly a fast-moving space and the following is based on data accessed in september ; an updated website is available at https://vac-lshtm.shiny apps. io/ncov_vacci ne_lands cape/ and the who has a vaccine tracker [ ] . as many of the vaccines under development are produced by commercial organizations, peer-reviewed publications concerning their development and efficacy are limited, as such some information has been taken from press releases which may be less robust in their scrutiny. published results from clinical trials are summarized in table . as with other pathogens, recombinantly produced viral surface proteins can safely be used as vaccines for covid- . although protein vaccines have a good safety profile they can have low levels of immunogenicity, which means that many require adjuvants to improve their efficacy. while bacterial protein vaccines can be made through the purification of whole pathogen preparations, viral subunit vaccines necessitate recombinant genetic engineering. the genes encoding the chosen antigens are cloned or synthesized, expressed and purified using a variety of expression systems, including insect, bacterial, yeast and mammalian cells [ ] . bacterial expression systems are often used because they have high levels of expression and are easy to scale-up, with fermenter repurposing relatively easy. however, for viral antigens, where post-translational modification can be important, the use of insect cells or mammalian cells may be preferential [ , ] . several protein subunit vaccination approaches were under development for sars-cov- and mers-cov [ ] [ ] [ ] . a subunit vaccine made up of sars-cov- spike protein fragments, expressed in escherichia coli, induced neutralizing pre-clinical (mice) [ ] phase i trials [ , , ] curevac pre-clinical (mice) [ ] phase i trial [ ] people's liberation army/ walvax antibodies in rabbits [ ] . neutralizing antibodies were induced in mice after immunization with transgenic plants [ ] or mammalian expressed [ ] several sars-cov- protein vaccines are in development; eight candidates are in clinical trials, but no data are yet available from these trials. two of the earliest to be announced are from clover biopharmaceuticals and the university of queensland. clover biopharmaceuticals has used 'trimer-tag' technology to make a mammalian cellexpressed, spike protein subunit trimer vaccine [ ] . this antigen can be recognized by antibodies in the sera of people who have recovered from sars-cov- [ ] . the vaccine will be given in conjunction with gsk's adjuvant as or cytosine-phosphate-guanosine (cpg)/alum during the phase i trial (nct ). the university of queensland, funded by the coalition for epidemic preparedness innovations (cepi) has developed a recombinant subunit vaccine using spike protein that has been 'locked' in prefusion conformation using the molecular clamp technique [ ] . this is currently being tested with mf (nct ). sanofi are developing a protein subunit vaccine against sars-cov- , expressed using a baculovirus platform, funded by the us biomedical advanced research and development authority (barda). this has been reported to be delivered in conjunction with as from gsk [ , ] or potentially one other adjuvant which has not been revealed. phase i clinical trials were initiated on september (nct ), with an aim to make the vaccine available in early [ ] . other protein candidates in clinical trials (table ) are from adimmune (baculovirus-derived, alum adjuvanted), anhui zhifei (rbd only), instituto finlay de vacunas (rbd), kentucky bioprocessing (tobacco-derived protein), medigen (alum/cpg adjuvanted) and vaxxine (advax adjuvanted). differences in cost of manufacturing, location of the manufacturer and impact of the adjuvant will determine which candidates progress beyond clinical trials. virus-like particles (vlps) are a subset of protein vaccines which are artificially produced nanoparticles that resemble viruses. rather than an individual protein, vlps are made up of some or all of the proteins that form the viral capsid [ ] . they have some similarities to live attenuated or inactivated vaccines, and can produce strong cellular and humoral immune responses with no risk of reversion, because they contain none of the genetic material of the virus. they are used for a wide range of viruses, including hpv, and a preclinical sars-cov- vlp has been tested [ ] . vlp nanoparticles are self-assembling protein particles, not necessarily derived from the virus capsid proteins. novavax, funded by cepi and us operation warp speed, have developed a recombinant nanoparticle vaccine (nvx-cov ) that displays the sars-cov- spike protein [ , ] . this is produced using engineered baculovirus to infect sf insect cells [ ] . for the clinical trial with nvx-cov novavax are using their own saponin-based matrix-m adjuvant (nct ), the data from which have recently been published [ ] . the vaccine was immunogenic, but required the addition of adjuvant to achieve % seroconversion; two doses were required for neutralising antibody in all individuals. immunized animal models develop spike proteinspecific antibodies that prevent the attachment of the spike protein to host cell ace- receptors and also neutralize the wild-type virus [ ] . another company (medicago) are using a plant-based system, nicotiana benthamiana, to produce a vlp [ ] which is currently in clinical trial in combination with cpg or as adjuvant (nct ). other groups at the preclinical stage include saiba ag, based in switzerland, who are using a cucumber mosaic virus vlp that is bound to sars-cov- rbd, which induced neutralizing antibody in mice [ ] . peptide vaccination is based upon the concept that, as induction of t cell responses can be achieved using a fraction of the entire protein [ , ] , only the minimal immunogenic peptide sequence needs to be included. by selecting conserved epitopes, peptide vaccines can potentially induce broad-spectrum immunity against multiple strains of a given pathogen [ , ] . peptides are easier to produce than whole protein antigens, as they can be produced synthetically and do not require folding into a tertiary structure. however, peptide vaccines are often weakly immunogenic. this is due to several factors, including the relatively small size of the peptide and differences in mhc processing; they therefore may require carrier proteins or adjuvants [ , ] . several groups are exploring the use of multi-epitope peptide vaccines against sars-cov- ; following bioinformatic and immune-informatic-based predictions of immunogenic epitopes [ ] [ ] [ ] [ ] , the studies are focusing upon t rather than b cell epitopes. ose immunotherapeutics have used a multiepitope peptide approach to induce t cell responses in mice [ ] . covaxx and the university of nebraska medical center have recently registered a phase i clinical trial for a multi-epitope peptide vaccine (nct ) as has the vector institute (nct ) currently in clinical trial. artificial antigen-presenting cells (aapc) are immunotherapeutic agents that can stimulate antigen-specific t cell responses [ ] they have been widely explored for cancer vaccines and have also been proposed for infectious disease vaccines. in the case of sars-cov- , aapcs are transfected with a lentivirus encoding the structural and protease proteins. the cells are then administered via subcutaneous injection [ ] . the shenzhen geno-immune medical institute in china are undertaking an ongoing phase i clinical trial with an aapc approach (nct ) and a modified dendritic cell platform (nct ). aivita biomedical inc. are following a similar platform (nct ). due to the need to isolate and purify cells and maintain them at gmp quality, this approach seems impractical for mass vaccination campaigns. isolating and then inactivating a virus, historically with formaldehyde, is one of the oldest methods of viral vaccination. inactivation of viruses has been effective for a range of different viruses. however, there have been major safety concerns relating to sars-cov- and mers-cov-inactivated vaccines, reminiscent of fi-rsv, and these concerns are also valid for sars-cov- . lung pathology of vaccinated animals on virus challenge has been seen for both a gamma-irradiated mers-cov vaccine [ ] and a uv irradiation-inactivated sars-cov- vaccine [ ] . the choice of both the adjuvant and the inactivating agent is important in shaping the immune response. for example, a formaldehyde inactivated mers-cov vaccine adjuvanted with alum and cpg demonstrated enhanced protection without inducing eosinophil-mediated vaccine-related pathology [ ] . there are four inactivated vaccine candidates in clinical trials. sinovac biotech are using a platform previously developed for sars-cov- [ ] . the virus is grown in vero cells and inactivated with beta-propiolactone. the inactivated vaccine was safe and immunogenic in rhesus macaques and offered complete protection against sars-cov- challenge, where no virus was detected in the pharynx or lungs [ ] . two different versions of this inactivated vaccine have been developed, adjuvanted with either alum or cpg . this vaccine has completed a phase ii human trial in healthy adults aged - years (nct ), with % seroconversion observed after the second dose of vaccine and some neutralizing antibody detected [ ] . it is interesting to note that the production method for the virus was changed between phases i and ii trials, and this may have increased immunogenicity. the vaccine has entered phase iii clinical trials in brazil (nct ) and indonesia (nct ). sinopharm, working with both the beijing institute of biological products and the wuhan institute of biological products, have also developed an inactivated vaccine. this vaccine has now been tested in a phases i/ii clinical trial (chictr ). no serious adverse effects were observed, and more than % of individuals seroconverted with detectable neutralizing antibody in the two different trials [ ] . the antibody was mainly observed after the second dose. two other organizations, bharat biotech (india) and the institute of medical biology/chinese academy of medical sciences, are running clinical trials of inactivated vaccines, but these are ongoing with no published data as yet. valneva, based in scotland, have just expanded their bsl manufacturing capacity and have signed a deal with the uk government for million doses of a formaldehyde-inactivated vaccine adjuvanted with cpg, based on their japanese encephalitis virus vaccine [ ] . the use of a live virus to prevent infection is the oldest vaccine approach. the original vaccine, cowpox, used exactly this approach to prevent smallpox. we are grouping two approaches under live viral vaccine platforms: attenuation of the virus or the use of a viral vector to deliver transgenes. live attenuated vaccines closely resemble natural infection. as a result, they are often immunogenic with a single administration without an adjuvant [ ] . one consideration is balancing attenuation and replication -over-attenuated vaccines may not replicate enough to be immunogenic, and this balance can vary between different individuals, especially the very young or immunocompromised. historically, serial passage for attenuating mutations has been used; for example, live attenuated influenza vaccine (laiv) is cold-adapted, restricting it to the upper airway. this method requires time and extensive testing: the yellow fever vaccine yf d was passaged more than times. alternatively, attenuated viruses can be generated by reverse genetics [ ] , introducing site-directed mutations into genes associated with virulence. the e protein has been targeted for both sars-cov- and mers-cov [ , ] . however, this method requires the identification of genes that would attenuate viral replication and the mutation(s) inserted to be phenotypically stable [ ] . a novel method of codon-pair de-optimization has been developed. the codon de-optimized virus is chemically synthesized to retain % amino acid sequence identical to the parent virus, but to contain an increased number of cpg and upa rna dinucleotides to up-regulate host responses. codon-pair de-optimization has been used for attenuating rsv [ ] . codagenix and the serum institute of india are developing a live attenuated sars-cov- vaccine, using codon de-optimization technology, building on previous experience with rsv and influenza [ ] . in vectored vaccines, the antigenic gene of interest is expressed from another micro-organism, either virus or bacteria. adenovirus, vsv and modified vaccinia virus ankara (mva) are some of the common viral vectors used [ ] . the vectors can either be replication-deficient, delivering a gene cargo but not growing themselves, or replication-competent, reproducing in the immunization site. the different platforms may alter the reactogenicity and immunogenicity of the vaccine. a recombinant mva expressing the sars-cov- s protein delivered via intranasal or intramuscular routes induced protective immunity in mice [ ] . an adenovirus vaccine against mers-cov offered complete protection against challenge in mice [ ] . as pre-existing immunity against human adenovirus is widespread and can hamper its clinical application as a vector [ ] , a chimpanzee adenovirus can be used. a recombinant chimpanzee adenovirus (chadox ) encoding the s protein, known as mers , was immunogenic in mice and safe in phase i clinical trials in humans [ ] . five non-replicating viral vectored vaccines are currently in clinical trials all based around adenoviral vectors. replication-deficient adenoviral vectors lack the e a and e b genes; these are the early genes which are essential for reproduction of the virus [ ] , and deliver the antigen gene without replicating in the vaccinated individual. building on experience with mers-cov, the university of oxford are developing a chimpanzee adenovirus vaccine vector expressing the wild-type s protein (chadox ncov- , also known as azd ). the azd vaccine was immunogenic in mice and pigs [ ] . in rhesus macaques it reduced viral load and pneumonia after challenge with sars-cov- [ , ] . the azd vaccine entered phase i clinical trial on april in volunteers aged - years (nct ). in this study, there were local and systemic reactions to the vaccine, controlled by paracetamol, but no severe adverse effects. the vaccine was immunogenic, with % participants having neutralizing antibody after one dose and % after two doses. interferon (ifn)-γ-producing t cells were also detectable [ ] . in partnership with astrazeneca, this vaccine received a further $ . billion from barda towards its global development, manufacturing and distribution. the vaccine has now progressed into phases ii/iii trials in the united kingdom (nct ), brazil (isrctn ) and the united states (nct ). cansino biologics (china) are developing a human ad vectored vaccine. in phase i trials (chictr ), the ad vectored covid- vaccine was tolerable and immunogenic at days post-vaccination [ ] . both humoral responses and specific t cell responses were observed in healthy individuals days after vaccination. transient and self-limiting adverse events such as severe fever, fatigue and muscle pain were reported in the high vaccine dose group. similar results were reported after the phase ii trial [ ] . the vaccine is now in a phases i/ii trial in canada (nct ). [ , , ] cansino biological subset had t cell responses analysed. [ ] janssen (part of j and j) are using an experimental, replication incompetent adenovirus vector (advac ® ) in their per.c ® cell line technology [ ] . this platform has been used for zika, rsv and hiv vaccine candidates. an ebola vaccine (ad .zebov) using the same platform has been proven safe and immunogenic, and has been used as part of efforts to contain democratic republic of the congo (drc) ebola outbreaks [ ] . the vaccine has been seen to be protective against sars-cov- challenge in rhesus macaques [ ] and is in phase i trials in the united states, belgium (nct ) and japan (nct ). one other vectored vaccine that has received a great deal of press attention is from the gamaleya research institute, which has been given the tradename sputnik v. this vaccine uses two different adenovirus vectors, ad and ad . to date, two clinical trials have been registered giving individually or as a prime-boost, either as a solution (nct ) or lyophilized formulation (nct ) in a total of people. the trial recorded mild-moderate systemic effects and mild local effects, including injection site pain; % seroconversion rate by binding enzyme-linked immunosorbent assay (elisa) was observed. interestingly, there was also some anti-vector antibody detected after immunization [ ] . the registration of this vaccine is presumably subject to larger efficacy trials, with a phase iii trial registered in september (nct ). an alternative to replication deficient vectors is to use a live attenuated vector. merck has recently acquired themis, who have developed an attenuated measles vector vaccine approach using an attenuated strain of measles derived from the original vaccine strain. themis have previously used this approach to develop a chikungunya vaccine, which was safe and immunogenic [ ] . in collaboration with institut pasteur and the university of pittsburgh they are now running clinical trials with these vaccines (nct and nct ). live and vectored vaccines may lend themselves to mucosal delivery which may achieve better local immunity and has been used for other vaccines; for example, intranasal live attenuated influenza vaccine (laiv). however, the enthusiasm for mucosal vaccines based on preclinical data has not always translated into clinical success. symvivo is using oral delivery of a probiotic bacteria, bifidobacterium longum, to deliver the spike transgene (nct ). the migal galilee research institute have adopted an existing vaccine against infectious bronchitis virus (ibv), which has been used in a preclinical veterinary trial inducing humoral, cellular and mucosal immunity [ ] to be delivered orally, but this is not yet in clinical trials. beijing wantai biological pharmacy and xiamen university have recently registered a phase i clinical trial using an influenza viral vector (chictr ). nucleic acid vaccines have been highlighted for their potential in pandemic situations due to their low cost and potential rapid development, although this potential has yet to be translated into a real-world vaccine [ ] . they utilize either plasmid dna or rna, encoding a target antigen. following delivery of the vaccine, the nucleic acid is taken up by the cells and the encoded antigen is expressed. conceptually, one facility can produce any required nucleic acid vaccine and production can be theoretically scaled-up to meet pandemic level demands. the covid- pandemic will serve as an important test case for nucleic acid vaccines, with six rna platforms and four dna platforms currently in clinical trial. most dna vaccines are constructed from plasmids that contain prokaryotic sequences that support the plasmids' propagation in e. coli, and a mammalian expression cassette that controls the expression of the target transgene in the vaccinated organism. the expression cassette contains an upstream promoter to drive transgene expression, a kozak sequence, the inserted transgene and a ′ polyadenylation (polya) tail. following delivery, the dna vaccine is taken up by host cells local at the immunization site or by migrating apcs [ ] . to induce an adaptive immune response the dna must enter the cell nucleus. in transiting to the nucleus the dna passes through the cytosol which is inflammatory, being sensed by intracellular pattern recognition receptors, for example sting [ ] or tbk [ ] , inducing an innate immune response. the triggering of innate immunity is essential for promoting adaptive immunity to dna vaccines. if apcs are transfected directly with a dna vaccine, they will load vaccine-encoded peptides onto both mhci and mhcii molecules and activate t cells [ ] . transfected stromal cells will generate antigen, which will be encountered by apcs and b cells following antigen release from cell exosomes or apoptotic bodies. transit of injected naked dna to the nucleus is highly inefficient, with a large majority of the dna failing to cross the cell membrane or nuclear envelope [ , ] . to mitigate this loss, dna vaccine programmes employ delivery platforms such as electroporation and bio-injection. preclinical animal studies have demonstrated that dna vaccines encoding the m, n, a or s proteins of the sars-cov- virus could elicit immune responses [ ] [ ] [ ] . a multivalent dna vaccine encoding s and m protein epitopes could protect from sars-cov- cytopathic effects. the s protein is the target of the only sars-cov- dna vaccine to progress to phase i clinical trial, delivered by bio-injector, and it was safe and induced neutralizing antibody responses [ ] . the leading dna vaccine against mers-cov (ino- ) was developed by inovio. phase i clinical trials were completed in , with the vaccine showing a good safety profile and inducing humoral immunity and polyfunctional cd + t cell responses [ ] . the inovio mers ino (gls- ) vaccine that was due to be taken to phase ii clinical trials (nct ) has now been redeployed as ino- (nct ) to begin clinical trials for protection against sars-cov- . in preclinical studies of the ino- vaccine, neutralizing antibody and t cell responses were observed in mice and blocking antibody responses in vaccinated guinea pigs [ ] and macaques [ ] . the phase i trial (nct ) is ongoing, but the data have not yet been published. genexine, in south korea (nct ), zydus cadila in india (ctri/ / / ) and osaka university in japan (nct ) have initiated phase i trials of dna vaccines. rna vaccines are based on the same premise as dna vaccines of expressing a vaccine antigen transgene in the host cell, but they are one step further along the expression pathway, skipping the transcription step. unlike dna vaccines, expression of rna vaccines begins once they enter the cell cytosol, which can increase the efficiency of expression. as with dna vaccines, the presence of 'foreign' rna is sensed in both the endosome and cytosol [ ] , giving rna vaccines a self-adjuvanting effect [ ] . however, the early triggering of type i ifn responses can downregulate protein expression [ ] . modified nucleosides can be incorporated into the mrna product to create a 'silenced' rna vaccine that avoids detection by tlrs and does not trigger a type i ifn response [ , ] , but there is a balance between antigen expression from the vaccine construct and triggering enough inflammation to activate the immune response. this balance may be altered by the formulation to deliver the vaccine and can be different between different animal species, making predictions from preclinical studies difficult. there are two primary types of rna vaccine mrna and self-amplifying mrna (sarna). non-replicating mrna vaccines are constructs engineered to encode the gene of interest, and typically have a ′ cap, utrs flanking the gene of interest and poly a tail. the ′ cap is essential for mrna to associate with the eukaryotic translation complex. utrs are selected to optimize rna protein expression, avoiding the inclusion of sequences that would hamper translation [ , ] . mrna vaccine constructs are made using bacteriophagederived rna polymerases and ntps to transcribe linearized dna in vitro. self-amplifying rna vaccines are alphavirus-derived rna replicons modified to encode the antigen of interest in place of rna structural proteins. the viral replicon also contains an open reading frame (orf) that encodes four alphavirus non-structural proteins (nsp - ) and a subgenomic promoter. the non-structural proteins form an rna-dependent rna polymerase (rdrp). the rdrp complex transcribes more copies of the vaccine in the transfected cell. as a result, sarna vaccines express protein at higher levels and persist for longer than non-replicating rna [ ] . as it is a newer technology, rna vaccines were not developed against sars-cov- . six rna vaccines are in clinical trials for sar-cov- . moderna fast-tracked their candidate vaccine mrna- and were first to begin clinical trials on march with the national institute of health's national institute of allergy and infectious diseases (niaid) (nct ). this phase i study involved patient volunteers, divided into three group cohorts, as a dose escalation: low ( µg), middle ( µg) and high ( µg) in a prime boost. a preclinical study using the same vaccines was protective in mice against viral challenge [ ] . the vaccine was immunogenic, with increasing antibody titres with increasing dose administered; of note, three individuals (of , %) in the -µg group reported severe adverse events, with severity increasing after the second vaccination [ ] . the vaccine is now in phase ii (nct ) and phase iii (nct ) trials, focusing on the -µg dose. biontech is collaborating with pfizer to develop four s protein vaccine candidates. they are using a nucleoside modified mrna. phases i/ii clinical trials are running in germany (nct ) and the united states (nct ), with a multi-site phase iii study planned. the vaccine induced both cellular and humoral responses in mice [ ] and induced neutralizing antibody in the clinical study [ ] . they have performed a further two clinical studies, observing similar responses in a second study with their initial construct (bnt b ) which encodes a rbd trimer [ ] . in a comparator study they observed similar levels of immunogenicity to a stabilised membrane anchored spike protein (bnt b ), but with lower levels of reactogenicity [ ] . both curevac and the people's liberation army have also developed mrna vaccines that are in clinical trials, but no results have been published as yet. imperial college london and its spin-out social enterprise, vacequity global health, are developing an sarna vaccine encoding the s protein. intramuscular injection with lnp formulation induced high neutralizing antibody titres in mice [ ] . tested doses of the preclinical vaccine ranged from · µg to µg, with a boost of the same dose at week post-vaccination. human trials of the vaccine with participants started in june (isrctn ). arcturus, based in singapore, are also developing an sarna vaccine encoding a prefusion spike, which is in phase i clinical trial (nct ). protein vaccines can have low levels of immunogenicity. this can be boosted by adjuvants [ ] . adjuvants enhance the immune response through multiple mechanisms, causing a depot effect; up-regulating the production of chemokines and cytokines; enhancing the cellular recruitment to site of injection; increasing antigen uptake and presentation by apcs and increasing inflammasome activation [ ] . adjuvants can also tailor the immune response, guiding it towards producing the most effective form of immunity against the specific pathogen being vaccinated against [ ] [ ] [ ] . a range of adjuvants have been proposed for use with sars-cov- protein vaccines. these include advax, alum, as (gsk), matrix-m (novavax), cpg (dynavax) and mf (csl). additional components are also included with nucleic acid vaccines to enhance uptake and immunogenicity. nucleic acids are combined with a range of formulations, including lipid nanoparticles (lnps), liposomes and polyplexes. such formulations are essential for rna vaccines, as 'naked' rna is susceptible to being degraded by extracellular rnases which will prevent efficient cell uptake. lnps have previously been used for other rna therapeutics and moderna, imperial college london, arcturus, curevac and biontech vaccines all utilize this technology. the stability of these formulations can be a concern and may necessitate vaccine storage at a lower temperature which might, in turn, impact access to the vaccine. for dna vaccines, delivery devices are often used to increase uptake. inovio uses an electroporation device (cellectra ® ), which delivers an electric current to the site of injection: in a study on acceptability, acute pain (six of on the vas score) was recorded for the first min after immunization, but this receded [ ] . a similar effect was observed in a study using a different electroporation device [ ] . genexine are also using electroporation in their trial, but they are also comparing with a needle-free biojector. biojector devices have been shown to increase the antibody response to dna vaccines [ , ] . one of the more experimental approaches proposed to reduce the impact of covid- has been the use of other live vaccines as non-specific vaccines [ ] . this has been proposed for bacille calmette-guérin (bcg), oral polio vaccine [ ] and measles, mumps and rubella (mmr) [ ] . the proposed mechanism is described as trained immunity, where exposure to one agent alters the epigenetic profile of innate immune cells, potentially increasing the production of cytokines. in preclinical models, bcg pretreatment has been shown to reduce influenza viral titres [ ] . early ecological data (in april ) suggested that countries with mandatory bcg vaccination had reduced mortality from covid- , but this analysis has a number of issues, mainly associated with demographics and the timing of when the virus reached different countries [ ] . a more recent study has supported this protective effect [ ] . remarkably, a number of randomized clinical trials have been set up to directly test whether bcg can reduce the burden of covid- . the covid- pandemic has led to a surge of different vaccines being rapidly moved to clinical trials. a number of these vaccines have been around for several years as promising preclinical platforms, but not necessarily been attractive enough to generate funding to support human trials. each of the approaches has advantages and disadvantages (table ): which aspects are the most important will only be identified following efficacy studies. live attenuated vaccines have a long track record of safety and efficacy, but they may not be feasible in the current pandemic due to the length of time it takes to generate a candidate and test for attenuation. inactivated vaccines also have a long track record of protective efficacy, and they have the advantage that they are fast to generate; however, they require highcontainment facilities to generate the virus stock. there is also a concern about vaccine-induced immunopathology with an inactivated vaccine, which has been seen for some other respiratory viruses and in preclinical models of sars-cov- ; whether this is the case for inactivated sars-cov- vaccines will only be seen after larger and longer phases ii/iii trials. recombinant protein vaccines have been in use since the s; they are a more targeted approach than using a whole virus, which may focus the immune response on a key antigen, but this may lose some breadth of protection. protein candidates were somewhat slower to enter clinical trials but may have a faster route to licensure, being a more known product than newer vaccines. one challenge is to use the correct conformation of the protein, spike is metastable and may be less protective if used in a post-fusion form. one peptide vaccine has registered a clinical trial (nct ) from the vector institute in koltsovo, russia; it is adjuvanted with alum. the cellular-based approaches, using aapc, do not seem to be practical for wide-scale rollout. nucleic acid vaccines, both dna and rna, have much potential in terms of speed of response and scale-up: this outbreak will be an important test for whether they can deliver on their promise. dna vaccines have historically been less immunogenic than other platforms, although with alternate delivery devices that may be overcome. rna vaccines have not been widely tested for infectious diseases; this is the first time an sarna vaccine has been trialled. rna may have a slight issue concerning heatstability, necessitating - ˚c storage. viral vectored vaccines are the furthest ahead in clinical trials, with three candidates in later phase clinical trials. they are known to be safe, but may be reactogenic at higher doses. historically, these approaches have had mixed results for efficacy and one concern is pre-existing immunity against the vector, especially when a human viral-derived vector such as ad is used. it will be of great interest to see which platforms and candidates are protective in efficacy trials. as of september , the furthest advanced candidates have completed phase i trials (table ) , although so far not all the organizations involved have published data from completed trials. all the data published so far indicate that the vaccines are safe, but there are more adverse events at higher doses: both the moderna [ ] and biontech [ ] vaccines had severe adverse effects at the highest doses, leading to a lower dose in later studies; some severe adverse effects were also recorded in the cansino [ , ] and university of oxford [ ] vectored vaccine trials. the vaccines all appear to be immunogenic, although it is hard to compare directly, as different groups will have used subtly different elisa and neutralization assays. a further complication for comparison is when data have been published as press releases rather than peer-reviewed papers. ultimately, vaccine efficacy in a randomized trial is the most important issue, but here again, different primary end-points are being explored. some studies are looking at reduction of disease, while others are looking at reduction of confirmed infections. the covid- crisis represents an opportunity for several experimental vaccine platforms to progress to clinical trials. however, there are considerations between a promising preclinical candidate and a global vaccination campaign, including trials, regulation and manufacture. clinical vaccine trials conventionally undergo four broad phases, from early safety in small numbers of volunteers (phase i) to wide-scale post-licensure monitoring (phase iv). usually, each of the phases take months or even years to complete before moving on. in a pandemic setting, there is need to speed up the transition between phases; this has been achieved with co-operation of regulatory agencies and research ethics committees. additionally, phases can be merged, with planning of phases ii/iii trials initiated before the phase i trial has even begun. there is the potential that data obtained from phase i will mean that planned later-phase trials are cancelled due to safety concerns or futility. ultimately, pushing a vaccine through the different stages of a clinical trial does not negate the need for complete safety data sets to be collected, but close co-operation with the researchers and regulators can accelerate a progress that could reduce years to months. one consideration is that due to the accelerated time-scale, postlicensure monitoring will be extremely important. another issue concerns the sample size required for efficacy studies; as cases fall, larger studies will be necessary or an alternate trial design. a ring trial was used in ebola [ ] , which allowed efficacy to be assessed in a few individuals. one of the major hurdles to a vaccine relieving the covid- pandemic is manufacturing enough doses to achieve global herd immunity. the number of doses needed to achieve global coverage depends upon the regime used, but is potentially as many as billion (assuming a prime boost regime with some contingency). to ensure licensure and prequalification status, good manufacturing process (gmp) standards must be upheld during up-scaled manufacturing and clinical studies. manufacturing a vaccine at a global scale in the timeframe required is a unique challenge. vaccines that are not only safe and effective but also highly scalable, to produce millions or even billions of doses, would be the most desirable tool for curbing the pandemic. logistics are a key consideration, including access to components to manufacture the vaccines -for example, nucleic acid vaccines require nucleotide triphosphates (ntps), which are also in high demand for the diagnostic tests. the vaccines also require plant and materials to fill and finish the final product; one bottleneck is exactly that: glass bottles. in parallel with the accelerated clinical trials, accelerated manufacturing scale-up is required. this telescoped manufacturing process means that investment in the next step is being made before the results of the previous step are known. this has considerable financial risk, especially in terms of setting up the necessary manufacturing plant if it cannot be repurposed, either for other pandemic vaccines or other biologicals. funding is a critical part of the vaccine development process. it remains to be seen whether the total costs of research, development and licensure of any novel vaccine platforms for sars-cov- is comparable to traditional vaccines, such as dengvaxia, which costed approximately $ . billion until licensure [ ] . a range of funding mechanisms have supported vaccine development. one of the major bodies co-ordinating the funding is cepi, which has received funding from multi-national sources, including governments and charities. other vaccine candidate teams are being supported by their governments 'fast-tracking' their candidates through clinical trials and streamlining their manufacturing. for example, operation warp speed in the united states, is supporting six candidates from astrazeneca (azd ), moderna (rna), pfizer (rna with biontech), merck (vectored vaccine with themis), johnson and johnson (vectored vaccine) and novavax (recombinant protein). a uk vaccine task force was announced on april [ ] to support uk-based candidates and reviewing government regulations to facilitate rapid and safe vaccine trials. several vaccine candidates have never progressed past phase i before, therefore manufacturing gmp material at scale poses new challenges. many of the vaccines are being developed by either academic groups or small-to mediumsized biotech companies, neither of which necessarily have capacity to manufacture at a large scale. one approach is outsourcing to contract manufacturing organizations (cmos), which can have licensing complications. some companies have invested in manufacturing capacity; for example in july , moderna opened a manufacturing facility in norwood (usa) [ ] , which produce rna up to the gram scale. however, they still need to work with external companies to complete formulation protocols. new manufacturing facilities are also being constructed elsewhere, including the vaccines manufacture and innovation centre (vmic) in the united kingdom, which has been accelerated and is planned to open in mid- , and valneva opened an expanded bsliii facility in scotland in august . larger companies may have more experience and capacity of manufacturing; for example, janssen and astrazeneca are making adenovirus vectors and sanofi is making a protein vaccine. in the initial stages of the outbreak there was relatively little publicized activity from the larger pharmaceutical companies, with most of the attention on smaller biotechs and academic groups. it is not clear why this was the case. one possible reason could have been exemption from liability, although vaccine manufacturers are exempt in the united states under the public readiness and emergency preparedness, or prep act. integrating national funding programmes with equitable global access to vaccines is vital. there is a concern that wealthy countries will monopolize initial production runs of vaccines, with preorders outstripping manufacturing capacity. alternative models of licensure, social enterprise and spoke and hub manufacture may be necessary. for example, the astrazeneca/oxford vaccine has been licensed to the serum institute of india, the largest global manufacturer of vaccines by doses produced, and imperial college london have established a social enterprise company to enable equitable access. vaccines require regulation to be introduced as a licensed product. there are a range of national and international bodies which cover this process; for example, any product trialled in the united kingdom will be considered for approval by the medicines and healthcare products regulatory agency (mhra). the product is then considered for prequalification by the who for multinational distribution, which has supported the regulation and distribution of vaccines for years [ ] . this process can normally take a substantial amount of time; however, during the covid- pandemic manufacturers and regulators are striving towards the delivery of a vaccine that is safe and effective within months from february . there is a precedent for accelerated licensure in the context of a pandemic, as seen with the approval of rvsv-zebov [ ] as a vaccine for ebola. the who supported the accelerated regulatory approval for rvsv-zebov-gp using an expedited prequalification review following its receiving conditional marketing authorization from the european medicines agency (ema) [ , ] . the who has issued guidance and recommendations for the regulation of sars-cov- vaccines [ ] . the issues faced during this pandemic and the vaccine platforms being developed to address it will be invaluable for future outbreak control. while, at this stage, it is not possible to say which platform is best, and what works best for one infection may not be best for all infections or for all populations. one of the biggest considerations of all the platforms is speed into trials versus ability to deploy the vaccine. while some of the high-speed platforms, for example rna, have entered into phase i trials faster than other approaches, their lack of track record means that approaches for global scale-up are potentially slower. older approaches may leapfrog the newer platforms in the scale-up and manufacturing stage. one observation of interest is the speed with which one of the oldest technologies for viral vaccines, inactivation, has been able to move forwards. prior to covid- , much of the attention for pandemic vaccine preparedness was on newer technologies; however, of the candidates in phase iii in august , two of four are inactivated vaccines. safety and efficacy data from these large studies will be critical. another consideration is that while distributed global manufacture is effective and appropriate for routine scheduled vaccination, local surge manufacturing capacity for new vaccines is important. this capacity may have to be maintained at a loss for large amounts of time, unless alternative commercial contracts can be found for the same facility that can then be replaced at short notice. this reflects a broader consideration that investment in public health, which may appear expensive to begin with, can save a considerable amount in the long term; one study estimated that every £ spent on public health saves £ in return [ ] . another consideration is for greater standardization of assays and end-points. comparisons of the different trials has been made significantly harder by the use of different methodologies. this is part of the broader global context of a true pandemic. collaborative worldwide action is required to control the virus, which necessitates leadership and a willingness to share by countries with more developed scientific research programmes. ultimately, while lessons will be learned from this pandemic, the next one will be different and sticking rigidly to a plan that controlled this coronavirus will not necessarily work for a different virus, as the german strategist helmuth von moltke 'sort of ' said: 'no plan survives contact with the enemy' . it is still far too early to know what the best approach will be to control covid- with vaccines. speculating as to which vaccine platform is 'best' , while academically enjoyable, is not of value here. that so many platforms, both new and old, are moving into efficacy study makes this an extremely exciting time for vaccinology. the outbreak will certainly be a test case for the novel vaccine platforms, particularly nucleic acid vaccines, which have promised much to date but not been licensed for human use. one issue is whether vaccines will play a role in reducing the burden of the pandemic. even at maximum speed, the first efficacy trials will start months after the start of the pandemic and the first licensed doses are unlikely to be ready for months, by which time the virus will have caused a large wave of mortality and a larger wave of global disruption. important questions remain (box ) regarding what is a successful vaccine, how should it be deployed and who should be prioritized. these will depend in part upon the results of the efficacy studies, although the who has produced some draft guidance [ ] . overall, it has been a remarkable chapter in vaccine development, with widespread collaboration and partnership in a race against the virus. a novel coronavirus from patients with pneumonia in china coronavirus envelope protein: current knowledge cryo-em structure of the -ncov spike in the prefusion conformation the sars-cov- spike protein has a broad tropism for mammalian ace proteins presumed asymptomatic carrier transmission of covid- real-time tracking of self-reported symptoms to predict potential covid- alterations in smell or taste in mildly symptomatic outpatients with sars-cov- infection a new coronavirus associated with human respiratory disease in china opensafely: factors associated with covid- death in million patients lessons for covid- immunity from other coronavirus infections t cell responses in patients with covid- cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd + t cells are important in control of sars-cov infection how immune t-cell augmentation can help prevent covid- : a possible nutritional solution using ketogenic lifestyle jri/vol without altering disease transmission work? how to achieve equitable global distribution? is human challenge part of the pathway to a licensed product? what is the risk of immunopathology? t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice virus-specific memory cd t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection characterization of sars-cov-specific memory t cells from recovered individuals years after infection targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals robust t cell immunity in convalescent individuals with asymptomatic or mild covid- intrafamilial exposure to sars-cov- induces cellular immune response without seroconversion reactive t cells in covid- patients and healthy donors the potential role of th immune responses in coronavirus immunopathology and vaccine-induced immune enhancement longitudinal analyses reveal immunological misfiring in severe covid- analysis of adaptive immune cell populations and phenotypes in the patients infected by sars-cov- . medrxiv two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome persistence of viral rna in stool samples from patients recovering from covid- potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sars-associated coronavirus kinetics of serologic responses to mers coronavirus infection in humans converting detachable wheelchair armrests into parallel bars transmission of merscoronavirus in household contacts a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease longitudinal evaluation and decline of antibody responses in sars-cov- infection the time course of the immune response to experimental coronavirus infection of man covid- re-infection by a phylogenetically distinct sars-coronavirus- strain confirmed by whole genome sequencing genomic evidence for a case of reinfection with sars-cov- identification of potential crossprotective epitope between a new type of coronavirus ( -ncov) and severe acute respiratory syndrome virus potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody analysis of a sars-cov- -infected individual reveals development of potent neutralizing antibodies with limited somatic mutation antibody in statspearls understanding sars-cov- -mediated inflammatory responses: from mechanisms to potential therapeutic tools the role of iga in covid- guillain-barré syndrome following vaccination in the national influenza immunization program, united states, - narcolepsy and influenza vaccinationthe inappropriate awakening of immunity comparison of pandemrix and arepanrix, two ph n as -adjuvanted vaccines differentially associated with narcolepsy development antigenic differences between as adjuvanted influenza a (h n ) pandemic vaccines: implications for pandemrix-associated narcolepsy risk antibodies to influenza nucleoprotein cross-react with human hypocretin receptor measles immunization with killed virus vaccine: serum antibody titers and experience with exposure to measles epidemic respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine effect of dengue serostatus on dengue vaccine safety and efficacy protection induced by virus-like particle vaccine containing tandem repeat gene of respiratory syncytial virus g protein alum adjuvant enhances protection against respiratory syncytial virus but exacerbates pulmonary inflammation by modulating multiple innate and adaptive immune cells intranasal nanoemulsion-based inactivated respiratory syncytial virus vaccines protect against viral challenge in cotton rats mechanisms for t cell receptor triggering immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms a potential molecular mechanism for hypersensitivity caused by formalininactivated vaccines formalintreated uv-inactivated sars coronavirus vaccine retains its immunogenicity and promotes th -type immune responses examination of the effects of virus inactivation methods on the induction of antibodyand cell-mediated immune responses against whole inactivated h n avian influenza virus vaccines in chickens immune responses and disease enhancement during respiratory syncytial virus infection vaccination strategies to enhance immunity in neonates vaccine-induced enhancement of viral infections immunopathogenesis of coronavirus infections: implications for sars a role for immune complexes in enhanced respiratory syncytial virus disease dengue viruses and mononuclear phagocytes. ii. identity of blood and tissue leukocytes supporting in vitro infection new insights into the immunopathology and control of dengue virus infection lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection molecular mechanism for antibodydependent enhancement of coronavirus entry a perspective on potential antibody-dependent enhancement of sars-cov- is there an ideal animal model for sars? lovastatin-mediated g arrest is through inhibition of the proteasome, independent of hydroxymethyl glutaryl-coa reductase caution urged on sars vaccines aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans lethal infection of k -hace mice infected with severe acute respiratory syndrome coronavirus development of animal models against emerging coronaviruses: from sars to mers coronavirus blocking transmission of middle east respiratory syndrome coronavirus (mers-cov) in llamas by vaccination with a recombinant spike protein humoral immunogenicity and efficacy of a single dose of chadox mers vaccine candidate in dromedary camels the pathogenicity of sars-cov- in hace transgenic mice sars-cov- infection in farmed minks, the netherlands susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus reinfection could not occur in sars-cov- infected rhesus macaques ethical guidelines for deliberately infecting volunteers with covid- human challenge studies to accelerate coronavirus vaccine licensure sars-cov- controlled human infection models: ethics, challenge agent production and regulatory issues effect of dexamethasone in hospitalized patients with covid- criner gjet al, for the gsusi. effect of remdesivir vs standard care on clinical status at days in patients with moderate covid- : a randomized clinical trial world health organization. key criteria for the ethical acceptability of covid- human challenge studies accelerating development of sars-cov- vaccines -the role for controlled human infection models vaccination-induced herd immunity: successes and challenges dissecting the indirect effects caused by vaccines into the basic elements novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov- compared to sars-cov structural basis for human coronavirus attachment to sialic acid receptors emergence of sars-cov- through recombination and strong purifying selection structure of rsv fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody prefusion f-specific antibodies determine the magnitude of rsv neutralizing activity in human sera human parainfluenza virus type expressing the respiratory syncytial virus pre-fusion f protein modified for virion packaging yields protective intranasal vaccine candidates a cysteine zipper stabilizes a pre-fusion f glycoprotein vaccine for respiratory syncytial virus development and characterisation of neutralising monoclonal antibody to the sars-coronavirus self-amplifying rna sars-cov- lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice interactions of sars coronavirus nucleocapsid protein with the host cell proteasome subunit p t cell-mediated immune response to respiratory coronaviruses prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov influenza vaccines and immunopathology the authors. clinical and experimental immunology published by john wiley & sons ltd on behalf of british society for immunology bioinformatic prediction of potential t cell epitopes for sars-cov- world health organization. draft landscape of covid- candidate vaccines recent advances in the production of recombinant subunit vaccines in pichia pastoris recombinant vaccines and the development of new vaccine strategies optimizing assembly and production of native bispecific antibodies by codon deoptimization optimization of antigen dose for a receptor-binding domain-based subunit vaccine against mers coronavirus the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a -residue region in the spike protein that efficiently elicits neutralizing antibodies searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design amino acids to in the s region of severe acute respiratory syndrome coronavirus s protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines clover initiates development of recombinant subunit-trimer vaccine for wuhan coronavirus ( -ncov) / vacci nes/clove r-initi ates-devel opmen t-of-recom binan t-subun it-trime r-vacci ne-for-wuhan -coron aviru s- -ncov.html clover successfully produced -ncov subunit vaccine candidate and detected cross-reacting antibodies from sera of multiple infected patients /vacci nes/clove r-succe ssful ly-produ ced- -ncov-subun it-vacci ne-candi date-and-detec ted-cross -react ing-antib odies the pandemic pipeline press release https:// www.gsk.com/en-gb/media/ press -relea ses/sanofi -and-gsk-tojoin-force s-in-unpre ceden ted-vacci ne-colla borat ion-to-fightcovid - virus-like particles as an instrument of vaccine production assembly of human severe acute respiratory syndrome coronavirus-like particles novavax awarded funding from cepi for covid- novavax recombinant protein nanoparticle vaccine technology phase - trial of a sars-cov- recombinant spike protein nanoparticle vaccine sars-cov- spike glycoprotein vaccine candidate nvx-cov elicits immunogenicity in baboons and protection in mice covid- : medicago's development programs development of a covid- vaccine based on the receptor binding domain displayed on virus-like particles synthetic peptide vaccines design and development of synthetic peptide vaccines: past, present and future comparing pooled peptides with intact protein for accessing cross-presentation pathways for protective cd + and cd + t cells dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and t-cell activation more than one reason to rethink the use of peptides in vaccine design peptide vaccine: progress and challenges preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies development of epitope-based peptide vaccine against novel coronavirus (sars-cov- ): immunoinformatics approach molecular and structural insights into covid- pandemic design of a peptidebased subunit vaccine against novel coronavirus sars-cov- tissue-resident memory cd t-cell responses elicited by a single injection of a multi-target covid- vaccine hla-class ii artificial antigen presenting cells in cd + t cell-based immunotherapy covid- vaccines: breaking record times to first-in-human trials a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge enhanced protection in mice induced by immunization with inactivated whole viruses compare to spike protein of middle east respiratory syndrome coronavirus immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine development of an inactivated vaccine candidate for sars-cov- immunogenicity and safety of a sars-cov- inactivated vaccine in healthy adults aged - years: report of the randomized, double-blind, and placebo-controlled phase clinical trial effect of an inactivated vaccine against sars-cov- on safety and immunogenicity outcomes: interim analysis of randomized clinical trials influence of elemental impurities in aluminum hydroxide adjuvant on the stability of inactivated japanese encephalitis vaccine, ixiaro® recent advances in the vaccine development against middle east respiratory syndrome -coronavirus live attenuated vaccines against influenza; an historical review severe acute respiratory syndrome coronaviruses with mutations in the e protein are attenuated and promising vaccine candidates engineering a replicationcompetent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate a codon-pair deoptimized live-attenuated vaccine against respiratory syncytial virus is immunogenic and efficacious in non-human primates codagenix and serum institute of india initiate co-development of a scalable, live-attenuated vaccine against the novel coronavirus, covid- . biospace viral vectors for vaccine applications severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice safety and immunogenicity of a candidate middle east respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, open-label, non-randomised, uncontrolled, phase trial adenovirus vectors for gene therapy, vaccination and cancer gene therapy evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored covid- vaccine candidate chadox chadox ncov- vaccine prevents sars-cov- pneumonia in rhesus macaques chadox ncov- vaccination prevents sars-cov- pneumonia in rhesus macaques safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind, randomised controlled trial safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, nonrandomised, first-in-human trial immunogenicity and safety of a recombinant adenovirus type- -vectored covid- vaccine in healthy adults aged years or older: a randomised, double-blind, placebo-controlled, phase trial patented technologies to advance disease research nson-johns on-recei ves-posit ive-chmp-opini on-for-janss en%e % % s-inves tigat ional -preve ntive -ebola -vacci ne-regimen single-shot ad vaccine protects against sars-cov- in rhesus macaques safety and immunogenicity of an rad and rad vector-based heterologous prime-boost covid- vaccine in two formulations: two open, non-randomised phase / studies from russia immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebocontrolled, active-comparator, first-in-man trial migal's coronavirus vaccine project using plasmids as dna vaccines for infectious diseases predominant role for directly transfected dendritic cells in antigen presentation to cd + t cells after gene gun immunization sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity inflammatory signals in dendritic cell activation and the induction of adaptive immunity in vivo kinetics and biodistribution of a hiv- dna vaccine after administration in mice high efficiency transformation by direct microinjection of dna into cultured mammalian cells the development of vaccines against sars corona virus in mice and scid-pbl/ hu mice humoral and cellular immune responses induced by a dna vaccines against severe acute respiratory syndrome (sars) or sars-like coronavirus in mice induction of specific immune responses by severe acute respiratory syndrome coronavirus spike dna vaccine with or without interleukin- immunization using different vaccination routes in mice a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase , open-label, single-arm, dose-escalation trial immunogenicity of a dna vaccine candidate for covid- intradermal-delivered dna vaccine provides anamnestic protection in a rhesus macaque sars-cov- challenge model rna sensors of the innate immune system and their detection of pathogens adjuvant effects of a sequence-engineered mrna vaccine: translational profiling demonstrates similar human and murine innate response induction of an ifnmediated antiviral response by a self-amplifying rna vaccine: implications for vaccine design suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna incorporation of pseudouridine into mrna yields superior nonimmunogenic vector with increased translational capacity and biological stability mammalian micrornas predominantly act to decrease target mrna levels au-rich elements and associated factors: are there unifying principles? self-amplifying rna vaccines give equivalent protection against influenza to mrna vaccines but at much lower doses sars-cov- mrna vaccine development enabled by prototype pathogen preparedness an mrna vaccine against sars-cov- -preliminary report a single immunization with nucleoside-modified mrna vaccines elicits strong cellular and humoral immune responses against sars-cov- in mice phase / study to describe the safety and immunogenicity of a covid- rna vaccine candidate (bnt b ) in adults to years of age concurrent human antibody and th type t-cell responses elicited by a covid- vaccine bnt b selected for a pivotal efficacy study adjuvanted influenza vaccines mechanisms of action of adjuvants vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens vaccine adjuvants: putting innate immunity to work evaluation of adjuvants for protein vaccines against tuberculosis in guinea pigs tolerability of intramuscular and intradermal delivery by cellectra(®) adaptive constant current electroporation device in healthy volunteers the safety and immunogenicity of gtu®multihiv dna vaccine delivered by transcutaneous and intramuscular injection with or without electroporation in hiv- positive subjects on suppressive art safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase clinical trials dna vaccine delivered by a needle-free injection device improves potency of priming for antibody and cd + t-cell responses after rad boost in a randomized clinical trial bcg-induced trained immunity: can it offer protection against covid- ? can existing live vaccines prevent covid- ? could an unrelated live attenuated vaccine serve as a preventive measure to dampen septic inflammation associated with covid- infection? nonspecific protection of mice against influenza virus infection by local or systemic immunization with bacille calmette-guerin is global bcg vaccination coverage relevant to the progression of sars-cov- pandemic? efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola Ça suffit!) a review of dengvaxia®: development to deployment government launches vaccine taskforce to combat coronavirus moderna manufacturing: why norwood? world health organization. standardization of vaccines for coronavirus disease (covid- ) the authors. clinical and experimental immunology published by john wiley & sons ltd on behalf of british society for immunology successful ebola vaccine provides % protection in trial roadmap for introduction and roll-out of merck rvsv-zebov world health organization. major milestone for whosupported ebola vaccine return on investment of public health interventions: a systematic review legal agreements: barriers and enablers to global equitable covid- vaccine access the authors thank dr ed parker, professor beate kampmann and the team at the vaccine centre at the london school of hygiene and tropical medicine for developing and maintaining the covid- vaccine tracker tool, which was invaluable for this review. b.f.p. is funded by the key: cord- -qfdxri authors: wack, andreas; rappuoli, rino title: vaccinology at the beginning of the st century date: - - journal: curr opin immunol doi: . /j.coi. . . sha: doc_id: cord_uid: qfdxri today, the main challenges for vaccinologists include improving vaccines against as yet undefeated pathogens, rapid identification and response to emerging diseases and successful intervention in chronic diseases in which ongoing immune responses are insufficient. reverse genetics and reverse vaccinology are now used to generate rapidly new vaccine strains and to mine whole genomes in the search for promising antigens. the rational design of adjuvants has become possible as a result of the discovery of the receptors that recognize microbial patterns and lead to dendritic cell activation. antigen-loaded dendritic cells, dna in naked, formulated or viral form, and other delivery systems are used to maximize immune responses. although work on the ‘easy’ vaccines has already been completed, it is hoped that a combination of conceptual and technical innovation will enable the development of more complex and sophisticated vaccines in the future. vaccinology at the beginning of the st century andreas wack and rino rappuoli today, the main challenges for vaccinologists include improving vaccines against as yet undefeated pathogens, rapid identification and response to emerging diseases and successful intervention in chronic diseases in which ongoing immune responses are insufficient. reverse genetics and reverse vaccinology are now used to generate rapidly new vaccine strains and to mine whole genomes in the search for promising antigens. the rational design of adjuvants has become possible as a result of the discovery of the receptors that recognize microbial patterns and lead to dendritic cell activation. antigen-loaded dendritic cells, dna in naked, formulated or viral form, and other delivery systems are used to maximize immune responses. although work on the 'easy' vaccines has already been completed, it is hoped that a combination of conceptual and technical innovation will enable the development of more complex and sophisticated vaccines in the future. although the eradication of smallpox in the s and of poliomyelitis hopefully in the coming years mark two of the most important milestones in medical history, we now face an unprecedented succession of new pathogens which jump species barriers to infect humans, and the frustration deriving from the inability to control devastating diseases such as hiv, malaria and tuberculosis. this review will cover the recent progress in vaccinology (figure ), largely resulting from dramatic technical innovation that is now reaching the clinic, as well as the huge challenges posed by old and new pathogens that are facing us. the three 'big killers', the pathogens that most heavily afflict global health, are hiv, mycobacterium and plas-modium. whereas the latter two represent long-term companions of the human species, hiv is a virus that spread in the human population only about years ago and has a nonhuman primate origin [ ] . in fact, most, if not all, of the recently emerging diseases go back to animal reservoirs, from which they infect humans through close contact during hunting and farming, in live animal markets or through food processing, preparation or consumption. an example is ebola virus, which, after three outbreaks between and , has appeared in the human population nine times since , with each outbreak resulting from the handling of dead gorillas, chimpanzees or duikers, which in turn are thought to have been infected by an unknown natural host [ ] . the variant creutzfeldt-jacob disease reached the population through the food chain, from 'rendered' sheep and cow carcasses fed to cows which were subsequently consumed by humans. the severe acute respiratory syndrome (sars)-coronavirus sequences from the earliest identified cases are identical to those found in palm civets and raccoon dogs in animal markets and farms [ ] , strongly suggesting an animal origin for this disease. rapidly changing ecosystems and human behavior, an ever-increasing density of human and (farmed) animal populations and their close vicinity, poverty, a high degree of mobility and many other factors contribute to the more frequent occurrence and often rapid dissemination of new diseases. another example of this is the arrival and persistence of west nile virus in the usa, which is probably a result of increased mobility, an exceptionally broad host range and climatic changes [ ] . among the reemerging diseases of the past few years, diphtheria and cholera should be mentioned [ ] , as well as the frequent appearance of multidrug-resistant bacteria and the cases of anthrax infection as a result of deliberate release in . in the case of influenza, concern for the advent of a new pandemic has been fuelled by reports of human infections by the avian h n virus strain (where h stands for hemagglutinin and n for neuraminidase, the two major surface glycoproteins of the virus) in , leading to deaths (http://www.who.int/csr/disease/avian_ influenza/en/). this was the third time in the space of a few years (after the previous outbreaks of infection in and ) that h n avian flu viruses caused disease and death in humans. an h n avian strain caused infection in humans in and . because the world's population has not been exposed to these strains, it is immunologically naïve, and these or any other avian flu strains with new surface glycoproteins would meet no possible intervention points for vaccine improvement. schematic view of a pathogen-or vaccine-induced immune response. vaccines or pathogens cross epithelial barriers and are taken up by antigen-presenting cells such as dcs. interaction between pattern recognition receptors (prrs) and their agonists activate dcs, resulting in increased antigen presentation, cytokine production and co-stimulation. cd + and cd + t cells recognize antigen presented by dcs and are activated. cognate interaction between primed cd + t cells and b cells activates the b cells, resulting in clonal expansion and antibody production. dc activation also leads to inhibition of the regulatory effect of cd + cd + treg cells. fully activated cd + cells can target tumor cells and pathogen-infected cells. the letters (a-i) indicate processes where improved vaccines can lead to more efficient immune responses: (a) the site of administration influences the type of immune response and enables the usage of lower vaccine doses [ , ] . (b) particulate antigen is taken up more easily by macrophages and dcs than soluble antigen [ ] . (c) tlr agonists and other immunostimulants binding to prrs increase the activation of dcs [ - , , , , ] . (d) dcs matured and loaded with antigen in vitro are efficient vaccines [ , , ] . (e) dna vaccination leads to efficient antigen presentation on mhc class i [ , , , , , ] . crosspresentation of antigen on mhc class i of host dcs is facilitated after vaccination with antigen-loaded dcs undergoing delayed apoptosis [ ] . (f) cytokines can be added in protein or dna form as natural adjuvants [ , ] . (g) recruited nk cells can be an early source of th -driving cytokines [ ] . (h) vaccines can break tolerance when the suppressive effect of cd + cd + treg cells is overcome [ ] . resistance provided by existing immunity and could rapidly expand. once an avian strain has developed a more efficient means of human-to-human transmission, devastating pandemics, such as those arising in , and , could ensue. it should be noted, however, that older studies found seroprevalence rates for avian flu strains among chinese rural populations to be between % and % for h viruses and between % and % for other avian strains [ , ] . thus, it might in fact be the quality of surveillance, rather than the frequency of outbreaks, that has increased over the past few years [ ] . however, the contact between humans and pathogens in animal reservoirs is likely to intensify, and, consequently, the emergence or re-emergence of diseases will occupy vaccinologists frequently in the future. one of the most important future challenges will be to respond promptly to emerging diseases such as those mentioned above. a striking example for rapid reaction was in the case of the sars outbreak, where the genome sequence was publicly available in less than a month after the virus was identified [ ] . this enabled the speedy development of diagnostic tools, as well as the identification and recombinant expression of targets for vaccines and therapeutic agents [ ] [ ] [ ] . for the influenza virus, in addition to the annual definition of the relevant strains to be included in the vaccine for the following season, the world health organization closely monitors cases of avian flu (for further information, see the world health organization website indicated above), and prototype vaccines for these strains are being developed. apart from almost complete lack of protection in the population, an additional threat of the avian flu h n isolate from is that it kills embryonated eggs, the traditional virus growth substrate used for influenza vaccine production. such problems can now be solved, owing to the discovery some years ago that influenza virus can be generated entirely from transfected dna (reverse genetics [ , ] ). in this particular case, webby et al. [ ] have used polymerase chain reaction-based mutagenesis to replace the hemagglutinin cleavage site (which was shown to be the cause of high pathogenicity) of the h n strain with the sequence from a nonpathogenic strain. vero cells were then transfected with plasmids encoding the neuraminidase and mutated hemagglutinin from the circulating strain, together with the plasmids encoding the remaining proteins from the laboratory-optimized pr strain. the resulting vaccine strain was successfully grown in eggs and shown to be nonpathogenic and stable. thus, the use of reverse genetics enables rapid production of a reference vaccine virus in response to the emergence of a new influenza variant [ , ] . in addition, reverse genetics can be used for more far-reaching vaccination strategies, such as the construction of 'consensus' strains expressing conserved amino acid sequences or more than one version of the surface glycoproteins, or additional immunoenhancing molecules, such as cytokines [ ] . much research effort is also being invested into the development of improved cell culture systems that can replace completely the use of embryonated eggs in vaccine production and would render the production process more flexible and controllable. several reports have addressed the question of how to stretch the available supply of vaccine doses in cases of shortage or in the face of a pandemic. two studies indicated that intradermal, rather than intramuscular, application of % [ ] the genomic revolution has opened up a completely new approach to vaccine discovery. for pathogens that do not grow in vitro, the availability of the genome sequence has enabled the development of recombinant vaccines, as has been carried out for hepatitis b virus (hbv) and is underway for hepatitis c. in regard to bacteria, group b meningococcus posed insurmountable obstacles to conventional vaccinology approaches; these were eventually overcome by mining the information from the sequenced genome [ ] . a total of potential vaccine candidates were predicted by computer analysis, of which were expressed and tested for immunogenicity [ ] . some of these candidates are now in clinical trials. this genomebased approach, called reverse vaccinology, is now used routinely in vaccine development, and is a major tool in the quest for vaccines against pneumococcus, group b streptococcus and chlamydia (see also update). recently, genome sequencing of both plasmodium falciparum [ ] and its main vector, anopheles gambiae [ ], has sparked off new hopes for an efficient vaccine against malaria. for the rodent models of this disease, subtractive cdna techniques were used to identify genes that are only expressed in pre-erythrocytic stages of the parasite [ , ]. plasmodium mutants deficient in one of these genes, uis , are blocked in their early liver-stage development and all subsequent stages, and therefore do not lead to disease. when uis -deficient sporozoites are used as genetically attenuated vaccines in mice, they confer long-lasting, stage-specific protection [ ] . this is a promising example of how molecular approaches are employed for the rational design of new vaccines. another example of encouraging progress towards a malaria vaccine was reported by alonso et al. [ ] . they describe phase iib trials of a subunit vaccine consisting of a recombinant protein (expressed in yeast) composed of the carboxy-terminal of the p. falciparum circumsporozoite protein and the hbv surface antigen. this fusion protein, together with unfused hbv surface antigen proteins, forms particles. the final vaccine formulation includes the adjuvant as a, an oil in water emulsion containing the immunostimulants monophosphoryl lipid a (mpl) and quillaja saponaria fraction . it had previously been shown that, indeed, a cd + t cell response to an epitope contained in the vaccine correlates with protection from infection and disease [ ] . in this trial in children, the vaccine showed an efficacy of % and % in preventing clinical episodes and severe episodes, respectively [ ] . the past ten years have changed our vision of the immune response to pathogens. it has become clear that the degree and type of antigen-specific, clonal b and t cell responses (acquired immunity) depend crucially on the prior action of a more ancient system of pathogen detection (innate immunity). this system relies on the activation of antigen-presenting cells such as dendritic cells (dcs) upon recognition of patterns common to viruses and bacteria and largely absent in mammals. with the discovery of the involved pattern recognition receptors, among which the toll-like receptors (tlrs) represent an important subgroup, immune-enhancing molecules or adjuvants can no longer be considered as the alchemistic 'immunologist's dirty secret' but have become amenable to rational design, providing a huge potential for manipulating the immune response. as different tlr agonists elicit different types of immune responses (reviewed in [ ] ), future adjuvants might be able to tailor the immune response so that optimal protection to a given pathogen is induced. in fact, the number of clinical trials involving tlr agonists as new adjuvants is ever increasing [ , ] (table ). the adjuvant function of nonmethylated cytidine-phosphate-guanosine (cpg) sequences, which are frequent in microbes but under-represented in humans and are agonists of tlr , has been extensively demonstrated in animal models [ ] and is currently being tested in several clinical trials. when cpgs are coadministered with licensed hbv or flu vaccines, the combination leads to increased antibody titers or increased (interferon-g) ifn-g production, as compared with the response to the vaccine alone [ , ] . an additional effect of cpg oligonucleotides appears to be the promotion of affinity maturation and, as a result, a higher overall affinity of the vaccine-specific antibody pool [ ] . similarly, the tlr agonist mpl has been shown in the past to enhance the immune response to hbv vaccination in humans [ ] . in addition, both the malaria subunit vaccine mentioned above [ ] and a licensed melanoma vaccine contain mpl. another experimental vaccine that includes a tlr agonist is described below, in the section on synthetic vaccines. activation of dcs increases their ability to process and present antigen and to attract and activate t cells through cytokine secretion; consequently, several cytokines are host-pathogen interactions table human tlr agonists used as adjuvants in vaccine formulations in clinical trials or licensed vaccines a . known currently being tested for their adjuvant function. as mentioned above, the way the innate immune system is activated influences the type of the ensuing acquired immune response. martin-fontecha et al. [ ] showed that the ability of adjuvants to elicit a th type response depends crucially on the recruitment of, and ifn-g production by, natural killer (nk) cells, which indicates a possible mechanism of the way in which adjuvants direct the type of adaptive immune response induced downstream. another vaccine approach is the use of heat shock proteins, which bind specifically and activate dendritic cells and, because they are loaded with endogenous peptides, can be purified from tumor cells and function as a combined antigen delivery system and adjuvant [ ] . because the main targets of adjuvants are dcs, it is a logical step to evaluate their direct use as a vaccine. despite the labor-intensive necessity of individual cell culture for each patient, this approach can be attractive where other approaches have failed, for instance as a therapeutic vaccine in hiv or cancer patients. when hiv patients were treated with autologous dcs loaded with autologous, inactivated hiv, both virus-specific cd + th and cd + responses were induced and the plasma viral loads were reduced [ ] . these results closely reflect previous findings from a similar vaccination of rhesus macaques [ ] , except for the lack of induction of neutralizing antibodies in the human study. dc vaccination is also being tested in a variety of cancer treatments [ ] . in a study comparing the immunogenicity of dcs transfected with cytopathic or noncytopathic viral rna, the former regimen was shown to be more efficient at inducing protective immune responses [ ] . this suggests that reprocessing of dying dcs by endogenous antigen-presenting cells enhances immunogenicity, either through additional danger signals triggered by the cell damage or by the increased level of crosspresentation by endogenous dcs. the same mechanism might be at work in mycobacterium infection of macrophages, where apoptosis was shown to enhance crosspresentation by bystander dcs [ ] . interestingly, following fractionation of the cytoplasm of dying cells, uric acid was identified as a highly efficient endogenous danger signal that enhances immunogenicity [ ] , and might explain the above results. the high expectations associated with dna vaccination, as a result of promising data obtained in mice, were somewhat tempered by disappointing early results when dna was tested as a vaccine in humans. therefore, the latest generation of dna vaccines rely on improved delivery either through use of microparticles [ ] or through viral vectors. a particularly promising approach is a heterologous prime-boost strategy, where adminis-tration of plasmid dna is followed by recombinant virus (modified vaccinia virus ankara [mva] or adenovirus) expressing the same antigen. this regimen induced strong t cell responses against p. falciparum in naïve adults [ ] and enhanced the response in gambian men who are constantly exposed to p. falciparum [ ] . although partial protection against challenge with a different p. falciparum strain was observed, no significant differences in the infection rate was found in the gambian trial [ ] . in spite of these setbacks, a very similar regimen has been shown also to be highly immunogenic against hbv, tuberculosis and hiv, and vaccination only with mva expressing the mycobacterium a antigen elicited strong t cell responses [ ] . because the induction of t cells is considered to be crucial in anti-tumor immune responses, a huge number of trials are presently being conducted to test dna vaccination regimens for anticancer treatment [ , ] . when viral vehicles (vaccinia or adenovirus) were compared with loaded dcs in terms of their ability to overcome established tolerance and induce immune responses in a transgenic mouse model, the viral formulations were able to do so, whereas dcs required repeated administration of tlr agonists or irrelevant virus, or removal of suppressive cd + cd + treg cells [ ] . such models of established tolerance might prove useful for testing the success of vaccines in the face of long-term antigen exposure, as in the case of cancer or chronic diseases. two studies analyzed in detail the t cell response after vaccination with a recombinant canarypox virus expressing melanoma-specific t cell epitopes [ , ] . focusing on the blood and metastases from a patient with complete regression, these studies reconfirmed earlier observations that the frequency of anti-tumor t cells can be relatively high. although vaccination leads to a slight increase in vaccine-specific t cells, these remain only a small fraction of total anti-tumor t cells. by contrast, t cells directed against epitopes not contained in the vaccine represent the vast majority of anti-tumor cells, and their frequency increases both in the blood and in metastases. thus, it appears that with the appropriate vaccine regimen, the inefficiency of pre-existing specific t cells to combat the tumor was reversed in an indirect manner, presumably by activating another subset of t cells. it remains to be clarified, however, how much of this reactivation is due to the action of vaccine-induced t cells and how much is a general, antigen-independent immune-enhancing effect. in any case, for the development of therapeutic vaccines, it will be vital to understand how the balance can be tipped back from a state of tolerance to a successful immune response. the development of vaccines aimed at the polysaccharide (ps) capsule of bacteria is one of the great achievements in vaccinology. so far, the ps used in largescale vaccine production has been purified from the pathogen itself, grown in large quantities -an approach that is costly and difficult to control. through great simplification of the carbohydrate chemistry involved, verez-bencomo et al. [ ] have now demonstrated the first large-scale production of an anti-haemophilus influenzae type b vaccine, consisting of synthetic ps conjugated to tetanus toxoid protein carrier. this vaccine has been shown to be as efficient as commercially available vaccines in inducing protective levels of antibody titers in infants. an entirely synthetic vaccine with a branched structure containing a tlr ligand, a cd + t cell epitope and either a cd + t cell or a b cell epitope has been shown to elicit strong cd + t cell and b cell responses, respectively [ ] . here, the minimal requirements for an efficient vaccine are met in a single molecule: targeting to and activation of dcs, t cell help and activation of antigen-specific cd + t cells or b cells. the world of vaccines is undergoing dramatic changes. never before have such sophisticated techniques and an in-depth knowledge of immunological processes been at hand to exploit fully the potential of protecting from, as well as curing, diseases through vaccination. a formidable task in the future will be the development of effective therapeutic vaccines, in situations where chronic antigen exposure by itself does not elicit a sufficiently strong immune response, as is the case in cancer and chronic infectious diseases. compared with vaccines against selflimiting infections, where the aim is to be as good as the real pathogen (but less harmful), this requires the development of vaccines that are better than the natural antigens in inducing immunity. all of our knowledge will be necessary to succeed in this challenge. a recent article describes the use of multigenome analysis and screening against a large panel of strains to identify a universal group b streptococcus vaccine. although none of the single antigens contained in the vaccine elicits protection against all strains, the combination of four proteins is able to cover a wide range of strains [ ] . mueller ak, labaied m, kappe sh, matuschewski k: genetically modified plasmodium parasites as a protective experimental malaria vaccine. nature , : - . in this study, reverse genetics was used to generate a plasmodium knockout strain with a developmental block at the early liver stage. if used as a live attenuated vaccine in a malaria mouse model, this strain confers protection against infectious sporozoite challenge. origin of hiv- in the chimpanzee pan troglodytes troglodytes multiple ebola virus transmission events and rapid decline of central african wildlife human ebola virus outbreaks consist of parallel epidemics caused by several different viral strains which appear to be transmitted to humans through handling of gorilla, chimpanzee or duiker carcasses molecular evolution of the sars coronavirus during the course of the sars epidemic in china the chinese sars consortium demonstrates that virus sequences from the earliest patients are identical to those found in palm civets, and seven out of cases had documented contact with wild animals. in addition, the isolated case in december was caused by a virus with highest sequence homology to palm civets west nile virus: where are we now? from pasteur to genomics: progress and challenges in infectious diseases pandemic influenza: a zoonosis? semin respir infect serological evidence of human infections with avian influenza viruses influenza: old and new threats sars -beginning to understand a new virus a dna vaccine induces sars coronavirus neutralization and protective immunity in mice severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus rescue of influenza a virus from recombinant dna generation of influenza a viruses entirely from cloned cdnas responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines this paper demonstrates the rapid generation of a genetically attenuated influenza h n reference vaccine strain by use of reverse genetics evaluation of a genetically modified reassortant h n influenza a virus vaccine candidate generated by plasmid-based reverse genetics serum antibody responses after intradermal vaccination against influenza see annotation to efficacy of the rts, s/as a vaccine against plasmodium falciparum infection and disease in young african children: randomised controlled trial this vaccine contains a yeast-expressed hybrid molecule composed of a part of the circumsporozoite protein and the hbv surface antigen, formulated in a complex adjuvant. significant efficacy, in terms of prevention of clinical episodes and of severe malaria a cd (+) t-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural plasmodium falciparum infection and disease using an enzyme-linked immunospot assay on t cells previously expanded in vitro, the authors identified a conserved cd + t cell epitope in the circumsporozoite protein with predictive value: an ifn-g response of cd + t cells to this epitope positively correlated with protection from malaria third meeting on novel adjuvants currently in or close to clinical testing world health organization-organisation mondiale de la sante th meeting on novel adjuvants currently in/close to human clinical testing world health organizationorganisation mondiale de la sante fondation merieux use of cpg oligodeoxynucleotides as immune adjuvants a phase i study of the safety and immunogenicity of recombinant hepatitis b surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant cpg , an immunostimulatory tlr agonist oligodeoxynucleotide, as adjuvant to engerix-b((r)) hbv vaccine in healthy adults: a double-blind phase i/ii study see annotation to co-administration of cpg oligonucleotides enhances the late affinity maturation process of human anti-hepatitis b vaccine response in these two clinical trials, cpg was shown to be an efficient adjuvant when used in combination with licensed vaccines the immunogenicity and reactogenicity profile of a candidate hepatitis b vaccine in an adult vaccine non-responder population induced recruitment of nk cells to lymph nodes provides ifn-gamma for t(h) priming the authors showed that the ability of some adjuvants to induce th priming correlates with the degree of recruitment of nk cells, which represent an early source of ifn-g. this might provide a rationale for the search for efficient th therapeutic cancer vaccines: using unique antigens therapeutic dendritic-cell vaccine for chronic hiv- infection this study followed a previous one in macaques [ ] and showed that injection of autologous dcs loaded with autologous inactivated hiv induces hiv-specific cd + and cd + t cell responses and leads to a prolonged tenfold reduction in virus therapeutic dendritic-cell vaccine for simian aids dendritic cells: a journey from laboratory to clinic dendritic cells transfected with cytopathic self-replicating rna induce crosspriming of cd + t cells and antiviral immunity the surprising observation in this study is that induction of immunity and crosspresentation of antigen is greatly facilitated by the death of the injected antigen-carrying dcs. this suggests that danger signals from dying cells might have an adjuvant effect in vaccinations, as well as in immune responses apoptosis facilitates antigen presentation to t lymphocytes through mhc-i and cd in tuberculosis molecular identification of a danger signal that alerts the immune system to dying cells microparticles for the delivery of dna vaccines enhanced t-cell immunogenicity of plasmid dna vaccines boosted by recombinant modified vaccinia virus ankara in humans safety and immunogenicity of dna/modified vaccinia virus ankara malaria vaccination in african adults a randomised, double-blind, controlled vaccine efficacy trial of dna/mva me-trap against malaria infection in gambian adults recombinant modified vaccinia virus ankara expressing antigen a boosts bcg-primed and naturally acquired antimycobacterial immunity in humans the authors observed antigen-specific ifn-g responses of short duration induced by vaccination with mva expressing the conserved mycobacterial antigen a. this t cell response is substantially higher and sustained in vaccinees who received bacillus calmette-guerin followed by mva three weeks later gene-based vaccines and immunotherapeutics dna vaccines to attack cancer persistent toll-like receptor signals are required for reversal of regulatory t cell-mediated cd tolerance this transgenic adoptive transfer model of tolerance enables the comparison of different vaccine regimens in their ability to break tolerance. viral vaccines are most efficient in overcoming established tolerance. dcbased vaccine strategies that are contrasting frequencies of antitumor and anti-vaccine t cells in metastases of a melanoma patient vaccinated with a mage tumor antigen high frequency of antitumor t cells in the blood of melanoma patients before and after vaccination with tumor antigens these two studies showed that in the blood and metastases of vaccinated cancer patients, t cells specific for the vaccine antigen might represent only a small fraction of total tumor-specific t cells, both before and after vaccination a synthetic conjugate polysaccharide vaccine against haemophilus influenzae type b the large-scale production of a conjugate vaccine containing synthetic polysaccharides has been achieved by simplification of the polysaccharide chemistry involved a totally synthetic vaccine of generic structure that targets toll-like receptor on dendritic cells and promotes antibody or cytotoxic t cell responses this elegant study showed how all of the components required for an efficient vaccine can be included in one synthetic molecule: a t helper epitope, a tlr agonist as an adjuvant and either a cd + t cell or a b cell epitope. the resultant vaccines confer protection and are able to break tolerance in mice variegation of the immune response with dendritic cells and pathogen recognition receptors identification of a universal group b streptococcus vaccine by multiple genome screen the use of different group b streptococcus strains for genome analysis and screening enables the development of a universal vaccine. the single vaccine components are not present in all strains, and do not protect against all strains we are grateful to giorgio corsi for artwork. papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest key: cord- -aozxvxxs authors: vermillion, meghan s.; klein, sabra l. title: pregnancy and infection: using disease pathogenesis to inform vaccine strategy date: - - journal: npj vaccines doi: . /s - - - sha: doc_id: cord_uid: aozxvxxs vaccination is the mainstay of preventative medicine for many infectious diseases. pregnant women, unborn fetuses, and neonates represent three at-risk populations that can be simultaneously protected by strategic vaccination protocols. because the pathogenesis of different infectious microbes varies based on tissue tropism, timing of infection, and host susceptibility, the goals of immunization are not uniform across all vaccines. mechanistic understanding of infectious disease pathogenesis and immune responses is therefore essential to inform vaccine design and the implementation of appropriate immunization protocols that optimize protection of pregnant women, fetuses, and neonates. vaccination significantly reduces the health burden of many infectious disease, especially in high-risk populations. pregnant women, unborn fetuses, and neonates represent three populations of high-risk individuals that can all be simultaneously protected from vaccine-preventable infectious disease with strategic maternal immunization protocols. infectious microbes that pose significant health risks during pregnancy can be divided into three broad categories, based on the pathogenesis and disease outcome (fig. ) , with some microbes falling within more than one category. first are maternal infections, which are defined by heightened disease severity in pregnant females, but with rare or inconsequential transmission and disease in the fetus. second are fetal or congenital infections, which are characterized by mild or no disease in pregnant females, but occasional vertical transmission and severe congenital disease in the fetus. third are neonatal and infant infections, which are not considered to pose significant risk to pregnant women or unborn fetuses, but can cause severe, and sometimes fatal disease in neonates and infants that lack protective maternal immunity following birth. the vaccination strategies employed differ for micobes within each of these categories and vary based on the at-risk individual (i.e., mother, fetus, and/or neonate/infant), the timing of the greatest risk of infection (i.e., early pregnancy, late pregnancy, or post-natal), and on the duration of protective immunity following vaccination. in this review, we discuss evidence to suggest that immunization strategies for pregnant women should be tailored to optimize protection for the mother, fetus, neonate, infant, or all individuals. we review vaccine-preventable infections during pregnancy and the current vaccination strategies employed to reduce the burden of infectious diseases, including influenza. further, we examine novel vaccine platforms and consider how their application may provide safe alternatives for enhancing protection of pregnant women. finally, we discuss vaccine development and prevention strategies for combatting emerging infectious diseases, including zika, that pose a threat to pregnant women and their fetuses. owing to physiologic and immunologic changes that support pregnancy and tolerance of a semi-allogenic fetus, pregnant women demonstrate increased susceptibility to certain infectious agents including hepatitis e, varicella zoster, and influenza viruses. infection with these viruses during pregnancy results in severe maternal disease, increased maternal mortality and associated pregnancy complications, which are observed most frequently during the third trimester and peripartum period. for example, the case fatality rate among pregnant women infected with hepatitis e virus is estimated to be - %, , compared with - % in the general population. approximately % of cases of varicella pneumonia in adults reported from - were from pregnant women ; and pregnant women infected with the pandemic h n influenza a virus (iav) were reportedly times more likely to be hospitalized or die than the general population. overall, vertical transmission of these viruses is relatively uncommon, but adverse pregnancy outcomes, including spontaneous abortion and pre-term birth can still occur as an indirect consequence of maternal inflammation. , reports during the h n pandemic in australia and new zealand indicated that among pregnant women who were hospitalized with suspected h n iav infection, % of their infants required intensive care, and % were either stillborn or died shortly after birth; only infants, however, had detectable h n infection. the primary goal of vaccination strategies for protecting against maternal infections is the generation of protective maternal immunity either prior to or during early pregnancy. optimally, vaccination should prevent or reduce disease by inducing sterilizing immunity (i.e., immunity that completely prevents infection). despite reported reductions in antiviral proteins during pregnancy, studies comparing vaccine responses between pregnant and nonpregnant women find no difference in either the magnitude or duration of antibody responses against influenza a viruses. [ ] [ ] [ ] in fact, surveillance data from taiwan reveal that influenza vaccination during pregnancy results in higher levels of seroprotection than does vaccination prior to conception, with no effect of gestational age on vaccineinduced antibody responses. as of , the centers for disease control (cdc) advisory committee on immunization practices (acip) categorizes pregnant women as a target population for receiving the inactivated influenza vaccine and recommends that pregnant women be immunized during any trimester. several studies evaluating adverse vaccine reactions in pregnant women have concluded that there is no link between pregnancy complications or adverse fetal outcomes among women who are vaccinated during pregnancy. [ ] [ ] [ ] [ ] although the live attenuated intranasal influenza vaccine is not recommended for pregnant women, accidental administration during pregnancy was not associated with an increased risk of adverse reactions. , despite the plethora of data that support the benefits and safety of influenza vaccination during pregnancy, coverage remains low, with a less than % maternal vaccination rate during the - influenza season in the united states. misconceptions about the safety and benefits of influenza vaccination represent the largest barriers to vaccine acceptance among pregnant women. varicella zoster virus (vzv) is another vaccine-preventable infection associated with increased severity during pregnancy. vzv is an alpha herpes virus and the causative agent of varicella or chickenpox. in temperate climates, seroprevalence among individuals over years of age is estimated to be %, with almost % of infections occurring prior to years of age. the first modified-live vaccine against varicella zoster virus was licensed in the united states in , and is now recommended for children over months of age. primary vzv infection during pregnancy is therefore uncommon, as most women of childbearing age have been either infected or immunized. in women who have not been previously exposed, however, primary vzv infection between weeks through of gestation is associated with a % risk of congenital transmission and disease in the offspring. because all licensed vzv vaccines contain live-attenuated virus, their use during pregnancy is contraindicated. instead, the cdc recommends that nonpregnant women of childbearing age be vaccinated against vzv at least one month prior to conception. as a herpes virus, infection with vzv is life-long, and reactivation occurs in approximately - % of individuals, which results in a painful skin condition known as shingles or herpes zoster. reactivated vzv, however, is not associated with increased disease severity or congenital infection during pregnancy. acute viral hepatitis caused by hepatitis e virus (hev) is an emerging infectious disease that causes severe disease in pregnant women, with a fatality rate of up to % in endemic regions. in addition to heightened maternal disease severity, hev infection during pregnancy is associated with increased rates of premature birth and prenatal mortality. although vertical hev transmission rates are high, with estimates between - %, the relative contributions of fetal hev infection to adverse perinatal outcomes is unclear. a recombinant hev subunit vaccine has been developed and proven safe and effective following completion of phase ii and iii clinical trials, but commercial use is currently limited to china. furthermore, the vaccine is not approved for use in pregnant women despite being % efficacious in participants receiving all three doses. additional hev vaccine candidates are being tested in preclinical pregnant animal models, and one recombinant hev vaccine has been shown to be safe and highly immunogenic in pregnant mice. additional studies in a susceptible animal model are needed to confirm efficacy following virus challenge. developing fetuses are extremely vulnerable to both infectious and noninfectious insults. certain infectious agents that are often clinically silent in healthy adults can cause severe birth defects if fig. infectious microbes that cause maternal, congenital, or postnatal complications. the infectious microbes are categorized according to the mechanism of transmission and disease, and the population at greatest risk for severe outcome during or after pregnancy. infection with some pathogens (e.g., sars coronavirus, hepatitis e virus, and ebola virus) during pregnancy cause severe disease in pregnant women, but are not transmitted to offspring. other infectious microbes (e.g., toxoplasma gondii, rubella virus, parvovirus b , cytomegalovirus, and zika viruses) infect and cause mild or asymptomatic disease in pregnant females, but can be vertically transmitted to the fetus and congenital complications. another category of microbes (e.g., bordetella pertussis, clostridium tetani, and respiratory syncytial virus) pose the largest risk to neonates after birth. many infectious microbes (e.g., listeria monocytogenes, plasmodium spp., hiv, vzv, influenza viruses, chlamydia trachomatis, gbs, treponema pallidum, and herpes viruses) may cause overlapping syndromes depending on the timing of infection during pregnancy. understanding the pathogenesis of infectious diseases during pregnancy should inform vaccine design and the implementation of appropriate immunization protocols that optimize protection of pregnant women, fetuses and neonates they breach the placental barrier during critical developmental periods during pregnancy. an increasing number of pathogens are being recognized for causing congenital disease, and what was originally designated as the torch complex (toxoplasma gondii, "other," rubella virus, cytomegalovirus, and herpes simplex virus) is now expanded to include other infectious agents including zika virus. development of congenital disease can depend on the timing of infection during gestation, the infectious burden, and the pathogenesis in the fetus. the congenital syndrome for each pathogen is characterized by a variety of different developmental abnormalities, and commonly impact hearing, vision, and central nervous system function. for many congenital infections, the timing of infection during gestation determines the relative risk to the fetus and dictates the spectrum of disease that results. for example, while infection with rubella virus during the first weeks of gestation is associated with an % risk of congenital rubella syndrome (crs), this is reduced to a % risk between - weeks, and minimal risk for infections occurring after weeks of gestation. in contrast, the risk of congenital toxoplasmosis has been demonstrated to be highest during third trimester pregnancy, which is hypothesized to be due to differential expression of placental toll-like receptors, including tlr , within first compared with third trimester trophoblast cells. similarly, maternal infection with listeria monocytogenes is typically associated with adverse pregnancy outcomes during the third trimester, though infection during the first trimester in nonhuman primates also leads to rapid fetal demise. the primary goal of vaccination strategies for protecting against fetal infections is generation of protective maternal immunity prior to pregnancy. because congenital infections can occur in the absence of maternal symptoms, vaccines against congenital agents should ideally provide complete sterilizing immunity. rubella is included in a live-attenuated combination vaccine for measles, mumps, and rubella (mmr), which confers lifelong protective immunity. because of the long duration of protective immunity following rubella vaccination, target populations include children and adolescent girls. however, incomplete vaccination coverage can lead to paradoxical increases in crs due to an increase in the average age of infection, and so it is also recommended that unvaccinated women of childbearing age be counseled to receive the rubella vaccine at least one month prior to conception. the implementation of large-scale rubella vaccination programs has resulted in sufficient population-level immunity, significant reductions in crs, and elimination of rubella virus from several developed countries, including the united states. following successful implementation of mmr vaccination programs, cytomegalovirus (cmv) has emerged as the most common congenital viral infection in the developed world. the incidence of congenital cmv varies based on geographic region and socioeconomic status, but overall birth prevalence is estimated to be . %, which is similar to the incidence of down syndrome and fetal alcohol syndrome. in contrast to rubella virus, however, there is currently no licensed vaccine available for cmv, and with seroprevalence approaching % in some developing countries, vaccine development has been identified as a priority public healthcare goal. , while cmv infection of healthy adults is usually asymptomatic, adaptive immune responses are insufficient to clear the infection, which results in lifelong latent infection of myeloid precursor cells. although latent or reactivated cmv is less likely to cause congenital infection than a primary cmv infection during pregnancy, , preconception immunity does not completely eliminate transplacental transmission and congenital disease. moreover, pregnant women with latent cmv infection are still susceptible to primary infection with different cmv strains, which have been shown to have distinct virulence patterns. , overcoming the challenges associated with latent infections and strain variability are significant hurdles in the development of an effective cmv vaccine, and despite significant advances in our knowledge of cmv pathogenesis, the precise immune targets that constitute fetal protection remain unknown. of the several cmv vaccine candidates that have been tested, none have provided complete protection against infection, and all have failed to protect against reactivation of latent cmv. more research on the pathogenesis cmv infection is needed to define immunological correlates of protection against cmv transmission during pregnancy to inform vaccine development. although not associated with congenital disease, hepatitis b virus (hbv) is another vaccine-preventable infection that can cross the placenta during pregnancy. mother-to-child-transmission remains the most common route of infection in endemic regions, and women with active viral replication have up to a % chance of vertical transmission. of those that are infected perinatally, up to % develop chronic hbv infection. since the initial recommendation of routine hbv vaccination of children in , the rate of new hbv infections has significantly declined in the united states, but chronic hbv remains prevalent in sub-saharan africa and east asia. although combined passive and active immunoprophylaxis of infants has significantly reduced perinatal hbv infection, perinatal transmission occurs in up to % of infected mothers. to augment neonatal prophylactic strategies, the cdc acip recommends that pregnant women who are identified as being at risk for hbv infection be vaccinated with the recombinant hbv vaccine. immunity following receipt of the hbv vaccine is long-lived, with anti-hbv antibodies persisting in most adults for at least years. because of the long-term protection conferred by the hbv vaccine, immunization is not necessary for pregnant women who have already been vaccinated and are at low risk of infection. owing to the limited exposure to foreign antigen and blunted innate immune responses in utero, the neonatal immune system is immature at birth, making neonates (i.e., less than one month of age) particularly susceptible infections. infectious diseases are responsible for over % of child mortality, and over % of these deaths occur within one month of age. during the neonatal period of immune system maturation, protection against pathogens relies primarily on passive immunity from maternal-derived igg antibodies. in humans, most maternal antibodies are transferred into the fetal circulation through the placenta prior to birth, which contrasts with most veterinary species, in which maternal antibody is transferred via colostrum immediately following birth. regardless of species, vaccination during pregnancy increases circulating maternal antibodies and enhances transfer to the fetus/neonate. the goal of vaccination strategies for protecting against neonatal infections is generation of robust maternal antibody responses during pregnancy to enhance placental transfer. further, because neonatal protection is exclusively conferred by maternal-derived antibody, vaccines aimed at protecting infants should prioritize induction of humoral over cellular immune responses, with the induction of igg being most important because this igg isotype is associated with the highest placental transport efficiency in females. moreover, the kinetics of maternal vaccine-induced antibody response, the efficiency of placental antibody transfer, and the half-life of the antibody in the neonate should inform the optimal timing of vaccination during pregnancy. because the peak antibody response is typically observed - weeks following immunization, vaccination during pregnancy as opposed to before conception is likely to result in the greatest benefit to the neonate. further, the efficiency of placental antibody transfer in females increases throughout gestation, with less than % maternal igg transferred to the fetus in the first weeks of gestation, significantly more transferred during the second and third trimesters, and at delivery fetal igg often exceeds maternal levels. vaccination of females during the second and third trimesters of pregnancy is most likely to generate the greatest level of protection in the neonate, but the precise timing for maximum protection is debated. controversy over the timing of pertussis vaccination in pregnancy has been reviewed elsewhere, with some reports claiming peak cord blood antibody concentrations following vaccination in the second trimester, and others reporting peak antibody concentrations following vaccination in the third trimester. antibody avidity also influences the efficiency of placental transfer, with higher avidity antibodies crossing the placenta with greater efficiently than low avidity antibodies. more consideration should be given to the development of high avidity antibodies in the timing of vaccination during pregnancy, as protective immunity in the infant depends on both the concentration and avidity of the maternal-derived antibody. the half-life of maternal antibodies in infants also must be considered in vaccine development and administration. maternalderived igg is reported to have a half-life of approximately days in serum, and depending on serum antibody titers present at birth, this translates into protective immunity for approximately the first - months of life for most infant pathogens. the half-life of the antibody also dictates the vaccination schedules for infants, as the presence of maternalderived antibody interferes with vaccine efficacy, and it is not until maternal-derived antibody has waned below a certain threshold that an infant can mount its own active vaccine response. the goal of the infant vaccine series is to time vaccination to coincide with the time that maternal-derived antibody drops below the threshold at which it can neutralize the vaccine antigen. because the precise timing of these events is unpredictable, infant vaccination schedules are designed so that vaccines are administered in a series that spans the duration of this window, and minimize susceptibility to natural infection. in the united states, infant vaccines are recommended at , and months of age. bordetella pertussis is a vaccine-preventable respiratory pathogen of significant public health importance, and it is a major cause of mortality in infants lacking protective maternal immunity. vaccination of women during pregnancy, however, significantly enhances the transfer of maternal antibody to the fetus, , and these newborns are times more likely to have protective antibody titers at birth compared with those born from women who were not vaccinated during pregnancy. inactivated pertussis antigen is combined with tetanus and diphtheria toxoids in a single vaccine (tdap), which the cdc acip recommends for all pregnant women, regardless of previous vaccine history. in contrast to vaccine formulations that contain killed whole b. pertussis organisms, the tdap vaccine contains only select antigens and confers relatively weak and only transient protective immunity that declines after year. vaccination of women either prior to conception or during early pregnancy does not provide adequate neonatal protection against pertussis. consequently, the cdc considers the third trimester to be the optimal time to administer the tdap vaccine to pregnant women. adverse events reported following tdap vaccination are generally mild, and there are no reported risks of adverse pregnancy outcomes related to tdap vaccination during pregnancy. despite consistent evidence that supports the benefit and safety of tdap vaccination during pregnancy, coverage remains low, with an estimated % of pregnant women receiving the tdap vaccine in the united states in . receipt of the tdap vaccine during pregnancy also confers protection against neonatal tetanus, which is associated with case fatality approaching % in the absence of medical care. disease is caused by the toxin produced by clostridium tetani, and infection occurs most commonly due to contamination of the umbilical stump following delivery. consequently, the incidence of disease is much greater in developing countries, where maternal vaccination is scarce and perinatal hygiene practices are poor. in , the world health assembly called for the elimination of neonatal tetanus, which has inspired an initiative to improve vaccination coverage and birth hygiene in countries with high disease prevalence. as part of this initiative, immunization standards have been expanded and recommend that pregnant women with unknown or inadequate vaccination history receive two doses of the toxoid-containing vaccine, administered one month apart. maternal anti-tetanus antibodies are passively transferred to the fetus, and it is estimated that maternal immunization reduces neonatal tetanus mortality by %. respiratory syncytial virus (rsv) is the most common respiratory viral pathogen of newborns and infants, and accounts for - % of acute bronchiolitis and - % of pneumonia cases in hospitalized children less than years of age. rsv is also reported to cause severe disease and hospitalization in pregnant women when infection occurs during the third trimester, , and therefore dually qualifies as a maternal infection as well. a licensed vaccine against rsv is currently unavailable, but several vaccine candidates have shown promise in various animal models. [ ] [ ] [ ] [ ] given the importance of this pathogen during early life, vaccine development strategies have focused on maternal immunization, with three maternal vaccines currently in clinical trials. maternal vaccination against rsv has direct and indirect benefits to the neonate; neonates are directly protected through passive transfer of maternal antibody through the placenta, and they are indirectly protected because a vaccinated mother is less likely to transmit the infection to her infant. vaccination of pregnant women is controversial, and immunization with live (i.e., replication-competent) viral or bacterial vaccines is generally contraindicated due to the theoretical risk of congenital infection and teratogenic effects from the vaccine strains. however, in a report of over pregnant women who were unknowingly immunized with live attenuated rubella vaccine, there were no cases of vaccine-associated congenital rubella infection, and live virus strains of influenza or yellow fever viruses administered to pregnant women also have no link with pregnancy complications. , vaccination with inactivated vaccines such as influenza and tdap during pregnancy have low uptake, with concerns of safety among both patients and their healthcare providers being a primary barrier. the safety of vaccine adjuvants is debated, and although neither the tdap nor seasonal influenza vaccine recommended during pregnancy contain adjuvants, retrospective studies evaluating safety of the adjuvanted pandemic h n influenza vaccine in pregnant women found no relationship with adverse pregnancy outcomes. the conservative approach to vaccination protocols for pregnant women stems from the lack of controlled safety and efficacy studies for this population. for ethical reasons, pregnant women are exempted from almost all clinical and vaccine trials, and heath care providers are less likely to endorse prophylactic treatments for which safety and efficacy profiles have not been adequately characterized. whereas study in pregnant women is not possible, pre-clinical testing in animal models may provide a useful alternative, and vaccine preclinical trials in pregnant animal models may provide information to inform healthcare policies for pregnant women. although there are some differences in the length of gestation, placental structure, and fetal development between humans and animal models, many structural and functional parallels exist, [ ] [ ] [ ] which serve as tractable platforms for evaluating the safety and efficacy of various therapies during pregnancy. similar to humans, pregnant mice, rats, and rabbits have a hemochorial placenta, and their relatively short gestation and large litters are advantageous for performing high throughput screening of candidate therapeutics for safety and efficacy. preclinical behavioral testing of rodent offspring has proven to be a promising avenue for identifying and predicting adverse effects associated with prenatal drug exposure in children. both rodent and rabbit models have been instrumental in testing teratogenic effects of artemisinin-based combination therapies for treating malaria in pregnant women. these studies concluded that drug-related teratogenic effects are limited to the first trimester, which supports the world health organization (who) recommendation that artemisinin may be administered only during the second or third trimester in pregnant women. , one limitation of mouse and rat models, however, is their inability to recapitulate certain elements of human congenital disease. for instance, because murine cmv is not transmitted vertically as it is in humans, other animal models, including guinea pigs and nonhuman primates, are required for studying this aspect of disease pathogenesis. studies in pregnant nonhuman primates have been instrumental for the identification of cd + t cell responses as critical for early control of cmv infection and transmission during pregnancy, and studies in guinea pigs have demonstrated that a single-cycle infectious cmv vaccine induces immune responses similar to natural infection and protects against congenital infection. guinea pigs are also a useful model of chlamydial genital infection in humans. experimental venereal infection with chlamydophila caviae mimics disease associated with c. trachomatis in humans, including both sexual and perinatal transmission. guinea pigs have therefore served as a useful model for testing candidate vaccines and treatments. rabbits continue to serve as an important model of venereal infection with treponema pallidum, the causative agent of syphilis, which is associated with congenital disease in humans. while natural infection in rabbits is associated with the species-specific t. paraluiscanuculi, rabbits can be experimentally inoculated with human t. pallidum, and have been instrumental in testing the efficacy of candidate vaccines. many mammalian species, including rodents, ruminants, and nonhuman primates, , are susceptible to infection with listeria monocytogenes and demonstrate similar fetal complications when infection occurs during pregnancy. studies in various animal models have uniquely contributed to our understanding of placental listeriosis and serve as a platform for evaluating prevention strategies. , finally, mice, cotton rats, guinea pigs, and sheep are all susceptible to infection with rsv, and vaccination of pregnant animals has facilitated the development and testing of maternal immunization strategies for protecting against neonatal rsv. based on preliminary studies in guinea pigs, an experimental rsv recombinant f nanoparticle vaccine is now being evaluated in third-trimester pregnant women (clintrials.gov, nct ). beyond the direct modeling of human congenital infection in animals, information can also be gained from the study of related veterinary pathogens. for example, bovine viral diarrhea virus (bvdv) is an important reproductive pathogen that infects cattle worldwide, and infection during pregnancy causes congenital infection and disease. persistently infected animals serve as reservoirs within a herd and can have a huge agricultural financial impact. as a result, significant resources have been dedicated to the development and optimization of bvdv vaccines and vaccine protocols, considering variables such as the type and timing of vaccination on immune response and protection against challenge. the information gleaned from these studies may inform vaccine development and optimization protocols for related pathogens in pregnant women, for which similar studies cannot ethically be performed. zika virus (zikv) is a unique flavivirus that causes mild or subclinical disease in pregnant women, , but can have devastating effects for the fetus and neonate. infection during pregnancy is linked with spontaneous abortion and a variety of birth defects, including microcephaly and impaired neurocognitive function. since its initial discovery in african macaques in , zikv has expanded its geographical range and evolved into separate virus lineages, with environmental pressures resulting in the emergence of virulent substrains, raising concerns about vaccine escape mutants once a vaccine is approved. the combination of its unique pathogenesis, diverse modes of transmission, and rapid global spread has increased efforts toward development and licensing of a zikv vaccine. a basic understanding of zikv biology and pathogenesis is essential for development of an effective vaccine against zikv. although we can extrapolate some biological information from related flaviviruses, such as yellow fever, west nile, and dengue viruses, zikv has unique characteristics following in vivo infection, which pose significant challenges to vaccine design. unlike other flaviviruses, zikv has a tropism for reproductive tissues, including the testes, semen, and sperm in males , and the placenta in pregnant females, [ ] [ ] [ ] which is hypothesized to contribute to sexual and vertical modes of transmission, respectively. in addition to unique tissue tropisms, zikv persists in reproductive tissue following clearance of systemic viremia. in males, zikv rna can be detected for months following recovery from symptomatic infection, , and virus persistence in the placenta of pregnant women is hypothesized to contribute to prolonged viremia in this population. evidence of virus persistence suggests that zikv may have evolved mechanisms for evasion of host immune responses when infection occurs in certain immune-privileged tissues. scenarios involving persistent zikv infections should be considered in developing and testing candidate vaccines. considering the potential for viral persistence in the semen, vaccinating men may serve as an additional strategy to reduce transmission to the fetus, as pregnant women may be infected by their sex partners. characterization of the immune response to zikv infection is essential for determining correlates of protection for vaccine efficacy. following infection of both humans and nonhuman animals, zikv induces neutralizing antibodies against the zikv e protein, which prevent fetal infection and demise when administered to pregnant mice. further, mhc class i epitopes have been identified that conferred protection against zikv challenge in immunocompetent mice. to date, candidate zikv vaccines have been developed, and as of february , nine have entered phase i clinical trials. vaccine candidates have been developed using diverse platforms, including dna, mrna, and purified inactivated and live-attenuated virus, many of which have been tested in non-pregnant mouse and nonhuman primate models for their ability to generate immune responses that mimic responses to natural infection and protected against zikv challenge. a candidate dna plasmid vaccine induced robust cellular immunity and neutralizing antibody responses in both nonhuman primates and immunocompetent mice, and conferred complete protection against lethal zikv challenge in type i interferon receptor deficient (ifnar −/−) mice. lnp-mrna vaccines induced similar protective immunity, which was characterized by high neutralizing antibody titers and sterilizing immunity against zikv challenge in non-pregnant mice and nonhuman primates. , whether these candidate vaccines induce protective immunity in pregnant females that is sufficient to prevent fetal and neonatal infections requires further evaluation. also, whether pre-existing immunity to other flaviviruses that co-circulate with zikv, including dengue virus and west nile virus, affects the efficacy of zikv vaccines in pregnant females should be considered in both preclinical animal models and human clinical trials. conventional vaccines are formulated from either live, attenuated pathogen strains or from inactivated pathogens, but there are notable disadvantages to each of these platforms. live, attenuated vaccines are replication-competent with the potential of becoming virulent and causing adverse effects in individuals with weakened immune systems. due to the unknown risk to the developing fetus, live virus vaccines are not recommended for use in pregnant women. inactivated vaccines, on the other hand, are not associated with a risk of reacquisition of virulence, but they tend to induce a weaker host immune response. efforts to balance safety with immunogenicity have led to the development of several novel vaccine technologies, including replicationdeficient nanoparticle-based vaccines and self-assembling recombinant virus-like particles (vlps), replication-competent recombinant viral vectors, and single-cycle infectious viruses that can infect, but not replicate in host cells. nanoparticle delivery platforms, including liposomes and synthetic polymers, can be engineered to enhance selective tissue homing for high potency, targeted delivery of antigen in its native conformation. recombinant vlps combine highly immunogenic surface viral proteins with encapsulated adjuvants that are devoid of infectious nucleic acid, but induce strong cellular and humoral immune responses. both nanoparticle and vlp vaccines are devoid of genetic material and are therefore replication incompetent, enhancing safety in vulnerable individuals. although current production yields and costs associated with these technologies may prohibit large-scale use, these platforms may be well-suited for targeted use in high-risk populations, including pregnant women. since the first nanoparticle-based vaccine was licensed for hepatitis b virus in , the technology has been applied to develop licensed vaccines against human papilloma virus, hepatitis e virus, and malaria. demonstration of safety and efficacy during pregnancy has not yet been documented. recombinant viruses can be engineered to combine the antigenic genes of one virus with the structural genes of another. this targeted manipulation of the virus genome is used to remove virulence genes to enhance safety and alter envelope proteins to change cell tropism. recombinant viruses can be engineered to retain the ability to infect and replicate in the host, while preserving infectious potential and enhancing the generation of innate and adaptive immune responses that mimic natural infection. recombinant virus vaccines, however, warrant careful consideration of the safety of the vector itself, especially in pregnant women. once the safety and efficacy profiles of a viral vector platform have been established, engineering new antigenic targets into the viral genome are relatively simple, and do not require extensive re-validation, as safety and efficacy is most influenced by the vector virus. this can significantly reduce the time to develop and manufacture vaccines against new viral pathogens. viruses from many different families can be used as vectors provided they can infect the host and elicit a productive immune response without causing disease. of note, poxviruses are practical vectors due to ease of growth and manipulation in vitro, wide host range, and robust induction of protective immune responses. although vaccinia virus vectors are contraindicated during pregnancy due to risk of disseminated disease, recent testing of a raccoonpoxvirus-vectored rabies vaccine in pregnant mice proved safe and effective. other novel vaccine platforms are based on genetic engineering and creation of targeted loss-of-function mutations in the viral genome. reverse genetics technology has contributed the identification of the sequences within the viral genomes that are essential for infection and replication in vivo. it is now possible to engineer recombinant virus vaccines with targeted mutations in a cost-and time-efficient manner. in contrast to inactivated vaccines, reverse genetics allows for controlled manipulation of the viral genome that targets a specific process in the virus life cycle. this enables production of a vaccine strain with maximum efficacy and safety, and represents another promising alternative to conventional attenuated or killed virus vaccines. among the safest recombinant vaccine approaches are the single-cycle infectious (sci) viruses, which have been developed from influenza a virus (iav) backbones by deleting or truncating viral proteins necessary for completion of the virus life cycle within the host. such genetic modifications render the virus replication-incompetent, but capable of infecting and inducing an immune response in the host. the infectious capacity of sciiav vaccines results in strong cellular and humoral immune responses, without the risks and adverse effects associated with live-attenuated vaccine strains. studies in nonpregnant mice have demonstrated that a single dose of sciiav vaccine confers protection against heterosubtypic lethal challenge without any adverse effects. [ ] [ ] [ ] [ ] [ ] [ ] similar safety and efficacy profiles of sciiav have been replicated in ferret and pig models, , but studies in pregnant animals have not been performed. compared with the risks associated with live virus vaccination and concerns of efficacy with inactivated vaccines, novel vaccine platforms, such as nanoparticle-based technologies, vlps and replication-deficient viruses have proven benefits. additional safety and efficacy studies in pregnancy models are warranted to validate and expand the use of these vaccine platforms for pregnant women. (table ) conclusions strategic immunization of women, either prior to or during pregnancy, can eliminate or substantially reduce the risk of maternal, fetal, and neonatal infection and disease. the effectiveness of an immunization protocol depends on both the efficacy of the vaccine in inducing protective immune responses and on the timing of vaccine delivery during pregnancy to synchronize the peak vaccine response with the period of greatest susceptibility in the host. optimization of vaccination protocols to achieve this goal requires an understanding of the mechanisms of infection and pathogenesis of disease during pregnancy. successful implementation of vaccine protocols for pregnant women requires consideration of additional challenges, such as the frequency of unplanned pregnancies and access to prenatal health care. human surveillance data provide correlative clues of the character of specific infections, but mechanistic understanding requires additional study in comparative animal model systems. pregnancy and pregnancy-associated hormones alter immune responses and disease pathogenesis incidence and severity of viral hepatitis in pregnancy clinical course and outcome of sporadic acute viral hepatitis in pregnancy hepatitis e virus chickenpox pneumonia: an association with pregnancy h n influenza virus infection during pregnancy in the usa maternal influenza infection causes marked behavioral and pharmacological changes in the offspring maternal influenza infection is likely to alter fetal brain development indirectly: the virus is not detected in the fetus australasian maternity outcomes surveillance, s. critical illness due to a/h n influenza in pregnant and postpartum women: population based cohort study impaired type i and iii interferon response to rhinovirus infection during pregnancy and asthma report of a controlled study during an outbreak of asian influenza antibody response to monovalent a/new jersey/ / influenza vaccine in pregnant women influenza vaccination of pregnant women protects them over two consecutive influenza seasons in a randomized controlled trial seropositivity of influenza a h ni in mothers and infants following maternal vaccination with trivalent seasonal influenza vaccine after the pandemic immunogenicity of trivalent inactivated influenza vaccination received during pregnancy or postpartum prevention and control of influenza. recommendations of the advisory committee on immunization practices (acip) adverse events in pregnant women following administration of trivalent inactivated influenza vaccine and live attenuated influenza vaccine in the vaccine adverse event reporting system trivalent inactivated influenza vaccine and spontaneous abortion monovalent h n influenza vaccine safety in pregnant women, risks for acute adverse events inactivated influenza vaccine during pregnancy and risks for adverse obstetric events maternal outcomes among pregnant women receiving live attenuated influenza vaccine & prevention. influenza vaccination coverage among pregnant women ---united states, - influenza season maternal influenza vaccination: evaluation of a patient-centered pamphlet designed to increase uptake in pregnancy varicella-zoster virus (chickenpox) infection in pregnancy seroprevalence of varicella in the french population centers for disease control and prevention pathogenesis and current approaches to control of varicella-zoster virus infections what does epidemiology tell us about risk factors for herpes zoster? hepatitis e and pregnancy: current state vertical transmission of hepatitis e virus sero-prevalence and mother-to-infant transmission of hepatitis e virus among pregnant women in the united arab emirates fetal and neonatal health consequences of vertically transmitted hepatitis e virus infection efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase trial lessons from hepatitis e vaccine design enhanced humoral response in pregnant mice immunized with liposome encapsulated recombinant neutralizing epitope protein of hepatitis-e virus risks associated with viral infections during pregnancy evaluation of pregnant women exposed to respiratory viruses do the placental barrier, parasite genotype and toll-like receptor polymorphisms contribute to the course of primary infection with various toxoplasma gondii genotypes in pregnant women? listeriosis in human pregnancy: a systematic review acute fetal demise with first trimester maternal infection resulting from listeria monocytogenes in a nonhuman primate model general recommendations on immunization ---recommendations of the advisory committee on immunization practices (acip) impact of birth rate, seasonality and transmission rate on minimum levels of coverage needed for rubella vaccination prevention of measles, rubella, congenital rubella syndrome, and mumps, : summary recommendations of the advisory committee on immunization practices (acip) surveillance for congenital rubella in australia since : cases reported between & prevention. elimination of rubella and congenital rubella syndrome--united states the "silent" global burden of congenital cytomegalovirus review and meta-analysis of the epidemiology of congenital cytomegalovirus (cmv) infection seroprevalence of cytomegalovirus among children to years of age in the united states from the national health and nutrition examination survey of vaccine development to prevent cytomegalovirus disease: report from the national vaccine advisory committee desirability and feasibility of a vaccine against cytomegalovirus latency and reactivation of human cytomegalovirus maternal immunity and prevention of congenital cytomegalovirus infection human cytomegalovirus (hcmv) replication dynamics in hcmv-naive and -experienced immunocompromised hosts strain variation and disease severity in congenital cytomegalovirus infection: in search of a viral marker cytomegalovirus (cmv)-encoded ul (truncated tumor necrosis factor receptor) and outcome of congenital cmv infection prospects of a vaccine for the prevention of congenital cytomegalovirus disease management of hepatitis b virus infection during pregnancy hepatitis b immunisation for newborn infants of hepatitis b surface antigen-positive mothers cesarean section to prevent motherto-child transmission of hepatitis b virus in china: a meta-analysis a comprehensive immunization strategy to eliminate transmission of hepatitis b virus infection in the united states: recommendations of the advisory committee on immunization practices (acip) part ii: immunization of adults persistence of antibodies y after vaccination with a combined hepatitis a and b vaccine role of innate host defenses in susceptibility to early-onset neonatal sepsis global, regional, and national causes of child mortality: an updated systematic analysis for with time trends since evolution of maternofetal transport of immunoglobulins during human pregnancy dynamics of immunoglobulins at the feto-maternal interface placental transfer of immunoglobulin g subclasses placental transfer of antibody and its relationship to vaccination in pregnancy placental transfer favours high avidity igg antibodies half-life of the maternal igg allotype in infants decay of passively acquired maternal antibodies against measles, mumps, and rubella viruses maternal immunization with tetanusdiphtheria-pertussis vaccine: effect on maternal and neonatal serum antibody levels effect of a prepregnancy pertussis booster dose on maternal antibody titers in young infants updated recommendations for use of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (tdap) in pregnant women--advisory committee on immunization practices (acip), . mmwr immunogenicity and reactogenicity of co-administered tetanus-diphtheria-acellular pertussis (tdap) and tetravalent meningococcal conjugate (mcv ) vaccines compared to their separate administration importance of timing of maternal combined tetanus, diphtheria, and acellular pertussis (tdap) immunization and protection of young infants infant outcomes after exposure to tdap vaccine in pregnancy: an observational study coverage with tetanus, diphtheria, and acellular pertussis vaccine and influenza vaccine among pregnant women -minnesota neonatal tetanus: a continuing challenge maternal and neonatal tetanus weekly epidemiological record tetanus toxoid immunization to reduce mortality from neonatal tetanus respiratory syncytial virus and parainfluenza virus maternal effects of respiratory syncytial virus infection during pregnancy clinical presentation and birth outcomes associated with respiratory syncytial virus infection in pregnancy maternal antibodies by passive immunization with formalin inactivated respiratory syncytial virus confer protection without vaccineenhanced disease preclinical assessment of safety of maternal vaccination against respiratory syncytial virus (rsv) in cotton rats maternal immunization with respiratory syncytial virus fusion protein formulated with a novel combination adjuvant provides protection from rsv in newborn lambs modeling maternal fetal rsv f vaccine induced antibody transfer in guinea pigs burden of paediatric respiratory syncytial virus disease and potential effect of different immunisation strategies: a modelling and cost-effectiveness analysis for england strategic priorities for respiratory syncytial virus (rsv) vaccine development pregnancy outcomes following rubella vaccination: a prospective study in the state of rio de janeiro, brazil & campinas group on yellow fever immunization during, p. the effects of yellow fever immunization ( dd) inadvertently used in early pregnancy during a mass campaign in brazil should expectant mothers be vaccinated against flu? a safety review comparative developmental anatomy of the murine and human definitive placentae comparative systems biology of human and mouse as a tool to guide the modeling of human placental pathology animal models to study placental development and function throughout normal and dysfunctional human pregnancy risk mitigation for children exposed to drugs during gestation: a critical role for animal preclinical behavioral testing antimalarial drugs in pregnancy: a review embryotoxicity of the artemisinin antimalarials and potential consequences for use in women in the first trimester maternal cd +t cells protect against severe congenital cytomegalovirus disease in a novel nonhuman primate model of placental cytomegalovirus transmission a novel non-replication-competent cytomegalovirus capsid mutant vaccine strategy is effective in reducing congenital infection the guinea pig as a model of infectious diseases syphilis: using modern approaches to understand an old disease listeriosis in the pregnant guinea pig: a model of vertical transmission experimental infection of pregnant ewes with listeria monocytogenes nonhuman primate model for listeria monocytogenesinduced stillbirths understanding how listeria monocytogenes targets and crosses host barriers conjugated action of two species-specific invasion proteins for fetoplacental listeriosis modelling the spread of bovine viral diarrhea virus (bvdv) in a beef cattle herd and its impact on herd productivity efficacy of bovine viral diarrhea virus vaccination to prevent reproductive disease: a meta-analysis zika virus outbreak on yap island, federated states of micronesia rapid spread of emerging zika virus in the pacific area congenital zika virus syndrome in brazil: a case series of the first livebirths with complete investigation mutational pressure in zika virus: local adar-editing areas associated with pauses in translation and replication zika virus in semen and spermatozoa zika virus infection damages the testes in mice intrauterine zika virus infection of pregnant immunocompetent mice models transplacental transmission and adverse perinatal outcomes zika virus infects human placental macrophages zika virus targets different primary human placental cells, suggesting two routes for vertical transmission presence and persistence of zika virus rna in semen potential sexual transmission of zika virus zika virus rna replication and persistence in brain and placental tissue neutralizing human antibodies prevent zika virus replication and fetal disease in mice analysis of the t cell response to zika virus and identification of a novel cd +t cell epitope in immunocompetent mice world health, o. who vaccine pipeline tracker in vivo protection against zikv infection and pathogenesis through passive antibody transfer and active immunisation with a prmenv dnavaccine modified mrna vaccines protect against zika virus infection zika virus protection by a single low-dose nucleoside-modified mrna vaccination a dtap-ipv//prp approximately t vaccine (pentaxim): a review of years' clinical experience beyond antigens and adjuvants: formulating future vaccines harnessing nanoparticles for immunomodulation and vaccines novel vaccine strategies against emerging viruses raccoonpoxvirus safety in immunocompromised and pregnant mouse models replication-defective viruses as vaccines and vaccine vectors reverse genetics approaches for the development of influenza vaccines protection against lethal influenza with a viral mimic a replication-incompetent virus possessing an uncleavable hemagglutinin as an influenza vaccine pseudotyped influenza a virus as a vaccine for the induction of heterotypic immunity pb amino acid at position affects replicative efficiency, but not cell tropism, of hong kong h n influenza a viruses in mice a novel bivalent vaccine based on a pb -knockout influenza virus protects mice from pandemic h n and highly pathogenic h n virus challenges a replicationincompetent pb -knockout influenza a virus vaccine vector an eight-segment swine influenza virus harboring h and h hemagglutinins is attenuated and protective against h n and h n subtypes in pigs pertussis antibody transfer to preterm neonates after second-versus third-trimester maternal immunization chlamydia trachomatis infection in pregnancy: the global challenge of preventing adverse pregnancy and infant outcomes in sub-saharan africa and asia perinatal infections and fetal/neonatal brain injury prevention of perinatal group b streptococcal disease. revised guidelines from cdc microbial vertical transmission during human pregnancy current perspectives on prevention of mother-to-child transmission of syphilis syphilis infection during pregnancy: fetal risks and clinical management diagnosing congenital malaria in a high-transmission setting: clinical relevance and usefulness of p. falciparum hrp -based testing treating severe malaria in pregnancy: a review of the evidence toxoplasmosis in pregnancy assessment of laboratory methods used in the diagnosis of congenital toxoplasmosis after maternal treatment with spiramycin in pregnancy neuropathogenesis of congenital cytomegalovirus infection: disease mechanisms and prospects for intervention congenital cytomegalovirus infection following first trimester maternal infection: symptoms at birth and outcome cytomegalovirus neutralization by hyperimmune and standard intravenous immunoglobulin preparations what obstetrician-gynecologists should know about ebola: a perspective from the centers for disease control and prevention ebola viral disease and pregnancy live neonates born to mothers with ebola virus disease: a review of the literature herpes simplex virus and epstein-barr virus infections in pregnancy: consequences of neonatal or intrauterine infection prospects and perspectives for development of a vaccine against herpes simplex virus infections incident hiv during pregnancy and postpartum and risk of mother-to-child hiv transmission: a systematic review and meta-analysis immunology of pediatric hiv infection parvovirus b infection in human pregnancy consequences of confirmed maternal rubella at successive stages of pregnancy severe acute respiratory syndrome (sars) in neonates and children sars during pregnancy, united states microbiology laboratory and the management of mother-child varicella-zoster virus infection initial description of the presumed congenital zika syndrome the authors thank the johns hopkins fisher center discovery program for funding. all authors (m.s.v. and s.l.k.) were involved in the conception and drafting of this review, and approved the manuscript before it was submitted by the corresponding author. competing interests: the authors declare no competing financial interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- - zrnfaad authors: giese, matthias title: types of recombinant vaccines date: - - journal: introduction to molecular vaccinology doi: . / - - - - _ sha: doc_id: cord_uid: zrnfaad the original scientific strategy behind vaccinology has historically been to “isolate, inactivate, and inject,” first invoked by louis pasteur. new vaccines and vaccination strategies are being developed including the use of attenuated live mycobacteria, recombinant microorganisms, and subunits, prime-boost strategies based on the successive administration of a certain mycobacterial antigen under two different vaccine vectors, and dna vaccines [ ] . the various types of vaccines differ in eliciting an immune response. live attenuated vaccines (lavs) mimic a natural infection without being virulent and trigger the activation of the innate immune system through pamps. following injections, lavs rapidly disseminate throughout the vascular network to the draining lymph nodes. therefore, the route of application of lavs does not specifi cally infl uence the immune response. lavs also don't need an adjuvant; they possess a natural intrinsic adjuvancy. safety concerns exist because of the replication competence and the possibility of recombination with a wild type. non-live vaccines, inactivated and most recombinant vaccines, whether containing proteins or carbohydrates (−conjugates), are less effective. in the absence of replication, vaccine-induced immune reactions remain more limited, and therefore the route of vaccination infl uences the effi cacy and the duration of the immune reaction. nonlive vaccines induce a lower antibody response and generally no cytotoxic t lymphocyte activation. compared to lav, all non-live vaccines are regarded as biologically safe ( fig. . ). dna vaccines entail the direct, in situ inoculation of dnabased eukaryotic expression vectors that encode the sequence of a pathogenic protein antigen. the constructed plasmids are then subsequently grown in bacteria like e. coli and highly purifi ed via chromatographic methods. lps contamination of plasmids has to be prevented because of the immunotoxic properties of natural lps. after purifi cation the circular double-stranded dna plasmids are ready for vaccination. the de novo production of the encoded antigens in the host results in the elicitation of both the antibody and the cellular response by activating cytotoxic t lymphocytes (ctls). vaccine proteins made by the host are natural proteins and contain important posttranslational modifi cations such as the correct glycosylation. but like subunit vaccines, dna vaccines must be adjuvanted. naked dna does not work. the unique advantage of dna vaccines is their ability to mimic the effects of live attenuated vaccines without the risk associated with the administration of infectious albeit attenuated material. dna vaccines are able to stimulate a complete, humoral and cellular immune response. peptide fragments are processed via the endogenous pathway, resulting in the presentation of antigen on the cell surface by mhc class i molecules. plasmid dna is very stable also beyond a cold chain. therefore, the storage, transportation, and distribution of dna vaccines are more practical and also cheaper [ ] . mostly all plasmid dna constructs ( fig. . ) used for vaccination share fi ve main characteristics: • strong promoter/enhancer sequence for driving the incorporated foreign gene • convenient cloning site for insertion of foreign genes • origin of replication for initiation of plasmid replication • polyadenylation/termination sequence for production of mature mrna • resistance/antibiotic marker for selection • immunomodulators, e.g., cpgs, interleukins, ubiquitin, etc. • (on the same plasmid or on extra plasmids) uptake of plasmid dna. some biological barriers have to be overcome by dna vaccines on the way to the cell nucleus where the plasmid dna is translated into cellular mrna. after delivery of plasmid dna to the target tissue, e.g., skeletal muscle or skin, lots of tissue nucleases attack . dna vaccines and degrade a large amount of the applicated dna. also the extracellular matrix with collagen and hyaluronic acid infl uences the passage from the application site to the cell membrane. only a small portion ( % estimated) of the still intact plasmid dna will cross the cell membrane by phagocytosis or pinocytosis. inside the cell the route toward the nucleus is also spiked with exo-and endonucleases so that probably only . % (estimated) is successfully and actively transported through the nucleus pore membrane (npc). small particles (<~ kda) are able to pass through the nuclear pore complex (npc) by passive diffusion; larger particles need the support of carrier proteins for effi cient passage through the complex. because of this enormous loss of plasmid dna (up to . %), various tools were developed to protect the plasmid dna and thus increase the effi cacy such as encapsulation into liposomes or binding of dna to dendrimers. figure . illustrates the passage of plasmid dna from the extracellular matrix (ecm) to the nucleus. whereas in human medicine clinical trials with dna vaccines are still ongoing without any registered product on the market, the fi rst approved dna vaccines for the veterinarian medicine are available since and are discussed now. the fi rst veterinarian dna vaccines were developed for horses (davis b.s., for wnv [ ] ; giese m., for eav [ ] ). today the number of current clinical trials worldwide with veterinary dna vaccines is unmanageable and probably all species are hit. canine malignant melanoma (cmm) typically begins in the mouth or around the toes and can spread within the body to the heart, lungs, intestines, and other organs. canine malignant melanoma is known for being one of the most aggressive cancers in dogs and deadly. cmm is most commonly seen in golden retrievers, scottish terriers, dachshunds, labradors, and poodles ( fig. . ). metastases of the tumors will be found very often in distant parts of the body. the overall biology of cmm is similar to the biology of human melanoma. however the melanomas in dogs have diverse biologic behaviors due to the race and a variety of factors. standardized treatments such as surgery, radiation, and chemotherapy are the common tools to fi ght canine malignant melanoma. these traditional tools have afforded minimal to modest stage-dependent clinical benefi ts. xenogeneic dna vaccine. the plasmid dna contains a cdna for the human tyrosinase, hutyr, a tumor antigen (ta). this is a non-mutated differentiation antigen and specifi c to melanoma. tyrosinase is a glycoprotein and essential in the process of melanin synthesis ( fig. . ). like other tas tyrosinase is overexpressed in tumor cells and therefore an ideal target in cancer therapy. normally there is no strong immune reaction against the body's own protein. but immunization of dogs with xenogeneic hutyr cdna can break the immune tolerance against this self tumor differentiation antigen and induce antibody and cytotoxic t cell response against melanoma cells [ ] . tyrosinase is highly conserved from dog to mouse to man. radiotherapy in cases with positive surgical margins or positive regional lymph nodes. one dose contains μg dna given in a volume of . ml by the transdermal route via a needle-free vaccination device. booster immunizations were given at -month intervals. in march the drug manufacturer received a conditional license for oncept from the usda and a full license in . the results of the xenogeneic immunization of dogs with hutyr cdna as an adjunct therapy for cmm demonstrate a signifi cant increase of survival time compared to the control group. none of the dogs developed systemic adverse reactions; no toxicity was seen. the overall safety of this dna vaccine is confi rmed. this vaccine development represents a tremendous milestone in dna science and technology. virus. west nile virus (wnv) is a mosquito-borne member of the family flaviviridae , genus flavivirus , and was fi rst identifi ed in in uganda, africa. it is a positive-sense, single-strand rna virus, (+)ssrna, of about kb that encodes a single polyprotein with seven nonstructural proteins and three structural proteins. the rna strand is held within a nucleocapsid. wnv replicates in the cytoplasm of infected cells. wnv is a zoonotic virus. the primary reservoir is birds with a signifi cant impact to spread the infection across countries and continents. more than different species are described as carrier of this virus. wnv is spread from bird to bird by mosquitoes when they bite, or take a blood meal, from birds that are infected with the virus. birds from some species get ill and die; others have no clinical signs and survive. mosquitoes are also capable of spreading the virus to horses, dogs, cats, mice, alligators, and lots of other mammals but also to humans. one-third of all horses bitten by carrier mosquitoes develop the disease and die or are so affected that euthanasia is required. the incubation period ranges from to days. horses that do become ill vary in symptoms: muscle trembling, skin twitching, ataxia, sleepiness, dullness, and listlessness. wnv may cross the placenta from mother to gestating foal. horses cannot spread the disease to humans. wnv produces different outcomes in humans like in horses: fever, headache, chills, diaphoresis, weakness, swollen lymph nodes, drowsiness, and pain in the joints comparable to symptoms of infl uenza. more severe neuroinvasive infection includes meningitis and encephalitis. wnv-dna vaccine. the surface envelope protein e is the main target for the antibody response. there are more than copies of the e protein in a mature wnv virion. the e function is the interaction between the cell surface and the fusion between virus and cellular membrane. the premembrane protein prm is cleaved during viral maturation into a smaller membrane m peptide. the expression of prm and e protein in cells results in the formation of virus-like particles, vlp. these vlp share many of the antigenic and structural properties of fully mature viruses and are of special interest for a vaccine development ( fig. . ) . the fi nal expression plasmid for immunization of horses contains the human cytomegalovirus early gene promoter, signal sequences from japanese virus, and a fusion gene of part of the fi shing industry is aquaculture, also known as aqua farming, but it can be contrasted with commercial fi shing, which is the harvesting of wild fi sh. aquaculture involves cultivating freshwater and saltwater fi sh and other populations (shrimp, oyster) under controlled conditions. salmon is one of the main food-producing fi sh in the world. a dna vaccine for fi sh must be not only safe for the animal but especially safe for the fi sh consumer. salmon is the major economic contributor to the world production of farmed fi sh, representing over u$ billion annually in the united states. salmon farming is also very big in norway, scotland, canada, and chile and is the source for most salmon consumed in the united states and europe. like all other animals also fi sh is threatened by viruses, bacteria, and parasites. one major problem for salmons is the infectious hematopoietic necrosis (ihn) virus [ ] . virus. infectious hematopoietic necrosis (ihn) virus is a common viral pathogen of both wild and farmed salmonids, in particular pacifi c salmonids, rainbow trout, and atlantic salmon. ihn virus is enzootic to the pacifi c northwest; however it has varying effects on different pacifi c salmonids. it is a negative-sense single-stranded, (−)ssrna virus that is a member of the rhabdoviridae family, genus novirhabdovirus . the rna genome is , nucleotides long and contains a leader (l) and trailer (t) sequences at its ′-end and ′-end, respectively. the coding regions are n, p, m, g, nv, and l genes. g encodes the surface glycoprotein, so-called spikes, main target for the immune response. transmission. ihnv is transmitted following shedding of the virus in the feces, urine, sexual fl uids, and external mucus and by direct contact or close contact with surrounding contaminated water. the virus gains entry into fi sh at the base of the fi ns. salmons are carnivorous and are currently fed a meal produced from catching other wild fi sh and other marine organisms -a permanent origin of possible infections with ihnv. clinical signs of infection with ihnv include anemia, skin darkening, bulging of the eyes, fading of the gills, and abdominal distension. infected fi sh commonly hemorrhage in several areas, like the mouth, the pectoral fi ns, muscles near the anus, and the yolk sac of fry. diseased fi sh weaken, eventually fl oating on the surface of the water. necrosis is common in the kidney and spleen and sometimes in the liver. mortality rates in older fi sh ( - kg) tend to range from to %; in smolts the mortality rate often exceeds %. the average cumulative mortality following an outbreak is estimated at %. ihnv-dna vaccine. the antigen is the viral surface glycoprotein (g) capable of eliciting neutralizing antibody and the production of a protective immune response. the g gene was cloned into a eukaryotic expression vector by insertion of an intermediate-early promoter and a polyadenylation signal. but the speciality of this vaccine is to be prepared as a two-component vaccine in a single vaccine, one plasmid or more. the second component is a portion of the nucleic acid sequence encoding a second peptide, derived from a fi sh pathogen other than the said rhabdovirus resulting in a fusion. this second pathogen can be any fi sh pathogen, e.g., isav, ipnv, iridovirus, nnv, spdv, svcv, vhsv, koi herpes virus, and more. the rationale behind this is that the presence of the ihnv g protein boosts the immune response to the second protein, resulting in a protective effect against infection by this fi sh pathogen. the vaccine is given intramuscularly with a dosage of only μg in μl on the left dorsal fl ank, in the area just below the dorsal fi n [ , ] . this fi rst dna fi sh vaccine was licensed in in canada by the veterinary biologics section (vbs), animal health and production division, canadian food inspection agency (cfia) and is also used now in studies in norway. there are many environmental stressors and diseases which infl uence and seriously threat the life of european honey bees, apis mellifera. the european honey bee is professionally managed worldwide for honey production and pollination. the bee was imported to the united states years ago with the fi rst european settlers and called "white man's fl y" by the native americans, the indians. first reported in the united states, a mysterious socalled colony collapse disorder (ccd) decimated the bee colonies there between and %, fi rst observed during the winters of - and then - and without interruptions up to now. a similar situation is also given in europe. about , - , bees live in a colony. the fi rst description originated from the s. in the early nineteenth century, the colony losses were known in england as "isle of wight disease," and the americans called this phenomenon "disappearing disease" in the s, whereas these colony losses in france in the late s were called "mysterious bee losses." where have all the bees gone? economic value. the huge loss of honey bees as pollinators has a dramatic impact on agricultural pollination. about crops, nuts, fruits, and vegetables are pollinated by a. mellifera , with an overall value of more than $ billion in the united states and more than € billion for the eu in . a bee colony produces some kg/ lb honey per day. in return, these bees have to pollinate - million fl owers. one should keep in mind that besides european honey bees, wild insects, among them , species of wild bees, have also a very great impact on pollination and seem to be more effi cient in pollination as managed honey bees [ ] . the industrial farming threatens also the natural biotope of wild insect pollinators. ecologic value. the total global economic value of honey bee pollination was calculated in to more than € billion or $ billion. the food and agriculture organization (fao) of the united nations estimates that there are million managed honey bee colonies worldwide. beside this professional agriculture, honey bees are irreplaceable for the biodiversity. this organism appeared during evolution with the fi rst fl ower plants and exists since million years as described in chap. , fig. . . after swine and cattle, bees are in europe and north america the third important farm animal and since formally listed as farm animal in switzerland. therefore, ccd is not only an economical but also an essential global ecological problem which urgently must be solved in the future. "the bee is more than honey." what is causing ccd? the colony collapse disorder of the last years seems to differ from past outbreaks: the worker bees disappear instead of dying in place, leaving behind the queen and young bees. high levels of bacteria, viruses, and fungi are measured in the gut of the remaining bees. collapses can occur within days. a complex problem. different theories are discussed about what is causing ccd. pesticide contamination, hotly debated to interfere with the nerve system affecting foraging behavior of bees, lead them to abandon their hives. fungal diseases such as nosema spp. is known for big bee losses in spain. monocultures or gene-manipulated crops. electro smoke (radio waves) caused by cell phones destroys the bee's compass. the rigors of travelling in trucks from crop to crop in the usa. down from february professional us beekeepers travel with their colonies through the country until december. thereby the bees must relocate up to times. in europe the bee colonies begin the winter sleep around september. also the climate change, the temperature sensitivity is discussed to have an impact on crop pollination. ccd is likely caused by a combination of factors [ , ] . varroa destructor . but in all ccd cases, an overload of bloodsucking varroa mites is detectable and varroa is currently considered the major threat for apiculture. the infection and disease is called varroosis. varroa destructor is an ectoparasite, has a reddish-brown fl at shape, and is - . mm long and . - mm wide, with eight legs. v. destructor infest worker bees and drones and its brood. the mite develops inside the brood cells. varroa is a real colossus compared to the size of bees as can be seen in fig. . . varroa mites belong to the scientifi c class of arachnida, subclass acari. there are , species described alone from mites. some mites prefer carbohydrates as food such as meal or crops. the house dust mites feed fl akes of shed human skin. varroa mites prefer fresh "blood" and the hemolymph of bees and can feed . mg/ . lb within h. varroa is transported into the hives via piggyback by worker bees. the female mite enters broad cells, preferentially drone cells. once the cell is capped, varroa lays eggs on the larvae. the development from egg to insect takes days. bee larvae and mites hatch in about the same time and the newborn varroa mites spread to other bees [ , ] . the lifetime of summer mites are - weeks, whereas fall mites can live for several months. varroa can only reproduce in honey bees and thus are considered harmless to other insects. varroa is more than a disease. it is a global pest having devastating effects on bees ( fig. . ). varroa as vector. varroa may be not considered as isolated agent for the disease. the mortality of adult bees and its brood must be considered in the context with secondary viral infections. at least various viruses are able to infect honey bees, mostly ssrna viruses. eight viruses are known to be associated with varroa mites: acute bee paralysis virus (abpv), black queen cell virus (bqcv), chronic bee paralysis virus (cbpv), deformed wing virus (dwv), kashmir bee virus (kbv), sacbrood bee virus (sbv), cloudy wing virus (cwv), and slow bee paralysis virus (sbpv) [ - ] . varroa control. a number of natural and synthetic chemicals are commercially available for the control of varroa infestations. the fi rst compounds were bromopropylate, fl uvalinate, or other pyrethroid insecticides. and to make a long story short, varroa mites became resistant not only against one product of a given chemical class; the resistance was against the entire class with several related synthetic products. also the use of natural products, such as formic acid, mineral oil, or thymol, is only partially and temporally effective and show adverse effects [ ] . there is no successful chemical treatment. mites will quickly develop resistance to all chemicals. the immune system of insects. the basic difference between insect and vertebrate immunity is the missing highly specifi c antigen response of the acquired immune system in insects. nevertheless, in the million years of evolution, insects developed a powerful defense strategy against bacteria, fungi, viruses, and parasites. only protected by this "primitive" immune system insects were so successful that they colonized all terrestrial ecosystems. the insect innate immunity shows many similarities to the vertebrate and to the human innate immunity, is multifaceted, and involves both humoral and cellular components [ ] . most insights on insect immunity are provided by drosophila melanogaster research. the key mechanism is also observed in honey bees. the humoral and systemic response to bacterial and fungal infections is controlled by antimicrobial peptides (amps). there are circulating receptors sensing a danger signal and activating the toll pathway, whereas membranebound receptors activate the imd pathway. both pathways lead to the translocation of nf-kb-like transcription factors and the production of amps. nf-kb response elements can be detected in the promoter region of the diptericin gene. the cellular immune response is mediated by specialized blood cells, the hemocytes, plasmatocytes, crystal cells, and lamellocytes [ ] . plasmatocytes represent % of the majority of hemocytes. they express phagocytic receptors and patrol through the body, clear microorganism and cell a b c debris, and signal infections to the fat bodies. the bee genome was completely sequenced in [ ] . the bee dna vaccine. an expression plasmid was constructed with a cmv promoter. surprisingly, no bee or other insect specifi c promoter was essential to drive the expression of the protein. the enhanced green fl uorescent protein (egfp) was chosen as reporter gene and inserted into the multiple cloning site, together with an sv enhancer element. the plasmid construct was produced in e. coli and highly purifi ed by standard techniques. european honey bees ( apis mellifera ) were obtained from local beekeepers and cultivated under lab conditions. varroa mites were collected from infested bees. the oral vaccination of the egfp plasmid was operated by feeding the bees with a mixed solution of sugar and plasmid dna (vaccine sugar). standard sugar solutions made by the beekeeper are the normal food for winter bees. results. over days after onset of feeding, we measured the expression of egfp by immunofl uorescence and western blot analysis with egfp antibodies. between day and , a clear egfp signal was detected in the thorax and especially in the malpighian tubules. control bees fed with dna lacking the reporter did not show any signal. in parallel, control experiments with transformed e. coli were done to study the possibility of egfp expression in gut bacteria instead of bee cells. no egfp signal was detected in transformed bacteria. most surprisingly, we found the egfp signal after days in varroa mites sucking hemolymph of bees which were fed by the vaccine sugar solution and no signals in control mites of infested control bees. feeding of plasmid dna results in expression of a reporter gene in different bee tissues over a period of several days, and fi nally varroa absorbs this protein via bloodsucking. the bee blood is not carried by arteries and veins but fl ows loosely around the body. no egfp signals were detected either in the honey stomach or in the feces. figure . illustrates the egfp passage through the bee body and toward the varroa mite. we started with the simple idea that the biochemistry in eukaryotic cells remains the same, irrespective of the organism. a difference is given in the confi guration of the immune system. that means, an insect can successfully fi ght against parasites and infections but with different weapons. no t cells, no b cells, and consequently no antibodies and no memory. we are able to stimulate targeted immune genes of bees and measure an insect typical immune response. a standard plasmid dna vaccine, fi rst developed for horses, bridges the evolution from fi sh to insects to mammals. no other vaccine type is able to do this job. how fascinating biology is! a protein subunit is based on a single protein molecule and able to stimulate a humoral immune response, but usually not a cellular response. after phagocytosis proteins are degraded by acid-dependent proteases in endosomes (endosomal or exogenous pathway), resulting in an mhc ii presentation of the antigenic peptides. a peptide is one form of a subunit. carbohydrates are also used as subunits with a poor and age-dependent immunogenicity. carbohydrate antigens induce a t cell-independent b cell response as discussed in chap. . therefore carbohydrates are mainly linked to a protein (conjugation) to enhance toe immune reaction as discussed here with the hib conjugate vaccine. conjugate vaccines. the polyribosylribitol phosphate (prp) capsule of haemophilus infl uenzae type b (hib) is a major virulence factor for the organism. prp is a t cellindependent antigen characterized by, e.g., induction of a poor antibody response in less than -month-old infants and children and the inability to induce a booster response. polysaccharide vaccines based on prp alone were developed in the s. by covalent linkage of prp with t cell dependent protein antigens, a conjugated vaccine was created to overcome the t cell independent characteristics of prp. at present three different licensed protein carriers are linked to prp: • hboc: diphtheria crm protein , mutant corynebacterium -linkage: no spacer • hbomp: outer membrane protein, omp, neisseria meningitidis -linkage: spacer • prp-d: diphtheria toxoid, d -linkage: spacer these hib conjugate vaccines differ by protein carrier, polysaccharide size, and method of chemical conjugation, including use of a spacer between the prp and protein carrier. a standard chemical conjugation between a polysaccharide and a protein is illustrated in fig. . . subunit vaccines, while offering greater safety, are intrinsically poorly immunogenic and strong adjuvants are essential to boost the activation of immune responses. serotype variability is dictated by modifi cations of the o-antigen portion of lps. o antigens vary in the number of oligosaccharide unit repeats, the types and distribution of carbohydrates, and the intra-and intermolecular linkages [ ] . in s. fl exneri , these genes are encoded in the bacterial chromosome. in contrast, s. sonnei , which shows no serotype variability, expresses plasmid-encoded o-antigen modifi cation enzymes. the o antigen is one of the major immunogenic components of shigella and is a virulence factor, in part, due to masking the exposure of type three secretion apparatus [ ] . the inclusion of conserved proteins in vaccine compounds potentially solves the issue of serotype specifi city, thus allowing the generation of a highly desirable pan-shigella vaccine. in addition, recombinant proteins usually have increased safety profi les. another important impact in shigella epidemiology that prompts vaccine development is the increasing frequency of antibiotic-resistant strains. antibiotic resistance is continually rising for this pathogen [ ] . shigella spp. as causative agent of shigellosis. first defi ned as a causative agent of bacillary dysentery by shiga in japan, shigella is a gram-negative bacillus that is noncapsulated and nonmotile. diagnosis is generally based on symptoms [ ] since bloody, mucoid stools are indicative of shigella infections. however, because several diarrheal infections caused by other microorganisms share these symptoms (enteroinvasive e. coli and campylobacter , among others), the sole analysis of symptoms is insuffi cient for an accurate diagnosis. therefore, clinical diagnosis must be complemented with microbiological isolation from culture. shigella invasion and pathogenesis. shigella is transmitted through the fecal-oral route by consumption of contaminated food and water. following ingestion, the acid-tolerant shigella passes through the stomach and small intestine into the large intestine [ ] (fig. . ). here, they are taken up by m cells, transcytosed to the basolateral face of the colonic epithelium, and presented to resident macrophages wherein ipab of the type three secretion system (t ss) induces apoptosis by caspase activation, thereby escaping killing by the macrophage [ ] . shigella then invades epithelial cells using its t ss to create a translocation pore in the host cell membrane to initiate an orchestrated fl ow of effectors into the host cell cytoplasm to induce actin rearrangements that ultimately result in uptake of bacteria. once inside, shigella quickly escapes its vacuole, replicates, and moves about the cytoplasm via actin-based motility. in a t ssdependent manner, the shigella then forms a protrusion into a neighboring uninfected cell with the resulting vacuole being quickly lysed to complete the process of intercellular spread. the genes associated with the t ss are encoded on a -kb plasmid which is highly conserved among the shigella species. at the heart of the t ss is the type three secretion apparatus (t sa) which is composed of a basal body similar to that of fl agellar systems and an extracellular needle [ ] . invasion plasmid antigen d (ipad) is a kda protein that forms a pentameric ring at the tip of the needle. it controls secretion of effector proteins and is the environmental sensor for mobilization of ipab to the t sa tip complex. ipab is a kda translocator that forms a ring atop the ipad ring and is responsible for host cell contact. this contact is required for mobilization of ipac to the needle tip and formation of a complete unidirectional conduit from the bacterial cytoplasm to the host cell cytoplasm. the initiation of infl ammation and invasion processes occurs exclusively at the basolateral side of host cells, highlighting the importance of the previous steps of macrophage subversion in shigella colonization of the gut. animal models. shigellosis is strictly a human disease. while the basis of this restriction is unknown, it complicates the ability to investigate the pathogenesis of shigella . however, several animal models have been developed to study the pathogenesis of shigella , the resulting immune response against shigella antigens, and the protection efficacy of candidate vaccines against shigellosis: [ ] . nonhuman primate (nhp) models : nhp models have been used to defi ne the ability of vaccines to elicit immune responses and protection (rhesus and cynomolgus monkeys) [ ] . the main advantage of this model is that shigella is able to colonize the large intestine and generate symptoms that these bacteria generate in human infection. o antigen/proteosome : o antigen represents the variable portion of shigella lps ( fig. . ). administration of lps or o antigen alone in animal models is not enough to elicit immune responses, making them ineffective immunogens. to solve this limitation, these molecules have been used in conjunction with different proteins as carriers. several variants of lps/oantigen mixtures have been developed and characterized. one of these protein combination approaches uses s. fl exneri and s. sonnei lps complexed with neisseria meningitidis outer membrane protein proteosomes [ , ] . lps is extracted from s. fl exneri or s. sonnei by hot phenol extraction and mixed with detergent-extracted outer membrane proteins from n. meningitidis . the complex was then separated from free lps present in the mixture by gel fi ltration chromatography. the concept behind this vaccine is that the proteins present in the n. meningitidis proteosome are able to act as carriers for t cell stimulation, thus allowing the recognition of lps. shigella outer membrane vesicles. outer membrane vesicles (omvs) are particles composed of lps, proteins, and nucleic acids. in a proposed vaccine formulation, these particles were purifi ed from liquid cultures of s. boydii by centrifugation with subsequent fi ltering ( fig. . ). the precise identity and amount of the proteins included in this preparation is not currently known, although the presence of proteins having the same mass as ipab, ipac, and ipad suggests its composition includes these proteins. when these omvs are administered orally to mice, antibodies are generated against omv lysates. this vaccine has the advantage of heterologous protection (as shown by challenge against strains from each shigella serogroup) and the absence of adjuvant dependency. in addi-tion, immunity can be passively transferred to offspring, suggesting that the protective mechanism involves antibodies and raising the possibility that this vaccine can be used in infants, which is the main target population for a shigella vaccine. the use of live, fully virulent shigella during its formulation process, the presence of lps, and lot-to-lot consistency are possible downsides of this preparation. invaplex. another vaccine candidate that uses t ss proteins and lps as part of the formulation is the invaplex [ ] . these complexes are obtained by aqueous extraction followed by ion exchange chromatography (fig. . ). the precise composition of these extracts has not been completely characterized but includes lps, ipab, and ipac [ ] . these complexes are able a b d c fig. . depiction of lps/o-antigen-based vaccines. lps ( a ) extracted from shigella fl exneri or sonnei is admixed with protein preparations from n. meningitides and used as a carrier. when this complex is administered orally and intranasally to mice and guinea pigs [ ] , serum igg and mucosal iga in intestines and lungs are vaccine com-pound ( b ). o antigen purifi ed from lps is delivered in combination with exoprotein a from p. aeruginosa ( c ). finally, o antigen from different shigella serogroups is combined with ribosomes from shigella and is depicted in ( d ) to elicit igg and iga responses against ipa proteins as well as lps in both mice and guinea pigs. in addition, they are protective against the shigella species/serotype used for extract generation [ ] in the mouse and guinea pig challenge models. two phase one studies have been performed using the invaplex vaccine on adult volunteers [ ] and showed no major side effects to delivery of intranasal doses of up to μg. the highest dose employed in these studies generated an asc response to lps in % of the volunteers. an advantage of this approach is that, other than the invaplex itself, no additional adjuvants need to be administered. a drawback of this vaccine consists in a challenging production process that includes cultures of virulent shigella as well as the presence of bacterial lps products in the intermediate steps and fi nal formulation. another possible caveat is the uniformity of protein composition in these complexes through manufacturing lots. finally, this vaccine was not designed to protect against multiple serotypes. a solution for this possible drawback, however, is the generation of formulations that include invaplex complexes generated from more than one particular serotype, which increases an already diffi cult manufacturing process. this would allow the generation of vaccine formulations specifi c for the serotypes prevalent in a particular region. a vaccine candidate that targets conserved shigella virulence proteins includes some of the t ss ipa proteins (fig. . ) . recombinant ipab and ipad can be expressed in e. coli at high levels. ipad is then easily purifi ed from the e. coli cytosol while ipab must be purifi ed as a complex with its cognate chaperone ipgc. the chaperone is needed to maintain the hydrophobic ipab in a soluble state and to provide stability for ipab from proteolytic degradation. ipab can then be further purifi ed after separation from ipgc in low concentrations of detergent. analyses have indicated that ipab is greater than % pure following this scheme. in its fi nal formulation, this ipa-based vaccine also contains a double mutant of heat-labile enterotoxin from e. coli (dmlt) [ ] as an adjuvant. the mechanism of protection for this vaccine has not yet been worked out. nevertheless, it was tested in the mouse lethal pulmonary model [ ] where it exhibited over % homologous protection (against s. fl exneri ) and greater than % heterologous protection (using s. sonnei during the challenge experiments). igg and mucosal iga were generated after intranasal administration along with antigen-specifi c ifn-γ-secreting cells. ompa. a -kda outer membrane protein (omp) was purifi ed from s. fl exneri a using ion exchange chromatography. incubation of macrophages with this -kda protein induced the production of nitric oxide and increased production of il- and tnf-α. this protein was delivered parenterally fi ve times in rabbits, giving protection against challenge by s. fl exneri in the rabbit cecal ligation model [ ] . subsequent work using a recombinant protein purifi ed by affi nity chromatography identifi ed this -kda omp as ompa, part of a family of immunomodulating proteins present in numerous gram-negative bacteria. this protein showed high protective effi cacy in the mouse lethal pulmonary model [ ] where it elicited serum igg and mucosal iga. ticks are widely distributed throughout the world, affecting % of the world's cattle population [ ] . the economic importance of ticks and tick-borne diseases (tbds) has been estimated by a number of studies; however they most likely represent an underestimation of the real impact of these arthropod vectors and their transmitted diseases. tick feeding has devastating effects including disease transmission, paralysis, toxicosis, and secondary infections of the tick-feeding site [ ] . the effect of ticks and tickborne diseases is particularly pronounced in the livestock sector where it is repeatedly rated highly for its impact on the livelihood of farmers, particularly in countries of the south which are heavily dependent on agricultural production. there are six genera of ixodid ticks of importance, namely, amblyomma , dermacentor , haemaphysalis , hyalomma , rhipicephalus , and ixodes . historically, tick and tick-borne disease control has focused on the control of ticks at tolerable levels through acaricide use and treatment of disease with appropriate drugs. in some cases acaricide-based tick control is often the only method of reducing tick populations without sacrifi cing productivity [ ] . acaricides are commercially available in a number of formulations that are applied either directly onto livestock or in dipping vats where multiple animals can be passed through at regular time intervals. acaricide application relies heavily on correct formulation and administration to be effective. a large number of chemical compounds have been found to be effective against ticks including arsenic (introduced ~ ), ddt (~ ), cyclodienes and toxaphene (~ ), organophosphates-carbamate group (~ ), formamides (~ ), and macrocyclic lactones (~ ). the potency and usefulness of many of the abovementioned compounds is gradually eroding with resistance developing in many tick species of rhipicephalus , amblyomma , and hyalomma . multiple acaricide-resistant tick stocks have been identifi ed, limiting or entirely excluding the use of many acaricides [ ] . in addition to resistance, chemical control through the guiding principle for anti-tick vaccination stems from early studies conducted on acquired host resistance to tick infestations. repeated exposure of hosts to ticks or tick organ homogenates induced resistance to tick re-infestation. while the degree of resistance may vary between different tick and host species, evidence strongly suggests that natural resistance against tick infestation develops based on adaptive immune response mechanisms [ ] . ticks feeding from hosts vaccinated with tick components take up effector molecules during feeding that mediate deleterious effects on the ticks. this effect manifests as reduction of feeding time, tick mortality (during or after feeding), reduced engorgement weights, and reduced reproductive capacity of adult females. eggs laid from ticks fed on vaccinated hosts may also show reduced hatching rates. the overall result culminates in reduction of tick populations and tick-borne diseases. many of the anti-tick vaccine targets have been identifi ed using conventional immune-screening techniques. immunization of vertebrate hosts with tick homogenates or purifi ed tick extracts generates immune sera. these sera are used to screen for tick antigens detected by the host. the identifi cation of tick proteins essential for tick survival is a useful method for more targeted antigen discovery, which is made increasingly possible as information is gathered on tick biology. with the availability of genome sequences for a number of tick species, the number of candidates for discovery is expanding through reverse vaccinology. the use of other techniques such as rna interference (rnai) has been useful in confi rming the importance of antitick vaccine candidates and is likely to play a role in future anti-tick vaccine antigen discovery [ ] . exposed or concealed antigens. anti-tick vaccine candidates have been classifi ed into two categories: exposed or concealed antigens. exposed antigens are secreted in tick saliva during attachment and feeding on a host while concealed antigens are normally hidden from the host immune response. molecular mimicry by ticks of host components has been observed, and vaccination may induce host sensitivity and autoimmune reactions when exposed antigens are used [ ] . one advantage of using exposed antigens is that natural boosting occurs through tick feeding. mechanistically, vaccination with exposed antigens is thought to induce a focal hostile environment unsupportive for tick attachment and feeding. concealed antigens do not come into contact with the host immune response during natural tick feeding. although often contained within the thoracic cavity of the tick, some salivary gland proteins can be characterized as concealed if they are not secreted into the tick-feeding site. one diffi culty in the development of concealed anti-tick vaccines is that the antigen must be accessible to the induced humoral vaccine response. this often limits the number of candidates to those coming into prolonged and direct contact with the blood meal or where the humoral response can be transported over the gut barrier into the hemolymph [ - ] . the second limitation of concealed antigens relates to natural boosting of the immune response. as the antigens do not come into contact with the immune response within the host, suffi ciently high antibody levels must be induced through repeated vaccination. as the blood meal acts as the carrier for the effector immune responses, the anti-tick effect can take place over a longer period of time compared to exposed antigens. this effect may even extend beyond the mere feeding period into the inactive stages where digestion and molting/egg laying takes place. bm anti-tick vaccine. the bm -based anti-tick vaccine remains the only anti-tick vaccine commercially produced and has become the benchmark for future anti-tick vaccine development and evaluation. the gut-associated bm glycoprotein was fi rst identifi ed in r. microplus although homologues in other tick species have since been identifi ed [ - ] . the biological function of bm remains unknown although it is thought to play a role in the digestion of the blood meal [ ] . in r. microplus , expression of bm is increased during embryogenesis, reaching the highest level in unfed larvae. expression decreases during feeding and molting with lowest levels of expression detected during the resting stages of the tick. bm has a translated coding sequence of amino acids and a size of . kda. the protein contains four potential n-linked glycosylation sites and a leader peptide suggesting transport to the cell surface. localization studies have shown the molecule is located predominantly on the microvilli of gut epithelial cells. a single c-terminal transmembrane sequence is present in the unprocessed protein which is replaced by a glycosylphosphatidylinositol anchor in the mature protein. the protein also contains multiple predicted egf repeats rich in cysteine residues. vaccination has been performed mostly with the whole molecule, and protective epitopes for bm have not been well determined. the site of a protective b cell epitope was defi ned and additional epitopes are likely to exist. overlapping cross-reactive immune-reactive epitopes have been found between bm and the r. decoloratus homologue, bd . vaccine effi cacy is directly related to anti-bm antibody titer and the ability to control tick populations is directly related to achieving a strong antibody response. substantial animal-to-animal variation has been observed in the ability to generate anti-bm antibody titers which is likely related to the mhc class ii haplotypes expressed. antibodies to bm and cattle complement system are taken up during the blood meal. antibody binding results in lysis of the gut epithelial cells culminating in impaired blood meal digestion. strong antibody responses may induce tick mortality due to blood leakage from the gut into the hemolymph and ticks may turn reddish instead of gray. the development of the antibody response in cattle [ ] after immunization with rbm is demonstrated in fig. . . recombinant expression of bm has been attempted in several expression systems including escherichia coli , aspergillus nidulans , aspergillus niger , and pichia pastoris . vaccine trials showed that bm vaccination targeted mainly the adult stage of r. microplus , particularly the number of adult females fully engorging and post-engorgement mortality. reproductive capacity of adult r. microplus females was affected in terms of egg-laying capacity and hatching of eggs [ ] . under fi eld situations, vaccination of cattle reduced tick numbers by % within a single generation and reduced the reproductive capacity by %. reversal of negative effects of tick feeding on live weight of vaccinated animals by an average increase in live weight of . kg over a -month period was observed. extensive fi eld trials in cuba, brazil, argentina, and mexico showed between and % control of r. microplus ticks within a -week period [ ] . importantly, complete control of acaricide-resistant ticks could be accomplished by integrating bm vaccination with acaricide use [ ] , showing that integrated control systems are effective in controlling tick populations. vaccination also decreased the amount of acaricides required to control tick populations and prolonged the time interval between cattle dipping. bm vaccination has been extensively evaluated for its ability to control other tick species. almost complete cross-protection against rhipicephalus annulatus has been reported [ ] . signifi cant protection against hyalomma anatolicum , h. dromedarii , and r. decoloratus has been observed; however no cross-protection was seen against r. appendiculatus or amblyomma variegatum . genetically attenuated microorganisms, viruses and bacteria, can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system. some experimental vector systems are summarized in work with vectors classifi ed as bsl- does not require biosafety program approval. work with vectors classifi ed as bsl- or higher requires approval by the local biosafety committee. safety concerns. as demonstrated for adenovirus (ad ), following i.m. injection, the vector persisted mainly near the injection site and in draining lymph nodes for up to months. low levels of integration into chromosomal dna were observed, with a calculated mutation rate of × − mutations per cell. the spontaneous mutation rate of a cell is × − and therefore tenfold higher. ad is classifi ed as biosafety level (bsl- ). live vectors are able to stimulate the mucosal as well as a systemic humoral and cellular immunity. a severe drawback of the vector technology is that, once used, the vector cannot be effectively used in the patient again because it will be recognized by antibodies. repeated booster immunization will fail. also preexisting immunity in the patient against the vector could render the vaccination ineffective. a heterologous prime-boost and vector priming as described in chap. could circumvent this barrier. disease lactic acid bacteria have generally recognized as safe (gras) status and have been developed in the past decade as potent adjuvants for mucosal delivery of vaccine. a platform technology based on lactobacillus plantarum ( fig. . ) was developed to deliver antigens against plague disease caused by yersinia pestis , an aerobic, nonmotile, gram-negative bacillus belonging to the family enterobacteriaceae , which is transmitted to humans via fl eabite or via aerosol droplet, causing bubonic or pneumonic plague, respectively [ ] . most human plague cases present as one of three primary forms -bubonic, septicemic, or pneumonic. secondary plague septicemia, pneumonia, and meningitis are the most common complications. the pathogenicity of y. pestis results from its impressive ability to overcome the defenses of the mammalian host and to overwhelm it with massive growth. plague is enzootic in rodents in africa, asia, south america, and north america. y. pestis is transmitted from host to host by fl eas via blood feeding, through consumption or handling of infectious host tissues, or through inhalation of infectious materials. y. pestis infects an astonishingly broad range of mammals and uses rats, squirrels, mice, prairie dogs, marmots, or gerbils as reservoirs and several arthropod vectors for transmission [ ] (fig. . ) . humans acquire this zoonotic infection via an atypical bite from animal fl eas, sometimes prompted by an animal's death from plague, after which the fl ea seeks a new source of blood. most infected fl eas come from the domestic black rat rattus rattus or the brown sewer rat rattus norvegicus. y. pestis cells spread from the site of the infected fl eabite to the regional lymph nodes, grow to high numbers causing the formation of a bubo, and spill into the bloodstream where bacteria are removed in the liver and spleen. growth continues in these organs, spreads to others, and causes septicemia. fleas feeding on septicemic animals complete the infection cycle. in humans, bubonic plague can develop into an infection of the lung (secondary pneumonic plague) that can lead to aerosol transmission (primary pneumonic plague) [ ] . multiple antibiotic-resistant strains of y. pestis occur naturally and they can be easily bioengineered. thus, plague is a category a bioterrorism agent in need for novel strategies for its prevention. bubonic plague is the classic form of the disease. patients usually develop symptoms of fever, headache, chills, and swollen, extremely tender lymph nodes (buboes) within - days of contact with the organism either by fl eabite or by exposure of open wounds to infected materials. primary septicemic plague is generally defi ned as occurring in a patient with positive blood cultures but no palpable lymphadenopathy. patients are febrile, and most have chills, headache, malaise, and gastrointestinal disturbances. primary pneumonic plague is a rare but deadly form of the disease that is spread via respiratory droplets through close contact ( - ft) with an infected individual. it progresses rapidly from a febrile fl u-like illness to an overwhelming pneumonia with coughing and the production of bloody sputum. the incubation period for primary pneumonic plague is between and days. in general, patients who develop secondary plague pneumonia have a high fatality rate. fig. . schematic of the plague cycle with small mammals as hosts and fl eas as vectors. arrows represent connections affected by climate with a color coding depending on the most infl uential climate variable on this link (i.e., precipitation, temperatures, and other variables indirectly depending on them such as soil characteristics and soil moisture). grey rectangles somewhat arbitrarily delimit epizootic, enzootic, and zoonotic cycles. note that despite their location at the far end of the cycle, humans often provide the only available information on plague dynamics the laboratory diagnosis of plague is based on bacteriological and/or serological evidence [ ] . samples for analysis can include blood, bubo aspirates, sputum, cerebrospinal fl uid in patients with plague meningitis, and scrapings from skin lesions, if present. staining techniques such as the gram, giemsa, wright, or wayson stain can provide supportive but not presumptive or confi rmatory evidence of a plague infection. lactic acid bacteria (lab) are a group of gram-positive, nonpathogenic, non-sporulating bacteria that include species of lactobacillus (fig. . ) , lactococcus , leuconostoc , pediococcus , and streptococcus . they have limited biosynthetic abilities and require preformed amino acids, b vitamins, purines, pyrimidines, and a sugar as a carbon and energy source. these nutritional requirements restrict their habitats to those in which the required compounds are abundant. thus, these highly specialized bacteria occupy a range of niches including milk, plant surfaces, the oral cavity, the gastrointestinal tract, and the vagina of vertebrates [ ] . lab have been consumed for centuries by humans in fermented foods and have an extraordinary safety profi le. these intrinsic advantages turn lab into excellent delivery vectors of novel preventive and therapeutic molecules for humans. a number of studies of oral vaccines generated from genetically engineered pathogenic or commensal bacteria have been reported [ , ] . live attenuated pathogenic bacteria, such as derivatives of mycobacterium , salmonella , and bordetella spp., are the most popular live delivery vectors used currently. they are particularly well adapted to interact with mucosal surfaces as they have specialized machinery to initiate the infection process. the major disadvantages of live vaccines include inadequate attenuation and the potential to revert to virulence. lactic acid bacteria-based vaccines act as live attenuated vaccines but without the safety concern. lab have a generally recognized as safe (gras) status and thus are not likely to cause harm. the production of a desired antigen by lab can occur in three different cellular locations: • intracellular , which allows the protein to escape harsh external environmental conditions (such as gastric juices in the stomach) but requires cellular lysis for protein release and delivery • extracellular , which allows the release of the protein into the external medium, resulting in direct interaction with the environment (food product or the digestive tract) • cell wall anchored , which combines the advantages of the other two locations (i.e., interaction between the cell wall-anchored protein and the environment, in addition to protection from proteolytic degradation) in this context, several studies have compared the production of different antigens in lab, using all three locations, and evaluated the subsequent immunological impact. these studies demonstrated that the highest immune response was obtained with cell wall-anchored antigens exposed on the surface of lab. therefore, most of the recent lab vaccination studies have selected surface exposure of the antigen of interest, rather than intra-or extracellular production [ ] . dendritic cells (dcs) play a central role in bridging the innate immune system with the adaptive immune system. dcs are found throughout the body and are especially common at mucosal surfaces. with only a single layer of epithelial cells separating the external from the internal world amid the constant need for particle exchange, intestinal dendritic cells (dcs) play a key role in maintaining intestinal homeostasis as well as governing protective immune responses against invading pathogens. to avoid activation of selfreactive t cells and to limit unnecessary responses, such as those against commensal fl ora, dcs can imprint tolerance onto t cells (fig. . ). immature-type dcs are enriched underneath the epithelium of mucosal inductive sites and are poised to capture antigens. they extend protrusions between epithelial cells, enabling direct sampling of luminal antigens [ ] . through upregulation of mhc and co-stimulatory molecules, matured dcs convert into highly effi cient antigen-presenting cells. successful antigen presentation to cd + t cells requires recognition of cognate peptide in the context of mhc class ii molecules, whereas epitopes presented on mhc class i molecules stimulate ag-specifi c cd + t cells. when antigen uptake occurs, these dcs change their phenotype by expressing higher levels of co-stimulatory molecules and move to t cell areas of inductive sites for antigen presentation. thus, dcs and their derived cytokines play key roles in the induction of antigen-specifi c effector th cell responses. in this regard, targeting mucosal dcs is an effective strategy to induce mucosal and systemic immune responses. lab persistence. the ability of some lab to persist in the gastrointestinal tract may be critically important in the effectiveness of lab-based vaccines. a comparison of a persisting lab strain, l. plantarum , with a nonpersisting lab strain, l. lactis , identifi ed l. plantarum to be more effective at eliciting antigen-specifi c immunity, suggesting that persistence promoted immunogenicity [ ] . furthermore, it has been shown that particular lactobacillus species induced critical infl ammatory cytokines and induced activation and maturation of dendritic cells [ ] . it has also been shown that immature dcs effi ciently capture lactobacillus species, and these bacteria activated human dcs, resulting in the production of pro-infl ammatory cytokines like il- , increased proliferation of cd + and cd + cells, and skewed t cell response toward a th pathway believed to be involved in effective clearance of microbial pathogens [ , ] ( fig. . ). evidence suggests that the peptidoglycan layer of some lab promote natural immuno-adjuvanticity [ ] , and antigen localization on the cell wall makes it more accessible to the immune system as compared to intracellular or secreted proteins. leader peptides mark proteins for translocation across the cytoplasmic membrane, and lipid modifi cation is of major importance both for anchoring exported proteins to the membrane and for protein function [ ] . it has been shown that lipidation at the fi rst amino acid of the mature borrelia burgdorferi ospa protein is essential to induce an immune response via tlr [ ] . the leader peptide of ospa targets the protein to the cell envelope of lactobacillus and that the cys [ ] is recognized by the l. plantarum cell wall-sorting machinery that lipidates and anchors the protein to the cell envelope. the end result is a delivery system that exerts a potent adjuvant effect [ ] . virus-like particles (vlps) that mimic the antigenic architecture of authentic virions, however, can be produced in insect, mammalian, and plant cells by the expression of the capsid protein. the particulate nature and high-density presentation of viral structure proteins on their surface render vlps as a premier vaccine platform with superior safety, immunogenicity, and manufacturability. therefore, this chapter focuses on the development of effective nov vaccines based on vlps of capsid proteins. the expression and structure of nov vlps, especially vlps of norwalk virus, the prototype nov, are extensively discussed. the ability of nov vlps in stimulating a potent systemic and mucosal anti-nov immunity through oral and intranasal delivery in mice is presented. gastroenteritis (ge) is a worldwide health problem that affects people of all ages. as its name implies, ge is characterized by infl ammation of the gastrointestinal tract and often associated with symptoms of diarrhea, nausea, vomiting, and abdominal cramping and pain. ge and its associated diarrheal diseases remain as one of the top causes of death in the world especially in developing countries and in young children with an estimated death toll of four to six million per year [ ] . ge can be caused by a variety of pathogens including viruses, bacteria, and parasites and by ingestion of noninfectious toxins or medications, with viruses as the most common offending agents. norovirus (nov) and rotavirus are the most common viruses that cause viral ge, while adenovirus, astrovirus, coronavirus, and parechovirus are also known to cause ge in humans. novs are a group of genetically diverse rna viruses that belong to the genus of norovirus in the caliciviridae family [ ] . they were fi rst discovered and characterized in their prototype virus, the norwalk virus (nv), in [ ] . studies of nv revealed that novs are non-enveloped viruses with a rna genome surrounded by a round capsid protein shell approximately nm in diameter. novs are divided into genogroups and clusters with clusters in genogroup i (gi), in gii, in giii, and each in giv and gv. within the fi ve genotypes, gi and giv strains are found to infect humans exclusively and gii are found in both humans and pigs, while giii and gv strains are animal viruses that infect cattle and murine species, respectively [ ] . currently, strains in cluster of gii (gii ) are the most prevalent novs in human population [ ] . the genome of novs, which was fi rst characterized in nv, contains a single-stranded positive-sense rna of . - . kb with three open reading frames (orfs) and a poly a tail at its ′ end [ ] (fig. . ). orf encodes a polyprotein that is processed by viral protease clpro into the rnadependent rna polymerase and approximately fi ve other nonstructural proteins including p , the nucleoside triphosphatase, p , vpg, and clpro. the two structural proteins, the major (vp ) and minor (vp ) capsid proteins, are encoded by orf and orf , respectively [ ] . structural analysis of nov has revealed that each viral capsid is composed of dimers of vp in a t = icosahedral symmetry. vp folds into two domains: a shell (s) domain that is responsible for initiating capsid assembly and icosahedral contacts and a protruding domain (p), containing two subdomains of p and p , that enhance the stability of the capsid by providing intermolecular contacts between vp dimers. studies of nv also indicate that the vp protein enhances the expression level of vp and stabilizes the vp in the viral capsid. novs are highly contagious and spread rapidly, and their outbreaks commonly occur in various social places and settings where people share common food and water sources or are in close physical proximity, such as cruise ships, schools, military units, nursing homes, daycare centers, hospitals, restaurants, and catered events . the life cycle of nov has not been fully understood due to the lack of an in vitro cell culture system and a small animal model of infection. the failure of nov replication in mammalian cell cultures is not due to the lack of host factors to support intracellular expression of nov rna. instead, the problem may lie in the steps of viral binding to cellular receptors, virus entry into cells. nov [ ] . while serum antibodies to nov can be readily detected, this method has little clinical relevance due to the cross-reactivity of antibodies. since there is no culture system available for nov, the detection of virus in stool samples has become the preferred method of diagnosis. traditionally, nov infection was diagnosed by detecting the virus by immune transmission electron microscopy (tem). tem offers the advantage of direct visualization of any potentially responsible virus particles in stool samples. however, it does have the disadvantage of requiring sophisticated and expensive equipment and highly specialized technicians for its operation. several enzyme-linked immunosorbent assays (elisas) that detect nov antigens were later developed for nov diagnosis. studies have shown that nov antigen-detecting elisas have high specifi city ( - %) but poor sensitivity ( - %), most likely due to the antigenic diversity of nov strains [ ] . similar to elisa-based assays, rt-pcr is rapid and robust, because it can process large numbers of samples simultaneously and results can be obtained within a working day. however, it requires rna extraction from fecal samples and needs expensive equipment and skilled workers to operate. therefore, rt-pcr is more labor intensive and less economical than elisas. overall, tem, elisa, and rt-pcr-based methods all have their advantages and challenges. the three methods detect different components of the virus and therefore are complementary to each other. immunology of nov infection. the immunological knowledge of nov is mostly obtained from human challenge studies and natural outbreaks due to the lack of small animal models. observations of repeat infections in adults suggest the scarcity of long-term immunity against these viruses. however, other studies showed that close to % of the genetically susceptible subjects were not infected by nov challenge, which support the possibility of long-term immunity [ ] . nov. the lack of a tissue culture system also impedes the development of vaccines against nov. fortunately, the discovery of the spontaneous assembly of expressed vp into virus-like particles (vlps) that are morphologically and antigenically similar to the native viruses has facilitated vaccine development. vlps combine the best traits of wholevirus and subunit antigens for vaccine development. vlps are noninfectious, therefore, safer than inactivated or attenuated virus due to the lack of viral nucleic acid genome. importantly, vlps can induce potent cellular and humoral immune responses without adjuvants and are more effective vaccines than other subunit antigens because their architectures mimic infectious viruses. vlps can be produced by recombinant technology in heterologous expression systems without requiring the ability to support viral replication. this is particularly important for nov because no such culture system has been developed to support the growth of these viruses. studies have demonstrated that viruses and corresponding vlps have a particle size ideal for dc and macrophage uptake to initiate antigen processing [ ] . thus, the particulate nature of vlps favors their targeting to relevant apcs for optimal induction of t cell-mediated immune responses. vlps can also be presented effi ciently to b cells and induce strong antibody responses. like live viruses, the quasicrystalline surface of vlps, with its arrays of repetitive epitopes, presents a prime target that vertebrate b cells have evolved to specifi cally recognize. this recognition triggers the cross-linking of surface membrane-associated immunoglobulins (ig) on b cells and leads to their proliferation and migration, t helper-cell activation, antibody production and secretion, and the generation of memory b cells. thus, vlps can directly activate b cells at much lower concentrations than other subunit antigens and induce high titer and durable b cell responses in the absence of adjuvants. these inherent advantages of vlps have made them one of the most successful recombinant vaccine platforms. for example, fi ve vlp-based vaccines for hepatitis b virus characterization of nov vlps. vlps of novs were fi rst produced in insect cells using baculovirus vectors [ ] and then in plants using tobamovirus and geminivirus vectors and in mammalian cells using the venezuelan equine encephalitis (vee) replicon system [ ] . these studies demonstrated that expression of the major capsid protein vp alone can drive the self-assembly of vlps that morphologically and antigenically resemble native virus particles (fig. . ) . vlps generated by all three expression systems are similar to each other. the structure of nov vlps is exemplifi ed by the vlp of nv capsid protein (nvcp). studies of insect cellbaculovirus-derived nvcp vlps by cryo-electron microscopy and x-ray crystallography reveal that the nv capsid is a nm icosahedral arrangement of copies of the kda capsid protein vp organized into dimers in a t = symmetry. while all dimers are formed from two identical nvcp monomers, two different dimer confi gurations are required to correctly form the complete assembled capsid [ ] . as in native nv particles, the nvcp also folds into two distinctive domains in vlps, with s domain forming the inner core of the shell and p domain protruding out from the capsid [ ] . similarly, the p subdomain is also the most surface-exposed region in nv vlps and may contain hbga and neutralizing antibody-binding sites and determinants of strains specifi city [ , ] . the similarity between vlps of nv and other novs including gii. viruses has been demonstrated [ ] . insect cell-baculovirus vector-produced nvcp vlps. it was shown that four oral doses of as little as μg nvcp vlps without any adjuvant triggered serum nv-specifi c anti-igg response in the majority ( / ) of vlpfed icd outbred mice [ ] . systemic igg response was observed after two oral dosages and the highest titer was induced by four doses of μg vlps. moreover, mice in the μg dosage group developed nv-specifi c intestinal iga in a level up to . % of total iga. inclusion of the mucosal adjuvant cholera toxin (ct) did not signifi cantly change the number of positive responders of serum igg or intestinal iga but signifi cantly enhanced the amplitude of serum igg response, especially for higher doses of vlps. thus, nvcp vlp is clearly a potent oral immunogen and can induce both systemic and gut mucosal antibody responses. nanovaccines: gas infections group a streptococcus ( streptococcus pyogenes) (gas) is an important human mucosal pathogen that is responsible for a wide spectrum of diseases with varying clinical manifestations and severity [ , ] : pharyngitis (strep throat) is a common minor complication of gas infection but when left untreated can lead to life-threatening diseases including the autoimmune sequelae rheumatic fever (rf) and rheumatic heart disease (rhd). rhd results in permanent damage to the heart tissues and valves. gas infections cause > , deaths each year mostly in developing countries and indigenous populations within developed nations where poor socioeconomic conditions and overcrowding contribute to the high rates of gas diseases. in developing countries, rf is the leading cause of heart disease among children [ ] . there is currently no available vaccine to prevent infection with gas and consequently prevent gas diseases. a successful mucosal gas vaccine would need to stimulate the appropriate humoral and cellular immunity for protection against gas infection ( fig. . ). this is especially diffi cult due to a lack of human-compatible mucosal vaccine adjuvants that are essential to boost immune responses. researchers have therefore focused mainly on parenteral gas vaccine delivery approaches, for which suitable adjuvants are available, designed to provide protection against systemic infection via the induction of opsonic igg antibodies. antigen. an effective gas vaccine needs to have broad antigenic reach because of the many different gas strains (> different m types) circulating in a population fig. . gas vaccination approaches. gas that breaches the physical barrier of the mucosal epithelium of the nasal-associated lymphoid tissue, functionally analogous to human tonsils, is purported to be transported to the underlying lymphoid tissue via association with membranous (m) cells. cells of the innate immune system sense gas and produce cytokines and chemokines to contain the infection to the mucosa. gas antigens are delivered to antigen-presenting cells such as dcs and b cells. iga-committed b cells are activated and initiate antigen-specifi c iga responses. dcs play a fundamental role in the development of immunity to gas and present antigen to t cells to induce a th response that is integral along with iga for mucosal defense against pharyngeal gas colonization. mucosal vaccination is designed to mimic these responses and effectively clear gas from the mucosal surface upon infection to prevent gas colonization and carriage. gas that escapes the host's defense mechanisms can disseminate into the lymphatics and blood, leading to systemic infection. mucosal vaccination is also able to induce a systemic immune response characterized by the induction of opsonic igg antibodies, which destroy the pathogen by opsonophagocytosis. parenteral gas vaccination induces serum igg but is not able to induce mucosal immunity and should not induce immune responses that are potentially cross-reactive with self tissue proteins. the gas m protein is the major protective antigen and an ideal target for vaccine development; however it contains heart tissue cross-reactive epitopes particularly in the conserved region [ ] . evidence suggests that cross-reactive t cells especially play a pivotal role in the pathogenesis of rhd. the m protein is an α-helical coiled-coil surface protein consisting of a hypervariable amino-terminal region and a highly conserved (> % sequence identity) carboxyterminal c-repeat region [ ] (fig. . ) . functionally, the m protein is important in preventing bacterial clearance by complement-mediated phagocytosis, which limits host defense mechanisms. previous studies indicate that protective immunity to gas can be evoked by opsonic antibodies to serotypic epitopes at the amino-terminal region that are m-type specifi c [ ] . nanovaccine. combined vaccine/adjuvant delivery systems offer the potential of mucosal vaccine delivery. for example, the lipid-core-peptide (lcp) system is a novel, synthetic, selfadjuvanted vaccine delivery system that incorporates the adjuvant (prr agonist), carrier, and antigenic peptides of a vaccine into a single molecular entity ( fig. . ). this system has been previously shown to effi ciently deliver gas vaccines and induce immunity [ ] . evidence suggests the adjuvant activity of lcp involves the induction of dc activation. preclinical developments. three approaches are currently being investigated in the development of a subunit gas vaccine based on the m protein: . multivalent gas vaccine . a multivalent approach employs a combination of amino-terminal protein fragments representing different m types and is designed to target prevalent gas strains in a population. using this approach, a recombinant multivalent gas vaccine containing m protein peptides from different gas serotypes prevalent in north america was demonstrated to evoke opsonic antibodies in animals [ ] . from epidemiological data, the -valent vaccine would cover the majority of pharyngitis and invasive gas diseases, including rf, invasive fasciitis, and toxic shock syndrome. recently, a new -valent gas vaccine was shown to be immunogenic in rabbits and evoked opsonic antibodies against "non-vaccine" serotypes [ ] potentially creating a vaccine with much broader coverage. this type of vaccine is population specifi c and therefore may not be effective universally. it may also need to be re-designed periodically to refl ect changes in the epidemiology of gas infections. (ch ) (ch ) ch ch ch fig. . the lipid-core-peptide (lcp) system. the lcp system contains an adjuvant component (lipid-core made of lipoamino acids) and a polylysine carrier onto which peptide epitopes are attached. the example shows a gas vaccine candidate containing j and an amino-terminal serotypic epitope called . the adjuvant has three -amino-dodecanoic ( n = ) lipoamino acids separated by glycine amino acid spacers . j vaccine . a gas vaccine that employs peptide epitopes from the conserved c-repeat region of the m protein is the second approach and has the potential in theory for greater coverage of m types. immunization of mice with a c-region peptide gas vaccine candidate called j conjugated to the carrier protein diphtheria toxoid (dt) and co-delivery with an appropriate adjuvant led to protection against systemic and mucosal gas infection [ ] (fig. . ). j also elicited protective immunity against gas when linked to lipopeptides [ , ] . other studies have shown that intranasal immunization of mice with c-region peptides conjugated to the experimental mucosal adjuvant cholera toxin b subunit (ctb) evoked protective immunity against gas at the mucosal level [ ] . ctb could possibly enter olfactory regions of the central nervous system and cause neuronal damage following intranasal delivery [ ] and therefore is not suitable for human use. vector delivery approaches have included expressing the c-repeat region on vaccinia virus [ ] , the commensal bacterium lactococcus lactis [ ] , or streptococcus gordonii [ ] . . j . the third combination vaccine approach uses both serotypic and conserved m protein peptide epitopes. initially, a heteropolymer gas vaccine construct was synthesized by free radical-induced polymerization of acryloyl peptides to combine seven serotypic epitopes and a highly conserved c-region peptide epitope called j [ ] . the m types that were targeted in the heteropolymer represented gas infections prevalent in the northern territory of australia -a region highly endemic for gas. immunization of mice with the heteropolymer demonstrated excellent immunogenicity and protection fig. . preclinical evaluation of gas vaccine candidates. intranasal immunization of mice with the j -dt vaccine candidate led to significantly greater survival after intranasal challenge with gas versus control groups but the mucosal adjuvant ctb was essential for protection ( a ). a multiepitope lcp-based gas vaccine candidate elicited protective immunity against mucosal gas infection even in the absence of ctb ( b ) against homologous and heterologous gas strains, indicating its potential to provide broad coverage. however, batch-to-batch variation led to altered immune responses, which limited its applicability for human use. the vaccine also required the addition of an adjuvant to be effective, further limiting its use as a mucosal vaccine due to a lack of safe and effective mucosal adjuvants. later, multiepitope gas vaccine candidates were synthesized based on the lcp system that induced highly opsonic antibodies following parenteral delivery to mice [ ] , as well as protection against mucosal gas infection following intranasal immunization [ ] (fig. . ). safety and effi cacy. the main concern when using large regions of the m protein in a gas vaccine is the potential for inducing an autoimmune response due to immunological cross-reactivity with host proteins. it is therefore important to identify protective antigenic determinants and to separate the biological relevant epitopes from those that are host tissue cross-reactive and potentially harmful. epitope mapping studies were used to identify the conserved gas vaccine candidate j , which contains a conformational protective b cell epitope and was designed to lack a human heart cross-reactive t cell epitope [ , ] . tb vaccines -state of the art and progresses dna-antiviral vaccines: new developments and approaches -a review west nile virus recombinant dna vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzymelinked immunosorbent assays stable and long-lasting immune response in horses after dna vaccination against equine arteritis virus vaccination with human tyrosinase dna induces antibody responses in dogs with advanced melanoma infectious haematopoietic necrosis epidemic ( to ) in farmed atlantic salmon salmo salar in british columbia naked dna vaccination of atlantic salmon salmo salar against ihnv effi cacy of an infectious hematopoietic necrosis (ihn) virus dna vaccine in chinook oncorhynchus tshawytscha and sockeye o. nerka salmon wild pollinators enhance fruit set of crops regardless of honey bee abundance a metagenomic survey of microbes in honey bee colony collapse disorder colony collapse disorder: a descriptive study the reproductive program of female varroa destructor mites is triggered by its host, apis mellifera brood cell size of apis mellifera modifi es the reproductive behavior of varroa destructor the transmission of deformed wing virus between honeybees (apis mellifera l.) by the ectoparasitic mite varroa jacobsoni oud detection of acute bee paralysis virus and black queen cell virus from honeybees by reverse transcriptase pcr varroa destructor is an effective vector of israeli acute paralysis virus in the honeybee, apis mellifera prevalence and transmission of honeybee viruses age-related changes in the behavioural response of honeybees to apiguard(r), a thymolbased treatment used to control the mite varroa destructor the immune response of drosophila drosophila hemopoiesis and cellular immunity c: insights into social insects from the genome of the honeybee apis mellifera structure and genetics of shigella o antigens optimization of virulence functions through glucosylation of shigella lps growing antimicrobial resistance of shigella isolates role of m cells in initial antigen uptake and in ulcer formation in the rabbit intestinal loop model of shigellosis ipab mediates macrophage apoptosis induced by shigella fl exneri molecular pathogenesis of shigella spp.: controlling host cell signaling, invasion, and death by type iii secretion mucosal lymphoid infi ltrate dominates colonic pathological changes in murine experimental shigellosis not available development of an improved animal model of shigellosis in the adult rabbit by colonic infection with shigella fl exneri a a challenge model for shigella dysenteriae in cynomolgus monkeys (macaca fascicularis) immunogenicity and effi cacy of oral or intranasal shigella fl exneri a and shigella sonnei proteosome-lipopolysaccharide vaccines in animal models enhancement of anti-shigella lipopolysaccharide (lps) response by addition of the cholera toxin b subunit to oral and intranasal proteosome-shigella fl exneri a lps vaccines isolation and characterization of a shigella fl exneri invasin complex subunit vaccine immunogenicity and effi cacy of highly purifi ed invasin complex vaccine from shigella fl exneri a development and evaluation of a shigella fl exneri a and s. sonnei bivalent invasin complex (invaplex) vaccine safety and immunogenicity of an intranasal shigella fl exneri a invaplex vaccine characterization of a mutant escherichia coli heat-labile toxin, lt(r g/l a), as a safe and effective oral adjuvant broadly protective shigella vaccine based on type iii secretion apparatus proteins purifi cation and characterization of an immunogenic outer membrane protein of shigella fl exneri a outer membrane protein a (ompa) of shigella fl exneri a, induces protective immune response in a mouse model global aspects of the management and control of ticks of veterinary importance the global importance of ticks chemical control of ticks on cattle and the resistance of these parasites to acaricides factors that infl uence the prevalence of acaricide resistance and tick-borne diseases the molecular revolution in the development of vaccines against ectoparasites rna interference for the study and genetic manipulation of ticks proteins in the saliva of the ixodida (ticks): pharmacological features and biological signifi cance comparison of the proteins in salivary glands, saliva and haemolymph of rhipicephalus appendiculatus female ticks during feeding immunoglobulin-binding proteins in ticks: new target for vaccine development against a blood-feeding parasite amblyomma americanum : specifi c uptake of immunoglobulins into tick hemolymph during feeding synthetic vaccine (sbm ) against the cattle tick rhipicephalus (boophilus) microplus : preservation of immunogenic determinants in different strains from south america cloning, expression and immunoprotective effi cacy of rhaa , the homologue of the bm tick vaccine antigen, from hyalomma anatolicum anatolicum differential transcription of two highly divergent gut-expressed bm antigen gene homologues in the tick rhipicephalus appendiculatus (acari: ixodida) cloning and expression of a protective antigen from the cattle tick boophilus microplus bovine immunoprotection against rhipicephalus (boophilus) microplus with recombinant bm -campo grande antigen tick vaccines and the transmission of tick-borne pathogens vaccination against ticks (boophilus spp.): the experience with the bm -based vaccine gavac immunity against boophilus annulatus induced by the bm (tick-gard) vaccine developing live vaccines against plague yersinia pestis--etiologic agent of plague yersinia: strategies that thwart immune defenses plague . in: crc handbook series in zoonoses . section a. bacterial, rickettsial, chlamydial and mycotic diseases mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria the induction of hiv gag-specifi c cd + t cells in the spleen and gutassociated lymphoid tissue by parenteral or mucosal immunization with recombinant listeria monocytogenes hiv gag mucosal immunization with surface-displayed severe acute respiratory syndrome coronavirus spike protein on lactobacillus casei induces neutralizing antibodies in mice lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and dna vaccines dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria protection against tetanus toxin after intragastric administration of two recombinant lactic acid bacteria: impact of strain viability and in vivo persistence lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells lactobacilli as natural enhancer of cellular immune response lactobacilli activate human dendritic cells that skew t cells toward t helper polarization instruments for oral disease-intervention strategies: recombinant lactobacillus casei expressing tetanus toxin fragment c for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. microbiol treponema pallidum and borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytic cells via a cd -dependent pathway distinct from that used by lipopolysaccharide immune response to lactobacillus plantarum expressing borrelia burgdorferi ospa is modulated by the lipid modifi cation of the antigen a review of viral gastroenteritis noroviruses: a comprehensive review biological properties of norwalk agent of acute infectious nonbacterial gastroenteritis norovirus classifi cation and proposed strain nomenclature mechanisms of gii. norovirus persistence in human populations sequence and genomic organization of norwalk virus norwalk virus genome cloning and characterization methods for the detection and characterisation of noroviruses associated with outbreaks of gastroenteritis: outbreaks occurring in the north-west of england during two norovirus seasons european multicenter evaluation of commercial enzyme immunoassays human susceptibility and resistance to norwalk virus infection t cellindependent type i antibody response against b cell epitopes expressed repetitively on recombinant virus particles threedimensional structure of baculovirus-expressed norwalk virus capsids expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons conformational stability and disassembly of norwalk virus like particles: effect of ph and temperature structural studies of recombinant norwalk capsids e. coliexpressed recombinant norovirus capsid proteins maintain authentic antigenicity and receptor binding capability c-terminal arginine cluster is essential for receptor binding of norovirus capsid protein structural basis for the recognition of blood group trisaccharides by norovirus pathogenesis of group a streptococcal infections group: a streptococcal infections and acute rheumatic fever multiple cross reactive epitopes of streptococcal m proteins streptococcal m protein: molecular design and biological behavior localization of protective epitopes of the amino terminus of type streptococcal m protein towards the development of a broadly protective group a streptococcal vaccine based on the lipid-core peptide system immunogenicity of a -valent group a streptococcal vaccine new -valent m protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group a streptococci protection against group a streptococcus by immunization with j -diptheria toxoid: contribution of j -and diphtheria toxoid-specifi c antibodies to protection intranasal vaccination with a lipopeptide containing a minimal, conformationally constrained conserved peptide, a universal t-cell epitope and a self-adjuvanting lipid protects mice from streptococcus pyogenes and reduces throat carriage immunisation of mice with a lipid core peptide construct containing a conserved region determinant of group a streptococcal m protein elicits heterologous opsonic antibodies in the absence of adjuvant epitopes of group a streptococcal m protein that evoke cross-protective local immune responses cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues protection against streptococcal pharyngeal colonization with a vaccinia: m protein recombinant mucosal vaccine made from live, recombinant lactococcus lactis protects mice against pharyngeal infection with streptococcus pyogenes clinical and microbiological responses of volunteers to combined intranasal and oral inoculation with a streptococcus gordonii carrier strain intended for future use as a group a streptococcus vaccine new multi-determinant strategy for a group a streptococcal vaccine designed for the australian aboriginal population immunization with a tetraepitopic lipid core peptide vaccine construct induces broadly protective immune responses against group a streptococcus intranasal administration is an effective mucosal vaccine delivery route for self-adjuvanting lipid core peptides targeting the group a streptococcal m protein mapping a conserved conformational epitope from the m protein of group a streptococci mapping the minimal murine t cell and b cell epitopes within a peptide vaccine candidate from the conserved region of the m protein of group a streptococcus key: cord- -zg akivn authors: chan, yinghan; ng, sin wi; mehta, meenu; anand, krishnan; kumar singh, sachin; gupta, gaurav; chellappan, dinesh kumar; dua, kamal title: advanced drug delivery systems can assist in managing influenza virus infection: a hypothesis date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: zg akivn outbreaks of influenza infections in the past have severely impacted global health and socioeconomic growth. antivirals and vaccines are remarkable medical innovations that have been successful in reducing the rates of morbidity and mortality from this disease. however, the relentless emergence of drug resistance has led to a worrisome increase in the trend of influenza outbreaks, characterized by worsened clinical outcomes as well as increased economic burden. this has prompted the need for breakthrough innovations that can effectively manage influenza outbreaks. this article provides an insight into a novel hypothesis that describes how the integration of nanomedicine, with the development of drugs and vaccines can potentially enhance body immune response and the efficacies of anti-viral therapeutics to combat influenza infections. apart from covid- , influenza is another infectious disease that ranks high as one of the deadliest, characterized by a remarkably high rate of transmission that could cause a rapid spread. it is estimated that influenza kills approximately thousand people yearly [ , ] . killed virus vaccine as an intramuscular injection and attenuated live vaccine as a nasal spray, are the two most widely known vaccines for this deadly virus [ ] . in the recent years, an increasing trend of influenza outbreaks have been observed, prompting medical researchers to design and develop suitable vaccines and novel therapeutic modalities [ ] . despite the availability of vaccines that may protect individuals from well-matched strains, it is well- known that the influenza virus has high mutation rates, resulting in frequent mismatches due to antigenic drift and shift, thereby, necessitating the development of a new vaccine every few years [ ] . however, the development of a new vaccine is time-consuming. in addition, vaccine-development remains mostly applicable to developed countries, attributing to the cost factor involved. moreover, long term use of standalone anti-influenza drugs and vaccines are often associated with adverse reactions and other shortcomings, that limit their effective clinical applications [ ] . for instance, although the neuraminidase inhibitor oseltamivir has been widely employed as an anti-influenza drug, it was found that the drug does not offer benefits in patients with pre-existing medical conditions [ , ] predict possible chronic and other unforeseen in-vivo effects [ , ] . hence, it is hoped that this hypothesis will trigger further exploration into nanomedicine-based approach to elucidate the in-depth mechanisms involved, along with their safety, to pave way for a paradigm shift in influenza management approaches. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. single- dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent h n , h n and h n viruses exploiting nanotechnology to target viruses universal influenza vaccines: from viruses to nanoparticles nanotherapeutic anti-influenza solutions: current knowledge and future challenges influenza antivirals and resistance: the next years? influenza viruses -antiviral therapy and resistance influenza vaccines: evaluation of the safety profile immunogenicity and protection of oral influenza vaccines formulated into microparticles celastrol-loaded liquid crystalline nanoparticles as an anti-inflammatory intervention for the treatment of asthma nanomaterials designed for antiviral drug delivery transport across biological barriers dramatically increase zanamivir absolute bioavailability in rats: implications for an nanotechnologies for boswellic acids the potential of sirna based drug delivery in respiratory disorders: recent advances and progress targeting neutrophils using novel drug delivery systems in chronic respiratory diseases emerging complexity and the need for advanced drug delivery in targeting candida species albumin nano-encapsulation of piceatannol enhances its anticancer potential in colon cancer via downregulation of nuclear p and hif- α. cancers (basel) nanomedicine for infectious disease applications: innovation towards broad-spectrum treatment of viral infections nanoparticles in influenza subunit vaccine development: immunogenicity enhancement. influenza other respi nanoparticle-based vaccines against respiratory viruses organic and inorganic nanoparticle vaccines for prevention of infectious diseases formulation and characterization of glibenclamide and quercetin- loaded chitosan nanogels targeting skin permeation patented therapeutic drug delivery strategies for targeting pulmonary diseases increasing complexity and interactions of oxidative stress in chronic respiratory diseases: an emerging need for novel drug delivery systems nanotechnology-based antiviral therapeutics rutin loaded liquid crystalline nanoparticles inhibit lipopolysaccharide induced oxidative stress and apoptosis in bronchial epithelial cells in vitro biopolymer encapsulated live influenza virus as a universal cd + t cell vaccine against influenza virus. vaccine anti- bacterial activity of inorganic nanomaterials and their antimicrobial peptide conjugates against resistant and non-resistant pathogens perspectives and advancements in the design of nanomaterials for targeted cancer theranostics microparticles as vaccine adjuvants and delivery systems investigation of tunable acetalated dextran microparticle platform to optimize m e-based influenza vaccine efficacy molecular assembly and application of biomimetic microcapsules hybrid inorganic-organic capsules for eficient intracellular delivery of novel sirnas against influenza a (h n ) virus infection biodegradable polyelectrolyte/silica composite microcapsules as carriers for small application of dendrimers for the treatment of infectious diseases nanoparticle vaccines against infectious diseases delivery vehicles for active phytoconstituents emerging trends in nanomedicine for topical delivery in skin disorders: current and translational oligonucleotide therapy: an emerging focus area for drug delivery in chronic inflammatory respiratory diseases emerging trends in the novel drug delivery approaches for the treatment of lung cancer interactions with the macrophages: an emerging targeted approach using novel drug delivery systems in respiratory diseases gene delivery by pamam dendrimer conjugated with the nuclear localization signal peptide derived from influenza b identification of biomarkers and genetic approaches toward chronic obstructive pulmonary disease inhibition of influenza a virus infection in vitro by saliphenylhalamide-loaded porous silicon nanoparticles enhanced inhibition of influenza virus infection by peptide-noble-metal nanoparticle conjugates development of an adjuvanted nanoparticle vaccine against influenza virus, an in vitro study pulmonary surfactant-biomimetic nanoparticles potentiate heterosubtypic influenza immunity protein nanoparticle immunization induces broad cross-protection against different influenza viruses in mice virus-mimetic polymer nanoparticles displaying hemagglutinin as an adjuvant-free influenza vaccine porous gold nanoparticles for attenuating infectivity of influenza a virus inhibition of h n influenza virus infection by zinc oxide nanoparticles: another emerging application of nanomedicine phase pivotal trial of nanoflu tm in older adults editorial: advances and challenges in nanomedicine nanoethics: from utopian dreams and apocalyptic nightmares towards a more balanced view the authors declare no conflict of interest, financial or otherwise. key: cord- -ouykx g authors: puig-barberà, j.; díez-domingo, j.; arnedo-pena, a.; ruiz-garcía, m.; pérez-vilar, s.; micó-esparza, j.l.; belenguer-varea, a.; carratalá-munuera, c.; gil-guillén, v.; schwarz-chavarri, h. title: effectiveness of the – seasonal influenza vaccine in preventing confirmed influenza hospitalizations in adults: a case–case comparison, case-control study date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ouykx g introduction: we estimated influenza vaccine effectiveness (ive) to prevent laboratory-confirmed influenza-related hospitalizations in patients years old or older during the – influenza season. methods: we conducted a prospective case-control study in five hospitals, in valencia, spain. study subjects were consecutive emergency hospitalizations for predefined conditions associated with an influenza-like illness episode < days before admission. patients were considered immunized if vaccinated ≥ days before influenza-like illness onset. cases were those with a real time reverse transcriptase polymerase chain reaction (rt-pcr) positive for influenza and controls were rt-pcr positive for other respiratory viruses. adjusted ive was estimated as × ( − adjusted odds ratio). to account for indication bias we computed adjusted ive for respiratory syncytial virus related hospitalizations. results: of eligible hospitalized patients, ( %) were influenza positive and considered cases, and ( %) were positive for other respiratory viruses and considered controls. adjusted ive was % ( % confidence interval, – %). by subgroup, adjusted ive was % ( – %) for those with high-risk conditions, % ( – %) for those ≥ years of age, and, % ( – %) for those ≥ years of age with high-risk conditions. no influenza vaccine effect was observed against respiratory syncytial virus related hospitalization. conclusion: influenza vaccination was associated with a significant reduction on the risk of confirmed influenza hospitalization, irrespective of age and high-risk conditions. yearly seasonal influenza epidemics are associated with excess morbidity and mortality [ ] . vaccination against influenza is considered the most effective strategy for preventing influenza [ ] . as a consequence of antigenic drift, influenza vaccines are to be a produced every year [ ] . despite this achievement, vaccine effectiveness varies from season to season and can be very low one in four influenza seasons [ ] . this is due to the unpredictable antigenic distance between vaccine's and the circulating strains [ ] . as a consequence, evidence on influenza vaccine effectiveness has been difficult to obtain and is disputed [ , [ ] [ ] [ ] . the reappraisal of the evidence on influenza vaccine effectiveness is possible by the availability of reverse transcriptase polymerase chain reaction (rt-pcr) to diagnose influenza infection [ ] . rt-pcr has allowed the development of the test-negative approach for measuring influenza vaccine effectiveness. in test-negative case-control studies, cases are rt-pcr positive for influenza, and controls those negative for influenza. this approach has been advocated for its practicability, comparability between cases and controls, and the use of laboratory confirmed outcomes [ , ] . various authors have used the test-negative casecontrol study [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . under conditions of concurrent circulation an appropriate test-negative control group are patients testing positive for other respiratory viruses, ensuring similarity on quality of sample collection and specificity of outcomes [ , , ] . using a prospective case-case comparison approach, we have estimated seasonal influenza vaccine effectiveness (ive) to prevent laboratory confirmed influenza-related hospitalizations in adults. during the - influenza season, we performed a prospective case-control study in five hospitals in valencia, spain. the five hospitals provided care to , inhabitants years of age or older. the influenza season was defined by the weeks with positive specimens for influenza on enrolled patients. it began on december (week ) and ended on march (week ) . patients with confirmed influenza by a rt-pcr test were considered cases and patients with an rt-pcr confirmed infection for other respiratory viruses were considered controls. influenza vaccines were offered free of charge to health district inhabitants older than months of age with high-risk conditions and to years old or older. three vaccine formulations were used. subunit trivalent non-adjuvanted vaccine (influvac ® , abbot-solvay, illinois, usa; batch numbers v , v , v ) offered to subjects less that years of age, virosomal trivalent subunit vaccine (inflexal ® -v, crucell, leiden, the netherlands; batch numbers , , ) offered to subjects years old or older, and an mf tm -adjuvanted trivalent subunit vaccine (chiromas ® , novartis vaccines and diagnostics, massachusetts, usa; batch numbers , , , ) offered by licensure requirements to those years old or older. subunit trivalent non-adjuvanted was offered in the five health districts included in the study; virosomal vaccine was used in two, and the mf tm -adjuvanted vaccine was used in three. the strains included in the influenza vaccine for the - season were a/california/ / (h n )-like, a/perth/ / (h n )like, and b/brisbane/ / -like [ ] . we established an active surveillance system. full-time field researchers identified, monday to saturday, patients who were hospitalized, coming from the emergency department, in the previous - h. patients whose indications for admission were any of a predefined set of conditions, described as possibly associated with a recent influenza infection [ ] , were invited to participate. patients were excluded if institutionalized, not permanent residents, with reported egg allergy, had been hospitalized in the previous days, or if they had had a previous laboratory confirmed influenza infection. patients were included if they reported an ili episode, defined as at least one of these four systemic symptoms (fever or feverishness, malaise, headache, myalgia) and at least one of these three respiratory symptoms (cough, sore throat, shortness of breath), sudden onset was not a requisite for inclusion [ ] , less than days preceding their arrival at the emergency department. the ethics research committee of the centro superior de investigación en salud pública (csisp) approved the study. all study subjects gave written informed consent before enrollment. a nasopharyngeal and a pharyngeal swab were obtained from each included patient. samples were introduced into vials with viral transport medium and kept at − • c until sent to the reference laboratory. four multiplex real-time rt-pcr/pcr qualitative amplifications were performed: multiplex # for influenza virus type a [ ] and influenza virus type b [ ] ); multiplex # for coronavirus, metapneumovirus, and bocavirus [ ] [ ] [ ] ; multiplex # for respiratory syncytial virus (rsv), adenovirus and parainfluenza virus [ ] ; and multiplex # for rhinovirus [ ] . negative results for viruses were only considered if human ribonucleoprotein gene amplification was positive. laboratory procedures to prevent pcr contamination were followed and a series of multiplex assays # to # negative controls without sample nucleic acid were included in all runs. information was obtained on age, sex, indications for inclusion, hospitalization date, time elapsed from symptoms onset to swabbing, presence of major underlying medical conditions, long-term treatments, contact with children, smoking habits, occupation, number of physician encounters in the last three months, number of hospitalizations in the last year, prescription of antivirals, intensive care unit admission, death in hospital and length of stay. functional status, measured by barthel index [ ] , was obtained in study subjects years old or older. social class was assigned according to occupation [ ] . influenza vaccination status was obtained by asking the patient if he or she had received the current season's influenza vaccine, on which month, and if the vaccine had been administered at least two weeks before the onset of symptoms. in addition, vaccination status was independently ascertained by a researcher blinded to patient characteristics, who consulted valencia's population-based vaccine information system. a patient was considered immunized with the - influenza vaccine if the vaccine was registered as administered or more days before the date of ili onset or if the patient recalled the month when the vaccine was administered and if it had been administered more than two weeks previous to current ili episode onset. information related to the administration of the - seasonal influenza vaccine, the a(h n ) pandemic vaccine and previous -valent polysaccharide plain pneumococcal vaccinations was obtained from the vaccine information system. ive was defined as × ( − adjusted odds ratio [or]) [ , ] . the adjusted or was obtained using a logistic regression model using stepwise background selection of the variables, with a criterion of p < . to remain in the model, starting with a fully saturated model, including being immunized with current season's vaccine, sex, age (in years of age intervals), socioeconomic class, number of high-risk conditions, obesity (imc ≥ ), smoking antecedents, number of physician encounters in the last three months, hospitalizations in the last year, pneumococcal vaccination, antiviral prescription, epidemiological week and time from symptoms onset to swabbing. we defined four groups for ive estimation: (a) all cases and controls enrolled (overall group); (b) all cases and controls with high-risk conditions regardless of age; (c) cases and controls years old or older and (d) cases and controls years old or older with high-risk conditions. to validate our estimates, we computed ive against rsv-related hospitalization following the same design and analysis strategy followed for influenza-related hospitalization, but in this instance cases were those positive for rsv and controls those positive for the other respiratory viruses, including influenza. the significance in differences in the distribution of covariates, between cases and controls, was estimated using the chi-squared, or fisher's test, for categorical variables; and the t-test, or kruskal-wallis test, for continuous variables; p < . was considered significant. all probabilities were -tailed. all analyses were performed with stata . (statacorp, college station, tx). we identified eligible patients, complied with all inclusion criteria, ( %) were positive for influenza and considered cases; ( %) were positive for other respiratory viruses and considered controls (fig. ) . swabs were performed days or less after onset of symptoms in % of study subjects. time from onset to swabbing was similar between cases and controls, median was in both instances four days, p = . . hospitalization rate associated with any of the respiratory viruses assessed was per , years old or older. by age group, hospitalization rate associated to respiratory viruses was , , , and per , - , - , - , and years old or older, respectively. influenza-related hospitalization rate was , , , and per , - , - , - and years old and older. type and number of virus identified were: h n pdm , ( %); rsv, ( %); rhinovirus, ( %); coronavirus, ( %); influenza b, ( %); parainfluenza virus, ( %); mixed infections, ( %); h n , ( %); and human metaneumovirus, ( %). including mixed infections, influenza accounted for % (n = ) of all respiratory viruses identified. influenza subtypes identified were h n pdm (n = ; %), influenza b (n = ; %), and h n (n = ; %). influenza viruses circulated concurrently with rsv, rhinovirus and coronavirus (fig. ). there were no differences regarding emergency admission diagnoses between cases and controls; with the exception of heart failure, that was % in cases compared to % in controls (p = . ) ( table ). in % of influenza patients the presenting complain was not for a respiratory condition (table ) . the duration of symptoms previous to admission was evenly distributed between cases and controls, p = . (table ). there were no differences in the percentage of sudden onset of symptoms, malaise, myalgia, headache, sore throat or shortness of breath; the only significant difference was the frequency of fever in influenza cases, p = . (table ) . when compared to controls, cases were younger, had fewer high-risk conditions, were of higher social class, more frequently smokers, and consulted their general practitioners or had been hospitalized in fewer occasions (table ). there were no differences between cases and controls aged years or more in their barthel index scores (table ) . when restricting the comparison, between cases and controls, by the presence of high-risk conditions, the differences that remained significant were age, -valent pneumococcal vaccination, and having been vaccinated with the previous or current season influenza vaccines (table ) . when restricted to those years old or older, age and influenza vaccination with the previous or current seasonal influenza vaccine remained as significant differences between cases and controls ( table ). compared to . % cases, . % controls were immunized with the influenza seasonal vaccine; p < . (table ) . controls also had more often been vaccinated with the -valent pneumococcal polysaccharide vaccine (p = . ), seasonal influenza (p < . ), and pandemic (p = . ) vaccines (table ) . when high-risk conditions or age were taken into account, no differences were observed in the percentage of cases and controls vaccinated with the pandemic vaccine, and only in those with table cases and controls a characteristics and vaccination history by group: overall, highrisk conditions, and years old or older. high-risk conditions, irrespective of age, there was a difference (p = . ) in pneumococcal vaccination ( % cases vs. % controls) ( table ) . this difference was not observed between cases and controls years old or older ( table ). according to the presence of high-risk conditions or age, the percentage of controls who had been vaccinated with the vaccine compared to cases remained significantly higher (table ) . current influenza season vaccination was highly associated with previous influenza-seasonal vaccination (p < . ) and age or older (p < . ). adjusted vaccine effectiveness to prevent confirmed influenzaassociated hospitalization was % ( %ci, - %) ( table ) . for the subgroup analysis, in those with high-risk conditions influenza vaccine effectiveness estimate was % ( %ci, - %); for those years old or older, it was % ( %ci, - %); and for those years old or older with high-risk conditions it was % ( %ci, - %) ( table ). the overall adjusted or of rsv-associated hospitalization of seasonal influenza vaccination was . ( %ci, . - . ); for those with high-risk conditions it was . ( %ci, . - . ); for those years old or older, . ( %ci, . - . ); and for those years old or older with high-risk conditions, . ( %ci, . - . ). subjects vaccinated experienced a risk of influenza-related hospitalization two times lower compared to the unvaccinated. the vaccine preventive effect was specific for influenza. three recent systematic reviews of studies reporting influenza vaccine efficacy or effectiveness [ , , ] reach the conclusion that evidence on ive to prevent influenza in older adults is scarce, elusive or non-existent. osterholm et al. [ ] looked for studies published between january and february with outcomes confirmed by rt-pcr or viral culture, as estimates based on serologic outcomes [ ] overestimate inactivated vaccine efficacy [ ] . they identified only two observational studies in older adults [ , ] . talbot et al. [ ] studied three consecutive seasons ( - ), using a test-negative case-control design, and reported a pooled adjusted ive of % for influenza-related hospitalizations; however, these estimates were only significant when pooled over three seasons. more recently, castilla et al. [ ] conclude that ive for preventing laboratory-confirmed influenzarelated hospitalization in adults years of age or older, during the - influenza season, is % to %. influenza vaccine effectiveness depends closely on the match of the vaccine strain to the circulating strain [ , ] . during the - influenza season, % of hospitalized subjects with confirmed influenza had been immunized with the seasonal vaccine. this was in clear contrast to what was observed during the - autumn pandemic wave, when, in presence of a good match between the circulating and the vaccine strain, vaccine failures were rare [ ] . the percentage of vaccine failures observed during the - influenza season can be interpreted considering that % of specimens collected in europe showed a reduced activity against the a/california/ / vaccine virus strain [ ] . we tried to minimize selection bias by an enrollment strategy based on an active surveillance system, the use of broad eligibility requirements for inclusion, completeness of inclusion, and enrolling subjects without previous knowledge of their vaccination or case-control status. we reduced classification bias by the use of two independent sources to ascertain vaccination, performing rt-pcr for influenza diagnosis, and by the case-case comparison. patients' recall is considered a valid source of influenza vaccination status [ ] , but is limited by recall bias and uncertainty on date of vaccine administration, or type of vaccine administered. record of vaccination reliably indicates immunization, but absence of record is not informative. we estimate electronic vaccine information system sensitivity as %, and specificity as %, during the - autumn pandemic wave [ ] . with the data collected in the present study, and using a capture recapture method [ ], completeness of ascertainment was % for electronic vaccine information system, % for patient recall, and % for both sources. we aimed to reduce classification bias considering a study subject as vaccinated or non-vaccinated adding the information provided by both sources. rt-pcr is the preferred diagnostic test for influenza [ ] ; but, case status misclassification may contribute to underestimation of ive because of false-negative rt-pcr results [ , , ] . to maximize rt-pcr sensitivity, we included patients with onset of symptoms seven or less days before hospitalization. pcr positivity is, with a similar swabbing strategy, % and %, at and days after symptoms onset [ ] . a non-differential misclassification of true positives as negatives cannot be ruled out and underestimation of vaccine effectiveness is to be expected if a test-negative design is used. this was minimized by case-case comparison [ , ] . although nasopharyngeal aspirate is considered the best specimen for detection of influenza viruses [ ] , we opted for pharyngeal (throat) and nasopharyngeal swabbing to reduce patients discomfort and performance easiness. in children nasal swabs are comparable to that of nasopharyngeal aspirates for the detection of all major respiratory viruses, except rsv [ ] . in adults, swabbing has been used to study respiratory virus disease [ , ] , and ive [ , , , , ] . we obtained a yield of positives similar to other studies on hospitalized adults [ , ] , and the timing of the epidemic wave and types and subtypes we identified were consistent with those reported by spain's surveillance system [ ] . swabbing is a reliable and convenient alternative to obtain specimens for rt-pcr testing [ ] , and accounting for days elapsed from symptoms onset to swabbing, should limit the effect of misclassification of true positives as negatives [ , ] . the case-case analysis approach design assures comparability of controls to cases [ , ] . in a case-case comparison approach, cases and controls should mainly differ in the exposure (and its correlates) associated to the outcome of interest [ ] . all this is even more plausible if influenza and other respiratory viruses cocirculate concurrently (fig. ) . . . impact of age as a confounder and age-related protection due to previous exposure age effect was taken into account by adjustment, and by performing an analysis restricted to those years old or older. vaccine effectiveness could be explained in the elderly by acquired protection due to distant exposure to similar h n strains. we consider this pre-existing protection bias in our results as debatable. first, we observed the third h n pdm wave, this repeated circulation levels exposure to h n pdm over the age range [ ] . second, age-specific h n pdm influenza-related hospitalization rates were in our population two to seven times higher in the years or more age group. third, seroepidemiology studies [ ] [ ] [ ] have described the persistence of protective antibody titers against h n pmd only in a small fraction of subjects years old or older [ ] [ ] [ ] . fourth, when t cell epitopes are compared between h n pdm and seasonal h n , % and % for cd + and cd +, respectively, are conserved [ ] , hence a less dependent on age protection for severe episodes should be expected in those years old or older [ ] . fifth, vaccine effectiveness did not differ when age was considered. the main weakness of our study was the number of influenzarelated hospitalization. although we were able to assess adjusted ive on large groups, this was done with broad confidence intervals, and we did not attain a sufficient number of cases to provide robust ive estimates by virus strain or vaccine type. we report ive estimates with a low probability of bias and the current vaccines provided a significant health benefit. any single ive study results are difficult to generalize. variability of the factors involved, such as circulating strains, vaccine types and composition, match between vaccine's and circulating strains, population characteristics, and outcomes measured are limitations to generalizability. future studies should be planned, after taking into consideration the strengths and limitations exposed, to attain the necessary statistical power to obtain robust ive estimates by virus antigenic subtypes, comparing the different vaccines available, and for relevant high-risk groups. the impact of influenza epidemics on hospitalizations influenza vaccines. who position paper the annual production cycle for influenza vaccine efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis influenza vaccine: the challenge of antigenic drift influenza vaccination: policy versus evidence a systematic review of the evidence on the effectiveness and risks of inactivated influenza vaccines in different target groups estimating the effect of influenza vaccines efficacy studies of influenza vaccines: effect of end points used and characteristics of vaccine failures influenza seasonal vaccine, preliminary mid-season effectiveness estimates: reason for concern, confounding or are we following the right track? effectiveness of inactivated influenza vaccines varied substantially with antigenic match from the - season to the - season association between the - seasonal influenza vaccine and pandemic h n illness during spring-summer : four observational studies from canada cyceva study team. estimating the influenza vaccine effectiveness in elderly on a yearly basis using the spanish influenza surveillance network -pilot case-control studies using different control groups and pandemic vaccines, to prevent influenza hospitalizations during the autumn influenza pandemic wave in castellón, spain. a testnegative, hospital-based, case-control study vaccine effectiveness against laboratory-confirmed influenza in healthy young children: a case-control study effectiveness of seasonal vaccine in preventing confirmed influenza-associated hospitalizations in community dwelling older adults effectiveness of h n / monovalent and trivalent influenza vaccines against hospitalization with laboratory-confirmed h n / influenza in australia: a test-negative case control study vaccine effectiveness in preventing influenza hospitalizations in navarre, spain, - : cohort and case-control study case-case comparisons to study causation of common infectious diseases recommended viruses for influenza vaccines for use in the - northern hemisphere influenza season influenza surveillance influenza case definition rapid multiplex reverse transcription-pcr typing of influenza a and b virus, and subtyping of influenza a virus into h typing (a/b) and subtyping (h /h /h ) of influenza a viruses by multiplex real-time rt-pcr assays clinical disease in children associated with newly described coronavirus subtypes evaluation of a new rapid antigen test using immunochromatography for detection of human metapneumovirus in comparison with real-time pcr assay real-time pcr for diagnosis of human bocavirus infections and phylogenetic analysis increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms new molecular detection tools adapted to emerging rhinoviruses and enteroviruses effectiveness of the mf -adjuvanted influenza vaccine in preventing emergency admissions for pneumonia in the elderly over years of age proposal for a social class measure, working group of the spanish society of epidemiology and the spanish society of family and community medicine proportion of disease caused or prevented by a given exposure, trait or intervention what does the odds ratio estimate in a case-control study? vaccines for preventing influenza in the elderly the efficacy of influenza vaccination in elderly individuals. a randomized doubleblind placebo-controlled trial i-move towards monitoring seasonal and pandemic influenza vaccine effectiveness: lessons learnt from a pilot multi-centric case-control study in europe community network of reference laboratories (cnrl) for human influenza in europe. influenza virus characterisation, summary europe evaluation of selfreported and registry-based influenza vaccination status in a wisconsin cohort validez del registro de vacunas nominal para conocer el estado de vacunación frente a la gripe en adultos ingresados en hospitales de la comunidad valenciana. • congreso de la asociación española de vacunología (aev) capture-recapture methods in epidemiology: methods and limitations viral loads and duration of viral shedding in adult patients hospitalized with influenza laboratory diagnosis of influenza comparison between pernasal flocked swabs and nasopharyngeal aspirates for detection of common respiratory viruses in samples from children respiratory syncytial virus infection in elderly and high-risk adults human metapneumovirus infections in adults: another piece of the puzzle the diagnosis of viral respiratory disease in older adults estimates of pandemic influenza vaccine effectiveness in europe, - : results of influenza monitoring vaccine effectiveness in europe (i-move) multicentre case-control study vigilancia de la gripe en españa, temporada - . (desde la semana / hasta la semana / ) incidence of pandemic influenza a h n infection in england: a cross-sectional serological study serologic survey of pandemic (h n ) virus, guangxi province high frequency of cross-reacting antibodies against pandemic influenza a (h n ) virus among the elderly in finland preexisting immunity against swine-origin h n influenza viruses in the general human population the authors thank the staff of the hospital la plana, in vila-real; arnau de vilanova, in valencia; la ribera, in alzira; san juan, in alicante; and hospital de elda, in elda. as well, we thank all the study participants and their families.we wish to express our recognition to prof juan garcía-de-lomas for his support and contribution on the laboratory methods section. we also acknowledge the dedication and commitment of researchers in the field begoña escribano-lópez, verónica alcarria-garcía, ester huet-trujillo, Ángela lópez-doménech, montserrat cano-armenteros m and consuelo calvo-mas.funding: the study was funded in part by contract code grt between sanofi-pasteur and centro superior de investigación en salud pública (csisp). sanofi-pasteur did not participate in the design or conduct of the study, collection, management, analysis, or interpretation of the data, writing the report, and the decision to submit the report for publication. key: cord- -axkdf vu authors: kim, shin-hee; samal, siba k. title: newcastle disease virus as a vaccine vector for development of human and veterinary vaccines date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: axkdf vu viral vaccine vectors have shown to be effective in inducing a robust immune response against the vaccine antigen. newcastle disease virus (ndv), an avian paramyxovirus, is a promising vaccine vector against human and veterinary pathogens. avirulent ndv strains lasota and b have long track records of safety and efficacy. therefore, use of these strains as vaccine vectors is highly safe in avian and non-avian species. ndv replicates efficiently in the respiratory track of the host and induces strong local and systemic immune responses against the foreign antigen. as a vaccine vector, ndv can accommodate foreign sequences with a good degree of stability and as a rna virus, there is limited possibility for recombination with host cell dna. using ndv as a vaccine vector in humans offers several advantages over other viral vaccine vectors. ndv is safe in humans due to host range restriction and there is no pre-existing antibody to ndv in the human population. ndv is antigenically distinct from common human pathogens. ndv replicates to high titer in a cell line acceptable for human vaccine development. therefore, ndv is an attractive vaccine vector for human pathogens for which vaccines are currently not available. ndv is also an attractive vaccine vector for animal pathogens. infectious diseases have been emerging and reemerging over millennia [ ] . human immunodeficiency virus (hiv), severe acute respiratory syndrome coronavirus (sars-cov), and the most recent pandemic h n influenza virus are only a few of many examples of emerging infectious pathogens in the modern world [ ] . each of these diseases has global societal and economic impact related to unexpected illnesses and deaths, as well as interference with travel, business, and daily activities. to overcome emerging, reemerging, as well as stable infectious diseases, the demand for development of efficient vaccines has greatly increased. historically, live attenuated vaccines have provided the most effective protection against viral infection and disease [ ] . however, there have been safety concerns with the risk of reversion to the wild-type pathogen phenotype as shown with some traditional live attenuated vaccines such as the polio vaccine. furthermore, development of live attenuated vaccines has not been successful for many important pathogens. on the other hand, inactivated vaccines are generally not very effective and require a high containment laboratory for cultivation of highly virulent pathogens. also, there is a risk of incomplete inactivation for inactivated vaccines. therefore, there is a need for an alternative approach for development of vaccines. replicating viral vector vaccines offer a live vaccine approach without requiring involvement of the complete pathogen or cultivation of the pathogen [ ] . replicating viral vectors have the ability to synthesize the foreign antigen intracellularly and induce humoral, cellular, and mucosal immune responses. specifically, vectored vaccines can have advantages for (i) viruses for which a live attenuated vaccine might not be feasible (i.e., hiv); (ii) viruses that do not grow well in vitro (i.e., human papillomavirus, hepatitis c virus, and norovirus); (iii) highly pathogenic viruses that present safety challenges during vaccine development (i.e., sars-cov and ebola virus); (iv) viruses that lose infectivity due to physical instability (i.e., respiratory syncytial virus (rsv)); and (v) viruses that can exchange genes with circulating viruses (i.e., coronaviruses, influenza viruses, and enteroviruses) [ ] . a vectored vaccine can be rapidly engineered against a newly emerging pathogenic virus by inserting the gene of the protective antigen of the virus into the genome of the viral vector. in general, the magnitude of the immune response to live viral vector vaccines is substantially greater and broader than that induced by vaccines based on subunit proteins or inactivated viruses. furthermore, manufacturing of vectored vaccines against highly pathogenic viruses do not require a high level of biosafety containment laboratories. newcastle disease virus (ndv) is a fast-replicating avian virus that is prevalent in all species of birds [ ] . in most avian species, ndv infections do not result in disease. in chickens, ndv causes a highly contagious respiratory and neurologic disease, leading to severe economic losses in the poultry industry worldwide [ ] . ndv strains vary widely in virulence. based on the severity of the disease in chickens, ndv strains are classified into three pathotypes: lentogenic strains which cause mild or asymptomatic infections that are restricted to the respiratory tract; mesogenic strains which are of intermediate virulence; and velogenic strains which cause systemic infections with high mortality [ ] . naturally occurring low-virulent ndv strains, such as lasota and b , are widely used as live attenuated vaccines to control newcastle disease in poultry. although ndv primarily infects avian species, many non-avian species have also been shown to be naturally or experimentally susceptible to infection. the advent of a reverse genetics system to manipulate the genome of ndv not only allowed us to study the molecular biology and pathogenesis of ndv but also to develop ndv as a vaccine vector against diseases of humans and animals. ndv vector has several advantages over other replicating viral vectors. avirulent ndv strains are highly safe in avian and non-avian species. ndv replicates well in vivo and induces a robust immune response. in contrast to adeno, herpes, and pox virus vectors whose genome encodes a large number of proteins, ndv encodes only seven proteins and is thus less competition for immune responses between vector proteins and the expressed foreign antigen. ndv replicates in the cytoplasm, does not integrate into the host cell dna, and does not establish persistent infection. recombination involving ndv is extremely rare. ndv has a modular genome that facilitates genetic manipulation. ndv infects via the intranasal route and therefore induces both mucosal and systemic immune responses. a wide range of ndv strains exists that can be used as vaccine vectors. ndv-vectored vaccine can also be used as a "differentiating infected from vaccinated animals" (diva) vaccine. in this review article, we have reviewed the biology of ndv, development of reverse genetic systems for generation of ndv-vectored vaccines, and use of ndv vector for development of human and veterinary vaccines. ndv is a member of the genus avulavirus in the family paramyxoviridae [ ] . ndv virions are pleomorphic, but mostly spherical with a diameter of nm. the virion is enveloped with a bilayer lipid membrane. the genome of ndv is a non-segmented, negative-sense, single-stranded rna of , to , nucleotides containing six transcriptional units ( -n-p-m-f-hn-l- ) (figure ) . the genome encodes a nucleocapsid protein (n), a phosphoprotein (p), a matrix protein (m), a fusion protein (f), a hemagglutinin-neuraminidase protein (hn), and a large polymerase protein (l). an additional protein called the v protein is produced by rna editing of the p gene. the beginning and end of each gene contain control sequences, known as gene-start (gs) and gene-end (ge), respectively. the viral rna-dependent rna polymerase begins transcription at the end of the genomic rna, in a sequential manner by a stop-start mechanism [ ] . the re-initiation of transcription at the gs is not perfect, thus leading to a gradient of mrna abundance with high levels of mrna transcription viruses , , of located at the end. the genome length of ndv must be an even multiple of six for efficient virus replication following the "rule of six" [ ] . in ndv, the hn and f proteins are the two integral membrane proteins. the hn protein is responsible for attachment of the virion to sialic acid containing cell surface receptors. the f protein mediates entry of the virus into the host cell by fusion of the viral envelope to the plasma membrane. the f protein is synthesized as a precursor (f ) that is cleaved by host cell protease into two biologically active f and f subunits. cleavage of the f protein is a pre-requisite for virus entry and cell-to-cell fusion. the amino acid sequence at the f protein cleavage site has been identified as the primary determinant of virulence [ , ] . virulent ndv strains have multibasic residues that conform to the preferred cleavage site of the intracellular protease furin present in most cell types. in contrast, avirulent ndv strains typically contain one or two basic residues at the f protein cleavage site and are delivered to the plasma membrane in an uncleaved form for cleavage by extracellular proteases, thus restricting viral replication to the respiratory and enteric tracts where secreted proteases for cleavage are available. infectious ndv can be recovered entirely from cloned cdna by transfecting cultured cells with plasmids encoding the viral components of a functional nucleocapsid, full-length antigenomic rna, and the major proteins involved in replication and transcription, i.e. the n, p, and l proteins under the control of bacteriophage t rna polymerase promoter [ ] (figure ). this method, which is also known as reverse genetics technique, is now available for all three pathotypes of ndv strains [ ] [ ] [ ] [ ] . in general, a foreign gene flanked by ndv gs and ge sequences is inserted into a ′ non-coding region of an ndv genome as an additional transcription unit. due to a polar gradient transcription, foreign genes are expressed more efficiently when placed closer to ′ end of the genome. although a foreign gene can be placed between any two genes of ndv, the insertion site between the p and m genes has been found optimal for efficient expression of the foreign protein and replication of ndv [ ] [ ] [ ] . the insertion of a foreign gene into ndv genome increases its genome length and gene number and often has a growth retardation effect on virus replication in vitro and in vivo [ ] . ndv accommodates foreign genes (at least . kb in length) with a good degree of stability [ ] . a single ndv vector can also express at least three different foreign genes. in ndv, the hn and f proteins are the two integral membrane proteins. the hn protein is responsible for attachment of the virion to sialic acid containing cell surface receptors. the f protein mediates entry of the virus into the host cell by fusion of the viral envelope to the plasma membrane. the f protein is synthesized as a precursor (f ) that is cleaved by host cell protease into two biologically active f and f subunits. cleavage of the f protein is a pre-requisite for virus entry and cell-to-cell fusion. the amino acid sequence at the f protein cleavage site has been identified as the primary determinant of virulence [ , ] . virulent ndv strains have multibasic residues that conform to the preferred cleavage site of the intracellular protease furin present in most cell types. in contrast, avirulent ndv strains typically contain one or two basic residues at the f protein cleavage site and are delivered to the plasma membrane in an uncleaved form for cleavage by extracellular proteases, thus restricting viral replication to the respiratory and enteric tracts where secreted proteases for cleavage are available. infectious ndv can be recovered entirely from cloned cdna by transfecting cultured cells with plasmids encoding the viral components of a functional nucleocapsid, full-length antigenomic rna, and the major proteins involved in replication and transcription, i.e., the n, p, and l proteins under the control of bacteriophage t rna polymerase promoter [ ] (figure ). this method, which is also known as reverse genetics technique, is now available for all three pathotypes of ndv strains [ ] [ ] [ ] [ ] . in general, a foreign gene flanked by ndv gs and ge sequences is inserted into a non-coding region of an ndv genome as an additional transcription unit. due to a polar gradient transcription, foreign genes are expressed more efficiently when placed closer to end of the genome. although a foreign gene can be placed between any two genes of ndv, the insertion site between the p and m genes has been found optimal for efficient expression of the foreign protein and replication of ndv [ ] [ ] [ ] . the insertion of a foreign gene into ndv genome increases its genome length and gene number and often has a growth retardation effect on virus replication in vitro and in vivo [ ] . ndv accommodates foreign genes (at least . kb in length) with a good degree of stability [ ] . a single ndv vector can also express at least three different foreign genes. [ ] [ ] [ ] . the insertion of a foreign gene into ndv genome increases its genome length and gene number and often has a growth retardation effect on virus replication in vitro and in vivo [ ] . ndv accommodates foreign genes (at least . kb in length) with a good degree of stability [ ] . a single ndv vector can also express at least three different foreign genes. avirulent ndv strains lasota and b are commonly used as vaccine vectors because of their proven track records of safety. mesogenic and velogenic ndv strains are not used as vaccine vectors because they are virulent in chickens. in an experimental study, the mesogenic strain beaudette c (bc) was evaluated as a vaccine vector in nonhuman primates [ ] . strain bc replicated to a higher titer and induced a substantially higher level of antibody response compared to strain lasota, indicating it would be an effective vaccine vector. ndv has several advantages for use as a vaccine vector in humans. ndv is safe in humans, due to a natural host range restriction. in nonhuman primates, the intranasal and intratracheal inoculation of african green and rhesus monkeys with . plaque-forming units (pfu) per site of ndv did not cause any disease symptoms and its replication was restricted to the respiratory tract [ ] . in humans, infection by ndv appears to be limited and benign based on both anecdotal observations with bird handlers and in clinical studies using ndv as an oncolytic agent [ ] . according to a clinical study for ndv as an oncolytic agent in humans, intravenous administration of pfu of ndv to humans was safe without causing adverse effects [ ] . ndv shares only a low level of amino acid sequence identity with known human paramyxoviruses and are antigenically distinct from common human and animal pathogens, and thus would not be affected by preexisting immunity in humans. ndv infects via the intranasal route and has been shown to induce humoral and cellular immune responses both at the mucosal and systemic levels in murine and nonhuman primate models [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . ndv is a strong stimulator of the host immune response, thus providing an adjuvant effect. the use of avirulent pathotypes of ndv in humans prevents the possibility of accidental spread of a virulent virus strain from treated patients to birds. ndv grows to high titers not only in embryonated eggs ( pfu/ml) but also in vero cells ( pfu/ml), which is acceptable for human vaccine development. in fact, ndv has been used to express protective antigens of various human pathogens and has shown promising results in nonhuman primates. ndv-vectored vaccines for several human pathogens are discussed as follows (table ) . the potential of recombinant ndv strain b as an effective vaccine vector for humans was first evaluated by expressing an influenza virus (a/wsn/ ) hemagglutinin (ha) protein [ ] . the expressed ha protein was incorporated into virions and appeared to be cleaved, indicating that the ha protein was accessible to proteolytic enzymes. in vitro growth kinetics and pathogenicity test in embryonated chicken eggs indicated attenuation of the recombinant ndv. intravenous administration of mice induced higher titers of antibody to influenza virus ha than intraperitoneal administration. further, immunized mice by the intravenous route were completely protected against a lethal dose of influenza virus, suggesting that ndv can be a safe and effective vaccine vector for possible use in mammalian and avian species. the potential of ndv as a vaccine vector for use in humans was first determined in nonhuman primates. two ndv strains lasota and bc were evaluated as vaccine vectors in nonhuman primates by inserting the hn protein of human parainfluenza virus type (hpiv ) as a protective antigen [ ] . two doses of immunization with ndv strains confirmed their restricted replication in african green monkeys (ndv-bc and ndv-ls) and in rhesus monkeys (ndv-bc only). however, the serum antibody response following the second dose exceeded that observed with hpiv infection, even though hpiv replicated much more efficiently than ndv in these animals. this is the first study to demonstrate efficacy of ndv-vectored vaccine in nonhuman primates. ebola virus (ebov) causes severe hemorrhagic fever in humans with a fatality rate of up to % (species zaire ebolavirus) of infected individuals [ ] . due to the limitation of inactivated vaccines, viral vectors based on common human pathogens have been used for ebov vaccine. to overcome the high seroprevalence against vectors based on common human pathogens in the adult human population, recombinant ndv strain lasota expressing the ebov gp envelope protein was generated to evaluate its potential as a vaccine for ebov [ ] . following one intranasal and intratracheal inoculation of rhesus monkeys with ndv/gp, titers of ebov-specific antibodies and serum ebov-neutralizing antibodies, were undetectable or low compared to those induced by hpiv /gp. however, a second immunization resulted in a substantial boost in serum immunoglobulin (ig) g enzyme-linked immunosorbent assay (elisa) titers, yet the titers remained lower than those induced by a second dose of hpiv /gp. in contrast, the elisa iga titers in respiratory tract secretions and the serum ebov-neutralizing antibody titers were equal to those induced after the second dose of hpiv /gp, showing that the efficacy of ndv vector can be comparable to that of hpiv vector by prime-boosting vaccination [ ] . ndv was evaluated as a vaccine vector for another important emerging pathogen, the severe acute respiratory syndrome-associated coronavirus (sars-cov) [ ] . two ndv vectors were constructed: mesogenic strain bc (ndv-bc) and lentogenic strain lasota in which the f protein cleavage sequence was modified to that of strain bc (ndv-vf) [ ] . these ndv vectors were engineered to express the sars-cov spike s glycoprotein, the major protective antigen. two dose immunizations of african green monkeys induced a robust neutralizing antibody response, resulting in reduction of virus shedding after challenge with sars-cov ( % tissue culture infective dose (tcid )). specifically, immunization with ndv-vf vector resulted in sars-cov titers of a -fold, -fold, and -fold reduction in nasal turbinate, trachea, and lung, respectively, compared with the control animals. the ndv-bc vector was even more effective, with average reductions in viral titer of -fold, -fold, and -fold in the nasal turbinate, trachea, and lung, respectively. this study demonstrated the safety and protective efficacy of ndv as a topical respiratory vaccine vector for sars-cov. the use of viral vectors expressing selected hiv antigens has been a promising vaccine strategy. the potential of ndv-vectored vaccine against hiv infection was first evaluated by generating recombinant ndv expressing simian immunodeficiency virus (siv) gag protein (rndv/sivgag) [ ] . the vaccine virus induced gag-specific cellular immune responses in mice. among intravenous, intraperitoneal, and intranasal immunization routes, intranasal administration induced the strongest protective immune response against a surrogate challenge virus (rvac/sivgag) following a booster immunization with recombinant influenza viruses expressing immunogenic portions of siv gag. specifically, this heterologous vaccination approach resulted in approximately, a -fold reduction in rvac/sivgag titers at day after challenge compared to titers of control mice injected with phosphate-buffered saline (pbs). the magnitude of the protective immune response also correlated with the levels of cellular immune responses to gag. these results suggest that ndv vector can be a suitable candidate vaccine against hiv. the hiv gag and env proteins have been expressed by ndv vector [ , [ ] [ ] [ ] . the expression level of gag protein was optimized using different insertion sites in the ndv genome. it was found that the codon-optimized gag inserted between the p and m genes of ndv induced the highest level of protein expression and an enhanced immune response against hiv gag in mice [ ] . in another study, expression of gp env protein by ndv vector lasota also induced systemic and mucosal antibody responses in guinea pigs [ ] . priming/boosting by the intranasal route was more immunogenic than by the intramuscular route. further, coexpression of gp env and p gag by vector lasota enhanced both env-specific and gag-specific immune responses in guinea pigs [ ] . this approach was efficient in inducing cellular and protective immune responses to challenge with vaccinia viruses expressing hiv- env and gag in mice. these results suggest that vaccination with a single ndv vector coexpressing env and gag represents a promising strategy to enhance immunogenicity and protective efficacy against hiv. in addition, heterologous prime (ndv expressing gp ) and boosting (purified gp protein) approach induced high neutralizing antibody titer in guinea pigs [ ] . these findings suggest that vaccination with multiple hiv antigens in combination can broaden antiviral immune responses. respiratory syncytial virus (rsv) is a major cause of severe lower respiratory tract disease in infants and elderly [ ] . the development of an effective vaccine against rsv is a high priority. in order to develop a vector vaccine against rsv, ndv strain b was used to express the fusion glycoprotein of rsv [ ] . ndv was chosen as a viral vector because of its ability to induce a strong interferon (ifn)-α/β response. the rsv f protein was more immunogenic when presented by ndv-f than by live rsv, and this correlated with an increased ability of ndv to activate antigen-presenting cells in vitro and to induce high levels of ifn-α/β in vivo. rsv f-specific, cd + memory t cells were present in greater numbers in ndv-f-primed balb/c mice than in animals previously infected with rsv. consequently, ndv vaccine virus provided protection from rsv challenge. this study also highlights the adjuvant effect of ndv vector mediated by the potent ifn induction. ndv has also been used as a vector to express the immunogens of a bacterial pathogen. lyme borreliosis is a prevalent vector-borne disease in the united states, europe and parts of asia. ndv was used to express the basic membrane protein a (bmpa) and the outer surface protein c (ospc) of the lyme disease pathogen borrelia burgdorferi [ ] . c h or balb/c mice that were immunized intranasally with the ndv vectors mounted vigorous serum antibody responses against the ndv vector, but failed to mount a robust response against either the intracellular or extracellular forms of bmpa or ospc. in contrast, a single immunization of hamsters with the ndv vectors via the intranasal, intramuscular, or intraperitoneal route resulted in rapid and rigorous antibody responses against the bmpa and ospc. challenged with b. burgdorferi ( cells/animal), immunization with vector-expressing bmpa provided a reduction of the pathogen load in the joints. this study showed the potential of ndv as a vaccine vector against bacterial pathogens. nipah virus (niv) is a deadly emerging zoonotic pathogen that causes fatal encephalitis in humans and pigs [ ] . the glycoprotein (g) and fusion protein (f) are two major niv surface glycoproteins that stimulate protective immune responses. ndv strain lasota expressing the niv g and f proteins (rla-nivg and rla-nivf, respectively) were evaluated for their immunogenicity in mice and in pigs [ ] . following the second dose of immunization, rla-nivg and rla-nivf induced niv-specific neutralizing antibodies in mice and long-lasting neutralizing antibodies in pigs (at least for weeks). this study also showed that rla-nivg induced higher levels of neutralizing antibodies than rla-nivf. although the protective efficacy of the vaccines was not evaluated in this study, the vaccine viruses showed the potential to be used for protecting humans and animals against niv infection. norovirus (nov) is the most frequent cause of viral gastroenteritis in people of all ages [ ] . the inability of nov to grow in the cell culture system has greatly hindered development of effective vaccines. to circumvent this obstacle, virus-like particles (vlps) produced by the baculovirus expression system have been commonly used as nov vaccine candidates. as a live vaccine vector, lasota and modified bc strains were used to express the capsid protein (vp ) of nov strain va (gii. ) and norwalk virus (gi. ) [ , ] . for the modified bc vector, the multibasic cleavage site sequence of the f gene was changed to that of strain lasota. the nov-expressed vp protein formed vlps in cell culture and in allantoic fluid of embryonated chicken eggs. the modified bc-vectored vaccine induced higher levels of serum, cellular, and mucosal immune responses than the baculovirus-expressed vlps in mice. these results suggested that ndv has great potential for developing a live nov vaccine. alternatively, vlps produced in large quantities in embryonated eggs or in cell culture by ndv can be a cost-effective method for producing a vlp-based vaccine for humans. this study also has implications for development of ndv-vectored vaccines for other non-cultivable pathogens of humans. ndv-vectored vaccines have been evaluated in several animal species (i.e., chicken, cattle, sheep, cat, mouse, pig, and dog) for veterinary use [ ] (table ) . ndv is a natural vaccine vector for poultry pathogens. live attenuated ndv vaccines are widely used all over the world. therefore, an ndv vector carrying the protective antigen of another avian pathogen can be used as a bivalent vaccine. such a vaccine will be economical for poultry farmers. as a bivalent vaccine, the ndv strain lasota was first used to express the host-protective immunogen vp of infectious bursal disease virus (ibdv), a birnavirus, which causes a highly immunosuppressive disease in chickens [ ] . the protective efficacy of lasota-expressing vp protein was evaluated by challenging vaccinated chickens with a highly virulent ndv strain texas gb or a virulent ibdv variant strain. vaccination with rlasota/vp provided % protection against ndv and ibdv. booster immunization induced higher levels of antibody responses against both ndv and ibdv and conferred complete protection against both viruses. these results indicate that the recombinant ndv can be used as a vaccine vector for other avian pathogens. infectious laryngotracheitis is a major respiratory disease in chickens and caused by infectious laryngotracheitis virus (iltv), a herpes virus [ ] . bivalent ndv-vectored vaccines against iltv have been developed to improve the safety of current live attenuated iltv vaccines [ , ] . the protective efficacy of ndvs expressing the three major iltv surface glycoproteins, namely, gb, gc, and gd was evaluated against iltv infection in chickens [ ] . particularly, rndv-expressing gd induced the highest level of neutralizing antibodies among the tested vaccine candidates and completely protected chickens against the challenge of virulent iltv and ndv, showing its potential as a bivalent vaccine. this protective efficacy of rndv gd vaccine was attributed to high levels of envelope incorporation and cell surface expression of gd compared to gb and gc. in another study, lasota viruses expressing gb and gd of iltv were generated and vaccination of chickens with the two viruses conferred protection against virulent iltv and ndv challenges [ ] . in addition, lasota with gb showed the protection of commercial broilers against clinical disease. discrepancy of vaccine efficacy between these two studies could be due to the different levels of gb and gd expressions by ndv vectors and experimental conditions. infectious bronchitis virus (ibv), a coronavirus, is an important avian pathogen, causing respiratory disease in broilers and poor egg production in breeders and layers worldwide [ ] . the spike polypeptide s gene was expressed by lasota (rls/ibv.s ) [ ] . the vaccine virus effectively elicited hemagglutination inhibition antibodies against ndv and protected chickens against lethal challenge with virulent strain ndv/ca . ibv heterotypic protection was assessed using a prime-boost approach with a commercially available attenuated ibv massachusetts (mass)-type vaccine. chickens primed ocularly with rls/ibv.s and boosted with mass were completely protected against challenge with a virulent ark-type strain. the protective efficacy of this heterologous vaccination was similar to that of priming and boosting with mass (mass + mass). based on clinical signs, both vaccinated groups appeared equally protected against challenge compared to unvaccinated challenged chickens. in shedding of challenge virus in the trachea, viral rna was detected in % of rls/ibv.s + mass-vaccinated chickens while chickens vaccinated with mass + mass and unvaccinated challenged controls showed % and % incidence of ibv rna detection, respectively. these results demonstrate the potential of ndv-vectored vaccine for ibv infection. ndv vector has also been used for the prevention of economically important livestock diseases. rift valley fever virus (rvfv), a bunyavirus, causes recurrent large outbreaks in humans and in livestock [ ] . ndv expressing the rvfv structural glycoproteins gn was generated (ndfl-gn) [ ] . immunization of calves via the intranasal route elicited no detectable antibody responses, whereas intramuscular immunization elicited antibodies against both ndv and the gn protein. in general, the titers of rvfv-neutralizing antibodies were modest, varying from to . to improve the efficacy of ndv-vectored vaccine, gn was coexpressed with another glycoprotein gc [ ] , which resulted in the formation of vlps and subsequent release from the producing cells. a homologous prime-boost vaccination of mice with this vaccine virus induced neutralizing antibodies and provided complete protection from a lethal rvfv challenge. the immunogenicity of the vaccine virus was further evaluated in lamb, the main target species of rvfv. a single intramuscular vaccination induced neutralizing antibodies, and this response was significantly boosted by a second vaccination. although coexpression of the gn and gc induced a good immune response, protective efficacy of this vaccine needs to be further evaluated. bovine herpesvirus- (bhv- ) is a major cause of respiratory tract diseases in cattle. since modified live bhv- vaccines can cause latent infection in immunized animals, ndv expressing the glycoprotein d (gd) of bhv- was generated as a vectored vaccine [ ] . a single intranasal and intratracheal inoculation of calves with ndv elicited mucosal and systemic antibodies specific to bhv- . challenge with bhv- showed reduced virus shedding and clinical signs in immunized calves compared to unimmunized claves. in addition, the titers of serum antibodies specific to bhv- were higher in immunized animals compared to unimmunized animals, indicating that the vaccines primed for secondary responses. this indicates that ndv can be used as a vaccine vector in bovines, and bhv- gd may be useful as a mucosal vaccine against bhv- infection. however, vaccination might require augmentation by a second dose or the inclusion of additional bhv- antigens. rabies virus (rv), a rhabdovirus, causes a fatal neurologic disease in humans and in animals [ ] . to generate an effective, safe, and affordable rabies vaccine, ndv strain lasota expressing the rabies virus glycoprotein g (rl-rvg) was evaluated. the safety of rl-rvg vaccine virus was confirmed in cats and dogs. intramuscular vaccination with rl-rvg induced strong and long-lasting protective neutralization antibody responses against rabies virus in dogs and cats. although three doses of vaccination were conducted, the second dose induced the highest levels of immune responses in both cats and dogs. vaccination dose of % embryo infective dose (eid) completely protected dogs from challenge after one year. this study demonstrated protective efficacy of ndv-vectored vaccine against rabies in dogs. this vaccine may also have potential use in high-risk human individuals to control rabies virus infections. canine distemper virus (cdv), a morbillivirus, infects many carnivores and cause several high-mortality disease outbreaks [ ] . the current cdv live vaccine cannot be safely used in some exotic species, such as mink and ferret. ndv strain lasota expressing envelope glycoproteins, hemagglutinin (h, rla-cdvh) and fusion protein (f, rla-cdvf), were generated as vaccine candidates. in immunized minks, rla-cdvh induced higher titers of neutralization antibodies against cdv than rla-cdvf neutralizing antibodies. further, rla-cdvh provided complete protection against virulent cdv challenge during the four weeks of observation. in contrast, all animals immunized with rla-cdvf developed clinical signs of distemper and virus shedding. this study suggested that recombinant ndv expressing the h protein of cdv is a safe and efficient candidate vaccine against cdv in mink. the efficacy of rla-cdvh virus also needs to be evaluated in other host carnivore species. highly pathogenic avian influenza virus (hpaiv) is an economically important pathogen of poultry worldwide. the outbreaks involving h n or h n influenza viruses resulted in lethal infections in poultry and the death of a limited number of people [ ] . therefore, vaccination of poultry against hpaiv could play an important role in reducing virus shedding and raising the threshold for infection and transmission [ ] . however, development of vaccines against hpaiv has been hampered due to poor immunogenicity of the virus [ ] . furthermore, inactivated vaccines are not commonly used because of the high cost due to the requirement of enhanced biosafety level containment and the difficulty in "differentiating infected from vaccinated animals" (diva). the use of live attenuated influenza viruses as vaccines in avian or mammalian species can also raise a major biosafety concern, because the vaccine viruses may become virulent through mutation or genetic reassortment with circulating strains. alternatively, ndv can be an ideal vaccine vector for development of an avian influenza vaccine. ndv infects via the intranasal route and therefore induces both local and systemic immune responses at the respiratory tract [ ] . therefore, it provides a convenient platform for rapid, efficient, and economical immunization. in fact, ndv has been most commonly used as a vaccine vector against aiv. protective efficacy of ndv-vectored vaccines has been evaluated and verified by many different vaccination studies [ , , [ ] [ ] [ ] , ] . for the generation of vaccines, a major protective antigen, hemagglutinin (ha) of hpaiv has been placed between the p and m genes or between the f and hn genes in lentogenic ndv strains lasota or b . to address a safety concern, an ndv-vectored vaccine was further generated by replacing the polybasic cleavage site in hpaiv ha with that from a low-pathogenicity strain of influenza virus [ ] . in addition, the ha gene has been modified to enhance its expression levels by ndv. specifically, elimination of an ndv transcription termination signal-like sequence located within the ha open reading frame of h enhanced expression levels of ha protein by ndv and completely protected chickens after challenge with a lethal dose of velogenic ndv or highly pathogenic aiv, respectively [ ] . in addition, the ectodomain of an h n or h n avian influenza virus ha was fused with the transmembrane and cytoplasmic domains derived from the f protein of ndv [ , ] . this approach resulted in enhanced incorporation of the foreign protein into virus particles and the protection of chickens against both hpaiv and a highly virulent ndv. these studies also demonstrated that ndv can be used to generate a bivalent vaccine. although use of avirulent ndv vectors has been effective in protecting chickens against clinical disease and mortality, some studies also found virus shedding in chickens after challenge with hpaiv [ ] . to enhance the replication of vaccine virus, attenuated mesogenic ndv strain bc has been generated by changing the multibasic cleavage site sequence of the f protein to the dibasic sequence of strain lasota [ ] . additionally, the bc, f, and hn proteins were modified in several ways to enhance virus replication. the modified bc-based vectors replicated better than lasota vector, and expressed higher levels of ha protein and provided complete protection against challenge virus shedding, suggesting its potential to be safely used as a vaccine vector. for effective human vaccines against hpaiv, the immunogenicity of ndv expressing the ha of h n was evaluated in african green monkeys by the intranasal route of administration [ ] . two doses of ndv-vectored vaccine ( ˆ pfu) induced a high titer of h n hpaiv-neutralizing serum antibodies in all of the immunized monkeys. moreover, a substantial mucosal iga response was induced in the respiratory tract, which can potentially reduce or prevent transmission of the virus during an outbreak or a pandemic. the intranasal route of administration is also advantageous for needle-free immunization and is thus suitable for mass immunization. the protective efficacy of vaccine viruses was evaluated in african green monkeys by the intranasal/intratracheal route or by the aerosol route of administration [ ] . each of the vaccine constructs was highly restricted for replication, with only low levels of virus shedding detected in respiratory secretions. all groups developed high levels of neutralizing antibodies against homologous (a/vietnam/ / ) and heterologous (a/egret/egypt/ -namru / ) strains of hpaiv and were protected against challenge with ˆ pfu of homologous hpaiv. this study demonstrated that needle-free, highly attenuated ndv-vectored vaccines were immunogenic and protective in a nonhuman primate model of hpaiv infection. newcastle disease virus (ndv) is an attractive vaccine vector for both human and animal pathogens. the live attenuated vaccine strains used as vaccine vectors have a proven track record of safety and efficacy. ndv vectors not only induce robust humoral and cellular immune responses but also induce mucosal immune response. therefore, ndv can be a vector of choice for mucosal immunization. the ability of ndv to infect a wide variety of non-avian species makes it a potential vector for other animals. ndv is also a promising vaccine vector for use in humans. one advantage is that most humans do not have pre-existing immunity to ndv. ndv-vectored vaccines have also become available commercially (i.e., h n hpaiv vaccine for poultry). emerging infectious diseases: threats to human health and global stability the perpetual challenge of infectious diseases caiv-t comparative efficacy study group. live attenuated versus inactivated influenza vaccine in infants and young children newcastle disease virus as a vaccine vector for humans newcastle disease and related avian paramyxoviruses newcastle disease and other avian paramyxoviruses rescue of newcastle disease virus from cloned cdna: evidence that cleavability of the fusion protein is a major determinant for virulence role of fusion protein cleavage site in the virulence of newcastle disease virus recovery of a virulent strain of newcastle disease virus from cloned cdna: expression of a foreign gene results in growth retardation and attenuation high-level expression of a foreign gene from the proximal first locus of a recombinant newcastle disease virus evaluation of the contributions of the individual viral genes to newcastle disease virulence and pathogenesis generation by reverse genetics of an effective, stable, live-attenuated newcastle disease virus vaccine based on a currently circulating, highly virulent indonesian strain recombinant newcastle disease virus as a vaccine vector optimization of human immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses recombinant newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication nonsegmented negative-strand viruses as vaccine vectors recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates phase i/ii trial of intravenous ndv-huj oncolytic virus in recurrent glioblastoma multiforme successful topical respiratory tract immunization of primates against ebola virus newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens immunization of primates with a newcastle disease virus-vectored vaccine via the respiratory tract induces a high titer of serum neutralizing antibodies against highly pathogenic avian influenza virus newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of h n highly pathogenic avian influenza virus protect against virus challenge in monkeys respiratory tract immunization of non-human primates with a newcastle disease virus-vectored vaccine candidate against ebola virus elicits a neutralizing antibody response newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous h n avian influenza viruses newcastle disease virus vector producing human norovirus-like particles induces serum, cellular, and mucosal immune responses in mice immunogenicity of newcastle disease virus vectors expressing norwalk virus capsid protein in the presence or absence of vp protein induction of cellular immune responses to simian immunodeficiency virus gag by two recombinant negative-strand rna virus vectors newcastle disease virus expressing human immunodeficiency virus type envelope glycoprotein induces strong mucosal and serum antibody responses in guinea pigs mucosal immunization with newcastle disease virus vector coexpressing hiv- env and gag proteins elicits potent serum, mucosal, and cellular immune responses that protect against vaccinia virus env and gag challenges enhanced immune responses to hiv- envelope elicited by a vaccine regimen consisting of priming with newcastle disease virus expressing hiv gp and boosting with gp and sosip gp proteins protection against respiratory syncytial virus by a recombinant newcastle disease virus vector newcastle disease virus-vectored nipah encephalitis vaccines induce b and t cell responses in mice and long-lasting neutralizing antibodies in pigs a host-restricted viral vector for antigen-specific immunization against lyme disease pathogen norovirus disease in the united states recombinant newcastle disease virus-vectored vaccines against human and animal infectious diseases a recombinant newcastle disease virus expressing vp protein of infectious bursal disease virus protects against ndv and ibdv recombinant newcastle disease viral vector expressing hemagglutinin or fusion of canine distemper virus is safe and immunogenic in minks a recombinant newcastle disease virus (ndv) expressing infectious laryngotracheitis virus (iltv) surface glycoprotein d protects against highly virulent iltv and ndv challenges in chickens infectious bronchitis virus s expressed from recombinant virus confers broad protection against challenge intramuscular inoculation of calves with an experimental newcastle disease virus-based vector vaccine elicits neutralizing antibodies against rift valley fever virus rift valley fever virus immunity provided by a paramyxovirus vaccine vector immunization of cattle with recombinant newcastle disease virus expressing bovine herpesvirus- (bhv- ) glycoprotein d induces mucosal and serum antibody responses and provides partial protection against bhv- . vaccine newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats newcastle disease virus expressing h hemagglutinin gene protects chickens against newcastle disease and avian influenza immunization of chickens with newcastle disease virus expressing h hemagglutinin protects against highly pathogenic h n avian influenza viruses engineered viral vaccine constructs with dual specificity: avian influenza and newcastle disease toward a comprehensive phylogeny for mammalian and avian herpesviruses newcastle disease virus (ndv) recombinants expressing infectious laryngotracheitis virus (iltv) glycoproteins gb and gd protect chickens against iltv and ndv challenges the avian coronavirus infectious bronchitis virus undergoes direct low-ph-dependent fusion activation during entry into host cells rift valley fever virus (bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention oie (world organisation for animal health) avian influenza safety and immunogenicity of an inactivated subvirion influenza a (h n ) vaccine contributions of the avian influenza virus ha, na, and m surface proteins to the induction of neutralizing antibodies and protective immunity modified newcastle disease virus vectors expressing the h hemagglutinin induce enhanced protection against highly pathogenic h n avian influenza virus in chickens author contributions: s.h.k. and s.k.s. wrote the manuscript. the authors declare no conflict of interest. key: cord- -wdpatr z authors: matoo, javaid jeelani; bashir, khalid; kumar, ajay; krishnaswamy, narayanan; dey, sohini; chellappa, madhan mohan; ramakrishnan, saravanan title: resiquimod enhances mucosal and systemic immunity against avian infectious bronchitis virus vaccine in the chicken date: - - journal: microb pathog doi: . /j.micpath. . . sha: doc_id: cord_uid: wdpatr z adjuvant enhancing mucosal immune response is preferred in controlling many pathogens at the portal of entry. earlier, we reported that a toll-like-receptor (tlr ) agonist, resiquimod (r- ), stimulated the systemic immunity when adjuvanted with the inactivated newcastle disease virus vaccine in the chicken. here, we report the effect of r- when adjuvanted with live or inactivated avian infectious bronchitis virus (ibv) vaccines with special emphasis on mucosal immunity. specific pathogen free (spf) chicks (n = ) were equally divided into six groups at two weeks of age and immunized with either inactivated or live ibv vaccine adjuvanted with or without r- . groups that received either pbs or r- served as control. a booster was given on days post-immunization (dpi). r- enhanced the antigen specific humoral and cellular immune responses when co-administered with the vaccines as evidenced by an increase in the antibody titre in elisa and stimulation index in lymphocyte transformation test (ltt) till dpi and increased proportion of cd (+) and cd (+) t cells on dpi in the flow cytometry. interestingly, it potentiated the iga responses in the tear and intestinal secretions when used with both live and inactivated ibv vaccines. the combination of ibv vaccine with r- significantly up-regulated the transforming growth factor beta (tgfβ ) transcripts in the peripheral blood mononuclear cells (pbmcs) than that of the respective vaccine per se. an enhanced secretory iga response is likely due to the up-regulation of tgfβ , which is responsible for class switching to iga. in conclusion, co-administration of r- with inactivated or live ibv vaccine enhanced the systemic as well as mucosal immune responses in the chicken. avian infectious bronchitis (ib) is an acute, highly contagious disease of all age groups of chicken and affects primarily the respiratory system with possible infection of renal and reproductive systems [ , ] . it is a disease of global importance and listed in the world organization for animal health (oie) diseases. infectious bronchitis virus (ibv), the causative agent of ib, belongs to the genus coronavirus, family coronoviridae, of order nidovirales [ ] . vaccination with live attenuated and inactivated vaccines is the mainstay tool to control the ib [ ] . as ibv enters the host through the mucosal surfaces and replicates in the epithelial cells, vaccines inducing mucosal immunity can prevent the entry of the pathogen before establishment of infection [ ] . studies indicate that local administration of attenuated ibv vaccines is effective as compared to systemic administration implying that respiratory mucosal immunity is essential for protection [ ] [ ] [ ] . in fact, an increase in the lachrymal-fluid iga levels in chickens increases the resistance against ibv infection [ ] and re-infection [ ] . further, in the inbred chicken lines, resistance to ibv was correlated with higher iga levels in the tear and saliva [ ] . limited number of adjuvants is available with the capacity to enhance antigen specific mucosal immunity. conventional mucosal adjuvants like heat-labile enterotoxin (lt) from e. coli and cholera toxin (ct) from vibrio cholerae are toxic to the host [ ] . owing to the immunostimulatory capacity, toll-like receptor (tlr) agonists are explored as an alternate and promising source for future adjuvant systems [ ] . [ , ] . emerging evidence supports the notion that the tlr agonists increase the secretory iga (siga) when used with the vaccine antigens [ ] [ ] [ ] [ ] . lps, a tlr agonist, when used with inactivated newcastle disease virus (ndv) vaccine significantly increased the mucosal and humoral immune responses [ ] . flagellin [ ] and combination of cpg and poly i:c [ ] increased the siga titres along with systemic immune responses in chicken with avian influenza virus vaccine. resiquimod (r- ) (tlr / agonist) and gardiquimod (tlr agonist) enhanced siga response besides cellular and humoral responses when used with virus like particles (vlps) based norwalk virus vaccine in the mice [ ] . the response seen is equivalent to that seen when cholera toxin is used as adjuvant with the same antigen. co-delivery of norwalk vlps with gardiquimod (tlr agonist) or cpg odn (tlr agonist) produced strong systemic as well as mucosal immune responses in the mice [ ] . recently, we reported the adjuvant potential of r- in the chicken when used with inactivated ndv vaccine [ ] . however, the effect of r- on the mucosal immune response is not explored in the chicken hitherto. accordingly, we investigated the systemic as well as mucosal immune responses of live or inactivated ibv vaccine in the chicken when adjuvanted with r- . specific pathogen free (spf) embryonated eggs were procured from venky's india private limited, pune, india and hatched at central avian research institute, izatnagar. birds were maintained following standard management practices and provided ad libitum sterile feed and water. the experiment was approved by the institute animal ethics committee. tlr agonist (resiquimod; r- ) was sourced from invivogen, california, usa. both live and inactivated massachusetts strain of ibv vaccines were purchased from the commercial sources. two week old spf chicks were immunized with live or inactivated ibv vaccines in the presence or absence of r- (table ) with a booster given on days post-immunization (dpi). ibv specific antibody levels were evaluated in the serum at weekly interval. levels of siga were checked in the tear and intestinal secretion of the experimental birds at weekly intervals after booster dose. cellular immune response was evaluated by lymphocyte transformation test (ltt) on , , and dpi and by flow cytometry on dpi. expression of tgf β transcripts in the peripheral blood mononuclear cells (pbmcs) of the experimental birds was quantified at and h post-booster dose by quantitative real time polymerase chain reaction (qpcr). the qpcr analysis of tgf β transcripts was also done in the spleen samples on dpi. birds of two week age were randomly divided into six groups (n = /group) and immunized withlive or killed ibv vaccine (table ) . a booster was given at the same dose and route on dpi and r- ( μg/bird) was given through intra-muscular route (i.m.). blood samples (n = /group on , , , dpi and n = /group on dpi) were collected to separate sera that was stored at − °c for the detection of ibv specific antibody levels on , , , and dpi. tear samples (n = /group on , dpi and n = /group on dpi) were collected from the experimental birds as reported elsewhere [ ] on , and dpi. briefly, a pinch of molecular grade sodium chloride crystals was sprinkled on either eye that induced lachrymation within - s. the tear was aspirated with a micropipette and stored in microcentrifuge tube at − °c for iga assay. birds (n = /group) were humanely sacrificed on , and dpi and spleen, intestinal secretions were collected as reported earlier [ ] . briefly, around cm long duodenum, jejunum and ileum were collected from each bird and pooled; the parietal surface was washed with pbs. longitudinal strip of intestine was prepared and placed in a graduated tube to which equal volume of pbs (v/v) containing ethylene diamine tetra-acetate (edta) mm, trypsin inhibitor μg/ml and phenyl methane sulfonyl fluoride (pmsf) . mg/ml was added. the samples were vortexed briefly for - min before centrifugation at × g for min at °c. the clear supernatant was aspirated and kept at − °c until further use. the pbmcs were isolated from the experimental birds (n = / group) at and h post-booster dose using ficoll hypaque (sigma, mo, usa) ( . g/ml) density gradient centrifugation as per the published protocol [ ] . the ibv specific antibody in the serum (n = /group on , , , dpi; n = /group on dpi) was quantified using commercial ibv antibody test kit (idexx laboratories, usa) following the manufacturer's instructions. an antibody titre of > or . log was considered positive for ibv. the levels of iga both in the tear (n = /group on , dpi; n = /group on dpi) and intestinal secretion (n = /group) were measured using the commercial ibv antibody test kit (idexx laboratories, usa) with certain modifications using anti-chicken iga-hrpo (bethyl, usa) as the secondary antibody. cellular immune response in the experimental birds was assessed by lymphocyte transformation test (ltt) and flow cytometry as reported earlier [ ] . blood was collected in a heparinized vial containing iu/ml on , , and dpi for ltt. the pbmcs (n = /group on , , dpi and n = /group on dpi) were suspended in rpmi complete medium containing % fetal bovine serum (fbs) and iu/ml penicillin, and μg/ml streptomycin. cell viability was determined by trypan blue dye exclusion method. cell suspension was adjusted to × cells/ml and μl of cell suspension was plated in each well of well cell culture plate. rpmi medium ( μl) with or without cona ( μg/ml), ibv antigen ( μg/well) was added to the wells in triplicate. the plates were incubated at °c, % co for h in a humidified chamber. at the end of incubation, mtt [ -( , dimethylthiazol- -yl)- , -diphenyl-tetrazolium bromide; sigma, usa] μl was added from the stock ( mg/ml). the plates were reincubated at the same condition for another h. culture supernatant table immunization plan followed in the spf chicken. vaccine preparation route ( μl) was discarded from each well and the formazan crystals were dissolved by adding dimethyl sulfoxide (amresco, usa) μl to each well and optical density (od) was taken at nm in a microplate elisa reader. blastogenic response was calculated by dividing the mean od of the stimulated well by the mean od of unstimulated well and expressed as stimulation index (si). flow cytometry analysis. the proportion of cd + and cd + t cell subsets in the pbmcs (n = /group) was measured on dpi by flow cytometry. for analysis, × cells were stained with antichicken cd /cd r-pe and cd -fitc labeled monoclonal antibodies (abcam, usa) and kept overnight at °c in the dark. subsequently, the cells were washed with pbs containing % fbs and were not fixed. the aliquots of × cells were analyzed per sample by bd facs tm calibur instrument (bd biosciences, uk). the unstained cells served as the negative control. the pbmcs ( × ) (n = /group) were collected from the experimental birds as mentioned above at and h post-booster and one ml of ribozol™ (amresco, usa) was added. similarly, one ml of ribozol™ was added to spleen tissue collected following sacrifice (n = /group). total rna extraction was done by phenol : chloroform and isopropanol method and the purity was checked by absorbance at and nm in a nanodrop uv spectrophotometer. total rna was used for the preparation of cdna employing revertaid™ first strand cdna synthesis kit (thermo scientific, usa), following manufacturer's instructions. quantification of the tgf-β gene was done by quantitect sybr green qpcr kit (qiagen, ca, usa) on cfx real time system (bio-rad, ca, usa) following the published report [ ] . β-actin served as the housekeeping gene. published primer sequences were used for βactin (f: ′ tatgtgcaaggccggttt ′, r: ′ tgtctttctggcccat accaa ′) [ ] and tgf-β (f: ′ cggccgacgatgagtggctc ′, r: ′ cggggcccatctcacaggga ′) [ ] genes. each sample was tested in triplicate on the same plate. expression of tgf-β was calculated relative to the β-actin gene and expressed as n-fold increase or decrease relative to the control. the data of real time pcr was calculated by −ΔΔct method [ ] . each experiment was repeated twice independently and data from the first experiment was used for analysis. the treatment effect at each time point was assessed by one way analysis of variance (anova) with duncan's multiple range test as post hoc test to find the significance of pair-wise mean difference. the minimum level of significance was set at %. results are presented as mean ± se. statistical software spss™ . (ibm corp., usa) was used for analysis while graphpad prism version . was used for generating the graph. effect of r- on vaccination induced ibv antibody titre in the sera is presented in fig. . the antibody titre in the pbs control and r- groups was consistently negative for ibv. there was no significant (p > . ) difference in the antibody response between the vaccinated and control groups on dpi. only live ibv vaccine with or without r- induced significantly higher antibody response than that of the control group on dpi. the combination of vaccine plus r- showed significantly higher (p < . ) antibody titre than that of the respective vaccine alone group after secondary immunization consistently (fig. ) . effect of r- on vaccination induced iga response in the tear and intestinal secretion is presented in figs. and , respectively. vaccine, either live or inactivated, induced a significantly higher iga response than that of the control group after secondary vaccination (p < . ). co-administration of r- with live or inactivated ibv vaccine significantly increased the iga response in the tear and intestinal secretion from dpi, which was maintained till dpi as compared to the vaccine alone group (p < . ). the peak iga response in the tear was observed in the live vaccine plus r- group followed by inactivated vaccine plus r- , live vaccine and inactivated vaccine groups. the iga response in the intestinal secretions was comparable with that of tear. the antigen specific lymphocyte proliferation following different treatment is depicted in fig. . live as well as inactivated ibv vaccine significantly increased the si as compared to the control at each time point studied. further, co-administration of r- significantly potentiated the si as compared to the vaccine alone groups (p < . ). the si was maximum in the live vaccine plus r- group, which was . ± . , . ± . and . ± . , . ± . on , , and dpi, respectively (fig. ). both the live and inactivated vaccines significantly increased the cd + and cd + t cells (%) as compared to the control (p < . ) as depicted in fig. . co-administration of r- with either type of vaccine showed a significant increase in the cd + and cd + t cells (%) indicating a immunomodulatory role in the adaptive immunity. combination of r- and live vaccine showed the highest percent increase of . ± . and . ± . , respectively in cd + and cd + t cells. the groups receiving vaccine along with r- showed significantly (p < . ) higher expression of tgf-β than that of the respective vaccine alone groups (fig. ) . the highest tgf-β transcripts was observed in the live vaccine plus r- group, which was . ± . and . ± . folds higher than that of the control group at and h post-booster immunization, respectively. the response seen in the inactivated vaccine plus r- group was almost equal to the live vaccine alone group. expression of tgf-β transcripts was comparable in both spleen (data not shown) and pbmcs. resiquimod, a tlr agonist, is an imidazoquinoline compound with tremendous immunodulatory capacity. the antiviral activity of tlr agonists has been reported against genital warts, herpes genitalias and molluscum contagiosum [ ] . tlr / agonists like imiquimod and r- are fda approved drugs for basal cell carcinoma, actinic keratosis and papilloma virus in the human [ ] [ ] [ ] [ ] . recently, we reported the enhanced antigen specific cellular as well as humoral immune responses in the chicken when resiquimod (r- ) was used with inactivated ndv vaccine resulting in complete protection against virulent ndv challenge [ ] . since the adjuvant potential is likely to vary with the type of vaccine, we studied the effect of r- with ibv vaccine. to the best of our knowledge, this is the first report on the effect of r- in modulating the mucosal immune response in the chicken. humoral immune response plays an important role in ibv infection [ ] . in the present study, antibody titre was highest in the spf chicken that received live vaccine with r- than other groups (fig. ) . the live vaccine virus stimulates more vigorous immune response as they replicate in the host and simulate the natural infection. adjuvanted live ibv vaccine induced higher immune responses than live vaccine alone [ ] , supports the concept of present study. further, the combination of r- withinactivated ibv vaccine showed higher antibody response than the vaccine alone group. these findings indicate the adjuvant capacity of r- with live as well as inactivated ibv vaccines in increasing the antibody response, which is supported by our earlier report on inactivated ndv vaccine in spf chicken [ ] . in addition to antibodies, cell mediated immunity also plays an important role in immunity against ibv. transfer of lymphocytes from birds on day post-ibv infection to naïve chicken completely eliminates the viral infection and clinical signs after challenge [ ] and viral load was reduced in the lung by increasing ibv-specific cytotoxic t cells (ctls) in spleen [ ] . in the present study, cell mediated immunity was analyzed by ltt as well as immunophenotyping. we found that spf chicken receiving live vaccine with r- mounted a strong antigen specific proliferation as compared to other groups (fig. ) , which is supported by the concomitant increase in the proportion of cd + and cd + t cells (fig. ) . similarly, an enhanced cellular immune response with inactivated ibv vaccine recorded in the present study is supported by the findings with inactivated ndv vaccine [ ] . there is a need for adjuvants that increase antigen specific mucosal immune response to curtail the infection at the entry level [ ] . despite the fact that the local ctls are essential for the virus clearance in early infection [ ] , siga of lacrimal origin is a good indicator of protection in ibv infection [ , , ] as mucosal antibody response resists reinfection [ ] . r- enhanced the siga response in the tear (fig. ) as well as intestinal secretions (fig. ) when adjuvanted with live or fig. . vaccination induced ibv antibody titre in the spf chickens in the presence or absence of r- . birds of week age were immunized with live or inactivated ibv vaccine in the presence or absence of r- ( μg/bird) with a booster days later. antibody response was monitored in the serum samples by commercial elisa kit (idexx laboratories, usa) at weekly interval till dpi (n = / group on , , dpi and n = /group on dpi). the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed at each time point by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at %. * indicate significant difference (p < . ) between the groups. $ ibv specific antibody titre > . log was considered positive. dpi: day post-immunization. fig. . vaccination induced ibv specific iga concentration in the tear of spf chicken in the presence or absence of r- . birds of week age were immunized with live or inactivated ibv vaccine in the presence or absence of r- ( μg/bird) with a booster days later. iga response was monitored after booster at weekly interval till dpi (n = / group on dpi , and n = /group on dpi). the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed at each time point by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at %. **indicate significant difference (p < . ) between the groups. dpi: day post-immunization. fig. . vaccination induced ibv specific iga level in intestinal secretions of spf chicken in the presence or absence of r- . birds of week age were immunized with live or inactivated ibv vaccine in the presence or absence of r- ( μg/bird) with one more dose of the same preparations days later. iga response was monitored after booster dose at weekly interval till dpi (n = /group). the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed at each time point by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at %. **indicate significant difference (p < . ) between the groups. dpi: day post-immunization. inactivated ibv vaccine. in support of the findings, it is reported that the administration of r- with vlps of norwalk virus enhanced the mucosal iga response in the mice [ ] . to explain the impressive siga response, we studied the relative expression of tgf-β as it has a major role in the induction of iga class switching [ ] . it is well known that tgf-β , which is the orthologue of tgf-β , is primarily responsible for class switching of b cells to produce iga in the mammals [ ] [ ] [ ] [ ] [ ] . a significant increase in the relative copy number of tgf-β (fig. ) suggests a role of r- in iga class switching in the groups that received live or inactivated ibv vaccine. in this study, the tgf-β transcripts were significantly higher in vaccine plus r- groups than that of the respective vaccine alone groups. activation of tlr by r- would initiate the signaling cascade through the myd -dependent pathway, which might have resulted in the up-regulation of tgf-β transcripts [ ] . studies by our group have shown that r- improves the vaccine response of ndv when adjuvanted [ ] and has prophylactic potential against ibdv [ ] . thus, the adjuvant effect of r- with ibv is needed to be tested following challenge studies. in conclusion, co-administration of r- with inactivated or live ibv vaccine enhanced the mucosal immunity by increasing siga which is likely mediated by tgf-β . the potential of r- to enhance mucosal immunity has translational significance in poultry vaccination. fig. . lymphocyte proliferation specific to ibv antigen in the pbmcs collected from the spf chickens following vaccination in the presence or absence of r- . birds of week age were immunized with live or inactivated ibv vaccine in the presence or absence of r- ( μg/bird) with a booster days later. pbmcs (n = /group on , , dpi and n = /group on dpi) were collected and stimulated with ibv antigen to assess lymphocyte proliferation using mtt dye at weekly interval from dpi . the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed at each time point by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at %. **indicate significant difference (p < . ) between the groups. dpi: day post-immunization. . cd + and cd + cells (%) in the pbmcs of spf chicken following ibv vaccination in the presence or absence of r- . birds of week age were immunized with live or inactivated ibv vaccine in the presence or absence of r- ( μg/bird) with a booster days later. the pbmcs (n = /group) were collected from the birds on dpi and analyzed by flow cytometry following addition of chicken specific monoclonal antibodies. the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at %. *indicate significant difference (p < . ) between the groups. dpi -day post-immunization. fig. . quantitative real time pcr analysis of tgf-β expression in the pbmcs of the spf chicken following vaccination in the presence or absence of r- . birds of week age were immunized with live or inactivated ibv vaccine in the presence or absence of r- ( μg/bird) with a booster days later. the pbmcs (n = /group) were collected from the birds on and dpi ( and h post-booster) and the tgf-β transcripts were analyzed by real time pcr using β-actin as the house keeping gene and expressed as n-fold increase or decrease relative to the control following −ΔΔct method ( ) . the experiment was repeated twice independently and data from the first experiment was used for analysis. treatment effect was analyzed at each time point by one way anova with duncan's multiple range test to compare the pair-wise mean difference. alpha error was set at %. **indicate significant difference (p < . ) between the groups. the authors declare that there is no conflict of interest with respect to the content of the manuscript. coronavirus avian infectious bronchitis virus an overview of infectious bronchitis virus in chickens virus taxonomy: classification and nomenclature of viruses poultry diseases function of mucosa-associated lymphoid tissue in antibody formation local antibody response in avian infectious bronchitis: virus-neutralizing antibody in tracheobronchial secretions transcriptome of local innate and adaptive immunity during early phase of infectious bronchitis viral infection molecular mechanisms of primary and secondary mucosal immunity using avian infectious bronchitis virus as a model system avian infectious bronchitis: specific lachrymal iga level and resistance against challenge infectious bronchitis the secretory antibody response of inbred lines of chicken to avian infectious bronchitis virus infection mucosal vaccine design and delivery immunostimulatory properties of toll-like receptor ligands in chickens the role of pattern-recognition receptors in innate immunity: update on toll-like receptors evolution of the chicken tolllike receptor gene family: a story of gene gain and gene loss adjuvanted intranasal norwalk virus-like particle vaccine elicits antibodies and antibody-secreting cells that express homing receptors for mucosal and peripheral lymphoid tissues an intranasally delivered toll-like receptor agonist elicits robust systemic and mucosal responses to norwalk virus-like particles evaluation of tlr agonists as potential mucosal adjuvants for hiv gp and tetanus toxoid in mice mucosal vaccine adjuvants update effect of lipopolysaccharide on intranasal administration of liposomal newcastle disease virus vaccine to spf chickens salmonella flagellin enhances mucosal immunity of avian influenza vaccine in chickens comparison of kinds of toll like receptor ligands for inactivated avian h n influenza virus intranasal immunization in chicken herbst-kralovetz, tlr and agonists are highly effective mucosal adjuvants for norovirus virus-like particle vaccines adjuvant potential of resiquimod with inactivated newcastle disease vaccine and its mechanism of action in chicken a comparison of methods of inducing lachrymation and tear collection in chickens for detection of virus-specific immuoglobulins after infection with infectious bronchitis virus intestinal mucosal immune response in chickens following intraocular immunization with liposome-associated salmonella enterica serovar enteritidis antigen identification of cpg oligodeoxynucleotide motifs that stimulate nitric oxide and cytokine production in avian macrophage and peripheral blood mononuclear cells synergy of lipopolysaccharide and resiquimod on type i interferon, pro-inflammatory cytokine, th and th response in chicken peripheral blood mononuclear cells prophylactic potential of resiquimod against very virulent infectious bursal disease virus (vvibdv) challenge in the chicken effects of lactobacilli on cytokine expression by chicken spleen and cecal tonsil cells a new mathematical model for relative quantification in real-time rt-pcr imiquimod and resiquimod as novel immunomodulators therapeutic response of basal cell carcinoma to the immune response modifier imiquimod % cream imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential imiquimod % cream for the treatment of actinic keratosis: results from two phase iii, randomized, double-blind, parallel group, vehicle controlled trials toll-like receptor promotes cross-priming to virus-infected cells effect of in ovo bursectomy on the course of an infectious bronchitis virus infection in line c white leghorn chickens efficacy of intranasal and spray delivery of adjuvanted live vaccine against infectious bronchitis virus in experimentally infected poultry adoptive transfer of infectious bronchitis virus primed αβ t cells bearing cd antigen protects chicks from acute infection cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry resiquimod and other immune response modifiers as vaccine adjuvants specific cytotoxic t lymphocytes are involved in in vivo clearance of infectious bronchitis virus humoral and cell-mediated immune responses to different doses of attenuated vaccine against avian infectious bronchitis virus heterologous live infectious bronchitis virus vaccination in day-old commercial broiler chicks: clinical signs, ciliary health, immune responses and protection against variant infectious bronchitis viruses tgfβ signalling in control of t-cell-mediated selfreactivity transforming growth factor beta specifically enhances iga production by lipopolysaccharide-stimulated murine b lymphocytes tgf-β induces germ-line transcripts of both iga subclasses in human b lymphocytes interleukin and transforming growth factor beta cooperate to induce anti-cd -activated naive human b cells to secrete immunoglobulin a transforming growth factor-β directs iga switching in human b cells tgf-β receptor controls b cell responsiveness and induction of iga in vivo the mucosal adjuvant cholera toxin b instructs non-mucosal dendritic cells to promote iga production via retinoic acid and tgf-β the authors thank the director, indian veterinary research institute for providing the facilities to carry out the work. key: cord- - t wpiqy authors: webby, rj; perez, dr; coleman, js; guan, y; knight, jh; govorkova, ea; mcclain-moss, lr; peiris, js; rehg, je; tuomanen, ei; webster, rg title: responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: t wpiqy background: in response to the emergence of severe infection capable of rapid global spread, who will issue a pandemic alert. such alerts are rare; however, on feb , , a pandemic alert was issued in response to human infections caused by an avian h n influenza virus, a/hong kong/ / . h n had been noted once before in human beings in and killed a third ( / ) of infected people. , the variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics. methods: we removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of a/hong kong/ / using influenza reverse genetics techniques. a reference vaccine virus was then produced on an a/puerto rico/ / (pr ) backbone on who-approved vero cells. we assessed this reference virus for pathogenicity in in-vivo and in-vitro assays. findings: a reference vaccine virus was produced in good manufacturing practice (gmp)-grade facilities in less than weeks from the time of virus isolation. this virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs. interpretation: the ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. the same technologies and procedures are currently being used to create reference vaccine viruses against the h n viruses circulating in asia. in february, , two family members were admitted to intensive care wards in hong kong special administrative region with influenza-like respiratory illness. avian-like h n influenza viruses were isolated from both patients, one of whom succumbed to infection. this was the first time since that h n viruses had been identified in human beings, and who responded by issuing a pandemic alert. candidate vaccines were immediately sought. the recent outbreak of severe acute respiratory syndrome (sars) had been a striking example of the rapid and global spread of an emerging infectious disease. however, even the effects of sars could be dwarfed by those that could arise with the emergence of an influenza pandemic. infection caused by the influenza a virus is a zoonosis, and the animal reservoir of this virus is the aquatic bird populations of the world. the compelling epidemiological link between the presence of the virus in poultry in live-bird markets and the appearance of h n in human beings in suggested that influenza a viruses can be transmitted directly from avian species to man and can cause severe respiratory disease. [ ] [ ] [ ] although control of the outbreak was achieved by culling millions of birds in the hong kong markets, this episode demonstrated that the capability for an effective global response to emerging influenza threats was poor because of technical, legislative, and infrastructural limitations. a disturbing finding that emerged from this event was that the scientific community was unable to produce an effective vaccine even after several years. the inactivated human influenza vaccines in use today are derived from essentially modified viruses. by exploiting the segmented nature of the influenza a genome, vaccine manufacturers and the laboratories of the who influenza network have produced a reassortant virus carrying the circulating virus's gene segments that encode haemagglutinin and neuraminidase, the major targets of neutralising antibodies. the remaining six-gene segments are supplied from pr , a laboratoryadapted avirulent h n strain. the resulting reassortant virus has the antigenic properties of the circulating strain and the safety and high-yield properties of pr . the first batch of inactivated material against the h n virus was not ready for clinical trial until months after the second case of human infection arose, and even today the effectiveness of vaccine against this virus has not been proven. a key reason for this delay in the production of an h n -specific vaccine was the nature of the virus itself. the h n virus is highly pathogenic in human beings and poultry. the agent must be handled only under conditions of at least biosafety level (bsl ), and it can kill fertilised chicken eggs, the standard medium for the reassortment and responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines propagation of influenza virus before its inactivation and formulation for use in vaccines. these same traits are present in the h n virus. the pathogenic nature of these h n viruses is linked to the presence of additional basic residues in haemagglutinin at the site of cleavage, a step required for haemagglutinin activation and, thus, for virus entry into cells. [ ] [ ] [ ] to overcome the high pathogenicity of the virus, polybasic aminoacids have to be eliminated. a rapid, reproducible system to achieve these modifications-ie, plasmid-based reverse genetics-has been developed only in the past - years [ ] [ ] [ ] the potential benefits of reverse genetics for the generation and attenuation of vaccine candidates against highly pathogenic and low pathogenic influenza viruses are enormous. [ ] [ ] [ ] however, the host specificity of the rna polymerase i promoter used in the influenza reverse-genetics systems and the required use of an approved cell line limits the practical options for the system's use in the manufacture of human vaccines. the vaccine-candidate reference virus stock described in this report has been produced entirely on a cell substrate licensed for the manufacture of human vaccine, and as such, is-to our knowledge-the first reverse genetically derived influenza vaccine suitable for testing in clinical trials. we describe the construction of a vaccine reference virus in good manufacturing practice (gmp)-grade facilities in less than weeks from the time of virus isolation. our findings highlight the speed with which new technologies can be implemented in response to influenza pandemic alerts. we obtained who-approved vero cells (who-vero, x , p ) from the american type culture collection (manassas, virginia, usa). passage- cells (five passages since their removal from a working cell bank) were used for the rescue of the vaccine-candidate virus. the plasmids containing the genes from pr have been described elsewhere. virus propagation, rna extraction, pcr amplification, and haemagglutinin and neuraminidase gene cloning we obtained a/hong kong/ / (h n ) that had been passaged in eggs from the who influenza network. the virus was isolated and propagated in -day-old embryonated chicken eggs. total rna was extracted from infected allantoic fluid with use of the rneasy kit (qiagen, valencia, ca, usa) in accordance with manufacturer's instructions. reverse transcription was carried out with the uni primer ( Ј-agca aaagcagg- Ј) and amv reverse transcriptase (roche, indiana biochemicals indianapolis, usa). the removal of the connecting peptide of the haemagglutinin was done with use of pcr with the following primer sets: ( ) bm-ha- ( Ј-tattcgtctcagggagcaa aagcagggg- Ј) and ⌬r ( Ј-taatcgtc tcgtttcaatttgagggctatttctgagcc- Ј); and ( ) ⌬f ( Ј-taatcgtctctgaaa ctagaggattatttggagctatagc- Ј) and bm-ns- r ( Ј-atatcgtctcgtattagtag aaacaagggtgtttt- Ј). we amplified the neuraminidase gene of a/hong kong/ / using the primer pair ba-na- ( Ј-tattggtctc agggagcaaaagcaggagt- Ј) and ba-na- r ( Ј-atatggtctcgtattagtagaaacaag gagtttttt- Ј). pcr products were purified and cloned into the vector phw as described previously. the rescue of infectious virus from cloned cdna was done under gmp conditions. vero cells were grown to % confluency in a cm flask, trypsinised (with trypsin-versene), and resuspended in ml of opti-mem i (invitrogen, carlsbad ca, usa). to ml of cell suspension we added ml of fresh opti-mem i; then, we added ml of this diluted suspension to each well of a six-well tissue culture plate (about ϫ cells per well). the plates were incubated at °c overnight. the next day, g of each plasmid and l of transit lt- transfection reagent (panvera, madison, wi, usa) were added to opti-mem i to a final volume of l and the mixture incubated at room temperature for min. after incubation, the medium was removed from one well of the six-well plate, l of opti-mem i added to the transfection mix, and this mixture added dropwise to the cells. h later, the dna-transfection mixture was replaced by opti-mem i. h after transfection, ml of opti-mem i that contained g/ml l-(tosylamido- phenyl) ethyl chloromethyl ketone (tpck)-treated trypsin (worthington biochemicals, lakewood, nj, usa) was added to the cells. about h after the addition of tpck-trypsin, the culture supernatants were harvested and clarified by low-speed centrifugation; we then injected l of the clarified supernatant into the allantoic cavity of individual -day-old pathogen-free embryonated research grade eggs (charles river spafas, north franklin, ct, usa). ten -week-old chickens received intravenous injections of · ml diluted virus (dilution ratio, / ). we monitored chickens for signs of disease for days using the intravenous pathogenicity index, approved by the office of international epizooites (oie). additionally, we took tracheal and cloacal swabs (in ml of media) days and days after infection, and we did assays for the presence of virus by injection of · ml into all of three -day-old embryonated chicken eggs. haemagglutination activity in the allantoic fluid of these eggs was assessed after incubation at °c for days. pathogenicity testing in ferrets we tested pathogenicity of the vaccine in five young adult male ferrets (marshall's farms, north rose, ny, usa) aged - months (weight about · kg) that were shown by haemagglutination inhibition assays to be seronegative for currently circulating human influenza a viruses (h n , h n ) and h n viruses. we anaesthetised the ferrets with inhaled isoflurane, and they were then infected intranasally with % egg infectious dose (eid )/ml of vaccine reassortant virus or wildtype virus. we monitored the ferrets once per day for signs of sneezing, inappetence, and inactivity, and we recorded rectal temperatures and bodyweights. , , and days after infection, the ferrets were anaesthetised with ketamine ( mg/kg), and we collected nasal washes using ml of sterile phosphatebuffered saline (pbs) containing antibiotics. we measured titres of virus in these washes with eid assays. to further assess the pathogenicity of the viruses, we collected tissue samples from lungs, brain, olfactory bulb, spleen, and intestine for virus isolation and histopathological analysis at the time of death or in the case of three ferrets, after euthanasia at day after infection. the tissues were fixed in % neutral buffer formalin, processed and embedded in paraffin, sectioned at g, stained with haematoxylin and eosin and examined by light microscopy in a blinded fashion. to test the stability of the vaccine virus on propagation, we made consecutive passages of the virus in embryonated chicken eggs. a - dilution of the virus was made in pbs, and · ml of the solution was injected into the allantoic cavities of all of four -day-old embryonated chicken eggs. eggs were incubated at ºc for · - days. after incubation, each egg was candled to determine embryo viability before chilling at ºc. we harvested ml of allantoic fluid from each egg harvested, and samples were pooled together, tested for haemagglutination activity, and then reinjected into another four eggs. the sponsor had no role in study design, in the collection, analysis, and interpretation of data, in the writing of the report or decision to submit this manuscript for publication. the first challenge we faced in producing a vaccine against a/hong kong/ / (h n ) was to attenuate the virus in preparation for mass production. previous experiences have shown that removal of the basic aminoacids at the haemagglutinin cleavage site substantially attenuates pathogenic influenza viruses. [ ] [ ] [ ] using a pcr-based mutagenesis approach, we replaced the cleavage site encoded by the haemagglutinin gene of a/hong kong/ / (h n ) with that of the avirulent a/teal/hong kong/w / (h n ) (figure ); this modified haemagglutinin gene and the neuraminidase gene of a/hong kong/ / (h n ) were cloned individually into the vector phw . the two resulting plasmids and the six plasmids encoding the remaining proteins of pr were transfected into whoapproved vero cells under gmp conditions to rescue the vaccine seed virus, ⌬ /pr . - h after transfection, isolated areas of cytopathic effect could be seen on the vero monolayers. although addition of further g aliquots of tpck-treated trypsin every h led to a proportional increase in the cytopathic effect, it was not required for successful virus rescue. the candidate vaccine strain grew to high titres on subsequent amplification in eggs (haemagglutination titres of - ) and did not cause embryo death. the vaccine seed virus was unable to form plaques on madin-darby canine kidney (mdck) cells in the absence of trypsin, a trait consistent with that of influenza viruses that lack the polybasic cleavage site, and was antigenically indistinguishable from the parental h n virus in haemagglutination inhibition assays. the rescued virus was fully sequenced and was identical to the plasmids used in its creation. to assess the pathogenicity of the h n vaccine seed virus, we compared the properties of this virus with those of the wildtype a/hong kong/ / (h n ) in ferrets and in chickens. by stark contrast with the wildtype virus, which was lethal to all chickens within h of infection, intravenous administration of a / dilution of ⌬ /pr did not result in any signs of infection in chickens, and we were unable to detect any virus in swabs of cloacae or tracheae from inoculated birds. compared with a/hong kong/ / (h n ), ⌬ /pr was attenuated in ferrets that had been inoculated intranasally with eid of virus. ferrets infected with a/hong kong/ / had inappetence and weight loss (figure ), with one infected animal dying days after infection and a second killed days after infection because of hind-limb paralysis. infection in these animals was characterised by viral shedding until days after infection and replication of virus in the lower respiratory tract and olfactory bulb (as determined by virus isolation). in the a/hong kong/ / infected animals, there was a mild mononuclear cell infiltrate in the meninges and tracheal submucosal mucous glands and an extensive bronchopneumonia. the pneumatic infiltrate progressed in severity from the bronchi to the pleura. the bronchi and bronchioles contained sloughed necrotic epithelial cells, numerous mononuclear cells, and a few neutrophils. the alveoli were consolidated with inflammatory cells and fibrin (figure ). by contrast, those ferrets infected with ⌬ /pr did not lose weight (figure ) and seemed to remain healthy during the study ( days) ( figure ) . virus was detected in the nasal washes of these animals at days but not days after infection, and virus was recovered from the upper respiratory tract only. by light microscopy, the meninges and trachea of the ⌬ /pr infected ferrets did not have an inflammatory infiltrate and only a few neutrophils were noted occasionally in pulmonary bronchi. our results clearly show that ⌬ /pr was attenuated. in view of our findings, this virus can be safely handled with standard precautions in bsl containment facilities. because the mechanisms and requirements for the accumulation of basic aminoacids at the haemagglutinin cleavage site are not entirely understood, we wanted to confirm that the altered cleavage site remained stable on multiple passages in embryonated chicken eggs. such passaging in eggs would occur in transition and amplification of the reference virus to vaccine stock. the rescued virus was stable on continued serial passage in embryonated eggs, and we did not detect any change in nucleotide sequence of the haemagglutinin cleavage site after passages. there was no evidence of changing pathogenicity of the virus and we noted only one dead embryo at passage . no haemagglutination activity was evident in this egg and no embryo death was seen in passage , which strongly suggests that the death was not related to virus replication. haemagglutination titres at each passage ranged from to with no apparent trend of increasing or decreasing titres in subsequent passages. the rapid response in terms of potential vaccine reference virus production to the h n outbreak differs strikingly from the response to the episode. this difference is attributable to the new scientific technology available in and, just as importantly, to the infrastructure for virus surveillance in hong kong developed since . the first case of h n influenza in hong kong was in may, ; yet several months elapsed before this virus was finally characterised as an h n virus. in , the causative agent was identified only hours after admission of the patients to the hospital. the increased awareness, surveillance, and availability of reagents to identify influenza viruses of all subtypes bode well for the rapid identification of viruses that arise from future interspecies transfer events and for the coordination of international vaccine development by who. the timely distribution of candidate viruses is a very important step in the development of vaccines for pandemic emergencies. despite the heightened security and documentation requirements for shipping and receiving potential bioterrorism agents, the h n and sars outbreaks have shown that in true emergencies, global distribution is feasible. although it is pertinent to prepare for future pandemics by stockpiling potential vaccine strains, the h n situation in -and the ongoing h n outbreaks throughout asia in (http://www.who.int)-have highlighted the fact that some of the focus of pandemic planning must go into the implementation of technology to rapidly produce vaccines from field isolates. although viruses similar to a/hong kong/ / (h n ) had been circulating in bird populations, these viruses were antigenically distinct, despite high genetic similarities (guan y and peiris js, unpublished data). that the aminoacid differences are on the globular head of haemagglutinin and seem to be responsible for much of the antigenic difference means that even a vaccine previously prepared from genetically similar precursor viruses might not provide adequate protection. we may well be faced with potential pandemic situations in the future and the rapid production of a matched vaccine will be needed-a point again highlighted by h n outbreaks in . although the reference virus described in this report was prepared from a virus isolated in a similar geographic region and only a year earlier, it shares only limited antigenic cross-reactivity to the h n viruses. hyperimmune sheep serum samples produced against the purified haemagglutinin of ⌬ /pr has at least a six-fold reduced haemagglutination inhibitory activity against a/vietnam/ / as compared with a/hong kong/ / . as our findings show, we have the technical capabilities to respond rapidly to outbreaks with a safe and stable reference virus, but there is still much to be accomplished before such viruses can be fully used in pandemic and interpandemic influenza vaccine production. the use of reverse genetics introduces a number of new processes into influenza vaccine manufacture that are not encountered with standard reassortment methods. one of the most obvious is the need for cultured cells. although both vero and mdck , cells are in development as substrates for the growth of influenza vaccine, there are additional requirements for the use of cells in reverse genetics. unfortunately, the number of suitable cell lines is very small. in addition to the regulatory requirements, the choice of cell is also limited by the technology. the plasmid based reverse-genetics systems necessitates the use of cells from primate origin. the vero cell line is probably the only option currently able to meet both regulatory and technical demands. we have shown that vero cells can be used to successfully rescue h n , h n , h n , and h n viruses on the pr backbone using the -plasmid system. others have demonstrated the suitability of vero cells for alternative influenza virus reverse-genetics systems. although cultures of vero cells are easily obtained, only cells from fully tested and licensed cell banks are likely to be acceptable for vaccine manufacture. this issue must be acknowledged and access to such cells must be incorporated as part of future pandemic plans. that future threats of influenza pandemics will be addressed by the use of the technology described in this report seems inevitable. despite the presence of low pathogenic surrogate strains, the recent human death from influenza-like illness caused by highly pathogenic h n virus in the netherlands reinforces the fact that future outbreaks will probably occur in which this reversegenetics technology provides the logical-and, possibly, the only-way to respond rapidly and effectively. although our response to the outbreak of h n influenza in has shown that current scientific capabilities are sufficient to respond to the threat, there are still legal and infrastructural barriers to be overcome. these barriers include licensing and intellectual property issues surrounding what is, essentially, a genetically modified organism. yet, these difficulties are not insurmountable and pandemic scares such as the and ongoing h n outbreaks are forcing commercial and regulatory parties to address these issues with some urgency. with the development of the h n vaccine reference virus, and ongoing attempts to create the same for the virus, the challenge in responding to a threat of an influenza pandemic must now be supported by the largescale manufacture of the vaccine and by clinical trials of a new vaccine manipulated by reverse genetics. r j webby, d r perez, j s coleman, j h knight, e i tuomanen, r g webster designed the study; r j webby did much of the construction of the vaccine seed virus; d r perez developed and constructed plasmid templates; y guan and j s peiris characterised and isolated the initial h n virus; j e rehg participated in the design and analysis of animal safety testing of the candidate h n vaccine seed virus; e a govorkova participated in the safety testing of the candidate h n vaccine seed virus; l r mcclain-moss participated in the preparation of gmp documentation of the process and was involved in the reconstitution of the vaccine seed virus. a pandemic warning? characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness characterization of avian h n influenza viruses from poultry in hong kong interspecies transmission of influenza viruses: h n virus and a hong kong sar perspective future influenza vaccines and the use of genetic recombinants developing vaccines against pandemic influenza the structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between ha and ha determines proteolytic cleavability and pathogenicity of avian influenza viruses molecular analyses of the hemagglutinin genes of h influenza viruses: origin of a virulent turkey strain rescue of influenza a virus from recombinant dna a dna transfection system for generation of influenza a virus from eight plasmids generation of influenza a viruses entirely from cloned cdnas eight-plasmid system for rapid generation of influenza virus vaccines plasmid-only rescue of influenza a virus vaccine candidates evaluation of a genetically modified reassortant h n influenza a virus vaccine candidate generated by plasmid-based reverse genetics recombinant influenza a virus vaccines for the pathogenic human a/hong kong/ (h n ) viruses preparation of a standardized, efficacious agricultural h n vaccine by reverse genetics development of a vero cellderived influenza whole virus vaccine influvac: a safe madin darby canine kidney (mdck) cell culturebased influenza vaccine safety and immunogenicity of a trivalent, inactivated, mammalian cell culture-derived influenza vaccine in healthy adults, seniors, and children generation of high-yielding influenza a viruses in african green monkey kidney (vero) cells by reverse genetics avian influenza a virus (h n ) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome pandemic influenza and the global vaccine supply we thank todd hatchette, katherine sturm-ramirez, and scott krauss for expert advice; ashley baker, christie johnson, yolanda sims, patrick seiler, jennifer humberd, and kelly jones for excellent technical assistance; julia hurwitz for access to the vero-cell banks. editorial assistance was provided by julia cay jones. these studies were supported by grant ai from the national institute of allergy and infectious disease, by cancer center support (core) grant ca from the national institutes of health, and by the american lebanese syrian associated charities (alsac). none declared. the corresponding author has had full access to all the data in the study and has had the final responsibility for the decision to submit this manuscript for publication. key: cord- - l w p authors: custers, jerome; kim, denny; leyssen, maarten; gurwith, marc; tomaka, frank; robertson, james; heijnen, esther; condit, richard; shukarev, georgi; heerwegh, dirk; van heesbeen, roy; schuitemaker, hanneke; douoguih, macaya; evans, eric; smith, emily r.; chen, robert t. title: vaccines based on replication incompetent ad viral vectors: standardized template with key considerations for a risk/benefit assessment date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: l w p replication-incompetent adenoviral vectors have been under investigation as a platform to carry a variety of transgenes, and express various antigens as a basis for preventive or therapeutic vaccine development. a replication incompetent adenoviral vector based on human adenovirus type (ad ) has been evaluated in several clinical trials. the brighton collaboration viral vector vaccines safety working group (v swg) was formed to evaluate the safety and features of recombinant viral vector vaccines. this paper reviews the biological features of the ad vectors, including tabulation of safety and risk assessment characteristics of ad vector-based vaccines. substantial information on immunogenicity, clinical safety, biological characteristics and manufacturing are reported. in the ad vector, deletion of the e gene, rendering the vector replication incompetent and providing space for transgene insertion, is combined with additional genetic engineering for vaccine manufacturability and transgene expression optimization. these vaccines are manufactured using the e -complementing per.c ® cell line, a continuous, human cell-line that can be cultured in serum-free medium in a suspension to high cell densities, providing an effective and flexible system for high-yield manufacturing. ad vector vaccines have favorable thermostability profiles, compatible with vaccine supply chains. safety data are compiled in the ad vaccine safety database version . , with unblinded data from ongoing and completed clinical studies for a total of participants in ebola, hiv, malaria, rsv and filovirus ad -based vaccine programs. overall, all ad -based vaccines have been well tolerated, with no significant safety issues identified from the available data in the current ad vaccine safety database. evaluation of ad -based vaccines to further characterize the safety profile is continuing, with more than , participants vaccinated as of st july (cut-off date). extensive evaluation of immunogenicity in humans shows strong and durable humoral and cellular immune responses. clinical trials have not shown meaningful impact of pre-existing immunity to ad on vaccine immunogenicity, even in the presence of ad neutralizing antibody titers or ad -targeting t cell responses at baseline. the first vaccine, against ebola virus, that makes use of the ad vector, received marketing authorization from ec on st july , as part of the ad .zebov, mva bn filo vaccine regimen. new developments based on the ad vector are underway, including a covid- vaccine, which is currently in clinical evaluation. the brighton collaboration (www.brightoncollaboration.org) v swg is an established collaboration who aim to enhance the science of vaccine safety research (http://cms.brightoncollaboration.org: /public/what-we-do/setting-standards/casedefinitions/process). the brighton v swg uses a standardized template to describe the key characteristics of novel vaccine vectors, compiled from the latest research, to facilitate scientific discourse among key stakeholders ( ). the adenovirus type (ad ) wild type virus was first isolated in from an anal specimen of a -month-old male child ( ) . as described in that study, different isolates were obtained from anal and throat swabs from different children, some of whom experienced mild selflimiting enteric infections. although, none of the illnesses could etiologically be associated with the isolated adenoviruses, it suggests that wild type ad can, presumably, cause asymptomatic or minor illness ( ) . human ad has been considered to be a low-prevalent adenovirus due to the low frequency of ad neutralizing antibodies in various populations compared with human adenovirus type . for example, a seroprevalence study of the human adenovirus serotypes known at the time showed that several serotypes from particularly subgroups b and d, including ad , were rare in a belgian population ( ) , suggesting that vectors (rad) derived from these serotypes might be useful alternatives to ad -based vectors for vaccine development, since for ad -based vectors it was shown that their high prevalence hampered their clinical use ( ) ( ) ( ) ( ) ( ) . more extensive seroprevalence and immunogenicity studies showed that while all of these vectors exhibited low seroprevalence, ad -based vaccine candidates were the most immunogenic in animals ( ) . further studies have shown that, depending on geographical location, %- % of people tested have neutralizing antibodies against ad . however, neutralization titers are low to intermediate compared with those observed for other adenovirus types ( - ). adenovirus genomes are linear, non-segmented double-stranded dna molecules with inverted terminal repeat (itr) sequences at each end. the vector system for replication-incompetent ad vaccine vectors consists of an adaptor plasmid and a cosmid ( ) . the adaptor plasmid contains the left end of the genome containing the left itr and the packaging signal. it also contains a transgene expression cassette in place of the e region and a ~ . kb fragment downstream of the e region to enable homologous recombination with the cosmid. the cosmid contains the majority of the ad genome, spanning from the pix sequence to the right itr, with a deletion of the e region and a modified e open reading frame (e orf ). transfection into a suitable packaging cell line (hek cells, per.c ® cells) and subsequent homologous recombination of the adaptor plasmid and cosmid results in the generation of a replicationincompetent e /e -deleted ad vector. packaging cell lines like the hek and per.c ® cell lines contain the e region of adenovirus serotype (ad ). because within the adenoviral life cycle, e protein k and e protein orf form a complex that is pivotal for high-level lategene expression, the e -orf sequence of ad is replaced by the corresponding sequence from ad in the vector. this modification has previously been shown to be necessary to allow for the efficient production of rad virus on ad e -complementing cells ( ) . finally, compensation for the loss of e is not needed since the e proteins are not essential for adenoviral growth in vitro but are involved in down regulating cellular immunological response mechanisms in an attempt of the adenovirus to escape the host immune system ( ) . most adenovirus serotypes use the coxsackievirus-adenovirus receptor for attachment to the target cell ( , ) . in contrast, ad has been reported to utilize cd as its primary cellular receptor ( , ) , but more recent reports indicated only a limited interaction between ad and cd , even showing evidence of a role for αvβ integrin for efficient transduction of epithelial cells, or interaction with sialic acid ( , ) . these data suggest that receptor usage by ad might be host cell-type dependent in vitro ( , ) . target cells in vivo in the natural host are not known, but ad virus can infect a variety of cell types in vitro. detailed studies dissecting the attachment, internalization and intracellular trafficking of adenoviral vectors have shown that ad , amongst others, accumulate in the late endosome to a larger extent and trigger innate immune pathways differentially compared with ad -based vectors ( ) . whether and how these differences may translate into differential profiles of adaptive immunity against the vaccine antigen is not known. ad vector-based vaccines are manufactured using the e -complementing per.c ® cell line, a continuous, human cell line capable of supporting the manufacturing of replication incompetent adenoviral vectors ( ) . one of the key strengths of this cell platform is that the cells can grow in suspension in serum-free media to very high cell densities. cell counts of million cells/ml, with a high percentage of viable cells, can be reached within days of cell culture. janssen has taken advantage of the ability to grow the per.c ® cell line at high cell densities in a so called "intensified process". cell-specific yields are in the same range as is generally achieved with other adenoviral vectors and e -complementing cell lines, therefore, due to the higher cell densities yields per volume unit are higher. the complete manufacturing process has now been scaled up to , l allowing manufacturing at a commercial scale. while lyophilized vaccines are generally more heat-stable than non-lyophilized alternatives ( ), liquid vaccine formulations may have several advantages over lyophilized vaccines, including ease of manufacture, packaging, and simple administration procedures ( ) . for ad -based vectors, progress in formulation development has allowed for long-term storage of product at - °c, enabling product distribution through existing vaccine supply chains. assessments of robustness during storage, handling and distribution conditions have shown that recombinant ad vectors can be maintained stable under frozen conditions or at - °c, and, furthermore, showed to be stable in-use with a syringe/needle, also when subjected to agitation or temperature excursions ( ). ad -based vaccines have been extensively tested in completed and ongoing clinical studies from multiple clinical programs ( figure ) the the brighton collaboration v swg authors declare that they have no known competing ad was rendered replication incompetent by deletion of early region (Δe ). a partial deletion of non-essential early region (Δe ) was made to create enough space in the genome for the transgene expression cassette (inserted in e region) also further attenuating the vector. • in gene therapy experiments n.a. • in any other special populations n.a. the ad vector is replication incompetent in non-e complementing cells and, as such, will be replication incompetent when administered to nonhuman species. in addition, insertion of foreign antigens is not expected to change the host-range of the vaccine vector. what is the risk of reversion to virulence or recombination with wild type virus or other agents? recombination of ad vaccine vectors with wildtype viruses would require sequence homology and presence of both the genome of ad vaccine vectors and wild-type adenoviruses to be present in the same cell(s). nonclinical biodistribution studies show that adenoviral vector dna of ad vaccine vectors did not distribute widely, as the vector dna was primarily detected at the site of administration in the muscle, the draining lymph nodes and, to a lesser extent, to the spleen. dna of intramuscularly administered replicationincompetent ad vaccine vector and wild-type adenovirus dna are unlikely to co-locate in the same body compartments. in the unlikely event that recombination occurs between vaccine vector and wild-type adenoviruses, the virulence can maximally be equal to the wild-type adenovirus already present in the tissue. the majority of the theoretical homologous recombination products are replication incompetent or attenuated forms of the ad . reversion of ad virulence by recombination is therefore highly unlikely. furthermore, reversion of virulence due to nucleotide mutations is impossible since deletion of the e gene from the ad vector cannot be restored by random mutations and or indels. recombination with other viruses has not been described and is considered highly unlikely due to the limited biodistribution and absence of sequence homology and replication. the dsdna genome of ad virus is relatively stable when compared to rna viruses. genetic stability of the vector is confirmed during manufacturing and upscaling by extended passaging and/or genetic stability testing. ( ) what is the potential for shedding and transmission to humans or other species? vector shedding is limited and transmission of the ad vector is highly unlikely in view of: i) the vector is replicationincompetent, and thus, allows only for one-time transduction of the target cell, ii) the limited shedding as observed in clinical studies, iii) the limited biodistribution profile as observed in nonclinical studies, and iv) the very low probability of regaining replication-competence through recombination with co-infecting wild-type virus. biodistribution studies in rabbits have shown that vector dna is not widely distributed, and clearance has been observed indicating that the vector is unlikely to persist in the tissues following intramuscular injection. in nature, wild-type adenovirus is known to be able to cause persistent infections. whether the ad vector can persist for longer time in humans is unknown. this will depend on the design of the antigen and the antigenic diversity of the pathogen. the transgene is inserted in the e region at the site of the e -deletion. the e region is deleted to render the vector replication incompetent and together with a deletion in the e region provide space for ( ) insertion of a transgene expression cassette. . . how is the transgene expression controlled (transcriptional promoters, etc.)? in general, expression of the antigen is regulated using the long human cmv immediate-early promoter, which is thought to be active in most mammalian cells, and an sv -derived polyadenylation sequence. the expressed antigen is not part of the viral particle and, as such, it is not expected that the phenotype of the vector nor the pathogenicity (the vector is replication incompetent) of the vector are altered. . . is the vaccine replicationcompetent in humans or other species? the ad vector is replication incompetent. . . what is the risk of reversion to virulence or recombination with wild type or other agents? see . . genetic stability of the vaccine vector is confirmed during manufacturing and upscaling by extended passaging and genetic stability testing. . . what is the potential for shedding and transmission to humans or other species? see . . the vaccine is replication incompetent and is unable to establish a productive infection. persistence/latency is not expected as the vaccine misses the e and e genes that code for proteins involved in countering the host immune system. in addition, nonclinicalbiodistribution studies of the vaccine have shown that the ad vaccine vector is cleared from vector positive tissues (see . .). . . does the vaccine replicate in the nucleus? the vaccine is replication incompetent. the vector genome (linear ds-dna) travels to the nucleus of the host cell where antigen expression occurs, in the absence of vaccine vector replication. . . what is the risk of integration into the human genome? see . . ( - ) . . list any disease manifestations caused by the vaccine in humans, the strength of evidence, severity, and duration of disease for the following categories: n.a. • in healthy people n.a. • in immunocompromised people n.a. • in neonates, infants, children n.a. • during pregnancy and in the unborn n.a. • in any other special populations n.a. see . . the ad vector is expected to have the same cell tropism as the wild-type ad virus. ( , ( ) ( ) ( ) . . what is known about the mechanisms of immunity to the vaccine? in general, the immune responses to the vaccine antigen encoded by the vector are characterized by a rapid increase in binding and in most cases neutralizing antibodies. in addition, induction of nonneutralizing, functional antibodies with effector functions, like antibodydependent cellular phagocytosis (adcp) and antibody-dependent cellular cytotoxicity (adcc), have been observed. induction of cellular immunity (both cd + and cd + t-cells) is also observed. . what is known about the effect of pre-existing immunity, including both natural immunity and repeat administration of the vector or the vaccine, on 'take', safety or efficacy in any animal model or human studies using this vector? data acquired to date, in more than , vaccinated human participants, have not revealed impact of preexisting vector immunity on the vaccine insert specific humoral or cellular ( , ) response. repeated administration with the ad vector leads to an increase in antigen specific humoral responses and a maintenance of cellular responses. with more than , participants vaccinated overall, no safety issues have been identified. . . . is the vaccine transmissible in humans or other species (including arthropods) and/or stable in the environment? the vaccine is replication incompetent, so no vaccine transmission is expected (see also . .). . . are there antiviral or other treatments available for disease manifestations caused by the vaccine? the vector is replicationincompetent; thus, no disease manifestations are expected besides local and systemic reactogenicity. therefore, there is no benefit for antiviral treatment. the ad vector showed a limited distribution following intramuscular injection in rabbits and clearance of the vector was observed, indicating that the vector does not replicate and/or persist in the tissues following intramuscular (im) injection. the ad vector did not induce any adverse effects in multiple glp repeat-dose toxicity studies in rabbits (and rats), irrespective of the transgene insert used. . . for replicating vectors, has a comparative virulence and viral kinetic study been conducted in permissive and susceptible species? (yes/no) if not, what species would be used for such a study? is it feasible to conduct such a study? not applicable, since the ad vector is replication incompetent . . does an animal model relevant to assess attenuation exist? not applicable, since the ad vector is replication incompetent . . does an animal model for safety including immuno-compromised animals exist? not applicable, since the ad vector is replication incompetent . . does an animal model for reproductive toxicity exist? not applicable, since the ad vector is replication incompetent the general (repeat-dose) toxicity studies conducted with the replication incompetent ad vector in rabbits (and rats) have not revealed any effects on male sex organs that would impair male fertility. in addition, the general (repeat-dose) and/or developmental and reproductive toxicity studies did not reveal any evidence of impaired female fertility nor did not indicate harmful effects with respect to reproductive toxicity in female rabbits. . . does an animal model for immunogenicity and efficacy exist? most mammalian species studied to date have shown induction of insert specific immunity after administration of ad based vaccines, dependent on the studied disease. several efficacy animal models exist -the most studied animal models have been mice, rabbits, ferrets, cotton rats and several nonhuman primate (nhp) species. . . does an animal model for antibody enhanced disease or immune complex disease exist? no . . what is known about biodistribution in animal models or in humans? biodistribution studies have been conducted in rabbits. following intramuscular administration, the ad vector did not widely distribute as vector dna was primarily detected at the site of injection, draining lymph nodes and (to a lesser extent) the spleen. over time, the number of animals with positive tissues and/or the vector copy number present in those positive tissues declined, indicating clearance of the ad vector. . . what is the evidence that vector derived vaccines will generate a beneficial immune response in: • small animal models? mice, cotton rats, rabbits and ferrets have been shown to ( , , , , , ( ) ( ) ( ) ( ) ( ) develop immune responses against a variety of vaccine inserts. • nonhuman primates (nhp)? nhp have been shown to develop protective immune responses against a variety of vaccine inserts. ( , , , , , , , , , ) • overall, no safety concerns were identified in elderly after vaccination with adeno-based vaccines. (cut-off: st july ; active only, estimate based on the study randomization ratios). no safety concerns were identified. • pregnancy and in the unborn in humans? the most recent aggregate review of pregnancy exposure data was performed in september ; this analysis of the current experience with pregnancies after exposure to the ebola candidate vaccines (ad .zebov, ad .filo) in female participants or partners of male participants did not reveal a safety concern. serious complications or saes during pregnancy were reported in out of a total of pregnancies reported in female study participants. none of these serious complications / saes were considered causally associated with the study vaccines by investigators or low risk; pregnancy is an exclusion criterion for all ad -based vaccine studies except study (described below). pregnancy tests prior to vaccination and the use of adequate contraception was mandatory for all female participants of childbearing potential. pregnant women are being enrolled in the ongoing ebola vaccination study in drc (drc-eb- /ebl ). ( ) the company. no apparent concernable pattern of aes is emerging from this review. no congenital malformations were reported to date in foetuses or newborns. spontaneous abortion was the most commonly observed sae ( out of pregnancies) with an incidence of . %, which is within the range of expected spontaneous abortion rates during the first trimester of gestation, even when considering that spontaneous abortion incidences vary significantly depending on geographical areas and individual risk factors (e.g. age, previous abortions) . • in any other special populations. not applicable . . what is the potential for shedding and transmission in risk groups? there is no significant risk for shedding and transmission of ad vectored vaccines across the risk groups (e.g. immunocompromised) who have received the vaccine vector. also designated v ) recombinant vesicular stomatitis virus pseudotyped with ebola zaire glycoprotein: standardized template with key considerations for a risk/benefit assessment four newly recognized adenoviruses replication-deficient human adenovirus type vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity safety and immunogenicity of a replication-incompetent adenovirus type hiv- clade b gag/pol/nef vaccine in healthy adults vaccine-induced immunity in the test-of-concept step study: a case-cohort analysis human adenovirus-specific t cells modulate hiv-specific t cell responses to an ad -vectored hiv- vaccine decreased preexisting ad capsid and ad neutralizing antibodies increase hiv- infection risk in the step trial independent of vaccination phase safety and immunogenicity evaluation of a multiclade hiv- candidate vaccine delivered by a replication-defective recombinant adenovirus vector comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups b and d international epidemiology of human pre-existing adenovirus (ad) type- , type- , type- and type- neutralizing antibodies: correlates of high ad titers and implications for potential hiv vaccine trials neutralizing antibodies to human and simian adenoviruses in humans and new-world monkeys international seroepidemiology of adenovirus serotypes , , , and in pediatric and adult populations novel replication-incompetent adenoviral b-group vectors: high vector stability and yield in per.c cells functions and mechanisms of action of the adenovirus e proteins the coxsackievirus-adenovirus receptor protein can function as a cellular attachment protein for adenovirus serotypes from subgroups a isolation of a common receptor for coxsackie b viruses and adenoviruses and adenovirus serotype utilizes cd as a primary cellular receptor and only transiently activates t lymphocytes following vaccination of rhesus monkeys alphavbeta integrin is required for efficient infection of epithelial cells with human adenovirus type adenovirus serotype utilises sialic acid bearing glycans as a primary cell entry receptor late endosomal trafficking of alternative serotype adenovirus vaccine vectors augments antiviral innate immunity new helper cells and matched early region -deleted adenovirus vectors prevent generation of replication-competent adenoviruses thermostability of vaccines opportunities and challenges of developing thermostable vaccines stability and suitability for storage and distribution of ad .zebov/mva-bn(r)-filo heterologous prime-boost ebola vaccine innovation: johnson & johnson announces donation of up to , regimens of janssen's investigational ebola vaccine to support outbreak response in democratic republic of the congo (drc) our company: johnson & johnson announces commitment to support republic of rwanda's preparedness against ebola outbreak european medicines agency. meeting highlights from the committee for medicinal products for human use (chmp) development of a replication-deficient adenoviral vector-based vaccine candidate for the interception of hpv -and hpv -induced infections and disease ad /mva therapeutic vaccination with tlr stimulation in siv-infected rhesus monkeys evaluation of a mosaic hiv- vaccine in a multicentre, randomised, double-blind, placebo-controlled, phase / a clinical trial (approach) and in rhesus monkeys (nhp - ) first-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype env vaccine (ipcavd ) characterization of humoral and cellular immune responses elicited by a recombinant adenovirus serotype hiv- env vaccine in healthy adults (ipcavd ) assessment of the safety and immunogenicity of novel vaccine platforms for hiv- prevention: a randomized trial our efforts to develop a vaccine and identify therapies for covid- immune responses to novel adenovirus type and modified vaccinia virus ankara-vectored ebola vaccines at year safety and immunogenicity of novel adenovirus type -and modified vaccinia ankara-vectored ebola vaccines: a randomized clinical trial induction of hiv- -specific mucosal immune responses following intramuscular recombinant adenovirus serotype hiv- vaccination of humans safety and preliminary efficacy of cs-based recombinant adenoviral serotype and serotype malaria vaccine candidates in prime-boost vaccination of healthy adults undergoing subsequent experimental p. falciparum sporozoite challenge (abstract) adenovirus transmission: national center for immunization and respiratory diseases, division of viral diseases perspective on adenoviruses: epidemiology, pathogenicity, and gene therapy new insights into stability of recombinant adenovirus vector genomes in mammalian cells progress in human gene therapy episomal vectors for gene therapy guideline on nonclinical testing for inadvertent germline transmission of gene transfer vectors conjunctivitis and enteric infection with adenovirus types and : responses to primary, secondary and reciprocal cross-challenges acute meningoencephalitis caused by adenovirus serotype adenovirus infections in immunocompetent and immunocompromised patients adenoviruses in the immunocompromised host quantifying adenovirus-neutralizing antibodies by luciferase transgene detection: addressing preexisting immunity to vaccine and gene therapy vectors new drug on the horizon for treating adenovirus division of viral diseases viral mutation rates a two-dose heterologous prime-boost vaccine regimen eliciting sustained immune responses to ebola zaire could support a preventive strategy for future outbreaks safety and immunogenicity of a -dose heterologous vaccine regimen with ad filo ebola vaccines: -month data from a phase randomized clinical trial in safety and immunogenicity of a -dose heterologous vaccination regimen with ad .zebov and mva-bn-filo ebola vaccines: -month data from a phase randomized clinical trial in uganda and tanzania packaging capacity and stability of human adenovirus type vectors ad and ad vaccine vectors induce potent and cross-reactive antibody and t-cell responses to multiple filovirus species recombinant low-seroprevalent adenoviral vectors ad and ad expressing the respiratory syncytial virus (rsv) fusion protein induce protective immunity against rsv infection in cotton rats ebovac-salone: lessons learned from implementing an ebola vaccine trial in an ebola-affected country similar epitope specificities of igg and iga antibodies elicited by ad vector prime, env protein boost immunizations in rhesus monkeys recombinant adenovirus serotype (ad ) and ad vaccine vectors bypass immunity to ad and protect nonhuman primates against ebolavirus challenge adenoviral vector type encoding zika virus (zikv) m-env antigen induces humoral and cellular immune responses and protects mice and nonhuman primates against zikv challenge correction: a prophylactic multivalent vaccine against different filovirus species is immunogenic and provides protection from lethal infections with ebolavirus and marburgvirus species in non-human primates protective efficacy of adenovirus/protein vaccines against siv challenges in rhesus monkeys a very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus low seroprevalent species d adenovirus vectors as influenza vaccines the th immune response to plasmodium falciparum circumsporozoite protein is boosted by adenovirus vectors and with a homologous insert alternative serotype adenovirus vaccine vectors elicit memory t cells with enhanced anamnestic capacity compared to ad vectors adenovirus vectorbased prime-boost vaccination via heterologous routes induces cervicovaginal cd (+) t cell responses against hpv oncoproteins immunogenicity without efficacy of an adenoviral tuberculosis vaccine in a stringent mouse model for immunotherapy during treatment vaccination with adenovirus serotypes , , and elicits higher levels of innate cytokine responses than adenovirus serotype in rhesus monkeys durability and correlates of vaccine protection against zika virus in rhesus monkeys adenoviral vaccine safety database v . . we thank the additional v swg members and participants for their support and helpful comments, and patrick zuber of the world health organization for bringing together many of the coauthors for completing the first draft of this template and for his support of the v swg.we would also like to thank wouter koudstaal (independent scientific writer) and kerstin luhn, ☐ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☒the authors declare the following financial interests/personal relationships which may be considered as potential competing interests:jerome custers, maarten leyssen, frank tomaka, esther heijnen, georgi shukarev, dirk heerwegh, roy van heesbeen, hanneke schuitemaker, and macaya douoguih, are current employees of janssen pharmaceuticals and potentially hold stock in j&j. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord- -rrsgl authors: beutels, philippe; scuffham, paul a; macintyre, c raina title: funding of drugs: do vaccines warrant a different approach? date: - - journal: the lancet infectious diseases doi: . /s - ( ) - sha: doc_id: cord_uid: rrsgl summary vaccines have features that require special consideration when assessing their cost-effectiveness. these features are related to herd immunity, quality-of-life losses in young children, parental care and work loss, time preference, uncertainty, eradication, macroeconomics, and tiered pricing. advisory committees on public funding for vaccines, or for pharmaceuticals in general, should be knowledgable about these special features. we discuss key issues and difficulties in decision making for vaccines against rotavirus, human papillomavirus, varicella-zoster virus, influenza virus, and streptococcus pneumoniae. we argue that guidelines for economic evaluation should be reconsidered generally to recommend ( ) modelling options for the assessment of interventions against infectious diseases; ( ) a wider perspective to account for impacts on third parties, if relevant; ( ) a wider scope of costs than health-care system costs alone, if appropriate; and ( ) alternative discounting techniques to explore social time preference over long periods. in many high-income countries, public funding of preventive vaccines is assessed based on the same criteria as the funding of curative pharmaceutical drugs. such routine drug assessment processes consider evidence on quality, safety, effi cacy, and cost-eff ectiveness. because of the increase in the number of diff erent vaccines available and advances in the science behind decision making, we have drawn on existing literature and practices to develop the arguments around potential disparities with other pharmaceuticals when assessing vaccines for public funding. these arguments revolve around vaccine-specifi c features of herd immunity and eradication, which are not evident in pharmaceuticals, and features for which the eff ects of quality-of-life losses in very young children, parental care and work loss, time preference, macroeconomics, and uncertainty substantially infl uence cost-eff ectiveness estimates. vaccines may increasingly be judged as unacceptable if these features are not acknowledged. we also illustrate these points for fi ve specifi c vaccines that are currently under consideration for widespread use in high-income countries. we use the term "cost-eff ectiveness" in a broad sense throughout this article, encompassing cost-utility and cost-benefi t analysis, although there are technical diff erences. in , australia was the fi rst country to make evidence on cost-eff ectiveness a mandatory part of funding decisions of drugs. the australian pharmaceutical benefi ts advisory committee is a rigorous and well-run system for evaluating drugs for acute care, chronic disease, palliation, and more recently vaccines. many other countries have adopted a similar philosophy towards cost-eff ectiveness considerations for funding pharmaceuticals (eg, belgium, finland, norway, canada [ontario] , portugal, sweden, netherlands, uk, and usa [some organisations]), but they deal with preventive public-health measures, such as mass vaccination, in diff erent ways. some countries have specifi c advisory groups to make funding recommendations on vaccinations (eg, uk joint committee on vaccination and immunisation, us advisory committee on immunization practices). often, cost-eff ectiveness evidence for vaccines is assessed in the same manner as for any drug. nevertheless, as we discuss below, vaccination has special features that make it particularly challenging to assess. furthermore, vaccination constitutes one of the largest preventive health programmes around the world, and increasing pressures on health-care budgets are as much a challenge for the use of vaccines as for other drugs. vaccines provide primary prevention of future morbidity and mortality. thus, unlike secondary prevention interventions, such as statins for cholesterol lowering, vaccines are targeted before, or in the initial stages of, the recipient's potential risk exposure. additionally, the recipient may or may not benefi t on an individual basis. vaccination may even harm some recipients through vaccine-associated adverse events (panel); for example, - % of varicella-zoster virus (vzv) vaccine recipients report a localised rash. the individual perception of risks of disease and risks of adverse events drives the demand panel: why many vaccines require a diff erent approach • primary prevention in healthy people, but with possibility of adverse events • unvaccinated or poorly vaccinated people may experience benefi cial or, more rarely, detrimental impact from herd immunity • many vaccines prevent short-lived illness in very young children, causing extra family care and work loss, for which evaluation methods lack credibility and acceptability • the cost-eff ectiveness of many vaccines is highly sensitive to the choice of discount method • some infections are eradicable • some emerging infections (eg, sars, pandemic infl uenza) would have a major macroeconomic impact that goes beyond lost productivity of sick people sars=severe acute respiratory syndrome. for vaccines, and may dominate the infl uence of other factors, such as price. the need to show protective effi cacy beyond the typical duration of clinical trials generally aff ects the assessment of vaccines more than therapeutic pharmaceuticals, primarily because the endpoints may not be immediate. in fact, the clinical endpoints might not show clinical effi cacy at the time of trial reporting because the numbers required can be extremely large. clinical endpoints of mortality or hospital admissions might require follow-up of thousands to millions of participants over as long as several decades. as such, some vaccines have been funded on the basis of immunogenicity data or intermediate endpoints alone (eg, meningococcal c conjugate vaccine and human papillomavirus [hpv] vaccine in several countries). , vaccination not only protects vaccine recipients, but reduces exposure of unvaccinated people to infection through herd immunity. herd immunity, in addition to lowering the incidence of infection in the unvaccinated, is well known to lead to an increased average age at infection. vaccination is therefore not always entirely benefi cial to public health because some childhood infections are more severe if contracted in adolescence or adulthood. furthermore, vaccination itself may modify vaccine eff ectiveness over time because of factors such as strain replacement and cross reactivity. some of these indirect eff ects improve the cost-eff ectiveness (eg, non-exposure of most of the unvaccinated, cross reactivity), whereas others may reduce the costeff ectiveness (eg, shift in the average age of infection, serotype replacement). for most vaccination programmes, the sum of these eff ects substantially improves cost-eff ectiveness, but sometimes the reverse may be true. , convincing evidence for the extent of herd immunity, and the duration of immunity, may only come from widespread use in another country, not from clinical trials. for example, the population impact of vaccinations against vzv and streptococcus pneumoniae in the usa are of major interest to other countries. appropriately parameterised dynamic transmission models could also provide credible estimates of herd-immunity eff ects. lieu and colleagues were the fi rst to estimate the costeff ectiveness of a vaccine based on dynamic model simulations. such models, which take into account the above indirect eff ects, are gradually becoming more widespread, but are not yet part of the traditional toolbox of epidemiologists or health economists. all these features add to the uncertainty under which vaccine funding decisions are made, as opposed to those of other drugs. for whatever the reason some people decline vaccination for their child, they may trade the uncertain value of direct protection for the certainty of avoiding the risk of vaccine-associated adverse events and the cost of vaccination, while potentially counting on a "free ride" from herd immunity induced by others being vaccinated. the risk perceptions driving this trade-off are distorted as a result of imperfect information. reductions in vaccine-preventable disease make people believe that their child's risk of disease has decreased. however, their risk is highly dependent on historical and future rates of exposure and vaccination in the rest of the population and can quickly rebound when uptake declines. , therefore, government intervention in the form of subsidies or public funding is required to ensure that vaccine uptake remains high enough to guarantee benefi cial herd immunity. the uk's recent struggle with the measles, mumps, and rubella vaccine uptake illustrates this point. for other pharmaceuticals, this kind of trade-off is not even conceivable. potential global eradication is another feature that sets some vaccines apart. for example, polio has been eliminated in high-income and middle-income countries. the risk of acquiring paralytic polio from the live oral polio vaccine is thus particularly sensitive to public scrutiny. however, replacing the oral vaccine with the risk-free inactivated polio vaccine is far more expensive, and would be judged unacceptable if cost-eff ectiveness were the only criterion under consideration. nevertheless, until polio is eradicated globally, vaccination must continue or polio will again become endemic, as shown by occasional outbreaks in unvaccinated communities. although not usually quantifi ed in cost-eff ectiveness analysis, , the prospect of eradication and concerns over the public's perception about the entire vaccination programme has led to the replacement of oral vaccine by inactivated vaccine in nearly all highincome countries. some infections have the capacity to aff ect not only patients and their direct contacts (ie, their family, health-care provider, employer) in terms of economic costs and medical eff ects, but they may also aff ect health-care use, and expectations and behaviour of consumers and investors. for instance, pandemic infl uenza is likely to lead to capacity problems within the health-care system, aff ecting the timely treatment of patients with infl uenza in addition to those with unrelated illnesses. additionally, it would have a macroeconomic impact that goes beyond lost productivity to employers of sick patients, because virtually everyone-employers, con sumers, and investors-would adapt their intentions under its perceived threat. , the latter was also shown in countries aff ected by the outbreak of severe acute respiratory syndrome. finally, affl uent countries pay much higher prices than poorer countries. this system of tiered pricing is not unique to vaccines, but might be most relevant for new vaccines (eg, rotavirus, pneumococcal, and hpv) and medications (eg, highly active antiretroviral therapy) with great lifesaving potential in poor countries. some economists argue that market prices set for high-income countries need to be much higher to suffi ciently stimulate personal view innovation through market mechanisms, rather than rely on publicly funded research. conversely, if a vaccine is added to a low-income country's national programme, it is likely to become cheaper for high-income countries through price discrimination mechanisms. clearly, decision making becomes more complex if such moral or opportunistic considerations are thought to be important. there are some methodological aspects to which the cost-eff ectiveness of vaccines is particularly sensitive. first, the defi nition of the analytical viewpoint is crucial. guidelines for economic evaluation, as used by most advisory committees, generally focus on direct health-care costs and do not consider indirect costs to society (eg, the value of lost productive and leisure time from illness or caregiving). these indirect costs can be very large for infectious diseases that aff ect virtually the entire population, even for generally benign illness. for example, the cost-eff ectiveness of childhood vzv vaccination is unlikely to be thought acceptable from the health-care budget perspective, but is possibly cost-saving from a societal perspective. , , second, the use of quality-adjusted life-years is widely advocated as the best measure currently available for valuing health states. however, standardised quality-oflife estimates for short-term diseases in young children are virtually non-existent, and the appropriate methods to measure them are subject to debate. [ ] [ ] [ ] additionally, the impact of a child's illness on the quality of life of caregivers can be substantial, just as it is for lifethreatening and severe chronic diseases in adults (eg, cancer). however, such indirect quality-of-life losses are typically not accounted for. these impacts have the potential to change decisions, for instance on rotavirus vaccine. finally, the peace of mind off ered through the reassurance of vaccine protection is a quality-of-life improvement of prevention programmes that is routinely ignored in economic evaluation. a third issue is the impact that discounting has in accounting for time preference. discounting is a technique that aims to put costs and benefi ts occurring at diff erent timepoints on the same basis of comparison. discounting scales down future events, such that, the further into the future they occur or the higher the discount rate, the less important they are to a decision maker in the present. in health economics, there is continued debate about whether the discount rate for health outcomes should be lower than or equal to that for costs. , for curative therapies, most benefi ts accrue immediately or shortly after the intervention is initiated, and the cost-eff ectiveness of these interventions is therefore largely independent of these methodological disagreements on discounting. conversely, the costeff ectiveness of most prevention programmes is highly sensitive to discounting because of the long time spans over which benefi ts accrue. a slight decrease in discount rate-eg, from % to %-could change the costeff ectiveness of vaccination from unacceptable to attractive. country-specifi c recommend ations on discount rates vary to the extent that a vaccine could be deemed cost eff ective in one country and cost-ineff ective in another for this reason alone (table ). in the standard discount procedure, as recommended in all guidelines known to us, the discount rate is constant, implying that preferences between outcomes are held constant through time and depend only on the length of the time interval between them. one can argue that discounting at a constant rate exaggerates the importance we give for the present over the future. [ ] [ ] [ ] this assertion is backed by psychological empirical evidence, which suggests that the diff erence between equidistant outcomes is thought less important the further into the future the outcomes occur. so-called "slow" discounting procedures could be used for cases in which the discount rate decreases and falls close to zero for the more distant future (eg, · % for years - , · % for years - , % thereafter), thus yielding a higher present value of benefi ts. , additionally, time preference may exist only to the time until risk exposure, and not the time until health consequences from risk exposure arise (eg, cervical cancer is the health consequence of a much earlier exposure to hpv). adjustment of the discount procedure to account for these aspects is not current practice, but would substantially improve the estimated cost-eff ectiveness of prevention versus cure. , currently, policy makers are presented with very wide cost-eff ectiveness ranges for preventive public-health actions when sensitivity to discounting is illustrated to them. in , baumol noted the "sorry spectacle" that economists provided through their diverging understandings on this subject, and his assertion that "little help is provided to the decision maker who is confronted with such an enormous range of finally, the equity impact of vaccination is far less predictable than for most drugs. generally, the less healthy or less wealthy are those least likely to be vaccinated, and thus more likely to experience the eff ect of herd immunity from other people receiving vaccination. as shown for measles in bangladesh, this eff ect is often equitable, but the reverse may also occur for poorly executed vaccination programmes. the redistribution eff ects on health and wealth are thus less straightforward in the prediction of decisions on vaccination compared with those used for therapeutic medicines. the fi rst generation of vaccines, such as measles, pertussis, and polio vaccines, were against serious childhood diseases that were common worldwide. little analysis was done before their introduction because their benefi ts were obvious and their costs were low in an era when there was less pressure on the health-care budget. new vaccines are much more expensive and often aimed at less common or less serious diseases, particularly in wealthy countries. thus, whether these vaccines are worth introducing is less clear. we will explain key aspects of the cost-eff ectiveness of current vaccines, while focusing on high-income countries. rotavirus is the commonest cause of dehydrating gastroenteritis in the world and accounts for most gastroenteritis hospital admissions in children under years of age. deaths are infrequent because of good medical care in high-income countries (eg, about three deaths per year in the uk). a challenge to the evaluation of both current oral rotavirus vaccines is the estimation of the part of the gastroenteritis disease burden specifi cally attributable to rotavirus, as well as assessing the extent to which these vaccines would invoke herd immunity. in high-income countries, the main benefi t of rotavirus vaccines is the prevention of parental care and productivity losses in virtually all households with infants or toddlers. however, as we have outlined, gains in quality-adjusted life-years in such young children and their parents, as well as parental care and work loss, are not standard features in cost-eff ectiveness analyses. given the current price setting (€ - per fully vaccinated child) and the recommended schedule for these vaccines (two doses rotarix [glaxosmithkline]; three doses rotateq [merck]), they are unlikely to be judged as cost eff ective unless these so-called "soft" benefi ts are also included. , [ ] [ ] [ ] but if they are, why should they not also be considered for all other health-care interventions, thus potentially reshuffl ing the comparison between all health-care programmes (including the other vaccines discussed here)? table describes potential consequences of including soft costs and benefi ts at various levels of government decision making. hpv vaccines are eff ective against the two hpv serotypes associated with most cervical cancers, and one of these vaccines also protects against two of the serotypes that cause genital warts. eff ectiveness against cervical cancer would have to be modelled based on the premise that hpv infection is a necessary condition for cervical cancer to develop, although often only decades later. the cost- programmes that prevent disease, with a proportionately larger aggregated impact on the quality of life and productivity of patients and/or their families, become more cost eff ective compared with other vaccination programmes ·· hpv=human papillomavirus. *costs and benefi ts arising to parties generally not considered relevant in guidelines for economic evaluation of pharmaceuticals for which public funding is sought. these third parties can consist of people not receiving the intervention, parents of patients, employers of patients, and employers in general. †cost-benefi t analyses do not routinely inform other sector decisions in many countries (eg, education, transport infrastructure, military, etc.). politics may dominate rational decision rules in other sectors more than in health care. ‡produced by glaxosmithkline. §produced by merck. ¶produced by wyeth. personal view eff ectiveness of hpv vaccines depends heavily on the choice of the discounting approach used. , furthermore, mathematical models for hpv vaccination ideally have to build in complexities related to herd-immunity eff ects from vaccinating cohorts of girls only and boys additionally, the optimum frequency of cervical cancer screening, and type-specifi c progressive infection and replacement, all over long time periods, which makes this a very complex programme to assess properly. , however, a more simple approach, based on static models, could give insights on the basic question: should we vaccinate girls before their sexual debut? such models would underestimate the benefi ts of hpv vaccination, and therefore would only be helpful for policy if they resulted in favourable cost-eff ectiveness ratios. the static models that have been published so far have tended to be favourable. , , policy makers could therefore quickly decide about vaccinating a limited number of cohorts before their sexual debut, and have reasonably confi dent cost-eff ectiveness evidence to support this decision. however, they cannot rely on such analyses to decide on more complicated aspects of the programme, such as the breadth of the programme in girls and boys. in view of the high costs of this programme (€ - per fully vaccinated individual), the uncertainty surrounding these more complicated decisions could unnecessarily postpone policy on the more basic issue. vzv childhood vaccination prevents chickenpox in vaccinated children and is likely to protect these vaccinees against shingles later in life. since chickenpox infects virtually all children by age years, the accumulated societal savings, including avoided parental care and productivity losses, are likely to be greater than the costs of vaccination at a price of € - per fully vaccinated person. however, childhood vzv vaccination increases the occurrence of shingles in adults and this may be such that it counteracts these societal savings and leads to adverse health eff ects. a further complication is that with single-dose infant vaccination many teenage breakthrough cases can still be expected, but the addition of a second dose to prevent this would make it a much less cost-eff ective programme. modifi ed vzv vaccine in adults was recently shown to prevent shingles, and was shown by static models to be cost eff ective. , finally, vaccination of susceptible pre-adolescents is an alternative strategy that has consistently been shown to be cost eff ective to the health-care budget, and is thus independent of the wider societal perspective. however, it is not advocated by public-health specialists, because it would only prevent a small part of all chickenpox disease, albeit the most severe proportion. , , clearly, the simultaneous modelling of all these strategies and considerations requires complex models and data from various sources to establish eff ectiveness. empirical studies alone cannot answer all these questions. infant infl uenza vaccination may be a cost-eff ective way of preventing seasonal infl uenza and pneumonia in young children directly and the elderly indirectly through herd immunity. , however, vaccinating a child partially to save a grandparent from experiencing serious illness does not only raise concerns over intergenerational equity, but also the eff ectiveness of such an approach could only be shown if put into practice on a large scale, or by applying an appropriately parameterised model. seasonal variations in incidence, severity of disease, and vaccine effi cacy are complicating factors that contribute to uncertainty. furthermore, preparing for pandemic infl uenza demands very large investments, and this can only be shown to be worthwhile by modelling. a policyrelevant approach to modelling the cost-eff ectiveness of pandemic infl uenza vaccination would entail considering macroeconomic impacts across sectors and across countries. clearly, deciding on the best options to prevent and control infl uenza requires an analytical framework and applied modelling work that substantially digresses from usual drug assessments. the currently available seven-valent pneumococcal conjugate vaccine (pcv ), which costs about € - per vaccinated child, is eff ective against invasive and non-invasive disease caused by seven serotypes of s pneumoniae. because of its high price, in the short term the cost-eff ectiveness of pcv depends in most high-income countries on the inclusion of positive herd-immunity eff ects in adults, which were observed after year of widespread use in the usa. if the long-term eff ect of its widespread use, consisting of a mix of herd immunity, serotype replacement, antibiotic resistance, and cross reactivity, remains benefi cial and if the cheaper three-dose schedule confers near-equivalent protection to the original four-dose schedule, pcv vaccination programmes are judged to be cost eff ective in high-income countries. to budget for this vaccine, european policy makers should accept imputations from herd-immunity eff ects observed in other countries in the short term as well as uncertainties with both positive and negative impacts of the programme in the longer term. advisory processes on drug funding can be generally eff ective at selecting which pharmaceuticals, and which subgroups of patients, should be subsidised to make the most of scarce health-care resources. vaccines are diff erent and more complex than most drugs assessed by such processes for the reasons we have outlined. this implies that such processes should be more fl exible in accepting the best available quantifi ed evidence of the unique features of vaccination programmes, and that decision makers and their advisers should be aware of these features if they cannot be quantifi ed. the best personal view available evidence depends on the type of infection and vaccine, and the time of its consideration. guidelines for economic assessment of pharmaceuticals dictate the approach to use to make such analyses acceptable for a country's decision makers. since economic evaluation is not an exact science, such guidelines are made on the basis of compromises between the people designing them and therefore can be changed (table ) . economic evaluation requires quantifi cation of the eff ects of interventions, as well as valuing these eff ects. in terms of quantifying the eff ects of vaccination, governments should adapt their guidelines to specify modelling options for the assessment of interventions against infectious diseases. this should enable submitters and drug-reimbursement committees to better understand which models are acceptable (or unacceptable) under which circumstances. crucially, drug-reimbursement committees must be represented by the required expertise to properly understand and evaluate complex vaccine models. in terms of valuing the eff ects of vaccination, we do not plead for a special case, but for a level playing fi eld. that is, we argue that not all aspects of ill health and time preference are currently captured by recommended techniques for economic evaluation, and that this may disadvantage the cost-eff ectiveness of interventions against diseases in children relative to interventions against diseases in adults, and prevention relative to cure. therefore, guidelines should also be adapted in general terms to allow for ( ) a wider perspective to account for eff ects on third parties, if these are aff ected substantially by specifi c interventions (eg, parents experiencing a quality-of-life impact through the illness of their child); ( ) a wider scope of costs to be included, if appropriate, than health-care system costs alone (eg, irrecoverable losses caused by modifi ed behaviour when faced with a large public-health threat); and ( ) alternative discounting techniques to deal with social time preference over long time periods. large uncertainties about the value and distribution of particular variables imply that timely vaccine decisions may need to be taken with more uncertainty than decisions on other drugs. this should not deter the widespread use of new safe and effi cacious vaccines, if-all things considered-these are unlikely to be judged cost-ineff ective relative to other interventions. furthermore, other criteria, including the programme's acceptability, feasibility, budget, and equity impact, are also important. a who guide for the standardisation of economic evaluations of immunisation programmes, which will become shortly available for public use, could be used as a starting point for governments to adapt their guidelines with respect to some of the issues mentioned here. economic evaluation of vaccination vaccine adverse events: separating myth from reality economic epidemiology and infectious disease the role of economic evaluation in vaccine decision making: focus on meningococcal group c conjugate vaccine human papillomavirus vaccination-reasons for caution the seroepidemiology of human papillomavirus infection in australia herd immunity: history, theory, practice impact of model, methodological, and parameter uncertainty in the economic analysis of vaccination programs evaluating the cost-eff ectiveness of vaccination programmes: a dynamic perspective eff ectiveness of sevenvalent pneumococcal conjugate vaccine against invasive pneumococcal disease: a matched case-control study cost-eff ectiveness of a routine varicella vaccination program for us children theoretical epidemiologic and morbidity eff ects of routine varicella immunization of preschool children in the united states impact of anti-vaccine movements on pertussis control: the untold story eradication versus control for poliomyelitis: an economic analysis compulsory vaccination and conscientious or philosophical exemptions: past, present, and future improving uptake of mmr vaccine cost-eff ectiveness analysis of changing from live oral poliovirus vaccine to inactivated poliovirus vaccine in australia poliomyelitis outbreak in an unvaccinated community in the netherlands, - cost-eff ectiveness of incorporating inactivated poliovirus vaccine into the routine childhood immunization schedule world wide experience with inactivated poliovirus vaccine partially wrong? partial equilibrium and the economic analysis of public health emergencies of international concern precautionary behavior in response to perceived threat of pandemic infl uenza the economic impact of sars: how does the reality match the predictions? tiered pricing of vaccines: a win-win-win situation, not a subsidy who benefi ts from new medical technologies? estimates of consumer and producer surpluses for hiv/aids drugs is g putting profi ts before the world's poorest children? economic evaluations of varicella vaccination programmes: review of the literature comments on the prosser et al approach to value disease reduction in children quality-adjusted life-years lack quality in pediatric care: a critical review of published cost-utility studies in child health preferences and willingness to pay for health states prevented by pneumococcal conjugate vaccine eff ect of pneumococcal vaccination on quality of life in children with recurrent acute otitis media: a randomized, controlled trial cost-eff ectiveness of rotavirus vaccination: exploring caregiver(s) and "no medical care" disease impact in belgium methodological issues and new developments in the economic evaluation of vaccines need for diff erential discounting of costs and health eff ects in cost eff ectiveness analyses discounting and cost-eff ectiveness in nice-stepping back to sort out a confusion recommendations of the panel on cost-eff ectiveness in health and medicine guidelines for the pharmaceutical industry on preparation of submissions to the pharmaceutical benefi ts advisory committee (pbac): including major submissions involving economic analyses. canberra: commonwealth department of health and ageing guide to the methods of technology appraisal. london: national institute for health and clinical excellence dutch health insurance board. guidelines for pharmacoeconomic research canadian agency for drugs and technologies in health. guidelines for the economic evaluation of health technologies: canada, rd edn. ottawa: canadian agency for drugs and technologies in health prescription for pharmacoeconomic analysis national institute for clinical excellence. guidance for manufacturers and sponsors pharmac responds to richard milne on discounting health benefi ts and costs a prescription for pharmacoeconomic analysis time preference, the discounted utility model and health anomalies in intertemporal choice: evidence and an interpretation the social rate of discount and the optimal rate of investment saving future lives. a comparison of three discounting models proportional discounting of future costs and benefi ts valuing prevention through economic evaluation: some considerations regarding the choice of discount model for health eff ects with focus on infectious diseases social rate of discount measles vaccination improves the equity of health outcomes: evidence from bangladesh epidemiology of rubella and congenital rubella syndrome in greece estimating the number of deaths with rotavirus as a cause in england and wales evaluating rotavirus vaccination in england and wales. part ii. the potential cost-eff ectiveness of vaccination the cost-eff ectiveness of rotavirus vaccination in australia cost-eff ectiveness and potential impact of rotavirus vaccination in the united states cost-eff ectiveness analyses of human papillomavirus vaccination the potential cost-eff ectiveness of prophylactic human papillomavirus vaccines in canada the epidemiological and economic impact of a quadrivalent human papillomavirus vaccine ( / / / ) in the uk modeling human papillomavirus vaccine eff ectiveness: quantifying the impact of parameter uncertainty varicella vaccination in england and wales: cost-utility analysis a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults the epidemiology of herpes zoster and potential cost-eff ectiveness of vaccination in england and wales an economic evaluation of varicella vaccination in italian adolescents infl uenza vaccine eff ectiveness in healthy -to -month-old children during the - season interdisciplinary epidemiologic and economic research needed to support a universal childhood infl uenza vaccination policy school-based infl uenza vaccination program reduces infl uenza-related outcomes among household members optimal allocation of pandemic infl uenza vaccine depends on age, risk and timing convincing or confusing? economic evaluations of childhood pneumococcal conjugate vaccination-a review the global burden of disease assessments-who is responsible? pb acknowledges funding from simulation models for infectious disease processes (simid), a strategic basic research project funded by the institute for the promotion of innovation by science and technology in flanders (project number ). we thank the anonymous referees for their helpful comments. key: cord- -uvnh s r authors: dube, taru; ghosh, amrito; mishra, jibanananda; kompella, uday b.; panda, jiban jyoti title: repurposed drugs, molecular vaccines, immune‐modulators, and nanotherapeutics to treat and prevent covid‐ associated with sars‐cov‐ , a deadly nanovector date: - - journal: adv ther (weinh) doi: . /adtp. sha: doc_id: cord_uid: uvnh s r the deadly pandemic, coronavirus disease (covid‐ ), caused due to the severe acute respiratory syndrome coronavirus (sars‐cov‐ ), has paralyzed the world. although significant methodological advances have been made in the field of viral detection/diagnosis with in vitro diagnostic tests receiving emergency use approval by the us‐fda, little progress has been made in identifying curative or preventive therapies. this review discusses the current trends and potential future approaches for developing covid‐ therapeutics, including repurposed drugs, vaccine candidates, immune‐modulators, convalescent plasma therapy, and antiviral nanoparticles/nanovaccines/combinatorial nanotherapeutics to surmount the pandemic viral strain. many potent therapeutic candidates emerging via drug‐repurposing could significantly reduce the cost and duration of anti‐covid‐ drug development. gene/protein‐based vaccine candidates that could elicit both humoral and cell‐based immunity would be on the frontlines to prevent the disease. many emerging nanotechnology‐based interventions will be critical in the fight against the deadly virus by facilitating early detection and enabling target oriented multidrug therapeutics. the therapeutic candidates discussed in this article include remdesivir, dexamethasone, hydroxychloroquine, favilavir, lopinavir/ritonavir, antibody therapeutics like gimsilumab and tjm , anti‐viral nanoparticles, and nanoparticle‐based dna and mrna vaccines. the entire world is facing the pandemic coronavirus disease of (covid- ) due to the outbreak of novel coronavirus ( -ncov; sars-cov- ) and an international health emergency has been declared. the disease has touched nearly every corner of the world. -ncov infection is highly contagious and containment efforts mostly deal with identification and quarantine of exposed and asymptomatic suspects, contact tracing, detection, and strict isolation of infected patients to limit its spread. [ ] [ ] [ ] [ ] world health organization (who), on th january , declared the disease caused by -ncov as the sixth international public health emergency. due to its rapid escalation across the globe, the who declared the -ncov outbreak a global pandemic on the th of march . so far, i.e., from st december to st september , more than individuals were afflicted by -ncov, and deaths have occurred worldwide. [ ] on the th of february , the who officially declared a name for the ongoing -ncov related disease pandemic as coronavirus disease whereas, the international committee on taxonomy of viruses (ictv) has renamed -ncov as severe acute respiratory syndrome coronavirus (sars-cov- ) based on its close genetic resemblance with another coronavirus sars-cov. [ , ] sars-cov- can transfer from an infected person with or without symptoms to another individual, with a high basic reproduction number (r ) value. although the initial r was estimated to be between . and . for sars-cov- , one estimate published in february indicated that the r value is as high as . to . , with the infected individual doubling time of . d. [ , ] the r value indicates that each individual can spread the infection to . - . individuals on average. more recent studies published in august indicated that covid- has a higher transmission rate than severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) with a basic reproduction number of . and an incubation period of . d. [ ] globalization and the convenience of country to country travel had additionally fueled the worldwide spread of the disease. [ , [ ] [ ] [ ] this article discusses sars-cov- nanostructure, the virus biology in connection to its epidemiology, clinical manifestations, and potential and future therapeutic options including repurposed drugs, vaccine/protein therapies, immune therapies, and nanotherapeutics. additionally, the recommended preventive and protective measures are presented (figure ). coronaviruses (covs) are a large family of viruses and are known causative agents for respiratory, hepatic, intestinal, and neurological diseases of altering severity in several animal species. tyrell and bynoe in were the first to cultivate and describe the covs from patients having common colds. [ ] covs are single-stranded ribonucleic acid (rna) viruses. morphologically, they are spherical virions, having a core of rna that is being surrounded by a membrane decorated with protein projections (called spike proteins) like a ring/crown (latin: corona = crown). nanomaterials with very complex structures. they are efficient nanovectors capable of transferring their genetic materials to the infected host cells, followed by hijacking host-cell machinery to express their proteins. [ ] many viruses have proven their worth in the field of drug, gene, and vaccine delivery. [ , ] each sars-cov- virion has an average diameter of - nm (figure ) . [ , , ] similar to other covs, sars-cov- carries four major structural genes encoding major structural proteins, known as the spike protein (s), an envelope protein (e), the membrane protein (m), and the nucleocapsid protein (n); n protein embraces the genome consisting of ssrna and the proteins s, e, and m constitute the envelope of the virus (figure ) . the s protein enables the virus to bind to and amalgamate with the host cell membrane. it is the key immunogenic antigen and has a decisive role in defining virulence, tissue tropism, host range, and protective immunity. [ ] the trimeric s protein belongs to a class i fusion protein consisting of s (receptor binding) and s (membrane fusion) subunits. the subunit s consists of a c domain carrying the rbd, and an n domain. s contains fusion peptide, the heptad repeat (hr- and hr- ), transmembrane, and cytoplasmic domains. the locking of the rbd to the ace receptor of the host cell initiates conformational modifications in the s subunit that further facilitates fusion among viral and host cell membranes. the s /s juncture is the cleavage site for proteases and is also required to trigger membrane fusion, viral entry, and in the formation of syncytium. [ , ] other structural proteins, like e, m, and n, are involved in the viral assembly. alternatively, nonstructural proteins constituting viral cysteine proteases such as papain-like protease (pl pro), chymotrypsin like protease ( cl pro), rna-dependent rna polymerase (rdrp), helicase, and other accessory proteins, participate in the viral transcription followed by replication. to counter the host immune response, the m protein and pl pro, antagonize the host interferon (ifn) response and help the virus to control in vivo replication efficacy and pathogenesis. [ ] alike sars-cov, sars-cov- ingress into target cells is also aided by the s protein. viral entry depends on i) the locking of s unit of s protein to host cell ace receptors; ii) the s protein priming by host cellular protease tmprss , which facilitates s protein cleavage right at the s /s juncture and the s ' site, facilitating the merger between viral and cellular membranes. following cellular entry, covs disassemble to release their genome (nucleocapsid and viral rna) into the cytoplasm of the infected host cell for initiating the translation of viral polyproteins and subsequent replication of genomic rna (figure ) . [ ] replicase polyproteins of sars-cov- are processed by pl pro and cl pro to form nonstructural proteins (helicase/rdrp). [ ] various cl pro inhibitors can be expected to repress viral replication by obstructing the cleavage function of the protein. rdrp is a fundamental enzyme of the rna synthesizing machinery of rna viruses, and it is highly conserved in all hcovs. therefore, it served as a prime target in various viral infections to halt genome replication and can be inhibited by various polymerase inhibitors (favilavir, remdesivir). [ , [ ] [ ] sars-cov- utilizes the ace receptors to gain entry into the host cells. [ ] ace is a counteractive component of the renin angiotensin system (ras) that is responsible for maintaining a balance between fluid volume and pressure by utilizing the cleavage products of angiotensin (agt) and their receptors. [ ] [ ] [ ] among these peptides, angiotensin ii (ang-ii) causes vasoconstriction and helps in sodium retention by means of agtr receptor and results in vasodilation and natriuresis by binding to the agtr receptor. the enzyme ace is responsible for producing ang-ii. after infecting host cells, the virus also uses its protease cl pro to suppress nfkappab by degrading the activating factor ikkgamma. [ ] since, nfkappab is involved in the induction of ace by attaching to its promoter and enhancing transcription. [ ] the virus thus reduces the expression of ace. ace further downregulates the expression of ace in-part by ang-ii, which is its catalytic product. [ ] thus, the viral infection results in upregulation of ace and downregulation of ace. it has been shown that, bradykinin, another important molecule that forms an important component of vasosuppressor system involved in inducing hypotension and vasodilation, is being regulated by aces. bradykinin is degraded by ace and its concentration is enhanced in presence of angiotensin - a peptide produced by ace . thus, the enhanced ace expression caused by the virus leads to increased levels of bradykinin causing "bradykinin storm." this is because sars-cov- increases the amount of bradykinin in the infected tissues. bradykinin causes vasodilation leading to swelling and inflammation of the tissue and induces pain. it also increases the production of hyaluronic acid. the bradykinin storm induced leakage of fluid and hyaluronic acid induced formation of hydrogel into the lungs may be the reason behind low oxygen uptake in severe covid- patients and generation of severe symptoms like hypokalemia associated with arrhythmia and sudden cardiac death. [ ] [ ] [ ] [ ] hence, the drugs which can take care of the bradykinin storm can also be considered as potential therapeutics for covid- . sars-cov- phylogenetic analysis showed that it is meticulously related to two bat-derived sars-like covs (bat-sl-covzc and bat-sl-covzxc ) found in horseshoe bats (rhinolophus) with ≈ % nucleotide sequence resemblance with bat-sl-covzc . it exhibits ≈ % nucleotide sequence resemblance with sars-cov and ≈ % with mers-cov. further, a . % nucleotide sequence resemblance to the partial rdrp gene of the bat-cov strain btcov/ was also observed. [ , ] gaining in-depth insight into the sars-cov- virus epidemiology, and characterizing its possible impact, is a pressing need of the current time in order to expand health care measures to tackle the pandemic. the overall impact of a pandemic is governed by the total number of infected persons, transmissibility, and its medical severity (asymptomatic, mild-to-severe symptomatic, requiring hospitalization, or fatal). [ ] covid- pandemic is continuously evolving, with massive number of cases and deaths each day. based on data from the initial outbreak and considering worldwide incidence, the trend of an increasing incidence of covid- principally follows exponential growth in the number of reporting cases. [ , ] the present mean incubation time for covid- is . d, and the median incubation time is . d, with the potential asymptomatic transmission. following sars-cov- infection, most patients (≈ . %) develop symptoms within . d and almost every patient shows symptoms within d. very few (≈ . %) sars-cov- infected patients develop symptoms within . d. [ , , , ] as of st september , countries/areas/territories/ regions have reported confirmed cases of the disease. usa has reported the largest number of confirmed covid- patients ( ), followed by india ( ), brazil ( ), ( ). [ ] sars-cov- can affect all age groups especially the higher risk groups which include: i) children < months, ii) pregnant women, iii) elderly, iv) people with chronic medical ailments (cardiac, pulmonary, hepatic, renal, metabolic, hematologic, neurodevelopmental diseases, or weak im-mune system), v) people with immunosuppressive conditions (hiv/aids, under chemotherapy or steroids), and vi) health care professionals. [ , , ] lower risk groups should self-isolate themselves at home, drink plenty of fluids, and follow general good-health guidelines to keep their immune system strong and healthy. sars-cov- infection is highly contagious, and containment efforts mostly emphasize on quarantine of exposed and asymptomatic suspects and strict isolation of infected patients during the incubation period to limit the disease spread. preliminary clinical feature of the sars-cov- infection in humans is pneumonia, which formed the basis of identification, detection as well as isolation of patients. sars-cov- infection manifests varied clinical features, extending from asymptomatic to a condition with acute respiratory distress syndrome (ards). [ ] in symptomatic patients so far, the clinical manifestations consist of fever > . °c (most common symptom), dry cough, nasal congestion, sore throat, dyspnea, headache, myalgia, fatigue, upper respiratory tract infections, smell/taste dysfunctions, and diarrhea. very few patients are reported with rhinorrhea. pneumonia mostly occurs by - weeks following the initial infection with prominent signs of hypoxemia and deviations in blood gas. [ , , , , ] visible changes observed in chest x-rays and computed tomography demonstrating deterioration in lung tissue are observed to be the ground glass irregularities of the disease. pulmonary consolidation, alveolar exudates, and interlobular involvements have also been observed. patients also show low white blood cell count (leukopenia), and elevation in inflammatory markers (c-reactive proteins, proinflammatory cytokines, etc.) and formation of blood clots in some cases. covid- patients seeking intensive care unit (icu) are particularly older and more likely to carry pre-existing comorbid conditions like hypertension and related heart diseases followed by diabetes. although, certain epidemiological features were identified, additional studies are still warranted. [ , , ] recent reports have also revealed the existence of asymptomatic infections and gastrointestinal symptoms, specifically among youngsters. [ ] the occurrence of asymptomatic individuals with the ability to spread the infection may be a great hurdle in the control and containment of the disease and are posing significant public health threats. however, detailed studies on the extent of asymptomatic persons, their viral loads, and share in transmission need further evaluation. this allows a better understanding of the viral pathogenesis and will aid policy makers to develop scientifically sound guidelines. [ ] currently, there is no specific approved therapeutic agent available to treat sars-cov- . treatment is supportive and based on the patient's medical condition to alleviate symptoms. proper public health measures to check the viral infections are of an urgent need to tackle the growing pandemic. additionally, stringent measures must be taken to check the human-to-human transmission to minimize secondary infections and prevent community transmission among near contacts, and health care professionals, and to prevent further spread. based on earlier knowledge of the mers and sars outbreak, the who recommends adaptation of various infection control measures to lessen the overall risk of the disease transmission and prevent overall spread. this includes avoiding close proximity with people showing symptoms of acute respiratory infections, washing hands with soap frequently (minimum for a span of s), using - % alcohol-based hand sanitizers after contacting infected people, and avoiding contact with infected inanimate surfaces and pet/wild animals without having proper protection. [ ] the practice of cough etiquettes by people suffering from acute respiratory tract infection, maintaining distance from these patients, proper covering of cough or sneezes, and washing hands frequently are few other disease preventive and control measures being recommended. the who further recommends proper and consistent adoption of environmental disinfection and cleaning methods like cleaning of supposedly infected surfaces with water, soaps, disinfectants, and detergents. people are recommended to stay at home, avoiding social contact (stay home stay safe). while the governments have imposed lockdown measures, travel bans, and ban on people gathering in parks and public places. people should leave their homes putting facemasks and only for essential/basic commodities or medical needs. people should avoid unnecessary journeys and travel to or from work only when they cannot work from home. people going outside should maintain a social distancing of more than m apart from anyone other than members of their own family. a person showing sars-cov- symptoms (fever of ≥ . °c, a persistent cough or breathing problems) should take extra precautions and self-isolate (quarantine) himself/herself for - d including his family members. [ ] although, significant methodological advances have been made in the field of viral detection of covid- with many companies receiving emergency use approvals for their in vitro diagnostic tests, molecular-based high complexity laboratory tests, or antibody tests as mentioned by the us-fda website (a total of as of september , ), little progress has been made in identifying curative or preventive therapies. [ ] thus, there is a definite need to find therapeutic agents directly targeting sars-cov- , and several scientists around the world are focusing on the development of fruitful prevention/treatment strategies and finding new drugs/antivirals/vaccines against sars-cov- , to halt the ongoing outbreak. the us-fda website lists dozens (a total of companies as of september , ) of companies involved in developing antiviral or vaccine therapies for covid- . [ ] antivirals can be broadly categorized into two classes, i.e., ) virus targeting antivirals, which target the viral life cycle, machinery and pathways or directly inactivate viral structural proteins; and ) host targeting antivirals, which either target the host cellular machinery important for viral infection or target the host's immune response pathways and cascades elicited toward the viral infection. [ , ] in the course of viral infection, various events in the viral life cycle and virus-host protein-protein interactions have been identified as the potential targets for antivirals. [ ] cellular events such as virion adsorption, intracellular transport, uncoating, genome and protein synthesis, and assembly inside infected host cells play a decisive role in the viral pathogenesis and targeting these can be a good strategy in the development of current therapies for tackling viral infections. the existence of a potentially small number of viral targets and fast mutating genes sometimes make the business of finding and developing novel and potent antivirals a challenging task. viruses offer limited intrinsic targets for engineering antivirals. [ ] as viruses are greatly dependent on the host cellular machinery for replication, [ ] targeting various host factor(s) [ ] and molecular pathways hijacked by the virus opens a pandora of opportunities to construct novel antivirals. [ , , ] potential therapeutic targets in sars-cov- : mostly various genes and their encoded proteins of sars-cov- can act as favorable diagnostic, therapeutic, or vaccine targets for the virus (figures and ) . mechanisms such as inhibition of viral enzymes (dna and rna polymerases, cl pro, tmprss , reverse transcriptase, neuraminidase, endonucleases, and other proteases) or processes such as ace cellular receptor inhibitors and endosomal acidification mediators prohibiting viral fusion; molecules interfering with glycosylation of the viral protein, viral assembly, new viral particle transport, and release, and immunomodulation of cytokine release can be potential targets in developing various antiviral drugs for the sars-cov- . [ ] [ ] [ ] tmprss inhibitors (camostat, nafamostat) and furin inhibitors can abrogate the s /s proteolytic cleavage, thereby blocking the viral entry. [ ] in addition, the viral entry can be curbed by using carbohydrate-binding protein inhibitors such as griffithsin, which bind to the spike glycoprotein. [ ] endosomal cell entry and s protein activation inside endosomes depend on the ph-dependent endosomal protease cathepsins and might be clogged using lysosomotropic agents like ammonium chloride, chloroquine, and cathepsin inhibitors. [ ] antibodies against the rbd and other viral proteins can effectively inhibit virus entry into the host cell. the enzyme cl pro, one of the essential proteases of the sars-cov life cycle, plays a crucial role in the proteolysis of various viral polyproteins, controlling viral replication. the cl pro enzyme is considered as a key drug target in the case of sars-cov and mers. [ ] recent studies revealed that sars-cov- cl pro is conserved and carries % nucleotide sequence identity with sars-cov cl pro. hence, cl pro inhibitors that impede the cleaving ability of cl pro might repress virus replication, rendering this enzyme an attractive therapeutic target against covid- . [ , ] however, most of the potential drugs/vaccines in the spotlight as potential covid- therapeutics are only experimental candidates, and vigorous clinical studies are warranted to verify their efficacy and safety. the antivirals being investigated may be toxic and many of them can merely alleviate certain conditions. to date, apart from the emergency use approval of the antiviral drug favilavir in china, india, russia, and parts of the middle east and the emergency use approval of remdesivir by the us-fda and japan in covid- patients, there are no approved therapeutic molecules to treat the covid- pandemic. [ , ] thus far, therapeutic modalities including various western, [ ] natural product-based (nct ), and traditional chinese medicines [ , ] with some potential activity against covid- have been briskly tested clinically, and they exhibited preliminary efficacy against covid- . [ ] drug repurposing (repositioning, or retasking) expedites drug product development by identifying new uses for existing approved or experimental drugs in the current global crisis. this www.advancedsciencenews.com www.advtherap.com strategy could significantly reduce the cost and duration of drug development compared to discovering and developing entirely new therapeutics. [ ] the following are the lists of various drugs/therapeutics under consideration and testing to be repositioned against covid- . favilavir (also known as favipiravir, t- ) a guanosine nucleotide analogue, is an antiviral with broad-spectrum activity. it is a pyrazine carboxamide derivative and is currently being mar-keted in china and japan as avigan for treating influenza. it became the first antiviral drug to gain approval from the national medical products administration of china for clinical trials in treating sars-cov- . favilavir is highly effective in treating rna virus infections by inhibiting the rdrp. favilavir, originally was formulated to battle catarrhal (inflammation in nose and throat), has shown efficacy in clinical trials carried out in covid- patients. chictr and chictr are ongoing clinical studies evaluating the safety profile and efficacy of favilavir. it has been approved in italy by italian medicines agency (aifa) for experimental use against covid- and clinical trials are underway (nct ). it is being administered to www.advancedsciencenews.com www.advtherap.com moderate covid- patients at mg twice a day on the first day, and thereafter mg thrice a day up to d; however, the dose may vary based on indications. favilavir is one of the potential treatment options currently being tried for possible use in the treatment of patients suffering from covid- . despite its potential effectiveness and mass production in china, favilavir is not yet approved as a drug product for covid- . nct is another ongoing clinical trial assessing the usefulness and safety of favilavir in combination with tocilizumab. vero e cells infected with ncov betacov/wuhan/wiv / exhibited a half-maximal effective concentration value (ec ) of . × − m against favilavir. [ ] remdesivir has been labeled as the most promising antiviral by the who for the ongoing sars-cov- caused covid- pandemic. [ ] remdesivir (development code gs- ), an adenosine nucleotide analogue, is a broad-spectrum antiviral drug. a study reported the ec value of remdesivir against vero e cells infected with ncov betacov/wuhan/wiv / to be around . × − m and also showed its antiviral effect against -ncov infected human liver cancer huh- cell line. [ ] it is a monophosphoramidate prodrug and is metabolized to the active form, gs- , which disguises and gets incorporated in new rna strand by viral rna polymerase, thereby escapes proofreading by viral exonuclease, halting genome replication. [ ] [ ] [ ] originally, remdesivir was developed and synthesized to battle ebola and was reported to have treated an american covid- patient, who has now fully recovered after receiving the drug. [ ] however, more clinical data are required before the drug can be approved and is considered as an effective and official drug to treat either sars-cov- or ebola virus. warren and group reported the ec of remdesivir against ebola virus infected macrophages, human endothelial and liver cells to be around . × − to . × − m, and demonstrated its therapeutic efficacy in an ebola virus infected monkey model of the disease. [ ] initially china planned phase iii clinical studies to estimate the safety profile and efficacy of remdesivir in covid- patients based on its promising preclinical data in sars-cov and mers-cov infections, [ , ] evaluating remdesivir in severe covid- (nct ) and in mild/moderate covid- patients (nct ). however, these trials were either terminated or suspended due to the unavailabilty of eligible covid- patients. similarly, in february , the u.s. national institute of allergy and infectious diseases (niaid) carried out a phase iii adaptive covid- treatment trial (actt) globally to assess the efficacy of various investigational molecules compared to the control arm (nct ). preliminary results from the trial indicated that remdesivir was superior as compared to placebo in shortening the recovery time in hospitalized covid- patients. [ ] another multicentric trial for remdesivir compared the drug with the standard of care treatment, which includes supplementary oxygen and ventilator support when indicated, in severe covid- (nct ) patients and patients with moderate covid- (nct ). in the trial, along with the standard care, a mg loading dose of remdesivir was administered on day , followed by mg dose administered as intravenous (iv) infusions every h for - or - d. [ ] results of the trials indicated no significant difference in clinical status of covid- patients compared to the standard of care. [ , ] other drugs like chloroquine or hydroxychloroquine are also under consideration in the global hunt to discover an effective covid- therapy [ ] after their approval for limited and emergency use for covid- by the us-fda. chloroquine a " -aminoquinoline" is an old drug used in the prevention and therapy of malaria. it is further prescribed in systemic lupus erythematosus (sle) and rheumatoid arthritis (ra) due to its anti-inflammatory activity. in preliminary studies, the drug has shown efficacy and tolerable safety profile against covid- associated pneumonia. [ ] wang and group reported effective in vitro inhibition of the virus, using chloroquine. vero e cells infected with ncov betacov/wuhan/wiv / and treated with chloroquine exhibited an ec of . × − m. [ ] chloroquine also acts as zinc ionophore, and allows passage of extracellular zinc to the cytoplasm and inhibits viral rdrp. [ ] [ ] [ ] it also interferes with viral entry by inhibiting host receptor glycosylation. administered to a patient orally at a dose of mg in to h for to d. [ ] hydroxychloroquine, a chloroquine derivative carrying a similar mechanism of action as well as therapeutic activity as chloroquine [ ] but with minimal adverse effects, has also been evaluated as an anti-covid- therapeutic. [ ] in a study by yao and group, hydroxychloroquine inhibited sars-cov- infected vero cells in vitro after h of growth with a lower ec value ( . × − m) as compared to chloroquine ( . × − m). [ ] multicentric clinical trials in china have also revealed that the drug shows a potent broad-spectrum antiviral effect. the mechanism of action may be increased endosomal ph, thereby interfering with the fusion process of the virus with the cell membrane. thus, the virus is incapable of releasing its genetic payload inside the cell and replicate further. unlike the us-fda, european regulators restricted the general use of chloroquine for covid- without significant data and limited their use to clinical trials only. therefore, clinical research at a large-scale is still looked-for to elucidate its mode of action and potential prophylactic/therapeutic efficacy against covid- . a study (chictr ) in china has tested the drug in covid- patients showing mild to moderate symptoms. all the patients received treatments like antiviral and antibacterial drugs, oxygen therapy, immunoglobulin as standard care, whereas only half of them received hydroxychloroquine ( mg per day) together with standard care up to d. clinical symptoms like the rate of recovery of the body temperature, the time required for cough remission were significantly shortened in patients receiving hydroxychloroquine. larger percentage of patients ( . %) with improved pneumonia symptoms in the treatment group compared to the control was reported ( . %). [ ] another phase ii clinical study (nct ) testing the efficacy of hydroxychloroquine in combination with vitamin c, d, www.advancedsciencenews.com www.advtherap.com and zinc toward the prevention of covid- infection is underway. a report by dr. raoult from france further demonstrated significant improvements in covid- patients administered with hydroxychloroquine at a dose of mg kg - for d along with azithromycin. [ ] however, who has pulled out the drugs from their solidarity trial due to lack of efficacy in treating covid- . [ ] . . . np- np- (ifenprodil-brand name cerocal), a potential therapeutic option for idiopathic pulmonary fibrosis (ipf), acute lung injury (ali), and persistent coughs may be repurposed for covid- . np- is n-methyl-d-aspartate (ndma) receptor glutamate receptor antagonist, which targets the nmda-type subunit b (glu nb). [ ] ifenprodil is a vasodilator, originally developed by sanofi as an oral medication to treat blood circulation disorders in french and japanese markets. it is no longer sold in france but is still marketed in japan. ifenprodil is an approved drug in countries like japan and south korea to treat certain neurological conditions. an independent animal study showing a considerable reduction in ali and enhanced survivability in avian h n infected mice encourages researchers to expand clinical programs to ali and ards associated with covid- infection. [ ] an adaptive phase iib/iii study assessing the safety and efficacy of ifenprodil ( / mg three times a day) together with standard of care in comparison to standard of care alone in the treatment of hospitalized covid- patients is underway (nct ). ifenprodil is already being tested in clinical trials in patients suffering from ipf and its associated cough (nct ). a repurposed drug combination treatment against sars-cov- , lopinavir and ritonavir (protease inhibitor combination; kaletra), is currently used as both first-and second-line antiretroviral medication for hiv. lopinavir is given in conjunction with ritonavir to increase its half-life. [ , ] several clinical trials of lopinavir/ritonavir, either alone or with various combinations, are underway. china launched a controlled trial (chictr ) to test the efficacy of lopinavir/ritonavir and ifn - b combination in patients hospitalized with covid- . the most commonly studied dosing regimen of lopinavir/ritonavir for covid- infection is mg/ mg orally twice daily for d. [ ] some other antiretrovirals were also screened for anti-sars-cov- activity. [ ] a randomized controlled phase iii trial (nct ) assessing the efficacy and safety of darunavir/cobicistat combination (prezcobix) for treatment of covid- is underway. however, due to the lack of efficacy in hospitalized covid- patients, who withdrew lopinavir/ritonavir from their solidarity trial. [ ] . . hoffmann and co-workers showed that sars-cov- infection relies on the host cellular factors, such as ace and tmprss , and could be successfully blocked by using protease inhibitors. [ ] ace enzyme is a protein recognized by various covs (sars-cov and sars-cov- ) to gain cell entry. [ , ] ace receptor is expressed by the epithelial cells covering organs like lung, intestine, and blood vessels. [ ] in addition to ace as the entry receptor, the virus also requires cellular proteases for the priming of s protein to enter the host cells. tmprss is a serine protease employed by covs for s protein priming. [ ] therefore, it is anticipated that the viral entry inside host cells can be obstructed by serine protease tmprss inhibitors. the marketed tmprss inhibitors, nafamostat and camostat have been demonstrated to be effective in blocking the sars-cov- cellular entry. [ ] a phase iia trial studying the impact of camostat mesilate (foipan) on covid- patients was initiated during march (nct ). nafamostat exhibits inhibitory effect against ncov betacov/wuhan/wiv / infected vero e cells with an ec value of . × − m. [ ] leronlimab (pro ) is used to treat breast cancer and hiv. leronlimab is a humanized monoclonal antibody (mab) capable of blocking ccr (ccr antagonist). it is in clinical trials for mild to moderate covid- patients (nct ) and it is expected to benefit patients showing respiratory complications by diminishing the cytokine storm. currently, leronlimab has received fast track approval by the us-fda as a combination therapy with antiretroviral therapy for hiv and triple-negative metastatic breast cancer and has completed nine clinical trials, including a phase iii trial in hivinfected patients. [ ] . . another promising repurposed antiviral drug is umifenovir, also known as arbidol which inhibits the virion membrane fusion to host cell by targeting the interaction between s protein/ace receptors. arbidol is currently approved in china and russia as prophylactic and treatment drug against influenza. in vitro cellular models showed antiviral activity against sars and therefore it is anticipated to have promising results against sars-cov- . the most studied dosing regimen of umifenovir for covid- infection is mg orally every h. ongoing clinical trials are further evaluating the efficacy of umifenovir. [ ] a randomized, placebo-controlled, phase iv clinical trial assessing the safety and efficacy of umifenovir as an adjuvant therapy to the combined therapeutic regimen of ifn a, lopinavir/ritonavir and hydroxychloroquine in moderate to severe covid- patients (nct ) is underway. our immune system relies on two vital pillars: ) the innate/general immunity; and ) the adaptive/specialized immunity. recent literature suggests that innate immunity can also influence the nature of adaptive responses apart from adv. therap. , figure . the immune responses generated against sars-cov- . mainly two types of immune responses are generated in the host against the virus. one is the normal or positive immune response, which leads to virus neutralization and ceasing of the disease progression and the other one is the abnormal or aggressive immune response that gives rise to disease associated complications like the ards during a severe covid- infection. protecting the host against viruses during early infection when the initial adaptive immune responses take effect. [ ] the synchronized activities of innate and adaptive immunity are vital for gaining overall protection against viruses. innate immunity gives early reactions by enabling the detection and destruction of pathogens quickly within hours and consists of the skin and mucous membranes in the body openings forming external barriers, phagocytic cells, natural killer (nk) cells, various substances in the blood and body fluids. [ ] adaptive immunity takes more time in the detection and destruction of pathogens, but it targets the pathogen more precisely and can patrol the body against antigens for months/years by producing memory cells. adaptive immunity consists of t cells, b cells, antibodies, and cytokines as soluble proteins in the blood and tissue. [ ] upon virus entry inside host cells, here most specifically the airway epithelial cells for sars-cov- , two types of immune responses are being observed (figure ) . one is the positive and healthy immune response and the other is the negative and defective immune response. in the case of rna viruses, after the virus entry and replication and further release inside the host cells, the infection is detected by a set of pattern/pathogen recognition receptors (prrs) of innate immune system comprising first-line of defense against viral infection. inside the cell, prrs like toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) sense the viral ss/dsrna genome and its replication intermediates. upon cellular entry, the virus is recognized by the endosomal ssrna sensor (tlr / ), the cytosolic dsrna sensor (rig-i, mda- ), and the cytosolic inflammasome sensor (nlrp ). subsequently, these different sensors recruit adaptor proteins including myeloid differentiation primary response (myd ) and mitochondrial antiviral signaling protein (mavs) respectively to further activate downstream signaling pathways. this causes activation of the transcription factors like nuclear factor kappa b (nf-b) and ifn regulatory factors (i.e., irf and irf ), and subsequently triggers the production of type i/iii interferons (ifn-and ifn-) and proinflammatory cytokines like interleukin beta (il- ), il- , and tumor necrosis factor alpha (tnf-), respectively. [ ] the antiviral activity of ifn-and ifn-is further amplified by the expression of ifn stimulated genes (isgs) such as ribonuclease l (rnase l) and the proinflammatory chemokine (cxcl ) and is vital in limiting the spread and replication of the virus and modulating the innate/adaptive immune responses. cytokines released by infected cells further modulate the adaptive immune response by recruiting and activating immune cells in eliminating the virus. this comprises the positive immune response, wherein the infection attracts t cells specific to the virus, and destroys the virus checking the infection. neutralizing antibodies are also produced against the viruses, which block the viral infection, and alveolar macrophages further clear the viruses by phagocytosis. [ ] the virus additionally activates the inflammasome sensor, nlrp , that lead to the secretion of highly inflammatory cytokine il- and the initiation of pyroptosis (highly reproduced with permission. [ ] copyright , invivogen. inflammatory form of programmed cell death). this comprises the negative immune response, wherein the host cells undergo pyroptosis, elicited against cytopathic viruses, in response to the viral release. then pathogen-associated molecular patterns (pamps) like viral m-rna and damage-associated molecular patterns (damps) like atp, nucleic acids, and asc oligomers are released from the host cells in reaction to the virus. these molecular patterns are then recognized by other neighboring cells, which include macrophages, epithelial and endothelial cells causing them to release various chemokines and proinflammatory cytokines like ifn gamma-induced protein (ip- ), macrophage inflammatory protein (mip- ), mip- , and mip- , and il- . these chemokines and cytokines attract immune cells like macrophages, monocytes, and t-cells to the infection site leading to further inflammation and additional production of ifn by t cells. in a defective response, higher accumulation of immune cells causes overproduction of the proinflammatory cytokines causing a cytokine storm that damages the organ being infected, and then it circulates to other organs causing multiorgan damage. it has also been observed that non-neutralizing antibodies produced against the virus by the activated b cells further enhance the damage by sars-cov- infection by a phenomenon called antibody-dependent enhancement. [ ] downregulation of the host ifn response can cause an unbalanced immune response producing high levels of proinflammatory cytokines and infiltration of inflammatory cells leading to hyperinflammation causing more severe clinical symptoms of covid- . covs have evolved many mechanisms to stop type i ifn induction and signaling. severe covid- patients demonstrated unusually impaired type i ifn signatures in comparison to mild or moderate cases. [ ] therefore the category of host immune response generated against the virus (figure ) defines the disease severity in the sars-cov- infection, and aggressive immune response against the virus causes significant damage to the lung airways. [ ] so, the adoption of immune therapeutic strategy against the virus could be either based on fortifying the positive immune response or minimizing the dysfunction immune response by means of immune-suppressive therapies. the process of vaccine development against the virus should be dealt with caution. following are some of the list of therapeutic molecules/drug candidates that are meant to target the immune response generated against the virus and under consideration in the present scenario. one promising vaccine candidate for covid- is the fusogenix dna vaccine (covigenix). fusogenix is a proteo-lipid vehicle (plv), formulated with well-tolerated neutral lipids and entos proprietary fusion-associated small trans-membrane www.advancedsciencenews.com www.advtherap.com proteins (fast proteins), which have a novel fusion mechanism to deliver therapeutic nucleic acid payloads directly into target cells, intact and unmodified. fusogenix dna vaccine utilizes plasmid dna, which encodes several protein epitopes (antigen) from key immunogenic sars-cov- proteins to develop an advanced therapeutic payload with maximum protection. these protein epitopes will help activate the body's inherent antibody production and elicitation of a protective immune response against covid- . further, in comparison to traditional vaccines, the dna-based vaccine avoids the use of the infectious agent, shows enhanced stability, easy large-scale production, and is expected to stimulate both b-and t-cell responses. in preclinical in vivo studies, covigenix showed high immunogenicity, efficacy, and safety. in phase i/ii human clinical trials, covigenix will be further evaluated for its safety, tolerability, immunogenicity, and efficacy. [ ] another vaccine candidate developed against sars-cov- is mrna- . it is a lipid nanoparticle (lnp) loaded with an mrna that encodes for a complete, prefusion stabilized form of the s protein of the sars-cov- . mrna- is in phase i human clinical trial to examine the safety and immunogenicity of its five dose levels ( , , , , and µg) on a double-dose vaccination schedule (nct ). on th may , mrna- was granted fast track designation by us-fda. data from the phase study (first clinical batch), with µg and µg dose levels, have shown the presence of well-tolerated neutralizing antibodies titers at or above convalescent sera. safety data obtained from the phase study led to the discontinuation of the µg dose level in the subsequent phase ii studies. [ ] phase iii trial was next launched to assess the efficacy, safety, as well as immunogenicity of the vaccine at a dose of µg in adult participants (nct ). another vaccine for covid- is based on an adenovirus and is named as ad -ncov. this vaccine is being developed by the chinese company cansino biologics in partnership with china's institute of biology at the country's academy of military medical sciences. results from a phase i safety (nct ) and phase ii (nct ) trials conducted for the vaccine, demonstrated strong immune response against sars-cov- . therefore, in an unprecedented move, the chinese military approved the vaccine as a "specially needed drug." [ , ] its phase iii trial in saudi arabia is underway (nct ). another rna based vaccine candidate developed against sars-cov- is bnt b . it is based on lnps loaded with nucleosidemodified messenger rna (modrna) that encodes for an optimized sars-cov- full length s protein and rbd. based on preclinical and phase i/ii studies (nct ) data and in consultation with the us-fda's center for biologics evaluation and research (cber), bnt b has received fasttrack designation by the us-fda and will be tested at a µg dose level (in a two dose regimen) in a global phase ii/iii study (nct ). [ ] another vaccine candidate against covid- is a purified inactivated sars-cov- virus vaccine called coronavac (formerly called as picovacc). preclinical studies for the vaccine showed the induction of specific neutralizing antibodies against sars-cov- in rats, mice, and macaques (nonhuman primates) those were able to neutralize representative sars-cov- strains. three immunizations with the vaccine at two different doses ( / µg per dose) provided partial or complete protection in non-human primates (macaques) against sars-cov- infection. [ ] preliminary results of phase i/ii clinical trials (nct and nct ), which was tested at three/two different doses in a two doses regimen either scheduled on day / or on day / or both. (antigen content of , , su/ . ml) showed promising immunogenicity and no severe adverse events. coronavac got an emergency approval for limited use by the chinese government. phase iii clinical trial (nct ) is underway to assess its safety and tolerability. the immunization schedule is two doses im ( su/ . ml) with a d interval. ino- dna vaccine is designed in such a way to directly deliver optimized dna plasmids into cells using inovio's trademarked device called cellectra. cellectra utilizes a transitory electric pulse to reversibly open tiny cellular pores, enabling the plasmids to enter. once inside the cell, the plasmids begin replicating along with the cell's dna and are translated into proteins (mabs), producing a specific immune response. this methodology has the potential to generate therapeutic mabs in vivo. clinical trials are underway to assess its safety, tolerability, and immunogenicity (nct ). smith and co-workers in a preclinical study testing humoral immunogenicity in both mice and guinea pigs have shown the successful generation of neutralizing antibodies and t cell immune responses against sars-cov- . these encouraging preclinical results further support its translation to large randomized clinical trials. [ ] an adenoviral vaccine candidate azd (provisionally named chadox ncov- ) based on the sars-cov- s protein that was originally developed to target mers is also a potential vaccine candidate for covid- . it is a weakened and safer form of a common cold adenovirus (chadox ) that cannot reproduce within the body but can produce cov s protein after vaccination resulting in the formation of antibodies against the s proteins. the vaccine's seed stock was in production at oxford university's biomanufacturing facility, uk, and phase i/ii clinical trials (nct /isrctn and nct ) assessing its safety, efficacy, and immunogenicity against sars-cov- are underway. chadox ncov- is being administered intramuscularly (im) either at a single dose of × viral particles (vp) or a single dose of × vp followed by a booster dose. the preliminary results from phase i/ii trial (nct ) showed an acceptable safety profile, and a second vaccine dose (boosting dose) further enhanced the antibody responses inducing both humoral and cellular immune responses. [ ] doremalen and co-workers in a study successfully demonstrated a robust humoral and cell-mediated response from a single dose of an investigational vaccine, azd . a single vaccination has prevented pneumonia caused by sars-cov- in six rhesus macaques by halting sars-cov- replication. [ ] the vaccine has progressed to phase ii/iii (nct ) as well as phase iii trials (isrctn ). on september , astrazeneca stopped global trials of the vaccine after finding a volunteer, who developed a type of inflammation called transverse myelitis. the british trial resumed on september , , but trials in other countries are still on hold. another vaccine in progress is based on virus-like particles (vlp). benthamiana. vlp mimic viruses without genetic material and enables the body's immune system to generate an immune response. vlps cannot replicate and are noninfectious. in this plant-based approach, the genetic sequence of a virus is inserted into agrobacterium, which then enters into the plant tissues. the plant begins to produce the protein that is used as a vaccine. in case the virus begins to mutate, the production may be updated in new plants. using plants and genetically engineered agrobacteria, the mass-production of vaccines is easy, faster, and inexpensive. the phase i trial (nct ) evaluating safety, tolerability, and immunogenicinity at dosages of . , . , or µg of the vaccine candidate alone or followed by a booster dose in healthy human volunteers is underway. medicago is also planning to initiate its phase ii/iii trials. [ ] a genetically modified infectious bronchitis virus (ibv) vaccine is also in the pipeline to treat covid- . ibv was originally developed to treat avian coronavirus. it is available in oral form and has shown efficacy in preclinical trials. the new vaccine is anticipated to turn sars-cov- infection into a very mild cold. [ ] another potential protein-based vaccine candidate in the pipeline is covid- s-trimer (scb- ). it is a recombinant subunit vaccine developed using clover's patented trimer-tag technology via a prompt mammalian cell expression system. s-trimer vaccine is based on the s protein of the sars-cov- . the company also identified the antibodies against the trimeric s protein in the serum of patients fully recovered from covid- . a randomized, placebo-controlled phase i trial of scb- has been launched and will be evaluating the safety, reactogenicity, as well as immunogenicity at multiple dose levels ( , , and µg) with and without the adjuvant (nct ). [ , ] vaast, oral recombinant vaccine tablets based on the genome covid- causative virus using vaxart's proprietary oral vaccine platform, are in preclinical trials. each vaccine constructs is a nonreplicating viral vector based on a different sars-cov- antigen combination. [ , ] adcovid is another potent single dose, intranasal vaccine candidate in preclinical trials to protect against covid- . it is an adenovirus-based vaccine expressing sars-cov- s protein. [ ] . . . sputnik v sputnik v a covid- vaccine was registered on th august and approved for early use by the russian ministry of health under the adopted emergency rules during the pandemic. it is a dual adenovirus vectors (rad and rad ) based vaccine loaded with a fragment of gene coding s protein of the sars-cov- . the use of two dissimilar forms of adenovirus vectors, for the first and second vaccination doses is a unique technology of the gamaleya national center for ensuring long lasting immunity. on st august , phase i/ii clinical trials of sputnik v (nct and nct ) have been completed and results suggested the induction of strong antibody and cellular immune responses. postregistration phase iii clinical trial comprising ≥ people in russia was expected to get started st august, onwards (nct ). other countries (uae, saudi arabia, philippines, india, and brazil) may also join the phase iii clinical trial locally. [ ] the key focus of developing therapeutics against the virus has been revolving around finding antivirals and vaccines. however, many reports point toward various patients associated complications such as cytokine storm syndrome (crs) and macrophage activation syndrome (mas), which lead to ards during severe covid- infection. covid- patients developing crs secondary to covid- pneumonia show increased production of various proinflammatory cytokines, chemokines, and other growth factors. therefore, treatment of hyperinflammation or the aggressive immune dysfunctions raised in response to the viral infection using immunosuppression mechanism can be advantageous and aid in reducing the mortality. [ , ] www.advancedsciencenews.com although immunomodulatory therapy is not recommended in covid- pneumonia in general, the compassionate use of immunomodulators might be advantageous in covid- patients exhibiting crs complications, decreasing the high levels of proinflammatory cytokines, and thereby preventing multiorgan failure. [ ] the following are some of the immunomodulators under consideration as anti-covid- therapeutics. gimsilumab (kin ) , a human mab, working by targeting granulocyte macrophage colony stimulating factor (gm-csf), is under clinical trials to prevent and treat ards in covid- patients. as gm-csf elevated levels in blood further augment the expression of other proinflammatory cytokines causing the progression of ards and serum from covid- patients have shown high levels of gm-csf; therefore, gimsilumab is anticipated to reduce the mortality rate by reducing lung damage. a multicenter, phase ii trial (nct ) is underway to assess gimsilumab's efficacy and safety profile in patients with ards/lung injury secondary to covid- . [ ] tjm (tj ) is a neutralizing antibody against human gm-csf. tjm shows a high affinity towards human gm-csf, thereby blocking gm-csf binding to its receptor. this will further prevent downstream signaling cascades resulting in response to gm-csf binding, halt target cell activation, resulting in inhibition of inflammatory responses causing reduced tissue inflammation and death. tjm is in clinical trials to study its efficacy in reducing the severity of complications associated with covid- . [ ] a phase i/ii, randomized, multicenter trial (nct ) will assess the efficacy of tjm under supportive care when administered as an iv infusion ( mg kg - or mg kg - ) in subjects with severe covid- . lenzilumab, another gm-csf neutralizing immunotherapy, is being studied in minimizing and treating the cytokine storm and associated lung dysfunction/ards in covid- patients. lenzilumab is humanigen's proprietary humaneered mab targeting gm-csf and is being approved by us-fda for sympathetic use in covid- patients. [ ] currently, it is being studied in a multicentric, phase iii trial in comparison to the current standard of care for taking care of respiratory failure and prevention of death in hospitalized covid- patients (nct ). namilumab (izn- , amg ) is another fully human immunoglobulin g mab targeting gm-csf, which has received compassionate use approval from us-fda in the treatment of critical as well as hospitalized covid- patients before their admission to icu. the double center compassionate use study of the drug will be conducted in bergamo and milan cities in italy. it is currently in later stages of clinical development programs against ra and ankylosing spondylitis. [ ] tzls- is another antibody in progress as a potential treatment for covid- . tzls- is a humanized mab targeting interleukin- receptor (anti-il- r). early clinical studies channeled in china suggest that anti-il- r mabs might be used clinically for treating covid- patients. tzls- binds to both forms of il- r (membrane-bound or soluble), thereby speedily depleting the levels of il- in blood. overproduction of il- leads to the elicitation of long-lasting inflammation, causing lung damage following sars-cov- infection. [ ] . . . at- another immunotherapeutic candidate explored for covid- is at- . at- is a human recombinant surfactant protein d (rhsp-d). initially, at- was developed for broncho-pulmonary dysplasia (bpd) in premature infants who requisite mechanical ventilation and oxygen therapy. [ ] tocilizumab (actemra) is a recombinant version of a humanized mab targeting the il- receptor, which binds to il- r, thereby inhibiting signal transduction. us-fda has approved it for the treatment of crs, rheumatoid arthritis, giant cell arthritis, polyarticular juvenile idiopathic arthritis, and systematic juvenile idiopathic arthritis. [ , ] xu and group from china in their pilot study used tocilizumab (single dose of mg iv) to treat a total of covid- patients. tocilizumab treated patients showed a significant reduction in fever and il- levels. lymphocyte counts in the patients returned to normal after treatment and they demonstrated an improvement in lung function. [ ] a multicentered randomized phase iii trial of tocilizumab is underway in china to assess its efficacy and safety in covid- patients with pneumonia and elevated il- (chictr ). another proof of concept study (tosca) in italy is underway for its efficacy and safety profile assessment in the treatment of patients with ards and crs secondary to covid- (nct ). another multicentric phase iii placebo-controlled clinical trial, called covacta (nct ), is underway to evaluate tocilizumab (maximum single dose of mg iv and one more dose only if clinical symptoms deteriorate/or exhibits no improvements) along with standard of care versus placebo along with standard of care in severe covid- patients globally. sarilumab (kevzara) is another il- receptor blocker under clinical trials for its ability to lessen the effect of the inflammatory adv. therap. , www.advancedsciencenews.com www.advtherap.com immune response against sars-cov- infection. it is a fully human mab against the il- receptor. it has been approved by us-fda toward the treatment of rheumatoid arthritis, but its use to treat covid- patients is investigational. sarilumab was developed using proprietary velocimmune technology (regeneron's) using a genetically engineered mouse equipped with a genetically humanized immune system to produce fully human antibodies. a multicenter, double blind, phase ii/iii trial (nct ) is underway to assess the efficacy and safety of a single iv dose of sarilumab compared to supportive care plus placebo in severe/critical covid- patients. in phase ii, patients were distributed into three groups at random, with two doses of sarilumab ( and mg) and placebo. preliminary data from the trial led to its immediate amendment to enroll only critically ill covid- patients (either on icu or requiring mechanical ventilation/high flow oxygenation) receiving a higher dose of the drug ( mg) or placebo. [ ] cytosorb is an extracorporeal blood purification technology based on hemo-adsorbent polymer to treat uncontrolled inflammation in patients those have undergone cardiac surgery. it consists of polymer beads with high porosity those can successfully adsorb and remove toxic substances and inflammatory mediators from blood and other bodily fluids. cytosorb has received us-fda's emergency use authorization in the us for use in severely ill covid- patients approaching or confirmed respiratory/multiple organ failure and are admitted to icu. it is approved in the european regions as an adjuvant therapy designed to lessen the complications associated with crs by absorbing a wide range of cytokines, pamps and damps, thereby reducing their levels in circulation. [ ] the use of convalescent sera is a promising treatment strategy against sars-cov- . it consists of patient derived serum enriched with passive neutralizing antibodies against the virus, obtained from covid- recovered patients. [ ] many reports from china suggest the use of convalescent serum as therapy for patients with covid- . china has offered convalescent plasma therapy to covid- patients (chictr ). convalescent plasma therapy is promising, but its safety and efficacy as a treatment modality for covid- are yet to be established. therefore, it is important to study and establish the safety as well as efficacy of convalescent plasma therapy in covid- patients in clinical trials. the us-fda based on encouraging results from early trials has given it the emergency use authorization against covid- . glucocorticoids may modulate inflammation-mediated lung injury in covid- patients and thereby reduce progression to respiratory failure and the resulting death. however, there is uncertainty about the effectiveness of glucocorticoids in covid- . dexamethasone is a corticosteroid used in a wide range of conditions for its anti-inflammatory along with immunosuppressant effects. preliminary findings of the phase ii/iii recovery trial (nct ), a randomized, controlled, open-label study conducted in the uk, noted a survival benefit with the use of dexamethasone (oral or iv at a dose of mg per day for up to d) as compared to standard care alone (invasive mechanical ventilation or oxygen) in hospitalized covid- patients. patients on ventilators showed one third and patients requiring only oxygen showed one fifth reductions in the day mortality. dexamethasone can also be used in both children and elderly, but in pregnancy/breast-feeding cases, the recovery trial used prednisolone ( mg orally) or hydrocortisone ( mg twice daily iv) instead of dexamethasone. [ ] among several drugs employed for the treatment of covid- , n-acetylcysteine (nac) is a potential therapeutic/preventive/adjuvant against sars-cov- . nac is a precursor of reduced glutathione (gsh). it has antiinflammatory, antioxidant, antiviral, and anticoagulant/plateletinhibiting properties. it is approved as an over-the-counter dietary supplement and is in the who list of essential medications. the us-fda has approved the usage of nac to treat acetaminophen overdose associated liver side effects, and also as mucolytic agent (due to its free sulfhydryl group) in cystic fibrosis or chronic obstructive pulmonary disease (copd). it has been also used as an antioxidant in a variety of disorders involving gsh depletion and oxidative stress. thiols can block ace receptors thereby blocking penetration of sars-cov- into cells. nac can act as a potential therapeutic agent in the treatment and/or attenuating the risk associated with covid- through a variety of potential mechanisms, including increasing glutathione level, improving t cell response, and modulating inflammation thereby preventing covid- patients from moving to icus. [ ] an ongoing phase ii clinical trial (nct ) is assessing the number of successful covid- patients discharged/transferred from critical care units after receiving nac ( g per day iv) in addition to supportive or other treatments as prescribed against covid- . other phase iii (nct ) and phase iv (nct ) clinical trials of nac are assessing nac's anti-inflammatory effect and its efficacy in minimizing the severity associated with covid- , respectively. [ ] cyclosporine a (csa) is a calcineurin inhibitor and is used to avert organ rejection and for treating t-cell associated autoimmune diseases. anti-inflammatory and immunosuppressive effects of csa are exerted due to its bonding to cyclophilin-a (cyp-a), thereby preventing the activation of nuclear factor of activated t-cell (nfat) and production of il- (a cytokine crucial for the proliferation of t-cells). csa has shown potent antiviral activity at noncytotoxic micromolar concentrations against many covs including sars-cov in cell cultures. csa may be successful in covid- due to the close resemblance between sars-cov- and sars-cov. this antiviral property might have resulted due to the inhibition of cyp-a-dependent viral assembly and inhibition of the nfat pathway. [ ] [ ] [ ] ongoing phase i safety study of csa (oral or iv) is assessing the tolerability and clinical effects in covid- patients requiring oxygen supplementation (nct ). a phase iia randomized clinical trial (nct ) is underway for the treatment of non-icu hospitalized covid- patients. patients will receive csa orally for up to d at . mg kg - body weight twice a day along with standard of care. another ongoing phase iv clinical trial (nct ) is assessing the efficacy and safety of csa plus standard treatment in comparison to standard treatment in hospitalized covid- patients. another corticosteroid in pipeline is inhaled budesonide. it may directly inhibit the viral replication or may exert the inflammatory effect. steroids reduce the effect of cytokines and thereby modulate immune response. budesonide may prevent the ards associated with covid- . inhaled corticosteroid's therapeutic effect against covid- is limited and still need to be explored. various clinical trials assessing their efficacy in covid- are ongoing (phase iv: nct ). vitamin d is a steroid hormone. vitamin d is being evaluated as an adjuvant therapy for covid- based on its immunomodulatory, anti-inflammatory, antifibrotic, and antioxidant effects. vitamin d exerts direct effect on immune cell proliferation and activity, and ace expression. [ ] the aim of one of its ongoing randomized phase iii trial (nct ) in covid- patients is to assess its efficacy at daily low dose ( iu) versus weekly high dose ( iu) for weeks and to determine the correlation between vitamin d deficiency and clinical characteristics. another phase iii trial (nct ) assessing the efficacy of vitamin d at high dose ( iu) in comparison to standard dose ( iu) is underway. apart from the currently available drugs, immunomodulators, and vaccine candidates, there are many other potent molecules like novel protein or peptide therapeutics and even nanoparticulate based strategies yet to be tested against the virus. these new therapeutics, once proven, can be the game changers in tackling the pandemic. below we list some of the potential future therapeutics for treating covid- patients. in the last decades, peptides as therapeutics and diagnostic molecules have gained massive interest in drug development due to the dynamic research conducted by pharmaceutical and biotech companies. [ ] currently, a large number of both natural as well as synthetic therapeutic peptides are in clinical development for various diseases ranging from cancer, neurodegenerative, cardiovascular, metabolic disorders, autoimmune disorders, hormonal deficiencies, to infectious diseases. [ , ] peptide based therapeutics offer numerous benefits over other molecules such as high selectivity and specificity, high efficacy and safety, tolerability, less immunogenicity, biocompatibility, low toxicity, easy, and cost-effective production. according to their source and method of production, peptide based therapeutics can be classified as: ) natural peptides (derived from peptide hormones, plant proteins, animals, and human-derived peptides); ) recombinant peptides; and ) synthetic peptides. [ ] [ ] [ ] peptides are relatively easy to modify structurally. improved therapeutic performances can be achieved by utilization of modified amino acids, cyclization, etc. many antiviral peptides are being explored for viral diseases like hepatitis, influenza, and hiv. [ ] peptides exhibit a reduced possibility of developing resistance during the treatment. antiviral activities of the peptides can be attributed to three major mechanisms: ) inhibition of viral attachment and penetration into host cells; ) inhibition of replication by interacting with viral polymerases, and ) disruption of the viral envelope. they can be presented as a new generation of antiviral agents with broad-spectrum activities. [ , ] potential peptide-based therapeutics which are in the pipeline against covid- include flowvax peptide vaccine from flow pharma, [ ] corvax signal peptide vaccine from vaxil bio, (vax-hit platform), [ ] epitope-based peptide vaccine from onco-gen (vaccinomics strategy), [ ] ii-key peptide vaccine from generex biotechnology (epivax's computational tools and li-key technology), [ , ] etc. peptide-based therapeutics can be effective alternatives for covid- prophylaxis. nanotechnology, as one of the ground-breaking approaches, can be really promising in offering remedial solutions toward fighting the on-going covid- outbreak and future pandemics. nanotechnology allows a number of tactics to fabricate highly advanced therapeutic options to cope with sars-cov- both outside and inside the host. the scope of nanotechnology against covid- includes conventional as well as advanced biomimetic approaches and engineered nanomaterials with versatile chemical functionalities. therefore, nano intervention will be extremely relevant in advancing covid- prophylaxis, diagnosis, and treatment (figure ) . in view of what we know so far about the sars-cov- structure, its pathophysiology, life cycle, and related immunological response, we envisage key steps where nanotechnologist and nanotechnology could play a pivotal part. herein, nanotechnology is discussed in terms of fabricating engineered nanocarriers to overcome the limitations associated with conventional antiviral and other biological therapeutics, nanomaterials as novel therapeutics, risk free immunization techniques, engineered nanomaterials in the development of point-of-care diagnostics, and alternative advanced disinfection methodologies. nanotechnology aims toward safe, effective, and targeted delivery of existing as well as future therapeutics, fabricating risk-free vaccines for safe and effective immunization, curbing the interactions between viruses and host cells, and permanent disruption of viral particles. [ ] [ ] [ ] in the recent past, many nanoparticulate systems have been tested for their efficacy and made their way to preclinical studies against several pathogenic viruses such as hiv, herpes simplex, human papilloma virus, and respiratory viruses. [ ] [ ] [ ] in the dearth of a specified/broad spectrum antiviral agent against sars-cov- , current therapy focusses on repurposing existing molecules. therefore, to make the repurposed therapeutics more effective and safer, an appropriate nanocarrier delivery system might be useful. successful delivery of therapeutics especially peptide/protein, dna/rna, and other biologicals to the target site is most often hampered due to their poor aqueous solubility, degradation, fast-clearance, low-bioavailability leading to subtherapeutic concentrations. nanomaterials with large surface area to volume ratios, versatile functionalities and surface modification, unique physicochemical properties, and suitable for various routes of administration can easily address the challenges associated with conventional therapeutics. biocompatible organic/inorganic nanoparticles can be potentially tuned to encapsulate therapeutics with large payloads, control release, minimize drug resistance, and improve pharmacokinetic/pharmacodynamic properties and patient compliance. further actively targeted nanocarriers can cross the biological barriers thereby reaching the protected target sites with higher specificity excluding the unwanted exposure to nontargeted areas resulting in dosage reduction, reduced systemic toxicity, improved bioavailability, and enhanced efficacy. nanotechnology further makes it possible to tailor the therapy according to individual needs. [ ] itani and group have demonstrated the potential role of different nanoparticles as efficient delivery agents for potential therapeutics or immunomodulators against covid- . [ ] nanoparticle based delivery of therapeutics (drugs, vaccines, rna/dna, and peptides) ensures targeted and optimized concentration in the desired site while minimizing adverse effects and unwanted degradation enhancing efficacy. donalisio et al. demonstrated nanotechnological approach for the tropical treatment of herpes simplex virus (hsv- and hsv- ) by encapsulating acyclovir in chitosan nanoparticles. the obtained gel formulation showed enhanced penetration across the porcine skin as compared to the commercial cream product, and revealed increased efficacy as compared to its free form. [ ] labauve and group used a lipid-coated mesoporous silica nanocarrier system to deliver antiviral molecule ml for inhibiting venezuelan equine encephalitis virus (veev), in order to improve its solubility and stability. the designed ml loaded nanosystem showed enhanced circulation time, biocompatibility, and viral inhibition in vivo in a murine model of veev infection. [ ] kumar et al. demonstrated the fabrication of zidovudine loaded lactoferrin nanoparticles. zidovudine is a highly bioavailable antiretroviral drug, which shows serious side effects and toxicity. results revealed higher efficacy and reduced toxicity of nanocarrier based formulation as compared to their free soluble counterparts. [ ] liu et al. demonstrated the fabrication of liposomes encapsulating cholesterol modified hydroxychloroquine for the treatment of bleomycin induced pulmonary fibrosis. the engineered nanosystem inhibited the proliferation of rat lung fibroblasts and reduced any toxicity associated with hydroxychloroquine thereby, providing evidence as a possible therapeutic agent for pulmonary fibrosis. [ ] encapsulation in a nanosystem enhances nucleic acid-based vaccine stability and offers a newer fusion mechanism to transport the genetic material loaded inside them directly to the cells. similarly, the efficacy of other mrna/sirna vaccines can be significantly enhanced via complexing them with lipid/polymerbased nanoparticles. these lipid/polymer-based nanoparticles are based on ionizable monomers, which complex negatively charged rna/dna and thus facilitate escape of the entrapped rna/dna from endosomes into the cytosol where the genetic material can be translated. [ ] the successful inhibition of viruses achieved with nanoplatforms could have extensive advantage in combatting various viral infections including sars-cov- . combinatorial therapy aims in suppressing more than one pathway simultaneously, thereby, producing synergistic effect. therefore, combinatorial therapy may represent another therapeutic approach against sars-cov- . it displays numerous advantages such as dose reduction of the individual therapeutics, leading to fewer adverse events. it may also overcome drug resistance. nanocarriers are very promising systems for the co-delivery of multiple drugs with different physicochemical properties. they enable encapsulation and sequential release of both hydrophobic and hydrophilic drugs in a single platform. nanoparticles can also be instrumental in attaining combinatorial multidrug therapeutics for controlling uncontrolled inflammation and manage disease severity. a very recent work by dormont et al. developed targeted multidrug nanoparticles composed of an en-dogenous lipid squalene, an immunomodulator adenosine and an antioxidant -tocopherol for treating acute viral inflammation (figure ). [ ] freeling et al. fabricated a lipid-drug nanoparticle system comprising of three antiretroviral drugs (lopinavir, ritonavir, and tenofovir) to overcome lymphatic drug insufficiency and reported -fold higher and sustained intracellular concentrations in lymph nodes compared to current oral therapy of free drugs. this combinatorial nanotherapy can be used to target sars-cov- . [ ] bearing in mind the risk of random mutations in sars-cov- genome, which may result in variation in antigenic protein, nanoparticle functionalization with a varied number of antigenic moieties/proteins concurrently will enhance the efficacy of vaccines against viral infections. these next-generation nanocarrier-based vaccines will not only help in guarding the antigenic proteins against metabolism/degradation but will also help in greater exposure of antigenic proteins to antigen-presenting cells, thereby improving their delivery and efficacy. nanomaterials have emerged as a favorable tool both as immune stimulators and immune suppressors for immune modulation. nanomaterial based nonviral vectored vaccines can successfully overcome two major limitations associated with conventional vaccines of weak immunogenicity and reversion risk associated with live/attenuated viral vectors, and can be a reliable alternative to viral vaccines. nanosystems can imitate the genome transfer ability of viral vaccines which show high pathogenicity and can also deliver molecular adjuvants simultaneously. [ ] recombinant protein and inactivated vaccines in general necessitate the use of adjuvants to enhance their immunogenicity. many nanomaterials depending on their functionalization possess an intrinsic adjuvant property and can amplify the immune response in a host. therefore, fabricating nanomaterials with intrinsic ability to amplify host's immune response will be very helpful in enhancing the efficacy of vaccines especially in patients with impaired immune system. nanosystems can also enhance the targeted delivery of immunosuppressants to both adaptive and innate immune systems. [ ] various nanoparticles like graphene oxide, silver, gold, copper, and zinc nanoparticles show intrinsic antiviral/antibacterial properties and can act as potential therapeutics against the disease. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] they may offer alternative methods to traditional disinfection protocols used in healthcare sites. nanoparticles can potentially sustain release, enhance cell entry, and reduce efflux of toxic metal ions. the use of metal nanoparticles would be particularly advantageous as antiviral agents because metals may attack a wide range of viral targets. also, there is a lower risk of developing drug resistance in the case of metals as compared to conventional antivirals. [ ] silver nanoparticles (agnps) have been investigated for their antimicrobial potency against bacteria, but many reports [ ] copyright , the authors. point toward their antiviral activities against viruses, which includes hiv, herpes simplex virus, respiratory syncytial virus, hepatitis b virus, etc. [ ] [ ] [ ] li and group have demonstrated efficient in vitro inhibition of h n infection utilizing agnps based codelivery of oseltamivir (neuraminidase inhibitor) by inhibiting viral attachment and release. [ ] the fact that agnps are less toxic and are nontoxic at lower concentrations to humans, vouch for their possible usage for therapeutic purposes. given the fact that silver ion itself attacks a broad range of targets in the microbial entry, microorganisms generally do not develop resistance against silver. [ ] nanocolloid agnps have also been shown to be effective in controlling viral proliferation in the respiratory tract after inhalation delivery, thus controlling the disease progression. [ ] another study by levina and group demonstrated in vitro antiviral activity of dna decorated titanium dioxide (tio ) nanosystem against different influenza a virus subtypes infected mdck cells. the nanosystem was found to be highly efficient in delivering nucleic acids directly inside the cells without the aid of any transfection agent with an ic value of . µg ml - ( × − m for dna). [ ] another nanostructured material, graphene, has shown promising antimicrobial/antiviral properties. [ ] [ ] [ ] recently, the efficacy of cu to inactivate hucov- e was demonstrated. it was found that the hucov- e ( plaque forming units) was inactivated irreversibly in less than min on brasses/alloys containing at least - % cu, whereas the virus was shown to persist on surfaces like glass, rubber, and stainless steel in an infectious state for at least d. [ ] dhanasezhian et al. fabricated biogenic gold and agnps using the seaweed sargassum wightii. their cytotoxic and antiviral activity against hsv- and hsv- strains on vero cells showed that functionalized metallic nanoparticles act as antiviral agents by hindering virion attachment and cellular entry, depending on their particle size. [ ] figure . schematic illustration for the selective naked-eye detection of sars-cov- rna facilitated by the suitably designed aso-capped au nanoparticles. reproduced with permission. [ ] copyright , american chemical society (acs). https://pubs.acs.org/doi/ . /acsnano. c technologies will accelerate patient management and identification and containment of infection hotspots. nanoparticle based disease monitoring mediated by various metallic nanoparticles carrying magnetic, fluorescence, and surface-enhanced raman scattering features would aid in tracking treatment response. [ , ] moitra and group reported the development of gold nanoparticles (aunps) based colorimetric assay for rapid, selective, and visual diagnosis of positive covid- patients within min after the isolation of viral rna. in the presence of targeted rna from the virus, thiol-modified antisense oligonucleotides against n protein of sars-cov- capped aunps exhibited selective agglomeration leading to altered surface plasmon resonance. subsequent addition of rnaseh further cleaved the rna from the rna-antisense oligonucleotides complex leading to the formation of a precipitate (figure ) . [ ] graphene sheets conjugated to antibodies can rapidly detect targeted viral proteins and viral targeting antibody functionalized graphenes can further be useful in the development of biosensors against covid- . [ ] similarly, utilizing theranostic nanoparticles, both therapy and diagnosis can be combined in order to detect and neutralize the virus in a single go, halting the virus to survive and reproduce within the host. the theranostic nanoparticles can efficiently complex with sars-cov- , detecting and disrupting their structure. [ ] potential nanoparticle based therapeutics which are in the pipeline against the current covid- outbreak include proteo-lipid nanovehicle based fusogenix dna vaccine, lipid nanoparticle-loaded mrna vaccine (mrna- ), plantbased virus-like particles (vlp), recombinant protein nanoparticle vaccine (nvx-cov ), [ , ] and nanomicelles based antiviral. [ ] analogous to cancer nanotherapeutics, covid- can also be managed successfully by exercising d nanotechnologybased approaches that include efficient detection, diagnosis, and delivery. the tissue and cell targeting principle achieved by targeting antibodies and peptides in handling other dreaded diseases like cancer and neurodegeneration can be adopted in the case of the antiviral nanoparticles for achieving targeted and effective therapy of infected patients. furthermore, theranostic nanoparticles can be developed to detect and kill the virus simultaneously. nanoparticle based kits can be developed to detect the virus at an early stage in asymptomatic patients with lesser viral loads. nanoparticle based antiviral material can be developed for fabricating superfine filters for face masks, personal protective equipment, and surface coatings. further, nanotechnology may assist in large-scale production of therapeutics and equipment addressing covid- and similar pandemics. thus, there are varieties of therapeutic molecules against covid- . the list of these potential therapeutic molecules under consideration for covid- is summarized in table . the exceptional speed, with which the sars-cov- pandemic is escalating across the world, is causing global anxiety to attain new heights. covid- has become the reason for the globally prevalent fear, stress, and concern. all these are natural and common reactions to the fast-changing and undefined situation that all of us find ourselves in. further, there is a noticeable degree of fear, stress, and apprehension in certain groups with the higher risk factor, such as children, older people, health care professionals, frontline workers, and people with pre-existing maladies (such as asthma, diabetes, heart diseases, and hypertension). the impact phase iii nct - [ ] linear dna vaccine dna based on sars-cov- spike (s) protein applied dna sciences/ takis biotech preclinical - [ ] zycov-d dna plasmid dna vaccine zydus cadila phase i/ii ctri/ / / [ , ] bacillus calmette-guerin (bcg) vaccine phase iii chictr - [ ] (continued) adv. therap. , phase iii - [ , ] covaxin inactivated coronavirus whole-virion inactivated sars-cov- vaccine (bbv ) phase i/ii nct - [ ] nvx-cov protein recombinant sars-cov- nanoparticle vaccine consisting of trimeric full-length sars-cov- spike glycoproteins and saponin based matrix-m adjuvant novavax phase i/ii nct - [ , ] flowvax peptide vaccine adjuvanted microsphere peptide vaccine targeting sars-cov- nucleocapsid adjuvanted microsphere peptide vaccine targeting sars-cov- nucleocapsid flow pharma preclinical ebola, marburg, hiv, zika, influenza, hpv [ ] signal peptide vaccine (vxl- , , ) signal peptide vaccine using vaxhit bioinformatics platform vaxil bio preclinical - [ ] epitope-based peptide vaccine peptide synthetic long peptide vaccine candidate for s and m proteins (vaccinomics strategy) oncogen preclinical - [ ] ii-key peptide peptide synthetic peptides that mimic the epitopes of the sars-cov- using epivax's computational tools and li-key technology generex/ epivax preclinical influenza, hiv, sars-cov [ ] monoclonal antibodies gimsilumab (kin ) human mab targets gm-csf roivant sciences phase ii nct - [ ] tjm (tj ) neutralizing antibody neutralizing antibody against human gm-csf phase i/ii nct - [ ] lenzilumab neutralizing antibody neutralizing antibody against human gm-csf humanigen phase iii nct compassionate use - [ ] namilumab (izn- , amg ) human mab targets gm-csf izana bioscience compassionate use ra and ankylosing spondylitis [ , ] tzls- human monoclonal antibody mab against il- receptor tiziana life sciences preclinical autoimmune diseases [ ] at- protein human recombinant surfactant protein d (rhsp-d) airway therapeutics preclinical bronchopulmonary dysplasia (bpd) in premature infants [ ] tocilizumab (actemra) human mab mab against il- receptor roche phase iii nct chictr ra, giant cell arthritis [ , ] sarilumab (kevzara) human mab il- receptor blocker sanofi and regeneron phase ii/iii nct ra [ ] convalescent sera passive neutralizing antibody passive neutralizing antibody developed in covid- recovered patients -p h a s e chictr phase ii nct - [ ] anti-inflammatory molecules glucocorticoid anti-inflammatory and immunosuppressant -phase ii/iii nct - [ ] (continued) adv. therap. , phase iv nct - [ , ] cyclosporine a calcineurin inhibitor anti-inflammatory and immunosuppressant phase iia nct phase iv nct [ ] [ ] [ ] budesonide corticosteroid anti-inflammatory -phase iv nct vitamin d steroid anti-inflammatory, antioxidant, immunomodulatory, -phase iii nct nct - [ ] of the crisis on people's mental health is also of major concern. as the new measures to prevent the infection are being introduced, such as quarantine (social isolation), cases of depression and loneliness are expected to rise. the disruptive effects of the covid- outbreak are dominating our daily lives; therefore, it is of prime importance that we should manage and react properly to this challenging situation unfolding fast in our lives. the world is in total chaos, lives have frozen, and everything around us has come to a standstill situation. nature has forced us to stay indoors. let us hope, together; we will be able to find a cure and curb the grave epidemic as soon as possible. many drug candidates are under investigation for the disease. initial reports suggest some of them are promising at this juncture. however, whether these drugs will act following virus dynamics and host response is a big question. how they will behave in a clinical trial is yet another long-term debate. besides, their affordability in developing countries is a significant concern. antiviral therapy shows a wide margin of ineffectiveness as many viruses possess extraordinary genetic adaptability, which endows them with the ability to escape antiviral repression. in some instances, the viruses reclaim advantage over the host by online mutagenesis, which creates novel strains with additional acquired resistance to the existing antivirals. therefore, finding new and multifaceted antiviral agents is the unmet need of the hour. an approach analogous to cancer nanotechnology, including early diagnosis, targeted delivery, and combinatorial approaches can be followed to tackle this deadly viral nanovector. us-fda's extraordinary speed in reviewing emergency use authorization (euas) and investigational new drug (ind) applications and their prompt approval to expedite anti-covid- therapeutics into the clinical development is highly appreciable. who and partners had launched an international clinical trial named solidarity, for comparing potential options against standard of care to find an effective treatment for covid- . the trial's aim is to expedite the discovery of any potential drugs that can slow down the disease progression or improve patient's survival rate. on th july , who has withdrawn hydroxychloroquine and lopinavir/ritonavir from their solidarity trial. the solidarity trial's committee found that these drugs were ineffective in treating the infection. similar findings were also reported from the uk's recovery trial. further, us-fda's approval for the emergency use of remdesivir and other drugs, and other medical countermeasures (mcms) and facilitating their availability is another noteworthy move in combating covid- and is an example of using science and technology at its best in protecting and rescuing the world from emerging chemical, biological, radiological and nuclear (cbrn) threats. with the current focused, rapid, worldwide discoveries in basic science, translational research, and clinical studies, and the expedited regulatory scrutiny, it is anticipated that preventive and curative therapies for covid- will be a reality in the near future. coronavirus disease (covid- ) weekly epidemiological update and weekly operational update world health organization naming the coronavirus disease (covid- ) and the virus that causes it proc. natl. acad. sci proc. natl. acad. sci summary of probable sars cases with onset of illness from epidemic and pandemicprone diseases (mers situation update sars-cov- life cycle: stages and inhibition targets who coronavirus disease (covid- ) dashboard infection control guidance for healthcare professionals about coronavirus (covid- coronavirus disease (covid- ) advice for the public emergency use authorization (eua) information, and list of all current euas covid- company directory clinical drug information drug discovery today favilavir: first approved drug to possibly treat coronavirus outline of trial designs for experimental therapeutics solidarity" clinical trial for covid- treatments biopharma products in development for covid- immunobiology: the immune system in health and disease predicted immune responses to sars-cov- coronavirus vaccine tracker israeli-made oral vaccine for coronavirus on track, but testing will take months proc. natl. acad. sci. usa catching up to coronavirus: top treatments in development therapeutic advances in infectious disease theranostics ieee th int. conf. on nanotechnology antibacterial and antiviral graphene-based coating for public settings key: cord- - bma authors: xu, yingying; yuen, pak-wai; lam, jenny ka-wing title: intranasal dna vaccine for protection against respiratory infectious diseases: the delivery perspectives date: - - journal: pharmaceutics doi: . /pharmaceutics sha: doc_id: cord_uid: bma intranasal delivery of dna vaccines has become a popular research area recently. it offers some distinguished advantages over parenteral and other routes of vaccine administration. nasal mucosa as site of vaccine administration can stimulate respiratory mucosal immunity by interacting with the nasopharyngeal-associated lymphoid tissues (nalt). different kinds of dna vaccines are investigated to provide protection against respiratory infectious diseases including tuberculosis, coronavirus, influenza and respiratory syncytial virus (rsv) etc. dna vaccines have several attractive development potential, such as producing cross-protection towards different virus subtypes, enabling the possibility of mass manufacture in a relatively short time and a better safety profile. the biggest obstacle to dna vaccines is low immunogenicity. one of the approaches to enhance the efficacy of dna vaccine is to improve dna delivery efficiency. this review provides insight on the development of intranasal dna vaccine for respiratory infections, with special attention paid to the strategies to improve the delivery of dna vaccines using non-viral delivery agents. majority of the current licensed vaccines for the prevention of infectious diseases are live-attenuated vaccines, inactivated vaccines, or subunit vaccines. each of them has its pros and cons. the live-attenuated vaccines can stimulate both cellular and humoral immune responses, and induce prolonged immunity that closely resembles natural infection. however, there are safety concerns associated with the use of the live attenuated virus or bacterial vaccines as they may revert to disease causing forms. it is also difficult to target multiple viral subtypes or pathogens using live-attenuated vaccines. inactivated and subunit vaccines are safer options as they cannot replicate and do not cause disease. they confer protection mainly through humoral immune responses with little or no cellular immunity. the induced immunity lasts for a shorter period of time; therefore, supplemental doses are always required. in recent years, dna vaccines have attracted considerable attention as an alternative vaccination method against infectious diseases, with the potential to provide broad immune responses similar to the live-attenuated vaccines without the risk associated with the replicating micro-organisms. dna vaccine approach relies on the in situ production of target antigens. plasmid dna encoding antigenic proteins is delivered to the appropriate tissues in the body, leading to the expression of the desired antigens, eliciting specific immunogenic responses and thereby inducing immune protection against the pathogens. since the host cells are responsible for antigen production, the natural glycosylation and folding of the protein are warranted. plasmid dna encoding different bacterial and viral antigens have been tested for their immunogenicity and protective efficacy in vivo, confirming their clinical potential [ ] [ ] [ ] [ ] . in addition, dna vaccines offer several distinct advantages over conventional vaccines. the double helical structure of dna is simple and stable at high temperature, allowing easy storage and transportation. large-scale manufacture of dna vaccines is convenient and relatively cheap. it only requires standard cloning of antigen coding sequence into plasmid vectors, avoiding the complex procedures of repeated culture and inactivation of infectious pathogens, or the purification of recombinant proteins. apart from their advantageous physicochemical properties, dna vaccines have the ability to generate the cellular immunity in addition to the humoral immunity. they are also highly flexible, encoding several types of genes including viral and bacterial antigens, as well as immunological proteins. the advantages of dna vaccines compared to conventional vaccines are summarized in table . the field of dna vaccination is developing rapidly. dna vaccines are currently approved for veterinary use against various infectious diseases [ ] [ ] [ ] [ ] . however, the results in clinical trials have been less encouraging. dna vaccines are generally safe and well tolerated in human, but the immune response is often too low to offer sufficient protection [ , [ ] [ ] [ ] . in early studies, dna vaccines alone were not able to generate t cell responses at a magnitude that was enough to protect against difficult diseases in humans [ , ] . recent attempts still failed to overcome this problem. a plasmid pthr dna hiv- vaccine candidate evaluation in phase i clinical trials on healthy volunteers showed that it had weak immunogenicity in human. no significant hiv- -specific cell-mediated immune response difference was found between vaccine recipients and placebo recipients, in addition to no hiv specific antibody production [ ] . another phase i trial of hiv vaccine using dna prime-virus vector vaccine boost strategy on healthy volunteers was proved effective in eliciting t-cell responses but incapable of inducing neutralizing antibody activities [ ] . in , a human hiv- gag dna vaccine with or without interleukin (il)- and/or il- plasmid cytokine adjuvant was reported to produce poor cellular immunogenicity with no vaccine-induced anti-gag humoral immune responses on healthy volunteers, which contrasted with the previous findings in macaques [ ] . several strategies have been introduced to optimize dna vaccines [ ] . one of them is to enhance the dna delivery efficiency, which is the focus of this review. dna delivery efficiency is dependent on the administration route and the delivery system used. mucosal surfaces are attractive sites of vaccine administration against infectious diseases as they are the portals of entry for many pathogens. vaccination at the mucosal sites where pathogens initiate infections can be more efficacious than parenteral administration as invading pathogens may be neutralized at the front lines before generating any systemic effect. in particular, intranasal vaccine has been extensively investigated in recent years. vaccination at nasal mucosa can stimulate respiratory mucosal immunity by interacting with the nasopharyngeal-associated lymphoid tissue (nalt) where large amounts of local lymphocytes are present. furthermore, intranasal delivery is a needle-free, non-invasive route of administration with the possibility of self-administration. intranasal dna vaccination has become a promising approach in offering immune protection against various pathogens that affect the respiratory system including tuberculosis, coronavirus infection, influenza and respiratory syncytial virus (rsv). in this article, the current developments of dna vaccine delivery systems that are specifically designed for intranasal administration against respiratory infectious diseases are discussed in detail. typically, dna vaccines are administered by intramuscular injection although other administration routes including intradermal, subcutaneous, oral and intranasal routes are also investigated. upon administration, somatic cells (e.g., myocytes or keratinocytes) and professional antigen presenting cells (apcs) are transfected. as the antigens are expressed intracellularly, both humoral and cell-mediated immunity can be activated to offer broad immune protection. the host-synthesized antigens become the subject of immune surveillance in the context of both major histocompatibility complexes (mhc) class i and class ii molecules of apcs. antigen expressed apcs then travel to the draining lymph nodes where they present the antigenic peptide-mhc complexes and stimulate t cells. alternatively, b cells are activated, initiating the antibody production cascades. the major advantage of dna vaccines is their ability to activate cd + t-cells, the cytotoxic t lymphocytes, which are important in controlling infections [ ] . this action is lacking in inactivated or subunit vaccines. the induction of cd + t-cells by dna vaccines can occur in two major ways: (i) direct dna transfection of the apcs such as dendritic cells (dcs); (ii) cross-presentation which occurs when somatic cells such as myocytes are transfected with dna and the expressed antigens are taken up by the apcs, or when the transfected apoptotic cells are phagocytosed by the apcs. the mechanisms of dna vaccines are illustrated in figure . it is interesting to note that immunization may occur rapidly before a dna vaccine expresses the antigens, and antigens expressed in somatic cells may not qualitatively be the major player in eliciting immune response. comparing to the secondary role of somatic cells, bone marrow derived antigen presenting cell (apc) activation is an important indicator for successful induction of dna vaccine [ ] . in this study, dna vaccine was delivered into the skin of mouse ears by gene gun. immune response was produced after the inoculation site (i.e., the ear) was rapidly removed after immunization before antigens were expressed, indicating that mobile cells are important in elaborating immunity. in another similar study, surgical removal of the injected muscle within min of dna vaccines administration did not affect the magnitude or longevity of antibody responses to the encoded antigen [ ] . again, the results confirmed the importance of apcs over somatic cells such as myocytes and keratinocytes for eliciting immune responses. early studies showed that dna vaccines are poorly immunogenic with low levels of antigen expression. to improve the immunogenicity of dna vaccines, cpg motifs are commonly employed in the construct of the plasmid backbone [ ] . bacterial or viral dna contains unmethylated cpg motifs, whereas in mammalian cells, cpg dinucleotide motifs are rare and are usually methylated [ ] . it has been demonstrated that the unmethylated cpg motifs have immunostimulatory effect and are considered by mammalian cells as pathogen-associated molecular patterns (pamps). unmethylated cpg activates innate immune cells through binding to toll-like receptor (tlr- ), which is constitutively expressed in the endosomal compartments of apcs and b cells. once bound to the dna that is rich in cpg motifs, tlr- activates the immune system by initiating pro-inflammatory response that result in the production of cytokines such as interferon (ifn)-γ and interleukin (il)- . however, it was found that tlr- deficient mice also responded to dna vaccines, suggesting that tlr- may not be the sole mediator of the adjuvant effect [ , ] . dna vaccines also interact with cytoplasmic dna sensors including tank-binding kinase (tbk ) [ ] and stimulator of ifn genes (sting) [ ] , activating tlr-independent pathways and inducing ifn-γ. these pathways are crucial in contributing to the immunogenicity of dna vaccines. although persistent antigen expression of dna vaccine is usually expected to provide long-term immune protection against infectious diseases, the effect of sustained expression of antigen must be carefully examined and controlled. it has been reported that prolonged expression of antigen may lead to the switch of type- ifn from an antiviral cytokine to an immunosuppressive cytokine [ ] , or may deplete the pool of memory t cells [ ] . to evaluate the efficacy of dna vaccine in humans, serum antibody titer or the enzyme-linked immunospot (elispot) assays are the commonly employed methods to measure the immunogenic response, although the induction of antigen-specific immune effectors by an immunization process does not imply that these antibodies or cytokines represent surrogates or correlates of vaccine efficacy. in early stage of vaccine development, in vitro serum antibodies and elispot assays are the direct detectable indicators of the clinical potential of a vaccine formulation. at later stage of development, morbidity and mortality (especially the improvement of survival rate after vaccination) in animals upon target pathogen challenge is a more certain way to confirm the protective efficacy of vaccines [ , ] , as the ultimate goal of vaccine is to prevent the targeted disease. the efficacy of vaccine such as influenza vaccine could be monitored in human during subsequent influenza epidemic season [ , ] or challenged with a controlled influenza virus [ ] . however, some lethal virus challenge studies are difficult to conduct directly on human. hence, the measurement of antibodies production and immune responses in humans remain the most direct way to assess vaccine efficacy. longer study is required to investigate if the vaccine is indeed able to prevent disease. safety is always a primary concern with any vaccine products. dna vaccines are generally considered to be safer than conventional vaccine approaches as they lack the risk of reversion to a disease causing state or secondary infection. similar to other gene therapy, the major safety issue related to dna vaccines is the risk of integration of the plasmid dna into the host genome, causing insertional mutagenesis, which may lead to the inactivation of tumour suppressor genes or activation of oncogenes, resulting in devastating adverse effects. according to preclinical and clinical studies, there is little evidence of genomic integration following dna vaccines administration, and the risk of integration is found to be significantly lower than the spontaneous mutation rate [ ] [ ] [ ] [ ] . another safety issue of dna vaccines is related to the development of anti-dna immune responses. animal studies showed that there is no increase in anti-nuclear or anti-dna antibodies after dna vaccination. in clinical trial studies, signs and symptoms of autoimmunity, and the markers of autoimmunity are sometimes monitored in the human subjects. there has been no evidence that autoimmunity is associated with dna vaccines [ , [ ] [ ] [ ] . it has been suggested that proper purification of e. coli can effectively prevent pathogenic anti-dna antibody production [ , ] . antibiotic resistance is another issue related to dna vaccines. typically, large-scale manufacture of plasmid dna involves the use of antibiotic-resistant marker. there is a safety concern that resistance to the same antibiotic might be introduced. in response to this issue, antibiotic-resistance genes in dna vaccine should be restricted to those that are not used to treat human infections. alternatively, the use of antibiotic-resistance genes should be avoided completely [ ] . another concern of dna vaccines is the development of tolerance to the encoded antigen, which appears to be age-related. newborns have immature immune system and are more likely to develop tolerance rather than protection when exposed to foreign antigens. in contrast, immunity instead of tolerance occurred when dna vaccines are administered to young animals [ ] [ ] [ ] . in recent years, there has been an increasing concern that vaccination in general may induce harmful systemic inflammation, which may lead to increase of cardiovascular risk [ ] [ ] [ ] [ ] [ ] . dna vaccine is still considered as a relatively new approach to vaccination but its potential to induce systemic inflammation must not be overlooked. it was reported that little or no local inflammatory infiltration was observed at the dna vaccine injection site, especially after the acute effects of the vaccination have disappeared [ ] . the first clinical trial of a dna-based vaccine for hiv- infection was published in in asymptomatic hiv-infected patients who were not using antiviral drugs. the immunization was well tolerated with neither local, systemic reaction nor laboratory abnormalities were detected after three doses of vaccines [ ] . in addition, no patient developed anti-dna antibody or muscle enzyme elevations. no consistent change of cd + or cd + lymphocyte counts occurred. another early experiment conducted on pigs showed that electroporation of dna vaccines was more efficient in enhancing immune response, but also stimulated inflammatory response and accompanying cellular infiltration, whereas the conventional intramuscular injection of dna vaccines only showed low gene expression and low inflammatory cell infiltration [ ] . it was suggested that improved antigen presentation was one of the possible mechanisms by which increased inflammatory cell infiltration may enhance immune responses to dna vaccines delivered with electroporation. however, the long-term safety effect was not investigated. overall, many recent preclinical studies and clinical trials have indicated that dna vaccines are generally well tolerated with good safety profile, and no systemic inflammation was reported [ , , [ ] [ ] [ ] [ ] . nonetheless, dna vaccines are relatively new vaccination approaches and yet to be approved in human use, the long term safety of their uses must be thoroughly evaluated for routine prophylactic and therapeutic use in human, especially when new delivery systems or adjuvants are introduced into the formulation. conventional vaccines are usually administered by parenteral injections which mainly target the systemic immune system, eliciting only weak mucosal immune response. when the vaccines are delivered directly to the mucosal site, mucosal immune response can be more efficiently potentiated. in particular, nasal mucosa has attracted considerable attention as the site of vaccination in recent years, including dna vaccines, due to several distinct advantages. however, there are also some formidable barriers that need to be overcome to allow successful development of intranasal dna vaccines. the intranasal route of drug administration has been frequently used to treat local conditions such as nasal congestion and allergy. intranasal administration is characterized by easy administration, rapid onset of action and avoidance of first-pass metabolism. the needle-free administration route is non-invasive and can avoid the risk of spreading blood-borne infections, which is a particular problem in developing countries. these desirable features lead to the exploration of the systemic delivery of polar drugs or biomolecules including vaccines that are not feasible in other administration routes. intranasal vaccination has been investigated for over a decade. the majority of currently available vaccines are administered by intramuscular, subcutaneous or intradermal injection. although these parenteral routes of administration are effective in inducing systemic immune responses, they are ineffective in inducing local immunity at mucosal sites. as many as % of pathogens infect human through the mucosal surfaces [ ] . mucosal vaccination could provide better protection than injectable vaccines against infectious diseases by inducing both systemic and mucosal immunity [ , ] . since the strongest immune response is usually induced at the vaccination site and the adjacent mucosal sites, intranasal immunization is able to elicit protective immune response effectively in the lungs and the upper respiratory tract [ ] . nasal mucosa appears to be an appropriate site of vaccine administration against respiratory infectious diseases, not only because the nasal cavity is the first site of contact with inhaled macromolecules and a common site of infection by respiratory pathogens, it can also stimulate respiratory mucosal immunity by interacting with the nalt. current, licensed intranasal vaccines include flumist ® , a live-attenuated vaccine that targets influenza types a and b [ ] ; and nasovac ® , a live-attenuated vaccine that targets h n influenza virus [ ] . apart from live-attenuated vaccine, intranasal route of administration is also favorable to protein-based vaccination, as evidenced by many studies including the intranasal pneumococcal protein immunization against pneumonia [ ] , and a recent study on the intranasal respiratory syncytial virus (rsv) vaccine based on a recombinant fusion protein [ ] . with the success of intranasal live-attenuated virus vaccine and the promising effect of protein-based vaccine, it is highly plausible that dna vaccines can adopt the same delivery route to achieve efficient immunization. it is well established that mucosal vaccination can induce humoral and cell-mediated immune response systemically as well as at mucosal sites [ , ] . immune response induced by mucosal vaccination is mainly initiated at specific mucosa-associated lymphoid tissue (malt). the malt lining the nasal cavity is known as the nasopharyngeal-associated lymphoid tissue (nalt), which include the waldeyer's ring of tonsils, adenoids and a collection of isolated subepithelial lymphoid follicles [ ] . the nalt is rich in immunocompetent cells, including b cells, t cells and phagocytic apcs such as macrophages and dcs [ ] . in addition, the overlying epithelium of mucosal follicles forms a specialized cell layer. these cells have microfolds on their apical surface and are known as microfold cells (m cells). m cells play a crucial role in the initial phase of induction of mucosal immune responses. therefore, m cell targeting is an important strategy to achieve mucosal immunity [ , ] . m cells are efficient in taking up particles from the epithelial surface, transporting them across the cells and releasing them to the underlying extracellular space. this process is known as transcytosis. at their basal surface, the cell membrane of m cells is extensively folded around the underlying immune cells including b cells, t cells and apcs, which take up the particles released from m cells and process them for antigen presentation. the initiation of mucosal immune response is summarized in figure . upon b cells activation following nasal vaccination, the production of antigen-specific secretory immunoglobulin a (siga) is triggered. siga is a critical component in the mucosal immune system. it is protease resistant, and can effectively bind and neutralize pathogens and their toxic products on the nasal mucosa surface despite the protease rich environment, thereby preventing the pathogens from breaching the mucosal barrier [ ] . local immunoglobulin g (igg) production is also detected after mucosal vaccination [ ] and partly contributes to the neutralization of pathogens. however, igg concentration is around - -fold lower than that of the siga due to its susceptibility to protease degradation [ ] . indeed, siga provides the first barrier to pathogens invasion, so induction of potent siga response is an important goal of mucosal vaccination. in addition, nasal immunization can also result in the production of serum iga and serum igg, which can potentially neutralize pathogens that enter the mucosa and prevent systemic spread. when the dcs at the mucosa are presented with antigens, the activated cells may migrate to the proximal draining lymph node and disseminate immune responses to other sites of the body. apart from the humoral immune response, cell-mediated immune response is also induced after mucosal vaccination. although the cytotoxic t cells in the mucosal tissues may not prevent pathogen entry, they are crucial for the clearance of pathogens [ ] . overall, cells of nalt are involved in the regulation of both humoral and cell-mediated immune responses locally and also systemically, offering a broad immune response. since the nasal mucosa is an important portal of entry for respiratory pathogens, the nasal route has become attractive for the administration of vaccines by reinforcing the nasal mucosal immune response. the defense mechanism of the nasal cavity presents a significant barrier to the entry of pathogens and potentially harmful substances; however, it has also become an important barrier to intranasal dna vaccines. the nasal mucosa, which constitutes the outmost layer of the nasal passage, consists mainly of ciliated columnar cells, non-ciliated columnar cells, goblet cells and basal cells. the proportions of these cells vary in different regions of the nasal cavity. nasal mucus, which is produced by goblet cells and submucosal glands, provides a protective physical barrier to foreign materials. it is a highly viscous, gel-like heterogeneous mixture that contains glycoproteins, enzymes, immunoglobulins, salts, proteins and lipidic components [ ] . dna vaccines that are administered to the nasal cavity have a propensity to be trapped by the nasal mucus, leading to enzymatic degradation. the effect of mucus depends on its viscosity and pore size, which affect the diffusivity of agents delivered to mucosal surfaces. in addition, the entrapped dna vaccines may also be removed by the mucociliary action of the ciliated cells that drives the overlying mucus layer continuously towards the nasopharynx, clearing the mucus from the nasal passage, resulting in short residence time at the mucosal surface. another challenge of intranasal vaccine is that the vaccine formulation may be diluted in mucosal fluids, and bulk fluid may limit effective deposition onto the epithelium of the mucosal system. to overcome these barriers, a safe and efficient dna delivery system must be developed. ideally such delivery system should target the mucosal apcs for antigens processing that lead to specific b and t cell activation. the ultimate goals of dna delivery systems are to facilitate the uptake of dna to the target tissues and cells, protect dna from enzymatic degradation, increase the residence time of the formulation in the nasal cavity, enhance the expression of the antigens and to increase the immune response without compromising safety. the different dna vaccine delivery systems currently being investigated for intranasal administration are discussed in detail in section . despite the numerous merits of intranasal immunization, the potential hazard of nasal vaccines must not be overlooked. the concern of the safety of intranasal vaccination was raised after an intranasal inactivated influenza vaccine called nasalflu, developed by berna biotech, was found to be associated with bell's palsy, a temporary neurological paralysis of one side of the face [ ] . nasalflu consists of influenza virosomes which are formulated to contain hemagglutinin (ha) and neuraminidase (na) antigens, as well as heat-labile enterotoxin (lt) from e. coli as mucosal adjuvant. since parenteral administration of inactivated influenza vaccine did not confer an increased risk of bell's palsy, nor the natural influenza virus infection, it was soon concluded that adjuvant lt from e. coli. was the culprit of these cases. for this reason, flumist ® and nasovac ® -both are intranasal live-attenuated influenza vaccines without enterotoxin as adjuvants-do not appear to confer an increased risk for this condition. understanding the pathogenesis of the bell's palsy that was connected to nasalflu has become an important research focus for vaccine development. following intranasal administration in mice, enterotoxins were found in the olfactory nerve and the olfactory bulb for an extended period. since the olfactory epithelium is the only part of the central nervous system (cns) that is exposed to the external environment, drugs and nanoparticles, including intranasal vaccines, may bypass the blood brain barrier and enter the cns through olfactory transmission. there is a reason to concern the neurotoxic effects of vaccine containing enterotoxin adjuvant for intranasal administration. while the nasal delivery of neuronal-binding lt-derived adjuvants is inadvisable [ ] , other toxin-derived adjuvants such as cholera toxin-derived cta-dd and double mutant cholera enterotoxin (ct) are claimed to be safe and effective adjuvant candidates without causing any inflammation or cns toxicity [ , ] . nevertheless, thorough evaluation must be performed with the use of toxin derivatives as intranasal vaccine adjuvants. tremendous efforts are now focusing on the development of alternative adjuvants with better safety profile. mankind has been haunted by respiratory infectious diseases for aeons. they have created public health concerns since ancient times. with the emergence of new or drug-resistant strains, it is becoming a challenge to protect the public from infections using conventional vaccine methods. dna vaccines have huge potential for the prevention of respiratory infections due to their ability to offer broad immunity, the relatively rapid process of designing new dna vaccine construct and the possibility of large-scale production in a short period of time. in this section, four pathogens that cause severe diseases in the airways are highlighted, including tuberculosis, coronavirus, influenza and respiratory syncytial virus, with a brief discussion of current dna vaccine development against these infections. tuberculosis (tb) is a bacterial infectious disease caused by mycobacterium tuberculosis which is transmitted by respiratory aerosols. tb has become a major public health problem that threatens the progress made in tb care and control in the world. the only tb vaccine currently available is an attenuated strain of mycobacterium bovis, bacillus calmette-guérin (bcg), developed in the s. however, its efficacy against adult pulmonary tb remains controversial [ ] . with the emergence of drug-resistant tb and increasing rates of hiv/aids and tb co-infection, a new effective tb vaccine is urgently in need. effective protective immunity to mycobacterium tuberculosis requires cell-mediated immune responses, including both cd + and cd + t cells [ ] [ ] [ ] . since dna vaccines have the ability to induce strong cellular immunity, it has become an attractive vaccine approach against tb. the first two studies that reported promising protective effect with dna vaccine against tuberculosis were conducted in mice using plasmid dna encoding antigen a (ag a) of mycobacterium tuberculosis [ ] and the kda heat-shock protein of mycobacterium leprae (hsp ) [ ] . other different antigens such as ag b esat- , il- , mpt , psts- and other fusion proteins have also been explored as dna vaccines against tuberculosis [ ] [ ] [ ] [ ] [ ] [ ] . most of these dna vaccines encode mycobacterial proteins that are either secreted in mycobacterial culture filtrate or exposed on the mycobacterial cell-wall surface. coronaviruses (cov) are potentially lethal pathogens, characterized by the presence of spike proteins on the viral surface. two new strains, severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov), have been identified. both of them could cause acute respiratory distress syndrome (ards) and are associated with high mortality rates [ ] . cov vaccines have historically exhibited poor capacity for cross-protection [ ] , the development of safe, broad spectrum and effective vaccines that can be rapidly made available during an emerging epidemic is required. currently, there are no approved vaccines for human cov infections, and most of the studies have focused on the sars-cov. spike and nucleocapsid proteins, which are the immunodominant cov proteins, are the antigens of interest for vaccine development [ , ] . dna vaccines that encode nucleocapsid protein induced strong cell-mediated immunity but are not protective after high titer of viral challenge [ , ] . in addition, nucleocapsid dna vaccine could induce delayed-type hypersensitivity even in the absence of an antibody response. this effect was not observed with spike protein dna vaccines. influenza is caused by orthomyxoviruses which are rna viruses that affect mainly the upper respiratory tract. in recent years, zoonotic or variant influenza has become a serious threat to human health, including the avian influenza virus h n and h n , and the swine influenza virus h n and h n . although these animal viruses are distinct from human influenza viruses and do not usually transmit between humans, they may still occasionally infect humans and cause severe pneumonia and even death. furthermore, if such a virus acquired the capacity to spread easily among people, it could start an epidemic or even a pandemic [ , ] . to protect the populations from influenza infection, highly effective, broad-spectrum influenza vaccines that could be prepared rapidly are in demand. current influenza vaccines mainly target the induction of antibodies against the viral glycoproteins, particularly surface glycoproteins hemagglutinin (ha) and neuraminidase (na). antibodies to ha neutralize the infectivity of the virus while antibodies to na prevent the release of the virus from the infected cells. apart from surface glycoproteins, internal proteins such as nucleoprotein (np) and matrix protein (m ), as well as the ion channel protein (m ), which are highly conserved between and within different subtypes, have also become very attractive target antigens for vaccines to provide broad, cross-strain protection [ , ] . dna vaccines can potentially solve the mismatch problem by shortening the lag time [ ] , which is particularly useful when facing influenza pandemic. in addition, the strategy of combined immunization with dna vaccines encoding surface protein (e.g., ha) and internal protein (np and m ) could offer better protection against influenza virus than single dna vaccine alone in mice and ferrets [ ] [ ] [ ] [ ] . with the success of dna vaccines in various animal models, several phase i & ii clinical trials on dna vaccine against influenza have been being carried out. results so far have been encouraging, demonstrating both safety and immunogenic response in human [ , , ] . respiratory syncytial virus (rsv) is a single stranded rna pneumovirus which belongs to paramyxoviridae family. it accounts for one of the leading pathogeneses of lower respiratory tract infections and hospitalization in infants and young children [ ] , as well as the elderly and high-risk population [ ] . immunity against rsv is dependent on the induction of antibody responses. in addition, cd + t cells responses have been shown to reduce disease severity [ ] . although maternal antibodies appear to protect infants against infection, their amount gradually decreases within the first few months of life. human rsv lacks an approved vaccine or an antiviral therapy. to prevent infant and childhood infection, vaccine should be able to induce immune responses rapidly after birth. this could be a challenging task because the immune system at the first few months of life is immature, and the persistence of maternal antibodies may limit the induction of infant antibodies responses. three rsv proteins, namely the fusion (f) protein, attachment glycoprotein (g) and matrix protein (m ), are the leading candidates for rsv vaccine development [ ] [ ] [ ] [ ] [ ] [ ] [ ] . delivery is one of the major barriers to dna vaccine. administration of naked dna is usually inefficient with only a small fraction of dna being taken up by the cells and subsequently expressed [ ] . this is because dna is a negatively charged, hydrophilic macromolecule; it is incapable of crossing the biological membrane unassisted. therefore, a safe and efficient dna delivery system is sometimes employed as adjuvant to facilitate efficient cellular uptake of dna vaccines, promote dna release inside the cells, induce high level of antigen expression and hence immune responses. physical method such as gene gun, also known as the particle-mediated epidermal delivery, has been studied to deliver dna to the skin [ , [ ] [ ] [ ] . gold beads coated with dna vaccines are discharged directly into the cytoplasm and nuclei of skin cells. this method of delivery has enjoyed some success, but is not applicable for intranasal administration. considerable efforts have been made to improve the efficacy by developing effective dna delivery systems for intranasal vaccines. formulation of dna vaccines in synthetic non-viral vectors such as polymeric nano-/micro-particles and liposomes has been reported to increase the uptake of plasmid dna by cells, increasing immunogenicity in animal models and humans. additional adjuvants may also be used to further improve the immunogenicity of these delivery systems. the elicitation of immune responses of dna vaccines mainly relies on professional apcs that present antigens to both b cells and t cells. to ensure good immune response, the dna vaccine delivery systems should be able to target apcs. in addition, m cells in nalt, which is a major site of pathogen entry, are also a target of dna vaccine. apcs are a heterogeneous group of immunocompetent cells that mediate immune response by processing and presenting antigens to the t cells. t cells recognize only antigenic peptide fragments on the surface of apcs through the t cell receptors. helper t cells recognize antigen in association with class ii mhc proteins, whereas cytotoxic t cells recognize antigen in association with class i mhc proteins. an additional co-stimulatory signal is then produced by the apcs, leading to the activation of t cells. non-professional apcs lack the co-stimulatory signaling, and therefore do not simulate t cells sufficiently. there are three types of professional apcs, namely dcs, macrophages and b cells. among them, dcs have the broadest range of antigen presentation and are considered as the most efficient cells for induction and regulation of immune responses. they play a central role in bridging the innate immune system with the adaptive immune system [ ] [ ] [ ] . to achieve efficient dna vaccination, it is logical to target the plasmid dna to dcs where the encoded antigen could be expressed endogenously. dcs express a large number of surface receptors such as c-type lectin receptors (clrs) and tlrs, which are engaged in the recognition of pathogens. it has been reported that targeting antigens to receptors on dcs can significantly enhance immune responses [ ] . these receptors could be exploited for dna vaccine targeting with the aid of antibodies or natural ligands. in particular, clrs are endocytic receptors which recognize carbohydrate structures that resemble pathogen cell-wall components. they are responsible for internalizing pathogens. one of the most commonly studied receptors for vaccine targeting is the dc c-type lectin receptor (dec- ) which is specifically expressed on dcs. however, ligands for dec- have yet to be identified. several studies have demonstrated the employment of anti-dec- antibodies to achieve dc targeting for dna vaccines, including intranasal immunization [ ] [ ] [ ] . another identified dc-specific target is c-type lectin domain family member a (clec a). activation of clec a leads to the stimulation of antibody production [ ] . alternatively, dcs could be targeted by using the natural ligands to the mannose receptors [ ] . however, the effectiveness of these targeting components in mucosal vaccines remains to be investigated. there are also several molecules being investigated to target apcs in general. one of the most widely studied molecules is the flt ligand. flt ligand is a growth factor that stimulates the proliferation of hematopoietic cells. it binds to the fms-like tyrosine kinase receptor flt . flt expression is, in hematopoietic tissue, restricted to cd + progenitors, including dc progenitors. in vivo treatment of flt ligand is found to up-regulate the number of dcs, but not their activation [ , ] . furthermore, flt ligand treatment could also enhance immune response when delivered via the mucosal route [ ] . it has been reported that when plasmid dna encoding flt ligand was co-administered with plasmids encoding protein antigens, effective immune responses were induced [ ] . in addition to its apcs targeting ability, flt ligand is an efficient and safe mucosal adjuvant that facilitated expansion of dcs following nasal administration [ , ] . as discussed in section . , improving m cells uptake is another strategy to enhance vaccine immunity. effective mucosal immunity often correlates with the uptake of antigen by mucosal inductive tissues, such as nalt in the upper respiratory tract following intranasal immunization. since m cells are responsible for antigen sampling on the mucosal surface for eventual antigen presentation to mucosal b and t cells, targeting of vaccines to m cells can be an effective method to achieve strong immune response. particle size is an important parameter for m cell uptake. a number of studies have been conducted to identify the optimal particle size for cellular uptake of the mucosal system. some studies suggest that particle size of less than μm is optimal for oral vaccine delivery for peyer's patch m cell uptake [ ] [ ] [ ] . another nalt nanoparticle uptake study also suggested that particles with sub-micron size are optimal for mucosal m cells uptake [ ] . it is generally accepted that nalt m cells can uptake nanosized particles rapidly with no definite size range being established [ ] . apart from cellular uptake, particle size also affects the kinetics of lymphatic drainage. it appears that nanoparticles less than nm are more readily transported by the draining lymph compared to larger particles [ ] . apart from controlling particle size to achieve specific targeting passively, inclusion of targeting ligand could also increase uptake by m cells [ , ] . a number of pathogens including reovirus [ ] , salmonella typhimurium [ ] and mycobacterium tuberculosis [ ] target m cells as a mode of entry into the host. by identifying the key molecules expressed by these pathogens that are crucial for their invasion, it would be extremely helpful to design an effective delivery system for m cells targeting [ ] . one example is related to reoviruses which target m cells using their surface protein sigma- (σ ). in this regard, wu et al. reported an m cell targeting dna vaccine delivery system consisting plasmid dna and the recombinant protein σ as targeting ligand which was covalently attached to poly-l-lysine (pll) for intranasal vaccination in mice. the results showed significant mucosal siga production as well as enhanced cell-mediated immunity [ ] . other ligands such as co- peptide [ ] , claudin targeting peptide [ , ] and m cell specific monoclonal antibody (nkm - - ) [ ] have been investigated for m cell targeting in mucosal protein vaccine, with the potential to be explored for mucosal dna vaccine delivery. however, mucosally induced tolerance may develop with m cell targeting delivery system. following nasal administration of protein σ genetically conjugated with ovalbumin, systemic unresponsiveness was induced instead of mucosal iga immunity [ ] . therefore, special attention must be paid with the development of m cell targeting delivery system. high versatility is one of the attractive features of polymer-based dna delivery systems. cationic polymers can form complexes (polyplexes) with nucleic acids through electrostatic interaction. polymer synthesis is relatively cheap and is easy to scale-up. particle size and surface properties of polymeric particles can be controlled by using different polymers and fabrication methods in order to optimize their cellular uptake and transfection efficiency. the polymeric particles can also be modified to include specific function groups or ligands to enhance immune responses. polyethylenimine (pei) (figure ) is one of the early generation polymers being investigated for gene delivery. it has high transfection efficiency and is frequently regarded as the gold-standard of non-viral gene delivery vectors. pei has high ph buffering capacity, which allows its cargo to escape from endosomal entrapment via a mechanism known as "proton sponge hypothesis" [ ] . transfection efficiency of pei depends on its molecular weight and the level of branching. shim et al. described the use of a simple method to prepare pei ( kda)-dna complexes for vaccine delivery [ ] . plasmid dna encoding sars-cov spike protein without transmembrane domain was employed in the study. mice that were immunized intranasally with the pei-dna vaccines produced significantly higher systemic spike protein specific igg and mucosal secretory iga in the lung compared to those immunized with naked dna. furthermore, cellular immune responses were detected with an improvement of specific t cell responses. in another study, torrieri-dramard et al. demonstrated the utilization of pei (in vivo-jetpei ® ) as dna vaccine carrier for intranasal administration [ ] . plasmid dna encoding ha from influenza a viruses was used. the intranasal administration of the pei/dna vaccines induced cellular and humoral immune response capable of providing protective immunity against a divergent virus of h n subtype in mice. the protection could be further improved by including the plasmid dna encoding na. although pei appeared as a promising vector for gene delivery including dna vaccines, the cationic pei is highly charged and non-biodegradable, and it often encounters toxicity problems which cannot be ignored. in this regard, many groups are developing low toxic or biodegradable pei derivatives for gene delivery application. mann et al. has developed a pei derivative, deacylated pei (dpei), as dna vaccine delivery agent [ ] . dpei is a nearly fully hydrolysed linear pei with % additional free protonatable nitrogen atoms, enabling more efficient binding with dna, reduced toxicity and high transfection efficiency. it is an effective dna vaccine carrier for pulmonary delivery to elicit both systemic and mucosal immune responses, and offers protection against influenza challenge in vaccinated mice. this system has a potential to be exploited for intranasal administration. (pei) (a,b) , chitosan (c) and poly(lactic-co-glycolic acid) (plga) (d). chitosan (figure ) is a natural polysaccharide that has been frequently studied for drug delivery. it is derived from chitin which is found abundant in crustacean. it is biodegradable and biocompatible with low toxicity [ , ] . the properties of chitosan can be tuned by changing its molecular weight and degree of deacetylation. because of its cationic nature, chitosan has strong binding affinity with nucleic acids, making it a suitable candidate for dna delivery agent. in addition, chitosan and its derivatives are found to display strong mucoadhesive property, making them particularly suitable to facilitate intranasal delivery [ ] [ ] [ ] . moreover, chitosan was also reported to have an immunostimulating effect, such as increasing the accumulation and activation of macrophages, promoting resistance to infections by cytokines, and enhancing cytotoxic t cell response [ , ] . a number of studies have described the use of chitosan nanoparticles to deliver dna vaccine formulation for intranasal administration. kumar et al. reported the exploitation of chitosan nanoparticles to deliver dna vaccine against acute respiratory syncytial virus (rsv) infection [ ] . a cocktail of plasmid dna encoding a number of rsv antigens was used to complex with chitosan to form nanoparticles. following the nasal vaccination in mice, high levels of serum igg and mucosal iga antibodies, as well as cytotoxic t cells responses were induced. there was also an elevated lung-specific production of ifn-γ with antiviral action. a single dose of dna vaccine was able to decrease the rsv titers by -fold on primary infection. similar study was performed by another group who described the use of chitosan nanoparticles to deliver plasmid dna encoding a t cell epitope from the m protein of rsv [ ] . it was found that intranasal administration of the formulation in mice induced specific cytotoxic t cell response that was comparable to those induced via intradermal immunization. following rsv challenge of the nasal immunized mice, the virus load in lungs was significantly reduced. in a recent study, raghwanshi et al. investigated a sophisticated dc targeted chitosan nanoparticle system for nasal dna immunization against sars-cov [ ] . the chitosan nanoparticles were surface functionalized with ligands to achieve dc selective targeted delivery. dec- receptor is c-type lectin receptor found in dcs for recognition and uptake of pathogens. the authors developed a bifunctional fusion protein (bffp) vector which consists of truncated core-streptavidin fused with anti-dec- single chain antibody. the core-streptavidin arm of the fusion protein binds with biotinylated chitosan nanoparticles while anti-dec- scfv imparts targeting specificity to dc dec receptor. plasmid dna encoding nucleocapsid protein of sars-cov was loaded into the chitosan nanoparticles. following intranasal administration of the dc targeted nanoparticles in mice, the levels of mucosal iga and systemic igg against nucleocapsid proteins were significantly enhanced, whereas no mucosal and systemic immune responses were detected when the naked plasmid dna was intranasally administered. the results showed that such dc targeting delivery system could be a promising strategy for low-dose nasal dna vaccines. to enhance the transfection efficiency of chitosan for intranasal administration, thiolated chitosan derivative has been introduced. thiolated chitosan derivative has strong mucoadhesive properties due to the formation of disulphide bonds between thiol groups of the modified polymer and cysteine-rich subdomains of glycoproteins in the mucus layer, leading to an improvement in mucoadhesion of up to -fold when compared to unmodified chitosan [ ] . improved and sustained gene expression could be achieved both in vitro and in vivo with thiolated chitosan derivative [ ] . this technology has a huge potential to be adopted for intranasal dna vaccine delivery. poly(lactic-co-glycolic acid) (plga) (figure ) is a synthetic biodegradable copolymer that has been extensively investigated for the delivery of different therapeutic agents including proteins and nucleic acids [ ] . due to its biocompatibility and excellent safety profile, plga is approved by the fda in various drug delivery systems for human use. since the degradation rate of plga can be adjusted by modifying the molecular weight of the polymer and the lactic acid to glycolic acid ratio, the rate of drug release can also be controlled accordingly. however, the negative charge and hydrophobic nature of plga limit its interaction with the negatively charged dna. cationic surface modification of plga micro/nanoparticles using polycations such as pei and chitosan can overcome this problem and allow efficient nucleic acids delivery [ ] [ ] [ ] [ ] . this strategy has also been applied to the delivery of dna vaccines. oster et al. first employed the use of micro-particles consisting plga and pei as dna vaccine carrier for injection [ ] . later on, wang et al. reported the use of chitosan coated plga nanoparticles to deliver plasmid dna encoding fmdv (foot-and-mouth disease virus) capsid protein together with il- as mucosal adjuvant for intranasal vaccination [ ] . chitosan coated plga nanoparticles were first prepared by emulsion-diffusion-evaporation technique [ ] , followed by the incorporation of plasmid dna to the nanoparticles by simple complexing. the samples were then freeze-dried with mannitol before use. after intranasal administration in guinea pigs and rats, both cellular and humoral immune responses were detected. il- was also found to be an effective mucosal adjuvant which significantly enhanced mucosal and systemic immune responses. more importantly, the vaccines could offer some immune protection to animals against fmdv challenge. liposomes are vesicles comprised of phospholipid bilayers. they have been extensively investigated for delivering dna into mammalian cells as well as vaccine adjuvants. the duo functions make them an excellent carrier system for dna vaccine [ ] [ ] [ ] [ ] . for dna delivery, the negatively charged plasmid dna can be absorbed to the surface of cationic liposomes through electrostatic interaction to form complexes. alternatively, dna can be encapsulated in the aqueous core of the cationic, non-ionic or anionic liposomes. in general, transfection efficiency of cationic liposomes is superior to their non-ionic or anionic counterparts [ ] , whereas anionic liposomes provided enhanced antibody responses [ ] . surfaces of the liposomes can be decorated with targeting ligands or antigenic components to improve the immune response in vaccine formulation [ ] . currently, there are at least two approved liposomal vaccine formulations on the market for antigen delivery, including inflexal ® v (influenza vaccine) and epaxal ® (hepatitis a vaccine). both formulations employed the virosomes technology (figure ) , in which the viral proteins are bound to the surface of a liposome in an array, similar to what is seen on viral particles [ ] . the idea is to create a safe viral-like particle that can induce strong protective immune response. similar technology could be applied to dna vaccines to improve immunogenicity. in fact, liposomes on their own could elicit immune response even in the absence of other antigens. it has been demonstrated by lay et al. that dotim (octadecenoyloxy-ethyl-heptadecenyl- -hydroxyethyl imidazolinium chloride)/cholesterol cationic liposome-dna complexes (jvrs- ), in which an empty plasmid dna vector was incorporated, were able to induce high levels of antibody and t cell immunity in mice and non-human primates [ ] . since lipid composition, particle size, surface charge and dna entrapment efficiency of the liposomes can affect their immunogenicity and potency, these parameters must be carefully characterized and controlled. the major limitation of liposome as dna vaccine carrier is long-term stability, as lyophilization of liposomes may not be always possible [ ] . [ ] . plasmid dna encoding ag a was complexed with the lipids. following intranasal immunization in mice, naked dna was completely ineffective, probably due to the degradation of the dna by mucosal nuclease. the lipid-dna formulation was capable of inducing a weak cell-mediated immune response, and no humoral immune response was detected. the co-lipid intranasal formulation was compared with another cationic lipid formulation, vaxfectin, which was used for intramuscular administration. it was found that the intramuscular formulation was able to induce a better immune response. however, combining intranasal and intramuscular administrations resulted in stronger immune responses in the lungs. considerable improvement is needed to further develop the formulation for intranasal use. rosada et al. developed another liposome-based formulation of dna vaccines against tb [ ] . the non-toxic, cationic liposome, epc/dope/dotap (egg phosphatidylcholine/ , -dioleoyl-snglycero- -phosphoethanolamine/ , -dioleoyl- -trimethylammonium-propane) was used. plasmid dna encoding the kda mycobacterial hsp was either entrapped inside or complexed with the cationic liposomes, and the intramuscular and intranasal routes of administration were compared in mice. when administered intramuscularly, both liposomal formulations were ineffective in preventing tuberculosis infection in mice even after two doses. on the contrary, the complexed liposomal formulation of dna vaccine was able to offer protection against infection even after a single dose, with a significant reduction of colony forming unit (cfu) in lungs after the immunized mice were challenged with mycobacterium tuberculosis. however, four doses of intranasal administration of naked dna vaccines failed to offer any protection. the authors reasoned that the intranasal vaccination enhance the immune response by stimulating the mucosal immunity, but the naked dna failed to cross the mucosal barriers in the nasal cavity, demonstrating the importance with the use of delivery carrier for intranasal dna vaccination. apart from tb vaccine, there are studies that reported the use of liposome to deliver influenza dna vaccine [ , ] . cationic liposomes dodac/dope/peg ( , -dioleoyl-sn-glycero- - phosphoethanolmine/dioleylphosphatidylethanolamine/polyethylene glycol) were used to encapsulate plasmid dna encoding influenza virus ha. after intranasal immunization in mice, the liposome system was effective at eliciting both igg and iga humoral responses systemically. local iga level was enhanced. cell-mediated immune response was also successfully induced. in addition, the immunized mice were able to withstand a lethal challenge of influenza virus. on the other hand, intramuscular immunization of the same system enhanced igg level only with no effect on iga level either locally or systemically. intranasal administration of naked dna failed to induce antibody response. the promising results demonstrated the potential of the intranasal liposomal dna vaccine system. to improve the dna vaccine delivery efficiency of liposomes for intranasal administration, khatri et al. modified the surface of liposomes by coating with glycol chitosan [ ] . the major function of glycol chitosan is to provide mucoadhesive and immune stimulating property [ ] . in the study, cationic liposomes, pc/dope/chol (phosphatidylcholine/dioleylphosphatidylethanolamine/cholesterol) were used to entrap plasmid encoding the s region of hepatitis b antigen, and the glycol chitosan was adsorbed on the liposome surfaces through electrostatic interaction and hydrogen bonding. following intranasal administration in mice, the surface modified liposomes could elicit both humoral mucosal and cell-mediated immune responses which were better than the uncoated liposomes. such system has the potential to be exploited for intranasal dna vaccine of respiratory infectious diseases. a number of studies have already demonstrated the potential of liposomal dna vaccine system for intranasal administration, and some could offer considerable immune protection against respiratory infections in animals. however, the lipid composition of different liposomal systems varied greatly, and currently there is a lack of knowledge of how the composition may affect the immune response. to enable the utilization of liposomal dna vaccine for clinical application and approval, a better understanding of how these factors govern the efficacy and immunity of the liposomal delivery system must first be sought. to further enhance the immune responses of the dna vaccines, adjuvants are included in the formulation in many studies. adjuvants are generally defined as agents that could enhance the immune response of the vaccinated subjects to an antigen. in dna vaccine, since the delivery of dna is a major hurdle, dna carrier system using bacterial, viral or non-viral vectors, as well as the cell specific targeting ligands, which are discussed above, are also considered as dna vaccine adjuvants. the summary of dna vaccine adjuvants being investigated are shown in table . in this section, proteins and other macromolecules with immunopotentiating properties but are not directly involved in the delivery of dna are discussed, especially those that are commonly employed for intranasal vaccination. enterotoxins are protein exotoxin released by pathogens that infect the gut. enterotoxin-based mucosal adjuvants are the most potent and well-established strategy for the induction of both mucosal and systemic immunity to co-administered protein antigens [ ] . heat-labile enterotoxin (lt) from e. coli and cholera enterotoxin (ct) are very potent adjuvants but they are too toxic to be used in human. therefore, detoxified mutants of enterotoxins have been produced by site-directed mutagenesis and they are extensively investigated as adjuvants for mucosal vaccine including intranasal vaccine. intranasal antigen immunization with lt mutant adjuvants can provide effective protection against infectious diseases in animals [ ] [ ] [ ] [ ] [ ] . it is suggested that the lt mutant adjuvants could induce potent cytotoxic t lymphocyte responses. the mechanism of action is believed to arise from enhanced permeation of antigens across epithelial barriers and a marked increase in antigen presentation by apcs [ ] . mutants of ct have also showed strong adjuvant activity [ , , ] , and could retain good adjuvant function when administered intranasally [ ] . it is expected that lt mutants and ct mutants have similar mechanisms of adjuvant activities [ ] . the major concern with the intranasal administration of mutant lt or ct is that these toxin derivatives may gain access into the central nervous system through the olfactory nerve. it has been reported that both native and mutant lt used as adjuvants were associated with the development of bell's palsy following intranasal delivery in humans [ , , ] . the risk of enterotoxin as mucosal adjuvant for intranasal administration is already discussed in section . . lipopolysaccharides (lps) are the major component of the outer membrane of gram-negative bacteria, and could elicit strong immune response. however, they are also highly toxic. in order to make them safe and suitable as vaccine adjuvants, lps derivatives are produced to reduce the endotoxic effect but to retain their immunostimulatory function. monophosphoryl lipid a (mpl) is one example of lps derivative that is investigated as vaccine adjuvant. mpl is prepared by removal of a phosphate and fatty acid group from the lipid a of salmonella minnesola. mpl is thought to interact with toll-like receptor (tlr- ) on apcs. it has been demonstrated that mpl could activate macrophages, increase their cytokine secretion and hence immune response [ ] [ ] [ ] [ ] [ ] . regarding safety, mpl appears to retain the immunogenic activity of lps but with significantly reduced toxicity [ ] . mpl has been extensively evaluated in the clinic as adjuvants for various diseases including infectious diseases with an acceptable profile of adverse effects [ ] . mpl has been used successfully as a mucosal adjuvant when formulated with liposomes or virosomes for intranasal administration in animals [ ] [ ] [ ] . cytokines are small proteins that are important in regulating immunological response by recruiting and stimulating t cells, or by directly acting on infected cells. they have potential to be natural adjuvants for dna vaccines [ ] . cytokines that have been evaluated as possible dna or intranasal vaccine adjuvants include il- , il- , il- , il- , il- and gm-csf [ , , , ] . in particular, il- is found to be an effective mucosal adjuvant [ , ] . lynch et al. demonstrated that intranasal administration of pneumococcal polysaccharide conjugate vaccine in the presence of il- was able to enhance systemic and mucosal immune responses to pneumococci in mice [ ] . however, cytokines have short half-life in vivo and poor stability. they are also expensive and are associated with dose related toxicity. therefore, these molecules may not be suitable for use as adjuvants in vaccines designed to protect against infectious diseases [ ] . nevertheless, intranasal administration of il- induced less toxicity than parenteral administration [ ] . alternatively, cytokines can be expressed from plasmid dna to allow long-lasting expression in vivo and to reduce the cost of production [ , ] . with the emergence of new or antimicrobial-resistant bacteria and viruses, and the ease of transmission, especially the respiratory pathogens, respiratory infections are becoming serious threats to human health. safe and effective vaccines are important to safeguard public health. intranasal dna vaccination appears to be a promising non-invasive approach to provide protection against various infectious diseases. evidence shows that intranasal dna vaccine could elicit strong and long-lasting humoral as well as cell-mediated immune responses in many animal models. dna vaccines are already successfully used in veterinary products for protection against infections, but their immunogenicity needs to be further enhanced to make them suitable for human use. improving dna delivery and formulation is one of the several strategies to enhance the immune response. various studies have demonstrated that significant improvement of immune response that could be achieved by the employment of dna carrier system, or to target the dna vaccines to apcs. dna vaccines generally have good safety profile, but the potential toxicity associated with dna delivery systems, especially when they are used at high concentration, must not be neglected. dna vaccines may circumvent many problems associated with conventional vaccines such as high costs of protein vaccine purification and bacterial/viral inactivated or attenuated process, the incorrect folding of antigen and viral mutation risk, thereby offering a safer alternative to benefit humans. in addition, mass manufacture of dna vaccine is easier and faster, and dna product is usually highly stable. once an effective intranasal dna vaccine delivery system is identified and optimized, a delivery technology platform could be established to allow the development of dna vaccine formulations for different infectious diseases in the future. dna vaccines: immunology, application, and optimization pulmonary dna vaccination: concepts, possibilities and perspectives dna vaccines: progress and challenges synthetic dna vaccine strategies against persistent viral infections dna vaccines: ready for prime time? dna vaccines in veterinary use immunologic responses to west nile virus in vaccinated and clinically affected horses efficacy of an infectious hematopoietic necrosis (ihn) virus dna vaccine in chinook oncorhynchus tshawytscha and sockeye o. nerka salmon dna and rna-based vaccines: principles, progress and prospects dna vaccines-a modern gimmick or a boon to vaccinology? safety and immunogenicity of an hiv- gag dna vaccine with or without il- and/or il- plasmid cytokine adjuvant in healthy, hiv- uninfected adults enhanced t-cell immunogenicity of plasmid dna vaccines boosted by recombinant modified vaccinia virus ankara in humans a human immunodeficiency virus (hiv- ) clade a vaccine in clinical trials: stimulation of hiv-specific t-cell responses by dna and recombinant modified vaccinia virus ankara (mva) vaccines in humans safety and immunogenicity of recombinant low-dosage hiv- a vaccine candidates vectored by plasmid pthr dna or modified vaccinia virus ankara (mva) in humans in east africa ev : a phase i trial to compare the safety and immunogenicity of hiv dna-c prime-nyvac-c boost to nyvac-c alone the future of human dna vaccines immune responses to dna vaccines: induction of cd t cells quantitative analysis of the immunopotency of genetically transfected dendritic cells differential dependence on target site tissue for gene gun and intramuscular dna immunizations cpg-oligonucleotides in vaccination: signaling and mechanisms of action cpg dna as a vaccine adjuvant tlr −/− and tlr +/+ mice display similar immune responses to a dna vaccine riffault, s. tlr pathway is involved in adjuvant effects of plasmid dna-based vaccines tank-binding kinase- delineates innate and adaptive immune responses to dna vaccines sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity blockade of chronic type i interferon signaling to control persistent lcmv infection class ii-independent generation of cd memory t cells from effectors feasibility of reconstructed ancestral h n influenza viruses for cross-clade protective vaccine development a single electroporation delivery of a dna vaccine containing the hemagglutinin gene of asian h n avian influenza virus generated a protective antibody response in chickens against a north american virus strain the efficacy of live attenuated, cold-adapted, trivalent, intranasal influenzavirus vaccine in children the role of live influenza vaccines in children dna vaccination protects against an influenza challenge in a double-blind randomised placebo-controlled phase b clinical trial potential dna vaccine integration into host cell genome biodistribution of dna plasmid vaccines against hiv- , ebola, severe acute respiratory syndrome, or west nile virus is similar, without integration, despite differing plasmid backbones or gene inserts plasmid dna vaccines: assay for integration into host genomic dna detection of integration of plasmid dna into host genomic dna following intramuscular injection and electroporation safety and immunogenicity of a gag-pol candidate hiv- dna vaccine administered by a needle-free device in hiv- -seronegative subjects first human trial of a dna-based vaccine for treatment of human immunodeficiency virus type infection: safety and host response safety, tolerability and humoral immune responses after intramuscular administration of a malaria dna vaccine to healthy adult volunteers immune responses to nucleic acid vaccines to rabies virus a novel antibiotic free plasmid selection system: advances in safe and efficient dna therapy the ontogeny of antigen-specific t cells successful nucleic acid based immunization of newborn chimpanzees against hepatitis b virus enhanced protection against influenza virus of mice immunized as newborns with a mixture of plasmids expressing hemagglutinin and nucleoprotein macrophagic myofasciitis: characterization and pathophysiology inflammation-related effects of adjuvant influenza a vaccination on platelet activation and cardiac autonomic function inflammatory responses to trivalent influenza virus vaccine among pregnant women residual adverse changes in arterial endothelial function and ldl oxidation after a mild systemic inflammation induced by influenza vaccination differential serum cytokine responses to inactivated and live attenuated seasonal influenza vaccines increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of dna vaccines phase safety and immunogenicity testing of dna and recombinant modified vaccinia ankara vaccines expressing hiv- virus-like particles phase clinical trials of the safety and immunogenicity of adjuvanted plasmid dna vaccines encoding influenza a virus h hemagglutinin safety and immunogenicity of tyrosinase dna vaccines in patients with melanoma dna priming and influenza vaccine immunogenicity: two phase open label randomised clinical trials antigen sampling across epithelial barriers and induction of mucosal immune responses mucosal vaccines: the promise and the challenge function of mucosa-associated lymphoid tissue in antibody formation recent progress in mucosal vaccine development: potential and limitations live attenuated influenza vaccine a review of its use in the prevention of seasonal influenza in children and adults a pandemic influenza vaccine in india: from strain to sale within months intranasal immunization of mice with a mixture of the pneumococcal proteins psaa and pspa is highly protective against nasopharyngeal carriage of streptococcus pneumoniae a protective and safe intranasal rsv vaccine based on a recombinant prefusion-like form of the f protein bound to bacterium-like particles nanoparticles for nasal vaccination bronchus-and nasal-associated lymphoid tissues m cell-targeted dna vaccination mucosal vaccine design and delivery interaction of antigens and antibodies at mucosal surfaces human immune responses to influenza virus vaccines administered by systemic or mucosal routes human respiratory mucus nasal vaccination, escherichia coli enterotoxin, and bell's palsy transient facial nerve paralysis (bell's palsy) following intranasal delivery of a genetically detoxified mutant of escherichia coli heat labile toxin the cholera toxin-derived cta -dd vaccine adjuvant administered intranasally does not cause inflammation or accumulate in the nervous tissues a second generation of double mutant cholera toxin adjuvants: enhanced immunity without intracellular trafficking current status of tb vaccines t cell mediated immunity to mycobacterium tuberculosis protective immunity against mycobacterium tuberculosis induced by dendritic cells pulsed with both cd + -and cd + -t-cell epitopes from antigen a tuberculosis vaccine development: the development of a novel (preclinical) dna vaccine. hum. vaccines immunogenicity and protective efficacy of a tuberculosis dna vaccine vaccination against tuberculosis by dna injection immunogenicity and efficacy of a tuberculosis dna vaccine encoding the components of the secreted antigen complex protection of mice with a divalent tuberculosis dna vaccine encoding antigens ag b and mpt a polyvalent dna vaccine expressing an esat -ag b fusion protein protects mice against a primary infection with mycobacterium tuberculosis and boosts bcg-induced protective immunity therapeutic effects of ag b and mpt dna vaccines in a murine model of mycobacterium tuberculosis infection plasmid interleukin- (il- ), but not plasmid il- , enhances the protective efficacy of a dna vaccine against mycobacterium tuberculosis infection immunogenicity and protective efficacy of a tuberculosis dna vaccine expressing a fusion protein of ag b-esat -hspx in mice a decade after sars: strategies for controlling emerging coronaviruses a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge sars coronavirus spike polypeptide dna vaccine priming with recombinant spike polypeptide from escherichia coli as booster induces high titer of neutralizing antibody against sars coronavirus a dna vaccine induces sars coronavirus neutralization and protective immunity in mice sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens immune responses against sars-coronavirus nucleocapsid protein induced by dna vaccine antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans update on avian influenza a (h n ) virus infection in humans dna vaccine expressing conserved influenza virus proteins protective against h n challenge infection in mice protection against multiple influenza a subtypes by vaccination with highly conserved nucleoprotein clinical applications of dna vaccines: current progress enhanced protection against a lethal influenza virus challenge by immunization with both hemagglutinin-and neuraminidase-expressing dnas preclinical efficacy of a prototype dna vaccine: enhanced protection against antigenic drift in influenza virus further protection against antigenic drift of influenza virus in a ferret model by dna vaccination comparing the ability of a series of viral protein-expressing plasmid dnas to protect against h n influenza virus epidermal dna vaccine for influenza is immunogenic in humans the two open reading frames of the k mrna of human respiratory syncytial virus: sequence comparison of antigenic subgroups a and b and expression in vitro respiratory syncytial virus infection in elderly and high-risk adults combining dna and protein vaccines for early life immunization against respiratory syncytial virus in mice the fusion glycoproteins of human respiratory syncytial virus of subgroups a and b: sequence conservation provides a structural basis for antigenic relatedness protection against respiratory syncytial virus infection by dna immunization immunogenicity and efficacy of codon optimized dna vaccines encoding the f-protein of respiratory syncytial virus plasmid dna encoding the respiratory syncytial virus g protein is a promising vaccine candidate resistance to respiratory syncytial virus (rsv) challenge induced by infection with a vaccinia virus recombinant expressing the rsv m protein (vac-m ) is mediated by cd + t cells, while that induced by vac-f or vac-g recombinants is mediated by antibodies enhanced delivery and expression of a nanoencapsulated dna vaccine vector for respiratory syncytial virus nano-encapsulated dna and/or protein boost immunizations increase efficiency of dna vaccine protection against rsv distribution of dna vaccines determines their immunogenicity after intramuscular injection in mice dna vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic t lymphocyte responses following epidermal delivery of nanogram quantities of dna advantage of gene gun-mediated over intramuscular inoculation of plasmid dna vaccine in reproducible induction of specific immune responses immunobiology of dendritic cells dendritic cells: specialized and regulated antigen processing machines dendritic cells in vivo: a key target for a new vaccine science targeting vaccines to dendritic cells a versatile bifunctional dendritic cell targeting vaccine vector dendritic cell targeted hiv gag protein vaccine provides help to a dna vaccine including mobilization of protective cd + t cells dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein the dendritic cell subtype-restricted c-type lectin clec a is a target for vaccine enhancement improving vaccines by targeting antigens to dendritic cells hematologic effects of flt ligand in vivo in mice dramatic increase in the numbers of functionally mature dendritic cells in flt ligand-treated mice: multiple dendritic cell subpopulations identified modulating dendritic cells to optimize mucosal immunization protocols enhancement of dna vaccine potency by linkage of antigen gene to a gene encoding the extracellular domain of fms-like tyrosine kinase -ligand nasal flt ligand cdna elicits cd c + cd + dendritic cells for enhanced mucosal immunity the nasal dendritic cell-targeting flt ligand as a safe adjuvant elicits effective protection against fatal pneumococcal pneumonia uptake and transport of copolymer biodegradable microspheres by rabbit peyer's patch m cells poly (lactide-co-glycolide) particles of different physicochemical properties and their uptake by peyer's patches in mice m-cell targeted biodegradable plga nanoparticles for oral immunization against hepatitis b claudin -targeted protein incorporated into plga nanoparticles can mediate m cell targeted delivery pharmaceutical aspects of intranasal delivery of vaccines using particulate systems exploiting lymphatic transport and complement activation in nanoparticle vaccines enhanced mucosal and systemic immune response with intranasal immunization of mice with hiv peptides entrapped in plg microparticles in combination with ulex europaeus-i lectin as m cell target intestinal m cells: a pathway for entry of reovirus into the host salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial m cells of the peyer's patches the m cell as a portal of entry to the lung for the bacterial pathogen mycobacterium tuberculosis novel vaccine development strategies for inducing mucosal immunity the m cell-targeting ligand promotes antigen delivery and induces antigen-specific immune responses in mucosal vaccination m cell targeting by a claudin -targeting peptide can enhance mucosal iga responses a novel m cell-specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses low-dose tolerance is mediated by the microfold cell ligand, reovirus protein sigma a versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine intranasal immunization with plasmid dna encoding spike protein of sars-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses intranasal dna vaccination induces potent mucosal and systemic immune responses and cross-protective immunity against influenza viruses pulmonary delivery of dna vaccine constructs using deacylated pei elicits immune responses and protects against viral challenge infection application of chitosan microspheres for nasal delivery of vaccines chitosan-based formulations for delivery of dna and sirna nasal vaccination: a non-invasive vaccine delivery method that holds great promise for the future chitosan and the mucosal delivery of biotechnology drugs biomedical applications of amino acid-modified chitosans: a review immune stimulating activity of two new chitosan containing adjuvant formulations intranasal gene transfer by chitosan-dna nanospheres protects balb/c mice against acute respiratory syncytial virus infection nasal delivery of chitosan-dna plasmid expressing epitopes of respiratory syncytial virus (rsv) induces protective ctl responses in balb/c mice thiolated chitosans thiolated chitosan/dna nanocomplexes exhibit enhanced and sustained gene delivery biodegradable nanoparticles for drug and gene delivery to cells and tissue plga-pei nanoparticles for gene delivery to pulmonary epithelium chitosan-coated plga nanoparticles for dna/rna delivery: effect of the formulation parameters on complexation and transfection of antisense oligonucleotides characterization of plasmid dna location within chitosan/plga/pdna nanoparticle complexes designed for gene delivery establishing chitosan coated plga nanosphere platform loaded with wide variety of nucleic acid by complexation with cationic compound for gene delivery cationic microparticles consisting of poly(lactide-co-glycolide) and polyethylenimine as carriers systems for parental dna vaccination intranasal delivery of cationic plga nano/microparticles-loaded fmdv dna vaccine encoding il- elicited protective immunity against fmdv challenge preparation and characterization of cationic plga nanospheres as dna carriers co-delivery of il- or liposomes augment the responses of mice to a dna vaccine for pseudorabies virus ie modulation of cellular immune response against hepatitis c virus nonstructural protein by cationic liposome encapsulated dna immunization augmentation of antigen-specific immune responses using dna-fusogenic liposome vaccine vaccine adjuvant formulations: a pharmaceutical perspective delivery of plasmid dna into mammalian cell lines using ph-sensitive liposomes: comparison with cationic liposomes surface modified liposomes for nasal delivery of dna vaccine cationic lipid/dna complexes (jvrs- ) combined with influenza vaccine (fluzone) increases antibody response, cellular immunity, and antigenically drifted protection improved tuberculosis dna vaccines by formulation in cationic lipids protection against tuberculosis by a single intranasal administration of dna-hsp vaccine complexed with cationic liposomes dna vaccination against respiratory influenza virus infection intranasal immunization with liposome-encapsulated plasmid dna encoding influenza virus hemagglutinin elicits mucosal, cellular and humoral immune responses glycol chitosan improves the efficacy of intranasally administrated replication defective human adenovirus type expressing glycoprotein d of bovine herpesvirus dendritic cell-targeting dna-based mucosal adjuvants for the development of mucosal vaccines improving vaccines by incorporating immunological coadjuvants mucosal adjuvants and delivery systems for protein-, dna-and rna-based vaccines mutants of escherichia coli heat-labile toxin act as effective mucosal adjuvants for nasal delivery of an acellular pertussis vaccine: differential effects of the nontoxic ab complex and enzyme activity on th and th cells intranasal immunization with pneumococcal polysaccharide conjugate vaccines with nontoxic mutants of escherichia coli heat-labile enterotoxins as adjuvants protects mice against invasive pneumococcal infections genetically detoxified mutants of heat-labile enterotoxin from escherichia coli are effective adjuvants for induction of cytotoxic t-cell responses against hiv- gag-p a dilemma for mucosal vaccination: efficacy versus toxicity using enterotoxin-based adjuvants mucosal vaccines: non toxic derivatives of lt and ct as mucosal adjuvants use of the inactivated intranasal influenza vaccine and the risk of bell's palsy in switzerland heat-labile enterotoxins as adjuvants or anti-inflammatory agents structure and function of cholera toxin and the related escherichia coli heat-labile enterotoxin the vaccine adjuvant monophosphoryl lipid a as a trif-biased agonist of tlr . science signal t-cell growth factor modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with dna immunogens il- expression plasmid enhances cell-mediated immunity induced by an hiv- dna vaccine control of viremia and prevention of clinical aids in rhesus monkeys by cytokine-augmented dna vaccination the role of cytokine dnas as vaccine adjuvants for optimizing cellular immune responses subsets of memory cytotoxic t lymphocytes elicited by vaccination influence the efficiency of secondary expansion in vivo co-immunization of plasmid dna encoding il- and il- with bacillus calmette-guerin vaccine against progressive tuberculosis cd positive t cells influence antigen-specific immune responses through the expression of chemokines dna vaccines encoding interleukin- and rantes enhance antigen-specific th -type cd + t-cell-mediated protective immunity against herpes simplex virus type in vivo plasmid chemokines and colony-stimulating factors enhance the immunogenicity of dna priming-viral vector boosting human immunodeficiency virus type vaccines recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid dna vaccines granulocyte-macrophage colony stimulating factor: an adjuvant for cancer vaccines recruitment of different subsets of antigen-presenting cells selectively modulates dna vaccine-elicited cd + and cd + t lymphocyte responses a phase i trial of dna vaccination with a plasmid expressing prostate-specific antigen in patients with hormone-refractory prostate cancer multigene/multisubtype hiv- vaccine induces potent cellular and humoral immune responses by needle-free intradermal delivery flt : receptor and ligand a novel adenovirus expressing flt ligand enhances mucosal immunity by inducing mature nasopharyngeal-associated lymphoreticular tissue dendritic cell migration enhancement of plasmid dna immunogenicity with linear polyethylenimine plga microspheres for improved antigen delivery to dendritic cells as cellular vaccines chitosan as a nasal delivery system: the effect of chitosan solutions on in vitro and in vivo mucociliary transport rates in human turbinates and volunteers cholesterol-fus in non-small-cell lung cancer transcutaneous immunization by lipoplex-patch based dna vaccines is effective vaccination against japanese encephalitis virus infection protective effect of dna-mediated immunization with liposome-encapsulated gra against infection of toxoplasma gondii cationic liposomal vaccine adjuvants in animal challenge models: overview and current clinical status bacterial cell wall products as adjuvants: early interferon γ as a marker for adjuvants that enhance protective immunity monophosphoryl lipid a as an adjuvant. past experiences and new directions structure-function relationships of bacterial endotoxins. contribution to microbial sepsis mechanism of action of clinically approved adjuvants modulation of adaptive immunity with toll-like receptors enhancement of antigen-specific immunity via the tlr ligands mpl adjuvant and ribi. adjuvant activity of monophosphoryl lipid a for nasal and oral immunization with soluble or liposome-associated antigen intranasal immunization with multivalent group a streptococcal vaccines protects mice against intranasal challenge infections efficacy and safety of an intranasal virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid a in mice and cotton rats chemical adjuvants for plasmid dna vaccines cytokines as immunological adjuvants cytokines: the future of intranasal vaccine adjuvants increased protection against pneumococcal disease by mucosal administration of conjugate vaccine plus interleukin- cytokine requirements for induction of systemic and mucosal ctl after nasal immunization delivery of il- intranasally leads to reduced il- -mediated toxicity the authors would like to thank aoe on control of pandemic and inter-pandemic influenza & centre of influenza research (aoe/m- / ) and health and medical research fund (hmrf ) for financial support. y.x. is supported by hong kong phd fellowship scheme, research general council (pf - ). the authors declare no conflict of interest. key: cord- - qlr authors: yu, wenzhou; lee, lisa a.; liu, yanmin; scherpbier, robert w.; wen, ning; zhang, guomin; zhu, xu; ning, guijun; wang, fuzhen; li, yixing; hao, lixin; zhang, xuan; wang, huaqing title: vaccine-preventable disease control in the people’s republic of china: – date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: qlr background: china's immunization program is one of the oldest and largest in the world. rates of vaccine-preventable diseases (vpd) are comparable to those in high-income countries. the program's evolution has been characterized by ambitious target setting and innovative strategies that have not been widely described. methods: we reviewed national and provincial health department archives; analyzed disease surveillance, vaccination coverage, and serosurvey data from through ; and, conducted in-depth interviews with senior chinese experts involved early vpd control efforts. results: widespread immunization began in the s with smallpox, diphtheria, and bacillus-calmette guerin vaccines, and in the s with pertussis, tetanus, polio, measles, and japanese encephalitis (je) vaccines. the largest drops in absolute vpd burden occurred in the s with establishment of the rural cooperative medical system and a cadre of trained peasant health workers whose responsibilities included vaccinations. from to , incidence per , population dropped % from . to . for diphtheria, % from . to . for pertussis, % from . to . for polio, % from . to . for measles, and % from . to . for je, averting an average of million vpd cases each year. until the early s, vaccines were delivered through annual winter campaigns using a coordinated ‘rush-relay’ system to expedite transport while leveraging vaccine thermostability. establishment of the cold chain system during in the s allowed bi-monthly vaccination rounds and more timely vaccination resulting in rates of diphtheria, pertussis, measles and meningitis falling over % from to , while polio and je rates fell – %. in the s, progress stalled as financing for public health was weakened by broad market reforms. large investments in public health and immunizations by the central government since has led to further declines in vpd burden and increased equity. during – , the incidence per , population was < . for measles and < . for pertussis, je, meningococcal meningitis, and hepatitis a. from to , the prevalence of chronic hepatitis b infection in children < years fell from . % to . %, a % decline. china was certified polio-free in and diphtheria was last reported in . conclusions: long-term political commitment to immunizations as a basic right, ambitious targets, use of disease incidence as the primary metric to assess program performance, and nationwide scale-up of successful locally developed strategies that optimized use of available limited resources have been critical to china's success in controlling vaccine-preventable diseases. china has one of the largest and oldest immunization programs in the world with over million infants vaccinated each year [ ] . incidence rates of vaccine-preventable diseases (vpd) are similar to those in high-income countries and vaccination coverage is uniformly high. these achievements are the result of enormous effort and evolving responses to new challenges and opportunities over the past years. when the people's republic of china was founded in , the new government faced immense health problems. most of the million population lived in rural areas and in extreme poverty with little access to health care. infant mortality exceeded per births and average life expectancy was only years [ ] . lack of united nations' recognition limited international support and scientific exchange. despite these challenges, the government set ambitious health goals and positioned immunizations as a central to their achievement. prior to liberation, vaccines were prohibitively expensive in china and largely inaccessible except to the very wealthy. in , constitutional principles of the communist party in the shaanxi-ningxia-gansu border region affirmed ''people's rights to freedom from ill health", and in line with this progressive policy, free mass vaccination campaigns against smallpox and cholera were implemented in liberated areas [ ] . this was the first time that large numbers of the rural poor in china benefitted from vaccination. since then, china's public health system has undergone numerous reforms with continued strengthening of vpd control efforts through sustained high-level political commitment to immunizations, strong supportive legal frameworks, increased public finance, dedicated efforts of many scientists and public health professionals and grass-roots health workers, and national scale-up of successful innovative delivery approaches that optimized use of available material and human resources. this article describes china's work on vpd control during to , which has not been widely described. annual vpd incidence from to was obtained from the national notifiable disease reporting system (nndrs). the nndrs is a compulsory reporting system established in for diseases with high epidemic potential including cholera, plague, smallpox, japanese encephalitis (je), meningococcal meningitis, poliomyelitis, diphtheria, pertussis, and measles. data on each case is limited to critical information including name, address, date of birth, date of disease onset, age, sex, and occupation. the nndrs has since been expanded to diseases, including hepatitis a and hepatitis b, tuberculosis, neonatal tetanus, mumps and rubella but the type of data collected has remained largely unchanged. hepatitis b burden, using hepatitis b surface antigen (hbsag) seropositivity as a marker for chronic infection, and hepatitis b vaccination coverage were estimated through national serosurveys conducted in , , and [ ] [ ] [ ] . all three serosurveys relied on random sampling of persons in the disease surveillance point (dsp) surveillance system, a nationally representative sample of rural townships and urban neighborhoods representing approximately % of the total population. vaccination coverage surveys were conducted in selected provinces during - while nationally representative surveys were conducted in , , , , and . all surveys utilized multi-stage probability of selection proportional to population size sampling based on world health organization (who) guidelines [ ] . as a proxy for coverage prior to the s, we searched for references to total numbers of persons vaccinated and total numbers of doses of vaccine produced or administered. a search was conducted for published and unpublished reports on vaccine development and vpd control in china from to , focusing on chinese-language documents not widely available in the published literature, and included manual searches through archived documents at the ministry of health (moh), china center for disease control and prevention, and provincial health departments [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . data on immunization expenditures was collected as part of national epi reviews conducted in , , and . because few reports on vaccination efforts were found prior to the s, we interviewed senior chinese experts who were closely involved in spearheading china's early immunization efforts using a structured open-ended questionnaire. persons interviewed included scientists involved in vaccine research and development; moh officials responsible for development of national policies and goals; and, provincial health staff responsible for planning and implementing vpd control activities. work on immunizations began in earnest as soon as the people's republic of china was founded and more than two decades before establishment of the world health organization's (who) expanded programme on immunizations (epi). in , only four small vaccine manufacturers existed in china (beijing, shanghai, lanzhou, changchun) and developing a secure vaccine supply was a high priority. manufacturing facilities were built in wuhan ( ) and chengdu ( ) to create a network of six regional manufacturers under moh supervision responsible for producing vaccines for the entire country. in , a plan was developed to eradicate smallpox nationwide through free mass compulsory vaccination of the entire population. by the end of , moh announced that over million people (about % of total population) had been vaccinated and smallpox rapidly disappeared from most of the country with the last case occurring in [ fig. ]. building on this success, the state council issued a directive in requiring establishment of a network of epidemic prevention stations (eps) at province, prefecture and county levels, with vpd control a main responsibility. directives on vaccination of children with diphtheria toxoid and bacillus calmette guérin (bcg) were issued in and , respectively, and research was accelerated to develop vaccines against diseases responsible for high mortality including polio, measles, and je. oral polio vaccine (opv) were developed in and was the first attenuated vaccine developed in china. the institute of medical biology, academy of medical sciences was established in kunming, to scale-up opv production. in , moh issued ''measures on implementing preventive vaccination" requiring that all provinces conduct annual winter campaigns to vaccinate children with smallpox, bcg, diphtheria, pertussis, and polio vaccines. in the early of s, combined diphtheria-tetanus-pertussis toxoid (dtp) was introduced. three attenuated measles vaccine strains, beijing- , shanghai- , and changchun- , were developed and introduced in . in contrast to the nationally synchronized smallpox campaigns, campaigns with other antigens were organized at the provincelevel and conducted during colder months since most vaccines were in liquid formulation with limited thermostability and there was no cold chain system. annual meetings were held by moh with provincial health departments and vaccine manufacturers to prioritize limited supplies. to expedite delivery before the vaccines lost potency, a highly coordinated ''rush-relay" transport approach was developed; when vaccines were shipped by the manufacturer, usually by train or truck, the provincial eps was notified by telephone or telegraph when the vaccines would arrive and to mobilize of all forms of transport (motor vehicles, bicycles, pack animals, health staff) to move the vaccines as rapidly as possible to the periphery. local cold storage solutions included use of refrigerators at food processing plants, wells, cellars, burial underground, and wooden boxes built with separate compartments for bottles of frozen water or pieces of ice. usually vaccines were transported from one administrative level to the next within a day and campaigns were completed in a single day. limited vaccine supplies precluded province-wide campaigns, so each province was divided into sections targeted on a rotating basis over a five to six-year period, with each campaign targeting all children under years old. as a result, by school entry most children had received only one or two doses each of diphtheria, pertussis and polio vaccines. while killed vaccines and toxoids could remain potent for up to days, opv and measles were live attenuated vaccines and lost potency within one week, limiting their use to urban areas and impacts. in , a measles epidemic coinciding with rural famine during the great leap forward affected nearly million persons and caused , deaths; during - , epidemic poliomyelitis spread throughout the country with , cases reported [ fig. ]. in , mao zedong's ''june directive" called for renewed focus on rural health. civil unrest at the start of the cultural revolution, however, disrupted vaccine work. during - , outbreaks of poliomyelitis, diphtheria, pertussis and measles started to recur in areas where the diseases had previously been controlled and incidence began to rise, and mass movement of students facilitated transmission of meningococcal meningitis group a resulting in an epidemic in - that affected over million persons and caused , deaths [ fig. ]. in the early s, a number of important public health reforms were instituted that had greatly strengthened vpd control efforts. in , the rural cooperative medical system (rcms) was established with commune-level health stations staffed by a new cadre of part-time peasants (''barefoot doctors") who received three months of training in delivery of basic medical and preventive services, including vaccinations. these grass-roots health workers were responsible for transporting vaccines from county and commune hospitals to their villages and administering vaccinations, which were essentially free with delivery costs covered by pooled rcms funds. vaccine production was increased and the frequency of campaigns were increased with most provinces conducting at least two or three province-wide campaigns each year; live vaccines in the fall and winter and killed in support of the united nations resolution on universal childhood immunization (uci), '' - " coverage goals were included in china's '' th -year plan for national social and economic development, - " setting targets of % percent coverage at province-level with bcg, dpt, opv and measles by months of age by , and % coverage at county-level by . while the uci goals were lower than china's coverage targets and excluded je and meningococcal meningitis, the target age for being fully vaccinated was much younger - months instead of years of age. administering the entire primary series of bcg, dpt, opv and measles vaccines during infancy would require at least six vaccination sessions per year nationwide, ability to delivery vaccinations year-round, and huge investments to extend the cold chain to township levels nationwide. the first coverage survey in china was conducted in provinces with assistance from who in highlighted the magnitude of the challenge [ table ]. to achieve the uci goals, a high-level inter-ministerial leading group consisting of moh, ministry of foreign economic relations and trade, ministry of tv and broadcasting, state education commission, state ethnic affairs commission, and the all china women's federation was formed to oversee planning and progress. april was established as ''national vaccination day" and huge investments were made in cold chain, training, social mobilization, and vaccines. by the early s most of the epi vaccines were lyophilized and more thermostable, enabling delivery to more remote areas. by , annual production had increased to million doses each of opv, dpt, and measles, and million doses of bcg. in , the national people's congress passed a law requiring health authorities at all levels implement a system of planned preventive immunizations that included issuing vaccination certificates to all children and establishing registers to monitor vaccination coverage at township levels and above. with tremendous effort, both '' - " targets were achieved despite large areas of the country still having no cold chain. the first nationwide coverage survey, conducted in to verify achievement of the uci goal documented coverage of % with all recommended bcg, dpt, opv and measles doses by months of age [ table ]. polysaccharide meningococcal meningitis group a and trivalent opv were introduced in and , respectively, and between and the incidence of meningococcal meningitis fell %, from . to . per , while the incidence of poliomyelitis fell %, from . to . per , [ fig. ]. incidences of other target diseases also continued to fall during this period. by , < cases of poliomyelitis were reported nationwide, although low levels of transmission persisted and the target for national elimination by was missed. in support of the world health assembly resolution to eradicate poliomyelitis globally by , the moh issued the '' - national plan for eradication of poliomyelitis", setting targets of < per million by , and zero cases by , with the main strategy supplementary campaigns with opv. during - , million opv doses were administered in province-wide campaigns. in , synchronized nationwide campaigns were approved by the state council targeting all children < years of age with two doses of opv, one dose on december and one dose on january for three consecutive years. the first campaign was conducted in winter / with million opv doses administered and the last case of polio occurred in september . despite epis high profile, financing for immunizations became progressively weaker starting in the s as a result of broad market reforms. farm collectives and the rcms were dismantled, and while epi vaccines were still provided for free, funding for vaccine delivery switched to fee-for-service, usually - rmb paid by parents to the village or township doctor for each dose administered. public health departments were largely left to generate their own operating expenses. in some regions, innovative financing mechanisms were developed to secure additional funding for immunizations, such as the ''epi contract", a lump-sum payment by parents to cover the cost of all epi vaccinations that was pooled and divided between village, township and county-levels to cover delivery costs, as well as an indemnity payment if a child developed a vpd against which he or she had been vaccinated. by , however, central government funds accounted for only % of total immunization expenditures while more than % of expenditures were at village and township levels, resulting in falling coverage and large disparities between more and less developed areas. in , coverage in children - months old with all recommended doses of bcg, dpt, opv and measles had fallen below % in nine ( %) provinces, including seven of the poorer western provinces. despite the challenges financing the immunization program, china was one of only two low-income countries (the other was cuba) to introduce hepatitis b vaccine into their epi when universal infant immunization with hepatitis b vaccine was endorsed by the world health assembly in . at that time, china had the largest disease burden of hepatitis b in the world with approximately % of the population chronically infected. adding hepatitis b vaccine would have tripled government vaccine costs per child from . to . rmb, so market strategies were also used to finance introduction of this expensive new vaccine. the government added hepatitis b to the epi but exceptionally allowed the vaccine cost to be passed to parents. this cost-recovery approach provided a strong incentive to health workers to deliver the vaccine and national coverage with three doses of hepatitis b vaccine in infants increased from % in , to % in , preventing millions of infections and averting hundreds of thousands of deaths. coverage, however, was predictably much lower in less developed areas, particularly the western provinces. requirements for all children to be fully vaccinated, and the regulation on vaccine circulation and immunization that included laws specifying that nationally recommended vaccines be fully funded by the government and administered completely free-of-charge. in , the immunization schedule was expanded to include new antigens (measles-mumps-rubella, attenuated hepatitis a), safer products (acelluar pertussis), and older vaccines that had been excluded from the original epi schedule (je, meningococcal meningitis) and epi vaccine procurement was centralized with moh responsible for procuring all epi vaccines. in , the government launched the new essential public health service package with service categories, including immunizations. government subsidies of rmb per person were provided to village and township-administrative levels to cover the cost of immunization services, and increased to rmb per person in , rmb in , rmb in , and rmb in . currently, all village and townships have the full-time health staff that provide vaccination services to all children at , delivery sites nationwide. there are approximately , epi managers at provincial, prefecture and county levels and , immunization staff at township and village levels. with the majority of china's population now living in urban areas, vaccination has shifted from pulse delivery by village-based health workers to predominantly daily delivery at fixed clinic sites. public health reforms during the past decade have strengthen financing for vaccines and vaccination services resulting in increased and more equitable coverage and historically low vpd burden [ table , fig. ]. in the national survey, coverage of recommended infant doses of bcg, dpt, opv, hepatitis b and measles vaccines was % in of provinces, and % in province, a major improvement from . from to , the incidence of pertussis fell % from . to . per million, measles fell % from . to . per , , meningococcal meningitis fell % from . to . per million, je fell % from . to . per million, and hepatitis a fell % from . to . per , . the last case of diphtheria was reported in . in , china was certified as having eliminated maternal and neonatal tetanus and an all-time low of measles cases were reported nationwide, although numbers of measles cases increased in subsequent years. from to , the prevalence of chronic hepatitis b infection in children under- years old dropped from . % to . %, a % decline. sustained political commitment to immunizations at the highest levels, ambitious targets, strong supporting legal frameworks, a vibrant domestic vaccine industry, and innovative financing and delivery strategies to maximize use of available resources have been key to china's many achievements in vpd control over the past years. immunizations has been a universal right since the founding of the people's republic of china, and have been supported at the highest political levels with passage of laws ensuring access and progressively increasing levels of public finance as the country has developed. goals for universal childhood immunization, smallpox eradication, and poliomyelitis eradication were established in china before comparable un global resolutions and before there was an established cold chain system. selfreliance has been a defining characteristic stimulating the development of innovative strategies to maximize use of limited resources, including mass training of village-based lay health workers to deliver vaccinations, cost-sharing strategies to finance introduction of new vaccines, large-scale social mobilization, use of rapid pulsedelivery approaches to leverage existing vaccine thermostability in areas without cold chain, support for domestic vaccine production, and rapid national scale-up of successful pilots. currently, there are seven state-owned and private vaccine manufacturers in china with annual production capacity of around billion doses, including acellular pertussis, influenza, rabies, yellow fever, japanese encephalitis, hepatitis a and b, rubella, varicella, typhoid, and live and inactivated polio vaccines (ipv). all vaccines recommended in the national immunization schedule are domestically produced. globally, vaccination coverage is stagnating with dpt coverage % since , and % in the african region [ ] . progress toward the global vaccine action plan % coverage target is off-track, and in , nearly million infants were not vaccinated [ ] . many of these children are socioeconomically marginalized, live in fragile or remote settings, and have limited access to health care. a delivery model that relies primarily on nurses and clinical officers to administer vaccinations at fixed sites and provide periodic outreach is costly, places a large burden on parents, and likely to be insufficient to achieve and sustain high vaccination coverage levels in resource-poor countries with large dispersed rural populations [ ] . china has more than half a century of experience successfully using community-based health workers to periodically come to clinics to collect vaccines in cold boxes to take back to their villages for pulse administration increasing rural access, community buy-in, and sustained high coverage levels. more widespread adoption of alternative service delivery approaches may be needed to close remaining coverage gaps. use of vaccine vial monitors that measure cumulative heat exposure and development of pre-filled injection devices further facilitate ease and safety of vaccination by community health workers and could also help address cold-chain challenges in remote areas [ ] . in , menafrivac Ò conjugate meningococcal meningitis a vaccine was the first vaccine prequalified by who for use outside the cold chain in controlled temperature chain (ctc); use of ctc for a mass vaccination campaign in chad would have reduced logistics costs by an estimated % [ ] . in china, in villages where village health workers were provided with hepatitis b vaccine with storage at ambient temperatures at the beginning of hepatitis b vaccine introduction, timely birth dose coverage in infants born at home increased from % to %, with no difference in antibody response compared to newborns who were vaccinated with hepatitis b kept in the cold chain [ ] . recent economic analyses indicate that ctc delivery of the hepatitis b birth dose would be cost-saving in most low and middle-income countries [ ] . disease control has always been the main goal of china's immunization efforts and disease incidence has always been the main metric used to guide development of immunization strategies and to assess immunization program performance. close monitoring of temporal, geographic, and demographic trends in vpd incidence has been critical in china for identifying under-immunized populations and for evaluating the effectiveness of delivery strategies, even when the majority of reported cases are primarily clinically confirmed. in contrast, epis in most low-income countries primarily rely on administrative coverage despite recognized problems with data quality and reliability [ , ] . the - ebola outbreak in west africa has spurred new initiatives to strengthen communicable disease surveillance and more effective use of surveillance data by immunization program managers, including district and sub-district mapping of disease incidence, could strengthen program monitoring and accountability. china's experience with hepatitis b vaccine is an interesting case study on use of cost-sharing to finance the introduction of new vaccines that may be of relevance to middle-income countries ineligible for gavi support. when who recommended universal infant immunization with hepatitis b vaccine in , gdp per capita in china was only us$ . adding hepatitis b vaccine to the infant schedule while allowing health workers to recover the vaccine costs from parents enabled early introduction without external or government financing, and provided a delivery incentive that achieved % coverage nationwide, preventing millions of infections. china's experience suggests that cost-recovery can be an effective interim option for countries to finance the early introduction of expensive new vaccines, particularly if the govern-ment can negotiate lower purchase prices, set caps on allowable charges, and provide subsidies for the poor. china also has much to learn from experience in other countries. challenges include strengthening delivery of immunizations within a larger package of integrated health services, balancing policies that make vaccines affordable against those providing incentives for new vaccine research and development, financing new vaccine introductions, and vaccine hesitancy. for many years, vaccination was one of the few preventive services that village and township doctors were required to deliver and china's immunization program is facing the challenges of navigating integration of immunization with delivery of a much wider range of other services. introduction of new expensive vaccines into the recommended schedule remains a challenge. the current infant schedule requires separate injections and there is urgent need for increased funding to develop and add combined products to the schedule, such as dpt-hepatitis b, dpt-hepatitis b-haemophilus influenzae type b (hib), and dpt-hepatitis b-hib-ipv. finally, while public trust in immunizations remains high, china has not been immune to problems of vaccine hesitancy. widespread internet access has facilitated rapid dissemination of often unfounded claims of harmful effects due to vaccination that have had negative effects on vaccination coverage [ ] . these and other challenges will require new approaches as china's immunization program continues moving forward. this work was supported by united nations children's fund for the literature review and the in-depth interviews of chinese immunization experts (yh - ). a -year history of disease prevention and control in china new china's achievements in health work chinese communist party. constitutional principles of the shaanxi-gansu-ningxia border region viral hepatitis in china. seroepidemiological survey in chinese population (part one) - . beijing science and technology press: beijing epidemiological serosurvey of hepatitis b in china -declining hbv prevalence due to hepatitis b vaccination prevention of chronic hepatitis b after decades of escalating vaccination policy world health organization. the epi coverage survey. geneva: world health organization, expanded programme on immunization regarding the launching of the autumn campaign for smallpox vaccination ministry of health of the people's republic of china. measures for implementing preventive vaccination ministry of health of the people's republic of china. regulations on acute infectious diseases, number ministry of health of the people's republic of china. report of the survey of the third % vaccination coverage target of the national expanded programme on immunizations ministry of health of the people's republic of china. compilation of national immunization documents: s national file ministry of health of the people's republic of china. report of the national review of the expanded programme on immunizations ministry of health of the people's republic of china. report of the national review of the expanded programme on immunizations. beijing: people's health publishing house state council of the peoples's republic of china. regulations of vaccine distribution and vaccination, number ministry of health of the people's republic of china. report of the national review of the expanded programme on immunizations national certification committee for the eradication of poliomyelitis in the people's republic of china. documentation for the certification of poliomyelitis eradication hubei province epidemic prevention station. expanded programme on immunization contract system tested study on financing the expanded program on immunizations in selected regions of china routine immunization services costs and financing in china progress and challenges with achieving universal immunization coverage. who/unicef estimates of national immunization coverage (data as of assessment report of the global vaccine action plan strategic advisory group of experts on immunization. geneva: world health organization the cost structure of routine infant immunization services: a systematic analysis of six countries can thermostable vaccines help address cold-chain challenges? results from stakeholder interviews in six low-and middle-income countries economic benefits of keeping vaccines at ambient temperature during mass vaccination: the case of meningitis a vaccine in chad hepatitis b vaccination of newborn infants in rural china: evaluation of a village-based, out-of-cold-chain delivery strategy costeffectiveness of the controlled temperature chain for the hepatitis b virus birth dose vaccine in various global settings: a modelling study tracking progress towards universal childhood immunisation and the impact of global initiatives: a systematic analysis of three-dose diphtheria, tetanus, and pertussis immunisation coverage the immunization data quality audit: verifying the quality and consistency of immunization monitoring systems loss of confidence in vaccines following media reports of infant deaths after hepatitis b vaccination in china the authors declare no conflicts of interest. key: cord- -v ruj bq authors: madsen, anders; cox, rebecca jane title: prospects and challenges in the development of universal influenza vaccines date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: v ruj bq current influenza vaccines offer suboptimal protection and depend on annual reformulation and yearly administration. vaccine technology has rapidly advanced during the last decade, facilitating development of next-generation influenza vaccines that can target a broader range of influenza viruses. the development and licensure of a universal influenza vaccine could provide a game changing option for the control of influenza by protecting against all influenza a and b viruses. here we review important findings and considerations regarding the development of universal influenza vaccines and what we can learn from this moving forward with a sars-cov- vaccine design. an influenza virus infection typically manifests as a fever, sore throat, cough, runny nose, myalgia, and headaches. people in certain risk groups (the elderly, children < months of age, pregnant women, and individuals with chronic conditions) have a higher chance of developing more severe illness with complications such as fulminant pneumonia. seasonal influenza causes around , to , respiratory deaths each year [ ] . however, this number can increase dramatically when, at unpredictable intervals, influenza pandemics occur. these global epidemics are characterized by the emergence of a novel influenza virus to which most humans are immunologically naïve [ ] . the emergence of the a/h n pdm virus ("swine flu") was the first influenza pandemic of the st century. this h n virus has subsequently continued to circulate in the human population as a seasonal virus, together with influenza a/h n and influenza b (yamagata-like and victoria-like) viruses. the current seasonal influenza vaccines contain strains from each of these viruses. the world health organization (who) decides biannually which strains should be included in the vaccine based on which viruses are most likely to circulate in the upcoming season. annual seasonal influenza vaccination remains the most cost-effective measure to reduce burden of the disease. however, the currently licensed influenza vaccines lack two essential attributes: firstly, they do not produce durable protective immunity. secondly, they do not produce a cross-reactive immune response that can neutralize diverse influenza virus strains. cross-reactivity is necessary because of the antigenic plasticity of the viral membrane protein hemagglutinin (ha), which leads to antigenically drifted viruses and potential mismatches with the vaccine strains [ ] . this shortcoming can be addressed by developing new vaccines targeting the more antigenically conserved regions of the influenza virus [ ] . in order to overcome these barriers, efforts have been made to develop next-generation influenza vaccines that provide robust, long-lasting protection against diverse influenza a subtypes (table ) [ ] . the ultimate goal is to develop a "universal" influenza vaccine that covers all influenza a and b viruses. [ ] . provide protection against all ha subtypes and at a minimum protection against h , h , h , h , h and h subtypes. provide a decade or more of protection. use manufacturing technology that can be readily transferred to developing-world countries. provide immunologic protection for those populations most at risk for severe disease and increased mortality use inexpensive manufacturing technology that permits rapid and highly scalable production, particularly to address emergence of a pandemic virus. offer heat stability, thereby eliminating the need to maintain a cold chain. rarely cause adverse events, and any adverse events are mild and temporary. do not require injection for administration. do not require injection for administration. the influenza virus is an enveloped virus with a segmented, negative-sense, single-strand rna genome ( figure ). there are four types of influenza: a, b, c, and d. influenza a and b are the only types that cause seasonal epidemics in humans. influenza a viruses are further subdivided into subtypes and strains depending on the characteristics of the most abundant surface proteins, ha and neuraminidase (na). ha is a trimeric glycoprotein responsible for the attachment of the virus to the surface of the host cell by binding to sialic acid receptors. there are subtypes of ha within the influenza a viruses. these can be divided into two distinct phylogenetic groups: group ha (h , h , h , h , h , h , h , h , h , h , h , and h ) and group ha (h , h , h , h , h , and h ). the ha protein comprises a head domain and a stalk domain, the latter being more antigenically conserved. antibodies targeting the ha head domain are usually strain-specific and neutralize influenza viruses by inhibiting the binding of ha to sialic acid receptors on the surface of the host cell. ha stalk-specific antibodies can provide heterosubtypic protection by blocking viral fusion with the host cell and by eliminating infected host cells through antibody-dependent cellular cytotoxicity (adcc) [ , ] . the stalk-specific antibodies are broadly cross-reactive within an ha group [ , ] . while ha remains the main target of current inactivated influenza vaccines (iivs), na has recently been recognized as a potential target for universal vaccines [ , ] . na functions by catalyzing the cleavage of sialic acids on the surface of an infected cell, leading to the release of newly formed viruses. several studies have shown that na antibody titers correlate with protection in humans [ - ]. na-specific antibodies function by inhibiting the enzymatic activity of na and thus preventing the spread of virions from infected cells. fc receptor-mediated effector functions such as adcc have also been reported for na antibodies [ , ] . the matrix protein ectodomain (m e) protein functions by transferring h + ions through the viral membrane as the virus is taken up by the endosomes. this leads to conformational changes of the ha molecule and fusion between the viral membrane and the endosome membrane. as a result, the viral rna is released into the cytosol. m e-specifc antibodies have been shown to be protective in animal studies, although the exact defense mechanisms are not fully understood. t-cell activation also plays an important role in targeting other conserved proteins that are generally not exposed on the outside of virus particles, such as the nucleoprotein (np) and matrix protein (m ) [ ] . these proteins are highly conserved between influenza a viruses. influenza-specific interferon-secreting t cells, cd + t cells, and cd + t cells play an important role in recovery from influenza in humans [ ] [ ] [ ] [ ] . there are currently three types of licensed influenza vaccines: inactivated, live attenuated, and recombinant ha ( figure ). inactivated influenza vaccines (iivs) are the most commonly used, partly because of their well-documented safety and low production costs. the iivs come in three variants: whole-virion vaccines, split-virion vaccines, and subunit vaccines, which contain purified ha and na. iivs are traditionally produced in embryonated chicken eggs. this egg-based approach has several drawbacks: firstly, the production time is relatively long. consequently, the producers have to start production months in advance of the flu season, which can lead to mismatches between the vaccine strain and the circulating strain [ ] . in addition, the prolonged production time becomes a crucial problem during influenza pandemics, where rapid production and distribution of vaccines are essential. another issue with the egg-based approach is the occurrence of egg-adapted mutations during the production process, which can lower vaccine effectiveness [ , ] . cell-based iivs have been licensed in europe and the united states as an alternative to the egg-based approach. another approach for overcoming egg-based vaccine production is the use of recombinant ha vaccines, which are based on a protein-expression system using baculoviruses and insect cells [ ] . these vaccines have been licensed in the united states since . however, as with the iivs, the protection appears to be strain-specific, with limited immunogenicity. some vaccine formulations include adjuvants in order to improve immunogenicity and vaccine effectiveness [ ] . adjuvants function by increasing antigen uptake and presentation in the local draining lymph nodes. during the h n pandemic, the use of the oil-in-water emulsion (as ) led to higher b and cd t-cell responses than vaccination with nonadjuvanted pandemic vaccines [ , ] . in contrast to iivs and recombinant ha vaccines, the live-attenuated influenza vaccines (laiv) contain live, cold-adapted viruses that are administered as a nasal spray, leading to a restricted viral replication in the upper respiratory tract of the recipient [ , ] . the laiv induces broader immune response, including t-cell and mucosal immunity, by mimicking natural infection [ ] [ ] [ ] . laiv is currently licensed in north america, europe, and india. it is generally not recommended for people younger than years of age due to increased risk of wheezing [ ] or for immunosuppressed individuals who are at greater risk of severe influenza illness as the vaccine may produce higher virus titers in these individuals, leading to severe side effects. recent scientific advances have made way for novel vaccine approaches, enabling a more targeted delivery of conserved antigens that can stimulate the innate and adaptive immune systems. here, we highlight three vaccine platforms that are being used in the development of next-generation influenza vaccines (figure ). virus-like particles (vlps) have similar morphological and structural features to viruses but lack the viral genome [ ] . they are a useful vaccine component as the immune system recognizes vlps similarly to viruses but without the risk of replication and recombination. a variety of immunogens have been tested on vlps, including ha, matrix protein (m ), and na [ ] [ ] [ ] . one of the main barriers to the use of this vaccine construct is the challenge of generating sufficient immunogenicity. however, the inclusion of adjuvants such as toll-like receptor ligands has led to improved vaccine effectiveness [ ] . in contrast to vlps, peptide-based vaccines focus on minimal epitopes of the influenza virus, such as t-cell-inducing np and m peptides [ , ] . however, as with vlp-based vaccines, they often require the use of adjuvants or particulate carriers for delivery in order to be sufficiently immunogenic. nucleic-acid-based vaccines use rna-or dna-sequences for the vaccines antigens. this technology is especially useful in disease outbreaks where rapid vaccine development is needed. using self-amplifying mrna, researchers were able to develop a vaccine candidate for the influenza a h n outbreak in china just days after a/shanghai/ / (h n ) sequences were released [ ] . this vaccine platform could be a viable option in the ongoing efforts to develop a sars-cov- vaccine. a phase clinical trial of a dna vaccine targeting the spike protein of sars-cov- found that the vaccine was safe and capable of eliciting neutralizing antibodies and cellular immune responses [ ] . viral vector vaccines use carrier viruses such as modified vaccinia virus ankara (mva) or adenovirus containing genes expressing influenza-proteins of interest [ ] . these vaccines allow for any influenza antigens to be expressed in their native conformation or with modifications, inducing both humoral and cellular immune responses [ ] [ ] [ ] . most viral vectors are replication-deficient in mammalian host cells and are therefore safe to use. issues with pre-existing immunity to some viral vectors have been reported [ ] , although other vectors, such as mva, remain immunogenic despite the presence of pre-existing immunity. influenza vaccines can be divided into two categories based on their immunological features: ( ) vaccines providing sterile immunity by eliciting ha head antibodies that prevent viral attachment to sialic acid receptors and ( ) vaccines that provide infection-permissive immunity by inducing cellular immunity or broadly reactive antibodies that function in the later stages of the viral life cycle (figure ) . many of the infection-permissive vaccines that are in development will probably not be universal vaccine candidates as stand-alone vaccines but could be combined with other approaches to increase immunogenicity. ha stalk antibodies have emerged as one of the leading strategies in the development of universal vaccine candidates [ ] . several approaches for avoiding the immunodominant ha head domain are under development. one strategy is to remove the globular head domain while maintaining the correct immunogenic conformation of the ha stalk domain [ ] . animal models have shown that vaccination with these "headless" ha proteins can induce heterosubtypic protection within group ha proteins [ ] [ ] [ ] [ ] . other strategies for refocusing the antibody response toward the ha stalk involve maintaining the full-length ha. krammer and colleagues have developed a method for directing the antibody response to the conserved ha stalk domain by sequential vaccination with chimeric ha proteins consisting of exotic ha head domains to which the recipient is immunologically naïve. the results of a phase clinical trial (nct ) provide evidence that group chimeric ha-based vaccines induce high titers of stalk-reactive immunoglobulin g (igg) that cross-react to other ha proteins within the same group [ ] . a trivalent vaccine comprising group , group , and influenza b virus chimeric constructs could potentially lead to a universal influenza vaccine. advances in the development of monoclonal antibodies (mabs) have shown that there are cross-reactive epitopes on the ha head domain that can be targeted to give heterosubtypic protection [ ] [ ] [ ] . a new vaccine approach called mosaic ha has been developed to combine the immunity of conserved epitopes of the ha stalk and head domains. mosaic ha vaccines are developed by replacing the immunodominant and strain-specific antigenic sites with sequences from exotic ha subtypes. through sequential immunization, the immune response is refocused toward more cross-reactive immunosubdominant epitopes of the ha stalk and head domains. cross-reactive ha stalk-and head-specific antibodies can also be induced by computationally optimized, broadly reactive antigens (cobras). this approach is based on developing novel ha proteins after multiple rounds of consensus sequence generation from a variety of different ha isolates. preclinical trials have demonstrated that cobra-based vaccines provide protection against multiple influenza viruses within a subtype in murine and ferret models [ ] [ ] [ ] . there has been increased recognition of na as a target for improved vaccines following the development of practical assays for measuring antibody responses to na [ ] . although antibodies targeting na can bind to conserved epitopes and provide heterologous protection [ ], they are not really induced by seasonal iivs. this is mostly due to the amount and stability of the na in the vaccines. enhanced na-antibody responses can be achieved by delivering na as a supplement to current iivs or vaccinating with recombinant or viral vector na in addition to the seasonal iivs [ ] [ ] [ ] [ ] [ ] . m e has been an attractive target in the development of the future universal influenza vaccine, as it is highly conserved between influenza viruses [ ] . because of the relatively small size of the m e, vaccines usually depend on carrier constructs such as vlps. preclinical trials have demonstrated that m e antibodies are protective in animal models, and some m e vaccines have advanced into clinical trials [ , ] . while the effectiveness of a stand-alone m e-based vaccine remains limited, recent studies have found promising results by combining an m e-based vaccine (m e x vlp) with other vaccine constructs such as laivs [ ] and ha vlps [ ] . most vaccine approaches targeting internal proteins such as m and np involve t-cell activation. t cells are important during an influenza infection because they limit viral replication and shedding by clearing influenza-infected cells. an ongoing phase clinical trial (clinicaltrials.gov: nct ) is testing a recombinant-peptide based vaccine consisting of conserved epitopes from ha, np, and m [ ] . other vaccine constructs use viral vectors expressing m and np [ , ] . these have been shown to induce cd + and cd + t cells in clinical trials. selecting the best approach for inducing t-cell-mediated immunity can be challenging due to the diverse cd t-cell repertoire in humans with different degrees of pre-existing immunity. however, with increasing insights into the quantity, functionality, and specificity of cd t-cell subsets, there is hope that new correlates of protection can be established in the near future, which would help in selecting the best vaccine candidates for clinical trials [ , , ] . some studies evaluating the t-cell response in various infection models have reported immunopathology with excessively large cd t-cell responses [ , ] . other studies have reported a narrowing of t-cell receptors during heterosubtypic challenges [ ] . additional studies that reflect pathogen encounters in humans are needed in order to select the best candidates for next-generation t-cell-based vaccines [ ] . there has been significant progress in the development of novel approaches for universal influenza vaccines. however, additional efforts are needed in order to translate more of these novel approaches into products ready for late-stage clinical trials. selecting the best candidates for clinical trials is a challenging process for several reasons: ( ) there is no uniform evaluation of novel influenza vaccines in preclinical trials. this is partly due to the variety of vaccine platforms and targets. in addition, there is no consensus on which animal model is best suited to accurately reflect human infection [ , ] . this is partly due to the existence of pre-existing immunity in humans, which is difficult to translate into animal models. other host factors to be considered include age, gender, and chronic diseases, which could affect vaccine efficacy. ( ) there are no established correlates of protection for broadly cross-reactive immune responses to influenza. assays measuring non-ha-head immune responses, such as ha stalk antibodies, na antibodies, and cellular immunity, are inadequately standardized. optimization and standardization of such assays are needed in order to assess the protective immunity contributed by broadly reactive or universal vaccines. ( ) the funding for universal vaccine development is limited. this leads to down selection of promising candidates before they advance to clinical trials. regulatory initiatives are needed to enhance advocacy for moving from an annually reformulated vaccine to a universal vaccine, despite the financial risks and inadequate incentives [ ] . as the search for an appropriate sars-cov- vaccine continues, comparisons can be drawn to influenza vaccine development. some of the next-generation influenza vaccines use vaccine platforms similar to those used by the sars-cov- vaccine candidates [ ] . given the urgency of moving forward with a sars-cov- vaccine, a nucleic-acid-based vaccine would be a convenient approach in order to facilitate rapid production. although this method has been tested for several influenza vaccine candidates, no dna or rna vaccines have yet been licensed for humans. the disadvantages of such vaccines include limited immunogenicity and reports of increased reactogenicity. another option is the use of viral-vector-based vaccines, which have shown promising results against mers-cov and other emerging viruses [ ] [ ] [ ] . some viral-vector-based vaccines focusing on the s protein of the sars-cov- virus are in the preclinical phase. however, it might be years until a nucleic-acidor viral-vector-based vaccines are licensed for human use since both of these vaccine platforms are novel and have not been extensively tested. other sars-cov- vaccines that are currently being tested include recombinant protein vaccines, live-attenuated vaccines, and inactivated vaccines. all of these platforms can be found among the licensed influenza vaccines, each with their own advantages and disadvantages. in the end, a sars-cov- vaccine or a universal influenza vaccine can only be successful if implemented effectively [ ] . efforts are needed to improve vaccine uptake by restoring widespread public confidence in vaccines. this is especially important in a pandemic setting. in addition, as the influenza a/h n pandemic demonstrated, there will likely be an issue of vaccine availability in low-and middle-income countries. therefore, strategies are needed to ensure sufficient and equitable vaccine distribution to these countries. encouraging progress has been made in the development of universal influenza vaccines. future universal vaccines will likely consist of a combination of different approaches to induce broad immunity to influenza. ultimately, the path forward depends on combined action from governments, industries, and the scientific community. estimates of global seasonal influenza-associated respiratory mortality: a modelling study in vitro and in vivo characterization of new swine-origin h n influenza viruses the future of influenza vaccines: a historical and clinical perspective development of a universal influenza vaccine a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases the compelling need for game-changing influenza vaccines an analysis of the influenza vaccine enterpris the role of fc-fcγr interactions in igg-mediated microbial neutralization broadly neutralizing anti-influenza antibodies require fc receptor engagement for in vivo protection highly conserved protective epitopes on influenza b viruses natural t cell-mediated protection against seasonal and pandemic influenza. results of the flu watch cohort study cytotoxic t-cell immunity to influenza preexisting influenza-specific cd + t cells correlate with disease protection against influenza challenge in humans influenza virus: dealing with a drifting and shifting pathogen h n influenza viruses in humans: viral mechanisms, evolution, and evaluation. hum. vaccines immunother efforts to improve the seasonal influenza vaccine safety, efficacy, and immunogenicity of flublok in the prevention of seasonal influenza in adults adjuvanted influenza vaccines. hum. vaccines immunother van der most, r. investigating the effect of as adjuvant on the plasma cell repertoire following ph n influenza vaccination adjuvant system as containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity recombinant cold-adapted attenuated influenza a vaccines for use in children: molecular genetic analysis of the cold-adapted donor and recombinants multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (flumist) derived from cold-adapted a/ann arbor/ / the role of nasal iga in children vaccinated with live attenuated influenza vaccine comparisons of the humoral and cellular immune responses induced by live attenuated influenza vaccine and inactivated influenza vaccine in adults immune responses after live attenuated influenza vaccination live attenuated versus inactivated influenza vaccine in infants and young children novel platforms for the development of a universal influenza vaccine multi-antigen avian influenza a (h n ) virus-like particles: particulate characterizations and immunogenicity evaluation in murine and avian models virus-like particles containing multiple m extracellular domains confer improved cross-protection against various subtypes of influenza virus an intranasal virus-like particle vaccine broadly protects mice from multiple subtypes of influenza a virus tlr-based immune adjuvants heterosubtypic influenza protection elicited by double-layered polypeptide nanoparticles in mice synthetic multi-epitope peptides identified in silico induce protective immunity against multiple influenza serotypes rapidly produced sam ® vaccine against h n influenza is immunogenic in mice a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial viral vector-based influenza vaccines. hum. vaccines immunother safety and immunogenicity of an oral, replicating adenovirus serotype vector vaccine for h n influenza: a randomised, double-blind, placebo-controlled, phase study preliminary assessment of the efficacy of a t-cell-based influenza vaccine, mva-np+ m , in humans safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine universal influenza virus vaccines that target the conserved hemagglutinin stalk and conserved sites in the head domain production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines a stable trimeric influenza hemagglutinin stem as a broadly protective immunogen influenza virus vaccine based on the conserved hemagglutinin stalk domain mini-hemagglutinin vaccination induces cross-reactive antibodies in pre-exposed nhp that protect mice against lethal influenza challenge hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection immunogenicity of chimeric haemagglutinin-based, universal influenza virus vaccine candidates: interim results of a randomised, placebo-controlled, phase clinical trial cross-neutralization of influenza a viruses mediated by a single antibody loop receptor mimicry by antibody f - facilitates universal binding to the h subtype of influenza virus heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity design and characterization of a computationally optimized broadly reactive hemagglutinin vaccine for h n influenza viruses a computationally optimized broadly reactive antigen (cobra) based h n vlp vaccine elicits broadly reactive antibodies in mice and ferrets a computationally optimized broadly reactive antigen subtype-specific influenza vaccine strategy elicits unique potent broadly neutralizing antibodies against hemagglutinin purified influenza a virus n neuraminidase vaccine is immunogenic and non-toxic in humans vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice vaccination with recombinant parainfluenza virus expressing neuraminidase protects against homologous and heterologous influenza virus challenge immunogenicity and protection against influenza h n in mice by modified vaccinia virus ankara vectors expressing influenza virus hemagglutinin or neuraminidase neuraminidase expressing virus-like particle vaccine provides effective cross protection against influenza virus m e-based universal influenza vaccines: a historical overview and new approaches to development immunopotentiation of trivalent influenza vaccine when given with vax , a recombinant influenza m e vaccine fused to the tlr ligand flagellin safety and immunogenicity of a recombinant m e-flagellin influenza vaccine (stf . xm e) in healthy adults enhancing the cross protective efficacy of live attenuated influenza virus vaccine by supplemented vaccination with m ectodomain virus-like particles influenza m virus-like particle vaccination enhances protection in combination with avian influenza ha vlps safety and immunogenicity of multimeric- -a novel universal influenza vaccine at cell-inducing influenza vaccine for the elderly: safety and immunogenicity of mva-np+ m in adults aged over years crtam determines the cd + cytotoxic t lymphocyte lineage nkg c/e marks the unique cytotoxic cd t cell subset, thctl, generated by influenza infection protective heterologous antiviral immunity and enhanced immunopathogenesis mediated by memory t cell populations memory cd t cells mediate severe immunopathology following respiratory syncytial virus infection t cell immunodominance and maintenance of memory regulated by unexpectedly cross-reactive pathogens past life and future effects-how heterologous infections alter immunity to influenza viruses workshop report: experimental animal models for universal influenza vaccines vaccines: status report. immunity immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type or vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus recent advances in the vaccine development against middle east respiratory syndrome-coronavirus improving influenza vaccines: challenges to effective implementation the authors declare no conflict of interest. key: cord- -vffa bu authors: richmond, heather; rees, natasha; mchale, sharon; rak, aaron; anderson, jonathan title: seasonal influenza vaccination during a pandemic date: - - journal: human vaccines & immunotherapeutics doi: . / . . sha: doc_id: cord_uid: vffa bu in the northern hemisphere, the persistence or reemergence of coronavirus circulation into the – influenza season threatens to overwhelm health-care resources and systems and increase mortality and morbidity. data from australia show that stay-at-home policies have reduced both influenza and coronavirus cases early in the season, thus “flattening the curve.” however, influenza vaccination is critical to ensure the reduction in co-infection. several policies, such as vaccination strategies to accommodate physical distancing measures, change population recommendations, and timing and location of vaccination have been implemented to increase influenza vaccine uptake during the pandemic. this commentary explores those policies. the persistence or reemergence of coronavirus circulation into the - influenza season threatens to overwhelm health-care resources and systems and increase mortality and morbidity. learnings from australia, where concomitant responses to both influenza and coronavirus are ongoing, offer insights that may assist influenza vaccination planning and implementation in the upcoming northern hemisphere influenza season and beyond. these include changes to the timing and location of vaccine administration to accommodate social distancing, policies to ensure optimal management of public demand, access and uptake of available vaccines across the season, and the need for communications to be clear, frequent, and aligned among all stakeholder groups. data from the southern hemisphere show that stay-at-home policies have reduced both influenza and coronavirus cases early in the season, thus "flattening the curve." however, as physical distancing measures such as shelter in place and lockdown restrictions are eased, influenza infections are likely to emerge and endure over several months. in recognition of this, the australian government introduced several policy changes to increase influenza vaccine uptake and implemented new vaccination strategies to accommodate physical distancing measures, changing not only who gets a vaccine but also when, where, and from whom. australia provides free influenza vaccine each season for atrisk populations, with strong uptake in these groups. at the beginning of the pandemic, the australian government recognized the importance of maintaining health-care visits and preventive care. additionally, the government issued a requirement that by may , , all individuals visiting, working, or living in an aged care/nursing/long-term care or assisted living facility must receive an influenza vaccine. the policy was also extended on april , , to apply to childcare centers. these policy changes were designed to further protect vulnerable populations and recognized health-care workers and caregivers as critical target populations for influenza vaccination. influenza vaccines are manufactured and delivered in a timeframe that allows providers to start vaccination prior to peak circulation of the influenza virus. policy changes in australia, together with strong communication from public health agencies and media reporting about the risk of coinfection and importance of influenza vaccination, generated significant demand for influenza vaccines early in the season. as a result, the australian government asked seqirus to go back into production requiring education to the public about the need to extend the duration of the immunization program and urging the need to continue vaccination late into the season. social and physical distancing requirements throughout the vaccination period in australia have yielded novel approaches to vaccine administration within the traditional clinic settings and through alternative models for administration supporting safe, effective vaccination. these locations have included pharmacies, carparks/parking lots, offices, church or community halls, or parks and outdoor areas that would allow people to maintain a safe distance while being vaccinated. planning for these activities occurred expeditiously, as local solutions, rather than being organized by public health authorities. immunization advisory groups, providers, and influenza vaccine manufacturers have worked together to share learnings and optimize vaccination during this pandemic. to achieve high immunization rates, the australian government worked with industry to align delivery dates of vaccine to immunization sites upon availability, although demand was so great at times that short-term shortages occurred. the centers for disease control and prevention (cdc), the white house coronavirus taskforce, and the who's european office have stated the need to use the influenza vaccine as a tool in the fight against co-infection. receiving an influenza vaccine reduces the number of infected patients, thereby helping to "relieve pressures" in hospitals treating patients with coronavirus. in the european union, seasonal influenza causes − million symptomatic cases each year. in the us, the cdc estimates that in any given influenza season, , - , hospitalizations occur due to influenza. the opportunity to increase influenza vaccination rates in the - season, to reduce the burden on health-care systems if a second coronavirus surge occurs, may lead to permanent, improved policy changes for influenza vaccination campaigns for years to come. as the influenza season approaches, public health stakeholders have already begun to consider the potential impact of concurrent influenza with coronavirus and potential changes to target populations. for example, the united kingdom and certain regions of italy are considering expanding influenza recommendations for people under y of age, while the german government is considering a recommendation for voluntary lifespan immunization. other countries are looking to increase the health and social care worker vaccination. with changes to vaccine recommendations or mandates, it is critical that recommending bodies (national immunization technical advisory groups -nitags) coordinate with medical and health professional societies to manage and adequately communicate these changes to all stakeholders involved in influenza vaccination. manufacturers are currently experiencing significant demand for influenza vaccines from many northern hemisphere countries. , meeting sudden, increased demand can be challenging as manufacturing preparations begin at least months before each season. nonetheless, manufacturers are responding by increasing and extending current production; however, the ability to dramatically increase supply in a short period of time is limited, and additional doses that are produced will require acceptance of later delivery compared to "normal" seasons. policy changes and strong public education can potentially optimize influenza vaccine coverage as doses become available throughout the season but need to be sustained into future seasons to maintain vaccine uptake and achieve the required on-time supply of doses, including influenza vaccines that are specifically designed for certain populations. clear and simple messaging about the influenza vaccine helps to play a role in spreading the word about the importance of influenza vaccination, particularly with the prospect of changes in demand, population recommendations, timing, and administration locations. in some countries, the coronavirus pandemic may result in many people seeking influenza vaccination who have not been vaccinated previously. these people may have new or additional questions that need to be considered. the opportunity for providers to communicate the right vaccine for the right individual within an opportune time frame may be advantageous for those at higher risk for co-infection. further, as we have seen in australia, the timing of vaccination will need to be extended well into the influenza season to align with vaccine availability, reaching as many as possible prior to the peak of influenza circulation. therefore, the message to vaccinate early -without creating perceived shortagesand throughout the season, is vital to the effectiveness of the - campaign. by working together, health officials, medical authorities, and medical journalists can help deliver effective messages about influenza vaccination during the pandemic, while being open to new and evolving questions based on the situation. clear, consistent, frequent, and honest communication from public health officials to all influenza vaccine providers, and to the public, is the only way to manage expectations and ensure that continued attempts to "flatten the curve" and protect patients from influenza and coronavirus are achieved. the case for seasonal influenza vaccination is very strong, yet vaccination rates remain suboptimal in most countries. influenza vaccine policy changes during the coronavirus pandemic have the potential to improve coverage but are limited by the ability of manufacturers to significantly increase supply doses in a short period of time. sustaining these policy changes will not only help to reduce seasonal influenzarelated deaths and hospitalizations but assist government preparedness for the next pandemic, particularly an influenza pandemic, by balancing vaccine supply/demand at higher levels. influenza disease is an annual public health threat with or without the concurrent pandemic. influenza vaccination is an ongoing public health tool to reduce disease now and in the future. the potential for coronavirus to persist or resurge during the upcoming influenza season is generating significant demand for influenza vaccines, which may outstrip available supply, particularly early in the season, with limited time and ability to produce additional vaccine. there is a current need to reconsider the timing of vaccination and differentiation among influenza vaccines to ensure that the best vaccine is available for each member of the population. increasing influenza vaccine uptake within the constraints of social distancing requirements may also require modification of timing, location, and provider -as the australian example illustrates. clear and succinct communications designed by multiple stakeholders (including industry), which are then delivered cohesively and with repetition, should be an annual component to influenza preparation. policy changes helping to drive increased demand are welcomed and should be sustained in the interests of public health and as an additional pandemic preparedness measure. planning for the - season must begin now given the lead times required for the manufacturing of influenza vaccine. no potential conflicts of interest were disclosed. stepping up flu vaccine supply -seqirus to supply additional two million flu vaccines to australia what if flu and covid- overlap? australia is trying to avoid that scenario. healthline. researchers report % covid- co-infection rate. center for infectious disease and policy (cidrap) european centre for disease prevention and control (ecdc): factsheet about seasonal influenza disease burden of influenza. estimated range of annual burden of flu in the u.s. since higher flu vaccination rates could help expose new viruses like covid- earlier, expert says. the guardian flu shot makers gear up-and get creative-for a critical vaccination season world health organization. regional office for europe. influenza vaccination coverage and effectiveness key: cord- - a u uux authors: moss, ronald b title: prospects for control of emerging infectious diseases with plasmid dna vaccines date: - - journal: j immune based ther vaccines doi: . / - - - sha: doc_id: cord_uid: a u uux experiments almost years ago demonstrated that injections of a sequence of dna encoding part of a pathogen could stimulate immunity. it was soon realized that "dna vaccination" had numerous potential advantages over conventional vaccine approaches including inherent safety and a more rapid production time. these and other attributes make dna vaccines ideal for development against emerging pathogens. recent advances in optimizing various aspects of dna vaccination have accelerated this approach from concept to reality in contemporary human trials. although not yet licensed for human use, several dna vaccines have now been approved for animal health indications. the rapid manufacturing capabilities of dna vaccines may be particularly important for emerging infectious diseases including the current novel h n influenza a pandemic, where pre-existing immunity is limited. because of recent advances in dna vaccination, this approach has the potential to be a powerful new weapon in protecting against emerging and potentially pandemic human pathogens. throughout recorded history, infectious diseases have plagued human existence. one effective approach to limiting these diseases has been vaccination. for example, in a recent report by roush and colleagues at the u.s. centers for disease control and prevention (cdc), ever since the introduction of vaccines the incidence of infectious diseases like diphtheria, mumps, pertussis, tetanus, hepatitis a and b, haemophilus influenza and varicella zoster has declined by more than % in the u.s [ ] . furthermore, after the introduction of vaccines, large scale transmission of measles, rubella, and polio has been eliminated in the u.s., while smallpox has been eradicated worldwide. however, new emerging infectious pathogens such as hiv (human immunodeficiency virus), sars coronavirus (severe acute respiratory syndrome virus), and highly pathogenic avian influenza (h n ) viruses have adapted strategies to rapidly change their genetic compositions. as the influenza pandemic of (h n ) killed approximately to million people worldwide, massive disease and death is similarly feared from newly emerging pathogens. in addition, the current novel swine derived h n pandemic further exemplifies the need for a rapid and effective vaccine against emerging pathogens [ ] . thus a vaccination strategy to control emerging diseases will require a more effective and rapid response than available from conventional approaches such as live-attenuated vaccines, inactivated vaccines, or protein subunit vaccines. plasmid dna vaccines, as reviewed in this article, may be an option to effectively combat current emerging infectious diseases. almost years ago, malone and felgner at vical incorporated, and wolff and colleagues at the university of wis-consin, demonstrated that mrna and closed loops of double-stranded dna (plasmids) injected into muscle tissue could be taken up by cells at the administration site (transfection) resulting in the production (expression) of proteins not normally made by the host cell [ ] . it was soon realized that this approach could be utilized for both gene therapy as well as vaccine applications, and thus the field of dna vaccines was born. shortly after these original observations, many groups including those led by liu and colleagues at merck research laboratories [ ] , weiner and colleagues at university of pennsylvania [ ] , johnston and colleagues at university of texas [ ] , robinson and colleagues at university of massachusetts [ ] , and hoffman and colleagues at naval medical research center [ ] , demonstrated that immunization with dna could result in the production of foreign proteins or antigens that stimulate the immune system resulting in protection from or amelioration of infectious diseases in animal models. development in this area has greatly advanced over the years and human clinical trials of dna vaccines have now been conducted against various infectious pathogens including the malaria parasite, dengue viruses, cytomegalovirus (cmv), ebola virus, seasonal influenza viruses, avian or pandemic influenza viruses, west nile virus (wmv), sars coronavirus, hepatitis b virus, and hiv. the precise mechanism of the induction of immunity after pdna vaccination is complex [ ] and multi-factorial ( figure ). it is thought that after immunization, transfected muscle cells may produce antigen or foreign proteins that then directly stimulate b cells of the immune system, which in turn produce antibodies. transfected muscle cells could possibly transfer the antigen to so-called antigen presenting cells (as demonstrated by cross priming) which then transport the proteins via distinct pathways (the mhc i for cd +t cells or mhc ii for cd +t cells) that result in the display of different processed fragments of expressed proteins (antigens). finally, direct transfection of antigen presenting cells (such as dendritic cells) with subsequent processing and display of mhc-antigen complexes may also occur. because the process of antigen production by host cells after dna vaccination mimics the production of antigens during a natural infection, the resulting immune response is thought to be similar to the type induced by pathogens. indeed, dna vaccination generates antigens in their native form and with similar structure and function to antigens generated after natural infection. a plasmid used in dna vaccination ( figure ) contains a gene encoding an antigen of the target pathogen (immunogen gene). expression of the protein antigen is "turned on" in the host cell by a promoter, and "turned off" by a terminator (a polyadenylation signal sequence, generally referred to aspoly-a). other genes such as the bacterial origin of replication sequence and an antibiotic resistance gene are incorporated for manufacturing purposes. the resulting plasmid is a stable, self-contained unit that can be manufactured by consistent and scalable bacterial fermentation and purification processes. production of dna vaccines starts with e. coli cells which are transformed with the plasmid of interest. these cells are grown and stored frozen in a stock of vials called a master cell bank. growth of the e. coli is typically done via a fermentation process similar to that used in the manufacturing of certain alcoholic beverages. the recovery process then requires lysis of the cells, in order to release the plasmid retained within the e. coli cells. dna is then purified using various chromatographic methods. dna vaccination has many advantages compared with conventional vaccine approaches (appendix ) particularly in the setting of protecting against potentially lethal emerging infectious diseases. protecting against a particular pathogen may require immunity to more than one component of the organism and may require stimulation of different components of the host immune system. traditionally, most preventive vaccines have relied on antibodies as the main correlate of protection, components that prevent infection or disease. however, t cells play an important role in controlling disease for established infection. conventional vaccines based on whole pathogens typically induce immune responses against a number of irrelevant components of the organism. subunit protein vaccines target individual components of the pathogen and usually only stimulate antibodies. dna vaccines can accommodate a combination of different genes that code for different antigens from one or more different pathogens. this can result in the generation of broad immunity to multiple protein antigens. dna vaccines have also been observed to stimulate both antibody and t cell arms of the immune system including those that are specialized to kill viruses or cancer cells (via cytotoxic or killer t cells). a significant advantage, especially for emerging pathogens, is that dna vaccines do not require the handling of potentially deadly infectious agents. in addition they have a significantly shorter production time, something paramounrt with an ongoing pandemic. overall, a well-tolerated safety profile has been observed in human clinical trials of dna vaccination [ ] . early in the development, there were numerous theoretical safety concerns regarding dna vaccines. in particular, there were concerns about the fate of the injected genes and the potential for insertion into the host dna possibly result- ing in the uncontrolled stimulation of other genes such as those that may cause cancer. these concerns have dissipated over the years based on thousands of human subjects who have undergone dna vaccination or plasmidbased gene therapy. furthermore, in pre-clinical safety studies in animals, the potential for plasmid integration into the host genome has been shown to be negligible and several orders of magnitude below the spontaneous mutation rate that occurs naturally in mammalian genes [ ] . there were also early concerns regarding the induction of autoimmune reactions, as dna may be considered as a self antigen to the host. increased rates of autoimmunity or anti-dna antibodies such as those observed in systemic lupus erythematosis have not been observed in clinical trials of dna vaccination [ ] . interestingly, as plasmid dna vaccines are noninfectious, significant immune responses are not developed against the plasmid itself. therefore only specific immune responses to the expressed antigen are stimulated with this approach. in contrast, with viral vectors such as adenoviruses, pre-existing and post vaccination immunity to the vector itself can be generated thereby limiting the pathogen specific immune response in contrast, dna vaccination can be repeated without diminishing the specific immune response. this may be a pertinent beneficial aspect of dna immunization, particularly in light of recent clinical trials in humans using viral vector-based vaccines, such as the common cold adenovirus, where immunity to the vector itself has been raised as a potential safety issue [ , ] . in summary, the potential risks of dna vaccines appear to be minimal based on safety data from human clinical trials in thousands of subjects. over the years, much progress has been made in optimizing dna vaccine immunogenicity. recent progress has targeted many different aspects of dna vaccination which has successfully resulted in improved immune responses (appendix ). with regard to the plasmid itself, significant advances have been made in optimizing the genetic sequence of the encoding gene as well as other related components. coadministration of the plasmid that encodes the pathogen along with other genes that encode for immune stimulating substances such as cytokines or chemokines has also been used to further enhance the immune response of certain dna vaccines [ ] . in some cases, once various components of the plasmid have been optimized, the encoded protein may be sufficiently immunogenic without the need for additional components (unformulated or "naked" dna) such as adjuvants. one of the earliest trials in humans of an unformulated or "naked" dna vaccine was one that targeted the malaria parasite. wang and colleagues at the naval medical research institute immunized subjects with a plasmid that encoded naturally occurring forms of malaria proteins [ ] . in this trial, more than half of the subjects were shown to have cells that can kill or lyse malaria infected cells (cytotoxic t cell or killer cells). in a more recent clinical trial, a dna vaccine optimized for human expression and encoding modified forms of west nile virus proteins was studied by martin and colleagues at the national institute of health (nih) and demonstrated that the vaccine stimulated antibodies that inhibited the virus (neutralizing antibodies) in all individuals receiving the vaccination regimen [ ] . this unformulated dna vaccine appears to induce a similar level of immune responses to those observed in vaccinated horses protected from wnv. in addition, a recent clinical trial by these same nih researchers tested an optimized but unformulated dna vaccine for sars coronavirus and demonstrated neutralizing antibodies in all subjects who received three doses of the vaccine [ ] . another area of research to enhance the immune response to dna vaccines is related to the route of administration. although intramuscular injection is the predominant route of vaccine delivery, other routes of administration have also been studied such as intradermal delivery. it is conceivable that intradermal delivery, which is a more superficial injection, may result in better transfection of antigen presenting cells, particularly a type of cell called dendritic cells. the mode of delivery may also be a pertinent factor in eliciting the proper immune response after dna immunization. needle and syringe is the predominant method to deliver both dna vaccines as well as conventional vaccines. however, roy and colleagues at powdermed (now pfizer incorporated) have used dna precipitated onto gold particles which are driven into to skin with a blast of pressurized gas, and called this approach "particle-mediated epidermal delivery" (pmed). in one clinical study by roy and collaborators using pmed, individuals exhibited potent antibody responses to a hepatitis b dna vaccine [ ] . in another study, drape and colleagues at pow-dermed also observed strong antibody responses to the influenza virus dna vaccine with the pmed approach [ ] . another approach for the delivery of dna vaccines is the use of needle-free devices. needle-free injection of dna vaccines has been utilized in numerous clinical studies and appears to be well-tolerated and may have some advantages of further augmenting the immune response to dna vaccination. for example, nabel and colleagues at the nih injected individuals intramuscularly with a sixplasmid unformulated dna vaccine for hiv (env a, env b, env c, subtype b gag, pol, and nef) with the needle-free device (biojector ® ) and observed hiv specific t-cell immune responses in over % of individuals [ ] . similarly, this same group at the nih completed a recent study of an ebola dna vaccine also using this same needle-free device. they demonstrated that ebola-specific antibody and cd + t-cell immune responses were elicited in all individuals who received the three-dose vaccination regimen [ ] . another novel mode of enhancing dna vaccines has been a more invasive technique called electroporation. this method involves administration of brief electrical pulses of various voltages, after injection of a dna vaccine, in order to enhance the uptake of dna, presumably through the formation of transient pores in the muscle cell membrane. many groups have shown encouraging results using electroporation in animals. one of the first groups to use this technique, ulmer and colleagues, at chiron corporation, demonstrated that antibody and t-cell responses to the hiv protein gag encoded in a dna vaccine were enhanced by electroporation in rhesus macaques [ ] . more recently, studies by pavlakis and colleagues at the nih have also noted that dna potency measured by t-cell responses to the hiv protein gag was augmented in nonhuman primates using electroporation [ ] . although encouraging in the strong magnitude of responses generated in animals, the wide clinical applicability of electroporation in humans in relation to tolerability remains to be determined. some researchers have used plasmid dna in what has been called a heterologous "prime-boost" vaccination approach. this method involves delivery of one or more plasmid dna vaccine priming doses followed by a boost with a viral vector (such as adenovirus) which codes for the same antigens. in the prime-boost setting, dna vaccination plays an important role in priming different types of t-cells (cd + and cd + t-cells) specific for various proteins. in studies, for example, by nabel and colleagues at the nih, dna vaccination with plasmids encoding hiv antigens followed by a boost with an adenoviral vector carrying the same hiv gene sequences, resulted in stronger t-cell responses compared with adenoviral vector or dna vaccine alone [ ] . similarly, pantaleo and colleagues at the university of lausanne have shown that a dna vaccine for hiv followed by a boost with a poxvirus vector (nyvac) resulted in stronger immune responses compared to immunization with pox vector alone [ ] . in another prime-boost study, jacobson and colleagues, at the university of california at san francisco, immunized subjects with a dna vaccine for cmv who were then boosted with a live-attenuated cmv virus (towne strain). faster and stronger virus-specific t-cell responses were observed in the group of subjects that received the dna and towne strain compared with a group of subjects that received the towne strain alone [ ] . an area of potentially paramount importance for dna vaccines is formulations and adjuvants. adjuvants are common to most licensed vaccines and are included to potentiate the immune responses elicited by vaccination. for dna vaccines, various delivery systems and adjuvants have been tested. one of the earliest promising adjuvants for plasmid dna vaccines was poly-lactide coglycolide (plg), cationic microparticles. ulmer and colleagues at chiron corporation evaluated hiv dna vaccines formulated with plg microparticles and found strong antibody and t-cell responses in macaques [ ] . poloxamers represent another class of adjuvants tested with dna vaccines. some poloxamers are nonionic block copolymers, and when combined with a cationic surfactant, bond with dna to form small particles. a study by wloch and colleagues at vical incorporated examined dna immunizations of human volunteers with cytomegalovirus (cmv) plasmids formulated with a specific poloxamer adjuvant. in that study cmv-specific t-cell responses were detected in a majority of cmv sero-negative individuals who were vaccinated [ ] . vaxfectin ® is another example of a delivery system and adjuvant for dna vaccines that has been recently tested in humans. vaxfectin ® is a cationic lipid-based adjuvant that bears a positive charge that binds electrostatically to negatively charged dna. studies in animals also demonstrated that vaxfectin ® -adjuvanted dna vaccines can be protective against lethal viral challenges. for example, webby and colleagues at st. jude children's research hospital and vical incorporated, immunized ferrets with three plasmids containing dna components of the h n pandemic influenza virus formulated with vaxfectin ® [ ] . after one or two immunizations, all animals were completely protected from lethal pandemic influenza virus challenge, while unvaccinated control animals died. similarly, griffin and colleagues at johns hopkins immunized juvenile and infant rhesus macaques by intramuscular and intradermal routes with measles antigen encoding plasmids formulated with vaxfectin ® [ ] . all of the vaccinated monkeys developed strong and durable neutralizing antibodies and they were challenged with high doses of measles virus after one year. all of the unvaccinated control animals developed viremia and became ill with rashes in contrast to the vaccinated ani-mals which remained healthy and had no detectable virus levels. lastly, the first human clinical trial of a dna vaccine formulated with vaxfectin ® has been completed with plasmids that encode pandemic influenza virus antigens (h n ). the preliminary results reported from this trial by smith and colleagues at vical incorporated suggest that vaccination with h dna plasmids formulated with this adjuvant were well-tolerated and stimulated strong h antibody responses in up to % of subjects [ ] . notably, the antibody response rate and safety profile observed in this trial are comparable to most conventional proteinbased vaccines for pandemic influenza. although dna vaccines have yet to be approved for human use, three have already been approved for animal health. as noted in appendix , the first dna vaccine approved for animal health was one that protected horses against wnv. wnv is a mosquito-borne virus, which causes encephalitis or inflammation in the brain of infected animals and humans. prior to approval of a vaccine, approximately one-third of the horses infected with wnv would die or be euthanized. the wnv dna vaccine, developed by fort dodge laboratories, was approved by the u.s department of agriculture (usda) in after the demonstration of safety and efficacy [ ] . a dna vaccine has also been recently approved to prevent a fatal viral disease that afflicts salmon, called infectious hematopoietic necrosis virus. in mid- , an epidemic occurred in atlantic salmon killing up to % of the fish. scientists at aqua health, a unit of novartis in canada, conducted a field trial by immunizing millions of salmons with a single dose of dna vaccine encoding for a protein of the virus [ ] . the vaccine was approved in based on the results of this trial that demonstrated that the vaccine, called apex-ihn, protected the fish from death without adverse effects. additionally, a therapeutic dna vaccine designed to treat dogs with skin cancer (melanoma) was granted conditional approval in . this vaccine was developed through a collaboration of memorial sloan-kettering cancer center (mskcc) and merial ltd. canine melanoma is an aggressive form of cancer. dogs with melanomas that have gone beyond initial stages typically have a lifespan of one to five months with conventional therapies. in addition, the cancer is often resistant to chemotherapy. in a study of the dna vaccine conducted by mskcc, many dogs who received the vaccinations lived beyond the average month survival [ ] . based on the significantly extended survival, the usda gave this dna vaccine conditional approval in . this is the first therapeutic vaccine approved by the u.s. government for the treatment of cancer in animals or humans. perhaps most relevant to emerging infectious diseases, dna vaccines have the distinct advantage of a rapid development time, are non-infectious, and have a well-defined manufacturing process. dna vaccines contain no infectious components and can be produced safely without the handling of hazardous infectious agents. furthermore, there is a well-defined analytical process for the manufacturing of all dna vaccines, which is universally applicable to any dna vaccine. vaccination is an important component of a response to potential pandemics such as avian influenza. according to the world health organization (who), since november , approximately cases of human infection with highly pathogenic avian influenza a (h n ) have been reported worldwide [ ] . pandemic (h n ) influenza virus has evolved into at least distinct clades or subclades. as noted earlier, the emergence of triple-reassortment swine influenza with limited cross reactivity antibody responses after vaccination with seasonal influenza vaccines suggests the need to rapidly produce new vaccines to this particular emerging virus [ ] . the manufacturing time of conventional protein-based vaccines may be excessive, as they typically require growth in egg or cell cultures, which involve a relatively slow production time. dna vaccines, in contrast, have estimated vaccine production times that can be months earlier, as only the dna sequence is required and the manufacturing process is standard (figure ) [ ] . dna vaccines therefore have a unique advantage of large scale production for human use in a relatively streamlined period of time. in the case of potentially fatal emerging pathogens, reducing the production time of an effective vaccine may be critical in preventing spread of infection and death. in summary, based on recent advances in enhancing protective immune responses and a well-tolerated safety profile in humans, plasmid dna vaccines have the potential to become an integral part of the arsenal dedicated to enhancing human health by preventing diseases through immunization in our ever changing microbial environment. the author declares that they have no competing interests. ability to immunize against multiple antigens and/or pathogens vaccine-preventable disease table working group: historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states implications of the emergence of a novel h influenza virus direct gene transfer into mouse muscle in vivo heterologous protection against influenza by injection of dna encoding a viral protein gene inoculation generates immune responses against humanimmunodeficiency virus type genetic immunization is a simple method for eliciting an immune response protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid dna plasmid dna malaria vaccine: the potential for genomic integration after intramuscular injection dna vaccination: antigen presentation and the induction of immunity preclinical and clinical safety studies on dna vaccines. huamn vaccines ensuring safety of dna vaccines karlsson hedestam gb: dna vaccines: recent developments and future possibilities human versus hiv: round defeat in aids vaccine development human immunodeficiency virus type vaccine development:recent advances in the cytotoxic t-iymphocyte platform "spotty business dna vaccines: ready for prime time? hoffman : induction of antigen -specific cytotoxic t lymphocytes in humans by a malaria dna vaccine a west nile virus dna vaccine induces neutralizing antibody in healthy adults during a phase clinical trial a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial induction of antigen-specific cd + t cells, t helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis b virus dna vaccine epidermal dna vaccines for influenza is immunogenic in humans study team: safety and immunogenicity of a gag-pol candidate hiv- dna vaccine administered by a needle-free device in hiv- -seronegative subjects a dna vaccine for ebolavirus is safe and immunogenic in a phase i clinical trial ulmer lb: enhancement of dna vaccine potency in rhesus macaques by electroporation increased immune responses in rhesus macaques by dna vaccination combined with electroporation vrc study team: phase i clinical evaluation ofa six-plasmid multiclade hiv-l dna candidate vaccine pantaleo giuseppe: an hiv- clade c dna prime, nyvac boost vaccine regimen induces reliable, polyfunctional, and long-lasting t cell responses a cmv dna vaccine primes for memory immune responses to live-attenuated cmv (towne strain) induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus dna vaccines safety and immunogenicity of a bivalent cytomegalovirus dna vaccine in healthy adultsubjects plasmid dna-based vaccines protect mice and ferrets against lethal challenge with anietharnl / (h nl) influenza virus use of vaxfectin adjuvant with dna vaccine encoding the measles virus hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques against measles virus vaxfectin-formulated pandemic influenza dna vaccines: preliminary clinical results. paper presented at: ibc life sciences' next generation vaccines; national harbor fort dodge animal health care products mmwr rapid-response vaccines-does dna offer a solution the author would like to thank my colleagues at vical incorporated including vijay samant, alain rolland, alan engbring, jenny chaplin, and larry smith for their helpful critical comments. key: cord- -h ma u authors: gellin, bruce g.; qadri, firdausi title: preparing for the unpredictable: the continuing need for pandemic influenza preparedness date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: h ma u nan preparing for the unpredictable: the continuing need for pandemic influenza preparedness ebola, zika, middle east respiratory syndrome (mers), chikungunya, severe acute respiratory syndrome (sars). the cadence of emerging infectious diseases that travel, transmit and stand ready to spark a public health emergency of international concern seems to be accelerating. these infectious diseases cause significant human suffering, can overwhelm unprepared health care systems, and significantly disrupt societies and economies. it is estimated that a severe pandemic similar to the ''spanish flu" could nowadays cost as much as % of global gross domestic product; the global cost of even a moderately severe pandemic could be as much as us$ billion, or . % of global income [ ] . given the health, social and economic impact of infectious diseases with pandemic potential, there has been much attention to these microbial threats and, not surprisingly, the first question is always, ''is there a vaccine?" but too often we find that the vaccines we need most are not available when we need them most. a number of efforts are focused on better anticipating the vaccines that we may need [ ] [ ] [ ] [ ] [ ] and a global emergency fund has been proposed to support accelerated vaccine development [ ] . the current headlines about zika virus have replaced last year's headlines about ebola. . .and next year's headlines will undoubtedly feature something new. imperfect crystal balls fail to guide us with certainty. but looming in the background is the ever present threat of influenza, the only microbe that causes regular widespread epidemics around the world. not so exotic and all too familiar -casually referred to as the ''flu" -influenza is the virologist's trojan horse, as it readily and rapidly mutates (drift), has the ability to swap genetic elements from influenza viruses with origins in other species (shift) and, as an rna virus lacks repair and self-correcting mechanisms that are the basis of its genetic instability and makes it both dangerous and unpredictable. a reassortment leading to an extremely virulent and readily transmissible pandemic virus is inevitable. of the many things that need to be in place to prepare for and respond to the next influenza pandemic, vaccines -together with the capacity to mount a timely global vaccination effort -are paramount. a pandemic can only be brought under control through a shift in the population from susceptibility to immunity. but as we learned in the influenza pandemic, although our response time has improved, a significant shift in approach is needed if an effective vaccine is to be in place before the next pandemic emerges. with that goal in mind, the science of influenza and of vaccine development needs to be looked at with fresh eyes [ ] . the holy grail is an influenza vaccine that provides a broader and more durable immune response than the natural infection and that can provide population immunity to pandemic influenza viruses before they emerge. a number of efforts are now focused on developing such a ''universal" influenza vaccine; however, the scientific challenges involved are enormous [ , ] . until such a universal influenza vaccine becomes available, global influenza vaccine production capacity needs to be ready to respond when the next pandemic emerges. vaccines obviously need to be safe and effective, but they must also be available and affordable. while vaccine supply is clearly essential for a large-scale vaccination programme, the logistics and supporting policies needed to conduct effective and efficient vaccination campaigns in a range of community settings also need to be addressed and the needs of high-risk and vulnerable populations and of low-and middle-income countries accommodated. ultimately, the success of a vaccination programme will hinge not only on the technical aspects of its implementation, but on the demand for it among the population; this underscores the need to understand the factors that promote vaccine acceptance [ , ] . within this broad framework, and with a focus on the role of vaccines in mitigating the impact of both seasonal and pandemic influenza, who established in the global action plan for influenza vaccines (gap) as ''a comprehensive strategy to reduce the present global shortage of influenza vaccines for seasonal epidemics and pandemic influenza in all countries of the world" [ ] . the papers in this special issue of vaccine, provide a foretaste of the in-depth review to come in november , when who will host the third and final consultation on gap as the programme, as it exists today, comes to close. the background papers prepared for this consultation are available on the who website (http:// www.who.int/influenza_vaccines_plan/news/gap _nov /en/). while this event is advertised as a programme review, in reality as an examination of the progress achieved during the decade, it should also serve to direct the global community on the path forward for the work that remains to achieve global pandemic influenza vaccine and vaccination preparedness. this review, combined with the current rethinking of global preparedness for emerging threats, the review of the pandemic influenza preparedness (pip) framework [ ] , and who's restructuring of its approach to global health emergencies the global action plan for influenza vaccines was structured around three broad objectives that underpin pandemic influenza vaccine and vaccination preparedness: -evidence-based increase in seasonal influenza vaccine use; -increase in influenza vaccine production capacity and regulatory capacity; -research and development for improved influenza vaccines. the articles contained in this issue not only relate to the gap objectives but also reflect the principles and goals of pip and highlight the synergy of these two efforts. pip's pandemic preparedness goal of increasing the access to vaccines and other pandemicrelated supplies by developing countries (and the critical importance of improving the sharing of influenza viruses with human pandemic potential toward that end), will ultimately depend on influenza vaccine production capacity in place when the next pandemic occurs. these articles present a variety of perspectives on the system that will develop, produce, regulate, distribute and evaluate pandemic vaccines and mass vaccination programmes. they have been selected to represent some of the many elements of the system that will be necessary for a pandemic vaccine response -including clinical trials of new vaccines, new vaccine production platforms, national regulatory systems, implementation of policy recommendations for vaccine use, and assessment of the sustainability of the global influenza vaccine manufacturing capacity that gap has supported. progress has already been significant and measurable. one clear example is that global production of seasonal influenza vaccine increased from less than million doses in to nearly . billion doses in [ , ] . yet, there is a pressing need for additional burden of influenza illness studies to provide the essential data that will be looked to by policymakers to assess the role of vaccines in national influenza control strategies. it is the implementation of these policies that will determine demand. without a concomitant increase in global demand for seasonal influenza vaccine, the capacity that will produce the world's pandemic vaccines that gap has stimulated cannot be sustained [ ] . at the end of the day there is the expectation that the efforts and investments made to prepare for a pandemic will result in the timely and equitable availability of a vaccine that is safe and effective, and that can be used in vaccination campaigns before the majority of the global population is exposed to the virus. however, a continuing dedicated effort is needed to ensure that all the elements of the system that will allow this to happen are in place before we need them. because we don't know when that day is, the global community must continue with speed and focus. and, as who develops a new structure to respond for the call to be prepared for all hazards, in its complex organizational chart pandemic influenza preparedness must remain front and center. the inclusive cost of pandemic influenza risk (working paper ) an r&d blueprint for action to prevent epidemics. accelerating r&d and saving lives. geneva: world health organization report from the world health organization's product development for vaccines advisory committee (pdvac) meeting antibiotic resistance threats in the united states the coalition for epidemic preparedness innovations (cepi) establishing a global vaccine-development fund (perspective) the compelling need for game-changing influenza vaccines edu/compelling-need-game-changing-influenza-vaccines> advances in the development of influenza virus vaccines induction of unnatural immunity: prospects for a broadly protective universal influenza vaccine (commentary) global vaccine action plan - . geneva: world health organization sage working group on vaccine hesitancy. how to deal with vaccine hesitancy? global action plan for influenza vaccines. geneva: world health organization pandemic influenza preparedness (pip) framework. geneva: world health organization melbourne: peter doherty institute for infection and immunity global action plan for influenza vaccines a literature review to identify factors that determine policies for influenza vaccination key: cord- -y ji k authors: connell, anna r.; connell, jeff; leahy, t. ronan; hassan, jaythoon title: mumps outbreaks in vaccinated populations—is it time to re-assess the clinical efficacy of vaccines? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: y ji k history illustrates the remarkable public health impact of mass vaccination, by dramatically improving life expectancy and reducing the burden of infectious diseases and co-morbidities worldwide. it has been perceived that if an individual adhered to the mmr vaccine schedule that immunity to mumps virus (muv) would be lifelong. recent mumps outbreaks in individuals who had received two doses of the measles mumps rubella (mmr) vaccine has challenged the efficacy of the mmr vaccine. however, clinical symptoms, complications, viral shedding and transmission associated with mumps infection has been shown to be reduced in vaccinated individuals, demonstrating a benefit of this vaccine. therefore, the question of what constitutes a good mumps vaccine and how its impact is assessed in this modern era remains to be addressed. epidemiology of the individuals most affected by the outbreaks (predominantly young adults) and variance in the circulating muv genotype have been well-described alluding to a collection of influences such as vaccine hesitancy, heterogeneous vaccine uptake, primary, and/or secondary vaccine failures. this review aims to discuss in detail the interplay of factors thought to be contributing to the current mumps outbreaks seen in highly vaccinated populations. in addition, how mumps diagnoses has progressed and impacted the understanding of mumps infection since a mumps vaccine was first developed, the limitations of current laboratory tests in confirming protection in vaccinated individuals and how vaccine effectiveness is quantified are also considered. by highlighting knowledge gaps within this area, this state-of-the-art review proposes a change of perspective regarding the impact of a vaccine in a highly vaccinated population from a clinical, diagnostic and public perspective, highlighting a need for a paradigm shift on what is considered vaccine immunity. muv is an enveloped, non-segmented, negative-sense, single stranded rna virus that varies between a spherical and pleiomorphic shape of ∼ nm ( - nm) ( , ) . muv is responsible for an acute viral infection, spread by respiratory droplets (via coughs, sneezes) and urine ( , ) . with an incubation period of - days, muv replicates in the nasopharynx and regional lymph nodes, with a secondary viremia occurring late in the incubation period ( , ) . muv can be detected from saliva up to days prior, and as late as days after clinical onset of parotitis ( ) . the muv genome of seven genes consists of , nucleotides, and encodes six structural proteins and at least two non-structural proteins; the nucleocapsid protein (np), v protein (v), phosphoprotein (p), matrix (m) protein, fusion (f) protein, small hydrophobic (sh) protein, hemagglutininneuraminidase (hn) protein, and large (l) protein. the role of the i protein is not known ( , , ) . the sh gene is the most variable region of the muv genome; a - % intravariation and - % inter-variation has been documented ( ) . this gene is used in molecular phylogeny for genotyping and to identify transmission patterns in populations ( ) . despite being serologically monotypic, muv genotypes (a to l) have been described to date (muv genotypes e and m are omitted, as the muv previously assigned to these groups were later re-assigned) ( , , ) . the geographic distributions of the muv genotypes varies worldwide but can co-circulate and thus drive temporal shifts in their distribution. genotype a was frequently isolated in europe until the 's. currently genotypes c, d, e, g, and h are prevalent in europe and the united states of america (usa) whereas genotypes b, f and i are more common in asian countries ( table ) ( , , , ) . since numerous mumps vaccines have been developed worldwide, varying in efficacy and safety profiles but primarily consisting of an attenuated live muv without an adjuvant ( , ( ) ( ) ( ) . currently in europe and for the majority of the g countries who have a mumps vaccine in their immunization schedule (table ) , the mumps vaccine is included as part of the trivalent measles, mumps rubella (mmr) vaccine, and is primarily administered in two doses ( , ) . the jeryl lynn (jl) vaccine, derived from the genotype a muv strain was first developed in the usa and has been used extensively in the united kingdom (uk), ireland and usa since it was licensed in ( ) . derived from a single clinical sample, and propagated in a chick embryo cell culture, two viral isolates (jl and jl ) are present, differing by ∼ nucleotides and amino acid changes ( ) ( ) ( ) . the rit mumps vaccine, developed from the dominant viral component (jl ) in the jl vaccine strain appears to have comparative safety and efficacy (seroconversion) profiles to the jl vaccine strain ( , ( ) ( ) ( ) . however, since no controlled clinical trials of efficacy have been published to compare the two doses of the two vaccines, the clinical significance of this observation is not known. despite the integration of the mmr vaccine into childhood immunization programs, cyclical outbreaks [defined as two or more cases linked by place and time ( ) ] of muv have been documented in several highly vaccinated populations such as ireland and the united kingdom ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . between august -and january , , mumps cases were notified in ireland, primarily affecting individuals between the ages of - years. of the % of cases that stated vaccination status, % had received two doses of the mmr vaccine ( ) . an upsurge of mumps cases has also occurred in states of the united states over the last decades, primarily affecting people between and years in close contact/shared settings ( ) . in indiana, . % of mumps cases ( . % of university affiliated and % of community cases) had documented evidence of mmr vaccination ( ) . this results in a significant resource burden for public health departments to control. several reviews, both observational and systematic have demonstrated the clinical benefit of a mumps vaccine ( , ) , the pathogenesis and genomic diversity of the muv ( , , ) and the epidemiology surrounding the outbreak ( , , ) . it is not clear why these mumps outbreaks occur, although it has been alluded to be due to a number of interrelated factors, such as sub-optimal vaccine uptake ( , , ) , primary or secondary vaccine failure or failure of the mumps vaccine to protect individuals from infection (vaccine efficacy) ( ) (figure ). history depicts the remarkable public health impact of mass vaccination. previously inevitable childhood diseases with potentially debilitating or deadly outcomes have seen their rates plummet worldwide or become successfully eradicated. immunizations of vaccine preventable diseases are estimated to prevent ∼ - million deaths per annum and increase life expectancy by ∼ years ( ) . more recently there has been a shift in the public and media perception of vaccines to their safety, which has facilitated outbreaks such as mumps ( ) . organized opposition to vaccinations has a long history; public outcry and resistance following the introduction of the smallpox vaccine in the nineteenth century led to the introduction in england of the vaccination act of ( ) . with one in eight children in the usa under the age of currently thought to be unvaccinated due to parental choice, the who now considers vaccine hesitancy as one of the ten threats to global health in ( ) . vaccine hesitancy, defined as a "delay in acceptance or refusal of vaccines despite availability of vaccination services" involves a multitude of social, political, cultural and emotional factors in highly vaccinated, western populations ( , ) . one of the main issues is the parental concerns regarding the perceived risk of a vaccine to their child (such as timing/schedules of vaccines, associated pain of administration, and potential adverse effects) vs. the disease morbidity and mortality associated with the vaccine preventable disease ( , ) . the retracted paper published in the lancet in ( ) and "anti-vaccination" opinions on social media have also contributed to the persistent and insistent misinformation ( ) , despite vast follow-up epidemiological studies showing no relationship between the mmr vaccine and autism, or differing cognitive development/intelligence ( ) ( ) ( ) . however, the resultant reaction of the public led to the uptake of the first mmr vaccine falling sharply from , with uptake falling to below % in ( , ) . the age demographic that are experiencing the most cases of mumps in ireland during the current ongoing outbreak would have been scheduled to have received the first mmr vaccine between and . nevertheless, no deductions can be made, due to the lack of vaccination status information provided with reported cases ( ) . frontiers in immunology | www.frontiersin.org heterogeneity of immunization coverage in specific populations or geographic locations of susceptibility is also becoming an important epidemiological issue in maintaining proficient population immunity for mumps ( , , ) . the who recommends a > % mmr vaccine coverage for herd immunity. maintenance of such coverage is well-demonstrated in finland, where a country-wide -dose mmr vaccination program initiated in the 's eliminated measles, mumps and rubella within years ( , ) . recent publications from around the world indicate that the level of mmr vaccine uptake is far lower than what is recommended [reviewed in ramanathan et al. ( ) ] ( , ( ) ( ) ( ) ( ) . of the g nations that implement a mumps vaccine within their vaccination schedule, only countries have maintained vaccine coverage levels of > % (table ) . however, poor uptake/incomplete vaccination alone may not be the only issue relating to mumps outbreaks. in the netherlands, mumps outbreaks still occurred with an overall herd immunity threshold of - %, and where and % received the first and second mmr at months and years, respectively ( , ) . the clinical presentation of mumps is pathognomic (bi-lateral parotitis); therefore supporting laboratory diagnosis was rarely employed in the past. as the classical symptoms of mumps are not always typical, there may have been a significant number of individuals in the past who may have been infected but were not identified as such. when mumps vaccination was introduced in , the criteria the vaccine had to meet was the proof that it was clinically effective, i.e., that it reduced the risk of disease in vaccinated individuals in real-world conditions over a set period. such an example was seen the usa; the reported cases (i.e., diagnosis of clinical symptoms) of mumps declined from > cases per , population before (pre-vaccine era) to cases per , population in , a reduction of % ( , , , ) . to note, clinical efficacy was probably based upon the reduction of the "classical bilateral presentation" rather than the milder mumps presentation. therefore, one could argue that the original vaccine efficacy for clinical manifestations was over estimated. currently the laboratory diagnosis of mumps infection in ireland is based upon two approaches: detection of mumps rna by reverse transcriptase pcr (rt-pcr) in a buccal swab containing saliva, throat swab or urine specimen, and serological detection of immunoglobulin m (igm) using a capture assay ( , ) . both approaches for diagnosis are impacted significantly by the quality and timing of sample collection post-onset of symptoms and also if the subject is mumps naïve or had received mumps containing vaccine ( , , , ) . there are challenges in using standard serological laboratory diagnostic methods to reliably confirm mumps re-infection of individuals who had been previously naturally infected or vaccinated ( , ) . briefly, vaccinated individuals re-infected with muv may only generate a weak or undetectable igm response ( ) . although a rise in igg titer may also not occur in vaccinated individuals ( , ) , numerous studies have documented a rapid, variable increase in mumps-specific igg levels, with neutralization antibody concentrations present up to months post-infection ( , , ) . therefore, reverse transcriptase-polymerase chain reaction (rt-pcr) is recommended ( , ) , and was formally introduced in as the principle diagnostic tool in ireland to detect mumps in oral fluids ( ) . rt-pcr can identify current mumps infection more effectively in vaccinated individuals than serological techniques alone as it identifies the presence of the muv vs. the immunological response (igg, igm), and has been previously shown to % correlate with viral culture results ( , ) . the case numbers of more recent mumps outbreaks should always be assessed with this question in mind; are the number of mumps cases increasing, or/and are we better at diagnosing an acute infection? the latter seems to be the most probable, as many individuals who are being tested do not present with classical symptoms. in addition to enhanced surveillance of mumps cases, further optimizations of technologies are also occurring; the utilization of next-generation sequencing demonstrated that by editing one -fold degenerate nucleotide in the forward primer and three -fold degenerate nucleotides in the probe sequence optimized the fluorescence intensity and clinical sensitivity of the real-time rt-pcr when compared to the cdc-developed and who-recommended rt-pcr target [(np) gene] leading to ∼ % increase in clinical sensitivity (i.e., ct values that were ∼ . cycles lower) ( ) . much is not known about the immunological response to the mumps vaccine strain. however, a number of young adults who were vaccinated as children over the last two decades have demonstrated an increased risk of muv infection with time, which is assumed to be related to a decline of antibodies to sub-protective levels of immunity ( , , , , ( ) ( ) ( ) ( ) . primary vaccine failure is defined as the lack of a sufficient initial antibody response to a vaccine in a recipient resulting in a lack of protective immune responses ( , ) . although this type of vaccine failure may be because of improper storage/handling or administration of the vaccine, impacting its efficacy, it may also be due to the initial immunological response of an individual to the vaccine, which is usually quantified by the presence of antibodies that should be detectable in the weeks following vaccination. primary vaccine failure was attributed to primaryschool outbreaks of both mumps and measles in ireland, which subsequently resulted in reducing the age for the second dose of mmr vaccine from - years in to - years of age ( ) . with the cyclical outbreaks occurring, it has been proposed that primary vaccine failure could again be a factor. how is a response to a vaccine determined? in pre-licensure studies of the jl and urabe mumps vaccines, high seroconversion and low failure rates were observed in children after the first vaccine dose (> and . %, respectively), demonstrating that the vaccine induced an antibody response ( ) ( ) ( ) ( ) ( ) ( ) . a more recent study by ong et al. demonstrated that a ≥ fold increase in mumps antibodies -days post-vaccination was considered to be an adequate response of immunity ( ) . vaccine effectiveness (i.e., seroconversion post-vaccination) of vaccine doses has only been conducted on the jl strain; studies provided a median vaccine efficacy of %. these studies have shown that doses of mmr were more effective (but not statistically significant) than a single mmr dose to combat the incidence of mumps infection ( , , , , , ) . mumps-specific antibodies have been detected - years postvaccination and without substantial decline for years after mumps vaccination, with the immunogenicity and efficacy of the mmr vaccine showing comparable immunogenicity levels to post-vaccination levels at years ( , ) . however, most studies of this vaccine (involving either a mumps-specific vaccine or a combined vaccine) only followed-up to - days postvaccination ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . despite few follow-up studies estimating post-vaccination antibody titers specific to the vaccine mumps strain, the evidence of seroconversion post-vaccination in a number of studies indicate that primary vaccine failure does not seem to be a significant contributor to the outbreaks that have been recently observed ( , , , , , ( ) ( ) ( ) ( ) . it has been noted that a small percentage of the population do not seroconvert post-vaccination; < % who received the mmr vaccine were seronegative - years after the first dose of mmr (n = ) ( ) . poor immune responses to primary vaccination has been shown to be a good indicator of infection susceptibility ( ) . this is in agreement with the correlation of pre-outbreak jl virus neutralization titres and elisa results being significantly lower in individuals who became infected compared to non-infected individuals ( ) . further studies of these individuals may provide insights of which immunological process are integral to develop immunity. the current methods used to determine immunity against mumps cannot discriminate between primary and secondary vaccine failure; only the timing of these tests can assess whether an individual ever mounted an immune response postvaccination or whether the response is detectable years postvaccination. primary vaccine failure encompasses the failure to mount an immune response to a dose of a vaccine, secondary vaccine failure refers to a more gradual loss of immunity after a successful initial response that occurs over a number of years post-vaccination ( ) . several factors have been proposed to be implicated with secondary vaccine failure, such as waning immunity, a lack of cross-neutralization, and natural boosting. waning immunity is defined as a decline in immunological protection proportional to time since vaccination. potential waning immunity has been documented in the current mumps outbreaks seen in europe and the usa, mostly affecting young adults within highly vaccinated populations attending tertiary education who have received two doses of the mmr vaccine in early childhood ( , , , , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . a number of studies from the usa, where a jl vaccine has been used since have demonstrated waning immunity within the population. the risk of developing clinical mumps was shown to increase by - % for every year post-mmr vaccination ( ) , with the rates of mumps infection rising from . cases per , in those who received the second dose of the vaccine within years of the outbreak, to . cases per , in those who received it over years prior. using a mathematical model with analytical limitations, a recent metaanalysis of six studies estimated that vaccine-derived immune protection to muv wanes about years post-vaccination ( ) . kennedy et al. ( ) also demonstrated a decrease of ∼ % in mumps neutralizing antibody titers over years. in contrast, other studies appear to contradict, these findings, showing no link between mumps protection and time elapsed following administration of mumps vaccine ( , , , , ) . lebaron et al. ( ) and gothefors et al. ( ) demonstrated that - % of individuals still had detectable anti-mumps antibodies ∼ years after initial vaccination. cohen et al. ( ) also demonstrated minimal antibody level decline after two mmr doses - years after second vaccination. neutralizing antibodies against the jl- vaccine strain has also been detected in ∼ % for age groups - years, % for age group - years; and % for age group + years ( ) . implementation of a third dose of the mmr vaccine has been shown to be effective as a stop gap measure in limiting disease spread in outbreak settings situations ( ) . individuals vaccinated for the third time had a % lower risk of contracting mumps, with a decreased attack rate of . vs. . cases per , when compared to those who received a second dose. more than % of those who received a third dose of the mmr vaccine showed a -fold increase in mumps antibody titers ( , , , ) . an increase in mumps igg humoral immunity was also observed post-vaccine administration. however, this immunity boost has been shown to be a transient effect, with mumps antibody titers returning to pre-third dose of mumps-vaccination levels year after vaccination. therefore, as waning immunity is thought to be an important factor facilitating mumps outbreaks, the emphasis placed on the quantity/quality of mumps-specific antibodies may need to be re-assessed. it is yet undetermined if the total loss of detectable antibodies correlates to a loss of clinical protection, as the minimal level of neutralizing antibody required for protection against mumps has not yet been defined ( ). antigenic variation and thus reduced cross-neutralization between the vaccine and circulating strains of different muv genotypes have been cited as possible explanations for mumps outbreaks in highly vaccinated populations ( , , ( ) ( ) ( ) . recent outbreaks in europe and northern america (including ireland) have shown the circulating muv during the current outbreaks to be genotype g ( , , , ) . this muv genotype was first identified in , and has demonstrated intra-genotype diversity of up to % (table ) ( , ) . the jl vaccine strain (genotype a), differs phylogenetically to the circulating muv (genotype g) ( ) . in vitro studies of the genotypic distribution and temporal shift of muv suggest that cross neutralization between wild type and vaccine genotypes may be approximately half the concentration measured against the vaccine strain ( ) . pre-infection neutralization titers in mumps positive cases were also significantly lower against genotype g vs. mumps vaccine strain, potentially due to amino acid differences in b-cell epitopes and/or n-linked glycosylation sites on the hn and also within the f protein ( ) . santak et al. ( , ) also demonstrated that conformational changes within the f protein may lead to immunological escape. despite the decline/scarcity of cross-neutralizing antibodies, different mumps vaccines used worldwide have been shown to prevent significant clinical mumps infection during outbreaks ( , ) . dependent on the strain, a - -fold variation of patient sample titers has been shown to be protective in in vitro plaque reduction neutralizations ( , , ) . although the sera of one of these studies, was collected only weeks after mmr vaccination, a time point that may not signify the concept of waning immunity and antigenic differences, several other groups have shown that the most divergent strains of muv can be neutralized in vitro with only slight variations in titers, supporting the concept that muv is serotypically monotypic ( , , , ) . epitopes of the muv that are presented to cd + t-cells have been shown to be present in not only the circulating strains of virus but also in a number of vaccine strains ( ) . in addition, lewnard et al. ( ) also found no evidence that recent mumps outbreaks were due to the emergence of muv strains escaping vaccine-driven immunological pressure. therefore, the limited data does not suggest that antigenic drift of the muv leading to diminished neutralization capacity of the vaccine strain could fully explain the recent outbreaks ( ) . further studies into the cross-neutralizing capacity of the mumps vaccine strain administered - years previously to the current circulating strain of muv in countries where outbreaks are being observed will allow better deductions to be made. it is possible that differences in the neutralization capacity of vaccine-induced antibodies against different muv strains may be more significant when levels of neutralizing antibody are low and become "overwhelmed" when the mumps viral load challenge is high ( ) . several prominent mmr/mumps vaccine studies were undertaken at a time when there was still a high prevalence of circulating wild type virus, which enabled sub-clinical boosting to occur in an individual. such natural boosting is illustrated in belarus, where a subpopulation of vaccinated individuals only had a small amount of their overall mumps igg antibody levels specific to the vaccine-strain ( ) . neutralization antibodies against iowa-g/usa (the circulating wild type virus) were also present in pre-infection plasma of all mumps cases during a recent outbreak in the us ( ) . this indicates that the mumps vaccine alone is not solely responsible for the high levels of mumps antibodies ( ) , and that longterm antibody persistence or protective efficacy data of the vaccines used may not truly reflect the current circumstance of viral transmission/circulating within a highly vaccinated population ( ) . herd immunity increases the chance for natural mumps boosting for an individual is at a minimum, reducing the potential of the frequency of mumps outbreaks ( , , ) . with less opportunity for subclinical boosting (asymptomatic response to the circulating virus), the impact of other elements of waning immunity may play an increasingly critical role in the re-emergence of mumps outbreaks ( , ) . additionally, as the heterogeneous uptake of vaccines in this modern era is leading to susceptible individuals within the community, future work will need to encompass genotyping of circulating muv to examine how impactful subclinical boosting was on early measures of vaccine efficacy in current populations. why do we consider antibodies to be the best measurement of vaccine efficacy? the evolution of an individual's immune response differs between natural infection and vaccination, in particular the difference in the affinity and specificity of an immunological marker such as antibodies ( ) . true correlates of mumps immunity after vaccination have been poorly characterized; to date, there are no reliable correlates of protection from either symptomatic mumps infection (clinical immunity), or individuals previously exposed to muv ( ) . therefore, a serological surrogate/ substitute is used ( ). mumps vaccine efficacy is quantified by a single measure, igg which may not suffice to evaluate the magnitude of the actual humoral response. borgmann et al. ( ) proposed an increase in mumps-specific igg titer in sera as a diagnostic criteria of mumps reinfection ( ) . it has been suggested that vaccinated individuals have modified b-cell responses to muv that allow for the rapid generation of igg antibodies and a blunted or absent igm response ( , ) . in addition, emerging data in simian immunodeficiency virus studies suggests that not all antibody responses are equal, and qualitative features of antibodies may be key to defining protective immune profiles ( ) . despite its use, the correlation to mumps-specific igg concentrations and neutralization titers against the jl virus is poor, suggesting that igg concentrations do not adequately represent a sufficient surrogate correlate of protection ( ) . this is demonstrated in finland; only % of vaccinees had no detectable mumps antibodies after years ( , ) . data from the european sero-epidemiology network (esen ) project in reported that mmr immunization uptake in ireland in was % ( ), however it was also suggested that only - % of -to -year-olds in ireland had detectable antibodies to muv by either natural immunity or immunization ( ) . in , vaccine coverage of medical students in germany was reported to be . % ( ) . in children between the ages of - years, where . % had been vaccinated with the mmr vaccine at least once, only . % showed prevalence of antibodies ( ) . however, . % showed a prevalence of antibodies to measles and rubella in the absence of mumps-specific antibodies. therefore, previous measurement of anti-mumps-specific igg that represented immunity induced by the mumps vaccine appears to be overestimated ( , ) . antibody levels of other components of the mmr vaccine have seen similar trends. waning rubella antibody titers have been observed, despite the number of acute rubella and congenital rubella syndrome cases not increasing. it has also been shown that college students who received rubella vaccination during childhood and had low/no antibody response were able to mount a secondary response when challenged with rubella indicating that an individual's low antibody levels are not always indicative of susceptibility to infection ( ) . measles antibodies can also be detected for up to a decade post-vaccination, with > % of individuals still measles igg positive at - years of age ( , ) . however, as with mumps and rubella, waning measles antibody titers have been observed ( , ) . despite this, a recent longitudinal study of up to years demonstrates how effective the mmr vaccine has been in preventing diagnosed measles cases during the 's/ 's ( ) . similarly, three doses of the hepatitis b (hbv) vaccine in a cohort of alaskan natives showed > % seroconversion in children and young adult post-vaccination and provided long term and durable protection against chronic hbv infection. although no increase of hbv prevalence were observed % individuals had low to undetectable antibody levels after years. these observations suggest that an individual's antibody levels do not indicate susceptibility to infection, that either an antibody titer lower than recommended guidelines is still protective, or/and is an ineffective surrogate of protection. this is emphasized in a study by amanna et al.; ( ) responses to non-replicating protein antigens (tetanus and diphtheria) were shown to have approximate antibody half-lives of - years. in comparison, antibodies following wild type infection were shown to have half-lives of years or more which was thought until recently to confer a more prolonged lifelong protection ( , , ) . however, reinfections observed in individuals that were previously naturally infected have demonstrated that the quantitative measurement of antibodies do not indicate sterile immunity ( ) . it is also important to stress that seroconversion rates due to immunization/natural infection only reflects a change of antibody status from negative to positive, but not necessarily the intensity of antibody response. in addition, there is no consistency in the timing of sample collected post-vaccination to test vaccine efficacy, and between the serological tests utilized for detecting mumps antibodies. as a result, documented seroconversion rates of the mumps vaccines used vary widely (jl: - %, rit strain: - %, urabe am : - %, rubini: - %). this highlights that the assays used to detect immunity to muv may not always detect an adequate post-vaccination response. only a small number of serological commercial assays such as the detection of hepatitis b surface antibody (anti-hbs) ( ) and rubella igg ( ) have been designed using who reference material as a standard for quantification. however, even utilizing this reference standard demonstrates significant differences in the determined quantification of either anti-hbs or rubella igg depending on the assays used; although a value for anti-hbs of iu/ml is regarded as protective against significant hbv infection, the detection of this anti-hbs is significantly influenced by which anti-hbs assays is used ( ) ( ) ( ) ( ) ( ) . therefore, it is possible that the current assays/tests mechanisms utilized to measure mumps antibodies are too insensitive/inappropriate/crude to identify nuances in the immune response which could correlate with immunity against mumps. in addition, variation within neutralization epitopes i.e., the quality of the antibody present could be a more important correlate than quantity ( , ) . though labor-intensive, neutralizing antibodies are considered to be a better correlate of mumps immunity. antibodies against the haemagglutinin-neuraminidase protein (hn) and nucleoprotein (np) have been shown to neutralize muv, however, repeated attempts to define a titer that provides a protective threshold titer have been inconclusive ( , ) . in older studies, during field evaluations of the jl vaccine, neutralizing antibody titers of : - : in unvaccinated individuals was considered seropositive and protective from mumps infection ( , , ) . using a more contemporary wild-type isolate (iowa-g/usa ), a : neutralizing titer cut off was defined between case patients and exposed patients, despite the fact that no cut-off could fully discern between the two groups ( ) . however, that these results are dependent on the challenge virus strain used in the assay. rasheed et al. demonstrated a fold lower neutralization titer to the g-genotype when compared to the jl vaccine strain in - year olds ( ) . this has also been seen between mumps vaccine strains vs. circulating strains in india and china ( , ) . despite studies in more highly vaccinated populations demonstrating that hn-inhibiting titers after natural disease were : compared to : post-vaccination, neither appeared to prevent reinfection ( , ( ) ( ) ( ) ) . there is increasing evidence that the mumps-specific antibody response is broader than neutralization alone ( ) . avidity testing for virus-specific igg has been proposed ( , , ) . individuals who lack measurable mumps-specific antibody levels may be susceptible to infection but protected from significant illness as they may be protected by cell-mediated immune memory. prolonged t-cell responses are reported after other vaccinations; - years after a single dose of the rubella vaccine ra / , a t-cell proliferative response to neutralizing antibodyinducing peptides suggest t helper and b-cell interactions. this indicates that full vaccine effectiveness could be dependent on mounting both an antibody and cell-mediated immune response ( ) . although cell mediated immunity has not been as wellassessed in mumps infection, a lymphoproliferative response was induced in infants vaccinated at , , or months of age was induced ( ) with antigen-specific t-cells reported to appear within month of infection ( ) . lymphoproliferative responses to measles and mumps vaccine viruses were shown to persist in two thirds of the population at least years after immunization ( ), with t-and b-cell immunity persisting for years post-immunization ( ) . low levels of mumps-specific memory b-cells have also been documented suggesting that mumps infection or vaccination may not generate a robust b-cell memory ( , ) . two principal mechanisms for maintaining long-term humoral immunity have been proposed and reviewed by amanna et al. ( ) : associations between memory b-cell levels and antibody may reflect an epiphenomenon in which serum antibody levels and memory b-cells are equally stable but independently maintained. if memory b-cells and plasma cells are independently regulated, then multiple re-exposures to antigens may cause divergence between memory b-cell levels and antibody levels ( ) . antigens with the highest rates of boosting through vaccination or latent viral infection coincidentally showed the weakest association between memory b-cell titers and antibody titers ( ). although the role and efficacy of t-cell immunity to mumps infection is unclear, there is a possibility that certain muv strains may be capable of escaping vaccine induced t-cell responses, which may not be considered of significance until b-cell waning immunity comes into play ( ) . in individuals who did not respond to vaccination (i.e., had a ≤ -fold of mumps antibody titers days post-vaccination), several genes including those implicated in antigen presenting, processing, t-cell response and function showed significantly increased expression, with mhc class ii hla-drb and hla-dra, and cd induced when compared to responders day post-mmr vaccination. this may indicate that the stimulation of a rapid adaptive immune response limits antigenic presentation and hence prevent the differentiation of memory b-cells to antibody-producing plasma cells ( ) . differences in predicted b-cell and t-cell epitopes between jl vaccine strain and other vaccine strains may also be implicated in the outbreaks witnessed ( ) . although, it has also been shown that natural mumps infection or vaccination do not always induce both cellular and humoral immunity. de wit et al. ( , , ) has shown the presence of th -type cd + t-cells recognizing a muv epitope in a hlr-dr restricted manner. in addition, the response of ifn-γ and tnf producing cd + t-cells specific to muv epitopes are lower in vaccinated individuals when compared to individuals who were naturally infected ( , , ( ) ( ) ( ) . utilizing current knowledge and new technologies may help define a better surrogate correlate of protection and potentially determine a cut-off between the immunity of a vaccinated individual and a secondary mumps infection. this may potentially move the diagnostic preference from serological tests to more comprehensive functional assays. despite the large resurgence of mumps outbreaks, there is insurmountable evidence highlighting the benefit of the mumps vaccine ( table ) . routine childhood mmr vaccination has resulted in a dramatic decrease in the incidence of mumps cases, and has shifted the peak age-specific attack rates from a young children (manifesting between and years) to one that affects young adults, in particular those who have close interaction with other young adults ( - years) ( , ) . additionally, clinical manifestations and severity of disease in vaccinated vs. unvaccinated individuals differ ( , ) . although muv can be clinically asymptomatic in about - % of those who become infected, the vaccine against mumps confers protection in a dose response manner; unvaccinated individuals saw an attack rate of based on the reduction seen upon the introduction of a mumps vaccine, it has been proposed that mmr vaccination also prevents the transmission of the virus. there is limited knowledge regarding the shedding and transmission of muv, but it is thought that close contact and transmission of a certain viral load may induce clinical symptoms ( , , ) . modeling data suggests that infectious muv shedding decreases rapidly after the onset of symptoms, however - % are patients are thought to still be virally shedding days after the onset of symptoms ( ) . this could be the reason why the transmission of muv can be exacerbated by close social situations within a heterogeneously vaccinated population. outbreaks generally occur in situations of intense contact such as college dormitories, boarding schools, and youth summer camps ( ) , with up to a third reporting some contact with a mumps case ( ) . evidence of lower levels of viral replication also suggests a clinical benefit of the vaccine ( , ) . viral load and presence of the mumps vaccine genome in areas of viral replication was lower in vaccinated individuals vs. unvaccinated individuals ( ) . in addition, patients who contracted mumps but had two doses of mmr have been shown to shed less muv in their urine, with fewer experiencing bilateral parotitis or orchitis than unvaccinated individuals ( ), this suggests that immunity induced by mmr vaccination limits virus transmission and complications ( , ) . it should be noted also that individuals who received two doses of mmr, and had a positive correlation between viremia, salivary viral loads and systematic clinical mumps infection may have an increased risk of transmitting virus. these individuals also lacked mature functional responses, with low neutralizing antibody titers and avidity indexes ( ) . overall, evidence demonstrates a clinical advantage to receiving a mumps vaccine ( table ) . currently no global consensus exists for the measurement of mumps antibodies, mumps avidity or neutralizing titers that correlate to vaccine response and protection in healthy individuals. if a biomarker is discovered, it could be utilized as an international diagnostic reference standard to allow global harmonization and evaluation of the relative effectiveness of the different vaccination programs worldwide. such an attempt was conducted by andrews et al. ( ) , who reported on the european sero-epidemiology network project which was established to harmonize the seroepidemiology of five vaccine preventable infections including measles, mumps, and rubella in eight european countries. the study concluded that the development of an international standard for mumps would help in the standardization and comparability of mumps antibodies in the different enzyme immunoassays used in laboratories. however, to date, no international reference standard for mumps has been established. in response to infection, the human immune system launches a series of immunological responses with the goal of controlling or eliminating the pathogen. if the pathogen circumvents the frontline defense of the innate immune system, an adaptive immune response specific for the pathogen will become activated to respond, with the intention to generate humoral-and cell-mediated immunity. humoral immunity, represented by antibodies secreted by b-cells are not effective against pathogens that invade host cells. therefore, cell-mediated immunity instructed by the vaccination aims to stimulate the host immunological process and formation of cell-mediated immunological memory via the use of live-attenuated or of inactivated/subunit vaccine components to promote a cell-mediated immune response. extensive knowledge gaps significantly hinder improvements to the mumps vaccine and prospects for mumps eradication and maintaining proficient population immunity ( , , ) . few studies have collected data that examines different aspects of mumps immunity and are limited in their predictive value for future outbreaks ( ) . for example, the importance of t and b-cell responses in protective mumps immunity and how memory/plasma cell numbers are homeostatically maintained post-infection or vaccination is relatively unknown ( ) . it should be acknowledged that the mechanism of protection of infection may not be the same mechanism of recovery from infection, which may make the identification of a common correlate of protection and recovery difficult ( ) . therefore, if a correlate or surrogate correlate is unobtainable to define an individual's protection to mumps, should we re-consider and re-focus efforts on optimizing the vaccine using available historical clinical and trial data? it has been suggested that wild-type infection could confer a "better quality, " broader and prolonged immuno-activation than vaccine-induced immunity. this is reflected in mean neutralizing antibody titers detected post-mumps vaccination, which were over five times lower than those detected following wild type infection. similarly, hemagglutination-inhibiting titers after natural disease were : compared to : post-vaccination ( , , ) . the use of a live-attenuated virus vaccine is intended to mimic immunological reactions and responses between the host and wild type virus ( ) . the current liveattenuated mmr vaccine is intramuscularly injected, a route that significantly differs from the natural infection mode of transmission. however, emphasized by differing immunological kinetics between immunized and naturally infected individuals when subjected to wild type pathogens, injectable vaccines are considered not to be the best inducer of antigen-specific mucosal immune responses for mucosal pathogens, especially if the mode of administration is not the natural route (the respiratory tract) ( , ) . improvements on a broader range of antigen delivery systems will improve vaccination strategies and potentially prolong the effect of a vaccination by producing a localized immunological response in the relevant tissues ( , ) . mucosal vaccines such as intra-nasal vaccination have advantages over traditional injectable vaccines as they can induce an effective, more robust immune response without any physical discomfort and more closely replicate the natural route of infection for mumps ( , ) . b-cells induced by the mucosal response are also capable of secreting iga class of antibodies in the lumen, where the interaction and neutralization of specific antigens form iga-antigen complexes are easily able to be entrapped in the mucus and eliminated by cilial epithelial cells ( ) . activated mucosal lymphocytes can also reach other mucosal sites via the lymphatic system and have the capability to transfer immunity ( ) . such an example is the intranasal immunization of inactivated influenza. with a - % similar efficacy between the injectable and intranasal influenza in healthy individuals this intranasal vaccine can elicit the secretion of haemagglutinin and neuraminidase specific iga antibodies in the upper respiratory tract, and corresponding igg antibodies ( ) . live, cold adapted attenuated nasal influenza vaccine has been routinely used in russia for over years ( ) . other liquid live-attenuated intranasal vaccines are available; "nasovac r " in india, and "flumist r " in the us, uk and new zealand ( , , ) . inactivated vaccines consisting of heat/chemical or liveattenuating monovalent or multivalent pathogens in animals/cell lines were developed to protect against disease causing microorganisms ( ) . less emphasis was placed on understanding the mechanisms related to conferring immunological memory; the focus lay on the availability, mass production and administration of the vaccine to introduce herd immunity into populations ( ) . currently, the least expensive and time effective method to licensure is the comparison of serologic responses of the new vaccine to an existing licensed vaccine, which can lead to a bias on the development of novel vaccines ( ) . this methodology also does not account for the fact that each vaccine developed elicits its own immunological signature and may need to be considered on an individual basis ( ) . raymond et al. ( ) has suggested that embryonated chicken egg-based vaccines may induce antibodies that are more preferential to egg adapted strains better than wild type virus. amino acid substitutions/differences in key antigenic targets due to the passage of the growing virus within this environment may optimize the growth of the virus, but could lead to differences over time that could affect the immunogenicity or potency of the vaccine ( , , ) . the jl vaccine contains two isolates of the jl strain (jl and jl ) and whilst no immunological differences have been documented, jl grows to higher titers than jl in embryonic eggs and also demonstrates significant sequence variability ( , ) . zost et al. ( ) also demonstrated that an egg selected mutation within a glycosylation site in the - influenza vaccine strain led to the production of poorer neutralizing antibodies to the vaccine strain compared to wild type influenza virus. vaccine rit strain derived from one of the two distinct virus subtypes of the jl vaccine (jl ) showed comparable seroconversion rates despite inducing a significantly lower geometric mean antibody titer when compared to recipients of the jl vaccine, but does not have any longitudinal trials investigating its efficacy, even though there are populations who are currently receiving it ( , ) . the significant time gap between pathogen emergence and vaccine licensure, could potentially lead to antigenic drift. there is potential that modern biotechnologies could be utilized to design novel vaccine platforms ( , , ). clinically derived recombinant muv lacking the expression of the immunomodulatory v or sh protein are currently being investigated ( ) . in china, a vaccine consisting of the prevalent wildtype virus genotype (f) has recently been produced and is currently undergoing trials ( ) . in addition, despite being extremely pleomorphic, utilizing mhc epitopes as potential b-cell and t-cell vaccine candidates are also being investigated ( , , ) . vaccine design has involved the utilization and templating of epitopes that previously induced a b-or t-cell response during natural disease that are considered to be immunogenic enough to induce similar responses if administered in a vaccine. however, the appropriate b-cell and t-cell epitope/peptide candidates to induce a protective immunological response can be difficult to correctly identify and synthesize, as it may differ to the immunodominant epitope and host presentation of that antigen ( , ). prediction of mhc-peptide binding and cleavage has demonstrated mismatches in both vaccine tcell and b-cell epitopes in vaccinated individuals highlighting small number of distinguishing amino acid changes of the jl major strain ( ) . the importance of understanding tand b-cell responses and how antigen-specific memory cells numbers are homeostatically maintained post-infection is crucial to understand to ensure successful vaccine development ( , ) . since the 's, significant progress has also been made in developing flexible, amplifiable, scalable, inexpensive, and cold-chain free rna vaccines, such as synthetic mrna molecules encoding only the antigen of interest and selfamplifying rna (sa-rna) ( ) . such examples include an experimental mrna vaccine candidate (mrna- ) which encodes a stable form of the sars-cov- spike protein and has been accepted as a trial candidate for clinical trials in healthy male and female individuals ( , ) . in addition, sa-rna viruses as gene delivery and vaccine vectors have also demonstrated therapeutic efficacy in a number of preclinical studies. in the context of influenza, sa-rna vaccines have shown comparable results of protection at lower doses than mrna vaccines ( , , ) . exponential developments in the "omic" area has enabled further vaccine development and understanding of the immunological response and challenges surrounding this area ( ) . systems vaccinology, which includes immunoformatics, dna/rnaseq, microarrays, mass spectrometry proteomics, transcriptomics, and metabolomics have all shown huge potential in elucidating differences in vaccine strains, vaccine growth and individual response in depth and on an epigenetic level allowing the identification of new vaccine antigens with increased speed and sensitivity ( , , ( ) ( ) ( ) . adjuvants, a group of biological and chemical compounds could also be considered to enhance and improve the longevity of the immune response of a vaccine such as the mmr. adjuvants have been successful in significantly reducing overall antigen dose in vaccine formulations as well as alter and broaden the host response through epitope spreading and qualitatively shaping the effector function of antibodies through subclass selection ( , ) . the re-purposing of live-attenuated vaccines as tibv are also being investigated. trained immunity based vaccines (tibv) elicit heterologous protective effects by inducing a broader, lasting priming of innate immune cells, in addition to the intended specific immunological response and memory of conventional vaccines [reviewed in ( ) ]. mmr and bcg vaccines have been considered as potential tibv in the context of the current coronavirus disease (covid- ) pandemic ( ) , however further research is needed. the mumps component of a vaccine is an unpurified product whose potency is measured through a biological assay for the substance rather than through evaluation of integrity of physical form (quantitative pcr after cell culture) ( ) . a monovalent mumps vaccine lot is used to characterize the performance of the mumps potency assay with international reference standards. degradation products are neither identified nor quantified ( ) . currently, the minimum potency of the mumps vaccine used varies between brands used [summarized by su et al. ( ) ] ( ) . however, this potency measurement differs to other mmr vaccines strains previously used [reviewed in ( ) ]. in addition, the maximum required potency is not usually specified. atrasheuskaya et al. ( ) demonstrated that the four out of lots of vaccine associated with six cases of viral transmission postvaccination to previously vaccinated contacts were in fact twice as potent as the lots that were not associated with viral transmission post-vaccination ( , ) . this may impact the use and efficacy of specific vaccines. due to their neurovirulence and increased incidence of aseptic meningitis and mumps cases, the urabe am and rubini mumps vaccine strains were discontinued in many countries ( , , ) . comparing alternative culturing technologies and defining a viral potency range for vaccines could help reduce variability within the mmr vaccine ( ) . ensuring the use of a reference sample that had similar replication rate and composition as the virus to be tested will allow accurate determination of the quantity of virus present per lot of vaccine. investigating novel vaccine candidates shown to induce a similar quantity but qualitatively different antibodies will help segregate and reveal potential correlates of protection ( ) . incorporating more modern technologies such as microarray technology or antibody pattern/profiling (rather than single antibody measures) to investigate biomarkers of neutralizing antibody response and/or correlates of protective immunity, in addition to incorporating what has been accomplished in finland will allow further understanding of mumps immunity ( , , , , , ) . the efficacy of a vaccine is defined by disease prevention (sterile immunity, establishment of primary infection and shedding of mature virus particle), or complications associated with infection (orchitis, neurological issues etc.) ( ) . despite the well-documented success of the global immunization programs demonstrating how vaccines significantly attenuate disease and onward transmission of infection, they are rarely totally efficacious (demonstrated in pre-licensure clinical trials) or effective (determined by practical use) ( , , ) . therefore, does "immunity" refer to sterile immunity or solely to protection from symptomatic infection? what defines an effective vaccine, or what constitutes vaccine failure? does the medical profession and the "pro-vaccine" message contribute to the public skepticism regarding immunization? is it time to shift the medical and public perception paradigm from "protection of infection following vaccination" to "protection from serious clinical mumps manifestation"? the lack of definition leads to misinterpretation by health professionals and media of what is truly occurring. such an example is currently observed with influenza; individuals who have recently being vaccinated against influenza and subsequently become infected with influenza, assume that the vaccine has "failed" even though there is a reduction in symptoms. the current assertion that vaccines "protect against" or "eliminate" the risk of infection may contribute to the misperception about what level of protection a vaccine actually provides (vaccination efficacy) perpetuated by the witnessing of visible clinical disease and outbreaks despite vaccination ( , , ) . therefore, definition and consensus of what is termed a true "vaccine failure" is required to inform both the clinical and public perception of what the function of a vaccine is. deciding what the clinical endpoint of a vaccine is i.e., infection with mild clinical symptoms vs. natural infection/disease with its associated complications and assessing the impact of the vaccine in a heterogeneously vaccinated population will allow a better consensus of what is required. a paradigm shift in what is considered to be a good vaccine i.e. one that provides protection against serious clinical sequalae, in addition to identifying a reliable laboratory marker for this protection is required ( ) . by focusing on, and acknowledging that vaccines may not prevent infection but will attenuate the clinical complications/consequences that arise from infection in addition to reducing onward transmission will provide a more realistic view of the benefits of vaccination ( ) . immunity is therefore beneficial but does not necessarily mean protection. if we can decide whether the end point of a vaccine is either the prevention of infection or protection against serious sequalae of infection, its efficacy and impact can be determined and will have enormous implications on how vaccine failure can be studied, quantified and interpreted. this teasing out of the immunological response to muv will ultimately provide potential correlates with robust predictive power, suggest directions for further vaccine improvement, and enable the discovery of potential biomarkers to help create a more efficient diagnostic assay that can discern between different infectious diseases and vaccination vs. disease status. the identification and incorporation of a correlate into diagnostic protocols which can be widely accessible may potentially allow global harmonization of criteria defining immunological protection against mumps. the medical and scientific field needs to inform the public more accurately about what a good vaccine consists of, which may result in a more positive attitude toward vaccines. in the majority of individuals, a vaccine can prevent serious clinical sequalae and associated complications following wild type infections, but also significantly reduce onwards transmission in particular to the cohorts who are not vaccinated due to a contraindication to vaccination. this is the positive and realistic view of vaccination which should be presented rather than the current flawed message of "get the vaccine and be protected from infection." the public deserves, and will appreciate, a more accurate and informed message. ac, jc, and jh contributed to the conception and design of the review. ac wrote the first draft of the manuscript. jc, tl, and jh contributed to manuscript revision. all authors have read and approved the submitted version. this work was funded by the national children's research centre, children's health ireland, dublin, ireland with grant number c/ / awarded to jh. mumps virus analysis of mumps vaccine failure by means of avidity testing for mumps virus-specific immunoglobulin g infectiousness of communicable diseases in the household (measles, chickenpox, and mumps) hearing loss due to mumps molecular epidemiological evaluation of the recent resurgence in mumps virus infections in ireland isolation of virus during the incubation period of mumps infection function of small hydrophobic proteins of paramyxovirus proposed criteria for classification of new genotypes of mumps virus mumps in the vaccination age: global epidemiology and the situation in germany measles resurgence in argentina: - outbreak available online at triple viral/double viral immunization: coverage data of mmr and mmr national vaccination calender everything you need to know about the new vaccination law in argentina molecular identification of mumps virus genotypes from clinical samples: standardized method of analysis genomic diversity of mumps virus and global distribution of the genotypes world health organization. mumps reported cases (as of austrailian government department of health. . mumps (last updated mumps laboratory case definitnion austrailian institute of health and welfare parental opinions towards the "no jab, no pay a protracted mumps outbreak in western australia despite high vaccine coverage: a population-based surveillance study immunogenicity and safety of the combined vaccine for measles, mumps, and rubella isolated or combined with the varicella component administered at -month intervals: randomised study calendários de vacinação sbim vacina sarampo, caxumba, rubéola e varicela (atenuada) outbreak of aseptic meningitis associated with mass vaccination with a urabe-containing measles-mumps-rubella vaccine: implications for immunization programs outbreak of aseptic meningitis and mumps after mass vaccination with mmr vaccine using the leningrad-zagreb mumps strain detection of a new mumps virus genotype during parotitis epidemic of - in the state of sao paulo, brazil reemergence of mumps in sao paulo, brazil-the urgent need for booster shot campaign to prevent a serious infectious disease guidelines for the prevention and control of mumps outbreaks in canada (archived) guidelines for the prevention and control of mumps outbreaks in canada. mumps-containing vaccine and immunization programs in canada immunization coverage report for school pupils in ontario. - school year. toronto, on: queen's printer for ontario investigation and management of a large community mumps outbreak among young adults in toronto guidelines: mumps in canada assessment of onedose mumps-containing vaccine effectiveness on wild-type genotype f mumps viruses circulating in mainland china mumps epidemiology and mumps virus genotypes circulating in mainland china during - importation of mumps virus genotype k to china from vietnam waning immunity against mumps in vaccinated young adults pediatric vaccines: global brands and country availability available online at information sheet observed rate of vaccine reactions. measles, mumps and rubella vaccines routine immunization: regional and country profiles summary of routine immunization and vaccine-preventable diseases surveillance data, based primarily on data for submitted through the who/unicef joint reporting form on immunization measles elimination efforts and - outbreak progress toward measles elimination in germany immunogenicity of mumps virus vaccine candidates matching circulating genotypes in the united states and china routine immunization: regional and country profiles; germany. summary of routine immunization and vaccine-preventable diseases surveillance data, based primarily on data for submitted through the who/unicef joint reporting form on immunization list of the names, pharmaceutical forms, strengths of the medicinal products, routes of administration, marketing authorisation holders in the member states (annex , article ) mumps and mumps vaccine: a global review live attenuated measles mumps and rubella vaccines: an over view the epidemiology of mumps in italy pediatric sentinel surveillance of vaccinepreventable diseases in italy available online at the law on compulsory vaccination in italy: an update years after the introduction mmr vaccination and autism european centre for disease prevention and control. : annual epidemiological report for ; mumps measles in mexico, - : interruption of endemic transmission and lessons learned detection of mumps virus genotype h in two previously vaccinated patients from mexico city missed opportunities for measles, mumps, and rubella (mmr) immunization in mesoamerica: potential impact on coverage and days at risk el programa nacional de vacunacion: orgullo de mexico progress toward measles elimination in the russian federation routine immunization: regional and country profiles; russian federation. summary of routine immunization and vaccine-preventable diseases surveillance data, based primarily on data for submitted through the who/unicef joint reporting form on immunization mumps vaccine failure investigation in safety evaluation of mmr vaccine during a primary school campaign in saudi arabia measles in saudi arabia: from control to elimination measles immunization in saudi arabia: the need for change ministry of health saudi arabia. ministries of health and education start a vaccination campaign against measles, mumps and rubella reemergence of mumps genetic characteristics of mumps viruses isolated in korea from to increasing mumps incidence rates among children and adolescents in the republic of korea: age-period-cohort analysis compulsory vaccines to be applied to baby after birth in turkey the national vaccination schedule in previously healthy children: the practical recommendations about additional vaccines routine immunization: regional and country profiles; turkey. summary of routine immunization and vaccine-preventable diseases surveillance data, based primarily on data for submitted through the who/unicef joint reporting form on immunization genotyping of mumps virus circulating in turkey in the - winter season risks of convulsion and aseptic meningitis following measles-mumps-rubella vaccination in the united kingdom nhs vaccinations and when to have them routine immunization: regional and country profiles; united kingdom of great britain and northern ireland. summary of routine immunization and vaccine-preventable diseases surveillance data, based primarily on data for submitted through the who/unicef joint reporting form on immunization proteomics for development of vaccine viral mumps: increasing occurrences in the vaccinated population. oral surg oral med oral pathol oral radiol centre for disease control and prevention. recommended child and adolescent immunization schedule for ages years or younger, united states vaccination coverage for selected vaccines and exemption rates among children in kindergarten -united states, - school year vaccination coverage among children aged - months -united states the molecular epidemiology of mumps virus the immunological basis for immunization series module : mumps. world health organization vaccination of human beings against mumps; vaccine administered at the start of an epidemic. i. incidence and severity of mumps in vaccinated and control groups preparation of mumps vaccines and immunization of monkeys against experimental mumps infection european centre for disease prevention and control. mumps: vaccine scheduler available online at: https://vaccineschedule.ecdc.europa.eu/scheduler/bydisease? live attenuated mumps virus vaccine. . vaccine development increased mumps incidence in the netherlands: review on the possible role of vaccine strain and genotype molecular differences between two jeryl lynn mumps virus vaccine component strains, jl and jl the jeryl lynn vaccine strain of mumps virus is a mixture of two distinct isolates health protection surveillance centre. definition of an outbreak estimation of the efficacy of three strains of mumps vaccines during an epidemic of mumps in the geneva canton (switzerland) comparative efficacy of rubini, jeryl-lynn and urabe mumps vaccine in an asian population estimates of mumps seroprevalence may be influenced by antibody specificity and serologic method mumps outbreak in a highly vaccinated student population mumps outbreak, england. emerg infect dis mumps outbreak in the republic of moldova mumps outbreak in jerusalem affecting mainly male adolescents ongoing mumps outbreak among adolescents and young adults mumps outbreak in a highly vaccinated university-affiliated setting before and after a measles-mumps-rubella vaccination campaign-iowa mumps outbreaks at four universities-indiana current status of mumps virus infection: epidemiology, pathogenesis, and vaccine mumps: an update on outbreaks, vaccine efficacy, and genomic diversity a report on the status of vaccination in europe mumps resurgences in the united states: a historical perspective on unexpected elements failure to vaccinate and vaccine failure the burden of disease and the changing task of medicine the politics of prevention: anti-vaccinationism and public health in nineteenth-century england a population-based cohort study of undervaccination in managed care organizations across the united states world health organization. meeting of the strategic advisory group of experts on immunization misinformation lingers in memory: failure of three pro-vaccination strategies delaying vaccination is not a safer choice reactogenicity and immunogenicity of a new live attenuated combined measles, mumps and rubella vaccine in healthy children measles, mumps, rubella vaccination and autism: a nationwide cohort study measles, mumps and rubella (mmr) vaccination has no effect on cognitive development in children-the results of the polish prospective cohort study available online at sustained transmission of mumps in a highly vaccinated population: assessment of primary vaccine failure and waning vaccine-induced immunity rapid effect on endemic measles, mumps, and rubella of nationwide vaccination programme in finland measles, mumps, and rubella in finland: years of a nationwide elimination programme knowledge gaps persist and hinder progress in eliminating mumps recent resurgence of mumps in the united states brief report: update: mumps activity-united states an outbreak of mumps in sweden mumps outbreaks in canada and the united states: time for new thinking on mumps vaccines dynamics of the serologic response in vaccinated and unvaccinated mumps cases during an epidemic challenges in interpretation of diagnostic test results in a mumps outbreak in a highly vaccinated population laboratory-based investigation of suspected mumps cases submitted to the german national reference centre for measles, mumps, and rubella comparison of the sensitivity of laboratory diagnostic methods from a wellcharacterized outbreak of mumps in new york city in enzyme-linked immunospot assay detection of mumps-specific antibodysecreting b cells as an alternative method of laboratory diagnosis mumps outbreak in a highly vaccinated school population: assessment of secondary vaccine failure using igg avidity measurements seroepidemiology of the recent mumps virus outbreaks in ireland laboratory testing and phylogenetic analysis during a mumps outbreak in ontario \sim:text=rt% dpcr% and % viral% culture,aid% in% diagnosing% mumps% infection diagnosis of acute mumps infection during an outbreak in a highly vaccinated population: mumps rna or mumps igm detection? monitoring viral genetic variation as a tool to improve molecular diagnostics for mumps virus persistence of mumps antibodies after doses of measles-mumps-rubella vaccine persistence of measles, mumps, and rubella antibodies in an mmr-vaccinated cohort: a -year follow-up mumps vaccine performance among university students during a mumps outbreak emerging mumps infection epidemiology of a mumps outbreak in a highly vaccinated island population and use of a third dose of measles-mumps-rubella vaccine for outbreak control-guam long-term follow-up for immunity after monovalent or combined live measles, mumps, and rubella virus vaccines persistence of antibody in human subjects for to years following administration of combined live attenuated measles, mumps, and rubella virus vaccines studies on live attenuated mumps vaccine. i. comparative field trials with two different live vaccines live attenuated mumps-virus vaccine. iv. protective efficacy as measured in a field evaluation live attenuated mumps-virus vaccine. . clinical and serologic aspects in a field evaluation experiences with jeryl lynn strain live attenuated mumps virus vaccine in a pediatric outpatient clinic genomic signature of early t-cell response is associated with lower antibody titer threshold for sterilizing immunity the effectiveness of the mumps component of the mmr vaccine: a case control study measles, mumps, rubella vaccine (priorix; gsk-mmr): a review of its use in the prevention of measles, mumps and rubella immunogenicity and safety of a measles-mumps-rubella vaccine administered as a first dose to children aged to months: a phase iii, randomized, noninferiority, lot-to-lot consistency study a new measles mumps rubella (mmr) vaccine: a randomized comparative trial for assessing the reactogenicity and immunogenicity of three consecutive production lots and comparison with a widely used mmr vaccine in measles primed children immunogenicity and safety of measles-mumps-rubella-varicella (mmrv) vaccine followed by one dose of varicella vaccine in children aged months- years or - years primed with measles-mumps-rubella (mmr) vaccine similar immunogenicity of measles-mumps-rubella (mmr) vaccine administrated at months versus months age in children immune response to the mumps component of the mmr vaccine in the routine of immunisation services in the brazilian national immunisation program immunogenicity and safety of measles-mumpsrubella vaccine delivered by disposable-syringe jet injector in india: a randomized, parallel group, non-inferiority trial safety and immunogenicity of human serum albumin-free mmr vaccine in us children aged - months immunogenicity and safety of early vaccination with two doses of a combined measles-mumps-rubella-varicella vaccine in healthy indian children from months of age: a phase iii, randomised, non-inferiority trial duration of the immune response to mmr vaccine in children of two age-different groups antibody persistence for years following two doses of tetravalent measlesmumps-rubella-varicella vaccine in healthy children a combined measles, mumps, rubella and varicella vaccine (priorix-tetra): immunogenicity and safety profile centers for disease control and prevention. prevention of measles, rubella, congenital rubella syndrome, and mumps, : summary recommendations of the advisory committee on immunization practices (acip) primary vaccine failure to routine vaccines: why and what to do? evaluation in young children of the urabe am strain of live attenuated mumps vaccine in comparison with the jeryl lynn strain safety and characterization of the immune response engendered by two combined measles, mumps and rubella vaccines horizontal transmission of the leningrad- live attenuated mumps vaccine virus mumps antibody levels among students before a mumps outbreak: in search of a correlate of immunity primary vaccine failure after dose of varicella vaccine in healthy children outbreak of mumps in a vaccinated child population: a question of vaccine failure? measles, mumps, and rubella-vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of mumps: recommendations of the advisory committee on immunization practices (acip) antibodies to measles, mumps and rubella in uk children years after vaccination with different mmr vaccines vaccine-induced measles virus antibodies after two doses of combined measles, mumps and rubella vaccine: a -year follow-up in two cohorts evaluation of cellular immunity to mumps in vaccinated individuals with or without circulating antibodies up to years after their last vaccination longterm persistence of mumps antibody after receipt of measles-mumpsrubella (mmr) vaccinations and antibody response after a third mmr vaccination among a university population immunity to mumps before and after mmr vaccination at years of age in the first generation offered the two-dose immunization programme vaccine waning and mumps reemergence in the united states differential durability of immune responses to measles and mumps following mmr vaccination antibody induced by immunization with the jeryl lynn mumps vaccine strain effectively neutralizes a heterologous wild-type mumps virus associated with a large outbreak mumps outbreaks in a highly vaccinated population: investigation of a neutralization titre against the current circulating wildtype genotype g mumps virus immunogenicity and reactogenicity of a new measles, mumps and rubella vaccine when administered as a second dose at y of age sera from different age cohorts in belgium show limited crossneutralization between the mumps vaccine and outbreak strains effectiveness of a third dose of mmr vaccine for mumps outbreak control antigenic relationships between six genotypes of the small hydrophobic protein gene of mumps virus serological and phylogenetic evidence of monotypic immune responses to different mumps virus strains remembering mumps phylogenetic analysis of clinical mumps virus isolates from vaccinated and non-vaccinated patients with mumps during an outbreak rt-pcr based diagnosis and molecular characterisation of mumps viruses derived from clinical specimens collected during the mumps outbreak in portugal mumps-specific cross-neutralization by mmr vaccine-induced antibodies predicts protection against mumps virus infection antigenic differences between vaccine and circulating wild-type mumps viruses decreases neutralization capacity of vaccine-induced antibodies identification of conformational neutralization sites on the fusion protein of mumps virus cross-neutralization between three mumps viruses & mapping of haemagglutininneuraminidase (hn) epitopes recent mumps outbreaks in vaccinated populations: no evidence of immune escape identification of naturally processed mumps virus epitopes by mass spectrometry: confirmation of multiple cd + t-cell responses in mumps patients seroprevalence of mumps in the netherlands: dynamics over a decade with high vaccination coverage and recent outbreaks investigation of mumps vaccine failures in minsk immune responses to mumps vaccine in adults who were vaccinated in childhood correlates of protection induced by vaccination correlations among measles virus-specific antibody, lymphoproliferation and th /th cytokine responses following measles-mumps-rubella-ii (mmr-ii) vaccination mumps virus infection in vaccinated patients can be detected by an increase in specific igg antibodies to high titres: a retrospective study laboratory diagnosis of mumps in a partially immunized population: the nova scotia experience mumps outbreak and laboratory diagnosis antibody fab-fc properties outperform titer in predictive models of siv vaccine-induced protection increase in mumps in ireland in late assessment of mumps virus-specific antibodies by different serological assays: which test correlates best with mumps immunity? seroprevalence of measles-, mumpsand rubella-specific igg antibodies in german children and adolescents and predictors for seronegativity cellular and humoral immunity after vaccination or natural mumps infection rubella specific cell-mediated and humoral immunity following vaccination in college students with low antibody titers measles, mumps, and rubella antibody patterns of persistence and rate of decline following the second dose of the mmr vaccine development and durability of measles antigen-specific lymphoproliferative response after mmr vaccination childhood mmr vaccination and the incidence rate of measles infection: a ten year longitudinal cohort study of american children born in the s duration of humoral immunity to common viral and vaccine antigens mumps outbreaks in vaccinated populations: are available mumps vaccines effective enough to prevent outbreaks? symptomatic mumps virus reinfections complete genome sequence of the who international standard for hepatitis b virus dna the establishment of surrogates and correlates of protection: useful tools for the licensure of effective influenza vaccines? duration of immunogenicity and efficacy of hepatitis b vaccine in a yupik eskimo population a longitudinal hepatitis b vaccine cohort demonstrates long-lasting hepatitis b virus (hbv) cellular immunity despite loss of antibody against hbv surface antigen antibody levels and protection after hepatitis b vaccine: results of a -year follow-up study and response to a booster dose protection provided by hepatitis b vaccine in a yupik eskimo population. seven-year results long-term efficacy of active postexposure immunization of infants for prevention of hepatitis b virus infection. united states-people's republic of china study group on hepatitis b mumps virus-specific antibody titers from pre-vaccine era sera: comparison of the plaque reduction neutralization assay and enzyme immunoassays decreased humoral immunity to mumps in young adults immunized with mmr vaccine in childhood mumps virus neutralizing antibodies do not protect against reinfection with a heterologous mumps virus genotype immune responses to measles and mumps vaccination of infants at , , and months correlates of lymphoproliferative responses to measles, mumps, and rubella (mmr) virus vaccines following mmr-ii vaccination in healthy children detection of mumps virus-specific memory b cells by transfer of peripheral blood mononuclear cells into immune-deficient mice are cases of mumps in vaccinated patients attributable to mismatches in both vaccine t-cell and b-cell epitopes?: an immunoinformatic analysis the human cd (+) t cell response against mumps virus targets a broadly recognized nucleoprotein epitope mumps infection but not childhood vaccination induces persistent polyfunctional cd (+) t-cell memory cellular immunity to mumps virus in young adults years after measles-mumps-rubella vaccination severity of mumps disease is related to mmr vaccination status and viral shedding mumps vaccination coverage and vaccine effectiveness in a large outbreak among college students-iowa characteristics of a large mumps outbreak: clinical severity, complications and association with vaccination status of mumps outbreak cases transmission of mumps virus from mumps-vaccinated individuals to close contacts the duration of mumps virus shedding after the onset of symptoms detection of rna of mumps virus during an outbreak in a population with a high level of measles, mumps, and rubella vaccine coverage notes from the field: absence of asymptomatic mumps virus shedding among vaccinated college students during a mumps outbreak-washington guidance for isolation precautions for mumps in the united states: a review of the scientific basis for policy change mumps clinical diagnostic uncertainty two major mumps genotype g variants dominated recent mumps outbreaks in the netherlands the european sero-epidemiology network: standardizing the enzyme immunoassay results for measles, mumps and rubella from vaccines to memory and back systematic review of measles and rubella serology studies how to determine protective immunity in the post-vaccine era challenges in mucosal vaccines for the control of infectious diseases correlates, surrogates, and vaccines aerosolized mmr vaccine: evaluating potential transmission of components to vaccine administrators and contacts of vaccinees vaccines against mucosal infections intranasal immunization with dry powder vaccines mucosal immunity and vaccines live attenuated pandemic influenza vaccine: clinical studies on a/ /california/ / (h n ) and licensing of the russian-developed technology to who for pandemic influenza preparedness in developing countries live attenuated influenza vaccine (flumist(r); fluenz): a review of its use in the prevention of seasonal influenza in children and adults proteomic contributions to our understanding of vaccine and immune responses advances in mrna vaccines for infectious diseases molecular signatures of antibody responses derived from a systems biology study of five human vaccines influenza immunization elicits antibodies specific for an egg-adapted vaccine strain influenza vaccine failure: failure to protect or failure to understand? sequence diversity of jeryl lynn strain of mumps virus: quantitative mutant analysis for vaccine quality control contemporary h n influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains stobart cc, moore ml. development of next-generation respiratory virus vaccines through targeted modifications to viral immunomodulatory genes self-amplifying rna vaccines give equivalent protection against influenza to mrna vaccines but at much lower doses immunogenicity of novel mumps vaccine candidates generated by genetic modification discovering protective cd t cell epitopes-no single immunologic property predicts it! characterization of peptides bound to the class i mhc molecule hla-a . by mass spectrometry identifying epitopes of hiv- that induce protective antibodies contrasting b celland t cell-based protective vaccines preclinical and clinical demonstration of immunogenicity by mrna vaccines against h n and h n influenza viruses safety and immunogenicity study of -ncov vaccine (mrna- ) for prophylaxis of sars-cov- infection (covid- ) kunjin virus replicons: an rnabased, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications rna viruses as tools in gene therapy and vaccine development vaccinomics: current findings, challenges and novel approaches for vaccine development vaccines for the st century the role of systems biology approaches in determining molecular signatures for the development of more effective vaccines adjuvants and alternative routes of administration towards the development of the ideal influenza vaccine trained immunity-based vaccines: a new paradigm for the development of broad-spectrum anti-infectious formulations could an unrelated live attenuated vaccine serve as a preventive measure to dampen septic inflammation associated with covid- infection? mbio potency estimation of measles, mumps and rubella trivalent vaccines with quantitative pcr infectivity assay evaluation of medicines for human use quality of biotechnological products: viral safety evaluation of biotechnology products from cell lines of human or animal origin a framework for research on vaccine effectiveness the urabe am mumps vaccine is a mixture of viruses differing at amino acid of the hemagglutininneuraminidase gene with one form associated with disease studies on live mumps virus vaccine. v. development of a new mumps vaccine "am " by plaque cloning highly parallel characterization of igg fc binding interactions the concept of vaccination failure effective messages in vaccine promotion: a randomized trial it's not all about autism: the emerging landscape of anti-vaccination sentiment on facebook key: cord- - a vjpvn authors: charlton hume, hayley k.; vidigal, joão; carrondo, manuel j. t.; middelberg, anton p. j.; roldão, antónio; lua, linda h. l. title: synthetic biology for bioengineering virus‐like particle vaccines date: - - journal: biotechnol bioeng doi: . /bit. sha: doc_id: cord_uid: a vjpvn vaccination is the most effective method of disease prevention and control. many viruses and bacteria that once caused catastrophic pandemics (e.g., smallpox, poliomyelitis, measles, and diphtheria) are either eradicated or effectively controlled through routine vaccination programs. nonetheless, vaccine manufacturing remains incredibly challenging. viruses exhibiting high antigenic diversity and high mutation rates cannot be fairly contested using traditional vaccine production methods and complexities surrounding the manufacturing processes, which impose significant limitations. virus‐like particles (vlps) are recombinantly produced viral structures that exhibit immunoprotective traits of native viruses but are noninfectious. several vlps that compositionally match a given natural virus have been developed and licensed as vaccines. expansively, a plethora of studies now confirms that vlps can be designed to safely present heterologous antigens from a variety of pathogens unrelated to the chosen carrier vlps. owing to this design versatility, vlps offer technological opportunities to modernize vaccine supply and disease response through rational bioengineering. these opportunities are greatly enhanced with the application of synthetic biology, the redesign and construction of novel biological entities. this review outlines how synthetic biology is currently applied to engineer vlp functions and manufacturing process. current and developing technologies for the identification of novel target‐specific antigens and their usefulness for rational engineering of vlp functions (e.g., presentation of structurally diverse antigens, enhanced antigen immunogenicity, and improved vaccine stability) are described. when applied to manufacturing processes, synthetic biology approaches can also overcome specific challenges in vlp vaccine production. finally, we address several challenges and benefits associated with the translation of vlp vaccine development into the industry. with the ability to integrate biological data and computational analysis, synthetic biology has significantly contributed to move vaccine development beyond the constraints of pasteur, assisting in the design of novel biological systems with enhanced efficacy and safety as well as reducing vaccine production times (ruder, lu, & collins, ) . with today's focus gearing towards using vlps, not only as vaccines of unmodified viral assemblies against parental viruses but also as scaffolds for display of heterologous antigens roldão et al., ) , synthetic biology has become a centerpiece in vlp vaccine engineering. seconded by a multitude of tools, such as omics technologies, structural biology, system immunology, and bioinformatics and computational biology, one can now screen for pathogen-specific antigens with high immunogenic potential and apply that information to rationally design modern vlp vaccines ( figure ). influenza is one of the best examples on how omics, more precisely genomics, is revolutionizing vaccine design. dedicated databases detailing complete and accurate influenza genomic information have been created and can be readily accessed worldwide (mchardy & adams, ). in the last decade, this vast wealth of data has been translated into knowledge, which, in conjunction with synthetic biology, has aided the design of vlp vaccine candidates (prabakaran et al., ; pushko et al., ; pushko et al., ) . these novel biological entities are undoubtedly safer with the potential to be more broadly reactive than their previous counterparts (i.e., traditional, commercially available live attenuated influenza vaccines). several genomic applications aid in the identification of novel antigens (liao et al., ) , namely, reverse vaccinology and comparative and functional genomics. reverse vaccinology combines genomics with proteomics and bioinformatics to identify virtually all potentially protective antigens from coding regions within the genome (bambini & rappuoli, ; rappuoli, ) . comparative genomics is primarily used to design broadly protective vaccines as it allows comparison of conserved and variable open reading frames within the same species. functional genomics allows the identification of protein function based on reverse genetic evaluation (mutations and knockouts) or gene expression analysis (transcriptomics; bagnoli et al., ; bambini & rappuoli, ; rappuoli, ; sette & rappuoli, ) . although genomics per se has contributed significantly to the design of many vaccine candidates, such as influenza (prabakaran et al., ; pushko et al., ; pushko et al., ) , malaria (draper et al., ; y. wu, narum, fleury, jennings, & yadava, ) , and human immunodeficiency virus (hiv; calazans, boggiano, & lindsay, ) , its full potential for vaccine development can only be realized when integrated with proteomics and immunomics (bagnoli et al., ) . moreover, the coordinated deployment of these omics technologies along with miniaturized bioprocess screening in, for example, microbioreactor and microfluidic cell culture systems can aid developability assessment leading to accelerated manufacture (bhambure, kumar, & rathore, ; gong & lei, ; hemmerich, noack, wiechert, & oldiges, ) . immunomics specifically addresses the interface between the host immune system and the pathogen proteome. it studies the subset of pathogen-derived proteins or epitopes that are recognized by the host immune system. this information can be used to validate antigens identified from in silico and/or in vitro approaches by evaluating whether they are targets of clinically relevant immune responses (i.e., they stimulate the production of specific cytokines or activate specific cell types; kuleš et al., ) . an example of immunomics application to vlp vaccine design is glaxosmithkline's rts,s vaccine, the most advanced malaria vaccine candidate. rts,s is a chimeric vlp in which a large portion of the c-terminal of plasmodium falciparum-derived circumsporozoite protein (csp) is displayed on a protein scaffold (hepatitis b surface antigen; y. wu et al., ) . in combination with systems biology (genomics and proteomics), immunomics was used to highlight regions within csp that were immunogenic and protective in humans and, thus, indispensable to incorporate into a chimeric vlp. from the many candidates identified, only one proved to be successful in phase iia/b efficacy trials (draper et al., ; kazmin et al., ) . structural information can be used to reconfigure and engineer conserved epitopes to expose areas that exhibit high immunogenicity or to insert multiple immunodominant epitopes within the same vlp platform (liljeroos, malito, ferlenghi, & bottomley, ) . these strategies can broaden the immune response or enhance the existing response to weak immunogenic antigens. identifying conformational epitopes and studying their interactions with the immune system can provide significant information for rational antigen design (anggraeni et al., ; mulder et al., ) . encouraged by recent results from hiv- vlps (c. zhao, ao, & yao, ) , structural biology is emerging as a powerful tool to assist in the rational design of a modern hiv vlp vaccine. novel protective epitopes can now be identified in conformational epitope mapping studies via structural biology (liljeroos et al., ; malito, carfi, & bottomley, ) . in addition, broad and potent hiv antibodies discovered in the pool of antigen-specific memory b cells using structural biology, highlight novel sites of vulnerability on hiv envelope glycoprotein epitope (j. huang et al., ) . when incorporated into hiv- vlps, these antigens can be strategically modified to insert the extended f , e epitope and membrane proximal external region (mper) of hiv- gp (zhai, zhong, zariffard, spear, & qiao, ) providing a better display of the conserved cd binding site and capturing broadly neutralizing antibodies (ingale et al., ) . structural biology is equally informative for vlp engineering as the capsid proteins forming each vlp need to be correctly folded to (a) (b) f i g u r e design tools for vlp vaccine engineering. multitude of tools and recent advances in synthetic biology enable screening for pathogenspecific antigens with high immunogenic potential and engineering of vlp function. ( ) omics technologies enable rapid identification and discovery of novel/potential vaccine antigens. ( ) structural biology and ( ) system immunology assist rational reconfiguration and engineering of epitopes/vlps for enhanced immunogenicity. ( ) bioinformatics and computational biology accelerate data analysis and translation into applicable knowledge. (a) engineering vlp function on different types of vlp. while nonenveloped vlps are commonly engineered using genetic engineering or chemical conjugation, enveloped vlps rely on pseudotyping for function engineering. (b) vlps can be engineered to offer broader immunogenicity, improved immunogenicity, or enhanced stability. broadly immunogenic vlps can be obtained by displaying multiple antigenically distinct epitopes (pushko et al., ; schwartzman et al., ) , highly conserved epitopes (krammer, ; wiersma et al., ) , or computationally optimized epitopes (carter et al., ) within a single vlp. improving vlp immunogenicity can be achieved by incorporating immunomodulatory agents, such as dendritic cells targeting antibodies into particles structure (rosenthal et al., ) . vlp stability can be enhanced by modulating particles formulation (collins et al., ; lua et al., systems immunology is an emerging area of research that uses a broad and integrated, multilevel approach to study the immune system to identify immune correlates of protection or immunogenicity signatures (davis, tato, & furman, ) . combined with recent technological advances in human immunology, systems immunology can provide guidance for rational vaccine design. systems immunology allows the assessment of most cell types of the immune system (including specialized b and t white blood cells), their state, function, signaling molecules, and encoding genes (bird, ) . this accumulation of data captures a snapshot of the human immune system, providing valuable information for the creation of human immune response models that may later be translated into improved vaccine design (davis et al., ) . a systems immunology approach was previously undertaken to investigate the immune response to the live attenuated yellow fever vaccine yf- d (hou et al., ; muyanja et al., ) . following immunization, comprehensive characterization of the immune system was performed providing insights into the vaccine's mechanism of action. a similar method was later applied to the malaria vaccine rts,s, where a systems-level approach led to the identification of molecular and cellular signatures associated with protection and immunogenicity unique to this vlp-based vaccine (kazmin et al., ) . systems immunology may also play an important role in designing vaccines capable of stimulating parts of the immune system not addressed by current vaccines (davis et al., ) . vlps are ideal testing candidates for systems immunology approach due to their ability to stimulate bcell-mediated immune responses, as well as cd proliferative responses and cytotoxic t lymphocyte responses (gause et al., ) . the correct identification of epitopes that stimulate an immune response is crucial for the design of novel immunogens. these epitopes are regions within the antigen that are recognized by b-and t-cell receptors (patronov & doytchinova, ) . potential b-and tcell epitopes can be mapped using bioinformatics and computational biology tools, however, without recent improvements in immunological characterization methods, the identification of epitopes that optimally stimulate the human immune response becomes challen- bioinformatics and computational biology tools have the potential to accelerate data analysis and translate results into applicable knowledge, fostering the discovery of new lead antigens by reducing the number of empirical experiments (he & xiang, ) . vaccine design is an inherently complex and laborious process but software, algorithms, and databases outlined below have the potential to streamline vaccine development via identification of candidate antigens that may otherwise have been overlooked. epitope mapping is essential for designing vaccines capable of mounting a robust t-and b-cell response. bioinformatics and computational biology can also assist in the discovery of conserved epitopes through sequence variability analysis. in/raghava/antigendb/) stores sequences, structures, origins, and epitopes of pathogen antigens (ansari, flower, & raghava, ) . the computationally optimized broadly reactive antigen (cobra) methodology was recently developed to overcome the challenges associated with antigenic diversity in influenza subtypes (carter et al., ) . this in silico approach, which uses consensus building to generate a number of antigen candidates termed cobra antigens, was used to identify ha antigens that were broadly protective against vlps are divided into two main groups, enveloped and nonenveloped. enveloped vlps are self-assembling capsids, which acquire a lipid layer when budding from their host cells. this layer is absent in nonenveloped vlps (mateu, ) . insertion of heterologous antigens (otherwise known as modularization) into nonenveloped vlps is mainly achieved through genetic fusion or chemical conjugation (peacey, wilson, baird, & ward, ) . the size of the insert has implications for vlp assembly and correct presentation of the antigen. small peptide epitopes are easily inserted into vlp structures without affecting vlp assembly, which can often occur when modularizing whole or large protein domains. genetic fusion is the most popular method despite being time-consuming and error-prone (mateu, ) . chemical conjugation supports the insertion of large antigens in preformed vlps and these modifications are performed on naturally occurring conjugation sites using chemical crosslinkers or enzymes (patel & swartz, ; tang, xuan, ye, huang, & qian, ) . the downside of chemical conjugation is the incurred cost as it requires the production of both the vlp and the epitope(s) as well as conducting the chemical conjugation (chackerian, ; smith, hawes, & bundy, ) . recently developed technologies have started to address the cost and technological challenges. an example is the "plug and display" system, a technology based on two proteins, "the tag" and "the catcher," which react irreversibly when in close proximity of each other. when "the catcher" is fused to a vlp and "the tag" is fused with a vaccine target, these form a two-component vlp vaccine ready to use (brune et al., ) . enveloped vlps can present heterologous membrane proteins (i.e., glycoproteins) in their native configuration on top of a selfassembling capsid protein(s) without the need to engineer both epitope and capsid structures in a process called pseudotyping (chua et al., ; kirchmeier et al., ) . with pseudotyping, one can alter the vlp stability or even its tropism (cronin, zhang, & reiser, ; k. palomares et al., ) . in addition, transmembrane domains within foreign viral envelope proteins can be replaced with transmembrane domains of specific viruses (e.g., vesicular stomatitis virus) to improve pseudotyping efficiency and immunogenicity (kirchmeier et al., ) . enveloped vlps, such as retro-and lenti-vlps, have shown promise as vaccines candidates against diseases, such as influenza, malaria, or dengue virus (chua et al., ; pitoiset, vazquez, & bellier, ) . the engineering of vlp has long been a complex process and often unsuccessful, as the insertion of small peptides can disrupt the vlp structure. the field has evolved and now vlp chimeras are used in both fundamental and applied research (mateu, ; murata et al., ) . a successful example of the insertion of large peptides is the vlp derived from flock house virus that was engineered to carry a receptor domain and could be used as an anthrax antitoxin as well as a vaccine (manayani et al., ) . circulating viruses that exhibit high antigenic variability and high mutation rates, such as influenza, pose substantial challenges for current vaccination strategies . high immunogenicity is a desirable vaccine attribute that potentially translates to protectivity against target infections. to enhance immunogenicity, modularized antigens must be strategically inserted to maximize their presentation to the immune system. has been previously reported (de filette et al., ; haynes et al., ). however, low surface expression or the inability of peptides to adopt native conformation can result in weak immunogenicity at these insertion sites (schödel et al., ) . platforms based upon hbcag, papillomavirus (bovine and human), flock house virus (fhv), and others, are thus engineered to take advantage of surfaceexposed loops that demonstrate enhanced immunogenicity (murata et al., ; slupetzky et al., ; ye et al., ) . peptides inserted into exposed loops protrude from the surface of vlps making them more accessible to the immune system. short peptides displaying relatively simple structures pose little problem for modularization within exposed loops, yet those with more complex structures require further platform engineering (anggraeni et al., ) . incorporation of epitope scaffolds into exposed loops of vlps display of whole protein domains on the surface of vlps allows presentation of multiple antigenic epitopes and increases the likelihood that epitopes will adopt their native conformation. their large size, however, can cause steric hindrance resulting in compromised vlp assembly (lua, fan, chang, connors, & middelberg, ) . as such, several strategies have been developed to specifically modularize large antigens. long, flexible glycine-rich linkers were engineered to flank the antigen in the c/e loop of the hbcag platform, allowing spatial separation and enabling proteins of up to aa to be inserted (kratz, bottcher, & nassal, ) . though effective, optimal linker length for different antigens needs to be empirically determined. steric hindrance can also be addressed in some cases by reducing the antigen content on the surface of the vlp. for example, peyret et al. ( ) engineered a single polypeptide composed of an unmodified hbcag fused to a chimeric hbcag. a dual expression construct was also devised to coexpress unmodified murine polyomavirus (mupyv) vp , with antigen-modularized vp displaying an kda rotavirus antigen tekewe, fan, tan, middelberg, & lua, ) . although such methods can lead to successful vlp assembly, it is possible that increasing the antigen mass, whereas reducing the antigen number may be reflected in lower immunogenicity, as is seen charlton hume et al. with the synthetically derived rts,s malaria vaccine (pitoiset et al., ) . nevertheless, this trade-off is inevitable as the mass of the antigen increases relative to the carrier, necessitating an understanding of the optimization domain. display of large antigens has been reported using novel platforms based upon the hpv l protein and the acinetobacter phage, ap (thrane et al., ; thrane et al., ; brune et al., ) . and mupyv vp proteins has led to the development of capsomere platforms (middelberg et al., ; schadlich et al., ) . removal of the carboxyl termini from each protein yields capsomeres incapable of forming vlps in vivo. mupyv capsomeres display increased stability, although less immunogenic than vlps when administered with adjuvant they are just as effective (middelberg et al., ) . furthermore, a quantitative process study reported that when produced in e. coli, the mupyv capsomere platform is capable of producing million vaccine doses in . days at low cost highlighting its suitability as a rapid response and low-cost vaccine platform . the safe and robust influenza m platform further illustrates the aforementioned, broadly immunoprotective capabilities of vlp platforms. multiple subtypes of ha antigens have been displayed on m -vlps either individually (schwartzman et al., ) or simultaneously (pushko et al., ; sequeira et al., ; tretyakova, pearce, florese, tumpey, & pushko, ) . m -vlp based vaccines afford protection against virus challenge in multiple species (liu, massare, et al., ; pyo et al., ) ( ) f i g u r e application of synthetic biology to vlp vaccine platforms. ( ) enhanced immunogenicity of peptides is achieved through their insertion into exposed loops of viral capsid proteins (murata et al., ; slupetzky et al., ; ye et al., ) . ( ) the structural properties of complex peptides are maintained through the incorporation of epitope scaffolds into exposed loops (schneemann et al., ) . ( ) large antigens are modularized onto vlp vaccine platforms using long flexible linkers to maintain structural separation between the viral capsid protein and the antigen (kratz et al., ) ; or onto preformed vlps using plug and play technologies, such as spycatcher/spytag (brune et al., ) and avitag (thrane et al., ) . ( ) dual expression of modified and unmodified viral capsid proteins reduces steric hindrance and permits vlp assembly (tekewe et al., ) . ( ) the splitcore system permits modularization of antigens with an extended structure through the coexpression of modified and unmodified hbcag core fragments (walker, skamel, and nassal, ) . (a) synthetic production of capsomeres minimizes host cell contaminants reducing required bioprocessing steps (chuan et al., ) . and a stable cell line can be adopted. such a strategy has proven already to be successful for the production of multivalent influenza vlps using an insect high five cell-based platform (sequeira et al., ) . vaccine production scale, cost, and purity vary depending on vlp purification processes. centrifugation, depth, and tangential flow filtration, used for initial clarification and concentration steps, are easily scaled. at the industrial level, chromatographic methods are used to remove host cell and dna impurities as density gradients and ultracentrifugation are not easily scalable. anion exchange and sec are time-consuming and costly, highlighting the need for more efficient and cost-effective methods. nonchromatographic strategies based upon aqueous two-phase systems (atps), a process presently used for enzyme production at the industrial level, are being developed for vlp purification. high yields of rotavirus vlps have been purified from insect cell supernatant using atps, although purity was relatively poor (benavides et al., ) . more recently, single and multistep atps were used to purify human b parvovirus-like particles from insect cell lysates (effio et al., ) . purity levels were greater than %, yet this appeared to be at the expense of yield. monolith technology presents a rapid and scalable method that offers distinct advantages over the classical packed-bed chromatography (vicente et al., ) . hbsag vlps from yeast homogenate was effectively purified using a hydroxyl derivatized monolith (burden, jin, podgornik, & bracewell, ) and when compared to density gradient centrifugation, an anion-exchange monolith yielded -fold more hiv- gag vlps from chinese hamster ovary (cho) cell supernatant (steppert et al., ) . sulfated cellulose membrane absorbers offer significant improvements over conventional ion exchangers membrane absorbers (carvalho, fortuna, et al., ) . as they are easily scaled and reduce the number of required processing steps they may qualify as a generic purification platform for vlp-based vaccines. these technol- (carvalho, moleirinho, et al., vlp vaccine platforms provide opportunities to overcome several limitations of traditional vaccine manufacturing. substantial variation between antigen characteristics of traditional vaccines and their infectious nature adds significantly to production cost as they demand specialized production facilities and tailored manufacturing processes (s. plotkin, robinson, cunningham, iqbal, & larsen, ) . the standardized production of noninfectious, generic vaccine platforms (used to display various antigens) would reduce process variation (i.e., platform remains constant) and provide grounds to establish multiproduct facilities. streamlining of upstream and downstream processes is likely to increase efficiency, reduce laboratory waste, and permit cost savings through bulk buying of equipment and reagents (konstantinov & cooney, ) . predictable bioprocesses may allow the standardization of facility infrastructure and the use of single-use technologies, already in use for some vlp vaccines (roldão et al., ) . disposable technologies offer several advantages over traditional stainless steel equipment including lower initial investment and operating costs, elimination of cross-contamination, and flexibility in terms of scale (lopes, ; shukla & gottschalk, ) . combining disposable technologies with vlp platforms offer advantages on multiple levels and would facilitate vlp translation into the industry. the increased cost and regulatory uncertainty associated with producing new vaccines have long been challenges for vaccine manufacturers (ulmer, valley, & rappuoli, ) . the intricacies of vaccine manufacturing demand a substantial amount of expertize and contribute significantly to cost (s. plotkin et al., ) . expenses associated with vaccine r&d, testing and manufacturing as well as a poor profit margin compared with current drug markets, have led to a dramatic fall in funding from profit-driven pharmaceutical companies (offit, ) . however, this decline is thought to be premature. as demonstrated throughout this review, vlp technologies are advancing rapidly with a strong focus on developing platform capabilities and improving vlp production yields to enable rapid production of safer and cheaper vaccines, particularly when combined with single-use bioprocessing systems. this progress is set to continue with financial investment provided from "not-for-profit," sources, such as national governments, international organizations, and philanthropic bodies. in particular, gavi, the vaccine alliance (balaji, ) and the bill & melinda gates foundation (www.gatesfoundation.org) are motivated to deliver affordable vaccines to third world countries that are the world's biggest vaccine market. to ensure vaccine purity, safety, efficacy, and stability, regulatory authorities govern every stage of vaccine manufacturing from raw materials and production processes to clinical trials and beyond. this includes the assessment and certification of safe and viable manufacturing processes (s. plotkin et al., ) . meeting regulatory requirements can be a complicated process, particularly when manufacturing vaccines for overseas markets or utilizing newly developed bioprocesses. by streamlining upstream and downstream processes and utilizing a generic vlp base, vlp platform technology is expected to reduce the regulatory load of individual vaccines given that regulations for the base and its purification will become well characterized. vlp platforms may even fast track vaccine delivery in response to pandemic circumstances. vaccinology is experiencing an impressive technological revolution, enabling to move vaccines development beyond the rules of pasteur (empirical approach), using data-rich disciplines, such as systems biology, immunology, or computational biology to assist rational vaccines design (modern approach). indeed, the last decade has witnessed a trend toward the use of alternative vaccine designs to attenuated pathogens, having vlps emerged as a powerful and versatile platform for their production. today, vlps are being used not only as vaccines of unmodified viral assemblies against parental viruses but also as scaffolds for displaying heterologous antigens. in addition, tremendous investments have been made to develop new technologies capable of (a) deciphering pathogen biology and vaccine mechanistic responses, and (b) storing and curating extracted data into biological references databases. machine learning algorithms can then use this information for epitope prediction and structure-based modular design. complemented with synthetic biology, this information will provide the basis for (a) engineering vlp functions and (a) developing generic vlp platforms offering not only the potential for streamlined bioprocesses, parallel infrastructure, and predictable biosafety but also the ability to manufacture vaccines against unrelated viruses or pathogens from other sources (i.e., bacteria and parasitic protozoans). although unable to deliver any marketable product to date, these technologies have massive potential to provide, in near future, solutions against untargeted infectious agents (e.g., antibiotic-resistant bacteria, hiv, malaria, or tuberculosis), and most importantly to reduce vaccine development times and manufacturing cost associated with current vaccine platforms. the authors acknowledge the support from australian research sensitivity of immune response quality to influenza helix antigen structure displayed on a modular viruslike particle antigendb: an immunoinformatics database of pathogen antigens expression and purification of virus-like particles for vaccination chimeric hepatitis b core antigen virus-like particles displaying the envelope domain iii of dengue virus type virus-like particles displaying envelope domain iii of dengue virus type induce virus-specific antibody response in mice designing the next generation of vaccines for global public health gavi and the vaccine fund-a boon for immunization in the developing world the use of genomics in microbial vaccine development plant-produced hepatitis b core protein chimera carrying anthrax protective antigen domain- rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems progress towards a universal influenza vaccine efficient induction of mucosal and systemic immune responses by virus-like particles administered intranasally: implications for vaccine design high-throughput process development for biopharmaceutical drug substances immune regulation: immune cell social networks the global threat of zika virus to pregnancy: epidemiology, clinical perspectives, mechanisms, and impact a chimaeric plant virus vaccine protects mice against a bacterial infection plug-and-display: decoration of virus-like particles via isopeptide bonds for modular immunization escherichia colibased cell-free synthesis of virus-like particles a monolith purification process for virus-like particles from yeast homogenate a dna inducing vlp vaccine designed for hiv and tested in mice design and characterization of a computationally optimized broadly reactive hemagglutinin vaccine for h n influenza viruses purification of influenza virus-like particles using sulfated cellulose membrane adsorbers bioorthogonal strategy for bioprocessing of specific-site-functionalized enveloped influenzavirus-like particles universal label-free inprocess quantification of influenza virus-like particles virus-like particles: flexible platforms for vaccine development induction of autoantibodies to mouse ccr with recombinant papillomavirus particles platform technologies for modern vaccine manufacturing immunoreactivity of hcv/hbv epitopes displayed in an epitopepresenting system chimeric hbcag virus-like particles presenting a hpv e epitope significantly suppressed tumor progression through preventive or therapeutic immunization in a tc- -grafted mouse model a novel platform for virus-like particle-display of flaviviral envelope domain iii: induction of dengue and west nile virus neutralizing antibodies virus assembly occurs following a ph-or ca +-triggered switch in the thermodynamic attraction between structural protein capsomeres the economics of virus-like particle and capsomere vaccines enhancing protective immunity to malaria with a highly immunogenic virus-like particle vaccine the ebola outbreak altering the tropism of lentiviral vectors through pseudotyping universal influenza a m e-hbc vaccine protects against disease even in the presence of pre-existing anti-hbc antibodies systems immunology: just getting started vaccine delivery system for tuberculosis based on nano-sized hepatitis b virus core protein particles recent advances in recombinant protein-based malaria vaccines predicting linear b-cell epitopes using string kernels murine polyomavirus virus-like particles carrying full-length human psa protect balb/c mice from outgrowth of a psa expressing tumor comparative immunogenicity evaluations of influenza a virus m peptide as recombinant virus like particle or conjugate vaccines in mice and monkeys pvs: a web server for protein sequence variability analysis tuned to facilitate conserved epitope discovery immunological principles guiding the rational design of particles for vaccine delivery advances in miniaturized instruments for genomics virus-like particles: passport to immune recognition significant productivity improvement of the baculovirus expression vector system by engineering a novel expression cassette development of a genetically-engineered, candidate polio vaccine employing the self-assembling properties of the tobacco mosaic-virus coat protein influenza-pseudotyped gag virus-like particle vaccines provide broad protection against highly pathogenic avian influenza challenge databases and in silico tools for vaccine design microbioreactor systems for accelerated bioprocess development a systems vaccinology approach reveals temporal transcriptomic changes of immune responses to the yellow fever d vaccine isolation of human monoclonal antibodies from peripheral blood b cells broad and potent hiv- neutralization by a human antibody that binds the gp -gp interface escherichia coliderived virus-like particles in vaccine development induction of mucosal and systemic antibody responses against the hiv coreceptor ccr upon intramuscular immunization and aerosol delivery of a virus-like particle based vaccine hyperglycosylated stable core immunogens designed to present the cd binding site are preferentially recognized by broadly neutralizing antibodies bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses bacterially produced recombinant influenza vaccines based on virus-like particles immunodrugs: therapeutic vlp-based vaccines for chronic diseases chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in africa characterization of non-infectious virus-like particle surrogates for viral clearance applications protein delivery using engineered virus-like particles systems analysis of protective immune responses to rts,s malaria vaccination in humans. proceedings of the national academy of sciences of the united states of america virus-like particle vaccine containing the f protein of respiratory syncytial virus confers protection without pulmonary disease by modulating specific subsets of dendritic cells and effector t cells enveloped virus-like particle expression of human cytomegalovirus glycoprotein b antigen induces antibodies with potent and broad neutralizing activity whole-chain tick saliva proteins presented on hepatitis b virus capsid-like particles induce high-titered antibodies with neutralizing potential white paper on continuous bioprocessing protective immunity against murine hepatitis virus (mhv) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an mhv epitope emerging influenza viruses and the prospect of a universal influenza virus vaccine native display of complete foreign protein domains on the surface of hepatitis b virus capsids new approaches and omics tools for mining of vaccine candidates against vector-borne diseases der p peptide on virus-like particles is safe and highly immunogenic in healthy adults biochemical and proteomic characterization of retrovirus gag based microparticles carrying melanoma antigens virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development high-throughput characterization of virus-like particles by interlaced size-exclusion chromatography downstream processing of virus-like particles: single-stage and multi-stage aqueous two-phase extraction inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus overcoming antigen masking of anti-amyloidbeta antibodies reveals breaking of b cell tolerance by virus-like particles in amyloidbeta immunized amyloid precursor protein transgenic mice epsvr and epmeta: prediction of antigenic epitopes using support vector regression and multiple server results integration of novel materials and advanced genomic technologies into new vaccine design structural and computational biology in the design of immunogenic vaccine antigens route of administration of chimeric bpv vlp determines the character of the induced immune responses enhanced production of porcine circovirus type (pcv ) virus-like particles in sf cells by translational enhancers recombinant virus-like particles elicit protective immunity against avian influenza a(h n ) virus infection in ferrets use of rotavirus virus-like particles as surrogates to evaluate virus persistence in shellfish single-use in the biopharmaceutical industry: a review of current technology impact, challenges and limitations. food and bioproducts processing bioengineering virus-like particles as vaccines synthetic biology design to display an kda rotavirus large antigen on a modular virus-like particle improved production efficiency of virus-like particles by the baculovirus expression vector system protein crystallography in vaccine research and development a viral nanoparticle with dual function as an anthrax antitoxin and vaccine a replication-incompetent rift valley fever vaccine: chimeric virus-like particles protect mice and rats against lethal challenge virus engineering: functionalization and stabilization frontline: a therapeutic vaccine for nicotine dependence: preclinical efficacy, and phase i safety and immunogenicity quantitative disassembly and reassembly of human papillomavirus type viruslike particles in vitro the role of genomics in tracking the evolution of influenza a virus human immunodeficiency virus type -neutralizing antibodies raised to a glycoprotein peptide expressed on the surface of a plant virus a microbial platform for rapid and low-cost virus-like particle and capsomere vaccines recombinant virus-like particles as a carrier of band t-cell epitopes of hepatitis c virus (hcv) toolbox for non-intrusive structural and functional analysis of recombinant vlp based vaccines: a case study with hepatitis b vaccine antigenic presentation of heterologous epitopes engineered into the outer surface-exposed helix loop region of human papillomavirus l capsomeres immune activation alters cellular and humoral responses to yellow fever d vaccine development of hepatitis b virus capsids into a whole-chain protein antigen display platform: new particulate lyme disease vaccines a fusion product of the complete borrelia burgdorferi outer surface protein a (ospa) and the hepatitis b virus capsid protein is highly immunogenic and induces protective immunity similar to that seen with an effective lipidated ospa vaccine formula cucumber mosaic virus as a presentation system for a double hepatitis c virus-derived epitope why are pharmaceutical companies gradually abandoning vaccines? middle east respiratory syndrome coronavirus vaccines: current status and novel approaches affinity selection of epitope-based vaccines using a bacteriophage virus-like particle platform protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing l capsid epitopes nipah virus envelope-pseudotyped lentiviruses efficiently target ephrinb -positive stem cell populations in vitro and bypass the liver sink when administered in vivo challenges for the production of virus-like particles in insect cells: the case of rotavirus-like particles virus like particle based strategy to elicit hivprotective antibodies to the alpha-helic regions of gp surface functionalization of virus-like particles by direct conjugation using azide-alkyne click chemistry t-cell epitope vaccine design by immunoinformatics towards the preparative and large-scale precision manufacture of virus-like particles versatile rhdv virus-like particles: incorporation of antigens by genetic modification and chemical conjugation the immune epitope database and analysis resource: from vision to blueprint tandem fusion of hepatitis b core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins enveloped virus-like particle platforms: vaccines of the future? the complexity and cost of vaccine manufacturing-an overview establishing a global vaccine-development fund neutralizing epitopes of influenza virus hemagglutinin: target for the development of a universal vaccine against h n lineages influenza virus-like particle can accommodate multiple subtypes of hemagglutinin and protect from multiple influenza types and subtypes virus-like particles displaying h , h , h hemagglutinins and n neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens pandemic h n influenza virus-like particles are immunogenic and provide protective immunity to pigs bridging the knowledge gaps in vaccine design reverse vaccinology . : human immunology instructs vaccine antigen design antibodies against a truncated staphylococcus aureus fibronectin-binding protein protect against dissemination of infection in the rat virus-like particles in vaccine development on the effect of thermodynamic equilibrium on the assembly efficiency of complex multi-layered virus-like particles (vlp): the case of rotavirus vlp pathogen-like particles: biomimetic vaccine carriers engineered at the nanoscale synthetic biology moving into the clinic predicting class ii mhc-peptide binding: a kernel based approach using similarity scores analysis of modified human papillomavirus type l capsomeres: the ability to assemble into larger particles correlates with higher immunogenicity a virus-like particle that elicits crossreactive antibodies to the conserved stem of influenza virus hemagglutinin syfpeithi: database for searching and t-cell epitope prediction an intranasal virus-like particle vaccine broadly protects mice from multiple subtypes of influenza a virus development of virus-like particles for diagnostic and prophylactic biomedical applications the position of heterologous epitopes inserted in hepatitis b virus core particles determines their immunogenicity a new epitope presenting system displays a hiv- v loop sequence and induces neutralizing antibodies combining stable insect cell lines with baculovirusmediated expression for multi-ha influenza vlp production reverse vaccinology: developing vaccines in the era of genomics single-use disposable technologies for biopharmaceutical manufacturing chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops a papillomavirus-like particle (vlp) vaccine displaying hpv l epitopes induces crossneutralizing antibodies to hpv reengineering viruses and virus-like particles through chemical functionalization strategies influenza virus-like particles containing m induce broadly cross protective immunity an overview of bioinformatics tools for epitope prediction: implications on vaccine development a vlp-based vaccine targeting domain iii of the west nile virus e protein protects from lethal infection in mice preclinical efficacy and safety of an anti-il- β vaccine for the treatment of type diabetes immunization with a chimeric tobacco mosaic virus containing an epitope of outer membrane protein f of pseudomonas aeruginosa provides protection against challenge with p. aeruginosa purification of hiv- gag virus-like particles and separation of other extracellular particles a preliminary evaluation of a recombinant circumsporozoite protein vaccine against plasmodium falciparum malaria. rts,s malaria vaccine evaluation group cobepro: a novel system for predicting continuous b-cell epitopes a malaria vaccine candidate based on a hepatitis b virus core platform dna vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration murine polyomavirus virus-like particles (vlps) as vectors for gene and immune therapy and vaccines against viral infections and cancer a single vaccination with polyomavirus vp /vp her virus-like particles prevents outgrowth of her- /neu-expressing tumors a rapid and simple screening method to identify conditions for enhanced stability of modular vaccine candidates integrated molecular and bioprocess engineering for bacterially produced immunogenic modular virus-like particle vaccine displaying kda rotavirus antigen a novel virus-like particle based vaccine platform displaying the placental malaria antigen var csa bacterial superglue enables easy development of efficient virus-like particle based vaccines effect of immunisation against angiotensin ii with cyt -angqb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase iia study versatile virus-like particle carrier for epitope based vaccines preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein intranasal vaccination with h , h and h hemagglutinins colocalized in a virus-like particle protects ferrets from multiple avian influenza viruses vaccine manufacturing: challenges and solutions large-scale production and purification of vlp-based vaccines the immune epitope database (iedb) . cucumber mosaic virus as the expression system for a potential vaccine against alzheimer's disease splitcore: an exceptionally versatile viral nanoparticle for native whole protein display regardless of d structure structural-based designed modular capsomere comprising ha for low-cost poultry influenza vaccination modular engineering of a microbially-produced viral capsomere vaccine for influenza protective efficacy of a bacterially produced modular capsomere presenting m e from influenza: extending the potential of broadly cross-protecting epitopes developing universal influenza vaccines: hitting the nail, not just on the head the second-generation active a β immunotherapy cad reduces amyloid accumulation in app transgenic mice while minimizing potential side effects expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector particlebased platforms for malaria vaccines chimeric virus-like particle vaccines displaying conserved enterovirus epitopes elicit protective neutralizing antibodies in mice through divergent mechanisms deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis b core particle-based anthrax epitope vaccine virus-like particles as drug delivery vectors bovine papillomavirus-like particles presenting conserved epitopes from membrane-proximal external region of hiv- gp induced mucosal and systemic antibodies current advances in virus-like particles as a vaccination approach against hiv infection. vaccines development of a candidate vaccine for newcastle disease virus by epitope display in the cucumber mosaic virus capsid protein synthetic biology for bioengineering virus-like particle vaccines key: cord- -cob ur authors: sharma, vaneet kumar; sharma, ity; glick, james title: the expanding role of mass spectrometry in the field of vaccine development date: - - journal: mass spectrom rev doi: . /mas. sha: doc_id: cord_uid: cob ur biological mass spectrometry has evolved as a core analytical technology in the last decade mainly because of its unparalleled ability to perform qualitative as well as quantitative profiling of enormously complex biological samples with high mass accuracy, sensitivity, selectivity and specificity. mass spectrometry‐based techniques are also routinely used to assess glycosylation and other post‐translational modifications, disulfide bond linkage, and scrambling as well as for the detection of host cell protein contaminants in the field of biopharmaceuticals. the role of mass spectrometry in vaccine development has been very limited but is now expanding as the landscape of global vaccine development is shifting towards the development of recombinant vaccines. in this review, the role of mass spectrometry in vaccine development is presented, some of the ongoing efforts to develop vaccines for diseases with global unmet medical need are discussed and the regulatory challenges of implementing mass spectrometry techniques in a quality control laboratory setting are highlighted. as the target populations for the vaccines are healthy individuals, pregnant women, or infants, vaccine safety is of paramount importance. appropriately, the vaccine landscape is changing from traditional vaccine approaches to cost-effective, highly scalable, and safe recombinant vaccines. , using recombinant dna technology, antigens are expressed in yeast, escherichia coli, baculovirus expression vector system (bevs), or mammalian cell lines. [ ] [ ] [ ] recombinant antigens are engineered to mimic the first step of virus attachment to the cell surface which is mediated by specific glycoproteins. the expressed recombinant antigens undergo multiple purification cycles to produce highly purified vaccines. , in order for recombinant vaccines to be acceptable to regulatory authorities, in-depth analytical characterization needs to be performed in this review, we focus on the expanding role of mass spectrometry in vaccine development, irrespective of the route of production. we also highlight the regulatory challenges and limitations of mass spectrometry-based techniques which constrain its further implementation as a quality control batch release assay in cgmp manufacturing. the role of mass spectrometry ( alternatively, an o-linked glycan can be formed by linking the oligosaccharide to a serine or threonine residue. glycosylation play a fundamental role in antigen conformation, folding, stability, solubility, and importantly, immune response. , plants, yeasts, and non-human cell lines generate glycans that are not compatible and bioactive within human hosts. thus, about % of all recombinant glycoproteins are produced in mammalian-based expression systems such as chinese hamster ovary (cho) cells. analytical characterization of a glycoprotein is challenging as there is inherent unpredictability associated with the glycans; they are either macroheterogeneous (potential glycan site in the protein not glycosylated) or microheterogeneous (different glycan structures found on the same site in the expressed protein). glycosylation analysis is performed to understand the nature of structural heterogeneity of glycans, quantify them, and more importantly to determine where glycosylation occurs (site specific analysis). mass spectrometry based techniques are valuable tool for detecting and investigating glycosylation ( figure ). although ms-based glycosylation analysis is not without limitations, the advances in ms instrumentation and glycan analysis software have led to increased resolution, automated identification, quantitative determination, and accurate structural characterization. , , another critical quality attribute (cqa) of glycoprotein is intramolecular disulfide bonds (s-s linkages); they ensure correct folding, functional activity and stability. incorrect formation of disulfide bonds can cause protein misfolding which tends to promote aggregation which could result in an unwarranted immune response. confirmation of correct disulfide bond formation in the recombinant first study analyzing the site-specific nglycosylation of gp . this report describes the structural characterization of the expressed gp . reversed phase hplc of the tryptic digest. peptides collected from rp-hplc were further identified by amino acid analysis (aaa) or nterminal sequencing analysis. leonard et al hiv- gp expressed in cho cells. mass spectrometric characterization of the glycosylation pattern. of hiv- gp . maldi and nanoesi-lc-ms/ms using a hybrid quadrupole-time-of-flight tandem mass spectrometer (q-tof) was used to assign glycosylation sites. zhu et al hiv gp jr-fl and con-s env expressed in cho and hek cells. a glycopeptide-based mass mapping approach was used to characterize the glycosylation of two env protein vaccine candidates in a glycosylation site-specific fashion. mass spectrometry based approach was used to map the complete glycosylation profile at every site in eleven hiv- env trimers. high-resolution lc-ms/ms was performed using an orbitrap velos pro hybrid mass spectrometer (thermo scientific) equipped with etd and coupled to an acquity uplc system (waters). go et al hiv gp monomers of the bg . sosip. global n-glycan site occupancy of hiv- gp by metabolic engineering and high-resolution intact mass spectrometry was performed. released n-glycan analysis was carried out using synapt g si ion mobility mass spectrometer. native highresolution mass spectrometry was performed on q exactive hybrid quadrupole-orbitrap mass spectrometer. as illustrated in the following section and in table , mass spectrometry-based techniques have been used to perform the structural characterization, glycosylation profiling and antigen quantitation during the development of the hiv, influenza, dengue, ebola, meningococcal, and other vaccines. the review also highlights that mass spectrometry-based methods such as glycan analysis has been used to analyze a specific envelope glycoproteins (env) and has broad applicability to any other glycoprotein-based vaccines. the quest for a safe and effective vaccine to protect against human one of the hypotheses being explored for protective immunity against hiv is to induce broadly neutralizing antibodies (bnabs) that target the highly glycosylated hiv- virion-associated envelop. a number of approaches are being pursued in the hiv- vaccine development field to establish protective immunity, including monomeric gp subunits and oligomeric gp and trimeric envelope glycoprotein to elicit bnabs. [ ] [ ] [ ] each of these envelop (env) immunogens have multiple exposed epitopes and are heterogeneously glycosylated molecules, with more than % of their mass consisting of glycans (∼ n-linked glycans). it is critically important to monitor this hiv env glycan shield during vaccine development. crispin and collaborators also extensively investigated the native glycosylation profile of envs, in particularly, trimeric immunogen bg sosip. . [ ] [ ] [ ] [ ] their studies led to an increased understanding of the glycan shield of the env and it is now largely accepted that to induce bnabs, the hiv vaccine candidate should have a glycan profile similar to the one present on the native env trimers. [ ] [ ] [ ] in a thorough study on trimeric env glycosylation, behrens et al influenza virus is a segmented, enveloped rna virus and is among the ic-idms-mrm method is truly an alternative to the approved srid method as it has dual purpose "potency and content determination", was found to be equivalent to the srid method and can also be used in response to a pandemic influenza threat. , additionally, a direct ultra-performance liquid chromatography (uplc)-idms method was reported for the rapid and accurate quantification of influenza na. label-free ms-based methods have also been reported for the simultaneous identification and quantification of ha and na in influenza vaccine with samples analyzed by lc-ms e on a waters synapt g mass spectrometer. quantification of proteins by labelfree lc-ms e is a powerful tool, in this method, alternating scans of low collision energy and elevated collision energy during lc-ms analysis to obtain both protein identity and quantity in a single experiment. quantification based on the experimental data showed that the signal intensity was proportional to concentration which allowed for the amount of any protein in the mixture to be estimated. lc-ms e utilizes parallel, multiplex fragmentation where all peptide precursors are simultaneously fragmented throughout the chromatographic the srid assay has been used for over years as a quantitative and potency method throughout the world despite issues regarding variability and availability of standard reagents. thus, even though new methods like hplc and mass spectrometry are being developed, it will take some time for these methods to be adopted worldwide. as the next influenza pandemic cannot be predicted, the health authorities' pandemic preparedness efforts include efforts to ensure expedited availability of pandemic vaccines. methods such as hplc and ms, with their ability to quantitate antigens without standard reference reagents, can become a cornerstone of pandemic influenza preparedness. the viral genus flavivirus, includes dengue virus, yellow fever virus, and and were glycosylated and, predominately, the n-glycan at asn was a high mannose-type and at asn was mainly a combination of complex-and hybrid-type glycans. this study provided important new insights for the role of glycans in the dengue virus-host cell interactions. in a separate study, accurate quantitation of the expressed four viral particles in the tetravalent dengue vaccine (cyd) was performed using targeted ms in selected reaction monitoring (srm) mode. the study described an orthogonal quantitation strategy (targeted ms in srm mode) and demonstrated that the variability of the ms method was low (between % and %) and the assay was linear between . and nmol/l. based on the reported method performance, it could be used to release future batches of the tetravalent dengue vaccine. ebola the data presented demonstrated that the n-glycan patterns were similar between gp , but o-glycan patterns were remarkably different on gp , the five ebola viruses. this study should serve as the foundation for future ebola viral entry and immunogenicity studies. to improve on the conventional approaches for absolute quantitation of gp in ebola virus-like particles (evlps), an isotope dilution full-scan liquid chromatography-high-resolution mass spectrometry method was developed using an ultimate hplc and an orbitrap elite hybrid ion trap-orbitrap mass spectrometer. the reported ms quantitation method provided not only a means to rapidly determine evlp batch quality based upon quantitation of antigenic gp but also ensured adequate preclinical/clinical dosing. chikungunya is a mosquito-borne viral disease, endemic in africa and southeast asia and has also recently emerged in the caribbean. currently there are no drugs or vaccines available for treatment or prevention. the name "chikungunya" derives from a makonde word meaning "to become contorted," and describes the stooped appearance of sufferers with joint pain (arthralgia ms-based approaches have been used to perform the physicochemical characterization for all of these vaccines. in one of the studies, researchers used lc-ms to characterize glycosylated lysine residues in menveo®. in another study a lc-ms method was used to determine the relative reactivity of lysine residues in crm to determine which of these amino acids were more susceptible to conjugation. lc-ms was also used to quantify the bexsero® vaccine which was the first vaccine developed by reverse vaccinology; a genome-based approach to vaccine development. in the field of vaccines, vera-velasco et al not only quantified the viral f, g and m proteins present in the viral particles but also analyzed the cellular proteomic composition in the niv vaccine candidate. traditionally, viral particles have been described as pure entities carrying only viral-derived proteins but the authors successfully analyzed the ratio between cellular and viral proteins in the niv vaccine candidate using lc-ms/ms. west nile fever is a flavivirus that causes a viral infection typically spread by mosquitoes. to date, no vaccine is available to prevent the west nile infection. partially purified virus-like particles were resolved by sds-page, and the coomassie blue-stained band corresponding to env protein was excised from the gel, destained, and analyzed by ms. microcapillary lc-ms was performed using a lcq deca ion-trap mass spectrometer (thermo finnigan) to identify the presence of env protein in virus-like particles to help in developing a potential vaccine. in should be a supplemental batch release test to ensure that the product is within specifications and without any unwarranted modifications. using ms techniques will ensure improved quality and increased safety for clinical trial participants. in conclusion, the evidence suggests that the emergence of ms-based techniques has advanced the field of vaccine development. history of vaccination vaccines, new opportunities for a new society research and development of new vaccines against infectious diseases recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production. hum vaccines immunother the challenge of emerging and reemerging infectious diseases emerging infectious diseases: threats to human health and global stability establishing a global vaccinedevelopment fund new vaccines against epidemic infectious diseases recombinant subunit vaccines: potentials and constraints recombinant therapeutic protein vaccines virus-like particles as vaccine viral vaccines and their manufacturing cell substrates: new trends and designs in modern vaccinology cell culture technology protein subunit vaccine purification cgmp production and analysis of bg sosip. , an extensively glycosylated, trimeric hiv- envelope glycoprotein vaccine candidate a systematic approach to protein glycosylation analysis: a path through the maze structural mass spectrometry in biologics discovery: advances and future trends impact of host cell line choice on glycan profile therapeutic glycoprotein production in mammalian cells analysis of protein glycosylation by mass spectrometry carbohydrates on proteins: site-specific glycosylation analysis by mass spectrometry recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry the quest for an antibody-based hiv vaccine developing an hiv vaccine vaccination with alvac and aidsvax to prevent hiv- infection in thailand new approaches to hiv vaccine development antigenicity and immunogenicity of rv vaccine aidsvax clade e envelope immunogen is enhanced by a gp n-terminal deletion comparable antigenicity and immunogenicity of oligomeric forms of a novel, acute hiv- subtype c gp envelope for use in preclinical and clinical vaccine research hiv- envelope glycoprotein immunogens to induce broadly neutralizing antibodies the hiv glycan shield as a target for broadly neutralizing antibodies assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type recombinant human immunodeficiency virus envelope glycoprotein (gp ) expressed in chinese hamster ovary cells comparison of hplc/esi-fticr ms versus maldi-tof/tof ms for glycopeptide analysis of a highly glycosylated hiv envelope glycoprotein glycosylation site-specific analysis of hiv envelope proteins (jr-fl and con-s) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopesʼ accessibility glycosylation site-specific analysis of clade c hiv- envelope proteins characterization of glycosylation profiles of hiv- transmitted/founder envelopes by mass spectrometry methods development for analysis of partially deglycosylated proteins and application to an hiv envelope protein vaccine candidate analysis of the disulfide bond arrangement of the hiv- envelope protein con-s gp Δcfi shows variability in the v and v regions characterization of host-cell line specific glycosylation profiles of early transmitted/founder hiv- gp envelope proteins glycosylation and disulfide bond analysis of transiently and stably expressed clade c hiv- gp trimers in t cells identifies disulfide heterogeneity present in both proteins and differences in o-linked glycosylation influences on the design and purification of soluble, recombinant native-like hiv- envelope glycoprotein trimers generation and characterization of a bivalent hiv- subtype c gp protein boost for proof-ofconcept hiv vaccine efficacy trials in southern africa glycosylation benchmark profile for hiv- envelope glycoprotein production based on eleven env trimers cell-and protein-directed glycosylation of native cleaved hiv- envelope composition and antigenic effects of individual glycan sites of a trimeric hiv- envelope glycoprotein molecular architecture of the cleavage-dependent mannose patch on a soluble hiv- envelope glycoprotein trimer glycosylation profiling to evaluate glycoprotein immunogens against hiv- envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens the glycan shield of hiv is predominantly oligomannose independently of production system or viral clade structural constraints determine the glycosylation of hiv- envelope trimers ion mobility mass spectrometry for extracting spectra of n-glycans directly from incubation mixtures following glycan release: application to glycans from engineered glycoforms of intact, folded hiv gp maldi-ms/ms with traveling wave ion mobility for the structural analysis of n-linked glycans ion mobility mass spectrometry for ion recovery and clean-up of ms and ms/ms spectra obtained from low abundance viral samples global nglycan site occupancy of hiv- gp by metabolic engineering and high-resolution intact mass spectrometry structural principles controlling hiv envelope glycosylation global site-specific nglycosylation analysis of hiv envelope glycoprotein comprehensive characterization of reference standard lots of hiv- subtype c gp proteins for clinical trials in southern african regions cell culture as a substrate for the production of influenza vaccines: memorandum from a who meeting current and emerging cell culture manufacturing technologies for influenza vaccines critical review of current and emerging quantification methods for the development of influenza vaccine candidates influenza vaccine: the challenge of antigenic drift confronting the next pandemic-workshop on lessons learned from potency testing of pandemic (h n ) influenza vaccines and considerations for future potency tests application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of pandemic influenza (h n ) vaccines at multiple manufacturing sites in china quantification of influenza virus hemagglutinins in complex mixtures using isotope dilution tandem mass spectrometry optimization of digestion parameters for protein quantification simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry quantification of viral proteins of the avian h subtype of influenza virus: an isotope dilution mass spectrometry method applicable for producing more rapid vaccines in the case of an influenza pandemic development of influenza a (h n ) candidate vaccine viruses with improved hemagglutinin antigen yield in eggs quantification of immunoreactive viral influenza proteins by immunoaffinity capture and isotope-dilution liquid chromatography-tandem mass spectrometry immunocapture isotope dilution mass spectrometry in response to a pandemic influenza threat quantification of influenza neuraminidase activity by ultra-high performance liquid chromatography and isotope dilution mass spectrometry label-free mass spectrometry-based quantification of hemagglutinin and neuraminidase in influenza virus preparations and vaccines quantification of proteins by label-free lc-mse absolute quantification of proteins by lcms e a virtue of parallel ms acquisition selective and quantitative detection of influenza virus proteins in commercial vaccines using two-dimensional high-performance liquid chromatography and fluorescence detection approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography, gel electrophoresis and mass spectrometry strain identification of commercial influenza vaccines by mass spectrometry simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by lc-ms e fast and highly selective determination of hemagglutinin content in quadrivalent influenza vaccine by reversed-phase high-performance liquid chromatography method advancing dengue vaccine development site-specific characterization of envelope protein n-glycosylation on sanofi pasteur's tetravalent cyd dengue vaccine absolute quantification of dengue virus serotype chimera vaccine candidate in vero cell culture by targeted mass spectrometry after ebola in west africaunpredictable risks, preventable epidemics identification of n-glycans from ebola virus glycoproteins by matrix-assisted laser desorption/ ionisation time-of-flight and negative ion electrospray tandem mass spectrometry comparison of n-and o-linked glycosylation patterns of ebolavirus glycoproteins development of a liquid chromatography high resolution mass spectrometry method for the quantitation of viral envelope glycoprotein in ebola virus-like particle vaccine preparations chikungunya virus: pathophysiology, mechanism, and modeling development and application of a reversed-phase high-performance liquid chromatographic method for quantitation and characterization of a chikungunya virus-like particle vaccine characterization of nglycosylation profiles from mammalian and insect cell derived chikungunya vlp label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group b outer membrane vesicle vaccine physicochemical characterisation of glycoconjugate vaccines for prevention of meningococcal diseases defined conjugation of glycans to the lysines of crm guided by their reactivity mapping quantification by lc-ms e of outer membrane vesicle proteins of the bexsero ® vaccine the dual role of lipids of the lipoproteins in trumenba, a self-adjuvanting vaccine against meningococcal meningitis b disease robust manufacturing and comprehensive characterization of recombinant hepatitis e viruslike particles in hecolin ® hepatitis e virus: a renewed hope with hecolin molecular and structural characterization of the l virus-like particles that are used as vaccine antigens in cervarix™, the as -adjuvanted hpv- and- cervical cancer vaccine viruslike particles in vaccine development virus-like particle-based human vaccines: quality assessment based on structural and functional properties status of vaccine research and development of vaccines for nipah virus proteomic composition of nipah virus-like particles production of immunogenic west nile virus-like particles using a herpes simplex virus recombinant vector characterization of human enterovirus virus-like particles used for vaccine antigens mass spectrometric characterization of the glycosylation pattern of hiv-gp expressed in cho cells glycan profiles of the n-glycosylation sites of the hiv envelope protein cn gp comparative analysis of the glycosylation profiles of membrane-anchored hiv- envelope glycoprotein trimers and soluble gp mapping the complete glycoproteome of virion-derived hiv- gp provides insights into broadly neutralizing antibody binding haemagglutinin quantification and identification of influenza a&b strains propagated in per. c ® cells: a novel rp-hplc method hplc-based quantification of haemagglutinin in the production of egg-and mdck cell-derived influenza virus seasonal and pandemic vaccines optimization and qualification of a quantitative reversed-phase hplc method for hemagglutinin in influenza preparations and its comparative evaluation with biochemical assays characterization of a recombinant influenza vaccine candidate using complementary lc-ms methods comparative glycomics analysis of influenza hemagglutinin (h n ) produced in vaccine relevant cell platforms glycosylation analysis of engineered h n influenza a virus hemagglutinins with sequentially added historically relevant glycosylation sites particle quantification of influenza viruses by high performance liquid chromatography design and expression of a qconcat protein to validate hi protein quantification of influenza vaccine antigens evaluation of haemagglutinin content by rp-hplc to generate pandemic influenza vaccine topological n-glycosylation and site-specific n-glycan sulfation of influenza proteins in the highly expressed h n candidate vaccines glycosylation characterization of an influenza h n hemagglutinin series with engineered glycosylation patterns: implications for structurefunction relationships cross reactive material glycoconjugate vaccines contain privileged conjugation sites quality by design approach in the development of an ultra-high-performance liquid chromatography method for bexsero meningococcal group b vaccine proteomics-driven antigen discovery for development of vaccines against gonorrhea production and purification of filovirus glycoproteins in insect and mammalian cell lines key: cord- -dmb x authors: lee, sujin; nguyen, minh trang title: recent advances of vaccine adjuvants for infectious diseases date: - - journal: immune netw doi: . /in. . . . sha: doc_id: cord_uid: dmb x vaccines are the most effective and cost-efficient method for preventing diseases caused by infectious pathogens. despite the great success of vaccines, development of safe and strong vaccines is still required for emerging new pathogens, re-emerging old pathogens, and in order to improve the inadequate protection conferred by existing vaccines. one of the most important strategies for the development of effective new vaccines is the selection and usage of a suitable adjuvant. immunologic adjuvants are essential for enhancing vaccine potency by improvement of the humoral and/or cell-mediated immune response to vaccine antigens. thus, formulation of vaccines with appropriate adjuvants is an attractive approach towards eliciting protective and long-lasting immunity in humans. however, only a limited number of adjuvants is licensed for human vaccines due to concerns about safety and toxicity. we summarize current knowledge about the potential benefits of adjuvants, the characteristics of adjuvants and the mechanisms of adjuvants in human vaccines. adjuvants have diverse modes of action and should be selected for use on the basis of the type of immune response that is desired for a particular vaccine. better understanding of current adjuvants will help exploring new adjuvant formulations and facilitate rational design of vaccines against infectious diseases. vaccines are the most effective and cost-efficient method for preventing diseases caused by infectious pathogens. despite the great success of vaccines, development of safe and strong vaccines is still required for emerging new pathogens, re-emerging old pathogens, and in order to improve the inadequate protection conferred by existing vaccines. one of the most important strategies for the development of effective new vaccines is the selection and usage of a suitable adjuvant. immunologic adjuvants are essential for enhancing vaccine potency by improvement of the humoral and/or cell-mediated immune response to vaccine antigens. thus, formulation of vaccines with appropriate adjuvants is an attractive approach towards eliciting protective and long-lasting immunity in humans. however, only a limited number of adjuvants is licensed for human vaccines due to concerns about safety and toxicity. we summarize current knowledge about the potential benefits of adjuvants, the characteristics of adjuvants and the mechanisms of adjuvants in human vaccines. adjuvants have diverse modes of action and should be selected for use on the basis of the type of immune response that is desired for a particular vaccine. better understanding of current adjuvants will help exploring new adjuvant formulations and facilitate rational design of vaccines against infectious diseases. [immune network ; ( ): [ ] [ ] [ ] [ ] [ ] [ ] [ ] keywords: vaccine, adjuvant, infectious disease, innate immunity, adaptive immunity vaccines infectious diseases remain the second leading cause of death worldwide after cardiovascular disease, but the leading cause of death in infants and children ( ) . vaccination is the most efficient tool for preventing a variety of infectious diseases. the ultimate goal of vaccination is to generate a pathogen-specific immune response providing long-lasting protection against infection ( ). despite the significant success of vaccines, development of safe and strong vaccines is still required due to the emergence of new pathogens, re-emergence of old pathogens and suboptimal protection conferred by existing vaccines. recent important emerging or re-emerging diseases were severe acute respiratory syndrome (sars) in , the h n influenza pandemic, and ebola virus in ( ) . last year, the most widespread epidemic of ebola virus caused significant mortality in several west african countries ( ) . as a result, we are aware of pursuing a new approach towards the rapid development of vaccines against emerging diseases. three different types of vaccine are currently used in humans: live-attenuated vaccines, inactivated vaccines and subunit vaccines ( ) . many of the most effective vaccines in use are live-attenuated vaccines. as an attenuated vaccine is composed of a virus or bacterium that can replicate within the host, this type of vaccine elicits robust humoral and cell-mediated immunity (cmi). examples of live-attenuated vaccine include mmr (measles, mumps, rubella), chicken pox, oral polio (sabin), influenza (the seasonal flu nasal spray and the table i . the benefits of adjuvants . decrease the dose of antigen needed (dose sparing) . decrease the number of vaccine doses needed . enhance vaccine efficacy in infants, the elderly and immunocompromised people . increase functional antibody titer . induce more rapid and long-lasting immune responses . induce robust cell-mediated immunity . provide broad protection (cross-reactivity) . facilitate mucosal immunity . overcome antigen competition in combination vaccines h n nasal spray), rotavirus and yellow fever vaccine. inactivated (killed) vaccines (e.g. inactivated polio -salk, hepatitis a) are either heat-inactivated or chemically inactivated particles of the pathogen. although these vaccines are safe and non-infectious, they stimulate only weak, short-lived and often insufficient immunity. thus, large and multiple doses of inactivated vaccine are required to confer protective immunity ( ) . in contrast to live-attenuated vaccines, inactivated vaccines elicit mainly humoral immunity, with little to no induction of cmi. purified or recombinant subunit vaccines derived from non-living vaccine antigens are poorly immunogenic and require the addition of some components to help stimulate protective immunity. in some cases, these vaccines utilize epitopes recognized and bound by antibodies or t-cells. because subunit vaccines contain only an essential part of the antigen instead of the entire microbe, the chances of adverse reactions to the vaccine are relatively low ( ) . subunit vaccines have been made for hepatitis b virus (hbv), influenza virus (injection) and pertussis (part of dtap combined immunization). recently developed subunit vaccines are less immunogenic and reactogenic than traditional vaccines such as live-attenuated, and inactivated vaccines. thus, repeated boost immunizations or the addition of adjuvant are necessary to enhance the efficacy of subunit vaccines. the word "adjuvant" is derived from the latin adjuvare, meaning "to help" or "to aid". adjuvants have been defined as agents added to vaccine formulations that enhance the immunogenicity of antigens and induce protection against infection. vaccines made from live-attenuated or inactivated pathogens can elicit robust protective immune responses because those vaccines contain naturally occurring adjuvants. in contrast, protein-based vaccines in most cases have limited immunogenicity although they have some advantages in terms of safety and cost-effectiveness. thus, adjuvants are necessary to help these proteins become effective vaccines by inducing strong and long-lasting protective immune responses. indeed, some protein-based vaccines have been successfully developed in use for human vaccines by mixing with aluminium salts (alum). however, new vaccine targets will require not only strong antibody responses but also robust cmi including t helper (th) cells and cytotoxic t lymphocytes (ctl). alum alone will be insufficient for such cases because it is a poor inducer of t cell responses. the use of appropriate adjuvants will allow for vaccine formulations that selectively trigger innate immunity and/or adaptive immunity to obtain a desired type of antigen-specific immune responses. we also describe the practical and functional reasons for why adjuvants are needed as a component in vaccines in table i . licensed adjuvants in use for human vaccines are listed in table ii . in , glenny et al. reported the adjuvant activity of aluminium compounds utilizing a suspension of alum-precipitated diphtheria toxoid (dt) ( ) . aluminium salts are the most widely used adjuvants in human vaccines. these adjuvants have been used in practical vaccination for more than years and are generally considered stimulators of th immunity ( , ) . until aluminium salt (referred to as "alum") adjuvants were the only ones contained in vaccines licensed for human use in the united states. alum is a component of licensed human vaccines such as hepatitis a virus (hav), hepatitis b virus (hbv), human papilloma virus (hpv), diphtheria, tetanus, haemophilus influenzae type b (hib) and meningococcal. although there are a number of adjuvants more potent than alum, they have not been used for human vaccine formulations due to high levels of toxicity. surprisingly, despite the wide use of alum adjuvants in licensed human vaccines, the mechanisms of action are not well characterized. the most well-known mechanism of action of alum is the "depot effect", first proposed by glenny in , whereby depot formation was cited to facilitate con- tinuous antigen release from the injection site ( ) . even though depot formation still remains somewhat controversial, recent studies have clearly demonstrated that depot formation is not required for alum adjuvanticity ( ) ( ) ( ) . alum has been shown to facilitate humoral immunity via th type immune responses (igg , ige, il- , il- and eosinophil) ( , ) . the advantages of alum are high safety record, antigen stabilization and augmentation of high and long-lasting antibody titer. however, alum does not have the ability to elicit th type immunity or cytotoxic t cell responses and vaccines containing alum adjuvant cannot be sterilized by filtration, frozen or lyophilized ( ) oil-in-water (o/w) emulsions: mf and adjuvant system (as ) emulsions are unstable two-phase systems consisting of at least two immiscible liquids, combined with a surfactant for stabilization. the major benefits of using emulsions are antigen dose sparing and enhancement of antibody titer. both mf (novartis) and as (glaxosmithkline) are squalene based oil-in-water emulsions ( , ) . mf has been approved for the h n pandemic influenza vaccine (fluad) and also for the h n influenza vaccine (focetria and celtura) in europe ( , ) . it recruits monocytes and macrophages into injection sites by the induction of local chemokine secretion ( ) . mf can also augment antigen uptake by dendritic cells (dcs) and activate cd t cells ( ) . as a result, mf generates high antibody titers with balanced igg :igg a responses. mf has been evaluated in conjunction with herpes simplex virus (hsv), human immunodeficiency virus (hiv), hbv and cytomegalovirus (cmv) vaccine trials ( ) . as is included in licensed h n and h n pandemic influenza vaccines. although both mf and as contain squalene oil, they have different compositions. as contains α-tocopherol. moral et al. demonstrated that as induced a non-specific activation of the immune system in mice in the presence of α-tocopherol ( ) . unfortunately, recent studies have reported a possible association between narcolepsy and the use of as adjuvanted h n influenza vaccine ( , ) . although oil-in-water emulsions seem to be very effective and promising adjuvants, further detailed characterization and analysis of components used in emulsion preparations need to be examined. a virosome is a reconstituted viral envelope possessing membrane lipids and viral glycoproteins, but devoid of viral genetic information ( ) . the virosome vaccine for influenza virus (inflexal v) is approved in europe and hepatitis a virus (epaxal) vaccine is approved in asia, europe and south america ( ). both vaccines utilize virosomes derived from influenza virus represented by immunopotentiating reconstituted influenza virosomes (iriv) harboring the influenza hemagglutinin (ha) protein ( ) . inflexal v is the only viroso- combination enhances antibody titer, th and th type immunity and cd t cell-mediated immunity. combined with saponin and phospholipid. phase mal adjuvanted influenza vaccine licensed for all age groups including children, adults and the elderly. as virosomal adjuvants present antigen via both major histocompability complex (mhc) i and mhc ii, virosomes are able to induce both humoral immunity and cmi ( , ) . major advantages of using virosomes in vaccines are: ) high quality and long-lasting antibody responses, ) conformational stabilization of antigen, ) protection of antigen from degradation, ) excellent safety profile, ) suitability to specific populations such as infants, immunocompromised patients, and the elderly ( ). toll-like receptors (tlr) are transmembrane signaling proteins, comprising a family of pattern recognition receptors (prr) ( ) . tlr agonists, the natural ligands which activate tlrs, are immunostimulatory adjuvants. advances in the design of efficient adjuvants based on the use of tlr agonists have been promising and some of these adjuvants have already been licensed for human vaccines. mpl, a tlr agonist, is a chemically detoxified derivative of the parent lipopolysaccharide (lps) from salmonella minnesota r strain ( ) . mpl increases the production of pro-inflammatory cytokines such as il- and ifn-γ, resulting in the generation of th immune responses ( ) . as is composed of mpl adsorbed to aluminium salts ( ) . two as -adjuvanted vaccines are licensed for human use: the hpv vaccine (cervarix) and hbv vaccine (fendrix) for haemodialised patients ( , ) . since mpl still retains the ability to activate innate immunity by interaction with tlr , it leads to activation of nf-κb signaling and production of pro-inflammatory cyto-kines and chemokines. subsequently, chemokines such as ccl and ccl recruit monocytes and macrophages, and activate dendritic cells (dcs) at the injection site ( ) . mature dcs that have migrated to the draining lymph node can interact with t-cells to stimulate cmi. a benefit of using as adjuvant in human vaccines is the effective induction of robust th -type immune responses by promoting il- and ifn-γ production, which cannot be achieved by using alum alone. a recent study showed that the antigen and as should be co-localized in lymph nodes in order to elicit an adjuvant effect on antigen presenting cells ( ) . table iii summarizes a subset of the adjuvants that have been tested in human clinical trials. all adjuvants listed in table iii are known as "immunostimulators" or "immune potentiators". tlrs provide a bridge between innate and adaptive immunity. a new class of effective vaccine adjuvant is based on the tlr pathway. here, we will focus on tlr , and which are in clinical trials of vaccines against infectious pathogens. tlr is one of the more advanced adjuvant candidates among tlr agonists ( ) . unmethylated cpg oligodeoxynucleotides (odn), a type of tlr agonist, enhance antigen-specific immune responses and induce proinflammatory cytokines such as tnf-α, il- , il- and ifn-γ. cpg odn are an example of immunostimmulatory sequences (iss) currently being evaluated for hbv vaccine (heplisav-b, dynavax) ( ) . polyriboinosinic acid-polyribocytidylic acid (poly i:c) mimics viral dsrna and is a promising candidate for a vaccine adjuvant against intracellular pathogens. poly i:c binds to tlr and enhances robust cmi and potent type i interferon response. however, the major draw-back of stability and toxicity issues need to be addressed before proceeding to clinical application of dsrnas. recently, a clinically safe dsrna, polyi:c analogue (ampligen), was investigated as an adjuvant for intranasal h n influenza virus vaccines ( ) . bacterial flagellin, a tlr agonist, is a known immunostimulator that induces high antibody titer, and mixed th and th type immune responses. the d portion of flagellin binds to tlr and can be expressed in a fusion protein with selected vaccine antigens. due to this characteristic of flagellin, a major advantage of the tlr -dependent adjuvant is that a fusion protein can co-deliver antigen and tlr agonist to the apc ( ) . thus, flagellin fusion proteins are suitable adjuvants for the development of vaccines to induce robust antigen-specific immune responses. indeed, a flagellin/ hemagglutinin-based vaccine (vax ) and a flagellin/matrix protein ectodomain (m e) vaccine (vax ) are in clinical trials of vaccines against influenza ( , ) . although further studies in humans are required, it appears that tlr agonists may be attractive candidates for use in human vaccines. iscoms are another promising lipid-based adjuvant formation. iscoms are spherical and ring-like structures spontaneously formed upon mixing antigens with saponin, cholesterol and phospholipid ( ) . the compound qs- , a potent immunostimulatory saponin, was extensively studied as an adjuvant in various vaccines, though it has not yet been approved for human vaccine use due to the toxicity of qs- . since iscom allows for the reduction in qs- dose, it is being considered as a new approach to overcome the issue of toxicity. the second type of iscom is called iscommatrix, which doesn't contain antigen. the major advantage of iscom and iscommatrix is their exceptional stability owing to the high affinity between saponin and cholesterol, therefore allowing them to be effective adjuvants for mucosal vaccines ( ) . main benefits of these adjuvants are induction of high and long-lasting antibody titer, induction of balanced th and th type immunity, and induction of cmi including cytotoxic t cell response ( ) . the adjuvant properties of iscom and iscommatrix are currently being evaluated in clinical trials of influenza, hcv and hpv. collectively, usage of iscom and iscommatrix as adjuvants could be an alternative approach in vaccine development against infectious pathogens. adjuvant systems (gsk) refer to various combinations of classical adjuvants such as aluminium salts, o/w emulsions, liposomes and immunostimulators designed to adjust the adaptive immune responses against pathogens ( ) . the challenge for this strategy is to define the best combination for an effective and safe formulation in which individual components can synergize with one another to elicit a more robust immune response. as described in table ii , as and as have been approved as adjuvants in several human vaccines. here, we will discuss as and as , which are in recent development. as is identical to as (α-tocopherol＋squalene) with the addition of mpl and qs . while as induces biased th type immune responses, as induces high antibody titer and dominant th type immune responses owing to the addition of mpl. although some local and systemic reactogenicity has been reported, as is in clinical trials for various vaccine applications, including malaria, hbv, hpv, tuberculosis, and hiv ( ) . as combines three components such as liposomes, mpl and qs ( ) . unlike as , as was designed to improve cd t cell responses. as induces robust th type immune responses, enhances antigen presentation to apc, and induces high antibody titer. recent studies demonstrated that an as malaria vaccine induces increased igg titers and polyfunctional cd t cells expressing il- , ifn-γ, tnf-α, or cd l ( ) . several clinical trials are in progress with as -containing vaccine candidates against infectious pathogens, including hiv, tuberculosis and malaria. the ultimate goal of vaccination is to generate potent and long-term protection against diseases. such protective immunity can be elicited by using vaccine formulations containing appropriate antigens and adjuvants. adjuvants are important components of vaccines and can affect the outcomes of vaccination. past approaches of vaccine formulation with adjuvants were focused on single-type adjuvants such as alum or emulsions. however, new vaccine targets require the induction of well-defined cmi in addition to high titer of antibody. consequently, new immunostimulant adjuvants in vaccine formulations are needed in order to stimulate robust immune responses including humoral immunity and cmi. as great progress has been made in the field of adjuvant research over last two decades, vaccinologists are now able to select an appropriate adjuvant from classical adjuvants, immunostimulants or combinations thereof to enhance vaccine efficacy. taken together, recent successful clinical studies conducted with new adjuvants suggest that a panel of novel immunostimulant adjuvants will be utilized for human vaccine formulations in a near future. the availability of these adjuvants in various combinations will facilitate the rational design of vaccines against infectious diseases. infectious diseases: considerations for the st century unmet needs in modern vaccinology: adjuvants to improve the immune response global capacity for emerging infectious disease detection ebola virus disease in west africa--the first months of the epidemic and forward projections vaccine adjuvants: key tools for innovative vaccine design active and passive immunity, vaccine types, excipients and licensing key roles of adjuvants in modern vaccines immunological notes. xvii-xxiv (how) do aluminium adjuvants work? aluminium adjuvants--in retrospect and prospect the antigenic effect of intravenous injection of diphtheria toxin antigen depot is not required for alum adjuvanticity mechanisms of action of adjuvants towards an understanding of the adjuvant action of aluminium new horizons in adjuvants for vaccine development aluminum compounds as vaccine adjuvants the mechanism of action of mf -an innately attractive adjuvant formulation the adjuvanted influenza vaccines with novel adjuvants: experience with the mf -adjuvanted vaccine impact of prior or concomitant seasonal influenza vaccination on mf -adjuvanted h n v vaccine (focetria) in adult and elderly subjects vaccine adjuvants alum and mf induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes the history of mf (®) adjuvant: a phoenix that arose from the ashes adjuvant system as containing alpha-tocopherol modulates innate immune response and leads to improved adaptive immunity designing and building the next generation of improved vaccine adjuvants formation of virosomes from influenza subunits and liposomes influenza virosomes as a combined vaccine carrier and adjuvant system for prophylactic and therapeutic immunizations applications of influenza virosomes as a delivery system toll-like receptors and innate immunity vaccine adjuvants: putting innate immunity to work bacterial cell wall products as adjuvants: early interferon gamma as a marker for adjuvants that enhance protective immunity glaxosmithkline adjuvant systems in vaccines: concepts, achievements and perspectives safety of human papillomavirus (hpv)- / as -adjuvanted vaccine for cervical cancer prevention: a pooled analysis of clinical trials safety and immunogenicity of a new hepatitis b vaccine for the protection of patients with renal insufficiency including pre-haemodialysis and haemodialysis patients as , an aluminum salt-and tlr agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity therapeutic potential of toll-like receptor activation the potential of iss adjuvant in hepatitis b vaccines: heplisav review development of mucosal adjuvants for intranasal vaccine for h n influenza viruses use of defined tlr ligands as adjuvants within human vaccines development of vax , a recombinant hemagglutinin (ha) influenza-flagellin fusion vaccine with improved safety and immune response safety and immunogenicity of a recombinant m e-flagellin influenza vaccine (stf . xm e) in healthy adults iscom, a novel structure for antigenic presentation of membrane proteins from enveloped viruses an overview of adjuvant formulations and delivery systems iscom technology-based matrix m tm adjuvant: success in future vaccines relies on formulation recombinant liver stage antigen- (lsa- ) formulated with as or as is safe, elicits high titer antibody and induces ifn-gamma immunomodulatory properties of the vaccine adjuvant alum alum adjuvanticity: unraveling a century old mystery recent advances in vaccine adjuvants: the development of mf emulsion and polymeric microparticles development and evaluation of as , an adjuvant system containing alpha-tocopherol and squalene in an oil-in-water emulsion adjuvants for pandemic influenza vaccines liposomes as vaccine delivery systems: a review of the recent advances influenza virosomes as vaccine adjuvant and carrier system role of as in human papillomavirus vaccine: mode of action and clinical profile the authors declare no conflict of interest. key: cord- - kdhf authors: lau, yuk-fai; tang, lay-hoon; ooi, eng-eong title: a tlr ligand that exhibits potent inhibition of influenza virus replication and has strong adjuvant activity has the potential for dual applications in an influenza pandemic date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: kdhf the appearance and spread of the h n highly pathogenic avian influenza (hpai) raise concern of a possible pandemic. current preventive measures include the development of a pre-pandemic influenza vaccine and stockpiling of neuraminidase inhibitors. however, their benefits can be significantly reduced by mutations in the hemagglutinin or neuraminidase resulting in antigenic changes and the appearance of drug-resistance, respectively. drugs that target the innate immune system to achieve a ‘heightened antiviral’ state represent another class of antiviral agents that could contribute to the control and treatment of influenza infection. in this study, pika (a stabilized dsrna) provides broad-spectrum prophylaxis against a number of influenza a viruses. in addition, when pika was admixed with influenza vaccine preparations, including a formalin-inactivated whole-virion h vaccine, significant adjuvanting effect leading to accelerated viral clearance was observed in a murine model. these biological effects appear to be mediated by the ability of pika to promote the maturation of dendritic cells, including up-regulation of co-stimulatory molecules, such as cd and cd , and the induction of various cytokines and chemokines. toll-like receptor (tlr ) was shown to recognize pika in a concentration-dependent manner. the potency and versatility in its activities make pika an attractive candidate for use in an influenza pandemic. the most effective tool for controlling an influenza pandemic is a suitable vaccine. for optimal effectiveness, the pandemic vaccine needs to be based on the causative strain and therefore, vaccine cannot be produced until the pandemic strain is identified. this criterion poses serious constraints in the capability of vaccine manufacturers to prepare pandemic vaccines in advance for stockpiles. in addition, a number of studies have shown that the hemagglutinin (ha) molecules of avian h viruses are poorly immunogenic [ , ] , where up to g of antigen ( times the normal dose of human influenza virus ha) was required to elicit potentially protective responses in a substantial number of subjects [ ] . this need for a larger antigen dose further reduces the availability of the 'limited' pandemic vaccines. in an attempt to increase the immunogenicity of the antigen, adjuvants like alum and mf- have been incorporated into vaccine formulations. although alum may enhance the magnitude of the antibody response to a high dose of antigen, it does not have significant antigen-sparing effect [ , ] . other adju-vants, such as mf or a proprietary oil emulsion adjuvant, have been tested and data indicate that the inclusion of these adjuvants in the vaccine preparation would significantly increase antibody titers [ , ] . given the likelihood that a suitable vaccine will not be available before the onset of a pandemic, initial protection in a pandemic may have to rely on antiviral treatment and prophylaxis. currently available anti-influenza drugs include neuraminidase inhibitors (oseltamivir and zanamivir) or ion-channel blockers (adamantanes). the success of these drugs, both for prophylaxis or therapeutics, is based on the susceptibility of the viruses to these drugs. recently reported studies, however, have shown that a number of h isolates are resistant to these drugs [ ] [ ] [ ] . these drugs or classes of drugs alone may thus be insufficient in protecting the human population from an influenza pandemic. an alternative, antigen-independent approach that could slow down the transmission of the virus and provide more time for vaccine production is thus highly desirable. the toll-like receptors (tlrs) are a collection of receptors that detect conserved molecular components encoded by microorganisms (reviewed in refs. [ , ] ) and stimulation of these receptors by specific ligands leads to the activation of the innate immune cells, such as dendritic cells (dc), macrophages and nk cells [ , ] , through a variety of signaling pathways, such as nf-b transcription factors, c-jun nh terminal kinase (jnk), mitogen-activated protein kinases (mapks), resulting in quantitative and qualitative changes in immunological functions, including antigen presentation. in addition, cytokines, such as ifn-␣, tnf-␣ and il- p , are produced in vivo shortly after tlr activation [ , ] . the production of these cytokines results in antiviral effects observed in a number of infection models. for example, administration of synthetic lipid a analogs (tlr agonists) protects mice against an influenza challenge [ ] . a possible explanation for this anti-influenza effect could be related to the production of type interferons, which induce the activation of ifn-stimulated genes including mx proteins, protein kinase r and ' oligoadenylate synthetase [ ] . the induction of these proteins increases the resistance to the replication of the influenza virus [ ] [ ] [ ] . the present study was designed to investigate the efficacy of pika, which is a synthetic stabilized form of double stranded rna, as a stand-alone agent for its antiviral effect and to evaluate the efficacy of pika as an adjuvant when co-administered with inactivated influenza vaccines, including an h n vaccine. our results show that pika has potent anti-influenza effect when administered h prior to or immediately after influenza virus challenge in mice. the antiviral effect is not strain-specific. when used as an immuno-adjuvant, pika was able to achieve significant antigen-sparing effect. when incubated with immature dc, pika altered the expression pattern of a number of immune genes. in addition, pika was capable of interacting with both human and mouse tlr , leading to the production of nf-b. thus, in the face of a potential influenza pandemic, pika could be a candidate as a prophylactic drug or could be used as an adjuvant to increase the population coverage when pandemic vaccines become available. specific pathogen-free (spf) male balb/c mice ( - weeks old) were purchased from the centre for animal resources (care), national university of singapore. the animal protocol used in this study was reviewed and approved by the institutional animal care and use committee (iacuc) of dso national laboratories, singapore. a/puerto rico/ / (h n ) and the reassortant virus mem , which bears the ha of a/memphis/ / (h n ) and the neuraminidase (na) of a/bellamy/ (h n ) are gifts from professor lorena brown, the university of melbourne. a/ws/ (vr- ) is from the american type culture collection (atcc). the reverse-engineered h n influenza virus was made based on the method described by hoffmann et al. [ ] , using an expression plasmid provided by dr brendon hanson (dso national laboratories, singapore). the ha sequence of a/indonesia/cdc l/ (cy , a clade , subclade virus which is closely related to a/indonesia/ / ) was downloaded from genbank and was synthesized by geneart ag (regensburg, germany). the viruses were grown in the allantoic cavity of -day-old embryonated chicken eggs for days at • c. allantoic fluids were pooled and divided into aliquots, and stored at − • c until use. the - south hemisphere seasonal influenza vaccine, fluvax, was from csl ptd ltd. (victoria, australia). virions of the reverse-engineered h n virus were concentrated by ultra-centrifugation and resuspended in pbs. formalin-inactivated purified whole-virion vaccine was prepared by inactivating the virions with . % formalin for days at • c followed by dialysis in pbs. the total protein concentration in the vaccine was measured by coomassie plus (pierce biotechnology, inc., il, usa). the amount of ha content was estimated to be % of the total protein content. complete and incomplete freund's adjuvant (cfa and ifa) were obtained from sigma-aldrich (st louis, usa). pika was obtained from newbiomed pika pte ltd. (singapore). for intranasal (i.n.) immunization, groups of mice were anesthetized with an intraperitoneal (i.p.) injection of ketamine and xylaxine followed by i.n. administration of immunogens in l. for immunization by the subcutaneous (s.c.) route, the immunogens were administered in l at the base of the tail. in selected experiments, g of pika was given to anesthetized mice i.n. in a volume of l. when given by the s.c. or i.p. route, g of pika was given in l volume per dose. pika contains dsrna that is greater than base pairs in length. three weeks after final vaccination, mice were challenged i.n. with . - . pfu of various strains of influenza virus in a volume of l. mice were sacrificed at various time-points and pulmonary viral titers were determined using previously described methods [ ] . mice were anesthetized by ketamine/xylaxine injection and blood samples were collected from the orbital plexus. sera were collected and stored at − • c until analysis. the titer of immunogen-specific antibodies were quantified by elisa, as previously described [ ] . avidity elisa was carried out using previously described method [ ] . hek cells were maintained in optimem (invitrogen) with % fetal calf serum and were transfected in flat-bottomed -well plates as previously described [ ] . each sample was tested in triplicate. six hours after the stimulation, luciferase and ␤-galactosidase activity in each sample was measured by commercial kits (promega corporation, wi, usa). the relative stimulation of nf-b was calculated by normalizing luciferase activity with ␤-galactosidase activity and was expressed using the readings from unstimulated wells as a baseline. the level of expression of various cytokine genes was determined using a lightcycler (roche, in, usa) using primers described in giulietti et al. [ ] . samples containing g of total rna from the abovementioned dc experiment were used for microarray analysis. piqor immunology microarrays (miltenyi biotech, gladbach, germany) were used. samples were amplified and labeled with different fluorochromes according to manufacturer's instructions. samples from pika-stimulated dc and unstimulated dc were labeled with cy and cy , respectively. after hybridization, the slides were scanned using the imagene software (biodiscovery). each gene was printed in quadruplicate on the array. net signal intensity, data normalization and calculation of the cy /cy ratios were performed by miltenyi biotech using the piqor analyzer software. one million d cells were stimulated with g of pika or g of lps ( :b , sigma-aldrich) overnight. the supernatants were collected and the cytokine protein levels measured using the bioplex protein array system (bio-rad, hercules, ca), in duplicates and against a standard curve according to manufacturer's instructions. the significance of any difference between any two different groups was assessed by the mann-whitney test using prism (graphpad software, ca, usa). reported p values < . are considered significantly different. in this study, the murine influenza model was employed to demonstrate the antiviral properties of pika. in this model, anesthetized mice were challenged with influenza virus i.n. this mimics a total respiratory tract infection and the pulmonary viral titer reaches its peak on day post-infection (pi) and remains at a high level till day pi (data not shown). using this model, groups of five mice were given g of pika i.n. h prior to challenge with plaque forming unit (pfu) of influenza/a/pr/ / . the pulmonary viral titer in the lungs was determined on day pi. as shown in fig. a , mice that received pika treatment at and h pi had the lowest pulmonary viral titer at day and this was significantly lower than the titer in control mice that received pbs (p = . ). in addition, significant reductions in pulmonary viral titers were also observed in mice that received only one dose of pika at h prior to infection. the difference in the pulmonary viral titer between the group that received daily treatment and the group that received only one dose of pika was not statistically significant (p = . ). after demonstrating that pika has an inhibitory effect on influenza viral replication in vivo, it was of interest to determine the optimal conditions for the treatment. to examine this, mice were given different concentrations of pika i.n. and were challenged with pfu of pr virus i.n. the mice were treated at and h and were sacrificed on day . a dose-dependent response was observed (fig. b) , in which mice given g of pika daily had the lowest mean viral titer among all the groups. an inhibitory effect of pika on viral replication was detected at g per dose but not at g per dose (p = . ). to determine whether pika could be used as a treatment option at the time of infection, mice were given pika immediately after infection with pfu of pr virus. as shown in fig. c , this treatment protocol was effective in reducing the pulmonary viral titer and the titer was not significantly different from the prophylaxis group that received pika h prior to infection (p = . ). however, it is apparent that daily treatment is preferable because the group that received only a single dose had a significantly higher titer of virus in the lungs compared to those that received daily doses (p = . ). intranasal administration of pika produced the most significant anti-influenza effect compared to s.c. or i.p. administration of the drug (fig. d ) though the pul-monary viral titers in the treated mice were still significantly lower than the titers of the pbs control group (p = . ). after demonstrating the effectiveness of pika in inhibiting the replication of pr virus in vivo, we sought to determine whether similar inhibition would be observed with different influenza viruses. groups of five mice were given pika treatment and were challenged with pfu of pr (h n ), mem (h n ) and a/ws/ (h n ), as previously described. on day pi, the viral titer of the pika-treated groups were significantly lower than the titers of the pbs group, regardless of the subtype of the challenge viruses ( fig. a -c, p = . , . , . ) respectively. in addition, mice were also challenged with higher doses of virus, or pfu ( and ld respectively) of pr . as shown in fig. a and b, the pika-treated mice had lower pulmonary titers than the pbs control group at these higher challenge doses (p = . ). in addition, when challenged with . pfu of mem , there was a % reduction in viral titer in pika-treated mice compared with the pbs control group (fig. c) . to demonstrate the therapeutic potential of the treatment, groups of five mice were challenged with pfu of pr i.n. and received pika treatment or h pi. on day pi, as shown in fig. d , significant reduction in pulmonary viral titers was observed in pika-treated mice (p = . , . ) compared with the pbs-treated group. therefore, in summary, we demonstrated that pika has the ability to inhibit the replication of several strains of influenza in vivo. although intranasal administration of the drug before the establishment of an infection provided the best protection, substantial viral reduction could still be achieved if the drug were given in the course of an established infection. since pika is an effective adjuvant for hepatitis b vaccine [ ] , we examined whether pika could enhance the immunogenicity of influenza vaccine. we sought to demonstrate that the enhancement of the humoral responses would lead to significant viral reduction in replication of challenge virus. previous studies have showed that when administrated at between . and g per dose, different forms of influenza vaccine could induce robust antibody responses without any external adjuvant [ ] [ ] [ ] with protection against viral challenge [ ] . as shown in fig. a , mice immunized with a single dose of the trivalent vaccine at . g s.c. had robust antibody responses which were comparable to those induced when the vaccine was admixed with cfa. when the vaccine dose was reduced by - fold ( . g) , there was about -fold reduction in the mag-nitude of the antibody responses. to mimic a vaccine shortage situation, a dose of . g, (∼ -fold reduction from the optimal dose) was selected and we evaluated whether the inclusion of pika could compensate for the reduction in antigen concentration. groups of five mice were vaccinated with a suboptimal dose of fluvax vaccine by the s.c. or i.n. route. three weeks after priming, sera were collected from the mice before they were boosted. sera were collected from the mice weeks after boost. as shown fig. . pika acts as a potent adjuvant by enhancing the immunogenicity of the seasonal influenza vaccine. (a) groups of four mice were vaccinated with either or l of fluvax influenza vaccine (containing . or . g of ha from each subtype) in pbs or in cfa by the subcutaneous route. on day , sera were collected and the antibody titer of the sera was determined by an elisa assay, using fluvax as the coating antigen. (b) groups of five mice were vaccinated with . l of fluvax influenza vaccine (containing ng of ha from each subtype) in pbs, either by the subcutaneous route at the base of the tail or by the intranasal route. some groups received the vaccine with additional adjuvant as indicated in the x-axis. for those that received pika as an adjuvant, g of pika was admixed with the vaccine prior to administration. on day post-vaccination, sera were collected and the mice received another boost by the same route and sera were collected on day . the antibody titer of the sera was determined by an elisa assay, using fluvax as the coating antigen. each dot represents the antibody titer of an individual mouse. the primary and secondary antibody titers are represented by the black and red dots respectively and the lines represent the geometric mean antibody titer of each group. the '*' symbol indicates that the difference between two groups is statistically significant (p < . ). (c) to determine the level of protection mediated by the antibody response, the mice were challenged with pfu of pr intranasally weeks after the boosting and the pulmonary viral titer was determined on day post-infection. closed circles represent the lung virus titer of an individual mouse and the line represents the geometric mean titer of the group of mice. the percent reduction in mean viral titer relative to the pbs control group is shown above each column of data. the '*' symbol indicates that the difference between the two groups was statistically significant (p < . ). (d) serum samples were diluted : and the avidity of the antibodies was tested in a urea-displacement elisa. the o.d. of the urea-treated plates was expressed as a percent of the untreated plates. data are represented as the mean and standard derivation of five mice. in fig. b , only two out of the five mice that received the vaccine without an adjuvant by the s.c. route had detectable amounts of anti-ha antibody in the primary response. for the cfa group, four mice had anti-ha antibody whereas all animals in the vaccine with pika group had detectable levels of anti-ha antibody. in the secondary responses, although all mice given vaccine alone developed anti-ha antibody, the antibody titers were significantly lower than those that were given vaccine with pika (p = . ). as for intranasal delivery of vaccine, none of the vaccine protocols was able to elicit detectable anti-ha responses after one dose. when two doses of vaccine were given with pika, the vaccine recipient group showed a low titer of anti-ha response. the response was substantially improved by the addition of pika to the vaccine (p = . ). interestingly, the titer of the sera attained by two doses administered i.n. was comparable to titers achieved by two doses given by s.c. injection (p = . ). in order to demonstrate that the enhanced antibody responses can lead to improved efficacy, vaccinated mice were challenged with pfu of pr i.n. and the pulmonary viral titers were determined days after challenge. as shown in fig. c , mice given two doses of the vaccine showed a % reduction in viral titer compared to the pbs control group (p = . ). although the cfa group had the highest titer of anti-ha responses (fig. a) , the group only showed a % reduction in viral titer, which was not statistically different from the group that received unadjuvanted vaccine (p = . ). on the other hand, the group given vaccine with pika the pulmonary viral titer was determined as previously described. the percent reduction in mean viral titer relative to the group treated with pbs is shown above each column of data. the '*' symbol indicates that the difference between the two groups was statistically significant (p < . ). showed the most potent viral reduction, with . % reduction in viral titer compared to the pbs control group, and this difference was statistically significant (p = . ). the group that received pika alone served as control to ensure that the observed reduction in pulmonary viral titer was due to the anti-ha antibody responses, instead of the direct inhibitory effects of pika on viral replication shown in the fig. . the enhancement in viral clearance was also observed in the group that received the vaccine by the i.n. route (fig. c) . mice that received either vaccine alone or vaccine with pika showed a . % and . % reduction, respectively, and the difference between the groups was statistically significant (p = . ). it is interesting to note that, although the group received the vaccine admixed with cfa had the highest antibody titers (fig. b) , however, the mice had higher pulmonary viral titers compared with mice received the vaccine administrated with pika (fig. c ). to investigate this discordance between the two parameters, the relative avidity of the antibodies of the two groups against the vaccine antigen was examined in a urea-displacement elisa. as shown in fig. d , the group received the vaccine with pika had antibodies with higher avidity to the surface glycoprotein of the virus as significantly more antibodies remained bound to the ha when exposed to m urea as compared to those induced with cfa as an adjuvant (p = . ). there is an accumulating body of data showing that h n virus vaccines are generally poorly immunogenic. we therefore proceeded to examine whether pika was an effective adjuvant for an h n vaccine. groups of five mice were inoculated by the s.c. route with a formalin-inactivated h vaccine, with or without an adjuvant, and weeks later, sera were collected to determine the antibody titer against the virus. as shown in fig. a , the whole-virion vaccine was capable of inducing a measurable titer of antibody after one dose. however, with the inclusion of pika, there was a ∼ -fold increase in antibody titer (p = . ), and this was comparable to the titer achieved in the group that received the vaccine with cfa. when challenged with the homologous virus i.n., however, this group of mice was the only group which showed significant reduction in pulmonary titer on day pi (fig. b, p = . ) . the efficacy was further boosted by a second dose of h vaccine with pika. mice in this group showed a further -fold reduction in viral titer (fig. c ). in addition, the vaccine-pika formulation was efficacious in reducing viral replication even when the vaccine was delivered intranasally. in contrast, in the absence of pika, the vaccine administered by the i.n. route failed to induce a significant reduction in viral titer compared with the pbs control (p = . ). in summary, we have demonstrated that the inclusion of pika in two different formations of influenza vaccine can achieve substantial antigen-sparing with robust humoral immune responses, leading to potent pulmonary viral titer reduction in vivo. in an attempt to elucidate the biological basis for the inhibition of viral replication and the adjuvant effects of pika, we sought to identify the receptor(s) that might be involved in these processes and to examine how cells of the innate immune system respond to pika stimulation. using a human tlr -expressing plasmid, we showed that pika is capable of interacting specifically with tlr in a dose-dependent manner (fig. ) . similar results were obtained when the experiment was carried out with mouse-tlr -expressing plasmid (data not shown). next, we investigated the outcome of stimulating dc in vitro with pika given that dcs are the most potent antigen presenting cells (apc), responsible for antigen capture and presentation to naïve t lymphocytes. using primary immature dc culture, d cells, we examined how dc responded to pika stimulation in vitro. pika was added into dc cultures for h following which total rna was extracted from the cells. the expression level of more than immunologically related genes between pika-stimulated and unstimulated samples were compared using microarray technology. as shown in fig. a , pika-stimulated dcs up-regulated expression of a number of cellular activation markers, such as cd and cd compared to unstimulated cells. a number of genes that have important immunological functions were also found to be up-regulated after pika treatment, including irf- (interferon regulatory factor ), il- , il- , il- and il- receptor. many chemokines, such as mip (macrophage inflammatory protein), rantes, mcp- (macrophage chemoattractant protein) and ip- (interferon gamma inducible protein) were also found to have increased expression. quantitative real-time pcr was used to confirm these findings. using cdna from unstimulated dc as the baseline, many genes were up-regulated in a dose-dependent manner (fig. b) . while certain genes, such as inos and il- , were up-regulated to a comparable level in lpsstimulated dc, other genes, such as cd l, mip and ifn-␥, were up-regulated more readily by pika stimulation. to demonstrate that the observed changes in gene expression lead to changes in these cytokines/chemokines, the level of these proteins in the culture supernatants was measured. consistent with the up-regulation of cytokine/chemokines genes, there was an increase in the production of these cytokines/chemokines in the supernatant from pika-stimulated dc compared with those from the unstimulated dc (fig. c ). it has been years since the first case of human infection with a highly pathogenic h n virus was identified in hong kong [ ] and the virus has now spread to many regions, including southeast asia, western china [ ] , africa [ ] , turkey [ ] and siberia [ ] . most human cases to date involve close contact with infected poultry and human-to-human transmission remains limited [ ] . nonetheless, the lack of anti-h immunity in humans, together with the continuous evolution of the virus in close proximity with the human population, threatens a pandemic. should the virus acquire the property of efficient transmissibility between humans, in combination with the high case-fatality rate resulting from infection, this virus has the potential of causing an influenza pandemic with damage on a scale similar to that achieved by the 'spanish' flu pandemic. therefore, there is an urgent need to develop effective prophylaxis and therapeutic strategies in preparation for a possible pandemic. in the current study, using a murine influenza model, we have evaluated the potential of pika for prophylaxis and treatment of influenza infection as well as an adjuvant for influenza vaccine. our data demonstrate that administration of pika intranasally prior to or shortly after an influenza infection can inhibit influenza replication, leading to a significant reduction in pulmonary viral titers. this effect is unlikely to be due to a direct antiviral activity, because pre-incubating influenza virus with pika did not inhibit virus infectivity for embryonated chicken eggs (lynn tang, unpublished data). instead, pika appears to act by stimulating the innate immune system (fig. ) , resulting in the production of several chemokines, cytokines and interferons and these, in turn, mediate the observed antiviral activity. myxovirus resistance (mx) proteins, protein kinase r and ' ' oligoadenylate synthetase are some of the products that have been reported to be up-regulated after type interferon production (reviewed in ref. [ ] ), achieving an antiviral state in the host [ ] . the fact that pika administration results in the stimulation of several antiviral proteins in the host, it decreases the likelihood that viruses will develop resistance through mutation. furthermore, as pika does not target a specific component of the virion for its antiviral activity, it is likely to be effective against multiple strains and subtypes of influenza viruses as demonstrated in this study. this broad-spectrum activity is not limited to influenza a viruses only. a number of studies have shown that tlr-ligands are capable of inhibiting a wide range of viruses, including herpes simplex virus- [ ] , cytomegalovirus [ ] , parainfluenza [ ] , west nile virus [ ] , severe acute respiratory syndrome coronavirus (sars-cov) [ ] and influenza virus [ , ] . the abovementioned advantages make this novel antiviral approach a compelling alternative to traditional antiviral drugs and vaccines especially since avian influenza viruses have rapidly developed resistance to antiviral drugs [ ] and some mutant viruses can maintain drug-resistance without losing virulence [ ] . in addition, thitithanyanont et al. showed that stimulating dc with poly(i:c) conferred the cells with resistance to the cytopathic effects of h n virus which might be important for the induction of virus-specific immune responses during the infection [ ] . apart from being an effective agent for prophylaxis and treatment, pika can also act as an effective adjuvant. using two different forms of influenza vaccine (split subunit vaccine and whole inactivated vaccine), the magnitude of the humoral responses elicited by the combination with pika was comparable to those formulated in freund's adjuvant. our observation is consistent with the report by shen et al. [ ] which showed that administering fig. . pika induces maturation of dendritic cells, with expression of a wide range of immunological genes. immature murine dendritic cells, d cells, were incubated with g per ml of pika or were unstimulated overnight. (a) total rna was harvested from the cells and converted into cdna. the cdnas derived from pika-stimulated dc and unstimulated dc were amplified and labeled with cy and cy dye respectively. the samples were hybridized overnight to a microarray chip and fluorescence signals were measured by an array scanner. each gene target was printed times on the array and the normalized mean cy /cy ratio and coefficient of variation (cv) of the four replicates were determined by the software. (b) rna was harvested from the dcs and converted into cdna. the expression level of each cytokine gene was determined by quantitative real-time pcr. the expression level was normalized with beta-actin, a house-keeping gene, and data were expressed relative to unstimulated samples. the bars and error bars represent the mean and standard deviation of triplicate samples and are representative of two independent experiments. (c) supernatants were harvested from dcs stimulated overnight with either g of pika, g of lps or unstimulated. fifty microliters of the supernatant was used to test for the presence of various cytokines/chemokines in the supernatants using the bioplex protein array system and were measured in duplicate. the bars and error bars represent the mean and standard error. pika with hepatitis b vaccine boosted the humoral response. we explored the concept further by demonstrating that a substantial antigen-sparing effect could be achieved by incorporating pika into vaccines. using the commercial split vaccine, the antigensparing effect was about -fold ( fig. a and b) . furthermore, we demonstrated that the augmented humoral response led to a func-tional reduction in viral titer (fig. c ). our data also showed that successful immunization could be achieved by a non-parenteral route. with two doses, the magnitude of the response achieved by intranasal immunization was comparable to that achieved by subcutaneous immunization and was associated with a similar level of viral clearance. our finding is consistent with the report from ichinohe et al. [ ] in which they showed that another tlr ligand, ampligen, was capable of enhancing the immunogenicity of a trivalent inactivated influenza vaccine when administrated intranasally. in their study, the enhancement effect was demonstrated using a higher dose of the trivalent vaccine ( g) and in this current study, we extended the observation further by showing that similar enhancement effect can be achieved even when the concentration of the immunogen was reduced to . g ( -fold lower). the antigen-sparing effect of pika might be an important strategy for maximizing vaccine coverage when vaccine supplies are limited. it is of interest to note that although the group that received the influenza vaccine with pika did not have the highest antibody titer, it had the lowest pulmonary viral titer (a -fold reduction compared with the cfa adjuvant vaccine group). the higher avidity of the antibodies induced by the vaccine with pika might allow more effective inhibition of viral replication in vivo which translated into lower pulmonary viral titers as observed in this study. this suggests that in addition to augmenting the amount of antibody produced, pika may also have an impact on the quality of the humoral responses, such as affinity maturation [ , ] . because pika is a stabilized form of double stranded (ds) rna that lacks direct anti-influenza activity, we evaluated its effect on the innate immune system in search of the mechanism underlying the observed activity. dsrna was shown to be a ligand of tlr [ ] and using a transient transfection system, we showed that pika maintained its tlr binding property. incubation of pika with primary immature murine dc in vitro resulted in maturation of dendritic cells, with marked up-regulation of co-stimulatory molecules, such as cd and cd and cytokines with potent immunostimulatory functions such as il- and il- . our observation is consistent with those observed by shen et al. with bmdc [ ] . the maturation process enables dc to act as a potent antigen presenting cells for effective activation of naïve lymphocytes [ , ] , which in turn, leads to a more robust immune response. in addition, a study conducted by perera et al. [ ] showed that a vaccinia virus expressing il- induced superior cellular and humoral immune responses compared to the parental strain. therefore, the induction of il- by pika could also have resulted in a more efficient induction of cd + t cell responses, translating to more robust humoral responses. lung tissue has recently been shown to constitutively express tlr in vivo in mice [ ] and intranasal administration of pika could directly stimulate these tlr- expressing cells, which is perhaps why pika worked most effectively when administrated intranasally (fig. d) . we believe that pika will have similar antiviral activity in humans, because we showed that human tlr- can recognize pika (fig. ) and a number of studies have demonstrated the expression of tlr- in various respiratory cell types, such as nasal epithelial cells [ ] , airway smooth muscle cells [ ] and airway epithelial cells [ ] . in addition, ds rna was found to be the most effective tlr ligand in activating airway epithelial cells in producing il- [ ] . although our experimental data are limited to tlr- , dsrna can also be recognized by rna helicase, rig- and mda , and can induce nf-b activation with type- ifns [ ] . it is possible that pika stimulates several pathways in vivo to achieve its antiviral and adjuvant activities. the knockout-mice, such as tlr k/o, mda k/o and rig- k/o mice, will be useful in shedding more light on the contribution of these receptors to the activity of pika. in summary, although poly(i:c) has been shown to be an effective mucosal adjuvant, its use in clinical trials was associated with side-effects that has limited its usage [ ] . in a recent toxicity trial, pika showed no significant toxicity in mice and a phase clinical study showed that the use of pika as an adjuvant was well-tolerated in humans without significant side-effects (peter brazier, unpublished data). the safety profile, together with the long shelf-life, makes pika an attractive complement to currently licensed antivi-ral drugs. in the early phase of an influenza pandemic, when a well-matched vaccine is unlikely to be available, pika can be used as an antiviral drug for temporary protection of the susceptible population, slowing down the rate of viral transmission and allowing more time for vaccine production. furthermore, global influenza vaccine production is likely to lag well behind the demand for pandemic influenza vaccine, so the inclusion of an effective antigensparing adjuvant such as pika can ensure that limited supplies of vaccine achieve maximum coverage. safety and antigenicity of non-adjuvanted and mf -adjuvanted influenza a/duck/singapore/ (h n ) vaccine: a randomised trial of two potential vaccines against h n influenza safety and immunogenicity of an inactivated subvirion influenza a (h n ) vaccine safety and immunogenicity of an inactivated split-virion influenza a/vietnam/ / (h n ) vaccine: phase i randomised trial vaccines for seasonal and pandemic influenza boosting immunity to influenza h n with mf -adjuvanted h n a/duck/ singapore/ vaccine in a primed human population antigen sparing and cross-reactive immunity with an adjuvanted rh n prototype pandemic influenza vaccine: a randomised controlled trial reduced sensitivity of influenza a (h n ) to oseltamivir oseltamivir resistance during treatment of influenza a (h n ) infection distribution of amantadine-resistant h n avian influenza variants in asia innate immune recognition: mechanisms and pathways type cytokine/chemokine production by mouse nk cells following activation of their tlr/myd -mediated pathways maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway synthetic toll-like receptor agonists stimulate innate resistance to infectious challenge interferons, interferon-like cytokines, and their receptors human and mouse mx proteins inhibit different steps of the influenza virus multiplication cycle host gene influences sensitivity to interferon action selectively for influenza virus interferon-induced antiviral actions and their regulation a dna transfection system for generation of influenza a virus from eight plasmids relative immunogenicity of the coldadapted influenza virus a/ann arbor/ / (a/aa/ / -ca), recombinants of a/aa/ / -ca, and parental strains with similar surface antigens antigenic determinants of influenza virus hemagglutinin. xii. the epitopes of a synthetic peptide representing the c-terminus of ha maturation of immunoglobulin g avidity after rubella vaccination studied by an enzyme linked immunosorbent assay (avidity-elisa) and by haemolysis typing myd -adapter-like) is required for toll-like receptor- signal transduction an overview of real-time quantitative pcr: applications to quantify cytokine gene expression pika as an adjuvant enhances specific humoral and cellular immune responses following the vaccination of mice with hbsag plus pika immune responses of mice to influenza subunit vaccine in combination with cia as an adjuvant intranasal immunisation with influenza-iscom induces strong mucosal as well as systemic antibody and cytotoxic t-lymphocyte responses crossprotection against h n influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine human influenza a h n virus related to a highly pathogenic avian influenza virus properties and dissemination of h n viruses isolated during an influenza outbreak in migratory waterfowl in western china confirmation of h n avian influenza in africa h n avian influenza: the turkish dimension h n influenza virus avian influenza a (h n ) infection in humans type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors posttherapy suppression of genital herpes simplex virus (hsv) recurrences and enhancement of hsv-specific t-cell memory by imiquimod in guinea pigs efficacy of s against guinea pig cytomegalovirus infection attenuation of virus-induced airway dysfunction in rats treated with imiquimod effect of interferon-alpha and interferon-inducers on west nile virus in mouse and hamster animal models evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice prophylaxis of acute respiratory virus infections using nucleic acid-based drugs nucleic acidbased antiviral drugs against seasonal and avian influenza viruses avian flu: isolation of drug-resistant h n virus neuraminidase inhibitor-resistant recombinant a/vietnam/ / (h n ) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo high susceptibility of human dendritic cells to avian influenza h n virus infection and protection by ifn-alpha and tlr ligands coadministration of cpg oligonucleotides enhances the late affinity maturation process of human anti-hepatitis b vaccine response enhancement of antibodies to the human immunodeficiency virus type envelope by using the molecular adjuvant c d recognition of double-stranded rna and activation of nf-kappab by toll-like receptor dendritic cell maturation is required for initiation of the immune response development of smallpox vaccine candidates with integrated interleukin- that demonstrate superior immunogenicity, efficacy, and safety in mice detrimental contribution of the toll-like receptor (tlr) to influenza a virusinduced acute pneumonia expression of toll-like receptors in cultured nasal epithelial cells toll-like receptor , , and expression and function in human airway smooth muscle activation of airway epithelial cells by toll-like receptor agonists the rna helicase rig-i has an essential function in double-stranded rnainduced innate antiviral responses a phase i-ii trial of multiple-dose polyriboinosic-polyribocytidylic acid in patieonts with leukemia or solid tumors we gratefully acknowledge kanta subbarao (niaid, nih) for helpful discussions and critical reading of the manuscript. this study was supported by future systems directorate, ministry of defence, the republic of singapore. key: cord- -b bm do authors: bernasconi, valentina; kristiansen, paul a.; whelan, mike; román, raúl gómez; bettis, alison; yimer, solomon abebe; gurry, céline; andersen, svein r.; yeskey, debra; mandi, henshaw; kumar, arun; holst, johan; clark, carolyn; cramer, jakob p.; røttingen, john-arne; hatchett, richard; saville, melanie; norheim, gunnstein title: developing vaccines against epidemic-prone emerging infectious diseases date: - - journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: . /s - - - sha: doc_id: cord_uid: b bm do today’s world is characterized by increasing population density, human mobility, urbanization, and climate and ecological change. this global dynamic has various effects, including the increased appearance of emerging infectious diseases (eids), which pose a growing threat to global health security. outbreaks of eids, like the – ebola outbreak in west africa or the current ebola outbreak in democratic republic of the congo (drc), have not only put populations in low- and middle-income countries (lmic) at risk in terms of morbidity and mortality, but they also have had a significant impact on economic growth in affected regions and beyond. the coalition for epidemic preparedness innovation (cepi) is an innovative global partnership between public, private, philanthropic, and civil society organizations that was launched as the result of a consensus that a coordinated, international, and intergovernmental plan was needed to develop and deploy new vaccines to prevent future epidemics. cepi is focusing on supporting candidate vaccines against the world health organization (who) blueprint priority pathogens mers-cov, nipah virus, lassa fever virus, and rift valley fever virus, as well as chikungunya virus, which is on the who watch list. the current vaccine portfolio contains a wide variety of technologies, ranging across recombinant viral vectors, nucleic acids, and recombinant proteins. to support and accelerate vaccine development, cepi will also support science projects related to the development of biological standards and assays, animal models, epidemiological studies, and diagnostics, as well as build capacities for future clinical trials in risk-prone contexts. background global trends, including increasing population density, urbanization, human mobility, and climate and ecological change, are leading to emerging infectious diseases (eids) that pose a growing threat to global health security [ ] . if a highly contagious and lethal airborne pathogen with the characteristics of the pandemic influenza were to emerge today, it is estimated that nearly million people might die in just months worldwide [ ] . the costs of eids are enormous, both in terms of lives lost and economic burden. a report prepared by the u.s. national academy of sciences in estimated that over years the global costs of epidemics could amount to us$ billion, or . % of global income. the cost of a severe pandemic like the influenza pandemic could total as much as % of global gross domestic product (gdp). even when the health impact of an outbreak is relatively limited, its economic consequences can quickly become magnified [ ] . liberia, for example, saw gdp growth decline % from to during the ebola outbreak in west africa, even as the country's overall mortality rate fell over the same period [ ] . from the beginning of the st century to the present, the world has experienced several outbreaks of eids, with considerable public health concerns: severe acute respiratory syndrome-related coronavirus ( [ ] [ ] [ ] [ ] [ ] . in each instance, it was not possible to predict the time, location, or identity of the causative pathogen beforehand [ ] . vaccination is a critical tool in the response to these unpredictable outbreaks as well as, eventually, in their prevention. however, the complete process for bringing a vaccine from the research laboratory to the population is long, complex, and expensive, typically requiring a capital investment of us$ million to us$ billion over a period of years [ ] . vaccination has been described as one of the most successful public health interventions to date [ ] . our modern vaccinology era started when edward jenner, an english general practitioner, conducted the first scientific investigation on smallpox prevention in [ ] . since then, vaccinations have reduced disease, disability, and death from a variety of infectious diseases all over the world [ ] . despite this success, there is still a great need for new vaccines that can prevent and reduce the impact of outbreaks of both endemic infectious diseases and emerging infectious diseases [ ] . in the case of eids, this is especially challenging due to the fact that the identity of the pathogen responsible for the disease, as well as the time and location of the next outbreak, cannot be accurately predicted using current means [ , ] . historically, vaccine development has been a long, risky, and costly endeavor. planning vaccination against eids is especially challenging: the potential market for vaccines against these diseases is limited, and testing such vaccines is difficult [ ] . several bottlenecks have been identified in the development of vaccines against eids [ ] . the first limiting factor is related to the preclinical discovery: understanding the pathogenesis mechanism, developing the appropriate animal-challenge models, and being able to screen, test, and generate the proof of concept (poc) for new antigens and delivery platforms is not trivial [ ] . moreover, preclinical development is a complex, multistep, and time-consuming process. this represents the second bottleneck in the vaccine development process and involves selection/screening of appropriate antigens and verification of efficacy in the an- imal models. this is followed by process development to ensure that a scalable, robust, and good manufacturing practice (gmp)-compliant process is established. material generated at the end of preclinical development can be used for animal toxicology studies and forms the basis of a clinical trial application [ ] . the traditional clinical trial phases require significant investment and resources to be executed, and the lengthy nature of the process could itself be described as a bottleneck. many eids are prone to sporadic outbreaks in which morbidity and mortality are high, and it is sometimes not possible to conduct traditional phase iii efficacy trials due to ethical considerations and the scale and unpredictable nature of eid outbreaks [ ] . to meet the unique challenges of vaccine development for eids, an innovative, efficient global system of vaccine research and development (r&d) for eids is needed [ ] . after the devastating west african ebola epidemic in - , which alone claimed the lives of more than , people and had a comprehensive economic and social burden estimated at over us$ billion (or more than $ . million per case), the global need for an organization that could finance and coordinate the development of vaccines against eids was recognized [ ] . in , although there was no licensed ebola vaccine available, approximately different vaccines were in preclinical development, including dna vaccines, virus-like particles (vlps), and viral vector-based vaccines [ ] . it took a year to initiate field trials of the first ebola vaccines, many of which had been under development for more than a decade. it became evident that an improved system for the development of vaccines against known and unknown epidemic threats was needed [ ] . the early ideas for establishing what became the coalition for epidemic preparedness innovations (cepi) were consolidated at the world economic forum annual meeting in davos in january , and cepi was launched year later to facilitate and fund coordinated, international, and intergovernmental planning to develop and deploy new vaccines to prevent and reduce the impact of eid epidemics. the coalition is an innovative global partnership between public, private, philanthropic, and civil society organizations, and its mission is to stimulate and accelerate the development of vaccines against eids and enable access to these vaccines for people affected by outbreaks [ ] . it was founded by the governments of norway and india, the bill & melinda gates foundation, the wellcome trust, and the world economic forum. from cepi has secured approximately us$ million of direct and aligned investments toward its us$ billion funding target, including multiyear funding from norway, germany, japan, canada, australia, the europeancommission, the bill & melinda gates foundation, and the wellcome trust. it has also received single-year investments from the governments of belgium and the uk (. table ; [ ] ). filling a critical gap in the vaccine "ecosystem" many organizations operate within the end-to-end space of vaccine funding and r&d implementation. however, several critical gaps have been identified, which cepi is designed to fill (. fig. ) . the r&d is complex, lengthy and expensive; the potential market for such vaccines against eids is very limited; and testing of such vaccines is difficult [ ] . cepi is designed to advance vaccines against known threats through poc and safety testing in humans and establishing investigational stockpiles to be used emergently at the beginning of an epidemic under a clinical trial regimen. it also funds new and innovative platform technologies that carry the potential to accelerate the development and manufacturing of vaccines against previously unknown pathogens. moreover, cepi coordinates activities to improve the collective response to epidemics, strengthening capacity in countries at risk and advancing the regulatory science that governs product development. cepi has three strategic objectives: preparedness, response, and sustainability, and it aims to advance safe and effective vaccines against eids; accelerate the research, development, and use of vaccines during outbreaks; and create durable and equitable solutions for outbreak response capacity [ ] . it offers a unique opportunity for investors to lead on global health security and, in partnership with other governments and international organizations, invest in solutions that aim to protect some of the most vulnerable people in the world while helping prevent the spread of epidemics [ ] . today's world is characterized by increasing population density, human mobility, urbanization, and climate and ecological change. this global dynamic has various effects, including the increased appearance of emerging infectious diseases (eids), which pose a growing threat to global health security. outbreaks of eids, like the - ebola outbreak in west africa or the current ebola outbreak in democratic republic of the congo (drc), have not only put populations in lowand middle-income countries (lmic) at risk in terms of morbidity and mortality, but they also have had a significant impact on economic growth in affected regions and beyond. the coalition for epidemic preparedness innovation (cepi) is an innovative global partnership between public, private, philanthropic, and civil society organizations that was launched as the result of a consensus that a coordinated, international, and intergovernmental plan was needed to develop and deploy new vaccines to prevent future epidemics. cepi is focusing on supporting candidate vaccines against the world health organization (who) blueprint priority pathogens mers-cov, nipah virus, lassa fever virus, and rift valley fever virus, as well as chikungunya virus, which is on the who watch list. the current vaccine portfolio contains a wide variety of technologies, ranging across recombinant viral vectors, nucleic acids, and recombinant proteins. to support and accelerate vaccine development, cepi will also support science projects related to the development of biological standards and assays, animal models, epidemiological studies, and diagnostics, as well as build capacities for future clinical trials in risk-prone contexts. nipah · mers-cov · chikungunya · rift valley fever · cepi nipah · mers-cov · chikungunya · rifttalfieber · coalition for epidemic preparedness innovation the world health organization (who) developed a list of diseases and pathogens to be prioritized for research and development under the who r&d blueprint for emerging infections. diseases were prioritized on the basis that they pose a public health risk due to their epidemic potential and that they have no, or insufficient, countermeasures against them [ ] . the who furthermore conducts an annual review of the blueprint priority list [ ] . ebola, marburg, lassa, mers-cov, nipah, and rift valley fever (rvf) viruses were among the viruses listed in [ ] . that same year alone, six of the priority pathogens listed in the who r&d blueprint caused outbreaks [ ] . "disease x" is also listed: it represents the fact that a serious international epidemic could be caused by a pathogen currently unknown to cause human disease, toward which it is important to enable cross-cutting r&d preparedness [ ] . cepi is prioritizing investments in two areas. the first is the development of vaccines against a set of high-priority pathogens, which currently include lassa, mers-cov, nipah, rvf, and chikungunya viruses. the second is the development of vaccine platform technologies that will enable rapid vaccine development and manufacturing to improve global capacity to respond to the emergence of an unknown pathogen with epidemic potential (disease x) [ ] . since its launch, cepi has announced three calls for proposals (cfp). the first and third cfp focused on cepi's priority pathogens, supporting candidate vaccines against mers-cov, nipah, lassa, rvf, and chikungunya viruses. the second cfp aims to advance rapid-response platforms against unknown pathogens. cepi has established multiple partnering agreements that make up its current portfolio of priority pathogen vaccine candidates and three rapid response platforms that reflect a potential investment of over us$ million. additional partnerships are under negotia-tion. . table provides some details of the cepi vaccine portfolio. these details are also provided on the cepi website (www.cepi.net). the cepi vaccine portfolio contains a wide variety of technologies, ranging across recombinant viral vectors, nucleic acid-based approaches, and recombinant proteins. given that vaccine development is largely an empirical science, it is difficult to determine in advance which technology is likely to succeed in the clinic. therefore, cepi has invested in developing multiple candidates for each of its priority pathogens. for these priority pathogen projects, cepi will seek to advance vaccine candidates through phase ii clinical trials and the generation of an investigational stockpile. such investigational stockpiles could be used during future outbreaks and in further clinical trials. for vaccine technologies enabling rapid response, cepi's initial investments will seek to demonstrate preclinical immunity to three pathogens and clinical (phase i) responses to two of these. in all cases, awards are made with stringent milestones and stage gates. the partnership arrangements that have been established also provide provisions ensuring that cepi's equitable-access goals can be achieved. currently, cepi has invested in five technologies for lassa fever vaccines using recombinant viruses and nucleic acid-based immunization. indeed, the first cepi-sponsored phase i clinical trial began in may using inovio pharmaceuticals' dna technology (nct ) [ ] . more recently, another phase i clinical trial for a lassa vaccine candidate was initiated by themis bioscience (nct ). there are four vaccine candidates under investiga- all three have the potential to produce a vaccine rapidly in the event of an emergency (. table ). additional investments in these areas will be announced shortly. international experts noted that for many of the diseases listed in the who r&d blueprint, there is not only a need for a vaccine but also for developing a broader knowledge base of the disease itself. basic and characterization research is needed, as well as epidemiological, entomological, and multidisciplinary studies; improved diagnostics; further elucidation of transmission routes; and social science research [ ] . the knowledge built will be fundamental in the process of vaccine development. to this purpose, cepi has identified a set of research activities needed to accelerate vaccine development, and it is currently focusing on several enabling science projects related to the development of biological standards and assays, animal models, epidemiological studies, diagnostics, clinical trial capacity, and sustainable manufacturing. although this list of research areas is not exhaustive, it represents a focused set of research activities and data collection priorities from a vaccine-development perspective. development of biological standards and assays is important for evaluating vaccine-elicited immune responses and promoting standardization, transparency, and comparability among the vaccine candidates. there are currently no available international antibody standards for lassa, mers-cov, or nipah, and there is a wide variety of intermediate standards currently used by lassa vaccine developers. cepi, in collaboration with international partners, is collecting serum from patients from endemic countries who recovered from the actual diseases for the development of reference antibody preparations and, ultimately, expert committee on biological standardization (ecbs)-endorsed international reference preparations (irps). it is the aim of cepi to make biological standards available to all cepi-funded vaccine developers as early as possible, and for this purpose cepi has established a working group on standards, assays and animal models, which is co-chaired by the who. in addition to this overarching group of experts, pathogen-specific task forces have also been established to obtain advice on specific topics related to standards, assays, and animal models. the task forces are instrumental in describing major needs for each disease, providing technical advice, and fostering collaboration across projects. these disease-specific task forces engage scientists from various geographic regions and from multiple disciplines. moreover, cepi also seeks to make pathogenspecific antigens available to relevant cepi-funded vaccine developers. when moving toward phase i/ii and, potentially, phase iii efficacy trials, access to common sets of reference standards will be crucial for the evaluation of the vaccine and the comparison of different vaccine candidates. as an example, in the past year cepi launched requests for proposals and signed several partnership agreements for the distribution of lassa virus-specific antigens and the development of a lassa antibody standard. in collaboration with the viral hemorrhagic feverconsortium (vhfc), the bernhard nocht institute for tropical medicine (bnitm), and the national institute of biological standards and control (nibsc), cepi is collecting serum from individuals who recovered from the disease in endemic countries (sierra leone, liberia, mali, and nigeria) for the development of reference antibody preparation and, ultimately, an irp available to all globally. due to the nature of eids, obtaining human efficacy data may prove challenging for the vaccines in cepi's portfolio. consequently, evidence of vaccine efficacy may need to rely, either in part or fully, on data from validated animal models acceptable to regulatory authorities. in the u.s. food and drug administration (fda) finalized the animal efficacy rule (also known as the animal rule), which applies to the development and testing of drugs and biologicals to reduce or prevent serious and life-threatening conditions caused by exposure to lethal agents for which human efficacy trials are not feasible or ethical [ ] . according to this rule, the fda relies on animal studies to provide substantial evidence of product effectiveness akin to a traditional phase iii clinical efficacy study. this route of licensure still requires human safety and immunogenicity, however. to rely on animal efficacy, much work needs to be done to build the foundation of data, such as natural history studies of one or more of the animal species selected, a reasonably wellunderstood mechanism for the toxicity of the pathogen, and pharmacokinetics and pharmacodynamics data sufficiently well understood to allow the selection of an effective dose in humans [ ] . therefore, cepi is planning to support animal model development/refinement and natural history studies that can serve as a basis for qualification of the model by regulatory agencies. it is aligning with the national centre for the replacement, refinement and reduction of animals in research (nc rs) guidelines to accelerate the development of models and tools toavoidtheuseofanimalswherepossible, reduce the number of animals used per experiment, minimize animal suffering, and improve welfare [ ] . cepi is currently mapping existing efforts and funding for such work and will explore collaborations and co-funding mechanisms as appropriate to avoid duplication of efforts in this space. the who has developed target product profiles (tpps) for many of the priority pathogens, and cepi uses the who tpps as guiding documents to make many of its decisions regarding the feasibility and intended use of funded vaccines [ ] [ ] [ ] . diagnostic tests can serve multiple functions, including epidemiological surveillance, diagnosis in efficacy trials, case detection, and outbreak response. cepi focuses on supporting the development of diagnostic tests to prepare for phase iib/iii clinical trials and identify cases of disease. its efforts are in mapping the needs around the development of diagnostic tools, without which cepi vaccine candidates cannot be advanced. cepi has limited funding for diagnostics-related activities; therefore, the diagnostic work is mainly accomplished through establishing partnerships and collaboration with potential product development partners. the foundation for innovative new diagnostics (find) and cepi have developed a partnership framework called cepi.dx to address diagnostic needs for priority pathogens, and cepi recently funded find with a total of us$ million to support the evaluation of serological assays (igg, igm elisa), clinical trial site development, and laboratory capacity strengthening in lassa-affected countries. cepi has also actively supported find's application for the mobilization of a total of € . million from the federal ministry for education and research (bmbf) of the german government. this funding has been used to support clinical evaluation of the altona realstar lassa virus rt-pcr kit . (altona diagnostics, hamburg, germany), strengthening outbreak surveillance, research capacity, and activities related to biobanking in lassa-affected countries. epidemiological studies are essential to understand the incidence and prevalence of eids, as well as their clinical characteristics and risk factors. these data are also essential to assess the feasibility of clinical field efficacy trials of promising vaccine candidates. some cepi-funded vaccine candidates have already entered testing in phase i clinical trials. if these initial trials are successful and vaccine candidates are deemed safe to proceed to the next stages of testing, further vaccine phase iia trials in affected countries, and potentially phase iib trials, will be conducted. to ensure the feasibility of efficacy trials and to support trial design, quality epidemiological data is needed. epidemiological research can also help strengthen site and investigator capacity to conduct clinical trials. therefore, cepi is providing grants for epidemiological studies that aim to collect data that can contribute to vaccine development in support of trial design, appropriate end points, and site capacity. to accelerate lassa vaccine development, cepi promoted an open call for research groups/consortia across nigeria, benin, sierra leone, guinea, and liberia to develop a core study protocol for a major multinational epidemiological study. this epidemiological study will be supported by an effort to develop and validate diagnostic assays in collaboration with find. moreover, clinical trial site development and the establishment of one fully accredited clinical trial site and two to three sites in nigeria, meeting good clinical laboratory practice (gclp) standards for reliable diagnosis of lassa fever cases, will be carried out to support future trials of vaccines. this will allow expanded sample collection and archiving to accelerate the research and development and regulatory approvals for new diagnostics and vaccines [ , ] . in addition, cepi will provide support with respect to the clinical development of vaccine candidates, as well as in exploring regulatory pathways. the aim is to conduct clinical trials in affected endemic countries as early in the development as possible. cepi will support the identification of clinical trial sites covering target populations and will engage in capacity building. it cooperates with the brighton collaboration, an international network of pharmacovigilance experts, to, among other activities, develop case definitions for potential adverse events of special interest (aesis), including, for example, sensorineural hearing loss for the safety evaluation of lassa vaccine candidates [ ] . moreover, the brighton collaboration will provide expertise in programspecific (upon request) and cross-program pharmacovigilance, for example by establishing a metadata safety monitoring board (meta-dsmb). as cepi's priority pathogens mainly result in outbreaks, it is essential to explore the feasibility of field efficacy trials (phase iib or iii). cepi will provide support in scenario planning, clinical trial design, capacity building, and so on for advanced-stage clinical trials to prove the vaccine candidate's efficacy against infection, disease, or both. these advancedstage clinical trials will have to be placed in an overall clinical development plan that is aligned and supported by the relevant regulatory authorities, as well as the who prequalification group. in this context, cepi will also explore alternative regulatory pathways in case vaccine efficacy cannot be demonstrated in field trials, for instance when there is rapid decline of the infectious disease outbreak or no ongoing outbreak. after the generation of an investigational stockpile for the candidate vaccines, a sustainable supply of vaccine will be critical. cost and time efficiency during manufacturing for future stockpiles, outbreak response, and routine use of new vaccines in endemic regions will be of great importance. since many of the vaccines cepi is developing will not find commercial markets to sustain them, cepi is exploring different approaches to provide for the long-term manufacturing of any successful vaccine candidates and is considering establishing advanced manufacturing partnerships with a limited number of public and private-sector manufacturing organizations. ongoing efforts to understand potential epidemic scenarios and to model supply chain and stockpile requirements will contribute to this effort. vaccines are a powerful tool with substantial potential to prevent and control outbreaks of eids. however, a key issue is to be able to manufacture and test a safe and efficacious vaccine for the immediate threat within a very short time frame. prior to cepi's establishment, vaccine development efforts for eids were fragmented, with no sustainable mechanism to support successful projects across the vaccine development life cycle nor to coordinate work toward the highest-priority global epidemic risks. cepi was created to fill these gaps and to stimulate, finance, and coordinate the development of vaccines against eids, especially in cases where market incentives alone were failing to drive needed development. from its creation, cepi developed a business plan to advance vaccine candidates to the poc stage (by supporting phase i and ii clinical trials) to enable clinical efficacy testing (phase iii) during outbreaks. it is also cepi's intent to contribute to technical and institutional platforms that can accelerate the r&d response to eids. cepi will continue to coordinate closely with the who and to work together with the international community to advance vaccine candidates against eids with epidemic potential, to establish and maintain investigational stockpiles of promising candidates, and to implement scientifically robust trials of these candidates during outbreaks. coalition for epidemic preparedness innovation (cepi) marcus thranes gate , oslo, norway gunnstein.norheim@cepi.net travel, migrationand emerging infectious diseases institute for disease modeling ?recordid= . accessed worldhealthorganizationworkinggroup on prevention of international and community transmission of sars ( ) public health interventions and sars spread an insight into the swine-influenza a (h n ) virus infection in humans outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study understandingebola: the epidemic zikavirusandpregnancyinbrazil: whathappened? funding vaccines for emerging infectious diseases the gold industry standard for risk and cost of drug and vaccine development revisited simply put: vaccination saves lives in celebration of the th anniversary of edward jenner's inquiry into the causes and effects of the variolae vaccinae new vaccines against epidemic infectious diseases thecurrentchallenges for vaccine development emerging infectious diseases: epidemiological perspective the complexity and cost of vaccine manufacturing-an overview process mapping of vaccines: understanding the limitations in current response to emerging epidemic threats the economic and social burden of the ebola outbreak in west africa clinical development of ebola vaccines ebola and fda: reviewing the response to the outbreak, to find lessons for the future coalition for epidemic preparedness innovation (cepi) infectious disease threats in the twenty-first century: strengthening the global response list of blueprint priority diseases why aren't we worried about the next epidemic? in: huffpost outbreak response as an essential component of vaccine development nih ( ) safety, tolerability and immunogenicity of ino- in healthy volunteers establishing efficacy of human products using animals: the us food and drug administration's "animal rule product development under the animal rule, guidance for industry who target product profile for lassa virus vaccine who ( )who targetproductprofilesformers-cov vaccines who target product profile for nipah virus vaccine acknowledgements. the authors would like to acknowledge all the staff members of the cepi secretariat for their invaluable contribution to cepi's mission.open access. thisarticleisdistributedundertheterms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. adverse events of special interest for this article no studies with human participants or animals were performed by any of the authors. all studies performed were in accordance with the ethical standards indicated in each case. key: cord- - a jxl authors: cohn, amanda c.; broder, karen r.; pickering, larry k. title: immunizations in the united states: a rite of passage date: - - journal: pediatr clin north am doi: . /j.pcl. . . sha: doc_id: cord_uid: a jxl today, vaccination is a cornerstone of pediatric preventive health care and a rite of passage for nearly all of the approximately , infants born daily in the united states. this article reviews the us immunization program with an emphasis on its role in ensuring that vaccines are effective, safe, and available and highlights several new vaccines and recommendations that will affect the health of children and adolescents and the practice of pediatric medicine in future decades. we must plan for the future, because people who stay in the present will remain in the past. -abraham lincoln in , when jenner showed successful inoculation of humans with cowpox to protect them from the devastation of smallpox, a revolution in science and medicine began [ ] . more than centuries later, immunizations were hailed as one of ''ten great public health achievements'' of the twentieth century [ , ] . today, vaccination is a cornerstone of pediatric preventive health care and a rite of passage for nearly all of the approximately , infants born daily in the united states. immunizations have had a profound impact on the health of children, adolescents, and adults in the united states (table ). the most extraordinary success of immunizations was the worldwide eradication of smallpox. declared in , smallpox eradication was achieved through an unprecedented collaborative international initiative, led by the world health organization, establishing an example for other vaccine-preventable diseases [ ] . vaccination since has led to elimination of wild-type poliomyelitis and indigenous measles in the united states, both major causes of pediatric morbidity and mortality in the prevaccine era [ , ] . an integral part of achieving these successes was establishment of a federal immunization infrastructure, which followed the introduction of polio vaccination in the s [ ] . immunization programs, legislation, and funding mecha- nisms are now in place to ensure that immunizations are accessible to all children. as a result, coverage levels for most routinely recommended childhood vaccines in the united states are approaching or have surpassed the us department of health and human services healthy people goal of % coverage [ ] . immunizations have changed the scope of pediatric practice in the united states. pediatric residents now infrequently encounter varicella, which in the s was commonplace. likewise, although haemophilus influenzae type b (hib) was the leading cause of meningitis in young children before availability of hib vaccines in , most newly trained pediatricians will never see a case of invasive hib [ ] . this article reviews the us immunization program with an emphasis on its role in ensuring that vaccines are effective, safe, and available and highlights several new vaccines and recommendations that will affect the health of children and adolescents and the practice of pediatric medicine in future decades. childhood and adolescent immunization schedule the centers for disease control and prevention (cdc), american academy of family physicians, and american academy of pediatrics (aap) annually publish a childhood and adolescent immunization schedule. the advisory committee on immunization practices (acip), with input from many liaison organizations, periodically reviews the schedule to ensure consistency with new vaccine developments and policies [ ] . the first combined immunization schedule was published in and recommended six vaccines containing antigens against nine infectious diseases [ ] : diphtheria and tetanus toxoids and whole-cell pertussis vaccine (dtp); tetanus and diphtheria toxoids (td); measles, mumps, and rubella vaccine (mmr); hib; oral polio vaccine (opv); and hepatitis b virus vaccine. ten years later in february , there were ten vaccines against [ ] . before a vaccination becomes part of routine clinical pediatric practice, three steps need to be taken: ( ) the fda must license the vaccine, ( ) the acip and the committee on infectious diseases of the aap and aafp must recommend the vaccine for use, and ( ) the vaccine must be subsidized to cover children without private health insurance. numerous government and partner organizations participate in bringing a vaccine from the bench into the clinic. table provides links where information about these organizations can be obtained. before fda licensure, a new vaccine goes through to years of preclinical testing and clinical trials, costing pharmaceutical companies millions of dollars in new development costs. before testing the vaccine in humans, a company files an investigational new drug application with the fda followed by three phases of clinical trials that are performed to study vaccine safety, immunogenicity, and efficacy (table ) [ ] . after completion of the prelicensure clinical trials, the manufacturer files a biologics licensure application (bla), and the fda, with input from its advisory committee, determines if data support vaccine safety, immunogenicity, and efficacy ( fig. ) [ ] . after licensure, monitoring for rare adverse events continues for some vaccines through formal phase iv trials conducted by the fda and manufacturer. after fda licensure of a new vaccine, information about the vaccine is reviewed by the acip. the acip comprises voting members appointed by the secretary of the department of health and human services. in addition, several professional medical and public health groups and industry representatives participate in acip discussions. to formulate recommendations, the acip establishes subject-specific working groups to review and synthesize data months to ensuring that all us children and adolescents, regardless of health insurance status or income level, have access to recommended immunizations requires a complex system of financing comprised of private and public funding mechanisms (table ). in , % of us children received vaccines purchased through the public sector, and % received vaccines purchased through the private sector. most of the public-purchase vaccines are financed through the vaccines for children (vfc) program, an entitlement program established in as part of the social security act [ , ] . other government funding mechanisms include section of the public health service act of , a federal grant program, and state and local government funding. these programs provide support for states to provide immunizations to children who do not qualify for the vfc program but who are not covered by private insurance. fourteen states, referred to as ''universal'' purchase states, use a combination of federal and state funding to purchase and distribute vaccines recommended for children to all immunization providers in private and public sectors. the remaining states purchase vaccines for uninsured and underinsured chil- immunizations in the us dren who are not eligible for vfc. in addition, insurance provides vaccines for children in the private sector. as the number of vaccines has increased and the scope of the immunization program has expanded, new challenges have emerged. the increasing cost of vaccines, vaccine shortages, and immunization safety are important concerns the immunization program will continue to address in coming years. the rising cost of fully immunizing a child in the united states is due to the increasing number of vaccines and the increasing price of existing vaccines. the estimated cost of completing the childhood immunization series through years of age in was $ . per child at the government-purchasing rate. the cost of immunizing a child through years of age in was $ per child for all vaccines, not including influenza vaccine [ ] . increasingly, state and local health departments are required to make difficult choices about which vaccines to purchase using public funds, including section grant funds. the recommendation in to vaccinate routinely with pcv doubled the cost of immunizing a child. section and state funding have not been adequate to cover pcv for underinsured children in many states, including of the universal purchase states. the addition of new, effective childhood and adolescent vaccines to the schedule has the potential to create serious funding challenges in the future. despite the increasing costs of immunization programs, numerous studies have shown that vaccination continues to be a cost-effective public health intervention. these studies show the need to continue to identify adequate funding sources to support immunization recommendations [ ] [ ] [ ] [ ] . an institute of medicine (iom) report on vaccine financing released in concluded, ''alternatives to current vaccine pricing and purchasing programs are required to sustain stable investment in development of new vaccine products and attain their social benefits for all'' [ ] . in addition to the increasing cost of vaccines, an unparalleled number of vaccine shortages in the united states has had a substantial impact on vaccine delivery. from through , vaccine shortages and changes in routine recommendations occurred for of the diseases for which childhood and adolescent vaccination is recommended (fig. ) [ ] [ ] [ ] [ ] [ ] [ ] . the shortages affected millions of children and health care providers, even triggering suspension of vaccine school entry requirements [ , ] . two vaccine shortages (pcv and tetanus and diphtheria toxoids [td]) lasted nearly years, one (pcv ) occurred twice [ ] , and one (inactivated influenza vaccine, - season) halved the us influenza vaccine supply virtually overnight [ , ] . the causes of these widespread vaccine shortages are multifactorial. one important long-term factor is the decrease in number of vaccine manufacturers of childhood vaccines routinely recommended in the united states. in , a federal immunization working group expressed concern about the stability of the us vaccine supply in the setting of ''a steady attrition of specific pharmaceutical manufacturers from the entire field of biologics'' [ ] . in , six manufactures produced the six vaccines. in , although four vaccines (pcv , varicella, influenza, and mcv ) have been added to the recommended schedule, the number of manufacturers decreased to five. in addition, there are single manufacturers for four of the childhood and adolescent vaccines (mmr, varicella, pcv , and mcv ). in response to concerns over fragility of the us vaccine supply, the general accounting office and national vaccine advisory committee conducted in-depth reviews of the vaccine shortages and concluded that future disruptions in vaccine supply are likely to continue, and proposed solutions [ , ] . vaccines are administered routinely to healthy children and must uphold a scrupulously high safety standard; however, no vaccine is completely safe. in , the national childhood vaccine injury act was passed creating a compensation program for families affected by childhood vaccine-associated adverse events. several other government programs and committees to ensure the safety of the vaccine supply also were created by this act ( table ) . as many vaccine-preventable diseases approach or reach elimination in the united states, continuing to balance the risks and benefits of each vaccine becomes increasingly important [ ] . opv, formerly recommended for routine use in the united states, was associated with vaccine-associated paralytic poliomyelitis ( case among . million vaccine doses distributed). this rare adverse event was no longer considered acceptable after elimination of polio in the united states [ ] . in , the acip recommended using ipv for all doses of polio vaccine. public perceptions of vaccine safety are a challenge to the continued success of the vaccination program. new parents and younger physicians grew up without appreciation of the morbidity and mortality of immunizations in the us several vaccine-preventable diseases. risk or perception of risk for adverse events becomes an important concern. two current prominent public vaccine safety concerns are the perceived causal association between mmr and autism and thimerosal-containing vaccines and autism. as a result of heightened concerns about safety, in the cdc and national institutes of health commissioned the iom of the national academy of science to convene an immunization safety review committee [ ] . between and , this independent expert committee published eight reports related to immunization safety concerns. the committee has made recommendations in the areas of public health response, policy review, research, and communications (box ). with respect to autism, the iom concluded that the body of epidemiologic evidence favors rejection of a causal relationship between the mmr vaccine and autism. the committee also concluded that there is no relationship between thimerosal-containing vaccines and autism [ ] . none of the eight iom reports recommended a policy review of the current vaccine recommendations or change in the immunization schedule. to help ensure safety of vaccines, a robust infrastructure consisting of several systems has been established to monitor vaccine safety after vaccine licensure. the vaccine adverse event reporting system, operated jointly by cdc and fda, is a national passive surveillance system used to detect early warning signals and generate hypotheses about possible new vaccine adverse events or changes in frequency of recognized events [ ] . intussusception associated with receipt of rotavirus vaccine, leading to the withdrawal of the vaccine from the market in , was an adverse event detected by the vaccine adverse event reporting system [ , ] . a third system is the vaccine safety datalink, which consists of large linked databases from health maintenance organizations. associations between serious medical events and immunizations can be evaluated through the vaccine safety datalink. the newest system is the clinical immunization safety assessment centers network, which consists of selected clinical academic medical centers in partnership with cdc to study the pathophysiology of vaccine reactions and develop clinical management protocols for affected patients [ ] . these systems are crucial to the vitality and strength of the us immunization program. since its inception, the major focus of the us immunization program has been on vaccinating infants and young children. of the vaccines routinely recommended for children and adolescents, only two, td vaccine and the mcv , are recommended for all adolescents [ ] . in , as a result of growing concern about morbidity associated with vaccine-preventable diseases in the hard-to-reach adolescent population, the acip recommended expanding efforts to immunize adolescents ( - years old) by establishing a routine vaccination visit at to years old [ ] . in addition to providing td and previously missed vaccinations, the report emphasized that this visit should be used to provide other important preventive health services. the anticipated addition of several new adolescent vaccines to the recommended schedule has stimulated a reappraisal of the approaches that most effectively and efficiently would increase the proportion of adolescents who receive newly recommended vaccines and develop ways to integrate these approaches with other adolescent health, education, and development programs. similar to all aspects of clinical medicine, immunization recommendations continuously change as new vaccines are licensed and new information becomes available. since , several new vaccination recommendations were implemented for existing and new vaccines. notable examples are pcv and the hepatitis b vaccine; new recommendations for both have affected children and health care providers. several vaccines with expected fda licensure in the near future likely will alter the us immunization program and preventive health care practices ( table ). vaccines with a pediatric or adolescent focus under review by the acip are relevant to the prevention of pertussis, human papillomavirus (hpv), influenza, varicella, and rotavirus. this section presents a summary of these vaccines in addition to information on pcv , hepatitis b vaccine, and mcv . the potential impact of vaccines on the distant horizon also will be highlighted. emphasis is on how recent and upcoming policy decisions might affect children and adolescents, health care providers, and society during the next decade. pcv was recommended for routine use in infants in the united states beginning in . before introduction of pcv , streptococcus pneumoniae (pneumococcus) was a leading cause of infectious morbidity in young children in the united states, annually causing approximately , cases of invasive disease in children younger than years old, including cases of meningitis and deaths. in addition, the burden of pneumonia without bacteremia, otitis media, and sinusitis was substantial [ ] . after introduction of routine pcv vaccination, the incidence of invasive pneumococcal disease declined dramatically, especially in children younger than years old [ ] [ ] [ ] . active us population-based surveillance data show that within years of pcv licensure, the rate of invasive pneumococcal disease in children younger than years old declined by % [ ] . in tandem with the decrease in invasive disease, data suggest the incidence of pneumococcal noninvasive disease, including otitis media, also decreased [ , ] . in addition to decreasing the burden of pediatric pneumococcal disease, pcv may have an impact on reducing pediatric antibiotic prescriptions and procedures such as blood cultures in young, febrile children [ ] . the decline in invasive pneumococcal disease is beyond what would be expected from childhood vaccination, given vaccine efficacy and pcv coverage data, suggesting that herd immunity may play a role in protecting unimmunized people from invasive disease [ ] . reduced nasopharyngeal carriage of vaccinecontaining serotypes in vaccinated children is believed to contribute to development of herd immunity against pneumococcus. rates of invasive pneumococcal disease seem to be declining among some unvaccinated groups after implementation of universal infant pcv vaccination. in addition, postlicensure surveillance data suggest a decrease in antibiotic-resistant strains of s. pneumoniae [ , ] . because pcv includes only of the more than serotypes of pneumococcus, there is theoretical concern that serotype replacement might occur in highly vaccinated populations. one study noted an increase in the proportion of cases of invasive pneumococcal disease resulting from nonvaccine serotypes, but the total number of cases was not changed [ ] . this study supports the need for continued pneumococcal surveillance in the post-pcv era [ ] . hepatitis b vaccine holds a unique place in the us immunization schedule because they are the only vaccines licensed for neonates and the only licensed vaccine that prevents cancer. the continued evolution of hepatitis b vaccine recommendations reflects many of the challenges associated with vaccines that will be licensed in the near future. before , an estimated , to , people in the united states were infected annually with hepatitis b virus, including approximately , children [ ] . although most vaccine-preventable diseases are spread via contact or airborne droplets, hepatitis b infection is spread via exposure to infected blood or blood products, sexual contact, and injection devices. much of the transmission of hepatitis b in adults is silent; there is no accompanying rash or symptoms. although adults have a % chance of developing chronic hepatitis b virus infection, infants infected perinatally who do not receive hepatitis b immunoglobulin and vaccine at birth have a % chance of developing chronic infection. twenty-five percent of these infections lead to hepatocellular carcinoma [ ] . the complexity of hepatitis b transmission required a vaccination strategy to protect infants and high-risk adults from infection. the first acip hepatitis b recommendation in was to vaccinate groups known to be at high risk for hepatitis b virus infection, such as health care workers, men who have sex with men, and intravenous drug users [ ] . in , the acip expanded recommendations to include infants born to mothers who were hepatitis b surface antigen (hbsag) positive. recognition of the difficulty in identifying mothers infected with hepatitis b led to a recommendation in to test all women for hbsag during the prenatal period. vaccinating high-risk groups continued to be difficult because no foundation existed to vaccinate adolescents and adults who already were participating in high-risk activities. in , a universal infant vaccination strategy was instituted to achieve the goal of reducing transmission of hepatitis b virus [ ] . it is recommended that the first dose be given at or before months of age with a preference for all infants to receive the first dose at birth. neonatal vaccination works by protecting the infant from contracting hepatitis after vertical or horizontal exposure. giving all infants the birth dose protects infants whose mothers were not tested for hbsag during pregnancy. infant vaccination eventually will provide protection against hepatitis b virus to adolescents who may engage in high-risk activities before exposure. from to , rates of hepatitis b virus infection in children and adolescents younger than years old declined more than % in the united states [ ] . from to , approximately to cases of invasive meningococcal disease occurred annually in the united states [ ] . the case-fatality ratio for meningococcal disease is approximately %, and severe sequelae (eg, neurologic disability, limb loss) occur in approximately % of survivors [ ] . nasopharyngeal carriage of neisseria meningitidis occurs in approximately % to % of the us population [ ] . transmission is through direct contact with respiratory tract droplets of infected individuals. infants younger than year of age have the highest rates of meningococcal disease, with an annual incidence of . cases per , population during [ ] . during the s, incidence rates of meningococcal disease increased among adolescents and young adults [ ] . evidence also showed that college freshmen living in dormitories have a modestly increased risk of meningococcal disease ( . cases per , ) compared with other persons the same age [ ] . a meningococcal polysaccharide (mps) vaccine containing the antigens of serogroups a, c, y, and w has been used in the united states since licensure in . this vaccine protects against the serogroups that cause approximately two thirds of meningococcal disease that occurs in persons to years old in the united states. more than half of cases in infants are due to serogroup b, however, for which a licensed vaccine does not exist in the united states [ ] . similar to other polysaccharide vaccines, mps induces a t cell-independent immune response resulting in poor long-term immunity and inconsistent immunogenicity in children younger than years old. an additional shortcoming is that mps does not reduce nasopharyngeal carriage or induce herd immunity [ ] . before february , mps vaccine was recommended for groups at high risk for meningococcal disease and for outbreak control. educating college freshmen about the potential for the mps vaccine to prevent severe infection also was recommended. some states required proof of vaccination or vaccine waiver for entry into colleges and universities [ ] . employing the same technology used to develop pcv , a meningococcal serogroup c conjugate vaccine was licensed in the united kingdom in . the vaccine was introduced into the routine infant schedule, with catch-up vaccination for older children and adolescents. in the years after introduction of infant meningococcal conjugate vaccine, the incidence of serogroup c meningococcal disease declined by % in vaccinated persons and at least % in unvaccinated persons, suggesting the vaccine produced herd immunity [ ] . in the united states, a quadrivalet meningococcal conjugate vaccine (serogroups a, c, y, and w- ) (mcv ) was licensed on january , , for use in persons to years old. during prelicensure clinical trials, immune responses to mps and mcv were similar in adolescents and adults. because mcv induces t cell-mediated immunity, the duration of protection is thought to be longer than immunity produced by mps. on february , , the acip voted to recommend that mcv be administered universally to adolescents ages to and around years of age, and college freshmen living in dormitories; a vfc resolution also was passed. with the addition of mcv to the immunization schedule, a new era of adolescent vaccination was launched. in the united states, meningococcal conjugate vaccines for use in children younger than years of age are under study. pertussis remains endemic in the united states despite high immunization coverage rates of infants and young children [ ] . immunity to pertussis wanes approximately to years after vaccination, and loss of immunity seems to play a major role in the continued circulation of pertussis [ ] . in and , , and more than , cases of pertussis were reported to the cdc, respectively [ ] . much of the reported increase is thought to be due to increasing physician recognition of pertussis as a nonspecific, persistent cough illness in adolescents and adults, coupled with increasing use of polymerase chain reaction testing for diagnosis of all age groups. how much of the reported increase is due to enhanced surveillance or improved diagnostic methodology is unclear. one study suggested that a true increase in the incidence of pertussis disease occurred in young infants in the united states between and [ ] . the burden of pertussis disease in adolescents is substantial. of the reported cases of pertussis in the united states in , % were in adolescents; the true number of cases is likely to be much higher (cdc, unpublished data). although pertussis is rarely life-threatening in adolescents, the morbidity and societal costs associated with adolescent pertussis disease are important [ ] . in a canadian study, % of adolescents with pertussis reported a cough duration of at least weeks [ ] . paroxysms, shortness of breath, posttussive vomiting, and difficulty sleeping occur commonly in adolescents with pertussis disease [ , ] . to reduce pertussis disease in adolescents, some countries have recommended an adolescent booster dose. in summer , two manufacturers submitted blas to the fda for use of adolescent and adult pertussis vaccines in the united states (tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine adsorbed (tdap). the bla indication for one vaccine includes persons to years old, and the other includes persons to years old. policymakers are reviewing several strategies for pertussis vaccination in adolescents and adults. a cost-benefit analysis in the united states found universal vaccination for persons to years old to be the most economic strategy [ ] . the expected impact of adolescent pertussis vaccination would be to reduce the risk of pertussis in vaccinated adolescents. ideally, another public health goal of pertussis vaccination is to reduce transmission to infants younger than months old who have not completed the primary vaccination series and are at highest risk of death from pertussis. the role of an adolescent vaccination program in reducing transmission to infants is unknown. vaccinating mothers and other close family members of young infants, referred to as a ''cocoon strategy,'' is one method under consideration to decrease pertussis transmission to infants. one study suggested that adult family members are the most frequently identified source for pertussis transmission to infants [ ] . universal replacement of the td booster given every years with tdap is another strategy being discussed. finally, vaccinating women during pregnancy and vaccinating neonates against pertussis have been raised as potential strategies to improve control of pertussis, although pertussis vaccines for these indications are unlikely to be licensed in the united states in the near future [ , ] . more than types of papillomaviruses have been recognized on the basis of dna sequence analyses [ ] . papillomaviruses are ubiquitous, have been detected in a wide variety of animals and humans, and are specific to their respective hosts. hpv is associated with a variety of clinical conditions that range from benign skin and mucous membrane lesions to cancer. most hpv infections are benign. clinical manifestations with the most frequently associated hpv type are as follows: skin warts (types , , , and ), recurrent respiratory papillomatosis (types and ), condyloma acuminata (types and ), and cervical cancer (types , , , , and ) . based on the association of hpv with cervical cancer and precursor lesions, hpvs can be grouped into low-risk and high-risk hpv types [ ] . in the united states and europe, hpv accounts for approximately % of the cases of cervical cancer, with types , , and accounting for an additional % to % of cases [ ] . hpv is one of the most common causes of sexually transmitted diseases in men and women worldwide, causing almost all of the morbidity and mortality associated with cervical cancer [ ] . epidemiologic studies have shown that the risk of contracting genital hpv infection and cervical cancer is related directly to sexual activity. several specific factors increase the risk of becoming infected with hpv, including multiple sexual partners at any time, having sex with a person who has had multiple sexual partners, sexual activity at an early age, presence of other sexually transmitted diseases, and hpv type. vaccination against high-risk hpv types could reduce substantially the incidence of cervical cancer. administration of hpv- vaccine has been shown to reduce the incidence of hpv- infection and hpv-related cervical intraepithelial neoplasia [ ] . in addition, a bivalent hpv vaccine was efficacious in preventing persistent cervical infections with hpv- and hpv- and associated cytologic abnormalities and lesions [ ] . currently, two hpv vaccines are in the final stages of phase iii testing. one vaccine contains hpv types , , , and , and the second hpv vaccine contains types and . applications for licensure are expected to be filed with the us fda in late or . rotavirus is a common cause of gastrointestinal tract illness in young children. by years of age, nearly all children test seropositive for rotavirus, indicating previous infection. in the united states, rotavirus disease leads to an estimated , clinic visits, , to , hospital admissions, and to deaths annually [ ] . the first rotavirus vaccine was licensed in the united states in and was removed from the market and from the immunization schedule in because of an association with intussusception. this vaccine was a tetravalent rhesus human reassortant vaccine [ , ] . currently, several other rotavirus vaccines are in different stages of development. two late-stage vaccines have completed phase iii clinical trials. one vaccine is derived from a monovalent human strain, and the other is a pentavalent bovine-human reassortant vaccine. large-scale phase iii trials did not show an association of these vaccines with intussusception, but postlicensure monitoring is planned. in january , mexico became the first country to make a new rotavirus vaccine available. the company has filed license applications in more than other countries outside the united states. the manufacturer of the second rotavirus vaccine plans to release it first in the united states after licensure by the us fda. after licensure in the united states, educational efforts that address identifiable barriers to achieving practitioner advocacy and patient acceptance will be necessary to ensure implementation of rotavirus vaccine recommendations [ ] . ensuring physician acceptance of the vaccine is critical to achieving high coverage levels [ ] . varicella vaccine, licensed for use in the united states in , is a live, attenuated virus vaccine developed from the vesicles of a healthy infected child with chickenpox. this vaccine is recommended as a single dose for children months to years old. susceptible persons years old or older should receive two doses administered weeks apart. before varicella vaccine became widely used, varicella was one of the most recognizable rashes seen by pediatricians and was associated with , hospitalizations and deaths in the united states each year [ ] . in , the vaccine had % coverage levels, resulting in a significant decrease in mortality, morbidity, and hospitalizations attributable to varicella [ ] . breakthrough varicella infections in vaccinated children occur in % of children exposed to varicella. breakthrough infections usually are mild (b lesions), however, with few complications [ ] . vaccinated children with mild disease were only one third as contagious as children with moderate-to-severe disease, whether they were vaccinated or unvaccinated. vaccine effectiveness for prevention of moderate disease (n lesions and complications requiring a visit to a clinician) was % [ ] . a second dose of varicella vaccine has been approved by the fda and is being considered for the routine childhood vaccination schedule. the impact of varicella vaccine on the incidence of zoster infections in adults in the united states is unknown. varicella vaccine may protect children receiving the vaccine from zoster when they become adults. studies suggest, however, that continued exposure to varicella protects latently infected adults [ ] . vaccination in children could lead to an increase in zoster incidence in unvaccinated adults because exposure to varicella-infected children has declined, but zoster surveillance is limited. a vaccine to prevent herpes zoster in adults is under investigation. since the worldwide influenza pandemic of that caused an estimated to million deaths, the control of influenza circulation has been a major challenge to clinicians and public health experts. the threat of an unpredictable influenza pandemic and the concern about avian influenza heighten the importance of preventing morbidity and mortality caused by epidemics of influenza disease in the united states, which cause more than , hospitalizations and more than , deaths annually [ ] . implementing the expansion of influenza vaccine recommendations to -to -month-old children and prioritizing vaccine during influenza vaccine shortages are important issues the us immunization program faces regarding influenza prevention. influenza virus contains eight major proteins, including hemagglutinin (ha), which controls viral penetration and attachment, and neuraminidase (na), which controls viral particle release and spread. influenza strains are identified by type (a, b, and c) and by subtype categorized by ha and na. there are different ha and different na subtypes. major changes in ha and na, called antigenic shifts, are associated with emergence of novel influenza viruses to which little or no immunity exists in the exposed population. antigenic shifts were the cause of the three influenza pandemics in the twentieth century (table ) [ ] . minor changes in ha and na, called antigenic drifts, define the influenza viruses that circulate each year. influenza vaccines are developed yearly based on antigenic drifts. worldwide surveillance established by the world health organization allows predictions to be made regarding antigenic drifts, which enables vaccine to be updated before the start of an influenza season. recommendations for which influenza strains should be included in the vaccine are made in early spring before influenza season. three influenza types are formulated and combined to make a new trivalent vaccine each year. two types of influenza vaccines are licensed for use in the united states. one is an inactivated vaccine recommended for persons months of age in highrisk groups and their close contacts. the second is a cold adapted, live, nasally administered vaccine licensed for healthy people to years of age, including close contacts of high-risk persons. the acip and aap recommended in to expand influenza vaccine recommendations to include all children to months old and household contacts of children up to months old as well as to continue immunization of all children in high-risk groups. this recommendation was made based on epidemiologic data showing that healthy children in this age group are at high risk of hospitalization from influenza, and that deaths in this age group occur [ ] [ ] [ ] [ ] . more than reports of pediatric influenzaassociated deaths during the - influenza season stimulated the addition [ ] . the influenza vaccine is unique to the recommended childhood and adolescent immunization schedule because it is the only vaccine that requires a visit during a certain time of year and that requires annual immunization. even if the circulating strains of virus are the same as the year before, an annual booster is necessary to retain immunity. in addition, children months to years old are recommended to have two doses of influenza vaccine administered month apart if they previously have never been vaccinated for influenza [ ] . adding influenza into the childhood schedule is challenging for public health officials and primary care physicians developing programs to attain high coverage rates in children to months old. vaccine development is expanding to include products against cancers, chronic diseases, and other infectious diseases. vaccines against inflammatory diseases for which an infectious cause has not been identified, such as multiple sclerosis and rheumatoid arthritis, are being developed as therapeutic vaccines. scientists effectively are using new biologic tools to improve existing vaccines. new technologies also are being used to improve vaccine delivery systems, producing better combination, oral, and intranasal vaccines. the science behind new vaccines continues to advance at a remarkable pace, driven by an evolving understanding of the cellular and molecular processes involved in different responses of the immune system [ ] . many infectious organisms have evolved over thousands of years to evade this immune response. adjuvants to vaccines are now being used not only to create an immune response, but also to focus the immune response down a desired path [ ] . dna vaccines, plasmids of dna encoding the desired antigen, also are being developed with the intention of simplifying vaccine production and eliminating the possible risk of organism reversion [ ] . as was true during the time of jenner, vaccines continue to push the frontiers of science and medicine. in , the iom published a report prioritizing development of vaccines to be used in the united states. the iom committee considered vaccines that could be licensed within years directed against conditions of domestic health importance [ ] . health benefits of these vaccines were measured by a standard health outcome measure, quality-adjusted life years gained. these vaccines were placed into categories of most favorable to least favorable (box ). since publication of this report, pcv has been licensed for infants beginning at months of age (most favorable category), and hpv vaccine (more favorable category) and rotavirus vaccine administered to infants (favorable category) are on the near horizon as discussed in this article. since release of this report, several organisms not included on the iom list have emerged or became larger public health threats, including west nile virus, metapneumovirus, methicillin-resistant staphylococcus aureus, the coronavirus associated with severe acute respiratory syndrome box . institute of medicine report on vaccines for the twenty-first century most favorable: vaccination strategy would save money cytomegalovirus vaccine administered to -year-olds influenza virus vaccine administered to the general population (once per person every years) insulin-dependent diabetes mellitus therapeutic vaccine multiple sclerosis therapeutic vaccine rheumatoid arthritis therapeutic vaccine group b streptococcus vaccine given to women during first pregnancy and to high-risk adults streptococcus pneumoniae vaccine given to infants and -year-olds more favorable: vaccination strategy would incur small costs (b$ , ) for each qaly* chlamydia vaccine administered to -year-olds helicobacter pylori vaccine administered to infants hepatitis c vaccine administered to infants herpes simplex virus vaccine administered to -year-olds hpv vaccine administered to -year-olds melanoma therapeutic vaccine mycobacterium tuberculosis vaccine administered to highrisk populations neisseria gonorrhoeae vaccine administered to -year-olds respiratory syncytial virus vaccine administered to infants and -year-olds favorable: vaccination strategy would incur moderate costs (n$ , but b$ , ) per qaly gained parainfluenza virus vaccine administered to infants and women during their first pregnancy rotavirus vaccine administered to infants group a streptococcus vaccine administered to infants group b streptococcus vaccine given to high-risk adults and low utilization in -year-olds or women during their first pregnancy (sars), and avian influenza virus (h n ). the ongoing outbreak of h n influenza in asia, associated with high mortality rates, has stimulated research of a vaccine that has the potential to thwart a possible major influenza pandemic. circulating h n viruses may adapt to humans through genetic mutation or reassortant with human influenza strains, allowing for human-to-human transmission, facilitated by the fact that most humans lack preexisting immunity owing to lack of previous exposure [ ] . these emerging infectious diseases and the need to prevent them add further complexity to immunization schedules of the future. until the twentieth century, approximately half of children in the united states died as a result of childhood illness. until the s, infectious diseases were the leading cause of death in the united states. in the first edition of the red book published by the aap in january , chapters dealt with infectious diseases, ranging from the common cold to smallpox. except for pertussis, diphtheria, and less favorable: vaccination strategy would incur significant costs (n$ , -n$ million per qaly gained) borrelia burgdorferi vaccine given to resident infants born in and immigrants of any age to geographically defined highrisk areas coccidioides immitis vaccine given to resident infants born in and immigrants of any age to geographically defined highrisk areas enterotoxigenic escherichia coli vaccine administered to infants and travelers epstein-barr virus vaccine administered to -year-olds histoplasma capsulatum vaccine given to resident infants born in and immigrants of any age to geographically defined highrisk areas neisseria meningitidis type b vaccine given to infants shigella vaccine given to infants and travelers or travelers only * quality-adjusted life year (qaly) takes into account quantity and quality of life generated by health care interventions. qaly is calculated by placing a weight on time in different health states. the cost per qaly is the cost required to generate year of perfect health. data from www.iom.edu/vaccinepriorities. tetanus, and smallpox, active immunization was not available for the other conditions in this edition. currently, active immunization exists for of the diseases contained in the eight pages of the red book, and one disease, smallpox, has been eradicated. since then, many other infectious disease have emerged or reemerged, including sars, hiv, west nile virus, metapneumovirus, avian influenza, and methicillin-resistant s. aureus. many of these conditions are expected to be controlled in the future by immunizations. as the recommended childhood and adolescent immunization schedule continues to expand, the us immunization program will be challenged to integrate novel immunization strategies into the current immunization infrastructure. the impact of future vaccines in the united states will be more difficult to calculate because they will prevent fewer deaths than vaccines in the past. the cost of these vaccines will continue to increase, and funding support will be challenged. the risk of adverse vaccine events will have to be weighed against the risk of the disease if not vaccinated. pediatric health care providers face a growing complexity of problems in children, including injury, obesity, asthma, and mental health and behavioral disorders. as the cost and complexity of the childhood and adolescent immunization schedule increase, considering the role of immunizations within the context of other preventive health interventions and overall societal values becomes increasingly important. immunizations are one of the most effective clinical preventive services in pediatric practice [ ] . despite the challenges facing the us immunization program, immunizations will likely remain on the list of great public health accomplishments of the twenty-first century, and the legacy of jenner will continue. smallpox and its eradication. geneva who ten great public health achievements, united states impact of vaccines universally recommended for children-united states measles elimination in the united states epidemiology of poliomyelitis in the united states one decade after the last reported case of indigenous wild virus-associated disease healthy people : national health promotion and disease prevention objectives haemophilus influenzae invasive disease in the united states, - : near disappearance of a vaccine-preventable childhood disease recommended childhood and adolescent immunization schedule-united states advisory committee on immunization practices, american academy of pediatrics, american academy of family physicians, national immunization program development of pediatric vaccine recommendations and policies vaccination policies and programs: the federal government's role in making the system work financing immunizations in the united states cost-effectiveness of a routine varicella vaccination program for us children projected cost-effectiveness of pneumococcal conjugate vaccination of healthy infants and young children priorities among recommended clinical preventive services cost-effectiveness of hepatitis b virus immunization strengthening the supply of routinely recommended vaccines in the united states: recommendations from the national vaccine advisory committee centers for disease control and prevention. shortage of tetanus and diphtheria toxoids update on the supply of tetanus and diphtheria toxoids and of diphtheria and tetanus toxoids and acellular pertussis vaccine notice to readers: decreased availability of pneumococcal conjugate vaccine shortage of varicella and measles, mumps and rubella vaccines and interim recommendations from the advisory committee on immunization practices experiences with obtaining influenza vaccination among persons in priority groups during a vaccine shortage-united states notice to readers: pneumococcal conjugate vaccine shortage resolved impact of vaccine shortages on immunization programs and providers variation in public and private supply of pneumococcal conjugate vaccine during a shortage limited supply of pneumococcal conjugate vaccine: suspension of recommendation for fourth dose report to congressional requesters: childhood vaccines: ensuring an adequate supply poses continuing challenges safety of immunizations: vaccine centers for disease control and prevention. poliomyelitis prevention in the united states: updated recommendations of the advisory committee on immunization practices (acip) immunization safety review: committee reports. washington, dc national academy of sciences understanding vaccine safety information from the vaccine adverse event reporting system withdrawal of rotavirus vaccine recommendation rotavirus vaccine for the prevention of rotavirus gastroenteritis among children: recommendations of the advisory committee on immunization practices (acip) safety of immunizations centers for disease control and prevention. immunization of adolescents: recommendations of the advisory committee on immunization practices, the american academy of pediatrics, the american academy of family physicians, and the american medical association preventing pneumococcal disease among infants and young children: recommendations of the advisory committee on immunization practices (acip) postlicensure surveillance for pneumococcal invasive disease after use of heptavalent pneumococcal conjugate vaccine in northern california kaiser permanente decrease of invasive pneumococcal infections in children among children's hospitals in the united states after the introduction of the -valent pneumococcal conjugate vaccine decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine population-based impact of pneumococcal conjugate vaccine in young children impact of the pneumococcal conjugate vaccine on otitis media pediatricians' self-reported clinical practices and adherence to national immunization guidelines after the introduction of pneumococcal conjugate vaccine potential impact of conjugate pneumococcal vaccines on pediatric pneumococcal diseases childhood hepatitis b virus infections in the united states before hepatitis b immunization clinical practice: prevention of hepatitis b with the hepatitis b vaccine centers for disease control and prevention. hepatitis b vaccination-united states acute hepatitis b among children and adolescents-united states opportunities for control of meningococcal disease in the us prevention and control of meningococcal disease and meningococcal disease and college students: recommendations of the advisory committee on immunization practices (acip) active bacterial core surveillance report, emerging infections program network changing epidemiology of pertussis in the united states: increasing reported incidence among adolescents and adults final: reports of notifiable diseases trends in pertussis among infants in the united states societal costs and morbidity of pertussis in adolescents and adults morbidity of pertussis in adolescents and adults evaluation of strategies for use of acellular pertussis vaccine in adolescents and adults: a cost-benefit analysis infant pertussis: who was the source? new pertussis vaccination strategies beyond infancy: recommendations by the global pertussis initiative pertussis vaccination strategies for neonates-an exploratory costeffectiveness analysis human papillomavirus and cervical cancer safety and immunogenicity trial in adult volunteers of a human papillomavirus l virus-like particle vaccine a controlled trial of a human papillomavirus type vaccine efficacy of a bivalent l virus-like particle vaccine in prevention of infection with human papillomavirus types and in young women: a randomised controlled trial intussusception among recipients of rotavirus vaccine: reports to the vaccine adverse event reporting system intussusception among infants given an oral rotavirus vaccine available at: www.pediatrics. aappublications.org/cgi/reprint/ / /e ?maxtoshow=&hits= &hits= &resultformat= &author =iwamoto&searchid= _ &stored_search=&firstindex= &sortspec= relevance&journalcode=pediatrics varicella disease after introduction of varicella vaccine in the united states contagiousness of varicella in vaccinated cases: a household contact study contacts with varicella or with children and protection against herpes zoster in adults: a case-control study influenza-associated hospitalizations in the united states enhanced virulence of influenza a viruses with the haemagglutinin of the pandemic virus risk factors associated with severe influenza infections in childhood: implication for vaccine strategy burden of interpandemic influenza in children younger than years: a -year prospective prospective study influenza and the rates of hospitalization for respiratory disease among infants and young children centers for disease control and prevention. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) update: influenza-associated deaths reported among children aged b years-united states, - influenza season vaccine technologies progress in immunologic adjuvant development financing vaccines in the st century: assuring access and availability genesis of a highly pathogenic and potentially pandemic h n influenza virus in eastern asia key: cord- - xo fiop authors: criscuolo, e.; caputo, v.; diotti, r. a.; sautto, g. a.; kirchenbaum, g. a.; clementi, n. title: alternative methods of vaccine delivery: an overview of edible and intradermal vaccines date: - - journal: j immunol res doi: . / / sha: doc_id: cord_uid: xo fiop vaccines are recognized worldwide as one of the most important tools for combating infectious diseases. despite the tremendous value conferred by currently available vaccines toward public health, the implementation of additional vaccine platforms is also of key importance. in fact, currently available vaccines possess shortcomings, such as inefficient triggering of a cell-mediated immune response and the lack of protective mucosal immunity. in this regard, recent work has been focused on vaccine delivery systems, as an alternative to injectable vaccines, to increase antigen stability and improve overall immunogenicity. in particular, novel strategies based on edible or intradermal vaccine formulations have been demonstrated to trigger both a systemic and mucosal immune response. these novel vaccination delivery systems offer several advantages over the injectable preparations including self-administration, reduced cost, stability, and elimination of a cold chain. in this review, the latest findings and accomplishments regarding edible and intradermal vaccines are described in the context of the system used for immunogen expression, their molecular features and capacity to induce a protective systemic and mucosal response. one of the ten greatest public health achievements of the last century was preventative vaccination [ ] . vaccines have successfully reduced the spread of diseases and mitigated mortality associated with infectious agents such as diphtheria, tetanus, polio, measles, mumps, rubella, and hepatitis b [ ] . in spite of the many successes achieved by vaccines, novel technologies and administration routes remain one of the main focuses in the vaccinology field. although many licensed vaccines are administered by injection, in certain cases, this administration route suffers from limitations. in particular, injectable vaccines require trained personnel for the administration of the vaccine and may require multiple doses or inclusion of an adjuvant. moreover, injectable vaccines may require specialized storage and transport conditions. from an immunological point of view, injectable vaccines are capable of eliciting robust systemic humoral responses while conferring weaker t cell-mediated immunity and mucosal protection [ , ] . importantly, t cell effector activity and mucosal immunity both contribute to prevention and control of infection from pathogens targeting the mucosa [ ] . to improve on this limitation, alternative vaccine delivery methods coupled with novel formulations and production systems have recently been proposed. numerous studies have focused on vaccines delivered to the mucosal interface or intradermally, demonstrating rapid and wide biodistribution of the antigen and the capacity to induce both protective mucosal (mainly mediated by secretory iga [siga] ) and systemic cellular and humoral responses [ ] [ ] [ ] . in this review, we discuss current advances and advantages of edible systems based on plants, algae, yeast, insect cells, and lactic acid bacteria and of the intradermal immunization route. . . the mucosal delivery and the immune response. the efficacy of the mucosal administration route is largely based on the fact that mucous membranes constitute the largest immunologic organ in the body. moreover, this interface is endowed with well-organized lymphatic structures, termed mucosa-associated lymphoid tissue (malt), containing both the innate and adaptive (t and b cells) arms of the immune system [ ] . furthermore, antigen-specific siga plays a pivotal role in protecting mucosal surfaces from both microbe adhesion and toxin activities [ ] . thus, the development of novel vaccine delivery platforms implementing the elicitation of pathogen-or toxin-specific siga, as well as systemic igg, is pivotal to improve vaccine effectiveness [ ] . to date, the most well-studied vaccine delivery platforms capable of eliciting both mucosal and systemic immunities are edible or intradermal vaccine formulations ( figure ). oral vaccines stimulate the generation of immunity in gut-associated lymphoid tissue (galt), which includes lymph nodes, peyer's patches (in which lymphocytes are the major component:~ % are b cells, while~ % are t cells), and isolated lymphoid follicles in the gastrointestinal tract (git). an effective immunization using oral vaccines is achieved when sufficient quantities of antigen are transported across the mucosal barrier by m cells into peyer's patches and subsequently presented to t cells by antigenpresenting cells (apcs) [ ] . briefly, professional apcs display peptide fragments of the antigen in the context of the major histocompatibility complex (mhc) on their surface, which leads to activation of cd + t cells [ ] . subsequently, activated cd + t cells support germinal center development, including b cell affinity maturation and class switching to iga, through providing cd /cd ligand interactions and cytokine secretion [ ] [ ] [ ] . moreover, through the expression of specific chemokine homing receptors (e.g., cxcr or ccr ), antigen-experienced b cells migrate to distant effector regions where they differentiate into plasma cells capable of secreting dimeric or polymeric figure : alternative methods of vaccine delivery. development of rationally designed vaccines starts with the identification of the gene encoding for the protective antigenic protein(s). subsequently, the antigen(s) can be incorporated into different edible systems, as plants, algae, insects, or yeasts, or used for intradermal formulations to induce a mucosal protective response. following the administration of the edible vaccine and the subsequent passage of the antigen(s) through the m cell compartment delivering it to dendritic cells, the individual's immune system triggers a response leading also to specific iga production and secretion. similarly, patches with coated microprojections or biodegradable needles activate langerhans cells and dermal dendritic cells in the skin dermis. these cells capture and present the antigen(s) to t and b lymphocytes, triggering both a mucosal and a systemic immunity. iga molecules that are transported into the intestinal lumen as siga [ , ] . in the context of edible vaccines aimed at eliciting pathogen-specific responses, it will be necessary to overcome mucosal tolerance. briefly, mucosal tolerance is achieved against certain foreign antigens, such as those contained in our food, and serves to prevent unnecessary and potentially detrimental immune responses in the gut mucosa. due to this phenomenon, an erroneous mucosal vaccine formulation could induce a t reg -based tolerogenic response instead of th -mediated protective immunity [ ] . this potential shortcoming can be circumvented using several strategies, including incorporation of an appropriate adjuvant in the vaccine formulation or using sufficiently high doses of antigen to promote induction of effector rather than regulatory cells [ , ] . moreover, in the context of edible-based vaccine immunizations, it will also be important to consider the characteristics of the git, in which several factors, including proteolytic enzymes, acidic ph, bile salts, and limited permeability, may hinder the induction of a protective immune response [ ] . to this end, conjugation of the vaccine antigen with specific ligands that enhance their uptake by m cells represents a focus of ongoing studies aimed at improving immunogenicity [ ] . moreover, antigen bioencapsulation avoids degradation and conformational alterations [ ] . in the following sections, we review the various strategies underlying the development of edible vaccines. in particular, we focused on plant, algae, insect cells, whole yeast, and lactic acid bacteria-based vaccines and describe the advantages and limitations of individual systems. plants have been extensively used for developing novel biopharmaceutical-producing platforms in recent years, as they promote proper folding of exogenous proteins and are economically sustainable [ , ] . this is also known in the context of "molecular farming," in which biomolecules of commercial value are produced in genetically engineered plants. there are several ongoing clinical trials using purified antigens transiently produced in tobacco plants (nicotiana benthamiana) for injectable vaccine formulations. for example, medicago recently completed a phase ii clinical trial using a plant-derived, virus-like particle (vlp) quadrivalent influenza vaccine and announced a phase iii clinical study in the last year (clinical-trials.gov identifier: nct ) [ ] . owing to the fact that plants are edible, the notion that they could serve as a delivery vehicle, as well as biofactories, led to their use for oral vaccination in the early s [ ] . in recent years, additional studies have sought to overcome the limitations of conventional vaccines through development of edible formulations [ , ] . since the inception of the idea, it has been evident that using plants to produce vaccines would have several advantages. first, plant vaccines would likely have a low production cost and could be easily scaled-up, as has been demonstrated by the biopharmaceutical industry. molecular farming gained visibility thanks to the success of zmapp, the experimental drug against the ebola virus that was produced in nicotiana plants [ ] . however, unlike biomolecule production, edible vaccine formulations do not need processing or purification steps before administration, which serves to further lower productionassociated costs. indeed, another advantage of this strategy is that plant cells would provide antigen protection due to their rigid cell wall. this is also known as the bioencapsulation effect and could increase bioavailability of antigenic molecules to the galts through preserving structural integrity of vaccine components through the stomach to elicit both a mucosal and a systemic immune response. additional strategies for antigen protection can be achieved through targeting biomolecule expression inside chloroplasts or other storage organelles [ ] or in the protein bodies of seeds [ , ] . this technology also offers advantages in terms of storage and cold chain-free delivery due to the high stability of the expressed antigens bioencapsulated within the plant and seed tissues. moreover, plant materials can be stored at elevated temperatures for longer periods and grown where needed, making this strategy even more attractive for developing countries [ ] . finally, plant-based oral vaccines are characterized by improved safety relative to traditional recombinant vaccine platforms, especially since contamination from mammalian-specific pathogens can be eliminated [ ] . indeed, some studies using lyophilized leaves have shown their advantages over fresh materials such as long-term stability, higher antigen content, and lower microbial contamination. as an example, freeze-dried ctb-ex -expressing (ctb: cholera toxin b subunit; ex : exendin- ) leaves were shown to be stable for up to months at room temperature, and lettuces expressing soluble antigen (pa; protective antigen from bacillus anthracis) were successfully stored for up to months at room temperature without showing antigen degradation [ ] . the antigen content in lyophilized leaf materials was also -fold higher than fresh leaves. an additional benefit of lyophilization was its ability to remove microbial contamination. while lyophilized lettuce had no detectable microbes, fresh leaves contained up to approximately cfu/g microbes when plated on various growing media [ ] . to date, vaccine antigens have been transformed into many edible species including lettuce, tomato, potato, papaya, carrot, quinoa, and tobacco [ ] . their proper folding and enhanced expression have also been tested in animal models, proving the immunogenicity of antigens produced in these systems [ , ] . to obtain high quantities of the protein of interest, both nuclear and chloroplast genomes have been successfully engineered. however, the latter option is preferred owing to some advantages including high levels of transgene expression (up to % of total soluble proteins (tsp)) [ , ] , bioencapsulation effect, and regulatory concerns since transgene containment is assured by the fact that plastids are not spread via pollen in most plants. moreover, incorporation of vaccine antigens into the chloroplast genome would also enable the expression of multiple genes in a single operon, which would be very attractive for multivalent vaccine development. likewise, this approach may enable the production of vaccines conferring protection against multiple infectious agents and would serve to further reduce costs associated with vaccine production and administration [ ] . unfortunately, there are some disadvantages undermining their applications. first, plastids are not suitable for production of antigens that require glycosylation for proper folding or those antigens in which a protective immune response requires glycan recognition. however, nuclear transformation represents a valid option. secondly, antigen expression can be either transient or stable in plants, but the second is preferred in order to obtain a stable genetic resource. in fact, transgenic seeds represent a constant resource to grow the transgenic plants and to extract proteins. however, stable transformation is time-consuming [ ] . moreover, expression in stable transformed crop plants suffers from low yields, typically less than % of tsp [ ] . on the other hand, transient expression technology using either agrobacterium or viral vectors is robust, quick, and easy to manipulate [ ] . however, this expression is typically unstable [ ] . another important challenge of plant-based oral vaccines is the lack of a proper dosing strategy because low doses may not be able to induce a sufficient immune response and high doses, as previously described, may lead to immune tolerance. to this end, freeze-drying methods were implemented to stabilize plant biomass, concentrate the antigen, and achieve an accurate dosage by quantifying the antigen in terms of dry biomass weight, as mentioned above [ , ] . to date, there are some plant-based vaccines for the hepatitis b virus (hbv), rabies virus, norwalk virus, enterotoxigenic e. coli, and vibrio cholerae in phase clinical trials (table ) . many others are still in preclinical development, including vaccines targeting a variety of pathogens such as avian influenza viruses (hpai h n ) [ ] , helicobacter pylori [ ] , and coronaviruses [ ] . . . algae-based vaccines. green microalgae, such as chlamydomonas reinhardtii, represent another viable option for vaccine production. however, some disadvantages of plantderived vaccines, such as low expression levels and improper glycosylation of antigen proteins, have been described [ ] . thus far, only chloroplast transformation is possible [ ] , and only a single organelle is present, even if it occupies half of the cell volume [ ] . stable transformed lines of green algae are easy to obtain and can lead to increased yield of expressed antigens. in fact, unicellular green algae have all the positive characteristics of plant systems, plus unique advantages over terrestrial plants. biomass accumulation is extremely fast and can be used in its entirety. their growth neither has seasonal constraints nor relies on soil fertility. cross-contamination of nearby crops cannot occur, as algae can be cultured with enclosed bioreactors [ ] . furthermore, in regard to regulatory aspects, green algae, such as c. reinhardtii, are generally recognized as safe (gras) by the fda. finally, algae can be easily lyophilized and, when dried, can be stored at room temperature for up to months without losing antigenic efficacy [ ] . in fact, the algae cell wall assures the bioencapsulation effect, as it was proven to prevent antigen degradation by enzymes of the git [ ] . collectively, these characteristics indicate that algae would be an ideal host for vaccine transport without a cold chain supply. thus, as already described for plant-derived edible vaccines, the low cost and simpler logistic in terms of manufacturing, storage, delivery, and administration of the algae-based technology make it an ideal system in the context of resource-limited settings compared to conventional vaccine formulations. there are no algae-based vaccines currently in clinical trials; however, preclinical formulations against human papillomavirus (hpv), hbv, and foot-and-mouth disease virus (fmdv) are under development [ , , ] to overcome some technical problems, such as a low expression level from the nuclear genome and lack of glycosylation following chloroplast expression [ ] . . . insect cell-based vaccines. insect cell systems have been widely adopted because of their capacity to produce high levels of proteins and to perform cotranslational and posttranslational modifications, including glycosylation, phosphorylation, and protein processing. this expression platform allows for generation of stable transformed cell lines or transient expression driven by recombinant baculovirus infection. the baculovirus-insect cell expression system, referred to as bevs, is one of the most well-known and used systems for large-scale production of complex proteins and, most recently, for the development of subunit vaccines [ ] . to date, there are three commercially available vaccines produced in insect cells for different indications: cervarix (gsk) for cervical cancer, flublok (protein sciences, now owned by sanofi pasteur) for influenza, and provenge (dendreon) for prostate cancer [ ] . importantly, the baculovirus expression system is not limited only to cultured cells. insect larvae or pupae can be used for protein production. in the context of edible vaccines using insect larvae or pupae, silkworm bombyx mori larvae or pupae have been commercially used for the production of recombinant proteins and also as a feasible delivery system for the vaccine [ , ] . as mentioned above, the baculovirus-silkworm expression system is able to perform cotranslational and posttranslational modifications and also able to produce large amount and multiple proteins. moreover, since baculovirus is unable to replicate in vertebral animals, it can be considered a gras. furthermore, the presence of protease inhibitors and biocapsule-like fat in the silkworms may protect recombinant proteins from enzymatic digestion in the git [ , ] . several vaccine prototypes are currently under evaluation, and strong systemic immune protective responses support the use of silkworm as a mucosal vaccine vector, as shown, for example, by high immunogenicity in mice of the urease b subunit of helicobacter pylori produced in silkworm [ , ] . while the data collected so far support the possible use of baculovirus-silkworm vaccines as a promising edible vaccine platform, it is only approved for food ingestion in a few asian countries. . . whole-cell yeast-based vaccines. the industrial usage of yeasts cells for production of heterologous proteins has been well described [ , ] . additionally, the capability of this system to perform posttranslational modifications, the gras status, and the cellular wall that could protect the antigen across the git make engineered yeasts an attractive vaccine delivery system [ , ] . in addition, the major drawback of this system is hyperglycosylation of recombinant proteins, but it has been already addressed by generating defective n-glycosylation strains of yeasts [ , ] . whole-cell yeast-based vaccines have been studied for their ability to elicit an immune response. remarkably, some preclinical studies based on orally administrated saccharomyces cerevisiae and developed for different infectious agents, such as hpv and actinobacillus pleuropneumoniae, showed that this delivery system is able to induce a protective mucosal and a systemic immune response [ ] [ ] [ ] . moreover, the increased immunogenicity of this delivery system could be explained by the adjuvant activity of β-glucans on the yeast cell wall, which demonstrates immunomodulatory and adjuvant effects through binding of innate pathogen receptors on macrophages, dc, and neutrophils [ ] . currently, two clinical trials have been developed: gs- for hbv treatment and gi- for hepatitis c virus (hcv) treatment (table ). regarding the clinical trial for gs- , despite the positive results obtained from phase [ ] , the second phase, conducted in virally suppressed, noncirrhotic patients with chronic hbv infection did not show a clinical benefit. however, other safety and efficacy studies have been conducted on another group of patients (in particular, in treatment-naïve patients with chronic hbv) [ ] . regarding the clinical trial for gi- , phases i and ii reported promising results [ ] . in particular, in this trial, gi- was used also in combination with peg-ifn/ribavirin. however, data on efficacy have not been published yet. lactic acid bacteria (lab) are gram-positive, nonsporulating, and nonpathogenic bacteria that have been used for decades for the production and preservation of food as well as for therapeutic heterologous gene expression, like the production of different anti-human immunodeficiency virus (anti-hiv) antibodies (scfv-m , dab-m , and dab-m . ) by lactobacillus jensenii and the production and functional expression of the antilisterial bacteriocin enta in l. casei [ ] [ ] [ ] . given these and the ability of lab to elicit a specific immune response against recombinant foreign antigens, these bacteria have been considered potential candidates as mucosal vaccine vectors. this delivery system can confer protection against antigen degradation and, thanks to its uptake at the git level, can activate both innate and adaptive immune responses [ , ] . many lab, in particular, lactobacillus spp and bacillus subtilis, were used in preclinical studies against different infectious diseases. different results have been obtained from these studies, but an elicited immune response was observed in all of them. as an example, the production of high levels of specific iga and systemic igg after immunization with bacillus spores expressing toxin a peptide repeat was reported [ ] , while in another paper, l. lactis expressing hev antigen orf vaccine was tested and a specific th -based cellmediated immune response was revealed as well as the production of specific mucosal iga and serum igg [ ] . another study reported a th /th immune response elicited after the immunization with csenolase-expressing bacillus subtilis [ ] . another example is the oral administration of b. subtilis spores expressing urease b of helicobacter pylori that provide protection against helicobacter infection [ ] . an important feature of lab is their natural adjuvanticity and their immunomodulatory effects, although the molecular mechanism of these capabilities is not completely understood [ ] . moreover, other studies reported an effect on dendritic cell maturation and an induction of cytokine secretion [ , ] . despite the promising characteristics of recombinant lab as mucosal vaccine vectors and given the encouraging results from murine studies, some aspects need to be taken into consideration, namely, the fact that vaccine strains cannot be considered avirulent, even if it could be listed as gras, due to potential transfer of antibiotic selection markers among microbes [ , ] . furthermore, other factors are important for the development of lab-based vaccines. as an example, the necessity of a suitable delivery system since different administration routes produce different immune effects. additionally, the role of specific adjuvants and the correct localization (intracellularly or on the bacterial surface) of each expressed antigen need consideration [ ] . overall, additional studies and clinical trials are needed for the development of efficient vaccines based on lab. a different carrier system based on nonrecombinant lactococcus lactis bacteria was recently developed. this system, called gram-positive enhancer matrix (gem), is composed of the rigid peptidoglycan (pgn) cell wall of the bacterium resulting in a nonliving particle that preserves the shape and the size as the original bacterium [ ] . gems are used in two different ways: mixed with vaccine antigens as adjuvants or as antigen protein carriers, with the antigens bound to the surface of gems. regarding the use of gems as adjuvants, because of their nature, gems are safer adjuvants compared to others. moreover, they retain the inflammatory properties of live bacteria and enhanced specific mucosal and systemic immune responses of the influenza subunit vaccine [ ] [ ] [ ] . therefore, the use of gems was further examined in a study investigating the immune response elicited by intranasal delivery of the influenza subunit vaccine coadministrated with gem (flugem). in detail, an influenza-specific memory b cell response and the presence of long-lived antibodysecreting plasma cells were reported. additionally, this immune response was able to confer protection from influenza infections [ ] . these important results obtained in murine studies have led to a phase i clinical trial which confirmed the positive preclinical data. systemic hemagglutination inhibition (hai) titers and local siga responses were reported. further studies will assess if this immune response confers protection against the influenza virus [ ] . gems have also been used as antigen protein carriers. in particular, antigens are bound to gem through the presence of a pgn-binding tag (protan) in the antigen. several works used this vaccination strategy, and the data support the potential of gems as safe vaccine delivery vehicles and their ability to elicit systemic antibodies [ ] [ ] [ ] [ ] . moreover, gems are also able to present several antigens at the same time which could be helpful for the preparation of multivalent vaccines [ ] . furthermore, the delivery of an adjuvant (gems) and an antigen together has been correlated with enhanced vaccine immunogenicity [ ] . lastly, as opposed to a vaccine based on lab, the absence of recombinant dna avoids its dissemination into the environment. however, the inability of gems to colonize any compartment does not allow the reduction of the number of vaccine doses. these promising premises allowed the development of a vaccine against respiratory syncytial virus (rsv). in particular, an intranasal formulation based on the trimeric rsv fusion protein coupled with gems and named syngem was developed. also, in this case, positive results from studies in mice and rats have been obtained, and as for flugem, vaccination with syngem resulted in the induction of a robust systemic and mucosal immune response as well as a balanced cytokine profile. these data supported further study of this vaccine in phase i clinical trial, which is currently ongoing [ ] . in conclusion, gems represent an interesting vaccination strategy either as adjuvant or as antigen protein carriers, but as in the case of vaccine based on lab, further studies are needed. immune response. another vaccine delivery route capable of triggering both systemic and mucosal immunities is the intradermal route, in which the antigen is delivered through the skin using recently developed self-administrable devices. in particular, the application of microneedle technology overcomes the skin permeation barrier imposed by the stratum corneum and facilitates antigen delivery. the efficacy of this new microneedle-based immunization approach is due to the presence of several types of immune cells (such as dcs, t lymphocytes, nk cells, macrophages, and mast cells) in the epithelium [ , ] . in fact, the cells that are responsible for triggering the inflammation cascade in the skin are the langerhans cells (comprising - % of epithelial cells). langerhans cells are a specific dc subset that migrates into the lymph node following antigen capture and aids in the initiation of an adaptive immune response [ ] . these cells are also efficiently stimulated by pathogen-associated molecular patterns (pamps) using an array of germlineencoded pattern recognition receptors (prr), including toll-like receptors (tlr) and langerin (cd ) [ ] . importantly, skin resident mast cells are also key drivers of the innate immune response in the skin through the release of granules containing inflammatory mediators [ ] . . . intradermal vaccination. using conventional syringes for intramuscular or subcutaneous vaccinations, large volumes of vaccine solution can be injected (≥ ml). thus, the choice of the muscle or hypodermis as vaccination targets is mainly based on convenience [ ] . intradermal immunization for vaccine delivery is an upcoming strategy showing significant advantages over conventional vaccination routes. in particular, in the last few years, intradermal vaccination has gained momentum as an alternative and more effective vaccine delivery route, both from a scientific and a commercial point of view (table ) . intradermal vaccination designates the delivery of an antigen directly into the dermis with a syringe, a needle, a microneedle, or a pressure injector. the standard intradermal immunization technique was invented by the french physician charles mantoux in , while he was developing the tuberculin test. this technique allows the injection of - μl of vaccine solution. however, mantoux's technique requires skilled medical personnel to be performed [ ] . recent advancements have led to the development of techniques and instruments that can overcome the difficulties associated with intradermal administration [ ] . in fact, different devices have been developed over the years for intradermal vaccination. among them, solid microneedles, particle injectors, and self-administrable patches with coated microprojections or biodegradable needles have been described [ ] . as previously mentioned, intradermal vaccination can induce mucosal and systemic immunities. these immunological capabilities, coupled with its ease of access, make the intradermal route an attractive vaccination delivery target [ ] . intradermal vaccination has been demonstrated to be very safe. in fact, novel devices involve the use of needles with a smaller size than the usual ( μm and mm) and make it possible to bypass the corneous layer of epidermis by creating transient micropores in the cutaneous tissues. even if some studies have shown that intradermal vaccination can be associated with a higher incidence of local reactogenicity, including primarily mild pain, swelling, and redness, systemic side effects have not been reported. in fact, the intradermal route limits the transfer of vaccine components to the blood circulation (and the risk of septic shock) and the possible toxicity due to hepatic first-pass effect [ ] . typically, when present, local effects resolve quickly, as reported in a study comparing the safety and immunogenicity of a large number of intradermal versus intramuscular influenza vaccines [ ] . another important aspect is the possibility of improving the immunogenicity of various vaccines in immunocompromised hosts as well as during pregnancy via the intradermal route [ , ] . as an example, it has been reported that the hbv vaccine fails to yield seroconversion in - % of recipients. however, a significant improvement was observed following intradermal vaccination [ ] . additionally, it has been demonstrated that in patients on dialysis or in hiv-positive subjects, the intradermal route was more immunogenic than the standard intramuscular route [ ] . from a commercial point of view, intradermal vaccination has been primarily explored for its ability to elicit equivalent antibody responses at lower doses, a phenomenon typically described as "dose sparing" [ ] . in this regard, the advantage of a low dose is most evident in high-surge situations, such as during pandemic and seasonal influenza waves, in which large populations are at an increased risk and large amounts of new antigen preparations are quickly required each year [ ] [ ] [ ] . above all, dose sparing is also important in a large-scale setting and in reducing the production-associated costs, especially in developing countries, where the price of the vaccine limits its use and coverage. in this regard, the u.s. food and drug administration (fda) approved the trivalent inactivated intradermal influenza vaccine for use in adults - years of age for use during the - season, and a quadrivalent formulation was subsequently approved in . however, similar to intramuscular vaccines, the formulation of these approved intradermal vaccines is liquid and thus still dependent on the cold chain and administered through a syringe. for these reasons, novel dried solid microneedle devices, while eliciting comparable immunogenicity to intramuscular-administered vaccines, represent an innovative approach to facilitate self-administration and allow vaccine storage at room temperature [ ] . infectious diseases represent a global concern, and the most effective strategy to reduce them is vaccination. unfortunately, not every disease can currently be prevented through vaccines. however, many strategies have been developed against infectious agents, such as the generation of neutralizing antibodies, antibiotics, and antiviral drugs [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and innovative approaches are currently under development [ ] [ ] [ ] . many vaccines have been developed and approved against various pathogens, and countless studies have been conducted to improve their efficacy by testing new adjuvants and performing the rational identification of the antigen formulations and pathogen contaminations [ ] [ ] [ ] . promising results have been also achieved by changing the delivery strategy. in fact, most of the approved vaccines are administrated by injection with intrinsic limitations like those concerning the immunological aspect. injected vaccines are able to elicit a strong humoral immunity but a weak cellular response. in addition, this type of administration is strongly associated with a systemic immunity but with a lack of mucosal response, which is helpful to block the early stages of infection since most pathogens infect through the mucosal membranes. for these reasons, new vaccination strategies have been proposed. in particular, edible vaccines, triggering the galt, and intradermal approaches, involving langerhans cells, are able to elicit both a mucosal and a systemic immune response. the increased knowledge of these approaches has led to the progression of many preclinical studies and several promising clinical trials (tables , , and ). moreover, these vaccine strategies are considered safe and cost-effective as no extensive antigen processing is needed [ , ] and they are easy to administrate (table ). in fact, due to the opportunity of self-administration and ease of distribution compared to an injection-based approach, these two vaccination systems could improve the overall coverage. there remain a number of obstacles and drawbacks associated with each antigen delivery platform that still need to be addressed (table ). presently there are no fda-approved compounds for edible vaccination, but new emerging technologies like nanoparticles (nps) could help to control antigen bioavailability to avoid mucosal tolerance. nps are particles with a mean size of - nm (up to nm), which can be used as carriers and/or adjuvants in vaccine preparation [ ] [ ] [ ] . moreover, nps can be targeted to specific cell populations. as an example, nps can be coated with antibodies recognizing a surface protein on dendritic cells [ , ] . this approach enabled a more accurate measurement of the quantity of antigen required to elicit an immune response [ ] . finally, a more efficient immunization was demonstrated using np-based approaches combined with an intradermal vaccine delivery [ ] , while oral delivery needed further investigations as they have been tested only in vitro [ , ] . in conclusion, novel approaches eliciting a stronger mucosal response are showing promising results both in preclinical and clinical studies. further studies are needed to implement and improve these delivery systems; however, mucosal delivery is becoming the most preferred mode of vaccination. ten great public health achievements-united states the intangible value of vaccination single mucosal, but not parenteral, immunization with recombinant adenoviralbased vaccine provides potent protection from pulmonary tuberculosis mucosal adjuvants and long-term memory development with special focus on cta -dd and other adp-ribosylating toxins recent progress in mucosal vaccine development: potential and limitations a prime-boost vaccination strategy using attenuated salmonella typhimurium and a replication-deficient recombinant adenovirus vector elicits protective immunity against human respiratory syncytial virus mucosal immunity and vaccines function of mucosa-associated lymphoid tissue in antibody formation experience with registered mucosal vaccines gut-associated lymphoid tissues for the development of oral vaccines current state and challenges in developing oral vaccines keynote review: intestinal peyer's patch m cells and oral vaccine targeting adaptive immune regulation in the gut: t cell-dependent and t cell-independent iga synthesis human iga-and igm-secreting intestinal plasma cells carry heavily mutated vh region genes diversification of memory b cells drives the continuous adaptation of secretory antibodies to gut microbiota antigen-driven evolution of b lymphocytes in coronary atherosclerotic plaques anatomical and cellular basis of immunity and tolerance in the intestine uptake through glycoprotein of fimh + bacteria by m cells initiates mucosal immune response green therapeutic biocapsules: using plant cells to orally deliver biopharmaceuticals transgenic plants: a -year update on oral antipathogen vaccine development plant-based vaccines against viruses preclinical and clinical development of plant-made virus-like particle vaccine against avian h n influenza food-grade organisms as vaccine biofactories and oral delivery vehicles plant-made oral vaccines against human infectious diseases-are we there yet? need of cost-effective vaccines in developing countries: what plant biotechnology can offer? the potential of plants as a system for the development and production of human biologics using storage organelles for the accumulation and encapsulation of recombinant proteins seed-based oral vaccines as allergen-specific immunotherapies the increasing value of plant-made proteins plant-produced vaccines: promise and reality oral delivery of bioencapsulated exendin- expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-tc cells disease prevention: an opportunity to expand edible plant-based vaccines? plastids: the green frontiers for vaccine production exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic the role of heterologous chloroplast sequence elements in transgene integration and expression chloroplast-derived vaccines against human diseases: achievements, challenges and scopes transient expression systems for plant-derived biopharmaceuticals expression of the b subunit of escherichia coli heat-labile enterotoxin as a fusion protein in transgenic tomato a plant-based system for rapid production of influenza vaccine antigens oral immunogenicity and protective efficacy in mice of a carrot-derived vaccine candidate expressing ureb subunit against helicobacter pylori severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato immunogenicity of recombinant lt-b delivered orally to humans in transgenic corn human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes expression in plants and immunogenicity of plant virus-based experimental rabies vaccine a plant-derived edible vaccine against hepatitis b virus immunogenicity in humans of an edible vaccine for hepatitis b a rice-based oral cholera vaccine induces macaque-specific systemic neutralizing antibodies but does not influence pre-existing intestinal immunity induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin b subunit vaccine without plant-associated sugar modification good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants mucoricecholera toxin b-subunit, a rice-based oral cholera vaccine, down-regulates the expression of α-amylase/trypsin inhibitorlikeprotein family as major rice allergens algae-based oral recombinant vaccines recent developments in the production of human therapeutic proteins in eukaryotic algae plant-derived vaccines and other therapeutics produced in contained systems heatstable oral alga-based vaccine protects mice from staphylococcus aureus infection recombination and expression of classical swine fever virus (csfv) structural protein e gene in chlamydomonas reinhardtii chroloplasts insect cell technology is a versatile and robust vaccine manufacturing platform non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules oral administration of a cholera toxin b subunit-insulin fusion protein produced in silkworm protects against autoimmune diabetes expression of ureb and hspa of helicobacter pylori in silkworm pupae and identification of its immunogenicity silkworm expression system as a platform technology in life science canine parvovirus vp protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity recombinant protein production in yeasts heterologously expressed aspergillus aculeatus β-glucosidase in saccharomyces cerevisiae is a cost-effective alternative to commercial supplementation of β-glucosidase in industrial ethanol production using trichoderma reesei cellulases whole pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize plasmodium antigens protease-deficient saccharomyces cerevisiae strains for the synthesis of humancompatible glycoproteins enhanced expression of heterologous proteins in yeast cells via the modification of n-glycosylation sites induction of antigen-specific immune responses by oral vaccination with saccharomyces cerevisiae expressing actinobacillus pleuropneumoniae apxiia whole recombinant pichia pastoris expressing hpv l antigen is superior in inducing protection against tumor growth as compared to killed transgenic leishmania oral immunization with whole yeast producing viral capsid antigen provokes a stronger humoral immune response than purified viral capsid antigen characterization and optimization of the glucan particle-based vaccine platform safety, tolerability and immunogenicity of gs- , a hepatitis b virus-specific therapeutic vaccine, in healthy subjects: a randomized study randomized phase ii study of gs- as a therapeutic vaccine in virally suppressed patients with chronic hepatitis b whole recombinant yeast-based immunotherapy induces potent t cell responses targeting hcv ns and core proteins expression of human immunodeficiency virus type neutralizing antibody fragments using human vaginal lactobacillus cloning strategies for heterologous expression of the bacteriocin enterocin a by lactobacillus sakei lb , lb. plantarum nc and lb. casei cect recombinant protein production in bacterial hosts recent advances in the development of live, attenuated bacterial vectors live-attenuated yersinia pestis vaccines immunization with bacillus spores expressing toxin a peptide repeats protects against infection with clostridium difficile strains producing toxins a and b oral immunization with recombinant hepatitis e virus antigen displayed on the lactococcus lactis surface enhances orf -specific mucosal and systemic immune responses in mice surface display of clonorchis sinensis enolase on bacillus subtilis spores potentializes an oral vaccine candidate expression of helicobacter pylori urease b on the surface of bacillus subtilis spores lactic acid bacteria -promising vaccine vectors: possibilities, limitations, doubts probiotic modulation of dendritic cell function is influenced by ageing immunomodulatory properties of lactobacillus plantarum and its use as a recombinant vaccine against mite allergy characterisation and transferability of antibiotic resistance genes from lactic acid bacteria isolated from irish pork and beef abattoirs lactic acid bacteria as mucosal delivery vehicles: a realistic therapeutic option novel surface display system for proteins on non-genetically modified grampositive bacteria influenza antigen-sparing by immune stimulation with gram-positive enhancer matrix (gem) particles intranasal delivery of influenza subunit vaccine formulated with gem particles as an adjuvant cta : purified and display onto gram-positive enhancer matrix (gem) particles as mucosal adjuvant lactic acid bacteria- years exploring their potential as live vectors for mucosal vaccination lactococcus lactis gem particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization development of lactococcal gem-based pneumococcal vaccines immunogenicity of a malaria parasite antigen displayed by lactococcus lactis in oral immunisations bacterium-like particles for efficient immune stimulation of existing vaccines and new subunit vaccines in mucosal applications lactic acid bacteria as a surface display platform for campylobacter jejuni antigens intradermal, epidermal and transcutaneous vaccination: from immunology to clinical practice disentangling the complexity of the skin dendritic cell network innate control of adaptive immunity: dendritic cells and beyond controversies in experimental dermatology: who is really in control of skin immunity under physiological circumstances -lymphocytes, dendritic cells or keratinocytes? intradermal vaccine delivery: will new delivery systems transform vaccine administration? cutaneous delivery of prophylactic and therapeutic vaccines: historical perspective and future outlook the success of microneedle-mediated vaccine delivery into skin noninvasive vaccination against infectious diseases vaccine allergy and pseudo-allergy a meta-analysis of intradermal versus intramuscular influenza vaccines: immunogenicity and adverse events microneedle patch for immunization of immunocompromised hosts microneedle patch delivery of influenza vaccine during pregnancy enhances maternal immune responses promoting survival and long-lasting passive immunity to offspring hepatitis b vaccine by intradermal route in non responder patients: an update efficacy of intradermal hepatitis b vaccination compared to intramuscular vaccination in hemodialysis patients dose sparing with intradermal injection of influenza vaccine the role of adjuvants in vaccines for seasonal and pandemic influenza influenza vaccine effectiveness in preventing hospitalizations and deaths in persons years or older in minnesota towards a universal influenza vaccine: different approaches for one goal safety, tolerability, acceptability and immunogenicity of an influenza vaccine delivered to human skin by a novel high-density microprojection array patch (nanopatch™) safety and immunogenicity of a quadrivalent intradermal influenza vaccine in adults safety and immunogenicity of a single oral dose of recombinant double mutant heat-labile toxin derived from enterotoxigenic escherichia coli assessment of acceptability and usability of new delivery prototype device for intradermal vaccination in healthy subjects a phase study of safety and immunogenicity following intradermal administration of a tetravalent dengue vaccine candidate randomized controlled clinical trial of fractional doses of inactivated poliovirus vaccine administered intradermally by needle-free device in cuba priming with a simplified intradermal hiv- dna vaccine regimen followed by boosting with recombinant hiv- mva vaccine is safe and immunogenic: a phase iia randomized clinical trial cloning of the first human anti-jcpyv/vp neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy entry inhibition of hsv- and - protects mice from viral lethal challenge neutralization activity and kinetics of two broad-range human monoclonal igg derived from recombinant fab fragments and directed against hepatitis c virus e glycoprotein monoclonal antibodies isolated from human b cells neutralize a broad range of h subtype influenza a viruses including swine-origin influenza virus (s-oiv) anti-hepatitis c virus e (hcv/e ) glycoprotein monoclonal antibodies and neutralization interference diverging effects of human recombinant anti-hepatitis c virus (hcv) antibody fragments derived from a single patient on the infectivity of a vesicular stomatitis virus/hcv pseudotype a phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments chimeric antigen receptor (car)-redirected t cells: is there a place for them in infectious diseases? chimeric antigen receptor (car)-engineered t cells redirected against hepatitis c virus (hcv) e glycoprotein adoptive t-cell therapy in the treatment of viral and opportunistic fungal infections peptide-based vaccinology: experimental and computational approaches to target hypervariable viruses through the fine characterization of protective epitopes recognized by monoclonal antibodies and the identification of t-cell-activating peptides epitope mapping by epitope excision, hydrogen/deuterium exchange, and peptidepanning techniques combined with in silico analysis elicitation of broadly protective antibodies following infection with influenza viruses expressing h n computationally optimized broadly reactive hemagglutinin antigens microneedle patches as drug and vaccine delivery platform microneedles-based transdermal drug delivery systems: a review non-carrier nanoparticles adjuvant modular protein vaccine in a particle-dependent manner vaccine delivery using nanoparticles antigen delivery via hydrophilic peg-b-page-b-plga nanoparticles boosts vaccination induced t cell immunity protein corona-mediated targeting of nanocarriers to b cells allows redirection of allergic immune responses biomedical nanoparticles: overview of their surface immunecompatibility nanovaccine: a novel approach in immunization intradermal delivery of vaccine nanoparticles using hollow microneedle array generates enhanced and balanced immune response interaction of cruciferinbased nanoparticles with caco- cells and caco- /ht -mtx co-cultures in vitro stability of cucumber mosaic virus nanoparticles carrying a hepatitis c virus-derived epitope under simulated gastrointestinal conditions and in vivo efficacy of an edible vaccine the authors declare that they have no conflicts of interest. e.c. and v.c. contributed equally to this work. key: cord- -cngz o authors: volz, a.; sutter, g. title: modified vaccinia virus ankara: history, value in basic research, and current perspectives for vaccine development date: - - journal: adv virus res doi: . /bs.aivir. . . sha: doc_id: cord_uid: cngz o safety tested modified vaccinia virus ankara (mva) is licensed as third-generation vaccine against smallpox and serves as a potent vector system for development of new candidate vaccines against infectious diseases and cancer. historically, mva was developed by serial tissue culture passage in primary chicken cells of vaccinia virus strain ankara, and clinically used to avoid the undesirable side effects of conventional smallpox vaccination. adapted to growth in avian cells mva lost the ability to replicate in mammalian hosts and lacks many of the genes orthopoxviruses use to conquer their host (cell) environment. as a biologically well-characterized mutant virus, mva facilitates fundamental research to elucidate the functions of poxvirus host-interaction factors. as extremely safe viral vectors mva vaccines have been found immunogenic and protective in various preclinical infection models. multiple recombinant mva currently undergo clinical testing for vaccination against human immunodeficiency viruses, mycobacterium tuberculosis or plasmodium falciparum. the versatility of the mva vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the middle east respiratory syndrome. recent advances include promising results from the clinical testing of recombinant mva-producing antigens of highly pathogenic avian influenza virus h n or ebola virus. this review summarizes our current knowledge about mva as a unique strain of vaccinia virus, and discusses the prospects of exploiting this virus as research tool in poxvirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology. host (cell) environment. as a biologically well-characterized mutant virus, mva facilitates fundamental research to elucidate the functions of poxvirus host-interaction factors. as extremely safe viral vectors mva vaccines have been found immunogenic and protective in various preclinical infection models. multiple recombinant mva currently undergo clinical testing for vaccination against human immunodeficiency viruses, mycobacterium tuberculosis or plasmodium falciparum. the versatility of the mva vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the middle east respiratory syndrome. recent advances include promising results from the clinical testing of recombinant mva-producing antigens of highly pathogenic avian influenza virus h n or ebola virus. this review summarizes our current knowledge about mva as a unique strain of vaccinia virus, and discusses the prospects of exploiting this virus as research tool in poxvirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology. poxviruses engineered to express foreign gene products are established tools for the development of novel vaccines and therapeutics in biomedical research. large packaging capacity for heterologous dna, strict virusspecific control of recombinant gene expression, lack of virus persistence in the host, immunogenicity and efficacy as vaccine, and ease of vector and vaccine production were important contributors to this success story. concerns about the safety of conventional vaccinia viruses as smallpox vaccine have been addressed by the study of replication defective viruses unable to produce infectious progeny in human cells. today, the highly attenuated vaccinia virus strain mva can be considered as one of the vaccine viruses of choice in preclinical and clinical research. mva is replication-deficient in cells of mammalian origin and fails to produce many of the virulence factors encoded by conventional vaccinia virus. because of its safety for the general environment mva can be handled under conditions of biosafety level (bsl- ). nonreplicating mva can enter any target cell and activate its molecular life cycle to express all classes of viral and recombinant genes. therefore, recombinant mva have been established as an extremely safe and efficient viral vector system for basic research and for the development of vaccines suitable for industrial scale production. here, we review the development of mva as product from serial tissue culture passage in chicken embryo fibroblasts (cef), its key biological properties, and recent accomplishments in vaccine research using recombinant mva. modified vaccinia virus ankara (mva) was developed by serial passage in chicken fibroblast tissue culture to serve as safer vaccine during the last years of the who smallpox eradication campaign. its ancestor virus is the vaccinia virus strain ankara which was originally propagated on the skin of calves and donkeys at the turkish vaccine institute in ankara for smallpox vaccine production. in , the vaccinia virus strain ankara was brought to munich and added to the strain collection of the institute for infectious diseases and tropical medicine at the university of munich. herrlich and mayr cultivated the virus on the chorioallantois membranes (cam) of embryonated chicken eggs and therefore named it as chorioallantois vaccinia virus ankara (cva) (herrlich and mayr, ) . at the bayerische landesimpfanstalt m€ unchen (bavarian state institute for vaccines), cva was grown on the skin of calves to manufacture smallpox vaccine for the vaccination campaigns in munich in munich in / . in addition, at the university of munich, cva was tested in passage experiments in various tissue cultures to study the genetic stability and the evolution of orthopoxviruses. mayr and munz ( ) reported that passages of cva in primary cef had resulted in the development of an infection phenotype with restricted host (cell) tropism and it was discussed that similar biological properties were known from poxviruses that are highly adapted to specific hosts, e.g., variola virus (varv) to humans or fowlpox virus to chicken. successive passage of vaccinia virus in minced chicken embryo tissue had been described as successful strategy for in vitro amplification of the smallpox vaccine virus in a culture system ward, , ) . the serial passage of cva in cef was further continued by anton mayr and colleagues and, in after the th passage on cef, the virus was renamed modifiziertes vakziniavirus ankara (mva) and provided to the bavarian state institute for vaccines to test its suitability for smallpox vaccine production (stickl and hochstein-mintzel, ). phenotypic changes relating to the repeated passage of the cva virus in cef cultures were first observed upon infection of the embryonated egg. for many years, cam inoculations were the gold-standard experimental system for the phenotypic study of various poxviruses (goodpasture et al., (goodpasture et al., , mayr et al., ) . mva infection is characterized by the formation of small proliferative lesions on the cam. in contrast, considerably larger cam lesions with variable size areas of central necrosis are typically found with cva or other conventional vaccinia viruses (herrlich and mayr, ) . interestingly, the cam lesions of mva were noted to closely resemble those induced by variola or fowlpox viruses following egg inoculation (mayr and munz, ; stickl and hochstein-mintzel, ). in addition, it was observed early on that mva had lost the capacity of vaccinia virus to cause prominent cytopathic effects and/ or to form plaques in first-generation tissue cultures such as cef, primary bovine, or porcine kidney cells, or human hela cells (mayr and munz, ) . the most characteristic changes in the in vivo behavior of the virus were reported from experimental inoculations of rabbits (german great white) (mayr et al., ; stickl and hochstein-mintzel, ). intradermal infections or cutaneous infections by scarification with conventional vaccinia virus (vacv) result in the formation of typical skin lesions. such lesions were totally absent following inoculation with mva suggesting a substantial loss of virulence upon in vivo infection. these data were confirmed by the finding that newborn (strain nmri; - days old) or adult mice ( - g) survived intracerebral inoculations of mva at doses that resulted in % mortality following cva infection (mayr et al., ; stickl and hochstein-mintzel, ). the inability of mva to induce primary reactions with pock lesions forming upon intradermal or cutaneous inoculation was also confirmed in the cynomolgus monkey model (macacus fascicularis). macaques tolerated intracranial inoculations with mva without obvious adverse effects, whereas animals injected with cva developed severe systemic disease. moreover, intradermal or intramuscular vaccination with about  infectious units (iu) of mva vaccine protected macaques from severe disease following intravenous challenge with varv strain madras (mayr et al., ; stickl and hochstein-mintzel, ). these data from early preclinical characterization in laboratory animals already suggested that mva had maintained immunogenicity as vaccine but demonstrated a dramatic loss of virulence. upon the th cef passage the virus was renamed mva and transferred to the bavarian state vaccine institute in munich for evaluation as safer smallpox vaccine in clinical trials. the first mva vaccine preparation to be tested in humans was produced in cef cultures and contained iu mva/ml (stickl and hochstein-mintzel, ). in first attempts, the use of this vaccine by scarification failed to induce any kind of skin reactions and the application by intracutaneous inoculation ( . ml vaccine suspension containing  iu mva) was chosen for first clinical testing and primary vaccination of individuals aged - years (stickl and hochstein-mintzel, ). after - days, minor local reactions developed at the site of injection with redness and swelling of the skin of the forearm. however, the investigators observed no pock lesions or other symptoms normally associated with smallpox vaccination and none of the individuals developed fever (body temperature ! °c). interestingly, it was noted that the mva application failed to induce circulation antibodies that inhibited the hemagglutination by vacv. thus, the efficacy of immunization was tested with secondary vaccination by scarification using vacv strain elstree in of the individuals that had received mva for primary vaccination. skin reactions typical for follow-up smallpox vaccinations were noted in of patients and suggested a successful primary immunization with the mva vaccine. these data supported further clinical development of mva as smallpox vaccine and, following the testing in more than patients (stickl et al., ) , the bavarian state vaccine institute in munich obtained the first marketing authorization for mva as primary prevaccine against smallpox in germany in (paul-ehrlich-institut, . . ) . until , the mva smallpox vaccine was given to more than , humans without documentation of severe adverse events otherwise associated with the use of conventional vacv vaccines (mahnel and mayr, ) . immunizations with this first licensed mva vaccine stopped with the end of the smallpox vaccination program in germany. only a few years later, targeted genetic modification of the vaccinia virus genome became possible and the concept to generate recombinant viruses for gene expression or vaccination also restored interest in mva. restriction mapping of the mva genome was based on the clonal virus isolate f from the nd cef passage of mva and revealed alterations in the mva genome that are the likely genetic basis for attenuation and growth restriction (meyer et al., ) . comparison to the genome maps of cva ancestor viruses revealed that the mva genome harbors large deletions and mutations affecting many genes with functions in virus-host interaction (fig. ) . first important observations included (i) the failure to rescue productive mva growth in cells of human origin by restoring the vacv host range gene k l despite presence of the second vacv host range gene c l with the mva genome; (ii) the absence of the gene for the major vacv a-type inclusion body protein and the lack of this protein among the polypeptides made in mva-infected cells; (iii) the demonstration that the coding sequences of the vacv hemagglutinin (ha) started right adjacent to deletion iii within the mva genome which a few years later allowed to identify the truncation of the ha promoter sequence explaining for the ha-negative phenotype of mva and the inability to detect ha-specific antibodies upon mva immunization (antoine et al., ) . notably, these early findings were further complemented by the elucidation of the fulllength sequence of the mva genome (antoine et al., ) and the discovery that mva lacks important immunomodulatory genes . the highly attenuated phenotype of the virus also encouraged the evaluation of mva as an expression vector. yet, mva cannot productively replicate in most cells of mammalian origin and high-level expression of recombinant genes was doubtful because other host range mutants of vacv are inhibited already early in their life cycle (drillien et al., (drillien et al., , . the coding sequences for the escherichia coli enzymes β-galactosidase and guaninephosphoribosyl-transferase served as the first heterologous genes to be inserted and expressed at the site of deletion iii in the mva genome (sutter and moss, ) . surprisingly, the production of early and late viral proteins turned out to be unimpaired in mva-infected human cells (fig. ) . indeed, the unique ability to efficiently express viral and recombinant genes supported its general application as exceptionally safe viral vector. to ascertain whether mva vectors would be applicable for immunization experiments with recombinant antigens, a first mva vector vaccine was constructed and tested that simultaneously expressed the ha and nucleoprotein (np) genes of influenza a virus (sutter et al., ) . the recombinant mva was found to be immunogenic, since vaccination of mice by various routes resulted in high levels of serum antibodies that inhibited hemagglutination by influenza a virus. moreover, the vector vaccine also elicited strong cytotoxic t cell responses directed to both influenza virus proteins. importantly, animals could be completely protected against a lethal respiratory tract influenza challenge following single inoculations with relatively low doses of the recombinant mva vaccine. to generate these first mva vectors, the foreign gene sequences were targeted precisely to the site of the naturally existing deletion iii in the mva genome. this strategy in designing the vector was to avoid unnecessary changes in the genotype and phenotype of the resulting recombinant mva. the development of other recombinant mva vaccines with heterologous genes from simian immunodeficiency virus (siv) or parainfluenza virus inserted in deletion iii rapidly followed (hirsch et al., ; wyatt et al., ) . also, the natural deletion ii within the mva genome was successfully used as insertion site to express recombinant bacteriophage t rna polymerase (sutter et al., ) . in addition, the thymidine kinase locus, a well-exploited insertion site in replication-competent vacv vectors, likewise, served to generate recombinant mva delivering antigens of hiv- or plasmodium berghei schneider et al., ) . poxviruses including the prototype orthopoxvirus vacv are excellent models to study virus-host interactions. vacv can efficiently antagonize the activity of interferons (ifns), cytokines, chemokines, and innate immune signaling (for review, see smith et al., ) . hereby, vacv exploits the expression of soluble-binding proteins and receptor antagonists. other viral immune evasion proteins work intracellularly to inhibit apoptosis or to interfere with host signaling pathways activating antiviral immune mechanisms. notably, of the many vacv genes involved in immune evasion, most are inactivated or truncated in the mva genome (antoine et al., ) . interestingly, early studies by mayr and coworkers had suggested distinct immune stimulatory activities associated with experimental mva inoculations (mayr et al., ) . e.g., intraperitoneal application of virus to mice enhanced the in vivo clearance of carbon marker particles from the blood of the animals suggesting an increased phagocytic activity of immune cells days after mva inoculation. in addition, intranasal delivery of mva to rabbits resulted within hours in the induction of "serum interferons" that efficiently inhibited sindbis virus replication in an in vitro infection assay. in the more recent past, various studies further elucidated the mechanisms of mva-mediated induction of type i ifns following in vitro and in vivo infections dai et al., ; delaloye et al., ; ishii et al., ; waibler et al., waibler et al., , . indeed, the lack of many immune evasion factors and the failure of mva infection to interfere early with key host (cell) defence mechanisms may explain the severe growth restriction of mva, its high attenuation upon in vivo infection and its immunogenicity when used as vaccine. moreover, due to the extensive loss of genetic information mva may serve as a particularly useful tool to study vacv host-regulatory genes. obviously, there are several pathways of the host (cell) defence that are targeted by multiple vacv proteins and the phenotype of a vacv single gene mutant may be masked by the complementary function of (an)other viral gene(s) (dobson and tscharke, ) . there are three obvious strategies using mva to elucidate on functions of selected vacv host-interaction factors: (i) the few remaining regulatory genes in the mva genome can be targeted for inactivation, (ii) candidate vacv genes missing in mva are reinserted in the genome to rescue a host-interaction phenotype, or (iii) a combination of these latter approaches may serve to investigate putatively complementary gene functions. in the following we describe some principles learnt by mva research that may help to better understand the regulation of virus-host interactions and likely influence safety and immunogenicity of vacv-based vaccines and therapeutics (fig. ). one most striking feature of mva is its inability to replicate in most cells of mammalian origin (carroll and moss, ; drexler et al., ; meyer et al., ) . in contrast, wild-type vacv has a broad cellular host range and productively infects various cell substrates (drillien et al., ) . it is noteworthy that poxvirus infections do not rely on the availability of specific cellular receptors but the viruses can efficiently bind to and enter many different cells from diverse animal species. after entry, however, the success of vacv replication depends on the functional activity of a subset of viral genes, the so-called "host range" genes (for review, see bratke et al., ; haller et al., ; mcfadden, ) . these viral genes encode regulatory proteins that control the intracellular host defence, e.g. by manipulation of the sensing of viral pathogen associated molecular patterns, the signal transduction, the cell cycle, or the onset of programmed cell death. some eminent viral regulators of intracellular host tropism are still functional in the mva genome and include the proteins c , k , e , f , and b ( k) (following the nomenclature as established for the genome of vacv strain copenhagen; goebel et al., ) . the vacv genes k l and c l (encoding the vacv proteins k and c ) are known to control virus replication in mammalian cells and both gene functions need to be inactivated to restrict the growth of wild-type vacv (gillard et al., ; perkus et al., ) . already early work by drillien and coworkers suggested that the replication defect of a vacv host range mutant (in the absence of k l and c l) is established at the level of viral gene expression (drillien et al., ) . however, the severe growth defect of the highly attenuated mva is not rescued when its genome is engineered to contain functional copies of both k l and c l suggesting that additional viral factor(s) await discovery (backes et al., ; meyer et al., ; sutter et al., ) . however, a c l-deleted mva lacks late gene expression in human and murine cells and induces phosphorylation of eukaryotic translation initiation factor α (eif α). the deficiency of late gene expression and the phosphorylation of eif α in the absence of c can be prevented by k as shown by the reinsertion of the k l gene into the genome of c l-deleted mva (backes et al., ) .the complementary function of k l and c l is intriguing because the two genes and encoded proteins are unrelated in sequence. interestingly, recent studies identified the human genes samd (sterile alpha motif domain-containing ) and wdr (tryptophan-aspartic acid repeat ) as host restriction factors for poxviruses and as targets of c and k (liu and mcfadden, ; sivan et al., ) . to date, rather little is known about the molecular functions of samd and wdr , but it can be assumed that they act in (an) innate antiviral defense pathway(s) awaiting further elucidation. the viral proteins k and e (encoded by k l and e l genes) are wellknown intracellular inhibitors of ifn-induced antiviral activities of the host cell (chang et al., ; davies et al., ) . k has homology to the alpha subunit of eif α and serves as a pseudo-substrate for double-stranded rna (dsrna)-activated protein kinase (pkr) to prevent virus-induced phosphorylation of eif α by pkr. in turn, e can sequester dsrna to inhibit the stimulation of pkr and to prevent activation of - oligoadenylate synthase and the endoribonuclease rnasel. in addition, e has been described to independently inhibit the phosphorylation of the transcription factors irf- and irf- and thus to block the production of type i ifns xiang et al., ) . these ifns evasion mechanisms provided by k and e substantially contribute to the broad cellular host range of vacv and numerous host range phenotypes of vacv mutants lacking e l or k l genes have been found (werden et al., ) . interestingly, cef-adapted mva has maintained fully active e l and k l sequences. indeed, a functional e l gene is required for mva replication in cef where e functions as an inhibitor of apoptosis and/or ifn induction to allow for unimpaired late protein synthesis (hornemann et al., ) . in human hela, hacat or t cells studies with an e l-deleted mva mutant (mva-Δe l) revealed that e is also essential to secure viral intermediate and late protein synthesis by counteracting host cell-specific activation levels of antiviral pathways pkr or rnasel . mva-Δe l also increased the type i ifn and/or chemokine production when compared to wild-type mva in infections of primary murine fibroblasts or murine bone marrow-derived dendritic cells which is in agreement with the known activation of transcription factors irf- and irf- in the absence of e (dai et al., ; ishii et al., ) . likewise, apoptosis induction in mva-Δe l infected murine fibroblasts required the proapoptotic cellular bh -only protein noxa which is well in line with the irf- /ifn-beta-mediated activation of noxa (delaloye et al., ; fischer et al., ) . cell death by apoptosis is an important defence mechanism to protect the host against viral infection. in turn, poxviruses have evolved specific viral proteins to inhibit the onset of programmed cell death and these regulatory proteins are relevant determinants of virus tropism at the cellular level (taylor and barry, ) . the vacv f protein (encoding gene f l) is an antiapoptotic protein with bcl- -like structure proposed to block apoptosis by binding to the proapoptotic family protein bak (postigo et al., ; wasilenko et al., wasilenko et al., , . f l is also conserved in the mva genome and f l-deficient mva (mva-Δf l) induces enhanced apoptosis in hela cells and in mouse embryonic fibroblasts (fischer et al., ) . additional work with mva-Δf l demonstrated that triggering of apoptosis predominantly requires the induction of the pro-apoptotic bh -only protein noxa which then activates proapoptotic bak (ferrer et al., ) . hereby, activation of noxa was linked to the recognition of viral rna and the upregulation of type i ifn signaling. thus, the noxa-dependent induction of apoptosis observed upon infections with mva-Δe l (fischer et al., ) may be explained by the failure to sequester viral rna in the absence of e resulting in strong activation of pro-apoptotic noxa (fig. ) . ankyrin repeat (ank) motifs are found in many poxvirus regulatory proteins that determine the cellular host tropism and/or counteract antiviral host responses controlled by nf-kb signaling (herbert et al., ) . otherwise ank motifs are described important for many proteinprotein interactions and cellular ank proteins are involved in diverse regulatory tasks including cellular transcription, cell cycle control, or cellular differentiation (mosavi et al., ) . the only ank-containing protein encoded by the mva genome is the -kda ankyrin-like protein ( kank; homologue to the vacv cop b r gene product). in addition to anks, k-ank contains an f-box-like pranc (pox protein repeats of ankyrin c-terminal) domain (mercer et al., ) . mva k-ank binds to cellular skp a and forms a cullin- -based scf ubiquitin ligase complex in an f-box-dependent manner (sperling et al., ) . a k-ank-deficient mva mutant shows reduced transcription of intermediate and late viral genes and suffers from a drastically impaired late protein synthesis in human and murine cells (sperling et al., ). thus, k-ank is essential for the completion of the mva molecular life cycle. interestingly, the f-box domain of k-ank is dispensable for rescue of the k-ank mutant phenotype suggesting multiple activities of this host-regulatory mva protein. mva is intensely used as vector vaccine being tested against a variety of infectious diseases (see section ) and some cancers. however, we still know very little about the influence on mva vaccine immunogenicity and vaccine efficacy with regard to these mva proteins regulating the host cell tropism. indeed, modulating the functional activity of these proteins could result in quite different outcomes. in one scenario, the inactivation of these regulators may further enhance mva-mediated activation of the innate immune system and may lead to improved protective immunity; another possible scenario is reduced mva vaccine efficacy because fig. role of the mva regulatory proteins e and f within the intracellular virus life cycle: e can efficiently sequester dsrna produced during infection to counteract the activation of the antiviral host enzymes oas ( - oligoadenylate synthetase)/rnasel (ribonuclease l) or pkr (protein kinase rna-activated). additional functions of the e protein are to prevent the activation of the host proapoptotic protein noxa and to block the phosphorylation of the transcription factors irf- and irf- inhibiting the production of type i interferons. the mva protein f can bind or prevent the activation of proapoptotic host proteins bax/bak or noxa and acts as efficient inhibitor of cytochrome c release (cyto c) and apoptosis induction at the level of host cell mitochondria. the deletion of some of these genes (e.g., e l, c l, k-ank) may drastically impair viral replication, the levels of gene expression and mvamediated antigen synthesis in addition to regulatory genes targeting the host tropism, the mva genome also contains a number of genes with known immunomodulatory functions. one interesting example is gene b r encoding the vacv interleukin- β receptor protein (il- βr). this viral cytokine-binding protein has highspecific affinity for il- β (alcami and smith, ; spriggs et al., ) and is thought to play an important role in the regulation of inflammatory responses following vacv infection (alcamí and smith, ) . synthesis of il- βr could be demonstrated in various mva-infected cultured cells zimmerling et al., ) . interestingly, primary murine myeloid dendritic cells are important il- β producer cells upon mva infection but free il- β can be detected only in the absence of il- βr using the deletion mutant mva-Δil- βr. immunizations with this mva deletion mutant led to significantly enhanced virus-specific cd + t-cell responses and increased protective capacity against lethal challenge infection with virulent vacv strain western reserve (wr) . in addition, the gene sequence encoding the vacv interleukin- (il- ) binding protein (il- bp; c l gene) is also functionally retained in the mva genome . vacv il- bp binds soluble il- and prevents it to reach its cellular receptor targeting the proinflammatory and antiviral function(s) of this cytokine (born et al., ; calderara et al., ; symons et al., ) . the possibility to enhance vaccine immunogenicity by inactivation of the il- bp gene in mva was investigated in two previous vaccination studies in mice. cottingham and coworkers found no significant difference in vacv-specific t cell responses when comparing vaccines based on mva mutated in c l or wild-type mva (cottingham et al., ) . a second study demonstrated functional activity of the mva il- bp and immunizations with the mva deletion mutant increased vacv epitope-specific cd + and cd + t-cell responses and protective capacity against a vacv challenge infection (falivene et al., ) . to diminish the inflammatory host response, another strategy of vacv is the production of secreted viral chemokine receptor proteins which prevent the chemokine-mediated recruitment of leukocytes ( fig. ) (alcamí et al., ; graham et al., ; ng et al., ) . interestingly, mva has lost much of this vacv immune evasion activity. mva efficiently induces chemotaxis and triggers the rapid immigration of leukocytes to the site of in vivo inoculation (lehmann et al., ). yet, mva still produces the secreted protein a which binds chemokines with relatively low affinity suggesting another functional mechanism than just blocking the binding of chemokines to their cellular receptors (bahar et al., ) . moreover, deletion of the a l gene in mva improved the cd + t-cell immunogenicity and the protective capacity of vaccination . vacv encodes an impressive variety of intracellular virus proteins that can inhibit the host signaling pathways for nf-kb and irf- (for review, see smith et al., ) . together these proteins serve to dampen the innate host response by blocking the induction of type i ifns, chemokines, and proinflammatory cytokines. mva fails to functionally produce a substantial number of these inhibitory proteins including a , b , c , c , k , m , and n . indeed, mva infection induces the activation of nf-kb (oie and pickup, ) and the reinsertion of the original vacv gene sequences into the mva genome allowed to further elucidate the function of k and m as nf-kb inhibitors (hinthong et al., ; shisler and jin, ) . other inhibitory genes, such as c l, k r, a r, and a r, are fully conserved in mva. interestingly, the removal of c l, k r, or a r from the mva genome is reported to contribute to higher frequencies of antigen-specific cd + and cd + t cells, enhanced polyfunctionality of t cells and higher antigen-specific antibody titers when tested in recombinant mvaproducing hiv candidate antigens (garber et al., ; garcía-arriaza et al., . a similar improved immunogenicity is also described for a recombinant mva-hiv virus deleted in the n l gene (garcía-arriaza et al., ) . these data suggest the functional activity of mva n despite the fact that the mva gene harbors an in-frame deletion causing the loss of five amino acids (aa - ). vacv n is an interesting regulatory protein because it acts as inhibitor of irf- activity within the nucleus of infected cells (fig. ) . shortly after the eradication of smallpox, vacv acquired a new mission as eukaryotic cloning vector for the expression of heterologous genes. homologous recombination that frequently occurs between vacv genomes within an infected cell was discovered to allow for efficient insertion of foreign dna into the vacv genome (mackett et al., ; panicali and paoletti, ; . this technology enabled the development of new recombinant vaccines that could be beneficial to decrease the impact of various infectious diseases. over the years replication-competent recombinant vacv has been successfully introduced for use in veterinary medicine as accomplished with the recombinant vacv vaccines against rabies (blancou et al., ; esposito et al., ; kieny et al., ) . the application of recombinant vacv for vaccination of humans was lagging behind restricted due to the well-known side effects of vacv smallpox vaccines. in these circumstances the availability of replication-deficient vacv such as nyvac or mva has spurred the establishment of exceptionally safe next-generation poxviral vectors (sutter and moss, ; tartaglia, ) . the technology for recombinant mva has been adapted from vacv. the still most frequently and effectively practiced strategy to generate recombinant poxviruses employs homologous dna recombination in infected cells, a relatively frequent event during vacv genome replication ( . %) (mackett et al., ; nakano et al., ) . recombination is typically directed by a gene transfer plasmid. for transcriptional control of foreign genes, various virus-specific promoters are used to achieve moderate to strong expression during the whole virus replication cycle (baldick et al., ; chakrabarti et al., ; davison and moss, ; di pilato et al., , wennier et al., ; wyatt et al., ) . usually, the plasmid carries the specific expression unit with a virus-specific promoter next to the multiple cloning site for insertion of various foreign gene sequences and selectable markers to facilitate the clonal isolation of recombinant mva (mackett et al., ; staib et al., ) . the foreign gene sequences and the marker gene are flanked on both sides by genomic mva sequences that direct the recombination of the expression cassette to favored loci in nonessential regions of the mva genome. in mva, the sites of major deletions are suited for the insertion of foreign gene sequences without affecting essential regions in the mva genome (sutter and moss, ; sutter et al., ) . until today, deletion site iii serves as one of the most frequently used insertion loci song et al., ; volz et al., ) . in addition, standard insertion sites used in conventional vacv, the thymidine kinase gene locus ( j r) and the ha gene locus (a r), were readily established for the construction of recombinant mva (antoine et al., ; schneider et al., ) . moreover, due to the high fidelity of homologous recombination also small nonessential regions between mva genes can be used to introduce heterologous dna for vector construction (wyatt et al., ). multiple insertion sites may allow for integration of various expression units for foreign genes in the same mva genome when opting for multivalent recombinant vaccines. for the generation of recombinant mva, cells are infected with mva and simultaneously transfected with the respective mva transfer plasmid to allow for homologous recombination (kremer et al., b; staib et al., ) . recombinant mva viruses are clonally isolated in repetitive cell culture passages screening for specific selection markers. different protocols have been established to allow differentiation between wild-type and recombinant mva. very first approaches took advantage of specific enzymes that allow for color discrimination. here, the transfer vector contains an antibiotic selection marker or a reporter gene allowing the screening due to a change in phenotype such as coexpression of the e. coli β-galactosidase and β-glucuronidase (carroll and moss, ; chakrabarti et al., ) . among the coexpressed antibiotic resistance markers, the e. coli gpt gene encoding the enzyme xanthine-guanine-phosphoribosyltransferase is frequently used for purification of recombinant viruses by dominant positive selection for resistance against mycophenolic acid (falkner and moss, ; isaacs et al., ) . staining procedures require additional time of tissue culture, supplementation of agar overlays, and the use of chromogenic substrates and antibiotics. complementation of a defect in virus production is a faster and more convenient method to obtain recombinant mva viruses. a first growth selection protocol was initiated using the vacv host range gene k l to rescue recombinant mva replication in rabbit kidney rk- cells (staib et al., ) . blasco and moss had introduced selection for vacv plaque formation through coinsertion of the f l gene which was adapted to isolate mva vector viruses (blasco and moss, ; sánchez-puig and blasco, ) . moreover, mva mutant virus and a matching complementing cell line enables for growth selection based on the essential d r gene function (ricci et al., ) . up to now, the generation of recombinant mva viruses is based on well-established techniques for isolation of clonal viruses that include the use of serum-free media and marker-free recombinant viruses. specific regulatory guidelines help to supervise the generation of recombinant vector vaccines suitable for applications in humans (ema, ). yet, in consequence, generation of recombinant mva is to a certain extent more limited to the use of some preferred methods (kremer et al., b) . e.g., one preferred scheme is the isolation of recombinant mva through screening for transient cosynthesis of marker proteins without enzyme activity and not related to an antibiotic or chemotherapeutic resistance phenotype. fluorescence proteins such as green or red fluorescent proteins are conveniently used as well-characterized inert marker proteins. alternative procedures to engineer poxvirus genomes have been pioneered more recently. the entire vacv and mva genomes were cloned as bacterial artificial chromosomes (bac), which can be engineered in e. coli by homologous recombination with bacteriophage lambda-derived enzymes (cottingham et al., ; moss, , ) . the modified bac clones can be used to produce pure recombinant poxvirus in mammalian cells with the initial assistance of a helper virus but without further requirements for plaque purification. another up-to-date method, yet to be adapted to the construction of recombinant mva, utilizes the crispr-cas system to insert alternative gene sequences into the vacv genome (yuan et al., ) . importantly, independent of the methodology used for generation of a recombinant mva virus any new vector virus has to be thoroughly quality controlled for genetic identity, purity, genetic stability, recombinant gene expression, and growth characteristics (kremer et al., b) . typically, the in vitro characterization already starts during the process of clonal isolation of mva vector viruses by plaque purification. a collection of straightforward methodologies has been established to confirm genomic identity and the correct insertion of the target gene sequence within the mva genome using finger-print pcrs to assess the insertion sites and the six naturally occurring deletion sites. levels and kinetics of recombinant protein synthesis, the stability, or the posttranslational modification of the foreign target proteins are typically monitored using standardized in vitro infection experiments and antigen-specific immune detection assays (e.g., western blotting). in this context, it is also important to analyze the growth capacities of recombinant mva. the well-known replication deficiency of mva in mammalian cells is a key biological characteristic of mva and allows the genetically modified virus to be handled in germany under conditions of bsl- with minimal potential biohazard to laboratory personnel, clinicians, patients, and the general environment (bundesamt f€ ur vebraucherschutz und lebensmittelsicherheit, ) . therefore, new recombinant mva are routinely tested for their growth characteristics in cells of human origin, preferably in cells with normal differentiation such as hacat cells (boukamp et al., ( jordan et al., ; lohr et al., ) . moreover, it is encouraging that some of these designer cell lines have already been used to manufacture first candidate vaccines being approved for clinical evaluation (genzel, ) . in medical research and development ongoing efforts focus on the study of candidate vaccines against infectious diseases that are more "complicated" to prevent, e.g., those caused by newly emerging pathogens such as severe acute respiratory syndrome coronavirus, west nile virus (wnv), or avian influenza virus. for decades, the availability of a safe and efficient vaccine against different influenza viruses has been one of the biggest demands of public health systems. until today, infection with seasonal influenza virus kills about , - , people every year. here, the main risk groups comprise the elderly, immunocompromised and children, with the highest incidence of fatal outcome (loubet et al., ) . moreover, in addition to the circulating seasonal influenza viruses, there is a continuous threat of new pandemic influenza viruses that might arise from pigs or birds herfst et al., ) . during the last century, there have been four pandemics that together caused more than million deaths (russell et al., ) . hence, it is not surprising that influenza was the target of the first recombinant mva vaccine. this vector virus was designed to codeliver the influenza virus a/pr/ / (h n ) antigens ha, and np (mva-ha-np) (sutter et al., ) . balb/c mice immunized with mva-ha-np mounted efficient levels of ha-specific antibodies as well ha-and np-specific cd + t cells. a single intramuscular immunization was sufficient to protect these mice against a lethal respiratory challenge with influenza virus a/pr/ / . subsequently, mva-ha-np was shown to induce mucosal immunity following oral immunization and to allow for partial protection against a heterologous influenza virus subtype h n challenge infection (bender et al., ) . after these results from testing a first recombinant mva-expressing influenza virus antigens, it took sometime to encourage a multitude of other experiments targeting influenza in veterinary as well as human medicine. breathnach and coworkers characterized a recombinant mva-producing ha or np from equine influenza a virus h n . vaccination of ponies with mva-ha induced robust levels of antibodies and protected against challenge with influenza virus h n a/equine/kentucky/ / . also mva-np-vaccinated ponies did not show severe clinical symptoms upon influenza virus challenge infection suggesting the induction of cellular immune responses with protective capacity (breathnach et al., ) . because of the zoonotic transmission and the pandemic potential of various highly pathogenic avian influenza viruses of the h subtype, the development of h -specific vaccines for humans is considered a priority since (de jong et al., ; yang et al., ) . vaccine development is complicated by antigenically different clades of influenza viruses within the h subtype. in this context, kreijtz (kreijtz et al., (kreijtz et al., , a . vaccination with either of the recombinant mva efficiently induced antibody responses against the homologous influenza virus. moreover, immunization with mva-ha-vn/ (clade ) was able to activate antibodies not only against the homologous virus but also to a/hk/ / (clade ) and to a lesser extent to a/indonesia/ / (clade . ). this potent activation of influenza virus-neutralizing h -specific antibodies correlated with protective efficacy against homologous and heterologous challenge infection (kreijtz et al., ) . interestingly, the exceptional immunogenicity of the ha antigen of influenza a/vietnam/ was confirmed in another study comparing recombinant mva vaccines expressing ha antigens from various influenza a viruses h n (clades , , . , . , . ) (hessel et al., ) . mva-ha-vn/ also proved immunogenic and protective when tested as single-shot vaccine in chickens against challenge with the highly pathogenic avian influenza virus a/duck/vietnam/tg - / (veits et al., ) . in cynomolgus macaques, prime-boost immunization with mva-ha-vn/ was able to protect the animals against homologous virus a/ vietnam/ / and infection with clade . influenza virus a/ indonesia/ / . the absence of virus infected cells in the lungs and the lack of fever and severe interstitial pneumonia highlighted the induction of solid protective immunity in vaccinated animals (kreijtz et al., b) . results from further evaluation in mice also demonstrated the efficacy of mva-ha-vn/ vaccines in dose-sparing and single-shot immunizations. here, minimal requirements for induction of protection against homologous and heterologous influenza virus challenge infections include a single immunization with pfu (plaque-forming units) mva-ha-vn/ or primeboost immunizations with pfu mva-ha-vn/ . of note, in some experiments, protection was observed in the absence of detectable ha-specific antibodies. these data look very encouraging with regard to pandemic risks where a vaccine has to protect rapidly and at minimal doses (kreijtz et al., a) . in this context recombinant mva expressing the ha from pandemic h n a/california/ / turned out to be highly immunogenic and protective when tested in the mouse model and ferrets (hessel et al., ; kreijtz et al., ) . in ferrets, prime-boost vaccination with mva-ha-ca/ efficiently activated virus-specific antibodies, reduced the clinical signs after challenge infection with a/netherlands/ / , and protected against severe histopathological changes in the lungs. two immunizations also significantly lowered the presence of infectious virus in the upper and lower respiratory tract (kreijtz et al., ) . due to these promising results from in vivo preclinical evaluation, a first-in-man phase i/iia clinical study of mva-ha-vn/ has been facilitated . the vaccine was safely tolerated and serious side effects were not observed. furthermore, this candidate vaccine proved to be immunogenic in all individuals enrolled in this study. mva-ha-vn/ efficiently activated antibodies cross-reacting with homologous and heterologous h subtype influenza viruses and with the recently emerging highly pathogenic avian influenza virus subtype h n (de vries et al., ) . of note, prime-boost applications via the intramuscular route induced levels of influenza h n -specific antibodies that raise expectations for protective capacity. interestingly, booster vaccinations allowed for a remarkable enhancement of h n specific antibodies when given months after primary immunization. these results firstly describe a major benefit of booster vaccinations with recombinant mva using an extended time period between primary and secondary immunization . future clinical studies using similar regimens might help to develop new vaccination strategies. overall, the induction of robust levels of influenza ha-specific antibodies can be expected to afford cross-protective immunity to viruses of the same influenza virus subtypes. in addition, mva-mediated delivery or codelivery of influenza virus t cell antigens is being considered to possibly confer broader protection against various subtypes (for review, see altenburg et al., ) . probably, most extensively tested as t cell vaccine is a recombinant mva vaccine expressing the influenza virus np and matrix (m ) proteins (mva-np+m ; lambe et al., ; . this candidate vaccine was also shown to induce influenzaspecific cd + t cells in phase i/iia clinical studies and to protect humans from an experimental influenza challenge infection (berthoud et al., ; lillie et al., ) . finally, aiming on broadly protective "universal" influenza virus vaccines kamlangdee and coworkers tested an innovative recombinant mva expressing an in silico generated synthetic h antigen representing a mosaic sequence of more than h n viruses (kamlangdee et al., ) . interestingly, this candidate vaccine protected mice against h n viruses from all clades but also against infection with influenza virus a/pr/ / (h n ). taken together these highly encouraging data support the clinical evaluation of existing mva candidate vaccines and the further development of novel recombinant mva against influenza. a safe and effective human immunodeficiency virus (hiv) vaccine is urgently needed to control the worldwide hiv epidemic. however, the development of a vaccine against aids represents a substantial scientific challenge related to hiv antigenic variability, the lacking understanding of immune correlates for protection, limitations of available animal models, and the enormous constraints associated with the probable need for multiple large-scale clinical trials in different parts of the world (for review, see excler et al., ) . moreover, the fragile immune system of hiv-infected individuals sets high standards to candidate vaccine safety. in the recent past, highly attenuated poxviruses continued to play a major role in the international search for an aids vaccine also taking advantage of established technologies for vector vaccine production at industrial scale. different recombinant mvaexpressing hiv proteins have undergone preclinical and clinical testing for the activation of protective immune responses against aids often in combination with dna-based and/or adenoviral vector vaccines (for review, see iyer and amara, ; ondondo, ) . recombinant mva vaccines targeting different hiv- subtypes continued to prove safe and immunogenic in additional clinical studies (goepfert et al., ; joachim et al., ; munseri et al., ; nilsson et al., ) . important new findings included data for the induction of high levels of antibody-dependent cellular cytotoxicity-mediating antibodies and months durability of the vaccine-induced hiv env-specific antibody responses. in this context, a necessary target antigen comprises the immunodeficiency virus envelope (env) protein, as it could be shown to elicit antibody responses with enhanced protective capacity in nonhuman primate chimeric simian/human immunodeficiency virus (shiv) or siv challenge infection models (barouch and michael, ; barouch and picker, ; roederer et al., ) . recent approaches are working on the induction of broadly neutralizing antibodies at the mucosal site of viral entry based on env-specific vaccines. other important hiv immunogens delivered by mva candidate vaccines include gag, pol, and nef proteins targeting the induction of hiv-specific cd + and cd + t cells . in addition, new synthetic hiv-specific "mosaic" immunogens are under evaluation as improved antigens for induction of cd + t cells (ondondo et al., ) . the large number of different hiv candidate vaccine necessitates the development of preclinical model systems to evaluate and select the most promising vaccine candidates. in many preclinical experiments, varying degree of protection against homologous immunodeficiency virus infection has been found, predominantly depending on the challenge virus and/or the animal model used for evaluation. however, hiv has an extraordinary genetic diversity and the "holy grail" aids vaccine would have to cross-protect against different hiv clades. a major scientific challenge is now to find appropriate antigens or epitopes that elicit a cross-protective immune response. for sometime, induction of cellular immunity was the primary focus of hiv vaccine development but the generation of broadly neutralizing antibodies is also believed to be indispensable (douek et al., ) . previous data from two studies in the macaque model showed that booster vaccinations with oligomeric or native env proteins enhance env-binding and virus-neutralizing antibody responses primed by recombinant mva vaccines, and suggest that such antibodies are indeed likely to play a role in vaccine-induced protection (earl et al., ) . currently, siv or shiv challenge infections in different nonhuman primates are considered to be the most appropriate animal models to test for immunogenicity and efficacy. these models very closely mimic the pathogenesis of hiv infections in humans concerning viremia, progressive depletion of cd + t cells, and the clinical manifestation of aids (van rompay, ). the thorough characterization of recombinant mva candidate vaccines in the siv model continues to further elucidate on the protective capacity of siv antigenspecific immune responses (chamcha et al., ; iyer et al., ; kwa et al., ; valentin et al., ) . thus, these nonhuman primate models might allow for the generation of proof-of-concept data on antiviral immunity that effectively inhibits immunodeficiency virus replication and disease development. in addition, mva vector vaccines have proven to be excellent candidates for vaccine development against other infectious diseases with global impact such as tuberculosis and malaria (for review mcshane and williams, ) . the incidence of disease caused by mycobacterium tuberculosis is steadily increasing often on the basis of poverty-impaired health services, widespread hiv infection, or the emergence of resistant m. tuberculosis. in recent efforts to elicit more potent antimycobacterial immunity, mva vector viruses served to identify new promising target antigens and resulted in the development of first subunit vaccines entering clinical testing (mcshane et al., ; sheehan et al., ) . here, the conserved mycobacterial antigen a turned out to be a promising immunogen for induction of antigen-specific t cells against m. tuberculosis. a recombinant mva candidate vaccine-expressing a under transcriptional control of the vacv early/late promoter p . (mva a) has been extensively tested in preclinical models and phase i to phase iib clinical studies. vaccination of balb/c mice with mva a induced both cd + and cd + t-cell responses and conferred protection against challenge with m. tuberculosis (mcshane et al., ) . importantly, in an approach to efficiently trigger activation of a-specific cellular immune responses by vaccination, recombinant mva a has been successfully evaluated for boosting the immunogenic effects of a mycobacterium bovis bcg primary immunization. preclinical studies in different animal models, including mice, guinea pigs, nonhuman primates, and cattle, confirmed the protective efficacy of the heterologous bcg prime-mva a boost vaccination. in these experiments protection was associated with a reduction of bacterial loads in the lungs rather than with sterilizing immunity (goonetilleke et al., ; williams et al., a,b) . unfortunately, when testing bcg-mva a immunizations in a large phase iib clinical trial in children in africa there was no improvement compared to the conventional bcg only immunization schedule (tameris et al., ) . mva a was well tolerated and immunogenic as shown by the induction of a antigen-specific cd + t cells. however, the immunizations demonstrated no significant efficacy against tuberculosis or m. tuberculosis infection. nevertheless, the study provides an important and encouraging pool of safety data for a recombinant mva candidate vaccine tested in more than infants. overall, the results obtained from the clinical testing of mva a support the development of improved recombinant mva a candidate vaccines against tuberculosis but also including additional antigens of m. tuberculosis. in , there were more than , estimated deaths and about million clinical illnesses due to malaria, the majority in central and southern africa. thus, an effective vaccine against malaria is urgently required (hoffman et al., ) . a variety of antigens from plasmodium falciparum has been expressed and tested with recombinant vacv. the pathogenesis of malaria involves a complex life cycle including different blood and nonblood life stages of the parasites in the human host as well as in the mosquito host. a major challenge is the choice of optimal target antigens mediating protective immune responses against the multiple phases of malaria. desirable targets should result in an efficient activation of humoral immunity as well as malaria-specific cd + t cells. in a landmark study, schneider and coworkers had evaluated recombinant mva vaccines in a mouse malaria model using p. berghei. the study demonstrated efficient induction of malaria-specific t cells that could be further enhanced by using dna prime in combination with recombinant mva as boosting agent. of note, this study demonstrated a need for antigen-specific cd + t cells to induce protection against p. berghei sporozoite challenge infection . first clinical trials have been initiated using recombinant mva expressing the important malaria liver stage antigens trap/csp alone or in a heterologous prime-boost schedule in combination with adenoviral vectors (gilbert et al., ) . further improvement of this antigen has been achieved by fusion of trap with a peptide sequence encompassing a plasmodium-specific multiple epitope (me-trap) (gilbert et al., ) . in detail, moorthy and coworkers generated a single polypeptide me-trap of amino acids which combines a multiple epitope string (me) consisting of cd + t-cell epitopes with three cd + t-cell epitopes from tetanus toxin, bcg, and pf-circumsporozoite protein (pfcsp) and two b-cell epitopes within the established trap antigen. here, dna prime and recombinant mva boost schedules have been evaluated for safety and immunogenicity in a phase i/ii clinical trial in humans. no severe adverse effects were observed after application of mva-me-trap alone or in combination with dna prime vaccination (moorthy et al., ) . in following clinical evaluations, immunogenicity and protection has been assessed in african adults in a malaria-endemic area. heterologous regimens using dna prime and mva boosting proved to be more immunogenic compared to homologous application of either vaccine used alone. however, despite measurable immunogenicity, robust protection against challenge with p. falciparum could not be detected (moorthy et al., (moorthy et al., , . another advancement was based on studies that tested different combinations of primary immunizations with recombinant fowlpox and recombinant mva vaccines . next steps included the clinical evaluation of these malaria vaccine candidates in heterologous prime-boost vaccinations. here, the booster immunizations resulted in an efficient activation of malaria-specific cd + t cells in adults and children webster et al., ) . however, efficacy testing in field studies in endemic areas failed to robustly protect against malaria infection at various stages (bejon et al., ) . another innovative approach of heterologous prime-boost vaccination has been conducted by rodriguez and colleagues in the murine p. berghei infection model. the alternative usage of porcine parvovirus-like particles delivering cs protein peptide sequences resulted in efficient priming of protective cd + t-cell responses following booster vaccination with recombinant mva expressing the cs antigen . more recently another heterologous prime-boost vaccination schedule has been thoroughly evaluated using chimpanzee-adenovirus vectors for priming and recombinant mva for booster vaccinations (for review, see sebastian and gilbert, ) . this combination of vector immunization induced high frequencies of me-trap-specific t-cell responses in humans as a promising approach to robustly protect against p. falciparum (ewer et al., ) . moreover, a recent study suggested the feasibility of developing malaria transmissionblocking vector vaccines to target p. falciparum within the mosquito. antibodies with high-level transmission-blocking activity could be elicited upon prime-boost immunizations of mice with recombinant chimpanzee adenovirus and mva-expressing candidate malaria antigens pfs -c and pfs (kapulu et al., ) . thus, future approaches in the development of mva vector vaccines against malaria aim at the induction of a more balanced immunity based on both efficient humoral and cellular immune responses. yet, complex clinical phase ii/iii studies in humans in endemic areas will be needed to evaluate the efficacy of these new promising approaches. newly emerging pathogens represent another global public health risk as they can suddenly and unexpectedly arise from a previously unknown ecological niche, in most cases an animal reservoir. thus, major concerns are zoonotic infections that are transmitted from animals to humans, which, when sufficiently adapted to the human host, may rapidly spread in the human population (kuiken et al., ; reusken et al., ; steffen et al., ) . during the past years, public health systems had been confronted with a multitude of new pathogens each demonstrating a different scenario of emergence. in this context, the year with the sudden occurrence of west nile fever in the western hemisphere (new york city, usa) is often marked as the beginning of a new era of epidemics (for review, see suthar et al., ) . wnv is a member of the genus flaviviruses and, as an arbovirus, continuously circulates between different mosquito species and birds as the major animal virus reservoirs. through the bite of an infected mosquito, wnv can be transmitted to mammalian hosts, primarily to humans or horses, causing the so-called west nile fever sometimes resulting in neuroinvasive disease with the potential for severe outcomes especially in elderly and immunocompromised humans. the virus had first been isolated in from a febrile woman in the west nile district in uganda (goldblum et al., ) . since then it was observed to be or become endemic in regions of europe, africa, and asia periodically causing wnv outbreaks in humans and/or horses. in , the virus was introduced into the new york city district of queens, supposedly by an infected mosquito or bird (lanciotti et al., ; murray et al., ; nash et al., ) . thus, wnv had relocated to a new, naïve population facilitating its impressive spread across the north american continent leading to a total of about , human infections and deaths between and (http://www.cdc.gov/westnile/statsmaps/). concurrently, the virus also increasingly emerges in european countries, resulting in outbreaks of severe disease in horses and humans. thus, a safe and effective wnv vaccine for humans is urgently needed in particular to protect at-risk populations. a recent study tested different recombinant mva vaccines delivering the wnv envelope protein (wnv-e) and fulfilling all the principal requirements to proceed to clinical testing in humans. vaccine immunogenicity, induction of neutralizing antibodies and e-specific cd + t-cell responses, and the capacity to protect against lethal challenge infections were evaluated in different mouse models. the mva-wnv candidate vaccines allowed to compare the performance of wnv-e antigens expressed in four different conformations. in mva-prm/me, wnv-e is produced together with the wnv membrane protein to induce the synthesis and the release of virus like particles (vlps) upon mva infection. mva-e sol produces a soluble version of wnv-e that is secreted from infected cells. mva-e tmv and mva-e tmc prominently expose wnv-e antigens on the cell surface by providing heterologous transmembrane domains derived from either vacv (tmv, transmembrane domain vaccinia virus) or chikungunya virus (tmc, transmembrane domain chikungunya virus). upon prime-boost vaccinations in balb/c mice, all four mva-wnv candidate vaccines elicited circulating serum antibodies binding to recombinant wnv-e protein and neutralizing wnv in tissue culture infections in addition, immunizations in hla-a . -/hla-dr -transgenic h- class i-/class ii-knockout mice efficiently induced wnv-e-specific cd + t-cell responses. finally, the mva-wnv candidate vaccines protected c bl/ mice against challenge infections with lineage and lineage wnv and activated crossneutralizing antibodies. thus, further studies are warranted to evaluate these recombinant mva-wnv vaccines in other preclinical models in an effort to select and develop an mva-wnv candidate vaccine for clinical testing in humans . recently, the family coronaviridae provided different new pathogens suddenly arising from an ecological niche. since two novel beta coronaviruses have been introduced into human populations causing acute atypical necrotizing pneumonia, the so-called severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) . in early , the sars epidemic initially occurred in southern china and rapidly spread to more than countries causing infections and deaths. bat species and civet cats were identified as animal reservoirs transmitting the virus to humans and/or other animals. the abrupt emergence of the sars-cov in china in november and its worldwide distribution until the end of is representative for the scenario of a pandemic public health emergency. luckily, further spread of the disease could be prevented and no further infections with sars-cov were detected since early . the successful containment of sars was likely due to multiple causes, e.g., the relative ease of clinical case isolation and the persistence of the pathogen in an animal reservoir rarely enabling transmission to humans. however, the lessons learned from sars include the need for the timely development of specific vaccines to counteract such an outbreak scenario with high morbidity and mortality rates and the lack of any specific treatment option. the most effective approach to deal with emerging pathogens is vaccination. mva with its excellent safety profile and well-established vector production platform holds great potential to rapidly develop new vaccines against such emerging pathogens ready to use in an immediate public health response. in an ideal scenario, mva emergency vaccines against selected pathogens of risk are being developed and undergo preclinical and phase i/ii clinical evaluations already in preepidemic times. in the case of a disease outbreak, these recombinant mva candidate vaccines could be immediately tested in efficacy studies when used to vaccinate people of special risk in endemic areas. this concept already spurred the development of a first set of recombinant mva candidate vaccines against highly pathogenic avian influenza viruses and the recently emerging mers-cov as current examples of new zoonotic agents. mers-cov was first described in september . in contrast to experiences with sars in the epidemic of / , the mers-cov continues to cause disease in humans in the fifth year after its first appearance in . at present, who reports a total of laboratory confirmed cases including deaths (http://www.who.int/emergencies/mers-cov/en/). the epidemiology of human mers-cov infections centers in the middle east, e.g., in qatar, saudi arabia, jordan, and united arab emirates. sporadically, mers-cov infections are also spread to other countries in europe, north-america, and asia due to transmission by travelers infected in the middle east. raj and coworkers identified the human cell surface amino peptidase dipeptidyl or cd as functional receptor of mers-cov-mediating entry into the cell (raj et al., ) . by now dromedary camels are known and generally accepted to be the critical animal reservoir responsible for spreading the virus to humans memish et al., ; meyer et al., ; raj et al., ) . these primary zoonotic infections can result in interfamilial or health care related secondary transmissions. the elderly and immunocompromised persons are among the people of risk to suffer most from severe and lethal mers-cov infections. other individuals at risk are health care workers and people with close contact to camels. all these groups are considered relevant target populations for potential mers-cov vaccines. so far, there is no licensed vaccine against mers-cov or sars-cov available. different approaches have been undertaken to develop effective means to prevent or cure these new infectious diseases. here, mva has been tested as viral vector vaccine against both beta coronaviruses. the envelope spike (s) protein has been proven to be a major target of sars-cov-neutralizing antibodies (he et al., ; sui et al., ) . indeed, a recombinant mva producing the sars-cov s antigen was evaluated in different animal models and demonstrated the induction of sars-cov-specific immune responses, including sars-cov-neutralizing antibodies and s-antigen-specific t cells in mice, rabbits, and rhesus macaques (bisht et al., ; chen et al., ) . prime boost immunization effectively inhibited sars-cov replication in cynomolgous monkeys after respiratory sars-cov challenge infection suggesting the induction of protective immunity. in the case of the mers-cov, a recombinant mva expressing the s protein of mers-cov (mva-mers-s) was generated rapidly after the discovery of mers in (song et al., ) . here, the mers-cov-s-encoding sequences were introduced into the deletion site iii of the mva genome. preclinical evaluation of mva-mers-s candidate vaccine confirmed the induction of mers-covneutralizing antibodies and mers-s epitope-specific cd + t cells in balb/c mice comparing different dosages and application routes (volz et al., ) . moreover, preclinical testing in a special mouse model allowed for the first demonstration of the protective capacity of this mers candidate vaccine. after adenovirus-mediated transduction with the human dipeptidylpeptidase receptor, balb/c mice are susceptible for respiratory mers-cov challenge infections and the monitoring of virus loads allows to determine the efficacy of experimental immunization (zhao et al., ) . as the virus is assumed to persist in dromedary camels, further preclinical analysis in dromedary camels demonstrated immunogenicity and protective efficacy of mva-mers-s . simultaneous immunizations by the intranasal and intramuscular routes resulted in efficient induction of virus-neutralizing antibodies in serum and nasal secretions of vaccinated animals. contrary to the mock vaccinated control animals, camels vaccinated with mva-mers-s revealed a significant reduction of excreted infectious virus and viral rna transcripts after mers-cov challenge infection. in addition, vaccination with mva-mers-s also induced the activation of orthopoxvirus-specific antibodies that readily cross-neutralized camelpox virus. camelpox virus causes severe systemic disease in camels with case fatality rates as high as %. clinical disease is characterized by papules and pustules that initially appear at the primary site of infection. this is then followed by the development of generalized rash and fever between day and postinfection. young camels are more susceptible to severe clinical disease. the activation of camelpox virusneutralizing antibodies suggested the potential dual use of this candidate mers-cov vaccine in dromedaries to efficiently protect against mers-cov and camelpox virus infection . these data further support the general safety and efficacy of the mva-mers-s candidate vaccine and introduce the possibility for application as veterinary vaccine with important implications to the one health concept. vaccination of dromedary camels in areas endemic for mers-cov could reduce the burden of virus excretions from the animal reservoir and thus inhibit the transmission of mers-cov to human populations. in addition, it is important to characterize the mva-mers-s candidate vaccine in humans and efforts are ongoing to start a first-in-man clinical evaluation of mva-mers-s as soon as possible. in this first clinical phase i/ii testing, safety and immunogenicity of the mva-mers-s candidate vaccine will be analyzed in healthy volunteers in germany. results from these phase i/ii clinical evaluations are prerequisite for testing the vaccine in larger phase ii studies in endemic countries. in addition, the availability of an investigational drug batch of mva-mers-s might allow for application of as emergency vaccine in the case of a suddenly occurring outbreak scenario, when the virus is rapidly transmitted and spread throughout a new geographic area. exemplary for this was the introduction of mers-cov to south korea on may (min et al., ) . a -year-old man returning from the middle east had been diagnosed with mers days after he initially visited the hospital for medical help. in the hospital further spread of mers-cov occurred by transmission to several health care workers and other patients. in this outbreak, a total of individuals have been infected, with a total of deaths. in this context, the government of the republic of korea began to implement intense case and contact management activities that in the end stopped the epidemic of mers-cov in a nonendemic region. however, the sudden and rapid spread of mers-cov in south korea again highlighted the global risks of newly emerging pathogens that might unexpectedly threaten a naïve population ( jeong-sun et al., ; kim et al., ) . another very recent example for the sudden reemergence of a highly infectious pathogen is the unprecedented ebola virus epidemic in west africa starting in and continuing for over years. during this devastating and most widespread ebola virus outbreak, the virus had caused more than , disease cases and , deaths mainly geographically linked to the africa but some cases were also diagnosed in travelers from africa to the united states, germany, france, spain, and great britain (de la vega et al., ; quaglio et al., ) . initially, this large epidemic began in the village of meliandou, gu eck edou prefecture, guinea in the end of . most likely bats transmitted the virus to humans followed by massive spread of the disease to other villages. moreover, the virus causing this outbreak was identified to be the most notorious member of the genus ebolavirus and the species zaire ebolavirus (ebov) (baize et al., ) . in consequence, the epidemic in west africa was associated with high morbidity and mortality rates that enabled an undamped transmission and perpetuation of the virus in the population for a rather long time period (gire et al., ) . this had not been observed for ebolavirus outbreaks in the past. so far, there are no efficient therapeutics available (choi and croyle, ) and there are also no vaccines licensed to protect against ebolavirus. however, since this very recent epidemic, research activities to develop and evaluate candidate vaccines against ebolavirus have been intensified. most promising candidates already advanced to clinical evaluation in humans, and include a recombinant vesicular stomatitis virus (vsv) delivering the ebolavirus zaire glycoprotein (vsv-ebov) and a chimpanzee-adenovirus (chad )-zaire ebola virus (zebov) also expressing the ebolavirus zaire glycoprotein as target antigen. with regard to the chad -zaire, the strategy is to use a heterologous prime-boost immunization schedule, boosting with an appropriate recombinant mvaexpressing ebolavirus zaire glycoprotein (ewer et al., ; tapia et al., ) . this strategy was supported by data from the preclinical evaluation of a chad -ebov candidate vaccine in nonhuman primates. here, primeboost vaccination with the chad -ebov vaccine alone did not confer protective immunity over a period of several months. however, robust protection against lethal challenge with ebov could be obtained following booster vaccination with a recombinant mva-ebov candidate vaccine coexpressing the ebolavirus zaire and sudan glycoproteins under the control of the p . vaccinia virus early promoter (stanley et al., ) . the approach of using a chad -ebov prime and mva-ebov boost vaccination has been further developed by the engineering of a multivalent recombinant mva coproducing the zebov and sudan ebola virus glycoproteins and other filovirus antigens (mva-bn-filo). a first phase ib clinical study demonstrated safety, tolerability, and immunogenicity of the chad -ebov prime and mva-bn-filo booster immunizations in adults in the united states and in mali. without any observation of severe side effects the combined chad -ebov and mva-bn-filo vaccinations proved to be highly immunogenic as it was shown by the activation of ebov-specific antibodies and cd + and cd + t-cell responses (tapia et al., ) . ewer and colleagues investigated another heterologous prime-boost vaccination schedule in healthy adult volunteers in oxford, united kingdom, using a chad vector vaccine and a monovalent recombinant mva encoding the surface glycoprotein of zebov. again, chad immunization boosted with mva-elicited b-cell and t-cell immune responses to zebov that were clearly superior to those induced by the chad vector vaccine alone (ewer et al., ) . nevertheless, a heterologous vaccination strategy with two different viral vector vaccines is a complex regimen to be applied in large field studies or in the case of emergency vaccination when whole populations have to be protected rapidly. thus, it should be interesting to evaluate the efficacy of a prime-boost vaccination scheme based on mva-ebov only. in summary, the highly versatile and safe mva vector platform should be particularly useful to effectively control newly emerging or reemerging infectious diseases. the vector system may be readily exploited in a plugand-play generic approach for the rapid generation of vaccine candidates suitable for rapid emergency immunization and, simultaneously, the clinical-stage development of a new licensable product. eradication of human smallpox has been achieved by prophylactic use of vacv to immunize humans, the only host reservoir of the causative agent varv. however, other members of the genus orthopoxvirus can cause zoonotic infections (kroon et al., ; mccollum and damon, ; vora et al., ) . moreover, recent fears that monkeypox virus (mpxv) or varv could be used as biological weapons have renewed the interest in safe vaccines against varv or other zoonotic orthopoxviruses (moss, ) . mva holds great promise for worldwide use as third-generation smallpox vaccine due to its well-established characteristics concerning safety and immunogenicity jones et al., ; knitlova et al., ; meseda et al., ; tree et al., ; wyatt et al., ) . in , an mva vaccine has been licensed in europe for active immunization against smallpox in adults and for use in situations where it is considered necessary to protect against smallpox in accordance with official recommendations (european medicines agency, ). data from multiple phase ii clinical studies have confirmed the safety and immunogenicity of the mva vaccine in patient populations considered at risk for conventional smallpox vaccination including individuals with atopic dermatitis or hiv infection (greenberg et al., ; overton et al., ; von sonnenburg et al., ) . in addition, mva has been successfully tested for cardiac safety in a large phase ii clinical trial (zitzmann-roth et al., ) . this is important because vaccines based on replication-competent vacv strains are associated with a high incidence of myo-/pericarditis, a severe cardiac complication. additional recent data from clinical studies addressed the meta-analysis of mva-induced immune responses in various patient populations and the comparative evaluation of inoculation routes or improved formulations of the mva vaccine troy et al., ) . today, with the eradication of smallpox, we lack an established human disease representing the pathogenesis of a systemic orthopoxvirus infection in humans. therefore, analyzing the protective efficacy of orthopoxvirus-specific vaccines is not straightforward. preclinical evaluations in animal models are required to test vaccine-mediated protection in relation to antigen-specific responses, and animal models must mimic the target poxvirus diseases in humans as closely as possible. varv infection in cynomolgus macaques can result in a lethal disease with similarities to smallpox wahl-jensen et al., ) . however, varv is a biosafety level agent and the handling with this virus is highly restricted. thus, several other animal models using the orthopoxviruses cowpox virus (cpxv), mpxv, ectromelia virus (ectv), and vacv have been developed (chapman et al., ; esteban and buller, ) . mva, as safe smallpox vaccine, has been tested in these different infection models. mva vaccination by the intramuscular or subcutaneous route protected mice against severe respiratory challenge infection with cpxv or vacv-wr (coulibaly et al., ; drexler et al., ) . of note, mva could also protect mice lacking several components of the immune system, resembling high-risk groups in the population that would require an alternative to the standard smallpox vaccine . in nonhuman primates, the challenge infection with mpxv is the most appropriate model to evaluate the preclinical efficacy of new candidate smallpox vaccines. here, a standard dosage of pfu mva robustly protected cynomolgous monkeys against intravenous or intratracheal mpxv challenges stittelaar et al., ) . in this context edghill-smith and coworkers demonstrated the essential need of antibodies for protection against fatal monkeypox disease (edghill-smith et al., ) . one major limitation in these orthopoxvirus infection models, using vacv, mpxv, or cpxv to test protective capacity of vaccination, is the high amounts of infectious virus (> pfu) required to induce lethal disease. in contrast, very few infectious particles of varv could generate fatal smallpox disease in naive humans. to overcome this shortcoming, the ectv infection of mice can serve as an additional highly appropriate model for the reassessment of the correlates of protective immunity (for review sigal, ) . ectv, the causative agent of mousepox, is a natural mouse pathogen causing a classical systemic poxvirus disease (esteban and buller, ) . very low amounts of ectv are sufficient for infection and induction of lethal mousepox disease in susceptible mouse strains. after an asymptomatic incubation period of - days following intranasal infection, mousepox disease starts in the respiratory tract followed by a systemic virus spread to internal organs such as liver and spleen (paran et al., ; parker et al., ) . specific signs of illness are characterized by severe bronchopneumonia and hepatitis. mice that survive this systemic phase of severe disease develop a characteristic pustular rash on the skin very reminiscent to that seen in human smallpox. the ectvmouse model offers the opportunity to study an orthopoxvirus pathogen in an experimentally easily accessible natural host. ectv is highly adapted to the mouse immune system and this allows to thoroughly analyze the mechanisms of viral immune evasion and vaccine-induced immune protection. several studies evaluated mva vaccine-mediated protection against lethal mousepox disease in more detail. coulibaly and coworkers demonstrated the usefulness of respiratory ectv challenge infections as improved model system for efficacy testing. here, intramuscular immunization with a single dose of pfu mva prevented severe disease and death in mice challenged weeks after vaccination. a single dose of robustly protected against any signs of illness and disease after the respiratory mousepox challenge. however, after vaccination with or pfu vacv wyeth, all mice suffered from severe lethal disease (coulibaly et al., ) . in the cpxv challenge model, these different candidate vaccines were equally protective underlining the impact of choosing a specific challenge virus. this study demonstrates the need for data from various preclinical models as key component in developing next-generation orthopoxvirus-specific vaccines for application in humans. in the case of an emergency with newly arising highly pathogenic orthopoxviruses, e.g., because of unintentional or intentional release of varv, vaccination protocols for rapid induction of protective immunity are urgently needed (reardon, ; sasse and gelderblom, ) . for smallpox vaccination there are historical reports that vacv application in a time window of up to days after exposure with varv may be protective. analysis of mva as emergency vaccine confirmed short-term protective capacity of vaccination in mice, latest when applied at the day of the lethal respiratory challenge with vacv strain wr. postexposure prophylaxis could not be achieved in the vacv-wr model, independent of dosage and application route (staib et al., ) . however, when testing emergency vaccination in the ectv-mouse model, a standard dosage of pfu mva robustly protected against lethal respiratory mousepox infection up to days before the challenge. even more, in this natural virus-host system, pfu mva also prevented death and severe disease when applied up to days after the lethal challenge. however, postexposure vaccination could not inhibit the onset of sign of disease including respiratory symptoms and body weight loss (paran et al., ) . in a follow-up study, kremer and colleagues analyzed the immune components mediating this rapid protection in more detail (kremer et al., a) . analysis of mva-induced protection in mice with defined deficiency in the innate or adaptive immunity identified cd + t cells to be essentially required to allow for mva-induced cd + t-cell expansion. interestingly, selected components of the innate immune system and b cellmediated responses were fully dispensable for prevention of fatal disease by immunization given days before challenge. analyzing protective capacity of mva immunization in ragÀ/À mice that lack t cells and b cells, these mice could not be protected against the lethal ectv challenge infection. these results underlined the prerequisite of adaptive cellular immunity for mediating a rapid protection with perforin-mediated cytotoxicity proven to be a key immune mechanism. in the case of emergency, when rapid induction of protective immunity by vaccination is desirable for prevention of morbidity and mortality, the instant activation of protective virus-specific immunity by single-shot vaccination, would be ideal. moreover, the possibilities of dose-sparing immunization regimens would increase the numbers of people that can be vaccinated. volz and coworkers assessed the minimal requirements for the induction of protective immunity against lethal ectv challenge infection days after immunization with mva . c bl/ mice had been intramuscularly vaccinated with tenfold increasing doses of mva starting with up to pfu, the mva standard dosage. interestingly, a minimal amount of pfu fully protected the mice against the lethal respiratory challenge infection with ectv. moreover, inoculations with pfu mva were still sufficient to prevent death of all vaccinated animals but could not protect against the induction of mousepox disease. analysis of immune responses again identified cd + t cells as the key components mediating the rapid protection in the low dose immunization model. moreover, mva immunization at low dosage also protected ifnarÀ/À mice, indicating efficient activation of cellular immunity even in the absence of type i ifn signaling. when monitoring for virus-specific cd + t-cell responses in mice vaccinated with the minimal protective dose of mva, we found significantly enhanced levels of antigen-specific t cells in animals that were mva vaccinated and ectv challenged compared to mice that were only vaccinated. the initial priming of naïve cd + t cells by mva immunization appears to be highly efficient and, even at low doses, mediates a rapid in vivo burst of pathogen-specific cd + t cells upon challenge. these findings define striking requirements for protective emergency immunization against severe systemic infections with orthopoxviruses. these data are of important practical relevance for public health, as producing sufficient amounts of vaccine is expected to be a major challenge should an outbreak occur. moreover, prevention of other infections may require similar immune mechanisms to elicit rapid protective immunity; hence, mva could be an extremely useful vaccine for delivering heterologous t cell antigens, particularly for infectious diseases that fit a scenario of emergency vaccination. thus, studies evaluating recombinant mva candidate vaccines as emergency vaccines might be promising for the development of new prophylactic or therapeutic approaches. today, almost years after its first licensing in germany, mva is well established as safety-tested, immunogenic, and efficacious thirdgeneration smallpox vaccine. in , the european medicines agency and canada health granted the marketing authorization of an mva vaccine to immunize against varv infection, in the absence of human smallpox or naturally occurring varv (european medicines agency, ). thus, efficacy data needed to be generated from animal models of orthopoxvirus infections considered to be representative for human smallpox. a similar licensing process by the us food and drug administration is ongoing and appears to be at an advanced stage. despite the eradication of varv more than three decades ago, these efforts are still important mostly due to the threat of varv being-accidently or intentionally-released into unprotected human populations. moreover, the use of a licensed mva vaccine should be ideal to protect individuals at risk-including laboratory workers-against other zoonotic orthopoxviruses such as cpxv and mpxv that continue to cycle in rodent reservoirs and can cause disease in humans. in addition, during years, mva has been continuously improved as an extremely safe and efficient viral vector system for the synthesis of high levels of foreign proteins in nonpermissive human cells. at the moment, recombinant mva viruses expressing various heterologous antigens are among the most promising vector candidates to develop innovative vaccination strategies to protect against complex infections such as aids, tuberculosis, or malaria, or against rare but threatening emerging diseases. desirable common characteristics of mva as viral vector vaccine include the genetic stability, the well-established production procedures and the general safety for the environment. results coming from clinical testing of various recombinant mva vaccines further emphasize its excellent safety record and its immunogenicity as vaccine in humans. in the context of recent clinical findings, it is noteworthy that repeated vaccinations with the same recombinant mva resulted in substantial booster induction of antigen-specific antibodies even in the presence of high levels of mva-specific immunity. these results together with other promising data gained from the combined application of mva with various other viral vector platforms greatly enhance the general acceptance of viral vectors as next-generation candidate vaccines. in addition to its promising characteristics concerning immunogenicity, the nonreplicating recombinant mva vaccines are also well positioned to satisfy very stringent requirements for a broad safety in various settings. mva vaccines appear highly suitable for use in immunocompromised individuals and in the elderly representing important target populations in ever-ageing populations in the industrialized world. at the same time, recombinant mva should be also safe to immunize persons with severe comorbidities, e.g., hiv infection, tuberculosis, or malaria, as it is often seen in developing nations. new vaccines that rapidly protect against threatening emerging pathogens are urgently needed. this it is further highlighted by the who-list of the "top most wanted" emerging diseases likely to cause major epidemics. here, the longstanding experience with the mva vector vaccine platform in preclinical and clinical research should lead to important contributions to the development of protective vaccination strategies against newly emerging pathogens. finally, the exciting results from ongoing fundamental research on the biology of mva spur the possibility to even improve the efficacy of future mva vaccines. here, an exemplary area of research targets the still unknown functions of host-regulatory genes remaining conserved in the mva genome. indeed, modulating the functional activity of these regulatory mva proteins could be beneficial in enhancing the immunogenicity of mva vaccines and activate innate and/or adaptive immune responses to heterologous antigens. a soluble receptor for interleukin- β encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection soluble interferon-γ receptors encoded by poxviruses blockade of chemokine activity by a soluble chemokine binding protein from vaccinia virus modified vaccinia virus ankara (mva) as production platform for vaccines against influenza and other viral respiratory diseases enhanced cd + t cell immune responses and protection elicited against plasmodium berghei malaria by prime boost immunization regimens using a novel attenuated fowlpox virus characterization of the vaccinia mva hemagglutinin gene locus and its evaluation as an insertion site for foreign genes the complete genomic sequence of the modified vaccinia ankara strain: comparison with other orthopoxviruses viral host-range factor c or k is essential for modified vaccinia virus ankara late gene expression in human and murine cells, irrespective of their capacity to inhibit protein kinase r-mediated phosphorylation of eukaryotic translation initiation factor α structure and function of a , a vaccinia virus chemokine binding protein emergence of zaire ebola virus disease in guinea mutational analysis of the core, spacer, and initiator regions of vaccinia virus intermediate-class promoters novel vaccine vectors for hiv- a phase b randomised trial of the candidate malaria vaccines fp me-trap and mva me-trap among children in kenya oral immunization with a replication-deficient recombinant vaccinia virus protects mice against influenza potent cd (+) t-cell immunogenicity in humans of a novel heterosubtypic influenza a vaccine, mvaÀnp+m severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice modified vaccinia virus ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine oral vaccination of the fox against rabies using a live recombinant vaccinia virus selection of recombinant vaccinia viruses on the basis of plaque formation identification and characterization of two members of a novel class of the interleukin- receptor (il-ir) family: delineation of a new class of il- r-related proteins based on signaling normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line a survey of host range genes in poxvirus genomes immunization with recombinant modified vaccinia ankara (rmva) constructs encoding the ha or np gene protects ponies from equine influenza virus challenge. vaccine , . bundesamt f€ ur vebraucherschutz und lebensmittelsicherheit orthopoxvirus il- binding proteins: affinities and antagonist activities e. coli beta-glucuronidase (gus) as a marker for recombinant vaccinia viruses host range and cytopathogenicity of the highly attenuated mva strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques strong, but age-dependent, protection elicited by a deoxyribonucleic acid/ modified vaccinia ankara simian immunodeficiency virus vaccine the e l gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded rna-dependent protein kinase animal models of orthopoxvirus infection recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region emerging targets and novel approaches to ebola virus prophylaxis and treatment deletion of gene a l enhances vaccinia virus immunogenicity and vaccine efficacy recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus ankara (mva) the nonreplicating smallpox candidate vaccines defective vaccinia lister (dvv-l) and modified vaccinia ankara (mva) elicit robust long-term protection modified vaccinia virus ankara triggers type i ifn production in murine conventional dendritic cells via a cgas/ sting-mediated cytosolic dna-sensing pathway the vaccinia virus k l gene product potentiates translation by inhibiting double-stranded-rna-activated protein kinase and phosphorylation of the alpha subunit of eukaryotic initiation factor structure of vaccinia virus late promoters a pandemic warning? ebolavirus evolution: past and present induction of influenza (h n ) antibodies by modified vaccinia virus ankara h n vaccine innate immune sensing of modified vaccinia virus ankara (mva) is mediated by tlr -tlr , mda- and the nalp inflammasome new vaccinia virus promoter as a potential candidate for future vaccines modification of promoter spacer length in vaccinia virus as a strategy to control the antigen expression redundancy complicates the definition of essential genes for vaccinia virus cloning the vaccinia virus genome as a bacterial artificial chromosome in escherichia coli and recovery of infectious virus in mammalian cells engineering of a vaccinia virus bacterial artificial chromosome in escherichia coli by bacteriophage [lambda]-based recombination the rational design of an aids vaccine highly attenuated modified vaccinia virus ankara replicates in baby hamster kidney cells, a potential host for virus propagation, but not in various human transformed and primary cells identification of vaccinia virus epitope-specific hla-a* -restricted t cells and comparative analysis of smallpox vaccines host range restriction of vaccinia virus in chinese hamster ovary cells: relationship to shutoff of protein synthesis host range deletion mutant of vaccinia virus defective in human cells comparison of vaccine strategies using recombinant env-gag-pol mva with or without an oligomeric env protein boost in the shiv rhesus macaque model smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus guideline on quality, non-clinical and clinical aspects of live recombinant viral vectored vaccines vaccinia virus recombinants expressing rabiesvirus glycoprotein protect against rabies ectromelia virus: the causative agent of mousepox imvanex: modifiziertes vacciniavirus ankara lebend protective cd (+) t-cell immunity to human malaria induced by chimpanzee adenovirus-mva immunisation hiv- vaccines improving the mva vaccine potential by deleting the viral gene coding for the il- binding protein escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors vaccinia virus protein n is a nuclear irf inhibitor that promotes virulence recombinant mva expressing secreted glycoprotein d of bohv- induces systemic and mucosal immunity in animal models modified vaccinia virus ankara protein f l is a novel bh -domain-binding protein and acts together with the early viral protein e l to block virus-associated apoptosis h n virus: transmission studies resume for avian flu comparison of lyophilized versus liquid modified vaccinia ankara (mva) formulations and subcutaneous versus intradermal routes of administration in healthy vaccinia-naïve subjects expanding the repertoire of modified vaccinia ankara-based vaccine vectors via genetic complementation strategies a candidate hiv/aids vaccine (mva-b) lacking vaccinia virus gene c l enhances memory hiv- -specific t-cell responses improving adaptive and memory immune responses of an hiv/aids vaccine candidate mva-b by deletion of vaccinia virus genes (c l and k r) blocking interferon signaling pathways deletion of the vaccinia virus n l gene encoding an inhibitor of irf improves the immunogenicity of modified vaccinia virus ankara expressing hiv- antigens designing cell lines for viral vaccine production: where do we stand? a protein particle vaccine containing multiple malaria epitopes enhanced cd t cell immunogenicity and protective efficacy in a mouse malaria model using a recombinant adenoviral vaccine in heterologous prime-boost immunisation regimes localization and sequence of a vaccinia virus gene required for multiplication in human cells specificity and -month durability of immune responses induced by dna and recombinant modified vaccinia ankara vaccines expressing hiv- virus-like particles systems analysis of mva-c induced immune response reveals its significance as a vaccine candidate against hiv/aids of clade c enhanced immunogenicity and protective efficacy against mycobacterium tuberculosis of bacille calmette-gu erin vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus ankara the t / kda family of poxvirus-secreted proteins bind chemokines and modulate leukocyte influx into virusinfected tissues a decade after sars: strategies for controlling emerging coronaviruses a multicenter, open-label, controlled phase ii study to evaluate safety and immunogenicity of mva smallpox vaccine (imvamune) in - year old subjects with diagnosed atopic dermatitis middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels poxviruses and the evolution of host range and virulence immunogenicities of intravenous and intramuscular administrations of modified vaccinia virus ankara-based multi-ctl epitope vaccine for human immunodeficiency virus type in mice airborne transmission of influenza a/h n virus between ferrets comparative experimental works on cow pox virus vaccines a pandemic influenza h n live vaccine based on modified vaccinia ankara is highly immunogenic and protects mice in active and passive immunizations vectors based on modified vaccinia ankara expressing influenza h n hemagglutinin induce substantial cross-clade protective immunity characterization of wild-type and mutant vaccinia virus m l proteins' abilities to localize to the endoplasmic reticulum and to inhibit nf-kappab activation during infection patterns of viral replication correlate with outcome in simian immunodeficiency virus (siv)-infected macaques: effect of prior immunization with a trivalent siv vaccine in modified vaccinia virus ankara the march toward malaria vaccines replication of modified vaccinia virus ankara in primary chicken embryo fibroblasts requires expression of the interferon resistance gene e l reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses a toll-like receptor-independent antiviral response induced by double-stranded b-form dna dna/mva vaccines for hiv codelivery of envelope protein in alum with mva vaccine induces cxcr -biased cxcr + and cxcr À cd t cell responses in rhesus macaques exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox middle east respiratory syndrome in persons potent functional antibody responses elicited by hiv-i dna priming and boosting with heterologous hiv- recombinant mva in healthy tanzanian adults mva vaccination provides long-term protection against nasal rabbitpox virus challenge a chemically defined production process for highly attenuated poxviruses broad protection against avian influenza virus by using a modified vaccinia ankara virus expressing a mosaic hemagglutinin gene comparative assessment of transmission-blocking vaccine candidates against plasmodium falciparum expression of rabies virus glycoprotein from a recombinant vaccinia virus middle east respiratory syndrome infection control and prevention guideline for healthcare facilities development of eczema vaccinatum in atopic mouse models and efficacy of mva vaccination against lethal poxviral infection recombinant modified vaccinia virus ankarabased vaccine induces protective immunity in mice against infection with influenza virus h n mva-based h n vaccine affords cross-clade protection in mice against influenza a/h n viruses at low doses and after single immunization recombinant modified vaccinia virus ankara expressing the hemagglutinin gene confers protection against homologous and heterologous h n influenza virus infections in macaques evaluation of a modified vaccinia virus ankara (mva)-based candidate pandemic influenza a/h n vaccine in the ferret model ankara-based influenza a h n vaccine: a randomised, double-blind phase / a clinical trial critical role of perforin-dependent cd + t cell immunity for rapid protective vaccination in a murine model for human smallpox easy and efficient protocols for working with recombinant vaccinia virus mva zoonotic brazilian vaccinia virus: from field to therapy pigs, poultry, and pandemic influenza: how zoonotic pathogens threaten human health cd l-adjuvanted dna/modified vaccinia virus ankara simian immunodeficiency virus (siv) vaccine enhances protection against neutralization-resistant mucosal siv infection immunity against heterosubtypic influenza virus induced by adenovirus and mva expressing nucleoprotein and matrix protein- modified vaccinia virus ankara triggers chemotaxis of monocytes and early respiratory immigration of leukocytes by induction of ccl expression preliminary assessment of the efficacy of a t-cell-based influenza vaccine, mva-np+m , in humans samd is an innate antiviral host factor with stress response properties that can be antagonized by poxviruses new avian suspension cell lines provide production of influenza virus and mva in serum-free media: studies on growth, metabolism and virus propagation factors associated with poor outcomes among adults hospitalized for influenza in france: a three-year prospective multicenter study role of viral factor e l in modified vaccinia virus ankara infection of human hela cells: regulation of the virus life cycle and identification of differentially expressed host genes doublestranded rna-binding protein e controls translation of viral intermediate rna, marking an essential step in the life cycle of modified vaccinia virus ankara vaccinia virus: a selectable eukaryotic cloning and expression vector general method for production and selection of infectious vaccinia virus recombinants expressing foreign genes experiences with immunization against orthopox viruses of humans and animals using vaccine strain mva ver€ anderung von vaccinevirus durch dauerpassagen in h€ uhnerembryofibroblastenkulturen experimental research on the s-antigen in the viruses of animal smallpox abstammung, eigenschaften und verwendung des attenuierten vaccinia-stammes mva poxvirus tropism a review of preclinical animal models utilised for tb vaccine evaluation in the context of recent human efficacy data protective immunity against mycobacterium tuberculosis induced by dendritic cells pulsed with both cd +-and cd +-t-cell epitopes from antigen a recombinant modified vaccinia virus ankara expressing antigen a boosts bcg-primed and naturally acquired antimycobacterial immunity in humans human infection with mers coronavirus after exposure to infected camels f-box-like domains are present in most poxvirus ankyrin repeat proteins percutaneous vaccination as an effective method of delivery of mva and mva-vectored vaccines mapping of deletions in the genome of the highly attenuated vaccinia virus mva and their influence on virulence antibodies against mers coronavirus in dromedary camels comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity safety and immunogenicity of dna/modified vaccinia virus ankara malaria vaccination in african adults malaria vaccine developments the ankyrin repeat as molecular architecture for protein recognition improved adjuvanting of seasonal influenza vaccines: preclinical studies of mva-np+m coadministration with inactivated influenza vaccine priming with a simplified intradermal hiv- dna vaccine regimen followed by boosting with recombinant hiv- mva vaccine is safe and immunogenic: a phase iia randomized clinical trial west nile virus and its emergence in the united states of america molecular genetics of vaccinia virus: demonstration of marker rescue the outbreak of west nile virus infection in the new york city area in equine vaccine for west nile virus hiv-dna given with or without intradermal electroporation is safe and highly immunogenic in healthy swedish hiv- dna/mva vaccinees: a phase i randomized trial cowpox virus and other members of the orthopoxvirus genus interfere with the regulation of nf-kb activation the influence of delivery vectors on hiv vaccine efficacy novel conserved-region t-cell mosaic vaccine with high global hiv- coverage is recognized by protective responses in untreated infection safety and immunogenicity of modified vaccinia ankara-bavarian nordic smallpox vaccine in vaccinia-naive and experienced human immunodeficiency virus-infected individuals: an open-label construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the dna of infectious vaccinia virus postexposure immunization with modified vaccinia virus ankara or conventional lister vaccine provides solid protection in a murine model of human smallpox mousepox in the c bl/ strain provides an improved model for evaluating anti-poxvirus therapies vaccinia virus host range genes interaction of f l with the bh domain of bak is responsible for inhibiting vaccinia-induced apoptosis ebola: lessons learned and future challenges for europe isolation of mers coronavirus from a dromedary camel cross host transmission in the emergence of mers coronavirus selection of recombinant mva by rescue of the essential d r gene cultivation of vaccine virus for jennerian prophylaxis in man further observations on the cultivation of vaccine virus for jennerian prophylaxis in man vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing cs of plasmodium immunological and virological mechanisms of vaccine-mediated protection against siv and hiv the potential for respiratory droplet-transmissible a/h n influenza virus to evolve in a mammalian host isolation of vaccinia mva recombinants using the viral f l gene as the selective marker lessons learnt from the german smallpox outbreaks after world war ii enhanced immunogenicity for cd + t cell induction and complete protective efficacy of malaria dna vaccination by boosting with modified vaccinia virus ankara recombinant modified vaccinia virus ankara-based malaria vaccines a phase i, open-label trial, evaluating the safety and immunogenicity of candidate tuberculosis vaccines aeras- and mva a, administered by primeboost regime in bcg-vaccinated healthy adults the vaccinia virus k l gene product inhibits host nf-kappab activation by preventing ikappabalpha degradation chapter six-the pathogenesis and immunobiology of mousepox identification of restriction factors by human genome-wide rna interference screening of viral host range mutants exemplified by discovery of samd and wdr as inhibitors of the vaccinia virus k l(À)c l(À) mutant ectromelia, vaccinia and cowpox viruses encode secreted interleukin- -binding proteins irf and irf phosphorylation in virus-infected cells does not require double-stranded rna-dependent protein kinase r or ikappa b kinase but is blocked by vaccinia virus e l protein vaccinia virus immune evasion: mechanisms, virulence and immunogenicity middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies the highly conserved orthopoxvirus k ankyrin-like protein is part of a cellular scf ubiquitin ligase complex the orthopoxvirus -kilodalton ankyrin-like protein is essential for dna replication and complete gene expression of modified vaccinia virus ankara in nonpermissive human and murine cells vaccinia and cowpox viruses encode a novel secreted interleukin- -binding protein transient host range selection for genetic engineering of modified vaccinia virus ankara construction and isolation of recombinant mva vaccinia virus and poxvirology inactivation of the viral interleukin β receptor improves cd + t-cell memory responses elicited upon immunization with modified vaccinia virus ankara short-term, but not postexposure, protection against lethal orthopoxvirus challenge after immunization with modified vaccinia virus ankara chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebola virus challenge henipavirus mediated membrane fusion, virus entry and targeted therapeutics intracutaneous smallpox vaccination with a weak pathogenic vaccinia virus mva-stufenimpfung gegen pocken modified vaccinia virus ankara protects macaques against respiratory challenge with monkeypox virus potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association west nile virus infection and immunity nonreplicating vaccinia vector efficiently expresses recombinant genes a recombinant vector derived from the host range-restricted and highly attenuated mva strain of vaccinia virus stimulates protective immunity in mice to influenza virus non-replicating vaccinia vector efficiently expresses bacteriophage t rna polymerase the vaccinia virus c l protein inhibits mouse il- and promotes virus virulence in the murine intranasal model safety and efficacy of mva a, a new tuberculosis vaccine use of chad -ebo-z ebola virus vaccine in malian and us adults, and boosting of malian adults with mva-bn-filo: a phase , single-blind, randomised trial, a phase b, openlabel and double-blind, dose-escalation trial near death experiences: poxvirus regulation of apoptotic death repeated high-dose (  tcid ) toxicity study, of a third generation smallpox vaccine (imvamune), in new zealand white rabbits sex difference in immune response to vaccination: a participant-level meta-analysis of randomized trials of imvamune ® smallpox vaccine comparative analysis of siv-specific cellular immune responses induced by different vaccine platforms in rhesus macaques protective efficacy of several vaccines against highly pathogenic h n avian influenza virus under experimental conditions rapid expansion of cd + t cells in wild-type and type i interferon receptor-deficient mice correlates with protection after low-dose emergency immunization with modified vaccinia virus ankara protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein immunogenicity and protective efficacy of recombinant modified vaccinia virus ankara candidate vaccines delivering west nile virus envelope antigens human infection with a zoonotic orthopoxvirus in the country of georgia differential immunogenicity of various heterologous prime-boost vaccine regimens using dan and viral vectors in healthy volunteers progression of pathogenic events in cynomolgus macaques infected with variola virus modified vaccinia virus ankara induces toll-like receptor-independent type i interferon responses vaccinia virus-mediated inhibition of type i interferon responses is a multifactorial process involving the soluble type i interferon receptor b and intracellular components vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis the vaccinia virus f l protein interacts with the proapoptotic protein bak and inhibits bak activation enhanced t cell-mediated protection against malaria in human challenges by using the recombinant poxviruses fp and modified vaccinia virus ankara a novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses chapter poxvirus host range genes boosting with poxviruses enhances mycobacterium bovis bcg efficacy against tuberculosis in guinea pigs an assay to compare the infectivity of mycobacterium tuberculosis isolates based on aerosol infection of guinea pigs and assessment of bacteriology tandem repeats within the inverted terminal repetition of vaccinia virus dna expression of the vaccinia virus genome: analysis and mapping of mrnas encoded within the inverted terminal repetition development of a replicationdeficient recombinant vaccinia virus vaccine effective against parainfluenza virus infection in an animal model highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge elucidating and minimizing the loss by recombinant vaccinia virus of human immunodeficiency virus gene expression resulting from spontaneous mutations and positive selection blockade of interferon induction and action by the e l double-stranded rna binding proteins of vaccinia virus human infection with a novel avian influenza a(h n ) virus efficiently editing the vaccinia virus genome by using the crispr-cas system rapid generation of a mouse model for middle east respiratory syndrome interleukin- β receptor expressed by modified vaccinia virus ankara interferes with interleukin- β activity produced in various virus-infected antigen-presenting cells key: cord- - ad fw z authors: monath, thomas p. title: vaccines against diseases transmitted from animals to humans: a one health paradigm date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ad fw z abstract this review focuses on the immunization of animals as a means of preventing human diseases (zoonoses). three frameworks for the use of vaccines in this context are described, and examples are provided of successes and failures. framework i vaccines are used for protection of humans and economically valuable animals, where neither plays a role in the transmission cycle. the benefit of collaborations between animal health and human health industries and regulators in developing such products is discussed, and one example (west nile vaccine) of a single product developed for use in animals and humans is described. framework ii vaccines are indicated for domesticated animals as a means of preventing disease in both animals and humans. the agents of concern are transmitted directly or indirectly (e.g. via arthropod vectors) from animals to humans. a number of examples of the use of framework ii vaccines are provided, e.g. against brucellosis, escherischia coli o , rabies, rift valley fever, venezuelan equine encephalitis, and hendra virus. framework iii vaccines are used to immunize wild animals as a means of preventing transmission of disease agents to humans and domesticated animals. examples are reservoir-targeted, oral bait rabies, mycobacterium bovis and lyme disease vaccines. given the speed and lost cost of veterinary vaccine development, some interventions based on the immunization of animals could lead to rapid and relatively inexpensive advances in public health. opportunities for vaccine-based approaches to preventing zoonotic and emerging diseases that integrate veterinary and human medicine (the one health paradigm) are emphasized. zoonoses (diseases transmissible from animals to humans) account for approximately % of all infectious pathogens of human beings and % of all emerging infectious diseases [ , ] . the origins of these diseases, and underlying factors (including man-made factors) in their emergence have been the subject of considerable interest [ , ] . certain zoonotic diseases have the potential for pandemic spread by human contagion, such as avian influenza, sars and the middle east respiratory syndrome coronavirus, and others for regional cross-border epizootics, such as yellow fever, venezuelan equine encephalitis and rift valley fever. the cost of a short list of zoonotic disease emergences in the interval between and , including bovine spongiform encephalopathy, sars, highly pathogenic avian influenza, west nile, and pneumonic plague (india), was estimated to exceed $ billion [ ] . of major emergencies that concerned public health (including hurricanes, earthquakes, and terrorist attacks) occurring between and , more than % were zoonotic disease outbreaks [ ] . however, the toll on public health is much greater than that caused by such dramatic outbreaks. it is estimated that different zoonotic diseases are responsible annually for . billion cases of human disease with . million deaths and substantial reductions in livestock production [ ] . animals, including livestock and companion animals, also suffer illness and death following infection with many zoonotic infections, and livestock and poultry are subject to large-scale intentional destruction as a means of preventing human infections, resulting in huge economic losses. wild animals, including endangered species, may also be mortally affected, examples being west nile disease in birds, yellow fever in neotropical monkeys, plague in black-footed ferrets, and ebola in the great apes. vaccines are an important means of prevention and control of zoonotic infectious diseases in humans and domesticated animals. however, the target for vaccination is, in almost all cases, the directly affected species, and there are few practically implemented illustrations of the potential for indirectly preventing human disease by immunizing the domesticated or companion animal sources of infection. the concept of immunizing wild animal reservoirs for the prevention of disease in humans or domestic animals is even more challenging and has received limited attention. moreover, human and animal health divisions of the biopharmaceutical industry are generally separate and segregated, and there is no organized approach to the development of new vaccines indicated for the prevention of spread of diseases from domestic or wild animals to humans. in addition, the major funding sources for research in human and animal diseases tend to be stove-piped into different government agencies, stifling cross-cutting approaches. the purpose of this review is to stimulate the science and policy communities to seek innovative ways to interdict zoonotic diseases by integrating human and veterinary medicine and vaccine development, and by creating new streams of funding aimed at the intersection of human and animal health. these aims are consistent with the one health initiative, which seeks to establish "collaborative efforts of multiple disciplines working locally, nationally and globally to attain optimal health for people, animals and our environment" [ ] . a framework for considering one health vaccine interventions is provided, as well as a brief review of past and current efforts, including a few successes. it will be obvious to the reader that this is a wide-open field with many opportunities that deserve more attention than they have received. the complexity, timeline, and cost of development of animal vaccines and the regulatory hurdles for product approval are far less than for human vaccines. thus some interventions based on the immunization of animals could lead to rapid and relatively inexpensive advances in public health. it is obvious that the benefit to human health deriving from vaccination of animals is far easier to justify when there is also a benefit to animal health, the latter often being closely linked to economic value. causative agents of zoonotic diseases, including viruses, parasites, bacteria, fungi, and prions, have extraordinarily varied life cycles and modes of transmission. some may persist between periods of active transmission in soil or invertebrate species. many have silent transmission cycles involving wild animals that have coevolved with the infectious agent and exhibit no signs of disease. some zoonotic diseases occur when a causative agent harbored by a wild animal reservoir jumps species to domesticated animals and thence to humans. others are primarily diseases of domesticated animal species. humans may be infected by direct contact with wild or domesticated animals, or indirectly by ingestion of contaminated milk or meat, inhalation of aerosolized secretions or excreta, fomites, or hematophagous insect or tick vectors. despite this complexity of epidemiological patterns, the opportunities for intervention often boil down to a few simple bottlenecks in the transmission process. for example, milk-borne diseases can be prevented by pasteurization, certain meat-borne diseases by inspection and animal husbandry improvements (e.g., trichinella, bovine tuberculosis), and other diseases avoided by limiting contact with known high risk species (e.g., tularemia, turtle-borne salmonellosis, exposure to bats carrying henipaviruses). where the risk of infection is high, or the resulting disease severe, vaccines may be the most efficient and cost-effective means of prevention and control. alternative methods for control of zoonotic diseases have generally employed trapping, poisoning, or other means of destroying the offending animal reservoir/vector; these methods have a mixed (often negative) record of success and are in any case becoming socially unacceptable. this review focuses on the use of vaccines for animals as an acceptable means of interdicting zoonotic disease in both animals and humans. three major epidemiological frameworks are identified for the control of zoonotic disease by means of vaccination of animals ( table ). the scope of this review encompasses only those diseases for which a strategy targeting vaccination of animals is actually used or is under development. there are many diseases of table frameworks for vaccine development and utilization as a means of preventing zoonotic diseases, and status of approval of existing vaccines for humans and animals. the first major framework includes zoonotic infectious diseases that affect both humans and economically important animals, where wild animals are the source of infection. livestock and humans are dead-end hosts, and neither contributes to the transmission cycle. in this case, vaccines are required to prevent disease in both humans and economically important animals, but do not interrupt transmission of the disease in nature. for framework i diseases, there is an important opportunity to accelerate the development of new vaccines by concurrent veterinary and human product research ( table ) . moreover the affected animal species represents a natural disease model of infection, pathogenesis and immunity that may be useful in testing efficacy and immunological correlates of protection of a new vaccine intended for human use, and thus provide data supporting regulatory approval under fda's animal rule [ ] . a list of framework i diseases and vaccines is shown in table , and an example is given below. west nile virus is a mosquito-borne, single strand, positivesense, enveloped rna virus (genus flavivirus, family flaviviridae), closely related to japanese encephalitis virus. west nile virus is a recognized cause of human disease ranging from mild feverrash-headache syndromes to lethal encephalomyelitis. horses, domesticated geese, farmed alligators (as well as a number of wild birds and mammals) are also susceptible to severe and fatal disease. although recognized as a cause of disease as early as the s, west nile became an increasing problem in the s, with outbreaks affecting humans and/or horses in northern africa, western europe, the eastern mediterranean, the black sea region and the volgograd oblast of russia [ ] . the most dramatic development was the introduction of west nile virus into north america in [ ] [ ] [ ] , the occurrence of large outbreaks in and , rapid spread across the us, and subsequent introduction to the caribbean and south america. horses and a number of wild bird species, notably crows and jays, were affected in addition to humans. between and , a total of , cases of west nile fever and , cases of neuroinvasive west nile disease were reported in the us, with the highest incidence in the middle of the country from texas north to the dakotas [ ] . the incidence of neuroinvasive disease in the us has varied between . and . per , in this interval [ ] . over , horses have been affected since , and in these animals the disease is more severe ( % case-fatality, % of survivors with neurological sequelae) than in humans ( - % case fatality, % of encephalitis survivors with sequelae) [ ] . moreover, the incidence of west nile in horses (∼ per , ) is substantially higher than in humans [ ] . the animal health industry rapidly responded to this veterinary emergency, and multiple west nile vaccines were rapidly developed, including a live vaccine (discussed below), whole virion inactivated, dna, and poxvirus vectored vaccines. the first vaccine for horses was marketed in , only years after introduction of the virus into the us. multiple human vaccine development programs were also initiated, but many of these efforts were discontinued or decelerated due to the high market risk associated with low incidence, a slackening of public concern with the disease, and the uncertain regulatory pathway for vaccine approval. although efficacy of a vaccine for equids is established [ ] , field trials to prove vaccine efficacy in humans would be large, expensive, and difficult due to the unpredictable occurrence of west nile outbreaks. the application of the animal rule to licensing a west nile vaccine, while plausible, has not been adjudicated by the fda with a sponsor. although approaches to developing veterinary and human vaccines against west nile were technologically similar, in only one case was a concurrent development program undertaken by a single company. this fact illustrates the current status of stove-piped and separate animal and human health biopharmaceutical industries. the exceptional case is of interest as a model of how vaccine development for zoonotic diseases could be improved. in , within months of the identification of west nile as the etiological agent of the initial outbreak affecting humans and horses in new york, acambis, a publically traded human-vaccine biotechnology company, applied its platform technology for yellow fever dvectored vaccines to the development of a west nile vaccine, with the intent to develop both vaccines for both horses and humans. this technology involved replacing the gene encoding the yellow fever d vaccine virus' envelope (e) protein with the corresponding gene of west nile virus. this chimeric vector vaccine platform had been previously used by the company to construct a chimeric vaccine against the closely-related japanese encephalitis virus [ ] ; at the time, that vaccine had proven to be highly effective, protecting non-human primates against intracerebral challenge with virulent japanese encephalitis virus [ ] . two new vectors were engineered, one with the wild-type west nile ny strain e protein gene and one with an e gene containing three attenuating mutations [ ] [ ] [ ] . the former was neurovirulent in mice, while the latter was more attenuated and thus deemed more suitable as a human vaccine. the principal question was whether the yellow fever d vector would infect and immunize horses, since yellow fever is a host-restricted primate virus. to find out, studies were sponsored by acambis in early at the colorado state university school of veterinary medicine. horses were vaccinated with the west nile/yellow fever chimeric virus or with yellow fever d vaccine, and viremia and antibody responses were determined [ ] . in addition, vaccinated and control horses were challenged by the intrathecal injection of a high dose of virulent west nile virus, following the same model referred to above for protection studies of the chimeric japanese encephalitis vaccine wherein monkeys were challenged by the intracerebral route. these studies showed that horses inoculated with chimeric vaccine developed neutralizing antibodies against west nile and were protected against a severe intrathecal challenge. a similar development plan was successfully followed for the mutated human vaccine version, using non-human primates as the test host. the veterinary and human vaccine candidates were developed side by side, and the data obtained in both programs were designed to support transition to advanced clinical trials in horses and humans. in , acambis licensed the veterinary vaccine technology to a major animal health company (intervet), and in the same year clinical trial materials were made for human trials. intervet completed development of the veterinary vaccine (prevenile ® ) [ ] , which was approved by usda in , and acambis brought the human vaccine (chimerivax-wn ) into clinical trials in the same timeframe [ ] , subsequently outlicensing the technology to sanofi pasteur. as expected, development of the veterinary vaccine and its progression through the regulatory pathway substantially outpaced the human vaccine; importantly, the veterinary application significantly informed the human vaccine table strengths and limitations of vaccination of animals as a means to control of zoonotic diseases. framework (see table development program in providing useful information on safety, durability of immunity and immune correlates or protection. co-development of the veterinary and human west nile vaccines is as a potential model for other vaccines. there are a few other examples where vaccines were co-developed for animals and humans, including vaccines against venezuelan equine encephalitis and rift valley fever (described below). certain issues arise, however, that may need to be considered in the context of such integrated efforts. first, a safety problem arising during use of a veterinary vaccine could provoke regulatory concerns or a liability problem for the human analog. although a safety issue did arise briefly due to anaphylaxis type reactions in horses, resulting in temporary withdrawal of prevenile ® from the market [ ], there were no repercussions for the human analog vaccine (chimerivax-wn ). this raises the interesting question whether the human and veterinary regulatory agencies are integrating information that might be of value, either during development of similar vaccines for different species or after marketing approval. this is an obvious overlooked area for application of one health principles. second, the immune response to veterinary vaccines generally should differentiate naturally infected from vaccinated animals (diva) on the basis of a laboratory test, which allows compliance with trade restrictions. this is, of course not a concern for regulatory approval of human vaccines, although it can be useful in seroepidemiological and vaccine coverage studies. an example of problems associated with diva is the off-label use of commercial horse vaccine to protect emus against eastern equine encephalitis (eee), since some combination vaccines against eee contain equine influenza antigen and such vaccines elicit cross-reactive antibodies to avian influenza resulting in quarantine. the chimeric west nile vaccine described above potentially allows for diva testing (using responses to the yellow fever nonstructural proteins expressed by the vector) [ ] . a number of important diseases are transmitted between domesticated animals and thence to humans. vaccination of domesticated animals has the potential to protect humans against these zoonoses, either indirectly by interrupting transmission where domesticated animals are amplifying hosts in the transmission cycle or directly by preventing spread from infected animals to humans. a list of framework ii diseases and vaccines is shown in table . selected examples are used to illustrate the role of vaccination of domesticated animals in preventing human disease. brucella spp. are facultative, intracellular gram-negative bacteria, pathogenic for domestic animals and humans. brucellosis, caused mainly by brucella melitensis (which infects sheep and goats), brucella abortus (cattle), and brucella suis (swine), occurs worldwide, with the highest prevalence in the middle east, asia, africa, tropical america, and the mediterranean region [ , ] . the annual incidence of human infections is estimated at , cases but the disease is widely acknowledged to be underreported [ ] . brucella canis, an infection of dogs, occurs worldwide with highest prevalence in tropical america. b. canis disease in humans has been reported, especially in persons handling breeding dogs and in immunosuppressed individuals. human brucellosis is acquired by contact or aerosol spread from infected animals, fomites, or ingestion of unpasteurized milk or undercooked meat. not surprisingly, brucellae survive for long periods in dust, animal excreta, soil, meat and dairy products. wild animals are also affected and can be the source of infection of livestock and humans. in animals, brucellosis causes epididymitis in males and abortion, placentitis, infertility and reduced milk production in female animals. the human disease is protean, manifested by chronic fatigue, relapsing fever, endocarditis, spondylitis, osteomyelitis, arthritis, and meningitis [ ] . prevention of human disease by control of brucellosis in livestock has long been a public health priority [ ] . control of brucellosis relies principally on surveillance, testing, removal of infected animals, import/export animal and animal product control provisions, protection from exposure to wild reservoirs (such as elk, deer, and bison), and vaccination. antibiotic treatment of animals is regulated and discouraged due to the large doses and long treatment required and concern about resistance. old, empirically developed live attenuated vaccines, b. melitensis rev vaccine for goats and sheep; b. abortus s and rb vaccines for cattle; and the oral b. suis s vaccine used widely in china for multiple species, elicit cellular immunity against the intracellular pathogen and are more effective than other types of vaccine [ ] . the rb vaccine (a spontaneous rifampin-resistant rough mutant) is approved in the us [ ] . however, the live vaccines have a number of drawbacks. the latter include lack of the ability to differentiate s and rev vaccine immunity from natural immunity (diva) and interference with surveillance and export control procedures; pathogenicity (especially abortion when animals are vaccinated during pregnancy); antibiotic resistance of the vaccine strains; and modest efficacy. live vaccines used in livestock can also cause illness in humans. vaccination as a stand-alone strategy has rarely been carefully evaluated, in large part due to concerns over the quality of the existing vaccines. however, vaccination is largely credited with elimination of brucellosis in the us [ , ] (the us was declared free of brucellosis in cattle in ) and for control of brucellosis in china [ ] . a recent study in greece showed that a mass vaccination program with the b. melitensis rev vaccine resulted in a decrease in human infections [ ] . a number of new vaccine approaches, including diva vaccines, designed to induce th oriented cellular immunity are under investigation, including safer rationally designed, mutated live vaccines [ ] ; recombinant, invasive escherischia coli [ ] ; recombinant subunit microencapsulated vaccines; and dna vaccines [ ] . experimental b. canis vaccines have been investigated in mice. where brucellosis-free status has been achieved, as in the us, wild animal reservoirs (especially bison and elk) threaten to reintroduce the disease. vaccination with existing vaccines is feasible, but delivery is challenging [ ] . this vero cytotoxin secreting gram-negative bacteria is an important cause of sporadic and epidemic food-borne illnesses of humans, including gastroenteritis and hemorrhagic colitis, with potentially lethal complications (hemolytic-uremic syndrome). cattle and sheep are the principal reservoirs of infection and transmission to humans occurs via food (meat, seeds and vegetables) contaminated with animal feces. undercooked ground beef is a source of infection in approximately one-third of human cases and recalls are a significant economic threat to the meat packing and distribution industry. animals concentrated at feed lots and slaughter that shed bacteria can produce lots of meat with high rates of o [ , ] , but there is considerable variability in the occurrence of contaminations [ ] . in developed countries, various sanitary measures and testing have been instituted to reduce the risk to consumers, but these remain imperfect. vaccination of feed lot cattle has been proposed as a measure to reduce the prevalence and duration of shedding and the risk to consumers. o -specific bacterial extract vaccines containing protective outer membrane proteins have been conditionally approved by usda (manufactured by epitopix, willmar, mn) and fully approved by the canadian food inspection agency (bioniche life sciences, belleville, ont.). feedlot cattle receiving or doses of the bioniche vaccine - weeks apart had - % reduction in colorectal colonization or fecal shedding and significant reduction in magnitude and duration of shedding [ ] [ ] [ ] . hurd and malladi [ ] modeled the impact of vaccinating cattle on human health outcomes. assuming % efficacy of the vaccine and % adoption rate, the model indicated a % reduction in the incidence of e. coli o -related human illness. the model also predicted significant reductions in the number of lots of contaminated ground beef and detection by usda, which would have substantial economic benefit to packers and distributors. vaccine effectiveness under conditions of field use will be highly dependent on adoption rate (vaccine coverage), and in part by whether cattle complete the -dose vaccination series on the proscribed schedule [ ] . an interesting question is: who will pay for vaccination of feedlot cattle? is the economic benefit there for the meat industry, and will vaccination reduce the cost of other preventive measures and testing or will vaccine costs simply be on top of other costs? will government agencies concerned with human health subsidize the cost, without empirical demonstration of a human health benefit? since ground beef only contributes about one-third of human infections, how could vaccines be used as a means of reducing other sources of food contamination? another issue for use of the current vaccines is the role of other enterohemorrhagic e. coli (e.g. o , o , and o ) in human disease. this disease is caused primarily by the gram-negative bacterium, bartonella henselae, transmitted between domestic cats by the agency of cat fleas, ctenocephalides felis. human infection occurs by contact spread from cats, including scratches by claws contaminated with blood or flea feces, or possibly by flea bite [ , ] . the prevalence of infection in household cats in the us is approximately %, but in stray animals it is % and high prevalence rates have been found in developing countries [ , ] . disease in humans is manifest by fever, a papule followed by a pustule at the site of infection and lymphadenopathy. rare complications include meningitis, encephalitis, endocarditis, glomerulonephritis, osteomyelitis, neuropsychiatric abnormalities, and relapsing fever and splenomegaly. human infections in immune-compromised individuals are particularly severe. approximately , - , cases and hospitalizations caused by cat scratch disease are estimated to occur annually in the us. in the late s, heska corporation, an animal heath company in colorado, initiated a program to develop a vaccine for household cats, with the goal of limiting the potential for transmission of the bacteria from cats to humans. unfortunately, the program was not completed and no vaccine is available at present. given the fact that cats rarely become ill with b. henselae, this would have been an unusual product providing protection to pet owners, with marginal if any real benefit to the target species. moreover, it would likely have been extremely challenging to demonstrate a health benefit to humans. rabies is a fatal infection of the central nervous system caused by rabies virus, a member of the lyssavirus genus, family rhabdoviridae. it is estimated that up to , - , cases of human rabies occur annually, and dog bite is the cause of over % [ ] . successful vaccination of dogs and humans against rabies was first demonstrated in by louis pasteur, using crude nerve tissue preparations. however, until the first decades of the th century in developed countries, and continuing in many developing countries today, the ancient practice of dog population reduction campaigns have been the main approach to rabies control, a method that has repeatedly proven to be ineffective. dog licensing and vaccination requirements were introduced in the united kingdom in , and gradually at the local and then state levels in the us beginning in the s, with national requirements attained by [ ] . many successful dog vaccination campaigns have been reported in latin american countries and asia [ ] . some countries have declared eradication of canine rabies, notably the united kingdom in , japan in , the us in , as well as malaysia, singapore, taiwan, hong kong. in , the first world rabies day event, the ultimate vision was promulgated of canine rabies elimination through systematic vaccination. however, rabies remains a significant public health problem, due to absent or incomplete dog vaccination in many areas of the world. more than . million post-exposure treatments with rabies vaccines are given annually, at a cost of over $ billion worldwide [ , ] . in china, due to the low prevalence of canine vaccination, the sales of rabies vaccines for human post-exposure prophylaxis outstrips any other human vaccine, accounting for % of all annual vaccine sales, i.e. million doses costing $ million [ ] . the requirement for human post-exposure vaccination at this scale represents an obvious failure of public health, since it plays no role in containing the spread of rabies in the canine vector. a very high rate of vaccine coverage in the dog population (exceeding %) is required for interruption of the rabies transmission cycle [ ] . the development of oral rabies vaccines has allowed vaccination of free-roaming dogs that could not be restrained and vaccinated by injection, as well as the vaccination of wild animal species that are the source of infection in dogs [ ] . oral bait vaccine for dogs has been successfully deployed in trials in many countries, including turkey, thailand, sri lanka, south africa, and the philippines [ ] ; the world health organization has supported this approach as a supplemental program where there are substantial populations of free-ranging or feral dogs [ ] . significant increases in canine vaccination coverage have been achieved when oral rabies vaccine was added to a program of standard parenteral vaccination. oral vaccination of dogs has been accomplished using commercial baits containing live modified rabies vaccines, such as the street-alabama-dufferin (sad) strain, variant b [ ] and the attenuated copenhagen strain of vaccinia expressing the rabies glycoprotein gene (g protein) [ ] . a more detailed review of oral rabies vaccines is provided below (framework iii). hendra virus disease, a severe and fatal infection of horses and humans in australia, has been noted as an example illustrating one health principles in disease prevention and control [ ] , and is especially relevant now that a new vaccine for horses has been introduced. hendra is a member of the henipavirus genus, family paramyxoviridae. the reservoir hosts are fruit bats (pteropus spp.), and the virus is spread from bats to horses by contact (including respiratory droplets), by food or fomites contaminated with bat urine, or by contact with sick horses. all reported human infections have resulted from contact with infected horses [ ] [ ] [ ] . hendra virus disease was first described in . human and equine cases have occurred in coastal queensland and new south wales and positive bats have been detected in the northern territory. a total of deaths in equids have been reported in outbreaks, with a very high case-fatality rate ( %), and cases ( fatal) have occurred in humans, including horse trainers and veterinarians, all of whom had contact with sick horses [ ] . the disease is manifested by severe systemic illness, respiratory symptoms or acute and relapsing encephalitis [ ] . swine appear to be susceptible to experimental infection. nipah virus, a closely related bat-borne agent in se asia has caused outbreaks of severe and fatal disease in swine and humans [ ] [ ] [ ] . in november , pfizer animal health launched equivac ® hev, an adjuvanted subunit protein vaccine for the prevention of hendra virus disease of horses in australia [ ] . since horses are a major source of contact spread of hendra virus to humans, the vaccine promises to make an important contribution to human health as well. fear of acquiring the disease has also constrained equine veterinary practice in australia [ ] , and the vaccine should mitigate this problem. development of equivac ® hev was a collaborative effort between pfizer and csiro's australian animal health laboratory. however, support for the development program was also provided by human medical researchers in the us, at the uniformed services university of the health sciences supported by the henry jackson foundation for the advancement of military medicine. a provisional approval for limited use of the vaccine was obtained in early , with full approval in november. the vaccine is a soluble, recombinant glycoprotein (g) of hendra virus, the ligand for cell attachment and antibodies to the protein neutralize cell receptor binding of the virus [ , ] . the vaccine protects horses and ferrets against experimental infection [ ] , and appears to cross-protect against nipah virus [ ] . the availability of equivac ® hev should lead to rapid uptake by horse owners in australia. the equine and horse racing industry in australia is large, contributing billions of dollars, and over % of total gross domestic product [ ] . hendra virus in horses is a notifiable disease in all australian jurisdictions; the property where the horse cases are located is quarantined and animals that are infected are euthanized. the occurrence of at least one hendra virus outbreak annually since , and the high lethality of the disease have raised considerable awareness in australia. since all human cases of this zoonosis have resulted from contact with infected horses, vaccination of horses against hendra virus promises to be a highly effective strategy for preventing human cases. rift valley fever is an enveloped, single-strand, segmented rna virus belonging to the phlebovirus genus, family bunyaviridae, occurring in africa, with intermittent extensions to the arabian peninsula. rift valley fever virus causes explosive and economically damaging outbreaks of disease in cattle and sheep, with stillbirth, abortion, and very high mortality of young animals; in adult animals, it causes % mortality in sheep, - % in cattle, and - % in goats [ ] . humans typically develop self-limited nonspecific febrile illness, but - % have a complicated course with hemorrhagic fever syndrome, encephalitis, hepatitis, renal failure, or retinitis, and case-fatality rates in severely ill and hospitalized patients is as high as % [ , ] . the virus is transmitted between livestock and from livestock to humans by the agency of mosquito vectors. in addition, humans commonly acquire infection by contact and aerosol routes when handling, treating, or butchering infected livestock, and the virus can persist in meat for weeks. the ecology of rift valley fever and the reasons behind its periodic emergences have been the subject of intensive study. in brief, the reservoir of infection is aedes mosquitoes, especially ae. linneatopennis, which maintain the virus by transovarial transmission [ ] . the dessication-resistant ova containing virus remain in depressions in the earth ('dambos') that are flooded during the rainy season, with the subsequent emergence of infected adult aedes mosquitoes. infected, viremic, livestock in turn served as source for amplified virus transmission by a variety of mosquito vectors [ , ] . there are no approved vaccines for prevention of rift valley fever in humans, although a number of candidates are in development by academic and government laboratories. vaccination of cattle and sheep represents a strategy for preventing disease in these species, and thereby for interrupting virus transmission to humans. rift valley fever epizootics are to some extent predictable based on rainfall patterns and surveillance of disease in livestock [ ] . surveillance provides opportunities for rapid intervention, particularly with a single-dose vaccine (most likely a live, attenuated vaccine) that would protect against viremia in cattle and sheep and prevent mosquito infection. in addition, routine vaccination of livestock in inter-epizootic periods with a product capable of inducing durable protective immunity is a long range goal supported conceptually by modeling [ ] . however, there are many obstacles to livestock immunization in africa, including access, policy, regulatory approval, cold chain, and commercial viability. additionally, diva requirements are driven by regulations prohibiting export of livestock or meat by countries experiencing rift valley fever [ ] . some of the obstacles to vaccination implementation could be overcome by development of improved vaccines. two old veterinary vaccines, the live smithburn vaccine, developed using techniques of serial passage in mouse brain similar to that applied to the early development of the french neurotropic vaccine against yellow fever [ , ] , and a formalin inactivated vaccine [ ] are commercially available from the onderstepoort institute in south africa. however, the smithburn vaccine, now produced in bhk- cells, is reported to cause teratogenicity and abortion when used in pregnant animals [ ] and is used only in countries endemic for rift valley fever due to concerns about reversion. the inactivated vaccine, also grown in bhk- cell culture, requires multiple doses to be effective and probably has relatively short durability [ ] , making it less desirable for the interventions proposed above. nonetheless, the inactivated vaccine was successfully used to interrupt a rift valley fever outbreak in south african sheep [ ] . indeed, following a large epidemic of rift valley fever in egypt in - , the veterinary serum and vaccine research institute (cairo, egypt) produced a formalin-inactivated vaccine in bhk- cells using the epidemic strain (zh ), which matched locally circulating strains compared to the south african strain [ ] . another inactivated vaccine (tsi-gsd ) developed by the us army for human immunization and grown in diploid fetal rhesus lung cells was tested clinically and shown to be well tolerated and immunogenic. ninety % of the subjects developed a neutralizing titer > , which was shown to be higher than the protective level in a passive immunization-challenge study in hamsters [ , ] . in addition to the requirement for multiple doses for primary and booster immunization, inactivated rift valley fever vaccines have the disadvantage of requiring high biocontainment facilities for manufacturing. in recent years, there has been substantial progress in development of newer vaccines, and some of these vaccine development projects have been collaborations between human and veterinary research groups. additionally, there has been a moderate level of support from the us government because rift valley fever is a credible threat of natural or intentional (bioterrorist) introduction. nevertheless, despite very promising technical results, there has been insufficient support from industry and government to propel any of these new vaccine candidates into use. because of the obvious advantages of rapid onset and durable immunity associated with live vaccines, development of an improved live vaccine has been the focus of research. us army investigators attempted to induce attenuating mutations in two rift valley fever virus strains isolated during the egyptian epidemic [ ] . mp- is a live vaccine that was developed from the virulent zh- strain by passages in mrc- cells in the presence of the mutagen, -fluorouracil, resulting in a temperature sensitive virus with amino acid mutations. attenuation was demonstrated in multiple animal models, and reassortment studies showed that attenuating mutations were redundant and resided in all three gene segments [ , ] . development of mp- was undertaken by the us army medical research institute of infectious diseases with the intention to produce a vaccine for both human and animal immunization. the vaccine was clinically tested in human volunteers and shown to be well-tolerated and highly immunogenic [ ] . army investigators, in collaboration with usda, conducted a number of studies of mp- vaccine in sheep and cows, including neonatal, pregnant and lactating animals. these studies showed that mp- caused a low viremia, but with no attendant clinical signs; there was no virus secretion in milk, and no abortions or teratogenicity when vaccine was given in in mid-to late term pregnancy. mp- was highly immunogenic and protected livestock against virulent challenge [ , ] . however, ewes vaccinated with mp- early in pregnancy showed a low incidence of abortion and teratogenicity, indicating some residual virulence of the vaccine [ ] . to improve genetic stability and safety of mp- , reverse genetic techniques were used to introduce deletions in the s and m rna segments in genes encoding, respectively, nss and nsm proteins [ , ] . two deletion mutants were evaluated for safety and immunogenicity in pregnant ewes [ ] and in calves (morrill jc personal communication, ) with positive results for the nsm deletant, making it an attractive candidate as a veterinary and human vaccine. there were no clear safety signals when ewes were inoculated in early-mid pregnancy. further safety studies are required to rule out the low incidence of abortion/teratogenicity seen with parental mp- in early-term ewes. unfortunately, human trials have not yet been performed with the rationally designed mp- derivative. a third live vaccine designated clone , is a plaque-derived clone of a central african strain of rift valley fever isolated from a human subject, and was found to be naturally attenuated for mice and to have an in-frame deletion of most of the nss gene [ ] . this observation was the basis for modifying the mp- vaccine by ns gene deletion, as described above. once again, a collaboration between the human and veterinary researchers led to a study in ewes, showing that clone was highly immunogenic but did not cause abortions [ , ] . another attenuated vaccine designated r , has been developed by reassorting clone and mp- so that it contains the s segment of clone and the l and m segments of mp- . this strain has attenuation domains from both parental vaccine candidates. a number of live, replicating, and non-replicating heterologous viral vectors expressing rift valley fever g and g glycoproteins and nonstructural proteins have been have been investigated in mice, elicited immune responses and protected against challenge. the vectors included lumpy skin disease (capripoxvirus) [ ] , alphavirus (vee and sindbis) replicons [ ] , newcastle disease virus [ ] , adenovirus, and modified vaccinia ankara. several of these constructs were used to immunize sheep and/or cattle (lumpy skin disease virus, newcastle disease virus, and sindbis replicons) with somewhat variable success. for a more comprehensive review see indran and ikegami [ ] and boshra et al. [ ] . live vectors are a promising approach for new rift valley fever vaccines, particularly veterinary vaccines, but may have problems for homologous boosting in light of anti-vector immunity. various prime-boost strategies have been proposed, as for plasmid dna vaccines (see below), but these would be exceptionally difficult to implement for immunization of livestock in the field, and are thus impractical. subunit protein produced in insect cells, virus-like particles [ ] , and dna vaccines [ ] against rift valley fever are also in early stage development. these approaches have potential advantages of safety and thermostability during storage and distribution, but may require multiple dosing and provide less durable immunity than live vaccines, and thus are less desirable products for framework ii implementation. overall, it remains to be seen which of the many rift valley fever vaccines in development progress to regulatory approval and whether an integrated veterinary and human health policy based on the immunization of livestock in africa together with predictive surveillance, can abort impending outbreaks, and lead to long range control of this important disease. vee is a mosquito-borne single strand, positive-sense, enveloped rna virus belonging to the alphavirus genus, family togaviridae. other medically important members of the alphavirus genus include eastern and western equine encephalitis viruses. there are vee virus subtypes identified by antigenic and genomic analyses, and a number of additional varieties. subtype iab and ic cause epizootic disease in equids and associated zoonotic infections of humans [ ] . during epizootics, horses and donkeys infected with these strains develop high viremias, serve as the primary hosts for infection of mosquito vectors and therefore are the indirect source of human infections acquired by mosquito bite. in contrast, the enzootic subtypes ii-vi, are maintained in nature in cycles involving rodent species and mosquitoes, are not amplified by equid viremic hosts, and cause sporadic illness in humans and equid dead-end hosts. epizootics of ic virus are the result of mutation and selection of virulent equine-competent viruses from enzootic strains, particularly the id variant [ ] . in the - s vee iab viruses caused large epizootics in south america, with associated human epidemics of encephalitis. between and , a series of major subtype iab and ic epizootics occurred in northern south america, and between and the virus spread north to central america, mexico, and texas [ ] . the cumulative economic and medical impact of vee outbreaks between and was devastating, with over , equid and , human cases. some of the vee iab epizootics are believed to have been spawned by the injection of horses with inactivated veterinary vee vaccines containing residual live virus [ ] . this likely occurred in trinidad in and again in nicaragua in , but probably was a widespread problem in the past. between and , vee ic re-emerged in venezuela and colombia, with an estimated equid deaths and over , human cases of which had encephalitis [ , ] . since vee causes an acute incapacitating illness in humans and the virus efficiently infects via the aerosol route, it was developed by both the us and soviet union as an offensive biological weapon [ ] . as part of these programs, vaccines for the protection of military personnel were also developed. in the us, a live, attenuated virus (tc- ) was developed by the us army medical research & development command (usamrdc) by empirical passages of the prototype trinidad donkey (subtype iab) virus in fetal guinea pig heart cell culture [ ] . the development of the live vaccine followed poor experiences with chemically inactivated vaccines; in animal models, only the live vaccine protected against aerosol challenge. however, tc- vaccine has a number of drawbacks as a human vaccine, including failure to immunize about % of vaccinees, and moderate-to-severe reactogenicity in about % of subjects. in humans, it remains an investigational product, used solely for the protection of laboratory workers [ ] , with approximately persons vaccinated since . the tc- virus acquired mutations during the empirical passage series in guinea pig heart cells, but attenuation appears linked to only of these, in the -noncoding region and the e envelope glycoprotein, and these substitutions appear to be subject to reversion in the vaccinated host [ ] . in addition, tc- has been isolated from mosquito vectors during field use, illustrating the potential for secondary spread and mutation and recombination events. an investigational formalin-inactivated tc- vaccine (designated c- ) was also developed by usamrdc and used following tc- priming to seroconvert tc- non-responders. since horses and related species are severely affected during epizootics and are the source of mosquito vectors infecting humans, there is an obvious need for a single dose veterinary vaccine that evokes rapid immunity. us army investigators explored the use of tc- live, attenuated human vaccine for immunization of equids beginning in [ ] , and there was limited field use of the vaccine in colombia in . however, when epizootic vee appeared for the first time in central america (guatemala) in may , and then spread southwards to costa rica and northwards to the us, there was considerable urgency to utilize a vaccine strategy for control of the disease in horses, donkeys and mules. in , the us military responded rapidly to requests for tc- vaccine from guatemala and el salvador. the vaccine had been produced and stockpiled at the merrell national laboratories, swiftwater pa under contract to the usamrdc for the purposes of biological defense. by , over million doses of tc- had been given to equidae in the us, mexico and central america [ ] . collaborative studies were also undertaken by agencies concerned with human and animal health (usamrdc, nih and usda) to fully explore the biology of the vaccine in horses [ , ] , ultimately leading to licensure and commercialized by the animal health industry, both as a live vaccine and then an inactivated vaccine combo with eastern and western equine encephalitis vaccines. tc- vaccine was credited with a rapid curtailment of the - outbreak. the history of vee exemplifies many one health principles, including the prevention of human cases through domesticated animal vaccination, use of a single vaccine product for animals and humans, and an agency (the us army) concerned with human health engaged in both veterinary and human vaccine development, and providing a solution for curtailing an emerging zoonosis. after the large epizootic in the s, tc- vaccine was again deployed during the epidemic in in colombia to create an immune barrier to spread of the virus. recent efforts have focused on development of improved vee vaccines for humans that are less reactogenic and more immunogenic than tc- , can be manufactured in a more acceptable substrate, and have a lower risk of reversion to virulence and of mosquito transmission [ ] . in addition, vaccines that crossprotect against the enzootic vee subtypes are needed. vee id is endemic in colombia, peru, venezuela, and ecuador, and the ie subtype circulates in southern mexico. aguilar et al. [ ] postulated that disease caused by vee is confused clinically with dengue, and that, in endemic areas, up to % of dengue cases may actually be due to vee enzootic subtype viruses. subtype id poses the ever-present risk of mutational change to produce high viremia and epizootic transmission in equids, as happened in the s. v vaccine is a rationally designed vaccine from the epizootic subtype iab genome, with insertion of a pe cleavage-signal mutation combined with an e gene resuscitating mutation. v had a good safety profile and was immunogenic and protective in laboratory animals, including nonhuman primates [ ] . while retaining a degree of neurovirulence for suckling mice, v is not virulent when inoculated intracranially in juvenile monkeys [ ] . v has also been evaluated in horses [ ] . the vaccine was safe and highly immunogenic, with subcutaneous doses as low as plaque-forming units shown to protect horses against challenge with virulent subtype iab virus. unfortunately, v proved to be too reactogenic for humans in a phase trial [ ] , and thus development for both human and veterinary use has stopped. the v virus was subsequently formalin inactivated and has been investigated with adjuvants replacement for the c- vaccine. other live and live vector approaches to improved vee vaccines have been investigated only in laboratory animals, including a chimeric virus constructed from nonstructural genes of sindbis and the structural genes from vee [ ] , vee replicon vaccines, and vaccinia recombinants. none of these approaches have reached advanced development. it is only a matter of time before another vee outbreak emerges in tropical america, and there is a substantial risk of cross-border spread. the prospects for vaccine interventions have diminished with dwindling support for new vaccines and increased concerns for vaccine safety. most zoonotic diseases are maintained in transmission cycles involving wild mammals or birds. however, because of the difficulties in vaccinating specific host species, wildlife immunization as a means of preventing spread to domestic animals and humans has been applied in only a few diseases. some of the barriers to implementing wild animal vaccination include (i) involvement of multiple species in natural transmission cycles; (ii) safety concerns for non-target species; (iii) high reproductive rates and population turn-over; (iv) fastidious feeding behaviors and difficulty in designing effective baits; (v) difficult delivery due to very high or, conversely, very low population densities of the target species; (vi) environmental concerns, and release of genetically modified organisms; (vii) difficulty in designing an effective formulation for oral immunization; (viii) instability of a vaccine or vector under prevailing environmental conditions; and (ix) requirement for low unit cost and government funding for vaccine purchase and delivery. nevertheless, targeted immunization of wild animal reservoirs is a subject of considerable interest for future research, not only for control of infectious agents affecting domestic animals and humans but also for control of wildlife diseases. one example of the latter was the effort to develop a means of immunizing great apes affected by ebola virus in central africa with vaccines previously developed for human use. aside from rabies vaccines delivered in oral baits, which is well-established, wildlife vaccination has had limited success. two promising examples of early-stage vaccine applications are described below (lyme disease and mycobacterium bovis). in addition experimental immunization and protection of prairie dogs (cynomys ludovicianus) using a raccoon poxvirus recombinant oral bait vaccine [ ] , and ballistic vaccination of bison against b. abortus [ ] have been described. plague, a global but localized zoonotic disease with rodent wildlife reservoirs, would appear to be a target of particular interest for future research [ ] . there are many other possible targets for new framework iii vaccines, and future research in this field is encouraged. in the united states, lyme disease is the most common vectorborne disease and the th most common infectious disease overall. it is also a major and increasing public health problem in europe. approximately , cases are reported in the us annually, and the number has doubled in the last years [ ] . however, at a meeting in august, , the centers for disease control and prevention (cdc) reported that the annual incidence of infection is believed to be -fold higher, i.e. , cases. although lyme disease occurs across the country, the incidence is highest in the northeast and north central states. in the us, lyme disease is caused by the spirochete borrelia burgdorferi, which is amplified each spring and summer in a cycle principally involving ixodes scapularis ticks and field mice. mice are persistently infected and represent the reservoir of infection in nature [ ] . b. burgdorferi is passed transtadially to nymphal and adult ticks which infect humans and dogs; these species develop clinical disease but are dead-end hosts. the human disease is manifested by a protean syndrome, starting with a localized skin infection (erythema migrans), and progressing to a multisystem disease variably with lassitude, arthritis, carditis, meningitis and other neurological manifestations [ ] . because of the increasing incidence and geographic expansion of lyme disease, the high incidence of tick exposure, and the difficulty in recognizing and removing attached ticks due to their small size, difficult differential diagnosis, troublesome and potentially severe clinical manifestations and medical controversies over treatment and chronicity of the disease, lyme disease has emerged as a high priority for public health interventions [ ] . vaccination of humans would appear to be a logical and cost-effective means to prevent the disease [ ] , and veterinary vaccines for dogs are widely used and have proven to be modestly effective [ ] . however, whereas safe and highly effective vaccines for humans have been developed, none is available for distribution today. glaxo smithkline's lymerix ® vaccine was approved in , but withdrawn in by the company, principally for commercial reasons, a decision that is lamentable given the increasing incidence of the disease [ , , ] . a new lyme disease vaccine for humans active against both b. burgdorferi and species causing lyme disease in europe developed by baxter bioscience is now in phase ii development, but it is uncertain whether it will reach the market. nevertheless, these vaccines established critical immunological principles; the human vaccines are composed of recombinant ospa protein, the dog vaccines of both ospa and ospc, and work via antibody-mediated mechanisms. ospa is expressed by the borrelia spirochete in the midgut of infected ticks. since the tick vector only begins to transmit borrelia - h after initiating blood feeding, ospa specific antibodies imbibed in the blood meal of a vaccinated host kill the bacteria and block transmission [ , ] . if a similar ospa antibody response could be evoked in the natural reservoir hosts of b. burgdoferi (peromyscus spp. field mice), it may be possible to interrupt the transmission cycle and reduce the prevalence of infected nymphal and adult ticks responsible for human and canine infections. proof of concept was obtained in a field study where peromyscus leucopus mice were trapped and vaccinated by subcutaneous injection of ospa; a reduction in the prevalence of b. burgdorferi in nymphal ticks was seen in the following year [ ] . however, practical vaccine delivery and effective immunization of mice in the wild, requires a thermostable oral bait vaccine matched to the high population density and rapid population turnover of the reservoir hosts, the effects of which are not diluted by non-targeted species that play a role in b. burgdorferi transmission [ ] . two promising live oral vaccine approaches have been investigated in the laboratory: a bacterial (e. coli) vector [ , ] and a viral vector (vaccinia) [ , ] expressing ospa. the e. coli vector contained in an oral bait formulation and ingested multiple times elicited anti-ospa antibodies and protected laboratory and wild p. leucopus mice against needle and tick challenge. a -year field study of the oral bait vaccine, sponsored by cdc, has been performed and results are anticipated with interest. a company, us biologics inc., is engaged in bringing this vaccine to market. the vaccinia technology, which rests on the shoulders of the successful oral bait vaccine against wildlife rabies (see below), has been tested in the laboratory. laboratory mice immunized by gavage with vaccinia expressing ospa were successfully immunized after a single dose and were protected against tick challenge. peromyscus consuming oral bait vaccine were also significantly protected against challenge with infected ticks. although the vaccinia vector looks promising, no commercial endeavor has yet emerged to support development. both the e. coli and vaccinia oral vaccines require specialized formulations in baits that incorporate the vaccine in the bait itself, rather than in a liquid sachet embedded in the bait used for delivery of rabies vaccines. many questions surround the application of an oral bait vaccine targeting the reservoir host, including efficacy of this approach in the field, the high density of baits required, cost and sustainability of local and state funded programs aimed at distributing baits, and the role of species not targeted by the vaccine in lyme disease maintenance cycles. if only partially effective, the risk of acquiring lyme disease may be reduced, but the public would still need to take precautions against tick bite. nevertheless, given the lack of a vaccine for humans, the high level of public concern about lyme disease, the high risk to children, the localized nature of b. burgdorferi transmission allowing geospatially focused control efforts, and the possibility that homeowners may be motivated to play an active role in distributing baits, the idea has appeal. m. bovis is the cause of tuberculosis in a wide array of domesticated and wild animals, and it remains a major veterinary health problem worldwide, causing severe economic losses from livestock disease, death and export restrictions. humans become infected by ingesting raw milk or undercooked meat, or by the aerosol route from infected animals or humans. in developed countries where pasteurization and test-and-slaughter programs have controlled the disease, zoonotic infections are relatively rare, accounting for . - . % of tuberculosis cases [ ] [ ] [ ] ; in developing countries which do not practice these measures, it remains more common, although few data on prevalence exist. wild animals are a major source of infection of domestic livestock [ ] . control measures aimed at control of m. bovis by culling wildlife reservoirs is problematic, with inconsistent results and ethical concerns. vaccination of wildlife is an attractive alternative control measure, especially since the traditional tuberculosis vaccine (bacille calmette-guerin, bcg) derived from m. bovis is effective when orally administered [ ] . examples of wildlife that serve as maintenance hosts of m. bovis and sources of infection in livestock, include white-tail deer in the us [ ] ; wild boar, red and fallow deer in europe [ ] ; badgers in the united kingdom [ ] ; african buffalo (syncerus caffer) in south africa [ ] ; and brushtail possums (trichosurus vulpecula) in new zealand [ ] . brushtail possums have been experimentally vaccinated using oral bcg and shown to be resistant to challenge with m. bovis [ ] . proof of concept has been provided by a field study in an endemic area of new zealand. possums were trapped, manually vaccinated using orally delivered bcg in a lipid matrix formulation, and vaccinated and control animals were recaptured at intervals [ ] . vaccinated animals received - vaccinations during the -year study. at the end of study, the ha study area was depopulated, and all animals assessed for clinical and subclinical m. bovis infections. vaccine efficacy against naturally acquired tuberculosis was - %. the authors concluded that oral vaccination of possums could be a practical strategy contributing to elimination of m. bovis in livestock. although the field study did not demonstrate control via freely consumed bait vaccine, captive possums have been shown to consume vaccine in flavored baits [ ] . rabies is transmitted between specific wild carnivore reservoir hosts, which serve as a source of spill-over infections of other wild carnivores, and infection of domesticated animals and humans. oral rabies vaccine was initially deployed in europe for control of rabies in the red fox (vulpes vulpes), using modified live virus vaccine [ , ] . the concept began in the s with the work of george baer at the cdc, which showed that foxes could be orally immunized with modified live virus [ ] . the live, attenuated evelyn-rokitnicki-abelseth (era) vaccine or street-alabama-dufferin (sad) viruses were employed in experimental and field studies. numerous studies in the s confirmed that captive and wild foxes could be orally immunized with a variety of baits containing vaccine [ ] . in , steck and colleagues initiated a wild fox rabies control program in the swiss alps using oral bait vaccine consisting of chicken heads with vaccine and tetracycline biomarker in a container made of polyvinyl chloride and aluminum foil inserted under the scalp [ ] . the trial demonstrated that % of foxes had ingested bait. over the next years, successful fox rabies control programs were carried out in many european countries, after the late s using baits distributed by fixed wing aircraft and helicopters rather than by ground [ ] , and resulting in elimination of terrestrial rabies in several countries [ , ] . for large scale distribution, the laborious chicken head method bait gave way to commercially manufactured molded or extruded baits of various kinds, consisting of fish meal or bone meal, fat, and a pouch or blister containing liquid vaccine virus [ ] . the vaccines currently used in europe are ( ) sag (e.g., rabigen ® , virbac laboratories, france), a modified live attenuated rabies virus derived from the original sad vaccine and having an additional mutation in the codon for amino acid of the rabies g protein, which increases genetic stability of the virus [ ] ; and ( ) recombinant vaccinia virus (copenhagen strain) expressing the era ® strain rabies g protein (raboral ® , merial corp.) [ ] . the rabies g protein gene has been inserted into the thymidine kinase gene of vaccinia, which results in further attenuation compared to the parental virus [ , ] . duration of oral rabies immunity, at least months in adult red foxes, is sufficient to provide herd immunity and reduce the reproductive rate (r ) to less than . the modified live virus vaccines are more thermolabile than vaccinia, require − • c storage, retain some pathogenicity for non-target species, and pose safety risks to humans exposed inadvertently. consequently, recombinant vaccinia is the only oral rabies vaccine approved for wildlife immunization in the us. this vaccine consists of fishmeal and fish oil bound by a polymer (ethylene vinyl acetate) and containing the vaccine in a plastic pouch held in place by a wax mixture. immunization with vaccinia or modified live virus occurs in the buccal mucosa and tonsils, and vaccines are poorly effective after ingestion [ ] . in one study, consumption by red foxes of a single bait containing vaccinia resulted in protection against virulent rabies in only half of the animals [ ] . this observation suggests that high bait densities and repeated vaccinations are important to effective control in the wild. bait densities distributed in europe generally range between and baits/km , resulting in - % of animals potentially immunized (positive for the tetracycline biomarker) [ ] . in addition to distribution density, feeding habits may also be important, since animals have been observed to pick apart baits and consume only the bait portion. while control of terrestrial wildlife rabies has been successful in parts of europe, it has been more challenging in other areas due to the diversity of carnivores involved in transmission of different rabies virus variants. in the arctic regions, a specific rabies variant is maintained in the arctic fox, with spill-over infections of red foxes, skunks, and raccoon dogs. where vaccination is not practiced in domesticated sledge dogs, these animals may be severely impacted by contacts with rabid foxes, and human dog owners placed at considerable risk. while experimental oral vaccination of arctic foxes has been successful, there is limited experience in the field [ ] . in ontario, canada control of arctic rabies variant in red foxes using oral bait vaccine has been successful, but the virus still occurs as a result of spill-over transmission to skunks, which are not efficiently immunized with recombinant vaccinia vaccine [ , ] . oral bait recombinant vaccinia vaccine has been primarily used to control raccoon rabies, which expanded beginning in the mid- s from enzootic areas in florida northwards and westwards to involve many states in the eastern us, as well as new brunswick and quebec, canada [ , ] . the control program relies on distribution of vaccine baits specific zones of rabies activity, particularly along the appalachian ridge, enhanced surveillance and ring vaccination with evidence of spread of the disease. judged from the absence of spread beyond the zones of vaccine distribution, the program has worked well, despite relatively low prevalence of rabies antibody (approximately %) in sampled raccoons [ ] . it is possible that pre-existing immunity to raccoonpox virus may interfere with immunization with vaccinia [ ] . the economics of large-scale oral vaccination programs in the us have been modeled and are generally favorable [ ] . in addition to control of raccoon rabies, successful use of the vaccine has been made in the control of gray fox (urocyon cinereoargenteus) variant rabies in west texas [ ] . in contrast to raccoons, a higher prevalence of rabies antibody ( %) attributed to vaccination is found in gray foxes. rabies in coyotes (canis latrans) was responsible for epizootic canine rabies in the s and s in parts of the us, and was controlled by an oral bait vaccination campaign, contributing to the elimination of canine rabies by [ ] . skunks remain a problematic species for vaccine control of rabies. skunks are an important spill-over host for the arctic fox, raccoon rabies, and big brown bat rabies virus variants [ ] . although the vaccinia vector vaccine is not sufficiently effective in skunks [ ] , promising results were obtained with a replicationcompetent adenovirus type vector expressing the rabies g protein [ ] . aerial distribution of this vaccine (onrab ® , artemis technologies, guelph) showed high rates of immunization of raccoons, and arctic foxes and modest seroconversion ( - % in different plots) in skunks, probably due to lower rates of bait acceptance [ , ] . onrab ® is approved by the canadian regulatory authorities for control of rabies in skunks, raccoons, and foxes, and is under investigation in the us. there are some notable failures of animal vaccination as a means to preventing zoonotic diseases of humans, and these illustrate some of the limitations of the approach shown in table . japanese encephalitis, a mosquito-borne flavivirus closely related to west nile virus, is endemic in asia, with nearly billion people at risk of infection [ ] . horses and humans are dead-end hosts, and framework i immunization of both is widely practiced in many parts of asia, with a long record of success. pigs are an important domesticated animal amplifying host for infection of rice paddy-breeding culex spp. vectors, resulting in spill-over infections of humans. moreover, infection of pregnant sows can lead to abortion and stillbirth, and infected boars may have reduced spermatogenesis and infertility [ ] . framework ii immunization of swine was previously a major initiative in japan, using live, attenuated vaccines. however, it was exceedingly difficult to vaccinate piglets born in spring and early summer during the narrow window between loss of interfering maternal antibody and contribution to virus amplification. the practice of vaccination was abandoned in favor of re-locating piggeries from areas of culex breeding and biting activity. elsewhere in asia, other obstacles precluded consideration of framework ii vaccination against japanese encephalitis, including the prevalence of small piggeries located near vector breeding sites and of feral swine, and the importance of wild ardeid birds and waterfowl in virus transmission. moreover in many areas of asia, less developed than japan, and undergoing rapid urbanization, the locations of pig holdings are not controlled and are often located near rice paddies and urban centers [ ] . consequently, the focus has long been on human vaccination as the primary means of prevention. this case study exemplifies some of the factors that can limit application of framework ii vaccination: ( ) the need to customize vaccination to the breeding and husbandry practices and timing of domesticated animal targets; ( ) the role of wild animals and feral domesticated animals as additional amplifying hosts in the transmission cycle; and ( ) difficult access given the very large scale and geographic complexity of domesticated animal populations. q fever is caused by the intracellular gram-negative bacterium, coxiellla burnetii and an important worldwide infection of ruminants, which serve as the source of infection of humans, especially where large numbers of animals are concentrated [ ] . q fever is a major occupational hazard of abattoir and farm workers, veterinarians, and persons involved in the handling and distribution of animals or animal products. a dramatic outbreak recently occurred in high-intensity goat farms in the netherlands ( ), with human cases and deaths [ ] . transmission of c. burnetii occurs between direct spread between domesticated animals, and from animals to humans. infected animals shed bacteria in urine, feces, vaginal secretions and products of conception, and in milk. ticks are also a reservoir of bacteria in nature and a source of infection of livestock. ruminants, particularly goats and sheep develop pneumonia, abortion, stillbirth, premature delivery, and delivery of weak offspring, and herds can be affected for prolonged periods, causing significant economic losses [ ] . there are two developmental stages of c. burnetii, a small-cell variant (scv, the extracellular form) and the metabolically active intracellular large-cell variant. the scv is highly resistant to degradation and can persist in the environment for long periods of time. infection of humans is acquired principally by the aerosol route via dust containing spore-like scv forms, with oral routes of infection (ingestion of unpasteurized milk) being secondary. the human disease is characterized by an influenza-like illness, and may be complicated by pneumonia, endocarditis, and (pregnant women) abortion and fetal death. person-to-person transmission occurs, but is rare. approximately % of infected persons may develop chronic infections, with various manifestations. prophylactic vaccines have played a role in the control of q fever in livestock, but the practice is not a reliable means of preventing human infections. in russia, a live, attenuated m- vaccine was used for many years in animals and humans, but causes a persistent infection and has not been considered safe for use elsewhere. in europe, coxevac ® , a formalin inactivated strain rsa /nine-mile phase bacterial form (smooth forms, with complete surface lipopolysaccharide) has been used in goats and cattle and reduced the incidence of shedding, but is not effective in pregnant or chronically infected animals [ , ] . the live and phase vaccines are also not diva, which presents substantial issues for export controls. in australia, an inactivated phase vaccine prepared from henzereling strain is marketed for humans by csl ltd (qvax ® ) [ ] . however, severe reactions occur in persons who have previously been naturally infected with c. burnetii, and skin testing to ensure absence of exposure is required [ ] . clearly, improved vaccines are needed for both livestock and humans, but various attempts at recombinant vaccines have been disappointing. the problem for framework ii vaccination against q fever is due to multiple factors, including imperfect vaccines, limited efficacy of vaccines in parous animals, the difficulty in recognizing the disease and intervening expeditiously, and the rapid and widespread contamination of the environment with scv forms. the last problem is the major reason that livestock vaccination is not a reliable means of protecting humans against exposure. a year study of sheep vaccinated with the phase vaccine showed that the proportion of animals shedding bacteria in feces was markedly reduced after year and then eliminated after years, but c. burnetii was still present in environmental samples [ ] . finally, live veterinary vaccines may pose a risk of inadvertent infection of humans handling the vaccine or vaccinated animals. examples include live brucella and orf (contagious ecthyma) vaccines. while this topic is beyond the scope of this review, it is important in several contexts, and indeed little is known about the risk of human pathogens to animals. immunization of swine and poultry workers against influenza as a means of preventing introduction of human influenza viruses to these animals has been emphasized as a means of preventing emergence of reassortant strains [ ] . immunization of humans to prevent spread of viruses to captive nonhuman primates is often practiced, including vaccines for influenza, hepatitis a and b, and measles. prevention of animal diseases and human diseases by use of vaccines is a well-established principle, and there are potential synergies that can be achieved in concurrent delivery of human and animal vaccines in developing country settings [ ] . for some diseases affecting both livestock and humans there is a clear commercial incentive to develop vaccine products (framework i vaccines) ( table ) . however, such development efforts are generally segregated in the animal and human health divisions in industry and academia, and have separate regulatory pathways. this results in a potential waste of resources and duplicated scientific endeavors. interestingly, when one company (akso nobel), an animal health company, decided in on a strategic move into human vaccine development, it drew on its veterinary scientists to staff the program. as pointed out in this review, there have been isolated successful examples, e.g. a west nile vaccine, of co-development of a vaccine for both veterinary and human indications, an obviously efficient strategy that broadened both the commercial and public health opportunity. future efforts along similar lines should be considered on a case by case basis, depending on medical need, but in general there is value in closer connections between human and veterinary vaccines and regulatory science, and in the application of domesticated animals as models for development of infectious disease and cancer vaccines. several issues related to diva requirements and liability concerns have been mentioned. prevention of zoonotic diseases of humans by means of vaccination of domesticated (framework ii) or wild (framework iii) animals is an attractive but under-exploited concept. an obstacle is that there may be limited commercial incentives (table ) . where a market exists, governments may be the principal customers, as is the case for the approved oral bait rabies vaccines and the reservoirtargeted lyme disease vaccine in development. thus, the public health gains for such an intervention need to be compelling and must offset the cost of development and implementation. the goal is far easier to justify when vaccination also prevents disease in an economically valuable animal species, there is a profit incentive for animal vaccination or a clear social gain from improved animal health, and when the public health spin-off is an added benefit. examples of the latter may include vaccines against hendra and nipah virus diseases, brucellosis, chlamydophila felis, rift valley fever, and venezuelan equine encephalitis. the potential for elimination of a disease through vaccination (employed together with other strategies), as has been demonstrated for brucellosis in the us and terrestrial rabies in some european countries is a compelling economic concept, though applicable in only selected cases. the cost of preventative programs is almost always lower than emergency response programs, as illustrated by the significantly lower cost of oral wildlife rabies programs over contingency actions to control epizootic spread [ ] . the recent announcement of a fold higher incidence of lyme disease than previously believed will lead to a reassessment of the economics of preventive strategies for this disease, including wildlife vaccines. economics represent the key determinant for development and utilization of framework ii and iii vaccine strategies. a low unit cost of such vaccines will always be a requirement. the economic barriers are particularly relevant when considering vaccines for developing countries. on the positive side, the cost of developing a new animal vaccine through licensure is a small fraction, approximately %, of the cost of a typical human vaccine (the latter being $ - million by one estimate [ ] , but often far higher) [ ] . the relatively lower cost and shorter timelines for developing animal vaccines reflects the simpler path to regulatory approval, and is driven by the significantly lower market potential for these vaccines. despite the lower cost of developing new veterinary vaccines, high and middle income countries still pay higher prices until the fixed costs of development are paid off, there is an over-supply of vaccine, or competing products enter the market. to redress the pricing barriers in the case of human vaccines, there has been strong advocacy for new approaches to secure vaccine supply and access for developing countries where the burden of infectious diseases is greatest [ ] . as part of this strategy, emerging market manufacturers provide access to low unit cost vaccines, and such manufacturers of veterinary vaccines could play a substantial role in a public health strategy for animal immunization. indeed all of the principles being applied to human vaccines could be extended to vaccines for animals, particularly if there is both a clear rationale for public health and the "pull" of a potentially expanded market or of guaranteed purchase agreements. up to now public-private financing for developing and distributing veterinary vaccines has represented a tiny fraction of support available for human developing-world vaccines, and has focused on vaccines for livestock as a means of improving animal production and protein supply rather than preventing zoonotic diseases [ ] . given the public health impact of zoonotic diseases described in the introduction to this review and the potentially lower cost of interventions targeting animals vs. humans, there should be a new emphasis on the public health improvements that could result from animal immunization. zoonotic diseases that merit consideration because they occur at high incidence or are poorly controlled, include rabies, zoonotic leishmaniasis, brucellosis, rift valley fever, m. bovis, lyme disease, and several enteric bacterial infections. as mentioned above, the regulatory pathway for animal vaccines is considerably simpler than for human vaccines [ ] . this is due to multiple factors, including less onerous requirements for manufacturing and control of veterinary products, the simpler and far less expensive clinical trial requirements for marketing approval, the ability to challenge animals to demonstrate vaccine efficacy, as well as an established regulatory mechanism for conditional approval allowing commercial sales while still gathering more definitive data. there is no requirement for large, statistically powered efficacy field trials to obtain marketing approval, as is the case for human vaccines. the development of veterinary vaccines is consequently far faster than for human vaccines. for example, west nile vaccine for horses was commercialized years after introduction of the virus into the u.s., whereas the first (phase ) human trial of a west nile vaccine was completed years after introduction. the lower costs and accelerated timeframe for development of animal vaccines represent an important rationale for novel investments in public health, particularly in developing countries. to justify investments in a framework ii or iii vaccine where an economic incentive for animal immunization is marginal and a public health benefit is a goal, it is important to plan for vaccine effectiveness studies showing that vaccination of animals actually reduces human disease prevalence, as was postulated in greece following vaccination against b. melitensis [ ] . such demonstrations would really drive the field forward. thus, while the deployment of tc- venezuelan equine encephalitis vaccine was credited with the curtailment of the human epidemic in - , no controlled study to demonstrate an effect on human disease incidence was actually performed. indeed, studies to confirm the attractive hypothesis that immunization of domesticated ruminants against rift valley fever in africa would prevent intermittent outbreaks of the disease in animals and humans, while leaving the mosquito reservoir of infection intact, would be difficult to design and carry out. because of its discrete epidemiology, hendra virus (albeit a low-incidence disease) presents a unique opportunity to demonstrate the effectiveness of animal immunization on the occurrence of a disease in humans. the increasing problem of emerging infections, the majority of which are the result of spill-over from animals to humans, is a compelling reason to consider novel vaccine interventions, and the collaborations between veterinary and human health institutions in the development of the hendra, west nile, vee and rift valley fever vaccines described in this review serve as examples of the power of this approach. other potential targeted vaccine interventions focused on animal reservoirs or intermediate hosts in order to control disease emergences include avian influenza, nipah virus disease, and, possibly, middle east respiratory syndrome. funding agencies and industry should be encouraged to seek integrated approaches to prevention of zoonotic diseases. the ultimate success of examples provided in this review, such as e. coli o vaccines for cattle, reservoir targeted lyme disease vaccines for field mice, and rift valley fever vaccines for livestock will require sustained efforts utilizing the one health paradigm. human-animal medicine. clinical approaches to zoonoses, toxicants, and other shared risks host range and emerging and reemerging pathogens the human/animal interface: emergence and resurgence of zoonotic infectious diseases monath tp, editors. one health-one medicine: linking human, animal, and environmental health the economics of one health research as a part of public health emergency response the multiple burdens of zoonotic disease and an ecohealth approach to their assessment health: a new professional imperative: avma one health initiative task force report fda experience with medical countermeasures under the animal rule epidemiology of west nile in europe and in the mediterranean basin west nile virus and its emergence in the united states of america west nile virus neuroinvasive disease incidence in the united states west nile virus: epidemiology and clinical features of an emerging epidemic in the united states long-term prognosis for clinical west nile virus infection a case-control study of factors associated with development of clinical disease due to west nile virus immunogenicity, genetic stability and protective efficacy of a recombinant, chimeric yellow fever-japanese encephalitis virus (chimerivax tm -je) as a live, attenuated vaccine candidate against japanese encephalitis chimaeric live, attenuated vaccine (chimerivax tm ) incorporating the envelope genes of japanese encephalitis (sa - - ) virus and the capsid and nonstructural genes of yellow fever ( d) virus is safe, immunogenic and protective in non-human primates yellow fever vector live-virus vaccines: west nile vaccine development chimerivax tm -west nile live-attenuated vaccine: preclinical evaluation of safety, immunogenicity and efficacy west nile vaccine efficacy of a live attenuated chimeric west nile virus vaccine in horses against clinical disease following challenge with virulent west nile virus, abstr. . in: suppl. proc. rd annu. meet comparative efficacies of three commercially available vaccines against west nile (wnv) in a short-duration challenge trial involving an equine wnv encephalitis model a live, attenuated recombinant vaccine against west nile virus differentiation of west nile virus-infected animals from vaccinated animals by competitive elisa using monoclonal antibodies against non-structural protein brucellosis: an overview the new global map of human brucellosis towards a brucella vaccine for humans clinical manifestations of human brucellosis: a systematic review and meta-analysis human health benefits from livestock vaccination for brucellosis: case study brucellosis: the case for live, attenuated vaccines rough vaccines in animal brucellosis: structural and genetic basis and present status estimating herd prevalence of bovine brucellosis in us states using slaughter surveillance vaccination strategies for managing brucellosis in yellowstone basin progress in brucella vaccine development incidence of human brucellosis in a rural area in western greece after the implementation of a vaccination programme against animal brucellosis invasive escherichia coli vaccines expressing brucella melitensis outer membrane proteins or or periplasmic protein bp confer protection in mice challenged with b. melitensis a combined dna vaccine provides protective immunity against mycobacterium bovis and brucella abortus in cattle efficacy of calfhood vaccination with brucella abortus strain rb in protecting bison against brucellosis risk factors for sporadic shiga toxin-producing escherichia coli o infections in foodnet sites escherichia coli o prevalence and enumeration of aerobic bacteria, enterobacteriaceae, and escherichia coli o at various steps in commercial beef processing plants animal-and truckload-level associations between escherichia coli o :h in feces and on hides at harvest and contamination of preevisceration beef carcasses bioniche food safety inc. econiche ® escherichia coli o bacterial extract vaccine use of a siderophore receptor and porin proteins-based vaccine to control the burden of escherichia coli o :h in feedlot cattle effects of a siderophore receptor and porin proteins-based vaccination on fecal shedding of esherichia coli o :h in experimentally inoculated cattle an outcomes model to evaluate risks and benefits of escherichia coli vaccination in beef cattle bartonella spp. in pets and effect on human health one health: the importance of companion animal vector-borne diseases arthropod-borne infectious diseases of the dog and cat zoonoses control in dogs how to eradicate canine rabies: a perspective of historical efforts mass vaccination campaign against rabies: are dogs correctly protected? the peruvian experience human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges re-evaluating the burden of rabies in africa and asia field evaluation of a dog owner, participation-based, bait delivery system for the oral immunization of dogs against rabies in tunesia world health organization. field application of oral rabies vaccines for dogs. geneva: world health organization field trial with oral vaccination of dogs against rabies in the philippines oral vaccination of foxes against rabies with sad b in europe, - : a review oral vaccination of dogs with recombinant rabies virus vaccines one health and hendra virus: a collaborative approach in action hendra and nipah viruses: different and dangerous pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission henipaviruses: emerging paramyxoviruses associated with fruit bats henipavirus: a review of laboratory animal pathology hendra vaccine success announced unexpected result of hendra virus outbreaks for veterinarians receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble g glycoprotein of hendra virus a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge a hendra virus g glycoprotein subunit vaccine protects african green monkeys from nipah virus challenge rift valley fever severe rift valley fever may present with a characteristic clinical syndrome rift valley fever epidemic in saudi arabia: epidemiological, clinical, and laboratory characteristics rift valley fever virus (bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention rift valley fever virus (family bunyaviridae, genus phlebovirus). isolations from diptera collected during an inter-epizootic period in kenya prediction of a rift valley fever outbreak breaking the chain: rift valley fever virus control via livestock vaccination rift valley fever: the neurotropic adaptation of the virus and the experimental use of this modified virus as a vaccine adverse response of nonindigenous cattle of european breeds to live attenuated smithburn rift valley fever vaccine an inactivated rift valley fever vaccine world health organization. the use of veterinary vaccines for prevention and control of rift valley fever: memorandum from a who/fao meeting zh -vsvri: is it still the best choice for vaccination against rift valley fever in egypt? immunogenicity of an inactivated rift valley fever vaccine in humans: a -year experience active and passive immunization against rift valley fever virus infection in syrian hamsters mutagen-directed attenuation of rift valley fever virus as a method for vaccine development use of reassortant viruses to map attenuating and temperature-sensitive mutations of the rift valley fever virus mp- vaccine mapping of the mutations present in the genome of the rift valley fever virus attenuated mp strain and their putative role in attenuation factors in the emergence of arbovirus diseases safety and efficacy of a mutagen-attenuated rift valley fever virus vaccine in cattle pathogenicity and immunogenicity of a mutagen-attenuated rift valley fever virus immunogen in pregnant ewes teratogenicity of a mutagenised rift valley fever virus (mvp ) in sheep rescue of infectious rift valley fever virus entirely from cdna, analysis of virus lacking nss gene, and expression of a foreign gene rift valley fever virus lacking the nss and nsm genes is highly attenuated, confers protective immunity from virulent virus challenge, and allows for differential identification of infected and vaccinated animals safety and immunogenicity of recombinant rift valley fever mp- vaccine candidates in sheep characterization of clone , a naturally attenuated avirulent isolate of rift valley fever virus, which is altered in the small segment rift valley fever evaluation of the efficacy and safety of the rift valley fever clone vaccine in sheep protective immune responses induced by different recombinant vaccine regimes to rift valley fever an alphavirus replicon-derived candidate vaccine against rift valley fever virus rift valley fever virus immunity pro-vided by a paramyxovirus vaccine vector novel approaches to develop rift valley fever vaccines rift valley fever: recent insights into pathogenesis and prevention vaccination with virus like particles protects mice from lethal infection of rift valley fever virus priming with dna plasmids encoding the nucleocapsid protein and glycoprotein precursors from rift valley fever virus accelerates the immune responses induced by an attenuated vaccine in sheep endemic venezuelan equine encephalitis in the americas: hidden under the dengue umbrella venezuelan encephalitis emergence mediated by a phylogenetically predicted viral mutation the health and economic impact of venezuelaqn equine encephalitis (vee) inactivated and live vee vaccines-a review epidemic venezuelan equine encephalitis in la guajira, colombia re-emergence of epidemic venezuelan equine encephalomyelitis in south america. vee study group medical aspects of chemical and biological warfare. textbook of military medicine, part i attenuation of venezuelan equine encephalomyelitis virus by in vitro cultivation in guinea pig heart cells long-term duration of detectable neutralizing antibodies after administration of live-attenuated vee vaccine and following booster vaccination with inactivated vee vaccine attenuation of venezuelan equine encephalitis virus strain tc- is encoded by the -noncoding region and the e envelope glycoprotein immunization of burros with living venezuelan equine encephalitis virus safety and efficacy of an attenuated vee vaccine for use in equidae field studies of an attenuated venezuelan equine encephalomyelitis vaccine (strain tc- ) vaccines for venezuelan equine encephalitis genetically engineered, live attenuated vaccines for venezuelan equine encephalitis: testing in animal models neurovirulence evaluation of venezuelan equine encephalitis (vee) vaccine candidate v in nonhuman primates venezuelan equine encephalitis virus vaccine candidate (v ) safety, immunogenicity and efficacy in horses evaluation of formalin inactivated v virus with adjuvant as a next generation vaccine candidate for venezuelan equine encephalitis virus recombinant sindbis/venezuelan equine encephalitis virus is highly attenuated and immunogenic protection of black-tailed prairie dogs (cynomys ludovicianus) against plague after voluntary consumption of baits containing recombinant raccoon poxvirus vaccine immune responses of bison to ballistic or hand vaccination with brucella abortus strain rb a baiting system for delivery of an oral plague vaccine to black-tailed prairie dogs vaccination against lyme disease of ticks, mice and men: understanding the dual-host lifestyle of lyme disease spirochaetes the emergence of lyme disease vaccines against lyme disease: what happened and what lessons can we learn? the cost effectiveness of vaccinating against lyme disease safety, efficacy, and immunogenicity of a recombinant osp subunit canine lyme disease vaccine prevention of lyme disease: a review of the evidence correcting a public health fiasco: the need for a new vaccine against lyme disease elimination of borrelia burgdorferi from vector ticks feeding on ospa immunized mice borrelia burgdorferi ospa is an arthropod-specific transmission-blocking lyme disease vaccine an ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the lyme disease cycle oral vaccine that breaks the transmission cycle of the lyme disease spirochete can be delivered via bait reservoir targeted vaccine for lyme borreliosis induces a yearlong, neutralizing antibody response to ospa in white-footed mice protective efficacy of an oral vaccine to reduce carriage of borrelia burgdorferi (strain n ) in mouse and tick reservoirs development of a baited oral vaccine for use in reservoir-targeted strategies against lyme disease human mycobacterium bovis infection in the united kingdom: incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis epidemiology of mycobacterium bovis disease in humans, the netherlands zoonotic tuberculosis due to mycobacterium bovis in developing countries classification of worldwide bovine tuberculosis risk factors in cattle: a stratified approach bcg moreau rio de janeiro-an oral vaccine against tuberculosis-review descriptive epidemiology of bovine tuberculosis in michigan ( - ): lessons learned risk factors associated with the prevalence of tuberculosis-like lesions in fenced wild boar and red deer in south central spain comparing badger (meles meles) management strategies for reducing tuberculosis incidence in cattle the epidemiology of tuberculosis in freeranging african buffalo (syncerus caffer) in the kruger national park, south africa directions and issues in bovine tuberculosis epidemiology and control in new zealand a new attenuated mycobacterium bovis vaccine protects brushtail possums (trichosurus vulpecula) against experimental tuberculosis infection oral vaccination reduces the incidence of tuberculosis in free-living brushtail possums lipid-formulated bcg as an oralbait vaccine for tuberculosis: vaccine stability, efficacy and palatability to new zealand possums (trichosurus vulpecula) epidemiology of fox rabies the use of commercially available vaccines for the oral vaccination of foxes against rabies oral immunization of foxes against rabies a review of the development of the oral vaccination technique for immunizing wildlife against rabies oral immunization of foxes against rabies. laboratory and field studies assessing anti-rabies baiting-what happens on the ground? efficacy of oral vaccination in the final stage of fox rabies elimination in switzerland a review of baits and bait delivery systems for free-ranging carnivores and ungulates vaccination against rabies: construction and characterization of sag , a double avirulent derivative of sadbern vaccination of wildlife against rabies: successful use of a vectored vaccine obtained by recombinant technology protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype the oral rabies immunization of foxes and dogs with sausage baits efficacy of three oral rabies vaccine baits in the red fox: a comparison arctic rabies -a review rabies challenge of captive striped skunks (mephitis mephitis) following oral administration of a live vaccinia-vectored rabies vaccine oral rabies vaccination in north america: opportunities, complexities, and challenges rabies surveillance in the united states during raccoon rabies: the re-emergence of an epizootic in a densely populated area potential effect of prior raccoonpox virus infection in raccoons on vaccinia-based rabies immunization tactics and economics of wildlife oral rabies vaccination, canada and the united states economic analysis of a large scale oral vaccination program to control raccoon rabies molecular inferences suggest multiple host shifts of rabies viruses from bats to mesocarnivores in arizona during human adenovirus type vectors expressing rabies glycoprotein aerial distribution of onrab baits as a tactic to control rabies in raccoons and striped skunks in ontario, canada high-density baiting with onrab ® rabies vaccine baits to control arctic-variant rabies in striped skunks in ontario effect of irrigated rice agriculture on japanese encephalitis, including challenges and opportunities for integrated vector management disorder of spermatogenesis and viral discharge into semen in boars infected with japanese encephalitis virus occurrence of japanese encephalitis virus mosquito vectors in relation to urban pig holdings q fever: current state of knowledge and perspectives of research of a neglected zoonosis the q fever epidemic in the netherlands: history, onset, response and reflection prévention de l'excrétion de coxiella burnetii à l'aide d'un vaccin phase i (coxevac en troupeaux bovines laitiers infectés) vaccine prophylaxis of q fever-a follow-up study of the efficacy of q-vax (csl) - prevention of coxiella bumetii infection: vaccines and guidelines for those at risk four-year evaluation of the effect of vaccination against coxiella burnetii on reduction of animal infection and environmental contamination in a naturally infected dairy sheep flock the importance of including swine and poultry workers in influenza vaccination programs human and animal vaccination delivery to remote nomadic families emergency response to raccoon rabies introduction into ontario how the research-based industry approaches vaccine development and establishes priorities current status of veterinary vaccines assembling a global vaccine development pipeline for infectious diseases in the developing world key: cord- -hksmt i authors: mclean, rebecca k.; graham, simon p. title: vaccine development for nipah virus infection in pigs date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: hksmt i nipah virus (niv) causes a severe and often fatal neurological disease in humans. whilst fruit bats are considered the natural reservoir, niv also infects pigs and may cause an unapparent or mild disease. direct pig-to-human transmission was responsible for the first and still most devastating niv outbreaks in malaysia and singapore in – , with nearly human cases and over fatalities. pigs can therefore play a key role in the epidemiology of niv by acting as an “amplifying” host. the outbreak in singapore ended with the prohibition of pig imports from malaysia and the malaysian outbreak was ended by culling % of the country's pig population with costs exceeding us$ million. despite the importance of niv as an emerging disease with the potential for pandemic, no vaccines, or therapeutics are currently approved for human or livestock use. in this mini-review, we will discuss current knowledge of niv infection in pigs; our ongoing work to develop a niv vaccine for use in pigs; and the pig as a model to support human vaccine development. nipah virus (niv) is an enveloped, single stranded, negative sense rna paramyxovirus, genus henipavirus. the natural hosts and wildlife reservoirs of niv are old world fruit bats of the genus pteropus ( ). both nipah and the related hendra virus possess a number of features that distinguish them from other paramyxoviruses. of particular note is their broad host range which is facilitated by the use of the evolutionary conserved ephrin-b and -b as cellular receptors ( ) . the niv attachment glycoprotein (g) is responsible for binding to ephrin-b /-b ( ). following receptor binding, the g protein dissociates from the fusion (f) protein. subsequently, the f protein undergoes a series of conformational changes which in turn initiates fusion of the viral and host membrane allowing entry ( ) . during viral replication, the f protein is synthesized and cleaved into fusion active f and f subunits. these subunits are subsequently transported back to the cell surface to be incorporated into budding virions, or facilitate fusion between infected and adjacent uninfected cells ( ) . this cell-to-cell fusion results in the formation of multinucleated cells called syncytia, and greatly influences the cyopathogenicity of niv as it allows spread of the virus, even in the absence of viral budding ( , ) . niv infection is currently classed as a stage iii zoonotic disease, meaning it can spill over to humans and cause limited outbreaks of person-to-person transmission ( , ) . niv outbreaks have been recognized yearly in bangladesh since as well as occasional outbreaks in neighboring india (figure ). these outbreaks have been characterized by person-to-person transmission figure | previous locations of henipavirus infection outbreaks. nipah and hendra virus distribution map highlighting the range of the natural wildlife reservoir, pteropus spp. bats [adapted from ( ) ]. and the death of over % of infected people ( , ) . in may , the first ever outbreak in southern india was reported. a total of niv cases, of which resulted in death, were reported in the state of kerala. pteropus giganteus bats from areas around the index case in kozhikode, kerala, were tested at the national high security animal diseases laboratory at bhopal. of these, % were found to be niv positive by rt-pcr ( ) . characteristics of niv that increase the risk of it becoming a global pandemic include: humans are already susceptible; many niv strains are capable of person-to-person transmission; and as an rna virus, niv has a high mutation rate ( ) . niv has been found to survive for up to days when subjected to various environmental conditions, including fruit bat urine and mango flesh ( ) . whilst survival time was influenced by fluctuations in both temperature and ph, the ability for niv to be spread by fomites could play a role in outbreak situations. the first and still most devastating niv outbreak occurred in peninsular malaysia from september to may ( , ) . the link to pigs in this outbreak was obvious as % of the infected patients had contact with pigs ( ) . if a niv strain were to become human-adapted and infect communities in southeast asia where there are high human and pig densities and pigs are a primary export commodity, infection could rapidly spread and humanity could face its most devastating pandemic ( , , ) . in september , there was an outbreak of severe febrile encephalitis among pig farmers in the state of perak, malaysia, that was associated with a high mortality rate. a total of cases of encephalitis, of which resulted in death, were confirmed. these deaths were initially thought to be due to japanese encephalitis (je), an endemic disease in malaysia. however, with most cases occurring in men who worked with pigs, the epidemiological characteristics of this disease were distinct from those of je, where ∼ % of cases occur in children aged - years ( ) ( ) ( ) . the epidemiological link was from fruit bats infecting pigs that then served as amplifier hosts, resulting in transmission to humans through close contact ( ) . as a result of movement of infected pigs and humans to other states in malaysia, by february similar diseases were recognized in both pigs and humans in new outbreak areas ( ). in the following month, there were cases of respiratory illness and encephalitis amongst singapore abattoir workers who had handled pigs imported from the outbreak areas in malaysia ( ) . due to this, the importation of pigs from malaysia ceased which in turn ended the outbreak in singapore. the outbreak in malaysia ended when . million pigs ( % of the country's pig population) were culled from outbreak and surrounding areas ( , ) . the niv outbreak incurred significant economic costs and long-term damage to the malaysian pig industry: us$ million in direct costs and lost market revenue, including us$ million in compensation to farmers for the . million pigs slaughtered and , jobs lost ( ) . to this date, malaysian pig farming is only permitted in "identified pig farming areas." pigs also suffered during the / malaysian outbreak, but this was only diagnosed as part of the investigation following the human cases. the severity of symptoms of niv infection in pigs varied with age. in suckling pigs (< weeks old), mortality could be high (up to %) and labored breathing and muscle tremors were evident. in growing pigs ( to months), an acute febrile (> . • c) illness was observed with respiratory signs ranging from increased or forced respiration to a harsh, loud non-productive cough, open mouth breathing, and epistaxis ( ) . in some cases these respiratory signs were accompanied by one or more of the following neurological signs: trembles, neuralgic twitches, muscle fasciculation, tetanic spasms, incoordination, rear leg weakness, or partial paralysis. pigs of this age had high morbidity and low mortality (< %) ( ) ( ) ( ) . some animals over months of age died rapidly (within h) without signs of clinical disease. respiratory signs were reported in adult pigs, as with younger animals, although these were less obvious (labored breathing, bloody nasal discharge, increased salivation) and neurological signs included head pressing, bar biting, tetanic spasms and convulsions. first trimester abortions were also reported ( ) ( ) ( ) . in an experimental infection study, pigs were inoculated subcutaneously with a niv isolate from the central nervous system of a fatally infected human patient. infection elicited respiratory and neurological symptoms consistent with those observed in naturally infected malaysian pigs, which included febrile illness, incoordination, mucosal nasal discharge, and persistent cough ( ) . pigs inoculated orally with the same dose did not show clinical signs although they still shed virus. in a second study, piglets were inoculated oronasally with a human niv isolate ( ) . all infected animals showed a transient increase in body temperature between and days post-infection. two of these animals developed transient respiratory signs, mild depression and a hunched stance. both these studies concluded that niv infection in pigs had no pathognomonic features i.e., the clinical signs observed were non-specific. this can make field diagnosis of niv infection in pigs difficult, as observed in the outbreak in malaysia ( , ) . the name proposed for the disease caused by niv infection of pigs was "porcine respiratory and neurological syndrome" (also known as "porcine respiratory and encephalitis syndrome"), or, in peninsular malaysia, "barking pig syndrome" ( ) . niv infection was included as the sixth pig disease notifiable to the oie world organization for animal health ( ) . the oie approve diagnostics and recommends preventative and control measures for a range of transboundary livestock diseases. despite the importance of niv as an emerging disease with the potential for pandemic, no therapeutics or vaccines are approved for use in humans or livestock species. due to the lethal nature of niv infection, producing a safe, live attenuated vaccine with no potential for reversion is difficult. however, recombinant niv mutants, attenuated in hamster and ferret models, have been shown to generate strong neutralizing antibody responses ( , ) . more commonly, niv vaccine approaches have focused individual candidate antigens delivered as subunit vaccines or using viral vectors. the most studied vaccine candidate is the soluble form of the g protein (sg) from the related hendra virus (hev). hev and the niv malaysia strain share between and % amino acid homology between their proteins; with f and g proteins sharing and % homology, respectively ( ) . both f and g envelope glycoproteins are regarded as vaccine candidate antigens since they are the targets of niv neutralizing antibodies ( ) . an adjuvanted hev sg protein subunit-based vaccine (equivac r hev, zoetis) has been licensed in australia to protect horses against hev and to reduce the zoonotic risk to humans ( ) . equivac r hev protects ferrets and african green monkeys (agms) after experimental challenge with niv, as well as hev ( , ) . surprisingly, this vaccine failed to protect pigs from experimental niv challenge ( ) . since the vaccine induced cross-neutralizing antibodies but not measurable t cell responses, the authors concluded that both arms of the adaptive immune response may be required for protection against niv and hev. these studies also potentially highlight that adjuvants can have species specific effects and tailoring of adjuvants to the target species may be required or considered in the context of preclinical models. the experimental viral vectored vaccine candidates for niv include vesicular stomatitis virus, rabies virus, canarypox virus (alvac strain), adeno-associated virus (aav), measles virus, newcastle disease virus (ndv) and venezuelan equine encephalitis virus ( ) . alvac expressing niv g or f (alvac-g and alvac-f) was found to protect pigs against niv challenge weeks after the second immunization ( ) . high titres of niv neutralizing antibodies were induced with the alvac-g vaccine, while despite the low levels of neutralizing antibodies induced by the alvac-f; all vaccinated pigs were protected against virulent niv challenge. recombinant attenuated ndv expressing niv glycoproteins have been shown to induce long lasting niv-specific nabs in pigs, with the vector expressing niv g performing better than niv f ( ). however, no challenge was performed in this study and it remains to be determined whether these paramyxovirus-based vaccine candidates are efficacious. compared to canarypox vectors, ndv-based vectors have a number of advantages including their high titer propagation in chicken eggs removing the requirement for cell culture ( , ) . despite these encouraging results and the continued threat posed by niv, no vaccine candidate has progressed toward market for either pigs or humans. the promising performance of experimental niv and hev vaccines in animal models and the licensure of equivac r hev, as a "one health" vaccine to safeguard animal and human health, strongly support the proposition that a safe and effective niv vaccine may be developed for pigs to reduce the severe economic consequences of niv outbreaks and the threat to public health. with partners, we have initiated a project that aims to develop such a vaccine. we are systematically analyzing the immunogenicity and protective efficacy of three niv vaccine candidates in pigs: ( ) an adjuvanted niv sg protein (orthologous to the equivac r hev vaccine), ( ) niv g protein delivered by a replication-deficient simian adenoviral vector (chadox niv g), and an adjuvanted, molecular clamp stabilized niv f (mcsf) protein. chadox is a multispecies vector with an established human and livestock safety profile ( ) . chadox offers the potential for both single dose efficacy and thermostabilization ( , ) . the molecular clamp is a proprietary stabilization domain that preserves the f protein in its native "pre-fusion" form, which should enhance immunogenicity and thermostability. in depth analyses of t cell and antibody responses are being conducted to identify correlates of vaccine-induced protection. we will examine the durability of niv-neutralizing antibodies and other immune responses associated with protection, including a comparison of a singleshot vs. homologous prime-boost immunization regimes. incontact animals will be introduced to assess transmission of challenge virus from vaccinates or unvaccinated control animals. the sporadic nature of niv outbreaks means that the commercial development of niv vaccines for use in pigs (other livestock or humans) is limited and animal health companies are of the opinion that niv vaccines will have limited marketability. our ongoing studies should help facilitate this by developing a safe and efficacious prototype niv vaccine that is amenable to "surge production" and discrimination of infection in vaccinated animals (diva) capability. subsequent development and licensure of this vaccine will require engagement with international, regional, and national agencies and the creation of dependable markets via the establishment of niv vaccine banks. the oie world fund manages vaccine banks and the delivery of vaccines for avian influenza, rabies, foot-andmouth disease, and peste de petit ruminants ( ) . vaccine banks ensure the procurement and delivery of high quality vaccines mass-produced in line with oie intergovernmental standards. critically these vaccine banks can be rapidly deployed when required and this model appears most appropriate in the context of reactive emergency vaccination programmes to aid niv outbreak control. vaccines can play a major component in an emergency response against emerging infectious disease, with the main aim to reduce virus spread between susceptible hosts ( ) . the precise decisions on control strategies will be complex and vary for different regions. factors such as: herd density, production systems, the presence of susceptible wildlife, the impact on export trade and current opinions on economic vs. ethical factors will likely play a role. one strategy to halt a niv outbreak would be to deploy a stockpiled vaccine for ring vaccination around the niv affected area. this approach was utilized in the ebola outbreak in guinea and showed great promise in terms of disease containment and elimination ( ) . for such a strategy, a vaccine with single-dose efficacy and a rapid onset of immunity preventing virus transmission would be preferential. this is likely to be best achieved with a viral-vectored ( ) or mrna vectored vaccine ( ) . the highly unpredictable nature of niv outbreaks means that it is highly unlikely that niv vaccines would be used routinely by pig producers. one strategy that could help ensure that immunity to niv is maintained in pig herds could involve the engineering of niv g into a live attenuated viral vaccine, such as pseudorabies, which are widely used in countries at-risk. the recent ebola and zika epidemics highlighted how poorly prepared we were to deal with these new and emerging diseases. there has therefore been a global drive to develop vaccines against these diseases and improve preparedness. the coalition for epidemic preparedness innovation's (cepi's) was established in with a mandate of financing and coordinating the development of new human vaccines to prevent and contain infectious disease epidemics. cepi selected niv, lassa virus and middle east respiratory syndrome-coronavirus, three pathogens from the who's list of priority diseases needing urgent r&d attention as its initial focus ( , ) . the who's list of priority diseases is part of the r&d blueprint, which identifies priority diseases and addresses gaps in the global scientific community to increase preparedness for future outbreaks. the main aim of the blueprint is to fast-track the availability of effective tests, vaccines, and medicines that can be used to save lives and avert large scale crises ( ) . in , the us food and drug administration (fda) established the "animal rule" for regulatory approval of vaccines and therapeutics for which efficacy testing in humans is impossible, therefore requiring relevant animal models that represent a disease model similar to that of the human disease ( ) . vaccine efficacy studies in animal models aim to identify specific vaccine-induced correlates of protection including neutralizing antibodies or cell-mediated responses ( ) . in , a vaccine to protect against anthrax was the first to be approved through the "animal rule" ( ) . the licensing pathway for the "animal rule" requires that immunogenicity results from clinical trials must be consistent with previously identified immune correlates associated with protection ( ) . therefore, identifying reliable markers of vaccine-generated immunity becomes critically important for pathogens such as niv. large animal models have been shown to more accurately predict vaccine outcome in humans in comparison to small animal models ( ) therefore defining correlates of vaccineinduced protection in pigs, may play an important role in supporting subsequent human vaccine licensure under the "animal rule." animal models can be validated for a particular disease according to a number of different criteria, which include "face" and "predictive" validity. for face validity there must be similarities in the pathology and clinical symptoms between the animal model and the human disease ( ). as discussed above, niv infection of pigs causes a similar respiratory and neurological syndrome as seen in human infections. although, disease severity in pigs may be considered lower than in humans. the predictive validity of a model means that clinically effective interventions demonstrate a similar effect in the animal model ( ) . no clinical trials of niv vaccine candidates have been reported to compare with vaccine performance in animal models, including the pig. as noted above, the success of the equivac r hev vaccine in horses and other animal models was not replicated in swine ( , ) , highlighting a potential issue of predicative validity when comparing niv vaccines between animal species, which may extend to humans. on the other hand, pigs have been used successfully as models to study many human infectious diseases ( ) ( ) ( ) ( ) ( ) ( ) ( ) , including niv infection ( ) . there is also a growing appreciation that pigs provide a superior animal model for influenza a virus infection and immunity and should play a more prominent role as a model for human influenza vaccine development ( ) . the success of the pig as an experimental animal model is partly due to their similarities with humans in terms of anatomy, immunology, and physiology, but also due to their manageable behavior and size, and by the general ethical acceptance of using pigs for experimental purposes instead of non-human primates ( , , ) . the niv outbreaks in malaysia and singapore demonstrated that pigs can play a key role in the epidemiology of niv by acting as an amplifier host. the region most at risk of niv infection has some of the highest pig population densities found anywhere in the world, which are rising fast due to the demand of a growing human population. this increases the risk of niv transmission to pigs and humans. the development of a niv vaccine for use in pig populations would decrease the major risk niv poses to the developing pig industries, as well as to the livelihoods of poor livestock keepers in southeast asia. the use of non-human animal models is crucial for vaccine development against diseases such as niv since efficacy testing in humans is impossible. the pig model may therefore contribute to human vaccine development, supporting human vaccine licensure under the animal rule. pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission functional studies of host-specific ephrin-b ligands as henipavirus receptors structural basis of nipah and hendra virus attachment to their cell-surface receptor ephrin-b unraveling a three-step spatiotemporal mechanism of triggering of receptorinduced nipah virus fusion and cell entry organ-and endotheliotropism of nipah virus infections in vivo and in vitro role of endocytosis and cathepsinmediated activation in nipah virus entry origins of major human infectious diseases the pandemic potential of nipah virus virus distribution map nipah virus encephalitis reemergence enhancing preparation for large nipah outbreaks beyond bangladesh: preventing a tragedy like ebola in west africa available online at nipah virus edits its p gene at high frequency to express the v and w proteins henipavirus susceptibility to environmental variables outbreak of nipah-virus infection among abattoir workers in singapore nipah virus outbreak in malaysia case-control study of risk factors for human infection with a new zoonotic paramyxovirus, nipah virus, during a - outbreak of severe encephalitis in malaysia pig production in cambodia, laos, philippines and vietnam: a review nipah virus: a recently emergent deadly paramyxovirus nipah virus encephalitis outbreak in malaysia japanese encephalitis and its epidemiology interdisciplinary approaches to understanding disease emergence: the past, present, and future drivers of nipah virus emergence new virus fingered in malaysian epidemic introduction to modern virology available online at the status, public response and challenges in overcoming emerging and exotic diseases -nipah virus disease experience. in: national congress on animal health and production: environmental care in animal production nipah virus infection of pigs in peninsular malaysia experimental nipah virus infection in pigs and cats bacterial infections in pigs experimentally infected with nipah virus infections and infestations in force the nonstructural proteins of nipah virus play a key role in pathogenicity in experimentally infected animals the immunomodulating v and w proteins of nipah virus determine disease course molecular characterization of nipah virus, a newly emergent paramyxovirus a treatment for and vaccine against the deadly hendra and nipah viruses hendra virus vaccine, a one health approach to protecting horse, human, and environmental health a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge a recombinant hendra virus g glycoprotein subunit vaccine protects nonhuman primates against hendra virus challenge protection against henipaviruses in swine requires both, cell-mediated and humoral immune response status of vaccine research and development of vaccines for nipah virus recombinant nipah virus vaccines protect pigs against challenge newcastle disease virusvectored nipah encephalitis vaccines induce b and t cell responses in mice and long-lasting neutralizing antibodies in pigs a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity potency of a thermostabilised chimpanzee adenovirus rift valley fever vaccine in cattle chimpanzee adenovirus vaccine provides multispecies protection against rift valley fever available online at emerging and neglected infectious diseases: insights, advances, and challenges efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial zika virus protection by a single low-dose nucleosidemodified mrna vaccination available online at research and development blueprint for action to prevent epidemics new drug and biological drug products; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible. final rule can ebola virus vaccines have universal immune correlates of protection? first vaccine approval under the fda animal rule large animal models for vaccine development and testing animal models in translational medicine: validation and prediction staphylococcal wound infection in the pig: part i a pig model of acanthamoeba keratitis: transmission via contaminated contact lenses animal models for gastric helicobacter immunology and vaccine studies the benefits of using diverse animal models for studying pertussis animal models of ventilator-associated pneumonia swine influenza h n virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h n influenza virus the pig: a model for human infectious diseases animal models of henipavirus infection: a review swine as a model for influenza a virus infection and immunity contribution of the swine model in the study of human sexually transmitted infections all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -f f h r authors: afrough, b.; dowall, s.; hewson, r. title: emerging viruses and current strategies for vaccine intervention date: - - journal: clin exp immunol doi: . /cei. sha: doc_id: cord_uid: f f h r during the past decade several notable viruses have suddenly emerged from obscurity or anonymity to become serious global health threats, provoking concern regarding their sustained epidemic transmission in immunologically naive human populations. with each new threat comes the call for rapid vaccine development. indeed, vaccines are considered a critical component of disease prevention for emerging viral infections because, in many cases, other medical options are limited or non‐existent, or that infections result in such a rapid clinical deterioration that the effectiveness of therapeutics is limited. while classic approaches to vaccine development are still amenable to emerging viruses, the application of molecular techniques in virology has profoundly influenced our understanding of virus biology, and vaccination methods based on replicating, attenuated and non‐replicating virus vector approaches have become useful vaccine platforms. together with a growing understanding of viral disease emergence, a range of vaccine strategies and international commitment to underpin development, vaccine intervention for new and emerging viruses may become a possibility. immunization is arguably the most appropriate way of preventing infectious disease. the control of many important viral pathogens by vaccination is perhaps one of the outstanding achievements of medical intervention. vaccine-induced immunity that is established in advance of virus infection relies primarily on adaptive immune responses for protective efficacy. critically, vaccination depends on the properties of antigen recognition, activation, expansion, memory, trafficking and the multitude of specialist functions of lymphocytes. the extent to which vaccine-induced immunity is successful also determines the spread and maintenance of a viral pathogen within a population. viral vaccines have had profound and enduring consequences for human and animal health; the worldwide eradication of smallpox and rinderpest are testament to their outstanding contribution to modern society. nevertheless, infectious diseases still pose one of the greatest threats to public health, and the past three decades have brought a constant barrage of new human pathogens. more than % of these infections are zoonotic [ , ] , entering either directly from wildlife reservoirs or indirectly via an intermediate domestic animal host [ , ] . hiv, avian influenza, hendra (hev) and nipah (niv) viruses, severe acute respiratory syndrome (sars) and middle east respiratory syndrome coronavirus (mers-cov), ebola and marburg filoviruses, lassa virus (lasv), rift valley fever virus (rvfv) and crimean-congo haemorrhagic fever (cchf) viruses are all examples of zoonoses currently emerging from wildlife. all these emerging zoonoses present a serious and increasing threat to health, biosecurity and economies worldwide. the mechanisms underlying disease transmission from animals to humans are becoming better understood [ ] with the emergence of pathogens from wildlife (which represents the greatest threat to global health) occurring in a non-uniform pattern, being localized to distinct geographic 'hotspots' in africa, asia and south america, and with each high-threat pathogen being weighted towards a key wildlife species (e.g. bats, rodents or non-human primates (nhps). it is clear that such diseases will continue to place a substantial burden on global health, especially in dense human populations where the pressures on environmental and economic resources are greatest. more than one billion cases of human zoonotic disease are estimated to occur annually, and emerging zoonoses result in enormous economic losses [ ] . increased urbanization, international travel, commerce and climate change increase the likelihood that emerging zoonosis will continue, if not worsen, in the future. when a zoonotic virus spills over into a susceptible new species, it often has the advantage that the new host has little or no pre-existing immunity, enabling attachment, entry and replication of the virus in receptive cells. the amplified virus may then evade clearance by host defences for long enough to be transmitted to another susceptible host, and the lack of herd immunity will result in a rapid dissemination of the virus, leading to disease in more virulent cases of infection. each step in the process represents an opportunity for vaccineinduced immunity and, through such intervention, transmitters and susceptible hosts are removed from a population by the pre-emptive development of protective immunity, so that the spread of infection becomes less likely. vaccination is therefore a powerful strategy for preventing and controlling emerging zoonotic infectious disease. the development of vaccines for such emerging infections, however, needs to contend with several key challenges associated with such viruses. an emerging infection may be a recently discovered virus and the result of a rare outbreak for which basic biological information such as correlates of protection, antigenic variability or immunodominance are unknown. there may be a lack of time to develop an appropriate animal model of disease in which to study viral immunology and evaluate vaccine candidates for preclinical assessment of protective efficacy and safety. additionally, many emerging viruses have high case fatality rates, spread easily and cannot be treated. these characteristics mandate that all experimental investigations with such infectious material be carried out at high levels of bio-safety, such as biosafety levels or . for such pathogens the availability of resource-heavy laboratory infrastructure is a bottleneck to basic research. moreover, the often standard vaccine approach of using attenuated strains or inactivated viral vaccines is not always a feasible option, because of the possibility of reversion to virulence or the requirement for large-scale culture and production in high containment facilities. in addition to these significant hurdles, the economic cost of novel human vaccine development for rare pathogens, which are unlikely to provide an effective payback on investment, has been a major impediment to progress. thus, basic research into many emerging pathogens has been neglected for years. in the unpredicted size, speed and reach of the ebola virus outbreak in west africa [ ] acted as a wakeup call for researchers, pharmaceutical communities and governments, emphasizing the importance of investment into the study of emerging pathogens. spurred on by this development and at the request of its member states in may , the world health organization (who) convened a broad coalition of experts to develop a research and development (r&d) blueprint for action to prevent epidemics. focusing on severe emerging diseases with the potential to generate public health emergencies, and for which no, or insufficient, preventive and curative solutions exist, the r&d blueprint specifies r&d needs, including vaccine research. through international governance, the programme aims to define r&d roadmaps for prioritized pathogens and to catalyse funding strategies [ ] . key to the development of successful and effective vaccines is the design of an antigen delivery system that optimizes antigen presentation and induces broad protective immune responses. recent advances in vector delivery technologies, immunology and basic virology have led to a deeper understanding of the molecular and cellular mechanisms by which vaccines should stimulate both arms of the adaptive immune response, thereby offering novel strategies of vaccination. here we discuss some current vaccine approaches for safe and effective vaccines encompassing recombinant virus technology, nucleic acid vaccines and self-disseminating vaccine approaches. advances in recombinant virology and virus reverse genetics have provided key insights into the replication and pathogenesis of a wide range of viruses. notably, these have facilitated the development of vectors for protein expression and vaccination. to date, several virus families have been exploited as vectors [ ] [ ] [ ] [ ] including many for vaccination [ ] [ ] [ ] [ ] fig . a basic advantage of viral vectored vaccines is that the choice antigen is expressed in the context of an active heterologous viral infection, which stimulates the full gamut of innate immune responses required for the development of adaptive humoral and t cell-mediated immunity [ ] . an important aspect of a virus-vectored vaccine for emerging viruses is that the characteristics, type and intensity of the immune response, as well as safety considerations and manufacturing techniques are determined predominantly by the vector and not the pathogen. therefore, developing and testing a vaccine against a newly discovered virus can be significantly shortened by the use of a viral vector platform with an extensive record of safety and efficacy. vaccines for emerging pathogens: from research to the clinic. part vesicular stomatitis virus (vsv), a negative sense rna virus of the rhabdoviridae family, has become a prominent replication-competent vaccine vector platform [ ] . vsv is non-pathogenic in humans and has an inherent ability to elicit strong cellular and humoral immune responses. one of the most useful aspects of this virus vector platform is its almost promiscuous ability to assemble recombinant vsv (rvsv), with many different types of heterologous viral glycoproteins. the platform is designed such that the vsv g protein is replaced with a heterologous envelope glycoprotein from, for example, an emerging virus; while this arrangement renders the rvsv replication competent, the recombinant viruses are generally highly attenuated [ ] . in , the attenuation of a yellow fever (yf) virus via successive rounds of serial passage led to the development of the yf vaccine termed d. the impact of this successful vaccine was recognized by the award of a nobel prize in [ ] and it has been widely adopted for human immunization for more than years [ ] . based on the utility of the vaccine, an infectious cdna clone of the yf d virus [ ] has enabled the development of a d platform that can be used to drive antigen delivery of pathogens of interest [ ] . the technology (licenced as chimerivax™) is well suited to similarly related flaviviruses and successful recombinants have been constructed, involving a simple swap of the prm/m-e genes of yf d for the same membrane envelope antigens of other emerging flaviviruses, such as japanese encephalitis, dengue and west nile. the resulting recombinant vaccines are efficiently delivered in a live-attenuated virus context with the safety profile afforded by the d non-structural genes [ ] . a licenced vaccine for japanese encephalitis (chimerivax™-je) using this technology has been developed by sanofi pasteur (lyon, france). other replication-competent platforms. the ease of direct manipulation of viral genomes together with a growing understanding of their biology has led to the development of attenuated virus vaccines with increased safety and immunogenicity. for example, a new vaccine candidate for rvfv has been developed in which a viral virulence factor has been deleted, resulting in a highly attenuated but immunogenic replicating virus [ ] . a similar recombinant approach has been used to attenuate the emerging and neglected pathogen mayaro virus (mayv) by swapping the subgenomic promoter of this alphavirus for an internal ribosome entry site [ , ] , which reduces the expression of mayv structural proteins. while this vaccines for emerging pathogens: from research to the clinic. part arrangement makes the virus unable to infect mosquito cells, replication in mammalian cells is still possible, resulting in a highly immunogenic profile. similar studies on infectious clones of other viruses [ ] have demonstrated that the genomewide de-optimization of codon usage dramatically reduces gene expression and can be used to attenuate otherwise pathogenic viruses. this strategy has been used for the prototype arenavirus lymphocytic choriomeningitis virus (lcmv) [ ] ; the study showed attenuation in an otherwise fatal mouse model of lcm disease and the ability to induce protective immunity. together with a maintained and robust ability to multiply in cell culture, this live attenuated approach may be suitable for similar viruses. however, work to underpin confidence in the genetic stability of such attenuated viruses is critical before further consideration can be given to using this approach for clinical intervention, especially considering the inherent high error rates of the arenavirus polymerase. for several years, recombinant adenoviruses have been adopted as promising tools for antigen delivery and vaccine efficacy [ ] . deleting the e gene from the adenoviral genome and supplying it in trans from a packaging cell line allows replication-deficient recombinant adenovirus to be produced, with the novel heterologous antigen gene of interest inserted at the e locus. early setbacks in this platform technology relating to pre-existing anti-adenovirus vector antibodies in humans [ ] have been resolved by adopting simian adenoviruses as vaccine vectors. a number of different replication-deficient vaccine vectors [ ] have recently been developed from simian adenoviruses and the platform has progressed work on emerging pathogens such as ebola virus, rvfv mers-cov and zika virus. modified vaccinia virus ankara (mva) is licensed as a third-generation vaccinia type vaccine against smallpox and serves as a potent vector system for the development of new candidate vaccines against a range of infectious diseases, including those caused by emerging pathogens. historically, mva was developed by serial tissue culture passage in primary chicken cells of vaccinia virus strain ankara, and clinically used to avoid the undesirable side effects of conventional vaccinia vaccines [ , ] . adapted to growth in avian cells, mva does not replicate in mammalian hosts and lacks many of the viral immune evasion genes [ ] . the features of mva, such as its capacity to accommodate large gene inserts [ ] , thermostability for application in remote regions without an established cold chain [ ] , ease of inexpensive manufacture to gmp standards and established regulatory package for development as an investigational new drug, make the recombinant mva platform [ ] highly versatile as a heterologous viral vector. in the context of emerging infections, the recombinant mva platform has shown encouraging preclinical efficacy against ebola, zika, chikungunya [ ] and cchf [ ] viruses. additionally, mva elicits a strong immunological response against a range of other orthopoxviruses (opxvs) (including variola), and vaccines based on this platform can be considered as providing added value, as human immunity to opxvs is low (after the cessation of the smallpox vaccination campaign) opening a gap for opxv emergence, as evidenced by the recent occurrence of monkeypox virus in west africa and onward cross boarder transmissions [ , ] . rna replicon systems are derived from either positive-or negative-strand rna virus genomes and embody disabled virus vectors that are non-pathogenic and unable to revert to virulence. driven by autonomous rna replication, rna replicons result in high-level, cytosolic expression of recombinant heterologous antigens stimulating both the humoral and cellular arms of the immune system. replicon vaccine approaches have closely followed technical developments to genetically manipulate viral genomes. for rna viruses, replicons based on positive-stranded picornaviruses were some of the first [ ] , and these were followed by those based on alphaviruses [ ] and negative-strand rna viruses [ ] . while a series of different vaccine replicon systems are available, new capabilities with negative-strand viruses have opened up opportunities against a wider range of emerging viruses. recent developments [ ] include the construction of lasv replicons packaged into lasv-like particles which allow a single round of replication and are able to confer protection against an otherwise lethal challenge of lasv in a guinea pig model of disease [ ] . while recombinant replicons are devoid of the viral glycoprotein gene, its incorporation into vlps is achieved by the use of a cell line that expresses the glycoprotein separately (in trans). this enables the scalable propagation of replicon particles in a way that aims to combine the safety of replicon-delivered lasv antigens with the convenience of simply and rapidly producing an attenuated virus. similar replicon approaches have been used to develop promising replicon-based vaccine candidates for ebola virus [ ] and rvfv [ ] . replicon approaches have the potency of live attenuated vaccines but are inherently safer, as their design ensures a single cycle of replication in contrast to a fully replicating, live attenuated vaccine virus. for live attenuated rna virus vaccines which incorporate error-prone polymerases, reversion to virulence is a distinct possibility after multiple rounds of replication. interest in the use of virus-like particles (vlps) as vaccines candidates stems from their ability to present ordered and highly antigenic structures to the immune system. at the same time, they lack a viral genome, potentially yielding safer vaccines, as there is no viral vaccines for emerging pathogens: from research to the clinic. part sequence that can revert to virulence. they induce strong b cell responses in the absence of adjuvants by efficiently cross-linking specific receptors on b cells [ ] , and they can also trigger t cell-mediated responses [ ] . the basis of ordered viral self-assembly from protein subunits that is noted in many different virus families provides the foundation for work with vlps, with more than different viruses that infect humans or other animals being identified as able to produce vlps. they are structurally diverse, having single or multiple capsid proteins, or a lipid envelope, although not all viruses are suitable vlp candidates. those which are can elicit a protective response without requiring multiple booster shoots, thus significantly reducing the vaccine costs. to date, vlp-based vaccines for human papilloma virus (hpv), hepatitis b virus (hbv) and hepatitis e virus (hev) have been licensed and are commercially available worldwide [ ] . several vlp-based vaccine candidates for human diseases are under clinical development, including those directed against norwalk virus, ebola and marburg viruses and hepatitis c virus. vlp vaccines combine many of the immunogenic advantages of whole-virus vaccines with the safety advantages of recombinant subunit vaccines. dna vaccines have emerged as a safer alternative to standard live and inactivated vaccines for treating human and animal infections [ ] . they exhibit several advantages over traditional strategies in terms of safety, stability, ease of manufacturing and immunogenicity [ ] . they offer potential advantages for vaccination against emerging viruses, in that plasmids expressing a viral antigen can be produced rapidly. furthermore, antigen is expressed in vivo and induces both humoral and cell-mediated immune responses. additionally, large quantities of dna can be produced in a short time at reduced cost, and dna preparations are more stable than other types of vaccines, which are desirable properties for a vaccine that may be used in remote areas. furthermore, dna vaccines are considered safe. however, the main limitation in the development of dna vaccines is their intrinsic low immunogenicity. work to improve this has focused on optimizing delivery approaches with the use of gene guns, or electroporation; targeting immune effector cells; and the use of potent adjuvants. dna vaccines are also frequently used in combination with other vaccine platforms in heterologous prime-boost strategies. a dna vaccine is currently licensed to immunize horses against west nile virus [ ] and has undergone phase i clinical trials in humans [ ] . dna vaccines have also been evaluated as candidates against many emerging viruses, including ebov [ ] , rvfv [ ] , dengue virus [ ] and chikv [ ] . synthetic peptide-based epitope-vaccines (evs) make use of short antigen-derived peptide fragments that can be presented either to t cells or b cells [ ] . evs offer several advantages over other forms of vaccines, particularly with regard to safety, ease of production, storage and distribution, without cold chain issues. they also offer the opportunity to vaccinate against several pathogens or multiple epitopes from the same pathogen. however, drawbacks include poor immunogenicity and the restriction of the approach to patients of a given tissue type [human leucocyte antigen (hla) haplotype] [ ] and, as such, they need to be tailored to accommodate the natural variation in hla genes. although initially this was thought to be a major impediment, new technologies have made this personalized-medicine approach feasible [ , ] . recently, bioinformatics tools have been developed to identify putative cd + t cell epitopes, mapped to the surface glycoproteins of the emerging viruses lasv, nipv and hendra [ ] . while these vaccine candidates still need to be experimentally tested, the approach represents an interesting and novel strategy that shows promise for vaccination and which could also address immunity in particular target populations. the induction of immune responses by the delivery of inactivated pathogens has been a standard and successful vaccination approach for many years, and licenced, inactivated vaccines for diseases such as poliomyelitis [ ] and rabies [ ] are commercially available. the long history of this approach is underpinned by a well-defined regulatory framework that can be readily applied to new disease targets [ ] . the major challenge for the inactivated virus approach is that infection is not established, and therefore a full adaptive immune response is generally not achieved. however, because of the absence of living pathogens, these types of vaccines are safe and a basic capability to prepare such vaccines, especially for emergency use, might be worthwhile as a stop-gap while alternative longer-term approaches are developed. in this regard, studies of virus inactivation with x-ray radiation (as a simple and cheap alternative to gamma irradiation by the use of radioactive isotopes), which maintain the tertiary antigenic structures of virus particles while destroying infectivity, have shown useful promise for a range of applications including vaccination (b. afrough, unpublished). vaccines play a pivotal role in host protection against infectious diseases and have significantly reduced mortality worldwide. however, many vaccine candidates for emerging diseases have failed to make it into clinical development. this is perhaps surprising, given the breadth of vaccine technology available and the nature of many of the diseases in question. a case in point is lassa fever, a viral haemorrhagic illness endemic to many parts of west africa responsible for more than cases of serious disease and approximately deaths each year since its discovery more than years ago. it is often in the headlines, being the most commonly exported vhf to other territories, including the united kingdom, which has received a disproportionate share of traveller-related cases (each incident placing substantial burden on public health resources). during , lasv caused an unusually large increase in cases in nigeria, which led the who to classify it as a grade public health emergency. however, despite the high burden of lassa fever and public attention, no vaccines have so far been approved. multiple approaches to develop a range of effective preclinical candidates have been made during the last three decades, including attenuated vaccines [ ] , replication competent vaccines, [ ] [ ] [ ] , non-replicating lasv vaccines [ ] , a rationally designed live attenuated vaccine [ ] and dna vaccines [ ] . additionally, many of these platforms have shown efficacy in animal models including nhps [ , , ] . these preclinical data illustrate that multiple vaccine technologies have the potential to yield protective lassa fever vaccines. therefore, the lack of a clinical vaccine after years since the disease was first described must be due to other factors, such as economic considerations or safety issues, perhaps connected with the growing burden of regulatory thresholds for human medical interventions. thus, bringing a lasv vaccine and, by analogy, other potential vaccines for emerging diseases to the clinic, may be very difficult -for non-technical reasons. although there is renewed interest from multiple international agencies to develop human vaccines for certain emerging pathogens [ , ] , it is prudent to consider other approaches to the control of emerging disease, including the feasibility of controlling infections at source. the pattern of disease emergence from viral pathogens into humans from wildlife reservoirs is a clear and present threat [ ] [ ] [ ] [ ] [ ] which will continue. this makes the task of identifying, controlling and preventing zoonoses a difficult and daunting goal, particularly when a new emerging pathogen may be completely unknown. while surveillance is an essential component of a successful control programme, effective containment of an emerging pathogen, before epizootics have the opportunity to spill over into human populations, has been achieved most effectively by large-scale culling or mass vaccination [ , ] of animals. nevertheless, the ability to contain even known emerging viruses such as ebola virus in wildlife is currently not possible. furthermore, the management of diseases that involve livestock, such as rvf and cchf, pose problems [ , ] in that conventional vaccines are not suited for use in these environments. a major limitation of conventional vaccination is the requirement for individual inoculation of each animal -a costly and impractical strategy for the target/reservoir species of those animals frequently involved in the emergence of high-risk pathogens [ ] . surveillance work focusing on epizootics that are, or may become, human pathogens is a useful goal. currently however, predicting which animal pathogens will become established as globally significant emerging human diseases is guesswork. nevertheless, in the early stages of a new zoonosis, pathogenic viruses are often poorly adapted to their new human host in terms of sustained human-tohuman transmission [ ] . this lag-phase in early zoonosis may, therefore, provide a window of opportunity to control the unrelenting zoonotic pressure of an emerging pathogen before it adapts further to humans. vaccine targeting of the pathogen within the animal transmission species could therefore bring useful advantages. self-disseminating vaccines, which aim to immunologically contain emerging viruses within their non-human reservoir hosts, offer an alternative to the conventional vaccine approach. they are designed to exploit the ability of replicating virus-based vectors to spread through animal host populations, so avoiding the need for direct inoculation of every animal. in this way, vaccination of a limited number of initiator animals is used for the introduction of the vaccine into a target population. the vaccine is engineered to express target antigens from the emerging pathogen of interest, so its transmission from vaccinated to non-vaccinated animals will result in the co-ordinated spread of specific immunity for the emerging pathogen throughout the targeted animal population. following early work to underpin a proof of principle for a disseminating vaccine against an animal pathogen [ , ] , a study targeting the human pathogen sin nombre orthohantavirus (snv) in its rodent reservoir -the deer mouse (peromyscus maniculatus) -proved effective. this approach used an engineered cytomegalovirus (cmv) vector (which causes a benign but transmissible infection in the host), expressing the snv envelope glycoprotein g [ ] . a similar approach is also being developed to interrupt zoonotic transmission of ebola virus [ ] , in this case disseminating cmv vaccines specific to great apes and expressing ebov antigens are being studied in african ape populations in the wild [ ] . interestingly, one of the goals of this work is to protect the great apes themselves from ebola virus disease, a major threat to the survival of these animals in the wild. also, as vaccines for emerging pathogens: from research to the clinic. part approximately % of human ebola virus outbreaks are known to have resulted from the direct handling of infected ape carcasses, the disseminating cmv-based strategy may be significant in protecting humans [ ] . clearly, further work needs to be conducted to assess the risks of live transmissible vaccines evolving into a pathogen with increased virulence. engineering such wild-life reservoir vaccines to be weakly transmitting, such that their reproduction number is below (r < ), making their transmission chains short so that they cannot be maintained, might be a way to address the justifiable safety risks. indeed, a mathematical model has recently demonstrated the value of such an approach [ ] . emerging pathogens represent one of the greatest risks to global health. there is already good evidence [ , ] that zoonotic pathogens will most probably be transmitted from a few key animal species in resource-poor areas of the world. based on recent history, it is probable that such pathogens have never been seen before. the global impact of the west african outbreak of ebola virus in underlines how stark differences in health-care infrastructure can impact upon human-to-human transmission of emerging pathogens. until basic health-care infrastructure in all countries can be raised to a level that enables early identification and control of high-risk pathogens at source, we will continue to respond to outbreaks of emerging disease long after epizootics have spilled over into human populations. innovative strategies are therefore urgently required to control such pathogens, vaccination is a proven approach. many novel vaccination strategies that have been developed during recent years have the potential to specifically address the growing threat of new and emerging disease. the use of well-defined vaccine vector platforms, with an extensive record of safety and efficacy against similar pathogens, can expedite the process of development, validation and production (table ) . accordingly, the design and licensure for particular platform vaccine technologies will help to accelerate the development of new vaccines, as only the simple substitution of a new antigen gene into the vector platform is required. this allows manufacturers to move to a new target disease with minimal changes in chemistry, manufacturing and controls. thus, new vaccine development can focus on the safety and efficacy of the inserted gene. in addition, the ability of platforms to target multiple pathogens helps to justify the investment required to build and maintain manufacturing infrastructure that specializes in one platform, because a single manufacturing facility can be ready to produce multiple vaccines at any time. in addition, further research into, and the development of, self-disseminating vaccines to control potential pathogens in their wild-life reservoirs should be encouraged. however, the progress of new vaccines through the necessary regulatory pathways to bring them to the clinic requires long-term investment by governments and international organizations. global trends in emerging infectious diseases emerging pathogens: the epidemiology and evolution of species jumps bats and emerging zoonoses: henipaviruses and sars spillover and pandemic properties of zoonotic viruses with high host plasticity ecology of zoonoses: natural and unnatural histories after ebola in west africa -unpredictable risks, preventable epidemics a research and development blueprint for action to prevent epidemics rna viruses: emerging vectors for vaccination and gene therapy immunologic basis of vaccine vectors viruses -from pathogens to vaccine carriers viral vectors as vaccine platforms: deployment in sight self-replicating alphavirus rna vaccines rapid development of vaccines against emerging pathogens: the replication-deficient simian adenovirus platform technology the evolution of poxvirus vaccines live virus vaccines based on a vesicular stomatitis virus (vsv) backbone: standardized template with key considerations for a risk/benefit assessment rhabdoviruses as vaccine vectors: from initial development to clinical trials. biology, pathogenesis of rhabdo-, filoviruses attenuated vesicular stomatitis viruses as vaccine vectors yellow fever and max theiler: the only nobel prize for a virus vaccine pathogenesis and pathophysiology of yellow fever transcription of infectious yellow fever rna from full-length cdna templates produced by in vitro ligation the yellow fever d virus as a platform for new live attenuated vaccines from research to hase iii: preclinical, industrial and clinical development of the sanofi pasteur tetravalent dengue vaccine creation of a recombinant rift valley fever virus with a two-segmented genome ires dependent replication of venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells ires-based venezuelan equine encephalitis vaccine candidate elicits protective immunity in mice modulation of poliovirus replicative fitness in hela cells by deoptimization of synonymous codon usage in the capsid region development of live attenuated arenavirus vaccines based on codon deoptimization adenoviruses as vaccine vectors pre-existing immunity against ad vectors: humoral, cellular, and innate response, what's important? a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity recombinant vaccinia viruses as vaccines mva vaccination against smallpox: clinical tests with an attenuated live vaccinia virus strain (mva) [author's translation] environmental risk assessment of clinical trials involving modified vaccinia virus ankara (mva)-based vectors infectious poxvirus vectors have capacity for at least base pairs of foreign dna long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass modified vaccinia virus ankara: history, value in basic research, and current perspectives for vaccine development vaccinia-based vaccines to biothreat and emerging viruses understanding orthopoxvirus host range and evolution: from the enigmatic to the usual suspects emergence of monkeypox as the most important orthopoxvirus infection in humans engineering poliovirus as a vaccine vector for the expression of diverse antigens selection of rna replicons capable of persistent noncytopathic replication in mammalian cells replicon rna viral vectors as vaccines use of a scalable replicon-particle vaccine to protect against lethal lassa virus infection in the guinea pig model replication-deficient ebolavirus as a vaccine candidate single-dose immunization with virus replicon particles confers rapid robust protection against rift valley fever virus challenge virus-like particles in vaccine development virus-like particles as particulate vaccines formulation and stabilization of recombinant protein based virus-like particle vaccines vaccines for the st century dna vaccines: recent developments and future possibilities dna vaccines -back in the saddle again? a west nile virus dna vaccine utilizing a modified promoter induces neutralizing antibody in younger and older healthy adults in a phase i clinical trial a dna vaccine for ebola virus is safe and immunogenic in a phase i clinical trial a dna vaccine encoding ubiquitinated rift valley fever virus nucleoprotein provides consistent immunity and protects ifnar(s/s) mice upon lethal virus challenge immunogenicity and protective efficacy of a vaxfectin-adjuvanted tetravalent dengue dna vaccine a dna vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates more than one reason to rethink the use of peptides in vaccine design hla class ii restriction of hiv- clade-specific neutralizing antibody responses in ethnic thai recipients of the rv prime-boost vaccine combination of alvac-hiv and aidsvax((r)) b/e the tubingen approach: identification, selection, and validation of tumorassociated hla peptides for cancer therapy innovative bioinformatic approaches for developing peptidebased vaccines against hypervariable viruses a bioinformatics tool for epitope-based vaccine design that accounts for human ethnic diversity: application to emerging infectious diseases polio vaccines: who position paper an overview of the immunogenicity and effectiveness of current human rabies vaccines administered by intradermal route a global regulatory science agenda for vaccines a live attenuated vaccine for lassa fever made by reassortment of lassa and mopeia viruses development of a new vaccine for the prevention of lassa fever yellow fever d-vectored vaccines expressing lassa virus gp and gp glycoproteins provide protection against fatal disease in guinea pigs vaccinia recombinant expressing lassa-virus internal nucleocapsid protein protects guinea pigs against lassafever development of live-attenuated arenavirus vaccines based on codon deoptimization of the viral glycoprotein enhanced efficacy of a codon-optimized dna vaccine encoding the glycoprotein precursor gene of lassa virus in a guinea pig disease model when delivered by dermal electroporation safety, immunogenicity, and efficacy of the ml reassortant vaccine for lassa fever in small non-human primates a dna vaccine delivered by dermal electroporation fully protects cynomolgus macaques against lassa fever who publishes list of top emerging diseases likely to cause major epidemics global partnership launched to prevent epidemics with new vaccines zoonosis emergence linked to agricultural intensification and environmental change impact of vaccines and vaccination on global control of avian influenza novel approaches to develop rift valley fever vaccines serological and virological evidence of crimean-congo haemorrhagic fever virus circulation in the human population of borno state, northeastern nigeria cross-species virus transmission and the emergence of new epidemic diseases isolation of an attenuated myxoma virus field strain that can confer protection against myxomatosis on contacts of vaccinates the current status and future directions of myxoma virus, a master in immune evasion replication and immunoactivity of the recombinant peromyscus maniculatus cytomegalovirus expressing hantavirus g glycoprotein in vivo and in vitro a cytomegalovirus-based vaccine provides long-lasting protection against lethal ebola virus challenge after a single dose ebola: outbreaks cause crisis for great apes and humans. toronto: the jane goodall institute of canada self-disseminating vaccines for emerging infectious diseases eradicating infectious disease using weakly transmissible vaccines the authors confirm that they have no competing interests in this work. key: cord- - nm tbig authors: moody, m. anthony title: modulation of hiv- immunity by adjuvants date: - - journal: curr opin hiv aids doi: . /coh. sha: doc_id: cord_uid: nm tbig purpose of review: to summarize the role of adjuvants in eliciting desirable antibody responses against hiv- with particular emphasis on both historical context and recent developments. recent findings: increased understanding of the role of pattern recognition receptors such as toll-like receptors in recruiting and directing the immune system has increased the variety of adjuvant formulations being tested in animal models and humans. across all vaccine platforms, adjuvant formulations have been shown to enhance desirable immune responses such as higher antibody titers and increased functional activity. although no vaccine formulation has yet succeeded in eliciting broad neutralizing antibodies against hiv- , the ability of adjuvants to direct the immune response to immunogens suggests they will be critically important in any successful hiv- vaccine. summary: the parallel development of adjuvants along with better hiv- immunogens will be needed for a successful aids vaccine. additional comparative testing will be required to determine the optimal adjuvant and immunogen regimen that can elicit antibody responses capable of blocking hiv- transmission. many hurdles remain for the development of a globally deployable hiv- vaccine. elicitation of a durable immune response that can prevent hiv- infection or disease will likely require the use of an adjuvant for some or all immunizations. at present in the usa there are only two licensed adjuvants, although other adjuvanted vaccines are licensed in other parts of the world, and many more have been tested in human and animal trials. this review will highlight recent work in adjuvant development for hiv- vaccines with particular emphasis on antibody responses. the word 'adjuvant' derives from the french adjuvant, which itself derives from the latin adjuvate that can be translated to 'helper'. the term was first used in a modern vaccine context by gaston ramon of institut pasteur in a series of papers in the s (e.g., [ && , , && ]) that established the use of adjuvants for eliciting high-titer antitoxin responses. since that time, many compounds and formulations have been tested for their ability to adjuvant a vaccine response, with the development of new adjuvants paralleling an increased understanding of pattern recognition receptors (prrs) and their role in recruiting and directing the immune system. an adjuvant is a compound, formulation, preparation, or delivery system that enhances or modifies the immunogenicity of the primary antigen in a vaccine. adjuvants perform this function in a variety of ways, but nearly all involve the triggering of prrs to stimulate the innate and adaptive arms of the immune system. this is accomplished in one of two ways -through the incorporation of active compounds in a vaccine formulation (e.g., formulating a protein immunogen in a liposome containing a tlr agonist) or by incorporating elements in the vaccine that result in the production of immune stimulants (e.g., addition of plasmids expressing cytokines in a dna vaccine regimen). these distinctions are not absolute, and some formulations incorporate elements of both approaches. the development of adjuvants has accelerated in the last years and has to some degree paralleled the development of hiv- vaccine candidates. during that time, a number of excellent reviews have been published [ && , , && , - ] that the reader may find useful. this review will focus on the historical context of adjuvant development since the discovery of hiv- , recent developments, and finally will highlight the lack of comparative data currently available. shortly after the discovery of hiv- , then secretary of health and human services margaret heckler held a press conference in which she predicted that vaccine trials against hiv- would be possible within years [ ] . the first vaccine trial began in [ , ] , and was followed by a series of attempts to develop an effective hiv- vaccine. early vaccine studies focused on leveraging strategies that had been successful for other vaccines including virus inactivation [ ] [ ] [ ] and subunit immunogens [ ] along with novel strategies such as recombinant viral constructs [ ] . although early subunit vaccine candidates were immunogenic [ ] , none of the follow-up efficacy trials showed protection [ , ] . concurrent with the development of vaccine candidates, numerous animal and human studies compared available adjuvants in head-to-head trials. no clearly superior regimen was identified, likely because of the lack of a consistent immunogen across trials along with differing immunization schemes and different outcome measures. for example, mannhalter et al. [ ] in reported the immunization of chimpanzees with a recombinant envelope (env) gp using alum, a water-inoil emulsion (termed lipid-based adjuvant), or alum plus deoxycholate. t-cell responses were best for the lipid-based adjuvant and were shown to last for months after the final immunization; antibody responses were not reported. niedrig et al. [ ] reported in on another group of chimpanzees immunized with formaldehyde-inactivated hiv- adjuvanted with alum, freund's incomplete adjuvant (an oil-in-water emulsion), or with a zinc hydroxide/lecithin-based adjuvant; in this study, antibody titers were best with the lecithin-based adjuvant, although proliferation and antibodydependent cell-mediated cytotoxicity (adcc) responses were similar between lecithin and alum arms. levi et al. [ ] reported in a comparison in rabbits of alum, iscom, iscomatrix, muramyl dipeptide (mdp), and freund's complete adjuvant with a recombinant gp as the immunogen. antibody titers were highest with freund's complete adjuvant and mdp. during the same time period, numerous mouse studies were published and nearly all demonstrated that one adjuvant was superior. these and other head-to-head studies of vaccines are shown in table [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . vaccine candidates deemed the most promising advanced to phase i and phase ii human trials. these studies tested proteins, peptides, and recombinant poxvirus vectors [ ] , and although none of the candidates produced overwhelming immunity, the vaccines were generally safe and well tolerated. without a stronger candidate available, a controversial decision was made to pursue a phase iii trial of poxvirus prime-gp boost vaccine strategy. the proposal had detractors [ ] and supporters [ ] , and ultimately demonstrated a modest and shortlived degree of efficacy [ , ] . the adjuvant used in that trial was alum, the only us food and drug administration (fda)-approved adjuvant at that time. studies are now being considered to examine the same immunization regimen using more potent adjuvants to see whether protection can be enhanced or prolonged. the remainder of this review will address more recent developments in adjuvant research. dna vaccines are attractive for eliciting cd þ t-cell responses, as protein production and antigen processing can occur without the need for an infectious vector. dna vaccines are generally not as potent at eliciting antibody responses, although evidence suggests that dna vaccines can prime for subsequent protein boosts [ , ] . numerous studies have reported the ability of immune modulators to provide an adjuvant effect for dna vaccines [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; most of these studies were performed in mice and few compared more than one regimen against an unadjuvanted control. there are no studies a wide variety of adjuvants have been tested for hiv- vaccines, but recent comparative data are limited. there are no 'universal' adjuvants, but adjuvants must be selected based on the desired response and carefully paired with the immunogen being tested. heterologous adjuvant strategies may be needed to balance efficacy and side-effects. comparing all available dna-encoded adjuvants, but a few smaller-scale studies have been reported. for example, testing of a series of dna adjuvants in mice suggested that one of the tested adjuvants was superior [e.g., granulocyte/macrophage colony stimulating factor (gm-csf) [ ] ], but studies in primates showed a more modest benefit [ ] . work is ongoing, but in the absence of a systematic study, at present, it is not clear whether any dna-encoded adjuvant is superior in eliciting desirable immunity for an hiv- vaccine. some studies have examined the effect of adding compounds to dna vaccines without having them encoded in a vector. mycobacterial extracts have been shown to enhance t-cell and antibody responses in mice [ ] as have tlr agonists [ ] . liposomes with mannan as a delivery vehicle for a dna vaccine enhanced fecal iga responses and altered subclass responses in mice [ ] . another toll-like receptor (tlr) agonist, imiquimod, applied topically adjuvanted a dna vaccine in mice, although the effect was similar to that of gm-csf [ ] . as with dna-encoded adjuvant molecules, it is unclear which of these strategies is superior. recent studies have suggested that physical adjuvants may be beneficial for dna vaccines. electrical current as an adjuvant has been tested in mice [ ] , rhesus macaques [ , ] , and humans [ & ]. the results suggest that electroporation alone is as effective as dna-encoded adjuvants, although side-effects were higher in electroporation groups [ & ]. electroporation almost certainly acts by increasing uptake of vaccine dna into cells and through minor tissue damage that stimulates damage-associated prrs that recruit an inflammatory response. further testing will be needed to determine whether electroporation can be implemented so as to reduce side-effects yet remain effective. immune stimulatory molecules can be encoded in viral or bacterial vectors that have sufficient room in their genomes (e.g., poxviruses, mycobacteria). as with dna vaccines, studies have tested different adjuvants with mixed results. for poxvirus vectors, cytokines [ , ] , soluble cd ligand [ ] , and cd [ ] have been tested in mice and each enhanced immune responses compared with controls. similar strategies have been tested for other viral vectors (e.g., rhabdovirus [ ] ). adjuvants can also be added with the vector but not encoded by it. for example, soluble cd ligand added to a dna-prime/poxvirus-boost strategy enhanced t-cell responses though the effect on antibody was variable [ ] . it remains to be seen if any of these strategies will ultimately prove useful for human trials. whether other adjuvant formulations can enhance recombinant vectors is under investigation. naito et al. [ ] demonstrated in a mouse model that tethering of liposomes to a poxvirus vector overcame previous immunity and could stimulate humoral and cellular immunity. many adjuvants, such as oil-in-water emulsions, can disrupt lipid membranes and so would be considered inappropriate for enveloped replicating vectors like poxviruses. in addition, as replicating vectors stimulate the immune system by the transient infection they cause, it is not clear that an adjuvant that is only transiently present at the site of injection would be useful. future studies will be needed to clarify these questions. for hiv- vaccine studies, the greatest variety of adjuvants have been tested for subunit/recombinant protein immunogens. as noted above, a large number of head-to-head trials were performed prior to (table ), but since that time, few large-scale direct comparisons have been published. older adjuvants continue to be explored to define those parameters critical for efficacy. alum is one of the most commonly employed adjuvants because of its long history of use in humans and the relative ease for regulatory approval; for this reason, research to optimize its utility is ongoing. hansen et al. [ & ] showed that the ability of alum to adsorb an env protein was important for immunogenicity, but that binding too tightly reduced immune responses after immunization. dorosko et al. showed that alternative methods of delivering alum can direct the immune response; injection of an alum-based peptide immunogen in the region of the supramammary lymph node of goats resulted in antibody secretion into colostrum [ ] . novel adjuvants continue to be studied in animal models. lipid-based adjuvants like the as x series have been shown to stimulate strong antibody responses in guinea pigs, although responses were similar to those elicited by an oil-in-water emulsion adjuvant [ ] . one of the adjuvants in this series, as b, elicited high-titered antibodies in rhesus macaques [ ] and was also used in a human hiv- clinical trial wherein it generated antibody and t-cell responses [ ] . another adjuvant in that series, as a, also elicited immune responses in humans [ ] , but which of the adjuvants in this series is the best for an hiv- vaccine is not yet established. oil-in-water emulsions as adjuvants have been used for many years, and include mineral oil-based formulations (e.g., freund's adjuvant) and more modern squalene-based preparations. they have also proved to be useful platforms for exploring the addition of immune stimulants and other compounds. tlr agonists like cpg oligodeoxynucleotides mixed with the squalene-based adjuvant mf appeared to enhance the adjuvant effect [ ] . the addition of carbopol to mf enhanced immunogenicity in rabbits to levels comparable with complete freund's adjuvant, likely because of the slower release of the immunogen [ ] . more recently, we reported that combinations of tlr ligands in a different squalene-based oil-in-water emulsion stimulated higher titers of antibodies and a greater breadth of functional responses, and that the combination of tlr / and tlr agonists was optimal in rhesus macaques [ ] . other adjuvant formulations have been studied as well. liposomes formulated with a modified polyethylene glycol elicited durable antibody responses to an env gp peptide; the proposed mechanism was persistence of the modified liposomes leading to a prolonged immune response [ ] . compounds derived from pathogens have also shown promise in initial studies. a protein derived from the worm onchocerca volvulus enhanced antibody responses in mice [ ] . there have been multiple human trials with env protein immunogens combined with different adjuvant formulations (table ) [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] . unfortunately, comparative data are lacking, especially head-to-head comparisons of adjuvants using the same immunogen and dosing schedule. the use of adjuvants in humans demonstrates promise; for example, the as a adjuvant formulated with a env gp immunogen was able to elicit similar titers of antibodies despite a -fold difference in the high and low immunogen dose groups, suggesting that the adjuvant might have a dose-sparing effect [ ] . additional studies will be needed to determine the best adjuvant-immunogen combinations for future large-scale trials. for eliciting mucosal responses, cholera toxin (ct) and other bacterial products have been extensively tested in animal models. ct combined with env gp elicited mucosal iga in rhesus macaques [ ] ; other studies in rhesus (albeit with a different form of ct) have elicited more mixed responses [ ] . ct has also been used to direct responses to the mucosa by combining it with agents that enhance retention at the mucosal surface, and has permitted dose sparing [ ] . in addition, modified ct combined with cytokines were able to elicit mucosal antibodies to a peptide immunogen when given to cynomolgus monkeys [ ] , suggesting that promising combinations identified by other vaccine strategies (e.g., dna vaccination) might be useful for mucosal immunization. other mucosal strategies are being investigated. interestingly, intranasal cytokines appear to be as effective as ct in eliciting mucosal antibodies in mice [ ] . a soybean oil nanoemulsion delivered intranasally with env gp elicited iga responses [ ] . other bacterial products, like mycoplasmaderived lipopeptides, have been shown to adjuvant vaccines in mice [ , ] . cranage et al. [ ] demonstrated that env gp administered with carbopol intravaginally resulted in better mucosal responses than systemic immunization. in mice, thymic stromal lymphopoietin has been shown to elicit mucosal antibody at levels similar to ct [ ] . finally, a strategy employing microneedles combined with a tlr agonist was able to elicit strong antibody responses including vaginal iga in mice [ & ]. it is possible that a successful hiv- vaccine strategy may involve heterologous immunizations as was used in the rv alvac-prime/aidsvaxboost trial [ ] . such strategies continue to be investigated in animal models. a regimen containing peptides adjuvanted with imiquimod and an oilin-water emulsion was able to prime for viral vector boosts in rhesus macaques, eliciting strong t-cell and modest antibody responses [ ] . similarly, an alphavirus-based particulate vaccine prime combined with a protein boost using mf in rhesus macaques resulted in a better response that was also somewhat protective against infectious challenge [ ] . side-effect considerations may also drive heterologous prime-boost regimens. a study in rabbits using an oil-in-water emulsion for the prime and alum for the boost showed that antibody responses were highest with the mixed regimen [ ] . the authors suggested that the regimen could be used to overcome the undesirable side effects of strongly adjuvanted vaccines by using less reactive adjuvants in subsequent steps. as edelman and tacket [ ] aptly stated in , 'the best adjuvant will never correct the choice of the wrong epitope.' for now, the aids vaccine field has not identified the best immunogen(s) and so work continues to find a strategy that will elicit durable and broad protection against infection. the work to find an optimal adjuvant strategy will continue to parallel these efforts. at present, the data to drive rational choices of adjuvants for an aids vaccine are lacking. this is partly because of the lack of a robust immunogen, but it is also because of the paucity of comparative data being published. in the last decade, few headto-head comparisons of adjuvant formulations using the same hiv- immunogen have been reported, especially when compared with the first years of the aids pandemic (table ) . a partial reason for this is that adjuvants are not licensed by themselves but only as part of the licensure of a vaccine product. that is an entirely appropriate regulatory hurdle, but it does mean that an adjuvant licensed or on track for licensure combined with a vaccine for a non-hiv pathogen could be put at risk. if an adjuvant is found to be superior for an hiv- vaccine candidate, by definition other adjuvants will be inferior for that hiv- vaccine. this does not mean that an adjuvant inferior for an hiv- vaccine is inferior for all other vaccines, nor would it render a licensed vaccine ineffective. however, it would create a perception that one company's adjuvant is 'better' than the others, putting vaccines using the 'inferior' adjuvants at risk. given that the vaccine market is small compared with blockbuster drugs [ ] , companies appear to be appropriately reluctant to put their investments at risk. in addition, other hurdles face adjuvant development. as preventive measures, vaccines should be well tolerated for the general population and ideally cause no side-effects to anyone. as vaccines require stimulation of the immune system, establishing a balance between stimulation and side-effects is paramount (fig. ) . however, no medical intervention is without risk and it is likely that a successful vaccine will cause some degree of side-effects in some recipients, and it will be important to determine the level of acceptable risk that balances with vaccine efficacy. public judgment of acceptable risk will depend on vaccine efficacy, that is, a highly effective vaccine against a present threat that has some side-effects will likely be more acceptable than a vaccine that is less effective or is against a pathogen perceived to be less of a threat. until an effective hiv- vaccine is available, work to find better adjuvants should continue. a wide variety of adjuvant formulations are available to enhance the response to hiv- immunogens and exciting new work suggests that formulations with better balances between safety and efficacy may be possible. however, there is much work remaining to determine the optimal adjuvant immunogen combination that will be effective in controlling the aids pandemic. the views expressed by m.a.m. are entirely his own and do not necessarily reflect those of duke university or the us government. conflicts of interest m.a.m. is supported by the center for hiv/aids vaccine immunology-immunogen discovery grant (chavi-id; grant um ai ). there are no conflicts of interest. papers of particular interest, published within the annual period of review, have been highlighted as: immune stimulation systemic inflammation safety fewer side effects ? figure . adjuvant activity balance. adjuvants need to be both well tolerated and effective, but the ability to stimulate an immune response is often associated with side effects. further investigation will be needed to determine whether efficacy can be achieved with a low side-effect profile or if some degree of inflammation will be necessary. key roles of adjuvants in modern vaccines this is an excellent review that contains timeline of adjuvant development and overview of mechanisms of adjuvant action. . barouch dh, letvin nl, seder ra. the role of cytokine dnas as vaccine adjuvants for optimizing cellular immune responses vaccine adjuvants: putting innate immunity to work this is an excellent review of adjuvants and the role of prrs in triggering the immune system. good figures and tables; has a useful summary about the kinds of innate immune cells recruited by different stimulatory triggers novel adjuvants for b cell immune responses vaccine adjuvants: mode of action cytokines as adjuvants for improving anti-hiv responses enhancement of human immunodeficiency virus type -dna vaccine potency through incorporation of t-helper molecular adjuvants a reflection on hiv/aids research after years a group specific anamnestic immune reaction against hiv- induced by a candidate vaccine against aids immunization against aids in humans immune response of chimpanzees after immunization with the inactivated whole immunodeficiency virus (hiv- ), three different adjuvants and challenge fusion-competent vaccines: broad neutralization of primary isolates of hiv immune response to immunostimulatory complexes (iscoms) prepared from human immunodeficiency virus type (hiv- ) or the hiv- external envelope glycoprotein (gp ) safety and immunogenicity of a fully glycosylated recombinant gp human immunodeficiency virus type vaccine in subjects at low risk of infection. national institute of allergy and infectious diseases aids vaccine evaluation group network bangkok vaccine evaluation group: randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein hiv- vaccine among injection drug users in rgp hiv vaccine study group: placebo-controlled phase trial of a recombinant glycoprotein vaccine to prevent hiv- infection immunization of chimpanzees with the hiv- glycoprotein gp induces long-lasting t-cell memory effects of adjuvants and multiple antigen peptides on humoral and cellular immune responses to gp of hiv- high-titer hiv- neutralizing antibody response of rhesus macaques to gp and env peptides saponin adjuvant enhancement of antigen-specific immune responses to an experimental hiv- vaccine adjuvant effect of liposomes and adamantylamide dipeptide on antigenicity of entrapped synthetic peptide derived from hiv- transmembrane region glycoprotein gp comparison of different adjuvants for inactivated hiv- split whole virus as antigen in mice. induction of titres of binding antibodies and toxicity of the formulations candidate hiv type multideterminant cluster peptide-p mn vaccine constructs elicit type helper t cells, cytotoxic t cells, and neutralizing antibody, all using the same adjuvant immunization development of a single-shot subunit vaccine for hiv- . . effect of adjuvant and immunization schedule on the duration of the humoral immune response to recombinant mn gp immunogenicity of hiv- lai gp and env peptides in squirrel monkey saimiri sciureus using alumine and experimental adjuvants comparison of nucleic acid and protein immunization for induction of antibodies specific for hiv- gp adjuvant is required when using env lipopeptide construct to induce hiv type -specific neutralizing antibody responses in mice in vivo comparison of immunity generated by nucleic acid-, mf -, and iscom-formulated human immunodeficiency virus type vaccines in rhesus macaques: evidence for viral clearance safety profile of phase i and ii preventive hiv type envelope vaccination: experience of the niaid aids vaccine evaluation group public health. a sound rationale needed for phase iii hiv- vaccine trials support for the rv hiv vaccine trial: new series vaccination with alvac and aidsvax to prevent hiv- infection in thailand risk behaviour and time as covariates for efficacy of the hiv vaccine regimen alvac-hiv (vcp ) and aidsvax b/e: a posthoc analysis of the thai phase efficacy trial rv intranasal administration of human immunodeficiency virus type- (hiv- ) dna vaccine with interleukin- expression plasmid enhances cell-mediated immunity against hiv- il- expression plasmid enhances cell-mediated immunity induced by an hiv- dna vaccine immunization of rantes expression plasmid with a dna vaccine enhances hiv- -specific immunity multimeric soluble cd ligand and gitr ligand as adjuvants for human immunodeficiency virus dna vaccines studies on gm-csf dna as an adjuvant for neutralizing ab elicited by a dna/mva immunodeficiency virus vaccine novel codon-optimized gm-csf gene as an adjuvant to enhance the immunity of a dna vaccine against hiv- gag enhancement of immune responses to an hiv gp dna vaccine by fusion to tnf alpha cdna augmenting the immunogenicity of dna vaccines: role of plasmid-encoded flt- ligand, as a molecular adjuvant in genetic vaccination macrophage inflammatory protein- alpha (mip- alpha) expression plasmid enhances dna vaccine-induced immune response against hiv- soluble multitrimeric tnf superfamily ligand adjuvants enhance immune responses to a hiv- gag dna vaccine interferon regulatory factor- acts as a powerful adjuvant in the dna based vaccination enhancement of immune responses to an hiv env dna vaccine by a c-terminal segment of listeriolysin o il- as memory t-cell adjuvant for topical hiv- dermavir vaccine potent cd þ t cell responses elicited by a bicistronic hiv- dna vaccine expressing gp and gm-csf comparative ability of various plasmidbased cytokines and chemokines to adjuvant the activity of hiv plasmid dna vaccines enhancement of hiv- dna vaccine immunogenicity by bcg-psn, a novel adjuvant augmentation of hiv- subtype c vaccine constructs induced immune response in mice by cpg motif -odn hiv- -specific cell-mediated immune responses induced by dna vaccination were enhanced by mannan-coated liposomes and inhibited by antiinterferon-gamma antibody topical delivery of imiquimod to a mouse model as a novel adjuvant for human immunodeficiency virus (hiv) dna recruitment of antigen-presenting cells to the site of inoculation and augmentation of human immunodeficiency virus type dna vaccine immunogenicity by in vivo electroporation high antibody and cellular responses induced to hiv- clade c envelope following dna vaccines delivered by electroporation combined effects of il- and electroporation enhances the potency of dna vaccination in macaques safety and comparative immunogenicity of an hiv- dna vaccine in combination with plasmid interleukin and impact of intramuscular electroporation for delivery only t-cell immunogenicity data are reported, but demonstrates that dna vaccination with electroporation is effective for eliciting responses in humans improving recombinant mva immune responses: potentiation of the immune responses to hiv- with mva and dna vectors expressing env and the cytokines il- and ifn-gamma il- delivery from recombinant vaccinia virus attenuates the vector and enhances the cellular immune response against hiv- env in a dose-dependent manner cd l expressed from the canarypox vector, alvac, can boost immunogenicity of hiv- canarypox vaccine in mice and enhance the in vitro expansion of viral specific cd þ t cell memory responses from hiv- -infected and hiv- -uninfected individuals the adjuvancy of ox ligand (cd ) on an hiv- canarypox vaccine interferon-beta expressed by a rabies virus-based hiv- vaccine vector serves as a molecular adjuvant and decreases pathogenicity multimeric soluble cd ligand (scd l) efficiently enhances hiv specific cellular immune responses during dna prime and boost with attenuated poxvirus vectors mva and nyvac expressing hiv antigens oral vaccination with modified vaccinia virus ankara attached covalently to tmpeg-modified cationic liposomes overcomes preexisting poxvirus immunity from recombinant vaccinia immunization effect of the strength of adsorption of hiv sf dv gp to aluminum-containing adjuvants on the immune response a demonstration of the 'goldilocks phenomenon' in which binding to alum must be neither too weak nor too strong for optimal immunogenicity induction of hiv- mpr( - )-specific iga and igg antibodies in caprine colostrum using a peptide-based vaccine characterization of antibody responses elicited by human immunodeficiency virus type primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants human immunodeficiency virus type env trimer immunization of macaques and impact of priming with viral vector or stabilized core protein strong and persistent cd þ t-cell response in healthy adults immunized with a candidate hiv- vaccine containing gp , nef and tat antigens formulated in three adjuvant systems durable hiv- antibody and t-cell responses elicited by an adjuvanted multiprotein recombinant vaccine in uninfected human volunteers neutralizing antibody responses to subtype b and c adjuvanted hiv envelope protein vaccination in rabbits mixed adjuvant formulations reveal a new combination that elicit antibody response comparable to freund's adjuvants tlr- / and agonists cooperate to enhance hiv- envelope antibody responses in rhesus macaques adjuvanticity of stealth liposomes on the immunogenicity of synthetic gp epitope of hiv- rov-asp- , a recombinant secreted protein of the helminth onchocerca volvulus, is a potent adjuvant for inducing antibodies to ovalbumin, hiv- polypeptide and sars-cov peptide antigens safety and immunogenicity of a genetically engineered human immunodeficiency virus vaccine safety and immunogenicity of env - , a human immunodeficiency virus type candidate vaccine, in combination with a novel adjuvant, mtp-pe/mf . niaid aids vaccine evaluation group a phase i trial in hiv negative healthy volunteers evaluating the effect of potent adjuvants on immunogenicity of a recombinant gp w d derived from dual tropic r x hiv- ach phase i/ii study of a candidate vaccine designed against the b and e subtypes of hiv- a novel adjuvant for mucosal immunity to hiv- gp in nonhuman primates cta -dd adjuvant promotes strong immunity against human immunodeficiency virus type envelope glycoproteins following mucosal immunization capric acid and hydroxypropylmethylcellulose increase the immunogenicity of nasally administered peptide vaccines a comparative evaluation of nasal and parenteral vaccine adjuvants to elicit systemic and mucosal hiv- peptidespecific humoral immune responses in cynomolgus macaques cytokines as adjuvants for the induction of antihuman immunodeficiency virus peptide immunoglobulin g (igg) and iga antibodies in serum and mucosal secretions after nasal immunization nasal immunization with a recombinant hiv gp and nanoemulsion adjuvant produces th polarized responses and neutralizing antibodies to primary hiv type isolates efficient mucosal delivery of the hiv- tat protein using the synthetic lipopeptide malp- as adjuvant efficient systemic and mucosal responses against the hiv- tat protein by prime/boost vaccination using the lipopeptide malp- as adjuvant antibody responses after intravaginal immunisation with trimeric hiv- cn clade c gp in carbopol gel are augmented by systemic priming or boosting with an adjuvanted formulation thymic stromal lymphopoietin (tslp) acts as a potent mucosal adjuvant for hiv- gp vaccination in mice microneedle mediated intradermal delivery of adjuvanted recombinant hiv- cn gp effectively primes mucosal boost inoculations this is an interesting study combining microneedles and adjuvants, and can be applied to skin or mucosal surfaces to elicit an immune response prime-boost regimens with adjuvanted synthetic long peptides elicit t cells and antibodies to conserved regions of hiv- in macaques antibody-mediated protection against mucosal simian-human immunodeficiency virus challenge of macaques immunized with alphavirus replicon particles and boosted with trimeric envelope glycoprotein in mf adjuvant a prime-boost regime that combines montanide isa and alhydrogel to induce antibodies against the hiv- derived multiepitope polypeptide tab bumps on the vaccine road key: cord- -moor dfc authors: richards, james; rodan, ilona title: feline vaccination guidelines date: - - journal: veterinary clinics of north america: small animal practice doi: . /s - ( ) - sha: doc_id: cord_uid: moor dfc the report of the american association of feline practitioners and academy of feline medicine advisory panel on feline vaccines was developed to help veterinary practitioners formulate vaccination protocols for cats. the current panel report updates information, addresses questions, and speaks to concerns raised by the report. in addition it reviews vaccine licensing, labeling, and liability issues and suggests ways to successfully incorporate vaccination protocol changes into a private practice setting. patients are healthy before vaccination. because vaccination alone does not completely protect animals from infection and disease, environmental conditions should be addressed and exposure to infectious agents should be minimized. the overall objectives of vaccination are to vaccinate the largest possible number of individuals in the population at risk, vaccinate each individual no more frequently than necessary, and vaccinate only against infectious agents to which individuals have a realistic risk of exposure and subsequent development of disease. kittens younger than weeks of age are generally more susceptible to infection than are adult cats and typically develop more severe disease. they represent the principal target population for vaccination. maternal antibody interference is the most common reason why some animals are not immunized after vaccination, and it is also the reason why a series of vaccinations is necessary for kittens younger than weeks of age. vaccination needs of adult cats should be assessed at least once yearly and, if necessary, modified on the basis of an assessment of their risk. it is recommended that administration sites for parenteral vaccine be chosen in accordance with the guidelines established by the american association of feline practitioners and adopted by the vaccine-associated feline sarcoma task force (table ) . use of multiple-dose vials is discouraged, because inadequate mixing may result in unequal distribution of antigen and adjuvant, possibly resulting in decreased efficacy or an increased likelihood of adverse events; iatrogenic contamination is an additional risk. the panel discourages the use of polyvalent vaccines other than those containing combinations of feline panleukopenia virus, feline herpesvirus- (fhv- ), and feline calicivirus (fev), exclusively. this opinion is based on the belief that as the number of antigens in a vaccine increases, so too does the probability of associated adverse events. additionally, use of polyvalent vaccines may force practitioners to administer vaccine antigens not needed by the patient. feline panleukopenia is caused by feline parvovirus (fpv). the virus remains infectious for months to years in the environment and is primarily spread via the fecal-oral route. fomites (e.g., cages, food bowls, litter boxes, health care workers) play an important role in the transmission of the organism. clinical signs of infection include lethargy, an-orexia, vomiting, diarrhea, fever, and profound panleukopenia; mortality is highest in young susceptible cats. in utero infection with fpv is a common cause of cerebellar hypoplasia. vaccination against fpv is highly recommended for all cats. immunity to 'feline panleukopenia is primarily through antibody response to natural infection, vaccination, or passive transfer of maternal antibodies from queen to kittens. maternal antibody may interfere with immunization when antibody titers are high during the neonatal period. maternal antibody titers generally wane sufficiently to allow immunization by weeks of age. immunity conferred by feline panleukopenia vaccines is considered to be excellent, and most vaccinated animals are completely protected from infection and clinical disease. serologic and challenge exposure data indicate that a parenteral fpv vaccine induces immunity that is sustained for at least years. s , after the initial series of vaccinations and revaccination year later, cats should be vaccinated no more frequently than once every years. modified-live virus (mlv) vaccines and adjuvanted inactivated virus vaccines for parenteral administration as well as an mlv vaccine for topical (intranasal) administration are available and effective. experimental studies have shown that intranasal administration of canine parvovirus- vaccines to puppies is less effective than parenteral administration in overcoming maternal antibody interference (ronald schultz, phd, personal communication, ). the most likely reasons are that fewer virus particles reach lymphoid tissue when the product is given intranasally compared with parenteral administration and viral replication in lymphoid tissue is required for immunization with mlv parvovirus vaccines. although studies have not been performed in cats, the same phenomenon may occur in this species. caution is appropriate when contemplating the use of intranasal fpv vaccines for primary immunization of kittens, especially those residing in environments where exposure to fpv is likely. it has been found recently that some cats with panleukopenia-like disease were infected with canine parvovirus- b. studies show that fpv vaccines provide excellent protection not only from fpv but also from canine parvovirus- b; thus, canine parvovirus infection should not be a concern for cats immunized as a result of vaccination with fpv vaccines. serious adverse events associated with fpv vaccines are rare. tumor formation at the site of a topically administered vaccine has not been reported. vaccination of pregnant queens with modified-live fpv vaccines may possibly result in neurologic disease in developing fetuses ; the same concern applies to kittens vaccinated at less than weeks of age. the use of mlv vaccines should be avoided in pregnant queens and kittens less than month of age. iimost often, the product approved for use annually is given for initial vaccination followed year later and every years after that by administration of the product approved for use every years; however, vaccination interval must comply with local and state statutes. ilfelv testing is recommended before vaccination; infected cats do not derive any benefit from vaccination. *'this product is not the same as the b. bronchiseptica vaccine approved for use in dogs; the product approved for use in dogs should not be used in cats. mlv = modified-live virus; felv = feline leukemia virus; sc = subcutaneously. feline viral rhinotracheitis caused by fhv- and fcv infection account for up to %. of all cases of infectious upper respiratory tract disease in cats. both viruses are shed in ocular, nasal, and pharyngeal secretions of infected cats. organisms are transmitted from cat to cat directly through sneezed macrodroplets or indirectly via contaminated fomitesy the disease is self-limiting; however, infected cats may develop chronic oculonasal disease. latent infection is lifelong for cats infected with fhv- ; reactivation can occur during periods of stress or after corticosteroid administration. some cats infected with fcv become persistently infected and shed virus for prolonged periods (months to years). although rarely serious in adult cats, disease caused by these viruses may be severe, and· sometimes fatal, in kittens. lameness and chronic oral inflammatory syndromes have been linked to calicivirus infection and vaccination with modified-live calicivirus vaccines. , , , , , risk of exposure to either fhv- or fcv is high, because both organisms are widespread tn the feline population. vaccination against fhv- and fcv is highly recommended for all cats. immunity is through humoral and cell-mediated immune responses to natural infection or vaccination or through passive transfer of maternal antibodies from queen to kittens. maternal antibody may interfere with induction of a systemic immune response; however, by the time that kittens are weeks of age, maternal antibody titers wane sufficiently to allow parenteral immunization. topically administered (intranasal, conjunctival) vaccines are capable of inducing a local immune response in the face of high maternal antibody titers. serologic and challenge exposure data indicate that parenteral fhv- and fcv vaccines induce protection that lasts at least years. , after the initial series of vaccinations and revaccination year later, cats should be vaccinated once every years. regardless of the route of administration, fhv- and fcv vaccines induce only relative but not complete protection. at best, these vaccines induce an immune response that lessens the severity of disease; vaccinates are not immune to infection nor are they protected from all signs of disease. o currently available fcv vaccines probably do not induce protection from all isolates of the virus. mlv and inactivated virus vaccines for parenteral administration and mlv vaccines for topical (intranasal and conjunctival) administration are available. if a susceptible cat is born into or is entering an environment in which viral upper respiratory tract disease is endemic (e.g., some catteries, boarding facilities, shelters), the use of a topical product may be advantageous. administration of such products to kittens as young as to days of age could be considered in these situations; however, products that also contain modified-live fpv antigens should not be administered to kittens younger than weeks of age. adverse events associated with vaccination against fhv-l and fev include mild transient fever, sneezing, conjunctivitis, oculonasal discharge, lameness, and, for parenteral products, pain at the injection site. , sneezing, conjunctivitis, oculonasal discharge, and ulceration of the nasal philtrum are believed to occur more frequently with vaccines licensed for topical use. tumor formation at the site of a topically administered vaccine has not been reported. rabies is transmitted mainly through bite wounds of infected mammals. more cats than dogs develop rabies in the united states ; although they are relatively resistant to rabies, both species serve as potential sources of infection for human beings. , treatment is ineffective in cats that develop clinical signs and should not be attempted because of the high potential for zoonotic infection. all instances of suspected or known rabies virus infection must be reported to local health department officials. proper precautions and quarantine procedures as outlined by local regulations and described in the compendium of animal rabies prevention and control, should be followed. although vaccine-associated sarcomas have been reported to develop in association with administration of a variety of vaccines, current data suggest that they are more frequently associated with administration of feline leukemia virus (felv) vaccines and adjuvanted rabies virus vaccines. inflammatory reactions are commonly observed at sites where adjuvanted rabies virus vaccines have been administered, and concern has arisen regarding the possible association between these reactions and vaccine-associated sarcomas. with the exception of a recently approved canarypox virus-vectored recombinant feline rabies virus vaccine (pure-vax feline rabies vaccine; merial limited, iselin, nj), all rabies virus vaccines currently on the market contain adjuvants. in rats, inflammation induced by the recombinant product seems to be minimal, but whether the use of this vaccine is associated with a reduced likelihood of vaccineassociated sarcoma formation in cats is not yet known. the recombinant product is currently licensed only for annual administration. rabies virus vaccination is highly recommended for all cats and is required by law in some states and municipalities. manufacturers are required by the us department of agriculture to establish, by means of experimental challenge exposure studies, the minimum duration of immunity for the rabies virus vaccines that they sell, and products approved for use every year or every years are available. statutes governing the administration of rabies virus vaccines vary considerably throughout the united states; veterinarians should comply with the legal requirements of their area. felv infects domestic cats throughout the world. transmission is through transfer of virus in the saliva or nasal secretions resulting from prolonged intimate contact (e.g., mutual grooming), biting, or sharing of food and water utensils. the virus may also be transmitted by transfusion of blood from an infected cat, in utero, or through the milk. exposure to virus persisting in the environment on fomites or in aerosolized secretions is not an efficient means of viral transmission. clinical signs of felv infection are primarily related to neoplasia, anemia, and diseases resulting from immunosuppression. kittens are the most susceptible to infection; resistance increases with maturity. experimental data demonstrate that kittens younger than weeks of age are most susceptible to infection, with cats older than this being relatively resistant. cats at greatest risk include outdoor cats (free-roaming pets, stray cats, and feral cats). also at risk are cats residing in open multiple-cat environments, cats living with felv-infected cats, and cats residing in households with unknown felv status. the decision to vaccinate an individual cat against felv infection should be based on the cat's age and its risk of exposure. vaccination against felv is recommended for cats at risk of exposure (i.e., cats not restricted to a closed, felv-negative, indoor environment), especially those younger than months of age. vaccination is not recommended for cats with minimal to no risk of exposure, especially those older than months of age. the ability of a particular vaccine brand to induce an immune response sufficient to resist persistent viremia varies from study to study.s because protection is not induced in all vaccinates, preventing exposure to infected cats remains the single best way to prevent felv infection. vaccination against felv does not diminish the importance of testing cats to identify those that are viremic. it is of critical importance that viremic cats not be in contact with other cats, especially those younger than months of age. as a result, the felv infection status of all cats should be determined. adverse events associated with vaccination against felv include local swelling or pain, transient lethargy or fever, and postvaccination granuloma formation. although vaccine-associated sarcomas have been reported to develop in association with administration of other vaccines, current data suggest that they are more frequently associated with administration of felv vaccines and adjuvanted rabies virus vaccines. if vaccination is deemed appropriate, annual revaccination is recommended. cats should be tested for felv infection before initial vaccination and when there is a possibility that they have been exposed to felv since they were vaccinated. the enzyme-linked immu-nosorbent assay is the preferred screening test; the indirect immunofluorescent assay is the preferred confirmatory test. individuals confirmed to be infected with felv need not receive felv vaccines, but they should be segregated from uninfected cats. chlamydia psittaci is a bacterial pathogen of the conjunctiva and respiratory tract of cats. transmission is through direct cat-to-cat contact; fomite transmission is less likely, because the organism is unstable in the environment. serous conjunctivitis, which may initially affect only one eye, is the most common clinical sign. sneezing or nasal discharge may develop, but if it does develop, it is usually mild. clinical signs are usually evident to days after infection and resolve with appropriate antimicrobial treatmenu isolation rates have been reported to range from a£proximately % for cats without signs of respiratory tract disease to approximately % for cats with concurrent upper respiratory tract disease. the highest rates of infection are reported for cats between weeks and months of age. immunity conferred by c. psittaci vaccines is similar to that conferred by fhv- and fcv vaccines in that vaccinates are protected from severe clinical disease but not from infection. the frequency of adverse systemic events associated with c. psittaci vaccines is higher than that associated with other commonly used vaccines; reactions include lethargy, depression, anorexia, lameness, and fever to days after vaccination. because signs of disease associated with c. psittaci infection are comparatively mild and respond favorably to treatment, and because adverse events associated with the use of c. psittaci vaccines are of greater concern than adverse events associated with the use of many other products, routine vaccination against c. psittaci infection is not recommended. vaccination may be considered for cats in multiple-cat environments, where infections associated with clinical disease have been confirmed. if vaccination is deemed appropriate, annual revaccination is recommended. feline coronaviruses (fcovs) vary considerably in pathogenic potential and have historically been grouped into two biotypes: feline enteric coronaviruses, which typically cause subclinical to mild enteric infections, and feline infectious peritonitis (fip) viruses, which cause fip. currently, pip viruses are believed to be generated as mutant variants in feline enteric coronavirus-infected cats. , fcovs are widespread in feline populations worldwide, with seropositivity rates highest in crowded multiple-cat environmentsy transmission of the virus is mainly via the fecal-oral route. in environments in which fcov infection is endemic (e.g., most multiple-cat environments), % to % of cats are shedding fcovs in the stool at any given time. , most infected cats remain healthy, although a few (usually between % and %) ultimately develop fip. affected cats rarely survive regardless of treatment. kittens are most often affected with fip, but the disease reportedly can develop in cats of all ages. a genetic predisposition has been suggested, with higher disease incidence in certain lines. , considerable controversy surrounds the ability of the currently available fip vaccine (primucell-fip; pfizer animal health, exton, pa) to prevent disease. some studies demonstrate protection from disease i , ; others show little benefit from vaccination. , antibody-dependent enhancement of disease in vaccinates has been demonstrated in experimental challenge exposuj'e studies, but it is uncertain whether antibody-dependent enhancement occurs in a natural setting. discrepancies between study results are probably attributable to differences in test methodology (e.g., strain and dose of challenge virus, genetic predisposition of the test animals). protection from disease has not been demonstrated in animals vaccinated when younger than weeks of age. most kittens born and reared in environments in which fco v infection is endemic are infected before reaching this age. l , in these instances, vaccination of infected cats has not proven beneficial. at this time, there is no evidence that the vaccine induces clinically relevant protection, and its use is not recommended. canis infection is not recommended. at the time of this writing, the product has not been independently evaluated for efficacy. based on studies conducted by the manufacturer, it is reasonable to consider vaccination as adjunctive treatment for individual infected cats months of age or older to hasten resolution of clinical signs. if the vaccine induces an immune response that accelerates lesion resolution, the number of infectious fungal spores produced by vaccinates may be reduced as well; thus, it is reasonable to consider vaccination as one component of a comprehensive treatment program in multiple-cat environments in which m. canis infection is endemic. nonetheless, the ability of this product to hasten elimination of endemic infections from such environments has not been evaluated. the revaccination interval is not stipulated on the label. major adverse events reportedly associated with the use of this product are pain, temporary hair loss, and formation of sterile abscesses or granulomas at the vaccine site. bordetella bronchiseptica is a small, aerobic, gram-negative coccobacillus long recognized as a respiratory tract pathogen of several species of animals. the natural route of transmission in cats is believed to be via the aerosol or intranasal route. experimental challenge exposure studies have shown that b. bronchiseptica can act as a primary pathogen in cats; inoculation of specific-pathogen-free kittens results in self-limiting disease characterized by variable degrees of fever, nasal or ocular discharge, sneezing, induced or spontaneous coughing, pulmonary rales, and submandibular lymphadenopathy. bronchopneumonia associated with naturally occurring b. bronchiseptica infection has been reported in kittens and adult cats. other factors, including nutritional status, overcrowding, coinfection with other agents such as fev or fhv- , and suboptimal hygiene, may influence the outcome of exposure. .s seroprevalence surveys suggest that exposure to the organism is common, with infection rates varying from population to population. the highest rates of seropositivity (often over %) are found among cats in rescue shelters and multiple-cat households, especially when there is a history of respiratory tract disease. the lowest rates are found among cats in households with few cats and no history of respiratory tract disease. • similarly, isolation rates vary. b. bronchiseptica was isolated from the oropharynx in of ( . %) asymptomatic cats and from the distal trachea in of ( %) asymptomatic cats from shelters in lousiana. s in a recent survey of cats in the united kingdom, none of the household cats were found to be infected, but % of cats from breeding colonies and % of cats from rescue shelters were found to be carrying the organism. s in the same survey, % of healthy cats and % of cats with respiratory tract disease tested positive for the organism. an additional finding was a strong positive association between oropharyngeal isolation of b. bronchiseptica and residence in households containing dogs with a recent history of respiratory tract disease. definitive diagnosis of disease associated with b. bronchiseptica infection may be difficult, in part, because signs of infection often mimic those associated with fhv-l or fev infection. isolation of b. bronchiseptica from a cat with respiratory tract disease is supportive of the diagnosis, but carriage of the organism in asymptomatic cats precludes establishing a direct cause-and-effect relation. resolution of disease with appropriately chosen antimicrobial medication might suggest a causative role for b. bronchiseptica, but the self-limiting nature of many cases of viral upper respiratory tract disease prevents attributing disease resolution solely to antimicrobial treatment. a vaccine (protex-bb; intervet) to prevent disease caused by infection with b. bronchiseptica has recently been licensed. the product contains a live reduced-virulence culture of b. bronchiseptica and is licensed for administration via the intranasal route in cats weeks of age and older. efficacy of the vaccine has not been independently evaluated; however, in studies conducted by the manufacturer to gain vaccine licensure, vaccinated -week-old specific-pathogen-free cats experienced less severe signs of disease than did unvaccinated controls when exposed to challenge weeks after vaccination. similar results were obtained when -week-old kittens were exposed to challenge hours after vaccination. as of this writing, studies to evaluate the duration of protection induced by the vaccine have not been completed and the revaccination interval is not yet stipulated on the label. routine use of this vaccine is not recommended. it is reasonable to consider vaccinating cats entering or residing in multiple-cat environments (e.g., shelters, catteries, boarding facilities) where disease associated with b. bronchiseptica infection has been confirmed. the ability of the product to reduce the prevalence of infection or the severity of disease in such environments has not been evaluated, however. infection of cats with the protozoan giardia lamblia is associated with acute or chronic gastrointestinal disease ranging in severity from subclinical to severe. , because infected cats shed cysts intermittently, diagnosis of g. lamblia infection is often cumbersome and usually requires multiple fecal examinations. several methods of diagnosis are available, including examination of a fecal smear, the zinc sulfate centrifugation method, and use of an enzyme-linked immunosorbent assay to test feces. there are currently no approved treatment methods for cats, and although treatment commonly controls signs of disease, it is uncertain that it clears infection. treatment effectiveness is highly variable, and resistant organisms are commonly encountered. , g. lamblia is transmitted via the fecal-oral route; cysts may be ingested from contaminated water, from direct cat-to-cat transmission especially in crowded environments (e.g., through mutual grooming), from exposure to contaminated litter boxes, and from consuming prey. d, giardiasis is a recognized zoonotic disease, but the role of cats in transmission of the organism is not well established. , , a vaccine has recently been licensed by the us department of agriculture (fel-o-vax giardia; fort dodge animal health) as an aid in the prevention of disease associated with c. lamblia infection and reduction in the severity of shedding of cysts. this vaccine is composed of quantified, homo gena ted, and chemically inactivated g. lamblia trophozoites, and it contains an adjuvant commonly found in other feline products from the manufacturer but different from the adjuvant in the manufacturer's canine product. the vaccine is approved for use in cats weeks of age and older. at the time of this writing, the vaccine has not been independently evaluated for efficacy, but in studies conducted by the manufacturer to gain vaccine licensure, vaccinates had a statistically significant reduction in severity of clinical signs (diarrhea), duration of cyst shedding, and prevalence of infection (percentage of cats with trophozoites at the end of the trial) compared with control animals. protection was demonstrated to persist for at least year after vaccination. routine use of this vaccine is not recommended, but because vaccinates had less severe clinical disease and shed cysts for a shorter time, it is reasonable to consider vaccination as part of a comprehensive control program in environments where exposure to g. lamblia is clinically significant. when parasite exposure is ongoing, revaccination at annual intervals is recommended. some vaccinates may shed cysts subsequent to c. lamblia exposure; thus, proper hygiene and sanitation practices should be implemented even with vaccinated cats. the ability of this product to aid in hastening elimination of endemic infection from multiple-cat environments has not been evaluated. a study of naturally occurring feline coronavirus infections in kittens enteric protozoal infections detection of feline calicivirus antigens in the joints of infected cats prevalence of antibodies against bordetella bronchiseptica in cats with a history of respiratory disease prevalence and risk factors for feline bordetella bronchiseptica infection lameness in kittens after vaccination studies on natural transmission of bordetella bronchiseptica in cats problems with respiratory virus vaccination in cats acute arthritis of cats associated with feline calicivirus infection investigation of vaccine reactions and breakdowns after feline calicivirus vaccination comments on cerebellar ataxia and its congenital transmission in cats by feline panleukopenia virus american association of feline practitioners and the academy of feline medicine recommendations for feline leukemia virus testing and recommendations for feline immunodeficiency virus testing report of the american association of feline practitioners and academi'of feline medicine advisory panel on feline vaccines the inheritance of susceptibility to feline infectious peritonitis in purebred catteries patterns of feline coronavirus infection and fecal shedding from cats in multiple-cat environments viral upper respiratory infection in cats infectious diseases of the respiratory tract protection against feline infectious peritonitis by intranasal inoculation of temperature-sensitive fipv vaccine immunoprophylaxis and immunotherapy infectious diseases of the dog and cat fecal shedding of feline coronavirus in adult cats and kittens in an abyssinian cattery feline leukemia virus infection: agerelated variation in response of cats to experimental infection independent evaluation of a modified-live feline infectious peritonitis virus-vaccine under experimental conditions (louisiana experience) isolation and characterization of bordetella bronchiseptica from cats in southern louisiana compendium of animal rabies prevention and control vaccination against feline viral rhinotracheitis in kittens with maternally derived feline viral rhinotracheitis antibodies epidemiologic evidence for a causal relation between vaccination and fibrosarcoma tumorigenesis in cats rabies surveillance in the united states during protozoal and miscellaneous infections giardia: diagnosis and treatment giardiasis in dogs and cats felv and non-neoplastic felv-related disease local postvaccinal reactions of a recombinant rabies vaccine the potential role of inflammation in the development of postvaccinal sarcomas in cats independent evaluation of a modified-live fipv vaccine under experimental conditions (university of liverpool experience) seroprevalence and isolation rate of bordetella bronchiseptica in cats in the uk dermatophytosis: advances in therapy and control in august jr canine parvovirus (cpv- b) infection in cats basic and clinical immunology feline husbandry: diseases and management in the multi-cat environment feline calicivirus infection an overview of feline enteric coronavirus and infectious peritonitis virus-infections feline panleukopenia and other enteric viral diseases acute and chronic faucitis of domestic cats. a feline calicivirus-induced disease feline sarcoma task-force meets diseases of the cat: medicine and surgery. philadelphia, wb saunders duration of immunity in cats vaccinated with an inactivated feline panleukopenia, herpesvirus, and calicivirus vaccine long-term immunity in cats vaccinated with an inactivated trivalent vaccine antibody-dependent enhancement of feline infectious peritonitis virus infection independent evaluation of a modified-live fipv vaccine under experimental conditions (cornell experience) hydranencephaly and cerebellar hypoplasia in two kittens attributed to intrauterine parvovirus infection feline leukemia virus-a review of immunity and vaccination bordetella bronchiseptica infection in the cat reaction rate in cats vaccinated with a new controlled-titer feline panleukopenia-rhinotracheitis-calicivirus-chlamydia psittaci vaccine prevalence of feline chlamydia psittaci and feline herpesvirus in cats with upper respiratory tract disease chronic oral infections of cats and their relationship to persistent oral carriage of feline calici-, immunodeficiency, or leukemia viruses a comparison of the genomes of fecvs and fipvs and what they tell us about the relationships between feline coronaviruses and their evolution feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses bordetella bronchiseptica infections in cats prevalence of chlamydia psittaci in different cat populations in britain other feline viral diseases consultations in feline internal medicine key: cord- -s gw k authors: capps, benjamin; lederman, zohar title: one health, vaccines and ebola: the opportunities for shared benefits date: - - journal: j agric environ ethics doi: . /s - - - sha: doc_id: cord_uid: s gw k the ebola virus outbreak in west africa, as of writing, is declining in reported human cases and mortalities. the resulting devastation caused highlights how health systems, in particular in west africa, and in terms of global pandemic planning, are ill prepared to react to zoonotic pathogens. in this paper we propose one health as a strategy to prevent zoonotic outbreaks as a shared goal: that human and great ape vaccine trials could benefit both species. only recently have two phase / ebola human vaccine trials been started in west africa. this paper argues for a conceptual change in pandemic preparedness. we first discuss the ethics of one health. next, we focus on the current ebola outbreak and defines its victims. third, we present the notion of a ‘shared benefit’ approach, grounded in one health, and argue for the vaccination of wild apes in order to protect both apes and humans. we believe that a creation of such inter-species immunity is an exemplar of one health, and that it is worth pursuing as a coextensive public health approach. ebola virus has devastated parts of west africa, and has caused alarm worldwide. it is one of a number of notable zoonotic emergent infectious diseases (zeid), also including influenza, coronaviruses like middle east respiratory syndrome (mers), and the now pandemic human immunodeficiency virus (hiv). the majority of all eids are caused by zoonoses ; and most of these are pathogens of wildlife origin that become endemic in localised non-human animal and human populations (jones ) . these pathogens are emerging at an alarming rate, reflecting changes in local topologies and the global climate, triggered by human and animal causative and adaptive activities (epstein ) . ebola is endemic to central africa, and is normally dormant in still unknown reservoirs. periodically however, it infects local human populations, causing extensive mortalities but then fading out before widespread contagion (hayden ; marzi and feldmann ; macneil and rollin ) . the ongoing outbreak in west africa surpasses all previous occasions, although at time of this writing, the endemic appears to be receded (who ebola response team ) . many have been dismayed by the global efforts to curtail the epidemic, questioning international resolve to respond timely and effectively (mitman ; spencer ) . in particular, many have been critical of the systematic neglect of public health infrastructure, and have identified strengthening health systems as the long term solution to the disease (dawson ; farmer ; gates ; rid and emanuel ) . the measures used during this outbreak are focused on human communities, and includes clinical case management (that to date lacks any curative treatment), quarantine and isolation, surveillance and contact tracing, a rapid and reliable laboratory service, safe and dignified burials, and social education (dawson ; macneil and rollin ; marzi and feldmann ) . critics have much to say about the importance of infrastructure and basic supplies needed, but less has been said about the limitations of ebola containment measures. although these previously worked well within geographically isolated communities where ebola periodically emerged, they were less likely to do so in a sustained and widespread outbreak. in light of the current catastrophe, it now compels us to consider also the limitations of traditional public health measures during an epidemic of this magnitude, which although they may bring an acute situation under control eventually, are challenging to enforce, strain medical and social networks, and provide limited prevention and no cure. indeed, although these measures have brought the emergency to its current abating state, it took a great deal of time and vast efforts, many still died, and infection resurgence is a possibility. the importance of biomedical countermeasures, such as vaccines, therefore cannot be understated. in this respect, it has been resolved that failures in advanced drug development and production must be tackled (who ) , especially the political and economic barriers that hamper development and deployment in places such as west africa, and which further emphasise the neglect of certain transmissible diseases in that region (marzi and feldmann ) . the current perspectives to zoonotic risks and pandemic planning have changed little despite the warnings from the 'swine flu' pandemic of that the opportunities for expedient vaccine production and sustainable clinical access still seem someway off (gates ) . our particular concern, however, is that while the ethical debate is being dominated by global human threats, other considerations about endemic zoonoses are being overlooked. using ebola as a case study, we apply one health (oh) as an ethical framework to make the case for strategic changes. in particular, the debate about vaccines plausibly could be extended to the concurrent need in primate populations. this paper therefore proposes the possibility of shared immunity between species that are equally affected by ebola. our proposal for a novel approach to vaccination that protects both human communities and the fauna they interact with and often depend upon is speculative, as technical issues are far from resolved. however, we have two further intentions: firstly, to highlight the oh in general, prevention and then containment of highly pathogenic eids is about slowing and limiting the contagion, while often treating patients to the degree possible and who are likely to die, thus allowing the existing infrastructure to operationalize and then keep up. traditional public health methods of infectious disease control are known to work up to a point, depending on various factors such as the pathogen, victim and context. in particular, these methods rely to a large degree on the trust of the populations effected to follow non-pharmaceutical precautions under conditions of immensurable suffering and burdens, and the dedication, training and supplies made available to health care and other workers who sustain the infrastructure (such as, in the ebola case, the highly risky and stressful job of digging and filling graves). confidence in these may have become complacent (putting aside the question of political negligence), as it was only a matter of time before ebola would befall upon a highly populated city for the first time. ''ebola emerged nearly four decades ago. why are clinicians still empty-handed, with no vaccines and no cure? because ebola has historically been confined to poor african nations. the r&d incentive is virtually non-existent. a profit-driven industry does not invest in products for markets that cannot pay'' (chan ) . these failings have become, for some, symbolic of the abject failures of a global system which does not allow new possibilities for pandemic planning, such as more effective and urgent vaccine production (capps and lysaght ) . that ebola is a neglected tropical disease cannot be disputed, meaning that it has failed to attract significant interest for deployment of pharmaceutical interventions (until its full pandemic potential came to light in the current outbreak) (macneil and rollin ). so far, local responses fall back on traditional public health measures; these measures do little to benefit non-human interests, as victims or by finding mutual solutions. we propose that a different approach to pandemic prevention should invest in such technologies as vaccines, but do so using a broad ecological scope. we use primate (clade haplorhini) to identify the non-human apes (hominidae) that are susceptible to the ebola virus; our analysis will proceed to discuss the great apes (genus gorilla and pan), as more is known about the effects of the virus on them as highly sentient and endangered species. initiative as a source of alternatives to pandemic planning, so that, secondly, in the spirit of oh collaboration, we can encourage further and broaden the ethical debate. the paper unpacks in the following way: first, we explain the ethics of oh as an approach that recognises an ecological perspective. second, we define and expand upon the victims of the ebola epidemic so as to consider a new oh-grounded agenda. third, we articulate a possible preventive measure to prevent ebola in both human and animal populations. we argue that, along with efforts to test ebola vaccines in humans, existing vaccines that have been proven safe and efficacious in primates should already be deployed in order to protect both species. our proposal supports the conjecture that focusing on broadly ecological factors, and understanding and reacting to the natural ecology of zoonoses, is central to future zeid planning . one health (oh) has come to signify the interdisciplinary effort to optimize the health of humans, non-human animals, and their ecosystems. as an approach to biomedical enquiry, it has been adopted as a broad heuristic for evidence-based policy involving the usual suspects from public health, as well as veterinarians, animal and plant biologists, ecologists, and environmental scientists (scoones ; leach and scoones ) ; and thereby, it has become a stimulus for collaborative research. thus, its trans-disciplinarily-across multiple disciplines, encouraging de-siloing of sectors, and engagement with partisan stakeholders-creates change by identifying and solving real-world ecological problems. it is thus an extensive ecological perspective to that of public health. however, there are those who have been critical of the oh agenda because, like some existing study or practice lenses, it excludes the humanities and social sciences (lapinski et al. ) , and that, in part, obstructs the development of an inclusive bioethics framework (thompson and list ) . while the first is largely an empirical point, and we can point to anthropologists, among others, expressing solutions, but perhaps being less heard, in respect to ebola (aaa ); the latter observation indicates oh's lack of a philosophical grounding. in fact, oh has no origins in any particular ethical theory. one explanation for this is that normative enquiries are outside of the purview of oh. the collaborative model, therefore, is not about a distinctive oh ethics per se, but an attempt to integrate ecological perspectives on the same terms as public health activism; to probe conventional wisdom to find innovative solutions. this is perhaps a practical consideration because oh otherwise would likely lose political traction under anything more concretely conceptual. the oh goal is to assemble a comprehensive set of data across a broad spectrum of expertise, and to thereby provide solutions that are of benefit to human wellbeing within ecological settings. most recently, this idea is being framed as effectiveness gains through dynamic cooperation in environmental contexts, and has the effect of raising environmental concerns on par with concurrent efforts in public health such as in disease surveillance and animal management. this might be enough to create a vision of oh ethics: van rensselaer potter, in his earliest definition of bioethics, talked about a system of human survival that included environmental, or ecological ethics (potter ). this could easily capture the idea of oh as broadening public health into diverse fields. potter, a pioneer in challenging parochial and non-secular ideas shaping the human condition, noted a schism between the medical-science domains and humanistic ethics, and that both were distanced from environmental ethics. the ethics of oh, therefore, may just be signalling the resurgence of bioethics as a unified endeavour (thompson and list ) , allowing for reflective and critical engagement with current pandemic measures, which up to now gave little credence to solutions outside the scope of public health ethics. a deeper appreciation of secular bioethics, however, also points to the intrinsic interests beyond those of human beings. in the developing oh literature, it is more commonly acknowledged that human beings are part of and dependent upon the biosphere. one way oh has developed is in a perspective that a 'healthy' environment entails healthy animals along with healthy people. it is not 'us versus them', then, but a problem of shared risk that is something concrete to act on, thus providing opportunities to maintain healthy or rescue unhealthy ecosystems (rabinowitz et al. ) . however, in practice, reactions to these risks, and solutions to pathogens, still prioritise human interests, because there is no fundamental sense in which non-human animals, or the environment, matter morally. sure, while oh in this sense creates the grounds for humans to express compassion towards animals and ecosystems and to engage in novel approaches to health problems, overall it often achieves the same goals of prevention and response so far already installed in public health; so oh, in this sense, adds nothing to the ethical debate except by broadening the factors considered in any human cost-benefit analysis. the difference oh makes is in engaging with alternatives: it questions public health ideas entrenched as the only way to solve such problems, and indicates the dangers of the unreflective or blinkered view (leach and scoones ) . its effectiveness in ethical discourse, much like the collaborative idea, is that it asks questions about ecological benefits without overstepping public health priorities. finally, there is the sense in which oh has an enabling effect in respect to grounding an ethical theory in environmental issues. what that theory is, however, is contested. in this paper, therefore, we will sketch the idea that oh ethics ought to contain two elements: ( ) a focus on the inclusive and shared determinants of health; and ( ) a unifying theory. by spelling out these elements better, one is able to assess those projects that profess to be oh; and this will be essential in judging our shared immunity proposal. health is often understood as being normative: implying something good or desirable. this might be applicable from an internal view (being healthy), or an external one, such as the view from public health that concerns community (that is, conditions for being healthy). an 'unhealthy' state can be explained by a pathogen or other kind of destabilising event that impacts or creeps into a biological system, resulting in an altered, often unwanted and endured state. this might be the presence of a virus in an individual, or even the conditions (opportunities and barriers) of healthy living. public health often takes a similar focus, aiming to create healthy circumstances and conditions for people by focussing on the determinants of health. in this respect, oh uses health as an inclusive determinant, such that it includes actions that are broad in orientation and scope, so that health activism ought not be limited to human agents. oh is therefore an investigation of the scientific, social, economic and ecological determinants of human, non-human and ecosystem health, but also a 'shared benefit' approach. our use of 'shared' points to ethical consistency; that actions that affect a broad spectrum of agents should be fairly applied. just as racism is paradoxical in human societies, some exclusionary actions between human beings and non-human animals might be similarly judged as speciesistic. this echoes ideas of equality, and the interests of minorities or the vulnerable being protected against parochial or vested interests. it also befits an examination of incongruity, need and fairness, and justice-these components of comprehensive doctrines are only knowable through ethical study, and in this respect, we are less confident in setting the oh agenda, for such a task requires far greater elucidation than is possible here. we can, however, offer a basic account of 'benefits' that will begin the conversation in earnest about oh as a unifying theory. human beings act in ways that affect non-human animals and the environment, and this raises the question as to how much we should either change such actions, or, indeed, make efforts to assist in the wellbeing of other species. the basic assumption in public health has been that we should interfere only to the extent that their collective welfare is at stake, because animals' interests are outweighed by human interests . thus, public heath applies welfare conditions for the health of animals, which only occasionally includes ethical considerations, such as the humane culling of disease vectors and hosts. however, without engaging in a lengthy debate about non-human moral status, there is also a condition of interspecies connectedness. in the case of preventing zoonotic pathogens, oh on this reading implores us to study the causes and roots of transmission, counting each being as an equal unit in this biological process. the wider study of biospheres, ecosystems, and social networks achieves this. what is ethically important is that this study is concerned with the health of the ecosystem in its entirety, not solely that of humans. oh, therefore, becomes a study of 'natural' environments, enriching public health with animal and ecological studies, and creates a whole new frame of evidence to better design effective responses. in turn, the emphasis turns to discovering and developing creative ways to recover and maintain healthy ecosystems. these hint at plausible strategies that draw on the humanities and social sciences, which can better comprehend the emergent contingencies beyond statistical confines (neyland ) . but what are the objects, goods, or benefits(and harms) that enable states of heath? one way we might extend ethical concerns to non-human interests is by securing universal goods (capps and lederman ) . these are the kinds of goods that reach beyond the needs of human communities, describing benefits as inclusive across species, and feature broadly in ecosystems and the environment. for example, ecosystems are necessary for life by providing the basic requirements (and even complex determinants of heath, in terms of social and cultural goods), and can therefore create 'unhealthy' lives by becoming unproductive and even toxic. these effects can be observed in stressed and challenged environments when they are misused, exploited and degraded. the ecosystem is, therefore, a foundation of universal goods-goods necessary for the health of multiple species, and these goods are likely shared through interspecies connectedness. primarily, then, universal goods extend terms of reference beyond the restrictions of public health purposes. one set of solutions would emanate from comparative medicine originating in human beings (this is the opposite of current comparative medicine studies where animal models are utilised for human health). human trials and treatments may well be useable in animal populations, benefiting them directly, and in some cases, where a pathogen is eliminated, it might reduce risks for human populations. a second possibility would be adapting biobanks, which, because of the terms of reference in providing public goods, are restricted to furthering human interests only (capps ). this does not make good scientific sense, because there is a welter of data being lost or overlooked simply because of intentional institutional design that arbitrarily excludes other contributions. for example, animal samples may well show up zoonotic risks sooner, or enable the natural history of a pathogen to be understood. a recent proposal to create an ebola biobank would do well to consider extending its remit to include the animals that are the essential links in zeids (hayden ) . these are intriguing possibilities because they also allow real environment information gathering and sharing, and not just the artificial data, for example, from de novo animal experiments (capps and lederman ) . there are, however, going to be more or less hard cases where conflict between public health goals and securing universal goods is more or less likely; and solutions are going to be less amicable between human interests and an ecological perspective. at this level of disagreement, a debate about animal or environmental interests or rights is to be had. but in our paper, we develop this idea of universal goods to give weight to the broadly inclusive and shared determinants that are affecting both humans and animals as victims of ebola. according to oh, it behooves us to consider the opportunities to improve the health of those directly affected by the virus, in the sense that operationalizing public health should be extended to other primates such as chimpanzees and gorillas; related not only in their level of evolutionary sentience, but also as victims of ebola. the current ebola outbreak, which started in a single index case in december , but was not reported as an outbreak until march , is the largest known in history (rio et al. ; yakubu et al. ) . both humans and great apes have been affected. at time of writing, there have been , reported confirmed, probable, and suspected cases in human infections, mainly in guinea, nigeria, liberia, and sierra leone. , confirmed patients have died. in great apes, the effect of ebola is likewise devastating. gorillas and chimpanzees are susceptible to the virus (bermejo et al. ; kaiser ) . ebola has killed roughly one third of the western lowland gorilla population in the past years, which, along with habitat loss and poaching, led the world conservation union to declare it a critically endangered species (walsh et al. ) . three interrelated enquiries interest us as advocates of oh. first, a significant question is 'why now?' (bausch and schwarz ; farmer ) . why only now has the ebola virus, which has previously emerged in isolated regions, become a regional endemic (olival and hayman ; olson et al. )? this question has been asked in the context of other zoonotic diseases, most prominently hiv and its analogous emergence from primates in africa. answers will likely become evident as our understanding of zoonoses encompasses the exponential amount of accumulated knowledge from across disciplines, including the study of the reservoir, host and effected animals, the ecologies they inhabit, and their natural responses to the virus. it is therefore not only a question of what humans might have done differently this time to create the tragedy, but also their ongoing interactions with the environment whence the virus came from. these insights will be significant in developing strategies for potential future ebola outbreaks, and paradigms for other zeids, including possible preventative measures. second, it has been debated as to whether medical interventions for eid should be deployed in animal species. according to one view, we should not interfere with natural systems at all; apes have lived with ebola for years without need for human intervention. yet the state of wild populations today is such that no environment is free from human effects, and therefore such groups must adapt to the 'anthropocene' (hockings et al. ) . in fact, the landscape has changed so significantly that human intervention is perhaps necessary for them to survive at all. although dissent has been voiced against interfering in 'natural systems' and the effectiveness of medical interventions relative to other conservation strategies (ryan and walsh ) , the magnitude and significance of the current ebola outbreak should at least question the premise of non-intervention. intervention, therefore, can be justified because the alternative is decimation across the biosphere, affecting human beings who rely on it, and the animals that live within it. if this can be considered as a universal good, then, we can start to envision medical strategies to protect both human and animal populations. the plausibility of vaccinating other species during significant endemics has been voiced before, often from the conservation angle (marzi and feldmann ; ryan and walsh ) , but never received any serious consideration, as far as we are aware. two reasons for this might be postulated: limited resources are to be used to address human needs, especially at times when endemics or potential pandemics are occurring; and vaccine safety in administering to potentially critically endangered species. recently, a vaccine trial for ebola was carried out on captive chimpanzees to inform future conservation (warfield et al. ) . third, what (at least partially) grounds the need to respond to the queries posed above in respect to the shared risk of zoonoses, is the fact that human beings and primates are equally affected by the virus. therefore, if an ethics of shared benefits is persuasive, then one can start to see how conceptual change is necessary in zeid planning. for example, standard public health policies prioritise human interests, and often, these interests are perceived to collide with and outweigh the conservation of the biosphere. examples would include devastating and often ineffective culling (johansen and penrith ; jenkins et al. ) , or ravaging biodiversity on the basis of 'at-risk-to-human' calculations. oh, however, starts to give rise to different opportunities: for example, developing data storage from veterinary and conservation studies that can benefit humans, and vica versa (capps and lederman ) ; or strategizing to create healthy ecologies that will concurrently present fewer risks to human beings. concomitantly, humans, who often receive better medical care, may serve as 'concurrent research participants' and adaptive public/veterinary health models. the scientific literature to support oh as an approach to coordinate pandemics of zoonotic origin is rapidly accumulating (rabinowitz et al. ), such that it should be gaining traction in pandemic planning. it has, however, yet to feature in the solutions to ebola. oh ought to have some quite significant implications for pandemic planning (against ebola and generally) in various chronological phases. . the natural ecology of the ebola virus. the animal origin of the current epidemic is perplexing. the virus tends to only occasionally emerge in isolated villages, rarely appearing in hospitals and other health facilities (garrett ) . in this regard, the current outbreak is unique. beyond human interference, the ecology of the virus itself undoubtedly plays a key part. there are a number of species that could be implicated as the host, such as bats, other large mammals, or primates; even insects and plant viruses have been implicated in its transmission to human beings (hayden ; monath ) . it is imperative to conduct studies to locate the reservoirs and the plausible transmission routes to human beings and primates (in terms of group-to-group interspecies and cross-species transmission) and other known and unknown species contagions, to explain risks and spillover events. wildlife conservation workers have been tracking ebola in gorilla and chimpanzee populations for some time; but these data rarely reach the attention of public health planners (walsh et al. ). . manage habitat disruption. there is a vast and largely uncharacterized pool of possible zoonotic pathogens, and increasing opportunities for infection caused by disruptive human activities and ecological encounters (morse ). the understanding ecologies of vector-borne pathogens reveals some intriguing events, such as how biodiversity and diverse species networks can buffer, dilute and 'soak up' pathogens (harris and dunn ; keesing et al. ). that is, comparative studies and the reverse data use of human trials to benefit animal populations, such as in veterinary application (yeates ) . an early report from the current outbreak hypothesized that the host was a bat colony living in a local hollowed out tree (saéz et al. ). development of industry, such as mining, can bring people into regular contact with zoonotic reservoirs and hosts (kangbai and koroma ). these industries employ local and international workers who then travel to and from wild territories (allouche ) . anthropocentric activity also disrupts normal animal behaviour, for example, changing fruit bat roosting and foraging ranges so that they move to proximate sites to human dwellings (looi and chua ) . further, evidence suggests that biodiversity is a key element in emergent zoonotic diseases, where, in some cases, there is a reversed correlation: less biodiversity, or even deprived ecologies, create more risks for human zeids spillover events (cardinale et al. ; jones ) . . prevention of zoonotic infections. non-pharmaceutical measures can work well in eid outbreaks, but are only practical considerations once the spillover event has occurred in humans (in other contexts, personal protection equipment might be used as biosafety measures). reactive pharmaceutical measures, such as vaccines, take time to develop to specific pathogens, and then are often hampered by politics and investment, biological limitations, errors, and logistics. prevention, as is central to public health, might therefore be considered key. currently, several types of ebola vaccines have been proven effective and safe in primates, but none has been approved in humans yet (see below). human trials however are ongoing; and several captive chimpanzee trials have been conducted warfield et al. ) . once an ebola vaccine is approved for use in humans, several strategies to increase coverage may be used, such as ensuring that eco-tourists are appropriately vaccinated before visiting at-risk primate populations, and introducing health programmes in mining and refining communities often located in remote areas and near to potential ebola hotspots, such as bat roosts. . monitoring of disease in animals. studies have identified stereotypic behaviours in animals when burdened with zoonotic disease. for example, gorillas faced with endemic ebola reacted in ways that point to a decrease in social cohesion and lower reproductive potential: females were significantly more likely to transfer from breeding groups to non-breeding groups and males were more likely to transfer from groups to solitary-living. in general, there was a decrease in formation of breeding groups. interestingly, during the post-epidemic period, immigration of breeders between groups returned to normal while immigration of non-breeders remained low. observable social dynamics, then, may be used as indicators to detect ebola outbreaks (genton et al. ). this is an example of how animals can act as sentinels for imminent human risk. . animal-to-human transmission. several routes of animal-human transmission of ebola exist, including ingestion of raw infected meat (bats, primates and other animals) , and exposure to hosts and reservoirs through daily life, professions and tourism (köndgen et al. ). these various routes are potentially causing more zeid spillover events. for example, 'bushmeat' is consumed in higher amounts due to population growth in some areas (wolfe et al. ) ; mining is a growing industry in many regions (see below); and local economies rely on the growth of ecotourism. presenting these as local and global issues is challenging: for example, the local population is unlikely to support the prohibition of eating specific species as part of infection control. moreover, to have ethical credence, it would be consistent to address concurrent risks in developed countries, such as reducing intensive farming that also drives zeids. as regional industrial growth is essential for creating sustainable development for all countries, a call to reduce anthropocentric activity in rich wildlife areas in order to meet expedient conservation efforts would likely be rejected because of the local economic losses (and the international desire to visit such areas). nonetheless, efforts should be aimed at education of locals and visitors about the modes of transmission of ebola (muyembe-tamfum et al. ). learning about the local ecology-animal behaviour, biology, anthropology-would point to innovative ways to adapt in order to reduce the risk of transmission. the accumulated knowledge, therefore, raises some intriguing possibilities for the study and feasibility of potential zoonotic control measures; and in particular, using and adapting the 'shared' biosphere as part of the solutions to endemic ebola. however, despite the obvious ecological links to human zeid outbreaks, the interest in devising such possibilities has, until now, had little traction in public health and extant pandemic planning. most pandemic plans mention little about the ecosystem beyond its risk potential and stipulate requirements to devastate the animal populations (culling and the like) as a means to limit future human-to-human transmission. the one health solution to endemic ebola: inter-species immunity vaccination is by far one of the most significant responses to eid in human beings, and in the context of ebola its importance is beyond doubt; since the outbreak was first detected, public health, clinical staff and allied workers struggled against quite immense odds to bring it to the current state. the case for human vaccination speaks for itself. however it should not be understated quite how important it is since the alternative is to fall back on the objective: ''not to dramatically increase the person's chance of survival, it's to contain the spread'' (fjeldsaeter ) . one can only imagine what advantages the early deployment of an effective vaccine would have been. putting aside questions of the economic inequality that provides little incentive for vaccines until worst case scenarios prevail (capps and lysaght ; dawson ; farmer ) , the current upscaling in research to find a vaccine for ebola illustrates the standard phased approach to innovation: invention, animal experiments, trials in humans before large scale production and delivery. as with human beings, the obvious advantage to animals affected by the disease, such as great apes, is immunity from the disease (and relationally, although not always the case, such as with endangered species, is exclusion from culling measures), and prevention of cross infection (walsh et al. ). the obvious benefit is also in terms of conservation. specifically the case of highly endangered gorillas (and other susceptible animals on wwf lists) is extremely significant (ryan and walsh ) . no ebola vaccine has yet to be approved for therapeutic use in human beings. however, ebola vaccine development has been an active field of research for several laboratories worldwide, and candidate vaccines were found some time ago to be safe and efficacious in mice (blaney et al. ) . several human-targeted vaccines have been proven safe and efficacious in trials in primates, including adenovirus type and , human parainfluenza virus type , and vesicular stomatitis virus geisbert and feldmann ; marzi and feldmann ; stanley et al. ). these prospective vaccines, however, raise different concerns, such as safety, price, effectiveness, delivery and side effects. in an attempt to aver safety in the use of replicating viruses (see below), warfield et al. ( ) tested the protective effects of a virus-like particle in captive chimpanzees using adenovirus as a vector. first, they demonstrated that the vaccine was safe for chimpanzees. second, they documented the development of a robust immune response in chimpanzees, evidenced by a detection of virus-specific glycoprotein and vp antibodies - weeks post-vaccination. third, they demonstrated that total igg fractions taken from the chimpanzees that were vaccinated had a protective effect in mice challenged with murine ebola: - % of mice in the study groups survived compared to none of mice in the control groups. similarly, blaney et al. ( ) developed a live-attenuated and inactivated rabies virus vaccine that expresses the ebola glycoprotein. the vaccine had no adverse effects in primate models, it induced humoral response to both rabies and ebola, and was shown to be protective against both viruses. while this is obviously an early-phase study, it has great potential in terms of resources and feasibility in conferring immunity in mammals against two lethal pathogens. so far, two vaccines have passed phase clinical testing: chimpanzee adenovirus -vectored vaccine encoding ebola surface glycoprotein (chad ) (rampling et al. ) , and vesicular stomatitis virus (vsv)-vectored vaccine also encoding for the outer protein of the zaire ebola strain (agnandji et al. ) . the prevail study is an ongoing phase / trial taking place in liberia that examines the safety and effectiveness of these two vaccines. concomitantly, the strive trial is taking place in sierra leone, where healthy volunteers will be given the vsv vaccine in order to test its safety and effectiveness. agricultural policy tends to follow vaccinating all of the exposed animals so that those not already infected will develop sufficient immunity. however, when time and resources permit, it is normal for all exposed animals to be slaughtered (kahn et al. ) . vp is a matrix protein that together with glycoprotein constitute the virus-like particle vaccine. occasionally, nucleoprotein is also present. vp is essential for cell expression of viral antigens to which the body responds by creating antibodies (escudero-perez et al. ; marzi and feldmann ) . see: http://www.niaid.nih.gov/news/qa/pages/ebolavaxresultsqa.aspx; accessed june . http://www.cdc.gov/media/releases/ /p -ebola-vaccine.html; accessed / ; accessed june . the vsv phase trial in geneva was halted due to safety concerns when several healthy participants developed different adverse effects such as arthritis. the trial was continued a month later, to understand contagion networks and possibilities for control, first we need to see the connections between vectors and victims, and by understanding these within shared ecologies we might be able to better safeguard communities-both animal and human. as these authors postulated: ''in addition to the protection of threatened nhps [nonhuman primates], vaccination of nhp populations in endemic areas might also offer an additional, critical benefit to humans. the interaction of humans and infected nhps has been associated with transmission of ebov to humans and initiation of subsequent outbreaks, so prevention of disease in nhps may also serve to limit ebov transmission into the human population'' (blaney et al. ) . concurrently with the race to develop and test vaccines on human beings, we argue that already now, we can and should (upon assuring the degree of safety) deploy vaccines in captive and wild primates with the aim of benefiting both primates and humans. the current strategies, we submit, are driven by a too narrow vision: we propose that oh espouses a 'shared benefit' approach that is complementary to the 'shared risk' approach (rabinowitz et al. ) . a 'shared benefit' approach seeks to actively maximize health in one species while in turn benefiting another species as well. specifically, we refer here to research and interventions in humans that benefit animals and vice versa. our proposal is to implement the notion of inter-species immunity. one of the identified risks for ebola, while not knowing for sure the reservoirs of the virus, is close proximity between human and primate populations (towner et al. ). our proposal is for direct action to administer vaccinations to humans through public health and research paradigms, and additionally to animals to stave off future outbreaks in both populations. such an approach, aimed at vaccinating animals in the first instance, would be preventative rather than reactive to an outbreak in human populations, by protecting across species and thereby creating a potential barrier to future occurrences of ebola in the fauna. our proposal is to co-develop vaccines for human and primate use in ebola endemic and at-risk sites in africa; and simultaneously, to deploy such vaccines to these sites in animal and (in due course) human populations. the delay in getting vaccines to the people in africa is in part due to the need to conduct proper clinical trials first and the troubling consequences of creating randomization (donovan ; shaw ) . however, captive primate populations could be enrolled in trials as benefiting vaccine development at a lower safety level (in comparison to the footnote continued upon approval by the review committee. the ongoing phase / vsv trials were modified according to the results in that study (agnandji et al. ) . one health, vaccines and ebola: the opportunities for… standard profiles for first in human trials, and additionally avoiding the later phased stages of human clinical trials). primates might be research subjects who can contribute to a longer-term ecologycentred strategy to vaccinate wild animal populations urgently. simply put, researchers are already injecting captive primate populations, and, if proven safe and efficacious in these trials (i.e. would not to our knowledge wipe out remaining primate populations), this approach provides a fast track to wild primate populations. an oh approach would potentially justify animal research on captive primates within parameters of participation of 'vulnerable' populations (i.e. the agents likely to be the first cases or most at risk in future outbreaks because of their situation and circumstances). the next stage would be vaccinating the same species in the wild for the protection both of the same species (primates) and other at-risk species (human beings). this is, firstly, an ethical enquiry involving the status of primates as sentient beings who possess moral value (fenton ) ; and secondly, a conceptualization of animals as vulnerable populations such that risky clinical trials, with conditions, can be ethical. in answering the first enquiry, we note that ebola and the recent chimpanzee trials happen at a time when the national institutes of health is planning to reduce significantly the use of chimpanzees in invasive research, and therefore raises the case of whether minimally invasive research on still captive or retired chimpanzees is ethical at all. we might see experimentation, however, as a parallel development to research and treatment in vulnerable human beings, such as children and other people who cannot consent (wendler ) . the idea here is that trials might benefit wild populations and therefore it might be possible to justify within human research ethics paradigms. in human clinical research, the acceptability of such study is a function of acceptable risk, and, when vulnerability is in question, so are the chances of direct benefit (the 'best interests' test) and the possibility of appreciating benefits for others of one's own kind (children suffering from the same condition, for example). in this sense, developing protocols with primates in captivity might be justified, including using those that have 'retired', to meet the conditions of expediency; but concurrently we must anticipate that there is a direct benefit-or a best interest in play. while potential 'secondary ecological risks' exist, such as accidental extinction of the animal species, there would be some important caveats scholars concerned with animal ethics will blame us here for putting the animals at increased risk compared to humans. however, given a vaccine that has been proven safe in the lab, and the significant risk ebola poses for apes, we believe that the risks posed by the vaccine are proportional to the benefit that might be accrued to the animals themselves. http://www.nih.gov/news/health/jun /od- .htm; news release; wednesday, june , ; accessed june . this perspective is different from the predominant study of exogenous factors such as habitat disturbance and climate change as drivers of ebola emergence, and links directly to the contribution of transmission between gorilla or chimpanzee social groups in the wild. if this equivalency were to remain within an oh approach: that the vaccine is safe enough to use in human phase trials concurrently (shared risk) and that wild apes would receive the treatment as part of the same strategy (shared benefit). if this shared benefit paradigm of securing universal goods is legitimate, then it goes some way in justifying our strategy as mutually benefiting from a single intervention. this raises feasibility problems, but some intriguing ecological repercussions warrant serious consideration of an oh vaccine approach. . one would have to possess extensive knowledge about the reservoirs, vectors and hosts, and hierarchical zoonotic bridges between species, to understand the impacts of vaccines in terms of safety, stability, and effectiveness. this will involve knowledge of human, human-animal, and animal-animal interactions (i.e. comprehensive studies of fauna and flora), and their linked activities within the biosphere. at present, pandemic planning is focussed on public health, and to a degree, anthropocentric studies of how we contract and spread the pathogens amongst our own kind. this focus, for instance, locates some major challenges of vaccine use, specifically high levels of distrust and ambivalence towards medical interventions in some african populations that would impede wide human community vaccination programmes (mark ; macneil and rollin ; mitman ) . one might therefore face resistance in deploying an effective vaccination programme. oh, therefore, helps planners look to other solutions that may complement communitybased interventions. firstly, vaccinating domestic animal populations, both companion animals and those in husbandry, could avoid collateral loss to families and livelihoods. these losses are substantial and as targets for public health intervention might gain widespread support. secondly, and which we focus on here, is developing a novel approach to research and deployment in the field as a protective measure that demands immediate attention. thus, following the approach we outlined above, the vaccine will increase the welfare of humans and wild apes, both as protection (eventually) and in conducting knowledge based trials. to address the expediency argument, we again note that both the chad vaccine and the vsv vaccine have proven to be safe and efficacious in non-human primates (stanley et al. ; geisbert and feldmann ) . the human trials will take time to conclude. clearly, with the primate trials already concluded, there is an while the context of animal vaccinations has been debated considerably in respect to farming practices (and risks to humans as pathogenic risks, food safety and economics), there has been little coverage of the benefits to the animals. the debate is now further sparked by the quarantine and killing of companion animals exposed to potential contagions by their owners, such as the spanish dog killed for the fear of ebola transmission (associated press ). we note that the ongoing debate is deliberate in its assessment to get vaccines into the field as soon as possible to protect health care workers and needed staffing (i.e. burial teams and cleaners). this is a separate, urgent debate which does not entirely equate to the stage wise proposal we make here. however, the design to get vaccines first to primates as a joint shared immunity strategy could expedite human benefits and use, with a focus on employing biologists, veterinarians and the like to target the animal populations. opportunity to deploy these vaccines right away to wild apes. in the short term, we might be seeing every primate that lives as a benefit of vaccine deployment. the long-term benefits are immunity, possibly extending across species and thus limiting the future scope for spillover events. furthermore, it will provide in situ data to be gathered from the wild populations. . achieving broad coverage to widely dispersed animals would be costly and logistically challenging but has been achieved in other settings using low interventional methods such as baiting in the case of rabies (morters et al. ) . one challenge is the difficulty in reaching entire ape populations. the dense tropical forests and the animals' nomadic tendencies would make effective immunization difficult. however, by use of local and interdisciplinary knowledge and expertise, and various vaccination methods such as hypodermic darts and synthetic baits, this obstacle may be overcome (ryan and walsh ) . one long-term strategy may be to create buffer zones around villages by vaccinating domestic and wild animals, that might be enough to minimise risks of future outbreaks, following the alreadyexisting use of designated zones in farmed animal populations elsewhere (kahn et al. ) . the approach would require an increased evidence base, of course, but effectiveness could be achieved by focusing on 'hot spots', localized risk maps (jones ) , and using targeted empirical data, such as weather patterns that are known to influence zoonotic spillover events (bausch and schwarz ) . ring vaccination is another strategy, where vaccines are delivered to animals found in the ryan and walsh ( ) counted different pathogens that are harmful for apes, of which have licensed vaccines. they claim that the major obstacle in dispensing these vaccines is the delivery to the animals (ryan and walsh ) . the authors point out that ''the high seroprevalence among children indicates the same source(s) of exposure [to eobla] as in adults, either inside or near villages''. moreover, because great ape infection is often lethal, and direct contact with humans is rare, some other animal, perhaps bat roosts near settlements, represent the most likely common animal source of exposure: ''these animals, previously identified as a potential reservoir, are abundant in the forest ecosystem and consume fruits on trees located in or around villages'' (nkoghe et al. ) . 'hotspot' maps highlight regions: ( ) where the risk of disease transfer between wild primates and from wild primates to humans is greatest; ( ) where there are cross-species transmission events between wild primates due to a high diversity of closely related primate species; and ( ) where it is most likely that human beings will come into frequent contact with their wild primate relatives. ''these areas also are likely to sustain a novel epidemic due to their rapidly growing human populations, close proximity to apes, and population centers with high density and contact rates among individuals'' (pedersen and davies ). this would have to be an ideal, managed area, additionally creating the rural populations in control of the solutions. there are two elements to achieving this: ( ) engagement, cf. ''far greater community engagement is the cornerstone of a more effective response. where communities take charge, especially in rural areas, and put in place their own solutions and protective measures, ebola transmission has slowed considerably'' (who ); and knowledgeable land use, cf. protecting ''threatened habitats by reminding nearby communities of all the benefits they derive from keeping these habitats intact. forests, meadows and marshes prevent floods, supply clean water, provide habitat for species that pollinate crops, put oxygen into the atmosphere and take carbon out, and otherwise make themselves useful. in some cases, conservation groups or other interested parties actually put down cash for these ecosystem services-paying countries, for instance, to maintain forests as a form of carbon sequestration'' (conniff ). proximity of a known outbreak. further, vaccination campaigns in animals are likely to be cheaper and possibly more temporally feasible than in humans (ryan and walsh ; macneil and rollin ) . . technical issues, including the use of live attenuated viruses as vectors. for example, live attenuated vaccines are more effective than killed vaccines in conferring long-term immunity, thus necessitating fewer vaccine shots and lower rates of compliance and coverage. moreover, using viruses that are replication-competent as vaccine vectors will increase the chances for herdimmunity and therefore the potential for inter-species immunity. however, one of the risks in using a live attenuated, replication-competent vaccine in wildlife is the activation of the attenuated virus and spread to other species, including humans. beyond using killed viruses or viral particles, one solution may be using as vector a species-specific virus. for example, a recombinant murine cytomegalovirus (cmv) that was genetically engineered to express ebola particles was found to be protective in mice. since cmv is highly species-specific, a cmv-based ebola vaccine will potentially spread rapidly in a wildlife population, such as gorillas, without any cross infection to other species (marzi and feldmann ) . the use of replication-competent vectors raises another problem: pre-existing immunity to the virus that is used as the vector will hinder spread of the ebola particles, thereby preventing immunity to be acquired. this challenge could be addressed by the development of vectors to which there is no pre-existing immunity among the specific population. for example, newcastle disease virus, to which there is no detectable pre-existing immunity in humans, was developed as a potential vector of ebola particles with some (limited) positive results. vsv was also used as a vector with little if any pre-existing immunity in humans, with even greater success (marzi and feldmann ; stanley et al. ). the existing challenge with vaccine development was captured by the ghana academy of arts and sciences technical committee. they enumerated that development of vaccine is notoriously tricky given pathogen diseases' drift and other factors that impact on their individual effectiveness with different strains; the possibilities of emergent side-effects and other unforeseen incidents; the distrust of trials originating from certain foreign organisations; and how all of this will affect uptake (both in terms of willingness and immunisation) in the target population. within a 'shared benefit' approach, however, one originating in a one health perspective, we coin the term inter-species immunity to conceptually re-think the notion of immunity within a community; specifically, to extend the goods of health, such as immunity strived for in human populations, to other species and vice versa. we suggest that ebola incidence may be prevented or reduced in one species population by inducing immunity against that pathogen in another species population. so far, the best example of the success of such an approach can be seen in the response to the hendra virus, where vaccination of horses prevented disease in both horses and humans (middleton et al. ). the key to success of inter-species immunity might be with other measures that look to adapt and benefit other ecologies, such as preparing protected ecological zones (removing food and perching areas for bats) based on planned and managed farming areas, and identifying timely and imminent risks to initiate human and animal vaccination in and around these zones. one could adopt already-used surveillance programmes in at-risks regions (these, as we noted earlier, should already be modified to include 'indicative' behaviour in animals of possible zoonoses infection): ''previous serosurveys, together with the geographic pattern of outbreaks, have highlighted the potential role of the ecosystem, and an increased risk among forest populations has previously been described. our study confirms that the forest, particularly the deep forest, is the environment most at risk. this is the area harboring animals susceptible to the virus, such as great apes and bats, the latter representing a viral reservoir'' (nkoghe et al. ). we could not say whether the remoteness and distances between villages could create conditions for regional immunity by lowering the chances that an affected host might infiltrate the buffered populations from long distances away. however, as mentioned, oh is about interdisciplinary collaboration, and solutions to extreme situations such as the current ebola outbreak require such an approach more than ever (middleton et al. ) . understanding the needs of the various stakeholders such as villagers and hunters, and the ecology of all the organisms involved, is without a doubt essential for the success of any viable long-term solution. at the moment, inter-species immunity is likely to involve a programme of trials in captive primate populations, early role out to wild populations (assuming they are safe), and then a concurrent programme to vaccinate human communities in at risk regions (this human challenge is already featured in the literature with respect to other pathogens). however, with the impending imh prohibition on some primate research in the united states, and paralleled restrictions in other countries, this is a window that is potentially closing. invasive great ape research rarely has scientific justification and primate research in general is falling out of favour, although it remains possible in many jurisdictions under strict conditions. invasive research on great apes-using chimpanzees in particular-is likely to be prohibited; but we suspect that monkey research will continue for some time. this might provide the necessary level to proceed to trials in human and great ape populations. so, one could also look at it through a shared vision-if human beings are willing to volunteer for phase one trials, which was highly evident in recent calls, then possibly retired chimpanzees could be coopted as well. at this stage, it will be envisioned that the vaccine is safe for human beings, so this might be an acceptable concurrent risk for primate populations. we surmise that this vaccination strategy, inspired by the ongoing ebola outbreak, might be replicated in future ones. we make the case for the vaccination of wild primates even prior to the completion of the ongoing human trials, with the conditions of optimizing their safety and welfare, and assuring mutual benefits to them and their future offspring, as well as to humans. phase trials of rvsv ebola vaccine in africa and europe-preliminary report ebola and extractive industry strengthening west african health care systems to stop ebola: anthropologists offer insights excalibur, spanish ebola patient's dog, is euthanised despite global outcry. the guardian outbreak of ebola virus disease in guinea: where ecology meets economy ebola outbreak killed gorillas inactivated or live-attenuated bivalent vaccines that confer protection against rabies and ebola viruses defining variables of access to uk biobank: the public interest and the public good introducing one health to the ethical debate about zoonotic diseases in south east asia one health and paradigms of public biobanking challenging the production of vaccines for a future influenza pandemic biodiversity loss and its impact on humanity who director-general addresses the regional committee for africa; director-general of the world health organization; address to the regional committee for africa, sixty-fourth session useless creatures ebola: what it tells us about medical ethics ebola, epidemics, and ethics-what we have learned climate change and emerging infectious diseases one health, vaccines and ebola: the opportunities for… shed gp of ebola virus triggers immune activation and increased vascular permeability the early spread and epidemic ignition of hiv- in human populations diary. london review of books ebola haemorrhagic fever. the lancet can a chimp say ''no''? reenvisioning chimpanzee dissent in harmful research ebola in sierra leone: battling sadness, fear and disgust on the frontline the coming plague: newly emerging diseases in a world out of balance the next epidemic-lessons from ebola recombinant vesicular stomatitis virus-based vaccines against ebola and marburg virus infections how ebola impacts social dynamics in gorillas: a multistate modeling approach species loss on spatial patterns and composition of zoonotic parasites the ebola question biobank planned for ebola samples apes in the anthopocene: flexibility and survival the duration of the effects of repeated widespread badger culling on cattle tuberculosis following the cessation of culling has culling been properly assessed as a valid and justified control intervention measure for zoonotic diseases? global trends in emerging infectious diseases confronting zoonosis through closer collaboration between medicine and veterinary medicine (as 'one medicine') vaccination against foot-and-mouth disease: the implications for canada changing epidemiology of west africa ebola outbreaks - impacts of biodiversity on the emergence and transmission of infectious diseases pandemic human viruses cause decline of endangered great apes recommendations for the role of social science research in one health the social and political lives of zoonotic disease models: narratives, science and policy lessons from the nipah virus outbreak ebola and marburg hemorrhagic fevers: neglected tropical diseases? fear and ignorance as ebola 'out of control' in parts of west africa. the guardian ebola virus vaccines: an overview of current approaches hendra virus vaccine, a one health approach to protecting horse, human, and environmental health. emerging infectious disease ebola in a stew of fear ecology of marburg and ebola viruses: speculations and directions for future research emerging infections: condemned to repeat? in microbial evolution and co-adaptation: a tribute to the life and scientific legacies of joshua lederberg: workshop summary. institute of medicine (us) forum on microbial threats evidence-based control of canine rabies: a critical review of population density reduction ebola virus outbreaks in africa: past and present an ethnography of numbers risk factors for zaire ebolavirus-specific igg in rural gabonese populations filoviruses in bats: current knowledge and future directions dead or alive: animal sampling during ebola hemorrhagic fever outbreaks in humans cross-species pathogen transmission and disease emergence in primates global bioethics the chimp and the river: how aids emerged from an african forest toward proof of concept of a one health approach to disease prediction and control from ''us vs. them'' to ''shared risk'': can animals help link environmental factors to human health? a monovalent chimpanzee adenovirus ebola vaccine-preliminary report ethical considerations of experimental interventions in the ebola outbreak ebola hemorrhagic fever in : the tale of an evolving epidemic consequences of non-intervention for infectious disease in african great apes investigating the zoonotic origin of the west african ebola epidemic towards a one world, one health approach randomisation is essential in ebola drug trials. the lancet having and fighting ebola-public health lessons from a clinician turned patient chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge ebola needs one bioethics isolation of genetically diverse marburg viruses from egyptian fruit bats catastrophic ape decline in western equatorial africa potential for ebola transmission between gorilla and chimpanzee social groups vaccinating captive chimpanzees to save wild chimpanzees should protections for research with humans who cannot consent apply to research with nonhuman primates? ebola situation in liberia: non-conventional interventions needed second who high-level meeting on ebola vaccines access and financing. summary report west african ebola epidemic after one year-slowing but not yet under control bushmeat hunting, deforestation, and prediction of zoonotic disease the ebola outbreak in western africa: ethical obligations for care animal welfare in veterinary practice acknowledgments capps conceived of the idea for this article and led its drafting. lederman was integral to developing the idea and contributed equally to the research for the paper. the authors would like to thank the following people for helpful comments: wang linfa, paul anantharajah tambyah, and sharon amit. key: cord- -vk ghjm authors: choi, kang-seuk title: newcastle disease virus vectored vaccines as bivalent or antigen delivery vaccines date: - - journal: clin exp vaccine res doi: . /cevr. . . . sha: doc_id: cord_uid: vk ghjm recent advances in reverse genetics techniques make it possible to manipulate the genome of rna viruses such as newcastle disease virus (ndv). several ndv vaccine strains have been used as vaccine vectors in poultry, mammals, and humans to express antigens of different pathogens. the safety, immunogenicity, and protective efficacy of these ndv-vectored vaccines have been evaluated in pre-clinical and clinical studies. the vaccines are safe in mammals, humans, and poultry. bivalent ndv-vectored vaccines against pathogens of economic importance to the poultry industry have been developed. these bivalent vaccines confer solid protective immunity against ndv and other foreign antigens. in most cases, ndv-vectored vaccines induce strong local and systemic immune responses against the target foreign antigen. this review summarizes the development of ndv-vectored vaccines and their potential use as a base for designing other effective vaccines for veterinary and human use. virulent ndv, the causative agent of newcastle disease (nd), results in devastating economic losses to the poultry industry worldwide, particularly in asia, the middle east, and africa [ ] . to control nd, poultry flocks are subjected to prophylactic vaccination with live attenuated and/or killed ndv vaccines [ ] . gallinaceous birds (e.g., chickens, quail, and turkeys) are highly susceptible to ndv, although the virus infects most bird species. the clinical severity in chickens varies greatly, depending on the virulence of the ndv isolate. based on their effects in chickens, isolates of ndv are classified into at least four pathotypes: velogenic (a highly pathogenic form), mesogenic (a moderately pathogenic form), lentogenic (a low pathogenic form), and avirulent (a subclinical form) [ ] . lentogenic (e.g., lasota and b ) and avirulent (e.g., vg/ga and v ) viruses are widely used as vaccines to prevent nd in poultry. ndv is an avian paramyxovirus type virus belonging to the genus avulavirus within the family paramyxoviridae [ ] . the virus contains a nonsegmented, negative-sense, singlestranded rna genome of , to , nucleotides in length, which encodes ( ´ to ´) six structural proteins: nucleocapsid (n) protein, phosphor (p) protein, matrix (m) protein, fusion (f) protein, hemagglutinin-neuraminidase (hn) protein, and rna-dependent rna polymerase (l) protein [ ] . the length of the ndv genome follows the "rule of six, " which allows efficient virus replication because the n protein binds effectively to six nucleotides [ , ] . fig. shows a schematic diagram of ndv morphology. the ndv f and hn glycoproteins are the major structural proteins and comprise a bilayer lipid membrane (envelope) that plays an important role in virus attachment, entry, and release. the m protein resides beneath the envelope and is involved in assembly and budding. the n protein binds to the genomic rna to form the nucleocapsid (nc) core, and the p and l proteins associate with the nc core to form the ribonucleoprotein (rnp) complex, which is essential for virus replication and transcription [ ] . two nonstructural proteins, v and w, can be produced by rna editing of the p gene: one (v) or two (w) g residues are inserted at a specific location within the mrna encoding the p gene [ ] . the v protein is an interferon (ifn) antagonist [ ] . the life cycle of ndv occurs entirely in the cytoplasm. first, ndv enters the cell cytoplasm via viral attachment and subsequent membrane fusion, which are mediated by the hn and f proteins, respectively. the viral nc or rnp complex is then released into the cytoplasm, whereupon the rnp complex transcribes the viral rna to produce viral mrnas, which are then translated into viral proteins. genome replication occurs when sufficient viral proteins (especially the n protein) are synthesized. the full-length anti-genomic rna (plus-strand) serves as the template for synthesis of the viral rna genome (minus-strand). the newly formed genomic rnas are then encapsidated by the n protein before associating with the polymerase complex. all viral components are then transported to the cell membrane, where they are assembled into progeny virus particles: the progeny acquire an envelope by budding under the direction of the m protein before being released from the cell surface by a process of detachment (elution), which is regulated by the hn protein (which has neuraminidase activity). because ndv is a negative-sense single-stranded rna virus, a functional viral rnp complex is required for viral transcription and replication. thus, the reverse genetics technology used to generate recombinant ndv (rndv) requires a fulllength complementary dna (cdna) clone and three helper plasmids encoding the ndv n, p, and l proteins under the transcriptional control of a t rna polymerase promoter [ , ] . once simultaneously transfected into permissive cells, the three helper plasmids express the n, p, and l proteins, which then bind to genomic or anti-genomic rna to form the rnp complex. in general, reverse genetics approaches used to rescue rndv are based on one of three different systems: ( ) a chicken fibroblast cell (or qm cell line) and fowl pox virus-t recombinant system [ ] ; ( ) a t rna polymerase expressed bsr t / (baby hamster kidney- cell clone) system [ ] ; or ( ) a human epidermoid carcinoma (hep- ) cell and a host-restricted recombinant vaccinia virus (modified vaccinia virus ankara [mva]) system [ ] . fig. depicts the reverse genetics system used in our own laboratory. briefly, hep- cells are infected with mva and then cotransfected with a ndv cdna clone (lasota backbone) and three helper plasmids (n, p, and l). the recovered viruses are purified on hep- cells in a plaque assay, and clones are selected and passaged several times in specific pathogen free (spf) embryonated chicken eggs. insertion of foreign genes into the full-length cdna clone is a pivotal step for generating rndv expressing foreign antigens. manipulating cdna clones to express foreign antigens must take several factors into account. the foreign gene of interest should be synthesized as a transcription unit, which comprises a gene start (gs) sequence, the foreign gene, and a gene end sequence (fig. ) . the transcription unit is then inserted into a noncoding intergenic region of the ndv genome [ ] . the number of nucleotides that make up the rndv genome must follow the "rule of six" to ensure integrity of the genome and efficient virus replication. the insertion site is one of the most important factors affecting efficient expression of the foreign gene. in general, genes closer to the ´ terminus are transcribed more abundantly than downstream genes (the ´ to ´ transcription gradient) due to imperfect restart of transcription at the gs. in other words, the foreign gene is expressed more abundantly and more efficiently when placed closer to the ´ end of the genome [ ] . while the foreign gene can be placed at any intergenic site within the ndv genome, the p/m intergenic site is preferred to ensure optimal expression of foreign genes [ , ] . the length of the inserted foreign gene also affects efficient expression of the foreign antigen. incorporating a foreign gene increases the size of a genome and the number of transcriptional units within that genome, which often leads to retarded growth of rndv in permissive cells [ ] . a single ndv vector can accommodate foreign genes up to . kb in length with a good degree of stability and express at least three different foreign genes [ , ] . ndv vaccine strains infect many mammals but are very safe due to restricted host tropism. nevertheless, ndv is a strong stimulator of humoral and cellular immune responses at both the local and systemic levels; it also has an adjuvant effect [ ] . importantly, ndv is antigenically distinct from known paramyxoviruses that infect mammals; thus it is unlikely to be affected by pre-existing immunity in mammals [ ] . ndv does not establish persistent infection in animals and humans because it replicates only in the cytoplasm [ ] . in addition, the rule of six means that genetic changes (e.g., insertions, deletions, and recombinations) affecting the integrity of the genome are rare [ ] . therefore, ndv vaccine strains are potential virus vectors that can be used to develop antigen delivery vaccines for use in animals and/or humans. ndv vaccines have been used to develop antigen delivery vaccines for use in cattle/sheep, dogs/cats, pigs, and horses (table ) . antigen delivery vaccines for veterinary use include those that protect against bovine herpesvirus (bhv- ) [ ] , bovine ephemeral fever virus (befv) [ ] , rift valley fever vihttp://www.ecevr.org/ https://doi.org/ . /cevr. . . . rus (rvfv) [ ] , vesicular stomatitis virus (vsv) [ ] , canine distemper virus (cdv) [ ] , rabies virus (rv) [ ] , nipah virus (niv) [ ] , and west nile virus (wnv) [ ] . bhv- is a major cause of respiratory tract disease in cattle. a major problem with the current modified live bhv- vaccines is their ability to cause latent infection, with the associated risk of subsequent reactivation [ ] . as an alternative strategy, khattar et al. [ ] developed a rndv vaccine (rlasota/gdfl) expressing the gd of bhv- . a single intranasal/intratracheal immunization with rlasota/gdfl ( . × pfu/ dose) induced marked mucosal and systemic antibody responses in calves. also, intranasal challenge with a high dose of virulent bhv- strain cooper ( × pfu) led to a marked reduction in viral shedding and more rapid clearance of chal- lenge virus when compared with mock-infected and challenged controls. these results suggest that ndv may have utility as a vaccine vector that can be used to develop a mucosal vaccine against bhv- infection of cattle. rvfv, a mosquito-borne bunyavirus that infects ruminants (cattle and sheep), causes severe economic losses to the livestock industry and has a high impact on public health in africa and the arabian peninsula [ ] . currently, a live attenuated vaccine (smithburn strain) and inactivated rvfv vaccines are available; however, although live attenuated vaccines are highly immunogenic, they are not safe for use in livestock [ , ] . inactivated vaccines are safer, but less effective, than live attenuated vaccines and require repeated vaccination [ ] . to improve efficacy and safety, kortekaas et al. [ ] developed a ndv-vectored vaccine (ndfl-gngc) expressing the gn and gc glycoproteins of rvfv. repeat vaccination (two times) of lambs with the ndfl-gngc vaccine ( . tcid /dose) elicited a high titer of neutralizing antibodies specific for rvfv. these results indicate that ndv has potential as a vaccine vector in sheep. rv, a rhabdovirus that infects dogs, causes a fatal neurologic disease (rabies) in humans and animals [ ] . more than , people die of rabies each year, with about % of deaths occurring in asia and africa [ ] . currently available live at- tenuated rv vaccines induce a weak humoral antibody response, which complicates evaluations of their efficacy [ ] . to generate a live attenuated vaccine with improved efficacy, a ndv-vectored rabies vaccine (rl-rvg) containing the rv g glycoprotein was developed [ ] . animal experiments demonstrated that rl-rvg induced production of rv neutralizing antibodies at levels comparable with those induced by a live attenuated rv vaccine (era strain) and provided complete protection against challenge with a potentially fatal virus strain (gx/ ) for more than year [ ] . ndv vaccine strains have been used as virus vectors to develop antigen delivery vaccines for use in humans ( [ ] , ebola virus (ebov) [ ] , niv [ ] , human norovirus (nov) [ ] , respiratory syncytial virus (rsv) [ ] , and human parainfluenza virus (hpiv- ) [ ] . currently, no live attenuated vaccine is available for hiv- . however, ndvs have been used as a vaccine vector to deliver the env and gag antigens of hiv [ , , ] . for example, khattar et al. [ ] developed two ndv-vectored hiv vaccines (rndv-gag/env and rndv-env/gag) expressing gp env and p gag, respectively (these genes were inserted between the p and m genes of ndv). intranasal immunization of guinea pigs with these vaccines induced long-lasting env-and gag-specific humoral immune responses. they also induced a marked and efficient cellular and protective immune response in mice after challenge with vaccinia viruses expressing hiv- env and gag. these results suggest that vaccination with a single ndv vector coexpressing env and gag is a promising strategy that increases vaccine immunogenicity and subsequent protective efficacy against hiv. ndv-vectored vaccines may be useful against human influenza viruses. nakaya et al. [ ] developed a ndv-vectored human influenza vaccine (rndv/b -ha) expressing the ha protein of a/wsn/ influenza virus (h n ). two intravenous inoculations of mice with the rndv/b -ha vaccine ( × pfu/dose) induced a strong anti-influenza virus antibody response and provided complete protection against lethal challenge with influenza a/wsn/ virus. recently, dinapoli et al. [ ] developed a ndv-vectored hpaiv vaccine (ndv-ha) expressing the ha protein of a/vietnam/ / (h n ) hpaiv for use in humans. its protective efficacy against hpaiv was evaluated in an african green monkey model. two oro-nasal administrations of ndv-ha ( × pfu/dose) induced a high serum titer of hpaiv neutralizing antibodies and a robust mucosal immunoglobulin a response in the respiratory tract. these results suggest that ndv-ha may be an effective human vaccine for hpaiv. ndv-vectored vaccines can also be used to develop vaccines against deadly emerging viruses. such viruses include sars-cov [ ] , ebov [ ] , and niv [ ] . for example, kong et al. [ ] developed one ndv-vectored niv vaccine expressing the niv glycoprotein (rla-nivg) and one expressing the f protein (rla-nivf), and evaluated their immunogenicity in a pig model. they showed that two intramuscular vaccinations with rla-nivf ( × eid /dose) elicited a high titer of niv-specific neutralizing antibodies (maximum titers of ) in pigs; these neutralizing antibodies persisted for at least weeks post vaccination, indicating potential utility as a vector vaccine that protects humans and pigs against niv infection. ndv can also be used as a vaccine vector to develop vaccines against human viruses for which no whole virus vaccines are available (due to the inability to grow target viruses [e.g., human papillomaviruses, hepatitis c virus, and nov] in cell culture). for example, kim et al. [ ] developed a ndvvectored nov vaccine (a modified rndv-vp vaccine) expressing the capsid protein (vp ) of nov strain va for human use. the modified rndv-vp grew to a high titer in embryonated chicken eggs (> pfu/ml) and produced high levels of vp protein in the allantoic fluid. the expressed vp protein self-assembled into virus-like particles. three inoculations of the modified rndv-vp vaccine ( × eid /dose) induced a strong igg a-mediated immune response, along with high levels of ifn-γ, tumor necrosis factor-alpha, and interleukin production by splenocytes, in mice; a strong fecal iga response was also noted. this suggests that the modified rndv-vp vaccine has potential as a live attenuated vaccine against nov in humans. in addition, ndv-vectored vaccines are also a promising strategy for other human respiratory viruses, including rsv [ ] and hpiv- [ ] . taken together, these results suggest that a primary vaccination with an ndvvectored vaccine expressing a foreign protein can be effective. however, the efficacy of an updated or new vaccine based on the ndv vector may be reduced by pre-existing ndv antibodies. this could limit the continuous use of ndv vector vaccines in human. http://www.ecevr.org/ https://doi.org/ . /cevr. . . . for several decades, the global poultry industry has used live attenuated or killed vaccines to control and prevent nd. conventional ndv vaccines are highly immunogenic and induce protective immunity in poultry. importantly, live attenuated ndv vaccines can be administrated in drinking water, eye drops, and sprays, and even via in ovo injection. this makes it possible to vaccinate poultry (especially young birds) on a large scale. ndv vaccines can be combined with other poultry vaccines (i.e., used as polyvalent vaccines) to control economically important poultry diseases. the production of ndv vaccines is highly cost-effective since they grow to very high titers in embryonated spf chicken eggs and in cell culture. this makes ndv vaccine strains attractive as viral vectors for the development of polyvalent vaccines against pathogens that could devastate the poultry industry [ ] . global epizootic episodes of h hpaiv in wild birds and poultry and their potential to cause a pandemic in humans have driven the development of effective h hpaiv vaccines [ ] , which aim to maintain food security and reduce viral load in the environment. since the mid- s, vaccination programs designed to protect poultry from h hpaiv have been implemented by several countries in which the virus is enzootic, including china (and hong kong), egypt, indonesia, and vietnam [ ] . in general, conventional highly pathogenic avian influenza (hpai) vaccines for poultry comprise killed virus prepared from a low pathogenic strain or, more recently, from a reverse genetic engineered low pathogenic reassortant virus that derives its hemagglutinin (ha) and neuraminidase genes from a field-virulent virus and its six internal genes from a/ puerto rico/ / (pr ) [ ] . killed avian influenza virus (aiv) vaccines have limitations, including high production costs, poor immunogenicity, and problems with mass vaccination. administration of live attenuated aiv vaccines to poultry is strictly prohibited due to the risk of emerging variants arising from reversion to virulence via antigenic drift or antigenic shift in the presence of circulating field viruses [ ] . ndv-vectored aiv vaccines are bivalent live attenuated vaccines that are suitable for mass vaccination of poultry on a commercial scale and have a proven safety profile. basically, ndv-vectored ai vaccines are designed to express the ha protein of aiv, which is critical for protection [ , ] (table ) . to date, ndv-vectored aiv vaccines have been administered to poultry in china and mexico. in china, approximately . billion doses of ndv-vectored aiv vaccine were used to prevent hpai during - [ ] . the ha gene from a/ goose/guangdong/ / (h n ) was initially incorporated into the ndv vector but was replaced with the ha gene from a/duck/anhui/ / (h n ) in , a/duck/guangdong/ s / (h n ) in , and, most recently, with a/chicken/guizhou/ / (h n ) [ , ] . approximately million doses of ndv-vectored ai vaccine were used in mexico (in the form of a live vaccine and a killed vaccine) to prevent h n lpai in poultry between july and october . the mexican vaccine expresses the ha gene from low pathogenic avian influenza virus a/chicken/mexico/ / (h n ) [ ] . ndv vector vaccines expressing the ha gene of hpaiv show high protective efficacy against hpaiv and virulent ndv in experimental spf chickens [ , ] ; however, under field con- ditions most breeders maintain a high level of immunity to ndv through multiple vaccinations, ensuring that progeny chicks have maternal antibodies to ndv for the first few weeks of life. however, the presence of maternal antibodies could interfere with the efficacy of the ndv-vectored aiv vaccine, resulting in poor antibody responses to ndv and aiv ha. as an alternative strategy, chimeric ndv-vectored vaccines, in which the ectodomains of f and hn proteins are replaced by those of another avian paramyxovirus serotype, have been developed [ , ] . bivalent ndv-vectored vaccines, which have been developed to prevent diseases of economic importance to the poultry industry, have advantages over traditional vaccines (table ) . examples include infectious bursal disease virus (ibdv) [ ] , infectious bronchitis virus (ibv) [ ] , infectious laryngotrachitis virus (iltv) [ , ] , and avian metapneumovirus (ampv) [ ] . ibdv, a birnavirus that infects chickens, is an important pathogen that causes severe immunosuppression and high mortality in young chickens. live attenuated vaccines of moderate virulence (especially widely used intermediate plus vaccines) are used widely to prevent infectious bursal disease (ibd); however, they can cause severe side effects (symptoms consistent with ibd) in young chickens. huang et al. [ ] developed a ndv-vectored ibdv vaccine (rlasota/vp ) expressing the vp gene of ibdv, which is responsible for protective immunity against ibdv. the vp gene is inserted into the ´end non-coding region of the ndv genome. the live ibv vaccine is very safe in young chickens and protects spf chickens against virulent ndv and virulent ibdv. ibv, a coronavirus that infects birds, causes respiratory disease and renal disorders (the nephropathogenic strain) in poultry and poor egg production in laying hens worldwide. currently available live attenuated ibv vaccines risk giving rise to new variants through recombination with field ibvs. this often reduces the efficacy of ibv vaccines. importantly, live ibv vaccines interfere with the live attenuated ndv vaccine. to overcome the limitations of currently available live vaccines, toro et al. [ ] developed a ndv-vectored ibv vaccine (rls/ibv.s ) expressing the s subunit of the ibv s glycoprotein. oculo-nasal immunization of chickens ( . × eid /dose) provided complete protection from clinical disease (mortality) after challenge with a lethal dose of virulent ndv (ca ). the protective efficacy of the rls/ibv.s vaccine was also assessed using a heterotypic protection approach based on priming with a live attenuated ibv mass-type vaccine followed by boosting with rls/ibv.s . the vaccine protected chickens against clinical disease after lethal challenge with a virulent ark-type ibv strain, leading to a significant reduction in virus shedding when compared with that in unvaccinated/challenged chickens. iltv, a herpesvirus that infects birds, causes respiratory disease in chickens. currently available live attenuated iltv vaccines are effective, but there are concerns about safety in chickens because of the risks of virulence acquirement and latent infections during bird to bird transmission [ , ] . bivalent ndv-vectored vaccines against iltv have been developed to overcome side effects associated with the live iltv vaccine. kanabagatte basavarajappa et al. [ ] developed a ndv-vectored iltv vaccine (rndv gd) expressing glycoprotein d (gd) of iltv. the protective efficacy of the rndv gd vaccine against challenge with virulent iltv and virulent ndv was then evaluated in spf chickens. immunizing chickens with rndv gd ( tcid /dose) via the oro-nasal route induced a strong antibody response and provided a high level of protection against subsequent challenge with virulent il-tv and ndv, indicating that rndv gd has potential as a bivalent vaccine. ndv vaccine strains show promise as a base from which to develop effective vaccines against pathogens that infect animals and humans. most ndv-vectored vaccines used for poultry are bivalent and provide protective efficacy against virulent ndvs and several foreign pathogens. ndv-vectored poultry vaccines have been developed to provide protection against hpaiv (a/h and a/h ), ibdv, iltv, ibv, and ampv. safe ndv-vectored vaccines have been developed as antigen delivery vaccines for veterinary and human use. such vaccines express the foreign target antigen and induce robust immune responses at both the local and systemic level as shown with ndv-vectored veterinary vaccines in cattle/sheep (e.g., bhv- , befv, rvfv, and vsv), dogs/cats (e.g., cdv and rv), pigs (e.g., niv), and horses (e.g., wnv). ndv-vectored human vaccines currently under development aim to provide protection against hiv, hpiv- , and rsv, newly emerging zoonotic viruses (e.g., hpaiv a/h , sars-cov, ebov, and niv), and noncultivable human viruses (e.g., human papillomavirus, hepatitis c virus, and nov). a primary vaccination by an ndv-vechttp://www.ecevr.org/ https://doi.org/ . /cevr. . . . tored vaccine expressing a foreign protein can be effective but the efficacy of an updated or new vaccine based on the ndv vector may be reduced by pre-existing ndv antibodies, which could limit the continuous use of ndv vector vaccines in human. in conclusion, ndv vaccine strains are attractive vectors that can be used to develop effective vaccines against pathogens that infect animals and/or humans; such vaccines are safe and efficient, and provide high levels of protective immunity, although there is a risk that previous vaccinations can reduce the efficacy of the vectored vaccine. kang-seuk choi http://orcid.org/ - - - recombinant viral vector vaccines for the veterinary use rna virus reverse genetics and vaccine design recombinant newcastle disease virus-vectored vaccines against human and animal infectious diseases avian paramyxovirus serotype- : a review of disease distribution, clinical symptoms, and laboratory diagnostics newcastle disease virus outbreaks: vaccine mismatch or inadequate application? therapeutic potential of oncolytic newcastle disease virus: a critical review newcastle disease: evolution of genotypes and the related diagnostic challenges newcastle disease virus as a vaccine vector for development of human and veterinary vaccines newcastle disease virus: current status and our understanding genome replication of newcastle disease virus: involvement of the rule-of-six newcastle disease virus v protein is a determinant of host range restriction recombinant newcastle disease virus as a vaccine vector rescue of newcastle disease virus from cloned cdna: evidence that cleavability of the fusion protein is a major determinant for virulence generation of recombinant lentogenic newcastle disease virus from cdna high-level expression of a foreign gene from the most '-proximal locus of a recombinant newcastle disease virus optimization of human immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses p and m gene junction is the optimal insertion site in newcastle disease virus vaccine vector for foreign gene expression newcastle disease and related avian paramyxoviruses immunization of cattle with recombinant newcastle disease virus expressing bovine herpesvirus- (bhv- ) glycoprotein d induces mucosal and serum antibody responses and provides partial protection against bhv- characterization of a recombinant newcastle disease virus expressing the glycoprotein of bovine ephemeral fever virus intramuscular inoculation of calves with an experimental newcastle disease virus-based vector vaccine elicits neutralizing antibodies against rift valley fever virus protective efficacy of a recombinant newcastle disease virus expressing glycoprotein of vesicular stomatitis virus in mice recombinant newcastle disease viral vector expressing hemagglutinin or fusion of canine distemper virus is safe and immunogenic in minks newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats newcastle disease virus-vectored nipah encephalitis vaccines induce b and t cell responses in mice and long-lasting neutralizing antibodies in pigs newcastle disease virus-vectored west nile fever vaccine is immunogenic in mammals and poultry phylogeography of rift valley fever virus in africa and the arabian peninsula adverse response of nonindigenous cattle of european breeds to live attenuated smithburn rift valley fever vaccine pathological studies on postvaccinal reactions of rift valley fever in goats pathogenesis of rabies geneva: world health organization optimization of human immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses newcastle disease virus expressing human immunodeficiency virus type envelope glycoprotein induces strong mucosal and serum antibody responses in guinea pigs mucosal immunization with newcastle disease virus vector coexpressing hiv- env and gag proteins elicits potent serum, mucosal, and cellular immune responses that protect against vaccinia virus env and gag challenges recombinant newcastle disease virus as a vaccine vector immunization of primates with a newcastle disease virus-vectored vaccine via the respiratory tract induces a high titer of serum neutralizing antibodies against highly pathogenic avian influenza virus newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens respiratory tract immunization of non-human primates with a newcastle disease virus-vectored vaccine candidate against ebola virus elicits a neutralizing antibody response newcastle disease virus vector producing human norovirus-like particles induces serum, cellular, and mucosal immune responses in mice protection against respiratory syncytial virus by a recombinant newcastle disease virus vector recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates success factors for avian influenza vaccine use in poultry and potential impact at the wild bird-agricultural interface avian influenza vaccines: a practical review in relation to their application in the field with a focus on the asian experience h n avian influenza vaccination in china newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous h n avian influenza viruses engineered viral vaccine constructs with dual specificity: avian influenza and newcastle disease immune responses of chickens inoculated with a recombinant fowlpox vaccine coexpressing ha of h n avain influenza virus and chicken il- avian influenza vaccines against h n 'bird flu recombinant viral-vectored vaccines for the control of avian influenza in poultry immunization of chickens with newcastle disease virus expressing h hemagglutinin protects against highly pathogenic h n avian influenza viruses chimeric newcastle disease virus protects chickens against avian influenza in the presence of maternally derived ndv immunity a recombinant newcastle disease virus (ndv) expressing vp protein of infectious bursal disease virus (ibdv) protects against ndv and ibdv infectious bronchitis virus s expressed from recombinant virus confers broad protection against challenge a recombinant newcastle disease virus (ndv) expressing infectious laryngotracheitis virus (iltv) surface glycoprotein d protects against highly virulent iltv and ndv challenges in chickens newcastle disease virus (ndv) recombinants expressing infectious laryngotracheitis virus (iltv) glycoproteins gb and gd protect chickens against iltv and ndv challenges a novel chimeric newcastle disease virus vectored vaccine against highly pathogenic avian influenza virus generation and evaluation of a recombinant newcastle disease virus expressing the glycoprotein (g) of avian metapneumovirus subgroup c as a bivalent vaccine in turkeys increased virulence of modified-live infectious laryngotracheitis vaccine virus following bird-to-bird passage latency and reactivation of infectious laryngotracheitis vaccine virus key: cord- -eeh a t authors: lambert, paul-henri; ambrosino, donna m.; andersen, svein r.; baric, ralph s.; black, steven b.; chen, robert t.; dekker, cornelia l.; didierlaurent, arnaud m.; graham, barney s.; martin, samantha d.; molrine, deborah c.; perlman, stanley; picard-fraser, philip a.; pollard, andrew j.; qin, chuan; subbarao, kanta; cramer, jakob p. title: consensus summary report for cepi/bc march - , meeting: assessment of risk of disease enhancement with covid- vaccines date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: eeh a t a novel coronavirus (cov), severe acute respiratory syndrome coronavirus (sars-cov- ), emerged in late in wuhan, china and has since spread as a global pandemic. safe and effective vaccines are thus urgently needed to reduce the significant morbidity and mortality of coronavirus disease (covid- ) disease and ease the major economic impact. there has been an unprecedented rapid response by vaccine developers with now over one hundred vaccine candidates in development and at least six having reached clinical trials. however, a major challenge during rapid development is to avoid safety issues both by thoughtful vaccine design and by thorough evaluation in a timely manner. a syndrome of “disease enhancement” has been reported in the past for a few viral vaccines where those immunized suffered increased severity or death when they later encountered the virus or were found to have an increased frequency of infection. animal models allowed scientists to determine the underlying mechanism for the former in the case of respiratory syncytial virus (rsv) vaccine and have been utilized to design and screen new rsv vaccine candidates. because some middle east respiratory syndrome (mers) and sars-cov- vaccines have shown evidence of disease enhancement in some animal models, this is a particular concern for sars-cov- vaccines. to address this challenge, the coalition for epidemic preparedness innovations (cepi) and the brighton collaboration (bc) safety platform for emergency vaccines (speac) convened a scientific working meeting on march and , of experts in the field of vaccine immunology and coronaviruses to consider what vaccine designs could reduce safety concerns and how animal models and immunological assessments in early clinical trials can help to assess the risk. this report summarizes the evidence presented and provides considerations for safety assessment of covid- vaccine candidates in accelerated vaccine development. since the identification of a novel coronavirus, sars-cov- , as the cause of pneumonia in patients from wuhan china, a pandemic has erupted, resulting in enormous health care, social and economic disruption to our global society [ ] . as of may , there have been , , cases and , deaths worldwide [ ] . in rapid response to the pandemic, academic and industry scientists from around the world have initiated efforts to develop vaccines and therapeutics for disease prevention and patient management. the coalition for epidemic preparedness innovations (cepi), a global partnership between public, private, philanthropic, and civil organizations, is funding work to develop sars-cov- vaccines using a variety of technology platforms. several vaccine candidates are already in phase studies with others likely to enter the clinic in the next few months [ ] . one of the challenges facing rapid vaccine development for sars-cov- is the need to adequately assure the safety of these vaccines. one such safety concern is disease enhancement syndrome that occurred in the s with inactivated rsv and measles vaccines. vaccinemediated disease enhancement is characterized by a vaccine that results in increased disease severity if the subject is later infected by the natural virus. during early trials with inactivated rsv vaccine, the vaccine did not prevent infection, % of those infected required hospitalization and two children died [ ] . lung pathology in patients showed an unexpected inflammatory response with both neutrophils and eosinophils, evidence of immune complex formation and complement activation in small airways [ ] . scientists later learned that the vaccine caused a similar disease enhancement in animals characterized by immunopathology and a t helper cell type (th ) biased response and antibody responses with poor neutralizing activity [ ] [ ] [ ] . since that time, the animal models have been relied upon to predict safety for new rsv vaccines that are developed. of note, the pathogenesis of rsv disease enhancement is distinct from antibody disease enhancement (ade) which occurs for macrophage tropic viruses, demonstrated most notably for dengue in humans and the coronavirus feline infectious peritonitis virus in cats, and is directly caused by non-neutralizing or sub-neutralizing antibodies leading to more efficient viral uptake via fcγ receptor binding [ ] . since pathology consistent with the rsv vaccine enhanced disease (and perhaps ade) has been demonstrated for some sars-cov- vaccine candidates in animal models, there is also a concern that a similar syndrome could occur in humans immunized with sars-cov- candidate vaccines. therefore, cepi and the brighton collaboration safety platform for emergency vaccines (speac) convened a scientific working meeting https://brightoncollaboration.us/brighton-collaboration-cepi-covid- -web-conference/) on march and , of experts in the field of vaccine immunology and coronaviruses to discuss current knowledge that could form the basis for the assessment of the risk of enhanced disease during sars-cov- vaccine development. this consensus report presents considerations for vaccine developers and can serve as a guide for the development and testing of vaccine candidates to avoid these safety concerns. ultimately, the door to clinical trials is controlled by regulators in the context of the risk/benefit for the entire dataset provided by developers and within the local trial context. microbiology and immunology and pediatrics at the university of iowa, both reviewed their work and that of others in animal models developed for sars-cov- and mers-cov. the lessons from these models can inform the development priorities for useful sars-cov- animal models to address both efficacy and safety. in inbred mouse strains, sars-cov- replicates efficiently in the respiratory tract and can cause pneumonitis, but clinical signs and pneumonia were only observed in old balb/c mice [ ] . subsequent passage of sars-cov- through mouse lungs resulted in the isolation of virus that caused severe disease in both young and old mice [ , ] . this virus was used in many subsequent studies. ferret models of sars-cov- also demonstrate virus replication in respiratory tracts with induction of a neutralizing antibody response but also demonstrated little evidence of clinical disease [ ] . hamsters, in contrast to mice and ferrets, demonstrate high levels of viral replication, develop pneumonitis, and can be shown to have clinical signs of disease [ ] . following the identification of human ace as the receptor for sars-cov- , transgenic murine models expressing human ace receptor (hace ) were developed and shown to develop mild pulmonary disease. of note, these mice also developed lethal viral encephalitis, attributed to viral spread through the olfactory nerve, despite the relative scarcity of hace expression in the brain which may have relevance to sars-cov- disease [ ] . efficacy of several sars-cov- vaccines was evaluated in these models with spike (s) protein based vaccines demonstrating neutralizing antibody and protection against pulmonary replication of the challenge virus in mice and hamsters [ ] . for dna vaccine studies, it was shown that candidate vaccines encoding the s protein conferred antibody mediated protection from challenge in mice and that vaccines encoding the n protein induced humoral and cellular immunity [ , ] . for vectored vaccines expressing sars-cov- proteins, it was shown that viral proteins were expressed in mice, ferrets, and hamsters. in these studies, neutralizing antibodies were elicited by b/hpiv , vsv, rabies, mva and adeno viruses expressing s protein, that protected against sars-cov- replication in lungs of challenged animals. however, one mva vaccine expressing the s-protein did not protect against infection [ ] . in contrast to sars-cov- , inbred mice were found to be resistant to mers-cov, thus infection was studied by creating models that expressed the mers receptor, human dpp (hdpp ). ad -hdpp transduced mice could be infected with mers virus but infection was associated with minimal clinical disease except in immunocompromised mice that developed weight loss after infection. of note, hdpp -transgenic mice developed lethal viral encephalitis with concurrent inflammatory changes on histopathological examination of the lung, similar to hace -tg mice with sars-cov- . subsequently, investigators developed mice "knocked-in" for expression of hdpp and after virus passage in these mice, identified mouse-adapted mers strains that caused more severe disease and increased histopathology with more pulmonary edema than those infected with the original mers strain [ ] . importantly, mice without functional t cells, such as rag -/-and tcr alpha-/-, had delayed viral clearance whereas mice that could not produce antibodies, mumt mice, did not show delay in clearance. similar models were developed by crispr/cas mutagenesis of two residues in the mouse ace molecule, followed by mouse adaptation with serial passage, leading to an ards model of lethal infection [ , ] . taken together this evidence supports the notion that t cells are important in viral clearance for mers [ ] . non-human primate (nhp) models have also been established for both sars-cov- and mers-cov. there was evidence of upper respiratory and lower respiratory tract sars-cov- replication in african green monkeys to a greater extent than in cynomolgus macaques, and least in rhesus macaques, with little evidence of clinical disease in all three species [ ] . of note, consistent with findings in older humans and mice, increased pathology has been documented in aged cynomolgus macaques with sars-cov- wild type infection [ ] . there is some controversy on the disease severity in the mers models with different groups seeing different levels of pathology. this has not been resolved [ , ] . both vaccine efficacy and safety have been studied in animal models with many sars-cov- candidate vaccines. the group of experts discussed how the vaccine models were utilized to characterize the response of specific vaccines and to examine both disease enhancement and antibody dependent enhancement (ade) signals. there is evidence for disease enhancement in vaccinated animals after challenge with live virus in multiple studies with sars-cov- vaccine candidates as summarized in table. we are limiting our comments in this report to data in animal models and not discussing in vitro data except to mention that there is some evidence of ade in human primary monocytes [ , ] . different animal models exhibit different pulmonary pathology but generally characterized by cellular infiltrates including eosinophils. in this summary, we provide an overview of the consensus opinion on vaccine related outcomes in animal models that were of concern for risk of disease enhancement and could guide assessments of sars-cov- vaccine candidates. in murine models, evidence for vaccine related disease enhancement has been demonstrated for inactivated whole vaccine (with and without alum), vectored vaccine expressing n protein (but not seen with vectored vaccine expressing s protein in same report), a replicon particle platform expressing s protein, and a vectored vaccine expressing s proteins. in general, the pathology described included pulmonary infiltrates often with eosinophils observed. th dominant responses were documented in some reports by expression of th driven cytokines [ ] [ ] [ ] [ ] [ ] . in a ferret model, hepatitis was demonstrated in animals vaccinated with a recombinant modified vaccinia virus ankara vaccine expressing s protein and then challenged with virus [ ] although questions have been raised about this study [ ] . [ , ] . non-human primate models have also produced evidence of enhanced disease after sars-cov- vaccine immunization. chinese macaques immunized with a modified vaccinia virus expressing s protein then challenged with sars-cov- did not develop clinical disease, but histopathology showed lung injury. this injury was characterized by decreased wound healing, and increased pro-inflammatory macrophages expressing il- , il- , and ccl [ ] . this report also demonstrated that passively administered anti-s antibody was associated with lung pathology after challenge with the live virus although the mechanism may not be through fc receptor and thus not classic "ade". of note, a second report similarly demonstrates the effect with certain anti-s antibody preparations and without fc involvement [ , ] . the relevance of these reports remains unclear as there are multiple studies with administration of neutralizing monoclonal antibodies to different models that did not induce disease enhancement. other investigators have reported absence of disease enhancement in both hamsters and monkeys immunized with a whole inactivated vaccine although these models differed in a number of ways, most notably by the use of bpl (β-propiolactone) instead of formalin for inactivation of the virus [ , ] . finally, we note that there has not be an agreed upon positive control applied in these animal studies and thus interpretations are hampered.ba animal models with sars-cov- are being rapidly developed by multiple research human ace transgenic mice (hace tg) aged - weeks and - months of age were studied and hace expression was observed in lung, heart, kidney and intestinal tissues. following intranasal inoculation with sars-cov- , weight loss was observed, and viral rna was detected in the lungs as well as in the intestine [ ] . gross pathology demonstrated swollen and enlarged lungs with moderate interstitial pneumonia. histological studies documented an accumulation of inflammatory cells including monocytes and lymphocytes in alveolar interstitium, with thickening of alveolar walls. sars-cov- s protein was detected by ihc in alveolar macrophages and epithelia [ ] . nhp were also infected with sars-cov- with rhesus macaques aged - years inoculated intratracheally and although no fever was observed, weight loss and asthenia were seen on multiple days. viral rna was detected from nasal and throat swabs and to a lesser degree in anal specimens, peaking on days to and lasting until day post infection. one animal was euthanized on day for necropsy and viral rna was detected in multiple organs including cns, skeletal muscle and heart. for the two surviving rhesus macaques, positive neutralization titers were documented by day post infection. there was radiographic evidence of multiple ground glass opacities in the lungs on days , and post infection. microscopically the lung lesions represented an acute interstitial pneumonia characterized by mild to moderate thickening of alveolar septum, increased number of macrophages, degeneration of pneumocytes and an inflammatory cell infiltration. presence of viral antigen was confirmed, predominately in alveolar monocytes and macrophages [ ] . analysis of blood samples showed a decline in counts of total white blood cells, lymphocytes and monocytes with no observed changes in percentages. a decrease in both cd +cd + and cd + cd + t-cell counts was observed. importantly, these hematological findings are similar to those seen in sars-cov- infected patients. this model could serve as a critical tool for detailed studies of pathogenesis and the evaluation of intervention strategies including vaccines. of note, following the meeting another group has confirmed sars-cov- infection in rhesus macaques with viral antigen detected in type i and type ii pneumocytes and diffuse pulmonary alveolar damage noted [ ] . experts agreed that these models and others under development should be utilized to evaluate vaccine candidates for any evidence of disease enhancement as specified in later sections. design barney the sars-cov- s protein structure was solved shortly after its emergence and shows similar structure and mobility as the sars-cov- s [ ] . the timing from first knowledge of sars-cov- to the beginning of the phase study was a remarkable sixty-five days. the advantages of mrna vaccines include ability to create a highly precise type of protein to elicit the correct antibodies, to elicit t cell responses that are th predominant, and the rapidity of manufacturing. of course, disadvantages include the novel nature of both mrna and dna vaccines without any licensed vaccine with either technology to date and lack of experience for mass production. therefore, multiple platforms for sars-cov- are under development that mitigate against some of the potential disadvantages of nucleic acid vaccines. although mrna and dna vaccines elicit t cell responses without adjuvants, adjuvants may be important for subunit and whole cell inactivated vaccines to increase their immunogenicity and drive an immune response that could limit the risk of disease enhancement. multiple sars-cov- vaccines are in development including vectored vaccines, whole cell inactivated vaccines, and recombinant protein vaccines. the experts discussed how the choice of adjuvants will be important for both efficacy and safety with these platforms. dr. arnaud didierlaurent from the centre of vaccinology at the university of geneva presented background on the effects of different adjuvants on animal and human immune responses. several adjuvants are now being used in commercial vaccines or are in clinical development [ ] . oil-in-water emulsions such as mf or as have been shown to increase the breadth of the antibody repertoire, binding affinity and affinity maturation when compared to unadjuvanted vaccines [ , ] in human studies with influenza vaccines, h n vaccine adjuvanted with mf (squalene-based emulsion) increased the levels of h -specific antibody for subclasses igg and igg and the binding to fcγr but not to fcγr when compared to alum adjuvanted vaccines. this demonstrates that the use of an adjuvant can skew the functionality profile of antigen-specific antibodies, with the potential to activate different innate effectors based on their fcγr expression [ ] . use of squalene-based emulsion vaccines for influenza have also been shown to increase cd + t cell response frequencies and crossreactivity. even if pre-existing cross-reactive antibodies are present prior to immunization, such adjuvants could activate naïve b cells and promote the adaptability of memory b cells [ ] [ ] [ ] [ ] . in addition to antibodies, adjuvants can promote cellular responses. human malaria challenge studies provided early evidence that the choice of adjuvants(combined with the malaria antigen rts,s) was critical in achieving optimal protection and highlighted the importance of cellular response [ ] . more recently, studies with hepatitis b surface antigen (hbs) vaccine adjuvanted with as , as , as or alum showed that vaccines formulated with as and as induced the highest antibody levels while as promoted best hbs-specific cd t cell response [ ] . these differences were associated with the magnitude of the initial inflammatory response triggered by the different adjuvanted formulations [ , ] . interestingly, although the level of cd t cell response was lower in the alum group compared to the as group, both adjuvants led to similar memory subset profiles and cytokine production profiles given the unprecedented demand for an effective vaccine, the use of adjuvants may be critical for subunit vaccines in providing antigen-dose sparing, increased immunogenicity, breadth and duration of response, potentially eliciting cross-protection against new cov strains and minimizing the risk of enhanced disease. following the presentations, attendees participated in discussion of the suggested consensus statements and all attendees were asked to comment on the draft statements available online. these comments were reviewed and discussed again on the second day of the meeting and resulted in the summary consensus statement that follows. • sars-cov- has a low affinity for murine ace receptor and murine models will require the use of hace transgenic mice, preferably with a 'knock-in' approach. preliminary data indicate the possibility of infecting hace transgenic mice with demonstration of viral replication and mild lung lesions. mouse adaptation of sars-cov- , as done with sars-cov- , will likely be required to obtain a virus that causes more severe disease in mice. models that develop acute lung injury with some lethality and that mimic the human condition will be important for evaluating vaccine safety. • previous studies from sars-cov- and mers-cov indicated that some vaccines, especially those using whole inactivated virus, could enhance the disease induced in mice challenged with sars-cov- or mers-cov. the lung lesions were highly inflammatory, with a dominance of eosinophil infiltration and occurred in animals despite presence of a neutralizing antibody response and reduced challenge virus replication in the lungs. such studies have not yet been completed for sars-cov . • in mice, this immunopathology was considered a consequence of a dominant th type response to the vaccine antigens. it was not seen after including adjuvants (e.g. cpg) in the vaccine or other vaccine formulations known to drive immune responses towards th . the timing of challenge after vaccination may be critical. it would be of major interest to explore the outcome following challenge at later timepoints when antibodies are significantly decaying. • one should be aware of the potential confounding effect of cell-culture excipients in the vaccine and challenge strain material. it is known that impurities such as fetal calf serum in the preclinical vaccine preparation may induce eosinophil influx in any mouse model if the challenge strain also contains the same excipients. • in these models, characterization of the immune response to the candidate vaccine (e.g., igg isotypes, th markers) may have some predictive value. • other small animal models which can be infected by sars-cov- can be considered (e.g. ferret, hamster). development of small animal models of severe disease will also inform studies of vaccine-enhanced disease. • passive transfer in nhps of human antibodies generated during phase trials, followed by viral challenge could be considered to assess the risk of disease enhancement. • challenge of immunized animals with a closely related heterologous cov strains may assess the risk of enhancement during future outbreaks. • in case of disease enhancement, in-depth studies in animal models may give some indications on the mechanism of immunopathology. they can inform human trial designers on the critical immunological risk markers to be monitored in phase trials. • given what will be the unprecedented demand for an effective vaccine, the use of adjuvants may be critical for sub-unit vaccines in providing increased immunogenicity, breadth of response, dose sparing, duration of response, potentially cross-protection against new cov strains, and possibly minimize the risk of enhanced disease. preference should be given to th -driving adjuvants with an established safety profile in humans. • understanding of the role of cross-reacting antibodies from prior coronavirus infections may have on natural disease caused by sars-cov- or influence the risk of enhanced disease following vaccination may inform vaccine design. • data are needed on whether antibody waning could increase the risk of enhanced disease on exposure to virus in the long term. it was the opinion of the experts that animal data to support clinical development could address: • post-vaccination (neutralizing) antibody responses, and t cell analysis to demonstrate a th response. • post-vaccination challenge data from nhps with careful evaluation for immunopathology and quantitative virology in the animals. • small animal data may also provide important supporting evidence of safety, and hamster, ferret and mouse models are likely to be available for developers. • where possible, immunopathology experiments with a positive control (e.g., formalin inactivated alum-adjuvanted sars-cov- or sars-cov- vaccine) and a mockimmunized negative control will provide best guidance. it was felt that it will be important to establish broadly accepted endpoints and scoring systems to allow comparison of various vaccine candidates. who is working on this issue. • for vaccine constructs likely to induce a predominant th response, the group felt that animal studies should be considered before entering human phase trials in more than one animal species including nhps where possible. it was noted that the absence of a th response does not eliminate the risk of enhanced disease. • since not all studies that have begun or are about to begin will prescreen to determine preimmunization serostatus of participants, although this shall be determined retrospectively, appropriate baseline blood specimens should be obtained and stored. because the virus is spreading rapidly, such specimens will allow assessment of the immune response in both seronegative and seropositive persons as both are likely to be vaccinated. • level of neutralizing antibodies and determination of the relative ratio of binding to neutralizing antibodies will be important to assess the potential risk of enhanced disease. also, detection of initial priming that includes cd t cells and/or a cd th biased response is likely to mitigate the risk of disease enhancement. determination of memory responses will be useful, particularly if sars-cov- continues to circulate. • consideration should be given to the use of post-vaccination sera from vaccinees which could be used for antibody transfer studies in animals to look for enhanced disease and for evidence of cross-protection against other coronaviruses. • monitoring for enhanced disease in immunized participants may require longer follow-up than is usual in phase trials but need not delay phase trials. • investigators on the call requested frequent updating with both preclinical and evolving clinical data that are being developed by the different academic and industrial developers to help in decision-making about the various vaccine clinical trials. creation of a central information hub was encouraged for this purpose. • participants on the call expressed the need for standardization of protocols, data collection forms, critical assays (including reagents) and biobanking of samples from initial clinical trials to allow future re-assay once standards are agreed to and enable comparison of results across trials • the group of experts considers that the demonstration of some disease enhancement with any candidate vaccine after viral challenge in animal models should not necessarily represent a no-go signal for deciding whether to progress into early trials in clinical development of a covid- vaccine. • continuous monitoring of this risk during clinical trials in an epidemic context will be needed. • each observed effect should be discussed by the developers with their regulators who will ultimately define the actual requirements for clinical studies. the considerations in this document should be interpreted as general scientific remarks based on current knowledge to inform a research agenda that could be beneficial for vaccines in development. these considerations are not of a regulatory nature and cannot in any sense replace the need for proper regulatory consultations on the requirements for vaccines clinical trials. vaccine developers are therefore encouraged to seek individual scientific advice from regulatory authorities. a novel coronavirus from patients with pneumonia in china covid- data center developing covid- vaccines at pandemic speed respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine a role for immune complexes in enhanced respiratory syncytial virus disease enhanced pulmonary histopathology induced by respiratory syncytial virus (rsv) challenge of formalin-inactivated rsv-immunized balb/c mice is abrogated by depletion of interleukin- (il- ) and il- pulmonary histopathology induced by respiratory syncytial virus (rsv) challenge of formalin-inactivated rsv-immunized balb/c mice is abrogated by depletion of cd + t cells lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease viral-induced enhanced disease illness aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice the sars-cov ferret model in an infection-challenge study severe acute respiratory syndrome coronavirus infection of golden syrian hamsters lethal infection of k -hace mice infected with severe acute respiratory syndrome coronavirus vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease a dna vaccine induces sars coronavirus neutralization and protective immunity in mice generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus mouse-adapted mers coronavirus causes lethal lung disease in human dpp knockin mice a mouse model for mers coronavirus-induced acute respiratory distress syndrome adaptive evolution influences the infectious dose of mers-cov necessary to achieve severe respiratory disease recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys exacerbated innate host response to sars-cov in aged non-human primates b -n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n / . virology intratracheal exposure of common marmosets to mers-cov jordan-n / or mers-cov emc/ isolates does not result in lethal disease anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine protease-independent fcgammar pathway antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets animal models and antibody assays for evaluating candidate sars vaccines: summary of a technical meeting - immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus a highly immunogenic, protective, and safe adenovirus-based vaccine expressing middle east respiratory syndrome coronavirus s -cd l fusion protein in a transgenic human dipeptidyl peptidase mouse model anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates correction: immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates immunogenicity and protective efficacy in monkeys of purified inactivated verocell sars vaccine. vaccine immunogenicity and protective efficacy in mice and hamsters of a betapropiolactone inactivated whole virus sars-cov vaccine the pathogenicity of sars-cov- in hace transgenic mice age-related rhesus macaque models of covid- comparative pathogenesis of covid- , mers, and sars in a nonhuman primate model immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen cryo-em structure of the -ncov spike in the prefusion conformation towards an evidence based approach for the development of adjuvanted vaccines mf adjuvant enhances diversity and affinity of antibody-mediated immune response to pandemic influenza vaccines as -adjuvanted h n vaccine promotes antibody diversity and affinity maturation, nai titers, cross-clade h n neutralization, but not h n cross-subtype neutralization selective induction of antibody effector functional responses using mf -adjuvanted vaccination superior antigen-specific cd + t-cell response with as -adjuvantation of a trivalent influenza vaccine in a randomised trial of adults aged and older correlates of adjuvanticity: a review on adjuvants in licensed vaccines investigating the effect of as adjuvant on the plasma cell repertoire following ph n influenza vaccination h n influenza vaccine formulated with as a induces strong cross-reactive and polyfunctional cd t-cell responses protective immunity induced with malaria vaccine, rts,s, is linked to plasmodium falciparum circumsporozoite protein-specific cd + and cd + t cells producing ifn-gamma impact of adjuvants on cd (+) t cell and b cell responses to a protein antigen vaccine: results from a phase ii, randomized, multicenter trial different adjuvants induce common innate pathways that are associated with enhanced adaptive responses against a model antigen in humans. front immunol systems analysis of human vaccine adjuvants innate transcriptional effects by adjuvants on the magnitude, quality, and durability of hiv envelope responses in nhps all authors attest they meet the icmje criteria for authorship key: cord- -aaokc authors: stanberry, lawrence r.; strugnell, richard title: vaccines of the future date: - - journal: perspectives in vaccinology doi: . /j.pervac. . . sha: doc_id: cord_uid: aaokc nan the advances made in vaccine technology since edward jenner vaccinated the young james phipps against smallpox have had a spectacular impact on human health over the last two centuries (see chapter e vaccine evolution). vaccines have been fundamental in the control and elimination of many debilitating and lethal diseases, and more diseases are currently targeted for eradication by vaccination. recent major breakthroughs in immunology, molecular biology, genomics, proteomics, biochemistry and computing sciences have driven vaccine technology forward, and will continue to do so. many challenges remain, however, including persistent or latent infections, pathogens with complex life cycles, antigenic drift and shift in pathogens subject to selective pressures, challenging populations and emerging infections. to address these challenges researchers are exploring many avenues: novel adjuvants are being developed that enhance the immune response elicited by a vaccine while maintaining high levels of tolerability; methods of protective antigen identification are iterated with every success; vaccine storage and transport systems are improving (including optimising the cold chain and developing temperature-stable vaccines); and new and potentially more convenient methods of vaccine administration are being pursued. high priority targets include life-threatening diseases, such as malaria, tuberculosis (tb) and human immunodeficiency virus (hiv), as well as problematic infections caused by ubiquitous agents, such as respiratory syncytial virus (rsv), cytomegalovirus (cmv) and staphylococcus aureus. non-traditional vaccines are also likely to become available for the management of addiction, and the prevention, treatment and cure of malignancies. this chapter is not meant as a compendium of all new-generation vaccines, but rather as an outline of the modern principles that will likely facilitate the development of future vaccines. as shown in figure . , there are several key elements that are likely to be understanding modern vaccines the foundation for the development of future vaccines. this chapter will illustrate these elements and provide examples that show promise. since the first use of an adjuvant in a human vaccine over years ago, adjuvant technology has improved significantly with respect to improving vaccine immunogenicity and efficacy. over the advances in adjuvant design have been driven by parallel advances in vaccine technology as many modern vaccines consist of highly purified antigens e with low non-specific reactogenicity which require combination with adjuvants to enhance the immune response. future developments in adjuvant technology are expected to provide stronger immune priming, enhance immune responses in specific populations, and lead to antigen sparing. adjuvants to date have demonstrated an ability new adjuvants must aim to drive the immune response that is associated with lifelong protection. new adjuvants and adjuvant combinations will play many roles in future vaccines as illustrated in figure . . adjuvants will need to be individually selected for specific vaccine targets in order to achieve the desired goal (ie enhanced immunogenicity, induction of specific immune profile etc). to deliver this aim, some adjuvants will be mixed with free antigens, while others will need to be covalently linked to the antigenic moiety as part of a complex molecule. some examples of new adjuvants that have been evaluated in humans or that are in clinical trials are listed in table . (also see chapter e vaccine adjuvants). modern approaches to antigen design tend to eschew classical trial and error techniques in favour of identifying the type of pathogenic structures (ie antigens) that are most likely to be important immunogens based on their structural signature or physical location within the pathogen (table . ) (see chapter e vaccine antigens). the t or b cell immune responses to an antigen are targeted to precise regions of the antigen (ie epitopes e either linear or three-dimensional conformational structures; in the case of protein antigens these are specific peptide epitopes). historically, simple, linear, synthetic peptide epitope vaccines have been poorly immunogenic because they lack a specific conformation and are easily degraded by a variety of extracellular and cell-surface proteases that serve to limit epitope presentation to t cells and/or result in destruction of the b-cell epitope. peptide vaccines need to union. every effort has been made to verify the information in the above table. the information included is not meant to be exhaustive but is intended to provide an overview of the subject matter. subunit and individual epitope vaccines need to be optimised to ensure adequate immunogenicity. novel strategies are being developed and exploited in order to identify antigens recognised by t and b cells, thus facilitating a more knowledge-based vaccine design. one of the most common ways to identify these antigens is to measure cellular proliferation (t or b cells) upon in vitro stimulation with antigen. high-throughput screening assays of candidate synthetic peptides that drive cellular proliferation help speed the rate of antigen discovery. reverse vaccinology combines knowledge of the pathogen's genome sequence with known protein sequences via computer analysis, to predict protein expression and post-translational modifications and identify likely vaccine candidates (see chapter e vaccine antigens; figure . ). the development of epitope-based vaccines is one example of reverse vaccinology where computer software combines prediction algorithms to suggest sequences similar to those for pathogenic components. epitope mapping, combined with the creation of more stable poly-epitope vaccines, may lead to the successful translation of this technology into products. mhc molecules exhibit widely varying binding specificities; a vaccine expressing a single peptide antigen would therefore only target a few mhc molecules and thus only be recognised by the t cells of individuals carrying a specific mhc phenotype. poly-epitope technology could be used to generate a synthetic protein carrying antigenic epitopes from multiple strains or pathogens. this would overcome the mhc restriction and afford protection in individuals carrying different mhc types. the screening of pathogen peptide libraries is another example of new approaches to antigen discovery. screening methods are used to identify antigens that can stimulate cd þ or cd þ t cells, or which bind to antibodies from humans known to have been infected with the relevant pathogen. where peptide screening uses antibodies, an additional consideration is the synthesis of antigens that contain the tertiary (folding/three-dimensional) structure of the native immunogen, since vaccine efficacy can be impacted by infidelities in the structure of the final product. incorrect protein folding may result in a less immunogenic antigen or an antigen that induces an immune response that differs from that of the native immunogen. the mimicking of the three-dimensional structure of the native immunogen is important during the synthesis of antigens that are being used to target b-cell responses. conversely, the requirement for folding is reduced for t cells since t cells bind only processed peptides, from degraded proteins. likewise, dna expression libraries using the pathogen genomic dna have been screened using animal model systems to identify genes encoding proteins that afford protection against infection or disease caused by the pathogen. one example is genocea's vaccine development programmes that are built around a broad platform for the rapid discovery of t-cell antigens. the process is explained in figure . . t-cell antigens, specifically antigens that stimulate cd þ and cd þ t cells, are critical to generating disease-specific cellular immune responses and long-term t-cell memory. stability of the final product is another important consideration. adverse environmental conditions can result in degradation of the vaccine, rendering it non-immunogenic. in order to maintain product integrity many vaccines (particularly live vaccines) must be stored at cold temperatures ( c). the maintenance of the vaccine at this temperature from production site to distribution site, and medical office or clinic, is referred to as the 'cold chain'. maintaining the cold chain is much less of a challenge in resource-rich countries, but can be a major barrier to vaccine implementation in resource-limited areas. ongoing research designed to increase our understanding of vaccine degradation may address the problems associated with cold chain management and lead to the development of thermostable vaccines. the level of antigen presentation which occurs with some current vaccines may sometimes be insufficient to drive long-lasting immune responses of high quality (see chapter e vaccine antigens). this may be due to inadequate exposure of the antigen to immature antigen-presenting cells (apcs) rapid or subimmunogenic degradation or sequestration of antigens, or lack of immunogenicity due to the physical presentation of the antigen. the discovery and modifying vaccine formulations to increase tolerance to temperature fluctuations is likely to increase the shelf-life of the product and reduce transport and wastage issues. understanding modern vaccines refinement of new and varied options for antigen presentation is expected to allow the design of vaccines to produce specific immune profiles. some of these technologies have been shown to facilitate oral delivery to target mucosal immune responses and also trigger both innate and adaptive immune systems, including t-and b-cell effector and memory responses. candidate viral vector vaccines utilise a non-pathogenic virus to carry and subsequently induce expression of genes that produce immunogenic foreign proteins at high levels in the host. these are taken up by immature apcs, and have been shown to lead to a robust, long-lasting immune response to the target antigen ( figure . ). viral vector vaccines, eg recombinant poxvirus vaccines, can be administered mucosally to stimulate mucosal immune responses. the attenuated modified vaccinia virus ankara (rmva) vectors are showing promise as mucosal delivery vectors. pre-existing immunity to the viral vaccine vector is an impediment to successful use of this approach. as ways to avoid anti-vector immunity, viruses can be attenuated or inactivated, by deleting or replacing pathogenic genes. figure . demonstrates how viral vaccine vectors are made. dna expressing an immunogenic transgene (the vaccine antigen) is inserted into the viral vector genome for expression following administration into the recipient; expression of the vaccine antigen can be boosted by using a variety of dna promoters. if the viral vector is no longer able to grow and replicate, the virus is grown using a cell line (a so-called complementing cell line) that has been engineered to produce the missing viral product. often, viral genes are removed in an effort to reduce or eliminate the pathogenicity of the vector and in some cases viral genes are removed to make the vector itself less immunogenic; an anti-vector immune response would greatly reduce the ability of the vector to induce an antigen-specific response. examples of viral vector candidate vaccines in clinical development are listed in table . . non-pathogenic bacterial vectors have many features that make them an attractive vaccine platform. bacterial vectors can be engineered for maximum safety (eg deletion of two or more genes from the same metabolic pathway), and to express large numbers of foreign antigens ( figure . ). two key issues affecting bacterial vaccine vectors are: a) to decide whether the optimal platform should be a bacterial vaccine in its figure . viral vectors for vaccines. viral vector vaccines exploit the natural ability of viruses to infect or otherwise enter (in the case of disabled viral vectors) host cells, and then deliver pathogen-specific antigens. antigen-encoding genes are isolated from the pathogen and inserted into the viral vector genome. the viral vector can then be used as a factory for production of large quantities of pathogen antigen in vivo, following introduction of the vector into the vaccine recipient, with the pathogen antigen then expressed on the surface of the infected/transduced host cells or exported out of the producer cell. mhc, major histocompatibility complex. own right or a bacterial vector system to deliver exogenous antigens; and b) to determine whether re-administration of the vector, either with the same or different target antigens, will fail because of the immune response to the bacterial vector vaccine at the time of its initial administration. initial assessments of the feasibility of using attenuated bacterial vectors for the delivery of foreign antigens have focused on salmonella species. bacterial vaccine vectors for humans, however, have been disappointing so far. it may be necessary to develop unique bacterial vaccine vectors for delivering exogenous antigens, in which case the vectors can be modified to allow for re-use. for example, if immunity against the vector, which is a major impediment to vaccine re-use, is determined by antibodies against the surface structures of the bacterium (such as lipopolysaccharide [lps]), the dedicated vaccine vector could be developed to lack expression of lps or to express truncated/ different forms of lps to the target, thereby avoiding priming of the immune response and allowing for re-use of the vector and/or vaccine. some potential options for live, attenuated bacterial vectors are shown in table . . dna vaccines are the result of the discovery in the early s that the gene, rather than the encoded protein, if delivered in an 'expressible' form, could induce an immune response (see chapter e vaccine evolution). the principle behind dna vaccines is that the antigenic molecule is produced within the host from the dna or rna that is injected, in contrast to more traditional vaccination where the antigen is supplied in the vaccine formulation. the gene(s) for target antigen(s) is/are usually encoded in a circular plasmid expression vector under the control of promoter sequences that direct gene expression in mammalian cells, which is achieved after injection into mammals. the dna vaccine process can circumvent some of the major issues resulting from recombinant protein administration. the construction and production of the plasmids carrying the gene of interest together with the promoter sequences is relatively simple; antigens expressed from plasmids retain their native conformation, the gene can be readily modified to produce tailored antigens, and bacterial plasmid dna is intrinsically immunogenic (subsequently shown to result from the pathogen-associated molecular patterns [pamps] it carries). additional desirable features include the ability to engineer and deliver genetic adjuvants in tandem or parallel with the antigen, the potential to deliver multiple antigen genes in one construct or within other constructs that encode adjuvanting protein(s), and the ability to induce both cellular and humoral immune responses. despite promising data in pre-clinical testing, dna vaccine candidates have shown only limited success in clinical settings so far. one of the current drawbacks of dna vaccines is the inefficiency of conventional delivery methods for the plasmid dna; however, understanding modern vaccines emerging proprietary particle-mediated delivery technology or electroporation technology seeks to improve this situation. with the electroporation method, brief electrical pulses are applied at the site of immunisation which causes a transient disruption of cell membranes. this results in an enhancement in uptake of the dna vaccine between e -fold. examples of dna candidate vaccines in clinical development are presented in table . . dendritic cell (dc) vaccines typically use monocytes harvested from the blood (in most cases from the individual who will receive the vaccine) to produce immature dcs in vitro. the monocytes are antigen-loaded and treated to induce their maturation into apcs and infused back into the patient. the first food and drug administration (fda)-approved dc vaccine, designed for the treatment of prostate cancer, was licensed in (sipuleucel-t); examples of other targets for dc vaccine therapy are presented in table . . dc vaccines offer an individualised approach to therapeutic vaccine development, but represent a specialised method of vaccination that is currently limited to aggressive cancers, and the treatment of serious, intractable infections. a comparison between the strengths and weaknesses of selected new vaccine platforms is presented in table . . developing administration techniques that place the vaccine directly at the site(s) where pathogens are most likely to initiate an infection (eg mucosal or respiratory sites) is likely to improve vaccine efficacy and safety. traditional methods of vaccine administration can potentially pose a number of limitations with respect to reactogenicity, immunogenicity, convenience, efficacy, safety and cost-effectiveness. the information included is not meant to be exhaustive but is intended to provide an overview of the subject matter. ongoing research on alternative experimental administration strategies includes ballistic delivery to skin (the gene gun), the transdermal patch and other intradermal methods, plus sublingual, aerosol, rectal and vaginal mucosal vaccines. the main advantages of alternative delivery strategies are the potential to induce immune responses at the common portals of pathogen entry (eg oral polio vaccine replicating in the gut), potential convenience (eg ease of use of the transdermal patch), potential combination of vaccines to reduce or simplify the vaccination schedule, and reduction or elimination of administration via standard hypodermic needle injection. despite the intuitive value of these approaches, few vaccines today are administered via non-im routes. this is for several reasons including feasibility, lack of proven efficacy and limited safety data. some problems have been observed with new routes of delivery, for example, after the launch of an inactivated intranasal influenza vaccine (a virosome formulation adjuvanted by heat labile enterotoxoid of escherichia coli), post-licensure data indicated a significantly increased risk of bell's palsy in vaccinees and forced its withdrawal from the market. this experience led to a higher level of caution in the development of intranasal vaccines. today, the only example of a licensed vaccine against a latent infection is the zoster vaccine; the vaccine formulation is the high potency (about -fold) version of the live, attenuated varicella zoster virus (vzv) vaccine. this vaccine has been used to boost the anti-vzv cell-mediated immune response in older subjects and has been shown to reduce the overall incidence of zoster by % in subjects aged years or older (oxman et al., ) . future vaccines may control persistent infections either by preventing the initial infection or disease (ie prophylactic vaccines) aerosol delivery: 'mass immunization of almost all susceptible children in a short period of time, has the potential of rapidly eliminating measles as a public health problem. immunization by inhalation of aerosolised measles vaccine provides a procedure that could make such a mass programme possible, especially in parts of the world where measles continues to be a serious problem.' (sabin et al., ) . administering the measles vaccine as an aerosol, either as nebulised vaccine or as an increased understanding of human immunology and of hostepathogen interactions should enable the identification of the type(s) of immunity required to effectively prevent or control persistent infections (see chapter e vaccine immunology). some examples of persistent infections are shown in table . . mycobacterium tuberculosis can persist in a latent state within the human host for years without causing disease (latent tb). protection against miliary (disseminated) tb in children is provided by the bacille calmetteeguérin (bcg) vaccine, developed through culture attenuation of mycobacterium bovis early in the th century, which is routinely given in many countries. the vaccine, however, provides only modest and often temporary protection against pulmonary tb, and provides lower efficacy in resource-limited regions closer to the equator. in addition, vaccination with live, attenuated mycobacterium bovis is a particular concern in hiv-positive individuals, especially those with advanced immune suppression; this population would particularly benefit from tb vaccination as tb is a leading cause of death worldwide for people with hiv/acquired immunodeficiency syndrome (aids). however, a recent phase iii trial demonstrated that protection against tb can be provided to individuals with hiv by using an inactivated whole-cell mycobacterial vaccine (von reyn et al., ) . the current state of tb vaccine development has been summarised in reviews by walker et al. ( ) and lambert et al. ( ) and examples of vaccines in development are shown in table . . cytomegalovirus, a herpes virus, establishes latent infection in cells in the bone marrow and peripheral blood. primary infection during pregnancy is associated with congenital infection that frequently causes a well-characterised spectrum of abnormalities and disabilities, which may be severe or fatal. reactivation in pregnancy is common, but is unlikely to cause severe congenital infection, although some manifestations, especially hearing loss, remain common. reactivation of cmv is of special concern in immunocompromised individuals, where severe and fatal pulmonary, hepatic and central nervous system infections are common. gastrointestinal disease and retinitis are common in association with hiv. a successful cmv vaccine has proved elusive for more than years. based upon the observation that antibodies to the cmv envelope glycoprotein b (gb) could pass et al., ) . a recent phase ii clinical trial in cmv-seronegative women year post-partum has shown the potential of gb/mf in decreasing incident cases of maternal and congenital cmv infection (pass et al., ) . this is the first evidence that a cmv vaccine can protect against infection. an alternative approach to the development of a cmv vaccine has been to utilise dna vaccination to induce host responses to cmv gb and phosphoprotein (pp is another viral target). recent studies have shown that injection of combinations of plasmids, formulated with an adjuvant, can induce vaccine-specific immune responses, and can prime for effective memory responses. the hallmark of herpes simplex virus types and (hsv- and hsv- ) is their ability to establish and maintain latent infection in to be exhaustive but is intended to provide an overview of the subject matter. sensory ganglion neurons. periodic reactivation of the latent infection results in recurrent infections. both hsv- and hsv- can cause myriad diseases but the greatest public health problem is genital herpes. genital hsv- infection increases the risk of hiv acquisition and transmission, and control of genital herpes has been predicted to significantly impact the hiv epidemic. given the complex natural history of hsv infections, vaccines could have a variety of possible risks and benefits (table . ). an effective hsv vaccine has been sought for more than years. recently, an hsv- glycoprotein d (gd ) candidate vaccine containing the as adjuvant (see chapter e vaccine adjuvants), was tested in three large, double-blind, phase iii controlled trials. the first two studies recruited volunteers with a partner with genital herpes disease and found the candidate vaccine was % effective against genital herpes disease in women seronegative for both hsv- and hsv- (stanberry et al., ) . trends towards protection against infection were also observed, but were not statistically significant. the candidate vaccine was not effective in hsv- seropositive women; or in understanding modern vaccines men, regardless of their hsv seropositivity status. these were the first studies to report a significant difference in vaccine efficacy between men and women. this finding could have important implications for other vaccines targeting sexually transmitted diseases. the basis for this difference could relate to differences in how men and women respond to novel adjuvants or may reflect differences in the acquisition and natural history of genital herpes in men and women. a third phase iii efficacy trial of the gd candidate vaccine in hsv- and hsv- negative women who thought themselves possibly at risk of acquiring genital herpes (a different risk population than in the original two trials) has been completed and is being analysed. an initial assessment of the results of the third trial showed that the vaccine had an acceptable safety profile but the primary trial endpoint, prevention of genital herpes disease, was not met (niaid, ) . although the development of the vaccine has been stopped, further analyses and comparison of the trials may guide researchers as they continue seeking vaccines to control hsv infections. as discussed in chapter e vaccine immunology, some pathogens have complex life cycles. one specific example is parasites, sometimes using more than a single host, where each development phase is marked by differential expression of major proteins, meaning that possible antigen targets are host-and development-phase specific. taenid worms aside, vaccines against parasites have been extremely difficult to develop and only a limited number have performed well in later-stage clinical trials. the protozoan parasite plasmodium falciparum, the most common cause of malaria, has a complex life cycle, as shown in figure . . the plasmodium parasite has a genome encoding more than proteins, and presents different allelic and immunogenic table ) . one of the furthest advanced of these new candidate vaccines is rts,s/as . the vaccine targets the pre-erythrocytic stage of the parasite (figure . ) . to be protective, a vaccine targeted at this phase needs to induce humoral immunity, to prevent parasites from invading the liver, and cell-mediated immunity to destroy hepatocytes that become infected in the face of the humoral immune response. the rts,s antigen, produced in saccharomyces cerevisiae, contains sequences of the p. falciparum circumsporozoite protein, linked to the hepatitis b surface antigen (hbsag). this chimeric protein spontaneously assembles into mixed polymeric particulate structures. in phase ii studies, the rts,s/as candidate vaccine induced a strong neutralising antibody response and cell-mediated immunity, and afforded protection against malaria (bejon et al., ; abdulla et al., ) . rts,s/as has been selected to proceed to phase iii clinical testing due to its higher efficacy compared with alternative formulations. if successful, the rts,s/as candidate vaccine could be the first licensed human vaccine against a parasite. other malaria candidate vaccines in development are shown in appendices, supplementary table . pathogens may mutate or recombine to change their antigenic profile. antigenic drift refers to a gradual process whereby point mutations in genes encoding antigenic proteins change the antigen sufficiently so that over time previously effective antibodies and vaccines no longer effectively control the pathogen and hence new vaccines need to be created. antigenic shift is a more dramatic event where there is a recombination of genes between different pathogen strains that gives rise to a new strain with a unique antigenic profile. in theory, pathogens are susceptible to selective pressure and an immunological environment that provides strong selective pressures should provide the 'bottleneck' that drives selection. this occurs with influenza viruses, where the high mutation frequency allows for the selection of mutants that are not neutralised. the risk of vaccine-mediated immune selection of pathogens, though certainly present, is difficult to demonstrate. moreover, peptide vaccines only use the antigenic epitope so the risk of pathogen evolution is theoretically increased. however, this phenomenon has not been regularly observed in experimental studies and may reflect the complex nature of most vaccine antigens and the presence of immune responses against multiple antigens and multiple epitopes within antigens. serotype replacement, where the distribution of specific microbial serotypes within communities changes after the introduction of vaccines, has occurred for some bacterial pathogens and may be a consequence of the use of capsular vaccines that address only a limited number of serotypes. similarly, since their introduction in the s, the use of antibiotics has exerted a selective pressure on bacterial strains leading to selection for common resistance alleles (eg the extended-spectrum beta-lactamase [esbl] resistance of enteric bacteria and beta-lactamase resistance in gonococci). to date, there has been no requirement to remodel a vaccine because of vaccine-mediated immune escape; however, new vaccines against the pneumococcus have been licensed, including additional capsular types, to expand the geographical coverage of most frequent types and, in part, to counter the observed phenomenon of serotype replacement. annual seasonal influenza infections are subject to natural antigenic drift which requires the reformulation of the vaccine when drifts occur, but there is no evidence that the deployment of the vaccine accelerates this drift. antigenic shift, while not the result of selective pressure, gives rise to viral strains containing a mixture of the surface antigens from the parent strains. pathogens that can undergo antigenic shift, including influenza viruses (figure . ), present major challenges for vaccine developers. chapter e vaccine adjuvants, there has been progress in the another approach to the problem of influenza genome shifts has been to target weakly immunogenic conserved antigens such as the influenza m e protein. one approach to addressing the weak immunogenicity of the antigen has been to link it to a potent toll-like receptor adjuvant such as flagellin, an approach developed by vaxinnate inc. during primary infection of a single individual with hiv, mutations in surface proteins of the virus lead to selection of a 'cloud' of antigenic variants that can evade the cell-mediated immune responses complicating the development of broadly effective vaccines. this propensity for mutation has given rise to many strains of hiv (figure . ). two types of hiv, hiv- and hiv- , have been identified, with hiv- being the most common. on a global scale, hiv- strains are differentiated according to their respective group and subtypes (or 'clades') within groups. the amino acid sequence of the viral envelope glycoprotein gp shows e % divergence between clades and up to % divergence within any given clade, which constitutes a formidable hurdle to vaccine development. this is made worse by recombination between clades of hiv- , which has produced circulating recombinant forms (crfs) which differ in antigenicity depending on the geographical region. since the initiation of hiv vaccine programmes, more than candidate vaccines have been tested in over phase i/ii clinical trials involving more than , healthy human volunteers. regrettably, all attempts to date have failed to yield a licensed hiv vaccine. questions remain concerning the immune mechanisms behind vaccines that achieve partial protection. regardless of the unknowns, the ability to prevent infection in at least some individuals still offers real hope that a globally effective hiv vaccine might be possible. current research is comparing the immune responses of subjects who are naturally protected against hiv with those who were infected, seeking to find the elusive immunological mechanisms of protection to help guide the design of future t-cell vaccines against the virus. infections of group a streptococcal serotypes (ie streptococcus pyogenes) account for approximately % of cases of uncomplicated bacterial pharyngitis and streptococcal invasive infections in north america. the m protein of group a streptococci is a major virulence determinant of these organisms and also functions as a major target for protective antibodies. one of several strategies for vaccine prevention of these infections is based on type-specific m protein epitopes. however, group a streptococcal vaccine development faces many obstacles: i) the widespread diversity of circulating m protein types; ii) immunological cross-reactivity between epitopes in the m protein and several human tissues introducing an autoimmune risk; and iii) animal models are of limited value because humans are the only hosts for group a streptococci. in an attempt to partially overcome some of these obstacles, a design strategy akin to that of the pneumococcal polysaccharide vaccines has been employed to generate a group a streptococci multivalent m protein-based vaccine containing type-specific determinants from different m serotypes. this multivalent vaccine is currently in clinical development. the 'prime-boost' approach the term 'prime boost' (or heterologous boosting) describes an approach to vaccination where one type of vaccine, such as a live-vector vaccine, is administered followed by a second type of vaccine, such as a recombinant subunit vaccine. this is in contrast with the traditional method of homologous boosting in which two or more doses of the same vaccine are given successively. the intent of prime-boost vaccination is to induce different types of immune responses and enhance the overall immune response, a result that may not occur if only one type of vaccine were to be given for all doses. this approach has been employed in trials with, for example, tb, cmv, malaria and hiv candidate vaccines. for example, in studies on new tb vaccines, subjects already primed with the live, attenuated bcg vaccine have been boosted with a subunit adjuvanted vaccine (see tuberculosis). respiratory syncytial virus is a common cause of bronchiolitis and pneumonia in infants, and exacerbations of chronic obstructive pulmonary disease in the elderly. the development of an effective vaccine has been challenging; natural immunity to rsv infection is incomplete and re-infections occur in all age groups. moreover, the primary target population for vaccination is newborns and young infants, and they are a challenging population as they have relatively immature immune systems and the presence of maternal antibodies may interfere with vaccination of the young infant (see chapter e vaccine immunology). the initial efforts to develop a formalin-inactivated cell culture-derived rsv vaccine resulted in an unanticipated enhancement of natural rsv disease in some of the rsv-naïve infants who received the vaccine in a clinical trial and subsequently were exposed to rsv. the exacerbated disease is thought to be due to an exaggerated t helper type cell immune response (see chapter e vaccine immunology). safety concerns regarding the potential of vaccines to trigger or prime for immunopathological responses has resulted in a cautious approach to the development of rsv vaccines. the vaccine candidates most advanced in clinical development use two different approaches e one uses a live, attenuated virus with a gene deletion deliberately targeted to minimise immunopathological responses. the other approach uses a live viral vector to deliver only a key rsv surface antigen, thereby avoiding the risk of an immunopathological response arising from exposure to the rsv virus itself. infectious illnesses exert a major burden of disease in developing countries. the greatest burden is caused by diseases for which we currently have no vaccines, eg taeniid cestode parasites are associated with high human morbidity and losses in livestock. global efforts to reduce these infections in humans are ongoing through the use of antihelminthics and the implementation of lifestyle changes, but this is having little effect. however, substantial progress has been made towards developing veterinary vaccines which encourages investigation of the potential use of similar vaccines in humans to prevent, for example, hydatid disease (arising from infection with echinococcus granulosus) and cysticercosis (from infection with taenia solium). relative to their burden on society, such diseases have a low priority for funding. unless comprehensive measures are taken to address the gaps in funding, research and global immunisation coverage, developing countries will continue to be overwhelmed by some of the most devastating diseases. in order to improve the situation, collaborative schemes are underway that bring together academic institutions, industry and public/charitable financing organisations. microbiome project is a national institutes of health initiative that seeks to determine the relationship between human health and changes in the human microbiome. by using revolutionary sequencing technologies to characterise the microbiology of five body sites e oral cavity, skin, vagina, gut and nasal tract/lung e an association may be made between the microbiomes associated with either the healthy body state or disease. characterising microbes associated with disease-related pathogens may allow for the development of new vaccines that preserve or protect the healthy microbiome and hence could protect human health. some of the areas of current research are outlined in the box, right. some conditions traditionally thought of as non-infectious may in fact have infectious origins (table . ); therefore, vaccination could be a strategy to prevent these diseases. other diseases may result from an interaction between the host's genetic background and a particular microbe (a so-called gene-environment interaction). some diseases have an established link with an identified infectious agent. for example, primary cmv infection is a known cause of congenital mental retardation; similarly the link between bacterial vaginosis and foetal prematurity is widely accepted. while some links have been established, others remain speculative (table . ). candidate vaccines are in development for the prevention and treatment of various types of addiction. the basic concept is to induce the production of antibodies which will bind the drug and impede its crossing the bloodebrain barrier to exert its psychoactive effects. several nicotine candidate vaccines have now entered clinical trials. a cocaine candidate vaccine has also shown some benefit in a phase iib clinical trial. the key issue to date for both nicotine and cocaine candidate vaccines has been to induce continued on next page high immunoglobulin (ig)g anti-drug antibody levels, which appear to be critical in achieving some degree of efficacy. candidate vaccines against methamphetamine addiction are also in early development. to date, the approach to developing prophylactic cancer vaccines has been to target infectious diseases that cause or contribute to the development of cancer such as hpv (cervical cancer) and hbv (hepatocellular carcinoma). examples of infectious diseases associated with cancer are shown in table . . the successful development of a nicotine vaccine would be expected to reduce cigarette smoking-related lung cancer. some cancers express tissue-specific antigens that can be targeted by the immune system. therapeutic cancer vaccines aim to target tumour-associated antigens (taa) with t-cell mediated immune responses. taa can be related to the genetic changes that drive the cancer (eg ras oncogene), or inappropriate up-regulation/ expression of genes (eg carcinoembryonic antigen). with such taa targets, vaccines aim to maximally stimulate a cytotoxic t-cell response and their design often includes adjuvants to enhance antigen presentation. tumours develop in a multistep process in the face of the host immune response and frequently evolve to escape immune control. mechanisms of evasion include genetic changes (loss of human leukocyte antigen/taa expression) and induction of immune regulatory systems (t-cell anergy due to the activity of t reg cells) which limit anti-tumour immunity. the key approach for therapeutic cancer vaccines is resetting the immune response to deliver anti-tumour immunity that alters or destroys cancer cells and hence eliminates or reduces the tumour. one strategy uses the patient's own tumour as the immunogen, thereby providing all the potential idiotypic changes that might act as taa, in conjunction with antigen-presenting dcs harvested from the same patient and activated in vitro (see dendritic cell vaccines). there are different types of therapeutic candidate vaccines currently undergoing clinical trials for numerous types of cancer (table . ). the most advanced candidates currently in phase iii are described in chapter e vaccine adjuvants. there has been some success in the development of therapeutic cancer vaccines, with the fda approval of the first dc vaccine oxford biomedica muc , mucin cell surface; hil , human cytokine interleukin- ; asci, antigen-specific cancer immunotherapeutics; mage, melanomaassociated antigen; ctl, cytotoxic t lymphocyte. every effort has been made to verify the information in this table. the information included is not meant to be exhaustive but is intended to provide an overview of the subject matter. vaccine approaches. however, this presents opportunities for the application of novel technologies and adjuvants. some of the considerations for vaccines designed for use in special populations include: immunosenescence in the elderly; the poor immunological response to traditional vaccines seen in immunocompromised individuals (patients with hiv, transplant recipients); the crossing of vaccine components into the foetal bloodstream when vaccines are administered to pregnant women; and the safety and immunogenicity concerns surrounding vaccines for neonates due to their naïve and immature immune system. cell-mediated immunity is depressed in pregnant women, leaving them at high risk of infection from pathogens, including those harmful to the foetus. most live, attenuated vaccines are contraindicated during pregnancy because of the theoretical risk of foetal infection from the vaccine. however, inactivated viral or bacterial vaccines can be administered. pregnant women can, therefore, be vaccinated against some infections, including several that pass from mother to foetus (such as hepatitis a and b), and against infections acquired by the infant in the first few months of life (often from close contact with the mother). in the latter case, the infant can be protected by transfer of maternal antibodies during late gestation. examples of diseases that can be prevented in pregnant women include influenza, tetanus, diphtheria and probably pertussis. other diseases, such as those caused by the so-called torch pathogens (toxoplasma, others including syphilis, cmv and hsv), are not yet preventable through vaccination though encouraging phase ii results have been presented for a vaccine to prevent group b streptococcus carriage in pregnant women (hillier et al., ; smith, ). the h n pandemic influenza outbreak posed an increased risk to pregnant women and vaccination was specifically recommended in pregnant women as one of the high-risk groups. the pandemic example has emphasised once more the importance of protecting pregnant women against influenza. seasonal influenza vaccination in pregnancy is well tolerated and the benefiterisk profile when administered to pregnant women supports its use during pregnancy. many public health authorities worldwide recommend seasonal influenza vaccination in pregnant women and this recommendation is motivated not only by the potentially severe course of influenza during pregnancy, but also by the need to protect vulnerable infants against influenza during their first months of life. boosting rsv immunity in pregnant women through vaccination may be another approach to protecting the newborn against rsv infection during the most vulnerable early months after birth. neonatal immunisation is a strategy to protect infants against infections during a particularly vulnerable period. a recent study showed that immunisation with an acellular pertussis vaccine at birth and month of age induces high igg anti-pertussis antibody titres by months of age (wood et al., ) . it is hoped that this approach may reduce death and morbidity from bordetella pertussis infection in the first months of life. the elderly respond poorly to vaccination as the immune system becomes more senescent with increasing age and, therefore, new vaccine technologies are needed to improve the response to vaccination in this population. in the late s, an influenza vaccine adjuvanted with the oil-in-water emulsion, mf Ô, was shown to be more effective at inducing high immune responses in the elderly (minutello et al., ) . alternative vaccine administration techniques have also been studied in the elderly. research showed that in subjects years of age or older, an influenza vaccine administered with an intradermal microinjection system induced significantly higher antibody titres compared with im vaccination (arnou et al., ). subsequently, a microinjection system influenza vaccine was licensed for use in europe, and a high antigen dose formulation has been licensed for the elderly in the usa. individuals with cancer, hiv infection or who are asplenic can be immunocompromised as a result of their condition. patients can also be immunocompromised as a result of therapy, eg when receiving an organ transplant, radiation therapy or immunosuppressive medication. such patients are therefore at an elevated risk of infection from pathogens such as herpesviruses (particularly cmv and epsteinebarr virus), hbv, hcv, pneumocystis and coinfections and represent a special population regarding immunisation. despite a likely reduction in the efficacy of vaccinations in immunocompromised individuals, immunisation remains a frequent recommendation in the hope that at least partial immunity will be achieved. eliciting a response from vaccination in immunocompromised patients may require an increase in the dose and/or number of doses; altering the dosing interval; selecting a different vaccine formulation; or administration via an alternative route. evidence in this patient population is lacking and guidelines are often based on theoretical assumptions. live vaccines are generally contraindicated in immunocompromised or immunosuppressed individuals due to the risk of an active and symptomatic infection resulting from the vaccine itself (non-controlled replication process). encouragingly, vaccine formulations with highly purified antigens and novel adjuvants or alternative deliveries have been shown to induce more effective immune responses than the classical inactivated vaccines in immunocompromised hosts, including patients with end-stage renal diseases in pre-haemodialysis and haemodialysis (see chapter e vaccine adjuvants), patients with hiv and those who have received haematological stem cell transplants. the future of vaccine development can build on the knowledge and experience gained over the last years, and at the same time can take advantage of the most cutting-edge technologies and understanding modern vaccines research. new approaches to antigen selection and production, antigen delivery, adjuvantation and vaccine administration will allow us to target established and emerging diseases, and populations with complex needs. vaccination has been one of the most successful and cost-effective health interventions ever conceived and is now expanding further into cancer and chronic diseases. this expansion of scope and the subsequent impact on human disease is likely to continue into the future in currently unforeseen ways, further increasing the importance of vaccine science and engineering in improving human health. safety and immunogenicity of rts,s/as d malaria vaccine in infants intradermal influenza vaccine for older adults: a randomized controlled multicenter phase iii study efficacy of rts,s/as e vaccine against malaria in children to months of age women receiving group b streptococcus serotype iii tetanus toxoid (gbs iii-tt) vaccine have reduced vaginal and rectal acquisition of gbs type iii new vaccines against tuberculosis safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with mf adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults a subunit cytomegalovirus vaccine based on recombinant envelope glycoprotein b and a new adjuvant vaccine prevention of maternal cytomegalovirus infection vaccination with alvac and aidsvax to prevent hiv- infection in thailand successful immunization of children with and without maternal antibody by aerosolized measles vaccine. i. different results with undiluted human diploid cell and chick embryo fibroblast vaccines associate clinical professor of medicine glaxosmithkline herpes vaccine efficacy study group. glycoprotein-d-adjuvant vaccine to prevent genital herpes prevention of tuberculosis in bacille calmette-guérin-primed, hiv-infected adults boosted with an inactivated whole-cell mycobacterial vaccine the second geneva consensus: recommendations for novel live tb vaccines acellular pertussis vaccine at birth and one month induces antibody responses by two months of age genocea technology developing cell culture-derived pandemic vaccines seeking new pathways for hiv vaccine discovery infectious diseases and global cancer control intrauterine infection and preterm delivery the nih human microbiome project alliance for case studies for global health. case studies for global health: building relationships alliance for case studies international aids vaccine initiative vaccines of the future national institute of allergy and infectious diseases (niaid). statement: study finds genital herpes vaccine ineffective in women world health organization's special programme for research and training in tropical diseases aidsvaxÔ is a trademark of global solutions for infectious diseases corporation; alvacÔ is a trademark of connaught technology corporation mf Ô is a trademark of novartis; montanideÔ is a trademark of seppic; nanostatÔ is a trademark of nanobio corporation. for details of trademarks not listed above please see the manufacturer's website (see section internet resources) vaccines of the future key: cord- -ep lzeo authors: pawelec, graham; mcelhaney, janet title: recent advances in influenza vaccines date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: ep lzeo seasonal influenza remains a major public health problem, responsible for hundreds of thousands of deaths every year, mostly of elderly people. despite the wide availability of vaccines, there are multiple problems decreasing the effectiveness of vaccination programs. these include viral variability and hence the requirement to match strains by estimating which will become prevalent each season, problems associated with vaccine and adjuvant production, and the route of administration as well as the perceived lower vaccine efficiency in older adults. clinical protection is still suboptimal for all of these reasons, and vaccine uptake remains too low in most countries. efforts to improve the effectiveness of influenza vaccines include developing universal vaccines independent of the circulating strains in any particular season and stimulating cellular as well as humoral responses, especially in the elderly. this commentary assesses progress over the last years towards achieving these aims. since the beginning of , an unprecedented international academic and industrial effort to develop effective vaccines against the new coronavirus sars-cov- has diverted attention away from influenza, but many of the lessons learned for the one will synergize with the other to mutual advantage. and, unlike the sars- epidemic and, we hope, the sars-cov- pandemic, influenza will not be eliminated and thus efforts to improve influenza vaccines will remain of crucial importance. despite the common perception that it is "only a flu", seasonal influenza is a powerful pathogen responsible for many hundreds of thousands of deaths every year, especially of elderly people. it is somewhat puzzling that a highly contagious pathogen responsible for an estimated average of half a million fatalities every year is faced with such insouciance by most people. indeed, globally, , to , people die from the consequences of respiratory complications of influenza each year, with a huge difference in mortality rates according to age: possibly a vanishingly small number of younger adults ( . %) compared with . % of people aged - but rising to . % of people over . although this may seem to be a low chance of death caused directly by influenza, indirect sequelae of influenza infection contribute to a deteriorated health status and frailty in the elderly. these long-term sequelae are not limited to the respiratory tract but are increasingly associated with systemic, especially cardiovascular , symptoms. vaccination is a highly effective, minimally invasive, and cheap protective measure. however, there are many unsolved problems associated with the current seasonal influenza vaccines. thus, advances in effective influenza vaccination must include increasing the protective efficiency of the vaccine, especially in the elderly. this review will focus on recent advances in the science and the r&d, but one should not forget that the sociology of enhancing acceptance and uptake is of paramount importance too. predicting the next season's predominant influenza strains is a major undertaking that is always fraught with difficulty and often incorrect. recent advances in surveillance, data exchange, and bioinformatics may help to mitigate this problem. unexpectedly, help may be at hand from the surveillance of social networking sites, which may yield more topical data than public health services . however, instead of chasing seasonal variations, it would be much more advantageous to develop vaccines that were effective against all influenza strains, hence the intensive efforts to develop "universal vaccines" that will protect regardless of the seasonal strain. many such efforts focus on directing antibody production away from targeting parts of the virus that are different from strain to strain (i.e. the highly variable hemagglutinin [ha] head structures) and towards generating antibodies against conserved antigens. these may be from the stem part of the molecule, which is not normally immunodominant. these antibodies should provide heterosubtypic protection, i.e. against multiple different strains . however, until recently, this approach was limited to one viral group, but modifying the glycosylation state of stem regions may increase antibody accessibility and broaden the range of strains targeted . it is not known whether these experiments in mice or even in ferrets reflect what would be effective in humans. in naturally acquired h n infection, at least, it seems that anti-head and not anti-stalk antibodies play a predominant role, as expected . nonetheless, in passive immunisation studies, monoclonal antibodies against stem antigens can be protective in human influenza challenge . other approaches include active immunisation with multi-epitope protein vaccines containing several from influenza a and b, common to multiple strains of influenza virus. there is some evidence in humans that vaccination in one season may confer protection in the next season against strains that were not circulating at the time of the earlier vaccination. an analogous approach employing mixtures of synthetic peptides is also being pursued by other companies, for example . an advantage of this approach, analogous to that in cancer vaccines, is to select epitopes stimulating both humoral and cellular immunity; indeed, an interesting aspect of the action of the m- vaccine is that it does not contain any ha head epitopes and stimulates predominantly cellular responses. another approach attempts to exploit an elegant idea to focus antibody responses on the stalk by vaccinating with stalk domains engineered onto different head domains, which were shown to be protective in mice . a very recent publication now reports the outcome of a phase i clinical trial concluding that high anti-stalk titres were induced and paves the way for further development of universal influenza virus vaccines . a major bottleneck in influenza vaccine production is the inability to generate large amounts of vaccine quickly. one problem here resides in methodology for producing the vaccine, which requires improvement. the technique still employed by most vaccine producers is to grow the virus in hens' eggs. alternatives are being energetically sought after, including cell culture approaches and genetic engineering of viral components. there are several reasons for this, not only to speed up the cumbersome process of growing the virus in eggs (not to mention allergy problems) but also because in some cases egg-adapted viruses used to make the vaccine are not identical to the wild-type pathogen in circulation that season and do not protect . thus, it was concluded that the quadrivalent vaccine "flucelvax" (grown in cultured canine kidney cells) was potentially superior to egg-based vaccines in real-life practise as well as in clinical trials , and importantly a similar trivalent vaccine was reported to be effective in individuals over years of age . an alternative approach dispensing with the need for using live viruses has now reached fruition in the "flublok" vaccine using cultured insect cells to produce the vaccine after their infection with genetically engineered baculovirus vectors. employing a direct comparison with egg-grown virus vaccines, researchers reported that the recombinant vaccine was more efficacious, also in older adults . the use of recombinant technology obviates the need for using live viruses, and once the sequence is known, production can be much more rapid, which is important for combating new and emerging strains. it may also be possible to simplify production even more by producing recombinant vaccines in plants, which can result in very rapid synthesis of viral proteins. plant-based vaccines have not yet entered clinical trials, but experiments in mice have suggested that such virus-like particles are protective, even in very old animals . some investigators go even further and propose that mrna itself can be used as a vaccine without the need to produce viral proteins outside of the host at all. this results in probably the most rapid pipeline for developing prophylactic vaccines. to stabilise the rna, it can be enclosed in liposomes, as in the mrna- phase i trials in miami and berlin, showing immunogenicity and good tolerance . adjuvants are non-immunogenic vaccine components that enhance immunity in different ways: by a depot effect or by stimulating antigen-presenting cells, for example . optimal vaccine formulations will of course depend on the route of administration. all of these considerations have been receiving a great deal of attention over the last years. new data on the use of well-established adjuvants such as m or even alum reflect the rather surprising relative paucity of information on their action , . work on developing new adjuvants, such as "self-adjuvanting" lipid nanoparticles including toll-like receptor (tlr) ligands, is beginning to deliver encouraging results . mouse models are revealing that responses to intranasal vaccines may also be enhanced by the inclusion of adjuvants such as the tlr ligand cpg . increasing attention is being paid to testing new adjuvants not only in young animals but also in those of advanced age, the most susceptible group . mouse models are suitable for testing the use of live attenuated viral vaccines, rather than the non-viable immunogens usually employed. thus, the effects of different interferons on outcomes can be assessed using genetically deficient mice, for example . however, mice are not people, and proof of the pudding must be rcts in humans, but there remain relatively few adjuvants in common use so far and this differs in different countries [ ] [ ] [ ] . developing better adjuvants is a high priority, especially for vaccination of the elderly . current discussions centre around the practicalities and ethics of human challenge models for assessing vaccine efficacy in the most relevant possible "model" . flu vaccines are mostly injected intramuscularly (i.m.), which may be suboptimal. alternative routes include intradermal (i.d.), oral, or inhaled. regarding the route of administration, oral vaccines using live attenuated viruses have not yet found widespread application since pilot studies in humans in , but in the meantime different approaches are being tested to protect various forms of immunogen from degradation in the stomach , . the expectation that i.d. administration might be more effective than the usual i.m. injection seems not to have been fulfilled, judging from the data thus far accumulated. for example, a comparison of recombinant vaccine followed by trivalent inactivated vaccine given i.d. or i.m. revealed no differences . there were some earlier data suggesting that for the elderly the i.d. route might be more efficacious, but differences were not large . other variant application routes are being examined in mice, for example, the so-called "primepull" strategy whereby i.m. priming is boosted by inhalation . a very recent study employed intranasal administration of a novel adjuvanted vaccine to mimic natural influenza infection and to activate cd + t cells in situ in the lungs. this sophisticated approach employed ', '-cyclic guanosine monophosphateadenosine monophosphate (cgamp) as an "adjuvant" to activate the innate immune sensor stimulator of interferon genes (sting) targeted to lung-resident alveolar macrophages and alveolar epithelial cells (aecs). moreover, encapsulation of cgamp with pulmonary surfactants enabled stimulation of sting in aecs while sustaining the barrier of the pulmonary surfactant appropriately. in this way, intranasally administered vaccine resulted in protection against multiple strains of influenza in mice and ferrets, associated with both humoral and cellular responses . current vaccines are licensed based on world health organization-approved centres that test efficacy solely in terms of standard measures of humoral immunity. subjects do not necessarily include elderly people, who may not respond as well as younger adults, and differences in responses between older men and women are not taken into account. measured parameters defining responses on the basis of which vaccines are licensed may not always be appropriate: for example, the requirement for an increase in antibody titre to two of three strains in the vaccine may miss the third one that could be critical that season. also, non-response could erroneously be attributed to subjects who already have a high (protective) titre of antibody pre-vaccination and cannot increase it more. these measures of vaccine responsiveness thus cannot predict clinical protection. in this context, advances in molecular analysis of the immune response may lead to insights on individual variability and guide vaccine design and application , . hence, better predictive biomarkers are required, particularly considering the essential component of t cell immunity, which is not usually measured in the assessment of vaccine efficacy prior to licensing. over the last years, the realisation that vaccines need to be formulated to take the importance of the t cell response into account has come to the fore. this is a crucial issue not only because t cells are required to eliminate infected host cells prior to viral release but also because the epitopes recognised by t cells tend to be conserved across viral strains . thus, efforts to develop new and improved vaccines increasingly aim to generate cellular as well as humoral responses . moreover, the type of cellular response achieved is likely to be of major importance , and this can be markedly influenced by the nature of the adjuvant as well as by multiple host factors including frailty and medication , . the impact of frailty is currently being intensively investigated, with some studies clearly documenting an important influence on effectiveness while others do not . the reasons for such discrepancies are likely to be multifactorial and are not yet clarified but may at least partly reside in definitions of frailty and pre-frailty as well as the population studied and subject selection . in particular, there has been some controversy regarding whether repeated annual vaccinations with the same antigens might result in decreased responsiveness , although recent studies suggest that this is not likely to be the case . confounding factors could also include exposures over the life-course that may have thus far under-investigated effects, such as exposure to ionising radiation . it has also been noted that obesity can dampen influenza vaccine efficiency (although, importantly, it did not decrease efficacy, i.e. the serological response) . other important host factors probably include the influence of infection with hiv, even when controlled , or with other persistent latent viruses. one of the latter is most notably cytomegalovirus (cmv), which is also likely to play a role, although its impact on humoral responses remains controversial . this may at least partly be due to the fact that cmv infection mostly affects t cell responses, with only knock-on effects on antibody levels, especially in the elderly . the main mechanism responsible may be cmv-driven impaired granzyme b responses in influenza-specific cytotoxic cd + t cells and higher levels of il , either human or cmv decoy derived . finally, an impact of the microbiota, usually taken to refer to the gut microbiota, is emerging as an important confounding factor in responsiveness to influenza vaccination . attempts to manipulate the microbiota to enhance responsiveness are being tested in clinical trials using probiotics or synbiotics , so far without resounding success but with some recent evidence of efficacy in the elderly . an extensive "super-meta-analysis" of studies published at the end of investigated the state of knowledge on the impact of "intravenous drug use, psychological stress, acute and chronic physical exercise, genetic polymorphisms, use of pre-/pro-/symbiotics, previous bacillus calmette-guérin vaccination, diabetes mellitus, vitamin d supplementation/deficiency, latent cmv infection and various forms of immunosuppression" on responses to influenza vaccination . this study concluded that "while the inhibiting effect of several immunosuppressive host factors was evident, the enhancing effect of pro/pre/symbiotics and chronic physical exercise was doubtful and virus type-specific (a but not b)" and that "studying the host-related correlates of the influenza vaccine-induced immune response could contribute to the production of new personalized vaccines and to the development of new patient-oriented vaccination strategies in a value-based public health perspective" . nonetheless, there are also detailed studies documenting improved cellular and humoral responses in elite athletes, so the degree of exercise and overall fitness may be crucial to seeing increased responsiveness . thus, these issues all remain a focus of intensive research which undoubtedly carry a great deal of relevance not only for influenza vaccination but for responses to other pathogens as well. moreover, benefits of effective vaccination to prevent influenza may be felt in unrelated areas of healthcare owing to their indirect impact on other diseases as diverse as lung cancer and in particular cardiovascular disease . while academic research teams are working on the development of "universal influenza vaccines" and other improvements and advances, one should not forget that the regulatory pathway to the approval of these new vaccines is often referred to as the "valley of death": many new vaccines never make it through the many steps in the approval process. the cost of making a new influenza vaccine is estimated at $ billion and takes - years. even the cost of annually refreshing the influenza strains contained in the current seasonal vaccines is estimated to be $ - million per year. these are significant non-scientific hurdles that need to be overcome when considering recent advances in influenza vaccines (https://www. wired.com/story/flu-vaccine-big-pharma/). nonetheless, a recent analysis suggests that despite the drawbacks of current seasonal influenza vaccines, there is a huge public health and public financial benefit to the use of influenza vaccines so that further improvements would make a big impact . as vaccines produced in cells rather than eggs become more generally available, some analyses are concluding that in addition to other potential advantages (see above), they may also be more cost-effective . as with other areas of medicine, a "one-size-fits-all" approach to influenza vaccination will never be optimal for every individual. in particular, the state of health and pre-exposures of the vaccinee will be highly influential in determining the success of the vaccine. in an ideal situation, prior to vaccination, the immunological history of the person would be assessed from a small blood sample. this would determine the state of humoral and cellular immunity as it pertained to influenza reactivity and the composition and nature of the vaccine modified accordingly. if the individual manifested problems regarding deficits in the presence and functions of antigenpresenting cells, steps would need to be taken to adjust adjuvants or vaccine antigens, or even replace defective antigen-presenting cells with artificial engineered constructs , which might one day be possible in vivo. similarly, if the t cell or b cell repertoire lacked cells with the appropriate antigen receptors, these could be engineered in. this scenario is admittedly highly unlikely, even in the not-so-near future, but technological progress in these areas, partly driven by efforts of cancer therapy researchers , has been so rapid that such an individualised approach may become feasible at some point. in the past years, there has seen steady progress in the science behind the development of improved influenza vaccines, but the main hurdles have not yet been overcome. these remain the continued necessity for producing seasonal vaccines rather than a universal vaccine, the mode of production (egg, cultured cells, recombinant products), the development of better adjuvants, the focus on humoral and under-appreciation of cellular immunity, and perceived problems of immunosenescence , , , as well as poor vaccine uptake in most countries. although practical progress has been slow, results from the last years encourage the belief that significant inroads will be made over the next years. estimates of global seasonal influenza-associated respiratory mortality: a modelling study impact of early life exposure to ionizing radiation on influenza vaccine response in an elderly japanese cohort. vaccine obesity impairs the adaptive immune response to influenza virus impact of aging and hiv infection on serologic response to seasonal influenza vaccination effect of latent cytomegalovirus infection on the antibody response to influenza vaccination: a systematic review and meta-analysis pubmed abstract | publisher full text | free full text cytomegalovirus seropositivity predicts a decline in the t cell but not the antibody response to influenza in vaccinated older adults independent of type diabetes status pubmed abstract | publisher full text | free full text influenza vaccine-mediated protection in older adults: impact of influenza infection, cytomegalovirus serostatus and vaccine dosage pubmed abstract | publisher full text | free full text human cytomegalovirus ul a and us gene products enhance the cxcl /cxcr signaling axis via distinct mechanisms pubmed abstract | publisher full text | free full text antibiotics-driven gut microbiome perturbation alters immunity to vaccines in humans pubmed abstract | publisher full text | free full text | f recommendation the influence of probiotics on vaccine responses -a systematic review impact of ageing and a synbiotic on the immune response to seasonal influenza vaccination; a randomised controlled trial pubmed abstract | publisher full text | free full text effects of lactobacillus coryniformis k cect on the immune response to influenza vaccination and the assessment of common respiratory symptoms in elderly subjects: a randomized controlled tria mapping host-related correlates of influenza vaccine-induced immune response: an umbrella review of the available systematic reviews and meta-analyses. vaccines (basel) elite athletes on regular training show more pronounced induction of vaccine-specific t-cells and antibodies after tetravalent influenza vaccination than controls effect of annual influenza vaccination on reducing lung cancer in patients with chronic obstructive pulmonary disease from a population-based cohort study pubmed abstract | publisher full text | free full text | f recommendation beneficial effects of vaccination on cardiovascular events: myocardial infarction a review of the cost-effectiveness of adult influenza vaccination and other preventive services editorial note on the review process are written by members of the prestigious . they are commissioned and f faculty reviews f faculty are peer reviewed before publication to ensure that the final, published version is comprehensive and accessible. the reviewers who approved the final version are listed with their names and affiliations.the reviewers who approved this article are: the benefits of publishing with f research:your article is published within days, with no editorial bias you can publish traditional articles, null/negative results, case reports, data notes and morethe peer review process is transparent and collaborative key: cord- -tzgql authors: masset, n.; meurens, f.; marie, m.; lesage, p.; lehébel, a.; brisseau, n.; assié, s. title: effectiveness of two intranasal vaccines for the control of bovine respiratory disease in newborn beef calves: a randomized non-inferiority multicentre field trial date: - - journal: vet j doi: . /j.tvjl. . sha: doc_id: cord_uid: tzgql bovine respiratory syncytial virus (brsv) and bovine parainfluenza- virus (bpi v) are major causes of bovine respiratory disease (brd) in newborn calves worldwide. vaccination is widely used to prevent brd, and intranasal vaccines for brsv and bpi v were developed to overcome interference from brsv and bpi v-specific maternally derived antibodies. many experimental challenge trials have demonstrated that intranasal vaccines for brsv and bpi v are efficacious, but effectiveness under field conditions has been demonstrated less often, especially for newborn beef calves. the objective of this field trial was to compare the effectiveness of a newly available commercial brsv-bpi v intranasal vaccine with that of a benchmarked one in newborn beef calves reared in a cow-calf system. a total of calves from farms were randomized into two vaccine groups (bovalto respi intranasal [vaccine a], n = ; rispoval rs + pi intranasal [vaccineb], n = ), and monitored during the in-house risk period up to three months after vaccination. non-inferiority analysis was performed by calculating the difference in brd prevalence between the two vaccine groups. no significant differences were observed between vaccines regarding clinical outcomes of morbidity, mortality, duration between vaccination and brd occurrence, or treatments required. because the upper limit of the -sided % confidence interval of the difference in brd prevalence between the two treatment groups ( . %) was less than the margin of non-inferiority (δ = %), a non-inferiority of vaccine a was concluded. in conclusion, vaccine a is at least as effective as vaccine b for the prevention of brd in newborn beef cattle in a cow-calf system under field conditions. bovine respiratory disease (brd) is one of the main health issues encountered in nonweaned beef calves, and can lead to high economic losses (assié et al., a; wang et al., j o u r n a l p r e -p r o o f ). viral infections generally initiate brd and predispose animals to secondary bacterial infections (mosier, ) . bovine respiratory syncytial virus (brsv), an orthopneumovirus of the pneumoviridae family, is a major virus involved in the brd complex and is highly prevalent in both dairy and beef herds (brodersen, ; sacco et al., ; valarcher and taylor, ) . likewise, bovine parainfluenza- virus (bpi v), a respirovirus of the paramyxoviridae family, is another virus involved in the brd complex, widely prevalent in herds (ellis, ) . vaccines against brsv and bpi v are widely used to control brd, especially in beef calves. in a french study of cow-calf herds in , / ( %) batches of beef calves were vaccinated against brsv ). the neonatal period is a significant risk period for brd. the immune system of newborn calves differs from that of adults in several respects (chase et al., ; cortese, ) . although functional at birth, the immune system of a calf remains immature until six months of age (hauser et al., ; tizard, ) , and the immune response during this time is weak, slow and more easily overcome by pathogenic microorganisms. in addition, maternally derived antibodies (mda), which are transmitted through colostrum and remain present for up to six months, can interfere negatively with immunization of newborn calves after vaccination (ellis et al., ; kimman et al., ) . to overcome interference between parenteral vaccines and mda, intranasal vaccination strategies using modified live vaccines for respiratory diseases have been developed and used widely for many years (windeyer and gamsjäger, ) . intranasal vaccination is able to induce protective immunity in newborn calves despite the presence of mda by priming mucosal immunization of the upper respiratory tract whereas protective immunity is inconsistent after parenteral vaccinations (osman et al., ) . veterinary vaccine efficacy is mainly evaluated in challenge trials under controlled conditions (knight-jones et al., ) . the efficacy of brsv intranasal vaccines has been proven in many challenge trials under controlled conditions even when vaccinations are performed in the presence of mda (ellis, ; osman et al., ) . however, these studies generally do not consider variations that occur under field conditions, such as exposure to other pathogens, or host and environmental factors. field trials are therefore needed to reliably evaluate vaccine effectiveness (knight-jones et al., ) . to our knowledge, only one study dedicated to brsv intranasal vaccination effectiveness has been carried out under field conditions in newborn dairy calves. in that study, no decrease in brd incidence or lung lesions associated with pneumonia was demonstrated, but an increase in average daily gain was observed (ollivett et al., ) . it should be noted, however, that the management of dairy calves is quite different from that of beef suckler calves. indeed, in cow-calf systems, animals of different susceptibilities to respiratory diseases or with different immune statuses are mixed in collective barns, whereas dairy calves are classically housed in individual pens during the first eight weeks of life before being sorted and mixed into groups of similar age in collective barns. one brsv-bpi v intranasal vaccine authorized for use in newborn calves to prevent brd has been available for over years in europe (vaccine b, rispoval rs + pi intranasal, zoetis). the efficacy and the safety of vaccine b have been demonstrated in several experimental studies (vangeel et al., (vangeel et al., , . with this vaccine, nasal shedding of brsv and bpi v in vaccinated calves with or without mda was reduced after challenges with brsv and bpi v respectively. additionally, the severity of clinical disease was also reduced after brsv in vaccinated calves with brsv mda. moreover, this vaccine has been widely used in europe and is now a benchmark for brsv-bpi v intranasal vaccines. recently, several new brsv intranasal vaccines have been launched in europe. our study aimed to compare the effectiveness of a new intranasal vaccine against brsv and bpi v (vaccine a, bovalto respi intranasal, boehringer ingelheim) with that of the benchmarked vaccine (vaccine b) in terms of decreasing brd morbidity in newborn beef calves reared in a cow-calf farming system. as these two vaccines were very similar in their composition (i.e. bivalent modified live vaccines against brsv and bpi v) and their indication for use (i.e. active immunization), a non-inferiority study was performed. the vaccines a and b contain brsv and bpi v strains administered as a single dose of ml with an intranasal applicator. a dose of vaccine a (evaluated vaccine) contains between . and . tcid of brsv bio /a strain and between . and . tcid of bpi v strain bio /a reconstituted with phosphate buffered saline. a dose of vaccine b (benchmarked vaccine) contains between . and . % tissue culture infective doses (tcid ) of brsv strain and between . and . tcid of temperature-sensitive mutant bpi v strain rlb reconstituted with saline. type of trial j o u r n a l p r e -p r o o f a randomized non-inferiority multicentre trial was carried out to assess whether vaccine a was at least as effective as vaccine b, with a pre-stated margin of non-inferiority (δ) for the prevention of brd in newborn beef cattle. the null hypothesis (h ) was that vaccine a was inferior to vaccine b in preventing brd, whereas the alternative hypothesis (ha) was that vaccine a was not inferior by more than the predefined non-inferiority margin. the hypothesis statements may be summarized as follows: where pbrd was the prevalence rate of calves treated for brd during the study period and δ the non-inferiority margin. due to the lack of published field trials of the effectiveness of vaccine b, it was not possible to determine the non-inferiority margin using a -step approach as described by freise et al. ( ) . however, efficacy and the safety of vaccine b has been demonstrated in controlled challenge trials, and this vaccine has been until now been considered as the benchmarked brsv-bpi v in vaccine. thus, based on clinical judgment, because % was the largest loss of effectiveness of vaccine a that would be considered clinically insignificant, the non-inferiority margin δ was defined as %. sample size was determined using the package 'trialsize' in r based on a noninferiority trial with the prevalence of calves treated for brd during the study period as the primary outcome. four hundred and forty-six ( ) calves per group were needed to demonstrate non-inferiority assuming α = . , β = . , δ = . and a prevalence of brd in the active control vaccine group b of %. as it was a stratified multicentre individually randomized trial with two equal group sizes (a/b) and an equal number of calves in the two groups on each farm, the inflating factor was defined as -ρ, with ρ the intraclass correlation coefficient giraudeau, , ) . after assigning this inflation factor with ρ = . (hendrick et al., ) and assuming % of loss to follow-up, it was decided to enrol at least calves in the study. forty cow-calf farms with ± calvings (mean ± standard deviation, sd), located in four areas of france (auvergne-rhône-alpes, bretagne, nouvelle-aquitaine, pays de la loire), were selected for this study. initial herd selection by veterinarians was based on the following criteria: ( ) pure charolais breed herds, ( ) at least calvings between december and april , ( ) calvings in stalls and a housing period as long as possible, ideally at least three months after vaccination, ( ) ability to detect and treat sick animals and to record health events, and ( ) no other brd vaccination program during the study period (from birth to months after enrolment). herds were then enrolled in the study after farmers agreed to participate. on each farm, to blocks of calves were enrolled ( , and farms had respectively , or blocks). as soon as there were more than calves over days of age, a clinical examination of each calf was performed by a veterinarian. only calves in good health (no intercurrent illness detected during the clinical examination) at the time of j o u r n a l p r e -p r o o f enrolment were included. if a calf had been ill and treated for brd or other diseases before the day of inclusion, enrolment was possible only after full clinical recovery of the animal. randomization was performed with a : allocation ratio. in each block of enrolled calves from each farm, calves were sorted by decreasing age. a predefined randomized spreadsheet was prepared for each block: a random drawing was performed using the rand function of ms excel to assign the vaccine to the oldest calf, then the following calves were vaccinated alternately with one of the two vaccines. vaccines were administered by one veterinary investigator with calves restrained by the farmer, while another veterinary investigator reconstituted vaccines and recorded data. the vaccines were administered according to the manufacturer's recommendations: for vaccine a, ml in each nostril with the specific intranasal applicator (cannula and disk) provided by the manufacturer; for vaccine b, ml in one nostril with the specific intranasal applicator (cannula) provided by the manufacturer. intranasal applicators were changed between each calf. due to the different administration modalities for the two vaccines, it was not possible to carry out a blinded vaccination. however, data were collected and registered by another veterinary investigator who kept randomization sheets and was not involved in the monitoring of calves after vaccination. to avoid potential bias in later husbandry, farmers were asked to not read the tag numbers of calves during vaccination. after vaccination, cow-calf couples were raised in the same pen according to the usual rearing conditions on each farm. outcomes and data collection the protocol started with vaccination and ended three months later. the primary outcome for analysis was brd events, defined by at least one respiratory sign (such as cough, dyspnea, and/or nasal discharge) associated with at least one general clinical sign (hyperthermia, depression, and/or anorexia). secondary outcomes included the time between vaccine administration and the occurrence of brd, calf mortality, and the number of calves treated with antibiotics or anti-inflammatory drugs during the study. were performed in agrivalys . calves with incomplete data, vaccinated before days or after days of age, or treated for brd without at least two clinical signs being recorded, or as part of a metaphylaxis protocol, or vaccinated after the beginning of the grazing period were excluded. the characteristics of calves assigned to the two vaccine groups were compared to where brdij is the occurrence of a brd case diagnosed during the study risk period for the calf i of the herd j with a probability of occurrence , is the intercept, is either vaccine a or b, is the duration of the in-house risk period, is sex, parity of j o u r n a l p r e -p r o o f dam and age at vaccination variables and is the random effect for herd j. herd random effect followed a normal distribution with mean and variance . the difference in brd prevalence between vaccine groups and its % confidence interval (ci) was calculated from the model. non-inferiority of vaccine a compared with vaccine b was concluded if the upper bound of the -sided % ci of the difference of brd prevalence between the two vaccines was smaller than the non-inferiority margin δ (fig. ) . secondary outcomes were compared between the two vaccine groups using the chi-squared test, fisher test or student's t-test. all analyses were carried out using r software and a statistical significance at p ≤ . was used. a total of , calves from farms were enrolled in the study. data from calves were excluded: because of incomplete data, for date of vaccination before days or after days of age, for being treated for brd without at least two clinical signs or in a metaphylaxis protocol, and for being vaccinated after the beginning of the grazing period (no housing period). thus, a total of calves from herds were used in the study analysis: ± calves (mean ± sd) per herd. the two experimental groups were homogeneous in regard to age at vaccination, duration of in-house risk period, parity of dams, sex ratio, and occurrence of diseases before vaccination (table ) . the occurrence of brd during the in-house risk period between the two vaccine groups was similar (table ). using least squares means of model outcome (brd events), the j o u r n a l p r e -p r o o f difference pbrd(vaccine a) -pbrd(vaccine b) was estimated at - . % with a % ci between - . % and . %. non-inferiority of vaccine a compared to vaccine b was concluded since the upper limit of the -sided % ci ( . %) of the difference in prevalence of calves diagnosed with brd between the two vaccines was smaller than δ (fig. ) . brd incidence in our study was . cases per , calf-days at risk (table ) . the two experimental groups were similar in regard to time between vaccination and occurrence of brd, treatments and mortality (table ). for the six calves which died during the study period, brsv was not detected from the samples collected during the necropsy procedure (table ). the objective of this study was to compare the effectiveness of the newly available brsv-bpi v intranasal vaccine to the benchmarked vaccine under field conditions in newborn beef calves reared in a cow-calf system. based on our results, non-inferiority of vaccine a compared to vaccine b was concluded. due to the lack of studies dedicated to the effectiveness of vaccine b compared to a placebo, the non-inferiority margin δ was defined as % only, based on a clinical judgment. this margin is narrow compared to the one used in most vaccine trials, with δ usually fixed at % as reviewed by donken et al. ( ) . choosing a more conservative non-inferiority margin required an increased sample size and improved the clinical significance of the trial. indeed, based on the experiences of the authors, an increase of more than % of brd events (i.e., the primary outcome) in the vaccine a group compared to the vaccine b group was considered unfavorable. in this study, calves from the two vaccine groups were mixed together in order to homogenize as much as possible environmental conditions and exposure to pathogens. this design is often chosen in field studies dealing with vaccine effectiveness (schunicht et al., ; stilwell et al., ; wildman et al., ) . moreover, in a cow-calf system, this design enables the absence of separation of paired calves of the two vaccine groups after randomization, and improves blind assessment of calf health in a single group. however, a bias in vaccine effectiveness evaluation could be introduced with this method. the reduction of virus shedding after vaccination of the calves of one vaccine group contributes to the protection of the calves of the other vaccine group reared in the same environment (smith, (smith, , stokka, ) . indeed, apparent effectiveness of the test vaccine could be improved if the comparison involved a reference vaccine with a better shedding reduction efficacy. moreover, vaccines a and b are both modified live vaccines. cross-immunization thus could occur between the two vaccine groups. in previous studies using commercial vaccines including the reference vaccine, nasal shedding of vaccine-origin viruses was detected by pcr in nasal swabs during days in most of the vaccinated calves after vaccination and was detected up to days post vaccination in a few calves (timsit et al., ; walz et al., ) . the study was designed to determine the effectiveness of two commercial vaccines under conditions as close as possible to those encountered by calves reared in beef herds. although the minimum age at vaccination recommended by the manufacturers is and days of age for vaccines b and a, respectively, under our conditions the mean age (± sd) at vaccination was ± days. this delay in administrating vaccines was due to the distribution of births on each farm and the packaging of the vaccines in -dose bottles. since both vaccines are available in a -dose bottle, calves had to be over days of age before j o u r n a l p r e -p r o o f being vaccinated in order to randomize them into two equal groups of five calves. however, this difference between the recommended and actual age at vaccination was the same in both groups and is common in french cow-calf systems. most of the efficacy studies for brsv and bpi v intranasal vaccination include a as observed in a previous study, brsv and bpi v infections were found in % and % of cow-calf farms respectively (assié et al., b) . furthermore, challenge trials do not reproduce the variability of host and environmental conditions that may be encountered in the field, such as variable passive immune transfer (raboisson et al., ) , variable calf housing contrary to challenge trials, the exposure of calves to pathogens, in particular to brsv and bpi v, is rarely controlled in a field study (ellis, ; ollivett et al., ) . however, the exposure of calves to respiratory pathogens in our study can be attested by the measurement of brd incidence, which was . cases per , calf-days at risk. although this brd incidence was low, it remains consistent with the incidence observed in another french study in a comparable breeding system in which respiratory vaccination was inconsistent: . cases per , calf-days at risk in farms (assié et al., a) . circulation of respiratory pathogens in the study farms can also be attested by the viruses (bpi v, bovine coronavirus) and bacteria (mannheimia haemolytica, pasteurella multocida, histophilus somni) identified at necropsy in dead animals. to overcome the variability in the exposure to pathogens under field conditions, our study would need to be repeated. the effectiveness of a newly available commercial brsv-bpi v intranasal vaccine to control brd has been demonstrated under field conditions. to the authors' knowledge, this is the first study under field conditions assessing brsv and bpi v intranasal vaccination effectiveness in newborn beef calves in a cow-calf system. data from challenge studies or from dairy calf field studies cannot be extrapolated to beef calves. beef cattle from different age groups with different immune statuses against respiratory pathogens are mixed together in a specific in-house environment, in contrast to dairy calves which are typically housed in individual pens or in collective pens with animals of the same age. this study was funded by boehringer ingelheim, which supports the european college of bovine health management (ecbhm) residency program of the first author. boehringer ingelheim played no role in the study design, in data collection, analysis or interpretation, or in the writing of the report and the decision to submit the manuscript for publication. none of the authors has any other financial or personal relationships that could inappropriately influence or bias the content of the paper. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f table comparisons of secondary outcomes between the two vaccine groups the ci includes δ and zero, the difference is non-significant but the result regarding noninferiority is inconclusive. i: if the ci is wholly above δ, vaccine a is inferior. vt is the representation of the main outcome of this non-inferiority trial. the black block indicates the difference in brd incidence between vaccine a group and vaccine b group. non-inferiority of vaccine a compared to vaccine b at a margin of % is demonstrated because the % ci lies to the left of δ (= %) and includes zero. management-and housing-related risk factors of respiratory disorders in non-weaned french charolais calves incidence of respiratory disorders during housing in non-weaned charolais calves in cow-calf farms of pays de la loire failure of passive immune transfer in calves: a meta-analysis on the consequences and assessment of the economic impact respiratory syncytial virus infection in cattle comparison of a multivalent viral vaccine program versus a univalent viral vaccine program on animal health, feedlot performance, and carcass characteristics of feedlot calves herd immunity. veterinary clinics of north america: food animal practice field epidemiology to manage brd risk in beef cattle production systems effect of a quadrivalent vaccine against respiratory virus on the incidence of respiratory disease in weaned beef calves prevention of respiratory disease in cow/calf operations. the veterinary clinics of north america: food animal practice bovine model of respiratory syncytial virus infection, in: challenges and opportunities for respiratory syncytial virus vaccines, current topics in microbiology and immunology detection by real-time rt-pcr of a bovine respiratory syncytial virus vaccine in calves vaccinated intranasally. the veterinary immunity in the fetus and newborn bovine respiratory syncytial virus infection efficacy of a modified live intranasal bovine respiratory syncytial virus vaccine in -week-old calves experimentally challenged with brsv efficacy of an intranasal modified live bovine respiratory syncytial virus and temperature-sensitive parainfluenza type virus vaccine in -week-old calves experimentally challenged with pi v design effect in multicenter studies: gain or loss of power? sample size calculation for multicenter randomized trial: taking the center effect into account virus detection by pcr following vaccination of naive calves with intranasal or injectable multivalent modified-live viral vaccines cost of bovine respiratory disease in preweaned calves on us beef cow-calf operations ( - ) a comparison of vaccination programs in feedlot calves at ultra-high risk of developing undifferentiated fever/bovine respiratory disease vaccinating calves in the face of maternal antibodies: challenges and opportunities the authors acknowledge the assistance of the veterinarians of the 'réseau cristal' and the farmers participating in this study. key: cord- -ew cwiw authors: su, hang; su, jianguo title: cyprinid viral diseases and vaccine development date: - - journal: fish shellfish immunol doi: . /j.fsi. . . sha: doc_id: cord_uid: ew cwiw in the past decades, global freshwater fish production has been rapidly growing, while cyprinid takes the largest portion. along with the rapid rise of novel forms of intensive aquaculture, increased global aquatic animal movement and various anthropogenic stress to aquatic ecosystems during the past century, freshwater fish farming industry encounter the emergence and breakout of many diseases, especially viral diseases. because of the ability to safely and effectively prevent aquaculture diseases, vaccines have become the mainstream technology for prevention and control of aquatic diseases in the world. in this review, authors summarized six major cyprinid viral diseases, including koi herpesvirus disease (khvd), spring viraemia of carp (svc), grass carp hemorrhagic disease (gchd), koi sleepy disease (ksd), carp pox disease (cpd) and herpesviral haematopoietic necrosis (hphn). the present review described the characteristics of these diseases from epidemiology, pathology, etiology and diagnostics. furthermore, the development of specific vaccines respective to these diseases is stated according to preparation methods and immunization approaches. it is hoped that the review could contribute to aquaculture in prevention and controlling of cyprinid viral diseases, and serve the healthy and sustainable development of aquaculture industry. aquaculture is an ancient practice and it is believed to date back to china at least years ago. there are also references of fish ponds in the old testament and in egyptian hieroglyphics of the middle kingdom. fish farms were common in europe in roman times and a recent study of land forms in the bolivian amazon has revealed a complex array of fish weirs that pre-date the hispanic era [ , ] . despite its ancient origin, aquaculture remained a low-level farming activity until the mid- th century when experimental husbandry practices for salmon, trout and a mass of tropical fish and shrimp species were developed. nowadays, aquaculture is commended to be a major global industry, with a total annual production exceeding million ton and estimated value of almost billion us dollar (food and agriculture organization of the united nations, fao, ). in the past three decades, aquaculture industry has made impressive progress and constitutes high quality protein for world population accounting for nearly % of the global food fish supply in . in addition to being an important form of nutrition, aquaculture is also a critical form of employment (fao, ) . aquaculture is a highly dynamic production and is characterized by enormous diversity in both the farmed species range and the production systems feature. over different species of aquatic animals are cultured in global in freshwater, brackishwater and marine environments. in , the yield of freshwater fish production reached . million ton (fao, ) . carps (cyprinidae) make up almost % of the global farmed freshwater fish production and are an important source of food in china and india where . % and . %, respectively, of farmed cyprinid are produced (fao, ) . economic aquaculture species belonging to cyprinid principally include grass carp (ctenopharyngodon idella), common carp (cyprinus carpio), crucian carp (carassius auratus), black carp (mylopharyngodon piceus), silver carp (hypophthalmichthys molitrix), bighead carp (aristichthys nobilis) and blunt snout bream (megalobrama amblycephala), etc. meanwhile, in the international ornamental fish trade, more than % of the freshwater fishes are farm bred, and about species of freshwater ornamental fishes are traded every year [ ] . goldfish (carassius auratus auratus) and koi (cyprinus carpio haematopterus) are the most extensively traded fish species [ ] . importantly, the global fish trade relocates large quantities of live fish species between countries, and therefore it can be a potential source for spread of exotic pathogens, particularly viral pathogens, which have been associated with high morbidity and mortality of farmed fishes [ , ] and their products, and various artificial pressures to aquatic ecosystems have led to the emergence and outbreak of numbers of diseases in fishes. the global expansion of fish farming industry and the consequent improvement in fish health monitoring have led to discovery of several new scientific viruses, which are almost endemic in local population and overflow opportunistically to infect fishes in aquaculture facilities, while other well-known fish viruses also cause significant losses in aquaculture. diseases caused by various fish viruses, bacteria, parasites and other pathogens pose great hazards to aquaculture industry and threat to food safety. as people pay more and more attention to the quality and safety of aquatic products and environmental pollution, the utilization of various chemical drugs to prevent diseases in aquaculture animals has been increasingly questioned. at present, prevention and control of aquaculture diseases mainly include methods of drug control and immune prevention. the potential risks of drug resistance, allergic reactions and poisoning reactions caused by drug residues have a serious impact on the environment, farmed animals and consumption of cultured products. immunoprevention can not only improve the specific immunity of fish, but also can enhance the ability to resist stress, meeting the requirements of no environment pollution and no residues in aquatic products. due to its ability to safely and effectively prevent aquaculture diseases, vaccine has become the mainstream technology for prevention and control of aquatic diseases in the world. vaccination has also become the normative production standard for international modern aquaculture. in the late s, the commercialization of fish vaccines developed rapidly and as a result, there are global approvals in , over in , and over in according to incomplete statistics [ ] . in the following sections, authors summarized the major emerging viral diseases that cause major losses in cyprinid fish farming, expanding in host or geographic scope due to the risk of spreading through the commercial trade of finfish. the respective pathogen-targeted vaccines were described subsequently. it is hoped that the information provided in this review could contribute to cyprinid farming industry decreasing the economic loss caused by viral disease and improving the quality of aquatic products. six cyprinid viral diseases are summarized in table . koi herpesvirus disease (khvd) caused by koi herpesvirus (khv) [ ] is a listed notifiable disease to the international office of epizootics (oie, ), spreading to most regions around the world due to the global fish trade and international ornamental koi shows [ , ] (fao, ). first detected in the late s, khvd has been found in different continents (europa, asia, north america and africa), leading to serious worldwide financial losses in common carp and koi culture industries. once fish are infected, the virus could persists for some period of time in a latent or carrier state without obvious clinical signs [ ] . molecular analysis shows that little variation among isolates is found, as it might be expected for a virus that is being rapidly disseminated by the global movement of infected fish [ ] . khvd is relatively host-specific, while only common carp and its ornamental subspecies, koi [ ] , are involved in the explosive losses reported globally [ ] . in addition to its negative economical and societal impacts, khvd has also a negative environmental impact by affecting wild populations of carp. in addition, hybrids of common carp and goldfish show partly susceptibility to khv infection, however the mortality rate is observed to be rather limited [ ] . cohabitation experiments indicate that some carp species such as goldfish, common bream (abramis brama), silver carp and grass carp, can carry khv asymptomatically and transmit it to wild carp [ ] [ ] [ ] [ ] [ ] . khvd is table cyprinid viral diseases. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] seasonal, occurring when water temperature is between °c and °c. it is highly contagious and extremely virulent with mortality rate up to %- %. fish infected with khv and kept at - °c die between and dpi (days post infection) with a peak of mortality between and dpi [ , , ] . carp of all ages, from juveniles upwards, are affected by khv, but younger fish ( - months, . - g) seem to be more susceptible to infection than mature fish ( year old, nearly g) [ ] . loss of osmoregulation of the gill, gut and kidney contributes to mortality during acute infection with khv [ ] . after infection, the initial clinical signs appear at - dpi, when fish become lethargic, lose appetite and lie at the bottom of the tank with the dorsal. in ponds, infected fish usually gasp at the surface of water gathering close to the water inlet or sides of the pond. gill necrosis coupled with extensive discoloration and increased mucus secretion appear as early as dpi. the skin exhibits different clinical signs depending on the stage of the infection. hyperemia particularly at the base of the fins and on the abdomen and pale, irregular patches on the skin associated with mucus hyper-secretion often appear at the beginning of infection. then, dead epithelium peels away lacking of mucus cover. appearance of epidermis surface with a sandpaper-like texture and herpetic lesions are observed at the following infection condition [ ] . in the later stages of infection, fin erosion and bilateral enophthalmia are observed. some fish show neurologic signs, getting disoriented and losing equilibrium in the final stage of disease [ , , [ ] [ ] [ ] [ ] . the enveloped virion of khv is formally classified as the species cyprnid herpesvirus (cyhv- ). cyhv- is a member of genus cyprinivirus, family alloherpesviridae, order herpesvirales [ ] . the alloherpesviridae is a newly designated family which regroups herpesviruses infecting fish and amphibians [ ] , being divided into four genera: cyprinivirus, ictalurivirus, salmonivirus and batrachovirus [ ] . the genus cyprinivirus contains viruses that infect common carp (cyprinid herpesvirus, cyhv- and cyhv- ), goldfish (cyprinid herpesvirus , cyhv- ) and freshwater eel (anguillid herpesvirus , anghv- ) (international committee on taxonomy of viruses, ictv, ). the genome of cyhv- is a typical herpesvirus containing a kb, linear, double stranded dna molecule consisting of a large central portion flanked by two kb repeat regions [ ] . so far, it is the largest genome among all sequenced herpesviruses [ ] . various cyhv- diagnostic methods have been developed based on detection of infectious particles, viral dna, transcripts or antigens. a series of molecular techniques for detection of viral dna fragments has been developed, such as dna hybridization, pcr, nested pcr, one-tube semi-nested pcr, semi-quantitative pcr, real-time taqman pcr, and loop-mediated isothermal amplification [ ] . cyhv- can be detected in environmental water, infected fish tissues and cell cultures by realtime pcr after viral concentration [ , ] , in carp serum by elisa aiming at the specific anti-cyhv- antibodies [ ] , in tissues and touch imprints of organs from infected fish by immunohistochemistry, immunofluorescence assays [ ] and monoclonal antibodies against cyhv- orf without cross-reaction against cyhv- and cyhv- [ ] . a cyhv- -detection kit (the fastest ® koi hv kit) allows detection of cyhv- in gill swabs in just min [ ] . cyhv- also attracts originally fundamental researches. it is phylogenetically distant from the vast majority of studied herpesviruses, thereby providing an original field of research. as the genome sequence published, it is revealed to be a fascinating virus with unique properties in the herpesvirales, such as an extremely large genome, a high number of genes which are not homologous to known viral sequences and genes that are normally found exclusively in the poxviridae [ ] . meanwhile, cyhv- genome analysis also reveals several genes encoding proteins potentially involved in immune evasion mechanisms [ ] . immune system of vertebrate recognizes virus particles by pattern recognition receptors (prrs), which senses particular protein ligand and virus nucleic acids [ ] . prrs detect viral pathogen associated molecular patterns (pamps) to trigger intracellular innate immune pathways and subsequent adaptive immune responses by producing cytokines, chemokines, t-cell stimulatory factors, etc [ ] . mammalian cells have the capacity to detect viral proteins using a series of receptors, such as cell surface toll-like receptor (tlr ) [ ] and a multiple set of cytoplasm receptors to viral dna including a dna dependent activator of interferon regulatory factors (dai) [ ] , the interferon-inducible protein (ifi ) [ ] and rna polymerase iii [ ] . in teleost, endosome tlr has been identified in numbers of carp species [ ] [ ] [ ] , but further information on receptors for viral dna remains unclear and the recognition mechanism of cyhv- by cyprinid cells need further research. the recognition of viral molecular patterns leads to the production of type i interferon (ifnei), which is crucial in antiviral response to many virus infections [ ] [ ] [ ] . ifns bind to specific cell-surface receptors, then trigger the jak/stat signal transduction pathway and subsequently activate interferon stimulated genes (isgs), which encode for a wide array of proteins with antiviral properties [ ] . in carp cells, cyhv- can be interfered by the activation of ifn-i. poly(i:c) stimulation in fibroblastic carp cells induces the expression of ifn-i, limiting cyhv- infectivity and replication, delaying the spreading of cyhv- in vitro [ ] . in carp infected by cyhv- , ifn-i response is detected in skin at h post infection [ ] and subsequently in spleen [ ] . ifn-i system triggers the expression of a quantity of genes encoding antiviral proteins, including grass carp reovirus-induced genes (gig- ) protein, ubiquitin like protein (isg ) and ubiquitin specific protease (usp ), inhibiting viral replication directly [ ] . furthermore, in cyhv- infected carp, genes encoding chemotactic cytokines of cc family are up-regulated [ ] . additional observation indicated that the induced inflammation may derive from the rapid prominent leukocytosis in the blood cycle of carps during the initial two days after cyhv- infection. complement system also participate in the reaction against cyhv- displayed as pathway triggering and complement hemolytic activity increasing in blood plasma [ ] . the activation of the complement system leads to neutralisation of viruses, phagocytosis of coated virus particles or elimination of virus infected cells by lysis. in conclusion, innate immune system including ifn-i, inflammation and complement are induced by cyhv- infection, while the importance for resisting the infection still needs to be addressed in greater detail. spring viraemia of carp (svc) is one of the ten piscine viruses listed by the oie (oie, ) as a notable animal disease. it is caused by a fish rhabdovirus, spring viraemia of carp virus (svcv). however, unlike infectious haematopoietic necrosis virus (ihnv) and viral hemorrhagic septicemia virus (vhsv), svcv is related to genus vesiculovirus of the family rhabdoviridae, order mononegavirales [ ] . it exhibits an enveloped, bullet-shaped virion and measures ∼ - nm in length and - nm in diameter [ , ] . it has a negative-sense, ssrna genome of ∼ kb, containing five orfs encoding five structural proteins: nucleoprotein (n), phosphoprotein (p), matrix protein (m), glycoprotein (g) and rna dependent rna polymerase (l). the five viral genes are organized in the typical order of rhabdoviruses: ′enep-m-g-l- ' [ ] [ ] [ ] . genotypes of svcv isolates from various locations form four major genetic clades [ ] . svcv isolates of the recent emergence and geographic range expansion link to the spread of the virus within china where common carp are reared in large numbers for food and koi are reared for export [ ] . it is responsible for the highly contagious svc, especially in common carp [ , , ] . svcv was first detected in yugoslavia in [ ] . it was initially believed to be endemic among common carp in eastern and western europe, and subsequently reported in the americas [ , ] and asia [ , , ] . svcv infection has now emerged in many european countries, including the uk, denmark, germany, the netherlands, austria, spain, france, the czech republic, georgia, belarus, moldova, ukraine and russia associated with numerous financial losses in common carp and koi [ , , [ ] [ ] [ ] [ ] . these outbreaks occur in both farmed and wild fishes suggesting the expansion of influenced range. the emergence of svc in region where is free of svc, such as north america, asia and in portions of europe, appears to be the consequence of both improved surveillance and the large volume of ornamental fish global shipment. outbreaks of svc usually occur in spring, which is why the disease is called so [ ] . studies in carp have shown that few adult fish are infected when the water temperature is over °c, but juveniles can be infected even at °c − °c [ ] . svcv infection is highly lethal in young fish, with mortality rate up to % [ ] . other risk factors associated with morbidity and mortality include fish density, geographical location, fish species and the immune status of susceptible fish [ ] . svcv infection can spread by fomites and parasitic in vertebrates. natural svcv infections have been reported in other cyprinid fish, including goldfish, koi, silver carp, crucian carp, bighead carp, grass carp, tench (tinca tinca) and orfe (leuciscus idus) [ , , ] . experimental infections have been reported in zebrafish (danio rerio), golden shiner (notemigonus crysoleucas), roach (rutilus rutilus), guppy (lebistes reticulatus), pumpkinseed (lepsomis gibbosus), northern pike (esox lucius), fathead minnow (pimephales promelas), emerald shiner (notropis atherinoides) and white sucker (catostomus commersonii) [ , [ ] [ ] [ ] . svcv infection is generally associated with non-specific symptoms, such as exophthalmia, abdominal distension and oedema of the vent region [ ] . petechial hemorrhages can be seen on the skin, gills, eyes and internal organs, particularly on the walls of the swim bladder [ , ] . other lesions may include degenerated gill lamellae, oedematous internal organs, swollen and coarse-textured spleen, hepatic necrosis, enteritis, and pericarditis [ , ] . the histological changes associated with hepatopancreas may range from perivasculitis to panvasculitis with a higher degree of oedematization and loss of structure of blood vessels walls. the hepatopancreas parenchyma show hyperaemia, multifocal necrosis and adipose degeneration [ ] , whilst the spleen is often hyperaemic, showing a co-significant hyperplasia of the reticuloendothelium [ ] . multifocal necrosis and non-purulent inflammation often occur in the pancreas of affected fish, and the heart shows pericarditis and discontinuous myodegeneration [ ] . the visceral and parietal serosa of the peritoneum show peritonitis. in the intestine, perivasculitis with subsequent atrophy of the villi is often observed [ , ] . the epithelial layer of the swim bladder changes into a discontinuous multilayer and hemorrhages are commonly observed in the submucosa [ ] . as svcv has a significant impact on carp aquaculture, its rapid detection and identification are crucial for effective control of the disease. conventional serological techniques include the virus neutralisation test, immunoperoxidase assay, indirect immunofluorescence assay and elisa. however, these techniques are laborious and time consuming [ ] [ ] [ ] [ ] . moreover, the indirect immunofluorescence assay and elisa appear to cross-react with other rhabdoviruses [ ] , leading to possible false-positive diagnoses. mabs are also useful tools for detecting svcv and studying the function of virus-specific proteins [ , ] . recently, a single-chain fragment variable antibody against svcv has been developed using phage display technology and employed for rapid detection of svcv [ ] . this antibody reacts specifically with svcv, not cross-react with other viruses. thus, this approach provides the basis for establishing simple and cost-effective way for the development of immunological detection assays for svcv. various pcr-based assays have also been used to detect svcv owing to their high sensitivity. these include reverse transcription (rt)epcr combined with nested pcr [ ] , multiplex real-time quantitative rt-pcr [ ] and one-step taqman real-time quantitative rt-pcr [ ] . these assays have clearly improved the specificity and sensitivity of detection [ ] . although pcr is generally considered impractical for routine diagnostics in the field owing to the need for specialized instruments, skilled operators and isolation of rna extracts, a recent report described an improved rt-pcr assay that is able to accurately detect svcv directly from fish tissues, indicating the potential application of this technology for svcv detection in infected fish in the field [ ] . a loop-mediated isothermal amplification (lamp) assay has been increasingly used for the detection of viruses [ ] [ ] [ ] [ ] . rt-lamp is a simple and effective technique to rapidly amplify specific nucleic acid sequences under isothermal conditions. moreover, it requires uncomplicated and inexpensive equipment easily manipulated at fish farms. this assay has been successfully applied for disease control in aquaculture [ , ] . two studies have used rt-lamp to detect svcv based on nucleotide sequences of the g and m genes [ , ] . in s, chinese experts found that sick grass carp suffer from bleeding symptoms, suspecting that the hemorrhage disease was caused by virus [ ] . grass carp hemorrhagic disease caused by grass carp reovirus (gcrv) leads to billions of rmb yuan of economic loss every year [ ] . the disease is identified as a viral etiology in and is the first fish viral disease studied in china [ ] . the mortality rate of grass carp can reach more than % at the fingerling stage, and it can also infect other species, such as black carp, making these fish die of bleeding symptoms [ ] . the prevalence of gcrv has a typical seasonality. the high incidence period of gcrv is mainly in summer when water temperature is °c- °c. the main reason is that the optimum temperature of the virus polymerase is °c. in many high density grass carp pools, when the temperature of the water exceeds °c, the virus rapidly proliferates and may result in serious dysfunction of the fish and death. when the water temperature is lower than °c, the virus proliferation will be inhibited, and even lose their infectivity. the clinical signs are organ hemorrhages showing spots or plate forms, in combination of some or all of the following signs: exophthalmia, body darkening, hemorrhage of the mouth cavity, hemorrhagic or pale gills, and hemorrhage at the base of fins and branchiostegites. internal hemorrhage may occur throughout the musculature, hepatopancreas, spleen, kidney, and intestines. present researches classify the disease into three types according to clinical signs: red muscles, enteritis, or red fin and branchiostegite [ ] . gcrv belongs to group c, aquareovirus genus in the spinareovirinae subfamily, reoviridae family. it is the first fish virus isolated in mainland china and is also known as the most virulent virus in aquareovirus [ ] . the genome of gcrv consists of segments of dsrna, encoding proteins, including seven structural proteins and five nonstructural proteins [ , ] . to date, more than thirty gcrv strains have been isolated from infected grass carp in global. based on the difference in vp genome constitution, gcrv could be mainly clustered into three types, and the representative strains of three types are gcrv- (type i), gd (type ii), and gcrv (type iii), respectively [ ] [ ] [ ] . as a consequence of the fast evolution, identities of amino acid sequences among types are less than % [ ] [ ] [ ] . among the three types, gcrv type ii is considered to be the most pathogenic and prevalent type currently in china and may be closer to orthoreovirus than any other known species of aquareovirus showed by phylogenetic analysis [ , ] . the virus identification by rt-pcr are the main method of surveillance and diagnosis of gcrv infection. primary diagnosis in fish farms is based on typical external and internal clinical signs, especially the "red muscles" that appears in some sick fish. epidemiological investigations are usually carried out in summer when it is easy to detect the virus from the carrier or sick fish. the virus can be identified by elisa or rt-pcr. at present, the antiviral immune responses against gcrv in grass carp are broadly researched. as a dsrna virus, gcrv activates the similar prr-pamp recognition mechanism of innate immune system, being sensed by different receptors. cytoplasm viral infection is primarily detected by rig-i-like receptors (rlrs), a prr family consisting of retinoic acid-inducible gene i (rig-i), melanoma differentiation-associated gene (mda ), and laboratory of genetic and physiology (lgp ) [ ] . rig-i preferentially binds to relatively short ′phosphorylated dsrna, while mda binds to long dsrna [ , ] . by gcrv or poly(i:c) challenge, mrna level of rig-i is up-regulated in cik cells [ ] . cpe assay and viral titter reveal the significant antiviral activity of full-length rig-i in response to gcrv infection. meanwhile, the expression levels of these three genes are significantly induced in spleen and hepatopancreas tissues after gcrv infection [ ] [ ] [ ] [ ] . lgp is found as a negative regulator for both rig-i and mda in resting state or early stage of gcrv infection. tlr signaling pathways also participate in the immune responses to gcrv infection. up-regulations of tlr , tlr , tlr , tlr , tlr and other tlrs are observed post poly(i:c) or gcrv challenge [ ] [ ] [ ] [ ] [ ] . the enhancement of nod and nod also occur in grass carp post gcrv infection [ , ] . all the members of hmgb family can respond to poly(i:c) and gcrv challenge in grass carp, delaying the cpe in cik cells, meanwhile modulating rlr and tlr pathway genes [ ] [ ] [ ] [ ] [ ] . similar to mammals, fish ifn antiviral responses are initiated through the pattern recognition of virus component by tlrs and rlrs [ ] . although antiviral immune researches in teleost have achieved great progresses and several fish specific immune genes have been identified, studies on the regulation mechanisms of fish antiviral immune signaling pathways remain far behind those in mammals. koi sleepy disease (ksd) is an emerging problem harmful to koi. it was first detected in japan in [ , ] . after its initial detection in koi populations in japan, the disease was limited to japan until when it was reported in both koi and common carp in numerous european countries, including germany, england, austria, poland, france and the netherlands [ ] [ ] [ ] [ ] . in addition, it also spreads to brazil, the united states, china and india [ ] [ ] [ ] . the disease is exclusively described in common carp and koi breeding facilities. ksd shows clinical symptoms when the water temperature is °c- °c [ , ] . clinical signs are seen in extreme association with stressful farm conditions, often occurring when fish are transferred from earthen to concrete ponds, causing a mortality of up to - % and prevalence to . %, especially in juvenile koi [ ] . the typical appearance, lying on the bottom of the ponds, leads to the name ksd. clinical signs are lethargy, skin hemorrhages with oedema in the underlying tissues, enophthalmos and pale swollen gills [ , , ] . in addition, gill hyperplasia and necrosis as well as lamellae clubbing, leading to dyspnoea and hypoxia are also seen in infected fish [ , , ] . carp edema virus (cev), a double-stranded dna virus, belonging to the family poxviridae, is the causative agent of ksd, based on transmission electron microscopy [ , ] . this mulberry-like enveloped member of the pox virus group has been identified in gill cells from diseased fish, and further morphological characterization is provided by electron microscopy [ , , ] . sequencing the known dna fragment encoding the core protein p a from infected fish in different locations in europe and asia shows a %- % of genetic diversity. it is classified into three genetic genogroups, called i, iia and iib [ , ] . viruses from genogroup i usually present in farmed carp, while cev from genogroup iia mainly in diseased koi [ , ] . infection experiments with both genogroups in koi and farmed carp confirmed biological differences between virus isolates [ ] . the genetic diversity and the observed differences in biology reveal that the virus has been present in the european carp population for a prolonged period and infections are diagnosed until recently. similarities of pathognomonic signs of viruses affecting carp in spring, such as cev, khv and svcv [ , ] , highlight the necessity for exploiting specific diagnostic procedures. although the disease has been successfully transmitted, using filtrated suspensions of gill homogenates obtained from sick fish [ ] , the virus has yet to be isolated successfully and grown in cell culture. ksd diagnosis relies on detection of viral dna by pcr [ , , ] . in the present reports, the diagnosis relied on clinical findings is confirmed by molecular detection of an undefined segment of the viral genome. a nested pcr protocol is initially designed in tokyo university of fisheries [ ] , recently is modified in cefas-weymouth laboratory, england, to improve reliability of detection. the latter method of detection is also used for an extensive phylogenetic analysis to reveal the presence of two potential main viral lineages. carp pox disease caused by cyhv- is probably the earliest recognized viral disease of fish, dating back to and had broken out in europe, north korea and japan [ ] . it has also been found in hubei, henan, hebei and many other provinces in china. cyhv- mainly infects common carp over year old and also has some effect on crucian carp, hybrids of common carp and goldfish, having no harm to black carp, grass carp, silver carp, bream [ ] . the proper temperature of carp pox is °c- °c. fish will recover without any administration when over °c. therefore, temperature control is an efficient method to fight against cyhv- . carp pox is an endemic chronic skin disease. in the early days after infection, the fish become thin and transparent. smooth gray and white patchy growth organisms appear and is covered with a thin layer of white mucus. with the development of the disease, white spots gradually expand and thicken, the number gradually increases and interconnects into pieces. smooth, creamy to brown appears as pink "paraffin-like or glass-like" pox and sores on blood vessels. these growth organisms are very tightly bound to the surface of infected fish body. the main component of the growth organisms is collagen fibers, which do not transfer but can naturally fall off and reappear in the original affected area. the back, head, fins and caudal peduncle are dense areas of pox and sores. the severely diseased fish is full of pox and sores, and the lesions often have bleeding. when the proliferation of organisms spreads to the bulk of the fish body, it will affect the growth and development of fish. the spine is damaged, the bones are softened, and the appetite diminishes, but generally no death. the occurrence and disappearance of pox can be controlled by changing certain environmental factors, such as improvement of water quality or rise of temperature leading to the disappearance of symptoms, but affecting the growth and value of the fish. inoculation of the pox with scratches on healthy common carp can produce the same symptoms as natural conditions [ ] . in natural conditions, when water quality deteriorates, toxic substances stimulate the surface of fish and tissues, secreting large amounts of mucus, and finally the mucus falls off. herpesviral harmatopoietic necrosis was firstly detected in farming goldfish in japan [ ] . it is also found in australia [ ] and new zealand [ ] , spreading to global. the disease caused a large-scale death of goldfish fry in taiwan in , and cyhv- is responsible [ ] . cyhv- infects crucian carp in many provinces in china [ , ] , commonly known as crucian carp gill hemorrhagic disease. the influenced species only include goldfish [ ] , crucian carp and its common variety [ ] , and hybrids of goldfish [ ] . cyhv- is harmful to fish eggs, fry, fingerlings, broodstock, etc., while juveniles are more susceptible to infection than adult fish, and are more likely to cause fulminant deaths of juveniles less than year old [ ] . in addition, like other herpesviruses, cyhv- can form latent infection and becomes a potential source of transmission [ ] ,while temperature is a key factor for virus replication. at the beginning of the disease, the fish appear dying, poor appetite and slow swimming, staying at the bottom of the pond or tank. after the skin is pale with mucus, pustules appear on fins. it appears part of the abdomen swells, the eyeballs protrude from both sides and there is freckle bleeding on the sputum [ ] , blisters on the fins [ ] , ascites, etc. [ ] . the hepatopancreas, spleen and kidney are pale and the spleen and kidney are swollen [ ] . hematopoietic cells in the head kidney and body kidney show obvious nuclear pyknosis and nuclear lysis necrosis, with spleen large areas of necrosis, sometimes accompanied by hemorrhage as well as pancreatic, thymus, intestinal development from multiple lesions to diffuse necrosis, epithelial cell proliferation, degeneration and necrosis of the oropharynx and epidermal cells, focal necrosis of the heart. other tissues and organs including muscle tissue, brain tissue are found no pathological changes [ , , , ] . the partial or complete nucleotide sequences of cyhv- such as helicase gene, dna polymerase gene, terminal enzyme gene, and capsid protein genes have been reported, but the complete genome sequence and gene map have not been determined yet, requiring further researches and improvement. crucian carp carassius auratus herpesvirus (cahv) was isolated and its genome was sequenced and analyzed, showing that cahv is most closely related to cyhv and clusters closely with cyhvs of the family alloherpesviridae [ ] . at present, pcr is a relatively accurate method for detecting cyhv- infection, even a very small amount of viral dna in tissue [ ] . conventional pcr method based on cyhv- helicase gene have high specificity, with no amplification of the genomic dna of cyhv- , cyhv- and ichv- . it can effectively detect cyhv- isolates in different regions. a real-time quantitative pcr is established [ ] , which has high specificity and high sensitivity. it can not only detect cyhv- from fish exhibiting clinical symptoms, but also detect carriers. the diagnosis can detect the sick fish in the incubation period in advance so that it can prevent the breakout in time and take appropriate measures to minimize the loss of the disease. the first successful use of inactivated aeromonas salmonicida for oral immunization in rainbow trout (oncorhynchus mykiss) in opens up a precedent for the utilization of vaccines in fish [ ] . in the following decades, many researchers make extensive explorations of the preparation of fish vaccines to prevent the sudden emergence of fish diseases that are difficult to control with drugs. according to incomplete statistics, the number of commercial vaccines for global commercial production has been more than by the end of [ ] . currently, there are more than aquaculture vaccines studied in china and nearly species of pathogens are involved, according to incomplete statistics [ ] . however, only aquatic vaccines have obtained the national new veterinary drug certificate up to now. according to the preparation method, aquaculture vaccines are classified into live vaccine, inactivated vaccine and gene engineering vaccine ( table ) . at present, most of the aquaculture live vaccines are prepared with attenuated or mutated attenuated virus strains whose pathogenicity has been greatly weakened, such as vhsv [ ] , canine coronavirus (ccv) [ ] , ihnv [ ] , and gcrv [ ] vaccines. the inactivated vaccine is a managerial method that inactivates pathogenic microorganisms, but it remains immunogenic, providing the ability to induce specific resistance of aquatic animals after inoculation. these vaccines include a variety of tissue fluid inactivated vaccines, vibrio inactivated vaccines, aeromonas hydrophila vaccines, and streptococcal vaccines, for example, vhsv vaccine [ ] which is commonly used in trout farming in europe and the united states. gene engineering vaccine is further divided into recombinant subunit vaccine, dna vaccine, gene deletion/mutant vaccine and livingvector vaccine. . . . . recombinant subunit vaccine. the protective antigen genes of pathogens is expressed in different expression systems in vitro using recombinant dna technology to produce pathogenic proteins that can induce protective immune responses in hosts. after separation and purification, recombinant subunit vaccine is completely prepared. recombinant subunit vaccines do not contain pathogenic virulence factors and are expressed by genetically engineered bacteria. at present, there are many studies on the preparation of subunit aquatic vaccines based on immunogenic components such as bacterial outer membrane proteins, lipopolysaccharides and other protective antigens, but most of them are still in the experimental stage and have not been commercialized. gene engineering vaccines in aquaculture include ihnv [ ] , infectious pancreatic necrosis virus (ipnv) [ , ] , among which the ipnv vp recombinant subunit vaccine is currently the only commercially available fish recombinant vaccine [ ] . a dna vaccine is a recombinant eukaryotic expression vector that encodes a certain protein antigen and is directly injected into animals. after being taken in by host cells, it expresses an antigen protein, thereby inducing non-specific and specific immune responses, playing an important role in immune protection effect. the research on aquatic dna vaccine was firstly reported in [ , ] . although the dna vaccine researches start late, encouraging achievements have been made [ ] . till nowadays, dna vaccines mainly focus on the prevention and control of infectious virus diseases such as ihnv [ ] , vhsv [ ] , svcv [ ] , and snakehead rhabdovirus (shrv) [ ] . . . . . gene deletion/mutant vaccine. gene deletetion or mutant vaccine is a type of vaccine made by genetic engineering removing a fragment of a virulence gene in a viral or bacterial genome, making it a defective strain. vaughan reported the first gene mutation vaccine, the attenuated strain of aeromonas salmonicida to prevent salmon furunculosis in [ ] . gene deleted/mutant vaccine could induce t cell immune responses in rainbow trout [ , ] . this type of vaccine is also researched in channel catfish against channel catfish virus (ccv) [ ] and in zebrafish against erwinia [ ] . live-vehicle vaccines are genetically engineered vaccines that use a non-pathogenic virus or bacteria to carry and express other highly pathogenic or protective immune-related antigen genes, creating a multivalent vaccine. under this conditions, attenuated pathogens have become excellent carriers of heterologous antigens in prevention and treatment [ ] . the effect of a bacterial live vector vaccine is mainly concentrated on whether it can express a sufficient amount of protein to cause the host immune responses and produce a protective immune response [ ] . the initial report of living-vehicle vaccine in aquatic organism was in , preventing salmonella in salmon [ ] . subsequently, the application of livingvehicle vaccine was reported to prevent streptococcus [ ] , edwardsiella tarda, a. hydrophila [ ] and ccv [ ] . common immunization delivery methods of aquaculture vaccines include injection, immersion and oral administration (table ) . global aquaculture vaccines are mainly immunized with injection. aquaculture vaccine injection mainly includes intramuscular injection and intraperitoneal injection. intramuscular injection is mainly used for vaccination of dna vaccines, while intraperitoneal injection is mainly used for inoculation of traditional inactivated vaccines and attenuated vaccines [ ] . there are four types of immersion immunization, direct immersion, hypertonic soak, spraying immunization and immersion bath. after the first successfully hypertonic immunized fish in [ ] , vibrio vaccine achieves success in soaking immunization in salmon [ ] and rainbow trout [ ] . however, the mechanism of fish vaccination during immersion immunization is still unclear, such as whether the vaccine enters the body through the skin, tendon, lateral line, or other sites, and whether the vaccine-induced immunity works through the blood circulation system or the mucosal system, etc. in addition, a variety of factors affect the host's uptake of soaked immune antigens, including vaccine concentration, immersion time, aquatic animal size, adjuvants, antigenic forms, water temperature and so on [ ] . traditionally, adjuvant vaccine, biofilm vaccine and antigenic component vaccine can be implemented by oral immunization. along with the development of new technology, new oral immunization more prefer to slow-releasing micro capsule vaccine, yeast carrier vaccine and transgenic plant vaccine [ ] . oral vaccines are considered to be the most operative immune modes for aquatic vaccines. the main consideration is that the living environment of aquatic animals is inseparable from the water body and the cultured animal scale is large. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] replication, got the patent granted to protect common carp and koi against khv indicating excellent immunoprotection capability [ ] . khv liposome oral vaccine can protect common carp from khv infection [ ] . abundant immune genes are found to participate in the antiviral responses against khv, indicating that the immune response to khv is largely controlled by the genetic factors of the host [ ] . there are various gcrv vaccines reported in the past decades. gcrv histoplasmic inactivated vaccine was attempted in china since s. until , chinese researchers exploited high-efficiency attenuated vaccine with good immune effect and long protection period. gcrv is detoxified by eucalyptus liquid and injected to immunize grass carp demonstrating long-term immunoprotection and up to % protection ratio [ ] . the immunogenicity of vp and vp , which are the major outer capsid protein of gcrv type i, are researched a lot to evaluate the potential of function as vaccine [ ] . recombinant of vp and vp vaccine is synergistically orally administrated to grass carp inducing good immunoprotection and less than % cumulative mortality [ ] . outer capsid protein vp in gcrv type ii is also used to prepare gcrv vaccine. the immune protection assay shows that igm and other antiviral immune responses are significantly triggered and % protection ratio is achieved [ ] . other researches on gcrv gene engineering vaccines mainly concentrate on the cell proliferation enhancing activity and immunoprotection of gcrv vp and vp recombinant plasmid [ ] [ ] [ ] [ ] . a gcrv vp dna vaccine obtained by baculovirus expression system can achieve a protection ratio of % [ ] . using bacillus subtilis as the carrier of gcrv vp , the recombinant vaccine is orally immunized grass carp. challenge experiment reveals that the oral recombinant subunit vaccine can protect %- % grass carp from infection and generate immunity against gcrv [ ] . gcrv vp expressed by escherichia coli is proved to be relative to virus infection and have the potential to produce subunit vaccine [ ] . the gcrv inactivated vaccine induces the ifn system of grass carp and strengthens the igm expression continuously [ ] . there are still hundreds of researches unlisted, working on the preparation of gcrv vaccine to improve the utility of vaccines to protect grass carp from gcrv infection. immune adjuvant is pivotal to enhance the potency of vaccines against gcrv. new type immune adjuvant like cpg [ , ] develops rapidly in the past decades. inactivated viral vaccine against svcv provides limited protection, whereas attenuated vaccine has not been pursued because of a number of limitations, including improper attenuation of the virus, lack of quantitative evaluation for the protection activity provided by the vaccine, and restrictive market and legal regulations [ ] . regardless of the obstacles of technologies and regulatory approvals [ ] , researches on dna vaccines against svcv has been steadily increasing in the last decade [ ] [ ] [ ] . the initial svcv dna vaccine did not exhibit an expected effectiveness like other fish dna vaccines against novirhabdoviruses, including ihnv and vhsv [ ] . therefore, multiple trials have been conducted to develop an efficacious svcv dna vaccine. the g gene of svcv encodes a surface glycoprotein which is considered to be a major antigen for inducing the primary host immune responses and is frequently used in dna vaccine preparation [ , ] . ten svcv dna vaccines carrying svcv g gene are tested in carp [ ] . the majority of treated groups show little protection, with relative survival ranging from % to %. the strongest protection ( % relative survival) is observed in a group injected with a combination of two constructs expressing the full-length g gene. however, the relative contribution of the two constructs to this protection rate is not determined [ ] . the efficacy of another dna vaccine expressing the g gene of a north american svcv isolate is investigated in ornamental koi and goldfish [ ] . in all trials, immunized fish demonstrate a strong protective response against svcv, with relative survival ranging from % to % [ ] . vaccination with an oral or injection subunit vaccine based on the baculovirus recombinant expression of transmembrane svcv-g protein in insect cells shows positive immune protection [ ] . taken together, these studies put forward the validation for the potential utilization of g gene for dna vaccine as a prophylactic therapy against svcv infection. although dna vaccination via the parenteral route has been used as an efficient strategy to elicit antiviral immunity, it is not convenient for large-scale immunization due to the handling stress on fish, high labour and production cost [ ] . furthermore, there is limited scientific literature about the condition of dna vaccines after injection into fish [ ] . intestine, gills, skin and other mucosal tissues of fish, are important for immune protection against pathogen invasion [ , ] . activation of mucosal immunity prevents pathogen infections and replications in mucosa, whereas intramuscular vaccines fail to do this [ ] . oral vaccination is an effective way to induce mucosal immunity [ ] and this strategy has shown a successful induction of the antiviral responses against viral diseases in different fish species [ ] . lactobacilli possess multiple properties to be suitable candidates for vaccine antigen delivery vectors [ ] . lactobacilli also play a non-specific immune adjuvant effect [ ] . a genetically engineered lactobacillus plantarum co-expressing svcv g protein and khv orf protein is investigated for protective immunity in carp and koi through oral vaccination [ ] . immunized carp and koi show effective protection rates of % and %, respectively [ ] . moreover, immunized carp shows significantly elevated expression of igm [ ] . these results demonstrate that the recombinant l. plantarum is able to induce a protective immune response in fish against svcv and khv, suggesting a practical approach for large-scale control of cyprinid viral disease [ ] . along with the rapid development of freshwater fish farming industry, increasing number of diseases break out, especially viral diseases. the huge economic losses and food and environment safety risks caused by the diseases lead to the urgent need of vaccine. the variation of pathogens and the diversity of antigens are the main obstacles of efficient vaccine development. at the same time, the immune response mechanisms of vaccines are still uncertain, which not only increases the difficulty of suitable programming, but also leads to the blindness of vaccine researches and applications. the use of fish vaccines lacks effective and convenient ways of administration as well as measures for enhancing vaccine immunity and effective vaccine efficacy assessment systems and methods. more factors restrict the utilization of vaccines, for example, water temperature, water quality, illumination and season. in addition, the organism conditions (age, weight, nutriture, physiological status, group effect) and vaccine quality (immunogenicity, adjuvant, preparation, storage, transportation, administration method, dosage and time) also have a great influence on the potency of vaccine. in the future, the studies on fish vaccine are needed to improve the theoretical basis. the tendency is to concentrate efforts to solve one or two severe aquatic animal diseases as a breakthrough and promote the development of fish vaccines. meanwhile, the development of classic and new fish vaccines need to cooperate with each other, with the help of human and veterinary vaccine researches. the research on the administration in relation to the effectiveness of fish vaccines is an important part of vaccine development. it is urgent to establish a simple and effective fish vaccine evaluation system and accelerate the construction of pilot test bases for fish vaccines. the developments of appropriate immunopotentiators and immune adjuvants also promote the potency of fish vaccines. as the worldwide rapid development of science and technology as well as the positive attempts in fish and other aquatic animals, aquaculture industry is showing the tendency to flourish. taking full advantage of the immune responses to diseases, vaccination is a vital strategy to prevent and control the potential diseases, solving the problems of food quality and safety as well as environmental pollution. an artificial landscape-scale fishery in the bolivian amazon piscinae: artificial fishponds in global trade in ornamental fish from an australian perspective: the case for revised import risk analysis and management strategies ornamental fish as trans-boundary vectors of viral diseases an etiological study on mass mortality of cultured colorcarp juveniles showing edema a survey of koi herpesvirus and carp edema virus in colorcarp cultured in niigata prefecture situation and tendency of the fishery vaccine development a herpesvirus associated with mass mortality of juvenile and adult koi, a strain of common carp current knowledge on koi herpesvirus (khv): a review khv, cngv or cyhv- , which is the koi/carp killer? reactivation of koi herpesvirus infections in common carp cyprinus carpio molecular comparison of isolates of an emerging fish pathogen, koi herpesvirus, and the effect of water temperature on mortality of experimentally infected koi susceptibility of koi crucian carp and koi goldfish hybrids to koi herpesvirus (khv) and the development of khv disease (khvd) susceptibility of koi carp, common carp, goldfish, and common carp hybrids to cyprinid herpesvirus- and herpesvirus- horizontal transmission of koi herpes virus (khv) from potential vector species to common carp do wild fish species contribute to the transmission of koi herpesvirus to carp in hatchery ponds? transmission of cyprinid herpesvirus- (cyhv- ) from goldfish to naiive common carp by cohabitation goldfish (carassius auratus auratus) is a susceptible species for koi herpesvirus (khv) but not for khv disease (khvd) common fish species in polyculture with carp as cyprinid herpes virus carriers resistance of common carp (cyprinus carpio l.) to cyprinid herpesvirus- is influenced by major histocompatibility (mh) class ii b gene polymorphism feeding cyprinus carpio with infectious materials mediates cyprinid herpesvirus entry through infection of pharyngeal periodontal mucosa epidemiological description of a new viral disease afflicting cultured cyprinus carpio in israel concentrations of a koi herpesvirus (khv) in tissues of experimentally infected cyprinus carpio koi as assessed by real-time taqman pcr cyprinid herpesvirus an interesting virus for applied and fundamental research the emergence of koi herpesvirus and its significance to european aquaculture cyhv- : the third cyprinid herpesvirus initial isolation and characterization of a herpes-like virus (khv) from koi and common carp (special issue: international symposium on koi herpesvirus disease) outbreaks of koi herpesvirus disease in rivers of kanagawa prefecture comparative genomics of carp herpesviruses phylogenetic relationships in the family alloherpesviridae genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in cyprinus carpio koi cyprinid herpesvirus quantification of cyprinid herpesvirus in environmental water by using an external standard virus development of mrna-specific rt-pcr for the detection of koi herpesvirus (khv) replication stage pathogenesis of acute viral disease induced in fish by carp interstitial nephritis and gill necrosis virus generation of monoclonal antibodies specific for orf of koi herpesvirus laboratory validation of a lateral flow device for the detection of cyhv- antigens in gill swabs pathogen recognition and innate immunity herpesviruses and immunity: the art of evasion human cytomegalovirus activates inflammatory cytokine responses via cd and toll-like receptor dai (dlm- / zbp ) is a cytosolic dna sensor and an activator of innate immune response innate immune sensing of dna viruses rna polymerase iii detects cytosolic dna and induces type-i interferons through the rig-i pathway herpes simplex virus type activates murine natural interferon-producing cells through toll-like receptor toll-like receptor -mediated recognition of herpes simplex virus- by plasmacytoid dendritic cells molecular cloning, characterization and expression analysis of tlr , myd and traf genes in common carp (cyprinus carpio) new insights into the evolution of ifns: zebrafish group ii ifns induce a rapid and transient expression of ifndependent genes and display powerful antiviral activities early viral replication and induced or constitutive immunity in rainbow trout families with differential resistance to infectious hematopoietic necrosis virus (ihnv) genetic resistance to rhabdovirus infection in teleost fish is paralleled to the derived cell resistance status early antiviral response and virus-induced genes in fish interferon type i responses to virus infections in carp cells: in vitro studies on cyprinid herpesvirus and rhabdovirus carpio infections cyprinid herpesvirus infection disrupts the skin barrier of common carp gene expression analysis of common carp (cyprinus carpio l.) lines during cyprinid herpesvirus infection yields insights into differential immune responses spring viremia of carp (svc) encyclopedia of virology determination of the complete genomic sequence and analysis of the gene products of the virus of spring viremia of carp, a fish rhabdovirus characterization of complete genome sequence of the spring viremia of carp virus isolated from common carp (cyprinus carpio) in china virus genomes and virus-host interactions in aquaculture animals nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four genogroups phylogenetic analysis of spring virema of carp virus reveals distinct subgroups with common origins for recent isolates in north america and the uk vaccination of fish in european pond culture: prospects and constraints svcv infection of carp (spring viraemia carp virus) infectious dropsy in carp-a disease complex comparison of multiple genes of spring viremia of carp viruses isolated in the united states infectious hematopoietic necrosis, fish viruses & fish viral diseases first report of a rhabdovirus affecting carp in spain fish rhabdoviruses: molecular epidemiology and evolution first report of spring viraemia of carp virus in moscow province phylogenetic analysis of spring viraemia of carp virus isolates from austria indicates the existence of at least two subgroups within genogroup id the influence of environmental temperature and infection route on the immune response of carp (cyprinus carpio) to spring viremia of carp virus (svcv) rhabdovirus carpio in herpivorous fishes: isolation, pathology and comparative susceptibility of fishes viral infection cycles in pike (esox lucius l.) comparative pathogenicity of two strains of pike fry rhabdovirus and spring viremia of carp virus for young roach, common carp, grass carp and rainbow trout pathogenesis of spring viremia of carp virus in emerald shiner notropis atherinoides rafinesque, fathead minnow pimephales promelas rafinesque and white sucker catostomus commersonii (lacepede) susceptibility of zebrafish (danio rerio) to a model pathogen, spring viremia of carp virus caspian white fish (rutilus frisii kutum) as a host for spring viraemia of carp virus serological techniques currently used in fish virology spring viraemia of carp virus (svcv): comparison of immunoperoxidase, fluorescent antibody and cell culture isolation techniques for detection of antigen rapid detection of svc virus antigen in infected cell cultures and clinically diseased carp by the enzyme-linked immunosorbent assay (elisa) enzyme-linked immunosorbent assay (elisa) for the detection of spring viraemia of carp virus (svcv) in tissue homogenates of the carp, cyprinus carpio l monoclonal antibodies against g protein of spring viremia of carp virus monoclonal antibody against m protein of spring viremia of carp virus selection and characterization of single-chain recombinant antibodies against spring viraemia of carp virus from mouse phage display library identification of spring viraemia of carp virus (svcv) by combined rt-pcr and nested pcr simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative rt-pcr assay development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time rt-pcr improved diagnosis of spring viremia of carp by nested reverse-transcription pcr: development of a chimeric positive control for prevention of falsepositive diagnosis development of an improved rt-pcr for specific detection of spring viraemia of carp virus reverse transcription loopmediated isothermal amplification assay for rapid detection of papaya ringspot virus challenging loop-mediated isothermal amplification (lamp) technique for molecular detection of toxoplasma gondii development of reverse transcription loop mediated isothermal amplification assay for rapid detection of bluetongue viruses a comparison of inhouse real-time lamp assays with a commercial assay for the detection of pathogenic bacteria rapid and sensitive detection of shrimp yellow head virus by loop-mediated isothermal amplification combined with a lateral flow dipstick a loop-mediated isothermal amplification assay for rapid detection of cyprinid herpesvirus in gibel carp (carassius auratus gibelio) detection of spring viraemia of carp virus (svcv) by loop-mediated isothermal amplification (lamp) in koi carp, cyprinus carpio l development and evaluation of a one-step loop-mediated isothermal amplification for detection of spring viraemia of carp virus recent developments and prospects of fish disease science in china advance in molecular biology of grass carp reovirus studies on the causative agent of harmorrhage of the grass carp (ctenpharyngodon idellus) ii. electron microscopic observation an improved rt-pcr assay for rapid and sensitive detection of grass carp reovirus histopathological studies on the hemorrage disease of grass carp complete characterisation of the american grass carp reovirus genome (genus aquareovirus: family reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses identification and genomic characterization of a novel fish reovirus, hubei grass carp disease reovirus, isolated in in china complete genomic sequence of a reovirus isolated from grass carp in china complete genome sequence of a reovirus isolated from grass carp, indicating different genotypes of gcrv in china development of a novel candidate subunit vaccine against grass carp reovirus guangdong strain (gcrv-gd ) innate immunity of finfish: primordial conservation and function of viral rna sensors in teleosts pattern recognition and signaling mechanisms of rig-i and mda in vivo ligands of mda and rig-i in measles virus-infected cells identification of a inducible gene i from grass carp (ctenopharyngodon idella) and expression analysis in vivo and in vitro identification and expression profiling analysis of grass carp ctenopharyngodon idella lgp cdna molecular cloning and immune responsive expression of mda gene, a pivotal member of the rlr gene family from grass carp ctenopharyngodon idella mda induces a stronger interferon response than rig-i to gcrv infection through a mechanism involving the phosphorylation and dimerization of irf and irf in cik cells identification, mrna expression and genomic structure of tlr and its association with gcrv susceptibility/ resistance in grass carp (ctenopharyngodon idella) genomic organization and expression analysis of toll-like receptor in grass carp (ctenopharyngodon idella) toll-like receptor regulates mx expression in rare minnow gobiocypris rarus after viral infection identification and expression profiles of grass carp ctenopharyngodon idella tlr in responses to double-stranded rna and virus infection teleost-specific tlr localizes to endosome, recognizes dsrna, recruits trif, triggers both ifn and nf-κb pathways, and protects cells from grass carp reovirus infection molecular characterization and expression analysis of nuclear oligomerization domain proteins nod and nod in grass carp ctenopharyngodon idella trunk kidney of grass carp (ctenopharyngodon idella) mediates immune responses against gcrv and viral/ bacterial pamps in vivo and in vitro characterizations of two grass carp ctenopharyngodon idella hmgb genes and potential roles in innate immunity two novel homologs of high mobility group box gene in grass carp (ctenopharyngodon idella): potential roles in innate immune responses two hmgb genes from grass carp ctenopharyngodon idella mediate immune responses to viral/bacterial pamps and gcrv challenge cytosolic sensing of viruses intracellular toll-like receptors insights into the antiviral immunity against grass carp (ctenopharyngodon idella) reovirus (gcrv) in grass carp a histopathological study on juvenile colorcarp, cyprinus carpio, showing edema a trial for the detection of carp edema virus by using polymerase chain reaction koi sleepy disease found for the first time in koi carps in the netherlands another potential carp killer? carp edema virus disease in germany carp edema virus/koi sleepy disease: an emerging disease in central-east europe carp edema virus in polish aquaculture-evidence of significant sequence divergence and a new lineage in common carp cyprinus carpio (l.) carp edema virus, an emerging threat to the carp (cyprinus carpio) industry in china movement of pathogens with the international trade of live fish: problems and solutions emergence of carp edema virus (cev) in cultured ornamental koi carp, cyprinus carpio koi in india histopathological and electron microscopy studies on sleepy disease of koi cyprinus carpio koi in japan comparison of pcr methods for the detection of genetic variants of carp edema virus carp edema virus from three genogroups is present in common carp in hungary experimental infections of different carp strains with the carp edema virus (cev) give insights into the infection biology of the virus and indicate possible solutions to problems caused by koi sleepy disease (ksd) in carp aquaculture concentration of carp edema virus (cev) dna in koi tissues affected by koi sleepy disease (ksd) prevention of carp pox herpesviral haematopoietic necrosis of goldfish, carassius auratus (l.) haematopoietic necrosis in a goldfish (carassius auratus) associated with an agent morphologically similar to herpesvirus development of a polymerase chain reaction assay to detect cyprinid herpesvirus in goldfish epizootic of herpes-like virus infection in goldfish, carassius auratus in taiwan mass mortality caused by cyprinid herpesvirus (cyhv- ) in prussian carp (carassius gibelio) in china disease prevention and control isolation of a cyprinid herpesvirus from goldfish, carassius auratus (l.), in the uk introduction of the family alloherpesviridae: the first molecular detection of herpesviruses of cyprinid fish in hungary a viral epizootic in cultured populations of juvenile goldfish due to a putative herpesvirus etiology herpesviral haematopoietic necrosis virus (cyhv- ) infection: case studies from commercial goldfish farms detection of the herpesviral hematopoietic necrosis disease agent (cyprinid herpesvirus ) in moribund and healthy goldfish: validation of a quantitative pcr diagnostic method herpesvirus haematopoietic necrosis in a goldfish (carassius auratus) in the uk complete genome sequence and architecture of crucian carp carassius auratus herpesvirus (cahv) the oral immunization of trout against bacterium salmonicida introduction of yersinia ruckeri biotype into finnish fish farms live vaccine of viral hemorrhagic septicemia virus (vhsv) for japanese flounder at fish rearing temperature of °c instead of poly(i:c) administration canine coronavirus vaccine attenuated infectious hematopoietic necrosis virus (ihnv) with rearranged gene order as potential vaccine the preparation and immune effect of attenuated live vaccine obtained through cell culture inactivated vaccine against viral hemorrhagic septicemia (vhs) emulsified with squalene and aluminum hydroxide adjuvant provides long term protection in olive flounder an oral dna vaccine against infectious haematopoietic necrosis virus (ihnv) encapsulated in alginate microspheres induces dose-dependent immune responses and significant protection in rainbow trout (oncorrhynchus mykiss) immunogenicity of lactobacillusexpressing vp and vp of the infectious pancreatic necrosis virus (ipnv) in rainbow trout genetic immunization of rainbow trout (oncorhynchus mykiss) against infectious hematopoietic necrosis virus introduction of foreign genes into the tissue of live fish by direct injection and particle bombardment dna vaccines encoding viral glycoproteins induce nonspecific immunity and mx protein synthesis in fish licensed dna vaccines against infectious hematopoietic necrosis virus (ihnv), recent pat vhsv g glycoprotein major determinants implicated in triggering the host type i ifn antiviral response as dna vaccine molecular adjuvants dna vaccination can protect cyprinus carpio against spring viraemia of carp virus dna vaccines encoding the glycoprotein genes of spring viremia of carp virus, snakehead rhabdovirus, or infectious hematopoietic necrosis virus induce protective immunity in rainbow trout (oncorhynchus mykiss) against an infectious hematopoietic necrosis v, drugs under exp an aromatic-dependent mutant of the fish pathogen aeromonas salmonicida is attenuated in fish and is effective as a live vaccine against the salmonid disease furunculosis a live (delta aroa) aeromonas salmonicida vaccine for furunculosis preferentially stimulates t-cell responses relative to b-cell responses in rainbow trout (oncorhynchus mykiss) potency testing of a live, genetically attenuated vaccine for salmonids the attenuated v strain of channel catfish virus possesses a deletion in orf coding for a potentially secreted glycoprotein search for live attenuated vaccine candidate against edwardsiellosis by mutating virulence-related genes of fish pathogen edwardsiella tarda improving live attenuated bacterial carriers for vaccination and therapy use of in vivo-regulated promoters to deliver antigens from attenuated salmonella enterica var. typhimurium recombinant infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in aeromonas salmonicida induce protective immunity in rainbow trout (oncorhynchus mykiss) identification and immunoprotective analysis of a streptococcus iniae subunit vaccine candidate a novel in vivo inducible expression system in edwardsiella tarda for potential application in bacterial polyvalence vaccine recombinant channel catfish virus (ictalurid herpesvirus ) can express foreign genes and induce antibody production against the gene product fish dna vaccine against infectious hematopoietic necrosis virus: efficacy of various routes of immunisation uptake of bovine serum albumin by rainbow trout from hypersmotic solutions: a model for vaccinating fish a field trial of vaccination against cold-water vibriosis in atlantic salmon (salmo salar l.) prolonged immersion improves the effectiveness of dilute vibrio vaccine for rainbow trout factors influencing the uptake of c-labelled vibrio anguillarum vaccine in direct immersion experiments with rainbow trout, salmo gairdneri richardson he, overview of new type oral fish vaccines koi herpesvirus vaccine oral immunization of common carp with a liposome vaccine fusing koi herpesvirus antigen single-walled carbon nanotubes as delivery vehicles enhance the immunoprotective effect of a dna vaccine against spring viremia of carp virus in common carp protection of grass carp, ctenopharyngon idellus (valenciennes), through oral administration of a subunit vaccine against reovirus the research about protective efficency of dna vaccine against hemorrhage disaese of grass carp enhanced expression of gcrv vp in cik cells by relative sequence optimization construction of a recombinant eukaryotic vector for a grass carp reovirus vp gene and its expression in vitro and in vivo protective immunity of grass carp immunized with dna vaccine encoding the vp gene of grass carp reovirus using carbon nanotubes as a carrier molecule evaluation of immune efficacy of gcrv vp dna vaccine prokaryotic expression and immunization analysis of grass carp reovirus hz strain outer capsid protein vp segment the use of an in vitro microneutralization assay to evaluate the potential of recombinant vp protein as an antigen for vaccinating against grass carp reovirus inactivated vaccine for hemorrhage of grass carp up-regulates the expressions of major immune-related genes in spleen of grass carp a specific cpg oligodeoxynucleotide induces protective antiviral responses against grass carp reovirus in grass carp ctenopharyngodon idella a plasmid containing cpg odn as vaccine adjuvant against grass carp reovirus in grass carp ctenopharyngodon idella dna vaccines-challenges in delivery protection of atlantic salmon against virus infection by intramuscular injection of ifnc expression plasmid a single dose of a suicidal dna vaccine induces a specific immune response in salmonids modulatory effect of cpg oligodeoxynucleotide on a dna vaccine against nervous necrosis virus in orange-spotted grouper (epinephelus coioides) dna vaccines for aquacultured fish dna vaccine protects ornamental koi (cyprinus carpio koi) against north american spring viremia of carp virus vaccination of carp against svcv with an oral dna vaccine or an insect cellsbased subunit vaccine development of an oral vaccine for immunisation of rainbow trout (oncorhynchus mykiss) against viral haemorrhagic septicaemia what happens to the dna vaccine in fish? a review of current knowledge the major portal of entry of koi herpesvirus in cyprinus carpio is the skin the mucosal immune system of fish: the evolution of tolerating commensals while fighting pathogens recent advances in mucosal vaccine development the probiotic bacterium lactobacillus casei induces activation of the gut mucosal immune system through innate immunity immunoadjuvant activity of oral lactobacillus casei: influence of dose on the secretory immune response and protective capacity in intestinal infections recombinant lactobacillus expressing g protein of spring viremia of carp virus (svcv) combined with orf protein of koi herpesvirus (khv): a promising way to induce protective immunity against svcv and khv infection in cyprinid fish via oral vaccination this work was supported by national natural science foundation of china ( ) and huazhong agricultural university scientific & technological self-innovation foundation ( rc ). key: cord- -mi v authors: wilder-smith, annelies title: dengue vaccine development by the year : challenges and prospects date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: mi v the first licensed dengue vaccine led to considerable controversy, and to date, no dengue vaccine is in widespread use. all three leading dengue vaccine candidates are live attenuated vaccines, with the main difference between them being the type of backbone and the extent of chimerization. while cyd-tdv (the first licensed dengue vaccine) does not include non-structural proteins of dengue, tak- contains the dengue virus serotype backbone, and the butantan/merck vaccine contains three full-genomes of the four dengue virus serotypes. while dengue-primed individuals can already benefit from vaccination against all four serotypes with the first licensed dengue vaccine cyd-tdv, the need for dengue-naive population has not yet been met. to improve tetravalent protection, sequential vaccination should be considered in addition to a heterologous prime-boost approach. dengue vaccine development has been hampered and delayed by remarkable challenges. the four genetically succinct but still closely related dengue serotypes are known to interact immunologically with potential for disease enhancement. as a tetravalent immune response is desired, when given a mixture of all four serotypes in a tetravalent live-attenuated vaccine, each component would need to independently result in four different monotypic immune responses that are solid to each serotype. this has unfortunately proven to be difficult to achieve. immune correlatestopredictprotectionversusdiseaseenhancement are still lacking [ ], and plaque reduction neutralization assays do not reliably differentiate between serotype-specific versus heterotypic antibodies [ , ]. other challenges include the lack of a reliable animal model. furthermore, dengue is primarily a disease of low and middle income countries, thus dengue research often does not receive the level of funding needed to accelerate vaccine development [ ] . unsurprisingly then, it has taken several decades to develop a vaccine. the first licensed dengue vaccine led to considerablecontroversy [ ] ,andto date,nodenguevaccine is in widespread use. nevertheless, we need to press on. dengue was identified as one of the threats to global health in by who, underlining the urgent need for a vaccine. the primary need for a dengue vaccine as a public health tool is the unpredictable nature of dengue outbreaks overwhelming already existing fragile health care systems, the extremely high annual incidence of at least million cases and the epidemic trajectory which shows a relentless increase over the past two decades [ , , ]. dengue infections in the communities, and even hospitalized dengue, lead to inappropriate antibiotic use in more than %of cases [ ] . dengue has also become a leading problem in international travelers [ ] [ ] [ ] [ ] [ ] . certain risk factors are predictive of more severe disease outcome such as young or old age, prior dengue infection, diabetes, sickle cell disease and underlying medical conditions [ , ]. all three leading dengue vaccine candidates are liveattenuated vaccines, with the main difference between them being the type of backbone and the extent of chimerization. first licensed dengue vaccine cyd-tdv, a tetravalent live attenuated with a yellow fever d backbone, is the first dengue vaccine to be licensed, under 'dengvaxia'. despite being first licensed in in mexico followed by other dengue endemic countries based on results from phase trials conducted in more than children and adolescents aged À , it was only introduced in two subnational public health programs in the philippines and brazil. the phase trials revealed a vaccine efficacy that differed by age, serostatus and serotype. in terms of cumulative incidence, cyd-tdv showed a population level benefit [ ] . further post-hoc retrospective analyses of the long-term safety data revealed an excess risk of severe dengue in those who were seronegative at baseline. serostatus refers to whether a person has had dengue infections in the past [ ] . this increased risk in seronegative subjects was observed starting from months after administration of the first dose. a plausible hypothesis is that cyd-tdv may trigger an immune response to dengue in seronegative persons that predisposes them to a higher risk of severe disease, analogue to what is seen in natural secondary dengue infections [ ] . a subsequent infection with the first true wild type dengue virus would then be a 'secondary-like' dengue illness. dengue non-structural proteins (ns) are absent from the sanofi dengue-yellow fever chimeric vaccine. given that ns may have toxinlike properties that disrupt the endothelial glycocalyx through either inflammatory-dependent or independent pathways [ ] [ ] [ ] [ ] , the absence of ns in cyd-tdv could also be a potential explanation for the limited vaccine performance. the world health organization (who) recommends that for countries considering cyd-tdv vaccination as part of their dengue control program, a pre-vaccination screening strategy, in which only dengue-seropositive persons are vaccinated, is the recommended strategy [ ] . in may , the us food and drug administration (fda) approved cyd-tdv for use in seropositive individuals through years of age living in endemic areas of the u.s. the european medicine agency also endorsed the use of this vaccine in seropositive individuals with a wider age range. the programmatic use of cyd-tdv, therefore, requires screening for serostatus before vaccination. igg-based enzyme-linked immunosorbent assays (elisas) or rapid diagnostic tests (rdts) can be used for pre-vaccination screening, also called a 'test and vaccinate' strategy. ideally, a screening test should be both highly sensitive and specific to minimize false positives and negatives to yield maximal population level benefit and minimize harm by correctly screening for seropositive individuals only [ ] . it should also be affordable, simple to use and provide rapid results. two recent comparative evaluations on currently available assays showed high specificity (> %) for all immunoassays apart from one rdt, but variable sensitivities (higher sensitivities observed for the elisas [ % and %] than the rdts [ - %]) [ , ] sensitivity appeared similar in samples from individuals with recent and remote virologically confirmed dengue (vcd). cross-reactivity to other flaviviruses was low with rdts (</ = %), but more significant with elisas (up to % for west nile and % for zika). cyd-tdv's public health utility is limited to seropositive persons. implementation research is now needed on how and for which settings (e.g. school settings) a prevaccination screening can be rolled out for programmatic national or subnational use [ ] . in private clinics and travel medicine settings [ ] , blood is often taken before hepatitis b vaccination to check for hepatitis b serostatus and other vaccine-preventable diseases; thus there is precedence for pre-vaccination screening. research is also needed to evaluate vaccine schedules with fewer doses, assess the need and timing for booster doses, and identify populations that will benefit most from this vaccine [ , , ] . lessons from the first licensed dengue vaccine for clinical trial designs lessons from the first dengue vaccine have shaped how second-generation dengue vaccines should be evaluated. the effect on cellular immunity needs to be studied, including the extent of truly neutralizing antibodies versus just transient cross-protective antibodies. vaccine trial designs should account for the known period of crossprotection between serotypes which can last up to one even two years before safety signals may appear in year and beyond. trial designs, therefore, need to be extended to include active surveillance of trial participants up to À years [ ] . furthermore, vaccine evaluation must include a-priori analysis plans for stratification by serostatus and serotype. to stratify by serostatus, baseline blood samples need to be taken from all study subjects. when interpreting efficacy results, one has to consider the predominance of a given serotype and the proportion of individuals who are seronegative in a population, which can vary from year to year and country to country [ ] . indeed, the vaccine trials for the secondgeneration live dengue vaccines take these trial specifications into account. non-structural proteins of the dengue virus backbone are at least partially present in second generation dengue vaccines. two chimeric live-attenuated dengue vaccines are now in phase trials: one developed by takeda (tak- ) and one by the national institute of allergy and infectious diseases (tv /tv ) (table ) . ( ) takeda's tak- takeda's live-attenuated tetravalent dengue vaccine candidate comprises an attenuated denv- strain plus chimeric viruses containing the prm and e genes of denv- , denv- and denv- cloned into the attenuated denv- 'backbone' [ ] . the difference to dengvaxia therefore is the presence of non-structural proteins due to the denv backbone. tad- induces crossreactive t cell-mediated responses that may be necessary for broad protection against dengue fever [ , ] . in agreement with who's prequalification requirements for dengue vaccines, takeda has manufactured a lyophilized formulation of tak- that allows stable storage at + degrees c to + degrees c. in a randomized, doubleblind, phase study (nct ) in healthy dengue-naive adults, - years of age, gmts and seropositivity rates to all four serotypes were achieved [ ] . a multi-color fluorospot (mcf) assay enabled quantitation of serotype-specific and cross-reactive individual memory b cells (mbcs) secreting denv-specific antibodies in a polyclonal mixture [ ] . using the mcf assay, type-specific and cross-reactive mbc responses were investigated; the results demonstrate that, unlike primary or secondary natural denv infection, tetravalent vaccination elicits tetravalent type-specific mbcs, and thus all four components of tak- contribute to the denv-specific mbc response following vaccination [ ] . the phase trials on immunogenicity showed that geometric mean titers (gmts) against denv , denv , and denv were lower in participants who were seronegative and receiving one primary dose than in those who received the two-dose primary series or one primary dose plus a -year booster [ ] . these immunogenicity results suggested that phase trials should be conducted with a two dose regimen to improve immunogenicity in seronegative individuals. at month , seropositivity rates were . %, . %, . % and . % for denv- , denv- , denv- and denv- , respectively [ ] . a phase trial in more than healthy children and adolescents - years of age to receive two doses of vaccine or placebo is currently being conducted in countries in asia and latin america. in summary, although the takeda vaccine appears much less serostatus dependent compared with cyd-tdv and efficacy data look promising, some complex nuances for serotypes and will require extended follow-up, and careful balancing by regulators and policy makers in determining the potential utility and safety of this vaccine [ ] . ( ) national institute of allergy and infectious diseases (tv /tv )/butantan this vaccine comprises full-length denv attenuated by one or more deletions in the untranslated region, while the fourth component is a chimeric virus in which the prm and e proteins of denv- replace those of denv- in the den d background [ ] . thus, this vaccine carries the full-genomic backbone of three dengue serotypes, except for denv . the capacity to elicit cd + cell responses closely mirrors those observed in a population associated with natural immunity [ ] . a single-dose induces robust tetravalent antibody and cellular t cell responses and resulted in a % efficacy in a human challenge study [ ] . developed by the u.s. national institutes of health (nih/niaid), it is currently in a phase trial in brazil through butantan, but was also licensed to merck for further development outside of brazil. the butantan institute has dengue vaccine development wilder-smith manufactured a lyophilized tetravalent live-attenuated dengue vaccine butantan-dv, which is analogous to the us national institutes of health (nih) tv admixture [ ] . seroconversion appears to be high for all four serotypes independent of serostatus, with the highest for denv , and , and the lowest for denv . to determine the ability of a single dose of the live attenuated tetravalent dengue vaccine tv to induce a suitable neutralizing antibody response, a placebo-controlled clinical trial was performed in healthy adults who received doses of vaccine or placebo administered months apart. evaluation of safety, vaccine viremia, and neutralizing antibody response indicated that a single dose is sufficient [ ] . thus, this vaccine has gone into phase trial with a single dose (in contrast to tak- with doses, and cyd-tdv with doses). the phase results remain unpublished to date, without known interim analyses. given that many endemic countries are popular tourist destinations, international travelers are increasingly at risk of dengue [ , , [ ] [ ] [ ] ] , with attack rates reported as high as . cases per travel-months [ ] . dengue is a frequent problem in travelers [ . ] , more frequent than 'traditional' travel-associated infectious diseases such as typhoid fever [ ] , rabies [ ] , and yellow fever [ , ] . geosentinel is an international network of travel medicine providers to monitor trends in travel-associated diseases [ ] [ ] [ ] which has reported a substantial increase of dengue over the past decade [ ] . dengue can affect tourist travelers, business travelers and expatriates [ , ] , migrants including those visiting friends and relatives (vfr) [ ] , and pilgrims [ ] ; both in adult and pediatric travelers [ , , ] . interruption of travel, hospitalization during or after travel, and out-of-pocket expenses can ensue [ ] . dengue is now much more frequent than many of the other travel-associated vaccine preventable diseases such as rabies, hepatitis a [ ] , yellow fever or japanese encephalitis [ , ] ; thus, vaccination against dengue would be an indication in the travel medicine context. the limitation of the currently only licensed dengue vaccine, cyd-tdv, is that it should only be used in seropositive travelers [ ] . however, most travelers are seronegative. furthermore, the dosing schedule of doses months apart for cyd-tdv renders the use of such a vaccine difficult in the travel medicine setting. a safe and efficacious vaccine that can be used regardless of serostatus is needed for travelers [ ] . until a vaccine becomes available that would benefit all travelers to dengue endemic countries, travelers should be advised to take day-time personal protective measures against mosquito bites [ ] and consider dengvaxia if they are seropositive [ ] . pre-travel advice for all travellers to dengue endemic countries need to include advice on the dengue risk [ , ] . while dengue-primed individuals can already benefit from vaccination against all four serotypes with the first licensed dengue vaccine cyd-tdv, the need for dengue-naive population has not yet been met. would sequential immunization induce stronger and broader immunity against four denv serotypes than tetravalent-formulated immunization and overcome the viral interference we have seen to date for the live attenuated dengue vaccine formulations? in a study in singapore mice were immunized with four dna plasmids, each encoding the pre-membrane and envelope from one denv serotype, either sequentially or simultaneously. the sequential immunization induced significantly higher levels of interferon (ifn)g-expressing or tumor necrosis factor (tnf)a-expressing cd + and cd + t cells to both serotype-specific and conserved epitopes than tetravalent immunization [ ] . moreover, sequential immunization induced higher levels of neutralizing antibodies to all four denv serotypes than tetravalent vaccination. in these animal data, sequential immunization resulted in more diversified immunoglobulin repertoire, and suggests that sequential immunization offers an alternative approach to potentially overcome the current challenges encountered with tetravalent-formulated dengue vaccines. another strategy to overcome viral interference for tetravalent dengue vaccines would be to use a heterologous prime-boost strategy. while tak- vaccine induces high protection against denv and to a lesser extent against denv in both dengue-seropositive and -seronegative individuals, cyd-tdv induces a high protection against denv and but to a lesser extent against denv and . furthermore, while tak- vaccine performance seems to be less serostatus dependent, an inconclusive relative risk > has been observed for denv in seronegative vaccinees and no conclusion could be drawn regarding denv [ ] . so what about combining both vaccines in a heterologous prime-boost regimen, leveraging upon the benefits of each vaccine and thereby minimizing safety concerns? priming with tak- followed by a cyd-tdv boost would initially ensure strong humoral and cellular responses against denv the weakest cyd-tdv serotype -, and then eventually strengthen responses against the other serotypes, in particular denv -the dominant cyd-tdv and weakest tak- serotype [ ] . heterologous cyd-tdv boost may also likely induce broader cross-reactive immune responses at both humoral and cellular levels. moreover, cyd-tdv possesses an yf- d backbone, decreasing the risk of being negatively impacted by initial tak- -induced denv -specific cellular responses. a denv - dominant vaccine followed by a denv - dominant vaccine may also better reflect the theoretical advantages of sequential infections as outlined above. it is likely that takeda will only investigate such a prime-boost strategy after their vaccine has been licensed. combining vaccine platforms developed by competing companies may pose challenges, but these can be overcome. next-generation dengue vaccines in development include dna, subunit, virus-like particles (vlp) and viral vector vaccines [ ] . two phase i clinical trials were conducted to evaluate the safety and efficacy of the tetravalent formulation purified inactivated vaccine combined with different adjuvants (e.g. aluminium hydroxide, as e or as b) [ , ] . all formulations were well tolerated and induced a balanced immune response against all four serotypes, with the highest mean antibody titers reached with as e and as b. a phase trial is currently evaluating a tetravalent purified inactivated dengue vaccine with as b to determine the most effective injection schedule ( - , - - , or - months) (nct ) the emergence of zika virus as a public health problem of international concern in early , a vector-borne virus with close genetic similarity to dengue viruses, was the first challenge to dengue vaccine development, followed by the emergence of another virus by late , not related to dengue, sars-cov- causing coronavirus related disease (covid- ). although dengue virus is not associated with severe pregnancy outcomes as zika [ ] , not thought to be sexually transmitted [ , ] and not as strongly associated with neurological complications such as guillain-barre syndrome [ ] , the emergence of zika has complicated dengue vaccine development because of the potential immunological interaction between these closely related viruses. by acquiring cytotoxic t-cell epitope-rich regions from culex-borne flaviviruses, zikv evaded denv-generated t-cell immune cross-protection [ ] . interestingly, pre-existing dengue immunity has minimal impact on the innate immune response to zika [ ] . primary and secondary denv elicit similar memory b-cell responses, but breadth to other serotypes and cross-reactivity to zika virus is higher in secondary dengue [ ] . immunity to denv only modestly shapes breadth and magnitude of enduring zikv antibody responses [ ] . while the evidence is mounting that preexisting high antibody titers to dengue virus were associated with reduced risk of zikv infection and symptoms [ ] , there is still lack of data on whether pre-existing immunity to zika protects against or enhances a subsequent dengue infection. these are data gaps that need to be addressed for dengue vaccine development. clearly, the presence of co-circulating arboviruses such as dengue and zika increases the chance of co-infection and demonstrates the importance of the differential diagnosis, especially during periods of arboviral outbreaks [ ] , and this needs to be taken into consideration for clinical trial design. the current covid- pandemic has placed immense pressure on health care and public health systems worldwide. covid- and dengue co-infections have been reported [ ] . the response to this pandemic unfortunately has diverted resources and finances; and pushed dengue vaccine development out of the international spotlight. a resurgence of dengue is a real threat during the covid- pandemic because the high burden of dengue related hospitalizations will further overwhelm already overwhelmed healthcare systems [ ] . the covid- pandemic therefore provides even more impetus to develop, license and roll out dengue vaccines for broader use. the first licensed dengue vaccine led to considerable controversy, and to date, no dengue vaccine is in widespread use. all three leading dengue vaccine candidates are live-attenuated vaccines, with the main difference between them being the type of backbone and the extent of chimerization. while cyd-tdv (the first licensed dengue vaccine) does not include non-structural proteins of dengue, tak- contains the dengue virus serotype backbone, and the butantan/merck vaccine contains three full-genomes of the four dengue virus serotypes. the four genetically succinct but still closely related dengue serotypes are known to interact immunologically with potential for disease enhancement. while dengueprimed individuals can already benefit from vaccination against all four serotypes with the first licensed dengue vaccine cyd-tdv, the need for dengue-naive population has not yet been met. to improve tetravalent protection, sequential vaccination should be considered in addition to a heterologous prime-boost approach. the ideal properties of a dengue vaccine should include the ability to induce long-lasting homotypic immune responses to all four serotypes in all age groups, regardless of dengue serostatus. the vaccine should have a schedule ideally with or fewer doses, should be able to prevent dengue outbreaks if used early at the onset of the outbreak, and should serve as prophylaxis in large populations to effectively prevent epidemics in the long term. aws wrote the review. none. severe dengue in travellers: pathogenesis, risk and clinical management dengue infection in international travellers visiting bali, indonesia imported dengue fever in east london: a -year retrospective observational study dengue fever among israeli expatriates in delhi, : implications for dengue incidence in delhi, india risk of severe dengue is higher in patients with sickle cell disease: a scoping review efficacy and long-term safety of a dengue vaccine in regions of endemic disease effect of dengue serostatus on dengue vaccine safety and efficacy long-term follow-up of all phase trial data globally including benefit risk assessment based on stratification based on baseline serostatus deliberations of the strategic advisory group of experts on immunization on the use of cyd-tdv dengue vaccine dengue virus ns protein activates cells via toll-like receptor and disrupts endothelial cell monolayer integrity dengue virus ns triggers endothelial permeability and vascular leak that is prevented by ns vaccination dengue virus ns cytokine-independent vascular leak is dependent on endothelial glycocalyx components dengue virus ns disrupts the endothelial glycocalyx, leading to hyperpermeability dengue vaccine: who position paper dengue prevaccination serology screening for the use of dengvaxia(r) cyd-tdv dengue vaccine performance by baseline immune profile (monotypic/ multitypic) in dengue seropositive individuals. clin infect dis . evaluation of several elisa and rapid diagnostic assays for determining prior dengue infection evaluation of rapid diagnostic tests and conventional enzymelinked immunosorbent assays to determine prior dengue infection pre-vaccination screening strategies for the use of the cyd-tdv dengue vaccine: a meeting report use of pre-travel vaccine-preventable disease serology as a screening tool to identify patients in need of pre-travel vaccination: a retrospective audit a: serostatus-dependent performance of the first licensed dengue vaccine: implications for travellers timing of administration of dengue vaccine in travellers with a recent confirmed dengue infection clinical development and regulatory points for consideration for second-generation live attenuated dengue vaccines a: evaluation of a tetravalent dengue vaccine by serostatus and serotype a recombinant, chimeric tetravalent dengue vaccine candidate based on a dengue virus serotype backbone immunogenicity and safety of one versus two doses of tetravalent dengue vaccine in healthy children aged - years in asia and latin america: -month interim data from a phase , randomised, placebo-controlled study immunogenicity and safety of lyophilized and liquid dengue tetravalent vaccine candidate formulations in healthy adults: a randomized, phase clinical trial characterization of the type-specific and crossreactive b cell responses elicited by a live-attenuated tetravalent dengue vaccine long-term safety and immunogenicity of a tetravalent dengue vaccine candidate in children and adults: a randomized, placebo-controlled, phase study efficacy of a tetravalent dengue vaccine in healthy children and adolescents efficacy of a tetravalent dengue vaccine in healthy children aged - years: a randomised, placebo-controlled, phase trial robust and balanced immune responses to all dengue virus serotypes following administration of a single dose of a live attenuated tetravalent dengue vaccine to healthy, flavivirus-naive adults human cd + t cell responses to an attenuated tetravalent dengue vaccine parallel those induced by natural infection in magnitude, hla restriction, and antigen specificity the human cd + t cell responses induced by a live attenuated tetravalent dengue vaccine are directed against highly conserved epitopes a -month-interval dosing study in adults indicates that a single dose of the national institute of allergy and infectious diseases tetravalent dengue vaccine induces a robust neutralizing antibody response paediatric dengue fever diagnosed through parents' epidemiologic report and preventive strategy during the acute phase of infection dengue in peace corps volunteers travel vaccine preventable diseases-updated logarithmic scale with monthly incidence rates what proportion of international travellers acquire a travel-related illness? a review of the literature prevention of enteric fever in travellers with typhoid conjugate vaccines perceptions of rabies risk: a survey of travellers and travel clinics from canada, germany, sweden and the uk pandemic yellow fever: a potential threat to global health via travelers recommendations for travellers during the yellow fever outbreaks in brazil- sentinel surveillance in travel medicine: years of geosentinel publications ( - ) illness among us resident student travellers after return to the usa: a geosentinel analysis business travel-associated illness: a geosentinel analysis geosentinel surveillance of illness in returned travelers illness in travelers visiting friends and relatives: a review of the geosentinel surveillance network dengue epidemic in touba, senegal: implications for the grand magal pilgrimage for travelers morbidity among israeli paediatric travellers fatal fulminant hepatitis a in a us traveller returning from peru japanese encephalitis: vaccine options and timing of pre-travel vaccination recent and historical trends in the epidemiology of japanese encephalitis and its implication for risk assessment in travellers what is the prospect of a safe and effective dengue vaccine for travellers? mosquito repellents for the traveller: does picaridin provide longer protection than deet? factors affecting pre-travel health seeking behaviour and adherence to pre-travel health advice: a systematic review sequential immunization induces strong and broad immunity against all four dengue virus serotypes combine dengue vaccines to optimize effectiveness this is an opinion paper that discusses heterologous prime-boost strategy by giving takeda's tak- first followed by sanofi pasteur's cyd-tdv status of current and under-development vaccines phase randomized study of a tetravalent dengue purified inactivated vaccine in healthy adults in the united states phase i randomized study of a tetravalent dengue dengue vaccine development wilder purified inactivated vaccine in healthy adults from puerto rico dengue, zika and chikungunya during pregnancy: preand post-travel advice and clinical management a: can dengue virus be sexually transmitted? dengue virus not detected in human semen incidence of guillain-barre syndrome (gbs) in latin america and the caribbean before and during the - zika virus epidemic: a systematic review and meta-analysis recombination of b-and t-cell epitoperich loci from aedes-and culex-borne flaviviruses shapes zika virus epidemiology comprehensive immunoprofiling of pediatric zika reveals key role for monocytes in the acute phase and no effect of prior dengue virus infection primary and secondary dengue virus infections elicit similar memory b-cell responses, but breadth to other serotypes and cross-reactivity to zika virus is higher in secondary dengue laboratory study that investigates the role of prior dengue infections on the immune response impact of pre-existing dengue immunity on human antibody and memory b cell responses to zika impact of preexisting dengue immunity on zika virus emergence in a dengue endemic region this paper discusses the immunological interaction between zika and dengue viruses co-infection between zika and different dengue serotypes during denv outbreak in brazil covid- and dengue co-infection in a returning traveller preventing dengue epidemics during the covid- pandemic nothing declared.disclaimer aws serves as consultant to the world health organization with regards to dengue vaccines. the views expressed in this article are those of the author and do not necessarily represent the decisions or policies of the world health organization. key: cord- -d j wpqk authors: kalita, parismita; padhi, adityak.; zhang, kam y.j.; tripathi, timir title: design of a peptide-based subunit vaccine against novel coronavirus sars-cov- date: - - journal: microb pathog doi: . /j.micpath. . sha: doc_id: cord_uid: d j wpqk coronavirus disease (covid- ) is an emerging infectious disease that was first reported in wuhan, china, and has subsequently spread worldwide. in the absence of any antiviral or immunomodulatory therapies, the disease is spreading at an alarming rate. a possibility of a resurgence of covid- in places where lockdowns have already worked is also developing. thus, for controlling covid- , vaccines may be a better option than drugs. an mrna-based anti-covid- candidate vaccine has entered a phase clinical trial. however, its efficacy and potency have to be evaluated and validated. since vaccines have high failure rates, as an alternative, we are presenting a new, designed multi-peptide subunit-based epitope vaccine against covid- . the recombinant vaccine construct comprises an adjuvant, cytotoxic t-lymphocyte (ctl), helper t-lymphocyte (htl), and b-cell epitopes joined by linkers. the computational data suggest that the vaccine is non-toxic, non-allergenic, thermostable, with the capability to elicit a humoral and cell-mediated immune response. the stabilization of the vaccine construct is validated with molecular dynamics simulation studies. this unique vaccine is made up of highly antigenic epitopes from three proteins that have a prominent role in host-receptor recognition, viral entry, and pathogenicity. we advocate this vaccine must be synthesized and tested urgently as a public health priority. severe acute respiratory syndrome coronavirus (sars-cov- ) belongs to the genus betacoronavirus of the coronaviridae family and is identified as the pathogen of coronavirus disease (covid- ) [ ] . the epicenter of the covid- coronavirus outbreak was the central chinese city of wuhan, from where it spread globally. on th january , the world health organization officially declared the covid- epidemic as a public health emergency of international concern. human to human transmission occurs through droplets, contact, and fomites. people with covid- show symptoms of fever, cough, muscle aches, headache, and diarrhea. the principal feature of the severe disease is acute onset of hypoxemic respiratory failure with bilateral infiltrates. the virus genome has been sequenced that allowed the development of diagnostic tests and research into vaccines and therapeutics [ , ] . a specific rt-pcr-based test has been developed that is in use for clinical diagnoses [ ] . the abundance of publications in the first three months of indicates the intensive scientific effort to address both molecular mechanisms and therapeutic routes for treating covid- [ ] . more than clinical trials are currently underway to test novel and repurposed compounds against sars-cov- [ , ] . certain drugs, including hydroxychloroquine, chloroquine, and remdesivir, are being tested in clinical trials [ ] [ ] [ ] . one small study reported that combination therapy of hydroxychloroquine with azithromycin reduced the detection of viral rna compared to control [ , ] . a recent openlabel trial with two protease inhibitors, lopinavir, and ritonavir, failed [ ] . several inactivated vaccines, viral vectored vaccines (adenovirus vector, ankara vector), nanoparticle-based vaccines, fusion-protein based vaccines, adjuvanted vaccines, recombinant protein, and dna vaccines, as well as live-attenuated vaccines, are also being developed and tested, but these vaccines are many months away from the market [ ] [ ] [ ] [ ] . a phase clinical trial of moderna's mrna-based sars-cov- candidate vaccine, mrna- , has started on march , [ ] [ ] [ ] . however, this is the first of several steps in the clinical trial process for evaluating the potential benefits of the vaccine. the sars-cov- consists of single, positive-stranded rna and four structural proteins: a spike glycoprotein (s), a membrane glycoprotein (m), an envelope protein (e), and a nucleocapsid protein (n) [ ] . to enter the host cells, the virus uses a densely glycosylated spike protein that binds to the angiotensin-converting enzyme (ace ) receptor with high affinity [ , ] . structural and biochemical studies suggest that the rbd has an ultra-high binding affinity to the human ace receptor [ ] . few groups have designed subunit vaccines against sars-cov- ; however, their workflow involved either use of single protein for vaccine design [ , ] or used only ctl epitopes without considering the importance of b-cell or htl epitopes [ ] . some subunit-vaccines are also in preclinical trials [ , ] . here, we focused on designing a multi-epitope-based subunit vaccine against sars-cov- using highly antigenic epitopes. we believe that experimental evaluation may result in a novel and immunogenic vaccine that may confer protection against sars-cov- infection. the protein sequences of sars-cov- were retrieved from the ncbi database (https://www.ncbi.nlm.nih.gov/nuccore/mn . /) for subunit vaccine development (table ) [ ] . each of these proteins was screened for their average antigenic propensity using the antigenic peptides prediction tool (http://imed.med.ucm.es/tools/antigenic.pl). proteins with an antigenic probability score of greater than . were considered for vaccine construction. the helper t-lymphocyte (htl) epitopes for the selected sars-cov- proteins were predicted using the mhc-ii epitope prediction tool from the immune epitope database (iedb, http://tools.iedb.org/mhcii/). selected epitopes had the lowest percentile rank and ic values. additionally, these epitopes were checked by the ifn epitope server (http://crdd.osdd.net/raghava/ifnepitope/) for the capability to induce th type immune response accompanied by ifn-ϒ production. cytotoxic t-lymphocyte (ctl) epitopes for the screened proteins were predicted using the netctl . server (http://www.cbs.dtu.dk/services/netctl/). b-cell epitopes for the screened sars-cov- proteins were predicted using the abcpred server (http://crdd.osdd.net/raghava/abcpred/). the prediction of the toxic/non-toxic nature of all the selected htl, ctl, and b-cell epitopes was checked using the toxinpred module (http://crdd.osdd.net/raghava/toxinpred/multi_submit.php). the vaccine subunit was designed by adding an adjuvant, htl, ctl, and b-cell epitopes connected by specific linkers to provide adequate separation of epitopes in vivo. eaaak linker was used to join the adjuvant and htl. intra htl, intra ctl, and b-cell epitopes were joined using gpgpg, aay, and kk, respectively. to enhance the immunogenicity of the vaccine construct, the tlr- agonist, human β-defensin (uniprot id: p ), was used as the adjuvant. the immunogenicity of the vaccine was determined using the vaxijen server (http://www.ddgpharmfac.net/vaxijen/vaxijen/vaxijen.html) and antigenpro module of scratch protein predictor (http://scratch.proteomics.ics.uci.edu/). the allergenicity of the vaccine was checked using allertop v . (http://www.ddg-pharmfac.net/allertop/) and algpred server (http://crdd.osdd.net/raghava/algpred/). the physiochemical characteristics of the vaccine were determined using the protparam tool of the expasy database server (http://web.expasy.org/protparam/). the secondary structure of the subunit vaccine construct was predicted using psipred . protein sequence analysis workbench (http://bioinf.cs.ucl.ac.uk/psipred/), while the tertiary structure was predicted by de novo structure prediction-based trrosetta modeling suite. trrosetta uses a deep residual neural network to predict the inter-residue distance and orientation distributions of the input sequence. then it converts predicted distance and orientation distributions into smooth restraints to build d structure models-based on direct energy minimization. the model of the vaccine construct with the best tm-score was validated by prochek v. . structures were saved at every ps for structural and dynamic analysis. the analysis from md simulation was performed as described earlier [ ] . briefly, the backbone java codon adaptation tool (jcat) (http://www.jcat.de/) was used for codon optimization of the vaccine sequence to test high-level expression of the vaccine in e. coli strain k . nebcutter (http://nc .neb.com/nebcutter /) was used for the selection of restriction enzyme cleavage sites, and the expression vector pet a(+) was selected. in silico clone of the vaccine was designed using the snapgene . . restriction cloning tool. the amino acid sequence of the three sars-cov- proteins, namely, nucleocapsid protein, membrane glycoprotein, and surface spike glycoprotein, were retrieved from the ncbi database (table ) . these proteins are known to have a prominent role in host receptor recognition, viral entry, and pathogenicity. the proteins with an antigenic score of greater than . (table ) were used further for the prediction of epitopes for subunit vaccine designing. a schematic representation of the methodology for the construction of the subunit vaccine candidate is shown in figure . helper t-lymphocytes are the key players of the adaptive immune response. they are involved in the activation of b-cells and cytotoxic t cells for antibody production and killing infected target cells, respectively. all three proteins were subjected to the iedb mhc-ii epitope prediction module for htl prediction. a total of six highest immunogenic epitopes of -mer were selected based on their percentile rank and ic values. also, all these epitopes showed positive scores on ifnepitope server output (table ) . b-cells are the main components of humoral immunity during the adaptive immune response that produces antibodies, which recognize antigens. therefore, it was necessary to predict b-cell epitopes before vaccine designing. abcpred was performed for predicting b-cell epitope, and a total of epitopes with top scores from the three proteins were considered for the vaccine (table ) . ctl epitopes are essential for inducing mhc-i cellular immune response by neutralizing virus-infected cells and damaged cells via releasing cytotoxic proteins like granzymes, perforins, etc. the ctl epitopes were predicted for all selected proteins using the netctl . server. here, a , a , and b supertypes were considered for prediction as they cover at least . % of the total ethnic population. eighteen epitopes with a combined score of > . were finally considered for the vaccine (table ). all the selected htl, ctl, and b-cell epitopes were subjected to the toxinpred module to screen for their toxicity. supplementary table shows that all epitopes chosen for the vaccine were non-toxic. a total of htls, ctls, and b-cell epitopes derived from the three proteins were used to design the subunit vaccine ( amino acid residues) against sars-cov- (supplementary figure ) . the human β-defensin ( amino acid residues) sequence was added as an adjuvant followed by the htl, ctl, and b-cell epitopes and linked by specific linkers. an important parameter of vaccine designing is ensuring that the constructed vaccine is immunogenic to induce a humoral and/or cell-mediated immune response against the targeted virus. the computational data suggest that our vaccine is antigenic with a probability score of . and . predicted by vaxijen v . and antigenpro servers, respectively. the allergenicity score was found to be − . in algpred prediction module. additionally, the vaccine was also found to be non-allergic using allertop v . . respectively, suggesting that the vaccine is thermostable and hydrophilic, respectively. the secondary structure was predicted using the psipred . server (supplementary figure ) . the tertiary structure of the vaccine was predicted using the trrosetta modeling suite. the d model generated by trrosetta modeling was subjected to the procheck server, where ramachandran plot statistics were generated. the output showed . % residues were present in the favored region, . % residues in the generously allowed region, and . % residues in disallowed regions. further, the z-score plot and energy plot was generated by the prosa web server. the calculated z-score (− . ) lies within the x-ray crystal structure range. the energy plot suggested that all the residues have low energy value in the modeled structure ( figure ). vaccine-receptor docking was performed to evaluate the binding energy of the vaccine with its tlr- receptor. cluspro analysis provided vaccine-receptor complexes with respective energy scores. the lowest energy complex with a binding energy of about − kj.mol - was selected and subjected to md simulation ( figure ). the binding modes, dynamics, and stability of the vaccine-tlr complex were evaluated using a ns md simulation study (figure ) . the atomic-level interaction between vaccine and tlr was determined, and root mean square deviation (rmsd), root mean square fluctuation (rmsf), radius of gyration (rg), hydrogen bond, and contact energy were calculated. the rmsd data suggests that the receptor-vaccine complex was stabilized after about ns until the end of the simulation ( figure a ). all the calculations were then done for the - ns md simulation trajectory. next, rmsf calculation, which gives information about the residue wise dynamics of a protein with respect to its initial position, was done. an average rmsf value of . nm was observed for the complex ( figure b ). our subsequent analysis of the changes in the rg for the vaccine-tlr complex during the simulation was also determined, and the average rg was found to be . nm, demonstrating the compactness of the tlr receptor with vaccine subunit during the simulations ( figure c ). we further analyzed the hydrogen bonds that typically, the largest associated eigenvalues define the essential subspace in which most of the protein dynamics occur. for this, the clusters of stable states of pca for the tlr -vaccine complex were visualized and analyzed. the trace value calculated from the covariance matrix of the tlr -vaccine was found to be . nm , suggesting that the complex exhibited compact behavior during the simulation (supplementary figure ) . lastly, the detailed interactions between tlr and the vaccine protein were computed from the starting structure of md simulations and the stabilized structure of the complex extracted from md simulated trajectory ( figure and table ). the higher total number of interactions in the stabilized complex suggests the stability and tighter binding of the vaccine with tlr . the codon optimization index ensures the relationship between codon usage and gene expression in a heterologous system. the jcat output was further analyzed in nebcutter, and at the n-and c-terminal ends of the optimized vaccine sequence, bamhi and ndei restriction sites were added that are non-cutters for the vaccine construct but are present in the multiple cloning site of the selected expression vector pet a(+). in silico clone was generated using the snapgene . . restriction cloning tool that resulted in a cloned product of bp ( figure ). some reports suggest that to % of recovered patients in wuhan test positive again; this indicates a possibility of a resurgence of covid- in places where lockdowns have already worked. as a consequence, the spread can also be caused by asymptomatic carriers [ ] [ ] [ ] . a positive re-test, however, may also be because the original test was false-negative, and the patient was not actually covid-negative. whatever may be the case, a vaccine is a better option for coronavirus management than drugs. the efforts to produce a vaccine against coronavirus are moving at a rapid pace. two candidate vaccines are in phase i clinical trials: i) an adenovirus type- vector-based vaccine, and ii) an lnp-encapsulated mrna vaccine. studies evaluating the safety and immunogenicity of these vaccines are underway. additionally, several vaccine candidates are under preclinical evaluation [ ] . though these trials are underway, there are known situations that vaccines have failed. recently few groups have tried designing subunit vaccines against sars-cov- ; however, their workflow involved either use of single protein for vaccine design [ , ] or used only ctl epitopes without considering the importance of b-cell or htl epitopes [ ] . we considered all of these points while designing the vaccine. based on extensive bioinformatics analysis, we used three proteins to design a multi-epitope subunit vaccine against novel coronavirus sars-cov- . these proteins are nucleocapsid protein (n), membrane glycoprotein (m), and the surface spike glycoprotein (s). the n protein is involved in packaging the viral genome into a helical ribonucleocapsid, and it plays a fundamental role during viral self-assembly [ ] . the m protein is responsible for the assembly and immunogenicity of virus particles. the s protein mediates the entrance of the virus to human respiratory epithelial cells by interacting with cell surface receptor ace . the s protein has two regions: s , for host cell receptor binding; and s , for membrane fusion. the s protein is a key target for the development of vaccines, therapeutic antibodies, and diagnostics for coronavirus [ , , ] . although the s protein is a promising immunogen for protection, optimizing antigen design is critical to ensure an optimal immune response. our vaccine contains a suitable adjuvant, htl, ctl, and b-cell epitopes that are joined by suitable linkers. furthermore, the epitopes were screened for their toxicity potential. the subunit vaccine was found to be thermostable, antigenic, and non-allergenic. molecular docking and md simulation provided insights about the interaction, stability, and dynamics of the vaccine-receptor complex. the data suggest constructive intermolecular interactions between the vaccine protein and the tlr- receptor. also, the in-silico cloning suggests the potential expression of the vaccine in a microbial expression system, thereby making it a potential vaccine against sars-cov- infection. the development of a vaccine is a lengthy and expensive process, with high failure rates, and it typically takes multiple candidates and several years to produce a commercial vaccine. upon optimization of the production process, the subunit vaccines can be rapidly tested and released in the market. they consist of only the antigenic portion of the pathogens that may directly elicit an immune response. additionally, the vaccine does not utilize live pathogen, thus, reducing the risk of pathogenicity reversal. hence, it can be used in immune-suppressed patients as well and elicit long-lived immunity. computational studies suggest that our multi-epitope based subunit vaccine has a probability of showing good protective efficacy and safety against sars-cov- infection in humans. we suggest the synthesis and experimental evaluation of this vaccine to determine its immunogenic potency. the authors acknowledge riken accc for the hokusai supercomputing resources. a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars-cov- early diagnosis of sars coronavirus infection by real time rt-pcr research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases network-based drug repurposing for novel coronavirus -ncov/sars-cov- ongoing clinical trials for the management of the covid- compounds with therapeutic potential against novel respiratory coronavirus hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro information for clinicians on therapeutic options for covid- patients hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial a trial of lopinavir-ritonavir in adults hospitalized with severe covid- the sars-cov- vaccine pipeline: an overview sars vaccines: where are we? immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic the covid- vaccine development landscape nih clinical trial of investigational vaccine for covid- begins the pandemic pipeline coronaviruses: an overview of their replication and pathogenesis structure of sars coronavirus spike receptor-binding domain complexed with receptor structural basis for the recognition of the sars-cov- by full-length human ace characterization of the receptorbinding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine design of multi epitope-based peptide vaccine against e protein of human -ncov: an immunoinformatics approach development of epitope-based peptide vaccine against novel coronavirus (sars-cov- ): immunoinformatics approach t cell epitope-based vaccine design for pandemic novel coronavirus -ncov the first coronavirus drug candidate is set for testing in china clover initiates development of recombinant subunit-trimer vaccine for wuhan coronavirus a pneumonia outbreak associated with a new coronavirus of probable bat origin development of multi-epitope driven subunit vaccine against fasciola gigantica using immunoinformatics approach mystery in wuhan: recovered coronavirus patients test negative then positive some recovered coronavirus patients in wuhan are testing positive again. slashdotmedia we need to be alert: scientists fear second coronavirus wave as china's lockdowns ease who. draft landscape of covid- candidate vaccines - t cell epitope-based vaccine design for pandemic novel coronavirus -ncov transient oligomerization of the sars-cov n protein--implication for virus ribonucleoprotein packaging the spike protein of sars-cov--a target for vaccine and therapeutic development immunogenicity of candidate mers-cov dna vaccines based on the spike protein the authors have declared no competing interest. pk and akp carried out the experiments. pk and tt conceived the study and participated in its design and coordination. pk, akp, kyjz, and tt analyzed the data and drafted the manuscript.all authors read and approved the final manuscript. figure : schematic representation of the multi-epitope subunit vaccine candidate designing using b-cell, ctl, and htl epitopes. • we present a multi-epitope subunit-based vaccine designed using an integrated immunoinformatics approach. • our vaccine is made up of highly antigenic epitopes from three vital pathogen proteins.• computational data predict that the vaccine is non-toxic, non-allergenic, and immunogenic.• an experimental evaluation of this vaccine is required to determine its practical immunogenic potency. the authors have declared no conflict of interest. key: cord- -l dehit authors: diaz-arévalo, diana; zeng, mingtao title: nanoparticle-based vaccines: opportunities and limitations date: - - journal: nanopharmaceuticals doi: . /b - - - - . - sha: doc_id: cord_uid: l dehit infectious diseases are the tip of the iceberg in the economic burden of the developing countries, due to the resistance of the pathogens to antibiotics and the lack of vaccines. the vaccines have become a big challenge in the last decades, where the attention has been focused on scientific challenges such as new vaccine development and adjuvants or delivery systems. the classical vaccines were developed from live-attenuated or killed organisms, such as influenza, smallpox, and bcg, as well as subunits such as hepatitis b. the attenuated vaccines carry the risk of regaining their pathogenicity under immunosuppression conditions. the development of subunit vaccines without risk are considered as an essential need in combination with adequate delivery systems to obtain desired cell and humoral immune responses against infectious diseases. in the last decades, the use of nanoparticles as a delivery system in vaccines has received special attention to improve vaccine efficacy. these nanoparticles could be composed of lipids, metal and nonmetal inorganics, several polymers, and virus-like particles, which have been tested in research; some of them have already been approved for human and animal use. the characteristics of the nanoparticles have allowed targeting desired antigen-presenting cells to improve immunization strategies to induce protection. the main characteristics of the nanoparticles are to protect the antigens from early proteolytic degradation, control antigen release, and help antigen uptake and processing by antigen-presenting cells, and they should be safe for human and veterinary use. in addition, the nanoparticles could be modified in their physicochemical properties to target specific cells and improve vaccine efficacy. this chapter focuses on the nanoparticle-based vaccine formulations and the approaches used to realize efficient delivery of vaccines in order to induce host protective immunity against infectious diseases. nanomedicine has seen increased interest by the medical community in recent years, as it has enabled modification of engineering devices for delivery to and interaction with cell environments. in particular, this technology has enabled advances in the design of delivery systems for drugs and vaccines [ ] . nanoparticles (nps) are now being engineered from different biodegradable materials, including natural and synthetic polymers (poly[lactic-co-glycolic] acid (plga) and polylactic acid (pla)), metals (gold, copper oxide, aluminum oxide, zinc oxide, iron oxide, and silver), or lipids (phosphatidylserine, phosphatidylcholine, and cholesterol) [ ] . nps and microparticles (mps) have been widely used to deliver drugs, especially cytotoxic drugs or immune-suppression treatments for transplantation. these nps are effective in the distribution of medications to target specific organs and control drug delivery. these drug carriers not only transport drugs to sites of cancer or other target diseased organs, preventing damage to healthy cells, but also protect the drug from degradation [ ] . some of the metal nanoparticles (e.g., gold, silver, or plga) themselves may have dual functions, working both as carriers and as targeted delivery systems using the membranes of cancer cells, which contain tumor-associated markers. in a recent study, fang et al. used a membrane from mouse melanoma cancer cells in the outer layer of plga nanoparticles. these nps were stable and persisted in the structure during cellular endocytosis, activating the maturation of dendritic cells (dcs) and presenting especially to t cells that have tcr binding to gp and inducing the production of ifn-g. moreover, the authors found that the plga covering membrane had receptors that allow interaction with cancer cells and delivery of the drug [ , ] . furthermore, the nps were used to deliver drugs to the specific site of organ transplantation to prevent rejection. systemic immunosuppression is a high risk for recipients of organ transplantation, as it uses high doses of immunosuppression agents that make them more susceptible to infections and in some cases cause death. previous studies found that diabetic mice were transplanted with islets into the eye to prevent islet graft immune rejection in vivo using rapamycin mps. the islets transplanted with the immunosuppressed drug mps survived more than month compared with the control (empty mps), which rejected the islets in the second week [ ] . work in last few decades has increased our knowledge of infectious diseases and the mechanisms of evasion of immune responses. however, new variants of antibiotic-resistant pathogenic microorganisms have emerged and are becoming a challenge for designing new vaccines and adjuvants. until now vaccines have been developed from live-attenuated microorganisms or killed pathogens (first-generation vaccines) [ ] , dna vaccines (third-generation vaccines), subunit vaccines [ ] , and synthetic peptides (second-generation vaccines). the last three vaccine types eliminate the risk of developing the disease, but they must be used in conjunction with an adequate adjuvant or delivery system (table . ). the combination of the vaccine with the adjuvant or delivery system should be safe, stable, and have the ability to induce long-lived memory b and t cell responses, preferably with a single dose and a maximum of two doses and be free from strict storage requirements [ ] . dna and rna vaccines are safe but need a second boost with recombinant protein or dna from another vector. nps have become an alternative to targeting vaccine delivery to immune cells, improving vaccine efficacy with slow release, easy antigen uptake, and induction of humoral and cellular responses [ ] . nps have played an important role in the activation of antigen-presenting cells (apcs), especially dcs, which may determine vaccine efficacy. although there is some cytotoxic effect of the nps [ , ] , the risk is low compared with the benefits of vaccine delivery [ ] . in this chapter, we will summarize the different nanocarrierbased vaccine formulations that achieve the desired host immunity against infectious diseases and cancer, and at the end, we will discuss on the limitations of the respective carriers. dcs are specialized apcs that coordinate the innate and adaptive immune responses to until now there have been various types of nps, composed of gold, dendrimers, carbon, polymers, and liposomes, that have been used to deliver vaccines. all can stimulate the production of cytokines and antibody responses [ e ]. the cargo is not limited to vaccines, and it is possible to add adjuvants and immune stimulatory molecules, including silica and iron, tlr agonist, and cytokines, to improve immunogenicity [ , ] . several vaccines have been tested on the different types of nps (table . ). liposomes are the second-most common type of np, and they self-assemble in water under special conditions. they are composed of lipids, which have a hydrophilic head and a hydrophobic tail that maintain hydrophilic inner and outer membranes, in lamellar lipid bilayers or in multilayers that simulate vesicles found within cells [ ] . liposomes can induce cellular responses or humoral responses, depending on the charge, size, and lipid composition. a previous study showed that charge had the main role in activation of the cellular or humoral immune responses. the authors' approach was to investigate the importance of the antigeneliposome interaction in immunogenicity and depot formation. they used subunit antigens ag be esat- (pi ¼ . ) from tb, and "cth " (pi ¼ . ), from chlamydia vaccines and a model antigen, lysozyme (pi ¼ ). the injection of cationic dimethyldioctadecylammonium using np platforms such as liposomes, emulsions, nanogels, and other substances. (b) nanovaccines can access the lymphatic drainage system for lymph node delivery while protecting their cargoes from environmental degradation. once at the lymph nodes, the nanocarriers deliver their cargoes to apcs for immune processing. (c) nanovaccine properties can be tuned to efficiently deliver their cargoes for maximum immune activation. for example, nps can be modified to target specific subsets of immune cells. they can also be delivered to specific intracellular compartments, where receptors for immune pathways can be triggered. to migrate to the secondary lymph nodes to activate the t and b cells, which the authors did not analyze in this manuscript [ ] . previous studies have shown that dda induces proinflammatory cytokines and chemokines after infiltration of monocytes and macrophages in vivo [ ] . one of the advantages of liposomes is mimicking properties of the pathogens, inducing humoral and cellular immune responses. the antigen's presentation to apcs depends on the membrane characteristics, size, and ligand-receptor binding. liposomes could induce th responses if the lipids are unsaturated whereas saturated lipids promoted th -type immune responses [ , ] . liposomes could be modified according to the needs of delivery to produce th , th , th , or tfh immune responses. cationic liposomes can increase the uptake of subunit vaccines as synthetic peptides and recombinant proteins; this can be explained by the interaction between positively charged liposome with the apcs that have a negative charge in their membrane [ , ] . another modification is the ph-sensitive fusogenic liposomes, which are stable in neutral ph . , but in acidic conditions, the antigen is released and presented by mhc class i or ii. these liposomes containing phosphatidylethanolamine and amphiphilic stabilizers allow pe-containing liposomes to form aggregates, due to the poor hydration of their headgroups, which can explain their high affinity to adhere to cell membranes [ , ] . another study designed a unique structure composed of inter-bilayer-crosslinked multilamellar vesicles (icmvs), which is stable in the extracellular environment but rapidly released in endosomes/lysosomes, thereby enhancing vaccine immune responses. the icmvs carried the antigen ova mixed with the adjuvant monophosphoryl lipid a (mpla). this mixture amplified vaccine responses and upregulated the costimulatory cells on splenic and bone marrow dcs. in addition, splenic dcs incubated with the ova þ vaccine mpla triggered proliferation of na € ive ot- cd þ t cells in vitro, suggesting that the icmvs enhanced cross-presentation of the antigen. the same results were obtained in vivo after the vaccination of mice with the mixture, and the icmvs elicited robust antibody titers that were w -fold greater than simple liposomes. these results could be attributed to activation enhancement of dcs and antigen cross presentation [ ] . the first use of liposomes as a delivery system for a malaria vaccine was in the s. ballou et al. synthetized peptides derived from the repetitive region of the circumsporozoite (cs) protein of plasmodium falciparum sporozoites. the synthetic peptides were conjugated to keyhole limpet hemocyanin (klh) proteins and were incorporated into liposomes. immunized mice and rabbits produced antibodies against the repeat region of the protein with biologic activity correlated with protection [ ] . rts, s is the only vaccine against malaria that is in phase three clinical trials in africa. the rts, s vaccine has the central repeat region of csp, and t cell epitopes localized in the c-terminal region are fused to hepatitis b surface antigen (hbsag) and expressed in saccharomyces cerevisiae yeast. this virus-like particle (vlp) vaccine also contains mpla and qs- , which enhances humoral and cd þ t cell responses in the first years, with a level of protection of %e %. protection decreased rapidly thereafter, with negative efficacy in some children. the problem is not rts, s nor the adjuvant system, as both induce potent immune responses. however, the csp part of rts, s is an antigenic polymorphism, and t cell epitopes present on the cs protein that are incorporated into the vaccine are also polymorphic. to address this problem, the newest generation of this vaccine is coformulated with matrix-m, a saponin-based liposomal adjuvant [ ] . however, the vaccine protection against p. falciparum has not been achieved due to the short-lived memory cells and the evasion mechanisms of the parasite [ ] . the first malaria vaccine was tested in in animals using plasmodium berghei s radiation-attenuated sporozoites, with a % of protection [ ] . hoffman et al. reported in that human volunteers were vaccinated with irradiated sporozoites of p. falciparum strain nf , and all of them were protected between to weeks [ ] . this vaccine will be used to vaccinate , children a year in three african countries (ghana, malawi, and kenya). the vaccine could prevent in cases according to previous clinical trials. virus-like particles (vlps) are composed of a self-assembling viral membrane maintaining viral surface proteins. vlps can be modified to express additional proteins of other microorganisms, which could be engineered by fusion of the proteins with membrane antigens or by endogenous expression of other antigens [ ] . gardasil is a four-component vlp-type vaccine specific against hpv. it contains the l major capsid protein of hpv types , , , and and is administered along with an aluminum adjuvant. this vaccine was administered to women and was followed up for incidence of persistent associated infection for months. the efficacy was % for preventing clinical disease. the hpv combination vaccine was immunogenic, inducing the production of long-lived antibodies [ ] . however, some problems with the vaccine in teenage girls were reported. the adverse events included dermatologic/ mucosa-allergic reaction ( %), rash ( %), and local/injection-site reaction ( %). however, some serious adverse events following immunization were reported ( . % of reports), including two incidents of anaphylaxis, two seizures, one incident of thrombocytopenia, and one death [ ] . inorganic metal nps are frequently used in drug delivery and bioimaging, especially in treating cancer patients. dna vaccines are more stable and protected from degradation when carried by a gold, silica, or silver np delivery system. the covalent attachment of chito to gnps increases the np molecular weight, enhancing dna binding and stability without compromising dna release and transfer. chito egnpedna (hbsag) complexes induce effective antibody and t cell responses after immunization of balb/c mice. by contrast, naked dna-primed hbsag induces antibodies after a series of four immunizations with mg of naked dna. hbsag-specific cd þ t cells eliminated p /balb target cells that had been sensitized with an hbsag ctl epitope peptide in vitro. these chimeric nps, employing a minimal amount of dna, induce effective immune responses when compared with naked dna [ ] . biodegradable polymers are of significant interest in the delivery of drugs and vaccines against infectious diseases. these polymers consist of either natural or synthetic monomers that are biodegradable, are nonimmunogenic, have low cytotoxicity, and are easy to prepare. there are several polymers, such as chitosan, plga, polyethylene glycol (peg), polycaprolactone, and dextran used as delivery systems [ ] . plga is approved for human use by the us food and drug administration (fda) and european medicines agency (ema), while plga has been used to deliver drugs for long periods. dcs play an important role in the activation of adaptive immune responses, in which b cells and t cells activate and differentiate into subpopulations that determine which humoral or cellular immune responses eliminate the microorganism. cruz et al. studied the delivery of nps and mps to dcs to create a biocompatible and biodegradable slow-release vaccine and effectively target the cells. the nps and mps (plgaepeg) were loaded with tetanus toxoid peptideefitc linked to a lyselys cathepsin cleavage site and an anti-dc-sign antibody. the dcs targeted plgabased vaccine nps but not mps. the nps were efficiently taken up with the help of anti-dc-sign antibodies and induced proliferation of t cells (fig. . ) . however, the mps were taken up for all apcs, including the dcs (fig. . ) . new research is needed to understand the biology, antigen processing, and presentation needed to induce long-lived memory b and t cells [ ] . botulism is a lethal neuroparalytic disease produced by clostridium botulinum toxins (a-h). ruwona et al. demonstrated that cationic plga nps can carry plasmid dna encoding the bont heavy-chain (hc) fragment and that its product is nontoxic. immunized mice produced high titers of antibodies after to weeks (fig. . ) . after four immunizations, specific igg had decreased, but igg a had increased, compared with the first immunization. after challenge, % of the mice vaccinated with plgae pvax/opt-bont/c-hc survived, while only % of the mice immunized with naked plasmid were protected (fig. . ) [ ] . modifications of polymeric nps have been used to deliver synthetic peptides or recombinant proteins to dendritic cells and macrophages. salvador et al., modified plga ms, : lactideeglicolide ratio, to produce cationic nps using polyethylene imine (pei), and bsa as antigen. in addition, the nps were modified encapsulating monophosphoryl lipid a (mpla) or polyinosinic-polycytidilic acid poly(i:c) and a-galactosyl ceramide to address their adjuvant effect. coumarin was incorporated into the oily phase for the preparation of fluorescent nps to evaluate in vitro assays. the nps were tested in vitro using monocytes and dendritic cells, and mice were immunized with the different np modifications to determine the immune responses in vivo. the cationic nps showed a noticeable enhancement of their uptake by the monocytes, mdcs, and pdcs in comparison to the classical nps. in addition, the cationic nps increased immunostimulatory effect since they induced the production of antibodies and th cell responses producing ifn-g [ ] . the interest of companies in nanoparticlebased vaccine delivery dramatically increased in the last decades. pevion biotech ltd., a swiss company founded in , developed the virosome-based technology platforms to make efficient and safe prophylactic/therapeutic vaccine candidates. for candida albicans that can cause vulvovaginal mucosal infections, pevion used the aspartyl-proteinase (sap ), which is an immunodominant antigen and virulence factor; this recombinant protein was assembled with virosomes (pev ). the evaluation in mouse model and the initial clinical trial on women showed that this candidate vaccine, intravaginally administered, has a therapeutic potential for the treatment of recurrent candidiasis [ ] . in addition, this company tested a malaria vaccine in africa. this vaccine had combination of ffm me-trapþpev a (ama- ) and was tested in phase i/iia clinical trials. although the vaccine did not show sterile protection, it induced responses on blood stage parasites and lower rates of parasite growth in human volunteers vaccinated with pev a, compared to unvaccinated controls [ ] . glaxos-mithkline plc is working to improve the coformulated rts, s vaccine using matrix-m, a saponin-based liposomal adjuvant [ ] . novavax is developing respiratory syncytial virus (rsv) f nanoparticle vaccine with aluminum. the purpose of the reported study was to evaluate the safety and efficacy of maternally transferred antibodies in preventing rsv in infants. the vaccinated healthy third-trimester pregnant women showed significant protection to newborn children from rsv challenge and reduced pulmonary inflammation, and the vaccine was safe and effective for maternal and . nanoparticle-based vaccines: opportunities and limitations adult vaccination [ ] . furthermore, this company is working in recombinant trivalent nanoparticle influenza vaccine with matrix m- . the vaccine can induce responses to one or more conserved ha head and stem epitopes. the antibody responses are able to neutralize against both homologous and heterologous strains [ ] . the nanoparticles have been used in recent years in only a few of advanced clinical trials. the vaccine against rsv infection has three studies using rsv f nanoparticle vaccine with aluminum in phase and trials. the first clinical study of rsv was rsv f dose-ranging study in women, which started october and finished may . the study showed that all formulations were well tolerated, without treatment-related serious adverse events. antibodies anti-f igg and palivizumab-competitive antibody responses were correlated and increased after both doses, while microneutralization assays increased significantly after the first dose, then plateaued [ ] . the second study was rsv f vaccine maternal immunization study in healthy third-trimester pregnant women, which started september and finished june . this study demonstrated that the vaccine is safe to infants and pregnant women. the third clinical study is in phase three trials and will finish in july . the vaccine is safe and effective for infants and pregnant women [ ] . the study "evaluation of the safety and immunogenicity of a recombinant trivalent nanoparticle influenza vaccine with matrix m- adjuvant (nanoflu)" was used to test protection in people older than age , but no results have been publicly released. the other study is the phase two trial entitled "dose and formulation confirmation of quad-niv in older adults." in this study, subjects were randomized to seven treatment groups to receive the vaccine or an active comparator. table . summarizes the clinical trials [ ] : phase ieiv clinical trials/vaccines/nanoparticles. engineered nps have revolutionized the delivery of drugs, monoclonal antibodies, and vaccines to target cells. nps have been designed from metals and nonmetals, polymeric materials, lipids, vlps, and bioceramics, giving distinctive physicochemical and electrical characteristics that specifically interact with a targeted cell or organ. however, nps can enter easily into the human body by crossing lipid bilayers and may interact with sensitive organs [ ] . the clearance and excretion of nps is mediated through the mononuclear phagocytic system, the renal urinary system, and by biliary clearance. macrophages phagocytize the nps and keep them in the secondary lymph organs and/or liver using the mertk (mer) receptor (the same receptor tyrosine kinase family as axl and tyro- ), which is responsible for promoting apoptotic cell engulfment and supports platelet aggregation and clot stability in vivo [ ] . in addition, kupffer cells, which are liver macrophages, sequester -nm nanoparticles [ ] . when the particles are nm in diameter, they are rapidly cleared from the circulatory system via renal filtration [ ] . on the other hand, some studies have shown that metallic nps (menps) have effects on innate immunity. specifically, in vitro assays showed that menps have some cytotoxicity and genotoxicity and interfere with cytokine production and gene expression due to receptor modifications. the response of the innate immune system to threats is mediated by inflammation and the production of cytokines, chemokines, free radicals (nitric oxide) [ ] , and other reactive oxygen species (ros). in this process, there is an accumulation of oxidized glutathione (gssg), generating stress from the proinflammatory signal (involving tlrs) on the menps and causing cell death and cancer [ ] . other studies have shown that adenovirus vlps combine with a gene to control gelsinger's ammonia metabolism (encoding ornithine transcarbamylase), which invades all the organs and induces severe reactions that can lead to death [ ] . other severe side reactions have been reported as carcinogenesis or germline alterations observed in animal models, in which the vlps can become widely dispersed in the body, and the viral vector could end up in the ovaries and testes [ ] . eudragit is a drug delivery polymer: poly (ethyl acrylate-comethyl methacrylate-cotrimethylammonioethyl methacrylate chloride) : : . . it has been reported that using nr macrophages cocultured with np/ers, the nps were close to the inner membrane of the mitochondria, as observed by transmission electronic microscopy. the authors also analyzed genes responsible for mitochondrial function by microarray and found that opa was reduced in expression. this gene helps to maintain network morphology and dynamics and in the regulation of the signaling pathways for cell death, and np/ers-treated cells reduced glutathione (gsh), stimulating ros production. due to unbalancing of oxidanteantioxidant homeostasis and changes of proteins implied in activation and autophagy, the mitochondria decay through phagophore and autophagosome formation, and mitophagy can occur [ ] . the nps are characterized for their size (< nm) with greater surface area per mass compared with larger-sized particles of the same chemistry causing nps more active biologically. the physicochemical properties of the material could increase the uptake by the antigen-presenting cells, but they can go to other tissues that can generate adverse biological effects such as apoptosis or necrosis. there are some materials that can be degraded easily. however, the nps (plga) had been modified to release antigens slowly to immunize only once and to increase the efficiency of protective adaptive immune responses. nevertheless, intranasal immunization with nps had been reported to cause lung injury through oxidative stress, which induced the production of cytotoxic cellular responses and inflammatory cytokines. another problem with the nps is the aggregation that may block the blood vessels in the host. modifications of polymeric nps with pei have been used to prevent the recognition to other cells such as epithelial cells in the lung. other technologies could be used as the singlechain fragment region of antibodies binding to nps, which recognizes the specific receptor on dendritic cells or macrophages that may deliver the antigens to target organ. other modifications in the nps have been used as plga covered with peg (plgaepeg) and formulated with tetanus toxoid peptideefitc linked to a lyse lys cathepsin cleavage site and an anti-dc-sign antibody, which help the delivery to dcs but not macrophages, with enhanced efficient proliferation of t cells [ ] . negatively charged nps prevent the activation of the immune system and induction of immunotolerance, therefore preventing the effect of exacerbated inflammatory responses. nanoparticles can also be engineered to be porous to increase the diffusion of intracellular proteases, resulting in earlier processing of apcs and presentation t cells [ ] . in addition, some nps carrying antigen and adjuvant in the same np are less efficient to induce the immune response than the ones with adjuvant separated from antigens in different nps. this may because of the competing effect of coactivation in several pathways [ ] . nps are widely explored in vaccine delivery and targeting apcs, especially dcs. despite advances in immunology, it is critical to understand the mechanisms of entry of nps into cells and activation of the adaptive immune response and related toxicity. some approaches inhibit the cytokine and chemokine cascade with dcs and t and b cells. nps have great potential in immunology, although in-depth study of toxicity is necessary. in addition, nps require molecules, such as antibodies or ligands, that bind to receptors on target cells to ensure that nps only adhere to those cells. any modification should be tested in cell lines or animals to prevent side effects or death. the synthesis of nps can be shifted to methods that generate reproducible nanoparticles in terms of size (< nm, allowing elimination by the kidney), shape, and composition (biodegradable materials) to reduce cytotoxicity. dna mediated vaccines delivery through nanoparticles nanoparticle vaccines against infectious diseases a ph-responsive mesoporous silica nanoparticlesbased multi-drug delivery system for overcoming multi-drug resistance cancer cell membrane-coated nanoparticles for anticancer vaccination and drug delivery biomimetic nanoparticle vaccines for cancer therapy local release of rapamycin by microparticles delays islet rejection within the anterior chamber of the eye influenza vaccination strategies: comparing inactivated and live attenuated influenza vaccines single low-dose un-adjuvanted hbsag nanoparticle vaccine elicits robust, durable immunity vaccine development strategies for improving immunization: the role of modern immunology cytotoxicity assessment of heparin nanoparticles in nr macrophages drug delivery by polymeric nanoparticles induces autophagy in macrophages design and evaluation of surface and adjuvant modified plga microspheres for uptake by dendritic cells to improve vaccine responses designing vaccines based on biology of human dendritic cell subsets the who, how and where of antigen presentation to b cells eleven years of inflexalÒ vda virosomal adjuvanted influenza vaccine potent functional immunogenicity of plasmodium falciparum transmission-blocking antigen (pfs ) delivered with nanoemulsion and porous polymeric nanoparticles induction of protective neutralizing antibody responses against botulinum neurotoxin serotype c using plasmid carried by plga nanoparticles designing improved poly lactic-co-glycolic acid microspheres for a malarial vaccine: incorporation of alginate and polyinosinicepolycytidilic acid intranasal delivery of cationic plga nano/ microparticles-loaded fmdv dna vaccine encoding il- elicited protective immunity against fmdv challenge nanoparticle-based vaccines: opportunities and limitations towards programming immune tolerance through geometric manipulation of phosphatidylserine administration routes affect the quality of immune responses: a crosssectional evaluation of particulate antigen-delivery systems nanoparticles as synthetic vaccines liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a cmi response t-helper and t-helper adjuvants induce distinct differences in the magnitude, quality and kinetics of the early inflammatory response at the site of injection interaction of dendritic cells with antigencontaining liposomes: effect of bilayer composition antigen chemically coupled to the surface of liposomes are cross-presented to cd þ t cells and induce potent antitumor immunity a new intranasal influenza vaccine based on a novel polycationic lipiddceramide carbamoyl-spermine (ccs): i. immunogenicity and efficacy studies in mice liposomes as nanovaccine delivery systems on the formulation of ph-sensitive liposomes with long circulation times interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses immunogenicity of synthetic peptides from circumsporozoite protein of plasmodium falciparum induction of plasmodium-specific immune responses using liposome-based vaccines malaria parasites: the great escape protective immunity produced by the injection of xirradiated sporozoites of plasmodium berghei protection of humans against malaria by immunization with radiation-attenuated plasmodium falciparum sporozoites chemical modification of viruses and virus-like particles prophylactic quadrivalent human papillomavirus (types , , , and ) l virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase ii efficacy trial adverse events following immunization in ontario's female school-based hpv program the effect of conjugation to gold nanoparticles on the ability of low molecular weight chitosan to transfer dna vaccine polymerbased drug delivery systems for cancer targeted plga nano-but not microparticles specifically deliver antigen to human dendritic cells via dc-sign in vitro candida vaginitis: virulence, host response and vaccine prospects evidence of blood stage efficacy with a virosomal malaria vaccine in a phase iia clinical trial efficacy of a respiratory syncytial virus vaccine candidate in a maternal immunization model novel hemagglutinin nanoparticle influenza vaccine with matrix-mÔ adjuvant induces hemagglutination inhibition, neutralizing, and protective responses in ferrets against homologous and drifted a (h n ) subtypes a phase randomized, observerblind, placebo-controlled, dose-ranging trial of aluminum-adjuvanted respiratory syncytial virus f particle vaccine formulations in healthy women of childbearing age current opinion on nanotoxicology a soluble form of the mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation can controversial nanotechnology promise drug delivery? sub- -nm pd nanosheets with renal clearance for efficient near-infrared photothermal cancer therapy nitric oxide. a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells engineered metal based nanoparticles and innate immunity gene therapy death prompts review of adenovirus vector trial halted after gene shows up in semen conformational changes and catalytic competency of hydrolases adsorbing on fumed silica nanoparticles: ii. secondary structure improving vaccine and immunotherapy design using biomaterials delivering adjuvants and antigens in separate nanoparticles eliminates the need of physical linkage for effective vaccination nanoparticle-based vaccines: opportunities and limitations key: cord- -swwz kzd authors: bramwell, vincent w.; perrie, yvonne title: the rational design of vaccines date: - - journal: drug discov today doi: . /s - ( ) - sha: doc_id: cord_uid: swwz kzd this review provides an insight into the various opportunities for vaccine intervention, analysis of strategies for vaccine development, vaccine ability to modulate immune responses and resultant rational vaccine design. in addition, wider aspects are considered, such as biotechnological advances, advances in immunological understanding and host–pathogen interactions. the key question addressed here is, with all our research and understanding, have we reached a new echelon in vaccine development, that of rational design? the goal of any vaccination is the induction of an appropriate and effective immune response, but precisely what constitutes an effective immune response for many diseases is still unclear. specific levels of antibody are considered protective in vaccine efficacy for vaccines against hepatitis, diphtheria and tetanus; for example, anti-hepatitis surface antigen antibody levels > miu/ml, anti-diphtheria antibody levels > miu/ml and anti-tetanus antibody levels > miu/ml are quoted as protective levels of antibody in humans [ , ] . however, for many other diseases, correlations of efficacy are less obvious or yet to be agreed. by definition of perceived need, we are most acutely aware of the requirement of effective vaccines against infectious agents, pathogens ancient, re-emergent and new, yet the opportunities for manipulation of immune responses offer potential in the prevention and treatment of a far larger diversity of diseases. from these, immunomodulation in cancer, allergy and autoimmune disease are also considered here. recent developments have brought vaccines against infectious diseases to the forefront of the scientific community. the 'wake up call' that came from sars (severe acute respiratory syndrome) has undoubtedly fuelled present fears of a flu pandemic. the spanish flu pandemic provides an extreme historical precedent, causing an estimated million deaths after infecting one billion people worldwide [ ] . as to the impact of such an outbreak, today there is serious concern over the capacity of the existing infrastructure to limit fatalities by providing suitable healthcare [ ] . although development of a suitable vaccine can take a minimum of six months, in an organized and prepared scenario this could provide the major weapon that will ultimately contain such a catastrophe. the latest polio outbreaks look set to delay the long hope for eradication of this disease [ ] . in the uk, the substitution of the live oral polio vaccine with an inactive vaccine should provide a step towards safe cessation of polio vaccination when the time is right. the live vaccine has the capacity to generate infectious polio virus through mutation, and altered immunological properties of mutants present concern about virus spread in immunized as well as non-immunized populations [ , ] . taken as a whole, vaccine research and development encompasses a wide diversity of potential applications, developmental strategies and opportunities. the ongoing elucidation of immunological mechanisms of pathogen recognition and adjuvant action, as well as the knowledge of pathogen mediated mechanisms of immune evasion and gene expression, have transformed our understanding of immunology and disease in recent years. in this complex but increasingly focussed environment of vaccine development, are there opportunities for new solutions for vaccine design? and can new and refined approaches succeed where more conventional approaches appear to have failed? there are several reasons as to why the bacillus calmette-guérin (bcg) vaccine against tuberculosis (tb) is perceived as ineffective, including manipulation of immune responses (as for virulent tb [ , ] ) and, interestingly, variation between propagated strains of bcg [ ] . in the case of tb, recent literature has correlated the production of interleukin (il- ) and il- δ (a splice variant of il- and an il- antagonist) with the propensity of a latently infected host to succumb to active tuberculosis. the production of il- δ is correlated with the control of the disease [ ] . focus on the type of immune response needed to engender protection against tb seems to be orientated (convincingly) towards suppression of il- and the long standing dogma advocating the importance of th type cytokines (such as interferon gamma, ifn-γ) in tb resistance [ , ] . in the case of hiv, only one hiv candidate vaccine has completed clinical trials phases i, ii and iii in more than years of the epidemic. the phase iii trial was based on the use of recombinant envelope proteins, with the aim of evoking virus neutralizing antibodies. however, results from this were disappointing. currently available vaccines are mostly effective against viral diseases that are normally cleared during natural infection [influenza, smallpox, polio and all three viruses in the measles, mumps and rubella (mmr) immunisation]. hiv, in common with epstein-barr virus, cytomegalovirus, hepatitis (b and c) and herpes simplex virus, normally establishes chronic infection. opinion is divided as to what type of immune response is required in the control of hiv. neutralizing antibodies might be efficient in blocking virus particles but poorly effective against cellassociated virus, whereas some cytotoxic t lymphocytes (ctls) are effective against virally infected cells but not against free virus particles. latent infection facilitates immune evasion and the rate of mutation inherent in hiv also facilitates evasion (by escape from responding t cells [ ] ). promising vaccine strategies will likely depend upon highly conserved epitopes and upon vaccine strategies that induce potent immune responses capable of driving cytotoxicity and producing broadly effective neutralizing antibodies [ ] . allergic and autoimmune diseases represent undesired hypersensitive immune responses to normally harmless environmental antigens, in the case of allergy, or normally tolerated self-proteins, in the case of autoimmunity. the understanding of the pathogenesis of allergic and autoimmune diseases has led to several proposed associations and causes for these diseases. it has been shown that highaffinity t cells can out-compete lower-affinity t cells during responses to antigen in vivo [ ] . competition between t cells provides the basis for the argument that maintaining a balanced peripheral immune system might also be a side effect of normal competition for shared resources within an intact immune system [ ] , a mechanism proposed to be responsible for the increased occurrence of these diseases, where hypersensitivity is normally passively controlled by expansion of immune cells specific for prevalent infectious agents. autoimmunity can have strong associations with exposure to crossreactive proteins, and genes encoding major histocompatibility complex (mhc) are strongly associated with some autoimmune diseases, but this is markedly less so for allergy. allergy and autoimmunity are thought to differ by virtue of their regulation through central and peripheral tolerance. genetic predisposition and environmental exposure are believed to play key roles in the development of asthma and atopy [ ] , with the significant increase in the incidence of these disorders thought to provide evidence for the involvement of various environmental factors, especially in developed countries [ ] . the rationale for immunological therapeutic intervention is strongly supported by the present lack of curative treatments for autoimmune and allergic disorders, a growing unmet need, where the largely palliative treatment is not without problems [ ] . evidence that pathology associated with allergy and autoimmunity is reversible is provided by transient and long-term resolution of disease as well as by the success of specific allergen immunotherapy, where subcutaneous or sublingual administration of the sensitizing protein(s) modifies immunity and reduces allergen sensitivity [ ] . serological identification of antigens by recombinant expression cloning has led to the identification of a multitude of new tumour antigens [ ] . it is thought that most or all paraneoplastic neurological disorders (neurological disorders remote from the site of a malignant neoplasm or its metastases) are immune mediated [ ] and this has been cited as evidence for the involvement of specific immune responses in cancer suppression [ ] . indeed, failure to find the relevant antigen in the cancer of a patient should prompt a search for a second cancer www.drugdiscoverytoday.com [ ] . immunotherapy has recently achieved much interest as a possible addition to chemotherapeutic treatment [ ] and this could open up a new and innovative avenue for clinical trials. effective chemotherapy against schistosoma mansoni with praziquantel is dependent on the presence of antibodies recognizing schistosome glycoprotein epitopes [ ] . another interesting approach is the therapeutic vaccination against cancer by targeting anti-apoptotic molecules [ ] . infectious agents are implicated as causes of cancer and contribute to a variety of malignancies worldwide. some of the major players in this role are shown in table and they account for several of the most common malignancies and up to % of malignancies around the globe [ ] . as such, these agents offer increased potential for prevention of cancer by prophylactic vaccination. administration of antisera as a treatment against snake venom dates back to the s and the work of albert calmette. although this article largely focuses on active immunisation and the generation of host antibodies by the host immune system itself, the strategy of passive immunisation is worth consideration, as epitopes identified using modern molecular biology techniques might be targeted by the use of passively administered monoclonal antibodies. examples of immunotherapy using monoclonal antibodies are campath ® (alemtuzumab), manufactured by genzyme for the treatment of b-cell chronic lymphocytic leukaemia, and rituxan ® (rituximab), a chimeric murine-human monoclonal antibody that targets the cd antigen found on the surface of normal and malignant b lymphocytes and used in the treatment of non-hodgkin's lymphoma. this strategy is not restricted to cancers; for example remicade ® , a chimeric monoclonal antibody specific for tumour necrosis factor α (tnf-α, is used in the treatment of autoimmune disease. the implementation of rational design was first evident when pasteur reproducibly manufactured attenuated cultures of chicken cholera vaccines, thereby routinely preventing cholera in vaccinated chickens. extrapolation of this strategy for anthrax vaccines in livestock in the s was a success with significant economic benefits. despite the elucidation of an attenuated vaccination strategy against rabies and other inactivated whole-organism vaccines towards the end of the nineteenth century, it wasn't until the cell-culture revolution in - that attenuated viral vaccines really took off, together with the isolation and use of extracts and subunits of infectious agents. pasteur's approaches of attenuation and inactivation still today provide the two poles of vaccine technology [ ] . a chronology of important developments and achievements in vaccine research and implementation is shown in figure . recent developments in vaccine technology and application include combination vaccines, new adjuvants and delivery systems, reverse vaccinology and vaccines against noninfectious diseases. the history of vaccine development is very much related to technological advances [ ] . subunit vaccines can be produced in bulk, safely and reproducibly, using recombinant dna technology. much research has already been accomplished in the development of suitable carrier systems able to engender enhanced immune responses to entrapped peptide and protein epitopes. the identification of potentially useful antigens is greatly facilitated by improved molecular biology techniques, such as microarray technology (discussed further below), but the elucidation of their full potential might rely on the development of effective delivery systems and adjuvants. many adjuvants are based on microbial components but others, traditionally aluminium salts and more recently emulsions and surfactant based formulations, exploit different mechanisms of action. particulate delivery can be mediated by polymer [often poli(lactideco-glycolide)] microparticles, immunostimulatory complexes, liposomes and virosomes [ ] . possible advantages of particulate delivery are that antigen and coadjuvant can be delivered to the same cell and particulates have excellent potential for targeting cells of the immune system [ ] . the extensive diversity of adjuvants and delivery systems is more comprehensively reviewed elsewhere [ ] [ ] [ ] [ ] and this review will, where appropriate, focus on the application and other aspects of this technology. the million doses of influenza vaccine needed annually worldwide require more than million chicken eggs and six or more months. the development of cell-culture technology that could replace the egg-based manufacturing process might facilitate faster production of much-higher antigen yields [ ] . live vaccines generally possess a natural adjuvant capability that is built on the evolved ability of our immune system to recognize many facets of potentially dangerous microbes and they have contributed immeasurably to the control of disease. although some live vaccines might have undesirable characteristics, such as the mutation of polio virus and persistence of viruses such as varicella zoster virus, their use has been, and indeed remains, able to significantly reduce the impact of associated diseases [ , ] . viruses and bacteria as carriers of heterologous antigens and genes have received considerable attention over previous decades, and generation of auxotrophic mutants, as well as the use of other replication incompetent vectors and of reassortant viruses generated by the addition of rna segments to viruses with segmented genomes, such as influenza and rotavirus, indicates the potential inherent in these efficient types of vaccine. in an interesting utilization of viral replication, a self-replicating rna vector encoding a model antigen (β-gal), which was shown to protect mice against subsequent tumour challenge with colon carcinoma cells engineered to express β-gal, was also shown to effectively induce apoptosis in transduced cells [ ] . the observed apoptosis also facilitated enhanced uptake by dendritic cells and was likely mediated by the presence of double-stranded rna in replication of the virally derived genome. double-stranded rna is recognized by toll-like receptor (tlr) binding. different tlrs recognize different surface and intracellular components of microorganisms. the interaction between a tlr and a microbial component triggers the activation of the innate immune system and www.drugdiscoverytoday.com the initiation of acquired immunity [ ] . the elucidation of these pathways sheds light on mechanisms of adjuvant action and provides a basis for the inclusion of such potent agents and their analogues in vaccine design. it is important to consider how we classify or view rational design in the context of vaccines. a picture springs to mind of the rapid analysis of pathogens, the identification and allocation of immunological interactions for specific genes, as well as the generation of a superimposed choreography of the infection or disease process, resulting in the identification of credible candidate target antigens. following this, appropriate formulation with adjuvants and delivery systems might elicit immune responses of the type and magnitude predictive of providing protection or therapeutic action. the truth is that we are still very far from this idyll. recent articles have intimated the minor contribution of our knowledge of immunology to the development of vaccines [ , ] . however, the development of improved and large-scale cellular immunity techniques for the analysis of immune responses, such as elispot for cytokine induction, tetramer staining for peptide specific ctls, along with analysis of the immunological basis for the efficacy of successful vaccines, facilitates the role of immunology in predicting the appropriate context for antigen delivery. extensive characterisation of adjuvant action and the potential to target desired immune responses through the exploitation of this knowledge is key for rational vaccine design [ , , , , ] . the interaction between different elements involved in rational vaccine design is outlined in figure . the use of microarray technology can allow identification of virulence genes and vaccine targets [ ] and is one of the most powerful tools for the study of the transcriptome, the complete set of transcripts of an organism. indeed, in conjunction with proteomics and comparative genome analysis, interpretation of whole-genome sequences through bioinformatics (or genomic mining) can be used to assign putative gene functions to each open reading frame on the basis of homology to known proteins [ ] . systematic identification of potential antigens of a pathogen using this information, without the need for cultivation of the pathogen, is termed 'reverse vaccinology' [ ] and represents a significant departure towards the idyll of rational vaccine design described above, at least in terms of dissection of potential pathogen-related antigens. knowledge of pathogen interactions during infection can provide an invaluable insight into potentially successful targets for vaccines. for example, knowledge of gene expression in mycobacterium tuberculosis enables us to see why vaccines based on early secreted proteins, such as ag and esat- , can generate effective pre-exposure vaccines and helps us to predict that an effective post-exposure vaccine will utilize dormancy induced proteins, such as α-crystallin, heat shock protein 'hspx' [ ] . in the case of hiv, the implication of lipid rafts in viral entry and budding processes might have provided a rationale for the evaluation of peptides, based on highly conserved caveolin- binding domains of hiv- glycoprotein gp in candidate vaccine formulations [ ] . encouragingly, this has resulted in peptides capable of the elicitation of neutralizing antibody responses able to inhibit different clades of hiv- . it is thought that the poor ability of some specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp epitopes [ ] . important factors in the rational design of vaccines and the cyclical generation of knowledge. the level of involvement of any of these factors depends on the aetiological agent of disease. for example, the level of protection required in a population (herd immunity) will be different and this could allow theoretical flexibility in vaccine efficacy.the application of molecular biology techniques can be crucial in the identification of new candidate antigens and subsequent determination of vaccine efficacy using adjuvants can feed knowledge back to correlates of protection in terms of immunological markers.this knowledge can then be used in choice of appropriate adjuvants and formulation.the key implication projected by this schematic is that for the greatest challenges in vaccine development the cyclical generation of knowledge provides a strong role for rational design. * can be protective or therapeutic. ** including reverse vaccinology and associated technologies. correlates of protection* immunological markers defining protective* antigens antibodies against the caveolin- binding domains are rare in many hiv infected individuals, possibly because of the presence of hypervariable immunodominant epitopes or the location and lack of exposure of conserved regions. the proposed opportunities for vaccines against hiv and tb are summarized in figure ; the rationale for elicitation of successfully neutralizing antibodies against hiv is centred upon increasing the immunogenicity of poorly immunogenic peptides, modification and/or removal of hypervariable regions and mimicking viral conformational changes. one of the hottest areas of interest regarding new vaccine development surrounds the phase iii efficacy trials and prospective licensure of two vaccines against human papillomavirus (hpv) [ ] . the vaccines, one from merck and one from gsk, are effectively vaccines against cervical cancer; the (now) unequivocal link between this common virus, which rarely causes serious disease in itself, and cervical cancer found credence as long ago as the s. the production of both vaccines (the merck vaccine produced in yeast administered with an aluminium adjuvant and the gsk vaccine in baculovirus with the adjuvant as ; aluminium and bacterial lipid already approved for use in europe) has its basis in work that elucidated the production and self-assembly of virus-like particles of the hpv outer l and l coat proteins [ , ] in various cell types. vaccines against most neisseria meningitidis serogroups have been developed using traditional approaches. however, group b meningococcus has represented a particular challenge because of the sequence variation of surface proteins and crossreactivity of the capsular polysaccharide proposed opportunities for vaccination against tb and hiv. in both cases, the utilization of adjuvants capable of driving the required type of immune response can be used.this can be based on our current understanding of tlr interaction, and studies that manipulate this understanding might also serve to elucidate complex interactions during infection and disease. (a) according to recent models, antigen specific cd + t cells, cd + t cells, γδ t cells and cd restricted t cells, all participate in protection against m. tuberculosis infection in response to antigen presentation by macrophages (mΦ) and dendritic cells (dc), probably also involving crosspriming, where mycobacterial antigens are transferred from infected mΦ to dc. it is unlikely that the induction of ifn-γ or the production of inducible nitric oxide synthase alone is enough to control tb infection, and evidence indicates that there might be another role for cd + t cells.the development of pre-and post-exposure vaccines will likely require antigens expressed by m. tuberculosis under pre-and post-exposure conditions, respectively. interestingly, nonpolymorphic cd restricted t cells are thought to have evolved as a result of prolonged interaction with mycobacteria, indicating the extent of the impact of mycobacteria on its mammalian host. (b) for vaccines against hiv, it seems increasingly likely that strategies will depend upon highly conserved epitopes and on vaccine strategies that induce potent immune responses capable of driving cytotoxicity, as well as broadly reactive, highly effective neutralizing antibodies.target antigens might very probably be different for each of these strategies. figure adapted, with permission, from ref. [ ] . adsorption of antigens; maintaining conformational integrity with host polysialic acid [ ] . in this case, reverse vaccinology was convincingly applied to overcome these problems by alternative means [ ] . screening of dna fragments, discarding likely cytoplasmic protein sequences and known neisseria antigens, extensive cloning and expression of identified candidate genes allowed selection of antigens that were found not only to offer significant potential for protection against group b meningococcus but also to facilitate crossprotection against heterologous strains. also worth mentioning is the technique of molecular breeding or gene shuffling, where reassembled chimeric genes are created from a selection of homologous gene sequences. the related sequences are denatured, annealed and subsequently extended in what is in effect a self-priming polymerase chain reaction (pcr) that takes advantage of crossovers, deletions, insertions, inversions and point mutations, as occurs in natural evolution (hence the term 'molecular breeding'). proteins and genes with enhanced activity are then selected for inclusion in further rounds of molecular breeding. this has a proven application for the generation of enzymes with markedly enhanced activity [ ] and the enhancement of the functions of a diverse range of proteins, such as green fluorescent protein [ ] and ifn-α [ ] . this technique is thought to be superior to other methods that employ random mutagenesis, such as error-prone pcr (hence the phrase 'the rational basis for irrational design' [ ] ), but the screening of candidate antigen genes is necessarily expensive in terms of resources and time if it is compared for example to the screening of an enzyme. their potential has been noted with relevance to allergy vaccines [ ] , as pre-existing immunity is the therapeutic target and screening of allergen variants might be somewhat more practicable. bringing together developmental strategies with the knowledge gleaned from host pathogen interactions represents a highly evolved design strategy. although each individual molecular biology technique appears to have something to contribute to vaccine development, it is the holistic application of a combined approach that is closest to a premeditated and planned rational vaccine design. pathogens such as streptococcus pneumoniae, porphyromonas gingivalis, staphylococcus aureus, chlamydia pneumoniae and bacillus anthracis have all been investigated using bioinformatics techniques in the context of reverse vaccinology [ ] , with continued interesting results. in terms of antigen identification, the national institute of allergy and infectious diseases (niaid) recently announced initiation of the large-scale antibody and t cell epitope discovery program; utilizing complementary methods for epitope discovery, it is designed to identify immune epitopes from selected infectious agents and will make this information freely available to scientists worldwide through the immune epitope database and analysis resource (iedb), currently under development [ ] . recent discoveries have resulted in a wealth of options, when considering what we can do in terms of vaccine design and production. mechanisms of adjuvant action have been elucidated, pattern recognition receptors including toll-like receptors, nod , nod , scavenger receptors, mannose and other receptors are becoming increasingly well defined [ ] and intracellular events related to these, such as activation of transcription factors and expression of inflammatory cytokines, shed further light on how these ligands and adjuvants that bind to pattern recognition receptors mediate their immunological actions. the ability of infectious agents to induce rapid, innate immune responses to molecular patterns provides the basis for adjuvant immunotherapy of cancers. studies, such as the recent work examining the adjuvant activity of bcg cell-wall skeleton, peptidoglycan and lipopolysaccharide by the induction of genes in dendritic cells [ ] , will very possibly help to achieve definition of efficient effector output with minimal toxicity. the easier and safer production of vaccines and vaccine components facilitated by modern biological techniques underpins a rational approach that is an integral part of the rational design of vaccines. the extensive knowledge of immunological mechanisms and host pathogen interactions is able to contribute to the design of effective vaccines in addition to the elucidation of the mechanisms of action of many candidate vaccine agents. in fact, rational design of vaccines represents a driving force born in the first revelations that components from or relating to a microbial pathogen can be used to protect against that disease and present throughout development of vaccines in the modern era, honing our understanding and preparing the way for the next steps in the battle to combat today's significant pathogens and diseases, such as cancer, hiv, tuberculosis and malaria. in this era, we begin to explore the very limits of immunotherapeutic and prophylactic intervention and the tools that have been developed to elucidate gene expression and function might have at last begun to find their application in our most significant of challenges. anamnestic response to administration of purified non-adsorbed hepatitis b surface antigen in healthy responders to hepatitis b vaccine with longterm non-protective antibody titres the safety, reactogenicity and immunogenicity of a -valent pneumococcal conjugate vaccine ( vpnc) concurrently administered with a combination dtap-ipv-hib vaccine vaccines in the public eye preparing for the next pandemic who's attempts to eradicate polio are thwarted in africa and asia spread of vaccinederived poliovirus from a paralytic case in an immunodeficient child: an insight into the natural evolution of oral polio vaccine long-term excretion of vaccine-derived poliovirus by a healthy child bacillus calmette-guerin shares with virulent mycobacterium tuberculosis the capacity to subvert monocyte differentiation into dendritic cell: implication for its efficacy as a vaccine preventing tuberculosis autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages bcg-different strains, different vaccines? healthy individuals that control a latent infection with mycobacterium tuberculosis express high levels of th cytokines and the il- antagonist il- δ disseminated tuberculosis in interferon gamma genedisrupted mice an essential role for interferon gamma in resistance to mycobacterium tuberculosis infection positive selection of hiv- cytotoxic t lymphocyte escape variants during primary infection requirement of diverse t-helper responses elicited by hiv vaccines: induction of highly targeted humoral and ctl responses t cells down-modulate peptide-mhc complexes on apcs in vivo t cell regulation as a side effect of homeostasis and competition recent findings on genes associated with inflammatory disease peptidebased therapeutic vaccines for allergic and autoimmune diseases analysis of the b-cell repertoire against antigens expressed by human neoplasms paraneoplastic syndromes involving the nervous system dna vaccines to attack cancer anti-hu-associated paraneoplastic encephalomyelitis: analysis of patients immunotherapy and chemotherapy-a practical partnership role of host antibody in the chemotherapeutic action of praziquantel against schistosoma mansoni: identification of target antigens regulators of apoptosis: suitable targets for immune therapy of cancer infectious agents and cancer: criteria for a causal relation vaccines: past, present and future six revolutions in vaccinology targeting the innate immune response with improved vaccine adjuvants particulate delivery systems for vaccines particulate delivery systems for biodefense subunit vaccines meeting on novel adjuvants currently in/close to human clinical testing: world health organization-organisation mondiale de la sante fondation merieux eradication and cessation of programmes: vaccination and public health care a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults cancer therapy using a self-replicating rna vaccine toll-like receptor signalling can successful vaccines teach us how to induce efficient protective immune responses? current status of dna vaccines and their route of administration dna content analysis on microarrays biotechnology and vaccines: application of functional genomics to neisseria meningitidis and other bacterial pathogens reverse vaccinology annulling a dangerous liaison: vaccination strategies against aids and tuberculosis the caveolin- binding domain of hiv- glycoprotein gp is an efficient b cell epitope vaccine candidate against virus infection access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp is sterically restricted on primary human immunodeficiency virus type public health. high hopes and dilemmas for a cervical cancer vaccine expression of human papillomavirus type l protein in insect cells: in vivo and in vitro assembly of viruslike particles expression of vaccinia recombinant hpv l and l orf proteins in epithelial cells is sufficient for assembly of hpv virion-like particles development of vaccines against meningococcal disease identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing rapid evolution of a protein in vitro by dna shuffling improved green fluorescent protein by molecular evolution using dna shuffling evolution of a cytokine using dna family shuffling directed evolution: the 'rational' basis for 'irrational' design molecular breeding of allergy vaccines and antiallergic cytokines a roadmap for the immunomics of category a-c pathogens gene-inducing program of human dendritic cells in response to bcg cellwall skeleton (cws), which reflects adjuvancy required for tumor immunotherapy the authors thank graham smith at aston university, birmingham b et, for his rendering of figure . key: cord- -c wcr m authors: rego, gabriel n. a.; nucci, mariana p.; alves, arielly h.; oliveira, fernando a.; marti, luciana c.; nucci, leopoldo p.; mamani, javier b.; gamarra, lionel f. title: current clinical trials protocols and the global effort for immunization against sars-cov- date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: c wcr m coronavirus disease (covid- ) is the biggest health challenge of the st century, affecting millions of people globally. the outbreak of severe acute respiratory syndrome coronavirus (sars-cov- ) has ignited an unprecedented effort from the scientific community in the development of new vaccines on different platforms due to the absence of a broad and effective treatment for covid- or prevention strategy for sars-cov- dissemination. based on current studies selected from the main clinical trial databases, this systematic review summarizes the global race for vaccine development against covid- . for each study, the main intervention characteristics, the design used, and the local or global center partnerships created are highlighted. most vaccine developments have taken place in asia, using a viral vector method. two purified inactivated sars-cov- vaccine candidates, an mrna-based vaccine mrna , and the chimpanzee adenoviral vaccine chadox are currently in phase iii clinical trials in the respective countries brazil, the united arab emirates, the usa, and the united kingdom. these vaccines are being developed based on a quickly formed network of collaboration. in december , in the chinese city of wuhan in china's hubei province an outbreak related to severe acute respiratory syndrome coronavirus (sars-cov- )-that is, a virus genetically close to bat-cov-ratg and bat-sl-covzc -emerged, leading to coronavirus disease . this infectious disease caused an unprecedented health emergency, which was declared a pandemic by the world health organization (who) on march [ ] . subsequently, new approaches to vaccine development or the adoptive transfer of immunity were rapidly implemented in preclinical and clinical studies. these studies aimed to avoid infection and prevent the symptoms of covid- , a disease of heterogeneous symptoms caused by sars-cov- . as of the end of june , this virus had affected about million people globally and killed about , individuals [ ] . currently, there is not yet a consolidated protocol or therapeutic strategy approved for covid- critical patients [ ] . at this moment, the best practices for the supportive management of respiratory acute hypoxemic failure must be used. the initial treatment of covid- was restricted to overall supportive care and critical care because no other appropriate therapies or vaccines existed. however, many clinical trials are investigating the effect of anti-inflammatories, antimalarials, antivirals, plasma therapy, cardiovascular drugs, antibiotics, cell therapy, and cancer drugs among other medications for covid- treatment [ , ] . dexamethasone and anticoagulant are suggested since some severe, critical, and deceased covid- patients have significant coagulation dysfunction with increased d-dimer concentration, decreased platelet counts, and the prolongation of prothrombin time [ ] . regarding the prophylactic use of bacilo calmette-guérin (bcg) (pasteur institute at lille, france) vaccine against sars-cov- , it was observed that previous bcg (lille, france) immunization correlates with a lower incidence and gravity of the covid- disease across different countries, even when the bcg (lille, france) immunization was performed in childhood. antimalarials such as chloroquine and hydroxychloroquine are being extensively investigated, but no strong evidence of their benefits has been released yet. thus, there are no specific mechanisms or robust evidence of the efficacy of these therapeutic procedures [ ] . thus, during the pandemic caused by covid- , several vaccine candidates with attenuated virus, encoding, or presenting sars-cov- antigens have been developed globally, reaching clinical trial phases i or ii for the evaluation of their safety and immunogenicity. these vaccines include those based on inactivated virus [ ] [ ] [ ] [ ] [ ] ; on nucleic acid platforms as messenger ribonucleic acid (mrna) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , self-amplifying rna (sarna) [ ] , and dna/plasmids [ ] [ ] [ ] [ ] [ ] [ ] ; recombinant adenovirus serotypes platforms as adenoviral vector [ ] [ ] [ ] [ ] [ ] , chimpanzee adenoviral vector chadox [ , ] , and combined serotypes vectors and [ , ] ; recombinant viral protein subunits [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; modified dendritic cells [ ] [ ] [ ] ; artificial antigen-presenting cells [ ] ; and virus-like particle (vlp) vaccine [ ] . six protocols are developing phase ii and/or iii clinical trials using the chimpanzee adenoviral vector chadox [ ] [ ] [ ] , purified inactivated sars-cov- vaccine [ , ] , and mrna- vaccine [ ] . several of these vaccines undergoing trials comprise new technology that has not been tested previously. the advent of nanotechnology enables different mechanisms to target viruses-for instance, through the use of acid functionalized multi-walled carbon nanotubes comprising photo-activated molecules [ ] , or natural (such as chitosan) or synthetic (such as polyethyleneimine (pei)) polymeric nanoparticles [ ] . these kinds of nanomaterials are able to work as gene carriers and have the potential to deliver small interfering rna (sirna) in covid- patients [ ] . in addition, nanotechnology has enabled the development of new vaccines, including vaccines based on recombinant protein nanoparticles with or without matrix m tm adjuvant [ ] , and mrna vaccines through the encapsulation of modified nucleoside mrna inside lipidic nanocapsules [ ] [ ] [ ] [ ] ] , which confers plasticity in terms of antigen manipulation and a potentially rapid effect. intense efforts are underway to expand the technological knowledge against covid- as part of a global scientific effort to battle this virus. however, to date no review has been conducted about this global development effort and the associated cooperation networks used to test new vaccines. this lack of information empowers the movements of anti-vaccine groups [ ] . nanotechnological mrna- arises in clinical trials just two months following sars-cov- sequence identification and is currently one of the most advanced vaccines already in phase iii clinical trial protocols (ctps) [ ] . viral vector platforms enable protein overexpression, and their long-term stability can induce robust immune responses with a single dose. at present, the chimpanzee adenoviral vector from oxford (chadox ) is another advanced vaccine in study phase (ii-iii) [ , ] and phase iii [ ] . in addition, recombinant protein subunits, as immunologic adjuvants that use a squalene (tlr ) agonist [ ] , previously licensed to be delivered as vaccines for other conditions, can provide apparatus for the necessarily large-scale production of the newly developed vaccines [ ] . the majority of the recently developed vaccines aim to stimulate antibody synthesis with the necessary potential to neutralize the sars-cov- spike protein (s protein) to prevent the uptake of the single-strand rna of this virus in ace receptor positive cells [ ] .the exceptions are the vaccines using an attenuated/inactivated whole virus, which would lead to broader immunization [ , , ] . the selected clinical trials focused on interventional approaches for active immunization through the application of the various types of vaccines currently being developed against sars-cov- ( table ) . the distribution of these ctps over the time by phase ( figure ) shows that the phase i studies started in february. the number of new studies by month ( figure a) shows that the number of ctps in phase i was triplicate and quadruplicate in march and april, respectively, as did the beginning of phase ii and phase ii-iii concomitant studies. in may, the number of studies at phase i reduced to a single ctp, and the i-ii phase studies declined by half compared to the previous month. in addition, a new study started phase iii. in june, the total number of new ctps was the same as presented in april, but half of the studies were phase i and the other half were phase i-ii. in july, as well as in june, there were six new studies at phase i-ii and a study in phase ii, but the number of ctps at phase i decreased by . %. there is a clear oscillation in the number of ctps at phase i over the months, less marked in ctp at phase i-ii. at the same time, there occurred an increased number of new studies in phase iii. a cumulative analysis of the number of ctps by phase ( figure b ) shows a continuous increase in phase i-ii ctps, which also occurred in phase i studies with a slight oscillation between april and may. on the other hand, cumulatively, the number of phase ii, ii-iii, and iii studies has remained constant since march, with a slight increase in phase iii ctps between june and july. regarding the ctps targeting vaccine development and application, their platforms display a variety of characteristics and properties ( figure ; table ). nucleic acid vaccines are the ones most tested in ctps at this moment ( %). they are based on a genetically engineered plasmid containing dna ( % of the ctps) or rna (as mrna and sarna, comprising % of the ctps) ( figure ) usually encoding a selected antigen (mainly s protein). the mrna was used in % of the selected clinical trials in different phases (i, i-ii, ii), including a phase iii mrna vaccine ctp [ ] . the mrna [ , , ] and biontech (bnt ) [ , , , ] vaccines used a lipid nanoparticle (lnp) encapsulated nucleoside modified mrna encoding the s protein of sars-cov- . regarding the ctps targeting vaccine development and application, their platforms display a variety of characteristics and properties ( figure ; table ). nucleic acid vaccines are the ones most tested in ctps at this moment ( %). they are based on a genetically engineered plasmid containing dna ( % of the ctps) or rna (as mrna and sarna, comprising % of the ctps) ( figure ) usually encoding a selected antigen (mainly s protein). regarding the ctps targeting vaccine development and application, their platforms display a variety of characteristics and properties ( figure ; table ). nucleic acid vaccines are the ones most tested in ctps at this moment ( %). they are based on a genetically engineered plasmid containing dna ( % of the ctps) or rna (as mrna and sarna, comprising % of the ctps) ( figure ) usually encoding a selected antigen (mainly s protein). the mrna was used in % of the selected clinical trials in different phases (i, i-ii, ii) , including a phase iii mrna vaccine ctp [ ] . the mrna [ , , ] and biontech (bnt ) [ , , , ] vaccines used a lipid nanoparticle (lnp) encapsulated nucleoside modified mrna encoding the s protein of sars-cov- . bnt by the biontech (bnt) and pfizer companies embrace different rna vaccine candidates, comprising two nucleoside modified mrna (modrna) vaccines, an uridine containing mrna (urna) vaccine, and a sarna vaccine. besides this, two of them codify full-length s protein, and the others codify s protein receptor binding domain (rbd) [ ] . initially, studies with bnt were developed with the four mentioned bnt vaccines; however, more recently a ctp with china as the recruiting country applied only the bnt b vaccine [ ] . other ctps of the same vaccine are recruiting patients in germany [ ] and the usa [ ] . bnt is being administered in a prime/boost regimen [ , , , ] , in which the same immunogen is applied in the prime, and the booster ctp objective is to observe the immunogenicity of the subjects against s protein and rbd through igg titration in healthy individuals [ , , , ] . the ctps using mrna by moderna tx inc are all being completely developed in the usa [ , , ] . this vaccine encodes full-length s protein. at phase i, this study tested different concentrations of mrna at , , , , and µg in different interventional arms [ ] . in the phase iii intervention, the established dose was µg via intramuscular administration in two doses days apart in healthy individuals over years old [ ] . the phase i german curevac´s novel coronavirus (cvncov) ctp [ ] and another mrna vaccine ctp recruiting in china [ ] encode an unspecific protein and an rbd s protein of sars-cov- , respectively, but there is no information about the delivery systems. recently, an vaccine based on self-amplifying rna encapsulated by lipid nanoparticle (lnp-ncovsarna) phase i used sarna encoding s protein in the united kingdom, which will produce more antigen per transfected cell due to the replicative element [ ] . dna vaccines [ ] [ ] [ ] [ ] [ ] [ ] were used in % of ctps with different delivery systems. plasmids encoding s protein (pgx ) are delivered with cellectra@ electroporation system in a phase i trial in the usa, with a . mg/dose and mg/dose [ ] , and in a phase i-ii trial in the republic of korea, with a mg/dose and mg/dose delivered in a population ranging from to years old in sequential assignment [ ] in two doses, as shown in table . the canadian bactrl-spike- vaccine uses a genetically modified probiotic bifidobacterium longum as a delivery system for a synthetic dna plasmid encoding s protein to host immune cells in a phase i study; this is the first time that bactrl has been delivered in humans orally with varied doses in parallel assignment containing , , or billion colony-forming units [ ] . the gx- vaccine plasmid in a phase i-ii trial in the republic of korea by genexine, inc. company (gyeonggi, korea) [ ] is applied via intramuscular administration in a dose-escalation protocol days apart. a second covid- indian vaccine zycov-d by cadila healthcare ltd. (also known as zydus cadila, ahmedabad, india) is testing the vaccine in two groups with different age ranges, one from to and another from to years old. in both groups, the doses of . ml are delivered intramuscularly days apart [ ] . the ctp for the japanese ag dna vaccine delivers the doses in the same period of time as zycov-d [ ] , with a population ranging from to years old. the second most used platform comprises different serotypes of non-replicating recombinant adenoviral vectors ( %) that comprise the chimpanzee adenoviral vector chadox ncov- by oxford university (figure ) [ , , [ ] [ ] [ ] , currently with two in phase ii-iii [ , ] and another in phase iii [ ] in the united kingdom and brazil, respectively. the adenovirus type (ad -ncov) by cansino biologics [ ] [ ] [ ] [ ] [ ] targeting the full-length s protein of sars-cov- displays two studies in phase ii in china [ , ] . the russian gam-covid-vac by gamaleya research institute, which combines ad and a recombinant adenovirus type (ad ) vectored system targeting the s protein of sars-cov- [ , ] , is in phase i-ii. in one of these ctps, the gam-covid-vac solution is lyophilized [ ] . the most advanced chadox ncov- protocol is delivered in a single dose of × viral particle [ , ] , which is being tested two times on days and [ ] . a clinical trial divided the groups accordingly to the age of the participants: children ranging from to , adults from to , and people older than years of age [ ] . in turn, the age used in two most advanced adn -ncov ctps is older than , with single doses of × and × in a study with crossover assignment [ ] and parallel assignment [ ] , both with three interventional arms, as shown in table . the inactivated sars-cov- vaccine was used in % of the selected clinical trials ( figure ) [ ] [ ] [ ] [ ] , , , ] . the vaccine developed by sinovac biotech corporation is classified in the ctps as purified inactivated sars-cov- and is an adsorbed vaccine. according to preclinical study information, this vaccine, named picovacc, was developed by propagating the cn strain of sars-cov- inside verocells and inactivated using the chemical agent β-propiolactone [ ] . this chinese vaccine is being tested in brazil in a phase iii study [ ] and in a phase iii ctp developed in the united arab emirates [ ] . another purified inactivated vaccine also propagated sars-cov- inside verocells [ , , ] , but there is no information about the strain or inactivating agent used in pre-clinical studies. a recent phase i-ii indian vaccine bbv will apply three inactivated whole-virion strains designated bbv a, bbv b, and bbv c [ ] . another two studies developing purified inactivated sars-cov- vaccine provides no information about the inactivation processes [ , ] . all these vaccines target multiple proteins of sars-cov- . the most advanced inactivated vaccine (phase iii) is applied in two doses days apart in the deltoid muscle [ ] in participants of years onwards, as shown in table . vaccines based on the recombinant protein subunit are being tested in % of the selected ctps ( figure ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , identified by different names according to their features. the most advanced of them (phase ii) apply sars-cov- protein subunits engineered, produced, and secreted by chinese hamster ovary (cho) cells [ , ] . phase i scb- vaccine is a recombinant sars-cov- trimeric s protein subunit vaccine, and is tested with or without lipid-based adjuvant system (as ) that increases the reaction of the innate immune system, or with a oligonucleotide sequence containing cpg motif (cpg ) adjuvants plus aluminum, with a ctp being developed in australia [ ] . another australian phase i vaccine is nvx-cov , a recombinant s protein nanoparticle vaccine tested with or without the novavax saponin-based matrix m tm adjuvant [ ] , covax- tm , a vaxine proprietary advax™ adjuvant technology combined with a recombinant s protein [ , ] and a s protein stabilized using the molecular clamp technology applied with mf immunologic adjuvant [ ] . a ctp is being developed based on the production of the rbd of s protein through fast-growing tobacco plant technology [ ] . the use of adjuvants increases the activity of t and b cells mediated by antigen-presenting cells (apcs) [ ] . another % of clinical trials (phase i-ii) are testing vaccines based on dendritic cells (dc) (figure ) [ ] [ ] [ ] . one of them, from the usa, is testing subcutaneously autologous dc loaded with × or . % antigens from sars-cov- with or without µg or µg of granulocyte-macrophage colony-stimulating factor (gm-csf), targeting non-specific proteins. this vaccine is developed with the isolation of monocytes from heparinized blood incubated with il- and gm-csf that are differentiated in vitro in dc, which in turn are incubated with sars-cov- antigens [ ] . china is testing subcutaneously a recombinant chimeric dc vaccine targeting an unspecified sars-cov- epitope. this vaccine, called lv-smenp dc, is applied subcutaneously ( × ) with intravenous antigen-specific cytotoxic t cells ( ) . the subjects will be analyzed once a week for four weeks. lv-smenp dc was developed with a technology that modifies these cells through the lentiviral vector system nhp/tyf [ ] . the same lentiviral vector system nhp/tyf was able to modify artificial antigen presentation cells (aapc) that represent % of ctps [ ] . all these lentiviral modified cells encode multiple proteins of sars-cov- , and both ctps are being developed in china [ , ] . besides this, a phase i study using vlp ( % of the selected ctps) is recruiting patients in canada ( figure ) [ ] . this vaccine is administered days apart via intramuscular injection. each dose is applied to the deltoid region of alternated arms. the doses being tested are initially . µg in a small number of the population (healthy adults to years of age) with dose-escalation to . µg and µg unadjuvanted or adjuvanted with either cpg or as , with a follow-up period of six months. after this time, the immunogenicity of the subjects will be tested. the vaccine development clinical trials are displayed by phase, country, and trial progression rate (tpr) according to the beginning and completion date of each study (table ) in figure . most of the clinical trials are in simultaneous phase i-ii ( %) [ ] [ ] [ ] [ ] [ ] , , , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] , ] . in china the tpr ranges from . % to % [ ] [ ] [ ] [ ] [ ] , , , ] , in germany the tpr of one study is . % [ ] , in russia it is . % [ , ] , in the united kingdom the tpr is . % [ ] , in canada the tpr is . % [ ] , in the usa it is . % [ ] , in south africa it is . % [ ] , in the republic of korea the tpr ranges from . % to . % [ , ] , in india it ranges from . % to . % [ , ] , and in japan it is . % [ ] . then, of the clinical trials in phase i ( %) [ , [ ] [ ] [ ] , [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] , ] , in china the tpr ranges from . % to . % [ , , , , , , ] , in the usa it is . % [ ] , in canada the tpr ranges from . % to . % [ , ] , in australia the tpr ranges from . % to . % [ , , ] , in germany it is . % [ ] , and in the united kingdom it is . % [ ] . four clinical trials ( %) are in phase ii [ , , , ] . in china, the trial has a tpr of . %; in the usa, the trial has a tpr of . %; two clinical trials ( %) are in simultaneous phase ii-iii [ , ] in the united kingdom, and the studies have a tpr ranging from . % to . %. four clinical trials ( %) are in phase iii [ , [ ] [ ] [ ] . in brazil, the tpr ranges from . % to . % [ , ] ; in the united arab emirates, the tpr is . % [ ] ; and in the usa, the trial has not yet been initiated [ ] . among all clinical trials involving vaccine development, so far only one study ( %) has been completed [ ] , % are recruiting patients [ , , , , - [ , ] , and % have classified their recruitment status as enrolling by invitation [ ] , as shown in table . regarding the clinical trial protocol studies design of vaccination against covid- ( figure and table ), % of the protocols were multicenter (mc) research trials and % were single center (sc); only two ctps did not report this information. the estimated enrollment of clinical trials in phase iii ( %) ranged from , to individuals [ ] ; in phase ii-iii ( %), there were , individuals [ , ]; in phase ii ( %), it ranged from to individuals [ , , , ] ; in phase i-ii ( %), it ranged from to individuals [ ] [ ] [ ] [ ] [ ] , , , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] , ] ; and in phase i ( %), it ranged from to individuals [ , [ ] [ ] [ ] , [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the number of volunteers estimated in each protocol was represented by the scale bar in figure . the ctp intervention design was mostly randomized ( %) and used some type of masking ( %), which varied from % single blinding (participant) to % double blinding (participant, investigator), % triple blinding (participant, investigator, outcomes assessor), and % quadruple blinding (participant, care provider, investigator, outcomes assessor). however, certain ctps have not adopted any techniques for minimizing the bias in allocations and blinding- % were nonrandomized; % did not report the strategy of allocation; % did not use masking (none), keeping the research open label; % did not report the strategy of masking. the intervention model was % in parallel-assignment design, following by % with sequential assignment design, % with single group assignment, % with crossover assignment, and % did not report the intervention model ( figure ). the number of intervention arms used in the clinical trial protocols were mainly under ( %), but % used between and arms; . % used between and ; and . % used more than arms of intervention, corresponding to the type of vaccine analyzed according to the different dose concentrations (low, medium, high), the number of doses (single or more than one), and the days of administration (single or double). in addition, the arms were distributed for different ages, including regarding the clinical trial protocol studies design of vaccination against covid- ( figure and table ), % of the protocols were multicenter (mc) research trials and % were single center (sc); only two ctps did not report this information. the estimated enrollment of clinical trials in phase iii ( %) ranged from , to individuals [ ] ; in phase ii-iii ( %), there were , individuals [ , ]; in phase ii ( %), it ranged from to individuals [ , , , ] ; in phase i-ii ( %), it ranged from to individuals [ ] [ ] [ ] [ ] [ ] , , , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] , ] ; and in phase i ( %), it ranged from to individuals [ , [ ] [ ] [ ] , [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the number of volunteers estimated in each protocol was represented by the scale bar in figure . the ctp intervention design was mostly randomized ( %) and used some type of masking ( %), which varied from % single blinding (participant) to % double blinding (participant, investigator), % triple blinding (participant, investigator, outcomes assessor), and % quadruple blinding (participant, care provider, investigator, outcomes assessor). however, certain ctps have not adopted any techniques for minimizing the bias in allocations and blinding- % were non-randomized; % did not report the strategy of allocation; % did not use masking (none), keeping the research open label; % did not report the strategy of masking. the intervention model was % in parallel-assignment design, following by % with sequential assignment design, % with single group assignment, % with crossover assignment, and % did not report the intervention model ( figure ). the number of intervention arms used in the clinical trial protocols were mainly under ( %), but % used between and arms; . % used between and ; and . % used more than arms of intervention, corresponding to the type of vaccine analyzed according to the different dose concentrations (low, medium, high), the number of doses (single or more than one), and the days of administration (single or double). in addition, the arms were distributed for different ages, including less than years old ( %), more than years old ( %), between and years old ( %), and more than years old ( %). the distribution of vaccine ctp networks between the sponsor and the collaborating institutions is highlighted in the figure map, focusing mainly on the ctps collaborating with more than five centers. among the vaccines leading multicenter ctps, there is one in the usa controlled by modernatx inc. at phase iii (green circle) coordinating collaborating institutions (green cylinders around the red bar of figure ) , with an estimated enrollment of , participants, not yet recruited [ ] (as detailed in tables and ). in addition, there is another ctp in the usa at phase ii (blue circle) recruiting volunteers from centers (blue cylinders of figure ) [ ] . two ctps in europe are controlled by oxford university in partnership with astrazeneca [ , ] , coordinating more than collaborating institutions (hospitals, labs, etc., represented by the yellow and purple cylinders in figure ) involving approximately , participants each, at phase ii or iii (green-blue circle). there is another ctp at phase i-ii (red-blue circle) that is recruiting volunteers at six centers in the united kingdom [ ] (gray cylinders in figure ). the ctp in south america is controlled by butantan institute in partnership with the sinovac life sciences co. (beijing, china) [ ] , and is at phase iii (green circle), coordinating centers in brazil (dark green cylinders in figure ) and the distribution of vaccine ctp networks between the sponsor and the collaborating institutions is highlighted in the figure map, focusing mainly on the ctps collaborating with more than five centers. among the vaccines leading multicenter ctps, there is one in the usa controlled by modernatx inc. at phase iii (green circle) coordinating collaborating institutions (green cylinders around the red bar of figure ) , with an estimated enrollment of , participants, not yet recruited [ ] (as detailed in tables and ). in addition, there is another ctp in the usa at phase ii (blue circle) recruiting volunteers from centers (blue cylinders of figure ) [ ] . two ctps in europe are controlled by oxford university in partnership with astrazeneca [ , ] , coordinating more than collaborating institutions (hospitals, labs, etc., represented by the yellow and purple cylinders in figure ) involving approximately , participants each, at phase ii or iii (green-blue circle). there is another ctp at phase i-ii (red-blue circle) that is recruiting volunteers at six centers in the united kingdom [ ] (gray cylinders in figure ). the ctp in south america is controlled by butantan institute in partnership with the sinovac life sciences co. (beijing, china) [ ] , and is at phase iii (green circle), coordinating centers in brazil (dark green cylinders in figure ) and recruiting participants. in asia, the ctp controlled by bharat biotech international ltd. is at phase i-ii of vaccine development, recruiting participants in centers in india [ ] . in africa, the vaccine center coordinated by the university of witwatersrand phase i-ii (red-blue circle) is recruiting volunteers in six centers in south africa [ ] . all these centers leading vaccine multicenter ctps are highlighted with enlarged figures around the map in figure . however, other centers for vaccines against covid- in development in europe, asia, africa, the americas, and oceania are highlighted in the map of figure . the global distribution of ctps, according to the execution phase, as shown in figure and table a at phase i, comprises of ( %) ctps [ , [ ] [ ] [ ] , [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] , ] . among these ctps, ( . %) are from china [ , , , , ] , ( . %) are from the usa [ , , , ] , ( . %) are from canada [ , ] , ( . %) are from australia [ , , ] , ( . %) is from germany [ ] , ( . %) is from the united kingdom [ ] , and one of the ctps is shared by germany and the usa [ ] , as shown in figure a . at phase i-ii, there are of ( %) ctps [ ] [ ] [ ] [ ] [ ] , , , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] , ] . among these ctps, most are in asia: ( . %) are from china [ ] [ ] [ ] [ ] [ ] , , , ] , are from the republic of korea ( . %) [ , ] , are from india ( . %) [ , ] , and is from japan ( . %) [ ] . then, in europe, two are in eastern europe from russia ( . %) [ , ] , two are from germany ( . %) [ ] , and one is from the united kingdom ( . %) [ ] . in north america, there are three ctps from the usa ( . %) [ , , ] . there is also one from south africa ( . %) [ ] , as shown in figure b . the ctps in phase ii display of ( %) [ , , , ] , and they are from china ( %) [ , , ] and the usa ( %) [ ] , as shown in figure c . there are two phase ii-iii ctps out of ( %) [ , ] , both located in the united kingdom ( figure d) . however, there are already of ( %) ctps at phase iii [ , [ ] [ ] [ ] . two of them are in brazil-one is testing the vaccine developed by the university of oxford [ ] at universidade federal de são paulo (unifesp), and other is testing the vaccine developed by sinovac life science co. at butantan institute [ ] ; one is in the united arab emirates, and is testing the vaccine developed by china national biotec group co. at beijing [ ] ; and one in the usa, testing the vaccine developed by modernatx, inc. [ ] , as shown in figure e . only of ctps ( %) comprise a network with other institutions located in different countries. these include the united kingdom and brazil [ ] , china and the united arab emirates [ ] , canada and china [ ] , the usa and the republic of korea [ ] , the usa and australia [ ] , and germany and the usa [ ] . four studies do not reveal about their partnerships [ , , , ] . the most ctps ( %) have entered into partnerships with other institutions within the same country. approximately four months after a pandemic was declared by the who and more than , deaths have occurred, a consolidated therapeutic drug strategy protocol approved for covid- still does not exist [ ] . at this moment, the epicenter of the outbreak is the american continent, mainly usa and brazil, and isolation or social distancing remain the central who recommendations to avoid infection and increasing numbers of deaths [ ] . unprecedented global initiatives and new partnerships are being established to accelerate the research and development of tests and therapies to control the spread of this pandemic around the world. however, vaccines that mitigate the damage caused by this infectious disease can take much longer to be available with proven safety and efficacy [ ] . the time frame for vaccine supply remains approximately four months after a pandemic was declared by the who and more than , deaths have occurred, a consolidated therapeutic drug strategy protocol approved for covid- still does not exist [ ] . at this moment, the epicenter of the outbreak is the american continent, mainly usa and brazil, and isolation or social distancing remain the central who recommendations to avoid infection and increasing numbers of deaths [ ] . unprecedented global initiatives and new partnerships are being established to accelerate the research and development of tests and therapies to control the spread of this pandemic around the world. however, vaccines that mitigate the damage caused by this infectious disease can take much longer to be available with proven safety and efficacy [ ] . the time frame for vaccine supply remains uncertain, but to date global labs and industries have registered vaccine ctps in leading research databases, using eight platforms based on inactivated virus [ ] [ ] [ ] [ ] , , , ] ; nucleic acid such as mrna [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] , sarna [ ] , and dna/plasmid [ ] [ ] [ ] [ ] [ ] [ ] ; recombinant adenovirus serotypes platforms, such as adenoviral vector [ ] [ ] [ ] [ ] [ ] , chimpanzee adenoviral vector chadox [ , , [ ] [ ] [ ] , and combined serotypes vectors adenovirus and [ , ] ; recombinant viral protein subunits [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; modified dendritic cells [ ] [ ] [ ] ; artificial antigen-presenting cells [ ] ; and vlp vaccine [ ] . the advantages and limitations of these platforms will be detailed ahead. the current landscape of covid- vaccine development shows that most ctps are being developed in asia (table a ). however, regarding former ctps, only of ( %; [ ] ) phase iii ctps are located in asia. there is one ctp recruiting in abu dhabi/united arab emirates, there is a protocol that was developed in china with an inactivated virus platform, and this protocol was also used in another phase iii ctp in brazil [ ] . the virus vector platform vaccine developed in the united kingdom (oxford university) is being tested in the most advanced studies (phase ii-iii), both in the united kingdom [ , ] and brazil [ ] (phase iii). these ctps ( % of total ctps) have used the same chimpanzee adenoviral vectored vaccine targeting the s protein of sars-cov- [ ] [ ] [ ] , in which the recent results show safety and a strong immune response [ , ] , but these ctps differ due to the number of interventional arms testing different vaccine doses (number and concentration), the group age of volunteers, the method of dosing analysis (abs , abs corrected for ps and qpcr), and other comparisons. lastly, the mrna vaccine from moderna tx, inc., (cambridge, ma, usa) is a usa phase iii ctp that is the biggest trial, with , subjects in recruiting centers (table a ) , but the recruiting is just starting [ ] . this vaccine platform used advanced technology to improve the drug delivery. the gold standard for the success of a vaccine is related to its broad and sustained immunogenicity, with adequate safety and efficacy. for this achievement, the antigen delivery systems and their eligibility are important factors [ , ] . due to the outbreak of covid- , it was essential to optimize and reduce the normal stages of the vaccine development process, but possible adverse effects may occur, especially for those belonging to high-risk groups, and this is necessary to take into account during the inclusion period of subjects in these trials [ ] . this comprising steps phenomenon is expressed by comprising stages (i-ii) [ ] [ ] [ ] [ ] [ ] , , , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] , ] and (ii-iii) [ , ] and shortcuts in % of the current vaccine ctps. however, in april, only [ , , , , ] of ctps were considered the most advanced candidate vaccines to initiate clinical development for (phase i) [ ] , and none of these ctps displayed clustered phases in their trials. on april , cohen [ ] identified eight different vaccine groups or "platforms" in preclinical and clinical development, classified as inactivated or attenuated whole viruses, genetically engineered proteins, and mrna technology. at that time, the most advanced studies were in phase i. after four months, there are the same eight different vaccine platforms and ctps ( . %), which include inactivated virus [ ] [ ] [ ] [ ] [ ] , non-replicating viral vectors [ ] [ ] [ ] [ ] [ ] , nucleic acids (rna [ , [ ] [ ] [ ] [ ] [ ] [ ] or dna [ ] [ ] [ ] ), protein subunits [ ] [ ] [ ] , but now most of these ctps are in phase ii-iii or iii. in this review, the non-replicating viral vector was the main technology/platform used ( %). the advantage of the vaccine based on viral vectors is the combined stimulation of the innate and adaptive immune response to the heterologous viral infection and against the antigen expressed by the vector, usually the s protein [ ] . the problem related to heterologous response is the previous exposure to some adenovirus serotypes by the human population, leading to a pre-existing anti-vector response, which makes the vector a disadvantage. the use of ad in immunological practice is well established and highly efficient, and exhibits simple manipulation and ease of purification. however, the specific response of the transgene can be mitigated by pre-existing adaptive immune responses to antigenic targets in the vector itself [ ] . the previous results of the ctp developed by cansino confirmed the activation of cd and cd t cells in the vaccine recipients. however, the vaccine-induced specific antibody or t cell response were partially diminished by the presence of pre-existing anti-ad immunity [ ] . on the contrary, the study by zhu [ ] in phase i and the study by folegatti [ ] used an ad vectored covid- vaccine that was very immunogenic, able to induce humoral and cellular responses in most trial participants, and the detectable immune responses was very rapid, with the t cell responses peaking at day after vaccination and the antibodies peaking at day . therefore, less prevalent adenoviral serotypes, such as ad and ad , or primate derivatives, such as chimpanzees, are more often used in vaccine development, as they mimic a natural infection at low risk of herologous response, and stimulate a significant immune response without additional adjuvants [ ] . previous studies using a replication-deficient adenovirus from chimpanzee (chadox ) led to the immunization of balb/c mice, cd mice [ ] , and rhesus monkeys [ ] against mers-cov. this adenovirus construct, now encoding the full-length s protein of sars-cov- , was able to generate high titers of neutralizing antibodies and a robust cd t cell response against this viral protein [ ] . dna or rna vaccine platforms, also known as synthetic vaccines, were identified in % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] of the selected ctps. vaccines with antigen-encoding dna/rna as a platform are relatively versatile, easy to manipulate, and relatively inexpensive to synthesize compared to other types of vaccines. these vaccines have similar strategies of action-for instance, they have coding for the synthesis of target antigens that will be expressed in immune cells. however, the target of each platform is different, in addition to the advantages or risks they present [ ] . mrna based-vaccines provide benefits compared to protein subunits, inactivated or attenuated viruses, and dna-based vaccines. these benefits were discussed by a study as follows [ ] . the first issue is safety, since mrna is a non-infectious, non-integrating platform, and shows no potential risk of infection or insertional mutagenesis. in addition, mrna is degraded by regular cellular processes, and its half-life in vivo can be regulated through modifications and delivery methods. the second issue is its efficacy; modifications can make the mrna more stable and highly translatable. third, mrna vaccines have the prospective for fast, inexpensive, and accessible manufacturing due to the high yields of in vitro transcription reactions. however, this platform is vulnerable in a high mutagenic potential virus [ ] . regarding the sars-cov- mutagenic potential, a study by phan [ ] revealed many mutations and deletions in coding and non-coding regions, mainly associated with the s protein. another study [ ] identified variations in the sars-cov- genome which were observed in over % of patient isolates from all over the world. therefore, this topic is still controversial. nonetheless, this vaccine platform, despite needing adjuvants (other technologies), is more agile and scalable than others. in the selected ctps, there are seven types of mrna vaccines: four named bnt [ , , , ] and three with mrna [ , , ] , the latter being at a more advanced stage of development. both types of vaccine consist of lnp encapsulated nucleoside modified mrna targeting the s protein of sars-cov- . the mrna- developed by the national institute of allergy and infectious disease (niaid) in the usa in collaboration with modernatx inc. [ , , ] produces the mrna of s protein in a stable form due to advances in carriers such as lnps, providing high-efficiency delivery. bacteria-derived plasmid-dna vaccines, in addition to encoding the target antigen, can also encode co-stimulating molecules. these are directed to the nucleus, and need to cross the plasma and nuclear membranes to be activated. furthermore, they have the possible disadvantage of persistent genomic integration in host cell dna, leading to subsequent deleterious effects. however, the need for specific delivery systems to achieve good immunogenicity remains a concern. different methods have been developed to enhance dna uptake, expression, and immunogenicity (i.e., encapsulation in containing cationic lipids or cholesterol nanoparticles, and adsorption to polymeric or biodegradable nanoparticles), such as the gene gun, needle-free injection devices (jet injection) [ ] , and in vivo electroporation. this is the case for the ino- vaccine against sars-cov- , currently in clinical trial (phase i) [ ] , which was administered by intradermal (id) injection followed by electroporation (ep) using a cellectra ® device in healthy adult volunteers [ ] . the synthesis process of this vaccine involved the alignment of four coding sequences of s protein and the addition of the n-terminal ige leader sequence. this highly optimized dna sequence was manufactured and cloned into pgx vector. the resulting plasmids were named pgx and pgx by inovio pharmaceuticals [ ] . the immunogenicity of ino- was pre-clinically evaluated, and the serum reactivity revealed igg binding against the s protein of sars-cov- . the serum from ino- -immunized balb/c mice neutralized two sars-cov- strains-wh- (neutralizing titer of antibody (nd : . ) and vic (nd : . )). in addition, the nd of . was observed in immunized guinea pigs. this study also generated robust s-specific t cell responses in these models, and the detected antibodies were able to block s protein binding to the host ace receptor [ ] . recently ( june ), another dna vaccine developed in south korea by genexine consortium entered phase i-ii clinical trials, in which a preventive gx- vaccine will be intramuscularly administrated in healthy volunteers from countries such as indonesia and thailand. the company managers expect preliminary data from the initial trial by september and hope to complete all phases of testing by the end of [ ] . to date, no preclinical studies or technical specifications for the gx- vaccine have been released. hence, dna and mrna vaccines are readily designed and can proceed into clinical trials very fast, in addition to outstanding targets for the development of vaccines against sars-cov- and other related epidemics in the future [ ] . attenuated or inactivated vaccines, known as conventional vaccines, require whole pathogen cultivation and propagation. thus, it is necessary to obtain a high biosafety level with specialized laboratories and biotechnological tools. moreover, there is the requirement for lineage cells accepted by regulatory authorities, such as vero cells, for the development of industrial-scale inactivated virus vaccines [ ] . a total of % of the ctps use inactivated virus vaccine platforms, including a phase iii ctp recruiting in abu dhabi/the united arab emirates [ ] , and a phase iii ctp in brazil [ ] . this approach has been used previously and led to smallpox eradication [ ] , and vaccines for other diseases such as polio, tetanus, diphtheria, and measles. the clinical trials developed by sinovac corporation [ ] cultivated the cn strain of sars-cov- in vero cells and chemically inactivated them using β-propiolactone. formaldehyde and uv light are other possible agents for virus inactivation [ ] . the process of viral inactivation is delicate and cannot destroy the major virus antigenic structures, which would interfere with their immunogenicity. depth filtration and optimized steps of chromatography allow vaccine purification [ ] . prior to ctps, picovacc initiated pre-clinical experiments and administered them with alum adjuvant in balb/c mice and rhesus macaques, a non-human primate species. this study demonstrated rapid rbd-igg development, accounting for half of the s protein-induced antibodies produced and possibly the dominant immunogenic part of this protein [ ] . furthermore, no antibody-dependent enhancement (ade)-mediated vaccine-induced infection aggravation was observed for any vaccinated rhesus macaques [ ] . two other ctps developed by chinese sinopharm corporation at the wuhan [ ] and beijing [ ] institutes of biological products also expanded sars-cov- in vero cells and are currently in trial. however, no pre-clinical studies or technical specifications of the last two vaccines have been disclosed to date. since protein subunit vaccines are restricted to specific epitopes of the virus, most developers have not found proteins other than the s protein used by sars-cov- to invade cells [ ] . eight clinical trial protocols are analyzing protein subunit vaccines [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] combined with different types of adjuvants to help individuals produce an immune response strong enough to protect them from the disease [ ] . however, the produced immune responses weaken over time, which means that an individual may require additional shots for booster immunizations throughout their life [ ] . the adjuvants used in the selected clinical trial protocols are licensed and approved by the food and drug administration (fda). the adjuvant cpg increases the body's immune response; the as enhances the vaccine antigen-specific adaptive immune response; and potassium aluminum sulfate (alum), one of the agents most used as an adjuvant, acts by creating a deposit at the injection site, thus allowing the slow release of antigen and prolonging the interaction time with apcs, in addition to acting on soluble antigens by converting them into particulate forms that are readily phagocyted [ , ] . the global vaccine r&d efforts against sars-cov- are unprecedented in terms of scale and speed [ ] . from examining all the ctps operating in the united states (clinicaltrials.gov), china (chictr.org.cn), and europe (clinicaltrialsregister.eu), it is evident that most covid- vaccine ctp activity is taking place in china, with ctps ( %). however, the greatest advance has been made in the united kingdom (oxford university), in conjunction with several collaborating countries (south africa and brazil, among others) and other recruiting centers/institutions in the uk (hospitals, universities, labs, etc.). this ctp has reached phase iii protocol [ ] , and will test , subjects. on july , its performance was . %. most ctps ( . %) have not collaborated with other external institutions due to the protocol phase; only of ( %) ctps have reached phase iii, for which it is necessary to expand the tests in territories with the outbreak, as seen with the oxford university protocol, and more recently with sinovac life sciences co., a chinese vaccine company. the distribution of collaboration networks between sponsors and the recruiting centers/institutions of the ctps is strongly concentrated in asia ( . %), particularly in wuhan city, china, where the pandemic started. in europe and the americas, the united kingdom, brazil, and the usa are the main centers, representing % of the global vaccine network or total subjects in vaccine tests. as we discussed earlier, these last countries are now the epicenter of the pandemic. furthermore, the development of vaccine platforms is occurring more rapidly in wide global networks compared to scientific reports or clinical trial databases. vaccine developers publish very few clinical trial articles, even regarding the safety or preclinical results. the pandemic caused by covid- has resulted in increased awareness of global threats to human health, particularly when caused by unknown and emerging pathogens. this pandemic has motivated science and health systems to be prepared for future outbreaks. in addition, global development has begun on vaccine platforms and improvements in public and private health systems to tackle the challenges of new outbreaks. new platforms or approaches, such as viral vectors, protein-like vaccines, and nucleic acids, meet the prerequisites for providing solutions to some of these challenges, representing highly versatile technologies that allow rapid vaccine manufacturing. each vaccine technology has its own advantages and disadvantages related to its ability to induce certain immune responses, manufacturing capacity, and safety for human use. the race of covid vaccine remains uncertain, but to date global labs and industries have registered vaccine ctps in leading research databases, using eight platforms: inactivated virus; nucleic acid, such as mrna, and dna/plasmid; recombinant virus vectors; recombinant viral protein subunits; modified dendritic cells; artificial antigen-presenting cell; and vlp vaccine. new groups and laboratories have changed the global vaccine development landscape, especially in asia. the most advanced trial does not mean to be the most efficient or safe, the collaborations between countries are still of little significance. the authors declare no conflict of interest. an overview on covid- : reality and expectation covid- pandemic data update immunogenicity and safety of a recombinant adenovirus type- -vectored covid- vaccine in healthy adults aged years or older: a randomised, double-blind clinical trials for the treatment of coronavirus disease (covid- ): a rapid response to urgent need current drugs with potential for treatment of covid- : a literature review is there a rationale for heparin use among severe covid- patients? einstein , , eed bacillus calmette-guérin vaccine, antimalarial, age and gender relation to covid- spread and mortality double-blind, placebo parallel-controlled phase i/ii clinical trial for inactivated novel coronavirus pneumonia vaccine (vero cells) a phase i/ii clinical trial for inactivated novel coronavirus ( -cov) vaccine (vero cells) safety and immunogenicity study of inactivated vaccine for prevention of sars-cov- infection(covid- ) safety and immunogenicity study of inactivated vaccine for prophylaxis of sars cov- infection (covid- ) safety and immunogenicity study of an inactivated sars-cov- vaccine for preventing against covid- a phase i clinical trial to evaluate the safety, tolerance and preliminary immunogenicity of different doses of a sars-cov- mrna vaccine in population aged - years and years and above a phase i clinical trial of novel coronavirus pneumonia (covid- ) mrna vaccine (bnt b ) in china study to investigate the safety and effects of vaccines in healthy adults a study to evaluate the safety, reactogenicity and immunogenicity of vaccine cvncov in healthy adults safety and immunogenicity study of -ncov vaccine (mrna- ) for prophylaxis of sars-cov- infection (covid- ) tolerability, immunogenicity, and potential efficacy of rna vaccine candidates against covid- in healthy adults a trial investigating the safety and effects of four bnt vaccines against covid- in healthy adults. . available online dose-confirmation study to evaluate the safety, reactogenicity, and immunogenicity of mrna- covid- vaccine in adults aged years and older clinical trial to assess the safety of a coronavirus vaccine in healthy men and women novel corona virus- -ncov vaccine by intradermal route in healthy subjects study of covid- dna vaccine (ag -covid ) tolerability and immunogenicity of ino- followed by electroporation in healthy volunteers for covid safety and immunogenicity study of gx- , a covid- preventive dna vaccine in healthy adults evaluating the safety, tolerability and immunogenicity of bactrl-spike vaccine for prevention of covid- tolerability and immunogenicity of ino- for covid- in healthy volunteers a phase i clinical trial for recombinant novel coronavirus ( -cov) vaccine (adenoviral vector) double-blinded, placebo-controlled phase ii clinical trial for recombinant novel coronavirus ( -ncov) vaccine (adenovirus vector) phase i/ii clinical trial of recombinant novel coronavirus vaccine (adenovirus type vector) in canada phase i clinical trial of a covid- vaccine in - healthy adults a phase ii clinical trial to evaluate the recombinant vaccine for covid- (adenovirus vector) vaccine (chadox ncov- ) trial in south african adults with and without hiv-infection a study of a candidate covid- vaccine (cov ) an open study of the safety, tolerability and immunogenicity of the drug "gam-covid-vac" vaccine against covid- an open study of the safety, tolerability and immunogenicity of interventional study to evaluate the safety and immune response of a vaccine against severe acute respiratory syndrome coronavirus (sars-cov- , the virus that causes infection) when given to healthy adult participants monovalent recombinant covid vaccine phase i clinical study of recombinant novel coronavirus vaccine kbp- covid- vaccine trial in healthy volunteers clinical study of recombinant novel coronavirus vaccine evaluation of the safety and immunogenicity of a sars-cov- rs (covid- ) nanoparticle vaccine with/without matrix-m adjuvant scb- as covid- vaccine. . available online therapeutic vaccine trial of covid- for severe acute respiratory syndrome coronavirus (sars-cov- ) infection. immunity and safety of covid- synthetic minigene vaccine phase ib-ii trial of dendritic cell vaccine to prevent covid- in adults a clinical study for effectiveness and safety evaluation for recombinant chimeric covid- epitope dc vaccine in the treatment of novel coronavirus pneumonia safety and immunity of covid- aapc vaccine tolerability and immunogenicinity of a coronavirus-like particle covid- vaccine in adults aged - years phase iii study to investigate a vaccine against covid- investigating a vaccine against covid- a phase iii clinical trial for inactivated novel coronavirus pneumonia (covid- ) vaccine (vero cells) clinical trial of efficacy and safety of sinovac's adsorbed covid- (inactivated) vaccine in healthcare professionals a study to evaluate efficacy, safety, and immunogenicity of mrna- vaccine in adults aged years and older to prevent covid- nanomaterials and nanotechnology-associated innovations against viral infections with a focus on coronaviruses optimizing use of theranostic nanoparticles as a life-saving strategy for treating covid- patients an evidence based perspective on mrna-sars-cov- vaccine development antivaccine forces gaining online emerging manufacturers engagements in the covid − vaccine research, development and supply the pandemic pipeline vaccines: status report. immunity convalescent plasma for patients with covid- : a randomized, open label, parallel, controlled clinical study. . available online a guide for systematic reviews: prisma. turk landscape of covid- candidate vaccines whole-virion inactivated sars-cov- vaccine (bbv ) for covid- in healthy volunteers safety and immunogenicity study of an inactivated sars-cov- vaccine for preventing against covid- in people aged years germany to begin first clinical trial of covid- vaccine candidate rapid development of an inactivated vaccine candidate for sars-cov- application prospect of polysaccharides in the development of anti-novel coronavirus drugs and vaccines treatment options for covid- : the reality and challenges health equity and covid- : global perspectives the covid- vaccine development landscape comparative analysis of simian immunodeficiency virus gag-specific effector and memory cd + t cells induced by different adenovirus vectors safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind emerging viruses and current strategies for vaccine intervention vaccine designers take first shots at covid- developments in viral vector-based vaccines viral vectors as vaccine platforms: from immunogenicity to impact safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial adventitious agents and live viral vectored vaccines: considerations for archiving samples of biological materials for retrospective analysis chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice a single dose of chadox mers provides protective immunity in rhesus macaques new vaccine technologies to combat outbreak situations mrna-based therapeutics-developing a new class of drugs preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies genetic diversity and evolution of sars-cov- an updated analysis of variations in sars-cov- genome engineered nanodelivery systems to improve dna vaccine technologies immunogenicity of a dna vaccine candidate for covid- vaccination strategies to combat novel corona virus sars-cov- a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses history of smallpox and its spread in human populations brief overview of all the covid- vaccines in the pipeline potential adjuvants for the development of a sars-cov- vaccine based on experimental results from similar coronaviruses development and evaluation of as , an adjuvant system containing α-tocopherol and squalene in an oil-in-water emulsion this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -kng z kf authors: quesenberry, katherine e.; de matos, ricardo title: basic approach to veterinary care of ferrets date: - - journal: ferrets, rabbits, and rodents doi: . /b - - - - . - sha: doc_id: cord_uid: kng z kf the approach to preventive medicine and basic veterinary care in ferrets is very similar to that used in dogs and cats. special equipment needs are minimal, and pet ferrets can be easily incorporated into a general small animal practice. this chapter describes the unique aspects of handling, restraint, and clinical and treatment techniques used in ferrets. the approach to preventative medicine and basic veterinary care in ferrets is very similar to that used in dogs and cats. special equipment needs are minimal, and pet ferrets can be easily incorporated into a general small animal practice. however, there are unique aspects of handling, restraint, and clinical and treatment techniques used in ferrets. for procedures not discussed here, modify techniques used in other small animals by using instrumentation appropriate for the ferret's small size and choosing appropriate sedation or anesthesia to facilitate the procedure while minimizing stress or discomfort in the ferret. most ferrets are docile and can be easily examined without assistance. however, an assistant is usually needed when taking the rectal temperature, when administering injections or oral medications, or if an animal tends to bite. young ferrets often nip, and nursing females and ferrets that are handled infrequently may bite. ferrets often bite without warning. therefore always ask the owner if the ferret bites before handling it and take precautions accordingly. obtain the rabies vaccination history before physical examination. be aware that rabies protocols for animal bites from vaccinated and unvaccinated ferrets differ by locale. ferrets that are prone to biting and are not currently vaccinated for rabies may need to be sedated for procedures requiring restraint. depending on the ferret's disposition, several basic manual restraint methods can be used for examination. for tractable animals, lightly restrain the ferret on the examination table. examine the mucous membranes, oral cavity, head, and skin. then pick the ferret up and support its body with one hand while using the other hand to auscultate the thorax and palpate the abdomen. for an active animal or one that bites, scruff the ferret and suspend it with all four legs off the table (fig. . ). most ferrets become relaxed with this hold, and the veterinarian can examine the oral cavity, head, and body; palpate the abdomen; vaccinate; and clean the ears. however, even scruffing may not work for fractious animals. to restrain a ferret for a procedure, hold it firmly by the scruff of its neck and around the hips without pulling the legs back. most ferrets struggle if their legs are extended by pulling on the feet. some animals can be distracted during a procedure by feeding a meat-based canned food or a small amount of a supplement such as ferretone ( -in- pet products, islandia, ny) by syringe. for very fractious or anxious animals or for procedures requiring lengthy restraint, light sedation or anesthesia may be indicated (see chapter ) . flexible digital thermometer that is well lubricated. the normal rectal temperature of a ferret is . °f to . °f ( . °c to . °c); however, a mean of °f ( . °c), with a wider range of °f to °f ( . °c to . °c), is also reported. physical examination of a ferret can be performed quickly and efficiently if a few simple guidelines are followed. observe the attitude of the animal. ferrets may sleep in the carrier in the veterinary office; however, once awakened for the examination, a ferret should be alert and active. assess hydration by observing the skin turgor of the eyelids, tenting of the skin at the back of the neck, and moistness of the oral mucous membranes. note that skin turgor can be difficult to evaluate in a cachectic animal. estimate the capillary refill time by digitally pressing on the gingiva. examine the eyes, nose, ears, and facial symmetry. cataracts can develop in both juvenile and adult animals. retinal degeneration occurs in ferrets and may be indicated by abnormal pupil dilation. inspect for nasal discharge, and ask the owner about any history of sneezing or coughing. the ears may have a brown waxy discharge, but excessive brown exudate may indicate infestation with ear mites. observe the facial symmetry. although uncommon, salivary mucoceles occur in ferrets and present as a unilateral swelling on the side of the face, usually in the cheek or temporal area (see chapter ) . the teeth should be clean and the gingiva pink. dental tartar is common in pet ferrets, possibly related to feeding kibble instead of natural prey. plaque buildup may be exacerbated by feeding a diet with a high mineral content. remove excess dental tartar by prophylactic techniques used in dogs and cats, and recommend measures to prevent tartar buildup. a pet toothpaste can be used to decrease the rate of calculus formation. , , gingivitis is a common sequela of excessive dental tartar. ferrets often break off the tip of one or both canine teeth, and the broken tooth may appear dark. however, ferrets rarely exhibit sensitivity associated with a fractured canine. if the ferret exhibits sensitivity when the tip of the canine is probed, recommend a root canal or extraction, depending on the degree of tooth damage (see chapter ) . bruxism often indicates gastrointestinal discomfort. palpate the submandibular, axillary, popliteal, and inguinal lymph nodes. nodes should be soft and may sometimes feel enlarged in overweight animals because of surrounding fat. any firmness or asymmetry warrants fine-needle aspiration or biopsy. if two or more nodes are enlarged and firm, a diagnostic workup is indicated. auscultate the heart and lungs in a quiet room. ferrets have a rapid heart rate ( to beats/min) and often a pronounced sinus arrhythmia. if a ferret is excited and has a very rapid heart rate, subtle murmurs may be missed. valvular disease, cardiomyopathy, and congestive heart failure are seen in ferrets, and any murmur or abnormal heart rhythm should be investigated further (see chapter ) . the ferret's normal respiratory rate is to breaths/min (see chapter ) . palpate the abdomen while either scruffing the ferret or supporting it around the thorax with one hand. this allows the abdominal organs to displace downward, facilitating palpation. if the history is consistent with an intestinal foreign body or urinary blockage, palpate gently to avoid causing iatrogenic injury, such as a ruptured bladder. palpate the cranial abdomen, paying attention to the presence of gas or any firm, irregularly shaped material in the stomach area, especially in ferrets with a history of vomiting, melena, or chronic weight loss. the spleen is often enlarged, which may or may not be significant, depending on other clinical findings (see chapter ) . a very enlarged spleen may indicate systemic disease or, very rarely, idiopathic hypersplenism, and further diagnostic workup is warranted. examine the genital area, observing the size of the vulva in females. vulvar enlargement in a spayed female is consistent with either adrenal disease or an ovarian remnant; the latter is rare. examine the preputial area and size of the testicles of male ferrets; preputial and testicular tumors are sometimes seen. check the fur for evidence of alopecia. tail tip alopecia is common and may be an early sign of adrenal disease. symmetric, bilateral alopecia or thinning of the fur that begins at the tail base and progresses cranially is a common finding in ferrets with adrenal disease. examine the skin on the back and neck for evidence of scratching. pruritus is common with adrenal disease and also may indicate ectoparasites (e.g., fleas or sarcoptes scabiei). palpate and visually examine the skin thoroughly for masses. mast cell tumors are common and are variable in size. often, the fur around a mast cell tumor is matted with dried blood from the animal's scratching. other types of skin tumors, such as sebaceous adenomas and basal cell tumors, are also common (see chapter ) . perform an excisional biopsy of any lump found on the skin. young, recently purchased ferrets need serial canine distemper vaccinations until they are weeks of age. rabies vaccines should be given annually beginning at months of age. ferrets should be examined annually until they are to years of age; then, older animals may need examinations twice yearly because of the high incidence of metabolic disease and neoplasia. annual blood tests are recommended for older animals. measure the blood glucose concentration twice yearly in healthy middle-aged and older ferrets; more-frequent monitoring is needed in ferrets with insulinoma. abdominal ultrasound scanning or an endocrine panel is indicated in ferrets with thinning fur on the tail or other clinical signs suggestive of adrenal disease (see chapter ) . testing for infectious diseases may be warranted, especially in new or young ferrets that will be introduced into a multi-ferret household or those that are taken to ferret shows. ferrets can be tested for aleutian disease virus and ferret enteric coronavirus by polymerase chain reaction testing (michigan state university, diagnostic center for population and animal health, www.animalhealth.msu.edu; veterinary molecular diagnostics, www.vmdlabs.com; zoologix, www.zoologix.com). serologic tests for aleutian disease by enzyme-linked immunosorbent assay (elisa) and counterimmunoelectrophoresis are also available (see chapter ). ferrets must be vaccinated against canine distemper virus (cdv). currently, one vaccine is approved by the u.s. department of agriculture for use in ferrets: purevax ferret (boehringer ingelheim animal health, duluth, ga). purevax is a canarypox-vectored recombinant vaccine that does not contain complete cdv or adjuvants; thus, post-vaccination risks are reduced. this product has a wide safety margin and has proved effective in protecting ferrets against cdv. although supply from the manufacturer has been intermittently problematic in the united states, the vaccine is available. canine distemper vaccines that were previously used in ferrets but are now discontinued include fervac-d (united vaccines, inc, fitchberg, wi), a modified-live virus vaccine propagated in avian cell lines, and galaxy d (schering-plough animal health/merck), a modified-live virus vaccine derived from the onderstepoort canine distemper strain and attenuated in a primate cell line. galaxy vaccines are now marketed under the nobivac (merck animal health, madison, nj) trade name. in a safety and efficacy study, galaxy d proved effective in preventing canine distemper in young ferrets challenged after serial vaccination. other canine distemper vaccines have been used off-label in ferrets in countries other than the united states or when purevax has been unavailable. recombitek cdv (boehringer ingelheim animal health) is also a recombinant canarypox vaccine approved for use in dogs that has been used in ferrets. this cdv vaccine is marketed in several multivalent combinations including cdv with parvovirus: a monovalent product is not available in the united states. nobivac puppy-dpv (merck animal health) is a modified live virus canine distemper vaccine combined with parvovirus vaccine, a virus that does not affect ferrets. although these vaccines have been used clinically in ferrets, their safety and efficacy in ferrets have not been studied. a cdv vaccine approved for use in mink (distemink; united vaccines inc, fitchberg, wi) is available in -dose vials only. because of the possibility of vaccine-induced disease, especially in immunosuppressed or sick ferrets, avoid using multivalent canine vaccines and do not use modified live cdv vaccines of ferret-cell or low-passage canine-cell origin. standard vaccination protocols for canine distemper in ferrets have been based on serial vaccinations of young ferrets at , , and weeks, with annual boosters. however, recent data suggest a modified vaccine schedule consisting of two initial vaccines with less-frequent boosters is effective. in an efficacy study of ferrets, % of ferrets that were initially vaccinated at weeks and given a booster vaccine between and weeks of age with one of three commercial vaccines (purevax ferret, fervac-d, or galaxy d) maintained protective antibody titers of > : for at least years. the three commercial vaccines did not differ in efficacy of eliciting protective titers. therefore an initial vaccination protocol of two vaccinations, starting at to weeks of age and separated by weeks, followed by a booster every years, should suffice in most cases (d. perpiñan, personal communication, may ). for ferrets at high risk of contracting canine distemper or when highly pathogenic strains of cdv are circulating, consider more-intensive vaccination protocols. if the ferret is first vaccinated after to months of age, a series of two vaccinations separated by weeks is sufficiently protective (d. perpiñan, personal communication, may ). all ferrets should be vaccinated against rabies. two inactivated (killed) rabies vaccines are approved for use in ferrets in the united states: imrab- or imrab- tf (boehringer ingelheim animal health) and defensor or defensor (zoetis, parsippany, nj). inactivated vaccines are effective at producing immunity for at least year. current recommendations are to vaccinate healthy ferrets at months of age at a dose of ml administered subcutaneously (sc). give booster vaccinations annually. titers develop within days of rabies vaccination. clinical signs of rabies in ferrets can vary. in studies of experimentally induced rabies in ferrets, clinical signs range from restlessness, apathy, and paresis to ascending paralysis, ataxia, cachexia, bladder atony, fever, hyperactivity, tremors, and paresthesia. , mean incubation period in experimental studies varies from to days. , virus is present in the brain tissue and salivary glands of inoculated ferrets, and virus is shed in the saliva to days after onset of illness. , ferrets are at least times less susceptible than skunks to rabies infection when fed mice carcasses infected with rabies virus. survival and clearance of rabies virus infection was reported in one ferret experimentally infected with rabies virus of skunk origin. the ferret initially exhibited hindlimb paralysis that resolved to paraparesis. no virus antigen was found at necropsy months after inoculation. ferrets are considered currently immunized days after the initial rabies vaccination and immediately after a booster vaccination. if a healthy pet ferret bites a person, current recommendations of the compendium of animal rabies prevention and control are to confine and observe the animal for days, during which the ferret should not be vaccinated. any illness that develops during observation should be reported immediately to the local health department. if signs suggest rabies, the ferret must be euthanized, and protocols for rabies evaluation followed. for a ferret with a current vaccine status exposed to a possible rabid animal, recommendations are to revaccinate the ferret within hours of exposure and then keep the ferret under the owner's observation and care for days. exposed ferrets that are overdue for a booster rabies vaccination should be evaluated on a case-by-case basis by the health department. an unvaccinated animal that is exposed to a rabid animal or a stray ferret that bites a person should be euthanized immediately and submitted for rabies testing. see the website of the centers for disease control and prevention (https://www.cdc.gov/ rabie s/specific_groups/veterinarians/) or the national association of public health veterinarians (http://www.nasphv.org/ documentscompendia.html) for specific guidelines. in ferrets, adverse events associated with vaccination are primarily type i hypersensitivity reactions or anaphylaxis. ferrets with mild reactions may exhibit pruritus and skin erythema. more severe reactions are typified by vomiting, diarrhea, piloerection, hyperthermia, cardiovascular collapse, or death. vaccine reactions are most common after canine distemper vaccination but may also occur after rabies vaccination. in a study of vaccine-associated adverse events in ferrets in the united states, the incidence of adverse events associated with rabies vaccine alone, canine distemper vaccine alone, and rabies and canine distemper vaccines together were . %, . %, and . %, respectively, with no significant difference among groups. however, occurrence of a vaccine-associated adverse event was significantly associated with the cumulative number of canine distemper vaccinations, with an % increase in risk of an adverse event with each additional distemper vaccine. the canine distemper vaccines used in this cohort of ferrets were purevax ferret and fervac-d; the two vaccines were grouped collectively in the analysis, and the incidence of adverse events associated with individual vaccines was not reported. all reactions occurred immediately after vaccination and most commonly consisted of vomiting and diarrhea. in another study of ferrets, the incidence of adverse events after administering either canine distemper ( . %) (fervac d), rabies ( . %) (imrab- ), or both vaccines ( . %) did not differ significantly between groups. in a report of vaccine reactions in ferrets reported to the u.s. pharmacopeia veterinary practitioners' reporting program, % involved administration of fervac d, % involved concomitant administration of fervac d and imrab , and % involved administration of imrab alone (purevax was not approved for use at the time these data were collected). according to merial's (now boehringer ingelheim) product information, the incidence of vaccine reactions with purevax is . %. no adverse events were reported in an efficacy study of vaccination of ferrets with either purevax ferret, fervac-d, or galaxy d. surveillance of vaccine-associated adverse events relies on voluntary reporting by practitioners. vaccine-associated adverse events can be reported to the center for biologics, u.s. department of agriculture (https:// www.aphis.usda.gov/aphis/ourfocus/animalhealth/veterinarybiologics/adverse-event-reporting). always follow the manufacturer's instructions for vaccine administration, and inform the owner of the possibility of an adverse reaction before vaccinating. because most reactions occur almost immediately after vaccination, have the owner monitor the ferret in the waiting area for minutes or more after vaccination with any product. if a ferret has an adverse reaction, administer an antihistamine (e.g., diphenhydramine hydrochloride, . to . mg/kg intravenously [iv] or intramuscularly [im]), epinephrine ( . mg/kg iv, im or intratracheally), or a short-acting corticosteroid (e.g., dexamethasone sodium phosphate, to mg/kg iv or im), and give supportive care. for any biologic product, veterinarians must assess risk versus benefit of vaccination. the treatment options for ferrets that have had a vaccine reaction are to administer diphenhydramine ( mg/kg orally [po] or sc) at least minutes before vaccination or not to vaccinate if exposure risk is minimal. vaccine injection-site sarcomas have been described in ferrets. , in one report, of fibrosarcomas in ferrets were from locations used for vaccination. fibrosarcomas had similar histologic, immunohistochemical, and ultrastructural features as those reported for feline vaccine-associated sarcomas. in cats, adjuvanted vaccines are most likely to be involved in tumor development; however, no definitive association was made between the fibrosarcoma and the type of vaccine in ferrets. ferrets appear less prone than cats to development of vaccine-induced tumors. gastrointestinal parasitism is most common in young or recently purchased pet ferrets and is relatively uncommon in mature ferrets in the united states. in a survey of ferrets that were either privately owned or in pet shops in italy, of ( %) were positive for ancylostomids (hookworms) and one ( %) was positive for sarcocystis. ferrets can be intermediate hosts or vectors of parasites from other natural hosts. protozoan parasites are occasionally seen. therefore perform routine fecal flotations and direct fecal smears for all young or recently acquired ferrets. coccidiosis (isospora species) usually is seen in young ferrets, which shed oocysts between and weeks of age. infection is often subclinical, although ferrets occasionally may have loose stool or bloody diarrhea. coccidiostats, such as sulfadimethoxine and amprolium, are effective and safe, and treatment should be continued for at least weeks. coccidia in ferrets may cross-infect dogs and cats; therefore check other pets in the household for coccidia and treat as needed. giardiasis is occasionally seen in ferrets. results of studies on molecular characterization and host specificity of giardia duodenalis isolates from pet ferrets vary. in one study, genetic sequences of giardia isolates from ferrets were similar to those of giardia associated with human infections. results of another study showed genetic sequences of giardia differed in ferrets and people and other mammals, suggesting that giardia isolates from ferrets may be host specific. giardia can be detected by identifying cysts or trophozoites in a fresh fecal smear or zinc sulfate flotation, or by fecal elisa. treat ferrets with giardiasis with metronidazole ( mg/kg po every hours for to days) or fenbendazole ( mg/kg po every hours for to days). cryptosporidiosis is described primarily in young ferrets. infection is associated with the ferret genotype of cryptosporidium parvum and therefore is an unlikely source of human infection. infection is usually subclinical and, although most ferrets recover within to weeks, can persist for months in immunosuppressed animals. oocysts of cryptosporidium are small ( - μm) and difficult to detect but can be found in fresh fecal samples examined immediately after acid-fast staining. , various drugs, including azithromycin, tylosin, and nitazoxanide, are used for treatment in dogs and cats, but efficacy in ferrets is not known. heartworms (dirofilaria immitis) can cause disease in ferrets. ferrets that are housed outdoors in heartworm-endemic areas are most susceptible to infection; however, all ferrets in endemic areas should be treated with heartworm preventive (see chapter and appendix). ear mites (otodectes cynotis) are common in ferrets, but affected animals may not exhibit pruritus or irritation. this mite species also infects dogs and cats, and animals in households with multiple pets can transmit mites to other animals. a red-brown, thick, waxy discharge in the ear canal and pinna characterizes infection. a direct smear of the exudate reveals adult mites or eggs. because ferrets normally have brown ear wax, the color or appearance of debris in the ear canal is not pathognomonic for mites. at the initial examination, check for ear mites and do follow-up checks at the annual examination in ferrets kept in multiple-pet households. several products, including selamectin, are effective in treatment (see chapter ) . flea infestation (ctenocephalides species) is most common in ferrets kept in households with dogs or cats. ferrets with chronic infestations can become severely anemic. check ferrets during the physical examination for signs of fleas or flea dirt. treat infested animals with products safe for use in cats, and institute flea control measures (see chapter ) . ticks are rarely seen on domestic ferrets, and lyme disease in ferrets has not been reported. ferrets can be hospitalized in standard hospital cages with some adaptations. ferrets are agile escape artists and often can squeeze through vertical cage bar openings on standard hospital cages. for housing ferrets, use only commercial cages with small spacing between bars or use cages with attached plexiglass fronts at least half the height of the cage door or higher to prevent escape. small animal intensive care cages or incubators also can be used to house ferrets and are especially useful for animals that need supplemental heat or oxygen. closely monitor the temperature in these cages when using supplemental heat to prevent hyperthermia or hypothermia. the cage should be large enough to accommodate a sleeping area and an area for defecation and urination. ferrets typically do not soil their sleeping area, even when very sick. all ferrets like to burrow and should be given opportunity to do so while hospitalized. clean towels or a mound of shredded paper make excellent burrowing material. take care with plastic-backed underpads, which ferrets may chew and ingest. small padded pet beds and fleece pet "pockets" work well as sleeping areas. provide water in either water bottles or small weighted bowls. before hospitalization, ask the owner which type of watering system the ferret is accustomed to. ferrets can be finicky eaters and should be fed their regular diet while hospitalized, if possible. otherwise, feed a very palatable ferret food or a premium high-protein cat/kitten kibble. for animals that are anorectic, force-feed a high-calorie semisolid food or supplement until the animal is eating on its own (see later discussion). most veterinary laboratories offer small mammal hematologic and biochemical panels that require . ml or less of blood. in-clinic, point-of-care analyzers require very small sample sizes (usually μl). the blood volume of healthy ferrets is approximately ml in average-sized females weighing g and ml in males weighing kg. up to % of the blood volume can be safely withdrawn at one time in a normal ferret; however, collect only the minimum amount needed for analysis. repeated blood drawing can contribute to anemia in sick animals hospitalized for long periods. obtaining a blood sample from a ferret is relatively easy. venipuncture usually can be done with manual restraint, but some veterinarians prefer sedating or anesthetizing especially active animals. several venipuncture sites are readily accessible; the technique and site chosen depend on how much blood is needed and the availability of assistants for restraint. anesthesia or sedation can be used if assistants are unavailable. if needed, ferrets often can be distracted during restraint for venipuncture by offering semisolid food or a product such as ferretone ( -in- pet products) by syringe. avoid using supplements with corn syrup or other sugars, because this will affect blood glucose levels, and collect blood for glucose determination or other fasting samples before offering food. the blood collection technique can affect hematologic test results. isoflurane anesthesia can cause decreases in all hematologic values beginning at induction of anesthesia and reaching maximal effects at minutes after induction. both isoflurane and sevoflurane can cause a decrease in packed cell volume. therefore hematologic results of blood samples collected while a ferret is anesthetized must be interpreted carefully (see chapter ) . two sites are commonly accessed to obtain large blood volumes in ferrets. jugular venipuncture can be accomplished by extending the ferret's forelegs over the edge of a table and the neck up, or by restraining the ferret in lateral recumbency (fig. . ) . have a second assistant restrain the ferret's hind end on the table to prevent twisting, or wrap the lower body in a towel. use a -gauge needle bent slightly to a -degree angle with a -to -ml syringe for venipuncture in most ferrets; a -gauge needle can be used in large males. shave the neck at the venipuncture site to enhance visibility of the jugular vein. the vein is relatively superficial and is located more laterally in the neck than it is in dogs or cats. once the needle is inserted, the blood should flow easily into the syringe; if the neck is overextended and the head is arched back, the blood may not flow readily from the vein. relax the hold on the head or gently "pump" the vein by moving the head slowly up and down to enhance blood flow into the syringe. with ferrets that resist limb extension, a towel-wrap technique can be used. scruff the ferret with its front legs extended caudally against the ventral thorax and wrap the animal's body firmly with a towel from the base of the neck down. have an assistant restrain the toweled ferret in dorsal recumbency while scruffing the neck or holding the head. however, with very fractious animals, even this technique may be difficult. the second venipuncture site from which to obtain large blood samples is the cranial vena cava. the site of venipuncture is the anterior vena cava or the right or left brachiocephalic trunk, depending on the point of entry and the depth of needle penetration (fig. . , a) . this technique is relatively safe in ferrets because of the long anterior vena cava and the caudal location of the heart in the thoracic cavity, which is approximately cm from the thoracic inlet. however, rare instances of hemorrhage into the anterior thoracic cavity can occur. for this technique, restrain the ferret on its back with the forelegs pulled caudally and the head and neck extended ( fig. . , b) . with manual restraint, two assistants are usually needed, one to restrain the forelegs and head and the other to restrain the rear just cranial to the pelvis. use a -gauge needle with an attached -ml or -ml syringe; insert it into the thoracic cavity between the first rib and the manubrium at an angle to degrees to the body. direct the needle toward the opposite rear leg or most caudal rib and insert it almost to the hub. pull back on the plunger as the needle is slowly withdrawn until blood begins to fill the syringe. if the ferret struggles, quickly withdraw the needle and wait until the ferret is quiet before making a second attempt. in very fractious or active ferrets, jugular venipuncture or use of tranquilization are safer choices to avoid lacerating the vessels. the lateral saphenous or cephalic vein can be used if only a small amount of blood is needed to measure a packed cell volume or blood glucose level. to prevent collapse of the vein during venipuncture, use an insulin syringe with an attached -or -gauge needle. the saphenous vein lies just proximal to the tarsus on the lateral surface of the leg; the cephalic vein is in the same anatomic location as in a dog. before venipuncture, shave the fur from the area to enhance visibility of the vein. venipuncture of the tail artery is possible but rarely used in pet ferrets. venipuncture at this site is painful and requires anesthesia. insert a syringe with a -gauge needle into the ventral midline of the tail directed toward the body. once the artery is entered to mm deep into the skin, slowly withdraw the plunger until blood fills the syringe. apply pressure to the venipuncture site for to minutes after withdrawing the needle. most commercial veterinary diagnostic laboratories provide laboratory-specific reference intervals for ferret hematologic and biochemical values (see chapter ) . published sources of reference intervals for both laboratory and pet ferrets are available. contains blood coagulation values determined in normal ferrets that can be used as guidelines in the absence of reference intervals specific to a laboratory or to coagulation analyzer equipment. indwelling intravenous catheters can be placed in the lateral saphenous or cephalic vein (fig. . ) in ferrets that are collapsed with poor blood pressure or in young or very small ferrets, attempts to place an intravenous catheter may be unsuccessful. an intraosseous catheter can be placed in these animals. the proximal tibia is the most common site for intraosseous catheter placement in small mammals, but the proximal femur can also be used and allows for a greater range of movement. unless the ferret is very depressed, anesthesia is required to place the catheter. sterilely prepare the insertion site and infiltrate the area with % to % lidocaine (maximum dose, mg/kg). insert a -or -gauge, . -inch spinal needle into the marrow cavity. alternatively, use a -or -gauge hypodermic needle with a surgical steel wire inserted into the lumen to prevent a bone plug from occluding the needle during insertion. the intraosseous catheter should occupy approximately % to % of the marrow cavity at the narrowest portion. administer drugs through intraosseous catheters in small volumes with minimal pressure to prevent leakage from the insertion site. if possible, change to an intravenous catheter as soon as the animal is rehydrated or blood pressure improves. intraosseous catheters should not be left in place for more than hours. vascular access ports can be placed when repeated vascular access is required for any reason, such as for chemotherapy. the technique used to place the catheter and port has been described and illustrated. hospitalized ferrets usually require fluid therapy to maintain hydration and correct dehydration. daily fluid requirements of ferrets have not been determined; however, calculating fluid requirements at a rate of to ml/kg per day appears to be adequate for maintenance. one source estimates daily water consumption of adult ferrets as to ml/day. provide additional fluids to compensate for ongoing fluid loss and to correct dehydration calculated as a percentage of the body weight. intravenous fluids are preferred in ill animals. if possible, administer intravenous fluids by continuous-rate infusion. alternatively, divide the calculated daily fluid volume into two or three doses and administer by a buretrol (baxter healthcare, glendale, ca) or syringe pump. depending on the clinical condition of the ferret, supplements and drugs can be added to fluids by using the same criteria and calculations as for dogs and cats. although subcutaneous fluids can be given, ferrets react painfully to subcutaneous administration, and adequate restraint is needed. administer subcutaneous fluids in the loose skin along the back and dorsal cervical area, dividing the calculated daily fluid volume into doses given two or three times daily. colloids are effective in improving intravascular fluid volume and oncotic pressure in ferrets that are hypoproteinemic or in shock. dosage and administration are like those used in other small animals (see chapter ). antibiotics and other therapeutic agents are usually given at dosages like those in cats per kg of body weight (see appendix). in hospitalized animals with an indwelling catheter, give medications intravenously if possible. however, monitor the leg where the catheter is placed carefully for phlebitis and cellulitis, particularly when caustic medications are administered. antibiotics can be administered im; however, if treatment continues over several days, subcutaneous administration is preferred in cachectic animals because of the limited muscle mass. pills are very difficult to administer to ferrets; therefore oral medications are most easily given compounded into liquid form. ferrets generally accept chicken and beef flavors of compounded medications but do not like the taste of fish flavors. pain management is important in the postoperative period, with diseases that create significant discomfort (such as gastrointestinal ulceration), and for traumatic injuries (see chapter ) . ferrets in pain may exhibit tachypnea, a stiff gait, a strained facial expression, teeth grinding, shivering, half-closed eyelids, aggression, focal muscle fasciculation, hiding, general malaise, bristling of tail fur, and appear "tucked" in the abdomen. , many analgesic agents are used effectively in ferrets, including opioids, α- agonists, nonsteroidal antiinflammatory drugs (nsaids), and local anesthetics. clinically, buprenorphine, butorphanol, or meloxicam or combinations thereof are most commonly used for hospitalized animals and outpatients (see chapter and appendix). although no studies have evaluated efficacy of meloxicam for analgesia in ferrets, a meloxicam dose of . mg/kg in ferrets achieves plasma concentrations considered effective in other companion animals. ferrets are sensitive to acetaminophen toxicity. the activity of uridine ′-diphospho (udp)-glucuronosyltransferase in their livers compares with that of cats. acetaminophen glucuronidation is slower in ferrets than in other nonfelid species. unlike cats, however, no genetic mutations are associated with this slow metabolism, and the exact cause is unknown. ibuprofen can be toxic in ferrets at high doses, and accidental ibuprofen ingestion of mg/kg can cause death. clinical signs of toxicosis are depression, coma, ataxia, recumbency, tremors, weakness, and death. therefore avoid acetaminophen in ferrets and use nsaids with caution. do not use nsaids in ferrets being treated with a corticosteroid for insulinoma or other disease. epidural administration of analgesics before surgery appears to be effective in controlling postoperative pain in ferrets undergoing procedures involving the abdomen, spine, pelvis, hind legs, tail, and perineum (see chapter ) . , the technique has been well described in ferrets and is similar to that used in other small animals. , epidural injection is contraindicated in cases of coagulopathy, sepsis, hypovolemia, skin infection, and local fractures. be careful in the immediate postoperative period when opioids, such as butorphanol or buprenorphine, are used. in cats, as well as in other species, opioids including hydromorphone, butorphanol, and buprenorphine are associated with an increase in body temperature. opioid analgesics also can cause pronounced sedation, and ferrets can remain very lethargic and immobile for long periods. because of these two factors, immobile ferrets can overheat quickly if a heating lamp or other focused heat source is used during anesthetic recovery. therefore closely monitor the body temperature of any immobile or lethargic ferret given opioids or sedatives to prevent overheating when using heat lamps or forced-air warming devices. many sick ferrets are either cachectic or hypoglycemic or have minimal body fat and require nutritional support. products formulated as recovery diets for carnivores are available and readily accepted by most ferrets (carnivore care, oxbow animal health, murdock, ne; emeraid carnivore, emeraid, cornell, il). ferrets can be syringe-fed meat-based soft foods marketed for hospitalized dogs and cats (maximum-calorie plus, the iams company, mason, oh; canine/feline a/d, hill's pet nutrition, topeka, ks). do not offer supplement gels that contain corn syrup because of the high sugar content. force-feed anorectic ferrets as much as they will accept comfortably, usually to ml syringe fed three or four times daily. ferrets that develop a taste for the food may eat it directly from a bowl. ferret owners often prepare homemade diets of "duck soup" or "chicken gravy" to nurse their pets at home. many different recipe variations are available, and most are based on whole chicken with additives ranging from beef fat, dog kibble, nutritional supplement gels, or brewer's yeast to echinacea capsules. the "duck soup" variations all provide a soft, porridge-consistency food that is usually readily eaten by sick and convalescing ferrets. some recipes are very high in fat and carbohydrates. unless the homemade diets are based on or used with a commercial ferret diet, they should not be used long term because of possible nutritional imbalances and deficiencies. although seldom used clinically, esophagostomy feeding tubes can be placed in ferrets to manage debilitated animals over the long term. the technique is like that used in cats. gastric feeding tubes have been placed in ferrets both experimentally and clinically. , in a study of ferrets, gastrostomy tubes were placed percutaneously by a nonendoscopic technique. a gastrostomy tube was placed and maintained successfully in a ferret after surgical repair of an esophageal perforation caused by an esophageal foreign body. total nutrient admixtures have been formulated to provide partial parenteral nutrition to ferrets. , depending on the osmolarity of the solution, parenteral nutrition solutions can be delivered via a central, peripheral, intraosseous, or intraperitoneal catheter; however, the relatively high protein requirements of ferrets usually result in a solution of to mosm/l, which should be administered into a large (central) vein. urine samples can be collected by cystocentesis or by free catch after natural voiding or gentle manual expression of the bladder. the techniques for manually expressing the bladder and cystocentesis are the same as those used in dogs and cats. anesthetize fractious ferrets to avoid trauma to the thin bladder wall. use a -gauge needle for cystocentesis. reference values for urinalysis are listed in table . . in one study, the reference range for urine ph in ferrets was reported as . to . ; however, urine ph can vary according to the diet. the normal urine ph in ferrets fed a high-quality, meat-based diet is approximately . . urinary catheterization is commonly indicated in male ferrets, but the procedure can be challenging. although techniques have been described for both sexes, in male ferrets, the urethral opening is very small and is located on the ventral surface of the penis, proximal to the j-hook in the end of the os penis. use a . -or . -fr polytetrafluoroethylene, silicone, or polyurethane urinary catheter (slippery sam tomcat urethral catheter, surgivet, smiths medical, dublin, oh; tomcat urethral catheter, surgivet; tomcat/small animal urinary catheter, mila international inc., florence, ky). alternatively, use a . -fr rubber feeding catheter; estimate the length of the catheter that must be inserted to reach the bladder before placing (see also chapter ). if using a long rubber catheter, use a stylet or flexible wire to stiffen the catheter if needed; the stylet can be retracted slightly while passing the catheter, but be very careful when rounding the pelvic flexure to avoid perforating the urethra. another option is to use a -gauge or -gauge, -inch jugular catheter with the stylet removed. to pass the urinary catheter, aseptically prepare the preputial area, locate the urethral opening, and introduce the catheter tip (fig . , a) . if the urethral opening is difficult to see, dilate the opening by passing a -gauge intravenous catheter just inside the tip of the urethra and flushing gently with saline solution (fig . , b) . slip the tip of the lubricated urinary catheter gently into the dilated urethral opening alongside the intravenous catheter and, while gently flushing with saline solution, pass the catheter into the bladder. often resistance is met at the pelvic flexure; if this occurs, try repeated gentle flushing and relubricating the catheter until it passes. once in place, if using a red rubber catheter, place butterfly tape strips around the catheter just as it enters the urethra and at another point to cm distal, and suture these to the skin (fig . , c) . if using a urethral catheter, suture the catheter hub to the prepuce (fig . , d) . attach a sterile urinary collection device and tape the tubing to the tail to further prevent tension. if needed, bandage the ferret's abdomen to minimize rotation of - - . ± . ph . ± . . - . . - . . ± . . - . the catheter and to restrict the ferret from traumatizing it. soft elizabethan collars are needed in some ferrets to prevent chewing at the catheter. maintain sterility of the collection system, and drain the bag by needle and syringe rather than opening the system (see also chapter ) . temporary tube cystostomy has been used successfully to manage male ferrets with urinary obstruction caused by adrenal disease. in four ferrets treated surgically, a -or -fr foley catheter was placed in the bladder at the time of adrenalectomy and left in place for to days. in these ferrets, immediate treatment of urinary blockage was by cystocentesis. a cystostomy catheter also can be placed by using interventional radiographic techniques. this is an option in obstructed ferrets in which a urethral catheter cannot be passed and surgery is considered high risk. a cystostomy catheter allows azotemic ferrets to be managed with diuresis before surgery; alternatively, in cases in which surgery is not an option, the catheter can remain in place pending response to medical management with leuprolide acetate. indirect blood pressure monitoring techniques, using both doppler ultrasonography and oscillometric methods, have been described in ferrets. , , , however, both methods poorly approximate direct arterial blood pressure measurements. indirect blood pressure readings may be inaccurate because the neonatal blood pressure cuff does not fit securely on the short legs of ferrets (doppler method), and pressure changes of the tail artery may not be sufficiently high to be detected by the oscillometric system. in one study, the indirect systolic blood pressure values measured by doppler on the tail of ferrets measured mmhg less than the direct arterial systolic blood pressure values. in a study using male ferrets that compared indirect blood pressure measurements on the tail, forelimb, and hindlimb using an oscillometric sphygmomanometer and a high-definition oscillometric monitor with direct arterial blood pressure measurements, measurements using the tail were considered most reliable. however, the oscillometric sphygmomanometer consistently overestimated the systolic arterial pressure. indirect measurements of systolic, mean, and diastolic arterial pressures were consistently higher during hypotensive states but substantially lower in hypertensive states than corresponding direct blood pressure measurements. in a study of healthy ferrets, sedation with butorphanol ( . mg/kg im) and midazolam ( . mg/kg im) was found optimal to allow indirect blood pressure measurement. reference values for indirect blood pressure measured with the cuff placed at the base of the tail were to mmhg systolic, to mmhg mean, and to mmhg diastolic arterial pressure. be aware of the above-described inconsistencies of indirect compared with direct blood pressure measurements and interpret clinical results carefully. in clinical situations, indirect blood pressure is most useful for evaluating changes in blood pressure from a known baseline or for surgical monitoring of general trends rather than for exact blood pressure measurement. the doppler method of indirect blood pressure measurement is most commonly used in ferrets. shave the hair on the ventral tail and place a no. ( to cm) cuff on the most proximal part of the tail and attach it to a sphygmomanometer. place the doppler transducer probe crystal with ultrasound gel on the shaved skin approximately cm distal to the cuff and tape or hold in place. alternatively, place the cuff just above the carpus or tarsus (overlying the radial artery on the front leg or the digital branch of the tibial artery on the rear leg, alternatively) or on the distal humerus. evaluating a bone marrow sample is a valuable diagnostic tool for many disease conditions, including anemia, thrombocytopenia, pancytopenia, proliferative abnormalities, and suspected hematopoietic malignancies. although the proximal femur is usually the most readily accessible site (fig. . ) , the iliac crest, tibial crest, and humerus can also be used to collect bone marrow samples. anesthesia is necessary to aspirate the bone marrow or perform a core biopsy. with the ferret in lateral recumbency, shave and aseptically prepare the area around the collection site. for the proximal femur, make a small incision over the greater trochanter. stabilize the femur with one hand while inserting a -gauge, . -inch spinal needle into the bone, medial to the greater trochanter. use steady pressure and an alternating rotating motion to advance the needle into the marrow cavity. withdraw the stylet and attach a -to -ml syringe to the needle to aspirate the marrow, stopping suction as soon as the sample is visible (to prevent blood contamination). to collect a core biopsy sample, use the same technique, but use a . -inch, -gauge needle in place of the spinal needle (see also chapter ) . collect samples from alternate sites by using the same basic technique. blood transfusions may be needed in ferrets that are anemic from chronic disease, blood loss, or estrogen toxicosis, or in ferrets that are thrombocytopenic. as in other species, evaluate the need for a transfusion based on the packed cell volume or platelet count and clinical status of the ferret. consider a transfusion if the packed cell volume is % or less in a ferret that exhibits clinical signs of anemia or requires surgery, or if a ferret is thrombocytopenic and exhibits ecchymosis, petechiation, or bleeding. ferrets lack detectable blood groups, and risk of transfusion reaction is minimal, even without cross-matching. because of a greater blood volume, large male ferrets are preferred over females as blood donors. depending on the size of the donor ferret, to ml of blood can be safely collected for transfusion. collect blood into an anticoagulant such as acid-citrate-dextrose or citrate-phosphate-dextrose-adenine (cpda) at a ratio of ml of anticoagulant to to ml of donor blood. ferret blood collected into cpda at a : ratio has a shelf life of days when stored at ° c; after days, deterioration of red blood cells (rbcs) causes a decrease in blood ph, glucose, and sodium and an increase in lactate and potassium. whenever possible, collect blood from donor ferrets shortly before transfusion. always use a filter when transfusing whole blood, and use at least a -gauge catheter to prevent cell lysis during the transfusion. intraosseous blood transfusions can be given to ferrets if an intravenous catheter cannot be placed. splenic aspiration is a common diagnostic technique in ferrets with enlarged spleens (see chapter ) . the technique is simple and can be done in unanesthetized ferrets; however, if a ferret is fractious, use an injectable sedative. an ultrasound-guided aspirate is preferred, especially if a splenic mass is present. alternatively, palpate the abdomen and immobilize the enlarged spleen next to the body wall and aspirate directly. a positive aspirate appears bloody. the most common findings on cytologic examination of a splenic aspirate are extramedullary hematopoiesis and lymphoma. analysis of cerebrospinal fluid (csf) as a diagnostic tool is occasionally indicated in ferrets that present with neurologic signs. the volume of csf fluid that can be removed safely is . ml/kg. for a csf tap, place the anesthetized ferret in lateral recumbency. using a -gauge, . -cm-long hypodermic needle, puncture the cerebromedullary cistern and allow free flow of the csf fluid. in clinically normal ferrets, reference values for csf tap are mean white blood cells (wbcs), . cells/μl (range - cells/μl); mean rbcs, cells/μl (range - , cells/μl; and mean protein concentration, . mg/dl (range - mg/dl). in a study of ferrets in which a csf tap was performed, rbcs of < cells/μl were obtained in % of cases, and the number of wbcs was significantly affected by blood contamination, but protein concentration was not. identification of genotypes of cryptosporidium parvum isolates from ferrets in japan molecular characterization of giardia duodenalis isolates from domestic ferrets antibody titers in domestic ferret jills and their kits to canine distemper virus vaccine parasites of domesticated pet ferrets susceptibility of carnivore to rabies virus administered orally percutaneous placement of a gastric feeding tube in the ferret coagulation values in normal ferrets (mustela putorius furo) using selected methods and reagents experimental rabies in the ferret (mustela [putorius] furo): susceptibility-symptoms-excretion of the virus simple technique for bleeding ferrets (mustela putorius furo) medical and surgical management of esophageal foreign body in a ferret acute ibuprofen toxicosis in a ferret meloxicam pharmacokinetics using nonlinear mixed-effects modeling in ferrets after single subcutaneous administration impact of diet on the dentition of the domesticated ferret compendium of animal rabies prevention and control. national association of state public health veterinarians acetaminophen udp-glucuronosyltransferase in ferrets: species and gender differences, and sequence analysis of ferret ugt a rabbit and ferret hemostasis first survey of endoparasites in pet ferrets in italy epidural anesthesia and analgesia in ferrets urine specific gravity values in clinically healthy young pet ferrets (mustelo furo) estimation of glomerular filtration rate and evaluation of renal function in ferrets (mustela putorius furo) esophagotomy feeding tube placement in the ferret normal clinical and biologic parameters incidence of adverse events in ferrets vaccinated with distemper or rabies vaccine: cases recovery from and clearance of rabies virus in a domestic ferret epidural analgesia in ferrets measurement of dietary and dentrifice effects upon calculus accumulation rates in the domestic ferret reference ranges for laboratory parameters in ferrets diagnosis and treatment of dental disease in ferrets viral diseases of ferrets anesthesia and analgesia in ferrets principles of transfusion medicine in small animals comparison of sevoflurane and isoflurane in domestic ferrets (mustela putorius furo) haematological and serum chemistry profiles of ferrets (mustela putorius furo) intraosseous catheterization of exotic animals a new type of urinary catheter for catheterization of the male ferret critical care monitoring comparative hemostasis in vertebrates reduction of calculus accumulation in domestic ferrets with two dentifrices containing pyrophosphate lack of detectable blood groups in domestic ferrets: implications for transfusion a technique for catheterization of the urinary bladder in the ferret effect of isoflurane on hematologic variables in ferrets use of behavior analysis to recognize pain in small mammals vaccine-associated adverse events laboratory management of the ferret for biomedical research incidence of and risk factors for adverse events associated with distemper and rabies vaccine administration in ferrets histology and immunochemistry of seven ferret vaccination-site fibrosarcomas vaccine injection-site sarcoma in a ferret pathogenesis of experimentally induced rabies in domestic ferrets viral excretion in domestic ferrets (mustela putorius furo) inoculated with a raccoon rabies isolate temporary tube cystostomy as a treatment for urinary obstruction secondary to adrenal disease in four ferrets evaluation of noninvasive monitoring techniques in domestic ferrets (mustela putorius furo) emergency and critical care of ferrets use of vascular access ports in exotic animals a technique for femoral bone marrow collection in the ferret occurrence and molecular typing of giardia isolates in pet rabbits, chinchillas, guinea pigs and ferrets collected in europe during assessment of a blood preservation protocol for use in ferrets before transfusion composition of cerebrospinal fluid in clinically normal adult ferrets effects of opioids and anesthetic drugs on body temperature in cats use of a vascular access system for administration of chemotherapeutic agents to a ferret with lymphoma cryptosporidiosis in ferrets parenteral nutrition support in rabbits and ferrets ibuprofen ingestion in ferrets: cases evaluation of an inactivated rabies vaccine in domestic ferrets update on the diagnosis and management of cryptosporidium spp infections in dogs and cats intra-arterial blood pressure in ferrets compared to peripheral blood pressure evaluation of epidural morphine for postoperative analgesia in ferrets (mustela putorius furo) hematology of the domestic ferret (mustela putorius furo) comparison of the blood coagulation profiles of ferrets and rats minimum protective dose (mpd) and efficacy determination of a recombinant canine distemper virus vaccine for ferrets the ferret, mustela putorius furo, as a new species in toxicology pain management in ferrets serum-neutralizing antibody responses to canine distemper virus vaccines in domestic ferrets (mustela putorius furo) non-invasive blood pressure measurement in sedated ferrets (mustela putorius furo): a study to find the optimal dosing regimen and reference ranges. faculty of veterinary medicine theses serologic evaluation, efficacy, and safety of a commercial modified-live canine distemper vaccine in domestic ferrets key: cord- -dcui lw authors: broadbent, andrew j.; boonnak, kobporn; subbarao, kanta title: respiratory virus vaccines date: - - journal: mucosal immunology doi: . /b - - - - . - sha: doc_id: cord_uid: dcui lw this chapter reviews the main viral pathogens of the respiratory tract, the immune responses they induce, currently available vaccines, and vaccines that are in development to control them. the main viruses responsible for acute respiratory infection in people include respiratory syncytial, influenza, human parainfluenza, human metapneumo-, human rhino-, corona-, and adenoviruses. licensed vaccines are available only for influenza virus, with vaccines against the other pathogens either in clinical trials or in preclinical stages of development. the majority of studies evaluating respiratory virus vaccines measure serum antibody responses, because, although both cellular and humoral responses contribute to the clearance of a primary infection, neutralizing antibodies are known to protect against secondary infection. humoral responses can be readily detected after vaccination with inactivated or subunit vaccines; however, fewer individuals seroconvert after vaccination with live vaccines. alternative immune mechanisms such as mucosal antibody responses are probably responsible for protection by live attenuated vaccines, and immune correlates of protection are under investigation. as we breathe, we sample an estimated l of air per minute (kohlmeier and woodland, ). the mucosa of our respiratory system is, therefore, in direct and continual contact with the environment and, as such, is highly exposed to microorganisms, some of which may be pathogenic. respiratory infections are among the leading causes of acute illness and mortality worldwide, being responsible for nearly million deaths annually, the majority of which occur in infants and children in developing countries (girard et al., ) . the main viruses responsible for acute respiratory infection include respiratory syncytial virus (rsv), influenza virus, human parainfluenza virus (hpiv), human metapneumovirus (hmpv), human rhinovirus (hrv), coronaviruses, and adenoviruses. however, despite the public health importance of these infections, licensed vaccines are currently available only for influenza viruses. protective immunity against respiratory virus infection is a complex interplay between systemic and mucosal responses. however, immune responses generated during a natural infection may not provide complete protection from reinfection and may actually contribute to the pathogenesis of disease (reviewed in sections pathogenesis and immune responses to respiratory virus infection). vaccine-induced immune responses must, therefore, aim to be more protective and less pathogenic than those induced naturally. in addition, our understanding of the relative contribution of mucosal and systemic immunity to protection remains incomplete. for example, it is well known that inactivated vaccines against influenza given intramuscularly (i.m.) are protective owing to the induction of systemic humoral immunity in the absence of a robust mucosal immune response, and current guidelines for vaccine licensure require influenza vaccines to induce systemic immune responses. however, it is also evident that some intranasal vaccines are protective owing to the induction of mucosal immunity, despite less impressive systemic immune responses (reviewed in section respiratory virus vaccines). unfortunately, standardized methods of measuring mucosal immune responses are lacking, and reliable correlates of protection for vaccines that protect through mucosal immunity have not been identified. in this chapter, we review the main viral pathogens of the respiratory tract, the immune responses they induce, current vaccines, and vaccines that are in development to control them. the viruses that infect the respiratory tract belong to various families and vary in their genome composition, the presence or absence of an envelope, and their replicative cycles. the majority of respiratory viruses that are responsible for acute respiratory infection belong to the paramyxoviridae family and include rsv, hpiv, and hmpv. these viruses infect cells lining the respiratory tract by first attaching to the cell through the interaction of viral envelope glycoproteins, with one or more cellular receptors in the host cell plasma membrane. for example, the hpiv envelope protein hn binds sialic acid residues extending from host cells (schomacker et al., ) , and the g protein of rsv and hmpv binds to glycosaminoglycans (gags) that comprise long chains of disaccharides that form part of the cellular glycocalyx (feldman et al., ) . the rsv and hmpv f protein are also known to bind gags, and findings indicate that the f protein of these viruses is involved in attachment by interacting with the cellular proteins nucleolin and integrin αvβ , respectively (tayyari et al., ; cseke et al., ) . upon binding to the host cell, the f protein undergoes a conformational change that exposes a hydrophobic fusion peptide, which is responsible for the fusion of the paramyxovirus envelope and the host cell plasma membrane. after viral fusion, the genome is released into the cytoplasm, viral genes are transcribed, and viral genomes are replicated (collins and crowe, ; collins and melero, ) . the paramyxovirus genome comprises single-stranded, negative-sense, nonsegmented rna. the viral rna (vrna) must first be transcribed into positive-sense messenger rna (mrna) before viral proteins can be translated by the host cell machinery. this is achieved by a viral rna-dependent rna polymerase (the large, l, protein) that is packaged into the virion and enters the host cell upon infection. the l protein is also responsible for genome replication, in which positive-sense complementary rna (crna) serves as an intermediate template for the production of vrna. an essential cofactor for the l protein is the phospho (p) protein, which tethers the polymerase so it can reach the bases in the vrna and also binds the n protein, which encapsidates the vrna and crna. there is also evidence that transcription is enhanced by the m - protein and that the switch from transcription to replication is mediated by the m - protein (collins and crowe, ; collins and melero, ) . once transcribed, viral structural proteins assemble and newly synthesized viral genomes are packaged into virions that bud from the plasma membrane. the matrix, or m protein, lines the inner surface of the viral envelope and may play a role in budding (henderson et al., ; teng and collins, ) . in addition, the hn protein of hpiv is also involved in budding and in clearing sialic acid residues from the plasma membrane (karron and collins, ) . to complete the replication cycle, paramyxoviruses have evolved multiple mechanisms to prevent the activation of cellular defenses in response to infection, such as the nonstructural (ns) proteins and of rsv (collins and crowe, ) and the c or v proteins of hpiv (karron and collins, ) . one additional protein found in paramyxoviruses is the short transmembrane glycoprotein (sh) that is anchored to the envelope and shares structural features with viroporins, a group of hydrophobic molecules that insert into the membrane of infected cells and increase their permeability to small molecules and ions (gonzalez and carrasco, ) . the orthomyxoviridae family includes influenza viruses, which bind to terminal sialic acid-galactose linkages by the hemagglutinin (ha) envelope glycoprotein. orthomyxovirus attachment to the host cell initiates receptor-mediated endocytosis and endosome acidification. protons are permitted to enter the influenza virion via the m ion channel, and acidification results in a conformational change in the ha protein, revealing the fusion peptide that initiates membrane fusion between the viral envelope and the endosome membrane (reviewed by palese and shaw ( ) ). the ha is synthesized as a precursor (ha ) that is cleaved into its active form (ha and ha ) by cellular proteases, and the amino acid sequence at the cleavage site determines the type of protease that is able to activate the ha. if trypsin-like proteases are required for cleavage, the virus is limited in its tissue tropism to the respiratory tract of humans and the gastrointestinal tract of birds; however, the presence of multiple basic residues at the cleavage site extends the range of proteases that can cleave the ha, resulting in a disseminated, often lethal, infection in poultry (wright et al., b) . once the virus envelope has fused with the endosome, the influenza genome enters the cytoplasm. the orthomyxovirus genome comprises seven or eight segments of singlestranded, negative-sense rna, and each segment encodes one or more proteins. each segment is encapsidated in nucleoprotein (np) and forms a panhandle comprising the ′ and ′ ends, to which a polymerase complex is attached. together, these are known as the viral ribonucleoprotein complex. in the nucleus, orthomyxovirus vrna is either transcribed into mrna or replicated by means of a positive-sense crna intermediate. viral mrna molecules exit the nucleus and are translated in the cytoplasm by the host cell machinery. structural proteins assemble at the plasma membrane, where newly synthesized viral genomes are packaged and virions bud (palese and shaw, ) . how the individual segments traffic to the plasma membrane and are packaged remain active areas of research. the matrix (m ) protein lines the virion beneath the envelope and may be important for morphology and viral assembly at the plasma membrane. in addition, the neuraminidase (na) protein permits budding by cleaving sialic acid residues from the host cell plasma membrane (palese and shaw, ) . to complete the replication cycle, influenza viruses inhibit interferon (ifn) production and signaling. this is achieved by the ns protein (palese and shaw, ) (section adenoviruses). the genomes of coronaviruses and rhinoviruses comprise positive-sense, single-stranded rna that can be translated by the host cell machinery in the cytoplasm (kennedy et al., ) . coronaviruses, which belong to the coronaviridae family, are enveloped and attach to host epithelial cells by the spike (s) envelope proteins (blau and holmes, ) . fusion occurs at the plasma membrane, or after endocytosis, and the genome is translated into a polyprotein, which is then posttranslationally processed into structural proteins that form viral particles and nonstructural proteins that aid in viral genome replication (lai et al., ) . rhinoviruses, which belong to the picornaviridae family, are not enveloped and instead have a capsid of icosahedral symmetry comprising four proteins, vp - (reviewed by greenberg ( ) , kennedy et al. ( ) ). the majority of rhinoviruses bind to intercellular adhesion molecule- (icam- ) (greve et al., ) , and binding leads to a conformational change in the capsid that creates a pore, through which the genome enters the cytoplasm to be translated and replicated (bella and rossmann, ) . adenoviruses are nonenveloped and possess a capsid of icosahedral symmetry. at each of the corners, a fiber protrudes from the capsid that makes contact with the host cell receptor to initiate receptor-mediated endocytosis. acidification of the endosome results in conformational changes in the capsid that lead to viral uncoating and the release of the double-stranded dna genome into the cell. the genome is transported into the nucleus, where it is transcribed into rna, which is alternatively spliced into monocistronic mrnas that are translated by the host cell machinery into early gene products. early gene products remodel the intracellular environment to favor viral replication and are responsible for viral replication. the late phase of the viral life cycle is concerned with the production of structural proteins in sufficient quantities to ensure adequate packaging of the newly synthesized genomes and maximizing the production of progeny virions, which are released by cell lysis (berk, ) . respiratory viruses can infect various parts of the respiratory tract and cause a range of illness. mild upper respiratory tract (urt) infection (urti) can be complicated by sinusitis or otitis media, and a lower respiratory tract (lrt) infection (lrti) can lead to bronchiolitis or pneumonia and possible postinfectious respiratory complications such as sensitization to asthma. the major public health impact is from lrtis, and rsv is responsible for the majority of cases in infants. hmpv, hpiv, and influenza can also lead to lrti, with hpiv and hmpv affecting infants almost as young as those afflicted with rsv, whereas hpiv , hpiv , and influenza are often diagnosed in children months of age or older. rsv and influenza are also recognized as an important cause of lrtis in the elderly and in those with cardiopulmonary disease or immunosuppression (schmidt, ) . moreover, influenza pandemics occur at irregular and unpredictable intervals with widespread morbidity and mortality and economic consequences. in addition, although there have been no cases of severe acute respiratory syndrome (sars) since , several novel coronaviruses have been identified, including the virus responsible for middle east respiratory syndrome (mers) (zaki et al., ) . given the clinical significance of these infections, and the fact that licensed vaccines are available only for influenza viruses, there is an unmet need for vaccines. humans are the only natural host for rsv, with infections occurring in annual epidemics during winter and spring months in temperate climates and the rainy season in the tropics (girard et al., ) . the virus is highly contagious, with most children being infected in the first year of life. the peak of severe disease usually occurs before months of age, with the peak incidence of hospitalization in -to -month-old infants (collins and melero, ) . reinfection is also common. in one study, among children who had been infected in their first year of life, % were reinfected in their second year, and % in their third year of life (glezen et al., ) . moreover, reinfection is independent of antigenic changes in the virus, implying that the protective immunity mounted during an infection does not protect against subsequent reinfection (collins and melero, ) . this is of note when attempting to induce protective immune responses by vaccination. globally, there were an estimated million cases of lrti caused by rsv in children under years of age in , with % requiring hospitalization (martinez et al., ) . in the united states, one study estimated that . million children under years of age require medical attention each year owing to rsv (botosso et al., ) , and another study estimated that rsv was responsible for , - , hospitalizations per year (girard et al., ) . in the united kingdom, the total annual incidence of hospitalization attributed to rsv was . per children under year of age and . per children between and years of age (waris, ) . more than half of the hospitalizations for rsv occur in previously healthy, fullterm infants, and children who experienced severe lrti caused by rsv were at increased risk of wheezing and asthma later in life (girard et al., ) . pediatric mortality from rsv in the united states was estimated to be between . per , per year in infants under year of age and . per , per year in children to years of age in one study (cooney et al., ) , and in another u.s. study, rsv was estimated to be responsible for - deaths per year (girard et al., ) . however, an estimated % of rsv-associated deaths occur in developing countries, possibly because of limited health-care resources (martinez et al., ) . in healthy adults, reinfection rates are approximately - % per year, though hospitalizations are rare. morbidity and mortality are more pronounced in the elderly and it has been estimated that rsv causes an average of , deaths annually in the united states, with % occurring in individuals over years of age (cooney et al., ) . in addition, the cost of caring for patients with severe lrti from rsv and its sequelae are substantial (girard et al., ) . rsv infection induces antibodies against the two main antigens, the f and g envelope glycoproteins. the g protein is the most variable protein in rsv and is the basis for the separation of strains into two antigenic groups (a and b). moreover, sites of positive selection that partially coincide with epitopes recognized by anti-gprotein monoclonal antibodies (mabs) suggest immunedriven rsv evolution (botosso et al., ) . however, most anti-g mabs do not neutralize infectivity (martinez et al., ) and the selection pressure is, therefore, weak. this favors a slow coevolution of several rsv lineages, and multiple genotypes within each group can cocirculate within the same season, with shifts in the predominance of groups a and b occurring in -to -year cycles (waris, ) . in contrast, the sequence of the f gene is highly conserved among rsv isolates, despite the identification of a number of neutralizing mabs against the protein that should impart a selection pressure for mutation (collins and melero, ) . this implies that the function of the f protein confers structural restrictions that may limit antigenic diversity. human parainfluenza viruses are also a common cause of acute respiratory infection, with % of children seropositive by years of age (cooney et al., ) . as with rsv, reinfection is common (schomacker et al., ) . there are four serotypes of hpiv (hpiv - ), with each serotype associated with a broad spectrum of upper and lower respiratory symptoms. hpiv and are, however, more frequently associated with croup (laryngotracheobronchitis), and hpiv is more likely to cause bronchiolitis, pneumonia, and lrti resembling disease caused by rsv (schomacker et al., ) . hpiv is a less frequent cause of clinically significant disease, though a study found hpiv in % of hpiv-positive samples in a day-care setting (fairchok et al., ) . hpiv lrti is a major cause of hospitalization in children under years of age, second only to rsv, though infection is usually self-limiting and rarely fatal, unless an individual is immunosuppressed. severe infection may have long-term effects on lung function, but this remains unclear (schomacker et al., ) . hmpv belongs to the same subfamily as rsv, and two major groups (a and b) and four minor subgroups (a , a , b , and b ) have been identified, based on sequence variability in the g and f glycoproteins (kroll and weinberg, ) . as with rsv, and hpiv, by years of age, most children will have been infected with hmpv, and reinfections are common (kroll and weinberg, ) . the virus also has a seasonal distribution, with the main occurrence in winter and spring (kahn, ) . hmpv typically leads to flu-like symptoms in otherwise healthy adults, but is responsible for - % of hospitalizations for lrti in children and can lead to severe disease in the elderly or immunocompromised hosts (papenburg and boivin, ; boivin et al., ) . influenza viruses are divided into types a, b, and c based on antigenic differences in the np and m genes. influenza a viruses are the most clinically significant and are divided into subtypes based on antigenic differences in the ha and na genes. to date, ha and na subtypes have been identified from waterfowl (palese and shaw, ) and th and th subtypes of ha have been identified from bats in guatemala and peru (tong et al., (tong et al., , . influenza viruses cause a spectrum of clinical illness associated with infection of the upper and lower respiratory tract, with more severe disease associated with lrti. the viruses are spread by respiratory droplets or direct contact. annual influenza epidemics have a seasonal distribution, with the main occurrence in winter months (seasonal influenza) in temperate climates (girard et al., ) . however, unlike rsv, hpiv, and hmpv, influenza a viruses have a broad host range that includes birds, pigs, dogs, horses, marine mammals, and humans, with the main reservoir for infection being aquatic birds (palese and shaw, ; wright et al., b) . this broad host range, together with the segmented nature of the influenza virus genome, makes the epidemiology of influenza complex and gives rise to zoonotic infections and pandemics. pandemic influenza can arise if a novel virus emerges that readily transmits from person to person and if the majority of the population is susceptible to infection. if an avian or animal virus crosses the species barrier to circulate in humans, the population will probably be immunologically naïve and, therefore, susceptible to infection. however, the virus must be able to transmit efficiently from person to person for a pandemic to occur. as the influenza virus genome is segmented, if a host is infected with two or more influenza viruses, the potential exists for the gene segments to reassort, such that a progeny virus containing genes from each parent virus can be produced (wright et al., b) . if a virus that circulates within the human population reassorts with one that is novel for humans, the resultant virus may possess genes that allow it to replicate efficiently in humans, but with glycoproteins to which the population is immunologically naïve, and a pandemic could occur. the introduction of a virus with a novel ha subtype into the human population is known as "antigenic shift" (wright et al., b) . three global influenza pandemics were recorded in the twentieth century from viruses of the subtypes h n , h n , and h n , respectively. in , another pandemic h n (ph n ) influenza virus emerged in mexico (girard et al., ) . this h n virus was antigenically unrelated to previously circulating seasonal h n viruses, and molecular studies revealed that it was a reassortant with genes derived from viruses that had been circulating in pigs: the north american h n triple-reassortant, the classical swine h n , and the eurasian "avian-like" swine h n viruses. in most countries, the median age of infection during the pandemic was estimated to be - years, and in most individuals, infection led to a mild, self-limiting urti. however, - % of confirmed cases in the united states and canada required hospitalization, and the case-fatality rate was . - . %. moreover, nearly one-third of the fatalities among hospitalized patients occurred in previously healthy individuals (girard et al., ) . after each pandemic, the newly emerged subtype became established and caused annual seasonal influenza epidemics. in the united states, it has been estimated that - million cases of influenza occur annually, with approximately , requiring hospitalization (lambert and fauci, ) . current vaccines are aimed at the circulating h n and h n subtypes of influenza a and the predominant circulating strain of influenza b and are therefore trivalent vaccines. however, two antigenically distinct lineages of influenza b viruses (victoria and yamagata) cocirculate, and the world health organization recommended that influenza vaccines should contain both of these lineages. clinical trials of quadrivalent vaccines containing the h n and h n influenza a viruses and the victoria and yamagata influenza b viruses have been conducted, and their use in the united states received an interim recommendation of the advisory committee on immunization practices for the - influenza season (dolin, ) . owing to the low fidelity of the rna-dependent rna polymerase of influenza, and immune selection pressure on the ha protein, viral replication can yield a quasi-species that may differ antigenically from the parent virus. therefore, each season, the predominant circulating strain may be antigenically distinct from the previous year. this phenomenon is known as "antigenic drift" and leads to a need to update the influenza vaccine annually (wright et al., b) . sporadic infections by h n , h n , h n , and h n subtypes of influenza have been reported in humans who were in close direct contact with infected animals. additionally, h n variant viruses have infected humans, the majority of whom were in close contact with pigs (epperson et al., ) . so far, there has been limited transmission of these viruses between people, though there is concern that they may acquire mutations or gene segments that allow efficient spread from person to person. coronaviruses are frequent causes of the common cold, causing urtis throughout the world, in all age groups, leading to millions of days of work and school absence, physician visits, and frequent inappropriate antibiotic use (greenberg, ) . coronaviruses are transmitted by respiratory droplets and are reported to cause - % of common colds, with a peak prevalence in late fall, winter, and early spring. the first human coronaviruses (hcov) to be recognized as significant respiratory pathogens, hcov- e and oc , were identified in the s (greenberg, ) . whereas infection with the majority of coronaviruses is associated with self-limiting urt symptoms in otherwise healthy individuals, a coronavirus was identified as the agent responsible for sars in (drosten et al., ; ksiazek et al., ) . the sars coronavirus (sars-cov) emerged in the guangdong province of china in november and spread to countries, leading to cases and deaths worldwide by the time the outbreak was brought under control in june (who, ) . subsequently, heightened international surveillance for coronaviruses led to the identification of the strains hcov-nl , nh, and hku in (greenberg, previously, hrvs were classified into two species, hrv-a (containing serotypes) and hrv-b (containing serotypes). in , a novel species, hrv-c, was identified, which contains at least serotypes (jacobs et al., ) . hrvs are spread by direct contact, hand-to-hand contact or aerosols. traditionally, they have been associated with a urti, causing between and % of common colds (makela et al., ) . however, they are increasingly recognized as a cause of lrti, particularly in patients with asthma and in infants, the elderly, and immunocompromised individuals (jacobs et al., ) . bronchiolitis is a common clinical manifestation in hospitalized children infected with hrv, and hrv is also a common pathogen in viral community-acquired pneumonia. hrv has also been associated with exacerbations of asthma and chronic obstructive pulmonary disease (jacobs et al., ) . as of this writing, serotypes and seven species of adenovirus have been identified. tissue tropism and clinical manifestations vary between the serotypes, and the viruses are responsible for both febrile respiratory disease and gastrointestinal illness (reviewed by lynch et al. ( ) ). adenoviruses are estimated to be responsible for - % of pediatric and - % of adult respiratory tract infections (ison, ; lee et al., ) . they are spread primarily via respiratory droplets, direct contact, or fomites, and more than % of cases are in children under years of age (lynch et al., ) . however, epidemics have also been described in children and adults, especially in military recruits in closed or crowded settings. most individuals develop a self-limiting urt infection that may be asymptomatic, but conjunctivitis, tonsillitis, otitis media, or croup can occur. infection can also spread to cause bronchiolitis or pneumonia or disseminate to cause viral meningitis or encephalitis that can be fatal (lynch et al., ) . there are many factors that determine the pathogenesis of disease and clinical outcome, including factors pertaining to the virus, host genetics, host immune responses, and the environment. the site of viral replication may influence the pathogenesis of disease and outcome of infection. for example, seasonal influenza viruses usually infect the epithelium of the urt, which is consistent with the most common clinical manifestations of seasonal influenza, whereas highly pathogenic avian influenza viruses of the h n subtype show a stronger tropism for the lrt than for the urt (kuiken et al., ) . h n viruses attach abundantly to "clara" or club cells lining the bronchioles, type ii pneumocytes lining the alveoli, and alveolar macrophages in the alveoli, consistent with the clinical manifestation of diffuse alveolar damage (kuiken et al., ) . rsv also targets both type i alveolar and nonbasilar epithelial cells and possibly alveolar macrophages, which may contribute to lrti (van drunen littelvan den hurk and watkiss, ). tissue tropism is determined, in part, by the receptor preference of the virus. for example, cells lining the urt of humans predominantly possess sialic acid residues with a terminal α , linkage to galactose, whereas cells lining the human lrt have both α , and α , linkages (shinya et al., ) . it is believed that the ability of an influenza virus ha to preferentially bind α , -or α , -linked sialosaccharides therefore partly determines the tissue tropism and hence the clinical outcome. when host cells are infected, type i ifn and proinflammatory cytokines are expressed, cellular translation is suppressed, and an antiviral state is induced (discussed in section immune responses to respiratory virus infection). however, most viruses that infect the respiratory tract modulate the host response to infection by blocking ifn activation and/or signaling and inhibiting apoptosis. this prevents the host from effectively clearing virally infected cells and inducing an antiviral state in neighboring cells, thereby promoting viral replication in infected tissues that may contribute to the observed pathology. rsv is the most effective paramyxovirus at subverting host cell responses, inhibiting apoptosis and type i ifn production and signaling by means of the ns and proteins, inhibiting nuclear factor-κb (nf-κb) activation through the binding and sequestration of cellular protein kinase r (pkr) by the viral n protein, and inhibiting the production of stress granules that can restrict viral replication (collins and melero, ) . influenza a viruses encode the ns protein that downregulates ifn production (palese and shaw, ) , and hpiv encodes either a c protein or a v protein that suppresses ifn induction and signaling (karron and collins, ) , whereas sars-cov encodes eight proteins that antagonize the ifn response by a variety of mechanisms (totura and baric, ) . in addition to blocking ifn activation and signaling, viruses employ other means of ensuring their replication in the face of host immunity. the rsv g protein is highly glycosylated, a feature that may inhibit the binding of antibodies, and the protein is highly variable, enabling a substantial population of immune-escape mutants to be produced during infection. additionally, a truncated, soluble form of the g protein is produced during infection, which acts as a decoy antigen that can bind rsv-specific antibodies, thus reducing their availability to neutralize virus. rsv also infects antigen-presenting cells (apcs), such as dendritic cells (dcs), and can affect their maturation and antigen-presentation functions and can lead to dysregulation of adaptive immune responses (van drunen littel-van den hurk and watkiss, ). a number of virulence determinants have also been identified in influenza viruses, including the ha and the polymerase complex. for example, the presence of a multibasic site in the ha gene renders viruses highly pathogenic in poultry, as they are able to replicate systemically (bosch et al., ; kawaoka and webster, ) . in addition, substitution of a glutamic acid (e) residue for a lysine (k) residue at amino acid position in the pb protein (e k) of the polymerase is associated with altered host range (subbarao et al., ) and virulence in humans and in mice that are experimentally infected with avian h n and h n viruses (hatta et al., ; munster et al., ; subbarao et al., ) . in the absence of the e k mutation, a pb d n mutation is also correlated with increased virulence (li et al., ) , and in the absence of both the pb e k and the d n mutations, pb s and r were found to contribute to ph n influenza virus replication and virulence (mehle and doudna, ). in addition, an n s mutation in the pb f protein has been shown to contribute to the virulence of h n and pandemic h n viruses (conenello et al., ) , and several virulence determinants have been identified in the ns protein, including d e, p s, l f, and i m, reviewed by kuiken et al. ( ) . there are several host factors that are known to contribute to the severity of respiratory virus disease and the outcome of infection. for example, it is known that the more severe rsv lrti in infants is associated with prematurity, chronic lung disease, congenital heart disease, and t cell immunodeficiency. other risk factors include low birth weight, multiple births, male gender, and low titers of maternally derived anti-rsv antibodies (groothuis et al., ; van drunen littel-van den hurk and watkiss, ) . additionally, low levels of vitamin d in cord-blood of healthy neonates is correlated with an increased risk of rsv lrti in the first year of life (belderbos et al., ) . infants are also at a greater risk of lrt disease from hpiv infection than older children; this has been attributed to smaller airways that are more susceptible to obstruction, immature immune responses, and the presence of anti-hpiv maternal antibodies that can suppress primary antibody responses (crowe and williams, ; karron and collins, ) . in adults, immunodeficiency, immunosuppression, or old age may lead to more severe illness (collins and melero, ) . in addition, a number of genetic polymorphisms have also been described in host genes that may affect the outcome of respiratory virus infection and disease severity. single-nucleotide polymorphisms have been identified in genes that encode surfactant proteins, such as surfactant proteins a, b, c, and d; pattern recognition receptors (prrs), such as toll-like receptors (tlrs); chemokines and cytokines, such as rantes, il- , - , - , - , - , - , - , and - , tnf-α, tgf-β, and ifn-γ; chemokine and cytokine receptors, such as ccr , il- ra, and il- ra; adhesion molecules, such as icam- , vcam- , and e-selectin; and hla molecules such as hla-a and -b, among others (miyairi and devincenzo, ; poland et al., ) . however, few consistent results have been obtained between studies, probably a result of differences in study design, sample size, etc., in addition to true variability. clearly the contribution of genetic polymorphisms to disease outcome is complex and remains an active area of research. as well as being responsible for viral clearance and protection against reinfection, the host innate and adaptive immune responses to respiratory viruses can lead to pathology and enhanced disease. this is especially important to bear in mind in vaccine development, as vaccine-induced immune responses must protect against infection without leading to immunopathology. excessive inflammation is an important component in the pathogenesis of respiratory virus infections. upregulation of il- leads to the recruitment of neutrophils to the site of infection, and although these cells may participate in virus elimination, in high numbers they can also cause pathology. upregulation of il- is known to correlate with rsv disease severity (goetghebuer et al., ) and, during an influenza infection, overproduction of cytokines such as tnf-α, il- , il- , and type i and ii ifns and chemokines can also result in the recruitment of immune cells to the site of infection and result in damage to lung tissue (cheung et al., ; de jong et al., ) . elevated il- /cxcl , mip α and β/ccl and , rantes/ccl , and cxcl have also been described in children with hpiv disease, with an association of more severe hpiv disease with high concentrations of il- and ip- (reviewed by schomacker et al. ( ) ). additionally, the rsv soluble g protein can lead to leukocyte recruitment by mimicking the chemokine fractalkine (tripp et al., ) and this can further exacerbate inflammation. pathogenesis can also be enhanced by an insufficiency of anti-inflammatory immune responses in the lung, such as the cytokines il- and tgf-β (carlson et al., ; lebouder et al., ; sun et al., ) , or insufficient numbers of immunosuppressive resident alveolar macrophages (rygiel et al., ; snelgrove et al., ) . dysregulation of adaptive immune responses can also lead to increased pathology, and a th -biased cellular immune response has been implicated in the immunopathogenesis of rsv disease (van drunen littel-van den hurk et al., ) . various defense mechanisms have evolved in the respiratory tract to prevent and control infection. currently, there is considerable effort to develop or improve vaccines against respiratory viruses. however, achieving this goal has been complicated by an incomplete knowledge of how the immune system recognizes, contains, and eliminates respiratory viruses. this section discusses the immune responses against respiratory virus infections, from the initiation of innate and adaptive responses following primary virus infection to the recall of immune responses during a secondary infection. in addition, advances in our understanding of respiratory mucosal immunity are discussed. a common feature of respiratory virus infections is that the initial infection is established in epithelial cells lining the respiratory tract. epithelial cells, as well as alveolar macrophages and dcs, are exposed to the contents of the airway lumen and detect the presence of an invading virus through prrs (holt et al., ) . the recognition of pathogen-associated molecular patterns (pamps) by these receptors initiates a cascade of signals that results in the production of cytokines and chemokines. the release of these inflammatory mediators into the surrounding environment establishes a local antiviral state. in addition, chemokines provide the necessary signals for the recruitment of leukocytes to the site of infection. finally, the combination of inflammatory cytokines and prrs initiates the process of dc maturation and trafficking that is required for the induction of adaptive immune responses (holt et al., ) . the best described of the prrs are those of the tlr family. with respect to respiratory viruses, tlr , , and recognize various products of viral replication (doublestranded rna, single-stranded rna, and unmethylated cpg dna, respectively) (alexopoulou et al., ; diebold et al., ; hagglund et al., ; lund et al., ) , and tlr recognizes the f protein of rsv (kurt- jones et al., ) . tlrs that recognize nucleic acids are located in late endosomes. this location optimizes the ability of tlrs to interact with viral nucleic acids while limiting their access to host-derived nucleic acids (heil et al., ; matsumoto et al., ) . although tlrs expressed on the cell surface or within the cell utilize different signaling pathways, each of these receptors can activate the transcription of ifninducing genes . viral rna is also recognized by cytoplasmic sensors such as rna helicases. the retinoic acid-inducible gene i protein interacts with ′-triphosphate rna and is important for early cytokine production in response to numerous rna viruses (hornung et al., ; pichlmair et al., ; yoneyama et al., ; kato et al., ; pothlichet et al., ; graham et al., ) . the melanoma differentiationassociated gene protein is a related helicase that recognizes polyinosinic polycytidylic acid and is crucial for innate recognition for picornaviruses and human metapneumovirus infection (banos-lara mdel et al., ) . similar to signaling through tlrs, the pathways utilized by rna helicases ultimately trigger ifn-regulatory factor and nf-κb activation (le goffic et al., ) . the key difference between these molecules and tlrs is that the rna helicases are localized throughout the cytosol, rather than being restricted to intracellular compartments. thus, pathogens such as paramyxoviruses that do not enter endosomes can trigger innate immune responses via rna helicases. the innate recognition of viral components through prrs described above leads to a program of gene expression that promotes a localized antiviral state and elicits the recruitment of inflammatory cells to the site of infection. type i interferons, including ifn-α and ifn-β, are most commonly associated with early antiviral responses in the lung, and numerous studies in mice and humans have shown that plasmacytoid dcs (pdc's) are the primary source of these cytokines after infection with viruses (asselin-paturel et al., ; mcgill et al., ) . however, there is a level of redundancy with respect to ifn production, with alveolar macrophages or pdcs predominating depending on the type of viral infection (pribul et al., ) . the precise contribution of pdcs to lung antiviral immunity is also controversial; several in vitro studies show that respiratory viruses, including influenza viruses, can infect pdcs and pdcs can activate virus-specific cd + t cells (wikstrom and stumbles, ; geurtsvankessel and lambrecht, ) , but evidence for a major role of pdcs in controlling influenza virus in vivo is absent. type i ifns produced after respiratory virus infection act in concert with prr signaling to form a feedback loop, by signaling through the ifn-α/β receptor to promote sustained production of proinflammatory cytokines such as tnf-α, il- , and il- by lung-resident innate immune cells (chan et al., ; nakajima et al., ; almansa et al., ) . these proinflammatory cytokines and prrmediated signals also prompt alveolar macrophages, dcs, and epithelial cells to initiate a coordinated program of chemokine production after viral infection. for example, dcs secrete successive waves of chemokines after influenza virus infection, beginning with those capable of recruiting inflammatory cells such as neutrophils and natural killer (nk) cells to the lung, followed by chemokines associated with the recruitment of monocytes and t cells (piqueras et al., ; marois et al., ; teijaro et al., ) . the cytokines il- β, il- , and il- activate monocytes, macrophages, and neutrophils and drive the development of cd + t cell adaptive responses in both mice and humans. cd + t cell differentiation in the presence of il- β, il- , or il- results in th , th , or th effector cells, respectively (chung et al., ; dinarello, ; lasiglie et al., ; ohno et al., ). these cytokines are processed as a result of caspase- activation, and activation of caspase- is regulated by the inflammasome, a large mutimeric structure (martinon et al., ) . a subgroup of the nucleotide-binding domain, leucine-rich repeat-containing proteins (nlrps) are key mediators of the inflammasome. activation of the inflammasome can be divided into two categories: activation driven by host-and environmentderived molecules and activation driven by pathogen-associated activators, including pamps derived from bacteria, virus, fungus, and protozoa. paramyxoviruses such as rsv and orthomyxoviruses such as influenza viruses can activate the nlrp inflammasome (kanneganti et al., ; segovia et al., ; komune et al., ) , which has been shown to play a critical antiviral role in influenza virus-infected mice (thomas et al., ; ichinohe et al., ; allen et al., ). after infection of the lrt, antigen-bearing mature dcs enter the lymph nodes draining the lung, where they form stable interactions with naïve t cells specific for that antigen through t cell receptors (tcrs). signals delivered by antigen recognition, in addition to accessory signals delivered through costimulatory molecules, result in t cell priming and the clonal expansion of antigen-specific effector t cells (moon et al., ; obar et al., ) . the instructions delivered by dcs during the initial expansion phase can have a dramatic impact on the survival and function of the responding t cells. for instance, expression of fasl on dcs after influenza infection has been shown to regulate the magnitude of the cd + t cell response (legge and braciale, ) . resident cd α + conventional dcs in the mediastinal lymph node also mediate the induction of protective immunity to influenza virus, and these cells have been found to cross-present viral antigens to cd + t cells without being directly infected by the virus (belz et al., ) . after activation and clonal expansion of antigen-specific effector t cells in the draining lymph nodes, the cells lose their preference for the lymphoid tissue and migrate via the bloodstream to the site of infection, where the antiviral mechanisms described below are exerted. analysis of chemokine expression in the lung during the adaptive phase of the immune response has shown elevated expression of numerous molecules associated with effector t cell trafficking (monick et al., ) . for instance, the trafficking of effector t cells during rsv infection is partially dependent on cxcr , and this chemokine receptor may play a role in other paramyxovirus infections (harcourt et al., ) . the appearance of antigen-specific effector t cells at the site of virus infection is first observed around - days after infection with influenza and parainfluenza viruses in mice (pommerenke et al., ; kohlmeier and woodland, ; lawrence and braciale, ; roman et al., ) . the continual migration of effector t cells from lymphoid tissues during acute infection results in a massive increase in the number of antigen-specific cells in the lung airways and parenchyma from to days after influenza virus infection (flynn et al., ) , and the arrival of effector t cells has an immediate and dramatic impact on the viral load (kohlmeier et al., ) . the adaptive immune responses induced after a respiratory virus infection are shown in figures - . upon arrival at the effector site (figure ), antigen-specific t cells first interact with apcs such as dcs (shen et al., ) . moreover, a subset of dcs, classed as cd c hi , present a crucial t cell survival factor (il- ) to antiviral cd + t cells in trans (mcgill et al., ) . immune responses are determined by the cytokine milieu in the respiratory tract, as well as the type and level of costimulatory molecules expressed by apcs. for instance, lung dcs are biased to promote th responses, most likely via the production of il- in the absence of the th -prone il p cytokine or through the production of leukotriene ltc (dodge et al., ; barrett et al., ) . effector t cells employ one of the following three antiviral mechanisms ( figure ). first, t cells can promote the lysis of infected cells by exocytosis of granules containing perforin and granzyme (trapani and smyth, ; hou and doherty, ) . second, t cells can induce apoptosis of infected cells by expressing cd (fas) ligand (fasl) hou and doherty, ) or tnf-related apoptosis-inducing ligand (trail) (brincks et al., ) . third, t cells can produce proinflammatory and regulatory mediators, such as ifn-γ, after an encounter with virally infected cells (hamada et al., ) . several studies suggest a crucial role for the cytolytic functions of cd effector t cells in influenza virus infection. the direct lysis of infected cells requires tcr-mediated recognition of processed viral antigens on the infected target cell (brincks et al., ; topham and doherty, ) . in contrast, the release of proinflammatory mediators such as ifn-γ by cd + t cells has only a modest impact on virus clearance and recovery. there is also evidence from models of influenza infection that infected alveolar epithelial cells may be eliminated by the host response through the action of macrophages capable of triggering apoptosis through a trail-dependent mechanism (herold et al., ) . effector cd + t cells have also been found to exhibit cytotoxic activity in vitro, but the contribution of this mechanism to virus clearance in vivo is modest (agrewala et al., ; graham et al., ) and is restricted to the cytolysis of cells that bear viral antigens presented by major histocompatibility complex (mhc) class ii molecules. such cells include cd + mononuclear phagocytic cells, and a few cd − lung parenchymal cells, such as type ii alveolar epithelial cells, that express mhc class ii molecules in either a constitutive or an inducible manner (debbabi et al., ) . the primary role of antiviral cd + t cells is to support the activation and differentiation of b cells, which leads to antibody production (topham et al., ; topham and doherty, ) as discussed below. another aspect of effector t cells is their potential to exert cytotoxic activity and simultaneously produce cytokines such as ifn-γ. for example, during an influenza infection, the interaction of primed cd + t cells with lung dcs elicits both cytokine production and cytotoxic phenotypes (hufford et al., ) . to summarize the cellular immune responses during a primary influenza infection, specific cd + and cd + effector t cells in the lung predominantly produce ifn-γ and figure upon virus infection, viral antigen from dying respiratory epithelial cells is transported from the lung to the lymph node by migratory antigen-presenting cells such as mouse cd + dendritic cells (dcs) or human monocyte-derived dcs that can stimulate both cd + and cd + t cell responses. viral antigen can also be transferred to lymph node-resident cd α + dcs and presented to naïve cd + t cells. in addition, plasmacytoid dendritic cells (pdcs) are an early source of antiviral type i interferon via recognition of viral rna. tnf-α, and cd + effector t cells also produce il- and il- (carding et al., ; pipeling et al., ; mayer et al., ) . cd + effector t cells localize to the respiratory epithelium and induce apoptosis of infected epithelial cells through fas-fasl interactions or the exocytosis of cytolytic granules containing perforin and granzyme tripp et al., ) . b cells are found interspersed in the lung interstitium and in the cervical and mediastinal/bronchial lymph nodes that drain the upper and lower respiratory tract, respectively. during a respiratory virus infection, a tertiary lymphoid structure also forms along the branching point of the bronchial tree, called the bronchus-associated lymphoid tissue (balt) (brandtzaeg, ) . the balt contains organized b cell areas, germinal centers, and antibody-forming cells (randall, ) . b cell responses can be classified into three categories: innate-like b cell responses, t-dependent b cell responses, and t-independent b cell responses. innate-like b cell responses consist of antibodies produced almost exclusively from b- cells, a small subset of b cells characterized by a unique developmental origin, phenotype, tissue distribution, and regulation, compared with conventional b cells (baumgarth et al., ; baumgarth, ) . t-dependent b cell responses are b cell responses that are facilitated by cd t cells, whereas t-independent b cell responses are not. it has been shown that cd t cell deficiency results in a drastically reduced humoral response to influenza virus infection in mice (mozdzanowska et al., ) . however, mice lacking cd and cd t cells are still protected from lethal infection mozdzanowska et al., ) , highlighting the importance of t-independent b cell responses. b cell responses are critical for viral clearance in primary respiratory virus infections, such as influenza (gerhard, ) . although control of early infection ( - days postinfection) is not impaired in b-cell-deficient mice, the mice fail to clear the virus and ultimately succumb to infection (graham and braciale, ; lee et al., ) . studies in influenza virusinfected mice also show that serum antibody titers are first detected around - days postinfection, at least days later than responses are detected in the respiratory tract. they steadily increase for about a month, after which relatively high figure during the effector phase of the response, influenza-specific cd + t cells elicit a cytotoxic response. tnf-α and nitric oxide (no) produced from a subset of dcs and neutrophils contribute to both viral clearance and immunopathology. lung cd c hi dcs present the crucial t cell survival factor il- to antiviral cd + t cells in trans and promote the production of chemokines. in draining lymph nodes, cd b + dcs contribute to the expansion of specific cd + t cells, and conventional dcs (cdcs) induce the th response. plasmablasts initially produce igm, and effector cd + t cells provide help for virus-specific b cell/plasmablast differentiation and proliferation. pdcs also play a detrimental role by eliminating virus-specific cd t cells in a process involving fasl. antibody titers are maintained for life. virus-specific antibodyforming cells reside transiently in the spleen, from around or days postinfection, and persist for the long term in the bone marrow (jones and ada, ; hyland et al., ) . after recovery from an infection (figure ), a state of immunological "memory" ensues, in which the individual is better able to control a subsequent infection with the same pathogen (ahmed and gray, ) . immunological memory is maintained by both t and b cell subsets, and there are profound differences in the generation, trafficking, and maintenance of t and b cell memory. antigen-specific memory t cells persist at increased frequencies, have a reduced requirement for costimulatory signals in comparison to naïve t cells, and respond quickly to antigenic restimulation (woodland et al., ) . in the case of influenza and parainfluenza (piv) virus infections, it has been clearly established that both cd + and cd + memory t cell subsets respond to and control secondary infection (woodland and dutton, ) . b cell memory is characterized by two distinct populations: long-lived plasma cells that continually secrete antibody and memory b cells that persist in a quiescent state (bachmann et al., ; slifka and ahmed, ) . the generation of long-lived plasma cells is dependent on cognate t-b cell interaction and cd signaling that occurs in the germinal center (noelle et al., ; lee et al., ) . antigen-specific igg and iga antibodies are maintained long after infection and may be protective against heterologous strains of virus. for example, up to % of people born between and in finland had preexisting antibodies to the ph n influenza virus, probably because of its relationship to the h n pandemic influenza virus that circulated in the first part of the twentieth century (ikonen et al., ; yu et al., ) . the presence of crossreactive antibodies contributed to the unexpectedly low numbers of the elderly with severe illness during the pandemic, compared with seasonal influenza virus strains (monsalvo et al., ; o'donnell et al., ) . although neutralizing antibodies directed against the ha globular head are highly efficient at preventing and clearing influenza virus infection, they can also figure in the memory phase, migratory lung dcs capture viral antigen retained on follicular dcs (fdcs) in tertiary lymphoid organs and present it to specific t cells in the respiratory draining lymph nodes. these stimulated t cells upregulate the expression of cd , causing them to be retained in the lymph nodes that drain the site of primary infection. resident dcs are able to reactivate memory responses in the lymph nodes. lung dcs can promote the production of iga in a process that depends on tgf-β. the generation of b cell memory, particularly the generation of long-lived plasma cells, is dependent on cognate t-b cell interaction and cd signaling that occurs in the germinal center. provide a selective pressure for viral immune evasion. crossprotection against various influenza a subtypes, termed heterosubtypic immunity, requires the immune system to recognize epitopes that are conserved between subtypes. such epitopes can be found in the membrane-proximal stalk region of ha (han and marasco, ) or in internal proteins such as nucleoprotein or the m protein. anti-stalk antibodies do not inhibit virion binding to mammalian host cells, but inhibit fusion between the viral envelope and the endosomal membrane. they have broadly neutralizing activity and passively protect mice from lethal challenge in vivo (okuno et al., ; throsby et al., ; sui et al., ). interestingly, cross-reactive anti-ha stalk monoclonal antibodies have been generated from the acute response to h n pandemic virus and also from healthy subjects vaccinated with inactivated virus (corti et al., ; sui et al., ; wrammert et al., ) . how to induce high-titers of anti-ha stalk antibodies in humans remains an active area of universal influenza vaccine research. cellular immune responses to cross-reactive epitopes (often expressed on internal viral proteins) also provide a substantial degree of protection against serologically distinct viruses (yewdell et al., ; rimmelzwaan and osterhaus, ) , and although these heterosubtypic cellular responses are not able to prevent reinfection, they can ameliorate disease by reducing the maximal viral load, mediating faster viral clearance, and providing a substantial degree of protection against challenge with a lethal dose of virus in animal models (hillaire et al., ) . there is also some epidemiological evidence that heterosubtypic cellular immunity plays a role in the response to infection with novel influenza viruses in humans; however, the protective effect appears to be weak and may wane over time (epstein, ; epstein and price, ) . it has, therefore, been suggested that protective cellular immunity could be sustained by reinfection or annual immunization. the effector mechanisms of heterosubtypic immunity remain ambiguous . in murine models of influenza a virus infection, heterosubtypic immunity is observed in the absence of antibodies that recognize influenza envelope glycoproteins and is thought to be mediated primarily by cd + t cells, with a relatively small contribution by cd + t cells mckinstry et al., ) . heterotypic influenza-specific cd + t cells have also been shown to lyse influenza virus-infected cells (nguyen et al., ) . however, heterosubtypic immunity has been observed in cd + t-cell-deficient mice, but not in mice lacking b cells , indicating that there is redundancy in the system. the same investigators found that the heterosubtypic immunity does not require ifn-γ (nguyen et al., ) , but does require a properly diversified antibody repertoire (nguyen et al., ) . the mucosal immune system has been described in detail elsewhere in this book; however, there are some features unique to the respiratory tract that are worth noting. like the immune system in general, the mucosal immune system of the respiratory tract uses innate and specific mechanisms to prevent and limit infection. innate defenses include physical and chemical factors such as the secretion of mucus, which traps microorganisms and antigens and facilitates their transport out of the body by mucociliary motion. mucosal secretions also contain chemically active substances, including acids, lactoferrin, and lysozyme, which inhibit the growth of microbes. in addition, the luminal side of the respiratory tract is physically protected by layers of epithelial cells that adhere to each other at tight junctions, using occludin and various members of the claudin family, and at adherent junctions using e-cadherin (tsukita et al., ) . in humans, the respiratory tract can be divided anatomically into the urt, which comprises the nose, mouth, and pharynx, and the lrt, which includes the trachea, bronchi, and lungs, with the lymphoid tissue of waldeyer's ring representing the line of demarcation. the unpaired nasopharyngeal tonsils (also called the adenoids) and the palatine and lingual tonsils constitute most of waldeyer's ring, with the tubal tonsils and lateral pharyngeal bands as less prominent components (dolen et al., ) . this lymphoid tissue is functionally comparable to the nasal or nose-associated lymphoid tissue (nalt) in rodents, which is composed of two paired lymphoepithelial structures beside the nasopharyngeal duct, dorsal to the cartilaginous soft palate (kuper et al., ; fukuyama et al., ) . however, rodents do not have tonsils. waldeyer's ring is more strategically situated than the nalt to generate mucosal immunity, because its elements are exposed to both airborne and alimentary antigens. in addition, human tonsils have deep antigen-retaining crypts, and tonsils express germinal centers shortly after birth, whereas the rodent nalt has a plain surface and requires an external stimulus to induce the expression of germinal centers (brandtzaeg, ) . tissue equivalent to waldeyer's ring has also been found in nonhuman primates (loo and chin, ; harkema et al., ) and horses (mair et al., (mair et al., , , but functional studies have not been performed in these species. the mucosa-associated lymphoid tissues (malt), including the nalt and balt, consist of follicle-associated epithelium and t-cell-and b-cell-enriched areas. the initiation of antigen-specific immune responses occurs at special gateways, which comprise microfold (m) cells located in the epithelium overlying the malt follicles. the cilia of the apical side of the m cells are shorter than those of conventional epithelial cells, and on the basal side, there is a large pocket-like structure that can hold immunocompetent cells required for the generation of immune responses such as t cells, b cells, and apcs. as lysosome development in m cells is poor, in most cases antigens pass through the cells unmodified and are taken up by dcs in the pocket (sato and kiyono, ) . upon encountering antigen, dcs migrate to the t cell region of the malt and present peptide antigen via mhc molecules to naïve t cells. antigen-specific t cells become primed, clonally expand, and leave the follicle to enter the circulation. they then home to effector sites to elicit mechanisms involved in viral clearance as described above. in the b cell region, a germinal center forms and antibody class switching occurs (cerutti, ) . a class switch to iga predominantly occurs in the malt owing to the action of the iga-associated cytokine family of tgf-β, il- , il- , il- , il- , and il- (mcghee et al., ; mestecky and mcghee, ; cerutti, ) . postswitched iga + b cells leave the malt through efferent lymph vessels under the control of the lipid mediator, sphingosine- -phosphate, and the cells then enter the circulatory system (gohda et al., ; lazarus et al., ) and home to effector sites found in unorganized lymphatic tissue spread over the lamina propria that underlies the mucosal epithelium. in the lamina propria, b cells differentiate into plasma cells and secrete iga, igd, igm, and igg antibodies, although iga is the major mucosal antibody isotype (shikina et al., ; shimoda et al., ) . surgical removal of the murine nalt or cervical lymph nodes does not, however, abrogate cellular or antibody immune responses to experimental influenza infection, suggesting that there may be additional inductive sites other than the nalt and demonstrating that dissecting the relative contributions of anti-influenza immunity is difficult. in the urt, iga is the major mediator of immunity to influenza. in mice that had recovered from an influenza infection, immunity to reinfection was abrogated by the intranasal instillation of anti-iga antibodies, but not anti-igg or igm (renegar and small, a) , and intravenous passive transfer of iga resulted in iga in nasal secretions that protected mice from intranasal challenge with influenza (renegar and small, b) . after passive transfer, nasal iga titers that conferred protection were at a concentration equivalent to that seen in convalescent mice, whereas igg transudation into the urt could be detected only after . times the normal convalescent serum titer had been passively transferred (renegar et al., ) . moreover, recombinant iga is sufficient to prevent influenza transmission in a guinea pig model (seibert et al., ) . in humans, iga is present in monomeric and dimeric forms. during transcytosis through mucosal epithelial cells, an extra polypeptide secretory component is added to dimeric iga and the resulting molecule is known as secretory iga (s-iga). dimeric and s-iga are - times more efficient than monomeric iga at neutralizing influenza viruses (renegar et al., ) . dimeric and s-iga are represented by two subclasses, iga and iga , with covalently or noncovalently joined dimers, respectively. both subclasses are detected in nasal secretions after an influenza infection; however, ha preferentially stimulates an iga response (brown et al., ) . s-iga is not considered to be inflammatory because the fc region is not available to activate immune cells or bind complement. s-iga is also resistant to proteolysis and can neutralize viruses inside epithelial cells and transport viruses that have passed the epithelial barrier to the lamina propria back to the lumen (sato and kiyono, ) . s-iga may therefore be useful in preventing viruses from breaching the mucosal barrier, while avoiding immunopathology by not activating inflammatory responses directly and by limiting the number of antigen-antibody complexes in the lamina propria that can trigger inflammation. it is currently thought that plasma igg serves as a backup for s-iga in the urt, whereas in the lrt, igg is the dominant antibody involved in protection (renegar et al., ) . the fc receptor for igg mediates transport of igg across epithelial barriers by transcytosis, permitting the transudation of igg from the serum into the lung where it is able to neutralize viruses (spiekermann et al., ) . this explains why passively transferred igg is effective at preventing severe disease from respiratory infections in experimental animals and why serum igg antibodies are the main correlate of protection for parentally administered inactivated influenza vaccines in humans (section respiratory virus vaccines). viruses that can cause repeated infection are typically characterized either by a failure to induce robust immunity or by significant antigenic diversity in the face of protective immune responses. influenza viruses can reinfect hosts because the antigenic sites evolve and drift to avoid neutralization by prior immunity. infection induces a strong homosubtypic neutralizing antibody response in healthy individuals that contributes to recovery and protection from repeat influenza virus infection with homologous virus or an antigenically similar virus (wrammert et al., ) . moreover, natural infection can lead to long-lasting immunity to the infecting virus. for example, when the influenza a h n subtype reemerged in , the most susceptible members of the population were those born after the time when similar h n viruses had previously circulated, the s (shortridge et al., ) . however, because influenza viruses undergo antigenic drift and shift, the effective period of protection may last only until an antigenic variant emerges. in contrast to the robust strain-specific protection after influenza virus infection, primary infection with rsv, hpiv, and hmpv provides only partial protection from reinfection. rsv commonly reinfects the host even though genetic diversity in the virus is not extreme. in healthy adults challenged every few months with the same strain of rsv, about % were infected each time and about half of those became symptomatic (hall et al., ) . these studies are now being reproduced with experimental human challenge infection (devincenzo et al., ) , and access to modern immunological techniques may provide some insight into the mechanism of immune evasion. most reinfections are limited to the upper respiratory tract, unless subjects are immunocompromised. reinfections may be the consequence of a highly prevalent and contagious virus, effective evasion of local and innate immunity, or a steep gradient for transudation of antibody from the serum to the nasal epithelium (graham, ) . alternatively, rsv infection may alter the characteristics of the adaptive immune effectors and memory. although rsv infection provides a sufficient antigenic stimulus to induce both antibody (shinoff et al., ) and t cell responses (heidema et al., ) , the durability of the antibody is poor (handforth et al., ; dakhama et al., ; collarini et al., ). the most efficient means of preventing respiratory virus infections is vaccination. however, among respiratory viruses, licensed vaccines are available only for influenza. it seems logical to consider live attenuated vaccines delivered intranasally for protection against respiratory viruses, as they would induce a mucosal immune response. however, a systemic immune response can be protective if it is sufficiently robust, such as that induced by inactivated influenza vaccines administered by the i.m. route. moreover, achieving an appropriate balance between sufficient attenuation and immunogenicity, especially in young infants who must be vaccinated in the face of maternal antibody, is a challenge. this section describes licensed vaccines as well as vaccines that are currently in development. vaccination remains the primary strategy for the prevention and control of influenza (lambert and fauci, ) . as described in section immune responses to respiratory virus infection, after an influenza infection, both cellmediated immunity and systemic and mucosal neutralizing antibodies are produced. whereas cell-mediated immunity contributes significantly to the clearance of a primary influenza virus infection, and can ameliorate disease caused by reinfection, neutralizing antibodies play an important role in preventing reinfection. the goal of vaccination is to prime the immune response to limit viral replication upon subsequent infection, and inactivated, recombinant hemagglutinin and live attenuated influenza vaccines (laivs) are licensed for use, with novel vaccines in varying stages of development. this section focuses on licensed vaccines and the immune responses they elicit, as determined from clinical trial data. owing to antigenic drift in circulating viruses (discussed in section virology), an influenza vaccine from one season may not be effective in subsequent seasons. each year, the strains that are to be included in the vaccine for the next influenza season are chosen and vaccine seed viruses are generated (lambert and fauci, ) . for inactivated vaccines, the influenza a seed viruses are reassortant viruses, with the ha and na gene segments derived from the circulating virus and the internal protein genes derived from a vaccine donor strain that is adapted for high yield in eggs (a/puerto rico/ / ; pr ) (kilbourne, ) . licensed laivs also contain the ha and na from the circulating virus, combined with the internal protein genes from temperature-sensitive, cold-adapted, attenuated master donor viruses that limit replication of the vaccine viruses to the cooler upper respiratory tract (maassab, ) . if the yield of the vaccine virus in eggs is poor, they may be "egg-adapted" through serial passage. vaccine viruses are then amplified in hundreds of millions of eggs and purified. the inactivated vaccine viruses are treated with formalin or β-propriolactone and "split" with detergents before being formulated for clinical use, with or without thimerosal as a preservative (fiore et al., ) . until , seasonal influenza vaccines were trivalent, containing two subtypes of influenza a viruses (h n and h n ) and one influenza b virus. however, from the winter season in the northern hemisphere, quadrivalent vaccines, containing two subtypes of influenza a viruses and two strains of influenza b viruses, have become available. it takes several months from the generation of a seed virus to the manufacture and distribution of a vaccine. typically, for seasonal influenza in the northern hemisphere, manufacturers amplify vaccine viruses and inactivate or purify them between february and late summer and formulate and distribute them for administration in the fall before the anticipated peak of the influenza season (lambert and fauci, ) . in , the h n pandemic virus emerged in april, when the manufacture of seasonal trivalent vaccines was already under way. a monovalent h n vaccine was produced as quickly as possible in addition to the seasonal vaccines, but the monovalent vaccine was not available for widespread use until after the pandemic had peaked in the northern hemisphere and was not available at all during the winter season in the southern hemisphere (broadbent and subbarao, ; skowronski et al., ) . in addition, current global vaccine production capacity does not cover the high-risk population around the world (girard et al., ) . one means of increasing capacity is to move toward using cell culture instead of eggs for vaccine production, and several companies are investigating this. additionally, the amount of ha in each vaccine dose could be reduced and used with an adjuvant such as mf or as . however, adjuvanted seasonal influenza vaccines are not yet licensed in the united states. trivalent inactivated vaccines (tivs) against seasonal influenza are administered i.m. and have an efficacy ranging from to % in preventing influenza morbidity and mortality in healthy adolescents and adults (osterholm et al., ) . a trivalent laiv consisting of an a or b ann arbor cold-adapted backbone together with the ha and na of the target viruses (flumist ® , medimmune), administered by a nasal spray, is licensed in several countries, including north america and europe. in the united states, the seasonal laiv is licensed for healthy individuals between and years of age, but it is currently not approved for use in children under years of age, and it is not approved for use in the elderly or in immunocompromised individuals (ambrose et al., ) . there is a substantial body of evidence from largescale randomized clinical trials demonstrating the effectiveness of laivs, and a number of clinical studies have also shown that the efficacy of laivs is equivalent or superior to that induced by i.m. vaccination with tivs in children (reviewed by luke et al. ( ) ). in adults, however, the majority of double-blind, randomized, placebo-controlled trials have shown tivs to have a greater efficacy than laivs monto et al., ; ohmit et al., ohmit et al., , . in the military, vaccine effectiveness was also found to be higher for tivs than for laivs, except in new recruits (eick-cost et al., ; wang et al., ). tivs primarily induce serum antibodies against the influenza ha glycoprotein, which are typically measured by hemagglutinin inhibition (hi) assays, which serve as a surrogate for virus-neutralization assays. in healthy individuals who are immunologically primed by previous infection or vaccination, influenza-specific antibodysecreting cells in the peripheral blood peak week after vaccination and serum antibody levels peak to weeks postvaccination. however, in unprimed individuals, for example, children, it may take weeks or longer for serum antibody levels to peak after vaccination (brokstad et al., ; cox et al., ; el-madhun et al., ) . the rise in serum antibody titer after tiv administration has been documented for multiple isotypes, including igm, iga, and igg, with a more pronounced rise in igg titers (moldoveanu et al., ) . in contrast, vaccination with an laiv leads to seroconversion more frequently in immunologically naïve individuals than in those who are immunologically primed. for example, in children, after a single dose of trivalent laiv, seroconversion rates of - %, - %, and - % have been reported against influenza a h n , h n , and b, respectively, which increased to % and % for h n after a second dose at day or , respectively (belshe et al., ; lee et al., ) . however, in healthy adults, serum antibody titers are lower after laiv than tiv vaccination (moldoveanu et al., ) , and in one study only % of laiv recipients had an increase in serum igg titer compared to % of tiv recipients . protection mediated by inactivated vaccines therefore correlates with serum neutralizing antibody titers, whereas other immune mechanisms contribute to protection mediated by laiv. a higher percentage of laiv recipients have mucosal antibodies and antibody-secreting cells than those receiving tiv. one study recorded that % of laiv recipients had increased influenza-specific iga in the mucosa, compared to only % of tiv recipients . moreover, levels of iga in nasal wash specimens correlated with protection against challenge with wild-type influenza viruses . mucosal iga in adults vaccinated with laiv declined months after vaccination . in addition to iga, igg has also been found in nasal secretions after laiv (moldoveanu et al., ) . as described (section immune responses to respiratory virus infection), iga is the major mediator of immunity to influenza infection in the urt, with igg serving as a backup. this also appears to be the case after vaccination with laiv. the majority of antibodies induced by influenza vaccines that are associated with protection are directed against the globular head of the ha. recently, neutralizing antibodies have also been identified that bind to a conserved epitope in the ha stem. the ha stem antibodies have been found to be broadly neutralizing, and there is much effort to generate vaccines that elicit these antibodies to produce a more broadly cross-protective vaccine (corti et al., ; ekiert et al., ; kashyap et al., ; okuno et al., ; sui et al., ). in addition, antibodies directed against the na protein are also generated after vaccination with both tiv and laiv (murphy et al., ) . anti-na antibodies restrict virus release from infected cells and reduce the severity of disease by limiting spread (murphy et al., ) . however, currently licensed inactivated vaccines are standardized to ha, but not na protein content. the amount of na protein varies from vaccine to vaccine, and the contribution of vaccine-induced anti-na antibodies to protection against influenza is not well understood (hassantoufighi et al., ) . in addition to humoral immunity, influenza-specific cd + cytotoxic t lymphocytes (ctls) are associated with accelerated clearance of virus and recovery from infection. however, the extent to which the cellular immune response is protective against infection is unknown because the recall response is likely to occur after the peak of viral replication (subbarao et al., ) , and cell-mediated immunity induced after vaccination has been less well studied than the humoral response, and the results are variable. studies have shown that immunization of healthy adults with whole-virus inactivated vaccine resulted in enhanced ctl responses in peripheral blood, whereas immunization with a subunit vaccine resulted in poor ctl responses, the duration of which varied from several months to years (ennis et al., ; mcmichael et al., ; powers and belshe, ) . in addition, an increase in ifn-γ-producing t cells was seen in children ages months to years of age who were vaccinated with inactivated vaccine; however, similar responses were not induced in adults (he et al., ) . h n -specific cd + t cells were also detected after a single dose of as -adjuvanted inactivated vaccine and was amplified by a second dose of vaccine (moris et al., ) . in addition, numerous studies have found a significant increase in ifn-γ-producing cd + and cd + t cells after vaccination with laiv in both adults and children (basha et al., ; forrest et al., ; he et al., a; hoft et al., ; lanthier et al., ) ; however, the role of cellular immune responses in laiv-mediated protection needs further investigation. more than % of annual influenza-related deaths in the united states occur in individuals years of age or older (thompson et al., ) . vaccinating elderly individuals is therefore a public health priority. however, a randomized placebo-controlled trial estimated the efficacy of the tiv to be % for the prevention of influenza in older adults (govaert et al., ) . elderly individuals often have a significantly reduced antibody response to influenza vaccination (goodwin et al., ) . in a quantitative review of elderly subjects, %, %, and % seroconverted to h n , h n , and influenza b vaccination, respectively, compared to %, %, and % in younger subjects (goodwin et al., ) . the impaired ability of the elderly to mount an adequate immune response to influenza vaccines has been attributed to immunosenescence. immunosenescence is the decline in the body's ability to fight infection, mount novel immune responses, and recall responses (targonski et al., ) , and both innate and adaptive responses are implicated. impaired function of costimulatory molecules, altered secretion of inflammatory cytokines, and diminished function of natural killer cells, macrophages, and neutrophils have been observed in the elderly, as well as a decreased proliferative capacity of b cells and impaired t cell memory recall (sullivan et al., ) . in addition, thymic involution and a decline in t cell output are features of advancing age. this, together with a lifetime of exposure to a variety of pathogens, leads to a reduction in the naïve t cell pool and a relative increase in the proportion of memory t cells in the elderly compared with young adults. the most pronounced functional changes are in the cd + t cell subset, in which progressive exhaustion occurs (pawelec et al., ) , whereas the cd + t cell subset is less affected by replicative senescence (czesnikiewicz-guzik et al., ) . the tiv is standardized on the basis of the amount of ha protein, with one dose of μg per strain being recommended in healthy, previously primed individuals. increasing the dose increases serum antibody response to the vaccine, and in an attempt to enhance immune responses to influenza vaccines in the elderly, a high-dose tiv ( μg ha protein per strain) was licensed in for use in persons ages years and older. postlicensure studies have shown enhanced immune responses in this age group, compared to the standard dose (sullivan et al., ) , and vaccine effectiveness studies are under way. in addition, an as -adjuvanted tiv (discussed below) containing μg ha protein per strain showed a % higher efficacy than a nonadjuvanted tiv in a phase randomized clinical trial in the elderly, but the difference was not statistically significant (mcelhaney et al., ) . laivs are not licensed for use in the elderly at present; however, they have been evaluated in clinical studies in persons years of age and older and are safe and well tolerated. in clinical trials, laivs were administered in addition to tivs, and coadministration was reported to enhance local ha-specific iga antibody responses. however, the efficacy of the combination was not greater than that of tiv alone (gorse et al., ; powers et al., powers et al., , treanor et al., ) . in individuals with chronic or immunocompromising conditions, serological responses to tiv vaccination are typically lower than in healthy adults. antibody responses against influenza were adequate in hiv-seropositive individuals who had no or minimal immunodeficiency or had responded well to antiretroviral therapy (chadwick et al., ; huang et al., ; madhi et al., ; staprans et al., ) . however, in individuals with advanced hiv disease and low cd t cell counts, tivs may not induce protective titers even after two doses (kroon et al., ; miotti et al., ) . laivs are not licensed for use in immunocompromised individuals. although there is a large amount of data available on the immune responses to seasonal influenza vaccines in humans, improved seasonal and pandemic influenza vaccines are evaluated first in animal models. the most commonly used models are mice and ferrets. mice immunized with inactivated influenza vaccines develop serum hi and neutralizing antibodies, the titers of which correlate with protection from subsequent challenge (luke and subbarao, ) . although cellular immune responses are mounted during a secondary influenza infection (woodland et al., ) , passively transferred antibodies protected immunosuppressed mice, suggesting that cell-mediated immunity is not essential for protection if sufficient antibody is present (virelizier, ) . the goal of parenteral immunization with inactivated influenza vaccines is to induce sufficient serum antibody titers to limit influenza disease. this protection is mediated by serum igg that transudes into the lower respiratory tract, neutralizing virus. passive transfer of immune serum to naïve mice reduced the replication of influenza virus in the lungs and protected recipient mice from lethal influenza pneumonitis, but did not prevent tracheitis or replication of the influenza virus in the urt (ramphal et al., ) . during a natural influenza infection of the urt, mucosal immune responses, including secretory iga antibodies, play an important role in controlling disease (section immune responses to respiratory virus infection). several studies have documented that higher levels of serum antibodies are required to provide protection against respiratory viruses in the urt compared to the lrt (prince et al., ; ramphal et al., ; takiguchi et al., ) . additionally, influenza in ferrets is primarily a urt infection and vaccination with killed or inactivated influenza viruses does not protect against influenza infection unless administered with an adjuvant (potter et al., a,b) . adjuvants are probably required for parenterally administered inactivated vaccines to elicit the higher levels of serum igg antibody that are needed to restrict viral replication in the urt, in the absence of robust mucosal immune responses. in mice, laivs induce a range of systemic and pulmonary immune effectors and protect animals against challenge virus replication (chen et al., ; lau et al., ) . the magnitude of the pulmonary iga and memory cd + t lymphocyte responses depends on the replication efficiency of the vaccine virus in the respiratory tract, but systemic immunity, such as serum antibody titers and memory cd + t lymphocytes in the spleen, does not . after one dose of an h n laiv that replicated in the lungs of mice and induced local immunity, influenza-specific lymphocytes in the lung contributed to the clearance of challenge virus from the lungs, whereas the contribution of serum antibody and splenic cd + t cells was negligible. after two doses, complete protection was achieved and was associated with maturation of the antibody response (lau et al., ) . taken together, the data suggest that laiv protects animals by inducing multiple arms of the immune response, including mucosal immunity and pulmonary and systemic antibody and cellular immune responses, in a manner similar to natural infection. however, inactivated vaccines aim to induce serum antibody alone. although this does not mimic a natural infection, if antibody titers are sufficiently high, the inactivated vaccine will protect against disease caused by influenza viruses. for inactivated influenza vaccines, serum anti-ha antibody titers correlate well with resistance to influenza infection in people as well as in animal models. lower antibody titers are associated with an increased risk of illness, though a specific antibody titer that can guarantee protection from infection has not been identified. an hi titer of : represents the level at which it is anticipated that approximately % of persons will be protected (hobson et al., ) , and this benchmark forms the basis of the licensing criteria for inactivated vaccines (cber, ) . although several papers refer to "seroprotection," there is insufficient evidence to support the use of this term for vaccines. the benchmark of an hi titer of : was defined in healthy adults who were experimentally challenged with an influenza virus (hobson et al., ) . however, antibody titers that correlate with protection in healthy adults may not translate to clinical improvements in influenza outcomes in the elderly (gorse et al., ) . in addition, laivs are effective despite inducing variable serum hi antibody titers. therefore, alternative correlates of protection are needed. as iga and cellular immune responses are generated in the lungs of mice vaccinated with laiv, in addition to systemic antibody and cellular responses, the extent to which the different arms of the immune response contribute to laiv-induced protection are beginning to be evaluated (chen et al., ; lau et al., lau et al., , . laiv-induced nasal wash igg and iga correlated with protection from virus replication, and in human challenge studies, either serum antibody or nasal wash iga was a predictor of protection (belshe et al., b) . cellular immune responses have also been investigated as a correlate of laiv-induced protection, and one study found a correlation between ifnγ-producing t cells (measured by elispot) and protection from culture-confirmed influenza illness in young children (forrest et al., ) . in addition, it has been shown that laivs alter the expression of ifn-related genes, whereas tivs do not, indicating that the innate immune response plays an important role in protection mediated by laivs (nakaya et al., ) . adjuvants are added to vaccine formulations to enhance immune responses to the antigen in the vaccine. aluminum salts (alum) are the most commonly and historically used adjuvants worldwide. they act by capturing antigens at the injection site, so the antigen is slowly processed and presented by the immune system (the so-called depot effect), and they cause mild cell damage and inflammation that promotes a th immune enhancement (tetsutani and ishii, ) . moreover, alum particles enter host cells and bind dna, introducing it into the cytoplasm of antigen-bearing dcs, where it engages receptors that promote both mhc class ii presentation and dc-t cell interactions (mckee et al., ) . oil-in-water adjuvants, such as mf (novartis) and as (glaxosmithkline), are more effective at inducing high-titer antigen-specific serum antibody responses than alum and have been used with inactivated split-virion influenza vaccines in europe. these adjuvants induce broad, cross-clade humoral responses and permit dose sparing, in which comparable immune responses are induced with a reduced amount of ha in the vaccine (galli et al., ; leroux-roels et al., ) . our understanding of the mechanism of action of mf and as remains incomplete. neither appears to act via a depot effect; instead they induce a local and transient proinflammatory cytokine and chemokine response at the injection site and draining lymph nodes that recruit immune cells from the circulation. in mice, as induced the cytokine il- and chemokine cxcl , which peaked locally by h postvaccination. the neutrophil-mobilizing cytokine csf and lymphocyte-mobilizing cytokines ccl , , and were induced by h postvaccination. in addition, the eosinophil-recruiting chemokine ccl and the cytokine il- β were induced at low levels, and ifn-γ, csf (gm-csf), and tnf-α were induced at levels that were only marginally above background. ifn-α and -β were not induced. the local cytokine response was paralleled by an enhanced recruitment of monocytes and granulocytes in the draining lymph node (morel et al., ) . mf also induces local upregulation of cytokines, chemokines, and other innate immunity genes, promoting the recruitment of immune cells such as monocytes, dendritic cells, and granulocytes. however, the mechanism of action of mf was found to be independent of the nlrp inflammasome, but required myd for a tlr-independent signaling pathway (seubert et al., ) . whereas the inactivated vaccine viruses are typically disrupted with detergents to make split-virion (subunit) vaccines, inactivated whole influenza virion (wiv) vaccines have also been developed, in which the virions are left intact. these are less widely used because of increased reactogenicity and adverse events (fiore et al., ) . however, mice immunized with wiv vaccines consistently showed higher hi titers and virus-neutralizing antibody titers than subunit vaccines, as well as an increased production of proinflammatory cytokines by dendritic cells and ifn-α by plasmacytoid cells, resulting in a desired th response (geeraedts et al., ; koyama et al., ) . the approach of using inactivated whole influenza vaccines is being revisited with pandemic influenza vaccines. whole-virion inactivated h n and h n vaccines administered with alum are immunogenic in humans (kulkarni et al., ; lagler et al., ; lin et al., ; nakayama et al., ) . dna vaccines encoding one or several proteins of influenza viruses induce an immune response targeting the encoded proteins (fynan et al., ; ulmer et al., ; wolff et al., ) . dna vaccines can be produced rapidly and at low cost; however, designing dna vaccines is complex. over the years, it has been shown that numerous factors play roles in the efficiency of expression, such as the promoter, the g/c content, supercoiling, polyadenylation, and codon optimization (laddy and weiner, ) . in addition, safety remains a concern, as there might be a risk of integration into the host genome (klinman et al., ) . numerous studies have evaluated dna vaccines expressing np, m , or ha proteins in animal models (fu et al., ; saha et al., ; tao et al., ; ulmer et al., ulmer et al., , a . in mice, the administration of dna vaccines encoding the np protein of influenza induces a strong ctl response, which correlates with protection against challenge with homologous or heterologous viruses (ulmer et al., ) . in addition, one study showed that delivering the vaccine by in vivo electroporation instead of the classical epidermal route also induces protective humoral and cellular immune responses in mice, ferrets, and nonhuman primates (laddy et al., ) . recently, a phase clinical trial with an adjuvanted plasmid dna vaccine encoding influenza h , ha, np, and m elicited t cell responses against ha in the majority of the subjects and against np and m in some (smith et al., ) . licensed influenza vaccines primarily elicit an immune response to the globular "head" region of the ha glycoprotein. however, immune selection pressure leads to antigenic drift (discussed in section clinical features and epidermology), so new influenza vaccines need to be selected for each season as well as when a pandemic emerges. it remains difficult to predict which strains will circulate in the upcoming influenza season, and rates of morbidity and mortality are greater in influenza epidemics when the virus and vaccine are "mismatched" (pica and palese, ) . the expectation is that vaccines capable of protecting against a broad(er) spectrum of influenza viruses would result in less frequent updating of seasonal influenza vaccines and would provide a degree of preexisting immunity if a novel strain emerges. vaccines that aim to provide broad cross-protection against multiple subtypes of influenza are known as universal vaccines, and several platforms are in development, including those that target the ha, m , np, m , and na proteins (subbarao and matsuoka, ) . unlike the head region of ha, the "stalk" domain has a high degree of sequence conservation between influenza viruses, and broadly neutralizing stalk-reactive antibodies have been identified. these antibodies may inhibit phinduced conformational changes in the ha required for cellular entry, prevent the cleavage of ha into ha and ha (discussed in section virology), or act via antibodydependent cell-mediated cytotoxicity (adcc) or through the activation of complement . however, the ha stalk is less immunogenic than the head, and several techniques have been employed to elicit stalk-reactive antibodies, including removing the head, novel prime-boost strategies, and sequential vaccination with chimeric ha molecules that contain the same stalk region but "exotic" head domains that aim to boost antibodies to the conserved epitopes within the stalk . these strategies have been tested in mice with varying degrees of success in generating protective immune responses against heterologous influenza viruses (subbarao and matsuoka, ) . taken together, ha stalkbased strategies show promise as universal influenza vaccine candidates. the influenza m protein extends beyond the viral envelope, and antibodies against the m extracellular domain (m e) may act via adcc, nk cell activity, or complementmediated lysis. in addition, the sequence of the m e is conserved among human influenza a viruses, making it an attractive target for universal vaccines. however, the peptide is poorly immunogenic, and techniques to improve immunogenicity are under investigation in mice, including fusing the peptide to an immunogenic carrier protein or delivering the antigen in a virus-like particle (vlp) (reviewed by subbarao and matsuoka ( ) ). the influenza np and m proteins are also conserved among influenza a viruses and have potential for use in a universal vaccine, but as they are not exposed on the surface of virions or infected cells, they mainly induce cellular immune responses. phase and clinical trials with modified vaccinia virus ankara (mva) expressing np and m proteins report a % reduction in laboratory-confirmed influenza infection after experimental challenge (lillie et al., ) ; however, larger studies are required to confirm this. immune responses directed against the influenza na protein do not protect against infection, but rather limit the spread of infection and reduce the severity of disease. recently, baculovirus-expressed vlps containing n na proteins were found to induce heterosubtypic na antibodies in mice that protected them against homologous and heterologous challenge (quan et al., ) . a universal vaccine may therefore benefit from anti-na immune responses. it may be useful to combine several components into a universal vaccine, for example, np and m to induce cellular responses and ha and m e to induce humoral responses. however, there are several challenges to the development of a universal vaccine. as outlined above, most of the viral proteins that potentially induce broad heterosubtypic immunity are poorly immunogenic and require a large dose of antigen, multiple doses, addition of adjuvants, fusion to immunogenic carriers, or the use of vectors or vlps. additionally, regulatory challenges include how to determine and define the potency of the vaccine and the need to identify immune correlates of protection and develop validated assays to measure them. in addition, clinical trials will be challenging because it is likely that efficacy will be measured in terms of amelioration of disease rather than preventing infection (subbarao and matsuoka, ) . given these hurdles, achieving a truly universal vaccine that protects against all types or subtypes of influenza will probably proceed in a stepwise manner, with the first step being a vaccine that is more broadly cross-protective than currently licensed vaccines. the primary target populations for rsv vaccination are young infants and the elderly, because hospitalization rates are the highest in these age groups and % of rsv-related deaths occur in individuals over years of age (thompson et al., ) . there is a large body of evidence that protection against infection is conferred mainly by neutralizing antibodies (collins and melero, ) ; however, multiple doses of vaccine might be necessary in young infants, because of the immature immune system and the presence of maternal antibodies. in addition, protective immunity mounted during infection does not protect against subsequent reinfection. reinfections are common and are independent of antigenic changes in the virus (collins and melero, ) . these factors present challenges to rsv vaccination and, as yet, vaccines have not been licensed. this section discusses clinical trial data from various approaches to rsv vaccination. the first approach to vaccination was the development of a formalin-inactivated rsv vaccine (f -rsv) that was evaluated in the s in infants and young children. a concentrated f -rsv vaccine, given i.m. with alum, was well tolerated and moderately immunogenic, but was poorly protective against infection. in fact, vaccinees who were exposed naturally to infection during the subsequent rsv season had immune-mediated enhancement of disease, with % of individuals requiring hospitalization and two fatalities (fulginiti et al., ; kapikian et al., ; kim et al., ) . subsequent studies revealed that vaccine-induced immune responses were different from those elicited after natural infection, with poor induction of neutralizing antibodies (murphy and walsh, ) and an exaggerated cd + t-cell response (kim et al., ) . vaccine-mediated enhancement of disease also occurred in murine models, with poor induction of neutralizing antibodies probably due to denaturation of antigen in the vaccine or a lack of antibody affinity maturation after poor tlr stimulation (delgado et al., ). in addition, an exaggerated th response and a loss of regulatory t cells contributed to disease (connors et al., (connors et al., , de swart et al., ; loebbermann et al., ; waris et al., ) . therefore, it is clear that in addition to a neutralizing antibody response, a balanced cd and cd t cell response is also desirable. after this experience, inactivated rsv vaccines were considered unsuitable for pediatric use, and alternative approaches were sought. in the s, a series of live attenuated vaccines (lavs) was developed by serial passage of rsv at suboptimal temperatures (cold passage, cp) or by growth in the presence of mutagens to produce temperature-sensitive (ts) mutants. the lav did not cause disease enhancement in animal models or in clinical trials (wright et al., a) . the most promising vaccine generated by this method, cpts / , was well tolerated and immunogenic in seronegative infants and children greater than months of age, but caused mild congestion in younger infants and was deemed to be insufficiently attenuated (wright et al., ) . several lavs have also been developed using reverse genetics techniques and were evaluated in clinical trials. the most promising, ra cp / / δsh (medi- ), was strongly ts and was well tolerated in infants . the first dose provided substantial reduction in replication of a second dose of vaccine given months later. however, the majority of individuals did not have increases in serum antibody titers, indicating that other immune mechanisms might play a role in the protection from replication of the second dose of vaccine. as with live attenuated influenza vaccines, establishing correlates of protection for live rsv vaccines is an active area of research, made more challenging by the weak immune responses in infants and limitations on sampling. as of this writing, clinical trials are under way to further monitor tolerability and immunogenicity and the ability of candidate vaccines to induce protection against natural rsv exposure in the community (collins and melero, ) . genetic changes have been employed to generate candidate lavs, including deletion of the m - coding sequence, which increases transcription and antigen synthesis at the expense of viral replication (bermingham and collins, ) , and deletion of the ns and ns genes, which increase ifn production and signaling and might limit viral replication and pathology, but increase immunogenicity (teng et al., ) . the use of attenuated parainfluenza virus as a vector to express rsv f and/or g protein has also been considered as a pediatric vaccine against both hpiv and rsv, as piv has the advantage of improved in vitro growth and stability compared with rsv. bovine piv is attenuated in humans and has been used as a vector into which the f and hn genes from human piv and the f and/or g protein of rsv are incorporated to make a bivalent vaccine against both hpiv and rsv (schmidt et al., ; tang et al., ) . one example of this approach is in clinical trials in children older than months of age and who are seronegative to rsv and hpiv (collins and melero, ) . the vaccine in this study comprises a bovine piv vector into which the human piv f and hn genes and the rsv f gene have been added (tang et al., ) . as an alternative to bovine piv , murine piv (sendai virus) is also being evaluated as a vaccine backbone into which rsv antigens are inserted (jones et al., ) . there is substantial antigenic crossreactivity between sendai virus and hpiv , and the virus may be attenuated in humans. other approaches to live vaccines for rsv include the use of alphaviruses (elliott et al., ) or replicationdefective adenoviruses (shao et al., ) as vectors for rsv antigens. in addition to an rsv vaccine in infants, there is also a need for rsv vaccines in older children, adults, and the elderly. however, these individuals are likely to have been previously infected with rsv, and preexisting immunity may restrict the replication of lavs. in addition, vectored vaccine approaches should not be based on common human pathogens, as preexisting immunity to the vector may limit vaccine virus replication and interfere with the development of immunity. the use of protein-based vaccines in these populations is currently being evaluated. disease enhancement has not been documented in adults who were previously exposed to rsv, and candidate rsv f protein-based vaccines are well tolerated in healthy adults, children over months of age, pregnant women, and the elderly (girard et al., ) . in a phase clinical trial involving children - years of age with cystic fibrosis, a purified f protein- (pfp- ) vaccine induced a fourfold rise in serum antibody titer, but was not associated with significant protection against lrti episodes compared to placebo . in another approach, a subunit vaccine of copurified f, g, and m proteins from rsv-a was tested in adults and found to induce neutralizing antibodies in - % of vaccinees, but titers waned after year, suggesting that annual immunization might be necessary with this vaccine (girard et al., ) . interestingly, in a phase clinical study of pregnant women vaccinated with pfp- , anti-f antibodies were persistently elevated in newborns of the vaccinated mothers, without enhancement of disease. maternal immunization could, therefore, be a strategy to protect infants under months of age, for whom the development of an rsv vaccine has been such a challenge (durbin and karron, ) . a recombinant postfusion form of the f protein with a deletion of the major hydrophobic regions was also produced. this subunit vaccine forms stable trimers that are recognized by neutralizing mabs. high levels of neutralizing antibodies were induced in the sera of vaccinated rodents, and the animals were protected from challenge. this may represent an improved subunit vaccine (mclellan et al., ; swanson et al., ) . another protein-based vaccine, bbg na, has been evaluated in clinical trials. bbg na consists of a fragment of the rsv g protein that contains a central conserved domain fused to the albumin-binding domain of streptococcal g protein, expressed in bacteria. however, this vaccine was not very immunogenic in clinical trials and was associated with hypersensitivity reactions (purpura) in some individuals (power et al., ) . vlps have also been evaluated for use as an rsv vaccine, and one such formulation (rsv-f particle vaccine) is currently in clinical trials (collins and melero, ) . it is the hope that improved methods of producing rsv antigens or using vlps will result in an effective vaccine that could be given to adults periodically, possibly with the annual influenza vaccine. however, in children, it remains to be seen whether a vaccine can be developed that is sufficiently attenuated, yet immunogenic enough to provide protection. however, even if complete protection is unrealistic, immunity that results in substantial reduction of virus replication may be sufficient to prevent severe disease. as % of children are seropositive for hpiv by years of age, and hpiv lrti is a major cause of hospitalization in this age group (schomacker et al., ) , young infants and children are the target population for hpiv vaccination. protective immunity against infection is mediated primarily by mucosal and serum neutralizing antibodies; however, reinfection is common owing to a difficulty in maintaining protective titers of s-iga and igg in the respiratory lumen, thus representing a challenge to vaccine development (glezen et al., ) . this section reviews data from clinical trials of inactivated, live, and vectored hpiv vaccines. the first approach taken to hpiv vaccination was the use of formalin-inactivated piv (fi-piv ) in an intramuscular vaccine. although vaccine-induced disease enhancement seen with formalin-inactivated rsv was not observed with fi-piv , there was no evidence of protection either (kim et al., ) . subsequent vaccine efforts, therefore, focused on the generation of live attenuated vaccines, and two approaches were investigated: the use of a ts hpiv strain and the use of a bovine piv strain. successive passage at lower temperatures has been shown to attenuate hpiv. the most promising vaccine candidate using this approach, cp , is designated by the number of times the virus was passaged at low temperature in african green monkey kidney cells (belshe and hissom, ; ray et al., ) . the vaccine was safe and immunogenic in adults, seropositive and seronegative children, and infants month of age or older (belshe et al., b) . in a phase clinical trial in -to -month-old children, the vaccine was well tolerated and % of previously seronegative vaccinees had a fourfold or greater rise in serum geometric mean antibody titer (belshe et al., b) . moreover, coadministration of an rsv and cp piv vaccine was successful, with little evidence of interference (belshe et al., a) . however, the cp piv vaccine was overattenuated in seropositive children and adults. bovine piv makes an attractive lav as it is known to be antigenically related to human piv, is attenuated in humans, and protects monkeys against challenge with hpiv . clinical trials were conducted in adults, seropositive children, seronegative infants, and children - months of age with residual maternal antibodies. in seropositive individuals, the vaccine was overattenuated, but in seronegative children and infants, the vaccine virus was highly infectious. despite replication of the vaccine virus, serum antibody responses to hpiv were low, consistent with an incomplete antigenic relatedness between the human and the bovine piv hn genes (greenberg et al., ; karron et al., ) . a phase clinical trial of children showed that seroconversion to bovine piv occurred in - % of vaccinated children after three doses, but that seroconversion to hpiv occurred in only - % of vaccinees (greenberg et al., ) . additionally, during the study, % of the placebo group experienced piv infection, highlighting the ubiquitous nature of the pathogen. after these studies, it was concluded that bovine piv might better serve as a backbone for the insertion of hpiv genes. as described above, at this writing, one such vaccine, with hpiv hn and f genes and the rsv f gene inserted into a bovine piv backbone, is being evaluated in clinical trials in seronegative children. this vaccine candidate was highly attenuated in seropositive children and adults. additional vaccine candidates include those generated by reverse genetics. one such vaccine contains mutations in the p/c gene, which reduces the virus's ability to inhibit type i ifn induction and signaling, and another contains ts and attenuating mutations from other paramyxoviruses (sato and wright, ) . by the age of years, most children will have been infected with hmpv. reinfections are common, and hmpv is responsible for - % of childhood hospitalizations for lrti. in addition, severe disease can also be seen in the elderly or immunocompromised individuals (feuillet et al., ) . despite this clinical need, hmpv vaccines have not yet entered clinical trials, and the data reviewed in this section are from studies in animal models. as with rsv, vaccination of macaques and mice with inactivated hmpv led to disease enhancement after challenge and a strong th -type immune response associated with a lack of neutralizing antibodies (herfst et al., a; hamelin et al., ) . therefore, alternative approaches have been evaluated, including soluble protein-based subunit vaccines, live attenuated vaccines, or dna vaccines encoding viral proteins. soluble f-protein subunit vaccines have been shown to generate high titers of neutralizing antibody in serum in golden syrian hamsters and cotton rats, associated with protection against subsequent infection (cseke et al., ; herfst et al., ) . however, stable and long-term immunity was not induced in monkeys (herfst et al., b) . live attenuated vaccines have been generated either by cold passage or by reverse genetics. temperature-sensitive viruses conferred complete protection against heterologous hmpv challenge in golden syrian hamsters (herfst et al., a) , and a recombinant hmpv lacking a region of the sh (Δ − sh), the g, or the m - protein induced high titers of neutralizing antibody and protected hamsters and african green monkeys against piv and hmpv challenge (biacchesi et al., ; buchholz et al., buchholz et al., , . a chimeric vaccine has also been developed in which the f subunit of the hmpv fusion protein is inserted into a backbone of bovine piv that also contains the hpiv f and hn genes (tang et al., ) . this b/hpiv /hmpv f vaccine induced neutralizing antibodies and protected hamsters and african green monkeys against piv and hmpv challenge (tang et al., . coronaviruses such as hcov- e and oc are frequent causes of respiratory illness throughout the world, and sars-cov and mers-cov represent significant public health threats. however, at present there are no licensed vaccines for human coronavirus infections, though a number of vaccines are licensed against animal coronaviruses, and several vaccine platforms have been developed for sars-cov that show great promise in preclinical studies. whole sars-cov particles inactivated with β-propiolactone, formalin, uv light, or a combination of two techniques have been evaluated in mice, ferrets, rabbits, and nonhuman primates. such vaccines have been administered by a variety of routes and tested in the presence or absence of various adjuvants. in general, vaccination elicited a serum igg response in animal models, with higher doses of vaccine eliciting higher igg antibody titers ( subbarao, ) . moreover, some studies demonstrated that inactivated sars-cov vaccines were efficacious in protecting mice (see et al., ; spruth et al., ; stadler et al., ) and nonhuman primates (qin et al., ; zhou et al., ) , although many studies did not investigate protective efficacy. in addition, an inactivated sars vaccine that was evaluated in clinical trials was safe and well tolerated and induced neutralizing antibodies (lin et al., ) . many subunit vaccines comprise purified, recombinant sars-cov s protein, as this protein is the target for protective neutralizing antibodies elicited by sars-cov infection. a truncated soluble form of the s protein that lacks the transmembrane domain also neutralized infectivity of sars-cov (bisht et al., ; he et al., b; zhou et al., ) and substantially reduced the titer of challenge virus replication in the respiratory tract of mice (bisht et al., ) . a trimeric spike protein vaccine (trispike) was also immunogenic in mice and hamsters and provided protection against challenge in hamsters, with reduced occurrence and severity of pneumonitis and no evidence of pulmonary consolidation or sars-cov-associated hepatic cellular necrosis (kam et al., ) . however, sera from animals immunized with trispike were associated with a -to -fold increase in virus entry into fcγrii-positive (ace -negative) human b cells, which has led to concerns about antibody-dependent enhancement of disease (kam et al., ) . vectored vaccines have been developed against sars-cov. in this approach, sars-cov proteins are expressed by venezuelan equine encephalitis virus replicon particles (vrps), chimeric bovine/human piv vectors, recombinant replication-defective human adenovirus- (ad- ), poxvirus mva, attenuated rabies viruses, or attenuated vesicular stomatitis virus (subbarao, ) . when animals were immunized with vectored vaccines expressing the s protein of sars-cov, neutralizing antibodies were induced, and vaccinated animals were protected from challenge. however, if other sars-cov proteins were expressed, animals were not protected from challenge. for example vrps expressing the sars-cov s protein provided protection from challenge in mice; however, vrp expressing the sars-cov n protein (vrp-n) did not (deming et al., ) . moreover, vrp-n vaccination resulted in a marked bronchiolitis, alveolitis, and interstitial accumulation of eosinophils and mononuclear leukocytes (deming et al., ) . in addition, chimeric bovine/ human (b/h) piv vectors expressing the sars-cov s protein or the s, m, and e proteins together also generated neutralizing antibodies in hamsters and protected animals from challenge, whereas b/h parainfluenza viruses expressing m, n, or e proteins were not protective . the route of administration is also important in determining protective efficacy of a vaccine. an ad- vector expressing the sars-cov s and n genes given i.m. had a limited effect in reducing pulmonary replication of challenge virus, despite eliciting higher serum igg titers and a greater cellular immunity than when the vaccine was given intranasally (i.n.). however, the vaccine administered i.n. induced higher mucosal iga responses than when given i.m. and provided protection from pulmonary replication, suggesting that mucosal immunity is important in mediating protection (see et al., ) . mice, rabbits, and monkeys immunized with a modified vaccinia virus mva-s vaccine were protected from replication of challenge virus (bisht et al., ; chen et al., ) . however, in ferrets, mva-s antibodies declined rapidly after vaccination, and the vaccine did not protect the animals from replication or spread of the challenge virus (weingartl et al., ) . a recombinant live attenuated sars-cov vaccine virus in which expression of the e protein was abrogated by point mutations and a deletion in the nucleotide sequence was restricted in vitro and attenuated in hamsters (dediego et al., ) . in addition, inactivation of the ′- ′ exonuclease attenuated sars-cov, and the resulting virus was protective in an aged immunocompromised mouse model of sars infection (graham et al., ) . dna vaccines expressing sars-cov s protein (either without the cytoplasmic domain or without the cytoplasmic and transmembrane domains) also induced humoral and cellular immunity in mice and protected animals from replication of challenge virus . in addition, priming with a dna vaccine and boosting with an inactivated vaccine resulted in an increase in cd + t cells and a stronger antibody response (zakhartchouk et al., ) . the dna vaccine was well tolerated and produced cellular immune responses and neutralizing antibody in a phase clinical trial (martin et al., ) . despite these successes in preclinical vaccine development, as the sars outbreak was declared over in , the impetus was not sustained long enough for many of these products to be evaluated in clinical trials. a cautionary note about sars-cov vaccines is the association of a variety of vaccine platforms with pulmonary immunopathology in mice after challenge with the virus, despite immunogenicity and protective efficacy (tseng et al., ) . the relevance of the rodent model to vaccines in humans remains uncertain. adenovirus epidemics have been described in adults, especially in military recruits in closed or crowded settings (lynch et al., ) . live attenuated, orally administered adenovirus vaccines against serotypes and , which cause respiratory disease, were used by the u.s. military starting in (top, ) . during this time the incidence of disease fell substantially. however, when the manufacturer ceased vaccine production in , epidemics reemerged in military facilities (lynch et al., ) . in , a new live attenuated adenovirus vaccine was issued to military recruits during basic training, and there has since been a reduction in the rate of febrile respiratory illness (armed forces health surveillance, ). the vaccine is not available to children or the civilian adult population at present, though adenoviruses are estimated to account for - % of pediatric and - % of adult respiratory tract infections (lynch et al., ) . in summary, the majority of studies evaluating respiratory virus vaccines measure serum antibody responses, because a large body of evidence indicates that, although both cellular and humoral responses contribute to the clearance of a primary infection, neutralizing antibodies protect against secondary infection. humoral responses can be readily detected after vaccination with inactivated or subunit vaccines; however, fewer individuals seroconvert after vaccination with live vaccines. alternative immune mechanisms such as mucosal antibody responses are probably responsible for protection by live attenuated vaccines, and immune correlates of protection are under investigation. because respiratory pathogens infect the host at mucosal surfaces, the induction of mucosal immunity by vaccination is desirable. vaccines that are administered by intramuscular or subcutaneous injection induce protective immunity in the systemic immune compartments, but are generally poor at inducing mucosal immune responses. mucosally delivered vaccines, in contrast, induce both mucosal and systemic immunity and are also more readily accepted because they do not require needles or syringes (levine and dougan, ; yuki and kiyono, ) . ongoing research into the molecular and cellular mechanisms of surface immunological barrier systems has provided practical strategies for the development of a new mucosal vaccine for the control of respiratory viruses. m cells, located in the follicle-associated epithelium of the malt, play a pivotal role in the uptake of luminal antigens in the respiratory tract and the induction of antigen-specific immune responses in both the systemic and the mucosal compartments . it is, therefore, logical and attractive to develop m-cell-targeted mucosal vaccines. several molecules have been found to bind preferentially to m cells, such as ulex europaeus agglutinin- (uea- ), which has specificity for α( , )-fructose (sharma et al., ) , and rho-protein derived from reovirus, which binds to a carbohydrate structure containing α , -linked sialic acids on the plasma membranes of m cells (helander et al., ) . moreover, intranasal vaccines conjugated to either uea- (manocha et al., ) or rho-protein induced not only strong antigen-specific plasma igg and mucosal iga responses, but also ctl immunity (wu et al., ) . in addition, a novel m-cell-specific monoclonal antibody has recently been identified that selectively recognizes m cells, but not goblet cells or epithelial cells. this has been used as a carrier for an m-cell-targeted mucosal vaccine (wu et al., ; nochi et al., ) . concerns have been raised about the potential induction of unwanted mucosal inflammation associated with m cell targeting (kuolee and chen, ) , and additional research is needed to better understand how m cells sample antigens and transcytose them to the basolateral membrane. however, most microparticles administered orally become trapped in the mucus, and only a small fraction of them enter mucosal inductive sites. new approaches that are being developed to transitionally or conditionally enhance the number and function of m cells (neutra and kozlowski, ) are likely to reduce these concerns. there is substantial interest in the exploitation of nanoparticle technology for drug and vaccine delivery. nanoparticles are solid particles ranging in size from to nm in diameter that are often made from biodegradable materials. an antigen payload can be dissolved, entrapped, adsorbed, attached, or encapsulated into the matrix of the particle and released as the particle degrades over a period of time, which may vary from days to weeks, depending on the formulation (adair, ) . several types of nanoparticles have been investigated for vaccine delivery and have proven to be safe, nontoxic, and suitable for loading with antigens. these include the polyesters (poly(lactic acid), poly(glycolic acid), and their copolymers), polyorthoesters, polyanhydrides, and polycarbonates (reddy et al., ) . these materials can protect antigens from degradation, and the particles can be prepared in a chemically reproducible manner within a narrow size limit. in addition, some biopolymers exhibit a natural adjuvant behavior, for example, poly(lactide co-glycolide) (plga) appears to activate the maturation of dcs, possibly by providing a necessary stimulatory signal, though the exact mechanism is not fully elucidated (bennewitz and babensee, ; yoshida and babensee, ) . surface modifications of nanoparticles that change their overall charge and hydrophobicity are aimed at improving mucoadherence properties (des rieux et al., ) . this can be achieved by coating with stabilizers, other polymers, or surfactants. polyethylene glycol has been used for its stabilizing properties and because it can enhance the affinity of the nanoparticles for mucosal surfaces. molecules such as lectins and chitosan can also increase interaction with and transport across the mucosal surface. interestingly, chitosan is reported to be able to open tight junctions between epithelial cells, facilitating the transport of encapsulated macromolecules across the epithelial layer . nanoparticles coated with mannose (mannan) have also been produced with the aim of targeting mannose receptors on apcs, thus improving cell adhesion and uptake (cui and mumper, ) . a plga-coated nanoparticle vaccine encapsulating influenza virus proteins has been developed. the influenza ha protein retained its antigenicity after encapsulation, and mice vaccinated with plga particles of . - . μm in diameter mounted both systemic and mucosal responses, which were protective against intranasal challenge with an h n influenza virus (amidi et al., ; moldoveanu et al., ) . chitosan-coated nanoparticles containing purified influenza virus have also been tested in human volunteers, in which a fourfold or greater increase in anti-ha antibodies was observed in > % of the volunteers . moreover, a single dose of the intranasal vaccine resulted in high titers of nasal iga antibody and strong systemic antigen-specific responses, which were greater than those induced after intramuscular inoculation with soluble influenza antigen (amidi et al., ) . nanoparticleconjugated rsv proteins have also been shown to generate strong systemic th immunity in mice, associated with protection against wild-type rsv challenge (kalkanidis et al., ; xiang et al., ) . in addition, plgacoated nanoparticles incorporating hpiv hn and f glycoproteins have been shown to induce virus-neutralizing antibodies, which were protective against challenge infection in hamsters (ray et al., ) . studies in animal models and humans have shown that the choice of adjuvant can dramatically affect the immediate immune response and long-term protective effect of a vaccine (ogra et al., ; galli et al., ) . in addition, the quality of the immune response, especially the development of high-affinity b cells, long-lived memory b cells, and plasma cells can be influenced by the choice of adjuvant (galli et al., ) . although the mechanisms have not yet been fully elucidated, mucosal adjuvants can be broadly classified into two categories: those that act as immunostimulatory molecules and those that facilitate vaccine delivery (o'hagan, ) . the former include adjuvants that are toxin-based or cytokine-based and molecules associated with innate immunity, for example, pamps. the latter contain immune-stimulating complexes, liposomes, live attenuated vectors, and chitosan (discussed above). from a mechanistic point of view, mucosal adjuvants modulate innate immune responses in the same way as parenterally administered vaccines (lambrecht et al., ) , and tlr agonists constitute a major category of mucosal adjuvants. these adjuvants are based on pamps and are often formulated as oil-inwater emulsions. virosomes are virus-like particles that have been investigated for their potential as vaccines bungener et al., ) . they closely resemble native virus; however, they are nonreplicating and consist of reconstituted viral envelopes, generated by treatment with a detergent. they have a diameter of - nm and, as such, fall into the size range of small particles. virosomal influenza vaccines are available commercially and have been shown to induce ha-specific antibodies after i.m. administration. phase clinical trials revealed the tolerability and immunogenicity of a matrix tm -adjuvanted virosomal h n dna vaccine (cox et al., ) . influenza virosomes that incorporate the rsv-fusion protein have also been constructed and have been shown to generate virus-specific iga in the urt and lrt after i.n. administration with adjuvant in mice (cusi et al., ) . immune stimulating complexes (iscoms) are nonreplicating particles of ∼ nm diameter, comprising viral glycoproteins complexed with saponin derived from the bark of the tree quillaia saponaria (gregory et al., ) . iscom antigens from a number of microorganisms, including viruses, bacteria, and parasites, have been shown to induce humoral and cell-mediated immunity; th immune responses appear to predominate . extensive studies have been carried out with influenza virus iscoms in several species, including mice and monkeys, and an equine influenza iscom vaccine is available commercially (mumford et al., ). an influenza iscom vaccine (sundquist et al., ) stimulated high levels of virus-specific igm and igg serum antibodies and proliferative t cell responses in macaques, and the animals were completely protected from intratracheal challenge with the virus (rimmelzwaan et al., ) . intranasal immunization of mice with rsv iscoms induced very high levels of long-lasting rsv-specific iga in both the urt and the lrt (hu et al., ) . bovine rsv iscoms inoculated subcutaneously into calves were completely protective against challenge with virulent bovine rsv, whereas calves immunized with a conventional bovine rsv vaccine developed moderate to severe respiratory disease after challenge (hagglund et al., ) . the iscom-vaccinated animals developed high titers of nasal and serum virus-specific igg as well as serum iga, which correlated with protection. detoxified derivatives of cholera toxin (ct) and heat-labile enterotoxin (lt) produced by enterotoxigenic escherichia coli are effective mucosal adjuvants that promote mucosal and systemic immunity to coadministered protein antigens via oral or nasal routes (freytag and clements, ; mestecky et al., ) . the results of murine studies demonstrated that both ct and lt induced the expression of b - and/or b - on apcs that deliver costimulatory signals to cd + t cells (freytag and clements, ; mestecky et al., ) , but ct acts in the presence of il- to induce predominantly th responses, whereas lt supports th responses with ifn-γ production. recent vaccine development efforts have focused on the nasal administration of antigen with ct and lt derivatives for the induction of mucosal iga (byun et al., ; fujihashi et al., ) . both ct and lt are ab -type molecules, consisting of one a subunit and five b subunits. chimeric molecules, made by the spontaneous association of the a subunit of ct with the b subunit of the lt, or vice versa, are effective mucosal adjuvants for protein vaccines. the type of t helper responses induced is dictated by the origin of the b subunit (takahashi et al., ) . one such example is the nasal administration of an influenza ha subunit vaccine together with a chimeric mutant (m) ct-a/lt-b adjuvant. in murine studies, this adjuvanted vaccine protected animals from influenza virus challenge (kweon et al., ) . however, there are some concerns about the safety profile of toxin-derived adjuvants. nasal administration of mct-a/lt-b targeted neuronal tissues, though it did not affect trafficking of coadministered vaccine antigens into the neuronal tissues (kweon et al., ) . taken together, these findings suggest that although recombinant chimeric toxin-based molecules show promise as a new generation of mucosal adjuvants, the safety of the ct adjuvant may need to be improved. the inactivated lt-adjuvanted intranasal influenza vaccine (nasalflu) was withdrawn because of concomitant facial nerve paralysis (bell's palsy) that was noted as a potential adverse event caused by the ct adjuvant kunzi et al., ; garner-spitzer et al., ). several cytokines associated with innate immunity and inflammation support the generation of antigen-specific s-iga and serum igg/iga and may be of use as mucosal adjuvants. mice nasally immunized with soluble antigens, including ovalbumin or tetanus toxoid (tt) plus il- α and il- β developed antigen-specific antibody responses that were similar to those induced by coadministered ct adjuvant. the cytokines il- α and il- β promoted antigen-specific igg antibody responses initially, followed by igg b, with minimal igg a antibody responses, a pattern associated with a predominantly th -type response (staats and ennis, ; thompson and staats, ) . furthermore, levels of antigen-specific s-iga antibody similar to those induced by ct were found in mucosal secretions of mice that received nasal il- . these results indicate that il- could be a useful mucosal adjuvant. apcs are known to contribute to the creation of an appropriate cytokine environment for the growth and development of th or th responses. two well-known cytokines secreted by apcs, il- and il- , can influence the outcome of th and th cell subset-mediated immune responses (vajdy et al., ; rincon et al., ) . when tt was used as an antigen, nasal coadministration of il- or il- induced antigen-specific serum igg antibody responses and promoted protective immunity against lethal challenge with tetanus toxin. interestingly, whereas nasal treatment with il- promoted mixed th /th -type responses, il- supported predominantly th -type responses. in addition, il- but not il- can induce antigen-specific mucosal iga antibody (boyaka et al., ) . these findings suggest that il- could be a potent mucosal cytokine for the upregulation of antigen-specific mucosal immune responses. type i ifn affects the differentiation and function of immune cells, including t cells and dcs, and efficiently enhances a primary antibody response (marrack et al., ; luft et al., ) . it was reported that type i ifn was effective as both a systemic and a mucosal adjuvant, promoting th -type immune responses (proietti et al., ) . nasal administration of influenza vaccine with type i ifn was effective at inducing serum antigen-specific igg a and mucosal iga antibody responses and at providing full protection against influenza virus challenge (proietti et al., ) . it is well known that i.n. vaccination stimulates immune responses in the nalt and is effective at inducing mucosal immunity in the respiratory tract (brandtzaeg, ; jabbal-gill, ) . intranasal administration of the live attenuated influenza vaccine flumist (medimmune) has proven effective in protection against seasonal influenza virus infection and protects children against drifted influenza virus strains (belshe et al., a; mendelman et al., ) . furthermore, owing to the development of novel technologies, both aerosol spray and droplet delivery of vaccines are attractive possibilities (jabbal-gill, ) . in addition to the i.n. route of vaccine inoculation, the sublingual route has been explored (shim et al., ; czerkinsky et al., ) . the sublingual epithelium harbors a dense lattice of dcs, and vaccine delivered via this route stimulates broad and disseminated mucosal and systemic immune responses, similar to intranasal inoculation . sublingual vaccination with soluble or particulate antigens promotes strong mucosal iga and systemic igg antibody responses as well as cd + t cell responses. overall, sublingual immunization was comparable to nasal immunization in the magnitude, breadth, and anatomic dissemination of the induced immune responses. importantly, sublingual administration did not redirect antigens and/or adjuvants to the brain . sublingual vaccination against influenza has been shown to protect mice against lung infection (song et al., ) . in addition, sublingual administration of inactivated influenza a/pr (h n ) vaccine virus together with a mucosal adjuvant such as ct (cuburu et al., ) or a nontoxic mct-a/lt-b adjuvant induced both systemic and mucosal virus-specific antibody responses as well as ctl responses with protective immunity after respiratory challenge with the a/pr virus (kweon et al., ; song et al., ) . these studies demonstrated that sublingual administration of an inactivated influenza virus with a toxin adjuvant such as ct did not migrate to or replicate in the central nervous system. moreover, using mucosal adjuvant such as ct mobilizes dcs within the sublingual epithelium. these observations suggest that sublingual immunization may be another attractive and safe mucosal route for the administration of respiratory virus vaccines. given the global burden of respiratory tract infection, there remains an unmet need for effective methods of intervention. the most efficient means of preventing respiratory virus infections is vaccination. however, among respiratory viruses, licensed vaccines are available only for influenza. systemic immune responses can be protective in the absence of mucosal immunity if they are sufficiently robust, such as that induced by i.m. inactivated influenza vaccines; however, many vaccines in development for respiratory viruses are live attenuated viruses that are mucosally administered. although live vaccines show promise, our understanding of the mechanism of protection at mucosal surfaces is incomplete, and there is currently a lack of adequate immune correlates of protection for these vaccines. in addition, immune responses mounted to a natural infection are often not sufficient to prevent reinfection and may also be involved in the pathogenesis of disease. achieving an appropriate balance between sufficient attenuation and immunogenicity, especially in young infants who must be vaccinated in the face of an immature immune system and maternal antibody, is a challenge. nanoparticle vaccines against respiratory viruses unique ability of activated cd + t cells but not rested effectors to migrate to non-lymphoid sites in the absence of inflammation immunological memory and protective immunity: understanding their relation recognition of double-stranded rna and activation of nf-kappab by tolllike receptor the nlrp inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna viral infection is associated with an increased proinflammatory response in chronic obstructive pulmonary disease an integrated, multistudy analysis of the safety of ann arbor strain live attenuated influenza vaccine in children aged - years n-trimethyl chitosan (tmc) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model incidence of acute respiratory illnesses among enlisted service members during their first year of military service: did the resumption of adenovirus vaccination of basic trainees have an effect? mouse type i ifn-producing cells are immature apcs with plasmacytoid morphology free recirculation of memory b cells versus antigen-dependent differentiation to antibody-forming cells critical role of mda in the interferon response induced by human metapneumovirus infection in dendritic cells and in vivo dectin- mediates th immunity through the generation of cysteinyl leukotrienes comparison of antibody and t-cell responses elicited by licensed inactivatedand live-attenuated influenza vaccines against h n hemagglutinin the double life of a b- cell: self-reactivity selects for protective effector functions innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system cord blood vitamin d deficiency is associated with respiratory syncytial virus bronchiolitis icam- receptors and cold viruses cold adaptation of parainfluenza virus type : induction of three phenotypic markers the efficacy of live attenuated, cold-adapted, trivalent, intranasal influenzavirus vaccine in children efficacy of vaccination with live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine against a variant (a/sydney) not contained in the vaccine correlates of immune protection induced by live, attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine evaluation of combined live, attenuated respiratory syncytial virus and parainfluenza virus vaccines in infants and young children phase evaluation of parainfluenza type cold passage mutant live attenuated vaccine in healthy children - months old distinct migrating and nonmigrating dendritic cell populations are involved in mhc class i-restricted antigen presentation after lung infection with virus the effect of the physical form of poly(lactic-co-glycolic acid) carriers on the humoral immune response to co-delivered antigen adenoviridae: the viruses and their replication the m - protein of human respiratory syncytial virus is a regulatory factor involved in the balance between rna replication and transcription recombinant human metapneumovirus lacking the small hydrophobic sh and/or attachment g glycoprotein: deletion of g yields a promising vaccine candidate severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice neutralizing antibody and protective immunity to sars coronavirus infection of mice induced by a soluble recombinant polypeptide containing an n-terminal segment of the spike glycoprotein human coronavirus hcov- e enters susceptible cells via the endocytic pathway an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between ha and ha determines proteolytic cleavability and pathogenicity of avian influenza viruses positive selection results in frequent reversible amino acid replacements in the g protein gene of human respiratory syncytial virus il- is an effective adjuvant for induction of mucosal immunity function of mucosa-associated lymphoid tissue in antibody formation potential of nasopharynx-associated lymphoid tissue for vaccine responses in the airways cd t cells utilize trail to control influenza virus infection the magnitude of the t cell response to a clinically significant dose of influenza virus is regulated by trail influenza virus vaccines: lessons from the h n pandemic parenteral influenza vaccination induces a rapid systemic and local immune response subclass distribution and molecular form of immunoglobulin a hemagglutinin antibodies in sera and nasal secretions after experimental secondary infection with influenza a virus in humans contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity deletion of m gene open reading frames and of human metapneumovirus: effects on rna synthesis, attenuation, and immunogenicity live vaccines for human metapneumovirus designed by reverse genetics virosome-mediated delivery of protein antigens in vivo: efficient induction of class i mhc-restricted cytotoxic t lymphocyte activity nasal immunization with e. coli verotoxin (vt )-b subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies activation of cytokine genes in t cells during primary and secondary murine influenza pneumonia transforming growth factor-beta: activation by neuraminidase and role in highly pathogenic h n influenza pathogenesis guidance for industry: clinical data needed to support the licensure of seasonal inactivated influenza vaccines the regulation of iga class switching serologic response to standard inactivated influenza vaccine in human immunodeficiency virus-infected children proinflammatory cytokine responses induced by influenza a (h n ) viruses in primary human alveolar and bronchial epithelial cells recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region seasonal influenza infection and live vaccine prime for a response to the pandemic h n vaccine induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? critical regulation of early th cell differentiation by interleukin- signaling development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza a virus vaccine serum and nasal wash antibodies associated with resistance to experimental challenge with influenza a wild-type virus potent high-affinity antibodies for treatment and prophylaxis of respiratory syncytial virus derived from b cells of infected patients respiratory syncytial virus and metapneumovirus progress in understanding and controlling respiratory syncytial virus: still crazy after all these years a single mutation in the pb -f of h n (hk/ ) and influenza a viruses contributes to increased virulence pulmonary histopathology induced by respiratory syncytial virus (rsv) challenge of formalin-inactivated rsv-immunized balb/c mice is abrogated by depletion of cd + t cells enhanced pulmonary histopathology induced by respiratory syncytial virus (rsv) challenge of formalininactivated rsv-immunized balb/c mice is abrogated by depletion of interleukin- (il- ) and il- the seattle virus watch. vi. observations of infections with and illness due to parainfluenza, mumps and respiratory syncytial viruses and mycoplasma pneumoniae heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins an early humoral immune response in peripheral blood following parenteral inactivated influenza vaccination evaluation of a virosomal h n vaccine formulated with matrix m adjuvant in a phase i clinical trial immunology of viral respiratory tract infection in infancy human metapneumovirus fusion protein vaccines that are immunogenic and protective in cotton rats integrin alpha-beta promotes infection by human metapneumovirus sublingual immunization induces broad-based systemic and mucosal immune responses in mice the effect of co-administration of adjuvants with a nanoparticle-based genetic vaccine delivery system on the resulting immune responses influenza virosomes are an efficient delivery system for respiratory syncytial virus-f antigen inducing humoral and cell-mediated immunity sublingual vaccination t cell subset-specific susceptibility to aging persistence of respiratory syncytial virus (rsv) infection and development of rsv-specific igg response in a guinea-pig model of acute bronchiolitis fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia immunization of macaques with formalin-inactivated human metapneumovirus induces hypersensitivity to hmpv infection primary type ii alveolar epithelial cells present microbial antigens to antigen-specific cd + t cells a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach a randomized, double-blind, placebo-controlled study of an rnaibased therapy directed against respiratory syncytial virus innate antiviral responses by means of tlr -mediated recognition of single-stranded rna il- : a th -inducing, proinflammatory cytokine and new member of the il- family il- production by pulmonary dendritic cells impedes th immune responses effector cd + and cd + t-cell mechanisms in the control of respiratory virus infections the hidden tonsils of waldeyer's ring the quadrivalent approach to influenza vaccination identification of a novel coronavirus in patients with severe acute respiratory syndrome progress in the development of respiratory syncytial virus and parainfluenza virus vaccines a randomized controlled trial of cold-adapted and inactivated vaccines for the prevention of influenza a disease effectiveness of seasonal influenza vaccines against influenza-associated illnesses among us military personnel in - : a case-control approach antibody recognition of a highly conserved influenza virus epitope systemic and mucosal immune responses in young children and adults after parenteral influenza vaccination alphavirus replicon particles encoding the fusion or attachment glycoproteins of respiratory syncytial virus elicit protective immune responses in balb/c mice and functional serum antibodies in rhesus macaques correlation of laboratory studies with clinical responses to a/new jersey influenza vaccines human infections with influenza a(h n ) variant virus in the united states prior h n influenza infection and susceptibility of cleveland family study participants during the h n pandemic of : an experiment of nature cross-protective immunity to influenza a viruses adjuvants in influenza vaccines a prospective study of parainfluenza virus type infections in children attending daycare identification of a linear heparin binding domain for human respiratory syncytial virus attachment glycoprotein g ten years of human metapneumovirus research inactivated influenza vaccines virus-specific cd + t cells in primary and secondary influenza pneumonia correlation of cellular immune responses with protection against culture-confirmed influenza virus in young children bacterial toxins as mucosal adjuvants dose dependence of ctl precursor frequency induced by a dna vaccine and correlation with protective immunity against influenza virus challenge a dilemma for mucosal vaccination: efficacy versus toxicity using enterotoxin-based adjuvants initiation of nalt organogenesis is independent of the il- r, ltbetar, and nik signaling pathways but requires the id gene and cd (−)cd (+)cd (+) cells respiratory virus immunization. i. a field trial of two inactivated respiratory virus vaccines; an aqueous trivalent parainfluenza virus vaccine and an alum-precipitated respiratory syncytial virus vaccine dna vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations fast rise of broadly cross-reactive antibodies after boosting long-lived human memory b cells primed by an mf adjuvanted prepandemic vaccine correlation between humoral and cellular immune responses and the expression of the hepatitis a receptor havcr- on t cells after hepatitis a re-vaccination in high and low-responder vaccinees whole inactivated virus influenza vaccine is superior to subunit vaccine in inducing immune responses and secretion of proinflammatory cytokines by dcs the role of the antibody response in influenza virus infection division of labor between dendritic cell subsets of the lung a review of vaccine research and development: human acute respiratory infections the a (h n ) influenza virus pandemic: a review parainfluenza virus type : seasonality and risk of infection and reinfection in young children risk of primary infection and reinfection with respiratory syncytial virus genetic predisposition to wheeze following respiratory syncytial virus bronchiolitis sphingosine -phosphate regulates the egress of iga plasmablasts from peyer's patches for intestinal iga responses antibody response to influenza vaccination in the elderly: a quantitative review immunity to influenza in older adults with chronic obstructive pulmonary disease the efficacy of influenza vaccination in elderly individuals. a randomized double-blind placebo-controlled trial biological challenges and technological opportunities for respiratory syncytial virus vaccine development resistance to and recovery from lethal influenza virus infection in b lymphocyte-deficient mice influenza virus-specific cd + t helper type t lymphocytes do not promote recovery from experimental virus infection a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease inflammatory response of mast cells during influenza a virus infection is mediated by active infection and rig-i signaling update on rhinovirus and coronavirus infections a bovine parainfluenza virus type vaccine is safe and immunogenic in early infancy vaccine delivery using nanoparticles the major human rhinovirus receptor is icam- prevention of serious respiratory syncytial virus-related illness. i: disease pathogenesis and early attempts at prevention bovine respiratory syncytial virus iscoms-protection in the presence of maternal antibodies immunity to and frequency of reinfection with respiratory syncytial virus multiple redundant effector mechanisms of cd + t cells protect against influenza infection enhanced lung disease and th response following human metapneumovirus infection in mice immunized with the inactivated virus structural basis of influenza virus neutralization basic epidemiology and immunopathology of rsv in children respiratory syncytial virus g protein and g protein cx c motif adversely affect cx cr + t cell responses nonolfactory surface epithelium of the nasal cavity of the bonnet monkey: a morphologic and morphometric study of the transitional and respiratory epithelium a practical influenza neutralization assay to simultaneously quantify hemagglutinin and neuraminidase-inhibiting antibody responses molecular basis for high virulence of hong kong h n influenza a viruses cellular immune responses in children and adults receiving inactivated or live attenuated influenza vaccines antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design phenotypic changes in influenzaspecific cd + t cells after immunization of children and adults with influenza vaccines generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters dynamics of human respiratory virus-specific cd + t cell responses in blood and airways during episodes of common cold the toll-like receptor (tlr )-specific stimulus loxoribine uncovers a strong relationship within the tlr , and subfamily the viral sigma protein and glycoconjugates containing alpha - -linked sialic acid are involved in type reovirus adherence to m cell apical surfaces sorting of the respiratory syncytial virus matrix protein into detergent-resistant structures is dependent on cell-surface expression of the glycoproteins immunization of syrian golden hamsters with f subunit vaccine of human metapneumovirus induces protection against challenge with homologous or heterologous strains immunogenicity and efficacy of two candidate human metapneumovirus vaccines in cynomolgus macaques lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed tnf-related apoptosis-inducing ligand eleven years of inflexal v-a virosomal adjuvanted influenza vaccine crossprotective immunity against influenza ph n viruses induced by seasonal influenza a (h n ) virus is mediated by virus-specific t-cells the role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza a and b viruses live and inactivated influenza vaccines induce similar humoral responses, but only live vaccines induce diverse t-cell responses in young children regulation of immunological homeostasis in the respiratory tract clearance of sendai virus by cd + t cells requires direct targeting to virus-infected epithelium the immunostimulating complex (iscom) is an efficient mucosal delivery system for respiratory syncytial virus (rsv) envelope antigens inducing high local and systemic antibody responses antibody responses after influenza and pneumococcal immunization in hiv-infected homosexual men the virosome concept for influenza vaccines antiviral cd + t cell effector activities in situ are regulated by target cell type respiratory virus infection of mice provokes a permanent humoral immune response inflammasome recognition of influenza virus is essential for adaptive immune responses high frequency of cross-reacting antibodies against pandemic influenza a(h n ) virus among the elderly in finland nasal vaccination: a non-invasive vaccine delivery method that holds great promise for the future chitosan as a novel nasal delivery system for vaccines adenovirus infections in transplant recipients nasal vaccine innovation human rhinoviruses persistence of influenza virus-specific antibody-secreting cells and b-cell memory after primary murine influenza virus infection human piv- recombinant sendai virus (rsev) elicits durable immunity and combines with two additional rsevs to protect against hpiv- , hpiv- , hpiv- , and rsv epidemiology of human metapneumovirus methods for nano-particle based vaccine formulation and evaluation of their immunogenicity antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro critical role for cryopyrin/nalp in activation of caspase- in response to viral infection and double-stranded rna an epidemiologic study of altered clinical reactivity to respiratory syncytial (rs) virus infection in children previously vaccinated with an inactivated rs virus vaccine parainfluenza viruses a live attenuated bovine parainfluenza virus type vaccine is safe, infectious, immunogenic, and phenotypically stable in infants and children identification of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants combinatorial antibody libraries from survivors of the turkish h n avian influenza outbreak reveal virus neutralization strategies cell type-specific involvement of rig-i in antiviral response differential roles of mda and rig-i helicases in the recognition of rna viruses sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells pathogenesis of rhinovirus infection future influenza vaccines and the use of genetic recombinants respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine cell-mediated immunity to respiratory syncytial virus induced by inactivated vaccine or by infection nalt-versus peyer's-patch-mediated mucosal immunity dna vaccines: safety and efficacy issues immunity to respiratory viruses type i interferons regulate cytolytic activity of memory cd (+) t cells in the lung airways during respiratory virus challenge measles virus v protein inhibits nlrp inflammasome-mediated interleukin- beta secretion plasmacytoid dendritic cells delineate immunogenicity of influenza vaccine subtypes influenza virus hemagglutinin stalk-based antibodies and vaccines human metapneumovirus antibody response after influenza vaccination in hiv-infected individuals: a consecutive -year study a novel coronavirus associated with severe acute respiratory syndrome pathogenesis of influenza virus infections: the good, the bad and the ugly safety and immunogenicity of an adjuvanted whole virion, inactivated a (h n ) influenza vaccine in young and elderly adults, and children plasma-cell homing ccr expression is a common feature of circulating and mucosal epithelial tissue iga ab-secreting cells immunogenicity and safety of low dose virosomal adjuvanted influenza vaccine administered intradermally compared to intramuscular full dose administration m cell-targeted delivery of vaccines and therapeutics the role of nasopharyngeal lymphoid tissue pattern recognition receptors tlr and cd mediate response to respiratory syncytial virus a nontoxic chimeric enterotoxin adjuvant induces protective immunity in both mucosal and systemic compartments with reduced ige antibodies from plasmids to protection: a review of dna vaccines against infectious diseases heterosubtypic protection against pathogenic human and avian influenza viruses via in vivo electroporation of synthetic consensus dna antigens pandemic whole-virion, vero-cell-derived, adjuvant-free influenza a h n vaccine in patients with solid tumors and hematologic malignancies receiving concurrent anticancer treatment: immunogenicity, tolerability, and acceptability during the pandemic situation coronaviridae influenza vaccines for the future mechanism of action of clinically approved adjuvants live attenuated influenza vaccine (laiv) impacts innate and adaptive immune responses role of il- beta in the development of human t(h) cells: lesson from nlpr mutated patients the magnitude of local immunity in the lungs of mice induced by live attenuated influenza vaccines is determined by local viral replication and induction of cytokines the contribution of systemic and pulmonary immune effectors to vaccine-induced protection from h n influenza virus infection activation, differentiation, and migration of naive virus-specific cd + t cells during pulmonary influenza virus infection a common mucosal chemokine (mucosaeassociated epithelial chemokine/ccl ) selectively attracts iga plasmablasts cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells immunosuppressive hla-g molecule is upregulated in alveolar epithelial cells after influenza a virus infection cd , but not cd , expression on b cells is necessary for optimal primary b cell responses measuring antibody responses to a live attenuated influenza vaccine in children cd t cell-independent antibody response promotes resolution of primary influenza infection and helps to prevent reinfection comprehensive serotyping and epidemiology of human adenovirus isolated from the respiratory tract of korean children over consecutive years ( - ) lymph node dendritic cells control cd + t cell responses through regulated fasl expression antigen sparing and cross-reactive immunity with an adjuvanted rh n prototype pandemic influenza vaccine: a randomised controlled trial optimism over vaccines administered via mucosal surfaces molecular basis of replication of duck h n influenza viruses in a mammalian mouse model preliminary assessment of the efficacy of a t-cell-based influenza vaccine, mva-np+m , in humans safety and immunogenicity of an inactivated adjuvanted whole-virion influenza a (h n ) vaccine: a phase i randomised controlled trial safety and immunogenicity from a phase i trial of inactivated severe acute respiratory syndrome coronavirus vaccine defective immunoregulation in rsv vaccine-augmented viral lung disease restored by selective chemoattraction of regulatory t cells lymphoid tissue in the nasal mucosa of primates, with particular reference to intraepithelial lymphocytes type i ifns enhance the terminal differentiation of dendritic cells the role of animal models in influenza vaccine research influenza vaccine-live toll-like receptor -mediated recognition of herpes simplex virus- by plasmacytoid dendritic cells adaptation and growth characteristics of influenza virus at °c trivalent inactivated influenza vaccine in african adults infected with human immunodeficient virus: double blind, randomized clinical trial of efficacy, immunogenicity, and safety the histological features of the immune system of the equine respiratory tract the distribution of mucosal lymphoid nodules in the equine respiratory tract viruses and bacteria in the etiology of the common cold enhanced mucosal and systemic immune response with intranasal immunization of mice with hiv peptides entrapped in plg microparticles in combination with ulex europaeus-i lectin as m cell target initial infectious dose dictates the innate, adaptive, and memory responses to influenza in the respiratory tract type i interferons keep activated t cells alive a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial antigenic structure of the human respiratory syncytial virus g glycoprotein and relevance of hypermutation events for the generation of antigenic variants the inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proil-beta subcellular localization of toll-like receptor in human dendritic cells the functional heterogeneity of type effector t cells in response to infection is related to the potential for ifngamma production as -adjuvanted versus non-adjuvanted inactivated trivalent influenza vaccine against seasonal influenza in elderly people regulation of iga synthesis and immune response by t cells and interleukins innate immune control and regulation of influenza virus infections il- trans-presentation by pulmonary dendritic cells promotes effector cd t cell survival during influenza virus infection host dna released in response to aluminum adjuvant enhances mhc class ii-mediated antigen presentation and prolongs cd t-cell interactions with dendritic cells memory cd + t cells protect against influenza through multiple synergizing mechanisms structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes the human cytotoxic t cell response to influenza a vaccination adaptive strategies of the influenza virus polymerase for replication in humans live attenuated influenza vaccine induces cross-reactive antibody responses in children against an a/fujian/ / -like h n antigenic variant strain immunoglobulin a (iga): molecular and cellular interactions involved in iga biosynthesis and immune response intestinal iga: novel views on its function in the defence of the largest mucosal surface the influence of hiv infection on antibody responses to a two-dose regimen of influenza vaccine human genetic factors and respiratory syncytial virus disease severity oral immunization with influenza virus in biodegradable microspheres human immune responses to influenza virus vaccines administered by systemic or mucosal routes respiratory syncytial virus synergizes with th cytokines to induce optimal levels of tarc/ccl comparative efficacy of inactivated and live attenuated influenza vaccines naive cd (+) t cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude iscom, a delivery system for parenteral and mucosal vaccination adjuvant system as containing alpha-tocopherol modulates innate immune response and leads to improved adaptive immunity h n influenza vaccine formulated with as a induces strong crossreactive and polyfunctional cd t-cell responses roles of cd + t-cell-independent and -dependent antibody responses in the control of influenza virus infection: evidence for noncognate cd + t-cell activities that enhance the therapeutic activity of antiviral antibodies antigenicity and immunogenicity of equine influenza vaccines containing a carbomer adjuvant the molecular basis of the pathogenicity of the dutch highly pathogenic human influenza a h n viruses formalin-inactivated respiratory syncytial virus vaccine induces antibodies to the fusion glycoprotein that are deficient in fusion-inhibiting activity association of serum anti-neuraminidase antibody with resistance to influenza in man pathological study of archival lung tissues from five fatal cases of avian h n influenza in vietnam systems biology of vaccination for seasonal influenza in humans alum-adjuvanted h n whole virion inactivated vaccine (wiv) induced igg and igg antibody responses in young children mucosal vaccines: the promise and the challenge influenza virus-infected epithelial cells present viral antigens to antigen-specific cd + cytotoxic t lymphocytes gamma interferon is not required for mucosal cytotoxic t-lymphocyte responses or heterosubtypic immunity to influenza a virus infection in mice heterosubtypic immunity to influenza a virus infection requires b cells but not cd + cytotoxic t lymphocytes heterosubtypic immunity to influenza a virus infection requires a properly diversified antibody repertoire a novel m cell-specific carbohydrate-targeted mucosal vaccine effectively induces antigenspecific immune responses a -kda protein on activated helper t cells binds cd and transduces the signal for cognate activation of b cells endogenous naive cd + t cell precursor frequency regulates primary and memory responses to infection vaccination strategies for mucosal immune responses recent developments in adjuvants for vaccines against infectious diseases prevention of antigenically drifted influenza by inactivated and live attenuated vaccines prevention of symptomatic seasonal influenza in - by inactivated and live attenuated vaccines caspase- , caspase- , and calpain are dispensable for il- release by macrophages a common neutralizing epitope conserved between the hemagglutinins of influenza a virus h and h strains protection against the mouse-adapted a/fm/ / strain of influenza a virus in mice by a monoclonal antibody with cross-neutralizing activity among h and h strains efficacy and effectiveness of influenza vaccines: a systematic review and metaanalysis effect of priming with h n influenza viruses of variable antigenic distances on challenge with pandemic h n virus orthomyxoviridae: the viruses and their replication the distinguishing features of human metapneumovirus and respiratory syncytial virus human immunosenescence: is it infectious? immunol toward a universal influenza virus vaccine: prospects and challenges rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates sequential annual administration of purified fusion protein vaccine against respiratory syncytial virus in children with cystic fibrosis differential cmv-specific cd + effector t cell responses in the lung allograft predominate over the blood during human primary infection upon viral exposure, myeloid and plasmacytoid dendritic cells produce waves of distinct chemokines to recruit immune effectors immunogenetics of seasonal influenza vaccine response global transcriptome analysis in influenza-infected mouse lungs reveals the kinetics of innate and adaptive host immune responses type i ifn triggers rig-i/tlr / nlrp -dependent inflammasome activation in influenza a virus infected cells immunity to influenza in ferrets. i. response to live and killed virus immunity to influenza in ferrets. ii. influence of adjuvants on immunization safety and immunogenicity of a novel recombinant subunit respiratory syncytial virus vaccine (bbg na) in healthy young adults effect of age on cytotoxic t lymphocyte memory as well as serum and local antibody responses elicited by inactivated influenza virus vaccine systemic and local antibody responses in elderly subjects given live or inactivated influenza a virus vaccines in elderly persons live attenuated influenza a virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory alveolar macrophages are a major determinant of early responses to viral lung infection but do not influence subsequent disease development quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats type i ifn as a natural adjuvant for a protective immune response: lessons from the influenza vaccine model influenza m vlps containing neuraminidase induce heterosubtypic cross-protection serum antibody prevents lethal murine influenza pneumonitis but not tracheitis bronchus-associated lymphoid tissue (balt) structure and function microencapsulated human parainfluenza virus induces a protective immune response characterization of a live, attenuated human parainfluenza type virus candidate vaccine strain targeting dendritic cells with biomaterials: developing the next generation of vaccines immunoglobulin a mediation of murine nasal anti-influenza virus immunity passive transfer of local immunity to influenza virus infection by iga antibody in vitro comparison of the biologic activities of monoclonal monomeric iga, polymeric iga, and secretory iga role of iga versus igg in the control of influenza viral infection in the murine respiratory tract cytotoxic t lymphocyte memory: role in cross-protective immunity against influenza? induction of protective immunity against influenza virus in a macaque model: comparison of conventional and iscom vaccines interleukin (il)- directs the differentiation of il- -producing cd + t cells cd effector t cell subsets in the response to influenza: heterogeneity, migration, and function lack of cd enhances pathological t cell responses during influenza infection a fused gene of nucleoprotein (np) and herpes simplex virus genes (vp ) induces highly protective immunity against different subtypes of influenza virus the mucosal immune system of the respiratory tract current status of vaccines for parainfluenza virus infections progress in respiratory virus vaccine development mucosal immunization of rhesus monkeys against respiratory syncytial virus subgroups a and b and human parainfluenza virus type by using a live cdna-derived vaccine based on a host range-attenuated bovine parainfluenza virus type vector backbone pathogenesis of acute respiratory illness caused by human parainfluenza viruses comparative evaluation of two severe acute respiratory syndrome (sars) vaccine candidates in mice challenged with sars coronavirus tlr / myd /nf-kappab pathway, reactive oxygen species, potassium efflux activates nlrp /asc inflammasome during respiratory syncytial virus infection recombinant iga is sufficient to prevent influenza virus transmission in guinea pigs adjuvanticity of the oil-in-water emulsion mf is independent of nlrp inflammasome but requires the adaptor protein myd immunogenic properties of rsv-b fusion (f) protein gene-encoding recombinant adenoviruses lectin histochemistry reveals the appearance of m-cells in peyer's patches of scid mice after syngeneic normal bone marrow transplantation antigen-bearing dendritic cells regulate the diverse pattern of memory cd t-cell development in different tissues iga class switch occurs in the organized nasopharynx-and gut-associated lymphoid tissue, but not in the diffuse lamina propria of airways and gut sublingual immunization with m -based vaccine induces broad protective immunity against influenza isotype-specific selection of high affinity memory b cells in nasalassociated lymphoid tissue young infants can develop protective levels of neutralizing antibody after infection with respiratory syncytial virus avian flu: influenza virus receptors in the human airway reappearance of h n influenza virus in man: evidence for the persistence of the virus in domestic chickens effectiveness of as adjuvanted pandemic h n vaccine: case-control evaluation based on sentinel surveillance system in canada long-lived plasma cells: a mechanism for maintaining persistent antibody production phase clinical trials of the safety and immunogenicity of adjuvanted plasmid dna vaccines encoding influenza a virus h hemagglutinin a critical function for cd in lung immune homeostasis and the severity of influenza infection sublingual vaccination with influenza virus protects mice against lethal viral infection receptor-mediated immunoglobulin g transport across mucosal barriers in adult life: functional expression of fcrn in the mammalian lung a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses il- is an effective adjuvant for mucosal and systemic immune responses when coadministered with protein immunogens sars vaccine protective in mice activation of virus replication after vaccination of hiv- -infected individuals sars vaccines the prospects and challenges of universal vaccines for influenza a single amino acid in the pb gene of influenza a virus is a determinant of host range development of effective vaccines against pandemic influenza structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses advances in the vaccination of the elderly against influenza: role of a high-dose vaccine effector t cells control lung inflammation during acute influenza virus infection by producing il- influenza virus iscoms: antibody response in animals structural basis for immunization with postfusion respiratory syncytial virus fusion f glycoprotein (rsv f) to elicit high neutralizing antibody titers new horizon of mucosal immunity and vaccines protective effect of serum antibody on respiratory infection of influenza c virus in rats effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two ' proximal genome positions of bovine/ human parainfluenza virus type on virus replication and immunogenicity parainfluenza virus type expressing the native or soluble fusion (f) protein of respiratory syncytial virus (rsv) confers protection from rsv infection in african green monkeys a host-range restricted parainfluenza virus type (piv ) expressing the human metapneumovirus (hmpv) fusion protein elicits protective immunity in african green monkeys development of a piv-vectored rsv vaccine: preclinical evaluation of safety, toxicity, and enhanced disease and initial clinical testing in healthy adults enhanced protective immunity against h n influenza virus challenge by vaccination with dna expressing a chimeric hemagglutinin in combination with an mhc class i-restricted epitope of nucleoprotein in mice immunosenescence: role and measurement in influenza vaccine response among the elderly identification of nucleolin as a cellular receptor for human respiratory syncytial virus memory cd t cells direct protective responses to influenza virus in the lungs through helper-independent mechanisms identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles recombinant respiratory syncytial virus that does not express the ns or m - protein is highly attenuated and immunogenic in chimpanzees the intracellular sensor nlrp mediates key innate and healing responses to influenza a virus via the regulation of caspase- cytokines: the future of intranasal vaccine adjuvants mortality associated with influenza and respiratory syncytial virus in the united states heterosubtypic neutralizing monoclonal antibodies cross-protective against h n and h n recovered from human igm + memory b cells a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses. plos pathog. control of adenovirus acute respiratory disease in u.s. army trainees cd + t cells clear influenza virus by perforin or fas-dependent processes clearance of an influenza a virus by cd + t cells is inefficient in the absence of b cells quantitative analysis of the influenza virus-specific cd + t cell memory in the absence of b cells and ig sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon functional significance of the perforin/ granzyme cell death pathway evaluation of a recombinant hemagglutinin expressed in insect cells as an influenza vaccine in young and elderly adults recruitment and proliferation of cd + t cells in respiratory virus infections cx c chemokine mimicry by respiratory syncytial virus g glycoprotein immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus tight junctionbased epithelial microenvironment and cell proliferation heterologous protection against influenza by injection of dna encoding a viral protein generation of mhc class i-restricted cytotoxic t lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells toward the development of dna vaccines protective cd + and cd + t cells against influenza virus induced by vaccination with nucleoprotein dna impaired mucosal immune responses in interleukin -targeted mice pathogenesis of respiratory syncytial virus immunopathology of rsv infection: prospects for developing vaccines without this complication host defenses against influenza virus: the role of anti-hemagglutinin antibody innate immunity to respiratory viruses live attenuated or inactivated influenza vaccines and medical encounters for respiratory illnesses among us military personnel pattern of respiratory syncytial virus epidemics in finland: two-year cycles with alternating prevalence of groups a and b respiratory synctial virus infection in balb/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant th -like cytokine pattern immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets summary of probable sars cases with onset of illness from middle east respiratory syndrome coronavirus (mers-cov) summary and literature update -as of mouse respiratory tract dendritic cell subsets and the immunological fate of inhaled antigens direct gene transfer into mouse muscle in vivo heterogeneity of cd (+) and cd (+) t cells antiviral memory t-cell responses in the lung rapid cloning of high-affinity human monoclonal antibodies against influenza virus evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy the absence of enhanced disease with wild type respiratory syncytial virus infection occurring after receipt of live, attenuated, respiratory syncytial virus vaccines orthomyxoviruses m cell-targeted dna vaccination pathogen recognition and development of particulate vaccines: does size matter? a dna vaccine induces sars coronavirus neutralization and protective immunity in mice influenza a virus nucleoprotein is a major target antigen for cross-reactive antiinfluenza a virus cytotoxic t lymphocytes the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses poly(lactic-co-glycolic acid) enhances maturation of human monocyte-derived dendritic cells neutralizing antibodies derived from the b cells of influenza pandemic survivors mucosal vaccines: novel advances in technology and delivery augmentation of immune responses to sars coronavirus by a combination of dna and whole killed virus vaccines isolation of a novel coronavirus from a man with pneumonia in saudi arabia immunogenicity, safety, and protective efficacy of an inactivated sarsassociated coronavirus vaccine in rhesus monkeys a recombinant baculovirus-expressed s glycoprotein vaccine elicits high titers of sars-associated coronavirus (sars-cov) neutralizing antibodies in mice key: cord- - wg wodr authors: dolzhikova, i. v.; grousova, d. m.; zubkova, o. v.; tukhvatulin, a. i.; kovyrshina, a. v.; lubenets, n. l.; ozharovskaia, t. a.; popova, o.; esmagambetov, i. b.; shcheblyakov, d. v.; evgrafova, i. m.; nedorubov, a. a.; gordeichuk, i. v.; gulyaev, s. a.; botikov, a. g.; panina, l. v.; mishin, d. v.; loginova, s. y.; borisevich, s. v.; deryabin, p. g.; naroditsky, b. s.; logunov, d. y.; gintsburg, a. l. title: preclinical studies of immunogenity, protectivity, and safety of the combined vector vaccine for prevention of the middle east respiratory syndrome date: journal: acta naturae doi: . /actanaturae. sha: doc_id: cord_uid: wg wodr the middle east respiratory syndrome (mers) is an acute inflammatory disease of the respiratory system caused by the mers-cov coronavirus. the mortality rate for mers is about . %. due to its high mortality rate, the lack of therapeutic and prophylactic agents, and the continuing threat of the spread of mers beyond its current confines, developing a vaccine is a pressing task, because vaccination would help limit the spread of mers and reduce its death toll. we have developed a combined vector vaccine for the prevention of mers based on recombinant human adenovirus serotypes and . studies of its immunogenicity have shown that vaccination of animals (mice and primates) induces a robust humoral immune response that lasts for at least six months. studies of the cellular immune response in mice after vaccination showed the emergence of a specific cd (+) and cd (+) t cell response. a study of the vaccine protectivity conducted in a model of transgenic mice carrying the human dpp receptor gene showed that our vaccination protected % of the animals from the lethal infection caused by the mers-cov virus (mers-cov emc/ , ld( ) per mouse). studies of the safety and tolerability of the developed vaccine in rodents, rabbits, and primates showed a good safety profile and tolerance in animals; they revealed no contraindications for clinical testing. the middle east respiratory syndrome (mers) is an acute inflammatory disease of the respiratory system that was first diagnosed in june in saudi arabia [ , ] . the disease is caused by the mers-cov coronavirus, a member of the genus betacoronavirus of the family coronaviridae. one-humped camels are the natural reservoir of the virus; human infection occurs through contact with camels and consumption of unpasteurized camel milk; an aerosol transmission of infection is also possible [ , ] . according to the who, a total of , laboratory-confirmed cases of mers had been registered by september , , of which resulted in a fatal outcome (a . % mortality rate) [ ] . most mers cases were registered in saudi arabia [ ] . however, the disease was also detected in other countries (the united arab emirates, south korea, yemen, etc.); cases of imported infection were reported in europe, north africa, and north america [ ] . because of the lack of effective preventive and therapeutic drugs for mers, the high mortality rate of the disease, and the widespread character of the infection reservoir, who experts classify mers-cov as a virus with the potential to cause a pandemic. there have been no cases of mers in russia. however, due to the high mortality of mers and the continuing threat that it could spread outside the endemic areas [ ] , development of a vaccine is an urgency. vaccination can limit the spread of mers and reduce its mortality [ ] . to date, several candidate vaccine preparations based on a protective antigen, mers-cov s glycoprotein and its derivatives (s subunit, receptor-binding domain), are known: vector vaccines (based on recombinant adenoviruses and vaccinia virus), a dna vaccine based on plasmid dna, as well as vaccines based on recombinant proteins and virus-like particles [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . since the formation of a humoral and cellular immune response is important to protect against mers-cov, the use of recombinant viral vectors for antigen delivery seems promising for the development of anti-mers vaccines. these vectors provide long-term expression of the antigen in the cells of the immunized organism, which results in a protective immune response as early as after the first or second immunization. repeated vaccination is effective in inducing the most pronounced and lasting immune response, while heterologous vaccination involving the use of different viral vectors for primary and secondary immunization is the most optimal regimen. this regimen was successfully implemented in the development of a vaccine against the disease caused by the ebola virus; the vaccine has been registered in the russian federation for medical use and already undergone post-registration clinical trials in the african republic of guinea [ ] . we have developed a combined vector vaccine for the prevention of mers based on recombinant human adenovirus serotypes and expressing mers-cov glycoprotein (mers-cov emc/ isolate). here, we present the results of a study of the post-vaccination humoral and cellular immune responses in mice and primates, as well as the results of preclinical studies of the safety of the developed vaccine against mers. both components are lyophilisates for the preparation of solutions for intramuscular administration. the drug was obtained in compliance with the conditions of biotechnological production at the medgamal branch of the gamaleya national research center for epidemiology and microbiology of the ministry of health of the russian federation. all experiments on animals were carried out in strict accordance with the recommendations of the national standard of the russian federation (gost r - , "principles of good laboratory practice"). sixweek-old female c bl/ mice ( - g) were purchased from the pushchino breeding facility (russia). transgenic f hybrid mice were obtained by crossing transgenic homozygous +/+ males carrying the human dpp receptor gene (hdpp ) (medical university of texas, usa) and non-transgenic c bl/ females (pushchino, russia). expression of the transgene in f hybrid mice was confirmed by immunoblotting. all mice had free access to water and food and were housed in an isocage animal housing system (tecniplast, italy). common marmosets (callithrix jacchus) were born and kept in a specialized animal facility at the chumakov federal scientific center for research and development of immune-and-biological products ras (moscow, russia). the animals were kept at the laboratory for modeling immunobiological processes with the experimental clinic of callitrichidae (chumakov federal scientific center for research and development of immune-and-biological products ras) in accordance with the requirements for housing laboratory primates. all experimental procedures with marmosets were carried out by a specialist who had received certification from the federation of european laboratory animal science associations (felasa) and completed a course on working with primates ("laboratory animal science for researchers: non-human primates," karolinska institute, stockholm, sweden). all animals were identified by a radio chip implanted subcutaneously and having a unique -digit code (globalvet, moscow). the mice were immunized intramuscularly using the widest possible dose range, × to v.p. per mouse. immunization was carried out twice successively with component and then component with a -day interval. mouse serum samples were collected at the following time points: and days, three and six months after immunization. the marmosets were immunized intramuscularly at a dose of v.p. per animal. immunization was conducted twice successively with component and then component with a -day interval. plasma samples were collected at the following time points: before immunization, seven and days, as well as three and six months, after immunization. the titer of glycoprotein-specific antibodies in serum/ plasma was determined by enzyme immunoassay. the following recombinant proteins were used: s glycoprotein ( -v b; sino biological, china) and rbd ( -v b ; sino biological). a pbs solution in . % tween- (pbs-t) containing % non-fat dry milk (a ; applichem, spain) was used for blocking. serum/plasma was titrated in two steps in a pbs-t solution containing % non-fat dry milk. anti-mouse igg horseradish peroxidase-linked secondary antibodies (nxa ; ge healthcare, usa) were used to detect mouse igg. serum of a rabbit immunized with marmoset igg and anti-rabbit igg horseradish peroxidase-linked secondary antibodies (na v; ge healthcare, usa) were used to detect marmoset igg. a tetramethylbenzidine solution (niiopik, russia) was used as a chromogenic agent. the reaction was stopped by adding m h so ; optical density was measured at nm (od ) using a multiskan fc microplate reader (thermo fisher scientific, usa). the igg titer was defined as the maximum serum dilution at which the od value of the serum sample from the immunized animal exceeded that of the control serum/plasma (serum/plasma of the control animal or animal before immunization) more than twofold. the titer of virus-neutralizing antibodies (vnas) in the plasma of immunized animals was determined in a neutralization reaction (nr) by suppressing the cytopathic effect caused by the mers-cov virus (mers-cov emc/ ) in the monolayer of vero b cells. the neutralization reaction was carried out in the "constant viral dose/serum dilution" mode. monkey plasma was incubated at °c for min to remove non-specific inhibitors. all serum samples were diluted in a dmem medium supplemented with % inactivated fetal bovine serum, starting from the : ratio, then with two-fold dilution to : , . dilutions of the mers-cov virus suspension were prepared in a dmem medium supplemented with % inactivated fetal bovine serum. the concentration of the mers-cov virus in the prepared dilution was , tcid /ml. a mixture of equal volumes of plasma and the virus suspension was incubated at °c for min. vero b cells were plated in -well plates at × cells per well at a volume of µl and then supplemented with µl of the mixture of plasma and the virus suspension. the cytopathic effect was assessed after four days. the vna titer of the studied plasma was defined as its highest dilution at which the cytopathic effect was suppressed in two out of three wells (compared to the control serum samples). the mice were euthanized on day eight after immunization. the spleens were collected and homogenized through a -µm sieve in sterile pbs. splenocytes were isolated by ficoll ( . g/ml; paneco, russia) density gradient centrifugation ( g, min). for t cell proliferation assay, the splenocytes were stained with carboxyfluorescein using the carboxyfluorescein succinimidyl ester (cfse) tracer kit (invitrogen, usa) as previously described [ ] . cells were seeded in -well plates at × cells per well in a rpmi medium and re-stimulated with the recombinant mers-cov s protein ( -v b; sino biological) at µg per well. after h, the media were collected for a ifn-gamma analysis and the cells were harvested, washed with pbs, stained with antibodies specific to cd , cd , and cd : allophycocyanin (apc)-labelled anti-cd , apc-cy -labelled anti-cd , and phycoerythrin-labelled anti-cd (bd biosciences, usa), and then fixed in % paraformaldehyde. proliferating cd + and cd + t lymphocytes were evaluated in the cell mixture using a bd facs aria iii flow cytometer (bd biosciences). the resulting percentage of proliferating cells (x) was determined using the following formula: x = %st -%, where %st is the percentage of proliferating cells after splenocyte re-stimulation with recombinant mers-cov s glycoprotein, and % is the percentage of proliferating cells in the absence of splenocyte re-stimulation (intact cells). the concentration of ifn-gamma in the medium was measured by elisa using a commercial kit (mouse ifn-γ elisa kit; invitrogen) according to the manufacturer's protocol. the increase in the concentration of ifn-gamma was determined using the following formula: x = cst/cint, where x is the fold increase in the concentration of ifn-gamma, cst is the concentration of ifn-gamma in the medium of stimulated cells (pg/ ml), and cint is the concentration of ifn-gamma in the medium of unstimulated (intact) cells (pg/ml). the protective efficacy of the vaccine was studied in a model of lethal infection in transgenic mice carrying the human dpp receptor gene and obtained by crossing homozygous transgenic hdpp +/+ males and non-transgenic c bl/ females. the animals were immunized intramuscularly twice successively with component and then component with a -day interval. seven days after the injection of component , the mice were infected intranasally with the mers-cov virus (mers-cov emc/ ) at a dose of ld per animal, and then the survival rate was analyzed for a period of days. preclinical studies of the safety of the combined vector vaccine against mers were conducted in collaboration with the autonomous non-commercial organization "institute of biomedical research and technology" and fsaei he i.m. sechenov first moscow state medical university, in compliance with the guidelines for preclinical trials of medicinal products [ ] and guidelines for experimental (preclinical) study of new pharmacological substances [ ] . the safety study included the analysis of the toxicity of a single and repeated administration, as well as sn assessment of the reproductive and ontogenetic toxicity, immunogenicity, and allergenicity. a total of mice, rats, rabbits, guinea pigs, and six common marmosets were used in the preclinical safety study. tolerability of the vaccine in primates was analyzed daily by assessing the physical condition of the animals and based on the presence of general symptoms of in-toxication, which included an assessment of behavior, appearance, and physiological functions. vaccine tolerance in marmosets was studied in the laboratory by monitoring the body weight, rectal temperature, and blood biochemical parameters: total bilirubin, direct bilirubin, aspartate aminotransferase (ast), alanine aminotransferase (alt), creatinine, total protein, and alkaline phosphatase (alp). the studies were carried out on fully automatic analyzers ca- and b- (furuno, japan) using diasys reagent kits (germany). statistical analysis of the data was performed using the graphpad . software. either the student's t-test for independent samples or the mann-whitney u-test was used for the analysis of the data of unpaired samples depending on the data distribution normality [ ] . either the student's t-test for paired samples or wilcoxon's test was used for the analysis of the data of related samples depending on the data distribution normality [ ] . distribution normality was determined using the generalized d'agostino-pearson test [ ] . in order to select an effective dose, mice were immunized intramuscularly with the vaccine at doses of - , × v.p. per mouse; serum samples were collected, and the titers of glycoprotein-specific antibodies were analyzed two and four weeks after immunization. next, the intensity of the post-vaccination humoral immune response was assessed based on the titer of glycoprotein-specific igg (fig. ) . analysis of the obtained results demonstrates a dose-dependent increase in the serum titer of glycoprotein-specific igg. the minimum dose of the combined vector vaccine required to induce a robust humoral immune response was v.p. per mouse for all vaccinated animals. analysis of the duration of post-vaccination humoral immunity showed that glycoprotein-specific antibodies were detected at a high titer in the mouse serum six months after immunization (the geometric mean titer was : , , fig. ) . next, we studied the level of humoral immunity in primates vaccinated with the developed vaccine. in order to determine the level of humoral immunity in common marmosets (c. jacchus), they were immunized with the combined vaccine according to the regimen intended for clinical use, i.e. successively with component (at a dose of v.p. per animal) and then com-ponent (at a dose of v.p. per animal) with a -day interval. further, plasma samples were collected from animals for the analysis of the titer of glycoproteinspecific igg seven and days, as well as three and six months, after the boosting of immunization (fig. a) . immunization of primates was shown to induce robust humoral immunity, which persists for at least six months. for instance, the titers of glycoprotein-specific igg in primates six months after immunization did not differ from the titers after three months, which is an indication of the induction of long-term immunity. analysis of the titer of neutralizing antibodies to the mers-cov virus in the plasma of immunized monkeys showed that vnas were detected in the animals as early as seven days after booster immunization, while virus-neutralizing antibody titers are shown on the ordinate axis; time after immunization is represented on the abscissa axis. individual titers for each studied animal and the geometric mean titer (n = ) are indicated the maximum vna titer was reached three and six months after immunization (fig. b) . no vnas were detected in the plasma of the control animals and the animals before immunization. thus, our analysis of the level of post-vaccination immunity showed that immunization of mice and primates induces a robust humoral immune response, which persists for at least six months after immunization. in order to assess the level of post-vaccination cellular immunity, the mice were immunized with the candidate vaccine against mers once at a dose of v.p. per mouse. spleens were collected from the animals days after immunization; splenocytes were isolated, and the number of proliferating cd + and cd + t lymphocytes was determined in the splenocyte culture in vitro after cell re-stimulation with the recombinant mers-cov s protein (fig. ) . the obtained data demonstrate that introduction of the combined vector vaccine induces the formation of s-specific cd + and cd + t cells. activation of cellular immunity was also analyzed by measuring the expression of ifn-gamma. the results of the study of an increase in the ifn-gamma concentration in an in vitro culture of mouse splenocytes after repeated stimulation of the cells with the recombinant mers-cov s protein are presented in fig. . administration of the vaccine increased the concentration of ifn-gamma in the medium upon stimulation of the splenocytes of immunized mice with the mers-cov s glycoprotein. the concentration of ifn-gamma in the medium increased by an average of times. summarizing the data of our analysis of the antigenspecific lymphoproliferative activity of cd + and cd + t cells and the level of ifn-gamma expression by restimulated splenocytes, we can conclude that immunization of animals with the combined vaccine against mers results in the formation of glycoprotein-specific cellular immunity. the study was carried out in a model of lethal infection caused by mers-cov in transgenic mice carrying the human dpp receptor gene. mice were immunized successively with component and then component with a -day interval. one week after administration of component of the vaccine, animals were infected intranasally with the mers-cov virus (mers-cov emc/ ) at a dose of ld per animal, and the survival rate was assessed during days. immunization of the animals with the combined vector vaccine was shown to protect % of animals from lethal infection caused by the mers-cov virus. all control (unvaccinated) animals died (fig. ) . irritation effect, immunotoxicity, allergenic properties, and reproductive toxicity) were evaluated in rodents (mice, rats, and guinea pigs) and large animals (rabbits). the combined vector vaccine against mers did not cause any toxic effects, did not have an allergenic or immunotoxic effect, did not affect the generative function, did not have a local irritation effect, and can be recommended for clinical studies. vaccine tolerability was also studied in primates. no abnormalities in the analyzed parameters of physical condition (behavioral reactions, appearance, and physiological functions) were found in the animals immunized with the combined vector vaccine against mers and the control animals during the observation period. rectal temperature, changes in body weight, and biochemical parameters were within the normal range for the species in all animals during the experiment (fig. ) . summarizing the obtained data, we can conclude that the combined vector vaccine against mers has shown good tolerability in the common marmoset model. currently, there are no specific prophylactic and therapeutic agents against the middle east respiratory syndrome in the world. intensive studies on the development of vaccines for this disease are currently underway in the united states, germany, korea, china, great britain, and other countries. among the prophy-lactic drugs with the highest efficiency demonstrated in preclinical studies, the following candidate vaccines can be mentioned: vaccines based on adenoviral vectors (ad , ad , chadox ) [ , ] , modified vaccinia virus ankara (mva) [ ] encoding mers-cov protective antigen s, as well as preparations of recombinant mers-cov protective antigen s [ , ] . two drugs are currently undergoing clinical trials: two vaccines based on recombinant viral vectors mers (based on chimpanzee adenovirus, phase ) [ ] and mva-mers-s (based on vaccinia virus, phase ) [ ] . clinical studies of the first phase of a vaccine based on plasmid dna (gls- ), as well as a vaccine based on a vaccinia virus (mva-mers-s), have been completed [ , ] . all vaccines that have reached clinical trials are based on mers-cov s glycoprotein. this glycoprotein performs one of the most important roles in the viral life cycle: it enables virus internalization via interaction with the dpp receptor on the cell surface. neutralization of this interaction limits penetration of the virus into the cell, thus decreasing its replication. since the formation of not only a humoral, but also cellular immune response is important for protection against mers, the development of vaccines based on recombinant viral vectors seems promising. such vectors effectively deliver antigen-encoding genetic material to the cells, which results in the cellular expression of the antigen and induction of a robust cellular and humoral immunity. an important property of recombinant viral vectors is that they induce protective immunity as early as after the first or second immunization, which is extremely important when developing a vaccine for the prevention of dangerous and extremely dangerous infections and is intended for use during an epidemic or in the case of an infection that spreads beyond non-endemic areas. we have conducted a study of the immunogenicity of various forms of mers-cov s glycoprotein: fulllength glycoprotein (s), full-length glycoprotein with the transmembrane domain of the g protein (s-g) of the vesicular stomatitis virus, a secreted glycoprotein receptor-binding domain (rbd), a secreted glycoprotein rbd fused to the fc fragment of human igg (rbd-fc), and the membrane form of the glycoprotein rbd (rbd-g) [ ] . the obtained data demonstrated that the membrane form of the rbd is the most effective in inducing a robust cellular immune response, while full-length glycoprotein is most efficient in inducing a robust cellular immunity. when choosing an immunization regimen, one should take into account the fact that repeated heterologous vaccination, which involves the use of two different recombinant viral vectors for primary and secondary immunization, is advisable for inducing long-term immunity. for this studies of the immunogenicity of the combined vector vaccine revealed the induction of long-term humoral immunity in mice, while the mean titer of glycoprotein-specific antibodies equaled : , two weeks after vaccination at a dose of v.p. per mouse. a similar antibody titer was observed by alharbi et al. in mice days after immunization with a vaccine against mers based on chimpanzee adenovirus chadox mers [ ] ; however, the authors used a dose of v.p. per mouse for immunization. in another study by munster et al. [ ] , immunization of transgenic mice carrying the human dpp receptor gene with a chadox mers vaccine at a dose v.p. per mouse was shown to protect % of the animals from a lethal infection with mers-cov. hashem et al. developed a rad -based drug carrying the mers-cov s sequence and demonstrated that repeated immunization of mice with the drug at a dose of v.p. per mouse induced a humoral immune response [ ] . the titer of glycoprotein-specific igg was : , three weeks after the second immunization (one and a half months from the beginning of immunization); the drug also provided % protection to animals from mers-cov infection [ ] . glycoprotein-specific antibodies were found at a titer range of : , to : , in the plasma of the animals as early as a week after the boosting of immunization. it is important to note that muthumani et al. [ ] detected glycoprotein-specific antibodies at a titer of : , for a period of six weeks in primates after long-term thrice immunization with a dna vaccine; the authors also showed that immunization of primates with the dna vaccine protects them from a mers-cov infection. the study of post-vaccination humoral immunity in mice and primates demonstrated that an intense humoral immune response persists in the animals for at least six months after vaccination. analysis of the cellular component of the immunity in the mice showed that administration of the developed vaccine induces a robust cellular response. it is important to note that not only a cd + but also cd + t cell response is observed, which can play an important role in protection against mers-cov [ , ] having completed studies of the immunogenicity of the combined vector vaccine in a model of lethal infection in transgenic mice carrying the human dpp receptor gene, we studied the protective effect of the vaccine. the vaccine was shown to provide % protection to animals from lethal infections of mers-cov. our series of preclinical studies of vaccine safety revealed no contraindications for the clinical testing of the developed vaccine. in this work, we have studied the immunogenicity and safety of a combined vector vaccine for the prevention of the middle east respiratory syndrome. the following conclusions were obtained: vaccination of animals with the vaccine induces a robust humoral immune response to the mers-cov s glycoprotein persisting for at least six months. vaccination of animals induces a robust cellular immune response to the mers-cov s glycoprotein. vaccination of animals induces a protective immune response, which protects % of animals from a lethal infection of mers-cov. preclinical studies of the vaccine safety did not reveal any contraindications to clinical testing. middle east respiratory syndrome coronavirus (mers-cov) annual review of diseases prioritized under the research and development blueprint) international multicenter study of the immunogenicity of medicinal product gamevac-combi. clinicaltrials.gov identifier: nct guidelines for preclinical trials of medicinal products. // moscow: grif i k guidelines for experimental (preclinical) study of new pharmacological substances // moscow: medicine // ekologiya cheloveka with the english safety and immunogenicity of a candidate mers-cov vaccine (mers ) phase ib study to assess the safety and immunogenicity of mva-mers-s_df- . clinicaltrials.gov identifier: nct url phase i, open label dose ranging safety study of gls- in healthy volunteers clinicaltrials.gov identifier: nct url proc. natl. acad. sci. usa. . v. . № key: cord- - ksahg o authors: cresswell, e.; brennan, m. l.; barkema, h. w.; wapenaar, w. title: a questionnaire-based survey on the uptake and use of cattle vaccines in the uk date: - - journal: vet rec open doi: . /vropen- - sha: doc_id: cord_uid: ksahg o background: vaccination is a widely used strategy for disease control in cattle in the uk and abroad. however, there has been limited research describing the uptake and use of cattle vaccines on uk farms. aim: to describe the current uptake and usage of cattle vaccines in the uk. design: a questionnaire, available in paper and online format, was distributed to cattle farmers by convenience sampling. participants: all uk cattle farmers were eligible to participate in the study. results: eighty-six per cent of respondents (n= / ) had vaccinated their cattle in the past year. diseases most commonly vaccinated against were bovine viral diarrhoea, leptospirosis and infectious bovine rhinotracheitis. vaccination compliance was limited in certain areas, for example only per cent of respondents stated that they administered the second dose in the primary course within the recommended timeframe, and per cent of respondents stated that they vaccinated earlier than the youngest recommended age. although outside the scope of this study, further work is needed to establish the extent of inadequate compliance and the effect this has on vaccine efficacy. the role of the veterinarian was highlighted as the main supplier of vaccines and preferred source of vaccination information. respondents preferred to receive recommendations regarding vaccination by face-to-face communication with the veterinarian. conclusions: the results provide a description of the current uptake and usage of cattle vaccines in the uk. uptake is generally high but there are areas of usage of vaccines which could be improved upon. the veterinarian plays a key role as supplier of vaccines and a source of information for the majority of farmers. although outside the scope of this study, further work is needed to establish the extent of inadequate compliance and the effect this has on vaccine efficacy. although the respondents in this study represent a biased population of farmers, the findings indicate areas for future investigation in order to improve vaccination strategies in cattle in the uk. vaccines have been used in veterinary medicine since the inoculation of lambs with sheep pox in the th century (mcvey and shi ) . the administration of vaccines is now commonplace and is considered one of the most important aspects of global disease control (tizard ). strategic implementation of vaccination is important to cattle health and welfare, as vaccination can help to control and eradicate disease, as demonstrated by the global eradication of rinderpest (normile ) , and control of rabies, foot and mouth disease and swine erysipelas (lombard and others ) . in order for disease control to be effectively achieved via vaccination, correct usage is required, which includes administering vaccines via the correct route, at the appropriate time and to a specified target group of animals (responsible use of medicines in agriculture alliance (ruma) ). incorrect administration may lead to breakthrough disease, rendering vaccination a wasteful, rather than beneficial exercise (salisbury and others ) . in the uk, vaccination may be carried out by anybody who is deemed capable by the person prescribing the vaccine. the person prescribing is a veterinarian, pharmacist or a 'suitably qualified person' (sqp), depending on whether the vaccine is categorised as a pom-v (prescription only medicine -veterinarian) or pom-vps (prescription only medicine -veterinarian, pharmacist or sqp). the ruma initiative provides guidance to farmers on correct vaccination protocols (ruma ) . each disease presents its own challenges, and therefore, the decisions on which animals to vaccinate and which vaccines to use are multifactorial. cost, logistical factors and side effects of vaccination need to be balanced with the potential benefits of improved animal health and increased production. factors such as worker health and safety and farm assurance requirements may also influence the decision whether to vaccinate. there is limited information in the literature about the uptake and usage of vaccination in cattle. a uk-based study on bovine viral diarrhoea virus (bvdv) vaccine use suggested that uptake and correct administration may be poor, and administration protocols may not be correct; one-third of farmers never referred to the vaccine product datasheet, and per cent of farmers administered the two doses in the primary vaccination course at the incorrect interval (meadows ) . the objective of this study was to gather data on the current usage and uptake of cattle vaccination in the uk using a farmer survey. a questionnaire was developed and distributed to uk farmers between september and november (available on request form authors). a paper version of the questionnaire was produced, and an identical online version was created using a proprietary survey tool (surveymonkey, palo alto, usa). the questionnaire was split in two parts: part a, involving questions about vaccination protocols on the farm, and part b, involving questions relating to the respondent and their farm. the questionnaire contained closed and open-ended questions and took approximately minutes to complete. eleven questions asked respondents about the protocol used for a specific vaccine the respondent nominated as being most familiar with. as protocols vary between vaccines, the interpretation of 'correct' or 'incorrect' use was made using information provided on the vaccine datasheet (national office of animal health (noah) ). pretesting of the questionnaire was carried out with two veterinary students, four veterinarians and three farmers. minor corrections were made, and paper copies for piloting were sent to five farmers. the online version was piloted with two veterinary students and two farmers. after minor typographical and lay-out adjustments following feedback from pilot participants, the questionnaire was finalised for distribution, and a cover letter included explaining the purpose of the study and offering the option of entering into a £ prize draw for agricultural stockist vouchers. farmers were asked to nominate which vaccines they had used in the past year, aided by images of the vaccines which were broadly categorised according to the diseases they target (table ) . multivalent vaccines, providing protection against more than one disease, were placed in multiple categories. all vaccines registered for use in cattle in the uk were included in the survey. bluetongue vaccines and one bvdv vaccine were excluded from the survey, because at the time the questionnaire was distributed, bluetongue vaccination was not permitted, and a bvdv vaccine (pregsure bvd, pfizer) had recently been taken off the market. the target population was any person in the uk who owned or worked with cattle. between september and november , participants were canvassed at events around the uk, including the dairy event and livestock show, royal berkshire show, dairy health events (dairy development council, wales), british mastitis conference, welsh dairy show and two local cattle markets (melton mowbray and market drayton); these were selected by convenience sampling (dohoo and others ) . paper copies were distributed to approximately cattle farmers at nine agricultural shows and meetings. questionnaires were completed at the events, or farmers were provided with reply address envelopes. the link to the online questionnaire was distributed by email to veterinary practices, and to staff and students at the school of veterinary medicine and science, university of nottingham, for participation or forwarding to potential respondents. the practices were selected by convenience sampling, via contacts established at the aforementioned events. the link was also placed on online fora (british farming forum, farmers weekly interactive, farmers guardian, farming forum, the cattle site, young farmers' club) and on two social networking sites (facebook and twitter). distribution and publicity regarding the survey was increased (by publishing information about the study in their newsletters or requesting participation from clients) with the help of veterinary practices, pharmaceutical companies and associations involved with cattle farming (xl vets, british cattle veterinary association, dairyco, animal health and veterinary laboratories agency, department for environment, food and rural affairs and dairy processors). awareness of the study was further increased through articles in farming and veterinary press such as veterinary newsletters, veterinary times, farmers guardian, and other online veterinary fora (www.vetsonline.com). a datasheet was created using microsoft excel (microsoft, redmond, usa) and questionnaire responses manually entered. ten per cent of the questionnaires were checked to detect data entry errors; no errors were observed. where appropriate, answers to open questions were categorised by the first author into themes using thematic analysis methodology (attride-stirling ). epitools epidemiological calculator (sergeant ) was used to perform χ tests on all biologically plausible combinations of categorical variables; only significant relationships are reported. continuous variables (age and herd size) were divided into three categories based on the range and distribution of data collected, taking into account information on age of farm 'holders' and herd size as reported by defra ( ). for questions, it was possible to select more than one answer; in those cases, the cumulative proportions could exceed per cent. the questionnaire was completed by respondents between september and november . peak responses were observed early in the study period when distribution at events was greatest. the response rate for the paperbased questionnaire was estimated at per cent (n= / ), with per cent (n= / ) of questionnaires being returned by post, and the remainder being completed at events. a response rate for the online questionnaire could not be determined, as it was distributed via public discussion fora. fifty per cent (n= / ) of respondents completed the questionnaire online (table ) . nonrespondents were not further investigated. eighty-six per cent (n= / ) of respondents indicated that they had vaccinated their cattle in the past year, with more dairy farmers vaccinating compared to beef farmers (table ). the most frequent reason for not vaccinating was that they did not perceive there to be a problem that required vaccination ( per cent). respondents from large farms (> adult cattle) were more likely to have vaccinated their cattle in the past year than respondents from small farms (< cows) (p= . ). the difference in vaccination uptake between small or large, versus average size farms was not significant in the χ analysis. as respondents indicated that they did not vaccinate (table ) , only a maximum of respondents could complete the questions on vaccine usage. the number of total responses for each question varied, which has been indicated in relevant tables and figures. the highest uptake of vaccination, for dairy as well as beef, was for bvdv vaccination, and there was a noticeable difference in uptake of lungworm vaccine between dairy and beef respondents (fig ) . uptake for other vaccines was: per cent for rotavirus, coronavirus and escherichia coli (trivacton , rotavec corona and lactovac), per cent for salmonellosis (bovivac s), per cent for ringworm (bovilis ringvac), and per cent for mastitis (startvac). specific questions were asked about usage of the vaccine that respondents had selected as the one they were most familiar with. thirty-three per cent of respondents excluded certain animals from vaccination (table ) . when prompted for further information, per cent of respondents indicated that they excluded pregnant animals, of which two respondents stated using a clostridial vaccine that is not recommended for use in animals in the first and second trimesters of pregnancy. fifty respondents stated that they used a vaccine which is not recommended for pregnant animals, but did not nominate any exclusions. sixteen of these respondents were dairy farmers, of whom indicated that they vaccinated calves up to six months, which may explain why they did not state to exclude pregnant animals from vaccination. the youngest age at which animals were nominated as first receiving a vaccine ranged from one week to months. of those respondents who had specified the vaccine they were most familiar with, per cent were vaccinating earlier than the youngest recommended age (table ). vaccines administered too early were against bvdv (n= ), lungworm (n= ), infectious bovine rhinotracheitis (ibr) (n= ), respiratory disease complexes (n= ), clostridial diseases (n= ) and leptospirosis (n= ). after the first dose was given, per cent of respondents were administering a second dose of the vaccine 'correctly' (i.e., at the recommended time, or not at all if not required). a second dose of vaccine was most commonly administered within the correct time frame for lungworm vaccines ( per cent), and least commonly administered within the correct time frame for vaccines against clostridial diseases ( per cent) ( table ) . several verbal and two written comments provided at the end of the questionnaire raised issues regarding the 'inconvenience of vaccines requiring two doses'. seventy-three per cent of farmers stated the correct route of administration for the nominated vaccines (intramuscular, subcutaneous, intranasal or oral routes). frequently mentioned incorrect routes were subcutaneous where intramuscular was indicated ( per cent, n = / ) and vice versa ( per cent, n = / ). for those vaccines where an injection site was recommended on the datasheet, per cent of respondents nominated the recommended site (table ). of those respondents not injecting at the recommended site, per cent were injecting in the gluteal region where the neck was recommended, and per cent were injecting elsewhere on the animal. the remainder of incorrect answers ( per cent) indicated more than one injection site. a recommended site for injection was not provided on the datasheet for vaccines; these were excluded from the analysis (n= ). vaccines were, on the majority of farms, administered by workers. twenty-three per cent of respondents did not read instructions as 'they did what they had done previously and did not need instructions' (table ) . eight per cent of respondents obtained the vaccine from an agricultural merchant (table ) , the majority of which were legal category pom-vps. three respondents obtained vaccines from an agricultural merchant which required a veterinary prescription (pom-v). sixty-six per cent of respondents indicated that they or somebody else on the farm had discussed the use of the vaccine with the person who had supplied it in the past year, and cost was the most common topic for dairy as well as beef farmers (table ) . most respondents ( per cent) sourced information regarding vaccinating cattle from their veterinarian ) . when asked for their preferred source of information, per cent of respondents preferred to receive information about vaccination from a veterinary practice, with the majority of respondents preferring face-to-face communication. email, websites and mobile telephone were more often preferred by beef farmers compared to dairy farmers (fig ) . this survey describes novel information on vaccine usage in the uk that could be beneficial to practitioners implementing vaccination protocols on farms. many farmers who did not use cattle vaccines did not perceive there to be a problem with disease on their farm, although few stated that they had actively monitored disease. the risk of introducing disease in naive herds can be high, particularly when buying in livestock. improved immunity through vaccination may reduce the risk of losses (stott and gunn ) . datasheets for most vaccines recommend that unhealthy animals should be excluded from vaccination, as vaccinating immunocompromised animals may lead to ineffective protection. in this survey, only a minority of farmers were excluding certain animals, including sick and injured cattle, and this could lead to less than adequate disease control. additionally, respondents who nominated using a vaccine which was contraindicated in pregnant cattle did not state they would exclude these animals from being vaccinated. this may be due to farmers being unaware of the risks of using these vaccines in pregnant animals, but may also be because there were no pregnant cattle on these farms, which is likely for respondents from beef farms with grower and/or finisher herds. other reasons for not excluding animals could be that a whole herd approach is being taken for management purposes, and all animals are being vaccinated, rather than selecting animals individually for vaccination. timing of vaccination may also be important for effective disease control, and the majority of respondents administered vaccines within the recommended timeframe on the datasheets. this was carried out correctly more frequently for first vaccinations ( per cent) than for the second dose within a primary course ( per cent). vaccine failure has been demonstrated in human beings where administration occurred below the recommended first age of vaccination (galil and others ) , and by not administering a second dose within the recommended time period (peltola and others ) . although these studies involve human patients, immunological responses to vaccines have been shown to be similar between bovine and human patients, as described in a study investigating immunological processes of tuberculosis in human beings and cattle (waters and others ) . this suggests that not administering vaccines to cattle within the recommended timeframe may lead to vaccine failure. the most common incorrect route of administration was the use of subcutaneous versus intramuscular injections. a study in human patients demonstrated that the same vaccine is immunogenic regardless of whether it is injected subcutaneously or intramuscularly (knuf and others ) . there is no data to support this finding in the veterinary literature with regards to vaccines. however, for other veterinary medicines, such as ivermectin and ceftiofur sodium, the efficacy of the drug was not deemed to be different when using intramuscular versus subcutaneous routes of administration (lifschitz and others , brown and others ) . understanding farmer motivators and barriers for vaccination may be useful to improve uptake and usage of vaccines. economic factors, such as vaccine cost and increased production profits associated with vaccination, could affect decision-making on whether to vaccinate. although nearly half the respondents had discussed cost with their vaccine supplier in the past year, cost was not frequently mentioned as a barrier for vaccination. however, a dutch study has suggested that economic factors are the main motivators (e.g., increased production profits) and barriers (e.g., vaccination costs) for farmers when deciding whether to vaccinate their livestock (elbers and others ) . other studies describe factors, such as worker satisfaction, as being equally as motivating as cost in farmer decision-making about cattle health (valeeva and others ) . the veterinarian was the main vaccine supplier, and also the preferred source of information for many respondents in this study, but only per cent of respondents had discussions about vaccine use with their supplier. the variation observed with respect to following recommended vaccination protocols may be explained by this lack of discussion on the use of vaccines. incorporating information such as ruma's 'checklist for vaccination' (ruma ), together with tailored farm advice during herd health visits provides an opportunity to discuss vaccination protocols with a client. this approach could improve adherence to recommended vaccination strategies, reduce vaccine failure on farms, and raise awareness of the potential benefits among those who are not vaccinating. veterinarians are valued by farmers as important discussion partners in the field of animal health (hall and wapenaar ) and can share expertise in addition to supplying vaccines. as out of the vaccines available require a prescription which can only be obtained from a veterinarian, it may be more convenient for farmers to obtain information and supplies from the same source, as opposed to agricultural merchants and internet pharmacies. supplying medicines forms an essential part of farm veterinary practice in the uk; it was recently reported that an average of per cent of income for farm veterinary practices is derived from medicine sales (veterinary development council ). although vaccines are available from agricultural merchants and internet pharmacies, per cent of respondents bought their vaccines from a veterinarian, highlighting the potential opportunity for the veterinarian to combine their role as supplier with their role as advisor about vaccination. veterinarians rarely carried out vaccination on farms, as this was mostly done by workers and senior staff. generally, no specific qualifications are required to be a farm worker; it may be that some of these workers carrying out vaccination have had no formal training in the correct administration of vaccines. further work to confirm lack of training as a cause of suboptimal vaccine usage may help to focus future knowledge transfer activities. a limitation of the study was that information provided by respondents may have differed from reality, particularly when answered from memory (recall bias). therefore, incorrect answers may have been provided by respondents who, in fact, are vaccinating correctly when able to refer to the recommended protocol. parts of the questionnaire responses could not be used for analysis as answers were not provided for some questions or were ambiguous. this was particularly apparent where respondents did not nominate a vaccine they were most familiar with, as a response was required for the correct interpretation of several of the subsequent questions. there was no apparent explanation for these missing responses. additionally, this study asked questions regarding the use of one specific vaccine they felt most familiar with which may not be representative of the usage of other vaccines on the farm. in this study, the proportion of farmers not vaccinating was likely to be underestimated. bias due to convenience sampling is probable; it is likely that farmers who vaccinate their cattle and were interested in vaccination were more inclined to participate in the survey than those who did not have any interest in vaccination. however, the demographic data of respondents, as described in table , resemble the demographic data collected by defra on the whole uk farming population (defra ), indicating that a reasonably representative crosssection of farmers participated in the study. the sample size of respondents limited the power of this study; increasing the number of respondents to consolidate our findings would further support the study results. however, the farming population in the uk is known to be challenging to engage in surveybased research, and increasing sample size will take considerable effort. our results, although only describing vaccination uptake and usage of respondents, are supported by other work performed in the uk (meadows ) and provide a basis for further studies to evaluate vaccine efficacy and disease control in situations with correct and less than ideal use of vaccines. a perhaps more important limitation of this study is the bias encountered because of the convenience sampling method used, which is difficult to counter using the voluntary survey method. one could hypothesise that this study underestimated vaccination compliance, as more engaged farmers may have participated, which makes it more likely to expect poorer compliance in nonrespondents. however, as the current published body of work in the field of uptake and usage of cattle vaccines is scarce, the findings of this study are important to describe current usage strategies on vaccination, and are therefore an important contribution to knowledge in this field. in conclusion, uptake of cattle vaccines in the uk was generally good, but improvements to usage would improve adherence to data sheet guidelines and could increase the efficacy of vaccination. a particular area where vaccination strategies could be improved would be correctly selecting animals for vaccination and administering vaccines within the recommended timeframes. the veterinarian was nominated as the main vaccine supplier and source of information on vaccines and, therefore, remains crucial to improve cattle vaccination strategies in the uk. contributors all authors made substantial contributions to the conception or design of the work. ec performed the data collection. the analysis of data was performed by ec and ww. all authors contributed to the interpretation of the data and approved the submitted version. funding school of veterinary medicine and science, the university of nottingham. ethics approval the study received ethical approval from the school of veterinary medicine and science ethics committee, the university of nottingham. provenance and peer review not commissioned; externally peer reviewed. data sharing statement additional unpublished data collected in the questionnaire which are not in this manuscript due to word limit restrictions, is available from the authors on request. ) ‡includes dairy, beef, mixed and unallocated respondents †responses for multivalent vaccines (n= ) were incorporated into multiple disease categories bvd thematic networks: an analytic tool for qualitative research comparison of plasma pharmacokinetics and bioequivalence of ceftiofur sodium in cattle after a single intramuscular or subcutaneous injection agriculture in the united kingdom veterinary epidemiologic research questionnaire survey about the motives of commercial livestock farmers and hobby holders to vaccinate their animals against bluetongue virus serotype in - in the netherlands younger age at vaccination may increase risk of varicella vaccine failure opinions and practices of veterinarians and dairy farmers towards herd health management in the uk safety, immunogenicity and immediate pain of intramuscular versus subcutaneous administration of a measles-mumps-rubellavaricella vaccine to children aged - months ivermectin disposition kinetics after subcutaneous and intramuscular administration of an oil-based formulation to cattle a brief history of vaccines and vaccination vaccines in veterinary medicine: a brief review of history and technology. the veterinary clinics of north america a study to investigate the use and application of bvdv vaccine in uk cattle compendium of data sheets for animal medicines. national office of animal health ltd rinderpest. driven to extinction the elimination of indigenous measles, mumps, and rubella from finland by a -year, two-dose vaccination program responsible use of medicines in agriculture alliance ( ) ruma cattle guidelines immunisation against infectious disease. rd edn. the stationery office epitools epidemiological calculators use of a benefit function to assess the relative investment potential of alternative farm animal disease prevention strategies veterinary immunology: an introduction. th edn motivation of dairy farmers to improve mastitis management the veterinary development council report - . british veterinary association tuberculosis immunity: opportunities from studies with cattle. clinical and developmental immunology key: cord- -df w l authors: rosales-mendoza, sergio; márquez-escobar, verónica a.; gonzález-ortega, omar; nieto-gómez, ricardo; arévalo-villalobos, jaime i. title: what does plant-based vaccine technology offer to the fight against covid- ? date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: df w l the emergence of new pathogenic viral strains is a constant threat to global health, with the new coronavirus strain covid- as the latest example. covid- , caused by the sars-cov- virus has quickly spread around the globe. this pandemic demands rapid development of drugs and vaccines. plant-based vaccines are a technology with proven viability, which have led to promising results for candidates evaluated at the clinical level, meaning this technology could contribute towards the fight against covid- . herein, a perspective in how plant-based vaccines can be developed against covid- is presented. injectable vaccines could be generated by using transient expression systems, which offer the highest protein yields and are already adopted at the industrial level to produce vlps-vaccines and other biopharmaceuticals under gmpc-processes. stably-transformed plants are another option, but this approach requires more time for the development of antigen-producing lines. nonetheless, this approach offers the possibility of developing oral vaccines in which the plant cell could act as the antigen delivery agent. therefore, this is the most attractive approach in terms of cost, easy delivery, and mucosal immunity induction. the development of multiepitope, rationally-designed vaccines is also discussed regarding the experience gained in expression of chimeric immunogenic proteins in plant systems. the coronaviruses (covs) are enveloped viruses having a positive-sense, single-stranded genomic rna [ ] and are grouped into four genera: α-covs, β-covs, γ-covs, and δ-covs. the ones that affect mammals are αand β-covs, while the other two genera infect birds and could also infect mammals [ ] . lately, the coronavirus has become a remarkable concern for global health after the diagnosis of a cluster of unknown pneumonia patients in december in wuhan, china. the outbreak was associated with workers in the wuhan wholesale seafood market, in which live exotic animals are sold [ ] . the pathogen was named sars-cov- given its similarity ( % of genomic similarity) to sars-cov- , which was responsible for the - severe acute respiratory syndrome epidemic (sars) . until now, the official and accepted name for this new coronavirus strain is covid- virus, an acronym for coronavirus infectious disease [ ] . a systematic genomic analysis has revealed amino acid substitutions between sars-cov- and sars-cov- , which is a starting point to study its functional and pathogenic divergence [ ] . presently, there is no specific treatment for covid- . as an emerging pathogen, several knowledge gaps exist about the sars-cov- virus and the coming months will be critical to refine clinical management and advancement in the validation of possible treatments. a myriad of efforts is ongoing to perform clinical trials and determine the efficacy of preexisting drugs, with hydroxychloroquine and remdesivir as the most promising candidates [ ] . receptor blockers are also under evaluation [ ] , along with the assessment and development of monoclonal antibodies. moreover, transfusions using plasma from individuals recovered from sars-cov- infection (containing neutralizing antibodies) are under exploration with promising findings [ ] . nonetheless, vaccination is the most effective approach to control and ultimately eradicate infectious diseases. since sars-cov- possesses high transmissibility (asymptomatic and presymptomatic virus shedding, which results in a high number of patients with mild symptoms), the development of vaccines is an urgent goal to fight against this pathogen. the straightforward path to generate vaccine candidates is the technology of inactivated vaccines, which can be formulated with sars-cov- virions previously inactivated by chemical or physical treatments. a vaccine based on live-attenuated virus is another possible approach [ ] . for sars-cov- and mers-cov, inactivated vaccine candidates have induced robust humoral responses leading to virus neutralization. alternatively, the construction of a chimeric attenuated virus constitutes an interesting path. for instance, the influenza virus can be used as a scaffold to expose sars-cov- antigens and generate a bivalent vaccine targeting two relevant pathogens causing respiratory diseases [ ] . similarly, adenoviral vectors could be applied in this field [ ] . vaccines based on whole viruses are associated with concerns about antibody-dependent enhancement of viral infection, reactogenicity, and strain reversion to pathogenic forms, among other safety issues [ ] . therefore, the path for vaccine development will require a detailed characterization, not only in terms of efficacy but also safety [ ] . although inactivated vaccines will perhaps be the main avenue for generating the first experimental vaccines, alternative approaches should be explored in parallel, namely the development of subunit vaccines. since the coronavirus spike (s) glycoproteins initiate entry into cells; they are considered the primary target for neutralizing antibodies (figure ). cytotoxic t-lymphocyte (ctl) responses are also of key relevance to protect against viral infections [ ] . under these principles, vaccine development against covid- has been initiated and efforts announced in this field include the development of rna-based vaccines by curevac [ , ] , and an rna vaccine candidate formulated with a novel lipid nanoparticle (lnp) carrying mrna encoding for a full-length, prefusion stabilized s protein [ ] . another candidate (developed by shenzhen geno-immune medical institute [ ] ) consists of a multiepitopic vaccine based on the generation of artificial antigen presenting cells through transduction as a way to express viral antigens and immune modulatory genes to ultimately activate t cells. vaccines , , x of these principles, vaccine development against covid- has been initiated and efforts announced in this field include the development of rna-based vaccines by curevac [ , ] , and an rna vaccine candidate formulated with a novel lipid nanoparticle (lnp) carrying mrna encoding for a fulllength, prefusion stabilized s protein [ ] . another candidate (developed by shenzhen geno-immune medical institute [ ] ) consists of a multiepitopic vaccine based on the generation of artificial antigen presenting cells through transduction as a way to express viral antigens and immune modulatory genes to ultimately activate t cells. figure . structure of the sars-cov- virus. the virus is formed by an envelope membrane associated with the following structural proteins: spike protein (s), which mediates binding to the host cell receptors and considered a critical target for the induction of antibodies capable of neutralizing the virus; hemagglutinin-esterase dimer (he), which acts as a potent mediator of attachment and destruction of sialic acid receptors on the host cell surface; a membrane glycoprotein (m), which is important to generate the virus; and the envelope protein (e), which adheres to the m protein to form the viral envelope. the viral structure also comprises a nucleocapsid protein (n) that, along with the rna genome, produces the nucleocapsid. although the conventional developmental paths for vaccines take several years, the vaccine against hand, foot, and mouth disease (hfmd) that was approved in china; demonstrates how joint efforts can lead to new vaccines that are accepted in relatively short periods after a pathogen emerges [ ] . similarities with the sars-cov- virus constitute a valuable reference for vaccine development, however it should be considered that the genetic variability of sars-cov- seems higher as more than two variants have been described. despite this, the cross protection of sars-cov- and mers experimental vaccines remains to be explored. thus far, there is evidence that some sars-cov- neutralizing antibodies cross react with sars-cov- , however extensive research in this regard is needed. the epitopes conserved among sars-cov- and sars-cov- have been identified and proposed for vaccine development [ ] . the race for generating fundamental knowledge on covid- is leading to rapid advances, which will be useful to refine vaccine design. for instance, walls et al. [ ] , reported cryo-em structures of the ectodomain trimer, which will be of critical importance for designing vaccines and viral entry blockers. they also found evidence of potent inhibition of sars-cov- entry into host cells by using murine polyclonal antibodies against sars-cov- , which suggests that conserved s figure . structure of the sars-cov- virus. the virus is formed by an envelope membrane associated with the following structural proteins: spike protein (s), which mediates binding to the host cell receptors and considered a critical target for the induction of antibodies capable of neutralizing the virus; hemagglutinin-esterase dimer (he), which acts as a potent mediator of attachment and destruction of sialic acid receptors on the host cell surface; a membrane glycoprotein (m), which is important to generate the virus; and the envelope protein (e), which adheres to the m protein to form the viral envelope. the viral structure also comprises a nucleocapsid protein (n) that, along with the rna genome, produces the nucleocapsid. although the conventional developmental paths for vaccines take several years, the vaccine against hand, foot, and mouth disease (hfmd) that was approved in china; demonstrates how joint efforts can lead to new vaccines that are accepted in relatively short periods after a pathogen emerges [ ] . similarities with the sars-cov- virus constitute a valuable reference for vaccine development, however it should be considered that the genetic variability of sars-cov- seems higher as more than two variants have been described. despite this, the cross protection of sars-cov- and mers experimental vaccines remains to be explored. thus far, there is evidence that some sars-cov- neutralizing antibodies cross react with sars-cov- , however extensive research in this regard is needed. the epitopes conserved among sars-cov- and sars-cov- have been identified and proposed for vaccine development [ ] . the race for generating fundamental knowledge on covid- is leading to rapid advances, which will be useful to refine vaccine design. for instance, walls et al. [ ] , reported cryo-em structures of the ectodomain trimer, which will be of critical importance for designing vaccines and viral entry blockers. they also found evidence of potent inhibition of sars-cov- entry into host cells by using murine polyclonal antibodies against sars-cov- , which suggests that conserved s epitopes among these viruses are relevant for immunization and perhaps preexisting immunization models will be useful in the fight against covid- . the characterization of other key targets for drug design has been accelerated (e.g., the protease x-ray structure has been recently described [ ] ). therefore, these recent advances will set the basis to accelerate the development of covid- subunit vaccines taking into consideration the sars-cov- vaccine development, especially the approach targeting the s protein. in parallel with these advances in vaccine design, defining vaccine platforms amenable for rapid and large scale production is required. these must keep in mind that low cost, safe, and easy distribution and delivery are the required attributes to implement broad coverage of vaccination campaigns; especially in the developing countries. genetically engineered plants constitute a consolidated platform for the manufacturing of biopharmaceuticals. plants have been used over the last three decades for this purpose. thus far, a diverse group of biopharmaceuticals has been functionally produced in plant systems that include antibodies, vaccines, growth factors, and cytokines [ ] . remarkably, a recombinant enzyme produced in carrot cells has been already approved by the fda for gaucher's disease treatment [ ] . the main advantages of plant-based platforms include the inability to replicate human pathogens (diminishing the risk of contamination and making the purification process less strident), use of unsophisticated bioreactors, and efficient synthesis of complex proteins (multimeric or glycosylated) [ , ] . the current expression approaches for recombinant proteins using the plant cell as host comprise stably-transformed plants at the nuclear or chloroplast levels and transiently-transformed plants. these expression modalities are summarized in table . the conventional approach for expression of transgenes in plants comprises transgene insertion into the nuclear genome. currently, agrobacterium-mediated transformation is the most popular method to achieve this modification since this bacterium has the ability to transfer large segments of dna with minimal rearrangement at high efficiency with low number of insertions. nonetheless, the transgene is randomly inserted into the genome, which often leads to positional effects that make expression levels unpredictable and interruption of endogenous genes a possibility. another limitation is the induction of silencing mechanisms that hamper productivity. nevertheless, it should be considered that new technologies are emerging to cope with these limitations by providing ways to achieve site-directed insertion through a number of mechanisms [ ] . the n-terminal fragment of the sars-cov- s protein (s ) was expressed in stably transformed tomato and low-nicotine tobacco plants, which induced iga and igg responses in mice. [ ] transient nuclear genome transformation rapid production; high productivity; implemented at the industrial level seed bank cannot be generated; requires purification of the antigen to eliminate toxic compounds from the host and ag-robacteria residues s protein; multiepitope vaccines a chimeric protein of gfp and amino acids - of the sars-cov- s protein (s :gfp) was transiently expressed in tobacco leaves and stably transformed in tobacco and lettuce. no immunization assays were performed the sars-cov- n protein was transiently expressed in nicotiana benthamiana, which induced in mice high levels of igg and igg a and up regulation of ifn-γ and il- in splenocytes. a chimeric protein of gfp and the sars-cov- s protein was transiently expressed in tobacco plants. no immunization tests were performed. the sars-cov- m and n proteins were transiently expressed in n. benthamiana. the n protein was antigenic but immunogenicity was not assessed. [ [ ] [ ] [ ] transplastomic technologies high productivity; multigene expression is possible; improved biosafety since the transgene is inherited maternally; site-specific insertion through recombination; not affected by silencing or position effects complex post-translational modifications are not performed; transformation protocols available for few species and the generation of lines takes long time. a chimeric protein of gfp and amino acids - of the sars-cov- s (s :gfp) was expressed in transplastomic tobacco plants. [ , ] vaccines , , in the case of transplastomic technologies, they typically comprise the site-directed insertion of the foreign dna into the chloroplast genome in an event induced by homologous recombination. the main advantage of this technology is the high protein yield, which is a consequence of the high copy number of the transgene and the fact that silencing events have not been reported thus far, and position effects are avoided due to the site-directed insertion. in addition, transgene confinement is achieved given the maternal inheritance shown by most plant species and several proteins can be produced from a single transformation event through the use of operon-like arrangements [ , ] . for more information regarding transplastomic technologies, readers are referred to reviews published by [ , ] . another approach to express heterologous protein in plants relies on the use of viral-based vectors, which exploit the efficient promoters, utrs, and dna/rna replication mechanisms found in plant viruses. interestingly, the agrobacterium-mediated delivery of viral vectors has consolidated as a highly efficient strategy to achieve rapid production of biopharmaceuticals with yield up to g of protein per kg of fresh plant biomass. the tobamoviruses, potexviruses, tobraviruses, geminiviruses, and comoviruses have served as a basis for the development of transient, efficient expression systems in plants [ ] . this technology, however, demands the purification of the target protein given the presence of toxic compounds (e.g., alkaloids) in the typical host (nicotiana benthamiana), as well as bacterial residues, especially endotoxins. therefore, at present, this technology is applied for the formulation of injectable or nasal vaccines. thus far, some plant-vaccine candidates have entered clinical trials, including candidates against swine influenza, rabies, and hepatitis b [ ] . the most promising candidates are the influenza vaccines developed by medicago inc. that rely on using a non-replicative vector carrying viral regulatory sequences to mediate the transient expression of hemagglutinin (ha) in n. benthamiana, which has led to injectable vaccine candidates [ , ] . overall, these vaccines have been seen as safe and their immunogenic properties have been positively evaluated using in vitro assays with human and mice cells. the trials conducted in volunteers revealed proper immunogenicity with no serious adverse effects (see table ). moreover, a group of respiratory diseases has been targeted through the plant-based production of antigens with promising immunogenicity in preclinical trials [ ] . both homologous and heterologous antigen-specific cd + t cells were elicited. additionally, production of ifn-γ, il- , and/or tnf-α was achieved upon ex vivo antigenic re-stimulation. ha from a/california/ / h n . vlps were evaluated in vitro using human monocyte-derived macrophages. the plant-made vlps were efficiently captured and subjected to endosomal processing and cross-presentation. [ ] ha from a/h n /california/ / (pdmh n ). the inactivated h n vaccine (iiv) was included as a reference vaccine. mice were i.m.-immunized twice. cd + (tnf-α, ifn-γ) and cd + (ifn-γ) t cell responses were higher for the plant-made vaccine than the iiv formulation. the plant-made vlp vaccine elicited stronger and more balanced immune responses than iiv. [ ] ha from a/california/ / (h n ) and a/indonesia/ / (h n ). in vitro assays were performed using mouse and human dcs. mice were immunized by the i.m. route using alhydrogel as an adjuvant. human dcs exposed to plant-made vlps showed high stimulation in terms of secretion of il- , il- , and tnfα and cd expression, along with activation of cd + and cd + t cells. vlps induced accumulation of both cdc s and cdc in the draining lymph nodes of test mice. lymphocytes from mice immunized with plant-made vlps secreted higher concentrations of pro-inflammatory cytokines (including il- , il- , and tnf-α) upon antigenic stimulation. [ ] ha from a/california/ / or a/indonesia/ / strains. in vitro assays were performed using mouse dendritic cells. the plant-made vlps trimmers were morphologically stable over time and interacted with activated antigen-presenting cells similar to the wild type virus. [ ] nonetheless, the ultimate goal for low-cost vaccine development could be achieved by generating oral formulations not requiring purification and being composed of freeze-dried biomass encapsulated in gelatin pills or tablets. under this focus, the goal is to trigger specific immune responses via the gut-associated lymphoid tissues (galt). for this purpose, edible plants lacking toxic metabolites should be used. perhaps the main disadvantage of this technology is the long time required to generate transformed lines of edible crops efficiently expressing the antigen of interest (e.g., rice or corn transgenic lines or transplastomic lines) [ ] . these oral vaccines will overcome the disadvantages of injectable vaccines such as painful administration and the risk associated with invasive delivery routes. in terms of costs, avoiding the requirements of sterile devices and trained personnel represent substantial savings. the fact that plant-based vaccine formulations will not require antigen purification will be, without a doubt, the main factor that will make them low-cost alternatives [ ] , which is necessary to provide wide vaccination coverage in developing and low-income countries. recent promising reports in the viability of formulating oral vaccines include the case of the hepatitis b virus surface antigen [ ] and models of immunotherapy against allergy with promising results [ ] , among other diseases. therefore plant-based vaccines are becoming a reality, although it should be recognized that the progress has been slower than initially expected. this is particularly true for the development of oral vaccines, with the main drawbacks being poor characterization of antigen stability, bioavailability, and reproducibility [ , ] . the currently available expression strategies for foreign proteins in plants allow projecting the generation of several vaccine candidates against covid- in the short term. the expression approach and targeted organelle will depend on the nature of the selected target antigen. in the following section, possible avenues for the development of plant-based vaccines are envisaged (table and figure ). the currently available expression strategies for foreign proteins in plants allow projecting the generation of several vaccine candidates against covid- in the short term. the expression approach and targeted organelle will depend on the nature of the selected target antigen. in the following section, possible avenues for the development of plant-based vaccines are envisaged (table and figure ). developmental paths for plant-based vaccines. vaccine antigens can comprise full length viral proteins or chimeric proteins rationally designed to serve as multiepitopic vaccines. this is based on the selection of t cell and b cell epitopes with the aid of immune bioinformatics tools and experimental data of their protective capacity. current genetic engineering technologies allow expressing target antigens either stably or transiently, which allow exploring distinct immunization approaches. transient expression typically requires purification of the antigen-which is useful for the formulation of injectable vaccines-whereas stable transformation, especially in edible crops, allows implementing oral immunization schemes without purification. combined schemes, using pure antigens for priming by parenteral administration and plant biomass containing the antigen for oral boosting, are likely ideal approaches capable of inducing long-term protection in several compartments, especially in mucous membranes. vaccine antigens can comprise full length viral proteins or chimeric proteins rationally designed to serve as multiepitopic vaccines. this is based on the selection of t cell and b cell epitopes with the aid of immune bioinformatics tools and experimental data of their protective capacity. current genetic engineering technologies allow expressing target antigens either stably or transiently, which allow exploring distinct immunization approaches. transient expression typically requires purification of the antigen-which is useful for the formulation of injectable vaccines-whereas stable transformation, especially in edible crops, allows implementing oral immunization schemes without purification. combined schemes, using pure antigens for priming by parenteral administration and plant biomass containing the antigen for oral boosting, are likely ideal approaches capable of inducing long-term protection in several compartments, especially in mucous membranes. one prominent approach for vaccine design is based on the use of virus-like particles (vlps), which are macromolecular complexes resembling viruses, but lacking their genome. in this way, vlps mimic the native structure of viruses, but are not infective. this avoids the disadvantages of vaccines formulated with attenuated or inactivated viruses that include reactogenicity and reversion to pathogenic forms [ , ] . a myriad of reports on the production of vlps can be found in the literature that comprise the cases of the influenza virus, human papillomavirus, human immunodeficiency virus, foot and mouth disease virus, norwalk virus, rift valley fever virus, and hepatitis b virus [ ] [ ] [ ] [ ] . the precedents of sars-cov- and mers antigens expressed in recombinant systems leading to the formation of vlps constitute important guides for the topic of covid- vaccine development. mortola and roy ( ) produced coronavirus vlps applying baculovirus/insect cells expression (sf cells), which was based on the following structural proteins: s (spike), e (envelope), m (membrane), and n (nucleocapsid) of sars-cov, either individually or simultaneously. simultaneous expression at high levels was achieved for s, e, and m proteins, leading to an efficient vlps assembly and release, evidenced by electron microscopy and immunofluorescence. the authors demonstrated that the formed vlps were similar in morphology to the sars-cov- virions [ ] . another group reported in the same year that m and e proteins were sufficient for the efficient formation of vlps in insect cells [ ] . in , the immunogenicity of sars-cov- vlps was first described by evaluating insect cell-made vlps based on the m, e, and s proteins. electron microscopy demonstrated vlps formation in co-infected insect cells. mice subjected to an immunization scheme consisting of four subcutaneous (s.c.) doses of vlps emulsified with freund's adjuvant showed high antibody titers against sars cov. furthermore, vlps elicited cellular immunity following increased production of ifn-γ and il- [ ] . subsequent in vitro assays using vlps designed with the bat-isolated coronavirus protein s and the sars-cov- proteins e and m; demonstrated ability to stimulate dcs in terms of cytokine induction, evidenced by il- and tnf-alpha production. furthermore, the study indicated that ifn-γ+ and il- + cd + t cells increased in co-culture with dcs pre-exposed to the vlps tested [ ] . given that immunization by mucosal routes is the most pertinent for vaccination, sars-cov- vlps were analyzed in a mouse model based on intraperitoneal or nasal immunization. both routes led to sars-cov- -specific igg responses, igg levels in the groups immunized intraperitoneally were higher. nasal immunization usually results in the induction of secretory iga responses at the genital tract, saliva, and lungs; a type of response that is not efficiently induced by systemic administration. in the mentioned study, iga production at the intestinal tract was higher for intraperitoneal administration when compared to intranasal administration, however only genital tract secretions from the i.n.-immunized group showed neutralization activity [ ] . another approach for the production of vlps consisted in co-expressing the sars-cov- s protein and the m influenza protein in the baculovirus/insect cell expression system. interestingly, the chimeric vlps showed similar size and morphology compared to the wild type sars-cov- . immunogenicity and protective efficacy of these chimeric vlps were tested in a mouse lethal challenge model. complete protection from death was observed in mice vaccinated intramuscularly or intranasally with the chimeric vlps, which contained . µg of the sars-cov- protein s [ ] . in the case of the middle east respiratory syndrome (mers), vlps with proteins s, e, and m were generated using the baculovirus/insect cell expression system by wang et al. ( ) [ ] . electron microscopy and immunoelectron microscopy revealed high structural similarity between the mers-cov vlps and the native virus. rhesus macaques inoculated with mers-cov vlps and aluminum adjuvant showed final virus neutralizing antibody titers reaching : ; inducing t-helper (th ) cell-mediated immunity. in a more recent study, the structural proteins of mers-cov were expressed in silkworm larvae and bm cells for the development of vaccine candidates. however, protein s was not detected in the form of vlps despite the fact that e and m proteins were secreted to the culture supernatant. surfactant treatment and mechanical extrusion using protein s or bm cells expressing structural proteins allowed producing nanovesicles exhibiting protein s with a diameter ranging from to nm as revealed by immuno-tem [ ] . although insect cells possess a protein processing capacity similar to that of higher eukaryotic cells, the protein processing pathways are not totally equivalent [ ] . in terms of n-acetylglycosylation, insect cells have the ability to assemble n-glycans to a nascent polypeptide. however, they have unusual final processing activity mediated by β-n-acetylglucosaminidase (soluble or membrane-bound form), which trims an intermediate product common to mammalian and insect pathways resulting in the insect-specific paucimannose as a final product that avoids the galactosylation process occurring in mammals [ ] . considering that vlps of enveloped viruses have been successfully expressed in plants by expressing the ha protein, it is anticipated that sars-cov- vlps could be assembled by expressing the s protein. in fact, although not published in the literature yet, medicago has described the generation of vlps in plants [ ] . for this purpose, nuclear expression targeting the trans-golgi secretion route is proposed to yield a protein subjected to the glycosylation and secretion machinery that could make possible the production of vlps [ ] . therefore, adoption of these works would be valuable during the development of covid- plant-based vaccines (see the section below). besides vlps resembling the sars-cov- virus, another possible approach is to adopt sars-cov- epitopes and express them in chimeric vlps. in this way, a vlp from an unrelated virus can serve as the scaffold to present the target sars-cov- epitopes. for this purpose, the hepatitis b core protein has been applied as scaffold to display unrelated antigens of some pathogens. in particular, this has been applied at the immunodominant c/e loop region, which allows displaying the target on the spike structures of the vlps [ ] . a recent review revealed that at least vaccine candidates have been developed based on plant viruses covering infectious agents, cancer, and autoimmune disorders [ ] . the glycosylation patterns of proteins that form vlps can impact their immunogenicity and protective capacity. interestingly, glycoengineering strategies have been successfully implemented for plants, which allows diversifying their application as hosts for the production of biopharmaceuticals. this is a relevant aspect considering that plants lack the ability to perform glycosylation, which is a characteristic of mammalian systems. for instance, plants perform beta , -xylosylation, core alpha , -fucosylation, and the addition of a second n-acetylglucosamine (glcnac) to the mannose core. moreover, plant glycans lack β ( , ')-galactose and sialic acid, as well as bi-antennary n-glycans [ ] . in some cases, such as antibody production, these differences on glycosylation can be associated to adverse effects that include generation of immunogenic activity, which can eventually lead to blocking antibodies against the therapeutic antibody. nonetheless, in the case of vaccines these differences could add more immunogenic potential and improve vaccine efficacy [ ] . in any case, it is of interest to comment that glycoengineering has allowed developing knock down n. benthamiana plants for beta , -xylosyltransferase (xylt) and alpha , -fucosyltransferase (fuct) genes, which showed significant reduction in the production of xylosylated and/or core alpha , -fucosylated proteins with no phenotypic alterations [ ] . these lines will be valuable tools to study the impact of glycosylation in the efficacy of covid- vlps, which will allow optimizing the plant-based vaccines. another approach that deserves attention is the development of multiepitopic vaccines, which offers the opportunity of achieving a fine rational vaccine design by selecting epitopes that potentially induce robust and protective immune responses. at the same time, this approach will discard those related to non-protective responses or even those inducing antibody-dependent enhancement of the disease. in this way, a highly efficacious and safe vaccine can be obtained. of special interest are the reports proving that specific s protein epitopes enhance the disease upon a pathogen challenge, which highlights the relevance of recurring to the rational design of vaccines to ensure not only immunoprotection, but safety [ ] . for the case of multiepitope vaccines against infectious agents, several reports have focused on selecting the most promising th, b cell, and t cell epitopes that might allow the rational design of a vaccine inducing robust protective responses, while at the same time avoiding deleterious or non-relevant responses. one key factor that adds importance to the development of multiepitopic vaccines against viral disease is genetic variability. in fact, it has been suggested that the sars-cov- virus evolved into two major types, l and s. the l type (∼ %) predominates versus the s type (∼ %), with the latter being proposed as the ancestral version. the l type is more aggressive than the s type and the analysis performed by tang et al. [ ] suggests that human interference influenced the prevalence of l and s types right after the outbreak appearance. another study has also suggested a rapid evolution of this coronavirus [ ] . therefore, the design of multiepitopic vaccines must adopt the selection of epitopes conserved among the viral variants with the capacity to induce neutralizing humoral responses as critical criterion. it should also be considered that ctl responses are relevant to cope with covid- [ ] . the selected epitopes are the targets to which adaptive immune responses should be induced, but they lack the required complexity to trigger robust immune responses. therefore, a carrier is required to increase antigen complexity and, through adjuvant effects, enhance potency of the induced immune response, making it properly polarized. among these carriers, the b subunit of the cholera toxin and the heat-labile enterotoxin from escherichia coli have been extensively used to carry unrelated antigens. the main advantage of these molecules is their adjuvant effects at the mucosal levels, as well as their ability to efficiently deliver the antigens to the submucosa thanks to a mechanism of translocation mediated by binding to the gm receptor at the epithelial cells in the mucosal surfaces [ ] . once at the submucosa, the antigen is efficiently captured and processed by antigen presenting cells, which subsequently induce robust mucosal and systemic th immune responses at the lymph nodes. the design of ltb/ctb-based chimeras carrying sars-cov- epitopes is an interesting approach that should be explored. proline-containing linkers have been used to properly display unrelated antigens without altering the ability of those carriers to form the pentameric structure responsible for gm binding [ ] . the accumulation of those chimeric proteins in the er is advisable, although toxicity has been reported for some plants accumulating high levels of those recombinant proteins [ ] . given that plant systems have been successfully adopted for the production of multiepitope proteins, the generation of such vaccines against covid- is considered highly viable [ ] [ ] [ ] . besides expressing chimeric proteins, multiple antigen expression can be achieved by transplastomic technologies (operon-like expression) or at the nuclear level using the picornaviral a sequence that allows releasing individual antigens from a single coding gene [ ] . the production of immune complexes (ics) in plants is another approach that renders highly immunogenic agents. ics consist of antigens complexed to antibodies recognizing them, constituting macromolecular entities that are efficiently captured and processed by antigen presenting cells [ ] . this results in the induction of robust humoral and cell-mediated immune responses [ , ] . taking advantage of the machinery of plant cells for protein synthesis and processing, they have been exploited as antibodies and ics factories [ ] . for instance, ics based on the tetanus toxin fragment c fused to a monoclonal antibody were produced in transgenic tobacco plants. these ics were highly immunogenic, inducing immunoprotective effects when administered without accessory adjuvants by the s.c. (subcutaneous) route in mice [ ] . this approach has also been applied to the case of the ag b and acr antigens from mycobacterium tuberculosis and the gp antigen from the ebola virus [ , ] . perhaps the main disadvantage of this approach is the requirement of defined antibodies targeting the antigen, which is not available yet for sars-cov- . however, it should be considered that the cross reactivity of anti-sars-cov- s protein antibodies with the counterpart of sars-cov- has been reported [ ] . the purification of antigens is a laborious and expensive activity as part of the production of injectable vaccines [ ] . one alternative to simplify purification relies on the fusion of elastin-like polypeptides (elp) that possess a unique property named reversible phase transition, which allows precipitating the protein of interest by changing the temperature. this approach is an alternative to the complex/expensive affinity chromatography [ ] that has been applied to develop plant-based vaccines candidates with relevant findings. for instance, the m. tuberculosis antigens ag b and esat- were fused to elp and expressed in transgenic tobacco [ ] , and the plant-made antigens were subcutaneously (s.c.)administered to mice and able to trigger long-lasting humoral immune responses. the elp did not negatively impact the immunogenic properties of the antigens. the haemagglutinin (ha) antigen from the influenza virus has also been expressed under this modality in tobacco, where antigen purification was simplified and the obtained product induced neutralizing antibodies in mice after two s.c. immunizations. elpylation did not alter the immunogenicity of ha [ ] . therefore, these precedents suggest that the elp technology is a possible approach to be explored for the production of antigens against the sars-cov- virus. the common steps to the above-mentioned antigen design will comprise determining antigen yields and antigenic activity, assessing the immunogenic activity in test animals under distinct administration routes, and validating the safety and stability of the vaccine. the latter will be critical to justify the application and get approval to conduct clinical trials. a key precedent for this field is the vaccine candidates already reported for sars-cov- and mers, which are both closely related to sars-cov- . following nuclear expression approaches, the n-terminal fragment of the sars-cov- s protein (s ) was expressed in tomato and low-nicotine tobacco plants using an agrobacterium-mediated method. mice orally immunized with this transgenic tomato revealed significantly increased levels of sars-cov- -specific iga. sera of mice parenterally primed with the transgenic tobacco showed the presence of anti-sars-cov- -igg [ ] . another study focused on expressing a chimeric protein of gfp and amino acids - of the sars-cov- spike protein (s :gfp) by using tobacco leaves subjected to transient expression. after microscopy localization, the fusion protein was observed in the cytosol and the periphery of the nucleus. stable transgenic tobacco and lettuce plants were generated by agrobacterium-mediated transformation. the expression of the chimeric antigen was also achieved in tobacco chloroplasts. however, no immunization assays were performed [ , ] . the sars-cov- nucleocapsid (rn) protein of -aa was transiently expressed in n. benthamiana. yields reached up to µg/g of leaf fresh weight (corresponding to . %- % of the total soluble protein, tsp) at dpi under silencing suppression conditions (p ). mice immunization revealed that three doses led to effective b-cell maturation and differentiation, achieving high levels of igg and igg a. ifn-γ and il- were up-regulated in splenocytes, while the expression of il- and il- was not [ ] . the nucleocapsid protein (n) and the membrane protein (m) were transiently expressed in n. benthamiana. yields reached up to - µg/g fresh leaf weight ( . % tsp) for the n protein, while the m protein yields were . %- . % tsp. the purified n protein reacted with human sera having n-specific antibodies. no immunization assays were performed [ ] . these studies constitute a valuable guide to select the most convenient expression platforms for specific sars-cov- antigens. although most of the vaccines against respiratory diseases are administered by parenteral routes, it should be recognized that immune profiles deserve improvements, especially in terms of immunogenicity and efficacy in the elderly [ ] . mucosal vaccines, especially intranasal-administered vaccines, are a promise for immunization against respiratory diseases given the protections induced in lungs and other mucosal tissues that are critical ports for pathogen entry. critical alternatives for the development of mucosal vaccines include guaranteeing antigen delivery and immunostimulating capacity at the mucosa, while at the same time being minimally reactogenic/toxic. among the immunopotentiator molecules that can be explored are bacterial toxin derivatives, toll-like receptor ligands, and cytokines [ , ] . although some companies have already started the production of plant-based vlps vaccines [ , ] using transient expression systems, these efforts are mainly directed to developing injectable vaccines. therefore, it is imperative to start developing alternatives related to mucosal immunization. for this purpose, generating plants stably expressing sars-cov- antigens will open new avenues for the exploitation of this technology. this approach will result in the production of edible plant material containing sars-cov- antigens that could be used for the development of oral boosting agents. interestingly, this concept has been assessed by some groups with relevant results. in the case of a vaccine targeting the hepatitis b virus, a scheme comprising intramuscular (i.m.) boosting with the pure hbsag antigen followed by double oral boosting with plant material containing hbsag at six week intervals led to a comparable response to that induced by the standard intramuscular (i.m.) vaccination scheme [ ] . moreover, in the case of poliomyelitis vaccination, a plant-based vaccine based on transplastomic plants expressing the vp antigen fused to a carrier has been assessed as oral boosting agents after priming with the inactivated polio vaccine administered subcutaneously. boosting with the plant-made vp -based antigen significantly enhanced vp -igg and vp -iga titers when compared to the response in animals not receiving boosts. the scheme also allowed inducing neutralizing antibodies for the three poliovirus sabin serotypes when two doses of ipv and plant-cell oral boosts were administered, whereas a single dose of ipv resulted in low neutralization potential [ ] . in this way, freeze-dried plant material can be used as a low-cost vaccine rendering thermostability and easy to administer features. similarly, pure antigens produced in plants have been assessed with promising results in schemes where a conventional vaccine is used as a priming agent and the plant-made antigen is administered by the oral route as boosting [ ] . exploring these schemes will be useful to achieve the best immune profile against sars-cov- in terms of safe and long-lasting protective immune responses. in fact, the potential of using prime-boosting schemes through different immunization routes has been proven as an efficacious approach to achieve the desired immune profiles [ ] . the emergence of covid- has led to a global emergency that demands the development of new biologics, especially vaccines, to counteract against this threat. in this scenario, a plant-made vaccine is a viable approach to rapidly respond to this need. the current expression technologies offer relevant paths for developing anti-covid- vaccines. vlps constitute an attractive approach for the development of efficient and safe vaccines, which is associated with high immunogenicity, preservation of the antigenic determinants, and lack of replicative capacity. thus, vlps based on the main sars-cov- structural proteins is an attractive approach for vaccine development against coronavirus infections. the transient expression systems based on deconstructed viral vectors and n. benthamiana as host will allow for immediate exploitation of plants as efficient biofactories of injectable vaccine candidates, which are expected to be implemented and entered into clinical trials in a year. the development of vaccines based on transplastomic lines or edible plant species transformed at the nuclear level and intended to result in oral vaccines (especially boosting agents to provide mucosal immunity) are considered long term goals. however, they have special importance given their low cost and potential to serve as effective boosting agents, which could lead to attractive immune profiles characterized by proper humoral response in the mucosal compartments and long-lasting protection, especially for the elderly. in parallel to these developments, the production of monoclonal antibodies in plants will provide another strategy to generate alternatives to the convalescent plasma transfusion, in which plant-made antibodies will constitute a low cost and safer intravenous treatment for critically ill patients. perhaps the main challenge envisioned for the development of covid- plant-based vaccines will be, as is typical for all vaccines, testing their efficacy in large clinical trials to validate their safety while fulfilling the requirements of regulatory agencies. the fact that there are precedents of a plant-made biopharmaceutical approved for human use and plant-made vaccines against influenza under clinical trials (with promising safety and efficacy) is encouraging. therefore, plant-based vaccines have a realistic potential to contribute to the fight against covid- . as the covid- epidemic advances, the exploitation of plant-made vaccines is a promise to generate low cost, easy to administer, and safe/effective vaccines to fight against this pandemic. the next few months will be critical to envision the real potential of this technology. the authors declare no conflict of interest. emerging coronaviruses: genome structure, replication, and pathogenesis potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody genome composition and divergence of the novel coronavirus ( -ncov) originating in china the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak coronavirus covid- global cases by the center for systems science and engineering characteristics of and public health responses to the coronavirus disease outbreak in china transmission routes of -ncov and controls in dental practice taking the right measures to control covid- use of antiviral drugs to reduce covid- transmission angiotensin receptor blockers as tentative sars-cov- therapeutics treatment of critically ill patients with covid- with convalescent plasma combination attenuation offers strategy for live attenuated coronavirus vaccines recombinant live attenuated influenza vaccine viruses carrying cd t-cell epitopes of respiratory syncytial virus protect mice against both pathogens without inflammatory disease adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques pathogenesis of oral type i feline infectious peritonitis virus (fipv) infection: antibody-dependent enhancement infection of cats with type i fipv via the oral route don't rush to deploy covid- vaccines and drugs without sufficient safety guarantees quantification of epitope abundance reveals the effect of direct and cross-presentation on influenza ctl responses advances in rna vaccines for preventive indications: a case study of a vaccine against rabies curevac focuses on the development of mrna-based coronavirus vaccine to protect people worldwide safety and immunogenicity study of -ncov vaccine (mrna- ) to prevent sars-cov- infection safety and immunity of covid- aapc vaccine ev vaccine, a new tool to control outbreaks of hand, foot and mouth disease (hfmd) a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov- structure, function, and antigenicity of the sars-cov- spike glycoprotein crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors molecular farming-the slope of enlightenment large-scale production of pharmaceutical proteins in plant cell culture-the protalix experience current status of viral expression systems in plants and perspectives for oral vaccines development mucosal immunology and oral vaccination. in genetically engineered plants as a source of vaccines against widespread diseases-an integrated view emerging genome engineering tools in crop research and breeding severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine accumulation of recombinant sars-cov spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines boosted expression of the sars-cov nucleocapsid protein in tobacco and its immunogenicity in mice antigen production in plant to tackle infectious diseases flare up: the case of sars. front vaccination via chloroplast genetics: affordable protein drugs for the prevention and treatment of inherited or infectious human diseases chloroplasts: state of research and practical applications of plastome sequencing production of polyhydroxybutyrate by polycistronic expression of bacterial genes in tobacco plastid plastid-based expression strategie. in genetically engineered plants as a source of vaccines against wide spread diseases-an integrated view when plant virology met agrobacterium: the rise of the deconstructed clones principles of plant-based vaccines techno-economic analysis of a plant-based platform for manufacturing antimicrobial proteins for food safety improving plant transient expression through the rational design of synthetic ' and ' untranslated regions plant-based vaccines against respiratory diseases: current status and future prospects prime-pull vaccination with a plant-derived virus-like particle influenza vaccine elicits a broad immune response and protects aged mice from death and frailty after challenge immunogenicity and safety of a quadrivalent plant-derived virus like particle influenza vaccine candidate-two randomized phase ii clinical trials in to and ≥ years old adults plant-derived virus-like particle vaccines drive cross-presentation of influenza a hemagglutinin peptides by human monocyte-derived macrophages a plant-derived vlp influenza vaccine elicits a balanced immune response even in very old mice with co-morbidities characterization of the innate stimulatory capacity of plant-derived virus-like particles bearing influenza hemagglutinin morphological characterization of a plant-made virus-like particle vaccine bearing influenza virus hemagglutinins by electron microscopy immunological aspects of using plant cells as delivery vehicles for oral vaccines magnifection-a new platform for expressing recombinant vaccines in plants plant lyophilisate carrying s-hbsag as an oral booster vaccine against hbv oral edible plant vaccine containing hypoallergen of american cockroach major allergen per a prevents roach-allergic asthma in a murine model clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond targeting of plant-derived vaccine antigens to immunoresponsive mucosal sites green therapeutic biocapsules: using plant cells to orally deliver biopharmaceuticals plant molecular farming of virus-like nanoparticles as vaccines and reagents highly immunogenic and protective recombinant vaccine candidate expressed in transgenic plants elastin-like polypeptides: a strategic fusion partner for biologics encompassing transition from the trivalent to bivalent oral poliovirus vaccine hitzeroth, i.i. substitution of human papillomavirus type l neutralizing epitopes into l surface loops: the effect on virus-like particle assembly and immunogenicity. front chimaeric rift valley fever virus-like particle vaccine candidate production in nicotiana benthamiana minimally processed crude leaf extracts of nicotiana benthamiana containing recombinant foot and mouth disease virus-like particles are immunogenic in mice efficient assembly and release of sars coronavirus-like particles by a heterologous expression system assembly of human severe acute respiratory syndrome coronavirus-like particles immune responses against severe acute respiratory syndrome coronavirus induced by virus-like particles in mice virus-like particles of sars-like coronavirus formed by membrane proteins from different origins demonstrate stimulating activity in human dendritic cells effect of mucosal and systemic immunization with virus-like particles of severe acute respiratory syndrome coronavirus in mice chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix efficiently form virus-like particles (vlps) that protect mice against challenge with sars-cov mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques preparation of virus-like particle mimetic nanovesicles displaying the s protein of middle east respiratory syndrome coronavirus using insect cells baculovirus as versatile vectors for protein expression in insect and mammalian cells insect cells contain an unusual, membrane-bound beta-n-acetylglucosaminidase probably involved in the processing of protein n-glycans covid- : medicago's development programs the molecular biology of coronaviruses recombinant expression of tandem-hbc virus-like particles (vlps) recent advances in the use of plant virus-like particles as vaccines n-glycosylation of recombinant pharmaceutical glycoproteins produced in transgenic plants: towards an humanisation of plant n-glycans n-glycosylation of cholera toxin b subunit in nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential production of a monoclonalantibody in plants with a humanized n-glycosylation pattern immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates on the origin and continuing evolution of sars-cov- genetic diversity and evolution of sars-cov- virus-specific memory cd t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection edible vaccine protects mice against escherichia coli heat-labile enterotoxin (lt): potatoes expressing a synthetic lt-b gene immunogenic properties of a lettuce-derived c (v ) multiepitopic hiv protein a multi-epitope plant-made chimeric protein (ltbentero) targeting common enteric pathogens is immunogenic in mice an env-derived multi-epitope hiv chimeric protein produced in the moss physcomitrella patens is immunogenic in mice expression of multiple taenia solium immunogens in plant cells through a ribosomal skip mechanism murine fc receptors for igg are redundant in facilitating presentation of immune complex derived antigen to cd + t cells in vivo cellular immune response to the antigen administered as an immune complex in vivo high level production of monoclonal antibodies using an optimized plant expression system plant-derived recombinant immune complexes as self-adjuvanting tb immunogens for mucosal boosting of bcg expression of an immunogenic ebola immune complex in nicotiana benthamiana vaccines elastin-like polypeptides revolutionize protein expression and their biomedical application expression and immunogenicity of the mycobacterial ag b/esat- antigens produced in transgenic plants by elastin-like peptide fusion strategy elpylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against h n viruses in mice use of gfp to investigate expression of plant-derived vaccines a plant-derived multi-hiv antigen induces broad immune responses in orally immunized mice influenza vaccination in older adults: recent innovations and practical applications mucosal vaccines and technology cold chain and virus-free chloroplast-made booster vaccine to confer immunity against different poliovirus serotypes production and immunogenicity of soluble plant-produced hiv- subtype c envelope gp immunogens. front prime-boost vaccine strategy against viral infections: mechanisms and benefits this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - yb b g authors: hammerschmidt, sven; hacker, jörg; klenk, hans-dieter title: threat of infection: microbes of high pathogenic potential – strategies for detection, control and eradication date: - - journal: international journal of medical microbiology doi: . /j.ijmm. . . sha: doc_id: cord_uid: yb b g abstract infectious diseases due to microbes of high pathogenic potential remain a constant and variable threat for human and animal health. the emergence of new diseases or the re-emergence of diseases that were previously under control complicates the situation to date. infectious disease research, which has undergone a dramatic progress in understanding disease mechanisms such as host–pathogen interactions, is now focusing increasingly on new strategies for prevention and therapy. significant progress has been achieved in the development of delivery systems for protective heterologous protein antigens and in veterinary vaccinology. a landmark of infectious diseases research is the chemical synthesis of genomes, a major new field of research referred to as “synthetic biology”, that to date has resulted in the chemical synthesis of the poliovirus and of phage φx genomes and their expression as infectious viruses. on the molecular level the evolution of pathogens and mechanisms of genome flexibility, which account for several pathogenic properties of infectious agents, have received increased attention. bacterial toxins are an additional threat to human health and their interference with host cells and cellular functions is receiving more attention. this report highlights some of the lectures that were presented during the international symposium 'threat of infection: microbes of high potential -strategies for detection, control and eradication' in july in wu¨rzburg (germany). this meeting was organized by the german academy of natural scientists leopoldina (deutsche akademie der naturforscher leopoldina, halle, germany), the acade´mie des sciences (paris, france) and the research center for infectious diseases of the university of wu¨rzburg together with other scientific societies (germany). the speakers and two poster sessions covered many aspects of infectious diseases, including political and social aspects. at the molecular and cellular levels, the action of virulence factors and toxins, the potential applications of rna interference and mechanisms of genome flexibility were discussed. the reservoirs and transmission of pathogens, novel detection methods and the exploitation of mechanisms of host-pathogen interaction were further discussed for the development of novel preventive and therapeutic approaches. a comprehensive summary with papers of each of the speakers who had been invited for an oral presentation during this symposium will be published soon in the series 'nova acta leopoldina' (http://www.leopoldina.uni-halle.de). infectious diseases are the biggest killer of children and young adults, accounting for more than million deaths a year and as many as one in two deaths in developing countries. almost % of deaths from communicable diseases are caused by six diseases: acute respiratory and diarrhoeal infections, aids, tuberculosis, malaria, and measles. david l. heymann (who, geneva) reminded that many deaths occur as a result of preventable diseases such as measles, hepatitis b, diphtheria, whooping cough and, most often, because of failure to implement vaccination programs. the burden of infectious diseases is not restricted to developing countries, since previously controlled infectious diseases such as tuberculosis and diphtheria have re-emerged in europe and other developed areas of the world. high mortality due to infectious diseases is alarming but so too is the fact that large populations in remote areas of the developing world are at risk of disabling diseases, such as polio, leprosy, lymphatic filariasis, and onchocerciasis. as further reported by david l. heymann these diseases cause a double economic burden by reducing the work force and undermining the financial security of already impoverished families. most infectious diseases can be prevented with drugs or vaccines that are already available, but inadequate funding of health care in developing countries or the inability to deliver the antimicrobial drugs and antibiotics to the entire population contributes to the devastating situation ( table ) . the emergence of severe acute respiratory syndrome (sars), which is one of diseases newly identified in recent decades, clearly demonstrated that every country is vulnerable and that the economic consequences, exaggerated by public fear of the unknown, can be felt around the world. today, diseases can be transported from one continent to another in a matter of hours. for these reasons public health care systems have received more attention. david l. heymann stated that global partnerships and strategies are required that increase the power of epidemic control such as real-time electronic communications. in considering novel vaccination strategies against microbes of high pathogenic potential, one has to distinguish between the case of bio-warfare and newly emerging agents with high pathogenic potential. we have to consider that, in the latter case, mass vaccination or even vaccination of high-risk individuals would be unrealistic especially since the overall risk of side effects will exceed the risk of disease. rapid protection of individuals at risk has to be given high priority. this causes various problems, including rapid diagnosis as well as prompt induction of a highly efficacious host response, both in naive and in infected individuals, in addition to prompt chemotherapy. therefore, classical vaccination strategies need to be complemented by generic vaccination strategies and immunomodulatory interventions. such strategies must include both innate and acquired immunity. in his presentation, stefan h. e. kaufmann (max-planck-institute for infection biology (mpi), berlin) suggested that the following vaccination strategies against intracellular bacteria deserve consideration: (i) attenuated viable strains, (ii) naked dna encoding protective antigens and (iii) protective antigens expressed by recombinant viable vectors (bacteria or viruses). extracellular bacteria are controlled by antibodies -typically opsonizing antibodiesand, hence, vaccination strategies are based on killed vaccines or subunit vaccines. protection against intracellular bacteria depends on t-lymphocytes rather than antibodies (winau et al., ) . although recent advances in genomics, basic immunology and molecular biology provide the guidelines for constructing novel vaccine candidates in research programs involving stretching from basic science to applied field studies. werner goebel (microbiology, wu¨rzburg, germany) highlighted the development of live virulence-attenuated bacteria as delivery systems for protective heterologous protein antigens. two different strategies were used: one that allows the release of the antigen from gramnegative bacterial carriers via type secretion system (t ss) (gentschev et al., ) and one that introduces antigen-determining plasmid dna or rna into mammalian cells including antigen-presenting cells via intracellular bacteria (dietrich et al., ; pilgrim et al., ; schoen et al., ) . a self-destructing variant of the gram-positive bacterium listeria monocytogenes, which activates a phage-derived lysine and lyses upon internalization, was used for efficient release of dna or mrna into the cytosol of host cells (pilgrim et al., ; schoen et al., submitted) . transfer of dna and rna was highly efficient not only into epithelial and fibroblast cell lines, but also into phagocytes including dendritic cells. the a-hemolysin (hlya) secretion system of e. coli was exploited as a t ss to deliver heterologous antigens. hlya can be genetically fused to almost any protein antigen without drastically affecting secretion, e.g. by virulence-attenuated salmonella strains including s. typhi ty (gentschev et al., ) . exploitation of type iii and auto-transporter secretion systems of gram-negative bacteria for recombinant vaccine delivery into the cytosol of mammalian cells has recently been demonstrated (ru¨ssmann, a, b; rizos et al., ) . werner goebel emphasized that these approaches might not only be useful in combating infectious diseases but might also open up new avenues in anti-tumor therapy by either designing anti-tumor vaccines or by delivering therapeutic agents. infected animals are not only a major economic factor in animal husbandry but also have a high impact as reservoirs of human pathogens. veterinary vaccines have to be cost-efficient, easy to administer and efficacious within a short period of time after, preferably, one immunization. this implies a rather exclusive reliance on live vaccines. fox rabies, a major threat for human health, will serve as an example. initiated by swiss researchers, oral immunization schedules have been devised which were initially based on the use of baits composed of chicken heads filled with blisters containing live-attenuated rabies vaccine (steck et al., ) . further research led to the development of industrially produced bait casings that are attractive enough to be eaten by foxes (schneider, ) . bait distribution over large areas eradicated rabies or drastically reduced its incidence in several european countries (po¨tzsch et al., ) . the successful oral immunization against rabies in foxes was followed by a similar approach against classical swine fever (csf) in wild boar. csf is a highly contagious disease of high pathogenic potential that is contained within the european countries by elimination of all pigs on infected holdings. most of the sporadic occurrences of csf in domestic pig can be epidemiologically linked to wild boar. therefore, elimination of csf virus from the wild boar population is a major prerequisite for protecting domestic pig farms. a corn-based bait that contains a blister filled with the live-attenuated csfv c-strain vaccine has all but eliminated csfv, which once was quite common in the german wild boar population (kaden et al., ) . veterinary vaccinology has recently achieved spectacular success by introducing the first genetically engineered live viral vaccines and marker technology able to differentiate between infected and vaccinated animals (diva or marker concept). thomas c. mettenleiter from the friedrich-loeffler-institute (insel riems, germany) illustrated the success of disease control by applying the diva concept. the diva concept (van oirschot, ) has been successfully employed for the elimination of aujeszky's disease (ad) in pigs caused by the alphaherpesvirus pseudorabies virus (prv; mettenleiter, ) . based on the initial finding that several live-attenuated prv vaccine strains lack a major surface antigen (glycoprotein e, ge) which is invariably present in all field strains (mettenleiter et al., ) , a simple elisa system has been developed that is able to specifically detect the presence or absence of anti-ge antibodies in the animal (van oirschot et al., ) . based on this concept, eradication programs allow the use of ge-deleted ('marker') vaccines combined with sero-surveillance using detection of anti-ge antibodies. using this approach, germany has been declared free of ad by february , (mu¨ller et al., ) . it is important to mention that during the ad eradication campaign, genetically modified live vaccines were used for the first time in europe on a wide scale. a similar eradication program has recently been started to eliminate bovine herpesvirus infections in cattle (beer et al., ; kaashoek et al., kaashoek et al., , . the less complex rna viruses generally do not specify a protein product like the glycoprotein e of prv and, therefore, require different diva approaches (scahaw (scientific committee on animal health and animal welfare), ). the diva concept is also applicable to the control of foot-and-mouth-diseases virus (fmdv), a picornavirus of the genus aphthovirus. only four virally encoded structural proteins are present within the mature virion, whereas in infected cells (and animals), non-structural proteins also act as antigens. thus, infected animals produce antibodies against both structural and non-structural proteins, whereas animals vaccinated with the inactivated virions will only produce antibodies against virion components. a recombinant swine fever virus (csfv) vaccine (de smit, ) with an antigenetically different e glycoprotein (from bovine viral diarrhea virus) induces excellent protection against csfv challenge (reimann et al., ) . likewise, a diva strategy is based on differential detection of antibodies against a heterologous neuraminidase in avian influenza. these examples, presented by thomas c. mettenleiter, illustrate the success of the diva concept in cost-efficient control and eventual elimination of specific infectious agents in livestock. a virgin population is particularly threatened by unintentional or intentional re-introduction of eradicated pathogens, such as variola virus. release of smallpox virus, the only virus to be successfully eradicated, would be catastrophic, since % of the population is susceptible to the disease and nobody under the age of years has ever been vaccinated (fenner et al., ) . the last natural case of smallpox occurred in somalia in . routine vaccination programs were stopped and vaccine producers ceased making vaccines. approximately million died from the disease prior to its eradication. the eradication strategy comprised widespread vaccination of at least % of the population, and surveillance for the early detection of cases and control of outbreaks. smallpox virus officially still exists in two laboratories of the who, one in atlanta, georgia, usa and one in novosibirsk, russia (and possibly unofficially in others). in these laboratories, research is continuing. the former deputy director of the soviet union biological weapons program declared in that smallpox would be developed as a weapon and that an annual stockpile of ton variola virus would be maintained (alibek and handelman, ) . bioterrorism has considerably dampened the hopes of positive medical and economic benefits resulting from the global eradication of a disease. in his presentation, reinhard kurth (robert-koch institute, berlin, germany) asked whether we can really eradicate infectious diseases. the development of a new vaccine or improvement of the old vaccine face challenges: the clinical efficacy of a new vaccine cannot be tested in humans (rosenthal et al., ) and the side-effects of the old one will be not accepted by the community. most countries, however, have no vaccine and will be dependent on others in the case of emergency as reported by donald l. henderson (johns hopkins center for civilian biodefense studies, baltimore, usa). poliomyelitis is the only disease worldwide that is currently being seriously targeted for eradication with a possibility of success. the number of cases decreased from over , cases in countries to cases in six countries between and . although in the majority of regions poliovirus, a human enterovirus of the picornaviridae, has already been eliminated (rasch et al., ) , the global eradication campaign will be in danger in those african countries which have for economic reasons reduced the vaccination campaigns after achieving polio elimination. the termination has led to an increase of susceptible persons. ninety percent of polio infections are asymptomatic. transmission can, therefore, easily occur without detection and even new outbreaks are at high risk. this problem is present in africa where sudden new cases are seen after many years of being polio-free. new problems will arise in the posteradication period, including those posed by the new threat of bioterrorism. the live attenuated virus used as a vaccine can be transmitted and recombine with other enteroviruses, most likely c-cluster coxsackie viruses. the result can be a vaccine virus -vaccine-derived poliovirus (vdpv) -with increased virulence and transmissibility. long-term circulation of such vdpv has, in the past, occurred in countries with suboptimal oral polio vaccine (opv) vaccination rates (kew et al., ) . this occurred independently in egypt ( ( - ( ), hispaniola ( , philippines ( ), and madagascar ( - . the live attenuated (sabin) opv was developed in the s. to minimize the circulation of vaccine viruses in, e.g. immunodeficient or unvaccinated individuals, it is essential that the who initiates an early and synchronized stop of live attenuated (sabin) opv vaccinations (maclennan et al., ) . after the confirmed global eradication it is up to the individual countries to stop vaccinating or to switch to using inactivated (salk) polio vaccine (ipv). at least for those countries in which ipv is produced, the manufacturers have to maintain large quantities of the wild-type virus and the corresponding seed virus stocks. even after the cessation of vaccination it will be essential to conserve the wild-type virus in case of emergency to fall back on these vaccines (henderson, ) . the problems with the containment of wild-type poliovirus would be reduced if the production of ipv could be successfully based on strains derived from the sabin vaccine virus (s-ipv, doi et al., ) . most of the basic mechanisms of poliovirus pathogenesis are still obscure. this is true for the replication of poliovirus in the gastrointestinal tract after oral infection and the mechanism by which the virus spreads to the central nervous system (cns). another mystery is the cunning preference of the virus to destroy motor neurons in the cns, especially in the spinal cord, a specificity leading to paralysis and possibly deathpoliomyelitis. a major determinant of poliovirus pathogenesis is the cd receptor (koike et al., ; mendelsohn et al., ) . analysis of human and primate specimens of the gastrointestinal tract revealed strong expression of cd in enterocytes, in follicularassociated epithelial cells and m-cells of peyer's patches (pps), which represent an important site of replication during first stage of infection (minor, ; iwasaki et al., ) . endothelial cells in the interfollicular region of pps were also found to express cd , an observation that leaves open the possibility that poliovirus may enter the bloodstream via vessel damage in these tissues (iwasaki et al., ) . entry into the cns can occur by passage through the blood-brain barrier or at neuromuscular junctions, followed by retrograde axonal transport to the motor neuron cell body. the latter is the route of cns invasion in ''provocation poliomyelitis'', e.g. injury enhanced disease progression (gromeier and wimmer, ) . a cargo protein of the dynein motor, tctex- , was discovered to have affinity to the c-terminal intracellular domain of cd . therefore, a new model of retrograde transport was proposed: once the virus has been internalized at the neuromuscular junction, it is transported to the cell body of motor neurons in cargo vesicles along axonal microtubules, driven by the dynein motor complex . the chemical synthesis of poliovirus in the absence of natural template (cello et al., ) indicated that a virus can no longer be considered extinct when its genome sequence is known. a poliovirus-specific double-stranded dna (''cdna'') that was assembled from synthetic desoxy-oligonucleotides was transcribed with an enzyme of bacteriophage t (t rna polymerase) yielding poliovirus rna. this rna was identical to the genomic rna of nucleotides except for the end (cello et al., ; van der werf et al., ) . the sequence of the synthetic poliovirus (spv) cdna was designed such that it contained ''silent'' mutations as identity markers (cello et al., ) . the spv rna was incubated in a cell-free extract of uninfected hela cells that directs virus specific translation, genome replication and virion assembly (molla et al., ) . this via synthetic cdna outside living cells generated polio virus has a plaque phenotype similar to that occurring in nature. the spv is growing in tumor cells but is highly attenuated (att) in cd transgenic mice (cello et al., ) . the first biologically active cdna synthesized was that of rna phage qb (taniguchi et al., ) . this landmark accomplishment heralded the birth of reverse genetics in virology. in contrast to cello et al. ( ) who synthesized virus from chemical information only, naturally occurring rna were required as templates for the enzymatic reactions preparing qb cdna. this is fundamentally different and, as proof of principle, has far reaching consequences as stated by eckard wimmer (stony brook, ny, usa). the chemical synthesis of genomes of viruses or bacteria is no longer a dream. this research referred to as ''synthetic biology'' has already become a new hot field. consequently, a paper by smith et al. ( ) described the synthesis of a chemical mixture of phage fx , bp long, in only weeks. the re-programming of microorganisms by existing functional modules (''lego biology'') may yield novel microbes capable of functioning as highly specific and efficient mini-factories (ferber, ) . genetic flexibility is a phenomenon that accounts for the pathogenic properties of many infectious agents. therefore, one focus of genomic research has been the characterization of mechanisms that are involved in genomic variability and evolution of bacterial pathogens. a new type of genomic entity -known as pathogenicity islands -has been shown to contribute to the rapid evolution of bacterial pathogens by horizontal gene transfer (for review, see dobrindt et al., ) . another flexible genetic element is represented by integrons, which were first identified as the primary mechanism for antibiotic resistance gene capture and dissemination among gram-negative bacteria. studies on yersinia and bartonella have further shown that numerous gene rearrangements, massive gene inactivations and loss of gene functions are at least as important determinants of pathogenicity as gene acquisitions. siv g.e. andersson (uppsala, sweden) presented that the deterioration of the a-proteobacterial human pathogen genomes, which could mean eliminations of a few thousand genes, shifted the bacterial lifestyle to intracellular animal environments and vector-mediated transmission pathways. a computational approach was used to infer ancestral gene sets and to quantify the flux of genes along the branches of the aproteobacterial species tree (boussau et al., ) . the analysis suggests that rickettsia and wolbachia represent the earliest diverging lineage in the tree, followed by caulobacter (boussau et al., ) . the analysis indicated a loss of more than genes prior to the divergence of rickettsia and wolbachia, followed by another loss of about genes prior to or associated with the emergence the typhus pathogen. r. conorii contains more than genes not present in r. prowazekii, the causative agent of epidemic typhus. eighty percent of the genes are orphans and not identified in any other species. genomic analysis suggests further a massive loss of more than genes immediately prior to the divergence of bartonella and brucella. this is followed by yet another major loss of some genes prior to the divergence of b. henselae and b. quintana. the genome sequence of the louseborne b. quintana comprises , , bp, and that of the zoonotic agent b. henselae comprises , , bp (alsmark et al., ) . a genome comparison elucidated a high degree of overall similarity between the two genomes. b. quintana and b. henselae, which cause trench fever and cat-scratch disease, respectively, are unique among bacteria in their ability to induce tumorlike lesions of the skin (bacillary angiomatosis) as well as the liver and spleen (bacillary peliosis) of immunocompromised individuals. both are transmitted by insect vectors, using mammalian reservoirs, and infecting similar cell types. however, b. quintana is a specialist, using only humans as a reservoir, whereas b. henselae is more promiscuous. the difference in gene content is mainly due to the presence of several genomic islands in b. henselae that are not present in b. quintana. around % of the unique genes in b. henselae were attributed to a prophage region of kb and three genomic islands of , and kb, respectively. the prophage is an evolutionary mosaic with genes of different origins that are interspersed with genes showing sequence similarities to a putative phage in wolbachia. potential virulence features located on the b. henselae genomic islands are multiple copies of hecb and fhab putatively coding for a hemolysin transporter and a filamentous hemagglutinin, respectively. these two proteins form a two-partner secretion system, where the hecb gene product may mediate the transport of filamentous hemagglutinin that is required for attachment to the host cell (alsmark et al., ) . the largest unique island in b. quintana is only kb and contains a gene with sequence similarity to yopp in yersinia enterocolitica and yopj in yersinia pseudotuberculosis. sequences in b. quintana located at positions corresponding to islands in b. henselae contain remnants of phage and island genes, suggesting that these were present in the common ancestor of the two species. in total, the siv g.e. andersson's group has identified pseudogenes and extensively degraded gene sequences in b. henselae (including tandem duplication remnants) as compared to fragmented genes in b. quintana. the rearrangements flank islands uniquely present in b. henselae, indicative of a link between rearrangement and insertion/deletion events. remnants of genes present in the b. henselae islands have been identified at the breakpoint positions in b. quintana. as reported by siv g. e. andersson both of the two bartonella genomes contain numerous scars of putative phage and plasmid integrations, indicative of exposure to genetic parasites in the evolutionary past. however, the human pathogen specialist b. quintana has lost most of these sequences and seems no longer to accumulate genetic parasites. thus, both of the two human pathogen specialists, r. prowazekii and b. quintana, have experienced an enhanced frequency of genome degradation as compared to their closest relatives with other host-vector reproduction strategies. interestingly, the high pathogenic potential of yersinia pestis is not solely attributable to the presence of two additional plasmids as shown by elisabeth carniel (institut pasteur, paris). a comparative analysis with the ancestor y. pseudotuberculosis revealed chromosomal genes in addition to the plasmids that represent the new genetic material in y. pestis. in contrast, other pseudogenes and genes absent from y. pestis were detected, indicating that as many as % of y. pseudotuberculosis genes no longer function in y. pestis (chain et al., ). it appears that loss of function is at least as important as gene acquisition in the evolution of y. pestis. high genetic variability is particularly dangerous if pathogens originate from animal reservoirs. an example is pandemic influenza which is a zoonotic disease caused by the transfer of influenza a viruses or virus gene segments from aquatic bird reservoirs to humans and domestic animals. in their natural reservoir these viruses are in evolutionary stasis -they are in equilibrium with their natural host and cause no disease. but after transfer to new hosts they evolve rapidly posing a constant threat to human and animal health as told by robert g. webster (st. jude children's research hospital, memphis, usa). widespread infections of poultry with h n viruses in asia have caused increasing concern that this subtype forms the basis for human-to-human spread and interspecies transmission. multiple opportunities for the successful mammalian transmission of h n influenza viruses are provided by their continuing evolution in asia, their propensity for re-assortment, the generation of multiple genotypes of h n viruses (matrosovich et al., ) , antigenic drift in the ha gene of h n viruses, and the acquisition of high pathogenicity for aquatic birds. if an opportunity for re-assortment with human influenza strains occurs, then the likelihood of successful transmission between humans is high. marburg and ebola viruses, family filoviridae, reemerge periodically from an unknown animal reservoir. they cause severe hemorrhagic fever in humans and non-human primates. disturbances of the blood tissue barrier, primarily controlled by endothelial cells, and immune suppression seem to be the key pathogenic factors of the disease as reported by heinz feldmann (national microbiology laboratory, winnipeg, canada). the endothelium is affected in two ways: directly by virus infection leading to activation and lytic replication and indirectly by a mediator-induced inflammatory response. these mediators originate from virus-activated cells of the mononuclear phagocytic system which are the primary target cells. immune suppression may result from lytic infection of circulating and sessile cells of the mononuclear phagocytic system, inactivation of neutrophils by secreted viral proteins, necrosis of antigen-presenting cells, and lymphoid depletion. in addition, the virion structural protein (vp ) has been identified as an interferon antagonist (basler et al., ) . despite being clearly immunosuppressive, there is good evidence for protective immunity during filovirus hemorrhagic fever. in contrast to survivors and asymptomatic cases that show igm and igg antibody responses to viral antigens, fatal infections usually end with high viremia and little evidence of a humoral immune response. neutralizing antibodies are directed against the transmembrane glycoproteins, which are responsible for virus entry. however, infectivity-enhancing antibodies directed against the transmembrane glycoprotein have also been described. the transmembrane glycoprotein can be used to provoke a protective immune response in animal models including nonhuman primates. several distinct approaches including dna vaccination or replication-deficient and replication-competent recombinant viruses can achieve this. thus, the transmembrane glycoprotein serves as the most promising vaccine candidate for filoviral hemorrhagic fever (feldmann et al., ) . yellow fever virus and lassa virus, an arenavirus, are zoonotic viruses that both cause , - , clinically apparent infections per year in west africa, carrying a mortality of - %. while the transmission, epidemiology and immunology of rodent-borne lassa fever is different from mosquito-borne yellow fever, the current re-emergence of both diseases is influenced likewise by the ecology of the viral reservoirs as well as the immunity of the susceptible human populations. herd immunity to yellow fever virus which was achieved in the s through mass-vaccinations is generally fading, since immunization programs have been discontinued. humoral cross-immunity due to heterologous flavivirus infections can mitigate severe disease, but cannot prevent yellow fever epidemics, which are again threatening the urban centers of west africa. no vaccine has ever been developed against the mainly t-cell-controlled lassa virus, but outbreaks of the disease have been rare because of focally restricted contact with infected rodents in west africa. migration of susceptible human populations and severe changes in the habitat of the rodent reservoir have now led to increased transmission of the virus and export of lassa fever cases to european countries (j. ter meulen, institute of virology, university of marburg, germany). the flavivirus family comprises about different viruses, most of which are transmitted to their vertebrate hosts by mosquitoes or ticks. several flaviviruses are important human pathogens -including the yellow fever, dengue, japanese encephalitis, west nile, and tick-borne encephalitis viruses -and some of them have proven to have the characteristics of newly emerging pathogens. as pointed out by franz x. heinz (institute of virology, medical university of vienna, austria) enormous progress has been made in recent years in the elucidation of the molecular structure of flaviviruses, which has greatly enhanced our understanding of specific functions during entry and assembly, viral pathogenicity, antigenic relationships, and immunogenicity. tick-borne encephalitis virus is endemic in many european countries and large parts of asia, including northern china and northern japan. three closely related subtypes can be discerned (european, siberian, far eastern), which circulate in their natural foci between different mammalian species and ticks (mainly ixodes ricinus and ixodes persulcatus). humans are infected only accidentally and do not play any role for maintaining the natural cycle of the virus. both the virus and the geographic distribution of natural foci are remarkably stable and exhibit relatively little variation, suggesting relative evolutionary stasis of the virus and a delicate balance of environmental factors required for this maintenance in nature. a highly purified formalininactivated whole virus vaccine has been in widespread use in europe and proven to be an excellent means for the prevention of tick-borne encephalitis in humans. the recent introduction of west nile virus to northern america is a dramatic example of the potential of flaviviruses to conquer new territories within a short period of time. although the same virus sporadically occurs in europe and also causes rare infections in humans, it has never been able to establish endemicity similar to what has occurred in america, where the virus has apparently found ideal ecological conditions for its maintenance and spread. so far human vaccines are not yet commercially available but are being tested in human trials. the four types of dengue viruses can cause severe forms of dengue fever (hemorrhagic fever and shock syndrome) and pose an enormous health threat to tropical and subtropical countries around the globe. these severe forms of the disease are apparently promoted during secondary infections with a certain dengue virus type by immunological enhancement phenomena triggered through the pre-existing immunity against a different type. this phenomenon has severely hampered the development of dengue vaccines, because of concerns that vaccination could induce a similar immunopathological state in vaccinated individuals. bacterial toxins represent important and highly efficacious virulence determinants of pathogens and are, therefore, a threat to human health in connection with infectious diseases (bradberry et al., ; o'loughlin and robins-browne, ; . elucidating the mode of action of toxins is important to understand pathogenicity and will help to prevent and treat diseases (karmali, ) . substantial progress has been made in elucidating the mechanisms by which bacterial toxins, such as tetanus, botulinum, anthrax and shiga toxin, invade cells and interfere with vital cellular functions. kirsten sandvig (institute for cancer research, norwegian radium hospital, oslo) introduced the pathways used by shiga toxin. the bacterial shiga toxin produced by shigella dysenteriae, shiga-like toxins (stxs) produced by escherichia coli, cholera toxin (vibrio cholerae), and certain plant toxins such as ricin act on cells after entry of an enzymatically active part of the molecule into the cytosol. in general, the toxins have one moiety which binds to cell surface receptors and another, enzymatically active moiety, which enters the cytosol and exerts the action of the toxin. studies of toxin entry into cells also provide us with basic knowledge about endocytosis mechanisms, intracellular transport and membrane translocation of proteins in cells (iversen et al., ; lauvrak et al., ; llorente et al., ; . shiga toxin was the first molecule found to move all the way from the cell surface to endosomes, and then retrogradely to the golgi apparatus and the er (sandvig et al., ) . a similar trafficking has been shown also for toxins such as cholera toxin, ricin and others . on the other hand a number of protein toxins travel a much shorter distance before being translocated to the cytosol (sandvig, ) . endocytosis is mediated via both a clathrin-dependent and -independent mechanism. the experiments with ricin indicated that the endocytosis can be independent of lipid rafts and can even under some circumstances operate without gtp hydrolysis (garred et al., ) . shiga toxin can also enter cells by clathrin-independent mechanisms (lauvrak et al., ; nichols et al., ; saint-pol et al., ) . however, the fraction of toxin exploiting the different pathways is likely to be cell-type dependent. similarly, cholera toxin can be endocytosed by different mechanisms (shogomori and futerman, ; singh et al., ; torgersen et al., .) and most probably cell-type-dependent differences exist. shiga toxin and ricin seem to be transported from the early endosomes by a rab -independent process, either directly to the golgi or via the perinuclear recycling compartment (iversen et al., ; johannes and goud, ; sannerud et al., ) . for shiga toxin rab is implied in the transport and clathrin is required for efficient endosome-to-golgi transport (johannes and goud, ; lauvrak et al., ; saint-pol et al., ) . both molecules are not required for ricin transport (iversen et al., ) . after arrival in the trans-golgi network the toxins are transported to the er. neither shiga toxin nor ricin contain a kdel sequence that could facilitate retrograde transport by the kdel receptor and the copi-coated vesicles. the kdel receptor-dependent transport has been shown for pseudomonas exotoxin a (jackson et al., ) . in the case of shiga toxin there is evidence for a rab , copi-independent pathway, but not all details are known (johannes and goud, ; sannerud et al., ) . in summary, kirsten sandvig indicated that more work is required to understand the retrograde transport and that the answers are important and might help to exploit the toxins in medical therapy. despite significant progress was made in controlling infectious diseases and understanding disease mechanisms with special emphasis on the molecular level, we face to date several new threats due to infectious agents. vaccination programs aimed to eradicate high pathogenic microorganisms with high mortality rates. nevertheless, the emergence of newly identified diseases such as sars and the re-emergence of diseases has become a great public health concern worldwide. a further threat is the dramatic increase of antimicrobial resistance of pathogens. antimicrobial resistance against antibiotics and other drugs has a deadly impact on the control of diseases such as tuberculosis, malaria, cholera, dysentery, and pneumonia. the basis to control infectious diseases and epidemics is the installation of worldwide accessible communication platforms by public health care systems. the interchange and storage of our current know-how and experience of successfully employed eradication programs and disease treatments is required to combat infectious diseases. a landmark in modern molecular microbiology and combinatorial chemistry is the chemical synthesis of microorganisms. this provides not only the possibility to construct novel delivery systems or vaccines but may also lead to an improved understanding of key molecules and events in the development of diseases. on the other hand, this so-called ''lego biology'' may represent a tool for bioterrorism in terms of constructing infectious agents with high pathogenic potential. the microbial evolution and genomic variability is accompanied with horizontal gene transfer. however, novel studies have indicated that not only gene acquisition but also loss of gene functions represents important determinants of pathogenicity. comparative genome analysis of microorganisms with special emphasis on intracellular pathogens will provide insights whether the evolution of these microorganisms is also associated with genome reduction by gene loss and resulted in a highly specified pathogenic potential. the developments in vaccinology in veterinary medicine highlight the potential and application of vaccine research. the successful disease control in animals by veterinary vaccination campaigns offers new perspectives to implement vaccination programs in low-or middleincome countries or even after bioterrorism attacks. veterinary vaccines have to be cost-efficient, easy to administer and efficacious within a short period of time. a single immunization is preferred. to reach and maintain high vaccination rates, novel vaccines have to fulfil these requirements. these vaccines should be ideally not based on live vaccines as used for veterinary vaccinology. instead, preferable oral immunizations with subunit vaccines and novel antigen-delivery systems have to be implemented in order to reduce the burden of infectious diseases worldwide. biohazard. random house the louse-borne human pathogen bartonella quintana is a genomic derivative of the zoonotic agent bartonella henselae the ebola virus vp protein functions as a type i ifn antagonist marker diagnostic for the eradication of bovine herpesvirus type : possibilities and limitations computational inference of scenarios for aproteobacterial genome evolution ricin poisoning chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template insights into the evolution of yersinia pestis through whole-genome comparison with yersinia pseudotuberculosis laboratory diagnosis, epizootiology, and efficacy of marker vaccines in classical swine fever: a review delivery of antigen-encoding plasmid dna into the cytosol of macrophages by attenuated suicide listeria monocytogenes genomic islands in pathogenic and environmental microorganisms progress with inactivated poliovirus vaccines derived from the sabin strains ebola virus: from discovery to vaccine smallpox and its eradication. who synthetic biology. microbes made to order reconstitution of clathrin-independent endocytosis at the apical domain of permeabilized mdck ii cells: requirement for a rho-family gtpase delivery of protein antigens and dna by attenuated intracellular bacteria use of the alpha-hemolysin secretion system of escherichia coli for antigen delivery in the salmonella typhi ty a vaccine strain mechanism of injuryprovoked poliomyelitis countering the posteradication threat of smallpox and polio endosome to golgi transport of ricin is independent of clathrin and of the rab -and rab -gtpases formation of clathrin-coated pits with long dynaminwrapped necks upon inducible expression of antisense to clathrin immunofluorescence analysis of poliovirus receptor expression in peyer's patches of humans, primates, and cd transgenic mice: implications for poliovirus infection the kdel retrieval system is exploited by pseudomonas exotoxin a, but not by shiga-like toxin- , during retrograde transport from the golgi complex to the endoplasmic reticulum facing inward from compartment shores: how many pathways were we looking for? a conventionally attenuated glycoprotein e-negative strain of bovine herpesvirus type is an efficacious and safe vaccine an inactivated vaccine based on a glycoprotein enegative strain of bovine herpesvirus induces protective immunity and allows serological differentiation oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in germany prospects for preventing serious systemic toxemic complications of shiga toxin-producing escherichia coli infections using shiga toxin receptor analogues circulating vaccine-derived polioviruses: current state of knowledge the poliovirus receptor protein is produced both as membranebound and secreted forms efficient endosome-to-golgi transport of shiga toxin is dependent on dynamin and clathrin induction of direct endosome to endoplasmic reticulum transport in chinese hamster ovary (cho) cells (ldlf) with a temperature-sensitive defect in epsilon-coatomer protein (epsilon-cop) failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man the surface glycoproteins of h influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily aujeszky's disease (pseudorabies. virus: the virus and molecular pathogenesis -state of the art pseudorabies virus avirulent strains fail to express a major glycoprotein poliovirus cell-free, de novo synthesis of poliovirus interaction of the poliovirus receptor cd with the dynein light chain tctex- and its implication for poliovirus pathogenesis eradication of aujeszky's disease in germany rapid cycling of lipid raft markers between the cell surface and golgi complex effect of shiga toxin and shiga-like toxins on eukaryotic cells bactofection of mammalian cells by listeria monocytogenes: improvement and mechanism of dna delivery summarizing the rabies situation in europe - from the rabies bulletin europe worldwide eradication of poliomyelitis an avirulent chimeric pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus autodisplay: efficacious surface exposure of antigenic urea fragments from helicobacter pylori in salmonella vaccine strains developing new smallpox vaccines yersinia outer protein e, yope. a versatile type iii effector molecule for cytosolic targeting of heterologous antigens by attenuated salmonella bacterial type iii translocation: a unique mechanism for cytosolic display of heterologous antigens by attenuated salmonella clathrin adaptor epsinr is required for retrograde sorting on early endosomal membranes transport of toxins across intracellular membranes membrane traffic exploited by protein toxins retrograde transport of endocytosed shiga toxin to the endoplasmic reticulum pathways followed by ricin and shiga toxin into cells retrograde traffic in the biosynthetic-secretory route: pathways and machinery diagnostic techniques and vaccines for foot-and-mouth disease, classical swine fever and avian influenza rabies control by oral vaccination of wildlife bacterial delivery of functional messenger rna to mammalian cells cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raftindependent mechanism selective caveolin- -dependent endocytosis of glycosphingolipids generating a synthetic genome by whole genome assembly: phix bacteriophage from synthetic oligonucleotides oral immunization of foxes against rabies qb dnacontaining hybrid plasmids giving rise to qb phage formation in the bacterial host internalization of cholera toxin by different endocytic mechanisms synthesis of infectious poliovirus rna by purified t rna polymerase diva vaccines that reduce virus transmission differentiation of serum antibodies from pigs vaccinated or infected with aujeszky's disease virus by a competitive enzyme immunoassay apoptosis paves the detour path for cd t cell activation against intracellular bacteria the authors wishes to express their appreciation for the helpful contributions and permissions of the speakers on (parts of) this report which gives an insight of presentations held during the symposium ''threat of infection'' in july in wu¨rzburg, germany. the authors are grateful to dr. anthony p. pugsley (paris) for helpful discussions and suggestions. key: cord- - qc c d authors: lamson, daryl m.; ramani, rama; kleabonas, matthew; metcalfe, maureen; humphrey, charles; st. george, kirsten title: an unusual case of influenza-like illness after yellow fever vaccination date: - - journal: journal of clinical virology doi: . /j.jcv. . . sha: doc_id: cord_uid: qc c d abstract yellow fever (yf) is an important public health concern in areas where the disease is endemic. for more than years a highly effective live attenuated vaccine has been available, its widespread use resulting in a dramatic decrease in the number of cases. on rare occasions, yf vaccine can cause mild to severe disease and rare adverse vaccine-associated events have been reported. additionally, an average viremia of – days after administration of the yf vaccine has been published. here we present a case where yf vaccine was isolated in cell culture from a respiratory swab collected from a patient presenting with influenza-like illness. to the best of our knowledge, this is the first report finding replicating yf vaccine in the respiratory sample of a post inoculated individual. yellow fever (yf) is an important public health concern in areas where the disease is endemic. for more than years a highly effective live attenuated vaccine has been available, its widespread use resulting in a dramatic decrease in the number of cases. on rare occasions, yf vaccine can cause mild to severe disease and rare adverse vaccine-associated events have been reported. additionally, an average viremia of - days after administration of the yf vaccine has been published. here we present a case where yf vaccine was isolated in cell culture from a respiratory swab collected from a patient presenting with influenza-like illness. to the best of our knowledge, this is the first report finding replicating yf vaccine in the respiratory sample of a post inoculated individual. © elsevier b.v. all rights reserved. influenza-like illness (ili) is a major health issue during winter months in temperate climates and can be caused by a large range of pathogens including influenza, rhinovirus, human metapneumovirus, parainfluenza viruses and some bacteria [ ] . the use of culture to detect respiratory pathogens became increasingly common in clinical virology laboratories during the last years with the commercial availability of cell cultures. however, with the introduction of commercially manufactured and fda-approved molecular assays, including multiplexed pcr assays, the preference for testing viral pathogens using cell culture systems is diminishing [ ] . in this case study we describe the utility of cell culture followed by electron microscopy (em) to identify an unknown pathogen causing ili in a healthy year old woman. moreover, the case highlights the importance of providing relevant case history in order to avoid unnecessary testing. a year old female was seen in a college student health clinic, presenting with fever of • f ( • c) for days, nausea, vomiting, cough and myalgia. the patient history did not note any recent travel, vaccination, animal contact or insect bites. ili was the initial diagnosis and a respiratory swab was sent for influenza testing. the testing algorithm included both molecular assays and conventional cell culture. the respiratory swab was inoculated into primary rhesus monkey kidney cells (diagnostic hybrids, athens, oh), human lung adenocarcinoma cells (a ), human embryonic lung cells (hel) and epithelial colorectal adenocarcinoma (caco- ) cells. all inoculated cell lines were examined for cytopathic effect (cpe) three times a week for weeks. in parallel, the primary sample was extracted using the biomérieux easymag, tested for influenza a and b using the centers for disease control and prevention (cdc) human influenza virus real-time rt-pcr diagnostic panel (unpublished protocol, details are available from the cdc on request), and for influenza a and b, respiratory syncytial virus a and b, human metapneumovirus, rhinovirus, enterovirus, parainfluenza viruses - , and human coronaviruses oc , e, nl and hku with the qiagen resplex ii kit (qiagen, germantown, md). multiple real-time (rt)-pcr assays were used for the detection of cytomegalovirus, epstein-barr virus, human herpesvirus , adenovirus, enterovirus, herpes simplex virus , herpes simplex virus , varicella zoster virus [ ] , rhinovirus [ ] , mycoplasma pneumonia [ ] , legionella pneumophila [ ] , and chlamydia pneumonia (unpublished assay provided by k. musser). cpe was observed at days in the caco- cells and all other cell lines exhibited no viral changes after a total of days incubation. further, the qiagen resplex ii assay and all of the real-time (rt)-pcr assays were negative. to further investigate the agent that had grown in the caco- cells, the isolate was extracted using the qiagen viral rna mini kit and tested with the same panel of molecular assays as those used for the primary sample; all results were negative. at this time the submitting laboratory was contacted to obtain additional patient information that may elucidate what could be propagating in cell culture. the clinician reported that there was nothing relevant of note in the history. the virus cultures were then prepared for shipment to the cdc for em. briefly, : mixtures of infected scraped cells and % glutaraldehyde were centrifuged at × g for min. supernatant of the centrifugate was adsorbed to formvar-carbon coated grids and negatively stained with % ammonium molybdate- % trehalose, ph . . subsequently, thin-section electron microscopy was performed on the centrifuged cell culture precipitate using routine methods, similar to those described previously [ ] [ ] [ ] . specimens were viewed within a tecnai biotwin electron microscope operating at - kv (fei company, hillsboro, or). images were captured digitally by using a k × k ccd camera (advanced microscopy techniques corp., woburn, ma). the images observed on em were consistent with either a togavirus or a flavivirus (fig. a and b) . consequent to this observation, nucleic acid from the caco- isolate was amplified using a pan-flavivirus conventional rt-pcr assay that targets a portion of the ns region [ ] . pcr products of the expected size were sequenced using the pcr primers. resulting sequences were combined and blast analyzed, demonstrating the highest sequence similarity ( %) to yf virus. the student health clinic was contacted for further discussion on the patient history. they provided additional information, including their administration of the yf vaccine to the patient days prior to the collection of the original respiratory specimen, and also that the patient had recovered. to further characterize this yf virus, published primers [ ] and primers designed in this study using geneious pro . (biomatters, san francisco, ca), were used to further sequence the membrane (m), and envelope (e) gene regions (table ). these targets further identified the virus as having the highest similarity to yf vaccine. multiple papers have been published on adverse events associated with the yf vaccine; however, the literature focuses on viscerotropic disease and multisystem organ failure that mimic wild-type disease progression [ ] [ ] [ ] . here we present a case suffering ili with fever and cough, eight days after receiving the yf vaccine. yf vaccine was isolated in caco- cells from a respiratory swab. the virus was further prepared, stained and imaged by em before it was identified by sequence analysis of the ns gene. additional sequence analysis of the e and m gene verified highest identity to yf vaccine (strain d), reversions to wild-type yf virus (asibi strain) were not present [ ] . yf vaccine is a highly effective vaccine that has the ability to greatly decrease the incidence of yf in people traveling to endemic areas. despite the possibility of an adverse event, the risk of a severe event post-vaccination is low (http://www.cdc.gov/vaccines/pubs/ vis/downloads/vis-yf.pdf). since replicating yf vaccine was isolated and propagated in cell culture, and in the absence of detection of other known infectious agents, the potential cause of the ili was the yf vaccine the patient received days previously. although a known viremia occurs - days after inoculation of the yf vaccine [ ] , this is the first report, to our knowledge, on isolation of yf vaccine from the respiratory sample of a recently vaccinated individual. this case highlights the importance of the collection and submission of an appropriate case history, and the ongoing availability of cell culture and electron microscopy to assist in the identification of unusual or unexpected agents. em and cell culture isolation have been utilized to identify many previously unknown and unsuspecting viruses associated with disease outbreaks [ ] ; such examples include zaire ebolavirus, reston ebolavirus, hendra virus, monkeypox virus, and sars coronavirus. in this case, like the other viruses mentioned, the pathogen was detected by cell culture and was subsequently identified by em. without the assistance of em the relevant molecular assay would not have been chosen to further identify the virus. funding was provided from within the wadsworth center to perform this study. masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during utilization of nucleic acid amplification assays for the detection of respiratory viruses molecular detection of viral causes of encephalitis and meningitis in new york state real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses development of a genomics-based pcr assay for detection of mycoplasma pneumoniae in a large outbreak in new york state design and implementation of a protocol for the detection of legionella in clinical and environmental samples principles and techniques of electron microscopy: biological applications same-day diagnostic electron microscopy of infected cell cultures by a modified protocol for conventional and microwave processing identification of enterically transmitted hepatitis virus particles by solid phase immune electron microscopy comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-pcr assay for detection of flaviviruses targeted to a conserved region of the ns gene sequences yellow fever outbreak, imatong, southern sudan fatal multiorgan failure due to yellow fever vaccine-associated viscerotropic disease viscerotropic disease following yellow fever vaccination in peru yellow fever vaccine -how does it work and why do rare cases of serious adverse events take place? comparison of the virulent asibi strain of yellow fever virus with the d vaccine strain derived from it development of viremia and humoral and cellular parameters of immune activation after vaccination with yellow fever virus strain d: a model of human flavivirus infection cell culture and electron microscopy for identifying viruses in diseases of unknown cause the authors would like to thank the wadsworth center applied genomics technology core for performing sequencing and the staff of the wadsworth center virology laboratory for testing of samples. special thanks to alan dupuis, dr. laura kramer and dr. paul masters for helpful scientific discussion and to dr. gino battaglioli for editorial comments. none declared. ethical approval was given and covered under the new york state department of health institutional review board study # - and # - . key: cord- -qiil gqk authors: tatlow, dean; tatlow, corinne; tatlow, scarlet; tatlow, savanah title: a novel concept for treatment and vaccination against covid‐ with an inhaled chitosan‐coated dna vaccine encoding a secreted spike protein portion date: - - journal: clin exp pharmacol physiol doi: . / - . sha: doc_id: cord_uid: qiil gqk a novel concept in dna vaccine design is the creation of an inhaled dna plasmid construct containing a portion of the coronavirus spike protein for treatment and vaccination. the secretion of a spike protein portion will function as a competitive antagonist by interfering with the binding of coronavirus to the angiotensin‐converting enzyme (ace ) receptor. the secreted protein binding to the ace receptor provides a unique mechanism of action for treatment to all strains of coronavirus in naïve patients, by blocking the ace receptor site. an inhaled plasmid dna vaccine replicates the route of lung infection taken by coronavirus with transfected cells secreting spike protein portions to induce immunity. unlike most dna vaccines with intracellular antigen presentation through mhc i, the current vaccine relies on the secreted proteins presentation through mhc ii as well as mhc i to induce immunity. lung specific production of vaccine particles by inhaled plasmid dna is appealing since it may have limited systemic side‐effects, and may induce both humoral and cytotoxic immunity. finally, the ease and ability to rapidly produce this plasmid construct makes this an ideal solution for managing the emerging threat of coronavirus. covid- is a disease which causes a contagious acute respiratory infection in humans by the coronavirus named, severe acute respiratory syndrome coronavirus (sars-cov- ). symptoms of infection varying from asymptomatic to critically ill with acute respiratory distress syndrome. cases of coronavirus infection have now spread to almost every country, with currently no effective treatment or vaccine available. this highlights the urgent need for the development of an effective treatment and/or vaccine. the genome of the novel sars-cov- was first released in late december , and since then a great deal of work has been done identifying the gene involved in infection. the genome of the novel sars-cov- virus encodes structural proteins that make up the outer layer which include; the spike, envelope, and membrane proteins. the key feature of sars-cov- is the surface spike protein, which plays a critical role in infection by mediating high specificity viral attachment to the host cell surface ace receptor allowing for viral entry. these findings of the identified spike proteins in the sars-cov- receptor binding domain and ace region may provide useful information for treatment or vaccine development. , various forms of vaccines have been used to fight against bacterial and viral disease. plasmid dna vaccines are of great interest, since they provide the ability to be rapidly constructed and manufactured. it was first shown that plasmid dna could protect mice from this article is protected by copyright. all rights reserved influenza challenge in the s. the challenge with plasmid dna is the optimal deliver of dna into the desired cells for the expression of proteins needed to generate an immune response. in this paper a series of inhaled plasmid dna vaccine construct containing various forms of the coronavirus spike protein sequence may provide potential treatment and vaccine options as revealed by yu et al. (fig .) . plasmid dna produced spike proteins will function to induce immunity as a vaccine candidate (fig. a. ) and work as a competitive antagonist against coronavirus host cell attachment (fig. b) . dna vaccination has emerged over the years as a novel approach for the prevention of bacterial and viral diseases. in recent years dna vaccination has made considerable progress with the development of many dna vaccines entering into human clinical trials, most notably zika virus phase and hpv infection phase trials. [ ] [ ] [ ] the dna vaccine for zika virus known as vrc was shown to be safe and well tolerated resulting in % of the participants generating humoral and cellular responses. , vrc is a plasmid dna constructed with zika virus genes prm, e(envelope) and a japanese encephalitis virus coding sequence to improve secretion of the zika vaccine particle. this article is protected by copyright. all rights reserved a major obstacle in dna vaccine development is low immunogenicity. administration of naked dna vaccines is usually inefficient, since its negative charge prevents it from crossing cell membranes because of charge repulsion with negative charge phospholipids. an important development in dna vaccination is the improvement in the delivery with nanoparticles of chitosan. chitosan nanoparticles have provided an effective means for prevention of dna vaccine degradation while the cationic nature enables binding to the negative charge of dna. chitosan has many favourable features for dna vaccine delivery to the mucosal surface such as mucoadhesion, highly soluble, inert and non-immunogenic. since mucosal surfaces are a major site for a majority of pathogens that infect humans, this article is protected by copyright. all rights reserved . three dna constructs are the s /s without the transmembrane (tm) and c-terminal (ct) domains, s and recptor binding domain (rbd) with trimerization taq. the other three dna constructs contain the jev tm and ct domains added to s /s , s and rbd. these dna constructs can be tested for protein secretion quantity, and effectiveness of blocking coronaviurs infection. other commonly used fusion partners include the constant domain igg (the fc region), maltose-binding protein (mbp), small ubiquitin-like modifier(sumo), and human serum albumin (hsa) tag. cells transfected by the dna construct will also secrete a portion of the spike protein as a competitive antagonist. it has been shown that the s protein fragment of the s protein binds with high efficiently to the ace receptor revealing the potential of a portion of the spike protein to work as a competitive antagonist once secreted. the mechanism of action of dna vaccination with these inhaled chitosan dna constructs involves encoding antigen(s) that are transfected into either antigen presenting cells (apc) or somatic cells. in one scenario intracellular processing of plasmid dna leads to vaccine derived endogenous peptide presentation on mhc-i molecules. , , this antigen is expressed to the cell surface with mhc i and presented to cytotoxic cd + t cells stimulating cell mediated immunity. in the other scenario antigens of exogenous origin or secreted protein from plasmid dna are loaded on mhc ii resulting in activation of helper cd + t cells which in turn contribute to b cell priming to yield a humoral immune response. in mucosal vaccination, the plasmid dna antigen is secreted into the extracellular space where m cells participate in antigen uptake and transport them to the extracellular space. at this point the antigen is released to apc cells for presentation to immune cells. these antigenic proteins expressed and secreted by plasmid dna vaccines access both the exogenous and endogenous pathways in the activation of both humoral and cellular mediated immune responses. this article is protected by copyright. all rights reserved there are studies that show a patient's immune response resulting from delivery of a vaccine to the lung may equal or exceeds the immune response created by vaccine injection. it has been proposed that a mucosal immune response may be created via particles of the vaccine material depositing on the upper respiratory tract. a systemic immune response may be created via particles of the vaccine material depositing into the deep lung. in a study by weaver there was a significant differences in antibody and t-cell response with im (intramuscular) immunized mice compared to inhaled immunization. , however, there was no significant difference between im and inhaled immunized mice response when challenged with lethal influenza virus amounts. these results suggest that vaccine efficacy of im and inhaled vaccines are almost identical, and inhaled vaccination may be adequate route of administration even though antibody and t-cell responses are low. in this paper the primary use of an inhaled plasmid dna vaccine containing the coronavirus spike protein dna sequence is for treatment. the small chitosan plasmid dna size of approximately .nm can reach deep into the lung, similar to that of the coronavirus with a size of nm. once in the lower respiratory tract or alveolar region of the lung deposited plasmid dna will be taken up and expression of coronavirus spike proteins by host cells such as pneumocytes will occur. this will create a competitive antagonistic environment whereby coronavirus will have few options available for binding to the ace receptor. thus, the expression of coronavirus spike proteins provides a protective barrier against coronavirus infection of type pneumocytes. a major concern with using an inhaled chitosan dna vaccine as a treament is obtaining sufficient production of secretion protein to compete with coronavirus. currently, there are few studies available using a dna vaccine by the respiratory route for treatment, a study by kodama et al., reveals the practicality of this route by using the luciferase this article is protected by copyright. all rights reserved gene to show gene expression in lung tissue by fluorescene intensty. this study revealed that high levels of inhaled dna vaccine expression can occur in lung tissue as seen by the increase in fluorsecence in lung samples, and in microscopic lung sections. these results indicate that high levels of protein expression may be generated by inhaled dna for competitive antagonism against coronavirus. a large dose of inhaled chitosan dna construct or multiple doses can easily be given when symptoms first appear when there is a lower viral load early in infection. in comparison to drug treatment approaches, inhaled dna treatment has many advantages, with a prolonged half-life, site-specific action, no metabolic concerns, and also ease of manufacture, administration and storage. another major concern with coronavirus vaccine development is the harmful inflammatory response known as antibody-dependent enhancement in which non neutralizing antibodies bind to coronavirus and enhance entry into cells. a recent study by yu et al., has shown protective immunity in rhesus monkeys with little side effects of dna vaccination by using various forms of the coronavirus spike protein. the dna vaccine constructs in the article by yu et al., reveals that both secreted and non secreted proteins were able to provide protective immunity. the mechanism of action of the inhaled dna construct may also work for treatment and vaccination of other respiratory viruses such as influenza where a ha expressed protein prevents binding of any strain of influenza. in addition, this novel method of treatment may be used for delivering dna encoding proteins to other tissues where secretion of proteins into the extracellular space may provide a therapeutic benefit. a dna vaccine encoding secretion of interferon beta protein to locally transfected tissue could be inhaled for deliver to lungs or oral deliver to the gastrointestinal tract. this article is protected by copyright. all rights reserved this dna construct has a great deal of versatility for use as a treatment or vaccine. many studies have proven dna vaccines to be safe and well tolerated. the use of plasmid dna provides an easy means of production which should provide a low cost method for delivering vaccines to the lung with some form of nebulizer or metered-dose inhaler. there may be many cons associated with inhaled dna vaccination such as autoimmunity and/or unseen problems with the quantity of vaccine particles produced. however, lung specific production of coronavirus spike proteins may be a safer alternative than systemic drug treatment. currently, plasmid dna vaccination is only licensed for veterinary use, hopefully further testing may allow for licensing in humans. characterization of the receptor-binding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein coronaviruses: an overview of their replication and pathogenesis heterologous protection against influenza by injection of dna encoding a viral protein a comparison of plasmid dna and mrna as vaccine technologies dna vaccine protection against sars-cov- in rhesus macaques safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase clinical trials zika virus vaccine development: progress in the face of new challenges therapeutic dna vaccines for human papillomavirus and associated diseases. hum intranasal dna vaccine for protection against respiratory infectious diseases: the delivery perspectives dendritic cells targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein maltose-binding protein (mbp), a secretion-enhancing tag for mammalian protein expression systems a -amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme airway m cells arise in the lower airway due to rankl signaling and reside in the bronchiolar epithelium associated with ibalt in murine models of respiratory disease dna vaccine that targets hemagglutinin to mhc class ii molecules rapidly induces antibody-mediated protection against influenza a dose effects of recombinant adenovirus immunization in rodents mucosal vaccination via the respiratory tract chitosan-dna nanoparticles enhanced the immunogenicity of multivalent dna vaccination on mice against trueperella pyogenes infection development of a dna vaccine for melanoma metastasis by inhalation based on an analysis of transgene expression characteristics of naked pdna and a ternary complex in mouse lung tissues thank-you dr. jordan b. peterson. there are no conflicts of interest to report. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord- -izdl f i authors: qin, ede; shi, huiying; tang, lin; wang, cuie; chang, guohui; ding, zhifen; zhao, kai; wang, jian; chen, ze; yu, man; si, bingyin; liu, jianyuan; wu, donglai; cheng, xiaojie; yang, baoan; peng, wenming; meng, qingwen; liu, bohua; han, weiguo; yin, xunnan; duan, hongyuan; zhan, dawei; tian, long; li, shuangli; wu, jinsong; tan, gang; li, yi; li, yuchuan; liu, yonggang; liu, hong; lv, fushuang; zhang, yu; kong, xiangang; fan, baochang; jiang, tao; xu, shuli; wang, xiaomei; li, changwen; wu, xiaohong; deng, yongqiang; zhao, min; zhu, qingyu title: immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: izdl f i background: in , severe acute respiratory syndrome (sars) resulted in hundreds of infections and deaths globally. we aim to assess immunogenicity and protective efficacy of purified inactivated vero-cell sars vaccine in monkeys. methods: the cultures of sars coronavirus (sars-cov) bj- strain infected vero cells were inactivated with β-propiolactone. sequential procedures, including ultrafiltration, gel filtration and ion exchange chromatography, were performed to obtain purified inactivated sars vaccine. the purified sars vaccine was analyzed with electron microscope, hplc and western blotting. we immunized three groups of cynomolgus macaques fascicularis with adjuvant-containing purified vaccine, purified vaccine and unpurified vaccine, respectively, and a fourth group served as a control. antibody titers were measured by plaque reduction neutralization test. the vaccinated monkeys were challenged with sars-cov bj- strain to observe protective efficacy. additionally, three groups of rhesus monkeys were immunized with different doses of the purified inactivated sars vaccine ( . , and μg/time/monkey) on days and , and the monkeys were challenged with sars-cov gz- strain. we assessed the safety of the sars vaccine and observed whether the antibody dependent enhancement (ade) occurred under low levels of neutralizing antibody in rhesus. findings: the purity of sars vaccine was . % by hplc identification and reacted with convalescent sera of sars patients. the purified sars vaccine induced high levels of neutralizing antibodies and prevented the replication of sars-cov in monkeys. under low levels of neutralizing antibody, no exacerbation of clinical symptoms was observed when the immunized monkeys were challenged with sars-cov. in this preliminary animal trial, no side effects were detected when monkeys were immunized with purified sars vaccine either at normal or large doses. interpretation: the purified inactivated sars vaccine could induce high levels of neutralizing antibody, and protect the monkeys from the challenge of sars-cov. the sars vaccine prepared in the study appeared to be safe in monkeys. severe acute respiratory syndrome (sars, also called infectious atypical pneumonia in china) is a new emerging respiratory infectious disease with rapid transmission and high mortality. according to statistics released by who on august , a total of clinically diagnosed cases were reported worldwide, causing deaths, an estimated mortality of . %. a novel coronavirus, sars coronavirus (sars-cov), was identified as the pathogen of sars [ ] , in , some new sars cases were reported in china, which suggest that sars may recure in the future. the current medical strategies mainly replying on non-specific anti-viral and supportive treatment are not sufficient. previous experience in controlling infectious diseases indicates that the most efficient protection against sars infection is mass immunization. effective vaccines for protecting the population are urgently needed. since the discovery of sars-cov as the cause of sars. the inactivated sars-cov has been proposed as one of the prophylactic vaccination approaches against sars. a number of studies about coronavirus vaccines have demonstrated that inactivated vaccines are effective methods, such as infectious bronchitis virus and bovine coronavirus. although, little information about pathogenesis of sars-cov is known, we postulated that it may be feasible to develop an inactivated vaccine for sars. the present study developed the techniques for the preparation of sars inactivated vaccine and investigated the immune response and protective effects induced by the vaccination in monkeys. our data suggest that this vaccine could stimulate monkeys to produce high levels of antibodies which could protect monkeys from sars-cov challenge. these results lay a solid foundation for our early phase human study. sars-cov bj- strain and gd- strain were isolated from autopsy lung tissues of sars patients from beijing and fushan, guangdong province, china in march [ ] . the complete sequences have been deposited in gen-bank, with accession numbers ay and ay , respectively [ ] . vero cells (atcc no. ccl- ) were provided by the national vaccine and serum institute, beijing, china. well-grown vero cells were inoculated by bj- strain at a m.o.i of . and maintained with serum-free mem at • c. the culture was harvested when cytopathic effects reached + [ ] , and passed through . m filter (polypure ® , in- /fin silicone) from pall cor., usa. the filtrate was collected and ␤-propiolactone was added at : (v/v). the shaken mixture was kept at - • c for more than h before incubating at • c for h to inactivate the virus completely. the inactivated virus was concentrated by omega ultrafiltration unit and suspended in pbs ( mm, ph . ). all procedures were performed at biosafety level . the concentrated inactivated virus was purified by gel filtration of sepharose fast flow (ff) (index / , amersham biosciences, sweden) at % column volume with elution buffer pbs ( mm, ph . ). the fractions from the outer void were collected. the eluate containing sars antigen was further purified by ion exchange chromatography. aktaexplorer chromatography system was used, the media was fractogel ® emd dwae(m), and the column was xk / . after being equilibrated by pbs ( mm, ph . ), the gel filtration purified inactivated viruses were loaded, then eluted by pbs containing . m nacl. the first elute peak containing the viral antigen was collected. the effluent was diluted according to the protein concentration of the antigen active peak to prepare the purified sars vaccine. the purity of the vaccine was analyzed with hplc analysis. two hundred microliters of purified sars vaccine was run on tskgel g sw column ( . mm × mm, f ) using . ml/min flow rate to acquire the hplc profile. the antigen content of inactivated sars vaccine was determined by double-antibody sandwich elisa. the rabbits were immunized with sucrose density gradient zonal centrifugation-purified virus antigen to prepare the anti-sars-cov antibody (first antibody), and horseradish peroxidase labeled rabbit anti-sars-cov igg was used as the second antibody. the protein content of the purified inactivated sars vaccine was determined by a modification of the lowry method. approximately l of purified sars vaccine was placed on a copper grid and then allowed to stand for min. after the residual fluid was removed by filter paper, the specimen on the grid was stained with % phosphotungstic acid for min. then the grid was dried at room temperature and observed under transmission electron microscope. sds-page was performed according to laemmli method [ ] . after electrophoresis, the protein band in the gel was transferred to polyvinyl fluoride (pvdf) membrane (millipore), and reacted with convalescent sera of sars patients in blocking buffer (containing % skimmed milk) at • c overnight. after washing, the membrane was incubated with horseradish peroxidase labeled rabbit anti-human igg (bio-rad) for h. the membrane was washed again, and the color was developed. cynomologus macaques (male, - years old, animal center of academy of military medical sciences, beijing, china) were divided into four groups, with five monkeys per group. group was immunized with purified vaccine and adjuvant (aluminium hydroxide, aluminium content: . mg/ml). group was immunized with purified vaccine. group was immunized with unpurified inactivated virus. all three experimental groups were immunized by deltoid muscle injection at g on days , , and . a further control group was injected with supernatants of vero cell culture ( . ml/monkey). all monkeys were blood sampled on days , , , , , and . the sera were isolated from the blood and inactivated at • c for min. the neutralizing antibody titers were then determined by the plaque reduction neutralization test. to observe the protective effect of the inactivated vaccine, monkeys in group (four monkeys, immunized with purified inactivated vaccine) and in the control group (four monkeys) were challenged with ml sars bj- strain (log tcid /ml = . ) by nasal route on day after the prime immunization. the specimens of blood, pharynx swab and stool were collected on days , , , and after the challenge. the neutralizing antibodies in sera were determined, and virus isolation and rt-pcr were also conducted [ , ] . all monkeys were sacrificed on day after the challenge, and lymph node, lung, spleen, liver and kidney tissues were collected for rt-pcr. the pathological changes of lung tissues were observed in slide slices by light microscope. safety evaluation tested the antibody dependent enhancement (ade) of the inactivated sars vaccine. three groups of rhesus (male, - years old, animal center of academy of military medical sciences, beijing, china) (three rhesus monkeys per group) were immunized with different doses of purified inactivated sars vaccine ( . , , g/time/monkey) on days and . three monkeys in the control group were injected with vero cell culture. blood was sampled on days , , and for neutralizing antibody determination. all monkeys were challenged with sars-cov gz- strain on day at the dose of . tcid /ml × and were euthanized on day after challenge. internal organs from the monkey were sampled and the systematically pathological changes were observed. to assess possible side effects of large doses of purified inactivated sars vaccine ( g) in major organs, the monkeys were immunized with the sars vaccine by deltoid muscle injection, and then the hematological, biochemical and pathological changes were observed. neutralizing antibodies in the sera of vaccined monkeys were tested by % plaque reduction neutralization test (prnt ) using vero cells [ , ] . in brief, heat inactivated ( • c min) sera were diluted serially and mixed with sars-cov bj- ( pfu/ . ml), then incubated at • c for min. the mixtures were added to a vero cell flask, and absorbed at • c for min, then covered with nutrient solution (containing agarose, calf bovine serum, streptomycin and dmem), and cultured at • c for h. after being dyed by . % neutral red, the flask was incubated at • c for another - h until the plaque was developed. vaccined monkeys were euthanized under ether anaesthesia. the lung, liver and kidney tissues were sampled and placed in % formaldehyde, followed by conventional dehydration and paraffin embed. then the embedded specimens were sliced (with the thickness of m), stained with hematoxyllin-eosine and observed under light microscope. when cytopathic effects in vero cells inoculated with sars-cov bj- strain reached +, the virus culture was harvested and clarified, then inactivated with ␤propiolactone. after concentrated by ultrafiltration, the inactivated virus culture was further purified by sepharose ff gel filtration chromatography and ion exchange chromatography to achieve higher purity. after two steps of purification, the residual bovine serum albumin and cellular dna in inactivated sars vaccine were less than . and ng/ml, respectively. the ratio of antigen activity/protein quantity increased from . to . u/g after purification. hplc analysis showed that the purity of sars vaccine was . %. the purified components of the inactivated viruses showed good antigenicity and immunogenicity in mice (data not shown). sars-cov-like particles could be observed in purified vaccine under electron microscope. as shown in fig. , the molecular weights of purified vaccine by sds-page analysis were consistent with the molecular sizes of the main structural proteins of sars-cov. western blot analysis showed that the protein bands reacted strongly with the convalescent sera of sars patients. to observe whether the sars inactivated vaccines induced sars-specific neutralizing antibodies, cynomolo- gus macaques were immunized with adjuvant-containing purified vaccine, purified vaccine and unpurified vaccine. their sera were sampled at different time to measure neutralizing antibody titers. as shown in fig. , weeks (on day ) after the first boost, large increases in neutralizing antibodies were observed in the sera of all three immunization groups. the neutralizing antibody titers in all immunization groups peaked week after the second boost ( weeks after the prime). antibody levels also increased rapidly after the last boost over a -week observation period. the results showed that the neutralizing antibody levels in the adjuvant-containing purified vaccine group were higher and lasted longer than those in other two groups. the quantity of neutralizing antibody production and the time for antibody level maintenance showed no apparent differences between the purified vaccine group (without adjuvant) and unpurified vaccine group, but the antibody levels in the unpurified vaccine group appeared to decrease somewhat faster. the results showed that both the purified and the unpurified sars vaccines can induce high levels of sars-cov specific neutralizing antibodies in monkeys, thus demonstrating high immunogenicity. fig. . anti-sars-cov neutralizing antibody titers of monkey sera elicited by different inactivated sars vaccine. cynomologus macaques were immunized with adjuvant-containing purified vaccine, purified vaccine, unpurified vaccine and supernatants of vero cell culture ( . ml/monkey) as control by deltoid muscle injection at g on days , , and . all monkeys were blood sampled on days , , , , and . the neutralizing antibody titers were then determined by the plaque reduction neutralization test, and presented as the geometric means (n = ). to observe the protective effect of the purified inactivated sars vaccine on monkeys, the aforementioned two animal groups (purified vaccine group and unvaccinated-control group, with four monkeys per group) were challenged with sars bj- strain by nasal route on day after the prime immunization. the results showed no sars-cov isolated from the monkeys in the immunization group. in the control group, sars-cov was isolated by pharynx swab inoculation in two out of four monkeys on days and after the challenge. rt-pcr amplification of the tissue samples from autopsy of monkeys on day after challenge showed that the lymph node, lung, spleen and kidney tissues from all four monkeys in the immunization group were negative. in the control group, the positive rates of rt-pcr amplification of the lymph node, spleen, lung and kidney tissues were % ( / ), % ( / ), % ( / ) and % ( / ), respectively. the results indicated that inactivated sars vaccine may inhibit the propagation of sars-cov in monkeys. further pathological examinations of the challenged monkeys showed that two out of four animals in the unvaccinatedcontrol group displayed moderate and mild interstitial pneumonia. two out of four from the immunization group were normal, and the remaining two immunized monkeys only showed mild interstitial pneumonia, without any pathological changes in other tissues. the results suggested that purified inactivated sars vaccine had protective effects in monkeys. as shown in fig. , the neutralizing antibody levels in the immunization group peaked between and on day after the last immunization (i.e., on day after the prime immunization), and decreased gradually thereafter. on day after the prime immunization, the neutralizing antibody titers in the sera of four immunized monkeys dropped to range of - . however, the antibody levels in the immunization group increased significantly week after the challenge, indicating that inactivated sars vaccine produced immune memory in monkeys. after rhesus monkeys were immunized with different doses of purified inactivated sars vaccines, hematological and biochemical indexes showed no change in any animal, including all nine monkeys in the immunization group and three monkeys in the unvaccinated-control group (data not shown). no fever was observed in any monkey , , , and h after the injection of the sars vaccine, and the appetite and mental state of all animals maintained normality. to examine whether different antibody levels produced by different doses of vaccines induced a corresponding antibody dependent enhancement, the monkeys in the immunization group and unvaccinated-control group were challenged with sars-cov gz- strain by nasal route on day after the prime immunization (fig. ) . the results showed that no clinical symptoms were detectable in immunized monkeys, and their body temperatures and hemograms were normal. on day after the immunization with different doses of inactivated vaccine, systematic pathological observation under light microscope showed no abnormity in the internal organs of monkeys. histopathology observations indicated that all monkeys in the unvaccinated-control group showed obvious interstitial pneumonia. interstitial pneumonia was also observed in the immunization group, but was obviously milder than in the unvaccinated-control group. among the monkeys in the low dose group, especially those with low levels of neutralizing antibodies, the pathological changes were milder than in unvaccinated-control group. the results showed that neither infection enhancement nor immunopathological exacerbation was observed under low neutralizing antibody titers. for monkeys immunized at higher doses ( g/time/monkey), body temperature, breathing, appetite, mental state and all biochemical indexes were normal (data not shown), and no abnormalities were observed in major organs such as lung, liver, kidney, etc. the above results indicated that the purified sars vaccine prepared in the present study was safe in monkeys. the isolation and propagation of high titers of sars-cov in vero cells provided a solid foundation for the development of inactivated sars vaccines. drawing on previous research, currently available technical platforms [ ] , and results from other inactivated viral vaccines [ ] , we developed concentration and purification techniques for a sars vaccine. our inactivated sars vaccine was produced with standard quality controls and showed high purity and immunogenicity. the vaccine could also induce high levels of neutralizing antibodies. to date, several approaches for developing sars vaccines have been reported, including inactivated vaccines [ , ] , recombinant adenovirus [ ] or vaccina virus [ ] , and dna vaccines [ ] [ ] [ ] [ ] [ ] . these vaccines could induce the production of specific anti-sars antibodies and activation of ctl responses, some could to induce protective immunity in mice. the recombinant adenovirus containing the genes encoding protein s fragments and full-length m and n were also proved to be able to induce protective immunity in monkey. our data and other studies about inactivated sars vaccines showed the highest specific abs titers in mice or monkeys, which suggest the high immunogenicities of inactivated vaccines. unlike their studies [ , ] on inactivated vaccines for sars, our inactivated sars vaccine was produced with standard quality controls. our observations of immunogenicity in monkeys showed that the unpurified inactivated sars vaccine induced almost the same level of neutralizing antibody as the purified vaccine. even so, the unpurified production could not be applied to clinical use. it had a high content of protein impurity that might induce underlying pyrogen fever. in this study it was also demonstrated that the vaccination with adjuvant of aluminium could induce higher antibody production of sars-cov, but clinical safety concerns about possible side effects in humans precluded use of the adjuvantcontaining purified vaccine to evaluate immune protection (efficacy testing). thus, we selected the purified inactivated sars vaccine for a detailed safety and efficacy study. in previous sars vaccine studies of other groups, challenges have been performed when monkeys were at high levels of neutralizing antibodies. this approach is not informative for population immunization. our study challenged monkeys at lower levels of neutralizing antibodies, which is more consistent with a general population vaccination context. the findings from the low neutralizing antibody stage challenges were promising. the results showed that sars-cov could replicate in relevant tissues, specifically in lymph node and spleen. further histopathological examinations of lung tissues showed that, moderate and mild interstitial pneumonia was developed in control monkeys, while no pathological evidence found in immunized monkeys. the dose-response relationship is an important criterion in all studies of efficacy of vaccines. the results of our neutralization tests, rt-pcr, and autopsies showed that higher level of neutralizing antibodies induced by higher dose vaccination could decrease the sars-cov viral load and pneumonic changes compared to control monkeys after challenge. more animal studies are needed to quantify the relationship between vaccination doses and protective efficacy accurately and to establish the thresholds for safety. some rna viruses are known to induce antibody dependent enhancement such as the exacerbation of diseases in immunized populations exposed to dengue virus infections. ade is therefore a significant obstacle in inactivated vaccine development. our study of safety and efficacy focused on the occurrence of ade in sars-cov. all immunized monkeys were challenged after their neutralizing antibodies decreased to low levels, mimicking the situation in immunized populations. the preliminary results showed that no obvious ade phenomena was detected in immunized monkeys after being challenged with sars-cov gz- strain. also, the levels of neutralizing antibodies in all immunized monkeys increased sharply after sars onset, indicating that immune memories were established in the vaccinated monkeys. the monkey is the most widely studied animal model in sars research. although our studies of purified inactivated sars vaccine were based on the monkey animal model, we are unable to conclude that the monkey is the ideal animal for sars vaccine evaluation. in our studies, some monkeys appeared lethargic for several days after inoculation. some developed diffuse alveolar damage similar to that in sars patients but without typical clinical symptoms. other animal models may elicit different reactions to vaccination. in this study, we used neutralizing antibody levels and interstitial pneumonia as two main indicators of the immunogenicity and protective efficacy of purified inactivated sars vaccine. the results indicated that the purified inactivated sars vaccine we developed could induce high levels of neutralizing antibody, protect monkeys after a sars-cov challenge, and be administered safely in monkeys. taken together, these findings provide a solid foundation for further clinical research of the inactivated sars vaccine. continued research on severe immunopathological reactions should be undertaken during future preclinical research and clinical trials. coronavirus never before seen in humans is the cause of sars isolation and identification of a novel coronavirus from patients with sars a complete sequence and comparative analysis of a sars-associated virus (isolate bj ) cleavage of structural proteins during the assembly of the head of bacteriophage t establishment of an analyzing method for a japanese encephalitis virus neutralization test in vero cells development of plaque method for sars virus bj strain determination of the partial polymerase gene sequence of novel coronavirus isolated from lung tissue of sars patients production of purified japanese encephalitis vaccine from vero cells with roller bottles development of vero cell-derived inactivated japanese encephalitis vaccine immunogenicity of sars inactivated vaccine in balb/c mice humoral immune responses in rabbits induced by an experimental inactivated severe acute respiratory syndrome coronavirus vaccine prepared from f strain effects of a sars-associated coronavirus vaccine in monkeys severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice characterization of humoral responses in mice immunized with plasmid dnas encoding sars-cov spike gene fragments dna vaccine of sars-cov s gene induces antibody response in mice a dna vaccine induces sars coronavirus neutralization and protective immunity in mice immune responses with dna vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in balb/c mice immune responses against sars-coronavirus nucleocapsid protein induced by dna vaccine conflict of interest statement: none declared. key: cord- - a i h authors: bittle, james l.; muir, susie title: vaccines produced by conventional means to control major infectious diseases of man and animals date: - - journal: advances in veterinary science and comparative medicine doi: . /b - - - - . - sha: doc_id: cord_uid: a i h publisher summary this chapter reviews the development of some of vaccines and their use in controlling such major diseases as diphtheria, rinderpest, newcastle disease, smallpox, pertussis, yellow fever, rabies, etc. park–williams number (pw ) strain is used to make diphtherial toxoid for vaccines. as a source of toxin, it is rendered nontoxic by incubation with formalin under alkaline conditions. the product's retention of antigenicity, enabling it to induce antitoxin antibodies, makes it an excellent pediatric vaccine. vaccine against rinderpest virus was developed by koch in by administering bile from infected cattle. animals that survived were permanently immune. formalin- and chloroform-inactivated vaccines were developed using tissues from the infected animals. for the control of newcastle disease, a number of attenuated live-virus vaccines have been developed which are widely used to control the disease. the bl strain, the lasota strain, and the f strain are used to immunize birds of all ages by different routes, including by addition to drinking water and by spraying. protection against rabies correlates with sn antibody, which can be assessed by a number of tests. pasteur's classical vaccine, developed from infected spinal cord tissue dried at room temperature for – days, was given in a series of – inoculations beginning with material dried the longest and progressing through material dried for only days. the first parvovirus shown to be a filterable agent was feline panleukopenia virus (verge and christoforoni, ) , which was not characterized until much later (johnson, a,b; johnson et al., ) . the virus affects most members of the family felidae, causing enteritis and bone marrow hypoplasia that results in severe leukopenia. the virus infects kittens in utero and postnatally causes cerebellar hypoplasia and ataxia (kilham and margolis, ) . immunization. the first panleukopenia vaccines were produced by infecting susceptible cats and harvesting their tissues. filtrates from these tissue suspensions were treated with chemical inactivants such as formalin. two inoculations of this type of vaccine produced longlasting protection (leasure et al., ; enders and hammond, ) . however, subsequent vaccines, produced in primary feline kidney cell culture and inactivated with formalin, proved safer and more effective (bittle et al., ; davis et al., ). an attenuated live-virus vaccine was also very effective in inducing immunity and protection (slater and kucera, ) . concern for the wide-scale use of an attenuated live-virus vaccine centers on the premise that shedding may spread the virus to other species, possibly allowing the emergence of pathogenic variants in these species. attempts to immunize cats orally with attenuated live-virus vaccines have not been successful, but intranasal aerosol application has been effective (scott and glauberg, ; schultz et al., ) . three members of the parvovirus family are known to infect dogs: minute virus of canines, a defective canine adeno-associated virus, and the pathogenic canine parvovirus. canine parvovirus is related to feline panleukopenia virus and may have originated from one of the mammalian parvoviruses. in young dogs, the virus causes leukopenia and severe intestinal disease with necrosis of crypt epithelium in the small intestine. myocarditis occasionally develops, causing sudden heart failure in young dogs. in an epizootic of this disease occurred in the united states, causing high mortality (eugester, ) . immunization. because of the close relationship between feline panleukopenia virus and canine parvovirus, inactivated feline panleukopenia vaccine initially was used to protect susceptible dog populations (appel et al., a) . later, inactivated and attenuated vaccines produced with the canine virus proved to be much more effective (pollack and carmichael, ; appel et al., b) . these parvovirus vaccines have been formulated with other vaccines, including canine distemper and canine adenovirus vaccines, and are administered routinely to young dogs. porcine parvovirus has a distant serologic relationship to other parvoviruses, but it causes disease only in swine. the virus, which is widespread, causes abortions, fetal death, and infertility in sows infected early in gestation. immune sows reinfected during gestation give birth to normal piglets. immunization. numerous reports have shown the effectiveness of inactivated porcine parvovirus vaccines in swine (suzuki and fujsaki, ; mengeling et al., ; wratthal et al, ; fujisaki et al., ; joo and johnson, ) . for example, mengeling et al. ( ) demonstrated the value of a vaccine in which the virus was inactivated with acetylethyleneimine. an attenuated live-virus vaccine has been described (paul and mengeling, ) in which a porcine isolate was attenuated by - passages in a swine testicular cell line. this vaccine effectively induced antibodies and protected challenged animals. the vaccine virus did not cross the placenta; however, it did kill fetuses when inoculated in utero. the virus was shed in feces, and so could be transmitted to unvaccinated animals. the inactivated vaccines now used in united states have been effective in controlling porcine parvovirus infection. vaccination is recommended for gilts and sows before breeding. adenoviruses cause significant disease in dogs, foxes, and man, but have also been isolated from cattle, swine, goats, sheep, horses, turkeys, and chickens, where they produce mild infections, mainly associated with the respiratory and intestinal tracts. there are at least different adenoviruses, of which occur in man. each adenovirus has a narrow host range. there are two canine adenoviruses. canine adenoviruses (cav- ) causes infectious canine hepatitis, which at one time was widespread, but now has been controlled by vaccination. infected dogs also develop corneal opacities following this infection, as a result of the formation of immune complexes and uveitis within the anterior chamber of the eye (carmichael et al., ) . in foxes, cav- produces a rapidly fatal encephalitis. canine adenovirus (cav- ) causes respiratory disease in dogs, but neither hepatitis nor encephalitis in dogs or foxes. the respiratory disease varies depending on the strain of virus and bacterial superinfection. cav- may be transmitted by aerosol, whereas cav- is spread by other direct means such as contact with urine or saliva from infected animals. cav- and cav- are oncogenic in experimentally infected hamsters (sarma et al., ; dulcac et al., ) . immunization. dogs that recover from natural cav- infection are immune for a long period. the first cav- vaccines were produced by formalin inactivation of tissue homogenates from infected dogs. cav- was first adapted to tissue culture by cabasso et al. ( ) and by fieldsteel and emery ( ) . the latter modified the virus by serial passage in porcine and canine tissue cultures; the resulting vaccine immunized dogs and did not produce clinical signs of infection except for occasional corneal opacity similar to that caused by natural infection. dogs immunized with cav- vaccines are also protected against cav- (appel et al., ) . the immunity produced by the attenuated live-virus cav- vaccines is long lasting and has drastically reduced the incidence of the canine disease. although cav- is closely related to cav- , when it is inoculated parenterally into dogs, it does not cause disease, although the virus is shed from the respiratory tract (appel et al., ) . such dogs become immune to both cav- and cav- . an attenuated live-virus vaccine containing cav- is now being widely used in place of older cav- vaccines (bass et al., ) ; this has resulted in a much lower incidence of corneal opacity in recipients. however, because of the oncogenicity of adenoviruses in other hosts, the respiratory shedding of cav- virus should be a concern. in man, adenoviruses mainly produce disease of the respiratory tract which varies in severity depending on the virus and the age of infected individuals. the viruses cause acute pharyngitis in infants and children, pharyngitis and conjunctivitis in children, and acute respiratory disease (ard) in military recruits and institutionalized young adults. pneumonia may also occur, expecially following ard. a vaccine consisting of adenovirus types , , and , grown in monkey kidney cell culture and inactivated with formalin, was introduced in for use in u.s. military recruits. the vaccine was effective in reducing ard in this population (sherwood et al., ) . however, the vaccine was withdrawn from use in because of concern for possible oncogenicity of the adenoviruses, and of the sv virus present in the monkey kidney cell culture. a subsequent adenovirus, type , was passed in human tissue and was, therefore, devoid of sv genomic material. this virus, encapsulated in enteric-coated capsules, proved to be a safe and effective vaccine edmonson et al., ). an adenovirus vaccine, prepared in the same way and given simultaneously with adenovirus vaccine, was equally effective (top et al., ) . the administration of a vaccine with only one of these viruses was not effective in controlling ard caused by any of several different adenoviruses, but formulation of a vaccine with both viruses proved to be broadly cross-protective and have had a major influence in controlling ard in u.s. military recruits. over herpesviruses produce disease in man and animals. these viruses have an affinity for epithelial tissues and nervous system tissues. these tropisms lead to specific disease syndromes involving the respiratory and urogenital tracts, and the central and peripheral nervous systems. the viruses often cause persistent infections, which can be latent and can be reactivated. herpesvirus disease are generally much more severe in young humans and animals. some herpes-viruses, including epstein-barr virus (ebv) in humans and marek's disease virus in fowl, are associated with malignancies. the presence of specific antibodies may prevent or modify the clinical disease but does not prevent infection. vaccines have been developed for those herpesviruses causing major diseases in animals; however, despite the seriousness of human herpesvirus diseases, including those caused by herpes simplex virus, ebv virus, cytomegalovirus, and varicella virus, progress has been slow in developing vaccines for humans. this stems from concern over possible persistence and oncogenicity of vaccine viruses. in the past few years, several attenuated live-virus varicella vaccines have been tested and found safe and efficacious (takahashi et al., ; asano et al., ; arbeter et al, ) . since the initial recognition of infectious bovine rhinotracheitis (ibr) in the early s and the later recognition of another manifestation of the disease, infectious pustular vulvovaginitis, this bovine herpesvirus has been acknowledged as a major problem in livestock. the respiratory disease varies from mild to severe, and herd mortality can be as high as % in an acute outbreak. the virus may cause abortions in pregnant cattle, and the genital disease results in a chronic vulvovaginitis but not abortions, apparently due to a lack of viremia. immunization. attenuated live-virus vaccines were developed by serial passage of field isolates in bovine kidney cell cultures. such vaccine viruses have reduced virulence when administered intranasally and do not produce disease when administered parenterally york et al., ) . vaccine virus does not spread from vaccinated to unvaccinated contact animals. the widescale use of such vaccines has controlled this disease effectively. when administered by the intranasal route (todd et al., ) , these vaccines had the advantage of producing more rapid protection, but long-term protection was no greater than with perenterally administered vaccines (mckercher and crenshaw, ) . because of the possibility that the attenuated live-virus vaccines given parenterally might cause abortion, their use has been restricted to nonpregnant animals. a formalin-inactivated vaccine has also been developed, but requires multiple inoculations, and the serum neutralizing (sn) antibody response is low (matsuaba et al., ) . however, inactivated vaccines are used especially in dairy cattle because of concern that the attenuated live-virus vaccines may cause abortion. four herpesviruses affect horses: equine rhinopneumonitis virus (ehv- ) causes abortion which may be epizootic, and also, occasionally, rhinopneumonitis; ehv- is cytomegalovirus found in buffy coat cells of most horses, but its role in causing disease is unknown; ehv- causes equine coital exanthema, a urogenital tract disease; and ehv- is the main cause of equine rhinopneumonitis. ehv- and - are related antigenically, whereas types and are distinct (sabine et al., ; studdert and blackney, ) . immunization. the first vaccine for equine rhinopneumonitis was developed by doll and bryans ( ) , who adapted an ehv- isolate to suckling hamsters. this vaccine produced a mild disease, but induced protective immunity against the more serious respiratory disease and abortion that occurs in older animals. however, this vaccine sometimes caused abortions, and the virus was known to spread from vaccinated to unvaccinated horses. a more attenuated strain of ehv- has been used widely and is considered to be reasonably effective (mayr, ) . this strain was attenuated by passage in hamsters and in pig kidney cell culture before adaptation to an equine cell line (gerber et al., ) . this vaccine induces low levels of sn antibodies, and protects against respiratory disease but not against abortion. a formalin-inactivated vaccine, emulsified in an oil adjuvant, has also been found safe and effective in preventing respiratory disease. this vaccine, in controlled field trials, lowered the abortion rate significantly (bryans, ; bryans and allen, ) . the attenuated live-virus and the inactivated vaccines contain only ehv- , but apparently there is enough cross-protection induced by repeated vaccinations to protect against the ehv- respiratory disease. this herpesvirus is unusual in that it occurs naturally in many species-cattle, sheep, goats, swine, dogs, cats, rats, and mice. it produces a fatal disease in all of these species except adult swine, in which the disease is mild. in each species except swine the primary sign is intense puritis resulting in the animal biting the affected area. infection rapidly spreads to the central nervous system, leading to paralysis and death. in adult swine, the signs of infection are mild, usually of a respiratory nature, but abortions follow in approximately % of pregnant sows. in young pigs, especially neonates, the infection may be fatal. since the disease is prevalent only in swine, this is the only animal for which a vaccine has been developed. in examining a virulent strain of pseudorabies virus, bartha and kojnok ( ) found two plaque sizes. the small plaque size variant, called k, occurred naturally and had reduced virulence for rabbits. further studies with this strain passaged in chick embryo or calf testicle cell culture produced a safe vaccine for swine. one dose induced partial immunity and a second dose yielded good immune responses in all recipients. mcferran and dow ( ) adapted the Κ strain to vero cells and showed that one dose of a vaccine prepared from this passage material was protective. pigs vaccinated with this vaccine did not shed the virus. although the vaccine did not prevent infection, it prevented clinical disease. this attenuated live-virus vaccine is used widely. feline rhinotracheititis virus, which was first isolated in , produces a widespread respiratory disease in cats (crandell and maurer, ) . the virus may also cause fetal death. immunization. an attenuated live-virus vaccine was developed by passage of a field isolate in feline kidney cells (bittle and rubic, ) . the vaccine is given parenterally and is safe and effective (scott, ) . low levels of sn antibodies persist for at least months. vaccinated cats are resistant to intranasal instillation of virulent virus; they may be reinfected, but do not develop clinical disease (bittle and rubic, ; kahn et al., ) . the vaccine has controlled this important respiratory pathogen and, when combined with a feline calicivirus vaccine, has drastically reduced the incidence of respiratory disease in this species. this herpesvirus is highly contagious and causes lesions in the larynx, trachea, and bronchi of infected fowl. the infection causes the formation of an exudate that produces the characteristic respiratory distress and rattling in severely affected birds. birds that recover from this disease are immune for a long period, but may remain as carriers and a source of virus for reinfection of flocks. immunization. the earliest method of immunization was developed by beaudette and hudson ( ) , who applied virulent virus from tracheal exudate to the mucosa of the bursa of fabricius and the cloaca with a stiff brush. this produced a local infection and a solid systemic immunity. the use of fully virulent virus caused occasional outbreaks of disease, particularly when the scarification was not properly done or insufficient virus was present, and birds did not become immune. the virus was propagated on the chorioallantoic membrane of embryonated eggs by burnet ( ) , and this became a source of vaccine material. other methods of vaccination involved intranasal instillation (benton et al., ) and feeding in drinking water (zamberg et al., ) . attenuated strains of ltv have been developed by serial passage in cell culture (gelenczei and marty, ) and by feather follicle passage (molgard and cavett, ) . attenuated strains isolated from outbreaks or selected from passage are now used in preference to virulent virus. marek's disease virus causes lymphoproliferative disease in chickens, occurring in three forms: neural, ocular, and visceral (the latter mainly in young birds) (sevoian and chamberlain, ; biggs and payne, ) . sevoian was the first to provide evidence that marek's tumors were caused by a virus and were transmissible. the virus has been established as a gamma herpesvirus (churchill and biggs, ; soloman et. al., ) . immunization. fatalities from marek's disease caused major economic losses in the poultry industry until a vaccine was developed for its control. this was accomplished by churchill et al. ( ) , who attenuated by serial passage a virus isolated from chickens that is parenterally administered at day of age. thereafter, okazaki et al. ( ) selected a herpesvirus from turkeys (hvt) that was relatively avirulent in chickens, and zander et al. ( ) and schat and calnek ( ) , selected a natural avirulent strain from chickens. these three vaccines are effective, but the hvt strain has been more widely used because it can be obtained from infected cells and can be lyophilized (calnek et al., ) . the vaccines are given parenterally to chicks at hatching and produce good protection ( - %) against virulent virus challenge (purchase et al., ) . viruses of the family poxviridae infect most domestic animals and man. from the standpoint of immunoprophylaxis, the most important poxviruses are: orthopoxvirus, smallpox (variola), mousepox (ectromelia); avipoxvirus, fowlpox, pigeonpox; capripoxvirus, sheeppox, goatpox; leporipoxvirus, myxomatosis virus; parapoxvirus, contagious pustular dermatitis virus. all these poxviruses cause serious disease in their primary host species and some may infect other species. each of the poxviruses causes characteristic vesicular lesions on the skin and mucous membranes, with the exception of myxomatosis virus which produces hyperplastic lesions in the form of myxomas and fibromas. ectromelia (mousepox) is caused by a virus closely related to vaccinia virus and produces a serious disease of laboratory mice. vaccination with vaccinia apparently will reduce the morbidity and mortality of mousepox in a colony, but it will not prevent infection and may act to maintain a silent reservoir of virus (buller and wallace, ) . sheeppox is one of the most serious pox diseases, occurring in europe, the middle east, and africa, but it is controlled by vaccination (aygun, ; sabban, ) . goatpox occurs mainly in the middle east and africa; a goatpox vaccine has been reported to also immunize against sheeppox (rafyi and ramyar, ) . contageous pustular dermatitis virus is unrelated to sheeppox, but causes a pox-like disease in young lambs in which vesicles form around the skin of lips, nostrils, and eyes. boughton and hardy ( ) showed that animals could be protected by scarification with dried contageous pustullar dermatitis virus material similar to that use with vaccinia. myxomatosis virus causes a fatal disease of domestic rabbits and may be spread by direct contact or by blood meals of insects such as mosquitos and fleas (myers et. al., ) . mckercher and saito ( ) developed a safe and efficacious attenuated live-virus vaccine by passage of the virus in rabbit kidney cell culture. this virus, once the cause of epidemics that decimated entire cities, has now been eradicated. the control was brought about by worldwide vaccination and isolation of infected persons. another factor in the control of smallpox was that variola virus had no other host except man. immunization. material from lesions of smallpox-infected individuals had been used for centuries to infect susceptible persons so they would develop a mild form of the disease and become resistant. this variolation was dangerous but much safer than natural exposure to the smallpox virus. jenner ( ) practiced an improved form of this method by using cowpox virus (vaccinia) to inoculate susceptible persons, as described in chapter . most vaccinia vaccines were produced by scarifying the skin of a calf with infected material and harvesting the lymph from the crusted lesions as aseptically as possible. this was stored in % glycerol to stabilize the virus and preservatives were added to destroy bacteria. sheep and rabbits were also used similarly for vaccine production. vaccinia virus was also adapted to grow in embryonated eggs (goodpasture et al., ) . vaccinia virus is very stable, can be produced at a low cost, and is simple to administer. these factors played a major role in allowing the wide-scale use of vaccinia for the eradication of smallpox. fowlpox virus occurs mainly in chickens and produces pox lesions on the wattles, comb, mouth, nostrils, and eyes. the disease is spread mainly by direct contact with infected birds and blood-sucking parasites such as mosquitoes. although it is a fairly resistant virus, it is not otherwise very transmissible. immunization. fowlpox vaccine was originally made by scarifying cockrel combs with virulent virus and harvesting the exudate. johnson ( ) demonstrated that dried exudate would produce immunity when scarified in the wing web or applied to the thigh skin free of feathers. fowlpox virus was later cultivated on the chorio allontoic membrane by goodpasture et al. ( ) and used as a source of vaccine material. later the virus was adapted to tissue culture. an attenuated live-virus fowlpox vaccine produced in cell culture may be used in -day-old chicks (siccardi, ) . another vaccine, an attenuated strain (hp- ) developed by mayr et al. ( ) , is given orally to -day-old chicks, then repeated after to months. hepadnaviridae is a new-found family of viruses containing hepatitis Β virus of man as well as three similar but distinct viruses that infect woodchucks, beechey ground squirrels, and pekin ducks. these viruses have many of the same ultrastructural, molecular, and biological features and their surface antigens cross-react to a small, variable degree. the host range appears to be specific for each virus. hepatitis Β infects man and certain higher primates, including the chimpanzee and gibbon. infection with these hepadnaviruses results in subacute hepatitis, which often becomes progressive and chronic. the most important of these viruses is hepatitis Β virus, which produces a chronic disease in man (blumberg et al., ; prince, ) . hepatitis Β virus is transmitted by blood, saliva, and semen, but also from mother to offspring, the latter route accounting for as much as one-third of persistent infections. the disease is usually selflimiting, but in - % of patients the infection becomes chronic, with the virus persisting in a carrier state. there are over million chronic carriers of this virus worldwide. a late sequela in chronic carriers is hepatocellular carcinoma. it is estimated that - % of malignancies in africa are the result of hepatitis b-induced oncogenesis. immunization. the development of a vaccine for hepatitis Β was hampered by the difficulty of growing the virus in cell culture. krugman et al. ( ) was the first to show that hepatitis Β virusinfected serum could be heat-inactivated and retain its antigenicity. they also showed that this inactivated serum given parenterally could protect subjects exposed to virulent hepatitis Β virus. this led to the use of plasma from infected but healthy virus carriers as the source of antigen. carriers produce large quantities of the hepatitis Β virus, along with its outer coat protein. by purifying and inactivating the coat protein, a safe and effective vaccine was developed (hilleman et al., ) . the coat protein, naturally formed into -nm particles, was purified by ammonium sulfate concentration, isopycnic banding, rate zonal separation and enzymatic digestion. the purified protein particles were then inactivated with : formalin. the particles induce good levels of protective antibody when given in a series of three injections (symuness et al., ) . however, the high cost of this vaccine has limited its use. newer vaccines produced by recombinant dna methods are now being used, as described in other chapters. the four genera in the family picornaviridae are: aphthovirus, rhinovirus, enterovirus, and cardiovirus. viruses in the first three genera cause important diseases in domestic animals and man, whereas viruses in the fourth infect rodents. the picornaviruses in general have a primary affinity for superficial tissues especially of the digestive and respiratory tracts. viruses in the first three genera also have an ability to mutate, thus yielding many serotypes. rhinoviruses cause clinical disease in man, horses, and cattle. no vaccines have been developed for the infections of humans because of the multiplicity of viral serotypes. the number of serotypes in horses (three) and cattle (two) is fewer; nevertheless, no vaccines are available. over enteroviruses exist; of these, vaccines have been developed only for poliomyelitis viruses, avian encephalomyelitis, and duck hepatitis viruses. other viruses in this group either are of low pathogenicity or the number of serotypes is so large as to preclude the development of vaccines. the exception is human hepatitis a virus, which causes a serious disease and has one serotype; the development of both inactivated virus and attenuated live-virus vaccines is in progress (hilleman et al., ; provost et al., ) . fmd was the first animal disease shown to be caused by a virus (loeffler and frosch, ) . fmd viruses cause one of the most economically important diseases of animals and its control is critical to the world's supply of animal protein. the viruses are widespread and occur in many cattle producing regions of the world. the viruses also affect other cloven-footed animals including sheep, swine, and goats. fmd virus infection produces vesicles on oral mucous membranes, including the tongue, gums, and dental pads, but also on the skin including the interdigital spaces and teats. these vesicles on the mucous membranes coalesce and erupt, leaving large denuded areas. the mucous membrane and skin lesions can incapacitate an animal for weeks, thus severely disrupting its productivity. the viruses are highly contagious and persist for long periods in infected animals. animals that recover from natural infection are immune for approximately one year. vallee and carre ( ) showed that there was more than one fmd virus; seven serotypes with over subtypes have now been identified, making the development of effective vaccines difficult. immunization. the first fmd vaccine for cattle was reported in and consisted of a formalinized emulsion of vesicular epithelium (vallee et al., ) . a similar but improved version called the schmidt-waldmann vaccine followed and contained vesicle material from the tongue epithelium of infected cattle. this material was treated with formalin and used with aluminum hydroxide (schmidt, ; waldmann and klobe, ) . another advance was described by frenkel ( ) , who infected superficial layers of bovine tongue epithelium in culture and inactivated the newly replicated virus with formalin to produce a more uniform product. although this method is used today in some areas of the world, most fmd vaccines are now produced by growing the virus in baby hampster kidney cells (mac-pherson and stoker, ; mowat and chapman, ; capstick et al., ) . the imines replaced formalin as an inactivant in most fmd vaccines after brown et al. ( ) showed that viral inactivation was more complete, and safer vaccines could be produced by this process. all inactivated fmd vaccines contain more than one serotype, including the serotypes most common in the area in which the vaccines are to be used. although inactivated vaccines that are produced and used properly can effectively lead to the control fmd, their stability could be improved, thereby lowering their cost. this is discussed further in the chapter by brown. attenuated live-virus fmd vaccines have been developed (henderson, ) but are not used because of the fear that the virus might persist in animals and in meat and milk products from animals (hyslop, (hyslop, - . there are three polio viruses and minor variants of each. the viruses infect man by entry into the upper alimentary tract, infecting cells, and spreading via the blood to the central nervous system, producing neuronal destruction in the medulla and spinal cord. the degree of paralysis that follows infection depends on such factors as virus strain and virus tropism. the vast majority of persons infected with wild polio viruses show no apparent clinical disease. paralysis occurs only in an estimated % of infected individuals; polio virus is responsible for at least % of cases. immunization. early attempts to develop inactivated poliovirus vaccines were hampered by not knowing that there are three distinct viruses. the differentiation of the three viruses by bodian ( ) and kessel and pait ( ) was a major step toward controlling the disease. enders et al. ( ) found that poliovirus would grow in extraneural tissues of human origin and thus laid the groundwork for the development of poliovirus vaccines. the first vaccine (salk) contained all three polio viruses grown in monkey kidney cell culture and inactivated with formalin (salk et al., ) . this vaccine, introduced in , reduced the incidence of paralytic poliomyelitis - % where it was used; however, multiple doses were required and intestinal tract infection was not prevented, thus allowing the virus to continue to spread. there was an intense effort in the s to develop an attenuated live-virus vaccine that could be administered orally, and could protect the intestinal tract, thus breaking the chain of transmission. koprowski, sabin, and cox each developed vaccine strains of reduced neurovirulence that underwent extensive laboratory and field studies (koprowski et al., ; sabin, ; cox et al., ) . the strains developed by sabin were finally licensed in the united states; they produced rapid immunity as well as protection of the intestinal tract while preventing spread to unvaccinated, susceptible persons in contact with vaccinées. this improved the overall level of immunity in communities. with the widescale use of oral poliomyelitis vaccine, the incidence of paralytic disease in the united states has dropped to less than cases per year. the occasional reaction to the attenuated vaccine is discussed in the chapter by hogle and filman. avian encephalomyelitis was first discribed and shown to have a viral etiology by jones ( jones ( , . the virus is widespread and affects young chickens ( - weeks old). characteristic clinical signs are ataxia and tremors of the head and neck. extensive neuronal degeneration occurs in the anterior horn of the cord and in the medulla and pons. the virus may affect older laying birds, causing a drop in egg production. flocks that have survived an outbreak or subclinical infection during the growing period are resistant to further infection (schaaf and lamoreux, ) . moreover, infected chickens - weeks of age undergo only mild disease, providing an opportunity for vaccination (schaff, ) . calnek and taylor ( ) successfully immunized immature birds with an attentuated live-virus vaccine delivered in drinking water. a number of vaccines have been developed including the strain (calnek, ) , the nsw- strain (westbury and senkovic, ) , the philips duphar strain (folkers et al., ) , and a strain grown in chicken pancreas cell culture (miyamae, ) . inactivated ae virus vaccines have been developed for use in susceptible breeding flocks that are in production (schaaf, ; calnek and taylor, ; butterfield et al., ; macleod, ) . two viruses in the family caliciviridae cause significant disease in animals, vesicular exanthema virus in swine and sea lions and feline calicivirus. caliciviruses have also been isolated from humans, calves, reptiles, nonhuman primates, birds, dogs, and fish, but do not produce significant disease in these animals. vesicular exanthema virus caused a disease in swine closely resembling fmd (traum, ) . eradication of this disease followed the discovery that the main source of contagion was uncooked infected meat in garbage fed to swine, and the consequent enforcement of garbage cooking laws. fastier ( ) first isolated a calicivirus from a domestic cat and showed that it produced an upper respiratory tract infection. a large number of viral isolates, with different neutralization patterns, were made from clinically ill cats (crandell et al., ; bittie et al., ) . these serotypes were later shown to have a common antigen and are now considered a single serotype (povey and ingersoll, ) . this virus is widespread, having been isolated from cats in many countries. the virus produces a disease that is generally mild, but, if allowed to progress, the lesions may extend from the upper respiratory tract into the lung causing pneumonia and death (kahn and gillespie, ) . immunization. cats that recover from natural infection and have neutralizing antibodies can be reinfected, but they do not have recurrent clinical disease. an attenuated live-virus calicivirus vaccine has been prepared by serial passage at low temperature; this vaccine virus produces only mild clinical signs in recipients (bittle and rubic, ) . this attenuated live-virus vaccine is administered parenterally; it induces high levels of neutralizing antibody and protects vaccinated cats challenged intranasally with both homologous and heterologous strains (kahn et al., ; scott, ) . immunity from vaccination persists for at least months and probably longer. the calicivirus is combined with feline rhinotracheitis and feline panleukopenia to make a multivalent vaccine that is routinely used in domestic cats (bittle and rubic, ) . inactivated vaccines have also been licensed and are used in multivalent vaccines. the family reoviridae is divided into three genera: reovirus, rotavirus, and orbivirus. infections cause by member viruses are common in mammals and birds. reoviruses are commonly isolated from dogs, cats, sheep, cattle, horses, mice, rats, rabbits, birds, and man. only in birds is the disease serious enough to warrant control with vaccines. the reoviruses are commonly found in sewage, and the mode of transmission is thought to be the fecal-oral route. avian reovirus. in chickens and turkeys, reoviruses produce a widespread disease called viral arthritis (tenosynovitis). this disease of the synovial membranes, tendon sheaths, and myocardium was first recognized by dalton ( ) and by olsen and solomon ( ) . viral arthritis occurs primarily in meat-producing birds, and in acutely affected flocks there is a high rate of condemnation. there are at least five avian reovirus serotypes, but they are antigenically unrelated to the mammalian reoviruses. immunization. vaccination of breeding stock is an effective way to control viral arthritis. van der heide et al. ( ) used an attenuated live-virus vaccine and cessi and lombardini ( ) used an inactivated vaccine in laying hens to protect chicks with maternal antibody. this eliminated transmission and protected susceptible day-old chicks. rotaviruses produce acute gastroenteritis in many species, especially in newborns, including newborn calves, foals, lambs, piglets, puppies, monkeys, and humans. the clinical signs are similar in all species; in each there is acute diarrhea followed by dehydration and rapid loss of weight. secondary bacterial infection may exaggerate the symptoms and also cause pneumonia. the viruses infect epithelial cells of villi, causing desquamation and loss of absorptive function, resulting in diarrhea, rapid dehydration, and emaciation. secretory antibody is very important in protecting the epithelial surface of the small intestine (snodgrass and wells, ) ; antibody contained in colostrum is protective when in high titer. a. bovine rotavirus. bovine rotovirus causes a rapidly spreading disease in neonatal calves (mebus, ) . the antigenic relationship of the bovine rotavirus and other rotaviruses isolated from children, calves, piglets, mice, and foals is very close (woode et al., ) . immunization. an attenuated live-virus vaccine developed by mebus et al. ( ) is administered in two doses to cows prior to calving. this is meant to stimulate colostral antibody, which is passed on to the nursing calves. this vaccine has also been combined with an attenuated live-virus coronavirus vaccine and entero-toxigenic e. coli vaccine to prevent calf scours. b. porcine rotavirus. leece et al. ( ) isolated a rotavirus from piglets with fatal diarrhea. additional reports of this disease showed that it was widespread and warranted the development of a vaccine for its control. immunization. early attempts to immunize pigs by oral administration of a bovine attenuated live-virus rotavirus vaccine were unsuccessful (leece and king, ) . presently an attenuated porcine rotavirus vaccine containing two serotypes, a and a , is licensed in the united states and is being used in combination with a transmissible gastroenteritis (tge) vaccine. the vaccines are administered to pregnant sows by both parenteral and oral routes. at least two doses of vaccine are given orally, and weeks before farrowing, and one dose is given parenterally week before farrowing. this induces antiviral colostral antibody for the protection of suckling piglets. the member viruses of this genus replicate in arthropods as well as in vertebrates. the most important viruses are the bluetongue viruses, and african horse sickness viruses. colorado tick fever virus, the only virus in this genus that infects man, is transmitted by the bite of infected ticks. the disease is usually benign, and infection produces long-lasting immunity. a. bluetongue virus. bluetongue viruses infect ruminants and are transmitted by culicoides gnats. the most serious disease is in sheep, which develop fever, depression, oral lesions, pneumonia, and lame-ness. mortality can be high, especially in lambs. ewes infected early in gestation may produce lambs with hydrocephalus and other congenital deformities. although cattle rarely have clinical bluetongue disease, in utero transmission can occur, resulting in congenital deformities. of the distinct bluetongue viruses, occur in the united states. infection with one bluetongue virus confers resistance to reinfection with that same virus for several months, but cross-protection against infection with other viruses is minimal. a multivalent attenuated live-virus vaccine developed in south africa by serial passage of several different viruses in sheep proved difficult to standardize. a more uniform vaccine was later developed by alexander et al. ( ) ; it contained strains of at least five viruses, attenuated by passage in chick embryos, and gave broad protection against the multiple viruses seen in the field. a similar vaccine was developed by mckercher et al. ( ) , who isolated bluetongue virus in the united states and also attenuated a strain of serotype by serial passage in chick embryos (mckercher et al., ) . recently mcconnell and livingston ( ) have been attempting to incorporate more bluetongue virus strains into multivalent attenuated live-virus vaccines. inactivated vaccines for bluetongue would have the advantage of greater safety than attenuated live-virus vaccines. they would eliminate the possible chance of reversion to virulence, and the chance of vaccine-associated abortion and birth defects. such vaccines are under development (stott et al., ) . . african horse sickness virus. african horse sickness virus causes an acute disease in equine animals in africa, the middle east, and asia. it was shown to be caused by a virus (mcfadyean, ) and has been more thoroughly characterized by breeze et al. ( ) . the viruses are transmitted to horses by culicoides species and affect principally the vasculature of the respiratory tract causing edema of the lungs, head, and neck. the viruses also cause cardiac lesions. immunization. some animals that recover from natural infection may be reinfected, so immunity is not permanent. a spleen pulp vaccine inactivated with formalin was made by dutoit et al. ( ) and administered in multiple doses. later, an attenuated live-virus vaccine was developed by serial intracerebral passage of a field isolate in mice (alexander and dutoit, ) . however, because there are nine african horse sickness viruses, it has been necessary to adapt each to mice and to combine them in a polyvalent vaccine. such vaccine has been effective in protecting horses. the virus that causes ibd was first isolated by winterfield and hitchner ( ) using embroyonated eggs. it causes a disease of chickens in commercial poultry-producing areas. the virus affects mainly young birds - weeks of age, with clinical signs of diarrhea and dehydration. the lesions arise in lymphoid tissues such as the bursa of fabricius, thymus, and spleen, producing immunosupression with the associated opportunistic infections. immunization. both attenuated live-virus and inactivated vaccines have been developed to control ibd. the vaccines are used mainly for immunizing breeder flocks to confer passive immunity through the yolk sac of the egg. maternal antibody protects chicks for - weeks. if breeder flocks are boosted with oil-adjuvant-inactivated vaccines, maternal antibody may last longer. there are several types of attenuated live-virus vaccines with varying degrees of virulence. these vaccines are administered in water, etc., to chicks from day to - weeks of age in broiler-breeder flocks, followed by vaccination with an inactivated product at approximately - weeks of age (lukert and hitchner, ). the four genera in the family togaviraidae are: alphavirus, pestivirus, rubivirus, and arterivirus. each of these genera contain important pathogens. the alphavirus genera include: all cause encephalitis in horses and humans. in horses, the mortality rate of eee in over %, and that from wee is about - %. the main mode of transmission is by culicine mosquitoes; however, vee has been transmitted from horse to horse by contact with body fluids. immunization. infections with togaviruses produce viremia, longterm humoral antibody responses, and immunity. early vaccines were made from formalin-inactivated infected animal brain tissue. the cultivation of both wee and eee in the chick embryo by higbee and howitt ( ) made possible the development of successful inactivated vaccines (beard et al., ) . more recent vaccines for wee and eee are produced in tissue culture. an attenuated live-virus vee vaccine, first developed for humans, is also used for horses in endemic areas (berge et al., ; mckinney et al., ) . the vaccine virus is grown in primary chick embryo cell cultures; it induces long-lasting immunity. an inactivated vee vaccine has also been developed and is combined with wee and eee vaccines in a trivalent formulation. a. hog cholera (hc) virus. hog cholera virus and bovine virus diarrhea (bvd) virus antigenically are closely related pestiviruses, but are specific in the diseases they cause in swine and cattle, respectively. hc virus produces an acute febrile disease marked by multiple hemorrhages, necrosis, and infarcts in internal organs. lethargy, vomiting, and encephalomyelitis are seen in a high percentage of infected animals during an outbreak and mortality is high. immunization. passive protection with convalescent swine serum from swine has been used effectively for short-term control of outbreaks for many years (dorset et al., ) . antiserum and either virulent or partially attenuated virus strains were also used to establish active immunity. although there is only one antigenic type of hc virus, variant biotypes have arisen that are more difficult to protect against with standard vaccines. the presence of neutralizing antibody correlated well with protection. an inactivated virus vaccine prepared from defibrinated swine blood taken during the acute phase of the disease and treated with crystal violet or phenol was safe, but required multiple doses (mcbryde and cole, ) . attenuation of hc virus was first accomplished by passage in rabbits (baker, ; koprowski et al., ) . tissue culture attenuated live-virus vaccines eventually replaced the rabbit vaccine; the latter produced a rapid and long-lasting immunity. a large number of different attenuated live-virus hc vaccines with different characteristics have been used over the years, but residual pathogenicity, shedding, and spread of vaccine viruses have remained problems. a vaccine containing bvd virus was tested in swine, based on evidence that this virus could block replication of hc virus (sheffy et al., ) . however, the bvd vaccine did not protect against all strains of hc virus (tamoglia et al., ) . formerly, control of hc was difficult because hc virus persists in infected meat scraps fed to swine in uncooked garbage. however, since no vaccines have been used in the united states. by controlling the transport of swine and cooking all garbage used as feed, the disease has been eradicated from the united states, and several european countries. b. bovine virus diarrhea (bvd) virus. bvd virus causes a widespread disease of cattle, especially in young stock. clinical signs, which vary in severity, include scouring, ulcerations of the oral cavity, and abortion. young animals that recover often remain stunted and unproductive for long periods. bvd viral strains differ in their cytopathic effects in tissue culture; cattle infected with noncytopathogenic strains during the first months of gestation can transmit the virus to the fetus, which may be born viremic and immunologically tolerant. later exposure to cytopathogenic strains, naturally or by vaccination, can cause offspring to develop the more severe form of the disease (bolin et al., ) . a cytopathogenic strain of virus isolated from a calf and designated oregon (c v) strain (gillespie et al., ) became less pathogenic for calves after passages in bovine kidney tissue culture . this has been the standard vaccine strain and has been used widely for many years. for cattle never exposed to bvd antigen, this vaccine strain is safe and effective; however, persistently infected cattle may react strongly to vaccination with the cytopathic strain of bvd virus, causing a mucosal disease syndrome. it is important to identify and eliminate persistently infected cattle from herds. rubella virus. in man, rubella virus causes a generally mild exanthematous disease, with malaise and respiratory symptoms. complications include arthritis, thrombocytopenia purpura, and encephalitis. gregg's observation ( ) that rubella virus produces fetal abnormalities if infection occurs early in pregnancy emphasized the destructive effects of this virus and the need to develop a means to protect against infection. immunization. natural exposure to rubella virus evokes nasopharyngeal antibody, which is important in preventing reinfection. antibody, especially igg antibody in mother's plasma, is important in preventing fetal infection. two groups isolated rubella virus in tissue culture (parkman et al., ; weller and nova, ) , allowing the first attempts to develop vaccines. both inactivated and attenuated live-virus vaccines were tried before the latter evolved as superior products. the attenuated live-virus vaccines were developed using different cell culture systems, including monkey kidney (parkman et al., ) , duck embryo (buynak, ), rabbit kidney (peetermans and huygelen, ) , canine kidney (musser and hilsabeck, ) , and human diploid cells (plotkin, ) . the vaccines now being used are more than % effective in inducing protective levels of antibody that persist for at least years. the annual incidence of rubella in the united states has dropped from , reported cases in to less than in . equine arteritis virus. equine arteritis virus was first isolated by doll et al. ( ) . the disease caused by this virus is characterized by edema of limbs, stiffness, and swelling in the tissues surrounding the eye, and abortion. immunization. horses that recover from infection develop longlasting immunity. an effective attenuated live-virus vaccine was developed by serial passage of bucyrus field strain virus in primary equine and rabbit kidney cell culture and an equine dermal cell line (doll et al., ; wilson et al., ; mccollum, ). the vaccine has been shown in challenge trials to protect recipients for as long as months (mccollum, ) ; it does not cause any clinical manifestations and it is not spread to susceptible horses in contact with vaccinated recipients. yellow fever virus causes acute hepatitis and hemorrhagic fever in man, characterized by jaundice, shock, and renal damage. transmission is by mosquitoes belonging to the aedes genus throughout tropical areas of south america and africa. the virus is maintained in a transmission cycle between mosquitoes and monkeys, with man being infected when he enters a territory in which the monkey-mosquito cycle exists. immunization. an attenuated live-virus yellow fever vaccine was developed by passage of the virulent asibi strain in mouse brain and cell culture until it had lost its pathogenicity for monkeys and man (theiler, ) . the vaccine virus, termed d, is propagated in embryonated eggs. the vaccine, given as a single dose, is extremely safe and efficacious, providing immunity for at least years. the family orthomyxovirus comprises the influenza viruses, the cause of acute, highly contagious respiratory disease in man, horses, swine, and birds. two structural viral proteins (nf and m) divide this family into three distinct genera: a, b, and c. the viruses, especially influenza a virus, undergo genetic réassortaient, which allows variant viruses to emerge. two viral proteins, the hemagglutinin and neuraminidase, both located on the surface of virions, are important in inducing immunity. vaccines have been widely used for controlling influenza with reasonably good success. since immunity is more closely related to local secretory iga antibody than to serum antibody, it is difficult to stimulate and maintain protection with the presently used inactivated vaccines. with human infections, the type a viruses undergo occasional antigenic shifts and drifts, after which the antigens in the vaccine may not be representative of those viruses found in the field. this potential antigenic variability, as well as animal reservoirs for this virus, are responsible for the pandemics associated with this virus. as an example, the acute respiratory disease of swine caused by a type a strain of virus was first recognized in during the human influenza epidemic and is believed to have been transmitted from swine from humans. both swine and humans are susceptible to the swine virus. however, the disease in swine occurs sporadically and has not been enough of a problem to warrant the use of vaccines in that species. influenza is a respiratory infection with systemic manifestations that include fever, chills, muscular aches, etc. the severity of the disease depends on the virus strain and the susceptibility of the population. persons that have recovered from an influenza infection are usually immune to rechallenge with the homologous virus. however, the change of a few amino acids in hemagglutinin may give rise to antigenic drift and reinfection of populations. immunization. because of the epidemic threat of influenza viruses, careful surveillance for new strains is carried out in many parts of the world. the strains that public health officials predict will be the cause of the next winter's epidemics are then scheduled for vaccine production. for vaccine production, virus is grown in the allantoic cavity of embryonated eggs and is purified and concentrated by zonal centri-fugation. the virus is inactivated with formalin, ß-propiolactone, or irradiation. the quantity of viral antigen per dose is standardized before use. the vaccine usually contains several type a viruses and a type Β virus. these inactivated whole virus vaccines produce protective levels of antibody in approximately % of primed recipients and in % of the unprimed recipients. antibody levels are maintained for approximately year in primed individuals. attenuated live-virus influenza vaccines have been used extensively in the ussr with varying results. problems of adverse reactions, inconsistent potency, and questionable appropriateness of strains make it difficult to evaluate the effectiveness of these vaccines. more recent attempts to develop attenuated live-virus vaccines involve genetic reassortment, a method that offers considerable promise (reviewed by wright and karzon, ) . influenza in horses resembles the disease in man and swine. the two type a influenza viruses of importance in the horse are: a/equi l/prague/ type and a/equi /miami/ type . the disease spreads rapidly through susceptible horses, and those that recover are protected for only a short time. recovery from infection with one virus type does not provide immunity against the other. immunization. vaccines for equine influenza are produced in essentially the same manner as human influenza vaccines. formalinactivated vaccines contain both equine type and viruses and one of several adjuvants, as described by bryans et al. ( ) . vaccines of this type are widely used and effectively control equine influenza. the family paramyxoviridae contains several viruses that cause significant disease in animals. the family is composed of three genera that include the following viruses for which vaccines have been developed. these viruses are transmitted by the respiratory route and are antigenically rather stable. parainfluenza viruses infect humans, rodents, swine, dogs, and cattle. these viruses, by themselves, cause mild upper respiratory tract disease, but when combined with other viral and bacterial pathogens may cause a more severe syndrome. parainfluenza types , , , and a/ b infect humans, especially young children. type is considered the most pathogenic, causing a bronchitis and pneumonitis. vaccines for parainfluenza of rodents (sendai virus infection), dogs (canine parainfluenza), and cattle (bovine parainfluenza) have been developed. there is no licensed parainfluenza vaccine for man. a. sendai virus. sendai virus, first isolated during attempts to recover human respiratory viruses in mice (kuroya et al., ) , is a parainfluenza type virus that causes respiratory disease in mice, rats, hamsters, and swine. the disease occurs either in an acute-short duration form or a chronic-persistent, clinically inapparent form. spread is by either direct contact or by aerosol. in mouse colonies, this disease is difficult to control because the virus is so highly infective. immunization. formalin-inactivated vaccines have been effective in controlling the disease in mice and rats (fukumi and takeuchi, ; eaton et al, ; tsukui et al, ) . additionally, a temperature-sensitive mutant strain of sendai virus has been used as an aerosol-delivered vaccine in mice. it suppresses virus replication, but the vaccine virus spreads throughout the colony and makes it difficult to monitor for wild virus strains (kimura et al, ) . b. canine parainfluenza virus. outbreaks of mild respiratory disease in laboratory dogs have been attributed to parainfluenza type virus (binn et al, ; crandell et al, ) . when other respiratory agents such as mycoplasma and bordetella bronchiseptica were given intranasally after exposure to this parainfluenza virus, more severe respiratory signs occurred (appel and percy, ) . this encouraged efforts to develop a vaccine for canine parainfluenza virus. immunization. an attenuated live-virus parainfluenza vaccine has been shown to protect dogs against aerosol challenge with virulent virus (emery et al, ) . the vaccine produces no untoward effects and has been combined with canine distemper and canine adenovirus vaccines in multivalent formulations. c. bovine parainfluenza virus. a parainfluenza type virus isolated from cattle can also cause mild respiratory disease (reisinger et al, ) . the virus, when combined with other respiratory pathogens including pasteurella and infectious bovine rhinotracheitis virus, causes the severe pneumonia syndrome, called "shipping fever." immunization. an attenuated live-virus parainfluenza type vaccine administered parenterally induces good levels of antibodies and affords protection against experimental challenge (mohanty and lillie, ; thorsen et al., ) . this vaccine has been combined with infectious bovine rhinotracheitis virus and bovine virus diarrhea vaccines in multivalent formulations. an inactivated vaccine, requiring two inoculations, induces high hemagglutination-inhibition titers and lessens the severity of the disease in cattle challenged with the same virus (gale et al., ) . mumps virus causes an acute infection in man with parotitis as the main clinical manifestation, although the central nervous system and other organs including the testes and ovaries can be affected. mumps virus has a limited host range; in addition to man, only certain monkey species and laboratory rodents can be infected. immunization. recovery from natural infection with mumps virus confers long-term immunity. early experiments with formalininactivated virus derived from infected parotid glands of monkeys showed that monkeys and humans could be immunized (enders et al., ; stokes et al., ) . habel ( ) found that chick embryo grown virus could be inactivated with ultraviolet light or formalin and would induce protection in monkeys. a similar vaccine was later shown to induce protection in man (habel, ; henle et al., ) . poor antibody responses to multiple inoculations of this type of vaccine encouraged the search for a more effective vaccine. a mumps virus strain ( jeryl lynn) has been attenuated by passage in chick embryos (weibel et al., ) . the vaccine is immunogenic in - % of subjects, and neutralizing antibodies persist for at least years. the annual incidence of mumps in the united states has been reduced from , cases in to less then , in by application of this vaccine. as presently used, mumps vaccine is combined with measles and rubella vaccines in a pediatric formulation called mmr. newcastle dibcctse is one of the most serious widespread diseases affecting poultry. the disease was first described by kranevelt ( ) and shortly thereafter by doyle ( ) , who named it after the area in england where an outbreak occurred and showed that its cause was a filterable virus. the disease has several forms causing mainly respiratory, enteric, and central nervous system manifestations. the morbid-ity and mortality vary depending on the virus strain. burnet ( ) described the hemagglutinating property of the virus, which has been very helpful in its quantitation and immunodiagnosis. immunization. a number of attenuated live-virus vaccines have been developed which are widely used to control the disease. the bl strain (hitchner and johnson, ) , the lasota strain (winterfield et al., ) , and the f strain (asplin, ) are used to immunize birds of all ages by different routes, including by addition to drinking water and by spraying. vaccines containing inactivated virus do not produce long-lasting immunity but may be used in certain situations when only short-term immunity is needed, such as when boosting immunity is needed in laying flocks. increasing the antigen content and using oil-emulsion adjuvants improves the quality of these inactivated vaccines (stone et al, ; zanella and marchi, ) . measles is a highly contagious disease of humans, occurring mostly in children, causing exanthemata and sometimes more serious manifestations including encephalomyelitis. the virus has two principal immunogens, the hemagglutinin and the fusion protein (norrby et al., ) . the immunity produced by natural infection is long lasting. immunization. the growth of measles virus in chick embryo fibroblasts by enders and peebles ( ) paved the way for the development of vaccines. a formalin-inactivated measles virus vaccine was shown to induce partial immunity. however, some vaccinated children later exposed to measles virus, either naturally or as attenuated live-virus measles vaccine, developed atypical measles (atypical rashes, edema of hands and feet, and respiratory disease). it was later found that formalin-inactivated vaccine failed to stimulate antibody to the fusion protein: consequently the virus could spread from cell to cell, causing the atypical manifestations of disease (norrby et al., ). an attenuated live-virus vaccine was developed from the edmonston strain of measles virus by passage first in human cell culture, then in the amnionic sac of embryonated hen's eggs, and finally in chick embryo cells (milovanovic et al., ) . this vaccine (enders passage level b) was effective in inducing immunity but produced some adverse effects (katz and enders, ; stokes et al., ) . further attenuation by growth at lower temperature yielded an equally effective vaccine that produced fewer side effects (schwarz, ; hilleman et al., ) . attenuated live-virus measles vaccine has been combined with mumps and rubella vaccines. measles vaccine usage has reduced the incidence of measles in the united states from , cases in to , in . canine distemper virus affects most carnivores, causing respiratory, gastrointestinal, and central nervous system disease. the mortality rate in dogs is about %. dunkin and laidlaw ( ) first described the disease in detail and confirmed the viral etiology proposed earlier by carre ( ) . immunization. dunkin ( , a,b) prepared a vaccine by treating virus derived from spleens of infected dogs with formalin. initial administration of this vaccine, followed weeks later with a small dose of virulent virus, usually produced only a mild disease with solid immunity. this approach was replaced with inactivated virus vaccines, given in multiple doses, which served as the main means of controlling the disease from to . green ( ) serially passaged canine distemper virus in ferrets and produced the first attenuated live-virus vaccine; however, this vaccine caused disease in some dogs. the adaptation of canine distemper virus to the chorioallantoic membrane of embryonated eggs by haig ( ) was a major step in developing an attenuated live-virus vaccine. cabasso and cox ( ) applied this method and after passages showed that the virus lost virulence for ferrets but retained its immunizing property for dogs. rockborn ( ) adapted a strain of canine distemper virus to cell culture, and this method is now widely used to produce attenuated live-virus vaccines. rinderpest is an acute highly contagious disease of ruminants characterized by erosions and necrosis of the intestinal mucosa. the disease is epizootic in parts of africa and asia, causing great losses of cattle and buffalo. immunization. koch in developed one of the first means of immunizing cattle against rinderpest by administering bile from infected cattle. animals that survived were permanently immune. formalin-and chloroform-inactivated vaccines were developed using tissues from infected animals. these vaccines were safe, but required two or three doses and protection lasted less than a year (walker et al., ) . rinderpest virus has been adapted to several foreign hosts, including goats (daubney, ) and rabbits (nakamura et al., ) and has been attenuated by passage in these animals. tissues of these animals have been used to produce vaccines in many countries. however, on continued passage, the seed strains tend to lose their immunogenicity, and vaccines become contaminated with adventitious agents from the foreign host. the kabete strain of rinderpest virus was adapted to grow on the chorioallantoic membrane of embryonated eggs, becoming attenuated for cattle after passages . this vaccine and another containing a lapinized strain of virus adapted to embryonated eggs have been used widely in africa (nakamura and miyamoto, ) . more recently, the kabete strain was adapted to bovine kidney cell culture and after passages became avirulent for cattle. this vaccine strain is safe and efficacious in most cattle breeds (plowright and ferris, ) . protection persists for at least years. over million doses of this vaccine have been used in africa, with good success (maurer, ) . bovine rsv produces a rhinitis and catarrhal bronchiolitis in cattle (mohanty et al., ; jacobs and edington, ; paccaud and jacquier, ) . the virus appears to be widespread having been isolated in europe, the united states, and asia. human and bovine virus are related antigenically (paccaud and jacquier, ) ; however, the cattle virus is not known to infect man. immunization. nasal secretory antibody is protective but the disease may occur in the presence of serum antibody. an inactivated bovine rsv vaccine is combined with vaccines for infectious bovine rhinotracheitis, bovine virus diarrhea, and bovine parainfluenza components in a multivalent formulation. the efficacy of the rsv component in this formulation is unclear. of the viruses in the family rhabdoviridae that cause disease in man and domestic animals, the most important is rabies virus. others include vesicular stomatitis viruses (vsv) and bovine emphemeral fever virus (befv). vsv occurs sporadically and epizootically, affecting horses, cattle and swine in the united states. there is a formalininactivated vaccine (gearhart et al., ) , but it is used rarely. befv is an arthropod-transmitted disease of cattle occurring mainly in africa, but also in asia and australia; it is controlled by immunization with attenuated live-virus vaccines (van der westhuizen, ; inaba et al, ; spradbrow, ; theodoridis et al, ) . rabies is an infection of the central nervous system; the disease can occur in most mammals and is usually fatal. there are only a few documented cases of human survivors. after isolating the virus, pasteur ( ) developed a vaccine for its control. historically rabies virus has been considered as a single serotype. but now shared antigens have been found in other viruses in africa of which two, mokola and duvenhage, may be associated with human disease (shope et al. y ) . mokola virus causes a rabies-like disease in dogs and cats in zimbabwe (foggin, ) . immunization. protection against rabies correlates with sn antibody, which can be assessed by a number of tests. pasteur's classical vaccine, developed from infected spinal cord tissue dried at room temprerature for - days, was given in a series of - inoculations beginning with material dried the longest and progressing through material dried for only days (pasteur ) . even though the last inoculum was virulent enough to cause rabies, the earlier inoculations conferred sufficient immunity to protect the recipients. this method of producing a vaccine was successful in most instances but caused the disease occasionally and was eventually replaced by chemically inactivated vaccines prepared from infected brain tissue. although effective, these vaccines gave rise to undesirable side-effects because they contained a myelin-related encephalitogen present in brains from mature animals. substitution of brain tissue from immature animals such as suckling mice, rats, and rabbits with their lesser myelin antigenic content greatly reduced these post-vaccination reactions. the adaptation of the flury strain of rabies virus to growth in chick embryos led to the development of attenuated live-virus vaccines produced in this tissue (leach and johnson, ; koprowski and cox, ; black, , ) . the growth of rabies virus in tissue culture has further improved rabies vaccines (kissling, ; cabasso et al., ; emery et al., ; brown et al., ; fenje, ; abelseth, ). yet, despite their benefits, the attenuated rabies vaccines occasionally caused rabies, particularly in cats. therefore, suckling mouse brain and tissue culture again became the substrates of choice to produce inactivated rabies virus vaccines for animals. in humans the requirement for a safe substrate is more exacting than in animals. for this reason, duck embryos proved better than brain tissue to produce rabies virus (peck et al., ) . after inactivation with ß-propriolactone (bpl), this virus was an improved product for humans, although the allergenic effects from duck embryo tissue still present a problem. therefore the adaptation of the pm rabies virus strain to human diploid cells and inactivation with bpl (wiktor et al., ) was a further improvement. this vaccine is less reactive and more effective for pre-and post-exposure use in humans than any other yet made (bahmanyar et al., ) . a similar vaccine produced in bhk cells is also beneficial in animals. retroviruses have an rna genome, a portion of which encodes the unique enzyme reverse transcriptase. this enzyme imparts to retroviruses the ability to make rna-directed dna copies of their genome, which can then act as a transposable element and can be integrated into the host cell dna. thus, once a cell is infected, it may escape immune surveillance and destruction and the host animal may be infected for life. the retroviruses thus constitute a considerable challenge to traditional vaccine approaches, as discussed further in the chapters by arlinghaus and by nathanson and gonzalez-scarano. many retroviruses infect mammalian species, from mouse to man. most notable are the c-type retroviruses, including the primate, murine, and feline leukemia viruses, as well as human t-cell leukemia viruses types i and ii; the b-type retroviruses, particularly mouse mammary tumor virus; and the lentiviruses, including caprine infectious anemia virus, visna virus, equine infectious anemia virus, feline immunodeficiency virus, bovine immunodeficiency virus, and human immunodeficiency viruses (hiv- and hiv- ). in recent years, as hiv has become a major threat, massive efforts have been directed to developing an efficacious vaccine. so far, all attempts have met with failure. in fact, there are only two retrovirus vaccines that have been proven effective: a formalin-inactivated whole virus preparation of the primate saids type d retrovirus, which is capable of protecting monkeys from a lethal challenge (marx et al., ) , and the commercially available vaccine for feline leukemia. felv commonly infects cats in urban areas, usually by the oralnasal route. kittens under months of age are particularly susceptible. about % of infected cats develop persistent anemia from which myeloproliferative disease and hypoplastic anemia may follow. the immunosuppression caused by felv infection may predispose to severe chronic opportunistic infections. because cats that develop neutralizing antibody are usually immune to infection, vaccines have been developed and tested with that goal in mind. immunization. the problem in developing a vaccine for feline leukemia was to find immunogens that could be used without exposing animals to oncogenic materials. early studies with inactivated whole virus were unsuccessful (yohn et al., ) . although attenuated live-virus vaccines induce sufficiently high levels of neutralizing antibodies to be protective ( jarrett et al., ; pedersen et al., ) , their oncogenic potential makes them unacceptable. efforts to develop vaccines containing only viral proteins, such as envelope protein, have had variable results. however, cultivation of felv in fl transformed cells, followed by treatment to release viral and cell proteins, yields a vaccine that stimulates antibodies to both viral and cell membrane components. a commercial vaccine using this method of antigen production has been approved for use in the united states; it is based on studies done by olsen and lewis ( ) . subsequently, the efficacy of this vaccine has been disputed (pederson and ott, ) . viruses in the family coronaviridae cause important diseases including avian infectious bronchitis, transmissible gastroenteritis of swine (tge), feline infectious peritonitis (fip), and human coronavirus infections. other coronaviruses may cause disease in calves, dogs, mice, rats, turkeys, horses, and parrots, but the diseases are of less importance. coronavirus diseases usually follow a similar pattern, except for fip. fip is a chronic debilitating disease manifested as fibrinous peritonitis and pleuritis. the infection may be inapparent, but is fatal in a small proportion of infected cats. the immune response to fip virus seems to mediate the disease; the immune response is not protective and antibody levels are higher in diseased animals. immune complexes have also been demonstrated in renal glomeruli of cats with fip. bovine coronavirus causes acute diarrheal disease in neonatal calves (mebus et al., ). an attenuated live-virus vaccine is being used in combination with an attenuated live-virus rotavirus vaccine to control calf diarrhea. the vaccine is administered to pregnant cows near the end of gestation and stimulates colostral antibodies that offer protection to nursing calves. with the exception of avian infectious bronchitis, most coronavirus infections have been difficult to control with vaccines. perhaps this is because primary lesions are in mucous membranes of the respiratory and gastrointestinal tracts, sequestered from immune reactivity. coronaviruses produce about % of common colds in man, second only to rhino viruses. there are two groups of human coronaviruses that are antigenically distinct. ibv is a highly contagious respiratory infection of young chickens. the virus may also infect older birds, causing a decrease in egg production. the disease was first shown to be caused by a virus by bushnell and brandley ( ) . beaudette and hudson ( ) propagated the virus in chick embryos, making possible the quantification of the virus and the means for attenuation. there are a number of serotypes of ibv, making the development of an effective vaccine difficult. immunization. immunity following natural infection may last up to year, depending on the serotype and the severity of challenge. van roekel et al. ( ) first developed an immunization procedure; he used a field strain of virus to infect -to -week-old birds before they start to lay, inoculating a few of the birds and allowing infection to spread naturally through the flock. today, there are a number of attenuated live-virus vaccines licensed in the united states. there is good protection ( - %) against homologous virus strains and about % against heterologous strains (hofstad, ) . reduced pathogenicity may be associated with reduced immunogenicity, so a balance must be maintained. the attenuated live-virus vaccines are administered by the usual labor-saving devices of spraying, dusting, or placing the vaccine in drinking water. the wide-scale use of ibv vaccines has significantly reduced the economic loss caused by this disease. tge is an often fatal disease of pigs under weeks of age. the main lesion is enteritis, resulting in malabsorption, diarrhea, and dehydration. tge virus is serologically related to fip virus, but the diseases have entirely different characteristics. there is one serotype of tge virus and one serotype of fip virus. high levels of maternal tge antibody in sows' colostrum protect piglets if fed continuously. immunity of this type has been produced by feeding sows tissues containing virulent tge virus several weeks prior to gestation. the effects of this virus are relatively mild in older animals. attenuated live-virus vaccines administered parenterally to pregnant swine in the latter part of the gestation period produce colostral antibodies. apparently, in sows previously exposed to tge, this vaccine produces sufficient immune responsiveness to be of value. the vaccine is also used orally in pigs - days old to induce local immunity. the effectiveness of this use of the vaccine has not been thoroughly demonstrated. the genus corynebacterium is a heterogeneous grouping with its species placed together largely on the basis of similar cell wall components (goodfellow and minnikin, ) . these species share a basic cell wall chemistry (barksdale, ) of which the mycolic acids (silva and ioneda, ) , especially trehalose dimycolate, are frequently used as potent adjuvants in immunization protocols. two corynebacteria-c. pyogenes and c. pseudotuberculosis-are important in veterinary medicine. the former is frequently associated with ruminate suppurative conditions and abscesses, but it rarely affects man. infections with this organism are sporadic, because it is an opportunist that gains entry through wounds and abrasions. it may also be seen as a secondary invader in devitalized tissues; e.g., vaccination site abscesses. the efficacy of vaccines, toxoids, and antisera against c. pyogenes is equivocal; little is known about immunity to the bacterium. corynebacterium pseudotuberculosis causes caseous lymphadenitis of goats and sheep. it is recognized as a worldwide problem and a serious cause of economic loss to the goat industry (burrell, ; ashfaq and campbell, ) . as with c. pyogenes, the responsible bacterium, c. pseudotuberculosis, is primarily an opportunist entering wounds or abrasions, where it causes local inflammation before settling in the regional lymph nodes. immunization. cell-mediated immunity is necessary for acquired resistance and protection against c. pseudotuberculosis (jolly, ; tashjian and campbell, ; irwin and knight, ) . killed and autogenous vaccines and a toxoid vaccine have been used in attempts to immunize against the bacterium (cameron, ; brogden et al., ; nairn et al., ; burrell, ; anderson and nairn, ; brown et al., ) ; however no one vaccine has proven highly efficacious. diphtheria, characterized by the formation of a tightly adherent pseudomembrane on the pharyngeal mucous membranes of the throat and trachea, is a highly contagious disease of man caused by the bacterium c. diphtheriae. the bacterium can also be isolated from the pharyngeal mucosa of normal individuals. the organism produces a lethal protein exotoxin (gill and pappenheimer, ; collier and kandel, ) . immunization. successful immunization against c. diphtheriae actually protects against the diphtherial exotoxin. because diphtheria toxin is produced in high yield by the park-williams number strain (pw ), pw is used to make diphtherial toxoid for vaccines. as a source of toxin it is rendered nontoxic by incubation with formalin under alkaline conditions. the product's retention of antigenicity, enabling it to induce antitoxin antibodies, makes it an excellent pediatric vaccine. it is commonly utilized in combination with antigens from c. tetani and b. pertussis. the most important species of the bacillus genus, b. anthracis, is the organism responsible for the disease anthrax in both man and animals. anthrax was the first bacterial disease ever to be reported, being described by davaine in . koch in reproduced the disease via animal inoculation and in pasteur successfully vaccinated against anthrax. in animals, natural infection usually occurs by ingestion of spores that germinate in the mucosa of the esophagus or the intestinal tract. herbivorous animals, especially cattle, horses, sheep, and goats, are highly susceptible to the disease, usually the result of grazing in infected pastures or consuming infected foods. in man, anthrax is manifested in three forms: cutaneous (malignant carbuncle), pulmonary (woolsorter's disease), and gastrointestinal with cutaneous being the most common. death results from the combined effects of an extracellular toxic protein complex (vodkin and leppla, ) comprised of three components: edema factor, protective antigen, and lethal factor (leppla, ; stephen, ) . effective vaccines require all three components. immunization. the attenuated pasteur vaccine has been supplanted in veterinary medicine by stable spore vaccines, carbo-zoo vaccine, or stern vaccine (jackson et al., ) prepared from avirulent, nonencapsulated variants of b. anthracis. the viable bacterial spores are suspended in % saponin. immunity is attributed to the development of antibodies to the toxins released from growing bacteria. vaccines of killed bacteria provide little immunity, since no bacterial toxins are produced; hence, no antitoxin antibodies are generated. purified protective antigen (complex toxin) is both antigenic and immunogenic and has been used as a vaccine for humans. it is prepared by aluminum potassium sulfate precipitation of sterile b. anthracis culture filtrates and has proven highly efficacious. erysipelothrix is found in soil, water, and decaying vegetative material and carcasses. the major species of interest is e. rhusiopathiae, which has serotypes (norrung, ) . the bacterium, most notable for causing swine erysipelas, is capable of invading the tissues of both man and animal. fatally affected animals develop welt-like, discolored cutaneous lesions, and numerous hemorrhagic lesions in thoracic and abominai viscera; chronic debilitating arthritis predominates in surviving animals. immunization. the principal vaccines used to control erysipelas are formalin-killed, alum-adsorbed, whole-cell culture vaccines. these combinations of soluble bacterial glycoprotein and whole killed bacterial cells are usually produced from strains of serotype , which possess highly antigenic soluble cell wall glycoproteins. animals immunized with cell-free culture fluids develop agglutinins to the whole bacteria (white and verway, ) . such vaccines are highly effective in controlling swine erysipelas. the pathogenic clostridia invade both man and many animal species of veterinary interest, in which they cause such diseases as tetanus (c. tetani), gas gangrene (c. perfringens, c. septicum, c. oedematous), botulism (c. botulinum), enterotoxemia, and dysentery (c. perfringens). the clostridia are widely distributed in soil and water and are common inhabitants of the intestinal tracts of animals and humans. additionally, the bacteria can often be isolated from infected wounds. vaccination is not routinely practiced against all clostridial organisms, notably c. botulinum. the toxins of c. botulinum, which exert their effects upon the nervous system (schantz and sugiyama, ) , are as potent as those of c. tetani. the lethal dose of the toxin, however, is less than that required to induce an antibody response. clostridium perfringens has five serotypes, a-e, classified according to the production of lethal exotoxins. types a and c are pathogenic for man, whereas all five serotypes can affect animals (see table i ). immunization. the exotoxins of c. perfringens are antigenic proteins that can be detoxified for use in vaccines. the existence of common capsular antigens, which elicit cross-reactions between the serotypes, demonstrates the considerable heterogeneity of this group. ewes and lambs are frequently vaccinated against c. perfringens enterotoxemias. effective vaccines employ type-specific alum- precipitation or formalinized toxoids (smith and matsuoka, ; kennedy et al, ) . clostridium tetani elaborates potent neurotropic exotoxins (tetanospasmin and tetanolysin) that may be lethal for susceptible species such as man, horses, mules, swine, cattle, and sheep. birds are not naturally susceptible to the bacterium. tetanus toxin is one of the most poisonous toxins known. it acts only on the nervous system and its effect characteristically causes spastic paralysis and generalized convulsions. immunization. protective antitoxin blood levels are obtained by immunizing both humans and susceptible animals with alumprecipitated or absorbed tetanus toxoid (chodnik et al., ) . ramon and lemetayer ( ) first introduced the concept of active immunization against tetanus when they used formalinized tetanus toxoid precipitated with alum to vaccinate horses. clostridium tetani vaccines are very effective at inducing long-lasting immunity in both man and domestic animal species. a serious, often fatal disease has been successfully controlled with these vaccines. clostridium novyi possesses four antigenic types, a, b, c, and d; type a is the most common clinical pathogen. types a and Β are responsible for gas gangrene both in man and animals (elder and miles, ) . in areas where sheep simultaneously carry a heavy liver-fluke infestation, exposure to c. novyi is often associated wtih hepatic necrosis and subcutaneous edema. migrating flukes produce foci of hepatic necrosis suitable for the germination of spores and the subsequent elaboration of lethal toxins (williams, ) . immunization. effective vaccinations for c. novyi in animals employ chemically inactivated, detoxified, and adjuvanted suspensions of alum-precipitated formalinized whole broth cultures. clostridium chauvoei, which is the etiologic agent for the disease "blackleg" and is pathogenic for animals only, occurs primarily in ruminant species. protective antigens and toxins with hemolytic and necrotizing activity are formed in susceptible animals ( jayaraman et al., ) . the necrotizing toxin may effect fatal toxemia with degenerative foci of myonecrosis. immunization. immunity to c. chauvoei can be produced via vaccination with its alum-precipitated formalinized cultures (chandler and gulasekhuram, ) . clostridium septicum, in contrast to c. chauvoei, is pathogenic for both man and animal. in man, it is associated with gas gangrene and in affected animals, primarily ruminants, it is the agent most closely identified with the diseases malignant edema and braxy. the organism produces four lethal necrotizing, hemolyzing toxins that cause an increase in capillary permeability and myonecrosis. immunization. immunity to c. septicum is induced with injection of formalinized bacterial cultures. the antitoxin provides homologous protection and additionally protects against c. chauvoei. animals are often vaccinated with mixtures of clostridial species; i.e., novyi, chauvoei, septicum, perfringens, and sordelli in one combination vaccine. these are highly efficacious vaccines and are routinely used in veterinary medicine. although infections with mycobacterium tuberculosis primarily occur through inhalation of the tubercle bacillus, ingestion of large numbers of the bacilli in contaminated milk or infectious sputum can readily produce disease in susceptible species. the bacterium is pathogenic in man, but can also cause disease in monkeys, pigs, and occasionally in cattle, dogs, and parrots. the disease may be asymptomatic or produce severe, debilitating pulmonary lesions. if infection is not restricted by the immune system, the disease may be fatal (comstock, ; bloom and godal, ) . since bacteriocidal mechanisms of the normal macrophage prevent m. tuberculosis from multiplying intercellularly (goren, ; goren et al., ) , protective immunity depends on cell-mediated immunity (lagrange, ) . mycobacterium bovis, closely related to m. tuberculosis, and m. avium causes disease primarily in cattle and birds. they can, however, be contagious to man, sheep, and pigs. immunization. immunoprophylaxis for tuberculosis is based on vaccination with an attenuated, relatively avirulent strain of m. bovis that does not produce lesions. the strain is known as bcg or the bacillus of guérin ( , ) . worldwide, this is one of the most widely used human vaccines, as it has proven efficacious in controlling a severe disease. additionally, bcg has been used for nonspecific enhancement of resistance against tumors and other infec-tions. the cell wall of m. tuberculosis is a potent immunostimulant when used in freund's adjuvant. although streptococci may be normal inhabitants of the gastrointestinal tract, they may also be pathogenic for both man and animals. on the basis of characteristic cell wall components, the streptococci are traditionally divided into lancefield groupings (lancefield, ) . streptococcus agalactiae, a streptococcus group Β organism, causes severe mastitis in the bovine species and has been identified as a major cause of serious neonatal infections in man (eickoff et al., ) . capsular antigens form the four major type-specific antigens (la, lb, ii, and iii), with type iii organisms being most commonly associated with neonatal meningitis. in infants, early-onset disease occurs within the first days of life and is characterized by sepsis and pneumonia. the mortality rate is high. late-onset disease occurs around month of age and is characterized by meningitis (einstein et al., ) . immunization. there is a direct correlation between the absence of maternal igg antibody to type iii antigen and the incidence of neonatal infection. thus, susceptibility to the bacterium is related to the absence of significant levels of maternal serum antibody being transferred transplacental^ to the fetus. current vaccine developments are directed toward maternal immunization with type iii antigen (einstein et al., ) . streptococcus pneumoniae, the etiologic agent of pneumococcal pneumonia in human infants and adults, may cause septicemia, meningitis, and inner ear infections. aerosol transmission of the bacterium, often in association with viral upper respiratory infections, is the major mode of transmitting s. pneumoniae infections. streptococcus pneumoniae possesses a capsular polysaccharide capable of deterring phagocytosis, thus enhancing the virulence of the bacterium. of over types of the bacterium identified, serotypes are most frequently associated with the disease. a polyvalent pneumococcal vaccine prepared from soluble purified capsular polysaccharides of the most predominant s. pneumoniae serotypes has proven effective in adults. the capsular polysaccharides are well-tolerated and highly immunogenic; signifi-cant rises in protective serum antibody titers are achieved following vaccination (kasper et al., ) . however, vaccination of infants has not proven beneficial, because they develop no higher antibody titers to the bacteria than do unvaccinated infants (ginsburg, ) . enterotoxigenic pathogenic strains of escherichia coli may cause severe, potentially fatal, diarrheal disease in both man and domestic species, particularly neonatal cattle and swine. the capsular (k) antigens are cell-surface proteins and/or polysaccharides associated with virulence. the k antigen mediates adhesion to the microvillus of intestinal epithelial cells; production and release of enterotoxin follow (bywater, ; lonroth et al., ) . escherichia coli neonatal enteritis of newborn calves is also a serotype-specific disease (myers and guinée, ) . ail important colostral antibodies in both swine and cattle are anti-k antibodies (usually k ) (myers and guinée, ; moon et ah, ) . immunization. vaccination of gilts, sows, heifers, and cows with vaccines prepared from the k or other pilus-associated antigens has reduced morbidity and mortality from e. coli nec natal enteritis of newborn piglets and calves (nagy et ah, ; rutter, ; kohler et ah, ; childrow and porter, ; myers and guinée, ) . to prepare the porcine vaccines, bacterial strains specific for the herds to be vaccinated are used to immunize animals weeks prior to parturition, thereby generating specific, protective colostral antibodies. recombinant dna technology, discussed in the chapter by collett has introduced the potential to construct e. coli vaccine strains that would afford considerably better protection than those currently available. salmonella species are a major cause of invasive enteric infections in humans and domestic animals, with domestic poultry constituting the largest reservoir of salmonella organisms in nature. normally, infection occurs through the oral route. salmonella is a facultative intracellular pathogen; therefore, cell-mediated immunity is more important than humoral immunity in resistance to the disease, salmonellosis (fields et ah, a,b; dougan et ah, ; woolcock, ) . salmonella typhi, the only salmonella species that has a capsular polysaccharide (vi antigen), is the etiologic agent of typhoid fever, a serious and common disease in underdeveloped areas (edelman and levine, ) . this pathogen infects humans only; there is no suitable animal model for typhoid fever. immunization. few vaccines have been developed for salmonella, and most are of low efficacy with undesirable side-effects. live vaccines are more effective than killed ones in promoting better immunity (levine et ah, ; dougan et ah, ; roantree, ) . with respect to s. typhi, vaccines containing the inactivated bacteria offer only limited and transient protection with undesirable side-effects (levine, et ah, ) . the attenuated strain of s. typhi, ty- a, requires multiple doses to achieve - % protection (hirschel et ah, ) . consequently, typhoid fever has not been controlled by immunization, although the vi antigen has recently been hailed as the agent of a preventative vaccine (robbins and robbins, ) without adverse side-effects (acharya et ah, ) . yersinia pestis is the etiologic agent of plague or "black death" in man, a highly fatal disease with fever and purulent lymphadenopathy. although not a disease of domestic animals, rats, ground squirrels, and other rodents may be affected. the bacterium is spread by the rat flea, xenopsylla cheopis, from rat to rat and from rat to man. immunization. the most widely used vaccine for the prevention of y. pestis infection is haffkine's vaccine, first developed in . this vaccine is prepared from heat and phenol-killed virulent cultures. formalin-killed virulent bacteria are also successful, as are living avirulent strains (grasset, ) . the is no evidence that any vaccine protects against pneumonic plague, the most contagious and fatal form of the disease. furthermore, vaccine protection is only recommended for plague research workers. disease control is primarily dependent upon eradication of rodent carriers of y. pestis. pasteurella multocida and p. haemolytica are common commensals of the mucous membranes of the respiratory tract and oropharynx of healthy cattle, sheep, swine, dogs, and cats. when the bacteria multiply unchecked, they can penetrate the oral and/or respiratory mucosa, where they quickly grow and overpower the host's defense systems. pasteur first described this bacterium as the etiologic agent of fowl cholera; it is also associated with bovine pneumonia, swine plague, and an epizootic hemorrhagic septicemia in ungulates. the bacterium's heat-stable antigens have been used as serologic indicators in the gel diffusion precipitin test to define its serotypes (brogden et al., ) . immunization. pasteur's successful bacterial vaccine to fowl cholera, and the first vaccine ever used, consisted of avirulent cultures of the p. multocida attenuated by prolonged growth on artificial medium. killed p. multocida vaccines are prepared from virulent immunogenic strains of the bacterium. the organisms are suspended in formalinized saline, incorporated into an adjuvant, and injected subcutaneously (heddleston et al., ) . these vaccines induce substantial immunity to fowl cholera. additionally, live vaccines for oral administration have been developed for use in the poultry industry (dougherty et al., ; heddleston et al., ; oison, ; bierer and derieux, ) . pasteurella multocida is usually mixed with modified live or killed bovine rhinotracheitis virus, parainfluenza virus, bovine viral diarrhea virus, and p. hemolytica bacteria in combination vaccines to protect against pasteurella pneumonia in cattle. bovine pneumonic pasteurellosis (shipping fever) is a severe fibrinous pneumonia of feedlot cattle usually associated with biotype a, serotype infections with this organism. immunization. administration of either killed or live vaccines has been of limited efficacy in controlling shipping fever. partial protection from experimental disease follows immunization of cattle with either live p. haemolytica by aerosol or parenteral routes confer et al., ) or lyophilized p. haemolytica vaccines consisting of chemically altered, streptomycin-dependent, or modified live organisms given intramuscularly or intradermally (confer et al., ) . humoral antibody responses appear to correlate strongly with protection against experimental disease (confer etat., ; mckinney et al., ) . for example, purified p. haemolytica lipopolysaccharide stimulates specific antibody formation and has protected calves challenged with the bacterium from developing the disease (hilwig et al., ) . these obligate parasites, restricted to respiratory and pharyngeal mucous membranes, cause important diseases in porcine (h. pleuropneumonia, h. suis, andi/. parasuis), equine (h. equigenitalis), bovine (h. somuns), and avian (h. gallinarum, h. paragallinarum) hosts. most hemophilus species require two factors, hemin (x) and nicotinamide adenine dinucleotide (v), for growth. antigens associated with protection and virulence have been described fori/, paragallinarum (yamamoto, ) , the etiologic agent of infectious coryza in chickens. birds that have recovered from natural infection possess varying degrees of immunity to re-exposure (page et al., ) ; immunity is serotype-specific. adjuvanted vaccines containing multiple bacterial serotypes are prepared from chicken embryos or formalinized bacterial broth cultures and are effective vaccines in preventing infectious coryza in chickens. this bacterium is the etiologic agent of the porcine disease, contagious pleuropneumonia, which is characterized by severe multifocal, necrotizing pneumonia with venous thrombosis and associated serofibrinous pleuritis (didier et al., ) . the disease is of considerable economic importance to the swine industry, being most prevalent in situations where swine are raised under intensive management conditions. hemophilus pleuropneumoniae possesses major and minor antigens that are both common and serotype specific (gunnarson et al., ; gunnarson, ; mittal et al., ) . since high antibody titer apparently provides little protection from the disease, cell-mediated immunity may be important in protection from infection (rapp and ross, ; rosendal et al., ) . immunization. no adequate immunoprophylaxis against contagious pleuropneumonia is currently available, although many vaccines have been tried (nielsen, ; henry and marstellar, ; christensen, ; masson et al., ) . prior infection with one serotype provides protection from heterologous serotypes (nielsen, ) . the bacterial strains used in vaccines are serotype specific and, while not preventing the disease, can reduce its severity (christensen, ) . hemophilus somnus is the cause of infectious meningoencephalitis, a disease with low morbidity but high mortality in cattle. whole or sonicated bacterial cells and bacterial protein are immunogenic (noyer et al., ) and efficacious bacterins foster protective immunity in calves (williams et al., ) . the bacterins of h. somnus, adjuvanted with aluminum hydroxide, are prepared from highly immunogenic strains of the bacterium and grown in serum-free media for use as vaccines. the species in this genera, b. pertussis, b. bronchiseptica, and b. parapertussis, can be either parasites or, as in swine and dogs, common inhabitants of the upper respiratory tract. these small, serologically related bacilli produce a dermonecrotic toxin. infection is by aerosol transmission with bacteria adherent to tracheal cilia (bemis et al., ) . local, not serum, antibody concentration is important in clearance of the infection. the etiologic agent of whooping cough, Β. pertussis, produces two distinct hemagglutinins, leukocytosis-promoting factor-hemagglutinin (lpf-ha) and filamentous hemagglutinin (fha), and various toxins (pertussis toxin [pt] and dermonecrotic toxin). fha is involved in bacterial adherence to the respiratory mucosa, whereas pt is believed to be the major protective (sato and sato, ) and pathogenic antigen (steinman et al., ) . immunization. although efficacious, the safety of the human vaccines currently in use, suspensions of killed whole cells containing protective antigens, is open to question (robinson et al., ) . undesirable side-effects such as screaming, collapse, encephalopathy, and other serious neurological complications have been reported in association with b. pertussis vaccinations (dick, ) . the potencies of whole cell vaccines correlate with the antigenic content of pt (reiser and germanier, ) . bordetella bronchiseptica is an obligate parasite of the upper respiratory tract of both dogs and pigs. in dogs the bacterium frequently invades the lungs as a sequela to canine distemper (caused by a morbillivirus), causing an often fatal bronchopneumonia. the bacterium is also associated with mild to severe tracheobronchitis, "kennel cough," in dogs (bemis et al., ) . in pigs, a deformation of the bony structures of the nasal area (atrophic rhinitis) and reduction of the total volume of nasal turbinates commonly follow the bacterial infection (ross et al., ) . degenerative changes in the osteoblasts and osteocytes may be caused by elaboration of a dermonecrotic toxin (dnt), which is released from b. bronchiseptica after colonization or multiplication of the organisms on the nasal mucosa (nakai et al., ) . the release of dnt from p. multocida type d is thought to exacerbate the disease. immunization. vaccines to control canine b. bronchiseptica infections are commonly incorporated into combination packages containing attenuated live-virus canine distemper, and canine adeno and parainfluenza viruses. to control atrophic rhinitis, avirulent live, or inactivated organisms alone or in combination with p. multocida, erysipelothrix rhusiopathiae and!?, coli have been utilized in vaccines. the organisms in this group, b. abortus, b. suis, Β. melitensis, Β. canis, and b. ovis, cause the disease brucellosis in domestic animals and man. the bacterium may localize in the reproductive tract which, in the female, can lead to fetal death with subsequent abortion. brucellosis, due to b. abortus or b. melitensis, is a zoonotic disease, readily transmitted from animal to man. the potentially severe consequences of brucellosis, fetal death and abortion, in the pregnant cow and epididymitis and sterility in the bull result in significant economic loss to the cattle industry. the primary source of infection is infected animals, whose mammary and/or genital secretions may contain the bacterium. calves can become infected in utero; however, the main portals of infection are oral mucosa, nasopharynx, and conjuctiva of exposed animals. immunity to b. abortus is dependent upon cell-mediated immunity, as the presence of serum antibodies, although a significant indicator of infection, does not correlate with the immune status of the host (fitzgeorge et al., ; kaneene, et al., ; swiderska et al., ; montaraz and winter, ) . most humans who contract brucellosis have been exposed either to b. melitensis, the etiologic agent of malta or mediterranean fever, or b. abortus. brucella melitensis, found in the milk of infected sheep and goats, may produce fatal disease when ingested by humans. brucellosis of sheep and goats mimics the disease as it is seen in cattle, with fetal death and abortion occurring in ewes and does and epididymitis in rams and billies. brucella ovis infects sheep, causing late fetal death and abortion in pregnant ewes and epididymitis in rams, such as b. abortus does in cattle. immunization. brucella abortus (strain ) is currently used as the vaccine of choice for control of brucellosis of cattle in the united states. this is a viable, smooth strain that, while posing virtually no threat for cattle, may cause disease in man. the major objections to the vaccine are this pathogenicity for humans and the difficulty of differentiating vaccinated from naturally infected animals since persistent serum antibodies are induced by the vaccine. killed b. melitensis in adjuvant or live avirulent strains have been used for vaccines to induce a high degree of immunity in sheep and goats. brucella ovis bacterins in adjuvant, as well as b. melitensis vaccines, have been used to protect animals from the disease b. ovis causes, since the antigens of these two pathogens are cross-protective (diaz et al., ) . pathogenic members of this genus are associated with venereal disease, fetal death, and abortion in cattle. this bacterium is transmitted to uninfected cattle by coitus or artificial insemination and is an obligate parasite of the genital tract. immunization. stimulation of opsonizing antibodies of the igg class by systemic vaccination with adjuvanted vaccines is effective in preventing natural infection in bulls (bouters et al., ) and infertility in cows (corbeil et al., ; hoerlin and kramer, ) , and prevents the carrier state (wilkie and winter, ). unlike c. fetus venerealis, this bacterium is contracted by ingestion and is not transmitted venereally. both sheep and cattle can be infected; however, the disease is most severe in pregnant ewes, which undergo a high percentage of abortions or premature births within a flock. immunization. in sheep, vaccination with polyvalent adjuvanted vaccines is efficacious in preventing disease (thompson and gilmour, ) . the most important organism of this genus, v. cholera, causes severe, acute enteritis in humans and nonhuman primates. the etiologic agent of cholera in humans, v. cholera, causes a potentially fatal diarrheal disease. infection results in the production of a powerful enterotoxin in the small intestine, which stimulates an increase in cyclic amp in intestinal epithelial cells and causes a profuse outpouring of isotonic fluids. vibrio possesses immunogenic heat-labile flagellar (h) protein and heat-stable (o) lipopolysaccharide somatic antigens. the cholera toxin is immunologically and functionally similar to the heat-labile enterotoxin of e. coli (yamamoto et al., ) . it consists of six light subunits (l) that assist in toxin adherence to intestinal cell receptors and one heavy subunit (h), which is the toxic entity. immunization. present cholera vaccines are administered by parenteral or oral routes. parenteral vaccines consist of formalin/phenolinactivated bacteria, whereas oral vaccines employ killed or live bacteria. vaccines given by either route provide protection for approximately - months. the predominant immune mechanism is antibacterial rather than antitoxic (levine et al., ) ; antibacterial antibodies prevent attachment of the bacterium, whereas antitoxic antibodies inhibit toxin adherence to cell receptors. because current vaccines often produce adverse side-effects (feeley, ) , synthetic and semi-synthetic vaccines are currently under investigation. for the latter, a nonpyrogenic, bivalent cell-surface protein-polysaccharide conjugate is being investigated (kabir, ) . h. leptospira spp. the pathogenic genera of this family can penetrate the gastrointestinal mucosa and abraded epidermis. leptospires are transmitted through contact with the urine of animal carriers, either directly or in contaminated water or soil. rodents are the primary reservoir of the bacteria which, due to its ability to synthesize urease, colonizes the renal nephron and subsequently is shed into the urine. leptospirosis causes economically serious disease in cattle and swine by causing fetal death, abortion, and infertility. recovery from infection with one serotype lends immunity only to that serotype. this immunity is predominantly humoral, since agglutinins (igm) are responsible for the initial clearance of the bacteria; neutralizing antibodies (igg) are also protective (hanson, ; negi et al., ) . canine leptospirosis infections may be severe, occurring more commonly in male dogs than in females. man is a dead-end host for leptospires; infection in man is accidental and usually related to occupational exposure. immunization. killed, multivalent, leprospira vaccines protect against clinical disease in cattle and swine; however, in pigs, immunity does not protect against renal colonization (stalheim, ) . dogs can be vaccinated successfully with formalin or phenol-killed vaccines that contain antigens from the two most common infecting serotypes, l. canicola and l. icterohemorrhagica (kerr and marshall, ) . vaccines for humans, prepared from chemically inactivated cells of leptospires, have been used extensively in certain areas of the world. neisseria meningitidis neisseria meningitidis is a frequent cause of endemic purulent meningitis in human infants and adults, although the incidence of the disease is substantially higher in young infants (hoffman, ) . bacterial invasion of the meninges is usually hematogenous from the upper respiratory tract and is a life-threatening affliction. neisseria meningitidis has been classified into at least nine groups (a, b, c, w- , x, y, z, l, -e) on the basis of its capsular polysaccharides (morse, ) . immunization. protection against meningococcus meningitis results primarily from the presence of antibodies against the capsular polysaccharide of n. meningitidis (frasch et al., ) . group a and c polysaccharide vaccines are especially effective against disease in children over two years of age and in adults. epidemic typhus fever has afflicted mankind since ancient times. it is an acute highly infectious disease with the potential for explosive epidemics in man. significant outbreaks of the disease have been intimately associated with war and famine. the disease is characterized by sustained high fevers, headache, panencephalitis, a diffuse maculopapular skin rash, and toxic vascular damage. the fatality rate may be high. the etiologic agent, r. prowazeki, is transmitted from person to person by the human body louse pediculus humanus corporis. infection is established by inoculating infected louse feces into the skin by scratching. immunization. although no etiologic relationship has been demonstrated between r. prowazeki and the bacterial strain proteus x , these two species share a common polysaccharide antigen (castaneda, ) . the sera of infected typhus patients agglutinates proteus x and this test is now standard (weil-felix reaction) for diagnosis of the acute disease. additionally, r. prowazeki has two major antigenic components-one heat labile and the other heat stable (craigie et al., ; topping et al., ) . typhus fever vaccine contains killed organisms propagated in the yolk-sac membranes of developing chick embryos (cox, ) . this vaccine not only diminishes the symptoms of typhus in immunized persons, but it also greatly reduces the mortality rate (gilliam, ) . the usefulness of the attenuated (madrid Ε strain) vaccine is hampered because, under appropriate conditions, the strain may revert to virulence (brezina, ) . the prokaryotes proc. world vet. congress, th proc. annu. conf. aust. vet. assoc tuberculosis in bacterial infections in humans: epidemiology and control symposium on rickettsial diseases vm/sac new trends and developments in vaccines vaccines ' : modern approaches to new vaccines principles and practice of cholera control vaccines ' proc. natl. acad the prokaryotes proc. natl public health rep viral hepatitis viral hepatitis proc. west. states food anim. conf vaccination against foot and mouth disease an inquiry into the cause and effects of the vanidae vaccinae vaccines ' a system of bacteriology the mycobacteria: a source book proc. natl. acad. sei. u.s.a. diseases of poultry bovine medicine and surgery proc. int. conf. equine infect. dis proc. annu. conf. aust. vet. assoc proc. natl. acad. sei. u.sa. proc. conf. res. work. anim. dis vaccines ' proc. th annu. meet. u.s. livestock sanit. assoc proc. natl. acad proc. th annu. meet. u.s. livestock sanit. assoc the virus in yellow fever proc. int. vet. congr vaccines ' diseases of poultry were useful references in writing this chapter. key: cord- - hto qn authors: schoch-spana, monica; brunson, emily k.; long, rex; ruth, alexandra; ravi, sanjana j.; trotochaud, marc; borio, luciana; brewer, janesse; buccina, joseph; connell, nancy; hall, laura lee; kass, nancy; kirkland, anna; koonin, lisa; larson, heidi; lu, brooke fisher; omer, saad b.; orenstein, walter a.; poland, gregory a.; privor-dumm, lois; quinn, sandra crouse; salmon, daniel; white, alexandre title: the public’s role in covid- vaccination: human-centered recommendations to enhance pandemic vaccine awareness, access, and acceptance in the united states date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: hto qn given the social and economic upheavals caused by the covid- pandemic, political leaders, health officials, and members of the public are eager for solutions. one of the most promising, if they can be successfully developed, is vaccines. while the technological development of such countermeasures is currently underway, a key social gap remains. past experience in routine and crisis contexts demonstrates that uptake of vaccines is more complicated than simply making the technology available. vaccine uptake, and especially the widespread acceptance of vaccines, is a social endeavor that requires consideration of human factors. to provide a starting place for this critical component of a future covid- vaccination campaign in the united states, the -person working group on readying populations for covid- vaccines was formed. one outcome of this group is a synthesis of the major challenges and opportunities associated with a future covid- vaccination campaign and empirically-informed recommendations to advance public understanding of, access to, and acceptance of vaccines that protect against sars-cov- . while not inclusive of all possible steps than could or should be done to facilitate covid- vaccination, the working group believes that the recommendations provided are essential for a successful vaccination program. since its first appearance in the united states in february , the novel coronavirus (sars-cov- ) has infected over . million americans and killed over , (as of october , ) [ ] . responses to the virus, including closing venues where person-to-person spread was likely, and requiring the use of masks and physical distancing measures when social contact could not be avoided, have reduced virus spread. at the same time, these protective actions have radically transformed social life and disrupted national and household economies [ ] . as the health crisis continues to linger and a sense of pandemic fatigue starts to take hold, political leaders, health officials, and the general public are seeking solutions [ ] . one of the most promising, if successfully developed and deployed, is vaccines. this technology could provide individual and population-level immunity, and through these the eventual conditions for the resumption of routine social and economic activities [ ] . to facilitate the development and dissemination of such vaccines, the us government has committed over billion dollars (via operation warp speed) with the aim of delivering million doses of a safe, effective vaccine by january [ ] . while this timeline is likely overly optimisticvaccine development, especially against a class of pathogens for which no licensed vaccine currently exists, typically takes - years [ ] -progress is being made. as of october , , vaccines are in preclinical evaluation, are in phase i and ii safety trials, have entered phase iii efficacy trials, and five vaccines have been approved for limited use: two in china, two in the united arab emirates, and one in russia [ ] . despite these promising developments, operation warp speed manifests a key social gap. the program rests upon the compelling yet unfounded premise that 'if we build it, they will come.' past experience in routine and crisis contexts demonstrates that, for a variety of reasons, not all segments of the public will accept medical countermeasures including vaccines [ ] [ ] . a recent poll in the us suggests this is already the case for sars-cov- (covid- ) vaccines. about half of us adults ( %) reported they definitely or probably would accept the vaccine, while % said they would not [ ] . in the same poll, only % of black americans indicated they would definitely/probably accept the vaccine compared to % of white americans. a human factor-centered vaccination campaign is needed to address these issues, but this campaign must be effectively planned and implemented. if poorly designed and executed, a covid- vaccination campaign could undermine increasingly tenuous beliefs in vaccines and the public health authorities that recommend them. at the same time, the broad impacts of a successful vaccination program would be considerable. immediate benefits would include interrupted disease transmission; fewer cases, hospitalizations, deaths, and chronic sequelae; and the beginning of reinstated social and commercial exchanges. longer term effects would include improved institutional capabilities to foster vaccine confidence among diverse communities, enhanced public understanding regarding vaccination's value to society, and heightened public trust in government, science, and public health. the purpose of this article, which is based on a report on the same topic [ ] , is to outline the major challenges and opportunities associated with a future covid- vaccination campaign and to provide empirically-informed recommendations to advance public understanding of, access to, and acceptance of vaccines that protect against sars-cov- . with the current lag time in vaccine availability, vaccination planners and implementers in the us and around the world have the opportunity to exercise foresight and take proactive steps to overcome potential hurdles to vaccine uptake and maximize public acceptance. these steps, however, must be taken now before this critical window of opportunity closes. the research and recommendations presented in this paper are a product of the -person working group on readying populations for covid- vaccine (table ) . this group was convened in april by principal investigators from the johns hopkins center for health security and the texas state university department of anthropology with support from the national science foundation-funded converge initiative [ ] . the purpose of the working group was to develop and disseminate recommendations informed by design thinking and evidence from social, behavioral, and communication sciences, that would support realistic planning for a us covid- vaccination campaign. members of the working group-listed as authors on this paper-included national figures in public health and social science with research, policy, and practice expertise in vaccinology, vaccine hesitancy/confidence, health disparities, infectious disease, bioethics, epidemiology, bioinformatics, public health law, pandemic mitigation, public health preparedness, mass vaccination campaigns, community engagement, and crisis and emergency risk communication. a combination of literature reviews on vaccination, pandemic planning, and health crisis communication; an assessment of current news and social media trends regarding covid- vaccines; and key informant interviews with each working group member focusing on their respective expertise formed the basis of the research presented in this article. this research was refined, and the recommendations were developed, through an iterative process involving the development of draft reports by a core working group, feedback from the entire working group via email and comments provided during a virtual meeting on may , , and subsequent rounds of revisions and feedback (including a second virtual meeting on june , ). the final report from the working group, which forms the basis of the recommendations and best practices below, was finalized on july , . envisioned largely as a biotechnology and logistics challenge, covid- vaccination also poses complex human factors challenges. such challenges have been observed during past emergencies. in , for instance, many americans rejected the h n vaccine due to safety concerns [ ] , despite the fact that the vaccine only involved a strain change (i.e., it was not a new technology) and was fully tested before release. the h n vaccine also amplified perceptions of bias. in los angeles, for example, distrust in public health-resulting from both prior experimentation on blacks (e.g. the tuskegee syphilis study) and long-term discrimination of blacks in health care settings [ ] [ ] [ ] -led local faith-based leaders, radio personalities, and other community representatives to advise black community members to avoid vaccination [ ] . even though the los angeles county health department actively sought to address these concerns, these suspicions coupled with a lack of convenient access to h n vaccines ultimately resulted in many blacks in this community remaining unvaccinated [ ] . despite the existence and importance of such challenges, funding for research on human factors related to vaccine acceptance is not commensurate with its significance for vaccination success [ ] [ ] . this type of inquiry-practical research of a social and behavioral nature on a medical technology-generally falls between the priorities of the national institutes of health ([nih] which rarely funds social science research) and the national science foundation (which does not fund applied public health research). funding from other sources including the centers for disease control and prevention (cdc) and private foundations has also historically been limited. in addition, the existing funding infrastructure is not outfitted for rapid response research during dynamic crises like sars-cov- . while initiatives are underway to develop communities of practitioners and a supportive infrastructure for disaster science in the us, including professional networks, streamlined institutional review board processes, and joint responder-researcher training [ ] [ ] , more progress is needed especially in regards to rapid funding opportunities. in the case of sars-cov- vaccination, for instance, while an nih funding opportunity award that could support research on human factors related to vaccine acceptance was made possible in june , the earliest project start date is september , a full nine months after operation warp speed plans for covid- vaccines to become available [ ] . to ensure a successful covid- vaccination campaign, it is necessary for sponsors to invest in time-critical investigations on human factors related to vaccine acceptance, and for public health authorities and other stakeholders to act on the social and behavioral findings of this research. such efforts include:  reconfiguring existing research investments to include social, behavioral, and communication science. one possibility for this is to set aside a small portion of the operation warp speed budget for research on human factors related to vaccine acceptance. such an approach has been used with great success in the past with other cutting-edge scientific initiatives such as the human genome project and manned space flight [ ] [ ] [ ] .  embeding rapid social, behavioral, and communication science within the covid- response, helping to deliver timely data and empirically based advice. by including social scientists in planning and implementation efforts, their people-centered methodologies and specialized knowledge can be integrated in a timely manner to maximize critical insights [ ] [ ] [ ] [ ] [ ] .  transforming the vaccine research enterprise by involving communities as active partners not passive subjects. traditional "one-sided, top down" approaches to community engagement are not always effective. community partnerships during the west africa ebola outbreak, for example, were necessary to overcome issues of trust and produce needed behavioral changes [ ] [ ] .  applying human-centered design principles (aka "design thinking") to the planning and implementation of the covid- vaccination program. user-focused approaches can result in more usable, acceptable, and effective interventions compared with traditional expert-driven methods [ ] [ ] . such an approach has been very successful in promoting hpv vaccination [ ] . vaccines typically require years of development and testing before licensure. nonetheless, us political leaders have publicly promised to accelerate covid- vaccine development at "an unprecedented pace," with the aim of delivering million doses of a safe and effective vaccine by january [ ] . although the use of new technologies can potentially accelerate vaccine production, public expectations around vaccine availability may not align with the practical realities of vaccine development, licensure, manufacture, and distribution. by failing to deliver sars-cov- vaccines as promised, the us government could frustrate pandemic-weary communities, siphon away trust, and suffer a major loss of institutional legitimacy. this situation is further complicated by public perceptions of the risks and benefits of sars-cov- vaccines. recent polling suggests that increasing numbers of americans plan to reject covid- vaccines, even if they are available and affordable [ , ] . a review of news reports, blogs, and other social media suggests a variety of potential causes for this result, including nonchalance about the disease and concern about vaccine safety. public perception, however, is a moving target. new developments, for example, an emergency use authorization (eua)-a power granted to the food and drug administration (fda) to make unlicensed drugs, vaccines, or other therapeutics available during a public health emergency, provided sufficient evidence that the countermeasure in question "may be effective"-for covid- vaccines, could engender additional uncertainties around vaccine safety due to the public's lack of familiarity with this complex regulatory mechanism. whatever the public's beliefs about vaccine benefits, risks, and supply, they cannot be separated from the current cultural milieu. in the us this is currently characterized by division, partisanship, and eroding public trust in government institutions-including the biomedical and public health agencies tasked with overseeing vaccine development, licensure, and distribution. in relation to the latter, for example, the intellectual independence of the fda has come under scrutiny for its ability to objectively assess vaccine safety and efficacy amid immense political pressure to quickly approve a sars-cov- vaccine [ ] . this complicated social environment poses a distinct and unprecedented complication to all vaccine promotion efforts in the us. amid this increasingly complex social landscape, there are several measures that us public health and healthcare practitioners, political leaders and policymakers, and communication experts can implement to prime the general public for sars-cov- vaccines including:  tempering expectations of vaccines as a "quick fix." because covid- vaccines will not immediately be available to everyone who wants them, and time will be needed to develop immunity (especially given the likelihood of two-dose regimens), communicators must prepare the public to continue implementing a mix of protective actions and harm reduction strategies.  forecasting a range of vaccine possibilities: from best case to worst case scenarios regarding vaccine supply and effectiveness. from a position of openness and transparency, public health communicators should address inevitable roadblocks and bottlenecks at every stage of vaccine testing, licensure, distribution, and administration, and convey to the public how this could affect vaccine availability. in addition, it will be necessary to reframe the dialogue about the value of vaccines, given that future sars-cov- vaccines may be not be the public's hoped for silver bullet. a vaccine, for example, may prevent the most severe disease but not prevent sars-cov- infection. in this scenario, vaccination could keep hospitals from being overwhelmed, prevent declines into frailty after severe bouts of disease, and avert medical bankruptcies that may arise with the longer-term impacts of covid- , but not provide the community immunity necessary to halt the spread of sars-cov- .  persisting in transparency around vaccine safety systems and actively work to protect their integrity. health authorities should focus existing vaccine safety infrastructure on the use of sars-cov- vaccines. in this vein, health authorities should develop a robust system for post-licensure surveillance, including ascertaining background rates of anticipated adverse events prior to vaccine rollout to enable comparison with post-rollout incidence of adverse events. independent oversight of vaccine safety, as occurred during the - h n pandemic, should also be used [ ] .  early on, seeking the counsel and input of communities of color that may have historic reticence towards public health. vaccine promotion efforts should engage these communities early and as frequently as possible. as partners in the task, they must also empathize with legitimate concerns around vaccine safety, medical experimentation, and inequalities in health care [ ] [ ] , while also identifying and sharing salient information that can help assuage unwarranted worry. a profusion of true and false information, which the who recently referred to as an "infodemic" [ ] , is now circulating around covid- . in this crowded information landscape, the veracity of information can be difficult to determine and key messages can be lost. in the us, public discourse on the pandemic currently incorporates a panoply of topics including science, public health, social disruptions, political divisions, and economic fallout [ ] , each of which can be a vehicle for misinformation-information that differs from expert consensus at the time it is shared [ ] . while many reasons exist for this flood of misinformation, including the widespread public adoption of social media platforms as a tool for information seeking, the uncertain nature around covid- as a novel infectious disease, and the presence of disinformation campaigns aimed at deflecting blame and pushing false narratives around the global covid- response [ ] [ ] [ ] , no easy solutions exist to stem the tide [ ] [ ] . regarding covid- vaccination specifically, while the first vaccine is minimally months away from materializing, the topic has already commanded immense public attention and generated its own pool of misinformation [ ] [ ] . this ranges from rumors questioning vaccine safety to more complicated narratives suggesting that future covid- vaccines were created alongside the virus and that major organizations are planning to use a covid- vaccination campaign for financial gain [ ] [ ] . while not the sole factor in determining behavior adoption, effective communication is necessary to address these issues and build public confidence in covid- vaccination [ ] . such communication will require addressing the enduring problem of how to best engage, exchange information, and empower audiences who have diverse beliefs and life circumstances. past communication experience with vaccines has shown the importance of engaging with key audiences to understand their concerns, values, attitudes, perceptions, and beliefs [ ] [ ] [ ] [ ] , and using this understanding to develop messages that resonate [ ] [ ] . messages that do not do this are often ineffective and, worse, can move audiences further away from the desired behaviors [ ] . given the diverse nature of social identities in the us, covid- vaccination communications will need to be tailored to meet the needs of specific audiences including essential workers, parents, groups with high comorbidity rates, and communities of color.  investing in qualitative research to identify specific community concerns and hopes in relation to covid- vaccination. qualitative research can provide insight into "how" and "why" participants feel, think, or behave a particular way [ ] [ ] . such insight, in turn, is the basis for developing more meaningful, trusted, and influential communication strategies [ ] . the current climate of racial, political, and economic division in the us has created a charged environment that necessitates both a fair vaccination campaign and widespread, public recognition of its fairness. an initial test of this will be how limited, initial doses of vaccines are allocated. in past public health emergencies, including the - h n pandemic, allocation strategies have been used to prioritize delivery of medical countermeasures to specific groups like critical health care workers and those who are at particular risk [ ] . pervasive racial biases in the us healthcare system, including lack of insurance and a lesser quality of care for non-white, rural, and low-income populations [ ] [ ] [ ] . such disparities have long-term consequences. black populations in the us, for example, experience increased morbidity and mortality compared to their white peers, sometimes in ways that cannot be accounted for by access to health care and income [ ] . public health authorities will need to anticipate and mitigate public discourse regarding vaccine allocation and distribution along with prejudicial ideas about social worth, explaining that vaccinating individuals residing in the us, regardless of social or legal status, is critical to the public's health as a whole. finally, politicization of the pandemic-both real and perceived-may prime expectations of a partisan-based vaccine allocation and distribution rather than an equitable one. some americans, for instance, perceive the use of masks as a slight against president trump by his detractors [ ] . likewise trump has signaled his preference for having a vaccine available prior to the election (a projection not in keeping with expert assessments), prompting concerns about whether he could turn a potential but inadequately tested vaccine into a campaign tool [ ] . such polarized views of covid- raise concerns about whether vaccine allocation and distribution can and will be judged as fair by the majority of americans. people will judge a covid- vaccination campaign's integrity not simply on biomedical merits, but on matters of fairness and equity-that is, have people received their just portion of health services, and has disease prevention, ultimately, been fairly distributed? past experience suggests the following steps may contribute to a fair process:  the us government taking steps to make the vaccine available at no cost to all americans and publicly pledge that everyone who wants covid- vaccines will get covid vaccines. removing cost as a barrier is among the most significant ways to assure that all individuals benefit from the life-preserving benefits of sars-cov- vaccines, and that the public can have the utmost confidence that public health needs and not economics will determine access. in the time that exists before vaccines are produced it is critical that safe and accessible vaccination sites are identified. this process will require ramping up the use of sites that are already available and accessible, but are used less frequently for vaccination efforts. community pharmacies, for example, are widespread and have been mobilized for past vaccination efforts [ ] . to fully utilize pharmacies in covid- vaccination efforts, however, it will be necessary to address state-level policies that may currently preclude pharmacists from administering these vaccines without standing orders from physicians. other nontraditional, potential vaccination settings that should be considered include grocery stores, senior citizen centers, workplaces, and schools [ ] [ ] [ ] [ ] [ ] . in some cases, it also may be acceptable and feasible to deliver vaccination via home visits by community health nurses when vaccination is bundled with delivery of other preventive health services; this approach has received a strong recommendation in the past from the community preventive services task force [ ] . for marginalized populations, including racial and ethnic minorities, additional consideration must be given to what constitutes a "safe" vaccination site. during the - h n pandemic, for example, mistrust and fear among marginalized communities posed a challenge. latino farmworkers were at greater risk for h n -related morbidity and mortality. however, reports of bullying and harassment within and outside of local healthcare settings led many members of this population to be fearful and hesitate to seek out h n vaccination [ ] . while national patterns may exist, assessments of what constitutes safe vaccination sites for marginalized populations should be conducted at local levels. once vaccination sites are identified, it will be essential for public health authorities to disseminate up-to-date, comprehensible, and trustworthy information about vaccination opportunities. much of this communication work will be done by local and state health departments, which may be challenging in light of budget cuts and strained local public health infrastructure. an additional complication will be the likely complex covid- vaccination environment, characterized by multiple manufacturers, multiple vaccine doses, and differently timed follow-up doses. making vaccines widely accessible is a complex endeavor. past experience suggests that this is possible with proactive, thoughtful coordination and clear communication like the following:  utilizing nontraditional vaccination sites like schools, pharmacies, places of worship, workplaces, grocery stores, health departments, mass vaccination clinics, senior centers, home visits, and others. utilizing these sites, as well as clinical sites that already serve vulnerable or underserved populations (e.g., free/low cost community health care clinics, std clinics, substance use treatment centers) will be important to improve uptake in populations that outreach efforts have failed in the past.  preparing, in advance, all necessary educational materials and training that may be needed for those tasked with vaccination at nontraditional sites. training may include information on how to look up immunization records in state immunization registries, how to safely store vaccines, and how to safely recommend vaccines for targeted populations, keeping in mind any contraindications.  anticipating hesitancy among marginalized populations who may be fearful or wary of seeking vaccination at sites that have historically caused mistrust, and plan to either expand sites to better serve these populations or engage these populations early to earn and build trust. this may require using novel sites to better serve marginalized populations (e.g., places of worship, schools, culturally specific community centers or senior centers, mobile clinics). these nontraditional settings will also require those administering vaccines to be culturally competent. vaccination sites should not be heavily policed or send any signals that they may be somehow unsafe for vulnerable persons.  fostering collaboration among interagency and nongovernment partners to make vaccination available alongside provision of other safety net services. bundling services that address individuals' broader needs during the pandemic (e.g., food security, rent assistance, workforce development) could be a way to build trust, streamline vaccine provision, and enhance more convenient access for community members. the protracted covid- pandemic has placed multiple stresses on the american people: the threat of illness and death, the isolating effects of physical distancing measures, and the uncertainties and hardships associated with disrupted economic and schooling activities. the public's patience is understandably wearing thin. operation warp speed is taking revolutionary steps to develop sars-cov- vaccines as swiftly as possible and, along the way, to inspire hope that relief from the pandemic's multiple burdens is coming. despite vaccination's promise of release from the confines of the pandemic, some members of the us public-including those most at risk of covid- 's impacts-are already reluctant to embrace this public health measure [ ] . likewise, current protests against nonpharmaceutical interventions to the sars-cov- crisis, including criticisms about government over-reach, encroachment on individual freedoms, and a clash of personal values, have the potential to further erode public trust in future sars-cov- vaccines. under these circumstances, bold measures are necessary to instill public trust and to change the reality and the perception that covid- vaccination is a top-down program administered without regard to public sentiment, concerns, or priorities. one potential solution to these issues is the formation of public oversight committees at state and, in large metropolitan areas like new york and los angeles, local levels. governance structures that incorporate public oversight and community involvement have the potential to inspire greater public confidence in, and a sense of ownership over, public health interventions. such "ownership" can fortify the intent to vaccinate and strengthen distribution systems to reach throughout communities, thus helping to assure the fitting and fair use of a public good. this type of community engagement entails the collaboration of affected and at-risk populations with policymakers and practitioners in the generation, implementation, and evaluation of measures to safeguard public health and safety [ ] [ ] [ ] . an accountability mechanism and metrics will be necessary to ensure that allocation is fair, target groups receive vaccine, and underserved populations that have been disproportionately affected during the pandemic are justly attended. while vaccines represent a promising solution to the covid- pandemic, the development of vaccines is only part of the answer. widespread acceptance of vaccines is also needed. this acceptance, in turn, requires more than just making safe and effective vaccines available. it is a complex social endeavor that necessitates deep engagement around the human element, and requires the efforts of us policymakers; federal, state, and local public health officials; private funders; professional and community organizations; university researchers; and nontraditional partners. while the content provided in this article is not all-inclusive of what can, or should, be done to support widespread acceptance of covid- vaccines, the recommendations and best practices outlined here are important for such a vaccination program to be successful. as experts in a wide variety of vaccination-related topics, we fear that unless these critical steps are taken, any future covid- vaccination campaign will be less than hoped for. a worst-case scenario would involve an inability to stop the ravages of the disease and its cascading social and economic effects; further erosion of public trust in government, public health, and vaccine science; and potential threat to other life-preserving and live-enhancing vaccination efforts. that said, a successful covid- vaccination endeavor promises an alternative future: a return to a sense of normalcy, major innovations in vaccine research and operations, and the investment of us society as a whole in making vaccines a public good in which all can share and derive value. establish public oversight committees to review and report on systems affecting public understanding, access to, and acceptance of covid- vaccines usg should sponsor a national panel entity (e.g., nasem) to review, synt best practices for engaging communi allocation, deployment, and commun achieve equity, solidarity, and good h each state (and the most populous cit committee that is demographically re incorporates diverse sectors of societ and faith communities. abbreviations: hcd = human centered design; nih = national institutes of health; nsf = national science foundation; cdc = centers for disease control and prevention; activ = accelerating covid- centers for disease control and prevention. coronavirus disease cases in the us the evidence and tradeoffs for a 'stay-at-home' pandemic response: a multidisciplinary review examining medical, psychological, economic and political impact of 'stay-at-home' implementation in america post-abc poll finds. the washington post department of health & human services. fact sheet: explaining operation warp speed the college of physicians of philadelphia. vaccine development, testing, and regulation coronavirus vaccine tracker. the new york times group on vaccine hesitancy. vaccine hesitancy: definition, scope and determinants understanding vaccine hesitancy around vaccines and vaccination from a global perspective: a systematic review of published literature widespread declines the shares who say they would get a covid- vaccine on behalf of the working group on readying populations for covid- vaccine. the public's role in covid- vaccination: planning recommendations informed by design thinking and the social, behavioral, and communication sciences converge. covid- working groups for public health and social sciences research the public's response to the medical apartheid: the dark history of medical experimentation on black americans from colonial times to the present more than tuskegee: understanding mistrust about research participation implicit racial/ethnic bias among health care professionals and its influence on health care outcomes: a systematic review pandemics and health equity: lessons learned from the h n response in los angeles county public trust in vaccines: defining a research agenda the need for a multi-disciplinary perspective on vaccine hesitancy and acceptance science during crisis: best practices, research needs, and policy priorities converge. converge training modules department of health and human services. nih director's emergency transformative research awards human genome project information archive - . ethical, legal, and social issues the ethical, legal, and social implications program of the national human genome research institute: reflections on an ongoing experiment national aeronautics and space administration preparedness for a high-impact respiratory pathogen pandemic protecting humanity from future health crises: report of the high-level panel on the global response to health crises will ebola change the game? ten essential reforms before the next pandemic. the report of the harvard-lshtm independent panel on the global response to ebola world health organization. implementation of the international health regulations: report of the review committee on the role of the international health regulations in the ebola outbreak and response community-centered responses to ebola in urban liberia: the view from below implementing a novel community engagement system during a clinical trial of a candidate ebola vaccine within an outbreak setting a design thinking approach to effective vaccine safety communication design thinking in health care user-centered design for developing interventions to improve clinician recommendation of human papillomavirus vaccination trump administration announces framework and leadership for 'operation warp speed cnn poll: most americans would be uncomfortable returning to regular routines today could trump turn a vaccine into a campaign stunt national vaccine advisory committee (nvac). h n vaccine safety risk assessment working group coronavirus disease (covid- ) situation report - coronavirus: the us resistance to a continued lockdown. bbc news defining misinformation and understanding its bounded nature: using expertise and evidence for describing misinformation european external action service strategic communications and information analysis division. eeas special report update: short assessment of narratives and disinformation around the covid- /coronavirus pandemic addressing health-related misinformation on social media weaponized health communication: twitter bots and russian trolls amplify the vaccine debate press freedom critical to countering covid- 'pandemic of misinformation': un chief surge of virus misinformation stumps facebook and twitter the new york times anti-vaccination leaders seize on coronavirus to push resistance to inoculation antivaccination activists are growing force at virus protests. the new york times there isn't a covid- vaccine yet, but some are already skeptical about it the epic battle against coronavirus misinformation and conspiracy theories anti-vaccine movement could undermine efforts to end coronavirus pandemic, researchers warn. nature news social construction of the valuebehavior relation the jossey-bass health series. health behavior and health education: theory, research, and practice a comparison of the theory of planned behavior and the theory of reasoned action vaccine education spectrum disorder: the importance of incorporating psychological and cognitive models into vaccine education preserving civility in vaccine policy discourse: a way forward boomerang effects in science communication: how motivated reasoning and identity cues amplify opinion polarization about climate mitigation policies researchers say that the debate over the coronavirus may become more violent focus group methodology: principle and practice qualities of qualitative research: part i american indian/alaska native and russian/ukrainian communities the impact of social networks on parents' vaccination decisions h n influenza vaccination campaign: summary of a workshop series fair allocation of scarce medical resources in the time of covid- the equitable distribution of covid- therapeutics and vaccines racial bias in pain assessment and treatment recommendations, and false beliefs about biological differences between blacks and whites racial disparities in exposure, susceptibility, and access to health care in the us h n influenza pandemic national research council (us) panel on race, ethnicity, and health in later life understanding racial and ethnic differences in health in late life: a research agenda state-of-healthcare-in-the-united-states/racial-disparities-in-health-care/; californians required to cover their faces in 'most settings outside the home'. the washington post cdc's h n vaccine pharmacy initiative in the united states: implications for future public health and pharmacy collaborations for emergency response safety and acceptability of pneumococcal vaccinations administered in nontraditional settings national survey of pharmacy-based immunization services safety of influenza vaccinations administered in nontraditional settings centers for disease control and prevention. national and state-level place of flu vaccination among vaccinated adults in the united states flu season polio: an american story increasing appropriate vaccination: home visits to increase vaccination rates stigma, health disparities, and the h n influenza pandemic: how to protect latino farmworkers in future health emergencies community engagement, organization, and development for public health practice community participation for transformative action on women's, children's and adolescents' health on behalf of the working group on community engagement in health emergency planning. community engagement: leadership tool for catastrophic health events key: cord- -jwjr po authors: mantel, carsten; cherian, thomas title: new immunization strategies: adapting to global challenges date: - - journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: . /s - - -x sha: doc_id: cord_uid: jwjr po immunization has made an enormous contribution to global health. global vaccination coverage has dramatically improved and mortality rates among children due to vaccine-preventable diseases have been significantly reduced since the creation of the expanded programme of immunization in , the formation of gavi, the vaccine alliance, in , and the development of the global vaccine action plan in . however, challenges remain and persisting inequities in vaccine uptake contribute to the continued occurrence and outbreaks of vaccine-preventable diseases. inequalities in immunization coverage by geography, urban-rural, and socio-economic status jeopardize the achievement of global immunization goals and call for renewed immunization strategies. these should take into account emerging opportunities for building better immunization systems and services, as well as the development of new vaccine products and delivery technologies. such strategies need to achieve equity in vaccination coverage across and within countries. this will require the participation of communities, a better understanding of vaccine acceptance and hesitancy, the expansion of vaccination across the life course, approaches to improve immunization in middle-income countries, enhanced use of data and possible financial and non-financial incentives. vaccines also have an important role to play in comprehensive disease control, including the fight against antimicrobial resistance. lessons learned from disease eradication and elimination efforts of polio, measles and maternal and neonatal tetanus are instrumental in further enhancing global immunization strategies in line with the revised goals and targets of the new immunization agenda , which is currently being developed. immunization is one of public health's most successful and cost-effective interventions, saving up to three million lives every year, according to un figures [ ] . the expanded program on immunization (epi) was initiated in with the goal of providing universal immunization with essential vaccines. among the initially targeted six vaccine-preventable diseases (vpds), diphtheria, pertussis, tetanus, measles, poliomyelitis, tuberculosis, a substantial reduction in the burden of preventable childhood illnesses and deaths was achieved in its initial years [ , ] . over the past decades, national immunization programmes (nips) have become substantially more complex, with vaccines now available to protect against more than infectious diseases, while health, societal, and political changes created additional volatility and ambiguity in often more uncertain environments (e.g., with the occurrence of conflicts, epidemics, or increasing vaccine hesitancy). gavi, the vaccine alliance, was established in primarilytoallownew vaccines to reach children in the poorest countries [ ] . stagnating global immunization coverage rates in many countries led global partners of the decade of vaccines collaboration to initiate the global vaccine action plan (gvap) in [ ] . this plan states in its mission "to extend, by and beyond, the full benefits of immunization to all people, regardless of where they are born, who they are, or where they live". the im- who, unicef, gavi, bmgf, niaid. portant focus on equitable immunization uptake and coverage is also reflected in the present gavi strategy - , and will likely be sustained in a subsequent gavi strategy leading up to [ ] and in the new immunization agenda (ia ) [ ] . major successes were accomplished during the decade. the mortality rate among children under years of age has been driven down from / to / children in only years between and [ ] . more children than ever ( . million) are receiving three doses of dtp before their first birthday, . million more than in . by , countries had reached at least % coverage of the third dose of dtp vaccine [ ] . country decision-making capabilities were strengthened, and many lifesaving vaccines were introduced primarily in the poorest countries [ ] . regional vaccine action plans were designed, a global immunization monitoring and evaluation framework was established, and efforts were made to shape vaccine markets and to improve vaccine price transparency. overall, the gvap helped to build political will and kept immunization visible on the global agenda [ ] . but challenges remain, and nips across the world need to adapt to these by continuously revising and updating their immunization policies and strategies. elimination and eradication goals for polio, measles, rubella, and neonatal tetanus have not been met everywhere and vaccine-derived polio virus and measles outbreaks are still circulating. importantly, not every child or eligible person is being reached everywhere [ , ] . inequities in vaccine uptake and disease persist across countries, and inequalities in routine immunization coverage can contribute to the continued occurrence and outbreaks of vpds. the global coverage of the third dose of diphtheria-pertussis-tetanus-pertussis (dpt) vaccine is stagnating at % with little change since . in some countries, progress has even gone into reverse. one in infants worldwide has still not received any vaccine as reflected in first dose dpt coverage and an estimated . million infants were not reached with routine immunization services such as three primary doses of dpt vaccine [ , ] . coverage with the full complement of doses, including booster doses, is far lower and immunization in the second year of life and beyond still broadly inadequate. an additional . million deaths could be avoided if global immunization coverage improves [ ] . within countries, geographical inequalities are apparent with district-level coverage varying substantially between easily accessible and more remote areas [ ] . social determinants of health, such as individual and household income and education, impact immunization uptake [ ] and areas of low coverage often overlap with those that are home to ethnic minorities, as well as marginalized or nomadic and migrant populations [ ] [ ] [ ] . in many situations, governments face challenges in reaching such underserved populations or lack the political will to do so. increasingly, there are also substantial inequities in vaccine use between urban and rural populations [ ] , with coverage in urban slum populations often as low as in remote rural communities. a preliminary sce-nario based on projected proportions of unimmunized children in rural and urban areas shows that almost half ( %) of the unimmunized and under-immunized children in the top countries that were home to more than half of the unimmunized and under-immunized children in lived in urban areas, and almost every fifth unimmunized child ( %) lived in a slum [ , ] . inequalities in the vaccine coverage by wealth quintiles can also reach enormous dimensions. a child from a rich family can be up to times more likely to be vaccinated for dtp than a child from a poor family in certain countries [ ] . these challenges call fora renewed and sustained global immunization strategy, taking into account emerging opportunities for building better immunization systems and services, but also for the development of new products and technologies. such a global strategy is presently being drafted as part of the ia [ ] . the following four immunization approaches and strategies important to achieving both the existing gvap and future ia goals are discussed: ( ) achieving equity in vaccination coverage; ( ) expanding vaccination across the life course; ( ) promoting integration of immunization in the health sector; and ( ) learning lessons from disease eradication and elimination efforts. expanding access and ensuring equality in uptake of vaccines requires strong immunization systems. in this context, the ongoing emphasis on new vaccine introduction, while exposing the fragility of these systems specifically in low-income countries, also created opportunities in several ways: it generated additional advocacy and visibility for immunization and provided opportunities for training and change in immunization practices. vaccine management and service implementation was improved in many countries, including planning, demand forecasting, vaccine supply, cold chain and logistics, and improved data quality through better recording, report-ing, and use of immunization data for programme monitoring [ , ] . however, strong service delivery systems alone are not enough to achieve optimal impact and need to be accompanied by appropriate policies and strategies that promote the equitable delivery of vaccination. the who global routine immunization strategies and practices (grisp) lays these out in a comprehensive manner [ ] . in this framework, delivery of immunization services through fixed health facilities and mobile outreach services is considered the most basic means for reaching all populations. this includes strategies for detecting and reaching marginalized and partially served populations, adjusting service availability and convenience, creating synergies with accelerated disease control activities, and creating demand for vaccination. these routine services may be supplemented by a strategy termed the periodic intensification of routine immunization (piri) as a mechanism to catch up individuals who may have missed their routine doses. another strategy to rapidly increase population immunity is to deliver additional doses of vaccines through supplementary immunization activities (sias) or vaccination campaigns. inclusion of communities and civil society is crucial to make sure the vaccines and their delivery are acceptable, appropriate, and sustainable. community-based organizations have stepped up their involvement in immunization and developed locally adapted solutions to removing social and cultural barriers, to re-create trust in immunization services, where it had waned, and to increase the locally adapted use of vaccines. in this context, important advances were also made in better understanding vaccine acceptance and hesitancy, which led to stagnant or declining vaccination coverage and resulted in outbreaks of vpds in some places. the who guide to tailoring immunization programmes (tip) helps to identify and prioritize vaccine hesitant populations and subgroups, diagnose the demand and supply-side barriers to vaccination in these populations, and design responses to vaccine hesitancy appropriate to the setting, context, and population [ ] . at the same time, efforts are being made to measure and publish data on inequalities in immunization coverage [ ] . the world health organization (who) and united nations children's fund (unicef) are encouraging all countries to conduct respective analysis to inform programme planning. a new who technical guide outlines in further detail how to progress from measurement of inequities to developing and implementing operational plans to achieve their reduction [ ] . there is also a growing interest in the use of incentives to attain higher immunization coverage and improved health outcomes. result-based financing (rbf) is a broad term that includes a variety of incentive schemes to provide resources to individuals, households, health workers, and district or local governments, whereby payment is conditional on the achievement of agreed-on and measurable outcomes. although there is potential to improve coverage with such programmes, the overall quality of evidence is still relatively low. countries with weaker management and monitoring and evaluation systems in place can face challenges in implementing rbf mechanisms, and the impact of rbf in countries with weak health systems is unclear [ ] . other approaches to improving immunization coverage include national vaccine legislation that mandates immunization and often includes the provision of a budget line for vaccine procurement [ ] . examples of successfully adapted national vaccination strategies are seen in india, where strong political will coupled with innovative and flexible immunization approaches and an appropriate monitoring framework (e.g., with geospatial mapping) led to a substantial increase in equitable coverage across the country [ , ] . in spite of their relatively higher gross national income (gni)/capita, middleincome countries (mics) that have been excluded from gavi support or are about to lose this support, increasingly recognize inequities in vaccine coverage as a se- bundesgesundheitsbl · : - https://doi.org/ . /s - - -x © springer-verlag gmbh deutschland, ein teil von springer nature immunization has made an enormous contribution to global health. global vaccination coverage has dramatically improved and mortality rates among children due to vaccine-preventable diseases have been significantly reduced since the creation of the expanded programme of immunization in , the formation of gavi, the vaccine alliance, in , and the development of the global vaccine action plan in . however, challenges remain and persisting inequities in vaccine uptake contribute to the continued occurrence and outbreaks of vaccine-preventable diseases. inequalities in immunization coverage by geography, urbanrural, and socio-economic status jeopardize the achievement of global immunization goals and call for renewed immunization strategies. these should take into account emerging opportunities for building better immunization systems and services, as well as the development of new vaccine products and delivery technologies. such strategies need to achieve equity in vaccination coverage across and within countries. this will require the participation of communities, a better understanding of vaccine acceptance and hesitancy, the expansion of vaccination across the life course, approaches to improve immunization in middle-income countries, enhanced use of data and possible financial and non-financial incentives. vaccines also have an important role to play in comprehensive disease control, including the fight against antimicrobial resistance. lessons learned from disease eradication and elimination efforts of polio, measles and maternal and neonatal tetanus are instrumental in further enhancing global immunization strategies in line with the revised goals and targets of the new immunization agenda , which is currently being developed. vaccines · global strategies · expanded programme on immunization · health systems · integration neue impfstrategien -anpassung an globale herausforderungen zusammenfassung impfungen haben in den letzten jahrzehnten deutlich zur verbesserung der globalen gesundheit beigetragen. im globalen durchschnitt haben sich die impfquoten signifikant verbessert, und die kindersterblichkeit aufgrund impfpräventabler erkrankungen ging seit beginn des "erweiterten impfprogramms" (expanded programme on immunization [ impfstoffe · globale strategien · erweitertes impfprogramm · gesundheitssysteme · integration rious problem in achieving their health goals [ ] . currently, more than two thirds of the world's poor live in mics and the largest fraction of children without access to vaccines is born in these countries [ ] . delays in vaccine procurement resulting in interruption in services, together with increasingly reported vaccine hesitancy, contribute to declining or stagnant coverage in some of these countries [ ] . in addition, there is persistent inequality in coverage in some socially disadvantaged populations and among the increasing number of migrants. in response to these equity challenges, mics with support from major immunization partners have developed strategies addressing a number of specific issues important to sustainably reach the underserved populations [ ] . these include support for decision-making and political commitment, financial sustainability, enhanced demand and eq- who, unicef, gavi, bmgf and others. uitable delivery, as well as affordable and timely supply [ ] . the new gavi strategy . also includes an additional provision for addressing immunization challenges in mics, specifically with regard to vaccine price, quality, and supply [ , ] . finally, further developments in vaccine delivery technologies could help in improving equitable vaccine coverage. a -year old technology for the administration of vaccines based on needle and syringe is still in use today. this technology is relatively complex to use, requires substantial training, is prone to programmatic errors, results in sharps waste, and may be a factor in the decreasing acceptability of vaccination [ ] . new technologies, e.g., microarray vaccine skin patches could allow less well-trained health workers to administer parenteral vaccines, reduce fear and hesitancy, simplify vaccine storage and transport, and improve vaccination coverage and reach [ ] . another important approach to reducing inequities in immunization is the extension of immunization beyond infant vaccination [ ] . with many more vaccines in the schedule and the need for administering booster doses, epi services need to increasingly achieve adequate coverage in all target populations across the life course. additional vaccines for all age groups are available or will likely become available in the near future targeting infants and young children (e.g., vaccines against measles [ nd dose], mumps, rubella, japanese encephalitis, group a and w meningococcus, varicella, hepatitis a, typhoid fever, malaria, dengue, enterotoxigenic e. coli, shigella, and group a streptococcus), older children and adolescents (tetanus adolescent dose, human papilloma virus [hpv] vaccine), pregnant women (influenza, pertussis, group b streptococcus, respiratory syncytial virus vaccines), and older adults (varicella-zoster vaccine). these new contacts with the health systems will also offer opportunities to provide missed vaccine doses that should have been administered earlier. new platforms for vaccine delivery are further explored, such as antenatal maternal services, pre-school and school-based delivery, adolescent health programs, as well as risk-based, targeted services for health workers and for specific patient groups in need of certain vaccines (e.g., diabetics). among all preventive healthprogrammes and initiatives, the epi traditionally has the greatest ability to reach infants, children, and pregnant women. with the ambitious goals set by the sustainable development goals (sdgs) and the who's th general programme of work, including a renewed emphasis on primary health care and universal health coverage, further synergies among different health programmes are being promoted [ ] . given the broader spectrum of diseases against which vaccines are or will soon be available, nips will need to work in a more cohesive way using integrated delivery platforms and broader collaboration in planning processes to achieve sustained equity and efficiencies. vaccines have an important place in comprehensive approaches to disease control. the use of vaccines against hepatitis b, pneumococcal, rotavirus, and hpv infection are vital interventions in the broader disease control efforts against chronic liver disease, acute respiratory infections, meningitis, severe diarrhoea, and cervical cancer [ ] . elimination of maternal and neonatal tetanus as a public health problem should be achievable in the near future in the remaining countries (as of july ) by applying integrated strategies and activities including the immunization of children, mothers, and women of reproductive age together with the promotion of clean deliveries and cord care practices. in this broader context, tetanus toxoid will successively be replaced with tetanus-diphtheria vaccines to ensure sustained protection against both diseases [ ] . the new who strategy to achieve the elimination of cervical cancer as a public health concern focuses on increasing the coverage of vaccination against hpv as one of three key targets besides screening and hpv testing [ ] . nips can also help to further increase equitable coverage of other health interventions, such as vitamin a supplementation, anti-helminthic treatment, nutrition interventions, insecticide-impregnated bed-nets, and intermittent preventive treatment against malaria. other important preventive interventions are being added, e.g., for adolescents [ ] . improving coordination and collaboration between immunization and other preventive and curative services can result in efficiency gains, save resources by offering one-stop services at the point of delivery, and increase the use of social mobilization to increase demand [ ] . in addition, non-governmental and private vaccinators can enhance the ability of programmes to deliver recommended vaccine doses, especially to clients who prefer them over public services. an added operational approach to increasing equitable immunization coverage is the reduction of missed opportunities for vaccination. unvaccinated and under-vaccinated children and pregnant women may access health services to seek care, and such a visit becomes a missed opportunity if immunization is not addressed. this could quite easily be rectifiedbyhealthfacilitystaffinbothnon-immunization and immunization services reviewing the vaccination status of all clients presenting or accompanying others and providing any missed doses [ , ] . recent work has shown that immunization coverage increases of %, and more can be achieved with relatively simple measures. chad, with % of eligible children leaving health facilities unvaccinated, made a quick evaluation and initiated a missed-opportunities-forvaccination strategy using cross-referral, which led to a sizable increase in the number of vaccinated children [ ] . a further example of using vaccines in an integrated approach to curb global health threats is the fight against antimicrobial resistance (amr), currently one of the most alarming issues for human health. drug resistant infections may already cause , deaths per year and it is estimated that million deaths due to amr may occur every year after [ ] . vaccines can impact antibiotic-resistant infections through a direct reduction in the resistant organisms and strains that are specifically targeted by the vaccine, as well as through a reduction in common illnesses that often lead to the use of antibiotics and therefore selection pressure on pathogens [ ] . the widespread use of haemophilus influenzae type b (hib) and pneumococcal vaccines have resulted in a dramatic reduction in disease burden and have been associated with decreased incidence of resistant strains. universal coverage with pneumococcal vaccination could potentially avoid more than million days of antibiotic use per year in children under [ , ] . a newer typhoid conjugate vaccine has a potential for high impact on prevention and control of antimicrobial resistant typhoid fever and was recently strategically used to control an extensively drug resistant typhoid outbreak in pakistan [ ] . but vaccines against viruses, e.g., influenza, can also exert a major impact here, due to the inappropriate prescription of antibiotics. vaccines offer a more sustainable approach to infection prevention, since pathogen resistance to vaccines is uncommon [ ] . the equitable use of vaccines is expected to avert substantial parts of the amrrelated fraction of disease, reduce antibiotic use, and-together with other interventions across human and animal health-add to the reduction in the economic and societal burden of amr [ ] . while the aspirational gvap goals to eradicate polio and eliminate measles and rubella will not be met by , substantial progress has been achieved in these areas. wild poliovirus (wpv) type was certified as eradicated in and wpv type has not been detected since . wp type is circulating in a mere two countries. at the same time, measles cases have been reduced through vaccination by % since , preventing . million deaths [ ] . important lessons informing enhanced immunization strategies can be learned from these efforts. the new polio endgame strategy for the years - uses intensified immunization approaches in afghanistan and pakistan, e.g., so-called hit-and-run vaccine delivery in conflict-affected and insecure areas [ ] . at the same time, strides are being made to integrate the delivery of polio vaccination and ded-icated surveillance networks fully into nips [ ] . measles, due to its high contagiousness, is often viewed as the "canary in the coal mine, " exposing even small areas of inequities that are not visible when just looking at inequalities in coverage. again, strategies to accelerate progress towards measles elimination comprise the enhanced use of surveillance platforms, which have been established in many countries to detect and respond to communicable diseases. in addition to conducting surveillance, the collection of detailed subnational coverage data and outbreak investigations are used to distinctly map areas and age groups with immunity gaps. specific approaches can then be used to fill these gaps in line with the measles and rubella strategic plan, e.g., through targeted sias [ ] . other epidemic-prone diseases periodically threaten the health and livelihoods of people [ , ] . outbreak risks are magnified by rapid population growth in areas with weak health systems, urbanization, human mobility, conflict, and the changing nature of pathogen transmission between human and animal populations [ ] . these drivers of disease emergence are likely to continue and intensify, while ecological changes, including climate change, can further amplify disease emergence risk [ ] . reducing the risk of epidemic-prone vpds involves several strategies: anticipating and preventing outbreaks using preventive vaccination campaigns (e.g., when immunity gaps accumulate or when natural disasters or other humanitarian emergencies increase risk); timely outbreak response to limit spread; postoutbreak initiatives to rebuild resilient health systems or address shortfalls that led to the outbreak. where possible, such initiatives should be combined with other vaccines or interventions, as appropriate, to maximize impact and achieve efficiency gains [ ] . capacities for the rapid deployment of vaccines and other interventions have been strengthened as a legacy of polio eradication and measles elimination efforts. major efforts for rebuilding systems with enhanced preparedness and response capacities were seen with the recent ebola outbreaks in west and central africa; this strategy was also pursued in the cholera outbreaks in conflict zones of syria and yemen or the diphtheria outbreaks among the rohingya refugees in bangladesh. outbreaks sometimes necessitate the use of new pre-licensure vaccines, as successfully implemented during the ebola outbreaks. a new entity, the coalition for epidemic preparedness innovations (cepi) was set up with the mission of stimulating and accelerating the development of vaccines against emerging infectious disease and enabling access to these vaccines for people during outbreaks [ ] . cepi is presently working on the preparedness and response against five priority pathogens: chikungunya, lassa virus, marburg virus, mers coronavirus, and nipah virus, thereby covering some of the who research and development 'blueprint' priority diseases [ ] . cepi's work includes manufacturing across the supply chain, stockpile establishment, and research to accelerate vaccine development with the aim to stop epidemics before they turn into large-scale health emergencies. firming up synergies between accelerated disease control efforts and routine immunization can contribute to achieving immunization goals. using the added attention on outbreaks, vaccination campaigns can also be used to actively look for and refer inadequately immunized persons to regular vaccination sessions, thus reinforcing overall immunization systems [ ] . extensive global progress has been made using immunization strategies specifically adapted to low-and lower middleincome countries and significant reductions in vpd and mortality were achieved. the many factors affecting vaccine implementation, ranging from weak health systems to humanitarian crises, outbreaks, hesitancy, and flagging demand for vaccination show the clear need for continued support of implementation of these strategies to sustain overall health gains and reach globally agreed immunization targets. equitable access to vaccines across and within countries is key to success. robust delivery mechanisms are needed if vaccines are to reach their full potential. to be effectively implemented, vaccines should be integrated into primary health care, so that vaccine schedules can be optimized for all age groups (e.g., by using school, adolescent, and other agespecific health services) and opportunities to vaccinate are not missed. immunization should be delivered in peoplecentered ways as part of primary health care in order to reach universal health coverage. in addition, vaccines are well placed for preparing for and responding to epidemics and may exert a major positive effect on amr. the next decade will see new and improved vaccines, opportunities, and technologies to build better immunization services. global immunization partners are presently developing the ia , which aims to exploit these opportunities by positioning immunization as a human right and as an investment to make the world healthier, safer, and more prosperous. it will include strategic priorities and worldwide goals for the new decade and will be accompanied by diseasespecific strategies, regional and country plans, immunization partner strategies, and an overall monitoring and evaluation framework. the strategy will be endorsed by the world health assembly (wha) in may and is designed to further evolve according to changing needs [ ] . meeting the challenges of immunization in the near future will require commitment and contributions from many stakeholders, including national governments, global agencies, development partners, regional bodies, and civil society, and such commitment is one of the crucial links for sustainability, delivering more vaccines to more along the full life course. united nations children's fund ( ) #vac-cineswork: vaccines are safe and save lives. un news global perspective expanded programme on immunization and primary health care protecting the world's children: the story of who's immunization programme national immunization programmes global vaccine action plan gavi the vaccine alliance ( ) gavi's strategy phase iv ( - ) and v immunization agenda . a global strategy to leave no one behind status of new vaccine introduction-worldwide immunization today and in the next decade-assessment report of the global vaccine action global vaccine action plan: report by the director-general. st world health assembly. who, geneva . world health organization ( ) who vaccine-preventable disease monitoring system. global summary children: reducing mortality fact sheets subnational immunization coverage data towards equity in immunisation jabs and jags: a qualitative exploration of barriers and facilitators to child and adult immunisation uptake among gypsies, travellers and roma immunization divide: who do get vaccinated in bangladesh? ethnicity and delay in measles vaccination in a nairobi slum inequalities in full immunization coverage: trends in low-and middle-income countries unimmunized children: urbanrural differences in top countries with un-and under-immunized children country urbanization profiles: a review of national health or immunization policies and immunization strategies global vaccine action plan: progress report the impact of new vaccine introductiononimmunizationandhealthsystems: a review of the published literature world health organization ( ) principles and considerations for adding a vaccine to a national immunization programme. from decision to implementation and monitoring global routine immunization strategies and practices (grisp): a companion document to the global vaccine action plan (gvap) commentaryto: guideto tailoring immunization programmes in the who european region world health organization ( ) global health observatory data. health equity monitor world health organization regional office for europe ( ) equity in immunization. a technical guide for addressing inequities in immunization in: an overview of research on the effects of results-based financing. knowledge centre for the health services at the norwegian institute of public health (niph) national legislation and spending on vaccines in latin america and the caribbean mission indradhanush makes vaccination progress in india national immunization programme-mission indradhanush programme: newer approaches and interventions the world bank in middle income countries middle income country task force ( ) the middle income country strategy-enhancing sustainable access to vaccines for populations in middle-income countriesi the assessment report of the global vaccine action plan challenges to sustainable immunization systems in gavi transitioning countries gavi's role in market shaping and procurement: progress, challenegs, and recommendations for an evolving approach challenges of vaccine presentation and delivery: how can we design vaccines to have optimal programmatic impact? vaccine exploring new packaging and delivery options for the immunization supply chain life-course immunization as a gateway to health promote health, keep the world safe, serve the vulnerable ending preventable child deaths from pneumonia and diarrhoea by . development of the integrated global action plan for the prevention and control of pneumonia and diarrhoea protecting all against tetanus: guid to sustaining maternal and neonatal tetanus elimination and broadening tetanus protection for all populations who leads the way towards the elimination of cervical cancer as a public health concern building on the success of the expanded programme on immunization: enhancing the focus on disease preventionandcontrol can vaccination coverage be improved by reducing missed opportunities for vaccination? findings from assessments in chad and malawi using the new who methodology enhancing immunization during second year of life by reducing missed opportunities for vaccinations in countries changing priorities in vaccinology: antibiotic resistance moving to the top impact of existing vaccines in reducing antibiotic resistance: primary and secondary effects vaccines to tackle drug resistant infections. an evaluation of r&d opportunities impactofpneumococcalconjugate vaccineoninfectionscausedbyantibiotic-resistant streptococcus pneumoniae extensively drug-resistant typhoid fever in pakistan call to action on antimirobial resistance r.t.g. ghana government, uk government approaches to vaccination among populations in areas of conflict global polio eradication initiative ( ) polio endgame strategy - : eradication, integration, certification and containment world health organization ( ) global measles and rubella strategic plan prediction and prevention of the next pandemic zoonosis global trends in emerging infectious diseases infectious disease threats in the twenty-first century: strengthening the global response assessing global preparednessforthenextpandemic: development and application of an epidemic preparedness index world health organization ( ) vaccination in acute humanitarian emergencies: a framework for decisionmaking coalition for epidemic preparedness innovations annual review of diseases prioritized under the research and development blueprint for this article no studies with human participants or animals were performed by any of the authors. all studies performed were in accordance with the ethical standards indicated in each case. key: cord- -sq mq f authors: ciabattini, annalisa; garagnani, paolo; santoro, francesco; rappuoli, rino; franceschi, claudio; medaglini, donata title: shelter from the cytokine storm: pitfalls and prospects in the development of sars-cov- vaccines for an elderly population date: - - journal: semin immunopathol doi: . /s - - - sha: doc_id: cord_uid: sq mq f the sars-cov- pandemic urgently calls for the development of effective preventive tools. covid- hits greatly the elder and more fragile fraction of the population boosting the evergreen issue of the vaccination of older people. the development of a vaccine against sars-cov- tailored for the elderly population faces the challenge of the poor immune responsiveness of the older population due to immunosenescence, comorbidities, and pharmacological treatments. moreover, it is likely that the inflammaging phenotype associated with age could both influence vaccination efficacy and exacerbate the risk of covid- -related “cytokine storm syndrome” with an overlap between the factors which impact vaccination effectiveness and those that boost virulence and worsen the prognosis of sars-cov- infection. the complex and still unclear immunopathological mechanisms of sars-cov- infection, together with the progressive age-related decline of immune responses, and the lack of clear correlates of protection, make the design of vaccination strategies for older people extremely challenging. in the ongoing effort in vaccine development, different sars-cov- vaccine candidates have been developed, tested in pre-clinical and clinical studies and are undergoing clinical testing, but only a small fraction of these are currently being tested in the older fraction of the population. recent advances in systems biology integrating clinical, immunologic, and omics data can help to identify stable and robust markers of vaccine response and move towards a better understanding of sars-cov- vaccine responses in the elderly. . , while the m:f ratio of deaths is around . , suggesting that, despite being more frequently infected, females are more capable of dealing with the infection [ , ] . it is widely reported that most deaths occurred among patients with at least one underlying disease, such as hypertension [ ] and diabetes mellitus [ ] . a meta-analysis of seven clinical studies performed in china identified chronic obstructive pulmonary disease (copd), cardiovascular disease, and hypertension as risk factors for severe disease and intensity care unit (icu) admission [ ] . analysis of risk factors associated with more than ten thousand deaths by covid- in the uk confirmed that age was linearly correlated with risk of death and that obesity, diabetes, severe asthma, respiratory disease, neurological disease (including stroke), recent (< years) hematological malignancy, and recent (< year) cancer diagnosis were all associated with higher death risk. as for hypertension, the hazard risk was higher only for the population < years old, even if hypertension itself was strongly associated with other risk factors such as obesity and diabetes [ ] . importantly, these epidemiological studies identify the categories of subjects who are at higher risk of developing severe sars-cov- infection and that should be prioritized in vaccine administration. the massive effort for the development of a vaccine against sars-cov- could be frustrated by the poor responsiveness to vaccination that characterizes a large proportion of the elderly population. in this rush against the time, we risk to pay a dear toll for the lack of knowledge in the response to vaccination of the elderly, a well-known issue, neglected notwithstanding its evident urgency and the annual reproposal through the seasonal influenza epidemic above all. interestingly, there is a consistent overlap between the factors hampering vaccination effectiveness in the elderly and those that boost the virulence and worsen the prognosis of sars-cov- infection. a common characteristic of the elderly people is the onset of a sterile low-grade increase of the basal inflammatory state named "inflammaging," which is considered a universal etiological agent of most of the age-related diseases [ ] . it is likely that some specific components of the inflammaging phenotype could both influence vaccination efficacy and then increase the risk of the early massive production of inflammatory cytokines, termed the "cytokine storm syndrome." this is a condition reported in severe covid- cases during which the patient's immune system spins out of control and starts damaging healthy organs owing to the increased vascular permeability, vascular paralysis, and hypovolemic shock [ ] . angiotensin-converting enzyme (ace ) has been identified as the receptor for sars-cov- , and it has been suggested that differential levels of ace in the cardiac and pulmonary tissues of younger versus older adults may be at least partially responsible for the spectrum of disease virulence observed among patients with covid- [ ] . here, we analyze the different aspects that tackle sars-cov- vaccination in the elderly population, considering immunologic, genetic, and socio-economic factors that impact on the age-related changes of immune responses. a view of the current available vaccine platforms with a special focus on the clinical trials including older adults is reported. how the elderly condition can affect covid- disease progression and the response to vaccination immunosenescence for many reasons, it is difficult to clearly define what immunosenescence is: (i) immunosenescence is quite complex and involves cellular and molecular changes occurring lifelong (from newborns to centenarians) in both the innate and the adaptive immune systems; (ii) these changes can be at the same time detrimental and beneficial/adaptive [ ] ; (iii) it is difficult to identify a unique common marker of immunosenescence, due to the overwhelming number of biological and non-biological factors that can impinge lifelong on the immune system of each individual; (iv) the changes occurring with age in the immune system are deeply correlated with the profound environmental, epidemiological, lifestyle, societal, medical, and public health changes, including vaccination policies and practices, that occurred in the last century. accordingly, immunosenescence is highly contextdependent [ ] , different in different geographical and historical settings and in men and women, correlated to socioeconomic position, and sensitive to psychological stressors. indeed, both the adaptive and the innate immune systems have the capability of "remembering" all immunological stimuli a person has been exposed to lifelong. we have conceptualized this situation with the term immunobiography, which should help in understanding the enormous heterogeneity of the immune phenotype in old people. this is also the reason why there is a sort of imprinting in the immune responses favoring those towards antigens that have been experienced early in life [ ] . the complex biological processes of aging are the result of alterations in gene regulation and protein expression, signaling pathways, and biological networks. complex changes, including pervasive epigenetic and metabolic modifications, affect most of the subsets of naïve, memory, regulatory effector t cells, and b cells [ ] [ ] [ ] . despite the challenging complexity, a universally observed hallmark of immunosenescence is the decrease of naive t cells (particularly cd + t cells) in peripheral blood [ ] consequent to thymic involution responsible for the early decline in the output of naïve t cells to the periphery and for the related shrinking of the t cell repertoire [ ] [ ] [ ] . other important aging-related alterations are (i) the shift in the bone marrow maturation of hematopoietic cells towards myelocytic differentiation [ ] , concomitant with a reduced lymphopoiesis, mainly due to changes in progenitor cells in the bone marrow [ , ] ; (ii) the increased numbers of memory cells owing to large clonal expansion towards epitopes of persistent viral infections (cytomegalovirus [cmv] and epstein barr virus [ebv]) [ , ] ; (iii) the compromised ability of cd + t cells to differentiate into functional subsets, resulting in a multitude of dysregulated responses, such as a reduced cognate help to b cells with consequent reduced humoral immunity, and the increased ratio of the proinflammatory th cells with respect to the immunosuppressive t regulatory cells, thus favoring a basal proinflammatory status [ , ] ; (iv) accumulation of differentiated exhausted t cells, induced by a repeated pathogen encounter during chronological aging, and end-stage differentiated senescent t cells, characterized by a progressive reduction of telomere length leading to a state of proliferative arrest [ ] . with aging, health conditions associated with immune senescence, comorbidities (particularly noncommunicable diseases such as heart disease, cancers, and metabolic and autoimmune diseases), and pharmacological treatments affect the immune responses to both vaccines and infectious diseases. overall, as a result of immunosenescence, the elderly population is more susceptible to infections, particularly to influenza, streptococcus pneumoniae rsv, and group b streptococcus but also to opportunistic, re-emergent chronic infections such as herpes zoster as well as antibiotic-resistant nosocomial pathogens. the reduced adaptive immune response, together with altered innate cell function, such as chemotaxis, phagocytosis, signaling pathways, and intracellular killing, prevents the appropriate control of the initial inflammatory response elicited upon viral infection. for rna virus, such as coronavirus, different pattern recognition receptors (prr) are triggered on the innate cells during the early phases of infection. these include the endosomic toll-like receptor and and the cytosolic rig-i/mda- molecules, which recognize viral rna [ ] , and the cgas-sting pathway, which recognizes cytosolic dna [ ] activated by cellular damage and mitochondrial dna release caused by viral infection [ ] . the stimulation of these prr leads to the expression of type i ifn, a factor that limits viral replication through the stimulation of interferon-stimulated genes, and other inflammatory cytokines [ ] . for middle east respiratory syndrome (mers)-cov, the timing of type i ifn production appears to dictate the outcome of infection in mouse models, and its administration within day after infection was protective against lethal infection, while a delay in ifn production caused an inability to control viral replication, leading to cellular damage of airway epithelia and the lung parenchyma and an eventual lethal inflammatory cytokine storm [ ] . the latter response often predominates in older individuals and in aged mouse models of sars-cov- infection [ , ] . induction of innate immune responses is a crucial step in the pathophysiology of covid- disease (fig. ) . on one hand, it triggers the anti-viral host defense mechanisms necessary for elimination of infection, but on the other hand, it may contribute to hyperinflammation and tissue damage during the later stages of the disease in a minority of patients [ ] . this can be particularly relevant in the elderly population in which inflammaging, the state of chronic low-grade sterile inflammation [ ] , characterized by high serum concentrations of c-reactive protein (crp), il- , il- , and tumor necrosis factor (tnf)-α, can be present. tissue damage in covid- is mainly mediated by an excess of immune response to the virus, which results in a cytokine storm, with activation of the il- signaling pathway. the pathophysiology of sars-cov- infection has strong similarities to other severe viral lung infections caused by sars-cov- and mers-cov. one of the first published studies on clinical features of covid patients hospitalized in wuhan showed that proinflammatory cytokines and chemokines, such as tnf-α, granulocyte-colony stimulating factor (g-csf), interferon g a m m a -i n d u c e d p r o t e i n - ( i p - ) , m o n o c y t e chemoattractant protein- (mcp- ), and macrophage inflammatory proteins -α (mip- α), were significantly higher in patients admitted to the intensive care unit (icu) compared to those who were not in icu [ ] . immune pathology in the form of vascular and cutaneous lesions has also been widely reported [ , ] . the role of a dysregulated inflammatory response was proven in an animal model of sars-cov- infection using aged macaques. aged animals are more prone to develop severe disease and activate more readily the innate response, in particular the nf-kb pathway and proinflammatory cytokines such as il- and il- β, while not inducing significantly ifn-β response. the innate immunity activation is not due to the viral load, which is comparable among young and aged macaques [ ] . transcriptomic analysis performed in samples from subjects with severe covid- revealed the presence of low levels of type i and type iii interferon genes together with elevated levels of proinflammatory cytokines and chemokines, such as il- , il ra, ccl , ccl cxcl , cxcl , cxcl , and cxcl [ ] . which type of cells elicits this cytokine storm and the virological mechanisms behind this inflammatory reaction are still unclear [ ] . lung epithelial cells, alveolar macrophages, dendritic cells, and endothelial cells can effectively release the proinflammatory cytokines and chemokines, thus attracting monocytes, macrophages, and t cells to the site of infection [ ] . the overproduction of proinflammatory cytokines in the lungs can damage the tissue infrastructure, recruit macrophages that infiltrate air spaces, and generate the respiratory failure from acute respiratory distress syndrome (ards), which is recognized as the leading cause of mortality. meanwhile, the direct attack on other organs by disseminated sars-cov- , the immune pathogenesis caused by the systemic cytokine storm, and the microcirculation dysfunctions together may lead to multi-organ damage, even though whether sars-cov- can directly target organs other than the lung and how it can happen are aspects that need to be further investigated [ ] (fig. ) . together with the hyperinflammatory response, a significant lymphopenia, mainly related to cd + t and cd + t cells, which correlates with the severity of viral infection, was reported [ ] [ ] [ ] . the causes of this adaptive immunity suppression are still unclear. pulmonary recruitment of immune cells from the blood and the infiltration of lymphocytes into the airways may explain the reduction in blood. the well- systemic and local (lung) immune responses and their pathological role, following sars-cov- entry into the host are schematically represented. induction of innate immune responses is a crucial step in the pathophysiology of covid- disease, contributing to hyperinflammation and tissue damage during the later stages of the disease. infiltration of immune cells in the lungs causes overproduction of proinflammatory cytokines, which eventually damages the lung infrastructure, accumulation of macrophages in the air spaces and diffuse alveolar damage leading to acute respiratory distress syndrome (ards). furthermore, elevated levels of circulating proinflammatory cytokines can cause septic shock and multi-organ dysfunction. together with the hyperinflammatory response, overt disseminated intravascular coagulation has been reported and a significant lymphopenia, mainly related to cd + t and cd + t cells, has been observed, possibly due to pulmonary recruitment of lymphocytes from the blood. a possible immunopathological role can be mediated by non-neutralizing antibodies produced by b cells, which may enhance sars-cov- infection through antibody-dependent enhancement (ade), further exacerbating organ damage known age-related alteration of the immune function of t cell and b cells could lead to insufficient control of viral replication, thus increasing the macrophage infiltration and the lung injury (fig. ) . finally, a possible immunopathological role can be mediated by non-neutralizing antibodies produced by b cells that may enhance sars-cov- infection through antibodydependent enhancement (ade), further exacerbating organ damage. it has recently been shown that sars-cov- and the mers-cov take advantage of non-or subneutralizing antibodies and enter cells via surface cd a receptors, an fc receptor expressed on the surfaces of monocytes and alveolar macrophages. the antibody-cd interaction facilitates viral entry and infection, and activates intracellular signaling to upregulate proinflammatory cytokines [ ] . the complex and still unclear immunopathological mechanisms of sars-cov- infection, together with the progressive age-related decline of innate and adaptive immune responses, and the lack of a clear correlate of protection, make the design of vaccination strategies for older people extremely challenging (fig. ). an emerging class of instruments in the aging research is the development of markers capable of assessing the speed of the aging process. age is a major risk factor for a high number of diseases, and in general, it affects the fitness of each individual, including the capability of responding to vaccine administration and counteracting a severe infection [ ] . however, it is also evident that the elderly population is extremely heterogeneous, so while chronological age is useful to identify macroscopic risk classes, it is poorly informative within age classes to get individual information. biological age is thus useful to evaluate clinical parameters and health risks on the basis of the individual aging pace, which tend to be more heterogeneous in the elderly population. several established biological age markers have been generated based on both classical anthropometric, clinical, and biochemical parameters as well as on innovative molecular characterizations such as dna methylation and the composition of the n-glycan shell of circulating proteins [ ] . such biomarkers have shown in a number of studies that the aging pace is higher in the vast majority of the different elderly conditions, thus demonstrating that biological age assessment should be a critical information in a broad spectrum of clinical practices and in the development of strategies to tackle healthcare burden and emergencies. the detailed description of available biological age markers is out of the scope of the present manuscript, and an extensive overview is available in the review by jylhävä et al. [ ] . to date, biological age has not been assessed in the sars-cov- clinical setting, but it is noteworthy that biological age has been associated with all the most important risk factors related to a poor prognosis of sars-cov- infection. the field of elderly vaccination could benefit from biological age information, but also in this case, the available data are rare. in a study from gensous et al. [ ] , the whole genome methylation profile of pbmc was assessed in a group of volunteers of different ages who underwent influenza vaccination. the relationship between the vaccination response and the methylation profile was studied. while no difference in terms of biological age emerged in the study, an agedependent epigenetic remodeling emerged in elder non-responders. the study is limited owing to the very low number schematic interconnection between the main immune mechanisms elicited by the vaccination process, with the peculiarity of the elderly immune system-affected by both inflammaging and immunosenescence-and the still undefined correlates of protection from sars-cov- infection. the complex and still unclear immunopathological mechanisms of sars-cov- infection, together with the progressive agerelated decline of innate and adaptive immune responses, and the lack of a clear correlate of protection make the design of vaccination strategies for older people extremely challenging of analyzed subjects but confirmed that dna methylation is an informative instrument to be exploited in vaccination studies and strategies. immunobiography refers to the comprehensive immunological, clinical, socio-economic, and geographical history of each individual, and accounts for the large heterogeneity observed in the elderly regarding their health status, mirrored by their large individual variation in the responsiveness to vaccines. a major advantage of immunobiography is that it incorporates the most advanced conceptualization of immunosenescence which, according to the most recent literature [ ] , has to be considered as a context-and population-dependent phenomenon. accordingly, in order to be properly interpreted, agerelated changes of immune parameters occurring in an elderly person necessitate a variety of other additional data regarding sex/gender, demographic cohort, population/country, individual immunological history, anthropometric parameters, socioeconomic status and education, cmv serostatus, morbidity and co-morbidity, among others. it is of critical importance taking in consideration the elderly vulnerability to direct the rational design of vaccines designed for this target population. gender is a critical issue in both vaccination of the elderly and in the sars-cov- pandemic. the pandemic epidemiological data show clearly that the risk of severe disease and mortality is sharply higher in men than in women. men's hospitalization exceeds women by about %, indicating a significantly higher susceptibility of men towards severe sars-cov- infection. available data show that men outnumber women to times in terms of icu admissions [ ] [ ] [ ] . these numbers are concordant with the fatality rate that ranges between . and . men deaths for one women death. moreover, this unbalanced pattern is mirrored by the vaccine uptake, responses, and outcome in older-aged individuals. elderly women are indeed more responsive than men for several vaccine protocols recommended in older-aged individuals such as those against influenza, tetanus, pertussis, shingles, and pneumococcal infections [ ] . on the other hand, an influenza vaccination study reported that aged men antibodies had higher affinity than those produced by women. moreover, men seem to respond better to pneumococcal vaccination in two independent studies [ , ] . there is an impaired vaccination response in both old men and women with sex-specific weaknesses. the most striking data, however, is related to infection and all-cause mortality: indeed, in a number of reports, vaccine administration produces a sharper decrease of specific and all-cause mortality in vaccinated women compared to men, indicating that women have higher benefit from vaccination in the elderly [ ] [ ] [ ] . these data indicate the need to consider sex-specific vaccination protocols for the elderly population [ , ] and that the lack of such instruments could be critical in the sars-cov- pandemic since old men are both the most susceptible to severe sars-cov- infection and are those less likely protected by a possible sars-cov- vaccine [ ] [ ] [ ] . another factor that could affect vaccine response is the intestinal microbiota that plays a crucial rule in the regulation of the immune system and is highly affected by age [ ] [ ] [ ] [ ] . microbial community composition indeed is influenced by age, environmental and socio-economic factors, diet, gender, chronic infections, immunosuppressive chemotherapy, antibiotic treatment, or probiotic use [ , [ ] [ ] [ ] . the improvement in the nucleic acid sequencing obtained in the last years hits massively the microbiological research and promotes the analysis of heterogeneous microbiological ecosystems such as those that reside in humans. the characterization of such ecological niches opens to the new conceptualization of humans as metaorganisms (organisms composed of different organisms) to stress the tight interdependencies between the host and the microbiological species residing in different anatomical districts. gut microbiota changes with age and that is likely an important contributor and modulator of the inflammaging phenotype [ , ] . elderly people have less diverse gut microbiota and reduced beneficial microorganisms [ ] . the general imbalance of gut microbiota, called "dysbiosis," is associated with both frailty, a geriatric syndrome leading to increased vulnerability for adverse health outcomes, and systemic inflammation. since a hyperinflammation status has been observed in most severe cases of sars-cov- infection, it is possible that gut dysbiosis may influence the clinical manifestation in covid- infection [ , ] . interestingly, the gut microbiota has been shown to also affect pulmonary health through a bidirectional cross-talk between the gut microbiota and the lungs [ ] . along this "gutlung axis," microbial products can reach the lung through blood and modulate pulmonary immune responses [ ] , while inflammation processes occurring in the lung can impact on the gut microbiota [ ] . some studies have demonstrated that respiratory infections are associated with a change in the composition of the gut microbiota [ ] and the antibiotic treatment of mice for removing some gut bacteria has led to increased susceptibility to influenza virus infection in the lungs [ ] . since one of the severe clinical manifestations of covid- is pneumonia and progression to acute respiratory distress syndrome (ards), especially in elderly and immunecompromised patients [ ] , it can be speculated that sars-cov- infection can affect this gut-lung cross-talk which might influence the outcome of the clinical manifestation [ ] . moreover, even though respiratory symptoms represent the principal clinical presentation of covid- , clinical evidence suggests that the intestine may be another viral target organ. indeed, a high expression of ace has been observed in the brush border of intestinal enterocytes [ ] and, using a human small intestinal organoid system, it has been demonstrated that sars-cov- readily replicates into the enterocytes, resulting in the production of large amounts of infective virus particles [ ] . some reports show that sars-cov- rna can be detected in the stool of some patients of covid- [ , ] , and patients often present gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain [ ] . therefore, the characterization of the gut microbiota in patients with active sars-cov- intestinal infection could represent a striking aspect to investigate. these considerations on inflammaging, immunobiography, biological age, gender, and microbiota pertain to every vaccination strategy, but are particularly relevant for the development of vaccines against sars-cov- since it more seriously affects the elderly population and immunopathology is a crucial factor for the severity disease. need for the design of a sars-cov- vaccination strategies tailored for the elderly sars-cov- vaccines are urgently needed, and their design should take into consideration that the elderly population is the main target population for vaccination. while older adults are most likely to be severely affected by covid- , they also may be less responsive to vaccination. efficacy of vaccination in the elderly is indeed strongly reduced compared to that of younger adults [ , ] . sars-cov- vaccination strategies, tailored for the elderly, should take into consideration the delicate balance between immunosenescence/ inflammaging and the immunopathological aspects of the covid- disease (fig. ) . vaccine adjuvants and vectors should be specifically designed for stimulating the elderly immune system without exacerbating the inflammatory status [ ] . despite these considerations, the elderly are rarely included in vaccine clinical trials; in the last decades, the vast majority of randomized control trials did not include older adults and in particular frail older adults who are mostly at risk. we currently do not have full knowledge on the mechanisms of immunity to protect this population from sars-cov- [ ] . the development of a sars-cov- vaccine is extremely challenging, since we are faced with a novel virus, just emerged in humans, and correlates of protection have not yet been fully identified, even though the induction of neutralizing antibodies is presumed to be a crucial target for an effective vaccination (fig. ) . protection in older individuals against influenza virus appears to require higher neutralization titers than in younger individuals [ ] , and this issue might need to be addressed for sars-cov- . the knowledge obtained from the vaccine development efforts for mers and sars-cov- can be of high value for sars-cov- , although no vaccines are licensed for these coronavirus strains [ ] . memory cd + t cells, induced by infections with other coronavirus and capable of responding to sars-cov- , have been detected in - % of sars-cov- unexposed donors [ , ] . the characterization of these cross-reactive t cells in the elderly and their impact on the immunogenicity of vaccine candidates should be taken into consideration in the ongoing covid- vaccination studies. sars-cov- vaccine candidates based on different vaccine platforms have been developed, and about candidates have been tested in pre-clinical experiments, according to the who landscape documents of covid- candidate vaccines (https://www.who.int/publications/m/ item/draft-landscape-of-covid- -candidate-vaccines) (fig. ) . information on the specific sars-cov- molecules selected as vaccine antigens is limited, even though most candidates aim to elicit neutralizing antibodies against the spike (s) protein and its receptor-binding domain (rbd), as already performed with the sars and mers vaccines. a wide range of both innovative and traditional technology platforms has been deployed, including nucleic acid (dna and rna), recombinant viral vectors (replicating and non-replicating), recombinant protein combined with adjuvants, and live attenuated or inactivated virus [ ] . some of these platforms were already tested in human studies for sars-cov- virus, such as inactivated virus, dna and soluble s proteins [ ] [ ] [ ] , or for mers-cov [ ] . the most advanced candidates for sars-cov- entered in human clinical testing with unprecedented rapidity employ nucleic acid (both mrna and dna), recombinant vaccine vectors (human or chimpanzee adenovirus vectors), subunit s protein combined or not with different adjuvants, and inactivated sars-cov- virus. other novel platforms based on the use of synthetic modified antigen presenting cells (apc) or cytotoxic t lymphocytes are also under study (fig. ) . the platforms using mrna, nonreplicating viral vectors, and inactivated sars-cov- virus have already reached the clinical trial phase iii. some of the different platforms used may be tailored for specific population subtypes, such as the elderly, children, pregnant women, or immunocompromised patients [ ] . in this regard, some of the ongoing clinical studies have specifically taken into consideration the older population, by including vaccination arms with people aged > years. a schematic diagram of the ongoing phase i and ii clinical trials that have included older adults is reported in fig. . enrolling older adult volunteers will help to better understand vaccination outcomes among the older population, who are most at risk of complications from covid- . the ongoing clinical studies based on mrna technology (mrna- from moderna n. nct , and bnt from biontech se, n. nct ) aim to evaluate the safety, tolerability, immunogenicity, and potential efficacy of different sars-cov- rna vaccine candidates in the adult population, with a specific attention to older people (n.-n. releases. nih clinical trial of investigational vaccine for covid- begins. . https://www.nih.gov/newsevents/news-releases/nih-clinical-trial-investigationalvaccine-covid- -begins). the lipid nanoparticleencapsulated mrna- vaccine, which encodes for the full-length s protein, is currently evaluated in a dose-ranging study in the adult population ( - years old), and in participants from to and > years of age (fig. ) . similarly, the large dose-finding study with the bnt biological component ( estimated participants) based on the administration of mrna coding for the full-length s protein, or for the two smaller receptor-binding domains, is going to test the immunogenicity in adults ( - years) and older adults ( - years) . an ongoing phase i/iia trial (n. nct ) is also aimed at evaluating the safety, tolerability, and immunological profile of the ino- vaccine that, exploiting the dna technology, contains a plasmid encoding the full-length s glycoprotein. the ino- vaccine is administered by intradermal injection followed by electroporation in healthy adults aged to years. another platform that is currently specifically tested in older people is based on the adenovirus type vector that encodes the s protein from the sars-cov- strain (trials n. - - ; pactr ; nct ; chictr and nct ; fig. ). different studies are ongoing, and one conducted in canada is a doseescalation designed study, from the younger adults ( to < ) to the older adults ( to < ). another huge phase / study (n. nct ) is aimed at determining the efficacy, safety, and immunogenicity of the candidate covid- vaccine based on the chimpanzee adenovirus vector (chadox ncov- ) in healthy uk volunteers, specifically divided in adults ( - years old), elderly (over the age of ), and children ( - years old). the chadox platform has already been shown to be effective in the established rhesus macaque model of sars-cov- infection [ ] . in this pre-clinical study, a single dose of chadox ncov- has protected six [ ] . moreover, the chadox has been used to develop investigational vaccines against several pathogens, including the closely related coronavirus responsible for the mers [ ] . adenovirus-based vectors are characterized by a broad range of tissue tropism that covers both respiratory and gastrointestinal epithelium, the two main sites that express the ace- receptor of sars-cov- , even though a possible immunodominance mediated by vector genes rather than the transgenes should always be considered [ ] . using the traditional recombinant protein technology to express the spike protein, a trial sponsored by clover biopharmaceuticals aus pty ltd. (n. nct ) is assessing the safety, reactogenicity, and immunogenicity of multiple doses of scb- administered with as adjuvant, or with cpg plus alum adjuvants. data will be separately analyzed on adult ( to years of age) and elderly ( - years of age) healthy subjects enrolled in the study. in another study, the s protein has been administered with the advax adjuvant (n. nct ), a potent and safe immunopotentiator composed of delta inulin [ ] . four trials are testing in the elderly population the inactivated sars-cov- virus (n. nct ; chictr ; chictr ), and one of these has been specifically performed only in people > years (n nct ; fig. ). numerous other vaccine developers have indicated plans to initiate human testing in . despite the several vaccine candidates (fig. ) , challenges including the need for optimizing antigen design and adjuvant formulation define the number of doses needed, induce the optimal immune response without exacerbating the inflammatory and antibody-dependent response involved in possible lung disease, and fully define correlates of protection and duration of immune responses have to be considered [ ] . finally, a general consideration for the sars-cov- vaccine development regards safety issues that could arise with covid- vaccines developed under the strong pressure of the pandemic situation. animal studies on vaccines for sars-cov- and mers-cov report possible adverse effects mediated by vaccine-induced antibodies that have poor or no neutralizing activity [ ] . safety and efficacy are two indissoluble properties of a vaccine to be administered to billions of people globally and need to be accurately evaluated for every sars-cov- candidate. the efforts in the development of covid- vaccines can benefit from the availability of most advanced tools and high-throughput technologies to decipher the effective immune responses in the older population and the correlates of protection. recent advances in systems biology integrating clinical, immunologic, and omics data can help to identify stable and robust markers of vaccine response and move towards a better understanding of sars-cov- vaccine responses in the elderly. machine/statistical learning applied to multi-omics data from clinical studies promises to revolutionize vaccine development by illuminating the mechanistic drivers of protective immunity. the high-performance data acquisition methods in molecular and cellular biology push the field of bioinformatics for the development and use application of the immunobiography approach could inform the stratification of elderly subjects and guide the implementation of vaccination strategies designed for specific elderly population clusters [ ] . mathematical modeling allows the combination of different networks involved in biological aging such as epigenetic networks, cell-cell networks, and population genetics and can allow to generate hypothesis on response to treatment or vaccination [ ] . recent progress in mathematical modeling can be utilized to generate biomarker models for prediction of disease and also response to vaccination taking into consideration biological age. currently, computational models have been applied to immunology data, for example, for the analysis of a high-dimensional dataset in vaccination studies [ , ] , but these models are limited to particular aspects [ , ] . there is the potential for these models to become more sophisticated and to predict how responses to pathogens and vaccines are affected by pre-disposing factors [ , ] . the systems vaccinology approach has been applied to characterize the immune response to different vaccines providing the proof-of-concept evidence of the capacity of systems approaches to delineate "molecular signatures" predictive of vaccine responses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this approach has also been applied to identify molecular signatures induced by immunization with the rvsv-zebov ebola vaccine, recently approved for human use. systems analysis has been conducted integrating clinical, immunologic, and omics data in clinical trials with different doses and in different continents (vianello et al. submitted, santoro et al. submitted). despite the great efforts made, unfortunately, many of the most useful clinical and multi-omics datasets are siloed in local databases to protect participant privacy and data confidentiality. creation of secure, faircompliant, federated learning databases in which predictive biological and mathematical models based on ai/ machine/statistical learning can be developed, refined, and tested on distributed datasets would have an enormous impact in supporting a rational vaccine development. sars-cov- vaccines are urgently needed, and their design should take into consideration that the elderly are the main target population for vaccination. the pandemic is stimulating the research on vaccine development, and this should be a tremendous opportunity to specifically include age and gender as critical factors for vaccination approaches and effectiveness. while older adults are most likely to be severely affected by covid- , they also may be less responsive to vaccination. in the ongoing tremendous efforts for covid- vaccine development, only a limited number of clinical trials have included the older fraction of the population in the study design, and the platforms used are not specifically designed considering the peculiarity of the elderly immune system. indeed, vaccination strategies tailored for the sars-cov- infection in the elderly should take into consideration the delicate balance of immunosenescence and inflammaging with the immunopathological aspects of the sars-cov- infection, such as the cytokine storm reported in severe covid- . therefore, the possible overlap between the factors hampering vaccination effectiveness in the elderly and those that boost the virulence and worsen the prognosis of sars-cov- infection should be carefully taken into consideration. thus, vaccine formulations, such as adjuvants and vectors, should be specifically designed for stimulating the elderly immune system without exacerbating the inflammatory status. the ongoing efforts in covid- vaccine development should fully exploit the availability of high-throughput technologies and recent advances in systems biology to decipher the effective immune responses in the older population and identify correlates of protection to guide towards sars-cov- vaccine strategies optimally designed to protect the older population. authors' contributions ac, pg, and dm, fs drafted the work; dm, rr, and cf revised it critically for important intellectual content; all the authors approved the version to be published. funding open access funding provided by università degli studi di siena within the crui-care agreement. this work was supported by commission of the european communities, horizon framework programme, grant number (transvac ), and russian ministry of science and education agreement no. - - - . conflict of interest the authors declare that they have no conflict of interest. consent for publication the authors are responsible for the correctness of the statements provided in the manuscript. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. estimates of the severity of coronavirus disease : a model-based analysis sex differences in immune responses sexx matters in infectious disease pathogenesis covid- with different severities: a multicenter study of clinical features risk and predictors of in-hospital mortality from covid- in patients with diabetes and cardiovascular disease predictive symptoms and comorbidities for severe covid- and intensive care unit admission: a systematic review and meta-analysis. public health opensafely: factors associated with covid- death in million patients inflamm-aging. an evolutionary perspective on immunosenescence covid- cytokine storm: the interplay between inflammation and coagulation covid- and immunity in aging populations -a new research agenda immunosenescence and inflamm-aging as two sides of the same coin: friends or foes? front immunol age-related changes of human bone marrow: a histometric estimation of proliferative cells, apoptotic cells, t cells, b cells and macrophages immunobiography and the heterogeneity of immune responses in the elderly: a focus on inflammaging and trained immunity senescence of t lymphocytes: implications for enhancing human immunity ageing, autoimmunity and arthritis: senescence of the b cell compartment -implications for humoral immunity the th /treg balance is disturbed during aging shortage of circulating naive cd (+) t cells provides new insights on immunodeficiency in aging successful and maladaptive t cell aging contributions of age-related thymic involution to immunosenescence and inflammaging age-related modifications of the human alphabeta t cell repertoire due to different clonal expansions in the cd + and cd + subsets inflamm-aging of hematopoiesis, hematopoietic stem cells, and the bone marrow microenvironment age-related changes in lymphocyte development and function mechanisms underlying t cell immunosenescence: aging and cytomegalovirus infection different contribution of ebv and cmv infections in very long-term carriers to agerelated alterations of cd + t cells human t cell immunosenescence and inflammation in aging ) t cells, aging and senescence ribose '-o-methylation provides a molecular signature for the distinction of self and non-semin immunopathol self mrna dependent on the rna sensor mda coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling dengue virus activates cgas through the release of mitochondrial dna immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic. asian pac ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection an interferon-gamma-related cytokine storm in sars patients trained immunity: a tool for reducing susceptibility to and the severity of sars-cov- infection clinical features of patients infected with novel coronavirus in an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study classification of the cutaneous manifestations of covid- : a rapid prospective nationwide consensus study in spain with cases exacerbated innate host response to sars-cov in aged nonhuman primates imbalanced host response to sars-cov- drives development of covid- sars-cov- and viral sepsis: observations and hypotheses the trinity of covid- : immunity, inflammation and intervention clinical and pathological investigation of patients with severe covid- longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov- infected patients hematological findings in coronavirus disease : indications of progression of disease the potential danger of suboptimal antibody responses in covid- biological age predictors the continuum of aging and age-related diseases: common mechanisms but different rates responders and non-responders to influenza vaccination: a dna methylation approach on blood cells does the human immune system ever really become "senescent baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region baseline characteristics and outcomes of patients with covid- admitted to intensive care units in vancouver, canada: a case series presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the new york city area sex and gender impact immune responses to vaccines among the elderly persistence of antibody response to pneumococcal capsular polysaccharides in vaccinated long term-care residents in brazil the immunogenicity of -valent pneumococcal conjugate vaccine versus -valent polysaccharide vaccine in adults aged - years study of the effectiveness of influenza vaccination in the elderly in the epidemic of - using a general practice database effectiveness of influenza vaccine in the community-dwelling elderly effect of influenza vaccine status on winter mortality in spanish community-dwelling elderly people during - influenza periods efficacy and costeffectiveness of influenza vaccination of the elderly in a densely populated and unvaccinated community systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination a single dose of unadjuvanted novel h n vaccine is immunogenic and well tolerated in young and elderly adults immune response following h n pdm vaccination: differences in antibody repertoire and avidity in young adults and elderly populations stratified by age and gender role of the microbiota in the modulation of vaccine immune responses the influence of the intestinal microbiome on vaccine responses antibiotics-driven gut microbiome perturbation alters immunity to vaccines in humans the impact of the microbiome on immunity to vaccination in humans the impact of diet and lifestyle on gut microbiota and human health antibiotic-induced changes in the intestinal microbiota and disease the microgenderome revealed: sex differences in bidirectional interactions between the microbiota, hormones, immunity and disease susceptibility inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty inflammaging: a new immune-metabolic viewpoint for agerelated diseases gut microbiome and aging: physiological and mechanistic insights diet, microbiota and gut-lung connection. front. microbiol frailty syndrome: an overview pulmonary-intestinal cross-talk in mucosal inflammatory disease gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis the role of the lung microbiota and the gut-lung axis in respiratory infectious diseases respiratory viral infection alters the gut microbiota by inducing inappetence collateral effects of antibiotics on mammalian gut microbiomes what we know so far: covid- current clinical knowledge and research gut microbiota and covid- -possible link and implications single cell rna sequencing of human tissues identify cell types and receptors of human coronaviruses sars-cov- productively infects human gut enterocytes prolonged presence of sars-cov- viral rna in faecal samples gastrointestinal manifestations of sars-cov- infection and virus load in fecal samples from a hong kong cohort: systematic review and metaanalysis covid- : gastrointestinal manifestations and potential fecal-oral transmission vaccination in the elderly: the challenge of immune changes with aging efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis hemagglutination inhibition antibody titers as a correlate of protection against seasonal a/h n influenza disease vaccines for sars-cov- : lessons from other coronavirus strains selective and cross-reactive sars-cov- t cell epitopes in unexposed humans sars-cov- -specific t cell immunity in cases of covid- and sars, and uninfected controls sars-cov- vaccines: status report safety and immunogenicity from a phase i trial of inactivated severe acute respiratory syndrome coronavirus vaccine vrc study team, a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial nih clinical trial of investigational vaccine for covid- begins safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase , open-label, single-arm, dose-escalation trial the covid- vaccine development landscape ) respiratory disease in rhesus macaques inoculated with sars-cov- chadox ncov- vaccination prevents sars-cov- pneumonia in rhesus macaques recent advances in the vaccine development against middle east respiratory syndrome-coronavirus a review of sars-cov- and the ongoing clinical trials advax adjuvant: a potent and safe immunopotentiator composed of delta inulin developing covid- vaccines at pandemic speed immunopathogenesis of coronavirus infections: implications for sars the human body as a super network: digital methods to analyze the propagation of aging. front from bivariate to multivariate analysis of cytometric data: overview of computational methods and their application in vaccination studies. vaccines computational analysis of multiparametric flow cytometric data to dissect b cell subsets in vaccine studies a perspective on the role of computational models in immunology computational modelling approaches to vaccinology sensitive detection of rare diseaseassociated cell subsets via representation learning multivariate models for prediction of human skin sensitization hazard systems biology approach predicts immunogenicity of the yellow fever vaccine in humans yellow fever vaccine induces integrated multilineage and polyfunctional immune responses systems biology of immunity to mf -adjuvanted versus nonadjuvanted trivalent seasonal influenza vaccines in early childhood early patterns of gene expression correlate with the humoral immune response to influenza vaccination in humans apoptosis and other immune biomarkers predict influenza vaccine responsiveness systems analysis of immunity to influenza vaccination across multiple years and in diverse populations reveals shared molecular signatures molecular signatures of antibody responses derived from a systems biology study of five human vaccines metabolic phenotypes of response to vaccination in humans defective t memory cell differentiation after varicella zoster vaccination in older individuals predicting rts,s vaccine-mediated protection from transcriptomes in a malaria-challenge clinical trial systems analysis of protective immune responses to rts,s malaria vaccination in humans expression of genes associated with immunoproteasome processing of major histocompatibility complex peptides is indicative of protection with adjuvanted rts,s malaria vaccine integrated analysis of genetic and proteomic data identifies biomarkers associated with adverse events following smallpox vaccination immunomonitoring of human responses to the rvsv-zebov ebola vaccine ebola vaccine r&d: filling the knowledge gaps correlates of vaccineinduced protective immunity against ebola virus disease systems vaccinology identifies an early innate immune signature as a correlate of antibody responses to the ebola vaccine rvsv-zebov merck ad /hiv induces broad innate immune activation that predicts cd + t-cell responses but is attenuated by preexisting ad immunity a cell-based systems biology assessment of human blood to monitor immune responses after influenza vaccination publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -n fs authors: ferreira, t b; alves, p m; aunins, j g; carrondo, m j t title: use of adenoviral vectors as veterinary vaccines date: - - journal: gene ther doi: . /sj.gt. sha: doc_id: cord_uid: n fs vaccines are the most effective and inexpensive prophylactic tool in veterinary medicine. ideally, vaccines should induce a lifelong protective immunity against the target pathogen while not causing clinical or pathological signs of diseases in the vaccinated animals. however, such ideal vaccines are rare in the veterinary field. many vaccines are either of limited effectiveness or have harmful side effects. in addition, there are still severe diseases with no effective vaccines. a very important criterion for an ideal vaccine in veterinary medicine is low cost; this is especially important in developing countries and even more so for poultry vaccination, where vaccines must sell for a few cents a dose. traditional approaches include inactivated vaccines, attenuated live vaccines and subunit vaccines. recently, genetic engineering has been applied to design new, improved vaccines. adenovirus vectors are highly efficient for gene transfer in a broad spectrum of cell types and species. moreover, adenoviruses often induce humoral, mucosal and cellular immune responses to antigens encoded by the inserted foreign genes. thus, adenoviruses have become a vector of choice for delivery and expression of foreign proteins for vaccination. consequently, the market requirements for adenovirus vaccines are increasing, creating a need for production methodologies of concentrated vectors with warranted purity and efficacy. this review summarizes recent developments and approaches of adenovirus production and purification as the application of these vectors, including successes and failures in clinical applications to date. the history of veterinary vaccine development starts with the well-known story of louis pasteur and his rabbit spinal cord vaccine and continues to this day with the demonstration of protection in animals by rabies virus reverse transcriptase dna plasmid vaccination. in between, in , frenkel used suspensions of the epithelium obtained from the tongues of recently slaughtered healthy cattle that were maintained in vitro and subsequently infected in a manner similar to that used today with baby hamster kidney cells to produce foot and mouth disease virus (fmdv). the frenkel procedure became the corner stone of vaccine production for many years and paved the way for modern biotechnology as we know it. more recently, recombinant pox viruses have been generated for vaccination against heterologous pathogens, using vaccinia-vectors, expressing the rabies virus glycoprotein and newcastle disease virus fusion and hemagglutinin (ha) glycoproteins, the first applications of genetically engineered vaccines. traditional vaccination involves the use of inactivated, live-attenuated or subunit vaccines. inactivated, or 'killed', vaccines consist of treated microorganisms that are unable to replicate; however, they do not elicit protein production in the cytosol and hence viral antigens cannot be presented by mhc class i molecules, thus cytotoxic cd t cells are not generated. liveattenuated vaccines are generally far more potent educing a greater number of relevant effector mechanisms, including cytotoxic cd t cells. nevertheless, these vaccines sometimes have residual pathogenicity, or a pathogenic virus strain may re-emerge by a further series of mutations. subunit vaccines would be as effective as live whole organisms, inherently safer than vaccines based on whole organisms, but they are not strongly immunogenic, being particularly difficult to obtain mhc class i specific responses. [ ] [ ] [ ] the development of vaccines therefore remains an important goal of immunology. moreover, there remains a need for further vaccines that can reduce the economic impact of disease in production animals. the ideal vaccine should be % efficacious in preventing infection, although this is totally unrealistic. therefore, the 'realistic' vaccine should provide greater than % efficacy in disease prevention within a few days up to a couple of weeks of a single administration. furthermore, in order to reduce the cost of regular reimmunizations and ensure receipt of a complete vaccination schedule, this protection should be of long duration, and should use a minimal number of doses. additionally, an 'ideal' vaccine would also stimulate mucosal immunity, since the majority of viruses enter via mucosal surfaces; thus, the 'perfect' vaccine should be designed to be delivered by mucosal routes, that is, intranasal or oral delivery is preferable. vaccines also need to be safe and not cause any adverse side reaction, such as immunosuppression or interference with immunity to other vaccines given simultaneously. finally, the vaccines need to be both genetically and thermally stable. genetic stability must be present to ensure the absence of reversion of the live vaccine to the virulent organism which might cause disease; and thermal stability is critical since the maintenance of the cold chain is not always guaranteed from manufacturing to delivery. a very important desired characteristic is to have vaccines that can be delivered to animals at a very young age and stimulate immunity in the presence of innate, passive immunity. , another very important criterion for an ideal vaccine in veterinary medicine is low cost; this is especially important in developing countries and for poultry vaccines, in which the vaccines must sell for a few cents a dose. unfortunately, such ideal vaccines are rare in the veterinary field; additionally, there are still severe diseases with no effective vaccines. the history of heterologous gene expression in adenovirus (ad) goes back to the discovery of simian virus (sv ) contamination in the s of inactivated ad strains - and vaccines during adaptation to growth in rhesus monkey kidney cells; it was observed that the sv t-antigen occasionally incorporated into the ad genome, which led to the realization that ad could be used to express heterologous genes. then recognition that purified replication-defective ads could be propagated on cells without helper viruses paved the way toward intentional production of genetically modified ads. the popularity of ad as a recombinant viral vector is largely due to the successful and safe immunization of millions of us military recruits in with enterically coated ad and ad as a prevention against acute respiratory disease (ard) outbreaks. following these first trials, a number of recombinant ad (rad) have recently been constructed and tested not only for humans but also for veterinary vaccination. , ads are highly efficacious vaccine carriers with strong immunogenicity. although the ability of ad vectors to elicit antigen-specific cd and cd t cells is well described, little is known about the kinetics or nature of the immune response following ad immunization. , however, yang et al observed that ad vectors have (i) the ability to deliver large amounts of antigen into the lymphoid tissues, (ii) the ability to induce rapid expansion and migration of cd t cells throughout the lymphatic system, and (iii) the ability to produce a sustained, high-level cd t-cell response, that may explain the strong immunogenicity of these vectors. the use of rad as a veterinary vaccine has many advantages: (i) they can infect a wide variety of dividing or nondividing cells; (ii) ad infections are ubiquitous and are normally without significant or severe clinical symptoms; (iii) they can be administered orally; (iv) ads have their genome well characterized; , (v) they can accommodate up to kb of foreign genetic material; (vi) their genome rarely integrates into the host chromosome; (vii) techniques are well established for the construction of rad vectors; and (viii) they have the ability to replicate at high titers in complementing cell lines. ad is a non-enveloped, icosahedral virus of - nm with a linear duplex dna genome of about kb. the genome is divided into early (e) and late (l) genes, expressed, respectively, before and after replication of the viral chromosome ( figure ). e gene products are involved in the control of viral gene transcription, shutoff of cellular proteins and cellular transformation. the e gene codes for proteins involved in viral replication, including a dna-binding protein involved in dna elongation (e a) and a dna polymerase (e b); e gene, dispensable for ad replication, codes for proteins that interfere with the host immune response against virus infection; finally, the e genes are involved in the transition from early to late gene expression, the shutoff of host-cell gene expression, the viral replication and the assembly of the virion (see russell and imler reviews). first-generation vectors are usually deleted in their e and e regions; however, when production is performed on cells, recombination between the left terminus of first-generation ad vector and partially overlapping e sequences in the cellular genome may result in the generation of e -positive, replicationcompetent ad (rca), a serious safety concern if nonreplicating vectors are desired. second-generation vectors are replicative-defective ad, which are further deleted in e a, e b or e , showing reduced immunogenicity and rca generation, but engineering of stable cell lines that complement these vectors can be cumbersome and lead to poor cell growth and viral titers. rad vectors have been developed either as replicationcompetent, with the expression cassette for the foreign antigen in place of the e genes, or as replication-defective viruses, when the antigen expression cassette replaces the e genes. despite the fact that it would be preferable to work with replication-defective virus, different studies have supported the use of rcas in veterinary vaccination, since the ability of the replication-defective form to induce mucosal immunity and to protect against a respiratory challenge, following immunization, was reported to be limited. , moreover, rca-based vaccines were known to have the potentiality to override maternal-derived immunity. , additionally, cattle vaccination with both rads against herpesvirus- showed that the use of the replication-defective form induced lower titers of antibody than the replication-competent form in intratracheal/subcutaneous immunization. currently, human rad represents one of the most efficient vector systems for delivery of vaccine antigens. figure schematic representation of the adenoviral genome organization. tb ferreira et al however, the use of human ad (had) as a vaccine delivery system in non-human animals is still limited. since non-hads are species specific, the development of animal-specific ad as a vaccine delivery system would be a logical choice. thus, ads isolated from species other than man have generated increasing interest, in particularly: (i) canine ad (cad), which has the ability to replicate and persist locally in the upper respiratory tract of puppies in the face of maternal-derived antibodies when administered via the intranasal route and use both depedent and independent coxsackievirus group b and ad receptor (car) pathays to enter the cells; , (ii) avian ad, the subject of numerous investigations, has a number of features that make them attractive to the poultry industry: ease of propagation, ease of administration (water, aerosol or injection) and a large range of serotypes that vary in virulence. chicken embryo lethal orphan (celo), classified as the type fowl ad (fad), is the most studied vector. this virus, like other typical avian ad, has a much larger genome than mammalian ad. despite its large scatter, it has never been associated with serious disease or economical losses in the poultry industry. it can be isolated from healthy chickens and does not induce clinical signs when experimentally inoculated in chickens. also this virus use both dependent and independent car pathways to enter the cells; , (iii) porcine ad (pad), which, depending upon serotype, can be grown in the pig kidney, testis, retina, thyroid cells, human kidney, canine melanoma and calf kidney cells, do not require adjuvants for the induction of proper immune responses. infection is generally sub clinical causing no adverse effects in producing pigs, with pad also isolated from apparently healthy pigs. pad is species specific having only been isolated from swine, reducing the possibility of its spread to other animal or man following administration. pad could induce both systemic and mucosal antibody responses; , and (iv) bovine ad serotype (bad ), which, like pad, do not require adjuvants for the induction of proper immune response, and has been used due to its lack of virulence. moreover, experimental infections of calves with bad failed to produce either clinical signs or gross lesions, but all animals seroconverted. the cell entry pathway of bad is not well understood. , applications adenovirus vaccination in companion animals feline immunodeficiency virus. the infection of cats with feline immunodeficiency virus (fiv) results in an immunosuppressive disease that is transmitted by blood and saliva. fiv invades and destroys the monocyte/ macrophage system and infects b cells. different approaches have been made trying to immunize cats against fiv; so far, no effective vaccine has been made. gonin et al, years ago, constructed a replicationdefective had , containing the envelope protein env gene of fiv; however, despite the fact that an antibody response to pseudorabies virus in cats showed the potential of rhad vectors to be used in this species, cats injected with . - . of % tissue culture infectious dose (tcid ) adjuvanted with montanide isa (water in nonmineral oil) or with montanide isa (double water/mineral oil/water) of this rad did not develop detectable antibody response against env. moreover, it was observed that even if high titers of antibodies against env products are induced, they could still be insufficient for protection. since then, no further work has apparently been performed concerning the use of ad vectors as a potential vaccine against fiv. (cdv) induces fatal diseases including encephalitis with demyelination, diarrhea and respiratory disorders in dogs. although conventional live modified vaccines are commercially available and widely used in the field, their efficacy is limited in the presence of maternally derived antibodies. thus, a new and improved cdv vaccine, which could overcome this limitation, would constitute a significant improvement. the construction and characterization of the first replication-competent rcad started recently. the genes that code for cdv ha , or fusion (f) proteins were inserted in two cad s and used as a candidate vaccine in puppies. it was reported that intranasal vaccination with a mixture containing . tcid of which rcad provided an excellent level of protection in seronegative puppies, inducing almost complete protection. in contrast, intranasal immunization of puppies born to cdv and cad immune dams failed to activate specific and protective immune responses. however, when the same puppies were vaccinated subcutaneously, significant seroconversion and solid protective immunity were triggered. furthermore, a significant priming of memory responses was evidenced immediately after challenge, this constituting an efficient strategy to overcome both passive and active ad-specific immunity in the dog. rabies virus. rabies still presents a health threat not only to humans but also to dogs and cats, dog being the only important vector for humans, especially in less developed nations where uncontrolled canine rabies often is endemic. a replication-competent and -defective had expressing a rabies glycoprotein (rg) has been developed, inducing immunity to rabies in rodent, canine, foxes and skunk when given by intramuscular, subcutaneous or intranasal routes with a dose of tcid /animal. however, oral immunization failed to induce a measurable antibody response to rabies virus (rv) using the same dose. , dogs previously vaccinated with commercially available vaccines, immunized with plaque forming unit (pfu)/animal of a replication-defective had expressing the rg, have developed higher titers of viral neutralizing antibodies against rv days after vaccination when compared with conventional vaccines under similar conditions. moreover, the immunization of dogs with the commercial non-ad vaccines is required yearly or, at best, every years, depending on the type of vaccine. on the other hand, the higher antibody titers obtained with ad vaccines against rv would reduce the frequency of dog immunization, reducing the costs for pet owners. one use of adenoviral vectors as veterinary vaccines tb ferreira et al important advantage of this recombinant vaccine is the fact that the immune response to the rg was shown not to be impaired by maternal immunity; thus, this ad vaccine is highly suitable for neonatal immunization. adenovirus vaccination in poultry avian infectious bronchitis virus. infectious bronchitis virus (ibv) is an highly contagious pathogen of poultry causing significant morbidity and mortality. depending on the strain, ibv can target the respiratory tract, kidney and oviducts, and result in nephritis and reduced egg production. in addition, more than ibv serotypes have been identified worldwide and new serotypic variants have been identified as a result of the widespread use of live attenuated vaccines. different approaches have been developed in order to generate a more efficacious vaccine against ibv. the expression of s , a glycoprotein involved in the attachment of cellular receptors, by a vaccinia virus was able to induce virus-neutralizing antibodies to ibv when delivered to mice; however, multiple injections are required to achieve a reasonable degree of protection. recently, a fad expressing the s of ibv has been developed. a single dose of tcid / animal was shown to be sufficient to obtain complete protection of chickens at the trachea, the primary site of infection by ibv. moreover, even in the face of fav maternal antibodies, a high level of protection was achieved. infectious bursal disease virus. infectious bursal disease virus (ibdv) induces an immunosuppressive disease of chickens by destruction of the b lymphocytes. current vaccination alternatives consist of either live virus or inactivated oil-emulsion vaccines, which induce serum antibody production in breeding hens after natural exposure; they are transferred to the progeny chicks via the yolk sac providing protection for the first critical weeks after hatching. the construction of an rfad containing the vp gene from ibdv has been described. this recombinant vaccine was shown to induce an immune response in chickens to vp after vaccination with pfu/animal. moreover, after challenge with ibdv, intravenously, intraperitoneally, subcutaneously or intramuscularly, vaccinated chickens were protected, although no protection was observed in conjunctival sac vaccinated chickens. adenovirus vaccination in swine classical swine fever virus. classical swine fever (csf), also known as hog cholera, is a serious and contagious viral disease of pigs with a high mortality rate, difficult to control in areas of high pig or wild boar densities, being the most economically important disease of swine in areas of intensive pig farming. for this reason, csf is included in the a list of infectious diseases of the highest importance for international trade. thus, it is highly relevant to develop efficacious vaccines for the control of csf virus (csfv) in domestic pigs and in wild boar. prophylactic vaccination is still carried out in many parts of the world. new vaccine developments have included a number of different strategies for delivering the major envelope glycoprotein, e , against which most neutralizing antibodies are directed. hammond et al, constructed the first rpad expressing the e protein and showed that a single dose of tcid /animal in tissue culture supernatant, when administered subcutaneously, is sufficient to completely protect pigs against subcutaneous challenge. later, the same authors showed that when the challenge is administered orally, only % of the animals were protected; more, recently, it was shown that pigs given two oral doses of tcid / animal of rpad were completely protected from the disease. pseudorabies virus. pseudorabies virus (prv) is an alpha herpesvirus, which causes the economically important and widespread aujeszky's disease (ajd) in pigs. prv is a highly neurotropic virus causing nervous and respiratory complications in pigs, the natural host, and in a variety of other animal species. vaccination against ajd is widely practised with live attenuated or killed whole virus vaccines. however, neonatal immunization is often limited in the presence of maternally derived antibody, which inhibits the immune response against both vaccines. recently, the use of rad vaccines carrying individual prv genes were constructed as a safe alternative. glycoproteins gd, gb and gc of prv were chosen on the basis of their role in eliciting a protective immune response against virus infection. it was shown that piglets vaccinated intramuscularly with . - . tcid /animal day after birth, with replicationcompetent had harbouring these three genes, developed similar neutralizing antibody responses independently of the presence or absence of maternal antibodies and were partially protected against challenge weeks later. for pigs that are slaughtered a few months after birth, one-shot vaccination at birth could provide protection of sufficient duration. finally, two doses of ml of clarified tissue culture supernatant containing . tcid of rpad expressing the prv gd gene were administered subcutaneously and showed to protect pigs after challenge. postmorten, gross lesions of pneumonia were found in the lungs of pigs given a single dose of vaccine; the lungs of pigs given two doses were free from disease. foot and mouth disease virus. foot and mouth disease (fmd) is a severe, clinically acute, vesicular disease of cloven-hoofed animals including domesticated ruminants, pigs and more than wildlife species. pigs are recognized as a significant factor in the spread of the disease since a single pig releases as much aerosol virus as cattle in a short period of time. they observed that vaccinated swine with  pfu/ animal in pbs developed antibodies against fmdv structural proteins and an fmdv-specific neutralizing antibody response, which seems to increase slightly by boosting the swine with a second inoculation of  pfu/animal in pbs at weeks post initial vaccination. this vaccination protocol was shown to offer a significant degree of protection to the pigs, as five of six pigs were completely protected, while the remaining animal had significantly reduced signs of disease. later, moraes et al showed that a single dose of  pfu/animal in pbs of a replication-defective had expressing the p coding region of fmdv strain a completely protected pigs against homologous challenge , or days after vaccination. since efficacious vaccination is strain dependent and the infection with one serotype does not confer protection against another, had bicistronic vector vaccines have been developed, decreasing the cost for multivalent adenoviral fmd vaccines. for the construction of these vectors, the p capsid coding region for both a and o strains and the c protease coding region of a strain were used. however, the neutralizing antibody response after vaccination with .  pfu/animal in pbs of the bicistronic vector was considerably lower than that induced by a commercial fmd vaccine or the monovalent ad-a vaccine. recently, a new strategy based on the fact that fmdv is highly sensitive to alpha/beta interferon (ifn-a/b) have been developed using a replication-defective had . , this strategy has been shown to completely protect pigs after vaccination with pfu/animal when challenged h later with virulent fmdv. porcine respiratory and reproductive syndrome virus. porcine respiratory and reproductive syndrome virus (prrsv) is the causative agent of an economically important pig disease, with a worldwide distribution, characterized by reproductive failure in sows and respiratory problems in unweaned and growing pigs. swine macrophage is the only cell type known to support prrsv replication, making commercial production impossible. direct contact between infected and naive pigs is the predominant route of prrsv transmission. moreover, pneumonia caused by prrsv infection is more severe in young pigs compared to adults and may be complicated by concurrent bacterial infection. gonin et al observed that prrsv-infected pigs present circulating antibodies responsible for viral neutralization mainly directed against gp , an envelope protein. gagnon et al constructed a replication-defective had expressing the gp protein and used this recombinant virus to immunize pigs using two intradermal injections with  -  pfu/animal in a mixture containing ml of pbs and ml of poloxamer sp at . %. it was observed that following challenge given intranasally days after the booster, pigs produced high antibody titers to gp protein. moreover, vaccinated pigs presented specific immune memory which, following a subsequent prrsv infection, resulted in a rapid clonal expansion of memory cells to the neutralizing epitopes of the authentic viral gp protein. the enteric and respiratory tissues of newborn piglets resulting in mortalities approaching %. the virus infects epithelial cells and, in some cases, lung macrophages. there are several commercially available tgev vaccines, both inactivated and attenuated; these do not fully protect piglets. several attempts have been made to develop efficacious recombinant tgev vaccines. the spike protein (s) was identified as the major inducer of tgev-neutralizing antibodies and it mediates binding of tgev to its cellular receptor. thus, had expressing the s protein was constructed and used to study the induction of antibodies providing protection in swine. it was observed that porcine serum, elicited by pfu/animal in pbs of this recombinant, when mixed with a lethal dose of virus prior to administration to susceptible pigs, prevented the replication of virulent tgev administered orally as virus-antibody mixtures and fully protected swine from clinical signs and death. moreover, the used dose did not produce any clinical symptoms in any of the more than animals inoculated up to weeks after inoculation, suggesting that this vector can be used as a live vaccine in swine without secondary complications associated with the vector. however, a pad vector could be more effective than had. thus, tuboly and nagy constructed a rpad expressing the tgev protein. it was observed that a single oral dose of  pfu/animal of the recombinant virus was sufficient to induce both a systemic and a local humoral immune response. unfortunately, challenge experiments were not carried out. is a widespread and important pathogen in species as diverse as poultry, swine, marine mammals and humans. in pigs, influenza can occur either as an enzootic problem in a herd or, more commonly, as explosive outbreaks of ard. although rarely fatal, swine influenza can be of substantial economic impact. in addition, there is growing concern for the potential for synergistic infections with influenza and prrsv. beyond the impact of influenza for the swine industry, pigs are also very important in the global ecology of influenza a viruses in humans. siv vaccines that are commercially available are inactivated, whole-virus or subunit vaccines. while these vaccines may decrease the incidence and severity of clinical disease, they do not consistently provide complete protection from virus infection. two replication-defective had were developed as potential vaccines against siv: rhad expressing the influenza virus h ha , inducing predominately a subtypespecific humural immune response; and rhad expressing the nucleoprotein (np), a group-specific stimulating cytotoxic t lymphocytes for crossreactive immunity to all influenza a subtypes. it was observed that the immunization of mice with  tcid / animal and pigs with  tcid /animal in pbs with the first recombinant, described above, developed high levels of virus-specific hemagglutination-inhibition antibody to siv by weeks post vaccination and the animals were partially protected. on the other hand, pigs vaccinated with  tcid /ml in pbs of both (bvdv) is responsible for reduced milk production, reduced reproductive performance and growth retardation. in addition, acute infection in adult cattle, congenital defects and increased neonatal mortality are also clinical manifestations of bvdv infection. finally, it was reported that bvdv may play an indirect role in immunosuppression. currently, inactivated and modified-live vaccines are used; however, both types of vaccines have significant shortcomings. a rhad expressing the nucleocapsid c protein (p ), which is highly conserved among many different pestiviruses, to which bvdv belongs, was constructed and shown to induced both humoral and cellular immune responses after vaccination with pfu/animal, in a mouse model. the e protein of bvdv, which is a major viral glycoprotein, was also used for mice immunization using the same vector and dose. a strong humoral immune response was detected as the presence of a strong memory response to the e protein. both strategies used an inducer/promoter allowing the generation of rad in which the production of the transgene may be toxic and permitting the control of the transgene expression in vitro and in vivo. finally, a rbad expressing bvdv e protein was constructed and shown to induce bvdv e -specific mucosal and systemic immune responses after intranasal immunization with pfu/animal of cotton rats. despite the fact that mice immunized with recombinant fowl pox virus or with a dna vaccine expressing e glycoprotein induced e -specific immune responses, it was also observed that the neutralization titers were low. bovine parainfluenza virus type . bovine parainfluenza virus type (bpiv ) has been isolated from normal cattle, cattle showing signs of respiratory disease and aborted fetuses. an intramammary inoculation of bpiv resulted in respiratory signs, fever and malaise. moreover, the milk showed a color change, an increased ph and increased numbers of glandular epithelial cells, neutrophils, lymphocytes and monocytes. currently, multivalent vaccines containing killed bpiv are used. breker-klassen et al constructed two rhad encoding either the f or the hn protein of bpiv . it was shown that intranasal vaccination with  pfu/animal in pbs of cotton rats produced a strong serological response within days, which increased with the second vaccination. moreover, f and hn proteins produced by rhad either alone or in combination were sufficient to induce a protective immune response. bovine herpes virus . bovine herpes virus (bhv- ) causes a variety of diseases in cattle including infectious bovine rhinotrachitis, infectious pustular vulvo-vaginitis, conjunctivitis and occasionally meningo-encephalitis. live attenuated and killed vaccines are partially effective in reducing the disease incidence and severity. the limitations with the existing vaccines have encouraged the development of alternative, cost-effective vaccination strategies such as recombinant live viral vector-based vaccines. mittal et al developed the first replicationcompetent and -defective had carrying the gd glycoprotein gene from the bhv- envelope. they showed that both forms induced immunity in cotton rats after vaccination with pfu/animal in pbs and no infectious bhv- virions were isolated from the trachea of these rats after challenge. the same strategy was used with a rbad and tested in its ability to induce mucosal and systemic immune response in cotton rats and calves with success. more recently, newborn lambs were immunized with  pfu/animal of replicationdefective had expressing the gd protein of bhv- . both humoral and cell-mediated gd-specific mucosal immune responses were detected in - -day-old lambs after a single immunization and these responses were qualitatively and quantitatively similar to those detected in - week-old lambs. moreover, gd-specific maternal antibody did not significantly alter either mucosal or systemic gd-specific immune response. in addition, bhv- glycoprotein gc was also tested in its ability to induce protective immunity to bhv- in cattle by intranasal administration of pfu/animal using a replication-defective had . however, it was observed that the highest bhv- neutralizing antibody titers were obtained with rhad expressing the gd protein followed by the live vaccine. rinderpest virus and peste des petits ruminants virus. because of their high mortality and high morbidity rates, rinderpest (rp) and peste des petits ruminants (ppr) are dreaded animal diseases that are drawing back the animal production in many developing countries (africa and asia). they affect both domestic and wild ruminants and belong to the list a of the office international des epizooties where highly contagious animal diseases with high economic importance are grouped. currently, attenuated tissue culture vaccines used to control rp and ppr viruses have been successfully used for many years. being safe and effective, they provide complete and lifelong protection with a single subcutaneous inoculation. , however, for vaccination programs, the oral route of administration would be more economical. moreover, the parenteral way of administration means that the current vaccine cannot be used in wildlife. in our laboratory, the production purification, and encapsulation processes for a replication-defective had expressing two different morbillivirus capsids proteins, the responsible agent of both diseases, is in progress. the aim is the development of heat stable rp/ppr oral vaccines better adapted for mass vaccination in hot climate developing countries. animal vaccination and challenge testes are ongoing with promising preliminary results. in summary, according to the numerous reported studies, live human or non-human rads, either infectious or not, appear to be attractive candidates for vaccination against several animal disease (table ) . success situations are clear against most pathogens reported and intermediate successes are apparent against cdv and rp virus (rpv)/ppr virus (pprv), whereas vaccination against fiv have, thus far, yielded poor or inconclusive results. nevertheless, this is not a comprehensive picture, since more than viral agents of veterinary importance are known ( table ) . tb ferreira et al process design is critical when developing cost-effective veterinary vaccines. the goal of minimizing the number of process steps is a prerequisite if industrial application is intended. in addition, veterinary vaccines do not impose the same final purity requirement as human vaccines, which should reflect on the purification schemes. the production of ad vaccines for veterinary applications is mainly based upon the use of continuous cell lines such as , mdbk, , vr bl, hela and a . during the cell proliferation step, various animal sera are used to enhance cell growth. however, supplementing cell culture media with such components presents several drawbacks like lot to lot variation, potential risk of contamination by viruses, mycoplasma, prions, etc. furthermore, regulatory authorities in europe (european medicines agency (emea)) and in the united states (food and drug administration (fda)) have encouraged biological manufacturers to reduce or eliminate the use of substances of animal origin in their manufacturing processes. recently, several serum and proteinfree culture media became commercially available. although the use of such defined media is still expensive, the large-scale production of ad vectors will require the use of this type of culture media to facilitate downstream processing and, in time, decrease the overall process cost. production methods differ according to the cell type used: adherent versus suspension cell culture. for manufacturing purposes, suspension adapted cell lines are more convenient for production at large scale and different operation modes have been developed for ad production ( figure ): (i) batch mode providing the easiest way to proceed as no extra feeding is required and the risk of contamination is lowered given the simplicity of operation; (ii) fed-batch mode, easy to operate and readily scalable, is employed to extend culture lifetime by supplementing limiting nutrients or reducing the accumulation of toxic metabolites; and (iii) perfusion mode, consisting in cell retention at a relatively high concentration inside the bioreactor, while fresh nutrient supply and metabolite removal takes place (see kamen and henry for a review). methodologies for production of concentrated ad vectors at low cost are mandatory as the market needs for ad are increasing. although ad has the advantage to be produced at high titers ( - pfu/ml), to obtain a good immune response in a large proportion of treated animals, particularly for mucosal vaccination, large doses and thus culture volumes are required. further process development aiming at higher yields of product is clearly necessary. improvements in volumetric production can be achieved by increasing the cell density at which cells can be infected without lowering the specific yield of the product; however, production of ad vectors that maintain a high specific yield in batch operation is limited to cell densities in the range of  cells/ml; several approaches have been made in order to overcome this so-called 'cell density effect': (hpi) together with periodical ph adjustments, allowed a sustained maximum specific productivity at .  cells/ml whereas nadeau et al, further improved this strategy by also adding essential amino acids at hpi thereby stabilizing volumetric productivity at cell densities above  and below  cells/ ml. such results hint at the existence of substrate limitation and/or byproduct inhibition at high cell densities. at production scale, medium exchange will increase the cost of the final product, which is even more critical when this is to be used in the veterinary field. perfusion mode operation has been attempted as a means to control the culture environment and remove toxic byproducts; , nevertheless, the cell-specific productivity could only be maintained by infecting cells at densities up to  cells/ml using high perfusions rates of two reactor volumes per day, at days postinfection, a very costly proposition. in our laboratory, by understanding kinetics and metabolism of infection, typical cell concentration at infection have already been doubled and further improvements seem possible using a simple and nonexpensive fed-batch mode. the importance of infection kinetics on ad production and the significance of variables such as multiplicity of infection (moi) and harvesting time in process optimization is also mandatory to increase production yields, to avoid rapid depletion of costly and certified master virus banks as well as to ensure that the infection kinetics is reproducible between different production scales. moreover, the use of low moi at large scale would be preferential since an intermediate step of virus inoculum production would be avoided. finally, veterinary products should be purified with a minimal number of steps and the unit operations employed should be simple and nonexpensive. traditionally, laboratory purification of rad was achieved using two rounds of cesium chloride (cscl) density gradient ultracentrifugation. however, cscl density gradient method is not scaleable. despite the fact that chromatographic purification is an expensive method, ion exchange, hydrophobic interaction, metal chelate and size-exclusion chromatography have been evaluated for capture and purification of had . kamen and henry developed a complex and expensive method for gene therapy application consisting of: (i) harvest of infected cells by continuous centrifugation, (ii) cell lysis by osmotic shock, (iii) dnase treatment with centrifugal/conditioning, (iv) filtration, (v) anionexhange chromatography, (vi) ultrafiltration/concentration and (vii) size exclusion chromatography. this protocol allows large-scale purification of ad vectors with purity comparable to the cscl gradient method; however, it dramatically increases the final product price, well beyond the reach of veterinary utilization. introgen developed a method consisting of a single ion chromatography run, after concentration/diafiltration and nuclease treatment, with a total recovery of the virus product of %. in our laboratory, a protocol to purify had from the bulk harvested directly from the bioreactor after lysis (without a concentration step) using an ion exchange chromatographic step, ultrafiltration and a final polishing step with gel filtration is in progress with promising preliminary results. to improve access to the veterinary marketplace, some hurdles still need to be overcome, as efficacy, safety, ease of delivery and cost of production. some of these factors are dependent from the emea and its committee for veterinary medical products (cvmp) who regulate vaccines developed in the european union (eu); meanwhile, the united states department of agriculture (usda) and its animal and plant health inspection service (aphis) regulate the development of animal vaccines in the united states (us). unfortunately, government regulators can often become extremely cautious when confronted with new technology and they not always follow a strictly science-based assessment approach, which should be the only criteria for regulation of products. the regulatory issues governing the use of ad vaccines on the farm and in the veterinary clinical are not yet well defined, although guidelines on live recombinant vector vaccines for veterinary use are available (emea/cvmp/ / ) in eu, while the rules governing medicinal products in the ue and the code of us federal regulations title ( cfr) are the main regulatory texts on animal veterinary vaccine production. the drawbacks of the use of ad vectors as veterinary vaccine is the rare but possible emergence of e -containing vectors, due to rare eventual minute homologous recombination events between the viral e -containing (permissive) mammalian host cell genome and the virus itself, restoring the e gene to the viral genome (rca). to reduce or eliminate the problem of rca, an e trasformed human cell line per.c was constructed. per.c was made by transformation of primary human embryonic retinoblasts with limited homology with viral sequences, expected to reduce or eliminate the emergence of rca during virus amplification. up to now, many viruses have been used as viral vectors for delivery of foreign antigens (see yokoyama et al for a review). the development of these recombinant vaccines may be of value in the veterinary field, especially when attenuation of the target pathogen is not possible, when immunity to one or more pathogens must be raised at the same time or when satisfactory quantities of the target pathogen or the live vaccine cannot be produced easily or safely. on the other hand, for their practical use, there might be still some potential problems that must be overcome, for example, safety issues such as reversion of virulence or recombination with field-type viruses spread in the environment and genetic stability. the selection of a good viral vector as recombinant viral vaccine should be determined by the host range of the vector, safety consideration, replication properties, stability, the amount of foreign dna, which can be inserted and the place where the primary immune reaction is induced by antigens expressed by the vector. exciting advances in biotechnology are enabling the development of new vaccines with enormous possibilities that were considered unthinkable. these newer vaccine approaches have benefited from an improved basic understanding of the immunology of antigen presentation and the factors important in sustaining memory immune responses. the continued improvement in our understanding of the nature of protective immunity against various infectious diseases should allow the rational design of vaccines and immunization use of adenoviral vectors as veterinary vaccines tb ferreira et al strategies that generate protective immune responses against infectious diseases for which no vaccines yet exist. applications of pox virus vectors to vaccination: an update risk of aseptic meningitis after measles, mumps, and rubella vaccine in uk children laboratory tests for live attenuated poliovirus vaccines recombinant viral vector vaccines for the veterinary use the application of nucleic acid vaccines in veterinary medicine dna-antiviral vaccines: new developments and approaches -a review novel viral vaccines for livestock bacterial ghosts as vaccine candidates for veterinary applications sv in adenovirus vaccines and adenovirus-sv recombinants characteristics of a human cell line transformed by dna from human adenovirus type the vaccine book recombinant adenoviruses as vaccines cytotoxic t-lymphocyte target proteins and their major histocompatibility complex class i restriction in response to adenovirus vectors delivered to mouse liver role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs detailed analysis of the cd + t-cell response following adenovirus vaccination update on adenovirus and its vectors adenoviruses as vectors for delivering vaccines to mucosal surfaces adenovirus vectors as recombinant viral vaccines packaging capacity and stability of human adenovirus type vectors frequency and stability of chromosomal integration of adenovirus vectors production of first generation adenovirus vectors: a review development and optimization of an adenovirus production process a single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent e -positive particles adenoviral vectors for gene transfer and therapy mucosal immunization with recombinant adenoviruses: induction of immunity and protection of cotton rats against respiratory bovine herpesvirus type infection vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge the effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein d of bovine herpesvirus type the immunogenicity and efficacy of replicationdefective and replication-competent bovine adenovirus- expressing bovine herpesvirus- glycoprotein gd in cattle canine adenovirus type attachment and internalization: coxsackievirus-adenovirus receptor, alternative receptors, and an rgd-independent pathway fowl adenovirus recombinant expressing vp of infectious bursal disease virus induces protective immunity against bursal disease vaccination of pigs with a recombinant porcine adenovirus expressing the gd gene from pseudorabies virus a recombinant e -deleted porcine adenovirus- as an expression vector a proposal for a new (third) genus within the family adenoviridae fiv vaccine development and its importance to veterinary and human medicine: a review fiv vaccine update and review immunization trial of cats with a replicationdefective adenovirus type expressing the env gene of feline immunodeficiency virus morbillivirus infections, with special emphasis on morbilliviruses of carnivores cytotoxic t-lymphocyte activity specific for hemagglutinin (h) protein of canine distemper virus in dogs immunogenicity of an e -deleted recombinant human adenovirus against rabies by different routes of administration a replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier adult dogs receiving a rabies booster dose with a recombinant adenovirus expressing rabies virus glycoprotein develop high titers of neutralizing antibodies the use of an e -deleted, replication-defective adenovirus recombinant expressing the rabies virus glycoprotein for early vaccination of mice against rabies virus a recombinant fowl adenovirus expressing the s gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus research on infectious bursal disease -the past, the present and the future classical swine fever -an update vaccinology of classical swine fever: from lab to field vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp (e ) gene protects pigs against classical swine fever oral and sub-cutaneous vaccination of commercial pigs with a recombinant porcine adenovirus expressing the classical swine fever virus gp gene protection of pigs against 'in contact' challenge with classical swine fever following oral or subcutaneous vaccination with a recombinant porcine adenovirus mechanistic bases for adverse vaccine reactions and vaccine failures single inoculation of replication-defective adenovirus-vectored vaccines at birth in piglets with maternal antibodies induces high level of antibodies and protection against pseudorabies the pathogenesis and diagnosis of foot-and-mouth disease foot and mouth disease in wildlife development of replication-defective adenovirus serotype containing the capsid and c protease coding regions of foot-and-mouth disease virus as a vaccine candidate immune responses and protection against footand-mouth disease virus (fmdv) challenge in swine vaccinated with adenovirus-fmdv constructs early protection against homologous challenge after a single dose of replication-defective human adenovirus type expressing capsid proteins of foot-and-mouth disease virus (fmdv) strain a recombinant adenovirus coexpressing capsid proteins of two serotypes of foot-and-mouth disease virus (fmdv): in vitro characterization and induction of neutralizing antibodies against fmdv in swine immediate protection of swine from foot-and-mouth disease: a combination of adenoviruses expressing interferon alpha and a foot-and-mouth disease virus subunit vaccine novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease porcine reproductive and respiratory syndrome seroneutralization of porcine reproductive and respiratory syndrome virus correlates with antibody response to the gp major envelope glycoprotein adenoviral-expressed gp of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions transmissible gastroenteritis construction and characterization of recombinant porcine adenovirus serotype expressing the transmissible gastroenteritis virus spike gene critical epitopes in transmissible gastroenteritis virus neutralization induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein tropism of human adenovirus type -based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus proinflammatory cytokines and viral respiratory disease in pigs genetic reassortment of avian, swine, and human influenza a viruses in american pigs recombinant adenovirus encoding the ha gene from swine h n influenza virus partially protects mice from challenge with heterologous virus: a/hk/ / (h n ) protection of weaned pigs by vaccination with human adenovirus recombinant viruses expressing the hemagglutinin and the nucleoprotein of h n swine influenza virus immunization of pigs with a particlemediated dna vaccine to influenza a virus protects against challenge with homologous virus protective cd + and cd + t cells against influenza virus induced by vaccination with nucleoprotein dna the evolution of bovine viral diarrhea: a review viral infections and bovine mastitis: a review induction of humoral and cellular immune responses against the nucleocapsid of bovine viral diarrhea virus by an adenovirus vector with an inducible promoter recombinant adenoviruses expressing the e protein of bovine viral diarrhea virus induce humoral and cellular immune responses recombinant bovine adenovirus type expressing bovine viral diarrhea virus glycoprotein e induces an immune response in cotton rats recombinant type adenoviruses expressing bovine parainfluenza virus type glycoproteins protect sigmodon hispidus cotton rats from bovine parainfluenza virus type infection induction of systemic and mucosal immune responses in cotton rats immunized with human adenovirus type recombinants expressing the full and truncated forms of bovine herpesvirus type glycoprotein gd construction and characterization of e -deleted bovine adenovirus type expressing full-length and truncated form of bovine herpesvirus type glycoprotein gd induction of immune responses in newborn lambs following enteric immunization with a human adenovirus vaccine vector induction of protective immunity to bovine herpesvirus type in cattle by intranasal administration of replication-defective human adenovirus type expressing glycoprotein gc or gd control of peste des petits ruminants: classical and new generation vaccines progress in the development of a heatstable recombinant rinderpest vaccine using an attenuated vaccinia virus vector animal cell culture: a pratical approach production of adenovirus vector for gene therapy scale-up of the adenovirus expression system for the production of recombinant protein in human s cells improvement of recombinant protein production with human adenovirus/ s expression system using fedbatch strategies insights into adenoviral vector production kinetics in acoustic filter-based perfusion cultures purification of a type recombinant adenovirus encoding human p by column chromatography method for the production and purification of adenoviral vectors. introgen therapeutics, inc.: virginia us patent nucleic acid vaccines: research tool or commercial reality new helper cells and matched early region -deleted adenovirus vectors prevent generation of replicationcompetent adenoviruses we acknowledge and appreciate the financial support received from the european commission (project ic-f a pr ) and from fundação para a ciência e tecnologia -portugal (project pocti/bio/ / ) and student grant (sfrh/bd/ / ). key: cord- -i czo a authors: johnson, alton r.; mcdonald, adam r.; malay, d. scot title: in pursuit of a sars-cov- vaccine date: - - journal: j foot ankle surg doi: . /j.jfas. . . sha: doc_id: cord_uid: i czo a nan recently, some of our patients have inquired about the availability of a vaccine for the prevention of covid- disease. currently, everyone wants to know when a safe and effective vaccine for the sars-cov- coronavirus will be ready to administer to the world's citizenry. in an effort to provide foot and ankle surgeons with information that they can use to accurately respond to their anxious patients, we are providing a synopsis of the current status or coronavirus vaccine development. in the united states, the food and drug administration (fda) is responsible for protecting the public health by assuring the safety, efficacy, and security of human and veterinary drugs, biological products, medical devices, our nation's food supply, cosmetics, and products that emit radiation. the fda is also responsible for advancing the public health by helping to speed innovations that make medicines more effective, safer, and more affordable and by helping the public get the accurate, science-based information they need to use medicines and foods to maintain and improve their health ( ) . like new drugs, new vaccines are developed through a process that entails a number of distinct phases [https://www.cdc.gov/vaccines/basics/test-approve.html]. in the preclinical phase, investigators conduct laboratory work in order to understand the basic science that underlies a vaccine's physical and chemical properties. thereafter, human testing begins with a phase i safety assessment, during which a small group of volunteer participants is administered the vaccine and followed in order to screen for potential adverse effects related to the vaccine. if the vaccine is determined to be safe, then its efficacy is assessed in a phase ii trial, wherein the primary aim is to determine whether or not the vaccine actually results in the development of antibodies to the target virus. if the vaccine is determined to be efficacious, then a phase iii comparison trial is conducted in order to see if the vaccine is better than or at least comparable to standard preventive measures. phase iv, the last and longest period of observation, monitors the safety and effectiveness of the vaccine over an extended period of time following widespread use of the vaccine. on may , , in the white house rose garden, united states president donald trump formally announced operation warp speed (ows). ows is a combined effort of both public and private sectors with oversight by the us department of defense, department of health and human services, fda, and the centers for disease control and prevention, for the safe development and distribution of sars-cov- vaccines. the mission objective of ows is to deliver million doses of safe and effective vaccine by january , ( ). this operation is unusual, in that the traditional process for vaccine development typically takes > months to complete; however, this mission's accelerated process aims to reduce the timeline to a duration of just months ( ). the reasons for such an effort are outlined by key goals of the mission: around the world, operations similar in scope to ows are also in progress. as of september , , worldwide efforts are directed toward a total of vaccines in human clinical trials, with another preclinical efforts in animal studies. including the united states, there are vaccines in phase i, in phase ii, in phase iii, and vaccines that have received approval for early or limited use; however, not a single vaccine has been approved for general use worldwide ( ) . these numbers are everchanging and will certainly be outdated by the time this editorial gets to press. on august , , russia announced approval of a vaccine, nicknamed "sputnik v," based on the results of patients in open, nonrandomized phase i/ii studies at hospitals in russia. the results of the combined phase trials were later published on september , and revealed good immunogenicity for both humoral and cell-mediated immune responses; however, further investigation is needed in order to determine how effective the vaccines will be in preventing covid- disease ( ) . two other vaccines developed by chinese companies have received emergency approval for use in china, where a number of clinical trials are underway ( ) . it is also not surprising that there have been some pitfalls associated with the human trials, with one volunteer in the astrazeneca/oxford trial in the united kingdom developing serious neurological symptoms after being administered a vaccine, causing the data safety monitor to temporarily halt the trial. that particular trial resumed days later ( ) . adverse effects such as this "unexplained illness" could impact just how swiftly such an effort of this magnitude is able to effectively and safely enable mass production and distribution of a sars-cov- vaccine. in the united states, notwithstanding the political implications of a deployable vaccine, most experts agree that a vaccine is likely to be approved in late with initial deployment to healthcare workers and vulnerable populations after completion of phase iii trials ( ) . manufacturing and organization of the vaccine deployment infrastructure has already begun in us-based facilities as a part of ows, an approach to rapid distribution known as fill-finish manufacturing, wherein millions of doses are quickly packaged and shipped upon approval ( ) . recently, both national institute of allergy and infectious diseases director anthony fauci, md, and centers for disease control and prevention director robert redfield, md, have stated publicly and in congressional testimony that they expect general deployment of sars-cov- vaccines in the summer of , with widespread immunizations and a "return to regular life" in late summer or fall ( ) . report of the science looking forward subcommittee. prepared for fda science board trump administration collaborates with moderna to produce million doses of covid- investigational vaccine trump administration's operation warp speed accelerates astrazeneca covid- vaccine to be available beginning to produce millions of covid- investigational vaccine doses hhs, dod collaborate with novavax to produce millions of covid- investigational vaccine doses in commercial-scale manufacturing demonstration projects hhs, dod partner with sanofi and gsk on commercial-scale manufacturing demonstration project to produce millions of covid- investigational vaccine doses government engages pfizer to produce millions of doses of covid- vaccine coronavirus vaccine tracker safety and immunogenicity of an rad and rad vector-based heterologous prime-boost covid- vaccine in two formulations: two open, non-randomised phase / studies from russia -vaccine-trial-paused-due-toillness-in-one-volunteer when will you be able to get a coronavirus vaccine? key: cord- -zv nbz p authors: tsikala vafea, maria; atalla, eleftheria; georgakas, joanna; shehadeh, fadi; mylona, evangelia k.; kalligeros, markos; mylonakis, eleftherios title: emerging technologies for use in the study, diagnosis, and treatment of patients with covid- date: - - journal: cell mol bioeng doi: . /s - - -w sha: doc_id: cord_uid: zv nbz p introduction: the covid- pandemic has caused an unprecedented health and economic worldwide crisis. innovative solutions are imperative given limited resources and immediate need for medical supplies, healthcare support and treatments. aim: the purpose of this review is to summarize emerging technologies being implemented in the study, diagnosis, and treatment of covid- . results: key focus areas include the applications of artificial intelligence, the use of big data and internet of things, the importance of mathematical modeling for predictions, utilization of technology for community screening, the use of nanotechnology for treatment and vaccine development, the utility of telemedicine, the implementation of d-printing to manage new demands and the potential of robotics. conclusion: the review concludes by highlighting the need for collaboration in the scientific community with open sharing of knowledge, tools, and expertise. , was first identified in the city of wuhan, in hubei province, china in december and has rapidly spread across the globe. the cov-id- pandemic has caused an unprecedented health and economic crisis. the magnitude of the crisis has called for an immediate response from governing officials, scientists, and medical professionals. with limited resources and immediate need for medical supplies, healthcare support and treatments, the crisis has demanded innovative solutions. novel uses of emerging technologies have been proposed in order to meet the rising demands. emerging technologies have aided in the study of covid- , the development of advanced diagnostic tools and treatments, and the response to medical supply shortages. the innovative use of emerging technologies continues to have a profound impact on our ability to respond to this global crisis and should continue to be utilized to help improve outcomes. the aim of this review is to describe the features and uses of new technological developments implemented to combat the covid- pandemic. the technologies in this review include: artificial intelligence (ai), machine learning and deep learning, nanomedicine, novel technologies for vaccines development and therapeutics, novel mathematical modeling, big data, internet of things (iot), telemedicine, robots, and d printing technology. ai has a wide array of features and applications that can be implemented to aid our response to covid- . researchers have used both machine learning and deep learning models to study, diagnose and treat covid- . machine learning tools enable the study of large datasets of viral genomes and can therefore increase our foundational knowledge of covid- . scientists utilized machine learning based models along with intrinsic genomic signatures to provide a timely and accurate taxonomic classification of the novel coronavirus sequences and to identify the potential origin. containment of the pandemic relies significantly on early and accurate diagnosis. mei et al proposed an ai system based on machine learning and deep learning models that combines demographic (age, sex) and clinical information (laboratory test results, reported symptoms, history of exposure etc.) with chest imaging findings for rapid identification of patients with covid- . major limitations of the proposed model for its adoption into clinical practice are the lack of generalizability to other patient populations and the difficulty on model training. imaging modalities has served as a valuable tool for detecting covid- , evaluating complications, and monitoring disease course. during the last years, ai has empowered conventional imaging approaches, including ct scans and x-rays, to meet the increasing challenges by providing detection accuracy and reliability. re-cently, deep learning models, the core algorithms of ai, have been used to develop a thoracic ct image analysis system, which can automatically detect covid- patients and quantify the disease burden. also, through volumetric measurement of the opacities burden, the ai-based image analysis tool allows for tracking of patient's disease progression. application of an independent ai software system decreased the need of radiologists' annotations of the lesions in ct images, lifting a significant burden from frontline medical staff. the above contribution of deep learning technology highlights the impact ai can potentially have during a pandemic, where workflow heavily relies on human labor and shortages of human resources are a common problem. deep convolutional neural networks (cnn), a widely used deep learning architecture, enabled distinguishing between covid- and other causes of pneumonia through chest x-ray image analysis. investigators used a neural network which was designed to learn covid- radiological pneumonia pattern and could yield accurate diagnostic results in a small cohort of infected patients. in addition to offering diagnostic accuracy and speed, ai can help reduce occupational exposure to the virus. frontline healthcare practitioners, including ct and mri technicians, are at high risk for contracting the virus given their frequent exposure to covid- patients. researchers proposed the use of a contactless workflow that can serve to protect the frontline workers and ensures that services continue to have adequate staffing. a visual ai empowered mobile ct platform allows the technician to determine the optimal scanning parameters such as identifying the pose and shape of the patient, and monitor the imaging procedure safely and efficiently. ai applications can also be utilized to predict the outcome of covid- infections. machine learning was implemented for development of a prognostic algorithm to estimate the mortality risk of a person with covid- . the prediction model was able to predict and help with the early identification of critically ill patients and through extraction of three key laboratory parameters: lactic dehydrogenase, lymphocyte count, and high-sensitivity c-reactive protein. the demand for intensive care unit-level care and ventilatory support provision has increased as a result of the current pandemic. the recognition of critical cases preemptively would enable appropriate evaluation of future demand, and thus allow institutions to be more prepared to react appropriately. an ai empowered model can predict the development of high mortality complications of covid- , such as acute respiratory distress syndrome (ards). researchers utilized such a model to identify features biomedical engineering society of initial disease presentation that are associated with later development of ards; these features included a mildly elevated alanine aminotransferase, the presence of myalgias and an elevated hemoglobin. emerging ai-based methods can potentiate epidemiological models for real-time forecasting of disease trends. modified auto-encoders, helped scientists predict the length of the progression and the transmission dynamics of the pandemic. an accurate forecasting method aids in the development of effective healthcare policies and helps address public health issues that have arisen with the covid- pandemic. ongoing efforts have been made to implement ai in the development of effective therapeutic strategies against covid- . machine learning principles can be applied to predict whether commercially approved medications can be used for disease treatment. this approach facilitates the repurposing of existing therapies in a time-efficient manner. a learning-based drug discovery model was also launched for the generation of a potential coronavirus main protease inhibitor. playing a central role in the viral replication, the main protease seems to be an attractive target for the new therapies. despite the potential of ai to be implemented in the fight against covid- , ai systems are still at a preliminary stage. a rigorous quality control to test their validity and to ensure patient benefit and safety is warranted. also, given the fact that ai algorithms need access to massive datasets, ethical concerns about patient privacy further hamper the clinical world adoption of ai tools in the healthcare setting. chloroquine is an example of a medication that has been studied within this field. chloroquine has inhibitory effects on the uptake of nanoparticles from cells. specifically, studies have found clinically significant doses of the medication are associated with reduced accumulation of synthetic nanoparticles (np) in cell lines and the mononuclear phagocyte system of mice. given the similarities in size and shape of the synthetic np and sars-cov- , nanoparticles can help in investigation for drug repurposing for covid- . researchers designed np-based peptides that mimic the virus-binding domain of ace protein. this particular domain facilitates viral entry into a cell; thus, a np mimetic could serve as an antagonist, inhibiting the virus from entering cells. investigators have postulated that inhalers containing the mimetic would be effective in preventing virus activation in the lungs. based on the association of covid- severity with the inflammatory response, scientists are developing nanoparticles that can help in inflam-mation control. lipid-based nanoparticles, loaded with immunomodulating and antioxidant molecules (adenosine and a-tocopherol respectively), can selectively deliver their therapeutic cargo to the sites of acute inflammation and therefore modulate oxidation stress and cytokine response. also, the use of peptide-based nanoparticles has been evaluated for the design of a sars-cov- vaccine. self-assembling peptide nanoparticles that were prepared from sars-cov- spike (s) protein, successfully induced neutralizing antibodies against the coronavirus, subsequently preventing infection of vero cells. novel vaccine development new technologies have enabled a possible covid- vaccine to be designed within just a few months of the initial outbreak. vaccine technology platforms under development include nucleic acid, recombinant viral vector (replicating and non-replicating), and protein-based (subunits or virus-like particles). advanced nucleic acid vaccine platforms are being investigated for sars-cov- . the naked viral dna enters the cell through electroporation, a technique that creates pores in cell membranes to increase nucleic acid uptake, and produces copies of s protein, the viral component that induces a neutralizing humoral immune response. messenger rna (mrna) technology is another approach for sars-cov- vaccine development. use of mrna, the intermediary molecule that cells use to translate genetic information into proteins, is an alternative to employing dna. mrnabased vaccines are designed to contain all of the necessary viral genetic information, allowing the virus to produce proteins-antigens of interest within the host cell, thus mimicking a natural infection. the use of mrna technology for vaccine development is promising, allowing for rapid vaccine production at a low cost. however, nucleic acid-based vaccines are not licensed for humans and thus their immunogenicity is not yet proven. in recombinant viral vectors vaccine approach, the sars-cov- s protein gene would be inserted in the genome of another virus, such as adenovirus or measles. these genetically engineered viruses can then deliver the genetic information for the production of sars-cov- proteins to the host, enabling an immune response. a novel vaccine candidate based on nonreplicating adenovirus vector (ad -ncov) carrying the full-length s protein gene, has already entered phase ii trials. viral vector vaccines, such those using weakened measles, are also a possible approach to developing a sars-cov- vaccine. the replication of viral vectors mimics a natural infection. as the vectors replicate within the infected cells, a complete immune response, including both innate and humoral immunity, is triggered. , a protein-based vaccines platform under evaluation, includes the direct injection of viral protein subunits, mainly s protein or a part of it called receptor binding domain (rbd). rbd is a key component of s protein useful in viral attachment and membrane fusion. however, addition of vaccine adjuvants as well as multiple booster injections might be required for an adequate immune response. virus-like particles, another protein-based vaccine approach, are molecules that closely resemble viruses but lack genetic material and therefore are not infectious. despite the potential to induce a strong immune response such vaccines are harder to produce. a glycoprotein-based approach to vaccine development has also been explored. this approach targets viral glycosylation of the s protein. glycosylation of viral spikes masks the immunogenic sites that are otherwise recognized by the host immune system. as such, glycosylation allows the virus to evade the host immune system. many viruses are known to use this process. investigators studying the glycan profile of sars-cov- , however, found that the glycan density of spikes is not high enough to effectively shield the virus from the host immune defenses. this vulnerability in virus glycan shield may be beneficial for induction of potent neutralizing antibodies and therefore be implicated in vaccine design. powerful technological platforms are used to determine if existing therapeutic modalities approved for other indications can also be efficacious in the treatment of covid- . computer-aided drug repurposing tools have identified several monoclonal antibodies that have potential to treat covid- . monoclonal antibodies have not traditionally been used for infectious disease management but due to in silico drug repurposing, they seem to be a promising addition to the therapeutic arsenal against covid- . novel coronavirus infection is characterized by a fulminant and fatal hyperinflammatory state, responsible for lung damage and multiorgan collapse. interleukin- (il- ) is considered to be a major driver of the cytokine storm and a predictor of fatality according to a recent study from china. antibodies directly targeting the il- receptor could theoretically suppress the overactivated immune system and optimize clinical outcomes. tocilizumab, a monoclonal antibody against il- receptor, is a licensed regimen for rheumatoid arthritis and cytokine release syndrome and recently approved for the treatment of covid- . while the clinical trials for this treatment are still under investigation, a report of patients who have undergone treatment suggests that tocilizumab is welltolerated and associated with rapid resolution of clinical symptoms and improved outcomes. other fda approved immunomodulatory medications are being investigated for potential use in the treatment of covid- include: sarilumab (il- receptor antagonist), bevacizumab (anti-vascular endothelial growth factor medication), eculizumab (antibody inhibiting terminal complement), and ulinastatin (tryspin inhibitor). in addition to monoclonal antibodies targeting key components of the immune response, monoclonal antibodies targeting the virus are also under development. researchers identified a human monoclonal antibody that targets an epitope that is conserved in both sars-cov and sars-cov- , and neutralizes the virus in cultured cells. such neutralizing antibodies can support viral clearance and help in post-exposure prophylaxis. also, due to the crossneutralizing properties, the antibody might be useful for potential future outbreaks caused by related coronaviruses. mathematical modeling has been at the forefront of predicting covid- transmission rates. these models have been essential for informed public policy decision-making that has prevented further spread of the virus. mathematical models comprise of a set of equations that mimic reality and can be refined to include new information about the virus. from simple to more complex mathematical models, researchers around the world are trying to capture the interplay between a plethora of factors, ranging from micropathogens and individual or population interactions, to macro-scale environmental, socio-economic and demographic conditions. that interplay is essential in order to explore possible scenarios of the epidemic spread. these generated scenarios are used to guide outbreak control strategies and prevention policies. , , many different types of models have been implemented. stochastic individual based models (ibm), as well as classic deterministic ''susceptible-infected-recovered'' (sir) and ''susceptible-exposed-infected-recovered'' (seir) models are examples of models that have been used historically for different infectious diseases. new models like the ''stereographic brownian diffusion epidemiology model'' are being developed during the covid- pandemic in an effort to better explain the virus spread. , , the interpretation of these models is of the utmost importance. given the mantra ''mathematical models are as good as the data they use,'' different models should be viewed as complementary rather than searching for a singular correct model that can answer all questions. furthermore, presenting model results alone is not enough. given the necessity for rapid responses, scientists should rapidly and openly share their codes, so results can be replicated, evaluated, and improved. it should be noted, that despite their usefulness, mathematical models in the covid- pandemic have also generated some concerns. significant information about the transmission of the virus remains unknown, thus limiting the precision of forecasts. as results of modeling projections are communicated online, they are over-interpreted as anticipated potentials. as a result, dynamic models developed with the known limitations of theoretical plausibility and mathematical probabilities, are misinterpreted as evidence of what is actually going to happen. with the creation of big data for analytics a wealth of information from around the world can be made available to scientists, doctors, epidemiologists and policymakers. big data has proven to be a useful tool for rapid real-time evaluations as a result. big data analytics has potential to play a key role in preventing covid- related hospital outbreaks. the storage and provision of accurate personal travel and contact history allows disease tracing and screening to be conducted for all patients and visitors before they enter a medical facility. in taiwan, the government has integrated and analyzed several big data from national health insurance administration (nhia), national immigration agency (nia) and taiwan centers for disease control. this has provided real-time information for outdoor quarantine station of each hospital and clinic in the state. as a result, all visitors are screened via personal identification cards and any suspected carrier is further examined before entering the building. this model could serve as way to further limit disease transmission. this same framework can be applied to allow for faster immigration clearance at airports and sea borders. for example, in taiwan, the nhia and the nia launched the entry quarantine system. this system uses the past -day travel history of an individual and their nhia identification card data to screen them for covid- . travel history data is created at the time of departure or arrival at a taiwan airport. at this time travelers are required to complete a health declaration form by scanning a qr code. a mobile health declaration pass is then sent via sms to phones using a local telecom operator. this system has allowed for faster immigration clearance for those with minimal risk. the internet of things (iot) is a system of interconnected computing devices that can transfer data over a network without human involvement. in typical daily functioning, examples of iot applications include home security systems and smart lighting arrangements which are controllable through smartphones. the internet of medical things (iomt) is the healthcare-specific version of iot and can be implemented to provide relief to medical staff, ensure quarantine implementation and trace epidemic origins. data collection can be done with the help of sensors incorporated in mobile phones, drones, robots, as well as self-sampling covid- tests. the data collected via these methods are sent to a central-cloud server for analysis. the analysis generated from such a server would better equip medical providers and government agencies to respond to the covid- crisis. with these data, medical providers would be able to provide patients with more tailored online health consultations. these online services would also enable patients to receive more adequate care while simultaneously limiting their own exposure and further transmission of the virus. government agencies, including local state health bureaus and centers for disease control and prevention (cdc), would be better able to allocate supplies, determine need for quarantine, monitor incidence, and implement emergency strategies with this information. large cities are already integrating these applications to mitigate the pandemic. for example, the shanghai public health clinical center is using body temperature sensors along with data transmission directly to nurse's station for real-time monitoring of covid- patients, thus reducing the potential exposure of healthcare staff. similarly, in boston, a robot currently used to take patient interviews is about to be deployed with sensors in order to measure patients' respiratory rate and body temperature. in singapore, a contact-tracing smartphone application uses wireless bluetooth technology in order to identify people who have been in close proximity with covid- patients. apple and google collaborate in contact tracing and tracking applications that will become available to many countries worldwide, and is expected to dramatically accelerate the identification and notification of individuals that have been unknowingly close to covid- patients. iomt would not only serve to help with the current pandemic, but also could be implemented to prevent future outbreaks as well. the covid- pandemic has created an unprecedented need for contact tracing, case detection, and isolation for containing. there have been two proposals addressing population screening for case detection: massive random screening and targeted screening. the latter comprises of three complementary testing strategies in order to maximize the number of infected people detected. the first strategy is to remove physician referral and cost restrictions for testing patients with symptoms suggestive of covid- . the second strategy is to select and test people in high-risk sentinel locations, like elderly in nursing homes and pregnant women presenting for delivery. the third strategy is to reserve a small number of tests for random screening in order to assess whether sentinel sites are efficiently detecting infected people. both population screening methods have to be followed by contact tracing and isolation of people testing positive. this could bring the reproductive number (ro) of covid- to less than unity and end the repeated cycles of imposing and releasing stay-at-home restrictions. aforementioned technologies could be implemented in comparing the efficacy and cost-effectiveness of the two proposals. telemedicine enables healthcare providers to care for patients while abiding by government shelter-in-place and social distancing orders. throughout this pandemic, online healthcare services have been utilized to provide care for patients at home with mild covid- as well as provide information about the symptoms and prevention of disease to all patients. telemedicine services have also recently expanded in developed countries for the medical management of mild non-covid- related issues. these services range from annual follow-ups to mental health services. mental health services in particular have increased in demand during this time as quarantine related isolation can lead to anger, frustration, fear, and increased stress. , telemedicine services are vital as they decreased the number of hospital visits and increase hospital resources, including hospital staff time. with the increased demand on hospital workers during the healthcare crisis, telemedicine helps off-load the burden on hospital workers, enabling them to focus efforts on those with more severe conditions. studies on real-world data have estimated that around % of us hospitals have adopted telemedicine. in massachusetts, minnesota, new hampshire, and wisconsin the proportion of hospitals adopting telehealth exceeds %. a study in a large healthcare system in new york has estimated that between march , and april , , telemedicine visits increased from . /day to . /day ( % increase) in urgent care and from . /day to . /day ( % increase) in non-urgent care. telemedicine can also be utilized to train staff in rural and remote areas to better respond to this healthcare crisis. increased capabilities of responders in rural areas helps to ensure affected patients have access to adequate care. this remote training platform can also be used to aid countries with fewer trained medical personnel. robotics can be used to minimize both patient and provider covid- risk. robotic surgery provides a safer alternative in terms of covid- exposure for procedures, compared to conventional laparoscopy and open surgery. if all high level precautions are taken, there is reduced number of directly exposed medical staff, as well as reduced patient hospital stay. as a result, there is a decreased patient risk of covid- infection and increased hospital capacity to treat covid- patients. robotics can be used to perform procedures and reduce medical staff exposure. autonomous or remotecontrolled robots can perform noncontact ultraviolet (uv) surface disinfection via intelligent navigation and identification of high-risk/high-touch areas. robots can also independently collect daily temperature measurements in inpatients with covid- as well the temperatures of patients at screening centers. in addition, robots can independently conduct nasopharyngeal swab testing. the use of robotics to perform these functions decrease required staff contact with those infected with covid- patients and would also allow staff to allocate their time towards other patient care tasks. finally, unmanned aerial vehicles (uav) or drones can identify potential covid- carriers. uav have the capacity to detect ground surface temperatures from a certain height above the ground and send the measured data, captured thermal images and optical images along with gps location, to a server. this technology can be adapted to capture body temperatures, enabling us to identify patients with fever, an early symptom of covid- . furthermore, uav technology can be deployed to distribute medical supplies and test kit equipment in areas difficult to reach, thus reducing mortality. three-dimensional printing is a quickly emerging field that can help in the design of medical equipment and can more readily supply needed materials at re-duced costs. the covid- pandemic has caused a worldwide shortage of medical supplies, including the personal protective equipment (ppe), necessary for the care of covid- patients. ppe includes facemasks, face shields and goggles. this equipment helps prevent droplet exposure and further spread of the virus. face masks need to be fitted appropriately in order to adequately prevent air and small droplets from entering around the edges of the mask. d laser scanning allows measurement of exact facial parameters, enabling the production of personalized masks. using open-source data, face shields can also be produced with biodegradable material, allowing at-home, on-demand production. utilization of d printing technology would increase access to these supplies and create more personalized equipment that can better protect medical personnel. three-dimensional printing technology can assist in the production of nasopharyngeal and oropharyngeal test swabs. the assistance of d printing in production of these materials would allow for widespread population testing. with increased access to testing, policy regarding carrier tracing and isolation to prevent the spread of the virus could be more conservative and effective. in countries impacted heavily by covid- there have been shortages in key components of the respiratory support equipment. in such settings, d-printing has been implemented to produce venturi valves. these valves were difficult to produce given their design being subject to copyright and patent covers. in addition, d printing can also be utilized for production of ventilator splitters with adjustable flow control valves. the adjustable flow control valves have further increased patient access to ventilators by enabling two patients with different oxygen requirements to use the same ventilator. utilization of d-printing can revolutionize equipment production in terms of efficiency, quantity and cost. large academic medical centers and hospitals should work together with the d printing community in order meet the rising demand of medical supplies. however, safety protocols should still be followed during this time to ensure medical supplies meet regulatory standards and are safe for use. emerging technologies can be effectively utilized to allow the medical community to rapidly respond to the increased demands and burden of covid- . technologies have been used in the study, diagnosis, and treatment of covid- . recent developments have proven the collaboration between medical researchers and engineers is essential for the development of expeditious and less expensive ways of addressing the pandemic. in the context of the rapid and worldwide disease spread, open access to knowledge, tools, and technology is essential for timely response. researchers and engineers must continue to collaborate and share expertise to continue to provide solutions in this time of crisis. although swift development and application of novel technologies is required, safety monitoring should not be overlooked. established standards regarding patient generated data, including confidentiality must be upheld. in addition, the safety standards for the production and distribution of supplies and services should continuously be monitored as new technologies are being used. treatments identified using emerging technologies should also undergo standard clinical trials. the community needs to strive to uphold all these safety standards to ensure best outcomes for patients. mtv, ea, and jg drafted the initial manuscript. fs, ekm, mk edited the manuscript. em reviewed and revised the manuscript. all authors read and approved the final manuscript as submitted and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. baharudin, h., and l. wong. coronavirus: singapore develops smartphone app for efficient contact tracing. h ttps://www.straitstimes.com/singapore/coronavirus-singap ore-develops-smartphone-app-for-efficient-contact-tracing . call for transparency of covid- models sbdiem: a new mathematical model of infectious disease dynamics the robot will see you now a boston hospital is using spot, the dog-like robot of internet fame, to screen for coronavirus the race for coronavirus vaccines: a graphical guide novel coronavirus (covid- ): leveraging telemedicine to optimize care while minimizing exposures and viral transmission big data integration and analytics to prevent a potential hospital outbreak of covid- in taiwan boosting the arsenal against covid- through computational drug repurposing covid- , an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics squalene-based multidrug nanoparticles for improved mitigation of uncontrolled inflammation logistics of aggressive community screening for coronavirus rapid ai development cycle for the coronavirus (covid- ) pandemic: initial results for automated detection & patient monitoring using deep learning ct image analysis computational design of ace -based peptide inhibitors of sars-cov- population-level interest and telehealth capacity of us hospitals in response to covid- : cross-sectional analysis of google search and national hospital survey data artificial intelligence in radiology insights from nanomedicine into chloroquine efficacy against covid- artificial intelligence forecasting of covid- in china. arxiv eprints on the responsible use of digital data to tackle the covid- pandemic applications of d printing technology to address covid- related supply shortages towards an artificial intelligence framework for datadriven prediction of coronavirus clinical severity key challenges for delivering clinical impact with artificial intelligence covid- vaccines: breaking record times to first-in-human trials robot assisted surgery during the cov-id- pandemic, especially for gynecological cancer: a statement of the society of european robotic gynaecological surgery (sergs) sphcc employs iot tech and wearable sensors to monitor covid- patients the covid- vaccine development landscape developing covid- vaccines at pandemic speed repurposing therapeutics for covid- : rapid prediction of commercially available drugs through machine learning and docking colder carras, and a. labrique. global preparedness against covid- : we must leverage the power of digital health covid- transforms health care through telemedicine: evidence from the field covid- : consider cytokine storm syndromes and immunosuppression artificial intelligence-enabled rapid diagnosis of patients with covid- pathological inflammation in patients with covid- : a key role for monocytes and macrophages automatic detection of coronavirus disease (covid- ) using x-ray images and deep convolutional neural networks artificial intelligence vs covid- : limitations, constraints and pitfalls apple and google's covid-tracing tech has been released to countries can mathematical modelling solve the current covid- crisis? mrna vaccines-a new era in vaccinology outbreak dynamics of covid- in china and the united states peptide nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine covid- and mental health: a review of the existing literature machine learning using intrinsic genomic signatures for rapid classification of novel pathogens: covid- case study a model society: maths, models and expertise in viral outbreaks replicating and non-replicating viral vectors for vaccine development pharmacologic treatments for coronavirus disease (covid- ): a review precise pulmonary scanning and reducing medical radiation exposure by developing a clinically applicable intelligent ct system: towards improving patient care review of artificial intelligence techniques in imaging data acquisition, segmentation and diagnosis for covid- internet of things (iot) applications to fight against covid- pandemic report proposes covid- national surveillance plan custommade d-printed face masks in case of pandemic crisis situations with a lack of commercially available ffp / masks mathematic modeling of covid- in the united states covid- and the role of d printing in medicine a human monoclonal antibody blocking sars-cov- infection response to covid- in taiwan: big data analytics, new technology, and proactive testing site-specific glycan analysis of the sars-cov- spike -d printed protective equipment during covid- pandemic rolling updates on coronavirus disease (covid- ) effective treatment of severe covid- patients with tocilizumab prediction of criticality in patients with severe covid- infection using three clinical features: a machine learning-based prognostic model with clinical data in wuhan combining point-of-care diagnostics and internet of medical things (iomt) to combat the covid- pandemic combating covid- -the role of robotics in managing public health and infectious diseases deep learning-based detection for covid- from chest ct using weak label publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - uuczmi authors: kidokoro, minoru; shida, hisatoshi title: vaccinia virus lc m ∆ as a vaccine vector for clinical applications date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: uuczmi the lc m strain of vaccinia virus, the active ingredient in the japanese smallpox vaccine, was derived from the lister/elstree strain. lc m is replication-competent and has been administered to over , infants and , adults with no serious adverse reactions. despite this outstanding safety profile, the occurrence of spontaneously-generated large plaque-forming virulent lc m revertants following passage in cell culture is a major drawback. we identified the gene responsible for the reversion and deleted the gene (b r) from lc m to derive lc m Δ. lc m ∆ is non-pathogenic in immunodeficient severe combined immunodeficiency (scid) mice, genetically-stable and does not reverse to a large-plaque phenotype upon passage in cell culture, even under conditions in which most lc m populations are replaced by revertants. moreover, lc m ∆ is > -fold more effective than the non-replicating vaccinia virus (vv), modified vaccinia ankara (mva), at inducing murine immune responses against pathogenic vv. lc m ∆, which expresses the siv gag gene, also induced anti-gag cd (+) t-cells more efficiently than mva and another non-replicating vv, dairen i minute-pock variants (dis). moreover, lc m ∆ expressing hiv- env in combination with a sendai virus vector induced the production of anti-env antibodies and cd (+) t-cells. thus, the safety and efficacy of lc m ∆ mean that it represents an outstanding platform for the development of human vaccine vectors. smallpox was eradicated worldwide in the s [ , ] . however, serious public health concerns due to the threat of bioterrorism [ ] and natural outbreaks of monkeypox [ , ] at the start of the st century have highlighted the necessity for a vaccinia virus (vv)-based smallpox vaccine. existing vaccine stockpiles have not been updated since the s; because these early vaccines are lymph-derived vaccines produced by propagating vaccine viruses in the skin of animals (i.e., first-generation vaccines ( table )), they do not meet good manufacturing practice (gmp) standards [ ] [ ] [ ] . therefore, they are at risk for adventitious microbial contamination. moreover, these vaccines occasionally caused serious adverse effects (e.g., progressive vaccinia, eczema vaccinatum and post-vaccinial encephalitis) due to the pathogenicity of the vaccine viruses used [ , ] . to address the issues outlined above, much effort has gone into developing safer smallpox vaccine candidates. some studies aimed to generate vaccines using a sterile cell culture technique to reduce the risk of contamination by adventitious agents (second-generation vaccines) (see table ) [ ] . for example, acam [ , ] was propagated in mrc- cells (diploid human lung fibroblasts) using a single clone vv isolated from a dryvax calf lymph vaccine (manufactured by wyeth laboratories using new york city board of health (nycbh)). acam was prepared in vero cells under serum-free conditions using acam as the seed virus [ , ] . the cell-cultured smallpox vaccine (ccsv), which was derived from a plaque-purified nycbh strain, was also prepared in mrc- cells [ ] . the elstree-bn vaccine was manufactured in chicken embryo fibroblasts (cef) using the lister/elstree (lister) strain, which was widely used as a lymph-derived vaccine in europe, africa and asia during the global smallpox eradication campaign [ ] . the manufacturing of vaccines in cell culture reduced the risk of vaccine contamination by extraneous agents. however, because second-generation vaccines were manufactured using first-generation vaccines or their isolates as seed viruses, their safety profiles were equivalent to those of the original lymph-derived vaccines, i.e., they caused the same adverse events [ , ] . as the global smallpox eradication campaign progressed and the risk of contracting smallpox infections diminished, developed countries began to raise concerns about the side effects associated with lymph-derived smallpox vaccines. this triggered new research to develop alternative attenuated vaccines (third-generation vaccines), such as the modified vaccinia ankara (mva) [ , ] , dairen i minute-pock variants (dis) [ ] and lister clone m (lc m ) [ ] [ ] [ ] [ ] . the main method used to attenuate the vvs was serial passage in primary cell culture or eggs. mva was attenuated by serial passage of the chorioallantois vv ankara strain in cef (> times). this resulted in the loss of approximately % of its genome, including host range-related genes, such as k l, and some immunomodulatory genes, thereby generating a phenotype that is unable to replicate in most mammalian cells [ ] [ ] [ ] . mva, which shows an extremely attenuated phenotype in animals and humans [ ] , was administered to about , individuals in western germany and turkey during the global eradication campaign with no apparent side effects [ ] . although its ability to protect against smallpox infection was not proven at that time, the need for a smallpox vaccine that was safe for use in immunocompromised individuals (including aids patients, patients treated with chemotherapy and transplant recipients) meant that mva was examined in a number of clinical trials [ ] [ ] [ ] [ ] [ ] . data from these clinical trials and some animal experiments suggest that although mva-derived vaccines have an extremely good safety profile; they are less immunogenic than replication-competent vvs, such as dryvax, lc m and lc m ∆ [ , [ ] [ ] [ ] . for example, data from animal models show that multiple and - log higher doses of mva are required to elicit antibody titers comparable with those elicited by replication-competent vvs [ ] [ ] [ ] . the dis strain originates from the dairen i strain, a smallpox vaccine strain in japan, and was isolated as a small-sized pock forming variant on chick chorioallantoic membrane (cam) after passages in one-day-old eggs [ ] . as is the case with mva, dis harbors a large deletion within the left terminal region of the genome, which contains the host range genes, k l and c l, and the immunomodulatory gene, k l [ ] ; consequently, dis lacks the ability to replicate in a number of mammalian cell types. although dis showed a good safety profile when tested in field trials involving japanese children, it was not adopted as a smallpox vaccine, because it was less immunogenic than lister clone (lc ). concerns regarding the side effects of first-generation smallpox vaccines, such as ikeda, dairen i and lister were becoming a problem in japan during the s. in response to demands for a safer (but still effective) vaccine, the chiba serum institute developed a highly attenuated strain, called lc m [ , ] . lc m , which forms minute pocks on the cam of embryonated eggs, was isolated from the lister (lister original, lo) strain via intermediate strains, such as lc and its derivative, lc mo [ , ] . tests in rabbit and monkey models showed that lc m was markedly less neurovirulent than first-generation vaccine strains, such as lo and dryvax; indeed, its virulence was comparable with that of replication-defective dis [ ] [ ] [ ] ] . moreover, lc m induced a much weaker dermal reaction in rabbits and humans and showed a lower rate of febrile reactions than lc mo (a direct parent of lc m ) in clinical trials [ , ] . lc m was administrated to approximately , infants without any serious adverse reactions and proved to be as immunogenic as the parental lo strain [ , ] . therefore, lc m was adopted as the favored vaccine strain in japan [ ] . a number of novel attenuation approaches involving direct modification of the vv genome using genetic engineering techniques were used to develop highly attenuated vv strains (fourth-generation vaccines), such as nyvac and lc m ∆ [ , , [ ] [ ] [ ] [ ] [ ] [ ] . these methods replaced classical attenuation methods based on serial passage in primary cell cultures or eggs. nyvac was derived from the copenhagen vv vaccine strain by deleting non-essential genes, which include c l and k l, the host range genes; the thymidine kinase gene, a gene related to viral dna synthesis and the i l gene encoding the large subunit of ribonucleotide reductase. thus, nyvac shows very restricted replication in mammalian cells and a highly attenuated phenotype in animals [ ] . however, since the replication of nyvac in non-permissive mammal cells is arrested at an early stage [ ] (as is the case for avipoxviruses, such as canary poxvirus and fowl poxvirus), it elicits weaker immune responses than mva or replication-competent vvs [ ] . lc m ∆ should be categorized as a fourth-generation vaccine, because it was obtained from the parental smallpox vaccine strain (lc m ) by deleting the b r gene, which is responsible for the reversion of lc m . consequently, it shows good genetic stability with very little (if any) reversion; however, it retains its ability to replicate in mammalian cells [ ] . takahashi-nishimaki et al. first identified the vv b r gene, which is responsible for large-plaque formation and replication in vero cells, during the course of investigating the mechanism of attenuation to generate lc m [ ] . lc m harbors a frameshift mutation due to a single base deletion in the middle of its open reading frame (orf); this mutation results in the loss of b r function. b r encodes a -kda glycoprotein (b protein), which is involved in packaging the intracellular mature virion (imv) within the trans-golgi membrane or endosomal cisternae to form an intracellular enveloped virion (iev) [ ] [ ] [ ] . the iev is transported along microtubules to the cell periphery [ , ] , where it adheres to the cell membrane as a cell-associated enveloped virion (cev). the b protein, in cooperation with the a and a proteins, also participates in the src kinase-dependent formation of actin-containing microvilli and the subsequent release of the cev from the cell surface to form an extracellular enveloped virion (eev) [ , ] . despite the relative paucity of whole progeny virions, eevs play an important role in dissemination within the host [ ] . since anti-b antibodies neutralize eev, b r-expressing vv has been proposed as an effective smallpox vaccine [ , [ ] [ ] [ ] [ ] . when generating and performing basic research on lc m , we found that the vaccine spontaneously reverted to large-plaque-forming clones (lpcs) [ ] . thus, we were concerned that lpc contamination might ruin the safety profile of a future lc m vaccine. therefore, we investigated the molecular mechanism(s) underlying the reversion, with a focus on the b r gene associated with the formation of large plaques [ ] . we isolated three lpc clones from a vaccine stock of lc m and examined their phenotypes in terms of plaque size, dermal reactions in rabbits and pathogenicity in severe combined immunodeficiency (scid) mice; these phenotypes were compared with those of lc m and the parental virus lc mo, which retains a fully-functional b r gene. all three lpc viruses showed phenotypes similar to that of lc mo, resulting in better growth in cell culture and greater virulence in scid mice than lc m [ ] . as expected, sequencing the b r in these lpcs revealed that the b r orf contained a single base insertion upstream of the nucleotide that was deleted from the lc m b r. it is noteworthy that the nucleotide insertion site in the lpc b r orf was different in each of the three clones, even though they originated from the same viral stock, which was prepared through only seven passages after the lc m cloning. these results strongly suggest that the reversion of lc m is a multi-clonal event and may occur frequently. to prevent the generation of lc m revertants, we decided to delete the entire b r gene from the lc m genome by homologous recombination to yield lc m ∆ [ ] . the phenotype of lc m ∆ (plaque size and dermal reaction in rabbits) was similar to that of lc m . intraperitoneal (i.p.) injection of pfu of lc m ∆ (a dose three logs higher than that needed to elicit protective immunity in balb/c mice) did not cause any symptoms in scid mice over an eight-week period (figure ). mva and plaque-purified lc m (which contains a very low level of revertants) were also nonpathogenic; however, lc mo and m b r (a derivative constructed by reintroducing the intact b r gene into lc m ) caused severe rashes and death in scid mice, even when administered at a dose two logs lower than lc m ∆ (figure ). the genetic stability of lc m ∆ was evaluated by serial passage in primary rabbit kidney (prk) cells, which were used to generate the lc m vaccine. no detectable lpcs emerged from lc m ∆ under any of the test conditions, including those used in vaccine production (passage in prk cells at °c). by contrast, lpcs emerged from lc m that was plaque-purified immediately before testing (figure ). it should be noted that once lpcs appeared in the cultures, the lpcs:lc m ratio increased rapidly with the passage number (figure ). [ ] . lpc, large-plaque-forming clone. the protective immune response elicited by lc m ∆ was compared with that elicited by dryvax, mva, lc m and lc m derivatives (m b r and m dtm, both of which express the b ectodomain at high levels) in a mouse model. this model, in which the immunized mice are challenged with a highly pathogenic vv (the western reserve (wr) strain), is one of the most popular methods of evaluating the efficacy of smallpox vaccines [ ] (figure ). we immunized each group of mice with a single dose ( , or pfu) of each vv via the intramuscular (i.m.) route. we found that the level of protective immunity elicited by lcm ∆ was comparable with that elicited by dryvax and superior to that elicited by mva. for example, the minimal dose ( pfu) of lc m ∆ or dryvax fully protected mice from lethal infection with wr, whereas mice immunized with mva, lc m , m b r or m dtm, lost weight and, in some cases, died. the maximum dose ( pfu) of mva resulted in prominent weight loss after wr challenge. it is noteworthy that immunization with lcm ∆ was more efficient than that with m b r or m dtm when compared at their minimal dose. in particular, m b r was significantly inferior to lc m ∆ (t-test, p = . ). these results suggest that b r does not play a major role in eliciting protective immune responses in these mice. in addition, lc m ∆ elicited protective immune responses in cynomolgus monkeys and fully protected them against lethal infection with monkeypox virus [ ] . taken together, these data suggest that lc m ∆ is as effective as the first-generation smallpox vaccine, dryvax. although several studies report that the b protein is the major target of eev-neutralizing antibodies, which are significant for protection against smallpox infection, immunization with b -deficient vaccine viruses protects animals against lethal challenge by pathogenic orthopoxviruses [ , [ ] [ ] [ ] [ ] . in addition, some reports show that smallpox vaccines do not always induce anti-b antibodies, and antibody response profiles against each viral protein are highly heterologous in humans [ ] [ ] [ ] . they also concluded that the key to inducing a strong neutralizing antibody response is to elicit antibodies that recognize multiple viral proteins; these antibodies then act synergistically to provide better protection. vv has been widely used as a vector for expressing foreign genes, because it has many excellent properties: high expression efficiency, a broad host range, a very large capacity for accepting foreign genes, heat stability and inexpensive vaccine production [ ] . therefore, vv vectors have been examined for use as live vaccines against both human and veterinary infectious diseases and cancers [ ] [ ] [ ] . however, concerns about the safety profile of vvs are a major barrier to developing recombinant vv vaccines for use in humans [ ] . most research has focused on replication-defective poxvirus vectors (which have better safety profiles) as vehicles for delivering antigens derived from human pathogens. for example avipox- [ ] , mva-and nyvac-based vectors expressing components of human pathogens, such as hiv- and tuberculosis, have been developed and evaluated in monkeys [ , ] and humans [ ] [ ] [ ] [ ] . however, although promising in animal models, these vaccines did not induce sufficiently strong immune responses in humans, nor did they protect humans from infection [ , ] . therefore, more effective vehicles are needed for human vaccine development. thus, a replication-competent vv that has been proven to be safe for human vaccination against smallpox could be a good candidate. the safety profile and strong antigenicity of lc m ∆, a genetically-stable variant of lc m , make it a promising vehicle for a vaccine against hiv or other human diseases. one concern regarding the use of viral vectors is pre-existing immunity against the vector virus, which has the potential to dampen specific immune responses. however, kohara et al. showed that a recombinant lc m vaccine expressing the sars coronavirus (sars-cov) spike protein elicited neutralizing antibodies against sars-cov in rabbits that generated a high titer of anti-lc m antibodies [ ] . another report shows that the vv lacking the b ectodomain induces a more potent immune response in vaccinia-immune animals than its wild-type counterpart [ ] . these results suggest that lc m ∆ would make a good vector virus for eliciting effective immune responses against foreign antigens in individuals pre-immunized with smallpox vaccines. previously, we developed the psfj - promoter, an a-type inclusion body (ati) complex promoter that comprises ten repeat units of the mutated early region of the p . promoter plus the ati late promoter [ ] . this complex promoter possesses strong activity in both the early and late phases of the vv infection cycle. indeed, the h protein of the measles virus and chloramphenicol acetyltransferase, the synthesis of which is driven by this promoter, comprised approximately % of total cellular protein [ , ] . moreover, we constructed lc m ∆vnc , a vector that harbors psfj - along with a multiple cloning site within the hemagglutinin (ha) gene, which can be used for the rapid production of recombinant lc m ∆ through in vitro ligation of the lc m ∆vnc genome with foreign dna [ ] . the foreign genes inserted were stably maintained in the lc m ∆ recombinants constructed by this technique and harboring the p . promoter after several passages in the rk cells, a standard cell line for the propagation of vvs. using this technique, we tested whether lc m ∆ is a better vector than non-replicating vaccinia virus for the expression of siv gag. the gag proteins of hiv- and siv are major antigens that elicit cytotoxic t lymphocyte (ctl) responses. the activity of anti-gag ctl inversely correlates with the viral load in hiv- -infected individuals [ ] . experimental infection of monkeys with siv suggests that the strength of the anti-gag ctl response correlates with the containment of siv [ ] . a m ∆/psfj/sivgag vector expressing the siv gag antigen under control of the psfj - promoter generated significantly more gag protein in vitro and elicited the production of anti-gag ifn-γ + t-cells in mice, more efficiently than the non-replicating vv dis strain (which harbors the gag gene under the control of the same promoter) [ ] . the dis strain is immunogenically similar to mva [ ] . we further optimized lc m ∆ for use as a vector by comparing the immunogenicity of siv gag proteins expressed under the control of either the psfj - promoter or the p . promoter, which is a classical early-late promoter [ ] with moderate activity (although weaker than that of psfj - ). preliminarily observations indicated that expressing too much foreign protein led to a reduction in vv propagation in vitro; therefore, the balance between the expression of a foreign antigen and viral propagation in vivo might be crucial for optimal immunogenicity. thus, we compared the immunogenicity and virulence of m ∆/p . /sivgag with that of m ∆/psfj/sivgag in the setting of a recombinant bacillus calmette-guerin (bcg) prime/recombinant lc m ∆ boost vaccination protocol. this setting was based on the observation that long-term maintenance of effector memory t-cells (tem) with the capacity to immediately attack siv-infected cells restricts infection by antibody-resistant siv at the site of virus entry. this was achieved using vaccine approaches that persistently express viral antigens in vaccinated macaques via the use of a cytomegalovirus (cmv) vector, thereby resulting in continuous immune stimulation [ , ] . since bcg persists in vaccinated individuals for long periods of time (up to years) without serious symptoms, vaccination with bcg expressing the gag protein may be expected to induce gag-specific cd + t-cells and to maintain immunological memory (via tem) for a long time. vaccination studies in mice revealed that m ∆/psfj/sivgag was less pathogenic and elicited gag-specific ifn-γ + , cd α + and cd + t-cells more efficiently than m ∆/p . /sivgag. tem were detected even at four months after boosting with m ∆/psfj/sivgag. therefore, lc m ∆ that express siv gag under the control of the psfj - promoter induced more efficient and long-lasting immune responses than lc m ∆ harboring the p . promoter [ ] . although inducing both anti-hiv- antibody and cytotoxic cd + t-cells is an effective way of preventing hiv- infection, it is often difficult to induce the production of anti-hiv- antibodies, particularly neutralizing antibodies, at a high titer. for example, only a low titer of anti-hiv- env antibodies was observed, even after repeated immunization with an mva-based vector [ ] . repetitive antigenic stimulation is required for affinity maturation, the process by which high avidity neutralizing antibodies against hiv- are generated. long-lasting expression of antigen by a replication-competent vector, such as lc m ∆, may enable repeated immunological presentation, which induces affinity maturation. we next examined the ability of lc m ∆ expressing the hiv- env gene to elicit anti-hiv- antibodies and cd + t-cells in mice in the setting of a recombinant lc m ∆ prime followed by a sendai virus vector boost. we found that this vaccination regimen led to the efficient induction of both env-specific cd + t-cells and anti-env antibodies, including neutralizing antibodies. these results are in sharp contrast to those reported by studies that used vaccine regimens based on priming with an env-expressing plasmid followed by a boost with the lc m ∆ or sev vector; such an approach mainly induced cell-mediated immune responses [ ] . despite its replication-competent phenotype, lc m ∆ is highly attenuated and shows no pathogenic effects in scid mice (similar to replication-defective vvs, such as mva). however, it is a comparably effective smallpox vaccine with respect to dryvax. moreover, lc m ∆-based vectors induce both antibody-and cell-mediated immune responses against foreign antigens more efficiently than non-replicating vv vectors. therefore, lc m ∆ is superior to non-replicating vv vectors and is suitable for use in humans. we also point out that lc m ∆ recombinants may be useful as a dual vaccine against both smallpox and pathogens targeted with the inserted genes. ladnyi, i.d.; organization, w.h. smallpox and its eradication; world health organization a reality in our time-certification of the global eradication of smallpox smallpox as a biological weapon: medical and public health management. working group on civilian biodefense the detection of monkeypox in humans in the western hemisphere the centers for disease control and prevention. multistate outbreak of monkeypox-illinois, indiana, and wisconsin vaccinia virus vaccines: past, present and future vaccinia viruses: vaccines against smallpox and vectors against infectious diseases and tumors a vaccinia virus renaissance: new vaccine and immunotherapeutic uses after smallpox eradication adverse events associated with smallpox vaccination in the united states neurologic adverse events associated with smallpox vaccination in the united states adventitious agents and smallpox vaccine in strategic national stockpile clonal vaccinia virus grown in cell culture as a new smallpox vaccine comparison of the safety and immunogenicity of acam , acam and dryvax ® in healthy vaccinia-naive adults acam clonal vero cell culture vaccinia virus (new york city board of health strain)-a second-generation smallpox vaccine for biological defense safety and immunogenicity of new cell-cultured smallpox vaccine compared with calf-lymph derived vaccine: a blind, single-centre, randomised controlled trial modified vaccinia virus ankara protects macaques against respiratory challenge with monkeypox virus mva vaccination against smallpox: clinical tests with an attenuated live vaccinia virus strain (mva) (in german) the smallpox vaccination strain mva: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defence mechanism immunogenicity of an attenuated strain of vaccinia virus on rabbits and monkeys special edition future of smallpox vaccination: everything about attenuated smallpox vaccines. basics of new attenuated smallpox vaccine strain lc m comparative studies of several vaccinia virus strains by intrathalamic inoculation into cynomolgus monkeys a comparison of neurovirulence of vaccinia virus by intrathalamic and/or intracisternal inoculations into cynomolgus monkeys properties of attenuated mutant of vaccinia virus, lc m , derived from lister strain mapping of deletions in the genome of the highly attenuated vaccinia virus mva and their influence on virulence marker rescue of the host range restriction defects of modified vaccinia virus ankara vaccinia virus host range genes successful intramuscular immunization against vaccinia and variola smallpox vaccination and bioterrorism with pox viruses safety and immunogenicity of imvamune, a promising candidate as a third generation smallpox vaccine clinical and immunologic responses to multiple doses of imvamune (modified vaccinia ankara) followed by dryvax challenge modified vaccinia ankara strain as an attenuated smallpox vaccine effect of vaccination with modified vaccinia ankara (acam ) on subsequent challenge with dryvax safety and immunogenicity of modified vaccinia ankara (acam ): effect of dose and route of administration genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia lc m enhanced immunogenicity and protective effect conferred by vaccination with combinations of modified vaccinia virus ankara and licensed smallpox vaccine dryvax in a mouse model highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge structural analysis of vaccinia virus dis strain: application as a new replication-deficient viral vector lc m : an attenuated smallpox vaccine smallpox vaccination of eczema patients with a strain of attenuated live vaccinia (cvi- ) vaccination research groups research report: ministry of health and welfare special research: postvaccination side effects and research regarding treatment of complications nyvac: a highly attenuated strain of vaccinia virus safe and effective poxvirus vectors-nyvac and alvac construction of a vaccinia virus deficient in the essential dna repair enzyme uracil dna glycosylase by a complementing cell line highly efficient induction of protective immunity by a vaccinia virus vector defective in late gene expression immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus ankara the nonreplicating smallpox candidate vaccines defective vaccinia lister (dvv-l) and modified vaccinia ankara (mva) elicit robust long-term protection cellular and biochemical differences between two attenuated poxvirus vaccine candidates (mva and nyvac) and role of the c l gene short-and long-term immunogenicity and protection induced by non-replicating smallpox vaccine candidates in mice and comparison with the traditional st generation vaccine regulation of plaque size and host range by a vaccinia virus gene related to complement system proteins the formation and function of extracellular enveloped vaccinia virus assembly of vaccinia virus: the second wrapping cisterna is derived from the trans golgi network vaccinia virus utilizes microtubules for movement to the cell surface kinesin-dependent movement on microtubules precedes actin-based motility of vaccinia virus visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-b r membrane protein chimera mutations in the vaccinia virus a r and b r envelope proteins that enhance release of extracellular virions and eliminate formation of actin-containing microvilli without preventing tyrosine phosphorylation of the a r protein src mediates a switch from microtubule-to actin-based motility of vaccinia virus extracellular release of enveloped vaccinia virus from mouse nasal epithelial cells in vivo neutralizing and protective antibodies directed against vaccinia virus envelope antigens four-gene-combination dna vaccine protects mice against a lethal vaccinia virus challenge and elicits appropriate antibody responses in nonhuman primates differential efficacy of vaccinia virus envelope proteins administered by dna immunisation in protection of balb/c mice from a lethal intranasal poxvirus challenge smallpox dna vaccine protects nonhuman primates against lethal monkeypox biological characterization of recombinant vaccinia viruses in mice infected by the respiratory route protective effects of improved smalpox vaccine lc m ∆ against a lethal monkeypox challenge in cynomolgus monkeys dna vaccination with vaccinia virus l r and a r genes protects mice against a lethal poxvirus challenge differential antigen requirements for protection against systemic and intranasal vaccinia virus challenges in mice lc m , a highly attenuated vaccinia virus vaccine lacking expression of the membrane protein b r, protects monkeys from monkeypox an attenuated lc m smallpox vaccine: analysis of full-genome sequence and induction of immune protection redundancy and plasticity of neutralizing antibody responses are cornerstone attributes of the human immune response to the smallpox vaccine humoral immunity to smallpox vaccines and monkeypox virus challenge: proteomic assessment and clinical correlations the heterogeneity of human antibody responses to vaccinia virus revealed through use of focused protein arrays vaccinia virus: a tool for research and vaccine development live attenuated vaccinia and other poxviruses as delivery systems a phase / comparative vaccine trial of the safety and immunogenicity of a crf _ae (subtype e) candidate vaccine: alvac-hiv (vcp ) prime with oligomeric gp ( th /lai-did) or bivalent gp (cm /sf ) boost attenuation of simian immunodeficiency virus sivmac infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing gag multispecific vaccine-induced mucosal cytotoxic t lymphocytes reduce acute-phase viral replication but fail in long-term control of simian immunodeficiency virus sivmac dna gag/adenovirus type (ad ) gag and ad gag/ad gag vaccines induce distinct t-cell response profiles induction of multifunctional human immunodeficiency virus type (hiv- )-specific t cells capable of proliferation in healthy subjects by using a prime-boost regimen of dna-and modified vaccinia virus ankara-vectored vaccines expressing hiv- gag coupled to cd + t-cell epitopes an hiv- clade c dna prime, nyvac boost vaccine regimen induces reliable, polyfunctional, and long-lasting t cell responses safety and efficacy of mva a, a new tuberculosis vaccine, in infants previously vaccinated with bcg: a randomised, placebo-controlled phase b trial toward an aids vaccine sars-cov spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus b -deficient vaccinia virus as a vaccine vector for the expression of a foreign antigen in vaccinia immune animals constructions of vaccinia virus a-type inclusion body protein, tandemly repeated mutant . kda protein, and hemagglutinin gene promoters support high levels of expression large-scale preparation of biologically active measles virus haemagglutinin expressed by attenuated vaccinia virus vectors cloning and characterization of the gene encoding the major protein of the a-type inclusion body of cowpox virus immunogenicity of newly constructed attenuated vaccinia strain lc m delta that expresses siv gag protein cd + t-cell responses to different hiv proteins have discordant associations with viral load cytotoxic t lymphocyte-based control of simian immunodeficiency virus replication in a preclinical aids vaccine trial recombinant vaccinia dis expressing simian immunodeficiency virus gag and pol in mammalian cells induces efficient cellular immunity as a safe immunodeficiency virus vaccine candidate general method for production and selection of infectious vaccinia virus recombinants expressing foreign genes effector memory t cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge profound early control of highly pathogenic siv by an effector memory t-cell vaccine immunogenicity and safety of the vaccinia virus lc m delta vector expressing siv gag under a strong or moderate promoter in a recombinant bcg prime-recombinant vaccinia virus boost protocol specificity and -month durability of immune responses induced by dna and recombinant modified vaccinia ankara vaccines expressing hiv- virus-like particles elicitation of both anti hiv- env humoral and cellular immunities by replicating vaccinia prime sendai virus boost regimen and boosting by cd lm we thank hanako yoshizawa and kazuyoshi suzuki for providing background information regarding the development of lc m . both authors contributed equally to this work. the authors declare no conflict of interest. key: cord- -cg wqc q authors: rappuoli, rino title: vaccines, emerging viruses, and how to avoid disaster date: - - journal: bmc biol doi: . /s - - - sha: doc_id: cord_uid: cg wqc q rino rappuoli is a graduate of siena university, where he also earned his phd before moving to the sclavo research center, the italian vaccine institute, also in siena. he then spent two years in the usa, mostly at harvard with john murphy and alwin pappenheimer working on a new diphtheria vaccine based on a non-toxic mutant of diphtheria toxin which has since become the basis for conjugate vaccines against haemophilus, meningococcus, and pneumococcal infections, before returning to the sclavo research center where he developed an acellular vaccine based on a mutant pertussis toxin. with many achievements in vaccine development to his credit, he is now global head of vaccines research and development for novartis vaccines in siena, and has most recently pioneered reverse vaccinology, in which the genome of the pathogen is screened for candidate antigenic and immunogenic vaccine components. we spoke to him about the potential for outbreaks of the kind we are now seeing with ebolavirus in west africa, and what can be done to prevent them. i know that there are 'flu vaccines, but are there licensed vaccines for any of the others? the answer is no -there are no vaccines for sars, which emerged in , there is no licensed vaccine for mers, there is no licensed vaccine for chikungunya, there is no licensed vaccine for ebola -so for most of these diseases there are no vaccines, because they are too rare to justify the economic investment. is that the only difficultly, or are there other difficulties in developing vaccines for these sorts of diseases? i realise it may be impossible to generalise. most of the time we do have the technologies to make these vaccines. i remember for example when sars came in / we made a vaccine pretty quickly and brought it to the stage where it was ready to go to phase , but when we got to phase the disease had gone away, there was no interest, no incentive to bring it even to phase , so we dropped it. for ebola i think that the technology to make a vaccine has been there for many years, but nobody wanted to invest in it because there was no market, no demand. do you think this is going to have to change in the light of what has happened with ebola? i hope that this humanitarian and health disaster is going to help us to become a little bit smarter in planning the future. i see these emerging infectious diseases as a constant threat. they pose a risk, just as in other areas we have a risk of earthquakes, we have a risk when we drive a car of car accidents, when we buy a house there is a risk of burning the house, and so on -and for all of those risks we prepare ahead of time. we have insurance for cars or we have insurance for houses and we build buildings that are earthquake-resistant. it looks as though the only place where we are not able to make investments ahead of time is health -we always have to wait for the disaster to happen and then we rush and usually with the vaccine we come too late and when the disease is no longer there then everyone forgets and we go from one disaster to another. how forward-thinking do we need to be? -how long does it actually take to develop a vaccine, generally? for a normal commercial vaccine the time is between and years. under very accelerated circumstances, like those we have now for ebola, you can take advantage of previous studies that have been done and try to rush the development of the vaccine, but we do take risks with safety and efficacy when we accelerate so much, so i think we will be wise to develop those vaccines ahead of time. it depends on the virus, so for ebola it is not a problem at all, for mers it does not seem to be a problem, for sars it does not seem to be a problem, for chikungunya it does not seem to be a problem. for influenza it is a problem and for hiv it is a big problem, so it depends on the virus. what about the possibility, aired in the press, that ebola might become transmissible in an airborne form? do you have any view on that possibility? i believe that it is extremely unlikely. this virus has been there for a long time in the wild in africa and it has never done it. it has had many opportunities to do it. i don't think that a small exposure to mankind right now is going to do it and the preliminary data from the genetics and genomics of the virus show very good stability so i don't think this is a risk in the short term. so a little bit of good news. but you say one of these diseases pops out about every six months. do you expect that we'll be continuing to see emerging infections we haven't seen before? oh yes, i believe that in six months we will have a new emerging disease. hopefully the ebola outbreak will be under control. it will not be in the news and there will be other news about another emerging infection and we will rush into the new one, and instead of doing that i think it would be nice to be able to plan ahead of time. i think we have technologies today by which we could prepare multiple vaccine platforms that could be tested for safety and efficacy against multiple diseases and where you could plug in an antigen from a new emerging infection quickly. we already effectively have this for influenza virus, for which as the virus mutates each year we just plug in the most prevalent haemagglutinin variants to the existing vaccine platform. if the regulatory authorities could approve platforms suitable for use against other pathogensthus already of proven safety and efficacywe could have a way of being as ready as possible for unexpected events. pandemic preparedness and response-lessons from the h n influenza of a decade after sars: strategies for controlling emerging coronaviruses chikungunya virus control: is a vaccine on the horizon? lancet ebola virus disease in west africa -clinical manifestations and management cite this article as: rappuoli r: vaccines, emerging viruses, and how to avoid disaster the author is a full-time employee of novartis vaccines. key: cord- - t bzc f authors: barclay, t.; petrovsky, n. title: vaccine adjuvant nanotechnologies date: - - journal: micro and nanotechnology in vaccine development doi: . /b - - - - . - sha: doc_id: cord_uid: t bzc f the increasing sophistication of vaccine adjuvant design has been driven by improved understanding of the importance of nanoscale features of adjuvants to their immunological function. newly available advanced nanomanufacturing techniques now allow very precise control of adjuvant particle size, shape, texture, and surface chemistry. novel adjuvant concepts include self-assembling particles and targeted immune delivery. these individual concepts can be combined to create a single integrated vaccine nanoparticle-combining antigen, adjuvants, and dc-targeting elements. in the process, the concept of an adjuvant has broadened to include not only immune-stimulatory substances but also any design features that enhance the immune response against the relevant vaccine antigen. the modern definition of an adjuvant includes not only classical immune stimulators but also any aspects of particle size, shape, and surface chemistry that enhance vaccine immunogenicity. it even includes purely physical processes such as texturing of particle surfaces to maximize immunogenicity. looking forward, adjuvants will increasingly be seen not as separate add-on items but as wholly integrated elements of a complete vaccine delivery package. hence, vaccine systems will increasingly approach the complexity and sophistication of pathogens themselves, incorporating highly specific particle properties, contents, and behaviors, all designed to maximize immune system recognition and drive the immune response in the specific direction that affords maximal protection. reduced immunogenicity and thereby efficacy is an unfortunate downside to vaccines based on highly purified or synthetic antigens. this requires the use of vaccine adjuvants to improve vaccine immunogenicity. the term adjuvant has traditionally referred to as any substance that when added to a vaccine antigen increases its immunogenicity. as such, the study of adjuvant action is made complicated by the wide diversity of agents including small molecule immune modulators, mineral salts, oil emulsions, and even whole viruses or bacteria. these agents all exhibit adjuvant action in vivo with no one common feature explaining this activity. almost certainly, these compounds work through many and varied different mechanisms to enhance immune responses to coadministered antigens. increasing use of small protein or peptide antigens allows greater control over vaccine design and synthesis but has further exacerbated issues of poor immunogenicity. this necessitates closer examination of how best to design vaccine delivery and adjuvant systems to maximize antigen immunogenicity, assist targeted immune delivery, and provide potent adjuvant activity. most importantly, this must all be done in a manner that does not compromise vaccine tolerability or safety. , the immune system has evolved to protect against invading pathogens. as pathogens present as nano-to microsized particles, it should not be surprising that the immune system has evolved strategies to specifically recognize and respond to particles of particular sizes and shapes. , this helps explain the boost to immunogenicity when antigens are presented to the immune system as virus-like particles (vlps) rather than as soluble proteins. particle size was shown to be important for uptake by dendritic cells (dcs), with spherical particles less than nm in diameter having better uptake than larger particles. size is also relevant to t cell maturation whereby poly(lactide-co-glycolide) (plga) microparticles modified with recognition and costimulatory ligands with similar dimensions to dcs were better able to induce t cell activation when compared with nanosized particles that were rapidly phagocytosed. while the idea that size is important to vaccine action is now well accepted, it is also likely that shape, texture, and surface chemistry are equally highly relevant to adjuvant action, albeit less well-explored. shape is known to be important to particle uptake in vivo, half-life, and biodistribution. , for example, cylindrical silicon particles exhibited strong liver accumulation, whereas discoidal particles accumulated away from the liver and spherical particles were distributed evenly across tissues. decreasing the diameter of spherical particles from to . µm resulted in increased accumulation in reticuloendothelial tissues. these tissues are rich in various immune cells, and consequently accumulation in the reticuloendothelial system is an advantage from a vaccine standpoint. notably, high-axial-ratio nanoparticles were shown to better target draining lymph nodes and thereby enhanced antigen presentation and memory b cell responses. , surface chemistry has also been shown to be important for adjuvant particle properties, with uptake of larger particles by dcs being enhanced by a positive surface charge. furthermore, while nm diameter polyhydroxylated and polymethoxylated poly(propylene sulfide) nanoparticles and polystyrene nanoparticles were all equally efficient in inducing dc uptake, only the polyhydroxylated particles induced high levels of dc maturation, an effect attributed to the polyhydroxylated surface activating complement thereby acting as an immune danger signal for dc activation. use of hydrophilic polymeric coatings such as polyethylene glycol and polyzwitterions with an overall neutral surface charge that bind water to the surface has also been shown to change particle behavior, with the hydrophobic regions of amphiphilic systems considered to act as immune danger signals that provide adjuvant effects. mechanical properties are yet another factor that need to be taken into account when assessing the immune effect of particles. macrophages and dc have been shown to preferentially phagocytose rigid particles even if these have identical surface chemistry to softer particles. hence making particles more flexible so that they mimic red blood cell properties was shown to increase their half-life in vivo, , , whereas making them more rigid should improve their uptake by dcs and thereby their immune action. increasingly sophisticated particle manufacturing approaches have enabled ever-tighter control of the size, shape, external chemistry, and mechanical properties of adjuvant particles. this has provided unique opportunities to modify vaccine and adjuvant particles with the aim to maximize their biological action. the remainder of this chapter will describe a wide variety of nano-and microparticulate vaccine and adjuvant systems, their method of manufacture, and how size, shape, and surface chemistry are all important to adjuvant action ( fig. . ). emulsions are composed of a dispersion of droplets of one liquid in another immiscible liquid, commonly described as either water-in-oil or oil-in-water emulsions. in use as adjuvants, the droplets in an emulsion act as particles and are perceived by the immune system as such. there are two different forms of nanoscale emulsions, both of which are defined as having droplet radii from to nm, which is sufficiently small to generate transparent, isotropic liquid media. the first form of nanoscale emulsion has historically been termed a microemulsion but is better described as a nanodimensional lyotropic liquid crystalline phase. in a microemulsion the structural organization of the two liquids is supported by a relatively large concentration of surfactant. formation is by spontaneous selfassembly with relatively gentle mixing, resulting in stable organization at thermodynamic equilibrium. , by contrast, what is termed a nanoemulsion generally uses significantly less surfactant and hence requires high shear forces to make a metastable structure. , emulsion adjuvants are thought to work through a depot effect, creating inflammation around the site of antigen injection and thereby enhancing humoral immune responses. early examples of emulsion adjuvants were water-in-mineral-oil emulsions. , however, such adjuvants were problematic as mineral oil is not biodegradable, is proinflammatory, and has long-term persistence in the body, thereby leading to major inflammatory side effects including injection site granulomas, pyrexia, and long-term safety issues. care must also be taken with any emulsion adjuvant to avoid any oxidation of the oil component as this could otherwise lead to increases in vaccine reactogenicity. generally, oil-in-water nanoemulsions using biodegradable oils have a better tolerability profile than water-in-oil formulations. a well-known example of an oil-inwater nanoemulsion adjuvant is mf , which is composed of squalene oil in a citrate buffer using nonionic surfactants. , the nanoemulsion is key to the adjuvant effect of mf as when tested individually the various components of mf failed to demonstrate adjuvant activity. mf -based vaccine formulations have been used extensively in research, and recent work has included tests as a cationic nanoemulsion delivery system for delivery of a self-amplifying mrna vaccine. nonetheless, the bulk of research has been for influenza vaccines, and while mf is licensed for this purpose in elderly humans and has extensive safety data in the elderly population, more limited safety data are available for use in children. of potential relevance, a pandemic influenza vaccine containing as , a squalene-based nanoemulsion that is similar to mf except for the addition of tocopherol, was associated with a rise in childhood cases of narcolepsy, , an autoimmune sleep disorder. this has put the question of pediatric safety of squalene-based emulsion adjuvants back under the spotlight. the immunogenicity of emulsion adjuvants can be increased further by coformulation with additional immune-stimulatory components. interestingly, the original mf squalene emulsion adjuvant was initially designed as a delivery system for muramyl tripeptide phosphatidylethanolamine (mtp-pe), a modified mycobacterial peptidoglycan with potent adjuvant activity. mtp-pe activates innate immune receptors including nucleotide-binding oligomerization domain-containing protein (nod ) and various toll-like receptors (tlr), leading to nuclear factor kappa-b (nfκb) activation and induction of inflammatory cytokines. however, the mtp-pe component was ultimately removed from mf because of excessive reactogenicity. a more recent version of a combined adjuvant emulsion and immunostimulator approach is the as adjuvant, which is an oil-in-water emulsion combined with qs saponin and the tlr agonist, monophosphoryl lipid a (mpl). intranasal delivery of vaccines provides advantages in terms of convenience and induction of mucosal immunity, a major benefit when protecting against respiratory or mucosal pathogens. , a nasal vaccine containing a soybean oil nanoemulsion adjuvant successfully enhanced humoral and cellular responses in mice, [ ] [ ] [ ] [ ] [ ] consistent with the utility of this approach. a library of more than intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions containing various cationic and nonionic surfactants were tested in mice and demonstrated that varying the physicochemical properties of the surfactant components (charge, surfactant polar head size, and hydrophobicity) and the surfactant blend ratio of the intranasal adjuvant formulations could modulate the strength and type of the immune response. this indicates that there may be considerable scope for further optimizing nanoemulsion adjuvants for intranasal use. however, the tolerability and safety in humans of this approach have yet to be confirmed. it was recently shown that nanoemulsion adjuvants induce epithelial cell death via activation of caspases , , and - . furthermore, the safety of nasal vaccine adjuvants has remained under a cloud since the experience of nasalflu, an intranasal influenza vaccine containing a detoxified enterotoxin adjuvant, that was associated with increased cases of facial nerve palsy in clinical trials. multiple or double emulsions, most often water-in-oil-in-water formulations, have also been used in vaccine research. however, water-in-oil-in-water multiple emulsion proved to be less stable and induced an excessive inflammatory response when compared with an oil-in-water microemulsion of isopropyl myristate, which successfully enhanced humoral responses to rabies vaccine in mice. these stability issues arise as there are two interfaces with high free energy to stabilize, generally requiring two surfactants that may interact, thereby interfering with stabilization. also, for multiple emulsions there is the potential for an osmotic pressure to develop, placing further strain on the system. even simple emulsions are not without issues. nanoemulsions not at thermodynamic equilibrium have poor stability, and microemulsions suffer from the need for high concentrations of surfactants, which may cause toxicity in vivo. furthermore, the stability of any microemulsion can be easily compromised by dilution, heating, or ph changes. various nanoparticles constructed from disordered, precipitated polymers, also described as nanobeads, have been used for vaccine delivery. poly-ε-caprolactone nanoparticles of two sizes were made, with mean diameters of and nm, by solvent evaporation. only the smaller diameter particles were found to have an adjuvant effect, eliciting both cellular and humoral responses in mice. polystyrene nanobeads covalently conjugated to ovalbumin were found to best activate cd t cells when they were - nm diameter when beads ranging in size from to nm diameter were compared. , another study found that larger beads with diameters from to nm were best at activating cd t cells thus, it may be possible to specifically tune nanoparticle size to induce either cellular or humoral immunity. in another system poly(γ-glutamic acid) grafted with l-phenylalanine ethyl ester on % of the carboxylic acid groups self-assembled upon the addition of water from organic solution into nanoparticles with diameters of - nm. in the presence of ovalbumin the nanoparticles encapsulated the antigen and targeted it to dcs, resulting in enhanced dc maturation and immunity. , inorganic nanoparticles have been used as vaccine delivery vehicles; aluminum oxide nanoparticles having distinctly better performance compared with other metal oxide nanoparticles. such aluminum oxide nanoparticles of - nm diameter elicited adjuvant effect when peptomers derived from an hiv antigenic sequence and retaining the native α-helical conformation were covalently attached to the external surface. , aluminum oxide nanoparticles were also functionalized with ovalbumin and shown to enhance dc activation of t cells via an autophagy-dependent mechanism. particle size was also shown to be important as while both and nm nanoparticles were effective in vitro, the nm aluminum oxide particles were much more effective in being transported to the draining lymph nodes in vivo. vlps can be made using recombinant protein techniques whereby antigenic proteins such as hbsag are synthesized in yeast, following which the antigen self-assembles into nm vlps. vlps are safe and immunogenic, making them ideal vaccine antigens. , however, cell-culture systems used for manufacture of vlps can be low yielding and expensive, and hence synthetic methods have been developed for vlp production. for example, short linear synthetic proteins with coiled-coil domains were used for self-assembly of α-helical motif-containing nanoparticles able to present repetitive epitopes on their surface. , these synthetic particles had a nm diameter and elicited strong immune responses to surfaceexpressed epitopes. [ ] [ ] [ ] a related nanoparticulate vaccine delivery system was also assembled from peptide chains containing a coiled-coil sequence to drive helical assembly. in this instance the peptide was modified at the n-terminus with a dual chain lipid and with synthetic epitopes at the c-terminus. upon dispersion in aqueous solution, the construct aggregated into ordered synthetic particles having a diameter of - nm and presenting multiple copies of the antigen on the surface that induced a humoral immune response. in another synthetic system polypeptides designed to self-assemble into β-sheet-rich nanofibers with epitopes attached to the c-terminus generated strong antibody responses without inflammation. similarly to vlp's, the self-assembled structure was critical to the adjuvant affect, the unassembled sequence having no adjuvant properties. it has been shown that the nanofibers are internalized by dcs and macrophages at the injection site, resulting in increased expression of the activation markers cd and cd and enhanced production of t follicular helper cells and germinal center b cells. nanostructuring of materials within tissue depots may also generate adjuvant activity. peptides having alternating hydrophobic and hydrophilic amino acids can undergo a sol-gel transition upon exposure to physiological conditions whereby they form a hydrogel made up of interconnected nanofibers. in this way a mixture of biphasic oligopeptide, antigen, and adjuvants can be injected with the peptides, subsequently undergoing postinjection self-assembly into hydrated nanofiber gel matrices, forming an antigen and adjuvant depot in the aqueous phase. a self-assembling nanofibrous hydrogel induced an antibody response when tested as a vaccine delivery platform, either alone or formulated with cpg adjuvant (tlr agonist) as a delivery system for recombinant hepatitis b surface antigen (hbsag). similarly, the self-assembling peptide q conjugated to a cd t cell epitope of ovalbumin elicited a strong antigen-specific cd t-cell response. liposome-based vaccines were an early form of particulate adjuvant formulation based on the use of cell-like spherical lipid bilayer vesicles containing antigens within the internal aqueous compartment, within the lipid bilayer, or attached externally. , for example, nm-diameter liposomes assembled from cationic lipid, cationic polymer, and plasmid dna were shown to target antigen to draining lymph nodes, resulting in enhanced dc activation and immunity. coating of these liposomes with a cholesterol-modified mannan further enhanced the therapeutic anticancer action of an oncoprotein vaccine. as liposomes themselves are generally poorly immunogenic, they may require combination with other immunostimulatory adjuvants to enhance their immunogenicity. for example, liposomal nanoparticles self-assembled from a mixture of phospholipids containing % pegylated lipid to prevent protein binding were used to target cyclic diguanylate as an agonist of stimulation of inf genes to draining lymph nodes, thereby enhancing vaccine potency. single bilayer liposomes have also been constructed using viral lipid envelopes, retaining the viral cell binding and fusion glycoproteins and resulting in nm-diameter virosomes that promote attachment to respiratory mucosal surface and subsequent fusion release of contents into the cytoplasm of endocytosing cells. further, the viral components enhance the efficiency of the interaction with antigen-presenting cells (apcs), making them an ideal vaccine delivery system. , more recent virosome research has included the incorporation of additional components to improve the particle formation and stability characteristics as well as enhance immunogenicity. for example, an amphiphilic saponin derivative (gpi- ) was incorporated into a virosome and provided potent immunogenicity to allow the use of very low antigen doses. electrostatic interactions are an important part of nanoparticle vaccine formulations, both between components of the formulation , and between the formulation and the anionic cell membranes of apcs. these charged interactions have demonstrated importance in various nanovaccines, including liposomal systems, but perhaps are best exploited in polymeric systems. a mixture of an anionic poly(γ-glutamic acid) acid grafted with phenylalanine ethylester and a cationic ε-polylysine was self-assembled from aqueous solution into nanoparticles with - nm diameter. when formed in the presence of ovalbumin, the particles were loaded with antigen and induced effective cellular and humoral immune responses in immunized mice. poly(γ-glutamic acid) has also been eletrostatically attached to complexes of dendrigraft poly-l-lysine with plasmid dna. in this instance the poly(γ-glutamic acid) moderates the cytotoxicity of the electrostatic complex to enable delivery and expression of genes in the spleen, suggesting application in vaccine formulations. this is related to a similar gene delivery formulation in which electrostatic interactions between positively charged polyethyleneimine and negatively charged plasmid dna and poly(γ-glutamic acid) were used to construct a nanoparticle dna vaccine. this formulation conferred protection against malaria infection in mouse models. standard plga particles have negative charge, and electrostatic interactions have been identified as a critical aspect of the binding to protein antigens. consequently, protein binding can be tuned through adjusting the ph of binding conditions to optimize the charge differential between components. plga nanoparticles have also been constructed with positive charge using print technology through the incorporation of cationic additives. these particles were specifically designed to electrostatically interact with commercial hemagglutinin antigens to generate an influenza vaccine with enhanced immune responses compared with the hemagglutinin alone. , lipid-like amphiphiles have been used in several self-assembling nanoparticulate-based self-adjuvanting antigen delivery systems because their structure mimics bacterial lipoproteins and as such can activate the immune system. this type of vaccine formulation includes a hydrophobic core covalently attached to a hydrophilic peptide antigen such that the antigen epitope is presented on the surface of the nanoparticle. a nontoxic, dendritic poly tert-butyl acrylate polymer core has been used and when conjugated to relevant peptide epitopes induced cellular responses able to kill human papillomavirus-infected cancer cells. epitope packing on the particle surface was dense enough to maintain a native conformation and enable proper recognition by the immune system. , in other examples the particle core was made up of either lipid-like peptides or lipidated peptides. , such formulations include a peptide epitope from a hookworm protein flanked by coil-producing sequences attached to a lipid-like peptide core. the native alpha helix structure of the epitope presented on the surface of the nanoparticle was able to generate antibodies in mice. as previously suggested by their use in emulsion and liposome adjuvants, saponins can provide potent immunogenicity to vaccine formulations. a commonly used adjuvant is the saponin qs , which is an acylated saponin at the -hydroxyl position on fucose with two linked , dihydroxy- -methyloctanoic acids. qs induces inflammatory cytokines and imparts a th bias in vaccine responses, , but because of its ability to lyse cell membranes, hemolysis and injection site pain are major limiting factors in its use. qs 's toxicity can be reduced by forming it into an immune-stimulating complex (iscom), which is a spherical particle of ∼ nm diameter that self-assembles from specific mixtures of cholesterol, phospholipids, and qs . [ ] [ ] [ ] an advantage of iscoms is that the toxic hemolytic effect of the saponin is moderated by its incorporation into the liposomal structure while its immune-stimulatory properties are retained. the liposomal particle also helps target the saponin adjuvant and antigen formulation to the draining lymph nodes. while initially thought important for immunogenicity that antigens were loaded inside the iscom structure, it is now recognized that adjuvant action can be achieved by simple admixture of the antigen with the iscoms, a major advantage as many antigens are difficult to load into the particles. many plant-based polysaccharides have been shown to have adjuvant activity, with the additional benefit that as sugar-based compounds polysaccharides are generally extremely safe and well tolerated. while some polysaccharides (eg, mannan) may have adjuvant activity when in soluble form, most are only adjuvant active when present as insoluble particles. this is clearly seen with the polysaccharide inulin (β-d- poly(fructo-furanosyl)-d-glucose), a natural plant-derived storage carbohydrate of plants of the compositae family that has no immunological activity when in the usual soluble form but has potent adjuvant activity when crystallized into the nanostructured delta inulin microparticles. [ ] [ ] [ ] [ ] delta inulin particles have been shown to enhance humoral and cellular immune responses to a wide variety of viral, bacterial, and protozoan antigens as well as toxins and allergens. for example, enhanced vaccine protection was seen in models of influenza, , japanese encephalitis, , west nile virus, hepatitis b, human immunodeficiency virus, anthrax, sars coronavirus, listeriosis, , and african horse sickness. its adjuvant effects are seen across a broad range of animal species including mice, rats, guinea pigs, rabbits, chickens, dogs, sheep, monkeys, horses, and camels. it has proved effective in human studies when combined with a recombinant pandemic influenza vaccine, a hepatitis b vaccine, or a bee sting allergy vaccine. an interesting feature of delta inulin is its ability to enhance adaptive immune responses even when injected a day prior to the antigen, a feature not shared by alum adjuvant. delta inulin does not work like other polysaccharide adjuvants, as it has not been found to activate innate immune receptors such as tlrs, dectin- , or the inflammasome. instead, delta inulin adjuvant appears to work by directly modulating dc function, thereby resulting in enhanced antigen presentation to memory t and b cells. this was recently shown in human subjects to translate into enhanced b cell receptor affinity maturation with upregulation of activation-induced cytidine deamidase in day postimmunized plasmablasts. notably, delta inulin has proved safe and effective when administered to pregnant dams or their -day-old mouse pups and, in a large animal model, to either pregnant mares or their foals, a feature that may be attributable to its noninflammatory mode of action. interestingly, a powder particle formulation of delta inulin has recently been shown to be safe and effective when administered with influenza vaccine directly into the lungs of mice, opening up a further novel vaccine application of this technology. other polysaccharide particles with adjuvant activity include dextran, β-glucan, lentinan, zymosan, mannan, and chitosan. these all work through the action of specific innate immune receptors expressed on apcs known as lectins that specifically recognize and respond to sugars. these include the β-glucan receptor, the mannan receptor, dectin , and tlr. activation of these innate immune receptors results in nfκb activation and production of proinflammatory cytokines that enhance adaptive immune responses. hence delta inulin currently appears to be the only polysaccharide particulate adjuvant that works through an alternative noninflammatory pathway. given the high expression of lectins on apcs, decorating the surface of vaccine or adjuvant particles with sugar groups can assist vaccine particle targeting to apcs. for example, mannose has been used to target plasmid dna-containing liposomes to macrophages. coating of cationic liposomes with mannan significantly enhanced the ability of a dna vaccine to induce hiv-specific cellular immunity and also enhanced the activity of a dna vaccine against melanoma. , mannosylated niosomes composed of span , cholesterol, and stearylamine (all coated with the modified polysaccharide o-palmitoyl mannan) have been used as orally administered dna vaccine carriers with a demonstration of enhanced mucosal immunity in mice. a similar approach using o-palmitoyl mannan coating was used to target niosomes to langerhan's cells in the skin after topical delivery. chitin, a linear β- - -linked polymer of d-glucosamine and n-acetyl-d-glucosamine extracted from shrimp, and chitosan, obtained by partial deacetylation of chitin, act by binding innate receptors including dectin- , macrophage mannose receptor, and tlr- . chitosan particles produced by cross-linking with a counter ion were used to entrap antigen and enhance its immunogenicity in mice. by virtue of their mucoadhesive qualities, particles coated with chitin and its derivatives have been extensively used as nasal adjuvants for delivery of inactivated or even live viral vectors. hence mucosal delivery of adenoviral vectors microencapsulated in a chitosan microparticle not only helped to protect and improve the viability of the viral vector but also made its release dependent on cell surface contact. given the ability of specific nanoparticles to target apcs, as discussed previously, such particles can also be loaded with immune stimulators to further increase their adjuvant potency. mdp (n-acetyl muramyl-l-alanine-d-isoglutamine), a mycobacterial peptidoglycan with potent adjuvant activity, binds and activates nod and tlr receptors, leading to nfκb activation, inflammatory cytokine production, and dc maturation. a modified form, mtp-pe, was the key immune stimulator for which the original mf emulsion adjuvant was designed as a delivery system, given the highly hydrophobic nature of mtp-pe. ironically, the combined mtp-pe in mf formulation was abandoned because of excess reactogenicity, but the mf vehicle was found to have some adjuvant activity in its own right, and hence its use in influenza vaccines was continued. in a similar fashion, liposomes formulated with dtp-gdp (n-acetylglucosaminyl-n-acetylmuramyl-l-ala-d-isoglu-l-ala-glyceroldipalmitate) had potent adjuvant activity and induced remission in human metastatic colorectal cancer, although reactogenicity with fever, chills, and hypotension was a problem at higher doses. , the combination of chitosan microparticles with the mucosal toxin-based adjuvant ltk significantly enhanced the immunogenicity of an intranasal group c meningococcal polysaccharide vaccine in mice. similarly, intranasal administration of alginate-coated chitosan nanoparticles loaded with antigen and cpg adjuvant enhanced antibody and cellular responses in mice. cpg has also been incorporated as the hydrophilic head group into a lipid-like adjuvant macromolecule, the hydrophobic tail composed of cholesterol, simple monoacyl, or diacyl lipidic groups. this formulation was used to successfully target cpg adjuvant and the antigen to lymph nodes, thereby helping decrease systemic cpg toxicity while enhancing vaccine immunogenicity. emulsions are not only useful as adjuvants, as discussed earlier, but they can also be used as templates to produce controlled-size solid adjuvant particles (fig. . ). using this method, the polysaccharide inulin was cosolubilized with an ovalbumin antigen into a water-in-oil emulsion with the inulin then precipitated by the slow addition of acetone. this resulted in formation of spherical . µm-diameter inulin particles encapsulating the ovalbumin with high efficiency. these antigen-loaded inulin microparticles improved apc uptake of ovalbumin and enhanced antiovalbumin antibody responses in mice. emulsions have similarly been used as templates to precipitate many other biodegradable polymeric nanoparticles for use in vaccine delivery, including the formation of plga nanoparticles from a nanoemulsion. the plga nanoparticles acted as an antigen delivery system although, unlike inulin particles, the plga particles themselves appeared to have no intrinsic adjuvant properties with an immunomodulator being required to create a strong immune response. , by contrast, poly(γ-glutamic acid)-based nanoparticles generated an immune response without the requirement of other adjuvants. emulsion polymerization is another method whereby polymeric particles of size defined by the emulsion can be generated. in this method the soluble polymer droplets within the emulsion are chemically cross-linked such that the droplet-sized particles remain solid when the emulsion is removed. poly(ortho ester) microparticles of µm diameter produced in this way were used to deliver plasmid dna antigen to apcs. the acidic conditions in the apc lysosome degraded the orthoester bonds holding the particles together, thereby releasing the dna. the dna was then transcribed into protein that was presented to t and b cells, resulting in a cellular and humoral memory response. in another example, size-tuneable cross-linked poly(propylene sulfide) nanoparticles with sizes ranging from to nm were prepared from emulsions, with monomers in the water phase stabilized by pluronic f- . particle size was then controlled by varying the concentrations of the monomer and surfactants in the emulsion. these particles presented pluronic hydroxyl groups at the surface, resulting in complement activation, dc maturation, and a strong adjuvant effect. such particles are unstable and release any attached drug under oxidative conditions, such as those found in the lysosome. , the ability to produce particles of different sizes enabled the effect of particle size on antigen delivery to be directly studied, revealing that small - nm nanoparticles delivered attached antigen to draining lymph nodes with -fold higher efficiency than nm particles. , other studies using these particles have shown that particle surface chemistry is important to the action of nanoadjuvants with, for example, polyhydroxylated surfaces performing much better than polymethoxylated surfaces despite both particles being targeted to the dcs. controlled production of defined-size adjuvant nanoparticles can also be achieved through particle replication in nonwetting template (print) technology. , print techniques use photolithography to create a template that is then transferred to a perfluoropolyether elastomer mold. the mold is filled with the desired material under pressure and then allowed to set. release of the material from the mold produces particles in the nano-or microscale that can have complex and highly conserved morphology. this technology has been used for a range of medical applications, including production of stimuli-responsive targeted drug delivery vehicles , and vaccine adjuvants. the size, shape, and chemistry of print nanoparticles can be optimized to maximize their transport to lymph nodes, enabling their use as an antigen delivery vehicle. this work found that anionic x nm cylinders covalently bound to ovalbumin through a -dalton polyethylene glycol spacer optimized delivery of antigen to lymph nodes and enhanced vaccine responses. this chapter highlighted the increasing sophistication of vaccine adjuvant design driven by the convergence of improved immunological understanding of the importance of nanofeatures to adjuvant function together with advanced nanomanufacturing techniques that allow very precise control of particle size, shape, texture, and surface chemistry. principles from antigen design (eg, particle self-assembly) are increasingly being incorporated into adjuvant nanoparticle design. these individual concepts are now being applied to the design of a single integrated vaccine particle, incorporating antigen, adjuvant, and apc-targeting strategies. in the process, the concept of what is an adjuvant needs to be broadened to not only include specific immune-stimulatory substances but also to include any vaccine design features that enhance immune responses against a relevant antigen. thus, the definition of an adjuvant could potentially now include aspects of antigen formulation such as nanoparticle incorporation; aspects of particle size, shape, and surface chemistry that enhance immunogenicity; or even purely physical processes such as texturing of the particle surface to maximize immunogenicity. looking forward, adjuvants will increasingly not be seen as separate add-on items but as wholly integrated elements of a complete vaccine delivery package. vaccine systems are steadily approaching the complexity and sophistication of the pathogens they mimic, with particle properties and behaviors designed to maximize long-term immune protection while maintaining a high safety profile. microparticulate polysaccharide adjuvants nanoparticle manufacturing methods . . emulsions for template-based particle assembly . . print-based adjuvant production vaccine adjuvants: current state and future trends new-age vaccine adjuvants: friend or foe? freeing vaccine adjuvants from dangerous immunological dogma particulate systems as adjuvants and carriers for peptide and protein antigens advances in vaccine adjuvants particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model a comprehensive platform for ex vivo t-cell expansion based on biodegradable polymeric artificial antigen-presenting cells engineering nano-and microparticles to tune immunity print: a novel platform toward shape and size specific nanoparticle theranostics nanofabricated particles for engineered drug therapies: a preliminary biodistribution study of print (tm) nanoparticles size and shape effects in the biodistribution of intravascularly injected particles rapid and persistent delivery of antigen by lymph node targeting print nanoparticle vaccine carrier to promote humoral immunity development of a nanoparticle-based influenza vaccine using the print® technology exploiting lymphatic transport and complement activation in nanoparticle vaccines peptide-based subunit nanovaccines fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform red blood cell-mimicking synthetic biomaterial particles in vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles carbohydrate-based immune adjuvants synthesis of nanomaterials in microemulsions: formation mechanisms and growth control nanoemulsions: formation, structure, and physical properties preparation of metal nanoparticles in water-in-oil (w/o) microemulsions formation and stability of nano-emulsions adjuvants designed for veterinary and hum vaccin sensitization and antibody formation after injection of tubercle bacilli and paraffin oil the mode of action of mineral-oil emulsion adjuvants on antibody production in mice outlining novel cellular adjuvant products for therapeutic vaccines against cancer design and evaluation of a safe and potent adjuvant for hum vaccin clinical and immunologic responses to human immunodeficiency virus (hiv) type sf gpl subunit vaccine combined with mf adjuvant with or without muramyl tripeptide dipalmitoyl phosphatidylethanolamine in non-hiv-infected human volunteers the adjuvant effect of mf is due to the oil-in-water emulsion formulation, none of the individual components induce a comparable adjuvant effect mf ™ as a vaccine adjuvant: a review of safety and immunogenicity a cationic nanoemulsion for the delivery of next-generation rna vaccines mf adjuvant: the best insurance against influenza strain diversity as adjuvanted ah n vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in finland increased incidence and clinical picture of childhood narcolepsy following the h n pandemic vaccination campaign in finland adjuvant activity of - -mycoloyl-nacetylmuramuyl-l-alanyl-d-isoglutamine muramyldipeptide and diaminopimelic acid-containing desmuramylpeptides in combination with chemically synthesized toll-like receptor agonists synergistically induced production of interleukin- in a nod -and nodl-dependent manner, respectively in human monocytic cells in culture enhancement of tlr-mediated innate immune responses by peptidoglycans through nod signaling systemic cytokine profiles in balb/c mice immunized with trivalent influenza vaccine containing mf oil emulsion and other advanced adjuvants enhancement of humoral response against human influenza vaccine with the simple submicron oil/water emulsion adjuvant mf evaluation of the immune response to rts,s/as and rts,s/as adjuvanted vaccines: randomized, double-blind study in malaria-naive adults development of immune response that protects mice from viral pneumonitis after a single intranasal immunization with influenza a virus and nanoemulsion efficacy, immunogenicity and stability of a novel intranasal nanoemulsion-adjuvanted influenza vaccine in a murine model nanoemulsion nasal adjuvant w ec induces dendritic cell engulfment of antigen-primed epithelial cells formulation and characterization of nanoemulsion intranasal adjuvants: effects of surfactant composition on mucoadhesion and immunogenicity safely and immunogenicity of a novel nanoemulsion mucosal adjuvant w ec combined with approved seasonal influenza antigens formulation, high throughput in vitro screening and in vivo functional characterization of nanoemulsion-based intranasal vaccine adjuvants nanoemulsionbased mucosal adjuvant induces apoptosis in human epithelial cells use of a sustained-release multiple emulsion to extend the period of radio protection conferred by cysteamine evaluation of water-in-oil-in-water multiple emulsion and microemulsion as potential adjuvants for immunization with rabies antigen multiple emulsions: technology and applications fabrication of nanoadjuvant with poly-ε -caprolactone (pcl) for developing a single-shot vaccine providing prolonged immunity size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors type and immunity following vaccination is influenced by nanoparticle size: formulation of a model vaccine for respiratory syncytial virus preparation and characterization of biodegradable nanoparticles based on poly(gamma-glutamic acid) with -phenylalanine as a protein carrier targeting of antigen to dendritic cells with poly( -glutamic acid) nanoparticles induces antigen-specific humoral and cellular immunity alpha-alumina nanoparticles induce efficient autophagy-dependent crosspresentation and potent antitumour response peptomer aluminum oxide nanoparticle conjugates as systemic and mucosal vaccine candidates: synthesis and characterization of a conjugate derived from the c domain of hiv- mngpl immunization of mice with peptomers covalently coupled to aluminum oxide nanoparticles synthesis and assembly of hepatitis b virus surface antigen particles in yeast priming of class i-restricted cytotoxic t lymphocytes by vaccination with recombinant protein antigens immunization of human hiv-seronegative volunteers with recombinant pl / p :ty virus-like particles elicits hiv- p -specific cellular and humoral immune responses nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine protective antibody and cd + t-cell responses to the plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine a nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria synthetic virus-like particles from self-assembling coiled-coil lipopeptides and their use in antigen display to the immune system the use of self-adjuvanting nanofiber vaccines to elicit high-affinity b cell responses to peptide antigens without inflammation a self-assembling peptide acting as an immune adjuvant controlled release of insulin from self-assembling nanofiber hydrogel, puramatrix (tm): application for the subcutaneous injection in rats vaccine self-assembling immune matrix is a new delivery platform that enhances immune responses to recombinant hbsag in mice antigenic peptide nanofibers elicit adjuvant-free cd (+) t cell responses liposomes as immunological adjuvants immunogenicity and protectivity of a new liposomal hepatitis a vaccine dileep padinjarae vangasseri a, leaf h. immunostimulation mechanism of lpd nanoparticle as a vaccine carrier coating of mannan on lpd particles containing hpv e peptide significantly enhances immunity against hpv-positive tumor nanoparticulate sting agonists are potent lymph node-targeted vaccine adjuvants adjuvant activity of immunopotentiating reconstituted influenza virosomes (irivs) virosome-mediated delivery of protein antigens to dendritic cells pharmaceutical aspects of intranasal delivery of vaccines using particulate systems influenza virosomes as vaccine adjuvant and carrier system influenza virosomes supplemented with gpi- adjuvant: a potent vaccine formulation for antigen dose sparing stabilization of polyion complex nanoparticles composed of poly(amino acid) using hydrophobic interactions immunogenicity of novel nanoparticle-coated msp- c-terminus malaria dna vaccine using different routes of administration nanoparticle vaccines liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a cmi response induction of potent adaptive immunity by the novel polyion complex nanoparticles biodegradable nanoparticles composed of dendrigraft poly-l-lysine for gene delivery an investigation of the factors controlling the adsorption of protein antigens to anionic plg microparticles handbook of biologically active peptides polyacrylate dendrimer nanoparticles: a self-adjuvanting vaccine delivery system selfadjuvanting polymer-peptide conjugates as therapeutic vaccine candidates against cervical cancer lipid core peptide system for gene, drug, and vaccine delivery structure-activity relationship of a series of synthetic lipopeptide self-adjuvanting group a streptococcal vaccine candidates a novel synthetic adjuvant enhances dendritic cell function peptidebased subunit vaccine against hookworm infection structure/function studies on qs- , a unique immunological adjuvant from quillaja saponaria structural and immunological characterization of the vaccine adjuvant qs- a strong cd + t cell response is elicited using the synthetic polypeptide from the c-terminus of the circumsporozoite protein of plasmodium berghei together with the adjuvant qs- : quantitative and phenotypic comparison with the vaccine model of irradiated sporozoites advances in saponin-based adjuvants the iscom: an immunostimulating complex iscoms and other saponin based adjuvants functional aspects of iscoms iscom, a delivery system for parenteral and mucosal vaccination inulin isoforms differ by repeated additions of one crystal unit cell the polysaccharide inulin is characterized by an extensive series of periodic isoforms with varying biological actions delta inulin: a novel, immunologically active, stable packing structure comprising beta-d-[ -> ] poly(fructo-furanosyl) alpha-d-glucose polymers inulin crystal initiation via a glucose-fructose cross-link of adjacent polymer chains: atomic force microscopy and static molecular modelling advax, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses delta inulin polysaccharide adjuvant enhances the ability of split-virion h n vaccine to protect against lethal challenge in ferrets an inactivated vero cell-grown japanese encephalitis vaccine formulated with advax, a novel inulin-based adjuvant, induces protective neutralizing antibody against homologous and heterologous flaviviruses je-advax vaccine protection against japanese encephalitis virus mediated by memory b cells in the absence of cd + t cells and pre-exposure neutralizing antibody an inactivated cell culture japanese encephalitis vaccine (je-advax) formulated with delta inulin adjuvant provides robust heterologous protection against west nile encephalitis via cross-protective memory b cells and neutralizing antibody a novel hepatitis b vaccine containing advax, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing induction of mucosal and systemic antibody and t-cell responses following primeboost immunization with novel adjuvanted human immunodeficiency virus- -vaccine formulations advax-adjuvanted recombinant protective antigen provides protection against inhalational anthrax that is further enhanced by addition of murabutide adjuvant severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology novel nanoparticle vaccines for listeriosis a gold glyco-nanoparticle carrying a listeriolysin peptide and formulated with advax delta inulin adjuvant induces robust t-cell protection against listeria infection improving the dromedary antibody response: the hunt for the ideal camel adjuvant randomized clinical trial of immunogenicity and safety of arecombinanthlnl/ pandemic influenza vaccine containing advax polysaccharide adjuvant immunogenicity and safety of advax, a novel polysaccharide adjuvant based on delta inulin, when formulated with hepatitis b surface antigen: a randomized controlled phase study immunotherapy- . a controlled study of delta inulin-adjuvanted honey bee venom immunotherapy delta inulin adjuvant enhances plasmablast generation, expression of activation-induced cytidine deaminase and b-cell affinity maturation in human subjects receiving seasonal influenza vaccine a single immunization with inactivated h n influenza vaccine formulated with delta inulin adjuvant (advax) overcomes pregnancy-associated immune suppression and enhances passive neonatal protection advax delta inulin adjuvant overcomes immune immaturity in neonatal mice thereby allowing single-dose influenza vaccine protection safety and immunogenicity of a delta inulin-adjuvanted inactivated japanese encephalitis virus vaccine in pregnant mares and foals enhanced pulmonary immunization with aerosolized inactivated influenza vaccine containing delta inulin adjuvant mannose receptor-mediated gene transfer into macrophages using novel mannosylated cationic liposomes hiv- -specific cell-mediated immune responses induced by dna vaccination were enhanced by mannancoated liposomes and inhibited by anti-interferon-gamma antibody development of an antigen-presenting cell-targeted dna vaccine against melanoma by mannosylated liposomes mannosylated niosomes as adjuvant-carrier system for oral genetic immunization against hepatitis b mannosylated niosomes as carrier adjuvant system for topical immunization chitosan-based systems for the delivery of vaccine antigens chitosan-based nanoparticles for improving immunization against hepatitis b infection the concomitant use of the ltk mucosal adjuvant and of chitosan-based delivery system enhances the immunogenicity and efficacy of intranasally administered vaccines encapsulation of adenoviral vectors into chitosan-bile salt microparticles for mucosal vaccination phase i trial of immther, a new liposome-incorporated lipophilic disaccharide tripeptide immunologic and toxicologic study of disaccharide tripeptide glycerol dipalmitoyl: a new lipophilic immunomodulator immune response by nasal delivery of hepatitis b surface antigen and codelivery of a cpg odn in alginate coated chitosan nanoparticles structurebased programming of lymph-node targeting in molecular vaccines development of soluble inulin microparticles as a potent and safe vaccine adjuvant and delivery system biodegradable nanoparticle delivery of a th -biased peptide for induction of thl immune responses molecularly engineered poly(ortho ester) microspheres for enhanced delivery of dna vaccines oxidation-sensitive polymeric nanoparticles incorporation controlled release of silyl ether prodrugs from print nanoparticles key: cord- -m ys z authors: kobinger, gary p.; meunier, isabelle; patel, ami; pillet, stéphane; gren, jason; stebner, shane; leung, anders; neufeld, james l.; kobasa, darwyn; von messling, veronika title: assessment of the efficacy of commercially available and candidate vaccines against a pandemic h n virus date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: m ys z background. the emergence and global spread of the pandemic h n influenza virus have raised questions regarding the protective effect of available seasonal vaccines and the efficacy of a newly produced matched vaccine. methods. ferrets were immunized with the – formulations of commercially available live attenuated (flumist; medimmune) or split-inactivated (fluviral; glaxosmithkline) vaccines, a commercial swine vaccine (flusure; pfizer), or a laboratory-produced matched inactivated whole-virus vaccine (a/mexico/indre / ). adaptive immune responses were monitored, and the animals were challenged with a/mexico/indre / after weeks. results. only animals that received the swine or matched vaccines developed detectable hemagglutination- inhibiting antibodies against the challenge virus, whereas a t cell response was exclusively detected in animals vaccinated with flumist. after challenge, all animals had high levels of virus replication in the upper respiratory tract. however, preexisting anti—pandemic h n antibodies resulted in reduced clinical signs and improved survival. surprisingly, flumist was associated with a slight increase in mortality and greater lung damage, which correlated with early up-regulation of interleukin- . conclusions. the present study demonstrates that a single dose of matched inactivated vaccine confers partial protection against a pandemic h n virus, and it suggests that a higher dose or prime-boost regimen may be required. the consequences of mismatched immunity to influenza merit further investigation. , confirmed cases and ∼ deaths worldwide, and the real numbers are likely to be considerably higher, because countries are now only required to confirm severe cases by laboratory diagnosis [ ] . even though most patients experience a disease similar to seasonal influenza, reports of severe cases are increasing [ ] [ ] [ ] . studies in different animal models reveal more efficient spread of the pandemic h n viruses to the lower respiratory tract and demonstrate increased virulence of some field isolates, suggesting that the genetic makeup of the respective strain may significantly contribute toward disease outcome [ , ] . this observation, in combination with reports of more frequent incidents of severe disease in the southern hemisphere [ ] , also increases concerns about the fall, which is typically the period of the most severe influenza activity in the northern hemisphere [ , ] . the rapid spread of the virus in countries with high seasonal influenza vaccine coverage suggests that there is little to no cross-protection conferred by these vaccines [ ] . at the same time, the presence of neutralizing antibodies and the generally milder course of dis-ease observed in individuals years of age are indicative of a protective effect of prior infection with antigenetically related viruses [ ] . as this pandemic unfolds, and especially in light of the emerging resistance to available antivirals [ ] , assessment of the safety and efficacy of available seasonal vaccines as well as matched candidate vaccines is becoming increasingly urgent. the current study evaluates in ferrets-the preferred preclinical animal model for influenza vaccine testing- commercially available influenza vaccines from the - season and fully matched laboratory-produced inactivated whole pandemic h n virus vaccine. immune responses were monitored, and the animals were challenged weeks after vaccination with a pandemic h n influenza isolate that exhibits moderate to high virulence in ferrets. viral loads, morbidity, mortality, and postchallenge immune responses were documented for weeks. groups of five -to -weekold ferrets without antibodies against circulating influenza strains were immunized with one of the seasonal inactivated split vaccines (fluviral; glaxosmithkline) or the cold-adapted live attenuated vaccine (flumist; medimmune), a swine influenza vaccine (flusure; pfizer), or a matched laboratory-produced inactivated vaccine (ph n inact). the latter vaccine consisted of a madin-darby canine kidney (mdck) cell-produced whole-virus preparation of a/mexico/ indre / (mx ; h n ) that was isolated during the ongoing h n influenza outbreak, purified by ultracentrifugation, subsequently inactivated by addition of formalin to a final concentration of . %, and incubated for days at Њc (fisher scientific). the animals received the recommended dose of the respective commercial vaccines or a dose containing mg of hemagglutinin (ha) of the experimental vaccine. with the exception of flumist, which was inoculated intranasally, all vaccines were injected in the gluteal muscle at the recommended dose for humans or pigs, respectively. five weeks later, the animals were challenged intranasally with % tissue culture infectious doses (tcid ) of mx . clinical signs, body temperature, and weight were assessed daily, and animals were euthanized based on clinical evaluation or at the end of the study on day . virus quantification and pathology. nasal washes were collected on days , , , , and after challenge, and virus titers were quantified by limiting dilution. in brief, -fold serial dilutions were incubated on mdck cells with replicates per dilution. at - h after infection, the plates were scored for cytopathic effect, and the tcid virus titers were calculated using the method of reed and muench [ ] . rna was isolated, and viral copy numbers were quantified using real-time reverse-tran-scription polymerase chain reaction (rt-pcr). tissues preserved in rnalater were homogenized using a bead mill homogenizer for extraction of total rna. rna was isolated from nasal washes and swabs, by use of the qiaamp viral rna mini kit (qiagen), and from tissues, by use of the rneasy mini kit (qiagen). the h n virus was detected by quantitative real-time rt-pcr performed using the lightcycler rna master hydrolysis probes (roche) assay targeting the ha gene (nucleotide position - ; genbank accession number gq ). reaction conditions were as follows: at Њc for min; at Њc for s; and cycles at Њc for s and at Њc for s with the use of a lightcycler (roche). the low detection limit for this h n assay is . pfu/ml. the primer sequences are as follows: haf, ggatcaagaagggagaatgaactatt; har, aatgcata-tctcggtaccactagattt; and hap, ccgggagacaaa-ataacattcgaagcaac. after euthanasia, necropsy was performed for all animals, and photographs of their lungs were taken before the lungs were harvested for histopathologic analysis. lungs were inflated by slow injection of ∼ ml of phosphate-buffered saline (pbs; invitrogen) in the trachea, and formalin-fixed and paraffinembedded tissue sections were stained with hematoxylin-eosin. immune response assessment. serum samples were collected on days , , , , and after vaccination and were analyzed for the presence of hemagglutination-inhibiting (hai) antibodies against mx and the seasonal h n strain a/brisbane/ / . hai antibody titers are expressed as the reciprocal of the highest serum dilution that inhibits hemagglutination of turkey red blood cells. on day after vaccination, heparinized blood was collected for proliferation assays. in brief, peripheral blood mononuclear cells were isolated by ficoll-hypaque (ge healthcare) gradient purification and cultivated in the presence of overlapping peptide pools covering the nucleocapsid (np), neuraminidase (na), and ha proteins of the related h n strain a/brevig mission/ . the proliferation response was measured by adding -bromo- -deoxyuridine (brdu) to the peptide-exposed peripheral blood mononuclear cells after h. the next day, cells were fixed, and brdu incorporation was quantified by immunostaining performed using a chemoluminescent substrate (roche). the proliferation index is expressed as the ratio of brdu incorporation measured for the respective influenza peptide pool and for an ebola virus peptide as negative control. messenger rna profiles of cytokines, including interferon (ifn)-a, ifn-g, interleukin (il)- , and il- , were generated from nasal wash rnas isolated on days , , , and after infection and from rna isolated from the right and left lung, respectively, of animals euthanized on day . the assays were performed using the primers and method outlined elsewhere [ ] . hai antibody titer kinetics were monitored for days after immunization, because titers of reciprocal dilutions remain the reference standard that is predictive of protective immunity elicited by influenza vaccine candidates [ ] . hai antibody titers against the pandemic h n mx isolate were detected at day in ferrets receiving the laboratory-produced matched vaccine ph n inact or the swine influenza vaccine flusure, reaching titers on day or , respectively. flusure or ph n inact did not generate detectable hai antibody titers against the seasonal h n strain a/brisbane/ / included in conventional seasonal influenza vaccines, such as fluviral or flumist. in contrast, between day and day , both seasonal vaccines elicited hai antibody titers against a/brisbane/ / that were , whereas no cross-reactive response to mx was detected before challenge ( figure b) . none of the vaccines elicited an ifn-g enzyme-linked immunospot response upon stimulation with overlapping peptides covering the np, na, and ha proteins of h n strain a/brevig mission/ . these proteins share %, %, and % amino acid identity with the respective mx proteins, which may have contributed to the weak response observed. however, increased proliferation activity in response to np peptide pools was detected in animals immunized with flumist, indicating a cross-reactive t cell response ( figure c) . correlation of presence of hai antibodies with milder disease and improved survival. upon intranasal challenge with mx , all vaccinated animals displayed a -day delay in the onset of fever, and they then followed a course comparable to that of nonvaccinated controls (figure a) . clinically, animals immunized with the swine vaccine flusure demonstrated more complete protection with mild and transient signs of disease, less weight loss in the first week than in the other groups, and % survival. the matched ph n inact vaccine also resulted in reduced weight loss, clinical signs of disease, and improvement of the survival rate from % to %, compared with observations in naive controls ( figure b-d) . in contrast, there were no statistically significant differences in recorded clinical signs of disease, weight loss, or survival rate between the animals given fluviral and the control groups, over the course of the experiment after challenge ( ). the group of ferrets vac-p . cinated with flumist showed a slight improvement in average body weight at days and after challenge. however, weight loss increased from day to day , at which point clinical signs of disease, including nasal seromucous exudates, shallow and labored breathing, and reduced activity forced euthanasia for of the animals given flumist, resulting in a small increase in the mortality rate, compared with that noted for the un- vaccinated control group. this increase in the mortality rate associated with mismatched flumist immunization was also observed in a second experiment with animals, although the increase was not statistically significant (data not shown). at the respective times of euthanasia, gross pathological evaluation of lungs demonstrated minimal lesions in all animals vaccinated with flusure, including animal euthanized on day , without reaching experimental end points to obtain, on a timely basis, tissue samples matched to those obtained from the other groups ( figure ). animals vaccinated with ph n inact showed a slight improvement, with smaller lesions noted in of the ferrets. three of control animals had severe lesions with hepatization, hemorrhages, and widespread alveolitis and bronchiolitis, which were comparable to lesions observed in / or / ferrets vaccinated with fluviral or flumist, respectively. all groups reached nasal infectious titers of ∼ tcid at day after challenge, with the exception of the animals immunized with the swine vaccine flusure, which had a -fold lower titer ( figure a ). the group vaccinated with flumist maintained relatively high levels of virus replication through day , whereas the other groups experi-enced titer decreases of у -fold. with the exception of one animal in the control group, no infectious viral particles were detectable by titration in the nasal washes of animals on day or later, although viral rna could be detected by real-time rt-pcr until the end of the experiment ( figure b ). animals immunized with ph n inact or flusure experienced the lowest ifn-a levels and up-regulation of ifn-g during the early stage of the infection. moderately elevated levels of il- were detected later in the course of disease-at day exclusively in these groups, which showed evidence of protection ( figure ). in contrast, the group vaccinated with flumist exhibited the highest average of mrna transcripts for ifn-a, up to day , and for ifn-g, at day , possibly reflecting a better cellular response. of interest, at day , levels of il- transcripts were significantly higher in the upper airway of animals vaccinated with flumist, and at day after infection, they were slightly increased in the lower airway, compared with observations in control animals and animals vaccinated with ph n inact or flusure ( figure ). no differences in tumor necrosis factor-a and il- levels were observed between the groups (data not shown). the availability of an efficient vaccine is essential to alleviate the effect of the ongoing influenza pandemic. a possible intervention strategy to mitigate the fall influenza season in the northern hemisphere was to initially perform mass immunization with the seasonal vaccine, followed by mass immunization with the fully matched pandemic h n vaccine as soon as would become available. to direct a concerted public health response to control the spread of the virus, the efficacy of a newly produced matched inactivated vaccine, as well as that of already available inactivated and live attenuated vaccines, has to be assessed. toward this end, we compared the antibody and cellular responses elicited by seasonal vaccines (fluviral and flumist), the commercial swine vaccine flusure, and a laboratory-produced matched inactivated whole-virus preparation. we found that only the swine and matched vaccines resulted in production of hai antibodies against the pandemic h n virus, whereas only flumist triggered a crossreactive cellular response. intranasal challenge with the virulent mexican isolate mx , similar to mx/ , which also leads to a % mortality rate among naive animals [ ] , revealed that none of the vaccines was able to confer complete protection after only one immunization. however, flusure was associated with the best reduction in morbidity and complete protection from mortality, whereas the matched inactivated vaccine resulted in moderate clinical improvement and reduced mortality. as was expected from undetectable hai antibody titers, animals vaccinated with fluviral or flumist did not experience a beneficial effect, compared with unvaccinated control animals. the partial protection observed in animals vaccinated with one dose of the matched inactivated vaccine, despite the detection of an hai antibody response within the protective range, indicates that protection from aggressive isolates may require more than a single immunization, which would put an additional strain on vaccine availability. the use of a more virulent challenge strain enables assessment of vaccine efficacy in a worst-case scenario. however, the disease severity associated with currently circulating pandemic h n strains in most patients is more similar to that associated with seasonal influenza [ ] . it is thus possible that a single -mg dose of a matched inactivated vaccine will be sufficient to confer protection against most pandemic h n strains, especially in individuals with some levels of cross-protection due to previous influenza infection. the efficiency of a commercially available swine vaccine indicates that this product would be adequate to protect animals, including pig herds, which could minimize interspecies transmission and maybe limit evolution of the virus. the flusure vaccine consists of an inactivated h n and h n type a field isolate formulated with amphigen (pfizer) as an adjuvant [ ] , indicating that the use of this and other adjuvants merits a more in-depth evaluation in the context of the development of improved human influenza vaccines. a curious observation is the more severe cases of disease and the higher mortality rate noted for animals vaccinated with flumist, which correlated with slightly more infectious virus in the nasal washes of this group at day . this correlation figure . relative quantification of cytokine messenger rna (mrna) induction. changes in cytokine mrna levels were determined by semiquantitative real-time reverse-transcription polymerase chain reaction in nasal wash rna or rna isolated from lung tissue harvested on day . ten nanograms of rna were used for each reaction, and the fold change was calculated using the comparative cycle threshold (ddct) method. columns denote the mean of all values obtained for the respective group, and error bars denote the standard error. ctl, nonimmunized control group; dpi, days post infection; ifn, interferon; il, interleukin. between the infectious viral load in the upper respiratory tract and the clinical outcome was in fact observed for all groups in the present study, confirming previous reports that the infectious viral load in the airway is predictive of levels of protection in vaccinated ferrets after challenge with respiratory viruses, including severe acute respiratory syndrome-associated coronavirus and influenza [ , ] . on the other hand, data from this study also indicate that weight loss was, on average, more severe for the unvaccinated control animals than for any other vaccinated group of animals. the present study was designed to evaluate protective efficacy after vaccination and not the possible subtle negative effects caused by immunization with mismatched influenza antigens. larger study group sizes will be necessary to conclusively address this question with an appropriate degree of statistical confidence. antibody-mediated enhancement of influenza infection, including subtype-crossreactive, nonneutralizing antibodies, has been previously described in cultured cells [ ] [ ] [ ] . this mechanism has never been directly associated with a worsened clinical condition in animal models of influenza infection, although a recent study reported that maternally derived antibodies possibly enhanced swine influenza virus-induced pneumonia in pigs [ ] . these observations further support the need for a more detailed evaluation of the efficacy of influenza vaccine in controlled exper-imental conditions where various levels of preexisting immunity to mismatched influenza antigens could be studied. all animals demonstrated a strong induction of the proinflammatory cytokine il- at day ; this level remained elevated at day only in the groups of ferrets showing noticeable protection. animals vaccinated with flumist, the only group that mounted a strong cross-reactive cellular response, had the highest ifn-g response. however, this response was not sufficient to control the disease. in fact, the strong expression of il- , an anti-inflammatory cytokine, detected in that group on day may have suppressed an appropriate inflammatory response, including il- expression, and temporarily favored virus replication, as previously demonstrated in pigs infected with foot-and-mouth disease [ ] . there are reports showing the negative effect of il- on influenza virus-infected mice and pigs [ , ] , and increased il- production correlated with a low antibody response in elderly individuals after influenza vaccination [ ] . evaluating the response of cytokines, including il- and il- , at early time points in patients may help predict unfavorable outcome and allow for better allocation of resources to individuals requiring more intensive clinical intervention. the present study reports the immune responses and protective efficacy of commercially available vaccines and one lab-oratory-produced matched vaccine with regard to prevention of pandemic h n infection in ferrets. the findings of this study may help to guide ongoing preparations for the influenza season in the northern hemisphere. world health organization (who) pneumonia and respiratory failure from swine-origin influenza a (h n ) in mexico swine influenza a (h n ) strikes a potential for global disaster severe respiratory disease concurrent with the circulation of h n influenza emergence and pandemic potential of swine-origin h n influenza virus transmission and pathogenesis of swine-origin a(h n ) influenza viruses in ferrets and mice influenza a(h n )v in the southern hemisphere-lessons to learn for europe? influenza activity in europe during eight seasons ( - ): an evaluation of the indicators used to measure activity and an assessment of the timing, length and course of peak activity (spread) across europe surveillance for influenza-united states, - , - , and - seasons emergence of a novel swine-origin influenza a (h n ) virus in humans oseltamivir-resistant novel influenza a (h n ) virus infection in two immunosuppressed patients a simple method of estimating fifty percent endpoints severe seasonal influenza in ferrets correlates with reduced interferon and increased il- induction a trivalent virus-like particle vaccine elicits protective immune responses against seasonal influenza strains in mice and ferrets adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques investigation of the biological indicator for vaccine efficacy against highly pathogenic avian influenza (hpai) h n virus challenge in mice and ferrets antibody-mediated growth of influenza a nws virus in macrophagelike cell line p d infection enhancement of influenza a nws virus in primary murine macrophages by anti-hemagglutinin monoclonal antibody subtype cross-reactive, infectionenhancing antibody responses to influenza a viruses the immune response and maternal antibody interference to a heterologous h n swine influenza virus infection following vaccination immunosuppression during acute infection with foot-and-mouth disease virus in swine is mediated by il- alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs il- deficiency unleashes an influenza-specific th response and enhances survival against high-dose challenge high interleukin- production is associated with low antibody response to influenza vaccination in the elderly we would like to thank eric poeschla, for providing the flumist doses, and naveed zafar janjua and nicholas svitek, for help with literature review or the cytokine real-time reverse-transcription polymerase chain reactions, respectively. key: cord- -lfn ggg authors: kenner, julie; cameron, fiona; empig, cyril; jobes, david v.; gurwith, marc title: lc m : an attenuated smallpox vaccine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: lfn ggg the frequency of moderate to severe adverse reactions associated with smallpox vaccines currently stockpiled in the us, and the continued threat of bioterrorism have prompted the development of effective vaccines with improved safety profiles. lc m , an attenuated, replicating smallpox vaccine derived from the lister strain of vaccinia, is currently licensed in japan where it was safely used in over , children in the s. it has been shown to have markedly less neurotoxicity than unattenuated vaccines in nonclinical studies. lc m is immunogenic after a single dose, and recent studies in two different animal models have demonstrated protective efficacy equivalent to that of the only fda-licensed smallpox vaccine. this article reviews the history and available scientific literature regarding lc m and provides comparisons to other smallpox vaccines. as efforts intensified internationally to eradicate smallpox in the s, health officials became increasingly concerned about adverse reactions to vaccinia vaccines. officials in countries with a low incidence of smallpox were particularly alarmed by the morbidity and mortality associated with postvaccination encephalitis and encephalopathy. while central nervous system (cns) adverse reactions were generally considered idiosyncratic events, the incidence seemed to vary by strain of vaccinia. this observation was eventually substantiated when the marennikova group of the viral pharmaceutical institute in russia published animal data that characterized over commonly used strains of vaccinia and showed a correlation between the degree of pathogenicity and viral replication in cns and other tissues [ , ] . according to the marennikova report, the ikeda strain of vaccinia was classified as having high pathogenicity, whereas the lister strain used by the world health organization (who) and the uk national health service (nhs) was rated as hav-ing medium pathogenicity, and the new york city board of health strain (nycbh, also manufactured under the trade name dryvax ® ) used in the us was rated as having low pathogenicity. in response to a growing concern about vaccinia-related adverse events (aes) in japan, the japanese ministry of health formed the smallpox vaccine research group (svrg) in [ ] . this organization, comprised of both clinical experts and basic scientists, was charged with providing and collecting detailed information on adverse reactions to vaccinia vaccines and advising the japanese public health authorities. at the time of the marennikova report, the strain of vaccinia used for smallpox vaccination in japan was ikeda, and the rate of encephalitis in japan was approximately per million per year [ ] . the highest risk of mortality was in primary vaccinees, particularly in infants. as one of its first tasks, the svrg investigated the cns toxicity of several strains of vaccinia in mice that were inoculated intracerebrally [ ] . the results from the mouse studies confirmed the greater neurotoxicity of the ikeda strain reported by marennikova and spawned several clinical trials in children comparing ikeda with lister and em- , a strain used in russia. after collecting data from several thousand primary vaccinees, it was found that all of the studied vaccines induced similar take rates, antibody responses, and febrile reactions, but that lister had the mildest local reactogenicity. while no direct conclusions could be made regarding the relative neurotoxicity of the studied vaccines, local reactogenicity was at that time regarded as an indicator of overall vaccinia toxicity, and was used to identify lister as the safest vaccine strain in the trials [ ] . in , the svrg recommended that routine smallpox vaccination in japan be conducted using lister instead of the ikeda strain [ , , ] . to further reduce vaccine-related morbidity and mortality, the svrg recommended that vaccinations be initiated at - months of age rather than at - months, and that only - punctures be administered with a bifurcated needle [ , ] . despite these changes, deaths from vaccine-related encephalitis (table ) continued in japan [ , ] . being at geographic risk of importing smallpox from india, the svrg was reluctant to discontinue vaccinations altogether as other industrialized nations had done in the early s [ , ] . therefore, a decision was made to search for a vaccine that reduced or eliminated cns risks. the search began with a review of study data from two attenuated smallpox vaccines that had undergone considerable development in other nations. the modified vaccinia ankara (mva) strain had been developed in germany in the s by passaging vaccinia ankara in chick embryo explants (cee) in excess of times [ ] [ ] [ ] . the resulting mutant was dramatically modified, having lost nearly % of the original genome [ ] as well as the ability to replicate in many mammalian cells [ ] . although clearly not neurotoxic in nonclinical studies and nonreactive in both normal and dermatologicallyor immune-compromised hosts, antibody response following single vaccination was poor [ , ] . since humoral immune response had become the leading criterion for determining vaccinia protective efficacy, further development of the mva vaccine in japan was abandoned. another attenuated strain, originally called the first revived strain and then later renamed cvi (cv- ) by parker et al. [ ] , had been developed in the us in the early s through repeated passage of the nycbh strain in rabbit testes, cee, and chorioallantoic membranes (cam) [ , , ] . the attenuated cvi- strain (cvi after passages), developed by kempe et al. at the walter reed army institute of research in the late s, was ultimately selected for development based on the mild dermatologic reactions it produced in rabbit and human studies [ ] . similar results were seen during numerous clinical studies where only mild local reactogenicity and fevers without serious adverse reactions were observed [ , , ] . additionally, cvi- appeared to offer a major advantage for dermatologically compromised individuals as kempe et al. confirmed the safety of this vaccine in children with eczema [ ] . nevertheless, two critical findings dampened enthusiasm for the cvi- vaccine. first, the neurotoxicity of cvi- following intracerebral inoculation of monkeys was shown to exceed that of the unattenuated ikeda, lister, and nycbh strains, suggesting a greater human risk for encephalitis with cvi- [ ] . second, it was found that the immune response to cvi- , particularly the neutralizing antibody response, was low when compared to unattenuated lister or ikeda strains, calling into question the protective efficacy of this vaccine [ , , ] . the svrg ultimately decided to sponsor the development of an entirely new attenuated strain. spearheaded by dr. hashizume of the chiba serum institute in japan, the latest technologies and understanding of vaccinia viruses were coupled to develop a variant of lister that did not produce neurotoxicity in animal models, yet maintained neutralizing antibody titers comparable to lister [ , , ] . in addition, cell culture techniques were employed to avoid contamination problems inherent to animal lymph-derived vaccines such as lister and nycbh, which may have contributed to toxicity, and to allow for mass production under controlled conditions. to produce the new strain, lister was initially passaged in primary rabbit kidney (prk) cells at low temperature ( • c) [ ] . at the th generation, clones were selected and evaluated for their ability to grow in vero cells. the clone with the lowest titer of growth, designated lc (lister clone ), was isolated and evaluated for growth in rabbit skin and the cns of rabbits and monkeys, where it compared favorably to lister. a small clinical trial was then conducted, comparing lc to lister in children [ ] . the vaccine was well tolerated and had a reduced fever rate compared to lister. however, a delay in pock formation and resolution was observed in children vaccinated with the attenuated strain. to lessen the chance of autoinoculation complications due to a prolonged pock reaction, a clone of lc that produced small pocks, a feature correlating with diminished growth capacity in mammalian tissues, was selected on cam [ ] . the virus was passaged six more times in prk at low temperature followed by growth on cam. medium-sized ( - mm) pocks were isolated and designated lc mo (lister clone medium pock size on cam original clone). finally, the lc mo clones were passaged three more times in prk cells and grown on cam where small ( . - mm) pocks were selected. the final attenuated clone was called lc m (clone ). formal in vitro comparisons were subsequently carried out to evaluate the replicative capacity of lc m , lc mo, lc , lister, and cvi- strains at , . , and • c [ ] . whereas all strains were able to grow at • c, lc m was unable to grow at temperatures above . • c, and lc and lc mo clones were unable to grow at temperatures above • c. in contrast, lister and cvi- grew at all temperatures tested. the neurotoxic effects of the parent lister strain and the three attenuated strains (lc , lc mo, and lc m ) were assessed by inoculating rabbits with these strains intracerebrally at a concentration of . tcid of each virus [ ] . all of the rabbits inoculated with unattenuated lister developed encephalitis, whereas none of the rabbits inoculated with the attenuated strains developed any adverse neurological symptoms. homogenates of the brains of rabbits inoculated with lc m had low levels of detectable virus, whereas brain homogenates of rabbits inoculated with comparators, most notably with the lister strain, contained higher levels of virus. a follow-up study was performed in cynomolgus monkeys [ ] . in this study, . ml of pfu of lc m or . tcid of lc mo was injected intrathalamically, with one monkey in each group euthanized on post-injection days and to evaluate cns tissue and virus titers. the growth of lc m in cns tissues was observed to be profoundly lower. in addition, fewer pathologic changes were observed in the brains and spinal cords of monkeys injected with lc m . to evaluate the ability of the attenuated virus strains to infect tissues beyond the skin, virus titers were assessed in the blood and brain of normal and immunosuppressed (cortisonetreated) mice [ ] . mice were inoculated intraperitoneally (ip) with . pfu of lister, lc , lc mo, lc m , or cvi- . five mice in each group were sacrificed daily for evaluation of viremia or cns viral growth. when inoculated with the lister or cvi- strain, virus was detected in the brain between days and in both normal and immunosuppressed animals, whereas the lc virus was only identified in the brains of immunosuppressed animals. in animals inoculated with either the lc mo or lc m strain, no virus was detected in the brains of either normal or immunosuppressed animals. however, lc m was detectable in the bloodstream for days in normal mice and days in immunosuppressed mice (fig. ) . lister, lc , lc mo, lc m , and cvi- were also compared for local reactogenicity by inoculating rabbits intradermally and scoring erythematous responses [ ] . the lc virus produced the largest erythematous reaction, followed by the lister strain, lc mo, cvi- , and finally lc m , which produced the smallest erythematous reaction. vaccine protective efficacy was extrapolated from rabbit studies that measured hemagglutination-inhibition (hi) and plaque reduction neutralizing titers (prnt) [ ] . as shown in table , both hi and prnt responses to lc m compared favorably with the responses to the lister strain and supported the clinical evaluation of lc m . in , sufficient doses of lc m and doses of lc mo were provided to health officials in prefectures throughout japan to vaccinate , and children, respectively [ ] . practitioners were instructed to use these vaccines or the standard lister vaccine for routine primary vaccination of children that year (predominantly - year olds, but some infants were included). exclusion criteria included known congenital or acquired heart problems, "wet eczema", known history of severe drug allergies, severe lister and lc mo: inoculated tcid /animal. lc m : inoculated pfu/animal. note: with respect to the units used to report titers, hi titers were done on two-fold dilutions of test sera, while prnt titers were done on four-fold dilutions of the test sera. source: hashizume et al. [ ] . kidney or liver disease, epilepsy, immunocompromise, and a history of convulsion in the past years [ ] . all vaccines were administered using an approximately l dose percutaneously at concentrations of . × to × . pfu/ml via bifurcated needles with - punctures in the upper deltoid region [ ] . within the large cohort of vaccinees, lc mo-and , lc m -vaccinated children were closely evaluated for local and systemic reactions [ ] . take rates were assessed at - days, with a physical exam performed and weeks after vaccination [ ] . since a vesicular or pustular reaction following vaccination had been used since the time of jenner as evidence that a vaccinee has developed protective immunity [ , ] , take rates were defined as a vesicular or pustular reaction at the site of vaccination; local redness or induration alone was recorded as a non-take. data for local and systemic reactogenicity were derived from surveys provided to the parents or guardians of vaccinees, and clinical assessments (objective and queried) were conducted during postvaccination clinic visits. endpoints assessed included fever or other systemic symptoms, lymphadenopathy, local redness and induration, take rates, and assessment of adverse reactions [ , ] . lc m produced take rates equivalent to other smallpox vaccines that had been used in japan (table ) . as with other vaccinia strains, the maximal adverse effects of lc m were reported around the time of peak pock reactions ( - days postvaccination) [ , ] . local induration at the site of vaccination, rates of fever, peak temperature, and fever duration were significantly less than with lister vaccination [ ] . in closely evaluated subjects receiving lc m , there were cases of urticaria, case of eczema vaccinatum (ev), cases of autoinoculation, cases of rash localized around the vaccine site, and benign febrile seizures of unclear etiology [ , ] . although the case of ev was mild and resolved without sequelae, details regarding any pre-existing skin condition in the vaccinated child are not known. several substudies were conducted within this large clinical trial. one involved the removal of excess vaccine at the site of vaccination by alcohol wipes to assess the possible impact on rates of autoinoculation and take rates [ ] . excess vaccine was removed at , , or min postvaccination. results showed that vaccination take rates were high ( - %) and autoinoculation rates were low when wiping the vaccination site within min. in contrast, when the vaccine was allowed to dry for min before wiping, autoinoculation rates were higher ( . %) [ ] . a second substudy examined a lister challenge vaccine administered at various timepoints, ranging from days to months post-primary vaccination [ , ] . japanese public health officials hoped that, by varying the interval between the primary and challenge vaccinations in this substudy and monitoring cutaneous effects, they would be able to gain insight on the speed with which the primary attenuated vaccine elicited an immune response as well as the robustness and longevity of that response. as shown in table , cross-protective immunity against lister challenge vaccination was induced early and in the vast majority of vaccinees (∼ %) [ , ] . findings with lc m were comparable to ikeda/dairen results [ ] . takeuchi reported that when children ( - year olds) were vaccinated with lc m and challenge-inoculated with lister days later, only / developed a small pustule; the remaining children had either no detectable reaction ( . %), or redness/induration or scab at the vaccination site [ ] . these findings suggested that cross-protection against other poxviruses, such as variola, could be mounted quickly following lc m primary vaccination, and be maintained with little variation during the first year post-lc m vaccination (the longest timepoint studied). however, other studies have shown that protection against a primary reaction following revaccination, although proposed historically for this purpose [ ] , is not a definitive measure of likely protective efficacy [ , ] . the relevance of a cutaneous reaction upon revaccination is therefore unclear. humoral immune responses at - months post-lc m vaccination were compared to data generated following vaccination with lc mo, cvi- , or lister vaccines [ ] . hi titers induced by the four vaccines did not differ significantly (table ) . although the numbers of lc m recipients assessed for prnt antibody generation were less than those sampled for hi ( versus ), titers following lc m vaccination appeared comparable to those generated following lister vaccination and superior to titers achieved with cvi- vaccination. although encephalitis had been rarely observed in subjects vaccinated with different strains of vaccinia, the investigators believed that strains with the potential to induce overt neurotoxicity might also produce transient brainwave anomalies. while the use of an electroencephalogram (eeg) to predict postvaccination encephalitis has never been validated, eegs were performed to document possible subclinical neurotoxicity in a cohort of children (aged - ) vaccinated with lc m [ ] . testing was done prior to vaccination, and , , and weeks postvaccination. findings were then compared to historical eeg data from children vaccinated with lister and cvi- (table ) [ ] . the combined clinical and immunological data convinced the svrg that lc m was a safe and effective vaccine. ninety-thousand doses of lc m vaccine were distributed for use in and , with no reports of severe aes [ ] . nevertheless, it is unclear how many of these doses were actually administered as the rapid eradication of smallpox in india led to the discontinuation of routine smallpox vaccination in japan in . shortly thereafter, the last known naturally acquired case of smallpox occurred in africa in . the lc m strain differs from the parent lister strain in a number of biological characteristics as summarized in table . the temperature sensitivity of lc m is due to restricted replication at temperatures above . • c, rather than thermal lability. the stability of the lc m phenotype on cam (small pock size), growth kinetics, and temperature sensitivity have been tested and confirmed by repeated passaging of virus in prk cells [ ] . with regards to replication kinetics, lister and lc m viruses are essentially the same in singlestep growth curves [ ] , suggesting that the small pock size of lc m on cam is not due to restricted intracellular replication but rather to the relative inability of the lc m virus to spread cell-to-cell. analysis of lister and lc m genomes by restriction endonucleases and cross-hybridization has revealed that lc m contains a new restriction site (xhoi), located in a hindiii d fragment near the terminus of the genome [ ] . recombinant studies, in which the hindiii d fragment of lister has been introduced into the lc m strain, have shown that a part of the genome containing the extra xhoi is responsible for small plaque and pock size, and restricted growth in vero cells [ ] . the gene in this fragment, originally called ps/hr for plaque size/host range, and now identified as b r, is related to the regulator of complement activation (rca) gene family in mammals that contain the characteristic short consensus repeat (scr) domains (fig. ) [ , ] . recent analysis of the full sequence of the lister, lc mo, and lc m genomes confirmed the -base deletion in the b r gene of lc m , which introduces a stop codon within the gene [ ] . as a result, while the full-length b r gene encodes for a -residue protein, the altered gene in lc m theoretically would encode for a -residue protein that, if processed correctly, would be secreted from infected cells as a -residue protein. all orthopoxviruses produce four forms of virus particles: the intracellular mature virus (imv), the intracellular enveloped virus (iev), cell-associated enveloped virus (cev), and the extracellular enveloped virus (eev) [ ] . deletion of b r generally results in decreased production of eev [ ] , which is critical for cell-to-cell transmission of virus within the infected host and plays an important role in disease pathogenesis [ , ] . b r is also a target for neutralizing antibodies [ ] , and anti-b r antibodies have been shown to protect mice against lethal infection [ ] . yet recent lethal poxvirus challenge studies in rabbits and mice [ , ] demonstrated that a deletion in the b r gene does not diminish lc m 's efficacy or its ability to induce eev antibodies. a primate study [ ] also suggested that the b r protein is not critical for smallpox vaccine efficacy. hooper et al. were able to show that a dryvax-vaccinated monkey with no detectable b r-specific neutralizing antibodies could survive a lethal monkeypox virus challenge. the serum of this particular monkey was able to neutralize monkeypox and the vv-connaught vaccine strain (derived from the nycbh strain) in prnt assays. it is possible that the protection conferred by lc m is b r-independent and other eev surface proteins may serve as epitopes for neutralizing antibodies [ , , ] . there is also evidence that smallpox vaccines induce cell-mediated immunity (cmi) [ ] [ ] [ ] [ ] [ ] [ ] . hence it is possible that neutralizing epitopes on the truncated b r protein might contribute to the overall protective immune response without being the primary mediator. lc m compared with that of other vaccines. recently it was shown that with prolonged passaging in cell culture, lc m undergoes a phenotypic reversion characterized by the production of plaques that are of intermediate size between wild-type lc m and the precursor virus, lc mo. this plaque-size heterogeneity can be mapped to the b r gene where some variants exhibit point mutations upstream of the known b r single-base deletion. these compensatory mutations correct the observed frameshift and lead to the reconstitution of a full-length b r gene. western blot analysis has confirmed a restored ability of these variants to produce a full-length b r protein. in a severe combined immunodeficiency (scid) mouse ld study, the pathogenicity of two revertant viruses and the lc mo precursor strain was similar. in addition, plaque-purified lc m and a construct of lc m lacking the b r gene were shown to have safety profiles comparable to that of mva in the same animal models and to confer protective immunity in a mouse/intranasal vaccinia (western reserve [wr] strain) challenge study [ ] . the specific clinical consequences that correlate with deleted or mutated genes in attenuated vaccinia strains are only just beginning to be studied. orthopoxviruses are known to use a wide array of immunomodulatory strategies to establish a rapid and ongoing infection within the host [ , ] . these mechanisms target the innate, humoral, and cellmediated immune pathways, using mechanisms as diverse as functional mimicry of host proteins, masking, and avoidance of innate antiviral pathways [ , ] . it is not yet known what immunomodulatory mechanisms are used or altered by the attenuation of lister to lc m . the complete genome sequences of the lc m , lc mo, and lister viruses have been published recently [ ] and studies are underway to compare the genomic and proteomic profiles of this group with profiles of other orthopoxviruses. in , two groups independently showed that vaccinia viruses could be modified to serve as cloning and expression vectors by inserting foreign dna into non-essential regions of the genome [ , ] . since then, numerous studies have demonstrated the utility of recombinant vaccinia viruses (rvvs) as effective vector systems due to their ability to produce robust cellular and humoral immune responses. with a carrying capacity for large dna fragments that rivals other vector systems (e.g., lentivirus, alphavirus, aav, adenovirus), rvvs may be a preferred strategy for the delivery of foreign antigens. both replication-deficient (e.g., mva) and replicationcompetent vector strategies have been employed, with safety being a major distinction between the two approaches. since fever, encephalitis, progressive vaccinia, and myopericarditis sometimes accompany vaccination with replication-competent vaccines, a replicating rvv vector system that is both safe and capable of eliciting a strong immune response is highly desirable. lc mo and lc m are attractive options for live rvvs because they are characterized by temperature sensitivity, restricted host range, and infrequent low-grade adverse events. moreover, lc mo has been found to be more immunogenic than its unattenuated parent, lister, and lc m , so it has generally been the preferred vector system [ ] . table highlights many of table history of lc mo-and lc m -based rvvs as vectors for antigen delivery and protein expression (modified and updated from [ ] the antigen delivery and protein expression applications that have used either lc mo or lc m as a vector system. a recent abstract [ ] showed particularly promising results for an lc m -vectored sars coronavirus (sars-cov) vaccine. it was observed that neutralizing antibody titers to the sars-cov spike protein increased -fold week after vaccination and increased an additional -fold weeks after a booster injection. interestingly, rabbits with preexisting antibodies to the vector (after being pre-immunized with lc m ) still generated an antibody response, suggesting that this vector system might produce vaccines suitable for populations that have undergone prior immunization with smallpox vaccine(s). work by kidokoro et al. [ ] demonstrated that lc mobased vectors provide a robust protein expression system that preserves the functional integrity of the expressed protein. these investigators were able to obtain yields of up to mg/l and purity of - % for the glycosylated measles virus hemagglutinin (h) protein. based on these promising results, further studies to determine the general utility of this lc mo vector for large-scale expression of other proteins are warranted. since animal challenge studies were not conducted during the development of lc m in the s, alternative measures of immunity that had been used to characterize other smallpox vaccines [ , ] were used to evaluate the efficacy of lc m in early clinical trials in japan. these included basic antibody and t-cell assays and monitoring of cutaneous responses to primary vaccination with lc m [ ] . however, current standards for determining vaccinia efficacy rely on more sophisticated measurements of humoral and cmi responses as well as lethal poxvirus challenges in vaccinated animal models [ , , , ] . one example of the latter is the lethal rabbitpox virus (rpxv) in rabbits, which mimics smallpox infection in humans [ ] since it is rapidly and widely disseminated, is highly infectious, and produces high levels of eev. the latter appears to play a prominent role in the pathogenesis of both rpxv and variola [ , ] , making the rpxv challenge model particularly suitable for testing the eev-neutralizing capability of lc m . hence, the protective efficacy of lc m was recently evaluated in the rabbit model using a rpxv challenge [ ] . in this comparative study, rabbits per group were vaccinated by scarification with approximately × pfu of lc m or dryvax, or with phosphate-buffered saline (pbs) as placebo. after days, the groups were divided in two, challenged with low or high intradermal doses of rpxv ( and pfu, respectively), and monitored for days. at days post-challenge, all vaccinated animals had survived, whereas / (low-dose challenge) and / (high-dose challenge) placebo (pbs) animals had died ( fig. ; only the antibody response was characterized at days , , and postvaccination and measured for the ability to bind imv as well as the ability to neutralize both imv and eev of rpxv in prnt assays. antibodies from the lc m vaccinated rabbits had a greater capacity to bind rpxv in an elisa (data not shown) as well as neutralize rpxv imv when compared to antibodies from dryvax-vaccinated rabbits (fig. ) . interestingly, antibodies from lc m -and dryvax-vaccinated animals showed comparable abilities to neutralize rpxv eev. fig. . antibody responses to lc m and dryvax in rabbits. sera obtained from rabbits and days after immunization were tested at a : dilution for neutralization of imv and eev forms of rpv by plaque-reduction assays. each data point represents the mean of serum samples. error bars indicate % confidence intervals. imv neutralization titers elicited by lc m were significantly greater than those elicited by dryvax (p < . , two-sided wilcoxon rank sum test). sera obtained from pbs-immunized animals demonstrated no neutralization. these data are not shown. finally, a plaquing virus assay used to measure virus in different tissues showed there was no detectable rpxv in the lung, liver, or spleen of both groups of vaccinated animals, whereas placebo animals had high titers of virus in these organs. in a second animal model, lc m was compared to dryvax for its ability to protect a/ncr mice from aerosolized ectromelia virus (ectv), the causative agent of mousepox [ ] . this model was chosen since it closely mimics the route in which smallpox might be delivered during a bioterrorist attack [ ] . ten mice per group were vaccinated by scarification with approximately × pfu lc m or dryvax, or with pbs as placebo. forty-nine ( ) days later, all animals were challenged with a dose approximately - times the lethal dose (ld ) of aerosolized ectv. at days following challenge, all vaccinated animals had survived, whereas % of the placebo mice had died (fig. ) . no significant clinical symptoms or weight differences were noted between the lc m -and dryvax-vaccinated mice post-challenge. serum samples were taken from all mice days after vaccination. each sample was assessed for recognition of orthopoxvirus using an elisa with vaccinia western reserve (wr) as the antigen. as was seen in the rabbit study, sera from lc m -vaccinated mice generated higher vaccinia-specific elisa titers than sera from dryvax-vaccinated mice (fig. ) . the rabbit and mouse data from the preceding studies are in agreement with recent work in a mouse-vaccinia wr intranasal challenge model by kidokoro et al. who reported that b r-deficient lc m was able to induce prnt antibodies, and that protection against lethal challenge was equivalent to that of the b r-containing dryvax [ ] . in the same report, these authors determined that, similar to mva and in contrast to dryvax, lc m did not induce mortality in scid mice, despite being given at doses -fold higher than the protective dose in this particular mouse model. fig. . geometric mean titer of vaccinia virus-specific antibodies in mice vaccinated with dryvax or lc m . sera obtained from mice days after immunization were tested for vaccinia virus-specific antibodies by elisa. the bars represent the geometric mean titers from dryvax-immunized animals and lc m -immunized animals. although mice were vaccinated with dryvax or lc m , serum volumes from some animals were insufficient for analysis. error bars indicate % confidence intervals. the geometric mean titer elicited by lc m were significantly greater than those elicited by dryvax (p < . , two-sided wilcoxon rank sum test). sera obtained from pbs-immunized animals did not contain detectable vaccinia virus-specific antibodies. these data are not shown. further confirmation of the protective efficacy of lc m in another mouse-vaccinia wr challenge model was reported by morikawa et al., who demonstrated that protection was equivalent to that of the b r-containing lister and lc mo vaccine strains [ ] . finally, it has recently been reported by saijo et al. that lc m and lister provided protection from disease in monkeys challenged with severe intranasal monkeypox [ ] . japan has retained a national stockpile of lc m since conditionally licensing it in (unconditional licensure was granted in ). the government decided to increase the volume of its lc m stockpile in in response to a resurgence of monkeypox in africa, and again in early in response to terrorist threats in the us and other regions of the world. in anticipation of increased lc m production, employees of the chiba serum institute (manufacturers of lc m until mid- ) volunteered for vaccinations in november and were followed for safety and immunogenicity [ ] . fifty-six ( ) of these were revaccinees (the strain and date of previous vaccination unknown) and were primary vaccinees. lc m was administered using a fivepuncture bifurcated needle inoculation technique, and symptom diaries were kept for weeks. the vaccination site was inspected by medical personnel - days postvaccination. clinical observation records for of the vaccinated workers are summarized in table . vaccination site symptoms were mild (mostly pruritis) and resolved within a week. a phase i/ii clinical trial of lc m is presently underway in the us to evaluate the safety and immunogenicity of lc m . preliminary data was recently presented and will be formally published shortly [ ] . the current us stockpile of smallpox vaccines is comprised of vaccines using the nycbh vaccinia strain, primarily the calf lymph derivatives (apsv "wetvax" and dryvax manufactured in the s and early s, respectively) and one of the more recently manufactured cell culture derivatives (acam ) [ , ] . although well proven with regard to protective efficacy against smallpox, a first-generation vaccine such as dryvax may produce rates of local and systemic adverse reactions that are unacceptable in a post-endemic smallpox era, particularly given the higher rates of immunosuppression and skin disease in today's population. common reactions to dryvax include fever, lymphadenopathy, and lymphadenitis, while more serious adverse events include auto/contact inoculation, generalized vaccinia, eczema vaccinatum, progressive vaccinia, and encephalitis [ ] . concern has also been expressed about the possible presence of adventitious agents in stockpiled dryvax and apsv since calf lymph-perpetuated smallpox vaccines were not screened for mycoplasma, viruses, or prions during manufacturing [ ] . deaths from dryvax vaccination in the us prior to the eradication of smallpox were frequently due to encephalitis, which occurred at a rate of approximately one per million primary vaccinees [ ] . in recent clinical trials evaluating dryvax, myopericarditis, a serious adverse event rarely reported in the past, was found to occur at high rates among primary vaccinees [ , ] . through careful evaluation of vaccinees for both clinical and subclinical symptoms, this reaction was identified in up to / primary vaccinees, prompting the addition of a black box warning to the package insert of dryvax in [ ] . this discovery has further fueled the drive to identify smallpox vaccines with improved safety profiles. initial attempts to improve vaccine safety focused on modernizing the manufacture of dryvax by growing the virus in cell culture and cloning single subpopulation strains of nycbh that had been characterized by protective efficacy and low neurovirulence in animal studies [ , , ] . clinical trials with the acambis-produced second-generation nycbh vaccines, acam and acam (derived from acam and grown in vero cells rather than mrc- cells), confirmed high take rates (∼ %) and immunogenicity [ ] . the quality, progression, and timing of pock reactions were similar to those seen with dryvax controls, as were most local and systemic reactions. apart from local reactions at the injection site, the most common adverse reaction to acam or acam was lymph node or axillary pain (seen in - % of primary vaccinees) [ ] . unfortunately, subclinical or overt myopericarditis was also found to occur at high rates in both the acam and dryvax control groups [ , ] . in addition, one vaccinee given the experimental vaccine experienced a new onset seizure days postvaccination [ ] . in another recent phase ii study, myopericarditis developed in one subject days after vaccination with . × pfu/ml of acam [ ] . ccsv (cell-cultured smallpox vaccine), a version of the nycbh vaccinia strain grown in mrc- cells by dyn-port vaccine company (dvc), was recently evaluated in a double-blinded dryvax-controlled clinical trial conducted in vaccinia naive and non-naive volunteers [ ] . as with the acambis products, rates of adverse reactions to ccsv were similar to dryvax, however, no serious adverse reactions or myopericarditis were reported during the trial. ccsvinduced prnt titers and seroconversion rates were generally lower than those induced by dryvax. given the disappointing safety findings of cell culturederived second-generation vaccines, focus has once again shifted to third-generation vaccines such as lc m and mva (imvamune, bavarian nordic; and mva , acambis) as a potential means of replenishing national stockpiles. the japanese government recently announced that it will significantly expand its national stockpile of lc m , affording protection for million people in the event of a bioterrorist attack [ ] . mva, with its extensive history of safety in humans (> , people have been vaccinated in germany although some with only a single dose of approximately pfu), is presently being reassessed in various clinical and nonclinical studies [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although the rationale for using this non-replicating vaccine in high-risk populations such as those with immunosuppression or skin disease is compelling [ ] , its efficacy against smallpox is unknown. most nonclinical studies evaluating the protective efficacy of mva indicate that multiple-dose regimens using higher concentrations of approximately pfu are required [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in one study of mice challenged intradermally or intranasally with replicating vaccinia, protective efficacy was observed only after two or three doses of mva had been administered [ ] . lc m is currently the only smallpox vaccine under investigation that is both attenuated and able to replicate in humans, potentially offering protection after a single dose. the available clinical and nonclinical data suggest that lc m merits further study as a potential vaccine for use in the future. however, development of any new smallpox vaccine is complicated by the evolving nature of the field. it is clear that historical data on protective antibody titers from the smallpox eradication era cannot be relied upon. with smallpox no longer an endemic disease, vaccine efficacy can only be inferred from animal challenge studies and corresponding immune responses in humans. the us fda has published specific requirements for proof of efficacy in animal models that every new smallpox vaccine must meet (the so-called "animal rule") [ ] . given the multifactorial nature of the immune response to smallpox vaccines, various markers for both humoral and cellular immunity are being studied with the aim of identifying objective measurements of efficacy [ ] [ ] [ ] [ ] [ ] [ ] . correlates of protection in humans have yet to be defined unequivocally, but may rely in part on the elicitation of neutralizing antibody responses that correspond to those demonstrated to be efficacious in animal models. although the threshold titer of neutralizing antibodies needed for protection is unknown [ , ] , it was recently shown that human vaccinia-specific antibodies passively transferred to nonimmunized macaques offered protection against a lethal monkeypox challenge [ ] . a lack of published data and the varying reagents used in plaquing virus assays make comparison of absolute prnt titers elicited by different smallpox vaccines difficult. however, published results suggest that cell-cultured nycbh vaccines produce immune responses similar to dryvax [ , ] . no recent data on prnt levels elicited by mva or lc m in humans have been published, but findings should be forthcoming from ongoing clinical trials. the duration of humoral and cell-mediated protective immunity afforded by smallpox vaccination remains unknown, yet there is both empirical [ , ] and experimental [ , [ ] [ ] [ ] evidence that some degree of immunity to smallpox persists for several decades postvaccination. in addition, hammarlund and colleagues recently reported that smallpox vaccination as much as years pre-exposure conferred immunity to humans exposed to monkeypox [ ] . no data on the duration of long-term immunity to lc m are currently available, however, immunity duration will be an important consideration for all new smallpox vaccine candidates. while the biodefense-focused quest for a safe and effective smallpox vaccine is urgent, only the eventual outcomes of controlled clinical and nonclinical studies of new smallpox vaccine candidates will ensure their suitability for use in humans should smallpox recur in the future. characteristics of virus strains for production of smallpox vaccines effort to improve smallpox vaccine clinical special edition: vaccination in future-everything about attenuated vaccine vaccination research group research report: ministry of health and welfare special research: postvaccination side effects and research regarding treatment of complications vaccination in japan comparison of virus production in chicken embryo fibroblasts infected with the wr, ihd-j and mva strains of vaccinia virus: ihd-j is most efficient in trans-golgi network wrapping and extracellular enveloped virus release successful intramuscular immunization against vaccinia and variola (author's translation) modified vaccinia ankara: potential as an alternative smallpox vaccine mapping of deletions in the genome of the highly attenuated vaccinia virus mva and their influence on virulence modified vaccinia virus ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine virulence and immunogenicity of a modified vaccinia virus (mva) further studies of the infectious unit of vaccinia smallpox vaccination of eczema patients with a strain of attenuated live vaccinia (cvi- ) smallpox vaccination of eczema patients with attenuated live vaccinia virus intracerebral inoculation of monkeys with several vaccinia strains: an approach to the comparison of different strains primary vaccination with an attenuated strain of vaccinia virus the rationale for elective prevaccination with attenuated vaccinia (cvi- ) in preventing some vaccination complications special edition future of vaccination: everything about attenuated vaccines. basics of new attenuated vaccine strain lc m properties of attenuated mutant of vaccinia virus, lc m , derived from lister strain. in: quinnan, editor. vaccinia viruses as vectors for vaccine antigens kaketsuken (chiba serum institute) documents on file results of experimental inoculation of attenuated lc m reducing effect of lc m strain pre-treatment smallpox and its eradication. geneva: world health organization immunologic responses to vaccinia vaccines administered by different parenteral routes laboratory and vaccination studies with dried smallpox vaccines studies with dried and glycerinated smallpox vaccines of full and diminished potencies immunogenicity of ultravioletirradiated, non-infectious, vaccinia-virus vaccine in infants and young children stability of recombinant vaccinia virus lc mo or lc m : preserved laboratory attenuation markers and conserved expression of hbsag gene genetic analysis of vaccinia virus lister strain and its attenuated mutant lc m : production of intermediate variants by homologous recombination gene structures of low-neurovirulent vaccinia virus lc mo, lc m , and their lister original (lo) strains regulation of plaque size and host range by a vaccinia virus gene related to complement system proteins a constitutively expressed vaccinia gene encodes a -kda glycoprotein related to complement control factors that forms part of the extracellular virus envelope functional analysis of vaccinia virus b r protein: essential role in virus envelopment is independent of a large portion of the extracellular domain an attenuated lc m smallpox vaccine: analysis of fullgenome sequence and induction of immune protection the formation and function of extracellular enveloped vaccinia virus significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia antibodies against the extracellular enveloped virus b r protein are mainly responsible for the eev neutralizing capacity of vaccinia immune globulin neutralizing and protective antibodies directed against vaccinia virus envelope antigens highly attenuated smallpox vaccine protects rabbits and mice against pathogenic orthopoxvirus challenge smallpox dna vaccine protects nonhuman primates against lethal monkeypox protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions vaccinia virus-specific cd + cytotoxic t lymphocytes in humans induction of human t cell-mediated immune responses after primary and secondary smallpox vaccination cutting edge: long-term b cell memory in humans after smallpox vaccination duration of antiviral immunity after smallpox vaccination differential regulation of granzyme and perforin in effector and memory t cells following smallpox immunization long-lived poxvirus immunity, robust cd help, and better persistence of cd than cd t cells genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia lc m poxvirus immunomodulatory strategies: current perspectives technical knockout: understanding poxvirus pathogenesis by selectively deleting viral immunomodulatory genes immunomodulatory proteins of orthopoxviruses vaccinia virus: a selectable eukaryotic cloning and expression vector construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the dna of infectious vaccinia virus characteristics of an attenuated vaccinia virus strain, lc mo, and its recombinant virus vaccines development of sars vaccine using recombinant vaccinia virus derived from lc m . abstract p large-scale preparation of biologically active measles virus haemagglutinin expressed by attenuated vaccinia virus vectors recombinant vaccinia virus lc mo or lc m that expresses hepatitis b surface antigen while preserving the attenuation of the parental virus strain improved recombinant lc mo or lc m vaccinia virus successfully expressing hepatitis b surface antigen effects and virulences of recombinant vaccinia viruses derived from attenuated strains that express the human t-cell leukemia virus type envelope gene induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express prem and e glycoproteins of japanese encephalitis virus induction of neutralizing antibodies against bovine leucosis virus in rabbits by vaccination with recombinant vaccinia virus expressing bovine leucosis virus envelope glycoprotein immunological and virological characterization of improved construction of recombinant vaccinia virus expressing rinderpest virus hemagglutinin immunisation of cattle with a recombinant vaccinia vector expressing the haemagglutinin gene of rinderpest virus analysis of canine herpesvirus gb, gc, and gd expressed by a recombinant vaccinia virus long-term protective immunity to rinderpest in cattle following a single vaccination with a recombinant vaccinia virus expressing the virus haemagglutinin protein clinical and immunological study of percutaneous revaccination in children who originally received smallpox vaccine subcutaneously standard percutaneous revaccination of children who receive primary percutaneous vaccination developing new smallpox vaccines cfr section . : approval of biological products when human efficacy studies are not ethical or feasible rabbit pox: an experimental study of the pathways of infection in rabbits an antigenic difference between intracellular and extracellular rabbitpox virus efficacy of oral active ether lipid analogs of cidofovir in a lethal mousepox model lc m , a highly attenuated vaccinia virus vaccine lacking expression of the membrane protein b r, protects monkeys from monkeypox reintroduction of smallpox vaccine with attenuated vaccine lc m timeline of arriving at manufacturing, clinical studies, and investigation of immunity safety and immunogenicity of attenuated smallpox vaccine, lc m : results from a phase study [poster e board of health strain)-a second-generation smallpox vaccine for biological defense adventitious agents and smallpox vaccine in strategic national stockpile deaths attributable to smallpox vaccination to and myopericarditis following smallpox vaccination smallpox vaccines: looking beyond the next generation clonal vaccinia virus grown in cell culture as a new smallpox vaccine safety and immunogenicity of new cell-cultured smallpox vaccine compared with calf-lymph derived vaccine: a blind, singlecentre, randomised controlled trial a novel, cell culture-derived smallpox vaccine in vaccinianaive adults government urged to stockpile smallpox vaccine. the daily yomiuri highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge immunogenicity of a highly attenuated mva smallpox vaccine and protection against monkeypox the future of smallpox vaccination: is mva the key? smallpox vaccination: risk considerations for patients with atopic dermatitis modified vaccinia virus ankara immunization protects against lethal challenge with recombinant vaccinia virus expressing murine interleukin smallpox vaccine does not protect macaques with aids from a lethal monkeypox virus challenge modified vaccinia virus ankara protects macaques against respiratory challenge with monkeypox virus identification of poxvirus cd + t cell determinants to enable rational design and characterization of smallpox vaccines identification of murine poxvirus-specific cd + ctl epitopes with distinct functional profiles profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus smallpox vaccinations: how much protection remains? smallpox in europe, - the persistence of neutralizing antibodies after revaccination against smallpox human cytotoxic t-cell memory: long-lived responses to vaccinia virus responses to smallpox vaccine multiple diagnostic techniques identify previously vaccinated individuals with protective immunity against monkeypox we would like to thank the following individuals for their contributions to this manuscript: hiroyuki yokote, munehiro hirayama, and so hashizume for permission to reproduce figures and tables from original lc m research reports; kouji matsushima for helpful discussions concerning the use of lc m as a vector system; catherine bolger for editorial assistance; and timothy corrigan for document translation support. in addition, we would like to express our grati- key: cord- -p zjxo h authors: malik, a. a.; mcfadden, s. m.; elharake, j.; omer, s. b. title: determinants of covid- vaccine acceptance in the u.s. date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: p zjxo h background:the covid- pandemic continues to adversely affect the u.s., which leads globally in total cases and deaths. as covid- vaccines are under development, public health officials and policymakers need to create strategic vaccine-acceptance messaging to effectively control the pandemic and prevent thousands of additional deaths. methods: using an online platform, we surveyed the u.s. adult population in may to understand risk perceptions about the covid- pandemic, acceptance of a covid- vaccine, and trust in sources of information. these factors were compared across basic demographics. findings: of the participants surveyed, ( %) said they would accept a covid- vaccine if it is recommended for them. males ( %), older adults ([≥] years; %), asians ( %), and college and/or graduate degree holders ( %) were more likely to accept the vaccine. when comparing reported influenza vaccine uptake to reported acceptance of the covid- vaccine: ) participants who did not complete high school had a very low influenza vaccine uptake ( %), while % of the same group said they would accept the covid- vaccine; ) unemployed participants reported lower influenza uptake and lower covid- vaccine acceptance when compared to those employed or retired; and, ) black americans reported lower influenza vaccine uptake and lower covid- vaccine acceptance than nearly all other racial groups. lastly, we identified geographic differences with department of health and human services regions (new york) and (chicago) reporting less than percent covid- vaccine acceptance. interpretation: although our study found a % acceptance of a covid- vaccine, there were noticeable demographic and geographical disparities in vaccine acceptance. before a covid- vaccine is introduced to the u.s., public health officials and policymakers must prioritize effective covid- vaccine-acceptance messaging for all americans, especially those who are most vulnerable. the coronavirus disease (covid- ) pandemic has spread across the world with millions infected and hundreds of thousands dead. , while most countries impacted have developed successful response strategies and observed significant improvements, the u.s. (as of may st, ) leads globally with · million cases and over , deaths. additionally, according to the centers for disease control and prevention (cdc), current data show a disproportionate burden of covid- infections and deaths among racial and ethnic minority communities. with the u.s. facing an economic disruption and the future remaining unknown, a vaccine to prevent covid- infection is perhaps the best hope for ending the pandemic. as misinformation about covid- has spread across media outlets, it is important for u.s. public health officials and politicians to begin planning for effective messaging and policies before a vaccine is introduced. the u.s. already struggles with reaching high rates of influenza vaccine coverage-with less than half of the adult population vaccinated in , resulting in over , vaccine-preventable deaths-therefore, covid- presents an imminent danger that requires immediate action. health communication must reach all communities, especially the most vulnerable, to educate americans about the safety of vaccines and prevent future infections and deaths. immunization programs are only successful when there are high rates of acceptance and coverage. to accomplish this, it is critical to understand americans' risk perceptions about covid- , acceptance of a covid- vaccine, and confidence in media sources, specifically those used to obtain information about the covid- pandemic. the purpose of our study is to describe the current vaccine acceptance landscape with aims to ) predict covid- vaccine acceptance using regularly available demographic information, ) identify the most vulnerable populations, and ) provide information for public health officials and politicians to develop messaging for all americans, while targeting communities most in need. data were collected using an electronic questionnaire via qualtrics® (qualtrics, provo, ut). in early may , participants completed a questionnaire on cloudresearch. cloudresearch is an online survey platform that allows for representative surveying. the goal of our sampling was to be representative of the u.s. general population based on age, gender, education, race and ethnicity. participants were eligible if they were years of age or older, could read english, and had a cloudresearch account with access to the internet via computer or smart phone. participants received compensation in the amount they agreed to with the platform through which they entered this survey; these rewards could include gift cards or donations to a participant selected charity. basic demographic information was collected as well as zip code, state of residence, and employment status. additionally, we asked participants if a covid- vaccine were available and recommended for them, would they would accept it ( -point likert scale: = strongly disagree to = strongly agree); this variable was dichotomized to covid- vaccine acceptance ( = strongly disagree/disagree/neutral; = agree/strongly agree). participants also completed the perceived risk scale (cronbach's α = · ) which had survey-items ( -point likert scale: = strongly disagree/disagree/neutral; = agree/strongly agree). of the participants, participants were removed from score calculation if they did not respond to one or more the of the items on the risk perception scale or selected "don't know," so this score was calculated for participants. we also asked participants if they had received the influenza vaccine in the previous months. finally, participants were asked about their confidence in media sources and the reliability of these sources regarding the covid- pandemic ( -point likert scale: = strongly disagree to strongly agree). yale university institutional review board approved this study (irb protocol number: ). participants provided informed consent prior to data collection. to assess the associations (odds ratios) of demographic factors with covid- vaccine acceptance, a logistic regression analysis was used. model selection using stepwise backward selection with a p-value of · was used to select the final, parsimonious model where age, gender, race, education, ethnicity, and employment status were included as explanatory variables. we computed area under the receiver operating curve (auc) for our final model to evaluate model performance. we used bootstrap resampling ( samples) for internal validation and to obtain an area under the curve value accounting for model optimism. , data were analyzed using stata version (statacorp, college station, texas). we invited , participants, of which ( %) completed the survey with ( %) females and ( %) years old or over. the majority of the participants were non-hispanic white (n = ; %) and had a college or graduate degree (n = ; %). the median risk perception score for covid- was (iqr: - ; mean: . ; sd: · ). table shows the demographics characteristics of the survey participants and the general u.s. population. of the participants surveyed, ( %) said they would accept a covid- vaccine if it is recommended for them. the vaccine acceptance differed by demographic characteristics with males ( %), older people ( years and above; %), asian ( %), and college or graduate degree holders ( %) more likely to accept the vaccine if it would be recommended for them (table ; figure ). the median risk perception score amongst those who would accept the vaccine was (iqr: - ) compared to a median of (iqr: - ) amongst those who would not accept the vaccine. this difference in risk perception score was statistically significant (p < . ). as sensitivity analysis, we carried out a weighted analysis for covid- vaccine acceptance using u.s. demographics. weighing by age and sex decreased the percent acceptance to % while weighing by age, sex, and race decreased the percent acceptance to %. vaccine acceptance for covid- also differed compared to the influenza vaccine with ( %) of the participants having received the influenza vaccine in the last eight months (chi sqr p-value < · ; figure ). notable demographic differences exist when comparing reported influenza vaccine uptake to reported acceptance of the covid- vaccine. for example, participants who did not complete high school, had a very low influenza vaccine uptake (n = ; %), but of that same group, % (n = ) said they would accept the covid- vaccine if it were available and recommended for them. another interesting demographic difference is that the part of the sample that reported being unemployed reported lower influenza uptake and lower covid- vaccine acceptance when compared to those who reported being employed or retired. additionally, black americans reported lower influenza vaccine uptake (n = ; %) and lower covid- vaccine acceptance (n = ; %) than nearly all other racial groups. a final demographic difference is that older adults reported higher influenza vaccine uptake (n = ; %) and higher covid- vaccine acceptance (n = ; %) than younger participants. there were notable geographic differences in covid- vaccine acceptance with dhhs region -denver having an acceptance rate of over % (total sample in region, n = ; number of participants within sample that would accept covid- vaccine, n = ) while region -new york (n = ; n = ), and region -chicago (n = ; n = ) had an acceptance rate of less than % (figure a). dhhs region -denver also had a higher influenza vaccine coverage with over % (n = ; n = ) of the participants having received the vaccine in the last months, while hhs regions -new york (n = ; n = ), -philadelphia (n = ; n = ), -dallas (n = ; n = ), and -kansas city (n = ; n = ) had less than % coverage (figure b). figure shows the percent covid- vaccine . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint acceptance against the percent influenza vaccine coverage by state. there was no statistical association between the two variables (coefficient: · ; % ci: - · - · ). the best model to predict covid- vaccine acceptance in our survey using demographic information that is readily available had age, gender, race, and education as explanatory variables with an area under the curve (auc) of % (table ; figure ). model optimism was estimated to be · % with optimism corrected auc being %. the participants reported the highest confidence in healthcare professionals (n = ; %), their own physician (n = ; %), cdc (n = ; %), state health departments (n = ; %), and local health departments (n = ; %). the participants also reported healthcare professionals (n = ; %) and health officials (n = , %) as the most reliable sources of information on covid- . comparatively, participants ( %) reported social media as a reliable source of covid- information. while a majority of our respondents from across the us would accept a covid- vaccine, that number may not be sufficient based on some of the estimates covid- herd immunity. with many covid- vaccines under development and substantial vaccination levels needed to achieve herd immunity, we must clearly understand the hesitancy and acceptance of a covid- vaccine to develop evidenced based interventions. this will allow healthcare professionals and health officials to develop messaging to best address concerns and educate all americans, especially vulnerable groups. our study shows that covid- vaccine acceptance can be predicted with relatively high accuracy by readily available demographic characteristics. since the beginning of the covid- pandemic in the united states, it has been clear that low-income and communities of color are at higher risk for infection and death from covid- . in fact, when looking at data by zip code from new york city, the dramatic inequality in covid- infections and deaths is evident. not only do the most affected zip codes include communities of color, but there are also significant income disparities, demonstrating the intersectionality between race, socio-economic status (ses), and health outcomes. the disparate health outcomes related to covid- occur not only in new york city, but also across the u.s. owen et al. discuss the racial and ethnic differences in covid- infections and deaths in chicago, illinois; charlotte, north carolina; milwaukee, wisconsin and across the states of michigan and louisiana. historical oppression and current disparities in care are linked to a mistrust of the healthcare system among some black americans and may result in these differences in health outcomes. supporting this, our study found that black americans were less likely to get the influenza vaccine and are less likely to accept a potential covid- vaccine. in addition to racial disparities, covid- vaccine acceptance differs based on education and employment. according to the u.s. bureau of labor statistics, as years of education increases, unemployment rates decrease, and income increases. related to this, our study found that as years of education increases, so does reported acceptance of the covid- vaccine. additionally, unemployed participants reported a lower acceptance rate of a covid- vaccine. these findings demonstrate that low income communities, which are disproportionately impacted by covid- , may be more susceptible to continued outbreaks, even if a vaccine is available. we need to use caution assuming that reported acceptance or intent translates into actual behavior. this is especially a concern when there is some time between the measurement of intention and the observation of behavior, which cannot occur until a covid- vaccine is available publicly. currently, the covid- pandemic is covered on the -hour news networks and dominates a great deal of online media. this media coverage may make the covid- pandemic more salient in daily life, especially when compared to influenza. additionally, during a pandemic and immediately around a new vaccine release, excitement about a vaccine is at its highest. another factor that could change salience is if a definitive pharmacological treatment is discovered that reduces duration of illness or deaths. aside from salience, there may be other unidentified factors that influence covid- vaccine acceptance and eventually vaccine uptake. for example, we found that hhs region -new york, which includes the epicenter for the covid- pandemic in the u.s., had the lowest reported covid- vaccine acceptance. therefore, covid- vaccine acceptance and eventual uptake may be influenced by more than just media coverage and direct exposure to the economic and health consequences. this means planning for a covid- vaccine should be comprehensive, with a focus on groups that are at high risk. . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint building confidence in a covid- vaccine is essential because the herd immunity threshold for sars-cov- , the virus causing covid- , is estimated to be between % and %, and we found that % of our sample would accept the vaccine. also, the number of americans who actually receive a covid- vaccine could be lower than those who claim they intend to vaccinate. thus, health professionals must be careful to encourage trust in vaccination and minimize misinformation. currently, opposition to vaccination overall may amplify outbreaks , like it did during the measles outbreak. opposition to vaccines, which occurs actively online, may influence covid- vaccine acceptance. however, according to larson, governments that deliberately release reassuring misinformation about covid- may also reduce covid- vaccine acceptance. in the united states, misinformation released by the government includes the country's testing capacity, the efficacy and safety of potential pharmacological interventions, and the speed at which a vaccine can safely be developed and produced. to counter this misinformation and improve trust, thoughtful and targeted messaging needs to be developed and tested now to build on the current public interest and continue the momentum past the release of a vaccine. messaging and education should focus the general american population as well as high risk groups, including lowincome individuals and communities of color. this emphasis on high risk communities indicates a need for cultural humility and community engagement. additionally, how these messages are made available to the public should be considered. we found that our participants had the most trust in covid- information from healthcare professionals and health officials; participants indicated that information from these sources are more reliable than social media. hence, health officials and healthcare professionals, including nurses and ancillary healthcare staff, should be engaged in community messaging to improve trust in a covid- vaccine and increase uptake. our findings may be influenced by possible selection bias because participants needed a cloudresearch account and access to smartphone/computer to participate. although we had a response rate of %, our data are fairly representative of the u.s. population. in addition to the representative sample, strengths of our study include timeliness and ability to stratify on demographic and geographic factors to predict covid- vaccine acceptance. most importantly, this is one of the first studies that looks at detailed covid- vaccine acceptance. our study found % of our sample from across the u.s. would accept a covid- vaccine. however, there were demographic and geographical variations in rates of acceptance that need to be carefully addressed. policymakers and stakeholders should focus on evidence-based community messaging to improve uptake and break the transmission dynamics. . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint author's contribution smm, aam, and sbo conceptualized the study. smm, and aam collected data under supervision from sbo. smm, aam, and je performed and reviewed the analysis, and wrote the initial draft of the manuscript. all authors helped interpret the findings, read and approved the final version of the manuscript. all authors declare no conflict of interest. . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint evidence before the study there are non-peer reviewed surveys suggesting that % of the u.s. population would get vaccinated against covid- , with % getting vaccinated soon after the vaccine is available. experience from the influenza vaccines and others shows that vaccine uptake is not optimal. although intent is high, intent does not always translate into behavior. we demonstrate demographic and geographic variations in vaccine intent for covid- with no relationship with the influenza vaccine. we also developed a predictive model to predict covid- vaccine acceptance by readily available demographic information. we found high level of confidence in covid- information received from healthcare professionals and health officials. targeted evidenced based community messaging through healthcare professionals and health officials will be required to increase covid- vaccine acceptance. . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint an interactive web-based dashboard to track covid- in real time covid- ): cases in the u covid- in racial and ethnic minority groups. estimated influenza illnesses, medical visits, hospitalizations, and deaths in the united states - - influenza season com: a versatile crowdsourcing data aquisition platform for the behavioral sciences clinical prediction models: a practical approach to development, validation, and updating correcting for optimistic prediction in small data sets population estimates by age, sex, race, and hispanic origin planning for a covid- vaccination program covid- and african americans new york city coronavirus map and case count. the new york times failing another national stress test on health disparities unemployment rates and earnings by educational attainment prospective prediction of healthrelated behaviors with the theory of planned behavior: a meta analysis epidemiological methods in immunization programs high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus a postmoder pandora's box: anti-vaccination misinformation on the internet blocking information on covid- can fuel the spread of misinformation key: cord- - qsirnk authors: dieffenbach, carl w; fauci, anthony s title: the search for an hiv vaccine, the journey continues date: - - journal: j int aids soc doi: . /jia . sha: doc_id: cord_uid: qsirnk nan as we commemorate hiv vaccine awareness day, we honour the thousands of study participants and the research teams that have played essential roles in the completed and ongoing vaccine clinical trials. we also recognize the scientists who have spent years dedicated to creating the tools and technologies to end the global hiv epidemic. on this vaccine awareness day, we must acknowledge that the hiv vaccine research field is quickly approaching a critical crossroads. this past decade has been spent launching improved vaccine concepts and immune-based strategies into trials. despite these advances, a safe and durably effective hiv vaccine has eluded us. in this regard, we must evaluate the current state of the field and make appropriate adjustments to help speed us to our goal. while this quest for a vaccine at times may feel extremely difficult, as evidenced by the recent disappointing results of a phase iii efficacy trial, hvtn , described below, we must steadfastly move forward to address critical research gaps and unanswered questions. as we reflect upon the status of hiv vaccine research, we would be remiss if we did not mention the current global pandemic caused by sars-cov- . it is important to acknowledge the global response to the covid- epidemic and to cite the range of vaccine platforms being used for anti-sars-cov- candidate vaccines that have their origin in hiv vaccinology. the hiv vaccine field is clearly assisting in addressing this newly emergent pandemic and the national institute of allergy and infectious diseases (niaid) is fostering these essential collaborations. for hiv vaccine awareness day in , we emphatically state that finding safe, effective and durable vaccines for hiv and covid- are niaid's top priorities. the world must have both. based upon the challenges that hiv biology presents: global sequence diversity, integration of the viral genome into the host cells and long duration of the symptom free period of infection as well as the fact that no single individual has been spontaneously cured, it is widely accepted that a vaccine must trigger responses that are qualitatively different from the immune responses to natural infection. after the step trial (also referred to as hvtn or merck v - ) was halted in , vaccine candidates that fulfilled these criteria have been prioritized [ , ] . over the past decade, three different vaccine approaches have been implemented, possible correlates of protection identified, and two have moved through clinical evaluation to advanced clinical trials. the first of these was based upon the correlates of risk that were generated from analysis of the rv trial conducted in thailand, which in , reported modest levels of efficacy [ , ] . rather than perform a direct repeat of rv in thailand, where the epidemic is driven by viruses from clades ae and b, a clade c-specific regimen was developed for testing in southern africa. when evaluated in the hvtn trial, the relevant correlate responses were detected and shown to be longer lasting than those of rv [ ] [ ] [ ] . with the go-no-go criteria for advancement met, the hvtn study moved ahead. however, no protection was observed, and the trial was terminated due to futility. while the field is disappointed with this outcome, it is important to understand the possible reasons for why this occurred. first, there were significant differences in the overall incidence between the study cohorts in hvtn and rv that could have overwhelmed a modestly protective response. additionally, it is conceivable that the correlates observed in rv are specific to the clades and population in thailand and not transferable to the southern africa epidemic. the remaining two vaccine concepts have reproducibly demonstrated efficacy in non-human primate studies. the most advanced concept is currently being evaluated in two studies, imbokodo (hvtn /hpx ) in women in southern africa and mosaico (hvtn /hpx ) in men and transgender persons who have sex with men in the americas and europe [ ] . analysis of the correlates of protection seen in the non-human primate studies point to qualitatively different responses than those observed in rv , and the trials are evaluating in silico designed immunogens to present the most globally conserved hiv sequences to trigger quantitatively superior cd + t cell responses [ , ] . the third approach, still in preclinical studies, is an siv vaccine delivered via the rhesus cytomegalovirus (rhcmv) vector platform. this strategy has been shown by picker and colleagues to protect percent of monkeys from sustained infection [ ] . efforts are currently underway to transition to a human cmv vector platform with phase clinical trials expected in . although humbled by the outcome of hvtn , we must continue to explore novel methods of inducing or providing protective immunity against hiv infection. one of the most exciting developments in hiv research has been the ability to isolate and produce broadly neutralizing monoclonal antibodies (bnabs) that neutralize nearly all of circulating virus strains from all clades [ , ] . these human antibodies prevent acquisition of shiv infection, in a dose dependent manner, in animal challenge models and when combinations of these antibodies are used in people living with hiv, strong antiviral activity has been demonstrated. the antibody mediated protection (amp) trials are currently evaluating vrc , the cd binding site targeted bnab, to determine the ability of this single antibody to prevent hiv infection in women in southern africa and msm and transgender persons in the americas [ ] . the amp studies have not been impacted by covid- , and results from the amp studies are expected in fall of . if protection is observed in the amp trials, an important correlate for future vaccine studies will be established. in the meantime, efforts are underway to define immunogens that specifically trigger neutralizing antibodies by targeting five specific epitopes, which are unique sites of vulnerability on the hiv env trimer glycoprotein [ , ] . the goal is to create a discrete set of immunogens that can reproducibly elicit the production of neutralizing antibody responses against two or more of these target sites. the collaborative hiv immunogen project [ ] is seeking to prospectively design and select the optimal combination of immunogens to be evaluated for the induction of broad neutralization of a range of hiv isolates. as we reflect on hiv vaccine awareness day and recent developments in the vaccine field, it is clear that much work remains to be done. going forward we must continue to engage community to ensure a shared understanding of our common goals. while we pursue a safe and effective hiv vaccine, we are also seeking ways of improving chemoprophylaxis. we remain optimistic that effective interventions that people will reliably use can be developed and brought to scale. on hiv vaccine awareness day, let us pause and reflect on how far we have come while continuing to focus on completing the work needed to achieve an effective vaccine. the authors collaboratively conceived the content of the paper. cwd wrote the first draft and asf edited the manuscript into this final form. the authors thank the trial participants, community members, activists and researchers who have so willingly participated in the challenging work of hiv vaccine discovery and development. the authors also thank robert gulakowski, thomas liang, mary marovich and sarah read for their careful review and comments on this manuscript. overview of step and phambili trial results: two phase iib test-of-concept studies investigating the efficacy of mrk adenovirus type gag/pol/nef subtype b hiv vaccine hiv vaccine research: the way forward vaccination with alvac and aidsvax to prevent hiv- infection in thailand immune-correlates analysis of an hiv- vaccine efficacy trial subtype c alvac-hiv and bivalent subtype c gp /mf hiv- vaccine in low-risk, hiv-uninfected, south african adults: a phase / trial safety and immune responses after a -month booster in healthy hiv-uninfected adults in hvtn in south africa: a randomized double-blind placebocontrolled trial of alvac-hiv (vcp ) and bivalent subtype c gp /mf vaccines immune correlates of the thai rv hiv vaccine regimen in south africa evaluation of a mosaic hiv- vaccine in a multicentre, randomised, double-blind, placebo-controlled, phase / a clinical trial (approach) and in rhesus monkeys (nhp - ) increased valency of conserved-mosaic vaccines enhances the breadth and depth of epitope recognition a live-attenuated rhcmv/siv vaccine shows long-term efficacy against heterologous siv challenge use of broadly neutralizing antibodies for hiv- prevention multiple roles for hiv broadly neutralizing antibodies safety, pharmacokinetics and neutralization of the broadly neutralizing hiv- human monoclonal antibody vrc in healthy adults hiv- neutralizing antibody signatures and application to epitope-targeted vaccine design key: cord- -f o aynz authors: samad, abdus; ahammad, foysal; nain, zulkar; alam, rahat; imon, raihan rahman; hasan, mahadi; rahman, md. shahedur title: designing a multi-epitope vaccine against sars-cov- : an immunoinformatics approach date: - - journal: journal of biomolecular structure & dynamics doi: . / . . sha: doc_id: cord_uid: f o aynz ongoing covid- outbreak has raised a drastic challenge to global public health security. most of the patients with covid- suffer from mild flu-like illnesses such as cold and fever; however, few percentages of the patients progress from severe illness to death, mostly in an immunocompromised individual. the causative agent of covid- is an rna virus known as severe acute respiratory syndrome coronavirus (sars-cov- ). despite these debilitating conditions, no medication to stop the disease progression or vaccination is available till now. therefore, we aimed to formulate a multi-epitope vaccine against sars-cov- by utilizing an immunoinformatics approach. for this purpose, we used the sars-cov- spike glycoprotein to determine the immunodominant t- and b-cell epitopes. after rigorous assessment, we designed a vaccine construct using four potential epitopes from each of the three epitope classes such as cytotoxic t-lymphocytes, helper t-lymphocyte, and linear b-lymphocyte epitopes. the designed vaccine was antigenic, immunogenic, and non-allergenic with suitable physicochemical properties and has higher solubility. more importantly, the predicted vaccine structure was similar to the native protein. further investigations indicated a strong and stable binding interaction between the vaccine and the toll-like receptor (tlr ). strong binding stability and structural compactness were also evident in molecular dynamics simulation. furthermore, the computer-generated immune simulation showed that the vaccine could trigger real-life-like immune responses upon administration into humans. finally, codon optimization based on escherichia coli k resulted in optimal gc content and higher cai value followed by incorporating it into the cloning vector pet +(a). overall, these results suggest that the designed peptide vaccine can serve as an excellent prophylactic candidate against sars-cov- . communicated by ramaswamy h. sarma coronavirus disease is an acute highly infectious disease. patients with covid- mostly feel like flu-like symptoms including cold and fever; a few percentages of the patients suffer from the respiratory tract infection leading to severe atypical pneumonia that eventually ends up in a case of fatality (boopathi et al., ; joshi et al., ; vankadari & wilce, ) . moreover, patients admitted to the intensive care unit were likely to report cardiovascular, respiratory disease, cerebrovascular, abdominal pain, endocrine, anorexia and digestive diseases (chan et al., ; . acute cardiac injury and acute respiratory distress syndrome are commonly observed in severe cases and is strongly associated with the mortality rate (abdelli et al., ; wu et al., ) . the infected patients can transmit the virus through coughs, sneezes, exhales and many other ways, hence, playing an essential role in human to human transmission (chan et al., ) . infection with the virus is sometimes asymptotic, which also plays a vital role in the transmission process (shen et al., ) . the covid- outbreak has already been taken place all over the world with a total of , , confirmed cases and , death cases ( june , : gmt) over countries and territories around the world (www.worldometers.info). as of june , the outbreak in bangladesh includes , confirmed and death cases, and the number of cases is increasing drastically day by day. the causative agent of the covid- outbreak is severe acute respiratory syndrome coronavirus (sars-cov- ) which is a positive-sense single-stranded rna virus that belongs to the family of coronaviridae (boopathi et al., ; umesh et al., ) , and genus beta-coronavirus li & de clercq, ) . this virus was first identified in patients with a cluster of pneumonia in the province wuhan of china on december (das et al., ; kumar et al., ; zhu et al., ) . the world health organization (who) country office in china announced the official declaration of this virus on december (calisher et al., ; heymann & shindo, ) . the new coronavirus was named 'sars-cov- ' by the international committee on taxonomy of viruses (ictv), and the disease caused by the pathogen was announced as covid- by the who on february (gupta et al., ; muralidharan et al., ; wu et al., ) . epidemiological investigations of the wuhan zoonotic virus revealed . % nucleotide similarity between sars-cov- and previously originated group of sars-like coronavirus vankadari & wilce, ; wu et al., ) . during the sars-cov- infection, the counts of cd þ and cd þ t-cells are increased in the peripheral blood, and cytotoxic granules are upturned with high concentration . the over-activation of t-cells causes injury to the immune system of the infected patients resulting in the characteristic feature of 'lymphopenia' increasing the disease severity. in contrast, less effective t-cell responses may allow the progression of viral pathology and thus increased mortality in sars-cov- infected patients (ahammad et al., ; . the cd þ and cd þ responses provide a long-lasting protection against covid- (enayatkhani et al., ) . moreover, antibody-mediated immune response along with cellular immunity plays a critical role to induce protectivity against these infections (enayatkhani et al., ) . in recent studies, it has been illustrated that the nucleotide structure of this virus particle has a similarity with sars-like coronavirus. their genome was encoded with different non-structural proteins and four main structural proteins, including spike (s), envelope (e), nucleocapsid (n), and membrane (m) proteins (hasan et al., ; sarma et al., ; wahedi et al., ; wu et al., ) . the s-protein formed the viral outer layer; the n-protein helps in the viral replication, genome construction and host cellular response; the mprotein determined the envelope shape and the e-protein functions in production and maturation of the sars-cov- (astuti & ysrafil, ) . the s-protein of the virus contains two subunit, s and s ; the s subunit of the s-protein recognizes the host t-cells while s subunit mediates fusion between the viral and host t-cells (astuti & ysrafil, ) and characterizes as a highly antigenic and surface exposure wrapp et al., ) . the cd þ and cd þ t-cells recognize viral epitopes presented by the major histocompatibility complex class i (mhc i) and class ii (mhc ii), respectively (abdellrazeq et al., ; borthwick et al., ) . the heterogeneity in t-cells responses to sars-cov- may, in part, be related to the capacity to recognize the viral antigens in the context of mhc i and mhc ii proteins (astuti & ysrafil, ) . it has been found that t-cell epitopes of sars-cov- spike protein elicit a t-cells immune response in patients who recover from the disease, and most of these immunogenic epitopes were localized to the s protein of the virus (astuti & ysrafil, ) . the s-protein has strong interactions and binding affinity to the human angiotensin-converting enzyme (ace ) receptor and facilitates viral entry into the target cell (sinha et al., ; . the s-protein of the sars-cov- is the major host interacting protein, which causes cell adhesion and virulence to the human host (vankadari & wilce, ; wu et al., ) . the virus s-protein entry is mediated by ace , and results in an inflammatory cascade initiation by the innate immune system of the host (astuti & ysrafil, ) . so, targeting s-protein can provide an immunogenic response in the human host, and has been chosen for designing a multi-epitopes vaccine candidate against the sars-cov- . the structural pattern of sars-cov- protein can be recognized by the transmembrane toll-like receptor (tlr ) which induces inflammatory cytokines or chemokines reaction. the tlr protein plays a vital role in the host pathogenesis. moreover, the involvement of the receptors has also been reported in various immune protective responses by the host. immune responses are a crucial step to the pathophysiology of the sars-cov- virus-related disease, and initiation of immune response targeting tlr can trigger the anti-viral host defense mechanisms necessary for the elimination of the covid- related infection (astuti & ysrafil, ) . vaccine is an immune-modulatory preparation that triggers a specific immune response against a foreign particle within the host body. a vaccine is now the primary demand to save millions of people from the covid- pandemic. the current world situation is releasing the necessity of an implausible and effectiveness of different anti-viral drugs or vaccine candidates against the sars-cov- . however, no effective drug or vaccine candidates have been developed that can fight against the sars-cov- (elfiky, ) . therefore, a multi-epitope vaccine consisting of potential tand b-cell epitopes can be an ideal approach for the prevention of covid- (astuti & ysrafil, ) . the vaccine can produce both cellular and humoral immune responses against specific pathogens without producing any immune complications. besides, it is very easy to control, cause the effectiveness of the vaccine to be regulated by choosing the specific and desired allelic interactions, which provide robust and diverse immune response over a large group of people (elfiky, ) . in multi-epitope vaccine, the biohazard risk is lower as compared to other types of immunizations. in this research, a multi-epitope vaccine has been constructed using the immunoinformatics approach. epitopes used for the vaccine construction were non-toxic, non-allergenic, highly immunogenic and antigenic. a sufficient number of linkers were used to combine those selected epitopes resulting in busting the immunogenic activity of sars-cov- vaccine (gaafar et al., ; li et al., ) . a flow chart representing the overall procedure from the antigen selection to vaccine construction and evaluation is illustrated in figure . for antigen selection, we collected available sars-cov- proteomes from the vipr (https://www.viprbrc.org/) database (pickett et al., ) . the outer membrane of the sars-cov- is formed by the spike glycoproteins. with the help of these glycoproteins, they adhere to the human host and enter into the host immune system . due to the direct involvement of glycoproteins in pathogenesis, we considered the spike glycoprotein of the sars-cov- for multiepitope vaccine design. initially, we isolated all the spike glycoprotein, and the selected protein sequences of the virus were downloaded in fasta format. the protective antigens of the surface glycoprotein were checked by vaxijen v . (http://www.ddg-pharmfac.net/vaxijen/) server (doytchinova & flower, ) and antigenpro (http://scratch.proteomics. ics.uci.edu/) server with a threshold value . was set for both of them (magnan et al., ) . finally, we selected the spike glycoprotein with the highest antigenic score for further investigations. cytotoxic t-lymphocytes (ctls) represent one of several types of cells of the immune system that have the capacity to kill other infectious cells directly . they go right away inside the virus-cell and play an important role in the host defense mechanism. for the prediction of ctls epitope, the sequence of the selected protein was submitted into the netctl v . server available at http://www.cbs.dtu. dk/services/netctl/ (larsen et al., ) . the predicted epitopes were further assessed through the vaxijen v . (doytchinova & flower, ) , mhc class i immunogenicity (http://tools.iedb.org/immunogenicity/) (calis et al., ) , toxinpred (http://crdd.osdd.net/raghava/toxinpred/) , and allertop v . (https://ddg-pharmfac.net/ allertop/) (dimitrov et al., ) servers. the default parameters of those servers were used for all the predictions. helper t-cells (htls) are an integral part of adaptive immunity that recognizes foreign antigens and activates b and cytotoxic t-cells resulting in destruction of the infectious pathogen . to determine the htl epitopes, we used the iedb's mhc class ii binding allele prediction tool, available at http://tools.iedb.org/mhcii/. the htl epitopes were selected based on a percentile rank of % using the consensus method (wang et al., ) . the predicted epitopes were further evaluated based on their antigenicity and cytokine, i.e. interferon-c (ifnc), interleukin- (il ) and interleukin- (il ) inducing abilities. the antigenicity was anticipated with the vaxijen v . server while ifnc, il , and il features were predicted using ifnepitope (http://crdd. osdd.net/raghava/ifnepitope/) , il pred (http://crdd.osdd.net/raghava/il pred/) and il pred (http://crdd.osdd.net/raghava/il- pred/) (nagpal et al., ) servers, respectively, with default parameters. b-cell epitopes are essential to induce humoral or antibodymediated immunity. b-cells consist of groups of amino acids that interact with the secreted antibodies and activate the immune system to destroy the pathogens (nain et al., ) . therefore, we predicted the linear b-lymphocyte (lbl) epitopes using the ibce-el server, available at http://www.thegleelab.org/ibce-el/ with default parameters (manavalan et al., ) . the predicted lbl epitopes were also evaluated using the vaxijen v . , toxinpred, and allertop v . servers. in computational vaccine design, the population coverage directly indicates the worldwide effectiveness of the vaccine by evaluating the prevalence of hla (human leukocyte antigen) alleles related to the epitope of interest. therefore, the population coverage was calculated using the t-cell epitopes with their respective hla binding alleles. to accomplish this, selected epitopes along their allelic information was submitted to the iedb population coverage tool (bui et al., ) . for modeling, the selected epitopes of ctl and htl were submitted into pep-fold v . (https://bioserv.rpbs.univ-parisdiderot.fr/services/pep-fold /) server. the sopep sorting scheme with simulations was selected for the operation (latysheva & babu, ) . by analyzing the epitope-wise hla binding alleles, allele hla-b à : and hla-c à : were considered for selected ctl epitopes, while drb à : and drb à : were selected for htl epitopes. the crystal structures of the hla alleles were retrieved from the protein data bank (pdb) (https://www.rcsb.org/) (berman et al., ) followed by processing with biovia discovery studio . for molecular docking, a grid-box around the active site of each hla allele was defined by the autodock tool. finally, molecular docking was performed between the epitopes and respective hla alleles using the autodock vina script (trott & olson, ) . the respective co-crystal ligands were used as the positive control to compare the epitope binding efficiency. the docked complex was visualized in biovia discovery studio . the vaccine construct was designed by using the selected ctl, htl, and lbl epitopes as well as a suitable adjuvant that was linked by the appropriate linkers (dorosti et al., ; nain et al., ) . here, we used tlr agonist as the adjuvant since tlr was recognized by viral glycoproteins, and the adjuvant is required for optimal translation and maximal rate of synthesis of the target vaccine candidate (olejnik et al., ; pandey et al., ) . therefore, s ribosomal protein l /l (ncbi id: p whe ) was considered as the adjuvant to improve the immunogenicity of the vaccine candidate. the adjuvant was linked to the vaccine front with a bi-functional linker eaaak that has the ability of several lengths of helixforming peptides to separate two weakly interacting b domains. in contrast, the selected ctl was linked with the help of ala-ala-tyr (aay) linkers, the htl was linked with gly-pro-gly-pro-gly (gpgpg) linkers and the lbl was linked with lys-lys (kk) linker (dorosti et al., ; nain et al., ) . the aay linker is a type of cleavage site of proteasomes that was used to influence protein stability, reduce less immunogenicity and enhance epitope presentation (abdellrazeq et al., ; borthwick et al., ) . the gpgpg, known as the glycine-proline linker, prevents the formation of 'junctional epitopes' and facilitates the immune processing, where the bi-lysine kk linker helps to preserve their independent immunogenic activities of the vaccine construct. the physiochemistry indicates the basic properties of a protein. the physicochemical features of the vaccine were anticipated using the protparam server available at https://web. expasy.org/protparam/ to understand the fundamental nature of the vaccine (gasteiger et al., ) . we also evaluated the immunological properties through vaxijen v . (doytchinova & flower, ) , mhc-i immunogenicity (calis et al., ) , allertop (dimitrov et al., ) , and solpro (magnan et al., ) servers. the two-dimensional ( d) structural features such as alphahelix, beta-turn, and random coils of the construct were identified by sopma (self-optimized prediction method with alignment) server at https://npsa-prabi.ibcp.fr/npsa/npsa_ seccons.html (geourjon & del eage, ) and psipred v . (psi-blast based secondary structure prediction) server at http://bioinf.cs.ucl.ac.uk/psipred/ (buchan et al., ) with default parameters. sopma has more than % prediction accuracy (geourjon & del eage, ) . the d structural features were retrieved and evaluated to understand the composition quality of the vaccine. . . homology modeling, d structure refinement and validation the constructed vaccine was submitted into i-tasser (iterative threading assembly refinement) online web portal (https://zhanglab.ccmb.med.umich.edu/i-tasser/) for threedimensional ( d) structure prediction (roy et al., ) . the i-tasser web produces the structure of the protein and its functions most accurately using a state-of-the-art algorithm in the form of a d structure (roy et al., ) . this web server can predict and determine the c-score, tm-score value, rmsd and top five models of the given protein sequence. the produced d structure was downloaded into the pdb format, which was chosen based on the c-score value. the server contains a c-score ranging from - to , where a higher value indicates a protein model with high confidence. the identified d structure was submitted into the galaxyrefine (http://galaxy.seoklab.org/refine) online web-based server for the refinement of the vaccine structure. this webserver was run by the casp refine technique (nugent et al., ) . the galaxyrefine website provides the rmsd, energy score and overall quality score. the refined structure was downloaded, and the selected structure was identified depending on the energy score of the lowest and highest rmsd value. the refined and identified structure was visualized using the pymol v . . software (delano, ) . the resulted d structure was evaluated depending on the ramachandran plot score (vaccine structure validity) and z-score value that determine the standard deviations from the mean value (z-score within the known native protein range indicating the good quality of the prepared model). the ramachandran plot was analyzed by the rampage server (http://mordred.bioc.cam.ac. uk/$rapper/rampage.php), which runs considering allowed and disallowed regions of amino acid (lovell et al., ; ramachandran et al., ) ; and z-score plot was analyzed by the prosa-web (https://prosa.services.came.sbg.ac.at/prosa. php) tool (wiederstein & sippl, ) . molecular docking studies can reveal the binding interactions between modeled protein and receptor molecules. for this purpose, we submitted the refined vaccine model as ligand and tlr protein as immunological receptor into the cluspro v . server, available at https://cluspro.bu.edu/, for molecular docking (kozakov et al., ) . the tlr receptor (pdb id: g a) was selected and downloaded from the pdb server (sussman et al., ) . initially, the receptor was prepared by separating the attached ligand from the protein, followed by the removal of waters and other chemicals. all these processes were performed in pymol v . . software (delano, ) . binding interactions and residues involved in the interacting plane were analyzed with discovery studio . for molecular dynamics (md) simulation, we used both software and server-based tools to evaluate the dynamics and stability of the vaccine-receptor complex critically. the stability of the docked complex was evaluated by a highly intuitive and accurate molecular dynamic simulation tool yasara, where parameters for macromolecules to facilitate simulations were generated using the amber force field . we evaluated the stability, fluctuation and compactness of the vaccine-receptor complex in terms of root mean square deviation (rmsd), root mean square fluctuation (rmsf), and radius of gyration (r g ) values, respectively. also, the complex was submitted to the imods server, available at http://imods.chaconlab.org/ (l opez- blanco et al., ) . based on the normal mode analysis (nma), this server provides eigenvalues, deformability, b-factors, and elastic network model to clarify the aggregate protein movement in the inside directions. to evaluate the possible immune response of the vaccine, the whole construct was submitted into c-immsim v . server available at http://www.cbs.dtu.dk/services/c-immsim- . /, and the generated responses were retrieved for detailed observation (rapin et al., ) . in this case, we considered a minimum interval period of days between two doses, as described earlier (castiglione et al., ) . in silico administration of three injections were given with time steps of , , and , respectively, where one-time step is equal to h in real life. the maximum value for simulation steps was set to , while the rest of the stimulation parameters were kept default. for the expression of a foreign gene in a host organism, codon optimization is necessary according to the specific host organism (grote et al., ) . therefore, the construct was submitted into the jcat server (http:/jcat.de/) for the codon adaptation. herein, we considered widely used e. coli k as the host, and the whole operation is carried out by avoiding the following three criteria: ( ) restriction enzymes cleavage sites, ( ) binding sites of the prokaryotic ribosome and ( ) rho-independent termination of transcription. the adapted sequence was evaluated based on the codon adaptation index (cai) value and guanine-cytosine (gc) content (grote et al., ) . finally, the adapted nucleotide sequence was used for in silico cloning into the pet a (þ) expression vector. the whole in silico cloning operation was executed in snapgene v . software (goldberg et al., ) . we found s-proteins from all the retrieved sars-cov- proteomes. based on the antigenicity, we selected a spike protein with an antigenic score of . (vaxijen) and . (antigenpro), which was the highest among all tested proteins. the length of the selected s-protein was amino acids long while the genbank accession was qic . the primary sequence of the selected protein was used for further analysis. a total of ctl epitopes, each with a length of amino acids, were predicted from the selected spike protein. assessment revealed that twenty-nine ctl epitopes were antigenic, immunogenic, non-toxic and non-allergenic (supplementary table s ). due to the high number of potential epitopes, we selected the top four ctl epitopes for the final vaccine construction based on the antigenicity score (table ) . a total of htl epitopes, each with a length of amino acids, were identified initially using the iedb server. among them, only htl epitopes were able to induce the evaluated three types of cytokines, such as ifnc, il , and il (supplementary table s ). likewise, we considered the top four htl epitopes for incorporating into the final vaccine construct based on the antigenic score (table ) . preliminary analysis revealed a total of lbl epitopes, each with a length of amino acids. later with further evaluation, epitopes were found as antigenic, non-toxic and non-allergenic (supplementary table s ). out of lbl epitopes, we selected the top four lbl epitopes for vaccine design purposes based on the antigenicity score (table ) . the selected ctl and htl epitopes, along with their binding alleles, were used to evaluate the population coverage, as shown in figure . both ctl and htl epitopes provided a high percentage ( . %) of population coverage across the world. the selected epitopes showed interactions with a high number of hla alleles from different countries such as the united states ( . %), north america ( . %), south korea ( . %), south asia ( . %) and india ( . %). this result suggests that the vaccine designed with these epitopes could be effective on most of the population in the world (figure and supplementary table s ). we used the docking method to validate the efficacy of selected epitopes in binding their respective hla alleles. the epitopes, along with their respective docking allele, binding affinities, interactions and residues involved in the hydrogen bonds, are described in table . the binding affinities of ctl epitopes were between - . and - . kcal/mol, while for htl epitopes, it was between - . and - . kcal/mol. the binding affinities were either very close or even higher than that of the positive control (table ). in addition to the tabulated details, we presented the best interacting ctl (vvflhvtyv) and htl (qyikwpwyiwlgfia) epitopes in figure . herein, the best ctl epitope produced a total of hydrogen bonds, in which were classical interactions involved with the active site residue lys , tyr , lys , val , thr , tyr , val , lys , asn , and thr . on the other hand, the best htl epitope showed nine hydrogen bonds, including six classical interactions while it interacted with ser , glu , asn , his , trp , ala , phe , tyr , ile , and ile residues. the vaccine construct was formulated using the previously selected epitopes belonging to three different classes ( ctl, htl, and lbl). the epitopes were added together with aay, gpgpg and kk linkers, respectively, as shown in figure . an adjuvant was added ahead of the construct to improve the immunogenicity. the tlr agonist s ribosomal protein l /l was linked to the first ctl epitope as an adjuvant by using eaaak linker. the final vaccine construct was amino acids long (figure ). the physicochemical properties of the vaccine construct were assessed as shown in table . the molecular weight of the construct was found to be , . da. at the same time, ifnc stands for interferon-gamma while il- and il- indicate the interleukin- and interleukin- , respectively. other properties such as theoretical isoelectric point (pi) was . , chemical formula was c h n o s , instability index was . , the aliphatic index was . , and grand average of hydropathicity was . . in addition, physicochemical features and the immunological potency of the construct were evaluated. for instance, the antigenicity of the construct was . , while immunogenicity was . . furthermore, the vaccine was non-allergenic and soluble, with a score of . out of (table ). the secondary structural features include a-helix, b-strand and random coils that were evaluated using two different servers. the sopma server predicted . % a-helix, . % b-strand and . % random coils in the construct (table , supplementary figure s ). on the other hand, the psipred server anticipated the features as . % a-helix, . % b-strand, and . % random coils (table , supplementary figure s ). in homology modeling, the i-tasser server used dd (pdb id) as the best template to generate the top five models. among the five models, we considered the model with the lowest c-score (- . ) as recommended by the server (supplementary figure s ) . a structural representation of the designed vaccine is provided in figure . after refinement, the vaccine (model ) showed . % residues in the favorable region in the ramachandran plot, with gdt-ha score . , rmsd value . , molprobity . , clash score . and poor rotamers score . (supplementary table s ). the refined d vaccine model was further validated with the rampage and prosa-web servers. before refinement, the ramachandran plot of the vaccine showed . % residues in the favorable region and . % in allowed regions, while . graphical map of the formulated multi-epitope vaccine construct. the vaccine constructs included (left to right) an adjuvant, ctl, htl and lbl epitopes are shown in the dark blue, red, olive green and green rectangular boxes. herein, the adjuvant and the first ctl epitope were linked by eaaak linker (blue), ctl epitopes were added together by ayy linkers (off-white), htl epitopes by gpgpg linkers (orange), and lbl epitopes by kk linkers (black). . % residues in disallowed regions (supplementary figure s ) . the ramachandran plot of the refined vaccine model showed . % residues in the favorable region and . % in allowed regions, while . % residues in disallowed regions (figure (a) ). likewise, the crude model showed a z-score value of - . , while the refined model provided a value of - . (figure (b), supplementary figure s ). the docking between the vaccine (ligand) and tlr (receptor) was performed to anticipate their binding affinity and interactions. in doing so, the cluspro v . server provided docked complexes with different poses. among them, we selected the complex with the least energy score and binding pose with functional interactions (supplementary table table s ). thus, model fulfilled the inclined criteria. hence, it was picked as the best vaccine-tlr complex, which had an energy score of - . ( figure ) . the selected complex was analyzed for binding interactions and involved in active site residues. the number of hydrogen and hydrophobic bonds present in the interaction plane was and , respectively. among the hydrogen bonds, were classical hydrogen bonds (chb). the interacting residues in the chb from the vaccine were ala , arg , asn , gln , gln , glu , glu , lys , met , pro , pro , thr , thr , thr , thr , leu , lys and ser . moreover, associated tlr active site residues were gln , asn , gln , lys , lys , gln , ser , asn , cys , asn , lys , gln , his , asn , glu , leu , thr and trp (figure ) . other hydrogen bond interactions were as follows: four were electrostatic salt bridges, two were carbon-hydrogen bonds and a single pi-donor hydrogen bond. we executed software-based md simulation where trajectories from ns long simulation showed structural stabilization around . ns and light fluctuation afterward ( figure (a) ). the calculated average rmsd value was . Å, while the average rmsf score was . Å. the fluctuation was higher in the vaccine part from aa to aa ( figure (b) ). furthermore, the average simulation energy was - , , . kj/mol. the average r g score was . that fluctuates between . and . (figure (c) ). the relatively higher pick at to amino acid residues was due to the flexible regions of the docked complex, which was the vaccine part. the md simulation was also carried out in the imods server, where nma assessment was applied to the internal coordinates of the complex. the deformability builds up the independent distortion of each residue portrayed by the method of chain hinges (figure (d) ). the eigenvalue determined for the complex was found to be . e- (figure (e) ). the variance of each typical mode was gradually decreased (figure (f)). all these results suggest stable binding interactions with compact conformation and minor fluctuations in the vaccine-tlr complex. the simulated immune response showed similar to actual immunological phenomena provoked by specific pathogens as shown in figure . for instance, secondary and tertiary immune responses were higher than the primary immune response (figure (a) ). secondary and tertiary responses showed higher levels of antibodies (i.e. igg þ igg , igm, and igg þ igm), which coincided with an antigen extenuation indicating the development of memory cells, thus, intensified antigen clearance upon successive exposures (figure (a) ). additionally, a prolonged period of viability in b-cells, cytotoxic t-cells and helper t-cells were noticed, indicating the class switching between immune cells and igm memory formation (figure (b-d) ). the elevated levels of ifnc, il- and il- were additionally apparent (figure (e)). the percentage (%) and amount (cells/mm ) of th type immune reaction were lower than the th type reaction (figure (f)). during the presentation, expanded macrophage movement was illustrated, while dendritic cell movement was predictable ( figure (g,h) ). we optimized the codons present in the vaccine construct according to the e. coli k in the jcat server to increase their translation efficiency. the peptide vaccine construct ( aa residues) produced lengths of nucleotide sequences. moreover, the adapted nucleotide sequence has gc content and cai value of . % and . , respectively. to insert the adapted sequence into the pet a (þ) vector, we selected xhoi and bamhi restriction sites as the start and end cut points, respectively. thus, the optimized vaccine construct was cloned into the pet a (þ) cloning vector with the snapgene software ( figure ). the final size of the cloning vector was nucleotide base pairs (bp). the present demonic appearance of covid- generates a life-threatening situation to the global public health (vankadari & wilce, ) , which influences us to design this multi-epitope vaccine applying immunoinformatics approach. the glycoprotein-based vaccine demonstrated an extraordinary significance ordained by immunoinformatics and revealed our attempt trustworthy. a vaccine is a safe and effective way to protect against infectious diseases (li et al., ) . it should have the ability to provide acquired immunity against contagious diseases (bol et al., ) . in this study, we designed an epitope-based vaccine that could provide a strong immune response against sars-cov- , thereby, preventing the covid- pandemic. a vaccine can prevent future outbreaks (melief et al., ) . however, in the absence of an effective vaccine, control and prevention of covid- infection and transmission are very difficult. besides, effective vaccination is yet to be developed in controlling the current situation. thus, a new strategy of vaccine development is a prime need that will contribute to finding a solution to solve this present life-threatening public health issue. since s-protein of sars-cov- plays a major role in immune invasion as well as the human to human transmission, our purpose was to design an epitope vaccine by targeting the s-protein. the location of the antigenic region of figure . molecular docking between the vaccine and the tlr receptor. the interacting residues from the vaccine were ala , arg , asn , gln , gln , glu , glu , lys , met , pro , pro , thr , thr , thr , thr , leu , lys , and ser while associated tlr active site residues were gln , asn , gln , lys , lys , gln , ser , asn , cys , asn , lys , gln , his , asn , glu , leu , thr , and trp . the surface of s-protein was evaluated so that this protein can be recognized by cellular and humoral immune systems. first, all potential ctl, htl and lbl epitopes were identified and evaluated. then, the vaccine was designed with the top four antigenic ctl, htl and lbl epitopes with their desired linkers. they were incorporated in vaccine construction as a part of the essential element that enhances the stability, folding and expression patterns of our vaccine candidate (shamriz et al., ) . the adjuvant was attached to the ctl epitope by eaaak linker, which helps to induce high levels of both cellular and immunogenic humoral responses for particular antigens, and amplify the vaccine's stability and longevity (bonam et al., ; lee & nguyen, ) . finally, the vaccine construction was found to accumulate amino acid residues long. solubility, a type of physicochemical property of a vaccine candidate, is counted as a vital characteristic of any recombinant vaccine (khatoon et al., ) . hence, the solubility of the vaccine construct was predicted by using a solubility assessing tool to determine the quality of being solvability of the construct inside the host e. coli, and the vaccine construct was found to be solvable inside the host e. coli. the nature of the vaccine determined by theoretical pi value was found to be acidic. instability index suggested by server tools indicate that the protein would remain stable after synthesis. in contrast, the gravy value and aliphatic index portrayed the vaccine to be the hydrophilic and thermostable, respectively. a favorable physicochemical property predicted for the vaccine and all the scores on different parameters relies on a high possibility to confer this vaccine as a valid candidate against sars-cov- . in our approach, we found the best population coverage all-over the world ( . %) for the combined results that make this vaccine construct a good candidate and significant weapon. the most cases of infection and mortality were found in the united states and the united kingdom, and we find the parentage of population coverage for those countries in a significant level (united states ( . %), europe ( . %)). after the d structure prediction (based on cscore), the identified models were refined and selected the best model (based on the lowest energy score). in the validation test of d structure, we found a good number of zscore (- . ) and the superior features of most favored, accepted and disallowed regions for the ramachandran plot. molecular docking between the peptide vaccine and virus glycoprotein binding favorable receptor of tlr with lowest energy score of À . confirmed the possibility of infection inhibitory activity of the vaccine and suggested a possible tight interaction between the modeled vaccine as a ligand and the tlr receptor surface. molecular dynamics simulation is a potential technique for accessing the physical ground of the protein structure and function of biological macromolecules. protein dynamic simulations can provide certain information regarding individual atomic movement as a function of time. for the dynamics evaluation of the vaccine candidate, ns dynamic simulations have been performed, and results have been analyzed based on the rmsd and rmsf score. rmsd value is used to compare different atomic conformations of a given molecular system. in this study, the rmsd value was used to determine the significant flexibility and departure of vaccine candidates from the receptor structure, where the rmsf of the complex structure was determined to measure the displacement of our particular vaccine candidate's atom relative to the receptor structure. the calculated average rmsd and rmsf value was . Å and . Å, respectively. the fluctuation was found higher in the vaccine part, but it smoothly became stable after ps suggesting possible stability of the modeled vaccine and the receptor. finally, we performed an immune simulation to observe the optimal behavior and cell density parameters for successful target clearance and find the best immunological response against the pathogen. the vaccine doses were upgraded immunological reaction causing memory b-cell (having a half-life of several months) and t-cell. sustained generation of ifn-c and il- were seen after immunization because of expanded aide t-cell initiation. in this manner, the vaccine effectively simulated a humoral immunological response to increasing immunoglobulin creation. the md simulation was performed to evaluate the stability of the vaccine candidate with the receptor, where codon optimization was performed to stabilize the construct vaccine within the host for optimum multi-epitope vaccine production. finally, the codon was optimized, and desired vaccine candidate in silico cloning was performed successfully into the pet a (þ) cloning vector of e. coli k expression host. in this study, a series of computational approaches led to the discovery of potential t-and b-cell epitopes in s-protein of sars-cov- that eventually embroidered into a multi-epitope vaccine. the newly designed vaccine has desired immunodominant properties with high population coverage. importantly, it was able to bind with the immune receptor tlr strongly as well as to elicit robust immune response upon sars-cov- infection. based on our findings, we believe that the vaccine candidate can be an important starting point for developing a potent vaccine against the etiological agent of covid- outbreak. moreover, the potential epitopes identified in this study can be used in future studies as well. however, further experimental assessments are required to confirm our formulated vaccine as an effective prophylactic against sars-cov- . in silico study the inhibition of angiotensin converting enzyme receptor of covid- by ammoides verticillata components harvested from the in silico cloning of the designed vaccine into the pet- a (þ) vector. herein, purple color represents the vector dna, while the red color indicates the adapted dna sequence of the designed vaccine. western algeria simultaneous cognate epitope recognition by bovine cd and cd t cells is essential for primary expansion of antigen-specific cytotoxic t-cells following ex vivo stimulation with a candidate mycobacterium avium subsp. paratuberculosis peptide vaccine contemporary strategies and current trends in designing antiviral drugs against dengue fever via targeting hostbased approaches severe acute respiratory syndrome coronavirus (sars-cov- ): an overview of viral structure and host response the protein data bank prophylactic vaccines are potent activators of monocytederived dendritic cells and drive effective anti-tumor responses in melanoma patients at the cost of toxicity an overview of novel adjuvants designed for improving vaccine efficacy novel coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment novel nested peptide epitopes recognized by cd þ t cells induced by hiv- conserved-region vaccines. vaccines scalable web services for the psipred protein analysis workbench predicting population coverage of t-cell epitope-based diagnostics and vaccines properties of mhc class i presented peptides that enhance immunogenicity statement in support of the scientists, public health professionals, and medical professionals of china combatting covid- how the interval between prime and boost injection affects the immune response in a computational model of the immune system a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster an investigation into the identification of potential inhibitors of sars-cov- main protease using molecular docking study pymol: an open-source molecular graphics tool prediction of il inducing peptides designing of interferongamma inducing mhc class-ii binders allertop -a server for in silico prediction of allergens vaccinomics approach for developing multi-epitope peptide pneumococcal vaccine vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines sars-cov- rna dependent rna polymerase (rdrp) targeting: an in silico perspective novel guanosine derivatives against mers cov polymerase: an in silico perspective reverse vaccinology approach to design a novel multi-epitope vaccine candidate against covid- : an in silico study immunoinformatics approach for multiepitope vaccine prediction from h, m, f, and n proteins of peste des petits ruminants virus protein identification and analysis tools on the expasy server sopma: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments salmonella persist in activated macrophages in t cell-sparse granulomas but are contained by surrounding cxcr ligand-positioned th cells jcat: a novel tool to adapt codon usage of a target gene to its potential expression host in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel in silico approach for predicting toxicity of peptides and proteins a review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme- and furin covid- : what is next for public health? the lancet clinical features of patients infected with novel coronavirus in wuhan. the lancet discovery of potential multi-target-directed ligands by targeting host-specific sars-cov- structurally conserved main protease targeting sars-cov- : a systematic drug repurposing approach to identify promising inhibitors against c-like proteinase and -o-ribose methyltransferase exploring leishmania secretory proteins to design b and t cell multi-epitope subunit vaccine using immunoinformatics approach the cluspro web server for protein-protein docking understanding the binding affinity of noscapines with protease of sars-cov- for covid- using md simulations at different temperatures large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction discovering and understanding oncogenic gene fusions through data intensive computational approaches recent advances of vaccine adjuvants for infectious diseases therapeutic options for the novel coronavirus ( -ncov) peptide vaccine: progress and challenges. vaccines imods: internal coordinates normal mode analysis server structure validation by calpha geometry: phi, psi and cbeta deviation solpro: accurate sequencebased prediction of protein solubility high-throughput prediction of protein antigenicity using protein microarray data ibce-el: a new ensemble learning framework for improved linear bcell epitope prediction therapeutic cancer vaccines computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov- protease against covid- computer-aided designing of immunosuppressive peptides based on il- inducing potential proteome-wide screening for designing a multi-epitope vaccine against emerging pathogen elizabethkingia anophelis using immunoinformatic approaches structural basis and designing of peptide vaccine using pe-pgrs family protein of mycobacterium ulcerans -an integrated vaccinomics approach evaluation of predictions in the casp model refinement category toll-like receptor in acute viral infection: too much of a good thing novel immunoinformatics approaches to design multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein transmission routes of -ncov and controls in dental practice vipr: an open bioinformatics database and analysis resource for virology research stereochemistry of polypeptide chain configurations computational immunology meets bioinformatics: the use of prediction tools for molecular binding in the simulation of the immune system i-tasser: a unified platform for automated protein structure and function prediction in-silico homology assisted identification of inhibitor of rna binding against -ncov n-protein (n terminal domain) effect of linker length and residues on the structure and stability of a fusion protein with malaria vaccine application the outbreak of sars-cov- pneumonia calls for viral vaccines diagnosis, treatment, and prevention of novel coronavirus infection in children: experts' consensus statement an in-silico evaluation of different saikosaponins for their potency against sars-cov- using nsp and fusion spike glycoprotein as targets protein data bank (pdb): database of three-dimensional structural information of biological macromolecules autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading identification of new anti-ncov drug chemical compounds from indian spices exploiting sars-cov- main protease as target emerging wuhan (covid- ) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd . emerging microbes & infections stilbene-based natural compounds as promising drug candidates against covid- a novel coronavirus outbreak of global health concern clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan peptide binding predictions for hla dr, dp and dq molecules prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins cryo-em structure of the -ncov spike in the prefusion conformation a new coronavirus associated with human respiratory disease in china evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission pathological findings of covid- associated with acute respiratory distress syndrome. the lancet a novel coronavirus from patients with pneumonia in china we extend our gratitude towards the deanship of scientific research (dsr) at king abdulaziz university and biological solution centre (biosol centre) for providing technical support. special thanks go to monokesh kumer sen, school of medicine, western sydney university, australia for his contribution in revising the whole manuscript. the authors declare no conflict of interest. key: cord- - dw xby authors: kathwate, gunderao h title: in silico design and characterization of multi-epitopes vaccine for sars-cov from its spike proteins date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: dw xby covid is disease caused by novel corona virus, sars-cov originated in china most probably of bat origin. till date, no specific vaccine or drug has been discovered to tackle the infections caused by sars-cov . in response to this pandemic, we utilized bioinformatics knowledge to develop efficient vaccine candidate against sars-cov . designed vaccine was rich in effective bcr and tcr epitopes screened from the sequence of s-protein of sars-cov . predicted bcr and tcr epitopes were antigenic in nature non-toxic and probably non-allergen. modelled and refined tertiary structure was predicted as valid for further use. protein-protein interaction prediction of tlr / and designed vaccine indicates promising binding. designed multiepitope vaccine has induced cell mediated and humoral immunity along with increased interferon gamma response. macrophages and dendritic cells were also found increased over the vaccine exposure. in silico codon optimization and cloning in expression vector indicates that vaccine can be efficiently expressed in e. coli. in conclusion, predicted vaccine is a good antigen, probable no allergen and has potential to induce cellular and humoral immunity. in december group of patients from wuhan city of china was found to have pneumonia like symptoms and diagnosed with the infection of beta coronavirus ( ) . later it spread across the china and now spread all over the globe. these infections were named as covid disease and the virus as sars-cov by who ( ) . on january due to its spread more than countries declared covid pandemic as a public health emergency of international concern. novelty of sars-cov is its rapid spread may be due asymptomatic patients ( , ) and highly sophisticated, time consuming diagnostic methods( - ). as of may , million confirmed cases with more than , deaths worldwide ( ) . in india due to lockdown positive cases are in control and spread is also marginal, but still after approximately four months' case reports are increases. as of may , a total of confirmed cases with deaths are reported by ministry of health and family welfare, govt. of india. till date, no antiviral drugs available to combat the sars-cov infections. there are few drug candidates in clinical trials but the process is time consuming ( ) . in south korea, lopinavir/ritonavir combination was found significantly effective in lowering of viral load to no detectible or little sar-cov titer( ). but in another group study same combination was totally ineffective beyond standard care ( ) . another drug pair drug, hydroxychloroquine, an antimalarial drug and azithromycin reported to found effectively associated with reduction of viral load ( , ) . but qt interval prolongation that may cause life threating arrhythmia ( ) and in large scale study both drugs and drug alone compared with neither drugs was not associated with mortality ( , ) . in a drug repurposing study, remdesivir, lopinavir, emetine, and homoharringtonine have significantly inhibited replication of sar-cov ( ) . but this was in vitro study, clinical trial reports are underway. similar determined attempts are going through the globe. to break the chain of infection prevention is much better option than treatments. several evidences suggest and support the importance of vaccine to eliminate covid epidemic ( ) ( ) ( ) . various epidemiological surveys provide the evidences that naturally acquired immunity can eliminate the sars-cov titer ( , ) . more than % patients develop mild and % develop severe symptoms of covid with zero case fatality rate ( ) . presently there is no vaccine available against covid disease. efforts are being taken for the development of effective vaccine all over the globe ( ) ( ) ( ) ( ) . all these vaccines are under development ( ) and may require at least to . year to hit the market ( ) . bioinformatics approach can accelerate the process by predicting potential candidate peptides. vaccines against mers, ebola, and human papilloma virus were designed and successfully developed by using bioinformatics approaches ( ) . sars-cov belong to beta coronoviridae family which includes four endemic viz hcov-hku , hcov-oc , hcov- e, hcov-nl , two epidemic viruses like middle east respiratory syndrome virus (mers), and sars ( ) . this is a non-segmented positive strand rna virus with envelop. envelop proteins are categorized into structural, non-structural and accessory proteins. structural proteins are involved in protection and bind to the host. several proteins of sar-cov are important for virulence and pathogenesis. for example nucleocapsid (n) protein n, is essential for rna binding and its replication and transcription ( ) . envelope (e) and membrane (m) proteins and instrumental in virus assembly and virulence promotion ( , ) . e and m proteins are effective in induction of immune response ( ) . spike (s), a structural protein is responsible for binding to receptor on host cell, angiotensin converting enzyme ( ) . thereby proteolytic cleavage by tmprss allows subsequent entry into cell through endocytosis ( ) . s protein also found to be involved in activation of t cell response ( , ) . entire s protein expressed in chimpanzee adeno (chad)-vector showed protection against sars-cov in mice and rhesus macaques ( ) . single dose of chadox ncov- vaccine is sufficient to elicit humoral and cell mediated immune response in both animals. viral load was found reduced compared to control animals and symptoms of pneumonia were absent. here we designed a multi-epitopes vaccine from s protein. this vaccine has all the ideal properties like stability at room temperature, immunogenic, antigenic and non-allergen. all the epitopes are good in stimulation of humeral as well and cell mediated immunity. complete spike protein sequence (p dtc ) of sars-cov was downloaded in fasta format from uniprot protein database. this sequence was used for further analysis and obtaining potential epitopes for b and t cell receptors. there are various tools used to predict the epitopes that could be presented to t cell receptors. potential epitopes of various bacteria and viruses are predicted by using such online epitopes predicting tools. following tools were used for prediction of tcr (t cell receptor) epitopes iedb mhc-i processing predictions, mhc-np, netctlpan . , rankpep, and netmhcpan . . all these tools were used to screen the potential peptides as epitopes for tcr. these tools have various methods to predict the epitopes. iedb (instructor/evaluator database) is a web-based epitope analysis resource includes tools for t cell epitope prediction, b cell epitope prediction and other analysis tools like epitope conservancy etc. this resource use methods based on artificial neural network (ann), stabilized matrix method (smm), and combinatorial peptide libraries(comblib), predicts the peptides way that processed naturally and presented by mhc i ( ) . immunogenicity, toxicity, allergen, conservancy, and antigenicity were analyzed for predicted tcr epitopes. iedb mhc class i immunogenicity server and conservancy tool were used for determination of immunogenicity and conservancy. immunogenicity of a peptide mhc complex (http://tools.immuneepitope.org/immunogenicity/ ) were assessed keeping all the parameters at default. protein sequence variants( ) used setting sequence identity % and other parameter default. toxipred (http://www.imtech.res.in/raghava/toxinpred/index.html ) online tool predicts the toxicity considering physicochemical properties of selected peptides( ). online server allergenfp v. . (http://ddgpharmfac.net/allergenfp/ ) was used to predict peptides as allergens ( ) . antigenicity of epitopes was analyzed by online server vaxijen v . (http://www.ddg-harmfac.net/vaxijen/vaxijen/vaxijen.html ). threshold value was set to . ( ) . this is alignment independent predictor based on auto-cross covariance (acc) transformation epitopes sequences into uniform vectors of principle amino acid properties. accuracy of this server varies in between to % depending on targeted organism. six different method were used for the prediction of b cell receptor (bcr) epitopes. all these methods generate fragments the protein. for the prediction of b cell epitopes, it is necessary to find linear sequence of b cell epitope in the protein sequence. bepipred linear epitope predication server (http://www.cbs.dtu.dk/services/bepipred/ ) uses a hidden markov model and propensity scale method. similarly, other properties are also being consider to predict good b cell epitopes ( ) . those properties are calculated by different methods at iedb server (http://tools.iedb.org/bcell/ )like kolaskar-tongaonkar antigenicity scale provide physiology of the amino acid residues( ), emini surface accessible score for accessible surface of the epitope( ), secondary structure of epitopes also has role in antigenicity. karplus-schulz flexibility score ( ) and chou-fasman β turns methods ( ) were used for flexibility and β turns prediction respectively. parker hydrophilicity prediction method ( ) was used for determination of in silico hydrophilicity of peptides. high scored and common peptides predicted by various tools were selected for deriving sequence of potential vaccine candidate. different epitopes for t cell and b cell receptors were linked together by gpgpg and aay. to enhance the immunogenicity, ompa (genbank: afs . ) protein was chosen as adjuvant and was linked through eaaak at n terminal site of the vaccine. antigenicity of chimeric vaccine was predicted by vaxijen v . (http://www.ddgpharmfac.net/vaxijen/vaxijen/vaxijen.html) and antigenpro. vaxijen . is alignment free antigenicity prediction tool utilizes auto cross covariance transformation of protein sequence into uniform vector vectors of principal amino acid properties ( , ) . antigenpro (http://scratch.proteomics.ics.uci.edu/) is also online tool utilizes protein microarray dataset for the prediction of antigenicity. based on cross validation experiment accuracy of this server is estimated to be around % using combined dataset. allertop v . and allergenfp were two online tools utilized for the prediction of allergenicity of chimeric protein. amino acid edescriptors, auto-and cross-covariance transformation, and the k nearest neighbours (knn) machine learning methods are basis of allertop v . (http://www.ddg-pharmfac.net/allertop) for allergenicity prediction ( ) . allergenfp, is descriptor-based fingerprint, alignment free tool for allergenicity prediction. the tool use four step algorithm. in first step, proteins are described based on their properties like hydrophobicity, size, secondary structures formation and relative abundance. in subsequent step, generated strings are converted into vectors of equal length by acc. then vectors are converted into binary fingerprints and compared in terms of the tanimoto coefficient. applying this approach to known allergen and non-allergens can identify the % of allergen/non-allergen with mathew's correlation coefficient of . ( ) . for solubility prediction of multi-epitopes chimeric vaccine, proso ii server (http://mbiljj .bio.med.uni-muenchen.de: /prosoii/prosoii.seam) was utilized ( ) . proso ii server works on an approach of classifier which utilizes difference between targetdb and pdb and insoluble proteins of targetdb. the accuracy of this server is % at default threshold . . protoparam, online web server was exploited for evaluation of physiochemical properties. the properties like amino acid composition, theoretical pi, instability index, in vitro and in vivo half-life, aliphatic index, molecular weight, and grand average of hydropathicity (gravy) were evaluated. psipred and raptorx property online servers were used to determine secondary structure of predicted vaccine. psipred is publicly available webserver, includes two feed forward neural networks works on output obtained from psi-blast. psipred . attains average q score of . % obtained using very stringent cross approval strategies to assess its performance. raptorx property is another web based server prediction secondary structure of protein without template ( ) . this server utilizes deepcnf (deep convolutional neural fields), a new machine learning model that predicts secondary structure and solvent accessibility and disorder regions simultaneously ( ) . it accomplishes q score of approximately for state secondary structure and approx. % for state solvent accessibility. tertiary structure of multi-epitopes chimeric vaccine candidate was built using i tasser online server (https://zhanglab.ccmb.med.umich.edu/i-tasser/). the i-tasser (iterative threading assembly refinement) web server utilize sequence-to-structure-to-function paradigm to build protein structure ( ) . it is top ranked d protein structure web server in community wide casp experiments ( ) . two web bases servers were used to refine the d structure of multi-epitopes chimeric protein. initially modrefiner server (https://zhanglab.ccmb.med.umich.edu/modrefiner/) and finally galaxyrefine server (http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=refine) was used. modrefiner server is an algorithm for atomic level structure refinement utilizes c alpha trace, main chain or atomic model. output structure is refined in term of accurate position of side chains hydrogen bon network and less atomic overlaps ( ) . on other hand galaxy server rebuild the d structure, performs repacking, and uses molecular dynamic simulations to accomplish overall protein structure relaxation. structure refined by galaxy server of best quality in accordance with community-wide casp experiments ( ) . prosa-web (https://prosa.services.came.sbg.ac.at/prosa.php), the errat server (http://services.mbi.ucla.edu/errat/) and rampage (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) web servers were utilized for d structure validation obtained after galaxy server refinement ( ) . prosa web server calculate the quality score as z score that should fall in a characteristic range ( ) . z score obtained for a specific input protein is in context with protein structure available in public domain. errata server analyses non bonded atom-atom interaction the refined d structure of protein compared to high resolved crystallographic protein structures ( ) . ramachandran plot displays energetically allowed and disallowed dihedral psi and phi angles of amino acids. the plot is calculated based on van der waal radius of the protein side chain. rampage server determines ramachandran plot for a protein that include percent residues in allowed and disallowed regions ( ) . more than % b cell epitopes are discontinuous that is they are present in small segments on linear protein and the brought to proximity while protein folding. discontinues b cell epitopes for designed vaccine were predicted by ellipro online tool (http://tools.immuneepitope.org/tools/ellipro) at iedb. in this tool three algorithms implemented to determine the discontinuous b cell epitopes. d structure of input protein is approximated as number of ellipsoid shapes, calculate protrusion index (pi) and clusters neighboring residues. ellipro defines pi score of each residue based on the center of mass of residue residing outside the largest possible ellipsoid. in consideration with other epitope predicting tools ellipro gave an auc value of . , best of all ( , ) . molecular docking server haddock (https://bianca.science.uu.nl/haddock . /) was used to see the interaction of designed vaccine and tlr . haddock (high ambiguity driven proteinprotein docking) is information derived flexible docking server ( ) . galaxyrefine d model of multi-epitopes chimeric proteins, adjuvant tlr (pdb id: nig) and tlr (pdb id: g a) were uploaded for docking at hddock server keeping all the parameter default. finally, top five models were downloaded and by prodigy (protein binding energy prediction) webserver (https://nestor.science.uu.nl/prodigy/) was utilized for prediction of binding affinities ( ) . in silico cloning of vaccine construct was predicted by c-immsim server (http:// . . . /c-immsim/index.php). c-immsim server is freely available web based server utilizes position specific scoring matrix (pssm) for the prediction of immune epitopes and machine learning techniques for immune interaction ( ) . jcat (java codon adaptation tool) server was utilized for reverse translation and codon optimization. codon optimization is carried out in order to express the construct in e. coli host. jcat (http://www.prodoric.de/jcat) output display sequence of nucleotide, other important properties of sequence that includes codon adaptation index (cai), percent gc content essential for to assess the protein expression in host ( ) . finally, vaccine construct was cloned in pet a (+) plasmid vector by adding xhoi and xbai restriction sites at c and n terminus, respectively. snapgene tool was used to clone the construct to ensure the cloning and expression ( ) . iedb recommend, mhc-np, netctlpan . , rankpep and netmhcpan . server predicted the potential candidate epitopes from the spike glycoprotein sequence of sars-cov . epitopes commonly predicated by at least four prediction tools were selected further for analysis parameter. there were four such epitopes with high score and predicted by four different tools (table a) . all the four epitopes were immunogenic and antigenic in nature, conserved %, and predicted to be non-allergen (table b) . immunogenicity scores for the predicted epitopes ranges from . to . . all the epitopes were with positive score hence considered for further analysis. all the peptides predicted to be nontoxic by toxipred online toxicity prediction tool. after provision of spike protein sequence to the bepipred server showed average score of . , with . maximum and . minimum scores (fig ). other properties like physiology of amino acid residues, accessible surface, hydrophilicity, fexibility and β turns were also determined ( table parker hydrophilicity method ( - ). three peptides high scored, predicted for t cell receptors and two peptides for b cell receptors were ligated together by gpgpg or aay. additionally, ompa (genbank: afs . ) was linked at n terminal end of the designed polypeptides by eaaak linker. for purification purpose sis histidine residues were added. final amino acid residues of designed vaccine were when all the five peptides, linkers and adjuvant were ligated (fig ) . antigenicity of the designed multi-epitopes vaccine predicted by vaxijen v . server was . with probable antigen annotation for virus as target organism at . threshold. antigenicity predicted for same sequence of vaccine by antigenpro server was . with . predicted probable solubility upon overexpression. antigenicity of peptide without adjuvant sequence was . with . probable solubility of peptides upon overexpression. the results obtained indicates that with and without adjuvant predicted vaccine sequence is potentially antigenic in nature. allergenfp and allertop servers predicted that both vaccine with adjuvant and without adjuvant are probable non allergen with . and . tanimoto similarity indexes, respectively. the computed molecular weight of designed vaccine was . kda and theoretical pi is . . predicted pi indicates the slight alkalinity of the vaccine ( table ). the estimated half-lives were hrs in mammalian reticulocytes (in vitro), > hours in yeast (in vivo) and > hours in escherichia coli (in vivo). vaccine is highly stable with instability index . that classify the protein as stable. estimated aliphatic index . indicating thermo-stability of the protein ( ) . the predicted gravy score is - . (table ) . negative gravy score indicates that protein is hydrophilic in nature and will interact with water molecules ( ) . secondary structure prediction: secondary structure predicted by online tool raptorx property includes % alpha helix region, % beta sheet region and % coil region. furthermore, solvent accessibility of amino acid residues predicted to be % exposed, % medium exposed and % buried. total residues ( %) are predicted as residues in disordered region. pictorial representation of secondary structure predicted of final protein by psipred is shown in figure . i-tasser predicted the top five d structure model of designed vaccine utilizing threading templates. z score for these templates was ranging from . to . indicating good alignment with sequence submitted. c score is critical in the quality of built model which is quantitatively measured. c score ranges in between - to signifies the best model quality. c score of top five model ranging from - . to - . . model with high c score i.e. - . , estimated tm score . ± . and estimated rmsd . ± . was chosen for further analysis ( fig a) ( ) . for refinement purpose tertiary structure predicted by i-tasser is initially submitted to modrefiner and finally to galaxyrefine. among top five refined models, model found to be the best based on various parameters like gdt-ha . , rmsd-. , molprobit- . and rama favored . (fig b) . quality of refined model was validated by ramachandran plot at rampage webserver. . % residues of modelled vaccine are in favored region, . % in allowed region and . % are in outlier region (fig d) . error in the model are analyzed by prosa web and errat web server. z score is - . ( fig e) and errat score index is . (fig c) . ramachandran plot z score and errat score showed that refined model is of good quality and can be used further. four discontinuous epitopes include of residues were predicted. the score of the predicted epitopes ranges from . to . . shortest and longest discontinuous b cell epitope is of and residues long respectively. haddock web bases server has been utilized to dock the designed vaccine to tlr and tlr . tlr / eliciting immune response toward designed vaccine was analyzed by conformational change with reference to adjuvant tlr / complexes. top models from each group with lowest haddock score were selected. haddock score for tlr /adjuvant and tlr /vaccine were, - . and - . kcal/mol, respectively. in addition, relative binding energies were - . (fig a, b) . similarly, tlr /adjuvant complex have charged-charged , charged polar , charged-apolar , polar-polar , polarapolar and apolar-apolar . while tlr /vaccine complex has charged-charged , charged-polar , charged-apolar , polar-polar , polar-apolar and apolar-apolar interface contacts were observed ( figure c, d) . enhance expression of nucleotide construct in escherichia coli, is reverse translated in jcat online webtool. total number of nucleotides in the constructs were . after codon optimization cai was . and average gc content of optimized nucleotide construct was . indicating good probable expression of vaccine in e. coli k . to clone the gene xho and xbai restriction sites are added at n and c terminus of the construct and gene was cloned in pet a+ plasmid, by using snapgene software (fig ) . c-immsim immune simulator web server was used for determining ability of vaccine to induce immunity. results obtained are consistent with the experimental results published elsewhere ( ) . within first week of vaccine administration primary response was observed by high level of igm. secondary and tertiary immune response clearly seen with increase in b cell number, level of igm, igg +igg , and igm+igg with decreasing concentration of antigen. different b cell isotypes population were also found increasing indicating isotype switching and memory formation (fig a) . b cell activities were also found high, especially b isotype igm and igg , was observed with prominent memory cell formation (fig b) . similarly, cell population of th and tc cells are found high along with memory development (fig c, d) . in addition, macrophage active was found to be increased consistently after each antigen shots and declined upon antigen clearance (fig e) . another cell from cell mediated immunity, dendritic cells were also found increased (fig f) . ifnγ and il expression was found high with low simpson index indicating sufficient immunoglobulin production, suggesting good humoral immune response ( fig g) . simpson index (d) is a measure of diversity. increase in d over time indicates emergence of different epitope-specific dominant clones of t-cells. the smaller the d value, the lower the diversity. till date there is no cure available for covid , suggesting urgent requirement of drug or vaccine to control the spread of sar-cov infections. advancement in bioinformatics tools, process of vaccine development can be facilitated by identifying potential epitopes for t and b cells that will lead to vaccine for prevention of covid . several reports and animal trials suggest that s-protein can be potential target for vaccine development ( , ( ) ( ) ( ) ( ) . there are several reports suggesting the importance of spike protein in activation of cells of immunity and vaccine development ( ) ( ) ( ) . hla-a restricted epitopes from s protein of sars-cov , elicit t cell specific immune response in sars-cov recovered patients ( ) . similarly serum samples of sars-cov recovered patients were sufficient to neutralize epitope rich region on the spike s protein from sars-cov ( ) . programing of live attenuated form of pathogen is commonly used strategy for the vaccine development. despite efficacy of such vaccines, reversion of virulence remains a challenge and not utilized in weak immune patients ( , ) . here in case sars-cov due to its high communal transmission vaccine research requires highly skilled workers, sophisticated instrumentation and biosafety level iii facility. this may likely the slow down the process of vaccine development, enhanced cost leading to restriction on availability of vaccine for mass population. advancement in the field of bioinformatics and molecular biology techniques have provided opportunity of development of vaccine with high efficacy, less time for development, low cost of production that may lead to low cost vaccine in time for large population. we designed a multi-epitopes vaccine construct from s-protein of sars-cov . several online tools were used for the designing of epitopes and thereby a vaccine. from various epitopes predicted by the online server based on common sequence and high score three tcr and two bcr epitopes were selected as part of covid vaccine. all the five tcr and bcr epitopes were linked by gpgpg and aay linker peptides (fig ) . to enhance the antigenicity of tcr/bcr linked epitopes peptide, adjuvant ompa were linked by eaaak linker sequence for a complete vaccine design which was found to restore antigenicity and nonallergen status. (table ). for successful vaccine candidate, secondary and tertiary structures characters play an essential role. secondary structure of designed vaccine contains % alpha helixes, % beta sheets and % coils. upon refinement tertiary structure of designed vaccine showed improvement to desirable level as indicated by ramachandran plot. designed vaccine showed high antigenicity score, no probable allergen. tlr are essential receptor proteins in activation of innate immune response. that recognize and respond by induction of immune reactions to pamps (pathogen associated molecular patterns). various tlrs have shown to activate immune response to virus through their interaction with nucleic acids and envelopes proteins. tlr and tlr / involved in nucleic acids detection, while tlr and tlr recognize envelope glycoproteins ( ) . interaction of s protein with tlr increase the production of il in human monocyte macrophages ( ) . tlr also involved in eliciting innate immune response upon recognition of several other virus components ( , ) . in a comparative binding efficacy of s-proteins to tlrs, tlr showed strongest proteinprotein interaction with hydrogen and hydrophobic bonds on extracellular domain ( ) . in molecular docking analysis, tlr and tlr showed stable protein-protein interaction with designed vaccine compared to adjuvant protein. tlr showed more stable interaction than tlr , which is consistent with previous report ( ) . stable interaction of designed vaccine indicates efficiency of vaccine for activation of tlrs, involved in dendritic cell activation, thereby subsequent antigen processing, and presentation on surface to t cells ( ) . immune simulation showed typical natural immune response pattern upon multiple exposure to same antigen. server predicted elevated level of b cell and t cell for longer time on repeated exposure of antigen. increased level of antiviral cytokine ifnγ and il indicate potential of subsequent activation of t-helper cell thereby high level of ig production, supporting humoral immune response ( ) . to validate the designed vaccine in the screening of immune reactivity by serological test, it is necessary to express the construct in preferred host like e. coli k for recombinant proteins. codon optimization of was performed to achieve high expression designed vaccine in e coli . codon adaptability index (cai= . ) and gc content ( . %) of the vaccine construct was optimum for high expression of recombinant protein in host. immediate step, here after is to express the protein in preferred host and validate results obtained in this report by performing the several immunological assays. inadequate number of drugs and time-consuming process of drug development, multiplication chain of sars-cov infection can be control only by vaccine. we utilized tools and techniques of immune informatics to design a potential vaccine candidate. this vaccine codes epitopes form s protein of sars-cov virus for t and b cell receptors. this is one more time showed that bioinformatics approach can effectively use to develop vaccines in short time and with incurred less cost. although in silico results point out the effectiveness of the vaccine, efficacy need to be analyzed by preforming laboratory experiments and animal model studies. properties of that peptides. in case of immunogenic properties peptide number has negative score for antigenicity hence neglected for further analysis. the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak-a n update on the status world health organization declares global emergency: a review of the novel coronavirus (covid- ) asymptomatic transmission, the achilles' heel of current strategies to control covid- presumed asymptomatic carrier transmission of covid- . jamanetwork improved molecular diagnosis of covid- by the novel, highly sensitive and specific covid- -rdrp/hel real-time reverse transcription-pcr assay validated in a rights reserved. n reuse, undefined. evaluation of covid- rt-qpcr test in multi-sample pools role of chest ct in diagnosis and management covid- )?: situation report the efficacy of lopinavir plus ritonavir and arbidol against novel coronavirus infection -full text view -clinicaltrials.gov a trial of lopinavir-ritonavir in adults hospitalized with severe covid- hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial covid- patients with at least a six-day follow up: a pilot qt interval prolongation and torsade de pointes in patients with covid- treated with association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state journal pre-proof no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro clinical characteristics of deceased patients with coronavirus disease : retrospective study epidemiology and transmission of covid- in cases and of their close contacts in shenzhen, china: a retrospective cohort study the effect of control strategies to reduce social mixing on outcomes of the covid- epidemic in wuhan, china: a modelling study covid- infection: the perspectives on immune responses allergy and immunology immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic. apjai-journal.org estimating case fatality rates of covid- preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development characterization of the receptor-binding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor identification of an hla-a* -restricted cd t-cell epitope ssp- of sars-cov spike protein draft landscape of covid- candidate vaccines sars-cov- vaccines: status report insilico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases covid- : living through another pandemic the nonstructural proteins directing coronavirus rna synthesis and processing the coronavirus e protein: assembly and beyond membrane binding proteins of coronaviruses sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor evidence that tmprss activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response coronavirus infections and immune responses targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals chadox ncov- vaccination prevents sars-cov- pneumonia in rhesus macaques properties of mhc class i presented peptides that enhance immunogenicity. ncbi.nlm.nih.gov in silico approach for predicting toxicity of peptides and proteins. ncbi.nlm.nih.gov allergenfp: allergenicity prediction by descriptor fingerprints. academic.oup vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines bepipred- . : improving sequence-based b-cell epitope prediction using conformational epitopes. academic.oup a semi-empirical method for prediction of antigenic determinants on protein antigens induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide downloaded from prediction of chain flexibility in proteins -a tool for the selection of peptide antigens prediction of the secondary structure of proteins from their amino acid sequence. ci.nii.ac.jp [internet new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites identifying candidate subunit vaccines using an alignmentindependent method based on principal amino acid properties allertop v. -a server for in silico prediction of allergens proso ii -a new method for protein solubility prediction raptorx-property: a web server for protein structure property prediction protein secondary structure prediction using deep convolutional neural fields. nature i-tasser: a unified platform for automated protein structure and function prediction in silico analysis of epitope-based vaccine candidates against hepatitis b virus polymerase protein improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization galaxyweb server for protein structure prediction and refinement. academic.oup data set for phylogenetic tree and rampage ramachandran plot analysis of sods in gossypium raimondii and g. arboreum prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. academic.oup verification of protein structures: patterns of nonbonded atomic interactions structure validation by calpha geometry: phi, psi and cbeta deviation a new structure-based tool for the prediction of antibody epitopes dec ; antibody-protein interactions: benchmark datasets and prediction tools evaluation the haddock . web server: user-friendly integrative modeling of biomolecular complexes prodigy: a web server for predicting the binding affinity of protein-protein complexes computational immunology meets bioinformatics: the use of prediction tools for molecular binding in the simulation of the immune system jcat: a novel tool to adapt codon usage of a target gene to its potential expression host salmonella persist in activated macrophages in t cell-sparse granulomas but are contained by surrounding cxcr ligand-positioned th cells thermostability and aliphatic index of globular proteins. academic.oup a simple method for displaying the hydropathic character of a protein i-tasser gateway: a protein structure and function prediction server powered by xsede development of an inactivated vaccine candidate for sars-cov- . science ( -) chadox ncov- vaccination prevents sars-cov- pneumonia in rhesus macaques neeltje van doremalen. biorxiv.org safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a doseescalation, open-label, non-randomised, first-in-human trial evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission subunit vaccines against emerging pathogenic human coronaviruses sars vaccine development. ncbi.nlm.nih.gov [internet hla-a * t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins b-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the s domain of the surface spike glycoprotein nonclinical development of novel biologics, biosimilars, vaccines and specialty biologics global+regulatory+guidelines+for+vaccines.+in+nonclinical+development+of+novel+b iologics,+biosimilars,+vaccines+and+specialty+biologics+&ots=uupokzupyo&sig=esm wpqt kubtszrvu lki zbq global regulatory guidelines for vaccines toll-like receptors and viruses: induction of innate antiviral immune responses. ncbi.nlm.nih.gov sars coronavirus spike proteininduced innate immune response occurs via activation of the nf-κb pathway in human monocyte macrophages in vitro innate immunity to respiratory viruses viruses and toll-like receptors in silico studies on the comparative characterization of the interactions of sars-cov- spike glycoprotein with ace- receptor homologs and human tlrs a theory on sars-cov- susceptibility: reduced tlr -activity as a mechanistic link between men, obese and elderly designing a multi-epitopic vaccine against the enterotoxigenic bacteroides fragilis based on immunoinformatics approach key: cord- -ep ezoko authors: gamble, lena j; matthews, qiana l title: current progress in the development of a prophylactic vaccine for hiv- date: - - journal: drug des devel ther doi: . /dddt.s sha: doc_id: cord_uid: ep ezoko since its discovery and characterization in the early s as a virus that attacks the immune system, there has been some success for the treatment of human immunodeficiency virus- (hiv- ) infection. however, due to the overwhelming public health impact of this virus, a vaccine is needed urgently. despite the tireless efforts of scientist and clinicians, there is still no safe and effective vaccine that provides sterilizing immunity. a vaccine that provides sterilizing immunity against hiv infection remains elusive in part due to the following reasons: ) degree of diversity of the virus, ) ability of the virus to evade the hosts’ immunity, and ) lack of appropriate animal models in which to test vaccine candidates. there have been several attempts to stimulate the immune system to provide protection against hiv-infection. here, we will discuss attempts that have been made to induce sterilizing immunity, including traditional vaccination attempts, induction of broadly neutralizing antibody production, dna vaccines, and use of viral vectors. some of these attempts show promise pending continued research efforts. since its discovery and characterization in the early s as a virus that attacks the immune system, leaving patients unable to fight off opportunistic infections, there has been an ebb and flow of effective treatments and hope as scientists continue to search for ways to eradicate human immunodeficiency virus- (hiv- ) from the human population similar to what has been accomplished in the case of smallpox. the majority of the effort and nearly all of the success has come in the area of patient treatment rather than inhibition of contraction or spread of the virus. a class of treatments, antiretroviral therapies (arts) and later highly active antiretroviral therapies (haarts), has been the mainstay of disease control during the last years. notwithstanding the increased life span of patients, increased time to full-blown aids, and decreased contraction of opportunistic infections and aids-related diseases (ie, non-hodgkin's lymphoma, kaposi's sarcoma, etc) by patients treated with haart, there are several reasons why development of an hiv- vaccine is still warranted. five of these reasons are as follows: ) nearly two-thirds of the patients who contract hiv- live in underdeveloped countries and cannot afford the expensive haart regimen, ) both the art and haart regimen are complex and are disruptive to patients' lives and diets, making long-term compliance an issue, ) the potential side effects of art/haart treatments negatively affect the long-term health of patients and include diabetes, cardiovascular disease, fractures, etc, - ) development of haart drug resistance, and ) the presence of latent hiv- reservoirs harboring viral strains that were produced through mutation throughout the duration of the infection of the host also play a role in the failure of haart. these reasons, as well as many others, underscore the need for a prophylactic hiv- vaccine. possibly, the strongest argument for development of a prophylactic vaccine may be the need for control of the virus spread worldwide. every day, patients worldwide are infected with hiv- . production of a vaccine that could inhibit infection, reduce spread, or both would aid in the reduction of the burden of aids and aids-related diseases. the expenses incurred by the aids epidemic can hardly be calculated. they range from tens of thousands of dollars per patient for the haart regimen, to millions of dollars required for building of orphanages by governments for children whose parents have succumbed to the disease, to the unknown cost of educational materials and condoms in the effort to prevent further spread of the disease. this public health challenge has not gone unnoticed and has been addressed by scientists' ongoing efforts to develop a safe and effective hiv- vaccine. a prophylactic hiv- vaccine would offer sterilizing immunity to patients, preventing infection upon presentation of the virus. a prophylactic vaccine must also be effective at all possible portals of hiv- entry, especially the mucosa. for this to occur, the vaccine must offer broad and durable immunity. several consortia have worked diligently to produce a vaccine that will induce broadly reactive neutralizing antibodies (nabs). these consortia include major international efforts as well as efforts of individual countries, regions, and institutions including, but not limited to: the international aids vaccine initiative neutralizing antibody consortium, the center for hiv-aids vaccine immunology, the hiv vaccine trials network, us military hiv research program, the collaboration for aids vaccine discovery, and the vaccine research center at the national institutes of allergy and infectious diseases of the national institutes of health. to date, however, no hiv- candidate vaccine has induced broadly reactive nabs. in the absence of a vaccine that can prevent infection of hiv- , there are still many benefits to be realized from production of a therapeutic vaccine. a therapeutic vaccine would be supremely valuable if it were able to increase the titer of virus necessary for infection, increase the time to clinical manifestation of virus, control viral load after infection, and reduce secondary transmission. [ ] [ ] [ ] [ ] [ ] a vaccine that could induce this type of response would invariably decrease contagiousness, decrease the need for costly and potentially dangerous art/haart, and decrease the number of opportunistic infections of patients. while the effect of controlling the normal hiv- pathology with therapeutic vaccines will be favorable for the individual patient as well as society, the effect of preventing hiv- infections in humans with a prophylactic vaccine is also broadly appealing. this potential for eradicating the hiv- virus from human hosts drives scientists to continue to find ways to circumvent the challenges presented by this unique virus in order to induce production of the nabs that are critical for sterilizing immunity. this review, therefore, will focus on the specific challenges presented by hiv- and strides that have been made toward creating a prophylactic vaccine, including past efforts that have failed and lessons that have been learned from those failures. we will also discuss novel vaccine options and some of the promising trials that are currently underway. while several problems face scientists who are attempting to create an hiv- vaccine, three problems in particular have posed extremely daunting challenges. these three problems are ) degree of diversity of the virus, ) ability of the virus to evade the hosts' immunity, and ) lack of appropriate animal models in which to test vaccine candidates. these three major problems will be discussed in more detail below. traditionally, prophylactic vaccines have been made by exposing some part of a pathogen's structure as an antigen to the host's immune system, and eliciting an immune response, resulting in the production of long-term memory lymphocytes that are capable of mounting a strong immune response upon later infection with the pathogen. the premise upon which this manipulation of the immune system is based is the ability of the immune system to make long-lasting antibodies to conserved structures on exposed proteins that are native to the pathogen. ideally, both humoral and cell-mediated immunity would be induced creating long-lasting immunity. traditional attempts to recreate this process using live attenuated simian immunodeficiency virus- (siv- ) viruses in an effort to vaccinate macaques against siv- have been proven safe and effective in macaques that were subsequently challenged with siv- . , however, an incidental study of the effect of liveattenuated hiv- (containing deletions of the nef gene and the long terminal repeat) was proven pathogenic in humans when three out of six treated patients developed late-onset immunosuppression. [ ] [ ] [ ] killed viruses have also been tested as a potential vaccine approach, but safety concerns have halted their use. these safety concerns include incomplete inactivation of the virus leading to potential residual infectivity during the vaccine preparation. due to the ineffectiveness of traditional vaccine approaches to date, scientists have attempted to use recombinant hiv- proteins to stimulate the production of nabs. these attempts failed due to their inability to induce a lasting, broad range of nabs that would inhibit infection in humans. [ ] [ ] [ ] [ ] perhaps these failures are a result of the inherent diversity of hiv- . this diversity has presented a major roadblock to development of a prophylactic vaccine. there are three main groups of hiv- (m, o, and n) as well as a recently discovered group, p. each group consists of several subtypes, clades. the various clades display biological differences with respect to transmission, replication, and disease progression. , these differences result in an inability to produce a generalizable vaccine that would induce the breadth of nabs necessary to counter an infection by a wide range of hiv- clades that may be encountered in a natural setting. the degree of diversity seen in hiv- is greater than that of any other virus observed. , this problem is being addressed by development of multiclade (multiple env and/ or subtype b gag, pol, nef) , and mosaic vaccines which incorporate sets of immunogenic proteins from different clades or bivalent proteins from clades b and c. [ ] [ ] [ ] there are proof of principle studies that illustrate immunological protection against hiv- in nonhuman primates that were passively treated with broadly reactive nabs. [ ] [ ] [ ] these studies show that protection against infection with hiv- can be conferred by the presence of broadly reactive nabs. the next step toward production of a prophylactic vaccine would involve induction of production of these or similar broadly reactive nabs by the host's immune system. the rate at which the hiv- virus mutates, due to the nature of the reverse transcriptase enzyme responsible for transcribing its rna, ensures that nearly every daughter virion will have a different genome than its parent. when these changes occur in the hiv- env protein that is needed for antibody recognition, they inhibit the immune system's ability to mount a sufficient response. one attempt to circumvent this problem has been to induce the production of nabs to the conserved regions of hiv- proteins. a major problem with this approach is that the conserved regions of hiv- proteins are often shielded from exposure to nabs within the hiv- envelope. the native structure of the envelope protein, reportedly the only hiv- protein susceptible to nabs, shields it from the immune system as a glycosylated trimer of heterodimers. the glycosylation of the envelope protein allows for the carbohydrates to masquerade as 'self' thereby forming an immunologically silent face and protects neighboring epitopes via an 'evolving glycan shield'. [ ] [ ] [ ] additionally, the gp coreceptor binding site, another conserved site, is not presented until primary binding to cd + has occurred. an attempt to create antibodies to the cd -binding region of the gp protein was made in rhesus macaques in and results indicated that vaccinated hosts were able to withstand challenge with shiv. other attempts to create an hiv- vaccine have focused on overcoming the ability of hiv- to escape immune surveillance through use of antibodies that are able to neutralize diverse isolates of hiv- . these antibodies include pg , pg , f , g , e , b , [ ] [ ] [ ] [ ] and most recently scd - b and others. identification of these antibodies gives hope that their induction or the induction of other such broadly reactive nabs may provide the basis for a prophylactic vaccine in the future. the use of animal models for development of therapeutics offers the benefit of thorough testing and validation prior to introduction of a vaccine in humans. in the past, vaccines were made by observing and then mimicking the immune response mounted by individuals who had recovered from a particular disease. to date, however, there are no known cases of individuals who have recovered from hiv- infection. however, data can be gathered from long-term nonprogressors -patients who have been infected with hiv- for at least years and do not display any hiv- -related symptoms. , another option that may be critical to the development of a prophylactic vaccine is the use of relevant animal models. such models will allow for analysis of the effect of a potential vaccine on an intact host prior to use in humans. one particular challenge with the use of animal models for development of a prophylactic hiv- vaccine is that there are very few naturally occurring disease models of hiv- . only a few nonhuman primates are susceptible to infection with hiv- and infected animals do not progress to aids. therefore, it is important to use other disease models that mimic the hiv-aids pathologic progression. one such potential model is feline immunodeficiency virus (fiv). fiv was discovered in and is known to cause an aids-like disease in domestic cats and mimics hiv-related dementia in humans. a vaccine for fiv was approved by the fda in . while the fiv model is potentially informative, its use is not sufficient as a basis for development of a prophylactic hiv- vaccine. an ideal animal model would display a pathological response to infection with hiv- that is very similar to the one that occurs in humans. unfortunately, hiv- does not cause pathology leading to the development of aids in any host other than humans. [ ] [ ] [ ] [ ] however, animal models have been developed and used that allow partial understanding of the pathology of hiv- , the natural immunological response to infection, and the response of the host to novel therapeutics. one of these models involves the simian immunodeficiency virus mac (siv mac ) that replicates and causes an aids-like disease in baboons, cynomolgus, and pigtailed macaques. while the similarities of siv mac to hiv- have allowed for insight into pathology, transmission, and immunological response of the infected host to the virus, the differences between siv mac and hiv- are still too great to be able to draw conclusions regarding potential human responses to an hiv- prophylactic vaccine. therefore, to broaden the scope of animal model usage, a chimeric shiv virus was engineered to incorporate both siv and hiv- proteins or genes. while macaques infected with shiv do go on to develop aids, the time to progression is much different from the time to progression to aids of hiv- -infected humans. infection of macaques with siv mac strain mimics hiv- infection in humans by leading to chronic, slow disease progression. route and dose required for infection, viral tropism, replicative capacity of the viruses, and pathology of siv/shiv-infected monkeys are all very different than these parameters in humans. , this distinction has been well characterized by the recent phase iib step trial, which involved healthy, uninfected volunteers. the result of this trial was termination at its first scheduled efficacy assessment due to its failure to suppress viral load in subsequently infected individuals and then-suspected increased hiv- infection due to interaction of the immune system with vaccine components. the vaccine, a recombinant adenovirus serotype (ad ) virus incorporating the gag, pol, and nef genes from hiv- , had been previously tested in an shiv model in macaques and the results of that experiment were not suggestive of the results of the human trial. this disparity underscores the need for animal models that more closely reflect the pathology seen in human infection with hiv- as well as identification of immunological correlates of protection that reflect control of hiv- viral load in human subjects. therefore, the search for an appropriate animal model or the appropriate use of current animal models in the search for a prophylactic hiv- vaccine continues. until a model can be derived that will allow for observation of each stage of infection, progression of disease, and response of the immune system in a way that is comparable to this process in humans, we will not be able to logically predict which vaccine candidates should be moved forward to clinical trials. several attempts to stimulate the immune system to provide protection against hiv infection have been attempted so far (table ) . hope for creating a prophylactic vaccine lies in the ability of the scientific community to identify and induce a broad neutralizing antibody response that would offer sterilizing immunity to vaccinated patients. to this end, several novel approaches are being studied. as mentioned in the previous section, there are several daunting problems facing scientists who are attempting to create an hiv- vaccine. in hopes of creating a vaccine which elicits sterilizing immunity to hiv- , researchers have focused their efforts on ( ) the use of plasmid dna vaccines, ( ) live recombinant vectors for vaccine development (expressing or presenting hiv antigens), and ( ) mucosal immunity. these critical topics will be discussed in more detail below. vaccines should elicit a robust immune response that is long lasting and is able to provide protection against various strains of a pathogen. plasmid dna vaccinations can induce a strong humoral and t-cell response. dna-based vaccination has been used as a powerful tool to fight against parasitic, fungal, bacterial, and viral infections. [ ] [ ] [ ] [ ] [ ] there are multiple advantages for using plasmid dna for vaccination: they are generally safe, nontoxic, and through the delivery of a gene encoding important immunogenic epitopes, the dnabased vaccine exploits biosynthetic machinery of the host cell. one such example was in , whereby wolff and colleagues illustrated protein expression after intramuscular (im) injection of plasmid dna into myocytes. despite these promising results, there had been speculation regarding dna vaccination strategies. for example, it was shown that protein production in response to dna plasmids that contained hiv inserts elicited substantial cellular response in mice and nonhuman primates. however, these products were poorly immunogenic in humans. one strategy to improve immune response of the plasmid dna vaccine strategy is by coadministration of dna plasmids coding for cytokines (eg, inf-g, il- , il- , il- , and il- ). [ ] [ ] [ ] [ ] a second strategy which has been utilized to improve plasmid dna vaccination has been the administration of plasmid dna with adjuvants (eg, cpg oligodeoxynucleotides), or the use of dna-delivery systems (eg, microparticles, cochleates, and linear polyenimines). [ ] [ ] [ ] [ ] a third strategy to improve vaccine efficacy involves the coadministration of plasmid dna in combination with viral vectors. for instance, research performed by harari and colleagues in demonstrated that vaccination by means of an hiv- clade c dna prime in combination with a pox vector (nyvac) boost induces a reliable polyfunctional and longlasting anti-hiv t-cell response in human participants. along these same lines, work recently published by jaoko and group demonstrated safety and immunogenicity of a multiclade hiv- ad-based vaccine alone or in combination with a multiclade hiv- dna vaccine in africa. these results also demonstrated that dna priming increased the frequency and magnitude of cellular and humoral responses; however, there was no effect of recombinant ad dosage on immunogenicity endpoints. the previously mentioned dna-delivery strategies have been used in combination with viral vectors or alone by means of a variety of immunization routes (eg, im, intravenous [iv], intradermal [id], intranasal [in] , oral, rectal, or vaginal). in the majority of reported studies, dna vaccines have been administered by the im and/or id routes. however, as it relates to hiv vaccination, mucosal immunity could potentially be an important factor to consider, with mucosal immunity being achieved optimally by in or oral routes of administration. the topic of mucosal immunity will be discussed in more detail in a later section within this review. after immunization, it is assumed that the dna vaccination immunogen is produced in the skeletal muscles, dendritic cells, and macrophages at the site of immunization. however, in adults, the skeletal muscles are not involved in a high level of protein synthesis as compared to the liver. therefore, the delivery of dna to cells, which are capable of high protein synthesis, such as hepatocytes, epithelia cells of the intestines, or salivary pancreas, may result in high levels of protein expression. the hepatocytes express enzymes involved in the formation of intrachain and interchain disulfide bonds required for proper folding and assembly of proteins. in addition, the liver expresses glycosyltranferases, which are essential for synthesis of both n-and o-linked glycan side chains; this may not be the case for other cell types, , the significance of this point being the fact that broadly crossclade nabs such as g recognize glycan moieties on the heavily glycosylated hiv- envelope antigens. , , another advantage of protein expression within the liver is that significantly lower amounts of dna are needed for protein expression of a particular antigen in the hepatocytes vs another cell type. for the immunization of humans, milligram quantities of dna are necessary to achieve adequate levels of immune response. any method whereby there would be a reduction in dna quantity needed to vaccinate humans would provide significant economic advantages. based on the previously mentioned reasons, it is not a surprise that the liver has been exploited extensively as a site for gene delivery due to its ability to produce proteins and glycoproteins. [ ] [ ] [ ] [ ] hydrodynamic delivery is the application of controlled hydrodynamic pressure in capillaries to enhance endothelial and parenchymal cell permeability; this methodology had its inception in the late s with investigations into intravascular injection of plasmid dna solution for gene delivery in whole animals. [ ] [ ] [ ] [ ] hydrodynamic plasmid dna delivery is well tolerated in mice. in , raska and colleagues demonstrated in mice that iv hydrodynamic vaccination with hiv- envelope dna injections resulted in high levels of expression of hiv antigen in the liver. in mice, immunological data illustrated that hydrodynamic administration of hiv- plasmid dna was superior to vaccination with dna by in, id, im, and intrasplenic routes. further results illustrated that after boosting, hydrodynamic vaccination yielded levels of hiv- -specific antibodies that were -fold higher than those elicited by other routes tested. however, this delivery scheme is not feasible in large animals and humans. as an alternative, receptor-mediated dna binding to hepatocytes could be a viable approach. molecules with terminal galactose residues covalently linked to dna are recognized by the hepatocyte-expressed galactosespecific asialoglycoprotein receptor for internalization. this alternative would avoid delivery through the hepatic system and the need for expansion of the blood volume. in addition, galactose-linked dna packaged in delivery vehicles such as liposomes, choleates, or microspheres can be given by oral administration, which would be absorbed by the intestine and ultimately delivered to the hepatic vein. as an additional alternative to hydrodynamic delivery in humans, it might be possible to express hiv antigens in the liver by means of plasmid dna delivery via viral vectors such as the ad. ads have been shown to transduce the liver efficiently in vivo by means of the hexon proteins. , in this regard, production of translation of hiv- proteins primarily in the liver might allow for the production of heavily glycosylated hiv- envelope antigens and thus the production of nabs. prophylactic vaccine for hiv- live recombinant vectors for vaccine development viral vectors are potent inducers of cellular and humoral response. viral vectors can express proteins from bacteria or viral pathogens to vaccinate against infectious diseases. there are several viral vaccine vectors that have been used successfully in models for vaccination. these vectors include alphaviruses, human rhinoviruses (hrvs), ads, picornaviruses, poxviruses, measles viruses, influenza, and vaccinia viruses. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] each of these vectors has its respective disadvantages and advantages with respect to vaccine development. some advantages of a few of these vectors include their ability to naturally infect a wide variety of cell types and tissues of interest. [ ] [ ] [ ] [ ] [ ] [ ] each respective vector has its own set of disadvantages. for instance, one disadvantage of using the poliovirus or the hrv as a vaccine vector is the insert size limit restriction of these vectors as compared to the large insert size (∼ kb) accommodation of ad vectors. the most common disadvantage of the majority of viral vaccine vectors is reduced vaccine efficacy due to vector preexisting immunity (pei). [ ] [ ] [ ] [ ] [ ] various strategies have been employed to circumvent the problems associated with vector pei. specifically, as it relates to ad vectors, pei is a tremendous problem. of the identified serotypes of ad vectors, human serotypes (ad ) and (ad ) have been the most extensively used for gene therapy protocols. ad has been used for hiv- vaccination protocols, most recently in the step study. as it relates to ad and ad , pei to these vectors may be found in up to % of the american population and up to % of the population of other countries. this ad pei can limit the effectiveness of ad-based vaccinations. [ ] [ ] [ ] to circumvent ad or ad pei, researchers have employed the use of vector chimeras, , use of alternative serotypes, [ ] [ ] [ ] [ ] [ ] [ ] [ ] and the use of nonhuman ads, such as chimpanzee ad. the chimpanzee ad virus was demonstrated to not be significantly neutralized by human sera, which gives chimpanzee ad an advantage for human vaccine development. [ ] [ ] [ ] other strategies have been used to reduce the immune response against ad vectors such as the use of helperdependent ad (hd-ads) vectors, - the use of ad delivery in combination with biochemical modifications such as pegylation, [ ] [ ] [ ] [ ] [ ] [ ] [ ] and the use of vector delivery by means of cell vehicles. , with respect to the hd-ads, these vectors were produced to further increase the safety and cloning capacity of first-generation ad vectors. hd-ads lack ad genes and contain only the packaging signals and end terminal repeats. these vectors were designed to avoid cellular immunity and diminish liver toxicity, thus promoting long-term transgene expression. [ ] [ ] [ ] [ ] the reduced immune response against hd-ads has allowed for transgene expression in mice and baboons for years. , , , this long-term transgene expression could be helpful for antigen production for an hiv vaccine, thus producing an opportunity to have increased protection against hiv, with reduced frequency of vaccinations. although nabs to ad may reduce the immunogenicity of ad -based vectors in animal model systems, their effect on the immunity in subjects with previous ad exposure is still largely unknown. as previously mentioned, the step trial, which tested a merck recombinant ad (rad ) vaccine (encoding hiv- gag, pol, and ne genes), failed to yield protection, either by lowering viral load or by decreasing acquisition of infection. analysis of data from this study aroused speculation that subjects with pre-existing nabs from wild-type ad infection had an increased risk of hiv infection after vaccination. one recent study has shown that there was no causative role for ad -specific cd + t cells in increasing hiv- susceptibility in the merck trial. in this regard, there are multiple studies ongoing to elucidate a concrete finding with respect to the role of ad pei and increased activation of cd + t cells in the mucosal milieu. , recently, there was a report by cheng and colleagues that attempted to characterize the specificity of rad nabs in ad immune subjects and determine the impact of ad exposure on immune responses elicited by ad -based vaccinations. cheng and colleagues reported that rad nabs were directed toward different components of the ad virion, depending on whether the ad infection was natural or from ad-based hiv vaccine trials. for example, ad nabs generated by natural infection are directed primarily to fiber components, while vector exposure elicits responses primarily to capsid proteins other than fiber. nabs elicited by natural infection significantly reduced the cd + and cd + cell responses to hiv gag after dna/rad vaccination. this report concluded that ad nabs differ based on the route of exposure and that previous ad exposure compromises ad vaccine-induced immunity to weak immunogens, such as hiv- gag. these results have a tremendous impact on hiv- vaccine trials and the design of next generation viral vaccine vectors. viral vectors such as ad, influenza, and polio have been used as vaccine vectors for many reasons. one important advantage of these vectors, which makes them attractive, is that they can provide mucosal immunity because they can easily infect the mucosal surfaces as well as act to induce cytokine and chemokine production at the mucosal entry sites. ad, influenza, and polio also have the advantage of being able to be delivered orally, without the use of needles. this is an important fact in developing countries where needle cost is prohibitive to vaccine administration. as it relates to hiv vaccine development, mucosal immunity is a debatable factor to consider. when deciding upon a vaccine agent, the importance of considering if the ultimate goal is to induce systemic immunity, mucosal immunity, or both is worth careful consideration. [ ] [ ] [ ] it is believed that % of hiv- i nfection will occur from heterosexual viral transmission and most of the rest will occur from homosexual or perinatal transmission. although the biology of sexual transmission is poorly understood, it is clear that the essential first step in the infection pathway is the transfer of infectious virus or hiv-infected cells through the mucosal surfaces. after hiv has entered a new host, the hiv or hiv-infected cells will soon encounter susceptible host target cells at the mucosal point of entry where the virus replicates and then invades local lymphatic tissues, initiating systemic hiv infection. on this basis, strong immunity is required to provide a protective immunological barrier at the most common point of entry, the mucosal surfaces of the reproductive tract. due to the compartmentalization of the secretory and systemic immune systems, parenterally administered antigens do not consistently stimulate mucosal immunity. therefore, it is important to consider a vaccine regime that induces mucosal immunity. since cd + ccr + memory t cells are the primary target of hiv infection in the gut and mucosa and rapid depletion of this subset occurs early after infection, , several studies have investigated the role of hiv mucosal immunity. previous studies have demonstrated the importance of a mucosal siv/hiv vaccine producing both strong mucosal antibody and cd + response capable of blocking the escape of virus from the intestinal mucosa into systemic lymphoid organs. , [ ] [ ] [ ] [ ] [ ] however, in other instances, the necessity of exclusive mucosal hiv immunity will be further debated based on the promising results found in a heterologous prime/boost regimen using dna/ . -expressing siv and hiv- transcripts , and modified vaccinia virus ankara (mva/ . )-expressing siv and hiv- transcripts under the control of vaccinia virus early/late promoter. in this case, either id or im dna/mva vaccination was able to provide protection against a intrarectal shiv- . challenge. along these same lines, recently, promising results were found by hessell and colleagues in . hessell and colleagues demonstrated that after an iv administration of monoclonal antibodies f or e to six monkeys followed by a shiv ba-l challenge, five out of six monkeys from either group showed complete protection and sterilizing immunity. a low level of viral replication could not be ruled out for the six monkeys in either group. replicative ad yields a robust immune response at the mucosal sites partly because ad is known to infect and replicate in epithelial cells. [ ] [ ] [ ] [ ] various strategies have been used to achieve mucosal immunity via the oral route. one such strategy embodies the development of replication-defective recombinant ad serotype (ad ) vector. serotype vectors are being currently used because ad has a natural tropism for the gut and causes no pathological disease outside of the gastrointestinal tract. ad vectors are likely to have a preferential tropism for the gut because ad appears to have a resistance to acidic ph and the capsid configuration of long and short fibers allows the ad virus to preferentially infect the gut. , live recombinant vectors for vaccine development engineered to express/present hiv- antigens as previously mentioned, viral vectors are potent inducers of cellular and humoral responses. of note, viral vectors have been practically used for human applications and have progressed treating a variety of disease contexts such as cancer and infectious diseases. [ ] [ ] [ ] [ ] traditional viral vector immunization embodies the concept that the vector uses the host cell machinery to express antigens, which are encoded as transgenes within the viral vector. cellular and humoral immune responses are generated against these antigens. over the last years, several viral vectors have been derived to express hiv- antigens for vaccine purposes. some researchers have taken an alternative approach to conventional transgene expression of antigens by means of viral vectors; this alternative approach embodies the capsid incorporation of antigens. this innovative paradigm is based upon the vector presenting the antigen as a component of the capsid rather than an encoded transgene. incorporation of immunogenic peptides into the vector capsid offers potential advantages. in this regard, the processing of the capsidincorporated antigen via the exogenous pathway should result in a strong humoral response similar to the response provoked by native ad capsid proteins. in this arrangement, potentially, hiv peptide antigens accrue the potent immunostimulatory effects of the native ad vector capsid proteins, which effectively perform an adjuvant function. on this basis, the immune response directed against vector capsid proteins with repetitive vector administration should achieve a booster effect against the incorporated antigen. most importantly, as it relates to hiv infection, this strategy yields the potential of generating antibodies to hiv proteins. recent crystallographic, cryo-electron tomography, and molecular modeling studies have provided valuable insight to molecular surfaces recognized by antibodies as well as assisted in rationale vaccine design of immunogens. [ ] [ ] [ ] [ ] [ ] these structural technologies can also potentially improve the abilities of scientists to advance the antigen capsidincorporation strategy. if the antigen capsid incorporation is effective, it can provide a way forward with respect to inducing sterilizing immunity. , , the antigen capsid-incorporation strategy has been used for ad-based vaccines in the context of many diseases. , [ ] [ ] [ ] [ ] [ ] one of the first examples where the antigen capsid-incorporation strategy was used was with research performed by crompton in . crompton and colleagues inserted an eight-amino acid sequence of the vp capsid protein of poliovirus type into two regions of the ad serotype hexon. one of the chimeric vectors produced grew well in tissue culture. in addition, antiserum raised against the ad with the polio insert specifically recognized the vp capsid of polio type . as it relates to ad serotype, wu and group demonstrated that his epitopes could be incorporated into ad hexon hypervariable regions (hvrs) - (now reclassified as - ) without perturbing viral viability and any major biological characteristics such as replication, thermostability, or native infectivity. this study by wu and colleagues demonstrated that his appeared to be surface exposed at these regions. with respect to peptide incorporation within ad hexon, hvr and hvr appear to be the most promising locales for peptide/antigen incorporation based on x-ray and peptide analyses along with molecular studies. our laboratory and others have focused on incorporations at hvr or single-site incorporations (such as fiber and pix). , - -, , , however, we recognized that the ability to place antigen within multiple sites of the ad capsid protein would hold important potential for presenting multiple epitopes/ antigens or several copies of the same epitope within a single ad vector-based vaccine. in an effort to create multivalent hiv vaccine vectors, our study explored the use of ad hvr and hvr in hopes of creating vectors which contained hiv antigenic epitopes at both locales. to compare the flexibility and capacities of ad hvr and hvr , we genetically incorporated identical epitopes of incrementally increasing size within hvr or hvr of ad hexon. we incorporated identical epitopes ranging from to amino acids within the ad hexon hvr or hvr region. viable viruses were produced with incorporations of amino acids plus a -amino acid linker at hvr or hvr . in addition, viable viruses were produced with incorporations of up to amino acids plus a -amino acid linker at hvr . with respect to identical antigen incorporations at ad hvr or hvr , hvr was more permissive allowing an epitope incorporation of amino acids in total. these model antigens were surface exposed via elisa analysis. in vivo immunization with these vectors illustrated an antigen-specific immune response. along these same lines, abe and colleagues evaluated the ability of ad -based vectors expressing an hiv transgene to induce antigen-specific immune responses under ad preimmune conditions. to overcome limitations that are generally experienced as a result of pei to ad , they constructed vectors that have a modification in the hvr . their study characterized various immunological parameters generated by these vectors such as vector neutralization, acquisition of adaptive immune response, and comparison of protective immunity. first, in order to evaluate the utility of the modified ad vector, they measured the neutralizing activity of sera by a modified ad vector. they administered ad-luc (luciferase protein expressed as a transgene in the ad e region), ad-hisluc (his epitope presented in hvr region and luciferase protein expressed as a transgene), or ad-end/ aaaluc vector (containing three amino acid mutations in hvr and expressing luciferase protein) to mice im. after administration of these vectors, neutralizing activity against ad was observed for - weeks. the hexon-modified vector (ad-hisluc) generated the lowest ad -specific neutralizing activity, which was significantly lower than what was generated by ad-luc at weeks and , and by ad-end/aaaluc vector at week . the individual neutralizing activity of ad-hisluc immunization was significantly lower than that of ad-luc immunization. additional studies performed by abe and colleagues support the concept that modified hexon thwarts ad nabs and promotes cellular immune responses. studies performed by this research group indicate that a change in the immunogenic epitope is necessary to avoid neutralization by pre-existing nabs. our recently published work exploits the antigen capsidincorporation strategy for hiv vaccination. our novel vectors were constructed in hopes of moving toward the goal of creating vectors that will provide cellular and humoral hiv immunity. our study is the first of its kind to genetically incorporate an hiv antigen within the ad hexon hvr alone or in combination with genomic incorporation of a gag transgene (ad /hvr -mper-l (gag)). in this study, we successfully incorporated a -amino acid epitope of hiv- within hvr . the hiv- region selected for hvr incorporation was the membrane proximal ectodomain region (mper) derived from hiv- glycoprotein (gp ). our rationale for choosing a portion of the mper (eknekel-leldkwaslwnwfditn) derived from gp was based on the fact that the gp envelope protein ectodomain is a target of three broadly neutralizing anti-hiv- antibodies. when the mper was incorporated into hvr in combination with transgene expression, we observed growth kinetics and thermostability changes similar to those of other capsid-incorporated vectors generated in other studies, , indicating that incorporation of the mper epitope within hvr was not dramatically detrimental to virological characteristics. , in addition, we demonstrated that the mper epitope is surface exposed within hvr . most importantly, we observed a humoral anti-hiv response in mice vaccinated with the hexon-modified vector. the mper-modified vector allows boosting compared to adcmvgag, possibly because the ad /hvr -mper-l (gag) ad elicits less anti-ad immune response. it is possible that the mper epitope reduced the immunogenicity of the ad vector. this finding is noteworthy because hvr has not been fully explored for antigen capsid-incorporation strategies. these vectors are currently being analyzed by cryo-electron microscopic analysis to determine the critical correlates related to antigen placement/configuration and immune response. in addition, with respect to hiv- vaccination, the antigen capsid-incorporation strategy has been evaluated within the context of hrv. research groups have constructed human rhinovirus:hiv- chimeras in an effort to stimulate immunity against hiv- . , in an effort to develop hiv- vaccines, researchers within this same group generated combinatorial libraries of hrv capsid-incorporated hiv- gp epitope. their results indicated that they were successful in eliciting antibodies whose activity can mimic the nab effect. commercial and clinical ad development of hiv- vaccines have progressed preferentially more than vector systems such as hrv because the flexibility of ad generally exceeds current rhinovirus systems. for example, because hrv is a relatively small rna virus, the hrv platform can display an array limited to copies of a single hiv- epitope. , in contrast, the ad vector platform allows incorporation of the hiv- mper epitope into three structurally distinct locales, including hvr , hvr , and protein ix (our unpublished data). in comparison, the ad mper antigen capsid-incorporation display platform could present an array of hiv- epitope copies within ad hexon and hiv- epitope copies within pix. if a multivalent ad vector is generated with hiv- epitopes within the hexon and the pix locales, this would represent hiv epitopes within one ad vector. another significant difference between the ad and hrv platforms is in the number of locales that have been successfully used for heterologous epitope insertion. finally, in contrast to the rhinovirus that lacks this capacity, the ad platform has sufficient coding capacity allowing for hiv- transgene expression in combination with presenting the same or a different antigen on the viral capsid surface. this latter finding is important because it provides the basis for constructing vectors that will provide cellular and humoral hiv- immunity. vectors which provide both cellular and humoral immunity may be the way forward with respect to prophylactic hiv vaccine development. recently, there have been encouraging developments regarding hiv vaccination. in the s, in thailand, there was a substantial increase in the prevalence of infection with hiv- . [ ] [ ] [ ] by first observation, these groups consisted of intravenous-drug users and commercial sex workers; this infected group then expanded to the general population. by the mid s, the overall seroprevalence of hiv- reached a peak of . % among members of the royal thai army and of . % among people from northern thailand. , the thai ministry of public health acted by starting an effective hiv-prevention campaign. with this effort, the number of new hiv- infections per year decreased from an estimated , in to , in . , [ ] [ ] [ ] although this decrease was promising, there was still a desire to do more to prevent hiv infection. to achieve this goal, an hiv phase iii study was begun. the thai phase iii hiv vaccine study, also known as rv , opened in the fall of . the placebo-controlled trial tested the safety and effectiveness of a prime-boost regimen of two vaccines: alvac-hiv vaccine (the prime), a modified canarypox vaccine, and aidsvax b/e vaccine (booster), a gp vaccine. the vaccines were based on the subtype e and b hiv- strains that commonly circulate in thailand. the subtype b hiv- strain is the most commonly found strain in the united states. the trial, conducted in the chonburi and rayong provinces of thailand, enrolled , women and men aged - years at various levels of risk for hiv infection. study participants received the placebo or alvac hiv vaccine at enrollment and again after , , prophylactic vaccine for hiv- and months. the placebo or aidsvax b/e vaccine was given to participants at and months. participants were tested for hiv- infection every months for years. during each clinic visit, study participants were counseled on how to prevent hiv- infection. the results showed that of placebo recipients became infected with hiv- compared with of participants who received the vaccine. this level of effectiveness in preventing hiv- infection was found to be statistically significant. the vaccine strategy had no effect, however, on the amount of virus in the blood of volunteers who acquired hiv- infection during the study. based on the final analysis of the study, the surgeon general of the us army, the trial sponsor, announced that the prime-boost investigational vaccine regimen was safe and % effective in preventing hiv- infection. with respect to an hiv- vaccine that can provide sterilizing hiv immunity, this is the best result in humans to date. however, the modest protection effect appeared limited to low-risk individuals, and there were data which suggest that this effect was confined to the first year following administration of the vaccine. efforts must continue to focus on evaluating the immune response induced by the vaccine to establish potential correlates of protection. over the last three decades, the world has been faced with the emergence and subsequent epidemic of hiv/aids. there has been much progress with respect to diagnosis and prevention. on the treatment front, there have been several significant advances with respect to drug development (ie, art/haart). however, there is a desperate need for an effective and safe vaccine. there has been tremendous difficulty with regard to developing a vaccine that provides sterilizing immunity. this has been the case due to some of the factors mentioned in this review such as hiv diversity, immune evasion, and lack of appropriate animal models. due to these obstacles, many researchers assumed that the control of hiv- viremia by vaccination would be a more realistic goal than the development of sterilizing immunity. the road to a safe and effective hiv- vaccine received a serious setback in the fall of with the premature termination of the merck-hiv- vaccine step trial due to the lack of efficacy and early speculation that the vaccine might have increased the risk of hiv infection in some populations of vaccinees. in late , promising results came in from thailand in response to their efforts to create a safe and effective vaccine against hiv- . a community-based, randomized, multicenter, double-blinded, placebo-controlled efficacy trial using a prime-boost combination showed % effectiveness in preventing hiv- infection. these results lend promise to the hope of producing an hiv- vaccine vector that yields sterilizing hiv- immunity. in the future, research scientists must work together to increase hiv- vaccine effectiveness beyond %. realization of this goal may be accomplished by some of the techniques mentioned in this review, such as acquisition of hiv mucosal immunity, development of effective prime-boost strategies, development of better animal models, better molecular antigen modeling and presentation, avoidance of pei (by the means of using novel vector serotypes in combination with pegylation), and/or induction of nabs (by means of capsid incorporation of hiv antigens within viral vectors). these are just a few considerations that scientists and clinicians must consider with respect to the development of an effective and safe hiv- vaccine. scientists and clinicians must also consider that one vector or scheme may not be sufficient with respect to providing effective hiv- immunity and some combination of the above-mentioned potential strategies may offer the most promising method of producing an effective hiv- prophylactic vaccine. submit your manuscript | www.dovepress.com dovepress dovepress report on the global aids epidemic adherence to antiretroviral therapy: a survey of factors associated with medication usage lipodystrophy, metabolic disorders, and human immunodeficiency virus infection: aquitaine cohort, france, . groupe d'epidemiologie clinique du syndrome d'immunodeficience acquise en aquitaine a syndrome of lipoatrophy, lactic acidaemia and liver dysfunction associated with hiv nucleoside analogue therapy: contribution to protease inhibitor-related lipodystrophy syndrome longitudinal evolution of bone mineral density and bone markers in human immunodeficiency virus-infected individuals the latent hiv- reservoir in patients undergoing haart: an archive of pre-haart drug resistance mucosal immunity to hiv: a review of recent literature hiv vaccine design and the neutralizing antibody problem aids/hiv. developing an aids vaccine: need, uncertainty, hope hiv- vaccine development: progress and prospects t cell vaccines for microbial infections the failed hiv merck vaccine study: a step back or a launching point for future vaccine development? protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells rapid development of vaccine protection in macaques by live-attenuated simian immunodeficiency virus long-term symptomless hiv- infection in recipients of blood products from a single donor genetic instability of live, attenuated human immunodeficiency virus type vaccine strains update on long-term symptomless hiv type infection in recipients of blood products from a single donor hiv vaccines: new frontiers in vaccine development placebo-controlled phase trial of a recombinant glycoprotein vaccine to prevent hiv- infection correlation between immunologic responses to a recombinant glycoprotein vaccine and incidence of hiv- infection in a phase hiv- preventive vaccine trial lessons from failure -preparing for future hiv- vaccine efficacy trials randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein hiv- vaccine among injection drug users in global and regional distribution of hiv- genetic subtypes and recombinants in a new human immunodeficiency virus derived from gorillas preferential in-utero transmission of hiv- subtype c as compared to hiv- subtype a or d cell reservoirs in lymph nodes infected with hiv- subtype e differ from subtype b: identification by combined in situ polymerase chain reaction and immunohistochemistry different rates of disease progression of hiv type infection in tanzania based on infecting subtype relation between chemokine receptor use, disease stage, and hiv- subtypes a and d: results from a rural ugandan cohort escape artist par excellence genetic subtypes, humoral immunity, and human immunodeficiency virus type vaccine development viral sequence diversity: challenges for aids vaccine designs standardized assessment of nab responses elicited in rhesus monkeys immunized with single-or multi-clade hiv- envelope immunogens a phase / study of a multiclade hiv- dna plasmid prime and recombinant adenovirus serotype boost vaccine in hiv-uninfected east africans (rv ) mosaic vaccines elicit cd (+) t lymphocyte responses that confer enhanced immune coverage of diverse hiv strains in monkeys hiv vaccines: mosaic approach to virus diversity mosaic hiv- vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys antibody protects macaques against vaginal challenge with a pathogenic r simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro human neutralizing monoclonal antibodies of the igg subtype protect against mucosal simian-human immunodeficiency virus infection protection of macaques against vaginal transmission of a pathogenic hiv- /siv chimeric virus by passive infusion of neutralizing antibodies evolutionary and immunological implications of contemporary hiv- variation the antigenic structure of the hiv gp envelope glycoprotein structure of an hiv gp envelope glycoprotein in complex with the cd receptor and a neutralizing human antibody antibody neutralization and escape by hiv- cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus antibodies to cd -induced sites in hiv gp correlate with the control of shiv challenge in macaques vaccinated with subunit immunogens broad and potent neutralizing antibodies from an african donor reveal a new hiv- vaccine target efficient neutralization of primary isolates of hiv- by a recombinant human monoclonal antibody. science a conserved neutralizing epitope on gp of human immunodeficiency virus type a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp of human immunodeficiency virus type cross-clade neutralization of primary isolates of human immunodeficiency virus type by human monoclonal antibodies and tetrameric cd -igg scd - b bifunctional protein: extremely broad and potent neutralization of hiv- env pseudotyped viruses from genetically diverse primary isolates analysis of memory b cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from hiv- -infected individuals long-term nonprogression in hiv infection: methodological issues and scientific priorities the immunology of hiv-infected long-term nonprogressors-a current view long-term observations of human immunodeficiency virus-infected chimpanzees simian and feline immunodeficiency viruses: animal lentivirus models for evaluation of aids vaccines and antiviral agents the feline model of neuroaids: understanding the progression towards aids dementia feline immunodeficiency virus model for designing hiv/aids vaccines human immunodeficiency virus infection of human-pbl-scid mice suppression of hiv infection in azt-treated scid-hu mice disseminated and sustained hiv infection in cd + cord blood cell-transplanted rag -/-gamma c-/-mice animal models of aids infection of cynomolgus monkeys with a chimeric hiv- /sivmac virus that expresses the hiv- envelope glycoproteins a chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type isolate env causes an aids-like disease after in vivo passage in rhesus monkeys highly pathogenic shivs and sivs target different cd + t cell subsets in rhesus monkeys, explaining their divergent clinical courses efficacy assessment of a cell-mediated immunity hiv- vaccine (the step study): a doubleblind, randomised, placebo-controlled, test-of-concept trial replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity importance of the nef gene for maintenance of high virus loads and for development of aids protective effects of a live attenuated siv vaccine with a deletion in the nef gene molecular and biological characterization of simian immunodeficiency virus macaque strain h proviral clones containing nef size variants macaques infected with live attenuated sivmac are protected against superinfection via the rectal mucosa immunization with a live, attenuated simian immunodeficiency virus (siv) prevents early disease but not infection in rhesus macaques challenged with pathogenic siv live, attenuated simian immunodeficiency virus sivmac-m , with point mutations in the env transmembrane protein intracytoplasmic domain, provides partial protection from mucosal challenge with pathogenic sivmac protection of macaques against simian immunodeficiency virus infection with inactivated vaccines: comparison of adjuvants, doses and challenge viruses. the european concerted action on 'macaque models for aids research' whole inactivated siv vaccine grown on human cells fails to protect against homologous siv grown on simian cells protection of monkeys by a split vaccine against sivmac depends upon biological properties of the challenge virus dual-subtype vaccine (fel-o-vax fiv) protects cats against contact challenge with heterologous subtype b fiv infected cats dual-subtype fiv vaccine protects cats against in vivo swarms of both homologous and heterologous subtype fiv isolates effect of dual-subtype vaccine against feline immunodeficiency virus infection dual-subtype fiv vaccine (fel-o-vax fiv) protection against a heterologous subtype b fiv isolate efficacy testing of recombinant human immunodeficiency virus (hiv) gp as a therapeutic vaccine in early-stage hiv- -infected volunteers. rgp hase ii vaccine investigators randomised trial of mnrgp hiv- vaccine in symptomless hiv- infection phase ii controlled trial of post-exposure immunization with recombinant gp versus antiretroviral therapy in asymptomatic hiv- -infected adults. vaxsyn protocol team repeated immunization with recombinant gp human immunodeficiency virus (hiv) envelope protein in early hiv- infection: evaluation of the t cell proliferative response a phase i evaluation of the safety and immunogenicity of vaccination with recombinant gp in patients with early human immunodeficiency virus infection. military medical consortium for applied retroviral research two double-blinded, randomized, comparative trials of human immunodeficiency virus type (hiv- ) envelope vaccines in hiv- -infected individuals across a spectrum of disease severity: aids clinical trials groups a nd a randomized, placebocontrolled study of the immunogenicity of human immunodeficiency virus (hiv) rgp vaccine in hiv-infected subjects with . or = / mm cd t lymphocytes improved cell-mediated immune responses in hiv- -infected asymptomatic individuals after immunization with envelope glycoprotein gp immunization with envelope mn rgp vaccine in human immunodeficiency virus-infected pregnant women immunogenicity of the yeast recombinant p /p :ty virus-like particles (p -vlp) in healthy volunteers immunization with recombinant p /p :ty virus-like particles in human immunodeficiency virus-infected persons safety and immunogenicity of a candidate therapeutic vaccine, p virus-like particle, combined with zidovudine, in asymptomatic subjects therapeutic vaccination with p -vlp and zidovudine augments hiv-specific cytotoxic t lymphocyte activity in asymptomatic hiv-infected individuals induction of potent humoral and cell-mediated immune responses following direct injection of dna encoding the hiv type env and rev gene products retroviral vectors for vaccine development: induction of hiv- -specific humoral and cellular immune responses in rhesus macaques using a novel mlv(hiv- ) pseudotype vector a single administration of lentiviral vectors expressing either full-length human immunodeficiency virus (hiv- )(hxb ) rev/env or codon-optimized hiv- (jr-fl) gp generates durable immune responses in mice rabies virus-based vaccines elicit neutralizing antibodies, poly-functional cd + t cell, and protect rhesus macaques from aids-like disease after siv(mac ) challenge highly attenuated rabies virus-based vaccine vectors expressing simian-human immunodeficiency virus . p env and simian immunodeficiency virusmac gag are safe in rhesus macaques and protect from an aids-like disease protection of rhesus monkeys against infection with minimally pathogenic simian-human immunodeficiency virus: correlations with neutralizing antibodies and cytotoxic t cells vaccination with alvac and aidsvax to prevent hiv- infection in thailand vector-mediated gene transfer engenders long-lived neutralizing activity and protection against siv infection in monkeys enhancement of humoral immunity to sivenv following simultaneous inoculation of mice by three recombinant adenoviruses encoding sivenv/poliovirus chimeras, tat and rev replication-defective adenovirus serotype vectors elicit durable cellular and humoral immune responses in nonhuman primates adenovirus vectored vaccines replication-deficient recombinant adenoviruses expressing the human immunodeficiency virus env antigen can induce both humoral and ctl immune responses in mice multi-envelope hiv- vaccine devoid of siv components controls disease in macaques challenged with heterologous pathogenic shiv cytotoxic t lymphocyte and antibody responses generated in rhesus monkeys immunized with retroviral vector-transduced fibroblasts expressing human immunodeficiency virus type- iiib env/rev proteins evaluation of a self-inactivating lentiviral vector expressing simian immunodeficiency virus gag for induction of specific immune responses in vitro and in vivo chimaeric hiv- subtype c gag molecules with large in-frame c-terminal polypeptide fusions form virus-like particles optimization of chimeric hiv- virus-like particle production in a baculovirus-insect cell expression system increased incorporation of chimeric human immunodeficiency virus type gp proteins into pr gag virus-like particles by an epstein-barr virus gp / -derived transmembrane domain recombinant human immunodeficiency pr gag virus-like particles presenting chimeric envelope glycoproteins induce cytotoxic t-cells and neutralizing antibodies development of hiv/aids vaccine using chimeric gag-env virus-like particles dna vaccines induction of gp -specific protective immune responses by genetic vaccination with linear polyethylenimine-plasmid complex dna vaccines: a review comparison of protective effect of protein and dna vaccines hsp in murine model of systemic candidiasis dna vaccines against human immunodeficiency virus type in the past decade direct gene transfer into mouse muscle in vivo enhancement of human immunodeficiency virus type -dna vaccine potency through incorporation of t-helper molecular adjuvants modulation of cellular and humoral immune responses to a multiepitopic hiv- dna vaccine by interleukin- dna immunization/ viral protein boost evaluation in macaques of hiv- dna vaccines containing primate cpg motifs and fowlpoxvirus vaccines co-expressing ifngamma or il- augmentation and suppression of immune responses to an hiv- dna vaccine by plasmid cytokine/ig administration cell-specific delivery of genes with glycosylated carriers organspecific gene expression in the rhesus monkey eye following intravenous non-viral gene transfer systemic linear polyethylenimine (l-pei)-mediated gene delivery in the mouse targeted gene delivery to cancer cells: directed assembly of nanometric dna particles coated with folic acid an hiv- clade c dna prime, nyvac boost vaccine regimen induces reliable, polyfunctional, and long-lasting t cell responses safety and immunogenicity study of multiclade hiv- adenoviral vector vaccine alone or as boost following a multiclade hiv- dna vaccine in africa differential effect of lentil feeding on proteosynthesis rates in the large intestine, liver and muscle of rats delivery of dna hiv- vaccine to the liver induces high and long-lasting humoral immune responses immunopathogenesis and immunotherapy in aids virus infections a role for carbohydrates in immune evasion in aids electrically mediated plasmid dna delivery to hepatocellular carcinomas in vivo tail-vein injection of mannan-binding lectin dna leads to high expression levels of multimeric protein in liver mechanism of plasmid delivery by hydrodynamic tail vein injection. ii. morphological studies mechanism of plasmid delivery by hydrodynamic tail vein injection. i. hepatocyte uptake of various molecules the efficient expression of intravascularly delivered dna in rat muscle hydrodynamics-based transfection in animals by systemic administration of plasmid dna high levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid dna hydrodynamic gene delivery: its principles and applications the asialoglycoprotein receptor: relationships between structure, function, and expression synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for cellular targeting adenovirus serotype hexon mediates liver gene transfer adenovirus serotype hexon is critical for virus infection of hepatocytes in vivo viral vectors for malaria vaccine development human rhinovirus type :human immunodeficiency virus type (hiv- ) v loop chimeras from a combinatorial library induce potent neutralizing antibody responses against hiv- broad neutralization of human immunodeficiency virus type (hiv- ) elicited from human rhinoviruses that display the hiv- gp eldkwa epitope use of adenovirus in vaccines for hiv development of nonhuman adenoviruses as vaccine vectors poliovirus vaccine strains as mucosal vaccine vectors and their potential use to develop an aids vaccine control of a mucosal challenge and prevention of aids by a multiprotein dna/mva vaccine recombinant poxviruses as mucosal vaccine vectors induction of neutralizing antibody responses to anthrax protective antigen by using influenza virus vectors: implications for disparate immune system priming pathways subcutaneous administration of a recombinant vaccinia virus vaccine expressing multiple envelopes of hiv- an adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism targeted adenoviral vectors viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics live attenuated measles vaccine as a potential multivalent pediatric vaccination vector infection of monocytes during measles vaccinology, immunology, and comparative pathogenesis of measles in the quest for a preventative against aids preexisting immunity to poliovirus does not impair the efficacy of recombinant poliovirus vaccine vectors strategies to overcome host immunity to adenovirus vectors in vaccine development neutralizing antibodies to adenovirus serotype vaccine vectors are directed primarily against the adenovirus hexon protein construction and characterization of adenovirus serotype packaged by serotype hexon hexon-chimaeric adenovirus serotype vectors circumvent pre-existing anti-vector immunity immune responses to adenovirus and adeno-associated virus in humans the impact of developmental stage, route of administration and the immune system on adenovirus-mediated gene transfer circumventing the immune response to adenovirus-mediated gene therapy hexon gene switch strategy for the generation of chimeric recombinant adenovirus sero-switch' adenovirusmediated in vivo gene transfer: circumvention of anti-adenovirus humoral immune defenses against repeat adenovirus vector administration by changing the adenovirus serotype circumvention of vectorspecific neutralizing antibody response by alternating use of human and non-human adenoviruses: implications in gene therapy generation of a novel replication-incompetent adenoviral vector derived from human adenovirus type : manufacture on per.c cells, tropism and immunogenicity dependence of adenovirus infectivity on length of the fiber shaft domain adenovirus type virions contain two distinct fibers human adenovirus type contains two fibers structure-based identification of a major neutralizing site in an adenovirus hexon characterization of a family of chimpanzee adenoviruses and development of molecular clones for gene transfer vectors replication-defective vector based on a chimpanzee adenovirus chimpanzee adenovirus vaccine protects against zaire ebola virus efficient flpe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination high-capacity, helperdependent, 'gutless' adenoviral vectors for gene transfer into brain regulatable gutless adenovirus vectors sustain inducible transgene expression in the brain in the presence of an immune response against adenoviruses immunization against the transgene but not the teton switch reduces expression from gutless adenoviral vectors in the brain comparison of replicationcompetent, first generation, and helper-dependent adenoviral vaccines protection against mucosal shiv challenge by peptide and helper-dependent adenovirus vaccines effects of shielding adenoviral vectors with polyethylene glycol (peg) on vector-specific and vaccine-mediated immune responses pegylated adenovirus for targeted gene therapy development of pegylated adenovirus vector with targeting ligand neutralizing antibody evasion ability of adenovirus vector induced by the bioconjugation of methoxypolyethylene glycol succinimidyl propionate (mpeg-spa) pegylated adenovirus vectors containing rgd peptides on the tip of peg show high transduction efficiency and antibody evasion ability evaluation of polyethylene glycol modification of first-generation and helper-dependent adenoviral vectors to reduce innate immune responses pegylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile vaccination against hepatitis c virus with dendritic cells transduced with an adenovirus encoding ns protein cell vehicle targeting strategies current strategies and future directions for eluding adenoviral vector immunity factors influencing therapeutic efficacy and the host immune response to helper-dependent adenoviral gene therapy in hemophilia a mice elimination of innate immune responses and liver inflammation by pegylation of adenoviral vectors and methylprednisolone administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons baseline ad serostatus does not predict ad hiv vaccine-induced expansion of adenovirusspecific cd + t cells translational mini-review series on vaccines for hiv: t lymphocyte trafficking and vaccine-elicited mucosal immunity route of adenovirus-based hiv- vaccine delivery impacts the phenotype and trafficking of vaccine-elicited cd + t lymphocytes differential specificity and immunogenicity of adenovirus type neutralizing antibodies elicited by natural infection or immunization distribution of poliovirus antibody in serum, nasopharynx and alimentary tract following segmental immunization of lower alimentary tract with poliovaccine local antibody response to experimental poliovirus infection in the central nervous system of rhesus monkeys strategies for optimizing targeting and delivery of mucosal hiv vaccines primary hiv- infection is associated with preferential depletion of cd + t lymphocytes from effector sites in the gastrointestinal tract cd + t cell depletion during all stages of hiv disease occurs predominantly in the gastrointestinal tract mucosal aids vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques impact of vaccineinduced mucosal high-avidity cd + ctls in delay of aids viral dissemination from mucosa a novel functional ctl avidity/activity compartmentalization to the site of mucosal immunization contributes to protection of macaques against simian/human immunodeficiency viral depletion of mucosal cd + t cells intranasal immunization is superior to vaginal, gastric, or rectal immunization for the induction of systemic and mucosal anti-hiv antibody responses prophylactic vaccine for hiv- comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women nucleocapsid protein zinc-finger mutants of simian immunodeficiency virus strain mne produce virions that are replication defective in vitro and in vivo an internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface broadly neutralizing monoclonal antibodies f and e directed against the human immunodeficiency virus type gp membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus shivba-l immunogenicity of recombinant human adenovirus-human immunodeficiency virus vaccines in chimpanzees long-term protection of chimpanzees against high-dose hiv- challenge induced by immunization protection against mucosal simian immunodeficiency virus siv(mac ) challenge by using replicating adenovirus-siv multigene vaccine priming and subunit boosting evaluation of combination dna/replication-competent ad-siv recombinant immunization regimens in rhesus macaques novel adenovirus vaccine vectors based on the enteric-tropic serotype human viral gastroenteritis unique physicochemical properties of human enteric ad responsible for its survival and replication in the gastrointestinal tract human enteric adenovirus type (tak) contains a second fiber protein gene adenovirus-based primeboost immunization for rapid vaccination against anthrax development of a preventive vaccine for ebola virus infection in primates adenoviral expression of a truncated s subunit of sars-cov spike protein results in specific humoral immune responses against sars-cov in rats adenoviruses as vectors for hiv vaccines characterization of a permissive epitope insertion site in adenovirus hexon challenges for structure-based hiv vaccine design designing immunogens to elicit broadly neutralizing antibodies to the hiv- envelope glycoprotein rational antibody-based hiv- vaccine design: current approaches and future directions molecular architecture of native hiv- gp trimers structural definition of a conserved neutralization epitope on hiv- gp one step forwards, one step back prospects for vaccine protection against hiv- infection and aids modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses protection against p. aeruginosa with an adenovirus vector containing an oprf epitope in the capsid optimization of capsid-incorporated antigens for a novel adenovirus vaccine approach epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity expression of a foreign epitope on the surface of the adenovirus hexon identification of sites in adenovirus hexon for foreign peptide incorporation structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods protective immunity to pseudomonas aeruginosa induced with a capsid-modified adenovirus expressing p. aeruginosa oprf rgd inclusion in the hexon monomer provides adenovirus type -based vectors with a fiber knob-independent pathway for infection adenovirus type with modified hexons induces robust transgene-specific immune responses in mice with pre-existing immunity against adenovirus type structure and mechanistic analysis of the anti-human immunodeficiency virus type antibody f in complex with its gp epitope genetic incorporation of a herpes simplex virus type thymidine kinase and firefly luciferase fusion into the adenovirus protein ix for functional display on the virion derivation of a triple mosaic adenovirus based on modification of the minor capsid protein ix genetic incorporation of hsv- thymidine kinase into the adenovirus protein ix for functional display on the virion hiv antigen incorporation within adenovirus hexon hypervariable for a novel hiv vaccine approach use of random systematic mutagenesis to generate viable human rhinovirus chimeras displaying human immunodeficiency virus type v loop sequences drug design, development and therapy the asian epidemic model: a process model for exploring hiv policy and programme alternatives in asia hiv/aids prevention in thailand: success and challenges the epidemiology of hiv infection and aids in thailand preventive hiv vaccine development in thailand impact of thailand's hiv-control programme as indicated by the decline of sexually transmitted diseases risk factors for hiv infection among young adult men in northern thailand hiv/aids preventive vaccine 'prime-boost' phase iii trial: foundations and initial lessons learned from thailand grant support was provided by national institutes of health grants r ai - , p ai - a , and p ca - s . we thank erin e thacker for her thoughtful insight. the authors report no conflicts of interest in this work. submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal drug design, development and therapy is an international, peerreviewed open-access journal that spans the spectrum of drug design and development through to clinical applications. clinical outcomes, patient safety, and programs for the development and effective, safe, and sustained use of medicines are a feature of the journal, which has also been accepted for indexing on pubmed central. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord- -ni gzg authors: wolf, jayanthi; bruno, samantha; eichberg, michael; jannat, risat; rudo, sharon; vanrheenen, susan; coller, beth-ann title: applying lessons from the ebola vaccine experience for sars-cov- and other epidemic pathogens date: - - journal: npj vaccines doi: . /s - - - sha: doc_id: cord_uid: ni gzg the world is experiencing an unprecedented global pandemic of coronavirus disease (covid- ) caused by a novel coronavirus, severe acute respiratory syndrome-coronavirus- (sars-cov- ). development of new vaccines and therapeutics are important to achieve long-term prevention and control of the virus. experience gained in the development of vaccines for ebola virus disease provide important lessons in the regulatory, clinical, and manufacturing process that can be applied to sars-cov- and other epidemic pathogens. this report outlines the main lessons learned by merck sharp & dohme corp., a subsidiary of merck & co., inc., kenilworth, nj, usa (msd) during development of an ebola zaire vaccine (ervebo®) and looks ahead to critical lessons beyond vaccine development. it highlights focus areas for public-private partnership and regulatory harmonization that can be directly applied to current vaccine development efforts for sars-cov- , while drawing attention to the need for parallel consideration of issues beyond development that are equally important to achieve global preparedness and response goals. first discovered in the democratic republic of the congo in , the highly lethal ebola virus has infected people in a number of african countries leading to sporadic outbreaks of ebola virus disease over the past years. while most of the outbreaks have been limited in geographic scope and number of cases, two of the outbreaks over the last years have been large, resulting in major loss of life and socioeconomic disruption in the region. in early , an outbreak of ebola virus disease caused by zaire ebolavirus started in west africa and was declared a public health emergency of international concern in august of that year. by the time the outbreak ended in , more than , people had died and more than , people were infected primarily in three countries, guinea, liberia, and sierra leone . in the decade prior to the west african outbreak, research efforts for biodefense purposes resulted in the identification of some promising ebola vaccine candidates that protected monkeys from a lethal challenge of wild-type ebola virus . these vaccine candidates were not taken into clinical development prior to the west african outbreak for several reasons, which included the inability to demonstrate clinical efficacy in the absence of an ongoing outbreak and lack of interest by the public health and vaccine development community to invest in the lengthy and costly process of vaccine development without a clear demand for an ebola vaccine . the west african outbreak changed this perspective and numerous vaccines entered clinical development during the outbreak resulting in the generation of safety and immunogenicity data for many of these novel vaccine candidates and the demonstration of efficacy for one vaccine . ervebo® is a live, attenuated, recombinant vesicular stomatitis virus (rvsv)-based, chimeric-vector vaccine, where the vsv envelope g protein was deleted and replaced by inserting only the envelope glycoprotein of zaire ebolavirus. extraordinary efforts were made to advance this vaccine candidate through phase , , and clinical trials and the data generated in the context of the west african ebola outbreak has supported its licensure by the us food and drug administration (fda), conditional authorization by the european medicines agency (ema) and several african countries, along with prequalification by the who. the period of years from the start of phase trials in oct to the approval of this vaccine in nov , was much faster than the typical - year timeline for vaccine development and approval . a timeline of the key activities in the development of this ebola vaccine is summarized in fig. and described in detail in the following sections of this article. through this ebola vaccine development effort a number of learnings have been identified, which are highly relevant for the current vaccine development efforts in response to the covid- pandemic. a summary of the key lessons learned can be found in fig. field response and non-governmental service organizations (such as medecins sans frontieres); global public health entities (such as the world health organization, wellcome trust, and gavi); universities (e.g., the university of geneva, dalhousie university) and private sector companies (such as newlink genetics, idt biologika, and msd). each of these entities had a specific role in the broad partnership, including conducting preclinical studies, manufacturing good manufacturing practices (gmp) trial materials, conducting clinical trials, and funding portions of the research and development. msd has a well-established history in manufacturing and clinical development of vaccines, with experience in taking novel vaccines through the regulatory process to approval and providing global access. however, msd did not have prior experience with ebola virus nor clinical development expertise in the african countries in which the ebola outbreak was taking place. therefore, msd relied on the expertise of organizations that were already involved in the public health response efforts to implement and complete nearly all the clinical trials. important lessons from this effort are that leveraging the expertise of different organizations can help to accelerate product development and that broad public-private partnerships require strong leadership and coordination to facilitate cooperation of diverse partners with different expertise and missions. furthermore, concerns over evaluation of any investigational product are present in less developed parts of the world and these concerns may be further exacerbated during an outbreak . therefore, clear and transparent communication with community leaders and the community at large, ethical review committees, local ministries of health, and other government agencies is critical to ensure that the purpose of the research is well understood and aligned with accepted local and international norms. failure to do so can result in delays or roadblocks to research on potentially life-saving products. ultimately the responsibility to convert the data generated by all the collaborators into a coherent regulatory submission to support licensure of a product lies with the marketing authorization holder. recognition and proactive planning for that eventuality can help to accelerate the regulatory submission to support product approval. examples of these types of activities include the following. data integration msd's role in advancing the product to licensure included integrating all the data generated by partners to support the marketing authorization applications. nine of nonclinical studies, of clinical assay validations, and of clinical trials were performed by partners. msd needed to obtain all the datasets and reports from these partners to include in the license applications. data from clinical trials that were performed by partners needed to be migrated into a central database that could be used for data integration, analysis, report-writing, and submission by msd to regulatory agencies. a lesson learned during this process is to account for the time needed to convert different formats and languages used to collect the data into standard formats required by regulatory agencies, such as use of the medical dictionary for regulatory activities (meddra) and study data tabulation model (sdtm). with the clinical trials all being designed and implemented in the context of the outbreak, it was not possible to harmonize the study designs. while this is not an issue a priori for the conduct of the trials, it introduced challenges when assembling the data to support licensure. for example, it was not possible to conduct extensive integration of the safety data for the product because the data collected and methods used were different for each trial. a key lesson learned during this process is that standardization of clinical trial protocols and data collection is preferred, and that to the extent possible late stage clinical trial designs should be defined prior to outbreaks. another challenge specifically related to conducting trials in response to emerging diseases is that there is a need to build clinical development capacities, laboratory and adverse event reporting infrastructure in outbreak-prone regions. this was a challenge taken on by individual trial sponsors in the affected countries and required significant and sustained support by the trial sponsors and their local partners. establishing mechanisms to maintain the capabilities established during outbreak response to provide experienced clinical trial sites for future research is another key lesson learned. examples of this in action include the ongoing partnership between the us and liberia and the partnership for research on ebola vaccination (prevac) consortium established in the wake of the west africa outbreak. clinical assays a key immunogenicity endpoint assay was developed and validated through a collaborative effort by private and public sector partners. the intent was to provide an assay available to all vaccine developers, which might ultimately allow comparison of different vaccine candidates. while the collaborative effort was successful, balancing the input of numerous partners took time. consequently, clinical assay validation took two years to complete, resulting in a back-log of samples from clinical trials that needed to be processed. a lesson learned is that whenever possible clinical immunogenicity sample collection should be standardized and assays should be validated prior to the start of late stage clinical trials. this requires knowledge of the target pathogen well in advance of clinical trials, which is a challenge for sars-cov- . expanded access use of the vaccine prior to approval with the rapid collection of efficacy data in support of the vaccine, it was recognized that there was the possibility that new ebola outbreak(s) could occur ahead of the licensure and availability of licensed doses and that a mechanism was needed to support access to the investigational product. for this scenario, it was necessary to manufacture doses to support outbreak response and establish regulatory frameworks needed to support vaccine deployment (e.g., compassionate use, expanded access, or emergency use). the who, msf, and other groups took the lead in implementing expanded access protocols in african countries, working closely with national governments and ministries of health. through this effort, more than , people were vaccinated during the - ebola outbreak in the democratic republic of the congo . a lesson learned is that the deployment of an investigational product requires careful consideration and thorough planning. the manufacturing process had to be rapidly scaled-up to handle the needs for emergency use and expanded access programs, in addition to establishing a final manufacturing facility. the approach taken by msd was to take the initial manufacturing process that was developed at a contract manufacturing organization, scale it up in a clinical manufacturing facility, and transfer it to a commercial manufacturing facility. technology transfer and the establishment of a new manufacturing facility has many steps and requires significant time to execute ( - years is typical for a new vaccine). there is limited understanding outside of vaccine manufacturers and regulators on the rigorous requirements leading to approval of a vaccine manufacturing facility. this broad lack of understanding can lead to misperceptions of "delays" when in fact timelines are driven by the elements required for licensure. for example, although clinical data were available in - , the vaccine could not be approved until the commercial manufacturing site was established due to expectations from regulatory agencies that the manufacturing process needs to be validated at the final manufacturing site. a lesson learned is that education is needed on how vaccines are manufactured and the time needed to establish and gain approval for a new manufacturing facility. this is particularly relevant for the clinical • exisƟng preclinical data on the vaccine plaƞorm can accelerate the start of phase trials during outbreaks. • clear communicaƟon with community leaders and government agencies is criƟcal to ensure the purpose of clinical research is well understood. • standardizaƟon of clinical trial protocols and data collecƟon is needed and late stage clinical trial designs should be defined prior to outbreak. • need to build and maintain clinical development capaciƟes, laboratory and adverse event reporƟng infrastructure in outbreak-prone regions. • clinical immunogenicity sample collecƟon should be standardized and assays validated prior to the start of late stage clinical trials. • deployment of an invesƟgaƟonal product requires careful consideraƟon and thorough planning. • it takes Ɵme to convert different formats and languages used to collect the clinical data into standard formats required by regulatory agencies. • educaƟon is needed on how vaccines are manufactured and the Ɵme needed to establish and gain approval for a new manufacturing facility. • aggressive Ɵmelines are enƟcing, but require "right-first-Ɵme" manufacturing execuƟon with low/medium probability of success for new vaccines. • global and regional harmonizaƟon is criƟcal for the efficient development and licensure of pandemic vaccines. • regulatory agency collaboraƟon is criƟcal for success. • clinical data requirements for licensure need to be clearly defined and harmonized across global regulatory agencies. • frequent discussions with regulatory agencies and expedited regulatory pathways can accelerate the program. • who's leadership is needed to facilitate innovaƟve review processes and collaboraƟve mechanisms to expedite global approvals. • addiƟonal regulaƟons for recombinant live virus vaccines can lead to delays in starƟng clinical trials, manufacturing, tesƟng and shipments. • harmonized labeling and packaging requirements would facilitate pandemic vaccines distribuƟon. • public-private partnerships help accelerate product development by leveraging experƟse from different organizaƟons. • policy, program, and system innovaƟons are needed to realize the full promise of new vaccines in advancing global public health preparedness. covid- outbreak where all aspects of vaccine development are being accelerated at an extraordinary pace with the goal of being able to produce unprecedented quantities of vaccine to address global needs. manufacturing site selection for vaccines is a complex decision, which is further complicated when seeking to move fast with incomplete information. the company needed to manage multiple factors, such as existing space, technical capability, infrastructure, and capacity. site selection also needed to account for the regulations in the country in which the manufacturing site is located and the permits and licenses necessary to support the efforts. the country's employment environment and labor laws can also impact the ability to hire qualified staff quickly where these factors need to be considered early. a lesson learned is that aggressive timelines are enticing for public health partners, but also require "right-first-time" execution to qualify the facility and manufacturing process, for which the probability of success for a new vaccine is likely medium to low. all parties involved must work to balance the desire to be ambitious (e.g., rapid development timelines) with execution realities and stakeholder expectations. parallel work and extensive collaboration between manufacturers will be needed in order to successfully bring a sars-cov- vaccine to the world. regulatory agency collaboration is critical for success from the start of the west african ebola outbreak, the us fda, ema, and health canada worked closely with each other and with the national regulatory authorities of the impacted west african countries, sharing information about candidate vaccines that were being tested and reviewing the clinical protocols, available data, and benefit-risk profiles. frequent conversations with manufacturers helped the agencies expedite the start of clinical trials simultaneously in different countries. a lesson learned is that existing preclinical data and availability of clinical supplies afforded the opportunity to start phase trials rapidly during the west african ebola outbreak. clinical data requirements for licensure need to be clearly defined placebo-controlled, randomized, double-blind studies are typically used to demonstrate the efficacy and safety for new vaccines. however, in the midst of the ebola outbreak, some countries considered it unethical to administer placebo to at-risk individuals. different study designs were implemented for the phase / trials and a cluster-randomized trial conducted by the world health organization in guinea was the only trial that demonstrated efficacy . efficacy data from this trial, together with safety data from trials and immunogenicity data with validated assays, were accepted for registration by regulatory agencies. a lesson learned is that the regulatory pathway to licensure for pandemic vaccines need to be clearly defined. novel trial designs might need to be implemented in order to maximize the possibility of evaluating efficacy during a waning outbreak, and alternative endpoints, such as using immune responses as a surrogate for efficacy, could be used to gain accelerated approval or conditional marketing authorization. frequent regulatory agency interactions are important based primarily on interim clinical efficacy data, the manufacturer applied for and was granted access to expedited regulatory pathways, such as priority medicines (prime) status from ema and breakthrough therapy designation (btd) from fda, which helped to obtain alignment on data requirements for product approval using frequent meetings. under btd, the fda accepted rolling submissions of portions of the biologics license application (bla), which resulted in approval in december , three months before fda's target approval date. the fda determined that a program specific advisory committee meeting was not needed. similar frequent discussions with ema under prime status, enabled the company to gain alignment on key aspects of the development program quickly. who leadership is needed to obtain rapid global approvals who is a recognized global public health leader with regional offices in many countries, and established connections with the african vaccines regulatory forum (avaref). in order to accelerate vaccine access to african countries, the world health organization's prequalification team (who-pqt) in collaboration with the ema and avaref developed an innovative facilitated process (roadmap) for decision making on the acceptability of the vaccine for registration . this allowed the company to make simultaneous submissions to ema, who-pqt, and regulatory authorities in african countries, with ema acting as the reference agency. following submission, the who-pqt and members from avaref, which represented the african countries, were invited to participate in teleconferences between msd and ema and in the inspection of the manufacturing facility. after a positive opinion from ema in october , the european commission granted a conditional marketing authorization on november th. within h, the who granted prequalification. one month later, african countries started to approve ervebo®, starting with burundi and the democratic republic of the congo. a lesson learned is that strong collaboration and willingness to share information with everyone involved resulted in registration nearly-simultaneously in the countries that needed the ebola vaccine. for sars-cov- vaccines, regulatory agencies should prepare for the simultaneous approval of candidates in multiple countries. who's leadership will be needed to facilitate innovative review processes and collaborative mechanisms to expedite approvals. recombinant viral vaccines need to overcome additional regulatory hurdles since this ebola vaccine was a recombinant virus, some countries required a detailed environmental risk assessment prior to the start of clinical trials and as part of the marketing authorization application. in some countries, the vaccine was considered a bio-safety level organism, requiring special handling and permits for the manufacturing and testing sites. these permits took a significant amount of time to obtain, which prevented rapid transfers of material. samples and technology, which even included parts of the marketing authorization application, required licenses for export because this vaccine is made from the genetic sequence of two viruses that are considered "dualuse" agents and subject to trade controls. a lesson learned is that these regulations led to delays and additional costs incurred in starting clinical trials, manufacturing, testing, and shipments. for manufacturers of sars-cov- vaccines, countries should consider waiving these requirements, or waiving the fees and expediting these processes. harmonized labeling and packaging requirements would facilitate pandemic vaccines distribution after approval, companies face challenges with product distribution due to the need to follow country-specific product labeling regulations covering package inserts, cartons, and artwork in addition to serialization requirements. this causes a supply chain issue for a vaccine product intended to be placed in a stockpile and diverted quickly to any country. a lesson learned is that these heterogenous requirements counter the goal of flexibility, speed, and cost-efficiency relevant to emergency preparedness, and there is an urgent need for harmonized solutions. electronic labeling and quick response (qr) codes could be a possible solution, but in most countries, legislation does not currently exist to support this option. there is an urgent need to find a solution to this problem for pandemic vaccines. in addition to considering lessons related to vaccine development for epidemic preparedness, it is equally important to consider the unique challenges and opportunities related to sustainable manufacturing, supply, access, and delivery of these vaccines at the scale and speed needed to achieve preparedness and response goals. such challenges include right-sizing manufacturing capacity and production while having to accommodate leadtimes, managing uncertain demand-and-supply dynamics, ensuring equitable allocation and access, and achieving operational and economic sustainability for all partners. producing and supplying any vaccine is inherently complex. it is exponentially more complex when there are high levels of uncertainty and unpredictability across nearly every dimension of the program: unpredictable disease, unpredictable and relatively low demand, unpredictable stakeholders and customers, unpredictable timing of need, and unpredictable geographies in which needs might arise. policy, program, and system innovation-in parallel to research and development innovation-needs to be addressed to realize the full promise of innovative, new vaccines in advancing global public health preparedness. emergency preparedness and response vaccines, such as ervebo®, are critical tools in the arsenal against pathogens that can cause pandemics. public-private partnerships are a powerful and effective approach to develop these vaccines. setting clear roles, expectations, and accountability enables each partner to bring their respective strengths to the effort. shared purpose, trust, flexibility, and cooperation are critical to ensure success. as highlighted in this report and in another recent article , the variability and complexity in regulatory processes and requirements interfere with the speed, flexibility, and efficiency needed to prepare for and respond to public health emergencies. there is an urgent need for global regulatory harmonization and standardization in the approach to the development of vaccines for emergency preparedness. regulatory agencies need to work together to remove roadblocks and put solutions in place to allow the rapid global development of vaccines for sars-cov- and other emerging pathogens. received: may ; accepted: june ; ebola: lessons on vaccine development accelerating vaccine development during the - west african ebola virus disease outbreak international federation of pharmaceutical manufacturers & associations. the complex journey of a vaccine ethics, emergencies and ebola clinical trials: the role of governments and communities in offshored research world health organization. ebola virus disease democratic republic of congo: external situation report efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola Ça suffit!) world health organization. roadmap for introduction and roll out of a licensed ebola vaccine developing covid- vaccines at pandemic speed. n. eng we thank all team members and management for their tireless efforts and contributions to this program. for their partnership and dedication, we thank external partners, collaborators, and funding organizations in addition to the study volunteers and study investigators. the authors also thank the who, fda, ema, avaref, and regulatory agencies that participated in the collaborative review procedure. we would also like to thank karyn davis of merck sharp & dohme corp., a subsidiary of merck & co., inc., kenilworth, nj, usa for editorial assistance. j.w. wrote the first draft of the manuscript. all co-authors provided critical inputs and revised the manuscript. all authors agreed to final submission. all authors are employees of merck sharp & dohme corp., a subsidiary of merck & co., inc., kenilworth, nj, usa (msd) and may own stock or stock options in the company. correspondence and requests for materials should be addressed to j.w.reprints and permission information is available at http://www.nature.com/ reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- - s ubbxl authors: drury, georgina; jolliffe, siobhan; mukhopadhyay, tarit k. title: process mapping of vaccines: understanding the limitations in current response to emerging epidemic threats date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: s ubbxl vaccination remains the most successful and effective mechanism of pathogen control. however, their development and deployment in epidemic settings have been limited, and the ebola outbreak in west africa identified several bottlenecks linked to a lack of investment in pathogen research, infrastructure or regulation. shortly after this outbreak, the uk government established the uk vaccine network to ensure the uk is better prepared to respond to pathogens outbreaks of epidemic potential. as part of their work, the network commissioned the creation of a vaccine development tool (http://www.vaccinedevelopment.org.uk/) to serve as a guide to the key stages in vaccine development. the tool also set out to capture the key, rate-limiting bottlenecks in the development of vaccines against emerging infectious disease such that corrective action could be taken, be it through research, funding, infrastructure and policy, both in the uk and internationally. the main research bottlenecks were related to understanding pathogen biology, identification of appropriate animal models and investment in the manufacturing sciences, especially into process development. infrastructure gaps in gmp manufacturing and fill-finish were also identified and limitations in gmo regulation and regulatory and ethical approvals, especially for outbreak pathogens required new policy initiatives. the uk vaccine network has since begun work to correct for these limitations with a series of funding calls and development programmes. this paper seeks to summarise the vaccine development tool and its key findings. timely response to epidemics requires the development, manufacture and distribution of vaccine. activities by the uk vaccine network (ukvn) and the who have seen the identification of several pathogens that have epidemic potential [ ] . however, the development of effective vaccines against these pathogens requires improved coordination between policy makers, funders, researchers, vaccine manufacturers, and regulators, if we are to develop and deploy vaccines to mitigate the spread of epi-or pandemics [ ] . set in the context of the ebola outbreak that began in , multiple stakeholders were mobilised to respond to the humanitarian crisis of this escalating outbreak. although there was no licenced ebola vaccine available, approximately fifteen different vaccines were in preclinical development, including dna vaccines, virus-like particles and viral vector [ ] . levine et al. [ ] described how these candidates had largely been developed as part of the bioterrorism preparedness programme, bioshield, in the united states. due to the relatively low geographic prevalence of infection there was a limited market and thus a poor case for industry to develop a vaccine for commercial purposes. however, concerns over the potential use of ebolavirus as a bioterrorism agent resulted in a number of stockpiled sources that had undergone testing in animal models [ ] . as the ebola epidemic worsened, the availability of small vaccine stockpiles, taken together with the threat of further spread, catalysed stakeholder engagement to test the vaccine in humans to determine safety for potential deployment. researchers, industrialists, regulators, and funders worked together to expedite the process of carrying out first-in-man trials of the ebola vaccine e-mail addresses: siobhan.jolliffe@homeoffice.gov.uk (s. jolliffe), ucbetkm@ucl. ac.uk (t.k. mukhopadhyay). unprecedented international consortium assembled to accelerate collaborative multi-site trials of candidate ebola vaccine, wellcome trust, th august (https://wellcome.ac.uk/press-release/unprecedented-international-consortiumassembled-accelerate-collaborative-multi-site). last accessed august . vaccine ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] contents lists available at sciencedirect vaccine j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e vaccines were deployed as part of the international response [ ] although this outbreak also served to highlight the necessity of effective public health systems and infrastructure, and community engagement in order to mitigate disease spread through cultural or social practices [ ] . in the case of ebola, it was fortuitous that several candidate vaccines were already in development and immense global efforts made the vaccine available. however, this also conspicuously highlighted that nations around the world need to be better prepared for outbreaks of all infectious diseases, particularly those with the potential for high levels of morbidity and mortality, and that other measures to control infection spread should also be implemented [ ] . the united kingdom (uk) has a strong international reputation for scientific research and innovation, and a long history in vaccinology. along with other countries, the uk government mobilised resources to manage the ebola outbreak, as well as played a role in testing ebola vaccines in the uk, and west africa. alongside these efforts, the uk chief medical officer for the department of health and social care (dhsc), dame sally davies, raised the question to government and related stakeholders -what could the uk do to be better prepared for future outbreaks of infectious diseases? a partnership was instigated across the dhsc, the uk medical research council (mrc) and the biotechnology and biological sciences research council (bbsrc), who then worked together to establish a 'uk vaccine network' (ukvn, www.gov.uk/government/groups/ uk-vaccines-network) bringing together a small number of specialists from industry, academia and relevant funding bodies to make targeted investments in specific vaccines and vaccine technology for infectious diseases with the potential to cause an epidemic. at its inaugural meeting in july the ukvn discussed potential priority areas for investment. members agreed that four working groups (wgs) with the following foci: wg : to prioritise - pathogens most likely to cause an epidemic or pandemic in the short to medium term. wg : to rationalise where and when vaccine development resources should be prioritised when intervention is possible. wg : to understand the challenges in vaccine development and the key rate limiting steps for any given vaccine in development. wg : to address manufacturing capacity for vaccines. this paper describes the activities undertaken by working group on vaccine development. the group created a visual process mapping tool to better understand the potentially ratelimiting bottlenecks associated in moving a vaccine candidate from discovery through to development and early phase clinical trials; then into clinical manufacture and phase trials. the primary purpose of the tool was to act as a visual aid for early discovery and development scientists, highlighting the major steps required to fully develop any experimental vaccine. the secondary, and crucial purpose of the tool, was to identify any generic bottlenecks that may slow down development. identifying such strategic limitations may identify corrective actions, that may in turn help to overcome potential delays or setbacks when expediting vaccine development during epidemics. actions could be taken by policy makers to correct for limitations in regulation, funders to identify limits in scientific knowledge that could be corrected with appropriate investment, and for government and trans-national organisations to consider local and global response. though the genesis of this tool was considered in the context of epidemic preparedness, the tool itself is sufficiently far-reaching that it can be utilized by the wider vaccine community. considering the global nature of vaccines, it is important to understand the interdependencies that exist between discovery, development and manufacture. this tool gives scientists vision of the considerations that will need to be made along the development process. in taking a much more holistic approach to vaccine development it is hoped that this can sup-port other international such as the global vaccine action plan [ ] , which relies on the continued supply of vaccines to meet immunization targets. thus, the tool serves multiple users and purposes, and we report on its main findings in this paper. after initial literature consultations, a basic schematic overview of the major steps was created, which then fell into four main areas: (i) pre-clinical discovery; (ii) pre-clinical development; (iii) manufacturing; and (iv) clinical trials. several subgroups were convened to discuss these areas; membership was drawn from the ukvn, a core group of specialists that made up wg and further expertise consulted according to need. process schematics were made, and members undertook interactive discussions to add in, remove, and reorganise steps. the ukvn secretariat led on organising a series of iterative workshops that repeated this process several times; each time a skills gap analysis was carried out to ensure appropriate expertise was representing different disease/vaccine interests, and from across the vaccine sector from academic and industry discovery, development, regulation, manufacturing and clinical trials. the repeated process of interactive mapping, refinement of core documents, and presentation of progress back to the subgroups stimulated dialogue on the map as a whole. this was a concerted effort to break down silos between disciplines and sectors, and address how choices made in early discovery may affect the later development pathway. once the iterative, interactive working subgroups had met to agree the final nodes and bottlenecks on the map, the final figures were transcribed into powerpoint. these images were then used as the basis for interactive webpages, which were constructed by the mrc regulatory support centre (mrc rsc, https://mrc.ukri.org/research/facilities-and-resources-for-researchers/regulatory-support-centre/). the mrc rsc offered designs for the main menu, layout and colour scheme and once agreed with the secretariat, all graphics for the tool were drawn using adobe illustrator then saved in a web-friendly format. the website was built in dreamweaver using html coding. efforts were made to make the site responsive to different devices, and while this was possible for the text it was not possible to do so for the maps due to the complexity of the subject. the final version can be seen at www.vaccinedevelopment.org.uk. the process mapping workshops culminated in production of a website, with standalone pages for maps that distinguished the following major subdivisions: ( ) target product profile (tpp); ( ) pre-clinical discovery; ( ) pre-clinical development; ( ) clinical development; and ( ) regulatory affairs and ethical approvals (fig. ). due to the mandate and remit of the tool to be used by the ukvn for the development of vaccines for outbreaks, wg agreed that the tool did not need to address steps for market access and authorisation, as outbreak vaccines would not normally undergo this process -instead being deployed in small populations according to need once necessary evidence on safety and immunogenicity had been achieved. the target product profile (tpp) is a descriptive document that sets out clear parameters for the characteristics of the desired end product, its intended use, and market. it is the designated entry point into the process map. this came about through repetitive discussions that prevailed on the importance of the tpp at all stages in development; in simple terms, to know what it is you are making, and for whom. wg agreed that the significance of the tpp and the relevance to even the earliest exploratory basic science work, should not be underestimated, therefore prominently placing the tpp, aimed to engage early discovery scientists to consider what the end product is, and how the choices made during pre-clinical discovery and development influence them. the consensus opinion within the working group was that the tpp should be seen as a strategic planning document that should be utilised as a benchmark as data emerges. during normal development, this is a tool for novel vaccine creation. however, in an outbreak situation, the tpp acts as an essential checklist under accelerated timescales. it is worth noting that that the importance of tpps is widely shared and recent work by the who and cepi includes creating standard tpps for priority pathogens. iterative workshops hosted ongoing discussion on what was involved in each step, and where might rate limiting steps slow down development. there was agreement that almost anything could be described as a bottleneck in that each step is necessary, and by the nature of discovery research and development, such endeavours take time. however, in order to scrutinize the bottlenecks that could potentially be ameliorated by corrective action or investments by governments, research funders and policy makers, wg agreed that the tool should only identify steps where corrective action could be taken that could potentially accelerate vaccine development. the main bottlenecks and the corrective action required are summarised in table . an additional important consideration, was that the identification of these bottleneck 'nodes' should be sufficiently generic so as to be applied to a range of vaccine types, and pathogens -thus wg refined the key steps to common denominators that were shared across approaches. overall, many of the bottlenecks focused around gaps in funding for scientific knowledge, training, and infrastructure. an additional item was harmonisation of regulation, especially with regards to the release of genetically modified organisms (gmos). the bottlenecks can readily be seen on the process map by clicking 'show bottlenecks' on each map page; below we describe the main bottlenecks highlighted at each stage. within pre-clinical discovery, understanding pathogen biology and developing the appropriate animal challenge models are seen as significant limiting factors, especially with emerging infectious disease. the ability to screen, test, and verify potential new antigens is of critical importance. increased research funding into standardised methodologies for in vitro and in vivo testing and work into human-livestock host immunology would be critical to increase capacity. as lead candidates are identified, ensuring that the work has the appropriate freedom to operate becomes increasingly important. a review of the intellectual property landscape including searches on the antigen, cell line, and biological methods employed to create them are required and time to completion could be as little as three months, but increases with complexity. conducting a freedom to operate review is an important step towards commercialisation. yet, in an outbreak scenario, special regulations such as compulsory licensure and step-in rights may allow this issue to be side-stepped, but would require further clarification in policy. pre-clinical development is a complex, multi-step, and timeconsuming process (fig. ) . it requires that vaccine candidates undergo strain development and confirmation of antigen presentation, verified by animal studies. this is followed by process development to ensure that a scalable, robust, and gmp compliant manufacturing process is created. material generated at the end of pre-clinical development can be used for animal toxicology studies and forms the basis of a clinical trial application. the first bottleneck encountered is in strain development. some development work can occur using a research cell bank, however research cell banks are generally not to gmp standards therefore delays at later stages can be avoided by ensuring banks are gmp compliant. this will often require re-cloning into traceable cell lines that are gmp certified and free of any adventitious agent. major bottlenecks are around process development to create a scalable, gmp compliant, manufacturing process and the determination of the critical process parameters. at this stage manufacturers must address if the vaccine can in fact be manufactured in a scalable process. there is no generic manufacturing process for vaccines, thus almost all processes are designed from the bottom up, which adds costs and delays to any future intervention plan. platform manufacturing technologies are a method to overcome this issue by streamlining manufacturing with generic operations into which your selected antigen(s) can be introduced. viral vectors are one such platform technology that was effectively employed during the ebola outbreak [ , ] . looking more broadly, recombinant sub-unit vaccine technologies are another platform technology that could be used in other epidemics and has been successfully demonstrated in context of influenza vaccines [ ] . other advances in nucleic acid based vaccines, such as dna and rna, offer a future, though as yet unrealised potential, for generic manufacturing and release testing of vaccines. the determination of the range of physical, chemical, biological, or microbiological properties that ensure the quality of the desired product, is described as 'critical quality attributes' (cqa) and requires welldefined analytics which in the long term can reduce quality control (qc) costs. finding suitable analytics that can resolve complex antigen structure, increase understanding of degradation pathways and formulation needs is a continuing bottleneck. funders must ensure that process development, manufacturing, and analytics are not overlooked in future research calls. further bottlenecks are encountered during animal in vivo studies; characterising immunogenicity and reactogenecity in relevant animal models is imperative for taking forward vaccine candidates into phase . with this model there are further potential bottlenecks associated with home office approvals, and containment level and facilities depending on the pathogen of study, and supply of animals. gmp manufacturing is an infrastructure bottleneck that requires funders and policy makers to examine the pressures upon the current manufacturing model. almost all vaccine manufacturers operate at capacity and are dedicated to production of a single product. ' step-in' rights in an emergency scenario will disrupt supply of an essential vaccine, but also cause delay. recruiting a cmo to the task may help to alleviate the situation, but essential training and know-how is required. furthermore, some sites may only be suitable for certain vaccines classes, which can range from protein sub-unit vaccines to live viral and bacterial vaccines. based on vaccine class, facilities may not have the appropriate containment level for bulk manufacture of the drug substance, or the fill-finish for drug product. indeed, many facilities do not have fill-finish operations co-located and fill-finish is also a bottleneck in clinical development due to the limited number of appropriate sites and the volume of vials required for phase and trials. the uk government, with the ukvn providing evidence and advice, has identified manufacturing infrastructure as a significant bottleneck, and it has sought to invest into a new vaccine development and manufacturing centre (https://www.gov. uk/government/news/medicine-and-vaccine-manufacturing-centres-apply-for-funding). other organisations, such as cepi [ ] , have also issued calls for proposals into new platform manufacturing technologies (cepi.net). the situation exists under the status quo that we may have a vaccine that can be utilised to fight a future epidemic, but not have the manufacturing capacity to enable production and distribution. these latest calls seek to reverse current limitations. many of the priority pathogens are prone to sporadic outbreaks, and due to potential lethality, it will not be possible to conduct full phase trials, and even phase studies may prove challenging in a human population due to the unpredictable nature of these outbreaks. nonetheless, the sequential clinical trial steps that would comprise the 'normal' steps in development of a vaccine being taken forward for licensure have been incorporated into the tool. clinical trials require great investment and resource to execute, and the lengthy nature of the process could itself be described as a bottleneck; for the purposes of the tool however, we highlight fives steps within clinical development. current gmo regulations do not distinguish vaccines based on viral vectors, or live, replication incompetent vectors based on bacteria and viruses. current regulation stipulates that use of gmo vaccines is done so under 'contained use' or 'deliberate release' protocols to prevent release into the environment. consequently, this may involve obtaining separate approvals, modifications to the trial site, modified procedures for sterilising, administering and disposing, separate storage facilities and special training of staff to handle gmos. overall, this whole area would benefit a revision of regulations and derogating the use of common vaccine vectors in separate regulations from the currently applicable gmo guidance would have a substantial beneficial impact in speeding up approvals. obtaining regulatory approval may be especially difficult in the context of an international trial, where local rules may differ from country to country, or the national regulatory authority (nra) itself may have insufficient experience and seek expert opinions, which can further delay the process. engagement with the nra at an early stage is essential, as seeking opinion on the proposed trial design can capture any 'in country' requirements. the availability of trial sites may also impede progress; particularly if sites are not approved to work with gmo vaccines, or there are other limitations such as lack of infrastructure, skilled personnel, and capacity. working with credible and experienced partners will help to minimise delay and several clinical trial networks already exists to facilitate clinical development, some of which are pathogen specific that can further minimise this bottleneck. to overcome these bottlenecks, finding clinical trial sites and experienced nras that can act as credible authorities is essential, and early discussions need to occur in tandem to development and production to avoid delays. the process mapping tool emphasises the importance of ethical and regulatory considerations throughout the entire process, as the iterative workshops continually highlighted the importance of early engagement so the appropriate advice and input shape the r&d. the tool provides an overview and signposting to other organisations and information sources, as many outbreak vaccines may never go through market authorisation due to there being no commercial incentive for the manufacturers. nevertheless, there will be several stages where necessary approvals must be obtained in order to proceed with developing and testing a candidate. for regular marketing authorization in the eu, vaccine developers must provide a full development dataset of pre-clinical and clinical trial results to evidence clinical safety and efficacy, as well as evaluation of a positive benefit-risk ratio. the mapping tool highlights that: to discuss the tpp the uk medicines and healthcare products regulatory agency (mhra) can be approached at any time. animal work is regulated under the animals (scientific procedures) act (aspa). a uk government home office licensure must be provided at an institutional, personal and project level. the use of gmo vaccines is regulated by separate legislation, and approval must be given by the health and safety executive (hse). to carry out experimental medicine studies of immune responses in humans the health research authority (hra) provides ethical approvals through the research ethics service (res) for projects in the nhs (led from england). clinical trial authorisation (cta) is made through a formal application to the mhra. trialists must provide information on the mode of action, nature of the target, animal model studies and an imp dossier with preclinical toxicology data, and any human mechanistic/proof of concept studies. though these regulations are specific for the uk, many other countries require a similar level of scrutiny. in the case of life-threatening infections, it is conceivable that many of the priority vaccines under development will ultimately never undergo the standard clinical trials process and be used in an outbreak situation under eual [ ] . in these circumstances, it is imperative to test novel vaccine candidates in well characterised animal models. previously applied to defence vaccines due to the limitations of clinical testing, the fda animal rule [ ] may provide a pathway to test novel vaccine candidates. in outbreak settings, as was the case with ebola, it is possible to undertake an emergency use protocol and expedite authorisation to ensure a promising vaccine candidate can be deployed for human use, if it is justified as major interest for public health and/or therapeutic innovation. once a generic framework that could be applied to vaccine r&d broadly was established, the working group undertook a series of meetings applying the process map to three case studies of different vaccine candidates under development for three of the ukvn's priority pathogens: mers viral vector vaccine, zika protein subunit/vlp vaccine (fig. ) , and the prokarium vaxonella Ò plague vaccine (fig. ) . the case studies can be accessed through the main landing page of the process map . mers was selected as there are several candidates in later stages of development, including the viral vectored vaccine candidate chadox mers. the viral vector approach overcomes a number of bottlenecks to speed up development, mainly impacting on the preclinical development stages to overcome delays that would be associated with masterseed bank production, pre-formulation work/in vitro and in vivo, and lead identification. antigen identification bottlenecks are also overcome, through straightforward selection of the spike protein (the major external antigen of coronaviruses) which elicits neutralising antibodies to the receptor binding domain [ ] . the bottlenecks that remains relevant from the generic tool are: basic understanding of human/livestock host immunology; using an appropriate animal challenge model (camels would be suitable, but few places have facilities for such a large model); and the three main bottlenecks in clinical development of obtaining ethical and regulatory approval, working with vaccines classified as gmos, as the viral vector platform has been altered genetically from its original form, and the availability of overseas trial sites. more bottlenecks were identified when applying the vaccine development process map to a novel zika vaccine. at the time of the ukvn formation, zika was little heard of, and had only modest investment as a potential emerging virus in uk research funding portfolios. as the work of the ukvn progressed, the zika outbreak unfolded, and funders in the uk and internationally were mobilised to increase investments. the ukvn immediately added zika to the priority pathogen list, and contributed to a number of funding streams to support vaccine r&d. applying the process mapping tool to such an emerging pathogen, allowed the visualisation of the significant number of unknowns and challenges when dealing with a little-known emerging disease without platform approaches (fig. ) . in the earliest pre-clinical discovery stage, pathogen biology and host immunology were both rate limiting, particularly in identifying regions of the virus that could be used as a possible antigen. in vitro studies and deploying an in vivo challenge model were also rate limiting as the generation of reagents and the appropriate assays need to be established. without a robust set of assays and generation of some antigen in the early discovery phase, the bottleneck would be overcome however identification of the surrogate/correlate of protection in the appropriate challenge models will be important. it should be noted that many vaccines have launched without the establishment of correlates of protection and will depend on the licensure sought and the severity of the disease and its outbreak. in pre-clinical development, due to the novelty of the vaccine, process development, assays, adjuvantisation and formulations were identified as bottlenecks. time could be saved if a platform technology was used for this vaccine, circumventing many steps in process development and formulation. in clinical development, deploying a human challenge model to study the response to a vaccine studies were considered rate-limiting, and it must be noted that in the case of emergency or experimental licensure, a strategy might be to establish some animal challenge data and seek to establish safety data in a phase trial before expansion into a phase a/b trial. however, demonstrating efficacy can only be one in endemic or outbreak situations and will require early engagement of the nra, and locating suitable overseas trial sites has the potential to be rate-limiting. a plague vaccine potentially has dual application: the development path could be in collaboration with government agencies in producing an anti-bioterror vaccine but could be expanded and developed as a vaccine against endemic disease in regions such as madagascar. this dual nature affects the way we think about the tpp and development path. as with the mers case study the plague vaccine exploited a platform technology -prokarium's vaxonella platform -thus many bottlenecks, particularly in preclinical development, were circumvented (fig. ) . the main bottlenecks are in the clinical phase and animal rule regulations necessary to follow for licensure and are particular to this pathogen and vaccine approach, and included: quality control; phase ii regulatory requirements for two animal models; fill finish; and in phase ii safety studies, meeting requirements for licensure regulations. the case studies have demonstrated how useful the process map can be in vaccine development, and how using platform technologies enables many development steps to be circumvented and thus overcome rate-limiting steps, which could be vital in an outbreak situation where speed of development must be minimised where possible. as an additional feature, the tool also presents some of the work completed by wg , which was undertaken to better understand the feasibility and challenges of developing vaccines for the major ukvn prioritised pathogens. the ukvn members agreed that to develop a new vaccine might not always be the most appropriate response in an outbreak setting, particularly if a candidate vaccine is early stage and presents significant r&d or manufacturing challenges. thus, it may be more appropriate to deploy other healthcare interventions to manage the disease, such a therapeutic antibodies or containment procedures. the prioritisation guide addresses the 'technical feasibility' of creating a vaccine together with 'public health value' based on pathogen severity and alternatives to vaccination. a final consideration was the time scale and cost of development, with the single criterion to address the available vaccine candidate(s), considering the stage in development of any existing candidates. this included manufacturing considerations, and whether any vaccine may already be stockpiled. each criteria has a red, amber or green status, and taken together will enable decision-making on the suitability of pursuing vaccine r&d in an outbreak setting. this tool represents a first step towards understanding the limiting factors in vaccine development and epidemic preparedness. however, there are still several key outstanding challenges it does not fully capture. the first is sustained funding for this problem, which is partly address through the creation of the coalition for epidemic preparedness innovation (cepi, www.cepi.net), however improved co-ordination is still required between industry, academia, government and international organizations, such as the who. this will be a key priority for policy makers in the future. infrastructure and manufacturing capacity needs to be accurately assessed, especially fill-finish capacity for the different vaccine types/classes. additionally, continued research is required to better understand the correlates of protection, appropriate animal models and, of course, the discovery of new antigens that can protect against these emerging pathogens. an extraordinary array of technologies are under development that have the potential to change our ability to respond to emerging infectious diseases. amongst these, viral vector and subunit vaccines are at the forefront, however future innovations with nucleic acid based vaccines are on the horizon. currently, there are no dna or rna vaccines licensed for human use. yet should this milestone be reached, many of the bottlenecks described currently will be eliminated. in the meantime, funders, governments and policy makers must all focus to overcome the existing bottlenecks identified and employ better co-ordination to overcome future epidemic threats. the author declares that there is no conflict of interest. . plague case study summary. the total number of bottlenecks is reduced due to the use of a live bacterial vector system being exploited as a platform technology. plague antigens are already known and well characterised, thus, many of the bottlenecks shift to clinical development and testing of the vaccine candidate. who's blueprint list of priority diseases the need for global r&d coordination for infectious diseases with epidemic potential clinical development of ebola vaccines how the current west african ebola virus disease epidemic is altering views on the need for vaccines and is galvanizing a global effort to field-test leading candidate vaccines a review of phase i trials of ebola virus vaccines: what can we learn from the race to develop novel vaccines lessons learnt from the ebola outbreak in west-africa cdc's evolving approach to emergency response collaborating to achieve global vaccine action plan goals ebola ça suffit ring vaccination trial consortium. the ring vaccination trial: a novel cluster randomised controlled trial design to evaluate vaccine efficacy and effectiveness during outbreaks, with special reference to ebola efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination clusterrandomised trial a fast track influenza virus vaccine produced in insect cells cepi-a new global r&d organisation for epidemic preparedness and response world health organization. emergency use assessment and listing procedure (eual) for candidate medicines for use in the context of a public health establishing efficacy of human products using animals: the us food and drug administration's ''animal rule chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice the authors would like to thank the members of the uk vaccine network, mrc, bbsrc, the mrc regulatory support centre, and the many subject matter experts who have helped put this tool together. key: cord- -cmbg ty authors: mühlebach, michael d.; hutzler, stefan title: development of recombinant measles virus-based vaccines date: - - journal: recombinant virus vaccines doi: . / - - - - _ sha: doc_id: cord_uid: cmbg ty this chapter describes the development of recombinant measles virus (mv)-based vaccines starting from plasmid dna. live-attenuated measles vaccines are very efficient and safe. since the availability of a reverse genetic system to manipulate mv genomes and to generate respective recombinant viruses, a considerable number of recombinant viruses has been generated that present antigens of foreign pathogens during mv replication. thereby, robust humoral and cellular immune responses can be induced, which have shown protective capacity in a substantial number of experiments. for this purpose, the foreign antigen-encoding genes are cloned into additional transcription units of plasmid based full-length mv vaccine strain genomes, which in turn are used to rescue recombinant mv by providing both full-length viral rna genomes respective anti-genomes together with all protein components of the viral ribonucleoprotein complex after transient transfection of the so-called rescue cells. infectious centers form among these transfected cells, which allow clonal isolation of single recombinant viruses that are subsequently amplified, characterized in vitro, and then evaluated for their immunogenicity in appropriate preclinical animal models. vaccines are the most effective way to prevent infectious disease in terms of safety and cost-benefit ratio. however, at present, the development of vaccines to the point of licensing for human use takes decades and sometimes has proven hardly possible as exemplified by the hiv pandemic. to minimize the time for vaccine development and to be safe, it is necessary to develop strategies that allow for the immediate initiation of standardized vaccine development, leading to successful and safe candidate vaccines in a minimal amount of time. one strategy is to use well-known, already authorized vaccines with exceptional safety and efficacy records as a platform to present critical antigens of the pathogen which is the focus of vaccine development. thereby, efficacious immune responses are triggered in immunized animals and patients not only against the vaccine vector, but also against the additionally present extra antigen. one of these potential vaccine platforms currently under development are recombinant vaccine strainderived measles viruses. unmodified live-attenuated measles virus (mv) vaccine strains are efficient replicating vaccines. besides revealing an excellent safety record, both humoral and cellular immune responses are elicited, which are responsible for long-lasting protection [ ] [ ] [ ] . therefore, the who targets eradication of measles by using these vaccines [ ] . the vaccine's manufacturing process is extremely well established [ ] and millions of doses can be generated quite easily and quickly. generation of recombinant mv from dna via reverse genetics became feasible [ ] and allows for the robust expression of different antigens, as outlined below, during replication of the modified recombinant mv vaccine viruses. thereby, generally robust immune responses against vector and foreign antigens are induced after vaccination of transgenic mv-susceptible ifnar −/− -cd ge mice [ ] , nonhuman primates, or human patients (see table ) indicating the high efficiency of the system. interestingly, pre-vaccinated animals with protective immunity against measles were still amendable to vaccination with the recombinant mv, since significant immune responses against the foreign antigen(s) are still induced [ , ] . also in human probands, levels of antibodies against chikungunya virus (chikv) env antigens did not display a negative correlation with preexisting antibody levels against the mv vector backbone [ ] . a plethora of different antigens of various viruses and of one bacterium have been cloned into recombinant mv genomes, and the respective recombinant viruses have already been rescued. these projects are summarized in the following table (table ) . as depicted, mainly structural proteins, especially viral envelope glycoproteins have been expressed as antigens by the yet generated recombinant mv-derived vaccines, simply due to the expectation that especially humoral immunity against these antigens may result in neutralizing antibodies with protective capacity. however, the vector system proved also capable to induce robust cellular immune responses. moreover, also secreted, cytoplasmic, or membrane bound markers, such as different luciferases, gfp, lacz [ ] , or cellular proteins such as carcinoembryonic antigen (cea) [ ] can be expressed by recombinant mv as demonstrated during its characterization and application as a potential oncolytic virus. even membrane pore proteins such as the sodium iodide symporter (nis) [ ] and up to three different transgene cassettes with a size potentially exceeding kb [ ] have been successfully expressed by recombinant mv, demonstrating the general versatility of this vector system. the targeted viruses span a couple of genera and families. also the developmental stage of the different vaccines is quite diverse, ranging from the demonstration of successful antigen expression by the recombinant mv, spanning the demonstration of humoral or cellular immunogenicity against the encoded antigen up to demonstration of the vaccine's protective efficacy in appropriate pre-clinical animal models. of note, recombinant mv encoding the glycoprotein antigens of chikungunya virus, mv-chikv, has already been tested in a clinical phase i study in human volunteers [ ] . after demonstrating efficacy in appropriate mouse and primate animal models [ ] , this recombinant vaccine delivered proof of triggered antigen-specific immune responses after immunization determined by measuring total antibodies (elisa), neutralizing antibodies (nabs), or reactive t cells determined by elispot or intracellular cytokine staining (ics) c protective capacity of vaccine-induced immune responses after challenge of the appropriate animal model determined by reduction of pathogen load or attenuation of etiopathology principle for safety and immunogenicity in human patients, irrespective of preformed anti-measles immunity [ ] . thus, the route for clinical development of such recombinant, mv-derived vaccines is open. on the one hand, these recombinant vaccines may be valuable to be used during primary measles vaccination to immunize children simultaneously against measles and a secondary pathogen of concern for the respective pediatric population without the need to vaccinate these children with two different vaccines. as an example, mv expressing hepatitis b virus small antigen (hbsag) may be used in regions with high hbv prevalence to protect children early on from this potentially chronic infection ("buy one, get one free" strategy). on the other hand, recombinant mv is one of the potential vaccine platforms that can be used for the (fast track) development of vaccines against emerging pathogens, for which fast availability of a vaccine may be critical. in this respect, our mv-derived vaccine against the corona virus responsible for the middle east respiratory syndrome (mers-cov) expressing the mers-cov glycoprotein s [ ] has been among the first and most progressed vaccine candidates, which were evaluated by the saudi arabia ministry of health [ ] and the who [ ] . . full-length mv genome plasmids such as pbr-mv vac -gfp(h) [ ] or p(+)polii-mv vac -atu(p) [ ] (see note ). . the mv genomes encoded on these plasmids contain the socalled additional transcription units (atus). to facilitate insertion of foreign orfs, usually single-cutter restriction endonuclease recognition sites are placed between the start and stop signals of the atu, in the above mentioned examples ´ mlui and ´ aatii (see note ). . t -promoter driven expression plasmids for mv proteins being components of the viral rnp complex (i.e., nucleocapsid protein n, phosphoprotein p, viral rna-dependent rna-polymerase l), such as pemc-na, pemc-pa, or pemc-la [ , ] , respectively. . cdna of the foreign antigen to be expressed by the recombinant vaccine. the orf has to be flanked ´ by mlui and ´ by aatii restriction sites (or alternative sites being part of the atu) such that the genome length of the putative recombinant vaccines obeys to the "rule-of-six" [ ] (see note ). . cell are cultivated in dmem supplemented with % fetal bovine serum and mm l-gln with additional . mg/ml of geneticin, when used for rescue as described in [ ] . . all cells were cultured at °c in a humidified atmosphere containing % co for no longer than months after thawing of the original stock. . g needles, one for each vaccine to be injected. . ml syringes with fine scale, one for each vaccine to be injected. . generation of gene segments encoding the desired foreign antigen of the target pathogen may be generated by gene synthesis. the (codon-optimized) orf encompassing start and stop codons is flanked by the respective restriction sites, which allow direct cloning of the gene segment into the atu of a fulllength mv genome plasmid. it may be necessary to include a specific number of extra nucleotides between stop codon and ´restriction site to obey the rule-of-six (see note ). alternatively, the desired orf is amplified on the basis of (plasmid) cdna or cdna directly isolated (eventually after reverse transcription) from the target pathogens' genomes using (rt-)pcr. for this purpose, specific primers have to be designed as outlined above, depending on the exact structure of the atu and the orf to be amplified. . the resulting cdna segments as well as the mv genome plasmids are then treated by the respective restriction endonucleases using the following standard reaction mix: - μg dna. - u per restriction enzyme. μl × neb buffer (depending on the enzyme used). μl × bsa (if required by the applied enzyme). fill up to μl with nuclease-free h o. dna digestions proceeds at the temperature required by the respective enzymes to allow thorough digestion of the dna, optimally overnight. . the desired segments are purified after restriction digestion by standard agarose gel electrophoresis (see note ). the purified genome-containing plasmid backbone and the antigen-orf containing insert are ligated using standard ligation conditions with at least tenfold molar excess of the insert: ng vector dna. ng insert dna. μl × dna dilution buffer. fill up to μl with nuclease-free water and mix. μl × t dna ligation buffer. . seed × t cells/well in ml complete dmem into six-well plates and incubate overnight (see note ). . the next day, cells should be approx. % confluent bevor starting transfection. . seed × vero cells/well in ml complete dmem into six-well plates and incubate overnight (see note ). . the next day, cells should be approx. % confluent bevor starting transfection. . infection: cells are infected with a t -encoding vaccinia virus such as mva-t at an moi of - (see note ). min after infection, the medium is replaced and cells are transfected. . transfection: . μg of the respective mv genomic cdna plasmid are mixed with . μg pemc-na, . μg pemc-pa, and . μg emc.la (helper plasmids) and transfected using a commercially available lipofection method as described above in the protocol using the polii-based rescue system. transfected cells are cultured and examined for syncytia formation, daily (see note ). . if you find syncytia, pick them as outlined below (see subheading . , step ). . if no syncytia are visible on day post overlay, split the overlay culture by passaging the culture : . for that purpose, seed × vero cells/ cm cell culture dish in ml complete dmem and incubate for h. aspirate the medium from the cm cell culture dish of the overlay and wash once with ml pbs. detach the cells by incubating with . ml trypsin-edta for min. after complete detaching (check by microscope) stop trypsin incubation by adding . ml complete dmem and suspend the cells by pipetting up and down. seed % of the cell suspension to the prepared cm cell culture dish. the remaining % of the cell suspension are transferred into a ml tube and snap-frozen in liquid nitrogen for min. the frozen cell suspension is thawed at °c in a water bath and the resulting cell debris is pelleted by centrifugation at × g for min at °c. transfer the supernatant to the cm cell culture dish dropwise and incubate overnight (see note ). if there is no syncytia formation wait for one additional day and if there is still no syncytia formation, repeat the rescue. . the cell suspension is transferred into precooled . ml reaction tubes and centrifuged at , × g for min at °c to remove the cell debris. . the protein containing supernatant is transferred into a fresh pre-cooled . ml reaction tube and stored at − °c. frozen cell lysates are thawed on ice for further western blot applications according to standard conditions using antibodies recognizing an mv protein, e.g., the nucleocapsid protein, to standardize for infection, and another antibody recognizing the foreign antigen to allow assessment of proper antigen expression by the recombinant vaccine. . four to days after booster immunization, splenozytes are harvested from immunized mice to assess abundance of antigen-or vector-specific t cells by elispot or intracellular cytokine staining after stimulation with respective antigens or peptides, either following standard protocols or using commercially available kits according to the manufacturer's instructions. . before immunization ("pre-bleed"), directly before the booster vaccination ("post-prime") or days after booster vaccination ("post-boost"), μl blood are taken from each mouse, and sera are separated to assess abundance of vector-or target-specific antibodies by titrating neutralizing titers or total antibody titers by elisa. . if the ifnar −/− -cd ge mouse strain is susceptible to the pathogen to which the vaccine is directed against, vaccinated mice can be challenged subsequent to immunization. the challenge relies on an established infection protocol of (unvaccinated) animals, which results in symptomatic infections or even death, thereby allowing to test the protective capacity of the vaccine. parameters such as course of disease or pathogen load in specific organs are assed (see note ). both examples pbr-mv vac -gfp(h) and p(+)polii-mv vac -atu(p) contain, as all genome plasmids for the rescue of recombinant mv, a full-length mv vaccine strain genome. pbr-mv vac -gfp(h) is derived from the plasmid backbone pbr (low copy) and expresses the viral rna antigenome under the control of a t -promoter, whereas p(+)polii-mv vac -atu(p) has a pbluescript (high copy) backbone and is polii-driven. plasmids containing full-length mv genomes tend to be a bit delicate to handling procedures. therefore, plasmids with a high-copy plasmid backbone should be amplified in e. coli at °c, whereas low-copy plasmids can be amplified at °c. in addition, it pays to directly pick clones or isolate plasmids from growing cultures instead of storing liquid bacteria cultures or colony plates with mv genomecontaining plasmids at °c for more than few hours (≪ overnight!). as an alternative, bacteria pellets can be stored frozen at − °c before plasmid isolation. . each mv gene cassette is flanked by conserved genetic elements representing start and stop signals for the viral polymerase complex. by duplicating these highly conserved intergenic sequences, a new transcription unit can be inserted in virtually any position of the virus genome, allowing insertion and expression of foreign genes such as antigen orfs. the relative genomic position of the atu allows regulation of the inserted gene's expression due to the transcriptional gradient found in mononegavirales. the further upstream the atu cassette is located, the higher the amount of mrna being transcribed in infected cells and the higher protein expression. however, if the encoded gene product interferes with mv replication, too high expression levels of the encoded antigen may be detrimental. . the number of nucleotides in mv genomes can be exactly divided by , presumably due to one n protein binding to six nucleotides. also recombinant genomes have to obey this rule; otherwise no recombinant virus can be rescued. therefore, it has to be considered that the length of the inserted gene segment can be divided with the same rest by as the length of the segment removed from the genome during cloning, if gene cassettes are inserted into genome plasmids. thereby, one makes sure that the length of the resulting full-length genome is multiple of , again. . due to the size of the genome-containing plasmid (approx. kda), voltage during agarose gel electrophoresis should be restricted to max. v. . while t -based rescue of mv, especially using the helper cell line - - , is the standard rescue system guaranteeing precise start and stop of the transcribed anti-genomic viral rna due to the precise start of t -driven transcription and stop due to specific termination signals in conjunction with the ribozyme flanking the viral genome sequences [ ] , efficiency of the rescue is quite variable and depends considerably on the status of the rescue cells. moreover, syncytia formation using - - cells after overlay is not too efficient [ ] , further limiting rescue efficiency especially of virus variants with limited fusion activity (unpublished own observation). vaccinia virus driven rescue allows usage of cell lines other than , which may be better suited for propagation of one specific mv variant, but depends on the quality of the used plasmids. polii-driven virus rescue is very efficient and usually results in recombinant mv with precise genomes, but the lower precision of start and stop of poll ii transcription may allow completion of virus from genomes not being in line with the rule of six [ ] . . prepare one more six-well plate than required for the rescue of the different mv. this additional well is used as transfection control. . seal six-well plate properly to avoid contamination of the cells. . if the recombinant mv will additionally express gfp or other fluorescent marker proteins, check for gfp-expression with the help of a fluorescence microscope to identify syncytia. if no easy marker protein for easy evaluation is encoded, check for syncytia formation via light microscope in phase contrast. . for transfection control, use μg of for example egfp expression plasmids such as pegfp-n without any mv cdna or helper-plasmids and transfect as described for mv cdna. . if using another vaccinia virus than mva for t pol expression, one has to make sure that the rescued recombinant mv can be separated from (co-) replicating vaccinia virus, which may be a tedious process due to the laborious methods needed to physically separate the different virus populations. step seems to be slightly more efficient in our hands, but it includes one additional freeze-thaw cycle. . after quick-freezing in liquid nitrogen, the tube containing the virus suspension can be stored at − °c for several days. . check thawing of the virus regularly, the virus is heat sensitive and incubating the virus at °c will lead to significantly reduced virus titers. . it is essential to slew the cell culture dish immediately as well as properly to avoid erratic infection of vero cells, which will lead to lower virus titers. . the growth rate is depending on the virus strain and might be modulated by the additional genetic information. therefore, check the virus growth regularly to develop a feeling for the growth rate. . scratch the cells to on edge of the cell culture dish and hold the plate inclined while aspirating the medium including all cell debris. . while mv vaccine strains authorized to be used as human vaccines are usually regarded as safe and accordingly being handled under bsl- conditions, recombinant viruses-even those bearing identical vaccine strain genomes-may be handled under bsl- or bsl- conditions depending on the locally responsible national or regional regulatory authorities. under very rare circumstances, single recombinant mv may be placed into bsl- , depending on the nature of the expressed foreign antigen. . try to remove the medium completely while following note . this will increase virus titers. . the number of aliquots can be modified, depending on freezerspace and experimental plans. . for passages >p it is sufficient to infect only one cm dish and store around five aliquots, because those viruses are not used for experiments, normally. . slew the plate gently a few times during incubation. . slew the plate vigorously a few times during incubation. . experimental mouse work has to be carried out in compliance with the regulations of the respective animal protection law. . mice are sacrificed or used for further studies dependent on the respective experimental schedule. . efficacy of the recombinant vaccines can be most directly assessed using a challenge experiment directly revealing protection by abolishing or attenuating the etiopathology. in absence of an appropriate challenge, quantitative abundance of antigen-specific (neutralizing) antibodies or t cell reactivity can indicate the protective efficacy of the recombinant vaccine. expand high-fidelity pcr system (roche) or standard pcr protocol measles virus persistence of measles, mumps, and rubella antibodies in an mmr-vaccinated cohort: a -year follow-up current overview of the pathogenesis and prophylaxis of measles with focus on practical implications who global eradication of measles. sixty-third world health assembly rescue of measles viruses from cloned dna measles virus spread and pathogenesis in genetically modified mice a single injection of recombinant measles virus vaccines expressing human immunodeficiency virus (hiv) type clade b envelope glycoproteins induces neutralizing antibodies and cellular immune responses to hiv a recombinant measles vaccine expressing chikungunya virus-like particles is strongly immunogenic and protects mice from lethal challenge with chikungunya virus immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-inman trial a vectored measles virus induces hepatitis b surface antigen antibodies while protecting macaques against measles virus challenge protective anti-hepatitis b virus responses in rhesus monkeys primed with a vectored measles virus and boosted with a single dose of hepatitis b surface antigen a recombinant measles virus expressing hepatitis b virus surface antigen induces humoral immune responses in genetically modified mice attenuated measles virus as a vaccine vector recombinant measles viruses expressing heterologous antigens of mumps and simian immunodeficiency viruses recombinant measles virus incorporating heterologous viral membrane proteins for use as vaccines measles vaccine expressing the secreted form of west nile virus envelope glycoprotein induces protective immunity in squirrel monkeys, a new model of west nile virus infection live measles vaccine expressing the secreted form of the west nile virus envelope glycoprotein protects against west nile virus encephalitis live attenuated measles vaccine expressing hiv- gag virus like particles covered with gp deltav v is strongly immunogenic immunogenicity of a recombinant measles-hiv- clade b candidate vaccine immunogenicity of a recombinant measles hiv- subtype c vaccine toxicology, biodistribution and shedding profile of a recombinant measles vaccine vector expressing hiv- antigens, in cynomolgus macaques a recombinant live attenuated measles vaccine vector primes effective hla-a -restricted cytotoxic t lymphocytes and broadly neutralizing antibodies against hiv- conserved epitopes induction of neutralising antibodies and cellular immune responses against sars coronavirus by recombinant measles viruses the successful induction of t-cell and antibody responses by a recombinant measles virusvectored tetravalent dengue vaccine provides partial protection against dengue- infection immunogenic subviral particles displaying domain iii of dengue envelope protein vectored by measles virus pediatric measles vaccine expressing a dengue antigen induces durable serotypespecific neutralizing antibodies to dengue virus pediatric measles vaccine expressing a dengue tetravalent antigen elicits neutralizing antibodies against all four dengue viruses protection from sars coronavirus conferred by live measles vaccine expressing the spike glycoprotein recombinant measles virus exp ressing single or multiple antigens of human immunodeficiency virus (hiv- ) in duce cellular and humoral immune responses immunogenicity of next-generation hpv vaccines in non-human primates: measles-vectored hpv vaccine versus pichia pastoris recombinant protein vaccine recombinant measles virus-hpv vaccine candidates for prevention of cervical carcinoma evaluation of a recombinant measles virus expressing hepatitis c virus envelope proteins by infection of human pbl-nod/scid/ jak null mouse broadly neutralizing immune responses against hepatitis c virus induced by vectored measles viruses and a recombinant envelope protein booster immunogenicity of attenuated measles virus engineered to express helicobacter pylori neutrophilactivating protein aik-c measles vaccine expressing fusion protein of respiratory syncytial virus induces protective antibodies in cotton rats recombinant measles viruses expressing respiratory syncytial virus proteins induced virus-specific ctl responses in cotton rats evaluation of measles vaccine virus as a vector to deliver respiratory syncytial virus fusion protein or epstein-barr virus glycoprotein gp a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform study of pathogenicity of nipah virus and its vaccine development recombinant measles aik-c vaccine strain expressing the prm-e antigen of japanese encephalitis virus non-invasive in vivo monitoring of trackable viruses expressing soluble marker peptides image-guided radiovirotherapy for multiple myeloma using a recombinant measles virus expressing the thyroidal sodium iodide symporter toward developing a preventive mers-cov vaccine-report from a workshop organized by the saudi arabia ministry of health and the international vaccine institute a roadmap for mers-cov research and product development: report from a world health organization consultation rescue of measles virus using a replication-deficient vaccinia-t vector rna polymerase ii-controlled expression of antigenomic rna enhances the rescue efficacies of two different members of the mononegavirales independently of the site of viral genome replication the rule of six, a basic feature for efficient replication of sendai virus defective interfering rna beitrag zur kollektiven behandlung pharmakologischer reihenversuche the authors are indebted to r. cattaneo for introduction into the exciting field of recombinant mv and thank u. schneider for providing the polii rescue system. the project was supported by federal ministry for education and research of germany (bmbf) grant a b (m.d. mühlebach). key: cord- -spt oea authors: bhardwaj, prateek; bhatia, eshant; sharma, shivam; ahamad, nadim; banerjee, rinti title: advancements in prophylactic and therapeutic nanovaccines date: - - journal: acta biomater doi: . /j.actbio. . . sha: doc_id: cord_uid: spt oea vaccines activate suitable immune responses to fight against diseases but can possess limitations such as compromised efficacy and immunogenic responses, poor stability, and requirement of adherence to multiple doses. ‘nanovaccines’ have been explored to elicit a strong immune response with the advantages of nano-sized range, high antigen loading, enhanced immunogenicity, controlled antigen presentation, more retention in lymph nodes and promote patient compliance by a lower frequency of dosing. various types of nanoparticles with diverse pathogenic or foreign antigens can help to overcome immunotolerance and alleviate the need of booster doses as required with conventional vaccines. nanovaccines have the potential to induce both cell-mediated and antibody-mediated immunity and can render long-lasting immunogenic memory. with such properties, nanovaccines have shown high potential for the prevention of infectious diseases such as acquired immunodeficiency syndrome (aids), malaria, tuberculosis, influenza, and cancer. their therapeutic potential has also been explored in the treatment of cancer. the various kinds of nanomaterials used for vaccine development and their effects on immune system activation have been discussed with special relevance to their implications in various pathological conditions. statement of significance: interaction of nanoparticles with the immune system has opened multiple avenues to combat a variety of infectious and non-infectious pathological conditions. limitations of conventional vaccines have paved the path for nanomedicine associated benefits with a hope of producing effective nanovaccines. this review highlights the role of different types of nanovaccines and the role of nanoparticles in modulating the immune response of vaccines. the applications of nanovaccines in infectious and non-infectious diseases like malaria, tuberculosis, aids, influenza, and cancers have been discussed. it will help the readers develop an understanding of mechanisms of immune activation by nanovaccines and design appropriate strategies for novel nanovaccines. since the advent of the first vaccine centuries ago, researchers have been trying to find the answer of most common pandemic diseases through immunotherapy. vaccination has successfully managed to eradicate a lot of prevalent diseases, but associated off-target responses, lack of prolonged protection across variable pathogenic strains and allergy, limits its horizon for the prevention or treatment of other globally prevalent diseases like tuberculosis, aids, malaria, influenza and cancer. though prophylactic vaccines use live-attenuated or inactivated pathogens, lack of response and protection against diverse pathogenic strains [ ] . in addition, nanoparticles have the ability to impart more stability with high antigen loading and less proteasomal degradation of antigenic subunits. small and more specific subunits often tend to be less immunogenic, which can be overcome by the use of adjuvants that produce co-immunostimulatory or immunomodulatory signals. side effects associated with such conventional adjuvants with individual-specific response, immunotolerance to the target antigen and unwanted reactions towards self-antigens limit their use [ ] . therefore, biocompatible nanoparticles can also be directly used as adjuvants and may mitigate the need of strong conventional adjuvants (e.g., alum). another reason associated with low subunit immunogenicity is their low cellular internalization and rapid clearance from the body. nanoparticles with tunable physicochemical properties can overcome this limitation by enhancing circulation time, bio-accumulation in lymphoid organs and efficient targeting of immune cells. they can also increase crosspresentation by antigen-presenting cells (apcs) and activate the immune system at much lower doses. for instance, poly (lactic-coglycolic acid) (plga)-based nanoparticles enhanced the cross presentation of ovalbumin through major histocompatibility complex-i (mhc-i) in bone marrow derived primary dendritic cells due to which t cells were stimulated at -fold lower concentrations as compared to soluble antigen [ ] . different kinds of nanoparticles (lipid-based, polymeric, inorganic and protein/peptide-based) have been extensively used as adjuvants, immunogens and antigen delivery vehicles for immune activation [ ] . most of the synthetic nanoparticles interact with apcs structurally to increase internalization rather than functionally as they lack specific binding locations for cell receptors. on the contrary, protein-based nanoparticles can interact both structurally and functionally as they can carry antigens as well as interact with the pattern associated receptors on apcs [ ] . nanovaccines have the ability to induce both the components of human adaptive immunity i.e. cell-mediated as well as antibody-mediated immunity with induction of memory response. prolonged release of antigens from nanoparticle depots can cause enhanced stimulation for a long period of time, alleviating the need for frequent booster doses and therefore, nanovaccines can be used as both prophylactic or therapeutic which involves its administration before or after the occurrence of the disease respectively. however, the safety, biodistribution and residence of the nanoparticles must be optimized to obtain preferred immune responses towards nanoparticle-based vaccines. in this review, the potential and pros and cons of nanoparticlebased vaccines are explored. the role of different nanovaccines in activating various arms of immunity with an intent to abate the use of frequent booster doses as vaccines for tuberculosis, malaria, hiv (human immunodeficiency virus), influenza, and cancer are discussed. the applications and advantages of nanovaccines against various infectious and non-infectious diseases are also further delineated in this article. the ultimate goal for vaccination is to generate safe and efficient primary as well as secondary immune responses in the body. in a simplified way, primary immune responses protect the body from potential damages that might occur on first exposure of the pathogen or antigen. on the other hand, secondary immune responses, which are comparatively rapid and stronger, are elicited depending on the immunological memory generated during first exposure and protect the body from future encounters of the same epitope [ ] . nanoparticle-based vaccine delivery emerged as an appealing platform for boosting both primary as well as secondary immune responses in the body [ , ] . there are multiple mechanisms by which nanovaccines help enhancing immune responses. nanovaccines facilitate uptake of antigens in dendritic cells (dcs), which can further be enhanced by decorating the particle surface with ligands that selectively recognize receptors on dcs (refer fig. ). for example, ligands including fc fragment, mannose and anti-dec- (mab) have been widely explored which selectively binds fc receptors, mannose receptors and dec- receptors respectively on dcs [ , ] . in addition to efficient internalization, ability to co-deliver multiple-antigens and adjuvants, inherent adjuvant property, rapid lymph node drainage and efficient antigen presentation on dc's surface are other predominant mechanisms by which nanovaccines help increase the duration as well as the magnitude of the immune responses in immunized animals [ , ] . interestingly, these properties of nanoparticles have been substantially engineered to achieve desirable immune response for developing either prophylactic or therapeutic vaccines [ ] . for designing a safe and effective nanovaccine, it is imperative to understand the mechanism by which the nanovaccine activates both innate and adaptive immunity in the body [ ] . prophylactic nanovaccines are generally administered subcutaneously, intramuscularly, or intranasally whereas therapeutic vaccines (e.g. for cancer treatment) are injected intravenously. depending on the route of immunization, nanovaccines first encounter immune cells including neutrophils, macrophages, dcs and natural killer (nk) cells which quickly recognize the nanovaccine (foreign particle) based on the pathogen associated molecular patterns (pamps) associated with them [ ] . pamps (e.g. bacterial lps, teichoic acid, lectins, oligonucleotides or material from the carrier nanoparticle etc.) serve as the ligands for prrs [e.g. toll-like receptors (tlrs), c-type lectin receptors (clrs), retinoic acid-inducible gene-i -like receptors (rlrs) etc.] abundantly present on these cells [ , ] . thus, interaction of pamps with prr trigger endocytosis of larger particles (usually > nm) preferentially by macrophages and smaller particles by dcs. engulfment of nanovaccines by macrophages results in gradual degradation of nanovaccine particles and the encapsulated antigens. to prevent this, nanovaccine particles have been engineered to survive the attack by macrophages and facilitate direct delivery to apcs [ , ] . additionally, depending on the type of pamps, neutrophils and macrophage secrete a variety of cytokines and chemokines which further activate apcs. besides pamps, there are several unknown mechanisms which can activate innate immunity for example, induction of immune responses (potential adjuvant effect) by glycolic acid component of plga co-polymer [ ] or by cationic liposomes [ ] . eventually, the secreted cytokines and chemokines stimulate maturation and activation of apcs (mainly dcs) which initiate strong adaptive immune response (refer fig. ). therapeutic nanovaccines developed for treatment of metastatic cancer directly target dcs for strengthening adaptive immune responses in body [ ] . activated dcs serve as the link between innate and adaptive immunity. adaptive immunity generally has two arms; cell-mediated and antibody-mediated immunity. adaptive immune responses are generated within few days to several weeks and last longer. essentially, both types of immunity are necessary for a protective and prolonged immune response. however, their requirement is different in both prophylactic and therapeutic nanovaccines. for instance, strong cell-mediated cytotoxic responses are more desirable in case of therapeutic nanovaccine in order to effectively kill the metastatic cancer cells [ ] . vaccination provokes cell-mediated immunity generated by b cells and t cells which serve to neutralize the pathogen/antigen and simultaneously stimulating the body for developing immunological memory [ ] . the cmi responses are triggered by migration of activated dcs (following uptake of nanovaccine particle) to lymphatic organs (spleen and lymph noted). activated dcs present their antigens via mhc class i receptor to cd + cytotoxic t lymphocytes (ctls) (refer fig. ). activated ctls elicit strong cmi and selectively kill target cells (infected altered cells or cancerous cell) by inducing apoptosis in them [ ] . killing target cells is a complex immunological process that attracts several compliment systems (cs) proteins and co-stimulatory molecules. therapeutic cancer nanovaccines are preferentially designed to strengthen ctls responses by ex vivo educating dcs via adoptive transfer of antigen (for example, sipuleucel-t, a therapeutic vaccine approved for treatment of metastatic contraction resistant prostate cancer) [ ] . on other hand, activated dcs also present antigens to cd + t-helper cells (th cells) via mhc-ii receptor. subsequently, effector th cells undergo activation and maturation leading to secretion of variety of cytokines signals (refer fig. ). depending on the type of cytokine secreted, th populations are divided into th cells and th cells subsets [ ] . th subset majorly secretes proinflammatory cytokines (e.g. inf-γ , tnf-α, il- ) which stimulates proliferation of ctls and strengthen the function of cmi. th subset secrete class of cytokines (e.g. il- , il- , il- , il- , il- and il- ) which stimulate b cell proliferation during antibody-mediated immune responses [ , ] . a subtle balance between th /th responses determines overall therapeutic or prophylactic vaccine potential of candidate nanovaccine [ ] . in some cases, nanovaccines also work by suppressing t-regulatory (treg) cells, which naturally suppress activation and proliferation of effector t cells in the body [ ] . depending on the potential of nanovaccines to generate strong th cytokine response, naïve b cells located in spleen and lymph nodes get stimulated and bind to soluble antigen via b cell receptors (bcrs). activated b cell population undergoes proliferation in the germinal center. further, by somatic hyper mutation, b cells becomes specific to particular epitope of an antigen and then only epitope-specific b cells are selectively (clonal selection) proliferated. at this stage, activated b cells either differentiate into antibody-secreting plasma cells and secrete soluble antibodies against target antigen or remain dormant as memory cells for a future encounter of the same antigen (refer fig. ) [ ] . plasma cells are short-lived and their number gradually declines over time, as do the corresponding antibody titers [ ] . in that case, memory cells (b cells and t cells) which were generated and stored in the lymphatic organs or bone marrow for several months, come into play and protect the body from reinfection of the same antigen. in such situations, memory b cells quickly proliferate and turn to antibody-secreting (mainly igg) plasma cells to neutralize the antigen. on the other hand, memory t cells (both types cd + and cd + ) serve to produce more cytokine and chemokine signals to stimulate enhanced cmi and antibody-mediated responses. however, in case the structure of the antigen (epitope) has undergone significant changes due to cleavage, aggregation or re-folding, even memory cells cannot provide the required protection [ ] . although, in some cases, primary immunization can offer up to % protection, however, as even the rest % can be disastrous, therefore booster doses are scheduled to increase protection up to % [ ] . booster doses promote generating of more population and a variety of memory cells. generally, booster doses are scheduled after the antibody titer from plasma cells has dramatically declined to allow more competitive binding of memory cells to the injected antigen [ , ] . in a re-lated context, the use of nanoparticles help recalling more robust immunological memory even at a low dose [ ] . nanoparticles (e.g. plga based nanovaccine) facilitate prolonged availability of intact antigen in blood due to sustained release that leads to a higher proliferation of b cells and hence more memory cells [ , ] . also due to small size, nanoparticles can travel through the narrow lymphatic system to lymph nodes inducing differentiation of more memory cells [ ] . for example, demento et al., reported that over alum-based (free antigen) or liposome-based (burst release) carriers, mice immunized with ova-expressing listeria monocytogenes (lm-ova) encapsulated in plga particles (sustained-release) showed the highest protection against a lethal dose of l. monocytogenes even after weeks post immunization. they observed that mice immunized with antigen in plga nanoparticles had a robust population of cd + t memory cells [ ] . similarly, kanchan et al., showed that encapsulation of tetanus toxoid (tt) inside poly(lactic acid) (pla) particles generated long lasting primary antibody response in mice and interestingly even after months re-exposing the animal using very low antigen dose induced heightened and robust b cell responses [ ] . nanomaterials can be efficiently used for designing nanovaccines for the enhancement of immune-modulation. based on their potential use, they can be employed as adjuvants, immunogens or nanocarriers for enhanced and prolonged antigenic delivery. these properties of a nanoparticle are strongly governed by its size, shape, composition, surface chemistry and route of administration. it plays an important role in deciding its capability to induce inflammatory response or expression of specific defenserelated genes. different types of nanoparticles like protein, lipid, polymer, or inorganic particles (such as gold, iron, carbon and silica nanoparticles) can induce immune stimulation and have been discussed below [ ] . adjuvants are used with vaccines to enhance their potency by combination of mechanisms. adjuvants activate innate immune responses that shape and trigger the adaptive immune responses to vaccines [ ] . traditionally used adjuvants such as alum, complete freund's adjuvant and as (combination of alum and monophosphoryl lipid a) are known to form local depots that induce secretion of cytokines, chemokines and recruitment of immune cells. absorption of antigen on surface of adjuvants enhances antigen uptake and presentation, thus promoting antigen transport to draining lymph nodes [ ] . activation of tlrs has been potentially used for designing vaccine adjuvants. tlr ligands or agonists have served as potential immunostimulatory molecules as tlrs activate innate immune responses by identifying pamps and down signaling the transcription factors responsible for immune responses such as nf-κb, irfs and lymphocyte activation [ ] . the co-delivery of antigen along with tlr ligands promotes antigen presentation to cd + t cells via mhc-ii signaling and crosspresentation to activate cd + t cells [ ] . tlr agonist such as cpg up-regulates expression of cd , cd , cd , cd , and mhc class ii molecules thus enhancing antigen processing and presentation in plasmacytoid dcs [ ] . nanovaccines generate amplified, specific and robust responses as compared to traditional vaccines despite having similar mechanism of immune activation. the exaggerated immune response generated by nanoparticle associated antigen is due to their ability to specifically target dendritic cells with enhanced cellular uptake and antigen presentation. moreover, nanoparticles can be used as adjuvants with specific antigens or as immunogen themselves. vaccine antigens are either encapsulated inside or surface decorated over nanoparticles to get delivered efficiently (refer fig. ). there are chances that free antigens may degrade and elicit local immune responses at the site of administration, therefore, encapsulating free antigen inside nanoparticles prevents their degradation and provides prolonged and controlled release at the target site. the controlled release of antigen prevents burst responses and eliminates the need of booster doses. surface decoration of nanoparticles with antigen also confers the advantage of presenting the antigen to cells in a similar way as presented by pathogens [ ] . polyanhydride-based nanoparticles encapsulating f -v antigen when administered intranasally induced an immune response that persisted for weeks and elicited a high anti-f -v igg antibody response post-vaccination and conferred long-lived protective immunity against yersinia pestis infections compared to recombinant f -v antigen [ ] . similar nanoparticles were functionalized with di-mannose targeted macrophage mannose receptor (mmr). these carbohydrate functionalized nanoparticles enabled sustained release of immunogen for days and enhanced the uptake of antigen by dendritic cells simultaneously upregulating the co-stimulatory molecule clr, cd , and cd in cells as compared to non-functionalized nanoparticles [ ] . apart from encapsulation, several other strategies have been exploited for the co-delivery of antigens and nanoparticle such as surface absorption, conjugation, and mixture (refer fig. ). nevagi et al., observed enhanced immune activation against gas (group a streptococcus ) antigen using tri-methyl chitosan (tmc) nanoparticle conjugated with b cell and t cell epitope as compared to antigen mixed with tmc [ ] . nanoparticles have also shown potential to be used as adjuvants because of their immunomodulating activities such as inflammasome activation, complement system activation and recruitment of immune cells [ ] . due to above-mentioned effects, nanoparticles act as a better adjuvant in comparison to complete freund's adjuvant and conventional aluminum containing salts by inducing higher immunogenic responses at low antigen doses [ ] . also, their small size facilitates internalization by dendritic cells and induces adaptive immune responses. studies by swaminathan et al., have proved enhanced immune responses by lipid nanoparticle (lnp) adjuvant formulations against ovalbumin (ova) and hepatitis b virus surface antigen (hbsag) in balb/c and c bl/ mice injected with hbsag along with lnps as compared to hbsag alone [ ] . co-administration of quarternized chitosan nanoparticles as adjuvants along with ovalbumin intranasally in female balb/c mice induced activation of apcs followed by enhanced lymphocyte proliferation and differentiation as compared to ova mixed with aluminum hydroxide gel [ ] . asgary et al., also reported that silver nanoparticles showed increased antibodymediated responses against cvs- rabies and reduced toxicity as compared to conventionally used adjuvants such as alum [ ] . although nanoparticles possess great adjuvant properties, their codelivery along with generally used adjuvants such as tlr agonists can provide very robust immune protection. the use of tlr agonists is restricted because of their instability, particularly while using rna as tlr agonists. nugyen et al., have designed lipidoids to effectively deliver immunostimulatory rna to activate innate and adaptive immune responses. these lipid-rna nanoparticles enhanced ifn-γ responses and anti-ova cell-mediated and antibodymediated responses. these lipidoids were superior to the conventionally used n-[ -( , -dioleoyloxy) propyl]-n,n,n-tri-methyl ammonium methyl sulfate-rna delivery system [ ] . similarly unmethylated cpg-rich oligodeoxynucleotides (cpg), a tlr agonist. these nanoparticles induced pro inflammatory responses and antigen-specific antibody and cell-mediated immunity. colocalization of mpla and cpg also induced functional memory cd + cells. these findings clearly demonstrated the ability of bacterial elements and tlr agonists towards developing a more effective vaccine design [ ] . in addition, tlr agonists have been used in designing disease-specific nanovaccines, which are discussed later in this review. layer by layer protein nanoclusters synthesized from recombinant trimeric hemagglutinin were immunogenic by themselves and induced protective antibody-mediated responses without the use of adjuvants. such high immunogenic potential of peptide nanoclusters is because of the high antigen density available on the surface of nanoparticles [ ] . various types of nanoparticles have been used for nanovaccine formulation and each type serves a different function, which has been elaborated in the next section. protein-based nanovaccine can serve both structural as well as functional purpose by carrying antigens and engaging pattern recognition receptors of apcs. the protein-based nanovaccine can be categorized as biomimetic (virus-like particles, cages, vaults), rationally designed (nanofibrils, self-assembled protein nanoparticles, nanoclusters) and micelles. biomimetic protein-based nanovaccines : biomimetic proteinbased nanovaccines are of viral origin with self-assembled viral capsid proteins devoid of genetic material and are categorized as virus-like particles (vlps) (refer fig. (e) ). on the other hand, those of prokaryotic or eukaryotic origin are called protein cages and vaults. cages and vaults are generally used for biocatalysis or molecular transport. they share common properties with vlps like nanoscale, symmetry and container like geometries. all these structures can be modified chemically or biologically with antigens for immune activation [ ] . vlps ( - nm) of salmonella typhimurium bacteriophage p were engineered to encapsulate two respiratory syncytial virus (rsv) protein antigens, matrix (m) and matrix (m ). mice vaccination and intranasal boosting showed antigen-specific cd + and cd + t cell responses. upon subsequent rsv challenge, -fold reduction in viral load and increased antigen-specific cd + t cells were observed in the lungs of vaccinated mice as compared to empty p vaccinated and unvaccinated animals [ ] . cages like nm e cage derived from the pyruvate dehydrogenase complex of bacillus stearothermophilus [ ] , nm cage of human heavy chain ferritin (an iron storage protein) [ ] , ~ nm encapsulins (class of icosahedral cage structures) of bacteria and archaea are used for the presentation of different antigens to elicit immune response after immunization [ ] . a recent study on nonviral e protein-based biomimetic nanoparticle-containing acidlabile cpg (dc activating molecule) and siinfekl peptide epitope conjugate has shown the potential to enhance the cpg mediated dc activation by -fold in vitro as compared to unbound cpg. interestingly, co-delivery of siinfekl peptide conjugated with cpg has shown elevated cross-presentation and cd + t cell activation by inducing a -fold greater display of siinfekl on mhc-i by dcs as compared to unbound peptide or peptide bound directly to e protein. this shows the ability of biomimetic protein-based nanoparticles to facilitate enhanced immune response by dc activation and cross-presentation [ ] . vaults are eukaryotic ribonucleoproteins assembly of a cage-like barrel-shaped structure (approximately nm × nm × nm) made from the major vault protein (mvp) [ ] . it has been shown that increased antigen-specific cd + t cell response with a reduction in genital bacterial load and inflammation was observed after chlamydia muridarum polymorphic membrane protein g- (pmpg) encapsulating vaults were administered intranasally [ ] . rationally designed protein-based nanovaccines: nanofibrils are generally made of engineered β-sheets or α-helical coiled coils. β-sheet nanofibrils can be formed with peptide sequences like q (qqkfqfqfeqq) and kfe (fkfefkfe) appended to c terminus of a peptide antigen with a short linker sequence (refer fig. (d)). similarly, α-helical coiled-coil can also be formed into fibrils from α-helical peptide-like coil [ ] . such peptide sequences undergo assembly into fibrils with the antigenic epitope sticking outward from the fibril surface [ ] . these fibrils have been assembled with various epitopes of plasmodium falciparum [ ] , mycobacterium tuberculosis [ ] , streptococcus aureus [ ] , t-helper cells epitope padre [ ] and ova epitopes [ ] . conjugation of e ep immunogenic epitope of chikungunya virus e glycoprotein with β-sheet self-adjuvanted amyloid assemblies showed cytocompatibility with enhanced macrophage uptake and robust igg response against e ep epitope [ ] . self-assembling protein nanoparticles (sapns) are - nm icosahedrons containing pentameric and trimeric helical coiledcoils conjugated with (glycine) linker (refer fig. (f)). antigens and adjuvants can be inserted depending on the expected structure from in silico modeling [ ] . subunit malaria [ ] , hiv [ ] , toxoplasma [ ] and severe acute respiratory syndrome [ ] . wahome et al., encapsulated two hiv protein epitopes ( e and f ) onto sapn surface and observed enhanced production of epitope-specific neutralizing antibodies. this indicates the potential of sapn as nanovaccine to activate immune response against hiv [ ] . nanoclusters have also emerged as a prominent vaccine platform prepared from desolvation of proteins and peptides. they are generally made up of antigens and crosslinkers with an intent to increase antigen loading and eliminate off-target sequences. intranasal vaccination of influenza matrix protein (m e) containing self-assembled protein nanoclusters elicited enhanced immune response and complete protection after lethal challenge with hetero and homo-subtypic virus [ ] . since the preparation of nanoclusters requires organic solvents, it may alter the secondary or quaternary structure of full protein antigens [ ] . micelles: antigenic molecules can be conjugated to micelles in two different ways. either they can be covalently linked onto the micellar surface or can be conjugated to the variety of alkyl tails to form amphiphilic peptide micelles (pams). micelles of different sizes and shapes affect their immune activation potential [ ] . studies have shown that cylindrical or spherical pams are efficiently able to reach lymph nodes and carry the required amount of antigen and adjuvant [ ] . studies have reported pam mediated increased immunogenicity of t cells [ ] and b cells epitopes [ ] . such peptide lipid conjugates also form nano-disc vaccines (refer fig. (g)). subcutaneous vaccination with neo-antigen adpgk nanodiscs showed enhanced cd + cellular response, apc uptake and increase survival after b f melanoma challenge as compared to intramuscular vaccination [ ] . peptide-polymeric micelles have also been explored as a vaccine platform by conjugating hydrophilic peptide antigen with dendrimer polymers for immunotherapy [ ] . the most common lipid-based nanovaccines getting explored from the past few decades are liposomal nanovaccines (refer fig. (b) ). they offer a distinct advantage over other nanovaccine systems in terms of being biodegradable, tolerable, less immunogenic and having tunable physicochemical properties. they provide a wide repertoire to load antigens of different philicity inside and outside of liposomes with easy surface conjugation [ ] . different physicochemical properties (like size [ ] , surface charge [ ] , surface modifications (peg) [ ] , bilayer composition and fluidity [ ] ) affect the immunological outcome of liposomal nanovaccines. size-dependent differential activation of immune response indicated the activation of th response by nm sized liposomes as compared to th response by liposomes ≥ nm [ ] . transdermal immunization of mice with dissolving microneedles loaded with opposite charged transferosomes indicated the enhancement of th immune response by cationic transferosomes as compared to anionic counterparts by exhibiting strong endocytic escape property with fecilitating antigen processing via mhc-i pathway and larger accumulation in lymph nodes [ ] . kaur et al., showed the effect of bilayer fluidity on immune activation by varying cholesterol concentrations. relatively lesser in vivo igg production and ifn-γ was observed by liposomal formulations containing high cholesterol [ ] . similarly, the effect of another important factor affecting bilayer fluidity on liposomal mediated immune activation was observed by preparing liposomes with phospholipids having different transition temperatures (tm). high tm phospholipids containing liposomes stimulated th immune response as compared to low tm phospholipids that induced th response in mice immunized with leishmaniasis rgp antigen [ ] . numerous clinical studies showed the use of liposomes as adjuvants in therapeutic vaccines. liposome encapsulated d. pteronyssinus vaccination reduced allergen bronchial provocation induced inflammatory changes and improved condition of asthma patient after sustained mite exposure [ ] . a phase iii study of tecetomide vaccine, containing blp lipopeptide antigen incorporated into dmpg:dppc:chol:mpl-liposomes was performed for stage iii non-small cell lung cancer after subcutaneous immunization (nct ) [ ] . however, the study was terminated due to no effect on survival and primary or secondary endpoint in a phase ii study [ ] . a phase i trial (nct ) showed safety and immunogenicity of caf (liposomal adjuvant system) given with a subunit vaccine against m. tuberculosis antigen h . this adjuvanted vaccine showed long-lasting t cell response with no antibody response observed in healthy adults [ ] . viral liposomes called "virosomes" have also been studied in the literature. virosome exhibits viral capsid proteins onto the li- posomal surface for effective fusing with the target cell membrane. recently, virosomes and liposomes mediated immune response was investigated on human respiratory tract triple culture model. both virosomes and liposomes were prepared from the same neutral phospholipids, dopc ( , -dioleoyl-sn-glycero- -phosphocholine) and pope ( -palmitoyl- -oleoyl-sn-glycero- phosphoethanolamine), but virosomes were also fused with influenza membrane protein. results showed enhanced internalization of virosomes in epithelial cells of triple culture as compared to liposomes of similar sizes [ ] . a recent study has shown the use of lipid-based - nm particles (iscomatrix particles) as adjuvant with chimera peptide vaccine containing gp , tax, gp and gag epitopes of human t cell lymphotropic virus type when given subcutaneously in mice. enhanced production of mucosal iga and igg a antibody titers along with ifn-γ and il- cytokines were observed as compared to vaccine formulation [ ] . another class of lipid-based nanocarrier includes 'hexosomes' and 'cubosomes' that can provide a high membrane surface area for antigen and membrane protein loading. hexosomes are generally made up of lipids known to form non-lamellar structure in hydrated systems with inverse hexagonal liquid crystalline phase. comparative adjuvant activity of mono mycoloyl glycerol- (mmg- ) immunopotentiator containing hexosomes and cationic liposomes (caf ) loaded with chlamydia trachomatis major outer membrane protein (momp) antigen in cb /f mice after vaccination was studied by rodrigues et al. mmg- hexosomes made up of lipid phytantriol induced a strong momp-specific antibodymediated immune response whereas mmg- cationic liposomes elicited much stronger momp-specific cell-mediated response. it indicated the immune activation by two lipid-based adjuvants via different mechanisms [ ] . in addition, cubosomes are generally made up of lipid bilayers forming continuous periodic cubic lattice structures with interwoven water channels and a stabilizing polymeric outer corona [ ] . phytantriol, pluronic f and propylene glycol-based cubosome formulation containing (imiquimod and monophosphoryl lipid a (mpl)) adjuvants and model ova antigen elicited robust ifn-γ production and cd + and cd + t cell proliferation as compared to similar adjuvant containing liposomal formulation and alum [ ] . such lipid-based self-assembled structures can provide high antigen and adjuvant loading with enhanced immunoactivation in comparison to conventional lipidbased carriers like liposomes. polymeric particles showed a great possibility to be used as nanovaccines due to their ease of preparation, biocompatibility and biodegradability, fine-tuning of surface properties and controlled release kinetics (refer fig. (a) ) [ ] . the most commonly used polymeric nanoparticle for antigen delivery is poly (lactic-coglycolic acid) plga or poly (lactic acid) pla that has been used to deliver a broad range of antigens like hepatitis b virus antigen [ ] , tetanus toxoid [ ] , bacillus anthracis antigen [ ] , mycobacterium tuberculosis antigen [ ] and ovalbumin [ ] . other natural polymers like chitosan [ ] , alginate [ ] , pullulans [ ] , and inulin [ ] have also been used as adjuvants and antigen carriers. chitosan alginate-based nanovaccines for hepatitis b virus [ ] and dna-based chitosan vaccines for mycobacterium tuberculosis ( mtb h rv ) showed a marked increase in immunologic and protective efficacy in immunized mice [ ] . hyperbranched polyglycerol (hbpg) globular polymer can be a potential candidate for inert dendrimer based multi-valent vaccine coupled with b cell epitope for tumor-associated antigen (muc ) and t cell epitope for tetanus toxin p . it showed enhanced immune response and igg antibodies against breast cancer cells. hence polymeric nanoparticles can be used for potential multivalent vaccines [ ] . several inorganic nanoparticles (gold, iron, carbon, silica nanoparticles) are being explored for vaccine delivery in different disease models (refer fig. (c) ). ease of antigen functionalization onto easily accessible surface groups, robust properties with reproducibility outweighs their low biodegradability and gives them an edge to be used as nanovaccines. they also increase antigen stability by preventing premature proteolytic degradation. however, there are reports showing dose and size-dependent clinical toxicities of these nanoparticles [ ] . amongst all the inorganic nanoparticles, gold nanoparticles have gained maximum attention in vaccine delivery. these particles have been used for the epitope delivery against hiv [ ] , influenza [ ] , malaria [ ] and tumor [ ] . effect of shape of gold nanovaccine on antibody production against wnv envelope e protein showed that rods induced only % of the antibody response as compared to spherical nanoparticles [ ] . multicopy multivalent nano-glycoconjugates of gold nanoparticles decorated with tn-antigen glycan generated strong long-lasting antibodies against breast cancer expressing aberrant mucin glycan [ ] . inorganic carbon spherical nanoparticles and nanotubes were used as adjuvants to increase immunogenicity and act as delivery vehicles for peptides and protein against various viral infections [ , ] . oral immunization of bovine serum albumin (bsa) loaded carbon nanoparticles showed effective stimulation of mucosal iga antibodies in intestinal, vaginal and salivary mucosa along with th and th responses [ ] . abundance of silanol groups over silica nanoparticle surface provides an advantage for easy conjugation of targeting moiety for various viral infections [ ] . silica vesicles adsorbing e glycoprotein of bovine viral diarrhea virus (bvdv) showed -month antibody-mediated and cellmediated immune response with no histopathological changes at the site of infection [ ] . the use of magnetic iron oxide nanoparticles for tumor-associated carbohydrate antigen (taca) through phospholipid functionalization of taca glycopeptide showed enhanced antibody titers against both human and mouse tumor cells expressing that glycopeptide [ ] . calcium nanoparticles have also been reported for their superior adjuvant property with dna vaccines and to confer mucosal immunity. they can also induce enhanced antibody production against viral antigens as compared to aluminum adjuvants [ ] . since different types of nanoparticles impart different properties to the developed nanovaccine, their vast use in immune activation against a variety of antigens have been observed. though clear demarcation or direct comparison across nanocarriers has not been studied but there are unique properties and advantages associated with each nanocarrier that gives them an edge over each other. biomimetic carriers like vlps are expected to possess inherent immunogenicity that can elicit antibody reaction against carrier antigens itself. this could lead to the competition with the target antigen and alter antibody repertoire or memory response against it. reactive toxicities or neutralization upon repeated dosing can also limit their multiple use in a patient [ ] . such problems can be avoided by the use of nanoclusters that are solely made up of single type of antigen. these protein-based biomimetic and rationally designed nanocarriers are most suitable for the incorporation of a highly hydrophobic antigens of varied sizes with an intent to preserve its native structure, whereas hydrophilic peptides can be well delivered using amphiphilic micelles [ ] . carrier specific immune response has also been observed for certain polymeric and lipid-based carriers but it can be avoided using natural or biomimetic components (e.g., cellular lipid isolates or extracellular matrix polymers) [ ] . control over spatiotempo- table . different type of nanovaccines can escalate the immunogenic potential of antigens by boosting the immunogenic responses and memory generated against antigens at low doses. immunogenic responses generated against certain antigens can be used in two different scenarios by varying the use of vaccines as prophylactic or therapeutic (refer fig. ) . prophylactic vaccines aim to develop protective immunity and are administered before the onset of disease. the goal of prophylactic vaccines is to develop immunogenic memory against certain infections. major challenge of prophylaxis is obtaining sufficiently long-lasting immunogenic memory. for instance, single-dose immunization of mice with polyanhydride nanoparticle containing pneumococcal surface protein a (pspa), a virulence factor of s. pneumonia activated protective immunity and enhanced the survival of animals upon challenge even at -fold reduction in dose. pspa based nanovaccine formulation performed comparable to protein adjuvanted with alum, with much less tissue reactivity at the site of immunization [ ] . prophylactic nanovaccines confer protective immunity mostly against infectious conditions at low doses of immunostimulating antigen and reduced need for adjuvants, thus mitigating associated toxicities. therapeutic vaccines are administered after the onset of diseases to alter the course of disease by encouraging the immune system to fight harder against the prevailing conditions. unique immune responses are generated against disease-specific antigens. generally, therapeutic vaccines are used for cancer treatment, but there are compelling evidences suggesting their potential use in autoimmune disorders also. the advantage of using therapeutic vaccines instead of conventional pharmaceutical treatment lies in their specificity [ ] . mono-specific, disease- phase ii [ ] relevant pmhc complexes when coated on nanoparticle surfaces triggered the selective expansion of memory-like autoregulatory cd + t cells that arise only in affected individuals. such engineered nanoparticles have the potential to become suitable vaccines with the capacity of resolving organ and disease-specific autoimmune responses. systemic delivery of type-i diabetes relevant peptide-mhc-nanoparticle complex triggered expansion of memory autoregulatory t cells and suppressed the autoimmune attack against insulin-producing beta cells, thus restoring glucose balance [ ] . in certain cases, nanovaccines can be formulated to achieve both prophylactic and therapeutic responses. poly ( γ -glutamic acid) based synthetic vaccine nanoparticles (svnps) loaded with ovalbumin and toll-like receptor agonist (poly (i:c)) were synthesized by kim et al., both of them enhanced the uptake of antigens by apcs and enhanced the secretion of inflammatory cytokines (tnf-α, il- ) and type i interferon (ifn-α, ifn-β). simultaneous injection of both svnp-ova and svnp-ic conferred both the protective as well as therapeutic immunities by inhibiting tumor growth in eg -ova tumor-bearing mice [ ] . it is clear from the above-mentioned examples that nanovaccines modulate the immune system either for providing prophylaxis or therapeutic responses against pathological conditions. majorly prophylactic vaccines are used to prevent infections ( table ) and therapeutic vaccines have been used for cancer ( table ) treatment. infections that can be prevented by vaccination still contribute to approximately half of the child morbidity each year [ ] .treatment and immunization of such infectious conditions is a greater challenge due to the rapid emergence of new pathogenic variants. in spite of advancements in vaccine development over the years, many vaccines are associated with the risk of regaining their pathogenicity under certain immuno-compromised conditions. so, there is an imperative need for the development of risk-free and effective vaccines that confers desired antibody-mediated and cellmediated immunity against infectious diseases [ ] . subunit nanovaccine against enterohemorrhagic escherichia coli (ehec) was developed using gold nanoparticle conjugated with ehec antigens and induced high titers of serum igg upon subcutaneous administration in mice. it has also prevented colonization of ehec in both the cecum and large intestine at days post-immunization [ ] . mice immunized against anthrax with a single dose of pad -np (plga-based protective antigen domain, nanoparticles) elicited robust antibody-mediated and cell-mediated responses with mixed igg /igg a subtypes and th /th response. the survival rate of mice immunized with pad -np was more than for pad immunized mice after a lethal challenge with bacillus anthracis spores [ ] . nanovaccines have also shown great prospects in mitigating parasitic invasion. chimeric peptides containing leishmania infantum epitopes (histone h , kinetoplastid membrane protein and cysteine peptidase a) were encapsulated in plga nanoparticles. stimulation with the peptide-based nanovaccine induced maturation of dcs and il- production subsequently promoting allogeneic t cell proliferation and production of ifn-γ by cd + and cd + t cell. on immunization of transgenic mice with this peptide-based nanovaccine induced peptide-specific ifn-γ producing cd + t cells and conferred protection against l. infantum infection [ ] . multiple self-assembling peptide-based nanovaccines containing epitopes of toxoplasma gondii specific antigens have been designed. immunization with self-assembled protein nanoparticles activated cd + t cells to produce ifn-γ and protected transgenic mice against subsequent challenge with type ii parasites [ , ] . some more example of nanovaccines for highly prevalent infectious diseases have been discussed below in detail. tuberculosis being the deadliest infectious disease, is the greatest cause of mortality worldwide. % of infected individuals develop the disease within one or two years of infection and the rest develop the disease in later stages of life when immune functions are compromised. co-infection of hiv patients with mtb ( mycobacterium tuberculosis ), further complicates the situation with % mortality each year [ ] . bcg (bacille calmette-guerin) is a widely accepted vaccine against tb (tuberculosis), but its protective efficacy is limited by age due to the absence of consensus genomic loci that are present in most of the virulent strains of mtb. it induces short term and variable immune responses from % to % [ , ] . there is an urgent requirement of booster vaccines that augment t cell immunity in the lungs of bcg-vaccinated individuals. to address this, ansari et al., have synthesized archeosomes encapsulating a t cell antigen rv c, that upon immunization elicited type- cytokine response in mice. also, it increased the production of igg a antibodies, antigen-specific t lymphocytes and enhanced jid: actbio [m g; april , ; : ] expression of co-stimulatory molecules on the surface of apcs. by the virtue of all these properties, vaccination with archeosomes containing antigen reduced mycobacterial burden in the lungs and spleen of animals upon challenging with mtb [ ] . similarly, selfassembled peptide nanofibers, when loaded with t cell epitope, also showed similar responses by inducing corresponding effector memory t cells and increasing cytotoxic t cell population in the lungs upon intranasal immunization. mice vaccinated with coassembly of nanofibers containing cd + t cell epitopes and tlr agonists in the lungs increased the expansion of multi-functional cytotoxic t cell population. on further challenge with mtb, significant reduction in lung bacterial load was observed in comparison to bcg primed mice [ ] . in another study, mtb lipid-based nanovaccines were used wherein chitosan nanoparticles bound with mtb lipids induced cell-mediated and antibody-mediated immunity by secretion of immunoglobulins (igg, igm) and prominent th and th cytokines in lymph node and spleen cells [ ] . the higher potency of nanoparticle encapsulated antigen preparation is because of their ability to form depots with a slow and steady release in the surrounding milieu and enhanced uptake of antigen by antigen-presenting cells [ ] [ ] [ ] . as shown in a study by diogo et al., liposome-encapsulated antigens elicited more profound antibody-mediated and cell-mediated responses than free antigens. mice were immunized with free antigens and liposomeencapsulated antigen, further challenging mice weeks post immunization with mtb, it was observed that mtb burden in the lungs and spleen was significantly less in mice immunized with liposome-encapsulated antigen as compared to free bcg vaccine [ ] . the potential of nanoparticles in augmenting the prophylactic activity of antigens against tb was well understood and human trials were initiated. first-in-man trial on novel liposome caf as an adjuvant along with the tuberculosis vaccine ag b-esat- (h ) is reported in [ ] . human volunteers were vaccinated with escalating doses of caf at and weeks. after immunization strong antigen-specific t cell responses persisted for weeks and did not cause local or systemic adverse effects [ ] . above discussed preclinical and clinical assessment of various antigen-loaded nanoparticles indicates the potential of nanovaccines in inducing robust and long-lasting cellular immunity against mtb and shows hope for the development of a different and more effective prophylactic regime against tb. it is clear that with the advent of nanotechnology, adjuvant and delivery vehicle associated problems have been reduced, but the effect of the host environment and lack of consensus genetic loci still remains an inevitable challenge while developing efficient vaccines against tb. malaria is known to affect almost million people annually, with half a million deaths worldwide [ ] . fighting malaria is challenging because of its multistage life cycle. various efforts have been made in designing vaccines against the pre-erythrocytic and blood stage of malaria [ , ] . similarly, nanovaccines have been used to target multiple stages of malaria. iron oxide (io) nanoparticles have been used for the delivery of merozoite surface proteins (rmsp ). immunization of mice with rmsp- loaded nanoparticles induced parasite inhibitory antibodies with high titers. macrophages and dendritic cells showed more than % internalization of io nanoparticles and enhanced the expression of cytokines and chemokines [ ] . immune responses generated against sexual stage antigens of malaria impairs its transmission and reduces the disease burden. pfs is one such malaria transmission-blocking vaccine antigen, but its immunogenicity is limited in humans. hence, pfs has been used along with nanoformulations to achieve desired im-mune responses. for example, codon harmonized pfs (chrpfs ) has been used with gold nanoparticles as adjuvants for the induction of immunity against transmission. strong transmissionblocking antibodies elicited by simultaneous delivery of chrpfs with gold nanoparticles could be due to the co-ingestion of nanoparticles and antigen by immune cells [ ] . similarly, recombinant polyhistidine-tagged (his-tagged) pfs when mixed with preformed co-porphyrin containing liposomes at the time of immunization resulted in spontaneous nanoliposome antigen particularization (snap). immunization of mice and rabbits with snap resulted in higher functional antibody generation as compared with other 'mix-and-inject' adjuvants. they have also been used for developing multi-antigen vaccines (apical membrane antigen- , pfg , pfs and the nanp peptide), targeting multiple stages of the plasmodium life cycle using liposomes [ ] . ovalbumin loaded carboxylated polystyrene nanoparticles acted as an adjuvant and induced il- and granulocyte colonystimulating factor, that affects the migratory and homing ability of dendritic cells. mice challenged with malaria two weeks after immunization with nanoparticles produced anti-malaria antibodies and create the state of immune readiness to a subsequent infectious challenge [ ] . stable nanomimic, which are polymersomes with receptors for parasite attachment were synthesized. they bind to host cells and block re-invasion of the parasite after their release from host cells in vitro . this strategy can be used in the future to modulate immune responses against malaria and in the efficient designing of the vaccine [ ] . thus, the increased role of nanovaccines in targeting multiple life cycle stages of malaria shows new avenues in building a robust vaccination regime for prevention against malaria infection. three types of influenza viruses a, b and c have different viral nucleoproteins and matrix proteins giving rise to variable antigenic differences among them. influenza virus a and b cause epidemics attacking both adults and children, causing around million infections annually [ ] . presently, two types of vaccines are used against influenza based on strain a and strain b that target and produce antibody-mediated protective responses against hemagglutinin and neuraminidase. these antigens are highly prone to antigenic shift and drift due to error-prone rna dependent rna replication in influenza [ , ] . due to environmental selection and antigenic variations, chances of influenza epidemic still persists [ ] . although animal influenza virus is distinct from the human virus, zoonotic animal viruses can still occasionally infect humans through direct/indirect contact. avian and swine influenza viruses have been known to infect humans in some countries. thus, there is an unmet need for producing more efficient vaccines that can provide both antibody-mediated and cell-mediated immunity to confront homologous and heterologous variants [ ] . association of various influenza antigens with nanoparticles have shown promising preliminary results in providing enhanced immune responses against variable influenza antigens. as the antigenic variability limits the use of a single vaccine against influenza a virus (iav), there are effort s to develop nanovaccines conferring protection against more than one serotype by using most conserved ectodomain of influenza matrix protein (m e). nanoparticles containing self-assembling recombinant cage of human heavy chain ferritin presented with -sequential repeats of m e protein was observed to elicit elevated levels of m especific igg and mucosal secretory iga antibodies with enhanced t cell response against h n and h n lethal infection after intranasal administration in mice [ ] . nanovaccines against the conserved ectodomains can confer protection against inter-species viral infection. self-assembled virus-like particles (vlps) containing multiple copies of m e protein inserted into capsid (cap) protein of porcine circovirus type (pcv ) have shown dual protection against iav and pcv lethal challenge in mice and pigs after generating m e specific and pcv neutralizing antibodies [ ] . hemagglutinin trimer has also been exploited as a potential antigen for inducing protective immunity against influenza virus. pham et al., immunized balb/c mice with nanodiamond conjugated trimeric h and observed a significant higher h specific igg production as compared to immunization with h trimeric antigen alone [ ] . similarly, ross et al., have shown that intranasal or subcutaneous immunization of balb/c mice with polyanhydride nanoparticles encapsulated h induced high titer neutralizing antibodies. also, significantly higher cd + t cell expansion was observed in vaccinated mice as compared to non-vaccinated mice, thus providing cell-mediated and antibody-mediated protective immunity to mice. mice were further challenged intranasally with a/h n vnh n -pr cdc-rg influenza virus at days post immunization and viral load was significantly reduced as compared to saline-administered mice [ ] . it has also been demonstrated that the polyanhydride nanoparticles give equivalent responses at -fold lower doses as compared to free antigen [ ] . also, encapsulation of antigen in polyanhydride nanovaccines exhibited stability of antigen for one year at room temperature, thus providing a major benefit for maintaining stocks of pandemic vaccines [ ] . as discussed above, the nanovaccines have been designed by various research groups as combinations of nanomaterials and different antigens, but they provide protection from a particular strain of the virus. due to antigenic variations, multiple strains of influenza virus have emerged over the years posing serious public threats, indicating the need for a universal vaccine, which includes broad cross-presentation against multiple strains of influenza to reduce community threats. insights into the structural properties of antigens or peptides can be further explored for the rational designing of broad-spectrum nanovaccines using multiple antigens. on similar lines, to increase the breadth and potency of nanovaccines, deng et al., have designed double-layered protein nanoparticles. the core of the protein nanoparticles ( mtg) contains four types of m e from human (hum e), swine (swnm e), avian (avim e), and domestic fowl (fwlm e) viral consensus sequences coated with ha variants hrh and hrh (refer fig. ). mice were immunized intramuscularly with uni mc (pnp), uni c (hrh -coated double-layer pnp) and uni c (hrh -coated doublelayer pnp) and uni c (cocktail of uni c and uni c ). robust antibody-mediated responses were generated and induced m e antibodies were strongly cross-reactive to diverse antigens. also, uni c group produced broad cellular responses by significantly inducing more ifn-γ secreting splenocytes after stimulation with diverse antigen peptides. naïve mice were vaccinated with sera from pre-immunized mice with dpbs, uni mc, or uni c and then challenged intranasally with pr or aic after hours. sera from uni c immunized mice prevented the mice from a viral infection, indicating the role of serum antibodies. also, antibodies specific to human m e, pr , and aic were viable for four months after the immunization in mice and implied long-lasting protection [ ] . from the examples discussed above, it is evident that with the help of structurally important antigens, smartly designed protein nanomaterials will have the potential of inducing long-lasting immunity against broad spectrum of antigens, thus providing platforms for producing efficient nanovaccines to combat pandemic viruses. hiv is the sixth leading cause of death worldwide. hiv infection causes systemic depletion of cd + t cells, hence compromising the activity of the immune system and the development of aids. immune functions can be preserved in the early stages of hiv by using antiretroviral therapy (art). it targets the hiv life cycle, but the major challenge is strict adherence to complex art regimens. this necessitates the urgent need for the development of a prophylactic vaccine against hiv. the most recent and promising hiv vaccine rv is in the phase iii trial [ ] . igg antibodies raised against v v loop were inversely correlated with the risk of infection in the clinical trial of rv [ ] . sapn-based vaccine reduces the need for essential glycosylation of the hiv- env v v loop to form a native-like structure to act as potential immunogen [ ] . although self-assembling vlps as vectors have shown great potential in the development of immunity against hiv, anti-vector immunity is a significant concern. here, synthetic nanoparticles offer versatile platform technologies that can induce strong adaptive immune responses while avoiding anti vector immunity and toxicity issues [ ] . plga based nanoparticles have been used for co-utilization of tlr agonist and the antigen. intradermal immunization of mice with hiv- p -nef/flic/plga (antigen/agonist/nanoparticle) enhanced igg production even at -fold lesser dose of antigen. nanovaccines shifted the immune response towards th polarization and enhanced the th cytokine pattern [ ] . recently, protease cleavage sites (pcs) has been utilized as a strategy to hamper the maturation and infectivity of hiv. due to low immunogenicity of peptide antigens, they have been crosslinked with chitosan and hyaluronic based nano-formulation. nanoparticles containing pcs showed enhanced activation of apcs and the production of anti-pcs antibody. also, these nanoparticles generated memory t cells at longer time points after the last booster dose, indicating their capacity to elicit a good immune response upon infection [ ] . silver nanoparticles coated with amantadine triggered the production of hiv specific ctls in the spleen of mice with -fold stronger tnf-α production in vivo . the co-culture of these hiv specific ctls with hiv infected cells in vitro enhanced the death of infected cells and reduced the production of hiv [ ] . similarly, hiv- peptide and oligosaccharide loaded aunps enhanced antigen presentation to isolated t cells from hiv- patients. treatment with aunp formulated vaccine increased proliferation of hiv-specific cd + and cd + t cells with increased secretion of pro-th cytokines and pro-inflammatory cytokines in vitro [ ] . malik et al., have shown the prophylactic potential of intravaginally delivered nanogold loaded formulation. poloxomer and hyaluronic based thermogels were loaded with gold niosomes and mannosylated gold niosomes along with efavirenz (efv). efv and gnps inhibited viral dissemination in pbmcs (peripheral blood mononuclear cells) host cells, but their individual potential in inhibiting pre-interaction viral dissemination was lower than that of the combination. a substantial . % increase in activity was seen when efv was given with gnps. both the moieties had a dual-faced attack wherein gnps inhibited hiv entry into the cell by causing denaturation of gp glycoprotein and efv inhibited transcriptase enzyme. the mannosylated efv-gnps (man(egnz)) showed further potential activity in inhibiting viral dissemination when the host was pre-exposed to them before viral infection. thermogels containing efv-gnp and man(egnz) showed . ± . % and . ± . % inhibition respectively in p antigen during anti-hiv prophylactic challenged study [ ] . various such formulations have been developed over the years that enhanced the prophylactic action of raltegravir and efavir [ ] , phthalate and efavirenz [ ] or efavirenz and saquinavir [ ] . bayon et al., have also shown the capacity of nano-lipid complexes (nlc) to induce p specific immune responses against hiv in non-human primates (nhp). four intradermal immunization over a period of months showed that nlc-loaded p- antigen elicited significantly higher p -specific antibodies as compared to the combination of free p antigen and cpg adjuvant [ ] . there have been advancements in understanding the role of nanovaccines in fighting hiv and development of nanovaccine loaded formulations for prophylaxis of hiv. with the emerging use of nanotechnology development of an efficient vaccine system for hiv prevention is not that far. despite constant efforts to develop effective cancer vaccines, their efficacy in clinics has always been limited. nonetheless, two prophylactic vaccines for hepatitis b virus-associated liver cancer and human papillomavirus virus-associated cervical cancer have been approved by us fda till now [ ] . the prophylactic vaccine development for cancer eradication is a challenge due to inefficient identification of tumor-specific antigens (neoantigens), rapid clearance with less accumulation in the lymphoid organs and generation of weak memory immune response. therefore, the use of nanoparticles to overcome these limitations would be an advantageous strategy. prolonged and sustained delivery of antigen would be extremely important for the generation of immunologic memory and booster dose reduction. immunization of mice with protein nanoparticles conjugated to melanoma-associated epitope gp and cpg adjuvant enhanced the production of melanoma-specific cd + t cells. pre-immunization with nanoparticles delayed onset of tumor growth and increased the survival rate to %, showing prophylactic potential of protein nanoparticles [ ] . luo et al., showed strong cytotoxic t cell response by a physical mixture of pc a polymeric nanoparticle and ova antigen. this was achieved by enhanced cytosolic delivery and antigen presentation in apcs of draining lymph node by pc a nps and activation of type-i interferon stimulating genes. nanovaccines showed tumor growth inhibition in melanoma, human papilloma virus e /e tumor and colon cancer models. the combination of anti-pd with pc a nps showed synergism with % survival in a tc- model over days. further, re-challenging of tumor-free mice with tc- cells after immunization with pc a nanoparticles and anti-pd showed complete tumor growth inhibition. this indicates the prophylactic response of nanovaccine by generating long-term antitumor memory [ ] . liu et al., developed a unique prophylactic nanovaccines using metallo-organic nanoparticles (mof) coated with fused membrane (fm) of dendritic cells and cancer cells. presence of whole tumor antigens and immunological co-stimulatory molecules on its cytomembranes impart them apc like properties for cancer specific t cell immuno-activation. since, fusion of dc and cancer membrane is accompanied by dc maturation, mof@fm nanovaccines possessed lymph node homing molecules. therefore, enhanced retention and homing of mof@fm in lymph nodes and spleen as compared to only cancer membrane fused (mof@cm) and dendritic cell membrane fused (mof@dm) nanovaccines was observed. pre-immunization of mice with mof@fm has significantly prevented the development of t tumors by promoting differentiation of cd + t cell precursors into cytotoxic t cells with enhanced generation of ifn-ɣ and il- immuno-stimulatory cytokines (refer fig. ) [ ] . tumor heterogeneity with differential expression of antigens makes it challenging to develop a prophylactic treatment strategy. therefore, most of the nanovaccines being developed for cancer are therapeutic. the detailed description of the same is given in the next section. unlike conventional therapeutics, therapeutic nanovaccines offer the prospect of eliciting antigen-specific responses without any additional non-specific responses. therapeutic vaccines have been used against autoimmune disorders by antigen-specific targeting of t-regulatory cells [ ] . clinical studies revealed the therapeutic efficacy of vlp in allergen-induced asthma. bacteriophage derived vlps were packed with cpg and tlr and induced th response in immune system. a total of asthmatic patients were treated with injections of vlps as a part of double-blind randomized trial and it was observed that the asthma was well-controlled in patients treated with vlps as compared to the placebo group [ ] . unlike cancer, nanovaccines showed promising results in pathogenic infections because of the presence of extremely potent foreign non-self antigens. therefore, vaccine-based therapy is still limited in cancer treatment but can provide an alternative to, or complement conventional cancer treatments. till now, cancer vaccines have failed to make a mark clinically as compared to other immunotherapies like t cell therapy and checkpoint blockade. the only us fda approved therapeutic cancer vaccine, provenge (sipuleucel-t), has shown a moderate increase in clinical efficacy in prostate cancer [ ] . the major problem in successfully developing a cancer vaccine is the selection of tumor-specific antigens called "neoantigens". conventionally, cancer vaccines were developed by targeting tumor-associated antigens (taas), which are overexpressed specifically in a particular tumor type across patients. however, taas are also present in normal tissues; therefore vaccines specific for them can induce peripheral or central immunotolerance leading to autoimmunity or low vaccination efficiency [ ] . neoantigens are tumor-specific antigens derived from random somatic mutations but absent in normal cells. hence, vaccines targeting such neoantigens using nanoparticles may enhance the outcome of cancer immunotherapy. cancer nanovaccines can present unique and highly immunogenic tumor-specific antigens with increased antigen loading and efficient delivery, controlled antigen presentation, and retention in lymphoid organs [ ] . a recent study showed the targeting of bone marrow and splenic dcs with peptide ty using mesoporous silica nanoparticles conjugated with ova/cpg for immune activation (msn-ty/ova/cpg). therapeutic application of (msn-ty/ova/cpg) in naïve c bl/ j b -ova mice enhanced dc activation and tumor elimination by eliciting tumor-specific cd + t cell response, with prolonged survival and less systemic toxicity [ ] . to overcome inadequate loading efficiency, weak immune response, and complex preparation process, dong et al., prepared antigen nanoparticles (ovalbumin nps) containing cpg adjuvant (onps-cpg) with high antigen and adjuvant loading for cancer immunotherapy. the in vitro and in vivo results showed enhanced immune response including dc maturation, t cell activation and ifn-γ production. onps-cpg induced remarkable anti-tumor efficacy in mouse lymphoma model [ ] . biomimetic-approach for developing cancer nanovaccines: overcoming cancer heterogeneity by using patient-derived cell membranes for the development of smart nanovaccines can be a promising biomimetic approach for personalized therapy. various strategies have been explored in the form of developing artificial antigen-presenting cells (aapcs) based nanovaccines, use of different cell membrane fused nanovaccines and membrane coated viruses for enhanced cross-presentation and immune stimulation (refer fig. ) . recently, kuai et al., have shown the anti-tumor efficacy of high-density lipoprotein (hdl) nanodiscs loaded with neoantigenic peptide and cpg t cell receptor agonist in b f and mc- colon carcinoma model. nanodiscs have delivered the neoantigen to draining lymph nodes and enhanced antigen presentation by apcs. it causes enhanced stimulation of cytotoxic t cell response that potentially inhibited tumor growth. delivery of nanodiscs with checkpoint blockade (anti-pd and anti-ctla- ) therapy also eradicated established b f and mc- colon carcinomas. this strategy showed a powerful therapy to generate personalized cancer medicine [ ] . zhang et al., developed a magnetic artificial antigen-presenting cell (aapc) for enhancing cd + t cell-mediated tumorigenic response. they developed fe o magnetic nanoclusters coated with leucocyte membrane antigens decorated with mhc-i loaded peptide (siinfekl) and anti-cd co-stimulatory ligand for eg- tumors. these biomimetic aapcs stimulated ot- cd + t cells and visually guided them to tumor magnetically, causing reduced tumor burden [ ] . this offers a great platform for t cell-based immunotherapy. the fusion of biological membranes with nanovaccines has emerged as an interesting concept towards biomimetic nanovaccines. a recent study showed biomimetic cancer-derived magnetosome with enhanced retention in lymph nodes after subcutaneous administration. toll-like receptor (tlr) agonist (cpg) loaded fe o magnetic nanoclusters camouflaged with cancer membrane and was decorated with anti-cd for preferential recognition by cd + dc population for cross-presentation to cd + t cells via mhc-i. enhanced stimulation of tumor-specific cd + t cells due to prolonged retention of magnetically guided nanocluster in lymph nodes showed anti-tumor efficacy with improved survival in different mice models [ ] . similarly, guo et al., developed an erythrocyte membrane coated plga nanoparticle-containing hgp - antigenic peptide and monophosphoryl lipid-a (mpl-a) tlr- agonist for enhanced anti-tumor immunity. intradermal injection in c bl/ had shown reduced tumor burden along with metastasis and prolonged tumor occurring time after re-challenge in metastatic models [ ] . studies exploiting cancer cell membranes as an envelope to impart a wide repertoire of cancer-specific antigens can offer an advantage to develop various personalized vaccines towards multiple tumor types. another interesting strategy for developing personalized biomimetic cancer nanovaccines is the use of cancer cell membrane coated virus for increased adjuvanticity, infectivity and oncolytic activities to generate a strong anti-tumor immune response. a recent study showed the prophylactic and therapeutic potential of a . ± . nm biohybrid artificially enveloped virus extracrad (extra conditionally replicating adenovirus). oncolytic adenovirus serotype containing a cpg island with a base pair deletion was encapsulated into b .ova cancer cell line membrane. coating of the cancer membrane was found to enhance the in vitro cytotoxicity of extracrad as compared to the naked virus indicating the enhanced infectivity of viral vaccine as compared to the naked virus in multiple cell lines. intra-tumoral injection of extracrad in c bl/ mice containing b .ova, b f and ll/ induced models showed in vivo therapeutic vaccine efficacy in facilitating reduced tumor proliferation as compared to the naked virus, individual vaccine component controls and virus membrane mixture because of enhanced cross-presentation and t cell activation. the prophylactic potential was explored by vaccinating mice times with extracrad.cmt .ova and extracrad.b f vaccine before cmt .ova and b f cells re-challenge, respectively. prolonged overall survival and enhanced inhibition of tumor proliferation in homologous membrane enveloped viral vaccinated groups as compared to the naked virus and heterologous membrane enveloped group indicated the strong potential of viral-based vaccine imparting wide repertoire of cancer-specific antigens as preventive vaccines [ ] . another viral protein-coated dendrimer nanovaccine containing e and e peptide epitope of hpv showed enhanced cytotoxic t cell stimulation followed by hpv infected cells eradication. tumor elimination was observed in % of treated mice with no recurrence up to months post initial challenge [ ] . therefore, to address the problems associated with cancer vaccine development, special emphasis has been given on biomimetic personalized nanovaccines. the use of an apcs and nanoparticles coated with the patient's own cancer cell membranes can provide effective antigenic diversity to overcome tumor heterogeneity and tumor-specific immune response. this provides a key strategy to train one's own immune system with appropriate antigens. research using such a biomimetic personalized approach can significantly improve the outcome of current treatment modalities. nanovaccines have shown potential in augmenting the therapeutic and prophylactic efficacy of cancer vaccines. in the past decade, research on vaccines has taken a big leap forward, exploring their vast potential to combat numerous diseases. specifically, strong interactions of nanoparticles with the immune system and the associated benefits have attracted atten- tion with the hope of producing less toxic and effective nanovaccines. physicochemical variations in nanoparticles are one of the prime factors affecting their fate as "nanovaccines" inside the body in terms of their clearance, specific bioaccumulation, adjuvanticity, antigenicity and toxicity. potent antigenicity with prolonged retention and release can cause nanovaccines to elicit both cellmediated and antibody-mediated antibody along with memory effector response, thus alleviating the use of frequent booster doses. although nanovaccines are still in their infancy because of its limited clinical trials but new generation nanovaccines possess enormous potential both as prophylactic or therapeutic. hence, exploring new avenues to increase nanovaccine safety and efficacy should be the driving force for further research in this domain of bionanotechnology. nanomedicine toxicity, scaling-up processes and lack of regulatory guidelines can be considered as the limiting factors in the production of nanovaccines. the nano-scale size can work as a double edge sword as pre-clinical and clinical reports showed their dosedependent acute and chronic toxicities with preferential bioaccu-mulation depending on the route of administration. few classes of nanoparticles possess inherent toxicity after prolonged exposure like inorganic nanoparticles (e.g., metallic nanoparticles). moreover, iscoms are being used in a number of animal vaccines, but toxicity associated with saponin-based adjuvants prohibited their use in humans. therefore, concerns over nanoparticle usage for vaccines are still in existence. scaling up is another major problem that has been minimized up to an extent because of technological advancements, but scale-up in a sterile environment is still a significant challenge [ ] . though, the conventional treatments in this era of modern medicine is saving countless lives. the steady rise in antibiotic resistance and lack of curative cancer therapy because of continuous causative strain variations and cancer heterogeneity respectively drives the focus towards personalized therapeutics. hence, on similar lines, vaccines against bacterial infections and tumors could benefit significantly, but the current vaccine's antigenic breadth and lack of potency are the limiting factors. therefore, researchers have recently shifted towards biomimetic nanotechnology to develop biomimetic nanovaccines as personalized medicines. such nanovaccines can be inherently immuno-stimulatory and multiantigenic. coating of bacterial outer membrane vesicles (omvs) jid: actbio [m g; april , ; : ] onto nanoparticles for anti-virulence vaccination to deliver and neutralize bacterial toxins outplays the pathogen by utilizing its own survival mechanism. this can prevent bacterial colonization effectively and reduce direct selective pressure to develop antibiotic resistance. a large number of such nano-toxoid formulations can be developed by varying the outer membrane coating of these biomimetic nanovaccines [ ] . despite the advancements in the field of cancer nano-based immunotherapy, the major challenge is the toxicological assessments of these nanovaccines. prior evaluation of autoimmune side effects generated from nanovaccine induced immune activation needs to be done. over-activation of antigen presenting cells by nanovaccine can prove to be detrimental as it may cause the death of dendritic cells [ ] . last but not least, increased complexity, cost of manufacturing, and commercialization hurdle related to nanovaccine therapy requires strategies to minimize these limitations for the successful translation of nano-based immunotherapy. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. a brief review of computer-assisted approaches to rational design of peptide vaccines peptide vaccine: progress and challenges principles of vaccine design-lessons from nature enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles advances and opportunities in nanoparticle-and nanomaterial-based vaccines against bacterial infections protein and peptide biomaterials for engineered subunit vaccines and immunotherapeutic applications immunological mechanisms of vaccination micro and nanoparticle-based delivery systems for vaccine immunotherapy: an immunological and materials perspective towards tailored vaccine delivery: needs, challenges and perspectives cd -targeted dendritic cell delivery of plga-nanoparticle vaccines induce potent anti-tumor responses dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting induction of anti-tumor cytotoxic t cell responses through plga-nanoparticle mediated antigen delivery innate and adaptive immune cells in the tumor microenvironment trained innate immunity as underlying mechanism for the long-term, nonspecific effects of vaccines pathogen-associated molecular patterns on biomaterials: a paradigm for engineering new vaccines vaccine delivery: a matter of size, geometry, kinetics and molecular patterns engineering nanoparticles to overcome barriers to immunotherapy poly(lactide-co-glycolide)-rifampicin nanoparticles efficiently clear mycobacterium bovis bcg infection in macrophages and remain membrane-bound in phago-lysosomes a therapeutic microparticle-based tumor lysate vaccine reduces spontaneous metastases in murine breast cancer enantiospecific adjuvant activity of cationic lipid dotap in cancer vaccine dendritic cell-based nanovaccines for cancer immunotherapy adoptive immunotherapy for cancer: harnessing the t cell response skwarczynski , multiantigenic peptide-polymer conjugates as therapeutic vaccines against cervical cancer provenge (sipuleucel-t) in prostate cancer: the first fda-approved therapeutic cancer vaccine dendritic cells and other innate determinants of t helper cell polarisation rosenberg , cancer immunotherapy based on mutation-specific cd + t cells in a patient with epithelial cancer how regulatory t cells work human memory b cells originate from three distinct germinal center-dependent and -independent maturation pathways rituximab specifically depletes short-lived autoreactive plasma cells in a mouse model of inflammatory arthritis the cell biology of antigen processing quantitative review of antibody response to inactivated seasonal influenza vaccines, influenza other respir single-injection vaccines: progress, challenges, and opportunities switched memory b cells maintain specific memory independently of serum antibodies: the hepatitis b example nanoparticle conjugation of cpg enhances adjuvancy for cellular immunity and memory recall at low dose role of sustained antigen release from nanoparticle vaccines in shaping the t cell memory phenotype polymeric particles in vaccine delivery exploiting lymphatic transport and complement activation in nanoparticle vaccines memory antibody response from antigen loaded polymer particles and the effect of antigen release kinetics applications of nanotechnology for immunology vaccine adjuvants: mode of action mechanisms of action of adjuvants toll-like receptor signaling pathways unleashing the potential of nod-and toll-like agonists as vaccine adjuvants activation with cpg-a and cpg-b oligonucleotides reveals two distinct regulatory pathways of type i ifn synthesis in human plasmacytoid dendritic cells vaccine delivery using nanoparticles design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases carbohydrate-functionalized nanovaccines preserve hiv- antigen stability and activate antigen presenting cells polyglutamic acid-trimethyl chitosan-based intranasal peptide nano-vaccine induces potent immune responses against group a streptococcus applications of nanomaterials as vaccine adjuvants nanovaccines and their mode of action a novel lipid nanoparticle adjuvant significantly enhances b cell and t cell responses to sub-unit vaccine antigens curdlan sulfate-o-linked quaternized chitosan nanoparticles: potential adjuvants to improve the immunogenicity of exogenous antigens via intranasal vaccination green synthesis and evaluation of silver nanoparticles as adjuvant in rabies veterinary vaccine lipid-derived nanoparticles for immunostimulatory rna adjuvant delivery artificial bacterial biomimetic nanoparticles synergize pathogen-associated molecular patterns for vaccine efficacy coated protein nanoclusters from influenza h n ha are highly immunogenic and induce robust protective immunity viruslike particles encapsidating respiratory syncytial virus m and m proteins induce robust t cell responses viral-mimicking protein nanoparticle vaccine for eliciting anti-tumor responses intranasal nanovaccine confers homo-and hetero-subtypic influenza protection encapsulins: microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science biomimetic protein nanoparticles facilitate enhanced dendritic cell activation and cross-presentation vault particles: a new generation of delivery nanodevices a protective vaccine against chlamydia genital infection using vault nanoparticles without an added adjuvant a supramolecular vaccine platform based on alpha-helical peptide nanofibers self-assembled peptide nanofibers raising durable antibody responses against a malaria epitope nanoscale peptide self-assemblies boost bcg-primed cellular immunity against mycobacterium tuberculosis titrating t-cell epitopes within self-assembled vaccines optimizes cd + helper t cell and antibody outputs intranasal delivery of adjuvant-free peptide nanofibers elicits resident cd ( + ) t cell responses synergistic sting activation by pc a nanovaccine and ionizing radiation improves cancer immunotherapy design and optimization of peptide nanoparticles protective antibody and cd + t-cell responses to the plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine conformation-specific display of e and f epitopes on self-assembling protein nanoparticles as a potential hiv vaccine protein nanovaccine confers robust immunity against toxoplasma peptide nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine nanoclusters self-assembled from conformation-stabilized influenza m e as broadly cross-protective influenza vaccines cytomembrane nanovaccines show therapeutic effects by mimicking tumor cells and antigen presenting cells a sting-activating nanovaccine for cancer immunotherapy self-assembled peptide amphiphile micelles containing a cytotoxic t-cell epitope promote a protective immune response in vivo peptide amphiphile micelles self-adjuvant group a streptococcal vaccination subcutaneous nanodisc vaccination with neoantigens for combination cancer immunotherapy comparison of the immune response against polio peptides covalently-surface-linked to and internally-entrapped in liposomes, asian pac the role of liposome size on the type of immune response induced in balb/c mice against leishmaniasis: rgp as a model antigen liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a cmi response manipulation of the surface pegylation in combination with reduced vesicle size of cationic liposomal adjuvants modifies their clearance kinetics from the injection site, and the rate and type of t cell response enhancement of immune response and protection in balb/c mice immunized with liposomal recombinant major surface glycoprotein of leishmania (rgp ): the role of bilayer composition a surface charge dependent enhanced th antigen-specific immune response in lymph nodes by transfersome-based nanovaccine-loaded dissolving microneedle-assisted transdermal immunisation antigen-specific vaccines for cancer treatment liposome-entrapped , pteronyssinus vaccination in mild asthma patients: effect of -year double-blind, placebo-controlled trial on inflammation, bronchial hyperresponsiveness and immediate and late bronchial responses to the allergen inspire: a phase iii study of the blp liposome vaccine (l-blp ) in asian patients with unresectable stage iii non-small cell lung cancer some-based adjuvants for subunit vaccines: formulation strategies for subunit antigens and immunostimulators a novel liposomal adjuvant system, caf , promotes long-lived mycobacterium tuberculosis-specific t-cell responses in human a triple co-culture model of the human respiratory tract to study immune-modulatory effects of liposomes and virosomes the novel immunogenic chimeric peptide vaccine to elicit potent cellular and mucosal immune responses against htlv- immune responses induced by nano-self-assembled lipid adjuvants based on a monomycoloyl glycerol analogue after vaccination with the chlamydia trachomatis major outer membrane protein cubosomes: the next generation of smart lipid nanoparticles? cubosomes containing the adjuvants imiquimod and monophosphoryl lipid a stimulate robust cellular and humoral immune responses in vitro protein release and degradation of poly-dl-lactide-poly(ethylene glycol) microspheres with entrapped human serum albumin: quantitative evaluation of the factors involved in protein release phases aerosolized pla and plga nanoparticles enhance humoral, mucosal and cytokine responses to hepatitis b vaccine enhancement of immune responses by co-delivery of a cpg oligodeoxynucleotide and tetanus toxoid in biodegradable nanospheres a single-dose plga encapsulated protective antigen domain nanoformulation protects mice against bacillus anthracis spore challenge role of trehalose dimycolate in recruitment of cells and modulation of production of cytokines and no in tuberculosis immune response by nasal delivery of hepatitis b surface antigen and codelivery of a cpg odn in alginate coated chitosan nanoparticles bioreducible alginate-poly(ethylenimine) nanogels as an antigen-delivery system robustly enhance vaccine-elicited humoral and cellular immune responses in vitro stimulation of cd and cd t cells by dendritic cells loaded with a complex of cholesterol-bearing hydrophobized pullulan and ny-eso- protein: identification of a new hla-dr -binding cd t-cell epitope a novel hepatitis b vaccine containing advax, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing enhanced immune response and protective effects of nano-chitosan-based dna vaccine encoding t cell epitopes of esat- and fl against mycobacterium tuberculosis infection a fully synthetic glycopeptide antitumor vaccine based on multiple antigen presentation on a hyperbranched polymer toxicologic effects of gold nanoparticles in vivo by different administration routes assembling different antennas of the gp high mannose-type glycans on gold nanoparticles provides superior binding to the anti-hiv antibody g than the individual antennas m e-immobilized gold nanoparticles as influenza a vaccine: role of soluble m e and longevity of protection nanovaccines for malaria using plasmodium falciparum antigen pfs attached gold nanoparticles in vivo gold nanoparticle delivery of peptide vaccine induces anti-tumor immune response in prophylactic and therapeutic tumor models gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo multicopy multivalent' glycopolymer-stabilized gold nanoparticles as potential synthetic cancer vaccines synthesis of a novel kind of carbon nanoparticle with large mesopores and macropores and its application as an oral vaccine adjuvant single-walled carbon nanotubes deliver peptide antigen into dendritic cells and enhance igg responses to tumor-associated antigens immunisation of mice by hollow mesoporous silica nanoparticles as carriers of porcine circovirus type orf protein silica vesicle nanovaccine formulations stimulate long-term immune responses to the bovine viral diarrhoea virus e protein lipopeptide-coated iron oxide nanoparticles as potential glycoconjugate-based synthetic anticancer vaccines a visible codelivery nanovaccine of antigen and adjuvant with self-carrier for cancer immunotherapy effect of preexisting neutralizing antibodies on the anti-tumor immune response induced by chimeric human papillomavirus virus-like particle vaccines room temperature stable pspa-based nanovaccine induces protective immunity reversal of autoimmunity by boosting memory-like autoregulatory t cells peptide-mhc-based nanovaccines for the treatment of autoimmunity: a "one size fits all" approach? synthetic vaccine nanoparticles target to lymph node triggering enhanced innate and adaptive antitumor immunity the contribution of vaccination to global health: past, present and future nanoparticle vaccines against infectious diseases development of a gold nanoparticle vaccine against enterohemorrhagic escherichia coli o :h a poly(lactic-co-glycolic) acid nanovaccine based on chimeric peptides from different leishmania infantum proteins induces dendritic cells maturation and promotes peptide-specific ifngamma-producing cd ( + ) t cells essential for the protection against experimental visceral leishmaniasis effectiveness of a novel immunogenic nanoparticle platform for toxoplasma peptide vaccine in hla transgenic mice adjuvanted multi-epitope vaccines protect hla-a * : transgenic mice against toxoplasma gondii multidrug and extensively drug-resistant tb (m/xdr-tb) rd antigen based nanovaccine imparts long term protection by inducing memory response against experimental murine tuberculosis vaccine delivery system for tuberculosis based on nano-sized hepatitis b virus core protein particles chitosan nanoparticles as an efficient delivery vehicle for mycobacterium tuberculosis lipids to induce potent cytokines and antibody response through activation of gammadelta t cells in mice immunisation with mycobacterium tuberculosis antigens encapsulated in phosphatidylserine liposomes improves protection afforded by bcg pre-erythrocytic malaria vaccines: identifying the targets progress and prospects for blood-stage malaria vaccines iron oxide nanoparticles as a clinically acceptable delivery platform for a recombinant blood-stage human malaria vaccine a malaria vaccine adjuvant based on recombinant antigen binding to liposomes plebanski , nanoparticles modify dendritic cell homeostasis and induce non-specific effects on immunity to malaria nanomimics of host cell membranes block invasion and expose invasive malaria parasites influenza vaccines: challenges and solutions influenza virus antigenic variation, host antibody production and new approach to control epidemics changing selective pressure during antigenic changes in human influenza h cellular immunity and memory to respiratory virus infections nanovaccine confers dual protection against influenza a virus and porcine circovirus type nanodiamond enhances immune responses in mice against recombinant ha/h n protein hemagglutinin-based polyanhydride nanovaccines against h n influenza elicit protective virus neutralizing titers and cell-mediated immunity single immunisation with a suboptimal antigen dose encapsulated into polyanhydride microparticles promotes high titer and avid antibody responses amphiphilic polyanhydride nanoparticles stabilize bacillus anthracis protective antigen double-layered protein nanoparticles induce broad protection against divergent influenza a viruses vaccine nanoparticles for protection against hiv infection glycosylation of the hiv- env v v loop to form a native-like structure may not be essential with a nanoparticle vaccine co-utilization of a tlr agonist and nano-formulation of hiv- vaccine candidate leads to increased vaccine immunogenicity and decreased immunogenic dose: a preliminary study polysaccharide nanoparticles can efficiently modulate the immune response against an hiv peptide antigen amantadine surface-modified silver nanorods improves immunotherapy of hiv vaccine against hiv-infected cells loading dendritic cells with gold nanoparticles (gnps) bearing hiv-peptides and mannosides enhance hiv-specific t cell responses efaverinz and nano-gold-loaded mannosylated niosomes: a host cell-targeted topical hiv- prophylaxis via thermogel system development and evaluation of a thermosensitive vaginal gel containing raltegravir + efavirenz loaded nanoparticles for hiv prophylaxis thermosensitive gel containing cellulose acetate phthalate-efavirenz combination nanoparticles for prevention of hiv- infection drug synergy of tenofovir and nanoparticle-based antiretrovirals for hiv prophylaxis overcoming immunogenicity issues of hiv p antigen by the use of innovative nanostructured lipid carriers as delivery systems: evidences in mice and non-human primates a virus-like particle vaccine candidate for influenza a virus based on multiple conserved antigens presented on hepatitis b tandem core particles a novel vaccine using nanoparticle platform to present immunogenic m e against avian influenza infection vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge the novel tlr- agonist qbg shows clinical efficacy in persistent allergic asthma gold nanoparticle-m e conjugate coformulated with cpg induces protective immunity against influenza a virus simultaneous surface display and cargo loading of encapsulin nanocompartments and their use for rational vaccine design development of a self-assembling protein nanoparticle vaccine targeting plasmodium falciparum circumsporozoite protein delivered in three army liposome formulation adjuvants a randomized trial assessing the safety and immunogenicity of as and as adjuvanted rts,s malaria vaccine candidates in children in gabon randomized, double-blind, phase a trial of falciparum malaria vaccines rts,s/as b and rts,s/as a in malaria-naive adults: safety, efficacy, and immunologic associates of protection evaluation of rts,s/as a and rts,s/as b in adults in a high malaria transmission area mechanisms of protective immune responses induced by the plasmodium falciparum circumsporozoite protein-based, self-assembling protein nanoparticle vaccine surface-engineered gold nanorods: promising dna vaccine adjuvant for hiv- treatment a randomized, controlled dose-finding phase ii study of the m /as candidate tuberculosis vaccine in healthy ppd-positive adults teyton , t cells control the generation of nanomolar-affinity anti-glycan antibodies immunogenicity and efficacy of flagellin-envelope fusion dengue vaccines in mice and monkeys encapsulation of an ep -conjugated ctl peptide vaccine in nanoscale biodegradable particles increases the efficacy of respiratory immunisation and affects the magnitude and memory subsets of vaccine-generated mucosal and systemic cd ( + ) t cells in a diameter-dependent manner chitosan-based nanoparticles for improving immunisation against hepatitis b infection bivalent mucosal peptide vaccines administered using the lcp carrier system stimulate protective immune responses against streptococcus pyogenes infection lipid core peptide/poly(lactic-co-glycolic acid) as a highly potent intranasal vaccine delivery system against group a streptococcus phase i study of a neisseria meningitidis liposomal vaccine containing purified outer membrane proteins and detoxified lipooligosaccharide preparation and efficacy of a live newcastle disease virus vaccine encapsulated in chitosan nanoparticles enhanced mucosal immune responses against tetanus toxoid using novel delivery system comprised of chitosan-functionalized gold nanoparticles and botanical adjuvant: characterization, immunogenicity, and stability assessment assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide immunisation with tumor neoantigens displayed on t phage nanoparticles elicits plasma antibody and vaccine-draining lymph node b cell responses engineering magnetosomes for high-performance cancer vaccination induction of immune response to the kda ompa burkholderia cenocepacia polypeptide and protection against pulmonary infection in mice after nasal vaccination with an omp nanoemulsion-based vaccine treg inducing adjuvants for therapeutic vaccination against chronic inflammatory diseases, front. immunol cancer immunotherapy: a treatment for the masses dendritic cell targeting peptide-based nanovaccines for enhanced cancer immunotherapy designer vaccine nanodiscs for personalized cancer immunotherapy biomimetic magnetosomes as versatile artificial antigen-presenting cells to potentiate t-cell-based anticancer therapy erythrocyte membrane-enveloped polymeric nanoparticles as nanovaccine for induction of antitumor immunity against melanoma artificially cloaked viral nanovaccine for cancer immunotherapy therapeutic vaccination using cationic liposome-adjuvanted hiv type peptides representing hla-supertype-restricted subdominant t cell epitopes: safety, immunogenicity, and feasibility in guinea-bissau ) fusion peptide and gm-csf dna elicits potent and prolonged cd ( + ) t cell-dependent anti-tumor immunity in mice de berardinis , vectorized delivery of alpha-galactosylceramide and tumor antigen on filamentous bacteriophage fd induces protective immunity by enhancing tumor-specific t cell response targeting mutated plus germline epitopes confers pre-clinical efficacy of an instantly formulated cancer nano-vaccine heat shock protein-peptide and hsp-based immunotherapies for the treatment of cancer pre-clinical development of listeria-based nanovaccines as immunotherapies for solid tumours: insights from melanoma antitumor humoral and t cell responses by mucin- conjugates of bacteriophage qbeta in wild-type mice intradermal delivery of vaccine nanoparticles using hollow microneedle array generates enhanced and balanced immune response effect of tlr ligands co-encapsulated with multiepitopic antigen in nanoliposomes targeted to human dcs via fc receptor for cancer vaccines tumor growth inhibition by msteap peptide nanovaccine inducing augmented cd ( + ) t cell immune responses, drug deliv heat-shock protein peptide complex- vaccination for recurrent glioblastoma: a phase ii, single-arm trial phase clinical trial of her -specific immunotherapy with concomitant her kinase inhibition enhanced intratumoral delivery of sn as a tocopherol oxyacetate prodrug using nanoparticles in a neuroblastoma xenograft model biomimetic nanotechnology toward personalized vaccines authors would like to thank iit bombay, ministry of human resource and development, government of india, for their research fellowships. key: cord- -yvgvyif authors: french, jeff; deshpande, sameer; evans, william; obregon, rafael title: key guidelines in developing a pre-emptive covid- vaccination uptake promotion strategy date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: yvgvyif this paper makes the case for immediate planning for a covid- vaccination uptake strategy in advance of vaccine availability for two reasons: first, the need to build a consensus about the order in which groups of the population will get access to the vaccine; second, to reduce any fear and concerns that exist in relation to vaccination and to create demand for vaccines. a key part of this strategy is to counter the anti-vaccination movement that is already promoting hesitancy and resistance. since the beginning of the covid- pandemic there has been a tsunami of misinformation and conspiracy theories that have the potential to reduce vaccine uptake. to make matters worse, sections of populations in many countries display low trust in governments and official information about the pandemic and how the officials are tackling it. this paper aims to set out in short form critical guidelines that governments and regional bodies should take to enhance the impact of a covid- vaccination strategy. we base our recommendations on a review of existing best practice guidance. this paper aims to assist those responsible for promoting covid- vaccine uptake to digest the mass of guidance that exists and formulate an effective locally relevant strategy. a summary of key guidelines is presented based on best practice guidance. as we work to develop a range of safe and effective covid- vaccinations, the anti-vaccination movement has already fired the first shots in what will be a global public health battle. research shows that general vaccine hesitancy (i.e., 'the delay in acceptance or refusal of vaccines despite the availability of vaccination services') is rising for several diseases, resulting in serious disease outbreaks. for example, european countries experienced more than cases of measles in [ ] . vaccine hesitancy has also steadily increased in more than % of countries since [ ] . given the potential to undermine vaccination coverage, all states must take steps to understand the extent and nature of hesitancy and to start promoting covid- vaccine uptake. as the who recommends, 'each country should develop a strategy to increase acceptance and demand for vaccination' [ ] . each country must consider the appropriate time to start promoting the uptake of covid- vaccines based on its specific trajectory of covid- infection and its ability to provide access to vaccination. as covid- vaccination uptake develops, governments should continue to promote other protective behaviors such as handwashing and physical distancing. this paper aims to set guidelines that governments and regional bodies across the world should take to enhance the impact of their pro-vaccination strategy. we base our summary on recommended best practice with the aim of assisting professionals to digest the mass of guidance that exists in the hope that the summary contained will inform the guidelines needed to maximize uptake of covid- vaccines. it is imperative that planning for a covid- vaccination uptake strategy begins in advance of vaccine availability for two reasons. first, countries will need to determine population sub-groups and build a consensus about the order in which these will get access to the vaccine. second, we should reduce fear and concern and create demand for vaccines. a key part of this strategy is to counter the anti-vaccination movement that is already promoting hesitancy and resistance. since the beginning of the covid- pandemic, we have witnessed a tsunami of misinformation and conspiracy theories that have the potential to reduce vaccine uptake. to make matters worse, sections of populations in many countries display low trust in governments, official information about the pandemic, and the official approach in tackling the epidemic. the who advocates a pre-emptive pro-vaccination strategy that psychologically inoculates the population and maximizes uptake of vaccines as they become available [ ] . this paper sets out the core elements of such a strategy. the paper explores key issues that relevant organizations must address and summarizes best practices that should be addressed when developing behavioral influence strategies to promote the uptake of covid- vaccines effectively, efficiently, and ethically as they become available. this paper does not set out a full review or commentary on the thousands of scientific papers and national and international guidance documents that already exist with respect to promoting vaccine uptake and reducing vaccine hesitancy. the volume and dispersed nature of this literature is, in some ways, an impediment to action as few people will have a full grasp of the multiple fields of research that inform it. the paper also does not attempt to set out a full planning model or a 'how-to' guide, as numerous well-tested examples already exist [ ] [ ] [ ] [ ] . the paper does not provide a comprehensive set of references; instead, it cites select evidence summaries and guidance documents to aid further reading. finally, we have not included a separate evaluation strategy, as each of the key guidelines will need an integrated monitoring and evaluation strategy to enable continuous improvement. context matters. each government and public health service face its own set of unique challenges. different countries also have differing resources, capacities, capabilities, assets, and constraints. regardless of such settings and challenges, governments and relevant bodies can action a number of key processes identified in the literature that will enhance vaccine uptake. we set out these key action areas in the guidelines below. see table . it is highly likely that in the coming months the who and other public health institutions will issue guidance about how to optimize the uptake of covid- vaccines. we present the guidelines set out in this paper as an ideal model based on the lessons learned from successful intervention programs to inform such guidance. organizations, however, should approach each action area in a locally relevant way. it is also clearly a big ask to address all the recommended guidelines identified, but the more of these actions that can be applied, the more likely it is that a successful uptake strategy will be delivered. it is important that a systematic approach to planning is adopted. there are numerous planning models from the fields of health promotion and social marketing that authorities can use to define objectives, design processes, and conduct monitoring and evaluation of efforts to promote vaccine uptake [ ] . the most crucial action is to set out a transparent (open access) and a logical plan that covers all the essential components contained in the guidelines included in this paper. however, a coordinated and a systematic approach will require strong leadership. behaviour change plans should also be informed by lessons from the fields of management, logistics, and emergency and disaster planning such as the highlight, audience, behaviour, intervention, test (habit) behaviour disaster change planning framework [ , ] . authorities should also consider lessons and tips set out in several detailed planning models and guides developed specifically for vaccine promotion efforts such as: who. guide to tailoring immunization programs (tip) for infant and child vaccination [ ] . the tip principles apply to communicable, non-communicable, and emergency planning where behavioral decisions influence outcomes [ ] https://www.who.int/immunization/programmes_ systems/global_tip_overview_july .pdf?ua= european centre for disease control (ecdc). technical guide to social marketing https://www.ecdc.europa.eu/en/publications-data/social-marketing-guide-public-healthprogramme-managers-and-practitioners who. improving vaccination demand and addressing hesitancy. https://www.who.int/ immunization/programmes_systems/vaccine_hesitancy/en/ ecom: effective communication in outbreak management (ecom) [ ] . the e.u. funded ecom project brings together multiple disciplines to develop an evidence-based behavioral and communication package for health professionals and agencies throughout europe in case of significant outbreaks of infectious diseases. http://ecomeu.info/ tell me. review of population behavior and communication during pandemics: https://www. tellmeproject.eu/ human center design for health. a comprehensive set of tools developed by unicef to apply the human-centered design approach to challenges facing health services, with a particular emphasis on demand for immunization and health services. https://www.hcd health.org/resources social science research for vaccine deployment in epidemic outbreaks. a practical guide to using social science research and insights to better understand social, behavioral, cultural, community and political dynamics as part of efforts to introduce vaccines in epidemic outbreak settings. https://opendocs.ids.ac.uk/opendocs/bitstream/handle/ . . / / pracapproach% .pdf?sequence= &isallowed=y further generic planning guidance can be found at: building better health: a handbook for behavioral change. "the handbook blends proven disease prevention practices and behavioral science principles into a one-of-a-kind, hands-on manual." [ ] (p. xiii). cdcynergy planning tool for social marketing. centers for disease control and prevention planning tool for social marketing, atlanta, ga. also available is cdcynergy "lite", intended for those who have previous social marketing experience and those who are familiar with the full cdcynergy edition. https://www.thecommunityguide.org/resources/cdcynergy applying behavioral insights-simple ways to improve health outcomes. a tool for the application of behavioral insights to improving health outcomes from the world innovation summit for health. https://www.imperial.ac.uk/media/imperial-college/institute-of-global-health-innovation/ behavioral_insights_report-( ).pdf if governments develop vaccine uptake programs based only on expert opinion, they are likely to be suboptimal [ , ] . what is required is an approach that seeks to gather as much understanding as possible about what people say will prevent, encourage, and assist them in taking up vaccines. authorities must understand what people value and what they fear when developing an effective promotional program. a targeted approach that uses a different intervention mix for different subsets of the population will be more effective. people do not respond uniformly to preventive interventions. for example, being older, female, and more educated is associated with a higher likelihood of adopting protective behaviors [ , ] . 'insight' data about citizens' attitudes, beliefs, wants, and behaviors should inform interventions. insights are 'deep truths' and understanding about why people act as they do. such insights can be developed from formative qualitative and quantitative survey research, observational data, demographic data, service use data, problem or issue tracking data, and epidemiological data. the development of deep insights into people's lives, with a focus on what will or will not motivate or enable people to take up vaccination, is a crucial investment that must be made to inform all aspects of vaccination promotion uptake strategy. a key component of behavioral planning is the setting of measurable behavioral objectives that are relevant and timely in relation to maximizing vaccine uptake. setting measurable goals related to uptake, attitudes, intention, understanding and beliefs will help focus behavioral planning and enable meaningful ongoing tracking and evaluation of impact [ ] . segmentation is key to success. segmentation is the identification of groups who share similar beliefs, attitudes and behavioral patterns. segmentation goes beyond demographic, epidemiological, and service uptake-based targeting. segmentation includes data about people's attitudes, values, understanding and observed behaviors. population segmentation models enable public heath planners to tailor interventions to specific audiences [ ] . fournet et al. have identified four unprotected and under-protected population groups that could form the basis for the development of a locally developed strategy [ ] : • 'the hesitant'-those who have concerns about perceived safety issues and are unsure about needs, procedures and timings for immunizing. • 'the unconcerned'-those who consider immunization a low priority and see no real perceived risk of vaccine-preventable diseases. • 'the poorly reached'-those who have limited or difficult access to services, related to social exclusion, poverty and, in the case of more integrated and affluent populations, factors related to convenience. • 'the active resisters'-those for whom personal, cultural, or religious beliefs discourage them from vaccinating. other segments that need dedicated foci are health and social care workers. studies have revealed that certain healthcare workers hesitate to vaccinate themselves or their family members [ , ] . the ecdc provides guides and toolkits for healthcare workers, immunization program managers, and public health experts, to support their efforts in addressing vaccine hesitancy [ ] . frontline workers can be a significant source of trusted advice and information but are often not optimally used in such roles. these workers lack training and support in advocacy roles and may also lack a full awareness of risks and safety issues associated with the disease and vaccination. governments and responsible agencies should facilitate support structures that increase worker awareness and willingness to act as public health advocates. to effectively promote and maintain demand for a covid- vaccine, governments and regional bodies must develop an insight-informed pro-vaccination strategy that includes action to reduce the impact of four kinds of competition: • active competition from the ani-vaccination movement • passive competition in the form of inaccurate media coverage • competition from negative social norms • competition in the form of structural and economic factors effective campaigning against vaccine misinformation should focus on the dangers of the disease as well as on the benefits of the vaccines, which can include highlighting protection. such approaches draw on the powerful motivator of fear of loss along with the possibility of gain of positive health [ ] . intervention designers should involve the target populations in building campaigns, and use data-supported insights about what will and what will not motivate them to take up vaccine programs and about how to frame the promotion of vaccination. a competition strategy that seeks to reduce the impact of those promoting hesitancy that emphasizes fact-checking and myth-busting may do more harm than good. such approaches often repeat misinformation as part of rebuttal strategies. engaging directly with conspiracies often spreads rather than closes down such views. people often exhibit what lord calls confirmation bias; they look and accept information that fits with their existing views and reject information that runs counter to their existing views [ ] . so, when repeating misinformation in order to debunk it, people may just hear the misinformation. a more effective approach is a combination of positive messaging that emphasizes the protective (individual, family, and community) benefits of the vaccine and the loss associated with not being vaccinated (death, poor health, loss of freedom and social solidarity, inability to travel, etc.) [ , ] . anti-vaccination advocates should not be left free to spread misinformation. public health authorities and their coalition partners, including both the traditional and digital media sectors, should proactively work together to reduce and remove at speed false content and misleading information. traditional media providers should be supported and briefed so that they are aware of anti-vaccination propaganda identified by public health authorities and do not repeat it. traditional media and social media sectors should also provide authorities with the information they have detected that anti-vaccination advocates are propagating so that information can be rebutted. public health agencies should seek protocols with media providers about the issue of how journalistic balance will be addressed. agreements should be put in place about how the media will identify and flag false and misleading anti-vaccination information and advocates. in this regard authorities and media channel providers should be alert to 'astroturfing' (anti-vaccination advocates disguising their views as coming from grass roots movements) and act swiftly to expose such tactics. finally, agreements should be developed about how and when misleading information and advocates of such information should be removed and flagged as being problematic on social media. distrust in elites and experts and political populism can also fuel antivaccination sentiment [ ] . social norms and cultural influences can have a significant effect on people's willingness at the population level to take up vaccine programs [ ] . as an initial step, authorities need to understand what informs social norms and beliefs. persuasive efforts should appeal to the values and beliefs that people already hold, such as a desire to protect family members, rather than a focus on factual or probabilistic messaging. validating people's existing motivations and using them to encourage behaviour is more effective than trying to shift people's world view. if, however, people hold incorrect opinions about the social norms prevailing in their community, for example, the erroneous belief that most people oppose vaccination, it can be helpful to inform them that a high percentage of people do in fact, support vaccination. subjective social norms, i.e., those that are informed by what we think key others in our social circles believe, are also crucial in promoting vaccine uptake [ , , ] . opinion leaders in the anti-vaccination community may hold negative attitudes and beliefs, so intervention organizers should also develop interventions with such key informants to address these concerns and seek to turn such informants into advocates for vaccination. previous reviews of vaccine demand campaigns using a systematic process, such as in the area of human papilloma virus (hpv) vaccine, have found that myths and misinformation, often prevalent in communities, can also pose significant barriers to vaccine adoption. evans et al. studied several hpv and cervical cancer awareness studies in low-and middle-income countries (lmics) [ ] . these studies confirm many widely reported barriers to hpv vaccination; these include myths (e.g., the vaccine causes infertility), beliefs that it will increase promiscuity, negative social norms within social groups, and concerns about safety and efficacy. solutions to these barriers include: • increasing knowledge about the risks prevented by the vaccine. promoting understanding that the community of interest is at risk; improving beliefs in vaccine safety, effectiveness, and community benefit. • dispelling unfounded myths. building a social norm that vaccination uptake is widespread and accepted in society (descriptive and injunctive normative beliefs). vaccine uptake strategy must address difficulties in accessing vaccines due to cost, lack of transportation to vaccination sites or clinics, and/ or a lack of a cold-chain network. authorities need to work with partners across government, ngos, communities, and the for-profit sector to reduce these barriers. poor access can reduce confidence in and demand for the vaccine. vaccine uptake promotion should thus facilitate availability and convenience. it is vital that countries review their public health finances early on to allocate funds to vaccinate their populations, as many countries already carry large debts. to inoculate the entire global community will require significant resources. countries with lower incomes will need to develop plans to access support from the international aid programs provided by governments, u.n. bodies and foundations, and other sources to secure adequate supplies of vaccines. promoters of the covid- vaccine should also consider that their efforts do not negatively impact on the availability and the uptake of other vaccine programs, predominantly for children. public health organizations rarely have sufficient resource capacity to develop, deliver, and maintain population-level change-focused programs. building and sustaining coalitions of organizations and individuals who can assist through the provision of resources, expertise, credibility and access is a crucial early action that needs to be addressed. critical asset identification and management falls into three main categories: government capacity coordination, private sector and ngo sector mobilization, and the mobilization of civil society. building alliances within government and across departments is a crucial aspect of asset identification and mobilization [ ] . there is a need to develop plans and structures to coordinate action between government agencies and departments and organizations such as hospitals, clinics and schools [ ] . an alliance or coalition team should also coordinate mechanisms and resources and set out chains of command and responsibilities. the ngo and private sectors can play a pivotal role in promoting the uptake of vaccines. partnerships with the pharmaceutical industry to develop, manufacture, promote, and distribute vaccines are underway across the world. many other for-profit organizations can also be harnessed to provide logistical and promotional support. the ngo sector is also well placed in terms of its reach, high level of understanding about local communities, and high levels of trust to act as a critical advocate and network for vaccine uptake. the third leg of the asset and capability resource base is civil society, represented by community groups and associations such as religious groups, community associations, recreational groups and community charities and volunteers. these groups and communities can play a crucial role in encouraging vaccine uptake and assisting with distribution and access. however, the part that civic society can play in promoting and helping with vaccine uptake is highly country-specific; therefore, local plans will need to reflect the role that such groups can play [ ] [ ] [ ] . developing and maintaining a vaccine promotion coalition of government, the private sector, the ngo sector, and civic society requires resources and staff with expertise in creating and managing stakeholder relationships. authorities need to identify the resources needed to undertake these essential tasks, set objectives, monitor progress, and provide feedback. well planned, evidence-based, and theory-informed health communication and health marketing can significantly impact behavior and vaccine uptake [ , , ] . well-designed campaigns, together with the application of behavioral science techniques, need to be supported by ease of access to vaccines, distribution networks and logistics, and taking notice of broader socio-economic and cultural factors [ , ] . those responsible for creating demand for the vaccine need to work with vaccine suppliers, administrators, and those delivering vaccination to bring together a full mix of demand-side and supply-side interventions. the intervention mix needs to include coordinated action in the fields of prioritization and access policy, supply systems, and promotions strategy. prioritization is especially critical, given insufficient availability, especially after the initial months of vaccine launch. more important than building general demand are building awareness and support for covid- vaccination prioritization plans and fostering high acceptance among people in priority groups. the key to promoting demand is a deep understanding of what will enable and encourage uptake. campaign managers should conduct formative research including secondary research based on published literature and case studies and primary research with interviews and surveys in each population to gain audience-specific insights. governments will need to deliver and communicate what mix of incentives and penalty interventions will be used to promote demand [ ] . demand strategy will also need to be supported by the development of a compelling, insight informed and segmented promotion that speaks to people's needs, values, and wants. health communicators must develop narratives that emphasize the positive personal, family, and community benefits associated with vaccine uptake. the demand strategy will need to include guidelines that reduce the influence of anti-vaccination advocates (see sections below for critical steps to consider when developing a competitor strategy). the demand strategy must also utilize positive narratives in both traditional and social media and apply behavioral influence tactics informed by behavioral sciences [ , ] . the who recommends that every country should include ongoing community engagement and trust-building programs. programs should be focused on confidence-building and active hesitancy prevention, together with regular national assessments of population concern and trust [ , [ ] [ ] [ ] . trust is built and maintained through transparency, constancy, active listening programs, and encouraging dialogue. agencies and governments need to share knowledge about certainty and uncertainty. governments and public health agencies also need to pre-empt and address any safety issues that are expressed or felt by the public or media [ ] . governments should also be transparent about vaccine licensing, manufacture, and prioritization planning. consistency of both messaging and policy directives is also crucial. the absence of these conditions will trigger confusion and reduce trust [ ] . anti-vaccination attitudes do not always relate to factors like level of education [ ] . instead, they are often related to anger and suspicion towards elites and experts and increasing support for anti-establishment political concerns. governments should listen actively and build dialogue, encouraging continuous feedback from citizens, key commentators, and influencers. regular proactive public media and influencer briefings should also form a central plank of trust-building strategy. the application of citizen-focused and human-centered design principles can also enhance program development and implementation [ ] . relevant agencies should realize the need for a coordinated mix of interventions to promote vaccine access, led by a strong leadership team [ ] . promoting uptake through the media and community advocates is a critical element of any pro-vaccination strategy, but it is not a panacea for convincing everyone reluctant to vaccinate. research shows that behavioral change is a complex process that entails more than having adequate knowledge about an issue. uptake and hesitancy are also related to cultural factors, attitudes, motivations and experiences, social norms, and structural barriers. understanding the multiple factors involved in people's decisions is, therefore, key to success. governments and public health authorities can enhance the effectiveness of their efforts by combining multiple strategies [ ] . for example, they could integrate financial and non-financial incentives, call and reminder interventions, along with penalties for non-compliance by imposing restrictions on travel, education, or employment [ ] . vaccine access information, requirements and support will need to reflect each country's vaccination implementation strategy. will it be mandatory? will there be penalties for non-compliance? communicators should deliver implementation and access strategies through a segmented approach that provides specific and relatable information to identified subgroups of the population about how and when they can have access to vaccination. call mechanisms will need to be established and monitored as part of this element of the strategy. with regard to vaccine selection, assuming that the medical fraternity has developed several safe and effective vaccines by , governments and public health authorities will need to explain to the population why they selected a particular vaccine in terms of its efficacy, safety, cost, etc. authorities will also need to explain their reasoning for the prioritization model for the vaccination that they adopt. for example, if a risk-based approach is adopted in which older people and care workers are prioritized over younger people and non-essential workers, this needs to be explained. governments and regional bodies need to explain and justify these decisions in terms of health protection, social and economic imperatives, safety and cost imperatives. schedules and timetables for total population vaccination should also be developed and shared before vaccination roll out begins so that everyone understands when they will get access. ideally authorities should share their plans for vaccine roll out prior to availability so that there is time for ethical and procedural issues to be publicly debated and a consensus reached. a coordinated national approach to communication will be successful among many groups, but not all [ ] . success depends on the nature and degree of immunization hesitancy and the degree of segmentation. tailored messages focusing on known motivators for specific groups are more likely to produce a desired behavioral response than a 'one size fits all' approach [ ] [ ] [ ] . to produce tailored messages, we recommend quantitative and qualitative formative research and ascertaining the efficacy of strategies with pre-test research before launch. as stated previously, there is a need to set out a compelling narrative that avoids 'backfire effects' [ ] , validates people's concerns, and addresses both fear of loss and the positive gain that will accrue from vaccine uptake. as tversky and kahneman have demonstrated, when confronted with choices we are averse to any that might result in perceived loss [ ] . we also do not like being confronted with complex choices. it follows that, if governments want to influence people to take up vaccination, they are more likely to be successful if the strategy emphasizes the positive gains accrued from vaccination, the loss that will occur if vaccination is refused, and that access to vaccines is easy. we know that the perceived attractiveness of options varies when communicators frame the same choice differently. therefore, the language used, the imagery, the messengers, and audio-visual effects are all important considerations that communicators should pilot test. as stated previously, authorities should tailor their promotional strategies by subgroups of the population, as each segment will respond differently to varied messaging and narratives. familiarity and trust in the messenger, as well as the message, is also a crucial success feature in tackling vaccine hesitancy [ , ] . authorities should determine which campaign face and voice should be used based on formative research with the target audience. messages that come from a variety of trusted sources are likely to make a vaccine promotion programs more successful. spokespeople recruited from trusted groups, including healthcare professionals and relatable members of the public, can enhance the effectiveness of campaigns. high-profile personalities can also be effective in communicating messages, as they lend their prestige and trust to the health communication activity. the use of religious leaders (like the cooperation offered by muslim religious leaders in india to communicate the importance of polio vaccination), community influencers and third-party advocates, such as teachers, can also improve support for vaccination uptake [ ] . as part of long-term public health strategy, governments and public health agencies should enhance media and digital literacy in schools and community settings, specifically related to health and vaccine topics [ ] . newly acquired literacy will equip the public to identify reliable sources of information and encourage reporting of misinformation to social media providers and regulating authorities. the news and general media can contribute significantly to address fears and risk perceptions, which can hurt vaccine uptake [ ] . it is, therefore, necessary to develop a proactive strategy for working with traditional media. any media management and engagement strategy that is developed will need to include proactive, rolling media briefings, story generation, editorial feeds, facilitating access to medical and other clinical and public health experts, advisers, and data. the media management and engagement strategy will also need to include / media monitoring and rebuttal/correction systems. communicators should mediate ongoing relationships between media contacts and experts who can provide accurate opinions on all aspects of vaccine promotion and safety. authorities should additionally monitor the strength of this relationship and address rapidly any conflicts that may arise. the responsibility of government agencies and others advocating for covid- vaccination is to communicate better, more visible, and more highly credible messages than the sceptics. successful media engagement is more likely when the public health system has developed a strong collaborative and open relationship with key editors, sub-editors and journalists. public health authorities and governments should continuously nurture trust and positive working relationships with media organizations so that the audience views the former as accessible and trustworthy. this will, however, require government authorities to be transparent, honest, and open regarding vaccine safety and effectiveness data that could be, or is, worrisome. anti-vaccination advocates abound on facebook, twitter, whatsapp, and youtube. social media platforms are already buzzing with misinformation about covid- vaccine safety, development, and planned rollout, months before vaccines are ready to be used at population level. it is encouraging to see such media platform owners starting to act against the anti-vaccination movement. for example, instagram avoids health misinformation in its explore page; youtube has demonetized anti-vaccination videos and gofundme has recently taken down anti-vaccination fundraising appeals. governments and their public health agencies need to develop a dialogue and joint strategy with social media platform providers to review and action against anti-vaccination misinformation and vaccine hesitancy promotion. governments and regional bodies should convince or regulate platform providers to remove misinformation. public health authorities need to build a proactive covid- vaccine trust capacity for active engagement in the social media space as part of their overall promotional strategy [ ] . social media platforms are now the primary information source and communication channel for a large and growing number of citizens. public health agencies need to invest in building teams of specialist staff trained and capable of understanding how to build and maintain social media presence. the key responsibilities of public health staff focused on social media are the development of and support for continuous positive story streams, nurturing multiple supportive voices, and amplification of pro-vaccination grassroots advocates. these dedicated staff need to support pro-vaccine influencers, advocates and social networks. public health staff can also assist in the identification of and responses to false social media posts. the team should address such negative posts instantly to prevent the decline of trust in public health authorities. we know, for example, that parents who are resistant to getting their children vaccinated are more likely to have based their decision on information obtained on the internet [ ] . the strategic and tactical guidance set out above provides a framework for promoting the uptake of covid- vaccines as they become available. this paper also acknowledges the importance of evidence and theory-driven behaviour change tools in addressing vaccine hesitancy. this is consistent with who's recent establishment of the technical advisory group on behavioral insights and sciences for health [ ] . key to the success of promoting vaccine uptake will be a significant and sustained strategic program, including strengthening of local capacities, to build and maintain confidence and trust [ ] . a crucial factor in the delivery of such a trust-building and demand building approach is the need for investment in communication, behavioral influence, and community engagement capacity and capability. communication and behavioral influence are often underfunded or under-resourced in public health organizations and within government ministries. building communication and behavioral influence capacity and expertise should be a priority. it is now often said that everything will be different in the post covid world; hopefully one difference will be a commitment to investment in developing and delivering the key action elements set out in this paper. this investment will need to be sustained over time in line with best practice requirements regarding risk communication and community engagement so that we are better prepared for inevitable future events [ ] . the authors acknowledge that countries, high-, low-and middle-income, have been using many of the guidelines described in this manuscript to foster high vaccination coverage. the challenges are not that they are unaware of the actions described here but rather: ( ) they have very limited resources (e.g., money, people) to implement all the actions at the scale the authors are recommending; and ( ) they are responsible for promoting and achieving compliance with vaccination schedules, not just a single vaccine. governments and relevant bodies should bear these limitations in mind as they consider our guidelines. world health organization. the guide to tailoring immunization programs; world health organisation vaccine hesitancy around the globe: analysis of three years of who/unicef joint reporting form data- - executive agency for health and consumers. project malmanagement in public health in europe a literature review on effective risk communication for the prevention and control of communicable diseases in europe behavioural change guide for the victoria emergency services. habit framework; ipsos diagnosing the determinants of vaccine hesitancy in specific subgroups: the guide to tailoring immunization programmes (tip). vaccine effective communication in outbreak management (ecom) building better health: a handbook for behavioral change the uk response to coronavirus is the greatest science policy failure for a generation influenza pandemic: perception of risk and individual precautions in a general population. cross sectional study demographic and attitudinal determinants of protective behaviours during a pandemic: a review factors associated with uptake of vaccination against pandemic influenza: a systematic review european center for disease control. technical guidance. social marketing guide for public health programme managers and practitioners measles still spreads in europe: who is responsible for the failure to vaccinate? eurosurveillance under-vaccinated groups in europe and their beliefs, attitudes and reasons for non-vaccination; two systematic reviews vaccine hesitancy and self-vaccination behaviors among nurses in southeastern france european centre for disease prevention and control. catalogue of interventions addressing vaccine hesitancy technical report; european centre for disease prevention and control guides/toolkits a psychological perspective on economic biased assimilation and attitude polarization: the effects of prior theories on subsequently considered evidence changes in suicide rates following media reports on celebrity suicide: a meta-analysis mass killings in the united states from to : social contagion or random clusters? suicide life-threat populist politics and vaccine hesitancy in western europe: an analysis of national-level data vaccine hesitancy: understanding better to address better social norms guidebook: a guide to implementing the social norms approach in the european centre for disease prevention and control. conducting health communication activities on mmr vaccination design and evaluation of a branded narrative-story based intervention to promote hpv vaccination in rwanda policy and system change and community coalitions: outcomes from allies against asthma social marketing and social movements: creating inclusive social change coalitions community coalitions for prevention and health promotion towards a million change agents. review of the social movement's literature, implications for large scale change in the nhs compilation of social marketing evidence of effectiveness. key references briefing paper.international social marketing association (isma) and affiliated national and regional associations effectiveness of social marketing interventions to promote physical activity among adults: a systematic review house of lords, science and technology select committee department of health and human services office of planning, research and evaluation strategies for addressing vaccine hesitancy-a systematic review leveraging behavioural insights to respond to covd- evidence-based community engagement in the development and humanitarian contexts european centre for disease prevention and control. a literature review of trust and reputation management in communicable disease public health risk communication and social mobilization in support of vaccination against pandemic influenza in the americas revealed: populists far more likely to believe in conspiracy theories. the guardian applying tools from human-centered design to social marketing planning insightful social marketing leadership combating vaccine hesitancy: teaching the next generation to navigate through the post truth era. front use of mass media campaigns to change health behaviour centres for disease control and prevention. gateway to health communication and social marketing practice government communication network and the central office of information. communications and behaviour change when corrections fail: the persistence of political misperceptions rational choice and the framing of decisions methods to overcome vaccine hesitancy social marketing in india health literacy and vaccination: a systematic review making a drama out of a crisis. a multidisciplinary study of news media coverage of a public health crisis and the role of emotion using social media to create engagement: a social marketing review a postmodern pandora's box: anti-vaccination misinformation on the internet technical advisory group on behavioural insights and sciences for health by failing to prepare, you are preparing to fail: lessons from the h n 'swine flu' pandemic this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: not applicable. the authors declare no conflict of interest. key: cord- -khm veuv authors: gonzález-mora, alejandro; hernández-pérez, jesús; iqbal, hafiz m. n.; rito-palomares, marco; benavides, jorge title: bacteriophage-based vaccines: a potent approach for antigen delivery date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: khm veuv vaccines are considered one of the most important bioproducts in medicine. since the development of the smallpox vaccine in , several types of vaccines for many diseases have been created. however, some vaccines have shown limitations as high cost and low immune responses. in that regard, bacteriophages have been proposed as an attractive alternative for the development of more cost-effective vaccines. phage-displayed vaccines consists in the expression of antigens on the phage surface. this approach takes advantage of inherent properties of these particles such as their adjuvant capacity, economic production and high stability, among others. to date, three types of phage-based vaccines have been developed: phage-displayed, phage dna and hybrid phage-dna vaccines. typically, phage display technology has been used for the identification of new and protective epitopes, mimotopes and antigens. in this context, phage particles represent a versatile, effective and promising alternative for the development of more effective vaccine delivery systems which should be highly exploited in the future. this review describes current advances in the development of bacteriophage-based vaccines, with special attention to vaccine delivery strategies. moreover, the immunological aspects of phage-based vaccines, as well as the applications of phage display for vaccine development, are explored. finally, important challenges and the future of phage-bases vaccines are discussed. the development of vaccines represents one of the greatest advances in medical fields which have saved a huge number of human and animal lives [ ] . nowadays, several research groups around the world have focused on the development of more effective, better, safer, inexpensive and with long-lasting immune response vaccines for medical applications [ ] [ ] [ ] . currently, different types of vaccines such as live-attenuated, inactivated, synthetic, among others have been successfully developed and tested for preventive purposes. conventional vaccines are made of inactivated or attenuated microorganisms. although significant improvements have been made regarding conventional vaccines, drawbacks as difficulties to culture microorganisms, low efficacies and potential risks related to pathogenic reversion or transmission to immunocompromised patients of these vaccines have been reported [ ] . therefore, the administration of specific and contaminant-free antigens has been proposed as a new strategy to overcome the limitations previously mentioned. thus, novel vaccines based on figure . simplified schematic representations of the three types of phage-based antigen delivery systems currently reported. an antigen delivery system based on a hybrid phage-dna vaccine combines the concepts of the other two systems. it is based on a phage displaying on its surface peptides with specific affinity towards antigen presenting cells (apcs) and at the same time, it harbors a dna plasmid encoding the therapeutic antigen in a eukaryotic expression cassette. phages are involved in a wide range of applications such as drug delivery, phage therapy, biosensors development and as vaccine delivery systems [ , , ] . many of these applications are possible as a result of the development of phage display technology, which lies on the manipulation of bacteriophages to present antigens on their surface. until now, phage display vaccines have been used for preventing or treating several diseases including cancer, viral, parasitic and fungal infection as well as their use in immunocontraception and drug abuse, among others [ , [ ] [ ] [ ] [ ] . vaccines targeting drug abuse consists on the use of phages particle displaying antibodies that block the effects of different drugs such as the cocaine [ ] . bacteriophages are a specific class of viruses that infect and replicate into bacteria and archaea and are incompetent for eukaryotic infection [ , ] . these viral particles are made by genetic material, dna or rna, packaged in either simple or elaborate protein-made structures known as capsids. phage display is a potent technology that consists on the expression of either peptides or proteins fused to coat proteins of the phage's surface [ , , ] . this technique has been used in biotechnology for several applications such as: directed evolution [ ] , identification of ligand binding sites (protein-protein, protein-ligand and protein-dna interactions) [ ] , selection of monoclonal antibodies [ ] , protein-based drug discovery [ ] , development of epitope-based diagnostic tools, identification of b-cell epitopes [ ] and for vaccine development [ , ] . filamentous phages (m , fd, and f ), lytic phages (t and t ) and the temperate phage lambda (λ) have been successfully used as display systems [ , , , ] . from all these systems, bacteriophage m is the most broadly used in phage display since its purification from an e.coli lysate is easier compared to other phages [ , , ] . filamentous phages are a group of non-lytic viruses with a circular single-stranded dna genome with a length around of . kb in the case of m phage [ , ] . as a result of the non-lytic nature of filamentous phages, these particles can be obtained in high titers with reduced bacterial figure . simplified schematic representations of the three types of phage-based antigen delivery systems currently reported. an antigen delivery system based on a hybrid phage-dna vaccine combines the concepts of the other two systems. it is based on a phage displaying on its surface peptides with specific affinity towards antigen presenting cells (apcs) and at the same time, it harbors a dna plasmid encoding the therapeutic antigen in a eukaryotic expression cassette. phages are involved in a wide range of applications such as drug delivery, phage therapy, biosensors development and as vaccine delivery systems [ , , ] . many of these applications are possible as a result of the development of phage display technology, which lies on the manipulation of bacteriophages to present antigens on their surface. until now, phage display vaccines have been used for preventing or treating several diseases including cancer, viral, parasitic and fungal infection as well as their use in immunocontraception and drug abuse, among others [ , [ ] [ ] [ ] [ ] . vaccines targeting drug abuse consists on the use of phages particle displaying antibodies that block the effects of different drugs such as the cocaine [ ] . bacteriophages are a specific class of viruses that infect and replicate into bacteria and archaea and are incompetent for eukaryotic infection [ , ] . these viral particles are made by genetic material, dna or rna, packaged in either simple or elaborate protein-made structures known as capsids. phage display is a potent technology that consists on the expression of either peptides or proteins fused to coat proteins of the phage's surface [ , , ] . this technique has been used in biotechnology for several applications such as: directed evolution [ ] , identification of ligand binding sites (protein-protein, protein-ligand and protein-dna interactions) [ ] , selection of monoclonal antibodies [ ] , protein-based drug discovery [ ] , development of epitope-based diagnostic tools, identification of b-cell epitopes [ ] and for vaccine development [ , ] . filamentous phages (m , fd, and f ), lytic phages (t and t ) and the temperate phage lambda (λ) have been successfully used as display systems [ , , , ] . from all these systems, bacteriophage m is the most broadly used in phage display since its purification from an e. coli lysate is easier compared to other phages [ , , ] . filamentous phages are a group of non-lytic viruses with a circular single-stranded dna genome with a length around of . kb in the case of m phage [ , ] . as a result of the non-lytic nature of filamentous phages, these particles can be obtained in high titers with reduced bacterial contamination, making their purification easier and cheaper [ ] . due to their inherent viral particle features, filamentous phages are considered vaccine carriers with high immunogenic potential [ , ] . these particles are flexible protein-based cylinders composed of five coat proteins (piii, pvi, pvii, pviii, and pix). from these, proteins piii, pvi, pvii and pix are the minor coat proteins, meanwhile pviii is the major coat protein which surrounds the whole particle. piii and pviii proteins are the most used for the display of fusion peptides due to their specific location on the phage structure (table ) [ , ] . short peptides ( or fewer amino acids) can be effectively fused to many copies of the protein pviii since the display level have been found to be dependent of the length and sequence of the peptide displayed [ ] . since the valency (protein copies per viral particle) has a direct relationship with the immune response to be generated, the proportion of immunogenic peptides displayed on protein pviii has been extensively used for vaccine development [ , , ] . on the other hand, protein piii is preferred for the expression of larger peptides since the phage has fewer copies and steric effects may be negligible [ , ] . furthermore, important advantages of using filamentous phages for the development of vaccines include that phages are stable under harsh conditions (ph and temperature), the phage size is determined by the length of the dna molecule it harbors and the phage genome capability to be used as cloning vector [ , , ] . although these features surely encourage the use of filamentous phages as antigen display systems, a strategy to address their inefficiency to display peptides above amino acids long on the major coat protein pviii is needed to maximize the display flexibility of the system [ ] . lytic phages t and t have also been employed in phage display for vaccine applications [ ] . the capsid proteins of phage t , hoc and soc [ ] , can be used to display larger proteins at high copy numbers more efficiently than filamentous phages (table ) [ ] . at the same time, t phage capsid proteins have demonstrated to promote an immune response in humans and mice [ ] . in addition, oral administration of whole wild type t phage to humans demonstrated to be highly safe in clinical trials [ ] . some advantages of using t phages as vaccine carriers include the high immunogenic activity exerted by their capsid proteins, plasmid compatibility to allow engineering of dual protein expression and the absence of toxic proteins secreted. based on these features, t phage has been preferred in some approaches as a display vector over filamentous phages commonly used [ ] . on the other hand, the carboxyl-terminus of protein b of phage t has been engineered to display heterologous peptides in antigen display strategies [ ] . some of the advantages of using the t phage for protein display include its high cloning capacity, high stability to harbor foreign gene inserts and its rapid propagation among other features [ ] . in addition, humoral and cellular immune responses have been reported to be activated following the administration of recombinant t phage particles in animal models [ ] . although filamentous phages are the most common vectors used in phage display, the λ phage has also been proposed as an antigen display platform [ ] . it has been reported that the λ phage can display properly folded antigenic peptides and - times larger fusion proteins than filamentous phages, offering a plausible alternative for complex antigens display [ , ] . for instance β-galactosidase, a protein larger than kda, have been properly displayed on the λ phage surface without affecting phage viability and morphology [ ] . moreover, it has been reported that the display of peptides using the λ phage generate particles with higher antigen densities when compared to the density of peptide displayed by filamentous phages [ , ] . impact vaccine development since the displayed proteins must be properly folded to exhibit the desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [ ] . desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [ ] . impact vaccine development since the displayed proteins must be properly folded to exhibit the desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [ ] . desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [ ] . desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [ ] . limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [ ] . protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [ ] . protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [ ] . in vivo and in vitro phage display systems. in an in vivo phage display system, the expression and fusion of the displayed protein occurs at the phage infection stage. although many large proteins have been successfully displayed in phage vectors using in vivo systems, limitations such as variations in intracellular expression, protein aggregation and deficient phage assembly have been described [ ] . such drawbacks can negatively impact vaccine development since the displayed proteins must be properly folded to exhibit the desired activity. on the other hand, in vitro display systems have demonstrated to overcome the limitations of in vivo display systems by promoting the correct fusion and further display of the protein of interest. this enhancement in protein expression and display efficiency in in vitro systems has been attributed to the use of purified components in a controlled environment [ ] . as mentioned in previous sections, phage display has been proposed as a potent tool for vaccine development [ , ] . this technology has been used for this purpose in two main strategies: ( ) to produce vectors displaying antigens and ( ) to identify new protective antigens [ ] . the use of phage vectors as antigen display agents is focused on both, the development and production stages of a vaccine. on the other hand, phage display can be employed in early stages of the vaccine design process for the identification of novel antigens through the use of genetic engineering to generate random proteins and peptides aimed to interact specifically with a target, allowing the selection of the best binding antigen. phage display vaccines relies on the successful expression of antigens fused to phage surface proteins to produce viral particles with specific immunogenic activity [ ] . table describes the phage display vaccines that have been currently reported. it is worth mentioning that anticancer vaccines successfully developed in recent years have already been used in cancer immunotherapy [ ] . administration of these anticancer phage vaccines stimulate the immune system of the host to produce antibodies against cancer antigens to reduce tumor cells proliferation [ ] . to identify the best immunogenic antigen for phage-based cancer vaccines, several candidates have been evaluated. for instance, some antigens reported are: epitopes from epidermal growth factor receptor (egfr) [ ] , melanoma antigen gene (mage), and fms-like tyrosine kinase (flt ) [ ] . since the discovery of the capacity of filamentous phages to exert anti-tumor activity in rabbits and mice [ ] , the use of phage particles as immunotherapy to treat melanoma tumors has become a very compelling research field. studies on the administration of m and t phages to mice tumor models have demonstrated that the mechanism by which anti-tumor phages induce tumor elimination is mediated by the activation of tumor-associated macrophages in a myeloid differentiation primary response (myd )-dependent way [ ] . interestingly, the effect of phage administration was observed with a single dose of × plaque forming units (pfu) injected subcutaneously. on the other hand, it has been reported that the administration of anti-tumor phages stimulates the infiltration of neutrophilic granulocytes, leading to the development of metastatic activity that results in damage of tumor tissue and reduction of viable tumor cells [ ] . in other study, ericksson et al. reported that the mechanism of action for the tumor degradation caused by bacteriophages is toll-like receptor (tlr) dependent. this study also reported effects on the production of proinflammatory cytokines, on the secretion of molecules necessary for antigen presentation by dendritic cells and co-stimulation of macrophages and t cells [ ] . these findings suggest the high capacity of phage particles to induce immunogenicity and prompts the development of phage-based vaccines for cancer immunotherapy. a phage-based vaccine aimed to treat amyloid plaque from alzheimer disease produced an increased igg response in a transgenic mice model, demonstrating the capacity of phage-based vaccines to activate humoral responses [ ] . immunocontraceptive vaccines have also gained attention for phage-based therapies. for instance, the gonadotrophin releasing hormone (gnrh) antigen was displayed on the surface of t phages for immunocastration in a mice model [ ] . antiviral phage-based vaccines have also been successfully developed. epitopes from hiv, hepatitis b, herpes simplex virus (hsv- ), hsv- [ ] , circovirus (pcv ) [ ] , human respiratory syncytial virus [ ] among others have been phage displayed. phage display has demonstrated to be an effective, inexpensive and fast technique for the identification of immunogenic proteins and peptides allowing the development of novel and more effective vaccines [ , , ] . this methodology incorporates principles of genetic engineering and combinatorial chemistry [ ] . it consists in cloning natural and random cdna sequence variants into the phage genome to be expressed on the phage surface to generate a population of phages carrying different candidate antigens. from these libraries, phages with more affinity to the desired target (antibody for antigen identification) are selected and the dna sequence encoding for the antigen is identified (figure ) [ ] . the high efficiency and low costs that have been reported for the design of several vaccines using phage display technology for antigen identification are highly relevant features that must be taken into consideration while choosing the best platform for drug design [ ] . phage display has demonstrated to be an effective, inexpensive and fast technique for the identification of immunogenic proteins and peptides allowing the development of novel and more effective vaccines [ , , ] . this methodology incorporates principles of genetic engineering and combinatorial chemistry [ ] . it consists in cloning natural and random cdna sequence variants into the phage genome to be expressed on the phage surface to generate a population of phages carrying different candidate antigens. from these libraries, phages with more affinity to the desired target (antibody for antigen identification) are selected and the dna sequence encoding for the antigen is identified (figure ) [ ] . the high efficiency and low costs that have been reported for the design of several vaccines using phage display technology for antigen identification are highly relevant features that must be taken into consideration while choosing the best platform for drug design [ ] . this technology has allowed the identification of epitopes, mimotopes, bacteria adhesins and vaccine components (table ) [ , ] . one of the greatest advantages of using this method is the rapid identification and isolation of phages expressing specific targets [ ] . at the same time, the protein piii from m has been the preferred protein for fusion since high affinity of specific targets towards the displayed antigens have been observed [ ] . this technology has allowed the identification of epitopes, mimotopes, bacteria adhesins and vaccine components (table ) [ , ] . one of the greatest advantages of using this method is the rapid identification and isolation of phages expressing specific targets [ ] . at the same time, the protein piii from m has been the preferred protein for fusion since high affinity of specific targets towards the displayed antigens have been observed [ ] . table . cases of epitope, mimotope and antigens identification through phage display describing the main findings in regards of the phage display protocol used for selection and the prophylactic and therapeutic results reported. phage display technology has been applied for the identification of new and more specific linear and continuous antigenic determinant epitopes which normally have a length of - amino acids [ , ] . for instance, in a recent published paper, chung-tao et al. reported the use of phage display to identify novel enhanced epitopes from the envelope protein of dengue virus which was used later for the development of a dna vaccine [ ] . authors concluded that the phage display methodology could be a key element for developing novel and more immunoprotective dengue vaccines [ ] . grabowska et al. demonstrated the applicability of phage display technology for the display and discovery of new protective virus hsv- epitopes [ ] . table summarizes some of the epitopes identified using phage display technology. altogether, this data supports the use of phage display for the development of new vaccines, diagnostic tools and immunotherapies [ ] . phage display libraries and biopanning have also been used for the identification of mimotopes of lipid and carbohydrate antigens which often present low immunogenicity [ , ] . mimotopes are short peptides that structurally mimics epitopes, enabling a conformational interaction with an antibody [ ] . these short amino acid sequences are used to produce antibodies against specific antigen epitopes and have several advantages over original antigens or epitopes since they are easy to produce for being short sequences, can mimic non-protein antigens, can be tested against undiscovered antigens and they have demonstrated to possess high bioactivity [ , ] . thus, several mimotopes displayed on phages have produced effective in vivo responses in murine and swine models (table ) . nowadays, mimotope-based vaccination is considered an effective immunotherapy approach to induce specific antibody responses [ ] . at the same time, use of phage display libraries have allowed the identification of amino acid sequences that mimics specific immunogenic and antigenic regions of toxins. by this approach, epitopes from botulinum neurotoxin a [ ] , the beta-mammal toxin cn and noxiustoxin (ntx) toxins have been discovered [ , ] . recently, jahdasani et al. used phage display technology for the identification of immunogenic epitopes from the scorpion venom of hemiscorpius lepturus to develop a preventive strategy against scorpion bites [ ] . successful results were obtained demonstrating the applicability of this tool for the development of novel vaccine candidates in the antivenom area. phage display technology has also been used for the identification of new anti-parasite antigens. in this regard, antigens from brugia malayi [ ] , plasmodium falciparum [ ] , leishmania major [ ] have been elicited through this platform. by using phage display to identify peptides with binding capacity to the tegument of schistosoma japonicum, liu et al. discovered the zl peptide which showed a potent antiparasitic activity [ ] . likewise, phage display for the identification of epitopes for taenia solium paramyosin to develop a sole epitope-based vaccine has been described [ ] . nowadays, there are no vaccines against pathogens transmitted by ticks, which have been described as a serious health risk [ ] . to overcome this problem, becker et al. used oligopeptide phage display for the identification of antigens present in the saliva of the black-legged tick, ixodes scapularis, which have been found to prevent pathogen transmission. authors identified the metalloprotease (mp ) as a potential candidate for the development of an anti-tick vaccine by testing mp immunogenicity in human sera [ ] . as can be seen, phage display, a more than years old technique, is an important tool for vaccine development and immunotherapy that had not been harnessed for these purposes until years ago. at the same time, phage display has been applied for the enhancement of antigen-antibody interactions leading to the design of more potent vaccines [ ] . the exploitation of this technique for vaccine development may lead to important advances for the prevention of several important infectious diseases [ , ] . dna vaccination consists in the direct administration of a foreign dna encoding an antigen to induce host immunity. for this approach, use of dendritic cells as transfection targets have demonstrated to increase immunity [ ] . dna vaccination has been proposed as a viable alternative when a protein or peptide vaccination is not viable or unsuccessful [ ] . several advantages of dna vaccines have been described, for instance: antigen is folded correctly inside the host's cell, downstream processing is not required which significantly decreases overall manufacture costs, null risk of vaccine to become pathogenic, dna can be obtained in large quantities and high purity at relatively low costs and no strict storage conditions are required for dna products [ , ] . moreover, since this approach have demonstrated to stimulate both cellular and humoral immune responses, dna vaccines have been proposed as promising immunotherapy treatments [ ] . unfortunately, standard dna vaccines have shown poor immunogenicity in large primate trials, meanwhile in human trials the use of adjuvants such as dendritic cells have been identified as mandatory [ , ] . the low stability and the poor distribution of naked dna vaccines diminish hugely the efficacy of the immune response, making imperative the use of a delivery vehicle [ ] . to note, currently, there are not commercial dna vaccines for human medical applications. however, novel vaccines based on nucleic acids have been rushed into clinical trials amid the sars-cov- pandemic, pointing out their promising efficiency as immune system stimulators [ ] . in that regard, since phage particles have shown adjuvant properties, they have been proposed as new and suitable vehicles for dna delivery. in concept, a bacteriophage dna vaccine is made of a eukaryotic expression cassette encoding a specific antigen controlled by target-specific promoters contained within a bacteriophage ( figure ) [ , ] . the expression cassette must have the necessary regulatory sequences to allow the correct gene expression and protein folding of the antigen. in this strategy, phage particles serve as passive carriers by allowing the transference of the dna encoded antigen to eukaryotic cells where the dna will be expressed [ ] . phage-dna vaccines are considered more advantageous than standard dna vaccines since the former are more stable for storage, transport and administration (mainly via oral route), since dna is protected from degradation by the capsid of phage particles. although y phage is the most common phage vector for dna vaccination, filamentous phages have also been tested [ , ] . an important feature to consider of filamentous phage-dna vaccines is the fact that phagemid vectors can efficiently contain multiple gene copies, which may allow immunization with various epitopes or antigens using a single delivery vector [ ] . table describes the phage-dna vaccines currently developed. furthermore, it has been demonstrated that phage-dna vaccines can induce a more effective immunoprotection when compared to naked-dna strategies [ , ] . for instance, march et al. reported a higher immune response by using a lambda-dna vaccine when compared to the effect of a naked plasmid vaccine even at lower doses [ ] . in other work, clark et al. reported a higher immune response when using a dna vaccine compared to the immune effect caused by the commercial vaccine engerix b, based on a recombinant protein-small surface antigen (hbsag) of hepatitis b. the enhanced performance of the dna vaccine was attributed to a better folding of the protein expressed in the host, better compatibility with post-translational modifications on the antigen, the adjuvant effect of the phage particle, the presence of lipopolysaccharides and lipid incorporation when the protein is secreted [ ] . notably, phage dna vaccines have exhibited more effectiveness when multiple doses ( × phages per dose at intervals of , and weeks) are administered. this effect has been attributed to poor dna expression in the first immunization since the host's immune response focuses on phage coat proteins incapacitating a proportion of the dna-carrying phages. however, the complete mechanism has not been elucidated [ ] . based on the favorable characteristics of phage particles and compared to other vaccine systems, production of phage dna vaccines is simple and low-cost, the viral particle do not carry antibiotic resistance genes, the phage genome is suitable for the insertion of large dna antigen sequences (up to kb in lambda vectors) and are quite stable compared to standard dna vaccines [ ] . in addition, phage dna vaccines are safer than dna vaccines using adenovirus vectors since the bacteriophage is unable to replicate into eukaryotic cells [ ] . hybrid bacteriophage vaccines are produced by the combination of phage display and phage dna vaccines. the most recent advances in phage-based vaccines have focused on improving the interaction and internalization of phage particles by antigen presenting cells (apc) to increase the immune response. this system relies on bacteriophages displaying proteins or peptides with high affinity to antigen-presenting cells or the antigen itself and at the same time they carry on their genome an eukaryotic expression cassette encoding a specific antigen with the final aim of increasing the immune response by combining both effects (figure ) [ ] . for such reasons, this strategy has been proposed to be implemented as a directed therapy based on its capacity of delivering antigenic genes to activators of the immune response such as dendritic cells. it has been reported that this kind of vaccines are capable to successfully induce cellular and humoral immune responses [ ] . unfortunately, the mechanism for dna delivery used by phages has not been completely elucidated [ ] . although the cellular internalization of phage particles seems to be quite efficient, the nuclear uptake efficiency must be improved. in , sartorius et al. developed a double-hybrid filamentous bacteriophage fd co-displaying peptides recognized by the major histocompatibility complex (mhc) class i and mhc class ii cell surface receptors and epitopes from the antigen mage aiming to enhance the anti-tumor immune activity based on ctl responses [ ] . by observing an increase in the ctl response through the administration of hybrid phages, sartorius et al. proposed this strategy as a potent tool for the development of more effective anti-cancer vaccines. ideally, a vaccine carrier should be easy to produce in large quantities, safe and capable to effectively present antigens to the immune system to induce a proper immune response. viral particles and more specifically bacteriophages have demonstrated to possess many of the properties previously mentioned. thus, in recent years bacteriophage-based vaccines have gained attention for medical applications considering their low production costs, relative simplicity to prepare and to genetically modify, easiness to produce at large scales, their adjuvant capacity, high stability under a wide range of ph and high temperature, resistance to nucleolytic and proteolytic enzymes and desiccation [ , , ] . the capability of phages to be genetically modified to be used as display or delivery systems represents an important aide in emergency situations, for instance outbreaks of infectious pathogens [ ] . in this context, recombinant phages can be produced in large amounts at low cost by basic methods making phage-based vaccines more cost-effective than standard or classical vaccine development strategies [ ] . according to munira et al. the cost of producing recombinant vaccines is estimated on average of usd per dose [ ] . on the other hand, in, torres-acosta et al. estimated the production cost for a phage-based vaccine was around $ . usd per dose ( pfu) [ ] . these estimations suggest a % reduction on the production costs of phage-based vaccines compared to recombinant-based vaccines, supporting the implementation of phages in vaccine development strategies. the high stability exhibited by filamentous phage preparations and their capacity to protect antigens from degradation makes bacteriophages ideal vaccine vehicles based on their storage and transportation resilience and delivery features. based on the discussion in previous sections of this review, phage-based vaccines are more suitable for immunization than dna naked or recombinant vaccines. in that regard, brigati and petrenko evaluated the stability of landscape phages-multivalent pviii-type filamentous-phage constructs with unique structures and properties-expressing peptides fused to protein pviii at high-temperature ( • c) and observed that these particles retained stability without affecting their binding sites [ ] . this capability of phages is quite remarkable and has to be taken into consideration for the development of vaccines that will be distributed to remote areas where no adequate storage conditions are found or for veterinary applications that requires vaccine administration in the native habitats of animals [ ] . the use of phages as antigen carriers has the advantage of increasing the half-life of the antigen in the blood stream facilitating the activation of t-helper cells, enhancing the immune response [ ] . to study the immunogenicity efficiency of phage display, wu et al. compared the immune response of recombinant antigens with phage-displayed antigens and reported that the antigens expressed on the phage surface exhibited a better refolding leading to a more efficient activation of the immune system [ ] . this results, along with the increase observed on cellular immunity by the administration of hybrid-phages displaying apcs-targeted peptides [ ] , demonstrates the remarkable properties of phage-displayed antigens bacteriophages are the most abundant organisms that inhabit the entire planet [ ] , suggesting their high capability to coexist with a plethora of micro and macro organisms. for such reason, it is not unusual that these particles have proved to be harmless in mammalian models even in oral administration trials in humans approved by the fda [ , , , ] . to reinforce even more the safety of phage administration, as any other viral particle used as vaccine, bacteriophages can be physically or genetically inactivated as well [ ] . furthermore, phage particles have also been used in therapeutic applications in humans without side effects being observed [ ] . thus, the safe application of phage-based vaccines have been demonstrated as bacteriophages are not capable to infect and replicate into eukaryotic cells and no side effects have been reported contrary to the outcomes observed in mammalian model using other viral vectors [ ] . although many proteins and peptides have been successfully displayed in bacteriophages, the correct display of molecules on the phage surface is still considered a potential drawback for the development of these vaccines. the issue relies on two related subjects; proper folding of the polypeptide sequences displayed, and the epitope density displayed. both features, correct folding and presence of enough active epitopes to generate a significative immune response have a direct effect on the overall efficiency of these vaccine [ ] . an equally important factor to have in mind while designing any kind of vaccine is the administration route. although phage particles have showed to be safe for animal and human use, phages administered by oral route may infect gut bacteria, potentially leading to a dysbiosis. the infection and further replication of phages could induce the release of endotoxins from infected bacteria, increasing the risk of host damage [ ] . this risk can be eliminated by using non-lytic bacteriophages such as the m phage. several authors have described that the natural immunogenic properties of phage particles relies mainly on their chemical structure (phage proteins and viral dna) [ ] . this capacity makes bacteriophages excellent candidates for their use as antigen delivery vectors. in recent years, although not completely, the immunogenic effects in hosts driven by phage-based vaccines have been described, allowing a more rational design of novel and better phage vaccines. several authors have reported the capacity of filamentous phages to act as natural vaccine adjuvants since these viral particles are capable to achieve an effective antigen presentation to immune cells [ ] . this notable feature allows phage-based vaccines to enhance the stimulation of the immune response, obtaining better results than conventional vaccines even at relatively low doses [ , ] . in addition, it has been reported that phage-based vaccines produce less variability between subjects than conventional vaccines [ ] . it has been suggested that filamentous phages evolved to induce low non-specific immune response in eukaryotic organisms [ ] . however, filamentous phages still can induce an effective activation of the immune machinery by themselves apart from activation of both cellular and humoral immune responses by being engineered to directly interact with apcs, giving them an advantage over other delivery systems [ ] . the immunogenicity exhibited by filamentous bacteriophages have been demonstrated to be caused not only by their repeating coat proteins but also mediated by the phage dna itself [ , ] . this was demonstrated by the administration of dna from m phage to mice in a strategy to induce in vivo heterologous expression of interferon (predominantly the beta type) to confer the mice protection from a vaccinia virus infection. the immune response was in part attributed to the presence of cpg motifs in the phage genome [ , ] . phage vaccination trials in mice have demonstrated that cpg motifs stimulates t helper type cells (th ) and b cells leading to an enhanced immune response [ ] [ ] [ ] . cpg motifs have been also described to be involved in the stimulation of the toll-like receptor (tlr ) signaling cascade, modifying the production of different immunoglobulin classes [ ] . in the case of the t phage, the major capsid protein gp and the highly immunogenic outer capsid protein (hoc) have shown high antigenicity [ ] . hashiguchi et al. investigated the immune response derived from the administration of m phages in an in vivo murine model. authors found that the administration of phages induced an equivalent immune response as the typical immunogen sheep red blood cells (srbc), reinforcing the theory that m phage may be an important vector for vaccine delivery [ ] . in another report, it was found that filamentous phages induce a stronger immune response than the carrier protein ovalbumin [ ] , demonstrating that bacteriophages can induce an immune responses by their own, supporting their use as promising vaccines carriers. interestingly, van houten et al. reported that reducing the complexity-quantity of different b-cell epitopes of immunodominant epitopes of filamentous coat proteins in phages enhanced the antibody response against synthetic peptides fused to the phage surface [ ] . thus, the adjuvant-like effect exhibited by phages in various vaccination studies along with their capacity to effectively present peptides or proteins to the immune system leading to the activation of cellular and humoral immune responses [ ] demonstrates that engineered phage particles are a proper strategy to enhance the efficacy and safety of viral particle-based vaccines. from a metabolic point of view, bacteriophages are considered inert antigen particles whose cell internalization mechanisms are still being studied. it is widely accepted that phages are internalized by endocytosis and processed by apcs [ ] . once phage particles are internalized by apcs, antigens are processed and presented through the major histocompatibility complex (mhc) class i and ii pathways (figure ) , stimulating cellular and humoral immune responses [ ] . phage-displayed antigens have been described to induce specific cd + and cd + t cells lymphocytes (ctls) responses, leading to the production of specific antibodies and activation of t helper cells without the utilization of supplementary adjuvants. also, it has been reported that macrophages are capable to internalize phages via phagocytosis [ ] . helper cells without the utilization of supplementary adjuvants. also, it has been reported that macrophages are capable to internalize phages via phagocytosis [ ] . antigen presentation via the mhc class i pathway have been demonstrated to activate the ctl through the interaction with cd + t cells (figure ). it has been suggested that standard vaccines (soluble exogenous antigen or inactivated pathogens) fail to stimulate the mhc class i pathway, and therefore are incapable to activate t cells, which are important to promote an optimal immune response [ ] . in that sense, it has been reported that filamentous phages displaying antigens are efficiently processed and presented by the mhc class i and class ii pathways, demonstrating their high potential as positive stimulants of the immune system [ , , ] . activation of the mhc class i pathway and further triggering of ctl responses have been reported to play a key role in figure . immunological mechanism of phage-based vaccines. phage-based vaccines can stimulate both humoral and cellular responses. phage particles displaying-antigens are taken up by apcs. the antigen is then processed and presented on mhc-i and mhc-ii molecules which leads to the activation of t cells. the direct recognition of the phage particle happens simultaneously, promoting the generation of plasma cells and further secretion of antibodies. antigen presentation via the mhc class i pathway have been demonstrated to activate the ctl through the interaction with cd + t cells (figure ). it has been suggested that standard vaccines (soluble exogenous antigen or inactivated pathogens) fail to stimulate the mhc class i pathway, and therefore are incapable to activate t cells, which are important to promote an optimal immune response [ ] . in that sense, it has been reported that filamentous phages displaying antigens are efficiently processed and presented by the mhc class i and class ii pathways, demonstrating their high potential as positive stimulants of the immune system [ , , ] . activation of the mhc class i pathway and further triggering of ctl responses have been reported to play a key role in anti-cancer and anti-viral drug approaches [ ] . in that regard, wan et al. reported that filamentous phages displaying an exogenous antigen successfully triggered a mhc class i-restricted cd + t cell response [ ] . in another work, sartorius et al. demonstrated the capacity of hybrid filamentous phages to induce t cell-dependent ctl responses when specific epitopes were displayed on the phage surface [ ] . moreover, phages can stimulate apcs for the secretion of costimulatory molecules required for t cell activation [ ] . phage display technology allows the design of engineered phages capable to express molecules that specifically targets presenting cells for the induction of a strong cellular response [ ] . iwagami et al. reported t cell activation of cd + and cd + with the co-expression of cd and cd markers after administration of a y phage-based vaccine displaying the peptidic antigen aspartate-β-hydroxylase (asph) [ ] . in the same study, the response also included an increase on the secretion of ifnγ and the asph antigen displayed in the y phage generated specific cd + and cd + responses in vitro [ ] . thus, phages capacity to induce a ctl response supports the use of bacteriophages as vaccine carriers capable to enhance the immune response produced in stand-alone antigen strategies. likewise coat proteins, bacteriophage dna has exhibited to possess an immunostimulatory effect. in that regard, cuesta et al. reported that a dna fragment of the domain i of the coat protein iii of filamentous phages is capable to enhance the t helper humoral and cellular immune responses when fused to a single-chain variable fragment (scfv) [ ] . antigen presentation to naïve cd + t cells via mhc-ii pathway has demonstrated to leads to the activation of th and th cells (figure ) [ ] . also, in vitro and in vivo studies have confirmed that recombinant filamentous phages are capable to trigger a strong immune response mediated by t helper cells. this response is mediated by phage components such as the coat proteins, phage dna as well as phage-associated lipopolysaccharide (lps) which can direct the activation of th and th cells [ , ] . to study the effects of phages in the immune system, iwagami et al. immunized mice using a y phage vaccine at a dose of pfu and observed an increased secretion of specific cytokines of th (ifn-γ and tnf-α) and th (il- , il- and il- ) [ ] . in the same study, authors observed that activation of th generated a ctl response, suggesting that phage particles may be great candidates to consider for the development of vaccines focused on ctl response activation. on the other hand, a phage-mimotope vaccine against fasciola hepatica showed a mixed th and th cells responses, advocating the approach of using phage particles in vaccination strategies targeting cellular responses [ ] . phage particles have demonstrated their capacity to induce humoral responses through the direct activation of b cells as well as through the activation of th cells (figure ) [ ] . it has been proposed that for a better b cell response, antigens must be presented in a repetitive and organized configuration [ ] . studies focused on the administration of phage display vaccines have also shown the induction of immune response in animal models injected with recombinant vaccines and mimotopes expressed and fused to coat proteins of phages at a dose of pfu [ ] . also, it has been reported that the antibody response induced by m phages is limited to recognition of the first n-terminal residues of protein pviii and to the external domains of protein piii [ ] . in , hashiguchi et al. studied the in vivo immune response induced by m phage with the aim to characterize this effect for vaccine development [ ] . the researchers administered a solution containing highly purified m phages ( pfu) to mice and observed the antibody response. they reported a strong primary response based on high production of igg antibodies which was attributed to a complete dependence on the activation of the myd pathway involved in innate immune response [ ] . other studies have suggested that bacteriophages may play an important role in the innate response activation by stimulation of tumor-associated macrophages (tams), which secretes immunomodulators that assists the recruitment of neutrophils [ ] . unfortunately, the complete molecular mechanism of the activation of innate immunity by phages has not been completely elucidated. in , thomas et al. produced a divalent subunit vaccine expressing green fluorescent protein (gfp) as model antigen and the hiv- tat protein to enhance cell uptake and thus establish a subunit λ-based vaccine [ ] . this platform allows the display of many epitopes at the same time, providing an easy and rapid approach to develop novel vaccines against emerging pathogens. authors reported a strong immune stimulation of splenocytes using the λ-based vaccine demonstrating the effectiveness of phage preparations to induce a strong adaptive immune response. cytokine profile was also evaluated, and the results showed a high ifnγ secretion by splenocytes, suggesting a th activation which lead to higher igg a isotype production. furthermore, it was observed that host's antibodies against the phage-based vaccine recognized more epitopes than those produced with naked dna vaccines. for long time, subcutaneous and intramuscular injections have been acknowledged as the most common routes for vaccine administration. wild-type and recombinant m phage particles have proved their capacity to cross the gastrointestinal barrier and remain stable, suggesting the use of phage nanoparticles as antigen delivery systems in oral route administration strategies [ , ] . nevertheless, based on their native capacity to recognize and infect bacteria, it has been reasonably speculated that oral administration of phages could cause an unbalance of the gut bacteria, compromising the host's health [ ] . fortunately, diverse strategies focused on improving the safety of phage-based vaccines administered by of oral route have been developed [ ] . a promising strategy could be the utilization of non-lytic phages, as the m , to diminish the risk of damaging the host's gut microbiome since these phages are not suited to destroy bacterial cells. another strategy is based on the use of viral particles with non-functional tail fibers, required for target recognition and further infection, to make them unable to infect bacterial cells without reducing their capacity to act as vaccine delivery systems [ ] . moreover, oral administration of phage-based vaccines has demonstrated strong immunostimulatory effects, reinforcing the idea of using phage particles as oral vaccine carriers [ , ] . despite the considerable amount of evidence supporting the use of bacteriophages as vaccines, their clinical application still requires further research and support. fortunately, u.s. food and drug administration (fda) has approved recently the first clinical trial for the use of phage therapy in humans. this phage therapy trial consists on the utilization of a bacteriophage combination to treat a resistant staphylococcus aureus infection. this step opens the opportunity to develop more effective clinical trials involving the use of phage particles for vaccine development. the current available knowledge describing the biology, physiology, and purification of phage particles should help to design adequate clinical trials. however, it is important to note that these clinical evaluations are quite expensive which limit their application to phage-based products. moreover, the wide diversity of phage-based vaccines could help in the development of combinatorial vaccination strategies that may surpass issues of current vaccination strategies. based on the wide range of applications that phage-based bioproducts may have and taking into consideration the effectiveness demonstrated of phage-based vaccines currently developed, the design of clinical trials should be taken as a priority for approval of these bioproducts [ ] . phage-based vaccines made without the insertion of foreign dna (in vitro display) into the phage genome can be considered as natural bioproducts since no risk of genetic transfer exists. these considerations should be acknowledged to accelerate the commercial use of phage-based vaccines. phage-based vaccines represent a promising approach for vaccine development since this approach offer important advantages over standard vaccine delivery systems. further studies are required for a better understanding of the immunological mechanism of phage vaccines in order to develop more specific antigen delivery systems. furthermore, immunization protocols aimed to test the effect of higher phage vaccine doses as those currently studied ( and pfu) should be implemented. future research of phage-based vaccines will focus on optimization the immunogenicity of the antigens displayed. finally, it has been suggested that the expression of mimotopes in a tandem configuration and fused to the pviii protein of bacteriophages could induce an enhanced effect on the immune response compared to the effect observed in other vaccination strategies. therefore, this approach should be thoroughly investigated to improve the efficacy of phage-based vaccine. the contribution of vaccination to global health: past, present and future inexpensive anti-cysticercosis vaccine: s pvac expressed in heat inactivated m filamentous phage proves effective against naturally acquired taenia solium porcine cysticercosis comparison of a bacteriophage-delivered dna vaccine and a commercially available recombinant protein vaccine against hepatitis b the development of veterinary vaccines: a review of traditional methods and modern biotechnology approaches influence of protein fold stability on immunogenicity and its implications for vaccine design therapeutic and prophylactic applications of bacteriophage components in modern medicine use of phage display technology in development of canine visceral leishmaniasis vaccine using synthetic peptide trapped in sphingomyelin/cholesterol liposomes dna vaccines-how far from clinical use? peptide vaccine: progress and challenges bacteriophage lambda display systems: developments and applications infective and inactivated filamentous phage as carriers for immunogenic peptides immunogenicity and epitope mapping of foreign sequences via genetically engineered filamentous phage recovery and primary purification of bacteriophage m using aqueous two-phase systems bacteriophages and their implications on future biotechnology: a review treating cocaine addiction with viruses reduction of β-amyloid plaques in brain of transgenic mouse model of alzheimer's disease by efrh-phage immunization cancer immunotherapy by a recombinant phage vaccine displaying egfr mimotope: anin vivostudy protective immunity against trichinella spiralis infection induced by a multi-epitope vaccine in a murine model recombinant bacteriophage-based multiepitope vaccine against taenia solium pig cysticercosis an overview on application of phage display technique in immunological studies phage-assisted continuous evolution of proteases with altered substrate specificity rapid isolation of single-chain antibodies by phage display technology directed against one of the most potent marine toxins: palytoxin targeting leishmania major parasite with peptides derived from a combinatorial phage display library an epitope-substituted dna vaccine improves safety and immunogenicity against dengue virus type phage display-a powerful technique for immunotherapy. hum. vaccines immunother m bacteriophage purification using poly(ionic liquids) as alternative separation matrices filamentous bacteriophage: biology, phage display and nanotechnology applications phage display: concept, innovations, applications and future dual expression system for assembling phage lambda display particle (ldp) vaccine to porcine circovirus (pcv ). vaccine filamentous bacteriophage fd as an antigen delivery system in vaccination engineering filamentous phage carriers to improve focusing of antibody responses against peptides plasmodium-mosquito interactions, phage display libraries and transgenic mosquitoes impaired for malaria transmission triple tandem mimotope peptide of epidermal growth factor receptordisplaying on the surface of m phage induces anti-tumor response in micetumor model immunisation with phage displaying peptides representing single epitopes of the glycoprotein g can give rise to partial protective immunity to hsv- peptide vaccination is superior to genetic vaccination using a recombineered bacteriophage λ subunit vaccine phage display as a technology delivering on the promise of peptide drug discovery evaluation of humoral and cellular immune responses against hsv- using genetic immunization by filamentous phage particles: a comparative approach to conventional dna vaccine m bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins human volunteers receiving escherichia coli phage t orally: a safety test of phage therapy inhibition of tumor angiogenesis in lung cancer by t phage surface displaying mvegfr vaccine immunogenicity studies of proteins forming the t phage head surface a bacteriophage t nanoparticle-based dual vaccine against anthrax and plague immunization with m e-displaying t bacteriophage nanoparticles protects against influenza a virus challenge advances in the t phage display system (review) immunogenicity of t bacteriophage nanoparticles displaying g-h loop of foot-and-mouth disease virus (fmdv) recombinant λ-phage nanobioparticles for tumor therapy in mice models lambda phage-based vaccine induces antitumor immunity in hepatocellular carcinoma in vitro binding of anthrax protective antigen on bacteriophage t capsid surface through hoc-capsid interactions: a strategy for efficient display of large full-length proteins bacteriophage t nanoparticle capsid surface soc and hoc bipartite display with enhanced classical swine fever virus immunogenicity: a powerful immunological approach simultaneous display of two large proteins on the head and tail of bacteriophage lambda phage display as a tool for vaccine and immunotherapy development phage display as a promising approach for vaccine development phage display-based nanotechnology applications in cancer immunotherapy immunotherapy of egfr-positive tumor based on recombinant egfr phage vaccine antitumor activity of endogenous mflt displayed on a t phage nanoparticle surface anticancer activity of bacteriophage t and its mutant hap in mouse experimental tumour models tumor-specific bacteriophages induce tumor destruction through activation of tumor-associated macrophages displaying of gnrh peptides on bacteriophage t and its immunogenicity in mice model protective immune responses induced by the immunization of mice with a recombinant bacteriophage displaying an epitope of the human respiratory syncytial virus the potential of phage display virions expressing malignant tumor specific antigen mage-a epitope in murine model induction of hepatitis b virus-specific cytotoxic t lymphocytes response in vivo by filamentous phage display vaccine de berardinis, p. the use of filamentous bacteriophage fd to deliver hla-a -restricted peptides and to induce strong antitumor ctl responses mutated and bacteriophage t nanoparticle arrayed f -v immunogens from yersinia pestis as next generation plague vaccines immunodiagnosis of human neurocysticercosis using a synthetic peptide selected by phage-display application of m phage display for identifying immunogenic proteins from tick (ixodes scapularis) saliva identification of immunotopes against mycobacterium leprae as immune targets using phdtm- mer phage display peptide library identification of immunogenic proteins and generation of antibodies against salmonella typhimurium using phage display identification and characterization of novel binding epitope of tetanus toxoid by phage display peptide library identification of the immunogenic epitopes of the whole venom component of the hemiscorpius lepturus scorpion using the phage display peptide library mimotope vaccination-from allergy to cancer mimotope vaccines: epitope mimics induce anti-cancer antibodies epitope mapping of neutralizing botulinum neurotoxin a antibodies by phage display potential of peptides selected from random phage-displayed libraries to mimic conformational epitopes: a study on scorpion toxin cn and the neutralizing monoclonal antibody bcf immunization with phage-displayed mimotopes novel phage display-based subtractive screening to identify vaccine candidates of brugia malayi identification of phage display peptides with affinity for the tegument of schistosoma japonicum schistosomula synthetic peptide-targeted selection of phage display mimotopes highlights immunogenic features of α-helical vs non-helical epitopes of taenia solium paramyosin: implications for parasite-and host-protective roles of the protein dna vaccines: roles against diseases genetic immunisation against hepatitis b using whole bacteriophage λ particles molecular and cellular mechanisms of dna vaccines the future of human dna vaccines safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind, randomised controlled trial phage displayed biomolecules as preventive and therapeutic agents a cost analysis of producing vaccines in developing countries economic evaluation of m bacteriophage production at large-scale for therapeutic applications using aqueous two-phase systems thermostability of landscape phage probes bacteriophage interactions with mammalian tissue: therapeutic applications selection of tumor-binding ligands in cancer patients with phage display libraries the influence of external factors on bacteriophages-review bacteriophages as vehicles for gene delivery into mammalian cells: prospects and problems pros and cons of phage therapy filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide interactions between bacteriophage, bacteria, and the mammalian immune system immunological basis of m phage vaccine: regulation under myd and tlr signaling anti-vaccinia virus effect of m bacteriophage dna cpg oligodeoxynucleotides with hepatitis b surface antigen (hbsag) for vaccination in hbsag-transgenic mice cpg motif in phage genome dna enhanced the efficacy of phage therapy by immunostimulation activation of human b cells by phosphorothioate oligodeoxynucleotides targeting toll-like receptor with cpg oligodeoxynucleotides enhances tumor response to fractionated radiotherapy cellular internalization mechanism and intracellular trafficking of filamentous m phages displaying a cell-penetrating transbody and tat peptide phage-phagocyte interactions and their implications for phage application as therapeutics a lipid based antigen delivery system efficiently facilitates mhc class-i antigen presentation in dendritic cells to stimulate cd + t vaccination with filamentous bacteriophages targeting dec- induces dc maturation and potent anti-tumor t-cell responses in the absence of adjuvants enhancement of dna vaccine potency through linkage of antigen to filamentous bacteriophage coat protein iii domain i induction of immunity in sheep tofasciola hepaticawith mimotopes of cathepsin l selected from a phage display library non-specific translocation of peptide-displaying bacteriophage particles across the gastrointestinal barrier identification of peptide sequences that induce the transport of phage across the gastrointestinal mucosal barrier arming filamentous bacteriophage, a nature-made nanoparticle, for new vaccine and immunotherapeutic strategies immunogenicity of filamentous phage displaying peptide mimotopes after oral administration orally delivered foot-and-mouth disease virus capsid protomer vaccine displayed on t bacteriophage surface: % protection from potency challenge in mice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors acknowledge the financial support of consejo nacional de ciencia y tecnología (conacyt) for the phd fellowship of alejandro gonzález mora (no. ) and the support from tecnológico de monterrey (bioprocess research group). the authors declare no conflict of interest. key: cord- - b mt a authors: garcía, leidy y.; cerda, arcadio a. title: contingent assessment of the covid- vaccine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: b mt a the covid- pandemic has not only had a negative impact on people’s health and life behavior, but also on economies around the world. at the same time, laboratories and institutions are working hard to obtain a covid- vaccine, which we hope will be available soon. however, there has been no assessment of whether an individual and society value ​​a vaccine monetarily, and what factors determine this value. therefore, the objective of this research was to estimate the individual’s willingness to pay (wtp) for a hypothetical covid- vaccine and, at the same time, find the main factors that determine this valuation. for this, we used the contingent valuation approach, in its single and double-bounded dichotomous choice format, which was based on a hypothetical market for a vaccine. the sample used was obtained through an online survey of n = individuals from chile. the main results showed that the wtp depends on the preexistence of chronic disease ([formula: see text]), knowledge of covid- ([formula: see text]), being sick with covid- ([formula: see text]), perception of government performance ([formula: see text]), employment status ([formula: see text]), income ([formula: see text]), health care ([formula: see text]), adaptation to quarantine with children at home ([formula: see text] and whether the person has recovered from covid- ([formula: see text]. according to our discrete choice model in double-bounded dichotomous format, it was concluded that the individuals’ wtp is us$ . (ci: . - . ; p < . ). this implies a social valuation of approximately us$ , million, corresponding to . % of the gnp per capita. format, which was based on a hypothetical market for a vaccine. the sample used was obtained through an online survey of n = individuals from chile. the keywords: covid- ; sars-cov- ; vaccine; acceptance; health economics; contingent valuation the severe acute respiratory syndrome coronavirus (sars-cov- ) as a pandemic coronavirus disease (covid- ) has had a negative impact on people's health and life and on economies around the world. for this reason, laboratories and institutions are working hard to obtain a covid- vaccine, which should be available in the future [ ] . such a vaccine is important in reducing mortality and the health costs of treating the disease. the vaccine is expected to be available free of charge to at least the poorest people with financing from the governments of each country, while the richest people could voluntarily seek vaccines in private clinics. however, the question arises of how much society and the individual value this vaccine. therefore, considering the adverse effects and high costs of the global pandemic generated by this syndrome, the aim of this research is to provide more information about the individual and social assessment of the vaccine. this assessment is important because it would yield useful information for the implementation of public policies aimed at improving health services. specifically, in terms of public health, governments should be designing potential vaccination campaigns against covid- ; however, due to logistical aspects and costs, in a first stage vaccination could target high-risk, socioeconomically vulnerable individuals , while other groups could paid for their vaccines. additionally, the individual and social assessment of the vaccine that we conduct in this paper, would help corporate research and development (r&d) laboratories to have an approximation of the expected benefits if they manage to develop the vaccine. furthermore, the acceptance rate of payment for a possible vaccine illustrates people's willingness to be vaccinated, so our findings would provide elements for discussion about the anti-vaccine movement. for this, it is important to understand the factors or variables that affect consumer demand and the decision to pay for a vaccine. this is addressed in this article through the estimation of a probabilistic model of the willingness to pay (wtp) for the vaccine. therefore, the objective of this research is to estimate an individual's wtp for a covid- vaccine and, at the same time, find the main factors that affect this valuation. the method used to estimate the wtp is the contingent valuation approach (cva), in its single and double-bounded dichotomous choice format. the double-bounded dichotomous choice format presents greater statistical efficiency by better estimating the variance and allows obtaining more precise confidence intervals for the mean of the wtp [ ] . cva is widely used in medical and health literature, which is based on a hypothetical market for a vaccine. specifically, wtp has been estimated for vaccines against diseases such as ebola [ ] , hepatitis b [ ] , chikungunya fever [ ] , dengue [ ] and diseases caused by meningococcus b [ ] and human papillomavirus [ , ] . there are no studies yet for covid- . the importance of the wtp for the vaccine is related to the model of provision and reliability of medical care. this study considers the chilean case, where individuals assume a significant part of the cost of preventive health care, including vaccines. specifically, chile has a mixed health system made up of public health insurance with groups according to health care coverage (ranging from % to %) and a private system where members pay according to the contracted plan and the type of hospital or clinic they wish to access. all workers must contribute to the health system with the equivalent of % of their taxable income and can choose to pay it to the public or private system. the indigent have free care in the public system. regarding vaccination, there is currently a national vaccination plan in chile that covers some vaccines for high-risk groups, while the rest of the population pays % of them. in the future, this could be replicated for covid- , due to the high costs that mass vaccination plans usually entail. the information was collected through a self-applied online questionnaire. we used a mixed sampling process (snowball and convenience sampling), but under an active recruitment system that allows access according to the demographic characteristics required to search for population representativeness. the target population were people years of age or older, with a medium and higher income level, who according to their health insurance would eventually have to finance the prevention costs of covid- , both in the public and private health systems (this target group corresponds to . % of the chilean population). people with lower incomes were not considered because they receive state aid. the survey was answered by individuals between april and may , . it is worth mentioning that chile is a high-income country according to the world bank [ ] because the it has a gross national income (gni) per capita of us$ , , where . % of the population has internet access [ ] and % uses social networks [ ] . the theory indicates that the wtp depends on individual preferences, income, attitude and perception towards the vaccine (as "good"), and the sociodemographic characteristics of the individual and their family [ ] . therefore, the questionnaire was structured in three sections that allowed capturing these aspects. in the first section, we asked about perception and individual context, previous chronic diseases, self-perception of the risk of contagion, and the general context of the pandemic, among others ( items). in the second section, the potential attributes of the vaccine and the context of contagion risk were presented; that is, we described the contingent market and asked about the wtp and the protest responses of individuals who are not willing to pay due to economic or moral reasons ( items). in the third section, the respondents were sociodemographically characterized according to age, gender, educational level, income, household composition, employment status, and health system ( items). the statistical validation of the instrument was performed with the traditional reliability indicators of the qualitative scales used. the cronbach's alpha indicator was . , indicating that there was internal consistency of the items of the perception scale (qualitative). the wtp measures the change in well-being as a result of the hypothetical acquisition of the covid- vaccine, and is expressed mathematically as follows: , where is a matrix of variables or =´+ characteristics of the individual observable under the hypothetical scenario with vaccine, is composed of the covariates in the scenario without vaccine (current scenario), such that: ; is the vector of = parameters that indicates the dependence of the wtp on the exogenous variables (x); and is the difference of the random components in both scenarios: which has a normal distribution, we used the relationship between wtp and its determining variables (x) to predict the probability of payment for the vaccine. this was obtained by comparing a given initial value with others that were above or below. these values were defined by the payment vector, such that: where and are the upper ( ) and lower ( ) limits of the wtp, and epsilon ( ) is the threshold of change between these that define the range of the wtp. it should be noted that wtp values were obtained from an open-format pre-survey of individuals. with these values, the distribution model of the payment ranges with equal selection areas was applied, assuming a normal distribution [ ] . this is an iterative technique that allowed us to find the minimum mean square error of the design of the payment vector, which in our case was limited to four initial values. with these values, the upper and lower payment vector of each of them were obtained at the same time, as the average of the contiguous values, which are presented in table and are used in equation ( ). the estimation of the wtp was made through a probabilistic model in which the dependent variable ( ) is a dummy that takes the value of if the individual is willing to pay and otherwise, depending on t he value of the assigned payment vector and the covariates that allow controlling for factors that may affect the wtp. thus, the probability of paying is: , where is a ( finite category selected by the individual under the hypothetical scenario, is a specific threshold for that category, is the probability of paying, and represents the standard normal density function, that is, we (.) defined a probit model. the estimations were made with the maximum likelihood method under the assumption that the errors distribute normally. through the analysis of the marginal effects we could identify the covariates or predictors of wtp for the covid- vaccine, which are determined as: these are the derivatives of the probability of paying a fixed value, given a change in a continuous explanatory variable ( ). subsequently, with the estimation of the vector ( ), the average value of the wtp per individual was obtained. the previous dichotomous method was applied to estimate two types of different formats: simple and double [ , ] . the simple format only considered the first response of the individual on whether ("yes" or "no") a given amount of money is paid that represents the value of the good. with this format, lower rejection rates are obtained, and the possibilities of guessing the response, the starting point bias and the induction of responses are reduced. however, this format has the disadvantage that it is a discrete indicator of the actual wtp and that the selection of the functional form can affect the results [ , ] . the double format solves this problem as the individuals are first asked whether they would pay a given amount, and according to the answer, they are offered a lower or higher value alternative. thus, the wtp is found in four types of possible intervals ("yes-yes", "yes-no", "no-no", and "no-yes"). with (table a ) . regarding the individual circumstances and perceptions of the covid- pandemic, most individuals considered that they were well informed about the disease caused by sars-cov- ( . %), most of them would be willing to pay for a vaccine ( . %), they believed that they will get sick ( . %), and only . % have or have had covid- . most individuals were quarantined at home ( . %), although most would prefer to work outside the home ( . %), which could be explained by the fact that they believed that it is "impossible to work with children" ( . %). regarding the context (table a ) the estimates of the models of discrete choice were made with two different groups of covariates. model contained the most relevant explanatory variables to predict the wtp for the covid- vaccine and model included the same variables as model plus the characteristics of the individual, which in conventional literature tend to be significant [ , , ] . this was done to avoid the statistical problem of omitted relevant variables [ ] . considering the bid vector estimates presented in table valor-p (pr > chi ) . . standard errors in parentheses = + p< . , * p< . , ** p< . , *** p< . . two observations from the sample (n= ) were dropped in the estimation process and were not considered because they were rejection responses. on the other hand, the variables that negatively influenced the wtp for the covid- vaccine were health care ( ), inability to work from home with children and △ - , %, ≤ . ( △ - , %, ≤ . ) covid- recovery . the first variable indicated that having a private health system ( △ - %, ≤ . ) that costs health expenses reduces the wtp for prevention measures by . % ( ). the second ≤ . variable showed that the negative perception of working from home with children, due to preventive or mandatory quarantine, could make the person less exposed to contagion; therefore, the wtp for a vaccine will be less ( . %, ). finally, the third variable indicated that those who have had covid- and △ -≤ . recovered, believe they have obtained greater immunity to the disease and that their risk of dying or worsening is less, so they would be less willing to pay for a vaccine ( %, ). it should be noted that both models have a good statistical fit. the goodness-of-fit, measured as the ability of the estimates to adequately predict the observed data, was high, as the probability predicted by the models was more than % (table ). the chi test indicated that the variables were significant together, that is, it allowed us to reject the null hypothesis that the regression parameters are zero, with a confidence level of % ( . = . ) of the sample, individuals ( . %) indicated that they were not willing to pay for a covid- vaccine (table a ). the self-declared reasons why they would not pay are presented in fig. . this shows that the main reasons for not paying are because they believe that the government should finance the cost of the vaccine ( . %) or they do not have the resources available to do so ( . %). the latter are individuals who may have a positive evaluation of the vaccine, but their budget constraint does not allow them to pay for it. in general, people would be willing to pay for a covid- vaccine, as . % of the individuals answered "yes" to the initial question of whether they would pay. however, this question is ambiguous because there could be people in that group who would only pay a penny. to solve this, the questionnaire had a double dichotomous design, which randomly presents values of the payment vector and reveals the true preferences of the individual. of the sample, % individuals answered that they would pay the initial value and that they would also pay a second value, higher than the first; whereas % answered "yes" to the first value, but "no" to the second higher value. however, the double format model is considered more appropriate in the technique because it is more efficient in estimating the variance of the parameter, therefore, its confidence interval is also more efficient [ , ] . this was evidenced through the difference between the upper and lower confidence interval values, which was smaller for the double dichotomous model ( . %, . , table ). additionally, when extrapolating these results to the population of legal age (over years), approximately million, and discounting the percentage of responses rejecting payment for the vaccine ( . %), it leaves a population of slightly more than million people. this value was multiplied by the estimated wtp that reached $ . per individual, which gives us the social assessment of us$ , million for the covid- vaccine, which represents . % and . % of chile's gross domestic product (gdp) and gdp per capita, respectively. the individual and social assessment of the covid- vaccine is key to defining prevention strategies, and allows visualizing the perceived benefit of the investment that research laboratories could have if they develop a vaccine, which is important considering the current global r&d activity focused on creating one [ ] . this study shows that there is a high individual wtp, with an average of us$ . under the most accurate estimation technique and with a reliability level of . %. these results are similar in magnitude to those found by [ ] for the meningococcal b vaccine (us$ . = au ) in australia. however, the values are higher than those found in other studies conducted with other serious diseases with risk of death [ , , ] . specifically, the evaluation of the covid- vaccine was far superior to that of the hepatitis b vaccine in malaysia (us$ [ ] ), zika in brazil (us$ . [ ] ), dengue in malaysia (us$ . [ ] ) and ebola in indonesia (us$ . [ ] ). this are upper/middle-income economies, except for malaysia, which is a low/middle-income country [ ] -thus, the high economic valuation of the covid- vaccine could be explained because sars-cov- has had a higher contagion rate, has spread more rapidly and has affected all countries in the world [ ] . the country's high per capita income is also a contributing factor. in fact, we found a relatively high vaccine acceptance rate ( . %), considering that other studies have found acceptance ranges that go from . % to . % [ , , ] . on the one hand, there could be a tendency in individuals to accept a payment or to say "yes" when they have less-formed preferences, which according to [ ] tends to occur with health-related goods and services. this could also explain the high approval rate with the payment vector thresholds presented. on the other hand, it could also be that individuals foresee a high risk of getting sick ( . %) and therefore would be more willing to pay, which has already been pointed out by other studies [ ] . this, especially considering that covid- could be perceived as "catastrophic" due to the health costs and the increased risk of death of vulnerable people (older adults and people with pre-existing chronic diseases), according to the results of [ , ] ; in addition to the high social and economic cost of this disease [ ] . furthermore, we precisely demonstrated that one of the main variables that determine the wtp is the pre-existence of chronic diseases, the level of knowledge of covid- and having covid- . thus, both the high approval rate for the vaccine ( . %) and the belief that one will eventually get sick ( . %) demonstrate a positive intention of individuals towards it, even without knowing the details of its real effects on health. this is an important argument against the so-called "vacillation" which, according to [ ] , becomes a rejection of vaccination. in other words, our results indicate that there are fewer people against vaccination in the case of covid- . additionally, we found that perception of government performance in managing the pandemic also influences the wtp. this variable had not been considered in previous studies related to vaccine evaluation, such as those carried out by [ , , ] . this is an important variable because the information provided by the government on the negative effects of covid- and the strategies applied to mitigate the pandemic (such as restriction on mobility or quarantine), are key to educating the population and affect the wtp for the vaccine. in fact, there are studies that indicate that education, information and communication can improve the willingness to vaccinate against respiratory viruses [ ] . therefore, it is important that the government executes credible measures, informs and educates clearly about the impact that sars-cov- contagion generates. it should be noted that the results of this study would be important to consider if the vaccine were to be introduced in a different country in the future, as they provide information to target available economic resources and many countries have budgetary constraints to deal with the sars-cov- health emergency. the results are applicable to countries that have mixed health systems (public and private provision) and that are based on copayments, such as some countries in north america and europe. in addition, considering that income is one of the important factors for the wtp for a vaccine, it is proposed that the government or the authorities in charge of public health carry out free covid- vaccination campaigns, especially for people with lower incomes, leaving private provision to households with higher incomes. this last strategy is endorsed by the literature [ ] . in this study, we found a high social and individual valuation for a covid- vaccine. the average value to pay per individual was us$ . , considering a discrete choice model in double dichotomous format, which implied a social valuation of approximately us$ , million. the variables that positively impacted the wtp were the pre-existence of chronic diseases, knowledge of covid- , being sick with covid- , perception of government performance, employment status, and income. the variables that negatively affected the wtp were belonging to a private health system, non-adaptation to working from home with children (due to quarantine) and having recovered from covid- . these latter variables could be used to define strategies for public health policy intervention to confront the covid- pandemic. additionally, if the vaccine will become a "public good" globally, there would still be costs associated with production and distribution, and the laboratory that develops it should be financially compensated. therefore, the wtp results from this study can serve as a compensation benchmark for vaccine developers. conceptualization, ac; methodology, formal analysis investigation, resources, writing-original draft preparation, ac. and lg; writing-review and editing, lg. all authors attest they meet the icmje criteria for authorship. this research received no external funding. the authors declare no conflict of interest. the covid- vaccine development landscape contingent valuation in practice willingness-to-pay for a hypothetical ebola vaccine in indonesia: a cross-sectional study in aceh willingness to pay for hepatitis b vaccination in selangor, malaysia: a crosssectional household survey consumer willingness to pay for a hypothetical chikungunya vaccine in brazil and the implications the acceptance and willingness to pay (wtp) for hypothetical dengue vaccine in penang, malaysia: a contingent valuation study adolescent, parent and societal preferences and willingness to pay for meningococcal b vaccine: a discrete choice experiment. vaccine parents willingness to pay for a human papillomavirus vaccine to protect their adolescent daughters mothers' preferences and willingness to pay for human papillomavirus vaccination for their daughters: a discrete choice experiment in hong kong world bank country and lending groups contingent valuation introduction to contingent valuation using stata systematic review of willingness to pay for health insurance in low and middle income countries error by omitted variables in regret-based choice models: formal and empirical comparison with utility-based models using orthogonal design data. transportmetrica a: transport science consumer willingness to pay for a hypothetical zika vaccine in brazil and the implications evolution of severe acute respiratory syndrome coronavirus (sars-cov- ) as coronavirus disease (covid- ) pandemic: a global health emergency valuing health care using willingness to pay: a comparison of the payment card and dichotomous choice methods covid- : risk factors for severe disease and death risk factors for disease severity, unimprovement, and mortality in covid- patients in wuhan, china attitudes to vaccination: a critical review knowledge, attitudes, and behaviors (kab) of influenza vaccination in china: a cross-sectional study in scope and magnitude of private sector financing and provision of immunization in benin, malawi and georgia. vaccine key: cord- - vlxa i authors: williamson, e. d.; westlake, g. e. title: vaccines for emerging pathogens: prospects for licensure date: - - journal: clin exp immunol doi: . /cei. sha: doc_id: cord_uid: vlxa i globally, there are a number of emerging pathogens. for most, there are no licensed vaccines available for human use, although there is ongoing research and development. however, given the extensive and increasing list of emerging pathogens and the investment required to bring vaccines into clinical use, the task is huge. overlaid on this task is the risk of anti‐microbial resistance (amr) acquisition by micro‐organisms which can endow a relatively harmless organism with pathogenic potential. furthermore, climate change also introduces a challenge by causing some of the insect vectors and environmental conditions prevalent in tropical regions to begin to spread out from these traditional areas, thus increasing the risk of migration of zoonotic disease. vaccination provides a defence against these emerging pathogens. however, vaccines for pathogens which cause severe, but occasional, disease outbreaks in endemic pockets have suffered from a lack of commercial incentive for development to a clinical standard, encompassing phase iii clinical trials for efficacy. an alternative is to develop such vaccines to request us emergency use authorization (eua), or equivalent status in the united states, canada and the european union, making use of a considerable number of regulatory mechanisms that are available prior to licensing. this review covers the status of vaccine development for some of the emerging pathogens, the hurdles that need to be overcome to achieve eua or an equivalent regional or national status and how these considerations may impact vaccine development for the future, such that a more comprehensive stockpile of promising vaccines can be achieved. globally, there are a number of emerging and re-emerging pathogens. some of these cause endemic disease in regions of the globe, where they are maintained in zoonotic reservoirs and transmitted to man either by direct or indirect contact. for most of the emerging pathogens there are no licensed vaccines available for human use, although there is ongoing research and development. however, given the extensive and increasing list of emerging pathogens and the time and investment required to bring vaccines into clinical use, the task is huge. overlaid on this task is the risk of anti-microbial resistance (amr) acquisition by micro-organisms which can endow a relatively harmless organism with pathogenic potential. furthermore, climate change also introduces a challenge by causing some of the insect vectors and environmental conditions prevalent in tropical regions to begin to spread out from these traditional areas, thus increasing the risk of migration of zoonotic disease. vaccination provides a defence against these emerging pathogens. however, to date, vaccines for pathogens which cause severe, but occasional, disease outbreaks in endemic pockets have suffered from a lack of commercial incentive for development to a clinical standard. while approval of vaccines for diseases caused by such pathogens would clinical and experimental immunology review article series editor: e diane williamson make a significant impact on disease outbreaks, taking niche vaccines into clinical development, including phase iii clinical trials for efficacy, requires a large investment in time and money. an alternative is to develop such vaccines to request us emergency use authorization (eua), or an alternative status in the united states, canada and european union (eu) making use of a considerable number of alternative regulatory mechanisms that are available prior to licensing, so that the products are deployable at the first indications of a disease outbreak. this review covers the status of vaccine development for some of the emerging pathogens, the hurdles that need to be overcome to achieve eua or an equivalent regional or national status and how these considerations may impact vaccine development for the future, such that a more comprehensive stockpile of promising vaccines can be achieved. pathogens which are classed as emerging or re-emerging are identified through global surveillance programmes and organizations such as the world health organization (who). although labelled 'emerging', most of these pathogens are not new and have either been quiescent in the environment until the conditions are opportune to emerge or have evolved from a parent organism to adapt to the prevailing conditions. thus, there is an intricate relationship between the environment, the climate, wildlife and human existence and lifestyle. the coronaviruses exemplify this point: the ancestral virus possibly existed approximately years ago [ ] . coronaviruses have a wide species range infecting birds, bats, chickens, pigs, dogs, cats and rodents [ ] . however, the first human coronavirus was described only in the s [ , ] , and the coronavirus causing severe acute respiratory syndrome (sars) was discovered only in [ ] [ ] [ ] , while that causing middle east respiratory syndrome (mers) first emerged in [ ] . it is likely that warm-blooded flying birds and bats have co-evolved with the coronaviruses to aid dissemination [ ] . for example, sars is thought to have first infected old world bats, then spreading to horseshoe bats [ ] , civets and finally to man [ ] . in the outbreak of sars in china and adjacent countries, phylogenetic analysis suggested that the virus spread from bats to humans, possibly through the intermediary civet species. neither bats or civets showed any clinical signs of infection, and it is thought that bats are the main zoonotic reservoirs for the virus [ ] . organizations such as the who, national institutes for allergy and infectious diseases (niaid) and the us centers for disease control (cdc) publish lists of emerging pathogens which may be viral, bacterial or rickettsial in nature. the who priority list contains viruses which have been prioritized as the most likely to cause epidemics and for which the who will establish a blueprint programme for accelerated research and development (r&d) [ ] . the list published in february is shown in table . niaid [ ] and cdc [ ] also publish lists of pathogens of priority which comprise bacteria and viruses, but categorize these into three groups depending on pathogenicity, accessibility and the availability of vaccines and therapies. all the viruses listed by the who also occur on these lists, alongside bacterial pathogens of concern. one of these is yersinia pestis, causative of bubonic and pneumonic plague, which is recognized by all three bodies (who, cdc, niaid) as a current priority following the exceptionally large and serious outbreak between september to april in madagascar [ ] , where the disease is endemic. in addition, niaid recognizes the added threat to human health posed by the acquisition of amr by pathogens and who has also published a priority list [ ] of bacterial species for which r&d is required to develop new antibiotics (table ) . globally, there are many regions of endemic disease which are maintained by reservoirs of zoonotic pathogens in the local wild animal species. these pathogens comprise viral, bacterial and rickettsial species and some have complex lifecycles, infecting an environmental (e.g. standing water) or animal reservoir and then being transmitted either by direct contact with man or indirectly, via an insect (e.g. mosquito) or mammalian (e.g. bat, civet, camel) vector, to man, from which they may be spread by human-tohuman transmission. (fig. ). an example of a serious and widespread bacterial infection is mosquito-transmitted plasmodium falciparum, causing malaria in many tropical regions. malaria causes significant morbidity and co-morbidity in these regions in which it is endemic. due to the complex life cycle of the causative bacterium, it has been challenging to achieve a vaccine for malaria. the most advanced candidate is the rts,s/as vaccine [ ] , which is undergoing a pilot implementation in three countries in sub-saharan africa, with a view to determining its impact on disease prior to a wider implementation [ ] . despite the availability of approved vaccines [ , ] , typhoid fever and cholera remain significantly debilitating enteric diseases in regions of the world where hygienic living conditions are poor and there is little access to health care. both infections are caused by bacteria (salmonella typhi and vibrio cholera, respectively) which exist in contaminated water and food. tuberculosis (tb) is a respiratory infection which is widespread globally. caused by mycobacterium tuberculosis, and transmitted to man from zoonotic reservoirs (badgers, cattle) with a high potential for subsequent human-to-human transmission, tb is prevalent in susceptible individuals living in overcrowded conditions [ ] . although the bcg vaccine has been in routine use for many years, variable efficacy has been reported, depending on region of use [ , ] with - % reported in the united kingdom but lower levels in equatorial countries. the emergence of multi-drugresistant tb (mdr tb) in recent years raises the bar for treatment of this disease and makes a high level of vaccine efficacy even more important [ ] . examples of zoonotic reservoirs which maintain endemic viral diseases are numerous and are summarized in table . in particular, viral endemic disease is caused by the coronaviruses (mers and sars) in saudi arabia and the eastern mediterranean countries; and by the lassa arenavirus, which is endemic in west african countries and caused a serious outbreak of lassa fever in nigeria in [ ] ; other viruses which are endemic and cause viral haemorrhagic fever include dengue, which is widespread in tropical and subtropical regions worldwide [ ] , the filoviruses (ebola and marburg) which have a zoonotic reservoir in bats in sub-saharan africa [ ] ; and yellow fever virus, which is prevalent in africa, central and south america and the caribbean [ ] . interestingly, the same mosquito (aedes aegypti) which spreads yellow fever virus also transmits the dengue, chikungunya and zika viruses [ ] . chikungunya virus has a widespread distribution in africa, asia, india and south america and with occasional cases in europe, and causes a debilitating, but rarely fatal, disease [ ] . zika virus emerged in brazil in [ ] , although it was first detected in monkeys in uganda as early as [ ] , and the first documented human case occurred in nigeria in [ ] . the large brazilian outbreak of zika viral disease culminated in , with thousands of cases reported [ ] . rift valley fever virus (rvfv) is another zoonotic virus which is endemic in sub-saharan africa and primarily infects cattle, sheep and goats, but can be transmitted to man by mosquito bite to cause an acute fever [ ] . an example of a bacterium which has been classed as a re-emerging pathogen and which is endemic in global regions is y. pestis, causative of plague. bubonic [ ] [ ] [ ] . over thousands of years, y. pestis has evolved away from the enteric yersinia species to become a lethal flea-transmitted bacterium [ ] . it is transmitted to man, typically from a zoonotic reservoir in infected rats or other rodents (e.g. ground squirrels or prairie dogs), by flea-bite to cause bubonic plague [ ] (fig. ). if not detected and treated, this can develop into either septicemic plague or the most serious form of all, a secondary pneumonic plague. in turn, individuals with pneumonic plague can transmit this by aerosol droplet to others, to establish a primary pneumonic plague infection. each year, a few cases of plague are also reported in the southwestern united states, where the disease has been endemic in the rodent population since the late s [ ] . as well as infecting the rodent population, infected fleas can spread y. pestis to other wildlife species (e.g. rabbits/hares or, rarely, domesticated animals [ ] ) which, in turn, raises the potential of transmission to man by inhalation to cause a primary pneumonic plague. in endemic areas, outbreaks of plague are often associated with seasonal environmental changes, causing rodents to stray closer to human habitation. the plague outbreak in madagascar during / was particularly serious, with an estimated cases and deaths ( · % fatality rate) [ ] . this outbreak was approximately sixfold greater than usual, with an unusual predominance of pneumonic, rather than bubonic plague. although y. pestis is susceptible to antibiotics, such as the aminoglycosides (gentamycin, streptomycin), the fluoroquinolones (e.g. ciprofloxacin) or tretracyclines (e.g. doxycycline) [ ] , these need to be administered very early to a suspected plague case, and ideally before symptoms emerge. additionally, there have been reported instances of antibiotic resistance including to multiple antibiotics [ , ] . hence, there is a clear and increasingly urgent need for an efficacious vaccine. other bacterial endemic diseases include melioidosis and glanders which, although not caused by zoonotic pathogens, are endemic in southeast asia where the bacteria reside in soil and are transmitted to humans through occupational exposure, e.g. working in paddy fields [ ] . another bacterial disease which is endemic in the northern hemisphere is tularaemia, caused by the bacterium francisella tularensis, which has zoonotic reservoirs in the rabbit, hare and rodent populations in the south, central and western united states and is transmitted to man by ticks and biting flies [ ] . rickettsial species, such as coxiella burnetii, causative of q-fever, comprise bacteria which exist within another cell, and as such q-fever is contracted when humans are exposed to aerosolized droplets from the urine, milk, faeces or birth detritus from infected sheep, goats and cattle [ ] . for all these pathogens, there is a requirement for efficacious approved vaccines to curtail or prevent regular disease outbreaks. for some of these pathogens (e.g. rvfv, cchf) there are vaccines [ ] , but these are not widely available [ ] or have been used and withdrawn for safety or regulatory reasons (e.g. the live vaccine strain for tularaemia) [ ] or they are not universally suitable, requiring a screening test prior to administration due to the potential for a hypersensitivity response (as for the vaccine for q-fever) [ ] . there is no readily available licensed plague vaccine, although a series of killed whole cell vaccines (kwcv) has been produced and used in the past, mainly in the biodefence context (reviewed in [ ] ). additionally, live attenuated plague vaccines, derived from an attenuated mutant strain as the ev series, have been used in the former ussr (fussr) and are still used in asia, notably in russia and china [ , ] . ev , the most commonly cited of these, provides protective efficacy against plague, but is licensed for human use only in countries of the fussr and is documented to cause serious adverse effects in non-human primates and malaise and adverse effects in human vaccinees [ ] . whatever the context, all these diseases would be positively impacted by the availability of efficacious and approved vaccines. however, to a greater or lesser extent they are all niche diseases with no major commercial incentives to drive vaccine development programmes. this is a space that non-governmental organizations (ngos) such as the coalition of epidemic preparedness innovations (cepi) [ ] and global vaccine alliance (gavi) [ ] have entered and they are supporting vaccine efforts for some of the diseases listed above. additionally, philanthropic funders such as the gates foundation are supporting vaccine r&d efforts [ ] . in the united kingdom, and subsequent to the ebola outbreak in west africa, vaccine networks for human and veterinary vaccines have formed to prioritize vaccine efforts in these respective contexts [ ], while the department of health, together with innovate uk, has supported r&d of vaccine candidates for the prioritized pathogens [ ] . as a result of these global initiatives, a number of candidate vaccines are being developed for emerging bacterial and viral pathogens, examples of which, although by no means exhaustive, are cited here [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . who reports ongoing global vaccine r&d efforts [ ] and also tracks the progress of clinical trials for emerging pathogens [ ] . here it may be worth drawing a distinction between prophylactic vaccination, i.e. general use prophylaxis (gup), given routinely and not necessarily in the face of specific, predicted outbreaks, in contrast to post-exposure prophylactic (pep) vaccination, to be given after a suspected exposure to a pathogen, or to ring-fence an outbreak or, indeed, post-exposure therapeutic (pet) vaccination, to be given after an actual exposure. in the pep and pet contexts, the benefit of vaccination vastly outweighs the risk of disease (i.e. the risk : benefit ratio is low), while in the prophylactic context the risk : benefit ratio may be greater than, or equal to, · ). for infections with short incubation times, e.g. less than h, as for pneumonic plague, pep or pet vaccination may only be useful if administered under antibiotic cover. in the united states, once a vaccine candidate has been thoroughly tested for safety in non-clinical models, and in an escalating-dose, statistically powered, phase i design in the clinic, from then on it may be possible to pursue approval for an eua, rather than pursue the full-length pathway to biological licensing authorization (bla). this can enable the earlier availability of vaccines for use in endemic regions. however, it should be noted that eua is only available through the food and drug agency (fda) in the united states. some alternative regulatory mechanisms that may be considered prior to licensing are outlined below. from the discovery phase to the clinic, vaccine development requires the completion of a series of steps represented as a generic outline in fig. . the regulatory agencies lay down specific guidance for these steps [ , ] and this review presumes no authority in this regard, but seeks to give a generalized overview of a generic vaccine development process. exit from the discovery and preclinical phases requires substantive data demonstrating immunogenicity and efficacy in at least one suitable animal model(s). where possible, efficacy in the animal model should be demonstrated by direct exposure to the pathogen concerned. technology transfer for manufacture under good manufacturing practice (gmp) will require a demonstration of known provenance of all essential materials required in the manufacture of the vaccine candidate. this includes seed stocks of cell lines from which recombinant proteins may be expressed, e.g. escherichia coli, human embryo kidney cells, baculovirus, tobacco mosaic virus; or seed stocks of attenuated vaccine vectors, e.g. viral vectors such as adenovirus, vesicular stomatitis virus, modified vaccinia ankara; or bacterial vaccine vectors, e.g. salmonella, listeria; the genetic constructs cloned into the cell line in question; all culture media and components and all formulations and excipients. transfer of the manufacturing process to gmp may include scale-up and conversion to, for example, fermentation conditions, or to plant-based, mammalian cell line or insect cell line expression on an expanded scale. this transfer will probably require the demonstration of consistency between consecutive batches at gmp, which will also enable the development of scaled-up downstream processing methodology. vaccine components (the drug substance) from these batches may be formulated (the drug product) as required and used in safety/toxicology studies and for immunogenicity/efficacy in an appropriate second animal model, which will be as representative as possible of the human response. the second animal model is often, but not necessarily, a non-human primate, and the selection of this second model will depend entirely on the vaccine indication. there will also be a requirement to generate sufficient stability data on both the drug substance and the drug product and to demonstrate that the drug product is stable for at least the duration of the intended phase i clinical trial under prevalent conditions in that location. clearly, extended stability testing under a range of conditions, including accelerated conditions (high ambient temperature and relative humidity), will also be required to progress the vaccine through development. as well as determining its stability, both the drug substance and product will require characterization for properties such as identity, purity, isoelectric point, osmolality, endotoxin levels and potency, to demonstrate consistency between batches and to allow for release of these for clinical trials. it is essential to ensure safety of the drug product before entering a clinical trial, and the drug product may be tested in suitable small animal models for the absence of adverse effects under conditions of repeated dosing at the anticipated human dose level, as well as in the intended human schedule, but at multiples of the anticipated human dose-level. the use of rodent models (mouse or rat) for this testing allows for sufficient statistical powering of such safety/toxicological testing. if the clinical trial is to be conducted in women of child-bearing age, it may be necessary to carry out reproductive toxicology testing of the drug product; in this case it may be necessary also to use a sensitive rabbit model and to administer the vaccine to pregnant rabbits to screen for adverse effects in the mother and potential teratogenic effects in the f generation. the national regulatory authority will expect to review the existing safety data and outlines of further protocols to be used. a review of all the data pertaining to the candidate vaccine will be required by the regulators in order to proceed to a clinical trial. depending on the specific requirements of the regulatory authorities in the country of origin/manufacture of the vaccine and also the intended location of the clinical trial, this may take the form of an investigator's brochure, protocol and summaries of manufacturing, non-clinical and clinical data in a clinical trial application (for the european medicines agency, ema) or an investigational new drug (ind) application (e.g. us fda). other international regulators (e.g. in canada, japan and china) will have variations on the documentation required. in the united states, the alternative approaches to full marketing approval that are available for vaccines under development and which may be considered through the fda are to either request an eua or to request expanded access (ea), sometimes called compassionate use. the eua is issued in support of potential and actual public health, military and domestic emergencies involving chemical, biological, radiological and nuclear agents (cbrn), including emerging infectious diseases, e.g. pandemic influenza. such fda-approved medical products which may be stockpiled for use in emergencies are referred to as medical countermeasures (mcm) and include biological products, e.g. vaccines, drugs and devices. specifically, the eua authority is separate and distinct from use of an investigational medical product held under an ind and must be able to treat serious or life-threatening diseases or conditions. in contrast, ea submissions are for products used under an ind or a protocol (treatment plan), or submitted as a protocol amendment to an existing or new ind. the ea categories cover use under either an individual patient ind or protocol, individual emergency ea use, or ea for an intermediate-size population or for widespread use (large populations). other us regulatory programmes for biologicals and drugs to treat serious or life-threatening conditions include fast-track designation, breakthrough therapy designation, accelerated approval pathway and priority review designation. in canada, access to drugs through special access programmes (saps) exists for serious and life-threatening conditions, when marketed alternative products are not available or unsuitable and evidence supports the intended use. priority review of marketing submissions and 'notice of compliance with conditions' for such products may also be considered and granted by health canada. in the european union, the ema supports early patient access to new medicines and which are eligible for a marketing authorization application under the centralized procedure and either target unmet medical needs or those which have a major public health interest. the ema has launched a priority medicines (prime) scheme to facilitate early dialogue and support the development of medicines that target unmet medical needs and which promotes an accelerated regulatory assessment. in addition, the prime scheme is intended for seriously debilitating or life-threatening diseases, compassionate use of unauthorized medicines for patients with an unmet medical need (when no satisfactory treatment is available in the european union) and also provides conditional marketing authorization. the regulation and rules of access to compassionate use programmes varies between national government authorities across eu member states. in the united kingdom, the early access to medicines scheme (eams) gives patients with life-threatening or debilitating conditions access to innovative medicines without marketing authorization, but for which there is a clear medical need. a two-stage evaluation process initially considers promising innovative medicine (pim) designation before an eams scientific opinion. also in the united kingdom, the potential supply of unlicensed medicinal products (specials) may be considered by the medicines and healthcare products regulatory agency (mhra) for the importation of medicines that have marketing authorization status from countries outside the united kingdom and european union. where there is an unmet need of priority to the who for a new vaccine, the who may establish a blueprint programme [ ] . as vaccine candidates for the specific blueprint programme advance through the development process, and particularly when they have attached clinical data, the who may run a prequalification check to determine whether the candidate meets their requirements on behalf of un agencies that will ultimately purchase vaccines [ ] . to do this, who will engage with subject matter experts to draft and publish a target product profile (tpp) for a vaccine requirement. who will use its scientific advisory group of experts (sage) to review vaccine candidate performance against the tpp and to prequalify a vaccine candidate(s) which fulfils the tpp. assuming that the preclinical safety and toxicology testing has been satisfactory, a protocol for phase i testing of the vaccine candidate may be submitted to the regulatory authorities for approval. the primary objective of the phase i clinical trial is to test the vaccine candidate for safety in informed and consenting adult volunteers who will be selected according to pre-agreed inclusion/exclusion criteria. as this is the first time the candidate is being used in man, the phase i trial is typically small and cautious. if a range of dose-levels is being tested, it may be necessary to dose a sentinel group of single subjects from each arm of the study to assess safety through week of immunization and starting at the lowest level before proceeding to the next level. the data from the sentinel group would be reviewed before proceeding with the main study. then the first cohort would be dosed at the lowest level before proceeding to the next level, and so on. volunteers will be closely monitored for adverse effects in the clinic at each dosing time-point and will typically maintain a diary at home to record any symptoms arising between time-points. an independent safety monitoring panel, including medically qualified personnel, will be required to monitor the reporting of adverse effects. volunteers may also be blood-sampled to assess vaccine immunogenicity from baseline and serial samples, and depending on vaccine type may be required to supply additional samples which can be collected non-invasively, e.g. saliva/stool samples. all volunteers may be followed up for a pre-agreed period on completion of the study, to check for latent adverse events and to monitor, e.g. for maintenance of circulating antibody titres or memory response to the vaccine. the second phase of clinical trials typically allows for an enlarged study to assess vaccine safety and to monitor immunogenicity. during this phase, significantly expanded cohorts of volunteers may be dosed with vaccine at doselevel(s) selected as optimum from the phase i trial and can be monitored for safety and a more detailed immunogenicity assessment, which may include assays of both serological and cellular memory responses. once again, volunteers may be blood-sampled for baseline and serial serum/plasma samples, and depending on vaccine type may be required to supply additional samples which can be collected non-invasively, e.g. whole blood/saliva/stool. in the event that a phase iii trial for efficacy is not feasible, for ethical or practical reasons, it may be necessary to plan a blood-sampling regimen in the volunteers which will enable sufficient sample volumes to be available for animal rule studies (discussed below). if sufficient safety data are gained from a phase ii trial, the regulatory authorities may be able to consider the vaccine for eua. the third phase of clinical trials is typically designed to assess efficacy. however, where this is not feasible for either practical/logistical or ethical reasons, phase iii could be regarded as a further, significantly enlarged trial for vaccine safety with adequate follow-up of vaccinated volunteers. a phase iii field trial of vaccine efficacy may be feasible in the event of an anticipated seasonal outbreak, in which case vaccination could be given prophylactically and well in advance. as long as effective biosurveillance programmes are in place to instigate a rapid response to new infections in an unvaccinated placebo cohort, the latter group may be included in a prophylactic efficacy trial. conversely, reactive mode vaccination in the context of an actual outbreak is likely to be more complicated, as it would be unethical to omit anyone at risk of infection from the vaccination programme. hence, sufficient safety data on the candidate vaccine would be required in case of need to administer it to pregnant/nursing mothers, children and elderly people as well as to the general adult population. additionally, it may be necessary to administer an adjunct to the vaccine, such as an appropriate antibiotic, to both cohorts (vaccinated and placebo). whatever the context, careful consideration of the trial design needs to be made and approved by the regulators to achieve adequate statistical powering and to determine the need to provide supplementary antimicrobial therapy, and also the need for a placebo cohort. after the anthrax letters incident in the united states in in which anthrax was released through the postal system, resulting in five fatalities and widespread anxiety [ ] , the fda issued the animal rule (fda cfr) [ ] to expedite the development of vaccines and therapies for biothreat agents for which it is neither feasible nor ethical to carry out phase iii efficacy studies in man. in this situation, the animal rule makes provision for the substitution of animal efficacy data for human efficacy data. the animal efficacy data should demonstrate a reasonable likelihood that the candidate vaccine or therapy would be efficacious in man [ ] . the animal efficacy data should be provided ideally from more than one animal model, or from one model only if that model is well-characterized and authentically represents the human disease syndrome [ ] . in addition, the fda may require supporting data to include pharmacokinetic/pharmacodynamic (pk/pd data) and a determination of the pathophysiological mechanism of action of the biothreat agent and a 'reasonable understanding' of the protective mechanism of the proposed vaccine or therapy. thus, if the candidate is a vaccine, a reasonable understanding of the protective mechanism(s) being invoked by it requires an identification of the immune correlates of protection. immune parameters which correlate with protection in the selected animal models may include the total titre of circulating antibody induced to the vaccine and the determination of a minimum cut-off titre required to confer protection. alternatively, the titre of functional or neutralizing antibody within that total may provide the correlate, particularly where the neutralization of specific virulence factors produced by the biothreat agent is identified and quantifiable [ ] . for some vaccines, the induction of a cellular memory response instead of, or in addition to, circulating antibody will provide the correlate. this is measurable by the ex-vivo recall response of peripheral blood mononuclear cells (pbmcs) on stimulation with the vaccine antigen(s) [ ] . in the absence of direct efficacy testing in the human vaccinee, the immune correlate identified above provides a surrogate marker of efficacy. if this is a functional antibody, the titre induced in man needs to be compared with the titre required in the animal model(s) to provide protection. this can be achieved, for example, by the in-vitro neutralization of a specific virulence factor, or by a competitive enzyme-linked immunosorbent assay (elisa), where the test antibody is competed with a known neutralizing antibody for binding to the target antigen or by the passive transfer of antibody from a human vaccinee into a naive animal, followed by pathogen challenge [ ] . whatever the immune correlate may be, its effect would be expected to follow the pattern shown in fig. , where as the value increases, the likelihood of death in the vaccinee decreases [ ] . thus, vaccine efficacy = relative reduction in risk of death = − (% of vaccinated subjects at < protective level) (% of unvaccinated subjects at< protective level) . although a number of immunotherapies and pretreatments have been licensed under the animal rule, the first vaccine to be licensed in these circumstances is biothrax for therapeutic vaccination in suspected exposure to anthrax [ ] . a case for the ancient origin of coronaviruses identification of alpha and beta coronaviruses in wildlife species in france: bats, rodents, rabbits and hedgehogs cultivation of novel type of commoncold virus in organ cultures a new virus isolated from the human respiratory tract identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome the aetiology of sars: koch's postulates fulfilled the emergence of the middle east respiratory syndrome coronavirus discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats, civets and the emergence of sars review of bats and sars naiad/nih priority list of pathogens. available at cdc priority list of pathogens. available at the plague outbreak in madagascar: data descriptions and epidemic modelling who priority list of bacteria rts,s malaria vaccine efficacy and immunogenicity during plasmodium falciparum challenge is associated with hla genotype malaria vaccine implementation programme (mvip) -programme advisory group vaccines for preventing typhoid fever global economic evaluation of oral cholera vaccine: a systematic review diagnosis and treatment of tuberculosis: latest developments and future priorities efficacy of bcg vaccine in the prevention of tuberculosis variation in protection by bcg: implications of and for heterologous immunity drug-resistant tb: deadly, costly and in need of a vaccine genomic analysis offers insight into nigeria lassa fever outbreak ecological niche modeling for filoviruses: a risk map for ebola and marburg virus disease outbreaks in uganda sep yellow fever in africa and the americas: a historical and epidemiological perspective zika, chikungunya and dengue: the causes and threats of new and re-emerging arboviral diseases chikungunya: bending over the americas and the rest of the world the zika virus epidemic in brazil: from discovery to future implications zika: the origin and spread of a mosquito-borne virus zika virus outbreak of zika virus infections the pathogenesis of rift valley fever ecologic features of plague outbreak areas, democratic republic of the congo mechanism study on a plague outbreak driven by the construction of a large reservoir in southwest china (surveillance from - ) the natural history and incidence of yersinia pestis and prospects for vaccination yersinia pestis, the cause of plague, is a recently emerged clone of yersinia pseudotuberculosis plague into the st century yersinia pestis: examining wildlife plague surveillance in china and usa cat-transmitted fatal pneumonic plague in a person who travelled from colorado to arizona cdc plague treatment transferable plasmid-mediated resistance to streptomycin in a clinical isolate of yersinia pestis resistance of yersinia pestis to antimicrobial agents melioidosis in thailand: present and future tularemia transmission to humans: a multifaceted surveillance approach airborne geographical dispersal of q fever from livestock holdings to human communities: a systematic review and critical appraisal of evidence current status of rift valley fever vaccine development the use of veterinary vaccines for prevention and control of rift valley fever: memorandum from who/fao meeting protection induced by a francisella tularensis subunit vaccine delivered by glucan particles standardized guinea pig model for q fever vaccine reactogenicity plague vaccine development: current research and future trends developing live vaccines against plague russian vaccines against especially dangerous bacterial pathogens cepi new vaccines for a safer world about gavi, the vaccine alliance vaccine delivery:bill and melinda gates foundation dual route vaccination for plague with emergency use applications immunogenicity and safety of subunit plague vaccine: a randomized phase a clinical trial rift valley fever mp- vaccine phase clinical trial: safety, immunogenicity and genetic characterisation of virus isolates development of vaccines against cchf virus a monovalent chimpanzee adenovirus ebola vaccine boosted with mva effect of dengue serostatus on dengue vaccine safety and efficacy non-neutralising antibodies elicited by recombinant lassa-rabies vaccine are critical for protection against lassa fever chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice vaccine and therapeutic options to control chikungunya virus chikungunya vaccines in the pipeline a bacteriophage t nanoparticle based dual vaccine against anthrax and plague who: health products in the pipeline for infectious diseases world health organization. who vaccine pipeline tracker yjurcmg xwo kvuyedybmzdcxqbyjgdczm/pubhtml# fda guidances for vaccine development vaccines for emerging pathogens: from research to the clinic. part regulatoryinformation/guidances/vaccines/default.htm ema guideline for vaccine development who guidance for clinical evaluation of vaccines who vaccine standards: vaccine prequalification. available at the anthrax vaccine and research: reactions from postal workers and public health professionals code of federal regulations (cfr) cfr . - . evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible predictive models and correlates of protection for testing biodefence vaccines first vaccine approval under the fda animal rule the authors declare no competing interests. key: cord- -x txhjkr authors: grech, victor; gauci, charmaine; agius, steve title: vaccine hesitancy among maltese healthcare workers toward influenza and novel covid- vaccination date: - - journal: early hum dev doi: . /j.earlhumdev. . sha: doc_id: cord_uid: x txhjkr introduction vaccine hesitancy is a chronic public health threat. this study was carried out to ascertain maltese healthcare workers’ hesitancy to a novel covid- vaccine and correlate this with influenza vaccine uptake. methods a short, anonymous questionnaire was sent out to all of malta’s government sector healthcare workers via the service’s standard email services ( - / / ). a total of , questionnaires were posted electronically, with . % response. results the proportion of maltese healthcare workers who will take the influenza vaccine increased significantly. doctors had the highest baseline uptake and highest likely influenza vaccine uptake next winter. the likely/undecided/unlikely to take a covid- vaccine were / / % respectively. males were likelier to take the vaccine. doctors were the occupation with the highest projected vaccine uptake. likelihood of taking covid- vaccine was directly related to the likelihood of influenza vaccination. concerns raised were related to insufficient knowledge about such a novel vaccine, especially unknown long term side effects. discussion the increased uptake of influenza vaccine is probably due to increased awareness of respiratory viral illness. doctors may have higher vaccine uptakes due to greater awareness and knowledge of vaccine safety. the proportions of who are likely/undecided/unlikely (half, quarter, quarter respectively) to take a covid- are similar to rates reported in other countries. the higher male inclination to take the vaccine may be due the innate male propensity for perceived risk taking. shared covid- with influenza vaccine hesitancy implies an innate degree of vaccine reluctance/hesitancy and not merely reluctance based on novel vaccine knowledge gap. vaccine hesitancy is a chronic public health threat. this study was carried out to ascertain maltese healthcare workers' hesitancy to a novel covid- vaccine and correlate this with influenza vaccine uptake. a short, anonymous questionnaire was sent out to all of malta's government sector healthcare workers via the service's standard email services ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) / / ). a total of , questionnaires were posted electronically, with . % response. the proportion of maltese healthcare workers who will take the influenza vaccine increased significantly. doctors had the highest baseline uptake and highest likely influenza vaccine uptake next winter. the likely/undecided/unlikely to take a covid- vaccine were / / % respectively. males were likelier to take the vaccine. doctors were the occupation with the highest projected vaccine uptake. likelihood of taking covid- vaccine was directly related to the likelihood of influenza vaccination. concerns raised were related to insufficient knowledge about such a novel vaccine, especially unknown long term side effects. the increased uptake of influenza vaccine is probably due to increased awareness of respiratory viral illness. doctors may have higher vaccine uptakes due to greater awareness and knowledge of vaccine safety. the proportions of who are likely/undecided/unlikely (half, quarter, quarter respectively) to take a covid- are similar to rates reported in other countries. the higher male inclination to take the vaccine may be due the innate male propensity for perceived risk taking. shared covid- with influenza vaccine hesitancy implies an innate degree of vaccine reluctance/hesitancy and not merely reluctance based on novel vaccine knowledge gap. hospital-acquired influenza has a high mortality, with an estimated median of % that rises up to % in high risk groups (e.g. transplant recipients and intensive care patients). ( , ) healthcare workers who carry the influenza virus have been frequently identified as sources of hospital-acquired outbreaks.( ) annual influenza vaccination is strongly recommended for all healthcare workers,( ) but vaccination rates remain poor,( ) despite models that show that a significant proportion of hospital-acquired burden of disease is vaccine preventable. ( ) in summer , the world remains in thrall to pandemic covid- , a respiratory virus with transmission characteristics similar to influenza. for this reason, vaccine development has accelerated at an unprecedented pace.( ) once a vaccine becomes available, it will be crucial to vaccinate healthcare workers so as to minimise nosocomial infections. it has been announced that in malta, frontliners (including all healthcare workers) will be given first priority for the first batch of vaccines that are anticipated to arrive in malta in december . ( ) an earlier study this year that surveyed maltese healthcare workers with regard to influenza vaccination showed that the proportion of workers who did not take the vaccine last year but who are likely to take the vaccine this winter halved from % to %. doctors had the highest baseline uptake ( % refused vaccination in ) and the highest likely uptake next winter ( % likely to refuse vaccination in ). ( ) this study was carried out in order to ascertain the degree of vaccine hesitancy in maltese healthcare workers vis-à-vis a putative novel covid- vaccine later this year, and correlate this with influenza vaccination uptake. a short, anonymous questionnaire was sent out to all of malta's government sector healthcare workers via the service's standard email services. the period for which the questionnaire was open was from / / to / / . the questionnaire was hosted via google forms and exported to bespoke excel spreadsheets for analysis. the questionnaire was sent to all healthcare workers in the main hospital (mater dei hospital), district primary care health centres, st. vincent de paul long term care facility, mount carmel mental health hospital, karin grech rehabilitation hospital and miscellaneous other smaller facilities. it commenced with the following introduction: malta has been fortunate to have the early allocation of a covid- vaccine later this year. the vaccine is licensed and approved and will have passed through phase trials. priority will be given to front liners and to the vulnerable, followed later by the rest of the population. this is totally anonymous and a very short, public health survey for healthcare workers, please fill completely. the questions, formatted in tick boxes, covered sex, occupation (medical, nursing, allied profession and other, with the latter including support staff such as in administration, ward clerks, cleaners, etc.), place of work (as above), age bracket, whether the influenza vaccine was taken last winter and whether it would be taken this coming winter (yes/no). the following text was inserted in the questionnaire followed by several questions on a likert scale of - . phase i, small groups of people receive the trial vaccine. in phase ii, the vaccine is given to people who have characteristics (such as age and physical health) similar to those for whom the vaccine is intended. in phase iii, the vaccine is given to thousands of people and checked for efficacy and safety. the covid vaccine that will arrive in malta will have gone through these phases and will be approved and licensed. based on this information, how likely are you to take the covid- vaccine? i am concerned as i don't know enough about the vaccine i am concerned about the short term side effects (e.g. fever etc) i am concerned about possible long term side effects i am concerned because i don't think the vaccine will be effective i am against vaccines in general for the first question in the list above, it was assumed that scores and were "unlikely", and were "likely" and a score of was regard as undecided. for the likert questions following the first, all were allowed to tick vaccines whatever their likelihood of taking the vaccine. chi tests and chi tests for trend were used except for one two by two table with small values wherein a fischer exact test was used. a p value ≤ . was taken to represent a statistically significant result. a total of , questionnaires were posted electronically, with ( . %) responses (table ) . the proportion of maltese healthcare workers who will take the influenza vaccine increased significantly across the board when compared to last year irrespective of sex, workplace or occupation (table ) . doctors had the highest baseline uptake and the highest likely influenza vaccine uptake next winter (table ) . with regard to a covid- vaccine, approximately half of respondents were likely to take the vaccine and a quarter each were undecided or unlikely to take the vaccine. males were likelier to take the vaccine than females (chi= . , p= . table ). doctors were also the likeliest group to take the covid- vaccine and when compared against all others this was a highly significant difference (chi= . , p< . table ). an analysis by age showed that there was a significant increase in the likely uptake of the influenza vaccine at all ages (first two columns of table with statistical analysis in next two columns). the covid- likelihood uptake pattern was as described above except for the over age group as none of these fell in the "unlikely to take" category. the proportion of those likelier to take the covid- vaccine was directly related to the likelihood of their taking the influenza vaccine (table : chi= . , p< . ). covid- vaccine concerns are shown in table . the issues raised were only very slightly related to vaccine avoidance in general but more related to insufficient knowledge about such a vaccine and any potential side effects especially those in the long term. the increased proportion of maltese healthcare workers who plan to take the influenza vaccine this year when compared to last winter is probably due to increased awareness of respiratory viral illnesses in general in the wake of the covid- pandemic. interestingly, it is the medical profession who had the highest baseline influenza vaccine uptake and the highest likely influenza vaccine uptake next winter and this may be due to greater awareness and knowledge of vaccine safety. the same applies for this profession with regard to the covid- vaccine. the proportions of those who are likely/undecided/unlikely (half, quarter, quarter respectively) to take a covid- are similar to rates reported in other countries.( ) the higher male inclination to take the vaccine may be due to a combination of factors which could include the innate male propensity for perceived risk taking in the face of a novel vaccine. ( ) the higher likely uptake of a covid- vaccine in the oldest age group is unsurprising as this is the most vulnerable group and therefore most likely, in their own selfinterest, to take this vaccine. vaccine hesitancy for covid- was similar to that for influenza implying an innate degree of vaccine reluctance/hesitancy and not merely a reluctance based on the concerns discussed below.( ) however, the concerns are, to some extent, valid. there are various types of vaccines in development and these include not only traditional vaccines but also next generation vaccines. ( ) non-vaccination and vaccine hesitancy our findings are unsurprising as the availability of a vaccine does not automatically equate to % aggregate uptake. for example, an h n influenza vaccine in had a population uptake of . - % across countries. ( ) the low acceptance and uptake of a safe vaccine for a high risk infection is well known and has been dubbed the "pandemic public health paradox".( ) this is a strong contributor to vaccine hesitancy and is a tragic public health outcome as vaccines only protect if a sufficient proportion of the population is vaccinated. ( ) non-vaccination has been quite extensively studied and table shows some of the commonest reasons for non-vaccination.( ) one specific example specifically related to this topic is the aforementioned h n influenza vaccine which was initially claimed to have had associated mortality using the vaccine adverse event reporting system(vaers) system which was eventually disproved, but not before undermining public confidence in this important vaccine. ( ) in , the world health organization named vaccine hesitancy as one of the top ten threats to global health. ( ) the reasons for hesitancy are varied and some common vaccine myths and their scientific rebuttals are summarised in table . ( ) clearly, the reasons for non-vaccination are complex but misconceptions pertaining to safety predominate. trends in hesitancy are overall not promising with a recent study showing that vaccine confidence in europe is low compared to other regions of the world, such as africa (strongly agreeing with vaccine safety range % in lithuania to % in finland). a drop in j o u r n a l p r e -p r o o f journal pre-proof confidence trend was linked to political instability and religious extremism, with rogue leaders sometimes promoting natural, unproven and ineffective alternatives to vaccines.( ) a representative sample of circa adults in the us questioned from - april with regard to a putative covid- vaccine replied: . % intended to be vaccinated, . % were uncertain and . % did not intend to be vaccinated. factors independently associated with vaccine hesitancy ("no"/"not sure") included younger age, black race, lower educational attainment, and not having received the influenza vaccine in the prior year. reasons specified for vaccine hesitancy included vaccine-specific concerns, a need for more information, antivaccine attitudes or beliefs, and a lack of trust.( ) overcoming hesitancy who advises a pre-emptive pro-vaccination strategy that psychologically impacts populations so as to maximize uptake when vaccines become available. ( ) in the case of covid- , national vaccination strategies must be in place in advance of vaccine availability so as to have a plan for population prioritisation for vaccination and to reduce the incidence of fear/concern vis-à-vis vaccination. ( ) a crucial part of the latter aspect is the countering of fake news and misinformation that already percolates (especially via social media) in this regard. ( ) suggested key guidelines/milestones are shown in table .( ) segmentation of target populations is vital and consists of the identification of groups who share similar beliefs/attitudes/behavioral patterns. this goes beyond easily pigeonholed fields such demographic/epidemiological data and greatly enables public heath planners to shape intervention/s to specific segment/s.( ) hesitancy already exists among healthcare workers with regard to ordinary vaccines, such as seasonal influenza vaccination. ( ) ( ) ( ) it is anticipated that the next challenge will be vaccination for covid- when this desperately-awaited vaccine becomes available. indeed, questionnaires in this regard already reveal novel covid- vaccine hesitancy among healthcare workers. ( , ) hesitancy is fuelled by social media, conspiracy theories and fake news, a topic about which entire volumes have been written. ( , ) public health and healthcare worker employers must do their best to ensure that the proportion of vaccinated workers is as close to totality as possible. clinicians, legislators and even ethicists are increasingly cognisant of this aspect of healthcare, and are progressively mandating seasonal influenza vaccination for healthcare workers in some countries. this is not being envisaged for malta. the society for healthcare epidemiology has recommended that annual influenza vaccination should be a condition of employment for healthcare workers,( ) a stance endorsed almost universally by professional bodies.( ) indeed, ethicists have averred that: "given the mounting evidence for the efficacy of influenza vaccination in infection control […] the provision of health care by non-vaccinated health care workers is not merely suboptimal health care, but it is also at variance with generally accepted principles of health care ethics." ( ) j o u r n a l p r e -p r o o f journal pre-proof this is because medical ethics upholds the dual principles of beneficence and nonmaleficence. the former infers the promotion of patients' well-being and the latter is primum non nocere. therefore "practicing without vaccination is maleficent because it falls below the standard of medical care".( ) it has in fact been shown that influenza vaccination of healthcare workers reduces influenza morbidity and mortality in influenza-vulnerable populations. ( ) ( ) ( ) ( ) the commonest reason for healthcare worker vaccination hesitancy is insufficient knowledge about its safety profile and irrational apprehension and it has been shown that improved information about the vaccine improves voluntary vaccine uptake. ( ) our study partially supports this contention in that doctors were more likely to take the influenza vaccine, both last year and with even greater likelihood next winter, and this may be due to greater knowledge in this group of healthcare workers than in the other groups i.e. allied health professionals, nurses and others. healthcare workers and their institutions are professionally responsible for the care and wellbeing of their patients following evidence-based practices.( ) this study may have been biased by the announcement that astrazeneca's vaccine trial was voluntarily paused as for a standard review process that was triggered by a "single event of an unexplained illness that occurred in the uk phase iii trial", a routine action "to allow an independent committee to review the safety data". ( ) vaccine passport several countries have suggested the introduction of covid- "immunity passports" following infection with the disease but this poses extensive scientific, practical, equitable, and legal challenges. ( ) on the other hand, the introduction of a vaccination passport or certificate to vaccinees could be used as additional incentive to take the vaccine by exempting holders from physical restrictions, social distancing etc. ( ) conclusions healthcare workers should be informed about and encouraged to take influenza vaccination. the introduction of a covid- vaccination "passport" may also be considered especially if it provides added benefit/s to the vaccinee. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f 'the unconcerned' -those who consider immunization a low priority and see no real perceived risk of vaccine-preventable diseases. 'the poorly reached' -those who have limited or difficult access to services, related to social exclusion, poverty and, in the case of more integrated and affluent populations, factors related to convenience. 'the active resisters' -those for whom personal, cultural, or religious beliefs discourage them from vaccinating. j o u r n a l p r e -p r o o f key concepts too many vaccines too soon. the number of immunologic components in vaccines have declined over time. the current vaccines on the united states schedule contain immunologic proteins in total, the smallpox vaccine contained . too many vaccines can "overwhelm" the immune system. epidemiologic data and biologic data show that cumulative increases in the number of vaccines have no effect on immune function. mmr vaccine causes autism. original study making this claim contained children, the paper was subsequently retracted due to evidence of misrepresented data. multiple large scale studies, including a study of half a million children have shown no association between receipt of mmr and risk of autism. hpv vaccine increases risk of autoimmune disease. more than million doses of hpv vaccine have been administered. repeated well-designed studies show no association between hpv and ai disease. influenza vaccine given in early pregnancy increases risk of miscarriage. a study of women showed no association between influenza vaccine and spontaneous abortion. j o u r n a l p r e -p r o o f influenza in the acute hospital setting influenza control in acute care hospitals a nosocomial outbreak of influenza during a period without influenza epidemic activity immunization of health-care personnel: recommendations of the advisory committee on immunization practices (acip) mmwr morb mortal wkly rep mandatory influenza vaccination for health care workers as the new standard of care: a matter of patient safety and nonmaleficent practice modeling the effects of influenza vaccination of health care workers in hospital departments developing a vaccine for covid- first delivery of potential covid- vaccine expected in december. times of malta influenza vaccination survey in maltese healthcare workers in the covid- era. malta medical school gazette attitudes toward a potential sars-cov- vaccine: a survey of u.s. adults age and gender differences in risk-taking behaviour as an explanation for high incidence of motor vehicle crashes as a driver in young males influenza a(h n )pdm vaccination policies and coverage in europe pandemic public health paradox media attention, risk perception and public reactions in european countries when a covid- vaccine is ready, will we all be ready for it? under-vaccinated groups in europe and their beliefs, attitudes and reasons for non-vaccination; two systematic reviews risk of fatal adverse events after h n influenza vaccine: limitations of passive surveillance data vaccine safety: myths and misinformation mapping global trends in vaccine confidence and investigating barriers to vaccine uptake: a large-scale retrospective temporal modelling study world health organization. the guide to tailoring immunization programs. world health organisation key guidelines in developing a pre-emptive covid- vaccination uptake promotion strategy measles still spreads in europe: who is responsible for the failure to vaccinate vaccine hesitancy and self-vaccination behaviors among nurses in southeastern france the continuum of influenza vaccine hesitancy among nursing professionals in hong kong knowledge and attitude towards vaccination among healthcare workers: a multicenter cross-sectional study in a southern italian region. vaccines (basel) vaccine hesitancy: the next challenge in the fight against covid- mistrust in biomedical research and vaccine hesitancy: the forefront challenge in the battle against covid- in italy social media and vaccine hesitancy: new updates for the era of covid- and globalized infectious diseases fake news and post-truth pronouncements in general and in early human development revised shea position paper: influenza vaccination of healthcare personnel influenza vaccination of health care workers in long-term-care hospitals reduces the mortality of elderly patients effects of influenza vaccination of health-care workers on mortality of elderly people in long-term care: a randomised controlled trial effectiveness of an influenza vaccine programme for care home staff to prevent death, morbidity, and health service use among residents: cluster randomised controlled trial effect of influenza vaccination of nursing home staff on mortality of residents: a cluster-randomized trial influenza vaccination acceptance and refusal rates among health care personnel covid- : oxford researchers halt vaccine trial while adverse reaction is investigated covid- immunity passports and vaccination certificates: scientific, equitable, and legal challenges the authors have no conflict of interest to declare. key: cord- -cik ino authors: corder, brigette n.; bullard, brianna l.; poland, gregory a.; weaver, eric a. title: a decade in review: a systematic review of universal influenza vaccines in clinical trials during the decade date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cik ino on average, there are – million severe cases of influenza virus infections globally each year. seasonal influenza vaccines provide limited protection against divergent influenza strains. therefore, the development of a universal influenza vaccine is a top priority for the nih. here, we report a comprehensive summary of all universal influenza vaccines that were tested in clinical trials during the – decade. of the studies found, eligible clinical trials, which investigated vaccines, were included in this review. information from each trial was compiled for vaccine target, vaccine platform, adjuvant inclusion, clinical trial phase, and results. as we look forward, there are currently three vaccines in phase iii clinical trials which could provide significant improvement over seasonal influenza vaccines. this systematic review of universal influenza vaccine clinical trials during the – decade provides an update on the progress towards an improved influenza vaccine. globally, seasonal influenza virus epidemics are estimated to cause - million cases of severe infection and result in , - , deaths annually [ , ] . mortality is increased in the elderly over years, children under years, and people in developing countries [ , ] . in the united states alone, influenza virus infects between . - . million people each year, leading to , - , hospitalizations [ ] . these annual influenza epidemics result in an estimated total economic loss of $ . billion each year due to direct medical costs and indirect costs such as projected lost earnings and loss of life [ ] . while the disease burden for seasonal influenza epidemics is substantial, this is significantly increased during influenza pandemics. for example, it is estimated that % of the worldwide population was infected during the h n swine influenza pandemic [ ] . a substantial challenge in the development of an effective influenza vaccine is the significant viral population diversity. the current influenza vaccine can be either trivalent or quadrivalent. the trivalent vaccine contains a h n , h n , and an influenza b strain, with the quadrivalent vaccine including both yamagata and victoria influenza b lineage strains [ , ] . the strains contained in the seasonal influenza vaccine are updated yearly to include those predicted to circulate in the upcoming influenza season. although the current influenza vaccine is effective at reducing morbidity and mortality due to seasonal influenza infections [ ] , vaccine effectiveness estimates only range from to % [ , ] . the vaccine effectiveness is lowest when there is poor antigenic match to the circulating influenza strains [ , ] . relevant information was extracted from the clinicaltrials.gov database including phase, vaccine target, vaccine platform, and results. if information was not available, relevant publications were analyzed. a summary of this data is reported in table . all data were analyzed using graphpad prism . software. figures were designed in adobe illustrator ( . . ). seasonal influenza vaccines provide limited protection and are updated annually to incorporate circulating strains. a vaccine which induces broad cross-protection against influenza remains a top priority for the national institute of health (nih). here we report a comprehensive review of universal influenza vaccine (uiv) clinical trials that were active between january and december . in the last decade, clinical trials investigating vaccines were performed ( figure ) . these trials include a variety of viral targets, vaccine platforms, and adjuvants to boost the immune response to vaccination. table reports a chronological summary for each uiv clinical trial. the unique id for each trial is used for identification in subsequent figures. importantly, several uivs were tested in up to clinical trials. since the trends may be skewed by these vaccines, we differentiate between vaccines and total clinical trials throughout this paper. influenza vaccines typically target specific viral antigens to maximize the immune response to vaccination. vaccination aims to induce a strong adaptive immune response which results in both t and b cell activation. these immune cells produce cytotoxic t cells and antibodies which can protect against future infection. vaccines targeting internal viral proteins, such as nucleoprotein (np) and matrix (m ), can induce strong t cell responses [ ] . viral surface (external) antigens, hemagglutinin (ha) and neuraminidase (na), are targeted by neutralizing antibodies [ ] . traditionally, a robust antibody response has been the goal of influenza vaccination and has been the basis upon which vaccines have been tested and licensed [ , ] . however, these antibodies provide limited protection against divergent influenza strains. since there are strengths for both internal and external strategies, many vaccines include multiple antigens to induce a strong humoral and cellular immune response. over the past decade, both internal and external influenza proteins were utilized in uiv clinical trials ( figure ). other strategies which target whole virus or attenuated virus through gene deletion have also been investigated. however, recent vaccines have focused on external proteins, specifically ha. internal influenza proteins are attractive vaccine candidates since they are more conserved than the external glycoproteins [ ] . this may result in broader cross-protection induced by the vaccine. one example is fp- . , a peptide-based vaccine which includes several cd + and cd + t-cell epitopes conjugated to a fluorocarbon chain. these epitopes are derived from internal influenza proteins including np, m , polymerase basic (pb ), and polymerase basic (pb ). four trials were performed in the past decade utilizing participants - years old. one phase i trial demonstrated that vaccination with fp- . induced strong cellular responses in % of participants with a median response of spot forming cells (sfc)/million peripheral blood mononuclear cells (pbmc) as measured using ifnγ elispot assay [ ] . this cellular response was activated against several heterologous h n and h n strains indicating broad cross-reactivity [ ] . another uiv targeting internal proteins is ovx , which is a recombinant np vaccine. in preclinical trials, vaccinated mice were protected against three lethal influenza a virus (iav) challenges and induced stronger immunogenicity than wild-type np alone [ ] . protection was further improved if mice were immunized with a combination of the seasonal inactivated vaccine and ovx . in the past decade, of the vaccines in uiv clinical trials targeted external glycoproteins. although the ha protein has a high amount of diversity in the globular head, the ha stalk region is more conserved [ ] . vaccines targeting the stalk region of ha have shown promise during development and are being investigated in several uiv clinical trials [ , , , , ] . one such vaccine is ch / n , h / n , which utilizes a prime-boost immunization strategy to promote an viruses , , of immune response towards the ha stalk domain. the phase trial included individuals between and years of age. two viruses were modified with chimeric ha containing a homologous ha stalk and heterologous ha heads. these were administered as live attenuated or inactivated vaccines and boosted with a heterologous ha head vaccine days later [ ] . an oil-in-water adjuvant, as , was included with the inactivated vaccine. after the prime vaccination, only the adjuvanted groups induced strong igg antibody responses. however, all groups demonstrated . to . -fold increases in ha stalk specific igg antibodies after a heterologous boost. these h ha antibodies were cross reactive to h , h , and h ha, indicating broad cross-protection against group ha [ ]. influenza vaccines typically target specific viral antigens to maximize the immune response to vaccination. vaccination aims to induce a strong adaptive immune response which results in both t and b cell activation. these immune cells produce cytotoxic t cells and antibodies which can protect against future infection. vaccines targeting internal viral proteins, such as nucleoprotein (np) and matrix (m ), can induce strong t cell responses [ ] . viral surface (external) antigens, hemagglutinin (ha) and neuraminidase (na), are targeted by neutralizing antibodies [ ] . traditionally, a robust antibody response has been the goal of influenza vaccination and has been the basis upon which vaccines have been tested and licensed [ , ] . however, these antibodies provide limited protection against divergent influenza strains. since there are strengths for both internal and external strategies, many vaccines include multiple antigens to induce a strong humoral and cellular immune response. over the past decade, both internal and external influenza proteins were utilized in uiv clinical trials ( figure ). other strategies which target whole virus or attenuated virus through gene deletion have also been investigated. however, recent vaccines have focused on external proteins, specifically ha. another vaccine utilized the full-length h ha protein in an oral recombinant adenovirus type (ad ) vectored vaccine, ad -h -vtn. three clinical trials have enrolled participants between and years of age to investigate this avian h influenza vaccine. three immunizations with ad -h -vtn resulted in low seroconversion, % for vaccinees and % for placebo [ ] . participants were boosted with an inactivated h n vaccine, which resulted in % seroconversion for vaccinees compared to % in the placebo group. vaccination with ad -h -vtn induced a significant t cell response after a single vaccination with a median sfc/million pbmc. no serious adverse events were reported although vaccinees experienced higher rates of self-limited abdominal pain ( . % vs. . %), diarrhea ( . % vs. . %), and nasal congestion ( . % vs. . %) compared to the placebo. na was included in a dna vaccine, vgx- x [ ] . the dna vaccine included plasmids containing na, ha, and m e-np from h n avian influenza. the vaccine was administered intramuscularly to over participants - years of age during clinical trials [ , [ ] [ ] [ ] . no results have been posted to date. interestingly, na was only investigated in combination with other influenza proteins. furthermore, besides the vgx- x vaccine, na was only included in whole inactivated and vlp vaccine strategies. the na protein should be further investigated for its cross-protective potential against influenza [ ] . antigens are presented to the immune system in different ways depending on the vaccine platform. most seasonal influenza vaccines utilize attenuated or inactivated wild-type viruses. these viruses display the external influenza proteins and stimulate strong antibody responses [ ] . although this strategy has been utilized since , it has consistently shown low efficacy for protection against mismatched influenza strains [ , ] . therefore, a variety of vaccine platforms were investigated during the last decade to further improve influenza vaccination. although many vaccine platforms have been investigated, no single platform has thus far been demonstrated to show superior protection against influenza. a common platform for uiv clinical trials is viral vectors ( . %), which utilize viral machinery to package, deliver, or display the vaccine antigen ( figure ). viral vectors have been commonly used as molecular biology tools and are approved for several gene therapies [ ] . one of these vaccines is mva-np+m , which is a modified vaccinia virus ankara (mva) viral vector expressing the nucleoprotein (np) and matrix protein (m ) genes from an h n influenza strain [ ] . in the past decade, nine clinical trials investigated mva-np+m enrolling over participants years or older. results from these trials report an increase in t cell response to vaccination, which remained significant above baseline for weeks in - -year-old participants. however, the response was only significant for weeks in subjects - years old and weeks for participants over years [ ] . a subsequent trial using healthy subjects reported no significant difference in t cell response days post-vaccination [ ] . the antibody response to vaccination was not reported. to further boost the immune response to mva-np+m , a heterologous boost with a simian adenovirus viral vector chadox -np+m was investigated [ ] . in this study involving participants, both the mva-np+m and chadox -np+m vaccines were shown to boost t cell responses when administered individually or together. a heterologous boost, regardless of the order, increased t cell responses -fold. another study investigating this heterologous strategy reported a significant increase in t cell responses at day after chadox -np+m vaccination, but a decreased response by day [ ] . vaccinees were boosted with mva-np+m , which again increased the t cell response; however, this response was not significant days following the boost vaccination. a recent viral vectored vaccine is nasovax, an intranasal adenoviral vectored vaccine. though no results have been posted for this clinical trial, data presented at the world vaccine congress reported strong immunogenicity and protection [ ] . indeed, vaccination with nasovax induced % seroconversion, which was maintained for over year. the newest vaccine platform utilizes nanoparticles to deliver viral antigens [ ] . one vaccine utilizing this method is val- and val- , which are mrna ha from h n and h n influenza strains delivered in a lipid nanoparticle (lnp) [ ] . two trials were performed utilizing participants aged - years. for the h n mrna vaccine, vaccination resulted in mild to moderate systemic adverse events including injection site pain ( . - . % vs. . - . %) and myalgia ( . - . % vs. . - . %) compared to the placebo. antibody responses were increased in a dose-dependent manner for the h n lnp vaccine reaching % seroconversion at µg compared to . % for the placebo group. these levels remained seropositive (hai ≥ ) for months after immunization. the h n lnp vaccine induced strong antibody titers for all vaccine doses with . % seroconversion for the µg dose group. participants vaccinated with the h vaccine displayed mild injection site pain compared to the placebo ( . - % vs. . - . %). mva-np+m and chadox -np+m vaccines were shown to boost t cell responses when administered individually or together. a heterologous boost, regardless of the order, increased t cell responses ~ -fold. another study investigating this heterologous strategy reported a significant increase in t cell responses at day after chadox -np+m vaccination, but a decreased response by day [ ] . vaccinees were boosted with mva-np+m , which again increased the t cell response; however, this response was not significant days following the boost vaccination. other nanoparticle vaccines include vrc-flunpf - -vp, which is a recombinant ha vaccine delivered in a ferritin nanoparticle, and vrc-flunpf - -vp, which is a ha stalk protein delivered in a ferritin nanoparticle. although neither trial has posted results, influenza ferritin nanoparticle vaccines have shown strong immunogenicity in mice and ferrets during preclinical trials [ ] . another common vaccine platform utilizes recombinant protein of a viral antigen ( . %). due to low immunogenicity, recombinant protein vaccines typically require the use of an adjuvant to enhance the immune response to vaccination [ , ] . panblok is a recombinant ha protein administered with a novel stable emulsion adjuvant. four clinical trials were performed which enrolled participants - years old. in one adjuvant dose-dependent trial targeting h influenza, results demonstrated that all adjuvanted vaccines ( . µg, . µg, or µg) increased seroconversion from % in the unadjuvanted group to % for participants who received an adjuvanted vaccine [ ] . however, another trial targeting h influenza reported low seroconversion regardless of the adjuvant dose [ ] . despite low antibody detection using hai assay, antibodies against h influenza were detected using elisa. passive transfer of these antibodies resulted in protection against lethal h challenge in mice. additionally, these antibodies were cross-reactive to h , h , h , h , h , and h , indicating broad immunity against both group and group influenza [ ] . some vaccines deliver conserved immunogenic peptides of viral antigens. one such vaccine is flu-v, a peptide-based vaccine containing conserved epitopes from influenza a and b viruses and adjuvanted with montanide isa- . over the past decade, clinical trials were performed involving participants between and years of age. vaccination with flu-v increased ifn-γ cellular responses -fold but did not induce antibody responses as expected [ ] . in another study, seronegative males were vaccinated with flu-v and then challenged with h n influenza virus [ ] . participants vaccinated with flu-v showed reductions in viral load and symptoms as well as an -fold increase in ifn-γ cellular responses. nonstructural protein (ns ) is an influenza protein that antagonizes the immune system by downregulating antiviral host proteins [ ] . several attenuated virus vaccines in clinical trials have deleted this viral gene to improve the immune response to vaccination. both ghb l and ghb l are intranasal live attenuated viruses with the ns gene deleted, but neither clinical trial has published results from these studies. matrix protein (m ) is an essential structural viral protein for influenza replication. the m sr vaccine includes a virus that lacks the m protein resulting in non-infectious viral progeny, essentially a single-cycle virus [ ] . all three m sr vaccine trials are currently active. an ideal uiv will provide highly effective and long-lasting protection. this can be difficult to achieve when targeting internal proteins or using poorly immunogenic vaccine platforms. adjuvants are compounds that stimulate the immune system and improve vaccine efficacy [ ] . this is commonly achieved by oil-in-water emulsions, which recruit immune cells to the site of vaccination [ ] . another common group of adjuvants are toll-like receptor (tlr) agonists. these adjuvants bind and activate cellular host pathways, which leads to increased immune activation [ ] . new adjuvants continue to be discovered and explored, but few are licensed for use in the united states [ ] . over the past decade, most adjuvants in uivs have been oil-in-water emulsions ( %) (figure ). m- is a recombinant protein vaccine that contains common b and t cell epitopes from the ha, np, and m influenza proteins. seven trials were performed over the past decade, which enrolled , individuals over years old. this vaccine was combined with an adjuvant, montanide isa vg, which increased igg titers -fold against the m- protein [ ] . strong t cell responses to m- were shown for all groups regardless of adjuvant inclusion. a subsequent trial reported m- could be used as a stand-alone or priming vaccine for the seasonal influenza vaccine [ ] . when compared to seasonal vaccination alone, participants primed with m- before seasonal vaccination showed elevated antibody responses for matched h n ( -fold vs. . -fold) and h n ( . -fold vs. . -fold), but not influenza b ( . -fold vs. . -fold). additionally, m- vaccination increased both cd + and cd + t cell responses to h n , h n , and influenza b strains compared to baseline. another unpublished clinical trial reported that % of m- -vaccinated participants had a -fold increase in hai titers compared to % for the control group [ ] . this vaccine has moved into phase iii clinical trials and was scheduled for primary completion in may . immunose flu is an inactivated split vaccine with a novel lipid adjuvant, endocine. the immunogenicity of immunose flu was not reported, but vaccination resulted in serious adverse events in of participants including erysipelas and gastroenteritis [ ] . mild to moderate adverse events were recorded in . % and . % of vaccinated participants compared to . % in the saline placebo control group. another common group of adjuvants are toll-like receptor (tlr) agonists. these adjuvants bind and activate cellular host pathways, which leads to increased immune activation [ ] . an example is vax , which is a recombinant ha protein fused to the tlr ligand, flagellin. four clinical trials were performed using participants over the age of . vaccine doses over µg resulted iñ -fold elevated hai titers, % seroconversion, and % seroprotection rates for h n influenza [ ] . however, dose escalation over µg and µg was stopped due to serious adverse events [ ] . vaccine doses ≥ . µg resulted in an average -fold increase in hai titer, % seroprotection, and % seroconversion against a matched h n influenza strain [ ] . viruses , , x for peer review of immunose flu is an inactivated split vaccine with a novel lipid adjuvant, endocine. the immunogenicity of immunose flu was not reported, but vaccination resulted in serious adverse events in of participants including erysipelas and gastroenteritis [ ] . mild to moderate adverse events were recorded in . % and . % of vaccinated participants compared to . % in the saline placebo control group. another common group of adjuvants are toll-like receptor (tlr) agonists. these adjuvants bind and activate cellular host pathways, which leads to increased immune activation [ ] . an example is vax , which is a recombinant ha protein fused to the tlr ligand, flagellin. four clinical trials were performed using participants over the age of . vaccine doses over µ g resulted in ~ -fold elevated hai titers, % seroconversion, and % seroprotection rates for h n influenza [ ] . however, dose escalation over µ g and µ g was stopped due to serious adverse events [ ] . vaccine doses ≥ . µ g resulted in an average -fold increase in hai titer, % seroprotection, and % seroconversion against a matched h n influenza strain [ ] . another tlr adjuvant is double-stranded rna (dsrna), which binds tlr and activates inflammatory pathways [ ] . vxa-a . utilizes this adjuvant by encoding dsrna and an h n ha transgene in a recombinant adenovirus type (ad ) vector. this oral vaccine has been studied in clinical trials with participants between and years of age. one trial reported increased antibody responses to matched h n strains with an average of . -fold increases in hai titers and -fold increases in microneutralization titers after vaccination [ ] . vaccination resulted in mild side effects at similar rates to the placebo group. phase clinical trial participants were immunized with vxa-a . or the seasonal qiv vaccine and then challenged with an h n influenza strain [ ] . vaccination with vxa-a . resulted in % protection compared to % with the seasonal vaccine. another tlr adjuvant is double-stranded rna (dsrna), which binds tlr and activates inflammatory pathways [ ] . vxa-a . utilizes this adjuvant by encoding dsrna and an h n ha transgene in a recombinant adenovirus type (ad ) vector. this oral vaccine has been studied in clinical trials with participants between and years of age. one trial reported increased antibody responses to matched h n strains with an average of . -fold increases in hai titers and -fold increases in microneutralization titers after vaccination [ ] . vaccination resulted in mild side effects at similar rates to the placebo group. phase clinical trial participants were immunized with vxa-a . or the seasonal qiv vaccine and then challenged with an h n influenza strain [ ] . vaccination with vxa-a . resulted in % protection compared to % with the seasonal vaccine. interestingly, although alum is one of the most commonly used fda-approved adjuvants, only one clinical trial in utilized this adjuvant [ ] . hai- is a recombinant h ha protein vaccine that is produced in a plant-expression system, nicotiana benthamiana [ ] . this trial enrolled individuals between and years of age and investigated the dose response of hai- with alum. interestingly, any combination of hai- ( , , and µg) with alum resulted in minimal antibody titers while hai- alone ( µg) induced the greatest antibody response ( . -fold increase). this suggests the hai- induced low immunogenicity that was not improved by the addition of an adjuvant. in the us, new drugs and vaccines must complete four phases of clinical trials to be licensed and marketed for public use. phase i trials investigate the safety and dosage of the vaccine. typically, phase i trials have limited numbers of participants and do not assess efficacy due to low statistical power [ ] . phase ii trials assess the dose response, efficacy, and side effects of the new vaccine. these trials include more study participants and can last longer than phase i trials. occasionally, phases i and ii can be combined into one clinical trial, phase i/ii. phase iii trials include a large sample size and assess participants for vaccine efficacy and adverse reactions. at this point, the new vaccine or drug may be approved for the market [ ] . lastly, phase iv clinical trials involve post-marketing surveillance of the efficacy and safety of the new vaccine. importantly, not all clinical trial results are reported or published. it is common for results to be posted several years after the completion of a trial ( figure ). over the past decade, only half of completed trials reported their findings ( figure e ). this delay is consistent regardless of clinical trial phase ( figure d ). one clinical trial in utilized this adjuvant [ ] . hai- is a recombinant h ha protein vaccine that is produced in a plant-expression system, nicotiana benthamiana [ ] . this trial enrolled individuals between and years of age and investigated the dose response of hai- with alum. interestingly, any combination of hai- ( , , and µ g) with alum resulted in minimal antibody titers while hai- alone ( µ g) induced the greatest antibody response ( . -fold increase). this suggests the hai- induced low immunogenicity that was not improved by the addition of an adjuvant. in the us, new drugs and vaccines must complete four phases of clinical trials to be licensed and marketed for public use. phase i trials investigate the safety and dosage of the vaccine. typically, phase i trials have limited numbers of participants and do not assess efficacy due to low statistical power [ ] . phase ii trials assess the dose response, efficacy, and side effects of the new vaccine. these trials include more study participants and can last longer than phase i trials. occasionally, phases i and ii can be combined into one clinical trial, phase i/ii. phase iii trials include a large sample size and assess participants for vaccine efficacy and adverse reactions. at this point, the new vaccine or drug may be approved for the market [ ] . lastly, phase iv clinical trials involve post-marketing surveillance of the efficacy and safety of the new vaccine. importantly, not all clinical trial results are reported or published. it is common for results to be posted several years after the completion of a trial ( figure ). over the past decade, only half of completed trials reported their findings ( figure e ). this delay is consistent regardless of clinical trial phase ( figure d ). as expected, most uiv clinical trials performed over the past decade were phase i trials ( . %) ( figure ). of the vaccines, have progressed past phase i ( . %); however, only vaccines ( %) have been tested in phase iii clinical trials. the first phase iii trial investigated inflexal v, a trivalent adjuvanted virus-like particle (vlp) vaccine [ ] . this study included children between and months and was completed in november [ ] . all participants were immunized with a single full dose ( . ml) or with two doses ( . ml) of the inflexal v vaccine. results suggest that both vaccine groups demonstrated improved seroprotection and seroconversion rates. participants who received two . ml doses weeks apart showed higher seroprotection rates for h n ( . ), h n ( . ), and influenza b ( . ). for h n and h n , the two-dose regimen resulted in higher seroconversion and geometric mean titer (gmt) fold increases than the single-shot regimen. half of participants from each group experienced non-serious adverse events including pyrexia, malaise, rhinitis, cough, otitis media acute, as well as adverse events at the injection site including erythema, induration, pain, or hemorrhage. the second uiv tested in a phase iii clinical trial was m- . this vaccine is a synthetic recombinant protein containing common linear influenza epitopes [ ] . as discussed above, the adjuvanted m- vaccine has shown promising immunogenicity and the phase iii trial was scheduled for primary completion in may [ , ] . the third vaccine tested in a phase iii clinical trial is nanoflu. this vaccine is a recombinant ha protein delivered in a nanoparticle with a saponin-based matrix-m adjuvant [ ] . although results for the phase ii trial have not been posted, a press release from novavax stated that nanoflu induced superior hai antibody responses against homologous and drifted strains compared to the seasonal influenza vaccine. a phase iii clinical trial involving participants over years of age was scheduled for primary completion in december . this systematic review documents uivs that were tested in clinical trials from january to december . although many papers have discussed strategies for uivs, few review papers address the translation of uiv strategies to clinical trials [ , ] . this is the first systematic review of uivs in clinical trials. the definition of a "universal" influenza vaccine is highly debated [ , ] . in , the niaid announced that a uiv should ( ) be at least % effective, ( ) protect against group i and ii iav, ( ) have durable protection that lasts at least year, and ( ) be suitable for all age groups [ ] . since this standard was put forward towards the end of the decade, our definition of a uiv remains broader than the niaid requirements. here, we have defined a uiv as a vaccine that aims to induce better cross-protection than seasonal influenza vaccines. therefore, "supra-seasonal vaccines" which cover a large subset of influenza strains and vaccines against specific subtypes of influenza have been included in this analysis. the influenza diversity targeted by each vaccine varied. only % of universal vaccines were designed to protect against both influenza a and b viruses. other strategies focused on iav ( %) or a single subtype of iav ( %). importantly, no vaccines focused on influenza b virus (ibv) alone. furthermore, the current niaid requirements for a universal influenza vaccine do not require cross-protection against ibv. notably, the cdc reports that ibv is responsible for % of influenza cases reported for children and young adults each year [ ] . overall, approximately % of annual influenza cases can be attributed to ibv [ ] . the significant burden of ibv should be addressed in the design of universal influenza vaccines. some limitations to this review should be noted. first, information about clinical trials can be limited until the results are published. specifically, not all clinical trial summaries include information on vaccine design and mechanism. in these cases, previous publications and press releases for the vaccines were consulted. additionally, most results reported safety information and homologous vaccine efficacy, providing limited information on the cross-reactivity of each vaccine. second, we searched clinical trials registered through clinicaltrials.gov, which could potentially exclude some studies. there are other clinical trial databases such as eu clinical trials register, however, the clinicaltrials.gov database reports more accurate and updated information for clinical trials [ ] . despite limited information, this review provides a comprehensive summary of the uivs tested in clinical trials. indeed, this is the first comprehensive review to also discuss efficacy and trends in vaccine development for influenza. the field of influenza vaccine development is ever progressing. this is reflected in new vaccine targets and platforms such as ha stalk and nanoparticles. researchers over the past decade have produced many promising influenza vaccines, each with strengths and limitations. the efficacy of a vaccine may induce strong protection against matched strains, but an effective uiv must induce strong cross-protection as well. this review identifies vaccines that report efficacy against matched strains alone. importantly, these vaccines may provide cross-protection if delivered in combination with vaccines targeting other influenza subtypes. however, this would require further research and investigation. influenza virus remains a major global pathogen despite the general widespread use of seasonal vaccines due to varying efficacy to drifted strains. a uiv remains a top priority for the nih and world health organization. this review provides an update on the progress towards a better influenza vaccine. with this information, researchers and clinicians can remain informed about the status and limitations of universal influenza vaccines. universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases influenza (seasonal) estimates of global seasonal influenza-associated respiratory mortality: a modelling study global role and burden of influenza in pediatric respiratory hospitalizations, - : a systematic analysis annual estimates of the burden of seasonal influenza in the united states: a tool for strengthening influenza surveillance and preparedness the annual impact of seasonal influenza in the us: measuring disease burden and costs estimating age-specific cumulative incidence for the influenza pandemic: a meta-analysis of a(h n )pdm serological studies from countries. influenza other respir prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices-united states deaths averted by influenza vaccination in the u.s. during the seasons / through / seasonal flu vaccine effectiveness studies immune history and influenza vaccine effectiveness a study to assess the safety and immunogenicity of a new influenza vaccine candidate mva-np+m in healthy adults potent cd + t-cell immunogenicity in humans of a novel heterosubtypic influenza a vaccine, mva-np+m coadministration of seasonal influenza vaccine and mva-np+m simultaneously achieves potent humoral and cell-mediated responses a t cell-inducing influenza vaccine for the elderly: safety and immunogenicity of mva-np+m in adults aged over years preliminary assessment of the efficacy of a t-cell-based influenza vaccine, mva-np+m , in humans a study to assess the safety and efficacy of a new influenza candidate vaccine mva-np+m in healthy adults university of oxford. a study to determine the safety and immunogenicity of co-administration of the candidate influenza vaccine mva-np+m and seasonal influenza vaccine a phase i study of candidate influenza vaccines mva-np+m and chadox np+m heterologous two-dose vaccination with simian adenovirus and poxvirus vectors elicits long-lasting cellular immunity to influenza virus a in healthy adults safety and immunogenicity of co-administration of candidate influenza vaccine mva-np+m and viroflu®seasonal influenza vaccine a study to determine the safety and immunogenicity of the candidate influenza vaccine mva-np+m safety and immunogenicity of the heterosubtypic influenza a vaccine mva-np+m manufactured on the age .cr.pix avian cell line. vaccines improved novel vaccine combination influenza study a phase iib study to determine the safety and efficacy of candidate influenza vaccine mva-np+m in combination with licensed inactivated influenza vaccine in adults aged years and above (invictus): a study protocol clinical network services (cns) pty ltd. efficacy of candidate influenza vaccine mva-np+m in adults learning lessons from mva a, a failed booster vaccine for bcg efficacy of mva-np+m in the influenza h n human challenge model a double-dose safety study of an influenza vaccine (multimeric- ) injected to elderly volunteers priming by a novel universal influenza vaccine (multimeric- )-a gateway for improving immune response in the elderly population further investigation of an intramuscular influenza vaccine (multimeric- ) back to the future: immunization with m- prior to trivalent influenza vaccine in / enhanced protective immune responses against / epidemic strain a study to assess the safety and immunogenicity of m- influenza vaccine as a primer to tiv in elderly volunteers biondvax pharmaceuticals ltd. phase ii study to assess safety & immunogenicity of multimeric- influenza vaccine assess the safety and immunogenicity of m- as a standalone influenza vaccine and as a h n vaccine primer in adults two doses of multimeric- (m- ) followed by influenza vaccine biondvax pharmaceuticals ltd. a pivotal trial to assess the safety and clinical efficacy of the m- as a standalone universal flu vaccine safety and immunogenicity of vax influenza vaccine in community-living adults >= years of age induction of a potent immune response in the elderly using the tlr- agonist, flagellin, with a recombinant hemagglutinin influenza-flagellin fusion vaccine (vax , stf .ha si) comparative safety and immunogenicity of vax a, vac b and vax c novel h n influenza vaccine in healthy adults development of vax , a recombinant hemagglutinin (ha) influenza-flagellin fusion vaccine with improved safety and immune response safety and immunogenicity of a novel h n influenza vaccine in healthy adults age - years superior efficacy of a recombinant flagellin:h n ha globular head vaccine is determined by the placement of the globular head within flagellin study of the safety and immunogenicity of a novel h n influenza vaccine in healthy adults age - safety and immunogenicity of an oral, replicating adenovirus serotype vector vaccine for h n influenza: a randomised, double-blind, placebo-controlled, phase study safety and immunogenicity of replication-competent adenovirus -vectored vaccine for avian influenza h n niaid); national institutes of health clinical center (cc) niaid); national institutes of health clinical center (cc) dose finding study of single dose ghb l in healthy adults phase b influenza vaccine study in healthy subjects synthetic influenza vaccine (flu-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled phase i trial influenza vaccine challenge study in healthy subjects caparros-wanderley, w. meta-analysis and potential role of preexisting heterosubtypic cellular immunity based on variations in disease severity outcomes for influenza live viral challenges in humans a synthetic influenza virus vaccine induces a cellular immune response that correlates with reduction in symptomatology and virus shedding in a randomized phase ib live-virus challenge in humans peptcell limited; national institute of allergy and infectious diseases (niaid) peptcell limited; seventh framework programme double-blind, placebo-controlled phase iib trial to test flu-v vaccine evaluation of the immunogenicity and safety of different doses and formulations of a broad spectrum influenza vaccine (flu-v) developed by seek: study protocol for a single-center, randomized, double-blind and placebo-controlled clinical phase iib trial study of vgx- x, h n avian influenza virus dna plasmid + electroporation in healthy adults inovio biomedical h n avian influenza dna vaccine receives korean approval to begin clinical trials. in first component of inovio's syncon(tm) universal flu vaccine to be tested in healthy volunteers inovio pharmaceuticals. study of vgx- , h n avian flu virus plasmid dna with electroporation device in healthy adult males a follow-on study with an h influenza vaccine for subjects who participated in study flu- a study of dna vaccine with electroporation for the prevention of disease caused by h and h influenza virus immune targeting systems ltd.; hammersmith medicines research. a study to evaluate the safety, tolerability and immunogenicity of a universal influenza a vaccine a novel peptide-based pan-influenza a vaccine: a double blind, randomised clinical trial of immunogenicity and safety tolerability and immunogenicity of two different formulations of an influenza a vaccine (fp- . ) immune targeting systems ltd. influenza a vaccine (fp- . ) formulated with and without adjuvant, in the presence or absence of a single administration of a trivalent inactivated influenza virus vaccine in older adults efficacy and immunogenicity of an influenza a vaccine (fp- . ) in healthy volunteers following virus challenge immunogenicity and safety of a single . ml dose of inflexal v with a . ml -dose regimen of inflexal v inflexal v a trivalent virosome subunit influenza vaccine: production. vaccine safety and immunogenicity of a recombinant h n vaccine in adults safety and immunogenicity of a plant-produced recombinant hemagglutinin-based influenza vaccine (hai- ) derived from a/indonesia/ / (h n ) influenza virus: a phase randomized, double-blind, placebo-controlled, dose-escalation study in healthy adults study of single dose ghb l trivalent influenza vaccine in healthy adults muster, t. phase i/ii trial of a replication-deficient trivalent influenza virus vaccine lacking ns protein sciences corporation. safety and immunogenicity of panblok influenza vaccine in healthy adults stable emulsion (se) alone is an effective adjuvant for a recombinant, baculovirus-expressed h influenza vaccine in healthy adults: a phase trial trial to evaluate the immunogenicity and safety of panblok®(h rha) in healthy adults aged and older vaccination with a recombinant h hemagglutinin-based influenza virus vaccine induces broadly reactive antibodies in humans australian respiratory and sleep medicine institute. recombinant h hemagglutinin influenza vaccine trial advax, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses panblok h vaccine adjuvanted with as or mf a phase i study to determine the safety and immunogenicity of the candidate influenza vaccine chadox -np+m clinical assessment of a novel recombinant simian adenovirus chadox as a vectored vaccine expressing conserved influenza a antigens high titre neutralising antibodies to influenza after oral tablet immunisation: a phase , randomised, placebo-controlled trial safety study of an oral vaccine to prevent seasonal influenza immunogenicity of seasonal influenza by delivery directly to ileum influenza vaccination via oral tablet is protective and induces a unique mucosal immune response a phase influenza a challenge study following oral administration of an h n ha ad-vector seasonal flu vaccine pharmacodynamic open-label trial with vxa-a . oral h vaccine in healthy adults a(h n ) vlp antigen dose-ranging study with matrix-m ™ adjuvant nova laboratories limited; the emmes company, llc. the safety, tolerance, and immunogenicity of mas- -adjuvanted seasonal inactivated influenza vaccine (mer ) mrna vaccines against h n and h n influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase randomized clinical trials tolerability, and immunogenicity of val- in healthy adult subjects tolerability, and immunogenicity of val- in healthy adult subjects safety and immunogenicity study of h n m sr monovalent influenza vaccine in healthy volunteers safety and immunogenicity study of an influenza vaccination strategy including a h n m sr prime followed by a seasonal quadrivalent inactivated vaccine boost in a pediatric population - years old flugen inc. safety and immunogenicity of the bris m sr and sing m sr h n monovalent influenza vaccines novel influenza vaccine m sr protects against drifted h n and h n influenza virus challenge in ferrets with pre-existing immunity study to assess the safety, tolerability and immune response following vaccination with immunose™ flu study to assess the safety, tolerability and immune response following vaccination with immunose™ flu in older adults nitto denko corporation. evaluation of the safety and immunogenicity of a sublingual influenza vaccine nsv in healthy male volunteers a study to evaluate the reactogenicity, safety and immunogenicity of glaxosmithkline (gsk) biologicals' investigational supra-seasonal universal influenza vaccines-inactivated (suivs) (gsk a) in healthy adults aged to years single-ascending-dose study of the safety and immunogenicity of nasovax safety and immunogenicity of nasovax, a novel intranasal influenza vaccine sybil tasker to present on april at : p.m. eastern time novavax. evaluation of the safety and immunogenicity of a recombinant trivalent nanoparticle influenza vaccine with matrix m- adjuvant (nanoflu). available online novavax announces positive phase nanoflu results in older adults phase pivotal trial of nanoflu™ in older adults immunogenicity of chimeric haemagglutinin-based, universal influenza virus vaccine candidates: interim results of a randomised, placebo-controlled, phase clinical trial icahn school of medicine at mount sinai; children's hospital medical center, cincinnati; duke university; the emmes company, llc.; glaxosmithkline. safety and immunogenicity of a live-attenuated universal flu vaccine followed by an inactivated universal flu vaccine influenza ha ferritin vaccine, alone or in prime-boost regimens with an influenza dna vaccine in healthy adults self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h n antibodies ferritin in the field of nanodevices vaccinate for the next h n pandemic now niaid); national institutes of health clinical center (cc). dose, safety, tolerability and immunogenicity of an influenza h stabilized stem ferritin vaccine, vrcflunpf - -vp, in healthy adults hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection safety and immune response of increasing doses of ovx after intramuscular or intranasal administrations in healthy subjects ovx a recombinant nucleoprotein vaccine inducing cellular responses and protective efficacy against multiple influenza a subtypes safety and immune response of one dose of ovx at two dose levels, in comparison to influvac tetratm, after intramuscular administration in healthy subjects aged - years health ministry of the russian federation. the study of the safety, reactogenicity and immunogenicity of the gamfluvac a double-blind randomized placebo-controlled study study of the safety, reactogenicity and immunogenicity of the gamfluvac influenza and memory t cells: how to awake the force host immune response to influenza a virus infection nucleoprotein of influenza a virus is a major target of immunodominant cd + t-cell responses emerging influenza viruses and the prospect of a universal influenza virus vaccine influenza infection in humans induces broadly cross-reactive and protective neuraminidase-reactive antibodies comparisons of the humoral and cellular immune responses induced by live attenuated influenza vaccine and inactivated influenza vaccine in adults history and evolution of influenza control through vaccination: from the first monovalent vaccine to universal vaccines viral vectors in gene therapy nanoparticle vaccines against infectious diseases recombinant vaccines and the development of new vaccine strategies. braz correlates of adjuvanticity: a review on adjuvants in licensed vaccines functions of the influenza a virus ns protein in antiviral defense mechanisms of action of adjuvants toll-like receptor agonists: are they good adjuvants? adjuvants help vaccines work better safety and immunogenicity of multimeric- -a novel universal influenza vaccine dsrna-activation of tlr and rlr signaling: gene induction-dependent and independent effects what are clinical trials and studies? available online the hurdles from bench to bedside in the realization and implementation of a universal influenza vaccine universal influenza virus vaccines and therapeutic antibodies impact of influenza b lineage-level mismatch between trivalent seasonal influenza vaccines and circulating viruses prevalence of clinical trial status discrepancies: a cross-sectional study of , trials registered on both clinicaltrials.gov and the european union clinical trials register key: cord- - wojtboi authors: abo, stéphanie m.c.; smith, robert title: modelling the daily risk of ebola in the presence and absence of a potential vaccine date: - - journal: infect dis model doi: . /j.idm. . . sha: doc_id: cord_uid: wojtboi ebola virus — one of the deadliest viral diseases, with a mortality rate around % — damages the immune system and organs, with symptoms including episodic fever, chills, malaise and myalgia. the recombinant vesicular stomatitis virus-based candidate vaccine (rvsv-zebov) has demonstrated clinical efficacy against ebola in ring-vaccination clinical trials. in order to evaluate the potential effect of this candidate vaccine, we developed risk equations for the daily risk of ebola infection both currently and after vaccination. the risk equations account for the basic transmission probability of ebola and the lowered risk due to various protection protocols: vaccination, hazmat suits, reduced contact with the infected living and dead bodies. parameter space was sampled using latin hypercube sampling, a statistical method for generating a near-random sample of parameter values. we found that at a high transmission rate of ebola (i.e., if the transmission rate is greater than %), a large fraction of the population must be vaccinated ( %) to achieve a % decrease in the daily risk of infection. if a vaccine is introduced, it must have at least % efficacy, and almost everyone in the affected areas must receive it to effectively control outbreaks of ebola. these results indicate that a low-efficacy ebola vaccine runs the risk of having vaccinated people be overconfident in a weak vaccine and hence the possibility that the vaccine could make the situation worse, unless the population can be sufficiently educated about the necessity for high vaccine uptake. trary to vsv-based vaccine vectors, meaning that the antigen is not present on the surface of the vector but is expressed at high levels once the vector enters the target cells of the vaccinated individual [ ] . the candidate cad -zebov is a non-replicative vector-based ebola vaccine (i.e., genes essential for the life cycle of the vector virus are deleted to restrict the transcription and replication) that encodes the glycoprotein from the zaire ebolavirus [ ] . the chimpanzee adenovirus type (chad ) is used as a carrier to deliver benign genetic material of the zaire strain [ ] . among the many studies on ebola virus, we note two recurring goals of most computational models: quantifying the effects of possible interventions on the incidence rate [ ] and developing predictive models to estimate the total number of infected cases, the reproduction number, the number of deaths and the duration of outbreaks [ , ] . some other studies focus on examining assumptions about how the disease spreads, such as uniform transmissibility vs. homogeneous mixing within a population [ ] . there are also several studies with the common goal of understanding the impact of public-health initiatives on the dynamics of an outbreak and control of the virus [ , ] . those studies often analyze the role of behavior change induced by health education or use consumer-centric models of public-health intelligence [ , ] . other models of ebola have focused on interventions such as quarantine, distribution of household kits, emergency treatment care (etc) expansion and accelerating case tracking. lewnard et al. suggested that the existing etc capacity necessary to decrease the severity of the current outbreak was impractical [ ] . legrand et al. provided a stochastic compartmental model incorporating transmission in the community, in the hospital and during burial ceremonies in order to suggest where interventions are best suited [ ] . rivers et al. [ ] investigated the efficiency of increased contact tracing, improved infection-control practices and a hypothetical pharmaceutical intervention to improve survival of hospitalised patients aimed at reducing the reproduction number of the ebola virus. chowell et al. [ ] used an seir model to determine r , the final size of the epidemic and performed a sensitivity analysis, showing that education and contact tracing with quarantine would reduce the epidemic by a factor of . fisman et al. [ ] used a two-parameter model to determine the degree to which the epidemic was being controlled, finding only weak control in west africa as of the end of august and essentially no control in liberia. webb and browne [ ] developed an age-structured model, tracking disease age through initial incubation, followed by an infectious phase with variable transmission infectiousness. browne et al. [ ] showed that, if contact tracing was perfect, then the critical proportion of contacts that need to be traced could be derived. bartlett et al. [ ] utilized a discrete-time model to represent the clinical progression of the ebola virus and estimate the number of infections and deaths in sierra leone. waife et al. [ ] used sensitivity analysis to show that an ebola vaccine could be effective if contact rates with unvaccinated individuals could be sufficiently lowered. risk equations have been used to determine contributing morbidity and mortality factors for a variety of infections, such as heart disease [ ] , kidney failure [ ] , diabetes [ ] and hiv [ ] . per-day risk equations to assess the potential effect of treatment have been used for vaginal microbicides as a potential hiv prophylaxis [ ] . although the use of such equations in infectious disease modelling is relatively new, this kind of risk assessment has become an accepted component of clinical guidelines and recommendations in cardiovascular medicine, for example [ ] our aim in this paper is to determine the daily risk of ebola infection during an epidemic, both before and after a potential vaccination using daily risk equations. we address the following research questions: . could vaccination result in a net increase in risk? . can we determine conditions that optimize vaccine efficacy? . which is more important to maximise, vaccination efficacy or use? in order to evaluate the potential effect of vaccination, we develop risk equations for the daily risk of ebola infection both currently and after vaccination. when hazmat suits, reduced contact with the living or dead or vaccination is employed, the probability that ebola is transmitted is reduced from the probability β to β (where β < β). we have modelled single exposure events, and thus the transmission probability of each event is significantly less than the transmission probability over the duration of the outbreak. if β is the probability of transmission during a single exposure event with a given protection type (hazmat suit, reduced contact, vaccination, any combination thereof, or no protection), then the probability of remaining uninfected during a single exposure event is ( − β ). the probability of remaining uninfected after n exposure events is thus ( − β ) n . thus the probability of ebola infection for an individual is where p i is the proportion of times a given protection protocol is employed (including no protection). the risk equations account for the basic transmission probability of ebola (β), the lowered risk due to various protection protocols: hazmat suits (e h ), reduced contact with the infected living (e c i ), vaccination (e v ) and reduced contact with dead bodies (e c d ). we denote the use of no protection by − β. our risk equations also include the proportions of use (individually or in combination) of these protection protocols. we denote these proportions by p i if applied before vaccination and q i if applied after. the proportion of use of all protection types sums to in each case. since ebola is a pervasive disease with extreme transmission rates, we focus our analysis on the higher range of transmissibility; i.e., when the basic risk of transmission (β) is between . and . as the exact transmission rate of the virus is still unknown, we have modelled low ( - %), moderate ( - %) and high ( - %) levels of transmissibility to analyze how the performance of different protection protocols can change across these levels of transmission. currently (when the only protection protocols are to reduce contact with the infected living and dead (e c i , e c d ), hazmat suits (e h ) and no protection ( − β)), the daily risk of contamination for a susceptible individual is where p , p , p , p , p , p and p are the proportions of use of protection protocols outlined in table . post-vaccine (when the available protection protocols are vaccine (e v ), reduced contact with the infected living and dead (e c i , e c d ), hazmat suits (e h ) and no protection ( − β)), the daily risk of contamination is where q , q , ..., q are the proportions of use of protection protocols available after the vaccine is introduced. parameter values are given in table . initially, we assumed a uniform use of all protection protocols available (including using no protection). there are eight possible combinations of protection protocols: p through p and the fraction of people using no protection ). as such, the upper bound of the range of values for each proportion is set to . (see table ). by equating the current daily risk of contracting the disease, r , and the daily risk after the introduction of the vaccine, r , we obtain an analytical expression for the threshold level of vaccine efficacy and use that would be necessary to j o u r n a l p r e -p r o o f induce a net decrease in the daily risk of infection: the risk ratio r * serves as a threshold indicating when the introduction of a vaccine can improve the situation. in particular, r * provides information on the levels of vaccine efficacy and use needed to reduce the daily risk of contamination. the benchmark value for r * is . at this value, the risk before the vaccine is identical to the risk after vaccination of catching ebola, which means that the introduction of a vaccine has no effect on the daily risk of infection. the vaccine will be favorable if r * > and detrimental (i.e., increase the daily risk) if r * < . we evaluated the impact of potential vaccination using latin hypercube sampling (lhs) and partial rank correlation coefficients (prccs) to explore the sensitivity of the daily risk of infection to parameter variations. lhs is a statistical sampling method that generates a quasi-random sampling distribution. it allows for an efficient analysis of parameter variations across simultaneous uncertainty ranges in each parameter [ , ] . prccs rank each parameter by the amount of effect on the outcome, regardless of whether that effect is positive or negative [ ] . we performed an uncertainty analysis using monte carlo simulations and multivariate sensitivity analysis to analyze our risk equations. we computed the risk over a wide parameter space to assess the variability of our dependent variable (risk per day of catching ebola r or r ), using lhs to sample parameter space. finally, we evaluated the potential risk-reduction effect of vaccination depending on the fraction of vaccinated people. a candidate vaccine will be beneficial (i.e., decrease the daily risk of infection) if the risk of transmission after the introduction of the vaccine is lower than the current risk. to show which parameters the daily risk is most sensitive to, we used plots of prccs for each of the independent parameters. parameters with prcc > increase a susceptible's daily risk as the parameter value increases, whereas parameters with prcc < decrease a susceptible's daily risk as the parameter value increases. j o u r n a l p r e -p r o o f figure illustrates the degree of sensitivity of the daily risk of infection before the vaccine is introduced. at relatively low transmissibility levels, reducing contact with infected individuals (e c i ) is the most effective alternative to decrease the daily risk of infection. when the rate of transmission is moderate and there is no vaccine, wearing hazmat suits and reducing contact with the dead has a better protective impact. as the transmission rate rises, a higher proportion of use of those protection protocols becomes necessary. when the transmission probability is high and there is no vaccine available, all protection protocols are similar in terms of their efficacy, but none has a substantial effect on protecting against the virus. it is here that the introduction of a vaccine will benefit populations at risk the most. the remainder of our focus is on the case of high transmissibility when β ranges from % to %. we analysed the impact of a potential vaccine with efficacy that ranged from moderate to high (e v ranges from . to ). figure illustrates the effect of the transmission rate (β) on the post-vaccination daily risk of infection (r ) when susceptibles use the vaccine as their sole protection protocol. the proportion of vaccine use q ranges from to (see table ). the red dots represent the current risk of infection, and the blue dots represent the risk after vaccination. as shown in figure , introducing the vaccine could result in one of two outcomes: in some cases, the vaccine may reduce the risk of contamination, but in others, we run the risk of having more cases following the introduction of an ebola vaccine. as the outcome is uncertain, it is important to understand which variables play a role in effectively decreasing the risk of infection and what levels are needed for these parameters. figure shows that a large fraction of the population must be vaccinated (> %) to achieve a % decrease in the daily risk of infection. figure illustrates the ideal situation where everyone in an area at risk gets vaccinated. in this case, q = . at any level of transmissibility, the daily risk of catching ebola is significantly less than without the vaccine. a strong vaccine (> % efficacy) leads to a % decrease in the daily risk of ebola if everyone receives it. we can reasonably infer that, with an effective vaccination campaign and more awareness about how ebola is transmitted, we can reach a stage of disease control if the vaccine is not too weak. figure illustrates three scenarios: currently (no vaccine), the vaccine being the sole protection protocol available (no protection is also an option) and with the vaccine being the only protection protocol used by everyone. without a vaccine, the daily risk of infection r remains above %. in the second scenario, the candidate vaccine is introduced, and people who initially used a protection protocol now replace it with the vaccine, while those who have not used protection continue to do so. the median post-vaccine risk of infection r is less than % in this case. however, the range of r is wide ( % ≤ r ≤ %), which implies a high volatility in the daily risk. in the third scenario, the ideal case of absolute vaccine uptake, the median per day-risk r drops to %, ranging from to %, a net decrease from the initial risk r of % and a decrease in volatility. this indicates that a high rate of vaccine uptake would be required to continuously maintain the risk of infection below its current value. figure shows prccs of the parameters that affect r * . for general vaccine uptake, the fraction of people who get vaccinated q is the only variable that has a significant impact on r * (figure (a) ). when the vaccine uptake is high ( . ≤ q ≤ ), figure (b) shows that the transmission probability β and vaccine efficacy e v affect r * significantly. figure shows that there is a threshold whereby the fraction of people who use the vaccine (q ) is guaranteed to increase the risk is if it sufficiently low (< %) but guaranteed to decrease the risk if it is sufficiently high (> %), regardless of the protective behavior or lack thereof. if a vaccine is introduced, it must have at least % efficacy, and at least % of people in the affected areas must receive it to start seeing an effective reduction in the risk of disease. to halve and control the risk of an ebola outbreak, at least % of people need to be vaccinated with a vaccine that is at least % effective. vaccine uptake has a stronger impact on disease control than vaccine efficacy. finally, we examined some possibilities for parameter ranges beyond the ranges given in table . in particular, we constrained the eight pre-vaccination proportions as ≤ p j ≤ . in order to consider all protection protocols equally. however, in order to stress test this, we examined three potential scenarios: (a) in the absence of hazmat suits, (b) if contact with the living was unchanged and (c) if contact with the dead was unchanged. these possibilities may arise if hazmat suits are not available, if people fail to follow social distancing or if funeral practices are unchanged. note that the absence of a protection protocol also implies its absence in any combination protocol. thus, for (a), we not only have p = but also p = p = p = , but we increased the range of the remaining protection protocols to ≤ p , p , p ≤ . . the results are illustrated in figure . in the absence of hazmat suits, the most significant protocol is p , the fraction of people reducing contact with the infected living and dead bodies. the next most significant parameters are e c i and e c d , the effectiveness of contact reductions with the living and the dead, respectively. that is, in the absence of hazmat suits, the combination of remaining protection protocols becomes crucial. likewise, if contact with the living is unchanged, then the most significant parameter is p , the fraction of people who use hazmat suits and reduce contact with the dead, followed by the efficacies of these two protection protocols. finally, if contact with the dead is unchanged, then p , the fraction of people who use hazmat suits and reduce contact with the living, becomes the most significant parameter, followed by the efficacies of these two protection protocols. according to our results, the parameters with the greatest effect on the ebola epidemic are the transmission probability (β), the fraction of people using the vaccine (q ), vaccine efficacy (e v ), reducing contact with dead bodies (e c d ) and wearing hazmat suits (e h ). the increased risk due to dead bodies is likely due to the fact that infectious dead bodies remain infectious for up to a week after death j o u r n a l p r e -p r o o f [ ] . safe burial methods enforced by the us centers for disease control (cdc) have proven useful in reducing case incidence rates and hence transmissibility [ ] . the transmission probability is the most influential parameter in our risk equations. our results indicate that any efforts to reduce this transmission, such as a vaccine, would have a significant effect on reducing the overall epidemic if populations can be sufficiently educated about the necessity for high vaccine uptake. in the absence of a vaccine, we also showed that if one of the protection protocols is not followed, then the combination of the remaining protection protocols becomes crucial. several interventions have already been used to reduce the spread of ebola including extensive awareness campaigns meant to educate susceptible individuals on transmission, symptoms and health risks of the disease. quarantine of individuals found through aggressive contact tracing and isolation of infected individuals while they are treated have also been effective methods to reduce the daily risk of infection [ ] . the cdc has employed safe burial teams, which are tasked with safely burying corpses [ ] . this results in less contact between susceptible individuals and infected dead bodies. there have been recent trials for an ebola vaccine, some of which have been promising [ ] . an excellent vaccine would reduce the case incidence rate; however, a reasonably good vaccine that can be administered to most people would likely lower the transmissibility and help to control disease spread. our model has several limitations, which should be acknowledged. initially, before a vaccine is introduced, we assumed a uniform use of all protection protocols available, including using no protection, which may not hold as populations become more aware of the disease and adjust their protective behaviours. we assumed the same transmission probability for the living and dead, which may not be true. after the recent outbreak in west africa, efforts to develop an effective vaccine against the ebola virus disease have increased. we have shown that a vaccine can control the epidemic, even when transmission rates are high (above %). we found that at a high transmission rate of ebola, a large fraction of the population must be vaccinated (> %) to achieve a % decrease in the daily risk of infection. if a vaccine is introduced, it must have at least % efficacy, and almost everyone in the affected areas must receive it to effectively control outbreaks of ebola. these results indicate that a low-efficacy ebola vaccine runs the risk of having vaccinated people be overconfident in a weak vaccine and hence the possibility of the vaccine making the situation worse, unless the population can be sufficiently educated about the necessity for high vaccination rates. the question of how effective and widely distributed a vaccine needs to be to have a beneficial impact on public health extends far beyond the application to ebola. given the economic costs and personal inconvenience of the mitigation strategies in use for covid- , for example, many of the existing strategies may end once a vaccine becomes available. hence, more modelling along these lines will provide further insights to other infectious diseases. j o u r n a l p r e -p r o o f table : parameter values before and after vaccine introduction. we assumed a uniform use of all available protection protocols (including no protection) before the vaccine. thus, there are eight possible combinations of protection protocols: p through p and no protection ( − p − p − p − p − p − p ). the upper bound of the range of values for each proportion is equal to . . after the introduction of a vaccine, we considered the extreme case where vaccination was the only available protection protocol ( < q < ) and that the vaccine was at least % effective. fraction of people using hazmat suits - . p fraction of people reducing contact with the infected living and dead bodies - . p fraction of people using hazmat suits and reducing contact with dead bodies - . p fraction of people using hazmat suits and reducing contact with the infected living - . p fraction of people using hazmat suits and reducing contact with the infected living and dead bodies - . q fraction of people reducing contact with dead bodies q fraction of people reducing contact with the infected living q fraction of people using hazmat suits q fraction of people reducing contact with the infected living and dead bodies q fraction of people using hazmat suits and reducing contact with dead bodies q fraction of people using hazmat suits and reducing contact with the infected living q fraction of people using hazmat suits and reducing contact with the infected living and dead bodies q fraction of people using the vaccine - q fraction of people using the vaccine and reducing contact with the infected living q fraction of people using the vaccine and wearing hazmat suits q fraction of people using the vaccine and reducing contact with dead bodies q fraction of people using the vaccine, wearing hazmat suits and reducing contact with dead bodies q fraction of people using the vaccine, wearing hazmat suits and reducing contact with the infected living q fraction of people using the vaccine and reducing contact with dead bodies and the infected living figure : prccs before vaccine introduction for low ( - %), moderate ( - %) and high ( - %) levels of transmissibility. the outcome variable is the daily risk of infection before vaccination, r . figure : sensitivity of the daily risk of ebola to transmission rates when the only protection protocol available is the vaccine. the red dots represent the current daily risk of infection while the blue dots represent the post-vaccine daily risk of contamination. the proportional vaccine uptake q ranges from to and the transmission rate β is between . and . . figure : sensitivity of the daily risk of ebola to the effectiveness of the vaccine e v , its proportion of use q and the fraction of people not using any protection. the proportional vaccine uptake q varies from to , and vaccination is the only available protection protocol. the transmission rate β is between . and . . figure : sensitivity of the daily risk of ebola to transmission rates and vaccine efficacy when all susceptible individuals get vaccinated. the red dots represent the current risk of infection while the blue dots represent the post-vaccine risk of ebola. this is an ideal case, and the proportion of use of the vaccine q is set to . the transmission rate β is between . and . . figure : boxplots of sampled values using the lhs ranges from table . the horizontal red line indicates the median value. we present the daily risk of ebola under three scenarios: before the vaccine, imperfect vaccine uptake ( < q < ) and perfect vaccine use (q = ). figure : prccs for parameters that affect r * . (a) the case for general vaccine uptake ( < q < ). (b) the case for high vaccine uptake ( . < q < ).the transmission rate β is between . and figure : sensitivity of the threshold level r * to (a) the fraction of people using the vaccine q and (b) the fraction of people not using any protection. the transmission rate β is between . and . inset: the range q > % that guarantees risk reduction. figure : prccs for some possibilities outside the ranges in table : (a) the absence of hazmat suits, (b) when contact with the living infected is unchanged and (c) when contact with dead bodies is unchanged. in each case, the most significant influence on the outcome is the combination of the remaining protection protocols, followed by the efficacies of those remaining protection protocols. estimating the reproduction number of ebola virus (ebov) during the outbreak in west africa mathematical modeling of the assessment of the risk of ebola virus transmission from bodily fluids and fomites sensitivity and uncertainty analysis of complex models of disease transmission: an hiv model, as an example predicting the unpredictable: transmission of drug-resistant hiv a tale of two futures: hiv and antiretroviral therapy in san francisco modeling contact tracing in outbreaks with application to ebola testing modeling assumptions in the west africa ebola outbreak public health intelligence: learning from the ebola crisis the basic reproductive number of ebola and the effects of public health measures: the cases of congo and uganda transmission dynamics and control of ebola virus disease (evd): a review the ebola outbreak, - : old lessons for new epidemics modeling the spread of ebola. osong public health and research perspectives ebola haemorrhagic fever. the lancet early epidemic dynamics of the west african ebola outbreak: estimates derived with a simple two-parameter model efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial an overview of ebola virus disease global cardiovascular risk in patients with hiv infection: concordance and differences in estimates according to three risk equations (framingham, score, and procam) understanding the dynamics of ebola epidemics ebola and marburg haemorrhagic fever viruses: major scientific advances, but a relatively minor public health threat for africa dynamics and control of ebola virus transmission in montserrado, liberia: a mathematical modelling analysis modeling the role of public health education in ebola virus disease outbreaks in sudan vesicular stomatitis virus-based ebola vaccines with improved cross-protective efficacy vesicular stomatitis virus-based vaccines for prophylaxis and treatment of filovirus infections immunomonitoring of human responses to the rvsv-zebov ebola vaccine. current opinion in virology validation of the atherosclerotic cardiovascular disease pooled cohort risk equations improving burial practices and cemetery management during an ebola virus disease epidemic -sierra leone increased risk of cardiovascular disease (cvd) with age in hiv?positive men: a comparison of the d: a: d cvd risk equation and general population cvd risk equations the survival of filoviruses in liquids, on solid substrates and in a dynamic aerosol postmortem stability of ebola virus modeling the impact of interventions on an epidemic of ebola in sierra leone and liberia wild animal mortality monitoring and human ebola outbreaks, gabon and republic of congo ebola virus: promising vaccine candidates ebola vaccine: how far are we? performance of the uk prospective diabetes study risk engine and the framingham risk equations in estimating cardiovascular disease in the epic-norfolk cohort evaluating the potential impact of vaginal microbicides in reducing the risk of hiv acquisition in female sex workers computational modelling and optimal control of ebola virus disease with non-linear incidence rate multinational assessment of accuracy of equations for predicting risk of kidney failure: a meta-analysis using sensitivity analysis to examine the effects of an ebola vaccine ebola vaccines in clinical trial: the promising candidates a model of the ebola epidemics in west africa incorporating age of infection single-vector, single-injection recombinant vesicular stomatitis virus vaccines against high-containment viruses report of an international commission ebola virus disease in nonendemic countries acknowledgements rs? is supported by an nserc discovery grant. for citation purposes, please note that the question mark in "smith?" is part of his name. key: cord- -biwnqa f authors: luke, anthony; d'hemecourt, pierre title: prevention of infectious diseases in athletes date: - - journal: clinics in sports medicine doi: . /j.csm. . . sha: doc_id: cord_uid: biwnqa f the sports medicine physician may face challenging issues regarding infectious diseases when dealing with teams or highly competitive athletes who have difficulties taking time off to recover. one must treat the individual sick athlete and take the necessary precautions to contain the spread of communicable disease to the surrounding team, staff, relatives, and other contacts. this article reviews preventive strategies for infectious disease in athletes, including immunization recommendations and prophylaxis guidelines, improvements in personal hygiene and prevention of spread of infectious organisms by direct contact, insect-borne disease precautions, and prevention of sexually transmitted diseases. a special emphasis on immunizations focuses on pertussis, influenza, and meningococcal prophylaxis. i nfections in sports can be serious medical problems. they can affect individual athletes, resulting in morbidity and decreased performance [ ] . they can also be spread to other athletes, putting them at risk for similar disease and complications. the sports medicine physician may face challenging issues regarding infectious diseases when dealing with teams or highly competitive athletes who have difficulties taking time off to recover. one must treat the individual sick athlete and take the necessary precautions to contain the spread of communicable disease to the surrounding team, staff, relatives, and other contacts. the authors aim to translate recommendations in public health to practice in a sports medicine setting. sports physicians have an opportunity to see athletes during adolescence and adulthood who might not come in for routine health maintenance except when related to their sports. primary prevention of infectious disease, the ideal goal, deals with avoiding the development of the disease before infection occurs. immunizations are an example of primary prevention and have been the most successful public health programs for disease prophylaxis. secondary prevention for infection control involves prevention of spread to others. athletes are often exposed to many different people, travel to compete in various environments locally and internationally, and engage in higher-risk activities, often in close contact with others [ ] . the authors discuss the preventive strategies for infectious disease in sport, including ( ) a review of immunization recommendations and prophylaxis guidelines, ( ) improvements in personal hygiene and prevention of spread of infectious organisms by direct contact, ( ) insect-borne disease precautions, and ( ) prevention of sexually transmitted diseases (stds). a special emphasis on immunizations focuses on pertussis, influenza, and meningococcal prophylaxis. athletes may share personal items (eg, towels, water bottles, and soap) and equipment (eg, weights). they may live in dormitories or in hotel rooms while traveling, which leads to close contact and high exposure to teammates. they participate in higher-risk activities [ , ] . fewer athletes practice safe sex, which can lead to stds in homosexual and in heterosexual individuals. athletes also use more illicit drugs and alcohol, which can place them at risk of intravenous needle exposure [ , ] . steroids, hormones, and vitamins are other substances that some athletes are injecting [ , ] . tattoos are popular among athletes, which are another source of needle infection risk. active immunization involves the administration of all or part of a microorganism or a modified product of that microorganism, such as an antigen or protein. this administration stimulates an immune response to develop protection against future exposure to the infection [ ] . although most vaccines are over % effective, they are not guaranteed to promote immune protection [ ] . immunizations typically involve inactivated vaccines or live, attenuated viruses. inactivated vaccines include killed virus or bacterial proteins to stimulate one's immune system to develop antibodies to any similar virus or bacteria. more side effects are associated with the live, attenuated viruses-typically local pain and, rarely, hypersensitivity reactions to vaccine constituents. a mild febrile illness, a recent exposure to an infectious disease, pregnancy, breastfeeding, nonspecific allergies, and family history of an adverse event after immunization, including seizures, are not contraindications for immunization [ ] . sports medicine physicians need to consider the following indications for immunizations (tables and ) : ( ) routine health maintenance; ( ) catch-up immunizations for failed or missed immunizations; ( ) immunizations of high risk groups (ie, splenectomy, chronic disease, immunocompromised); ( ) travel to an endemic area; ( ) close contact with an infected individual, or ( ) recent potential exposure to an infectious agent. when doing preparticipation physical examinations, it is sometimes assumed that athletes have received all their immunizations. proof of immunizations is required by schools and colleges, although exceptions can be given to individuals refusing to receive immunizations. in a study surveying , minnesota children who entered kindergarten in , by months of age, % of students had received the measles, mumps, and rubella (mmr) vaccine, and only % had received their fourth dose of diphtheria, tetanus, and pertussis vaccine (dtap) [ ] . vaccination rates can vary substantially by age, race/ethnicity, and neighborhood [ ] . white, non-hispanic students usually have higher vaccination rates than children of other racial/ethnic groups. it has been estimated that % of whites, % of hispanics, and % of african americans are fully immunized [ ] . although mmr vaccines have been administered for many decades and incidences of disease are presently low [ ] , the diseases can still occur in the adult population. between and , % of the measles cases occurred in college students [ ] . enzyme-linked immunosorbent assay tests for antibodies to mmr are available to detect for immunization status [ ] . in a series of students, ( %) were found to be seronegative to measles alone, ( %) were seronegative to rubella alone, and ( %) were seronegative to measles and rubella. eighty-six percent of the individuals seronegative to measles had previously received a dose of measles vaccine. following a second injection, conversion to seropositive status rose to % and % for measles and rubella, respectively. these data support the need for a two-dose vaccine schedule [ ] . pneumococcal vaccine is administered to prevent streptococcus pneumoniae infections. a conjugate heptavalent is given to during the first years of life. a polysaccharide vaccine is provided to high-risk individuals older than years against types of streptococcus pneumonia that account for % of invasive disease [ ] . high-risk groups include patients who have asplenia, sickle cell disease, diabetes mellitus, cirrhosis, immunocompromised states, chronic cardiac or pulmonary disease, or age years or older. immunity following vaccination is successful for periods of to years, requiring booster injections [ ] . hepatitis b is a blood-borne virus transmitted through sexual contact and parenteral exposure to blood and blood components [ ] . hepatitis b has a greater risk for transmission in sports than hiv. the risk of hiv transmission is estimated to between in million games and in million games [ , ] . the risk arises if bleeding and skin exudates from an infected individual come into contact with open wounds in another athlete, particularly during contact and collision sports. there are no confirmed cases of spread of hiv through sports [ ] ; however, out of high school sumo wrestlers at one club developed hepatitis b [ ] . another case series reported on of american football players who developed hepatitis b over a period of months [ ] . contact through open wounds, cuts, and abrasions were the suspected routes of transmission. although hepatitis a is a considered immunization in athletes who are traveling to endemic areas, routine vaccination for hepatitis b is recommended for all individuals after birth using single or combination vaccines [ ] . a three-dose immunization schedule is typically used after years of age, with injections at months, month, and months, although there is an optional four-dose schedule [ ] . the licensed vaccines have had % to % efficacy of preventing hepatitis b, with immunity lasting years or longer [ ] . immunizations for hepatitis b should be checked during the preparticipation physical examination, and catch-up immunizations recommended to the individual (see table ). if individuals are uncertain about their immunization status, serologic testing for antibody to hepatitis b surface antigen can determine immunity. when athletes are known to be infected with hepatitis b, secondary prevention includes education on personal hygiene, appropriate management of open wounds, proper use of protective equipment, safe sex practices using a condom, and avoidance of intravenous blood transmission (eg, through needle sharing and illicit drug use). bordetella pertussis, which is responsible for whooping cough, is a gram-negative coccobacillus transmitted by way of airborne droplets [ ] . although tetanus and polio have been controlled well with the use of vaccines [ ] , the rate of pertussis cases has been increasing in adolescents and adults despite routine immunizations [ ] . most cases occur in patients years or older [ ] . the infection is most concerning for infants because immunity is not complete until older ages. the spread to infants is typically from adults. pertussis usually presents with nonspecific upper respiratory tract infection symptoms for to weeks (catarrhal stage), after which the paroxysmal and sometimes uncontrollable cough develops [ ] . the cough is not necessarily always followed by the classic ''whooping'' sound, and pertussis should be considered with any persistent, prolonged cough. the whole-cell pertussis vaccine is estimated to be approximately % effective [ ] . this vaccine is still recommended for use in the routine immunization of young children; however, the immunity provided begins to decline at to years following vaccination, which makes adolescents and adults susceptible [ ] . rare adverse reactions from the vaccine include hypotonic, hyporesponsive episodes, high fever, seizures, and anaphylaxis [ ] . two acellular vaccines have been introduced that are as effective as whole-cell vaccines and have fewer adverse reactions [ ] . these vaccines are combined with tetanus toxoid and reduced diphtheria toxoid (dtap). the centers for disease control and prevention (cdc) recommends use of these dtap boosters rather than the tetanus-diptheria (td) booster starting after to years of age [ ] . for pertussis, individuals are most contagious during the first to weeks during the catarrhal stage but should be considered contagious until weeks after the paroxysmal stage ends or after taking antibiotics for days [ ] . diagnosis of pertussis infection is best performed through polymerase chain reaction assay (sensitivity, %; specificity, %) or through direct fluorescent antibody testing (sensitivity, %; specificity, %). nasal swab cultures (sensitivity, %; specificity, %) are routinely performed; however, they have high false negative rates and take to days to yield results [ ] . physicians in the united states are legally required to report cases of pertussis to state public health departments [ ] . it is estimated that % of susceptible household contacts will be infected after close contact [ ] . antibiotic prophylaxis is recommended for close contacts of persons who have pertussis to prevent outbreaks [ ] . preferred drugs are azithromycin for days, clarithromycin for days, or trimethoprim-sulfamethoxazole or erythromycin for days, which are similar for prophylaxis and treatment [ ] . influenza presents with constitutional symptoms of fever, chills, malaise, fatigue, and myalgia in addition to upper respiratory tract symptoms of a sore throat, cough, and rhinitis. rarely, more serious conditions can occur, including encephalopathy, transverse myelitis, myocarditis, and pericarditis [ ] . immunogenicity is determined by hemaglutinins and neuraminidases on the virus surface. antigenic drift can occur that can mutate the virus into different strains. transmission occurs by way of respiratory droplets. the virus has an incubation period of usually days (range, - days), and adults are infectious from the day before symptoms begin to approximately days after the illness starts [ ] . symptoms usually last a week, although less likely, symptoms can last longer than weeks. these symptoms can be very disruptive for treatment and challenging for the athlete to keep training and competing. a case series of students, mostly healthy adolescents at a ski school in austria, reported a severe outbreak of influenza a, leading to an attack rate of %, with % becoming ill within days of the outbreak. two students were hospitalized with pneumonia and died [ ] . influenza vaccines contain strains of antigenically equivalent strains of influenza similar to those annually recommended: influenza a (h n ), influenza a (h n ), and a b virus. depending on the emergence and spread of new strains, other virus strains can be added to update the vaccine [ ] . the efficacy of influenza vaccine is approximately % to % for individuals under age years [ ] . vaccination for influenza should occur in the fall (october or november), at the beginning of the flu season (box ) [ ] . antibodies develop approximately weeks after vaccination [ , ] . inactivated influenza vaccine is generally appropriate for all populations requiring influenza vaccine. three influenza vaccines were available in the united states for the to season: fluzone (manufactured by sanofi-pasteur); fluvirin (manufactured by novartis); and fluarix (manufactured by glaxosmithkline). the typical dose is . ml administered intramuscularly, usually in the deltoid muscle. live, attenuated influenza vaccine (laiv) is approved for use in healthy, nonpregnant individuals aged to years. the laiv is administered by way of a nasal spray once in each nostril (flumist, manufactured by medimmune). individuals who have a hypersensitivity or anaphylactic reaction to components of the flu vaccine or to eggs should not be vaccinated [ ] . adults reported having a % reduction in severe febrile illnesses after laiv compared with placebo [ ] . side effects from laiv increased in adults within days of immunization compared with placebo and consisted mainly of nasal congestion ( . % versus . %) and sore throat ( . % versus . %), which lasted, on average, days. less common complaints were tiredness, cough, and chills. there was no significant difference in the number of mild febrile illnesses between immunization and placebo groups [ ] . injections can be scheduled to occur at the optimum time during the athlete's competitive schedule to minimize concern about side effects. when inactivated influenza vaccine shortages occurred in previous years, the vaccine was recommended for high-risk groups as priority; however, the general recommendation now is to offer the immunization annually to anyone who wishes to reduce the likelihood of being ill with influenza or transmitting the virus if they should become infected [ ] . although this policy cannot be directly translated into a benefit for the athlete, depending on the level of athlete, the use of the laiv may also be beneficial to prevent lost time from sport. influenza vaccine has been suggested for competitive athletes and essential personnel, especially before international events occurring during the influenza season [ , ] . treatment with antiviral medications can reduce the duration of uncomplicated influenza a and b illness by approximately day when administered within days of illness onset [ , ] . recommended antiviral treatment should be given for days [ ] . four antiviral agents are currently available: amantadine, rimantadine, zanamivir, and oseltamivir [ ] . the influenza a virus, however, has become resistant to amantadine and rimantadine, which are presently not recommended to be used as first-line drugs [ ] . zanamivir (ralenza, dry powder taken by orally inhaled route) and oseltamivir (tamiflu, capsule or oral suspension) are neuraminidase inhibitors and can be used to treat patients and to control influenza outbreaks in closed settings. although typically used in nursing homes, an outbreak in a dormitory may require chemoprophylaxis [ ] . there are limited data to suggest that serious complications from influenza, such as lower respiratory tract infections, may be reduced [ ] . the use of antiviral medications for prophylaxis of influenza is unclear and is not yet recommended for routine seasonal control [ ] . the use of oseltamivir, however, has been recommended in specific cases, especially if there is high risk of spread such as household contacts and if individuals have not been immunized [ ] . oseltamivir was used to treat of patients, including athletes during the salt lake city winter olympics, with medications given to close contacts, which was believed to limit the spread of influenza [ ] . clinical history and physical examination are still the mainstays for diagnosing influenza. rapid swab tests are available and take approximately minutes to detect the influenza virus. the tests are less sensitive ( %- %) and specific ( %- %) than the traditional viral cultures [ ] . they have moderate sensitivities for influenza antigens and are more likely to produce false negative rather than false positive results [ , ] . direct and indirect fluorescent antibody staining tests are also available, but they are ordered more in hospitals because they take to hours to obtain results [ ] . viral cultures are still the ''gold standard'' for confirming the presence of influenza and identifying the strains and subtypes [ ] . neisseria meningitides is a serious concern, especially for the adolescent and college populations. an alarming trend during the s demonstrated an increase in meningitis deaths in college students, with living in dormitories being a risk factor. the disease can be spread by asymptomatic carriers. students living in dormitories were to times more likely of getting infected than those living in other types of accommodations [ ] . freshmen who lived in dormitories had an elevated risk of meningococcal disease (odds ratio, . ; % confidence interval, . - . ; p ¼ . ) compared with other college students [ ] . aside from the risk of death, % to % of survivors of meningitis have serious sequelae such as neurologic disability, limb loss, and hearing loss [ ] . routine vaccination with meningococcal vaccine is recommended for college freshmen living in dormitories and for other populations at increased risk. the cdc advisory committee on immunization practices recommends routine vaccination of young adolescents ( - years old) with meningococcal vaccine (mcv ) at the preadolescent health care visit [ ] . therefore, sports medicine physicians may be faced with higher frequency of checking for meningococcal immunization status for high school and college athletes. a tetravalent conjugate vaccine (menactra, sanofi pasteur) is available against neisseria meningitidis isolates a, c, y, and w- in a . -ml single-dose vial. over the age of years, % of the meningococcal infections are caused by strains c, y, or w- (cdc, unpublished data, ) [ ] . another vaccine, menomune (aventis pasteur limited), has been licensed since and has a similar immunogenicity profile to menactra and is delivered subcutaneously as a . -ml dose. menactra and menomune have serum bactericidal protection ranging from . % and . % for strain w- and . % and . % for strain y, respectively [ ] . revaccination may be necessary for individuals at high risk after years [ , ] . common side effects with mcv were local pain in just over % of patients, followed by swelling, induration, and redness in approximately . % to . %. fever was reported in . % of children years old or younger and in . % of adults [ ] . close contacts are at high risk and should be treated with chemoprophylaxis ideally within hours of identifying the index patient [ ] . the goal of treatment is to reduce nasopharyngeal carriage of n meningitidis. after more than days after the onset of illness in the index patient, chemoprophylaxis is not necessary [ ] . a single dose of ciprofloxacin ( mg orally) or ceftriaxone ( mg by intramuscular injection), or rifampin ( mg twice a day for days) is recommended for adults. children between month and years old may take rifampin ( mg/kg every hours for days), or ceftriaxone ( mg intramuscularly) if younger than years [ ] . one dose of azithromycin ( mg) was also shown to eradicate n meningitidis and may represent another treatment option [ ] . human papillomavirus (hpv) is associated with % of cervical cancers and anogenital, head and neck, and nonmelanoma skin cancers. it is an std and can be diagnosed by abnormal cervical cell changes seen on pap smear [ ] . this is a common infection, especially in sexually active adolescents and university students [ ] . primary prevention is now possible with two new vaccines: a bivalent vaccine against hpv types and and a quadravalent vaccine against types , , , and . the vaccines have a three-dose schedule: , , and months (bivalent vaccine) and , , and months (quadravalent vaccine). at . years, the bivalent vaccine was effective for producing a persistent antibody response against hpv and , with more than % seropositivity and . % effectiveness ( % confidence interval, . - . ) in reducing the number of reported abnormalities on pap smear, colposcopy, or both [ ] . routine vaccination with three doses of quadrivalent hpv vaccine is recommended for girls to years old but can be started in girls as young as years. girls and women aged to years who have not been vaccinated previously or who have not completed the full vaccine series are recommended to receive a catch-up series. the vaccine is intended to be administered before potential exposure to hpv through sexual contact [ ] . secondary prevention involves checking the affected individual's partners for signs of genital warts and other stds. regular cervical screen is recommended. use of condoms and education on spread is important. hpv infection persists for life; however, the degree and duration of contagiousness is yet unknown [ ] . athletes traveling need to consider the endemic diseases in the geographic location where they are competing. they should be aware of risks of acquiring common diseases, their accommodations (urban versus rural), local foods, and customs. immunizations should ideally be planned or more months in advance to allow for adequate time to administer vaccines (table ). there are many resources for information about prevention of infectious diseases for travelers (table ) . mosquito-borne disease a number of arthropods, such as mosquitoes and ticks, can transmit diseases. mosquito-vector diseases include west nile virus, yellow fever virus, and dengue virus. west nile virus, a flavivirus, has demonstrated a seasonally endemic epidemiology with geographic variation in the united states, especially in california, arizona, and colorado [ , ] . this disease typically presents between july and october, although cases have presented between april and december. the prevention of mosquito bites is the cornerstone of prevention. an athlete in an endemic area should wear an insect repellant such as deet (n,n-diethyl-mtoluamide), picaridin (kbr- ), or oil of lemon eucalyptus (p-menthane- , diol). deet and permethin may be applied to the clothing [ ] . if a sunscreen is used concomitantly, the insect repellant should be applied on top of this and removed at the end of the day. long-sleeved shirts that are tucked into long pants are also useful. tick-borne diseases include rickettsial diseases, lyme disease, babesiosis, tickborne relapsing fever, and occasionally, tularemia and q fever (table ) . certain athletes who participate in rural outdoor activities are more susceptible to tick bites. these sports include cross-country running, training in multiple sports in rural areas, and recreational outdoor sports such as fishing and hiking. children are more at risk to tick bites. three more common rickettsial illnesses are rocky mountain spotted fever, human monocytotropic ehrlichiosis, and human granulocytotropic anaplasmosis [ ] . the infectious organisms responsible for these illnesses maintain their lifecycles in mammals and ticks. their prevalence reflects the geographic locations and the seasonality of the tick abundance. their season is usually from april to september, but they can present throughout the year. newer rickettsial diseases are emerging. these potentially lethal diseases are difficult to diagnose because they often mimic viral syndromes. as many as % to % of patients are initially misdiagnosed [ , ] . with rocky mountain spotted fever, more than % of cases are reported in the five states of north carolina, south carolina, tennessee, oklahoma, and arkansas [ ] . the presentation most often manifests as a sudden febrile illness with headache, myalgia, and a maculopapular rash that spreads in a centripetal pattern. rickettsia rickettsii has a predilection for endothelial cells and can cause a diffuse vasculitis and an untreated mortality of %. the diagnosis is based on clinical presentation, with epidemiologic, geographic, and seasonal considerations. laboratory testing may be supportive with thrombocytopenia and mild liver enzyme elevation. serologic testing is supportive only on a delayed basis with acute and convalescent titers. human monocytotropic ehrlichiosis and human granulocytotropic anaplasmosis can also present with acute headache, fever, and myalgia. laboratory evaluation often demonstrates leukopenia, thrombocytopenia, and transaminase elevation. common tick-borne illnesses that have been reported in the northeast united states are lyme disease and babesiosis, which are transmitted by the tick ixodes scapularis [ ] . babesiosis can cause a febrile illness and possibly life-threatening anemia and thrombocytopenia. lyme disease is a rickettsial disease caused by borrelia burgdorferi. as such, concurrent disease may be caused by the same tick bite (see table ). there are no proven vaccines for these tick-borne illnesses, but all are preventable by careful vigilance and protection. the key to prevention is to understand the regional epidemiology and seasonality of the diseases. vaccination for lyme disease (lymerix) was originally approved; however, the manufacturer took the vaccine off the market due to declining sales. there was a % efficacy after two doses and a % efficacy after three doses [ ] ; however, the protection diminished after years. ticks thrive in a wooded environment and at the edge of woods with surrounding high vegetation. ticks are uncommon in well-mowed lawns. relative tick-free zones can be created by placing wood chips or gravel around recreational areas to separate the woods [ ] . other landscape management tips include removing clippings and leaves, keeping stone walls clean of leaves, and restricting the use of groundcover, such as pachysandra, where pets and children may play. widening woodland trails andkeeping in the center of the trail while walking may be helpful. when traveling in wooded areas, light-colored clothing is helpful to identify the tick. long pants tucked into tightly woven socks and closed shoes minimize exposure. deet at % to % should be applied to the skin. permethrin may be applied only to the clothing. clothes should be removed and cleaned and dried after exposure. the clothes dryer is effective in killing ticks. one should carefully check for ticks in the nymphal phase that may be the size of a pin head. careful inspection should be done of the hair, ears, axilla, belly button, and legs. children and pets should also be checked. it is also important to monitor pets that may travel in the woods and return indoors. the technique of tick removal is critical. tweezers with fine tips should be used close to the skin and pulled directly away. squeezing the body may allow contamination of the disease into the host [ ] . lyme disease is not contracted until at least hours of tick adherence [ ] ; however, ehrlichiosis may transmit in less than hours. preventive antibiotics are generally not indicated because less than % of bites are lyme infected, especially with a flat tick. after a high-risk exposure (when the tick has been engaged for more than hours and is engorged), a single dose of mg of doxycycline is believed to be effective [ ] . most infectious diseases are spread from contact with the microorganism directly or indirectly from the infected individual. athletes frequently interact with teammates, coaches, athletic trainers, and physicians and share equipment, water bottles, towels, and supplies. this interaction is particularly a concern, with the recent outbreaks of methicillin-resistent staphylococcus aureus (mrsa) infections among sports teams [ , ] . three categories of potential risk factors for spreading infection have been suggested: ''sharing'' (sharing soap/towels/water bottles with teammates), ''skin injury'' (cuts, abrasions), and ''close contact'' (locker adjacent to infected teammate, living on-campus) [ ] . good personal hygiene can help reduce colonization of bacteria. bacterial counts can range from to million colony-forming units per square centimeter on the hands [ ] . universal body fluid precautions-for example, using disposable gloves when examining the oral cavity or wounds and frequent hand washing-can reduce the risk of infection. mrsa is transmitted from an infected patient to the gloves of a health care worker in approximately % ( %- %). physicians, in particular, have a low compliance for using gloves and washing their hands [ ] . proper surgical hand washing is recommended to be to seconds with soap, a -second rinse with water, followed by complete drying with a towel. the use of rinses and gels with concentrations of % to % alcohol take seconds to use and are effective at killing organisms [ ] . the use of chlorhexidine soap has been useful for reducing mrsa infections. viruses and bacteria can exist on equipment. mrsa was found in the taping gel and whirlpool in the training facilities of a professional football team [ ] . using diluted bleach ( part bleach in parts water) to cleanse training areas and equipment is recommended [ ] . routine cleaning schedules for shared equipment should be established and recorded. for upper respiratory tract infections, isolation of those who have had close contact with someone who has a confirmed or suspected infection, especially those who have active symptoms such as persistent fever and cough, is an effective and practical method of avoiding contact [ ] . any athlete who has a scratch, abrasion, or laceration or who has potentially infectious skin lesions such as vesicular or weeping skin lesions should be removed from play until the area can be securely covered with occlusive bandages or dressings to prevent leakage of blood or serous fluid [ ] . uniforms with fresh blood should be removed and replaced immediately after stopping any bleeding. bleach diluted with tap water in a : ratio can be used to wash equipment that has had contact with blood or body fluid. body substance precautions should be taken by health care professionals at all times when treating open wounds. one type of bacteria that has become more common in the hospital and a community-acquired infection is mrsa. although contact sports such as wrestling and football have been commonly associated with mrsa spread, this infection has also been discovered in minimal-contact sports such as fencing [ ] . three factors are associated with mrsa spread in sports. first, even with sports that have minimal contact, there are often abrasions and chaffing from clothing and hot environments. second, equipment is often shared and there is potential for transmission of bacteria. third, many sports have sufficient skin-to-skin contact to transmit organisms. subsequently, health care providers should strongly encourage good overall and hand hygiene in addition to covering all wounds and limiting shared equipment. it is crucial to have an ample supply of soap and water and alcohol-based hand cleansers. athletes, staff, and coaches should be educated in proper first aid for wounds, in recognition of wounds that are potentially infected, and in seeking medical attention for lesions that have concerning signs, especially large pustules or boils. athlete's foot, tinea pedis, is a common ailment not only during the hot summer months but also during the winter months with indoor sports. a number of prevention items include washing feet daily; drying between the toes; wearing cotton, nonsynthetic socks; wearing bathing shoes in public showers; and wearing sandals in warmer weather. jock itch, tinea cruris, is best prevented by showering immediately after athletic endeavors and wearing cotton briefs. a good talc powder may be used for prevention of athlete's foot and jock itch. ring worm, tinea corporis, is best prevented by avoiding contact. contact athletes such as wrestlers should not participate until any lesions have cleared or can be safely and effectively covered. athletes may manifest risk-taking behavior and subsequently be at increased risk for stds [ ] . the preparticipation examination affords the opportunity for the clinician to address these concerns. the cdc has emphasized the five intervention strategies [ ] , which include education on sexual behavior, identification of asymptomatic individuals, diagnosis and treatment of infected individuals, counseling of sexual partners of persons who have an std, and pre-exposure vaccination when applicable. individuals at risk should be questioned about partners regarding number and same or opposite sex. information about the type of sexual activity, the use of protection, and history of previous stds should also be identified. preventive measures for an std include abstinence if an individual or partner is actively infected and undergoing treatment. pre-exposure prophylaxis is relevant in several situations. hepatitis b vaccine is recommended in all individuals potentially exposed to stds. hepatitis a vaccine is recommended for all men who have sex with men or users of illicit drugs (injectable and noninjectable). for girls and women aged to years, the new quadrivalent vaccine for hpv types , , , and is recommended due to the higher associated risk of cervical cancer. most condoms are made of latex and are quite effective in std prevention. in one study of partners of hiv-infected individuals, partners were % less likely to seroconvert than those who did not use condoms [ ] . the male condom can also reduce the transmission of gonorrhea, chlamydiosis, and trichomiasis [ ] . there may be some added protection against herpes simplex virus and a % reduction of hpv transmission [ , ] . when an individual is allergic to latex, certain polyurethane condoms are likely just as effective, although they may break more readily. conversely, natural-membrane condoms such as lambskin are too porous to be used for std prevention. only water-based lubricants should be used with latex condoms because oil bases will weaken the latex. the female condom is a double-ringed polyurethane sheath that is used vaginally and during anal receptive intercourse that is effective in limited trials in preventing hiv/stds [ , ] . spermicides and nonbarrier contraception have no role in std prevention. finally, providers should encourage patients who have stds to notify their partners. often, this notification is pursued by the public health department. in the event of exposure to hiv by sexual exposure or needle stick, hiv prophylaxis is often undertaken and should be immediate. prophylactic treatment usually involves the hospital infectious disease division to determine the best combination therapy. education is paramount in public health and in the prevention of infectious diseases. athletes are a high-risk population often due to their increased exposure to different people and environments and, sometimes, their outgoing lifestyle behaviors. primary prevention can be promoted through accurate immunizations; appropriate, planned health maintenance; good hygiene practices; and behavior modification to minimize high-risk activities. secondary prevention can be achieved through vigilant surveillance for reportable illnesses, proper education and containment for reducing spread if an illness occurs, and timely prophylaxis with medications and immunizations in certain cases. virus infections and sports performance-a prospective study lifestyles and health risks of collegiate athletes: a multi-center study an outbreak of measles at an international sporting event with airborne transmission in a domed stadium influenza vaccination for athletes? pneumococcal vaccine for olympic athletes and visitors to spain. barcelona olympic organizing committee medical and public health services at the atlanta olympic games: an overview summary of notifiable diseases-united states severe acute respiratory syndrome and sport: facts and fallacies prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) sports participation and health-related behaviors among us youth aids in a bodybuilder using anabolic steroids hiv infection associated with injections of anabolic steroids blood borne infections in sport: risks of transmission, methods of prevention, and recommendations for hepatitis b vaccination the infectious complications of anabolic-androgenic steroid injection active and passive immunization a statewide survey of immunization rates in minnesota school age children: implications for targeted assessment and prevention strategies swanson's family practice review: a problem-oriented approach evaluation of a two-dose measles, mumps, and rubella vaccination schedule in a cohort of college athletes antibody response of pneumococcal vaccine: need for booster dosing? bleeding injuries in professional football: estimating the risk for hiv transmission an outbreak of hepatitis b in members of a high school sumo wrestling club horizontal transmission of hepatitis b virus among players of an american football team a comprehensive immunization strategy to eliminate transmission of hepatitis b virus infection in the united states: recommendations of the advisory committee on immunization practices (acip) part : immunization of infants, children, and adolescents red book: report of the committee of infectious diseases pertussis: a disease affecting all ages duration of immunity against pertussis after natural infection or vaccination summary of notifiable diseases-united states randomised controlled trial of two-component, three-component, and five-component acellular pertussis vaccines compared with wholecell pertussis vaccine. ad hoc group for the study of pertussis vaccines systematic review of the effects of pertussis vaccines in children preventing tetanus, diphtheria, and pertussis among adolescents: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccines recommendations of the advisory committee on immunization practices red book: report of the committee of infectious diseases comparison of pcr, culture, and direct fluorescent-antibody testing for detection of bordetella pertussis recommended antimicrobial agents for the treatment and postexposure prophylaxis of pertussis: cdc guidelines influenza a outbreak among adolescents in a ski hostel effectiveness of influenza vaccine in health care professionals: a randomized trial parenteral influenza vaccination induces a rapid systemic and local immune response safety, efficacy, and effectiveness of live, attenuated, cold-adapted influenza vaccine in an indicated population aged - years effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial study indicates influenza vaccine beneficial for college athletes safety and efficacy of the neuraminidase inhibitor zanamivir in treating influenza virus infection in adults: results from japan. gg group effectiveness of neuraminidase inhibitors in treatment and prevention of influenza a and b: systematic review and meta-analyses of randomised controlled trials adamantane resistance among influenza a viruses isolated early during the - influenza season in the united states impact of oseltamivir treatment on influenza-related lower respiratory tract complications and hospitalizations antivirals for influenza in healthy adults: systematic review treatment and prevention of influenza: swedish recommendations evaluation of diagnostic tests for influenza in a pediatric practice influenza diagnosis and treatment in children: a review of studies on clinically useful tests and antiviral treatment for influenza prevention and control of meningococcal disease. recommendations of the advisory committee on immunization practices (acip) risk factors for meningococcal disease in college students control and prevention of meningococcal disease: recommendations of the advisory committee on immunization practices (acip) effectiveness of meningococcal serogroup c conjugate vaccine years after introduction azithromycin compared with rifampin for eradication of nasopharyngeal colonization by neisseria meningitidis risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females genital human papillomavirus infection: incidence and risk factors in a cohort of female university students sustained efficacy up to . years of a bivalent l virus-like particle vaccine against human papillomavirus types and : follow-up from a randomised control trial acip provisional recommendations for the use of quadrivalent hpv vaccine red book: report of the committee of infectious diseases the incidence of ehrlichial and rickettsial infection in patients with unexplained fever and recent history of tick bite in central north carolina the epidemic of west nile virus in the united states laboratory evaluation of mosquito repellents against aedes albopictus, culex nigripalpus, and ochierotatus triseriatus (diptera: culicidae) physician knowledge of the diagnosis and management of rocky mountain spotted fever: mississippi rocky mountain spotted fever: clinical, laboratory, and epidemiological features of cases rocky mountain spotted fever in the united states, - endemic babesiosis in another eastern state: new jersey vaccination against lyme disease with recombinant borrelia burgdorferi outer-surface lipoprotein a with adjuvant. lyme disease vaccine study group prevention of lyme disease effect of tick removal on transmission of borrelia burgdorferi and ehrlichia phagocytophila by ixodes scapularis nymphs duration of tick bites in a lyme disease-endemic area prophylaxis with single-dose doxycycline for the prevention of lyme disease after an ixodes scapularis tick bite a clone of methicillin-resistant staphylococcus aureus among professional football players methicillin-resistant staphylococcus aureus in a high school wrestling team and the surrounding community recurring methicillin-resistant staphylococcus aureus infections in a football team on washing hands an investigation of contact transmission of methicillin-resistant staphylococcus aureus transmission of blood-borne pathogens during sports: risk and prevention methicillin-resistant staphylococcus aureus infections among competitive sports participants-colorado sexually transmitted diseases treatment guidelines effectiveness of condoms in preventing sexually transmitted infections a randomized controlled trial to reduce hiv transmission risk behaviors and sexually transmitted diseases among women living with hiv: the willow program effect of condoms on reducing the transmission of herpes simplex virus type from men to women condom use and the risk of genital human papillomavirus infection in young women use-effectiveness of the female versus male condom in preventing sexually transmitted disease in women use of reality ''female condoms'' for anal sex by us men who have sex with men. hivnet vaccine preparedness study protocol team key: cord- - qrht uq authors: van der zeijst, bernard a.m. title: infectious diseases know no borders: a plea for more collaboration between researchers in human and veterinary vaccines date: - - journal: vet j doi: . /j.tvjl. . . sha: doc_id: cord_uid: qrht uq nan infectious diseases know no borders: a plea for more collaboration between researchers in human and veterinary vaccines our present society would not be sustainable without vaccination, which is an essential and cost effective strategy to prevent, control and even eradicate infectious diseases in man and animals. a special report in the economist published on rd may entitled the world goes to town noted that > % of mankind and our companion animals now live together in big cities. moreover, animals for meat production are farmed in ever larger groups, and the movement of humans and animals has increased tremendously. under these circumstances, infectious diseases (that know no borders anyway) spread easily and vaccination are needed to stop or slow down serious disease outbreaks. despite the global importance of vaccines, the knowledge and experience to produce and develop them is limited to an estimated , specialists worldwide. most of these experts work for pharmaceutical companies or specialised research institutes and strive to translate a proof of concept from academic research into a vaccine that is not only commercially viable but also safe, efficacious and can be produced consistently under proper quality regimens, such as good manufacturing practice. following many mergers in recent years there are now only a handful of major commercial players still producing and developing veterinary and human vaccines, largely reflecting the complexity of the vaccine business and the escalating rigour of the quality standards. some vaccines approved in the past would certainly not be acceptable under the current regulatory framework. the development of a vaccine typically requires years and will cost hundreds of millions of euros. for human vaccines, the first . years and around % of the investment are needed for preclinical development (struck, ) . when successful, the result is a 'proof of principle', which means that the prototype vaccine has demonstrated that it protects in an animal model. the remainder of the time is then needed to show that the vaccine also protects the target animal (in this case man), that it is safe and can be produced consistently and eco-nomically. for veterinary vaccines the procedure is comparable, but it is usually possible to test the prototype vaccine directly in the target animal. all the results are summarised in a dossier that is then submitted to the regulatory authorities, which decide whether the vaccine may be admitted to the market. there are separate regulatory authorities for veterinary and human vaccines. theoretically, the same vaccine against a zoonotic disease could be used both in animals and man, but two different filings would be necessary. only / attempts to develop a vaccine is successful (struck, ) . this, of course, affects the choice of vaccine projects by industry. since there is an urgent need to recoup the investment, vaccine projects with the best expectations for profit will be at the top of the list. vaccines against neglected diseases affecting the poorer regions of the world will inevitably therefore be low on the priority list. the development of new vaccines depends on the collaboration of academic researchers and vaccine specialists from industry. essential for good teamwork and synchronous working is that both parties understand the complete process of vaccine development. a paper published in this issue of the veterinary journal by jacco heldens and his colleagues does an excellent job in explaining this process (heldens et al., ) and should be required reading for every academic researcher who ventures into vaccine development. the paper also outlines new scientific developments in vaccine r&d. in this field there is so much to gain by more collaboration between academia and industry which could lead to improved methods, for example in the field of genomics to predict protection capability and make vaccine development faster and less expensive. shortened timelines for vaccine development would be an enormous asset for the development of vaccines against new or re-emerging viral diseases. vaccination is virtually the only way to control these diseases, but a development time of years is far too long for a www.elsevier.com/locate/tvjl available online at www.sciencedirect.com the veterinary journal ( ) - the veterinary journal contemporary threat such as severe acute respiratory syndrome (sars) or pandemic influenza. two lesser known examples underline this dilemma: bluetongue, a viral disease of ruminants (especially sheep), and chikugunya virus in humans are both transmitted by insects. in both cases the region where these insects roam has increased considerably in recent years. in the case of bluetongue, warmer temperatures have enlarged the area where the midges that transmit the virus will survive. with chikugunya, the virus first mutated and was then transmitted by another mosquito species that is present over a far wider area (enserink, b) . in both cases some vaccine development has been undertaken that will hopefully reduce the -year timeline, but additional work will be needed to get these products on the market. my final plea is for more collaborative research between the medical and veterinary fields. human and veterinary medicine have drifted apart (kahn, ; enserink, a) , and although there are many good arguments for breaking down the walls between the two disciplines (michel, ) , progress is slow. something must be done, however, as further delay in the field of vaccine development would be inexcusable (marano et al., ) . perhaps commonsense can prevail? bernard a.m. van der zeijst netherlands vaccine institute, leiden university medical center, p.o. box , al bilthoven, the netherlands e-mail address: ben. van.der.zeijst@nvi-vaccin.nl initiative aims to merge animal and human health science to benefit both chikungunya: no longer a third world disease veterinary vaccine development from an industrial perspective confronting zoonoses, linking human and veterinary medicine vaccines for emerging infections comparative clinical science: the medicine of the future vaccine r&d success rates and development times key: cord- - g a s z authors: shih, hsin-i.; wu, chi-jung; tu, yi-fang; chi, chia-yu title: fighting covid- : a quick review of diagnoses, therapies, and vaccines date: - - journal: biomed j doi: . /j.bj. . . sha: doc_id: cord_uid: g a s z the covid- pandemic caused by a novel coronavirus, sars-cov- , has infected more than . million individuals and resulted in over , deaths globally. the rapid spread of the virus and the precipitously increasing numbers of cases necessitate the urgent development of accurate diagnostic methods, effective treatments, and vaccines. here, we review the progress of developing diagnostic methods, therapies, and vaccines for sars-cov- with a focus on current clinical trials and their challenges. for diagnosis, nucleic acid amplification tests remain the mainstay diagnostics for laboratory confirmation of sars-cov- infection, while serological antibody tests are used to aid contact tracing, epidemiological, and vaccine evaluation studies. viral isolation is not recommended for routine diagnostic procedures due to safety concerns. currently, no single effective drug or specific vaccine is available against sars-cov- . some candidate drugs targeting different levels and stages of human responses against covid- such as cell membrane fusion, rna-dependent rna polymerase, viral protease inhibitor, interleukin blocker, and convalescent plasma may improve the clinical outcomes of critical covid- patients. other supportive care measures for critical patients are still necessary. advances in genetic sequencing and other technological developments have sped up the establishment of a variety of vaccine platforms. accordingly, numerous vaccines are under development. vaccine candidates against sars-cov- are mainly based upon the viral spike protein due to its vital role in viral infectivity, and most of these candidates have recently moved into clinical trials. before the efficacy of such vaccines in humans is demonstrated, strong international coordination and collaboration among studies, pharmaceutical companies, regulators, and governments are needed to limit further damage due the emerging sars-cov- virus. severe acute respiratory syndrome coronavirus (sars-cov- ), which emerged in wuhan, china in late , has rapidly spread throughout china and globally. although belonging to the same family, sars-cov- has different clinical and epidemiological characteristics from sars-cov and middle east respiratory syndrome coronavirus (mers-cov). its high transmissibility resembles that observed for pandemic influenza viruses; however, tools for diagnosis, treatment, and vaccines still need tremendous work to achieve the levels needed to respond to a pandemic flu. although other measures designed to respond to and control a pandemic such as surveillance, quarantine, and social distancing work efficiently to flatten the curve at a major cost to the economy, the development and deployment of effective tests, drugs, and vaccines to protect lives and limit disease spread are still urgent. emergency use authorizations (eua) expedite the availability of drugs to prevent serious or life-threatening diseases or conditions when there are no adequate, approved, and available alternatives. for many drugs that are already marketed for other conditions, off-label use can increase access for patients who need them. currently, thousands of clinical trials are ongoing to test clinical outcomes. adequate clinical trials will soon confirm or refute the usefulness of several candidate drugs and vaccines in treating and preventing covid- . here, we review the state of diagnostic tests, initial clinical experience on accessible drugs and convalescent plasma administered to patients with covid- , and updated information on vaccine development against covid- . for covid- patients, fever and cough are the two most common symptoms, and some patients might also suffer from sputum production, sore throat, headache, myalgia/arthralgia, rhinorrhea, and diarrhea [ ] . shortness of breath and dyspnea occur in cases that have progressed to pneumonia. of note, a substantial proportion of patients reported olfactory and gustatory disorders, and thus sudden anosmia or ageusia may represent a clinical screening tool to identify covid-patients [ ] . most patients had normal or decreased leukocyte count, lymphopenia, and elevated c-reactive protein, and some also had thrombocytopenia and elevated d-dimer, lactate dehydrogenase, and alanine aminotransferase [ ] . in patients with pneumonia, ground-glass opacity is the typical radiological finding on chest computed tomography (ct) scan, and it may be obscure on chest x-ray; in patients with severe pneumonia, local or bilateral patchy consolidation has also been seen on ct images [ ] . however, these clinical, laboratory, and imaging findings are nonspecific and cannot differentiate covid- from other viral respiratory infections; viral diagnostic methods specific for sars-cov- should be applied for disease confirmation. the current standard diagnostics for covid- are based on detection of the sars-cov- rna by nucleic acid amplification tests (naats), usually through real-time reverse transcription polymerase chain reaction (rt-pcr) with conformation by sequence analysis when necessary [ ] . reverse transcription loop-mediated isothermal amplification (lamp) assays also appear to be a simple and sensitive diagnostic tool without a requirement high-level facilities and instruments [ ] . samples recommended for testing are those from the lower respiratory tract, including sputum, bronchoalveolar lavage (bal), and endotracheal aspirates when possible [ ] . sputum, nasopharyngeal swab (np), and oropharyngeal swabs (op) are the most common sample types taken from patients with mild to moderate illness. if both np and op are collected, they can be placed in the same tube and tested simultaneously to save reagents [ , ] . in general, bal showed the highest positive rates, followed by sputum, np, and op in order of decreasing sensitivity [ ] [ ] [ ] . throat gargling samples are an alternative specimen, although they are less sensitive than sputum [ ] . the laboratory confirmation of cases in regions without covid- virus circulation requires detection of two different genetic targets of the covid- viral genome, while in regions with established covid- virus circulation, confirmation through detection of a single genetic target is considered sufficient [ , ] . many national laboratories have established and published their diagnostic protocols, which are summarized on the world health organization (who) website [ ] . for example, the charité protocol from germany recommended detecting the e (envelope) gene for screening, followed by confirmation of e gene-positive samples through detection of the rna-dependent rna polymerase (rdrp) gene, where the e assay is specific for all sars-cov related viruses (i.e., sars-cov, sars-cov- , and bat-derived sars-related cov) and the rdrp assay using the p probe only detects sars-cov- [ ] . pharyngeal virus shedding is very high during the first week of symptoms, and viral rna shedding from sputum persists even after resolution of symptoms and seroconversion [ , ] . in a study with most samples (> %) taken from the lower respiratory tract, the median duration of rna detection was days (interquartile range, - days) after illness onset, and independent risk factors for prolonged sars-cov- rna shedding (> days) included male sex, delayed hospital admission, and invasive mechanical ventilation [ ] . however, viral rna does not equate to a live virus, and more data are needed to realize whether viral rna load correlates with infectivity [ ] . it has also been noted that a negative naat result does not mean that covid- is absent, since several factors can lead to false-negative results. these factors include inappropriate sample collection or transportation, sample collection at time when the patient was not shedding sufficient virus, and technical reasons [ , ] . periodically sequencing the evolving viruses is also suggested to monitor any mutations in the regions targeted by the assays that might affect test performance [ , ] . moreover, the presence of a non-sars-cov- pathogen does not preclude the possibility of covid- ; approximately one-fifth of specimens positive for sars-cov- were positive for one or more additional common respiratory viruses [ ] . virus isolation is essential to obtain isolates for characterization and to support the development of antivirals and vaccines. however, although sars-cov- can be cultured in selected cell lines, such as vero cells and llc-mk cells, in a biosafety level- laboratory (bsl- ), viral isolation is not recommended as a routine diagnostic procedure due to biosafety concerns and time constraints [ , , ] . studies have indicated that infectious virus can be readily isolated during the first week of symptoms, with sputum having a higher culture yield than np or op; however, infectious virus could not be isolated from samples taken days after onset despite ongoing high viral load detected by rt-pcr [ ] . it appears that virus isolation success depends on viral load, as culture failed to yield virus when samples contained < copies per ml or per sample [ ] . further studies elucidating the duration of culture-positivity would provide a rationale for proposing strategies of isolation of infected patients. serological antibody detection is the other broad category of tests to diagnose covid- , and this method detects igm, igg, or total antibodies (typically in the blood) against sars-cov- . techniques used for antibody detection include virus neutralization assay, enzyme-linked immunosorbent assay (elisa), immunochromatographic assay, chemiluminescent immunoassay, etc. [ , [ ] [ ] [ ] [ ] . most tests are designed to capture antibodies, which recognize the nucleocapsid (n) protein and the s subunit and receptor biding domain (rbd) of spike (s) proteins, as n and s proteins are the two major coronavirus immunogens [ , ] . rbd-specific monoclonal antibodies derived from two b cell clones of one covid- patient have demonstrated impressive binding and neutralizing activity against live sars-cov- [ ] . of importance, these serologic tests should not cross-react with other seasonal coronaviruses. nevertheless, the use of antibody tests is limited to settings of acute illness because it takes time for hosts to mount an adequate immune response [ ] . studies indicate that the majority of patients have seroconversion weeks after symptom onset [ , , ] . less than % of patients had detectable antibodies within week of onset, but this percentage rapidly increased to . % (igm) and . % (igg) days after onset, with the median seroconversion time of and days, respectively [ ] . igm began to decline weeks after symptom onset, while igg remained at high levels after weeks [ , ] . based on the time course of seroconversion, serological tests could be used as complementary tools to identify patients presenting late in their illness [ ] . as mentioned earlier, seroconversion has not usually been followed by a rapid decline in viral rna load [ ] . serological tests may also aid in (i) contact tracing, (ii) assessment of prior infection and immunity to sars-cov- (if there is protective immunity), (iii) determining the extent of the pandemic with seroprevalence data, and (iv) vaccine evaluation studies [ , ] . to date, it is not known whether antibodies elicited by sars-cov- provide protective immunity against reinfection and how long the protective immunity lasts. a rhesus macaque study does suggest protective immunity after recovery from primary infection, since reinfection did not occur in convalescent monkeys rechallenged with the same dose of sars-cov- strains [ ] . further studies are necessary to elucidate the situation in humans. rapid antigen-detection methods using immunoassays targeted at n or s proteins are under development, although with the same challenge of low sensitivity observed in influenza virus antigen tests. to date, a number of laboratory-developed assays and commercially available kits (mostly naats and serological antibody tests) have been granted an eua by the us food and drug administration (fda), which greatly strengthens the diagnostic capability of frontline clinical laboratories [ ] . currently, there are no drugs or other therapies approved by the us fda to treat covid- . the major clinical treatment and management approaches emphasize the importance of life supportive care and relief of complications. oxygen-based therapy has been applied when patients experience dyspnea, and advanced sepsis management has been warranted for patients who progressed to severe sepsis and acute respiratory distress syndrome. no existing antiviral drugs have sufficient evidence that they efficaciously treat covid- pneumonia. some drugs have been selected to treat covid- pneumonia patients ( table ) ; most of these drugs were designed for other purpose such as ebola, influenza, parasites, human immunodeficiency virus (hiv) infections, and immune therapy for some autoimmune and inflammatory diseases. clinical trials have been conducted in which potential antiviral therapy targets were tested, such as blocking viral entry to human cells, inhibiting viral enzymes that were responsible for genome replication. others focus on the human immune system to boost the innate response and inhibit the inflammatory process to relieve rapid progressed acute lung injuries. global, large-scale, randomized clinical trials are still ongoing to test the safety and clinical outcomes of these drugs. here, we summarize the mechanisms of potential therapeutic options that may combat the emerging sars-cov- ( figure) . chloroquine/hydroxychloroquine chloroquine and hydroxychloroquine have a long-standing history in the prevention and treatment of malaria and the treatment of chronic inflammatory diseases such as systemic lupus erythematosus and rheumatoid arthritis. chloroquine and hydroxychloroquine block viral entry into cells by inhibiting the glycosylation of host receptors, proteolytic processing, and endosomal acidification. immunomodulatory effects through attenuation of cytokine production and inhibition of autophagy and lysosomal activity in host cells have also been reported [ , ] . the experiences of the us, china, and europe have indicated clinical effects of chloroquine and hydroxychloroquine in the early phase but limited effects in the late phase [ ] . an earlier study demonstrated that hydroxychloroquine was significantly associated with viral load reduction/disappearance in covid- patients and its effect was strengthened by azithromycin [ ] . in addition, a recent meta-analysis indicated hydroxychloroquine alone, or in combination with azithromycin had benefits in positive-to-negative conversion of sars-cov- and reduction of progression rate though being associated with higher mortality [ ] . nevertheless, an us study which evaluated hospitalized patients with covid- in metropolitan new york showed no significant difference in in-hospital mortality among patients treated with hydroxychloroquine, azithromycin, both, and neither of them [ ] . safety data and data from larger, randomized, placebo-controlled trials with longer follow-up are urgently needed. hydroxychloroquine can prolong the pr, qrs and qtc intervals, especially in patients with underlying risk factors or use in combination with other qt-prolonging drugs; cautious monitoring of ecg changes during treatment is important. chloroquine and hydroxychloroquine are substrates of cyp c and cyp a ; therefore, co-administration with moderate and strong cyp c and cyp a inhibitors may result in increased plasma concentrations of hydroxychloroquine. experiences and interim guidelines for clinical management of sars-cov- infection in taiwan advised that early administration of hydroxychloroquine may be considered to be given for days after thoroughly evaluating potential risks/benefits and ethical issues [ ] . lopinavir, an hiv type aspartate protease inhibitor, has in vitro inhibitory activity against sars-cov [ ] . ritonavir is combined with lopinavir to increase its plasma half-life through the inhibition of cytochrome p . an open-label study published in suggested, by comparison with a control group that received only ribavirin, that the addition of lopinavir-ritonavir ( mg and mg, respectively) to ribavirin reduced the risk of adverse clinical outcomes (acute respiratory distress syndrome or death) and viral load among patients with sars [ ] . lopinavir also has activity, both in vitro [ ] and in an animal model [ ] , against mers-cov, and case reports have suggested that the combination of lopinavir-ritonavir with ribavirin and interferon alfa resulted in virologic clearance and survival [ ] . a randomized controlled trial enrolled covid- patient with dyspnea and desaturation in china and suggested that treatment with lopinavirritonavir was similar to standard care in the time to clinical improvement. gastrointestinal adverse events were more common in the lopinavir-ritonavir group, but serious adverse events were more common in the standard-care group. lopinavir-ritonavir treatment was stopped early because of adverse events such as nausea, diarrhea and hepatotoxicity [ ] . additionally, as a cyp a inhibitor, significant drug-drug interactions have been reported before. ivermectin is an fda-approved broad spectrum anti-parasitic agent. it was identified as an inhibitor in vitro and has been demonstrated to limit infection by a broad spectrum of rna viruses such as dengue virus (denv), west nile virus, venezuelan equine encephalitis virus, and influenza by binding to inhibitors of importin α/β-mediated transports of proteins and rna during infection. in the phase iii clinical trial in thailand in - against denv infection with a single daily oral dose, ivermectin was observed to be safe and resulted in a significant reduction in serum levels of viral ns protein, but no change in viremia or clinical benefit was observed [ ] . ivermectin has been shown to reduce viral rna up to , -fold after h of infection with sars-cov- [ ] . this drug is extensively metabolized in human liver microsomes by cyp a . for patients with liver function abnormality, drug effects and interactions should be closely monitored during treatment. remdesivir remdesivir, a nucleotide analogue prodrug that inhibits rdrp, results in premature termination of the viral rna chain and consequently halts replication of the viral genome. it has shown broad spectrum activity against members of several virus families, including filoviruses (e.g., ebola) and coronaviruses (e.g., sars-cov and mers-cov), and has shown prophylactic and therapeutic efficacy in nonclinical models of these coronaviruses [ ] [ ] [ ] . remdesivir in vitro testing has also shown that remdesivir has activity against sars-cov- [ ] . a compassionate-use study reported % oxygen improvement and % overall mortality. a total of patients ( %) reported adverse events during follow-up. the most common adverse events were increased hepatic enzymes, diarrhea, rash, renal impairment, and hypotension, and some of the patients discontinued remdesivir treatment prematurely. higher improvement rate in chest imaging in the favipiravir arm ( . % versus %) [ ] . another multicentered randomized clinical study also suggested that the day clinical recovery rate increased in the favipiravir treatment group, along with shorter defervescence time and cough in patients with hypertension and/or diabetes [ ] . although few adverse effects were reported during treatment, potential drug and drug interactions should be kept in mind because favipiravir undergoes metabolism in the liver by aldehyde oxidase and xanthine oxidase to produce an inactive oxidative metabolite and is excreted by the kidney [ ] . no hepatic or kidney adjustments are recommended at this time, but initiation is not recommended in patients with an estimated glomerular filtration rate less than ml/min. the humoral immune response mediated by antibodies is crucial for preventing viral infections. therefore, the development of specific surface epitope-targeting neutralizing antibodies is a more long-term, albeit more specific, approach to target covid- [ ] . sars-cov- -specific neutralizing antibodies (nab) have been detected in patients from day - after the onset of the disease and remained thereafter. the titers of nab among these patients correlated with the spike-binding antibodies targeting the s , rbd, and s regions. studies in china indicated that one or two doses of ml of convalescent plasma derived from recently recovered donors with neutralizing antibody titers above : were well tolerated and could significantly increase or maintain the neutralizing antibodies at a high level; this outcome leads to disappearance of viremia, improvement of clinical symptoms, and absorption of lung lesions in radiological examination when administered, in addition to supportive care and antiviral agents such as lopinavir/ritonavir and favipiravir. the donors must be symptom-free for at least days, have a negative sars-cov- pcr test after recovery to provide abo-compatible and test negative plasma for major blood-borne diseases at the time of blood donation. known general reactions such as transfusion-associated circulatory overload and transfusion-associated acute lung injury in patients with already severe lung damage still exist and need to be closely monitored [ ] [ ] [ ] [ ] . evidence suggests that cytokine release syndrome (crs) might play a major role in severe covid- [ , ] . inflammatory cytokines and chemokines, including il- , il- β, induced protein and monocyte chemoattractant protein- , are significantly elevated in covid- patients, especially in severe patients with life-threatening multiple organ dysfunction. in covid- patients with elevated inflammatory cytokines, postmortem pathology has revealed tissue necrosis and interstitial macrophage and monocyte infiltrations in the lung, heart, and gastrointestinal mucosa [ ] . moreover, severe lymphopenia with hyperactivated proinflammatory t cells [ ] and decreased regulatory t cells [ ] is commonly seen in critically ill patients, suggesting dysregulated immune responses. tocilizumab is a recombinant humanized monoclonal anti-il- receptor antibody. it binds both soluble and membrane-bound il- r to inhibit il- -mediated cis-and trans-signaling [ ] . given the efficacy of tocilizumab in crs and the pivotal role of il- in covid- , clinical use should consider evaluation of patients with the following criteria: (i) h score, a diagnostic score for hlh, to discriminate patients with crs; (ii) chinese guidelines for covid- grade patients into mild, moderate, severe, and critical by vital signs, radiographic findings, and complications; (iii) il- measurement, as il- levels are significantly elevated in covid- patients, especially in icu patients [ ] . crp, an acute-phase inflammatory protein synthesized by il- -dependent hepatic biosynthesis, is a reliable marker of il- bioactivity and is used to predict crs severity and monitor il- blockade efficacy. most studies suggested that elevated crp levels are associated with severe covid- [ , , ] . clinicians should add antivirals and cautiously evaluate the possibility of secondary infection thereafter [ ] . adverse hepatic effects have been reported previously, and clinicians should consider discontinuation of drug in cases of marked elevation of liver enzymes and hyperbilirubinemia. other drug-drug interactions should be monitored because tocilizumab interferes with the serum concentration of cyp a substrates. arbidol hydrochloride (umifenovir) umifenovir targets the s protein/ace interaction and inhibits membrane fusion of the viral envelope [ ] . the agent is currently approved in russia and china for the treatment and prophylaxis of influenza and is being tested in some clinical trials treating covid- based on in vitro data suggesting activity against sars [ ] . the current dose of mg orally every hours for influenza is being studied for covid- treatment [ ] . limited clinical experience with umifenovir for covid- in china in a nonrandomized study of patients with covid- showed that treatment with umifenovir for a median duration of days was associated with lower mortality rates ( % [ / ] vs % [ / ] ) and higher discharge rates compared with patients who did not receive the agent [ ] . another randomized chinese study compared umifenovir (arbidol) ( mg* /day) and favipiravir ( mg* /first day followed by mg* /day) and indicated that favipiravir, compared to umifenovir, did not significantly improve the clinically recovery rate at day [ ] . camostat mesylate is an inhibitor of the cellular serine protease tmprss [ ] , which is used by sars-cov- for s protein priming [ ] . it was developed to treat chronic pancreatitis ( mg daily in three divided doses) and reflux esophagitis ( mg daily in three divided doses after each meal). sars-cov- is surrounded by an envelope composed of a lipid bilayer and envelope proteins. sars-cov- initiates human cell entry after the s protein present on the envelope binds to a cell membrane receptor called ace . the s protein is cleaved into two subunits, s and s , by a human cell-derived protease, which is thought to be furin. s then binds to its receptor, ace . the other fragment, s , is cleaved by tmprss , a human cell surface serine protease, resulting in membrane fusion. both ace and tmprss are therefore thought to be essential in airway cells for sars-cov- infection. an in vitro study found that nafamostat and camostat suppressed sars-cov- s protein-initiated fusion in ft cells (derived from the human fetal kidney) ectopically expressing ace and tmprss . nafamostat, a new developed iv form drug, was found to inhibit sars-cov- s protein-initiated fusion at a concentration less than one-tenth of that needed by camostat. adverse effects included mild gastrointestinal upset, dizziness, skin rash, thrombocytopenia, and elevated liver enzymes [ ] . vaccination probably offers the best option for blocking infectious disease circulation. table ) ; they are further discussed in the following section. platform mrna-based vaccines comprise mrna that encodes a protein antigen. conventional mrna-based vaccines encode the antigen of interest and contain ′ and ′ untranslated regions, whereas the virally derived, self-amplifying rnas encode not only the antigen but also the viral replication machinery that enables intracellular rna amplification and abundant protein expression [ ] . recent mrna vaccine designs have improved the stability and protein translation efficiency for enhanced innate and adaptive immunogenicity [ ] . delivery of the mrna vaccine has been optimized by use of lipid nanoparticles for intramuscular or intradermal administration [ ] . additionally, unlike conventional vaccines, which are made from either inactivated pathogens or the small subunit of live pathogens, no infectious virus needs to be handled for mrna vaccines. therefore, testing is relatively safe, efficient, cost effective, and rapid. an mrna vaccine for covid- , mrna- , was the first to advance to a phase i clinical trial in the us, which is currently recruiting healthy volunteers aged between to years to assess the safety, reactogenicity, and immunogenicity. dna vaccines, another type of nucleic acid-based vaccines, consist of plasmid-dna encoding one or several antigens that will be expressed in host cells. dna vaccines can be produced rapidly and at low cost. however, the need for specific delivery systems to achieve good immunogenicity and possible genomic integration and persistence in host cells is a remaining concern [ ] . dna generation of an inactivated whole-virus (iwv) vaccine is the quickest approach for vaccine production following a new outbreak. such vaccines have successfully been developed for influenza virus and enterovirus [ , ] . iwv vaccines are usually made by exposure of a virulent virus to chemical or physical agents, e.g., formaldehyde or gamma irradiation, to destroy infectivity while retaining immunogenicity. the need to use large amounts of antigen to elicit an adequate antibody response and the possibility of causing th -bias hypersensitivity are major concerns for iwv vaccines [ ] . one inactivated vaccine candidate that displayed good cross-neutralization to different covid- strains has received approval for testing in human trials [ ] . viral vector vaccines are also potential tools for vaccine development. these vaccines can specifically deliver genes to target cells, enhance immunogenicity without an adjuvant, and induce a robust cytotoxic t cell response to eliminate virus-infected cells. although the results of viral vector-based vaccines have been encouraging in animal models, some obstacles need to be overcome before use in humans. these obstacles include genetic stability, ability to evade pre-existing immunity, and genotoxicity. adenovirus serotype (ad ) is the most widely used vector because this vector can be easily produced and has high levels of transgene expression and a broad range of viral tropism [ ] . the ability to enhance mucosal immunity through targeting epithelial cells of the upper respiratory tract and gut, two main sites that express high levels of the ace receptor for sars-cov- , makes ad an advantageous viral vector against covid- . recombinant ad vector-based vaccines have been examined in clinical trials against infectious diseases [ , ] . the work for covid- has been accelerated based on the experiences of previous trials. a candidate vaccine known as ad -ncov, which encodes a full-length s protein of sars-cov- , is the first demonstrated to be safe for humans and to proceed to a phase ii clinical trial in china. another viral vector vaccine, chadox ncov- , is composed of a nonreplicating chimpanzee adenovirus vector and genetic sequence of s protein. the vector represents an attractive alternative to the human adenoviral vector due to its good safety profile and lack of pre-existing immunity in human population [ ] . the vaccine candidate has entered a phase i/ii clinical trial. lentivirus vector (lv) systems represent an attractive technology for vaccine development. in addition to their ability to effectively deliver genes or antigens of interest into cells and to generate humoral and cellular mediated immune response against the encoded transgenes [ ] , lvs can transduce antigen presenting cells (apcs), the main cell types mediating the immune response, at high efficiencies with little to no cytotoxicity [ ] . through up or downregulation of immune modulatory genes in apcs by lvs, the genetically modified apcs may potentially activate a strong protective immunity against infections [ ] . two vaccine candidates, covid- /aapc vaccine and lv-smenp-dc vaccine, which were made by modifying artificial apcs and dendritic cells with lvs expressing multiple viral genes and immune modulatory genes, act as 'trojan horses' against sars-cov- virus. clinical trials are currently underway to evaluate their safety and immune reactivity. bifidobacterium is one of the domestic, nonpathogenic anaerobic bacteria found in the intestine of humans. these organisms are believed to have health-promoting properties for their host, including increasing the immune response and protecting the host against viral infection [ ] . as vaccine vectors, they offer several advantages including low cost, low resistance to antibiotics, noninvasive administration, and high safety levels. the most attractive feature is that bifidobacterium tends to elicit high levels of mucosal antibodies against the expressed foreign antigen following uptake via the mucosal immune system [ ] . some strains of bifidobacterium have been used as a delivery vector for the development of vaccines against hepatitis c virus and enterovirus [ , ] . the bactrl-spike vaccine candidate contains live bifidobacterium longum, which contains synthetic plasmid dna encoding the s protein of sars-cov- . the ongoing trial is designed to evaluate the safety and tolerability of orally delivered bactrl-spike vaccine in healthy adults. antibody-dependent enhancement (ade) is a condition in which subneutralizing or nonneutralizing antibodies are produced following primary infection or vaccination, and they enhance the infectivity of subsequent infections [ ] . ade modulates the immune response and elicits sustained inflammation, lymphopenia, and/or cytokine storm [ ] . ade has been observed for a variety of viruses, most notably flaviviruses (e.g., dnev) [ ] . ade also occurs in sars-cov infection [ , ] . diluted anti-sera against sars-cov promotes sars-cov infection, and this enhancement is significantly mediated by anti-s protein antibodies [ , ] . similarly, vaccination with recombinant s protein of sars-cov elicits both neutralizing and ade-inducing igg antibodies [ ] . as described above, many potential vaccine candidates against sars-cov- have focused on the full-length s protein. attributed to the taxonomic and structural similarities between sars-cov and sars-cov- , ade is a critical issue that should be considered seriously during the practical application of sars-cov- vaccines. three approaches have been suggested to mitigate the adverse effects of ade. the first one is shielding the nonneutralizing epitopes of the s proteins by glycosylation. the second approach is immunofocusing, which aims to direct the adaptive immune responses to target only the critical neutralizing epitope to elicit a more robust protective immunity. supporting evidence for the latter is that a vaccine candidate based on the shorter rbd induced higher neutralizing activity than based the full-length s protein [ , ] . the third approach is eliminating epitope sequences that mediate enhancement of infection. protein sequences responsible for ade have been identified at s − of the sars-cov s protein [ ] , a region that is also conserved in sars-cov- [ ] . thus, vaccines against covid- could be engineered to minimize ade via elimination of the epitope. bacillus calmette-guérin (bcg), the most commonly administered vaccine worldwide, contains a live attenuated strain of mycobacterium bovis to protect against tuberculosis (tb). universal vaccination at birth with a single dose of bcg is recommended in many countries where tb is highly endemic or where there is high risk of exposure to tb, such as japan, china, and taiwan. other countries, such as spain, france, and switzerland, have discontinued their universal vaccine policies because of the declining incidence of tb infection and the proven variable effectiveness in preventing adult tb. countries such as the united states, italy, and the netherlands have yet to adopt universal vaccine policies [ ] . although developed to prevent severe forms of tuberculosis in children, bcg vaccination has been shown to induce heterologous or nonspecific immune effects against nonmycobacterial pathogens, a phenomenon termed 'trained immunity'. trained immunity refers to the ability of innate immune memory to mount an enhanced subsequent response to diverse microbes [ ] . favorable effects of bcg have been observed in mouse and human studies for distinct viral pathogens [ , ] . epidemiological studies have also linked bcg vaccination to the reduction in all-cause mortality in neonates and respiratory infections in elderly [ , ] . nod -and mtor-mediated changes in the epigenetic landscape of immune cells is proposed to underly such protection to increase the secretion of pro-inflammatory cytokines, particularly il- β, and enhance anti-viral immunity [ , ] . recent preprint studies suggested significant associations of bcg vaccination with prevalence, progression of disease, and mortality due to covid- [ , ] . the authors indicated that countries without universal policies for bcg vaccination have been more severely affected compared to countries with routine use of the vaccine in neonates. the national immunization program in taiwan has included neonatal bcg vaccination since , and the coverage rate has remained at % since [ ] . as of may , , a cumulative total of covid- cases were confirmed in taiwan with a case fatality rate was of . %. the low morbidity and mortality rate are attributed to the government's quick response, border control, case identification, containment, and resource allocation to protect public health [ ] . it is not known whether bcg vaccination plays a protective role against covid- infection in taiwan. in addition to bcg, live attenuated influenza vaccine has been shown to promote nk cellmediated heterologous immunity [ ] . previous studies also suggest that the heterologous beneficial effects of bcg vaccination may vary by bcg formulation, age, and route of administration [ , ] . although these vaccines may bridge the gap until a vaccine specifically for sars-cov- is available, their protective effects and clinical relevance need to be further characterized. clinical trials have been initiated to study the effects of bcg vaccination given to healthcare workers who are at the frontline of the covid- pandemic ( table ) . before the evidence is available, the who is not likely to recommend bcg vaccination for the prevention of covid- [ ] . in the face of a pandemic, the rapid development, production, and deployment of diagnostic tools, drugs and vaccines are critical. scientific advancements since the sars and mers pandemics have accelerated our understanding of the epidemiology, pathogenesis, and diagnosis of sars-cov- , as well as the development of therapies to treat viral infection. rigorous and adequate clinical trials for drug safety and effectiveness in randomized, controlled trials remain fundamental measures to protect the public from drugs that are ineffective, unsafe, or both. some available candidate drugs targeting different levels of human responses to covid- , such as cell membrane fusion, rna-dependent rna polymerase, viral protease inhibitor, il- blocker and convalescent plasma, may improve the clinical outcomes of critical covid- patients. as no effective treatment against sars-cov- is currently available, the best action is to develop vaccines to prevent the infection. some potential vaccine candidates have progressed to phase i and ii clinical trials, but a year and a half are likely to pass before an effective vaccine is vetted through trials and is ready for marketing for humans. therefore, considerable efforts should be given to limit or hinder the spread of the virus. in addition, pandemics will generate simultaneous demand for drugs and vaccines around the world. the elderly and those with underlying diseases or chronic comorbidities are at greater risk of severe disease or mortality. clinical and serologic studies will be needed to confirm which populations remain at the highest risk once effective treatments or vaccines are available. strong international coordination and collaboration among studies, pharmaceutical companies, regulators, and governments are needed to ensure that promising therapies or vaccines can be manufactured and supplied successfully. the first wave of this pandemic has created devastating social, economic, and political threats. it is time for us to work together, share experiences, and move forward to fight covid- . although this virus persists, there is light at the end of the tunnel. the authors declare that there are no competing interests. clinical characteristics of novel coronavirus infection in china olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid- ): a multicenter european study laboratory testing for coronavirus disease (covid- ) in suspected human cases, interium guidance rapid and visual detection of novel coronavirus (sars-cov- ) by a reverse transcription loop-mediated isothermal amplification assay pan american health organization and world health organization. laboratory guidelines for the detection and diagnosis of covid- virus infection report from the american society for microbiology covid- international summit sars-cov- viral load in upper respiratory specimens of infected patients detection of sars-cov- in different types of clinical specimens prolonged virus shedding even after seroconversion in a patient with covid- the sars-cov- receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement virological assessment of hospitalized patients with covid- factors associated with prolonged viral rna shedding in patients with covid- rates of co-infection between sars-cov- and other respiratory pathogens serological immunochromatographic approach in diagnosis with sars-cov- infected covid- patients severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease patients antibody responses to sars-cov- in patients of novel coronavirus disease profile of specific antibodies to sars-cov- : the first report potent human neutralizing antibodies elicited by sars-cov- infection evaluation of nucleocapsid and spike protein-based elisas for detecting antibodies against sars-cov- reinfection could not occur in sars-cov- infected rhesus macaques diagnostic testing for severe acute respiratory syndrome-related coronavirus- : a narrative review covid- : a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? lymphopenia predicts disease severity of covid- : a descriptive and predictive study hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial systematic review and meta-analysis of the effectiveness and safety of hydroxychloroquine in covid- association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state interim guidelines for clinical management of sars-cov- infection role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture treatment with lopinavir/ritonavir or interferon-beta b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset combination therapy with lopinavir/ritonavir, ribavirin and interferon-alpha for middle east respiratory syndrome a trial of lopinavir-ritonavir in adults hospitalized with severe covid- efficacy and safety of ivermectin against dengue infection: a phase iii, randomized, double-blind, placebo-controlled trial the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro prophylactic and therapeutic remdesivir (gs- ) treatment in the rhesus macaque model of mers-cov infection experimental treatment with favipiravir for covid- : an open-label control study favipiravir versus arbidol for covid- : a randomized clinical trial ebola virus infection: review of the pharmacokinetic and pharmacodynamic properties of drugs considered for testing in human efficacy trials perspectives on therapeutic neutralizing antibodies against the novel coronavirus sars-cov- deployment of convalescent plasma for the prevention and treatment of covid- effectiveness of convalescent plasma therapy in severe covid- patients treatment of critically ill patients with covid- with convalescent plasma favipiravir versus arbidol for covid- : a randomized clinical trial plcgamma /tmem dependent pathway in myeloid cells modulates the pathogenesis of cytokine storm syndrome pathological findings of covid- associated with acute respiratory distress syndrome dysregulation of immune response in patients with covid- in wuhan, china fda approval summary: tocilizumab for treatment of chimeric antigen receptor t cell-induced severe or life-threatening cytokine release syndrome effectiveness of convalescent plasma therapy in severe covid- patients diagnostic utility of clinical laboratory data determinations for patients with the severe covid- can we use interleukin- (il- ) blockade for coronavirus disease (covid- )-induced cytokine release syndrome (crs)? tocilizumab treatment in covid- : a single center experience structural basis of influenza virus fusion inhibition by the antiviral drug arbidol antiviral activity of arbidol and its derivatives against the pathogen of severe acute respiratory syndrome in the cell cultures borneol and poly (ethylene glycol) dual modified bsa nanoparticles as an itraconazole vehicle for brain targeting antimicrobial properties of benzalkonium chloride derived polymerizable deep eutectic solvent simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor mrna vaccines -a new era in vaccinology new vaccine technologies to a dna vaccine induces sars coronavirus neutralization and protective immunity in mice safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase , open-label, single-arm, dose-escalation trial inactivated whole virus influenza a (h n ) vaccine formaldehyde-inactivated whole-virus vaccine protects a murine model of enterovirus encephalomyelitis against disease immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus rapid development of an inactivated vaccine for sars-cov- . biorxiv developments in viral vector-based vaccines efficacy trial of a dna/rad hiv- preventive vaccine a human type adenovirus-based tuberculosis vaccine induces robust t cell responses in humans despite preexisting anti-adenovirus immunity a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity integrase defective lentiviral vector as a vaccine platform for delivering influenza antigens alteration of t cell immunity by lentiviral transduction of human monocyte-derived dendritic cells feeding of bifidobacterium bifidum and streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus lactobacilli and bifidobacteria enhance mucosal b cell responses and differentially modulate systemic antibody responses to an oral human rotavirus vaccine in a neonatal gnotobiotic pig disease model oral administration of genetically modified bifidobacterium displaying hcv-ns multi-epitope fusion protein could induce an hcv-ns -specific systemic immune response in mice oral immunization of mice using bifidobacterium longum expressing vp protein from enterovirus antibody-dependent enhancement of ebola virus infection by immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms antibody-dependent enhancement of severe dengue disease in humans evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates an effective cancer vaccine modality: lentiviral modification of dendritic cells expressing multiple cancer-specific antigens the bcg world atlas: a database of global bcg vaccination policies and practices trained immunity: a program of innate immune memory in health and disease bcg vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity non-specific effects of bcg vaccine on viral infections heterologous immunological effects of early bcg vaccination in low-birth-weight infants in guinea-bissau: a randomized-controlled trial the efficacy of bacillus calmette-guerin vaccinations for the prevention of acute upper respiratory tract infection in the elderly long-lasting effects of bcg vaccination on both heterologous th /th responses and innate trained immunity correlation between universal bcg vaccination policy and reduced morbidity and mortality for covid- : an epidemiological study association of bcg vaccination policy with prevalence and mortality of covid- tokyo- bcg vaccination complications response to covid- in taiwan: big data analytics, new technology, and proactive testing influenza vaccination generates cytokine-induced memory-like nk cells: impact of human cytomegalovirus infection bacille calmette-guerin vaccine strain modulates the ontogeny of both mycobacterial-specific and heterologous t cell immunity to vaccination in infants antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro key: cord- -wawui fd authors: tulchinsky, theodore h.; varavikova, elena a. title: communicable diseases date: - - journal: the new public health doi: . /b - - / - sha: doc_id: cord_uid: wawui fd publisher summary in a world of rapid international transport and contact between populations, systems are needed to monitor the potential explosive spread of pathogens that may be transferred from their normal habitat. the potential for the international spread of new or reinvigorated infectious diseases constitute threat to mankind akin to ecological and other man-made disasters. public health has addressed the issues of communicable disease as one of its key issues in protecting individual and population health. methods of intervention include classic public health through sanitation, immunization, and well beyond that into nutrition, education, case finding, and treatment, and changing human behavior. the knowledge, attitudes, beliefs, and practices of policy makers, health care providers, and parents is as important in the success of communicable disease control as are the technology available and methods of financing health systems. together, these encompass the broad programmatic approach of the new public health to control of communicable diseases. important for all health providers and public health personnel so as to be able to cope with the scale of these problems and to absorb new technologies as they emerge from scientific advances and experience, and their successful application. lived. it was to be observed, indeed, that it did not come straight on toward us; for the city, that is to say within the walls, was indifferently healthy still; nor was it got over the water into southwark; for though there died that week , of all distempers, whereof it might be supposed above died of the plague, yet there was but in southwark, lambeth parish included; whereas in the parishes of st. giles the agent-host-environment triad, discussed in chapter , is fundamental to the success of understanding transmission of infectious diseases and their control, including those well known, those changing their patterns, and those newly emerging or escaping current methods of control. infection occurs when the organism successfully invades the host body, where it multiplies and produces an illness. a host is a person or other living animal, including birds and arthropods, who provides a place for growth and sustenance to an infectious agent under natural, as opposed to experimental, conditions. some organisms, such as protozoa or helminths, may pass successive stages of their life cycle in different hosts, but the primary or definitive host is the one in which the organism passes its sexual stage. the secondary or intermediate host is where the parasite passes the larval or asexual stage. a transport host is a carrier in which the organism remains alive, but does not develop. an agent of an infectious disease is necessary, but not always sufficient to cause a disease or disorder. the infective dose is the quantity of the organism needed to cause clinical disease. a disease may have a single agent as a cause, or it may occur as a result of the agent in company with contributory factors, whose presence is also essential for the development of the disease. a disease may be present in an infected person in a dormant form such as tuberculosis, or a subclinical form, such as poliomyelitis or hiv. the virulence or pathogenicity of an infective agent is the capacity of an infectious agent to enter the host, replicate, damage tissue, and cause disease in an exposed and susceptible host. virulence is indicated by the severity of clinical disease and case fatality rates. the environment provides a reservoir for the organism, and the mode of transmission, by which the organism reaches a new host. the reservoir is the natural habitat where an infectious agent lives and multiplies, from which it can be transmitted directly or indirectly to a new host. the reservoir refers to the natural habitat of the organism, which may be in people, animals, arthropods, plants, soil, or substances in which an organism normally lives and multiplies, and on which it depends for survival or in which it survives in a dormant form. contacts are persons or animals who have been in association with an infected person, animal, or contaminated inanimate object, or environment that might provide an opportunity for acquiring the infective agent. persons or animals that harbor a specific infectious agent, often in the absence of discernible clinical disease, and who serve as a source of infection or contamination of food, water, or other materials, are carriers. a carrier may have an inapparent infection (a healthy cartier) or may be in the incubation or convalescent stage of the infection. communicable diseases may be classified by a variety of methods: by organism, by mode of transmission, by methods of prevention (e.g., vaccine preventable, vector controllable), or by major organism classification, that is, viral, bacterial, and parasitic disease. a virus is a nucleic acid molecule (rna or dna) encapsulated in a protein coat or capsid. the virus is not a complete cell and can only replicate inside a complete cell. the capsid may have a protective envelope of a lipid containing membrane. the capsid and membrane facilitate attachment and penetration of a host cell. inside the host cell, the nucleic molecule may cause the cell's chromosomes to be changed in its own genetic material or so that there is cellular manufacture and virus replication. viroids are smaller rna structures without capsids which can cause plant disease. prions are recently discovered (stanley prusiner, nobel prize, ) variants of viruses or viroids which are the infective agents cause of scrapie in sheep, and similar degenerative central nervous system diseases in cattle and in man (mad cow disease or creutzfeld-jakob disease in humans). bacteria are unicellular organisms that reproduce sexually or asexually, grow on cell-free media, and can exist in an environment with oxygen (aerobic) or in one lacking oxygen (anaerobic). some may enter a dormant state and form spores where they are protected from the environment and may remain viable for years. bacteria include a nucleus of chromosomal dna material within a membrane surrounded by cytoplasm, itself enclosed by the cellular membrane. bacteria are often characterized by their coloration under gram's stain, as gram-negative or gram-positive, as well as by their microscopic morphology, colony patterns on growth media, by the diseases they may cause, as well as by antibody and molecular (dna) marking techniques. bacteria include both indigenous flora (normal resident) bacteria and pathogenic (disease causing) bacteria. pathogenic bacteria cause disease by invading, overcoming natural or acquired resistance, and multiplying in the body. bacteria may produce a toxin or poison that can affect a body site distant from where the bacterial replication occurs, such as in tetanus. bacteria may also initiate an excessive immune response, producing damage to other body tissues away from the site of infection, e.g;, acute rheumatic fever and glomerulonephritis. parasitology studies protozoa, helminths, and arthropods that live within, on, or at the expense of a host. these include oxygen-producing, flagellate, unicellular organisms such as giardia and trichomonas, and amoebas such as entamoeba important in enteric and gynecologic disorders. sporozoa are parasites with complex life cycles in different hosts, such as cryptosporidium or malarial parasites. parasitic disease usually refers to infestation, with fungi, molds, and yeasts that can affect humans. helminths are worms that infest humans especially in poor sanitation and tropical areas. transmission of diseases is by the spread of an infectious agent from a source or reservoir to a person (table . ). direct transmission from one host to another occurs during touching, biting, kissing, sexual intercourse, and projection via droplets, as in sneezing, coughing, or spitting, or by entry through the skin. indirect transmission includes via aerosols of long-lasting suspended particles in air, fecal-oral transmission such as food and waterborne as well as by poor hygenic conditions with inanimate materials, such as soiled clothes, handkerchiefs, toys, or other objects. vector-borne diseases are transmitted via crawling or flying insects, in some cases with multiplication, and development of the organism in the vector, as in malaria. the subsequent transmission to humans is by injection of salivary gland fluid during biting, e.g., congenital syphilis, or by deposition of feces, urine or other material capable of penetrating the skin through a bite wound or other trauma. transmission may occur with insects as a transport mechanism, as in salmonella on the legs of a housefly. airborne transmission occurs inderectly via infective organisms in small aerosols that may remain suspended for long periods of time and which easily enter the respiratory tract. small particles of dust may spread organisms from soil, clothing, or bedding. vertical transmission occurs from one generation to another, or from one stage of the insect life cycle to another stage. maternal-infant transmission occurs during pregnancy (transplacental), delivery, as in gonorrhoea, breast-feeding, e.g., hiv, with transfer of infectious agents from mother to fetus or newborn. resistance to infectious diseases is related to many host and environmental factors, including age, sex, pregnancy, nutrition, trauma, fatigue, living and socioeconomic conditions, and emotional status. good nutritional status has a protective effect against the results of an infection. vitamin a supplements reduce complication rates of measles and enteric infections. tuberculosis may be present in an individual whose resistance is sufficient to prevent clinical disease, but the infected person is a cartier of an organism which can be transmitted to another or cause clinical disease if the person's susceptibility is reduced. immunity is resistance to infection resulting from presence of antibodies or cells that specifically act on the microorganism associated with a specific disease or toxin. immunity to a specific organism can be acquired by having the disease, that is, natural immunity, or by immunization, active or passive, or by protection box . vaccines and prevention "the greeks had two gods of health, aesculapius and hygeia, therapy and prevention, respectively. medicine in the twentieth century retains those two concepts, and vaccination is a powerful means of prevention. what follows is information on the vaccines that together with sanitation, make modem society possible, and that if wisely used will continue to bestow on mankind the gift of prevention, which according to proverb is worth far more than cure." source: plotkin, s. a., mortimer, e. a. . vaccines. second edition. philadelphia: wb saunders (with permission). infectious agent: a pathogenic organism (e.g., virus, bacteria, rickettsia, fungus, protozoa, or helminth) capable of producing infection or an infectious disease. infection: the process of entry, development, and proliferation of an infectious agent in the body tissue of a living organism (human, animal, or plant) overcoming body defense mechanisms, resulting in an inapparent or clinically manifest disease. antigen: a substance (e.g., protein, polysaccharide) capable of inducing specific response mechanisms in the body. an antigen may be introduced into the body by invasion of an infectious agent, by immunization, inhalation, ingestion, or through the skin, wounds, or via transplantation. antibody: a protein molecule formed by the body in response to a foreign substance (an antigen) or acquired by passive transfer. antibodies bind to the specific antigen that elicits its production, causing the infective agent to be susceptible to immune defense mechanisms against infections e.g., humoral and cellular. immunoglobulins: antibodies that meet different types of antigenic challenges. they are present in blood or other body fluids, and can cross from a mother to fetus in utero, providing protection during part of the first year of life. there are five major classes (igg, igm, iga, igd, and ige) and subclasses based on molecular weight. anfisera or antitoxin: materials prepared in animals for use in passive immunization against infection or toxins. source: jawetz, melrick, and adelberg, medical microbiology, . through elimination of circulation of the organism in the community. immunity may be by antibodies produced by the host body or transferred from externally produced antibodies. the body also reacts to infective antigens by cellular responses, including those that directly defend against invading organisms and other cells which produce antibodies. the immune response is the resistance of a body to specific infectious organisms or their toxins provided by a complex interaction of antibodies and cells including a. b cells (bone marrow and spleen) produce antibodies which circulate in the blood, i.e., humoral immunity; b. t cell-mediated immunity is provided by sensitization of lymphocytes of thymus origin to mature into cytotoxic cells capable of destroying virusinfected or foreign cells; c. complement, a humoral response which causes lysis of foreign cells; d. phagocytosis, a cellular mechanism which ingests foreign microorganisms (macrophages and leukocytes). surveillance of disease is the continuous scrutiny of all aspects of occurrence and spread of disease pertinent to effective control of that disease. maintaining ongoing surveillance is one of the basic duties of a public health system, and is vital to the control of communicable disease, providing the essential data for tracking of disease, planning interventions, and responding to future disease challenges. surveillance of infectious disease incidence relies on reports of notifiable diseases by physicians, supplemented by individual and summary reports of public health laboratories. such a system must concern itself with the completeness and quality of reporting and potential errors and artifacts. quality is maintained by seeking clinical and laboratory support to confirm first reports. completeness, rapidity, and quality of reporting by physicians and laboratories should be emphasized in undergraduate and postgraduate medical education. enforcement of legal sanctions may be needed where standards are not met. surveillance of infectious diseases includes the following: . morbidity reports from clinics to public health offices; . mortality reports from attending doctors to vital records; . reports from selected sentinel centers; . special field investigations of epidemics or individual cases; . laboratory monitoring of infectious agents in population samples; . data on supply, use, and side effects of vaccines, toxoids, immune globulins; . data on vector control activities such as insecticides use; . immunity levels in samples of the population at risk; . review of current literature on the disease; . epidemiologic and clinical reports from other jurisdictions. epidemiologic monitoring based on individual and aggregated reports of infectious diseases provide data vital to planning interventions at the community level or for the individually exposed patient and his contacts, along with other information sources such as hospital discharge data and monitoring of sentinel centers. these may be specific medical or community sites that are representative of the population and are able to provide good levels of reporting to monitor an area or population group. a sentinel center can be a pediatric practice site, a hospital emergency room, or other location which will provide a "finger on the pulse" to assess the degree and kind of morbidity occurring in the community. it can also include monitoring in a location previously known for disease transmission, such as hong kong in relation to influenza. epidemiologic analysis provided by government public health agencies should be published weekly, monthly, and annually and distributed to a wide audience of public health and health-related professionals throughout the country. feedback to those in the field on whose initial reports the data are based is vital in order to promote involvement and improved quality of data, as well as to allow evaluation of the local situation in comparison to other areas. in a federal system of government, national agencies report regularly on all state or provincial health patterns. state or provincial health authorities provide data to the counties and cities in their jurisdictions. such data should also be readily available to researchers in other government agencies, universities, and other academic settings for further research and analysis both on internet and hard-copy publications. notifiable diseases are those which a physician is legally required to report to state or local public health officials, by reason of their contagiousness, severity, frequency, or other public health importance (table . ). public health laboratory services provide validation of clinical and epidemiologic reports. they also pro- vide day-to-day supervision of public health conditions, and can monitor communicable disease and vaccine efficacy and coverage. in addition, they support standards of clinical laboratories in biochemistry, microbiology, and genetic screening. nosocomial or hospital-acquired infections constitute a major health hazard associated with care in institutions. in the united states, they occur in - % of hospital admissions and are the cause of lengthening of hospital stay and an estimated , deaths per year. in developing countries, nosocomial infection rates may occur in up to % of hospitalizations. this category of infectious disease most commonly includes infections of the urinary tract, surgical wounds, lower respiratory tract (pneumonias), and blood poisoning or septicemias. in the united states, up to % of hospital-acquired infections are caused by multidrug resistant organisms. staphylococcus infections resistant to many current antibiotics, for example, methicillin and vancomycin, are a notable cause of prolongation of hospitalization or even death. the increasing number of immunodeficient patients has increased the importance of prevention of nosocomial infections. where standards of infection control are lacking, in both developed and developing countries, hospital staff are vulnerable to serious infection. in developing countries, deadly new viruses, such as ebola and marburg viruses mainly affect nursing, medical, and other staff as secondary cases. surveillance and control measures are important elements of hospital management. hospital epidemiologists and infection control staff are part of modem hospital staffing. the cost to the health system of nosocomial infections is a major consideration in planning health budgets. reducing the risk of acquiring such infections in hospital justifies substantial expenditures for hospital epidemiology and infection control activities. with diagnostic related group payment for hospital care (by diagnosis rather than by days of stay) the good manager has a major incentive to ensure that the risk of nosocomial infections is minimized, since they can greatly prolong hospital stays, raising patient dissatisfaction and health care costs. an endemic disease is the constant usual presence of a disease or infectious agent in a given geographic area or population group. hyperendemic is a state of persistence of high levels of incidence of the disease. holoendemic means that the disease appears early in life and affects most of the population, as in malaria or hepatitis a and b in some regions. an epidemic is the occurrence in a community or region of a number of cases of an illness in excess of the usual or expected number of cases. the number of cases constituting an epidemic varies with the disease, and factors such as previous epidemiological patterns of the disease, time and place of the occurrence, and the population involved must be taken into account. a single case of a disease long absent from an area, such as polio, constitutes an epidemic, and therefore a public health emergency because a clinical case may represent a hundred carriers with nonparalytic or subclinical poliomyelitis. in the s, two to three or more cases of measles linked in time and place may be considered sufficient evidence of transmission and presumed to be an epidemic. a pandemic is occurrence of a disease over a very wide area, crossing international boundaries, affecting a large proportion of the population. each epidemic should be regarded as a unique natural experiment. the investigation of an epidemic requires preparation and field investigation in conjunction with local health and other relevant authorities. verification of cases and the scope of the epidemic will require case definition and laboratory confirmation. tabulation of known cases according to time, place, and person are important for immediate control measures and formulation of the hypothesis as to the nature of the epidemic. an epidemic curve is a graphic plotting of the distribution of cases by the time of onset or reporting, which gives a picture of the timing, spread, and extent of the disease from the time of the initial index cases and the secondary spread. epidemic investigation requires a series of steps. this starts with confirmation of the initial report and preliminary investigation, defining who is affected, determining the nature of the illness and confirming the clinical diagnosis, and recording when and where the first (index) and follow-up (secondary) cases occurred, and how the disease was transmitted. samples are taken from index case patients (e.g., blood, feces, throat swabs) as well as from possible vectors (e.g., food, water, sewage, environment). a working hypothesis is established based on the first findings, taking into account all plausible explanations. the epidemic pattern is studied, establishing common source or risk factors, such as food, water, contact, environment, and drawing a time line of cases to define the epidemic curve. how many are ill (the numerator) and what is the population at risk (the denominator) establish the attack rate, namely, the percentage of sick among those exposed to the common factor. what is a reasonable explanation of the occurrence; is there a previous pattern, with the present episode a recurrence or new event? consultation with colleagues and the literature helps to establish both a biological and epidemiologic plausibility. what steps are needed to prevent spread and recurrence of the disease? coordination with relevant health and other officials and providers is required to establish surveillance and control systems, document and distribute reports, and respond to the public's fight to know. the first reports of excess cases may come from a medical clinic or hospital. the initial (sentinel or index) cases provide the first clues that may point to a common source. investigation of an epidemic is designed to quickly elucidate the cause and points of potential intervention to stop its continuation. this requires skilled investigation and interpretation. epidemiologic investigations have defined many public health problems. rubella syndrome, legionnaire's disease, aids, and lyme and hantavirus diseases were first identified clinically when unusually large numbers of cases appeared with common features. the suspicions that were raised led to a search for causes and the identification of control methods. a working hypothesis of the nature of an epidemic is developed based on the initial assessment, the type of presentation, the condition involved, and previous local, regional, national, and international experience. the hypothesis provides the basis for further investigation, control measures, and planning additional clinical and laboratory studies. surveillance will then monitor the effectiveness of control measures. communication of findings to local, regional, national, and international health reporting systems is important for sharing the knowledge with other potential support groups or other areas where similar epidemics may occur. the centers for disease control and prevention (cdc), originally organized in as the office for malaria control in war areas, is part of the u.s. public health service. as of , the cdc had a budget of $ . billion, and employees include epidemiologists, microbiologists, and many other professionals. the cdc includes national centers for environmental health and injury control, chronic disease prevention and health promotion, infectious diseases, prevention services, health statistics, occupational safety and health, and international health. the epidemic intelligence service (eis) of the cdc in the united states is an excellent model for the organization of the national control of communicable diseases. young clinicians are trained to carry out epidemiologic investigations as part of training to become public health professionals. eis officers are assigned to state health departments, other public health units, and research centers as part of their training, carrying out epidemic investigation and special tasks in disease control. the cdc, in cooperation with the who, has developed and offers free of charge, a personal computer program to support field epidemiology, including epidemic investigations (epi-info), which can be accessed and down-loaded from the worldwide web. this program should be adopted widely in order to improve field investigations, to encourage reporting in real time, and to develop high standards in this discipline. cdc's morbidity and mortality weekly report (mmwr) is a weekly publication of the cdc's epidemiologic data, also available free on the internet. it includes special summaries of reportable infectious diseases as well as noncom- although an infectious disease is an event affecting an individual, it is communicable to others, and therefore its control requires both individual and community measures of protection. control of the disease is a reduction in its incidence, prevalence, morbidity, and mortality. elimination of a disease in a specified geographic area may be achieved as a result of intervention programs such as individual protection against tetanus; elimination of infections such as measles requires stoppage of circulation of the organism. eradication is success in reduction to zero of incidence of the disease and presence in nature of the organism, such as with smallpox. extinction means that a specific organism no longer exists in nature or in laboratories. public health applies a wide variety of tools for the prevention of infectious diseases and their transmission. it includes activities ranging from filtration and disinfection of community drinking water to environmental vector control, pasteurization of milk, and immunization programs (see table . ). no less important are organized programs to promote self protection, case finding, and effective treatment of infections to stop their spread to other susceptible persons (e.g., hiv, sexually transmitted diseases, tuberculosis, malaria). planning measures to control and eradicate specific communicable diseases is one of the principal activities of public health and remains so for the twenty-first century. treating an infection once it has occurred is vital to the control of a communicable disease. each person infected may become a vector and continue the chain of transmission. successful treatment of the infected person reduces the potential for an uninfected contact person to acquire the infection. bacteriostatic agents or drugs such as sulfonamides inhibit growth or stop replication of the organism, allowing normal body defenses to overcome the organism. bacteriocidal drugs such as penicillin act to kill pathogenic organisms. traditional medical emphasis on single antibiotics has changed to use of multiple drug combinations for tuberculosis and more recently for hospital-acquired infections. antibiotics have made enormous contributions to clinical medicine and public health. however, pathogenic organisms are able to adapt or mutate and develop resistance to antibiotics, resulting in drug resistance. wide-scale use of antibiotics has led to increasing incidence of resistant organisms. multidrug resistance constitutes one of the major public health challenges at the end of the twentieth century. antiviral agents (e.g., ribovarin) are important additions to medical treatment potential, as are "cocktails" of antiviral agents for management of hiv infection. antibiotic use is a health problem requiting attention of clinicians and their teachers as well as the public health community and health care managers, representing the interaction of health issues across the entire spectrum of services. organized public health services are responsible for advocating legislation and for regulating and monitoring programs to prevent infectious disease occurrence and/or spread. they function to educate the population in measures to reduce or prevent the spread of disease. health promotion is one of the most essential instruments of infectious disease control. it promotes compliance and community support of preventive measures. these include personal hygiene and safe handling of water, milk, and food supplies. in sexually transmitted diseases, health education is the major method of prevention. each of the infectious diseases or groups of infectious diseases have one or more preventive or control approaches (table . ). these may involve the coordinated intervention of different disciplines and modalities, including epidemiologic monitoring, laboratory confirmation, environmental measures, immunization, and health education. this requires teamwork and organized collaboration. very great progress has been made in infectious disease control by clinical, public health, and societal means since in the industrialized countries and since the s in the developing world. this is attributable to a variety of factors, including organized public health services; the rapid development and wide use of new and improved vaccines and antibiotics; better access to health care; and improved sanitation, living conditions, and nutrition. triumphs have been achieved in the eradication of smallpox and in the increasing control of other vaccine-preventable diseases. however, there remain serious problems with tb, stds, malaria, and new infections such as hiv, and an increase in multiple drug-resistant organisms. vaccines are one of the most important tools of public health in the control of infectious diseases, especially for child health. vaccine-preventable diseases ta b l e . annual incidence of selected vaccine-preventable infectious diseases in rates per , population selected years, united states, - disease the body responds to invasion of disease-causing organisms by antigenantibody reactions and cellular responses. together, these act to restrain or destroy the disease-causing potential. strengthening this defense mechanism through im-box . definitions of immunizing agents and processes vaccines: a suspension of live or killed microorganisms or antigenic portion of those agents presented to a potential host to induce immunity to prevent the specific disease caused by that organism. preparation of vaccines may be from: a. live attenuated organisms which have been passed repeatedly in tissue culture or chick embryos so that they have lost their capacity to cause disease but retain an ability to induce antibody response, such as polio-sabin, measles, rubella, mumps, yellow fever, bcg, typhoid, and plague. b. inactivated or killed organisms which have been killed by heat or chemicals but retain an ability to induce antibody response; they are generally safe but less efficacious than live vaccines and require multiple doses, such as polio-salk, influenza, rabies, and japanese encephalitis. c. cellular fractions usually of a polysaccharide fraction of the cell wall of a disease-causing organisms, such as pneumococcal pneumonia or meningococcal meningitis. d. recombinant vaccines produced by recombinant dna methods in which specific dna sequences are inserted by molecular engineering techniques, such as dna sequences spliced to vaccinia virus grown in cell culture to produce influenza and hepatitis b vaccines. toxoids or antisera: modified toxins are made nontoxic to stimulate formation of an antitoxin, such as tetanus, diphtheria, botulism, gas gangrene, and snake and scorpion venom. immune globulin: an antibody-containing solution derived from immunized animals or human blood plasma, used primarily for short-term passive immunization, e.g., rabies, for immunocompromised persons. antitoxin: an antibody derived from serum of animals after stimulation with specific antigens and used to provide passive immunity, e.g., tetanus. munization is one of the outstanding achievements of public health, as treatment of infectious diseases by antimicrobials is a major element of clinical medicine. immunization (vaccination) is a process used to increase host resistance to specific microorganisms to prevent them from causing disease. it induces primary and secondary responses in the human or animal body: a. primary response occurs on first exposure to an antigen. after a lag or latent period of - days (depending on the antigen) specific antibodies appear in the blood. antibody production ceases after several weeks but memory cells that can recognize the antigen and respond to it remain ready to respond to a further challenge by the same antigen. b. secondary (booster) response is the response to a second and subsequent exposure to an antigen. the lag period is shorter than the primary response, the peak is higher and lasts longer. the antibodies produced have a higher affinity for the antigen, and a much smaller dose of the antigen is required to initiate a response. c. immunologic memory exists even when circulating antibodies are insufficient to protect against the antigen. when the body is exposed to the same antigen again, it responds by rapidly producing high levels of antibody to destroy the antigen before it can replicate and cause disease. immunization protects susceptible individuals from communicable disease by administration of a living modified agent, or subunit of the agent, a suspension of killed organisms or an inactivated toxin (see table . ) to stimulate development of antibodies to that agent. in disease control, individual immunity may also protect another individual. herd immunity occurs when sufficient persons are protected (naturally or by immunization) against a specific infectious disease reducing circulation of the organism, thereby lowering the chance of an unprotected person to become infected. each pathogen has different characteristics of infectivity, and therefore different levels of herd immunity are required to protect the nonimmune individual. the critical proportion of a population that must be immunized in order to interrupt local circulation of the organism varies from disease to disease. eradication of smallpox was achieved with approximately % world coverage, followed by concentration on new case findings and immunization of contacts and surrounding communities. for highly infectious diseases, such as measles, immunization coverage of over % is needed to achieve local eradication. immunization coverage in a community must be monitored in order to gauge the extent of protection and need for program modification to achieve targets of disease control. immunization coverage is expressed as a proportion in which the numerator is the number of persons in the target group immunized at a specific age, and the denominator is the number of persons in the target cohort who should have been immunized according to the accepted standard: vaccine coverage = no. persons immunized in specific age group • no. persons in the age group during that year immunization coverage in the united states is regularly monitered by the national immunization survey by a household survey in all states, as well as selected urban areas considered to be at high risk for undervaccination. an initial telephone survey is followed by confirmation, where possible, from documentation from the parents or health care providers. the survey for july -june examined children born between august and november (i.e., aged - months, median age months). the results show improving coverage, with % having received three or more doses of dpt (diphtheria, pertussis, and tetanus), % with three or more doses of opv (oral polio vaccine), % with three or more doses of haemophilus influenzae, type b (hib), but only % with three or more doses of hepatitis b. however, only % had received all recommended vaccines at the recommended ages. eases that still cause millions of deaths globally each year. other important infectious diseases are still not subject to vaccine control because of difficulties in their development. in some cases, a microorganism can mutate with changes. viruses can undergo antigenic shifts in the molecular structure in the organism, producing completely new subtypes of the organism. hosts previously exposed to other strains may have little or no immunity to the new strains. antigenic drift refers to relatively minor antigenic changes which occur in viruses. this is responsible for frequent epidemics. antigenic shift is believed to explain the occurrence of new strains of influenza virus necessitating, for example, annual reformulation of the influenza vaccine associated with large scale epidemics and pandemics. new variants of poliovirus strains are similar enough to the three main types so that immunity to one strain is carded over to the new strain. molecular epidemiology is a powerful new technique used to specify the geographic origin of organisms such as poliomyelitis and measles viruses, permiting tracking of the source of the virus and epidemic. combinations of more than one vaccine is now common practice with a trend to enlarging the cocktail of vaccines in order to minimize the number of injections, and visits required. this reduces the number of visits to carry out routine immunization saving staff time and costs, as well as increasing compliance. there are virtually no contraindications to use of multiple antigens simultaneously. examples of vaccine cocktails include dpt (diphtheria, pertussis, and tetanus) in combination with haemophilus influenzae b, poliomyelitis, and varicella, or mmr (measles, mumps, and rubella) vaccines. interventions in the form of effective vaccines save millions of lives each year and contribute to improved health of countless children and adults throughout the world. vaccination is now accepted as one of the most cost-effective health interventions currently available. continuous policy review is needed regarding allocation of adequate resources, logistical organization, and continued scientific effort to seek effective, safe, and inexpensive vaccines for other important diseases such as malaria and hiv. new technology of recombinant vaccines, such as that of hepatitis b, holds promise for important vaccine breakthroughs in the decades ahead. internationally, much progress was made in the s in the control of vaccinepreventable diseases. at the end of the s, fewer than % of the world's children were being immunized. who, unicef, and other international organizations mobilized to promote an expanded programme on immunization (epi) with a target of reaching % coverage by . immunization coverage increased in the developing countries, preventing some million child deaths annually. bacillus calmette-gu rin (bcg) coverage rose from to %; poliomyelitis with opv (three doses) from to %, and tetanus toxoid for pregnant women from to %. since , there has been a decline in coverage in some parts of the world, mainly in sub-saharan africa. the challenge remains to achieve control or eradication of vaccine-preventable diseases, thus saving millions of more lives. part of the hfa stresses the epi approach, which includes immunization against diphtheria, pertussis, tetanus, po-liomyelitis, measles, and tuberculosis. an extended form of this is the epi plus program which combines epi with immunization against hepatitis b and yellow fever and, where appropriate, supplementation with vitamin a and iodine. the success in international eradication of smallpox is now being followed by a campaign to eradicate poliomyelitis and other important infectious diseases. diphtheria. diphtheria is an acute bacterial disease of the tonsils, nasopharynx, and larynx caused by the organism corynebacterium diphtheriae. it occurs in colder months in temperate climates where the organism is present in human hosts and is spread by contact with patients or carriers. it has an incubation period of - days. in the past, this was primarily an infection of children and was a major contributor to child mortality in the prevaccine and preantibiotic eras. diphtheria has been virtually eliminated in countries with well-established immunization programs. in the s, an outbreak of diphtheria occurred in the countries of the former soviet union among people over age . it reached epidemic proportions in the s, with , cases ( - ) with deaths in in russia alone. this indicates a failure of the vaccination program in several respects: it used only three doses of dpt in infancy; no boosters were given at school age or subsequently; the efficacy of diphtheria vaccine may have been low, and coverage was below %. efforts to control the present epidemic include mass vaccination campaigns for persons over years of age with a single dose of dt (diphtheria and tetanus) and increasing coverage of routine dpt vaccines to four doses by age years. the epidemic and its control measures have led to improved coverage with dt for those over years, and % coverage among children aged - months. who recommends three doses of dpt in the first year of life and a booster at school entry. this is considered by many to be insufficient to produce long-lasting immunity. the united states and other industrialized countries use a four-dose schedule and recommend periodic boosters for adults with dt. pertussis. pertussis is an acute bacterial disease of the respiratory tract caused by the bacillus bordetella pertussis. after an initial coldlike (catarrhal) stage, the patient develops a severe cough which comes in spasms (paroxysms). the disease can last - months. the paroxysms can become violent and may be followed by a characteristic crowing or high pitched inspiratory whooping sound, followed by expulsion of a tenacious clear sputum, often followed by vomiting. in poorly immunized populations and those with malnutrition, pneumonia often follows and death is common. pertussis declined dramatically in the industrialized countries as a result of widespread coverage with dpt. however, because the pertussis component of the vaccine caused some reactions, many physicians avoided its use, using dt alone. during the s in the united kingdom, many physicians recommended against vaccination with dpt. as a result, pertussis incidence increased with substantial mortality rates. this led to a reappraisal of the immunization program, with insti-tution of incentive payments to general practitioners for completion of vaccination schedules. as a result of these measures, vaccination coverage, with resulting pertussis control, improved dramatically in the united kingdom. pertussis continues to be a public health threat and recurs wherever there is inadequate immunization in infancy. a new acellular vaccine is ready for widespread use and will be safer with fewer and less severe reactions in infants, increasing the potential for improved confidence and support for routine vaccination. use of the new vaccine is spreading in the united states and forms part of the u.s. recommended vaccination schedule. tetanus. tetanus is an acute disease caused by an exotoxin of the tetanus bacillus (clostridium tetani) which grows anaerobically at the site of an injury. the bacillus is universally present in the environment and enters the human body via penetrating injuries. following an incubation period of - days, it causes an acute condition of painful muscular contractions. unless there is modem medical care available, patients are at risk of high case fatality rates of - % (highest in infants and the elderly). antitetanus serum (ats) was discovered in and during world war i, ats contributed to saving the lives of many thousands of wounded soldiers. tetanus toxoid was developed in . the organism, because of its universal presence in the environment, cannot be eradicated. however, the disease can be controlled by effective immunization of every child during infancy and school age. adults should receive routine boosters of tetanus toxoid once very decade. newborns are infected by tetanus spores (tetanus neonatorum) where unsanitary conditions or practices are present. it can occur when traditional birth attendants at home deliveries use unclean instruments to sever the umbilical cord, or dress the severed cord with contaminated material. tetanus neonatorum remains a serious public health problem in developing countries. immunization of pregnant women and women of childbearing age is reducing the problem by conferring passive immunity to the newborn. the training of traditional birth attendants in hygienic practice and the use of medically supervised birth centers for delivery also decreases the incidence of tetanus neonastorum. elimination of tetanus neonatorum by the year was made a health target by the world summit of children in . in that year, the number of deaths from neonatal tetanus was reported by unicef as , infants worldwide, declining to , in . immunization of pregnant women increased from under % in to % in - . despite progress, coverage is still too low to achieve the target of elimination. poliomyelitis. polio virus infection may be asymptomatic or cause an acute nonspecific febrile illness. it may reach more severe forms of aseptic meningitis and acute flaccid paralysis with long-term residual paralysis or death during the acute phase. poliomyelitis is transmitted mainly by direct person-to-person contact, but also via sewage contamination. large-scale epidemics of disease, with attendant paralysis and death, occurred in industrialized countries in the s and s, engendering widespread fear and panic and thousands of clinical cases of "infantile paralysis". growth of the poliovirus by john enders and colleagues in tissue culture in led to development of the first inactivated polio vaccine by jonas salk in the mid- s and gave hope and considerable success in the control of the disease. the development of the live attenuated oral poliomyelitis vaccine by albert sabin, licensed in , added a new dimension to its control because of the effectiveness, low cost, and ease of administration of the vaccine. the two vaccines in their more modern forms, enhanced strength inactivated polio vaccine (eipv), and triple oral polio vaccine (topv), have been used in different settings with great success. oral polio vaccine (opv) induces both humoral and cellular, including intestinal, immunity. the presence of opv in the environment by contact with immunized infants and via excreta of immunized persons in the sewage gives a booster effect in the community. immunization using opv, in both routine and national immunization days (nids) has proven effective in dramatically reducing poliomyelitis and circulation of the wild virus in many parts of the world. use of the enhanced strength ipv (eipv) produces early and high levels of circulating antibodies, as well as protecting against the vaccine-associated disease. in rare cases opv can cause vaccine-associated paralytic poliomyelitis (vapp), with a risk of case per , with initial doses, and case per over million with subsequent doses. approximately eight to ten cases of vapp occur annually in the united states, with clinical, ethical, and legal implications. use of ipv as initial protection eliminates this problem. experience in gaza and the west bank in the s and s, and later in israel, showed that a combination of ipv and opv is effective in overcoming endemic and imported poliovirus. opv requires multiple doses to achieve protective antibody levels. where there are many enteroviruses in the environment, as is the case in most developing countries, interference in the uptake of opv may result in cases of paralytic poliomeylitis among persons who have received or even doses of adequate opv. controversy as to the relative advantages of each vaccine continues. the opv program of mass repeated vaccination in control of poliomyelitis in the americas established the primacy of opv in practical public health, and the momentum to eradicate poliomyelitis is building. a combined schedule of ipv and opv would eliminate the wild virus and protect against vaccine-associated disease. the sequential use of ipv and opv was adopted as part of the routine infant immunization program in the united states in , but ipv alone was adopted in . there are concerns that exclusive use of either vaccine alone will not lead to the desired goal of eradication of polyomyelitis. progress in global eradication of polio has been impressive. global coverage of infants with three doses of opv reached % in as compared to % in . the african region of who had an increase in opv coverage from % in to % in . national immunization days (nids) were conducted in countries in and in , covering million children in . mopping up operations to reinforce coverage of children in still endemic areas is proceeding along with increased emphasis on acute flaccid paralysis (afp) monitoring. confirmed polio cases reported continued at - , per year in - . with continued national and international emphasis, and support of who, rotary international, unicef, donor countries, and others, there is a real prospect of a world without polio, if not by the year , then or shortly thereafter. measles is an acute disease caused by a virus of the paramyxovirus family. it is highly infectious with a very high ratio of clinical to subclinical case ratio ( / ). measles has a characteristic clinical presentation with fever, white spots (koplik spots) on the membranes of the mouth, and a red blotchy rash appearing on the rd- th day lasting - days. mortality rates are high in young children with compromised nutritional status, especially vitamin a deficiency. the measles virus evolved from a virus disease of cattle (rinderpest) some - years ago, becoming an important disease of humans with high mortality rates in debilitated, poorly nourished children, and significant mortality and morbidity even in industrialized countries. in the prevaccine era, measles was endemic worldwide, and even in the late s it remains one of the major childhood infectious diseases. it is one of the commonest causes of death for school age children worldwide. despite earlier predictions that measles deaths would be halved to , by , who reported . million measles deaths in that year and over million in . eradication in the first decade of the next century is a feasible goal, provided that there is an adequate international effort. measles immunization increased from under % worldwide in to % in - , but % in sub-saharan africa. single-dose immunization failed to meet control or eradication requirements even in the most developed parts of the world. a live vaccine, licensed in , was later replace by a more effective and heat stable vaccine, but still with a primary vaccination failure rate (i.e., fails to produce protective antibodies) of - %, and secondary failure rate (i.e., produces antibodies but protection is lost over time) of %. a two-dose policy incorporates a booster dose, usually at school-age, in addition to maximum feasible infant coverage of children in the - month period (timing varies in different countries). catch-up campaigns among schoolage children should be carried out until the routine two-dose policy has time to take full effect. nearly universal primary education in developing countries, offers an opportunity for mass coverage of school age children with a second dose of measles and a resulting increase of herd immunity to reduce the transmission of the virus. the two-dose policy adopted in many countries, should be supplemented with catch-up campaigns in schools to provide the booster effect for those previously immunized and to cover those previously unimmunized, especially in developing countries. the cdc considers that domestic transmission in the united states has been interrupted and that most localized outbreaks were traceable to imported cases. south america and the caribbean countries are now considered free of indigenous measles, based on their successful use of nids, although a large epidemic occurred in in brazil. it now appears that eradication has become a feasible target during the early part of the next century, with a strategy of levels of coverage in in-fancy with a two-dose policy, supplemented by catch-up campaigns to older children and young adults, and outbreak control. mumps. mumps is an acute viral disease characterized by fever, swelling, and tenderness usually of the parotid glands, but also other glands. the incubation period ranges between and days. orchitis, or inflammation of the testicles, occurs in - % of postpubertal males and oophoritis, or inflammation of the ovaries, in % of postpubertal females. sterility is an extremely rare result of mumps. central nervous system involvement can occur in the form of aseptic meningitis, almost always without sequelae. encephalitis is reported in - per , cases with an overall case fatality rate of . %. pancreatitis, neuritis, nerve deafness, mastiffs, nephritis, thyroiditis, and pericarditis, although rare, may occur. most persons born before are immune to the disease, because of the nearly universal exposure to the disease before that time. the live attenuated vaccine introduced in the united states in is available as a single vaccine or in combination with measles and rubella as the measlesmumps-rubella (mmr) vaccine. it provides long-lasting immunity in % of cases. mumps vaccine is now recommended in a two-dose policy with the first dose of mmr given between and months of age and a second dose given either at school entry or in early adolescence. mmr in two doses is now standard policy in the united sates, sweden, canada, israel, the united kingdom, and other countries. the incidence of mumps has consequently declined rapidly. local eradication of this disease is worthwhile and should be part of a basic international immunization program. rubella. rubella (german measles) is generally a mild viral disease with lymphadenopathy and a diffuse, raised red rash. low grade fever, malaise, coryza, and lymphadenopathy characterize the prodromal period. the incubation period is usually - days. differentiation from scarlet fever, measles, or other febrile diseases with rash may require laboratory testing and recovery of the virus from nasopharyngeal, blood, stool, and urine specimens. in , norman gregg, an australian ophthalmologist, noted an epidemic of cases of congenital cataract in newborns associated with a history of rubella in the mother during the first trimester. subsequent investigation demonstrated that intrauterine death, spontaneous abortion, and congenital anomalies occur commonly when rubella occurs early in pregnancy. congenital rubella syndrome (crs) occurs with single or multiple congenital anomalies including deafness, cataracts, microophthalmia, congenital glaucoma, microcephaly, meningoencephalitis, congenital heart defects, and others. moderate and severe cases are recognizable at birth, but mild cases may not be detected for months or years after birth. insulin-dependent diabetes is suspected as a late sequela of congenital rubella. each case of crs is estimated to cost some $ , in health care costs during the patient's lifetime. prior to availability of the attenuated live rubella vaccine in , the disease was universally endemic, with epidemics or peak incidence every - years. in unvaccinated populations, rubella is primarily a disease of childhood. in areas where children are well vaccinated, adolescent and young adult infection is more apparent, with epidemics in institutions, colleges, and among military personnel. a sharp reduction of rubella cases was seen in the united states following introduction of the vaccine in , but increased in , following rubella epidemics in - . a further reduction in cases was followed by a sharp upswing of rubella and crs in [ ] [ ] [ ] . an outbreak of rubella among the amish in the united states, who refuse immunization on religious grounds, resulted in cases of crs in . it is now thought that vaccination of sufficient numbers in the united states reduced circulation of the virus and protected most vulnerable groups in the population. in the past, immunization policy in some countries was to vaccinate school girls aged to protect them for the period of fertility. the current approach is to give a routine dose of mmr in early childhood, followed by a second dose in early school age to reduce the pool of susceptible persons. women of reproductive age should be tested to confirm immunity before pregnancy and immunized if not already immune. should a woman become infected during pregnancy, termination of pregnancy previously recommended is now managed with hyperimmune globulin. the infection of pregnant women during their first trimester of pregnancy is the primary public health implication of rubella. the emotional and financial burden of crs, including the cost of treatment of its congenital defects, makes this vaccination program cost-effective. its inclusion in a modem immunization program is fully justified. elimination of crs syndrome should be one of the primary goals of a program for prevention of vaccine-preventable disease in developed and developing countries. adoption of mmr and the two-dose policy will gradually lead to eradication of rubella and rubella syndrome. viral hepatitis. viral hepatitis is a group of diseases of increasing public health importance due to their large scale worldwide prevalence, their serious consequences, and our increasing ability to take preventive action. viral hepatic infectious diseases each have specific etiologic, clinical, epidemiologic, serologic, and pathologic characteristics. they have important short-and long-term sequelae. vaccine development is of high priority for control and ultimate eradication. hepatitis a. hepatitis a (hav) was previously known as infectious hepatitis or epidemic jaundice. hav is mainly transmitted by the fecal-oral route. clinical severity varies from a mild illness of - weeks to a debilitating illness lasting several months. the norm is complete recovery within weeks, but a fulminating or even fatal hepatitis can occur. severity of the disease worsens with increasing age. hav is sporadic/endemic worldwide. improving sanitation raises the age of exposure, with accompanying complications. it now occurs particularly in persons from industrialized countries when exposed to situations of poor hygiene, or among young adults when traveling to areas where the disease is en-demic. common source outbreaks occur in school-aged children and young adults from case contact, or from food contaminated by infected handlers. hepatitis a may be a serious public health problem in a disaster situation. prevention involves improving personal and community hygiene, with safe chlorinated water and proper food handling. hepatitis a vaccine has been recently licensed for use in the united states, and will probably soon be recommended for routine vaccination programs, as well as for persons traveling to endemic areas. hepatitis b. hepatitis b (hbv) once called serum jaundice, was thought to be transmitted only by injections of blood or blood products. it is now known to be present in all body fluids and easily transmissible by household and sexual contact, perinatal spread from mother to newborn, and between toddlers. however, it is not spread by the oral-fecal route. hepatitis b virus is endemic worldwide and is especially prevalent in developing countries. carrier status with persistent viremia varies from < % of adults in north america to % in some parts of the world. carders have detectable levels of hbsag, the surface antigen (i.e., australian antigen), in their blood. high risk groups in developed countries include intravenous drug users, homosexual men, persons with high numbers of sexual partners, those receiving tattoos, body piercing or acupuncture treatments, and residents or staff of institutions such as group homes and prisons. immunocompromised and hemodialysis patients are commonly carders of hbv. hbv may also be spread in a health system by use of inadequately sterilized reusable syringes, as in china and the former soviet union. transmission is reduced by screening blood and blood products for hbsag and strict technique for handling blood and body fluids in health settings. hbv is clinically recognizable in less than % of infected children but is apparent in - % of infected adults. clinically hbv has an insidious onset with anorexia, abdominal discomfort, nausea, vomiting, and jaundice. the disease can vary in severity from subclinical, very mild to fulminating liver necrosis, and death. it is a major cause of primary liver cancer, chronic liver disease, and liver failure, all devastating to health and expensive to treat. hepatitis b virus is considered to be the cause of % of primary cancer of the liver in the world and the most common carcinogen after cigarette smoking. the who estimates that more than billion people alive today have been infected with hbv. it is also estimated that million persons are chronic carriers of hbv, with an estimated - . million deaths per year from cirrhosis or primary liver cancer. this makes hepatitis b control a vital issue in the revision of health priorities in many countries. strict discipline in blood banks and testing of all blood donations for hbv, as well as hiv, and hepatitis c, is mandatory, with destruction of those with positive tests. contacts should be immunized following exposure with hbv immunoglobulin and hbv vaccine. the inexpensive recombinant hbv vaccine should be adopted by all countries and included in routine vaccination of infants. catch-up immunization for older children is also desirable. immunization programs should include those exposed at work, such as health, prison, or sex workers and adults in group settings. hbv immunization has been included in who's epi-plus expanded program of immunization. hepatitis c. first identified in , and previously known as non-a, non-b hepatitis, hepatitis c (hcv) has an insidious onset with jaundice, fatigue, abdominal pain, nausea, and vomiting. it may cause mild to moderate illness, but chronicity is common going on to cirrhosis and liver failure. the cdc estimates that million americans are chronically infected with hcv, with - , resulting deaths per annum, and the main cause of liver transplants. hcv is transmitted most commonly in blood products, but also among injecting drug users ( % of intravenous drug users were hcv positive in a vancouver study in ), and is also a risk for health workers. the disease may also occur in dialysis centers and other medical situations. person-to-person spread is unclear. prevention of transmission includes routine testing of blood donations, antiviral treatment of blood products, needle exchange programs, and hygiene. the who in has declared hepatitis prevention as a major public health crisis, with an estimated million persons infected worldwide ( ) , stressing that this "silent epidemic" is being neglected and that screening of blood products is vital to reduce transmission of this disease as for hiu hcv is a major cause of chronic cirrhosis and liver cancer. no vaccine is available at present, but an experimental vaccine is undergoing field trials. interferon and ribavirin treatment is reportedly effective in % of cases. hepatitis d. hepatitis d virus (hdv) also known as delta hepatitis, may be self-limiting or progress to chronic hepatitis. it is caused by a viruslike particle which infects cells along with hbv as a coinfection or in chronic carriers of hbv. hdv occurs worldwide in the same groups at risk for hbv. it also occurs in epidemics and is endemic in south america, africa, and among drug users. prevention is by measures similar to those for hbv. management for hdv is by passive immunity with immunoglobulin for contacts and high risk groups, and should include hbv vaccination as the diseases often coincide. there is currently no vaccine for hdv. hepatitis e. hepatitis e virus has an epidemiological and clinical course similar to that of hav. there is no evidence of a chronic form of hev. one striking characteristic of hev is its high mortality rate among pregnant women. the disease is caused by a viruslike particle with an incubation period of - days and is most common in young adults. sporadic cases as well as epidemics have been identified in india, pakistan, burma, china, russia, mexico, and north africa. hev results from waterborne epidemics or as sporadic cases in areas with poor hygiene, spread via the oral-fecal route. it is a hazard in disaster situations with crowding and poor sanitary conditions. prevention is by safe management of water supplies and sanitation. disease management is supportive care; passive immunization is not helpful and no vaccine is currently available. teria which causes meningitis and other serious infections in children under months of age. before the introduction of effective vaccines, as many as in children developed invasive hib infection. two-thirds of these had hib meningitis, with a case fatality rate of - %. long-term sequelae such as hearing impairment and neurological deficits occurred in - % of survivors. the first hib vaccine was licensed in , based on capsular material from the bacteria. extensive clinical trials in finland demonstrated a high degree of efficacy, but less impressive results were in seen in postmarketing efficacy studies. by , a conjugate vaccine based on an additional protein cell capsular factor capable of enhancing the immunologic response was introduced. several conjugate vaccines are now available. the conjugate vaccines are now combined with dpt as their schedule is simultaneous with that of the dpt. although the hib vaccine has been found to be cost-effective, despite initially being as costly as all the basic vaccines combined (i.e., dpt, opv, mmr, and hbv). for this reason, its use thus far has been limited to industrialized countries. the vaccine is a valuable addition to the immunologic armamentarium. it showed dramatic results in local eradication of this serious early childhood infection in a number of european countries and a sharp reduction in the united states. impressive field trials in the gambia showed a sharp reduction in mortality from invasive streptococcal diseases. the price of the vaccine has also fallen dramatically since the mid s. as a result, in , the world health organization recommended inclusion of hib vaccine in routine immunization programs in developing countries. influenza. influenza is an acute viral respiratory illness characterized by fever, headache, myalgia, prostration, and cough. transmission is rapid by close contact with infected individuals and by airborne particles with an incubation period of - days. it is generally mild and self-limited with recovery in - days. however, in certain population groups, such as the elderly and chronically ill, infection can lead to severe sequelae. gastrointestinal symptoms commonly occur in children. during epidemics, mortality rates from respiratory diseases increase because of the large numbers of persons affected, although the case fatality rates are generally low. over the past century, influenza pandemics have occurred in , , , and , while epidemics are annual events. the influenza pandemic of caused millions of deaths among young adults, by some estimates killing more than had died in world war i. it was the fear of recurrence of this pandemic which led the cdc to launch a massive immunization program in the united states in to prevent swine flu (the virus was a strain antigenically similar to that of the pandemic influenza) from spreading from an isolated outbreak in an army camp. the effort was stopped after millions of persons were immunized with an urgently produced vaccine when serious reactions occurred (guillain-barre syndrome, (i.e., a type of paralysis), and when no further cases of swine flu were seen. this demonstrated the difficulty of extrapolating scenarios from a historical experience. each year, epidemiologic services of the who and collaborating centers such as the cdc recommend which strains should be used in vaccine preparation for use among susceptible population groups. these vaccines are prepared with the current anticipated epidemic strains. the three main types of influenza (a, b, and c) have different epidemiological characteristics. type a and its subtypes, which are subject to antigenic shift, are associated with widespread epidemics and pandemics. type b undergoes antigenic drift and is associated with less widespread epidemics. influenza type c is even more localized. active immunization against the prevailing wild strain of influenza virus produces a - % level of protection in high risk groups. the benefits of annual immunization outweigh the costs, and it has proven to be effective in reducing cases of influenza and its secondary complications such as pneumonia and death from respiratory complications in high-risk groups. pneumococcal disease. pneumococcal diseases, which are caused by streptococcus pneumoniae, include pneumonia, meningitis, and otitis media. the capsular types of pneumococci selected out of known types of the organism for the vaccine are those responsible for % of pneumococcal pneumonia cases and - % of all pneumonia cases in the united states, and are responsible for some , deaths per year. this vaccine has been found to be cost-effective for high risk groups, including persons with chronic disease, hiv carriers, patients whose spleens were removed, the elderly, and those with immunosuppressive conditions. it should be included in preventive-oriented health programs, especially for long-term care of the chronically ill. because pneumococci cause bacterial meningitis, pneumococcal vaccine may be a future candidate for use in routine immunization programs for children (over age ). varicella is an acute, generalized virus disease caused by the varicella zoster virus (vzv). despite its reputation as an innocuous disease of childhood, varicella patients can be quite ill. a mild fever and characteristic generalized red rash lasts for a few hours, followed by vesicles occurring in successive crops over various areas of the body. affected areas may include the membranes of the eyes, mouth, and respiratory tract. the disease may be so mild as to escape observation or may be quite severe, especially in adults. death can occur from viral pneumonia in adults and sepsis or encephalitis in children. neonates whose mothers develop the disease within days of delivery are at increased risk with a case fatality rate of up to %. long-term sequelae include herpes zoster or shingles with a severely painful, vesicular rash along the distribution of sensory nerves, which can last for months. its occurrence increases with age and it is primarily seen in the elderly. it can, however, occur in immunocompromised children (especially those on cancer chemotherapy), aids patients, and others. some % of a population will experience herpes zoster during their lifetimes. reye's syndrome is an increasingly rare but serious complication from varicella or influenza b. it occurs in children and affects the liver and central nervous system. congenital varicella syndrome with birth defects similar to congenital rubella syndrome has been identified recently. varicella vaccine is now recommended for routine immunization at age - months in the united states, with catch-up for children up to age years and for occupationally exposed persons in health or child care settings. varicella vaccine is also recommended for nonpregnant women of child bearing years. cost-benefit studies indicate a : ratio if both direct and indirect costs are included (see chapter ). varicella vaccine is likely to be added to a "cocktail vaccine" containing dpt, polio (ipv), and hib. meningococcal meningitis. meningococcal meningitis, caused by the bacterium neisseria meningitides, is characterized by headache, fever, neck stiffness, delirium, coma, and/or convulsions. the incubation period is - days. it has a case fatality rate of - % if treated early and adequately, but rises up to % in the absence of treatment. there are several important strains (a, b, c, x, y, and z). serogroups a and c are the main causes of epidemics, with b causing sporadic cases and local outbreaks. transmission is by direct contact and droplet spread. meningitis (group a) is common in sub-saharan african countries, but epidemics have occurred worldwide. during epidemics, children, teenagers, and young adults are the most severely affected. in developed countries, outbreaks occur most frequently in military and student populations. in , meningococcal meningitis spread widely in the "meningitis belt" in central africa. epidemic control is achieved by mass chemoprophylaxis with antibiotics (e.g., rifampin or sulfa drugs) among case contacts, although the emergence of resistant strains is a concern. vaccines against serotypes a and c (bivalent) or a, c, w, and y are available. their use is effective in epidemic control and prevention institutions and military recruits, especially for a and c serogroups. vaccination is one of the key modalities of primary prevention. immunization is cost-effective and prevents wide-scale disease and death, with high levels of safety. despite the general consensus in public health regarding the central role of vaccination, there are many areas of controversy and unfulfilled expectations. a vaccination program should aim at % coverage at appropriate times, including infants, school children, and adults. immunization policy should be adapted from current international standards applying the best available program to national circumstances and financial capacities (table . ). public health personnel with expertise in vaccine-preventable disease control are needed to advise ministries of health and the practicing pediatric community on current issues in vaccination and to monitor implementation and evolution of control programs. controversies and changing views are common to immunization policy, so that discussions must be conducted on a continuing basis. policy should be under continuing review by a ministerially appointed national immunization advisory committee, including professionals from public health, academia, immunology, laboratory sciences, economics, and relevant clinical fields. bduring , the recommendation for polio virus was changed to doses of ipv in infancy. vaccine supply should be adequate and continuous. supplies should be ordered from known manufacturers meeting international standards of good manufacturing practice. all batches should be tested for safety and efficacy prior to release for use. there should be an adequate and continuously monitored cold chain to protect against high temperatures for heat labile vaccines, sera, and other active biological preparations. the cold chain should include all stages of storage, transport, and maintenance at the site of usage. only disposable syringes should be used in vaccination programs to prevent any possible transmission of blood-borne infection. a vaccination program depends on a readily available service with no barriers or unnecessary prerequisites, free to parents or with a minimum fee, to administer vaccines in disposable syringes by properly trained individuals using patientoriented and community-oriented approaches. ongoing education and training on current immunization practices are needed. incentive payments by insuring agency or managed care systems promote complete, on-time coverage. all clinical encounters should be used to screen, immunize, and educate parents/guardians. contraindications to vaccination are very few; vaccines may be given even during mild illness with or without fever, during antibiotic therapy, during convalescence from illness, following recent exposure to an infectious disease, and to persons having a history of mild/moderate local reactions, convulsions, or family history of sudden infant death syndrome (sids). simultaneous administration of vaccines and vaccine "cocktails" reduces the number of visits and thereby improves coverage; there are no known interferences between vaccine antigens. accurate and complete recording with computerization of records with automatic reminders helps promote compliance, as does co-scheduling of immunization appointments with other services. adverse events should be reported promptly, accurately, and completely. a tracking system should operate with reminders of upcoming or overdue immunizations; use mail, telephone, and home visits, especially for high risk families, with semiannual audits to assess coverage and review patient records in the population served to determine the percentage of children covered by second birthday. tracking should identify children needing completion of the immunization schedule and assess the quality of documentation. it is important to maintain up-to-date, easily retrievable medical protocols where vaccines are administered, noting vaccine dosage, contraindications, and management of adverse events. all health care providers and managers should be trained in education, promotion, and management of immunization policy. health education should target parents as well as the general public. monitoring of vaccines used and children immunized, individually and by category of vaccination can be facilitated by computerization of immunization records, or regular manual review of child care records. where immunization is done by physicians in private practice, as in the united states, determination of coverage is by periodic surveys. inspection of vaccines for safety, purity, potency, and standards is part of the regulatory function. vaccines are defined as biological products and are therefore subject to regulation by national health authorities. in the united states, this comes under the legislative authority of the public health service act, as well as the food, drug and cosmetics act, with applicable regulations in the code of federal regulations. the federal agency empowered to carry out this regulatory function is the center for drugs and biologics of the federal food and drug administration. litigation regarding adverse effects of vaccines led to inflation of legal costs and efforts to limit court settlements. the u.s. federal government enacted the child vaccine injury act of . this legislation requires providers to document vaccines given and to report on complications or reactions. it was intended to pay benefits to persons injured by vaccines faster and by means of a less expensive procedure than a civil suit for resolving claims. using this no-fault system, petitioners do not need to prove that manufacturers or vaccine givers were at fault. they must only prove that the vaccine is related to the injury in order to receive compensation. the vaccines covered by this legislation include dtp, mmr, opv, and ipv. development of vaccines from jenner in eighteenth century to the advent of recombinant hepatitis b vaccine in , and of vaccines for acellular pertussis, varicella, hepatitis a, and rotavirus in the s, has provided one of the pillars of public health and led to enormous savings of human life. vaccines for viral in-fections in humans for hiv, respiratory syncytial virus, papilloma, epstein-barr virus, dengue fever, and hantavirus are under intense research with genetic approaches using recombinant techniques. the potential for the future of vaccines will be greatly influenced by scientific advances in genetic engineering, with potential for development of vaccines attached to bacteria or protein in plants, which may be given in combination for an increasing range of organisms or their harmful products. recombinant dna technology has revolutionized basic and biomedical research since the s. the industry of biotechnology has produced important diagnostic tests, such as for hiv, with great potential for vaccine development. traditional whole organism vaccines, alive or killed, may contain toxic products that may cause mild to severe reactions. subunit vaccines are prepared from components of a whole organism. this avoids the use of live organisms that can cause the disease or create toxic products which cause reactions. subunit vaccines traditionally prepared by inactivation of partially purified toxins are costly, difficult to prepare, and weakly immunogenic. recombinant techniques are an important development for production of new whole cell or subunit vaccines that are safe, inexpensive, and more productive of antibodies than other approaches. their potential contribution to the future of immunology is enormous. molecular biology and genetic engineering have made it feasible to create new, improved, and less costly vaccines. new vaccines should be inexpensive, easily administered, capable of being stored and transported without refrigeration, and given orally. the search for inexpensive and effective vaccines for groups of viruses causing diarrheal diseases led to development of the rotavirus vaccine. some "edible" research focuses on the genetic programming of plants to produce vaccines and dna. vaccine manufacturers, who spend huge sums of money and years of research on new products, tend to work on those which will bring great financial rewards for the company and are critical to the local health care community. this has led to less effort being made in developing vaccines for diseases such as malaria. yet industry plays a crucial role for continued progress in the field. since the eradication of smallpox, much attention has focused on the possibility of similarly eradicating other diseases, and a list of potential candidates has emerged. some of these have been abandoned because of practical difficulties with current technology. diseases that have been under discussion for eradication have included measles, tb, and some tropical diseases, such as malaria and dracunculiasis. eradication is defined as the achievement of a situation whereby no further cases of a disease occur anywhere and continued control measures are unnecessary. reducing epidemics of infectious diseases, through control and eradication in selected areas or target groups, can in certain instances achieve eradication of the disease. local eradication can be achieved where domestic circulation of an organism is interrupted with cases occurring from importation only. this requires a strong, sustained immunization program with adaptation to meet needs of importation of carriers and changing epidemiologic patterns. smallpox was one of the major pandemic diseases of the middle ages and its recorded history goes back to antiquity. prevention of smallpox was discussed in ancient china by ho kung (circe ao ), and inoculation against the disease was practiced there from the eleventh century ad. prevention was carried out by nasal inhalation of powdered dried smallpox scabs. exposure of children to smallpox when the mortality rate was lowest assumed a weakened form of the disease, and it was observed that a person could only have smallpox once in a lifetime. isolation and quarantine were widely practiced in europe during the sixteenth and seventeenth centuries. variolation was the practice of inoculating youngsters with material from scabs of pustules from mild cases of smallpox in the hope that they would develop a mild form of the disease. although this practice was associated with substantial mortality, it was widely adopted because mortality from variolation was well below that of smallpox acquired during epidemics. introduced into england in (see chapter ) it was commonly practiced as a lucrative medical specialty during the eighteenth century. in the s, variolation was also introduced into the american colonies, russia, and subsequently into sweden and denmark. despite all efforts, in the early eighteenth century smallpox was a leading cause of death in all age groups. toward the end of the eighteenth century an estimated , persons died annually from smallpox in europe. vaccination, or the use of cowpox vaccinia virus to protect against smallpox, was initiated late in the eighteenth century. in , a cattle breeder in yorkshire, england, inoculated his wife and two children with cowpox to protect them during a smallpox epidemic. in , edward jenner, an english country general practitioner, experimented with inoculation from a milkmaid's cowpox pustule to a healthy youngster, who subsequently proved resistant to smallpox by variolation (see chapter ). vaccination, the deliberate inoculation of cowpox material, was slow to be adopted universally, but by , over , persons in england were vaccinated. vaccination gathered support in the nineteenth century in military establishments, and in some countries which adopted it universally. opposition to vaccination remained strong for nearly a century based on religious grounds, observed failures of vaccination to give lifelong immunity, and because it was seen as an infringement of the state on the rights of the individual. often the protest was led by medical variolationists whose medical practice and large incomes were threatened by the mass movement to vaccination. resistance was also offered by "sanitarians" who opposed the germ theory and thought cleanliness was the best method of prevention. universal vaccination was increasingly adopted in europe and america in the early nineteenth century and eradication of smallpox in developed countries was achieved by the mid twentieth century. in , the soviet union proposed to the world health assembly a program to eradicate smallpox internationally and subsequently donated million doses of vaccine per year as part of the million needed to promote vaccination of at least % of the world population. in , who adopted a target for the eradication of smallpox. a program was developed which included a massive increase in coverage to reduce the circulation of the virus through person-to-person contact. where smallpox was endemic, with a substantial number of unvaccinated persons, the aim of the mass vaccination phase was % coverage. increasing vaccination coverage in developing countries reduced the disease to periodic and increasingly localized outbreaks. in , countries were considered endemic for smallpox, and another experienced importation of cases. by , the number of endemic countries was down to , and by only countries were still endemic, including india, pakistan, bangladesh, and nepal. in these countries, a new strategy was needed, based on a search for cases and vaccination of all contacts, working with a case incidence below per , . the program then moved into the consolidation phase, with emphasis on vaccination of newborns and new arrivals. surveillance and case detection were improved with case contact or risk group vaccination. the maintenance phase began when surveillance and reporting were switched to the national or regional health service with intensive follow-up of any suspect case. the mass epidemic era had been controlled by mass vaccination, reducing the total burden of the disease, but eradication required the isolation of individual cases with vaccination of potential contacts. technical innovations greatly eased the problems associated with mass vaccination worldwide. during the s, there was wide variation in sources of smallpox vaccine. in the s, efforts to standardize and further attenuate the strains used reduced complication rates from vaccinations. the development of lyophilization (freeze-drying) of the vaccine in england in the s made a heat-stable vaccine that could be effective in tropical field conditions in developing countries. the invention of the bifurcated needle (bernard rubin ) allowed for easier and more widespread vaccination by lesser trained personnel in remote areas. the net result of these innovations was increased world coverage and a reduction in the spread of the disease. smallpox became more and more confined by increasing herd immunity, thus allowing transition to the phase of monitoring and isolation of individual cases. in the last case of smallpox was identified in somalia, and in the who declared the disease eradicated. no subsequent cases have been found except for several associated with a laboratory accident in the united kingdom in . the who recommends that the last stores of smallpox virus should be destroyed in . the cost of the eradication program was $ million or $ million per year. worldwide savings are estimated at $ billion annually. this monumental public health achievement set the precedent for eradication of other infectious diseases. the world health assembly decided to destroy the last two remaining stocks of the smallpox virus in atlanta and moscow in . destruction of the remaining stock was delayed in to because of concern that illegal stocks may be held by some states or potential bioterrorists for potential use in weapons of mass destruction, concern regarding the appearance of monkeypox and a wish to use the virus for further research. in , the who established a target of eradication of poliomyelitis by the year . global immunization coverage with three doses of opv increased from some % in to over % in , with a slight decline in the period - . support from member countries and international agencies such as unicef and rotary international has led to widescale increases in immunization coverage throughout many parts of the world. the world health organization promotes use of opv only as part of routine infant immunization or national immunization days (nids). this strategy has been successful in the americas and in china, but india and the middle east remain problematic. eradication of wild poliomyelitis by the year will require flexibility in vaccination strategies and may require the combined approach, using opv and ipv, as adopted in the united states in to prevent vaccine-associated clinical cases. the combination of opv and ipv may be needed where enteric disease is common and leads to interference in opv uptake, especially in tropical areas where endemic poliovirus and diarrheal diseases are still found. the world bank estimated that achievement of global eradication would save $ million annually in the united states alone. since the eradication of smallpox, discussion has focused on the possibility of similarly eradicating other diseases, and a list of potential candidates has emerged. some of these have been abandoned because of practical difficulties with current technology. diseases that have been under discussion for eradication have included measles, tb, and tropical diseases such as malaria and dracunculiasis. eradication of malaria was thought to be possible in the s when major gains were seen in malaria control by aggressive case environmental control, case finding, and management. however, lack of sustained vector control and an effective vaccine has prevented global eradication. malaria control suffered serious setbacks because of failure in political resolve and capacity to continue support needed for necessary programs. in the s and s, control efforts were not sustained in many countries, and a dreadful comeback of the disease occurred in africa and asia in the s. the emergence of mosquitoes resistant to insecticides, and malarial strains resistant to antimalarial drugs, have made malaria control even more difficult and expensive. renewed effort in malaria control may require new approaches. use of community health workers (chws) in small villages in highly endemic regions of colombia resulted in a major drop in malaria mortality during the s. the chws investigate suspect cases by taking clinical histories and blood smears. . scientific feasibility a. epidemiologic vulnerability; lack of nonhuman reservoir, ease of spread, no natural immunity, relapse potential; b. effective practical intervention available; vaccine or other primary preventive or curative treatment, or vectoricide that is safe, inexpensive, long lasting, and easily used in the field; c. demonstrated feasibility of elimination in specific locations, such as an island or other geographic unit. . political will/popular support a. they examine smears for malaria parasites and a diagnosis is made. therapy is instituted and the patient is followed. quality control monitoring shows high levels of accuracy in reading of slides compared to professional laboratories. in the late s, there was widespread discussion in the literature of the potential for eradication of measles and tb. measles eradication was set back as breakthrough epidemics occurred in the united states, canada, and many other countries during the s and early s, but regional eradication was achieved combining the two-dose policy with catch-up campaigns for older children or in national immunization days, as in the caribbean countries. tuberculosis has also increased in the united states and several european countries for the first time in many decades. unrealistic expectations can lead to inappropriate assessments and policy when confounding factors alter the epidemiologic course of events. such is the case with tb, where control and eradication have receded from the picture. this deadly disease has returned to developed countries, partly in association with the hiv infection and multiple-drug-resistant strains, as well as homelessness, rising prison populations, poverty, and other deleterious social conditions. directly observed therapy is an important recent breakthrough, more effective in use of available technology and will play a major role in tb control in the twenty-first century. a decade after the eradication of smallpox was achieved, the international task force for disease eradication (itfde) was established to systematically evaluate the potential for global eradicability of candidate diseases. its goals were to identify specific barriers to the eradication of these diseases that might be surmountable and to promote eradication efforts. the subject of eradication versus control of infectious diseases if of central public health importance as technology expands the armamentarium of immunization and vector control into the twenty-first century. the control of epidemics, followed by interruption of transmission and ultimately eradication, will save countless lives and prevent serious damage to children throughout the world. the smallpox achievement, momentous in itself, points to the potential for the eradication of other deadly diseases. the skillful use of existing and new technology is an important priority in the new public health. flexibility and adaptability are as vital as resources and personnel. selecting diseases for eradication is not purely a professional issue of resources such as vaccines and manpower, organization and financing. it is also a matter of political will and perception of the burden of disease. there will be many controversies. the selection of polio for eradication while deferring measles when polio kills few and measles kills many may be questioned. the cdc published criteria for selection of disease for eradication are shown in box . . the who, in a review of health targets in the field of infectious disease control for the twenty-first century, selected the following targets: eradication of chagas' disease by ; eradication of neonatal tetanus by ; eradication of leprosy by ; eradication of measles by ; eradication of trachoma by ; reversing the current trend of increasing tuberculosis and hiv/aids. in , a conference in atlanta, georgia, reviewed the subject, which is still very much in a state of flux. table . summarizes the selection of diseases which are presently seen as controllable and those considered to be potentially eradicable. the subject will be under review in the years ahead. mycobacterium tuberculosis in humans and m. bovis in cattle. the disease is primarily found in humans, but it is also a disease of cattle and occasionally other primates in certain regions of the world. it is transmitted via airborne droplet nuclei from persons with pulmonary or laryngeal tb during coughing, sneezing, talking, or singing. the initial infection may go unnoticed, but tuberculin sensitivity appears within a few weeks. about % of those infected enter a latent phase with a lifelong risk of reactivation. approximately % go from initial infection to pulmonary tb. less commonly, the infection develops as extrapulmonary tb, involving meninges, lymph nodes, pleura, pericardium, bones, kidneys, or other organs. untreated, about half of the patients with active tb will die of the disease within years, but modern chemotherapy almost always results in a cure. pulmonary tb symptoms include cough and weight loss, with clinical findings on chest examination and confirmation by findings of tubercle bacilli in stained smears of sputum and, if possible, growth of the organism on culture media, and changes in the chest x-ray. tuberculosis affects people in their adult working years, with - % of cases in persons between the ages of and . its devastating effects on the work force and economic development contribute to a high cost-effectiveness for tb control. the tubercle bacillus infects approximately . billion people in the world today, causing over million cases and nearly million deaths in . during , new cases of tb included . million ( %) in southeast asia and the western pacific regions of who, with . million cases in india, and . million in indonesia. by , the incidence of tb may increase to . million new cases per year, a % increase over . between and , who estimates there were million new cases of tb, of which million cases were in association with hiv infection. during the s, an estimated million persons died of tb, including . million with hiv infection. a new and dangerous period for tb resurgence has resulted from parallel epidemiologic events: first, the advent of hiv infection and second, the occurrence of multiple drug resistant tb (mdrtb), that is, organisms resistant at least to both isoniazid (inh) and rifampicin, two mainstays of tb treatment. mdrtb can have a case fatality rate as high as %. hiv reduces cellular immunity so that people with latent tb have a high risk of activation of the disease. it is estimated that hiv negative persons have a - % lifetime risk of tb; hiv positive people have a risk of % per year of developing clinical tuberculosis. drug resistance, the long period of treatment, and the socioeconomic profile of most tb patients combine to require a new approach to therapy. directly observed treatment, short-course (dots), has shown itself to be highly effective with patients in poor self-care settings, such as the homeless, drug users, and those with aids. the strategy of dots uses community health workers to visit the patient and observes him or her taking the various medications, providing both incentive, support, and moral coercion to complete the needed to month therapy. dots has been shown to cure up to % of cases, at a cost of as little as $ per patient. it is one of the few hopes of containing the tb pandemic. in , who released a new strategy for control of tuberculosis over the next decade. the plan calls for new guidelines for control, new aid funds for developing countries, and enlistment of ngos to assist in the fight. the new guidelines stress short-term chemotherapy in well-managed programs of dots, stressing strict compliance with therapy for infectious cases with a goal of an % cure rate. even under adverse conditions, dots produces excellent results. it is one of the most cost-effective health interventions combining public health and clinical medical approaches. tuberculosis incidence in the united states decreased steadily until , increased in , and has declined again since. from to , there was an excess of , cases over the expected rate if the previous decline in case incidence had continued. this rise was largely due to the hiv/aids epidemic and the emergence of mdrtb, but also greater incidence among immigrants from areas of higher tb incidence, drug abusers, the homeless, and those with limited access to health care. this is particularly true in new york city, where mdrtb has appeared in outbreaks among prison inmates and hospital staff. from to , tb incidence in the united states declined by % and in some states, including new york, by % or more. this turnaround was due to stronger tb control programs that promptly identified persons with tb and initiated and ensured completion of appropriate therapy. aggressive staff training, outreach, and case management approaches were vital to this success. concern over rising rates among recent immigrants and the continued challenge of hiv/aids and coincidental transmission of hepatitis a, b, and c among drug users and marginal population groups show that continued support for tb control is needed. bacillus calmette-gurrin (bcg) is an attenuated strain of the tubercle bacillus used widely as a vaccination to prevent tb, especially in high incidence areas. it induces tuberculin sensitivity or an antigen-antibody reaction in which antibodies produced may be somewhat protective against the tubercle bacillus in % of vaccinees. although the support for its general use is contradictory, there is evidence from case-control and contact studies of positive protection against tb meningitis and disseminated tb in children under the age of . in some developed, low-incidence countries, it is not used routinely but selectively. it may also be used in asymptomatic hiv-positive persons or other high risk groups. the bcg vaccine for tuberculosis remains controversial. while used widely internationally, in the united states and other industrialized countries, it is thought to hinder rather than help in the fight against tb. this concern is based on the usefulness of tuberculin testing for diagnosis of the disease. where bcg has been administered, the diagnostic value of tuberculin testing is reduced, especially in the period soon after the bcg is used. studies showing equivocal benefit of bcg in preventing tuberculosis have added to the controversy. while those in the field in the united states continue to oppose the use of bcg, internationally it is still felt to be of benefit in preventing tb, primarily in children. a metaanalysis of the literature of bcg carried out by the technology assessment group at harvard school of public health concluded: on average, bcg vaccine significantly reduces the risk of tb by %. protection is observed across many populations, study designs, and forms of tb. age at vaccination did not enhance predictiveness of bcg efficacy. protection against tuberculous death, meningitis, and disseminated disease is higher than for total tb cases, although this result may reflect reduced error in disease classification rather than greater bcg efficacy. [colditz et al., jama, .] box . control of tuberculosis . identifying persons with clinically active tb; . diagnostic methods--clinical suspicion, sputum smear for bacteriologic examination, tuberculin skin testing, chest radiograph; . case finding and investigation programs in high risk groups; . contact investigation; . isolation techniques during initial therapy; . treatment, mainly ambulatory, of persons with clinically active tb; . treatment of contacts; . directly observed treatment, short-course (dots), where compliance suspect; . environmental control in treatment settings to reduce droplet infection; . educate health care providers on suspicion of tb and investigation of suspects. currently, the who recommends use of bcg as close to birth as possible as part of the expanded programme of immunization (epi). tuberculosis control remains feasible with current medical and public health methods. deterioration in its control should not lead to despair and passivity. the recent trend to successful control by dots despite the growing problem of mdrtb suggest that control and gradual reduction can be achieved by an activist, community outreach approach. the who in made tb control one of its major priorities, expressing grave concern that the mdr organism, now widely spread in countries of asia, eastern europe, and the former soviet union, may spread the disease much more widely. the disease constitutes one of the great challenges to public health at the start of the new century. acute infectious diseases caused by group a streptococci include streptococcal sore throat, scarlet fever, puerperal fever, septicemia, ersypelas, cellulitis, mastoiditis, otitis media, pneumonia, peritonsillitis (quinsy), wound infections, toxic shock syndrome, and fasciitis, the "flesh eating bacteria." streptococcus pyogenes group a include some serologically distinct types which vary in geographic location and clinical significance. transmission is by droplet, person-to-person direct contact, or by food infected by carriers. important complications from a public health point of view include acute rheumatic fever and acute glomerulonephritis, but also skin infections and pneumonia. acute rheumatic fever is a complication of strep a infection that has virtually disappeared from industrialized countries as a result of improved standards of living and antibiotic therapy. however, outbreaks were recorded in the united states in , and an increasing number of cases have been seen since . in developing countries, rheumatic fever remains a serious public health problem affecting school age children, particularly those in crowded living arrangements. longterm sequelae include disease of the mitral and aortic heart valves, which require cardiac care and surgery for repair or replacement with artificial valves. acute glomerulonephritis is a reaction to toxins of the streptococcal infection in the kidney tissue. this can result in long-term kidney failure and the need for dialysis or kidney transplantation. this disease has become far less common in the industrialized countries, but remains a public health problem in developing countries. the streptococcal diseases are controllable by early diagnosis and treatment with antibiotics. this is a major function of primary care systems. recent increases in rheumatic fever may herald a return of the problem, perhaps due to inadequate access to primary care in the united states for large sectors of the population, along with increased social hygiene problems. where access to primary care services is limited, infections with streptococci can result in a heavy burden of chronic heart and kidney disease with substantial health, emotional, and financial tolls. measures to improve access to care and pub-lic information are needed to assure rapid and effective care to prevent chronic and costly conditions. zoonoses are infectious diseases transmissible from vetebrate animals to humans. common examples of zoonoses of public health importance in nonindustrialized countries include brucellosis and rabies. in industrialized countries, salmonellosis, "mad cow disease" and influenza have reinforced the importance of relationships of animal and human health. strong cooperation between public health and veterinary public health authorities are required to monitor and to prevent such diseases. brucellosis is a disease occurring in cattle (brucella abortus), in dogs (br. cahis), in goats and sheep (br. melitensis), and in pigs (br. suis). humans are affected mainly through ingestion of contaminated milk products, by contact, or inhalation. brucellosis (also known as relapsing, undulant, malta, or mediterranean fever) is a systemic bacterial disease of acute or insidious onset characterized by fever, headache, weakness, sweating, chills, arthralgia, depression, weight loss, and generalized malaise. spread is by contact with tissues, blood, urine, vaginal discharges, but mainly by ingestion of raw milk and dairy products from infected animals. the disease may last from a few days to a year or more. complications include osteoarthritis and relapses. case fatality is under %, but disability is common and can be pronounced. the disease is primarily seen in mediterranean countries, the middle east, india, central asia, and in central and south america. brucellosis occurs primarily as an occupational disease of persons working with and in contact with tissues, blood, and urine of infected animals, especially goats and sheep. it is an occupational hazard for veterinarians, packinghouse workers, butchers, tanners, and laboratory workers. it is also transmitted to consumers of unpasteurized milk from infected animals. animal vectors include wild animals, so that eradication is virtually impossible. diagnosis is confirmed by laboratory findings of the organism in blood or other tissue samples, or with rising antibody titers in the blood, with confirmation by blood cultures. clinical cases are treated with antibiotics. epidemiologic investigation may help track down contaminated animal flocks. routine immunization of animals, monitoring of animals in high risk areas, quarantining sick animals, destroying infected animals, and pasteurizing milk and milk products prevents spread of the disease. control measures include educating farmers and the public not to use unpasteurized milk. individuals who work with animals (cattle, swine, goats, sheep, dogs, coyotes) should take special precautions when handling animal carcasses and materials. testing animals, destroying carriers, and enforcing mandatory pasteurization will restrict the spread of the disease. this is an economic as well as public health problem, requiring full cooperation between ministries of health and of agriculture. rabies is primarily a disease of animals, with a variety of wild animals serving as a reservoir for this disease, including foxes, wolves, bats, skunks, and raccoons, who may infect domestic animals such as dogs, cats, and farm animals. animal bites break the skin or mucous membrane, allowing entry of the virus from the infected saliva into the bloodstream. the incubation period of the virus is - weeks; it can be as long as several years or as short as days, so that postexposure preventive treatment is a public health emergency. the clinical disease often begins with a feeling of apprehension, headache, pyrexia, followed by muscle spasms, acute encephalitis, and death. fear of water ("hydrophobia") or fear of swallowing is a characteristic of the disease. rabies is almost always fatal within a week of onset of symptoms. the disease is estimated to cause , deaths annually, primarily in developing countries. it is uncommon in developed countries. rabies control focuses on prevention in humans, domestic animals, and wildlife. prevention in humans is based on preexposure prophylaxis for groups at risk (e.g., veterinarians, zoo workers) and postexposure immunization for persons bitten by potentially rabid animals. because reducing exposure of pets to wild animals is difficult, immunization of domestic animals is one of the most important preventive measures. prevention in domestic animals is by mandatory immunization of household pets. all domestic animals should be immunized at age months and revaccinated according to veterinary instructions. prevention in wild animals to reduce the reservoir is successful in achieving local eradication in settings where reentry from neighboring settings is limited. since , the use of oral rabies immunization has been successful in reducing the population of wild animals infected by the rabies virus. rabies eradication efforts, using aerial distribution of baits containing fox rabies vaccine in affected areas of belgium, france, germany, italy, and luxembourg, have been underway since . the number of rabies cases in these affected areas has declined by some %. switzerland is now virtually rabies-free because of this vaccination program. the potential exists for focal eradication, especially on islands or in partially restricted areas with limited possibilities of wild animal entry. livestock need not be routinely immunized against rabies, except in high risk areas. where bats are major reservoirs of the disease, as in the united states, eradication is not presently feasible. salmonella, discussed later in this chapter under diarrheal diseases, is one of the commonest of all infectious diseases among animals and is easily spread to humans via poultry, meat, eggs, and dairy products. specific antigenic types are associated with food-borne transmission to humans, causing generalized illness and gastroenteritis. severity of the disease varies widely, but the diseases can be devastating among vulnerable population groups, such as young children, the elderly, and the immunocompromised. epidemiologic investigation of common food source outbreaks may uncover hazardous food handling practices. laboratory confirmation or serotypes helps in monitoring the disease. prevention is by maintaining high standards of food hygiene in processing, inspection and regulation, food handling practices, and hygiene education. bacillus anthracis causes a bacterial infection in herbivore animals. its spores contaminate soil, worldwide. it affects humans exposed in occupational settings. transmission is cutaneous by contact, gastrointestinal by ingestion, or respiratory by inhalation. it has gained recent attention (iraq, ) as a highly potent agent for germ warfare or terrorism. limited supplies of vaccine are available. creutzfeld-jakob disease is a degenerative disease of the central nervous system linked to consumption of beef from cattle infected with bovine spongiform encephalopathy. it is transmitted by prions in animal feed prepared from contaminated animal material and in transplanted organs. this disease was identified in the united kingdom linked to infected cattle leading to a ban on british beef in many parts of the world and slaughter of large numbers of potentially contaminated animals. the tapeworm causing diphyllobothriasis (diphyllobothrium latum) is widespread in north american freshwater fish, passing from crustacean to fish to humans by eating raw freshwater fish. it is especially common among inuit peoples and may be asymptomatic or cause severe general and abdominal disorder. food hygiene (freezing and cooking of meat) is recommended; treatment is by anthelminthics. leptospiroses are a group of zoonotic bacterial diseases found worldwide in rats, raccoons, and domestic animals. it affects farmers, sewer workers, dairy and abattoir workers, veterinarians, military personnel, and miners with transmission by exposure to or ingestion of urine-contaminated water or tissues of infected animals. it is often asymptomatic or mild, but may cause generalized illness like influenza, meningitis, or encephalitis. prevention requires education of the public in self protection and immunization of workers in hazardous occupations, along with immunization and segregation of domestic animals and control of wild animals. vector-borne diseases are a group of diseases in which the infectious agent is transmitted to humans by crawling or flying insects. the vector is the intermediary between the reservoir and the host. both the vector and the host may be affected by climatic condition; mosquitoes thrive in warm, wet weather and are suppressed by cold weather; humans may wear less protective clothing in warm weather. the only important reservoir of malaria is humans. its mode of transmission is from person to person via the bite of an infected female anopheles mosquito (ronald ross, nobel prize, ) . the causative organism is a single cell parasite with four species: plasmodium vivax, p malariae, p falciparum, and p ovale. clinical symptoms are produced by the parasite invading and destroying red blood cells. the incubation period of approximately - days, depending on the specific plasmodium involved. some strains of p vivax may have a protracted incubation period of - months and even longer for p ovale. the disease can also be transmitted through infected blood transfusions. confirmation of diagnosis is by demonstrating malaria parasites on blood smears. falciparum malaria, the most serious form, presents with fever, chills, sweats, and headache. it may progress to jaundice, bleeding disorders, shock, renal or liver failure, encephalopathy, coma, and death. prompt treatment is essential. case fatality rates in untreated children and adults are above %. an untreated attack may last months. other forms of malaria may present as a nonspecific fever. relapse of the p ovale may occur up to years after initial infection; malaria may persist in chronic form for up to years. malaria control advanced during the s- s through improved chlovaquine treatment and use of ddt for vector control with optimism for eradication of the disease. however, control regressed in many developing countries as allocations for environmental control and case findings/treatment were reduced. there has also been an increase in drug resistance, so that this disease is now an extremely serious public health problem in many parts of the world. the need for a vaccine for malaria control is now more apparent than ever. the world health organization estimated that, in , sub-saharan africa (ssa) had million new malaria cases, with % of children up to age . over million deaths occur annually from malaria more than two-thirds of them in ssa. large areas, particularly in forest or savannah regions with high rainfall, are holoendemic. in higher altitudes, endemicity is lower, but epidemics do occur. chloroquine-resistant p. falciparum has spread throughout africa, accompanied by an increasing incidence of severe clinical forms of the disease. the world bank estimates that % of all disability-adjusted life years (dalys) lost per year in ssa are from malaria, which places a heavy economic burden on the health systems. in the americas, the number of cases detected has risen every year since , and the who estimates there to have been . - . million cases in . the nine most endemic countries in the americas achieved a % reduction in malaria mortality between and . southeast asian region reports some . million cases of malaria in and deaths from tb. this accounts for more than one-third of all non-african malaria cases. there is an increase in resistant strains to the major available drugs and of the mosquitoes to insecticides in use. vector control, case finding, and treatment remain the mainstay of control. use of insecticide-impregnated bed nets and curtains, and residual house spraying, and strengthened vector control activities are important, as are early diagnosis and carefully monitored treatment with monitoring for resistance. control of malaria will ultimately depend on a safe, effective, and inexpensive vaccine. attempts to develop a malaria vaccine have been unsuccessful to date due to the large number of genetic types of p. falciparum even in localized areas. a colombian-developed vaccine is being field-tested with partial effectiveness. research in vaccines for malaria has also been hampered by the fact that it is a relatively low priority for vaccine manufacturers because of the minimal potential for financial benefit. research on malaria concentrates on the pharmacological aspects of the disease because of increasing drug resistance. in , who has initiated a new campaign to "roll back malaria" and maintain the dream of eradication in the future. effective low technology interventions include community-based case finding, early treatment of good quality, insecticide use, and vector control. the use of community health workers in endemic areas, has shown promising results. local control and even eradication can be achieved with currently available technology. this requires an integration of public health and clinical approaches with strong political commitment. the rickettsia are obligate parasites, i.e., they can only replicate in living cells, but otherwise they have characteristics of bacteria. this is a group of clinically similar diseases, usually characterized by severe headache, fever, myalgia, rash, and capillary bleeding causing damage to brain, lungs, kidneys, and heart. identification is by serological testing for antibodies, but the organisms can also be cultured in laboratory animals, embryonic eggs, or in cell cultures. the organisms are transmitted by arthropod vectors such as lice, fleas, ticks, and mites. the diseases caused millions of deaths during war and famine periods prior to the advent of antibiotics. these diseases appear in nature in ways that make them impossible to eradicate, but clinical diagnosis, host protection, and vector control can help reduce the burden of disease and deal with outbreaks that may occur. public education regarding self-protection, appropriate clothing, tick removal, and localized control measures such as spraying and habitat modification are useful. epidemic typhus, first identified in , is due to rickettsia prowazekii. spread primarily by the body louse, typhus was the cause of an estimated million deaths, i.e., during war and famine, in poland and the soviet union from - . untreated, the fatality rate is - %. typhus responds well to antibiotics. it is currently largely confined to endemic foci in central africa, central asia, eastern europe, and south america. it is preventable by hygiene and pediculicides such as ddt and lindane. a vaccine is available for exposed laboratory personnel. murine typhus is a mild form of typhus due to rickettsia typhi, which is found worldwide and spread in rodent reservoirs. scrub typhus, also known as tsutsugamushi or japanese river fever, is located throughout the far east and the pacific islands, and was a serious health problem for u.s. armed forces in the pacific during world war ii. it is spread by the rickettsia tsutsugamushi and has a wide variation in case fatality according to region, organism, and age of patient. rocky mountain spotted fever is a well-known and severe form of tick-borne typhus due to rickettsia rickettsii, occurring in western north america, europe, and asia. q. fever is a tick-borne disease caused by coxiella burnetii and is worldwide in distribution, usually associated with farm workers, in both acute and chronic forms. regular anti-tick spraying of sheep, cows, and goats helps protect exposed workers. protective clothing and regular removal of body ticks help protect exposed persons. arthropod-borne viral diseases are caused by a diverse group of viruses which are transmitted between vertebrate animals (often farm animals or small rodents) and people by the bite of blood-feeding vectors such as mosquitoes, ticks, and sandflies and by direct contact with infected animal carcasses. usually the viruses have the capacity to multiply in the salivary glands of the vector, but some are carried mechanically in their mouthparts. these viruses cause acute central nervous system infections (meningoencephalitis), myocarditis, or undifferentiated viral illnesses with polyarthritis and rashes, or severe hemorrhagic febrile illnesses. arbovirus diseases are often asymptomatic in vertebrates but may be severe in humans. over antigenetically distinct arboviruses are associated with disease in humans, varying from benign fevers of short duration to severe hemmorhagic fevers. each has a specific geographic location, vector, clinical, and virologic characteristics. they are of international public health importance because of the potential for spread via natural phenomena and modem rapid transportation of vectors and persons incubating the disease or ill with it, with potential for further spreading at the point of destination. arboviruses are responsible for a large number of encephalitic diseases characterized by mode of transmission and geographic area. mosquito-borne arboviruses causing encephalitis include eastern and western equine, venezuelan, japanese, and murray hill encephalitides. japanese encephalitis is caused by a mosquito-borne arbovirus found in asia and is associated with rice-growing areas. it is characterized by headache, fever, convulsions, and paralysis, with fatality rates in severe cases as high as %. a currently available vaccine is used routinely in endemic areas (japan, korea, thailand, india, and taiwan) and for persons traveling to infected areas. tick-borne arboviruses causing encephalitis include the powassan virus, which occurs sporadically in the united states and canada. tickborne encephalitis is endemic in eastern europe, scandinavia, and the former soviet union. an epidemic of mosquito-borne encephalitis in new york city in included cases and deaths, due to the west nile fever virus, never before found in the united states. other insect vectors. it affects animals and humans who are in direct contact with the meat or blood of affected animals. the virus causes a generalized illness in humans with encephalitis, hemorrhages, retinitis and retinal hemorrhage leading to partial or total blindness, and death ( - %). it also causes universal abortion in ewes and a high percentage of death in lambs. the normal habitat is in the rift valley of eastern africa (the great syrian-african rift), often spreading to southern africa, depending on climactic conditions. the primary reservoir and vector is the aedes mosquito, and affected animals serve to multiply the virus which is transmitted by other vectors and direct contact with animal fluids to humans. an unusual spread of rvf northward to the sudan and along the aswan dam reservoir to egypt in - caused hundreds of thousands of animal deaths, with , human cases and deaths. rvf appeared again in egypt in . this disease is suspected to be one of the ten plagues of egypt leading to the exodus of the children of israel from egypt during pharaonic-biblical times. in , an outbreak of rvf in kenya, initially thought to be anthrax, with hundreds of cases and dozens of deaths, was related to abnormal rainy season and vector conditions. satellite monitoring of rainfall and vegetation is being used to predict epidemics in kenya and surrounding countries. animal immunization, monitoring, vector control, and reduced contact with infected animals can limit the spread of this disease. arboviruses can also cause hemorrhagic fevers. these are acute febrile illnesses, with extensive hemorrhagic phenomena (internal and external), liver damage, shock, and often high mortality rates. the potential for international transmission is high. yellow fever. yellow fever is an acute viral disease of short duration and varying severity with jaundice. it can progress to liver disease and severe intestinal bleeding. the case fatality rate is < % in endemic areas, but may be as high as % in nonendemic areas and in epidemics. it caused major epidemics in the americas in the past, but was controlled by elimination of the vector, aedes aegypti. a live attenuated vaccine is used in routine immunization endemic areas and recommended for travelers to infected areas. determining the mode of transmission and vector control of yellow fever played a major role in the development of public health (see chapter ). in , the who reported , cases and , deaths from yellow fever globally. dengue hemorrhagic fever. dengue hemorrhagic fever is an acute sudden onset viral disease, with - days of fever, intense headache, myalgia, arthralgia, box . dengue fever and dengue hemorrhagic fever, dengue fever, a severe influenza-like illness, and dengue hemorrhagic fever are closely related conditions caused by four distinct viruses transmitted by aedes aegypti mosquitos. dengue is the world's most important mosquito-borne virus disease. a total of , million people worldwide are at risk of infection. an estimated million cases occur each year, of whom , need to be hospitalized. this is a spreading problem, especially in cities in tropical and subtropical areas. major outbreaks were reported in colombia, cuba, and many other locations in . source: world health organization. . world health report gastrointestinal disturbance, and rash. hemorrhagic phenomena can cause case fatality rates of up to %. epidemics can be explosive, but adequate treatment can greatly reduce the number of deaths. dengue occurs in southeast asia, the pacific islands, australia, west africa, the caribbean, and central and south america. an epidemic in cuba in included more than , cases, and deaths. vector control of the a. aegypti mosquito resulted in control of the disease during the s- s, but reinfestation of mosquitoes led to incresased transmission and epidemics in the pacific islands, caribbean, central and south america in the s and s. outbreaks in vietnam included , cases in , another , cases in , and a similar sized outbreak in . indonesia had over , cases in with deaths, and in over , cases (january-may) with at least deaths. in , epidemics of dengue were reported in fiji, the cook islands, new caledonia, and northern australia. the who estimates , deaths and . million cases worldwide in . monkeys are the main reservoir, and the vector is the a. aegypti mosquito. no vaccine is currently available, and management is by vector control. lassa fever. lassa fever was first isolated in lassa, nigeria, in and is widely distributed in west africa, with , - , cases and deaths annually. it is spread by direct contact with blood, urine, or secretions of infected rodents and by direct person-to-person contact in hospital settings. the disease is characterized by a persistent or spiking fever for - weeks, and may include severe hypotension, shock, and hemorrhaging. the case fatality rate is %. marburg disease. marburg disease is a viral disease with sudden onset of generalized illness, malaise, fever, myalgia, headache, diarrhea, vomiting, rash, and hemorrhages. it was first seen in marburg, germany, in , following ex-posure to green monkeys. person-to-person spread occurs via blood, secretions, organs, and semen. case fatality rates can be over %. ebola fever. ebola fever is a viral disease with sudden onset of generalized illness, malaise, fever, myalgia, headache, diarrhea, vomiting, rash, and hemorrhages. it was first found in zaire and sudan in in outbreaks which killed more than persons. it is spread from person to person by the blood, vomitus, urine, stools, and other secretions of sick patients, with a short incubation period. the disease has case fatality rates of up to %. an outbreak of ebola among laboratory monkeys in a medical laboratory near washington, d.c., was contained with no human cases. the reservoir for the virus is thought to be rodents. an outbreak of ebola in may in the town of kikwit, zaire, killed persons out of cases ( % case fatality rate). this outbreak caused international concern that the disease could spread, but it remained localized. another outbreak of ebola virus occurred in gabon in early , with cases, of whom had direct exposure to an infected monkey, the remainder by human-to-human contact, or not established; of the cases died ( %). this disease is considered highly dangerous unless outbreaks are effectively controlled. in zaire, lack of basic sanitary supplies, such as surgical gloves for hospitals, almost ensures that this disease will spread when it recurs. lyme disease is characterized by the presence of a rash, musculoskeletal, neurologic, and cardiovascular symptoms. confirmation is by laboratory investigation. it is the most common vector-borne disease in the united states, with , cases reported between and . it primarily affects children in the - age group and adults aged - . lyme disease is preventable by avoiding contact with ticks, by applying insect repellant, wearing long pants and long sleeves in infected areas, and by the early removal of attached ticks. several u.s. manufacturers produced vaccines which are approved for animal and human use. in the mid s, a mother of two young boys who were recently diagnosed with arthritis in the town of lyme, connecticut, conducted a private investigation among other town residents. she mapped each of the six arthritis cases in the town, cases which had occurred in a short time span among boys living in close proximity. this suggested that this syndrome of "juvenile rheumatoid arthritis" was perhaps connected with the boys playing in the woods. she presented her data to the head of rheumatology at yale medical school in new haven, who investigated this "cluster of a new disease entity." some parents reported that their sons had experienced tick bites and a rash before onset of the arthritis. a tick-borne, spiral shaped bacterium, a spirochete, borrelia burgdorferi, was identified as the organism, and ticks shown to be the vector. cases repond well to antibiotic therapy. in over , cases ( . per , ) were reported from states, an increase from , in and , in . cases were mainly located in the northeast, north central, and mid-atlantic regions. the disease accounts for over % of vector-borne disease in the united states and was the ninth leading reported infection in . lyme disease has been identified in many parts of north america, europe, the former soviet union, china, and japan. a newly licensed vaccine is effective for people exposed to ticks but not general usage. personal hygiene for protection from ticks and environmental modification are important to limit spread of the disease. source: cdc, , mmwr, : - ; and cdc, , mmwr, , no. . lyme disease website http://www.cdc.gov/ncidad/disease/lyme/lyme.htm medically important parasites are animals that live, take nourishment, and thrive in the body of a host, which may or may not harm the host, but never brings benefit. they include those caused by unicellular organisms such as protozoa, which include amoebas (malaria, schistosomiasis, amebiasis, and cryptosporidium), and helminths (worms), which are categorized as nematodes, cestodes, and trematodes. public health continues to face the problems of parasitic diseases in the developing world. increasingly, parasitic diseases are being recognized in industrialized countries. giardiasis and cryptosporidium infections in waterborne and other outbreaks have occurred in the united states. parasitic diseases are among the most common causes of illness and death in the world, e.g., malaria. milder illnesses such as giardiasis and trichomoniasis cause widespread morbidity. intestinal infestations with worms may cause of severe complications, although they commonly cause chronic low-grade symptomatology and iron deficiency anemia. echinococcosis (hydatid cyst disease) is infection with echinococcus granulosus, a small dog tapeworm. the tapeworm forms unilocular (single, noncompartmental) cysts in the host, primarily in the liver and lungs, but they can also grow in the kidney, spleen, central nervous system, or in bones. cysts, which may grow up to cm in size, may be asymptomatic or, if untreated, may cause severe symptoms and even death. this parasite is common where dogs are used with herd grazing animals and also have intimate contact with humans. the middle east, greece, sardinia, north africa, and south america are endemic areas, as are a few areas in the united states and canada. the human dis-ease has been eliminated in cyprus and australia. while the dog is the major host, intermediate hosts include sheep, cattle, pigs, horses, moose, and wolves. preventive measures include education in food and animal contact hygiene, destroying wild and stray dogs, and keeping dogs from the viscera of slaughtered animals. a similar, but multilocular, cystic hydatid disease is widely found in wild animal hosts in areas of the northern hemisphere, including central europe, the former soviet union, japan, alaska, canada, and the north-central united states. another echinococcal disease (echinococcus vogeli) is found in south america, where its natural host is the bush dog and its intermediate host is the rat. the domestic dog also serves as a source of human infection. surgical resection is not always successful, and long-term medical treatment may be required. control is through awareness and hygiene as well as the control of wild animals that come in contact with humans and domestic animals. control may require cooperation between neighboring countries. tapeworm infestation (taeniasis) is common in tropical countries where hygienic standards are low. beef (taenia saginata) and pork (t. solium) tapeworms are common where animals are fed with water or food exposed to human feces. freezing or cooking meat will destroy the tapeworm. fish tapeworm (diphyllobothrium latum) is common in populations living primarily on uncooked fish, such as inuit people. these tapeworms are usually associated with northern climates. toddlers are especially susceptible to dog tapeworm (dipylidium caninum), which is present worldwide, and domestic pets are often the source of oral-fecal transmission of the eggs. the disease is usually asymptomatic. similarly, dwarf tapeworm (hymenolepis nana) is transmitted through oral-fecal contamination from person to person, or via contaminated food or water. rat tapeworm (hymenolepis diminuta) also mostly affects young children. onchocerciasis (fiver blindness) is a disease caused by a parasitic worm, which produces millions of larvae that move through the body causing intense itching, debilitation, and eventually blindness. the disease is spread by a blackfly that transmits the larva from infected to uninfected people. it is primarily located in sub-saharan africa and in latin america, with over million persons at risk. control is by a combination of activities including environmental control by larvicidal sprays to reduce the vector population, protection of potential hosts by protective clothing and insect repellents, and case treatment. a who-initiated program for onchocerciasis control started in is sponsored by four international agencies: the food and agriculture organization (fao), the united nations development program (undp), the world bank, and who. it covers countries in sub-saharan africa, focusing on control of the blackfly by destoying its larvae, mainly via insecticides sprayed from the air. prevalence in was reported by who as over million persons. the program has been successful in protecting some million persons and helping . million infected persons to recover from this disease. who estimates that the program will have prevented , cases of blindness by the year and has freed million hectares of land for resettlement and cultivation. the program cost $ million. this investment is considered by the world bank to have a return of - % in terms of large scale land reuse and improved output of the population. a who program, the african program for onchocerciasis control (apoc), started in , uses a new drug (ivermectin) and selective vector control efforts by spraying. this involves countries in africa, and in a similar program in south america. see website http://www/who.int/ocp and is financed by many donor countries, internation organizations, merck & company, and ngos. dracunculiasis (guinea worm disease) is a parasitic disease of great public health importance in india, pakistan, and central and west africa. it is an infection of the subcutaneous and deeper tissues caused by a large ( cm) nematode, usually affecting the lower extremities and causing pain and disability. the nematode causes a burning blister on the skin when it is ready to release its eggs. after the blister ruptures, the worm discharges larvae whenever the extremity is in water. the eggs are ingested in contaminated water and the larva released migrate through the viscera to locate as adults in the subcutaneous tissue of the leg. incubation is about months. the larva released in water are ingested by minute crustaceans and remain infective for as long as a month. prevention is based on improving the safety of water supplies and by preventing contamination by infected persons. education of persons in endemic areas to stay out of water sources and to filter drinking water reduces transmission. insecticides remove the crustaceans. chlorine also kills the larvae and the crustaceans which prologue larval infectivity. there is no vaccine. treatment is helpful, but not definitive. dracunculiasis was traditionally endemic in a belt from west africa through the middle east to india and central asia. it was successfully eliminated from central asia and iran and has disappeared from the middle east and from some african countries (gambia and guinea). the world health organization has promoted the eradication of dracunculiasis. major progress has been made in this direction. worldwide prevalence is reported to have been reduced from million cases in to million in , , in , and , cases in . eradication was anticipated for the year , and in the who established a commission to monitor and certify eradication in formerly endemic areas. india's reported cases fell from , in to in , and the country was free of transmission in . in , formerly high prevalence countries such as kenya reported no cases in , while chad, senegal, cameroons, yemen, and the central african republic less than cases each. eradication of this disease appears to be imminent. the who eradication program was developed successfully as an independent program with its own direction and field staff, but further progress will require the integration of this program with other basic primary care programs in order to be self-sustaining as an integral part of community health. community-based surveillance systems for this disease are being converted to work for monitoring of other health conditions in the community. schistosomiasis (snail fever or bilharziasis) is a parasitic infection caused by the trematode (blood fluke) and transmitted from person to person via an intermediate host, the snail. it is endemic in countries in africa, south america, the caribbean, and asia. there are an estimated million persons infected worldwide and more than million at risk for the disease. the clinical symptoms include fever, nausea, vomiting, abdominal pain, diarrhea, and hematuria. the organisms schistosoma mansoni and s. japonicum cause intestinal and hepatic symptoms, including diarrhea and abdominal pain. schistosoma haematobium affects the genitourinary tract, causing chronic cystitis, pyelonephritis, with high risk for bladder cancer the ninth most common cause of cancer deaths globally. infection is acquired by skin contact with freshwater containing contaminated snails. the cercariae of the organism penetrate the skin, and in the human host it matures into an adult worm that mates and produces eggs. the eggs are disseminated to other parts of the body from the worm's location in the veins surrounding the bladder or the intestines, and may result in neurological symptoms. eggs may be detected under microscopic examination of urine and stools. sensitive serologic tests are also available. treatment is effective against all three major species of schistosomiasis. eradication of the disease can be achieved with the use of irrigation canals, prevention of contamination of water sources by urine and feces of infected persons, treatment of infected persons, destruction of snails, and health education in affected areas. persons exposed to freshwater lakes, streams, and rivers in endemic areas should be warned of the danger of infection. mass chemotherapy in communities at risk and improved water and sanitation facilities are resulting in improved control of this disease. leishmaniasis causes both cutaneous and visceral disease. the cutaneous form is a chronic ulcer of the skin, called by various names, e.g., rose of jericho, oriental sore, and aleppo boil. it is caused by leishmania tropica, l. brasiliensis, l. mexicana, or the l. donovani complex. this chronic ulcer may last from weeks to more than a year. diagnosis is by biopsy, culture, and serologic tests. the organism multiplies in the gut of sandflies (phlebotomus and lutzomi) and is transmitted to humans, dogs, and rodents through bites. the parasites may remain in the untreated lesion for - months, and the lesion does not heal until the parasites are eliminated. prevention is through limiting exposure to the phlebotomines and reducing the sandfly population by environmental control measures. insecticide use near breeding places and homes has been successful in destroying the vector sandflies in their breeding places. case detection and treatment reduce the incidence of new cases. there is no vaccine, and treatment is with specific antimonials and antibiotics. visceral leishmaniasis (kala azar) is a chronic systemic disease in which the parasite multiplies in the cells of the host's visceral organs. the disease is characterized by fever, the enlargement of the liver and spleen, lymphadenopathy, anemia, leukopenia, and progressive weakness and emaciation. diagnosis is by culture of the organism from biopsy or aspirated material, or by demonstration of intracellular (leishman-donovan) bodies in stained smears from bone marrow, spleen, liver, or blood. kala azar is a rural disease occurring in the indian subcontinent, china, the southern republics of the former u.s.s.r., the middle east, latin america, and sub-saharan africa. it usually occurs as scattered cases among infants, children, and adolescents. transmission is by the bite of the infected sandfly with an incubation period of - months. there is no vaccine, but specific treatment is effective and environmental control measures reduce the disease prevalence. this includes the use of antimalarial insecticides. in localities where the dog population has been reduced, the disease is less prevalent. sleeping sickness. sleeping sickness a disease caused by trypanosoma brucei, transmitted but the tsetse fly, primarily in the african savannahs, affecting cattle and humans. some million persons are at risk in sub-saharan africa. who reported , new cases, a total prevalence of , cases, and , deaths from this disease in . prevention depends on vector control, and effective treatment of human cases. chagas disease is a chronic and incurable vector and blood transfusion borne parasitic disease (trypanosoma cruzi) which causes disability and death. it affects some million persons mainly in latin america, with some , new cases and , deaths occurring annually. about % of affected persons develop severe heart disease. brazil, which accounts for % of the cases prevalent in latin america, achieved elimination of transmission in , after uruguay ( ) and venezuela ( ) and followed by argentina ( ) . elimination of transmission is projected by who by the year . control is difficult, but control measures include reducing the animal host and vector insect population in its habitat by ecological and insectiside measures, education of the population in prevention by clothing, bednets, and repellents, and with chemotherapy for case management. amebiasis. amebiasis is an infection with a protozoan parasite (entamoeba histolytica) which exists as an infective cyst. infestation may be asymptomatic or cause acute, severe diarrhea with blood and mucus, alternating with constipation. amebic colitis can be confused with ulcerative colitis. diagnosis is by microscopic examination of fresh fecal specimens showing trophozoites or cysts. transmission is generally via ingestion of fecal-contaminated food or water containing cysts, or by oral-anal sexual practices. amebiasis is found worldwide. sand filtration of community water supplies removes nearly all cysts. suspect water should be boiled. education regarding hygienic practices with safe food and water handling and disposal of human feces are the basis for control. ascariasis. ascariasis is infestation of the small intestine with the roundworm ascaris lumbricoides, which may appear in the stool, occasionally the nose or mouth, or may be coughed up from lung infestation. the roundworm is very common in tropical countries, where infestation may reach or exceed % of the population. children aged - years are especially susceptible. infestation can cause pulmonary symptoms and frequently contributes to malnutrition, especially iron deficiency anemia. transmission is by ingestion of infective eggs, common among children playing in contaminated areas, or via the ingestion of uncooked products of infected soil. eggs may remain viable in the soil for years. vermox and other treatments are effective. prevention is through education, adequate sanitary facilities for excretion, and improved hygienic practices, especially with food. use of human feces for fertilizer, even after partial treatment, may spread the infestation. mass treatment is indicated in high prevalence communities. pinworm disease or enterobiasis. pinworm disease (oxyuriasis) is common worldwide in all socioeconomic classes; however, it is more widespread when crowded and unsanitary living conditions exist. the enterobius vermicularis infestation of the intestine may be symptomless or may cause severe perianal itching or vulvovaginitis. it primarily affects schoolchildren and preschoolers. more severe complications may occur. adult worms may be seen visually or identified by microscopic examination of stool specimens or perianal swabs. transmission is by the oral-fecal ingestion of eggs. the larvae grow in the small intestine and upper colon. prevention is by educating the public regarding hygiene and adequate sanitary facilities, as well as by treating cases and investigating contacts. treatment is the same as for ascariasis. mass treatment is indicated in high prevalence communities. ectoparasites. ectoparasites include scabies (sarcoptes scabiei), the common bed bug (cimex lectularius), fleas, and lice, including the body louse (pediculus humanis), pubic louse (phthirius pubis), and the head louse (pediculus humanus capitis). their severity ranges from nuisance value to serious public health hazard. head lice are common in schoolchildren worldwide and are mainly a distressing nuisance. the body louse serves as a vector for epidemic typhus, trench fever, and louse-borne relapsing fever. in disaster situations, disinfection and hygienic practices may be essential to prevent epidemic typhus. the flea plays an important role in the spread of the plague by transmitting the organism from the rat to humans. control of rats has reduced the flea population, but during war and disasters, rat and flea populations may thrive. scabies, which is caused by a mite, is common worldwide and is transmitted from person to person. the mite burrows under the skin and causes intense itching. all of these ectoparasites are preventable by proper hygiene and the treatment of cases. the spread of these diseases is rapid and therefore warrants attention in school health and public health policy. legionnelae, a gram-negative group of bacilli, with species and many serogroups. the first documented case was reported in the united states in , and the first disease outbreak was reported in the united states in among participants of a war veterans convention. general malaise, anorexia, myalgia, and headache are followed by fever, cough, abdominal pain, and diarrhea. pneumonia followed by respiratory failure may follow. the case fatality rate can be as high as % of hospitalized cases. a milder, nonpneumonic form of the disease (pontiac fever) is associated with virtually no mortality. the organism is found in water reservoirs and is transmitted through heating, cooling, and air conditioning systems, as well as from tap water, showers, saunas, and jaccuzzi baths. the disease has been reported in australia, canada, south america, europe, israel, and on cruise ships. prevention requires the cleaning of water towers and cooling systems, including whirlpool spas. hyperchlorination of water systems and the replacement of filters is required where cases and/or organisms have been identified. antibiotic treatment with erythromycin is effective. leprosy (hansen's disease) was widely prevalent in europe and mediterranean countries for many centuries, with some , leprosaria in the year . leprosy was largely wiped out during the black death in the fourteenth century, but continued in endemic form until the twentieth century. leprosy is a chronic bacterial infection of the skin, peripheral nerves, and upper airway. in the lepromatous form, there is diffuse infiltration of the skin nodules and macules, usually bilateral and extensive. the tuberculoid form of the disease is characterized by clearly demarcated skin lesions with peripheral nerve involvement. diagnosis is based on clinical examination of the skin and signs of peripheral nerve damage, skin scrapings, and skin biopsy. transmission of the mycobacterium leprae organism is by close contact from person to person, with incubation periods of between months and years (average of - years). rifampicin and other medications make the patient noninfectious in a short time, so that ambulatory treatment is possible. multidrug therapy (mdt) has been shown to be highly effective in combating the disease, with a very low relapse rate. treatment with mdt ensures that the bacillus does not develop drug resistance. mdt is covering % of known cases in , according to who reports, as compared to only % in . the increase has been associated with improved case finding. bcg may be useful in reducing tuberculoid leprosy among contacts. investigation of contacts over years is recommended. the disease is still highly endemic primarily in five countries, india, brazil, indonesia, myanmar, and bangladesh, and is still present in some countries in southeast asia, including the philippines and burma, sub-saharan africa, the middle east (sudan, egypt, iran), and in some parts of latin america (mexico, colombia) with isolated cases in the united states. world prevalence has declined from . million cases in , . million in , to less than million cases in . the world health organization expects to eliminate leprosy as a public health problem by the year , defined as prevalence of less than per , population, or less than , cases. trachoma is currently responsible for million blind persons or % of total blindness in the world. the causative organism, chlamydia trachomatis, is a bacteria which can survive only within a cell. it is spread through contact with eye discharges, usually by flies, or household items (e.g., handkerchiefs, washcloths). trachoma is common in poor rural areas of central america, brazil, africa, parts of asia, and some countries in the eastern mediterranean. the resulting infection leads to conjuncfival scarring and if untreated, to blindness. who estimates there are million cases of active disease in endemic countries. hygiene, vector control, and treatment with antibiotic eye ointments or simple surgery for scarring of eyelids and inturned eyelashes prevent the blindness. a new drug, azithromycin, is effective in curing the disease. the who is promoting a program for the global elimination of trachoma using azithromycin and hygiene education in endemic areas. chlamydia (chlamydia pneumonia) is suspected of playing a role in coronary artery disease by intraarterial infection, with plaque formation and occlusion of the artery by thrombi consisting mainly of platelets. if borne out, this will provide potential for low cost intervention to reduce the burden of the leading worldwide cause of death. sexually transmitted diseases (stds) are widespread internationally with an estimated million new cases per year, with . million new cases, over million total cases, and . million deaths ( ), aids has captured world attention over the past decade. the global burden of stds is enormous (table . ), and the public health and social consequences are devastating in many countries. sexually transmitted diseases, especially in women, may be asymptomatic, so that severe sequelae may occur before patients seek care. infection by one std increases risk of infection by other diseases in this group. syphilis is caused by the spirochete treponema pallidum. after an incubation period of - days (mean - ), primary syphilis develops as a painless ulcer or chancre on the penis, cervix, nose, mouth, or anus, lasting - weeks. the patient may first present with secondary syphilis - weeks (up to weeks) after infection with a general rash and malaise, fever, hair loss, arthritis, and jaundice. these symptoms spontaneously disappear within weeks or up to months later. tertiary syphilis may appear - years after initial infection. complications of tertiary syphilis include catastrophic cardiovascular and central nervous system conditions. early antibiotic treatment is highly effective when given in a large initial dose, but longer term therapy may be needed if treatment is delayed. gonorrhea (gc) is caused by the bacterium neisseria gonorrhoeae. the incubation period is - days. gonorrhea is often associated with concurrent chlamydia infection. in women, gc may be asymptomatic or it may cause vaginal discharge, pain on urination, bleeding on intercourse, or lower abdominal pain. untreated, it can lead to sterility. in men, gc causes urethral discharge and painful urination. treatment with antibiotics ends infectivity, but untreated cases can be infectious for months. drug resistance to penicillin and tetracycline has increased in many countries so that more expensive and often unavailable drugs are necessary for treatment. prevention of gonococcal eye infection in newborns is based on routine use of antibiotic ointments in the eyes of newborns. chancroid. chancroid is caused by haemophilus ducreyi. in women chancroids may cause a painful, irregular ulcer near the vagina, resulting in pain on in-tercourse, urination, and defection, but it may be asymptomatic. in men it causes a painful, irregular ulcer on the penis. the incubation period is usually - days, but may be up to days. an individual is infectious as long as there are ulcers, usually - months. treatment is by erythromycin or azithromycin. herpes simplex. herpes simplex is caused by herpes simplex virus types and and has an incubation period of - days. genital herpes causes painful blisters around the mouth, vagina, penis, or anus. the genital lesions are infectious for - days. herpes may lead to central nervous system meningoencephalitis infection. it can be transmitted to newborns during vaginal delivery, causing infection, encephalitis, and death. cesarian delivery is therefore necessary when a mother is infected. anti-viral drugs are used in treatment, orally, topically, or intravenously. chlamydia. chlamydia is caused by chlamydia trachomatis. in women, it is usually asymptomatic but may cause vaginal discharge, spotting, pain on urination, lower abdominal pain, and pelvic inflammatory disease (pid). in newborns, chlamydia may cause eye and respiratory infections. in men, chlamydia causes urethral discharge and pain on urination. the incubation period is - days and the infectious period is unknown. treatment for chlamydia is doxycycline, azithromycin, or erythromycin. chlamydia infection, not necessarily venereal in transmission, may be transmitted to newborns of infected mothers. chlamydia pneumoniae, presently under investigation as a possible cause or contributor to coronary heart disease, and is widespread in poor hygenic conditions. trichomoniasis. trichomoniasis is caused by trichomonas vaginalis. the incubation period is - days (mean = ). in women, trichomoniasis may be asymptomatic or may cause a frothy vaginal discharge with foul odor, and painful urination and intercourse. in men, the disease is usually mild, causing pain on urination. treatment is by metronidazole taken orally. without treatment, the disease may persist and remain infectious for years. (hpv). it is a sporadic disease which may be associated with cervical neoplasia and cancer of the cervix. hpv includes many types associated with a variety of conditons. the search for a hpv vaccine to prevent cancer of the cervix looks promising. in areas where a full range of diagnostic services is lacking, a "syndromic approach" is recommended for the control of stds. the diagnosis is based on a group of symptoms and treatment on a protocol addressing all the diseases that could possibly cause those symptoms, without expensive laboratory tests and repeated visits. early treatment without laboratory confirmation helps to cure persons who might not return for follow-up, or may place them in a noninfective stage so that even without follow-up they will not transmit the disease. std incidence between and is shown in table . , with decline overall except around , with subsequent further fall in incidence. screening in prenatal and family planning clinics, prison medical services, and selected years - disease syphilis ( [ ] [ ] [ ] [ ] [ ] [ ] and subsequent decline by more than % in reported cases includes all three stages of the disease as well as congential syphilis. rates are cases per , population, rounded. in clinics serving prostitutes, homosexuals, or other potential risk groups will detect subclinical cases of various stds. treatment can be carried out cheaply and immediately. for instance, the screening test for syphilis costs $ . and the treatment with benzathine penicillin injection costs about $ . in . partner notification is a controversial issue, but may be needed to identify contacts who may be the source of transmission to others. control of stds through a syndrome approaach based on primary care providers is being promoted by who. health education directed at high risk target groups is essential. providing easy and cost-free access to acceptable, nonthreatening treatment is vital in promoting the early treatment of cases and thereby reducing the risk of transmission. promoting prevention through the use of condoms and/or monogamy requires long-term educational efforts that are now fostered by the hiv/aids pandemic. increased use of condoms for hiv prevention is associated with reduced risk of other stds. training medical care providers in std awareness should be stressed in undergraduate and continuing educational efforts including personal protection as care givers. human immunodeficiency virus (hiv) is a retrovirus that infects various cells of the immune system, and also affects the central nervous system. two types have been identified: hiv , worldwide in distribution, and the less pathogenic hiv , found mainly in west africa. hiv is transmitted by sexual contact, exposure to blood and blood products, perinatally, and via breast milk. the period of communicability is unknown, but studies indicate that infectiousness is high, both during the initial period after infection and later in the disease. antibodies to hiv usually appear within - months. within several weeks to months of the infection, many persons develop an acute self-limited flulike syndrome. they may then be free of any signs or symptoms for months to more than years. onset of illness is usually insidious with nonspecific symptoms, including sweats, diarrhea, weight loss, and fatigue. aids represents the later clinical stage of hiv infection. according to the revised cdc case definition ( ), aids involves any one or more of the following: low cd count, severe systematic symptoms, opportunistic infections such as pneumocystis pneumonia or tb, aggressive cancers such as kaposi's sarcoma or lymphoma, and/or neurological manifestations, including dementia and neuropathy. the who case definition is more clinically oriented, relying less on often unavailable laboratory diagnoses for indicator diseases. aids was first recognized clinically in in los angeles and new york. by mid- it was considered an epidemic in those and other u.s. cities. it was primarily seen among homosexual men and recipients of blood products. after initial errors, testing of blood and blood products became standard and has subsequently closed off this method of transmission. transmission has changed markedly since the initial onslaught of the disease, with needle sharing among intravenous drug users, heterosexual, and maternal-fetal transmission becoming major factors. comorbidity with other stds apparently increases hiv infectivity and may have helped to convert the epidemiology to a greater degree of heterosexual transmission. the disease grew exponentially in the united states (table . ), but incidence of new cases nas declined since . aids has become a major public health problem in most developed and developing countries, reaching catastrophic proportions in some sub-saharan african countries affecting up to % of the population. hiv-related deaths were the eighth leading cause of all deaths in in the u.s., the leading cause among men aged - years of age, and the fourth leading cause for women in this age group. by , aids had been diagnosed in , persons and , had died. it is estimated that up to million persons are hiv infected in the united states. globally, deaths from aids totalled . million in , with an estimated . million person having died from this pandemic up to . in , an estimated . million person were hiv infected with . million new infection in . the declining incidence of new cases in the industrialized countries may be the result of greater awareness of the disease and methods of prevention of transmission. improving early diagnosis and access to care, especially the combined therapy programs that are very effective in delaying onset of symptoms, are important parts of public health management of the aids crisis. until an effective vaccine is available, preventive reliance will continue to be on behavior risk-reduction and other prevention strategies such as needle and condom distribution among high risk population groups. throughout the world, hiv continues to spread rapidly, especially in poor countries in africa, asia, and south and central america. the united nations reports that million persons are living with hiv/aids, % of them in developing countries, where transmission is % by heterosexual contact. every day, more than persons are infected, including children. in thailand, person in is now infected. in sub-saharan africa person in is infected, and in some cities as many as person in carries the virus. estimations of new infections per year in sub-saharan africa range from to million persons, while in asia the range is from . to . million new infected persons per year. lessons are still being learned from the aids pandemic. the explosive spread of this infection, from an estimated , people in to an anticipated million persons hiv infected, shows that the world is still vulnerable to pandemics of "new" infectious diseases. enormous movements of tourists, business people, truck drivers, migrants, soldiers, and refugees promote the spread of such diseases. widespread sexual exchange, traffic in blood products, and illicit drug use all promote the international potential for pandemics. war and massive refugee situations promote rape and prostitution, worsening the aids situation in some settings in africa. hiv has arrived in almost every country. however, there is the somewhat hopeful indication that the rate of increase, has slowed in the united states. this may be an indication either of higher levels of self-protective behavior, or that the most susceptible population groups have already been affected and the spread into the general population is at a slower rate. it is also possible that this may yet prove to be only a lull in the storm, as heterosexual contact becomes a more important mode of transmission. the eleventh international conference on aids, held in vancouver, canada, in july , reported signs that combinations of several drugs from among a number of antiretroviral medications are showing promise to suppress the aids virus in infected people. at a current annual price of $ , - , per patient, these sums well beyond the capacity of most developing countries. development of methods of measuring the hiv viral load have allowed for better evaluation of potential therapies and monitoring of patients receiving therapy. in developed countries, transmission by blood products has been largely controlled by screening tests; transmission among homosexuals has been reduced by safe sex practices; transmission to newborns has been reduced by recent therapeutic advances. safe sex practices and condom use may have helped in reducing heterosexual transmission. further advances in therapy and prevention with a vaccine are expected over the next decade. the hiv/aids pandemic is one of the great challenges to public health for the st century due to its complexity, its international spread, its sexual and other modes of transmission, its devastating and costly clinical effects, and its impact on parallel diseases such as tuberculosis, respiratory infections, and cancer. the cost of care for the aids patient can be very high. needed programs include home care and community health workers to improve nutrition and self-care, and mutual help among hiv carriers and aids patients. the ethical issues associated with aids are also complex regarding screening of pregnant women, newborns, partner notification, reporting, and contact tracing, as well as financing the cost of care. diarrheal diseases are caused by a wide variety of bacteria, parasites, and viruses (table . ) infecting the intestinal tract and causing secretion of fluids and dis- solved salts into the gut with mild to severe or fatal complications. in developing countries, diarrheal diseases account for half of all morbidity and a quarter of all mortality. diarrhea itself does not cause death, but the dehydration resulting from fluid and electrolyte loss is one of the most common causes of death in children worldwide. deaths from dehydration can be prevented by use of oral rehydration therapy (ort), an inexpensive and simple method of intervention easily used by a nonmedical primary care worker and by the mother of the child as a home intervention. in , diarrheal diseases were the cause of almost million child deaths, but by this had declined to . million, largely under the impact of increased use of ort. diarrheal diseases are transmitted by water, food, and directly from person to person via oral-fecal contamination. diarrheal diseases occur in epidemics in situations of food poisoning or contaminated water sources, but can also be present at high levels when common source contamination is not found. contamination of drinking water by sewage and poor management of water supplies are also major causes of diarrheal disease. the use of sewage for the irrigation of vegetables is a common cause of diarrheal disease in many areas. salmonella are a group of bacterial organisms causing acute gastroenteritis, associated with generalized illness including headache, fever, abdominal pains, and dehydration. there are over serotypes of salmonella, many of which are pathogenic in humans, the most common of which are salmonella typhimurium, s. enteritidis, and s. typhi. transmission is by ingestion of the organisms in food, derived from fecal material from animal or human contamination. common sources include raw or uncooked eggs, raw milk, meat, poultry and its products, as well as pet turtles or chicks. fecal-oral transmission from person to person is common. prevention is in safe animal and food handling, refrigeration, sanitary preparation and storage, protection against rodent and insect contamination, and the use of sterile techniques during patient care. antibiotics may not eliminate the carrier state and may produce resistant strains. shigella are a group of bacteria that are pathogenic in man, with four groups: type a = shigella dysenteriae, type b = s. flexneri, type c = s. boydii, and type d = s. sonnei. types a, b, and c are each further divided into a total of serotypes. shigella are transmitted by direct or indirect fecal-oral methods from a patient or carrier, and illness follows ingestion of even a few organisms. water and milk transmission occurs as a result of contamination. flies can transmit the organism, and in nonrefrigerated foods the organism may multiply to an infectious dose. control is in hygienic practices and in the safe handling of water and food. escheria eoli e. coli are common fecal contaminants of inadequately prepared and cooked food. particularly virulent strains such as o :h can cause explosive outbreaks of severe (enterohemmorhagic) diarrhoeal disease with a hemolytic-uremic syndrome and death, as occurred in japan in with cases and deaths due to a foodborne epidemic. other milder strains cause travellers diarrhoea and nursery infections. inadequately cooked hamburger, unpasturized milk, and other food vectors are discussed under food safety in chapter . cholera is an acute bacterial enteric disease caused by vibrio cholerae, with sudden onset, profuse painless watery stools, occasional vomiting, and, if untreated, rapid dehydration, and circulatory collapse, and death. asymptomatic infection or carrier status, and mild cases are common. in severe, untreated cases, mortality is over %, but with adequate treatment, mortality is under %. diagnosis is based on clinical signs, epidemiologic, serologic and bacteriologic confirmation by culture. the two types of cholera are the classic and el tor (with inaba and ogawa serotypes). in , a large scale epidemic of cholera spread through much of south america. it was imported via a chinese freighter, whose sewage contaminated shellfish in lima harbor in peru (box . ). the south american cholera epidemic has caused hundreds of thousands of cases and thousands of deaths since . prevention requires sanitation, particularly the chlorination of drinking water, prohibiting the use of raw sewage for the irrigation of vegetable crops, and high standards of community, food, and personal hygiene. treatment is prompt fluid therapy with electrolytes in large volume to replace all fluid loss. oral rehydration should be accomplished using standard ort. tetracycline shortens the duration of the disease, and chemoprophylaxis for contacts following stool samples may help in reducing its spread. a vaccine is available but is of no value in the prevention of outbreaks. viral gastroenteritis can occur in sporadic or epidemic forms, in infants, children, or adults. some viruses, such as the rotaviruses and enteric adenoviruses, af- in the s, peruvian officials stopped the chlorination of community water supplies because of concern over possible carcinogenic effects of trihalomethanes, a view encouraged by officials of the u.s. environmental protection agency (epa) and the u.s. public health service. in january , a chinese freighter arrived in lima, peru, and dumped bilge (sewage) in the harbor, apparently contaminating local shellfish. consumption of raw shellfish is a popular local delicacy (ceviche) and associated with cases of cholera seen in local hospitals. contamination of local water supplies from sewage resulted in the geometric increase in cases, and by the end of the pan american health organization (paho) reported an epidemic of , cases and deaths. the epidemic spread to countries, and in there were a further , cases and deaths spreading over much of south america, continuing in . in the united states, cases of cholera were reported in ; of these, cases and death were among passengers of an airplane flying from south america to los angeles in which contaminated seafood was served. in , cases of cholera were reported in the united states which were unrelated to international travel. these occurred mostly among persons consuming shellfish from the gulf coast with a strain of cholera similar to the south american strain, also possibly introduced in ship ballast. cholera organisms are reported in harbor waters in other parts of the united states (promed, , promed, . fect mainly infants and young children, and may be severe enough to cause hospitalization for dehydration. others such as norwalk and norwalk-like viruses affect older children and adults in self-limited acute gastroenteritis in family, institution, or community outbreaks. rotaviruses cause acute gastroenteritis in infants and young children, with fever and vomiting, followed by watery diarrhea and occasionally severe dehydration and death if not adequately treated. diagnosis is by examination of stool or rectal swabs with commercial immunologic kits. in both developed and developing countries, rotavirus is the cause of about one-third of all hospitalized cases for diarrheal diseases in infants and children up to age . most children in developing countries experience this disease by the age of years, with the majority of cases between and months. in developing countries, rotaviruses are estimated to cause over , deaths per year. the virus is found in temperate climates in the cooler months and in tropical countries throughout the year. breastfeeding does not prevent the disease but may reduce its severity. oral rehydration therapy is the key treatment. a live attenuated vaccine was approved by the fda in and adopted in the u.s. recommended routine vaccination programs for infants. adenoviruses. adenoviruses, norwalk, and a variety of other viruses (including astrovirus, calcivirus, and other groups) cause sporadic acute gastroenteritis worldwide, mostly in outbreaks. spread is by the oral-fecal route, often in hospital or other communal settings, with secondary spread among family contacts. food-borne and waterborne transmission are both likely. these can be a serious problem in disaster situations. no vaccines are available. management is with fluid replacement and hygienic measures to prevent secondary spread. giardiasis. giardiasis (caused by giardia lamblia) is a protozoan parasitic infection of the upper small intestine, usually asymptomatic, but sometimes associated with chronic diarrhea, abdominal cramps, bloating, frequent loose greasy stools, fatigue, and weight loss. malabsorption of fats and vitamins may lead to malnutrition. diagnosis is by the presence of cysts or other forms of the organism in stools, duodenal fluid, or in intestinal mucosa from a biopsy. this disease is prevalent worldwide and affects mostly children. it is spread in areas of poor sanitation and in preschool settings and swimming pools, and is of increasing importance as a secondary infection among immunocompromised patients, especially those with aids. waterborne giardia was recognized as a serious problem in the united states in the s and s, since the protozoa is not readily inactivated by chlorine, but requires adequate filtration before chlorination. person-to-person transmission in day-care centers is common, as is transmission by unfiltered stream or lake water where contamination by human or animal feces is to be expected. an asymptomatic carrier state is common. prevention relies on careful hygiene in settings such as day-care centers, filtration of public water supplies and the boiling of water in emergency situations. cryptosporidium. cryptosporidium parvum is a parasitic infection of the gastrointestinal tract in man, small and large mammals and vertebrates. infection may be asymptomatic or cause a profuse, watery diarrhea, abdominal cramps, general malaise, fever, anorexia, nausea, and vomiting. in immunosuppressed patients, such as persons with aids, it can be a serious problem. the disease is most common in children under years of age and those in close contact with them, as well as in homosexual men. diagnosis is by identification of the cryptosporidium or-ganism cysts in stools. the disease is present worldwide. in europe and the united states, the organism has been found in < to . % of individuals sampled. spread is common by person-to-person contact by fecal-oral contamination, especially in such settings as day-care centers. raw milk and waterborne outbreaks have also been identified in recent years. a large waterborne disease outbreak due to cryptosporidium occurred in milwaukee in described in chapter . management is by rehydration and prevention is by careful hygiene in food and water safety. helicobacter pylori. helicobacter pylori, first identified in , is a bacterium causally linked to duodenal ulcers and gastritis, contributing to high rates of gastric cancer (chapter ). it is an important example of the link between infection and chronic disease. this has enormous implications for prevention of cancer of the stomach, chronic peptic ulcers and large-scale use of hospitals and other medical resources (see chapter ). the control of diarrheal diseases requires a comprehensive program involving a wide range of activities, including good management of food and water supplies, education in hygiene, and, particularly where morbidity and mortality are high, education in the use of oral rehydration therapy (ort). oral rehydration therapy (ort) is considered by unicef and who to have resulted in the saving of million lives each year in the s. proper management of an episode of diarrhea by ort (table . ), along with continued feeding, not only saves the child from dehydration and immediate death, but also contributes to early restoration of nutritional adequacy, sparing the child the prolonged effects of malnutrition. the world summit for children (wsc) in called for a reduction in child deaths from diarrheal diseases by one-third and malnutrition by one-half, with em- phasis on the widest possible availability, education for, and use of ort. this requires a programmatic approach. public health leadership must train primary care doctors, pediatricians, pharmacists, drug manufacturers, and primary care health workers of all kinds in ort principles and usage. they must be backed by the widest possible publicity to raise awareness among parents. oral rehydration therapy is an important public health modality in developed countries as well as in developing countries. diarrhoeal disease may not cause death as frequently in developed countries, but it is still a significant factor in infant and child health and, even under the most optimal conditions, can cause setbacks in the nutritional state and physical development of a child. use of ort does not prevent the disease (i.e., it is not a primary prevention), but it is excellent in secondary prevention, by preventing complications from diarrhoea, and should be available in every home for symptomatic treatment of diarrheal diseases. an adaptation of ort has found its place in popular culture in the united states. a form of ort, marketed as "sports drinks," is used in sports where athletes lose large quantifies of water and salts in sweat and insensible loss from the respiratory tract. the wider application of the principles of ort for use in adults in dry hot climates and in adults under severe physical exertion with inadequate fluid/salt intake situations requires further exploration. management of diarrheal diseases should be part of a wider approach to child nutrition. the child who goes through an episode of diarrheal disease may have a faltering in growth and development. supportive measures may be needed following the episode as well as during it. this involves providing primary care services that are attuned to monitoring individual infant and child growth. growth monitoring surveillance is important to assess the health status of the individual child and the child population. supplementation of infant feeding with vitamins a and d, and iron to prevent anemia are important for routine infant and child care, and more so for conditions affecting total nutrition such as a diarrheal disease. in the developing world, respiratory infections account for over one-quarter of all deaths and illnesses in children. as diarrheal disease deaths are reduced, the major cause of death among infants in developing countries is becoming acute respiratory infections (aris). in industrialized countries, aris are important for their potentially devastating effects on the elderly and chronically ill. they are also the major cause of morbidity in infants in developed countries, causing much anxiety to parents even in areas with good living conditions. cigarette smoking, chronic bronchitis, poorly controlled diabetes or congestive heart failure, and chronic liver and kidney disease increase susceptibility to aris. aris place a heavy burden on health care systems and individual families. improved methods of management of such chronic diseases are needed to reduce the associated toll of morbidity, mortality, and the considerable expenses of health care. acute respiratory infections are due to a broad range of viral and, to a lesser extent, bacterial infections. it is the latter which can progress to pneumonia with mortality rates of - %. acute viral respiratory diseases include those affecting the upper respiratory tract, such as acute viral rhinitis, pharyngitis, and laryngitis, as well as those affecting the lower respiratory tract, tracheobronchitis, bronchitis, bronchiolitis, and pneumonia. aris are frequently associated with vaccine-preventable diseases, including measles, varicella, and influenza. they are caused by a large number of viruses, producing a wide spectrum of acute respiratory illness. some organisms affect any part of the respiratory tract, while others affect specific parts and all predispose to bacterial secondary infection. while children and the elderly are especially susceptible to morbidity and mortality from acute respiratory disease, the vast numbers of respiratory illnesses among adults cause large-scale economic loss from work absence. bacterial agents causing upper respiratory tract infection include group a streptococcus, mycoplasma pneumonia, pertussis, and parapertussis. pneumonia or acute bacterial infection of the lower respiratory tract and lung tissue may be due to pneumococcal infection with streptococcus pneumoniae. there are known types of this organism, distinguished by capsule characteristics; account for % of pneumococcal infections in the united states. an excellent polyvalent vaccine based on these types is available for high risk groups such as the elderly, immunodeficient patients, and persons with chronic heart, lung, liver, blood disorders, or diabetes. opportunistic infections attack the chronically ill, especially those with compromised immune suystems, often with life-threatening aris. mycoplasma (primary atypical pneumonia) is a lower respiratory tract infection which sometimes progresses to pneumonia. tb and pneumonocytis carynia are especially problematic for aids patients. other organisms causing pneumonias include chlamydia pneumoniae, h. influenza, klebsiella pneumonia, escherichia coli, staphylococcus, rickettsia (q fever), and legionella. parasitic infestation of lungs may occur with nematodes (e.g., ascariasis). fungal infections of the lung may be caused by aspergillosis, histoplasmosis, and coccidiomycosis, often as a complication of antibiotic therapy. access to primary care and early institution of treatment are vital to control excess mortality from aris. in developed countries, aris as contributors to infant deaths are largely a problem in minority and deprived population groups. because these groups contribute disproportionately to childhood mortality, infant mortality reduction has been slower in countries such as the united states and russia than in other industrialized countries. the continuing gap in mortality rates between white and black children in the united states can, to a large extent, be attributed to aris and less access to organized primary care. children are brought to emergency rooms for care when the disease process is already advanced and more dangerous than had it been attended to professionally earlier in the process. many field trials of ari prevention programs have been proved successful involving parent education and training of primary care workers in early assessment and, if necessary, initiation of treatment. this needs field testing in multiple settings. reliance on vaccines to prevent respiratory infectious diseases is not currently feasible. aris are caused by a very wide spectrum of viruses, and the development of vaccines in this field has been slow and limited. the vaccine for pneumococcal pneumonia has been an important breakthrough, but it is still inadequately utilized by the chronically ill because of its limitations, costs, and lack of sufficient awareness, and it is too expensive for developing countries. improvements in bacterial and viral vaccine development will potentially help to reduce the burden of aris. a programmatic approach with clinical guidelines and education of family and care givers is currently the only feasible way to reduce the still enormous morbidity and mortality from aris on the young and the elderly. the success of sanitation vaccines and antibiotics led many to assume that all infectious diseases would sooner or later succumb to public health and medical technology. unfortunately, this is a premature and even dangerous assumption. despite the longstanding availability of an effective and inexpensive vaccine, the persistence of measles as a major killer of million children per year represents a failure in effective use of both the vaccine and the health system. the resurgence of tb and malaria have led to new strategies, such as managed or directly observed care, with community health workers to assure compliance needed to render the patient noninfectious to others and to reduce the pool of carriers of the disease. current successes in reducing poliomyelitis, dracunculiasis, onchocerciasis, and other diseases to the point of eradication has raised hopes for similar success in other fields. but there are many infectious diseases of importance in developed and developing countries where existing technologies are not fully utilized. oral rehydration therapy (ort) is one of the most cost-effective methods of preventing excess mortality from ordinary diarrheal diseases, and yet is not used on sufficient scale. biases in the financing and management of medical insurance programs can result in underutilization of available effective vaccines. hospital-based infections cause large-scale increases in lengths of stay and expenditures, although application of epidemiologic investigation and improved quality in hospital practices could reduce this burden. control of the spread of aids using combined medical therapies is not financially or logistically possible in many countries, but education for "safe sex" is effective. community health worker programs can greatly enhance tuberculosis, malaria, and std control, or in aids care, promote prevention and appropriate treatment. in the industrialized and mid-level developing countries, epidemiologic and demographic shifts have created new challenges in infectious disease control. prevention and early treatment of infectious disease among the chronically ill and the elderly is not only a medical issue, it is also an economic one. patients with chronic obstructive lung disease (copd), chronic liver or kidney disease, or congestive heart failure are at high risk of developing an infectious disease followed by prolonged hospitalization. public health has addressed, and will continue to stress the issues of communicable disease as one of its key issues in protecting individual and population health. methods of intervention include classic public health through sanitation, immunization, and well beyond that into nutrition, education, case finding, and treatment, and changing human behavior. the knowledge, attitudes, beliefs, and practices of policy makers, health care providers, and parents is as important in the success of communicable disease control as are the technology available and methods of financing health systems. together, these encompass the broad programmatic approach of the new public health to control of communicable diseases. in a world of rapid international transport and contact between populations, systems are needed to monitor the potential explosive spread of pathogens that may be transferred from their normal habitat. the potential for the international spread of new or reinvigorated infectious diseases constitute threat to mankind akin to ecological and other man-made disasters. the eradication of smallpox paved the way for the eradication of poliomyelitis, and perhaps measles, in the foreseeable future. new vaccines are showing the capacity to reduce important morbidity from rubella syndrome, mumps, meningitis, and hepatitis. other new vaccines on the horizon will continue the immunologic revolution into the twenty-first century. as the triumphs of control or elimination of infectious diseases of children continue, the scourge of hiv infection continues with distressingly slow progess an effective vaccine or cure for the disease it engenders. partly as a result of the hiv/ aids, tb staged a comeback in many countries where it was thought to be merely a residual problem. at the same time an old/new method of intervention using directly observed short-term therapy has shown great success in controlling the tb epidemic. the resurgence of tb is more dangerous in that mdrtb has become a widespread problem. this issue highlights the difficulty of keeping ahead of drug resistance in the search for new generations of antibiotics, posing a difficult challenge for the pharmaceutical industry, basic scientists as well as public health workers. the burden of infectious diseases has receded as the predominant public health problem in the developed countries but remains large in the developing countries. with increases in longevity and increased importance of chronic disease in the health status of the industrial and mid-level developing nations, the effects of infectious disease on the care of the elderly and chronically ill is of great importance in the new public health. long-term management of chronic disease needs to address the care of vulnerable groups, promoting the use of existing vaccines and antibiotics. most important is the development of health systems that provide close monitoring of groups at special risk for infectious disease, especially patients with chronic diseases, the immunocompromised, and the elderly. the combination of traditional public health with direct medical care needed for effective control and eradication of communicable diseases is an essential element of the new public health. the challenge is to apply a comprehensive approach and management of resources to define and reach achievable targets in communicable disease control. access to e-mail and the internet are vital to current practice of public health and nowhere is this more important than in communicable diseases. there are many such information sites and these will undoubtedly expand in the coming years. several sites are given as examples. the internet has great practical implications for keeping up to date with rapidly occurring events in this field. outstanding encyclopedia database on infectious diseases (available via mdcassoc@ix.netcom.com at reduced price for promed users, and free to sub-saharan african sites) promed is an excellent, free report on current events in communicable diseases internationally; join via owner-promed @usa recommended readings centers for disease control. . update: international task force for disease eradication addressing emerging infectious disease threats: a prevention strategy for the united states. executive summary update: trends in aids incidence--united states one thousand days until the target date for global poliomyelitis eradication tuberculosis morbidity--united states measles--united states, . morbidity and mortality weekly report national adult immunization awareness week--october - , recommended readings ; and influenza and pneumococcal vaccination levels among adults aged --- years impact of the sequential ipv/opv schedule on vaccination cover-agemunited states advances in global measles control and elimination: summary of the international meeting recommended childhood immunization schedulemunited states impact of vaccines universally recommended for childrenmunited states progress toward global poliomyelitis eradication global disease elimination and eradication as public health strategies childhood immunizations rotavirus vaccines: who position paper. weekly epidemiologic record infectious diseases of humans: dynamic and control vaccines and world health: science, policy, and practice control of communicable diseases manual jawetz, melnick and adelberg's medical microbiology, twenty-first edition preventive medicine and public health, second edition efficacy of bcg vaccine in the prevention of tuberculosis. meta-analysis of the published literature manson's tropical diseases vaccination and world health principles and practice oflnfectious diseases immunization of adolescents: recommendations of the advisory committee on immunization practices, the american academy of pediatrics, the american academy of family physicians and the combination vaccines for childhood immunization: recommendations of the advisory committee on immunization practices, the american academy of pediatrics, the american academy of family physicians and the poliomyelitis prevention: revised recommendations for use of inactivated and live oral poliovirus vaccines diphtheria outbreakmrussian federation rubella and congenital rubella syndrome~united states compendium of animal rabies control, : national association of state public health veterinarians progress toward elimination of haemophilus influenzae type b disease among infants and children in the united states tetanus surveillance~united states, - recommendations and reports--vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of measles: recommendations of the advisory committee on immunization practices national, state and urban area vaccination coverage levels among children aged - months~united sates varicella related deaths among children--united states progress toward global poliomyelitis eradication ten great public health achievements--united states a ten-year experience in control of poliomyelitis through a combination of live and killed vaccines in two developing areas measles control in developing and developed countries: the case for a two-dose policy integration of vitamin a supplementation with immunization. weekly epidemiological record update cholera--western hemisphere, . morbidity and mortality weekly report isolation of vibrio cholerae o from oystersmmobile bay, - estimates of future global tuberculosis morbidity and mortality arbovirus disease--united states ~:~ other communicable diseases update: outbreak of legionnaire's disease associated with a cruise ship rift valley fever--egypt the role of bcg vaccine in the prevention and control of tuberculosis in the united states: a joint statement by the advisory council for the elimination of tuberculosis and the advisory committee on immunization practices update: trends in aids incidence--united states case definition for infectious conditions under public health surveillance guidelines for treatment of sexually transmitted diseases primary and secondary syphilis--united states global tuberculosis incidence and mortality during the th century pandemic: need for surveillance and research escherichia coli o :h diarrhoea in the united states: clinical and epidemiologic features the state of the world's children the rational use of drugs in the management of acute diarrhoea in children world health organization. . the malaria situation in aids: images of the epidemic. geneva: who. world health organization progress toward the elimination of leprosy as a public health problem the world health report : fighting disease, fostering development the world health report health for all in the twenty-first century. eb / . geneva: who. world health organization. . the world health report : life in the twenty-first century: a vision for all world health organization. . the world health report : making a difference key: cord- -f iuy e authors: wehling, martin title: calling for an exponential escalation scheme in vaccine development for covid- date: - - journal: eur j clin pharmacol doi: . /s - - -w sha: doc_id: cord_uid: f iuy e purpose: covid- as a pandemic calls for rapid development of vaccines. methods: here a proposal of a seamless, adaptive, phase – trial for accelerated vaccine development is described. results: starting at , the number of vaccinated volunteers would exponentially increase by tenfold at an interval of weeks; close surveillance of antibody responses, safety and efficacy is necessary. after only weeks, general vaccination would be feasible if supply meets the demand. conclusion: a covid- vaccine would be rapidly available at a slightly increased risk for undetected late side effects or insufficient efficacy if compared with standard vaccine development schemes. covid- is a pandemic that may kill millions of people. obviously, the only true solution is a successful vaccine, but the timing of its realization is at least as crucial as its existence. a successful intervention has to be realized under an enormous developmental time pressure. the biggest threat to the timely development of a vaccine is to adhere to common rules of clinical development, mainly characterized by subsequent phases ( - ) of clinical testing before marketing approval. this process may take - years. some accelerated testing schemes have been suggested in recent weeks [ ] , but as a clinical pharmacologist engaged in translational medicine and in the design of smart early clinical trials [ ] , i would like to propose an even more radical deviation from normal standards. the pre-requisite for human trials of a vaccine is its successful testing in animals with three major goals: . the induction of desired antibodies known to be induced by the real infection . absence of antibody-dependent enhancement meaning non-neutralizing antibodies that otherwise may enhance disease-related damage . absence of structural homologies of antibody targets with protein structures normally present in humans, analogous to the still disputed induction of narcolepsia by the influenza vaccine pandemrix due to a structural homology with the hypocretin receptor [ ] i suggest exponential exposure starting with healthy volunteers, testing of antibody responses and safety after days; if no stoppers [lack of adequate (see points - above) antibody response, intolerable side effects] occur, the number of vaccinated people will be increased by tenfold in each subsequent step, in this case to the next cohort of people. each transition may include a modification of dosing, reflecting the antibody response measured. only weeks after the first vaccination, one million people would have been vaccinated; at this point, side effects have been monitored in people for , in people for , in people for and in , people for weeks. as a rule of thumb, a rare side effect occurring within weeks would have been detected at a likelihood of / × = cases. thus, even rare (though not very rare or late) side effects would have been noticed, stopping further escalation. for safety reasons, this scheme should be restricted to inactivated vaccines as the risks for live-attenuated vaccines may be greater. vaccination efficacy must be monitored in parallel; ideally, the early stages of the study should be placed in countries with low risk of infection. at stage + ( + people) when primary efficacy has been established at the antibody level and safety is no issue to this point, volunteers living in countries with high infection rates should be vaccinated. if spontaneous infection rates drop below average in vaccinated people, this would be an early sign for efficacy. such efficacy results may be visible already at the end of the , - , people level, meaning after - weeks. this seamless, adaptive phase - study design relies on continuous observation, and testing of all participants may be up the , , people level. obviously, at week ( months, million people) or even earlier if results are compelling, pandemic (unlimited) vaccination could be performed. thus, this vaccine would be available worldwide weeks after the end of successful animal testing. participating people would have to be informed that: this design is relatively insensitive to late side effects occurring beyond - weeks. the escalation may put people at risk before side effects or insufficient efficacy may become visible. obviously, many ( +) vaccine projects have been started, and such escalation schemes could be performed competitively for those fulfilling the above criteria. studying various vaccines in parallel to identify the best candidate is possible as the covid pandemic is still rapidly expanding in several countries, in particular in brazil, for example. a recent proposal to infect healthy people even in the placebo group by covid- [ ] is truly heroic given the fact that even young people may die of the disease and would be unnecessary under the auspices of this proposal. it has to be kept in mind that not only regional political interests but also difficulties in producing large quantities of vaccines may interfere with the presently most urgent goal of medical care around the planet: rapidly vaccinating a large share of the global population against covid- . political discussions about vaccine distribution and provision of production capacities should be preemptively addressed as otherwise we may witness another ethical tragedy: a successful vaccine could have been developed, but will not be available for all countries, and shortage of supply may aggravate the disaster of politically driven restrictions of distribution. we applaud covid heroes, e.g. in the health care system. vaccine development is not possible without the participation of consenting volunteers. if the extreme time pressure leads to accelerated clinical trials, volunteers may face a slightly increased risk (see above). we have to ask them if they would be willing to take this risk for ethical reasons, in that they may save the lives of many covid victims. thus, volunteers in vaccine studies should be applauded as well. should scientists infect healthy people with the coronavirus to test vaccines? principles of translational science in medicinefrom bench to bedside antibodies to influenza nucleoprotein cross-react with human hypocretin receptor key: cord- - afsk ix authors: ward, jeremy k.; alleaume, caroline; peretti-watel, patrick title: the french public's attitudes to a future covid- vaccine: the politicization of a public health issue date: - - journal: soc sci med doi: . /j.socscimed. . sha: doc_id: cord_uid: afsk ix as covid- spreads across the world, governments turn a hopeful eye towards research and development of a vaccine against this new disease. but it is one thing to make a vaccine available, and it is quite another to convince the public to take the shot, as the precedent of the h n influenza illustrated. in this paper, we present the results of four online surveys conducted in april in representative samples of the french population years of age and over (n = ). these surveys were conducted during a period when the french population was on lockdown and the daily number of deaths attributed to the virus reached its peak. we found that if a vaccine against the new coronavirus became available, almost a quarter of respondents would not use it. we also found that attitudes to this vaccine were correlated significantly with political partisanship and engagement with the political system. attitudes towards this future vaccine did not follow the traditional mapping of political attitudes along a left-right axis. the rift seems to be between people who feel close to governing parties (centre, left and right) on the one hand, and, on the other, people who feel close to far-left and far-right parties as well as people who do not feel close to any party. we draw on the french sociological literature on ordinary attitudes to politics to discuss our results as well as the cultural pathways via which political beliefs can affect perceptions of vaccines during the covid- pandemic. as covid- spreads across the world, governments turn a hopeful eye towards research and development of a vaccine against this new disease (yamey et al., ) . in the past century, vaccination has progressively been seen not only as one of "medicine's greatest life-savers" but also as the ideal form of intervention against infectious diseases (allen, ; holmberg et al., ; moulin, ) . the special status of vaccination is manifest in contemporary pandemic preparedness and management as it has been institutionalized in the past years. in most countries in the global north, pandemic preparedness plans highlight the importance of devoting special resources to vaccine research and development as well as fast-tracking market approval procedures (torny, ) . the hopes put in vaccination have not been dampened by the experience of the latest main pandemic in the global north: the h n influenza scare. but while governments succeeded in rolling out vaccines before the main wave of influenza cases hit the northern hemisphere, they did not obtain high vaccination coverage in the public. in most countries, this vaccination campaign was a resounding failure. sweden, canada, the usa, the netherlands, hungary, and norway were the only countries to achieve more than % coverage. in france, only % of the population was vaccinated (setbon and raude, ) . it is one thing to make a vaccine available, and it is quite another to convince the public to take the shot. in the case of a putative future covid- vaccine, it is crucial to take into consideration another development of the past ten to twenty years. for more than a decade now, public doubt about vaccines has become an increasingly important global issue (dubé et al., ; larson et al., ) . this has recently led the world health organization to include "vaccine hesitancy" -i.e. negative attitudes towards vaccines that do not amount to a radical refusal of any form of vaccinationin its list of "ten threats to global health in ". when a vaccine will be available, will it be widely used? in countries where vaccine hesitancy was widespread before the covid- epidemic, will it affect the coronavirus vaccination campaign? in this paper, we present the results of four online surveys conducted in april in representative samples of the french population years of age and over (n = ). these surveys were conducted during a period when the french population was on lockdown and the daily number of deaths attributed to the virus reached its peak. we found that if a vaccine against the new coronavirus became available, almost a quarter of respondents would not use it. we also found that attitudes to this vaccine were correlated significantly with political partisanship and engagement with the political system. these results are interesting not only because one would expect hesitancy toward this particular vaccine to be weak given the strength of the international mobilization, the stringency of containment measures, and the number of deaths rising rapidly. they are also striking because, at the time, no prominent politician had questioned the safety or efficacy of the future covid- vaccine. this is in sharp contrast with previous studies of the relationship between politicization and vaccine hesitancy which have focused on vaccines for which there have been much political investment in mainstream news -such as the mmr and hpv vaccines in the united states of america (baumgaertner et al., ; featherstone et al., ; joslyn and sylvester, ; kahan, ) . we set these results against the backdrop of the recent transformations of the french political landscape and discuss the cultural pathways via which political beliefs can affect perceptions of vaccines during the covid- pandemic. during each week of april , we conducted a cross-sectional online survey among a sample representative of the french population aged and over (n = from th to th april, n = from th to th, n = from th to th, n = from th april to th may, global sample n = ). for each survey, participants were randomly selected from an online research panel of more than , nationally representative households of the french general population developed and maintained by ifop (paris, france), a survey research firm (https://www.ifop.com/). random sampling was stratified to match french official census statistics for gender, age, occupation, size of the population in the area of residence and region. the study design was approved by the ethical committee of the university hospital institute méditerranée infection (# - ). in addition to background socio-economic variables (gender, age, educational level), we computed each respondent's equivalized household income per month, taking the size and composition of the household into account, using the organization for economic cooperation and development's scale. then we built a three-item indicator: 'low income' refers to the first quartile of the household income per consumption unit (hicu), 'intermediate income' to the second and third quartile, 'high income' to the last quartile. regarding the covid- pandemic, participants were also asked whether they have been diagnosed with covid- and to what extent they were concerned about being infected with it. they had to mark their level of concernvfrom (no concerned at all) to (very concerned) and we re-coded their answer into a binary outcome: 'veryconcerned' for marks and , 'less concerned' for lower marks. regarding partisanship, we followed a standard practice in contemporary political science research in france (fillieule et al., ) . respondents were asked to which french political party they felt the closest (among a quite comprehensive list of parties), and responses were encoded into a four-item outcome: far-left, green party, left/-center/right governmental parties, far-right. for those who answered they felt close to no party, we aimed to assess their degree of distance toward the political system. we therefore considered their voting behavior at the first round of the presidential election, and we regrouped them into three categories: no current preference but voted in , no current preference and abstained in , no preference and other (for those who did not respond to the question related to the election or were too young to vote). regarding vaccination, respondents were asked whether they would agree to get vaccinated if a vaccine against the covid- was available: 'certainly', 'probably', 'probably not', 'certainly not'. responses were merged into a binary outcome: 'covid- vaccine refusal' equaled if participants answered probably or certainly not, otherwise the value was . finally, in case they answered probably or certainly not, they had to indicate why. three non-exclusive reasons were proposed: being against vaccination in general, thinking that a vaccine produced in a rush is too dangerous, and finally considering the vaccine useless because of the harmless nature of the covid . respondents could also elaborate about other motives. we first used bivariate analyses and a logistic regression to investigate factors associated with covid- vaccine refusal, using respondents' socio-economic background, covid- diagnosis and concern, and political preferences as covariates. we repeated these analyses for each of the three pre-coded reasons for refusal. as the corresponding sample sizes were smaller, we used a forward stepwise selection method (entry threshold p < . ) to retain statistically significant covariates only. among the individuals surveyed, almost a quarter declared that they would refuse "certainly" ( . %) or "probably" ( . %) the coronavirus vaccine if it were available. first, we compared the two groups of people who would refuse the coronavirus vaccine. no difference was found according to gender, age, and covid- -related concern. however, other differences were observed as people with an educational level under the high school degree, those with a low or intermediate level of household income per consumption unit (hicu), and those feeling close to a far-right party, were more numerous to be certain they would refuse the vaccine. table displays the results of descriptive and multivariate analyses highlighting factors associated with refusing the coronavirus vaccine ("certainly" or "probably"). therefore, refusing the vaccine was found to differ according to sociodemographic characteristics such as gender, age, and level of hicu: women, young people (aged under years old), and those with a lower level of hicu were more likely to refuse the vaccine. by contrast, no difference was observed across educational levels. surprisingly, no difference was found between people who were diagnosed with covid- ( , % of our sample) and those who were not. however, covid- -related concern seemed to have a strong influence on intentions to vaccinate: those who were highly concerned about being infected with the disease were less likely to refuse the vaccine compared to others ( . % against . %, p < . ). after adjustment for gender, age, education level, hicu, and covid diagnosis in a logistic model, partisan preference remained significantly associated with refusing the coronavirus vaccine. indeed, respondents who felt close to radical parties and those who did not feel close to any party and did not vote at the last presidential campaign were significantly more likely to refuse the vaccine compared to respondents with no partisan preference but who still voted in ( three main, but not exclusive reasons, were given to refuse the coronavirus vaccine: being against vaccination in general (reason chosen by . % of refusers), thinking that a vaccine produced in a rush is too dangerous ( . %), and finally considering the vaccine useless because of the harmless nature of covid- ( . %). moreover, around eight percent of refusers declared another reason to reject this vaccine, including a general lack of trust (about politics, about medicine, about science, about the pharmaceutical industry or unspecified), doubt about the efficiency of the vaccine (because of the mutation process of the virus, comparisons with the influenza vaccine), but also the belief that the respondent was already immunized against the virus. but these responses were too diverse to be pooled into a new category. analysis of these three main reasons highlighted the differentiated effects of the factors identified as associated with refusing the coronavirus vaccine according to the reason given. table shows for instance that men were more likely to refuse the vaccine because of the harmless nature of the disease whereas women were conversely more likely to be against the vaccine (this one specifically or because they are against vaccination in general). these logistic models did not identify any strongly different effects of partisan proximity according to the reason for refusing the vaccine. however, some of those effects seem stronger according to the reason given. far-right parties related effect was higher when people refused the vaccine because they were against vaccination in general (aor = . versus . in the full model), while far-left parties related effect was higher when people refused the vaccine because of the harmless nature of the disease (aor = . versus . in the full model). also, people who did not feel close to any party and did not vote at the last presidential campaign were more likely to refuse the coronavirus vaccine following one main reason: they thought that a vaccine produced in a rush is too dangerous. finally, people who felt close to governing parties (left-center-right), were much less prone to refuse the vaccine for the first two reasons (being against vaccination in general: aor = . ; thinking that this vaccine is too dangerous: aor = . ; all reasons confounded in the full model: aor = . ). we showed that almost a quarter of french adults would not get vaccinated against covid- and that the main reason for this reticence was the idea that this vaccine would not be safe. this result is coherent with previous studies showing that, in france, reticence towards vaccines tends to be vaccine-specific rather than targeted at vaccination in general (ward et al., ) . this tendency is not limited to the french public as the literature on vaccine hesitancy has shown in the past ten years (attwell, ; dubé et al., ) . but our main finding was that partisanship was an important determinant of attitudes to this future vaccine. to our knowledge, this is the first study of the effect of politicization on attitudes to vaccines in france, one of the most vaccine-hesitant countries in the world ( politicization in vaccine hesitancy has mostly been studied in the united states of america where political polarization has increasingly become an object of concern in the past years. several studies found that conservative ideology or republican partisan identity is associated with various forms of vaccine scepticism (baumgaertner et al., ; featherstone et al., ; hornsey et al., ; joslyn and sylvester, ; rabinowitz et al., ) . other american studies have found no impact of politicization (kahan, ; pew research center, ) . recently, polls have suggested that republicans are more likely to believe conspiracy theories relative to a future covid- vaccine or to refuse it (apnorc, ; cnn, ; yahoo! news, ) . in australia, one study found little effect of partisan identity on the propensity to believe vaccines are unsafe (only people who would vote for the green party were slightly more likely to believe that) (smith et al., ) while another found that negative attitudes to vaccines were linked to "minor political parties" (rozbroj et al., ) . in our study, we found that attitudes towards this future vaccine do not follow the traditional mapping of political attitudes along a left-right axis. we found an opposition between people who feel close to governing parties (right, centre and left) on the one hand, and, on the other, people who feel close to far-left and far-right parties as well as people who do not feel close to any party (with the highest reticence for those among them who did not vote in ). these results underline the two main transformations of the french political system of the past years: a rise in abstention and a possible current crisis of the left-right partisan dichotomy. let us start with non-partisanship and abstention. in france, the main explanation for abstention is not that it reflects a conscious rejection of the political system in its present form (braconnier, ; buton et al., ) . there is evidence to suggest that dissatisfaction toward the political system is also very prevalent among those who do vote. abstaining would rather reflect an absence of political socialization: the development of a taste for political issues as well as the presence in everyday life of the type of cultural practices (such as following the news) that lead people to maintain this interest as well as prompt them to participate in political elections. the fact that non-partisanship is associated with refusal of a future vaccine -especially when combined with abstentionsuggests that it reflects at least some dissatisfaction and distrust of institutions. this might be because abstention is more prevalent among the poor and marginalized, groups most at risk of having difficult interactions with the various institutions of the french state or to feel abandoned by them. this interpretation is supported in part by our finding that refusal increases as income decreases. nevertheless, this interpretation is weakened by the fact that non-partisanship was such a significant factor even after controlling for income. as for the rift in attitudes between people who feel close to governing parties and those who feel close to far-right and far-left parties, this could reflect an on-going transformation of the french political landscape. this transformation is partly due to the evolution of the main far-right party: the "rassemblement national" (rn). since , it has been engaged in a strategy of normalisation or "de-demonization" by rebranding their xenophobic nationalism as a defence of the hard workers against the pro-european elites (crépon and mayer, ; dézé, ) . this strategy has allowed the fn-rn to gain in popularity to the point that it is considered the main opposition party since the presidential election. the emergence of emmanuel macron's centrist party la republique en marche (larem) is another important evolution as former left-wing voters moved to this centrist party while others moved towards the far-left party france insoumise. the emergence of larem and the rise of the fn-rn have also affected the main right-wing party les républicains which has struggled to respond to this increased competition on both sides. these phenomena combined with the emergence of new radical political media, and with the development of social media has led many to believe that we are witnessing a process of polarization "à la française": a "vertical" polarization where the opposition is not so much between the left and the right but between institutionalist actors (governing parties, mainstream media …) and anti-elite actors (radical parties, social movements and media) (institut montaigne, ) . but how can these political opinions affect representations of a population: respondents for the waves / , / , / , / of the coconel survey (n = ). variable put in the models and unselected in all of them by the stepwise procedure: education level and being diagnosed with covid . putative future vaccine against covid- ? indeed, most issues related to health are not commonly perceived as politicized. how do individuals come to include a vaccine in the list of objects and decisions upon which they apply their political understanding of the world? in his work on motivated reasoning, dan kahan suggests two pathways via which worldviews and ideology come to play in people's perceptions: a) the person spontaneously perceives the issue as warranting a politicalcultural interpretation, and b) the sources of information provide cues signalling the political-cultural nature of the issue at hand (kahan, ; kahan et al., ) . activists and politicians have a crucial role in foregrounding politics in people's perception of issues. in the case of the putative future vaccine against covid- , it is important to note that, to our knowledge, at the time of writing this paper, no major political figure had voiced concern over these future vaccines. this constitutes a sharp difference with other studies of the effect of politicization on vaccine hesitancy which have tended to focus either on attitudes to vaccination in general or on attitudes to intensely debated and politicized vaccines such as mmr and hpv. this particularity suggests that the reticence we recorded is at least in part due to the spontaneous understanding of this issue using a political lens. but the question remains of what, in this idea of a future covid- vaccine, constitutes a partisan cue for our respondents. we suggest two non-exclusive interpretations. firstly, this politicized reticence could reflect the mobilizations in the past years of far-right and far-left activists against certain vaccines or vaccination in general. these mobilisations could have sensitized far-right and far-left leaning people to vaccine-related issues in general. but if we follow this interpretation, we should find the strongest reticence among people feeling close to the green party since environmental activists have been among the most visible figures of vaccine criticism in the past ten years (ward, ) . the fact that we only found a relatively small over-representation of reticence among them could reflect the diversisty within its activist base and electorate. the party attracts both moderate reformers who advocate for more sustainable development and for alliances with governing parties and radical activists who advocate for a more significant overhaul of institutions (boy, ; ollitrault, ) . the issue of vaccination seems to have fallen spot on these lines and has been the object of much internal and public debate in the past years. those who pushed forward the issue of vaccination could only represent a dissenting minority within this movement. secondly, this reticence could reflect the increasing politicization of debates surrounding the pandemic. during the period covered by our surveys, members of the far-right and the far-left have severely criticised the government on many issues. this was particularly the case of the rassemblement national which seems to have adopted a strategy of systematic criticism, while france insoumise has opted for more targeted attacks (le monde, ) . both have presented the various difficulties faced during this pandemic and the errors made by public authorities as reflecting the ideology of "macronism". in doing so, they may have fostered a general distrust of public health authorities and decisions regarding anything related to covid- -including future vaccines. this brings us to our final point. whether this hesitancy will spread or shrink will depend on the evolution of knowledge on the virus and its spread in france. it will also depend on the mobilisations of vaccine critical activists, especially on social media, which some studies suggest have intensified since the beginning of the pandemic (ball, ) . while we could not assess the extent of french vaccine critics' mobilisations on social media and their effect on the wider public's attitudes, such phenomena should be investigated further to better understand the origins of the reticence towards a future covid- vaccine. but how governments will anticipate possible reticence in the future and whether they manage to avoid vaccines becoming part of political debates constitutes another crucial factor. the choice to make the future vaccine mandatory or not is also likely to bear on the public's perception, as studies suggest that political polarization is stronger on the issue of legal mandates than it is on the issue of vaccine safety (blank and shaw, ; kahan, ) . it is crucial to guarantee that all the necessary precautions are taken before marketing the vaccine and to communicate transparently on the process as we have argued elsewhere (coconel, ) . vaccine: the controversial story of medicine's greatest lifesaver expectations for a covid- vaccine the politics of picking: selective vaccinators and population-level policy. ssm -population health anti-vaccine movement could undermine efforts to end coronavirus pandemic, researchers warn the influence of political ideology and trust on willingness to vaccinate does partisanship shape attitudes toward science and public policy? the case for ideology and religion l'électorat écologiste et l'élection présidentielle l'ordinaire du politique: enquêtes sur les rapports profanes au politique cnn poll: most americans would be uncomfortable returning to regular routines today a future vaccination campaign against covid- at risk of vaccine hesitancy and politicization les faux-semblants du front national: sociologie d'un parti politique. presses de sciences po que sait-on du front national vaccine hesitancy: an overview relationship of people's sources of health information and political ideology with acceptance of conspiratorial beliefs about vaccines . sociologie plurielle des comportements politiques. presses de sciences po wellcome global monitor . how does the world feel about science and health? wellcome trust the politics of vaccination: a global history donald trump and vaccination: the effect of political identity, conspiracist ideation and presidential tweets on vaccine hesitancy media polarization 'à la française'? comparing the french and american ecosystems. institut montaigne the determinants and consequences of accurate beliefs about childhood vaccinations cultural cognition as a conception of the cultural theory of risk vaccine risk perceptions and ad hoc risk communication: an empirical assessment motivated numeracy and enlightened self-government the state of vaccine confidence : global insights through a -country survey coronavirus : la france insoumise et le rassemblement national veulent profiter de la colère militer pour la planète: sociologie des écologistes. presses universitaires de rennes more americans now see 'very high' preventive health benefits from measles vaccine beliefs about childhood vaccination in the united states: political ideology, false consensus, and the illusion of uniqueness psychosocial and demographic characteristics relating to vaccine attitudes in australia factors in vaccination intention against the pandemic influenza a/h n majority acceptance of vaccination and mandates across the political spectrum in australia de la gestion des risques à la production de la sécurité rethinking the antivaccine movement concept: a case study of public criticism of the swine flu vaccine's safety in france vaccine hesitancy and coercion: all eyes on france new yahoo news/yougov poll shows coronavirus conspiracy theories spreading on the right may hamper vaccine efforts ensuring global access to covid- vaccines this work was supported by grants from the agence nationale de la recheche (anr- -covi- - ) and the cnrs (momentum, to jkw). the funding sources had no role in the design of the study, analysis of the data or writing of the paper. key: cord- -z nigtb authors: bradbury, jane title: custom-made vaccines at speed date: - - journal: drug discovery today doi: . /s - ( ) - sha: doc_id: cord_uid: z nigtb the concept of reverse genetics has enabled researchers to construct an experimental vaccine against a potential pandemic influenza strain in less than a month. a concept called reverse genetics has recently enabled researchers at the st jude children's research hospital (http://www.stjude.org) to construct an experimental vaccine against h n , a potential pandemic influenza strain, in less than a month. a similar approach could be used to develop vaccines for severe acute respiratory syndrome (sars). vaccines against new strains of well-known viruses or emerging infectious diseases used to take many months to develop. however, the increasing availability of genome sequences for pathogens is now expediting the development of safer, more effective vaccines. 'to virologists,' explains richard webby, a postdoctoral fellow in the department of infectious diseases at st jude, 'reverse genetics means the production of a virus from cloned dna'. a team led by yoshihiro kawaoka, professor of virology at the university of wisconsin-madison (http://www.vetmed.wisc.edu) was the first to achieve this feat for influenza, a segmented negative-sense rna virus, in [ ] . the classic method of making a vaccine to a new flu variant, explains webby, is to co-infect hens eggs with the field isolate and a vaccine strain and then to genetically select for reassorted viruses containing the desired mix of genome fragments, a process that can take many weeks. 'because of its pathogenicity, traditional reassortment was never an option for h n ,' webby continues. instead, to develop a vaccine against h n , the st jude team isolated the h n genome segments encoding haemagglutinin and neuraminidasethe surface glycoproteins against which neutralizing antibodies are raised. they then engineered haemagglutinin so that it was less pathogenic and mixed plasmids carrying these two fragments with six plasmids carrying the remaining flu genome fragments from h n , a standard vaccine strain (fig. ) . 'in four weeks, we went from a newly isolated, potentially pandemic flu strain to an experimental vaccine,' says webby. this flu vaccine will be the first produced by reverse genetics to go into clinical trial, and the speed with which it was produced, comments rino rappuoli, vice president of vaccine research at chiron corporation (http://www.chiron.com) 'could be critical if h n turns out to be the next, long-overdue pandemic strain to emerge from the far east'. reverse genetics is being used to develop vaccines for many other viruses, including respiratory syncytial viruses and parainfluenza viruses [ ] . and if the update news definition of the technology is broadened to include expression of proteins from engineered genes, then its future applications are virtually boundless. 'even years ago,' says rappuoli, 'genetics was used minimally in vaccine development. nowadays, we would not even try to develop a vaccine without using all the genetics tools available. ' speed is only one aspect of this revolution. even more important is the flexibility that is now available for vaccine design [ ] . for example, says kawaoka, 'many companies are planning to use reverse genetics to produce attenuated flu strains to be used as live vaccines that should give more protection than current inactivated vaccines'. in other cases, says rappuoli, 'this technology has facilitated the development of previously elusive vaccines. for example, for years we failed to develop an effective vaccine for meningococcus b. with reverse genetics we went from getting its genome sequence through identifying new antigens to starting clinical vaccine trials in less than four years.' the earliest known cases of sars were reported in mid november . by may , probable cases and deaths had occurred in more than countries. in response to this threat to global health, researchers have moved fast. 'in early march, no-one knew what this disease was caused by,' explains rappuoli. 'in late march somebody suggested it could be a coronavirus. by mid april we knew its genetic code. at chiron, we are already expressing sars envelope proteins to try to develop a vaccine,' he continues. 'we will also be using the information available on animal coronavirus to introduce attenuating mutations into the sars virus to make a live vaccine.' for the latter approach, recombinant virus will have to be generated, not an easy task given the size of the coronavirus rna genome. but, says luis enjuanes, research professor at the centro nacional de biotecnologia (http://www.cnb.uam.es), since , systems have been available to do this [ ] . several laboratories are engineering coronavirus genomes as vectors for vaccine development and gene therapy, says enjuanes, so it should be possible to make vaccines for sars by adapting the available infectious cdna clones. however, enjuanes warns that these will be the vaccines of the future. for now, other types of vaccine -based on single viral components, for example -can be made more quickly, he says. other experts agree -the use of reverse genetics to make recombinant sars vaccine could ultimately be the most effective and adaptable system for vaccine development, but will require considerable research set-up time. for now, most emphasis is on using inactivated sars virus as a vaccine. this is the approach that the us national institute for allergy and infectious diseases (http://www.niaid.nih.gov) will initially focus on although niaid director anthony fauci says that 'other approaches will soon follow… as more knowledge about the cause of sars and its etiology becomes available'. this classic approach will not be fast -fauci estimates that it will be least a year before a vaccine is ready for human testing -but 'it is probably the fastest way to go,' concludes kawaoka. a decade after the generation of a negative-sense rna virus from cloned cdna -what have we learned? live-attenuated virus vaccines for respiratory syncytial and parainfluenza viruses: applications of reverse genetics reverse vaccinology coronavirus derived expression systems key: cord- -rmvqf s authors: schmidt, harald title: vaccine rationing and the urgency of social justice in the covid‐ response date: - - journal: hastings cent rep doi: . /hast. sha: doc_id: cord_uid: rmvqf s the covid‐ pandemic needs to be considered from two perspectives simultaneously. first, there are questions about which policies are most effective and fair in the here and now, as the pandemic unfolds. these polices concern, for example, who should receive priority in being tested, how to implement contact tracing, or how to decide who should get ventilators or vaccines when not all can. second, it is imperative to anticipate the medium‐ and longer‐term consequences that these policies have. the case of vaccine rationing is particularly instructive. ethical, epidemiological, and economic reasons demand that rationing approaches give priority to groups who have been structurally and historically disadvantaged, even if this means that overall life years gained may be lower. t he covid- pandemic needs to be considered from two perspectives simultaneously. first, there are questions about which policies are most effective and fair in the here and now, as the pandemic unfolds. these polices concern, for example, who should receive priority in being tested, how to implement contact tracing, or how to decide who should get ventilators or vaccines when not all can. second, it is imperative to anticipate the medium-and longer-term consequences that these policies have. the case of vaccine rationing is particularly instructive. ethical, epidemiological, and economic reasons demand that rationing approaches give priority to groups that have been structurally and historically disadvantaged, even if this means that overall life years gained may be lower. as social-distancing measures were implemented across the country in recent months, people differed in their responses. new york city is the nation's single largest hotspot. it can serve as a useful case study, as it magnifies countless of the dynamics at work in implementing our collective covid- response. as the pandemic unfolded, many affluent people moved to their vacation homes. but many people needed to stay put because their work (whether in formal or informal employment) could not be done remotely or they could not afford not to work. analyses of transit data at the end of march showed that subway ridership in new york city was significantly reduced, albeit unequally in geographic terms. in manhattan, which has the highest median household income of the five boroughs at $ , , morning commute ridership fell by around percent. but in the bronx, which has the lowest median income at $ , and the highest poverty rate of all boroughs, there was only a percent drop. these differences are plausibly explained by differences in people's ability to prioritize protecting their health over income opportunities. lower-wage workers are more likely to be exposed to environmental risks associated with more affordable mass transit, and their risk of exposure is oftentimes compounded by less-safe housing and the nature of their formal or informal employment. similar disparities can be observed in more directly healthrelated measures. at the end of april, covid- -related deaths were almost twice as high in the bronx, compared to manhattan ( versus per , residents). deaths also differed across racial groups. twice as many black/ african american residents died when compared to white new yorkers, and hispanic/latino people fared almost as badly ( versus versus per , ). when it comes to testing, preliminary analyses suggest that access is the inverse: the vast majority of the thirty zip codes that had the highest rates of testing (per capita) were whiter and wealthier (or both), compared to city averages. along with other data at the national level indicating that low-income communities and communities of color are at higher risk of serious illness if infected, these disparities bear out that historically and structurally disadvantaged populations incur a far larger share of the morbidity and mortality burden while being far less able to absorb financial and other costs. it is unclear at what point a vaccine will be available. the u.s. government's operation warp speed aims to deliver one by the end of , though many experts believe it will take at least two or three times longer. what is clear is that even when a vaccine has been found, production limits will pose another major bottleneck. for some period that seems to be not just a matter of days or weeks, demand will outstrip supply, and rationing will become necessary. the federal government indicated in the naming of the vaccine production initiative that a timely response to covid- is desirable. yet the government has not yet adapted existing national pandemic vaccine guidelines that would set out who receives vaccines first. but clarity on who should receive vaccines when not all can is as critical as having a vaccine in the first place. across national guidelines and scholarly perspectives, there is consensus that front-line health workers and those deemed essential workers should receive priority. in the absence of clear federal guidance, it seems at least plausible to assume that the group of essential workers will be similar to those identified in the national pandemic guidelines and in state-based characterizations as part of lockdown measures. essential workers would therefore centrally include those without whom health care facilities would no longer be able to function as required, and likewise comprise critical infrastructure workers (for transport, waste, water, and so forth) and workers in core manufacturing, services, and retail (such as food, pharmaceuticals, and communications). when it comes to the general population, both guidelines and academic commentary are less clear. part of the reason is that there is lack of clarity about what principles should guide us in situations of absolute scarcity. authors of an influential multicriteria model argue that the value of maximizing benefits should be the most important one (and more important than, respectively, treating people equally, promoting instrumental value, or prioritizing the worst off ). they view maximizing benefits as constituted by two principles: saving the most lives and saving the most life years. for covid- , both principles should receive "the highest priority." saving the most life years is supposed to refer to "how long the patient is likely to live if treated." roughly, the thought is that when choosing between giving a scarce resource to someone likely to live thirty more years as opposed to someone who is estimated to live only five more years, the former should receive it, as this would contribute to producing more life years. when it comes to vaccines, the authors note that covid- outcomes have been much worse in older people and that, here, saving the most lives should trump the rationale of saving the most life years. that is, older people should receive priority right after health care workers and first responders. in addition, when supplies are "insufficient for patients in the highest risk categories-those over years of age or with coexisting conditions-then equality supports using random selection, such as a lottery, for vaccine allocation." younger people, whom the overall framework otherwise generally favors (as they stand to gain more life years, and societal investments such as education would otherwise be wasted) should be prioritized only if "epidemiologic modeling shows that this would be the best way to reduce viral spread and the risk to others." using a lottery for allocating scare vaccines in the general population, as proposed here, is one way of treating people equally, and it is certainly superior to a first-come-firstserved approach (or, perhaps more accurately, a let-me-usemy-connections-and-pointy-middle-class-elbows approach) that likely explains why better-off and whiter groups typically get tested more frequently for covid- than lowerincome people and people of color, as noted above. but a lottery also implies equality at baseline. and when it comes to health and life expectancy, there is no such equality. life expectancy and health differ widely. in new york city, residents in zip code can expect to live to fifty-nine years. those in zip code live, on average, to ninetyfour. similarly wide differences are found in many other larger american cities, as well as between better-off suburban residents when compared to poorer inner-city or rural people, and within socioeconomically stratified areas within these regions. the reasons that some people die thirty years younger than others are, at one level, very easy to understand: a larger number of risk factors such as smoking, obesity, high blood pressure, or higher levels of stress lead to deadly diseases that shorten life, along with unexpected injuries and accidents. at another level, however, it is quite hard to accept that earlier death is not distributed equally across all population groups but occurs disproportionately in particular racial, ethnic, insurance status, and income groups, all of which closely track geographic area. the centers for disease control's statistics "health, united states," and the cdc's health disparities & inequalities report (last issued ) provide extensive, detailed, and uncontroversial evidence. for example, while percent of whites lack insurance, percent of non-hispanic blacks have no insurance, and percent of hispanics lack it: this means groups differ in their ability to receive health care when they need it. blacks have a percent increased cancer death rate, compared to whites. people with less than a high school education are twice as likely to have diabetes than those with college or higher education ( percent versus percent). four in ten non-hispanic blacks have high blood pressure, compared to three in ten of non-hispanic whites ( percent) and hispanics ( percent versus percent versus percent). asthma is significantly more common among adults with incomes below percent of the federal poverty line than among adults percent above it ( %). a recent survey complements these data by finding that hispanic and black respondents were more concerned than whites about getting infected by covid- and requiring hospitalization ( percent versus percent versus percent). likewise, lower-income respondents were far more likely to have this concern than were middle-and high-income respondents ( percent versus percent versus percent). blacks were more than twice as likely as whites and hispanics to personally know someone who has been hospitalized because of or died from covid- . the covid- pandemic is in the process of assuming generation-defining significance-especially for many of the most vulnerable populations. there is a substantial risk that our current collective response will add to many people's-and particularly african american's-sense that they are "always . . . getting skipped over." so, while a vaccine lottery can be appealing in giving everyone a fair chance at securing a future health benefit (and can certainly be based on promoting equality in this sense), this approach would not reflect the fact that, prior to being entered into the lot-tery, some population groups would already have received far more of what is to be distributed-health benefits-than others. nor would a lottery that gives everyone an equal chance be sensitive to the urgent need to avoid, as far as possible, a longer-term impact of our covid- response that could rival the injustices of the tuskegee syphilis study, the henrietta lacks case, and comparable events in the collective memory of many. insofar as lotteries are used, at the least, they should therefore be weighed to reflect levels of underlying disadvantage so that worse-off population groups stand higher chances than better-off ones. a normatively superior approach would go further by directly prioritizing residents of disadvantaged neighborhoods. how, then, do we get the right vaccine to the right person? more or less in the same way as we would send them a letter. the area deprivation index, based on a measure created by the health resources and services administration and refined and adapted by amy kind and her research team at the university of wisconsin-madison, does not use zip codes as its reference frame but uses american community survey data in combining income, education, employment, and housing quality data to rank neighborhoods by socioeconomic status disadvantage. it has already been used to target program delivery of the everyone with diabetes counts initiative, established by the centers for medicare and medicaid services and the quality improvement organization. like any measure, it has methodological limitations, and as in many other cases, each unit of analysis will show differences (here: neighborhoods are never completely homogeneous). however, for the present purposes, the perfect should not be the enemy of the good. the adi is a helpful proxy for identifying those areas where vaccines should first be made available-whether in hotspots like new york city or elsewhere in the country. operationally, what is known as a reserve system can provide administrators with a reference frame to establish priority groups, building on precedent in areas such as school choice and immigration visas. prioritizing people in neighborhoods scoring low on the adi is ethically fitting, as the measure provides a sufficiently fine-grained composite proxy for the "worst off " that is otherwise unduly recognized (note that the above-cited framework expressly states that "sickest first" should be used only "when it aligns with maximizing benefits" ). it is the best way of guarding against charges of perceived or real disparate treatment and impact. further, prioritizing the worst off matters in economic and epidemiological terms. due to lower savings and higher dependency on wages and other income from work, people in lower-income groups are far less able to stay home than wealthier people are. when they do, they are much more likely to be sharing their living quarters with several people, often of multiple generations. as the subway example illustrated, lower-income populations are considerably less able to reduce their commutes. while completely understandable in economic terms, epidemiologically, the risk from unabated mobility is that controlling the epidemic becomes even harder-and, conversely, easier, if these groups receive priority access. the case of new york city is no anomaly here: the basic dynamics play out in the same way in other socioeconomically stratified urban, suburban, and rural areas across the united states. arguably, two of the most important general lessons from covid- are to realize the existential importance of universal health coverage and, outside of the health care system more narrowly conceived, to work toward dramatic improvements in social and structural determinants of health. progress in both areas would reduce the need to consider disadvantage in rationing. hopefully, one silver lining of the pervasive fear of a covid- infection that currently grips rich and poor (even if to different extents) is that health policy will be salient in voting booths in years to come. but these more complex and longer-term reforms aside, we must seize the opportunity to allocate vaccines in a way that is both just in the here and now and recognizes the enormous symbolic importance for the collective memory of structurally and historically marginalized groups that comes with being placed first, or last, in line. in the allocation of initially scarce vaccines, the firstpriority group should be health care and other essential workers, particularly those whom epidemiological modeling suggests are more likely to spread the infection due to their work and living profiles. a sad irony is that, in many cases, these groups will often be found in areas with high deprivation, for, as important as we evidently currently deem the services of nursing home aides, garbage collectors, supermarket checkout workers, and others designated essential, their pay often does not reflect this appreciation, and health insurance benefits are typically patchy. when it comes to allocating vaccines among the general population, economic, ethical, and epidemiological considerations urge us to prioritize the worse off, as identified by measures such as the adi. rudolf virchow had it right in every way when he suggested that medicine "is a social science, and politics is nothing more than medicine on a grand scale." when it comes to allocating vaccines among the general population, economic, ethical, and epidemiological considerations urge us to prioritize the worse off. they can't afford to quarantine. so they brave the subway association of county-level socioeconomic and political characteristics with engagement in social distancing for covid- variation in covid- hospitalizations and deaths across new york city boroughs most nyc coronavirus testing done in whitest and wealthiest zip codes low-income and communities of color at higher risk of serious illness if infected with coronavirus the black plague interim updated planning guidance on allocating and targeting pandemic influenza vaccine during an influenza pandemic fair allocation of scarce medical resources in the time of covid- university of wisconsin school of medicine public health, department of medicine university of wisconsin school of medicine public health, department of medicine. . ibid.; city health dashboard; robert wood johnson foundation online database when is discrimination wrong? association of county-level socioeconomic and political characteristics with engagement in social distancing for covid- foundation online database i am grateful for discussion of key points with moti gorin and govind persad. key: cord- -dqikrmk authors: greenberg, harry b.; dormitzer, philip r. title: vaccination against viruses date: - - journal: encyclopedia of immunobiology doi: . /b - - - - . - sha: doc_id: cord_uid: dqikrmk most vaccines in use today are the result of empirical development. the mechanism of action of many vaccines in common use remains incompletely understood. understanding how such vaccines protect is an ongoing subject of study using increasingly sophisticated immunological tools, such as b cell and t cell repertoire and transcriptome analysis. such tools are also being applied to the design of vaccines against those viral targets that have evaded vaccine-mediated protection thus far. as basic immunological science intersects with the practicalities of assuring vaccine safety, tolerability, efficacy, and consistency in the clinic, the practical utility of more sophisticated immunological measures for vaccine development may be determined by whether they can be reduced to simply executed, highly standardized, reproducible assays with outcomes that have clear interpretations for vaccine development and use. basic immunology, empirical vaccine testing, and regulatory science are all necessary contributors to developing the next generation of vaccines, including vaccines effective against the pathogens for which vaccines are not currently available. immunization against viruses has achieved some of the greatest successes of medicine, such as the eradication of smallpox worldwide and the control of polio in most of the world. yet, there are major viral pathogens, such as the human immunodeficiency virus (hiv), hepatitis c virus (hcv), respiratory syncytial virus (rsv), and human cytomegalovirus, for which no effective vaccines are available, despite decades of research and development. table lists the viral agents for which licensed vaccines are now available in the united states. with a singular exception, these viruses show limited antigenic diversity and have undergone little antigenic change over time. that exception, influenza virus, defines the current limits of our ability to immunize effectively in the face of substantial antigenic diversity and change. on the list, there are few vaccines developed using the tools of modern molecular biology or sophisticated immunological methods. many of the vaccine antigens (such as those in vaccines against influenza virus, japanese encephalitis virus, rabies virus, and poliovirus) are produced by growing viruses and chemically inactivating them. other viral vaccines are produced by propagating live viruses that have been attenuated by experimental passage in culture or in nonhuman hosts (influenza virus, yellow fever virus, measles virus, mumps virus, and rubella virus), by using virus strains originating in nonhuman hosts ('jennerian' vaccinesrotavirus, vaccinia virus), or by nonrecombinant genetic manipulation (genome segment reassortment through coinfection for rotavirus and influenza virus). some preparations of vaccines still in use (against influenza virus and yellow fever virus) are produced by virus propagation in fertilized chicken eggs rather than in cell culture. exceptions to the generally low technology approaches to producing vaccine antigens include recombinantly produced vaccine antigens for hepatitis b virus (hbv), human papillomavirus (hpv), and influenza virus. the hpv and hbv vaccine antigens, which are expressed in yeast or insect cells, self-assemble into particles that resemble those released from virus-infected cells (zeltins, ) . similarly, most vaccines in use today are either unadjuvanted or adjuvanted with alum, which has been used to enhance immune responses to vaccines since the s (baylor et al., ) . there are a few exceptions. one licensed hpv vaccine includes a toll-like receptor agonist adjuvant based on monophosphoryl lipid (mpl) a, and some influenza vaccines available in the european union and other countries include oil-in-water emulsion adjuvants (del giudice and rappuoli, ; garcon et al., ) . vaccines are more complex in their mechanism of action than almost all other pharmaceutical products. rather than acting directly through a well-defined, single ligand-receptor interaction, vaccine antigens and adjuvants have a series of complex interactions with multiple components of the immune system at or near the site of immunization and sometimes at systemic sites as well. these interactions prime the host for an anamnestic response to a subsequent infectious challenge, often at another anatomic site in the host, at a time remote from the immunization. the antigens in the viral challenge may have differences from those in the vaccine. in some cases, the host may have experienced infection with an antigenically related virus prior to immunization, adding an additional element of complexity to the vaccine immune response. the complex immunology that underlies protection from disease by immunization makes it a fascinating subject for scientific investigation. however, for practical vaccine development, straightforward, definitive, easily executed, reproducible, and readily interpreted immunological assays are needed to characterize and define the response to vaccines more fully. the intersection between the rich complexity of vaccine immunology and the practical need for simplicity in regulatory science contributes to a gap between the science and practice of vaccinology. for some vaccines, simple models of the determinants of protection have proven sufficient for practical decision making. for example, if the hbv vaccine elicits a postvaccination-binding titer against the hbv surface antigen (sag) miu/ml, a subject is considered to be protected (jack et al., ) . even if the anti-sag titer falls to < miu/ml after having been miu/ml, substantial levels of protection persist (west and calandra, ) . similarly, for the yellow fever vaccine, a plaque neutralization serum antibody titer of . neutralization index (equivalent to the difference in titer, log , between pre-and post-vaccination serum) is strongly associated with protection (mason et al., ) . neutralization of virus by antibody is generally considered the dominant mode of protection against those viruses whose pathogenesis requires viremia. examples include poliovirus, smallpox virus, yellow fever virus, hbv, varicella zoster virus (for chicken pox, not zoster), measles virus, mumps virus, and rubella virus. this is likely an oversimplification of the true situation, but it has proved a useful simplification for vaccine development and monitoring (plotkin, ) . assigning reliable correlates of protection can be more challenging for vaccines against viruses that predominantly infect and replicate at a mucosal surface. for inactivated influenza vaccines, a serum hemagglutination inhibition (hi) titer of : has long been accepted as a correlate of protection, based primarily on studies in young adults (de jong et al., ) . however, considerably higher antibody levels may be required to protect children, with hi titers of : conferring % protection after immunization with an adjuvanted, inactivated influenza vaccine (black et al., ) . in the elderly, measures of cell-mediated immunity, such as granzyme b levels in virus-stimulated peripheral blood mononuclear cells, may correlate better than serum antibody titers with vaccine-elicited protection (mcelhaney et al., ). in addition, the correlates of protection after immunization with inactivated influenza vaccines do not predict protection after immunization with live attenuated influenza vaccines, which offer more protection than would be predicted based on the elicited serum hi or neutralization titers (beyer et al., ) . whether cell-mediated effector mechanisms, mucosal antibody, or some other factor is primarily responsible for protection by live attenuated influenza vaccines or natural infection remains controversial despite several decades of study. a reliable correlate of protection is even more elusive for rotavirus, which causes diarrhea by infecting the small intestinal epithelium. based on studies in animal models, homotypic neutralizing antibodies were thought to play a key role in protection, making viral serotype an important distinction. yet, in humans, a monovalent live attenuated rotavirus vaccine provides substantial heterotypic protection (leshem et al., ) . there are a number of candidates for the mechanism of the greater breadth of protection, including heterotypic neutralizing antibody, heterotypic nonneutralizing antibodies that block intracellular replication, and cellmediated effector mechanisms, with no mechanism widely accepted as responsible. cell-mediated effector mechanisms are thought to play a significant role in vaccine-mediated protection from a few viruses, such as varicella zoster virus (in adults) and influenza virus (with live attenuated vaccines) (forrest et al., ; weinberg et al., ) . eliciting an effective t helper (th) response is essential for mounting an affinity-matured, durable humoral response to vaccines. the control of more changeable viruses and the harnessing of cell-mediated effector mechanisms for prevention of viral diseases in humans are, therefore, two of the chief challenges in modern vaccinology. increasingly sophisticated tools of b cell repertoire analysis, transcriptome analysis, and advanced functional imaging are being used to understand vaccine mechanism of action (tsang et al., ; jiang et al., ; karlsson et al., ) . yet, as is the case for the technologies used to manufacture most current human vaccines, the core immunoassays used to bring viral vaccines to licensure are generally simple, as exemplified by antibody-based viral neutralization assays, enzyme-linked immunosorbent assays, and multiplex binding assays. this restricted range of assay types largely reflects a need to standardize assays rigorously (guidance for industry. bioanalytical method validation (draft) ) and practical limitations on the sophistication of assays that can be used as primary endpoint in the large-scale clinical trials (often with tens of thousands of subjects) required to bring novel vaccines to licensure. more sophisticated measures of cellmediated immunity have had a prominent role in vaccine research but a much more minor role in vaccine licensure. the difficulty of standardizing such assays and assigning a threshold assay value that indicates protection has limited their use as correlates of protection for vaccine trials being used for registration. similarly, although mucosal immunity surely plays a major role in determining vaccine-mediated protection, challenges in sample collection and assay consistency have the consequence that serum antibody levels rather than measures of mucosal immunity are favored as correlates of protection for vaccine licensure. nevertheless, measures of cell-mediated immunity, such as enzyme-linked immunosorbent spot assays and fluorescence-activated cell sorter-based t cell functional analyses, are routinely used as exploratory endpoints during vaccine development. recently, investigators have also added systems biology approaches, with transcriptional array, mass cytometry-based cell sorting, and luminex binding assays to their analytical tool box for investigating the early innate immune responses to vaccination (o'gorman et al., ; tsang et al., ) . to date, none of these interesting approaches have proven sufficiently robust or predictive to be used as licensure criteria. standardization of such assays could lead to a more prominent role for assays of cell-mediated, mucosal, and innate immunity in vaccine development. antigenic diversity and antigenic change remain key challenges for the development of effective vaccines for several important agents. influenza vaccines must change composition on an almost annual basis to track antigenic drift or shift, and the vaccine varies considerably in effectiveness from year to year depending in part on the accuracy of the antigenic match between the vaccine and circulating strains. for pathogens that are more variable than influenza, such as hiv and hcv, attempts at immunization have thus far proven largely ineffective despite the identification of viral targets of broadly neutralizing antibodies (burton and mascola, ; kong et al., ; throsby et al., ; whittle et al., ) . evasion of vaccine-mediated immunity, for example, by replication in privileged reservoirs, poses another challenge for vaccine development. hiv evades vaccine-mediated immunity not only by selection of resistant variants in response to immune pressure, but also by replication in immune-privileged sites, such as cells of the central nervous system (churchill and nath, ) . the latency of herpes viruses has posed a challenge for vaccine development, though not an insurmountable challenge, as evidence by the efficacy of varicella zoster vaccines against reactivation disease (shingles) (lal et al., ; oxman et al., ) . the duration of immunity elicited by viral vaccines also varies. in the best case, a single immunization with the live attenuated yellow fever vaccine appears to confer lifelong protection (although current recommendations are for a booster every years for those at risk) (gotuzzo et al., ) . at the other extreme, the need for annual immunization against influenza does not appear to be solely due to antigenic changes in circulating viruses, as the duration of immunity elicited by nonadjuvanted, inactivated influenza vaccines appears to be limited, with vaccine strain-specific effectiveness decreasing over the course of a season more than can be accounted for by the antigenic drift of circulating viruses (kissling et al., ; pebody et al., ) . special populations pose a particular challenge for viral vaccines. the neonatal response to immunization differs from responses later in life, with preferential elicitation of memory b cells rather than plasma cells and a more th -biased t cell response (wood and siegrist, ) . passively transferred maternal antibodies can interfere with vaccine immunogenicity, particularly in infants below months of age (crowe, ) . safety is a critical issue for neonatal immunization. for example, findings of excess wheezing and hospitalization prevent a live attenuated influenza vaccine from being used in infants under years of age despite its utility in older children (prutsky et al., ) . to overcome these factors and to take advantage of prime-boost effects, infant immunization is generally carried out with multiple doses, often including remote boosters. for rsv, a major cause of the infant disease for which there is currently no licensed vaccine, there is insufficient time to elicit an active immune response before the peak incidence of severe disease at approximately months of age. this early peak of disease is a chief rationale underlying proposals for maternal immunization against rsv (anderson et al., ) . immunizing pregnant women can increase the levels of pathogen-specific antibodies transferred transplacentally or in breast milk to newborns. immunization of pregnant women is gaining increasing acceptance, in part due to the experience of immunizing pregnant women during the influenza pandemic (which caused disproportionately severe illness in pregnant women) (fell et al., ) , evidence of improved birth outcomes with influenza immunization during pregnancy (steinhoff et al., ) , and the success of maternal immunization to prevent neonatal tetanus (blencowe et al., ) . at the other end of the age spectrum, immune senescence creates challenges for immunizing the elderly. high-dose formulations of inactivated influenza vaccine and varicella zoster vaccine and an adjuvanted formulation of inactivated influenza vaccine have been used to increase vaccine effectiveness in the elderly (diazgranados et al., ; mannino et al., ; oxman et al., ) . immunodeficiencies also pose a challenge for immunization. for example, renal failure patients on hemodialysis are at particular risk for the acquisition of hepatitis b. yet, the hbv vaccine is less likely to elicit protective antibody titers in this population. this has led to the licensure of a hepatitis b vaccine adjuvanted with an mpl-analog adsorbed on aluminum phosphate for those with renal insufficiency in the european union (kundi, ) . there are also safety concerns around the use of live attenuated vaccines in the immunodeficient, with some live attenuated vaccines (such as those targeting smallpox virus, yellow fever virus, or rotavirus) causing clinical illness due to infection with the attenuated vaccine virus in those with immunodeficiency (bakare et al., ; reed et al., ; seligman, ) . the potential for vaccine-mediated disease enhancement has substantially hindered the development of an rsv vaccine (anderson et al., ) . in the s, a formalin-inactivated rsv vaccine candidate primed immunized infants for more severe rsv disease upon natural rsv challenge (kapikian et al., ) . a preponderance of evidence suggests that an immunopathologic th -biased cellular response coupled with a minimal neutralizing antibody response to the vaccine was responsible for the enhanced disease, although the mechanism of the vaccine-mediated disease enhancement is still not completely understood (graham, ) . observations of vaccine-enhanced disease have also been made in humans after immunization with an inactivated measles vaccine (fulginiti et al., ) and in a ferret model with experimental immunization against the severe acute respiratory syndrome coronavirus (weingartl et al., ) . as is evident from the preceding comments, there is ample opportunity to apply state-of-the-art immunology as well as more modern manufacturing practices to vaccines. there remains a large gap between the large diversity of immunization approaches in research and early development and the limited application of new science and technology to vaccines that have been licensed or that are in late-stage development. in part, this reflects the challenges of replacing sufficiently effective, old vaccines with new ones. the old vaccines have a long track record of safe administration; for the new vaccines, knowledge of safety, efficacy, and effectiveness is necessarily more limited, no matter what theoretical advantages new technologies may offer. in addition, many of the old, established vaccines still in use are remarkably inexpensive, undermining the economics that would permit replacement with much more expensive new vaccines, burdened by new development costs. nevertheless, key viral pathogens, such as hiv, rsv, cmv, herpes simplex virus, dengue virus, and hcv have resisted successful vaccine development using conventional techniques and are prime targets for innovative approaches. more sophisticated application of immunology to vaccine development could address some of the challenges in the clinical development of vaccines. for example, increased use of immunological markers of vaccine safety, immunogenicity, and efficacy could limit clinical trial sizes and durations. more intensive study of smaller numbers of subjects in early clinical trials could lead to the testing of more vaccine candidates and better informed decisions on whether to progress vaccine candidates to larger-scale clinical testing or to switch to more promising alternative vaccine candidates (rappuoli and aderem, ) . for example, cd þ t cell responses observed weeks after immunization with a h n influenza vaccine predict neutralizing antibody responses months after immunization, potentially enabling more rapid answers about immunogenicity from clinical trials of such vaccines (galli et al., ) . although analysis of biomarkers and more sophisticated immunological analyses may enhance our ability to assess some aspects of vaccine safety and immunogenicity in smaller trials, it is less likely that such approaches will enable the detection of the unanticipated, idiosyncratic adverse events that large phase iii trials and postlicensure surveillance are designed to detect. examples of such idiosyncratic events include the guillain-barre syndrome observed after influenza immunization following the swine flu outbreak (schonberger et al., ) , the narcolepsy associated with aso -adjuvanted pandemic influenza vaccine in northern european adolescents (ahmed et al., ) , or the intestinal intussusception associated to greater or lesser degrees with different live attenuated rotavirus vaccines (rosillon et al., ) . however, if a core set of safety and immunogenicity parameters could be assessed in smaller trials through more intensive study of immunological and other biomarkers, more of the burden of detecting rare events could be shifted from large prelicensure trials to postlicensure surveillance systems, decreasing the barriers to advancing vaccines through licensure to meet the unmet medical needs. b cell repertoire analysis has provided a wealth of new information on the diversity of antibodies that collectively make up a serum antibody response to a vaccine. a key finding has been that, among the neutralizing antibodies elicited in response to influenza virus, hiv, or rsv infection or immunization, some have remarkably broad specificity (burton and mascola ; corti et al., corti et al., , . to date, b cell repertoire analysis coupled with the functional analysis of antibodies and the structural analysis of antigen-antibody complexes has defined the antibodies we want our vaccines to elicit and the target structures on viruses recognized by such antibodies. progress has also been made in our ability to design epitope-focused antigens that elicit such antibodies (correia et al., ; schickli et al., ) . however, to date, such engineered antigens have not proven more effective at eliciting high titers of broadly neutralizing antibodies than the intact glycoprotein domains that contain the epitopes of interest. iterative improvement of such rationally designed antigens does, however, hold promise for improved vaccines that target patches of conservation on variable, multiepitope antigens. vaccines based on conserved t cell epitopes or internal protein antigens that are the targets of t cell-based effector mechanisms are an area of active research. theoretically, vaccines that target the conserved nonsurface proteins of viruses that are expressed in infected cells could overcome the antigenic diversity of surface neutralization determinants. for example, in a small human live viral challenge study, the vectored expression of the influenza internal matrix protein and nucleoprotein together significantly reduced the number of days of virus shedding during laboratory-confirmed influenza infection (lillie et al., ) . it remains to be determined whether such mechanisms of protection will prove effective and durable enough to be the primary basis for vaccine-mediated protection, however. even absent the ability to suffice as the primary basis for influenza vaccines, conserved targets of t cell effectors may prove useful supplements to increase the breadth of coverage by vaccines that also include targets of more potently protective, strain-specific humoral immunity (antrobus et al., ) . as t cell effector mechanisms involve the killing of host cells, priming for increased t effector responses does carry some risk of exacerbating disease even as it accelerates viral clearance. increased insights into innate immune mechanisms have informed the search for new generations of rationally designed adjuvants. the adjuvants in currently licensed vaccines, aluminum salts, oil-in-water emulsions, and mplanalogs have been the products of largely empirical development efforts (o'hagan and fox, ) . the elucidation of toll-like receptors as key sentry molecules that detect potential pathogens and recruit antigen-presenting cells for a subsequent antigen-specific response has enabled the rational design of a new generation of potential vaccine adjuvants (wu et al., ) . using well-established principles of small-molecule screening and optimization for ligand binding, new toll-like receptor agonists are now reaching clinical testing. similarly, the ability to target vaccineprimed responses to mucosal tissues through the use of retinoic acid as an adjuvant, thus far demonstrated in preclinical testing, provides an additional mode of action for adjuvants to shape the character of vaccine-mediated immune responses (tan et al., ) . it is important to note, however, that demonstration of safety, especially as it applies to rare idiosyncratic events, remains a critical hurdle to the licensure of vaccines containing novel adjuvants. largely empirical approaches to vaccine development have provided tremendous public health benefits. the understanding of vaccine immunology has advanced greatly since many vaccines still in use were originally developed. applying this new knowledge could improve current vaccines and lead to the development of vaccines for viral diseases that have resisted vaccine-mediated protection to date. cell activation: antigen acquisition in vivo and its role in b cell activation extrafollicular antibody responses; germinal centers; long-lived plasma cells marginal zone b cell responses to antigens regulation of b cell migration and location in response to antigens somatic hypermutation and class switch recombination; t cell-dependent b cell activation; the humoral immune response to t cell-independent antigens cells of the innate immune system: dendritic cells and dendritic cell subsets. cytokines and their receptors: inflammasomes; viral anticytokine strategies. immunity to bacterial, parasitic and fungal infections: immune-driven pathogen evolution immunology of bacterial and parasitic diseases: an overview immunology of central nervous system pathogens. immunity to viral infections: activation of cd t lymphocytes during viral infections antibody responses and functions in defense against viral infection cd t cell immunity to viral infection cd t cell memory to pathogens; chemokines and viral infections dendritic cells in viral infection human genetic defects resulting in increased susceptibility to viral infections immunity to hiv; immunity to oncogenic viruses; innate cytokine responses and their functions during viral infections; intrinsic cellular defenses (trims) in modulating viral infection and immunity; nk cells in antiviral defense; nk cells in hiv- infection protective and pathogenic t cell responses to virus infections sensors of viral infection; t cell responses during acute respiratory virus infection. mhc structure and function: immune epitope database and analysis resource; ligand selection and trafficking for mhc i ligand selection and trafficking for mhc ii; origin and processing of mhc class ii ligands; origin and processing of mhc-i ligands; structure of classical class i mhc molecules structure and function of diversifying receptors: aid in somatic hypermutation and class switch recombination; error-prone mismatch and base excision dna repair in somatic references ahmed strategic priorities for respiratory syncytial virus (rsv) vaccine development coadministration of seasonal influenza vaccine and mva-npþm simultaneously achieves potent humoral and cell-mediated responses severe combined immunodeficiency (scid) and rotavirus vaccination: reports to the vaccine adverse events reporting system (vaers) aluminum salts in vaccines-us perspective cold-adapted live influenza vaccine versus inactivated vaccine: systemic vaccine reactions, local and systemic antibody response, and vaccine efficacy. a meta-analysis hemagglutination inhibition antibody titers as a correlate of protection for inactivated influenza vaccines in children tetanus toxoid immunization to reduce mortality from neonatal tetanus antibody responses to envelope glycoproteins in hiv- infection where does hiv hide? a focus on the central nervous system proof of principle for epitopefocused vaccine design cross-neutralization of four paramyxoviruses by a human monoclonal antibody a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins influence of maternal antibodies on neonatal immunization against respiratory viruses efficacy of high-dose versus standard-dose influenza vaccine in older adults h n influenza vaccination during pregnancy and fetal and neonatal outcomes correlation of cellular immune responses with protection against culture-confirmed influenza virus in young children altered reactivity to measles virus. atypical measles in children previously immunized with inactivated measles virus vaccines inactivated and adjuvanted influenza vaccines adjuvanted h n vaccine induces early cd þ t cell response that predicts long-term persistence of protective antibody levels role of as in human papillomavirus vaccine: mode of action and clinical profile efficacy and duration of immunity after yellow fever vaccination: systematic review on the need for a booster every years biological challenges and technological opportunities for respiratory syncytial virus vaccine development bioanalytical method validation (draft). us food and drug administration haemagglutination-inhibiting antibody to influenza virus what level of hepatitis b antibody is protective? lineage structure of the human antibody repertoire in response to influenza vaccination an epidemiologic study of altered clinical reactivity to respiratory syncytial (rs) virus infection in children previously vaccinated with an inactivated rs virus vaccine visualizing real-time influenza virus infection, transmission and protection in ferrets low and decreasing vaccine effectiveness against influenza a(h ) in / among vaccination target groups in europe: results from the i-move multicentre case-control study structural basis of hepatitis c virus neutralization by broadly neutralizing antibody hcv new hepatitis b vaccine formulated with an improved adjuvant system efficacy of an adjuvanted herpes zoster subunit vaccine in older adults distribution of rotavirus strains and strain-specific effectiveness of the rotavirus vaccine after its introduction: a systematic review and meta-analysis preliminary assessment of the efficacy of a t-cell-based influenza vaccine, mva-npþm , in humans effectiveness of adjuvanted influenza vaccination in elderly subjects in northern italy yellow fever vaccine: direct challenge of monkeys given graded doses of d vaccine granzyme b: correlates with protection and enhanced ctl response to influenza vaccination in older adults the split virus influenza vaccine rapidly activates immune cells through fcg receptors new generation adjuvants -from empiricism to rational design a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults vaccine effectiveness of / trivalent seasonal influenza vaccine in preventing laboratory-confirmed influenza in primary care in the united kingdom: evidence of waning intra-seasonal protection complex correlates of protection after vaccination assessing the evidence: live attenuated influenza vaccine in children younger than years. a systematic review a vision for vaccines against hiv, tuberculosis and malaria eczema vaccinatum risk of intussusception after rotavirus vaccination: meta-analysis of postlicensure studies palivizumab epitope-displaying virus-like particles protect rodents from rsv challenge guillain-barre syndrome following vaccination in the national influenza immunization program, united states, - risk groups for yellow fever vaccine-associated viscerotropic disease (yel-avd) neonatal outcomes after influenza immunization during pregnancy: a randomized controlled trial retinoic acid as a vaccine adjuvant enhances cd þ t cell response and mucosal protection from viral challenge heterosubtypic neutralizing monoclonal antibodies cross-protective against h n and h n recovered from human igm þ memory b cells global analyses of human immune variation reveal baseline predictors of postvaccination responses complete list of vaccines licensed for immunization and distribution in the us varicella-zoster virus-specific immune responses to herpes zoster in elderly participants in a trial of a clinically effective zoster vaccine immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets vaccine induced immunologic memory for hepatitis b surface antigen: implications for policy on booster vaccination broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin neonatal immunization: where do we stand? rational design of small molecules as vaccine adjuvants construction and characterization of virus-like particles: a review key: cord- - yxurnev authors: green, manfred s; leduc, james; cohen, daniel; franz, david r title: confronting the threat of bioterrorism: realities, challenges, and defensive strategies date: - - journal: lancet infect dis doi: . /s - ( ) - sha: doc_id: cord_uid: yxurnev global terrorism is a rapidly growing threat to world security, and increases the risk of bioterrorism. in this review, we discuss the potential threat of bioterrorism, agents that could be exploited, and recent developments in technologies and policy for detecting and controlling epidemics that have been initiated intentionally. the local and international response to infectious disease epidemics, such as the severe acute respiratory syndrome and west african ebola virus epidemic, revealed serious shortcomings which bioterrorists might exploit when intentionally initiating an epidemic. development of new vaccines and antimicrobial therapies remains a priority, including the need to expedite clinical trials using new methodologies. better means to protect health-care workers operating in dangerous environments are also needed, particularly in areas with poor infrastructure. new and improved approaches should be developed for surveillance, early detection, response, effective isolation of patients, control of the movement of potentially infected people, and risk communication. access to dangerous pathogens should be appropriately regulated, without reducing progress in the development of countermeasures. we conclude that preparedness for intentional outbreaks has the important added value of strengthening preparedness for natural epidemics, and vice versa. the biological weapons convention prohibits the manufacture and use of biological weapons. it came into force in , and has undergone periodic reviews, the last being in . to date, countries are signatories to the convention. unfortunately, terrorist groups or rogue governments are unlikely to feel bound by international agreements. the potential for bioterrorism is of particular concern, since it can cause disease, death, and panic-in great disproportion to the resources expended. there have been a few well documented cases of bioterrorism. in , a religious sect in the usa deliberately contaminated restaurant salad bars with salmonella typhimurium, intending to disrupt local elections. the attack resulted in several hundred cases of salmonellosis and no deaths. the anthrax letters incident in in the usa resulted in cases of inhalation anthrax, with five deaths, and another cases of cutaneous disease. extensive circumstantial evidence strongly suggests that the perpetrator was a civilian employee of the us military. however, no evidence of a clear motive was found. thousands of workers received prophylactic or post-exposure therapy, and affected buildings were decontaminated at huge expense. , in , a cult in japan carried out an attack using anthrax spores with no physical casualties, but later, evidence of post-traumatic stress syndromes was found in victims of the attack. the perpetrators were apparently planning to use other agents such as q fever bacteria, botulinum toxin, and ebola viruses, but they were detained before they could implement further attacks. in this review, we discuss the threat of bioterrorism, potential perpetrators, and general preparedness principles. we examine the special characteristics of biological agents that could potentially be used for bioterrorism, advances in prevention and treatment of diseases caused by these agents, and the remaining deficiencies in the management and control of possible bioterrorist outbreaks. in all respects, the ways in which the resources developed for bioterrorism preparedness could be used for controlling naturally occurring epidemics remain a guiding principle. • preparedness for intentional outbreaks will strengthen the response to naturally occurring epidemics • high level leadership should be maintained with responsibility and authority • health-care providers should maintain awareness of biological agents with bioterrorism potential and consider the presence of unknown pathogens • emergency room and community physicians should be updated regularly about the clinical manifestations of diseases caused by potential bioterrorist agents and emerging infectious diseases. • personal protective equipment should be improved to become more user friendly • improved surge capacity (the ability to rapidly gear up the health system to cope with a sudden, large increase in patients with a serious, contagious disease) is required, particularly in peripheral areas • the capacity of general and reference laboratories should be increased, to keep developing faster, more reliable diagnostic tests • new and improved vaccines (pre-exposure and post-exposure) and treatment regimens should be developed • clinical and environmental surveillance needs to increase • syndromic surveillance systems can be maintained to register suspicious or confirmed cases reported by physicians, and the data can be used to improve risk communication programmes and to monitor the progress of an outbreak • an adequate stockpile of vaccines and medications should be maintained, both nationally and internationally • to improve preparedness for natural and bioterrorist outbreaks, international cooperation should include joint exercises involving multiple countries and constant improvement in the exchange of information on potential bioterrorism threats and management following the breakup of the former soviet union, there was concern that loss of control of their biological weapons programme could allow terrorist groups to gain access to both the weapons and scientific expertise. additionally, in the past few years, developments in the field of microbial genetics have heightened concern about the possible abuse of new technologies. since there are so many unknowns, it is extremely difficult to assess the risks and threats of bioterrorism. , the most likely perpetrators could be disgruntled individuals, terrorist organisations, or rogue countries that are believed to support international terrorism. whereas individual attackers are unlikely to cause mass casualties, terrorist organisations could pose a substantial threat if they gain access to sophisticated biological weapons, materials, or scientific expertise. although regulations and safeguards for securing dangerous pathogens in research laboratories now exist in most countries, the scope of these regulations and the extent of the safeguards vary. rogue countries have the necessary capabilities for a bioterrorist attack but might be restrained by the threat of the response of a unified global community. knowledge gained from legitimate research that could also be applied to bioterrorism is considered dual-use. as a result, the regulation of legitimate research on infectious diseases has increased. there will always be a risk of the "insider threat", , which typically involves a single individual, so it is important to assure that new regulations truly increase security and have minimal negative effect on legitimate research. the cost of regulations applied to research on infectious diseases, in terms of missed opportunities for international collaboration, exchange of pathogens, and sharing of novel agents, is often intangible and overlooked. it is essential to promote healthy organisational cultures to enhance both safety and security in laboratories. since a bioterrorist attack is a low-risk, high-impact event, effective and sustained preparedness is an essential component in both the deterrence and management of an attack. a bioterrorist attack has a lot in common with naturally occurring public health emergencies resulting from infectious diseases. however, there are some important differences. since it is a deliberate act to cause harm, there are the obvious security considerations. the resulting outbreak differs in some important ways from naturally occurring epidemics-for instance, it is more likely to be a point source outbreak initiated by simultaneous exposure to many people. the infectious agent used is likely to be uncommon and possibly not endemic to the region, might have been modified genetically to make it resistant to current medications and vaccines, and produced in a way that enhances its transmission or virulence. therefore, early clinical symptoms and signs after infection with a bioterrorist agent might be unusual, complicating both recognition and management of the disease. these factors could create greater public panic. despite the many similarities with naturally occurring outbreaks of infectious disease, preparedness for bioterrorist attacks is more complex. in many aspects, a bioterrorist attack has the characteristics of a mass casualty event, and thus preparedness involves strengthening of the specialised infrastructure that is required for treatment of seriously ill patients over a very short period of time. new prophylactic and treatment regimens for unusual diseases are required, to ensure their accessibility when needed, along with clear standards for the handling and study of dangerous pathogens. when the proportion of available resources poor international preparedness • delay before the who declared a public health emergency of international concern • delay in implementing coordinated international assistance • logistic challenges in delivering support to assist epidemic response • shortcomings in who's regional and country-level capacity exposed • lack of global plans to address an epidemic of a high-risk pathogen in the least developed urban centres • evaluation of promising vaccine and therapeutic interventions came too late and ebola virus. the local and international responses to the west african ebola virus epidemic revealed shortcomings that could allow highly contagious epidemics of infectious disease to spread widely before they are terminated (panel ). during the cold war, agents that could potentially be used as biological weapons were identified on the basis of the following characteristics: pathogenicity for humans, animals, or plants; ability to cause disability or death; stability and infectivity as small particle aerosols; and capability of being readily and rapidly produced and weaponised in munitions or delivery systems. more characteristics have been added, to include other features of biological agents such as the relative ease of medical prevention or treatment and the likelihood of harm to the perpetrator. the us centers for disease control and prevention (cdc) identified bacteria, viruses, and toxins that could potentially be weaponised (panel ). in , they categorised them into three groups-a, b, and cdepending on ease of dissemination, severity of illness caused, and ability to cause death. biological agents can be infectious and contagious, infectious but not usually contagious, or toxins if they are neither. category a agents were considered the greatest risk to public and national security. the more recent classification of tier select agents and toxins is similar to the category a classification (table ) . other agents, such as naturally occurring pathogens, produce diseases that are considered of intermediate risk to the public (eg, brucellosis, glanders, q fever). they are moderately easy to disseminate, and include emerging and re-emerging infectious diseases. however, genetic modifications could make them more virulent, produce uncharacteristic clinical signs, increase their resistance to treatment and vaccines, and even change their transmissibility or host range. genetic modifications could be made using the tools of synthetic biology; such activities might be an example of dual-use research. , for instance, in , the spanish influenza pandemic virus was reconstructed, and the poliovirus was synthesised nearly years ago. the addition of an immuno-modulatory gene to the mousepox virus genome in , rendered a mousepox vaccine ineffective, and this technology could potentially be applied to the smallpox virus. the recent synthesis of the extinct horsepox virus has been a reminder that the smallpox virus could be reconstructed, and that the regulations that have been put in place to prevent the misuse of powerful, cheap, and globally available tools must be reconsidered. this possibility has also raised the issue of whether research results should sometimes be censored, or even refused publication, if the potential to cause harm is too high. although bioterrorist agents could be disseminated through multiple routes, the aerosol route would likely maximise exposure. contagious agents could produce a large number of second and later generation cases, depending on the number of people initially exposed, the series average number of people who acquire the disease from one infected individual (r ), and the disease generation time in humans. for instance, the r of pneumonic plague has been estimated to be around · , whereas the r for smallpox is likely to be around . for diseases that are not contagious, such as inhalational anthrax, the number of cases of disease will depend almost entirely on the size of the population exposed and the timing of post-exposure antibiotic prophylaxis. aerosolised agents remain the threat of most concern, but safety and security of food [ ] [ ] [ ] and water supplies are also important components of primary prevention (panel ). new methods to detect toxins in food, such as antibody based assays, are being developed. rapid diagnostics take on additional urgency in a bioterrorist event, because of both health and security concerns. since the anthrax letters, there have been major advances in diagnostic capabilities. some of the greatest advances in the past decade have been in the speed and reduced cost of sequencing capabilities. , highly sensitive and specific pcr-based systems, coupled with modern sample preparation technologies, have enabled sequencing technologies to become less costly, more portable, and multiplexed. with fieldable patient-side diagnostics and sequencing outputs directly connected via cloud-based networks, health-care providers globally can make decisions more rapidly and respond more quickly for individual care or outbreak detection. a rapid, cartridge-based assay for francisella tularensis has been developed for use at point of care. a system that uses a sensitive microsphere technology to detect both antibodies and antigens is now available to diagnose infections with ebola virus and lassa virus. although diagnostic elisa tests are available for anthrax antibodies, , a compact system (genexpert) that includes both sample processing and pcr amplification can produce a result in about minutes. a rapid and sensitive method to detect smallpox virus has been developed for use at point of care, based on antibody immuno column for analytical processes (abicap) immunofiltration, that produces results in about minutes. however, diagnostic electron microscopy is also still considered a fast and efficient method to identify smallpox and other viral agents. ebola virus was rapidly sequenced during the outbreak in sierra leone to link sporadic cases with the transmission chains. advanced proteomics are also being developed as reference assays and a new method for simultaneous immunodetection of anthrax, plague, and tularaemia from blood cultures has recently been reported, using multiplexed suspension arrays. next generation safety and security of food [ ] [ ] [ ] and water supplies are important components of primary prevention: • intentional contamination of food should be considered, particularly during a large foodborne epidemic with a common source • salmonella and shigella species, enterohaemorrhagic escherichia coli (all serotypes), vibrio cholerae, cryptosporidium parvum, and noroviruses are all potential candidates for intentional contamination of food • contamination of water with biological agents should still be considered, even though it is unlikely to be the major target of bioterrorism, due to chlorination, dilution, and the need for large quantities of the agent to cause a substantial outbreak • c parvum and noroviruses are more resistant to chlorination than other agents, so can be a threat to the water supply • food-borne or water-borne dissemination of these biological agents might lead to higher rates of morbidity and case fatality than previously observed, if the population has been exposed to substantially higher infectious doses • algorithms could be developed to measure the likelihood that outbreaks of disease were a consequence of intentional contamination of food or water, using descriptive, analytical, and molecular epidemiologic tools (none are known to be available so far) increased networking and collaboration of laboratories will also improve the response to intentional outbreaks. effective global surveillance of infectious diseases is essential to control both intentional and naturally occurring epidemics. surveillance data can be used to monitor the progress of an outbreak, and for risk communication. to obtain information rapidly, the ongoing collection of health-related data (termed syndromic surveillance) has been introduced, to monitor patterns of symptoms and signs that are suggestive of an outbreak. [ ] [ ] [ ] [ ] although it was hoped that syndromic surveillance would be a more sensitive method for early detection of an epidemic, frequent reports of unusual increases in incidence of non-specific illnesses can desensitise and paralyse the system. in fact, early detection will depend largely on alert, prepared clinicians. for example, when the anthrax attacks occurred, an astute clinician identified the index case. emergency room and community physicians should be updated regularly on the clinical signs and symptoms associated with the most common bioterrorist agents. the syndromic surveillance system would be more useful after suspicious or confirmed cases have been reported by physicians. a focused analysis of the surveillance data against non-specific, background disease rates could detect changes and provide information about the dynamics of the disease. special legislation might then be necessary, to gain access to medical records. the internet facilitates other potential forms of surveillance and communication about infectious diseases. , one such system is promed, which was established by the user community and has proven effective in connecting clinicians and scientists around the world; it has already served as an early warning system standard contact and airborne precautions should be taken. supportive therapy and antibiotics can be provided for secondary infections. there is some evidence of the potential efficacy of thiosemicarbazones. cidofovir has shown in vitro efficacy against variola, and has shown efficacy against other diseases caused by human orthopoxviruses, notably diseases caused by vaccinia viruses. it has also shown efficacy in animal models of orthopoxvirus infections. since , the us food and drug administration (fda) has approved drugs and biologic agents developed under the animal rule. this rule allows for approval of a drug that cannot be tested for efficacy in humans, but is effective in animals and safe in humans. the first drug approved under this rule was the monoclonal antibody raxibacumab for treatment of inhalation anthrax. tecovirimat is a drug that inhibits all orthopoxviruses tested in vitro. it was found to be highly effective in treating monkeypox and rabbitpox in animals and is considered safe in humans. tecovirimat is being considered by the fda for approval for use in humans to treat smallpox under the animal rule. standard and droplet precautions should be taken. ciprofloxacin, levofloxacin, and doxycycline have been approved for the treatment of pneumonic plague. streptomycin and gentamicin have been found to be effective in treatment, although there is some evidence of the development of multiple resistance. , tularaemia isolation of the patients is not necessary and standard precautions should be taken. ciprofloxacin, levofloxacin, and doxycycline are all approved for the treatment of tularaemia. streptomycin and gentamicin have been found to be effective. , haemorrhagic fevers standard contact and airborne precautions should be taken until diagnosis is confirmed. subsequently, droplet precautions can be considered. supportive care and treatment of secondary infections can be provided. ribavirin is now approved for treatment of lassa fever and it also appears to be effective against new world arenaviruses and crimean-congo haemorrhagic fever. protective n respirators and clothing should be provided to health-care personnel. clothing of patients should undergo decontamination and thorough handwashing. supportive therapy is available, with antibiotics such as ciprofloxacin, doxycycline and ampicillin. if the bacteria are resistant to some of the antibiotics, the treatment regimen will depend on sensitivity testing. the regulations require immediate reporting of serious health risks by all member countries. additionally, who has established the global outbreak alert and response network, and the european union has a programme called bichat to improve cooperation between member states in preparedness and response to biological and chemical attacks. they operate the early warning and response system for outbreaks of communicable diseases. the world organisation for animal health has developed plans to identify and deal with a bioterrorism attack on populations of food-producing animals. canada has established the global public health intelligence network for worldwide monitoring of threats to public health. a major benefit of a less formal global collaboration is the development of networks of trust among knowledgeable scientists and clinicians, who are considered early warning posts for both natural and intentional outbreaks. the one health initiative encourages collaboration between health professionals and is as important for bioterrorism preparedness as it is for management of emerging infectious disease and the global spread of antimicrobial resistance. the management of patients that have been infected during incidents of bioterrorism can be challenging. precautions and treatment regimens for several bioterrorist agents are summarised in panel . although supportive care serves as the basis of management for all agents, treatments for some of the relevant diseases have substantially progressed. the management of inhalation anthrax has advanced since the attack, , , with improvements in critical care, and in treatment of acute lung injury and acute respiratory distress syndrome, severe sepsis, and septic shock. pleural effusions are routinely drained and there are more options for antimicrobial therapy. antibiotics are still recommended for days after exposure or diagnosis, together with the anthrax vaccine. if the vaccine is given concurrently with antibiotic treatment, the period of treatment could be shortened. antibiotics for treatment of other bacterial infection are usually given for shorter periods, since the causative agents do not sequester spores. tularaemia is treated with ciprofloxacin or doxycycline. for smallpox, antivirals such as cidofovir or a related acyclic nucleoside phosphonate analogue appear to be more effective than post-exposure vaccines in preventing mortality, according to experiments in non-human primates infected with monkeypox virus. this suggests that antivirals might play an important role when preparing for a smallpox outbreak. for viral haemorrhagic fevers, ribavirin might have some efficacy in post-exposure prophylaxis. a small-molecule antiviral drug, gs- , has been developed that appears to be effective in treating ebola virus infection. for intentional and other sudden outbreaks of contagious disease, isolation of patients and controlling risks to health-care workers remains extremely challenging, as was noted in the mers coronavirus, sars, ebola virus disease, and avian influenza epidemics. hospital units adequately equipped for isolation are needed, similar to those equipped to care for filovirus-infected patients, with negative pressure air filtration. if facilities are too small for the number of patients, a lower level of isolation with strict barrier nursing should be implemented, and in the event of a very large outbreak, there might be a need to set up isolation facilities in public buildings. in regions with poor infrastructure, it might be necessary to consider treating patients in their homes. people who have died should be regarded as infectious and handled with the same precautions used for patients. burial procedures might have to be modified, but every effort should be made to respect religious practices and traditions of the local culture. quarantining of people who might have been exposed to the infectious agent can be problematic, as was the case in the sars and west african ebola virus epidemics. since the quarantined population includes both people who were exposed and people who were not, the risk of disease transmission is higher. during the ebola virus outbreak in west africa, suspect cases were held until they could be cleared as negative, which took about days pending the pcr results. on a national level, reducing the movement of populations is a sensitive issue, potentially interfering with commerce. closure of schools is an important means of achieving social distancing to reduce spread. whether the public should use masks during an outbreak of a contagious disease is less clear, and the efficacy of the masks is extremely variable. the most important reasons for variable efficacy are differences in facial shape, incorrect application, and duration of use. a substantial proportion of the cases and fatalities in the sars and ebola virus epidemics were among healthcare workers. clear guidelines specific to each agent are available to health-care personnel, public health workers, and emergency workers for the use of masks and personal protective equipment. the national ebola virus training and education center has been established in the usa to train health-care workers and assist hospitals in preparing for patients infected with high hazard virus in the usa and other countries. laboratory workers must be trained to work with dangerous pathogens and wear protective gear. designated threat pathogens must be stored, handled, and transported under a different set the role of vaccines in pre-exposure and post-exposure prophylaxis measures should be in place to protect the population from biological agents likely to be used in an attack before an incident occurs. however, since a bioterrorist incident is likely to be caused by biological agents not covered by routine immunisation, pre-exposure prophylaxis is generally confined to vaccines for military forces, health-care workers, and emergency response personnel. to the majority of the population, only postexposure prophylaxis is relevant. post-exposure prophylaxis for an intentional outbreak would include both those people known to be exposed during the incident and those people who were infected by others. the prophylaxis itself might consist of both vaccines and antimicrobials. when using live, attenuated vaccines, the relatively large proportion of the population who has some form of immunodeficiency has to be taken into account. , monoclonal antibody preparations are now being considered for prophylaxis in select, high risk groups. table summarises pharmacological prophylaxis for tier pathogens. currently, the vaccines that would most likely be used for pre-exposure or post-exposure prophylaxis are the smallpox and anthrax vaccines. since routine vaccination against smallpox was stopped in the s, less than % of the world's population has been vaccinated, and antibody titres usually decline markedly after to years. residual cellbased immunity can persist for many years. , postexposure prophylaxis involves ring vaccination, which requires intensive tracing and vaccination of primary contacts, followed by vaccination of secondary contacts, and finally, vaccination of all people in a defined affected region. for post-exposure prophylaxis of those directly exposed during the incident, vaccination can be effective if given within to days of exposure. since it might take that amount of time to detect the first cases after exposure, the vaccine will generally only be effective for secondary and subsequent contacts. immune-boosting adjuvants and toll-like receptor agonists have the potential to improve the immune response to post-exposure vaccination. serious side-effects are relatively rare , but can affect com pliance. in those cases, lower doses of vaccine might be administered, to provide adequate protection with fewer side-effects. newer smallpox vaccines are in development, including those that could immunise people who have atopic dermatitis. because it is produced in small quantities by collecting antiserum from immunised humans, there might be a shortage of vaccinia immune globulin, used to treat people who would have serious side-effects with the vaccine. one possible solution to this shortage would be to use antibodies against other poxviruses, such as cowpox and monkeypox, because of their cross-protective properties. since naturally occurring inhalation anthrax is extremely rare, there have been safety and immunogenicity profiles of the anthrax vaccine in humans, but the efficacy has only been tested in animal models, and not in clinical trials. [ ] [ ] [ ] the current anthrax vaccine is made from culture filtrates of a toxigenic, avirulent, nonencapsulated mutant of the bacillus anthracis vollum strain, and is administered in five intramuscular doses, followed by annual boosters. the protective, antigen specific memory b cells persist for many years after vaccination and are associated with humoral immunity. serum igg response to the vaccine has been % after the fourth dose. for post-exposure prophylaxis in unvaccinated people, the vaccine should be administered as a three-dose subcutaneous series (at , , and weeks), in conjunction with a -day course of appropriate antimicrobial drugs. it has been given to thousands of us military personnel, and notable adverse events have been rare. the anthrax vaccine is not recommended for pregnant women, although one study with women in the us military, inadvertently vaccinated during pregnancy, did not show evidence of an increase in birth defects. in this study, women received the vaccine in the first trimester and in the second and third trimeseter. more effective anthrax vaccines that require fewer doses are constantly being tested, , such as the neat protein anthrax vaccine, a dual purpose influenza vaccine that protects against anthrax, and a combined anthrax-plague vaccine. apart from the d yellow fever live attenuated vaccine and the junin virus vaccine, no vaccines for haemorrhagic fevers have been licensed. since the west african ebola virus epidemic, new ebola virus vaccines that have been long under development are being used successfully in the epidemic in the democratic republic of the congo. essentially no other licensed vaccines are available for other tier select agents. the previously licensed, formalin-inactivated, whole-bacilli plague vaccine has not proven effective against primary pneumonic plague in non-human primate models, series us military. the vaccine has not been used widely for preexposure prophylaxis and has no place in post-exposure prophylaxis. new vaccines against tularaemia are under development , including one that might provide crossprotection between plague and tularaemia. the investigational pentavalent (abcde) botulinum toxoid vaccine was provided by the cdc for laboratory workers at high risk of exposure to botulinum toxin and it has also been given to military members that are at risk. the botulinum toxoid vaccine produces effective immunity after several months and has no value for postexposure prophylaxis because of the short latent period of the toxins. this vaccine was discontinued in , because of declines in immunogenicity and adverse events. new recombinant botulism vaccines are being developed, in addition to vaccines against glanders and rift valley fever. [ ] [ ] [ ] efforts are ongoing to greatly shorten the time required to develop and produce new vaccines and other immune approaches. improved technologies are important for rapid scale-up and production of new treatment regimens, particularly following an attack with a contagious agent. the largely unpredictable nature of an epidemic initiated intentionally is likely to increase uncertainty and reduce public trust in the authorities. public education and effective risk communication are essential to increase public confidence and improve cooperation and compliance with recommended medical counter-measures. the anthrax vaccine in military populations has caused considerable scepticism regarding the need for, safety, and efficacy of the vaccine. [ ] [ ] [ ] clinicians and public health personnel should have access to up-to-date information, and the general public should be provided with nontechnical information and simple instructions on how to act during an emergency. sandman has proposed that "one should not over-reassure, ack nowledge uncertainty, and share dilemmas". this behaviour would only cause overreaction or panic when new information about the risk is made public. risk communication will be necessary at all stages: before a bioterrorist incident occurs, when an incident is suspected, when it is confirmed, while it is taking place, and in the aftermath. credible and trusted spokespersons, including respected clinicians, scientists, and public servants for a country, should be adequately informed before an incident. during an outbreak, there could be unexpected events, such as atypical presentation of cases, varying responses to treatment (including unusual side-effects), and false positive and false negative diagnoses. the public might lose trust in the authorities if apparently unexposed people become ill. the advent and global distribution of social networking increases the risk of the dissemination of false or misleading information. lastly, a major infectious disease incident will also require flexibility and possible changes of established government policy. environmental detection of biological agents is another area of research that should be developed. to date, most systems of environmental detection have focused on anthrax, as a result of the anthrax attacks. , however, a sensitive and specific set of recombinase polymerase amplification assays for fast screening, detection, and identification of b anthracis in a field setting has recently been developed. the rare occurrence and likely small effect of an aerosol bioterrorist attack limits the practical use of environmental detection to special event venues, public transportation systems, and possibly some government buildings thought to be likely targets. many countries have national stockpiles of drugs and vaccines, for use in the event of a biological or chemical attack, or for serious outbreaks that might achieve epidemic proportions. the usa, for instance, maintains a strategic national stockpile of vaccines and other medical countermeasures. global stores of smallpox vaccines are held by who, in addition to stores held by individual countries. some countries have undertaken active vaccination programmes against smallpox and anthrax in the military and first responder populations. preparedness for bioterrorist incidents requires constant re-evaluation of policies. although there is no evidence that the h n influenza pandemic or the - ebola virus epidemic in west africa were initiated intentionally, the local and international responses revealed strengths and weaknesses in the current state of preparedness for bioterrorist incidents. the ebola virus epidemic spread to a number of countries, with more than cases reported worldwide and a case fatality rate of more than %. imported cases of ebola virus disease were identified in the usa and spain. locally, in the affected countries in west africa, % of the people who died because of ebola virus disease were health-care workers. various shortcomings in the response to the epidemic have been identified since (panel ). accurately predicting the intentional misuse of a biological agent to cause harm is difficult without intelligence data, but several attempts have been made to rationally predict the categories of risk: man-made, natural, accidental, contagious, and non-contagious. series risks. the risk of bioterrorism has called into question some of the dogmas related to eradication of diseases such as poliomyelitis and measles. for example, if polio is successfully eradicated, universal vaccination might have to continue because of the risk of poliovirus being used as a bioterrorist agent. [ ] [ ] [ ] emerging and reemerging infectious diseases will continue to be a threat, but preparedness for bioterrorism is, in many ways, similar to preparedness for naturally emerging disease. all countries should collaborate to address the root causes of terrorism, and develop appropriate preventive strategies. effective preparedness is, in itself, a deterrent to bioterrorism, since it reduces the incentive to use biological weapons by making a country or region a hard target. it is also the cornerstone of consistent and effective responses to naturally occurring epidemics. the abuse of biological agents can be further reduced or discouraged with reliable intelligence and an effective response if it does occur. national and regional resources and capabilities will vary, but all will require infrastructures that are capable of recognising and dealing with a variety of biological agents. the needs of specific populations, such as the paediatric population, pregnant women, elderly people, and people with immunological disorders, must also be addressed. funding for biodefence is crucial to adequate preparation and response to bioterrorist threats. international preparedness for bioterrorism has the dual benefit of strengthening the infrastructure for responding to naturally occurring epidemics of highly pathogenic organisms. lessons from the west african ebola virus epidemic show that health-care providers must always be watchful for unusual presentations of disease, and new and improved approaches must be developed for early detection and response. health-care providers need more effective means of isolating infected patients, and better methods to control the movement of potentially infected people outside of the affected areas. personal protective equipment should be inexpensive and effective, and available to use with minimal training and under harsh environments. protection of health-care workers against infection remains particularly prob lematic, and should be a focus of research and development. the ebola virus epidemic has highlighted the importance of improving the logistics of moving human and material resources in areas with relatively poor infrastructure. risk communication and public education before and during an outbreak need to be improved. more clinical trials should be fast-tracked during development of new vaccines and antiviral drugs. preparedness for a low-risk, high-impact event that is bioterrorism should be monitored constantly, tested in tabletop exercises, , and integrated into the routine functioning of the health system. here it would serve the dual purpose of ensuring that countries are prepared to meet the challenges of controlling epidemics of emerging and re-emerging infectious diseases. msg designed the review, did the literature search and was responsible for writing the manuscript. jld assisted in the literature search, revised the manuscript, and supplied technical expertise. dc assisted in the literature search and revised the manuscript. drf helped design the review, contributed source material, did the literature search, and helped write and revise the manuscript. we declare no competing interests. we searched pubmed and google scholar using the terms "bioterrorism", "sustainable bioterrorism preparedness", "all-hazards infectious disease preparedness", "biological threat agents", "bioterrorism preparedness", "emerging infectious diseases", "smallpox", "anthrax", "plague", "tularemia", "botulism", "hemorrhagic fevers", and "risk communication". we searched national and international reports from the who and us centers for disease control using the terms "bioterrorism", "bioterrorism preparedness", and "emerging infectious diseases". we also completed web searches for "disease surveillance", "infectious disease diagnostics", "medical countermeasures", "emergency healthcare delivery", and "risk management". we focused on academic literature in english and restricted most of our searches to documents published since , with an emphasis on those published after , and included mainly those published since . federal funding for health security in fy a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars investigation of bioterrorism-related anthrax, united states, : epidemiologic findings remediation of bacillus anthracis contamination in the u.s. department of justice mail facility total decontamination cost of the anthrax letter attacks bacillus anthracis incident post-traumatic stress disorder symptoms in victims of tokyo subway attack: a -year follow-up study biological warfare and bioterrorism: a historical review the threat of bioterrorism: identifying the unknown. ffi focus biodefense in the st century department of health and human services (hhs). possession, use, and transfer of select agents and toxins-addition of bacillus cereus biovar anthracis to the hhs list of select agents and toxins. interim final rule and request for comments destruction of microbial collections in response to select agent and toxin list regulations implementing the select agent legislation: perfect record or wrong metric? gaps remain in china's ability to detect emerging infectious diseases despite advances since the onset of sars and avian flu health system resource gaps and associated mortality from pandemic influenza across six asian territories ebola: lessons learned and future challenges for europe public health assessment of potential biological terrorism agents responsible conduct by life scientists in an age of terrorism mitigating the risks of synthetic biology. council on foreign relations characterization of the reconstructed spanish influenza pandemic virus chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox labmade smallpox is possible, study shows the de novo synthesis of horsepox virus: implications for biosecurity and recommendations for preventing the reemergence of smallpox epidemiologic determinants for modeling pneumonic plague outbreaks risk assessment and risk communication strategies in bioterrorism preparedness. nato security through science series a: chemistry and biology estimating time and size of bioterror attack threat of a biological terrorist attack on the us food supply: the cdc perspective a large food-borne outbreak of group a streptoccocal pharyngitis in an industrial plant: potential for deliberate contamination german outbreak of escherichia coli o :h associated with sprouts water and bioterrorism: preparing for the potential threat to u.s. water supplies and public health a proteomics assay to detect eight cbrn-relevant toxins in food index case of fatal inhalational anthrax due to bioterrorism in the united states rapid outbreak sequencing of ebola virus in sierra leone identifies transmission chains linked to sporadic cases real-time pcr to identify variola virus or other human pathogenic orthopox viruses pocket dna sequencers make real-time diagnostics a reality sensitive detection of francisella tularensis directly from whole blood by use of the genexpert system comparison of magpix assays and enzyme-linked immunosorbent assay for detection of hemorrhagic fever viruses the early humoral immune response to bacillus anthracis toxins in patients infected with cutaneous anthrax specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin g antibodies to anthrax toxin protective antigen rapid detection of bacillus anthracis bloodstream infections by use of a novel assay in the genexpert system rapid and sensitive point-of-care detection of orthopoxviruses by abicap immunofiltration rapid viral diagnosis of orthopoxviruses by electron microscopy: optional or a must? striking against bioterrorism with advanced proteomics and reference methods simultaneous immunodetection of anthrax, plague, and tularemia from blood cultures by use of multiplexed suspension arrays the changing face of pathogen discovery and surveillance public health surveillance and infectious disease detection evaluation of a syndromic surveillance system using the wsare algorithm for early detection of an unusual, localized summer outbreak of influenza b: implications for bioterrorism surveillance syndromic surveillance during pandemic (h n ) outbreak internet-based surveillance systems for monitoring emerging infectious diseases digital surveillance for enhanced detection and response to outbreaks if syndromic surveillance is the answer, what is the question? clinical recognition and management of patients exposed to biological warfare agents public health. ethics and the conduct of public health surveillance digital disease detectionharnessing the web for public health surveillance factors influencing performance of internet-based biosurveillance systems used in epidemic intelligence for early detection of infectious diseases outbreaks the internet and the global monitoring of emerging diseases: lessons from the first years of promed-mail social and news media enable estimation of epidemiological patterns early in the haitian cholera outbreak geneva: world health organization the global public health intelligence network and early warning outbreak detection: a canadian contribution to global public health with the changing biological threat…smart international engagement policy would lower cost and increase national security animals as sentinels of bioterrorism agents clinical management of potential bioterrorism-related conditions wisconsin department of health services. infection control and prevention-standard precautions oral tecovirimat for the treatment of smallpox plague: recognition, treatment, and prevention the sanford guide to antimicrobial therapy new therapeutic approaches for treatment of tularaemia: a review centers for disease control and prevention expert panel meetings on prevention and treatment of anthrax in adults antitoxin treatment of inhalation anthrax: a systematic review consequences of delayed ciprofloxacin and doxycycline treatment regimens against francisella tularensis airway infection a single cidofovir treatment rescues animals at progressive stages of lethal orthopoxvirus disease therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys risks to healthcare workers with emerging diseases: lessons from mers-cov, ebola, sars, and avian flu implementing a negative-pressure isolation ward for a surge in airborne infectious patients large-scale quarantine following biological terrorism in the united states: scientific examination, logistic and legal limits, and possible consequences ebola and fda: reviewing the response to the outbreak, to find lessons for the future assessment of the potential for international dissemination of ebola virus via commercial air travel during the west african outbreak transmission of ebola viruses: what we know and what we do not know estimating the size of the u.s. population at risk of severe adverse events from replicating smallpox vaccine influence of population immunosuppression and past vaccination on smallpox reemergence next-generation monoclonal antibodies: challenges and opportunities analysis of historical data suggests long-lasting protective effects of smallpox vaccination duration of neutralizing antibody persisting in thai individuals after childhood vaccination against smallpox a model for a smallpox-vaccination policy can postexposure vaccination against smallpox succeed? immune-boosting adjuvants tlr and tlr agonists improve postexposure vaccination efficacy of live smallpox vaccines risks of serious complications and death from smallpox vaccination: a systematic review of the united states experience adverse events following smallpox vaccination with acam in a military population factors associated with healthcare worker acceptance of vaccination: a systematic review and meta-analysis reducing the dose of smallpox vaccine reduces vaccine-associated morbidity without reducing vaccination success rates or immune responses comparing new-generation candidate vaccines against human orthopoxvirus infections long-term safety of replication-defective smallpox vaccine (mva-bn) in atopic eczema and allergic rhinitis cross-neutralizing and protective human antibody specificities to poxvirus infections randomized, double-blind, placebo-controlled, safety and immunogenicity study of formulations of anthrax vaccine adsorbed plus cpg (av ) in healthy adult volunteers select human anthrax protective antigen epitope-specific antibodies provide protection from lethal toxin challenge protective antigen-specific memory b cells persist years after anthrax vaccination and correlate with humoral immunity lethal factor antibodies contribute to lethal toxin neutralization in recipients of anthrax vaccine precipitated serum igg antibody response to the protective antigen (pa) of bacillus anthracis induced by anthrax vaccine adsorbed (ava) among u.s. military personnel an overview of adverse events reported by participants in cdc's anthrax vaccine and antimicrobial availability program safety of inadvertent anthrax vaccination during pregnancy: an analysis of birth defects in the u.s. military population progress and novel strategies in vaccine development and treatment of anthrax host immunity to bacillus anthracis lethal factor and other immunogens: implications for vaccine design progress toward the development of a neat protein vaccine for anthrax disease a dual purpose universal influenza vaccine candidate confers protective immunity against anthrax a bivalent anthrax-plague vaccine that can protect against two tier- bioterror pathogens, bacillus anthracis and yersinia pestis experimental treatment of ebola virus disease with tkm- : a single-arm phase clinical trial plague vaccines: current developments and future perspectives plague vaccines: status and future intranasal delivery of a protein subunit vaccine using a tobacco mosaic virus platform protects against pneumonic plague protective immunity against lethal f. tularensis holarctica lvs provided by vaccination with selected novel cd + t cell epitopes francisella tularensis live vaccine strain deficient in capb and overexpressing the fusion protein of igla, iglb, and iglc from the bfr promoter induces improved protection against f. tularensis respiratory challenge yopp-expressing variant of y. pestis activates a potent innate immune response affording cross-protection against yersiniosis and tularemia notice of cdc's discontinuation of investigational pentavalent (abcde) botulinum toxoid vaccine for workers at risk for occupational exposure to botulinum toxins recombinant botulinum neurotoxin hc subunit (bont hc) and catalytically inactive clostridium botulinum holoproteins (cibont hps) as vaccine candidates for the prevention of botulism burkholderia pseudomallei and burkholderia mallei vaccines: are we close to clinical trials? safety and immunogenicity of a mutagenized, live attenuated rift valley fever vaccine, mp- , in a phase dose escalation and route comparison study in humans bioterrorism risk communication policy why do uk military personnel refuse the anthrax vaccination? a longitudinal study of uk military personnel offered anthrax vaccination: informed choice, symptom reporting, uptake and pre-vaccination health notes from the field: compliance with postexposure prophylaxis for exposure to bacillus anthracis among u.s. military personnel-south korea rapid detection of bacillus anthracis spores using immunomagnetic separation and amperometry rapid detection of viable bacillus anthracis spores in environmental samples by using engineered reporter phages sensitive and specific recombinase polymerase amplification set of assays for fast screening, detection and identification of bacillus anthracis in a field setting the pitfalls of bioterrorism preparedness: the anthrax and smallpox experiences pandemic preparedness and response-lessons from the h n influenza of global catastrophic biological risks: toward a working definition expert views on biological threat characterization for the u.s. government: a delphi study the risk of bioterrorism re-analysed risks of paralytic disease due to wild or vaccine-derived poliovirus after eradication eradicating polio: a balancing act vaccination against polio should not be stopped vaccines should be kept even if polio is wiped out ebola and zika: cautionary tales medical countermeasure development since : a long way yet to go preparing for biological threats: addressing the needs of pregnant women safety of live vaccinations on immunosuppressive therapy in patients with immune-mediated inflammatory diseases, solid organ transplantation or after bone-marrow transplantation-a systematic review of randomized trials, observational studies and case reports biosecurity: assessing the bioweapons threat a plague on your city: observations from topoff shining light on "dark winter key: cord- -zpi uis authors: roberts, anjeanette; wood, john; subbarao, kanta; ferguson, morag; wood, david; cherian, thomas title: animal models and antibody assays for evaluating candidate sars vaccines: summary of a technical meeting – august , london, uk date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: zpi uis abstract severe acute respiratory syndrome (sars) emerged in the guangdong province of china in late and spread to countries. by the end of the outbreak in july , the cdc and who reported cases with a . % case fatality rate. the disease was caused by a previously unrecognized coronavirus, sars-cov. drawing on experience with animal coronavirus vaccines, several vaccine candidates have been developed and evaluated in pre-clinical trials. available data suggest that vaccines should be based on the the kda viral spike protein, s, the only significant neutralization antigen capable of inducing protective immune responses in animals. in the absence of clinical cases of sars, candidate vaccines should be evaluated for efficacy in animal models, and although it is uncertain whether the united states food and drug administration's “animal rule” would apply to licensure of a sars vaccine, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. this report summarizes the recommendations from a who technical meeting on animal models and antibody assays for evaluating candidate sars vaccines held on – august in south mimms, uk, provides guidance on the use of animal models, and outlines the steps to develop standard reagents and assays for immunological evaluation of candidate sars vaccines. severe acute respiratory syndrome (sars) is a severe respiratory illness caused by the sars coronavirus (sars-cov) [ ] . the disease emerged in the guangdong province of china in late [ ] and spread to countries mostly within asia, although europe and north america were also affected, notably toronto, canada. the epidemic was finally controlled by july through strict implementation of prevalence for sars-cov antibody, although they had no history of sars-like disease. sars-cov-like viruses that were isolated from civets and raccoon dogs had more than % homology with human sars-cov, with major differences found in orf , whose deletion has been suggested to represent a sign of adaptation to humans [ ] . only four amino acid residues in the receptor glycoprotein ace binding domain of the viral spike protein differ between the human epidemic sars-cov strains and civet strains, but they cause more than a -fold difference in binding affinity to the ace molecule [ , ] . although a high prevalence of sars-like coronaviruses were found in chinese horseshoe bats [ , ] , their great genetic diversity makes it difficult to identify which one might be the ancestor of sars-cov and to decide with certainty whether bats indeed are the animal reservoir of the virus. sars-cov infection exhibits a wide clinical course characterized mostly by fever, dyspnea, lymphopenia and lower respiratory infection, often with concurrent gastrointestinal symptoms including diarrhea [ , ] . pathology in sars patients has been associated with diffuse alveolar damage, epithelial cell proliferation and multinucleated giant cell infiltrates of epithelial or macrophage origin, suggestive of syncytium-like formation in the lung. the virus can be recovered from peripheral blood mononuclear cells, respiratory secretions, stools, urine and even sweat (for a review, see [ ] ). sars vaccine development efforts were initiated very rapidly after the identification of the etiologic agent, even though the immune correlates of protection were not known. research efforts to identify protective antigens and to develop animal models were undertaken in parallel with efforts to develop candidate vaccines [ ] , drawing on experience with animal coronavirus vaccines and using several vaccine strategies, including inactivated virus vaccines, purified subunit vaccines, plasmid dna and viral vector-based vaccines as well as virus-like particles. much effort has been made to identify appropriate animal models for sars-cov replication and pathogenesis. several research groups have shown that mice [ , ] , ferrets [ ] , hamsters [ ] and nonhuman primates [ ] [ ] [ ] [ ] [ ] [ ] support replication of sars-cov with varying degrees of associated disease. these animal models were used for the evaluation of candidate vaccines, and the common conclusion that has emerged from the evaluation of several vaccines is that the kda viral spike protein, s, is the only significant neutralization antigen [ ] [ ] [ ] [ ] and the only one to elicit protective immunity in animal models [ , [ ] [ ] [ ] [ ] [ ] [ ] . the s protein can be divided into two domains by analogy with other coronavirus spike proteins: an n-terminal s domain, which contains the receptor-binding site and neutralization epitopes and a cterminal s domain which forms the membrane-anchored stalk region and contains a putative fusion peptide followed by two heptad repeats predicted to form a six-helix coiled-coil bundle [ ] . in the absence of clinical cases of sars, candidate vaccines will have to be evaluated for efficacy in animal models. the united states fda "animal rule" states that, when efficacy studies in humans are not feasible, vaccines may be approved based on animal data alone, provided the pathophysiological mechanism of the disease is reasonably wellunderstood as is its prevention or reduction by the vaccine. moreover, the protective effect of the vaccine should be demonstrated in more than one animal species expected to react with a response predictive for humans. the endpoint of animal studies should be clearly related to the desired benefit in humans (i.e. enhancement of survival or reduction in major morbidity), and the data generated should allow selection of an effective dose in humans. at the present time it is uncertain whether the "animal rule" would apply to licensure of a sars vaccine. however, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. scientists at the who technical meeting on animal models and antibody assays for evaluating candidate sars vaccines held on - august in south mimms, uk, discussed many aspects of research pertaining to the use of animal models in vaccine development including available animal models, suitability of the various models, correlates of protection, critical components of potential vaccines, and the potential for disease enhancement in vaccinated animals following exposure to sars-cov. in addition, standardization of antibody assays and the establishment of a who international standard for sars-cov antibody were also discussed. this report endeavors to summarize the recommendations from this meeting, based on consensus agreement. recommendations for use of each animal model are given in section below. correlates of protection, an overview of vaccine development, and observations pertaining to potential disease enhancement are summarized in the following sections - . in selecting animal models for vaccine evaluation, it is important to remember the principle underlying the so called "animal rule", where data from more than one animal species is often required: each animal species should contribute something different to our understanding of disease and protection. at this time, no single model offers a direct reproduction of what was seen in humans with sars. pathology (including pneumonitis, alveolar edema, and diffuse alveolar damage (dad)) in humans is probably the most difficult element to reproduce in an animal model. attention also should be given to the reduction of viral shedding because this would likely correlate with decreased risk of further spread of the disease among humans. in using animal models to study aspects of sars-cov infection, it must be emphasized that the kinetics of viral replication and appearance and resolu-tion of pathological findings are much more rapid in animal models than in humans. whichever animal model is employed, special consideration should be given to the presence of co-existing pathogens, the age of the animals and the route(s) of infection. a sufficient number of time points and large enough number of animals should be used to allow statistical evaluation. the strain of sars-cov used also could be of importance. it should be emphasized that different species may prove useful for studying different aspects of sars-cov. whereas vaccines or antivirals may be addressed in many models, pathogenesis is best evaluated in those animal models for which immunological tools and reagents are available for detailed analysis of the immune response to the vaccine. this includes inbred mice and rhesus and cynomolgus macaques. it may actually be worthwhile to enhance the virulence of a sars-cov isolate by serial passages in an animal model to produce a challenge virus stock for vaccine studies that would elicit more reproducible disease in the animals. if a highly virulent host-adapted virus were to become available, such as a mouse-adapted or a monkey-adapted sars-cov strain, demonstration of the capacity of vaccines to protect against challenge with these more virulent strains would provide an almost ideal animal model. different models may also need to be employed to evaluate pathogenesis versus immunogenicity. for pathogenesis studies in animal models, mortality is not required as a readout. it would be ideal to develop animal models with comparable levels of mortality to that seen in humans (∼ % overall), including the increased mortality at increased age (∼ % > age ). the optimal result would be to demonstrate efficacy of vaccines or antivirals in sars-cov animal models that mimic human morbidity and mortality and that show protection without vaccine-associated immunopathology. inbred mouse strains (balb/c, c bl/ , svev, stat −/−) support sars-cov replication and can develop pneumonitis ( s), but pneumonia and clinical symptoms are only observed in older balb/c mice [ ] . the mouse model is suitable for immunogenicity and efficacy studies of vaccines. prolonged viral replication, dissemination of virus to liver and spleen and accompanying pathology are seen in stat −/− mice; these mice, therefore, also are suitable for studies of pathogenesis and evaluation of antiviral drugs. specific pathogen-free (spf) animals must be used. their age can be either - weeks or over months and should be specified. the number of animals included must be sufficient for statistical analysis, and should include mock-infected controls. a variety of sars-cov strains has been tested in mice, including urbani, frankfurt, hku- , tor- , and the mouse-adapted sars-cov strain ma- . these were inoculated by the intranasal (in) route under light anesthesia [ , ] , using a dose of tcid in l/mouse. critical time points for specimen collections are days (peak titer) and post-infection (p.i.) for quantitative virology, days and p.i. for the study of interstitial pneumonitis and dad in aged mice (inflate lungs with % neutral-buffered formalin), and day for resolution. golden syrian hamsters are an excellent animal model because they demonstrate high levels of sars-cov replication and develop pneumonitis. hamsters are suitable for vaccine efficacy, immunoprophylaxis and treatment studies [ ] . in contrast with balb/c mice, in which the virus is detected only in the respiratory tract and is cleared by day p.i., hamsters demonstrate a longer duration of viral shedding from the upper respiratory tract, some transient viremia, spread of the virus to liver and spleen and, most significantly, inflammation of respiratory tissues [ , ] . spf animals older than weeks of age should be used in sufficient number for statistical analysis and study design should include mock-infected animals. the animals can be housed in pairs or by three if the experiment is to last less than - weeks. reserve space should be available to separate the animals in case of fights. males and littermates tend to fight less. virus strains that have been tested in hamsters are urbani, frankfurt and hku- . virus should be administered by the in route under light anesthesia, using a dose of tcid in l/hamster. as an outcome of efficacy studies, quantitative virology should be preferred over quantitative pcr. for pathology studies, one can grade pathology as none, mild, moderate or severe as per roberts et al. [ ] . critical time points for specimen collection are days or p.i. (peak viral titer) and (clearance) for quantitative virology studies; and days p.i. (consolidation and pneumonitis) and or (resolution) for pathology studies (lung). there is evidence from one study that ferrets support sars-cov replication and develop pulmonary lesions [ ] but according to another study, the animals remain asymptomatic, in the presence of sars-cov replication [ ] . in view of these conflicting data, the ferret model needs to be further studied to determine its optimal utility for vaccine efficacy and immune prophylaxis studies. additional studies are needed to define the extent of biological variability of the model and the possible role of co-pathogens that may contribute to the variability observed between different laboratories. animals aged months or older should be used. although not well documented, more consistent viral replication, pathology and clinical symptoms seem to be observed in older animals. the animals should be screened for viral co-pathogens: aleutian mink disease, respiratory viruses, hepatitis viruses, and others. the route of inoculation may be in or it, but not iv. the dose of virus (strains tor- , hku- ) sufficient to ensure reproducibility of infection in all animals is likely to be pfu or more/ferret. again, quantitative virology is preferred over qrt-pcr. for pathology studies, the same recommendations apply as for nonhuman primate studies (see below): slides should be shared between pathologists to develop a scoring system. regarding specimen collection of respiratory tissues, further studies are needed to establish how much variation occurs in samples from different lobes of the lungs. critical time points are day p.i. for quantitative virology and days - p.i. for pathology studies (pneumonitis). nhps support sars-cov replication and develop pneumonitis with a variable degree of clinical symptoms depending upon the species employed. no single nhp species is preferred at this time. the number of animals in a given study needs to be large enough to account for animal-toanimal variability: a sample of or animals is not sufficient. in view of the cost of the experiments, challenge studies should be limited to those vaccine candidates that are most promising, using larger sample sizes ( - animals/group) and avoiding animals with free-range periods in life if possible. immunological responses are best studied in species for which microarrays and reagents for identifying immune components are available, such as rhesus or cynomolgus macaques. however, the limited viral replication observed in cynomolgus macaques might be a disadvantage in selecting this species for studies. other recommended nhp species are the common marmoset and african green monkeys (agms, chlorocebus aethiops sabeus). the country of origin may play an important role and should be specified, e.g. philippines (cynomolgus macaques, macaca fascicularis); china (rhesus, macaca mulatta); brazil (marmosets, callithrix jacchus), etc. prior to the experiment, the animals should be housed indoor to limit exposure to potential co-pathogens. they should be screened for parasites (strongyloides, pneumonyssus simicola (lung mites)) and for possible viral co-pathogens (retroviruses, respiratory viruses, adenoviruses). the sars-cov strains tested in nhp models are hku- (cynos), pumc (rhesus) and urbani (marmosets and agms). these were inoculated by the respiratory route (in, it) at a dose of pfu or more/nhp. here again, quantitative virology is preferred over qrt-pcr. for pathology studies, it would be an obvious advantage that laboratories share pathology slides for review by different pathologists in order to develop a scoring system. specimens of respiratory tissues should be collected, but further studies are needed to establish how much variation occurs in samples from different lobes of the lungs, as was done and reported for african green monkeys (agms) [ ] . critical time points are days - p.i. for quantitative virology in cynomolgus macaques and agms, and later than day p.i. for rhesus macaques of chinese origin. for collection of tissues for histopathological analyses, days - p.i. are optimal for cynomolgus macaques and agms, and later than day p.i. for rhesus macaques. due to limitations of immunological reagents (including microarray assays now available), research may be limited to rhesus and cynomolgus macaques. data on other animal models are insufficient for consideration for use in sars-cov vaccine and antiviral evaluations [ ] [ ] [ ] . any additional model other than the four listed above (section . . to section . . ) would require thorough characterization including viral replication data and histopathological analysis of sars-cov-infected and mockinfected animals of the same age and gender. viral replication and histopathological data in any new animal model should be reminiscent of at least some aspect of sars in humans. although all the correlates of protection from sars associated disease have not been identified in human infections, neutralizing antibodies are present in convalescent human serum. antibodies to sars-cov spike (s) protein have been shown to prevent virus entry and neutralize virus infectivity in vitro [ , ] . prophylactically administered monoclonal antibodies and passively transferred sars-cov hyper-immune sera have been shown to prevent sars-cov infection and associated disease following sars-cov challenge of naive mice and hamsters [ , , [ ] [ ] [ ] . monoclonal antibodies administered therapeutically (i.e. post-infection) also have been shown to limit viral replication and reduce associated disease in hamsters [ ] . although cell mediated immunity may have a protective role in viral clearance or resolution of disease, work in animal models shows that antibody alone is effective for prevention and treatment of sars. thus, mice immunized with live-recombinant vaccines expressing the sars-cov spike protein, using rabies virus [ ] , vesicular stomatitis virus (vsv) [ ] , adenovirus (ad ) [ , ] or attenuated vaccinia virus mva [ , ] as a vector, as well as mice immunized with dna vaccines expressing the s gene [ , ] , developed neutralizing antibodies to sars-cov and were protected against sars-cov challenge. similar findings were reported after mucosal immunization of hamsters and agms using a bovine parainfluenza virus type (bpiv ) vector expressing the sars cov s gene [ , ] . several whole inactivated virus and recombinant protein candidate vaccines also have been developed and shown to elicit a neutralizing antibody response that provided protection against infectious challenge [ ] [ ] [ ] [ ] [ ] [ ] . in addition, passively administered sera from vaccinated animals prevented sars-cov infection upon subsequent challenge of naïve mice, demonstrating that antibodies induced by these vaccines did confer protection [ , ] . the neutralizing antibody titer that is necessary to achieve protection in humans exposed to sars-cov is, however, still not known. it was recommended that when evaluating vaccine efficacy in future animal experiments, the challenge virus should be administered at two different time-points, once when postimmunization neutralizing antibody titers are high, and later when neutralizing antibody titers have waned or are low. it also was suggested that viral titers and pathology should be evaluated at two different time points. specific times points for sample collection are given for each animal model in section above. previous observations of disease enhancement have been reported for human viral pathogens and shown to be due to antibody-mediated enhancement of virus entry (for reviews see [ , ] ). enhanced disease and mortality have been observed in kittens immunized against or infected with a type-i coronavirus, feline infectious peritonitis virus (fipv), when subsequently exposed to fipv infection [ ] [ ] [ ] . aggravated fip is apparently a result of enhanced viral entry into macrophages mediated by sub-neutralizing antibody levels [ ] . children vaccinated with inactivated respiratory syncytial virus (rsv) vaccines developed serious disease on subsequent exposure to rsv [ ] [ ] [ ] . individuals exposed to one of the four serotypes of dengue virus developed severe disease when subsequently infected with a second, different serotype [ , ] . enhanced disease following rsv vaccine or dengue infection occur by different mechanisms than fipv. in view of such examples of enhanced disease following infection in a vaccinated host, there has been heightened concern that a similar phenomenon could occur with sars-cov vaccines. it was highly recommended, therefore, that known mechanisms of disease enhancement observed with other viruses and especially with other coronaviruses should be examined in sars-cov infections, especially in vaccinated animals. although none of the studies to date have shown enhanced respiratory disease following sars-cov challenge in previously immunized animals, further studies in this area are warranted in view of some of the available in vitro data. antibodies against human sars-cov isolates were shown to enhance the entry of pseudo-typed viruses expressing the civet sars-like cov-spike protein into a human renal adenocarcinoma cell line ( -o) . enhancement was only demonstrated at the level of entry, but not of replication [ ] . this phenomenon was seen with pseudo-typed lentiviruses expressing sars-cov spike protein of civet sequence specificity, but not with pseudo-typed viruses expressing spike proteins of human sars-cov isolates. it also was not observed with human isolates of sars-cov. the role of enhanced viral entry, as observed in these in vitro studies, has not been related to any known component of human disease or infection in vivo. however, given that sars-cov may replicate, albeit poorly, in human pbmcs [ , ] , in vitro experiments looking for antibody-enhancement of sars-cov replication in human cells (e.g. macrophages and b-cells) should be performed. several groups have studied sars-cov infection in animals in the presence of neutralizing and sub-neutralizing levels of sars-cov anti-sera or anti sars-cov s-protein monoclonal antibodies, but no evidence of enhanced respiratory disease has been observed. however, foci of hepatic necrosis were noted following sars-cov challenge in mva-sars-s immunized ferrets [ ] . although these findings are worrisome, several questions were raised regarding the significance of the observation. the mva-sars-s vaccine used in these experiments was poorly immunogenic in the ferrets. the question of whether there could have been any co-pathogen in the animals was raised. it also would be important to know if the observed phenomenon depends on the mva vector or on the animal model. it was strongly urged, therefore, that the experiment be repeated in ferrets. additional experiments, in nonhuman primates and hamsters, looking for evidence of enhanced respiratory and hepatic diseases upon vaccination and challenge were also encouraged. several candidate sars vaccines that are at various stages of pre-clinical and clinical development are being developed worldwide. in china alone, three companies have been given regulatory approval for the clinical evaluation of a candidate sars vaccine. it is important, therefore, to be able to compare data from each of the candidate vaccines, which, in turn, requires international standardization of the immunological assays used for the evaluation of these vaccines. the accepted method of international standardization is to employ a who international standard (is), which allows comparison of results from different laboratories [ ] . this is essential for establishing international requirements for vaccines, diagnostics or therapeutics. an is is prepared from material bearing a close resemblance to the samples being assayed; the material is distributed in glass ampoules with high precision and reproducibility and then freeze dried. it is important that a sufficient number of ampoules ( - ) be prepared so as to provide for about years of use, and that the activity of the contents remain stable over this period. the process of establishing a who is involves an international collaborative study, in which the candidate is is compared with other samples. if the results of the tests are suitable, the candidate is assigned a provisional arbitrary unitage, a report is distributed for approval by study participants and for eventual approval by the who expert committee on biological standardization (ecbs). the preparation, storage and distribution of over % of is have been undertaken by the national institute for biological standards and control (nibsc) at south mimms (uk), which is a who laboratory for biological standards. nibsc has developed in the past several who iss for calibration of the antibody response against virus vaccines, including iss for antibodies against dengue, hepatitis a virus (hav), [ ] hepatitis b virus (hbv), measles, [ ] polio, rabies, [ ] rubella, and smallpox. a candidate standard against human papilloma virus- (hpv- ) is under evaluation. the corresponding is's were used for a variety of antibody assays including virus neutralisation (vn), haemagglutination-inhibition, single radial diffusion, enzyme-linked immunoassay and radio-immunoassay. antibody iss are most useful in epidemiological studies and in clinical trials. their use allows correlates of immunity and potency requirements of prophylactic and therapeutic products to be expressed in international units (ius). data from several collaborative studies demonstrate that use of an is generally reduces the level of variability between assay results. however, there may be problems in using iss due to the complex array of antibody populations in each serum and the different sensitivity of different assay systems. examples of potential problems can be found in hbv and parvovirus b studies [ ] , which showed that different assay kits gave different results even when the is was included in the assays. another issue is the degree of antigenic homology between the viral antigen used for the preparation of the is and the virus used in the assays. in a jev collaborative study, a candidate antibody is, which had been prepared from the sera of vaccinees immunized with an inactivated vaccine that was antigenically different from some of the viruses used in vn assays, demonstrated that the response to at least some inactivated vaccine is strain specific and the candidate is was consequently not established by the who ecbs. whether a panel of monoclonal antibodies to sars-cov could be used to prepare an is is an attractive alternative which should be explored. significant progress with standardisation of sars-cov antibody assays had been made in china with the development of a national antibody standard. in order to develop the national standard, sera were collected from convalescent sars patients who were found to have sars-cov vn antibody titers ranging from undetectable to : . one serum sample was selected for further evaluation based on crossreactivity with four sars-cov strains and on western blot analysis. this serum was freeze dried in . ml aliquots and was then assessed for stability by vn assays. the chinese standard was assigned a vn titer of . with % confidence limits of . - . . a further important development in china was the preparation of human immunoglobulin for treatment of sars patients. national guidelines have been prepared for collec-tion of plasma, quality control testing and standardisation of assays. three chinese manufacturers have been licensed for preparation of sars immunoglobulin. the source material was plasma from convalescent patients at more than days after infection. all were in good health and their plasma tested negative for blood-borne agents. plasma samples were processed by a combination of cold ethanol fractionation and ultrafiltration. in september , three lots of igg were produced and assayed by nationally-agreed procedures. the stock of immunoglobulin currently available is sufficient to treat patients. monoclonal antibodies have not yet been considered for treatment purposes in china. the importance of assessing immunogenicity of candidate sars-cov vaccines using vn assays is well acknowledged, but the variety of vn tests in use is a significant problem since there is at this time no consensus on the most sensitive, specific, and reproducible assay system. it is therefore desirable to establish an antibody is to serve as a basis of comparison in all vn assays. the most important activity at this time is to obtain a suitable source of antibody. a number of options can be considered, such as convalescent human sera, post-immunization human sera, monoclonal antibodies or hyperimmune animal sera. as an example, the availability of a suitable source of serum from convalescent patients in hong kong needs to be explored, although antibody levels in these individuals are probably quite low by now. it also would be important that other assays than vn be included in the collaborative study, and that the impact of sars-cov strain variation be examined by using different sars-cov strains and/or sera with different specificities. the centralized facility for aids reagents (cfar), which is based at nibsc, could be a suitable model for a sars-cov repository [ ] . the cfar was established in to support aids vaccine research and it is now eu-funded [ ] . there are currently reagents available including peptides, recombinant proteins, human sera/plasma, monoclonal and polyclonal antibodies, expression systems, cdna clones and viruses. a comparison can be drawn between sars-cov and hiv vn assays. currently, there are several different hiv neutralization assays formats under consideration and a lack of agreement on the most suitable assay. the cfar is supporting a joint who/eu project (neunet) to evaluate and standardize hiv vn assays in an international collaborative study. in the usa, a sars-cov repository has been established on behalf of the american type culture collection (atcc) in order to meet the needs of biodefence and the threat of emerging infections [ ] . the type of reagents stored includes viruses, peptide arrays, monoclonal antibodies and proteins. it is hoped that an active collaboration can be established between niaid and nibsc in order to meet the expanding needs of the sars research community. based on the discussion at the meeting, the following recommendations were made with respect to standardization of the immunological assays for sars vaccine evaluation: . a who repository for sars-cov reagents ought to be developed. collaboration between niaid and nibsc is recommended to achieve this goal. . consensus must be reached for the reagents to be given priority in the repository. needed. . the most suitable source of antibody for the is is convalescent human sera, but post-vaccination human sera could also be used. . a protocol for an international collaborative study aimed at validating the is should be developed and distributed to prospective participants. . collaborative study participants should be asked about their assay capabilities, e.g. number of sera, virus strains handled, etc. . . . the proposed is collaborative study should include a core set of antibody preparations to be distributed and assayed in each laboratory (e.g. monoclonal antibodies, animal sera, other human sera). . tests should be conducted using the same strain of sars-cov in each laboratory, but the different genetic lineages of sars-cov should also be represented in the study. biosafety issues associated with sars-cov vaccine development stem from the reports of laboratory-acquired infections in china. sanofi pasteur has adopted bsl practices and bsl equipment (e.g. class or microbiological safety cabinets with respiratory protective equipment) for the preparation of sars-cov vaccines. of note is the fact that sars-cov appears to be quite resistant to normal methods of virus decontamination (jf saluzzo, personal communication). who has developed guidance, both general [ ] , and specific for handling sars specimens [ ] . the rapid success in the development of immunogenic and protective vaccines against sars using a variety of platforms is encouraging, but should be tempered with concerns about the possibility of enhanced disease following exposure in vaccinated individuals [ ] . concerns mainly stem from reports of enhanced disease in fipv-immunized or -infected kittens [ , ] , from observations that antibodies elicited against certain coronaviruses mediate antibody-dependent enhancement of viral entry [ ] , and from the observation of inflammatory foci in liver tissue following sars-cov challenge in mva-sars-s vaccinated ferrets [ ] . candidate sars vaccines will need to be evaluated in more than one animal model. they also will need to be thoroughly evaluated for the duration of the antibody response they induce, as well as for the breadth of their protective effi-cacy against different strains of sars-cov. the implications of the sequence heterogeneity among sars-cov strains are difficult to test at this time because the most divergent strains (civet sars-like viruses) have not been recovered in culture. validation and international standardization of immunological assays for the evaluation of candidate sars vaccines are essential to compare data across different trials. this requires the establishment of international standards for sars-cov antibody and a repository for sars-cov reagents, with an international collaborative study to validate the iss. the establishment of the repository by who in collaboration with nibsc and niaid was recommended. severe acute respiratory syndrome who investigates china's fall in sars cases who says sars outbreak is over, but fight should go on summary of probable sars cases with onset of illness from identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage the genome sequence of the sars-associated coronavirus sars coronavirus: a new challenge for prevention and therapy isolation and characterization of viruses related to the sars coronavirus from animals in southern china the aetiology, origins, and diagnosis of severe acute respiratory syndrome structure of sars coronavirus spike receptor-binding domain complexed with receptor adaptation of sars coronavirus to humans bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study clinical features and short-term outcomes of patients with sars in the greater toronto area coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus sars immunity and vaccination prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans virology: sars virus infection of cats and ferrets severe acute respiratory syndrome coronavirus infection of golden syrian hamsters koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome effects of a sars-associated coronavirus vaccine in monkeys replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys macaque model for severe acute respiratory syndrome an animal model of sars produced by infection of macaca mulatta with sars coronavirus an exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies identification of an antigenic determinant on the s domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity evaluation of human monoclonal antibody r for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants development and characterisation of neutralising monoclonal antibody to the sars-coronavirus severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice a dna vaccine induces sars coronavirus neutralization and protective immunity in mice immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars adenoviral expression of a truncated s subunit of sars-cov spike protein results in specific humoral immune responses against sars-cov in rats identification of the membrane-active regions of the severe acute respiratory syndrome coronavirus spike membrane glycoprotein using a / -mer peptide scan: implications for the viral fusion mechanism mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice symptoms of infection caused by sars coronavirus in laboratory mice and guinea pigs civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates infection of sars-cov on juvenile and adult brandt's vole microtus brandtii potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters a single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (sars-cov) s protein results in the production of high levels of sars-cov-neutralizing antibodies long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice characterization of humoral responses in mice immunized with plasmid dnas encoding sars-cov spike gene fragments a subcutaneously injected uv-inactivated sars coronavirus vaccine elicits systemic humoral immunity in mice inactivated sars-cov vaccine prepared from whole virus induces a high level of neutralizing antibodies in balb/c mice immunogenicity of sars inactivated vaccine in balb/c mice augmentation of immune responses to sars coronavirus by a combination of dna and whole killed virus vaccines immunogenicity, safety, and protective efficacy of an inactivated sars-associated coronavirus vaccine in rhesus monkeys a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses antibody-dependent enhancement of virus infection and disease antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly localization of antigenic sites of the s glycoprotein of feline infectious peritonitis virus involved in neutralization and antibody-dependent enhancement monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus a review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (rsv) or purified f glycoprotein and challenged with rsv - months after immunization cotton rats previously immunized with a chimeric rsv fg glycoprotein develop enhanced pulmonary pathology when infected with rsv, a phenomenon not encountered following immunization with vaccinia-rsv recombinants or rsv a human respiratory syncytial virus (rsv) primate model of enhanced pulmonary pathology induced with a formalin-inactivated rsv vaccine but not a recombinant fg subunit vaccine antibody-mediated enhancement of viral disease neutralization and antibody-dependent enhancement of dengue viruses evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses sars-coronavirus replicates in mononuclear cells of peripheral blood (pbmcs) from sars patients sars-coronavirus replication in human peripheral monocytes/macrophages recommendations for the preparation, characterization and establishment of international and other biological reference standards (revised ) a immunoglobulin: an international collaborative study to establish the second international standard the st international standard for anti-measles serum calibration of a replacement preparation for the international standard for rabies immunoglobulin report of a collaborative study to calibrate the second international standard for parvovirus b antibody the importance of standardisation of laboratory evaluations in hiv vaccine trials biodefense and emerging infections research resources repository (bei resources world health organization. laboratory biosafety manual who biosafety guidelines for handling of sars specimens caution urged on sars vaccines the authors thank dr. marc p. girard and dr. marie-paule kieny for their invaluable assistance in preparing the manuscript. the contributions of anjeanette roberts and kanta subbarao were supported in part by the intramural research program of nih/niaid. key: cord- - t uxmj authors: lamphear, barry j.; jilka, joseph m.; kesl, lyle; welter, mark; howard, john a.; streatfield, stephen j. title: a corn-based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: t uxmj recombinant plant expression systems offer a means to produce large quantities of selected antigens for subunit vaccines. cereals are particularly well-suited expression vehicles since the expressed proteins can be stored at relatively high concentrations for extended periods of time without degradation and dry seed can be formulated into oral vaccines suitable for commercial applications. a subunit vaccine candidate directed against porcine transmissible gastroenteritis virus and expressed in corn seed has been developed for oral delivery to swine. here, we show that this vaccine, when administered to previously sensitized gilts, can boost neutralizing antibody levels in the animals’ serum, colostrum and milk. thus, this vaccine candidate is effective at boosting lactogenic immunity and is appropriate to pursue through large-scale field trials preceding commercialization. oral administration of vaccines has the potential to greatly cut the cost and increase the safety of vaccine delivery. in the case of human vaccines, avoiding the use of needles reduces equipment costs, removes the requirement for trained medical personnel to supervise delivery and eliminates safety concerns associated with needle disposal. the economic benefits of oral over parenteral delivery are also apparent with animal vaccines, where equipment and labor costs can be substantially reduced. also, in the cases of farmed animals destined for meat markets, carcass quality may be compromised by repeated injections and oral vaccines overcome this concern. subunit vaccines are generally considered to have a low safety risk since they are well defined and do not contain attenuated or inactivated pathogens with the potential for adverse affects. thus, oral delivery of subunit vaccines is a particularly attractive option for safe, inexpensive vaccination programs. however, oral delivery generally requires very high levels of specified antigens to be administered in order to attain efficacy. this is presumably because of degradation of the selected proteins in the digestive tract and only a small proportion of the relatively intact molecules being presented to the immune system in a manner favoring an immunogenic response. several recombinant systems are being utilized to generate large amounts of subunit vaccines, including, for example, the use of yeast to produce the surface protein of hepatitis b. however, despite the development of such recombinant vaccines, the economic production of large quantities of desired antigens is severely limited. recently, certain recombinant plant expression systems have begun to offer a means to produce very large quantities of proteins in a sufficiently concentrated form to make oral delivery feasible for a wider array of antigens. levels of expression have been achieved with various antigens in plants that allow practical oral delivery of a sufficient dose to elicit desired immune responses in humans and target animals (reviewed in [ ] ). the approaches followed to achieve these high levels of expression include the use of tissue-specific promoters to direct expression to tissues well suited to the stable storage of proteins [ ] and the targeting of the expressed proteins to sub-cellular locations conducive to their accumulation [ ] . many plant-based oral vaccine candidates have been tested in animal studies and several responses have been noted, including the generation of serum and mucosal antibodies (reviewed in [ ] ) and raised cytokine levels [ ] . protective efficacy has also been recorded with some of these vaccine candidates in model species trials (reviewed in [ ] ). a few plant-based vaccine candidates have advanced into early phase human clinical trials or target animal trials. among human vaccines, these include those directed against travelers' diarrhea and norwalk virus delivered in potato tubers [ , ] , against hepatitis b virus delivered in lettuce leaves [ ] and against rabies virus delivered in spinach leaves, themselves infected with a recombinant plant virus [ ] . immune responses were observed during these trials, and although there were some reports of nausea, presumably resulting from the administration of up to g of unprocessed, unpalatable plant material, the vaccine candidates were generally well tolerated. in the case of farmed animals, a corn-based vaccine directed against transmissible gastroenteritis virus (tgev) can induce protective immunity in piglets [ , ] . a key issue in producing plant-based oral vaccines is the selection of plant material that both expresses high levels of a chosen subunit vaccine candidate and is also suitable for extensive storage and oral delivery. the chosen plant material must also be ready for direct administration or must be suitable for inexpensive processing into an appropriate form for oral delivery to the target species. much of the work to date on plant expression systems has been conducted using tobacco leaf tissue (discussed in [ ] ). however, tobacco leaves are inedible, and therefore protein extraction is required prior to delivery. several edible options have also been pursued, including the tubers and leaves of certain vegetable crops such as potato and lettuce, respectively [ ] [ ] [ ] . some fruits, such as bananas, are also being considered. however, perishable items are not practical for extended storage and expression levels can vary considerably between, for example, potato tubers taken from a single harvest [ , ] . cereal seeds are particularly well-suited systems for the oral delivery of subunit vaccines. they have low water contents and naturally store proteins over long periods of time without degradation. corn (zea mays) is an especially attractive option because of its intensively studied genetics and the availability of established transformation procedures. several vaccine candidate antigens have been expressed at high levels in corn seed and the proteins are stable when stored in this tissue for periods of at least a year and probably for much longer, obviating the requirement for a cold chain during distribution and storage [ ] . furthermore, the antigen concentration is uniform across a corn grain harvest, facilitating even dosing [ ] . a wide range of processing alternatives have been developed by the food and feed industries to convert corn grain into readily edible forms and pilot-scale processes have been developed that ensure antigens are not degraded during processing [ ] . in the case of farmed animals, such processing is unnecessary since the livestock can consume corn grain directly. here, we focus on the development of a corn seed-based subunit vaccine directed against swine tgev. this virus causes a contagious enteric disease that is particularly severe for piglets. it results in severe diarrhea and vomiting and is associated with high mortality rates among piglets under weeks of age [ ] . tgev is a coronavirus and has a large surface glycoprotein referred to as the spike (s) protein displayed on its surface [ ] . the tgev vaccine candidate assessed here comprises the s protein expressed in corn seed. feeding studies have been conducted with this vaccine candidate delivered orally to piglets. the animals showed a priming of their immune system and were protected against infection [ , ] . here, we extend these swine feeding studies to assess the potential for this oral tgev vaccine candidate to boost immunogenic responses in gilts (young sows) previously sensitized with a commercially available modified live viral vaccine. we focus particularly on the level of antibodies in the colostrum and milk as a guide to whether immunity could be acquired passively by piglets through suckling. the subunit vaccine candidate comprised milled yellow grain corn expressing the s protein of tgev. a single dose corresponded to kg of corn containing mg of the antigen. the placebo for the study comprised kg of non-transformed milled yellow grain corn. a commercially available modified live tgev vaccine (intervet inc., millsboro, de) with a titer of . tcid (tissue culture infectious doses)/ ml was used to prime all animals and to provide booster treatments to a positive control group. this vaccine was administered according to label directions. a total of specific pathogen free gilts of suitable age for breeding were included in the study. they were taken from a low disease incidence herd and were seronegative for tgev at the outset of the study. the gilts were randomized into six treatment groups with from five to eight animals in each group (table ) . duplicate ear tags were used for identification purposes. all gilts in all groups were orally administered the modified live tgev vaccine on the day of breeding ( days before farrowing) and also days before farrowing. they were then administered the tgev modified live vaccine by intramuscular injection days before farrowing. the subsequent immunization regimen for each group is outlined in table . all animals were fasted overnight prior to oral administrations of the corn-based vaccine to test groups. standard lactation gestation rations were administered to all gilts throughout the study. during the period comprising the three administrations of modified live virus to all gilts, the groups were housed together and allowed pen-to-pen contact. prior to days before farrowing gilts were separated into their separate groups and during subsequent vaccine administrations, animals were individually isolated. blood samples were collected from gilts on the day of breeding ( days prior to farrowing), and days prior to farrowing and on the day of farrowing. blood was allowed to clot and was sedimented by centrifugation, so allowing the serum to be collected. tgev neutralizing titers were determined by incubating a specific dilution of tgev with multiple serum dilutions for h at • c. these mixtures were then inoculated onto a swine testicular cell line and the capacity of the serum to interfere with the viral infection was assessed after days. sample titers were calculated using a spearman-karber % endpoint table. colostrum samples of at least ml were collected on the day of farrowing and at least ml milk samples were collected , , and days after farrowing. the samples were sedimented by centrifugation, and the central region was collected. tgev neutralizing titers were determined as with serum samples. for all tgev neutralization data, geometric mean titers were compared and differences in excess of four-fold were considered to be significant. the generation of transgenic corn containing the s protein of tgev has been previously described [ , ] . in brief, sequence encoding the s protein was synthesized with optimal codon usage for expression in z. mays. an n-terminal cell surface targeting signal was included to direct accumulation of the protein to the cell wall. dna encoding the s protein was introduced into immature zygotic z. mays embryos by agrobacterium tumefaciens mediated transformation and selection was imposed for transgenic callus. transgenic plants with sequence encoding the s protein integrated into the nucleus were regenerated, and those expressing the highest levels of the s protein were taken through a plant-breeding scheme to increase and stabilize expression levels. this culminated in a large-scale grain harvest in which the s protein was present at mg kg − , as determined using a sandwich enzyme linked immunosorbent assay [ ] . at this concentration a practical antigen dose of - mg can be delivered in an amount of corn material easily consumed at a single feeding. all animals were seronegative for tgev at the time of breeding. subsequent serum neutralization titers are summarized for each study group in fig. . the modified live virus vaccine, which was administered twice orally and then once intramuscularly resulted in gilts in all groups having similar tgev serum neutralizing titers days prior to farrowing. analysis of serum samples taken from gilts at days prior to farrowing showed that animals that had received the oral corn-based tgev vaccine (groups a-c) had notably higher serum neutralization titers than those that had received no material at this stage (groups d and f). the difference between the test and control groups was significant in all cases except for that of a single administration of corn-based vaccine (day − to group c) over group d. animals that had received the modified live virus vaccine as a single intramuscular boost (day − to group e) responded to an almost identical level to those that had received a single oral administration of the corn-based vaccine (group c). although more oral administrations of the corn-based vaccine appeared to increase the neutralization titer, differences between the treatment groups (a-c) were not significant and none of the treatments induced a significantly stronger response than the intramuscular boost of modified live vaccine delivered to group e. similarly, at the time of farrowing the tgev serum neutralization titers in gilts administered the corn-based tgev vaccine as a boost (groups a-c and f) were raised over those observed with animals that had received the corn placebo (group d). this difference was significant in all but the case of gilts that had received six administrations of the corn-based vaccine (group b) compared to those that had received the placebo (group d). animals given intramuscular administrations of the modified live virus vaccine as a boost (group e) responded similarly to those that received the oral corn-based vaccine, again with two administrations of either vaccine giving almost identical results (groups c and e). gilts administered a boost of the corn-based tgev vaccine only during the second week before farrowing (group f) showed the most marked increase in the serum neutralization titer at the time of farrowing, although differences between the groups that received the corn-based vaccine (groups a-c and f) were generally not significant. interestingly, for groups that received two blocks of booster administrations (a-c and e), in no case did the second set of treatments elevate the serum neutralization titers over those observed with the first set. indeed, neutralization titers appeared to decline with the second set of administrations, although in no case was the drop statistically significant. colostrum and milk neutralization titers are summarized for each study group in figs. and , respectively. each of the groups of gilts that were orally administered the corn-based tgev vaccine as a booster (groups a-c and f) showed a greater level of neutralizing antibodies than did gilts administered two intramuscular injections of the tgev modified live virus vaccine as a booster (group e). however, these differences were not sufficient to be considered significant, and therefore all of the booster treatments with either the corn-based oral vaccine or with the modified live intramuscular vaccine are considered similarly effective. all of the booster regimens with the corn-based vaccine (groups a-c and f) resulted in significantly greater neutralizing antibody levels than those observed among animals that were administered the control corn placebo material (group d). gilts in all groups that received an oral corn-based tgev vaccine boost (groups a-c and f) showed similar levels of neutralizing antibodies in their milk, with levels trailing off steeply between and days after farrowing and continuing to decline thereafter. these levels correspond closely to those observed with the modified live tgev vaccine delivered intramuscularly (group e). with all groups that received a corn-based oral booster treatment (groups a-c and f) the neutralizing antibody titer in milk days after farrowing is considerably higher than for the group that received the control corn placebo (group d). this difference is significant in all cases except that of the group that received two blocks, each of seven consecutive days, of the corn-based vaccine (group a). by days after farrowing differences in the neutralization titers between the placebo (group d) and other groups are not significant. the corn-based tgev vaccine candidate described here shows great potential for expediting the administration of an efficacious vaccine to large herds of swine. the current standard regimen for administrating a vaccine comprises both priming and boosting stages. during the priming phase of the regimen the modified live tgev vaccine is often administered along with other swine vaccines. thus, at this point no reduction in labor costs is achieved through delivering an oral corn-based tgev vaccine separately. however, replacing subsequent injections of the modified live tgev vaccine with oral corn vaccine boosters would clearly save considerable time and effort. the orally administered corn-based tgev vaccine is effective in boosting the serum neutralizing titer response in animals previously sensitized to tgev using the modified live virus vaccine. when administered as a booster to gilts the corn-based vaccine also results in increased levels of neutralizing antibodies in the colostrum and early milk. milk antibodies of the igg class have typically disappeared within h of farrowing, so the neutralizing antibody activities observed in milk samples collected days after farrowing most likely reflect iga levels. protection against tgev amongst nursing piglets has been linked to iga levels [ ] , indicating that the corn-based tgev vaccine is inducing an immune response with the potential to confer protection. in this regard, a potato-based vaccine candidate directed against rotavirus, and assessed in a mouse feeding study, has been shown to confer passive immunity to pups when administered orally to dams [ ] . protective efficacy has previously been demonstrated with a corn-based oral vaccine directed against tgev and administered to piglets [ , ] . the neutralizing antibody levels achieved here in the colostrum and early milk of gilts extends the scope for how this vaccine candidate can be deployed. future studies with this tgev oral vaccine candidate will focus on optimizing the administration reg-imen for maximum responses and on conducting larger scale trials. these will include an assessment of whether the lactogenic immunity observed here results in protection being conferred to piglets. the results presented here successfully demonstrate a commercial application for a corn-based vaccine and indicate that there is great promise for plant-based vaccines that can be easily administered to large farmed animals by oral delivery. plant-based vaccines expression of a synthetic e. coli heat-labile enterotoxin b sub-unit (lt-b) in maize corn as a production system for human and animal vaccines a plant-based multicomponent vaccine protects mice from enteric diseases immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes a plant-derived edible vaccine against hepatitis b virus expression in plants and immunogenicity of plant virus-based experimental rabies vaccine plant-based vaccines: unique advantages delivery of subunit vaccines in maize seed medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants congress symposium on molecular farming: development of an edible subunit vaccine in corn against enterotoxigenic strains of escherichia coli molecular biology of transmissible gastroenteritis virus immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine key: cord- -uup bhm authors: murphy, frederick a.; osburn, bennie i. title: adventitious agents and smallpox vaccine in strategic national stockpile date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: uup bhm in keeping with current standards, we urge that old smallpox vaccines that were made in animal skin and are still a key part of our strategic national stockpile be tested for adventitious infectious agents. the advisory especially applies to viruses that have the potential for zoonotic transmission to human vaccine recipients. testing of new smallpox vaccine stocks produced for biodefense purposes, we were surprised that the largest part of our national vaccine stockpile, the wyeth vaccine dryvax produced in - and the connaught (now aventis-pasteur) vaccine wetvax produced in the s, has never been scrutinized by modern methods. of particular concern is the fact that these stocks have never been subjected to testing for adventitious agents, whereas a new vaccine intended to supplement the existing stockpile has been thoroughly tested ( ) . testing of these old stocks met the standards of the day. however, if these old vaccines are to be considered valid parts of our national stockpile we should expect not only continuing testing of potency and sterility but also testing for adventitious agents with methods that reflect the standards of today. this is not to say that the finding of adventitious agents must result in removal of these vaccine stocks. that issue must be a matter for formal risk analysis and consideration by the same experts who review data on new vaccines (e.g., the advisory committee on immunization practices of the centers for disease control and prevention) and by officials of the food and drug administration (fda). however, as greater concern emerges about the potential pathogenicity of infectious agents that might be present in old vaccine stocks, prudence dictates caution and testing. such concerns are amplified by the memory of how these old smallpox vaccines were made. such vaccines were made in the skin of calves and sheep, and seeds and stocks were passaged in tissues of calves, sheep, and rabbits (especially used for seed lot production). equally important is the fact that for many decades preceding the development of standardized manufacturing methods in the th century, the vaccine virus (vaccinia virus) was propagated by serial passages in animals without precise knowledge of the passage history and without use of a seed lot system that stabilized passage level. this uncontrolled system could have allowed amplification of any passenger viruses and could have increased the possibility of untoward changes in their genetic makeup. in addition, since the crude manufacturing methods (figure) allowed direct contact of the materials harvested from animals with human operators, possibilities existed for contamination of the resulting product with pathogenic human viruses. it was not uncommon practice before the widespread acceptance of vaccine manufacture in animals (at the end of the th century) to passage vaccinia virus arm-to-arm between humans ( ). standardized methods for manufacture in animal skin were not initiated until . such concerns are further amplified by infectious agents that are targeted by modern vaccine testing protocols. when materials of animal skin origin are used, such agents are brucella spp., mycobacterium spp., bacillus anthracis, clostridium spp., and other bacteria and fungi. old stocks of vaccine were tested for microbes, and the presence of specific pathogenic species was the basis for rejection of vaccine lots. the specifications for approval of dermal vaccines produced in calves and sheep allowed for the presence of a low concentration of nonpathogenic bacteria and fungi. however, the dermal vaccines produced from the s to the s and currently in the national stockpile were not tested for mycoplasma or viruses. in the case of vaccines manufactured in calves, the agents of concern include several bovine viruses ( . we were unable to find a comprehensive list of possible adventitious agents when ovine materials are used, as is the case for the lister strain smallpox vaccine produced in europe and old vaccine stocks held by some european countries for biologic defense. sheep harbor several members of the same virus groups found in cattle, but they also carry other viruses. rabbits were sometimes used for intermediate passaging of vaccinia virus stocks and for seed virus production, particularly in europe. possible rabbit viruses that could contaminate vaccinia stocks include endogenous retroviruses, papillomavirus, herpesviruses, and leporipoxviruses. because of research and development of specific pathogen-free swine as special organ and tissue donors for human xenotransplantation over the past decade, the list of possible adventitious agents in materials derived from swine is quite comprehensive ( ) . when materials of human origin are used, adventitious agents include hiv- and hiv- ; human t cell lymphotropic virus type i (htlv-i) and htlv-ii; hepatitis a, b, and c viruses; human cytomegalovirus; epstein barr virus; human herpesviruses , , and ; human parvovirus b ; reoviruses; polyoma (jc/bk) viruses; v virus; human coronaviruses; human papillomaviruses; influenza a, b, and c viruses; various human enteroviruses; human parainfluenza viruses; and human respiratory syncytial virus. as mentioned, there is a risk that a human virus could have been introduced into smallpox vaccine seed or vaccine stocks during manufacturing, since barrier methods such as those currently used in all phases of vaccine production were not in place. although the ability of such human viruses to be propagated in subsequent vaccine lots is uncertain, many human viruses are capable of replicating in animal cells. when materials from any animal source are used, special consideration is given to exogenous and endogenous retroviruses (e.g., bovine immunodeficiency virus), lymphocytic choriomeningitis virus, adenoassociated viruses, minute virus of mice, and other viruses that are notorious contaminants. however, these special considerations fail to include many infectious agents that should raise concern. however, for old smallpox vaccine stocks, it is enough to question whether any of the infectious agents specifically cited in fda and european commission regulations, recommendations, and guidelines are present. current regulations, recommendations, and guidelines on testing for adventitious microbial and viral agents from various national and international agencies require nonspecific screening and relevant specific tests. regulations requiring tests for mycoplasma and viruses came into effect long after old stocks of smallpox vaccine were manufactured. nonspecific screening tests include classic culture tests for bacteria and fungi (sterility tests), special culture and animal tests for mycobacterium spp., cell culture tests for the presence of certain cytocidal viruses (by inoculation of and blind passage in vero, mrc- , hela, rk, and a cells with observations for cytopathology and tests for hemadsorption and hemagglutination at the end of the culture period), and animal inoculation tests for certain viruses (suckling and adult mice, guinea pigs, and embryonated hen eggs). electron microscopy is often used to find adventitious agents in cell culture banks. in the united states, federal regulations specify that products of bovine origin (such as virus preparations, cell lysates, cultured cells, or other reagents) intended for use in the production of human biologics be tested for the presence of specific bovine viruses in accordance with c.f.r. . . specific tests for adventitious infectious agents are conducted using various polymerase chain reaction (pcr)-and immunochemical-based assays. the extraordinary sensitivity of these assays has served to raise the bar of expectation of test veracity, while improving practicality and containing costs. in many cases, these assays have been validated, that is, proof-tested using salted vaccine and vaccine substrate materials. since companies exist that conduct these specific tests for vaccine developers and manufacturers, such testing on old smallpox vaccine stocks is eminently feasible. the new cell culture-based smallpox vaccine has been tested by using these methods ( ) . however, considering more advanced pcr-based tests for unknown or unrecognized adventitious agents (e.g., representational difference analysis, use of various degenerate primers) would require extensive research and add substantially to overall costs. for the purpose at hand, we are suggesting only that the battery of specific tests now used on all modern vaccine materials be used. concerns about the possible presence of adventitious agents in old smallpox vaccine stocks are amplified further by current concerns about prions and the zoonotic potential of prion diseases. old smallpox vaccine stocks might not be contaminated by bovine spongiform encephalopathy (bse) prions, but lister vaccine stocks that were produced in sheep and vaccine seeds that had been passaged in sheep could be contaminated by scrapie prions. regulations and guidelines for modern vaccines state that all materials used must come from bse-free regions but say nothing about scrapie-free regional status. testing of old vaccines for prions is beyond the sensitivity of any present in vitro prion test, but this issue should be considered ( ) . since no problems related to contamination have been recognized during the long history of smallpox vaccines, or during the intensified program to eradicate smallpox, one might argue that little risk for humans is posed from adventitious agents in old stocks of vaccine. however, it is unlikely that low-incidence untoward events temporally related to adventitious agents have been recognized. it is equally unlikely that diseases that appeared at long intervals after smallpox vaccination would have been associated with the vaccine. furthermore, since most smallpox vaccine was used in children, we may have less data on its use in adults than we would want. of note is the recent observation that myopericarditis is a relatively common serious adverse event following smallpox vaccination, but that this complication was not recognized during the era of routine vaccination ( ) . today, the senior guiding document for manufacturers of first-generation smallpox vaccines, i.e., vaccines produced in animal skin, is the world health organization (who) recommendations for production and control of smallpox vaccine, revised ( ) . a definitive version of this document will be published in the who technical report series (the working version is available from http://www.who.intlbiologicals/smallpox_final.pdf). fda documents on this subject are much more general and say little or nothing about adventitious agents ( ) . the who document represents continuation of a series that started in . several points from the who document ( ) are of particular interest here (the chosen points are not meant to be comprehensive or reflect the overall sense of this document). first, adventitious agent testing for viruses in vaccine virus seeds and product intermediates is complicated and might give ambiguous results. therefore, newer, more specific tests are planned for the future. second, testing for viral adventitious agents of animal skin vaccine should take into consideration the source country of the animals. guidelines for transmissible spongiform encephalopathy testing should be followed. third, the concentration of nonpathogenic bacteria and fungi in vaccines produced in animal skin may be very difficult to validate, and consistent sterility of the finished product may be difficult to achieve. the use of a nonsterile final product may be justified since smallpox vaccine is administered by scarification rather than by intramuscular or intravenous inoculation, and because its use over many years did not cause problems. fourth, the general method for testing a live viral vaccine strain for contaminating viruses is to neutralize the vaccine virus and test for adventitious viruses both in vitro and in vivo. however, it is recognized that vaccinia virus is very difficult to neutralize to the extent required for such studies. additional testing such as nucleic acid amplification techniques for specified viruses and reverse transcriptase assays for retroviruses should complement nonspecific tests. fifth, in preparing master seed lots, procedures should be used that help remove extraneous agents. since removal or inactivation of microbial contaminants is unlikely at any downstream level, the presence of extraneous agents in seed lots during the production process must be avoided. sixth, the absence of specific human pathogens should be confirmed by additional testing, e.g., bacterial and fungal cultures, virus culture, pcr testing for viral agents. taken together, these points from the who document make it clear that members of the who expert committee on biological standardization had difficulty dealing with the exceptional problems posed by the firstgeneration smallpox vaccines, i.e., vaccines produced in animal skin. their difficulty in producing guidelines also pertained to old lots of such vaccines, such as those that are part of the us and european strategic stockpiles. this seemed most obvious regarding testing for adventitious agents. the limits of such testing seem clear, but so are the practicalities. standard testing for adventitious agents is practicable and would provide important evidence for riskbenefit considerations if or when old vaccines are used in an emergency situation. time will eventually obviate such considerations as modern smallpox vaccines replace old vaccines in national stockpiles, but for the present we see the who document as another basis for suggesting comprehensive testing of old vaccines. since old smallpox vaccine stocks have been in the public domain for many years, we would expect that comprehensive testing would be funded by the same public agency (the us department of health and human services) that intends to distribute the vaccines should the need arise. we believe that the testing should be fully transparent, that is, fully open to public scrutiny. acam clonal vero cell culture vaccinia virus (new york city board of health strain) -a second-generation smallpox vaccine for biological defense development of smallpox vaccine in england in the eighteenth and nineteenth centuries the public health risk of animal organ and tissue transplantation into humans myopericarditis following smallpox vaccination world health organization. recommendations for production and control of smallpox vaccine regulatory requirements for historical and new smallpox vaccines. g + workshop as of january , summaries of emerging infectious disease conferences will be published online only.summaries submitted for online publication may include illustrations and relevant links. for more information on conference summary requirements, please refer to author guidelines at http://www.cdc.gov/ncidod/eid/instruct.htm.submit conference summaries at http://www.eid.manuscriptcentral.com key: cord- -hqgwj vs authors: fehr, daniela; holznagel, edgar; bolla, stefania; hauser, beat; herrewegh, arnold a.p.m.; horzinek, marian c.; lutz, hans title: placebo-controlled evaluation of a modified life virus vaccine against feline infectious peritonitis: safety and efficacy under field conditions date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: hqgwj vs abstract a modified live virus vaccine against feline infectious peritonitis (fip) was evaluated in a double blind, placebo-controlled field trial in two high-risk populations. the vaccine was found to be safe and efficacious in one population of cats that had low antibody titre against feline coronavirus (fcov) at the time of vaccination. although clinically healthy at the time of vaccination, retrospectively some vaccinees that later came down with fip were found to be rt-pcr positive for fcov in plasma and showed changes in blood parameters consistent with early stage of fip. it is concluded that vaccination can protect cats with no or low fcov antibody titres and that in some cats vaccine failure was probably due to pre-existing infection. feline infectious peritonitis (fip) is a normally fatal disease of cats caused by infections with feline coronaviruses (fcov) which are antigenically related to a respiratory coronavirus strain of man (hcv e), transmissible gastro-enteritis virus (tgev) of swine and canine coronaviruses '. in switzerland, infections with fcov in domestic cats are widespread. about % of the cattery cats and % of all cats with access to outdoors were found to be seropositive . five to % of these develop lethal fwt. certain cat populations seem to be more susceptible to fip. young cats are especially prone: % of all fip cases affected cats younger than months of age and % cats younger than year?. a genetic disposition in certain breeds and in cheetahs was described -', and cats living in multiple-cathouseholds such as catteries or cat shelters and cats with access to outdoors are more likely to get exposed to fcov and develop fip than animals from single-cathouseholds'. clinical signs include the effusive or the non-effusive, granulomatous form of fip; both can also appear together. characteristic laboratory findings in fip ; accepted december ) are anaemia, neutrophilia, lymphopenia, increase of total serum protein, hyperglobulinemia and decreased albumin . in , a low virulent fcov type, called feline enteric coronavirus (fecv), which caused only mild gastrointestinal and respiratory diseases mainly in kittens, was described'. antibodies to these fecv and the virulent fip-causing viruses (feline infectious peritonitis virus: fipv) do crossreact. these authors formulated the hypothesis that most of these seropositive cats are actually infected with fecv and that fipv is just a mutant of fecv which has the ability to infect macrophages. at this time, no molecular or immunological differences are known between fecv and fipv which can explain the difference of virulence between these coronaviruses". therefore, it appears to be justified to generally designate them as fcov and to consider every fcov infection in cats as a potential risk ,". several observations point out the important role of the cell mediated immunity (cmi) in fip patho-genesis - , but the detailed immune mechanisms for controlling fcov infection remain unknown. under experimental conditions humoral immunity does not lead to protection. on the contrary, after experimental fip infection seropositive cats develop fip after a much shorter incubation period than seronegative control cats '- . this antibody dependent enhancement (ade) is thought to occur when virus-antibody complexes are formed and bound to the fc receptors of macrophages. macrophages are then more efficiently infected by the fc receptor-mediated endocytosis than by the virus alone + . when we initiated this study, this modified live virus vaccine (primucell fip@) was already commercially available in the usa, but many questions concerning safety and efficacy under field conditions were still unanswered. the safety of the vaccine was confirmed under experimental and field conditions , but vaccinated cats showed ade when challenged with a high dose of heterologous virus strain . the efficacy of the vaccine was assessed only under experimental conditions. with this trial the safety and efficacy of the vaccine was evaluate under field conditions in two high risk populations. a preliminary report has been presented at the fecv/fipv-workshop in davis, ca in and published in the proceedings". the study was performed as a placebo-controlled double blind assay. neither the investigators, nor the cat owners, knew which of two colour coded vials contained the vaccine. the code was not opened to the investigators, veterinarians and cat owners, until all cats terminated the month observation period. two populations with a high risk for fcov infection and fip were included in this trial. the first population consisted of cats from catteries with fip problems. in all of these catteries, fip cases had occurred in the last months prior the beginning of this trial either in the cattery itself or in kittens which had been re-homed to new owners. we expected that some of these cats had been already exposed to fcov. the second population consisted of cats < months of age, which were vaccinated by veterinarians in switzerland. as already mentioned, this ge group is more susceptible to fip than older cat?,-. the cats of each population were further subdivided into two groups, vaccine and placebo, respectively, which were comparable regarding age, sex, breed and living conditions. only clinically healthy cats older than weeks of age were vaccinated and pregnant queens were excluded from the study. in week and - weeks later the cats were vaccinated intranasally with either the coded vaccine or the placebo. after the vaccination the two coded groups were kept separately for h to prevent spread of the vaccine virus to cats of the placebo group. in both populations a blood sample was collected before vaccination (week ) and tested for felv and fcov-antibodies. in cattery cats only, haematology and clinical chemistry were done in week , and and in cats each of the vaccinated and of the placebo group, cd +/cd +-t-cells were measured in week , and . fiv-tests were carried out in week in the cattery cats. of sick cats, a blood sample was collected and haematology, clinical chemistry, felv and fcov-antibodies were determined. the modified live virus vaccine was developed by gerber et a .' briefly, fipv-df was attenuated in cell culture passages on the norden laboratories feline kidney (nlfk) cell line. passages - were propagated at °c. the th passage was exposed to ultraviolet irradiation. the vaccine has been shown to induce iga antibodies in the mucosa and to stimulate the cell mediated immune response '. the serial of the vaccine used in this study was a commercial batch (serial number ) with a titre of .' tcid,,. the placebo consisted of supernatant of non-infected nlfk cell culture. the vaccine and placebo were provided by the manufacturer in identical vials coded with coloured labels. the code was not broken to the veterinarians and the cat owners until all cats had finished the months observation period. the characteristics of the cattery cats and young pet cats are summarized in table . animals of the placebo and the vaccine groups in both, the cattery cats and the young pet cats, did not differ significantly with respect to age, sex, breed and living conditions. antibody titres to fcov were measured by indirect immunofluorescence using pd- cells of swine origin infected with tgev as antigen. plasma dilutions of : , : , : and : were tested. plasma samples of all cats were examined for circulating feline leukemiavirus (felv) p antigen and plasma samples of the cattery cats were also examined for antibodies to feline immunodeficiency virus (fiv) by indirect immunofluorescence using fiv-infected fl- cells as antigen . o samples with positive fluorescence results were subjected to western blotting for confirmation '. in cattery cats cd +/cd +-t-cells were measured by flow cytometry as described *. of all cats dying of fip, ~ of plasma samples taken at the time of first vaccination were retrospectively examined for presence of fcov-rna by polymerase chain reaction (pcr) . the mean of the haematological and clinical chemistry parameters between the vaccine and placebo group were analysed for significant differences by the mann-whitney u test, changes of laboratory values obtained from different cats over time were examined by the wilcoxon test. frequencies of fcov antibody titres in the placebo and vaccine group were compared using the x test. to determine differences in the frequencies of fip in the vaccine and placebo group, the exact test of fisher was performed . the results are presented separately for the cattery cats and the population of the young pet cats. the side-effects reported after the vaccination in the cattery cats are summarized in table . during the - months of observation, cats of the vaccine group and of the placebo group died due to various causes. five cats of the vaccine group and six cats of the placebo group died due to non fip-related causes. all cases, except one cat of the vaccine group which died months after the vaccination with liver problems and two cats of the placebo group which died and months after vaccination due to an accident and joint problems in a -year-old cat, respectively, were submitted to necropsy and fip was excluded. fip cases occurred in six catteries. the characteristics of all cattery cats which died of fip are summarized in table . some of these cats, though clinically healthy, showed changes in blood parameters at the time of vaccination. to our knowledge, the safety of the vaccine in breeding cats has not been investigated so far neither under experimental nor under field conditions. therefore, all data collected from queens which had kittens after the vaccination are summarized in table . no differences were found between the parameters evaluated. with respect to the laboratory parameters no differences were found between those in the vaccine group and the placebo group at the different time points (haematology, clinical chemistry, cd +/cd +lymphocytes). however, in both the vaccine and placebo groups, changes in some of the laboratory parameters were observed at the different time points. both groups showed a decrease in albumin in weeks and compared to week and an increase in plasmaprotein in week compared to weeks and (pco. ) (data not shown). at the beginning of this trial, all cattery cats had tested negative for felv and fiv-antibodies, but . % and . % of the cats in the vaccine and placebo group showed fcov antibody titres of or higher. the frequency of the fcov titres in cats of the vaccine and placebo group at different time points after vaccination (weeks , and ) is shown in figure . there was no statistically significant difference in the distribution of the fcov antibody titre in the vaccine and. placebo group at the different time point, but the vaccine group as well as the placebo group showed a transient increase of titres in week compared to week (pco. ) followed by a decrease in week compared to week (pco. ). retrospectively, plasma samples collected from cats at the time of first vaccination, which were stored frozen, were submitted for rt-pcr for fcov (table ) . of plasma samples tested, three were positive. the side-effects reported in the population of the young pet cats are summarized in table . the observation period in this population was between and months. the health condition of the cats at the end of the observation period is summarized in table . thirteen cats of the vaccine group and cats of the placebo group died from fip. all, except one in each group, were confirmed by necropsy (table ) . two cats in the vaccine group were already ill at the time of (table ). of samples tested, were found positive. in one cat shelter with high fip incidence, cats were vaccinated (placebo cats, vaccine cats), of which cats developed fip (placebo , vaccine ). the frequency and distribution of antibody titres to fcov at the time of first vaccination is presented in figure . more than % of these clinically healthy young cats had already been exposed to fcov in the first year of life. the distribution was identical in the vaccine and placebo group. domestic shorthair cats showed statistically significantly lower fcov antibody titres than pure-bred cats of the same age (p<o.ool) (figure ) . cats of the placebo group, which showed a titre of or higher, had a significantly higher risk for developing fip in the next months ( . %) than cats with a titre of or lower ( . %) (pzo. ) (figure ) . though seven cats of the vaccine group and six of the placebo group were felv positive at the time of first vaccination, three more cats of each group tested positive during the observation period. one of these felvpositive cats of the vaccine group and three of the placebo group died due to fip which was confirmed by necropsy. the aim of this study was to evaluate the efficacy and safety of a modified live virus vaccine in a double-blind study under field conditions in two cat populations with higher risk for fip. not all cats infected with fcov develop lethal fip and the incidence of fip in a cat population can hardly be predicted'. in this study, the placebo and vaccine group were indistinguishable regarding distribution of age, sex and living conditions and no statistically significant difference in laboratory parameters (haematology, clinical chemistry, serology, cd +/cd +-lymphocyte subsets) were observed at the time of first vaccination. therefore, cats in the vaccine and placebo group had about the same probability to become infected with fcov and to develop fip, and differences in the frequency of fip cases or changes in laboratory parameters must be attributed to the vaccine. the vaccinated cats did not show an accelerated form of fip (tables and , figure ). one cattery cat aged . months was euthanized days after vaccination, but this cat, though clinically inconspicuous, showed growth retardation compared to litter mates, anaemia (pcv: %), increase of total serum protein and viremia with fcov at the time of first vaccination. two young pet cats were euthanized and days after vaccination. cat , which died days after vaccination had shown chronic nasal discharge and anorexia and had been treated with antibiotics and corticosteroids the last weeks before vaccination. the fcov antibody titre was high ( ) at the time of vaccination and in retrospect this cats was positive for fcov in the rt-pcr. cat , which died days after vaccination had also been anorectic for some time and had been treated by the veterinarian. this cat too had a high titre at the time of vaccination ( ) and was pcr positive. these two cats were vaccinated by mistake and should not have entered the study in the first place. they remained in the study for the sake of completeness and because our study represent the real situation in the field where cats with obvious clinical signs may be vaccinated by mistake. it can be argued that in these three cats vaccine induced acceleration of fip may have happened. however, if the incidence of fip cases during the first days after vaccination were compared in vaccinated vs placebo cats, there is absolutely no difference in that cats in the vaccine group and cats in the placebo group died during this time. in the population of the cattery cats where prevalence of fcov antibodies was especially high ( %) the average time of death after vaccination was . weeks in the vaccine group and weeks in the placebo group. from this, the lack of side-effects and effect on fertility it was concluded that the vaccine is safe. as expected, most of the cattery cats showed antibodies to fcov, indicating prior infection. not only the vaccine group, but also the placebo group showed an increase of titres in week compared to week and a decrease in week compared to week (figure ). increase of antibodies could be explained by vaccination in the vaccine group, but not in the placebo group. as animals of the vaccine and placebo groups were separated for h after the vaccination, spreading of the vaccine virus to cats of the placebo group seems unlikely. during the weeks between vaccination and the collection of a second blood sample, antibody titres increased in cats of catteries, where fip cases occurred. a similar rise of antibody titres was observed in cattery cats where no fip cases were seen during this time period. it is well known, that stress can influence the immune system. therefore, we speculate that the stress caused by the handling during vaccination and the collection of blood samples led to a weakening of the cm and to a reactivation of a persistent fcov infection and an increase of the antibody titres. surprisingly % of the clinically healthy pet cats younger than months showed antibodies to fcov (figure ) . pure-bred cats showed higher titres and were more frequently seropositive for fcov than domestic cats (figure ) , which is probably due to different living conditions. pure-bred cats are normally raised in multiple-cat-households with close contact of the cats to each other, whereas domestic cats are often kept in single-cat-households or in small groups. when vaccination was performed according to the recommendations of the manufacturer, the vaccine showed no overall efficacy in these cattery cats. in view of the fact that in all catteries fip cases had occurred during the last months prior this study, it has to be concluded that some of the cattery cats, which later died of fip during the observation period, had already been vaccine volume number field trial of an fip vaccine: d. fehr et al. infected with virulent fcov and were vaccinated during the incubation period. this conclusion is also supported by the facts that all cats showed antibodies to fcov, that some cats showed changes in blood parameters consistent with fip (anaemia, low serum albumin, high serum protein, high bilirubin), and that retrospectively some cats were rt-pcr positive for fcov at the time of first vaccination (table ) . vaccination itself did not inauence laboratory parameters such as haematology, clinical chemistry or cd +/cd +-lymphocyte subsets (data not shown). in the population of the young pet cats no reduction of fip cases in the vaccinated cats was observed in the first days after infection ( figure ). however, after this time point until the end of the observation period, the vaccinated cats showed significantly less fip cases in one cat shelter the incidence of fip was % in the vaccine group (six out of cats) and % in the placebo group (nine out of cats). it can be concluded that the exposure to fcov was extremely high for these cats. as these animals were vaccinated after they had already been housed in the shelter for some time, it can be concluded that most of the vaccine failures in this population were due to previous infection with fcov. if cats with high fcov antibody titres at the time of first vaccination ( or higher) were excluded and only cats with titres of or lower at the time of first vaccination were examined in our study, vaccination significantly decreased the incidence of fip (p=o.o ) ( figure ). this observation suggests that low antibody titres are related with some degree of immunity, probably through a strong cell-mediated immune response via the thl pathway , and presumably with a lower virus load compared to cats with titres of or higher. this interpretation would also be in agreement with the finding that high titres (in the placebo group) are associated with a higher risk for the development of fip (figure ) . it can be concluded that vaccination of cats, which were already infected with fcov, cannot prevent or alter the course of the disease. however, vaccination of cats with no or low antibody titres seems to be efficient. these findings confirm the results of a placebocontrolled trial in which seronegative animals were vaccinated, before they were entered into a cat shelter with fip problems . to increase the efficacy of the vaccine changes in husbandry and management conditions may be initiated that lead to a decrease in coronaviral load in the kittens ,". under experimental conditions, vaccination of fcov naive cats was also found to reduce the incidence and the severity of infections with the low virulent viruses called fecv . since every fcov infection has to be considered a potential risk for fip ,", reducing these infections by vaccination may also help to reduce the fip incidence. cats of the placebo group with a titre of showed a significantly higher risk for developing fip in the following months ( . %), than cats with lower titres ( . %) (pzo. , figure ). this finding and the observation that many of these cats were pcr positive at the time of first vaccination suggest that they were already infected with fcov and were vaccinated during the incubation period. this would explain why vaccination showed a low efficacy in the young pet cats. however, it is difficult to apply the classic term of an incubation period to fip. according to pedersen and co-workers more virulent mutants of fcov capable of infecting macrophages can emerge spontaneously any time during an fcov infection . as mutations in viruses occur randomly, it can be concluded that a high virus load increases the probability that such mutants arise. a functioning immune system can control the virus load at a low level. therefore, all factors which suppress the immune response, may increase the virus load and the genesis of virulent mutants. thus, a cat can develop fip many months or even years after an fcov infection. in conclusion, the use of a live modified vaccine against fip was found to be safe in high risk populations under field conditions. efficacy was clearly shown in cats with low fcov antibody titre, but overall was rather low in high risk populations. to optimize its efficacy, changes in management and husbandry should help to prevent fcov exposure prior to vaccination. our laboratory for their help as well as to the cat breeders, veterinarians and cat owners that participated in this trial. cornell vet. , , scott, f.w., corapi, w.v. and olsen, c.w. evaluation of the safety and efficacy of primucell-fip vaccine. feline hlth top. , , fehr, d., holznagel, e. and bolla, s. et a/. evaluation of the safety and efficacy of a modified live fipv vaccine under field conditions. feline pratt. , , pedersen, n.c. feline infectious peritonitis and feline enteric coronavirus infections. part i, feline enteric coronavirus. feline pratt. , , the biology and pathogenesis of coronaviruses antigenic homology among coronaviruses related to transmissible gastroenteritis virus das feline immunschwlchevirus in der schweiz: klinik und epidemiologie im vergleich mit dem leukbmie-und dem coronavirus a study of naturally occurring feline coronavirus infections in kittens die diagnostik dir felinen infektioesen peritonitis (fip): retrospektive und prospektive untersuchungen feline coronavirus infections of cheetahs field safety studies of an intranasal fipv vaccine serologic studies of naturally occurring feline infectious peritonitis an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis a comparison of the genomes of fecvs and flpvs and what they tell us about the relationships between feline coronaviruses and their evolution risk of feline infectious peritonitis in cats naturally infected with feline coronavirus role of thymus-dependent lymphocytes and antibodies in feline infectious peritonitis after oral infection role of circulation antibodies and thymus-dependent lymphocytes in production of effusive type of feline infectious peritonitis after oral infection virologic and immunologic aspects of feline infectious peritonitis virus infection delayed-type hypersensitivity skin response associated with feline infectious peritonitis in two cats evaluation of immunity to feline infectious peritonitis in cats with cutaneous viral-induced delayed hypersensitivity immunologic phenomena in the effusive form of feline infectious peritonitis early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization a study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection in feline macrophages by monoclonal antibodies characterization of an attenuated temperature sensitive feline infectious peritonitis vaccine virus protection against feline infectious peritonitis by intranasal inoculation of a temperature-sensitive fipv vaccine monoclonal antibodies to three epitopic regions of feline leukemia virus p and their use in enzyme-linked immunosorbent assay of ~ felines t-lymphotropes lentivirus (ftlv): experimentelle lnfektion und vorkommen in einigen laendern europas. kleintierpraxis specificity assessment of feline t-lymphotropic lentivirus serology detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr two types of murine helper t cell clones. i. definition according to profiles of lymphokine activities and secreted proteins thl and th cells: different patterns of lymphokine secretion lead to different functional properties vaccination against naturally occurring fip in a single large cat shelter the potential use of a modified live fipv vaccine to prevent experimental fecv infection experimental studies with three new strains of feline infectious peritonitis virus: fipv-ucd , fipv-ucd , and fipv-ucd the study was supported by a grant from pfizer animal health inc. the authors thank drs alasdair wiseman and serge martinod from pfizer animal health inc. for their assistance. we are indebted to the technicians of key: cord- - dfaqsv authors: moore, anne c.; dora, emery g.; peinovich, nadine; tucker, kiersten p.; lin, karen; cortese, mario; tucker, sean n. title: pre-clinical studies of a recombinant adenoviral mucosal vaccine to prevent sars-cov- infection date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: dfaqsv there is an urgent need to develop efficacious vaccines against sars-cov- that also address the issues of deployment, equitable access, and vaccine acceptance. ideally, the vaccine would prevent virus infection and transmission as well as preventing covid- disease. we previously developed an oral adenovirus-based vaccine technology that induces both mucosal and systemic immunity in humans. here we investigate the immunogenicity of a range of candidate adenovirusbased vaccines, expressing full or partial sequences of the spike and nucleocapsid proteins, in mice. we demonstrate that, compared to expression of the s domain or a stabilized spike antigen, the full length, wild-type spike antigen induces significantly higher neutralizing antibodies in the periphery and in the lungs, when the vaccine is administered mucosally. antigen-specific cd + and cd + t cells were induced by this leading vaccine candidate at low and high doses. this fulllength spike antigen plus nucleocapsid adenovirus construct has been prioritized for further clinical development. the emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus (sars-cov- ), the causative agent of covid- disease, in , has led to a global pandemic and significant morbidity, mortality and socio-economic disruption not seen in a century. coronavirus disease (covid- ) is a respiratory illness of variably severity; ranging from asymptomatic infection to mild infection, with fever and cough to severe pneumonia and acute respiratory distress . current reports suggest that asymptomatic spread is substantial , and sars-cov- infection induces a transient antibody response in most individuals . therefore, development of successful interventions is an immediate requirement to protect the global population against infection and transmission of this virus and its associated clinical and societal consequences. mass immunization with efficacious vaccines has been highly successful to prevent the spread of many other infectious diseases and can also prevent disease in the vulnerable through the induction of herd immunity. significant effort and resources are being invested in urgently identifying efficacious sars-cov- vaccines. a number of different vaccine platforms have demonstrated pre-clinical immunogenicity and efficacy against pneumonia , . several vaccines have demonstrated phase i or phase ii safety and immunogenicity [ ] [ ] [ ] . however, at this time, no vaccine has demonstrated efficacy in the field. the most advanced sars-cov- vaccine candidates are all given by the intramuscular (im) route, with some requiring - °c storage. this is a major barrier for vaccine dissemination and deployment during a pandemic in which people are asked to practice social distancing and avoid congregation. the ultimate goal of any vaccine campaign is to protect against disease by providing enough herd immunity to inhibit viral spread, not to make a set number of doses of vaccine. an injected solution takes a long period of time to administer and distribute and requires costly logistics, which means dose availability does not immediately translate to immunity. further, systemic immunization can induce immunity in the periphery and lower respiratory tract. however, these vaccines cannot induce mucosal immunity in the upper respiratory tract. mucosal iga (with the polymeric structure and addition of the secretory component), creates more potent viral neutralization , can block viral transmission , , and in general, is more likely to create sterilizing immunity given that this is the first line of defense for a respiratory pathogen. mucosal vaccines can induce mucosal immune responses, antibodies and t cells at wet surfaces. we are developing oral vaccines for multiple indications, including influenza and noroviruses, delivered in a tablet form for people. our vaccine platform is a replication-defective adenovirus type- vectored vaccine that expresses antigen along with a novel toll-like receptor agonist as an adjuvant. these vaccines have been well tolerated, and able to generate robust humoral and cellular immune responses to the expressed antigens [ ] [ ] [ ] . protective efficacy in humans was demonstrated against a respiratory virus days or more post vaccination, as shown in a well characterized experimental influenza infection model . furthermore, the vaccine also has the advantage of room temperature stability and needle-free, ease of administration, providing several advantages over injected vaccine approaches with respect to vaccine deployment and access. here, we describe the pre-clinical development of a sars-cov- vaccine based on vaxart's oral adenovirus platform. the key approach was to develop several vaccine candidates in parallel, in order to create premanufacturing seeds while initial immunogenicity experiments were in progress. given that the vaccines were made during the pandemic, rapid decisions were required to keep the manufacturing and regulatory timelines from slipping. we assessed the relative immunogenicity of four candidate vaccines that expressed antigens based on the spike (s) and nucleocapsid (n) sars-cov- proteins. these proteins have been well characterized as antigens for related coronaviruses, such as sars-cov and mers (reviewed in yong, et al., ) and, increasingly, for sars-cov- spike. the aim of our vaccine is to induce immunogenicity on three levels; firstly, to induce potent serum neutralizing antibodies to s, secondly to induce mucosal immune responses, and thirdly to induce t cell responses to both vaccine antigens. this three-fold approach aims to induce robust and broad immunity capable of protecting the individual from virus infection as well as disease, promote rapid dissemination of vaccine during a pandemic, and to protect the population from virus transmission through herd immunity. here, we report the induction of neutralizing antibody (nab), igg and iga antibody responses, and t cell responses in mice following immunization of rad vectors expressing one or more sars-cov- antigens. initially, three different rad vectors were constructed to express different sars-cov- antigens. these were a vector expressing the full-length s protein (rad-s), a vector expressing the s protein and the n protein (rad-s-n), and a vector expressing a fusion protein of the s domain with the n protein (rad-s -n). the n protein of rad-s-n was expressed under control of the human beta actin promoter, which is much more potent in human cells than mouse cells. an additional construct where the expressed s protein was fixed in a prefusion conformation (rad-s(fixed)-n) was constructed at a later date as a control for exploring neutralizing antibody responses. these are described in figure . expression of the various transgenes was confirmed following infection of cells using flow cytometry and monoclonal antibodies to the s or n protein (supplemental figure ) . the primary objective of the initial mouse immunogenicity studies was to determine which of the rad vectors induced significant antibody responses to s, and to obtain those results rapidly enough to provide a gmp seed in time for manufacturing. we and others have observed that transgene expression by vaccine vectors orally administered to mice can be suppressed in their intestinal environment, so immunogenicity was assessed following intranasal (i.n.) immunization. animals were immunized i.n. and the antibody titers were measured over time by igg elisa. all three rad vectors induced nearly equivalent anti-s igg titers, at weeks and and the igg titer in all animals was significantly boosted by the second immunization (p < . mann whitney t-tests) ( fig. a) . however, the vector expressing full-length s (rad-s-n) induced higher neutralizing titers compared to the vector expressing only s (fig b) . this was measured by two different neutralizing assays, one based on sars-cov- infection of vero cells (cvnt) and one based on a surrogate neutralizing assay (svnt). furthermore, rad-s-n induced higher lung iga responses to s and unsurprisingly, to s (fig. c ) compared to rad-s -n two weeks after the final immunization. notably, neutralizing titers in the lung were also significantly higher when rad-s-n was used compared to the s -containing vaccine (rad-s -n) (fig. d ). this demonstrated that the rad-s-n candidate induced greater functional responses (nab and iga) s s tm cmv s s furin furin compared to the vaccine containing the just the s domain. because the n protein is much more highly conserved than the s protein, and is a target of long term t cell responses induced by infection , the vector rad-s-n was chosen for gmp manufacturing. c d three dose levels of rad-s-n were then tested to understand the dose responsiveness of this vaccine. the antibody responses to both s (fig. a) and s (fig. b) were measured. similar responses were seen at all three dose levels at all timepoints. responses to s and s were significantly increased at week compared to earlier times, in all groups. the induction of s-specific t cells by rad-s-n at different doses was then assessed. induction of antigen-specific cd + and cd + t cells that produced effector cytokines such as ifn-g, tnf-a and il- was observed two weeks after immunizations ( fig a) . notably, little il- was induced by this vaccine and only in cd + t cells; providing a level of assurance that the risk for vaccine dependent enhancement of disease was very low. furthermore, immunization with rad-s-n induced double and triple positive, multi-functional ifn-g, tnf-a and il- cd + t cells (fig. b) . a second dose response experiment was performed to focus on t cell responses to the s protein, weeks after the final immunization (week of the study). splenocytes were stimulated overnight with a peptide library to the s protein, divided in two separate peptide pools. t cell responses in the two pools were summed and plotted (fig. c ). animals administered the e iu and the e iu dose levels had significantly higher t cell responses compared to the untreated animals but produced a similar number of ifn-g secreting cells to each other, demonstrating a dose plateau at the e iu dose. notably, this t cell analysis was conducted weeks after the second immunization, potentially after the peak of t cell responses. balb/c mice were immunized, in, on days and with x iu, x iu or . x iu of rad co-expressing full length s and n (rad-s-n). the amount of igg specific for s (a) and s in serum diluted / , was evaluated using a mesoscale binding assay. points represent the mean and lines represent the standard deviation. a. c. rad-expressed wild-type s induces a superior neutralizing response compared to stabilized/pre-fusion s. an additional study was performed to compare rad-s-n to a vaccine candidate with the s-protein stabilized and with the transmembrane region removed (rad-s(fixed)-n). a stabilized version of the s protein has been proposed as a way to improve neutralizing antibody responses and produce less non-neutralizing antibodies. the s protein was stabilized through modifications as described by amanat et al., . rad-s-n induced higher serum igg titers to s (fig. a ) at both timepoints tested, although these were not statistically significant at week by mann-whitney (p = . ). however, rad-s-n induced significantly higher neutralizing antibody responses (fig. b ) than the stabilized version (p = . ). these results suggest that a wild-type version of the s protein is superior for a rad based vaccine in mice. the endgame to the covid- pandemic requires the identification and manufacture of a safe and effective vaccine and a subsequent global immunization campaign. a number of vaccine candidates have accelerated to phase iii global efficacy testing and, if sufficiently successful in these trials, may form the first generation of an immunization campaign. however, all of these advanced candidates are s-based vaccines that are injected. such an approach will unlikely prevent virus transmission, but should prevent pneumonia and virus growth and damage in the lower respiratory tract and periphery, as evidenced in macaque challenge studies , . one key constraint in a global covid- immunization campaign will be the cold chain distribution logistics and a bottleneck of requiring suitably trained health care workers (hcws) to inject the vaccine. current logistics costs, including cold chain and training, can double the cost of fully immunizing an individual in a low-middle income country (lmic) . implementing a mass immunization campaign, requiring trained hcws for injection-based vaccines, will have a significant impact on healthcare resources in all countries. the need for cold chain, biohazardous sharps waste disposal and training will result in increased cost, inequitable vaccine access, delayed vaccine uptake and prolongation of this pandemic. these costs will be magnified if vaccines are unable to provide long-term protection (natural immunity to other beta-coronaviruses is short-lived ), and annual injection-based campaigns are needed. vaxart's oral tablet vaccine platform provides a solution to these immunological as well as logistic, economic, access and acceptability problems. in this study we demonstrate, in an animal pre-clinical model, the immunogenicity of a sars-cov- vaccine using vaxart's vaccine platform; namely the induction of serum and mucosal neutralising antibodies and poly-functional t cells. mouse studies were designed to test immunogenicity of candidate vaccines rapidly in the spring of , before moving onto manufacturing and clinical studies critical to addressing the pandemic. vaxart's oral tablet vaccine platform has previously proven to be able to create reliable mucosal (respiratory and intestinal), t cell, and antibody responses against several different pathogens in humans , , , . we know from our prior human influenza virus challenge study that oral immunization was able to induce protective efficacy days post immunization; on par with the commercial quadrivalent inactivated vaccine . these features provide confidence that the adoption of the platform to covid- could translate to efficacy against this pathogenic coronavirus and could provide durable protection against virus infection. finally, a tablet vaccine campaign is much easier because qualified medical support is not needed to administer it. this ease of administration will result in increased vaccine access and potentially, acceptability, as has been evidenced by the success of easy-to-administer, oral polio vaccine, in the elimination of polio virus . these features could be even more important during sars-cov- immunization campaigns compared to other vaccines, as substantially more resources may be required to ensure uptake of this vaccine, given the global levels of covid- denialism, mistrust and increased vaccine hesitancy , . the tablet vaccine does not need refrigerators or freezers, does not require needles or vials, and can potentially be shipped via standard mail or by a delivery drone. these attributes significantly enhance deployment and distribution logistics, even permitting access to isolated regions with fewer technical resources. finally, from an immunological perspective, oral administration of this adenovirus is not compromised by pre-existing immunity to adenoviruses nor creates substantial anti-vector immunity , ; issues that have been shown to cause significantly decreased vaccine potency in an rad based sars-cov vaccine and can prevent durable increased immunity when the same adenovirus platform is re-administered by the im route . the choice of antigen can be difficult during a novel pandemic, a time in which key decisions are needed quickly. the s protein is believed to be the major neutralizing antibody target for coronavirus vaccines, as the protein is responsible for receptor binding, membrane fusion, and tissue tropism. when comparing sars-cov- wu- to sars-cov, the s protein was found to have . % identity . both sars-cov and sars-cov- are believed to use the same receptor for cell entry: the angiotensin-converting enzyme receptor (ace ), which is expressed on some human cell types . thus, sars-cov- s protein is being used as the leading target antigen in vaccine development so far and is an ideal target given that it functions as the key mechanism for viral binding to target cells. however, the overall reliance on the s protein and an igg serum response in the vaccines could eventually lead to viral escape. for influenza, small changes in the hemagglutinin binding protein, including a single glycosylation site, can greatly affect the ability of injected vaccines to protect . sars-cov- appears to be more stable than most rna viruses, but s protein mutations have already been observed without the selective pressure of a widely distributed vaccine. once vaccine pressure begins, escape mutations might emerge. we took two approaches to address this issue; firstly to include the more conserved n protein in the vaccine and secondly to induce broader immune responses, namely through mucosal iga. high expression levels of ace are present in type ii alveolar cells of the lungs, absorptive enterocytes of the ileum and colon, and possibly even in oral tissues such as the tongue . transmission of the virus is believed to occur primarily through respiratory droplets and fomites between unprotected individuals in close contact , although there is some evidence of transmission via the oral-fecal route as seen with both sars-cov and mers-cov viruses where coronaviruses can be secreted in fecal samples from infected humans . there is also evidence that a subset of individuals exist that have gastrointestinal symptoms, rather than respiratory symptoms, are more likely to shed virus longer . driving immune mucosal immune responses to s at both the respiratory and the intestinal tract may be able provide broader immunity and a greater ability to block transmission, than simply targeting one mucosal site alone. blocking transmission, rather than just disease, will be essential to reducing infection rates and eventually eradicating sars-cov- . we have previously demonstrated that an oral, tableted rad-based vaccine can induce protection against respiratory infection and shedding following influenza virus challenge as well as intestinal immunity to norovirus antigens in humans . furthermore, mucosal iga is more likely to be able address any heterogeneity of the s proteins in circulating viruses than a monomeric igg response. miga has also been found to be more potent at cross reactivity than igg for other respiratory pathogens . iga may also be a more neutralizing isotype than igg in covid- infection, and in fact neutralizing iga dominates the early immune response . notably, we saw a higher ratio between neutralizing to non-neutralizing antibodies in our lung versus serum antibody results in our mouse study as well, which supports the concept that iga may have more potency compared to igg. polymeric iga, through multiple binding interactions to the antigen and to fc receptors can turn a weak single interaction into a higher overall affinity binding and activation signal, creating more cross-protection against heterologous viruses . our second strategy to mitigate this potential vaccine-driven escape problem was to include the n protein in the vaccine construct. the n protein is highly conserved among b-coronaviruses, (greater than % identical) contains several immunodominant t cell epitopes, and long-term memory to n can be found in sars-cov recovered subjects as well as people with no known exposure to sars-cov or sars-cov- , . in an infection setting, t cell responses to the n protein seem to correlate to increased neutralizing antibody responses . all of these reasons led us to add n to our vaccine approach. the protein was expressed in a cells (supp fig ) . however, as the human beta actin promoter is more active in human cells than mice, we did not explore immune responses in balb/c mice, but will examine them more carefully in future nhp and human studies. the optimum sequence and structure of the s protein to be included in a sars-cov- vaccine is a subject of debate. several labs have suggested that reducing the s protein to the key neutralizing domains within the receptor binding domain (rbd) would promote higher neutralizing antibody responses, and fewer non-neutralizing antibodies , . we made a vaccine candidate composed of the s domain, which includes the rbd, in an attempt to promote this approach. although the s based vaccine produced similar igg binding titers to s , neutralizing antibody responses were significantly lower compared to the full-length s antigen. other gene-based vaccines have also shown the reductionist approach to s does not work so well, demonstrating that the dna vaccine expressing the full-length s-protein produced higher neutralizing antibodies than shorter s segments . in agreement with these macaque studies, we observed that the sequence of the adenovirus encoded antigen had a significant impact on antibody function, here with respect to neutralization. while reducing the potential for exposing non-neutralizing antibody epitopes seems reasonable in theory, this might reduce the t cell help that allows for greater neutralizing antibodies to develop. indeed, of the spike protein t cell responses, which make up % of the responses to sars-cov- , only % map to receptor binding domain . stabilizing the s protein might be important for a protein vaccine, but not necessarily for a gene-based vaccine. the former is produced in vitro and it is produced to retain a homogenous, defined structure, ready for injection. in contrast, the latter, is expressed on the surface of a cell, in vivo, like natural infection, substantially in a prefusion form, and the additional stabilization may be unnecessary for b cells to create antibodies against the key neutralizing epitopes. we directly compared a stabilized version of s to the wild-type version. the wild-type version was significantly better at inducing neutralizing antibody responses. of interest, this was also observed in a dna vaccine study in nhps, where the stabilized version appeared to induce lower neutralizing antibody (nab) titers compared to the wild-type s . a slightly different result was observed in studies of rad vectors by mercado, et al in nhps, where expressing a stabilized version of the s protein appeared to improve nab but lower t cell responses . in summary, stabilization doesn't universally improve the immune responses in gene-based or vectorbased vaccines. multiple vaccine candidates are in, or are about to begin, clinical testing. due to known safety and immunogenicity for epidemic pathogens such as ebola virus, two leading candidate vaccines are based on recombinant adenovirus vectors; university of oxford's chadox -ncov and janssen pharmaceutical's advac platforms [ ] [ ] [ ] [ ] . we saw stronger serum igg and nab titers in our study compared to a chadox -ncov in balb/c mice , however, this might reflect differences in assay components. a rad vaccine study was performed by hassan, et al., where doses of e vp were given by intranasal delivery . the results were significant from the standpoint of blocking lung infection in a mouse sars-cov- challenge model. they reported titers of serum antibody titers of e above the background titers, similar to our results, despite using doses -to -log fold higher viral doses compared to our study. indeed, in our study, equivalently strong t cell and antibody responses were observed using e iu and e iu by the intranasal route. using these doses, we observed high percentages of cd + t cell responses (up to %) secreting ifn-g and tnf-a and strong cd + t cells after peptide restimulation. although we did not evaluate the trafficking properties of these antigen-specific t cells, we know that oral administration of this ad-based vaccine in humans induces high levels of mucosal homing lymphocytes , . a proportion of the antigenspecific cd + and cd + t cells were polyfunctional in this mouse study. vaccine-induced t cells possessing multiple functions may provide more effective elimination of virus subsequent to infection and therefore could be involved in the prevention of disease. however, it is uncertain at this time what is the optimum t cell phenotype required for protection against disease. in summary, these studies in mice represent our first step in creating a vaccine candidate, demonstrating the immunogenicity of the construct at even low vaccine doses and the elucidation of the full-length spike protein as a leading candidate antigen to induce t cell responses and superior systemic and mucosal neutralizing antibody. future work will focus on the immune responses in humans. for this study, four recombinant adenoviral vaccine constructs were created based on the published dna sequence of sars-cov- publicly available as genbank accession no. mn . . specifically, the published amino acid sequences of the sars-cov- spike protein (s protein) and the sars-cov- nucleocapsid protein (n protein) were used to synthesize nucleic acid sequences codon optimized for expression in homo sapiens cells (blue heron biotechnology, bothell, wa). these sequences were used to create recombinant plasmids containing transgenes cloned into the e region of adenovirus type (rad ), as described by he, et al , using the same vector backbone used in prior clinical trials for oral rad tablets , . as shown in fig , c. rad-s -n: rad vector using a fusion sequence combining the s region of sars-cov- s gene (including the native furin site between s and s ) with the full-length sars-cov- n gene. d. rad-s(fixed)-n: rad vector containing a stabilized s gene with the transmembrane region removed under the control of the cmv promoter and full-length sars-cov- n gene under control of the human beta-actin promoter. the s gene is stabilized through the following modifications: a) arginine residues at aa positions , , were deleted to remove the native furin cleavage site b) two stabilizing mutations were introduced: k p and v p c) transmembrane region was removed following p and replaced with bacteriophage t fibritin trimerization foldon domain sequence (gyipeaprdgqayvrkdgewvllstfl) all vaccines were grown in the expi f suspension cell-line (thermo fisher scientific), purified by cscl density centrifugation and provided in a liquid form for animal experiments. studies were approved for ethics by the animal care and use committees (iacuc). all of the procedures were carried out in accordance with local, state and federal guidelines and regulations. female - week old balb/c mice were purchased from jackson labs (bar harbor, me). because mice do not swallow pills, liquid formulations were instilled intranasally in µl per nostril, µl per mouse in order to test immunogenicity of the various constructs. serum was acquired by cheek puncture at various timepoints. elisas. specific antibody titers to proteins were measured similarly to methods described previously . briefly, microtiter plates (maxisorp: nunc) were coated in carbonate buffer ( . m at ph . ) with . ug/ml s protein (genscript). the plates were incubated overnight at °c in a humidified chamber and then blocked in pbs plus . % tween (pbst) plus % bsa solution for h before washing. plasma samples were serially diluted in pbst. after a -h incubation, the plates were washed with pbst at least times. antibodies were then added as a mixture of anti-mouse igg -horseradish peroxidase (hrp) and anti-mouse igg a-hrp (bethyl laboratories, montgomery, tx). each secondary antibody was used at a : , dilution. the plates were washed at least times after a -h incubation. antigen-specific mouse antibodies were detected with , =, , =-tetramethylbenzidine (tmb) substrate (rockland, gilbertsville, pa) and h so was used as a stop solution. the plates were read at nm on a spectra max m microplate reader. average antibody titers were reported as the reciprocal dilution giving an absorbance value greater than the average background plus standard deviations, unless otherwise stated. to measure responses to both s and s simultaneously, a multi-spot® -well, -spot plate (mesoscale devices; msd) was coated with sars cov- antigens. proteins were commercially acquired from a source (native antigen company) that produced them in mammalian cells ( cells). these were biotinylated and adhered to their respective spots by their individual u-plex linkers. to measure igg antibodies, plates were blocked with msd blocker b for hour with shaking, then washed three times prior to the addition of samples, diluted : . after incubation for hours with shaking, the plates were washed three times. the plates were then incubated for hour with the detection antibody at μg/ml (msd sulfo-tag tm anti-mouse igg). after washing times, the read buffer was added and the plates were read on the meso quickplex sq . neutralizing antibodies were routinely detected based on the sars-cov- surrogate virus neutralization test (svnt) kit (genscript). this elisa-based kit detects antibodies that hinder the interaction between the receptor binding domain (rbd) of the sars-cov- spike glycoprotein and the ace receptor on host cells, and is highly correlated to conventional virus neutralizing titers for sars-cov- infection of vero cells . the advantage of this approach is that the assay can be done in a bsl- laboratory. sera from mice immunized with the candidate vaccines was diluted at : , : , : , : , : and : using the provided sample dilution buffer. sera from non-immunized mice was diluted : . lung samples were diluted : , : , and : . positive and negative controls were prepared at a : volume ratio following the provided protocol. after dilution, sera or lung samples were individually incubated at a : ratio with hrp-rbd solution for minutes at °c. following incubation, µl of the each hrp-rbd and sample or control mixture was added to the corresponding wells in the hace -precoated capture plate and once again incubated at °c for minutes. afterwards, wells were thoroughly washed and µl of the provided tmb ( , =, , =tetramethyl-benzidine) solution was added to each well and left to incubate for minutes at room temperature ( - °c). lastly µl of stop solution was added to each well, and the plate was read on a spectra max m microplate reader at nm. the absorbance of a given sample is inversely related on the titer of anti-sars-cov- rbd neutralizing antibody in a given sample. per test kit protocol, a cut-off of % inhibition when comparing the od of the sample versus the od of the negative control was determined to be positive for the presence of neutralizing antibodies. samples that were negative at the lowest dilution were set equal to ½ of the lowest dilution tested, either for sera or . for lung samples. additional neutralizing antibodies responses were measured in some studies using a cvnt assay at visimederi under bsl conditions. the cvnt assay has a readout of cytopathic effect (cpe) to detect specific neutralizing antibodies against live sars-cov- in animal or human samples. the cvnt/cpe assay permits the virus to makes multiples cycles of infection and release from cells; its exponential grow in few days (usually hours of incubation) causes the partial or complete cell monolayer detachment from the surface of the support, clearly identifiable as cpe. serum samples are heat inactivated for min at °; two-fold dilutions, starting from : are performed then mixed with an equal volume of viral solution containing tcid of sars-cov- . the serum-virus mixture is incubated for hour at ° in humidified atmosphere with % co . after incubation, µl of the mixture at each dilution are added in duplicate to a cell plate containing a semiconfluent vero e monolayer. after hours of incubation the plates are inspected by an inverted optical microscope. the highest serum dilution that protect more than % of cells from cpe is taken as the neutralization titer. two weeks after the final immunization (day of the study), mice were sacrificed and bled via cardiac puncture. lungs were removed and snap frozen at - c. on thawing, lungs were weighed. lungs were homogenized in µl dpbs using pellet pestles (sigma z ). homogenates were centrifuged at rpm for minutes and supernatants were frozen. the total protein content in lung homogenate was evaluated using a bradford assay to ensure equivalent amounts of tissue in all samples before evaluation of iga content. antigen-specific iga titers in lungs were detected using a mouse iga elisa kit (mabtech) and pnpp substrate (mabtech). briefly, maxisorp plates (nunc) were coated with s or s (the native antigen company; ng/well) in pbs for overnight adsorption at °c and then blocked in pbs plus . % tween (pbst) plus . % bsa (pbs/t/b) solution for h before washing. lung homogenates were serially diluted in pbs/t/b, starting at a : dilution. after hours incubation and washing, bound iga was detected using mt a-alp conjugated antibody ( : ), according to the manufacturer's protocol. plates were read at nm. endpoint titers were taken as the x-axis intercept of the dilution curve at an absorbance value x standard deviations greater than the absorbance for naïve mouse serum. non-responding animals were set a titer of or ½ the value of the lowest dilution tested. spleens were removed and placed in ml hanks balanced salt solution (with m hepes and % fbs) before pushing through a sterile strainer with a ml syringe. after rbc lysis (ebiosolutions), resuspension, and counting, the cells were ready for analysis. cells were cultured at e cells/well with two peptide pools representing the full-length s protein at µg/ml (genscript) overnight in order to stimulate the cells. the culture media consisted of rpmi media (lonza) with . m hepes, x l-glutamine , x mem basic amino acids, x penstrep, % fbs, and . e- mole/l beta-mercaptoethanol. antigen specific ifn-g elispots were measured using a mabtech kit. flow cytometric analysis was performed using an attune flow cytometer and flow jo version . . , after staining with the appropriate antibodies. for flow cytometry, e splenocytes per well were incubated for hours at ºc with peptide pools representing full-length s at either or ug/ml, adding brefeldin a (thermofisher) for the last hours of incubation. the antibodies used were apc-h conjugated cd , fitc conjugated cd , bv conjugated cd , percp-cy . conjugated ifn-y, bv conjugated il- , pe-cy conjugated tnfa, apc conjugated il- , alexa fluor conjugated cd , and pe conjugated cd l (bd biosciences). clinical features of patients infected with novel coronavirus in wuhan, china transmission and clinical characteristics of asymptomatic patients with sars-cov- infection clinical and immunological assessment of asymptomatic sars-cov- infections chadox ncov- vaccine prevents sars-cov- pneumonia in rhesus macaques dna vaccine protection against sars-cov- in rhesus macaques safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind, randomised controlled trial immunogenicity and safety of a recombinant adenovirus type- -vectored covid- vaccine in healthy adults aged years or older: a randomised, double-blind, placebo-controlled, phase trial an mrna vaccine against sars-cov- -preliminary report in vitro comparison of the biologic activities of monoclonal monomeric iga, polymeric iga, and secretory iga blocking interhost transmission of influenza virus by vaccination in the guinea pig model safety and immunogenicity of an oral tablet norovirus vaccine, a phase i randomized, placebo-controlled trial high titre neutralising antibodies to influenza after oral tablet immunisation: a phase , randomised, placebo-controlled trial. the lancet infectious diseases oral administration of an adenovirus vector encoding both an avian influenza a hemagglutinin and a tlr ligand induces antigen specific granzyme b and ifn-gamma t cell responses in humans efficacy and immune correlates of protection induced by an oral influenza vaccine evaluated in a phase , placebo-controlled human experimental infection study recent advances in the vaccine development against middle east respiratory syndrome-coronavirus firewalls prevent systemic dissemination of vectors derived from human adenovirus type and suppress production of transgene-encoded antigen in a murine model of oral vaccination long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients a serological assay to detect sars-cov- seroconversion in humans projections of costs, financing, and additional resource requirements for low-and lower middle-income country immunization programs over the decade projecting the transmission dynamics of sars-cov- through the postpandemic period systemic and mucosal immune responses following oral adenoviral delivery of influenza vaccine to the human intestine by radio controlled capsule high titer neutralizing antibodies to influenza following oral tablet immunization: a randomized, placebocontrolled trial the lancet infectious diseases systematic review of mucosal immunity induced by oral and inactivated poliovirus vaccines against virus shedding following oral poliovirus challenge disinformation, misinformation and inequality-driven mistrust in the time of covid- : lessons unlearned from aids denialism mistrust in biomedical research and vaccine hesitancy: the forefront challenge in the battle against covid- in italy safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, nonrandomised, first-in-human trial first-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype hiv- env vaccine (ipcavd ) structural, glycosylation and antigenic variation between novel coronavirus ( -ncov) and sars coronavirus (sars-cov) receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus contemporary h n influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains high expression of ace receptor of -ncov on the epithelial cells of oral mucosa report of the who-china joint mission on coronavirus disease (covid- ) enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov- possible? digestive symptoms in covid- patients with mild disease severity: clinical presentation, stool viral rna testing, and outcomes comparison of antiviral activity between iga and igg specific to influenza virus hemagglutinin: increased potential of iga for heterosubtypic immunity iga dominates the early neutralizing antibody response to sars-cov- cross-protection against influenza a virus infection by passively transferred respiratory tract iga antibodies to different hemagglutinin molecules sars-cov- -specific t cell immunity in cases of covid- and sars, and uninfected controls detection of sars-cov- -specific humoral and cellular immunity in covid- convalescent individuals a novel receptor-binding domain (rbd)-based mrna vaccine against sars-cov- development of a covid- vaccine based on the receptor binding domain displayed on virus-like particles selective and cross-reactive sars-cov- t cell epitopes in unexposed humans single-shot ad vaccine protects against sars-cov- in rhesus macaques safety and immunogenicity of a -dose heterologous vaccine regimen with ad .zebov and mva-bn-filo ebola vaccines: -month data from a phase randomized clinical trial in safety and immunogenicity of a -dose heterologous vaccination regimen with ad .zebov and mva-bn-filo ebola vaccines: -month data from a phase randomized clinical trial in uganda and tanzania clinical assessment of a novel recombinant simian adenovirus chadox as a vectored vaccine expressing conserved influenza a antigens a single intranasal dose of chimpanzee adenovirus-vectored vaccine confers sterilizing immunity against sars-cov- infection a simplified system for generating recombinant adenoviruses very fast folding and association of a trimerization domain from bacteriophage t fibritin systemic and mucosal antibody responses following retroductal gene transfer to the salivary gland a sars-cov- surrogate virus neutralization test based on antibody-mediated blockage of ace -spike protein-protein interaction the authors would like to thank alessandro torelli, laura palladino, emanuele montomoli and the team at vismederi for running neutralizing antibody titers (bsl ) and susan johnson for critically reviewing the manuscript. authorship acm, mc, and snt designed the experiments; acm and snt wrote the manuscript; np, kpt, and kl performed immunological assays; acm, np, kpt, kl, mc, and snt analyzed the data. egd and snt designed the vaccine vectors. egd, nd, kpt, klm mc and snt are current employees and/or own stock options in vaxart, the sponsor of the studies. egd and snt are named as inventors covering a sars-cov- (ncov- ) vaccine. snt is named as an inventor on patent covering the vaccine platform. acm declares no competing interest. key: cord- - nz qioh authors: carabineiro, sónia alexandra correia title: applications of gold nanoparticles in nanomedicine: recent advances in vaccines † date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: nz qioh nowadays, gold is used in (nano-)medicine, usually in the form of nanoparticles, due to the solid proofs given of its therapeutic effects on several diseases. gold also plays an important role in the vaccine field as an adjuvant and a carrier, reducing toxicity, enhancing immunogenic activity, and providing stability in storage. an even brighter golden future is expected for gold applications in this area. it is well known that gold is inert in the bulk stable and is only active as a catalyst in the form of nanoparticles [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as the name suggests, nanoparticles have small (nano) size and corresponding large surface/volume ratio. examples are shown in figure . due to these characteristics, au nanoparticles can have an important role, not only in catalysis, but also in nanomedicine, suitable for several biological applications. in fact, it has been argued that au nanoparticles could be used in almost all medical applications, from diagnostics to therapy, prevention, and hygiene [ ] [ ] [ ] [ ] [ ] [ ] [ ] . gold nanoparticles have remarkable properties and can also play an important role in vaccine research for several diseases, as showed in several recent reviews [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . vaccines have been a significant advancement in public health, preventing the death of many millions of people every year [ ] . the main purpose of vaccination is to introduce an antigen from a vaccines have been a significant advancement in public health, preventing the death of many millions of people every year [ ] . the main purpose of vaccination is to introduce an antigen from usually nanoparticles are synthesized in colloidal solution by reduction of chloroauric acid (haucl ) by variations of the so-called turkevich method [ ] . their characterization is carried out by ultraviolet-visible spectroscopy, their diameters determined by transmission electron microscopy. then, they can be further functionalized with the desired molecules, according to their specific needs. one example is given in scheme , showing a procedure carried out by saha et al. [ ] . gold nanoparticles were synthesized in aqueous medium by an ultra-sound assisted green method using black pepper extract and chitosan as reducing agent and a stabilizing agent, respectively. the biopolymer-inspired biogenic method (scheme ) was found to be stable and with enhanced bioactivity. the developed nanomaterial boosts the production of reactive oxygen species and misbalances the antioxidant parameters of detoxification enzymes of filarial parasites, such as gst, which besides its immune prophylactic potency, is also a promising candidate vaccine as shown in s. cervi and w. bancrofti [ ] . gold nanoparticles, in general, have remarkably high surface-to-volume ratio, are biocompatible and inert, and can be easily functionalized with several molecules; thus, they can also play an important role in the vaccine field as adjuvants, reducing toxicity, enhancing immunogenic activity, and providing stability of vaccine in storage, and have great potential as carriers for the development of a great diversity of fully synthetic vaccines [ ] [ ] [ ] [ ] [ ] , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . their shape and size can affect immunological responses in vivo and in vitro [ , [ ] [ ] [ ] [ ] . moreover, they are able to penetrate blood vessels and tissue barriers and to be targeted to a specific cell by means of specifically functionalized molecules [ ] . moreover, gold nanoparticles can be packaged inside virus-like particles generated by heterologous expression of viral structural genes that are powerful tools in vaccine development [ ] . anionic gold nanoparticles increased the half-life of a green fluorescent protein expressing adenovirus from similar to h to days at °c (from to > days at room temperature) [ ] . promising cd + t cell results were obtained with polyelectrolyte multilayers assembled on gold nanoparticles [ , ] . a positively charged antigen and a negatively charged immuno-adjuvant on gold nanoparticles resulted in a new vaccine platform. usually nanoparticles are synthesized in colloidal solution by reduction of chloroauric acid (haucl ) by variations of the so-called turkevich method [ ] . their characterization is carried out by ultraviolet-visible spectroscopy, their diameters determined by transmission electron microscopy. then, they can be further functionalized with the desired molecules, according to their specific needs. one example is given in scheme , showing a procedure carried out by saha et al. [ ] . gold nanoparticles were synthesized in aqueous medium by an ultra-sound assisted green method using black pepper extract and chitosan as reducing agent and a stabilizing agent, respectively. the biopolymer-inspired biogenic method (scheme ) was found to be stable and with enhanced bioactivity. the developed nanomaterial boosts the production of reactive oxygen species and misbalances the antioxidant parameters of detoxification enzymes of filarial parasites, such as gst, which besides its immune prophylactic potency, is also a promising candidate vaccine as shown in s. cervi and w. bancrofti [ ] . gold nanoparticles, in general, have remarkably high surface-to-volume ratio, are biocompatible and inert, and can be easily functionalized with several molecules; thus, they can also play an important role in the vaccine field as adjuvants, reducing toxicity, enhancing immunogenic activity, and providing stability of vaccine in storage, and have great potential as carriers for the development of a great diversity of fully synthetic vaccines [ ] [ ] [ ] [ ] [ ] , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . their shape and size can affect immunological responses in vivo and in vitro [ , [ ] [ ] [ ] [ ] . moreover, they are able to penetrate blood vessels and tissue barriers and to be targeted to a specific cell by means of specifically functionalized molecules [ ] . moreover, gold nanoparticles can be packaged inside virus-like particles generated by heterologous expression of viral structural genes that are powerful tools in vaccine development [ ] . anionic gold nanoparticles increased the half-life of a green fluorescent protein expressing adenovirus from similar to h to days at • c (from to > days at room temperature) [ ] . promising cd + t cell results were obtained with polyelectrolyte multilayers assembled on gold [ ] ). an immunostimulatory nanocomposite (cpg-au@hbc vlp), designed by self-assembling engineered virus-like particles encapsulating cpg-gold nanoparticle conjugates through electrostatic interactions, showed an increase in cd + and cd + t cell numbers, inducing important cellular and humoral immune response [ ] . ultrasmall graphene oxide-supported gold nanoparticles (usgo-au) were obtained from reduction of chloroauric acid using usgo and then decorated with ovalbumin antigen (ova) through physical adsorption to obtain usgo-au@ova and used immune adjuvants [ ] . it was shown that usgo-au@ova can efficiently stimulate raw . cells to secrete tumor necrosis factor (tnf-), a mediator for cellular immune response. usgo-au@ova can also promote robust ova specific antibody response, cd + t cells proliferation, and secretion of different cytokines. fluorescent au nanoclusters were synthesized using ovalbumin-cpg oligodeoxynucleotides (odns) conjugates as templates [ ] . the nanoclusters can act as self-vaccines to assist in generation of high immunostimulatory activity. gold-based nanovaccines were synthesized using a self-assembling conjugation method [ ] . dendritic cells uptake gold nanovaccines with minimal toxicity and are able to process the vaccine peptides on the particles to stimulate cytotoxic t lymphocytes (ctls). these high-peptide density au nanovaccines can stimulate ctls better than free peptides and have great potential as carriers for various vaccine types [ ] . hydrophilic gold nanodots were used to control lipopolysaccharide assembly to ease the formation of stable endotoxin nanovesicles, which are stable precursors of cubosomes and hexosomes with specific immunological effects that might be useful in vaccine development [ ] . needle-free vaccine delivery systems efficiently transport powdered or particulate dna and protein vaccines into the epidermal tissue [ ] [ ] [ ] [ ] . it can be used to directly transfect antigen presenting cells by formulating dna or protein vaccines onto - µm gold particles (particle-mediated immunization). a solid-in-oil dispersion of gold nanorods can also enhance transdermal protein delivery and skin vaccination [ ] . plasmid dna (pdna)-coated gold nanoparticles were successfully delivered into ex vivo murine and porcine skin at low inlet pressures using parallel arrays of microchannels [ ] . it was shown that full-length pdna was preserved after each particle preparation and jetting procedures. below, several examples are given on the role of gold nanoparticles in the attempts of producing vaccines for several diseases. cancer is one of the main causes of death worldwide, that can affect people at all ages, even small children and foetuses, the risk usually increasing with age [ , ] . the present treatments consist of surgery, chemotherapy, or radiotherapy, which can have adverse side effects, due to a lack of specificity for tumors [ ] . an ideal treatment should aim only at the target tumor cells and have limited detrimental effects on normal cells [ , ] . [ ] ). an immunostimulatory nanocomposite (cpg-au@hbc vlp), designed by self-assembling engineered virus-like particles encapsulating cpg-gold nanoparticle conjugates through electrostatic interactions, showed an increase in cd + and cd + t cell numbers, inducing important cellular and humoral immune response [ ] . ultrasmall graphene oxide-supported gold nanoparticles (usgo-au) were obtained from reduction of chloroauric acid using usgo and then decorated with ovalbumin antigen (ova) through physical adsorption to obtain usgo-au@ova and used immune adjuvants [ ] . it was shown that usgo-au@ova can efficiently stimulate raw . cells to secrete tumor necrosis factor (tnf-), a mediator for cellular immune response. usgo-au@ova can also promote robust ova specific antibody response, cd + t cells proliferation, and secretion of different cytokines. fluorescent au nanoclusters were synthesized using ovalbumin-cpg oligodeoxynucleotides (odns) conjugates as templates [ ] . the nanoclusters can act as self-vaccines to assist in generation of high immunostimulatory activity. gold-based nanovaccines were synthesized using a self-assembling conjugation method [ ] . dendritic cells uptake gold nanovaccines with minimal toxicity and are able to process the vaccine peptides on the particles to stimulate cytotoxic t lymphocytes (ctls). these high-peptide density au nanovaccines can stimulate ctls better than free peptides and have great potential as carriers for various vaccine types [ ] . hydrophilic gold nanodots were used to control lipopolysaccharide assembly to ease the formation of stable endotoxin nanovesicles, which are stable precursors of cubosomes and hexosomes with specific immunological effects that might be useful in vaccine development [ ] . needle-free vaccine delivery systems efficiently transport powdered or particulate dna and protein vaccines into the epidermal tissue [ ] [ ] [ ] [ ] . it can be used to directly transfect antigen presenting cells by formulating dna or protein vaccines onto - µm gold particles (particle-mediated immunization). a solid-in-oil dispersion of gold nanorods can also enhance transdermal protein delivery and skin vaccination [ ] . plasmid dna (pdna)-coated gold nanoparticles were successfully delivered into ex vivo murine and porcine skin at low inlet pressures using parallel arrays of microchannels [ ] . it was shown that full-length pdna was preserved after each particle preparation and jetting procedures. below, several examples are given on the role of gold nanoparticles in the attempts of producing vaccines for several diseases. cancer is one of the main causes of death worldwide, that can affect people at all ages, even small children and foetuses, the risk usually increasing with age [ , ] . the present treatments consist of surgery, chemotherapy, or radiotherapy, which can have adverse side effects, due to a lack of specificity for tumors [ ] . an ideal treatment should aim only at the target tumor cells and have limited detrimental effects on normal cells [ , ] . it was first postulated by coley in that the immune system was able to recognize and set a response against tumors [ ] . later, it was shown that immunization of mice with mutated tumor cells could induce a protective anti-tumor immune response against non-immunogenic tumor [ ] . this led to cancer immunotherapy research and to the development of cancer vaccines capable of generating an active tumor-specific immune response. cancer vaccines have high specificity for tumor cells and long-lasting immunological memory that may safeguard against recurrences, and can be used to either prevent (prophylactic) or treat established cancer (therapeutic) [ , ] . therapeutic cancer vaccines (also called active immunotherapy) work to enable the immune system of a patient to eradicate cancer cells [ ] . identification of tumor-associated antigens (taas) and tumor-specific antigens (tsas) has led to increased efforts to develop vaccination strategies. vaccines may be composed of whole cells or cell extracts, genetically modified tumor cells, dendritic cells (dcs) loaded with taas, immunization with soluble proteins or synthetic peptides, recombinant viruses or bacteria encoding tumor-associated antigens, and plasmid dna-encoding tsas or taas in conjunction with appropriate immunomodulators [ , ] . all of these vaccination approaches aim to induce specific immunological responses and are localized into taas, destroying only the tumor cells and leaving the majority of other cells of the body undamaged. an efficient delivery to cells is needed and gold nanoparticles have proved to be excellent carriers [ ] [ ] [ ] [ ] [ ] [ ] [ ] . gold nanoparticles enabled efficient antigen delivery to dendritic cells and then activated the cells to facilitate cross-presentation, inducing antigen-specific cytotoxic t-lymphocyte responses for effective cancer therapy [ ] . moreover, they show several advantages over other nanoparticulate-based carriers, such as the ease with which they control their size for different applications, the variety of antigens and adjuvants that can be easily linked to and displayed on their surface, the fact that they can be detected using non-invasive imaging techniques, providing clinicians with information on where or whether the vaccines have been delivered, which helps to predict or evaluate therapeutic efficacy, and the biocompatibility and non-toxicity of gold [ , , ] . au nanoparticle immunotherapies are well suited for synergistic combination therapy with existing cancer therapies such as photothermal ablation [ , ] . all these features suggest that gold nanoparticle-based antigen delivery systems may be a useful vaccine technology that is able to prevent and/or treat cancer. the several functionalities of gold nanoparticles make them promising vehicles for immune therapies, especially for combinatorial treatment approaches that target multiple immune pathways (figure ) . a polymerizable version of the tn-antigen glycan was synthesized and the polymers were conjugated to gold nanoparticles, producing nanoscale glycoconjugates [ ] . these nanomaterials generated a strong and long-lasting production of antibodies, selective to the tn-antigen glycan and cross-reactive toward mucin proteins displaying tn, and thus represent a simple approach to the synthesis of anticancer vaccines. gold nanoparticles obtained by a modified turkevich method were functionalized with mucin- (muc- ) glycoprotein using clealand's reagent [ ] . (muc- is involved in fundamental biological processes that can be found over-expressed and with a clearly changed glycan pattern on epithelial tumor cells, and thus is a promising target structure in the search for effective carbohydrate-based cancer vaccines and immunotherapeutics [ ] ). the obtained au-muc- nano-construction has been shown to be an important macrophage activator, leading to the release of cytokines such as tnf-alpha, il- , il- , and il- on peritoneal macrophages isolated from mice [ ] . au nanoparticle immunotherapies are well suited for synergistic combination therapy with existing cancer therapies such as photothermal ablation [ , ] . all these features suggest that gold nanoparticle-based antigen delivery systems may be a useful vaccine technology that is able to prevent and/or treat cancer. the several functionalities of gold nanoparticles make them promising vehicles for immune therapies, especially for combinatorial treatment approaches that target multiple immune pathways (figure ) . a polymerizable version of the tn-antigen glycan was synthesized and the polymers were conjugated to gold nanoparticles, producing nanoscale glycoconjugates [ ] . these nanomaterials generated a strong and long-lasting production of antibodies, selective to the tn-antigen glycan and cross-reactive toward mucin proteins displaying tn, and thus represent a simple approach to the synthesis of anticancer vaccines. gold nanoparticles obtained by a modified turkevich method were functionalized with mucin- (muc- ) glycoprotein using clealand's reagent [ ] . other authors also reported on the synthesis of muc -glycopeptide antigens and their coupling to gold nanoparticles of different sizes and developed a new dot-blot immunoassay to test the potential antigen-antibody binding [ ] . a glycopeptide sequence derived from muc- glycoprotein and the t-cell epitope p sequence were immobilized gold nanoparticles attached to polyethylene glycol chains [ ] . after immunization, mice showed significant mhc-ii mediated immune responses, and their antisera were able to recognize human mcf- breast cancer cells. gold nanoparticles designed this way have the potential to be used in the development of anticancer vaccines. gold nanoparticles proved to be efficient in facilitating the delivery of the ovalbumin (ova) peptide antigen and the cpg adjuvant and enhance their therapeutic effect in a b -ova tumor model. gold nanoparticle-mediated ova peptide delivery can have important therapeutic benefits without the need of an adjuvant, showing that gold nanoparticles are effective peptide vaccine carriers, having the potential to allow the use of lower and safer adjuvant doses during vaccination [ ] . the use of gold nanoparticles in hiv vaccine development has been recently reviewed by several authors [ ] . after more than years since the discovery of hiv in , no effective vaccine is yet available [ , [ ] [ ] [ ] . some histocompatibility complex molecules expressed on the surface of hiv are potential targets for neutralizing antibodies [ ] , as shown in figure . among the few broadly neutralizing hiv monoclonal antibodies, g is the only carbohydrate-directed that is able to recognize a cluster of high-mannose glycans on the viral envelope glycoprotein gp [ ] . this type of glycan is thus envisaged as a target to develop an hiv vaccine capable of eliciting g -like antibodies. gold nanoparticles coated with self-assembled monolayers of synthetic oligomannosides, which are present in gp , are able to bind g with high affinity and to interfere with g /gp binding [ ] . thiol-terminated oligosaccharides have been attached on gold nanoparticles and have been used in attempts to develop an hiv vaccine [ ] . two nanometer gold glyconanoparticles were coated with synthetic partial structures of several mannosides [ ] . the assembly of the antennas of the gp high-mannose type glycan on gold glyconanoparticles provided superior binding to the anti-hiv antibody g , which could help in the design of a carbohydrate-based vaccine against hiv. it was shown that conjugation to negatively charged gold glyconanoparticles could stabilize either the alpha-helix or beta-strand conformation of the third variable region (v peptide) of the hiv- gp [ ] . the peptide on the nanoparticles shows more stability toward peptidase degradation compared to the free peptide. v beta-glyconanoparticles produce antibodies in rabbits that recognize a recombinant gp , and the serum showed consistent neutralizing activity. these results potentially allow for the design of new fully synthetic hiv vaccine candidates [ ] . gold nanorods modified with poly(diallydimethylammonium chloride) or polyethyleneimine can significantly promote cellular and humoral immunity, as well as t-cells proliferation, through activating antigen-presenting cells, when compared to naked hiv envelope plasmid dna treatment in vivo [ ] . this makes gold nanorods promising dna vaccine adjuvants for hiv treatment. the use of gold nanoparticles in hiv vaccine development has been recently reviewed by several authors [ ] . after more than years since the discovery of hiv in , no effective vaccine is yet available [ , [ ] [ ] [ ] . some histocompatibility complex molecules expressed on the surface of hiv are potential targets for neutralizing antibodies [ ] , as shown in figure . among the few broadly neutralizing hiv monoclonal antibodies, g is the only carbohydrate-directed that is able to recognize a cluster of high-mannose glycans on the viral envelope glycoprotein gp [ ] . this type of glycan is thus envisaged as a target to develop an hiv vaccine capable of eliciting g -like antibodies. gold nanoparticles coated with self-assembled monolayers of synthetic oligomannosides, which are present in gp , are able to bind g with high affinity and to interfere with g /gp binding [ ] . thiol-terminated oligosaccharides have been attached on gold nanoparticles and have been used in attempts to develop an hiv vaccine [ ] . two nanometer gold glyconanoparticles were coated with synthetic partial structures of several mannosides [ ] . the assembly of the antennas of the gp high-mannose type glycan on gold glyconanoparticles provided superior binding to the anti-hiv antibody g , which could help in the design of a carbohydrate-based vaccine against hiv. it was shown that conjugation to negatively charged gold glyconanoparticles could stabilize either the alpha-helix or beta-strand conformation of the third variable region (v peptide) of the hiv- gp [ ] . the peptide on the nanoparticles shows more stability toward peptidase degradation the use of gold nanoparticles in designing vaccines against tick-borne encephalitis (tbe) was proposed for the first time by demenev et al. [ ] . the vaccine was prepared by conjugating colloid gold with a soluble tbe antigen. in animals immunized with the experimental vaccine, the protection coefficient and mean survival time were, respectively, . - . times and - % higher than in mice immunized with a commercial vaccine. moreover, the mean survival time was . - . times longer in animals injected with antibodies from mice immunized with the experimental vaccine, compared with the commercial one. in a more recent study, colloidal gold particles were used as carriers of protein antigen of the capsid of the tbe virus in the antiviral vaccine [ ] . after two inoculations, the mice in that study were found to resist challenge with , times the % lethal dose (ld ) of jev (beijing- strain), even when immunized with a relatively small dose of . mug of plasmid dna. japanese b encephalitis vaccine is an important vaccine to prevent this serious mosquito-borne disease caused by the virus [ , ] . there are some semiquantitative methods to determine this vaccine, such as plaque forming unit and the animal testing, but they have low sensitivity, are time-consuming, and have a high cost. a label-free amperometric immunosensor for fast and sensitive assay of japanese b encephalitis vaccine was reported with a specific response in the range . × − to . × − lg·pfu/ml (pfu/ml is plaque forming unit and lg is common logarithm) with a detection limit of × − lg·pfu/ml [ ] . an immunosensor based on the immobilization of antiserum of japanese b encephalitis on a gold electrode modified by [nano-au/co(bipyridine) + ] /nano-au/l-cysteine has also been reported [ ] . it exhibited fast potentiometric high sensitivity and long-term stability. these works are promising test methods for biological products. hepatitis is inflammation of the liver that may originate a yellow color in the skin and eyes and abdominal pain, among other symptoms that can be caused by viruses or from the abuse of alcohol and certain medications. there are several types of viral hepatitis: types a, b, c, d, and e. hepatitis a and e are often spread by contaminated food and water, while hepatitis b is mainly sexually transmitted but may pass from mother to child during pregnancy. types b and c are usually spread through figure shows a comparison between the viruses. hepatitis is inflammation of the liver that may originate a yellow color in the skin and eyes and abdominal pain, among other symptoms that can be caused by viruses or from the abuse of alcohol and certain medications. there are several types of viral hepatitis: types a, b, c, d, and e. hepatitis a and e are often spread by contaminated food and water, while hepatitis b is mainly sexually transmitted but may pass from mother to child during pregnancy. types b and c are usually spread through infected blood. hepatitis d can only infect people already infected with hepatitis b. figure shows a comparison between the viruses. concerning hepatitis b vaccine research, hbsag-dna can be introduced into the host by intramuscular or intradermal route using a needle with syringe. an alternative is using gene-gun, biolistic ® , powderjecttm, accell ® , or particle-mediated dna delivery, when dna-coated microscopic gold particles are transported using a needle-free device directly into the epidermic cells [ ] . micron-size gold particles were used as particulate adjuvants and coinjected intradermally with plasmid dna encoding the hbsag into mice [ , ] . the presence of gold particles accelerated the antibody response significantly, increased the percentage of responding animals, and shortened the time taken to reach maximal antibody titers by two weeks. these immunizations were effective in protecting mice against tumor challenge with cancer cells, expressing hbsag as a surrogate cancer antigen. good results were also obtained with minipigs [ ] . electrically activated plasmonic au nanoparticles can drive vibrational and dipole-like oscillations that are able to disrupt nearby cell membranes, allowing enhanced cell poration and facilitating the uptake of a model hepatitis c virus dna vaccine was recently reported [ ] . immunized mice showed up to -fold higher gene expression compared to control treatments (without nanoparticles) and exhibited significantly increased levels of both antibody and cellular immune responses. a new method of preparation of a vaccine for hepatitis e (heva) using in situ easy growth of gold clusters in the vaccine was recently reported [ ] . the prepared heva/au enhances its immune responses in vivo and reduces the potential toxicity of heva. the fluorescence of gold clusters enables the heva to be traceable, which may open a way to track the dynamic behavior of vaccines and further help to optimize an individual therapeutic regimen for immunotherapy [ ] . gold has been used in several other vaccines. some examples are reported below. gold nanoparticles were conjugated to a synthetic peptide of the vp capsid protein of the foot-and-mouth disease virus, which causes an acute, highly contagious infection of domestic and wild animals, transmittable to humans, for which the existing vaccines are not much effective [ ] . the resulting conjugate (with or without the use of complete freund's adjuvant) was compared to a commercial vaccine and to the native peptide, in immunization of guinea pigs. the titer and sensitivity of the antibodies were maximal for the combination comprising the nanoparticle-peptide conjugate and complete freund's adjuvant. gold nanoparticles were evaluated as vaccine carriers for enhancing the antibody response against a resembling foot-and-mouth disease virus peptide [ ] . particles with - nm in diameter stimulated the highest antibody levels and accumulated at the highest numbers in the spleen of mice. recently, the potential of chitosan functionalized gold nanoparticles (csaunps) for the transmucosal delivery of tetanus toxoid vaccine was shown [ , , ] . excellent stability was found for the formulation at recommended storage conditions. csaunps were also used as a carrier for the tetanus toxoid (tt) antigen along with the immunostimulant quillaja saponaria extract (qs) [ ] . the synthesized csaunps were spherical, around nm in size and conferred protection to tt against gastric hydrolysis. tt-qs-csaunps induced up to -fold immune responses compared to tt and tt-qs controls, after oral administration in mice. the delivery of tt and qs with functionalized csaunps might be a good approach for oral vaccine delivery [ ] . gold glyconanoparticles proved to be good carriers for a synthetic streptococcus pneumoniae type conjugate vaccine [ ] . a gold nanorod construct that displayed the major protective antigen of the respiratory syncytial virus (which causes pneumonia and wheezing in children and the elderly), the fusion protein (f) was reported, which can be a candidate vaccine preparation by the covalent attachment of viral protein using a layer-by-layer approach [ ] . gold nanoparticles of nm were conjugated to the matrix protein (m e) of influenza a virus m e through thiol-gold interactions [ ] . this virus ( figure ) is often responsible for seasonal epidemics and is also a high risk for pandemics [ ] . vaccination of mice with m e-au conjugates induced m e-specific igg serum antibodies, which significantly improved by addition of cpg as adjuvant, as mice immunized that way were fully protected against lethal pr [ ] . the same authors also showed that the inclusion of excess soluble m e antigen along with m e immobilized on au nanoparticles is vital for inducing high levels of antibody response, and in providing complete protection [ ] . also showed that the inclusion of excess soluble m e antigen along with m e immobilized on au nanoparticles is vital for inducing high levels of antibody response, and in providing complete protection [ ] . a recent study showed that animals immunized with a transmissible gastroenteritis virus, conjugated with colloidal gold nanoparticles, produced antibodies with a higher titer than those produced in response to the native virus [ ] . a gold nanoparticle antigen delivery approach was used together with a novel polysaccharide nanoparticulate adjuvant, and an effective t-cell vaccine was developed providing protection in animal models of listeriosis (a fatal infection for fetuses and newborns that causes meningitis and cutaneous lesions) [ ] . this antigen delivery approach against listeriosis, using gold nanoparticles and bacterial peptides, elicits protective cellular immunity, independent of the adjuvant used, either as a carbohydrate such as advax [ ] or a tlr- agonist such as dio- [ ] . gold glyconanoparticles loaded with listeriolysin peptide - (llo - ) or glyceraldehyde- -phosphate dehydrogenase - peptide (gapdh ) were successfully administrated to pregnant female mice as a vaccination for this disease [ ] . neonates from vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice showed clear brain affections and cutaneous diminishment of melanocytes. amphiphilic surface ligand-coated au nanoparticles able to target myeloid dendritic cells in lymph nodes were used as a peptide antigen carrier, increasing the efficacy of a model vaccine in b -ova melanoma mouse models [ ] . gold nanoparticles were coated with the f -antigen of yersinia pestis (bacterium responsible for the plague), using n-hydroxysuccinimide and n-( -dimethylaminopropyl)-n′-ethylcarbodiimide a recent study showed that animals immunized with a transmissible gastroenteritis virus, conjugated with colloidal gold nanoparticles, produced antibodies with a higher titer than those produced in response to the native virus [ ] . a gold nanoparticle antigen delivery approach was used together with a novel polysaccharide nanoparticulate adjuvant, and an effective t-cell vaccine was developed providing protection in animal models of listeriosis (a fatal infection for fetuses and newborns that causes meningitis and cutaneous lesions) [ ] . this antigen delivery approach against listeriosis, using gold nanoparticles and bacterial peptides, elicits protective cellular immunity, independent of the adjuvant used, either as a carbohydrate such as advax [ ] or a tlr- agonist such as dio- [ ] . gold glyconanoparticles loaded with listeriolysin peptide - (llo - ) or glyceraldehyde- -phosphate dehydrogenase - peptide (gapdh( - )) were successfully administrated to pregnant female mice as a vaccination for this disease [ ] . neonates from vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice showed clear brain affections and cutaneous diminishment of melanocytes. amphiphilic surface ligand-coated au nanoparticles able to target myeloid dendritic cells in lymph nodes were used as a peptide antigen carrier, increasing the efficacy of a model vaccine in b -ova melanoma mouse models [ ] . gold nanoparticles were coated with the f -antigen of yersinia pestis (bacterium responsible for the plague), using n-hydroxysuccinimide and n-( -dimethylaminopropyl)-n -ethylcarbodiimide hydrochloride coupling chemistry, and administrated to mice, triggering an igg antibody response to the f -antigen [ ] . the sera raised against f -antigen coupled to au nanoparticles were able to competitively bind to recombinant f -antigen, displacing protective macaque sera. target antigens identified in plasmodium falciparum, the parasite responsible for malaria, include surface proteins pfs and pfs / in male and female gametocytes and pfs expressed in zygotes and ookinetes. the codon-harmonized recombinant pfs in escherichia coil (chrpfs ) is able to produce malaria transmission-blocking antibodies in mice. chrpfs was also investigated, with gold nanoparticles of different shapes, size and physicochemical properties as adjuvants for induction of transmission blocking immunity, causing transmission blocking antibodies [ ] . these results show that gold nanoparticle-based formulations can be developed as nanovaccines to enhance the immunogenicity of vaccine antigens. gold nanoparticles were conjugated to n-terminal domains of pseudomonas aeruginosa flagellin recombinant protein through direct interaction of thiol molecules of the cysteines with gold and formation of au-s bond [ ] . mice that received aunp-flagellin(( - )) with freund's adjuvant produced high titers of anti-flagellin(( - )) antibodies that effectively recognized the native flagellin on the bacteria. synthetic virus-like particles (svlps) were prepared by incubating nm gold nanoparticles in a solution containing an avian coronavirus spike protein [ ] . after removing the free proteins, antigen-laden particles were recovered and showed morphological semblance to natural viral particles. vaccination with the svlps showed enhanced lymphatic antigen delivery, stronger antibody titers, increased splenic t-cell response, and reduced infection-associated symptoms, and provided superior antiviral protection when compared to a commercial whole inactivated virus vaccine [ ] . burkholderia mallei are bacteria responsible for the glanders disease, which is recently classified as a tier agent, since they can be weaponized for aerosol release, cause high mortality rates, and show multi-drug resistance, and for which there is so far no vaccine available [ ] . gold nanoparticles were covalently coupled with one of three different protein carriers (tethc, hcp , and flic) followed by conjugation to lipopolysaccharide (lps) generated significantly higher antibody titers compared with lps alone. in another study, a gold nanoparticle glycoconjugate composed of burkholderia thailandensis lps conjugated to flic was evaluated for the first time as a candidate vaccine for glanders on rhesus macaques (macaca mulatta) [ ] . it has recently been shown that the pichia pastoris yeast expression system was adequate for the production of recombinant-truncated proteins, and their apparent bioactivity suggests that torf , torf c, and torf d are potential candidate vaccines against cyprinid herpes virus infection. found in farmed gibel carp carassius gibelio, it is an infectious disease that recently emerged in china that has been troubling the aquaculture industry [ ] . colloidal gold-labelled immunoglobulin was used to confirm rv c protein localization on mycobacterium tuberculosis [ ] . these results, along with those obtained for other proteins, might lead to the discovery of select peptides that could have the potential to be included in a subunit-based vaccine for tuberculosis, an infectious disease that remains lethal around the world. biocompatible gold nanoparticles were complexed with anti-dengue virus small interfering rnas (sirna), and were able to enhance the sirna delivery and stability, becoming a novel strategy against a virus that causes flu-like symptoms, haemorrhagic fever, and death [ ] . escherichia coli as a model pathogen for the design of an antibacterial vaccine [ ] . the bacterial outer membrane vesicles were collected and successfully coated onto gold nanoparticles with a nm diameter. the resulting bacterial membrane-coated gold nanoparticles, when injected subcutaneously, induced rapid activation and maturation of dendritic cells in the lymph nodes of the vaccinated mice, generated durable antibody responses, and induced a high production of interferon gamma and interleukin- , but not interleukin- , indicating its capability of generating strong th -and th -biased cell responses against the used bacteria. a monoclonal antibody raised against human cytomegalovirus (cmv, a herpes virus) surface glycoprotein (gb) was chemically conjugated with gold nanoparticles [ ] . the gb-gold nanoparticles blocked viral replication, virus-induced cytopathogenic effects, and virus spread in cell culture without inducing cytotoxicity, and cells treated with them gained resistance to cmv infection. as discussed above, research on gold has indicated its potential for several applications in nanomedicine and its (bio)medical applications, including vaccines, and, as shown in this review, has been increasing. the overall conclusion is that the potential of gold for stimulating research in vaccines is considerable. the results will certainly lead to more practical and commercial applications, the full extent of which has yet to be envisaged. certainly, the use of gold carries added costs. as newer and more expensive vaccines are introduced and attempted to reach people of different ages and in new settings, the logistics systems must be strengthened and optimized, as recently highlighted and reviewed by zaffran et al. [ ] . catalysis by gold nanoparticles when gold is not noble: catalysis by nanoparticles gold catalysis catalysis by gold catalytic applications for gold nanotechnology anisotropic gold nanoparticles: preparation and applications in catalysis gold nanoparticles in biology and medicine: recent advances and prospects gold nanoparticles in biomedical applications: recent advances and perspectives biological applications of gold nanoparticles particulate inorganic adjuvants: recent developments and future outlook gold nanoparticles and their applications in biomedicine applications of nanotechnology in the agriculture, food, and pharmaceuticals use of nanoparticles to deliver immunomodulatory oligonucleotides current status of multiple antigen-presenting peptide vaccine systems: application of organic and inorganic nanoparticles vaccine delivery using nanoparticles applications of nanomaterials as vaccine adjuvants. hum. vaccines immunother oral delivery of nanoparticle-based vaccines synthetic nanoparticles for vaccines and immunotherapy gold nanoparticles and vaccine development gold nanocluster-based vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides gold nanoparticles (aunps): a new frontier in vaccine delivery design of nanomaterial based systems for novel vaccine development recent advances in synthetic carbohydrate-based human immunodeficiency virus vaccines advanced nanobiomaterials: vaccines, diagnosis and treatment of infectious diseases recent advances in subunit vaccine carriers nanomaterial-based vaccine adjuvants understanding the immunogenicity and antigenicity of nanomaterials: past, present and future immunological properties of gold nanoparticles role of metallic nanoparticles in vaccinology: implications for infectious disease vaccine development vaccine technologies: from whole organisms to rationally designed protein assemblies review: cancer therapy and vaccination biomedicine: new insights from plant viruses nanovaccines and their mode of action glycodendrimers: versatile tools for nanotechnology. braz functional nanomaterials can optimize the efficacy of vaccines nanomedicine in the management of microbial infection-overview and perspectives nanomedicine scale-up technologies: feasibilities and challenges nanoparticle based tailoring of adjuvant function: the role in vaccine development the potential of nanoparticles for the immunization against viral infections learning from vaccines and progressing to antigen-specific immunotherapies advances in multifunctional glycosylated nanomaterials: preparation and applications in glycoscience nanoparticle-based modulation of the immune system real-time assessment of nanoparticle-mediated antigen delivery and cell response a study of the nucleation and growth processes in the synthesis of colloidal gold development of chitosan based gold nanomaterial as an efficient antifilarial agent: a mechanistic approach gold colloidal solution is used as an adjuvant, provides reducing toxicity of vaccines, to enhance their immunogenic activity and to provide stability of vaccine in storage. patent ru -c new adjuvant composition comprising colloidal gold, used for therapeutic vaccine or for inducing immune response gold nanoparticles as carriers for a synthetic streptococcus pneumoniae type conjugate vaccine gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo injectable nanomaterials for drug delivery: carriers, targeting moieties, and therapeutics high-density sub- -nm peptide-gold nanoparticle complexes improve vaccine presentation by dendritic cells in vitro gold nanoparticle conjugates: recent advances toward clinical applications polymeric nanoparticles potent vectors for vaccine delivery targeting cancer and infectious diseases tracking targeted bimodal nanovaccines: immune responses and routing in cells, tissue, and whole organism protection of non-human primates against glanders with a gold nanoparticle glycoconjugate vaccine synthesis of tumor-associated muc -glycopeptides and their multivalent presentation by functionalized gold colloids metal based frameworks for drug delivery systems gold nanoparticles: recent advances in the biomedical applications additives for vaccine storage to improve thermal stability of adenoviruses from hours to months assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide structure function attributes of gold nanoparticle vaccine association: effect of particle size and association temperature intracellular accumulation and immunological properties of fluorescent gold nanoclusters in human dendritic cells different-sized gold nanoparticle activator/antigen increases dendritic cells accumulation in liver-draining lymph nodes and cd +t cell responses yeast-expressed bacteriophage-like particles for the packaging of nanomaterials polyelectrolyte multilayers assembled entirely from immune signals on gold nanoparticle templates promote antigen-specific t cell response impact of dose, route, and composition on the immunogenicity of immune polyelectrolyte multilayers delivered on gold templates construction and immunological evaluation of cpg-au@hbc virus-like nanoparticles as a potential vaccine ultrasmall graphene oxide supported gold nanoparticles as adjuvants improve humoral and cellular immunity in mice engineered cpg-antigen conjugates protected gold nanoclusters as smart self-vaccines for enhanced immune response and cell imaging endotoxin nanovesicles: hydrophilic gold nanodots control supramolecular lipopolysaccharide assembly for modulating immunological responses epidermal powder immunization induces both cytotoxic t-lymphocyte and antibody responses to protein antigens of influenza and hepatitis b viruses powder and particle-mediated approaches for delivery of dna and protein vaccines into the epidermis recent advances in hepatitis b vaccination nanoparticulate mediated transcutaneous immunization: myth or reality a solid-in-oil dispersion of gold nanorods can enhance transdermal protein delivery and skin vaccination a microarray mems device for biolistic delivery of vaccine and drug powders cancer: facts, causes, symptoms and research application of nanostructured drug delivery systems in immunotherapy of cancer: a review end results in hodgkin's disease and lymphosarcoma treated by the mixed toxins of erysipelas and bacillus prodigiosus, alone or combined with radiation in vitro administration of gold nanoparticles functionalized with muc- protein fragment generates anticancer vaccine response via macrophage activation and polarization mechanism glycopolymer-functionalized gold nanoparticles: a new strategy toward synthetic anticancer vaccines development of a novel cancer vaccine based on multivalent presentation of tumor-associated carbohydrate antigens on gold nanoparticle scaffolds design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines imageable antigen-presenting gold nanoparticle vaccines for effective cancer immunotherapy in vivo gold nanoparticle mediated cancer immunotherapy gold nanoparticles displaying tumor-associated self-antigens as a potential vaccine for cancer immunotherapy current status and future directions of nanoparticulate strategy for cancer immunotherapy gold nanoparticles as an antigen and as an adjuvant multicopy multivalent' glycopolymer-stabilized gold nanoparticles as potential synthetic cancer vaccines glycopeptide-functionalized gold nanoparticles for antibody induction against the tumor associated mucin- glycoprotein in vivo gold nanoparticle delivery of peptide vaccine induces anti-tumor immune response in prophylactic and therapeutic tumor models role of nanotechnology in hiv/aids vaccine development gold nanoparticles coated with oligomannosides of hiv- glycoprotein gp mimic the carbohydrate epitope of antibody g gold manno-glyconanoparticles for intervening in hiv gp carbohydrate-mediated processes enhancing dendritic cell activation and hiv vaccine effectiveness through nanoparticle vaccination immunization with recombinant hla classes i and ii, hiv- gp , and siv p elicits protection against heterologous shiv infection in rhesus macaques design and synthesis of thiol-terminated oligosaccharides for attachment on gold nanoparticles: toward the development of an hiv vaccine assembling different antennas of the gp high mannose-type glycans on gold nanoparticles provides superior binding to the anti-hiv antibody g than the individual antennas negatively charged glyconanoparticles modulate and stabilize the secondary structures of a gp v loop peptide: toward fully synthetic hiv vaccine candidates surface-engineered gold nanorods: promising dna vaccine adjuvant for hiv- treatment perfection of methodical approaches to designing vaccines against tick-borne encephalitis inoculation of plasmids encoding japanese encephalitis virus prm-e proteins with colloidal gold elicits a protective immune response in balb/c mice a label-free amperometric immunosenor based on multi-layer assembly of polymerized o-phenylenediamine and gold nanoparticles for determination of japanese b encephalitis vaccine layer-by-layer self-assembly of films of nano-au and co(bpy)( )( +) for the determination of japanese b encephalitis vaccine accelerated immune response to dna vaccines enhancement of the effectiveness of electroporation-augmented cutaneous dna vaccination by a particulate adjuvant the assessment of local tolerance, acute toxicity, and dna biodistribution following particle-mediated delivery of a dna vaccine to minipigs electrically oscillating plasmonic nanoparticles for enhanced dna vaccination against hepatitis c virus assembly of hepatitis e vaccine by 'in situ' growth of gold clusters as nano-adjuvants: an efficient way to enhance the immune responses of vaccination use of a synthetic foot-and-mouth disease virus peptide conjugated to gold nanoparticles for enhancing immunological response gold nanoparticles as a potential carrier for transmucosal vaccine delivery enhanced mucosal immune responses against tetanus toxoid using novel delivery system comprised of chitosan-functionalized gold nanoparticles and botanical adjuvant: characterization, immunogenicity, and stability assessment quillaja saponaria extract as mucosal adjuvant with chitosan functionalized gold nanoparticles for mucosal vaccine delivery: stability and immunoefficiency studies gold nanorod vaccine for respiratory syncytial virus gold nanoparticle-m e conjugate coformulated with cpg induces protective immunity against influenza a virus m e-immobilized gold nanoparticles as influenza a vaccine: role of soluble m e and longevity of protection immunostimulatory effect of gold nanoparticles conjugated with transmissible gastroenteritis virus novel nanoparticle vaccines for listeriosis. hum. vaccines immunotherapeut a gold glyco-nanoparticle carrying a listeriolysin o peptide and formulated with advax™ delta inulin adjuvant induces robust t-cell protection against listeria infection pregnancy vaccination with gold glyco-nanoparticles carrying listeria monocytogenes peptides protects against listeriosis and brainand cutaneous-associated morbidities high-throughput quantitation of inorganic nanoparticle biodistribution at the single-cell level using mass cytometry conjugation of y. pestis f -antigen to gold nanoparticles improves immunogenicity nanovaccines for malaria using plasmodium falciparum antigen pfs attached gold nanoparticles induction of humoral immune response against pseudomonas aeruginosa flagellin( - ) using gold nanoparticles as an adjuvant synthetic virus-like particles prepared via protein corona formation enable effective vaccination in an avian model of coronavirus infection a gold nanoparticle-linked glycoconjugate vaccine against burkholderia mallei protective immunity in gibel carp, carassius gibelio of the truncated proteins of cyprinid herpesvirus expressed in pichia pastoris rv c protein peptide inhibiting mycobacterium tuberculosis h rv entry to target cells delivery of antiviral small interfering rna with gold nanoparticles inhibits dengue virus infection in vitro modulating antibacterial immunity via bacterial membrane-coated nanoparticles inhibition of cytomegalovirus infection and photothermolysis of infected cells using bioconjugated gold nanoparticles the imperative for stronger vaccine supply and logistics systems key: cord- -eeg k a authors: detoc, maëlle; bruel, sébastien; frappe, paul; tardy, bernard; botelho-nevers, elisabeth; gagneux-brunon, amandine title: intention to participate in a covid- vaccine clinical trial and to get vaccinated against covid- in france during the pandemic date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: eeg k a introduction the world is facing the covid- pandemic. the development of a vaccine is challenging. we aimed to determine the proportion of people who intend to get vaccinated against covid- in france or to participate in a vaccine clinical trial. methods we conducted an anonymous on-line survey from the th of march to the th of april . primary endpoints were the intention to get vaccinated against covid- if a vaccine was available or participate in a vaccine clinical trial. results three thousand two hundred and fifty nine individuals answered the survey; women accounted for . % of the respondents. according to their statements, . participants ( . %, % ci . - %) will certainly or probably agree to get vaccinated against covid- . older age, male gender, fear about covid- , being a healthcare worker and individual perceived risk were associated with covid- vaccine acceptance. vaccine hesitancy was associated with a decrease in covid- vaccine acceptance. one thousand and five hundred and fifty respondents ( . % % ci . - . %) will certainly or probably agree to participate in a covid- vaccine clinical trial. older age, male gender, being a healthcare worker and individual perceived risk were associated with potential acceptance to participate in a covid- vaccine clinical trial. vaccine hesitancy was associated with refusal for participation in a covid- vaccine clinical trial. conclusions nearly % and % of the survey respondents were respectively likely to accept vaccination or participation in a clinical trial against covid- . vaccine hesitancy will be the major barrier to covid- vaccine uptake. coronavirus: the severe acute respiratory syndrome-coronavirus (sars-cov ). the world is now facing a pandemic, more than millions of people have become infected worldwide, cases are described in more than hundred countries, and more than , people died [ ] . in , the world health organization identified ten threats to global health [ ] . among these threats, vaccine hesitancy, the risk of a global influenza pandemic, and the risk of emergence of highthreat pathogens such as middle-east respiratory syndrome and/or severe acute respiratory syndrome were identified. since this statement, covid- had emerged. developing covid- vaccines is a crucial challenge, and several candidates are currently under basic development [ ] . some of them will be tested in phase i trials in the weeks to come [ ] . the time it takes to develop a vaccine is estimated to be or . years as different steps are necessary during the clinical development of a vaccine [ ] . recruitment of volunteers in a vaccine clinical trial is a real challenge [ ] , and some trials are stopped due to difficulties in recruitment. vaccine hesitancy may also have an impact on recruitment in covid- vaccine clinical trial [ ] . after its clinical development, a covid- vaccine will also face the challenge of acceptance by the general population in a post-crisis context. . the impact of the current pandemic on the intention to participate in a covid- vaccine clinical trial and on the intention to get vaccinated against covid- vaccine is not obvious. this is a particular concern in france, which has been shown to be the leader-country of vaccine hesitancy [ ] . the aim of this study was to evaluate the intention to get vaccinated against covid- among the general population and healthcare personnel, in the context of the current pandemic. we conducted an anonymous online survey (lime survey®) from the th of march to the th of april among adult general population and adult patients. the survey was proposed to individuals via social networks (facebook, twitter), shared by e-mail, on the website of the university hospital of saint-etienne (france), and in centers for covid- diagnosis and in medical centers. we developed a standardized questionnaire based on a literature review. the questionnaire addressed: ( ) demographical characteristics (age, chronic medical conditions), ( ) fears about covid- , ( ) history of vaccination against pandemic h n influenza and seasonal influenza, ( ) intention to get vaccinated if a covid- vaccine was available, ( ) vaccine hesitancy. according to the who strategic advisory group of experts on immunization, vaccine hesitancy refers to delay in acceptance or refusal of vaccines despite availability of vaccination services. vaccine hesitancy is complex and context specific, varying across time, place and vaccines. it is influenced by factors such as complacency, convenience and confidence [ ] . we evaluated participants' self-reported vaccine hesitancy according to the who definition using three previously adapted questions: "have you ever refused a vaccine for yourself or a child because you considered it as useless or dangerous?" "have you ever postponed a vaccine recommended by a physician because of doubts about it?" "have you ever had a vaccine for a child or yourself despite doubts about its efficacy" [ ] . if a participant answered yes to one of these proposals, he or she was considered to be "vaccine hesitant". the questionnaire included items to be answered on a -level likert scale including a "don't know" option to evaluate intention to participate in a clinical trial and to get vaccinated if a vaccine was available. we combined survey responses into two categories (strongly agree/agree, don't know/disagree/strongly disagree) and ran ordinal regression models to examine demographic and attitudinal factors predictive of respondents' willingness to get vaccinated against covid- . to identify suitable candidate variables for regression models, we first conducted univariate analysis using a chi-squared test. candidates that were significant at p< . in univariate analyses were then included in a multivariable regression model. data were analyzed using spss version . . the protocol complied with the data privacy laws of the national commission for informatics and civil liberties and was approved by the institutional review board with the number during the study period, , people opened the web links for the online survey, according to their statements, , participants ( . %, % ci . factors associated with covid- vaccine acceptance are displayed in table . in multivariable analysis, older age, male gender, fear about covid- , be healthcare workers and individual perceived risk remained associated with covid- vaccine acceptance. vaccine hesitancy was associated with lower acceptance of a covid- vaccine. one thousand and five hundred and fifty two respondents ( . % % ci . - . %) will certainly or probably be willing to participate in a covid- vaccine clinical trial. among the . men, ( . % % ci . - . %) will probably accept to participate in a covid- vaccine clinical trial, this proportion is significantly greater than women ( . % % ci . - . %, p< . ). the percentage of potential participants in a covid- vaccine clinical trial was . % ( . - . %) in the - years age group, and . % ( % ci . - . %) in the - years age group. healthcare workers are more prone to participate in a vaccine clinical trial than non-healthcare workers ( . % vs . %, p< . ). factors associated with covid- vaccine clinical trial acceptance are displayed in table . in this online survey, we observed that nearly three quarters of the respondents would accept a vaccine against covid- although % of the respondents were qualified as "vaccine hesitant". moreover, around a half of the respondents would accept to participate in a clinical trial for a covid- vaccine. concerning the intention to get vaccinated, our results are similar to the results of the longitudinal coconel study conducted by the observatoire régional de santé provence alpes côte d'azur [ ] and to results of investigations conducted in the usa [ , ] . similar to coconel, we observed that men were more prone to get vaccinated than women. women accounted for the vast majority of our study respondents, suggesting that in real-life settings, covid- vaccine acceptance could be greater. in addition, older individuals are more prone to get vaccinated in both studies, this is probably due to a greater perceived risk of getting infected and developing a severe disease in older people. we analyzed factors associated with perceived risk of sars-cov- infections, and with fear about covid- . male gender and older age were not associated with perceived individual risk but were associated with fear about covid- (data not shown). in the same vein, healthcare workers were more prone to get vaccinated or to participate in a vaccine clinical trial than the others. we hypothesized that healthcare workers perceived a greater risk to get infected. healthcare workers are particularly vulnerable and accounted for % of the infected people in italy, and in greece [ , ] . on the contrary, expecting a low infection risk is associated with a lower willingness to get vaccinated [ ] . seventy-eight percent of healthcare workers considered themselves at-risk to get infected, however after adjustment on age, gender, and comorbidities, being a healthcare worker was not associated with perceived risk to get infected, but with fear about covid- (data not shown). around the half of the respondents will accept to participate in a covid- vaccine clinical trial. in the context of a clinical trial, men were also more prone to participate. fears about covid- were not associated with willingness to participate in a clinical trial. however, individuals who considered themselves at-risk for covid- infection were more prone to accept to participate in a clinical trial for a vaccine. outside the pandemic context, self-perceived risk for a disease was not the main motivation to participate in a vaccine clinical trial [ ] . moreover, older age was associated (except in the group over years) with willingness to participate in a covid- vaccine clinical trial, this observation contrasts with a previous study about willingness to participate in a vaccine clinical trial [ ] . this observation suggests that in the pandemics context, individuals are more prone to participate in a clinical trial for a vaccine. we also observed that a part of respondents who will certainly accept to participate in a vaccine clinical trial will not certainly get the vaccine if available. further studies are needed to explore indepth the differences in barriers and motivations to participation in a vaccine clinical trial, and to the acceptance of the vaccine. next steps will be the construction of multi-component interventions to facilitate recruitment of volunteers in clinical trials, and to increase covid- vaccine acceptance. communities should be involved in the development of these interventions. the rush to develop a covid- vaccine may jeopardize public confidence in scientists [ ] . the proportion of french people favorable to a covid- vaccine can appear low, but we have to put this in perspective with the fact that the vaccine would be a new vaccine, and that vaccine coverage against h n pandemic influenza was only . % in france [ ] . however, a greater proportion of respondents to our survey declared they had been vaccinated against h n pandemic influenza, so this observation may suggest that the respondents are more pro-vaccine than the general population in france, and more often healthcare workers. we observed a similar prevalence of vaccine hesitancy than in a recent study in france, which identified a prevalence of % [ ] . one limitation of our work may be the use of social media to recruit study participants. social media users who use platforms, such as facebook and twitter, as health information sources are more prone to get vaccinated against seasonal influenza in the united states of america [ ] . furthermore, a great number of healthcare workers answered the survey and we observed that healthcare workers were more prone to get vaccinated or to participate in a vaccine clinical trial independently of the perceived risk to get contaminated. however, vaccine hesitancy also affects healthcare workers [ ] [ ] [ ] . in our study sample, vaccine hesitancy affects around % of the healthcare workers and % of the non-healthcare workers. our work suffers from limitations. first, our sample is not completely representative of the french general population, and the great amount of hcws may overestimate the proportion of individuals with intentions to get vaccinated. in a recent report, in european countries, the proportion of individuals with intention to get vaccinated against covid- was . %, but in france, it was only % [ ] . this survey was also conducted on line but a priori in a representative panel of . individuals per country. secondly, we did not precise in our survey, the type of clinical trials, and intention to participate may change between early and later phases clinical trials [ ] . in conclusion, during the pandemics, around % of the french people would agree to get vaccinated. due to the burden of the disease, and the potential natural immunity, it may well be possible that this proportion would be enough to obtain a herd effect [ ] . antecedents of vaccine hesitancy may affect vaccine acceptance even in a context of a pandemic due to an emerging pathogens. around fifty percent will agree to participate in a covid- vaccine clinical trial; we can hope that vaccine trials would not be stopped because of recruitment difficulties. co-authors have no conflict of interest to declare for this work. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: dr amandine gagneux-brunon on the behalf of all co-authors covid- map. johns hopkins coronavirus ten health issues who will tackle this year n developing covid- vaccines at pandemic speed the covid- vaccine development landscape human challenge studies to accelerate coronavirus vaccine licensure public interest in medical research participation: differences by volunteer status and study type barriers and motivations for participation in preventive vaccine clinical trials: experience of clinical research sites the state of vaccine confidence : global insights through a -country survey who | improving vaccination demand and addressing hesitancy vaccine hesitancy in the french population in , and its association with vaccine uptake and perceived vaccine risk-benefit balance a majority of vaccine skeptics plan to refuse a covid- vaccine, a study suggests, and that could be a big problem planning for a covid- vaccination program covid- : protecting healthcare workers is a priority cov- infection in healthcare personnel with high-risk occupational exposure: evaluation of sevenday exclusion from work policy future pandemics and vaccination: public opinion and attitudes across three european countries the rush to create a covid- vaccine may do more harm than good social media use and influenza vaccine uptake among white and african american adults vaccine hesitancy among general practitioners: evaluation and comparison of their immunisation practice for themselves, their patients and their children discrepancies between general practitioners' vaccination recommendations for their patients and practices for their children vaccine hesitancy and self-vaccination behaviors among nurses in southeastern france once we have it, will we use it? a european survey on willingness to be vaccinated against covid- herd immunity and herd effect: new insights and definitions key: cord- -mn r x authors: hodgins, douglas c.; chattha, kuldeep; vlasova, anastasia; parreño, viviana; corbeil, lynette b.; renukaradhya, gourapura j.; saif, linda j. title: mucosal veterinary vaccines: comparative vaccinology date: - - journal: mucosal immunology doi: . /b - - - - . - sha: doc_id: cord_uid: mn r x infections of mucosal surfaces are major causes of morbidity, mortality, and economic loss in species of veterinary interest, and a concern for animal welfare. vaccines are used extensively in veterinary medicine, and innovative vaccine technologies such as recombinant dna-vectored and distinguishing infected from vaccinated animals (diva) vaccines and automated in ovo vaccination (of embryonated chicken eggs) have been rapidly adopted commercially. immunological research using outbred, nonrodent animal models has contributed to a broader understanding of mucosal defenses, and has provided the initial impetus for investigation of the common mucosal immune system. studies of the potential of novel adjuvants to improve vaccine efficacy against genetically unstable, immune-subverting rna viruses, such as porcine reproductive and respiratory syndrome virus in pigs, should assist in the control of pathogens with similar characteristics in other species. successful development of vaccines to prevent and treat ascending infections of the reproductive tract of cattle set a precedent for applications in other species including humans. studies of mucosal adjuvants and delivery systems continue at the interface between passive and active immunity, with the goal of inducing the earliest possible protection against enteric and respiratory pathogens of neonates. despite advances in nutrition, genetics, housing, and therapeutics, diseases of the respiratory, reproductive, and gastrointestinal tracts of domestic animals and poultry continue to be major causes of morbidity and mortality. although vaccines have been developed and licensed for prevention of many of these diseases, there is a need for improvement in vaccine efficacy and for new vaccines. reasons for low vaccine efficacy include inappropriate, unstable, or outdated antigens in vaccine preparations (e.g., in vitro expressed antigens instead of in vivo expressed antigens), inappropriate immune responses (e.g., systemic instead of mucosal, or th type instead of th type or vise versa), and inappropriate use of otherwise efficacious vaccines (e.g., inappropriate timing of vaccination) (tizard, ). an overview of some of the vaccines currently licensed for vaccination of domesticated animals and wildlife by mucosal routes is provided in table . many of these vaccines consist of pathogens attenuated by traditional methods, but engineered virus-vectored vaccines are now used extensively (by both mucosal and parenteral routes) in veterinary medicine. the majority of current poultry vaccines are attenuated agents delivered in ovo, orally, intranasally (in) or by other mucosal routes, for reasons of ease of administration, economy, and protective efficacy. in comparison, fewer vaccines for domestic mammals are delivered by mucosal routes. management practices for mammals differ from those for poultry; mass vaccination techniques for mucosal delivery have been developed for poultry, but have not been pursued as zealously for mammals. recently however, a number of attenuated live vaccines for in administration have been developed for respiratory tract infections, using traditional methodologies. improved protective efficacy and rapid onset of immunity compared to killed vaccine products have led to widespread acceptance. attenuated live oral vaccines for enteric diseases have also been marketed, but in many cases efficacy has been disappointing due to lack of potency in adults or interference by maternal antibodies in suckling animals. better strategies for induction of immunity in the gastrointestinal tract are needed, especially for neonates in the presence of maternal antibodies. in contrast, effective parenteral vaccines for the most common diseases of the reproductive tract in veterinary species have been available for years, and there has been little motivation to develop mucosal vaccines. many of the diseases of the respiratory and gastrointestinal tracts are most devastating in the neonatal period. for these diseases active immunization may not provide protection before natural exposure to the pathogen. maternal vaccination to enhance passive immunity has been widely used in veterinary medicine, especially for control of enteric diseases. practical difficulties arise, however, with diseases such as parvovirus enteritis in puppies in which a smooth transition must be made from protection by passive maternal antibodies to protection by active immunity, without permitting a window in between of disease susceptibility. this transition is difficult to achieve because induction of active immunity is commonly inhibited by maternal antibodies. various strategies are used to address this problem, but improved vaccines, adjuvants, and antigen delivery systems would improve the reliability of neonatal immunization. although progress is being made in disease prevention in veterinary species, ever changing management practices (e.g., earlier weaning of piglets, larger animal operations) generate new patterns of disease, requiring new control strategies. the emergence of new pathogens (e.g., porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus- ) provides new challenges for vaccine research before some of the older challenges have been met. in this chapter, we focus on mucosal veterinary vaccines and vaccine concepts related to selected pathogens of economic importance. our intent is to highlight progress, to review existing and future vaccination strategies, and to acknowledge the unique contributions of this research to our understanding of mucosal vaccines and immunology. respiratory tract infections are a major cause of morbidity and mortality in farm animals, poultry, and pets. disease conditions are intensified by current management practices such as mixing of recently weaned, stressed beef calves from multiple sources in auction barns. certain disease conditions, such as atrophic rhinitis of pigs, result from the interplay of several pathogens, and multiple agents must be represented in vaccines. some respiratory pathogens such as mycoplasma hyopneumoniae in young pigs are causing new patterns of disease as management practices change (e.g., weaning at an earlier age), requiring changes in vaccine strategies. other pathogens such as prrsv have only recently emerged and improved vaccines await advances in understanding of the agent and of disease pathogenesis. a discussion of respiratory vaccines for even the major pathogens of veterinary species is beyond the scope of the present review. this section will focus on vaccines for prrsv infections in pigs and influenza in horses to illustrate general principles. porcine reproductive and respiratory syndrome (prrs) is a chronic reproductive and respiratory disease of pigs caused by prrsv, a member of the family arteriviridae (benfield et al., ) . signs associated with prrs are anorexia, fever, lethargy, pneumonia, red/blue discoloration of the ears and vulva, delayed return of sows to estrus after weaning, abortion, fetal mummification, stillborn or weak born piglets, and high preweaning mortality (mengeling et al., ) . prrsv is prevalent in swine-producing countries worldwide. annual economic losses to the pork industry in the united states due to prrs have been estimated at $ million (holtkamp and kliebenstein, ) . according to the american animal and plant health inspection service (aphis) report of january , . % of unvaccinated pigs in the united states are seropositive to prrsv. infected pigs excrete prrsv in saliva, nasal secretions, urine, milk, colostrum, and feces at low levels or intermittently, and also in semen of infected boars (rossow et al., ) . in the absence of control measures, prrsv is spread by aerosols, fomites, and personnel. prrsv is divided into two distinct genotypes, type i (european) and type ii (north american). each genotype contains subtypes and strains, which are genetically diverse and vary in virulence and pathogenicity (kim et al., ) . immunity to one genotype of prrsv may provide partial or no protection against reinfection, reflecting the complexity of prrsv genetics and immunological variation among strains (botner et al., ) . swine are the only known species susceptible to prrsv. alveolar macrophages (mΦs) are the primary permissive cells to prrsv; mΦs present in pulmonary intravascular spaces, lymph nodes, thymus, heart, spleen, placenta, and umbilical cord may also be infected by the virus (halbur et al., ) . the most efficient and rapid host response against viruses consists of production of type i interferons (ifns) (ifnα and ifnβ). in prrsv-infected pigs, innate ifnα secretion is significantly suppressed (albina et al., ) and the virus dampens innate nk cell-mediated cytotoxicity as early as day postinfection (renukaradhya et al., ; dwivedi et al., ) . in pigs, a significant correlation has been observed between the prrsv infection and an increased expression of il- , associated with reduced expression of ifnα, ifnγ, il- , and tnfα (gómez-laguna et al., ) . induction of il- is mediated by interaction between prrsv proteins and mΦs/dendritic cells (dcs). the prrsv nucleocapsid protein induces il- production by peripheral blood mononuclear cells (pbmcs) and alveolar mΦs (wongyanin et al., ) , delaying the onset of protective cell-mediated immunity (cmi) to prrsv (dwivedi et al., a . dysregulated immune responses in infected pigs affect viral pathogenesis, disease severity, and susceptibility to secondary microbial infections (thanawongnuwech et al., ; renukaradhya et al., ) . immune responses against prrsv are inadequate to completely clear the virus. viremia disappears in - weeks, but the virus persists in the tonsils and lymphoid tissues for several months (moyron-quiroz et al., ) . prrsv infection in both germ-free and conventionally raised pigs is associated with polyclonal b cell activation, hypergammaglobulinemia, lymphoid adenopathy, renal lesions, and lymphoid hyperplasia (cooper et al., ) . polyclonal lymphoplasia also occurs in mice infected with lactate dehydrogenase-elevating virus, a related virus (grossmann et al., ) . primary antibody responses occur promptly after prrsv infection (loemba et al., ) , but the majority of secreted antibodies are autoantibodies or prrsv nonneutralizing antibodies (lemke et al., ) . the virusneutralizing antibody (na) response is weak, and delayed (mateu et al., ) . both killed and modified live virus (mlv) prrsv vaccines are available commercially for intramuscular or intradermal administration (charerntantanakul, ) . mlv vaccines confer protection against genetically homologous prrsv, but incomplete protection against heterologous viruses (mengeling et al., ) . in both prrsv-infected and mlv-prrsv-vaccinated pigs, virus-specific cell-mediated immune responses are either delayed or dampened . reversion to virulence is a major concern with modified live vaccines. there are numerous reports of reversion of prrsv vaccine virus to virulence and of the presence of reverted vaccine strain prrsv in unvaccinated sows and pigs . vaccine-derived virus has been isolated from fetuses, stillborn, and dead piglets, indicating the spread of disease from vaccinated pigs. identification of mutations in multiple vaccine-derived isolates at identical positions of the viral genome suggests a strong selective pressure on critical viral proteins in field situations. available killed prrsv vaccines fail to protect even against homologous virus, do not elicit na, and induce weak cell-mediated immune responses (bassaganya-riera et al., ) . pregnant sows and gilts and breeding boars are not protected from prrsv due to a lack of safe and protective vaccines. control of prrsv in breeding stock is critical in preventing vertical and horizontal transmission of the virus. early attempts to develop killed prrsv vaccines using recombinant prrsv proteins, plasmids expressing viral genes, and inactivated prrsv administered with or without adjuvants have been unsuccessful ( charerntantanakul, ) . however, recent studies show promise. killed prrsv vaccine coadministered in with cpg oligodeoxynucleotide adjuvant (a tlr- ligand) induced antibodies, virus-specific t cells, and secretion of ifnγ and il- (zhang et al., ) . in another study, prrsv inactivated by uv light or binary ethylenimine induced na and protected pigs against homologous viral challenge. the suppressive effect of live prrsv on ifnα production was lost when the virus was inactivated by uv irradiation (vanhee et al., ). an oral immunization strategy using prrsv nucleocapsid protein genetically fused to cholera toxin (ct) stimulated prrsv antibody responses in the intestines, but no detectable response in vaginal secretions (hyland et al., ) . lack of success in developing protective killed prrsv vaccines may reflect an inability to present killed prrsv antigens effectively to the pig immune system, or (like live prrsv) killed virus may induce immunosuppressive effects. in addition, the antigenic mass used in killed vaccines may be insufficient, suggesting the need for potent adjuvants or novel delivery systems. attempts have been made to improve mlv-prrsv vaccines using adjuvants. in one study, pigs were injected with recombinant porcine il- , il- , il- , or ct within week of intramuscular administration of mlv-prrsv. il- and ct enhanced ifnγ and gp antibody responses, respectively (foss et al., ) . however, use of these adjuvants did not reduce the severity of viremia. unfortunately, prrsv (na) responses were not assayed, and protection against heterologous challenge was not assessed in this study. prrsv gains entry through respiratory and reproductive mucosal surfaces and causes disease primarily at mucosal sites. therefore, a mucosal vaccine against prrsv may be an effective strategy for controlling the disease. until recently, attempts to elicit protective mucosal immunity against prrsv have been unsuccessful, probably due to a lack of appropriate adjuvants. because prrsv infection rapidly subverts the host immune responses, an effective adjuvant must overcome immunosuppression caused by vaccine virus and simultaneously potentiate virus-specific adaptive immunity. a panel of bacterial preparations derived from mycobacterium, vibrio, and streptococcus species, which were potent adjuvants in rodent models, were tested in with mlv-prrsv (bonavida et al., ; barral and brenner, ) . based on mucosal and systemic immune responses, three of the preparations, mycobacterium tuberculosis whole cell lysate (m. tb wcl), ct b subunit, and ok- (a product of streptococcus pyogenes), were selected for viral challenge trials. only m. tb wcl significantly dampened prrsv immunosuppressive effects, and enhanced virus-specific adaptive responses (renukaradhya et al., ; dwivedi et al., b) . historically, heat-killed mycobacteria are recognized to have as potent adjuvant effects as components of freund's complete adjuvant (fca) and have been used extensively in experimental animals. the use of fca in humans and food animals is unacceptable due to severe granulomatous inflammatory reactions to toxic cell wall components ( bekierkunst, ). however, a nontoxic water-soluble purified fraction of m. tb wcl (werner et al., ) has adjuvant properties in rodents, guinea pigs, and rabbits. pigs inoculated in with m. tb wcl have no detectable signs of toxicity locally or systemically (dwivedi et al., a,b) . a recombinant vaccine (prrsv gp and m expressed in bacillus calmette-guerin (bcg)) is reported to reduce clinical signs of prrs with decreased viremia and viral load in the tissues (bastos et al., ) . cross-protective immunity against prrsv has been evaluated in pigs receiving mlv-prrsv and m. tb wcl in, by challenging with virulent heterologous prrsv virus (kim et al., ) . reduced clinical disease, viremia, and virus-mediated immunosuppression were noted. in addition, secretion of ifnγ and il- by lung and blood lymphocytes in response to prrsv m and nucleocapsid proteins was enhanced (dwivedi et al., a) . guillonneau et al. ( ) reported similar findings in mice vaccinated in with adjuvanted influenza vaccines. enhanced virus-specific cytotoxic t cell and memory responses to internal viral proteins were noted, as well as cross-protective immunity. studies of passive protection of pigs by prrsv na have indicated that modest na titers (≤ / ) protect pregnant sows against reproductive failure, block placental transmission of infection, prevent viremia in piglets, and provide sterilizing immunity (osorio et al., ) . pigs immunized in with mlv-prrsv alone or with m. tb wcl developed na titers < / and / , respectively, postimmunization (dwivedi et al., b) . in pigs immunized (mlv-prrsv + m. tb wcl) and challenged with prrsv mn , an na titer > / persisted until pid (dwivedi et al., a) . relatively low numbers of circulating virus-specific ifnγ secreting cells have been reported for pigs vaccinated in with mlv-prrsv without adjuvant, ( - cells per million pbmcs) (dwivedi et al., a) . in contrast, meier et al. ( ) have reported - ifnγ secreting cells per million pbmcs in pigs vaccinated against aujeszky's disease virus. pigs immunized in with mlv-prrsv with m. tb wcl and challenged with prrsv had greater than ifnγ secreting cells per million pbmcs, and more than a twofold higher frequency of cd + cd + t cells (dwivedi et al., a) . the literature concerning mucosal immune responses in the pig respiratory tract is limited compared to studies of rodents and humans. recent research highlights the advantages of activating the mucosal immune system using vaccines delivered with potent adjuvants. induction of adequate immune responses in the respiratory and reproductive tract will be essential for control of prrs in swine. in-delivered, potent mucosal vaccines generate better cross-protective immunity against genetically variable prrsv field viruses. efforts to control prrs outbreaks using conventional parenterally delivered vaccines have had limited success; development of an alternative approach of generating immunity in the respiratory tract should be a priority. in particular, mucosal adjuvants based on components of mycobacterial species show promise. respiratory tract disease affects virtually every aspect of equine husbandry, including working, pleasure, and race horses. considerable efforts are expended to prevent epizootics of respiratory disease in stables, fairs, shows, and race tracks. equine influenza virus will be discussed in detail as an example of past, current, and future vaccine approaches. equine influenza virus causes epizootics of upper and lower respiratory tract disease almost worldwide. until equine influenza was excluded from the continent of australia through import restrictions and quarantine. in august of that year, importation of an infected horse led to an explosive epizootic of equine influenza with an estimated , horses infected over a -month period (callinan, ) , demonstrating the potential of the virus to spread in a naïve, unvaccinated population. infection can occur in horses of all ages, but epizootics often involve younger animals (van maanen and cullinane, ) . clinical signs include high fever, a persistent dry cough, nasal discharge, anorexia, and depression. secondary bacterial pneumonia may complicate the clinical picture. equine influenza viruses are classified as type a influenza. antigenic differences in the hemagglutinin (h) and neuraminidase (n) glycoproteins define the two recognized subtypes, a/equine/ (h n ) and a/equine/ (h n ) (wilson, ) . two lineages of a/equine/ , american and european, have been identified; multiple virus strains are included in vaccines since protection against heterologous strains is incomplete (yates and mumford, ) . antigenic drift is sufficient to require regular reappraisal of strains included in vaccines (mumford and wood, ) . natural infection induces iga antibodies in nasal secretions, igga and iggb antibodies in serum nelson et al., ) , and circulating cytotoxic t lymphocytes . protection against reinfection persists for at least a year (hannant et al., ) . vaccination with inactivated virus vaccines induces serum igg (t) antibodies without detectable iga in nasal secretions (nelson et al., ) and without cytotoxic t cell activity (van maanen and cullinane, ) . two or three doses of vaccine are typically administered in the primary series, with booster doses at least once a year thereafter. more frequent vaccination is advised for horses at high risk of infection (wilson, ) . protection is typically incomplete and of limited duration. improved adjuvants can enhance the level and duration of antibody responses to inactivated virus vaccines (mumford et al., c) . suppressive effects of maternal antibodies on responses to inactivated vaccines have led to recommendations not to vaccinate foals before months of age (van oirschot et al., ) . a subunit equine influenza vaccine based on the immune stimulating complex (iscom) adjuvant technology has been licensed and marketed in europe since (newmark, ) . antibody responses to iscom-based vaccines typically are of higher titer and are more persistent than for conventional inactivated vaccines (mumford et al., a) . protection has been demonstrated against experimental challenge, months after a three dose vaccination series (mumford et al., b) . protection may be due in part to the ability of iscom-adjuvanted vaccines to induce cytotoxic t lymphocytes (morein et al., ) . although iscom-based vaccines can induce iga antibody responses following in administration (hu et al., ) , the commercial influenza iscom vaccine is administered parenterally. in administration of inactivated equine influenza virus with ct b has been reported to induce local iga antibodies, and protection against experimental challenge (hannant et al., ) . a cold-adapted, temperature-sensitive live vaccine for in administration is available commercially . protection against experimental challenge has been demonstrated months after a single vaccination . this is a notable improvement in efficacy and practicality over conventional killed vaccines. experimental plasmid vaccines encoding the hemagglutinin gene of equine influenza have been examined in horses. three doses of plasmid administered to the skin and mucosal sites (tongue, conjunctiva, and third eyelid) induced protection against clinical disease and partial protection against viral shedding. protection against clinical disease was reduced if plasmid was administered only to the skin (lunn et al., ) . a canarypox-vectored equine influenza vaccine, expressing hemagglutinins from two strains of h n equine influenza, has been available commercially (intramuscular administration) since (toulemonde et al., ) . in the australian epizootic of , this vaccine was used extensively during the government program to eradicate equine influenza. the vectored vaccine was chosen for this program because of its diva (distinguishing infected from vaccinated animals) characteristics ( kirkland and delbridge, ) . infected horses mount antibody responses to the viral nucleoprotein in addition to the viral hemagglutinin, whereas vaccinated horses respond only to the hemagglutinins expressed by the vaccine vector. a recent report indicates that adequate antibody responses are generated with as little as days between primary and secondary vaccination (el-hage et al., ) . protection against experimental challenge has also been noted weeks after a single dose of vaccine in ponies ( toulemonde et al., ) . for some respiratory pathogens (e.g., m. hyopneumoniae in pigs) the critical antigens associated with protective immune responses have not been identified. for other pathogens (e.g., prrsv) there is also a need to identify the appropriate type of immune response (th or th ) needed for protection. for some complex disease conditions (e.g., bovine respiratory disease complex) there is continuing uncertainty whether all of the relevant contributing pathogens have been identified. although many parenteral vaccines are efficacious in reducing lower respiratory tract disease, there is a need to investigate whether induction of mucosal immunity in the upper airways, in combination with systemic immunity, can further reduce infection rates, transmission of pathogens, and economic losses. finally, there is a need to devise and implement changes in management procedures to reduce disease exposure (by nonimmunological methods), and to optimize immune interventions by improved timing of vaccinations. vaccines to prevent reproductive tract disease have received much emphasis in veterinary medicine. this is especially true of food-producing animals because reproductive failure is a major economic problem. although vaccines to prevent reproduction are of interest for abandoned pets or deer in areas of overpopulation, this section will deal only with vaccines designed to prevent infectious disease of the reproductive mucosa. infections causing adverse pregnancy outcome can be classified by route of infection: hematogenous or ascending. several hematogenous infections have a predilection for the gravid uterus resulting in early to late abortions . these include leptospirosis, chlamydial infection, and brucellosis in several animal species, histophilus somni infection in cattle and sheep and neospora caninum in cattle. although vaccines are available for several of these infections, the vaccine for brucellosis has been available since the s and is the most well known. several brucella species cause abortion or epididymitis/orchitis in the primary hosts ( brucella abortus in cattle, brucella suis in swine, brucella melitensis in goats, brucella ovis in sheep, brucella canis in dogs, and brucella marinum (or brucella delphini) in marine mammals). infection may be acquired via the gut mucosa or the conjunctiva/upper respiratory mucosa and localizes in the reticuloendothelial system and endometrium/placenta by systemic spread. thus, systemic vaccines are effective. a modified live b. abortus vaccine, along with a "test and slaughter" eradication program, has been successful in controlling bovine brucellosis in north america. the modified live vaccine (b. abortus strain ) is very effective in stimulating cmi that is critical for protection against this facultative intracellular pathogen. strain now has been largely replaced by a new attenuated strain, rb , which does not stimulate an antibody response known to interfere with diagnostic serologic assays. there is considerable information on mechanisms of systemic immunity to brucellosis. neospora caninum also causes abortion by a hematogenous route and a vaccine is available (weston et al., ) . histophilus somni can infect either by the hematogenous route or by an ascending genital route to cause abortion or infertility and vaccines are available. since the focus of this volume is mucosal immunity, no more will be said concerning protection against hematogenous infections of the genital tract. ascending local infections of the reproductive tract are usually transmitted sexually. the two best examples of vaccines for sexually transmitted infections of animals are campylobacter fetus subsp. venerealis (formerly vibrio fetus subsp. venerealis) and tritrichomonas foetus. both are host-specific bovine sexually transmitted diseases (stds) that only infect the reproductive mucosa. both are extracellular pathogens that do not invade the mucosa of the reproductive tract but may be found in the placenta and fetus. the localized nature of these infections and transmission limited to coitus suggest that mucosal immunity must be important. because a vaccine has been available for c. fetus subsp. venerealis for several decades and its use has controlled the disease in developed countries, that vaccine will be discussed first. vibriosis (or campylobacteriosis) is a chronic bacterial genital infection with no overt clinical signs other than reproductive failure (corbeil et al., ) . after months of infection, the uterus is cleared first, followed by the vagina. convalescent immunity is partially protective for a limited time. antibody is effective in protection against this extracellular pathogen as demonstrated by systemic passive immunization (berg et al., ) . the antibody response to infection is primarily iga in the vagina and igg in the uterus (corbeil et al., ) . systemic immunization with a whole cell vaccine results in both igg and igg antibody responses to surface antigen in serum, and uterine and vaginal secretions (corbeil et al., ) . this response prevents infection and can rapidly clear infected cows (corbeil and winter, ) . that is, the vaccine can be used prophylactically and even therapeutically. immunization is efficacious even though surface antigenic variation occurs in the face of a local immune response (corbeil and winter, ) . presumably, immune clearance occurs when the dynamic interaction between protection and evasion is shifted in the favor of the host. this appears to occur earlier when the response is primarily igg than when iga predominates (corbeil et al., ) . this may be related to the ability of the igg antibodies to mediate opsonization and intracellular killing of the bacterium, an ability which iga antibodies lack (corbeil and winter, ) . although this work was done many years ago, it sets a precedent for systemic immunization for prophylaxis and therapy of reproductive mucosal infections. trichomoniasis is a similar chronic genital mucosal infection of cattle. it is caused by the protozoan, t. foetus, and results in pregnancy loss. trichomonas vaginalis causes a human std also associated with adverse pregnancy outcome. thus, bovine trichomoniasis serves as a model for immune prevention of both bovine and human reproductive mucosal infections. because of the economic significance of bovine trichomoniasis and because no chemotherapy is approved, investigations have focused on immunoprophylaxis and immunotherapy. t. foetus colonizes the vaginal and uterine or preputial surfaces for months, like c. fetus subsp. venerealis infection. in fact, mature bulls are often infected for life whereas young bulls may clear the infection with time (cobo et al., ) . this is probably related to innate immunity. trichomonads are anaerobic parasites and are found deep in uterine glands and epithelial crypts of the penis and prepuce (rhyan et al., ) where the oxygen tension is probably lowest. older bulls have deeper epithelial crypts, which may have lower oxygen tension. in order to understand protective acquired immune responses, monoclonal antibodies (mabs) with putative protective functions were chosen for immunoaffinity purification of a highly glycosylated surface antigen (bondurant et al., ; corbeil et al., ) . analysis of many isolates of t. foetus indicated that two mabs recognized different epitopes of the same antigen, which was conserved in all isolates tested. this glycosylated surface antigen was later shown to be a lipophosphoglycan (lpg)/protein complex. systemic immunization with the immunoaffinity-purified lpg-containing surface antigen, followed by intravaginal challenge with t. foetus, resulted in significantly earlier clearance of the parasite from vaccinated animals than from controls (bondurant et al., ; corbeil et al., ) . more importantly, clearance of immunized animals most often occurred before weeks of infection. parsonson et al. ( ) showed that significant inflammation accompanied by reproductive failure did not occur until after weeks of infection, so the vaccine should protect against fetal loss. analysis of vaccine-induced antibody responses demonstrated predominantly igg responses in the serum and iga plus igg antibodies in secretions (corbeil et al., , . ige antibodies were also increased during infection. as ige antibodies increased, mast cells degranulated and clearance of t. foetus occurred. these studies raised several questions. first, why is the systemic response to the lpg/protein complex skewed toward igg (a th response in cattle) and not igg (a th response)? this question is under investigation. second, are igg or iga antibodies to the lpg-containing antigen most protective and how can that ig isotype be enriched to enhance protection? to address the latter questions, preliminary studies were done in mice to determine the best routes and adjuvants to enrich for igg or iga in genital secretions . subcutaneous priming with the immunoaffinity-purified lpg-containing surface antigen (called tf . antigen) in quil a adjuvant and subcutaneous boosting with whole cells enriched for igg anti-tf . antibodies in genital secretions whereas subcutaneous priming and intravaginal boosting greatly enriched for iga antibodies in genital secretions. when cattle immunized by these two methods were challenged intravaginally with t. foetus, those with predominantly iga or predominantly igg anti-tf . antibodies in genital secretions were equally protected. later studies with similar in immunizations showed that stimulation of the common mucosal immune system gave results similar to those of intravaginal immunization . this raised the question of inductive sites for local immune responses in the genital tract. others have suggested that the genital tract is not an inductive site because m cells and mucosa-associated lymphoid tissue (malt) are not present. this is true of cattle as well as mice and people. however, even though control cows did not have histologically demonstrable malt in the uterus and vagina, cows experimentally infected with t. foetus did . similar lymphoid nodules and follicles under a modified epithelium were detected in preputial and penial surfaces of bulls infected with t. foetus (rhyan et al., ) . immunostaining of parallel sections with mab to tf . antigen indicated uptake of antigen by epithelial cells and large mΦ or dc under the basement membrane near the lymphoid follicles (rhyan et al., ) . similar antigen uptake has been detected in the infected female uterine and vaginal mucosa (corbeil et al., ) . thus, even though the parasite is noninvasive, released tf . antigen appears to be taken up by epithelial cells. rat uterine epithelial cells can present antigen to t helper cells (wira and rossol, ) . also, mΦ/ dc positive for antigen should be antigen-presenting cells (apcs). detection of igg and iga antibodies in genital secretions of infected bulls (cobo et al., ; rhyan et al., ) and cows and the histologic demonstration of follicles and putative apcs suggests that inductive sites in the genital tract are formed in response to antigenic stimulation. like c. fetus subsp. venerealis, t. foetus has mechanisms for evasion of immune responses. these include coating of the surface with ig nonspecifically , epitope variation (ikeda et al., ) , and cleavage of igg , igg , and complement component by extracellular cysteine proteinase (talbot et al., ; kania et al., ) . studies with cysteine proteinase inhibitors demonstrated decreased cytotoxicity of t. foetus for bovine trophoblast cells and decreased infectivity in a mouse model, confirming the likely role of cysteine proteinases in pathogenesis (cobo et al., ) . even with these mechanisms for immune evasion, as with c. fetus, it is clear that the dynamic interaction between host and parasite can be made to favor the host by systemic or local immunization. the usefulness of whole cell vaccines in preventing t. foetus infection and reproductive failure in cows has been demonstrated in clinical trials (kvasnicka et al., ) . first-generation whole cell vaccines are now commercially available for prevention of trichomoniasis in cows. earlier, clark et al. ( ) demonstrated efficacious immunization of bulls with whole t. foetus cells or crude membrane glycoproteins that probably contained tf . antigen. this study indicated that vaccination of bulls could both prevent infection and clear already established infection. so vaccines for bovine trichomoniasis are both immunoprophylactic and immunotherapeutic. recent studies showed that vaccination of bulls with a commercially available whole cell killed t. foetus vaccine in oil adjuvant resulted in protection against challenge and high levels of igg antibodies in serum and preputial secretions (cobo et al., ) . igg antibodies to whole cell t. foetus antigens predominated but fairly high levels of igg antibodies were also detected. the above studies with c. fetus subsp. venerealis and t. foetus show that: . stds can be prevented or even cured by systemic vaccination of both males and females. . at least for one std, igg and iga of the same antigenic specificity are equally protective at the mucosal surface. either systemic immunization or mucosal (in or intravaginal) immunization with systemic boosting is protective. . inductive sites are formed in the mucosa of infected male and female genital tracts even with noninvasive pathogens. . strong and appropriate immune responses will clear microbial infection from the genital tract even when the microbe has multiple immune evasive mechanisms. . the fact that protection against two stds has been demonstrated in the natural outbred host (cattle) has advantages over murine models of std vaccines. in the latter, the human pathogen is usually inoculated into the unnatural murine host and the disease does not mimic the human infection. furthermore, although inbred mice provide a homogeneous experimental animal, they do not represent the variation in immune responses seen in the outbred human population. the work on bovine vibriosis and trichomoniasis demonstrates protection under field conditions for two stds that cause adverse pregnancy outcome in an outbred host. this is an encouraging precedent for control of human stds and related adverse pregnancy outcomes. future needs include identification of the protective antigens for most stds. for antibody-mediated protection of the genital mucosa, several questions have not yet been addressed. as far as we know, the role of ige in the genital tract is largely unstudied even though it does seem to be involved in clearance of trichomonads from the bovine genital tract. lastly, manipulating genital immune responses to enhance th or th type responses is an unexplored research area. enteric disease is a major cause of mortality and morbidity in animals. agents causing diarrhea in animals include viruses (e.g., adenoviruses, pestiviruses, caliciviruses, coronaviruses, parvoviruses, rotaviruses, toroviruses), bacteria (e.g., campylobacter spp., clostridium spp., diarrheagenic escherichia coli, salmonella spp., yersinia spp.), and parasites (e.g., coccidia spp., cryptosporidium parvum). these infections occur most commonly in suckling animals or in poultry under weeks of age, but may also be common postweaning and in susceptible seronegative or stressed adult animals (saif and jackwood, ) . induction of local secretory iga (s-iga) antibodies (prevent attachment, invasion, and neutralize toxins or infectious agent) and mucosal cellular immune responses (against intracellular bacteria and viruses) by vaccines are essential for protection from enteric pathogens. peyer's patches (pp) and mesenteric lymph nodes are two important organized gut-associated lymphoid tissues (galt) in domestic animals and serve as the induction site for gut immune responses. ileal pp in some domestic animals (sheep, cattle, pigs, dogs) differ from jejunal pp, serving as a primary lymphoid organ similar to the bursa of fabricius in chickens, unlike in humans and rodents where pp are secondary lymphoid organs (chu and liu, ; yasuda et al., ) . cryptopatches or clusters of lymphoid cells in the basal lamina propria occur in mice, but are absent in pigs (pabst et al., ) . mucosal lymphoid tissues and lymphoid cells in domestic animals and humans differ in toll-like receptor (tlr) expression and function (after binding their ligand) when compared to mice (iwasaki and medzhitov, ; tohno et al., ; guzylack-piriou et al., ) . these differences in immune components suggest that vaccine studies carried out in mice may fail to translate to domestic animals or humans. enteric pathogens have different characteristics related to their intestinal tropism and replication, requiring different vaccination strategies. enteric viruses have predilections for replication in distinct vertical and longitudinal regions of the small or large intestines. cytolytic infection of enterocytes leads to varying degrees of villous loss and fusion, reduced small intestinal absorptive capacity, and a malabsorptive, maldigestive diarrhea (saif, a) . secretory diarrhea is also induced by some viruses such as rotavirus (rv), which involves the viral enterotoxin, nonstructural protein (nsp ), and/or stimulation of the enteric nervous system (ball et al., ; lundgren et al., ) . thus, viral diarrheas can be of variable severity and act via multiple mechanisms that differ from those of enteric bacteria, most of which cause secretory diarrhea mediated by enterotoxins (fairbrother and gyles, ) . enteropathogenic viruses can be divided into three types (types i, ii, and iii) according to their preferred site of replication in the intestine (reviewed by saif ( a) ). type i (transmissible gastroenteritis virus (tgev) and rv) and ii virus infections can be prevented by inducing local intestinal immunity, whereas type iii viruses (parvovirus), which infect crypt enterocytes basolaterally, can be prevented by inducing systemic immunity. the enteropathogenicity of bacteria is determined by their virulence factors including adhesion factors (fimbriae or pili) and enterotoxins; therefore, bacterial vaccines generally need to prevent attachment and toxin action within the intestine (fairbrother and gyles, ) . in the following sections, we will review vaccine strategies for these different types of enteric infections. tgev and rv vaccines in pigs will be reviewed to illustrate findings using domestic outbred animals instead of inbred laboratory rodent models. neonates can be protected from enteric infections by providing passive lactogenic immunity, which can be achieved by immunizing mothers preparturition. pioneering work done by bohl and saif in the early s with live oral virulent tgev in pigs was the foundation for the gut-mammary gland-s-iga immunologic axis, which later became the basis for the concept of the common mucosal immune system. their studies showed that in tgev seronegative sows, only oral immunization with virulent tgev induced high rates of protection in suckling neonates, which was associated with high titers of s-iga antibodies in colostrum and milk, whereas systemic immunization induced mainly igg antibodies saif et al., ) . rotavirus is a major cause of dehydrating diarrhea in young livestock, infants, and poultry (saif and fernandez, ) . multiple rv serogroups, based on common inner capsid vp antigens (a-h), and multiple g (vp , glycoprotein) and p (vp , protease-sensitive) serotypes (based on neutralizing epitopes) or genotypes (based on sequence analysis) for group a rvs have been detected in humans, sheep, swine, cattle, horses, dogs, cats, poultry, and wildlife (estes and kapikian, ; martella et al., ) . among the distinct rv serogroups and g/p serotypes/genotypes, cross-protection is limited. the antigenic divergence among different sero/genotypes of rvs presents a challenge for the design of vaccines that are capable of inducing heterotypic protection. commercial modified live and killed rv vaccines for rv diarrhea in livestock and poultry are limited to group a rvs (saif and fernandez, ) . mebus et al. ( ) developed the first oral rv vaccine for calves in ( year prior to the discovery of human rv) using a cell cultureadapted neonatal calf diarrhea rv strain. although a significant reduction in morbidity and mortality was observed in a field trial among vaccinated calves, in the majority of herds (compared to previous years), subsequent field studies revealed variable efficacy. experimental studies suggested that maternal antibodies interfered with live oral vaccine replication and suppressed development of active immunity (saif and fernandez, ) . the neonatal gnotobiotic pig model has been used to investigate immune responses to rv vaccines and infection for nearly three decades (saif et al., , yuan and saif, ; gonzalez et al., ) . gnotobiotic pigs are free of maternal antibodies (placental transfer of igs does not occur in swine), but they are immunocompetent at birth. they are maintained aseptically and are free of exposure to extraneous wildtype rvs, assuring that exposure to a single pathogen can be analyzed. initial studies were conducted to mimic natural rv infection (bohl et al., ) and to study immune correlates of protection gonzalez et al., ; yuan et al., ) . understanding the sequence and kinetics of immune responses stimulated by virulent rvs allows for the determination of markers of protective immunity and pathogenicity, which can then be used to design vaccines that will stimulate protective immunity without inducing pathology. gnotobiotic pigs orally inoculated with virulent porcine or human rvs were completely protected from homotypic but not heterotypic (distinct g/p types) rv challenge (hoshino et al., ; saif et al., ) . significant correlations were observed between the protection to rv-induced diarrhea and shedding and the following immune parameters: the number of intestinal iga rv antibody-secreting cells (ascs), serum and intestinal iga rv antibody titers, and the frequency of intestinal rv-specific ifnγ producing cd + and cd + t cells yuan et al., ; to et al., ; yuan and saif, ; yuan et al., ) . these immune parameters were significantly higher in virulent rv inoculated pigs than in those inoculated with attenuated or inactivated rv (ward et al., b) . pigs inoculated with two or three doses of attenuated rv were moderately protected against diarrhea ( %- doses, %- doses) and virus shedding ( %- doses and %- doses) after homotypic challenge, suggesting a need for multiple doses and suitable mucosal adjuvants to enhance the efficacy of rv vaccines yuan et al., yuan et al., , yuan and saif, ) . gnotobiotic pigs inoculated with virulent rv developed an acute proinflammatory serum cytokine profile (il- , tnfα) coinciding with peak diarrhea and viremia, followed immediately by increased th (il- , ifnγ) cytokines and convalescent th (il ) and tr (il- ) cytokine responses. gnotobiotic pigs inoculated with one dose of attenuated rv showed lower early ifnγ and proinflammatory cytokine responses compared to virulent rv inoculated pigs. both attenuated and virulent rv inoculated pigs developed th /th /tr cytokine-secreting cell (csc) responses at - weeks postinoculation; however, attenuated rv inoculated pigs developed lower intestinal ifnγ and higher intestinal and splenic il- cscs compared to virulent hrv inoculated pigs (azevedo et al., ) . virulent rv inoculated pigs had significantly higher protection rates against rv challenge ( % and % against diarrhea and shedding, respectively) compared to one dose of attenuated rv inoculated pigs ( % and % against diarrhea and shedding, respectively) (azevedo et al., ) . these findings suggest that higher protection rates are associated with early proinflammatory and th cytokine responses, which promote cytotoxic t cell activity and viral clearance, and later th induced cytokines, which are important for protective s-iga antibody responses. thus, protection against rv in pigs requires balanced th /th /treg responses (azevedo et al., ; gonzalez et al., ) . infection of gnotobiotic pigs with virulent rv causes early recruitment of innate apcs (monocytes/mΦ and dcs) and γδ t cells into the ileum, and enhanced tlr , tlr , and tlr expression among apcs in spleen (zhang et al., c; wen et al., wen et al., , . in virulent rv-infected pigs, plasmacytoid dcs are major producers of serum ifnα and, along with other innate immune cells (γδ t cells and apcs) and cytokines (tnfα, ifnγ and il- ), are important for controlling early rv viral replication and subsequent development of protective adaptive immune responses . development of attenuated rv vaccines with mucosal adjuvants that mimic immune responses to virulent rv may improve existing vaccines. immunogenicity and protective efficacy of rv vaccine formulations (attenuated replicating virus, inactivated virus, and recombinant baculovirus-expressed virus-like particles (vlp)), administration routes, and adjuvants have also been evaluated using the gnotobiotic pig model (bohl et al., ; yuan and saif, ; gonzalez et al., ) . inactivated oral or intramuscular rv vaccines failed to protect against virulent rv challenge, despite high igg antibody responses induced in serum and systemic lymphoid tissues. serum igg antibodies did not correlate with protection. however, a recent study showed that an inactivated reassortant rv strain (cdc- strain) formulated with aluminum phosphate and administered systemically in gnotobiotic pigs resulted in induction of serum igg antibody titers, coinciding with partial protection against shedding and diarrhea, suggesting that adjuvant may have stimulated local specific (gut iga antibodies) or nonspecific immune responses, which were not assessed in this study (wang et al., ) . rotavirus subunit vaccines consisting of double-layered vlp composed of rv inner capsid proteins vp and vp ( / -vlps) administered in or orally with mutant heat-labile toxin of e. coli (mlt) or iscoms as adjuvants (yuan and saif, ) induced igg asc responses in systemic lymphoid tissues and low or no iga asc responses in intestinal lymphoid tissues and also failed to mediate protection, contrary to results in adult mouse studies (yuan and saif, ) . the failure of in or oral / -vlp vaccines suggests that protective immunity to rv diarrhea in neonatal pigs requires mainly the presence of systemic or intestinal iga antibodies to the outer capsid rv proteins, vp and vp , each of which induces na. however, when / -vlps adjuvanted with mlt or iscom were used as in or oral booster doses in pigs orally primed with attenuated rv, the protective efficacy increased significantly. an oral attenuated rv prime and in / -vlp-iscom boost regimen (attrv/ / -vlp) induced the highest numbers of intestinal iga ascs and serum and intestinal iga antibody titers, and protection rates were similar to or higher than those induced by three oral doses of attenuated rv (gonzalez et al., azevedo et al., ) . interestingly, priming with two doses of / -vlp (in or oral) followed by live attenuated rv was ineffective for inducing iga antibodies or protection. thus the use of a replicating vaccine to prime at one inductive site (galt) followed by boosting with a nonreplicating vaccine at a second mucosal inductive site (nasal-associated lymphoid tissue, nalt) is an effective strategy for stimulating protective mucosal immune responses, which can be applied to other enteric viral vaccines. furthermore, efficacy of the prime/boost strategy (replicating/nonreplicating vaccine, attrv/ / -vlp) was examined in the presence of high and low titers of passive antibodies to mimic neonatal pigs receiving maternal antibodies (nguyen et al., a,b) . similar to attrv/ / -vlp prime/boost vaccine studies, plasmid dna containing vp was used as a booster subsequent to oral attenuated rv vaccine priming. this regimen showed high protection against shedding, but poor efficacy against rv diarrhea (yuan et al., ) . collectively these findings suggest that mucosal boosters are effective in enhancing iga antibody titers to rvs in orally primed animals . studies have shown that immunogenicity and efficacy of mucosal vaccines can be improved by the use of appropriate strains of probiotics that modulate mucosal and systemic immune responses, by interaction with epithelial cells and the underlying intestinal immune cells (fukushima et al., ; sanz and de palma, ) . certain probiotic strains are reported to enhance immune responses to rv vaccines in children; others reduce rv diarrhea severity, but the mechanisms are not well defined (fang et al., ; holscher et al., ) . supplementation with lactobacillus acidophilus in attenuated rv vaccinated pigs is reported to enhance intestinal ifnγ-producing cd + t cells, intestinal iga and igg rv ascs, and serum iga and igg rv antibody titers (zhang et al., b) . these findings suggest that probiotics are an alternative, cheap, and safe adjuvant for enhancing efficacy of oral attenuated rv vaccines in animals and potentially in humans. colonization of pigs by two different strains of lactic acid bacteria (lab, l. acidophilus and lactobacillus reuteri) and subsequent virulent rv infection resulted in higher and balanced th /th /treg cytokine responses (il- , ifnγ, il- , il- , tgfβ), higher total intestinal iga-secreting cells, total serum igm, and intestinal igm and igg titers, although the numbers of iga rv ascs and serum and intestinal rv antibody titers did not differ compared to virulent rv-only infected pigs. no overall difference in protection rates was noted when compared to virulent rv-only inoculated pigs, which was likely because of the short interval (only days) between lab colonization and virulent rv challenge (zhang et al., a; azevedo et al., ) . dual colonization of the aforementioned lab strains also modulated innate immune components in virulent rv inoculated pigs as follows: ( ) enhanced frequency of γδ t cells in the intestine and their distribution; ( ) enhanced tlr and tlr -expressing cd + apc, and tlr -expressing cd − apcs in the blood, but reduced tlr -and tlr expressing cd − apcs in the spleen, and ( ) reduced frequency of mΦ and cdcs in the spleen. collectively, these findings suggest that probiotics may reduce systemic inflammatory responses induced by virulent rv. effects on tlr expression on apcs in the ileum were not determined (zhang et al., c; wen et al., wen et al., , . probiotics may not only modulate immune responses to rvs or other enteric vaccines, but may also directly ameliorate diarrhea/infection by enhancing gut barrier integrity and maintaining intestinal permeability, by stimulating nonspecific immune responses, by changing gut microbial populations, and by aiding in the regulation and prevention of apoptosis ( madsen et al., ; yan and polk, ; preidis et al., ) . using the tgev model for evaluation of active protection against diarrhea in pigs, researchers also delineated compartmentalization in the common mucosal immune system and its impact on mucosal vaccine strategies and protection (vancott et al., (vancott et al., , . the natural occurrence of a deletion mutant of tgev with exclusive respiratory tropism, referred to as porcine respiratory coronavirus (prcv), provided a unique opportunity to study asc responses and protective immunity to two antigenically related porcine coronaviruses with enteric (tgev) versus respiratory (prcv) tropism. oral immunization of pigs with tgev induced high numbers of iga asc in the intestine and provided complete protection against tgev challenge, whereas in immunization of pigs with prcv induced mainly systemic responses (igg asc) and provided only partial protection against tgev challenge. thus, the in prcv alone failed to elicit sufficient intestinal iga asc to provide full protection against the enteric pathogen, tgev. findings from this study and rv vaccine studies suggest that the use of multiple mucosal inductive sites in a prime/boost vaccination regimen may be an effective approach for overcoming compartmentalization in the common mucosal immune system. canine parvovirus (cpv) infects crypt enterocytes causing hemorrhagic gastroenteritis in pups (saif and jackwood, ) . because cpv is likely disseminated to the basolateral surface of crypts by the hematogenous route, serum na (derived maternally or actively produced) is protective against the disease. pollock and carmichael ( ) demonstrated that pups with hemagglutination inhibition (hi) serum antibody titers of > : were immune to oronasal cpv type challenge. cpv is highly stable in the environment and pups were susceptible to infection as soon as maternal antibodies declined to hi titers of : - : . a maternal hi antibody titer as low as : severely affected the efficacy of a live low titer cpv vaccine ( - th passage in culture) in generating active immune responses (carmichael et al., ) , which resulted in a window of susceptibility for pups. others have shown that an hi titer of less than : in cpv- (low passage, high titer) vaccinated pups did not severely affect active antibody responses (burtonboy et al., ; hoare et al., ) , suggesting that increasing the dose or reducing the attenuation of the virus may help to overcome inhibitory effects of maternal antibodies. studies with modified live variant cpv- b strain (low titer) have shown that these vaccines, when given either parenterally ( th passage) or in ( th tissue culture passage), elicited almost % protection in pups with maternal antibody titers of : to : and even % protection in pups with antibody titers of : . higher efficacy of these vaccines can be attributed to either strong inherent immunogenicity of these new vaccines or antigenic differences among cpv- and cpv- a and cpv- b (pratelli et al., ) . overall, during the past four decades of cpv vaccine development, modified live viruses have proven superior to inactivated intramuscular (im) vaccines (appel, ) . in brief, various strategies such as the use of less attenuated virus (low serial passage), high titer vaccines, multiple doses, or the use of the in route of immunization have been attempted to overcome inhibitory influences of maternal antibodies and to reduce the window of susceptibility in pups. recently new variant cpv- c has emerged, which is highly pathogenic and causes more severe diarrhea in dogs. currently used vaccines (cpv- or cpv- b strains) are effective in protecting dogs against challenge with cpv- c virus under experimental conditions (spibey et al., ) , although efficacy in the field is unknown (reviewed by decaro and buonavoglia ( ) ). antigenic differences between original cpv- and its variants may reduce the efficacy of current cpv vaccines, which is supported by in vitro virus-neutralization tests conducted on vaccinated animals that showed low cross-reactivity between heterologous cpv variants (cavalli et al., ) . however, this may not represent actual cross-protection in clinical cases. there is a need not only to make current vaccines effective in the presence of maternal antibodies, but also to update them based on continuous epidemiological surveillance studies. dna plasmids expressing vp (jiang et al., ) , replicon-based cpv dna vaccine expressing vp (dahiya et al., ) , b cell epitope ( l peptide of vp ) fused to ct b subunits expressed in transgenic tobacco chloroplasts (molina et al., ) , chimeric virus particles expressing cpv peptide (different prime/boost strategies) (nicholas et al., ) , and recombinant vlps formed by baculovirusexpressed vp (casal, ) have been evaluated in dogs or rodents without maternal antibodies and have demonstrated good immunogenicity and/or protective efficacy. further efficacy tests in pups with maternal antibodies are needed to assess their commercial potential. oral vaccines for the induction of active immunity against bacterial diarrhea are not commonly used in livestock, although e. coli diarrhea is an important problem in neonatal and postweaning calves and pigs. f (k ) and f are the major fimbrial adhesins present on swine enterotoxigenic e. coli (etec) and are major targets for e. coli vaccines (fairbrother et al., ) . whole bacteria vaccines are routinely administered parenterally to pregnant cattle, sheep, and swine to protect their suckling neonates against etec infections (moon and bunn, ) . such vaccines are practical and effective because: ( ) fimbriae are required for the adhesion-colonization of bacteria early in the pathogenesis of the disease; ( ) most fatal etec infections in farm animals occur in the neonatal period; ( ) more than % of the etec in farm animals belong to a small family of fimbrial antigen types, ( ) and mothers are seropositive to etec so booster responses are elicited. recent vaccine studies have focused on administration of purified bacterial subunits (transgenically expressed adhesin of f fimbriae in plants) and mucosal adjuvants (verdonck et al., ; joensuu et al., ) and have shown promising results. overall, the vaccine strategy used, parenteral vaccination of field-exposed seropositive mothers, to induce lactogenic immunity is the same as that shown to be effective for parenteral application of rv vaccines in rv seropositive mothers (bohl et al., ; saif and jackwood, ; saif and fernandez, ) . studies of live oral enteric vaccines in animals have clarified the mechanisms of induction of protective immunity against enteric disease and contributed to our understanding of the common mucosal immune system. however, commercial live oral vaccines often have shown inadequate or inconsistent efficacy under field conditions (saif and jackwood, ; saif and fernandez, ) . major obstacles to improved efficacy of oral vaccines include: ( ) maternal antibodies in the intestine of neonates (mainly colostrum and milk antibodies), which interfere with live vaccine replication; ( ) qualitative and quantitative limitations in the neonatal immune system, although neonates are immunocompetent; ( ) the inability of attenuated vaccine strains to adequately infect (too high attenuation) or stimulate s-iga antibodies in the intestine; ( ) the use of inappropriate (or unstable) antigens or route for subunit vaccines; ( ) the lack of oral delivery vehicles or mucosal adjuvants for subunit vaccines; and ( ) infection by pathogens prior to vaccination. studies of neonatal pigs indicate that protection rates against rv diarrhea upon challenge correlate with the magnitude of iga asc and memory b cell responses in intestinal lymphoid tissues gonzalez et al., ; yuan and saif, ) . studies conducted in immunodeficient or specific gene knockout adult mice have shown that neither cd + or cd + t cells nor antibodies were essential for induction of protective immunity to rv infection, but usually one of these effectors (t or b cells) was necessary for elimination of primary rv infection (mcneal et al., (mcneal et al., , . in pigs, recent studies have shown that cd + and cd + ifnγ producing t cells play a role in protection against rv diarrhea and infection (yuan et al., ) , but it is difficult to create genetically modified pigs similar to knockout mice, to assess the contribution of b cells and t cells. the redundant nature of immune responses to rv in mice, the multiple immunologic and possibly nonimmunologic pathways to resolve rv infections (franco and greenberg, ; ward, ) , the age factor and host differences in rv pathogenesis in mice and pigs (saif et al., , ward, ) , and the use of inbred mouse strains contribute to the discrepancies seen between the adult mouse and the neonatal pig models. the majority of pathogens enter and initiate infection at mucosal surfaces, making mucosal sites relevant targets for vaccines to prevent infection. to develop effective mucosal vaccines, it is important to determine correlates of protection against enteric pathogens. generally for localized gut infections, s-iga antibodies and intestinal t cells play an important role. improved vaccines that induce high levels of intestinal iga antibodies against the appropriate microbial antigens can be achieved by choosing the proper vaccine formulation and delivery method. vaccines should also induce: ( ) heterotypic protection; ( ) active immunity in the presence of maternal antibodies; and, ( ) long-lasting immunological memory. novel vaccines (e.g., vlps, transgenic plants), adjuvants (e.g., mlt, iscoms, tlr ligands (e.g., cpg), mycobacterial extracts, α -dihydroxyvitamin d , retinoic acid, probiotics, and cytokines), and vaccine delivery systems (e.g., recombinant plant or animal viruses or bacterial vectors, genetically engineered probiotics, iscoms, liposomes, and nanoparticles) should be explored and evaluated in relevant animal models to further enhance the efficacy of current or new vaccines. recent studies have shown that the targeting of antigens directly to apcs (via surface receptors) is an effective way to tailor immune responses to optimize protection and should be explored further in large animals (trumpfheller et al., ) . the passive transfer of maternal immunity provides essential protection in newborns. although most neonatal immune systems are competent to mount primary immune responses against pathogens, primary responses often do not develop quickly enough to prevent disease. maternal immunologic transfer provides a critical (though temporary) aid to survival for neonates. the enhancement of passive immunity through vaccination of the mother has been a successful disease prevention strategy in domesticated animals. vaccinated mothers develop higher levels of specific antibodies in colostrum and milk and increased levels of immunity in their offspring (glezen, ; saif and fernandez, ) . passive immunity can also be enhanced by oral administration of immune milk or heterologous antibody preparations (e.g., chicken egg yolk igy (ikemori et al., ; kuroki et al., ) or monoclonal antibodies) or by parenteral administration of hyperimmune plasma (becu et al., ) . recent studies using monoclonal, single-chain antibodies (variable heavy domain (vhh) nanoantibodies) of llama origin open new possibilities for providing passive immunity to humans and animals (garaicoechea et al., ) . immunoglobulins of the igg and igg isotypes of camelids consist of heavy chains without associated light chains (hamers-casterman et al., ) . the distinctive biochemical characteristics and binding qualities of vhh antibodies overcome some key limitations of conventional antibodies (see below). unfortunately, passive antibodies often interfere with active immunization of young animals and birds. various vaccination strategies have been developed to minimize the suppressive effects of maternal antibodies, but improved adjuvants and antigen-delivery systems are needed to facilitate efficient induction of active immunity in the presence of maternal antibodies. this section will address past, current, and future approaches for enhancing passive immunity in veterinary species. the transfer of systemic passive immunity from mother to offspring can occur prenatally, via the placenta or yolk sac, or postnasally via ingestion of colostrum and milk, depending on the species. the main ig isotype transferred in most mammalian species is igg. in avian species igy, the functional equivalent of mammalian igg, is transferred to the yolk to passively protect the developing chick (kovacs-nolan and mine, ) . in primates and rabbits, maternal igg is transferred across the placenta to the fetus. in rodents, transplacental transfer occurs in combination with prolonged ( - days) postnatal transfer by means of colostrum and milk (husband, ) . in dogs and cats transfer of igg occurs by a combination of prenatal and postnatal mechanisms, with % to % of total transfer occurring before birth (tizard, ) . in ruminants, horses, and pigs, offspring are born virtually agammaglobulinemic and transmission of ig occurs only via colostrum for a limited time after birth (tizard, ) . after the transition from production of colostrum to milk, ig are no longer absorbed from the intestines and only act locally in the gastrointestinal tract. immunoglobulin absorption in neonates of large domestic species is facilitated by the presence of protease inhibitors in colostrum and its efficiency declines rapidly after birth, with maximal absorption occurring in the first h. the cessation of absorption of intact macromolecules is termed "gut closure," and occurs at different ages in different species. in calves and pigs closure normally occurs by - h after birth. absorption of colostrum ig can be highly effective, supplying the newborn with serum ig at concentrations similar to those of the dam. failure of passive transfer (fpt) is a common problem, however, in newborn calves and foals (besser and gay, ; tyler-mcgowan et al., ) . fpt may occur because of the production of low quantities of colostrum, because of production of colostrum with low concentrations of maternal antibodies, because of ingestion of low quantities of colostrum, or because of inefficient absorption (quigley and drewry, ) . colostrum supplements, colostrum replacers, and plasma products have been developed commercially to address this problem, with variable success. vaccination of the dam in late pregnancy can enhance antibody titers in colostrum and after suckling in the serum of the offspring (saif and fernandez, ) . the benefits of vaccination of the dam for enhancing passive immunity can be lost if colostrum is of low quality or if absorption of colostral ig is inefficient. the half-life of ig varies considerably among species of domestic animals and with the ig isotype. neonatal receptor for fc of igg (fcrn) is involved in homeostasis of serum levels of igg in general, but distinct mechanisms may function in neonates. the main route of clearance of passively acquired maternal igg in calves is transfer from the serum to the intestine (besser et al., ) . approximately % of passively acquired igg is eliminated by this route. if titers of passive circulating antibodies are high enough, the transfer of antibodies from the circulation to the intestinal lumen can mediate short-term protection against rotavirus diarrhea (besser et al., ) . the same mechanism may be functional in piglets (saif and wesley, ; parreño et al., ; ward et al., a) . the persistence of titers of circulating maternal antibodies is generally considered in designing vaccination strategies for young animals because of suppressive effects of maternal antibodies on active immune responses. induction of immune memory can occur in the absence of a detectable serum antibody response (boersema et al., ; parreño et al., ) . the presence of passive antibodies in the intestines can interfere with mucosal immune responses to natural infection as well as to vaccination parreño et al., ; nguyen et al., a,b) . experiments in colostrum-deprived lambs (jones et al., ) and calves (mosier et al., ) have demonstrated the ability of parenterally administered immune antisera to mediate protection following experimental challenge with mannheimia haemolytica. parenteral administration of hyperimmune plasma raised against rhodococcus equi has been shown to protect against pneumonia in young foals in experimental (hooper-mcgrevy et al., ) and field studies (becu et al., ) . hyperimmune plasma is available commercially for use in foals. prepartum vaccination of beef (van donkersgoed et al., ) and dairy (hodgins and shewen, ) cows has been demonstrated to increase antibody titers to m. haemolytica in their colostrum and in the serum of their calves. vaccination of broiler breeder chickens can be used to provide passive protection against respiratory/septicemic disease caused by avian pathogenic e. coli (kariyawasam et al., ) . rodents have been a popular model for the study of passive protection by milk antibodies. however, rats and mice actively transport igg from the gut into the circulation during the first - weeks of life. thus, antibodies in ingested milk contribute to both local and systemic immunity in rodents, in contrast to the strictly local effects occurring in humans and most domestic animals. in pigs, horses, dogs, and cats, igg is the most abundant ig in colostrum but iga predominates in milk. parenteral vaccination, by enhancing serum igg antibody titers, contributes to igg antibodies in colostrum but has limited effects on iga antibodies in milk. milk antibodies provide passive protection to the neonatal intestinal tract by immune exclusion preventing the attachment of viruses, bacteria, and parasites and by neutralizing viruses or enterotoxins. s-iga antibodies, because of their resistance to cleavage by digestive enzymes, and higher levels in milk are more efficient in mediating protection in the gut of pigs and other monogastrics (saif and jackwood, ) , but high persisting levels of passive igg antibodies are also protective. in ruminants igg antibodies, relatively resistant to proteolytic enzymes (brock et al., ) and predominant in milk, have functions similar to those of s-iga. numerous vaccines are marketed for vaccination of cows and sows to provide lactogenic immunity to rotavirus, coronavirus, and e. coli in suckling offspring. vaccine efficacy has been variable. ideally, suckling animals become subclinically infected with enteric pathogens while receiving adequate passive antibodies to prevent disease, and develop active immunity to prevent subsequent diarrhea. this balance between passive immunity and disease can be disrupted in intensive animal production systems by exposing animals to pathogens in confined, contaminated environments. earlier weaning practices reduce intake of milk antibodies. maternal enteric vaccines are commonly used in two populations of pregnant animals. to control epidemic infections, they are used in naïve, seronegative animals to induce primary immune responses. to control endemic infections (such as rotavirus and e. coli), booster vaccines are used in seropositive, field-exposed animals to stimulate anamnestic memory responses. virulent tgev given to pregnant sows stimulates high levels of iga antibodies in milk and passive protection (saif and jackwood, ) . oral attenuated tgev vaccines, which replicate poorly in sows, induce lower iga milk antibody titers and low or variable efficacy in the field (moxley and olson, ) . parenteral killed tgev vaccines induce only low igg milk antibody titers and have the lowest protection rates. attempts to develop maternal tgev recombinant subunit vaccines based on the surface tgev spike (s) protein that induces na, or live vector vaccines expressing the s protein, have also been of limited success in tgev seronegative swine (saif and wesley, ) . however, prime/boost strategies such as im administration of tgev s protein following oral/in priming with attenuated tgev have shown promise as a means of enhancing iga milk antibody titers (park et al., ) . booster vaccination strategies are required to enhance lactogenic immunity to endemic enteric pathogens, such as rotavirus and e. coli, because antibody titers in milk decline dramatically during lactation. breast milk iga antibodies are increased in women endemically exposed to cholera following parenteral boosting with a cholera vaccine (svennerholm et al., ) . in pigs infected with rotavirus, iga memory b cells initially reside in the ileal pps but are subsequently present in substantial numbers in spleen (yuan et al., ) . systemic stimulation of such iga memory b cells by parenteral booster vaccines can yield dimeric iga antibodies in serum for transport to mucosal secretions via the polymeric ig receptor. under field conditions, antibodies to endemic intestinal pathogens are also common in bovine colostrum and milk, but without the boosting effect of highly immunogenic vaccines, antibody titers are often too low to protect calves (besser and gay, ; saif and fernandez, ) . thus vaccines are marketed for prepartum vaccination of cows against rotavirus, coronavirus, and e. coli to enhance passive immunity in their calves, but the field efficacy of these vaccines has been questioned (waltner-toews et al., ) . vaccination of pregnant dairy cows with modified live or binary ethyleneamine inactivated rotavirus or recombinant / / / vlps has been shown to increase igg and virus na titers to rotavirus in colostrum and milk and mediate passive protection in calves against oral rotavirus challenge (saif and fernandez, ; kim et al., ) . prepartum vaccination of cows and sows with bacterins prepared from enteropathogenic (epec) e. coli for prevention of diarrhea in their offspring is also commonly practiced. under modern farming practices, dairy and veal calves rarely are fed whole milk from their dams for more than or days. thus vaccine efficacy is based on antibodies absorbed from colostrum or retained temporarily in the gut, rather than on a continuing supply of immune milk. piglets, in contrast, continue to receive immune milk until weaning at - weeks of age. the importance of a continuous supply of passive antibodies for protection against tgev has been demonstrated (saif and wesley, ) . numerous commercial ig preparations with antibody activity against specific enteric pathogens have been marketed. products intended for prevention of e. coli enteritis in calves include dried bovine colostrum and whey, hyperimmune sera raised in horses, and mouse monoclonal antibodies to the k (f ) antigen of e. coli. these products are administered orally in the first h of life to prevent adhesion of epec e. coli. orally administered bovine colostral whey containing rotavirus antibodies also passively protects piglets against rotavirus (schaller et al., ) . immunization of chickens shows promise as an efficient method for producing polyclonal antibodies for passive protection. specific antibodies of the igy isotype are induced by vaccination and are concentrated in egg yolk. laying hens can produce about g of igy per year. yolk antibodies with virus na provide calves with partial protection against diarrhea caused by rotavirus (kuroki et al., ; vega et al., ) , and etec e. coli (ikemori et al., ) . in a recent study, supplementation of the milk ration of neonatal calves with egg yolk containing igy antibodies to rotavirus for days provided % protection against rotavirus diarrhea after challenge, and also enhanced mucosal asc numbers in the duodenum (vega et al., ) . egg yolk lacking rotavirus specific igy did not provide clinical protection, but did enhance asc responses, suggesting the presence of immune modulators in egg yolk. protective effects of yolk antibodies are dependent on antibody titers in oral preparations (marquardt, ) . development of better means to protect yolk antibodies from digestive processes will improve both the efficacy and the economic viability of yolk antibodies for clinical applications (kovacs-nolan and mine, ). for many diseases of newborns and neonates, passive immunity is the only practical means of providing timely protection. unfortunately, it is well documented that maternal antibodies can suppress active immune responses following vaccination. this effect has been observed with both live and nonreplicating vaccines, and for both systemic and mucosal responses (siegrist et al., ; hodgins et al., ; parreño et al., ; nguyen et al., a,b) . antibody responses especially are affected; t-lymphocyte responses may not be suppressed (siegrist et al., ) . titers of maternal antibodies are maximal for most species of interest in the first week of life and then decline gradually over the next few months, but variability of titers among individuals is high. with many vaccines, a "window of disease susceptibility" of variable duration occurs when titers of maternal antibodies are too low to mediate protection, but are too high to permit effective vaccination. a number of strategies are used to cope with this problem. some veterinary vaccines for cattle are sold with the disclaimer that "animals vaccinated before months of age should receive a booster dose of vaccine at months of age." this provides little solace for the many diseases of cattle occurring in the first weeks or months of life. a common strategy for vaccines of dogs and cats is to administer a series of doses of vaccine from an early age (at which time only a few individuals will be responsive) and to continue vaccinating until an age at which virtually all can respond to vaccination. this strategy has economic disadvantages for the pet owner. some manufacturers produce low passage, high virus titer vaccines especially for use in situations where high titers of maternal antibodies and high pathogen exposure are anticipated. this is similar to a strategy once (but no longer) approved by the world health organization for vaccination of children in developing countries against measles (gellin and katz, ) . preliminary evidence suggests that incorporation of vaccine antigens in highly structured iscoms or in application of vaccines can enhance immune responses in the presence of maternal antibodies (van binnendijk et al., ; brockmeier et al., ) . immunoglobulin g in most mammalian species is composed of two heavy chains, covalently linked by disulfide bonds, and two light chains. "conventional" heavy chains consist of one variable domain and three constant domains (ch , ch , and ch ); light chains are composed of a variable and constant domain. in contrast camelid species produce "heavy chain immunoglobulins" that lack light chains and the first constant heavy domain (hamers-casterman et al., ) . the antigen-binding site of these unusual heavy chain antibodies is formed by a single domain, designated vhh in camelids. vhh are easily produced as recombinant proteins, designated single domain nanoantibodies or nanobodies ® and represent the smallest molecule in nature capable of binding a specific antigen. the cdr region of these nanobodies has the capacity to form long loops that can extend into cavities on antigens, e.g., the active site crevice of enzymes. other advantageous features of nanobodies include their high solubility, thermal stability (resist pasteurization), refolding capacity, and optimal tissue penetration in vivo (reviewed by vanlandschoot et al. ( ) ). nanobodies have demonstrated efficacy as agents of passive immunity for infectious diseases. vhh specific for rotavirus inner capsid protein vp are able to broadly neutralize rotaviruses independently of serotype, and in mouse experiments provide passive protection against challenge with human rotavirus (pant et al., ; garaicoechea et al., ) . nanobodies against other viral diseases with veterinary impact have been developed (foot-and-mouth disease, influenza, rabies (vanlandschoot et al., ) ) and represent a promising next-generation biologic platform for passive immunity. maternal vaccination to enhance passive immunity is already widely used in veterinary medicine. some of these vaccines, especially vaccines against enteric viruses, have limited efficacy. new approaches to enhance immunogenicity are promising, but await commercial development. a clearer understanding of protective mechanisms and immune modulation mediated by passive antibodies would contribute to more effective interventions. there is an urgent need for adjuvants and delivery systems capable of reliably inducing active immunity in neonates despite the presence of maternal antibodies. the ability to provide continuity of immune protection from birth, by combining passive immunity with active immunization, would have a major impact on neonatal morbidity and mortality. the rapid expansion of commercial fish farming (aquaculture) in many countries in recent decades has been accompanied by an urgent need to develop vaccines to prevent (infectious) fish diseases that were previously unknown or obscure. added to the difficulties involved in identifying the pathogens responsible, there has been the challenge of delivering vaccine efficiently with minimal stress to very large numbers of fish. some commercial vaccines for fish are administered by intraperitoneal or im injection, but mucosal vaccines are also widely used (reviewed by brudeseth et al. ( ) ). fish are routinely vaccinated by immersion in tanks of diluted bacterin, modified live bacteria or viruses, with exposure times ranging from s up to min, depending on the vaccine and age of the fish. it is unclear whether the main route of antigen uptake is oral or via the mucosal surface of the gills. several commercial vaccines are now available that consist of recombinant viral proteins for mixing into the feed (evensen and leong, ) ; days of feeding is recommended by the manufacturer. research on mucosal veterinary vaccines has contributed new concepts to the field of mucosal immunity. investigations of pathogen-host interactions in outbred animals have illustrated the complexity of these interactions. early studies of an enteric coronavirus infection of swine (tgev) led to the concept of the gut-mammary gland s-iga immunologic axis and provided part of the basic tenet for a common mucosal immune system. later studies of tgev and a deletion mutant of tgev with respiratory tropism (prcv) revealed functional compartmentalization within the common mucosal immune system whereby in inoculation of pigs with prcv failed to elicit sufficient intestinal iga antibody to fully protect against the enteric pathogen tgev (vancott et al., (vancott et al., , . subsequent studies have explored new prime/boost mucosal immunization strategies to elicit intestinal immunity to rotavirus in naïve pigs (saif, b; yuan and saif, ; gonzalez et al., ) . in these studies only oral priming with attenuated virus led to successful in booster responses using nonreplicating (vlp) vaccines combined with mucosal adjuvants such as iscom or mlt. thus use of a replicating vaccine to prime lymphocytes at a major mucosal inductive site (galt) followed by boosting with a nonreplicating vaccine at a second inductive site (nalt) effectively stimulated intestinal iga antibodies and induced active protection against rotavirus diarrhea. although there is progress in developing safe and effective nonreplicating vaccines to boost mucosal immune responses (saif and fernandez, ) , the dilemma remains to develop effective vaccines to prime for mucosal immunity. mucosal adjuvants (mlt, iscom, cpg, cytokines) and new delivery systems (replicating vectors, microparticles) have shown promise in animal studies reviewed in this chapter. however, their economical production and final evaluation under field conditions, including in the presence of maternal antibodies as relevant, are needed. considerable research effort has been devoted to development of vaccines for respiratory diseases of domestic animals. in some instances attenuated organisms delivered by mucosal routes have demonstrated improved efficacy over nonreplicating antigens given by systemic routes. for many respiratory diseases, however, further progress in the development of mucosal vaccines will have to await advances in understanding of disease pathogenesis and identification of protective antigens. in contrast, studies of ascending infections of the reproductive tract in cattle have demonstrated the efficacy of systemic vaccination to clear established infections, and highlight the possibility of therapeutic vaccines. finally it is important to realize that there are considerable species differences in mucosal immunity. for example, the primary ig in mammary secretions of ruminants is igg which is actively transported to the mammary gland from serum and provides effective passive immunity to the nursing offspring against enteric pathogens. thus parenteral immunization of the dam stimulates passive immunity in ruminants against enteric pathogens. in contrast, in monogastrics, iga predominates in milk and iga lymphoblasts that traffic to the mammary gland originate in the intestine. therefore oral vaccines in monogastrics may provide a more effective vaccine strategy to induce iga antibodies in milk (saif and fernandez, ) . by applying the aforementioned vaccine concepts with new and effective mucosal adjuvants, delivery systems, and bioengineered vectors expressing appropriate microbial antigens, it is likely that a new generation of veterinary vaccines will emerge to better cope with existing and emerging mucosal pathogens. interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus forty years of canine vaccination an oral versus in prime/boost regimen using attenuated human rotavirus or vp and vp virus-like particles with immunostimulating complexes influences protection and antibody-secreting cell responses to rotavirus in a neonatal gnotobiotic pig model cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus lactobacillus acidophilus and lactobacillus reuteri modulate cytokine responses in gnotobiotic pigs infected with human rotavirus agedependent diarrhea induced by a rotaviral nonstructural glycoprotein cd antigen presentation: how it works impact of immunizations with porcine reproductive and respiratory syndrome virus on lymphoproliferative recall responses of cd + t cells immune response of pigs inoculated with mycobacterium bovis bcg expressing a truncated form of gp and m protein of porcine reproductive and respiratory syndrome virus immunoprophylaxis of rhodococcus equi pneumonia in foals acute granulomatous response produced in mice by trehalose- , -dimycolate characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) effects of passively and actively acquired antibody on bovine campylobacteriosis (vibriosis) the importance of colostrum to the health of the neonatal calf passive immunity to bovine rotavirus infection associated with transfer of serum antibody into the intestinal lumen the transfer of serum igg antibody into the gastrointestinal tract in newborn calves silent memory induction in maternal immune young animals antibody responses in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus isolation and serotyping of porcine rotaviruses and antigenic comparison with other rotaviruses mechanism of nk activation by ok- (streptococcus pyogenes). i. spontaneous release of nkcf and augmentation of nkcf production following stimulation with nk target cells immunization of virgin cows with surface antigen tf . of tritrichomonas foetus appearance of acute prrs-symptoms in sow herds after vaccination with a modified live prrs vaccine the effect of limited proteolysis by trypsin and chymotrypsin on bovine colostral igg successful pseudorabies vaccination in maternally immune piglets using recombinant vaccinia virus vaccines status and future perspectives of vaccines for industrialized fin-fish farming performance of high titre attenuated canine parvovirus vaccine in pups with maternally derived antibody equine influenza -the a modified live canine parvovirus vaccine. ii. immune response use of parvovirus-like particles for vaccination and induction of multiple immune responses evaluation of the antigenic relationships among canine parvovirus type variants a new modified live equine influenza virus vaccine: phenotypic stability, restricted spread and efficacy against heterologous virus challenge adjuvants for porcine reproductive and respiratory syndrome virus vaccines porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy, and safety aspects morphological and functional comparisons of peyer's patches in different parts of the swine small intestine therapeutic immunization of bulls with the membranes and glycoproteins of tritrichomonas foetus var. brisbane immunity to infections in the lower genital tract of bulls preputial cellular and antibody responses of bulls vaccinated and/or challenged with tritrichomonas foetus effect of vinyl sulfone inhibitors of cysteine proteinases on tritrichomonas foetus infection renal lesions associated with experimental porcine reproductive and respiratory syndrome virus (prrsv) infection animal model for the study of genital immune mechanisms: venereal vibrosis of cattle immunoglobulin binding by tritrichomonas foetus uterine mast cells and immunoglobulin-e antibody responses during clearance of tritrichomonas foetus bovine trichomoniasis as a model for development of vaccines against sexually-transmitted disease immunity in the female bovine reproductive tract based on the response to campylobacter fetus an oral sindbis virus replicon-based dna vaccine containing vp gene of canine parvovirus delivered by escherichia coli elicits immune responses in dogs canine parvovirus -a review of epidemiological and diagnostic aspects, with emphasis on type c evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions crossprotective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant intranasal delivery of whole cell lysate of mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs accelerated vaccination schedule provides protective levels of antibody and complete herd immunity to equine influenza rotaviruses dna vaccines against viral diseases of farmed fish escherichia coli infections escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies dosedependent effect of lactobacillus rhamnosus on quantitative reduction of faecal rotavirus shedding in children adjuvant danger signals increase the immune response to porcine reproductive and respiratory syndrome virus immunity to homologous rotavirus infection in adult mice effect of a probiotic formula on intestinal immunoglobulin a production in healthy children llama-derived single-chain antibody fragments directed to rotavirus vp protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice measles: state of the art and future directions maternal vaccines cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus innate immune responses to human rotavirus in the neonatal gnotobiotic piglet disease model intestinal and systemic immunity to rotaviruses in animal models and humans antibody responses to human rotavirus (hrv) in gnotobiotic pigs following a new prime/boost vaccine strategy using oral attenuated hrv priming and intranasal vp / rotavirus-like particle (vlp) boosting with iscom autoimmunity induced by lactate dehydrogenase-elevating virus: monoclonal autoantibodies against golgi antigens and other subcellular elements combined nkt cell activation and influenza virus vaccination boosts memory ctl generation and protective immunity type-a cpg oligonucleotides activate exclusively porcine natural interferon-producing cells to secrete interferon-α, tumour necrosis factor-α and interleukin- immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs naturally occurring antibodies devoid of light chains cell mediated immune responses in ponies following infection with equine influenza virus (h n ): the influence of induction culture conditions on the properties of cytotoxic effector cells equine mucosal immune system: intranasal vaccination with inactivated equine influenza virus protects from infection duration of circulating antibody and immunity following infection with equine influenza virus antibody isotype responses in the serum and respiratory tract to primary and secondary infections with equine influenza virus (h n ) immunogenicity of a lowpassage, high-titer modified live canine parvovirus vaccine in pups with maternally derived antibodies preparturient vaccination to enhance passive immunity to the capsular polysaccharide of pasteurella haemolytica a effects of maternal antibodies on protection and development of antibody responses to human rotavirus in gnotobiotic pigs bifidobacterium lactis bb enhances intestinal antibody response in formula-fed infants: a randomized, double-blind, controlled trial pork checkoff study: prrs costs industry $ million annually evaluation of equine immunoglobulin specific for rhodococcus equi virulence-associated proteins a and c for use in protecting foals against rhodococcus equi-induced pneumonia infection immunity of piglets to either vp or vp outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen immunostimulating complexes (iscoms) for nasal vaccination passive transfer of immunity oral immunization induces local and distant mucosal immunity in swine conservation of a protective surface antigen of tritrichomonas foetus protection of neonatal calves against fatal enteric colibacillosis by administration of egg yolk powder from hens immunized with k -pilated enterotoxigenic escherichia coli toll-like receptor control of the adaptive immune responses nucleic acid immunization protects dogs against challenge with virulent canine parvovirus f (k ) fimbrial adhesin faeg expressed in alfalfa reduces f + enterotoxigenic escherichia coli excretion in weaned piglets protection of lambs against experimental pneumonic pasteurellosis by transfer of immune serum degradation of bovine complement c by trichomonad extracellular proteinase resistance of broiler chickens to escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies effect of genotypic and biotypic differences among prrs viruses on the serologic assessment of pigs for virus infection lactogenic antibody responses in cows vaccinated with recombinant bovine rotavirus-like particles (vlps) of two serotypes or inactivated bovine rotavirus vaccines use of a blocking elisa for antibodies to equine influenza virus as a test to distinguish between naturally infected and vaccinated horses: proof of concept egg yolk antibodies for passive immunity passive protection against bovine rotavirus in calves by specific immunoglobulins from chicken egg yolk clinical evaluation of the efficacy of inoculating cattle with a vaccine containing tritrichomonas foetus lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea antibody responses to dna vaccination of horses using the influenza virus hemagglutinin gene equine influenza virus infections: an update probiotic bacteria enhance murine and human intestinal epithelial barrier function control of intestinal diseases in pigs by feeding specific chicken egg antibodies zoonotic aspects of rotaviruses evolution of orf of spanish porcine reproductive and respiratory syndrome virus strains from to effector functions of antibody and cd + cells in resolution of rotavirus infection and protection against reinfection in mice cd t cells are the only lymphocytes needed to protect mice against rotavirus shedding after intranasal immunization with a chimeric vp protein and the adjuvant lt(r g) neonatal calf diarrhea: results of a field trial using a reo-like virus vaccine gradual development of the interferon-γ response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (prrs) virus isolated from field cases of "atypical strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus induction of na by a tobacco chloroplast-derived vaccine based on a b cell epitope from canine parvovirus vaccines for preventing enterotoxigenic escherichia coli infections in farm animals iscom: a delivery system for neonates and for mucosal administration passive protection of calves with pasteurella haemolytica antiserum clinical evaluation of transmissible gastroenteritis virus vaccines and vaccination procedures for inducing lactogenic immunity in sows the function of local lymphoid tissues in pulmonary immune responses who/oie meeting: consultation on newly emerging strains of equine influenza antigenicity and immunogenicity of experimental equine influenza iscom vaccines duration of protective efficacy of equine influenza immunostimulating complex/tetanus vaccines antigenicity and immunogenicity of equine influenza vaccines containing a carbomer adjuvant bovine herpes virus infections in cattle local and systemic isotype-specific antibody responses to equine influenza virus infection versus conventional vaccination bark extract amplifies vaccine high titers of circulating maternal antibodies suppress effector and memory b-cell responses induced by an attenuated rotavirus priming and rotavirus-like particle-immunostimulating complex boosting vaccine regimen low titer maternal antibodies can both enhance and suppress b cell responses to a combined live attenuated human rotavirus and vlp-iscom vaccine effect of priming/booster immunisation protocols on immune response to canine parvovirus peptide induced by vaccination with a chimaeric plant virus construct experimental inoculation of late term pregnant sows with a field isolate of porcine reproductive and respiratory syndrome vaccinederived virus passive transfer of virusspecific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea immune response of sows vaccinated with attenuated transmissible gastroenteritis virus (tgev) and recombinant tgev spike protein vaccines and protection of their suckling pigs against virulent tgev challenge exposure modulation by colostrum-acquired maternal antibodies of systemic and mucosal antibody responses to rotavirus in calves experimentally challenged with bovine rotavirus serum and intestinal isotype antibody responses to wa human rotavirus in gnotobiotic pigs are modulated by maternal antibodies early pathogenesis and pathology of tritrichomonas foetus infection in virgin heifers maternally derived immunity to canine parvovirus infection: transfer, decline, and interference with vaccination immunization of pups with maternally derived antibodies to canine parvovirus (cpv) using a modified-live variant (cpv- b) probiotics stimulate enterocyte migration and microbial diversity in the neonatal mouse intestine nutrient and immunity transfer from cow to calf pre-and postcalving porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective demonstration of tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally-infected bulls experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and -week-old pigs comparative pathogenesis of enteric viral infections of swine enteric viral infections of pigs and strategies for induction of mucosal immunity group a rotavirus veterinary vaccines enteric virus vaccines: theoretical considerations, current status, and future approaches transmissible gastroenteritis and porcine respiratory coronavirus isolation of porcine immunoglobulins and determination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies the gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses comparative studies of the pathogenesis, antibody immune responses, and homologous protection to porcine and human rotaviruses in gnotobiotic piglets gut microbiota and probiotics in modulation of epithelium and gut-associated lymphoid tissue function prevention of human rotavirus-induced diarrhea in gnotobiotic piglets using bovine antibody influence of maternal antibodies on vaccine responses: inhibition of antibody but not t cell responses allows successful early prime-boost strategies in mice canine parvovirus type vaccine protects against virulent challenge with type c virus boosting of secretory iga antibody responses in man by parenteral cholera vaccination cleavage of proteins of reproductive secretions by extracellular proteinases of tritrichomonas foetus differential production of proinflammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model veterinary immunology, an introduction serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease toll-like receptor and are expressed and functional in gut-associated lymphoid tissues of presuckling newborn swine efficacy of a recombinant equine influenza vaccine against challenge with an american lineage h n influenza virus responsible for the outbreak in the united kingdom efficacy of a cold-adapted, intranasal equine influenza vaccine: challenge trials dendritic cell-targeted protein vaccines: a novel approach to induce t-cell immunity failure of passive transfer in foals: incidence and outcome on four studs in new south wales protective immunity in macaques vaccinated with live attenuated, recombinant, and subunit measles vaccines in the presence of passively acquired antibodies contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge isotype-specific antibody-secreting cells to transmissible gastroenteritis virus and porcine respiratory coronavirus in gut-and bronchus-associated lymphoid tissues of suckling pigs effects of various vaccination protocols on passive and active immunity to pasteurella haemolytica and haemophilus somnus in beef calves development of an experimental inactivated prrsv vaccine that induces virus-neutralizing antibodies nanobodies ® : new ammunition to battle viruses maternal antibodies against equine influenza virus in foals and their interference with vaccination egg yolk igy: protection against rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves cholera toxin improves the f (k )-specific immune response following oral immunization of pigs with recombinant faeg a field trial to evaluate the efficacy of a combined rotaviruscoronavirus/escherichia coli vaccine in dairy cattle inactivated rotavirus vaccine induces protective immunity in gnotobiotic piglets possible mechanisms of protection elicited by candidate rotavirus vaccines as determined with the adult mouse model rotavirus vaccines: how they work or don't work role of maternally derived circulating antibodies in protection of neonatal swine against porcine group a rotavirus pathogenesis of an attenuated and a virulent strain of group a human rotavirus in neonatal gnotobiotic pigs toll-like receptor and innate cytokine responses induced by lactobacilli colonization and human rotavirus infection in gnotobiotic pigs development of γδ t cell subset responses in gnotobiotic pigs infected with human rotaviruses and colonized with probiotic lactobacilli adjuvant and immunostimulating activities of water-soluble substances extracted from mycobacterium tuberculosis (var. hominis) efficacy of a neospora caninum killed tachyzoite vaccine in preventing abortion and vertical transmission in dairy cattle comparative field evaluations of in ovo applied technology equine influenza antigen-presenting cells in the female reproductive tract: influence of estrous cycle on antigen presentation by uterine epithelial and stromal cells role of porcine reproductive and respiratory syndrome virus nucleocapsid protein in induction of interleukin- and regulatory t-lymphocytes (treg) probiotic bacterium prevents cytokine-induced apoptosis in intestinal epithelial cells the sheep and cattle peyer's patch as a site of b-cell development equine influenza vaccine efficacy: the significance of antigenic variation induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model mucosal and systemic antibody responses and protection induced by a prime/boost rotavirus-dna vaccine in a gnotobiotic pig model short-term immunoglobulin a b-cell memory resides in intestinal lymphoid tissues but not in bone marrow of gnotobiotic pigs inoculated with wa human rotavirus systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease virus-specific intestinal ifn-γ producing t cell responses induced by human rotavirus infection and vaccines are correlated with protection against rotavirus diarrhea in gnotobiotic pigs intranasal administration of cpg oligonucleotides induces mucosal and systemic type immune responses and adjuvant activity to porcine reproductive and respiratory syndrome killed virus vaccine in piglets in vivo influence of probiotic lactobacilli colonization on neonatal b cell responses in a gnotobiotic pig model of human rotavirus infection and disease probiotic lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs key: cord- -xhm wy authors: rinaldi, andrea title: rna to the rescue: rna is one of the most promising targets for drug development given its wide variety of uses date: - - journal: embo rep doi: . /embr. sha: doc_id: cord_uid: xhm wy the race for a vaccine against sars‐cov‐ has accelerated research on rna‐based therapeutics. beyond vaccines, rna also shows great potential for cancer therapies.[image: see text] a fter a long incubation time, rnabased therapeutics are coming of age and offer a big hope for an urgently needed sars-cov- vaccine. as the covid- outbreak is ravaging communities worldwide and exacting a heavy toll in terms of lives and economic costs (https://corona virus.jhu.edu/)-with nothing to counter its spreading but social distancing-many put their hopes on the new kid among the therapeutics: rna. whether targeting nucleic acids or proteins, rna therapies are emerging in the form of a large platform rather than a single approach, with great expectations for a new wave of drugs to treat a range of ailments. infectious diseases, cancers, rare genetic defects, metabolic disorders, degenerative diseases, you name it: rna seems to be the solution for each and every one. the metaphor of the swiss army knife is often abused, but it fits rna like a glove given the multiple roles played by this key molecule: messenger rna (mrna), microrna (mirna), or small interfering rna (sirna). the beginning of the rna therapies universe can be safely placed in , with the discovery by andrew fire and craig mello of rna interference (rnai) as a fundamental mechanism for the regulation of gene expression. in brief, rnai works through gene silencing, degrading the mrna for a specific protein through the coordinated action of double-stranded rna and a complex biochemical machinery it activates (fig ) . the possibility of harnessing the newly identified gene silencing mechanism for developing innovative therapeutics was soon evident and is now finally becoming reality, notably in the form of sirnas, synthetic rna duplexes usually - nucleotides long. however, the road to success was a bumpy one. after the initial excitement, with lots of biotech companies sprouting around rnai discovery, it became soon evident that it was not an easy task. delivering intact sirnas into target cells and tissues proved to be particularly challenging. a whirl of disinvestments, mergers, and discounted acquisitions followed (lawrence, ) . despite all the setbacks, the first therapy based on rnai gene silencing was approved in by the fda. the key molecule is patisaran (marketed as onpattro), a double-stranded sirna to treat a rare and often fatal hereditary condition known as transthyretin-mediated amyloidosis (hattr). this progressive degenerative disease is caused by the misfolding of the liver protein transthyretin (ttr), which leads to the accumulation of amyloid deposits in the peripheral nervous system but also in the heart, kidneys, eyes, and gastrointestinal tract. the drug uses lipid nanoparticles as delivery system and acts by inhibiting the synthesis of ttr, thus reducing the resultant amyloid deposits in the tissues (akinc et al, (wu & turnbull, ) . aptamers are oligonucleotides able to bind proteins with high affinity, and pegaptanib is specifically directed against vegf- , an isoform of the vascular endothelial growth factor that plays a key role in amd vascular pathology. two other rna drugs gained fda approval in , nusinersen (spinraza) and eteplirsen (exondys ), used to treat spinal muscular atrophy and duchenne muscular dystrophy, respectively. both are examples of antisense rna, single-stranded rna that binds to the mrna coding a protein involved in disease pathology, and blocks mrna translation or alters its splicing (wu & turnbull, ) . the future of rna drugs might be bright, but it will be expensive. when an rna virus infects the cell, it injects its genome consisting of double-stranded rna. rna interference destroys the viral rna, preventing the formation of new viruses. a. synthesis of many proteins is controlled by genes encoding microrna. after processing, microrna prevents the translation of mrna to protein. in the research laboratory, dsrna molecules are tailor-made to activate the risc complex to degrade mrna for a specific gene. c. rna interference (rnai) is an important biological mechanism in the regulation of gene expression. of embo reports : e | ª the author treatment will need to be maintained for lifetime. exondys , that benefitted from an accelerated approval process by the fda, can cost up to us$ million a year. "one of the top explanations that drug companies give for the high cost of their products is that it's expensive to get drugs through clinical trials and across the regulatory finish line. when those requirements are truncated, the resulting drugs ought to cost less, not more", commented the editorial board of the new york times in an opinion article, asking for price caps for drugs approved with an expedite pathway ( money will not be a problem for developing a vaccine against sars-cov- (fig ) . as the pandemic spreads across the globe, funds are pouring from both public and independent sources. the coalition for epidemic preparedness innovations (cepi) estimated the cost to develop such a vaccine within the next - months at us$ billion (https://cepi.net/covid- /). the usual time frame to develop a vaccine is between and years; to get a covid vaccine to market, so rapidly will therefore require regulatory compression, like shortening animal tests. in mid-june this year, the world health organization (who) issued a "draft landscape of covid- candidate vaccines", listing scores of products (the number is growing by the day) at various stages of clinical development (https://www.who.int/who-documents-deta il/draft-landscape-of-covid- -candidate-vac cines). the aspirant vaccines were grouped in "platforms", including protein subunits, dna, rna, and non-replicating viral vectors. several of the entries in the who list, including two of the eleven in phase / all the others are still in pre-clinical research with a handful expected to enter clinical trials in the next months-are mrna-based vaccines. the spearhead is mrna- , which encodes a prefusion-stabilized form of the spike (s) protein of sars-cov- , developed at an unusually fast rate (first volunteer injected on mach , only days after the publication of the virus sequence) by moderna, a biotech based in cambridge, massachusetts (https://www.modernatx.c om/). in mid-april this year, the company announced receiving us$ million from the us biomedical advanced research and development authority (barda), to speed up the development of its candidate. "if supported by safety data from the phase study, the company intends to begin a phase study of mrna- . . ..in the second quarter of ", reads a relevant press release (https://investors.modernatx.c om/news-releases/news-release-details/mod erna-announces-award-us-government-agencybarda- -million). "subject to data from these studies and discussions with regulators, a phase study could begin as soon as fall, ". in mid-may, moderna announced positive interim clinical data for mrna- (https://investors.modernatx. com/news-releases/news-release-details/mod erna-announces-positive-interim-phase- -dataits-mrna-vaccine). another mrna covid- vaccine program that has recently started human trials is developed by the german biotech company biontech, in collaboration with pfizer (https://investors.biontech.de/newsreleases/news-release-details/pfizer-and-bio ntech-dose-first-participants-us-part-globalcovid). four vaccine candidates will be studied, two of which include a nucleosidemodified mrna (modrna), one a uridinecontaining mrna (urna), while the fourth vaccine candidate utilizes self-amplifying mrna. the mrna vaccines against sars-cov- are not the first of their type, but rather the last shoot in a rapidly growing tree (jackson et al, ) (fig ) . although there is still no product on the shelves, a number of pre-clinical and clinical trials have shown promising results against a range of infectious diseases, including zika, ebola, dengue, and papillomavirus. the main advantage of the mrna approach to vaccine development is rapid and scalable manufacturing, a very attractive feature in times of epidemic emergency. moreover, rna vaccines may be able to elicit a stronger and broader immune response since they stimulate both the adaptive and innate immune systems. in addition, rna vaccines are cell-free, non-infectious, and not-integrating, thus eliminating the risks of infection and insertional mutagenesis that comes with viral vectors. multi-component proteins that would be impossible to target with other systems are accessible with rna technology, as recently demonstrated by the generation of a multi-antigenic mrna vaccine encoding human cytomegalovirus glycoproteins gb and pentameric complex (john et al, ) . instability and inefficient delivery, two of the main hurdles that hampered rna vaccines success so far, have been largely overcome by recent improvements (pardi et al, ) . "mrna platforms can be designed very fast to express an optimized version of the gene of interest and their production is much easier than the traditional vaccine platforms. in addition, you can also combine mrnas coding for multiple antigens to increase the chance of inducing both production of neutralizing antibodies and resident memory cytotoxic t cells", explained chantal pichon, a molecular and cell biologist at the french national centre for scientific research (cnrs) in orléans. "this could be done through different mrnas coding those antigens or a polycistronic mrna coding the multiple sequences for those antigens". however, gaps still exist in our knowledge on how different mrnas are processed and sensed once they are injected, which will require more basic studies, pichon warns. "the fundraising obtained by companies, mostly in the us, has invigorated both basic ª the author embo reports : e | and translational research around this approach, which can be applied beyond vaccination", pichon concludes. "for sure, mrnas hold a real promise as biopharmaceuticals for the years to come". however, despite the urgency dictated by the covid- pandemic and various calls for international collaboration and data sharing in order to "reduce inefficiencies and duplication of effort" (https://www.who.int/ne ws-room/detail/ - - -public-statementfor-collaboration-on-covid- -vaccine-deve lopment), the landscape looks much fragmented, with probably too many candidate vaccines being pushed forward, and a risk of dispersing much needed resources (berkley, ) . the consequences of failure would be dreadful. "novel infectious diseases resulting from rna viruses subject to mutation and genetic recombination, as well as cross-species transmission, will continue to present a serious global health threat, as exemplified by covid- ", recently remarked cynthia liu and colleagues at the american chemical society (liu et al, ) . "despite two former major outbreaks of coronavirus infections causing the sars and mers respiratory illnesses, the world remains underprepared to effectively manage the current covid- outbreak, as evidenced by the fact that covid- has resulted in thousands of deaths worldwide". another key area, where rnas are expected to have a significant impact, is cancer therapy. here, again, mrna vaccines are bound to play a major role in the future. the idea behind many ongoing clinical trials is to elicit an antitumor immune response in patients by challenging them with mrnas encoding tumor-specific antigens (pastor et al, ; pardi et al, ) . these mrnas can be directly injected as naked rna or loaded into patient-derived dendritic cells (fig ) . the identification of patient-specific tumor antigens can also allow for personalized mrna cancer vaccines. "rna-based cancer vaccines have the advantage of providing transient expression of tumor antigens at high levels", said kenneth lundstrom, an expert in cancer vaccines at pantherapeutics, lutry, switzerland. "especially, application of rna replicons from self-amplifying rna viruses provides the means for direct antigen mrna amplification in the cytoplasm leading to extremely high levels of antigen expression for optimal induction of antibodies". most such therapeutics are still in phase / , but there are promising pre-clinical indications that this approach might trigger an anti-cancer immune response. cv , for example, is a mrna encoding six antigens usually expressed in non-small-cell lung cancer (nsclc), developed by curevac, a biotech based in tübingen, germany (https://www.curevac.com/), in collaboration with boehringer ingelheim. the mrna has been complexed with the cationic protein protamine to protect it from degradation and increase immunogenicity; cv is currently being investigated in a phase / trial in metastatic nsclc patients (https://c linicaltrials.gov/ct /show/nct ?term =nct &draw= &rank= ). other forms of cancer are also in the crosshairs. "melanoma seems like a good target, which has shown promise in clinical trials", said lundstrom. "liposome and polymer encapsulation of rna can further provide protection against degradation and host immune recognition and can target and improve rna delivery". needless to say, success is not guaranteed. in april , argos therapeutics discontinued a phase trial of its dendritic cells-loaded mrna vaccine (rocapuldencel-t) against metastatic renal cell carcinoma after disappointing results (https://www.targetedonc.com/ view/phase-iii-rcc-trial-stopped-after-rocapuld encelt-falls-short). "mrna is the only vaccine format that is highly robust in terms of productionany sequence can be produced with the exact same manufacturing processand very reliable in terms of efficacy, as it naturally induces an innate immune response on its own and at the same time an adaptive immune response against the encoded protein", commented steve pascolo, a molecular biologist at the university of zurich, switzerland. "therefore, its current evaluation in different formats such as naked or formulated in particles, self-amplifying or non-replicating, or different site of injection and design (e.g., coding wild type proteins, modified antigens or epitopes), will with no doubt lead to commercialization of superlative vaccines against cancer and infectious diseases". the scenario of cancer rna immunotherapeutics is rapidly expanding, with clinical studies of known molecules being carried out and new classes of compounds emerging. the potential of rna aptamers (see above), to target cancer-specific proteins, has been exploited by noxxon pharma (berlin, pharma). their nox-a (olaptesed pegol), a pegylated l-oligoribonucleotide that binds and neutralizes cxcl , a chemokine promoting tumor proliferation, yielded encouraging results from a phase / trial in metastatic pancreatic and colorectal cancer and is now being tested for other oncological applications (https://www. noxxon.com/downloads/pressrel/ - - _gbm_cohort_ _dsmb_pr_en.pdf). london-based mina therapeutics, a biotech graduated from the imperial college's translation & innovation hub, is on a more innovative path. the company's technology hinges around small activating rnas (sarna), short oligonucleotides similar in structure to sirnas, that upregulate the expression of their target gene. mina's lead, mtl-cebpa, promotes the expression of cebpa, the product of a tumor suppressor gene that is downregulated in various cancers. this approach is being tested in patients with hepatocarcinoma and is now entering a phase / b clinical study as a treatment for advanced solid tumors. "mtl-cebpa has shown real promise as a combination agent in patients with liver cancer and pre-clinical studies suggest that mtl-cebpa may also be an attractive agent to enhance the benefits of checkpoint inhibitors", commented ruth plummer, newcastle university centre for cancer, and leading scientist of the new study (https://minatx.com/wp-content/uploads/ / / _mina_timepoint-enrolm ent_final.pdf). "we are looking forward to evaluating this highly innovative combination treatment in the upcoming phase i trial. we hope this combination could become an effective new treatment option for patients with solid tumour cancers". with the first results from anti-sars-cov- mrna vaccines just weeks way, rna therapeutics have their once-in-a-lifetime opportunity to ultimately prove their value. to expand our possibilities to treat a variety of serious diseases, and to be ready, for when the next virus will spill over into humans. two categories of mrna constructs are being actively evaluated. non-replicating mrna (nrm) constructs encode the coding sequence (cds) and are flanked by and untranslated regions (utrs), a -cap structure and a -poly-(a) tail. the self-amplifying mrna (sam) construct encodes additional replicase components able to direct intracellular mrna amplification. ( ) nrm and sam are formulated in this illustration in lipid nanoparticles (lnps) that encapsulate the mrna constructs to protect them from degradation and promote cellular uptake. ( ) cellular uptake of the mrna with its delivery system typically exploits membranederived endocytic pathways. ( ) endosomal escape allows release of the mrna into the cytosol. ( ) cytosol-located nrm constructs are immediately translated by ribosomes to produce the protein of interest, which undergoes subsequent post-translational modification. ( ) sam constructs can also be immediately translated by ribosomes to produce the replicase machinery necessary for self-amplification of the mrna. ( ) self-amplified mrna constructs are translated by ribosomes to produce the protein of interest, which undergoes subsequent post-translational modification. ( ) the expressed proteins of interest are generated as secreted, trans-membrane, or intracellular protein. ( ) the innate and adaptive immune responses detect the protein of interest. reproduced from (jackson et al, ) , with permission. ª the author embo reports : e | the onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs covid- needs a big science approach it's time for scientists to shout about rna therapies the promise of mrna vaccines: a biotech and industrial perspective multi-antigenic human cytomegalovirus mrna vaccines that elicit potent humoral and cell-mediated immunity rna interference therapies could be on the cusp of success research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases recent advances in mrna vaccine technology an rna toolbox for cancer immunotherapy current trends in rnabased therapeutic development figure . personalized mrna-based antitumor vaccines normal cells are compared to identify tumor-specific mutations. point non-synonymous mutations, gene deletions, or rearrangements can give rise to neoantigens. several bioinformatics tools are used to predict major histocompatibility complex (mhc) class i and class ii binding (necessary for recognition by t cells) and rna expression presence of the mutated antigen among tumor cells (clonality) key: cord- - xxu orq authors: bozkurt, hayrunnisa bekis title: is the impact of childhood vaccines on coronavirus disease , which is moderate in pediatric patients, possible? date: - - journal: clin exp vaccine res doi: . /cevr. . . . sha: doc_id: cord_uid: xxu orq nan with covid- are vaccinated and live in an area with high vaccine hesitation. finally, we think that childhood vaccines can be effective in observing covid- infection as a mild disease for pediatric population. however, we need more knowledge about the pandemic considering that pediatric cases may be helpful in shedding light on the treatment of the disease. hayrunnisa bekis bozkurt https://orcid.org/ - - - clinical features in pediatric cov-id- are children less susceptible to covid- ? measles, immune suppression and vaccination: direct and indirect nonspecific vaccine benefits protection from sars coronavirus conferred by live measles vaccine expressing the spike glycoprotein nonspecific effects of vaccines and the reduction of mortality in children key: cord- -p wp yvo authors: de groot, anne s; einck, leo; moise, leonard; chambers, michael; ballantyne, john; malone, robert w; ardito, matthew; martin, william title: making vaccines “on demand”: a potential solution for emerging pathogens and biodefense? date: - - journal: hum vaccin immunother doi: . /hv. sha: doc_id: cord_uid: p wp yvo the integrated us public health emergency medical countermeasures enterprise (phemce) has made great strides in strategic preparedness and response capabilities. there have been numerous advances in planning, biothreat countermeasure development, licensure, manufacturing, stockpiling and deployment. increased biodefense surveillance capability has dramatically improved, while new tools and increased awareness have fostered rapid identification of new potential public health pathogens. unfortunately, structural delays in vaccine design, development, manufacture, clinical testing and licensure processes remain significant obstacles to an effective national biodefense rapid response capability. this is particularly true for the very real threat of “novel pathogens” such as the avian-origin influenzas h n and h n , and new coronaviruses such as hcov-emc. conventional approaches to vaccine development, production, clinical testing and licensure are incompatible with the prompt deployment needed for an effective public health response. an alternative approach, proposed here, is to apply computational vaccine design tools and rapid production technologies that now make it possible to engineer vaccines for novel emerging pathogen and wmd biowarfare agent countermeasures in record time. these new tools have the potential to significantly reduce the time needed to design string-of-epitope vaccines for previously unknown pathogens. the design process—from genome to gene sequence, ready to insert in a dna plasmid—can now be accomplished in less than h. while these vaccines are by no means “standard,” the need for innovation in the vaccine design and production process is great. should such vaccines be developed, their -d start-to-finish timeline would represent a -fold faster response than the current standard. pathogens. the design process-from genome to gene sequence, ready to insert in a dna plasmid-can now be accomplished in less than h. while these vaccines are by no means "standard," the need for innovation in the vaccine design and production process is great. should such vaccines be developed, their -d start-to-finish timeline would represent a -fold faster response than the current standard. according to the commission on the prevention of weapons of mass destruction (wmd) proliferation and terrorism, medical counter-measures such as vaccines are critically important for protecting first-responders and noncombatant (civilian) populations from the consequences of a bioterror attack. in , bob graham (d-fl) and jim talent (r-mo), chairs of the wmd commission and authors of world at risk, reported that the united states was "seriously lacking" in this vital capability. the h n influenza pandemic highlighted continued weaknesses in the national preparedness system; as a consequence, graham and talent gave us bio-defense preparedness an "f" in their follow-up report, published in . the governmental accounting office (gao) also reported poor inter-agency coordination on biodefense. , as a result of renewed emphasis on biodefense, the united states recent reports of a novel h n avian influenza virus emerging in china have led to even greater scrutiny of methods used to respond to infectious disease public health threats and have, in turn, provided for a "live fire" assessment of novel approaches. in - , the fastvax group began to discuss whether existing tools and vaccine production platforms could be used to accelerate the development of vaccines for emerging infectious diseases, as illustrated in figure . traditional vaccine development for previously unknown pathogens takes place on the time scale of years. the accelerated process, as proposed by our group, would begin with analysis of the genomic sequence of an emerging pathogen with immunoinformatics tools, followed by rapid design of an epitope-based vaccine containing the most immunogenic components, using an integrated in silico approach illustrated in figure . once the vaccine is designed, production and testing would involve a four-step process undertaken by the fastvax consortium arrangement, as described below. several constraints affecting the proposed approach bear mentioning; each of these is addressed in turn. t cell epitope-based vaccines provide the minimal, essential information required for protective immunity t cell epitopes are critical mediators of cellular immunity. they are derived from a pathogen's proteins via two pathways: ( ) intracellular proteins are processed, and their constituent peptides are loaded onto major histocompatability complex (mhc) class i molecules; and ( ) exogenous proteins are processed in the proteolytic compartment, and their constituent peptides are loaded onto mhc class ii molecules. mhc class i and class ii-peptide complexes are then transported to the surface of an apc, where they are exposed to interrogation by passing t cells (cd + and cd + t cells, respectively). from these different antigen processing and presentation pathways, two distinct t cell responses are generated: ( ) a cd + cytotoxic t lymphocyte immune response that is critical for pathogen clearance, and distributed in different regions of the country that are capable of producing millions of doses of protein-based vaccines. unfortunately, despite these important advances in the strategic preparedness of us agencies for biodefense, vaccine design remains a significant obstacle to national biodefense. this is particularly true for the very real threat of as-yetundetermined pathogens for which little is known about their critical antigenic determinants and correlates of immunity, the key parameters used in vaccine design for conventional pathogens. government has expended substantial resources on protecting the nation against a potential bioterror attack, creating specialized units for planning and preparedness within the departments of health and human services, defense, homeland security, agriculture, commerce and state. vaccine production infrastructure has also improved due to significant investments by the federal government. for example, there are now several federally subsidized "advanced development and manufacturing" production facilities we applied the genome-to-vaccine approach to developing an epitope-based vaccine for avian h n influenza. the design project started on april , and was completed h later. for vaccine production, the genome-derived vaccine sequences would be sent by secure email to a plasmid dna production facility to manufacture a dna vaccine (step ); following scale-up and production, the vaccine would be distributed in a microneedle patch or another easy-to-distribute formulation (step / ). *ics = immunogenic consensus sequences. on whether comparisons have to be performed to other existing genomes and epitopes. tools for carrying out the task have been applied to the development of vaccine candidates for sars, h n pandemic influenza, smallpox, and a number of other emergent and biowarfare agents, such as west nile virus, h. pylori and burkholderia. , [ ] [ ] [ ] most recently, the tools were applied in may to the design of a vaccine for h n , an emerging avian-origin influenza (fig. ) . the integration of epitope mapping into a step-by-step vaccine design process makes it possible to design vaccines in the shortest time possible once the dna sequence from the emerging infectious disease or biowarfare pathogen is available. should errors later be found in the sequence, they may impact one or two epitopes. for an epitope-based string of beads vaccine, the overall impact would be minimal, since t cell epitopes are linear; in contrast, sequence variations may compromise the structural integrity of a whole protein vaccine with negative effects on immunogenicity. how many epitopes? available evidence from animal studies suggests that the number of vaccine components (epitopes) required for full protection against disease is a small and definable subset that can be discovered using state-of-the-art computer programs such as the ones described and validated by epivax. , we have proposed that any fastvax vaccine would include a minimum of broadly reactive t cell epitopes in several strings, designed to induce multi-functional immune responses that are essential for protective immunity. careful selection of the vaccine components, comprising epitopes covering most common hla, can provide greater than % coverage of diverse human populations. need for adjuvants? currently, mf and as , both oil-in-water emulsions, and virosome, a liposome formulation, are three adjuvants licensed for use in seasonal, pre-pandemic and pandemic influenza vaccines. no influenza vaccines containing adjuvant are fda approved. t cell epitope vaccine responses may be enhanced through genetic immunization. dna vaccines are self-adjuvanting through co-encoded sequences, and thus epitope-driven vaccines offer distinct advantages that should contribute to a reconsideration of the current vaccine approval process for emergency use. multiple epitopes derived from more than one antigen can be packaged together in a single cassette. in this way, a broad-based immune response directed against multiple antigenic proteins associated with the pathogen can be elicited without the need to manufacture and administer large quantities of protein, much of which will be immunologically irrelevant or potentially even reactogenic. this is likely to reduce formulation challenges, decrease cost and accelerate the development process. the use of epitopes also helps to mitigate potential safety concerns stemming from the use of intact recombinant proteins that may have undesired biological activity (e.g., enzymes, immunomodulators, cross-reactivity, toxins, etc.). for example, the np protein of lassa has been associated with immune-suppressive activity. genome sequencing, immunoinformatics tools and the epitope-driven approach now make it possible to develop vaccines on demand in response to emerging pathogens. step one: genome-derived, epitopedriven vaccine strategy (gd-edv). the first step to making "faster vaccines" is to design vaccine immunogens directly from pathogen genomes. for example, for emerging influenza strains, the vaccine "payload" is constructed in silico using the pathogen genome sequence provided by the world health organization (who) or posted on gisaid (http:// platform.gisaid.org/). t cell epitopemapping algorithms that are integrated in a "vaccine design toolkit" developed by martin and de groot are applied to the genome sequences. these tools derive and concatenate those epitopes that have a high likelihood of driving an effective t cell response into a "string-of-beads" format for insertion into a vaccine delivery vehicle. the process can be performed in less than h; the exact length of time required for the analysis depends ( ) a cd + t helper immune response that is essential for robust and sustained antibody and cytotoxic t lymphocyte responses. after initial exposure to pathogen, memory t cells are established that respond more rapidly and efficiently upon subsequent exposure. because epitopes provide the essential information needed to trigger a protective immune response, epitope-based vaccines can be developed to recreate this response. given the lengthy process that is usually associated with the development of killed, live-attenuated and whole-subunit vaccine approaches, an epitope-based strategy is one rational alternative, particularly when no vaccine exists and an emerging pathogen threatens human health on a global scale. t cell epitopes do not protect against infection; however, they may protect against disease there is published evidence demonstrating that epitope-based vaccines can be protective. vaccination with peptide epitopes stimulates protective immune responses in a range of animal models, including complete protection of balb/c mice against rsv challenge, partial protection of balb/c mice against plasmodium yoelii sporozoite challenge, partial protection of balb/c and cba mice against encephalitis following intracerebral challenge with a lethal dose of measles virus, complete protection of balb/c mice from intraperitoneal hsv challenge, high degree of protection of balb/c mice against infection with malaria or influenza a virus, full protection of sheep against blv, and full protection of horses against west nile virus. furthermore, experts are generally in agreement that cross-reactive t cell epitopes were responsible for the limited morbidity and mortality associated with pandemic h n in . [ ] [ ] [ ] the absence of t cell epitopes may be contributing to the rapid spread and significant mortality rate of h n in china. t cell epitoperelated immune responses appear to be critically important for reducing morbidity and mortality in human infectious disease. no "fast track" to vaccine-on-demand approval is currently possible under existing fda regulations such is the case with lassa fever, ebola, the encephaloviruses, and a number of other "category a, b and c" biodefense pathogens. in some cases, correlates of protection are unknown, and either an antibody-focused or a t cell-driven vaccine may prove effective. where antibodymediated immunity is critically important, t cell-driven vaccines still merit attention as potential adjuncts to more traditional whole-antigen (b cell-driven) approaches, since t cell help drives higher titer, higher affinity antibody responses. especially in settings where challenge studies cannot be performed in advance of use in humans, licensure may be possible by means of the "two animal rule" in lieu of a human correlate. rapid clinical testing can be achieved using existing commercial clinical research organizations and clinical site networks such as the medical countermeasures clinical studies network currently envisioned by aspr/ barda. emergency use authorization approval can be based on achievement of "correlates" such as induction of broadly protective t cell or antibody responses, provided an allowed investigational new drug (ind) application is in hand. one problem facing t cell-driven vaccines that are designed to stimulate hlarestricted human immune responses is that testing for correlates of immunity as described in the "two animal rule" may not demonstrate the true efficacy of the product. thus alternative approaches may need to be considered. the mimic assay, a comprehensive measurement of localized reactogenicity, could be utilized for initial safety studies and to qualify release of the actual vaccine intended for emergency use. additionally, in pandemic response simulations, "mock up" or example vaccines (in a specific dna plasmid backbone) and patch delivery system could be submitted for approval by the fda, and this formulation would be evaluated in the clinic for immunogenicity that recapitulates the influenza correlates of protective immunity already defined by cber and ema. correlates of protective immunity for currently approved influenza vaccines will not serve as a basis for regulatory approval of a dna vaccine. the fda would require correlates to be determined for a new the dna vaccine delivery platform and rational in silico design provide for a strong safety profile. the dna vaccine manufacturing process, particularly the efficient and stringent release criteria, allow for a highly pure and well-characterized final product. rational design permits in silico analysis of the vaccine sequence for identification of potential unfavorable immune responses including regulatory sequences or cross-reactive immune responses. a fundamental principle of rapid biodefense vaccine production is that safety and speed are paramount for eliciting a protective immune response prior to the epidemic. delivery vehicle. the bulk vaccine product would then be coated onto premanufactured micro-needle patches that provide direct delivery to the dermis, or would be delivered using another skinbased method such as "scarification." a number of self-applied patch delivery systems have already been developed. these would be optimal in bioterror and pandemic scenarios, because patches can be pre-manufactured and stored in bulk and do not require refrigeration for delivery or trained practitioners for administration. vaccination centers would not be required, which would minimize transmission of the biothreat organism between patients and health care providers. alternatively, previously approved electroporation delivery methods could be used, though this would take more time and increase the need for vaccine administration personnel training, leading to an escalation of the vaccine administration expense and more protracted timelines. step three: clinical trials. while there are no phase iii or fda-approved dna vaccines, there are more than phase ii trials listed in clinicaltrials.gov. fda approval of a dna vaccine appears to be on the horizon, but until then, the fastvax dna vaccine may encounter an additional fda-associated barrier. implementation of a previously untested vaccine is only possible after rapidly completing initial clinical testing to the point that "emergency use authorization" can be invoked by the secretary of health and human services (hhs). in some biodefense scenarios, approximate correlates of protection may have been previously identified; many such vaccines do not incorporate traditional adjuvants in their final formulation. a number of strategies that are currently being evaluated may improve dna vaccine potency for humans, including use of more efficient promoters and codon optimization, addition of traditional or genetic adjuvants, electroporation and intradermal delivery. step two: manufacturing and production. reliable, reproducible methods for producing vaccines are currently available. the fastvax consortium favors dna vaccines because production is scalable, the vaccines are stable at room temperature, manufacturing can be easily distributed to different geographic locations, and the production method is more rapid than many other vaccine manufacturing technologies. alternative scalable and rapid production methods for accelerated vaccine production include plant-derived vaccines, phage-based vaccines and recombinant vaccines produced in cell culture. proteins produced using each of these systems have been approved by the fda for use in humans. rapid production of dna vaccines. the initial vaccine sequence designed in silico can be electronically provided to a production facility, where a cassette representing the vaccine genetic construct(s) is then synthesized and inserted into a standardized dna vaccine plasmid. a cgmp seed lot of bacteria containing the vaccine plasmid with cassetted payload can be rapidly produced and vialed using existing sops for release and characterization assays. an initial manufacturing lot of plasmid vaccine would be produced from the seed lot and used to initiate safety studies. to reduce time to produce sufficient vaccine product, multiple scale-up facilities could be located in different regions of the us. using current methods of dna vaccine development, seed lot production would take one to three weeks. scale-up for dna production is much more rapid than traditional vaccine designs; only three to four weeks would be required to produce one million doses per facility. see below for discussion of biological agents research defense agency (barda) appropriations for the construction of distributed vaccine production facilities. foreground with lessons applicable to influenza t cell-driven vaccine development. perhaps the most prominent example of this new focus is the expanding use of t cell-driven immunotherapy as an adjunct to cancer therapy. many of the barriers to effective t cell-driven vaccine development are being addressed and surmounted in clinical cancer trials. for example, dendritic-cell pulsing vaccines using tumor antigens have moved into clinical use. , outcomes of these types of vaccination protocols have improved as mhc class ii epitopes (cd + t cell help) were included and antibodies against cytotoxic t lymphocyte antigen- (anti-ctla- ; see ref. ) and other anti-t regulatory cell (treg) agents have been added to the conditioning regimen. quite a few t cell-driven vaccines are currently in human clinical trials (reviewed by gilbert in ; see ref. ). while it is true that infectious disease t cell-driven vaccines have lagged behind t cell-driven vaccines for cancer, the regulatory pathway for t cell vaccines is improving, since more than cancer vaccines that are based on t cell-driven immune responses are in clinical trials. a furthermore, recent challenge studies have shown that humoral immunity is not required for protection against all human pathogens. this was demonstrated in the case of influenza, following vaccination of study participants with a multi-antigen vaccine. following exposure to live influenza virus, two of vaccinees and five of control subjects developed laboratoryconfirmed influenza (symptoms plus virus shedding). symptoms of influenza were less pronounced in the vaccinees and there was a significant reduction in the number of days of virus shedding in those vaccinees who developed influenza (mean of . d in controls, . d in vaccinees, p = . ) , for a final efficacy of %, which is better than many vaccines currently available. this is a major milestone for t cell vaccines for infectious disease, as it is one of the first vaccines to reach a phase clinical trial and none have reached phase . while one cannot directly extrapolate from this trial nor the many cancer t cell-driven immunotherapy trials to state that the approach will work for all types involve redundancy and higher costs, it would allow for the rapid production and scale-up of vaccines at any given moment. each site would need to utilize the same manufacturing process to ensure consistency across vaccine batches, and entities would need to be willing to share their specific methodologies to harmonize an approach. one site would create the master cell bank (mcb), and then generate the manufacturer's working cell bank (mwcb) for distribution to all other sites. in order to reduce production time by two weeks, this step would be performed "at risk," meaning mwcbs would be distributed prior to the completion of testing on either the mcb or mwcb. sequencing on the mcb could likely be completed before the mwcb goes into fermenter starters. assuming that a dose would constitute . mg of dna vaccine and that each site has several l fermenters (either as back-ups or for parallel growth), one million doses ( g) per site could be produced in a three-to four-week period. barda recently invested hundreds of millions of dollars in distributed influenza vaccine production; adapting these facilities for dna vaccine production would be an added but not insurmountable expense (as compared with the initial investment). an in vitro assay like the mimic system could serve as a release characteristic of the multi-site lots that would run in parallel with the patch loading, preventing a single problematic dna vaccine batch from impeding the release of patches generated with other batches. if the backbonehost system is proven to be rugged with virtually any type of insert, a pilot run would no longer be necessary. conversely, if the system is not shown to be rugged, then pilot runs would be important, as some inserts can greatly influence stability and growth characteristics. such pilot runs would need to be undertaken at every facility, most likely with different methods tested, to maximize the likelihood of determining the best method for production. a number of technological advances are moving t cell-driven vaccines to the influenza vaccine and will not rely on related, but different, vaccines already approved. advance trials will establish correlates of protection for a fastvax influenza vaccine to serve as a basis for regulatory review in an emergency. in a pandemic, a novel fastvax sequence composition might be rapidly tested in a small, swiftly completed safety and immunogenicity trial, much like ema precedence for annual influenza vaccine updates. step four: approval and emergency use authorization. one means of obtaining initial fda review, experience and oversight for the fastvax vaccine-on-demand system would be to firmly establish the immunogenicity of an existing, clinicaltrial-ready dna influenza virus vaccine in a patch or scarification delivery system. demonstration that the vaccine candidate meets influenza correlates of protection criteria with an acceptable profile in human trials would inform regulatory review for products of similar composition, much as current regulatory policy supports annual marketing re-authorization despite changes in influenza subunit vaccine composition (from trivalent to quadrivalent) to reflect seasonal shifts and drifts. timely approval by the fda to allow distribution of product in response to a rapidly emerging threat would require close cooperation between the vaccine manufacturer and the agency. the manufacturer can assist by providing clinical safety and efficacy data for a variety of vaccine products based on standardized vaccine platform, manufacturing, specifications, operating procedures and method of delivery. if the manufacturer can establish predictable immunogenicity of epitopes in a demonstrated safe and reproducible vaccine platform and rapidly perform phase i and phase ii trials establishing safety and immunogenicity in terms of a surrogate endpoint that predicts clinical benefit, the agency may be able to provide a rapid review and emergency use allowance/authorization; release of the vaccine would then be possible through emergency use authorization by the hhs secretary. scale up. to reduce the time to vaccine production, manufacturing sites could be pre-inspected and maintained at a state of operational readiness. while this would acknowledge that there is a potential conflict of interest related to their employment and attest that the work contained in this research report is free of any bias that might be associated with the commercial goals of the companies. strategic [ ] [ ] [ ] over the past five years, the authors of this report have advanced a number of t cell-driven vaccines described to the point of formulation and delivery studies. vaccines for many of the high-priority biodefense pathogens and emerging or re-emerging infectious diseases under development are not currently available, and evidence that t cell-mediated immune response is critically important for protection against these pathogens is emerging. , [ ] [ ] [ ] [ ] [ ] members of the fastvax consortium are well aware that there are many obstacles to overcome before the proposed "rapid response" or fastvax platform for biodefense vaccines can be implemented. nonetheless, there is a critical national need for an accelerated vaccine design, development and production process that can be accomplished in weeks, not months, in the event of a serious infectious disease outbreak or biowarfare attack. the development of a rapid response to emerging infectious disease threats, using bestin-class technologies to provide a first line of defense, will contribute to greater biodefense preparedness and a significant improvement in the ability of the us to protect its citizens against pandemic infectious diseases. the need for new vaccines for protecting against bioterror pathogens and emerging infectious disease is great, and we would argue that, for the reasons cited above, the time to advance these vaccines to the clinic is now. adg and wdm are senior officers and majority shareholders at epivax, inc., a privately owned immunoinformatics and vaccine design company located in providence, ri, usa. lm is an employee and holds stock options in epivax. le and rwm have been paid consultants of epivax on vaccine development programs. jb and mc are employees and stockholders at aldevron, inc. the authors of vaccines against infectious disease, successful implementation of the t celldriven approach in a range of contexts suggests that it is worth pursuing. immunome-mining (computational immunology) tools have played a major role in the design and development of t cell-driven vaccines for infectious diseases. the process was first termed "vaccinomics" by brusic and petrovsky in , then "reverse vaccinology" by rappuoli in , and more recently, "immunomederived or genome-derived vaccine design" by pederson, de groot and martin, and doytchinova, taylor, and flower. the concept behind these descriptors is that a minimal set of antigens that induces a competent immune response to a pathogen or neoplasm can be discovered using immunoinformatics, and that administration of these epitopes in the right delivery vehicle and with the correct adjuvant will result in a degree of protection against infection by the pathogen. in short, the t cell-driven approach to developing vaccines is based on these fundamental principles: payload + adjuvant + delivery vehicle = vaccine. t cell-driven vaccines also offer some significant advantages over conventional vaccines for infectious diseases. for example, despite strain-to-strain variation at the protein level, immunoinformatics tools can be used to identify highly conserved t cell epitopes that are immunogenic and broadly representative or universal, covering a wide range of variant strains; our group has published results for tb, hiv, smallpox, hcv and h. pylori, , [ ] [ ] [ ] [ ] [ ] [ ] [ ] and additional evidence can be found in literature published by other gene-to-vaccine researchers (e.g., sette and newman, brusic, petrovsky, reche, and he). concatenation of multiple epitopes, either from a single organism or from multiple pathogens in a single delivery vehicle, has been shown to elicit broad-based immune response directed at the epitopes and is associated with improved efficacy when compared with the whole organism (lysate) in animal challenge studies. , furthermore, epitope-based vaccines limit the antigenic load, diminishing the need to manufacture and administer large quantities of immunogen, much of which is immunologically irrelevant. in an world at risk prevention of wmd proliferation and terrorism report card - sp: homeland security/law enforcement microneedle-based vaccines therapeutic and prophylactic dna vaccines for hiv- a predictive biomimetic model of cytokine release induced by tgn and other therapeutic monoclonal antibodies united states of america department of health and human services support for advancing influenza vaccine manufacturing in the developing world immunotherapy for castration-resistant prostate cancer sipuleucel-t (provenge) autologous vaccine approved for treatment of men with asymptomatic or minimally symptomatic castrate-resistant metastatic prostate cancer targeting cd (+) t-helper cells improves the induction of antitumor responses in dendritic cell-based vaccination ctla- blockade in tumor models: an overview of preclinical and translational research t-cell-inducing vaccines -what's the future examination of influenza specific t cell responses after influenza virus challenge in individuals vaccinated with mva-np+m vaccine preliminary assessment of the efficacy of a t-cell-based influenza vaccine, mva-np+m , in humans phase i trial of a cd + t-cell peptide epitope-based vaccine for infectious mononucleosis reverse vaccinology and genomics the immunome from immunome to vaccine: epitope mapping and vaccine design tools proteomics in vaccinology and immunobiology: an informatics perspective of the immunone immunoinformatics: the next step in vaccine design from immunome to vaccine: epitope mapping and vaccine design tools how the sars vaccine effort can learn from hiv-speeding towards the future, learning from the past immunoinformatic comparison of t-cell epitopes contained in novel swine-origin influenza a (h n ) virus with epitopes in - conventional influenza vaccine dna-prime, peptideboost multi-t-cell epitope poxvirus vaccine, induces protective immunity against vaccinia infection by t cell response alone rapid determination of hla b* ligands from the west nile virus ny genome analysis of chimerivax japanese encephalitis virus envelope for t-cell epitopes and comparison to circulating strain sequences helicovax: epitope-based therapeutic helicobacter pylori vaccination in a mouse model low immunogenicity predicted for emerging avian-origin h n : implication for influenza vaccine design a consensus epitope prediction approach identifies the breadth of murine t(cd +)-cell responses to vaccinia virus putting immunoinformatics to the test immunization with hiv- gag protein conjugated to a tlr / agonist results in the generation of hiv- gag-specific th and cd + t cell responses nine major hla class i supertypes account for the vast preponderance of hla-a and -b polymorphism enhanced priming of multispecific, murine cd + t cell responses by dna vaccines expressing stress protein-binding polytope peptides technologies for enhanced efficacy of dna vaccines a subdominant cd (+) cytotoxic t lymphocyte (ctl) epitope from the plasmodium yoelii circumsporozoite protein induces ctls that eliminate infected hepatocytes from culture ctl epitopes identified with a defective recombinant adenovirus expressing measles virus nucleoprotein and evaluation of their protective capacity in mice immunization with a leaps heteroconjugate containing a ctl epitope and a peptide from beta- -microglobulin elicits a protective and dth response to herpes simplex virus type recombinant sindbis viruses expressing a cytotoxic t-lymphocyte epitope of a malaria parasite or of influenza virus elicit protection against the corresponding pathogen in mice vaccine-induced cytotoxic t lymphocytes protect against retroviral challenge west nile virus recombinant dna vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays immunoinformatic comparison of t-cell epitopes contained in novel swine-origin influenza a (h n ) virus with epitopes in - conventional influenza vaccine pre-existing immunity against swine-origin h n influenza viruses in the general human population coupling sensitive in vitro and in silico techniques to assess crossreactive cd (+) t cells against the swine-origin h n influenza virus low immunogenicity predicted for emerging avian-origin h n : implication for influenza vaccine design t-cell-inducing vaccines -what's the future structure of the lassa virus nucleoprotein reveals a dsrna-specific ' to ' exonuclease activity essential for immune suppression vaccines against lyme disease: what happened and what lessons can we learn? t cell vaccines for microbial infections a dominant cd (+) t-cell response to helicobacter pylori reduces risk for gastric disease in humans identification of class i hla t cell control epitopes for west nile virus development of burkholderia mallei and pseudomallei vaccines signatures of t cells as correlates of immunity to francisella tularensis dna-prime, peptideboost multi-t-cell epitope poxvirus vaccine, induces protective immunity against vaccinia infection by t cell response alone epitope-based therapeutic h. pylori vaccination in a mouse model of gastric cancer the two-faced t cell epitope: examining the host-microbe interface with janusmatrix the two-faced t cell epitope: examining the host-microbe interface with janusmatrix immunogenicity and immune modulatory effects of in silico predicted l. donovani candidate peptide vaccines developing an epitope-driven tuberculosis (tb) vaccine hiv vaccine development by computer assisted design: the gaia vaccine efficacy of novel plasmid dna encoding vaccinia antigens in improving current smallpox vaccination strategy immunogenic consensus sequence t helper epitopes for a pan-burkholderia biodefense vaccine coupling sensitive in vitro and in silico techniques to assess crossreactive cd (+) t cells against the swine-origin h n influenza virus conservation of hiv- t cell epitopes across time and clades: validation of immunogenic hla-a epitopes selected for the gaia hiv vaccine further progress on defining highly conserved immunogenic epitopes for a global hiv vaccine: hla-a -restricted gaia vaccine epitopes funding to support the discussions leading to the development of the fastvax consortium can be attributed to the nih u grant ai (to adg). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institute of allergy and infectious diseases or the national institutes of health.endnote a there are clinical trials for "t cell vaccines" currently reported at http:// www.cancer.gov/clinicaltrials/search. key: cord- -upsc cf authors: taylor-robinson, andrew w title: a vaccine effective against zika virus is theoretically possible but may not be delivered anytime soon date: - - journal: res rep trop med doi: . /rrtm.s sha: doc_id: cord_uid: upsc cf following the first report in may of the unexpected emergence of zika in north east brazil, there has been an explosive epidemic of this infection across latin america. the outbreak has caused alarm among social and news media as to the virulence and transmission potential of the aedes mosquito-borne virus. this debate is heightened by the proximity, both in time and distance, to the forthcoming olympic games to be held in rio de janeiro this august, provoking fears for the safety of athletes and spectators alike. the threat, real or perceived, is exacerbated by the movement between nations in the same or separate continents of persons who act unwittingly as asymptomatic carriers. pregnant females are considered at greatest risk because microcephaly in newborn infants is linked to, if not yet proven as caused by, zika infection. in february this year, the world health organization declared that further to the then unconfirmed association between the virus and the clinical manifestations of microcephaly and also guillain-barré syndrome, the zika epidemic was a “public health emergency of international concern”. no anti-zika therapy, vaccine or drug, is currently available and while the production of the former has now been prioritized by multiple funding agencies, the history of infectious disease vaccine development indicates that this may take several years to reach the market place. the fact that zika is a close relative of yellow fever and japanese encephalitis viruses, for both of which there are already effective vaccines, provides a rational basis for the fast-tracked laboratory-based preparation of a candidate vaccine. however, undertaking clinical trials on pregnant females provides ethical and practical hurdles to overcome before licensure is granted for public administration. meanwhile, public health management strategies, including mosquito control programs to reduce breeding, are needed to limit the global spread of this re-emerging disease. the aedes mosquito-transmitted viral disease of humans, zika, was originally identified in and is named after the rainforest in uganda, east africa, where it was first isolated from rhesus macaques. for close on years, the prevalence of zika infection was very low, such that prior to now it has attracted little interest apart from arbovirus and tropical medicine specialists. in the last several months, this scenario has changed dramatically, however, subsequent to a major zika epidemic in over countries in latin america and the caribbean. this includes an estimated , , clinical cases in brazil, from where the outbreak arose in early , although questions regarding the accuracy of reporting have been raised. furthermore, the world health taylor-robinson organization (who) predicts that by the end of this year up to million people across the americas may be infected with the zika virus. the impact on the vast majority of those people will be minimal, but particularly in infants the effect may be profoundly debilitating and long-lasting. such is the extent of the issue, real and predicted, and the degree of concern that it has engendered globally, that on february , the international health regulations emergency committee of the who declared the zika epidemic as "a public health emergency of international concern" and highlighted the importance of aggressive measures to reduce infection, especially of pregnant females and those of childbearing age. subsequently, the us centers for disease control and prevention moved zika to level activation, the highest available state of response. similar to the etiological agents of yellow fever, japanese encephalitis and dengue, zika is a member of the flavivirus genus of enveloped, positive sense, single-stranded rna viruses. compared to each of these close relatives, infection with which can be severely incapacitating for a person of any age, it is thought that approximately % of adults infected with zika show no clinical manifestations. hence, for several days after being bitten by an infectious mosquito, they may serve as asymptomatic carriers of infection. if a person is ill, the main symptoms may last for up to week and are similar to but less severe than other related febrile illnesses -a mild headache, fever, myalgia, arthralgia, conjunctivitis, and maculopapular rash. the principal possible consequence of zika infection, for which there is now claimed to be a causal link, occurs via congenital transmission from a pregnant female to her fetus in utero or newborn baby, , the effects of which can be severe. in brazil alone, the virus has been associated with over , cases of microcephaly, a previously uncommon condition that as a result of aberrant brain development produces babies with abnormally small heads and, in most cases, neurological impairment. in march , panama registered a baby born with microcephaly linked to the zika virus, in what is thought to be the first such case outside brazil during the current outbreak. the baby died within hours and at postmortem examination the virus envelope protein was detected by enzyme-linked immunosorbent assay in the baby's umbilical cord. rarely, zika is also associated with, but still not proven to be causally linked to, guillain-baré syndrome, a neural demyelination syndrome that is considered to be an autoimmune sequela of infectious disease. , the zika epidemic is arousing apprehension among the general public just months before the world's attention will focus on rio de janeiro for the summer olympic games. at present, the advice to pregnant females from non-endemic regions is to not travel to brazil and surrounding countries. aside from this immediate concern in latin america, the increasing media frenzy has fed misinformation with regard to the possible global spread of zika, and the impact this may have on public health in regions that are currently not affected. as a consequence of global climate change the geographical distribution of a. aegypti, the species of mosquito that is considered to act as the principal vector of zika transmission, is expanding gradually in tropical and subtropical zones. for non-endemic industrialized nations in north america, europe, and australasia, the critical issue is whether or not zika virus-permissive mosquito populations can adapt for persistence in temperate climates. a more rapid spread of the virus via the intercontinental travel of infected persons is an additional concern, although for zika to become established in a location distant to an endemic area requires local transmission of the initially imported focus of infection; this is dependent on the availability of the vector. vertical, sexual, and blood-borne transmission of zika has been suggested, , which, if confirmed, may provide auxiliary routes to enable viral persistence in regions that are not endemic for aedes mosquitoes. thus, one recommendation to prevent disease, especially microcephaly, is to practice safe sex in those territories where zika is reported as endemic. importantly, the risks should be considered in family planning until such time as the apparent association between viral infection and microcephaly is either confirmed or refuted. , another re-emerging infectious disease the zika epidemic comes hard on the heels of other notable infection outbreaks by pathogenic viruses around the world in recent years, each of which has posed a threat to human wellbeing. these include the coronaviruses that cause severe acute respiratory syndrome and middle east respiratory syndrome, the h n swine influenza, h n and h n avian influenza, and the filovirus ebola. each in turn has led epidemiologists to consider transmission cycles and zoonoses, infectious zika vaccine development disease immunologists to improve emergency measures such as pathogen detection and containment, and vaccinologists to aim to develop effective immunization regimens. , in much the same way as the upsurge of ebola cases in west africa in , zika may be considered as a re-emerging infectious disease; that is one which is established but displayed in a novel setting. while the reasons for the current zika epidemic in south america are multifactorial, inadequate vector control of an unexpected population explosion of mosquitoes is a suspected cause. this raises the question as to what guidance is appropriate to provide to combat zika, especially in industrialized nations where there has been the loudest call for anti-zika prophylaxis, such as that which could be provided by sterilizing immunization. in the short term, as anti-viral therapies are yet to be realized alternative means should be used, including low technology measures that focus on vector control such as insecticide fogging, limiting mosquito breeding, and providing protection from mosquito bites. the delivery of different interventions, singularly or in combination, should be informed by, and thereby tailored to, local settings. what should not be overlooked in the current furore over zika is that a. aegypti is also responsible for transmitting yellow fever, japanese encephalitis, dengue, and chikungunya, viruses that have arguably far more impact on global public health. the implementation of any program to limit or eliminate a. aegypti as a result of concern over the spread of zika will have the added benefit of coincidentally reducing the risk of incidence of these other important viral human pathogens. , it is also possible that a species of mosquito other than a. aegypti is the main vector fueling the current epidemic of zika in a continent where this virus has not been established previously, although this too would be susceptible to identical means of vector control. until now there has been little in the way of research into zika, both with regard to the virus and the pathologies that it causes. hence, there is a knowledge gap surrounding infection transmission and clinical manifestations of disease. since there has been no pressing demand before the present epidemic, no prophylactic vaccine or therapeutic drug is currently available with which to treat zika infection. for any infectious disease, the vaccine development pathway from candidate design, via preclinical screening, through phase i-iii clinical trials to final approval for public administration is long, demanding, and expensive. while this has now been prioritized by international funding agencies for zika, it may take several years for a vaccine to come to commercial fruition. as efficacious vaccines have been prepared against yellow fever and japanese encephalitis viruses, it is anticipated that a similar therapeutic is feasible for zika. nevertheless, in spite of ring-fenced funds to support research into developing a vaccine, a note of caution should be advised. it is feasible that the laboratory-based design and preclinical screening of a candidate vaccine may be fast-tracked in a matter of months, perhaps by the end of . this is particularly so if a dna-based construct is developed. both the indian pharmaceutical company bharat biotech and the us national institute of allergy and infectious diseases have plans to produce a vaccine. however, in all probability, it will take several years to perform any ensuing clinical trials and to attain full approval from national regulatory bodies for public administration. , although to gain ethical approval for, and to conduct, tests of vaccine safety and efficacy in humans necessitates diligence and caution at all times, the due process for any candidate vaccine that is dispensed to pregnant females is naturally subject to extremely exacting analysis. , this would relate especially to zika as, allegedly, the gravest manifestation of infection, microcephaly, affects pregnancy. the positive news is that the latest clinical trial of an experimental dengue vaccine, tv , has proved % effective, albeit a small scale volunteer challenge study of only the dengue- serotype performed under highly controlled conditions. while this is no doubt a significant breakthrough in the ongoing fight against dengue, as an offshoot of this success it is hoped that a modified version of the live attenuated construct may be developed in order to treat the related zika virus. this head start may accelerate production of a candidate zika vaccine but will not truncate its potentially arduous journey through clinical trials. during this time, scientists, clinicians, and health care professionals all should be attentive to stress the standard caveats and qualifications to the outcomes of any study, so not to augment the plethora of misinformation that concerns zika by implying, however unintentionally, that a vaccine is forthcoming anytime soon. , the recent striking emergence of cases of zika in latin america poses a threat for a worldwide outbreak of this mosquito-borne flavivirus infection. the public health challenges presented by zika will remain unless and until the taylor-robinson current deficit of knowledge relating to its epidemiology, pathology, and immunology is addressed. current measures to combat the spread of infection rely on vector control programs, which evidently require improved operation and integrated management. a specific anti-zika therapy does not exist, but in response to the ongoing epidemic research funds have been committed to develop a vaccine. while prior success in producing effective vaccines against closely related human viral pathogens provides a proof of principle for this approach, vaccine design and clinical testing can be laborious and the timescale long. in the short term, therefore, optimism regarding delivery of a zika vaccine should be tempered by realism regarding a delivery date. the author reports no conflicts of interest in this work. zika virus. i. isolations and serological specificity zika virus in the americas -yet another arbovirus threat pan american health organization. zika virus infection. washington dc: paho/who concern over zika virus grips the world who director-general summarizes the outcome of the emergency committee regarding clusters of microcephaly and guillain-barré syndrome zika virus, what cdc is doing? centers for disease control and prevention full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses zika without symptoms in returning travellers: what are the implications zika virus -symptoms, diagnosis & treatment zika virus and birth defects--reviewing the evidence for causality rapid risk assessment. zika virus epidemic: potential association with microcephaly and guillain-barré syndrome zika virus and pregnancy: what obstetric health care providers need to know zika: panama has 'first microcephaly case outside brazil zika virus, french polynesia, south pacific cdc broadens zika virus travel alert for pregnant women zika virus: rumours and theories fuel 'information war the global spread of zika virus: is public and media concern justified in regions currently unaffected? probable non-vector-borne transmission of zika virus potential sexual transmission of zika virus zika virus: your questions answered possible association between zika virus infection and microcephaly -brazil detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study novel vaccine strategies against emerging viruses a systems biology approach for diagnostic and vaccine antigen discovery in tropical infectious diseases temporal and spatial analysis of the - ebola virus outbreak in west africa mosquitoes and their control local transmission of zika virus infection is possible in australia but should be contained by current vector control measures introducing new vaccines in developing countries a review of successful flavivirus vaccines and the problems with those flaviviruses for which vaccines are not yet available the guardian bbc news here's why we don't have a vaccine for zika (and other mosquito-borne viruses) pregnancy in the time of zika: addressing barriers for developing vaccines and other measures for pregnant women dengue vaccine aces trailblazing trial. protection against virus raises hopes of vaccine development for dengue and even zika key: cord- - qfe kok authors: xia, yufei; fan, qingze; hao, dongxia; wu, jie; ma, guanghui; su, zhiguo title: chitosan-based mucosal adjuvants: sunrise on the ocean date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: qfe kok mucosal vaccination, which is shown to elicit systemic and mucosal immune responses, serves as a non-invasive and convenient alternative to parenteral administration, with stronger capability in combatting diseases at the site of entry. the exploration of potent mucosal adjuvants is emerging as a significant area, based on the continued necessity to amplify the immune responses to a wide array of antigens that are poorly immunogenic at the mucosal sites. as one of the inspirations from the ocean, chitosan-based mucosal adjuvants have been developed with unique advantages, such as, ability of mucosal adhesion, distinct trait of opening the junctions to allow the paracellular transport of antigen, good tolerability and biocompatibility, which guaranteed the great potential in capitalizing on their application in human clinical trials. in this review, the state of art of chitosan and its derivatives as mucosal adjuvants, including thermo-sensitive chitosan system as mucosal adjuvant that were newly developed by author's group, was described, as well as the clinical application perspective. after a brief introduction of mucosal adjuvants, chitosan and its derivatives as robust immune potentiator were discussed in detail and depth, in regard to the metabolism, safety profile, mode of actions and preclinical and clinical applications, which may shed light on the massive clinical application of chitosan as mucosal adjuvant. vaccination has arguably been the most successful and effective medical intervention for preventing infectious diseases, resulting in decreased mortality, extended life expectancy, and improved quality of life [ ] [ ] [ ] . more than million vaccine formulations are now administered by subcutaneous or intramuscular injections, according to who records [ ] . however, some shortcomings have persisted throughout the development of the vaccine industry, such as needle-stick injuries, the potential for contamination, and the need for highly trained personnel [ ] . furthermore, most pathogens invade the body at mucosal sites. the mucosal immune system, comprising epithelial cells, mucosa-associated lymphoid tissues (malts), and lamina propria, contains almost % of the immunocytes in the human body [ ] and constitutes the first line of defense against infection [ ] . unfortunately, vaccination via injection fails to achieve acceptable immune protection at mucosal sites [ ] . needle-free vaccination offers immense potential in terms of patient compliance and ease of administration, and has attracted great attention recently [ , [ ] [ ] [ ] . the significance of mucosal immunity and its potential for improving global health has inspired research demonstrating that mucosal vaccines could be an attractive alternative to parenteral administration [ , ] . nonetheless, the administration of antigens via mucosal routes normally leads to poor immunogenic stimulation. to overcome this, live pathogens have been employed in commercial vaccines such as oral polio [ , ] , oral cholera [ ] [ ] [ ] [ ] , oral rotavirus [ ] [ ] [ ] and nasal influenza (flumist tm [ ] [ ] [ ] , nasovac tm [ ] ), but these still retain some potential hazards for large-scale application. hence, rational design of an appropriate adjuvant system with a potent capacity to stimulate immunity is essential [ ] . modern manufacturing technology is obliged to meet the following requirements: ( ) stability of the antigen in an acidic, alkaline, or enzymatic environment [ ] [ ] [ ] ; ( ) retention of the antigen despite constant clearance at mucosal sites [ , ] ; ( ) potent induction of both systemic and mucosal immunity [ ] ; and ( ) a balance between enhanced mucosal immunity and the possibility of immune tolerance when using high doses of antigen [ ] . as a promising solution, chitosan possesses well-defined properties including biocompatibility, low cost [ ] , high levels of tolerance, a broad antibody response, and the capacity to stimulate robust th responses [ , ] . chitosan has been demonstrated to have distinct mucoadhesivity, which prolongs the antigen http://dx.doi.org/ . /j.vaccine. . . - x/© elsevier ltd. all rights reserved. retention time at mucosal sites [ ] , as well as the ability to open the tight junctions between epithelial cells to intensify the transport of antigen to sites of immune induction and effector responses [ , , , ] . moreover, the versatility of chitosan enables a diversity of formulations for chitosan-based delivery systems, such as solutions, powders, micro/nanoparticles, hydrogels, and microneedle patches, to meet the various needs of novel vaccination methods [ , , , ] . recent research has proposed several promising chitosan-based formulations that are highly suitable as mucosal adjuvants with outstanding safety profiles. this article aims to provide an up-to-date review of pre-clinical and clinical applications for these novel adjuvant systems. although chitosan has been investigated for many years as a basic material for mucosal adjuvants, it is not yet present in any marketed vaccine formulation. the main obstacles may be a lack of understanding of its metabolism and concerns regarding safety issues. hence, a more comprehensive evaluation of its metabolism, safety profile, modes of action, and clinical applications is also presented in the following sections. chitosan, a natural cationic polysaccharide, is made up of copolymers of glucosamine (␤( - )-linked -amino- -deoxyd-glucose) and n-acetylglucosamine ( -acetamido- -deoxy-dglucose), and is derived by the partial deacetylation of chitin obtained from the shells of crustaceans or the mycelium of fungi [ ] . a wide range of chitosan polymers is available with diverse molecular weights (mw), degrees of de-acetylation (dd), and modifications, all of which may impact on their solubility, biocompatibility, mucoadhesive behavior, and degradation, and thus influence their immunogenicity [ ] . scherließ et al. successfully showed that three commercialized chitosans with different mw and dd possessed varied levels of adjuvant activity, and the impurities (protein contaminants) in the chitosan were found to play only a minor role. they also pointed out that these diverse adjuvant effects cannot be attributed to a single factor but rather an interplay between molecular weight, degree of deacetylation, particle size, and solubility [ ] . to overcome the poor solubility of chitosan at neutral ph [ ] , various chitosan derivatives have been developed by chemical modification of hydrophilic groups or by simply grafting on water-soluble polymers (e.g., polyethylene glycol) [ ] [ ] [ ] . the derivatization of chitosan not only enhances its water solubility, but also improves the cationic nature of the material, which enhances its interactions with antigens or cell membranes, boosting antigen adsorption/encapsulation and cellular internalization [ ] , as well as facilitating the residence time at mucosal sites [ ] . among the water-soluble chitosan derivatives, n-trimethyl chitosan (tmc) and n-( -hydroxy) propyl- -trimethyl ammonium chitosan chloride (htcc) are the most commonly used mucosal adjuvants. the chemical structures and main properties are summarized in table [ ]. a comprehensive understanding of the metabolism of a polymer is a prerequisite for its development as a novel adjuvant [ ] . as a natural polysaccharide, chitosan is usually regarded as a nontoxic, biologically compatible polymer [ , ] . in vitro studies have proven that chitosan can be degraded efficiently by lysozyme, resulting in approximately a % decrease in the weight of the chitosan hydrogel within days [ ] . three enzymatically active forms of human chitinases have been identified in investigations of the degradation of chitosan and its derivatives [ ] , namely acidic mammalian chitinase (amcase), di-n-acetylchitobiase, and chitotriosidase, which have been found in the gastrointestinal tract, liver, and plasma, respectively [ ] [ ] [ ] [ ] . recent research has revealed that the degradation kinetics depend strongly on both the molecular weight and the degree of deacetylation [ , ] . typically, a higher enzyme-reaction rate was found for the degradation of high-mw chitosan. low-dd chitosan was degraded more rapidly, and the remaining to acetyl groups may serve as essential contacts in the active sites of chitosanases [ ] . tmc was degraded by lysozyme at a similar rate to native chitosan, but the degradation rate was independent of ph [ ] . in a study of in vivo degradation, chitosan underwent partial digestion and uptake by the gastrointestinal tract, and was then deposited in tissues, or circulated in the plasma to eventually be eliminated from the body [ ] . limited data on the degradation and distribution of chitosan have been presented, because of difficulties tracking trace amounts of chitosan in the living body [ ] . however, some conclusions can be deduced. first of all, the in vivo degradation and distribution of chitosan is largely dependent on the molecular weight. fitc-labeled chitosan with a small molecular weight ( . kda) was absorbed by the gastrointestinal tract to give a significant plasma concentration. however, chitosan with a mw higher than kda (with similar dd) was excreted in the feces without any uptake [ ] , which indicated that it was not chitosan polymers but chitosan oligosaccharides that could be adsorbed in the gut. in addition, the optimal mw of chitosan for renal clearance was estimated to be kda. if the mw of the adsorbed chitosan oligomers was larger than that, they would undergo further biodegradation [ ] . the failure of intranasal formulations in clinical trials suggests that toxicity may be caused by the uptake of a degradation product by the brain and irritation of the nasal mucosa during degradation. this may lead to serious local symptoms or neurological disorders such as bell's palsy, which is a facial nerve paralysis caused by the intranasal administration of a heat-labile enterotoxin used as an adjuvant. in the case of chitosan-based mucosal adjuvants, understanding the distribution and degradation of chitosan is vitally important for dispelling safety concerns. recently, smith et al. pointed out that high-dd chitosan was degraded much more slowly under the local ph ( . - . ) of the nasal cavity and might guarantee that the administered chitosan would be cleared by mucociliary movement and swallowed into the alimentary tract, which might protect the body from severe adverse effects and toxicological responses [ ] . chitosan with a high dd can probably serve as an ideal raw material for developing intranasal vaccines. most of the above results have concentrated on the metabolic behavior of chitosan polymers. on the other hand, the metabolism of chitosan powder and smaller particles may be a very different story. although trimethyl-chitosan oligomers/dna nanoparticles were shown to be absorbed in the gastric tract [ ] , there is still insufficient evidence to support the deduction that other chitosan formulations would be degraded in a similar manner. with regard to the safety profile of chitosan, cytotoxicity is dependent on the concentration, molecular weight, and degree of de-acetylation, with a half-maximal inhibitory concentration (ic ) of . - . mg/ml in most cell models [ , , ] . in addition, different forms of chitosan have shown similar cytotoxic effects [ , ] . a number of studies have shown the good tolerance and safety performance of chitosan administered via oral routes. in table currently employed chitosan derivatives as mucosal permeation enhancer. vivo, very few adverse effects have occurred in both mouse and rat models after oral intake of chitosan in doses of - g/kg/day for months [ ] . in addition, no relevant clinical signs were found in healthy human volunteers after taking oral doses of up to . g/day [ ] . moreover, people with shrimp allergies have shown no allergic reactions after oral administration of shellfishderived glucosamine [ ] . archimedes and other groups have conducted a large number of preclinical or clinical studies on chitosan administered via the intranasal route. they concluded that chitosan could be well tolerated with no adverse effects or only mild symptoms such as erythema, itchy nose, rhinorrhea, and sore throat [ ] . in view of the above facts, chitosan is generally thought to be a biodegradable and biocompatible biomaterial with no or minor toxicity and offers the potential to be a safe pharmaceutical material for clinical vaccine formulations [ , ] . consequently, the raw material should be selected carefully with regard to the molecular weight, degree of deacetylation, and purity. moreover, it is worth noticing that the physiochemical and biological properties of chitosan are also influenced by the original raw material, the manufacturing procedure, and even by the season and weather. batch-to-batch consistency and stability of the raw material are other challenges when selecting chitosanbased adjuvants for clinical trials. the greatest difference between vaccine adjuvants and drug delivery systems is that vaccines are administered to healthy people, especially populations at high risk of contracting infectious diseases such as infants and the elderly, which means any side effects must be avoided [ ] [ ] . nevertheless, the truth about adjuvants is that "the nearer they get to killing you, the more effective they are". there is an appropriate level of inflammatory response that is undesirable in a drug-delivery system but obligatory for immune stimulation. future research must manage the balance between toxicity and adjuvanticity, in order to advance the clinical application of chitosan-based mucosal adjuvants. chitosan-based formulations have been found to orchestrate both cellular and humoral responses, and represent the most promising candidates for adjuvant and delivery systems for mucosal vaccines [ ] . therefore, it is essential to understand the mechanisms of mucosal immunity and the mode of action of chitosan-based adjuvants, to further the rational design of a chitosan-based adjuvant system with a profound ability to stimulate immunity and a distinct safety performance [ ] . numerous studies have demonstrated the potent induction of cellular responses, activation of the innate immune system, and positive effects on mucosal adjuvanticity [ , , ] . in this section, general strategies for developing chitosan into a promising mucosal adjuvant are addressed in detail with reference to recent reports. the innate immune system not only initiates immediate and consistent responses towards infection, but also directly triggers the adaptive immune system. recognition by the innate immune system requires the activation of pattern recognition receptors (prrs), such as toll-like receptors (tlrs, one of the membranebound prrs) and nucleotide-binding domain leucine-rich repeat containing receptors (nlrs, one of the cytoplasmic prrs), which are specific for conserved ligands associated with cellular damage (danger associated molecular patterns, damps) or invading pathogens (pathogen associated molecular patterns, pamps) [ ] . upon stimulation by danger signals, dcs undergo activation/maturation to an active antigen-presenting phenotype [ ] , with up-regulation of co-stimulatory membrane proteins such as cd and cd , and secretion of proinflammatory cytokines such as tnf (tumor necrosis factor), interleukin (il)- , or il- . recent evidence has showed that all three signals are key factors for tlymphocyte activation [ ] . villiers et al. have demonstrated that chitosan induces the activation of dcs at the level of membranereceptor recognition. chitosan promoted the activation of dcs via a tlr -dependent mechanism, with up-regulation of membrane proteins such as cd and cd [ ] . however, it failed to elicit the robust cytokine production that activates t cells, which indicates that chitosan alone was unable to evoke a t-cell response. nevertheless, when combined with antigen, chitosan and its derivatives stimulated the expression of major histocompatibility complex (mhc) class i molecules, which profoundly up-regulate the expression of co-stimulator molecules, namely cd , cd , and cd , as well as the secretion of the cytokines il- , il- , mip- , and tnf-␣. [ ] this led to stronger cd + cells proliferation and cytotoxic t lymphocyte (ctl) responses [ ] . as one of the best characterized nlr family members, the nlr family pyrin domain containing (nlrp ) was shown to be associated with potent inflammatory responses, which were activated by a multi-protein complex composed of nlrp , the adaptor apoptosis-associated speck-like protein (asc), and the protease caspase- (fig. ) . currently, chitosan particles are proven triggers of a potent adaptive inflammatory response through the nlrp pathway, which facilitates the recruitment of neutrophils [ ] [ ] [ ] the protonation of the amino groups ("proton sponge effect") leads to an extensive inflow of ions and water into the lysosome, which caused the osmotic swelling and deconstruction of the lysosome. the entrapped components (csp and ag) were released and finally presented onto mhc i, by cytoplasm degradation (with proteosome and er involved); after the rupture, lysosome enzymes, cathepsin b, was also leaked into the cytoplasm, which induced the assembly and activation of nlrp complex. the capacity of tlr stimulation of csp also played an important role in the intracellular synthesis of pro il- ␤ and triggered the secretion of il- ␤ and inflammatory responses, together with nlrp activation. (ag: antigen; csp: chitosan particle.) and maturation of apcs, and creates a pro-inflammatory environment characterized by the secretion of cytokines such as il- ␤ and il- [ , ] . as shown in fig. , the disruption of lysosomes is thought to trigger the chitosan-particle-induced nlrp response. after rupture, the leakage of lysosomal enzymes such as cathepsin b, which can prompt the assembly and activation of nlrp complex, stimulates the secretion of il- ␤ and subsequent inflammatory responses [ ] [ ] [ ] . recent research has suggested that the activation of the inflammasome by chitosan particles (csps) is dependent upon the physiochemical properties of csps, such as particle size, charge [ , ] , and surface texture [ ] . neumann et al. found that only positively charged chitosan nanoparticles and alum induced significant il- ␤ secretion after pre-incubation with lps to activate tlr in vitro [ ] . cellular immunity, which is caused by cd + t-cell activation, is vital for the success of prophylactic and therapeutic vaccines [ ] . typically, exogenous antigens are endocytosed by professional antigen presenting cells (apcs), and are then degraded into short peptides within their endosomes. these degraded peptides are loaded onto mhc class ii molecules to present antigen to cd + helper t cells, which leads to humoral immunity. only some of the intracellular antigens would escape from the endosome and be presented to cd + t lymphocytes through the mhc class i pathway, to stimulate cellular immune responses. traditional adjuvant systems using alum, mf , and as , failed to elicit potent cellular immunity [ ] . on the other hand, chitosan particles possess a robust capacity to elicit cross-presentation of exogenous antigens and induce potent cellular immune responses. cross-presentation can be induced by several means such as tlr activation [ , ] , mannose receptor-mediated uptake [ ] , and up-regulation of the expression of heat shock proteins [ , ] . in the case of csps, tuning the cellular trafficking of antigen is triggered by the "lysosome escape" pathway, which indicates that the internalized chitosan particles can disrupt the lysosome [ , ] and escape into the cytoplasm through a "proton sponge effect" [ ] . as shown in fig. , the protonation of amino groups present on the chitosan chains leads to an extensive inflow of ions and water into the lysosome. when the "proton sponge effect" becomes strong enough, the entrapped components (csp and antigen) are released by rupture of the lysosomes [ , ] . the escaped exogenous antigen can be cross-presented through the mhc class i pathway, like endogenous antigens, to prime cd + t cells and evoke robust ctl responses [ , ] . briefly, mucosal surfaces are constructed of a single layer of epithelial cells joined by tight junctions [ ] , which acts as a natural barrier to protect the body from the outside world. when pathogens frequently breach the epithelial barrier, epithelial tissues are activated and serve as sensors for detecting danger signals through pattern-recognition receptors such as tlrs [ , ] . they respond by releasing chemokines and cytokines to recruit malt-resident immunocytes and trigger innate and adaptive immune responses [ ] . recently, chitosan-based adjuvant systems have been proven to have mucoadhesive qualities and the unique ability to open the tight junctions between epithelial cells at natural portals, which stimulates a robust mucosal immune response by facilitating the transport of antigens via an alternative yet effective route for antigen delivery [ , , , , ] . numerous studies have confirmed the mucoadhesive properties of chitosan that prolong the residence time of formulations at mucosal sites, including the nasal mucosa [ , , , ] , gastrointestinal surfaces [ ] , and rectal and genital tracts [ ] . our group employed an in vivo imaging system to evaluate the ability of a thermo-sensitive htcc hydrogel to prolong the residence time of cy labeled h n antigen in the nasal cavity. as shown in fig. a , no fluorescent signal was detected in the pbs/h n group h post administration. however, strong fluorescence intensity could be observed in the htcc/h n group. more than % of the administered antigens persisted in the nasal cavity after h (fig. b) , which shows the ability of chitosan to offer a sustained antigen-mucosal interaction without being cleared away [ ] . the distinguishing trait of chitosan to mimic pathogen entry by opening tight junctions between epithelial cells has also been investigated in recent research [ , ] . artursson's group suggested that the ability to open junctions was due to the positively charged amino group on the c- position of chitosan, which could interact with the negatively charged mucosal surface [ ] . in another study, they compared differences in the transport of [ c]mannitol, among three chitosan-derivative solutions, including chitosan hydrochloride, chitosan glutamate, and tmc, all with a similar degree of de-acetylation. in the intestinal epithelial cell line caco- , the transport of [ c]-mannitol was increased -fold by chitosan hydrochloride, -fold by chitosan glutamate, and -fold by tmc at . % w/v concentrations in vitro, compared with the administration of mannitol alone [ ] . by determining the change in transepithelial electrical resistance to indicate the tightness of the junctions and using confocal laser scanning microscopy in vitro, they further demonstrated the interactions between chitosan and zona occludens (zo)- protein, which is regarded as the target of chitosan and its derivatives [ ] [ ] [ ] . hsu et al. elucidated the signaling mechanism underlying the opening of tight junctions by chitosan in caco- cell monolayers. as shown in fig. , the cascade of tight-junction opening is accompanied by the clustering of integrins (␣ v ␤ ) along the cell periphery, which led to the phosphorylation of focal adhesion kinase (fak, also known as protein tyrosine kinase , ptk ) and src (a family of non-specific tyrosine kinases). this also resulted in the regulation of tight-junction permeability via the redistribution of cldn (a tight-junction protein) from the cell membrane to the cytosol and its eventual degradation in lysosomes [ , ] . in pre-clinical and clinical research, chitosan and its derivatives have demonstrated low-toxicity, mucoadhesive capabilities, the ability to enhance the internalization of antigens by apcs, and immune-stimulatory properties, which makes them promising candidates for effective mucosal adjuvants [ ] . the current evidence supporting chitosan as a potent mucosal adjuvant is summarized and recapitulated in table , in a multitudinous array of vaccine formulations, different chitosan types, dosage forms, model animals, and routes of administration. in the following section, applications for adjuvant systems based on chitosan . the signaling mechanism of cs-mediated tj opening [ ] . and its derivatives are reviewed with regard to their routes of administration. since the adoption and widespread administration of the sabin oral polio vaccine (opv) [ ] , oral delivery has been considered the preferred route of administration for vaccines, especially for children [ ] . recently, chitosan has been extensively studied as a promising candidate for oral administration to promote the internalization of antigen and activate both systemic and mucosal immunity [ ] . several studies have demonstrated that associating the antigens with csps can enhance their uptake by m cells and reduce the risk of vaccine degradation. van der lubben et al. reported that the size of microparticles (mps) should be m for efficient uptake by m cells [ ] . they also found that fluorescently labeled chitosan mps can be taken up by m cells in peyer's patches at intestinal surfaces [ ] . in later research, van der lubben et al. further showed that antigen was released from mps after its uptake by m cells [ ] . moreover, a dose-dependent immune reaction was revealed for mice vaccinated with different doses of antigen associated with chitosan mps [ ] , which proved the ability of chitosan to promote the internalization of vaccines. with the aid of an enteric coating, chitosan delivery systems can be protected from stomach acid and released from within the enteric coating after reaching the intestine [ ] . hori et al. developed ovalbumin (ova)-containing chitosan mps (chi-ova), and coated them with eudragit l (er-chi-ova). compared to an uncoated chi-ova delivery system, significantly higher intestinal mucosal iga and systemic igg responses were detected after oral administration of coated chi-ova. [ ] . in another study, cho et al. presented a mucoadhesive and ph-sensitive thiolated eudragit-coated chitosan microparticle, with an enhanced capacity for retaining the structural integrity of the encapsulated protein [ ] . recently, hbsag-loaded chitosan microparticles were formulated. to overcome the limitations imposed by enzymatic degradation and permeation barriers, bacitracin was introduced as a protease inhibitor, which resulted in better protective levels of immunity after oral administration compared with aprotinin as a protease inhibitor. moreover, the mps remained stable at room temperature for up to four months [ ] . the nasal route is an excellent alternative to conventional forms of vaccination, and is effective at inducing systemic and mucosal immunity in not only the upper and lower respiratory tracts, but also the gastric and genital tracts via cross-protection [ , ] . as mentioned above, the chitosan-based adjuvant system can provide effective immune stimulation through potent enhancement of antigen retention, mucosal permeability, cellular uptake, and protection of the antigen [ ] , as well as a good safety profile [ ] . the past few years have witnessed the wide application of chitosan-based adjuvants in pre-clinical research on intranasal vaccines. vila et al. prepared tetanus toxoid-encapsulated chitosan nanoparticles as an intranasal antigen delivery system, which resulted in an increasing and long-lasting humoral immune response (igg and iga levels) [ ] . the controlled release of antigen by chitosan particles is another important factor contributing to adjuvanticity. mokarram et al. has demonstrated the effect of peg (polyethylene glycol) modifications on the immune-stimulating performance of chitosan nanoparticles, using diphtheria toxoidloaded chitosan nanoparticles with or without peg modifications (cs-peg and cs). in the study, cs-peg and cs nanoparticles both elicited profoundly higher iga and igg levels compared with fluid antigen. in addition, the performance of cs-peg as an adjuvant was significantly better, which may be due to the controlled releasing of antigen [ ] . scherließ et al. proposed a novel spray-dried and ␤-irradiated sterilized vaccine with outstanding stability for intranasal administration against influenza a(h n )pdm in nonhuman primates, and employed chitosan and a stabilizing and protecting solution (sps). the chitosan in the formulation not only served as an inducer of immunity, but also stabilized the antigen, especially in combination with sps. the seroconversion results revealed that the effectiveness of the pilot formulation was comparable to pandemrix ® . moreover, the hemagglutination-inhibition titer of the group administered sterilized vaccine showed no difference from the group administered unsterilized vaccine [ ] . chitosan derivatives, such as tmc [ ] , cg [ ] , and chitosan hydrochloride salt [ ] , have also been employed in the intranasal delivery of antigen. our group has developed a thermosensitive hydrogel formulated from the chitosan derivative htcc and ␣,␤-glycerophosphate (␣,␤-gp) as an intranasal vaccine delivery system for the h n split antigen [ ] . this hydrogel was designed to gelate in a short time at body temperature on the surface of the nasal mucosa, which significantly reduced mucociliary clearance and prolonged the antigen residence time in the nasal cavity. using confocal laser scanning microscopy and scanning electron microscopy, we observed the stained nasal epithelia after intranasal administration of chitosan hydrogel, and have provided significant evidence for the disorganization of zo- protein in nasal epithelial tissue. the thermo-sensitive hydrogel facilitated the transportation of antigen into the sub-mucosa via the paracellular pathway rather than the endocytosis pathway (fig. ) . when employed in the intranasal delivery of h n split antigen [ ] and adenovirus antigen [ ] , the hydrogel system enhanced robust serum and mucosal antibody levels, the secretion of cytokines, and a potent response by antigen-specific memory cd + t cells. when chitosan and its derivatives were added to a hybrid system, an increase in the adjuvanticity of other biodegradable polymers was observed. jaganathan et al. reported that intranasal administration of chitosan hydrochloride-modified plga mps led to significantly higher anti-hbsag igg and iga titers and levels of cytokines (il- and ifn-␥) in spleen homogenates than unmodified plga mps [ ] . in another study, pawar et al. prepared uncoated plga nanoparticles, chitosan-modified plga np (cs-plga), and glycol chitosan-modified plga np (gc-plga) for encapsulation of the antigen, and administered these via the nasal tract. the results showed that gc-plga exhibited the highest level of mucus adhesion and elicited the highest igg and iga titers, as well as the secretion of il- , il- , and inf-␥ [ ] . vicente et al. developed a polymer/oil-based core-corona nanocapsule, formulated with an oily core of miglyol ® , a neutral oil composed of triglycerides of medium chain fatty acids, and a positively charged shell of chitosan [ ] . through co-delivery of rhbsag antigen and the tlr agonist imiquimod, the vaccine orchestrated greater secretion of pro-inflammatory cytokines (il- ␣, il- , and tnf-␣), increasing igg levels over time and boosting both cellular and humoral immunity [ ] . chitosan-based adjuvant systems have also been employed in mucosal vaccination via other routes. chen et al. developed a novel transdermal delivery system consisting of embeddable chitosan microneedles with a supporting array composed of a polylactic acid (pla) polymer, for the sustained delivery of encapsulated antigen, which in this case was ova. the chitosan microneedle remained in the epidermis and dermis (at a depth of approximately m) for days as the chitosan degraded (fig. ) . the antibody levels were significantly higher in the microneedle group than the intramuscular-administration group [ ] . for pulmonary administration, microparticles containing diphtheria toxoid (dt) and chitosan were prepared using a spray-drying technique with a supercritical mixture of carbon dioxide (co ) and ethanol. after immunization, a strong immune response was observed in the group receiving chitosan-dt, with higher igm, igg, igg , and igg titers than the group receiving alum-absorbed dt, which supports the potential of chitosan as a potent delivery system for pulmonary vaccines [ ] . in the food industry, chitosan has been designated generallyrecognized-as-safe (gras) in the us, japan, and italy [ ] . however, chitosan has still not been utilized in any marketed vaccine formulations. only a few studies have been published on the clinical applications of chitosan as an adjuvant. mills et al. have reported the safety and immune-stimulatory capacity of a chitosan-glutamate intranasal delivery system for the diphtheria toxoid antigen crm , among healthy volunteers. in phase a of the study, three groups of subjects were administered intranasal diphtheria vaccine ( g of crm with . mg of chitosan glutamate , and . mg of mannitol), antigen alone (on day and ), or alum-adsorbed diphtheria toxoid vaccine via intramuscular injection (single administration on day ). serum igg and iga from nasal wash were collected on days and . the results suggested that the chitosan intranasal formulation was well tolerated with only transient mild-to-moderate adverse effects (aes), such as nasal discharge, blockage, and discomfort. there were no significant differences in the circulating antibody titers of the groups after first administration, but the antibody levels of the group receiving crm and chitosan intranasally were boosted after a second vaccination [ ] . norovirus infection is the most common cause of viral gastroenteritis in humans [ , ] . parra et al. developed a norovirus virus-like particle (vlp) vaccine, which orchestrated distinct immune responses in the rabbit after intranasal vaccination [ ] . the vaccine (nv-vlp) was formulated as spray-dried powder, which was composed of chitosan (chisys tm ) and norovirus vlp antigen, with mpl (monophosphoryl lipid) as an immune enhancer. chisys tm is a drug delivery technology based on chitosan, which is provided by archimedes development limited. in phase i clinical studies, two randomized, double-blinded, controlled studies of nv-vlp vaccine have been performed. healthy volunteers ( - years old) were randomized to receive two intranasal administrations of nv-vlp or act as controls (with a interval of days). in study , g, g, and g dosages of norwalk antigen and chitosan alone were evaluated. in study , four groups of healthy subjects were administered mpl/chitosan ( or g vlp per dose, as a powder), chitosan only, or placebo (puff of air). the symptoms were recorded for days after vaccination, and the safety evaluation was followed up for days. samples of serum and peripheral blood mononuclear cells were also collected. the results indicated that the most common symptoms were nasal stuffiness and discharge, and sneezing. no vaccine-related serious adverse effects (saes) were observed. the most significant immune response was found for the -g dose, with igg and iga titers increased by . and . -fold, respectively. all subjects who received the vaccine dose of or g produced iga antibodysecreting cells (ascs), with surface molecules that are associated with homing to mucosal and peripheral lymphoid tissues (cd +, cd +, cd l−, integrin ␣ /␤ +, cd +, cd +, cd l+, and integrin ␣ /␤ +). the results indicated that intranasal administration of nv-vlp may evoke iga secretion by intestinal mucosal tissues (the entry site of norovirus) [ ] . in other studies, healthy adults ( - years of age) were randomized and received primary and booster injections of either vaccine ( g vlp per dose) or placebo, administered weeks apart. three weeks after the second injection, the subjects were challenged with norovirus. serology data were collected and the clinical efficacy was assessed by the occurrence of infection, the onset severity, and the duration of the illness caused by norovirus. aes of nasal stuffiness, nasal discharge, and sneezing were also reported in this trial, and occurred with similar frequency among vaccine and placebo recipients. a significant iga response (a -fold increase) was detected in % of vaccine recipients. moreover, the occurrence of norovirus-induced gastroenteritis ( % of placebo recipients vs. % of vaccine recipients, p = . ) and virus infection ( % of placebo recipients vs. % of vaccine recipients, p = . ) was significantly reduced by nv-vlp vaccination [ , ] . chitosan-based adjuvant systems have been developed with versatility in formulation, excellent tolerance, distinct mucoadhesivity, and a robust capacity to stimulate cellular and humoral immunity in both clinical and pre-clinical studies. application of these adjuvant systems would significantly improve vaccine development to meet the needs of reduced cost, increased stability, and easier administration. however, only a few chitosan adjuvant systems have been approved for clinical trials ( on-going clinical experiments), and none of them have yet been included in a licensed vaccine product. numerous challenges are still hindering the development of chitosan-based mucosal adjuvant systems in humans. diverse types of chitosan at different concentrations with various dd and mw are now being studied by separate research groups using a diverse range of antigens (table ) , which could also lead to controversy and confusion regarding its potential application in human medicine. future research is needed to investigate the mode of action of chitosan, based on its different aforementioned properties, and establish criteria for advancing chitosan development. we envisage a booming future for chitosan as an adjuvant in novel mucosal vaccine formulations. although there are hurdles that still need to be overcome, it is reasonable to expect that it will not be very long before chitosan-based delivery systems for vaccines are released onto the market. vaccine preventable dis t historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states the intangible value of vaccination new horizons in adjuvants for vaccine development vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens chitosan: a promising safe and immuneenhancing adjuvant for intranasal vaccines mucosal immunity and vaccines mucosal vaccines: the promise and the challenge design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review needle-free jet injection for administration of influenza vaccine: a randomised non-inferiority trial recent progress in mucosal vaccine development: potential and limitations needle-free influenza vaccination immunology of gut mucosal vaccines adjuvants and inactivated polio vaccine: a systematic review considerations for development of whole cell bacterial vaccines to prevent diarrheal diseases in children in developing countries oral vaccines against cholera optimism over vaccines administered via mucosal surfaces enteric vaccines for the developing world: a challenge for mucosal immunology effect of human rotavirus vaccine on severe diarrhea in african infants rotaviruses: from pathogenesis to vaccination safety and efficacy of a pentavalent human-bovine (wc ) reassortant rotavirus vaccine live attenuated influenza vaccine influenza vaccine immunology effectiveness of inactivated influenza vaccine in children aged months to years: an observational cohort study a pandemic influenza vaccine in india: from strain to sale within months immunization without needles chitosan-based gastrointestinal delivery systems cyclodextrins in nasal drug delivery polymeric hydrogels for oral insulin delivery jabbal-gill i. nasal vaccine innovation use of the inactivated intranasal influenza vaccine and the risk of bell's palsy in switzerland chondroitin sulfate, hyaluronic acid and chitin/chitosan production using marine waste sources: characteristics, applications and ecofriendly processes: a review advances in chitosan-based drug delivery vehicles chitins and chitosans as immunoadjuvants and non-allergenic drug carriers chitosan and its derivatives in mucosal drug and vaccine delivery chitosan-based systems for the delivery of vaccine antigens recent advances in nanocarrier-based mucosal delivery of biomolecules chitosan-based delivery systems for protein therapeutics and antigens functional characterization of chitin and chitosan in vivo evaluation of chitosan as an adjuvant in subcutaneous vaccine formulations chitosan-based delivery systems for mucosal vaccines chemical modification of chitosan part : synthesis of novel chitosan derivatives by substitution of hydrophilic amine using n-carboxyethylchitosan ethyl ester as an intermediate challenges in evaluation of chitosan and trimethylated chitosan (tmc) as mucosal permeation enhancers: from synthesis to in vitro application utilization of carboxymethyl chitosan in cosmetics mono-ncarboxymethyl chitosan (mcc) and n-trimethyl chitosan (tmc) nanoparticles for non-invasive vaccine delivery thermal-sensitive hydrogel as adjuvant-free vaccine delivery system for h n intranasal immunization chitin and chitosan: functional biopolymers from marine crustaceans challenges in evaluation of chitosan and trimethylated chitosan (tmc) as mucosal permeation enhancers: from synthesis to in vitro application chitosan derivatives as novel potential heparin reversal agents adjuvants-a balance between toxicity and adjuvanticity an introduction to biodegradable materials for tissue engineering applications oral drug absorption enhancement by chitosan and its derivatives chitosan hydrogel as sirna vector for prolonged gene silencing biodegradation, biodistribution and toxicity of chitosan acidic mammalian chitinase is secreted via an adam /epidermal growth factor receptor-dependent pathway and stimulates chemokine production by pulmonary epithelial cells acidic mammalian chitinase in asthmatic th inflammation and il- pathway activation chitotriosidase: the yin and yang x-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family chitinases in vitro degradation of chitosan by bacterial enzymes from rat cecal and colonic contents influence of the degree of acetylation on the enzymatic degradation and in vitro biological properties of trimethylated chitosans interaction of bile-acids, phospholipids, cholesterol and triglyceride with dietary-fibers in the small-intestine of rats influence of molecular weight on oral absorption of water soluble chitosans biodegradation and distribution of water-soluble chitosan in mice chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: results of an in vitro and in vivo study effect of salt forms and molecular weight of chitosans on in vitro permeability enhancement in intestinal epithelial cells (caco- ) chitosans as absorption enhancers for poorly absorbable drugs: influence of molecular weight and degree of acetylation on drug transport across human intestinal epithelial (caco- ) cells transmucosal macromolecular drug delivery a comparative study of the potential of solid triglyceride nanostructures coated with chitosan or poly(ethylene glycol) as carriers for oral calcitonin delivery the safety of chitosan as a pharmaceutical excipient chitosan-based drug nanocarriers: where do we stand? do shrimpallergic individuals tolerate shrimp-derived glucosamine chitosan a promising safe and immuneenhancing adjuvant for intranasal vaccines evaluation of the effectiveness and safety of chitosan derivatives as adjuvants for intranasal vaccines challenges in evaluation of chitosan and trimethylated chitosan (tmc) as mucosal permeation enhancers: from synthesis to in vitro application novel vaccine delivery system induces robust humoral and cellular immune responses based on multiple mechanisms a polymer/oil based nanovaccine as a single-dose immunization approach the effects of chitosan oligosaccharide on the activation of murine spleen cd c+ dendritic cells via toll-like receptor toll-like receptors: key mediators of microbe detection cd t cell clonal expansion and development of effector function require prolonged exposure to antigen, costimulation, and signal cytokine from secretome analysis to immunology: chitosan induces major alterations in the activation of dendritic cells via a tlr -dependent mechanism the effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells contribution of il- to resistance to streptococcus pneumoniae infection role of interleukin- and myd -dependent signaling in rhinovirus infection airway epithelial myd restores control of pseudomonas aeruginosa murine infection via an il- -dependent pathway cutting edge inflammasome activation by alum and alum's adjuvant effect are mediated by nlrp interleukin- promotes coagulation, which is necessary for protective immunity in the lung against streptococcus pneumoniae infection the nlrp inflammasome: a sensor of immune danger signals uptake of particulate vaccine adjuvants by dendritic cells activates the nalp inflammasome chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis surface charge and cellular processing of covalently functionalized multiwall carbon nanotubes determine pulmonary toxicity tuning innate immune activation by surface texturing of polymer microparticles: the role of shape in inflammasome activation activation of the nlrp inflammasome is not a feature of all particulate vaccine adjuvants key roles of adjuvants in modern vaccines vaccine adjuvant formulations: a pharmaceutical perspective systemic activation of dendritic cells by toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity intracellular mechanisms of antigen cross presentation in dendritic cells distinct pathways of antigen uptake and intracellular routing in cd and cd t cell activation heat shock protein (hsp ) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells heat shock proteins as regulators of the immune response engineering synthetic vaccines using cues from natural immunity steady-state antigen scavenging, cross-presentation, and cd (+) t cell priming: a new role for lymphatic endothelial cells effect of chemical functionalities in poly(amido amine)s for non-viral gene transfection designing polymeric particles for antigen delivery improving chitosan-mediated gene transfer by the introduction of intracellular buffering moieties into the chitosan backbone endosomal escape pathways for delivery of biologicals epithelial cells as sensors for microbial infection immunity, inflammation, and allergy in the gut a thermosensitive hydrogel based on quaternized chitosan and poly(ethylene glycol) for nasal drug delivery system local and systemic activity of the polysaccharide chitosan at lymphoid tissues after oral administration chitosan-coated liposomes for topical vaginal therapy: assuring localized drug effect oral transmucosal drug delivery for pediatric use effect of chitosan on the permeability of monolayers of intestinal epithelial cells (caco- ) comparison of the effect of different chitosan salts and n-trimethyl chitosan chloride on the permeability of intestinal epithelial cells (caco- ) in vitro permeability across caco- cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth transport properties are not altered across caco- cells with heightened teer despite underlying physiological and ultrastructural changes oral delivery of peptide drugs using nanoparticles self-assembled by poly(gamma-glutamic acid) and a chitosan derivative functionalized by trimethylation mechanism and consequence of chitosan-mediated reversible epithelial tight junction opening elucidating the signaling mechanism of an epithelial tight-junction opening induced by chitosan low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice preparation and evaluation of chitosan nanoparticles containing diphtheria toxoid as new carriers for nasal vaccine delivery in mice n-trimethyl chitosan (tmc) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model the potential of mannosylated chitosan microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization diphtheria toxoid-containing microparticulate powder formulations for pulmonary vaccination: preparation, characterization and evaluation in guinea pigs dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein mechanistic study of the adjuvant effect of biodegradable nanoparticles in mucosal vaccination strong systemic and mucosal immune responses to surface-modified plga microspheres containing recombinant hepatitis b antigen administered intranasally immune response by nasal delivery of hepatitis b surface antigen and codelivery of a cpg odn in alginate coated chitosan nanoparticles development and characterization of chitosan coated poly-(varepsilon-caprolactone) nanoparticulate system for effective immunization against influenza in vitro and in vivo study of n-trimethyl chitosan nanoparticles for oral protein delivery covalently stabilized trimethyl chitosan-hyaluronic acid nanoparticles for nasal and intradermal vaccination co-delivery of viral proteins and a tlr agonist from polysaccharide nanocapsules: a needle-free vaccination strategy development and characterization of surface modified plga nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity evaluation of the immune response following a short oral vaccination schedule with hepatitis b antigen encapsulated into alginate-coated chitosan nanoparticles a single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge fully embeddable chitosan microneedles as a sustained release depot for intradermal vaccination first in vivo evaluation of particulate nasal dry powder vaccine formulations containing ovalbumin in mice novel thermal-sensitive hydrogel enhances both humoral and cell-mediated immune responses by intranasal vaccine delivery chitosan microparticles for oral vaccination: preparation, characterization and preliminary in vivo uptake studies in murine peyer's patches transport of chitosan microparticles for mucosal vaccine delivery in a human intestinal m-cell model chitosan microparticles for mucosal vaccination against diphtheria: oral and nasal efficacy studies in mice chitosan chemistry and pharmaceutical perspectives evaluation of eudragit-coated chitosan microparticles as an oral immune delivery system ph-sensitive and mucoadhesive thiolated eudragit-coated chitosan microspheres formulation, characterization and optimization of hepatitis b surface antigen (hbsag)-loaded chitosan microspheres for oral delivery bioadhesive-based dosage forms: the next generation chitosan as a novel nasal delivery system for vaccines induction of protective immunity against h n influenza a(h n )pdm with spray-dried and electron-beam sterilised vaccines in non-human primates biodistribution and lymph node retention of polysaccharide-based immunostimulating nanocapsules chitosan gras notice protective levels of diphtheria-neutralizing antibody induced in healthy volunteers by unilateral priming-boosting intranasal immunization associated with restricted ipsilateral mucosal secretory immunoglobulin a human susceptibility and resistance to norwalk virus infection characterisation of a gii- norovirus variant-specific surface-exposed site involved in antibody binding immunogenicity and specificity of norovirus consensus gii. virus-like particles in monovalent and bivalent vaccine formulations adjuvanted intranasal norwalk virus-like particle vaccine elicits antibodies and antibody-secreting cells that express homing receptors for mucosal and peripheral lymphoid tissues norovirus vaccine against experimental human norwalk virus illness intranasal vaccination with an adjuvanted norwalk virus-like particle vaccine elicits antigen-specific b memory responses in human adult volunteers we thank the financial support provided by national key key: cord- -m vazpq authors: barello, serena; nania, tiziana; dellafiore, federica; graffigna, guendalina; caruso, rosario title: ‘vaccine hesitancy’ among university students in italy during the covid- pandemic date: - - journal: eur j epidemiol doi: . /s - - -z sha: doc_id: cord_uid: m vazpq the debate around vaccines has been in the spotlight over the last few years in europe, both within the scientific community and the general public debate. in this regard, the case of the italian vaccination debate is particularly worrying given that italy has been one of the european countries with the highest number of measles cases in the recent past. according to this scenario, we conducted a cross-sectional study on a convenience sample of italian university students aimed at: ( ) exploring their attitudes towards a future vaccine to prevent covid- and; ( ) evaluating the impact of the university curricula (healthcare vs. non-healthcare curricula) on the intention to vaccinate. descriptive analysis on the students that answered to the question on the intention to vaccinate showed that ( . %) students reported that they would choose to have a vaccination for the covid- coronavirus; on the other side, ( . %) students reported that they would not or be not sure to vaccine (low intention to vaccinate). this means that in our sample more than one student out of shows low intention to vaccinate (vaccine hesitancy). furthermore, when running analysis comparing healthcare students versus non-healthcare students we found no significant differences in responses’ percentage distribution (p = . ). understanding the student’s perspective about the future covid- vaccine and supporting their health engagement and consciousness may be useful in planning adequate response and multidisciplinary educational strategies—including the psychological perspective on vaccine hesitancy underlying factors - in the post-pandemic period. we read with great interest the essay by harrison and wu [ ] on vaccine confidence in the time of covid- . they wondered if the impact of the covid- global health emergency will solve the problem of vaccine refusal that has worried the global public health community for the last several decades. indeed, the concept of 'vaccine hesitancy' has been considered by the world health organization (who) as "one of the top-ten threats to global health" [ ] . the debate around vaccines has been in the spotlight over the last few years in europe, both within the scientific community and the general public discourse. in this regard, the case of the italian vaccination debate is particularly worrying given that italy has been one of the european countries with the highest number of measles cases in the recent past [ ] . this issue led to the introduction of laws prescribing mandatory vaccinations for children to attend school in december , which gave rise to an active debate [ ] . in line with harrison and wu [ ] , we wonder if the covid- experience will fix the problem of vaccine hesitancy in italy. according to this scenario, we conducted a cross-sectional study on a convenience sample of italian university students aimed at: ( ) exploring their attitudes towards a future vaccine to prevent covid- and; ( ) evaluating the impact of the university curricula (healthcare vs. nonhealthcare curricula) on the intention to vaccinate. university students could be considered as an insightful population to investigate their attitudes to accept new vaccination because they are open-minded, educated, and supposed to respond quickly to public health issues. our sample was not designed to be representative of the italian university students, but to provide an initial and insightful description of the investigated phenomena. of the contacted, students responded (response rate = %) to the online questionnaire sent through the participating universities' mailing lists: students ( . %) attended healthcare curricula (i.e., medicine, nursing) and students ( . %) attended non-healthcare curricula (i.e. economics, law, engineering, physics, math, human sciences). females were the majority ( . %); mean age was . years (standard deviation = . ). the majority of the respondents who indicated their provenience (n = ) lived in one of the most hit italian regions -lombardy -(n = ; . %), followed by respondents from lazio (n = ; . %). descriptive analysis on the students that answered to the question on the intention to vaccinate showed that ( . %) students reported that they would choose to have a vaccination for the covid- coronavirus; on the other side, ( . %) students reported that they would not or be not sure to vaccine (low intention to vaccinate). this means that in our sample more than one student out of shows low intention to vaccinate (vaccine hesitancy), a percentage that reflects the international literature on general influenza vaccine hesitancy [ ] [ ] [ ] [ ] . all the comparison analysis showed that responders who chose not to disclose their intention to vaccinate did not significantly differ from the others on demographic and social characteristics. furthermore, when running analysis comparing healthcare students versus non-healthcare students we found no significant differences in responses' percentage distribution (p = . ). these results requires particular attention. first, we expected that the intention to vaccinate would have been higher in students attending healthcare curricula due to higher literacy on health-related issues. second, these findings shed light on the potential risks liked to healthcare students vaccination hesitancy because vaccination of healthcare workers is a key measure in the prevention of healthcare-associated infections due to their close contact with high-risk patients [ ] . although preliminary, this finding suggests that vaccination attitude is not only influenced by the students' level of health knowledge, but probably by other motivational and psychological factors, including the sense of individual responsibility for population health and the common sense about the value of civic life and social solidarity, as demonstrated by other studies on the covid- pandemic and previous emergencies [ , ] . not only the general public, but also those studying to become health professionals, seem to struggle to keep up with a growing body of evidence and increasingly complex information and contrasting feelings about vaccines. therefore, public health information campaign should be also supported by other actions aimed at raising students' consciousness regarding the crucial role of individuals' engagement in safeguarding their own and others' health through vaccination. students are a good target for educational campaigns as they are still in their training period and are open to changing their habits. understanding the student's perspective about the future covid- vaccine and supporting their health engagement and consciousness may be useful in planning adequate response and management strategies in the post-pandemic period [ ] . we think that the strategy to achieve efficient synergy between healthcare professionals and the general public is to better improve medical education of students during university and beyond introducing dedicated multidisciplinary curriculum about vaccinations and preventive behaviours for all university students and in particular to those attending healthcare curricula, an issue that requires increased attention to mitigate and control the covid- pandemic. unfortunately, population health and clinical prevention (i.e. vaccine behaviours) have long been relegated as a secondary topics in university curricula of most universities worldwide [ ] [ ] [ ] . moreover, evidence demonstrated how multidisciplinary educational interventions are the preferred strategy to improve students' adherence, attitude and knowledge about vaccinations [ ] [ ] [ ] . that's because a multidisciplinary approach to teaching could contribute to look beyond the traditional approach to health information. teaching complex health-related issues -through appropriate educational program across multiple disciplines, including social, psychological and behavioural sciences to complement the clinical and epidemiological ones -is key to better educate students about the multi-layer drivers and barriers of trust in vaccination, and defining how best to engage patients in preventive behaviours and to understand their behavioural and social reactions to immunization [ ] . we think that our findings deserve attention in light of the particularly intense experience that the italian health services had with covid- . we expected that the population living in this country would have higher levels of intention to vaccinate. the fact that this expectation does not find confirmation in our data makes our results warranted of public health attention and merits follow up studies designed for better understanding of the nature of the hesitancy among university students in italy. public health science should carefully consider the need to implement target and cultural-specific actions for university students, as influencing attitudes at an earlier stage of life can be more advantageous. people involvement around vaccines needs to be broad and multifactorial, with engagement at multiple levels [ ] . these include policymaking, sensitizing programs' design, the development of risk communication strategies and psychosocial research aimed to understand people perspectives on vaccination behaviours in order to dialogue with them in an appropriate way. in this arena, educational initiatives-even not standalone interventions-are certainly an important step to ignite appropriate consciousness of individuals and communities vaccine confidence in the time of covid what should we do about vaccine hesitancy? italy's response to vaccine hesitancy: an innovative and cost effective national immunization plan based on scientific evidence quantifying population preferences around vaccination against severe but rare diseases: a conjoint analysis among french university students an observational study of university students of healthcare area: knowledge, attitudes and behaviour towards vaccinations understanding influenza vaccine perspectives and hesitancy in university students to promote increased vaccine uptake uptake of influenza vaccination, awareness and its associated barriers among medical students of a university hospital in central saudi arabia influenza vaccination of healthcare workers and vaccine allocation for healthcare workers during vaccine shortages factors associated with pandemic influenza a (h n ) vaccination acceptance among university students from india during the post-pandemic phase using social and behavioural science to support covid- pandemic response teaching vaccine safety communication to medical students and health professionals clinical prevention and population health: curriculum framework for health professions prevention for the st century: setting the context through undergraduate medical education improvement in vaccination knowledge among health students following an integrated extra curricular intervention, an explorative study in the university of palermo improvement in attitudes toward influenza vaccination in medical students following an integrated curricular intervention mandatory influenza vaccination of health care workers: translating policy to practice missed opportunities? locating health promotion within multidisciplinary public health. public health a multidisciplinary research agenda for understanding vaccine-related decisions conflict of interest the authors declare no conflict of interest. key: cord- -k dqe lk authors: hoelzer, karin; bielke, lisa; blake, damer p.; cox, eric; cutting, simon m.; devriendt, bert; erlacher-vindel, elisabeth; goossens, evy; karaca, kemal; lemiere, stephane; metzner, martin; raicek, margot; collell suriñach, miquel; wong, nora m.; gay, cyril; van immerseel, filip title: vaccines as alternatives to antibiotics for food producing animals. part : new approaches and potential solutions date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: k dqe lk vaccines and other alternative products are central to the future success of animal agriculture because they can help minimize the need for antibiotics by preventing and controlling infectious diseases in animal populations. to assess scientific advancements related to alternatives to antibiotics and provide actionable strategies to support their development, the united states department of agriculture, with support from the world organisation for animal health, organized the second international symposium on alternatives to antibiotics. it focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the development and licensure of alternatives to antibiotics. this article, the second part in a two-part series, highlights new approaches and potential solutions for the development of vaccines as alternatives to antibiotics in food producing animals; opportunities, challenges and needs for the development of such vaccines are discussed in the first part of this series. as discussed in part of this manuscript, many current vaccines fall short of ideal vaccines in one or more respects. promising breakthroughs to overcome these limitations include new biotechnology techniques, new oral vaccine approaches, novel adjuvants, new delivery strategies based on bacterial spores, and live recombinant vectors; they also include new vaccination strategies in-ovo, and strategies that simultaneously protect against multiple pathogens. however, translating this research into commercial vaccines that effectively reduce the need for antibiotics will require close collaboration among stakeholders, for instance through public–private partnerships. targeted research and development investments and concerted efforts by all affected are needed to realize the potential of vaccines to improve animal health, safeguard agricultural productivity, and reduce antibiotic consumption and resulting resistance risks. alternatives to antibiotics can help minimize the need for antibiotics by helping to prevent and control infectious diseases in animal populations. as such, safe and effective alternatives are crucially important to the future success of animal health and production. to assess scientific advancements in the research and development of alternatives to antibiotics, highlight promising research results and novel technologies, assess challenges associated with their commercialization and use, and provide actionable strategies to support their development, the united states department of agriculture (usda), with support from the world organisation for animal health (oie), organized the second international symposium on alternatives to antibiotics. the symposium focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the licensure and development of alternatives to antibiotics. this two-part manuscript synthesizes and expands on the scientific presentations and expert panel discussions from the symposium regarding the use of vaccines as alternatives to antibiotics that can reduce the need for antibiotic use in animals. part synthesizes and expands on the expert panel discussions regarding the opportunities, challenges and needs related to vaccines that may reduce the requirement for use of antibiotics in animals, while part two focuses on highlighting new approaches and potential solutions. a general discussion of the importance of antibiotic resistance and the opportunities, challenges and needs related to vaccines as alternatives that may reduce the need for use of antibiotics in animals is provided in part of this review, including a discussion of the properties of ideal vaccines, how current vaccines compare to these ideal vaccines, and how investment decisions around research and development of vaccines are made. this second part of the manuscript will highlight specific research advancements in the area of veterinary vaccines. as mentioned in part one of this manuscript, most pathogens invade the host at the mucosal surfaces, such as the gastro-intestinal (gi) tract. the gi tract constitutes the largest surface area of the body and is exposed daily to vast numbers of foreign antigens derived from feed, the microbiota and pathogens [ ] . within the intestine a complex cellular network has evolved to prevent unwanted immune responses to innocuous antigens, for instance feed or microbiota, while allowing swift protective responses against agents that cause infectious disease. key to keeping enteric pathogens at bay is the presence of protective pathogen-specific secretory iga (siga) at the site of entry, which prevents the adhesion of micro-organisms to the intestinal surfaces and neutralizes their enterotoxins. triggering robust and protective intestinal siga responses usually requires the local administration of vaccines [ ] . although live attenuated oral vaccines have had tremendous success, resulting for instance in the near global eradication of poliovirus [ ] , concerns on the dissemination of vaccine strains into the environment and rare cases of reversion to virulence, leading to vaccine-induced disease, have driven oral vaccine development to nonliving or vectored vaccines [ ] . however, oral vaccination is challenging due to several hurdles imposed by the cellular and molecular architecture of the gut: (i) the harsh environment of the stomach and small intestine, including the low ph, digestive enzymes, and bile salts, required to digest feed also easily destroys vaccines, (ii) a poor uptake of vaccine antigens by the intestinal epithelial barrier and (iii) the tolerogenic mechanisms that pervade the intestinal tissues, leading to peripheral and oral immune tolerance upon oral administration of antigens via the induction of foxp + regulatory t cells. this often results in a low immunogenicity of oral vaccines and requires innovative strategies to deliver the vaccine antigens to the intestinal immune system as well as the inclusion of adjuvants that promote innate and adaptive immunity [ ] . the mucosal immune system in the gut can be divided in inductive sites, where sampled antigens stimulate naive t and b cells, and effector sites, where effector cells perform their functions, e.g. assisting in the production of siga. in the small intestine, the inductive sites comprise the gut-associated lymphoid tissues (galt) and the mesenteric lymph nodes, while the effector sites constitute the lamina propria and the surface epithelium [ ] . the galt itself is composed of peyer's patches (pp), appendix and isolated lymphoid follicles. the presence of other galt-like structures, such as lymphocyte-filled villi (rat, human) and cryptopatches (mouse) is dependent on the species. interestingly, while in birds and most mammals pp or their equivalent are scattered throughout the small intestine, in pigs, ruminants and dogs the pp in the distal small intestine (ileum) are continuous. fish and reptiles on the other hand lack pp and the intestinal immune system in these species is composed of epithelial leukocytes . . next generation anticoccidial vaccines autogenous vaccines to reduce the need for antibiotic use conclusions references and rare, small non-organized lymphoid aggregates. it remains largely unknown how these species-specific differences might affect the efficacy of oral vaccines. from their entry point, which is typically the oral cavity, to their delivery site, most commonly the small intestine, the integrity of delivery systems and the stability of vaccine components are at risk. lysozyme in saliva, the low gastric ph together with pepsin and intestinal proteases can degrade oral vaccines. enteric coating of vaccine components with ph-responsive polymers with a dissolution threshold of ph might protect against gastric degradation and results in the release of their contents in the small intestine [ ] . in this context, ruminants pose an additional problem to vaccine stability as their polygastric gastro-intestinal tract effectively degrades substances including vaccines. site-specific delivery of oral vaccines to the small intestine is favorable as the mucus layer covering the small intestinal epithelium consists of only one layer, which is loosely adherent, less thick and patchy as compared to the colonic mucus layers and might promote their access to the intestinal epithelium. in addition, the small intestine is less densely populated by the microbiota, which might further disrupt the integrity of the delivery systems and the stability of vaccine components. underneath the mucus layer, a single layer of intestinal epithelial cells prevents uncontrolled access of the luminal content to the underlying intestinal tissues, further restricting uptake of oral vaccine antigens. crossing of the epithelial barrier by vaccines could be enhanced by exploiting antigen sampling routes in the small intestine or by adopting strategies used by enteric pathogens to colonize or invade the host [ ] . the best-known sampling route in the gut is associated with microfold (m) cells. these specialized intestinal epithelial cells reside within the follicle-associated epithelium covering the peyer's patches and take up macromolecules, particulate matter and microorganisms [ ] . many enteric pathogens hijack m cells to invade the host by binding to apical receptors. for instance, the invasin protein of yersinia species interacts with β integrin on m cells, leading to infection [ ] . likewise, gp marks m cells in many species and binds to fimh, a subunit of type i pili on escherichia coli and salmonella enterica. this interaction results in uptake of fimh + bacteria and initiates mucosal immunity [ ] . although many groups have focused on improving antigen uptake by targeting oral vaccines to m cell-specific receptors, these cells represent only a small, species-specific percentage of the total intestinal epithelial cell population. although m cell numbers increase from the cranial to caudal small intestine and m cell targeting strategies work quite well in rodent models, they mostly fail in larger animals due to the long passage time needed to reach the distal small intestine, where the gut-associated immune system is most pronounced. besides m cells, sampling of luminal antigens also occurs by intestinal mononuclear phagocytes via transepithelial dendrites. this sampling mainly occurs by cd c + cx cr + macrophages, which transfer the antigens to cd + dendritic cells (dcs). these dcs then drive the differentiation of regulatory t cells (tregs), which subsequently induce tolerance to these proteins [ ] . in the steady state, goblet cells can also transport small soluble proteins (< kda) across the epithelium to tolerogenic dcs via so-called goblet cell-associated antigen passages [ ] . absorptive intestinal epithelial cells or enterocytes, constituting > % of the small intestinal epithelium, may also sample the luminal content through receptor-mediated transcytosis. for instance, the neonatal fc receptor (fcrn), an mhc class i-like fcγ receptor, is expressed on the apical surface of enterocytes and transcytoses igg, immune complexes or fc-coated nanoparticles from the lumen to the basolateral surface of the epithelium [ ] . similar to m cells, it might be worthwhile to target apical receptors exploited by enteropathogens on small intestinal enterocytes to promote uptake of antigens by the epithelial barrier. a potential candidate would be aminopeptidase n (anpep), a zinc-dependent peptidase present in the brush border of small intestinal enterocytes, which serves as an entry receptor for several coronaviruses and also binds f fimbriae, a colonisation factor produced by porcine-specific enterotoxigenic e. coli. anpep also transports f fimbriae as well as microparticles functionalised with anpep-specific monoclonal antibodies across the intestinal epithelial barrier, resulting in robust intestinal siga responses, at least in piglets [ , ] . although the selective targeting of vaccine antigens to apical receptors might promote their uptake by the epithelium via transcytosis, this process is in itself insufficient to trigger protective intestinal immunity upon oral vaccination and explains the need to include adjuvants. these adjuvants should act on antigen presenting cells as well as intestinal epithelial cells to promote the induction of protective siga and cell-mediated immune responses. indeed, enterocytes not only provide a physical barrier separating the intestinal lumen from the host tissues, but also relay information on the luminal content to the underlying immune cells through the secretion of inflammatory or tolerogenic mediators. for instance, during the steady state, enterocytes produce thymic stromal lymphopoëtin (tslp) and transforming growth factor (tgfβ), which imprint a tolerogenic phenotype on intestinal dendritic cells [ ] . in contrast, upon infection enterocytes secrete il- and il- [ ] . this probably facilitates a switch from a tolerogenic to an immune-inductive environment, allowing activation of intestinal antigen presenting cells. as yet the most effective adjuvants for oral application are the enterotoxins from vibrio cholera (ct) and enterotoxigenic e. coli (etec) (lt). due to inherent toxicity, dmlt was developed, a nontoxic lt mutant retaining its adjuvanticity. this dmlt triggered intestinal memory responses upon oral vaccination with a nonliving etec vaccine and seems a promising candidate to be included as adjuvant in oral vaccines [ , ] . similarly promising strategies have been reported for eimeria [ ] . recent studies have shown that eimeria-induced il- production is critical in the initiation of early innate immune response in coccidiosis and blocking of il- production by exogenous il- -neutralizing antibody reduced both the intracellular development of eimeria and the severity of intestinal lesion [ ] [ ] [ ] . in summarizing this part, future design of oral vaccines should be tailored to the needs of the target species, focus on the selective delivery of vaccines to epithelial receptors to promote their transport across the epithelial barrier, induce protective immune response in the target tissues, and should include a mucosal adjuvant able to trigger memory siga responses. endospores, or spores, are produced by many bacteria as a response to nutrient deprivation. the spore is a dormant entity about μm in size that can germinate, allowing a nascent cell to emerge and enter vegetative cell growth [ ] . the spore carries remarkable resistance properties, being typically resistant to high temperatures (typically - °c), desiccation, irradiation, and exposure to noxious chemicals [ ] . the two principal sporeforming bacterial genera are bacillus and clostridia with the latter being exclusively anaerobic. members of the bacillus genus are being used as probiotics, that is, microorganisms that are added to the diet to improve the balance of microbial communities in the gi-tract and are therefore beneficial to human or animal health [ , ] . typical species include bacillus clausii, bacillus coagulans and bacillus subtilis. for a long time, it has been assumed that bacillus spores are soil organisms yet the evidence supporting this is actually rather sparse. instead, spores are found in the soil in abundance but live, vegetative cells, are rarely if ever found other than in association with plants or in the animal gut. mounting evidence shows that spores, although found in the soil, are mostly dormant and are shed in the feces of animals, which are their natural hosts [ ] . the consumption of spores associated with soil-contaminated plant matter enables spores to enter the gi-tract, transit the gastric barrier unscathed and then germinate and proliferate in the intestine before excretion as dormant spores [ ] . evidence suggests that spore forming bacteria comprise as much as % of the gut microbiota, indicating that the ability to form spores enables bacteria to survive in the environment as well as entering and transiting the gastric barrier of animals [ ] . the extraordinary resistance properties of bacillus spores coupled with their ease of genetic manipulation, and their successful use as probiotics, makes them attractive candidates for the delivery of heterologous antigens for vaccination. spores have been used as vaccine vehicles in a number of ways, differing principally in whether spores are genetically modified or not. in all cases b. subtilis has been utilized due to the excellent genetics available. using genetic modification, a chimeric gene consisting of a fusion between a b. subtilis anchor gene and an open reading frame encoding a putative protective antigen is first constructed. the next step is introduction of the chimera into the b. subtilis chromosome using a gene transfer technique, typically dna-mediated transformation, a process in b. subtilis that is straightforward. typically, the anchor is the ′-end of a gene encoding a spore coat protein such that the chimera is displayed on the spore coat. surprisingly, heterologous antigens displayed on b. subtilis spores are mostly stable and do not appear to suffer extensive degradation. using this approach a number of candidate antigens have been displayed and then evaluated in animal models. for example, spores displaying a tetanus antigen ttfc conferred protection to a lethal dose of tetanus toxin when administered orally [ , ] . mice dosed orally with spores expressing part of the alpha toxin of clostridium perfringens were protected to challenge with alpha toxin [ ] . a more recent example is that of clostridium difficile where a c-terminal fragment of the toxin a (tcda) could be stably expressed and when administered orally to hamsters conferred protection to c. difficile infection [ , ] . this particular vaccine has now entered clinical evaluation in humans [ ] . using a non-genetically modified organism (gmo) approach it has been shown that spores can adsorb antigens efficiently onto their surface and surprisingly this is both strong and stable, and reflects the unique biophysical properties of the spore [ ] . for the adsorption approach, it has been shown that the gastric barrier is particularly corrosive and adsorbed antigens are labile, but for intranasal delivery this method appears satisfactory. using this approach inactive (killed) spores can be used and success has included studies showing protection to influenza (h n ) [ ] and significant reduction in lung counts of animals challenged with mycobacterium tuberculosis [ ] . a unique feature of spores is their ability to enhance immune responses and this adjuvant effect has been characterized in depth [ - ]. however, the use of spores as mass-delivery vehicles for vaccines has several limitations. oral delivery clearly is the preferred approach but appears to work effectively only for the gmo approach. oral delivery also raises issues of tolerance and may prove to be a limiting factor. sublingual delivery has also been explored; this approach appears to provide levels of protection that are equivalent to oral delivery, but requires more doses [ , ] . nasal delivery is suitable but raises potential safety issues. for animal vaccines, spores are attractive since they are currently used as feed probiotics but also because they can survive the high temperatures used for feed production and may offer long-term utility. as mentioned already, spores have been manipulated for protection against c. perfringens but there now exists the opportunity to develop spores for protective vaccination to necrotic enteritis, an important poultry disease caused by c. perfringens that has been identified as a high vaccine research priority by the oie ad hoc group (see additional file in http://doi.org/ . /s - - - ). one application that is particularly promising is the use of spore vaccines in aquaculture. with intensive fish farming, bacillus spores are being used as probiotic feed supplements. for shrimp farming, viral diseases have devastated the industry and one of the most important shrimp pathogens is white spot syndrome virus (wssv) that causes seasonal outbreaks of disease [ ] . a number of groups have developed b. subtilis spores that display the vp capsid protein of wssv and when administered in feed appears to protect against white spot disease [ - ]. the mechanism for protection is intriguing; even though shrimp are not thought to produce antibodies, it is clear that presentation of the viral antigens does produce some level of specific immunity. despite the progress being made with spore vaccines one key issue remains: the containment of gmos. because spores are dormant with the potential to survive indefinitely in the environment, the use of recombinant spores in spore vaccines is likely to raise environmental concerns and successful regulatory approvals may be slow or impossible to secure. for human use, it is likely that a case can be made that the recombinant spore vaccines addresses an unmet clinical need, but for animal use devising a method for biological containment will be crucial. technological advancements now make it possible to genetically engineer bacteria and other microorganisms that deliver heterologous antigens in a way that can stimulate mucosal as well as humoral and cellular systemic in veterinary medicine, oral vectored vaccines have been instrumental in the eradication or control of rabies in wildlife reservoirs [ , ] . oral vectored vaccines have also been developed for several other veterinary applications, including some economically important diseases of food-producing animals that are associated with considerable antibiotic use such as porcine circovirus type- (pcv- ); in some cases, the vaccine vector is a chimera containing parts of multiple microorganisms-for instance, an attenuated live vaccine may be used as the vector-and the resulting vaccine simultaneously confers protection against multiple diseases, for instance marek's disease and infectious bursal disease or newcastle disease and avian influenza [ , ] . the development of some vaccine vector systems has been very successful and numerous veterinary vaccines have been developed based on them; the canarypox virus vector system alvac, for instance, has been used for the development of a range of veterinary vaccines including against rabies, influenza, and west nile virus [ ] . similarly, adenovirus vectors have also been widely used in veterinary medicine, both in companion and food-producing animals [ ] . vaccine platforms such as these are particularly valuable as they can allow for the rapid development of vaccine candidates in response to emerging vaccine needs, but the possibility of anti-vector immunity can restrict their usefulness [ ] . research and development of additional vaccine vector platforms is therefore needed. salmonella strains that express foreign antigens, either chromosomally or plasmid-based, have yielded promising results in several species including mice, humans, pigs and chicken [ ] [ ] [ ] [ ] [ ] [ ] . diseases for which these salmonella vectored vaccines were investigated include influenza, brucella abortus, post-weaning diarrhea and heterologous strains of salmonella [ ] [ ] [ ] [ ] . the use of pasteurellaceae as vectors for modified live vaccines against shipping fever in calves is currently under investigation, with promising preliminary findings [ ] . use of this vector system for other diseases including pinkeye has been suggested [ ] . in-ovo vaccination is a mass-vaccination strategy that is mainly used in broiler chickens albeit occasionally also in broiler-breeder and layer chickens [ ] . eggs are injected in the hatchery, typically during the third week of embryonic development around day or . to vaccinate, a small hole is made in the shell at the blunt end of the egg and the vaccine is injected below the chorion-allantoic membrane into the amniotic cavity or directly into the embryo. commercial in-ovo vaccination systems that automatically inject the eggs have been available since the early s. more than % of broiler chickens in the us are vaccinated in ovo, and in brazil that fraction equals % [ ] . the most common use of in-ovo vaccination is for marek's disease, potentially combined with vaccines against other diseases such as gumboro or newcastle disease. the ability to deliver a clearly defined vaccine dosage to every single chick and to invoke early protection in the chicks is among the main benefits of this technology, but it is labor-intensive, causes stress for the chicks, and high sanitary standards need to be followed during vaccine preparation and injection to manage infection risks [ , ] . in addition, the location of the vaccine injection is critical for efficacy. it has been shown, for instance, that if marek's disease vaccine is accidentally deposited into the air cell or allantoic fluid, adequate protection is not achieved [ ] . the stage of embryonic development can have profound effects on vaccine safety and efficacy [ ] . one study, reported that vaccination of - dayold embryos with herpes virus of turkeys (hvt) led to pronounced lesions and embryonic deaths, while vaccination on days did not cause detectable lesions [ ] . embryonic age at vaccination has also been shown to be correlated with antibody titers [ ] . maternal antibody titers actually increase after the typical age for in-ovo vaccinations and peak just after hatch [ ] . this can interfere with proper vaccine responses. however, evidence suggests that some vaccine strains are more affected by maternal antibodies than others [ ] . deliberate vaccine development may therefore limit the often disruptive effects that can be caused by maternal antibodies [ ] . other factors that need to be considered in the development of a successful in-ovo vaccination program include the characteristics of the vaccine or vaccines to be used, the type of incubator in which the eggs are housed in the hatchery, and the breed and age of the parent flock [ ] . in-ovo vaccination strategies are promising means of reducing antibiotic use in poultry production and have been the subject of intense research. importantly, they can provide robust and early protection against immune suppressive diseases such as infectious bursal disease [ , ] and vaccines against multiple diseases have been successfully combined. for instance, studies have shown that in-ovo vaccination strategies can simultaneously confer protective immunity against marek's disease, infectious bursal disease, newcastle disease, fowl poxvirus, coccidiosis, and necrotic enteritis [ , ] . other combination vaccines under investigation include vectored vaccines that simultaneously provide protection against newcastle disease and infectious bursal disease [ ] . in-ovo vaccination strategies have also been explored for other poultry diseases with promising results. this included an avian influenza vaccine based on a non-replicating human adenovirus vector [ ] , a recombinant viral vector vaccine against infectious laryngotracheitis [ ] , recombinant protein eimeria vaccines [ , , ] and a fowl adenovirus vectored vaccine against inclusion body hepatitis [ ] , among many others. a mycoplasma gallisepticum vaccine for in-ovo vaccination of layer chickens has also recently been evaluated, even though high chick losses at hatch were reported for the medium and high doses of the vaccine that were investigated [ ] . therefore, in-ovo vaccination strategies are capable of controlling several economically important poultry diseases. many of these diseases are viral, but can predispose animals to secondary bacterial infections. therefore, in many cases, in-ovo vaccines are promising alternative approaches to the use of antibiotics. clostridium perfringens is widespread in the environment and in the gastrointestinal tract of most mammals and birds. however, this bacterium is also one of the most common pathogens of food-producing animals, causing disease only under circumstances that create an environment which favors growth and toxin production, such as stress, injury, or dietary changes [ ] . the bacterium itself is not invasive, but causes disease through the production of a wide array of toxins and enzymes. however, no single strain produces this entire toxin repertoire, resulting in considerable variation in the toxin profiles and disease syndromes produced by different toxinotypes of this bacterium [ ] . while some of these toxins act only locally, other toxins which are produced in the gut exert their action in other internal organs or can act both locally and systemically [ ] [ ] [ ] . to date, efficacious vaccines are only available for the diseases caused by systemic action of the toxins and vaccination against enteric diseases still remains a challenge. however, some of these enteric diseases caused by c. perfringens are of major economic importance and lead to considerable use of antibiotics. amongst them are necrotic enteritis in broilers and necro-haemorrhagic enteritis in calves. despite the fact that much research is being directed to the development of novel vaccines against these c. perfringens-induced enteric diseases, several key barriers still have to be overcome. in general, clostridial vaccines require multiple doses to achieve full immunity. unfortunately, parenteral booster immunizations are impossible in the broiler industry, where mass parenteral vaccination is only feasible at the hatchery, either in ovo or on day-old chicks. because single parenteral vaccination at day of hatch offers no protection, other delivery methods need to be developed [ ] . oral vaccines can more easily be administered to birds, without the need of individual handling of the chicks and are therefore recommended. however, some questions arise when developing an oral vaccine as compared to the parenteral administration route. in addition to the fact that maternal antibodies can block the immune response in young chicks, also the induction of oral tolerance has to be circumvented and an efficient way to present the antigens to the mucosal immune system has to be developed. oral tolerance is a common problem in mammals and fish when developing oral vaccines. this is in contrast to chickens, where oral tolerance is age-dependent, and only an issue in -to -day-old chicks. after that age, protein antigens have been shown to induce a robust immune response and oral vaccination schemes are thought to be feasible [ ] . one appealing strategy for the delivery of vaccine candidates to the mucosal immune system is the use of attenuated or avirulent bacteria as antigen vehicles [ ] . attenuated recombinant salmonella strains which express c. perfringens antigens have been tested in several studies as oral vaccine vectors, leading to some promising results. however, the amount of protection afforded by these vaccines is not as high as compared to multiple doses of parenteral vaccination, and seems to depend on the colonization level and persistence of the vaccine strain [ ] [ ] [ ] [ ] . this indicates that the use of live vectors to express antigens derived from c. perfringens strains in the gut of broilers is a promising approach, but the vaccine delivery strategy still needs to be optimized to achieve optimal antigen presentation to the mucosal immune system and provide improved protection. alternatives to attenuated salmonella strains can be bacillus subtilis spores or lactobacillus casei, which both have a gras status and have the potential to be used as vaccine carriers for clostridium antigens [ , ] . b. subtilis has the advantage that the heat-stable spores can easily be incorporated in the feed and l. casei has known probiotic effects that facilitate the development of mucosal immunity. however, these types of vectors still have to be tested for their capacity to induce a good immune response, in particular against heterologous antigens, in broilers and whether they are able to provide protection against necrotic enteritis. another issue to be addressed when developing a vaccine against c. perfringens-induced enteric diseases is the choice of the antigens to be included in the vaccine. c. perfringens-induced diseases are the result of the toxins and enzymes that are produced and vaccination of chicks with c. perfringens supernatants provides protection against experimental necrotic enteritis [ , ] . however, the protective capacity of the supernatants depends on the strain used for supernatant preparation, indicating that full protection might be determined by an effective combination of different bacterial immunogens [ ] . in order to elucidate the optimal mixture of antigens to protect against necrotic enteritis, challenge trials are being performed mostly using parenteral vaccination schemes. once the ideal combination of antigens is known, this will have to be adapted to oral delivery strategies. several c. perfringens antigens have been evaluated as potential vaccine candidates. the tested antigens include both c. perfringens toxins (e.g. alpha toxin and the netb toxin) and highly immunodominant proteins identified in postinfection serum from birds immune to necrotic enteritis [ ] . in general, immunization studies of broilers with a single antigen all resulted in some level of protection against experimental necrotic enteritis. remarkably, immunization with netb toxin, which is essential to cause disease in broilers, does not afford higher levels of protection than vaccination with other toxins or proteins. however, when birds were vaccinated either via the parenteral or the oral route, with a combination of both netb toxin and alpha toxin, higher levels of protection were obtained [ , ] . in order to obtain full protection against c. perfringens-induced enteric diseases, not only antibodies that inhibit toxin activity might be needed; a combination of antigens targeting also bacterial proliferation, colonization and/or nutrient acquisition could be more efficient than either one of the individual approaches. indeed, in a recent study disruption of the putative adhesin-encoding gene cnaa resulted in a reduced ability to colonize the chicken intestinal mucosa and to cause necrotic enteritis [ ] . this strengthens the idea that vaccine antigens that target bacterial colonization might be indispensable to obtain a working vaccine against c. perfringens-induced enteric diseases. additional vaccine targets might be enzymes that aid in breakdown of the host tissue and nutrient acquisition, such as, amongst others, mucinases, collagenases and hyaluronidases. in contrast to the extensive efforts to develop a vaccine against necrotic enteritis in chickens, considerably less research has been directed to vaccination against necro-haemorrhagic enteritis in calves. the recent demonstration of the essential role of alpha toxin in necro-haemorrhagic enteritis and the proposition of a pathogenesis model will allow for the more targeted development of a vaccine [ , ] . in calves as in chickens, protection against c. perfringens-induced necrosis can be obtained by antibodies against a mixture of toxins, at least in an experimental model for bovine necro-haemorrhagic enteritis [ ] . furthermore, antibodies against alpha toxin alone, which is essential to cause intestinal disease in calves, are not sufficient to provide the same level of protection as antibodies directed against a mixture of c. perfringens proteins, indicating that a mixture of different antigens will be needed to provide full protection [ ] . in order to fully protect calves against c. perfringens-induced enteric diseases, antigens that target bacterial colonization and proliferation might be of equal importance as antigens targeting the toxin activities. next, it has to be explored whether parenteral vaccination is sufficient to induce a protective immune response or if a combination of systemic and mucosal immunity is needed when not only the bacterial toxins but also bacterial colonization is targeted. as administration of multiple parenteral doses of a vaccine to calves is more feasible than for chicken, it may be assumed that the development of a vaccine against necro-haemorrhagic enteritis is more straightforward and that c. perfringens supernatants can be used as a vaccine preparation. however, native toxins cannot be used as vaccine antigens due to safety issues. inactivation of clostridial toxins is generally achieved by formaldehyde treatment, which risks residual formaldehyde in the vaccine preparation, incomplete inactivation of the toxins, and batch-to-batch variation. moreover, formaldehyde inactivation can induce changes in the tertiary protein structures of relevant antigens and influence the immunogenicity of the vaccines. indeed, vaccination of both chickens and calves with formaldehyde inactivated c. perfringens supernatants or toxins have resulted in a good antibody response, but these are unable to protect against intestinal disease [ , ] . to overcome the need of chemically inactivating the c. perfringens toxins, current research focusses on the use of recombinant toxoids to develop a vaccine against c. perfringens-induced diseases. while this may be a good strategy to obtain a safe and protective vaccine on a laboratory scale, the production process is more laborious and time-consuming than production of conventional toxoids, especially because of the required purification steps [ ] . therefore, recent studies have explored the use of efficient low-cost alternatives, such as non-purified recombinant clostridial toxins and even recombinant bacterins, with success [ ] [ ] [ ] . in summary of this section, considerable progress has recently been made in the development of efficacious vaccines against c. perfringens-induced enteric diseases. the main issue that hampers a breakthrough in this field is the identification of a defined combination of antigens that is able to provide full protection against disease. these antigens will most likely target both the bacterial toxins and the bacterial colonization and proliferation. for the broiler industry, once the ideal vaccine antigens have been identified, development of an oral vaccine is needed. coccidiosis, an enteric disease cause by protozoan parasites of the genus eimeria, remains a major economic and welfare concern for the poultry industry globally. seven species (eimeria acervulina, e. brunetti, e. maxima, e. mitis, e. necatrix, e. praecox and e. tenella) are known to infect chickens, and at least six others infect turkeys [ , ] . the costs associated with coccidial disease are difficult to calculate, but have been estimated to exceed billion us dollars for the chicken industry alone, worldwide [ ] . because coccidiosis is a predisposing factor for the occurrence of necrotic enteritis, the true economic burden is likely even higher. all eimeria species can cause disease but the severity and clinical symptoms vary among species, and there is little or no cross-protection across species or some strains [ , ]. modern poultry production systems require effective control of coccidian parasites, typically through the routine use of anticoccidial drugs in feed or water. in the european union, eleven different anticoccidial drugs are currently licensed and between and tonnes are sold for use in animals for markets such as the uk every year [ ] . anticoccidial drugs can be divided into two groups, synthetic or chemical anticoccidials and ionophores, which are products of fermentation [ ] . in some countries such as the us, ionophores are classified as antibiotics, albeit with low human medical importance. the ionophores currently dominate the anticoccidial drug market, largely because they provide incomplete protection, even against naïve field strains without any drug resistance. low levels of parasites survive and induce protective immunity against the prevailing local parasite strains, without causing clinical disease [ ] . anticoccidial drugs provide an efficient means of controlling coccidial parasites and are highly cost-effective. however, drug resistance is widespread and increasing consumer concerns related to drug use in livestock production and residues in the food chain encourage the use of alternatives such as vaccination. notably, because coccidiosis is a predisposing factor for necrotic enteritis and other secondary bacterial infections, efficient control of this parasite is important to minimize the use of medically important antibiotics, including those deemed critically important for human health, in poultry production. the first anticoccidial vaccine was marketed in [ ] . it is a live parasite vaccine which includes multiple wild-type (i.e., non-attenuated) eimeria species. exposure to limited levels of such non-attenuated parasites permits the induction of a natural immune response in the chicken, resulting in protection against subsequent coccidial challenge. however, because protective immune responses against eimeria are fully species specific, the inclusion of each individual target species is necessary if comprehensive protection is to be achieved, which results in relatively complex vaccine formulations. such vaccines commonly include between three and eight parasite species or strains. the approach has been highly successful, although the lack of attenuation has been associated with reduced flock performance following vaccination and occasional clinical disease (reviewed elsewhere [ ]). in response to this limitation, a second generation of live eimeria vaccines has been developed using attenuated parasite lines. for most of these vaccines, attenuation was achieved by selecting for so-called precocious strains, which typically exhibit reduced pathogenicity with fewer and/or smaller rounds of asexual replication. these attenuated strains retained their ability to immunize. the first live attenuated anticoccidial vaccine was launched in , and several similar vaccines have been developed since using the same approach [ ]. nonattenuated and attenuated anticoccidial vaccines have become popular in the breeder and layer sectors, but are less widely used in the much larger broiler sector due to their relatively high cost compared to anticoccidial drugs and their limited availability. because eimeria cannot replicate effectively in vitro, the production of these live vaccines can only be achieved in eimeria-free chickens and separate chickens have to be used for each species or strain to be included in a vaccine. despite these production concerns billions of anticoccidial vaccine doses are sold every year, but more would be required to fully meet the growing demand. efforts to improve on first and second generation live anticoccidial vaccines have included extensive attempts to identify antigens that are appropriate for use in subunit or recombinant vaccines. in addition, progress has been made on the preparation of novel adjuvants and some promising results have been obtained, although data on their use in poultry has so far remained fairly limited [ ] . as an example, one vaccine is formulated from a crude mix of affinity purified e. maxima gametocyte antigens [ ], although the levels of protection achieved have remained controversial and production of the vaccine still requires parasite amplification in chickens. numerous studies have suggested that defined antigens such as apical membrane antigen , immune mapped protein , lactate dehydrogenase and so are highly promising vaccine candidates (reviewed elsewhere [ ] ). studies of eimeria field populations have reported limited diversity in many of these antigens, indicating that recombinant vaccines for eimeria may succeed even though antigenic diversity has undermined equivalent vaccines for related parasites such as plasmodium [ , ] . however, at present no recombinant anticoccidial vaccine is close to reaching the market. one of the biggest remaining challenges is how to deliver the antigens in an affordable, effective, and, most importantly, scalable manner. a range of vectored expression/delivery systems have been suggested including fowlpox virus (fwpv), hvt, salmonella typhimurium, yeasts such as saccharomyces cerevisiae and the tobacco plant nicotiana tabacum, with several showing promise [ ] . most recently, it has been suggested that eimeria itself might function as an expression/delivery vector for vaccine antigens [ ] [ ] [ ] . the ability to express and deliver anticoccidial vaccine antigens from multiple parasite species in a single transgenic line could provide an opportunity to streamline anticoccidial vaccine production from as many as eight lines to just one or two. using an attenuated vector species such as e. acervulina can improve productive capacity enormously and reduce vaccine cost. the parasite vector may also provide some ability as an adjuvant and methods for on-farm delivery are well established [ ] . in summary of this section on new coccidiosis vaccines, as pressure to reduce antibiotic drug use in livestock production increases it is clear that the demand for coccidial vaccines is stronger than ever. in the us, approximately - % of broiler companies use programs that include vaccination to control coccidiosis [ ] . this trend is primarily driven by demands to produce "no antibiotics ever" poultry products. however, it has also been shown that some coccidial vaccines provide an opportunity to replace drug-resistant field parasites in a poultry house with susceptible vaccine strains. while current european attenuated vaccines are limited by their lower reproductive potential, live vaccines do retain considerable unexplored potential. a better understanding of the underlying immune mechanisms through which these nontraditional approaches operate is needed to allow further progress. ultimately, it is clear that novel vaccines must be cost-effective, compatible with high standards of animal welfare, scalable and easy to deliver. autogenous vaccines (av) are also known as emergency, herd-specific or custom made vaccines. although the legal basis and exact definition differs from country to country, avs are used worldwide (e.g. eu, usa, canada, brazil, china, indonesia, australia, egypt) and have a long history of use. the use of avs for the control of fowl cholera has been well-documented [ , ] . as a common definition, all avs are made from inactivated bacterial or viral strains which were isolated from the same flock in which the vaccine is to be used. the use of avs is only allowed if no licensed vaccine is available, or it is respectively ineffective or does not cover the current pathogen strains in the flock. the definition of a flock varies and may include integrated concepts of production chains in different places; to address the issue, the concept of an epidemiological link has recently been proposed by the co-ordination group for mutual recognition and decentralised procedures [ ] . licensed vaccines have advantages compared to avs, including obligatory good manufacturing practice (gmp) production. licensed vaccines are also produced in bigger batches with defined strains and a high level of quality which makes their efficacy and safety predictable. however, licensed vaccines are not available in all cases. to generate avs, selected bacterial or viral strains are usually combined with a proper adjuvant. several viral or bacterial species can be used in a combination vaccine and different serotypes can also be combined in a polyvalent vaccine. the combination of inactivated viruses and bacteria is also an option. bacterial avs are accepted in all countries of the economic european area, whereas viral avs are not allowed in european countries including france, denmark and spain [ ] . a critical role in the successful production and use of an av falls to the isolation of vaccine strains. therefore diagnostic samples must be carefully obtained, based on appropriate choices regarding which sick and untreated animals to select for sample collection, which necropsy material to select, and which cultivation conditions and strains to use after results from sero-, toxo-or virulencetyping. for that purpose several methods like pcr, maldi-tof ms, slide agglutination or dna sequencing are available. because of the fundamental importance of the strain choice for the production of an adequate av, close collaboration between diagnostic laboratory and vaccine production is critical. each production is custom-made and numerous adjuvants, viral and bacterial isolates, including serotypes, toxins and species, provide countless combinations. this underlines the importance of experience as the basis in the production of high quality avs. the veterinarian also has obligations regarding diagnosis, ordering and responsibility for the administration of the vaccine. a variety of bacterial components are often used in avs. these include for poultry: depending on the animal species and age at vaccination different adjuvants can be used. as a standard adjuvant with good safety and efficacy, aluminium hydroxide is often used for production. polymer and other gel-like adjuvants are also available for production in aqueous mixtures. oily adjuvants, especially for water-in-oil emulsions, require a more sophisticated mixing procedure because of the need of a stable emulsion. furthermore oily vaccines might pose safety concerns. however, these induce a promising long lasting immune response because of a depot effect. in the case of organic animal production use of plant oil might be an option in order to avoid unwanted hydrocarbons. the risk of adverse effects, which depend on the adjuvant-antigen combination, can be decreased by standardization of the protocols. more data regarding the efficacy and safety of avs in field studies should be collected because clinical safety and efficacy is not regulated. . further steps in quality control include the inactivation test, endotoxin content or stability tests. some producers offer gmp production, and gmp production is required in some countries such as finland or sweden [ ] . in most countries gmp is only recommended. this example shows the vast differences in national legislation regarding the definition and interpretation of avs. because of worldwide circulation of animals and their pathogens a harmonization of manufacture, control and use of immunological veterinary medicinal products like av is important, and the aim at the economic european area [ ] . in summary, avs are a valuable option in certain situations where commercial vaccines are either not available or expected to lack efficacy because of a mismatch between circulating and vaccine strains. the selection of adequate clinical isolates and vaccine formulations requires considerable expertise and the effective use of avs depends on adequate manufacturing and appropriate veterinary oversight. regulatory differences among countries create a highly fragmented legal landscape that would benefit from further harmonization. vaccines are proven strategies for the prevention or control of infectious diseases in animal populations. therefore, they are promising alternatives that can reduce the need to use antibiotics in food-producing animals and their direct mitigating impact on antibiotic consumption has been demonstrated in a number of studies, even though the relationship between antibiotic use and vaccination is not in all cases clear-cut. the ideal vaccine is safe, effective against a broad range of pathogens, and easily adapted to mass-application. at the same time, it is cheap to produce and use, easy to register across key jurisdictions, and generates durable protection, ideally after a single administration. existing vaccines still fall short of these ideals. in fact, many current vaccines have a number of shortcomings with regard to safety, efficacy and/or user-friendliness that limit their ability to replace antibiotic use. overcoming these challenges will take close collaboration and innovative new approaches. public-private partnerships represent one promising governing structure for assuring such close collaboration across public and private sectors. investments in basic and applied research are equally needed to overcome these challenges, and research needs will have to be prioritized to ensure scarce resources will be preferentially dedicated to areas of greatest potential impact. research to characterize and quantify the impact of vaccination on antibiotic use is equally needed. yet, some data demonstrating the ability of vaccines to reduce antibiotic consumption are already available. similarly, key research breakthroughs and a number of highly promising vaccination approaches are already in development. these include new oral vaccines based on bacterial spores, live vectors, or new delivery strategies for inactivated oral vaccines; they also include new vaccination strategies in-ovo, combination vaccines that protect against multiple pathogens, the use of recent biotechnological advances, and comprehensive approaches to manage diseases caused by ubiquitous pathogens. therefore, further reductions in the need for antibiotic use through the use of new vaccines are all-but-certain, and investments in research and development of new vaccines will be vital for the sustained success of animal agricultural production around the world. intestinal epithelial cells: regulators of barrier function and immune homeostasis induction of secretory immunity and memory at mucosal surfaces vaccines: the fourth century crossing the barrier: targeting epithelial receptors for enhanced oral vaccine delivery delivery strategies to enhance oral vaccination against enteric infections terminology: nomenclature of mucosa-associated lymphoid tissue development of an enteric-coated pellet formulation of f fimbriae for oral vaccination of suckling piglets against enterotoxigenic escherichia coli infections antigen sampling in the small intestine intestinal m cells m-cell surface β integrin expression and invasin-mediated targeting of yersinia pseudotuberculosis to mouse peyer's patch m cells uptake through glycoprotein of fimh+bacteria by m cells initiates mucosal immune response oral tolerance can be established via gap junction transfer of fed antigens from cx cr + macrophages to cd + dendritic cells goblet cells deliver luminal antigen to cd + dcs in the small intestine transepithelial transport of fc-targeted nanoparticles by the neonatal fc receptor for oral delivery etec vaccination in pigs ) β-glucan microparticles targeted to epithelial apn as oral antigen delivery system intestinal epithelial cells promote colitis-protective regulatory t-cell differentiation through dendritic cell conditioning enterotoxigenic escherichia coli (k ) induce proinflammatory responses in porcine intestinal epithelial cells evaluating the a-subunit of the heat-labile toxin (lt) as an immunogen and a protective antigen against enterotoxigenic escherichia coli (etec) induction of long term mucosal immunological memory in humans by an oral inactivated multivalent enterotoxigenic escherichia coli vaccine induction of protective immunity against eimeria tenella, eimeria maxima, and eimeria acervulina infections using dendritic cell-derived exosomes il- a regulates eimeria tenella schizont maturation and migration in avian coccidiosis chicken il- f: identification and comparative expression analysis in eimeria-infected chickens downregulation of chicken interleukin- receptor a during eimeria infection roles of bacillus endospores in the environment resistance of bacillus endospores to extreme terrestrial and extraterrestrial environments the use of bacterial spore formers as probiotics joint fao/who (food and agriculture organization/world health organization) working group report on drafting guidelines for the evaluation of probiotics in food. food and agriculture organization defining the natural habitat of bacillus spore-formers the intestinal life cycle of bacillus subtilis and close relatives culturing of 'unculturable' human microbiota reveals novel taxa and extensive sporulation bacterial spores as vaccine vehicles efficacy, heat stability and safety of intranasally administered bacillus subtilis spore or vegetative cell vaccines expressing tetanus toxin fragment c recombinant bacillus subtilis expressing the clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis immunization with bacillus spores expressing toxin a peptide repeats protects against infection with clostridium difficile strains producing toxins a and b mucosal antibodies to the c-terminus of toxin a prevent colonization of clostridium difficile mucosal delivery of antigens using adsorption to bacterial spores killed bacillus subtilis spores as a mucosal adjuvant for an h n vaccine mice protected by oral immunization with lactobacillus reuteri secreting fusion protein of escherichia coli enterotoxin subunit protein efficacy of a novel trivalent inactivated vaccine against the shedding of salmonella in a chicken challenge model immunization of chickens with live escherichia coli expressing eimeria acervulina merozoite recombinant antigen induces partial protection against coccidiosis oral immunization with lactococcus lactis-expressing espb induces protective immune responses against escherichia coli o : h in a murine model of colonization oral vaccination of fish: lessons from humans and veterinary species fenner's veterinary virology current status of veterinary vaccines use of adenoviral vectors as veterinary vaccines viral vectors as vaccine platforms: deployment in sight salmonella enterica serovar typhi live vector vaccines finally come of age pre-existing immunity against vaccine vectors-friend or foe? a vaccine candidate for post-weaning diarrhea in swine constructed with a live attenuated salmonella delivering escherichia coli k ab, k ac, feda, and fedf fimbrial antigens and its immune responses in a murine model oral immunization of mice and swine with an attenuated salmonella choleraesuis [delta cya- delta(crp-cdt) ] mutant containing a recombinant plasmid swine immunity to an attenuated salmonella typhimurium mutant containing a recombinant plasmid which codes for production of a -kda protein of brucella abortus recombinant orally effective vaccine platforms expressing putative conserved antigens for reduced antimicrobial usage in poultry pasteurellaceaeoral vaccine vector for economically important diseases of cattle in ovo vaccination for everyone benefits of in ovo vaccination available to smaller hatcheries american college of poultry veterinarians ( ) from basics to field applications: poultry vaccination and immunity field evaluation of the accuracy of vaccine deposition by two different commercially available in ovo injection systems comparison of in ovo and posthatch vaccination with particular reference to infectious bursal disease. a review evaluation of incubation yield, vaccine response, and performance of broilers submitted to in-ovo vaccination at different embryonic ages ovo vaccination technology efficacy of hvt-ibd vector vaccine compared to attenuated live vaccine using in-ovo vaccination against a korean very virulent ibdv in commercial broiler chickens advances in vaccine research against economically important viral diseases of food animals: infectious bursal disease virus in ovo vaccination of specificpathogen-free chickens with vaccines containing multiple agents in ovo vaccination using eimeria profilin and clostridium perfringens netb proteins in montanide ims adjuvant increases protective immunity against experimentally-induced necrotic enteritis novel in-ovo chimeric recombinant newcastle disease vaccine protects against both newcastle disease and infectious bursal disease protective avian influenza in ovo vaccination with non-replicating human adenovirus vector protection against infectious laryngotracheitis by in ovo vaccination with commercially available viral vector recombinant vaccines in ovo vaccination with the eimeria tenella etmic gene induces protective immunity against coccidiosis effects of coccidiosis vaccination administered by in ovo injection on the hatchability and hatching chick quality of broilers , , immune responses to in ovo vaccine formulations containing inactivated fowl adenovirus b with poly [di (sodium carboxylatoethylphenoxy)] phosphazene (pcep) and avian beta defensin as adjuvants in chickens layer chicken embryo survival to hatch when administered an in ovo vaccination of strain f mycoplasma gallisepticum and locations of bacteria prevalence in the newly hatched chick clostridial enteric diseases of domestic animals clostridium perfringens: toxinotype and genotype clostridial enteric infections in pigs recent progress in understanding the pathogenesis of clostridium perfringens type c infections clostridium perfringens toxins involved in mammalian veterinary diseases day-of-hatch vaccination is not protective against necrotic enteritis in broiler chickens oral tolerance in birds and mammals: digestive tract development determines the strategy vaccine discovery and translation of new vaccine technology oral immunization of broiler chickens against necrotic enteritis with an attenuated salmonella vaccine vector expressing clostridium perfringens antigens a live oral recombinant salmonella enterica serovar typhimurium vaccine expressing clostridium perfringens antigens confers protection against necrotic enteritis in broiler chickens recombinant attenuated salmonella enterica serovar typhimurium expressing the carboxy-terminal domain of alpha toxin from clostridium perfringens induces protective responses against necrotic enteritis in chickens assessment of salmonella enterica serovar typhimurium-based vaccines against necrotic enteritis in reducing colonization of chickens by salmonella serovars of different serogroups oral immunization of mice against clostridium perfringens epsilon toxin with a lactobacillus casei vector vaccine expressing epsilon toxoid variable protection after vaccination of broiler chickens against necrotic enteritis using supernatants of different clostridium perfringens strains progress and problems in vaccination against necrotic enteritis in broiler chickens protection against necrotic enteritis in broiler chickens by regulated delayed lysis salmonella vaccines variable protection against experimental broiler necrotic enteritis after immunisation with the c-terminal fragment of clostridium perfringens alpha-toxin and a non-toxic netb variant the adherent abilities of clostridium perfringens strains are critical for the pathogenesis of avian necrotic enteritis rethinking the role of alpha toxin in clostridium perfringens-associated enteric diseases: a review on bovine necro-haemorrhagic enteritis van immerseel f ( ) the c-terminal domain of clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis van immerseel f ( ) toxin-neutralizing antibodies protect against clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis recombinant alpha, beta, and epsilon toxins of clostridium perfringens: production strategies and applications as veterinary vaccines potency against enterotoxemia of a recombinant clostridium perfringens type d epsilon toxoid in ruminants potential protective immunogenicity of recombinant clostridium perfringens alpha-beta -beta fusion toxin in mice, sows and cows protective potential of recombinant non-purified botulinum neurotoxin serotypes c and d host specificity of turkey and chicken eimeria: controlled cross-transmission studies and a phylogenetic view we would like to thank the organizers, sponsors, and participants of the nd international symposium on alternatives to antibiotics on which this manuscript is based, in particular the united states department of agriculture (usda) and the world organisation for animal health (oie). the authors declare that they have no competing interests.authors' contributions kh, fvi, and cg planned the manuscript. kh led the drafting of the manuscript. lb, dpb, ec, smc, bd, eev, eg, kk, sl, mm, mr, mcs, nmw, cg, and fvi provided additional information and contributed to writing the manuscript including drafting selected sections and reviewing the manuscript. fvi and cg revised the manuscript. all authors read and approved the final manuscript. key: cord- -eg hajzl authors: jamrozik, euzebiusz; heriot, george s.; selgelid, michael j. title: coronavirus human infection challenge studies: assessing potential benefits and risks date: - - journal: j bioeth inq doi: . /s - - -x sha: doc_id: cord_uid: eg hajzl human infection challenge studies (hcs) have been proposed as a means to accelerate sars-cov vaccine development and thereby help to mitigate a prolonged global public health crisis. a key criterion for the ethical acceptability of sars-cov hcs is that potential benefits outweigh risks. although the assessment of risks and benefits is meant to be a standard part of research ethics review, systematic comparisons are particularly important in the context of sars-cov hcs in light of the significant potential benefits and harms at stake as well as the need to preserve public trust in research and vaccines. in this paper we explore several considerations that should inform systematic assessment of sars-cov- hcs. first, we detail key potential benefits of sars-cov- hcs including, but not limited to, those related to the acceleration of vaccine development. second, we identify where modelling is needed to inform risk-benefit (and thus ethical) assessments. modelling will be particularly useful in (i) comparing potential benefits and risks of hcs with those of vaccine field trials under different epidemiological conditions and (ii) estimating marginal risks to hcs participants in light of the background probabilities of infection in their local community. we highlight interactions between public health policy and research priorities, including situations in which research ethics assessments may need to strike a balance between competing considerations. human infection challenge studies (hcs)-i.e., experiments involving the intentional infection of research participants-have improved scientific understanding and public health responses to multiple infectious diseases. in particular, hcs are a key method of accelerating or improving vaccine development, and hcs results have contributed to the recent licensure or approval of vaccines for cholera and typhoid (jamrozik and selgelid a; tacket et al. ; jin et al. ) . in response to the covid- pandemic, challenge studies have been proposed as a means of accelerating the development of sars-cov- vaccines plotkin and caplan ; schaefer et al. ) . novel research designs, particularly where such studies might be controversial as in the case of sars-cov- hcs, require especially careful ethical evaluation including rigorous risk-benefit assessments as well as timely, thorough, public engagement ( who working group for guidance on human challenge studies in covid- ; bambery et al. ; hope and mcmillan ; nhmrc and arc ) . in this article, we argue that risk-benefit assessment of sars-cov- hcs should include thorough consideration of the potential benefits and risks of such studies in comparison to other kinds of studies-and that estimates of relevant benefits and risks should, insofar as possible, be informed by mathematical modelling. section of this article describes the potential public health benefits of hcs, section illustrates how these benefits should be systematically evaluated in light of comparisons to vaccine field trials in different epidemic settings, and section evaluates risks to participants as compared to background risk in different epidemic settings. recent academic and public debates surrounding sars-cov- hcs have largely focused on the potential for hcs to accelerate vaccine development through more rapid demonstration of candidate vaccines' efficacy (or lack thereof) plotkin and caplan ; schaefer et al. ; cohen ) . widespread use of a safe and effective vaccine for sars-cov- could have enormous global public health benefits, and the sooner mass vaccination commences the greater these benefits would be. one proposal for the use of challenge studies to accelerate sars-cov- vaccine development involves replacing a traditional phase iii vaccine efficacy field trial with a challenge study, followed by safety testing in a larger cohort and application for licensure or emergency use authorization for the vaccine (if it is shown, via such studies, to be safe and effective) (fig. , option ) . additional safety and/or efficacy data would then be collected during and after the introduction of the vaccine into public health use (who working group for guidance on human challenge studies in covid- ). the acceleration of phase iii efficacy testing is not the only way that hcs might plausibly accelerate or improve vaccine development (see fig. and table ). given that many of the more than sars-cov- candidate vaccines might ultimately need to be tested in humans, hcs would presumably provide the most efficient way to prioritize (or "select") the most promising vaccine candidates worthy of further investigation in field trials ( fig. , option ). this could accelerate vaccine development, for example by permitting early investment in manufacturing capacity for the most promising candidates based on rapid results from challenge studies. decisions have already been made to develop production capacity for vaccine candidates that have not yet been tested for efficacy (gates ) . by providing early estimates of efficacy, challenge studies would enable some manufacturing decisions to be made sooner, to be ready to increase production earlier than would be possible if such decisions were delayed until large field trials were completed-hcs might also make it more likely that investment in production capacity is not wasted on vaccines with low or zero efficacy and instead allocated to those most likely to be highly efficacious. the potential to compare the efficacy of multiple vaccine candidates in a standardized challenge study design would also obviate multiple parallel or sequential field trials that would plausibly require many months or years, tens of thousands of participants, and enormous financial costs (jamrozik and selgelid b) . direct comparative efficacy testing of multiple vaccines in hcs could also increase the likelihood that a more efficacious vaccine is ultimately deployed, for example, by comparing the first licenced vaccine with other candidates. without conducting hcs, comparative estimates of vaccine efficacy are extremely difficult to obtain (lurie et al. ), yet even small but statistically significant differences in efficacy (e.g., of per cent) might result in large differences in public health outcomes when vaccines are used by millions of people at substantial risk of infection (see fig. , option ). similarly, challenge studies might obviate more complex ethical and study design choices regarding optimal methods for comparing a new vaccine to one with early evidence of efficacy . the fact that a particular vaccine happens to be tested first or has stronger financial and/or political support should not preclude other vaccines being tested, especially where there is reason to think that this might result in a safer or more effective vaccine ultimately being deployed for widespread public health use. challenge studies could improve vaccine development in other ways-for example by determining vaccine-related correlates of immune protection that could be used to inform several aspects of vaccine development, including the use of vaccines in populations not involved in efficacy testing (who working group for guidance on human challenge studies in covid- ; memoli et al. ) . immunological correlates of protection can be used in immune bridging studies as a simplified measure to assess whether vaccination of older individuals, children, and pregnant women (who are often excluded from standard efficacy trials) is likely to result in protection against infection. once correlates have been determined, protection can be measured via the relevant correlate (i.e., immunological blood test), rather than measuring efficacy in terms of reduced rates of infection in a field trial or challenge study. similarly, hcs provide a unique opportunity to validate the performance of serological assays in predicting protective immunity to infection (memoli et al. )-more accurate tests would help to inform both future research ) and immediate public health practice (see table ) (phelan ). finally, hcs may provide unique or otherwise difficult to obtain results regarding (i) the characteristics of asymptomatic infection, (ii) the route(s) and risk of transmission during the early phases of illness (and the effect of different types of protective equipment) (hilding ) , and (iii) risks of re-infection after repeat challenge (a study design previously conducted with lower virulence coronaviruses (callow et al. ). the ethical evaluation of a particular challenge study (or programme of studies) should include comparison of the expected benefits and risks with those of other feasible research designs. since animal challenge studies with sars-cov- to date do not appear to be highly generalizable to humans (lurie et al. ) , the key comparator for hcs would be human vaccine field trials. vaccine field trials for sars-cov- might be more difficult (and onerous) than usual because of requirements for intensive testing for asymptomatic infection among participants, although study designs incorporating such testing have been proposed (kahn et al. ) . another key consideration is the effect of local epidemiology on research design and assessment, which we discuss below. challenge studies can be used for phase iii efficacy trials of vaccines in at least three ways. option describes a standard use: the selection among multiple candidates for the most promising vaccines to enter field trials. option describes a type of proposal where efficacy data from a challenge study plus safety data from a larger cohort could be submitted in an application for vaccine licensure or emergency use. whether regulators would accept these data alone, and how this would affect the design of post-market surveillance, remains to be confirmed. option describes an iterative process where challenge studies could be used to compare a licensed vaccine with a novel vaccine. assessments of potential vaccine research strategies should include direct comparisons between hcs and field trials regarding (i) the time required to produce results estimating vaccine efficacy, (ii) the number of participants involved, and (iii) relevant benefits and risks of research. these comparisons will be complex and may require epidemiological modelling to inform ethical assessments with quantitative data. a higher population incidence of infection, a larger field trial, and a more effective vaccine all allow a field trial to report in a shorter period of time than would otherwise be the case. conversely, field trials may be infeasible in low incidence populations where public health interventions are successfully suppressing or have eliminated disease transmission-for example, at time of writing (june ) field trials would not be feasible in australia or new zealand. in such populations, hcs would be the only way to estimate vaccine efficacy (heriot et al. ), yet there are few hcs research centres in australia and new zealand and, to our knowledge, none have recently conducted respiratory virus hcs. in some settings, field trial participants might be recruited from higher risk groups, and healthcare workers (hcws) might be considered candidates for such studies. however, where hcws have access to adequate protective equipment (and use it appropriately), they may not face significantly higher risks of infection with respiratory viruses than others in the wider community (bandaranayake et al. ) . the recruitment of hcws with poor access to protective equipment without remediating this basic deficiency might be considered unjust and/or exploitative, even though such individuals might benefit from participation in cases where an experimental vaccine turns out to be effective. field trials will also be infeasible in inter-epidemic periods. this issue was illustrated by the - zika epidemic: a prominent report on zika hcs recommended that vaccine efficacy be tested quickly during the epidemic using field trials without the need to expose research participants to challenge infection (shah et al. ) . however, by the time vaccines were ready for field trial testing, the epidemic had waned, incidence was low, and herd immunity levels were high in affected communities (as a result of widespread prior infection)-thus making zika vaccine field trials infeasible (vannice et al. ) . similar conditions will presumably occur in some communities already significantly affected by covid- . the practical difficulty of timing a field trial with an epidemic peak is joined by justice concerns, including that the burdens of field trials would be concentrated in communities already managing a high burden of disease (which may be at least partly attributable to injustice and/or public health policy failures) and also that there would be little community benefit of post-trial access to a vaccine (if shown to be effective in a field trial) due to high levels of herd immunity (jamrozik and selgelid b; wenner ). the benefits of any vaccines proven to be safe and effective via field trials would largely accrue to other populations in which public health measures had more successfully controlled disease transmission until such time as an effective vaccine became available. elsewhere, we have provided epidemiological models of the conditions under which vaccine field trials could be conducted as quickly as hcs and/or the extent to which populations in which field trial research is conducted might stand to benefit from vaccines tested in this way (heriot et al. ) . such models should arguably be used to inform practical decisions about what type(s) of trial(s) could be conducted in particular settings as well as inform ethical judgements about when and where they should ideally be conducted (e.g., relative to local epidemic conditions and public health policies). it is also important to compare hcs and field trials with respect to the risks to participants. both hcs and field trials involve exposing participants to the risks related to experimental vaccines including, in some cases, risks of vaccine-enhanced disease (jamrozik and selgelid b). on the one hand, hcs involve exposing smaller numbers of participants to such risks and often involve especially close monitoring and early provision of medical care (if required). on the other hand, because of small sample sizes, hcs might fail to detect less common risks of vaccines that could be more easily detected in larger studies such as field trials (jamrozik and selgelid b) . key additional risks of hcs relate to the challenge infection itself. to be ethically acceptable, sars-cov- hcs would need to have multiple risk minimization strategies in place, including (i) selection of low risk participants (e.g., healthy young adults ), (ii) careful strain selection and development, (iii) careful titration of viral dose, (iv) early diagnosis and availability of all necessary medical care, (v) long-term follow-up of participants, (vi) compensation for any lasting harms, and (vii) stringent infection control including measures to protect and screen research staff for infection (who working group for guidance on human challenge studies in covid- ). infection with sars-cov- would nevertheless involve residual risks, even for young healthy adults aged - . risks of hospitalization and fatal infection in this age group (whether healthy or not) have been estimated to be around . - per cent and . - . per cent respectively (verity et al. ; salje et al. ) . however, significant uncertainty remains, and more data are needed to clarify these risks and determine any risks of lasting harms from sars-cov- infection (e.g., longer-term lung function deficits, which sometimes occur after other respiratory tract infections (krzyzanowski et al. ) ). while the absolute risk to participants related to the challenge infection is one important consideration, in cases where participants face a background risk of infection (e.g. as a result of a local covid- epidemic), it is also important to assess (via modelling) and consider the marginal risks to participants (i.e., related to the additional probability of being infected as a result of study participation, relative to background risk). the relevance of background risk to judgements regarding risks to participants is sometimes controversial, especially if background risks are attributable to injustice (jamrozik and selgelid c) . however, background risk has been considered one reason in favour of the ethical acceptability of early challenge studies with yellow fever (lederer ) ; more recently, it has been considered in favour of zika hcs (shah et al. ) and hcs in endemic settings more generally-even though, in the case of zika and (other) diseases endemic to lmics, one might think that higher incidence of certain infections in particular locations is at least partly due to injustice (jamrozik and selgelid a; lederer ) . marginal risk depends not only on one's probability of being infected and suffering a bad outcome after infection from community transmission (i.e., background risk) but also on the degree to which one might develop immunity as a result of hcs participation (whether from challenge infection or an experimental vaccine) and the probability of having access to a vaccine developed via the participation of others (whether in hcs or field trials). since the infection fatality risk (ifr) of sars-cov- infection in healthy - year old individuals (e.g., those who might plausibly be eligible to volunteer for hcs) is likely around . - . per cent to begin with (verity et al. ; salje et al. ) , marginal risk in locations with significant background risk would be a fraction of this (i.e., extremely low). an estimate of marginal fatality risk can be determined by multiplying the ifr by the probability where current data provide grounds for reasonable confidence that certain individuals are at significantly higher risk of participation, such individuals should be excluded from participation. there should nonetheless be acknowledgement that even rigorous selection criteria do not preclude there being rare serious outcomes. one potential concern might be the exclusion of certain groups (e.g. ethnic groups) from vaccine research, which might result in the loss of information important for achieving public health benefits for these groups. such concerns could be remedied by (i) inclusion of lower risk individuals in these groups where such individuals can be confidently identified and/ or (ii) inclusion of such groups in other relevant lower risk research (e.g., immune-bridging vaccine studies). of in-study infection (which might be reduced from - per cent to - per cent in a : randomized hcs, depending on the degree to which the vaccine being tested prevents infection) and subtracting the background risk of infection (which might be low in the short term under public health lockdown but potentially greater than per cent in the longer term in communities with prolonged/recurrent high incidence epidemics) (heriot et al. ; salje et al. ) . under certain conditions, an individual's marginal risk (if there is significant background risk and/or significant vaccine efficacy in the trial) might range from half the ifr to zero or less (i.e. a net benefit of participation). the relative marginal risk reduction due to background risk would be high during high incidence epidemics, although the absolute risk reduction might be low because risks of infection for young healthy adults are small in any case. still, because rare severe outcomes may nevertheless occur, and because public trust might depend on challenge trials being conducted to especially high standards, risk minimization remains an essential part of study design (who working group for guidance on human challenge studies in covid- ). ethical assessments of the benefits and risks of sars-cov- hcs should be particularly systematic and must take into consideration the pandemic setting in which these studies might occur. assessments should include a thorough evaluation of the potential public health benefits and marginal risks of hcs as compared to vaccine field trials or other methods of vaccine development. this will help to ensure that informed judgements are madeincluding decisions about trade-offs between different ethical considerations, which may vary between settings. the justification of sars-cov- hcs might therefore be context specific, and local decision-makers should be informed by the best available evidence including modelling of the benefits and risks of possible research strategies under different epidemiologic conditions. ethical criteria for human challenge studies in infectious diseases seroprevalence of the influenza a (h n ) pandemic in new zealand. institute of environemntal science and research limited, national centre for biosecurity and infectious diseases the time course of the immune response to experimental coronavirus infection of man speed coronavirus vaccine testing by deliberately infecting volunteers? not so fast, some scientists warn. science magazine ethical comparators in coronavirus vaccine trials human challenge studies to accelerate coronavirus vaccine licensure responding to covid- -a once-in-a-century pandemic? tensions between public health and vaccine research priorities: a comparative modelling assessment of the risks and benefits of sars-cov- vaccine field trials versus human challenge studies literature review: the common cold challenge studies of human volunteers: ethical issues ethical issues surrounding controlled human infection challenge studies in endemic low-and middle-income countries efficacy and immunogenicity of a vi-tetanus toxoid conjugate vaccine in the prevention of typhoid fever using a controlled human infection model of salmonella typhi: a randomised controlled, phase b trial analyzing vaccine trials in epidemics with mild and asymptomatic infection longitudinal analysis of the effects of acute lower respiratory illnesses on pulmonary function in an adult population walter reed and the yellow fever experiments antibody testing will enhance the power and accuracy of covid- -prevention trials developing covid- vaccines at pandemic speed evaluation of antihemagglutinin and antineuraminidase antibodies as correlates of protection in an influenza a/h n virus healthy human challenge model covid- immunity passports and vaccination certificates: scientific, equitable, and legal challenges extraordinary diseases require extraordinary solutions estimating the burden of sars-cov- in france covid- vaccine development: time to consider sars-cov- challenge studies ethical considerations for zika virus human challenge trials randomized, double-blind, placebo-controlled, multicentered trial of the efficacy of a single dose of live oral cholera vaccine cvd -hgr in preventing cholera following challenge with vibrio cholerae o el tor inaba three months after vaccination demonstrating vaccine effectiveness during a waning epidemic: a who/nih meeting report on approaches to development and licensure of zika vaccine candidates estimates of the severity of coronavirus disease : a model-based analysis palgrave macmillan and springer nature. who working group for guidance on human challenge studies in covid- . key criteria for the ethical acceptability of covid- human challenge studies publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -hw silv authors: hilton, shona; hunt, kate; langan, mairi; bedford, helen; petticrew, mark title: newsprint media representations of the introduction of the hpv vaccination programme for cervical cancer prevention in the uk ( – ) date: - - journal: soc sci med doi: . /j.socscimed. . . sha: doc_id: cord_uid: hw silv in september , the human papillomavirus (hpv) immunisation programme was introduced in the uk for schoolgirls aged between and years of age. the vaccine shows high efficacy in preventing infection against hpv types and responsible for % of cervical cancer. however, to be most effective, the vaccine needs to be administered before exposure to the viruses and therefore, ideally, before young people become sexually active. the introduction of any new vaccine, and perhaps particularly one given to young teenage girls to prevent a sexually transmitted cancer-causing virus, has the potential to attract a great deal of media attention. this paper reports on content analysis of articles published between january and december in uk newspapers. it includes both manifest and latent analysis to examine newsprint media coverage of the introduction of the hpv vaccination programme and its role in hpv advocacy. we concluded that the newspapers were generally positive towards the new hpv vaccination and that over the years period the newsworthiness of the hpv vaccination programme increased. in two events dominated coverage, firstly, the introduction of the hpv programme in september and secondly, in august the diagnosis on camera of cervical cancer given to jade goody, a year old mother of two, who gained fame and notoriety in the uk through her participation in several reality television shows. there are two conclusions from this study. firstly, the positive media coverage surrounding the introduction of the hpv vaccination programme is to be welcomed as it is likely to contribute towards influencing public perceptions about the acceptability and need for hpv vaccination. secondly, the focus on prevalence rates of hpv infection among women and on women's sexual behaviours, in relation to hpv vaccination ‘encouraging’ promiscuity, is an unhelpful aspect of media coverage. the introduction of cervical screening has reduced invasive cervical cancer incidence and mortality by more than % (bray et al., ; peto, gilham, fletcher, & matthews, ) . nevertheless, cervical cancer still accounts for approximately cases and deaths per annum in the uk (http://info.cancerresearchuk.org/ cancerstats/types/cervix/). in september , a new immunisation programme against strains of the sexually-transmitted human papillomavirus (hpv) was introduced in the uk in order to further reduce incidence and mortality from cervical cancer. the programme targets girls aged - years, and in the first two years is to be supplemented by a 'catch up' campaign for girls aged - years (uk department of health, , october ) . the vaccine chosen (cervarixÒ) offers protection against two strains of the sexually transmitted virus (types and ) which are responsible for % of cervical cancer and may offer cross protection against other hpv strains (von krogh et al., ) . in a randomised control trial of women with no prior evidence of exposure or infection with hpv, the vaccine showed high efficacy in preventing infection with hpv types and for at least . years (harper et al., (harper et al., , . ongoing trials are assessing the duration of efficacy, but it is anticipated that immunity may persist for up to years. to be most effective it is recommended that young people are given the vaccine before they become sexually active (jit, choi, & edmunds, ; jit, vyse, et al., ) . according to a study by the health protection agency in which they took blood samples from girls and women between the ages of and to identify hpv infection, the risk of girls contracting hpv rises 'substantially' after the age of , with one in ten teenage girls having been infected by the age of and around % of girls having contracted the virus by the time they reach years of age (jit, choi, et al., ; jit, vyse, et al., ) . in view of this the joint committee for vaccination and immunisation recommended giving hpv vaccination to girls aged - (joint committee for vaccination and immunisation statement on human papillomavirus vaccines to protect against cervical cancer). to maximise the success of the hpv immunisation programme it needs to achieve a high uptake rate, particularly among girls from more deprived groups who experience the highest rates of cervical cancer (parikh, brennan, & boffetta, ) . research in scotland found that cervical cancer rates amongst women living in the most deprived areas were more than two and a half times of those women living in the least deprived areas (harris, sandridge, black, brewster, & gould, ) . a recent economic evaluation estimated that for the programme to be cost effective the hpv programme needs to achieve uptake rates in excess of % (jit, choi, et al., ; jit, vyse, et al., ) , well above the . % coverage reported in a study conducted to pilot the acceptability of the hpv vaccine (brabin et al., ) . whilst the pilot figures fall short of desired uptake rates, it has been suggested that this uptake should be viewed as 'encouraging' because the pilot was conducted 'in the absence of national publicity' (waller & wardle, , p. . provisional figures on uptake in england up to the end of august for girls aged - (year ) rd dose is . % (see: http://www.immunisation.nhs.uk/ publications/hpv_vaccineuptake_aug .pdf). the introduction of any new vaccine, and perhaps particularly one given to young teenage girls to prevent a sexually transmitted cancer-causing virus, has the potential to attract a great deal of media attention. recent experience shows that the mass media has a key role to play in the perceived desirability and acceptability of vaccines, and hence a key determinant of the uptake of hpv vaccination programme will be the media coverage it receives. adverse publicity about the safety of the mmr vaccine (beggs, ramsay, white, & bozoky, ) and whooping cough vaccine (church, ) caused large numbers of parents to refuse immunisation for their children. studies of the mmr controversy highlight how important it is to address any gaps in public understanding of the benefits and risks of a vaccine in order to anticipate and plan future public health messages (bellaby, ; hilton, hunt, & petticrew, ; smith, yarwood, & salisbury, ) . in a recent analysis of canadian and us national newspaper articles that compared the level of fear-inducing messages about hpv, cervical cancer and hpv vaccination, it was found that some factors that elicit fear about the hpv vaccine and cervical cancer were common to both countries, but that the frequencies of others may have varied based on the cultural and political environments. the authors suggest that: ''communicating health risk information in the mass media is not separate from social and political contexts and that health communication efforts may be overshadowed by negative media coverage'' (abdelmutt & hoffman-goetz, , p. ). on the other hand, positive media campaigns to promote immunisation have been shown to improve uptake rates by increasing public knowledge of the dangers of vaccine-preventable diseases (wallace, corben, turahui, & gilmour, ) . to date, studies investigating levels of knowledge about hpv among the british public suggest a scant awareness of its role in the aetiology of cervical cancer. for example, in a survey of female university employees just % had heard of hpv and only % knew of the link between hpv and cervical cancer (pitts & clarke, ) . similarly, in a questionnaire survey of women attending a well woman clinic in london (uk) (n ¼ ) about % recognised hpv only in name. on further questioning, less than half knew of the link with cervical cancer and there was confusion about whether condoms or oral contraceptives could prevent hpv infection (waller et al., ) . in a more recent study of a representative sample of british women (n ¼ ), a quarter of respondents were aware of hpv, and awareness was lower in those with less formal education . a similar disparity in knowledge was evident in a study investigating knowledge about other preventable cancers such as skin and lung cancers (viswanath et al., ) . this may reflect what tichenor, donohue, and olien ( ) proposed as the 'knowledge gap hypothesis' in which they believe that information in society is not acquired evenly by individuals. their hypothesis is based on the 'apparent failure of mass publicity to inform, the public at large' (tichenor et al., , p. ) . in short they suggest that people with higher socioeconomic status tend to have better ability to acquire information and the nature of the mass media itself is that it is geared towards persons of a higher socioeconomic status. this leads to a division of two groups: a group of better-educated people who know more about most things, and those with low education who know less. further, concerns have been expressed that one consequence of increasing public awareness that a sexually transmitted virus (i.e. hpv) causes cervical cancer might lead to increased stigmatisation of cervical cancer patients (mccaffery, waller, nazroo, & wardle, ) . conversely, waller and colleagues have argued that an emphasis on the high prevalence of hpv in the population may help to normalise the infection and reduce feelings of shame and stigma . if people perceive that the likelihood of contracting hpv infection is high this may also increase the acceptability of hpv vaccination. key factors known to influence whether parents will accept vaccination for their children include parents' perceptions of the severity and likelihood of contracting the disease, and the safety of the vaccine. when parents perceive the risk of contracting a serious disease is remote or when they have concerns over its safety the incentive to vaccinate is lessened. conversely, if parents perceive the risk of contracting a serious disease is high or if they perceive the vaccine to be safe the incentive to vaccinate is increased (smailbegovic, laing, & bedford, ) . whist studies have shown the public know little about hpv and its link with cervical cancer, any vaccine reported as preventing cancer is likely to generate interest since cancer has been described as the most feared of modern diseases. sontag draws parallels between the current language used to describe cancer and past descriptions of tuberculosis (tb) where contracting tb was generally viewed as ''tantamount to hearing a sentence of death''. she goes on to suggests that over the years the lurid metaphors with which diseases such as cancer have been described have led to a situation where in the popular imagination ''cancer equals death'' (sontag, ) . indeed, portrayals of cancer in the media have led it to being associated with long debilitating treatments, fear, hopelessness and death (clarke & everest, ) . a recent example of this is the high profile media coverage surrounding reality television celebrity jade goody's 'battle' with terminal cervical cancer. it has been suggested that the media coverage of her case has raised public awareness about the hpv programme particularly among younger women from lower socioeconomic groups, like goody, who are most at risk from developing cervical cancer (cassidy, ) and least likely to utilise cervical screening services (baker & middleton, ) . it has been suggested that the prevalence of co-factors such as smoking, sexual promiscuity and multi-parity among women from socially disadvantaged backgrounds explains the higher incidence of cervical cancer among these women (currin, jack, linklater, & mak, ). more broadly, the mass media play a key role in setting the agenda for some health issues and news stories are often constructed to take one perspective or another. dominant frames are often constructed to define what issues are viewed as important and these constructs can be particularly powerful when they are consistent over a long period of time (menashe & siegel, ) . these perspectives or frames influence what are included or excluded from stories and can be misleading in relation to the scientific evidence they present. researchers at the cardiff university school of journalism have investigated media coverage of three scientific issues with social policy implications: climate change; cloning and genetic medical research; and the mmr vaccine. in relation to the mmr controversy, they found that attempts to balance claims about the risks of the mmr vaccine tended merely to indicate that there were two competing bodies of evidence rather than offer more substantive evaluations of the case for or against a causal link between autism and mmr (hargreaves, lewis, & speers, ) . these perspectives can also be hugely influential on health behaviours. in some cases the role of the media has been shown to contribute towards promoting harmful health behaviours. for example wakefield and colleagues summarised the results of empirical studies on cigarette advertising and promotions, anti-smoking advertising, product placement in movies, on television and in music media and news coverage about smoking. they found that the media act as a source of observational learning by providing models which teenagers may seek to emulate and that exposure to media messages about smoking also provides direct reinforcement for smoking (wakefield, flay, nichter, & giovino, ) . on the other hand, the media can be hugely influential in changing health behaviors and improving health. taveras and colleagues conducted a cross-sectional mailed survey of , boys and girls, between the ages of - years. they found that wanting to look like figures in the media was associated with higher physical activity levels among older children and adolescents, independent of other personal and social influences. these data suggest that television, movie, and magazine industries should be encouraged to cultivate and reinforce realistic and healthy norms of physical activity and body image (taveras et al., ) . thus a key determinant of the acceptability of hpv vaccination to the public will be how the newsprint media construct and frame messages about the new hpv vaccination programme. this research aims to examine the role the newsprint media have played in hpv advocacy by identifying key messages about the risks and benefits associated with hpv vaccination and hpv infection, and how these stories were constructed and framed for different readership groups. we selected uk newspapers with high circulation figures and a range of readership profiles (www.abc.org.uk, www.nrs.co.uk) for this study. our sample consisted of national newspapers and regional scottish newspapers widely read in scotland where the hpv programme is first being introduced in the uk. the sample comprised of 'serious' papers (previously called broadsheets before the paper sizes changed) (times, guardian, telegraph, independent, sunday times, observer, herald and scotsman), 'middlemarket tabloid' papers (daily mail and express) and 'tabloid' papers (daily record, mirror, sun, sunday people, news of the world). fig. shows the readership profiles of these newspapers by age and social class, illustrating the tendency for the 'serious' newspapers to be read by people from higher socioeconomic groups and conversely the 'tabloid' papers to be read by people from lower socioeconomic groups (www.nmauk.co.uk). this typology has been used by williams and colleagues to select a broad sample of newspapers with various readership profiles and political orientation when examining the print media discourses on a particular debate (williams, seale, boden, lowe, & steinberg, ) . they suggest that uk newspapers are well suited to studies which aim to differentiate mass media discourses by intended readers' age and social class because the uk newspaper market is powerfully segmented into 'tabloid' and 'serious' genres catering for distinct readership groups of class and age. this also enables a comparison of newspaper genre to examine whether the educational content supports the knowledge gap theory which is particularly relevant since cervical cancer is known to be socially patterned. our search period was from st january to st december . we selected this time frame to encompass the period when the findings from hpv vaccine trials began to gain public and media coverage and the months immediately following the roll-out of the programme in september . articles from our target publications were identified using the electronic database newsbank, adopting the single search term 'hpv' in 'all text'. each article was scrutinised (ml) to establish whether its content included reference to: hpv infection, hpv vaccination, hpv and cervical cancer or hpv and cervical cancer screening. we identified a total of articles of which were excluded because: they were duplicate articles; they were published in irish editions of the papers (because the hpv vaccination has not been introduced in ireland); they focused on hpv and other health outcomes (e.g. throat cancer, genital warts); or they referred to cervical cancer screening but did not mention hpv. initially a random selection of articles were examined (sh) to identify the key discourses around hpv vaccination; these became thematic categories in an initial coding frame. this initial coding frame was tested against a further articles (i.e. every tenth article in the sample). three researchers (sh, kh, ml) independently coded each article. following discussion of any discrepancies, the coding frame was revised (including more extensive definitions of coding categories) and two researchers (sh, ml) independently coded another randomly selected articles. at this stage no further thematic categories were identified and there was complete agreement between the coders. the final coding framework recorded the publication, date, headline, whether there was any reference to thematic categories and a section assessing the tone of the articles towards hpv vaccination. the tone was employed primarily to assess whether, from a public health perspective, hpv vaccine was being supported or advocated. thus, if the text was generally favourable towards cervical cancer prevention and hpv vaccination, the tone was recorded as 'positive'. if the text was judged as generally unfavourable towards cervical cancer prevention and hpv vaccination, the tone was recorded as 'negative'. articles which included both positive and negative messages without favouring either side of the debate were coded as 'mixed'. neutral articles tended to be informative articles which lacked any rhetoric encouraging or discouraging hpv vaccination. when assessing tone, particular attention was paid to the headline, subheadline and lead paragraph as these areas act to anchor the most newsworthy aspect of the story, its main trajectory and encapsulate what the journalist might consider most important (chapman & chapman, ) . newspaper articles were analysed for both manifest content and latent content (altheide, ) . manifest content refers to what is explicitly stated and thus draws on the objective and replicable qualities of quantitative methods. clarke suggests that latent content includes the investigation of deeper and perhaps unintended themes (clarke & everest, ) . this involves more indepth interpretive analytical qualities of qualitative methods to make inferences from the data. a main feature of this textual analysis involved comparing, contrasting and categorising a corpus of data (schwandt, ) . first, in order to systematically quantify the manifest content of the articles, each article was read line by line and coded to indicate whether or not each of thematic categories in the coding frame was mentioned. these data were entered into spss . we formally tested using chi-square tests (c ) whether these aspects of coverage of the introduction of the hpv vaccine were differentially mentioned in the three different types of publication ('serious', 'middle-market tabloid', and 'tabloid'). for the latent and inductive analysis all the text pertaining to each category was transcribed and imported into the qualitative software package nvivo to aid management of the data. all the text was re-read and re -coded to discover patterns and anomalous ideas. written summaries of these thematic categories and the constant comparative method (glaser & strauss, ; lincoln & guba, ) informed the interpretation of the data across the articles and different genres of newspaper to consider what the key messages were and how messages were being framed at different readership groups. over the period from january to december , news articles referred to coverage of the hpv virus and the introduction of the new hpv vaccine and satisfied our inclusion criteria in the newspapers that we examined. more than half ( . %, n ¼ ) were published in 'serious' newspapers, and roughly equal proportions of the remainder appeared in 'middle-market tabloid' ( . %, n ¼ ) and 'tabloid' ( . %, n ¼ ) papers (see table august (n ¼ ) and secondly, the introduction of the hpv programme in september (n ¼ ). more generally, of the articles, . % referred to the development of the vaccine, including reference to vaccine trials and introduction of the hpv programme in the uk (see table ). it was also common for articles to make some reference to: the epidemiology of cervical cancer ( . %), the target age and rationale for the vaccine to be administered ( . %), hpv infection as being causally associated with cervical cancer ( . %), and that the virus is sexually transmitted ( . %). just under half reported that promiscuity increased the risk of contracting an hpv infection ( . %) and reported on safety and efficacy of hpv vaccines ( . %). over one third of the articles explicitly mentioned the benefits of hpv vaccination ( . %) and anti-immunisation sentiments featured in less than one in five ( . %). the newspapers were generally positive towards the new hpv vaccination programme, with . % of articles in 'serious' newspapers positively toned, . % of articles in 'middle-market tabloid' positively toned, and . % of articles in 'tabloid' newspapers positively toned (p ¼ . ). none of the 'tabloid' newspapers were categorised as negatively toned, and only two 'middle-market tabloid' and three 'serious' newspapers articles were negatively toned. the 'tabloid' newspapers were less likely to offer mixed messages about hpv vaccination. they tended not to report opposing views towards the new hpv vaccination and only % were rated as mixed toned compared with the 'middle-market tabloid' ( . %) and 'serious' ( . %) newspapers, which were more likely to report on the competing views about hpv vaccination (see table ). in terms of content, anti-immunisation sentiments towards the introduction of the hpv programme were slightly more likely to be represented in the 'serious' newspapers, for example, the guardian and independent compared with the tabloid newspapers the mirror and the sun. likewise in scotland the 'serious' newspapers the scotsman and herald were more likely than the daily record to include anti-immunisation sentiments. only one article ( . %) out of the 'tabloid' newspaper articles contained any anti-immunisation sentiment compared to ( . %) of the 'serious' newspapers and ( . %) of the 'middle-market tabloid' newspapers (see table ). some issues were much less likely to be included in articles on hpv and hpv vaccination in the 'tabloid' newspapers (although in part this is a function of the length of the article as most tabloid articles were considerably shorter). overall the 'tabloids' were significantly less likely to mention cost, anti-immunisation sentiments, pharmaceutical companies, vaccine development, health professionals' role, safety and efficacy of the vaccine, promiscuity, getting the vaccine privately, and whether vaccination should be compulsory or voluntary (see table ). it was common for newspaper headlines to convey a message of a scientific breakthrough and for this dominant discourse to be a key feature of articles. the scientists behind the development of the vaccine were given a particularly high profile role in the media reports. direct quotes from scientists were used to lend credibility to the development of the hpv vaccines and the ongoing safety and efficacy trials. these direct quotes added to an overall sense that the evidence was irrefutable and that hpv vaccines were a safe scientific advance towards preventing cervical cancer. it was common for the quotes to detail the magnitude and significance of the scientific breakthrough by describing the scientists' emotions and feelings of celebration. for example, it was widely reported across the newspapers that professor frazer, was quoted at saying: ''.it probably was the most exciting thing, scientifically, that's happened in my lifetime'' (scotsman, oct , ) . similarly, professor margaret stanley of cambridge university was described in the times (oct , ) as ''euphoric.'' another element of the discourse was to present the development of the hpv vaccines as a 'genuine' medical breakthrough which would 'revolutionise cervical cancer prevention'. for instance, the daily mail (jul , ) reported: ''we are today on the brink of a world and lifechanging breakthrough in the fight against cancer through (guardian, apr , ) . the language of the scientific breakthrough extended to commentary on the results of efficacy and safety trials. dramatic language was employed to report on the high efficacy rates from the hpv vaccines trials on cervarixÒ (glaxo-smithkline, uk) and gardasilÒ (merck, us) published in scientific journals. across the different newspapers these trial results were reported as ''revolutionary'' (daily mail oct , ) , ''scientifically brilliant'' (guardian, mar , ) and as a ''tremendous opportunity'' (daily mirror, apr , ) . using personal testimonies to convey messages across the different newspapers there were many instances of compelling, personal testimonies and human interest stories appearing in both the 'tabloid' and 'serious' newspapers from women who had been diagnosed with cervical cancer. these stories tended to be lengthy accounts and most of the articles appeared in the daily mail, perhaps reflecting the newspaper's intention of appealing to a female readership. there was little difference between the main characters that featured in these stories. in total women featured in stories. on average the women featured in the 'serious' and 'tabloid' newspapers were about the same age (serious years, tabloids years), although the women in the 'serious' newspapers were less likely to be described as single mothers (serious , tabloid ) and more likely to have professional occupations mentioned (serious , tabloid ). these subtle differences in the framing of these key characters may reflect attempts on behalf of the newspaper to select women who may appeal to their readership. in common, these discourses drew on tragic stories of young women with cervical cancer and were primarily used to illustrate the risks associated with hpv infection and cervical cancer. for example, the following excerpt from a story run in the daily mirror (dec , ) started with: ''attractive, bright, with a loving family, rachel was planning her wedding and seemed to have everything she could want out of life. but rachel, was diagnosed with cervical cancer.'' ''rachel died aged just , six weeks before her wedding''. other stories acted as warnings to women. the daily mirror (jun , ) reported the case of a woman who had always had normal smear test results, or so she thought, but had in fact abnormal test results which had been overlooked, which was described as ''a terrible error that cost the -year-old her life''. the most high profile personal testimony was that of jade goody's 'battle' with cervical cancer. discourses about goody tended to closely mirror the emotional stories of ordinary women. it is of note that the dominant use of women with cancer as main characters to anchor the risks posed by cervical cancer and benefits of the new hpv vaccine silenced most other discourses. there were only a few stories that used interviews with parents and girls to examine and debate the dilemmas about hpv vaccine decision-making and only one case of an interview with a mother and daughter who allegedly became paralysed from the waist down after receiving hpv vaccination. this story ran in the daily mail (dec , ) , the daily telegraph (dec , ) and sunday times (dec , ) . military metaphors were liberally used in both the headlines and body of the article. analysis of the use of this language revealed it was employed to primarily to convey the importance of the development of a vaccine to 'fight' cancer, and secondly to portray cancer as the 'enemy'. freeman-halton test found association between the genre of newspaper and tone p ¼ . . 'the green light to sex' few differences were evident in the framing of messages about promiscuity across newspaper genres. in striving to offer 'balance' in reporting, some newspapers gave a voice to religious groups/ objectors. often their quotes were balanced against opposing views. for example, in one article the director of the christian institute charity referred to the hpv vaccine as: ''.basically a sex jab, encouraging the view that girls can be sexually available. it is a disease that you can only get through being sexually promiscuous. the thing we should be doing is trying to stop kids being sexually active'' (daily telegraph, jun , ) . the following day the daily telegraph (jun , ) reassured readers, stating that the ''sex jab'' referred to is: ''.actually a vaccine that gives girls immunity against human papilloma virus (hpv) which is responsible for almost all cases of cervical cancer. experts have recommended that every girl aged has the vaccine, in order to afford them protection against one of the most deadly and devastating of diseases''. generally comments from religious groups tended to question hpv vaccination. one exception reported in the guardian (jun , ) is that of a spokesman for the catholic church who supported hpv advocacy suggesting: ''the vaccination programme should be supported by the promotion of sex within marriage and that the promotion of marriage should remain the number one social policy priority". in a similar vein, several newspapers then ran stories about the vaccine under inflammatory headlines. arguments were made in direct opposition to this stance: ''this vaccination is about protecting a woman's health, not about giving a green light to sex (mirror, jun , ) . however, hpv vaccination giving 'the green light to sex' was a more dominant discourse as illustrated by the following excerpts from the times newspaper: "anyone giving this drug to a girl is telling her i think you are a slag'' (spokesperson christian voice) and '' it will make girls think it's ok to pick up. boys and sleep around'' (spokesperson islamic medical association) (times, oct , ) . these excerpts typify the tendency in all articles to focus on promiscuity in relation to the behaviours of young women but not men. one feature article written by dr thomas stuttaford, attempted to redress the balance: ''some of the unreasonable stigma that surrounds carcinoma of the cervix may stem from the belief that it is her fault.'' he goes on to state: ''.men who are multipartnered are more likely to hand it on. for example, it is found that cancer of the cervix is diagnosed most commonly in women in societies where although they are expected to be abstinent it is acceptable for men to use brothels'' (times, feb , ) . it was common for all the newspapers to mention that there is a high prevalence of hpv infection in the community and therefore that the likelihood of contracting hpv infection once sexually active is high. the times (oct , ) wrote: ''about per cent of sexually active women can expect to have an hpv infection at some point in their lives'' and the daily mirror also reported: ''up to per cent of women contract hpv at some point'' (may , ) . across the newspapers the high prevalence of hpv infection rates was reported for women and not men. the framing of different messages to target audiences the 'tabloid' newspapers were more likely to simplify messages and directly advocate hpv vaccination than the 'serious' newspapers which tended towards advocating hpv vaccination whilst offering a critical commentary and competing views. for example, the daily mail's (oct , ) headline recommended: ''girls of 'should be given sex disease jab','' whilst a sun (apr , ) headline reported: ''sex jabs 'can foil cancer''' and the express (oct , ) stated: ''girls' anti-cancer jabs will stop dying every year''. in contrast, the 'serious' newspapers were more likely to debate the issues and highlight areas where the evidence could be misinterpreted or misrepresented, rather than offer unconditional hpv advocacy. for example, it was more likely for concerns to be debated in the serious newspapers about the lack of data on longterm safety of the new hpv vaccines and whether the benefits of immunisation would compensate for any potential risks associated with it. for instance, one article published in the independent on nov , examined the merits of the programme. the article was written by jerome burne (who has written previous antiimmunisation articles). he states that he will not be letting his two daughters have hpv vaccination and goes on to say: ''it's a public health initiative that is unnecessary, reckless and ridiculously expensive. worse, serious doubts about its wisdom have not been properly presented to the public. instead, children and parents have been bombarded with publicity -''a totally life-saving, revolutionary vaccine'' -while the media have largely parroted official assertions that it is ''safe, proven and effective'', all of which are unfounded.'' similarly, the guardian (mar , ) ran a story in which the columnist anne karpf stated: ''this is what concerns me. not just that gardasil works on only four strands of hpv when there are between and that can cause cancer. or that some serious adverse reactions include convulsions and numbness. no, what's really disquieting about this public health campaign is that most cases of hpv clear up of their own accord -a healthy immune system knocks it on its head.'' despite the success of immunisation in the reduction or eradication of many once serious diseases there has been growing adverse media publicity about the risks associated with immunisation (beggs et al., ) (church, ) . nevertheless, although some view the hpv vaccine as a controversial vaccine because it is given to young teenage girls to prevent a sexually transmitted disease, uk newsprint coverage has generally been positive. over the -year period of this study the newsworthiness of the vaccination programme increased as the date for the introduction of the hpv programme in september neared and there was also growing public interest in cervical cancer because of publicity generated by jade goody's diagnosis of, and treatment for, cervical cancer. although this study was conducted prior to the media frenzy leading up to goody's death from cervical cancer on march nd , it is likely that her story may become a major factor in securing high hpv vaccination coverage. indeed, it has been widely suggested that jade goody's legacy will be the publicity generated about the potential for cervical screening and hpv vaccination to save lives, particularly among those 'hard to reach' groups of girls/ women who tend to default on screening and vaccination services (cassidy, ) . the use of personal testimonies in newspapers was particularly evident in the newspapers such as the daily mail whose readership tends to be women. these testimonies served to draw the reader's attention to the importance of the hpv vaccine and provided a powerful incentive towards hpv vaccination. karpf describes these media portrayals as a powerful way of universalising and personalising human experience which is beyond political and economic factors (karpf, ) . o'dell and brownlow argue that it is because of the levels of disbelief in government and concern that corporate issues are more important than the 'truth' that the experiences of everyday people become so powerful and persuasive (o'dell & brownlow, ) . another feature of the stories was the use of direct quotes, particularly from scientists as a means of lending credibility to the development of the hpv vaccines and the ongoing safety and efficacy trials. these quotes were framed to add to a sense that the evidence was irrefutable and that hpv vaccines were a safe scientific advance towards preventing cervical cancer. it was common for the quotes to detail the magnitude of the scientific breakthrough by using celebratory language. in general the 'tabloid' newspapers offered the most simplistic messages and were most likely to advocate hpv vaccination unconditionally, whereas the 'serious' newspapers tended to advocate hpv vaccination but also offer some level of critical commentary and competing views. it seems likely that this was to be seen as a means of offering balance (hargreaves et al., ) . this reflects what tichenor and colleagues called the 'knowledge gap hypothesis', in which the information in society is not acquired evenly by individuals nor targeted evenly by the mass media with people of higher socioeconomic status being offered more informed and critical commentary. the fact that tabloid newspapers have offered their readership who may have the most to gain from hpv vaccination unambiguous messages advocating hpv vaccination, may encourage a higher uptake among these girls. however, such uncritical journalism also serves to socially exclude these readers from the wider tapestry of arguments which may leave them relatively less informed about the issues surrounding hpv vaccination. the dominant discourses mapped in this study suggest the hpv vaccine was reported as a 'good news story' and hailed as a safe and effective step towards preventing cervical cancer. on the whole, the newspapers keenly presented the benefits of the new hpv vaccination programme and the vaccine was frequently portrayed as offering hope for future generations of young women against developing cervical cancer. articles were generally very informative about the safety and efficacy trials and rationale for the policy on the target age of girls to be vaccinated. militaristic language was liberally used to help convey the importance of the vaccine and add a sense of urgency to the need to 'wipe out' cervical cancer. since the emergence of hiv/aids in the s, social scientists and sociologists of health and illness have explored metaphorical framing of diseases and have identified the use of militaristic language to permeate discourses of immunology, bacteriology and infection for at least a century (sontag, ) . such language has been observed more recently in studies of newsprint media representations of how the sars (severe acute respiratory syndrome) disease threat was depicted (wallis & nerlich, ; washer, ) . washer examined the phenomena of 'emerging and re-emerging infectious diseases' over the past or so years and suggests that these have impacted on the faith once widely held that western biomedicine could 'conquer' infectious disease. there was little mention of any specific risks associated with the hpv vaccine itself. the main risk presented was that the vaccine might 'signal a green light to sex'. this finding is consistent with a recently published study examining the content of newspaper articles on the issue of adolescents engaging in risky sexual behaviour following hpv vaccination (forster et al., in press ). forster and colleagues found that that newspapers provided parents with broadly positive descriptive norms about vaccination; however, the issue that adolescents will engage in risky sexual behaviours following hpv vaccination was regularly discussed in the national press and they suggest it has the potential to increase parents' concerns about vaccination. similarly, our study found that the issue of promiscuity was raised across all the newspapers, and religious and ethical views were presented in juxtaposition to those of scientists, parents and journalists. concerns about the vaccine sexualising young girls and encouraging promiscuity among girls were commonplace. the focus of these excerpts was almost entirely focused on the behaviours of young women rather than men, which is likely to add to a sense of stigma that many women already feel about cervical cancer (mccaffery et al., ) and calls into question whether a gender-specific vaccination programme for a sexually transmitted disease add to the heavy burden of responsibility women already have for sexual health. whilst waller and colleagues' suggestion that emphasising the high prevalence of hpv in the population may help to normalise the infection and reduce feelings of shame and stigma, as yet the sole focus on high prevalence rates among women might suggest 'the problem' lies entirely with them, and not men. in conclusion, adverse media publicity about vaccination programmes can have serious public health consequences as seen in the case of the pertussis scare (church, ) and controversy surrounding the safety of the mmr vaccine (beggs et al., ) . the positive media coverage which has surrounded the introduction of the new hpv vaccination programme is likely to play a crucial role in influencing public perceptions about the acceptability of hpv vaccination in the early stages of the programme at least and may contribute towards reducing cervical cancer among future generations of young women. whilst this positive news coverage is to be welcomed, our analysis has uncovered some discourses that may have wider implications for the promotion of equality in sexual health and relationships among young people. to date, some newsprint reporting has tended to present hpv vaccination as encouraging promiscuity among girls with little mention of boys' sexual behaviour and reports have tended to focus on high prevalence of hpv infection among women with little mention of rates among men. further, although rates of cervical cancer are known to be high in women from less advantaged socioeconomic backgrounds this discourse was absent, which may lead the general public to conclude that that higher prevalence of cervical cancer is wholly due to the sexual behaviours of women from these backgrounds. whilst newspapers have a wide readership and undoubtedly act as an important information source on health and influence on health behaviours the fact that the newspapers read by more affluent people contained more useful educational information is likely to contribute to increasing the gaps in knowledge between members of society. further, there is a need to actively challenge possible negative messages which centre upon women and plan more balanced future public health messages about hpv infection and vaccination. risk messages about hpv, cervical cancer, and the hpv vaccine gardasil: a content analysis of canadian and u.s. national newspaper articles creating fear: news and the construction of crisis cervical screening and health inequality in england in the s media dents confidence in mmr vaccine communication and miscommunication of risk: understanding uk parents' attitudes to combined mmr vaccination uptake of first two doses of human papillomavirus vaccine by adolescent schoolgirls in manchester: prospective cohort study incidence trends of adenocarcinoma of the cervix in european countries jade, class, and cervical cancer framing pub smoking bans: an analysis of australian print news media coverage return of whooping cough cancer in the mass print media: fear, uncertainty and the medical model inequalities in the incidence of cervical cancer in south east england - : an investigation of population risk factors passport to promiscuity or lifesaver: press coverage of hpv vaccination and risky sexual behavior the discovery of grounded theory towards a better map: science, the public and the media efficacy of a bivalent l virus-like particle vaccine in prevention of infection with human papillomavirus types and : randomised controlled trial sustained efficacy up to . years of a bivalent l virus-like particle vaccine against human papillomavirus types cancer registration statistics gaps in parental understandings and experiences of vaccine-preventable diseases. a qualitative study. child: care, health and development economic evaluation of human papillomavirus vaccination in the united kingdom joint committee for vaccination and immunisation statement on human papillomavirus vaccines to protect against cervical cancer doctoring the media the reporting of health and medicine naturalistic inquiry public awareness that hpv is a risk factor for cervical cancer social and psychological impact of hpv testing in cervical screening: a qualitative study the power of a frame: an analysis of newspaper coverage of tobacco issues -united states media reports of links between mmr and autism: a discourse analysis meta-analysis of social inequality and the risk of cervical cancer the cervical cancer epidemic that screening has prevented in the uk human papillomavirus infections and risks of cervical cancer: what do women know? qualitative inquiry: a dictionary of terms why do parents decide against immunization? the effect of health beliefs and health professionals tracking mothers' attitudes to mmr immunisation illness as a metaphor and aids and its metaphors the influence of wanting to look like media figures on adolescent physical activity mass media flow and differential growth in knowledge hpv vaccine recommended for nhs immunisation programme cancer knowledge and disparities in the information age european guideline for the management of anogenital warts role of the media in influencing trajectories of youth smoking the role of television advertising in increasing pneumococcal vaccination coverage among the elderly the association between knowledge of hpv and feelings of stigma, shame and anxiety awareness of human papillomavirus among women attending a well woman clinic hpv vaccination in the uk disease metaphors in new epidemics: the uk media framing of the sars epidemic representations of sars in the british newspapers medicalization and beyond: the social construction of insomnia and snoring in the news we have no competing interests. this study was funded by the medical research council's, population health science research network (wbs. u . . . . ). hb has been reimbursed in the past (not in the past five years) by several vaccine manufacturers, for attending and speaking at conferences and conducting research. sh participated in the design, data collection and analysis, and drafted the manuscript. kh, mp and hb participated in the design and kh helped in drafting the manuscript. ml participated in data collection and analysis. all authors approved the final manuscript. key: cord- -guciupc authors: hajj hussein, inaya; chams, nour; chams, sana; el sayegh, skye; badran, reina; raad, mohamad; gerges-geagea, alice; leone, angelo; jurjus, abdo title: vaccines through centuries: major cornerstones of global health date: - - journal: front public health doi: . /fpubh. . sha: doc_id: cord_uid: guciupc multiple cornerstones have shaped the history of vaccines, which may contain live-attenuated viruses, inactivated organisms/viruses, inactivated toxins, or merely segments of the pathogen that could elicit an immune response. the story began with hippocrates b.c. with his description of mumps and diphtheria. no further discoveries were recorded until a.d. when the smallpox vaccine was described. during the eighteenth century, vaccines for cholera and yellow fever were reported and edward jenner, the father of vaccination and immunology, published his work on smallpox. the nineteenth century was a major landmark, with the “germ theory of disease” of louis pasteur, the discovery of the germ tubercle bacillus for tuberculosis by robert koch, and the isolation of pneumococcus organism by george miller sternberg. another landmark was the discovery of diphtheria toxin by emile roux and its serological treatment by emil von behring and paul ehrlih. in addition, pasteur was able to generate the first live-attenuated viral vaccine against rabies. typhoid vaccines were then developed, followed by the plague vaccine of yersin. at the beginning of world war i, the tetanus toxoid was introduced, followed in by the pertussis vaccine. in , the expanded program of immunization was established within the who for bacille calmette–guerin, polio, dtp, measles, yellow fever, and hepatitis b. the year witnessed the launching of the international aids vaccine initiative. in , the who passed a resolution to eradicate polio by the year and in ; the first vaccine to prevent cervical cancer was developed. in , “the decade of vaccines” was launched, and on april st , the united nations launched the “shot@life” campaign. in brief, the armamentarium of vaccines continues to grow with more emphasis on safety, availability, and accessibility. this mini review highlights the major historical events and pioneers in the course of development of vaccines, which have eradicated so many life-threatening diseases, despite the vaccination attitudes and waves appearing through history. vaccines constitute one of the greatest success stories within the health sector. they form part of a multifaceted public health response to the emergence of pandemics. this review is general in nature. it highlights the major historical cornerstones in the development and progress of various types of vaccines since the beginning and through the ages until today. it recognizes the major pioneers whose work has made a difference in the advancement of this vital health field, despite all the anti-vaccination movements that appeared through the ages. multiple reviews were encountered during our literature search; however, each of those reviews dealt with a specific aspect of vaccination like effectiveness of a particular vaccine, or side effects of another or even attitudes toward vaccines. consequently, this work tried to put together the major achievements through history stressing the importance, continuous vital role, and the need for immunization for health prevention and protection as well as its impact on human experience. the physiological mechanisms behind vaccination are well established. vaccination activates the immune system and induces both innate and adaptive immune responses thus leading to the production of antibodies, in the case of a humoral response, or to the generation of memory cells that will recognize the same antigen, if there is a later exposure. periodic repeat injections can improve the efficacy and effectiveness of inoculations ( ) . the approval of a vaccine abides by a set of well-established international rules and regulations. prior to their approval by the respective health authorities, scientists test vaccines extensively in order to ensure their efficacy, safety, and effectiveness. next to antibiotics, vaccines are the best defense that we have to date against infectious diseases; however, no vaccine is actually % safe or effective for everyone. this is attributed to the fact that each body reacts to vaccines differently ( ) ( ) ( ) . significant progress has been made over the years to monitor side effects and conduct research relevant to vaccine safety. in addition, vaccine licensing is a lengthy process that may take years or longer. the food and drug administration (fda) and the national institute of health (nih) require that vaccines undergo the required phases of clinical trials on human subjects prior to any use in the general public. this process is becoming more complex as more caution and care is being allocated to the quality of the market product. furthermore, vaccines can be divided into different categories depending on the way that they are prepared including liveattenuated vaccines, inactivated vaccines, subunit vaccines, conjugate vaccines, and toxoids. live-attenuated vaccines are used more frequently for viruses rather than bacteria, since the former contain a lesser amount of genes and can be controlled more easily ( ) . the most common method in formulating live-attenuated vaccines involves passing the virus through successions of cell cultures to weaken it. this will produce a form of the virus that is no longer able to replicate in human cells. however, it will still be recognized by the human immune system, hence protecting the body from future invasions. examples of such vaccines are measles, rubella, mumps, varicella (more commonly known as chickenpox), and influenza. the disadvantage of using this technique is that the virus may transform into a more virulent form due to a certain mutation and cause illness once injected into the body. although this rarely occurs, it must always be taken into consideration ( ) . by using heat, radiation, or certain chemicals, one can inactivate a microbe. the microbe will no longer cause illness but can still be recognized by the immune system. poliovirus and hepatitis a are common examples of inactivated vaccines. this type of vaccine has the disadvantage of being effective for a shorter period of time than live-attenuated vaccines. multiple boosters of the vaccine are sometimes required to improve effectiveness and sustainability ( ) . a subunit vaccine contains only portions of the microbe that can be presented as antigens to the human immune system instead of the microbe as a whole. the antigens or the microbe portions that best activate the immune response are usually selected. an influenza vaccine in the form of shots is an example. in addition, a recombinant subunit vaccine has been made for the hepatitis b virus. hepatitis b genes are injected into maker cells in culture. once these cells reproduce, the desired antigens of the virus are produced as well, and these can be purified for use in vaccines ( ) . conjugate vaccines are designed from parts of the bacterial coat. however, these parts may not produce an effective immune response when presented alone. hence, they are combined with a carrier protein. these carrier proteins are chemically linked to the bacterial coat derivatives. together, they generate a more potent response and can protect the body against future infections. vaccines against pneumococcal bacteria used in children are an example of conjugate vaccines ( ) . some bacteria release harmful toxins that cause illness in infected individuals. vaccinations against such types of bacteria are prepared by inactivating or weakening the toxin using heat or certain chemicals. this will help prepare the immune system against future invasion. the vaccine against tetanus caused by the neurotoxin of clostridium tetani is a good example of a toxoid ( ) . the generation of vaccine-mediated protection is a complex challenge. effective early protection is conferred primarily by the induction of antigen-specific antibodies. the quality of such antibody responses has been identified as a determining factor of efficacy. efficacy requires long-term protection, namely, the persistence of vaccine antibodies and/or the generation of immune memory cells capable of rapid and effective reactivation upon subsequent microbial exposure ( ) . the exponential development of new vaccines raises many questions about their impact on the immune system. such questions related to immunological safety of vaccines as well as triggering conditions such as allergy, autoimmunity, or even premature death ( ) . such issues were always looked for and monitored and some vaccines were even stopped because of these issues. recent vaccine models rely on both a cell-mediated response and a humoral immune response with highly specific antibodies and have shown an adequate amount of success. this, however, has not been the case for a few diseases such as tuberculosis where the humoral immunity mounted by the bacille calmette-guerin (bcg), the only currently used human vaccine, is inefficient in conferring proper immunization ( ) . however, t cells do take part indirectly in the production of antibodies and of secreted biological molecules (e.g., interferon) for protection. it seems that a proper mounted immunity is better achieved by vaccineinduced antibodies, whereas a t cell immune response is needed for disease attenuation. hence, a robust understanding of b and t cell function is needed for proper immunization ( ) . multiple determinants modulate the primary vaccine antibody response in healthy individuals; they include the vaccine type, live versus inactivated, protein versus polysaccharide, and use of adjuvants ( ) . they also include the nature of the antigen and its intrinsic immunogenicity ( ) , the dose of the antigen, the route of administration, the vaccine schedule, and the age at administration ( ) . in addition, genes play a direct role in the body's response to vaccination even in healthy individuals ( , ) . for each of the above determinants, there might be a particular mechanism involved and is further influenced by other factors including extremes of life, acute or chronic diseases, immunosuppression, and nutrition status ( ) . early life immune responses are limited by ( ) limited magnitude of antibody responses to polysaccharides and proteins, ( ) short persistence of antibody responses to protein, ( ) influence of maternal antibodies, and ( ) limited cd + t cell and interferongamma responses. such factors are difficult to study in human infants due to neonatal immune immaturity and the inhibitory influence of maternal antibodies, which increase with gestational age and wane a few months post-natal ( ) . on the other hand, in elderly persons, the immune system undergoes characteristic changes, termed immunosenescence, which leads to increased incidence and severity of infectious diseases and to insufficient protection following vaccination ( ) . vaccines induce both innate (non-specific) and adaptive (specific) immune responses, which decline substantially with age thus leading to the decreased efficacy of vaccines in elderly persons. in the elderly, the innate immune response will witness a reduced phagocytic capacity of neutrophils and macrophages, a decrease in their oxidative burst, and impairment in the up-regulation of mhc class ii expression among other parameters ( ) . in addition, persistent inflammatory processes occur with increasing age and may reduce the capacity to recognize stimuli induced by pathogens or vaccines. for the elderly, improved special antigen delivery systems are needed to overcome these limitations ( ) . furthermore, the adaptive immune response is functionally defective in the elderly. the involution of the thymus with aging leads to a decrease in content and in output of mature naïve t cells into the periphery, which hampers the induction of adaptive immune responses to neoantigens. in the context of primary vaccination, this causes reduced response rate ( ) ( ) ( ) ( ) ( ) ( ) . b cells also undergo age-related changes that aggravate the functionality of b cells colonies. as effector b cells accumulate, naïve b cells decrease in number and this leads to a reduction in the diversity of antibody responses. in brief, vaccines tailored to the needs of the elderly will have to be developed, taking into consideration these limitations in order to improve protection in this population. in , weinberg and szilagyl eloquently approached the issues of efficacy and effectiveness clarifying the road to correctly answer the relevant but complex question: "how well does the candidate vaccine prevent the disease for which it was developed?" they highlighted clearly the distinction between efficacy (individual level) and effectiveness (population level), which are often confused terms that fit well into the new paradigm of translational research ( ) . at about the same time, curns et al. elaborated on the distinction between the epidemiologic concepts of vaccine efficacy and effectiveness within the context of translational research ( ) . such concepts were also addressed earlier, but slightly differently, by clemens and co-workers in two separate publications in and , and also by orenstein et al. in ( - ) . accordingly, vaccine efficacy is measured as the proportionate reduction in disease attack rate when comparing vaccinated and unvaccinated populations. vaccine efficacy studies always have rigorous control for biases through randomized prospective studies and vigilant monitoring for attack rates ( ) . in addition to proportionate reduction in attack rates, these studies can furthermore assess outcomes through hospitalization rates, medical visits, and costs. despite the complexity and expenses that arise from the initial trials, they are needed to establish vaccine efficacy ( ) . on the other hand, the related but distinct concept of vaccine effectiveness has always been compared to a "real world" view of how a vaccine reduces disease in a population. as such, it can evaluate risks versus benefits behind a vaccination program under more natural field conditions rather than in a controlled clinical trial. vaccination program efficiency is proportional to vaccine potency or efficacy in addition to the degree and success of immunization of the target groups in the population. in brief, it is influenced by other non-vaccine-related factors that could influence the outcome. the "real world" picture provided by vaccine effectiveness data is desirable in planning public health initiatives, an advantage that makes these studies attractive. translating research data into real public health application are a process that has been reengineered by the nih as part of a road map for future research. consequently, a new expanded definition of translational research, consisting of four steps was proposed, which fits nicely within the continuum of vaccine research ( ) . in this new process of phase i to phase iv clinical trials, safety, immunogenicity, efficacy, and post-licensure effectiveness of a particular vaccine are assessed ending up in phase iv with the burden of the disease ( ) . vaccines stood the test of time and many techniques have been introduced into the world of vaccination. practitioners used to write articles about their vaccinating instruments and techniques. according to john kirkup, vaccinators and physicians used various instruments and techniques to inject the vaccinating material into the human body. more than different vaccinating instruments have been recorded in british, american, german, and french catalogs between the years and ; most of them are out of use nowadays ( ) . there are multiple major landmarks in the history of vaccines. it was reported that the origin goes as far back as hippocrates, the father of modern medicine, b.c. he described mumps, diphtheria, and epidemic jaundice among other conditions ( ) . the earliest methods of immunization and protection against smallpox date back to about a.d., and are attributed to the chinese. it has been said that the son of a chinese statesman was inoculated against smallpox by blowing powdered smallpox sores into his nostrils ( ) . another method used for inoculation was the removal of fluid from the pustules of an infected individual and subsequently rubbing it into a skin scratch of a healthy individual. this procedure was later introduced into turkey around , long before reaching europe ( ) . it took six centuries for variolation to be introduced to great britain, in ( ) . the eighteenth century was marked by several major events that started with the spread of variolation from turkey and china to england and america, followed, in the late eighteenth century, by edward jenner's breakthrough of vaccination. variolation, derived from the latin word varus, meaning "mark on the skin, " or inoculation, derived from the latin word inoculare, meaning "to graft, " are two words that were used interchangeably in describing the aforementioned immunization process. by , variolation was introduced to england after the pursuit of an english aristocrat, lady mary wortley montague, who had been personally inflicted with an episode of smallpox. after being informed of the method of variolation, she made the embassy surgeon, charles maitland, perform the procedure on her year-old son in in turkey. in , dr. charles maitland performed the first english variolation on lady montague's year-old daughter after their return to london ( ) . lady montague became a great proponent of the procedure and worked thoroughly on advocating this process for its ability to protect against the spread of smallpox. data from the u.s. national library of medicine and the nih showed that - % of those variolated died as compared to % of those who contracted the disease naturally. correspondingly, rev. cotton mather and dr. zabdiel boylston introduced variolation in america and were also great advocates of this procedure especially since, in the same year, there was a smallpox epidemic in boston that killed hundreds ( ) . however, lady montague, rev. mather, and dr. boylston faced great opposition regarding their promotion of variolation even with the presentation of the comparative analysis of fatality rates, which reached % for those variolated compared to % for the naturally occurring disease ( ) . despite some variolation-related deaths, the word of inoculation kept spreading along with data suggesting that variolation was still the safeguard against the spread of smallpox. in addition, benjamin franklin, who lost his son in , wrote: "i long regretted that i had not given it to him by inoculation, which i mention for the sake of parents who omit that operation on the supposition that they should never forgive themselves if a child died under it; my example showing that the regret may be the same either way, and that therefore the safer should be chosen" ( ) . in , dr. william heberden, at his own expense and with the support of benjamin franklin, wrote a pamphlet entitled "some account of the success of inoculation for the small-pox in england and america: together with plain instructions by which any person may be enabled to perform the operation and conduct the patient through the distemper" ( ) . toward the late eighteenth century came jenner's breakthrough in finding a safer immunizing technique than variolation, which is vaccination. the method of variolation had low yet significant death rates; therefore, physicians were on the quest of finding a new and more secure method of immunization with minimal or no death rates. on this basis, an english physician named edward jenner ( - ) searched for a cure for smallpox, a debilitating disease that rendered the world helpless. jenner became interested in certain individuals who were immune to smallpox because they had contracted cowpox in the past. he personally witnessed this when he learned of a dairymaid that was immune to smallpox due to her previous infection with the cowpox virus, usually transmitted from infected cattle. during that time, an english farmer named benjamin jesty personally took charge of inoculating his wife and children with fresh matter from a cowpox lesion in one of his cows out of fear of having his wife and children become victims of the smallpox epidemic. he applied this method after having contracted cowpox himself and believing he was immune to smallpox. he never published his results even though his wife and children did not show symptoms after being exposed to smallpox ( ) . during these years, there were still outbreaks of smallpox. george washington, after surviving smallpox, ordered mandatory inoculation for his troops in ( ) . after many speculations on the role of cowpox and its immunizing effect against smallpox, jenner, in , inoculated an -year-old boy named james phipps using matter from a fresh cowpox lesion on the hands of a dairymaid named sarah nelms who caught them from her infected cattle. after several days, jenner inoculated the boy again but this time with fresh matter from a smallpox lesion and noted that the boy did not acquire the disease proving that he was completely protected ( ) . a few years later, word of his success circulated among the public, and jenner wrote "an inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western counties of england, particularly gloucestershire and known by the name of cowpox, " after adding several cases to his initial achievement with the boy phipps. at first, his publication and achievement did not stir any interest in his community, but with time, word of jenner's breakthrough began spreading ( ) . the late eighteenth century was characterized by the implementation of the new process of immunization, vaccination, which required the inoculation of fresh matter from cowpox lesions into the skin of healthy individuals. the nineteenth century was a major landmark in the history of vaccines since it witnessed discoveries made by louis pasteur, the father of microbiology, and robert koch, the scientist who discovered the germ responsible for tuberculosis ( ) . in the beginning of the nineteenth century, the term "vaccination" was introduced by richard dunning from the latin word for cow "vacca. " after becoming aware of the fact that vaccination was more secure than variolation, several physicians initiated movements against the use of variolation and advocated for its eradication. dr. jean de carro, for example, aided in the elimination of variolation and its substitution with vaccination. some of the major efforts implemented in america were initiated by dr. benjamin waterhouse, who received the vaccine from edward jenner and vaccinated his own family. he later proved that they acquired immunity when they remained asymptomatic after he infected them with smallpox. waterhouse worked effectively on making vaccination universal in the u.s. unfortunately, like any other medical breakthrough, problems arose both because waterhouse aimed at making profit and the public was not ready to implement these procedures. however, after breaking his initial monopoly, waterhouse accepted to share his vaccines and made the supplies available to other physicians ( ) . despite all these efforts, smallpox epidemics continued to occur and jenner stated in a pamphlet that he wrote, "the annihilation of the small pox, the most dreadful scourge of the human species, must be the final result of this practice. " eradication was finally achieved years later. the time it took could be attributed to the fact that jenner did not think of the necessity of revaccination nor of the instability of vaccines, which made them unable to handle different environmental conditions, including countries other than england ( ) . the late nineteenth century was distinguished by pasteur's achievements that made him the father of vaccines after creating the first laboratory vaccine. louis pasteur ( - ), a french chemist and microbiologist, was the first to propose the "germ theory" of disease in addition to discovering the foundations of vaccination ( ) . he studied chicken cholera and received strains of bacteria causing anthrax and septic vibrio. pasteur started his experiments by intentionally infecting chickens by feeding them cholera-polluted meals and then recording the fatal progression of the illness. at first, pasteur was using fresh cultures of the bacteria to inoculate the chickens, most of which did not survive. during that time, pasteur had to go on a holiday, so he placed his assistant in charge of injecting the chickens with fresh cultures. however, his assistant accidentally forgot to perform the injections, and the bacterial cultures were left in a medium that was exposed to room air for about a month. later, the attendant injected the chickens with the now "attenuated" strain of bacteria resulting in mild, nonfatal symptoms. pasteur later re-injected these chickens, but this time with fresh bacteria. to his surprise, they did not get ill. ultimately, pasteur reasoned that what made the bacteria less deadly was exposure to air, mainly oxygen. pasteur used the french verb "vacciner" during the years and to describe how he was able to provide total body immunity through vaccination by inoculation of an attenuated virulence which was the first vaccine made by a human in the laboratory ( ) . pasteur also developed the anthrax vaccine in his laboratory, not long after performing his studies on chicken cholera. in , pasteur used his own anthrax vaccine, which contained attenuated live bacterial cultures in addition to carbolic acid, and demonstrated that all vaccinated animals survived while the control group died ( ) . during the same year, louis pasteur in france and george miller sternberg in the u.s. almost simultaneously and independently isolated and grew the pneumococcus organism. later in , pasteur successfully fought rabies that was endangering the european livestock by using his attenuated rabies vaccine obtained from desiccated brain tissue inactivated with formaldehyde, which provided immunity to dogs against rabies in his experiments ( ) . he reported his success to the academy of sciences in france, and a year later, he applied his original vaccine h after a -year-old boy was bitten several times by a rabid dog. the boy survived after being first inoculated with the most attenuated organisms, then subsequently with less attenuated organisms each day for days ( ) . in , the pasteur institute was established as a rabies treatment center as well as an infectious diseases research and training institute. after pasteur's successful live vaccines, a new type of vaccine was introduced in the last few years of the nineteenth century. these were killed vaccines, which were directed against three chief bacterial causes of human morbidity: cholera, typhoid, and the plague. the first cholera vaccine used to immunize humans was actually a live vaccine developed by jaime ferran ( - ), which provided a high level of protection during the epidemic in spain. however, the first killed vaccine for cholera was developed in by wilhelm kolle ( - ) and was used in japan in with over % efficiency. the credit for developing the killed typhoid vaccine during the s goes to both richard pfeiffer and almroth wright who made great contributions. wright was later credited for carrying out the "first large-scale vaccination using a killed typhoid vaccine" ( ) . finally, the killed vaccine for plague was first developed in by haffkine, who was one of pasteur's followers, when an epidemic struck bombay. during this period, vaccine production was taken over by factorytype laboratories, which formed the precursors of the biological products supply houses. many types were produced. paul ehrlich ( - ), a german physician and scientist who worked under a contractual collaboration with behring, noted the existence of toxoids in the late s. he also promoted enrichment and standardization protocols. these protocols enabled the exact determination of quality of the diphtheria antitoxins. in , it was demonstrated that toxoids could be used to durably immunize guinea pigs. it is crucial to briefly address the historical background of the bacterial infections that led to some of the earliest and most successful use of toxoids, inactivated forms of bacterial toxins, for the purpose of immunization. until the twentieth century, diphtheria, tetanus, and pertussis proved to be significant causes of illness and death with no effective treatments or prevention in sight. fortunately, advances in improved the prognosis of numerous future patients ( ) . at the end of the nineteenth century, especially in and , the cholera and typhoid vaccines were developed, followed by the introduction of the plague vaccine. the latter was preceded by the preparation of anti plague horse serum at the pasteur institute by alexandre yersin. yersin demonstrated disease protection in animals. later, he went to china to try his vaccine on humans during a plague epidemic ( ) . diphtheria is a potentially fatal disease that primarily involves tissues of the upper respiratory tract and kills its victims slowly by suffocation. in , a german physician, edwin klebs ( - ), was able to successfully isolate the bacteria that proved to be the etiological agent of the disease. it was later proved that toxin production is initiated only after the bacteria are themselves infected by a specific virus or a bacteriophage carrying the toxin's genetic instructions ( ) . in france, during the year , emile roux discovered the diphtheria toxin. his discovery led to the development of passive serum therapies through the scientific contributions of many, including emil von behring and paul ehrlich ( ) . similarly, the etiological agent of pertussis, commonly known as the "whooping cough, " was found to be a bacterium isolated from infected patient tissues in ( ) . tetanus was similarly a significant cause of mortality usually resulting from dysfunction of the autonomic nervous system or the respiratory muscles ( ) . in , another german scientist, arthur nicolaier ( - ), correlated tetanus with an anaerobic soil bacterium found in wounds. a few years later, the japanese investigator shibasaburo kitasato ( - ) was able to isolate this bacterium ( ) . at the beginning of world war i in , the tetanus toxoid was introduced following the development of an effective therapeutic serum against tetanus by emil von behring and shibasaburo kitasato. the rabies and typhoid vaccines were then licensed in the u.s. as the etiology of these destructive diseases was slowly being uncovered, by shibasaburo kitasato along with emil von behring ( ) . they discovered that the serum of animals that had been exposed to sub-lethal doses of the bacteria involved in tetanus and diphtheria was protective against the lethal effects associated with these pathogens by having an antitoxin effect when injected into another animal. additionally, this discovery, which earned behring the inaugural nobel prize for physiology and medicine in , was the concept of passive transfer in addition to serum therapy. he proved that serum could be acquired from immune animals and transferred to others as protection ( ) . once this concept made its way to clinical practice in , technical problems were faced while developing the right antitoxin concentration and potency. as a result, in the early twentieth century, the u.s. congress enacted the biologics control act legislation "to regulate the sale of viruses, serums, toxins, and similar products" to ensure medication quality control. nevertheless, with the increasing use and popularity of antitoxins derived from animal serum, scientists began to observe a syndrome now called serum sickness, or a reaction to immune-complexes formed from combining high concentrations of antigens with antibodies. this eventually led to the use of human rather than animal serum in order to decrease the frequency of adverse events; still, serum therapy was not perfect in preventing disease due to the frequency of adverse events and its brief duration of action. later on, combining diphtheria toxin and antitoxin in the same syringe proved much more effective in decreasing mortality rate. this combination became commercially available in . this was the first step in the shift from passive to active immunization ( ) . in , gaston ramon ( - ), a french veterinarian working at the pasteur institute, used a diphtheria toxoid produced by formalin and heat inactivation without the use of antitoxin to safely induce active immunity in humans. this product, termed anatoxine, was the basis for the novel and clinically effective toxoid vaccine against diphtheria. experiments followed to improve the durability of the protective response of the vaccine, and in , the importance of aluminum salts as an adjuvant added to the vaccine to augment the immune response to the antigen, became apparent ( ) . this was discovered by alexander thomas glenny ( - ) who proved that toxoid alone produced a lower level of antibody and immunity than desired, whereas better immunity was achieved when an inflammatory reaction was triggered. with these significant improvements, tetanus and diphtheria toxoids became routinely used across america and europe in the s and s ( ) . since then, refinements have been made to these vaccines to yield higher purity and reduce the number of booster doses. nowadays, widespread childhood vaccination is reducing the burden of these diseases. while this is a huge advantage, vaccines may potentially produce adverse effects that can discourage their acceptance by some populations. this has led to numerous safety movements which culminated in the congressionally legislated national childhood vaccine injury act in the s created to compensate families for selected adverse events potentially related to mandatory childhood vaccinations ( ) . nevertheless, global recommendations continue to call for routine immunization of children against diphtheria, tetanus, and pertussis with the combined dtp vaccine to sustain immunity in childhood and adolescence. dtp has, therefore, become one of the most widely used vaccines to achieve widespread immunity across age groups ( ) . tuberculosis, otherwise known as the "great white plague, " is another disease that started spreading as an epidemic once industrialization began. this disease caused approximately % of deaths in the eighteenth and nineteenth centuries across all socioeconomic groups ( ) . a french physician named jean antoine villemin ( - ) demonstrated that the mode of transmission of disease is through the respiratory system. robert koch ( - ) , known as the founder of modern bacteriology, revealed in that the causative agent of the disease is mycobacterium tuberculosis, which later became known as koch's bacillus ( ) . following this discovery, koch created what later came to be known as koch's postulates, which listed the criteria necessary for proof of bacterial causality: "the organism must be present in diseased tissues; it must be isolated and grown in pure culture; and the cultured organisms must induce the disease when inoculated into healthy experimental animals" ( ) . in , two bacteriologists working in the pasteur institute in lille, albert calmette ( - ) and camile guerin ( - ), announced their discovery of mycobacterium bovis, which is a strain of tubercle bacilli that could be used to create a vaccine against tuberculosis. this occurred after it became evident that different forms of the bacterium were required to prevent or treat tuberculosis, including non-pathogenic, attenuated, or killed tubercle bacilli from different sources, including human, bovine, and equine. this strain had an attenuated virulence while maintaining its antigenicity and became known as bcg ( ) . bacille calmette-guerin vaccinations proved to be successful in animal studies in and were soon used as an oral vaccine to immunize humans against tuberculosis. in , the bcg vaccine, constituted by the live-attenuated m. bovis, was first used in newborns. it has become the most widely administered of all vaccines in the who expanded program for immunization, but has been estimated to prevent only % of all potentially vaccinepreventable deaths due to tuberculosis ( ) . despite its imperfections, bcg remains the only effective vaccination for protection against human tuberculosis ( ) . yellow fever is a highly fatal infection caused by a small, enveloped, single-stranded rna virus and results in renal, hepatic, and myocardial injury, along with hemorrhage and shock ( ) . unlike previously mentioned diseases, the history of yellow fever is highly uncertain and filled with misconceptions. early work on immunization against the disease began with carlos finlay in the s and s when koch's postulates were becoming increasingly accepted. finlay proposed that mosquitos carried the yellow fever "germ. " he attempted to prove it by feeding mosquitos that had fed on yellow fever patients. however, it was later revealed that his process failed due to the lack of an incubation period within the mosquito, which is a transmission requirement that finlay was unaware of ( ). since , significant advances have been made in creating a vaccine by the yellow fever commission, which was originally led by walter reed ( - ) along with jesse lazear, aristedes agramonte, and james carroll. reed's experiments took finlay's discovery one step further by adding an incubation period of approximately weeks and achieved the same positive results. when mosquitos bite non-immune individuals after feeding on individuals who had yellow fever, none of the non-immune subjects died and very few suffered disease. this led the commission of investigators to a major discovery, namely, the identification of the asibi strain, which is the parent strain of the present d vaccine, obtained via continuous indirect passage through the aegypti mosquitos and direct passage through monkeys. in addition to identifying the etiological agent of the disease, the commission also identified rhesus monkeys as susceptible hosts, hence providing a means for testing future vaccine attempts. this paved the way for max theiler and other rockefeller foundation scientists to develop a successful live-attenuated vaccine for yellow fever in . "the most important experimental passage seriesdesignated d -used a virus that had been subcultured eighteen times in whole mouse embryos, followed by passages in wholeminced chick embryo cultures, after which the virus was passed in minced chick embryo depleted of nervous tissue. " theiler himself was actually one of the first individuals to be successfully vaccinated. the vaccine was quickly implemented, and alternative vaccines shown to be more dangerous were discontinued ( ) . influenza has proved to be very difficult to trace back in history due to its non-specific symptoms and features. it was not until the early twentieth century that influenza outbreaks began to be systematically studied due to well-documented clinical descriptions and epidemiological data. in , the "spanish flu" influenza pandemic was responsible for - million deaths worldwide and more than one-half million in the u.s. this virus was unusual because it spread so quickly, was so deadly ( ) . richard e. shope ( - ) , a physician who conducted his research in the department of animal pathology at the rockefeller institute in princeton, was the first to isolate influenza virus; a member of the orthomyxovirus family, from a mammalian host in ( ) . he was able to induce the syndrome of swine influenza in pigs by applying respiratory secretions intranasally. he also isolated a bacterium from the respiratory tract of infected pigs called haemophilus influenzae suis. when this bacterium was combined with a filterable agent and inoculated, the pigs developed the clinical manifestations of swine influenza. these two agents seemed to act synergistically with the virus to damage the respiratory tract hence creating the suitable environment needed for the virus to exercise its pathological effects. in , scientists from the british national institute for medical research including christopher andrews, wilson smith, and patrick laidlaw successfully isolated and transmitted the influenza virus from humans. throughout this year, "burnet has successfully cultivated the organism in chick embryos; other influenza types had been recognized; neutralizing antibodies had been identified and quantitated; and viral surface glycoproteins, h and n had been described" ( ) . these discoveries led scientists to introduce the inactivated vaccine in the mid- s that is still used to this day ( ) . the influenza a/b vaccine was initially presented to the armed forces epidemiological board in . it was licensed following the war and used for civilians in in the u.s. starting , a series of vaccines were licensed for haemophilus influenza type b (hib) polysaccharide vaccines. these vaccines are recommended routinely for children at and months of age. the vaccine was, however, not consistently immunogenic in children < months of age. in , the protein-conjugated hib vaccine was licensed and in the next years, it became available. during , a combined vaccine hib conjugate and hepatitis b was licensed. later on, in , the first nasally administered influenza vaccine was licensed. this live influenza a and b virus vaccine was indicated for healthy, non-pregnant persons ages - years. the contracts to develop vaccine against the h n avian influenza virus were awarded to aventis pasteur and to chiron in . during the following year, an inactivated, injectable influenza vaccine was licensed. it was indicated for adults years of age and older. during the same year, the fda approved afluria, a new inactivated influenza vaccine, for use in people aged years and older. two years later in , the department of health and human services, supported the building of a facility to manufacture cellbased influenza vaccine. it also directed toward development of a vaccine for novel influenza a (h n ). during the same year, the fda approved four vaccines against the h n influenza virus high-dose inactivated influenza vaccine (fluzone high-dose) for people aged years and older. in , the fda approved several vaccines: hibmency a new combination of meningococcal and hib vaccine for infants; flucelvax, which is the first seasonal influenza vaccine, manufactured using cell culture technology and a quadrivalent formulation of fluarix ( ) . unfortunately, one of the difficulties in dealing with influenza is the continuous mutability of the viral genome necessitating annual reassessments and reformulations of the vaccine. this has led to a suboptimal effectiveness of influenza vaccines, which are only successful against strains included in the vaccine formulation or strains of homogenous subtype. several pandemics were caused by the influenza virus: during the years - , the "asian" influenza pandemic caused by h n influenza virus resulted in an estimated , deaths in the u.s. alone and in the years - , the "hong kong" influenza pandemic caused by an h n influenza virus induced roughly , deaths in the u.s. ( ) . future studies should focus on producing vaccines protective against variant strains and creating surveillance systems to detect novel strains in time to formulate the proper vaccines. poliomyelitis, or polio, is an intestinal infection spread between humans through the fecal-oral route. it is a disease of the developed nations striking younger individuals most frequently in warmer weather. one of the most famous polio victims, president franklin d. roosevelt, founded the national foundation for infantile paralysis in , later known as the march of dimes ( ) . it is well established that better hygiene decreases childhood exposure to the disease, when infection would usually be milder since protective maternal antibodies are present ( ) . in , the nobel prize in medicine was awarded to john enders, thomas weller, and fredrick robbins for their discovery of the ability of poliomyelitis viruses to grow in tissue cultures ( ) . two major lifelong competitors were involved in the race for the polio vaccine, jonas salk and albert sabin . salk took a more traditional route using a killed-virus approach, which did not involve natural infection in acquiring immunity. instead, his approach involved a fully inactivated virus that still had the ability to induce protective antibodies. sabin, on the other hand, set out to create a live-virus vaccine based on the belief that this would trigger natural immunity and provide a lasting protection. salk had speed, simplicity, and safety on his side since a killedvirus did not have the ability to revert to virulence, whereas the live-virus vaccine could be given orally, establish longer lasting immunity, and offer passive vaccination through the excreted weakened virus potentially immunizing a large portion of nonvaccinated communities ( ) . not surprisingly, salk's vaccine was the first to make it to the population. following successful clinical trials in , six companies began mass production of the vaccine. unfortunately, salk's vaccines were soon suspended and recalled when contaminated samples were found in the market due to poor monitoring and control in some laboratories leading to serious health consequences and national panic. the first cutter polio vaccine incident was reported on april , with more cases reported just a day later with the number eventually rising to of those vaccinated and in of their close contacts. on april , the laboratory of biologics control requested that cutter laboratories recall all vaccines and the company did so immediately. on may , the surgeon general recommended that all polio vaccinations be suspended pending inspection of each manufacturing facility and thorough review of the procedures for testing vaccine safety. the investigation found that live polio virus had survived in two batches of vaccines produced by cutter laboratories. large-scale polio vaccinations resumed in the fall of ( ) . at the same time, sabin had been making great advances with his live-virus vaccine since . after successful clinical trials conducted in the soviet union that left polio virtually wiped out with no safety issues, it soon became the vaccine of choice in the west. the polio vaccination assistance act was enacted by congress and was the first federal involvement in immunization activities. it allowed congress to appropriate funds to the communicable diseases center [later the centers for disease control and prevention (cdc)] to help states and local communities acquire and administer vaccines. at the beginning of the s, the oral polio vaccine types , , and as well as the trivalent product were licensed in the u.s. the first were developed by sabin and grown in monkey kidney cell culture, while the trivalent oral polio was developed to improve upon the killed salk vaccine ( ) . as a result, in the late s, the cdc recommended switching back to salk's killedvirus polio vaccine, while the who also advocated the switch for polio-free nations and the continued use of the favored live-virus vaccine for routine immunization ( ) . the last two cases of wild type polio were reported in an unvaccinated amish in and in a -year-old boy from peru in ( ) . in , the enhanced-potency inactivated poliovirus vaccine was licensed. following successful developments in the polio vaccine, attention soon shifted to three other common viral diseases of childhood: measles, mumps, and rubella. the measles virus is an rna virus from the genus morbillivirus belonging to the paramyxooviridae family. it causes an acute illness that includes fever, cough, malaise, coryza, and conjunctivitis, in addition to a maculopapular rash. in general, measles is a mild disease but, like many others, has the potential to cause serious complications. in addition, measles is known to be one of the most contagious human diseases causing major outbreaks to occur very often. until the year , measles was still the leading cause of vaccine-preventable childhood deaths worldwide ( ) . john enders ( enders ( - , known as the "father of modern vaccines" had a particular interest in revealing the virus responsible for measles. he isolated the edmonston strain of the virus in , which was named after the child from whom it was isolated. a formalin-inactivated measles virus vaccine derived from this strain was subsequently licensed in the u.s. in . however, following the discontinuation of this vaccine in due to short-lived and incomplete immunity, over further attenuated vaccines were developed and used throughout the world, most of which were also derived from the edmonston strain ( ) . the first live-virus measles vaccine, rubeovax, was licensed in . other live-attenuated virus measles vaccines were eventually licensed in the u.s. in . the recommended age for routine administration was changed from to months of age. the first national measles vaccine campaign was launched in . the world recorded a % decreased incidence compared to the pre-vaccination years. in , a second live, further attenuated measles virus vaccine was also licensed. in , both the advisory committee on immunization practices (acip) and the american academy of pediatrics (aap) issued recommendations for a routine second dose of the measles vaccine. during the midto-late s, a high proportion of reported measles cases were in school-aged children ( - years) who had been appropriately vaccinated. these vaccine failures led to new national recommendations of a second dose of measles-containing vaccine ( ) . mumps is another acute viral illness. it is the only virus known to cause epidemic parotiditis in humans accompanied by fever, anorexia, headache, and malaise. k. habel and john enders isolated the virus in ( ) , and trials of formalin-inactivated mumps vaccine in humans began the same year by joseph stokes and colleagues and by enders. this approach was abandoned in the s due to short-lived immunity, and work began to develop live-attenuated mumps vaccines in by the vaccinologist maurice hilleman ( hilleman ( - and colleagues ( ) . hilleman isolated the wild type virus from his daughter, jeryl lynn, who contracted the virus at the age of and was recovering from it. it became known as the jeryl lynn strain of mumps virus. the mumps livevirus vaccine was licensed in december , ( ) . trials with this attenuated virus resulted in % protective efficacy and the vaccine was licensed in the u.s. in . this strain is still used to produce mumps vaccines until this day. it is given as part of the measles, mumps, and rubella (mmr) vaccine ( ) . rubella is a rash disease in children and adolescents caused by a filterable virus. it poses a severe threat to pregnant women and their children by potentially causing congenital deafness and cataracts. in , a rubella epidemic swept the u.s. resulting in . million cases of rubella infection, with an estimated , newborns having congenital rubella syndrome (crs), along with fetal and neonatal deaths in the thousands ( ) . the rubella virus was detected and isolated by two groups of scientists, thomas weller and franklin neva at harvard medical school, in addition to paul parkman and colleagues at the walter reed army institute of research (wrair). similar to measles and mumps, inactivated whole virus vaccines proved ineffective, so efforts turned to discovering a live-attenuated vaccine ( ) . in , paul parkman left wrair and joined harry meyer jr. at the nih division of biological standards, and the pair developed the first live-attenuated rubella vaccine in , hpv- , which was subsequently included in the initial mmr vaccine used in the u.s. in the s ( ) . maurice hilleman discovered the superior ra / vaccine that became the only vaccine used outside of japan starting in the late s. this vaccine maintained its preference due to many factors including increased durability and harmlessness to fetuses of inadvertently vaccinated pregnant women ( ) . in , three rubella virus strains were licensed in the u.s.: hpv- strain grown in dog-kidney culture, hpv- grown in duck-embryo culture, and cendehill strain grown in rabbit-kidney culture. a decade later, in , the ra / (human diploid fibroblast) strain of rubella vaccine (meruvax ii) by merck was licensed. all other strains were discontinued. merck's combined trivalent mmr as well as the combined measles and rubella vaccine (m-rvax) developed by maurice hilleman and colleagues, was licensed by the u.s. government in ( ) , and is still in use today. moreover, the age for routine vaccination with mmr vaccine was changed from to months in the year of . the next vaccine that combined measles, mumps, rubella, and varicella antigens (proquad) was licensed in . it was indicated for use in children months to years. in response to the association of this vaccine with autism, in , the eighth and final report of the immunization safety review committee was issued by the institute of medicine concluded that the body of epidemiological evidence favors rejection of a causal relationship between the mmr vaccine and autism ( ) . combination vaccines hold many advantages including reduced need for several injections, therefore, reducing the incidence of vaccination site reaction ( ) . the etiological agent of clinical hepatitis, identified by its distinguishing yellow jaundice, was found to be infectious in the early s. the different hepatitis strains, a and b, were first differentiated in ( ) . in the mid- s, blumberg and coworkers and prince discovered hepatitis b surface antigen in the circulating blood of carriers of the infection. deinhardt et al. soon followed this discovery with that of the hepatitis a virus ( ) . provost et al. successfully prepared a killed hepatitis a vaccine in , which proved to be safe and highly effective in extensive clinical trials. the first inactivated hepatitis a vaccine (havrix) was licensed in . the following year, a second inactivated vaccine (vaqta) also became available ( ) . hepatitis b, on the other hand, rarely causes any severe risk as a primary infection. however, those who develop a chronic persistent infection may continue to have severe disease for the rest of their lives. this may even lead to cirrhotic destruction of the liver due to host immune response to the virus. the discovery of the surface antigen particles of the hepatitis b virus by blumberg and colleagues in the plasma of human carriers was followed by attempts to create a vaccine. in , a killed hepatitis b vaccine was developed and clinical trials began in proving the safety and efficacy of the vaccine. merck and pasteur institute subsequently independently licensed the plasma-derived vaccine in ( ). on july rd , the recombinant hepatitis b vaccine (recombivax hb) was licensed. using recombinant dna technology, merck scientists developed a hepatitis b surface antigen subunit vaccine. three years later, on august th , the recombinant hepatitis b vaccine (engerix-b) was licensed. a decade later in , the fda approved a two-dose schedule of hepatitis b vaccination for adolescents - years of age using recombivax hb (by merck) with the -μg (adult) dose at and - months later. at the beginning of the new millennium, in , a combined hepatitis a inactivated and hepatitis b (recombinant) vaccine, twinrix was licensed. the following year, a vaccine combining diphtheria, tetanus, acellular pertussis, inactivated polio, and hepatitis b antigens (pediarix) was licensed ( ) . in conclusion, fortunately, both hepatitis a and b are now preventable due to the discovery of these highly effective vaccines that proved to maintain long-term immunity in vaccinated individuals ( ) . in , the world health assembly called for global smallpox eradication, which was launched the following year. during the first year of the program , cases of polio were reported in countries that were endemic to smallpox. four years later, the cdc recommended discontinuation of routine vaccination for smallpox in the u.s. following a greatly reduced risk of disease ( ) . during the s, especially in , the expanded program on immunization was created within who, in response to poor immunization levels in developing countries (< % of children in ). the following vaccines were used by the expanded program on immunization: bcg, polio, dtp, measles (often mmr vaccine), yellow fever (in endemic countries), and hepatitis b. three years later, in october , the last case of naturally acquired smallpox occurred in the merca district of somalia. in the same year, the first pneumococcal vaccine was licensed, containing serotypes (of the known serological groups) that composed % of all bacteremic pneumococcal infections in the u.s. ( ) . on may , the world health assembly declared the world free of naturally occurring smallpox. on the other hand, in july , two enhanced pneumococcal polysaccharide vaccines (pneumovax and pnu-imune ) were certified. these vaccines included purified capsular polysaccharide antigens of streptococcus pneumoniae and replaced the -valent polysaccharide vaccine licensed in . a few years later, in , the world health assembly passed a resolution to eradicate polio by the year ( ) . later on, in , the japanese encephalitis (je) inactivated virus vaccine (je-vax) was licensed. during the year , the expanded program for vaccine development and the vaccine supply and quality program were merged creating the global program for vaccines and immunization. during the same year, the western hemisphere was finally labeled as "polio-free" by the international commission for the certification of polio-eradication. the was another monumental year with the launching of the international aids vaccine initiative (iavi) that called for the speedy development of a human immunodeficiency virus (hiv) vaccine for use worldwide. this in turn led to the introduction of the scientific blueprint for aids vaccine development. iavi was funded by several ngos and foundations. it is a collaborating center of the joint united nations program on hiv/aids (unaids) whose efforts led finally lead to the first possible vaccine against hiv (aidsvax) which reached phase iii trials, the largest recorded human hiv vaccine trial at that time. the trial involved volunteers from the u.s., canada, and the netherlands, the majority of whom were men who have sex with men ( ) . preliminary results from the trial of aids vax (vaxgen) vaccine were reported in early . while the vaccine was shown to be protective amongst non-caucasian populations, especially african-americans, the same effect was not reproducible in caucasians ( ) . during the same year, the children's vaccine program was established at who's program for appropriate technology in health (path). the program's goal was to provide vaccines to children in the developing world and to accelerate research and development of new vaccines. the first vaccines purchased were hib, hepatitis b, rotavirus, and pneumococcal, which were not commonly used in the developing world ( ) . at the beginning of the new millennium, the western pacific region of the world was certified as polio-free. during the next years, the european region also became certified as polio-free. in , the fda licensed the first vaccine developed to prevent cervical cancer (gardesil), precancerous genital lesions and genital warts due to human papillomavirus (hpv) types , , , and . the first smallpox vaccine for certain immune-compromised populations was delivered under project bioshield on july th . the following year , the who declared the "decade of vaccines" and in , the united nations foundation launched shot@life campaign ( ) . varicella ("chickenpox") is caused by the varicella zoster virus (vzv). michiaki takahashi, professor of virology at the research institute for microbial diseases at osaka university, successfully produced the oka vaccine strain of live, attenuated varicella vaccine in the s. takahashi was able to make this remarkable advance at a time when very few viruses had been attenuated to produce efficacious live-virus vaccines including yellow fever, polio, measles, mumps, and rubella as previously mentioned. the vzv vaccine is the first and only licensed live, attenuated herpesvirus vaccine in the world. numerous trials in the early s continued to prove the safety and efficacy of the vaccine in both healthy and immunocompromised, high-risk individuals. as a result of these successful trials, the live varicella virus vaccine (varivax) was licensed in for the active immunization of persons months of age and older ( ) . about years later, in , varizig, a new immune globulin product for post-exposure prophylaxis of varicella, became available under an investigational new drug application expanded access protocol ( ) . as a herpesvirus, vzv possesses the unique ability to establish latent infection subsequent to primary infection. zoster results from reactivation of latent vzv that spreads through nerves to the skin. therefore, one fear associated with this vaccination was the possibility that it could increase the incidence and/or severity of zoster when compared to natural disease. conversely, it was actually shown that following vaccination, zoster is less common than after natural infection ( ) . in , the fda licensed a new vaccine to reduce the risk of shingles in the elderly. the vaccine, zostavax was approved for use in people aged years of age and older ( ) . rotavirus is the leading cause of severe diarrhea and vomiting (severe acute gastroenteritis) among young infants and children worldwide. no significant difference was found in the incidence of rotavirus in industrialized and developing countries, suggesting that vaccination may be the only way to control the impact of this severe disease. dr. ruth bishop and colleagues were the first to describe rotavirus in humans in . it was clear, early on, that a naturally acquired first infection, whether symptomatic or asymptomatic, was the most effective defense against severe reinfection, and subsequent infections progressively created greater protection. therefore, the goal was to create a vaccination that mimicked the effectiveness of naturally acquired immunity following infection. the development of live, attenuated, oral, safe, and effective rotavirus vaccines was then attempted starting in the mid- s. dr. albert kapikian and colleagues, at the nih, developed the rrv strain that was subsequently used to develop the rrv-tv, or the rotashield, live oral, and tetravalent vaccine licensed in to be used in infants at , , and months of age ( ) . however, due to several reported cases of vaccine-associated intestinal intussusception, rotashield was pulled off the market in the u.s. months after its introduction on the th of october, . in , the national institute of allergy and infectious diseases (niaid), part of the nih, awarded a new license agreement for rotashield to biovirx, inc. of minneapolis, mn, usa, which planned its global commercialization. in , history of intussusception was added as a contraindication for rotavirus vaccination ( ) . clark, offit, and plotkin then produced the rotateq vaccine by merck based on their bovine strain wc in , which was licensed in by the u.s. fda. this vaccine, live oral and pentavalent, is destined for use in infants ages - weeks ( ) . another vaccine, rotarix, was also licensed in . it is a liquid given in a two-dose series to infants from to weeks of age. before being licensed, both vaccines were shown to be safe and effective in rigorous clinical trials ( ) . during the past two decades, improvements in environmental health have contributed tremendously to disease vector control. however, substantial challenges remain in dealing with the newly emerging diseases such as severe acute respiratory syndrome (sars), h n , h n , and h n influenza, middle east respiratory syndrome (mers-cov), rotavirus, ebola virus, and a variety of other viral, bacterial, and protozoal diseases ( ) . the role of vaccines in the control and protection from the above mentioned emerging diseases cannot be overemphasized. actually, the importance of inducing protective immunity through vaccination came out to be the most powerful tool and effective strategy to prevent the spread of emerging viruses among populations, in particular, among people that are immunologically naïve and susceptible hosts. such emerging diseases represent a major public health concern; they affect livestock and humans thus threatening the world's economy and public health. vaccine strategies for emerging pathologies are limited by sudden appearance of the pathogen and the delayed time consuming traditional vaccine development process. novel methods to rapidly develop vaccine are being experimented, whereby investigators are working to achieve a better understanding of the nature of the interactions between the immune system and a panel of novel harmful microbes. on this basis several novel strategies have been developed and applied. such strategies included the use of ( ) recombinant proteins, or nanoparticles like in sars-cov and mers-cov, ( ) synthetic peptides like the case of influenza virus, in managing or even in preventing the emergence of new infectious diseases, a plan should be developed to strengthen surveillance and promote a multi-partners response within local, national, and global programs. with the high burden of emerging infectious diseases (eid) it becomes an essential part to find an effective method of either preventing or controlling their spread, that is where the role of vaccines prevails. it is significant to mention that the average case fatality rate for ebola is around % and outbreaks are affecting both developed and developing countries. another emerging disease, mers-cov, has caused the death of around % of people reported to have contracted the disease. another disease with high health and economic burden would be rotavirus which was estimated to have annual direct and indirect costs of around $ billion with "more than , physician visits, more than , emergency department (ed) visits, , to , hospitalizations, and to deaths each year in children younger than years" (cdc, ) . these are few of the facts regarding the affliction of eid most of which have no approved vaccine yet. on the other hand, the influenza virus which was estimated to cause an average of , deaths annually with a $ billion cost of an epidemic, showed that with the introduction of its vaccine, studies proved it to be % effective in preventing death ( ) . these figures have managed to influence many governmental and non-profit organizations to intervene either through governmental funding of vaccines where the congress provides yearly international eid funding to several u.s. governmental agencies or through international non-profit organizations which are the leaders in global health innovation ( ). vaccines remain among the most reliable and effective medical interventions in providing the means to fight debilitating and preventable diseases thus ensuring the continuity of mankind and saving lives. through reviewing the factsheets provided by the world health organization, which provide statistical data on the mortality and morbidity percentages before and after the introduction of the vaccines, one can comprehend the vital role vaccines have played up till this day. some of the figures that depict the impact of vaccines in decreasing mortality and morbidity include more than % decrease in polio cases since , with cases reaching , from over endemic countries down to cases as reported in with only endemic countries, measles vaccine has prevented the death of around . million children during - , in general vaccines prevent around million deaths annually worldwide ( ) . this success in providing better public health does not negate the economic burden of vaccination. vaccination programs require excessive funding to ensure proper handling and maintenance of vaccines, adequate staffing and ongoing provision over efficacy and safety of vaccines and the development of newer vaccines ( ) . nevertheless, the economic and social burden related to the expenses in hospitalizing affected unvaccinated people still outweighs the aforementioned burden. moreover, better health in the society would promote economic growth and productivity. consequently, public awareness and public efforts agree on the importance of vaccination and the implementation of policies regarding mandatory vaccinations as a way to decrease outbreaks of preventable diseases and improve global health and prosperity. as early as the introduction of vaccines, campaigns against vaccination were raging. as with any new medical intervention there are safety concerns that arise which might be deleterious to the public health. concerns regarding vaccines often follow a path that starts with the hypothesis of a potential adverse event that is impulsively announced to the public without having reproducible studies to confirm this hypothesis, and thus it would take the public several years to regain trust in the vaccine. a notable example in the recent history would be of the paper published by andrew wakefield in the british medical journal the lancet in , linking the mmr vaccine to autism. however, his research was discredited and the paper was retracted from the journal after it was proven that actually there is no link between mmr vaccine and autism as per the systemic review by the cochrane library ( ) . the battle against vaccines did not reach a halt, and there are still ongoing campaigns that come from religious, political, community-based, and even individual-based grounds raising even ethical issues regarding the mandatory vaccinations proposed by the government. according to the cdc, this year % of the children were vaccinated in the u.s., leaving % unvaccinated due to religious and philosophical exemptions or even parental refusal due to the fear of vaccine's side effects and concerns regarding autism from vaccines ( ); this is still a critical number since the unvaccinated would pose risk of outbreaks even among the immunized, which necessitates the need for additional awareness campaigns regarding the importance of vaccination since vaccines remain the only plausible measure of protection against preventable diseases. actually, a trend was reported in the health news lately, in the u.s., that pediatricians refuse to offer medical care for children whose parents declined their vaccination. vaccination has been of great importance throughout centuries (tables and ). people started with inoculation techniques dating back to a.d. with the chinese, turks, and asians. with every century and with every curious physician, inoculation techniques improved gradually giving rise to newer vaccination techniques with edward jenner and later on, louis pasteur and others. however, there is still plenty of room for improvement with the presence of ongoing epidemics and the spread of newly emerging diseases. one important goal is to strengthen the science base for vaccine development and for public health action and disease prevention. despite the common belief that infectious diseases were virtually eliminated by the middle of the twentieth century, new and reemerging infections are appearing along with drug resistant infections in the past two decades in the various parts of the world and whose incidence threatens to increase in the near future, due to changes in human demographics and behavior, immigration, and speed of international travel among other things ( ) ( ) ( ) . the importance of vaccine safety continued to grow throughout the twenty-first century, with the development and licensure of new vaccines added to the already robust immunization armamentarium. scientists also perfected new ways of administering immunizations including edible vaccines and needleless injections. however, formulated or delivered, vaccines will remain the most effective tool we possess for preventing disease and improving public health in the future. despite the antivaccination campaign and the association of vaccines with some side effects, vaccines continue to remain a cornerstone in global health. the distinctions between national and international health problems are losing ground and could be misleading, the "world is a village. " clinicians and public health workers need to interact on regular basis with veterinarians and veterinary public health. actually, good examples of the necessity of such collaboration is the emergence of sars-cov and mers-cov, it shows clearly how coronaviruses can spillover from animals into humans at any time, causing lethal diseases. foodborne diseases could lead to regional and international outbreaks which might constitute a threat to national and global security. centers for disease control and prevention. vaccines and immunizations: the basics epidemiology of vpds, vaccine safety the complicated task of monitoring vaccine safety vaccine safety: current and future challenges niaid. topics: vaccines the college of physicians of philadelphia. articles -different types of vaccines general aspects of vaccination the immune response to bcg vaccination of newborns the methodology for determining the efficacy of antibodymediated immunity aluminum hydroxide adjuvant induces macrophage differentiation towards a specialized antigen presenting cell type twin studies of immunogenicity-determining the genetic contribution to vaccine failure biology of immune responses to vaccines in elderly persons the influence of genetic factors on the immune response as judged by pneumococcal vaccination of mono-and dizygotic caucasian twins genetic regulation of immune responses to vaccines in early life vaccine epidemiology: efficacy, effectiveness, and the translational research roadmap the aging innate immune system reduction in acute gastroenteritis hospitalizations among us children after introduction of rotavirus vaccine: analysis of hospital discharge data from us states resolving the pneumococcal vaccine controversy: are there alternatives to randomized clinical trials? evaluating new vaccines for developing countries. efficacy or effectiveness? assessing vaccine efficacy in the field. further observations translational research and pediatrics the evolution of surgical instruments the history of pediatric infectious diseases the college of physicians of philadelphia. history of vaccines timelines. the college of physicians of philadelphia ( ) surgeons, smallpox, and the poor: a history of medicine and social conditions in nova scotia the immunization action coalition. vaccine timeline. the immunization action coalition jenner and the history of smallpox and vaccination experience in massachusetts and a few other places with smallpox and vaccination the medical side of benjamin franklin history of vaccine development (smallpox eradication: the vindication of jenner's prophesy chapter history of vaccine development. (pasteur and the birth of vaccines made in the laboratory chapter the history of anthrax vaccines: a biography (rabies) vaccines: a biography (killed vaccines: cholera, typhoid, and plague) vaccines: a biography (toxoid vaccines) historical review of pertussis and the classical vaccine preventing tetanus, diphtheria, and pertussis among adolescents: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccines recommendations of the advisory committee on immunization practices (acip) a brief history of vaccines and vaccination vaccines: a biography (tuberculosis and bcg chapter the history of tuberculosis yellow fever: a disease that has yet to be conquered vaccines: a biography (yellow fever chapter a history of influenza vaccines: a biography (influenza chapter vaccines: a biography (polio chapter a brief history of polio vaccines vaccines: a biography (measles, mumps, and rubella chapter the history of vaccines and immunization: familiar patterns, new challenges vaccines in historic evolution and perspective: a narrative of vaccine discoveries history of vaccine development (three decades of hepatitis vaccinology in historic perspective. a paradigm of successful pursuits chapter vaccines: a biography (varicella and zoster chapter vaccines: a biography (rotavirus chapter pandemic preparedness and response -lessons from the h n influenza of emerging infectious diseases: a cdc perspective vaccination greatly reduces disease, disability, death and inequity worldwide historical comparisons of morbidity and mortality for vaccinepreventable diseases in the united states vaccines for measles, mumps and rubella in children vaccination coverage among children in kindergarten -united states historic dates and events related to vaccines and immunization ih is the first author. she provided the idea and followed along with aj the execution of the work and final editing. nc and sc did the literature search for the eighteenth and nineteenth century and the respective preliminary writing about this period. ss and rb did the literature search for the twenty-first century, and about general aspects of vaccination and the respective preliminary writing about this period.mr did the literature search regarding vaccine efficacy and effectiveness in the context of the transnational research map and regarding vaccination instruments and inoculating techniques. ag did the literature search and the preliminary writing for the early history of vaccination and wrote a draft of a manuscript about this period. al edited thoroughly and commented on the final manuscript. aj supervised the whole process from inception to the final submission and edited the whole manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -qkfxfmxb authors: ampofo, william k.; baylor, norman; cobey, sarah; cox, nancy j.; daves, sharon; edwards, steven; ferguson, neil; grohmann, gary; hay, alan; katz, jacqueline; kullabutr, kornnika; lambert, linda; levandowski, roland; mishra, a. c.; monto, arnold; siqueira, marilda; tashiro, masato; waddell, anthony l.; wairagkar, niteen; wood, john; zambon, maria; zhang, wenqing title: improving influenza vaccine virus selectionreport of a who informal consultation held at who headquarters, geneva, switzerland, – june date: - - journal: influenza other respir viruses doi: . /j. - . . .x sha: doc_id: cord_uid: qkfxfmxb • for almost years, the who global influenza surveillance and response system (gisrs) has been the key player in monitoring the evolution and spread of influenza viruses and recommending the strains to be used in human influenza vaccines. the gisrs has also worked to continually monitor and assess the risk posed by potential pandemic viruses and to guide appropriate public health responses. • the expanded and enhanced role of the gisrs following the adoption of the international health regulations ( ), recognition of the continuing threat posed by avian h n and the aftermath of the h n pandemic provide an opportune time to critically review the process by which influenza vaccine viruses are selected. in addition to identifying potential areas for improvement, such a review will also help to promote greater appreciation by the wider influenza and policy‐making community of the complexity of influenza vaccine virus selection. • the selection process is highly coordinated and involves continual year‐round integration of virological data and epidemiological information by national influenza centres (nics), thorough antigenic and genetic characterization of viruses by who collaborating centres (whoccs) as part of selecting suitable candidate vaccine viruses, and the preparation of suitable reassortants and corresponding reagents for vaccine standardization by who essential regulatory laboratories (erls). • ensuring the optimal effectiveness of vaccines has been assisted in recent years by advances in molecular diagnosis and the availability of more extensive genetic sequence data. however, there remain a number of challenging constraints including variations in the assays used, the possibility of complications resulting from non‐antigenic changes, the limited availability of suitable vaccine viruses and the requirement for recommendations to be made up to a year in advance of the peak of influenza season because of production constraints. • effective collaboration and coordination between human and animal influenza networks is increasingly recognized as an essential requirement for the improved integration of data on animal and human viruses, the identification of unusual influenza a viruses infecting human, the evaluation of pandemic risk and the selection of candidate viruses for pandemic vaccines. • training workshops, assessments and donations have led to significant increases in trained laboratory personnel and equipment with resulting expansion in both geographical surveillance coverage and in the capacities of nics and other laboratories. this has resulted in a significant increase in the volume of information reported to who on the spread, intensity and impact of influenza. in addition, initiatives such as the who shipment fund project have facilitated the timely sharing of clinical specimens and virus isolates and contributed to a more comprehensive understanding of the global distribution and temporal circulation of different viruses. it will be important to sustain and build upon the gains made in these and other areas. • although the haemagglutination inhibition (hai) assay is likely to remain the assay of choice for the antigenic characterization of viruses in the foreseeable future, alternative assays – for example based upon advanced recombinant dna and protein technologies – may be more adaptable to automation. other technologies such as microtitre neuraminidase inhibition assays may also have significant implications for both vaccine virus selection and vaccine development. • microneutralization assays provide an important adjunct to the hai assay in virus antigenic characterization. improvements in the use and potential automation of such assays should facilitate large‐scale serological studies, while other advanced techniques such as epitope mapping should allow for a more accurate assessment of the quality of a protective immune response and aid the development of additional criteria for measuring immunity. • standardized seroepidemiological surveys to assess the impact of influenza in a population could help to establish well‐characterized banks of age‐stratified representative sera as a national, regional and global resource, while providing direct evidence of the specific benefits of vaccination. • advances in high‐throughput genetic sequencing coupled with advanced bioinformatics tools, together with more x‐ray crystallographic data, should accelerate understanding of the genetic and phenotypic changes that underlie virus evolution and more specifically help to predict the influence of amino acid changes on virus antigenicity. • complex mathematical modelling techniques are increasingly being used to gain insights into the evolution and epidemiology of influenza viruses. however, their value in predicting the timing and nature of future antigenic and genetic changes is likely to be limited at present. the application of simpler non‐mechanistic statistical algorithms, such as those already used as the basis of antigenic cartography, and phylogenetic modelling are more likely to be useful in facilitating vaccine virus selection and in aiding assessment of the pandemic potential of avian and other animal influenza viruses. • the adoption of alternative vaccine technologies – such as live‐attenuated, quadrivalent or non‐ha‐based vaccines – has significant implications for vaccine virus selection, as well as for vaccine regulatory and manufacturing processes. recent collaboration between the gisrs and vaccine manufacturers has resulted in the increased availability of egg isolates and high‐growth reassortants for vaccine production, the development of qualified cell cultures and the investigation of alternative methods of vaccine potency testing. who will continue to support these and other efforts to increase the reliability and timeliness of the global influenza vaccine supply. • the who gisrs and its partners are continually working to identify improvements, harness new technologies and strengthen and sustain collaboration. who will continue in its central role of coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support efforts to improve the vaccine virus selection process, including through the convening of periodic international consultations. • for almost years, the who global influenza surveillance and response system (gisrs) has been the key player in monitoring the evolution and spread of influenza viruses and recommending the strains to be used in human influenza vaccines. the gisrs has also worked to continually monitor and assess the risk posed by potential pandemic viruses and to guide appropriate public health responses. • the expanded and enhanced role of the gisrs following the adoption of the international health regulations ( ) , recognition of the continuing threat posed by avian h n and the aftermath of the h n pandemic provide an opportune time to critically review the process by which influenza vaccine viruses are selected. in addition to identifying potential areas for improvement, such a review will also help to promote greater appreciation by the wider influenza and policy-making community of the complexity of influenza vaccine virus selection. • the selection process is highly coordinated and involves continual year-round integration of virological data and epidemiological information by national influenza centres (nics), thorough antigenic and genetic characterization of viruses by who collaborating centres (whoccs) as part of selecting suitable candidate vaccine viruses, and the preparation of suitable reassortants and corresponding reagents for vaccine standardization by who essential regulatory laboratories (erls). • ensuring the optimal effectiveness of vaccines has been assisted in recent years by advances in molecular diagnosis and the availability of more extensive genetic sequence data. however, there remain a number of challenging constraints including variations in the assays used, the possibility of complications resulting from non-antigenic changes, the limited availability of suitable vaccine viruses and the requirement for recommendations to be made up to a year in advance of the peak of influenza season because of production constraints. • effective collaboration and coordination between human and animal influenza networks is increasingly recognized as an essential requirement for the improved integration of data on animal and human viruses, the identification of unusual influenza a viruses infecting human, the evaluation of pandemic risk and the selection of candidate viruses for pandemic vaccines. • training workshops, assessments and donations have led to significant increases in trained laboratory personnel and equipment with resulting expansion in both geographical surveillance coverage and in the capacities of nics and other laboratories. this has resulted in a significant increase in the volume of information reported to who on the spread, intensity and impact of influenza. in addition, initiatives such as the who shipment fund project have facilitated the timely sharing of clinical specimens and virus isolates and contributed to a more comprehensive understanding of the global distribution and temporal circulation of different viruses. it will be important to sustain and build upon the gains made in these and other areas. • although the haemagglutination inhibition (hai) assay is likely to remain the assay of choice for the antigenic characterization of viruses in the foreseeable future, alternative assays -for example based upon advanced recombinant dna and protein technologies -may be more adaptable to automation. other technologies such as microtitre neuraminidase inhibition assays may also have significant implications for both vaccine virus selection and vaccine development. • microneutralization assays provide an important adjunct to the hai assay in virus antigenic characterization. improvements in the use and potential automation of such assays should facilitate large-scale serological studies, while other advanced techniques such as epitope mapping should allow for a more accurate assessment of the quality of a protective immune response and aid the development of additional criteria for measuring immunity. • standardized seroepidemiological surveys to assess the impact of influenza in a population could help to establish well-characterized banks of age-stratified representative sera as a national, regional and global resource, while providing direct evidence of the specific benefits of vaccination. • advances in high-throughput genetic sequencing coupled with advanced bioinformatics tools, together with more x-ray crystallographic data, should accelerate understanding of the genetic and phenotypic changes that underlie virus evolution and more specifically help to predict the influence of amino acid changes on virus antigenicity. • complex mathematical modelling techniques are increasingly being used to gain insights into the evolution and epidemiology of influenza viruses. however, their value in predicting the timing and nature of future antigenic and genetic changes is likely to be limited at present. the application of simpler non-mechanistic statistical algorithms, such as those already used as the basis of antigenic cartography, and phylogenetic modelling are more likely to be useful in facilitating vaccine virus selection and in aiding assessment of the pandemic potential of avian and other animal influenza viruses. • the adoption of alternative vaccine technologies -such as live-attenuated, quadrivalent or non-ha-based vaccines -has significant implications for vaccine virus selection, as well as for vaccine regulatory and manufacturing processes. recent collaboration between the gisrs and vaccine manufacturers has resulted in the increased availability of egg isolates and high-growth reassortants for vaccine production, the development of qualified cell cultures and the investigation of alternative methods of vaccine potency testing. who will continue to support these and other efforts to increase the reliability and timeliness of the global influenza vaccine supply. • the who gisrs and its partners are continually working to identify improvements, harness new technologies and strengthen and sustain collaboration. who will continue in its central role of coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support efforts to improve the vaccine virus selection process, including through the convening of periodic international consultations. the historic initiative to establish a global network to detect and identify new and potentially dangerous influenza viruses predates the adoption of the who constitution in . with memories of the - influenza pandemic still vivid, and the ever-evolving threat posed by influenza recognized, the who global influenza surveillance and response system (gisrs) was formally established in . influenza thus became one of the first diseases to highlight the importance of international monitoring and collaboration in protecting human health. following the re-emergence of human cases of highly pathogenic avian h n influenza in and the adoption of the international health regulations ( ), the gisrs was strengthened and its role in protecting public health enhanced. in addition to tracking the course and impact of annual influenza epidemics and monitoring the evolution of seasonal influenza viruses, the gisrs also acts as a global alert mechanism for the emergence of influenza viruses with the potential to cause a human pandemic. the network provides support to both seasonal and pandemic influenza preparedness and response activities in areas such as diagnostics, vaccine development, virological surveillance and risk assessment. it also acts as the focus of who efforts to former who global influenza surveillance network (gisn), which has been renamed as who global influenza surveillance and response system (gisrs) since may , when the world health assembly resolution wha . was adopted. assist member states in strengthening their national capacity for the surveillance, diagnosis, characterization and sharing of influenza viruses. as a key player in global influenza risk assessment and response, the gisrs continues to evolve and expand, and as of december consisted of national influenza centres (nics) in countries, six who collaborating centres (whoccs), who h reference laboratories and four who essential regulatory laboratories (erls). the gisrs also works to ensure the successful coordination of who activities with those of external agencies such as the global outbreak alert and response network (goarn), national regulatory authorities, academic and veterinary institutes, and the pharmaceutical industry. the first formal who recommendations on influenza vaccine composition were issued in . since , separate and appropriately timed recommendations for the northern and southern hemispheres have been issued each year in february and september, respectively. these biannual recommendations are based upon the virological and epidemiological information generated by the gisrs and play a crucial role in the development, production and availability of effective influenza vaccines. the continuing threat posed by avian h n , the aftermath of the h n pandemic, the increased knowledge of influenza, and the development and availability of new technologies provide a timely opportunity to review the complex processes and issues involved in influenza vaccine virus selection and to identify potential areas for improvement. this who informal consultation represents the latest step in an ongoing process of gisrs strengthening and was convened with the following objectives: • to review the current vaccine virus selection process, including its constraints and limitations; • to identify opportunities for improving influenza surveillance and representative virus sharing; • to assess the potential for improving the assays and technologies used for vaccine virus selection; and • to assess the potential impact of new vaccine technologies on the vaccine virus selection process. participants were drawn from a broad and highly diverse range of institutes and sectors including the following: whoccs, nics, who erls, who h reference laboratories, national regulatory authorities, public health agencies, academia, influenza vaccine manufacturers, and veterinary laboratories and organizations. the primary goal of the gisrs vaccine virus selection process (annex ) is to generate and analyse the data needed to recommend the influenza vaccine viruses that will most closely match the influenza viruses likely to be circulating during forthcoming influenza seasons. current vaccine technologies and production schedules mean that decisions on vaccine composition have to be made almost a full year in advance of the peak of seasonal influenza activity. as a result, the process relies upon the earliest possible detection of emerging antigenic variants and the most up-to-date information on their potential future epidemiological significance. information must therefore be collected year round on the continuous evolution and global circulation of human influenza viruses to provide a sound basis for the biannual who recommendations on the composition of influenza vaccines for use in the northern and southern hemispheres. for countries in equatorial regions, epidemiological considerations influence which recommendation (february or september) individual national and regional authorities consider more appropriate. national influenza centres (nics) play a vital role in this complex process. their core activities include collating epidemiological information, diagnosing cases of influenza a and b infection, and identifying the subtype or lineage of the viruses responsible. the primarily molecular diagnosis of infection using rt-pcr techniques is based upon standardized primers and probes provided by the gisrs. viruses must also be isolated to allow their antigenic identification using the type-and subtype-specific reference reagents provided in annually distributed who kits. further detailed characterization may include sequence analyses to monitor genetic changes and assessment of virological traits such as resistance to antiviral drugs. sequence data are shared within the gisrs using public databases such as genbank and gi-said epiflu. nics in some settings then attempt to relate potentially important virological changes observed with clinical and epidemiological information and trends and may even conduct serological studies to evaluate the immune status of the population. weekly reports on the virological characteristics and epidemiology of circulating viruses are submitted to the who flunet -an internet-based data-query and reporting tool. information on the virus subtypes and lineages is collated, together with observations of potential clinical or epidemiological importance, and regular summaries of the geographical spread, intensity and impact of influenza are produced by who. if human infection with an avian or other animal influenza virus is suspected, a suitably equipped nic or other national influenza reference laboratory can conduct preliminary diagnostic testing using rt-pcr protocols and ⁄ or reagents for h , h and h subtypes provided by who. such rt-pcr testing does not require high-level biocontainment facilities. however, it is expected that the detection of any unusual influenza a virus distinct from known circulating viruses, especially one suspected to be of animal origin or unsubtypable using current who reagents, will immediately be reported to who and collaboration urgently initiated with a who collaborating centre (whocc). if the required laboratory biosafety facilities and procedures are not available, then virus isolation should not be attempted in the national laboratory and the sample should be promptly sent to a whocc. the routine and timely sharing of representative circulating influenza viruses and unusual viruses with a whocc is an essential step in the vaccine virus selection process. the criteria for forwarding viruses include their temporal, geographical and age-group distribution, severity of cases and virological characteristics such as unidentified subtype and antiviral drug resistance. whoccs are then responsible for the systematic antigenic characterization of the thousands of viruses forwarded each year by nics and other laboratories, and for the detailed genetic characterization of a selected subset. such detailed antigenic and genetic characterization is a necessary step in monitoring virus evolution and detecting any distinct antigenic variants that may necessitate updating the seasonal vaccine composition. the process also allows for the identification and characterization of animal viruses causing sporadic human infections, assessment of the risk they pose and the potential development of candidate vaccine viruses as part of pandemic preparedness. of prime importance in immunity to influenza is the production of antibodies to the virus haemagglutinin (ha) protein. such antibodies can neutralize the infectivity of viruses, and their level in the blood has been shown to correlate with the level of protection against infection with a homologous virus. as a result, influenza vaccine virus selection has primarily been based upon the antigenic characterization of virus ha using the haemagglutination inhibition (hai) assay. hai tests provide a visual readout of the ability of specific antibodies to prevent the attachment of ha to red blood cells (rbcs) and thus prevent their agglutination. antigenic drift in the ha of circulating viruses in response to host immunity reduces the effectiveness of vaccines and is therefore the major consideration when recommendations are made on the composition of influenza vaccines. the hai test is likely to remain the assay of choice for the antigenic characterization of virus ha for the foreseeable future. strain-specific antisera are produced by infecting previously unexposed ('naive') ferrets with either vaccine viruses, reference viruses representative of circulating viruses or viruses that appear in hai tests to be potential antigenic variants. the resulting sets of reference viruses and antisera are then used to evaluate the antigenic characteristics of the has of recent isolates. where antigenic differences are detected, these are likely to affect human immunity against the new variants. the hai test is a surrogate for the more complicated and time-consuming virus neutralization assay used to clarify antigenic relationships when observed variations in hai titre reflect, for example, changes in receptor binding rather than differences in antigenicity. a subset of between % and % of all viruses received is selected for genetic sequencing and more detailed analysis -principally of their ha and na components. this subset is selected to include representative circulating viruses, as well as apparent antigenic variants and viruses from severe or fatal cases. phylogenetic analyses are carried out to better understand the evolution of circulating viruses, their degree of genetic heterogeneity and the emergence of new genetic clades. antigenic or other phenotypic variants may thus be defined in terms of separate genetic clades with distinct amino acid signatures. relating the locations of amino acid substitutions to antigenic, receptor-binding or glycosylation sites on the d structure of the ha molecule then helps to identify the individual substitutions associated with phenotypic (antigenic) changes. identifying such amino acid signatures also facilitates global monitoring of the emergence, distribution and impact of different genetic variants. this is particularly helpful when data on emergent variants are limited at the time of a who vaccine consultation. comparisons of the sequences found in clinical specimens and virus isolates are also useful in revealing amino acid substitutions which result from passage in different substrates, mainly mdck cells and eggs. up-todate sequence data are shared within gisrs and made publically available via the gisaid epiflu database. complete genome sequencing is necessary to identify animal (including avian) viruses causing human infection and is important in detecting the emergence of reassortant viruses among co-circulating human viruses or between human and animal viruses. whoccs maintain panels of reference reagents for all influenza a subtypes. these include h (especially h n ), h and h avian viruses and various h n and h n swine viruses, as well as viruses present in other animals such as horses and dogs. whoccs also collaborate with the who erls in serological studies of representative human sera from previously vaccinated individuals. sera are provided by vaccine manufacturers and are used in hai tests to assess whether or not the antibodies induced by current vaccines are likely to be effective against currently circulating viruses. the results the principal criteria used to decide whether or not to recommend changes to influenza vaccine components include: • the emergence of an antigenically and genetically distinct variant among circulating viruses (including a novel influenza a virus with the potential to cause a pandemic); • evidence of the geographical spread of such a distinct variant and its association with outbreaks of disease, indicating its future epidemiological significance; • the reduced ability of existing vaccine-induced antibodies to neutralize the emergent variant; and • the availability of suitable candidate vaccine viruses. to facilitate collaborative studies by the whoccs and who erls and ensure that appropriate potential candidate vaccine viruses are identified in advance of the who vaccine composition consultation, the most recent virological and epidemiological data are shared and discussed via teleconferences held and weeks before the who consultation. a summary of each teleconference is promptly distributed to keep all nics and vaccine manufacturers informed of the developing situation. in addition, potential candidate vaccine viruses are provided to manufacturers. during the formal biannual consultations, the technical advisory group considers the cumulative antigenic and genetic data on the viruses characterized by whoccs. the data are set against the broader epidemiological context collated by who and are supported by serological data from whoccs and who erls, as well as by additional information provided by nics. hai data obtained in the different centres using a wide variety of reference viruses and ferret antisera are correlated using common reference reagents. in recent years, antigenic cartography has been used to collate and statistically visualize the degree of antigenic variation. the interpretation of hai data may, however, be complicated by the influence of changes in the receptor-binding properties of natural viruses or by the selection of variants during isolation and passaging in different cell or egg substrates. comparisons with sequence data are made to relate any differences in antigenicity with specific ha genetic clades and to more precisely define the identity of antigenic variants. the results of virus neutralization tests, which usually correspond to those of hai tests, are used to clarify the true antigenic relationships between different viruses. if the antigenic data, supported by genetic and serological data, indicate that a new antigenic variant is spreading globally, then a change in that component of the seasonal vaccine is considered to be warranted. the implementation of a rec-ommendation to update a vaccine component is, however, contingent upon the availability of suitable vaccine viruses. only after all the factors have been taken into account is a decision taken on whether or not to recommend a change in influenza vaccine virus composition. the decision is announced at an information meeting immediately following each who consultation and published on the who web site and in the who weekly epidemiological record. since the re-emergence of human cases of highly pathogenic h n avian influenza in , who has also regularly reviewed the available antigenic and genetic data on human and avian viruses in relation to the epidemiology of h n influenza among birds. to support the development of safe and effective human h n vaccines, who has coordinated the development of a number of candidate attenuated vaccine viruses (annex ) and made them available to vaccine producers. clinical trials have been conducted to evaluate the immunogenicity of different h n vaccine formulations and the breadth of antibody responses elicited. in addition, as part of pandemic preparedness, who has coordinated the ongoing development and updating of an inventory of h , h and h candidate vaccine viruses. important constraints on the vaccine virus selection process include the tight timelines involved (annex ), particularly in the northern hemisphere, where since recent years seasonal influenza activity tends to start increasing in middle or late january in general. as a consequence, decisions often have to be made relatively early in the influenza season. in addition, post-infection ferret antisera against potential antigenic variants are urgently required to define their antigenic relationships to previously circulating viruses. panels of recent isolates must also be prepared to assess the degree to which they are neutralized by antibodies in the sera of previously vaccinated individuals. finally, potential new candidate vaccine viruses must be prepared and evaluated for their suitability in vaccine production. ensuring the timely availability of viruses with suitable growth properties is a crucial step in ensuring that sufficient quantities of vaccine can be produced in time for administration prior to the next influenza season. although cell culture has steadily replaced the use of embryonated eggs for the primary isolation of viruses, candidate vaccine viruses must still be isolated directly in eggs according to current regulatory requirements. the limited availability of egg isolates, particularly of recent h n viruses which generally grow poorly in eggs, has led to the establishment of cooperative research and development agreements (cra-das) and similar agreements between the vaccine industry and a number of whoccs to increase the availability of egg isolates for vaccine use. the gisrs vaccine virus selection process necessarily involves a series of collaborative steps, including the selection of prototype antigenic variants and suitable vaccine viruses, and the provision of standardizing reagents by the who erls. the process thus impacts directly upon the subsequent authorizing of vaccine composition by national and regional regulatory authorities and upon the large-scale production of vaccine by manufacturers. mismatches have occasionally occurred as a result of the emergence of variant strains shortly after the recommendations have been made, highlighting one of the unavoidable consequences of current vaccine development and production constraints. nevertheless, retrospective studies have shown that with very few exceptions who vaccine virus recommendations have closely matched the influenza viruses that have circulated during the following influenza season. in addition, following the out-of-season emergence of the pandemic a(h n ) virus, this closely integrated system demonstrated its unique ability to very rapidly orchestrate the development and provision of appropriate (suitably attenuated) candidate vaccine viruses for pandemic vaccine production. global influenza surveillance has always presented a major challenge as it is a highly demanding public health need with a significantly uneven distribution of surveillance capacity worldwide. since the outbreak of severe acute respiratory syndrome (sars) in , the re-emergence of h n infection in humans and the h n pandemic, it has become ever clearer that surveillance and the prompt sharing of viruses and information are central to the broad range of influenza preparedness and response activities. although the known impact and the awareness of seasonal influenza vary in different parts of the world, the threat posed by avian h n viruses has galvanized influenza surveillance efforts in all countries. improving surveillance and acquiring the capacity to detect and report unusual cases of influenza are essential components of global pandemic planning and are enshrined in the international health regulations ( ) . successful efforts to increase the capacity of nics and other laboratories have been made, and in a number of settings the development, revision and adoption of guidelines on strengthened national, regional and global surveillance and collaboration is under way. global influenza surveillance has also been strengthened through expanded geographical coverage and the collection of more data of better quality. for example, in africa there are now influenza laboratories in countries, including recognized nics, almost all of which have the capacity to conduct rt-pcr diagnosis of influenza infection. in less than two years, the percentage of african countries with an nic increased from % to % with the number of countries with no influenza laboratory markedly decreasing. global, regional and national training workshops, assessments and donations have all led to significant increases in trained personnel, equipment procurement and laboratory capacity, resulting in the increasingly widespread use of molecular techniques such as real-time rt-pcr and genetic sequencing. recent who capacity-building activities have included bsl- training courses for nics to promote safe practices when working with highly pathogenic influenza viruses, and courses on virus isolation, gene sequencing and antiviral resistance detection. increased participation in both internal and external quality assurance programmes such as the who external quality assessment project (eqap) has contributed to marked improvements in laboratory proficiency. these and other efforts enabled a more effective response to the emergence of the h n pandemic in many countries. however, the pandemic also revealed significant limitations in the analysis and integration of epidemiological and virological surveillance data. in addition, few early seroprevalence surveys were conducted to allow for the timely assessment of the extent and impact of the pandemic. the pandemic also revealed significant gaps in laboratory infrastructure and personnel, equipment procurement and funding, particularly in developing countries. improvements and training in areas such as web-based integration and analyses of clinical, epidemiological and virological data are being implemented but care must be taken to ensure that such activities are not conducted at the expense of detection, characterization and virus-sharing activities in less well-resourced settings. identified research priorities in influenza surveillance and response include evaluation of the temporal and geographical circulation of influenza viruses and of the burden of influenza. in all settings, establishing a sound evidence base will support the development or updating of national, regional and global policies, plans and guidelines. this in turn could lead to greater acceptance of the use of influenza vaccines, particularly seasonal vaccines, and assist in the development of vaccination policies. the primary requirement of nics will remain the prompt diagnosis of influenza infection and the timely sharing of clinical specimens and virus isolates -especially those obtained from unusual, severe or fatal cases -backed up by appropriate epidemiological and clinical information. procedures should be in place to ensure that the increasingly predominant use of molecular diagnostic techniques, particularly real-time rt-pcr, does not adversely affect the timely isolation and for-improving influenza vaccine virus selection ª blackwell publishing ltd, influenza and other respiratory viruses, , - warding of viruses. improved communication between nics and whoccs on how best to facilitate prompt virus sharing, including discussion of the constraints faced, could improve coordination and avoid potential delays. a more systematic approach to engaging nic information and expertise would also lead to significant benefits. such an approach is likely to be facilitated by a number of developments in the use of who web-based tools. for example, nics with enhanced capabilities currently strengthen the collaborative characterization of viruses and aid early assessment of the significance of genetic and antigenic changes by sharing detailed virological information (especially ha sequences) on selected viruses, either directly or via public databases. as technologies advance, national patterns of seropositivity to circulating influenza viruses may also become available on a more timely basis and could thus guide vaccine use. this is particularly important given the increasing emphasis now placed on assessing vaccine effectiveness. comprehensive nic summary reports forwarded just prior to each who consultation also provide highly beneficial additional data to inform who recommendations on vaccine composition. to overcome logistical and other obstacles to the safe and efficient shipping of clinical specimens and virus isolates to whoccs, a who shipment fund project was established. the project provides support to nics and other influenza laboratories in all countries by arranging the transport of specimens and isolates along a guaranteed cold chain, especially in settings where there are severe financial and infrastructural constraints. as a direct result of the project, and associated 'infectious substances shipping' workshops conducted in all who regions, there has been a significant increase in the number of countries sharing specimens and isolates, especially following the outbreak of the h n pandemic. furthermore, the expansion and harmonization of the information currently provided in the accompanying standard shipping form to include information such as clinical outcome, patient vaccination status or recent travel history would greatly enhance understanding of the epidemiological context associated with the spread of viruses. a better understanding of the diversity and evolution of animal influenza viruses is essential for evaluating the pandemic risk posed by subtypes currently causing sporadic human infections (such as h n and h n ) and informing the selection of candidate vaccine viruses. the emergence of h n in particular led to the establishment in of the oie-fao network of expertise on animal influenza (of-flu) -a worldwide network of approximately laboratories and institutions that coordinates the global surveillance of animal influenza. a number of joint who-offlu tech-nical initiatives on influenza at the human-animal interface have been conducted (including successful collaboration during the h n pandemic) and reciprocal participation in annual meetings has taken place. there remains, however, considerable scope for improved coordination and collaboration with the animal influenza surveillance sector, especially in the collection and analysis of antigenic and genetic data, the timely exchange of representative viruses and reference reagents, and the conducting of serological studies of human exposure to zoonotic infection. influenza is an important disease of many avian and mammalian species with serious economic consequences for livestock industries and has potential adverse impacts on human food supplies. despite this, animal influenza surveillance coverage is limited with a shortage of epidemiological data on the circulation of various viruses in different countries. efforts are now under way to establish triggers for initiating enhanced surveillance that go beyond animal disease notification and sporadic human infections. although there is increasing understanding of the interrelationships between animal and human influenza and the need for 'integrated' surveillance, full collaboration at both national and global levels is currently constrained by a number of practical, funding, regulatory and policy issues. maintaining a regular dialogue based upon the mutual interests of the different networks will be an important public health activity and may also help to enhance the sustainability of animal influenza surveillance in particular settings. a more formal collaborative mechanism might allow for the improved integration of animal virus data into the who candidate vaccine virus selection process. increased awareness of the content and extent of use of animal influenza vaccines would also aid understanding of their impact on virus evolution. a range of laboratory assays and other techniques provide the complementary information on changes in the antigenic and genetic characteristics of influenza viruses needed to select the most appropriate influenza vaccine viruses. however, inherent limitations in the biological assays used and significant variations in the results obtained by different laboratories complicate the collation and definitive interpretation of data. because the hai test outlined previously is a simple, rapid and reproducible surrogate assay for virus neutralization, it is widely used to measure the antigenic relationships between different viruses as well as antibody responses to infection or vaccination. in addition, the test provides the basis of the only current quantitative correlate of protection against infection (serum hai antibody titre ‡ ) used to standardize inactivated vaccines. however, variations in the physical characteristics of rbcs obtained from different species and differences in the receptor-binding properties of different viruses influence both the sensitivity and the utility of the assay. furthermore, changes in receptor-binding affinity or specificity associated with adaptation, antigenic drift or the isolation and passage of viruses in eggs and cell culture may also affect hai titres. standardization between laboratories has also proved difficult, and the assay is currently not suitable for use in a fully automated system. a range of practical refinements such as attempts to develop 'synthetic' rbcs (for example using glycan-coated beads) have been unsuccessful. given the currently limited knowledge of the principal natural receptors for influenza viruses, such approaches are unlikely to circumvent the virus-dependent shortcomings of assays based upon natural rbcs which are therefore likely to remain the primary approach to antigenic characterization for the foreseeable future. recent developments based on the use of panels of recombinant ha do offer alternative or supplementary microtitre or microarray binding-assay formats for assessing antibody specificity and antibody inhibition of the ha-glycan receptor interaction. although such approaches are relatively expensive and require a high degree of skill to implement, they are potentially highly suited to automation and in time may reduce the need for virus isolates. in addition, such formats can readily be adapted to incorporate biosensor technologies to provide more quantitative analyses of binding characteristics. a number of such assays are currently being validated using ferret and human antisera. the contribution of antibodies against virus na in conferring protection following natural infection or vaccination is still not well understood. studies of na antigenic variation have been limited, and the na content of influenza vaccines is not currently standardized. although neuraminidase inhibition (nai) assays were conducted more routinely in the past, these were cumbersome to perform and were complicated by the relatively low levels of antibodies against na in post-infection ferret sera and by interference from antibodies against ha. a number of different nai microtitre assay formats have recently been developed. these have been used to correlate antigenic changes with sequence variations in the na component, provide more precise information on the evolution of na and assess na antibody responses following vaccination. improved understanding of antigenic drift in na and of the role of anti-na antibodies in conferring immunity might have significant implications for both vaccine virus selection and vaccine development. microneutralization (mn) assays -based on measuring virus replication, cell viability or na activity -provide an important adjunct to hai tests in antigenic characterization. mn assays are generally more sensitive and measure a broader repertoire of functional antibodies that neutralize viral replication, with potential advantages in the evaluation of human serological responses. in addition, comparisons of mn and hai tests for measuring antibody responses in vaccinated individuals have shown a consistent degree of correlation and have confirmed the utility of mn assays in analyses of human antibody responses to h vaccine components. techniques for simplifying assay formats and making them more readily applicable to the routine testing of low-titre viruses are under investigation, and efforts are under way to use mn assays for h and b viruses. this should facilitate the use of mn assays to overcome the variable nature of interactions between viruses and rbcs, and hence in interpreting 'anomalous' hai results which complicate vaccine virus selection. pseudotype virus neutralization assays may also offer some advantages in scale and standardization over conventional mn assays for measuring serological responses to particular viruses, especially highly pathogenic viruses. furthermore, ongoing improvements in automation will potentially enable the more labour-intensive mn assay to be applied to large-scale serological analysis. epitope mapping using genome fragment phage display libraries provides another powerful technique for further dissecting the fine specificity of antibody responses to vaccination and infection and should allow for a better assessment of the quality of a 'protective' immune response and aid the development of additional correlates of immunity. to encourage the performance of seroepidemiological surveys to assess the impact of influenza in a population, countries should be supported in establishing well-characterized serum banks of age-stratified representative sera as a national, regional and global resource. current advantages of the gisrs serological activities undertaken in support of vaccine virus selection include the use of shared serum panels and common antigens, with frequent consensus obtained from participating whoccs and who erls. limitations include the large variability of hai data, a requirement for antibody standards and a need for mn or other assays to resolve inconsistencies. the availability of antibody standards would not only enhance the comparability of serological data generated in different laboratories and countries but also facilitate the comparison of antibody responses to different vaccines. increasing attention to influenza vaccine effectiveness studies will lead to the availability of more real-time data for comparing clinical benefit with the degree of antigenic improving influenza vaccine virus selection ª blackwell publishing ltd, influenza and other respiratory viruses, , - relatedness of vaccine and circulating viruses. such studies, especially those based upon laboratory-confirmed outcomes, should provide evidence of the specific benefits of vaccination. consistent studies providing estimates of vaccine efficacy over successive influenza seasons should improve understanding of the effects of small rather than major antigenic differences between vaccine and circulating viruses on clinical outcomes and should help to allay concerns arising from a perceived vaccine mismatch caused by the emergence of virus clades exhibiting little or no antigenic drift. recent advances in high-throughput genetic sequencing could potentially lead to a greatly enhanced understanding of the genetic changes occurring in influenza viruses and the evolutionary interactions that occur between co-circulating viruses. in-depth analyses of the precise mechanisms involved in the evolution and epidemiology of influenza would require advanced bioinformatics tools to comprehensively mine the data produced. such an approach should reveal, for example, the broader genetic changes that underlie antigenic variation in ha and thus allow for a better understanding of the relationship between genetic evolution and antigenic drift. increased information from x-ray crystallography on the structural features of the has of recent viruses and specific mutants, together with developments in computer modelling, should assist in attempts to predict the likely influence of amino acid substitutions on the antigenic and receptor-binding properties of new variants. further development of high-throughput laboratory systems for integrated and automated genetic and phenotypic analyses -from initial sample accession to data management -offers the intriguing prospect of a futuristic standardized virtual network for virus characterization in an epidemiological context. as such systems will have broad implications, not only for vaccine virus selection, but also for the organization and conduct of global influenza surveillance, it is extremely important that their development and deployment are integrated with the activities of the who gisrs. numerous mathematical modelling techniques have now been used to gain insights into the mechanisms that underlie both the evolution and the epidemiology of influenza viruses. for example, exploratory models have been developed to generate and test various hypotheses to explain the relatively restricted diversity of influenza viruses in terms of constrained antigenic repertoire, and to explore the underlying nature of immunity. they have also been used to improve understanding of the extent of between-subtype and between-type competition and of the potential conse-quences of such interactions for trends in the incidence of seasonal influenza viruses. phylogenetic models have also been used to identify changes in selective constraints in relation to antigenic drift and inter-species transmission. when based upon the amino acid substitutions associated with mammalian host adaptation, such models may aid assessment of the pandemic potential of avian and other animal viruses. phylodynamic modelling based upon available sequence data, supplemented with antigenic data, has already been successfully used to trace the emergence of new antigenic and genetic variants and track their geographical spread. however, in the absence of greatly improved understanding of the underlying evolutionary and biological mechanisms and other processes involved, the capacity of current mathematical modelling techniques to predict the timing and nature of future antigenic and genetic changes is limited. the intrinsically stochastic nature of influenza evolution may make such predictive modelling extremely challenging. where changes occur over short time scales, the application of simpler non-mechanistic statistical algorithms, such as those used as the basis of antigenic cartography, is likely to be more useful in facilitating vaccine virus selection than attempts to develop predictive models from the existing complex dynamical models of influenza evolution and transmission. such predictive models might presently be better suited for use in understanding the possible long-term effects of vaccination, optimizing the timing and location of focused surveillance efforts and predicting the possible consequences of the emergence of a novel virus. eventually, these models should be able to take advantage of integrated immunological and antigenic surveillance data to develop predictions of short-term dynamics in specific locations. all new influenza vaccine technologies have implications for vaccine virus selection and for regulatory and manufacturing processes. however, any potential requirement to tailor the virus selection process to specific types of vaccine is unlikely to be a crucial issue, especially if advances in vaccine technology and speed of production lead to greater flexibility in the timing of recommendations. although live-attenuated vaccines are not yet universally licensed, the current vaccine composition recommendation process is used. however, antibody response is not a good correlate of protection for such vaccines and the identification of a true correlate might affect the requirement for annual updating. several quadrivalent vaccines are also now under development that contain representative strains of the two influenza b virus lineages (b ⁄ victoria and b ⁄ yamagata) together with influenza a(h n ) and a(h n ) viruses. this raises a number of issues that could affect vaccine supply, including the possibility of two poorly growing vaccine viruses; the likely variable impact of a fourth component on vaccine yields and timing of manufacture; the prioritization of influenza b lineage viruses in the context of both trivalent and quadrivalent vaccine production; and the need for a fourth set of reagents. adjuvanted vaccines have been licensed with the primary aims of inducing better immune responses in certain age groups and allowing 'antigen sparing'. although there has been no specific intention to provide a broader spectrum of immunity to circumvent the need for annual vaccine updates, different products are likely to show a different breadth of response. providing recommendations in relation to product-specific cross-reactivity over successive influenza seasons is unlikely to be a feasible option for the who gisrs. in addition, various types of recombinant vaccines are now under development, including protein subunit, dna, vector and vlp vaccines -none of which are presently licensed. in the case of non-ha-based vaccines, different guidelines will apply and all such vaccines are likely to impact the current vaccine virus selection process in various ways depending upon their precise type and mechanism of protection. the level of protection afforded by immunity to na is receiving continued interest. currently, this component is included as part of the candidate vaccine virus and is selected on the basis of its sequence but not antigenicity. standardization of the na component would require antigenic characterization during the virus selection process, while antigenic changes in na in the absence of a corresponding change in ha antigenicity may on its own necessitate the updating of vaccine composition. for all such vaccines, ha variant selection may become less crucial than it is for current vaccines. although high-growth reassortants have been used to manufacture influenza a vaccine components for many years, their yields have been variable and there is continued need to identify the molecular determinants of high yield to engineer a more reliable and reproducible production process. reverse genetics, now used in the united states to produce virus reassortants for live-attenuated vaccines, has also been used to produce attenuated candidate h n vaccine viruses suitable for inactivated vaccine manufacture. this approach was, however, less successful than classical reassortment in obtaining a suitable h n pandemic vaccine virus, emphasizing the need for further investigation of the applicability of reverse genetics in the routine provision of suitable vaccine viruses. following the licensing of cell culture vaccines, the feasibility of isolating seasonal vaccine viruses in qualified cell lines is being evaluated in a collaboration involving a number of whoccs and who erls under cradas with vaccine manufacturers. these studies should provide the basis for the introduction of a universal qualified cell culture system for providing mammalian cell-derived seasonal influenza candidate vaccine viruses. this would result in a greater choice of candidates, especially for recent h n viruses, and may provide greater flexibility in responding to the 'late' emergence of a variant necessitating a vaccine composition change. such virus isolates would not be subject to undesirable egg-selected changes and would potentially provide a better match to the natural virus. however, the relative merits of egg and cell culture candidate vaccine viruses have still to be rigorously evaluated. guidance on quality assurance aspects has already been published by the european medicines agency (ema). the finalization of new ema regulatory guidelines may be accompanied by a who technical document on harmonizing regulatory approaches worldwide and the engagement of other regulatory authorities in vaccine-manufacturing nations. vaccine manufacturers and the who erls are also collaborating in an evaluation of cell culture-based reagents for use in single radial immunodiffusion (srid) potency testing, due for completion in early . in addition, despite international consensus on the key quality specifications for h n pandemic influenza vaccines, reagents to calibrate the majority of candidate vaccines using conventional potency tests only became available immediately prior to the initiation of clinical trials. in some cases, candidate vaccines were available ahead of the reagents. although national authorities proved flexible in accepting the use of validated alternative potency tests to allow clinical trials to proceed, newer methods such as high-performance liquid chromatography (hplc) and mass spectrometry are now being evaluated. the gisrs has a long history of success in recommending influenza vaccine compositions that have closely matched the combination of viruses circulating during subsequent influenza seasons. based upon the voluntary participation of its many constituent partners, the gisrs enjoys strong institutional and governmental support. global influenza surveillance is the foundation of the vaccine virus selection process. efforts to enhance and strengthen national, regional and global laboratory capacity for virological surveillance and representative virus sharing must continue. as part of this, improved integration of virological and disease surveillance data will be a key aim and will help to build the foundations for future studies of the impact and burden of influenza worldwide. to strengthen the pandemic influenza preparedness, collaboration between the gisrs and veterinary laboratories and organizations such as offlu in relation to zoonotic influenza infection has been greatly enhanced and has included the development of appropriate candidate human vaccine viruses from animal viruses. however, there remains considerable scope for improvement in this area, including the more timely exchange of information, viruses and reagents, and strengthened technical collaboration at all levels. although antigenic characterization of promptly forwarded virus isolates will remain the central criterion for selecting influenza vaccine viruses in the foreseeable future, technological developments (such as advanced recombinant dna and protein technologies, and highthroughput sequencing and advanced bio-informatics tools) will inevitably impact current gisrs surveillance and virus selection activities. in the interests of global public health, it will be important to integrate into the gisrs system appropriate information and data generated by various networks using emerging technologies. antigenic cartography has been adopted by the gisrs in recent years as a means of integrating hai data from different laboratories to allow for statistical comparison and visual display. the development of new statistical algorithms to complement the use of antigenic cartography may further facilitate vaccine virus selection. greater emphasis should be placed on conducting human serological studies which incorporate the use of antibody standards to improve the comparability of results. such studies would improve current understanding of the prevalence and spread of influenza, and complement the development of improved epidemiological models. greater collaborative effort is needed to generate randomly sampled, representative and integrated serological, epidemiological and evolutionary data that provide snapshots of host and viral populations suitable for modelling hypotheses on virus evolution and host immunity. the application of advanced techniques for dissecting the fine specificity of antibody responses to vaccination and infection should also lead to improvements in understanding the quality of a 'protective' immune response and aid in the development of additional correlates of immunity. recent collaboration between the gisrs and external partners including academic institutions and vaccine manufacturers has resulted in the increased availability of egg isolates and high-growth reassortants. new approaches to the generation of high-growth vaccine viruses involving the use of reverse genetics and qualified cell cultures will continue to be evaluated and developed, as will alternative methods of vaccine potency testing. who will continue to support these and other efforts to increase the reliability and timeliness of global influenza vaccine supply. new vaccine types currently under development may allow more flexibility in the timing of recommendations on vaccine virus composition. conversely, alterations to the virus selection process and additional information may be needed in relation to new-generation vaccine types with different compositions and mechanisms of protection. the who gisrs vaccine virus selection process lies at the heart of global efforts to address the constantly evolving threat posed by influenza. for decades, this highly collaborative and complex process has ensured a continued supply who writing group of effective seasonal vaccines and was able to respond very rapidly to the emergence of the h n pandemic. if the current limitations and constraints inherent in the process are to be overcome, ongoing efforts by the who gisrs and its partners must continue to identify improvements, harness new technologies and strengthen collaboration. who will continue in its central role of developing and coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support this process through the convening of periodic international consultations on improving influenza vaccine virus selection. annex : process of influenza vaccine virus selection and development the diagram shows that the individual steps in the selection of candidate vaccine viruses and development of standardizing reagents for seasonal influenza and for a potential h n influenza pandemic are essentially equivalent. for seasonal vaccines the timelines are: • steps - : the collection, isolation and thorough antigenic and genetic characterization of recent virus isolates continues throughout the year; • step a: comparisons of the recognition of representative recent viruses by vaccine-induced antibodies in human sera are conducted - weeks before the biannual who vaccine consultation meetings; • steps , a and a: candidate viruses for vaccine use are reviewed and selected, and high-growth reassortants prepared and characterized following identification of (potential) antigenic variants -these steps are not solely dictated by the recommendations of the who biannual vaccine virus consultations. • step : evaluation of their growth properties is conducted in a timely manner around the time of the who vaccine virus consultations and prior to authorization of vaccine composition by national authorities. • step a: preparation of the standardizing reagents for new vaccine components is initiated once the particular vaccine virus has been selected following the who recommendation. the who informal consultation for improving influenza vaccine virus selection, - june , was organized by the virus monitoring and vaccine support (vmv) unit of who, with participation from who collaborating centres on influenza, erls, national influenza centres, national control laboratories, national regulatory authorities, academic and veterinary institutions, influenza vaccine manufacturers and other collaborating organizations. in accordance with who policy, all members of the writing group that assisted who in the development of this meeting report had completed the who declaration of interests for who experts. these declarations were then evaluated by the who secretariat prior to the consultation. the members of the writing group declared the following personal current or recent (within the last years) financial or other interests relevant to the subject area: group on data needs). plos curr influenza (revised december ). rrn . northern hemisphere season. writing committee of the world health organization consultation on northern hemisphere influenza vaccine composition for public workshop on immune correlates of protection against influenza a viruses in support of pandemic vaccine development use of antigenic cartography in vaccine seed strain selection influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses who. a description of the process of seasonal and h n influenza vaccine virus selection and development. draft version antigenic and genetic characteristics of influenza a(h n ) and influenza a(h n ) viruses and candidate vaccine viruses developed for potential use in human vaccines strategies to improve global influenza surveillance: a decision tool for policymakers global influenza surveillance network: laboratory surveillance and response to pandemic h n improving the process of vaccine virus selection determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the octet rapid semiautomated subtyping of influenza virus species during the swine origin influenza a h n virus epidemic in milwaukee, wisconsin a practical influenza neutralization assay to simultaneously quantify hemagglutinin and neuraminidase-inhibiting antibody responses development of rapid, automated diagnostics for infectious disease: advances and challenges high-throughput dna sequencingconcepts and limitations high-throughput laboratories for homeland and national security current challenges in implementing cell-derived influenza vaccines: implications for production and regulation establishment of retroviral pseudotypes with influenza hemagglutinins from h , h , and h subtypes for sensitive and specific detection of neutralizing antibodies new tests for an old foe: an update on influenza screening ecological and immunological determinants of influenza evolution population dynamics of rapid fixation in cytotoxic t lymphocyte escape mutants of influenza a epochal evolution shapes the phylodynamics of interpandemic influenza a (h n ) in humans rambaut a et al. the genomic and epidemiological dynamics of human influenza a virus the generation of influenza outbreaks by a network of host immune responses against a limited set of antigenic types h n influenza pandemic: insights from modelling. the who informal network for mathematical modelling for pandemic influenza h n (working ne win immunization, vaccines and biologicals (ivb) global influenza programme (gip) who regional office for south-east asia (searo) global influenza programme (gip) who regional office for europe (euro) global influenza programme (gip) global influenza programme (gip) keiji fukuda, health, safety and environment (hse) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) immunization, vaccines and biologicals (ivb) global influenza programme (gip) global alert and response (gar) global influenza programme (gip) global influenza programme (gip) who regional office for the americas (amro) laszlo palkonyay, immunization, vaccines and biologicals (ivb) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) ludy suryantoro, health, safety and environment (hse) global influenza programme (gip) immunization, vaccines and biologicals (ivb) global influenza programme (gip) acknowledgements ª blackwell publishing ltd. the world health organization retains copyright and all other rights in the manuscript of this article as submitted for publication. key: cord- - xk w d authors: flower, darren r; macdonald, isabel k; ramakrishnan, kamna; davies, matthew n; doytchinova, irini a title: computer aided selection of candidate vaccine antigens date: - - journal: immunome res doi: . / - - -s -s sha: doc_id: cord_uid: xk w d immunoinformatics is an emergent branch of informatics science that long ago pullulated from the tree of knowledge that is bioinformatics. it is a discipline which applies informatic techniques to problems of the immune system. to a great extent, immunoinformatics is typified by epitope prediction methods. it has found disappointingly limited use in the design and discovery of new vaccines, which is an area where proper computational support is generally lacking. most extant vaccines are not based around isolated epitopes but rather correspond to chemically-treated or attenuated whole pathogens or correspond to individual proteins extract from whole pathogens or correspond to complex carbohydrate. in this chapter we attempt to review what progress there has been in an as-yet-underexplored area of immunoinformatics: the computational discovery of whole protein antigens. the effective development of antigen prediction methods would significantly reduce the laboratory resource required to identify pathogenic proteins as candidate subunit vaccines. we begin our review by placing antigen prediction firmly into context, exploring the role of reverse vaccinology in the design and discovery of vaccines. we also highlight several competing yet ultimately complementary methodological approaches: sub-cellular location prediction, identifying antigens using sequence similarity, and the use of sophisticated statistical approaches for predicting the probability of antigen characteristics. we end by exploring how a systems immunomics approach to the prediction of immunogenicity would prove helpful in the prediction of antigens. vaccines are agentseither molecular or supramolecular -which can stimulate protective immunity against microbial pathogens and the diseases they cause. protective immunity is a specific and enhanced adaptive immune response to subsequent re-infection or, when luck holds, infection by related organisms. such augmented immunity is mediated by the exacerbation of immune memory, which militates against the effects of infectious organisms. the word vaccine itself is derived from vacca (latin for cow). [ ] [ ] [ ] . it is a thing of near universal agreement that mass vaccination -synergising as it does with the herd immunity it helps engender -is the most effective and efficacious prophylactic treatment currently available for contagious disease. humankind is commonly affected by over seventy infectious diseases, many of which are or will be targets for vaccines. there are in excess of fifty licensed vaccines, half of which are deemed to be in common use. most vaccines prevent childhood infections or are used by travellers to tropical or subtropical regions; only a minority combat disease in third-world countries. as recently as the late s, there were over million cases of smallpox spread through countries, with about two million deaths a year, yet now smallpox is wholly and totally eradicated. poliomyelitis or polio is the other key global disease close to eradication. in , the pan american health organization eliminated polio from the western hemisphere. in the first world, the annual death rate arising for contagious diseases such as polio, diphtheria, or measles is less than one in a thousand. the global polio eradication program has now greatly reduced the prevalence of polio in the rest of the world. only cases of polio were reported in . nevertheless, polio remains endemic in nigeria, afghanistan, pakistan, and india. despite such outrageous and egregious success, many major issues persist. no licensed vaccines exist for hiv and malaria, two of the world health organization (who)'s three big global killers, and there are no realistic hopes for such vaccines appearing in the short to medium term. bacille calmette guérin (bcg), the only vaccine licensed currently for the third major world disease, tuberculosis, has only limited efficacy [ ] . add to this the new, previously unknown infectious diseases identified in the past years: hiv, marburg's disease, sars, dengue, west nile, and over human infections with the potentially pandemic h n influenza. it is commonly believed that new contagious diseases will continue to emerge in the st century. the world of the st century is threatened by parasitic diseases and emerging zoonotic infections; antibiotic-resistant bacteria; and bioterrorism [ ] the vaccine arena has long been neglected, partly as a consequence of the extraordinary success just adumbrated, but activity within it is now feverish and febrile [ , ] . dozens of vaccine candidates have passed through phase ii clinical trials, and during the past decade, vaccines in late development have numbered over . unlike antibiotics, resistance to vaccines is negligible. in the same way that vaccines target many kinds of disease, themselves caused by microbial pathogens of all types, so there are many types of fundamentally distinct vaccine. see figure . these include attenuated or inactivated whole pathogens, subunit vaccines, peptide vaccines, and vaccines based on carbohydrates, amongst others. historically, the most successful and prevalent types vaccines have been those based on attenuatedthat is "weaken" or noninfective -whole pathogen vaccines, for example bcg for tb or sabin's polio vaccine. safety concerns have fomented other vaccine strategies to develop, which focus on antigens and latterly epitopes as the intrinsic component of single or composite vaccines. hepatitis b vaccine is an antigen -or subunitbased vaccine. while many epitope-based vaccines have now entered clinical trials, they are yet to fulfil their potential, medically or commercially. antigens, immunogenicity, and subunit vaccines subunit vaccines are typically but not exclusively protein molecules and their discovery is often based around a somewhat haphazard, essentially empirical search for antigenic or immunogenic protein antigens. the word antigen has several meanings in immunology and thus in vaccinology. the oxford english dictionary defines an antigen as a "…foreign substance which, when injected into an organism, stimulates antibody production." they trace its first use to , and by the middle of the century the word had found its way into common technical dictionaries. an immunogen is on the other hand: "any substance capable of eliciting an immune response", although the word had other, earlier definitions. its first use in its modern rendering is traced to , but was doubtless in use before then. figure a schema summarising the components of a generic vaccine. the immunogenic component will typical be an attenuated or chemically-inactivated microbe, a protein antigen, or a poly-epitope or conjugate. this component will be combined with an appropriate delivery mechanism and an adjuvant. delivery mechanisms and adjuvants overlap in their ability to increase the immunogenicity of a weaklyactive vaccine. dictionary definitions seldom capture the scientific meaning of scientific terms particularly well. so, to be more precise, an immunogen, that is a moiety exhibiting immunogenicity, is any substance able to elicit a specific immune response, while an antigen -a moiety exhibiting antigenicity -is a substance which is recognized by an existing immune response, and its associated underlying molecular moieties such as t cells or antibodies, in a recall response. immunogenicity is the most important and interesting property for vaccine design and discovery. it is this property that allows a molecular moiety to induce a significant immune response. for the purposes of this review, our use of the terms antigen and immunogen shall be restricted to a single meaning; that of a protein, specifically one from a pathogenic microorganism, that evokes a measurable immune response. currently, the prophylactic responses of almost all effective vaccineswith the exception of bcg which is mediated by the quadrille of interacting antigen presenting cells (apcs), t-cells, and other phagocytotic cells that characterise cellular immunityare mediated by humoral immunity and antibodies. the pathogenesis of most diseases for which vaccines are not currently available are typically if not exclusively mediated by cellular immune mechanisms. thus, when seeking to identify immune responses for untreated disease, the immunogenicity sought need not be mediated by antibodies; mediation by cellular mechanisms or a combination of humoral and cellular mechanisms is equally valid. in what follows, we will endeavour to explore the availability of informatic tools and techniques for the identification of candidate subunit vaccines. even today, in the early years of the st century, many experimental scientists still retain an atavistic distrust of any and all computational approaches. the very suggestion that algorithms can help them, saving effort and time is an anathema to them and their way of thinking, undermining all that they hold dear. they are deeply distrustful of the reliability of computer methods, preferring what they perceive as the infallible reliability of the experiment. not that they would admit to views so philosophically -and practicallynaive; but deep down this is how they think and how they feel. yet things have changed and changed dramatically, and things will continue so to change. slowly but surely, reverse vaccinology is becoming a discernibly more prevalent means of identifying subunit vaccines; and slowly but surely the contribution made by computation to the practice of reverse vaccinology is becoming ever more significant and ever more obvious [ ] . see figure . conventional laboratory-based, experimental microbiological approaches to antigen identification typically start with the cultivation of the target pathogenic micro-organism under laboratory conditions, then dissecting them into their component proteins, assaying these in some cascade of in vitro and in vivo assays, leading ultimately to the identification of proteins which display requisite protective immunity. it would indeed be wonderful if the identification of candidate vaccines was really and consistently this simple and systematic? unfortunately, it is often so much more complex, confusing, and confounding. it is not always possible to cultivate a particular pathogen outside of the host organism. many proteins are only expressed transiently during the course of infection. nor are all proteins easily expressed in sufficient quantities in vitro. thus many potential candidate vaccines may be missed. reverse vaccinology [ ] [ ] [ ] [ ] is, by contrast, able to analyze a genome to identify potential antigens. initially, the pathogenic genome is scanned for "open reading frames" or orfs. once all orfs have been identified, proteins are selected on the basis that they will be accessible to immune system surveillance, usually using some form of informatic-based prediction methodology or, more likely, set of methdologies. reverse vaccinology was established by a group studying neisseria meningitidis, which is responsible for sepsis and meningococcal meningitis. vaccines are available for all serotypes except subgroup b. potential orfs in n. meningitidis genome were identified [ ] [ ] ; proteins were identified, expressed in vitro, and were surface exposed. seven proteins conferred immunity over a broad range of strains. another good example is streptococcus pneumoniae, a major cause of sepsis, pneumonia, and meningitis [ ] [ ] . potential orfs were identified, of which could be expressed; six proteins were found to induce protection. another example is porphyromonas gingivalis, a gram-negative anaerobic bacterium found in subgingival plaques present in chronic adult inflammatory gum disease periodontitis. of the orfs identified in the genome [ ] , protein sequences were predicted to be open to surveillance by the immune system, were positive for one or more sera. when used to vaccinate mice, two antigens exhibited clear protection. this very brief survey highlights the potential for vaccine discovery using this approach. however, the number of potential antigens is still high, particularly when we explore all potential difficulties in expressing and characterising them. both experimental and computational methodologies may thus omit potentially important antigenic proteins from analysis, albeit for different reasons, and both require continual optimisation. in particular, bioinformatics support for preclinical vaccine discovery has yet to flourish; but, as interest in vaccines waxes, this situation is changing: expanding from the nursery to at least the kindergarten. there are two main forms of support for vaccine discovery provided by bioinformatics. the first is technically indistinguishable from support for more general target discovery, and includes genomic annotation for both host and pathogen sequences and immunotranscriptomics, the application of microarray analysis to the immune system. the other type kind of support is focussed on immunoinformatics: it addresses problems such as the accurate prediction of epitopes. currently, prediction of t cell epitopes is essentially confined to predictions of varying accuracy of peptide binding to major histocompatibility complex. binding of peptides to class i mhc are reasonably accurate, at least for well characterised alleles [ ] . however, several comparative studies have shown recently that the prediction of class ii mhc binding prediction t-cell epitopes is typically poor [ ] [ ] [ ] , and likewise for structure-driven prediction of mhc-mediated epitopes [ ] [ ] . similarly, both structure- [ ] and data-driven [ ] prediction of antibody-mediated epitopes is suboptimal. while the prediction of b cell epitopes remains primitive, or depends on an oft elusive knowledge of protein structure, methods for t cell epitopes prediction displaying not inconsiderable algorithmic sophistication have been and continue to be developed. despite this, and for different reasons, reliable and consistent prediction remains somewhat elusive; this regrettable decision will doubtless continue so for some time. ultimately, all methods, however sophisticated, are severely circumscribed by the data used to propagate and parameterise them. no data-driven method can go beyond the data used to train it; all methods are likewise much superior in their ability to interpolate than their ability to extrapolate. it is only by compiling extensive, high-quality data that we can aspire to create excellent models of general applicability. what we require are sets of data that are complete, thorough, and properly thought through. such sets would be able to explore both efficiently and effectively the complex and confounding multi-dimensional phase space of properties evinced by the molecules we target. iedb [ ] [ ] [ ] [ ] [ ] [ ] is a step-change in this direction. iedb is the dominant force in current immunoinformatics and stands as one of the principal achievements of the field. the number and quality of epitopes within it are an enormous improvement over all that has gone before. alex sette and his co-workers are to be congratulated on their achievement, but, like all dominant things, iedb has tended to choke most other efforts in the field, and even iedb has its limitations. data is usually multi-dimensional, and each dimension will typically be correlated, to a greater or lesser extent, with each other dimension. together, these many dimensions chart a space: a space of structural variation or variation of properties. if the data we use is itself of sufficient quality and provides a good enough coverage figure whole antigen discovery. taking a reverse vaccinology dynamic, the process of discovering candidate subunit vaccines begins with a microbial genome, perhaps newly sequence, progresses through an extensive computational stage, ultimately to deliver a shortlist of antigens which can be validated through subsequent laboratory examination. the computational stage can be empirical in nature; this is typified by the statistical approach embodied in vaxijen [ ] . or this stage can be bioinformatic; this involves predicting subcellular location and expression levels and the like. or, this stage can take the form of a complex mathematical model which uses immunoinformatic models combined with mathematical methods, such as metabolic control theory [ ] , to predict cell-surface epitope populations. of the space, then straightforward methods drawn from, say, computer science -of which there are indeed very many -are now of satisfactory accuracy to build models of high predictive accuracy. as we have said, there is an on-going need for the quality, quantity, and availability of data to improve and increase. prediction is enthral to its underlying data. bias within the data places strict limitations upon the interpretability and generality of models from which it is derived. in general, for mhc-peptide binding experiments, the sequences of peptides studied are very biased in terms of amino acid composition, often favouring hydrophobic sequences. this arises, in part, from preselection processes that result in self-reinforcement. binding motifs are often used to reduce the experimental burden of epitope discovery. very sparse sequence patterns are matched and the corresponding subset of peptides tested, with an enormous reduction in sequence diversity. irrespective of their poor performance in prediction, several other problems exist, albeit different for t-and b-cell epitope prediction. for t-cell prediction, the key issue is the availability of data. it has recently been shown that t-cell epitopes, which were previously thought to be short peptides of - amino acids, can be up to amino acids or perhaps even more. the existence of these longmer epitopes has greatly expanded the repertoire of peptides open to inspection by t-cells [ ] [ ][ ] [ ] . problematic as this seems, the situation is made worse by the fundamental logistic constraints of sampling within the specificity exhibited by a single allele. the number of possible sequences that a nonameric peptide might possess is in the region of billion; considering that a single model is built from at most a few hundred peptides, the sampling undertaken is infinitesimally small. similarly, the + different mhc alleles known to exist in the global human population indicates the extraordinary potential for distinct peptide specificities within the potential patient cohort. for b-cell prediction, explaining the poor performance of b-cell epitope prediction algorithms may point to a fundamental misinterpretation of extant epitope data. pepscan is perhaps the most abundant data available currently but may not be what it seems. experimentally derived epitopes are identified by being assayed against pre-existing antibodies with affinity for whole antigens. however, if such "epitopes" are mapped on to their parent antigen structure they are randomly located throughout the protein and do not equate to clear surface patches, as one might expect if they simply mimic discontinuous epitopes identified by crystallography; in situ these antigenic regions can be completely buried, and thus inaccessible to antibody binding, rather than exposed. if we compare the conformation of antibodybound peptides with those from the intact antigens, they are usually quite different. however, b-cell epitopes in intact antigen and in whole antigen-antibody complexes are similar. it may be that the preformed antibody recognizes denatured antigen in vivo or that the isolated peptide adopts a conformation able to mimic the surface features of a discontinuous epitope. moreover, there is also evidence that the responsiveness of the immune system to pathogenic proteins is only poorly correlated with the possession of t cell epitopes, and that many potential epitopes have been deleted in proteins regularly accessible to immune surveillance, perhaps as an evolutionary counter measure in the war between host and pathogen [ ] . taken together, these factors suggest that methods which rely solely on the possession of epitopes are unlikely to be fully effective when tasked with identifying antigens or immunogens. this conjecture is confirmed by what information there is, which indicates that there is little simple correspondence between antigens selected on this basis and experimentally verified antigenic or protective proteins. thus we must seek alternative methods for selecting, and prioritising within that selection, proteins likely to be antigenic and protective. we shall below examine three key approaches: subcellular location prediction, sequence similarity, and empirical statistical approaches, typified by vaxijen. for a protein to be accessible to surveillance by the immune system, it is often assumed to be physically external to the microbial organism or at least present on its surface rather than being sequestered away far from the roving eye of the immune system. for bacteria this means it must be secreted or located on -or in -the outer membrane surface. in this context, immune surveillance is undertaken by a variety of cellular and molecular actors, but chiefly by neutralising antibodies. thus the goal of identifying antigens through the use of subcellular location prediction is principally to identify surface proteins accessible to binding by neutralizing antibodies. unlike the relatively straightforward and well-studied task of identifying orfs, selecting the subcellular location of proteins can be challenging to the point of confusion. nonetheless, and notwithstanding the obvious caveats inherent in this strategy, this has become a favoured approach taken by many when tasked with selecting vaccine candidates. we should remember, however, that antigens are not epitopes, and epitopes are not antigens. so possession of t-cell and b-cell epitopes is in certain senses independent of sub-cellular location, and they may in principle be possessed by any protein. as we have said, there is evidence for t-cell epitope depletion in pathogenic proteins [ ] ; or it may be that just picking immune-accessible proteins naturally favours proteins rich in antibody and helper t-cell epitopes, as opposed to cytotoxic t-cell epitopes, at least for membrane proteins. as data accumulates on the specific responses made to differently located accessible proteins we can build these recondite subtleties into prediction strategies. there are two basic kinds of prediction method: manual construction of rules of what determines subcellular location and the application of data-driven machine learning methods, which determine factors that discriminate between proteins from different known locations. accuracy differs markedly between different methods and different compartments, mostly due to a paucity of data. data used to discriminate between compartments include the amino acid composition of the whole protein; sequence-derived features of the protein, such as hydrophobic regions; the presence of certain specific motifs; or a combination thereof. databases, such as prodom [ ] , pfam [ ] , and pro-site [ ] , contain within them the capacity to identify sequence motifs characteristic of certain protein families; this can in turn help us to predict if a protein belongs to an family of proteins which are typically extracellular. however, gross sequence similarity alone is neither a necessary nor a sufficient condition for a protein to share a common sub-cellular location. very similar sequences may be located quite differently, while lack of similarity is no guarantee that proteins will not be co-located. moreover, many proteins can and do exist in several locations within the cell, and often evince different if related functions in these different locations [ ] . likewise, different organisms, and specifically eukaryotes versus prokaryotes, have quite different locations, both in number and in physical nature. the number of such compartments used in prediction studies varies considerably, and few papers address the full rich complexity of cell biology. for example, a common schema reduces eukaryotic cells to compartments (cytoplasmic, extracellular, mitochondrial, and nuclear) and prokaryotic to compartments (cytoplasmic, extracellular, and periplasmic), though some papers use over compartments for eukaryotes. in fact, compartments is an under-estimate, and the richness and the complexity of sub-cellular location is daunting, since any attempt to predict it must account for transient and permanent location, multiple locations, and both membrane organelles and multi-protein complexes as potential locales. among binary approaches, arguably the best method is signalp, which employs neural networks and predicts n-terminal secretion signals cleaved by signal peptidase-i-and their associated cleavage site [ ] [ ] [ ] . the signal predicted is the type ii signal peptide common to both eukaryotic and prokaryotic organisms, for which there is a wealth of data, in terms of both quality and quantity. a recent enhancement to signalp is the implementation of a hidden markov model (hmm) which seeks to discriminate uncleaved signal anchors from cleaved signal peptides. one of the limitations of signalp is overprediction, as it cannot reliably discriminate between several very similar yet distinct signal sequences, regularly predicting membrane proteins and lipopteins as type ii signals. many other kinds of signal sequence exist. a number of methods have been developed to predict lipoproteins, for example. the prediction of proteins that are translocated via the tat-dependent pathway is also important but has yet to be addressed in any depth. many programs and servers have been devised and developed able to predict the subcellular location of gene products and proteins. psort is a well-known, well-used example; it is knowledge-based, multicategory prediction method, composed of several programs [ ] [ ] [ ] [ ] . psort i predicts different subcellular compartments, while psort ii predicts locations. there are several specialized versions of psort. ipsort deals specifically with secreted, mitochondrial, and chloroplast locations. psort-b only predicts bacterial sub-cellular locations. it reports precision values of . % and recall values of . %. another effective program is hensbc [ ] . this constructs a hierarchical ensemble of classifiers by applying a series of if-then rules. hensbc is able to assign proteins to one of four different types (cytoplasmic, mitochondrial, nuclear, or extracellular) with approximately % accuracy for gram-negative bacterial proteins. another program is subloc [ ] , a client/server suite which offers an interface for the prediction of prokaryotic subcellular location in three compartments. another still is gpos-ploc [ ] , which fuses many basic classifiers, engineered using the optimized evidence-theoretic k-nearest neighbours rule. other programs include tatp . [ ] , lipop . [ ] , and phobius [ ] . this list goes ever on and on. a comparison of a subset of these programs, which used a test set of mycobacterial proteins [ ] , indicated that subcellular localization methods generally had high predictive specificity and recognised true negatives reliably. we have also developed a range of programs specifically aimed at addressing the prediction of subcellular location within bacteria. building on a set of algorithms able to predict membrane proteins, tat secretion, and lipoproteins [ ] [ ] [ ] [ ] [ ] [ ] [ ] , a set of bayesian networks, which were able to make individual predictions for specific subcellular locations was implemented in three pipelines with different architectures: a parallel implementation with a confidence level-based decision engine and two serial implementations with a hierarchical decision structure [ ] . these two hierarchies were rooted differently, one by prediction between membrane types and the other by soluble versus membrane prediction. the parallel pipeline outperformed the serial pipeline, but took twice as long to execute. the soluble-rooted serial pipeline outperformed the membrane-rooted predictor. assessment using genomic test sets was more equivocal, as many more predictions are made by the parallel pipeline, yet the serial pipeline identifies more of the proteins of known location. this work is indicative of a clear direction that the development of future subcellular prediction strategies might take. currently, the richness of subcellular structures is yet to be integrated into location prediction. many cellular organelles are not included in such studies, nor are proteins with multiple locations, nor are the different routes by which proteins can reach the same compartment. the list of such caveats continues. clearly, if we can develop high specificity predictors for each such compartment, then combining them appropriately [ ] , must be the way forward. there is then a need for a more sophisticated phase of supervised learning, where better and more complete annotation is built into any approaches taken. many difficulties remain however, most notably the lack of validated, high quality datasets where the location of proteins has been established unambiguously. this paucity is particularly acute for certain important but less well-studied kinds of secreted protein, such as those secreted by the type iii secretion system. while databases such as dbsubloc [ ] have been developed, their utility is open to question. so too is the extraction of datasets directly from swiss-prot. here the inherent uncertainty in some of the available annotation can be confounding or at least frustrating. likewise, much more work on the prediction of the subcellular location of viral proteins is required; while some studies have been undertaken [ ] [ ] , the complexity of their interactions with their host organism must be factored into future analyses. generally, we can make the observation that ignoring this complexity is, in trying to simplify matters, more likely to render any analysis simplistic. fortunately, subcellular location prediction, useful though it can be, and quibbles and cavils notwithstanding, is not the only method available to us. there are other weapons in our arsenal, many with an equal provenance and of equal utility. one of the most obvious ways to identify new potential antigens in newly sequenced microbial genomes is through similarity searching. assuming that we know the sequence of one or more extant antigens, we can make use sequence searching programs of various complexity and sophistication, such as blast or fasta, to identify similar sequences in the target genome. this set of selected candidate antigens can then be prioritised for further theoretical and ultimately experimental validation. obviously, all the same caveats that exist for any sequence search hold here also: which thresholds are appropriate? are apparently high-scoring matches real or an artefact of search methodology? this process also presupposes that enough known antigens are available so that such searches can be comprehensive and thus effective. such compilation is the role played by the database. in the last decade, factory-scale experimentation allied to extensive literature mining has generated many functional immunology databases. databases, such as syf-peithi [ , ] , which focus on properties of cellular immunology, and look primarily at data relevant to mhc processing, presentation, and t-cell recognition have existed since the mid s. arguably, the best such database is the hiv molecular immunology database [ ] , although clearly the depth of the database is at the expense of breadth and generality. it archives cd + and cd + t-cell and b-cell epitopes derived from the virus. it also includes detailed biological information regarding the response to the epitope, including its impact on long term survival, common escape mutations, whether an epitope is recognized in early infection, and curated alignments summarizing the epitope's global variability. other recent databases include mhcbn [ , ] , which contains , mhc-binding peptides, , mhc nonbinding peptides, , tap binders and nonbinders, and , t-cell epitopes. epimhc [ ] is a relational database of naturally occurring mhc-binding peptides and t-cell epitopes. presently, the database includes distinct peptide sequences from various sources, including tumor antigens. two databases in particular, warrant special attention, albeit for different reasons. they are antijen [ ] , formerly known as jenpep [ , ] , and iedb [ ] . antijen seeks to integrate a wider range data than is archived by other databases. implemented as a relational postgresql database, antijen is sourced from the primary literature and contains over , entries; it includes quantitative kinetic, thermodynamic, functional, and cellular data within the context of immunology and vaccinology. as well as t-cell and mhc binding data, antijen holds over , entries for linear and discontinuous b-cell epitopes, and includes measurements of peptide interactions with tap transporter and peptide-mhc complex interactions with t-cell receptors (tcr), as well as immunological protein-protein interactions. iedb is a database lavishly funded by the nih, which addresses issues of biodefence. as we have said, it is on a much larger scale than any other similar database, and benefits significantly from the input of dedicated epitope sequencing projects. these exist, in part at least, to populate the database. iedb has largely eclipsed other efforts in functional immune databases. however, it does not, as a priority, address antigenicity at the whole protein level. at this point it is worth discussing the distinction between functional annotation and the objective discussed here. generally speaking, the function that a protein performs within the context of its organism of origin is irrelevant to its status as an antigen. here the ubiquity and multiple meanings of the word antigen are of little if any help. a protective antigen is a protein which is recognised and recalled by the host. this characteristic does not seem to be linked to the fact that a protein is an enzyme or a dna binding protein, nor does logic require such a link. thus identifying function is not a necessary condition for a protein to be an antigen, though the unequivocal identification of certain functions, such as being a virulence factor for example, greatly increases the probability that it will be such. concerning antigens, however, these databases, although replete with information concerning individual b cell epitopes, t cell epitopes, and major histocompatibility complex (mhc) binding peptides, remain otherwise partial and incomplete. their focus is on the epitope, not the antigen. there are many antigens for which specific epitope or mhc binding information is not currently available, yet many such antigens are known experimentally to induce either or both innate or adaptive immune responses. fortunately for the future of vaccine design and discovery specific antigen-orientatedrather than epitope-orientated -databases are now becoming available. arguably, the clearest and most unequivocal example of an antigenic protein is the so-called virulence factor (vf). these proteins are able to undertake the colonization of a host organism and/or induce disease. they are the front-line weapons in the pathogenic arsenal. analysis of known pathogenic species, such as vibrio cholerae or streptococcus pyogenes, has enabled the recognition of recurrent "systems" of vfs and toxins that may total or more distinct proteins. these may exist as discrete pathogenicity islands or be spread more widely in the genome. traditionally, classification of vfs has categorised them as belonging to several thematic groups: adherence/colonization factors, invasions, exotoxins, transporters, iron-binding siderophores, and miscellaneous cell surface factors. a broader definition groups vfs into three classes: ( ) "true" virulence factors; ( ) vfs associated with the expression and regulation of class vf genes; and ( ) vfs required for the colonization of the host [ ] . a number of databases that archive vfs have been reported. the virulence factor database (vfdb; url: http://www.mgc.ac.cn/vfs/) contains characterized bacterial genomes with an emphasis on functional and structural biology and can be searched using text, blast, or functional queries [ , ] . tvfac (los alamos national laboratory toxin and virulence factor database; url: http://www.tvfac.lanl.gov/) contains genetic information on over organisms and separate records for thousands of virulence genes and associated factors. the fish pathogen database (url: http://www. fishpathogens.eu/vhsv/index.php), set up by the bacteriology and fish diseases laboratory, has identified over virulence genes using fish as a model system. pathogens studied include aeromonas hydrophila, edwardsiella tarda, and many vibrio species. candida albicans virulence factor (candivf) is a small species-specific database that contains vfs, which may be searched using blast or a hla-dr hotspot prediction server [ ] . phi-base is a noteworthy development as it integrates vfs into a cohesive whole a variety of pathogens of plants and animals [ ] . obviously, antigens need not be vfs, they need only be accessible to immune surveillance and need not be directly or indirectly involved in infectivity. because of this, other types of database are required, able to capture and contain a wider tranche of relevant data. in the recent past, another database has been developed: anti-gendb [ ] contains a compilation of over antigens drawn from the literature and other immunological resources. it marks a new beginning in immunoinformatics, signalling a switch away from the peptide epitope and toward the whole protein antigen. these antigens come from important pathogenic species. in antigendb, a database entry contains information regarding the sequence, structure, origin, etc. of an antigen with additional information such as b and t-cell epitopes, mhc binding, function, gene-expression and post translational modifications, where available. anti-gendb also provides links to major internal and external databases. antigendb will be updated on a rolling basis, with the regular addition of antigens from other organisms. this database will form the core of future attempts to predict antigens both by sequence similarity and using more recondite analysis. at this point it is worth mentioning the issue of thresholds. clearly, when one runs a sequence search, using blast for example, one might generate huge lists of near-identical proteins or get no hits at all; and, for that matter, we could also obtain almost any intermediate result. the issue is to judge which result is useful and which is not. this typically equates to setting a threshold, above which we anticipate usefulness and below which we might expect a lack of utility. setting such thresholds is however no easy task. they are dependent on the nature of the family in question. for the lipocalin family [ ] [ ] [ ] , for example, hits are still valid in terms of structure and function at levels that would simply be noise for most other protein families. thus thresholds are family dependent, as well as problem dependent. empirically-selected cut-offs are thus the order of the day, but much thought and experimentation is needed in order to select appropriate values. as well as seeking similarity to known antigens, there is another, quite prevalent, idea that is deserving of comment: that the immunogenicity of protein is determined solely by its lack of similarity to the host. what we search for is some quantitatively-meaningful measure of the "foreignness" of a protein which correlates highly with its immanent immunogenicity. in this context, what we mean by the word "foreignness" is the evolutionary distance between the hosta man or a cow or mouse -and the candidate antigen, or the organism that produced it, or both. some consider this to be the prime factor determining the potential immunogenicity of a protein [ , ] . clearly, since we are dealing with proteins and carbohydrates and the like, this evolutionary distance must be specified in terms of their molecular structures, or more likely their sequences, rather than in terms of morphological features. the potential importance of such a concept is supported by the observation that immune systems are actively educated to lack reactivity when presented with self-proteins [ ] , a processoften called immune tolerancewhich is generated via epitope-specific mechanisms including clonal anergy, receptor editing, and clonal deletion [ ] [ ] . but how can we progress beyond this rather inexactly-specified philosophical standpoint to something which is practically useful when we select vaccine candidates? a potentially more useful way to express this conjecture is that the likelihood that a protein is immunogenic is solely a function of that protein's dissimilarity to the whole host proteome at the sequence level. or, to be more precise, how close in terms of sequence similarity is the candidate to the closest or most similar member of the host proteome. most sequence software is well suited to solving this problem, since it is precisely this problem that they were written to address. more difficult is assessing average dissimilarity to the whole proteome, a problem compounded when we use the similarity of overlapping peptide fragments rather than looking at the similarity at the level of global sequence alignment. in terms of choosing the length of such fragments, the epitope would seem to be the most logical choice, since this immunological quantum is the moiety most likely to be recognised during the immune response. yet even at the level of the epitope, a peptide of say amino acids, even one mismatch in an otherwise perfect match can be significant, since such sequence differences, comprising a single amino acid, may exacerbate or abrogate neutralizing antibodies binding to a particular antigenic protein. moreover, the crossreactivity of a single high-affinity monoclonal antibody is rather different in nature to the cross-reactivity of large set of less affine polyclonal antibodies, and so too their ability to tolerate amino acid mismatches. it will also vary between individuals, since the immunization history of each organism will dictate to a large extent the recognition of epitopes. our understanding of epitopes can inform our understanding of mismatch tolerance, since the affinity of t-cell epitopes is more dependent on the possession of suitable anchor residues than it is on the possession of non-anchor residues. having said, the dogma of anchordependent affinity was long ago debunked, since all residues make some kind of contribute to affinity, entropic or enthalpic, although generally it is right to say that socalled anchors do make more significant contributions. our understanding of the structural-basis that determines the affinity of antibody-mediated epitopes is much less assured and complete, and the underlying thermodynamic causes of affinity, if strict causes they are, typically only become clear when high resolution structural data combines with measured thermodynamic metrics. likewise, when one looks not at a representative individual, but at the whole population, then the deletion of a single protein, within one host versus another, can render candidates previously valid and immunogenic as suddenly neither. again, these are difficult issues; as yet, they remain unresolved. given the hypothesis that immunogenicity is in some sense mediated by the level of similarity between a pathogenic protein and the host proteome, we have, in as yet-unpublished work, sought to bench-mark sequence similarity analysis as a means of quantifying the differences between populations of antigens and non-antigen. to that end, we identified sets of known antigenic and non-antigenic protein sequences derived from a variety of sources: allergens, bacteria, fungi, parasites, tumours and viruses. these were compared to the human and mouse genomes using standard sequence similarity searching protocols. whole pathogen proteomes were also aligned to these host proteomes. most antigenic and non-antigenic sequences were observed to be non-redundant; this implies a lack of clear homologues between pathogens and the human or mouse proteomes, although a number of parasite antigens were found to have a much higher level of similarity. these proteins comprised heat shock proteins, catalases, and enzymes involved in hydrolysis. these protein families are structurally conserved, though they might display significant functionality diversity. we also used statistical approaches such as the nonparametric mann-whitney test to assess if comparisons between the two populations were significant. the statistical null hypothesis was accepted in most cases, signifying that the effect presumably resulted solely from chance. the mann-whitney test supported the observations from sequence similarity analysis. we were unable to determine a threshold or cut-off based on the hypothesis of non-redundancy to the host's proteome. these results suggest that this is not in itself a solution to the identification of antigens. a variant based on fragments may be more successful, and this is clearly an issue crying out for further, deeper research. there are, of course, many other ways to approaches identifying antigenic proteins. one notable way, is looking for the horizontal transfer of so-called pathogenicity islands, clusters of pathogenic proteins acquired by transfer between micro-organisms. detection of such islands, which are typically large gene clusters with an atypical yet characteristic g + c content, can in turn lead to the identification of antigenic proteins [ ] [ ] [ ] [ ] . analysis at the nucleic acid level rather than at the protein level can facilitate the discovery of virulence proteins, perhaps using similar approaches to that used to identify cpg islands [ ] [ ] [ ] [ ] . however, rather than look at nucleic acid sequences, or at protein sequences directly, a new approach, based upon alignment-free techniques, has been developed which shows significant potential; we examine this next. most in silico approaches to predicting antigens make use of bioinformatics tools of one kind or another. such tools can identify membrane proteins, signal peptides, or lipoproteins with some success, yet most algorithms still rely on sequence alignment to identify characteristic sequence relationships or motifs characteristic of antigens. this may prove problematic in several ways. some proteins formed through divergent or convergent evolution lack obvious sequence similarity, although they may share similar structures and/or biological properties [ , ] . in such a situation, alignment-based approaches may produce ambiguous results or fail spectacularly. many methods presuppose a direct sequence relationship such as that which can be revealed by simple sequence search techniques, such as blast. this is not always the case. immunogenicity, as a property, may instead be encoded within the sequence and structure of a protein in such a cryptic and subtle manner as to go beyond the conventional limitations imposed when one seeks direct identification by sequence alignment protocols. likewise, the discovery of truly novel antigens will be frustrated by their lack of similarity to antigens of known provenance. as a departure from alignment-dependent techniques, which dominate immunoinformatics as much as they do bioinformatics, we have implemented an approach which seeks to discriminate between candidate vaccines and nonantigens, using an alignment-free sequence representation [ , ] . rather than concentrate on epitope and nonepitope regions, the method uses data on immunoprotective antigens derived from pathogenic sources to derive statistical models for predicting wholeprotein antigenicity. in attempting to overcome the limitations imposed by alignment-dependent sequence similarity methods, we have implemented a novel alignment-independent method for antigen identification based on auto cross covariance (acc) transformation of protein sequences into uniform equal-length vectors. the acc transform is a protein sequence mining method originally devised by wold et al. [ , ] , which has found much application to the quantitative structure-activity relationships (qsar) study of peptides and also for the classification of proteins [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the principal properties of the amino acids were represented by z descriptors [ ] [ ] [ ] , which describe amino acid hydrophobicity, molecular size and polarity. the acc transformation accounts for neighbour effects, i.e. the lack of independence between different sequence positions. initially, we sought to develop models able to distinguish immunoprotective proteins from the general microbial proteome. we applied acc pre-processing to sets of known bacterial, viral and tumour antigens and developed alignment-independent models for identifying antigens. in a separate paper, extra models were added which address fungal and parasite antigens. for bacterial, viral and tumour antigens, models had prediction accuracies in the % to % range [ , ] [ ] . for the parasite and fungal antigens, models had good predictive ability with % to % accuracy under internal cross validation in groups. under external validation, they gave % sensitivity ranking the true immunoprotective proteins in the first % of their proteomes. the models were implemented in a server for the prediction of protective antigens and subunit vaccines, which we have christened vaxijen [ ] (url: http:// www.darrenflower.info/vaxijen). the accuracy values noted above are indicative of an approach that is more accurate than has been seen before; for example, for b-cell epitope prediction. vaxijen is an imperfect beginning; future research will yield significantly more insight as the number of known protective antigens increases [ ] . there are several bioinformatics problems unique to immunology: the foremost and greatest is the challenge of accurately predicting immunogenicity. the successful computational prediction of immunogenicity may manifest itself at one extreme in the identification of a t-cell or b-cell epitope. this is mediated by straightforward if not uncomplicated molecular recognition events, which can be understood through properly exploring relationships between fundamental physical propertieshydrogen bonding and lipophilicity for exampleand apparent biological activity. establishing such structureactivity or property-activity relationships is a considerable concern of current immunoinformatics. at the other extreme, we encounter an altogether more complex phenomenon, where we see immunogenicity made manifest in the accurate estimation of antigenicity at the whole protein level. this is a system property; that is a property of the whole immune system rather than an individual molecular recognition event. at present, this is very much a secondary aspect of modern day immunoinformatics. the task of predicting whole protein immunogenicity is probably several orders of magnitude more complex and demanding than say predicting the binding of peptides to an mhc or even of predicting the binding of a pmhc complex to a tcr. this is not to say that such calculations are in any way trivial or lacking difficulty, but rather than that such complex prediction exercises are themselves subsumed in the task of predicting immunogencity at the system level. in our view, and in the view of other commentators, the clinical manifestation of the immunogenicity of a vaccine antigen, as opposed to the immunogenicity of an isolated epitope, arises as the very complex amalgam of many intertwining intrinsic and extrinsic factors, operating at various length-scales and at various different rates. such factors include host-side properties and pathogen-side properties, as well as protein-side properties that arise more or less exclusively from the protein itself. see figure . so-called host-side properties are properties intrinsic to the immune system of the host. they include the possession of b-cell epitopes or t-cell epitopes, as recognised by the adaptive immune system, or the possession of pathogen-associated molecular patterns or pamps, which are recognised by pattern recognition receptors, such as toll-like receptors (tlrs), part of the innate immune system. pathogen-side properties are properties intrinsic to the pathogen as a whole organism. they include the expression level evinced by an antigen, the time-course of its expression and secretion, and also its subcellular location. protein-side properties include the state of aggregation exhibited by a candidate vaccine and any post-translational danger signals that the protein might possess. some of these properties have been discussed before. thus one would expect, at least naively, that a bona fide vaccine antigen would be both highly expressed and available for immune surveillance, as well as possessing epitopes that the host can recognise, where as a protein which is not immunogenic would lack such characteristics. identifying and predicting this diverse tranche of properties is a problem and thus a challenge. indeed, each component part is a challenge unto itself. consider the prediction of a t cell epitope: the best understood and most accurate of immunoinformatic prediction problems. such an epitope needs to be processed properly into a population of different peptides, and for the processed peptides to exist in a reasonable amount, and then be bound by an mhc with reasonable affinity, before being recognized appropriately within the context of a pmhc complex by a tcr. this then is a complex and contingent process, similar to a generic markov process, comprised of many stages which are themselves dependent on preceding steps. in terms of both mechanism and what steps-within-steps there may be, many of these steps are less than adequately understood. however, each step remains amenable to statistical evaluation. all stages are inherently predictable given an appropriate accumulation of relevant data. the situation is very similar but not identical for biopharmaceuticals. in this case, we can dispense with the pathogen and replace it with several classes of extrinsic factors which include amongst others product manufacture, patient health, and medication strategy. acute illness, particularly systemic illness, or latent bacterial or viral infections such hcv, can have a most profound effect on the manifestation of immunogenicity. likewise, a patient's genetics may alter profoundly the immune reaction to biopharmaceuticals, most obviously, the mhc haplotype will affect host t-cell responses. the nerve program is an expert system for vaccine antigen discovery, which addresses in a practical way some of the issues of prioritisation discussed above. the program has been developed to help automate the process of reverse vaccinology [ ] . the identification and prioritising of potential orfs using nerve comprises stages. firstly, the prediction of subcellular localization; then whether the protein is an adhesin; followed by the identification of transmembrane domains; then the protein is compared with both the human and pathogen proteomes; after which the protein is finally assigned a suggestive function. vaccine candidates are thus filtered and ranked, and a list of proteins is produced graded by precedence and the probability that it will be an immunogenic antigen. but nerve and other extant programs, such as dynavacs [ ] , tasked with such a confounding task necessarily fall far short of what is needed or indeed what is possible. there needs to be a concerted and farreaching effort to address this issue. the most direct line of attack is to address first the subcellular location issue, as we discussed above. likewise, we can deconstruct the antigen presentation pathway, building models for each step and then integrating them into a fully functional model. we can develop empirically-based, statistically-grounded approaches -of which vaxijen [ , , ] is merely the vanguardthat have their basis in emerging antigen databases. yet all of this is just the beginning. we need to factor in b-cell and antibody mediated issues using structural data. we need to properly assess the role of aggregation, of expression levels, of post-translational danger signals and the possession of pathogen-derived pamps, as well as the ability of large molecule and small molecule adjuvants to raise the intrinsic immunogenicity of candidates to useful levels. we should note in passing that many hostderived molecules are also labelled pamps; these include fibronectin, hsp , and heparan sulphate, amongst others. however, these molecules are endogenous activators of innate immunity and not relevant to this discussion, though they may figure in auto-immune scenarios. vaccines are a good thing; putting caveats and cavils to one side, no one sensible really doubts the truth of this statement. vaccines have proved their worth time and again, but for them to continue to prove their worth we must find new ways of making them. almost all present-day vaccines are mediated by antibodies, and the majority target viral diseases. unfortunately, we are now running short of target diseases which fit this restrictive bill. many of the pathogens responsible for current recalcitrant and emergent diseases are, and are set to prove, much more demanding and difficult to target. in many senses, the low hanging fruit has been cut down and now the fruit we most desire is well out of reach. many diseases, including the who's big three diseases: hiv, tb, and malaria, are much more complicated functionally and/or structurally, and cellular, as opposed to humoral, immunology is tasked with defence against these dark arts. peptide vaccines and those based on apcs are important new if as yet unspectacular directions for research in vaccine discovery, yet modern strategies for vaccine figure factors underlying immunogenicity as elaborated in the text, the phenomenon of immunogenicity can be explored through the diversity of underlying individual factors contributing to the instigation of the immune response. these factors can be assigned to the host (epitope recognition), the pathogen (location and expression level), and also factors intrinsic to the protein antigen itself, such as the possession of post-translational danger signals. development hinge primarily upon the effective search for vaccine antigens, at least for the proactive search for vaccines targeting long-standing but untreated diseases rather than the reactive response to so-called pandemics. such antigens, once discovered, will, in time, and with careful manipulation and an appropriate delivery system and/or appropriate adjuvant, become first candidate subunit vaccines and, after proper clinical evaluation, the vaccines of tomorrow. there are, as we have outlined, many competing ideas, thoughts, and concepts that can help us in our quest. certain of these hypothesis we have had cause to outline, are indubitably persuasive, even compellingly convincing, yet in execution many such methods fall far short of our desires. no single approach, however promising, is able to deliver on its promise. the reason for this failure is comparatively simple if uncompromisingly unsatisfying; as we are dealing with over simplified abstractions, we cannot hope to capture what is necessary for prediction by looking in a relatively superficial way at a single contributory factor, since protein immunogenicity arises from many, many factors. this is not a bioinformatics problem that is easily solved; instead it is like protein secondary structure prediction which has resisted all attempts over many decades, so obscure and recondite and far removed from direct experience a problem as it is. factors mediating protein immunogenicity are many and include host-side properties -possession of b or t cell epitopes for example -and pathogen-side propertiesprotein expression levels and sub-cellular location -as well as its aggregation state and the possession of posttranslational danger signals. a candidate vaccine should be highly expressed, available for immune surveillance, and possess epitopes that the host recognises. predicting such diverse properties remains challenging, though several contributing factors can be reliably predicted. yet, no one factor is itself enough. what we need is an integrative, systems biology approach to the problem. as the poet maya angelou so neatly puts it: "we all should know that diversity makes for a rich tapestry, and we must understand that all the threads of the tapestry are equal in value no matter what their colour." no one method is universally applicable and successful; rather we need to integrate several equally-valid, equally-partial methods and draw from their synergy, useful, helpful data. this is the as yet unattained goal; yet as these are issues of such importance even a partial solution would be prize enough. computer-aided biotechnology: from immunoinformatics to reverse vaccinology new vaccines against tuberculosis computer-aided biotechnology: from immuno-informatics to reverse vaccinology harnessing bioinformatics to discover new vaccines the use of genomics in microbial vaccine development post-genomic vaccine development microbial genomes and vaccine design: refinements to the classical reverse vaccinology approach biotechnology and vaccines: application of functional genomics to neisseria meningitidis and other bacterial pathogens complete genome sequence of neisseria meningitidis serogroup b strain mc identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing use of a whole genome approach to identify vaccine molecules affording protection against streptococcus pneumoniae infection identification of a universal group b streptococcus vaccine by multiple genome screen identification of vaccine candidate antigens from a genomic analysis of porphyromonas gingivalis prediction of mhc-peptide binding: a systematic and comprehensive overview in silico tools for predicting peptides binding to hla-class ii molecules: more confusion than conclusion on evaluating mhc-ii binding peptide prediction methods evaluation of mhc-ii peptide binding prediction servers: applications for vaccine research a critical cross-validation of high throughput structural binding prediction methods for pmhc limitations of ab initio predictions of peptide binding to mhc class ii molecules antibody-protein interactions: benchmark datasets and prediction tools evaluation benchmarking b cell epitope prediction: underperformance of existing methods the immune epitope database . the curation guidelines of the immune epitope database and analysis resource immune epitope database analysis resource (iedb-ar) the immune epitope database and analysis resource: from vision to blueprint the design and implementation of the immune epitope database and analysis resource the immune epitope database and analysis resource: from vision to blueprint t cell receptor recognition of a 'super-bulged' major histocompatibility complex class ibound peptide high resolution structures of highly bulged viral epitopes bound to major histocompatibility complex class i. implications for t-cell receptor engagement and t-cell immunodominance have we cut ourselves too short in mapping ctl epitopes? a long, naturally presented immunodominant epitope from ny-eso- tumor antigen: implications for cancer vaccine design proteins accessible to immune surveillance show significant t-cell epitope depletion: implications for vaccine design are bacterial vaccine antigens t-cell epitope depleted? the prodom database of protein domain families: more emphasis on d the pfam protein families database prosite, a protein domain database for functional characterization and annotation single proteins might have dual but related functions in intracellular and extracellular microenvironments locating proteins in the cell using targetp, signalp and related tools improved prediction of signal peptides: signalp . a comprehensive assessment of nterminal signal peptides prediction methods wolf psort: protein localization predictor secreted protein prediction system combining cj-sphmm, tmhmm, and psort psort-b: improving protein subcellular localization prediction for gram-negative bacteria psort: a program for detecting sorting signals in proteins and predicting their subcellular localization predicting protein subcellular locations using hierarchical ensemble of bayesian classifiers based on markov chains subloc: a server/client suite for protein subcellular location based on soap gpos-ploc: an ensemble classifier for predicting subcellular localization of gram-positive bacterial proteins prediction of twinarginine signal peptides prediction of lipoprotein signal peptides in gram-negative bacteria advantages of combined transmembrane topology and signal peptide prediction-the phobius web server validating subcellular localization prediction tools with mycobacterial proteins toward bacterial protein sub-cellular location prediction: single-class discrimminant models for all gram-and gram+ compartments multi-class subcellular location prediction for bacterial proteins alpha helical transmembrane proteins: enhanced prediction using a bayesian approach beta barrel transmembrane proteins: enhanced prediction using a bayesian approach a predictor of membrane class: discriminating alpha-helical and beta-barrel membrane proteins from non-membranous proteins tatpred: a bayesian method for the identification of twin arginine translocation pathway signal sequences lippred: a web server for accurate prediction of lipoprotein signal sequences and cleavage sites combining algorithms to predict bacterial protein sub-cellular location: parallel versus concurrent implementations dbsubloc: database of protein subcellular localization predicting the subcellular localization of viral proteins within a mammalian host cell virus-ploc: a fusion classifier for predicting the subcellular localization of viral proteins within host and virus-infected cells syfpeithi: database for searching and t-cell epitope prediction syfpeithi: database for mhc ligands and peptide motifs hiv sequence databases mhcbn . : a database of mhc/tap binding peptides and t-cell epitopes mhcbn: a comprehensive database of mhc binding and non-binding peptides epimhc: a curated database of mhc-binding peptides for customized computational vaccinology antijen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data jenpep: a novel computational information resource for immunobiology and vaccinology jenpep: a database of quantitative functional peptide data for immunology bacterial virulence: can we draw the line? vfdb release: an enhanced webbased resource for comparative pathogenomics vfdb: a reference database for bacterial virulence factors candivf -candida albicans virulence factor database phi-base: a new database for pathogen host interactions antigendb: an immunoinformatics database of pathogen antigens the lipocalin protein family: structure and function structure and sequence relationships in the lipocalins and related proteins the lipocalin protein family: structural and sequence overview epitopic peptides with low similarity to the host proteome: towards biological therapies without side effects peptimmunology: immunogenic peptides and sequence redundancy primer: mechanisms of immunologic tolerance recent advances in immune modulation cutting edge: contributions of apoptosis and anergy to systemic t cell tolerance a computational approach for identifying pathogenicity islands in prokaryotic genomes identification and characterization of pathogenicity and other genomic islands using base composition analyses identification and characterization of specific sequences encoding pathogenicity associated proteins in the genome of commensal neisseria species prediction of pathogenicity islands in enterohemorrhagic escherichia coli o :h using genomic barcodes cpgcluster: a distance-based algorithm for cpg-island detection cpgif: an algorithm for the identification of cpg islands identifying cpg islands by different computational techniques cpg_mi: a novel approach for identifying functional cpg islands in mammalian genomes structure and sequence relationships in the lipocalins and related proteins structural relationship of streptavidin to the calycin protein superfamily vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines identifying candidate subunit vaccines using an alignment-independent method based on principal amino acid properties dna and peptide sequences and chemical processes multivariately modeled by principal component analysis and partial least-squares projections to latent structures principal property-values for nonnatural amino-acids and their application to a structure activity relationship for oxytocin peptide analogs peptide binding to the hla-drb supertype: a proteochemometrics analysis proteochemometrics mapping of the interaction space for retroviral proteases and their substrates proteochemometrics analysis of substrate interactions with dengue virus ns proteases generalized modeling of enzyme-ligand interactions using proteochemometrics and local protein substructures rough set-based proteochemometrics modeling of gprotein-coupled receptor-ligand interactions improved approach for proteochemometrics modeling: application to organic compound-amine g protein-coupled receptor interactions melanocortin receptors: ligands and proteochemometrics modeling proteochemometrics modeling of the interaction of amine g-protein coupled receptors with a diverse set of ligands peptide quantitative structure-activity-relationships, a multivariate approach multivariate parametrization of coded and non-coded amino-acids new chemical descriptors relevant for the design of biologically active peptides. a multivariate characterization of amino acids bioinformatic approach for identifying parasite and fungal candidate subunit vaccines. the open vaccine journal nerve: new enhanced reverse vaccinology environment dynavacs: an integrative tool for optimized dna vaccine design fell da: enzymes, metabolites and fluxes computer aided selection of candidate vaccine antigens we should like to thank the referees for their helpful an insightful comments. this article has been published as part of immunome research volume supplement , : computational vaccinology: state-of-the-art assessments. the full contents of the supplement are available online at http://www.immunome-research.com/supplements/ /s . the authors declare no competing financial interests. key: cord- - y fedcr authors: chang, christopher title: unmet needs in respiratory diseases: “you can’t know where you are going until you know where you have been”—anonymous date: - - journal: clin rev allergy immunol doi: . /s - - - sha: doc_id: cord_uid: y fedcr the care of patients with respiratory diseases has improved vastly in the past years. in spite of that, there are still massive challenges that have not been resolved. although the incidence of tuberculosis has decreased in the developed world, it is still a significant public health problem in the rest of the world. there are still over million deaths annually from tuberculosis, with most of these occurring in the developing world. even with the development of new pharmaceuticals to treat tuberculosis, there is no indication that the disease will be eradicated. respiratory syncytial virus, severe acute respiratory syndrome, and pertussis are other respiratory infectious diseases with special problems of their own, from vaccine development to vaccine coverage. asthma, one of the most common chronic diseases in children, still accounts for significant mortality and morbidity, as well as high health care costs worldwide. even in developed countries such as the usa, there are over , deaths per year. severe asthma presents a special problem, but the question is whether there can be one treatment pathway for all patients with severe asthma. severe asthma is a heterogeneous disease with many phenotypes and endotypes. the gene for cystic fibrosis was discovered over years ago. the promise of gene therapy as a cure for the disease has fizzled out, and while new antimicrobials and other pharmaceuticals promise improved longevity and better quality of life, the average life span of a patient with cystic fibrosis is still at about years. what are the prospects for gene therapy in the twenty-first century? autoimmune diseases of the lung pose a different set of challenges, including the development of biomarkers to diagnose and monitor the disease and biological modulators to treat the disease. the study and management of lung diseases is of concern to allergist/immunologists and pulmonologists. the most common chronic lung disease is asthma, but there is an immunological basis for many other lung diseases, which span a vast clinical spectrum of lung disorders ranging from infectious lung disease to cancer. several significant challenge areas include the diagnosis and treatment of certain specific infectious lung diseases, including viral lower respiratory infections caused by respiratory syncytial virus, rhinovirus, metapneumovirus, coronovirus, and enterovirus. severe acute respiratory syndrome (sars) coronavirus (sars-coa) was responsible for the outbreak of severe acute respiratory syndrome in the early s that originated in asia and led to significant mortality in those afflicted. other viruses such as bird flu and swine flu have the ability to cause respiratory tract disease as well. tuberculosis is another primarily respiratory infection that has been resistant to eradication. new strains of multidrug-resistant tuberculosis have emerged. other challenges involve the genetic diseases cystic fibrosis and alpha- -antitrypsin deficiency, autoimmune lung diseases, lung diseases that are part of a systemic autoimmune disease, and chronic obstructive pulmonary disease (copd). the diagnosis and treatment of hypersensitivity pneumonitis still poses problems to clinicians. biomarkers are continually being developed in lung and other cancers, but more research is needed [ ] . the genetics of cystic fibrosis are now well elucidated, but the development of a successful gene therapy has been unexpectedly slow (table ) . for many of these conditions, it was and still is expected that the ongoing development of new waves of molecular biology techniques, coupled with computerized automated analysis that can provide transcription signatures, will help identify differences between patients with these diseases and spur on the development of relevant and effective treatments. the subsequent discussion takes on some of the more interesting and challenging issues in respiratory disease over the past two decades, with a focus on what to expect in the future as well. asthma is one of the most common chronic diseases worldwide. the who estimates that there are over million asthmatics worldwide and , deaths per year. in the usa, there are million asthmatics, . million of whom are children. until recently, the mortality rates from asthma had been steadily increasing. it is only in the past - years that the mortality rates have begun to stabilize and are perhaps even on a decline. the increasing incidence of asthma has been attributed to various theories, perhaps the most popular of which is the "hygiene hypothesis" [ ] . this attributes the rise in incidence to the reduced rate of infectious diseases, cleaner living environments, and, in general, those features that accompany a higher standard of living. but, conversely, the annual death rates per , asthmatics tend to be higher in the economically poorer countries. this emphasizes the most significant global concern regarding asthma care, which is access to care and medications. in developed countries, this problem is less prevalent, and in countries such as the usa, we are primarily focused on asthma education and compliance issues. in both situations, severe asthma is still a problem in that it impacts quality of life and still contributes to considerable morbidity and mortality. one of the problems of severe asthma is related to defining the disease. asthma is inherently very heterogeneous, and many phenotypes of severe asthma have been described. it is increasingly clear that there is no single medication that works for all patients with severe asthma. while patient compliance is an issue, we frequently fall into the trap of believing the problem is always patient compliance, when in fact, it may be that patients are not taking their medications because they perceive that they do not work. nevertheless, the explosive growth in the availability of new pharmaceutical interventions provides us with an arsenal of controller medications, at least in the developed world. more medications are being developed every day, including the newer class of biological modulators against cytokines and chemokines [ ] , such as anti-il [ , ] and anti-il rα [ , ] . the next challenge will involve the identification of suitable patients for these new and existing treatments. but how will this be done? researchers over the years have attempted to define asthma phenotypes, trying to categorize asthma patients based on demographic and clinical characteristics in an attempt to define certain patient groups for certain preferential treatments [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in general, this has led to more confusion as the different research groups do not necessarily come up with the same classifications. moreover, asthma in children is also significantly different from that in adults, and the response to medications varies based on multiple and not a single factor. the choice and exclusion of confounding variables has been a thorn in the side of those attempting to define a specific asthma phenotype. it has also not been found that specific childhood asthma phenotype will develop into a specific adult asthma phenotype. the concept of asthma endotypes has now been the subject of much research because endotypes address the underlying mechanism of a disease [ ] [ ] [ ] [ ] . however, this is also confusing because of the highly redundant inflammatory pathways that can lead to disease. the use of biomarkers to identify those patients who are at high risk for severe, lifethreatening exacerbations has not been entirely fruitful. the recent commercial introduction of fractional exhaled nitric oxide (feno) to identify patients with eosinophilic inflammation is a potentially useful tool, but more is needed to guide treatment. the identification of a gene for asthma has revealed that this is more complex than one might have expected. there are now over genes that have been attributed to asthma. some of the more likely candidates include adam , ormdl , dennd b, filaggrin, chi l , and il- . moreover, the existence of shared genes between asthma and other comorbid conditions such as obesity has also been demonstrated [ ] . ultimately, asthma, like many other diseases, is a heterogeneous disease with many phenotypes and endotypes [ , ] . not one single treatment will fit all, and the art of medicine may be to find the right peg for the corresponding hole [ ] . the twenty-first century has ushered the concept of personalized or genomic medicine, which utilizes advancements in molecular biology, such as proteomics, metabolomics, and genomics, to identify the optimal therapy for each individual patient. cystic fibrosis-what happened to gene therapy? it has now been years since the identification of the gene responsible for cystic fibrosis (cf) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the initial study describing the gene named "cystic fibrosis transmembrane conductance receptor (cftr)" was first published in the late s by tsui et al. [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this discovery led to a localization of the gene to chromosome , leading to widespread belief that a cure for cf utilizing gene therapy was right around the corner. the landmark development of a molecular biology technique which became known as genetic targeting in mice, pioneered by capecchi [ ] [ ] [ ] [ ] , evans [ , ] , and smithies [ ] [ ] [ ] [ ] [ ] , promised gene replacement in humans in a similar way to that performed in mice. twentyfour years later, there are new advancements in the management of cystic fibrosis with regard to pharmacological and supportive respiratory treatment, but still no cure. these advancements have prolonged the longevity of patients with cf, but their life expectancy still only averages to be about years. so, what happened to gene therapy? what happened to all that promise that buoyed the cystic fibrosis community back in the s? the idea behind gene therapy is to introduce a missing of malfunctioning gene into the cells of a patient using a "harmless" virus that can be manufactured to carry a normal copy of the diseased gene. this technique can either target cells globally or be restricted to a certain group or location of cells. but gene therapy has not progressed as smoothly or as quickly as anticipated. previously unrecognized barriers became apparent [ ] . these included the fact that studies were initially focused on molecular or biochemical outcomes and not on clinical efficacy. moreover, the administration of the gene would need to be repeated due to epithelial turnover. repeated administration leads to host recognition, which may inhibit gene expression. questions on which cells to target in order to achieve efficacy has slowed research. the use of viral material, including viral dna and liposomes, could potentially lead to inflammatory responses. the point is that unexpected consequences were discovered as research proceeded, and this has had an impact on the progress of gene research. in , a group of british investigators began large-scale trials of gene therapy delivered by encompassing the gene in fat globules and delivering the gene by nebulization. future methodologies will utilize a viral delivery strategy, but this is still several years into the future. gene therapy for cystic fibrosis is not dead, but certainly moving at a far slower pace than originally expected. current treatments are not curative for cystic fibrosis. gene therapy and stem cell transplants are two techniques that are still under investigation, and it may turn out that only certain mutations may be candidates for gene therapy. mutations in the cf gene have in fact been subclassified into six classes [ ] , each with its own pathogenic or physiologic characteristics. for example, the common f del mutation results in reduced amounts of cftr channel expression, which leads to exacerbation of the disease [ ] . respiratory syncytial virus-how to develop an effective vaccine? respiratory syncytial virus is an infection of the lower respiratory tract that causes significant morbidity and mortality in infants and young children [ ] . globally, there are over million new lower respiratory tract infections per year and approximately , deaths per year in children fewer than years of age. the majority of these deaths occur in lowincome countries where access to care may be limited. the search for a vaccine for respiratory syncytial virus (rsv) has been ongoing for many years, but like the previous case of gene therapy in cystic fibrosis, this also has been a challenge to achieve. in the absence of a vaccine, researchers have developed passive immunization to the virus in the form of a monoclonal antibody to rsv, named palivizumab. the current global strategies for the development of an rsv vaccine now target four areas: infants < months of age; infants > months of age and young children; pregnant women for whom passive immunization can be implemented; and the elderly, in whom rsv can also have significant morbidity [ ] [ ] [ ] . the main challenges that have prevented the development of an effective vaccine so far revolve around the fact that even natural infection to rsv does not provide long-term protection from reinfection. an earlier study actually found that the vaccine may actually accentuate the disease, especially in young infants who may not have a fully mature immune system and may be unable to effectively engage in somatic mutation, leading to a suboptimal b cell repertoire [ , ] . this may also apply to older infants or young children who may still be rsv-naive. the issues in adults are just the opposite as most adults have been exposed to rsv and thus have rsv antibodies. the effectiveness of passively immunizing pregnant women in order to deliver igg across the placenta to the fetus must be weighed against possible adverse effects of such a "vaccine" on the fetus. in the elderly, the challenge has been the ability to boost immunity through active immunization in an individual who is becoming immunosenescent. there are now several vaccine development programs that pursue a variety of vaccine strategies. a live virus vaccine trial has shown that a particular vaccine candidate, medi- , is safe, although effectiveness has not been proven [ ] . other strategies include subunit rsv vaccines, the use of dnaconjugate vaccines to boost antigen presentation, and further development of passive antibody prophylaxis that is focused on f or g proteins [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . an additional challenge with all of these strategies is that effective vaccine studies in animals have not been translated into successful human trials. tuberculosis is a global health problem. tuberculosis is an infection caused by the bacterium mycobacterium tuberculosis, which primarily affects the lung, but can also affect other tissues, including bone and the nervous system. tuberculosis is believed to be one of the oldest infections to reportedly affect mankind, with archeological and anthropological studies showing evidence of infection in humans over , years ago. it continues to top the charts of mortality and morbidity in developing nations. it is estimated that in , there were . million new cases of tb, as well as a prevalence of . million. the . million deaths in from tb in patients without hiv and , deaths in patients with hiv is a shocking statistic that shows how far we have come, yet how far we still are from effectively eradicating the disease [ ] . two very effective drugs have been developed to treat tuberculosis, isoniazid (inh) and rifampin. however, tuberculosis continues to be a global health problem. tuberculosis is second to hiv/aids as the infection with the highest mortality globally. more recently, an increase in multiple drug-resistant tuberculosis (mdr-tb) has been observed, with most cases from india, china, and russia. there were , reported cases of mdr-tb in the world in . of these, it is estimated that . % fall into the category of xdr-tb, which is an even more resistant form. thus, drug resistance is a significant challenge in the treatment of tb in the twenty-first century. another challenge stems from the fact that about one third of patients with hiv/aids are infected with tb, although many may not yet be symptomatic. the who defines six core functions and six strategic approaches to combat tb (table ) . overall, % of tb cases occur in just countries, and % of cases occur in china. however, as overall healthcare accessibility and technology improves, there have been significant declines in the rate of tb in some of the asian countries, including cambodia and china. there is no dispute that the dots and "stop tb strategy," as outlined in table , have contributed to significant progress in the control of tb, but significant challenges remain. one of the areas that are being focused on now has to do with the identification of biomarkers. biomarkers can play several roles in the improvement of treatment of tb in the world. the main areas of focus for research in biomarkers are to identify those that can perform the following functions: ( ) prediction of a curative state, ( ) prediction of reactivation of tb, and ( ) prediction of immunity to tb. in the past, biomarkers for tb would normally involve culturing for the organism, but the development of molecular biology techniques has afforded us the use of non-culture biomarkers. the types of approaches, as described by wallis et al. in [ ] , categorize biomarker development into functional categories ( table ) . non-culture biomarkers include cytokine levels, quantifiable genetic measures of the presence of tb, other serological tests, imaging techniques, gene transcription profiles, and micro-rna profiles. the development of a vaccine has been an ongoing challenge in the quest for a cure for tb, and limitations in these studies include the lack of valid biomarkers to assess protection in clinical trials. the cytokines interferon-γ and il- have been studied as biomarkers for latent tb infection. a tuberculosis-stimulated interferon-γ release assay can detect sensitization to tb antigens, but, unfortunately, cannot differentiate between resolved and persistent latent infection. interleukin- is a marker of innate immunity. elevated levels of plasma interleukin levels seen in tb patients indicate increased activation of innate immunity, which was not observed in controls. a chemokine that was observed to be elevated in tb were ccr [ ] , while the levels of the apoptosis inhibitor bcl expression were reduced [ , ] . il- levels actually correlated with the radiographic extent of the disease [ , ] . hiv appears also to modify cytokine production in patients with tb [ ] . other cytokines and chemokines have been profiled as potential biomarkers for tb disease activity [ ] [ ] [ ] [ ] [ ] [ ] , although to this date, none has been shown to be useful for widespread clinical application. fifty years ago, it was the introduction of pyrazinamide and rifampicin that reduced the duration of therapy from months to months. the value of biomarkers may be reflected in the implications of being able to predict cure and latency of infection, as well as active infection. from a therapeutic standpoint, this may mean the difference between the current regimen of months of treatment versus a possible reduction in the duration of therapy for certain patient groups. but this depends on the biomarker being significantly accurate as to not lead to inadequate treatment of affected individuals. though a half century has passed since the development of effective treatment for drug-susceptible tb, the period of treatment is still at least months of standard therapy, i.e., rifampicin and isoniazid for months and pyrazinamde and ethambutol during the initial months of treatment. efforts to reduce the length of treatment are dependent on the discovery of more effective treatment as well as being able to monitor the treatment. at the same time, more and more patients with tb are demonstrating multiple drug resistance forms including mdr or xdr tb. the addition of second-line drugs for the treatment of these more difficult to treat patients is plagued by longer periods of treatment, higher cost, and reduced efficacy. the drugs often used for second-line treatment include aminoglycosides, terizidone, protionamide, capreomycin, fluroquinolones, cycloserine, and ethionamide. the mortality of mdr and xdr tb in hiv patients is particularly high, and researchers have focused on novel approaches to combat this global health issue, including revamping of efforts to develop a vaccine for tb [ ] [ ] [ ] [ ] [ ] [ ] , treatment of latent infection to reduce reservoir pools of infection, and the development of predictive biomarkers. one should not forget that for years we have had a vaccine for tb, the bcg vaccine named after albert calmette and camille guerin. while this vaccine has been used primarily in underdeveloped countries and has been administered to over billion people worldwide, the limitations include an inability to protect against adult tb and adverse effects when used in hiv-positive tb patients. further research is ongoing to develop an improved replacement or enhancement vaccine for bcg [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . therefore, as we develop new biomarkers and new treatment strategies, one should remember that the infective landscape of tb is not a static target either. the emergence of multidrug-resistant strains as mentioned above means that the disease may become more difficult to treat and that the development of biomarkers must take new medication use into consideration. moreover, the development of biomarkers requires the availability of large patient samples, such as those that can be found in biobanks of large high-volume treatment centers. the willingness of patients to contribute to research, and the regulatory landscape for the collection of samples for research, could potentially negatively impact the ability to conduct large-scale studies on biomarkers. for a comprehensive review of biomarkers, diagnostics, drug treatment, and vaccine development, the reader is referred to an excellent series of articles published in lancet in [ , , ] . the incidence of pertussis appears to be increasing over the past years. this trend flies in the face of an availability of, first, the killed whole organism pertussis vaccine dtwp and, subsequently, the acellular vaccine (dpap). the acellular vaccine was less reactogenic because it incorporated a step during manufacturing that removed endotoxin, which was a major reason for the adverse side effect profile. it was clear that vaccination against pertussis afforded much improved prevention of the disease, as illustrated by the -fold decreased rate for pertussis between the s and s. dtwp vaccines were used until the s, when technological development in the s and s led to the introduction of commercially available acellular pertussis vaccines [ ] . the continuing increase in pertussis cases leads to an obvious question. is this a failure of an immunization program? earlier, we discussed several examples that illustrate the difficulty of bringing an effective vaccine to market (tb and rsv). the problems associated with pertussis are clearly different. there are several reasons that may explain why we have not eradicated the disease. it is well documented that the dtap vaccine is not as immunogenic as the whole organism dtwp vaccines. studies have indicated that the overall [ ] efficacy of dtap vaccines is below %. we do not normally test for pertussis titers following vaccination, except in studies. but there are those who suggest that the increase in pertussis cases is mainly due to the increased ability to diagnose the disease and to an increased awareness. yet another challenge may be that pertussis has mutated over the years, rendering the vaccine less effective. because our current vaccine is < % effective, the ability of mutant strains to evade destruction would lead to their preferential survival. it has been shown that pertussis vaccine which contains more antigens, such as the five-component vaccine that contains pertussis toxin (pt), filamentous hemagglutinin (fha), pertactin (prn), and fimbriae (fim / ), is superior to those containing fewer antigens. it has also become apparent that the balance of antigens in the vaccine may play a role in determining efficacy. for example, a whole-cell pertussis vaccine with antigenic components pt, fha, and fim was compared to an acellular pertussis vaccine in children. the whole-cell vaccine produced high levels of antibodies to prn and fim, but low levels to pt and fha, in contrast to the acellular pertussis vaccine which produced high levels of antibody to pt and fha. more than one study has suggested that the vaccine components may antagonize each other. it has been found that the antibody levels to fha do not correlate with the efficacy of the vaccine; furthermore, it has been suggested that adding fha to a vaccine may actually suppress efficacy. linked epitope suppression is another concept to explain the failure of pertussis vaccines. while one would expect that the presence of antigens that do not deliver a brisk vaccine response should not affect the response to other vaccine antigens, this may not be the case. if there were a linkage suppression between two epitopes on the pertussis vaccine, then this could be a significant consideration in vaccine design as one would then need to exclude certain antigens from the vaccine component mix. suffice to say that vaccine development is a very complex topic, and it involves various techniques, including the use of classical adjuvants such as aluminum salts, emulsions or liposomes, or novel adjuvants such as toll-like-receptor agonists, saponins, or immune stimulating complexes [ ] . one cannot discuss challenges in pertussis without commenting on the fear of immunizations held by many families, even in developed countries such as the usa. the refusal of many parents to immunize their children, for whatever reason they may have, plays a significant role in our inability to eradicate several diseases, including pertussis, measles, rubella, and so on. this phenomenon is not limited to the uneducated population as many highly educated individuals appear to choose not to subject their children to the relatively miniscule real or imagined risks of immunizations. certainly, one has an astronomically higher risk of being killed in a car accident than dying from a vaccination. and yet, people who refuse to vaccinate their children do not stop driving or riding in cars. the problem with all this is that achieving "herd" immunity is important in total eradication. this means that if only a very small portion of the population do not immunize their children, there is still a possibility of eradicating a disease, but as more people choose to remain unimmunized, the likelihood of successful eradication becomes less and less. unfortunately, people who choose to remain unimmunized fail to understand that in order that everyone not have to get immunized in the future (as in the case of smallpox), everyone must be immunized in the present [ ] . severe acute respiratory syndrome, or sars, was an infection that reached epidemic proportions during the early part of the s. it originated in china and rapidly spread to neighboring countries, and eventually, it was reported in many other distant nations, including europe and the usa. although the number of cases was far fewer, by several orders of magnitude, than the common flu, it caught the attention of healthcare professionals and public health officers because of its high mortality, with , cases and deaths reported between and , for a mortality rate of . %. sars was later identified to be caused by a coronavirus and was named sars-coa. travel histories of infected people were instrumental in tracing the origin of the disease [ ] . but where did this virus come from and how did it suddenly cause so much havoc? initially, in may of , the virus was traced to civets, a cat-like mammal that is occasionally consumed as food by the chinese. however, these creatures were eventually dismissed as the original source because of the lack of appearance of further infected civets. in , reservoirs of sars-like viruses were found in chinese horseshoe bats. these were often brought to market, and it was thought that the virus was passed to humans at that time. phylogenetic studies further provided evidence that genetically, it was likely that the sars coronavirus evolved from viruses that infected the horseshoe bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . so can sars happen again? in fact, smaller outbreaks that do not attract as much attention may already be occurring in a manner that mimics sars. a middle east respiratory syndrome or mers has been reported to also be caused by a coronavirus. the mortality in this case is high as well, of cases, or > % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . mers is also thought to have originated in bats. interestingly, many other viruses are thought to be transmitted by bats, including ebola, hendra, and rabies. the cellular receptor for mers is cd , or dipeptidyl peptidase [ ] . lessons learned from the sars epidemic included the fact that transmission can often be facilitated by the very medical workers who are trying to save patients [ , ] . strict infection control measures are critical in limiting the spread of the disease, including the use of negative pressure rooms, n masks, and gowns. the optimal dosing of antiviral medications and the timing of supportive measures of respiratory care are still unknown, but previous experience should guide us to be more vigilant if this ever occurs in a large-scale fashion in the future. autoimmune lung diseases-biomarkers and biologics-sparing the steroids? anti-neutrophil cytoplasmic antibody (anca) was discovered in [ , ] . the antigenic targets of anca include myeloperoxidase or proteinase- , and the antibodies against these antigens were for a long time referred to as p-anca and c-anca, respectively, the p and c indicating their cellular staining pattern (perinuclear or cytoplasmic). this latter nomenclature has since been discouraged, and the antibodies should be referred to in the context of their specific antigenic target [ , ] . an understanding of the pathogenesis of anca-associated vasculitis (aav) would help in the development of more effective drugs to treat this group of diseases [ ] , which include granulomatosis with polyangiitis (formerly wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (egpa or churg-strauss disease), and microscopic polyangiitis [ ] . for toxin-mediated aav, a meta-analysis has shown a link between crystalline silica and aav [ ] . silica is an inflammasome activator and is believed to trigger the activation of pro-inflammatory cytokines leading to the disease. other triggers include drugs, most commonly hydralazine, propylthiouracil, and penicillamine (all of which are known to stimulate b lymphocyte activation), and infectious trigger such as staphylococcus aureus. avoidance of exposure may prove to be a useful methodology to prevent disease, but as of this time, the triggers of aav are too diverse and too controversial to recommend any particular avoidance strategy. another disease state that affects millions, perhaps billions, of people worldwide is allergies. allergies that affect the respiratory tract can take the form of allergic asthma or simply allergic rhinitis and conjunctivitis. the hygiene hypothesis would indicate that because of our cleaner living environments, we are developing higher rates of sensitivities to common allergens, both indoor and outdoor, as well as higher rates of autoimmune diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one of the main avenues of treatment has always been avoidance, but we have not really been able to define what exactly constitutes effective avoidance. many of the studies done on dust mite and animal dander avoidance have not been able to identify a single avoidance measure that can make a significant impact on outcome, although perhaps a comprehensive avoidance plan may provide some relief. studies are needed to ( ) determine whether indeed effective avoidance is possible in the face of indoor and outdoor exposures to allergens and ( ) the extent to which avoidance of allergens, if possible, provides a significant benefit to patients. the diagnosis and management of respiratory diseases is plagued by numerous unmet needs, affecting both common diseases such as asthma to uncommon occurrences such as sars. some of the problems are common and ultimately involve improving our understanding of the mechanisms of disease. only then can we develop tools to help us manage these patients. the advent of molecular biology has allowed us to develop genetic analyses of patients, and this has led to a realization that these diseases are frequently not one disease, but many [ ] . thus, the genomic and personalized medicine acts of and are important legislatures that will hopefully divert increased funding into studies that will help us identify which treatment should optimally be used on which patient [ , ] . understanding the pathogenesis of these diseases is another significant unmet need which requires commitment of funding and resources [ ] . the respective contributory roles of genes, epigenetics, and the environment in the pathogenesis of lung diseases must be better elucidated [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . with better understanding of disease comes better diagnostic markers and better treatment. other unmet needs circle around the availability of biomarkers to both diagnose disease and vaccines and other novel treatments to manage disease. new techniques for vaccination which will hopefully alleviate concerns of vaccines among vaccine-apprehensive patients may help lead to the eradication of infectious respiratory disease such as tb, pertussis, and rsv. there is much work yet to be done, and it is important to remember that diseases are not static either, especially in the context of pathogen-related diseases as pathogens are also very adept at evading our efforts to destroy them. the challenges outlined in this article are far from comprehensive, and specific unmet needs exist for a variety of other respiratory diseases, including alpha- -antitrypsin deficiency, hypersensitivity pneumonitis, chronic obstructive pulmonary disease, lung cancer, pulmonary hypertension in the newborn, and others. while each disease state may have its own unique set of challenges, in general, one can categorize these into three major areas of need, namely, diagnosis, treatment, and prevention (fig. ). more specifically, these would include the identification of biomarkers to provide less invasive methods for the diagnosis and monitoring of the disease, as well as determining prognosis, the development of new drugs and treatment modalities, and also the development of vaccines or other environmental controls to reduce the incidence of the disease. autoantibodies to tumor-associated antigens as biomarkers in cancer immunodiagnosis early life exposure to antibiotics and the risk of childhood allergic diseases: an update from the perspective of the hygiene hypothesis chemokines and their receptors in the allergic airway inflammatory process profile of reslizumab in eosinophilic disease and its potential in the treatment of poorly controlled eosinophilic asthma mepolizumab in eosinophilic disorders inhibiting interleukin- and interleukin- in difficult-to-control asthma dupilumab in persistent asthma with elevated eosinophil levels the practical understanding and treatment of asthma asthma in children and adolescents: a comprehensive approach to diagnosis and management asthma: developments in targeted therapy asthma and pregnancy the patient with asthma in the emergency department occupational asthma the critically ill asthmatic-from icu to discharge the challenge of asthma in minority populations the adult asthmatic characterizing wheeze phenotypes to identify endotypes of childhood asthma, and the implications for future management multiple atopy phenotypes and their associations with asthma: similar findings from two birth cohorts methylation of il- promoter at birth alters the risk of asthma exacerbations during childhood asthma endotypes: the right direction towards personalized medicine for asthma analyses of shared genetic factors between asthma and obesity in children symptom-adjusted therapy in asthma: it is time to listen to our patients physical localization of two dna markers closely linked to the cystic fibrosis locus by pulsed-field gel electrophoresis identification of the cystic fibrosis gene: genetic analysis the search for the cystic fibrosis gene tracing the mutations in cystic fibrosis by means of closely linked dna markers progress towards cloning the cystic fibrosis gene in situ hybridization of two cloned chromosome sequences tightly linked to the cystic fibrosis locus molecular approaches to the cystic fibrosis gene cystic fibrosis locus defined by a genetically linked polymorphic dna marker a polymorphic dna marker linked to cystic fibrosis is located on chromosome cystic fibrosis: analysis of linkage of the disease locus to red cell and plasma protein markers cystic fibrosis: progress in mapping the disease locus using polymorphic dna markers. i genetic analysis of cystic fibrosis using linked dna markers mapping of the cystic fibrosis locus on chromosome progress towards cloning the cystic fibrosis gene gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century how close are we to implementing gene targeting in animals other than the mouse? targeted gene replacement the new mouse genetics: altering the genome by gene targeting production of a severe cystic fibrosis mutation in mice by gene targeting disruption of the cystic fibrosis transmembrane conductance regulator gene in embryonic stem cells by gene targeting gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension targeted gene duplication and disruption for analyzing quantitative genetic traits in mice toward an animal model of cystic fibrosis: targeted interruption of exon of the cystic fibrosis transmembrane regulator gene in embryonic stem cells gene conversions and their relation to homologous chromosome pairing direct alteration of a gene in the human genome gene therapy for the treatment of cystic fibrosis assessing the disease-liability of mutations in cftr progress in cystic fibrosis and the cf therapeutics development network respiratory syncytial virus-a comprehensive review the path to an rsv vaccine strategic priorities for respiratory syncytial virus (rsv) vaccine development influenza and respiratory syncytial virus (rsv) vaccines for infants: safety, immunogenicity, and efficacy understanding respiratory syncytial virus (rsv) vaccine-enhanced disease o l s z e w s k a w ( ) immunopathogenesis of vaccine-enhanced rsv disease nonclinical phenotypic and genotypic analyses of a phase pediatric respiratory syncytial virus vaccine candidate medi- (ra cp / / deltash) at permissive and non-permissive temperatures rsv vaccine in development: assessing the potential costeffectiveness in the dutch elderly population translational sciences approach to rsv vaccine development a protective and safe intranasal rsv vaccine based on a recombinant prefusion-like form of the f protein bound to bacterium-like particles implication of respiratory syncytial virus (rsv) f transgene sequence heterogeneity observed in phase evaluation of medi- , a live attenuated parainfluenza type vectored rsv vaccine the recent progress in rsv vaccine technology new insights for development of a safe and protective rsv vaccine challenges in developing a pediatric rsv vaccine protective effect of a rsv subunit vaccine candidate g f/m was enhanced by a hsp -like protein in mice development of a piv-vectored rsv vaccine: preclinical evaluation of safety, toxicity, and enhanced disease and initial clinical testing in healthy adults immunogenicity and efficacy of recombinant rsv-f vaccine in a mouse model effects of alveolar macrophage depletion on liposomal vaccine protection against respiratory syncytial virus (rsv) evaluation of the live attenuated cpts / rsv vaccine in combination with a subunit rsv vaccine (pfp- ) in healthy young and older adults replacement of the f and g proteins of respiratory syncytial virus (rsv) subgroup a with those of subgroup b generates chimeric live attenuated rsv subgroup b vaccine candidates immunological properties of plaque purified strains of live attenuated respiratory syncytial virus (rsv) for human vaccine current approaches to the development of vaccines against disease caused by respiratory syncytial virus (rsv) and parainfluenza virus (piv). a meeting report of the who programme for vaccine development tuberculosis: a global health problem biomarkers and diagnostics for tuberculosis: progress, needs, and translation into practice expression of cxc and cc type of chemokines and its receptors in tuberculous and nontuberculous effusions identification of probable early-onset biomarkers for tuberculosis disease progression immunohistochemical analysis of cytokines and apoptosis in tuberculous lymphadenitis serum il- levels in tuberculosis: comparison with pneumonia, lung cancer and healthy controls il- production in human pulmonary and pleural tuberculosis hiv alters plasma and m. tuberculosisinduced cytokine production in patients with tuberculosis production of tnf-alpha, il- (p ) and il- can discriminate between active tb disease and latent infection in a west african cohort nk cell activity in tuberculosis is associated with impaired cd a and icam- expression: a regulatory role of monocytes in nk activation impaired interleukin- beta converting enzyme (ice) activity in patients with pulmonary tuberculosis profiles of ifn-gamma and its regulatory cytokines (il- , il- and il- ) in peripheral blood mononuclear cells from patients with multidrug-resistant tuberculosis stimulation of dendritic cells via cd enhances immune responses to mycobacterium tuberculosis infection depressed interleukin- (il- ), but not il- , production in response to a -or -kilodalton mycobacterial antigen in patients with active pulmonary tuberculosis toward novel vaccines against tuberculosis: current hopes and obstacles ten years of the global alliance for vaccines and immunization: challenges and progress new vaccines for tuberculosis novel tuberculosis vaccines on the horizon tuberculosis: current state of knowledge: an epilogue : the year in review. part ii: tuberculosis and lung disease history of bcg vaccine genome sequencing and analysis of bcg vaccine strains tuberculosis vaccine trials tuberculosis vaccine trials the current state of tuberculosis vaccines vaccine development for tuberculosis: current progress tuberculosis vaccines: time to reset the paradigm? bettering bcg: a tough task for a tb vaccine global tuberculosis drug development pipeline: the need and the reality pertussis: challenges today and for the future unmet needs in modern vaccinology: adjuvants to improve the immune response immunization coverage levels among -to -month-old children in diverse, medically underserved areas of the united states severe respiratory syndromes: travel history matters detection of coronaviruses in bats of various species in italy ecology, evolution and classification of bat coronaviruses in the aftermath of sars isolation and characterization of a bat sars-like coronavirus that uses the ace receptor clinical management and infection control of sars: lessons learned severe acute respiratory syndrome (sars): lessons learnt in hong kong tracing the sars-coronavirus from sars coronavirus to novel animal and human coronaviruses middle east respiratory syndrome coronavirus (mers-cov): a perpetual challenge middle east respiratory syndromecoronavirus infection: an overview a novel human coronavirus: middle east respiratory syndrome human coronavirus update: recommendations for middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome: new disease, old lessons middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread middle east respiratory syndrome coronavirus (mers cov): update first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide from sars to mers: years of research on highly pathogenic human coronaviruses molecular basis of binding between novel human coronavirus mers-cov and its receptor cd anti-neutrophil cytoplasmic antibody (anca)-associated vasculitis: where to go? pathogenesis of anca-associated vasculitis, an update antineutrophil cytoplasmic autoantibodies: how are they detected and what is their use for diagnosis, classification and follow-up? treatment of anca-associated vasculitis, where to go? pathogenesis of ancaassociated vasculitis the association between silica exposure and development of ancaassociated vasculitis: systematic review and meta-analysis hydroxychloroquine: from malaria to autoimmunity the hygiene theory harnessing helminths and their ova to treat autoimmunity hygiene hypothesis and autoimmune diseases the role of the environment in the development of pediatric inflammatory bowel disease do infections facilitate the emergence of systemic sclerosis? air pollution in autoimmune rheumatic diseases: a review the role of environmental estrogens and autoimmunity systemic lupus erythematosus one disease or many? the genomics of autoimmune disease in the era of genome-wide association studies and beyond the genomics and personalized medicine act of genes, epigenetic regulation and environmental factors: which is the most relevant in developing autoimmune diseases? the influence of sex and gender on the immune response genes, tolerance and systemic autoimmunity the x chromosome and immune associated genes twin studies in autoimmune disease: genetics, gender and environment genetics of asthma susceptibility and severity the genetic and environmental causes of pulmonary fibrosis epigenomics of idiopathic pulmonary fibrosis who's new stop tb strategy key: cord- -wfakzb w authors: trovato, maria; sartorius, rossella; d’apice, luciana; manco, roberta; de berardinis, piergiuseppe title: viral emerging diseases: challenges in developing vaccination strategies date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: wfakzb w in the last decades, a number of infectious viruses have emerged from wildlife or re-emerged, generating serious threats to the global health and to the economy worldwide. ebola and marburg hemorrhagic fevers, lassa fever, dengue fever, yellow fever, west nile fever, zika, and chikungunya vector-borne diseases, swine flu, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and the recent coronavirus disease (covid- ) are examples of zoonoses that have spread throughout the globe with such a significant impact on public health that the scientific community has been called for a rapid intervention in preventing and treating emerging infections. vaccination is probably the most effective tool in helping the immune system to activate protective responses against pathogens, reducing morbidity and mortality, as proven by historical records. under health emergency conditions, new and alternative approaches in vaccine design and development are imperative for a rapid and massive vaccination coverage, to manage a disease outbreak and curtail the epidemic spread. this review gives an update on the current vaccination strategies for some of the emerging/re-emerging viruses, and discusses challenges and hurdles to overcome for developing efficacious vaccines against future pathogens. since the start of this century, a certain number of new or neglected pathogens have emerged from wildlife reservoirs and spilt over into human populations, causing severe diseases ( - ). factors such as urbanization, globalization, travels, international commerce, aging, and climate changes have contributed to favor emergence, spread, and transmission of pathogens. contacts among humans and potential zoonotic reservoirs are increasing, the number of travelers and their movements is growing, the aged population are more susceptible to infections, and the geographic distribution of pathogens within a previous endemic zone is changing ( , ) . during the last decades, the global community faced several outbreaks of emerging and reemerging infectious diseases, with high threats to the health security, biodefense, and economy worldwide ( , ) . the occurrence of significant disease outbreaks-such as sars (severe acute respiratory syndrome) originating in china in ( ) , the h n swine flu pandemic from mexico ( ) , mers (middle east respiratory syndrome) that occurred in saudi arabia in ( ) , the west african outbreak of ebola virus (ebov) in late ( ) , the zika virus (zikv) outbreak originating in brazil in ( ) , the health emergence in nigeria caused by lassa virus ( ) , and the ongoing coronavirus disease (covid- ) pandemic ( ) -has renewed interests in developing strategies to faster prevent, treat, and/or control emerging and re-emerging viruses with high epidemic potential. usually, there is little or no knowledge about identity, epidemiology, and pathogenesis of a new infectious agent appearing for a first time in a certain geographic area (as in case of novel coronaviruses or new influenza variants), as well as the potential to spread out from the zoonotic reservoir, making hard to predict if, where, and when a disease outbreak will occur. the world health organization (who) and the national institutes for allergy and infectious diseases (niaid) published a list of pathogens to be prioritized for research and development, given their epidemic potential. this non-exhaustive list comprises viruses, bacteria, protozoa, and fungi, causing diseases for which efficient countermeasures do not actually exist, or require new therapeutics ( , ) . as proven by historical records, vaccination has played a pivotal role in reducing morbidity and mortality from devastating infectious diseases, successfully leading to disease eradication (i.e., smallpox), and generally decreasing infectious disease burdens. even in presence of therapeutic options, vaccines are the valuable means to prevent infections and overall represent the much wanted achievement. however, even with worldwide efforts, getting a vaccine to the public takes time, and side effects, dosing issues, and manufacturing problems can all cause delays. thus, we have to use this time with great concern. generally speaking, in case of newly emergent diseases, conventional strategies might raise some issues. the unpredictable identity of largely unknown emerging pathogens, the lack of appropriate experimental animal models, and the time and costs for faster developing, producing, licensing, and globally distributing effective vaccine candidates are some of the major challenges to overcome in case of pandemic threats. hence, new and/or alternative approaches in vaccine design and development are required to rapidly face outbreak situations ( ) . this review will discuss the current vaccination strategies for some of the emerging and re-emerging viruses, as well as the approaches that might be suitable in face of global pandemic threats. emerging and re-emerging pathogens represent a constant epidemic threat to humanity not only for the public health consequences but also for the economic, social, and political effects they may globally provoke. therefore, a major public awareness and preparedness would be fundamental in fighting emerging infectious diseases. the terms "emerging and reemerging infectious diseases" mainly refer to two major categories of infectious diseases: newly emergent infections, caused by novel pathogens; and re-emerging infectious diseases, caused by microbes reappearing after previous control, and/or eradication ( ). almost % of emerging infectious diseases are zoonoses, with the great majority of them originating in wildlife, and the number is constantly increasing. climate changes have been related to the emergence of vector-borne diseases in severe environmental conditions, but this is a most debated issue, as well as the contribution of agricultural practices ( ) . in addition, the chances of infectious disease spreading could also include livestock/wildlife animal markets and consumption of those. a study where australia was used as a model of urbanization has proposed a relation among increasing pandemic threats and urbanization: it ascribes the increased threat of pandemic to the high number of major city residents, the exponential intensification of international air traffic, and the commuter mobility network ( ) . basically, an epidemic is an event that occurs when there is an increase, often sudden, in the frequency of a disease above what is normally expected in that population, in that area; while pandemic (from: παν = all, and δεµoσ = people) refers to an epidemic that spreads over several countries or continents at the same time, usually affecting a large number of people ( ). in the last decades, a certain number of viruses came to light for the first time or reappeared, giving rise to significant epidemics and pandemics (figure and table ) . epidemic outbreaks of viral diseases were mostly caused by flaviviruses generally transmitted by vectors, including west nile virus (wnv) ( , ) , zikv ( , ) , yellow fever virus (yfv) ( , ) , and dengue virus (denv) ( , ) . vector-borne diseases, including chikungunya fever caused by chikungunya alphavirus (chikv) ( , ) , are extremely difficult to eradicate because viruses are maintained in nature by propagation among vectors and hosts, without human-human contact. moreover, dry and hot climate conditions seem to foster mosquitoes to bite humans than animals, increasing the risk of spreading diseases with a devastating impact ( ) . today, most areas of the world are endemic for at least one flavivirus, with denv being the most prevalent, and approximately - million people are infected each year. among viral hemorrhagic fevers, lassa fever (lf) is a rodent-borne acute disease caused by lassa virus (lasv) ( ) , endemic in many west african countries, including nigeria that experienced a high mortality rate in the outbreak ( ) . ebola virus disease (evd) and marburg virus disease (mvd) are caused by members of the filoviridae family, ebov ( ), and marburg virus (marv) ( ), respectively. the - ebola outbreak in west africa was the largest since the virus was first discovered in , with a case fatality rate for zaire ebolavirus of % ( ) , while the largest recorded mvd outbreak occurred in angola in ( ) . concerning pandemics, flu pandemics were reported three times during the twentieth century; genome analysis of pandemic influenza viruses dated (h n ), (h n ), and (h n ) demonstrated that all viral strains fully or partially originated from non-human reservoirs, and that the ultimate origin of ha (hemagglutinin) genes are from avian influenza viruses ( ) , with the strain likely being the ancestor of the subsequent epidemic variants. hence, the influenza pandemic has been called the mother of all pandemics ( nhps, non-human primates. frontiers in immunology | www.frontiersin.org during the first months, after the first reported case ( ) . the age of deceased people was below years in almost % of cases, a peculiarity compared with the seasonal influenza epidemic. the mortality rates observed in and flu were of . % and - % due to h n and h n , respectively, and ranging from . and . % in ( ) . the h n pandemic has been commonly referred to as swine flu for the swine origin of the virus, first isolated in mexico and united states in april . the viral genome sequencing indicated that the virus contains a combination of genes never reported in swine or humans before. it has been demonstrated that the swine has become a reservoir of h viruses with the potential to cause future pandemics ( ) . a (h n )pdm virus monovalent vaccine was produced in late ( ) , but the virus has not been eradicated and it continues to circulate as a seasonal variant, causing hospitalization, and death ( ) . in november , a first case of sars was reported in guangdong (china), and after months, the coronavirus (cov) causing the disease, named sars-cov, spread in countries, giving rise to lower respiratory tract infections, with a poor outcome in % of cases ( , ) . middle east respiratory syndrome-cov, the causative agent of mers, was isolated in . the coronavirus has caused isolated mers outbreaks thereafter, becoming endemic in arabian peninsula, with a case fatality rate of . % ( ) ( ) ( ) . on march , , who has declared the coronavirus disease (covid- ) outbreak a global pandemic. the disease is caused by a novel coronavirus, known as sars-cov- , that shares almost % of the genome with that of sars-cov ( , ) . actually more than . million (as of august , ) of people are infected, with an overall case fatality rate of . - . % ( ) . in case of global public health emergencies, governmental and private organizations, vaccine developers, and regulatory authorities should all massively collaborate in selecting and funding the most suitable vaccine platform and strategy to quickly act and curtail disease outbreaks. at the outset of a disease outbreak, gaps in knowledge of identity, pathogenesis, epidemiology of the new emerging pathogen, time required to study the immune responses correlating with the outcome of the viral infection, and the lack of appropriate preclinical models susceptible to infection for testing a vaccine candidate pose several barriers and impediments to expedite vaccine design and development, and thus to ensure global vaccination coverage in time. in the fight against newly emergent viruses, vaccine design might benefit from a range of platform technologies, including nucleic acid vaccines, viral-vector vaccines, and recombinant protein-based vaccines (likely to be administered with adjuvants) ( , ) . compared with conventional vaccines, such as live attenuated and inactivated vaccines, molecular-based platforms might offer a more versatile tool against new emergent viruses, allowing a more fast, low-cost, and scalable vaccine manufacturing. essentially, these platforms rely on the use of a system to deliver and present a new antigen (or a synthetic gene) to rapidly target an emergent pathogen. theoretically, once a platform has previously met safety and efficacy requirements to be moved and advanced into the market, a candidate vaccine against a new virus might profit from the same system, production, and purification protocols, only replacing the disease target antigen (or inserted gene), thus streamlining the vaccine discovery. in inactivated vaccine, the virus is rendered uninfectious using chemicals, such as formaldehyde or heat. this technology, conceived in the nineteenth century, is used for few vaccines still in use (i.e., inactivated polio, whole cell pertussis, and hepatitis a) ( ) . live attenuated vaccines are obtained by passing the virus through animal or human cells until it picks up mutations that make it unable to cause the disease (i.e., measles, mumps, chickenpox, etc.); the attenuated smallpox was used for the massive vaccination campaign that successfully eradicated the infection ( ) , and currently, attenuated influenza viruses are used as vaccines against the seasonal influenza ( ). the advantages of live attenuated vaccines are the intrinsic adjuvant properties, the ability to infect cells (figure ) , and to activate the innate immune response. interestingly, a safe sars-cov- inactivated vaccine (picovacc) has been recently described as being able to induce specific neutralizing antibodies (nabs) in experimental animal models ( ) , and a phase iii clinical trial (nct ) will soon assess efficacy and safety of this candidate in health care professionals ( table ) . nucleic acid vaccines include either mrna or plasmid dna (pdna) vaccines (figure ) . two types of mrna vaccines were developed: conventional non-replicating mrna vaccines and self-amplifying vaccines (or viral replicons). the in vitro enzymatic transcription (ivt) of a dna template plasmid, containing the promoter sequence for the dna-dependent rna polymerase, provides a mature mrna molecule, with the open reading frame that encodes the target antigen, the and flanking untranslated regions (utrs), the cap, and the terminal poly(a) tail. self-amplifying rna (sam) vaccines are commonly based on alphavirus genomes, where genes coding for the structural proteins are replaced with that encoding the target antigens, while the rna replication machinery sequences are conserved, allowing intracellular antigen-encoding rna amplification and higher antigen expression levels than the conventional mrna vaccines ( , ) . once the mrna vaccine is delivered to the host cells and reaches the cytoplasm, it is translated in vivo by the host cellular machinery, providing the corresponding posttranslationally modified antigen (figure ) , thus mimicking the in vivo natural infection. mrna vaccines activate the innate immune system, triggering host immune sensing receptors, and successively promoting adaptive immune responses ( ) . several figure | platforms for vaccine manufacturing: a graphical overview. nucleic acid, viral-vector, protein-based, live attenuated and inactivated vaccines are schematically illustrated. nucleic acid vaccines: conventional non-replicating mrna vaccine, containing the target gene sequence, can be encapsulated into a delivery system to aid its cellular uptake. once released from endosome into the cytosol, it is translated by the host cellular machinery into the target antigen. a pdna carrying a gene target reaches the nucleus to achieve transcription and translation into the cytosol. pdna can be internalized by somatic cells (i.e., myocytes) and then the secreted antigen can be taken up by apcs or naïve b cell, priming immune responses. viral vectored vaccines: defective viral vector, carrying a transgene cassette, can be employed as a system to deliver a transgene and allow the expression of the heterologous antigen within the infected cell. a recombinant replicating viral vector retains the ability to replicate and produce progeny virus particles that can then infect cells, leading to transgene expression and ag processing and presentation. protein-based vaccines: recombinant subunit vaccine or a vlp can be taken up by apcs for mhc presentation and b-cell recognition through bcr. virus vaccines: compared with an inactivated virus, a live attenuated virus retains the ability to replicate and infect cells, mimicking the natural infection. apcs, antigen-presenting cells; mhc, major histocompatibility complex; ag, antigen; pdna, plasmid dna; ep, electroporation; bcr, b-cell receptor; and vlp, virus-like particle. technological innovations have allowed to overcome some of the concerns associated with instability, half-life, inefficient in vivo delivery, and high innate immunogenicity of mrna platform ( ) . mrna vaccines do not produce infectious particles and potentially do not integrate into the host genome, reducing safety issues, and no anti-vector immunity is elicited. they can be quickly produced (likely within the time required to get genomic information from the new emergent virus), saving time and cutting costs. thus, the mrna platform offers a promising attractive alternative to conventional vaccines, should a disease outbreak occur. no rna vaccine has been yet licensed for humans, but encouraging results from preclinical and human clinical trials have shown that mrna vaccines are able to induce safe and long-lasting immunity against different infectious viral diseases, including zika ( ), influenza ( - ), ebola ( ), dengue ( ) , and other viral diseases ( ) . a sars-cov- mrnabased vaccine entered clinical phases just months after the identification of the viral genome sequence (nct ), and a phase iii study (nct ) will assess its effectiveness to prevent covid- ( ) ( ) ( ) . a clinical study (nct ) is currently evaluating a similar vaccine in healthy adults ( table ) . the dna-based strategy, like the mrna-based technology, offers a valuable platform to design and deliver any target of choice, due to safety profile, stability, ease of gene manipulation, and large-scale vaccine manufacturing, in short ( , ), on cellular uptake and in vivo long-term gene expression, potentially providing advantages over mrna vaccines in terms of protein coding capacity, and amount and extent of antigen production. unlike mrna, pdna needs to cross both plasma and nuclear membranes to enter into a cell target, reach the nucleus, and achieve transcription (figure ) . advances in pdna delivery devices (i.e., use of gene gun; in vivo electroporation, ep), and delivery systems (i.e., encapsulation in lnps; adsorption to polymers), have greatly enhanced molecular stability, delivery efficiency, uptake, and antigen expression. in addition, the use of optimized pdna formulations and encoding molecular adjuvants, to be administered in prime-boost strategies or simultaneously with other vaccine platforms, has generally improved the low protective immunestimulatory profile of pdna ( ) . however, some potential safety concerns should be considered, including long-term persistence upon administration, which could eventually lead to genomic integration events, antibodies against bacteria-derived plasmids that could potentially trigger autoimmune diseases, and unwanted side effects due to encoded and co-delivered molecular adjuvants ( , ) . even though no dna vaccine has been yet licensed for use in humans (four for veterinary use), this platform has shown great promise for several emerging viral diseases, including ebola and marburg ( ), mers ( ), west nile ( ), dengue ( ), chikungunya ( ) , and other viral diseases ( ) , and more recently for covid- ( ) . currently, dna-based vaccine candidates, encoding the s protein from sars-cov- , have moved into clinical phase i/ii development ( , , ) (table ). recombinant viral vector-based platform employs either live replicating often attenuated or non-replicating viruses as vector vaccines (figure ). viral vector vaccines represent the biotechnological evolution of live attenuated and inactivated vaccines: a viral backbone devoid of the replication machinery to be used as a shuttle to express in vivo the chosen target antigen. several viral backbones have been exploited to generate viral-vector vaccines. targeted deletion of replication genes represents the non-empirical way of virus attenuation, allowing the generation of a wide array of viral vectors, engineered by insertion of a transgene cassette. the modified virus ankara (mva) is an attenuated form of the vaccinia virus (vacv), derived from more than passages in chick embryo fibroblasts, a method that empirically modifies the viral genome, without affecting the immunogenicity ( ) . it is able to infect multiple cell types but cannot replicate inside the infected cells, ruling out the safety concerns related to the use of live vaccines. one of the drawbacks in the use of a viral vector vaccine is that multiple immunizations lead to the host response against the structural viral proteins, limiting the efficacy of vaccination, as demonstrated in a study based on cellular immune response. to overcome this limitation, the heterologous prime-boost regimen has been introduced in several clinical trials, where two different viral vectors or a pdna prime-viral vector boost were tested ( ) . risks of integration into the host genome do potentially exist, as some viral vectors enter to the nucleus of cells to achieve transcription and replication. a major restrain in the production of viral vector vaccines is the time-consuming manufacturing; several attempts to accelerate vaccine production are in development, like selecting cell lines with higher yield or choosing the best promoter for transgene expression to reduce vaccine doses ( ) . among the available viral vectors, the adenoviruses are the most used in priming the immune response, being able to induce humoral and cellular responses ( ) . a pre-existing anti-vector immune response jeopardized the vaccine response in adenoviral-based clinical trials ( ) . to avoid pre-existing immunity, adenoviral vectors of non-human origin or rare serotypes have been used as vaccine platform. the use of chimpanzee adenoviral vectors proved to be safe and effective in clinical trials conducted against ebola ( ) and respiratory syncytial virus (rsv) ( ) . vesicular stomatitis virus (vsv), a single-stranded negative sense rna virus that naturally infects livestock, represents an attractive safe alternative over other viral vectors due to low risk of pre-existing immunity, lack of dna molecules during replication, and ability of vsv-based vaccines to induce effective humoral responses ( ) . for humans, two viral-vector vaccines are available: imojev, a japanese encephalitic virus (jev) vaccine; and dengvaxia, a dengue vaccine (both from sanofi pasteur). both are produced using the chimeric yfv as vector: two of the yfv genes have been replaced by genes encoding the pre-membrane (prm) and the envelope (e) protein of jev or denv, and the chimeric viruses are propagated in cell culture ( , ) . conversely, several viral vector vaccines have been licensed for veterinary use because of the less stringent regulatory requirements ( ) . to face covid- , an adenovirus type vector expressing sars-cov- s protein (ad -ncov) has been advanced into phase ii trial (nct ), while a phase iii study (isrctn ) is currently investigating the chimpanzee adenoviral vector (chadox ncov- ), expressing the same protein ( , , ) ( table ) . recombinant protein-based vaccines consist of immunogenic proteins from the target pathogen. once identified, recombinant proteins can be produced on a large scale, in bioreactors, using heterologous expression systems, like bacteria, yeast, plants, insect, or mammalian cell lines, depending on the posttranscriptional pattern of modification required ( ) . vaccines based on recombinant proteins represent a safe platform because they do not contain pathogen-derived genetic information, and the manufacturing does not require manipulation of live pathogens. they might represent a platform of choice when a fast response to an epidemic is on demand, as the vaccine production can start once the genome of the new virus has been sequenced, even before the virus isolation. protein-based vaccines can be obtained producing recombinant virus subunits (suvs) that can be administered in combination with adjuvants to improve the host immune response against the recombinant viral antigens ( ) . recombinant proteins derived from viral capsid can selfassemble into virus-like particles (vlps), high ordered and repetitive structures devoid of the viral genome. vlps display antigenic epitopes in their original conformation in high copy number, they retain the size and geometrical organization of the original virus (mainly icosahedral or rod shape), preserving the viral immunogenicity due to the ability to crosslink b cell receptor on b cell surface ( ) , and to be taken up by antigenpresenting cells (apcs) ( , ) (figure ) . several strategies have been proposed to improve dendritic cell (dc) uptake, by expressing targeting molecules such as antibodies directed against endocytic receptors, and to augment immunogenicity, through simultaneous delivery of maturation stimuli, like tlr agonists ( , ). when not able to self-assemble into a vlp, the selected antigen can be expressed as chimeric protein: several vlp platforms are available for the display of heterologous antigens on the viral coat proteins. recombinant vlps from plant virus, like tobacco mosaic virus ( ) , or alpha mosaic virus ( ) , are easily produced, competing for speed and cost of production with vlp platform based on mammalian viruses ( ) . the most used vlp platform is the hbcag-vlp, the core antigen from hepatitis b virus (hbv) ( ) . it is also possible to chemically attach the heterologous antigen to a preformed vlp by using conjugation methods ( ) . although this strategy could increase the manufacturing costs, it might be suitable when the expression of recombinant antigens affects the vlp assembly. to table ) . it is worth mentioning that kim and colleagues designed and developed a sars-cov- subunit vaccine within weeks of the identification of sars-cov- s protein n-terminal domain s sequence. delivery of recombinant subunit vaccines by microneedle array resulted in potent antibody response in mice ( ) , and vaccination with a sars-cov- spike s -fc fusion protein induced antibody responses in small animal models and nabs in monkeys ( ) . in table are listed the vaccine candidates that currently moved into clinical trials for preventing the viral infectious diseases discussed in the following section. west nile virus includes five lineages; among them, lineage was classified as the most virulent, while lineage is considered more attenuated. however, during a serious outbreak in hungary in , the sequencing of lineage showed some genetic mutations that demonstrated the increased virulence of this strain and its explosion throughout the central europe ( , ) , causing renewed interest in the development of a vaccine against wnv. years after the epidemic that hit the united states, no wnv vaccine has been yet released for human use, while four vaccine formulations are on the market for veterinary use, three based on the whole inactivated virus (wn innovator, vetera wnv, and prestige wnv), and one on recombinant vaccine expressing wnv prm/e into a canarypox backbone (recombitek equine wnv) ( , ) . these vaccines completely protect horses from viral infection but require subsequent administrations and several booster doses overtime. for the development of a vaccine for humans, many different platforms were used in preclinical studies, and many of them entered into phase i/ii trials, including hydrogen peroxideinactivated whole virus (hydrovax) vaccine (nct ) ( ), a recombinant truncated form of wnv e protein ( ), recombinant chimeric live attenuated viral vectors, employing yfv ( ), or mva ( ) delivering wnv prm/e proteins (nct ), pdna vaccines encoding prm/e (nct ) ( , ) . all the envelope-based vaccines induced nabs against both wnv lineages and , but some candidates are unable to generate long-lasting antibody responses, requiring multiple administrations ( , , ) . thus, further improvements are needed for the development of next-generation vaccines ( ) . recently, a wnv replicationdeficient vaccine candidate with a deletion of the non-structural protein ns has been shown to protect mice from a highly lethal viral challenge, after a single dose, without adverse effects ( ) . during the outbreak in brazil, an abnormal microcephaly number and other birth defects in newborns were reported ( ) . for this reason, vaccination of pregnant and of reproductiveage women became an urgency. shan and colleagues developed a candidate vaccine, using a live attenuated viral strain containing a deletion in the region of the virus genome. this vaccine induced strong and protective antibody response, after a single injection in mice and macaques, and reduced viral rna in placental and fetal tissues in infected mice ( ) . the immunized mice also developed a robust t-cell response ( ) . although promising, this attenuated virus-based formulation does not meet the safety standards required to be used to vaccinate pregnant women, whose prophylaxis requires a vaccine that fulfill higher safety standards. a number of different replication-deficient viral vectors have been recently developed and are currently under evaluation. immunization of mice with a vaccine based on mva delivering the zikv prm and the e structural proteins (mva-zikv) elicited nabs and potent zikv-specific cd + t-cell responses, mainly with an effector memory phenotype ( ) . a rhesus adenovirus serotype vector (rhad ), expressing zikv prm and e proteins, induced high titer of zikv-specific antibodies after the first prime, offering complete protection against subcutaneous zikv challenge, in mice ( ) , and rhesus monkeys ( ) . these adenoviral-based vaccines induced antibodies that were also maternally transmitted ( ) . in addition, abbink et al. using the rhesus macaque model demonstrated that a complete anti-zikv immunity can only be achieved through vaccination with a combination of different vaccine platforms ( ) . zikv vaccine candidates currently in phase i clinical trials include inactivated and live attenuated vaccines, mrna and pdna vaccines, and recombinant viral-vectored vaccines, mainly targeting the prm and e proteins ( ) . a dna-based vaccine encoding the prm signal sequence from jev and zikv e proteins moved into phase ii (nct ), showing immunogenicity and safety in humans ( ) . a protective and efficacious vaccine against yfv is currently available. to date, the main type of yf vaccine produced on a large scale is based on the live attenuated d virus vaccine. this vaccine is obtained after numerous passages of asibi virus strain in mouse and chicken embryo that generate a strain with accumulated mutations in the envelope protein. these mutations affect the virus binding to the host receptor, reducing its neurotropism and vicerotropism, and mosquito transmissibility ( ) . because the vaccine is produced in chicken embryo, there are issues related to manufacturing costs and vaccine availability. the interruption of vaccination coverage against yf in endemic countries has caused major outbreaks in africa and south america in and , which exhausted the d vaccine stockpiles leading to the use of an emergency "fractional dose" campaign in the democratic republic of congo ( ) . thus, the fluctuating demand for doses during outbreaks makes the accessibility to the vaccine still a problem to be solved. the need for a vaccine against denv has become an urgency only in recent decades. dengue fever is caused by four distinct virus serotypes, denv - , able to circulate simultaneously in endemic areas, making extremely difficult the development of a broad protective vaccine. recently, the food and drug administration approved the first dengue vaccine by sanofi-pasteur, named cyd-tdv or dengvaxia ( , ), a tetravalent live attenuated virus vaccine on yfv backbone, whose release has generated controversy due to evidence that the administration can increase the risk of a more severe form of the illness in people with a pre-existing immunity toward other denv strains ( , ) . for this reason, the use of dengvaxia is strictly limited, depending on age (between and ) and serostatus of recipients to vaccinate (exclusively individuals who had a previous denv infection), generating concerns about its costbenefit balance. studies for the development of a safer vaccine are still ongoing, and candidate vaccines include a tetravalent dengue purified inactivated virus vaccine, currently in phase i/ii clinical trial (nct ), and two live attenuated tetravalent chimeric tdv (denvax), and tvd / (tetravax-dv) vaccines, currently in phase iii clinical trials (nct ; nct ) ( ). no vaccine is actually available to prevent chikv infection. among the candidates in ongoing studies, two of them achieved and completed phase i or ii trials: vla and mv-chik vaccines. vla candidate (by valneva) is a live chikv (la réunion isolate lr opy ) attenuated by a partial deletion of the gene encoding the non-structural replicase complex protein. this vaccine induced immunity lasting over months after a single shot immunization (nct ). mv-chik vaccine is a live attenuated measles-vectored chikv vaccine that induced chikv-specific nabs and shown to be well tolerated by all the participants (nct ) ( ) . recently, moderna therapeutics tested a vaccine based on engineered mrna encoding chikv structural polyproteins (mrna- ) in a phase i clinical trial. as shown in preclinical studies, this formulation induced strong immune responses after one single injection, totally protecting mice from developing the disease ( ) . several vaccine platforms have been tested in preclinical animal models and shown to be able to protect animals from marv infection and to induce both humoral and cellular immune responses. these include vlps ( ) , dna vaccines ( ), recombinant adenoviral vectors ( ) , and rvsv ( , ) . many works have emphasized the use of a multivalent vaccine formulation to achieve protection against different filoviruses. vaccination with a single dose of a trivalent formulation based on rvsv expressing glycoproteins from ebov, sudan ebolavirus (sudv), and the angola strain of marv elicited antibodies specific for the three glycoproteins in non-human primates (nhps) and a balanced t-cell response sufficient to protect against the viral challenges ( ) . similarly, vlps delivering a trimeric hybrid glycoprotein from marv, ebov, and sudv fully protected vaccinated animals from marv challenge, inducing specific nabs ( ) . using an enhanced dna-based platform encoding the envelope glycoprotein from marv and ebov, shedlock and colleagues showed that a polyvalent-filoviral vaccine candidate, delivered by in vivo ep, elicited in preclinical models robust nabs and cytotoxic t cells, completely protecting animals from the viral challenge, after a single dose administration ( ) . actually, a multivalent phase i study (nct ) is evaluating safety and immunogenicity of two heterologous and two homologous prime-boost regimens using a mva multi-filo and ad zaire ebola (ad .zebov) vaccines ( ) in healthy volunteers, with the aim to analyze the protective response to different filoviruses. coronaviruses are a group of single-stranded rna viruses that have been present in humans for at least - years and all originated in bats ( , ) . earlier than , six coronaviruses had been known to cause diseases in humans: hcov- e, hcov- , hcov-nl , hcov-hkn , sars coronavirus (sars-cov), and mers coronavirus (mers-cov) ( ) . in late and early , a novel coronavirus was discovered to be the cause of a rapidly spreading outbreak of respiratory disease, including potentially fatal pneumonia, in wuhan, china. the virus, provisionally designated -ncov and later given the official name sars-cov- , owing to its similarity to sars-cov (then named sars-cov- ), was isolated and the viral genome sequenced. sars-cov- was characterized as a beta-coronavirus ( ) . the disease caused by the virus was officially named coronavirus disease (covid- ) by who. coronaviruses are capable of adapting quickly to new hosts through the processes of genetic recombination and mutation in vivo. point mutations alone are not sufficient to create a new virus. however, this may occur when the same host is simultaneously infected with two coronavirus strains, enabling recombination of genomic fragments of hundreds or thousands of base pairs long and thus making a new virus ( , ) . this susceptibility enabled the emergence, in approximately two decades, of three new human coronavirus species with epidemic potential: sars-cov- , mers-cov, and sars-cov- . coronaviruses enter cells via binding to a host receptor followed by membrane fusion. the angiotensin-converting enzyme (ace ) was identified as the cell receptor for sars-cov ( ) , and recently also for the new sars-cov- ( ), while mers-cov binds the dipeptidyl peptidase (dpp ) receptor, also known as cd ( ) . the s protein is used for virus-cell receptor interaction during viral entry ( ) . transmission of the virus during the viremic stage of disease is primarily via respiratory secretions (droplets) or direct contact. sars-cov- is extremely contagious, with an estimated basic reproduction number (r ) of . - . ( ) . in contrast, the r for both sars-cov- and mers-cov is less than ( ). it soon became apparent that infected individuals might be capable of transmitting the virus during the prodromal period ( ) . social distancing strategies (quarantine and community containment) represent the only efficacious means of controlling coronavirus spread in the absence of effective drugs or vaccine against the pathogens. of importance, for preventing the spread of the disease caused by contact with patients or contaminated fomites, hygiene measures are also mandatory, such as washing hands with soap and water or with alcohol-based preparations. indeed, coronaviruses are able to survive on various surfaces for few days but can be inactivated by disinfection ( ) . finally, because it has been demonstrated that the overlap between human and animal ecosystems have given to coronaviruses the opportunity to cross the species barrier, to prevent future zoonotic diseases, a coordination with veterinary experts as well as stricter laws governing the trade of wild animals would be necessary. humans are extremely exposed to these pathogens because these viruses had not previously circulated in the human population, as testified by the absence of antibodies against coronavirus in healthy people. in addition, the innate immune response has demonstrated to be insufficient in controlling coronavirus infection because decreases in viral load are coincident with the specific antibody response ( , ) . in this context, vaccines represent a much expected resource. a hopeful premise is represented by the successful containment of coronavirus epidemics in farm animals by vaccines, based on either killed or attenuated virus ( ) , and concerning sars-cov- by the finding that specific antibodies are detectable in % of patients with covid- , - days after symptom onset ( ) , and that the magnitude of antibody titers positively correlated with viral neutralization potency ( ) . after the sars outbreak, several vaccines were formulated based on various strategies, as recombinant s protein-based vaccines, attenuated and whole inactivated vaccines, as well as vectored vaccines. pre-clinical data showed animal protection from challenge with sars-cov- . however, sterilizing immunity was not always achieved ( ) . in few cases, the use of live virus as a vaccine resulted in complication including lung damage, eosinophil infiltration, and liver damage in animal models. moreover, a study of vaccination with inactivated sars-cov- in nhps reported enhancement of disease caused by specific epitopes on the s protein [reviewed in ( ) ]. another issue is related to the length of a protective immune response. both humoral and cellular responses have been found important for lasting protection. in long-term studies of recovered sars patients, antibody responses waned after approximately years, while t-cell responses persisted, suggesting that the latter is required for long-lasting immunity. concerning mers-cov, the vaccines proposed target the s protein ( ) ( ) ( ) , including mucosal vaccine for intranasal administration ( ) . however, cases of enhanced lung diseases were also reported in preclinical models of vaccination in mice ( ) . new mers-cov vaccines in development also include live attenuated, protein subunit, and dna vaccines ( , ) . recently, a small animal model that replicates mers-cov transmission has been developed ( ) and will help the preclinical studies. following the alarming data and casualties provoked by covid- , a strong effort by the research community is going on at the moment, and who has been informed of dozens of vaccines in preparation using different platforms, as mentioned in section "vaccine platforms." some of these candidate vaccines are already in phase i/ii clinical trials, while others have been advanced to phase iii studies ( , , ) (table ). however, it is possible that a sars-cov- vaccine will not be available for another - months. recently, a rhesus macaque model that recapitulates sars-cov- infection has been developed to study immunopathogenesis and test vaccine candidates ( , ) . therapy based on passive administration of anti-coronavirus antibodies, isolated from patient sera, also represents a much wanted option for the treatment of coronavirus diseases ( ) , and a global effort is pursued in this direction to treat patients before the achievement of a validated vaccine. in addition, researchers are trying to produce in laboratory specific and protective anti-coronavirus antibodies. in the case of sars outbreak, a monoclonal antibody (mab) with neutralizing activity, being able to block receptor association, was identified and described ( ) . moreover, neutralizing mabs have also been produced to fight mers-cov infection. in a collaborative study by us and chinese researchers, mabs targeting the receptor (cd /dpp ) binding domain of mers-cov spike glycoprotein were reported ( ) . japanese researchers have also investigated anti-cd mab for mers-cov and have identified the humanized mab ys as a promising candidate ( ) . finally, in the case of sars-cov- outbreak, dutch researchers claimed the identification of a human mab named d able to block sars-cov- infection ( ). recently, a mab able to cross-neutralize sars-cov- has been identified from memory b cells of a sars-cov-infected individual. the antibody, named s , engages the s receptor-binding domain, recognizing a highly conserved protein/glycan epitope distinct from the receptor-binding motif ( ) . more recently, other potent neutralizing antibodies were isolated by different research institutions ( ) ( ) ( ) . amidst the gamut of high-affinity antibodies with the potential to neutralize human pathogenic viruses, single-domain antibodies, referred to as nanobodies or nbs ( kda), and nanobody-based human heavy chain antibodies ( kda) derived from camelids might be harnessed as useful therapeutics for the ongoing covid- pandemic ( ) . camelid heavy-chainonly antibodies (hcabs) are composed of two heavy chains with a single variable domain (vhh) as the target-binding module. recombinant vhhs, devoid of the effector domains, act as single-domain antibodies and harbor advantageous features over conventional antibodies (higher thermal and chemical stability, higher solubility, smaller size, lower susceptibility to steric hindrances, ease of manufacturing, and simple structure) to have been recently proposed as prospective therapeutic candidates against various infectious pathogens ( ). vhhs isolated from a llama subcutaneously immunized with perfusionstabilized sars-cov- and mers-cov s proteins have been recently characterized and shown to be able to neutralize s pseudotyped viruses in vitro, interfering with the host cell receptor binding ( ) . interestingly, sars-cov- s-directed vhh cross-reacted with sars-cov- receptor binding domain (rbd) and neutralized sars-cov- s pseudoviruses in vitro as a bivalent human igg fc-fusion format, underscoring the potential of vhhs to treat coronavirus diseases ( ) . because of the global spread of diseases caused by flaviviruses, understanding the cross-reactivity of anti-viral immunity among these viruses is of crucial importance for predicting the evolution of viral disease outbreaks. recently, the analysis of pbmcs isolated from individuals infected by denv or vaccinated with denv tv or yf d vaccines, and pulsed with a pool of antigens from autologous and heterologous flaviviruses, indicated that both cd and cd t-cell responses were specific, with little or no cross-reactivity, despite the high level of homology ( ) . individuals preexposed to denv infection developed t-cell responses against non-structural zika proteins rather than structural envelope protein, suggesting that previous flaviviral infections biased the t-cell response toward more cross-reactive non-structural epitopes ( ) . studies enrolling mothers who gave birth to microcephalic babies after zikv infection, showed serological evidence of a pre-existing anti-dengue response, suggesting that vaccination against denv does not protect against zikv microcephaly ( ) . however, cross-reactive antibodies between zikv and denv have been described, mainly targeting the structural dimeric envelope protein ( , ) . the antigenic sequences are both linear and quaternary, with nabs mainly recognizing the latter. the high-conserved e protein fusion loop induces cross-reactive but weak nabs that can be a marker of worst outcome during subsequent flaviviral infections ( ) . a research concerning zikv-specific b-cell responses in three denv-experienced donors showed that months after the infection, the pool of antibodies comprised both poorly nabs derived from pre-existing denv-induced memory b cells, associated with an enhanced zikv infection in vitro, and potent zikv-specific antibodies originated de novo ( , ) . the possibility that wnv-specific antibodies may drive the infection by other flaviviruses is still controversial, even if cross-reactivity was demonstrated. plasma samples from convalescent human wnv patients were shown to enhance zikv infections by antibody-dependent enhancement (ade) phenomenon ( ) ; conversely, mice previously infected with zikv and challenged with wnv showed enhanced protection toward the second infection ( ) . the immunological flavivirus cross-reactivity, the ade phenomenon (discussed below), genetic mutations that increase the virulence, potential pre-existing immunity concerns, combined with the necessity to increase cost-effectiveness of marketable products are among the issues that have limited the development of successful vaccines until now. the use of t-cell inducing vaccines or proteins with mutations into conserved envelope fusion-loop epitopes might be useful to overcome the cross-reactivity hurdle ( ) . known as ade of viral infection, ade is a phenomenon occurring when antibodies facilitate virus entry into the host cells, driving viral replication and increasing infectivity, with subsequent severe outcomes. among the several stumbling blocks in realizing a safe vaccine, ade is a phenomenon largely underestimated, but that can produce severe adverse effects, rendering vaccinated individuals more predisposed to develop harsh symptoms after infection ( ) . the first report of ade dates ( ) . the molecular mechanisms disclosed the involvement of fcγr ( ) and complement receptors ( ) . when an antiviral antibody (induced by vaccination or viral infection) with no neutralizing or sub-neutralizing activity is produced, it can act like a bridge between the virus and the fcγr expressed on the surface of immune cells, leading to viral uptake (figure ) , as demonstrated for denv, zikv, wnv, influenza, sars-cov, mers-cov, and ebov ( ) . the role of complement receptor has been demonstrated in ebov response: two antibodies directed against epitopes in close proximity bind the c q, forming an immune complex able to enhance the virus entry into a target cell ( ) , whereas in an animal model of mers-cov, c a and c protein level increase was observed after passive immunization ( ) . the first licensed vaccine against denv (cyd-tdv-dengvaxia) caused hospitalizations in two large multicenter phase iii trials; after result revision, it has been estimated that in seronegative individuals, it can produce adverse effects ( ) , and who recommendations are to vaccinate only seropositive individuals in endemic areas of age older than years. using a mathematical model of denv transmission to formulate hypothesis on vaccine trial results, it was speculated that "seronegative recipients gain transient protective cross-reactive immunity akin to that observed for natural infection, " increasing the risk of severe disease after infection, while vaccination of seropositive subjects results in boosting the immune response, producing a protection comparable with the one obtained in individuals who has had two natural infections ( ) . the most severe adverse effect after vaccination was registered when a formalin-inactivated vaccine against rsv produced an increase of severe illness in vaccinated infant (hospitalization: % rsv vaccinated vs. % vaccinated against parainfluenza) and two deaths ( ) . afterward, a role for the th response was hypothesized in generating the rsv-mediated ade ( ) , and it was demonstrated that the formalin-inactivated virus produced ade in monkeys ( ) , suggesting that the carbonyl groups on formaldehyde-inactivated rsv were responsible for the th response in mice ( ) . moreover, the observation that formalin inactivation produced an alteration of antigens, leading to the production of non-nabs, whose avidity did not mature, and the activation of complement were also reported for a measles vaccine ( ) . the low-avidity non-nabs are produced in absence of tlr activation (and affinity maturation), and they figure | antibody-dependent enhancement on dengue infection. antibodies generated from a previous denv infection can recognize but do not neutralize another denv serotype and can lead to antibody-dependent enhancement (ade) of entry of the latter virus into host cells. the pre-existing non-(or sub-) neutralizing antibodies bind denv through the fab domains and mediate viral entry into fcγr-expressing cells. on engagement by the fc domains, the virus-antibody immune complex is internalized by the activating fcγriia within the endosome. co-ligation of fcγriia and lilrb (leukocyte immunoglobulin-like receptor-b ) to opsonized denv drives the inhibitory signal cascade via immunoreceptor tyrosine-based inhibition motif (itim) pathway, abrogating the expression of isgs (interferon stimulated genes). ligation of fcγriia to immune complex also increases th cytokine production and reduces ifnγ, inhibiting the jak/stat signaling pathway, overall resulting in the suppression of the antiviral response and increase of viral replication. nabs, neutralizing antibodies; and itam, immunoreceptor tyrosine-based activation motif. trigger complement activation ( ) , enhancing viral infection. to induce potent nabs, the tlr activation has been obtained using a th -polarizing adjuvant ( ) , in association with the candidate vaccine exposing the epitopes of interest. antibody-dependent enhancement has been reported also in many studies focusing on the development of sars and mers vaccines, demonstrating that vaccination with the whole s glycoprotein can increase the susceptibility to viral infection with a mechanism not linked to the virus receptor expression on the host cells ( ) , and especially when antibodies are induced with low titer ( ) . while for many flaviviruses the mechanism of ade has been explained through evidences that antibodies developed during a primary infection can enhance entry of a heterologous virus via fc-receptor during a secondary infection, for mers-cov and sars-cov, it has also been proposed that nabs that strongly bind the rbd region of the s surface protein can induce conformational changes that enhance the virus entry via canonical viral-receptor-dependent pathways, mimicking viral receptor binding ( , ) , and antibodies targeting a specific region of the s protein enhanced the viral infection in a sars model of nhps ( ) . the high sequence homology and the similarity in structure shared among sars-cov, mers-cov, and sars-cov s glycoproteins raises reasonable concerns about the development of covid- vaccines based on the s protein. in potential pandemic settings, the clinical development of vaccines is the main aim. however, apart from technical reasons, the vaccine production might be delayed also for economic considerations and safety issues. other strategies may be based on self-disseminating vaccines and induction of trained immunity. to control zoonosis, the formulation of self-disseminating vaccines acts at the level of animal, insect, or environmental reservoir, to directly interfere within the animal-to-human transmission ( ) . they are essentially based on replicating viral vectors engineered to express the disease antigen and to target a certain animal population ( ) . global vaccination of animals could be achieved to effectively contain an emerging pathogen within the wildlife reservoir, avoiding its global spread. feasibility concerns, costs, and safety issues should be considered when using this strategy to control reservoirs linked to the emergence of high-risk pathogens. in addition, which animal pathogen will cause a human disease is generally unpredictable. it is interesting to underline that a vaccination of great apes with an engineered specific cmv-based vector has been proposed as a strategy to potentially interrupt (or at least decrease) the zoonotic transmission of ebola virus to humans, being able to protect animals from the lethal viral challenge ( , ) . trained immunity-based vaccines (tibv) might be formulated to stimulate broader anti-infectious responses compared with conventional vaccines for their capacity to increase innate immunity and enhance adaptive responses ( ) . this strategy exploits the ability of innate immune cells (monocytes, macrophages, nk cells) to undergo extensive metabolic and epigenetic reprogramming, following certain vaccinations or infections, and to become primed for a quite long period of time to respond more potently to autologous or heterologous re-infection, mounting the so-called "innate immune memory." triggering of pattern recognition receptors (prrs) by microbial effector stimuli results in increased production of pro-inflammatory cytokines and/or reactive oxygen species, and in enhanced immune responses, regardless the primary stimulation ( ) . many infectious stimuli are considered potent activators of trained innate immunity, including β-glucan and chitin (components of fungal cell wall), lps (a component of the cell wall of gram-negative bacteria), and the bacille calmette-guérin (bcg) vaccine ( ) . thus, tibv should contain pathogen-associated molecular patterns (pamps) to target prrs and subsequently induce trained immune cells. bcg vaccine, vacv, and live attenuated influenza vaccines, together with immunostimulants, could be ascribed to this category of vaccines ( ) . it is worth mentioning that a whole-cell killed bacterial vaccine might have played a role in preventing pneumonia and mortality during the influenza pandemic ( ) . recently, a work by berg and colleagues showed that bcg vaccination is associated with the flattening of the curve in the spread of covid- , suggesting that bcv vaccine might serve as a protective factor against the disease ( ). however, it should also be noted that an enhanced immune response mediated by reprogrammed immune cells could contribute to the development or maintenance of inflammatory, neuroinflammatory, and chronic metabolic disorders ( ) . the phenomenon of "trained immunity" occurring in the brain is known as microglial priming. exposure of primed microglial cells to a second stimuli can cause an augmented inflammatory response, leading to neuroinflammation and production of neurotoxic molecules. the hyperglycemia condition that characterizes type diabetes could long term affect the cellular metabolism of monocytes and macrophages, leading to increased cytokine production and subsequent diabetes complications, including atherosclerosis. an augmented activation of innate cells may also result in the induction and maintenance of chronic inflammatory disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, or sarcoidosis ( ) . the covid- pandemic experience, combined with the previous viral disease outbreaks, should give blueprints for rapidly responding to the emergence of high-risk pathogens in the future. it is a common belief that vaccines would be the only means of providing long-term immunity and preventing viral diseases. despite the great progress made in vaccine research, we are still unable to produce successful vaccines in a timely manner. human trials take a long time and given a huge list of vaccine candidates, it is hard to choose the most promising one. while the who proposed a solidarity vaccine trial to test all the candidates in rolling trial until they fail, to increase the chances of succeeding, some vaccine stakeholders are considering extreme alternatives for emergency use: intentionally infect young healthy volunteers at low risk in controlled "human challenge trials" to define which vaccine will work ( ) . although these approaches are already used for studying influenza ( ) and dengue diseases ( ) , it is hard to ethically accept this option without a validated therapy. vaccines go through regulatory pathways before the final approval and licensure. in epidemic or pandemic settings, we need to carefully develop a vaccine, as quickly as possible, that adequately proved to be safe and effective ( ) . scientists need to fill the gaps in understanding the epidemiology of novel viruses, to identify potential zoonotic reservoirs or spill-over hosts, and the way of transmission of pathogens. once the pathogen is identified, preclinical models need to be developed to study virus-host interactions and early test vaccine candidates, defining the immune correlates of protection. pathogen-specific epitopes need to be identified to guide structure-based vaccines that will elicit protective antibodies, minimizing the induction of non-or weakly nabs that would promote ade of viral infection ( ) . moreover, data sharing and collaboration among academia, government, and companies will be essential to coordinate a strategic approach in face of next pandemic threats ( ) . all authors equally contributed to this work and read and approved the final manuscript. this work was supported by prin "nanotechvax tackling biological barriers to antigen delivery by nanotechnological vaccines" prot. zeccm, and consiglio nazionale delle ricerche (cnr), italy: laboratori congiunti bilaterali internazionali (scienze biomediche), project: "new vaccines against poverty-related and neglected tropical diseases." mt was supported by postdoctoral fellowship from cnr, italy: laboratori congiunti bilaterali internazionali (scienze biomediche), project: "new vaccines against poverty-related and neglected tropical diseases." references . nii-trebi ni. emerging and neglected infectious diseases: insights, advances, and challenges infectious disease threats in the twenty-first century: strengthening the global response the epidemic of -novel-coronavirus ( -ncov) pneumonia and insights for emerging infectious diseases in the future major factors affecting the emergence and re-emergence of infectious diseases climate change and multiple emerging infectious diseases infectious disease and economics: the case for considering multi-sectoral impacts emerging infectious diseases: a proactive approach identification of a novel coronavirus in patients with severe acute respiratory syndrome characterizing the epidemiology of the influenza a/h n pandemic in mexico middle east respiratory syndrome ebola virus disease - outbreak in west africa: an analysis of the epidemic spread and response first report of autochthonous transmission of zika virus in brazil metagenomic sequencing at the epicenter of the nigeria lassa fever outbreak the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak -an update on the status world health organization r&d blueprint national institutes for allergy and infectious diseases (nih) niaid emerging infectious diseases/pathogens new vaccine technologies to combat outbreak situations global trends in emerging infectious diseases centers for disease control and prevention (cdc) principles of epidemiology in public health practice. an introduction to applied epidemiology and biostatistics the epidemic of west nile virus in the united states nile in europe: an increasing public health problem zika virus, vectors, reservoirs, amplifying hosts, and their potential to spread worldwide: what we know and what we should investigate urgently microcephaly case fatality rate associated with zika virus infection in brazil: current estimates yellow fever: a reemerging threat yellow fever in the americas: the growing concern about new epidemics. f res the emergence of arthropod-borne viral diseases: a global prospective on dengue, chikungunya and zika fevers history, epidemiology and diagnostics of dengue in the american and brazilian contexts: a review atypical chikungunya virus infections: clinical manifestations, mortality and risk factors for severe disease during the - outbreak on réunion growth of cities could boost mosquito-borne diseases lassa fever in west african sub-region: an overview epidemiologic and clinical features of lassa fever outbreak in nigeria transmission of ebola virus disease: an overview forty-five years of marburg virus research the ebola outbreak, - : old lessons for new epidemics how severe and prevalent are ebola and marburg viruses? a systematic review and meta-analysis of the case fatality rates and seroprevalence antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans influenza: the mother of all pandemics estimated global mortality associated with the first months of pandemic influenza a h n virus circulation: a modelling study predictors of fatality in pandemic influenza a (h n ) virus infection among adults estimates of pandemic influenza vaccine effectiveness in europe, - : results of influenza monitoring vaccine effectiveness in europe (i-move) multicentre case-control study centers for disease control and prevention (cdc) h n pandemic (h n pdm virus sars and mers: recent insights into emerging coronaviruses a tug-of-war between severe acute respiratory syndrome coronavirus and host antiviral defence: lessons from other pathogenic viruses world health organization middle east respiratory syndrome coronavirus (mers-cov) the proximal origin of sars-cov- genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding available online at challenges and solutions in the development of vaccines against emerging and neglected infectious diseases history of vaccination history of smallpox and its spread in human populations influenza vaccination strategies: comparing inactivated and live attenuated influenza vaccines rapid development of an inactivated vaccine candidate for sars-cov- mrna as a transformative technology for vaccine development to control infectious diseases advances in mrna vaccines for infectious diseases mrna vaccines -a new era in vaccinology modified mrna vaccines protect against zika virus infection nucleoside-modified mrna immunization elicits influenza virus hemagglutinin stalk-specific antibodies preclinical and clinical demonstration of immunogenicity by mrna vaccines against h n and h n influenza viruses self-amplifying mrna vaccines expressing multiple conserved influenza antigens confer protection against homologous and heterosubtypic viral challenge dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h n influenza, and toxoplasma gondii challenges with a single dose a tetravalent alphavirus-vector based dengue vaccine provides effective immunity in an early life mouse model the covid- vaccine development landscape sars-cov- vaccines: status report covid- : what do we know so far about a vaccine? molecular mechanisms for enhanced dna vaccine immunogenicity engineering dna vaccines against infectious diseases induction of broad cytotoxic t cells by protective dna vaccination against marburg and ebola a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates a west nile virus dna vaccine utilizing a modified promoter induces neutralizing antibody in younger and older healthy adults in a phase i clinical trial immunogenicity and protective efficacy of a vaxfectin-adjuvanted tetravalent dengue dna vaccine a dna vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates dna vaccine protection against sars-cov- in rhesus macaques who draft landscape of covid- candidate vaccines modified vaccinia virus ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine viruses as vaccine vectors for infectious diseases and cancer viral vectors as vaccine platforms: from immunogenicity to impact effective induction of high-titer antibodies by viral vector vaccines hiv- vaccine-induced immunity in the test-of-concept step study: a casecohort analysis chimpanzee adenovirus vector ebola vaccine chimpanzee adenovirus-and mva-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults novel viral vectors in infectious diseases immunogenicity and safety of a single dose of a live attenuated japanese encephalitis chimeric virus vaccine in vietnam: a single-arm, single-center study efficacy and long-term safety of a dengue vaccine in regions of endemic disease current state and recent advances in biopharmaceutical production in escherichia coli, yeasts and mammalian cells application of recombinant dna techniques in the development of viral vaccines the influence of antigen organization on b cell responsiveness vaccine delivery: a matter of size, geometry, kinetics and molecular patterns hiv- gag p presented as virus-like particles on the e scaffold from geobacillus stearothermophilus induces sustained humoral and cellular immune responses in the absence of ifnγ production by cd + t cells hepatitis c vlps delivered to dendritic cells by a tlr targeting lipopeptide results in enhanced antibody and cell-mediated responses cd l-containing virus-like particle as a candidate hiv- vaccine targeting dendritic cells malarial epitopes expressed on the surface of recombinant tobacco mosaic virus antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and hiv- the production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza phase i trial of an alhydrogel adjuvanted hepatitis b core virus-like particle containing epitopes of plasmodium falciparum circumsporozoite protein engineering virus-like particles as vaccine platforms microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development recombinant sars-cov- spike s -fc fusion protein induced high levels of neutralizing responses in nonhuman primates explosive spread of a neuroinvasive lineage west nile virus in central europe genetic determinants of virulence in pathogenic lineage west nile virus strains equine vaccine for west nile virus a west nile virus (wnv) recombinant canarypox virus vaccine elicits wnv-specific neutralizing antibodies and cell-mediated immune responses in the horse an observer blinded, randomized, placebo-controlled, phase i dose escalation trial to evaluate the safety and immunogenicity of an inactivated west nile virus vaccine, hydrovax- , in healthy adults immunogenicity and protective efficacy of a recombinant subunit west nile virus vaccine in rhesus monkeys preclinical and clinical development of a yfv d-based chimeric vaccine against west nile virus immunogenicity and protective efficacy of recombinant modified vaccinia virus ankara candidate vaccines delivering west nile virus envelope antigens a west nile virus dna vaccine induces neutralizing antibody in healthy adults during a phase clinical trial twenty years of progress toward west nile virus vaccine development a : recombinant subunit west nile virus vaccine for protection of human subjects. united states patent us advanced oxidation technology for the development of a next-generation inactivated west nile virus vaccine replication-defective west nile virus with ns deletion as a new vaccine platform for flavivirus zika virus associated with microcephaly a single-dose live-attenuated vaccine prevents zika virus pregnancy transmission and testis damage a live-attenuated zika virus vaccine candidate induces sterilizing immunity in mouse models a vaccine based on a modified vaccinia virus ankara vector expressing zika virus structural proteins controls zika virus replication in mice construction and evaluation of novel rhesus monkey adenovirus vaccine vectors protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys preventative vaccines for zika virus outbreak: preliminary evaluation recent advances in zika virus vaccines safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase clinical trials comparison of the virulent asibi strain of yellow fever virus with the d vaccine strain derived from it live attenuated yellow fever d vaccine: a legacy vaccine still controlling outbreaks in modern day development of sanofi pasteur tetravalent dengue vaccine from research to phase iii: preclinical, industrial and clinical development of the sanofi pasteur tetravalent dengue vaccine pathogenesis of dengue: challenges to molecular biology antibody-dependent enhancement of severe dengue disease in humans dengue: status of current and under-development vaccines immunogenicity, safety, and tolerability of the measles-vectored chikungunya virus vaccine mv-chik: a double-blind, randomised, placebo-controlled and active-controlled phase trial recent progress in vaccine development against chikungunya virus effective vaccine for lassa fever vaccine platforms for the prevention of lassa fever vaccines inducing immunity to lassa virus glycoprotein and nucleoprotein protect macaques after a single shot isolation of marburg-like virus from a case of haemorrhagic fever in zaire vaccines against ebola virus a review of phase i trials of ebola virus vaccines: what can we learn from the race to develop novel vaccines viruslike particle vaccination protects nonhuman primates from lethal aerosol exposure with marburgvirus (vlp vaccination protects macaques against aerosol challenges) vector choice determines immunogenicity and potency of genetic vaccines against angola marburg virus in nonhuman primates live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses protection against marburg virus using a recombinant vsv-vaccine depends on t and b cell activation singledose trivalent vesiculovax vaccine protects macaques from lethal ebolavirus and marburgvirus challenge cross-protection conferred by filovirus virus-like particles containing trimeric hybrid glycoprotein stability and suitability for storage and distribution of ad .zebov/mva-bn r -filo heterologous prime-boost ebola vaccine human coronavirus oc cl protease and the potential of ml as a broad-spectrum lead compound: homology modelling and molecular dynamic studies epidemiology, genetic recombination, and pathogenesis of coronaviruses available online at a novel coronavirus from patients with pneumonia in china mers: emergence of a novel human coronavirus molecular pathology of emerging coronavirus infections the spike protein of sars-cova target for vaccine and therapeutic development receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc coronavirus infections and immune responses severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study who scientific and technical advisory group for infectious hazards aerosol and surface stability of sars-cov- as compared with sars-cov- clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study serology characteristics of sars-cov- infection since exposure and post symptom onset efficacy of genogroup based porcine epidemic diarrhea live vaccine against genogroup field strain in japan antibody responses to sars-cov- in patients with covid- rapid generation of neutralizing antibody responses in covid- patients sars vaccines: where are we? from sars to mers, thrusting coronaviruses into the spotlight searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design receptor-binding domain-based subunit vaccines against mers-cov intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus development of middle east respiratory syndrome coronavirus vaccines -advances and challenges middle east respiratory syndrome vaccine candidates: cautious optimism sars-cov- infection protects against rechallenge in rhesus macaques the convalescent sera option for containing covid- potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies inhibition of middle east respiratory syndrome coronavirus infection by anti-cd monoclonal antibody a human monoclonal antibody blocking sars-cov- infection cross-neutralization of sars-cov- by a human monoclonal sars-cov antibody potent neutralizing antibodies directed to multiple epitopes on sars-cov- spike structural basis for neutralization of sars-cov- and sars-cov by a potent therapeutic antibody longitudinal isolation of potent near-germline sars-cov- -neutralizing antibodies from covid- patients nanobodies: prospects of expanding the gamut of neutralizing antibodies against the novel coronavirus, sars-cov- structural basis for potent neutralization of betacoronaviruses by singledomain camelid antibodies t cell responses induced by attenuated flavivirus vaccination are specific and show limited cross-reactivity with other flavivirus species two is better than one: evidence for t-cell cross-protection between dengue and zika and implications on vaccine design. front immunol strong cd t cell responses to zika virus antigens in a cohort of dengue virus immune mothers of congenital zika virus syndrome infants structural basis of potent zika-dengue virus antibody crossneutralization cross-reactivity, and function of antibodies elicited by zika virus infection cross-reactive immunity among flaviviruses zika virus activates de novo and cross-reactive memory b cell responses in dengue-experienced donors dengue virus sero-cross-reactivity drives antibodydependent enhancement of infection with zika virus enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity zika virus infection confers protection against west nile virus challenge in mice nile virus vaccines -current situation and future directions viral-induced enhanced disease illness enhancement of the infectivity of arboviruses by specific antisera produced in domestic fowls the enhancement of virus infectivity by antibody complement receptor mediates enhanced flavivirus replication in macrophages antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody benefits and risks of the sanofi-pasteur dengue vaccine: modeling optimal deployment respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine priming immunization determines t helper cytokine mrna expression patterns in lungs of mice challenged with respiratory syncytial virus antibodydependent enhancement, a possible mechanism in augmented pulmonary disease of respiratory syncytial virus in the bonnet monkey model a potential molecular mechanism for hypersensitivity caused by formalin-inactivated vaccines a role for nonprotective complement-fixing antibodies with low avidity for measles virus in atypical measles lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease a novel respiratory syncytial virus (rsv) f subunit vaccine adjuvanted with gla-se elicits robust protective th -type humoral and cellular immunity in rodent models antibodydependent infection of human macrophages by severe acute respiratory syndrome coronavirus antibodydependent sars coronavirus infection is mediated by antibodies against spike proteins molecular mechanism for antibody-dependent enhancement of coronavirus entry unexpected receptor functional mimicry elucidates activation of coronavirus fusion immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates emerging viruses and current strategies for vaccine intervention self-disseminating vaccines for emerging infectious diseases a cytomegalovirus-based vaccine provides long-lasting protection against lethal ebola virus challenge after a single dose trained immunity-based vaccines: a new paradigm for the development of broad-spectrum anti-infectious formulations trained immunity: a program of innate immune memory in health and disease infectious agents as stimuli of trained innate immunity efficacy of whole-cell killed bacterial vaccines in preventing pneumonia and death during the influenza pandemic mandated bacillus calmette-guérin (bcg) vaccination predicts flattened curves for the spread of covid- trained innate immunity not always amicable scores of coronavirus vaccines are in competition -how will scientists choose the best? the future of flu: a review of the human challenge model and systems biology for advancement of influenza vaccinology challenge accepted: human challenge trials for dengue tortoises, hares, and vaccines: a cautionary note for sars-cov- vaccine development rational vaccine design in the time of covid- a strategic approach to covid- vaccine r&d a dictionary of epidemiology the authors declare that the research was conducted in the key: cord- - phlylce authors: felberbaum, rachael s. title: the baculovirus expression vector system: a commercial manufacturing platform for viral vaccines and gene therapy vectors date: - - journal: biotechnol j doi: . /biot. sha: doc_id: cord_uid: phlylce the baculovirus expression vector system (bevs) platform has become an established manufacturing platform for the production of viral vaccines and gene therapy vectors. nine bevs‐derived products have been approved – four for human use (cervarix®, provenge®, glybera® and flublok®) and five for veterinary use (porcilis® pesti, bayovac csf e ®, circumvent® pcv, ingelvac circoflex® and porcilis® pcv). the bevs platform offers many advantages, including manufacturing speed, flexible product design, inherent safety and scalability. this combination of features and product approvals has previously attracted interest from academic researchers, and more recently from industry leaders, to utilize bevs to develop next generation vaccines, vectors for gene therapy, and other biopharmaceutical complex proteins. in this review, we explore the bevs platform, detailing how it works, platform features and limitations and important considerations for manufacturing and regulatory approval. to underscore the growth in opportunities for bevs‐derived products, we discuss the latest product developments in the gene therapy and influenza vaccine fields that follow in the wake of the recent product approvals of glybera® and flublok®, respectively. we anticipate that the utility of the platform will expand even further as new bevs‐derived products attain licensure. finally, we touch on some of the areas where new bevs‐derived products are likely to emerge. the baculovirus expression vector system (bevs) is far from new. for thirty years researchers have been using this platform to express recombinant proteins, and thousands of proteins have been successfully expressed and purified. however, for much of this time, bevs was rele-gated to the ranks of research tool. what we have seen in the last decade is the elevation of bevs from research tool to an established manufacturing platform for production of novel biologic products. ten years ago there were only two commercial products manufactured using the bevs manufacturing platform and both of these were veterinary vaccines to prevent classical swine fever in pigs. since then, seven new products have been licensed, four of which are for humans, including vaccines and therapeutics, and many more products are in development (table ) [ , ] . we have passed a tipping point where bevs-derived products are becoming mainstream, and the bevs platform is being actively utilized by major players in the biotechnology industry to develop new products. although the bevs platform has distinct features that make it an attractive platform for the production of many biologics, it is not ideally suited for all products. factors such as protein complexity, post-translational modification, scale and cost must be considered collectively when selecting a manufacturing platform; these have been extensively reviewed elsewhere [ ] [ ] [ ] . in this review, we explore the bevs platform by looking at how the system works and the advantages and limitations of the platform from a manufacturing and regulatory perspective. the opportunities to develop new products using bevs are abundant, and we review the latest developments in the gene therapy and influenza vaccine fields as examples. finally, we consider some newer areas where bevs-derived products show promise for the future. the bevs platform has been previously described [ , , [ ] [ ] [ ] [ ] . the platform takes advantage of baculoviruses' natural propensity to infect insect cells. in nature, there are more than different types of baculoviruses, all of which have a host range restricted to invertebrates [ ] . in the laboratory and for manufacturing purposes, the most commonly used baculovirus is autographa californica multiple-capsid nuclear polyhedrosis virus (acmnpv), a virus with a double-stranded dna genome of approximately kb [ ] . the large size of its genome gives the baculovirus ample capacity to accommodate large amounts of foreign dna, including multiple genes, an advantage over other expression vectors such as vaccinia and adenovirus [ ] . to begin the bevs process, a recombinant baculovirus is constructed comprising the desired gene(s) of interest (goi) (fig. ) . first, the goi is cloned into a transfer plasmid, typically behind the strong polyhedrin or p promoter that can drive protein expression to high levels in insect cells [ , ] ; notably, these promoters are not very active in e. coli and, therefore, can be stable expression cassettes. the goi is flanked by acmnpv dna, e.g. the polyhedrin promoter on one side and a portion of the essential gene orf on the other. insect cells are then co-transfected with a mixture of the transfer plasmid and parental acmnpv dna that has been linearized such that the parental polyhedrin gene and portion of orf are missing, rendering it non-infectious [ ] . the plasmid and parental dna undergo homologous recombination to generate de novo recombinant baculoviruses. these baculoviruses are plated and individual plaques purified to isolate a single, pure plaque of recombinant baculovirus. this plaque is subsequently passaged through multiple rounds of insect cell infection to generate a high-titer stock and establish a working virus bank (wvb) that can be utilized for protein production. for manufacturing purposes, it is important that wvbs be stable and retain integrity as virus passages are scaled up. laboratory kits such as bac-to-bac ® (life technologies) that employ bacmid technology have been developed that allow researchers to quickly and easily construct recombinant baculoviruses in e. coli rather than [ ] . long-term stability of the baculovirus is an important consideration if large-scale protein production is envisioned. once a high-titer wvb has been established it is used to infect insect cells and stimulate protein production. cells are seeded in culture flasks (for small-scale production) or bioreactors (for large-scale production) and the wvb added to infect the insect cells when they are in their logarithmic growth phase. the baculoviruses reprogram the cellular machinery to produce the recombinant protein(s). following protein expression (typically - hours post-infection), the cells and/or supernatant are harvested, depending on whether the product is intracellular or secreted, respectively, and the proteins are purified according to standard techniques such as ultracentrifugation or column chromatography. many different insect cell lines have been used for bevs but the most common are derived from ovarian cells of the fall army worm, spodoptera frugiperda (e.g., sf- , sf- and expressf+ ® [protein sciences corporation]), and the cabbage looper, trichoplusia ni (high five tm cells, life technologies) [ , , ] . high five cells are used to manufacture the licensed human papillomavirus vaccine, cervarix ® (glaxosmithkline), and sf- cells are used to produce the antigen used in the prostate cancer immunotherapy, provenge ® (dendreon). expressf+ cells are used to manufacture three licensed products: flublok ® influenza vaccine (protein sciences corporation), glybera ® gene therapy for the treatment of familial lipoprotein lipase deficiency (uniqure), and ingelvac circoflex ® veterinary vaccine to protect against porcine circovirus type (boehringer ingelheim vetmedica). large scale manufacturing and commercial production require specific cell line characteristics such as scalability, high yields, the ability to grow in low-cost, serum-free media, and qualification to meet regulatory agency (e.g. fda, ema, etc.) requirements for purity and safety. characteristics of the bevs platform are summarized in fig. . these features as well as other important considerations about the technology are discussed below. the choice of an expression system for manufacturing is a complex decision that must balance many facets, including attainment of specific product features, the demands of the therapeutic indication, and the needs of the manufacturer. comparisons of the bevs platform to other expression systems such as bacteria, yeast, mammalian cells and plants have been made and are useful to consider when selecting a platform [ ] [ ] [ ] . bevs employs recombinant technology, giving researchers and product developers a level of control over the production process that is not possible with other techniques. a good example is found with vaccine manufacturing. traditionally, vaccines are made by cultivating large volumes of the pathogen against which protection is desired to generate the "raw materials" required for the product. this can be accomplished by infecting substrates such as embryonated chicken eggs or mammalian cells, both of which are used to manufacture the majority of the vaccines recommended for routine immunization [ , ] . the pathogen is either weakened and administered live as a live attenuated vaccine, or is killed or inactivated with reagents such as formalin or heat prior to being formulated into vaccine. these methods yield safe and effective vaccines; however, important shortcomings have been observed. the first concerns specificity. for example, traditional influenza vaccine production involves virus propagation in eggs. as influenza viruses are rna viruses, they have a tendency to mutate to optimize their figure . the bevs platform. the bevs platform is an efficient process for producing a wide variety of proteins in a streamlined manner. a gene of interest (goi) is cloned into a transfer plasmid behind a strong promoter (green arrow) and surrounded by dna homologous to the parent baculovirus (yellow and green boxes). a library of recombinant baculoviruses (rbv) can be made using standard cloning techniques and varying the goi. construction of an rbv takes eight days. to generate protein, the appropriate rbv is scaled up (taking on average two to five weeks) and used to infect insect cells, which programs the cells to generate large quantities of recombinant protein that can subsequently be purified to high levels using standard techniques. a single insect cell line can be used to produce all proteins. protein production averages three to five weeks and yields highly pure, biologically active products. growth [ ] . generally, the changes introduced into the virus sequence have little impact on vaccine efficacy. however, since the virus receptors for birds and mammals differ, these changes can be meaningful, and it has been documented that in some cases the changes have rendered egg-based influenza vaccines ineffective [ ] . the bevs process does not involve pathogen growth and, therefore, avoids this complication. rather than cultivate a pathogen to collect "raw materials", a recombinant baculovirus is constructed that codes for the antigen required for protection. since accommodations for growth do not need to be made, the antigen can be an exact sequence match to the human pathogen. this has the potential to solve the ineffectiveness observed for some egg-adapted vaccines as was described by skowronski et al. ( ) [ ] . moreover, specific point mutations can be introduced to enhance features such as stability. for example, it has recently been reported that purified hemagglutinin protein, the protective antigen in influenza vaccines, appears unstable in the srid potency assay due to crosslinking of specific cysteine residues in the protein [ ] . by mutating these cysteine residues to non-thiol residues, it was possible to prevent cross-linking and enhance stability [ ] . a second shortcoming with traditional vaccine manufacturing is process length. again, influenza serves as a good example. influenza epidemics occur every year and annual influenza vaccination is recommended by the centers for disease control and prevention [ ] . in addition, pandemic outbreaks occur occasionally, the most recent of which was the h n swine flu (a/california/ / ) [ , ] . timeliness of vaccine manufacture is critical to ensure that adequate vaccine supply is available. egg-based influenza vaccine manufacturing takes on average about six months, as the process of creating a high-producing, egg-adapted seed virus is slow [ ] . this means that manufacturing of seasonal influenza vaccines must begin the winter prior to an epidemic, before that season's strain prevalence is definitively known, to guarantee adequate supply, and this has resulted in seasons where the available influenza vaccines have not matched the circulating influenza strains [ , ] . moreover, the president's council of advisors on science and technology (pcast) reported that pandemic influenza vaccine in was not readily available until after the pandemic peaked, a major concern for public health and safety (www.whitehouse.gov/sites/default/files/microsites/ostp/ pcast-influenza-vaccinology-report.pdf). pcast identified recombinant technology and freedom from virus growth and egg adaptation as the solution to this problem. bevs-derived influenza vaccines do not require seed viruses and can be produced in as little as days [ ] . in addition to specificity and speed, recombinant technology offers advantages with respect to purity and product design. with respect to purity, recombinant products are free of pathogens, eggs and many chemicals (such as formalin and antibiotics) that can be undesirable or allergenic [ ] . for product design, recombinant techniques make it possible to engineer proteins with desired features, such as fusion proteins that increase immunogenicity or include multiple antigens and truncated proteins with deleted domains to improve yields and ease purification [ ] [ ] [ ] [ ] [ ] . one example of this application is the antigen used in provenge immunotherapy. the antigen is a fusion glycoprotein consisting of prostatic acid phosphatase (pap) linked to granulocyte-macrophage colonystimulating factor (gm-csf) and is used to stimulate autologous antigen presenting cells [ ] . the gm-csf activates the antigen presenting cells and enhances cell viability while the pap serves as the target antigen. another example is the cervarix ® vaccine manufactured by glaxosmithkline. the vaccine is a bivalent virus-like particle (vlp) comprised of the human papillomavirus l proteins (strains and ) . by expressing c-terminally truncated l proteins, the manufacturers are able to prevent intracellular vlp self-assembly, which would complicate purification. instead, the l subunit proteins are purified to a high degree and vlp assembly is achieved in vitro [ ] . the bevs platform has inherent safety measures built in that are attractive from a regulatory perspective. baculoviruses have a narrow host range restricted to specific insects and are considered safe to use as biological pesticides with no negative impact on plants, mammals, birds, fish or non-target insects [ ] . people are exposed to baculoviruses daily by consuming fresh vegetables. for instance, a serving of coleslaw may contain hundreds of millions of baculoviruses [ ] . baculovirus vectors have also been explored as gene therapy vectors. these studies have demonstrated that baculoviruses cannot repli-cate in mammalian cells and cannot express a gene cassette unless it is driven by a mammalian promoter [ , [ ] [ ] [ ] [ ] . a major consideration for regulatory agencies when evaluating a novel cell substrate is the potential to harbor adventitious agents that could threaten patient safety. there are very few adventitious agents that can replicate in both insect and mammalian cells. a notable exception is arboviruses that can be transmitted to humans via insect bites and can cause complications such as encephalitis and hemorrhagic fever [ ] . to mitigate this risk, cells from non-biting insects such as the fall armyworm and cabbage looper have been used with bevs as noted above. nonetheless, adventitious agents have been detected in some insect cell lines. for instance, the trichoplusia ni high five cell line, bti-tn- b - , used to make cervarix ® , was found to be latently infected with an alphanodavirus that was induced by recombinant baculovirus infection [ ] . in addition, other insect cell lines, such as those generated from drosophila melanogaster, have been shown to harbor innate retroelements derived from retroviruses that could potentially be infectious [ ] . more recently, a possible insect-specific virus sf-rhabdovirus was identified in spodoptera frugiperda cells [ ] . although studies showed that this virus could not enter or replicate in human cell lines and, therefore, is unlikely to be a risk, novel cell lines will most likely need to be characterized and monitored for the presence of this virus as they are for nodaviruses, retroviruses and others. in general, because adventitious agents are a potential threat, cell substrates of all origins (including insect and others) must be thoroughly tested for the presence and infectivity of such agents before they are allowed by regulatory agencies for manufacturing use. in addition to the safety considerations just discussed, there are two important regulatory features associated with the bevs platform that should be considered. the first is that bevs is a transient protein expression system; the recombinant baculoviruses used vary based on their foreign gene cassettes but a single cell line can be used for the expression of all proteins (fig. ) . qualifying a cell line is no small feat. it can take years of work to adequately ensure purity and safety in addition to high productivity. stable cell lines have to be independently qualified each time their genetic composition changes [ ] . in contrast, cell lines used with bevs and other transient expression systems remain constant and, consequently, need to be qualified just once. a second feature is the growing number of bevsderived products that have been approved by regulatory agencies worldwide ( table ) . as is the case for all technologies, prior regulatory approvals remove barriers for future product approvals. the technology becomes more mainstream and less novel with each approval, and the safety database of patients that are administered products without complication continues to grow. nine bevsderived products have been licensed, providing regulators confidence with the platform. the following characteristics make the bevs platform appealing for commercial manufacturing: scalability, biosafety, flexibility and existing manufacturing capacity. insect cells are grown in suspension, so if the cells and baculoviruses have been optimized for large scale and multiple passages, the culture size is only limited by the size of the bioreactor. for example, expressf+ cells have been used to produce recombinant proteins at scales ranging from two to l [ ] . the cost of goods for bevs production is largely dependent on capital costs and yields. as discussed by cox ( ), vast global bioreactor capacity (~ l) already exists and presents the opportunity to minimize the investment needed to establish bevs manufacturing facilities [ ] . moreover, opportunities for yield improvements are abundant and include genetic and fermentation-based approaches; these are described elsewhere [ ] . unlike some other production facilities, bevs facilities can be multi-purpose and used to produce a variety of bevs-derived products, especially when disposable or single-use technology is employed [ ] . this is because a single cell line can be used for production of different products. this feature is especially meaningful for regions of the world where limited manufacturing capacity exists. a single bevs facility could, for example, be used to produce vaccines for diseases endemic to a region and quickly be converted to produce pandemic influenza vaccine in an emergency. finally, because the bevs platform does not require the handling of live, potentially dangerous pathogens, requirements for biocontainment that can be very costly are reduced. the bevs platform is a versatile technology useful for the manufacture of many products; however, other platforms may be better suited for the production of certain proteins. for instance, small proteins that do not require post-translational modifications are best made in e. coli that can quickly generate high yields at low cost [ ] . yeasts such as s. cerevisiae can also produce high yields of protein at low cost and are capable of some post-translational modifications [ ] . insect cells are capable of many post-translational modifications but proteins that require complex post-translational modifications and folding may best be made in mammalian expression systems. for www.biotecvisions.com www.biotechnology-journal.com example, the glycosylation patterns produced by insect and mammalian cells are related, and glycoproteins produced in insect cells are often correctly folded, biologically active and immunogenic [ ] . however, insect cells generate less complex n-glycans than mammalian cells and this can negatively impact biological function [ , ] . some developments have been made to address this limitation, including engineering transgenic insect cell lines that stably express mammalian glycosylation enzymes or co-expressing such enzymes with the gene of interest in a single baculovirus [ ] [ ] [ ] [ ] . whether this is required must be assessed on a protein by protein basis. licensure of the first bevs-derived products has paved a regulatory pathway, reducing regulatory uncertainty. in this section we discuss opportunities in the areas of gene therapy and influenza vaccines spawned by the two most recent bevs-derived product approvals, glybera ® (licensed by the ema in ) and flublok ® (licensed by the fda in ), respectively. the bevs approach to gene therapy has largely involved the production of recombinant adeno-associated viruses (raavs) that house therapeutic dna. the use of raavs as a gene delivery vector has gained popularity for several reasons, including long-term gene expression, lack of pathogenicity and ability to transduce a wide variety of cells, both dividing and non-dividing [ , ] . nine different raav serotypes ( - ) are most commonly used for raav-based gene therapy, each serotype with a different propensity for tissue-specific infection and infection kinetics [ ] . recombinant aav-based gene therapies have been in development and shown promise for some time; however, a major limitation to their implementation had been the inability to scale up the manufacturing process to produce sufficient quantities of raavs. the original raav vectors were produced in mammalian tissue culture using adherent cells such as hek cells, which required about -cm flasks to produce enough material for a large animal study or human clinical trial (~ raav particles) [ ] . to overcome this limitation, scientists adopted and optimized the bevs platform for production of large scale, high titer raavs [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . by adjusting parameters such as multiplicity of infection, cell density and fermentation mode, raav yields on the order of vector genomes per liter have been reported [ ] . the traditional bevs production strategy for raavs requires the co-infection of insect cells with three differ-ent recombinant baculoviruses: bac-rep that expresses the major aav replication enzymes (rep and rep ); bac-cap that expresses the aav virion coat proteins; and bac-goi that expresses the gene of interest flanked by the aav inverted terminal repeat elements that are required for the rescue, replication and packaging of the gene [ , ] . no adenovirus helper is needed as is required for mammalian cell raav production [ , ] . due to some genetic instability of the bac-rep construct, a streamlined two-baculovirus system has further been developed where rep and cap proteins are expressed from a single baculovirus (bac-repcap) and rep and rep are transcribed from a single mrna species that enhances stability [ ] . alternatively, genetic modifications have been made to the original bac-rep and bac-cap constructs to enhance stability and improve expression [ ] . an important regulatory hurdle was overcome in when glybera received marketing authorization in europe, making it the first gene therapy product approved in the western world and launching the bevs platform into the spotlight as a preferred platform for raav manufacture. glybera (alipogene tiparvovec) is comprised of the human gene lpl s x in a bevs-derived raav serotype vector and is used for the treatment of patients with lipoprotein lipase (lpl) deficiency [ ] . clinical studies have shown glybera to be safe and effective [ , ] . glybera will likely be the beginning when it comes to raav-based gene therapy. the approach is relevant to the estimated thousands of monogenic diseases [ ] . treatments are actively being investigated in a diverse array of therapeutic areas and dozens of product candidates are in clinical development (summarized in table ). hemophilia is one area where progress has been made (reviewed in [ ] ). there are four ongoing human clinical trials involving raav serotypes or , all designed to express factor ix for the treatment of hemophilia b (table ). factor viii raav-based therapy is a target for the treatment of hemophilia a, the most common severe inherited bleeding disorder, and only a modest increase in plasma factor viii levels is expected to be required to be clinically relevant [ ] . raav-based treatments for retinal degeneration, including macular degeneration and leber's congenital amaurosis type , are another area of intense investigation [ , ] . retinal treatments are ideal because of cell accessibility through intravitreal and subretinal injections and the ability to assess structure and function noninvasively. serotype is most commonly used for these therapies but types , , and - are all capable of infecting photoreceptors, the most prominent cell type for retinal degenerations [ ] . diseases of the central nervous system (cns), such as parkinson's and alzheimer's diseases, are also actively being tested with raav-based therapies that are promising. aavs exhibit a strong preference for neuronal transduction, making them a popular gene delivery vehicle for cns therapies but vector improvements are still needed to optimize treatments (discussed in [ ] ). final-ly, duchenne muscular dystrophy (dmd) is a therapeutic target where progress is being made. although the single gene affected in dmd (dystrophin) has long been known, its large size has made gene therapy a challenge. recent treatment approaches to overcome this include exonskipping, trans-splicing and micro-and mini-dystrophin delivery strategies [ , ] . for fifty years influenza vaccine manufacturing technology remained largely stagnant. that changed with the licensure of flublok ® in . flublok ® is a trivalent bevs-derived vaccine for seasonal influenza composed of µg of recombinant hemagglutinin (ha) derived from the two a and one b influenza viruses selected for inclusion in the annual influenza vaccine by the world health organization; the vaccine is licensed by the fda for adults and older [ ] . influenza vaccines are standardized to contain a specific amount of ha, the major surface glycoprotein on the influenza virus [ ] . bevs-derived recombinant ha forms trimers that in turn oligomerize into immunogenic rosettes [ ] . these proteins can be purified to high levels resulting in a vaccine that has been shown to be safe and effective in clinical studies [ , [ ] [ ] [ ] [ ] . the advantages of recombinant bevs vaccines for pandemic influenza are especially important. vaccines for avian influenza viruses such as the h , h and h subtypes are urgently needed because of these viruses' high pathogenicity and mortality rates in humans and the fact that h has previously demonstrated pandemic potential and human-to-human transmissibility [ , ] . a monovalent variation of the flublok ® vaccine called panblok ® has been developed. in a phase ii study of h panblok ® (a/indonesia/ / ), a two-dose schedule of vaccine at doses of . - µg ha formulated with the adjuvant glucopyranosyl lipid a/stable emulsion (gla/se) had an acceptable safety and reactogenicity profile and elicited serologic responses meeting seroconversion criteria in adults - years old [ ] . moreover, an earlier study showed that people administered h panblok ® (a/hong kong/ / ) in were primed for an enhanced immune response following administration of an antigenically variant vaccine strain in [ ] . evaluated a different bevs-derived h subunit vaccine candidate (a/goose/guangdong/ / ) and showed that it protected against a lethal challenge in balb/c mice and in specific pathogen-free and commercial chickens, suggesting it could be useful as both a human and animal vaccine [ ] . multi-component vlp vaccines for pandemic influenza are under development that are composed of recombinant ha, neuraminidase (na) and matrix (m ) proteins produced in sf- cells [ ] [ ] [ ] . evaluation of an h n (a/indonesia/ / ) vlp vaccine in a phase i/ii study of adults - years old showed that two doses of unadjuvanted vaccine at , or µg ha/dose were generally well-tolerated and resulted in seroconversion [ ] . similarly, a phase ii study of an h n (a/california/ / ) vlp vaccine in adults - years old showed it was safe at doses of , or µg ha/dose and elicited high rates of seroprotection ( - %) [ ] . more recently, an h n vlp vaccine was developed that was comprised of ha and na proteins matched to a/anhui/ / (h n ) and m protein matched to a/indonesia/ / (h n ) [ ] . this vaccine candidate was tested with or without saponin-based iscomatrix adjuvant in balb/c mice and was shown to protect against a lethal challenge. antibodies against both ha and na were elicited, with -to -fold higher responses in the iscomatrix groups. although the data are promising, a challenge for the development of multi-component vlp vaccines will be standardizing the vaccines for each of their components (e.g., quantity of ha vs. na vs. m ). an advantage of using recombinant technology for vaccine design is the opportunity to modularly add or subtract antigens to a formulation. inclusion of recombinant na in vlp vaccines has been shown to induce formation of anti-neuraminidase antibodies [ ] . it has been noted that different vaccine compositions (e.g., vlp vs. subunit vs. whole virion) induce different immune profiles in balb/c mice [ ] . while the advantages of these various profiles are not yet clear, the flexibility of the bevs platform enables catering towards different outputs. besides inclusion in vlps, recombinant na can be individually produced via bevs and may serve as a potentially efficacy-enhancing additive to influenza vaccines [ ] . na immunity is infection-permissive but can reduce infection severity and duration [ ] . the potential benefits of including recombinant na in influenza vaccines has recently been reviewed [ ] . other opportunities have emerged with bevs as researchers begin pursuit of a so-called universal influenza vaccine. licensed influenza vaccines offer limited cross protection to heterologous influenza viruses and, thus, there is the need for annual update of seasonal influenza vaccines and concern over pandemic preparedness. a successful universal influenza vaccine would offer long-lasting and broad protection against a range of different influenza virus strains. approaches to universal influenza vaccine design include ha stalk-based constructs and chimeric ha-based vaccines that are composed of conserved stalk domains fused to "exotic" heads, usually of avian origin (these approaches are reviewed elsewhere [ ] ). the bevs platform, being based on recombinant technology, offers the flexibility and genetic control required for the design and manufacture of these universal vaccine candidates. in this review we have taken a close look at the bevs platform, describing how the platform works, outlining the features and limitations of the technology and highlighting the growth opportunities that emerged from the two most recent bevs-derived product approvals. we expect this growth to continue and expand as future bevsderived products attain regulatory approval. the approvals of bevs-derived cervarix ® and flublok ® vaccines have broadened the acceptance of the platform beyond its initial veterinary borders to use in healthy adolescents and adults. human therapeutics is another area of use and gene therapy in particular is a growing area of interest; the approval of glybera drew major attention to bevs-derived raavs. baculoviruses themselves can also be used as gene delivery vectors, and other recombinant protein complexes produced using bevs are being explored for the delivery of various peptides and antigens, such as in the form of the newly characterized vault particles [ ] . the speed at which recombinant proteins can be produced using bevs makes it a particularly attractive platform to design safe and effective vaccines to be available to timely combat new infectious pathogens as they arise. success with this approach has been demonstrated for influenza and can be applied to broader areas. for example, coronaviruses have been plaguing both people and animals especially in the last decade with lethal outbreaks of severe acute respiratory syndrome (sars) in , middle east respiratory syndrome (mers) in late , and porcine epidemic diarrhea virus (pedv) in [ ] [ ] [ ] . all coronaviruses share a similar structure that includes the presence of the spike glycoprotein on the viral envelope that is the dominant immunogen [ ] [ ] [ ] . multiple coronavirus bevs-derived vaccine candidates have been developed that include recombinant spike protein either alone as a subunit vaccine or together with recombinant envelope and membrane proteins as vlps and have demonstrated efficacy in animal models [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the spike protein has been shown to be immunogenic both as full length protein and as a truncated protein containing only the extracellular domain [ , ] , and multiple routes of vaccine administration have been examined [ ] . two recently emerged threats are chikungunya virus and ebola virus, both of which can be addressed with bevs-derived vaccines. chikungunya virus causes a serious disease that involves severe joint pain and can be fatal; a recent outbreak appeared in saint martin in december and has since made its way to more than countries or jurisdictions in the americas, including the continental united states [ ] . chikungunya is an arbovirus, for which there are many opportunities to develop bevs-derived vaccines (reviewed in [ ] ). both subunit and vlp vaccine candidates expressing the chikungunya viral envelope glycoproteins have been developed using bevs, with vlps demonstrating higher immunogenicity in mice [ ] [ ] [ ] . ebola virus is a filovirus that causes lethal hemorrhagic fever in humans and is devastating africa in an ongoing outbreak [ ] . the ebola virus glycoprotein has been shown to be the protective antigen and could be produced similar to a chikungunya virus vaccine [ ] . in addition to glycoprotein-based vaccines, the bevs platform has shown early promise for the production of biotechnology journal toxin-based vaccines. for example, the c-terminal heavy chain domain of clostridial botulinum neurotoxin, a highly toxic protein that causes botulism, has been produced with bevs and has demonstrated immunogenicity and challenge protection in mice [ , ] . recombinant toxin vaccines for other diseases such as clostridium difficile could also be possible [ , ] . in conclusion, bevs is a versatile platform whose potential is just beginning to be realized. the technology offers speed, flexibility, specificity and safety, and the use of a single cell line to manufacture multiple products makes bevs an attractive platform to adopt. bevs can be used to develop a wide variety of products and is especially well suited for combating rapidly emerging and dangerous pathogens. with new threats continually on the rise, tools such as bevs offer an important defense. the author works for protein sciences corporation, which has a financial interest in bevs and manufactures flublok ® influenza vaccine. recombinant protein vaccines produced in insect cells vaccines for viral and parasitic diseases produced with baculovirus vectors commercial production in insect cells. one company's perspective production of recombinant proteins by microbes and higher organisms a fast track influenza virus vaccine produced in insect cells insect cell technology is a versatile and robust vaccine manufacturing platform opportunities and challenges for the baculovirus expression system baculovirus-insect cell expression systems baculovirus as a highly efficient expression vector in insect and mammalian cells the complete dna sequence of autographa californica nuclear polyhedrosis virus production of human beta interferon in insect cells infected with a baculovirus expression vector a baculovirus dual expression vector derived from the autographa californica nuclear polyhedrosis virus polyhedrin and p promoters: co-expression of two influenza virus genes in insect cells a method for producing recombinant baculovirus expression vectors at high frequency spontaneous excision of bac vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells the establishment of two cell lines from the insect spodoptera frugiperda (lepidoptera; noctuidae) screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system traditional and new influenza vaccines molecular mechanisms enhancing the proteome of influenza a viruses: an overview of recently discovered proteins low - influenza vaccine effectiveness associated with mutation in the egg-adapted h n vaccine strain not antigenic drift in circulating viruses mechanism of a decrease in potency for the recombinant influenza a virus hemagglutinin h antigen during storage modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevents cross-linked multimer formation and potency loss prevention and control of seasonal influenza with vaccines antigenic and genetic characteristics of swine-origin a(h n ) influenza viruses circulating in humans pandemic novel h n influenza: what have we learned? semin. respir impact of influenza b lineagelevel mismatch between trivalent seasonal influenza vaccines and circulating viruses influenza vaccine: the challenge of antigenic drift molecular and structural characterization of the l virus-like particles that are used as vaccine antigens in cervarix, the as -adjuvanted hpv- and - cervical cancer vaccine a recombinant baculovirus-expressed s glycoprotein vaccine elicits high titers of sars-associated coronavirus (sars-cov) neutralizing antibodies in mice a designer hyper interleukin (h ) is a biologically active cytokine a vaccine of l epitope repeats fused with a modified igg fc induced cross-neutralizing antibodies and protective immunity against divergent human papillomavirus types baculovirus vector as an avian influenza vaccine: hemagglutinin expression and presentation augment the vaccine immunogenicity provenge (sipuleucel-t) in prostate cancer: the first fda-approved therapeutic cancer vaccine the presence of nuclear polyhedrosis viruses of trichoplusia ni on cabbage from the market shelf in vitro survey of autographa californica nuclear polyhedrosis virus interaction with nontarget vertebrate host cells baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells efficient gene transfer into human hepatocytes by baculovirus vectors baculovirus-mediated gene transfer into mammalian cells arbovirus vaccines; opportunities for the baculovirus-insect cell expression system latent infection of a new alphanodavirus in an insect cell line gypsy endogenous retrovirus maintains potential infectivity in several species of drosophilids identification of a novel rhabdovirus in spodoptera frugiperda cell lines mammalian stable expression of biotherapeutics technology transfer and scale-up of the flublok ® recombinant hemagglutinin (ha) influenza vaccine manufacturing process vaccine process technology thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology baculovirus as versatile vectors for protein expression in insect and mammalian cells transforming lepidopteran insect cells for improved protein processing n-glycosylation engineering of lepidopteran insect cells by the introduction of the beta , -n-acetylglucosaminyltransferase iii gene a transgenic insect cell line engineered to produce cmp-sialic acid and sialylated glycoproteins sweetbac: a new approach for the production of mammalianised glycoproteins in insect cells recombinant adeno-associated virus transduction and integration new developments in the use of gene therapy to treat duchenne muscular dystrophy analysis of aav serotypes - mediated gene expression and tropism in mice after systemic injection insect cells as a factory to produce adeno-associated virus type vectors. hum a simplified baculovirus-aav expression vector system coupled with one-step affinity purification yields high-titer raav stocks from insect cells improving adeno-associated vector yield in high density insect cell cultures successful production of pseudotyped raav vectors using a modified baculovirus expression system economized large-scale production of high yield of raav for gene therapy applications exploiting baculovirus expression system removal of empty capsids from type adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression toward exascale production of recombinant adeno-associated virus for gene transfer applications producing recombinant adenoassociated virus in foster cells: overcoming production limitations using a baculovirus-insect cell expression strategy. hum large-scale production of recombinant adeno-associated viral vectors novel tools for production and purification of recombinant adenoassociated virus vectors adeno-associated virus as a gene therapy vector: vector development, production and clinical applications immune responses to aav-vectors, the glybera example from bench to bedside safety profile of recombinant adeno-associated viral vectors: focus on alipogene tiparvovec (glybera ® ) identification of sequence variants in genetic disease-causing genes using targeted next-generation sequencing current status of haemophilia gene therapy advances in aav vector development for gene therapy in the retina preclinical safety evaluation of aav -sflt -a gene therapy for agerelated macular degeneration adeno-associated virus (aav) gene therapy for neurological disease full-length dystrophin reconstitution with adeno-associated viral vectors. hum recombinant trivalent influenza vaccine (flublok ® ): a review of its use in the prevention of seasonal influenza in adults pandemic influenza vaccine: characterization of a/california/ / (h n ) recombinant hemagglutinin protein and insights into h n antigen stability protective efficacy of a trivalent recombinant hemagglutinin protein vaccine (flublok ® ) against influenza in healthy adults: a randomized, placebo-controlled trial evaluation of the safety, reactogenicity and immunogenicity of flublok ® trivalent recombinant baculovirus-expressed hemagglutinin influenza vaccine administered intramuscularly to healthy adults - years of age safety and immunogenicity of a baculovirus-expressed hemagglutinin influenza vaccine: a randomized controlled trial comparative immunogenicity of recombinant influenza hemagglutinin (rha) and trivalent inactivated vaccine (tiv) among persons > or = years old global alert to avian influenza virus infection: from h n to h n receptor specificity and transmission of h n subtype viruses isolated from the pandemic of evaluation of safety and immunogenicity of recombinant influenza hemagglutinin (h /indonesia/ / ) formulated with and without a stable oil-inwater emulsion containing glucopyranosyl-lipid a (se+gla) adjuvant immune responses of healthy subjects to a single dose of intramuscular inactivated influenza a/vietnam/ / (h n ) vaccine after priming with an antigenic variant a subunit vaccine candidate derived from a classic h n avian influenza virus in china protects fowls and balb/c mice from lethal challenge h n virus-like particle vaccine elicits cross-reactive neutralizing antibodies that preferentially bind to the oligomeric form of influenza virus hemagglutinin in humans safety and immunogenicity of a virus-like particle pandemic influenza a (h n ) vaccine in a blinded, randomized, placebo-controlled trial of adults in mexico development of influenza h n virus like particle (vlp) vaccine: homologous a/anhui/ / (h n ) protection and heterologous a/chicken/jalisco/cpa / (h n ) cross-protection in vaccinated mice challenged with h n virus influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin baculovirus-expressed influenza vaccine. a novel technology for safe and expeditious vaccine production for human use influenza neuraminidase as a vaccine antigen advances in universal influenza virus vaccine design and antibody mediated therapies based on conserved regions of the hemagglutinin development of the vault particle as a platform technology middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution severe acute respiratory syndrome vs. the middle east respiratory syndrome porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines the sars-cov s glycoprotein current advancements and potential strategies in the development of mers-cov vaccines drug targets for rational design against emerging coronaviruses baculovirus surface display of sars coronavirus (sars-cov) spike protein and immunogenicity of the displayed protein in mice models antigenic and immunogenic characterization of recombinant baculovirusexpressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design immune responses against severe acute respiratory syndrome coronavirus induced by viruslike particles in mice virus-like particles of sarslike coronavirus formed by membrane proteins from different origins demonstrate stimulating activity in human dendritic cells vaccination of mice with recombinant baculovirus expressing spike or nucleocapsid protein of sars-like coronavirus generates humoral and cellular immune responses effect of mucosal and systemic immunization with virus-like particles of severe acute respiratory syndrome coronavirus in mice chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix efficiently form virus-like particles (vlps) that protect mice against challenge with sars-cov assembly and immunogenicity of coronavirus-like particles carrying infectious bronchitis virus m and s proteins purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice chikungunya at the door -deja vu all over again? effective chikungunya virus-like particle vaccine produced in insect cells chikungunya virus-like particles are more immunogenic in a lethal ag mouse model compared to glycoprotein e or e subunits enhanced production of chikungunya virus-like particles using a high-ph adapted spodoptera frugiperda insect cell line estimating the ebola epidemic antibodies are necessary for rvsv/zebov-gp-mediated protection against lethal ebola virus challenge in nonhuman primates easy expression of the c-terminal heavy chain domain of botulinum neurotoxin serotype a as a vaccine candidate using a bi-cistronic baculovirus system rapid immune responses to a botulinum neurotoxin hc subunit vaccine through in vivo targeting to antigen-presenting cells development of a recombinant toxin fragment vaccine for clostridium difficile infection recombinant antigens based on toxins a and b of clostridium difficile that evoke a potent toxin-neutralising immune response reingard grabherr key: cord- -intxyu authors: chaudhry, sundas nasir; hazafa, abu; mumtaz, muhummad; kalsoom, ume; abbas, saima; kainaat, amna; bilal, shahid; zafar, nauman; siddique, aleena; zafar, ayesha title: new insight on possible vaccine development against sars-cov- date: - - journal: life sci doi: . /j.lfs. . sha: doc_id: cord_uid: intxyu in december , a novel virus, namely covid- , caused by sars-cov- , developed from wuhan, hubei territory of china, which used its viral spike glycoprotein receptor-binding domain (rbd) for the entrance into a host cell by binding with ace- receptor and cause acute respiratory distress syndrome (ards). data revealed that the newly emerged sars-cov- affected more than , , people with , deaths worldwide. until now, no licensed immunization or drugs are present for the medication of sars-cov- . the present review aims to investigate the latest developments and discuss the candidate antibodies in different vaccine categories to develop a reliable and efficient vaccine against sars-cov- in a short time duration. besides, the review focus on the present challenges and future directions, structure, and mechanism of sars-cov- for better understanding. based on data, we revealed that most of the vaccines are focus on targeting the spike protein (s) of covid- to neutralized viral infection and develop long-lasting immunity. up to phase- clinical trials, some vaccines showed the specific antigen-receptor t cell response, elicit the humoral and immune response, displayed tight binding with human-leukocytes-antigen (hla), and recognized specific antibodies to provoke long-lasting immunity against sars-cov- . immune medical institute), and ino- (inovio) presenting the significant results in initial trials and soon will be tested on human [ ] . spike protein (s) of coronavirus are targeted by these vaccines and help in the stimulation of neutralizing antibodies [ ] . different subunits of spike proteins like the s and s subunits, and the receptor-binding domain (rbd) are the critical elements for the formation of a vaccine against the newly emerged virus that helped in producing t cell responses and protective immunity against sars-cov- [ ] . furthermore, two recombinant proteins that carry a receptor-binding domain (see glossary), and recombinant vectors that code for the receptor-binding domain can also play a useful role in developing a corona-vaccine. besides, the monoclonal antibodies of recovered corona-patients showing better results in neutralizing the antibodies by targeting the specific domains of sars-cov- [ , ] . however, with increasing the cases and deaths of covid- day by day, the development of vaccines against sars-cov- is also required on an urgent basis. the present review investigated the new insight parameters and components to develop a vaccine against newly emerged covid- by targeting the spike protein and epitopes. the present study also aims to share the candidate antibodies and latest development in the formulation of a vaccine against sars-cov- . further, current challenges and future perspectives, the structure, mechanism, immune response, and different components of covid- are studied in the present review for a better understanding. cathepsins b and l in sars-cov- [ ] . the evidence revealed that the cellular protease, like tmprss (see glossary) plays an essential role during the entry and circulation of sars-cov- . however, the antiviral or vaccine could be a potential therapeutics against covid- by blocking the regulation of tmprss enzyme [ ] . however, the entry of the virus is cell type and protease specific dependent. six hbs are framed by the communication of heptad repeats and (hr and hr ), and the areas present in a spike protein formed by the embedment of peptides in the layer of endosomes [ ] . as a result, an envelope of virus and plasma film is synthesized by the golgi intermediate compartment. finally, the newly formed virus rna genome is discharged from the cell through exocytosis. the detailed pathway is presented in fig. . to reduce the binding of virus with the host receptor known as ace , the subunit immunizations for both sars coronaviruses depend on inspiring a resistant reaction contrary to the spike protein [ ] . to prevent the viral infection, there is a need for the natural killer cells to perceive the entrance of the virus into a host cell. according to accumulated data, only limited researches are presented yet on the innate immune response to sars-cov- infection. recently, an ongoing project (consist of cases) in buali hospital, iran revealed a significant reduction of % ( u/l) in lymphocytes, improvement in total neutrophil ( %), c-reactive protein ( %), and serum il- ( %). the results also reported that the amount of lymphocytopenia and neutrophilia are correlated with infection mortality and disease severity [ , ] . similarly, another report based on cases in wuhan, china, claimed the % reduction in total lymphocytes, , , and % enhancement in neutrophils, c-reactive proteins, and serum il- , respectively [ ] . the immune responses associated with the diseases of the lung may have resulted in a large amount of j o u r n a l p r e -p r o o f cytokines (see glossary) production, which provides the st line defense against viral infection [ ] . in addition, the humoral cells of the body are responsible for antibodies production against viral, which could play a significant role in preventing viral infection by killing them [ ] . the innate immune system could effectively identify the situation during the incursion of the virus, usually called pathogen-associated molecular patterns (pamps). however, the endosomal rna receptors, rig, cytosolic rna sensor, tlr , and tlr quickly recognized these pamps (present in the form of ssrna on the viral genome) during the replication of the virus and cause stimulation of numerous signaling pathways and transcription factors including nf-κb, irf- , ap- and irf- [ , ] . ultimately, these nuclear factors, especially ap- and nf-κb, activated the expression of genes that encoded the chemokines (cxcl and ccl ), and cytokines (il- and tnf) that lead to the inflammatory responses. similarly, other nuclear factors, namely irf and irf , increased the production of inf-type (ifnα and infβ), which significantly helped in the reduction of viral dissemination and replication (see fig. ) [ ] . based on the increasing incidences of newly emerged covid- , there is necessary to make a successful and safe vaccine against sars-cov- to control its present pandemic and future recurrence. the present lethal coronavirus (covid- ) shared the structure homology with previously reported beta-coronaviruses, including mers-cov ( %) and especially sars-cov ( . %) [ ] . based on their structure homology, the genome accession number (genbank), [ ] . accumulated data reported that the formation of a vaccine falls into one of the following types; viral vectors ( projects and one is under clinical trial), dna and mrna-j o u r n a l p r e -p r o o f based ( projects and one from each type is under clinical trial), protein-based ( projects and mostly on s protein), virus-like particle (vlp) ( projects), inactivated or live-attenuated virus ( projects and two are under clinical trial), and epitope vaccine [ , ] . antibodies are the best and conservative means to anticipate and control desirable infections. till more than pharmaceutical industries and scholarly foundations all over the world have propelled their plans on immunization improvement against sars-cov- [ ] . herein, we discussed the new insights and latest developments in each class of vaccines to formulate a strong and effective vaccine against sars-cov- . the vaccine development gained special attention during the last few decades, including the development of rna, dna, and protein-based vaccines against several viruses like the influenza virus, and ervebo virus. the proteins are the important constituents in the structural activities of coronavirus, which are involved in the transmission, entry, and replication of viruses. the data suggested that proteins could be ideal targets for the development of vaccines [ , ] . the emerging evidence revealed that the viral spike (s) protein vaccine showed the higher neutralizing titers (see glossary) against sars-cov than other vaccine candidates. based on accumulated data, the s protein is the preferred site of vaccine formation against previously emerged coronaviruses (mers and sars-covs) because s protein could easily be encountered and allow the body to make an immune response more efficiently than other proteins [ , ] . based on these points, the potential parts of s protein, which are used as antigens in immunization improvement, integrate the entire extent of this protein and vaccine development [ ] . pallesen et al. [ ] reported that the s ectodomain of coronavirus with g showed a glycosylated loop variation in a structure of the virus, and observed the four major j o u r n a l p r e -p r o o f conformational sites in a trimer for the binding of each rbd either relatedly or tightly to a receptor accessible confirmation. however, the structure-based design of coronavirus could provide a prospective way for the formulation of a vaccine. many pharmaceutical industries and universities start working for the development of vaccines against covid- . recently, the astrazeneca and the university of oxford began to combined effort to develop a spike protein (s) vaccine, namely azd against newly emerged sars-cov- in chimpanzee to generate the dna for the spike antigen and to grow the vigorous t cell and b cell responses for better prophylaxis with less dose [ ] . besides, another company who made a vaccine against ebola virus, namely cansino biologics (china) started a project to create a safe and efficient s protein antigen-based vaccine (ad -ncov) against sars-cov- that is undergoing the phase- clinical trial on individuals aged between - years under clinicaltrial.gov: nct [ ] . similarly, the two top vaccine producer companies, including glaxosmithkline and sanofi start working on the formation of spike protein-based vaccine against covid- to trigger the immune response, and they reported that they would begin phase- clinical trials later this year [ ] . besides, the subunits of s protein, including receptor-binding domain (rbd), n-terminal domain (ntd), and c-terminal domain (ctd) also consider as a vital target for the progress of a vaccine. the study reported that antibodies that are attached to the n-terminal domain of the s subunit of coronavirus demonstrated an active killing movement, which indicates that ntd is applicable in balance for vaccine development. jiaming and his research group [ ] experimented on balb/c immunized mice at two different doses ( and µg) to investigate the effect of recombinant ntd (rntd) for vaccine development and immunogenicity. they revealed that high administration of µg significantly reduced the lung infection caused by a j o u r n a l p r e -p r o o f coronavirus in a recombinant vaccinated mouse model. they also observed a robust t-cell immune retort in the serum of vaccinated mice (rntd) with cpg and aluminum adjuvant. geo et al. [ ] experimented on balb/c mice (n= ) to check the rbd, s, and n-protein antibody response after - weeks of first immunization at µg dose (accessed by eliza). they observed that sars-cov- linked rbd and s-protein immunoglobulin g (igg) (see glossary) instantly start to develop inside the immunized mice at th week with the peak titer of , ( µg/ml) and , ( µg/ml) respectively. interestingly, the peak titer of n-specific igg in antibody response was about times fewer than rbd in vaccinated mice. but recently, another study stated that the n-specific igg is reported as one of the most effective proteins in covid- recovered patients as a diagnostic marker and vaccine development [ ] . the study revealed that the rbd of specific igg showed about the half of the value of antibody response than s protein, and suggested that the rbd is a meticulously associated immunogen to the recovered patients of covid- [ ] . however, the s protein and rbd of sars-cov- might be an innovative possible target for the formulation of the vaccine. the inactivated vaccine (known as whole killed virus: wkv) is an important vaccine that possesses the ability to cease the replication cycle of the virus, and it is generally prepared by neutralizing the virus through radiation or heat, and chemicals like formaldehyde. according to emerging evidence, the inactivated virus vaccine is one of the safe and effective vaccines, which can easily be prepared with much fewer cost as compared to nucleic acid vaccines [ ] . the wkv vaccine could significantly target the subunits of viruses, including s and e proteins, orf, matrix (m), and boost the immune response against viruses [ ] . the wkv vaccine has attained j o u r n a l p r e -p r o o f special attention over the decades due to its several advantages against viruses, particularly sars-cov, that could easily neutralize the virus antibodies [ ] . most recently, a study experimented on non-human primates (rhesus macaques), rat, and mice (balb/c) at two different doses ( and µg) for the investigation of protective potential and immunogenicity of purified inactivated virus vaccine candidate (picovacc). they revealed that picovacc significantly neutralized the effect of ten sars-cov- strains and demonstrated the partial or complete protective potential against sars-cov- in macaques deprived of any development of the antibody-dependent disease. the data suggested that the inactivated virus vaccine candidate (picovacc) could be proved as a novel option for a vaccine development against covid- [ ] . another study demonstrated that inactivated virus vaccine also showed a protective behavior against previously emerged coronavirus, namely sars-cov- , but phase- clinical trials were dried due to several reasons including a low number of cases and lack of funds, but few neutralizing monoclonal antibodies (see glossary) development by inactivated virus vaccine like cr that could be a potent cross-protection against covid- [ , ] . based on the accumulated data, the live attenuated and inactivated virus vaccines are reported as one of the safest and effective vaccines against previously emerged viruses, namely influenza virus [ ] . the live attenuated virus vaccine could be developed by diagnosing the newly emerged transmitted sars-cov- through a possible fewer risk of pathogenesis like enhanced anti-inflammatory cytokines, minimum lung infection, and fewer neutrophil influx as compared to wild type virus [ ] . the inactivated and live attenuated virus vaccines consider the whole virus as a vaccine. a study evaluated the performance of live attenuated vaccine, by initiating alteration (y h) into orf a/b polyprotein (nsp ) of mice, which showed the significant replication reduction behaviors of coronavirus in mice after day five intracerebral inoculation j o u r n a l p r e -p r o o f [ ] . similarly, a recent study suggested that the development of oral live attenuated virus vaccine could be a potential target to diminish the lung infections triggered by sars-cov- , due to its initial infection in guts which resultingly boosted up the mucosa that associated with immune system during the early immune response against covid- [ ] . another study also reported that the live attenuated virus vaccine could easily be administrated and serve as a community spread to rapidly develop herd immunity against the pathogens of covid- [ ] . against sars-cov- that is under phase- clinical trial [ ] . however, few limitations could also be manifested during the expansion of inactivated or live attenuated vaccines against sars-cov- . first, the live attenuated vaccines could regain its virulent effect in an in vivo or cell culture. second, the coronavirus could prevent seepage from the immunity developed by these vaccines through quick progression. thus, the manufacturer should be aware and careful during the development of these (inactivated and live attenuated) vaccines against sars-cov- [ ]. antibodies that are made up of dna contained circular dna molecules that encoding at least one foreign particle, and these antibodies are considered as better, as compared to other antibodies [ , ] . the dna-based vaccine could effectively target several variants of coronavirus including the s domain, and prefusion stabilized ectodomain with furring cleavage, spike protein, receptor-binding domain (rbd), cytoplasmic tail, and transmembrane domain [ , ] . the dna-based vaccine composition is an expensive and sophisticated method, which consists of double-stranded plasmids that are usually designed with the help of a computer/smart device to generate an immune retort against the virus. a specific device, named cellectra is used to generate the electric pulse under the skin to usher the dna-based vaccine [ , ] . the results revealed no serious adverse effect and immune response was dose-dependent, at mg the candidates showed the maximum immune response against mers-cov. however, future study is required to check the efficiency of dna-based vaccine (dls- ) against newly emerged sars-cov- , that could be a novel target. the previously reported studies revealed that the computation strategy is one of the effective techniques for the development of vaccines against many lethal infections, including malaria, cancer, and dengue. this technique worked by recognition of novel t cell epitopes, and mhc and molecules (see glossary) for the development of specific vaccines associated with the transporter of antigen presentation (tap) agents [ , ] . this vaccine is made up of antibodies related to the segments of immaculate foreign particles and is generally arranged by j o u r n a l p r e -p r o o f substance amalgamation procedures. these antibodies are simpler in readiness and also have efficient control [ ] . based on its previous tremendous application in other diseases like dengue, the epitope-based virus could be an essential option in the formulation of vaccines against sars-cov- . currently, many projects are undergoing vaccine formation. khan subunit immunizations include at least one antigen with solid immunogenicity to prepared to efficiently vaccine that activates the host immune system. this kind of antibody is more secure and simpler to create because it did not contain any live virus for the development of a vaccine. however, it frequently requires the development of adjuvants (see glossary) to induce a solid defensive safe reaction. but like other vaccines like inactivated or live attenuated vaccine, the subunit vaccine is less immunogenic that could be improved by adding the suitable adjuvants [ , ] . up until few foundations have started working on the formulation of a subunit-based vaccine to target the virus. the university of queensland has begun work to build up this type of subunit body, which depends upon the molecular lamp methodology [ ] . similarly, clover biopharmaceuticals inc. started the initial validation in building up an antibody competitor against sars-cov- by utilizing the "trimer-tag" innovation [ ] . liu and his research group designed a novel s -subunit based nano-vaccine against sars-cov- and stated that nano-vaccine showed the strong immune and humoral response and effectively elicited the t-cell response by triggering cd + and cd + cells, which to minimize the viral load in affected persons. the results also revealed that the nano-vaccine provoke the iga antibody, which provides the mucosal protection against viral load [ ] . kalita the recombinant protein is known as one of the emerging fields for the development of a vaccine against viruses due to several properties including tight binding to specific ace- receptor, provoke immune protection against viral infections, increase antibody-dependent viral entry, and promote antigenicity against virus like sars-cov [ ] . the accumulated data stated that the recombinant ace- (race- ) receptor exerted the potential targets against the protection of many diseases, including acute ang -induced hypertension, and severe lung injury [ , ] . besides, race- showed the rapid cure rate with a half-life of about min in mice as well as humans [ ] . lei et al. [ ] suggested that this vaccine could be a new hope against newly emerged sars-cov- due to structure homology with sars-cov- [ ] . table illustrates some essential vaccine candidates that are presently under clinical trials for the formulation of an active vaccine against the pandemic of sars-cov- . in contrast, fig. explained the general mechanism of action of different vaccines to provoke the humoral and immune retort against sars-cov- . j o u r n a l p r e -p r o o f based on the present pandemic situation caused by newly emerged sars-cov- , there is a need to make a vaccine on urgent bases. after examination, it is revealed that the calculated ro value of sars-cov- is . . this indicates that one contaminated person could contaminate more . persons. however, by using different precautionary measures, isolation, and quarantine activities, the ro value could be decreased [ ] . no doubt, the vaccine formulation process is not only expensive and time taking but also very complicated that sometimes gives a very low success rate. currently, more than pharmaceutical industries and various international institutes are working for the development of several types of vaccines. still, all are under phase- clinical trials, and no vaccine has been licensed yet [ ] . vaccines elicit the immune response against viruses, but currently, one of the major concerns is that no one knows what kind of immune response is required against sars-cov. one of the basic alarms is the disadvantages of several vaccines, including the unstable under physiological condition, low immunogenicity, organ failure, required high dose, and more adjuvants. however, these disadvantages need to be addressed and improved on urgent bases in the next version of vaccine development [ , ] . however, the delivery of vaccine both in immunization and modality modes are also significant issues which also needs to be revised to minimize the off-target effects. based on data, it is suggested that the aerosol route or oral administration could be a better way for vaccine delivery that might significantly induce a mucosal response. similarly, dna plasmid or vector could also be used for the delivery of the vaccine to its target site [ , , ] . previously due to negligence and improper attention of government towards this task, no proper improvement in vaccine development is seen especially against mers and sars-covs. the government and health departments do not have a proper budget to manage it. in years the sars-cov is considered to be rd epidemic war [ ] . based on accumulated data, we suggested that the development of the ideal vaccine should possess some major properties, including ) the perfect vaccine should be well-coordinated and strongly elicit the t-lymphocyte immunity that could lead to the stimulation of cytotoxic t- based on the increasing incidences of covid- , there is urgently needed to formulate a vaccine against sars-cov- . based on emerging evidence, we recommended several vaccine categories including subunits, epitopes, mrna, protein, especially s protein, recombinant adenovirus, and fusion proteins, inactivated and live attenuated virus for the formulation of a timely required vaccine against sars-cov- . more than pharmaceutical industries and institutes are started works on more than projects for the development of different vaccines against covid- . these vaccines significantly neutralized the sars-cov- and developed a strong immune and humoral response against the virus. we suggested that the receptor-binding j o u r n a l p r e -p r o o f domain (rbd), a component of s protein, might show a noteworthy role in neutralizing the antibodies because it inhibits the entrance of the virus into the host by blocking the attachment of the virus to the host membrane and also block the virus membrane fusion which could help in vaccine development. but the site-specific glycan defense creates difficulties in targeting the specific antigen. however, the vaccine development will be a challenging task, and it did not guarantee the success against sars-cov- , because no efficient and reliable vaccine is developed yet against the aids/hiv even after the years of occurrence. it is recommended that further study is needed to discover the more target sites instead of to only s protein for better inside into understanding and also suggested that cryptic epitopes based studies should be conducted on urgent bases to identify more antibody targets to generate a better long-lasting and stable immunity against sars-cov- . this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. for this type of study informed consent is not required. covid- spike-host cell receptor grp binding site prediction presumed asymptomatic carrier transmission of covid- overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and -ncov a rapid advice guideline for the diagnosis and treatment of novel coronavirus ( -ncov) infected pneumonia (standard version) hospitalization rates and characteristics of patients hospitalized with laboratoryconfirmed coronavirus disease -covid-net, states a systematic review of covid- epidemiology based on current evidence the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak the broad-spectrum antiviral recommendations for drug discovery against covid- clinical features of patients infected with novel coronavirus in the trinity of covid- : immunity, inflammation and intervention emerging coronaviruses: genome structure, replication, and pathogenesis covid- in singapore-current experience: critical j o u r n a l p r e -p r o o f journal pre-proof global issues that require attention and action a new coronavirus associated with human respiratory disease in china a review of coronavirus disease- (covid- ) a novel coronavirus from patients with pneumonia in china evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission covid- infection: origin, transmission, and characteristics of human coronaviruses single-cell rna expression profiling of ace , the putative receptor of wuhan -ncov a pilot clinical trial of recombinant human angiotensin-converting enzyme in acute respiratory distress syndrome cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease situation report patients of covid- may benefit from sustained lopinavir-combined regimen and the increase of eosinophil may predict the outcome of covid- progression green synthesis and biological evaluation of novel -fluorouracil derivatives as potent anticancer agents assessment of knowledge, attitude and practice of adverse drug reaction reporting among healthcare professionals in secondary and tertiary hospitals in the capital of pakistan engineering a stable cho cell line for the expression of a merscoronavirus vaccine antigen phylogenetic analysis and structural modeling of sars-cov- spike protein reveals an evolutionary distinct and proteolytically-sensitive activation loop a fusion peptide in the spike protein of mers mrna vaccines-a new era in vaccinology preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies adjuvant system as : helping to overcome the challenges of modern vaccines genome composition and divergence of the novel coronavirus ( -ncov) originating in china cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer characterization of the receptor-binding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus public health responses to covid- outbreaks on cruise ships-worldwide functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses host cell proteases: critical determinants of coronavirus tropism and pathogenesis covid- : coronavirus replication, pathogenesis, and therapeutic strategies structural basis of receptor recognition by sars-cov- vaccination against toxoplasma gondii: an increasing priority for collaborative research? immune responses and pathogenesis of sars-cov- during an outbreak in iran: comparison with sars and mers a pneumonia outbreak associated with a new coronavirus of probable bat origin pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic coronavirus infections and immune responses sars and mers: recent insights into emerging coronaviruses coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses the outbreak of sars-cov- pneumonia calls for viral vaccines research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases the race for coronavirus vaccines: a graphical guide the socio-economic implications of the coronavirus and covid- pandemic: a review sars-cov- vaccines: status report structure of the sars-cov- spike receptor-binding domain bound to the ace receptor structure of the sars-cov- spike receptor-binding domain bound to the ace receptor immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen covid- vaccine development pipeline gears up sars-cov- /covid- : viral genomics, epidemiology, vaccines, and therapeutic interventions the recombinant n-terminal domain of spike proteins is a potential vaccine against middle east respiratory syndrome coronavirus (mers-cov) infection development of an inactivated vaccine candidate for sars-cov- a preliminary study on serological assay for severe acute respiratory syndrome coronavirus (sars-cov- ) in admitted hospital patients vaccination strategies to combat novel corona virus sars-cov- the orf b protein of severe acute respiratory syndrome coronavirus (sars-cov) is expressed in virus-infected cells and incorporated into sars-cov particles potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices-united states, - influenza season an oral live attenuated vaccine strategy against severe acute respiratory syndrome coronavirus (sars-cov- / -ncov) evolution and containment of transmissible recombinant vector vaccines progress and prospects on vaccine development against sars-cov- , vaccines potential of live pathogen vaccines for defeating the covid- pandemic: history and mechanism mrna as a transformative technology for vaccine development to control infectious diseases optimization of lipid nanoparticles for intramuscular administration of mrna vaccines race for a coronavirus vaccine: thanks in part to institutional support moderna therapeutics, and other developers are exploring diverse approaches against sars-cov- sars-cov- mrna vaccine development enabled by prototype pathogen preparedness leveraging mrnas sequences to express sars-cov- antigens in vivo the novel coronavirus originating in wuhan, china: challenges for global health governance pre-fusion structure of a human coronavirus spike protein a dna vaccine induces sars coronavirus neutralization and protective immunity in mice characterization and preclinical evaluation of the cgmp grade dna based vaccine, av- d to enter the first-in-human clinical trials engineering dna vaccines against infectious diseases dna vaccine protection against sars-cov- in rhesus macaques immunogenicity of a dna vaccine candidate for covid- safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase , open-label, single-arm, doseescalation trial design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin- against influenza viruses the role of the proteasome in generating cytotoxic t-cell epitopes: insights obtained from improved predictions of proteasomal cleavage a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster design of an epitope-based peptide vaccine against the severe acute respiratory syndrome coronavirus- (sars-cov- ): a vaccine informatics approach application of in-silico reverse vaccinology for designing multiepitope vaccine against coronavirus multi-epitope vaccine design using an immunoinformatics approach for novel coronavirus in china (sars-cov- ), biorxiv the latest advancements in zika virus vaccine development the potency of an anti-mers coronavirus subunit vaccine depends on a unique combinatorial adjuvant formulation the sars-cov- vaccine pipeline: an overview, current tropical medicine reports clover initiates development of recombinant subunit-trimer vaccine for wuhan coronavirus a translatable subunit nanovaccine for covid- design of a peptide-based subunit vaccine angiotensin-converting enzyme protects from lethal avian influenza a h n infections angiotensin-converting enzyme inhibits lung injury induced by respiratory syncytial virus pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme in healthy human subjects potent neutralization of novel coronavirus by recombinant ace -ig protection of rhesus macaque from sars-coronavirus challenge by recombinant adenovirus vaccine comparative replication and immune activation profiles of sars-cov- and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid- potential rapid diagnostics, vaccine and therapeutics for novel coronavirus ( -ncov): a systematic review a decade after sars: strategies for controlling emerging coronaviruses new vaccine technologies to combat outbreak situations early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia safe staphylococcal platform for the development of multivalent nanoscale vesicles against viral infections a snapshot of the global race for vaccines targeting sars-cov- and the covid- pandemic rapid covid- vaccine development s national library of medicine sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor infection and rapid transmission of sars-cov- in ferrets key: cord- - iq s n authors: atzrodt, cassandra l.; maknojia, insha; mccarthy, robert d.p.; oldfield, tiara m.; po, jonathan; ta, kenny t.l.; stepp, hannah e.; clements, thomas p. title: a guide to covid‐ : a global pandemic caused by the novel coronavirus sars‐cov‐ date: - - journal: febs j doi: . /febs. sha: doc_id: cord_uid: iq s n the emergence of the sars‐cov‐ strain of the human coronavirus has thrown the world into the midst of a new pandemic. in the human body, the virus causes covid‐ , a disease characterized by shortness of breath, fever, and pneumonia, which can be fatal in vulnerable individuals. sars‐cov‐ has characteristics of past human coronaviruses, with close genomic similarities to sars‐cov, the virus that causes the disease sars. like these related coronaviruses, sars‐cov‐ is transmitted through the inhalation of droplets and interaction with contaminated surfaces. across the world, laboratories are developing candidate vaccines for the virus – with vaccine trials underway in the us and the united kingdom ‐ and considering various drugs for possible treatments and prophylaxis. here, we provide an overview of sars‐cov‐ by analyzing its virology, epidemiology, and modes of transmission while examining the current progress of testing procedures and possible treatments through drugs and vaccines. human transmission, causing the number of cases to skyrocket and outweigh both mers and sars [ ] . the r for sars is about , mers to be . , and early estimates placed this value for covid- at . to . [ , , ] . while both sars and covid- appear to have similar r , covid- has a higher viral load in the nose and throat of patients before symptoms develop whereas sars has a presence more directly tied to symptoms [ ] . this suggests that covid- can be transmitted before the development of symptoms, making it harder to isolate and control [ ] . in over % of cases of covid- , symptoms present as a mild fever, dry cough, and shortness of breath. severe cases displayed symptoms such as dyspnea (shortness of breath) in % of patients, hypoxia (oxygen depletion in body tissues) in about % of patients, and a high fever in around % of total patients [ ] [ ] [ ] [ ] . depending on the age, hospitalization rates in the united states range from . % for ages - to . % for others years or older with five percent of total cases experiencing critical conditions such as shock and multi-organ failure [ , ] . there are two main clinical symptoms that appear for critically ill patients with covid- : low levels of oxygen due to poor breathing (acute respiratory distress syndrome) and fevers [ , ] . this lack of oxygen is usually addressed through mechanical ventilation in non covid- patients [ ] . additionally, it is thought that later phases of covid- brings a lack of oxygen due to low long compliance and sedation has been suggested in support of mechanical ventilation due to the large demand of drugs such as oxycodone and hydromorphone [ ] [ ] [ ] [ ] . however, in some cases, those confirmed with covid- are asymptomatic yet can still spread the disease [ ] . for this reason and the long incubation period of up to two weeks, covid- has demanded extreme safety precautions to be implemented to minimize transmission and morbidity. this "flattens the curve" of the projected number of infections and aims to stop healthcare services to be overwhelmed with so many new cases at once. such precautions include social distancing (upholding m distance from others), which would reduce the median estimated number of infections this article is protected by copyright. all rights reserved by % by some models [ , ] . despite the implementation of these measures, many countries are still experiencing infections at an exponential rate. notably, infection and death rates are not the same between countries, age groups, or even races [ ] . despite the worldwide mortality rate of . %, death rates range from ~. % in chile and israel to % in italy (note these are estimates based on numbers reported by the who as of may th , and doesn't take account for asymptomatic or those infected who have not been tested) [ ] . countries are in various stages of their outbreaks, but there are still many factors that lead to the infection and death rate disparity such as population density, healthcare system, testing policy, and age structure of the country. testing alone has proved to be a major determinant of the success of a country's response to this outbreak. the decision on whether to engage in mass testing has contributed to the quick control of the outbreak in countries like germany and south korea, whereas the reluctance to provide mass testing of those exhibiting symptoms of infection done in the uk and united states has, arguably, prolonged the outbreak [ , ] . a nation's testing system is especially pertinent given the possibility of disease transmission through asymptomatic carriers [ ] . moreover, the severity and onset of covid- varies dramatically for different ages with worsening symptoms with age [ ] . the lowest risk is observed in those under years of age with a mortality rate ranging from to . %, whereas those to years of age have mortality rates ranging from . to . %. the highest risk is seen in those years old or older, with a mortality rate of . % to . % [ ] . underlying health conditions including diabetes, cardiovascular disease, or a suppressed immune system also increase the fatality rate [ ] . considering its high mortality rate in certain groups and high transmission rate, without proper treatment, the thousands of reported deaths from this virus might turn into millions without a coordinated and strategy-based, world-wide response. this article is protected by copyright. all rights reserved like other coronaviruses, sars-cov- is a single-stranded, positive-sense rna virus that uses spike proteins to bind to human lung epithelial cells (fig. ) [ ] . the structure of the receptor-binding region that the virus binds to, the angiotensin-converting enzyme (ace ) receptor (fig. ) , is the same target of the sars-cov outbreak of [ ] . it's these receptors that act as docking sites for the spike proteins of sars-cov- to bind to, allowing the viral and cellular membranes to fuse (fig. ). from there, the virus "hijacks" the cell; it integrates its rna into the cell's own replication machinery, facilitating propagation of the virus. the virus is then able to proliferate throughout the body, creating immune responses, and causing the person to become infected [ , ] . a commonality amongst coronaviruses is the use of their envelope-anchored homotrimeric spike glycoprotein (s protein) to mediate binding with host cell receptors ( fig. ) [ ] . the s protein is cleaved into two subunits during cell infection -the s subunit, which contains two receptor-binding domains (rbds) that allow the virus to bind to its host cell, and the s subunit, which is critical for membrane fusion [ ] . the host cell receptor for the s subunit of the s protein, ace , is a transmembrane protein located on the cells of epithelial tissue in the lungs, heart, kidneys, and intestine [ ] . its primary physiological function is to regulate the maturation of the peptide hormone angiotensin, which helps regulate vasoconstriction and blood pressure [ ] . thus, the expression of ace has antiinflammatory effects that can protect against lung injury, while its downregulation by the binding of sars-cov or sars-cov- has pro-inflammatory effects, which promote the severe acute lung injury symptomatic of infection by these viruses [ , ] . beyond its potential role in the pathology of covid- , this receptor is directly linked to the infectivity and transmissibility of the sars-cov- virus. the fact that infectivity and transmissibility of sars-cov- are higher than that of sars-cov is a result of the -to -fold higher binding affinity of the sars-cov- rbd to the ace receptor due to differences in the amino acid sequence of the sars-cov- rbd region which allow more interactions between the s protein and the host cell receptor [ , ] . the binding affinity of the virus to host cell ace receptors also dictates the pathway of intermediate host organisms that the virus this article is protected by copyright. all rights reserved could infect before being transmitted to humans and, therefore, which organisms could be used to study the virus [ , ] . after the s subunit binds to the ace receptor of the target cell, the heptad repeat (hr ) and (hr ) domains of the s subunit combine to create a six-helix bundle core that brings the viral and host cell membranes within close proximity of each other for fusion to take place [ , ] . upon membrane fusion, the rna of the coronavirus genome is released into the host cell cytoplasm via an early endosome -unlike sars-cov, which employs a late endosome and therefore must cross higher barriers of antiviral host immunity -where it is translated into a replication-translation complex that in turn translates sub-genomic rna into accessory and structural proteins (fig. ) [ - ]. these proteins form viral particle buds that are exocytosed to spread the virus to surrounding cells, thus spreading the infection throughout the host organism. between infected hosts, covid- is primarily transmitted through contact with droplets that contain viral particles [ ] . droplets are any medium in which a human can release the virus, such as coughs, sneezes, and mucous. they generally cannot travel more than meters from their origin, though simulations investigating the effects of aerodynamics on the spread of these droplets suggest that fast physical activity, such as running or cycling, increases the distance they can travel [ , ] . while it is generally thought that droplets do not linger in the air, in one study, sars-cov- was found to last in the air for hours in experimental conditions [ ] . this disease can also spread by a person touching contaminated surfaces then subsequently touching their facial area (fomite-mediated transmission). depending on the material, the surfaces were shown to be infectious from several hours on cardboard to days on plastics or stainless steel [ ] . despite these similarities to previous outbreaks and viruses, the ability of sars-cov- to stay on surfaces and affected individuals to not immediately display symptoms has made it difficult to contain and trace the virus, leading to the current global situation. an understanding of the molecular mechanisms of this virus not only explains its increased transmissibility and the subsequent symptoms of infections, but also opens the door for a wide range of testing and treatment mechanisms that target the entry mechanism or epitopes of the virus. the most prevalent form of testing right now utilizes quantitative reverse transcription polymerase chain reaction (rt-qpcr) [ ] . this form of testing is widely used to combat the outbreak in places like the us, hong kong, germany, italy, and south korea [ ] [ ] [ ] . the test requires a nasopharyngeal swab to gather genetic material that will reveal whether or not the patient has the virus. the testing mechanism first requires the isolation of rna, then the production of a singlestrand complementary dna (cdna) copy of the rna [ ] . finally, pcr is performed to amplify the cdna for analysis, which, in all, takes hours to complete [ ] . an important consideration for this testing method is navigating potential false negative and false positive results, which generally result from sample contamination [ ] . in some instances, cases that have been suspected to be positive for covid- via computed tomography (ct) images were not diagnosed as positive by rt-qpcr [ ] . china has employed a high-resolution ct to supplement their testing to ward out false negatives [ ] . the challenge with carrying out this procedure on a global scale is that the current supply for swabs is limited and hard to distribute. swab production facility locations can lead to delays and shipment issues, inhibiting the volume of testing [ ] . some countries, including the us and iceland are struggling to find suppliers that will provide authorized nasal swabs for mass testing [ ] . recently, molecular testing has been made more accessible to organizations that are outside of the traditional hospital setting through the us-based biotechnology company abbott and the german accepted article technology company bosch healthcare solutions. the abbott realtime sars-cov- assay, which is their new automation systematic machine, reduces processing time from a few days to minutes, because it reduces hands-on time and increases patient analysis flexibility. each test conducted for this machine is one cartridge and each cartridge elicits one test, which means that there will be a low throughput [ , ] . the vivalytic vri (viral respiratory tract infections) covid- test system pioneered by bosch and randox laboratories is similar to the abbott realtime sars-cov- assay in that it reduces hands-on time and can confirm a positive test within . hours with a reported % accuracy [ ]. one of the most common and fastest forms of testing is the lateral flow immunoassay, which takes a genetic sample from the patient and measures whether or not they have antibodies against a virus [ ] . this test, however, is not for determining whether the patient has the disease currently, but instead it measures whether an individual has antibodies for a disease, which would indicate that they have mounted an immune response against the pathogen in the past. companies like abbott also have antibody test and recently england has approved a serological test pioneered by the swiss biotechnology company roche diagnostics [ , ] . serological tests have a much higher success rate with these companies both boasting successes above % accuracy [ ] . another viable option is the enzyme-linked immunosorbent assay (elisa)-based testing, because it is sensitive and is amenable to high-throughput assays [ ] . however, some of these serological assays introduced early in the pandemic lacked specificity, which may result in the generation of false positives, leading to overestimates of rates of infection [ ] . more specific assays have now emerged that are proving very useful in providing a fuller picture of the rates of asymptomatic or mild sars-cov infection, through detection of anti-viral antibodies that persist for months and even years after the virus has been cleared [ ] . altogether, serological tests can serve as an indicator for how widespread the virus is, while rt-qpcr tests can show who currently has the disease. this article is protected by copyright. all rights reserved clustered regularly interspaced short palindromic repeats (crispr) technology has promise as a tool for disease detection [ ] . two enzymes that work in conjunction with crispr, cas and cas , can be activated when specific sequences of rna are detected. once the enzymes are activated, they exhibit local rnase or dnase behavior, respectively, breaking down strands of rna or singlestranded dna nearby; in a test scenario, this is usually measured with a fluorescent signal or activity on a lateral flow strip, which acts similarly to a pregnancy test. if the crispr-cas complex is combined with a reporter molecule, the enzyme breaks down the nearby reporter molecule, triggering a response. when cas is used, a process called sherlock can detect the presence of an rna virus such as sars-cov- [ ]. cas is activated when it is guided to the specified sequence and starts to breakdown nearby rna strands. when a rna sequence reporter molecule is combined with the crispr-cas sequence, cas will break it down, signaling the presence of a specific rna strand, such as a virus, through fluorescence or a lateral flow strip. cas can also be used, in a process very similar to sherlock, called detectr, that shows the presence of a virus by activating cas to break down a dna-based reporter molecule [ ] . detectr technology is faster than sherlock, but both are being researched and tried as future testing methods. recently, the company sherlock biosciences has received fda emergency-use approval (eua) for its sherlock based covid- test, which the first eua with crispr technology [ ] . currently, there is no available cure or vaccine for covid- and the fastest timescale for development of a vaccine is estimated to be between - months [ ] . in the absence of these long-term and sustainable treatments, doctors and healthcare professionals are working on managing critical respiratory symptoms (dyspnea and hypoxia) from covid- through the use of mechanical ventilation [ ] . in a case study on hospitalized patients in new york, about % of the this article is protected by copyright. all rights reserved patients required ventilation and the mortality among those receiving ventilation was % [ ] . the repurposing of drugs has also shown promising results for a possible treatment of covid- symptoms: the who has outlined four drugs as potential therapeutic candidates for covid- in their solidarity project: remdesivir, lopinavir/ritonavir, interferon beta- a, and hydroxychloroquine/chloroquine [ ] . of these, remdesivir currently shows the most promise [ ] . however, further studies have shown the promise of other drugs -eidd- and favipiravir [ , ] . analyzing the pathways of these drugs not only provides a greater depth of understanding of the virus, but also reveals possible targets for the creation of an antiviral drug. a drug originally created to be used against the ebola virus, remdesivir has recently been discovered to be effective in preventing the proliferation of sars-cov and mers-cov [ ] . the structure of remdesivir prevents the propagation of viruses by blocking a crucial piece of rna duplication machinery -the rna-dependent rna polymerase (rdrp) [ ] . in a study of remdesivir, the drug was administered to mouse cell lines infected with sars-cov, mers-cov, and sars-cov- , and was able to inhibit the spread of the sars-cov- virus [ ] . for this reason, there is excitement about the potential of this drug. currently, several phase ii/iii trials are underway with one highly powered (large sample size) trial sponsored by the national institute of allergy and infectious diseases (niaid) in the us, reporting patients had a % faster time to recover when taken compared to a placebo [ , ] . this has been one of the few trials highly powered with a use of a placebo to generate positive results leading to its emergency authorization use by the fda [ ] . as a result, remdesivir has become one of the most promising drugs to fight covid- , being tested around the world by countries such as china, germany, italy, south korea, among others [ ] . further studies and results will be needed to determine its efficacy. favipriavir (also known as avigan) similarly selectively inhibits rdrp through preventing it from binding to its energy source (atp), thus making it non-functional [ ] . its efficacy is promising, this article is protected by copyright. all rights reserved with china's science and technology ministry calling it an "effective treatment" with "a high degree of safety", as many symptoms such as fever and cough subsided quicker when taking the drug [ ] . even given these positive results, japan is one of few countries heavily evaluating the drug with fujita health university performing phase ii trials on the drug [ ] . hydroxychloroquine and chloroquine are two safe, inexpensive drugs known mainly for their fdaapproved treatment against malaria and autoimmune diseases, but also been found to have antiviral activity [ , ] . viruses usually enter the body and infect cells through endocytosis, which packages the virus and brings it into the cell via an endosome. however, hydroxychloroquine/chloroquine increases the endosome's ph, making it too basic for other viruses to survive and replicate [ ] . given its hydrophobic structure, the drug can circulate throughout the blood and the effects can spread throughout the body including the lungs, making it a strong candidate for treatment against covid- [ ] . tests of hydroxychloroquine and chloroquine against covid- in china and france have proven promising but only studied small sample sizes [ ] . nevertheless, due to the minimal known side effects of the drug, the fda has authorized for emergency use by healthcare professionals [ ] . there are future hopes that this is a drug that everyone could take to prevent covid- (prophylaxis approach) and federal regulators in the us have removed many regulations that have allowed for testing to hit "warp speed" [ ] . however, there are other studies suggesting no/limited effect of chloroquine, or even severe side effects [ ] [ ] [ ] [ ] . some hospitals have also stopped using this drug for these reasons. lopinavir and ritonavir, referred to as kaletra when combined, are mainly used to combat human immunodeficiency virus (hiv) infection by acting as a protease inhibitor that would prevent the cutting of certain proteins needed for hiv replication [ ] . after initial reports of success from china and south korea, scientists were excited by the potential of lopinavir/ritonavir as a treatment against covid- [ ] . yet, when tested on hospitalized infected adults in isolation from other this article is protected by copyright. all rights reserved drugs, lopinavir/ritonavir showed no significant benefit/improvement, indicating that the protease inhibition was not the same for sars-cov- as for hiv, ceasing future testing of the drug [ , ] . interferons are a natural component of the immune system that activate antiviral machinery in order to inhibit replication and increase immune response [ ] . this process can be sped up via interferon injection [ ] . given its ability to stimulate active immunity, previous studies have tested alpha and beta types of interferons against mers and sars [ ] . it was identified that the type interferon of a beta subtype was most effective against coronaviruses as it upregulates anti-inflammatory cells in the lungs [ ] . early testing of interferons in human epithelial cell lines showed a reduction of viral concentration by injection and a reduction in infection rates by administration of interferons by spray, in support of interferon-based prophylaxis [ , ] . currently, interferon beta- a is a specific type of interferon available in the market to help mitigate the symptoms of multiple sclerosis, but further research is needed to see its effects in humans [ ] . eidd- can both serve as a preventable and treatable medicine through oral intake. in contrast to remdesivir, which may treat covid- by inhibiting rdrp [ ] , eidd- induces lethal mutagenesis by accumulating deleterious transition mutations in the viral rna while having many off-target effects [ ] . the results of eidd- have shown the greatest promise out of all these drugs, having been shown to reduce the concentration of sars-cov- as well as other coronaviruses (mers and sars) when taken prophylactically [ ] . while these results have only been observed in mice, its ability to be easily taken orally and as a potential preventative treatment makes it a strong candidate for continued research. as of april th , , the fda has given the company responsible for eidd- approval to perform human trials [ ] . overall, the antiviral activities of these drugs, with the exception of lopinavir/ritonavir, have shown some level of efficacy against covid- using different pathways. eidd- , remdesivir, and favipriavir all mitigate sars-cov- by stopping the rna-dependent-rna-polymerase from replicating the virus further. by contrast, hydroxychloroquine/chloroquine targets endosomes, while this article is protected by copyright. all rights reserved interferon beta- a takes advantage of the body's innate immunity increasing inflammatory responses. trying to evaluate the level of success these drugs can be against covid- is difficult due to their different points of research; however, remdesivir is the one of the few drug to have a high-powered experiment that favors positive results, which has led it to become one of the more promising and frequently tested drug around the world [ ] . while there is no definite answer yet, testing of these drugs has garnered greater understanding of the viral mechanism that could further aid other drug repurposing or synthesis for a possible cure. crispr along with possible drugs, new technologies have been utilized preemptively to counter the symptoms of sars-cov- . crispr-cas has been utilized to target essential parts of the sars-cov- virus, through an approach called pacman (prophylactic antiviral crispr in human cells) [ ] . similar to sherlock-based virus detection, pacman utilizes cas , which has rnase activity that can be used for both the detection (see above) and the destruction of sars-cov- ( fig. ) [ ] . this aspect of cas can be utilized to destroy the virus with pinpoint accuracy, while also knocking out the rdrps. the main roadblock to using this technology is delivery. some of the problems associated with the delivery of crispr-cas have been researched and possible liposomal delivery systems have been established [ ] . while these treatments still require further testing, there is promising research in the use of crispr as a means of targeting the virus and stopping symptoms, as well as utilizing pacman to prevent future pandemics. while antibodies indicate that a person has developed an immune response to the virus, there is no evidence that shows how long the person will be protected, if they are protected at all, from reinfection of covid- . however, in a study of patients that recovered from covid- , it was found using elisa that antibody concentration was associated with neutralizing activity [ ] . this has led healthcare professionals to consider blood transfusion of convalescent plasma as a possible treatment. in a case study of five critically ill patients treated with plasma, four out of the five patients this article is protected by copyright. all rights reserved exhibited normalized body temperatures and decreased viral load, suggesting that the antibodies have antiviral activity [ ] . using elisa, scientists from utrecht university in the netherlands were able to identify a reactive human monoclonal antibody ( d ) that blocks sars-cov- infection [ ] . similar to the antibodies found in the blood plasma transfusions, d antibody is able to reduce viral activity, while also having the capability to be synthesized in a lab. this leads to significant advantages compared to plasma as nothing has to be stored and there isn't a limit to availability. with the recent evidence of d 's efficacy in cells, pharmaceutical companies are in a rush to produce a monoclonal antibody treatment as the world waits for an answer. the vaccine development processes is known to take years for full approval for mass production. however, many leaders in this area such as dr. anthony fauci, the director of the us national institute of allergy and infectious diseases, has recently suggested that this process could take less than a year given the current the need to combat covid- . meanwhile, researchers at oxford university's edward jenner institute for vaccine research have started testing a vaccine in humans and could have results of its efficacy by june [ , ] . for most vaccines, this rapid progression would be seen as unrealistic, but the pressure needed to put this pandemic to halt has led to many regulatory agencies to speed up their approval process. typically, vaccine trials are three-phase processes that are run over the course of several years. the first phase is meant to assess the safety of a vaccine, and so it is injected into a small group of healthy volunteers. next, in the second phase, the efficacy of the vaccine is tested in several hundred volunteers, including at-risk groups. finally, the third phase moves to testing in thousands of patients if the results are promising and only then can a vaccine be mass-produced. however, governments worldwide have encouraged companies to launch production of vaccine candidates despite the fact this article is protected by copyright. all rights reserved that none of them have completed the clinical trial process [ ] . there are several different types of vaccines that scientists are researching to combat covid- . this type of vaccine aims to provoke an immune response in vaccinated people by introducing a weakened version of the virus into their bodies. these live, attenuated vaccines are the closest thing to a natural infection, and so they are very effective at creating an immune response [ ] . the vaccines for measles, mumps, and chickenpox use this approach. however, children and immunocompromised individuals can have difficulties handling the live attenuated vaccines and evoking an immune response [ ] . another method of vaccination is to use a dead or inactivated virus, like in the polio vaccine. one upside to these vaccines is that there is no concern about the virus regaining pathogenicity and causing disease. however, booster shots are needed for continued protection because these vaccines trigger smaller immune responses [ ] . rather than include the entire virus, subunit vaccines work by including only parts of the virus' structure, or subunits [ ] . for example, a subunit vaccine for a coronavirus strain may only include the s protein or parts of the s protein (fig. ) . similar to the inactivated vaccine, side effects are less common but booster shots are needed. additionally, subunit vaccines may not be the most costeffective method of acquiring immunity against the coronavirus since their production is very costly. the previously mentioned approaches to vaccine development are labor-intensive, and require scientists to work long hours cultivating and modifying the specific virus in the lab. due to the urgency for a sars-cov- vaccine, researchers are trying novel approaches to vaccine development, with one such being a nucleic acid vaccine. this article is protected by copyright. all rights reserved one of the first covid- vaccines to be approved for clinical trials was a rna vaccine first tested in seattle [ ] . rather injecting a weakened or other altered form of the virus into patients, this vaccine works by injecting mrna directly into the patient. the mrna then codes for viral proteins that would lead to an immune response from the body and thus giving individual protection from the disease should they come into contact with the virus again. other types of nucleic acid based vaccines code for material that interfere protein translation that is essential to the virus' propagation [ ] . however, despite the fact that this technology has been around for about years, nucleic acid vaccines have not yet earned approval since an effective delivery system is needed in order for these vaccines to match the efficacy of a live vaccine [ ] . in clinical trials, research is also being conducted for viral vector vaccines for covid- [ ] . in this particular vaccine, scientists engineer non-pathogenic viruses -or a viral vector -with the particular sequence that codes for the virus' antigen. this approach is being used by researchers at oxford university, who engineered a non-replicating chimpanzee adenovirus with the spike sequence of the sars-cov- spike protein [ ] . this vaccine is particularly promising since it can elicit a strong immune response [ ] . however, patients may have pre-existing immunity to the vector, rendering the vaccine useless. even with all of these options available, developing a vaccine that can be distributed in mass to combat covid- is not without its challenges. vaccines are developed from scratch for each target and vaccines against similar coronaviruses cannot be simply repurposed for new uses. it is important to note that while vaccines were developed for both the sars-cov and mers-cov outbreaks, they were not utilized in the public sphere because they did not complete clinical trials. both of the sars-cov and mers-cov outbreaks were largely controlled due to containment, patient isolation, and social distancing. nonetheless, as the sars-cov- continues to spread farther than its predecessors, the need for a vaccine only becomes more urgent. this article is protected by copyright. all rights reserved the sars-cov- pandemic has sparked advancement in coronavirus research. data from previous coronavirus outbreaks has been used to enhance the study of treatment and prevention measures for covid- . studying the structure of the virus has allowed scientists to design testing processes rapidly, and new systems of testing are currently being designed to increase the speed and specificity of the process. the testing process is rapidly improving, yielding greater knowledge of who is or has been infected. this data could greatly advance regional decision-making concerning when to lift restrictions and deciding when individuals can resume their usual day-to-day activities. treatments for covid- are also being rapidly developed to treat the symptoms of the disease, since no vaccine has currently been approved for mass distribution. out of all the drugs tested, remdesivir has shown the most promising results. this drug has shown potential in low-powered studies, but hopefully further research will shine light on its efficacy in the short term. vaccine production is underway, and many types of vaccines are currently being researched and tested so that a vaccine can be available as soon as possible. there are many options for possible treatments of covid- , and, ultimately, it boils down to what is most effective, efficient, and available in the near future. the combination of increased testing and efficient symptom treatment is an encouraging response to the pandemic. currently, the spread of this virus shows promising decline, but this data will become more accurate as more people are tested and more time has passed. it is essential for governments to consider these factors before initiating the return to normal life. this article is protected by copyright. all rights reserved cla, im, rdpm, tmo, jp, ktlt, and hes reviewed the literature, wrote the text, and revised the manuscript. cla, im, rdpm, tmo, jp, ktlt, and hes contributed equally to this manuscript. tpc carefully revised and co-wrote the manuscript. this article is protected by copyright. all rights reserved the merging of the viral and cellular membranes, causing the viral genomic rna to be inserted into the host cell and thus allowing the virus to replicate. the binding of sars-cov- to the ace receptor causes a down-regulation of this receptor, disrupting its normal function in maintaining immune homeostasis and leading to pro-inflammatory effects that can cause lung injury. figure . the top of the drawing represents the dna encoding for the cas protein and the crispr array, which contains the targets for cas cleavage and subsequent degradation. this array is transcribed into pre-crrna. the cas protein turns this transcript into mature crrnas, forming a crrna-cas complex that in turns searches along existing rna transcripts for matching sequences known as protospacers. once this complementary protospacer is found, cas undergoes a conformational change to enhance binding and activates the rna cleavage activity of cas , which can then be used to degrade foreign viral rna entering the cell as shown on the right side of the image. adapted from [ ] . leung gm & feng z ( ) early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia coronaviridae study group of the international committee on taxonomy of viruses ( ) the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- accepted article this article is protected by copyright. all rights reserved a pneumonia outbreak associated with a new coronavirus of probable bat origin covid- in singapore-current experience: critical global issues that require attention and action covid- and italy: what next? world health organization coronavirus simulation suggests that rapid activation of social distancing can arrest epidemic development due to a novel strain of influenza the trinity of covid- : immunity, inflammation and intervention history and recent advances in coronavirus discovery epidemiology, genetic recombination, and pathogenesis of coronaviruses cdc ( ) coronavirus | human coronavirus types | cdc the molecular biology of coronaviruses recently discovered human coronaviruses accepted article this article is protected by copyright. all rights reserved epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method complexity of the basic reproduction number (r ) further notes on the basic reproduction number the final size of an epidemic and its relation to the basic reproduction number the estimation of the basic reproduction number for infectious diseases epidemiologic clues to sars origin in china a review of coronavirus disease- (covid- ) the sars (severe acute respiratory syndrome) pandemic in hong kong: effects on the subjective wellbeing of elderly and younger people coronavirus as a possible cause of severe acute respiratory syndrome public health interventions and sars spread the reproductive number of covid- is higher compared to sars coronavirus world health organization covid- situation reports pattern of early human-to-human transmission of wuhan sars-cov- viral load in upper respiratory specimens of infected patients hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease -covid-net, states stavropoulou c in patients of covid- , what are the symptoms and clinical features of mild and moderate cases? auwaerter coronavirus covid- (sars-cov- ) | johns hopkins abx guide clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study covid- in critically ill patients in the seattle region -case series testing recommendation for covid- (sars-cov- ) in patients planned for surgery -continuing the service and 'suppressing' the pandemic asymptomatic transmission, the achilles' heel of current strategies to control covid- case-fatality rate and characteristics of patients dying in relation to covid- in italy cdc people who are at higher risk for severe illness | cdc rna viruses: rna roles in pathogenesis, coreplication and viral load cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor coronaviruses: an overview of their replication and pathogenesis mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation severe acute respiratory syndrome coronavirus (sars-cov- ): an overview of viral structure and host response structure, function, and antigenicity of the sars-cov- spike glycoprotein structural basis for the recognition of sars-cov- by full-length human ace angiotensin-converting enzyme in lung diseases cdc ( ) coronavirus disease (covid- ) -transmission. cent dis control prev & marchal t towards aerodynamically equivalent covid . m social distancing for walking and running aerosol and surface stability of sars-cov- as compared with sars-cov- reverse transcription-polymerase chain reaction molecular testing of cytology specimens: pre-analytic and analytic factors risk of nosocomial transmission of coronavirus disease : an experience in a general ward setting in hong kong ackermann n & sing a ( ) rapid establishment of laboratory diagnostics for the novel coronavirus sars-cov- in bavaria a "one-health" approach for diagnosis and molecular characterization of sars-cov- in italy quantitative rt-pcr: pitfalls and potential rapid pcr of str markers: applications to human identification real-time rt-pcr in covid- detection: issues affecting the results small solitary ground-glass nodule on ct as an initial manifestation of coronavirus disease (covid- ) pneumonia accepted article this article is protected by copyright. all rights reserved from iceland -covid- screening swab shortage continues comparison of two commercial molecular tests and a laboratory-developed modification of the cdc -ncov rt-pcr assay for the qualitative detection of sars-cov- from upper respiratory tract specimens | medrxiv assay (eua) | abbott molecular vri multiplex test vivalytic lateral flow based immunobiosensors for detection of food contaminants abbott launches covid- antibody test roche's covid- antibody test receives fda emergency use authorization and is available in markets accepting the ce mark diagnostics poon ll & peiris m ( ) serological assays for severe acute respiratory syndrome coronavirus benucci m & manfredi m ( ) serological assays for sars-cov- infectious disease: benefits, limitations and perspectives a serological assay to detect sars-cov- seroconversion in humans crispr-cas a target binding unleashes indiscriminate single-stranded dnase activity sherlock biosciences receives fda emergency use authorization for crispr sars-cov- rapid diagnostic • sherlock biosciences cdc ( ) coronavirus disease (covid- ) situation summary. cent dis control prev qiu m & zanos tp ( ) presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the race to find covid- treatments accelerates remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro an orally bioavailable broad-spectrum antiviral inhibits sars-cov- in human airway epithelial cell cultures and multiple coronaviruses in mice favipiravir (t- ), a broad spectrum inhibitor of viral rna polymerase accepted article rna-dependent rna polymerase by remdesivir gilead sciences gilead announces results from phase trial of investigational antiviral remdesivir in patients with severe covid- some fda approved drugs exhibit binding affinity as high as kcal/mol against covid- main protease (mpro): a molecular docking study indiarxiv major ongoing clinical trials for covid- treatment and studies currently being conducted or scheduled in japan. glob health med advpub cdc ( ) coronavirus disease (covid- ). cent dis control prev new insights into the antiviral effects of chloroquine breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies fda emergency use authorization | fda pre-exposure prophylaxis with hydroxychloroquine for covid- disease review of current evidence of hydroxychloroquine in pharmacotherapy of covid- . medrxiv hydroxychloroquine versus covid- : a rapid systematic review and meta-analysis no evidence of clinical efficacy of hydroxychloroquine in patients hospitalized for covid- infection with oxygen requirement: results of a study using routinely collected data to emulate a outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- lopinavir/ritonavir: a review of its use in the management of hiv infection a trial of lopinavir-ritonavir in adults hospitalized with severe covid- interferon pathway in sle: one key to unlocking the mystery of the disease type interferons as a potential treatment against covid- sars-cov- is sensitive to type i interferon pretreatment halford b an emerging antiviral takes aim at covid- development of crispr as a prophylactic strategy to combat novel coronavirus and influenza molecular mechanisms of rna targeting by cas -containing type vi crispr-cas systems accepted article this article is protected by copyright. all rights reserved ng ac temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study a human monoclonal antibody blocking sars-cov- infection kuznia r the timetable for a coronavirus vaccine is months. experts say that's risky. cnn kamisar b oxford scientist says its vaccine is making headway, could show efficacy by wsj news exclusive | drugmaker moderna delivers first experimental coronavirus vaccine for human testing long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine live bacterial vaccines -a review and identification of potential hazards. microb cell factories immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine accepted article this article is protected by copyright. all rights reserved induction of th type response by dna vaccinations with n, m, and e genes against sars-cov in mice recent advances in mrna vaccine delivery sars-cov- vaccines: status report about the covid- vaccine trials univ oxf covid- oxf vaccine tral vaccine delivery methods using viral vectors cdc ( ) international locations with confirmed covid- cases. cent dis control prev rna-targeting crispr-cas systems and their applications before this pandemic occurred, all of these authors were undergraduate students in a discovery-based research lab focused on projects centered on performing novel crispr-cas genetic knockouts in zebrafish under the discretion of senior lecturer dr. thomas clements at vanderbilt university.when the spring semester went online, the class decided to focus their efforts on learning everything they could about covid- . we would like to thank dr. james pask, the introductory biology lab coordinator, for creating and fostering an environment that encourages us to explore these endeavors. additionally, we are grateful for the biological sciences department at vanderbilt university for funding our experiments. lastly, we would like to thank the ta family for helping revise the citations. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord- -p o zkhf authors: tizard, ian r. title: feline vaccines date: - - journal: vaccines for veterinarians doi: . /b - - - - . -x sha: doc_id: cord_uid: p o zkhf many of the decisions regarding the vaccination of cats relate to the animal’s lifestyle. vaccination requirements for the solitary indoor cat are very different than those for feral or free-roaming cats. core vaccines include those directed against feline herpesvirus, feline parvovirus, and feline caliciviruses. other important vaccines include the mandated rabies vaccination and also vaccination against feline leukemia. one significant issue with respect to feline vaccination is the development of injection site sarcomas. although the prevalence of these is low and should not inhibit the use of vaccines, they are impossible to predict and very difficult to treat. as with other companion animal species, informed vaccination decisions for cats depend on multiple factors. in general, healthy cats should be vaccinated but mild concurrent illness may not be disqualifying. issues such as immunosuppression by feline retroviruses, other immunodeficiencies, nutritional status, and chronic stress must be considered. the age of the cat is critical considering the persistent effects that maternal antibodies have on early vaccination, whereas old age presents other significant issues. the other key issue is, of course, lifestyle. the opportunities that a cat has to encounter other infected cats will vary greatly ranging from the housebound pet, to cats in shelters, to feral cat populations. it is self-evident that some vaccines are of greater importance than others. the choice of a vaccine will depend not only on the factors stated earlier, but also on the prevalence and severity of a disease. thus the core vaccines for cats in north america are considered to be those directed against feline panleukopenia caused by a parvovirus, feline rhinopneumonitis caused by feline herpesvirus- , and feline calicivirus infection. rabies presents a special case because its use is governed by regulation and is mandatory in certain jurisdictions. vaccines that may be considered as noncore include those against bacterial diseases caused by chlamydia felis and bordetella bronchiseptica, in addition to the infections caused by feline leukemia virus (felv), feline immunodeficiency virus, and feline coronavirus (box . ). feline respiratory disease is caused by multiple agents. the most important ones are feline herpesvirus (fhv- ), feline calicivirus (fcv), chlamydia felis, and mycoplasma felis, or combinations of these. as in other species it is likely that the viral infection predisposes to secondary bacterial infections such as those by bordetella bronchiseptica. licensed vaccines containing multiple antigens are most widely employed. they usually contain calicivirus, feline herpesvirus, and parvovirus. some may also incorporate chlamydia, leukemia, or rabies vaccines. b. bronchiseptica is primarily a dog pathogen (chapter ), and vaccination should only be considered if there is contact between a cat and dogs with a recent or current history of respiratory disease. routine use of this vaccine is not recommended. this pathogen is not consistently isolated from cats with respiratory disease. it should only be vaccinated against if it has been identified as a problem by a diagnostic laboratory. it is usually prevented by the use of combined vaccines containing other pathogens such as feline viral rhinotracheitis (fvr) or feline parvovirus (fpv). this virus causes an upper respiratory tract infection. symptoms include nasal discharge, rhinosinusitis, tracheitis, conjunctivitis, keratitis, oral ulceration, fever, malaise, and loss of pregnancy. cats of all ages are susceptible, and it is especially common in multicat households and shelters. as with other herpesviruses, infected cats become lifelong latent carriers. at times of stress, the virus may become reactivated in these latent carriers. in such cases, it may cause clinical disease or be transmitted to susceptible, in-contact animals. for example, the stress of parturition may cause queens to shed the virus. many inactivated adjuvanted vaccines are available against fhv, usually in combination with multiple other respiratory pathogens. these vaccines do not induce strong immunity, and as a result assessment of duration of protection is difficult. modified live vaccines are available for either intranasal or intraocular administration. intranasal vaccines may be combined with a calicivirus vaccine. owners should be warned that cats vaccinated by the intranasal route may sneeze frequently for four to seven days after vaccination. although antibodies may be detected three years after vaccination, these antibodies do not correlate well with protection. as with all herpesviruses, cell-mediated immunity is critical. cats at low risk may be vaccinated every three years, but cats in catteries are at high risk and may be vaccinated more frequently at the veterinarian's discretion. if a cat is to be moved to a boarding facility it should be revaccinated one to two weeks before the move, especially if its vaccines are not current. feline calicivirus is ubiquitous in cats worldwide. it causes infections that range from subclinical to oral and upper respiratory tract disease and has been considered to have high morbidity and minimal mortality. affected cats develop oral ulcers, sneezing and a nasal discharge. recently however, some highly virulent calicivirus biotypes have emerged. virulent systemic feline caliciviruses strain fcv-ari causes fever, jaundice, hemorrhage, skin necrosis, vomiting, edema, and death. guidelines for feline vaccination have been published by: the american association of feline practitioners (aafp) at: https://www.catvets.com/guidelines/ practice-guidelines/feline-vaccination-guidelines. the world small animal veterinary association (wsava) at: https://www.wsava.org/wsava/ media/pdf_old/wsava-vaccination-guidelines- -full-version.pdf. the advisory board for cat diseases (abcd) at: http://www.abcdcatsvets.org/vaccines-andvaccination-an-introduction/. -feline vaccines calicivirus vaccines are usually administered in combination with vaccines against other respiratory pathogens. multiple inactivated vaccines are available. because of concerns regarding the antigenic diversity of calicivirus strains some manufacturers produce vaccines containing more than one strain. most modified live vaccines currently contain the fcv-f strain. some are designed for intranasal use whereas others are injectable. because of the genetic diversity of caliciviruses however, f vaccines may differ in their ability to protect against heterologous strains. although fcv-f is still broadly effective against current circulating strains it may not protect well against newly emerged systemic virulent strains such as fcv-ari. it may be necessary to add additional avirulent strains to the vaccine to maintain broad coverage. virus neutralizing antibodies develop in about a week after vaccination and correlate well with protection. however, vaccination does not prevent infection and vaccinated cats can become persistently infected. both cell-mediated immunity and mucosal immunoglobin (ig)a also contribute to resistance. duration of immunity is at least four years for inactivated products and about seven years for modified live virus (mlv) vaccines. intranasal vaccines may induce respiratory signs such as sneezing for several days after vaccination in some individuals. this may result in shedding of the vaccine virus. however, the intranasal vaccines require only a single dose and trigger the rapid onset of immunity. they also are better able to overcome inhibition by maternal antibodies in kittens. fpv causes panleukopenia. infected cats develop a fever followed by vomiting and possibly diarrhea. they become dehydrated, followed by hypothermia, septic shock, intravascular coagulation, and death. in addition to fpv, some canine parvovirus variants (cpv- a, - b, and - c) may cause disease in cats. fpv vaccines may afford some protection in these cases. inactivated adjuvanted fpv vaccines are invariably given with calicivirus and rhinopneumonitis vaccines. the safety of the killed fpv vaccines means that these are the preferred vaccines used in wild felids, pregnant queens, and cats immunosuppressed by retroviral infections. both parenteral and intranasal modified live vaccines are available in combination with the other core vaccines, fcv and fhv- . as with other such vaccines the mlv vaccines induce protection rapidly, probably because of interferon release. they are also more effective in overcoming the blocking effect of maternal antibodies. mlv vaccines should not be administered to cats infected with the immunosuppressive retroviruses fiv and felv. they should not be used in pregnant queens nor should they be given to neonatal kittens (, - weeks of age) to avoid possible encephalitis and cerebellar damage. the duration of protection after natural infection is long and probably lasts at least seven years after mlv vaccination. after the preliminary series cats should be revaccinated every three years. the presence of antibodies is correlated with protection. otherwise vaccine administration schedules are the same as for herpesvirus vaccines. (they are usually given as combination vaccines.) feline panleukopenia virus may persist in the environment for at least a year, a fact that makes fpv vaccination absolutely essential. rabies in cats is prevented by similar or identical vaccines to those used in puppies. both inactivated and canarypox vectored recombinant vaccines are available. because of concerns regarding the development of injection site sarcomas, many veterinarians prefer nonadjuvanted vaccines. vaccination intervals and use may be governed by local regulations. before vaccinating against feline leukemia, kittens should be tested to ensure that they are not already infected. there is no benefit to vaccinating an infected cat. although this is a noncore vaccine, it is often unknown where a young kitten may eventually be housed. it is possible that they may end-up in a high-risk environment. it is therefore recommended that kittens receive at least the initial vaccination series and their first annual booster. the protection provided by priming outweighs the risk of adverse effects. after the first year, revaccination should depend on the cat's lifestyle and risk factors. uninfected cats in a household with infected cats should also be vaccinated. most vaccines are directed against strain felv-a. there is no evidence that incorporating other strains, (felv-b and -c) in a vaccine provides any benefit. a vectored vaccine is available that contains recombinant felv glycoprotein (gp ) plus part of the transmembrane protein expressed in an escherichia coli vector. this was the first genetically engineered vaccine used in companion animals. also available is a canarypox-vectored vaccine expressing the genes for the envelope glycoprotein, gp , and the nucleocapsid protein, p . because of the great diversity in felv vaccines, the question of relative effectiveness is often asked. unfortunately, few comparative studies have been performed and there have been great variations in the strains and protocols used to measure protection. studies on available vaccines suggest that most have preventable fractions (pfs) ranging from % to %. whole cell vaccines appear to provide the most consistent protection. like all other vaccines, none of these are % protective nor do they protect against transient viremia or induce sterile immunity. however, low levels of viremia are not considered clinically important. duration of immunity following feline leukemia vaccination appears to be about three years, therefore cats in high-risk situations should be boosted annually or every two years. although these are safe vaccines, with a very low prevalence (, %) of adverse events, mainly injection site swelling or transient lethargy, felv vaccines are also associated with the development of injection site sarcomas. feline infectious peritonitis (fip) is caused by feline enteric coronavirus (fcov). fip is a major problem in catteries but is less of an issue among pet cats. the virus causes a fatal granulomatous peritonitis of wild and domestic cats. there are two distinct genotypes of feline enteric coronavirus, avirulent and virulent. the avirulent genotype prefers to replicate within intestinal epithelial cells, whereas the virulent genotype prefers to replicate within macrophages. macrophages also spread the virus throughout the body. fcov tends to infect relatively young cats between six months and three years of age. the course of the infection depends on the nature of the immune response to the virus-a phenomenon also seen in several bacterial diseases. immunity to fcov is entirely cell mediated, and a helper t cell (th ) response is protective. a cat that mounts a good th response will become immune, regardless of the amount of antibodies it makes. some cats, however, mount a th response to the viral spike proteins. in these animals, the antibodies enhance virus uptake by macrophages. virus-laden macrophages accumulate around the blood vessels of the omentum and serosa. antibodies also generate immune complexes that are deposited in the serosa, causing pleuritis or peritonitis, and in glomeruli, leading to glomerulonephritis. cats with preexisting high levels of antibodies against fcov develop effusive fip rapidly on challenge. administering antiserum to fcov before challenge may also enhance the peritonitis. a modified live intranasal vaccine is available against fcov (felocell fip, zoetis). the vaccine contains a temperature-sensitive mutant of the fcov strain df -fipv that replicates in the upper respiratory tract and so induces a local iga response in the mucosa. ideally this acts in the oropharynx at the site where fcov enters the body. this local mucosal response should prevent fcov invasion without inducing high levels of serum antibodies. this vaccine, however, will only be effective if administered before coronavirus exposure. in highly endemic situations where kittens are infected at a young age, vaccination at weeks of age may be too late to prevent infection. the american association of feline practitioners does not recommend this vaccine. a vaccine containing an inactivated fiv-infected-cell associated virus containing subtypes a and d has been marketed in the united states. it gave % protection against fiv subtype a. however, this protection did not correlate well with antiviral antibody titers and was not effective against other fiv subtypes. superficial fungal infections-ringworm, caused by fungi belonging to the genera trichophyton and microsporum-occur in both companion and food animals. under some situations these may be controlled by vaccination. fungi have a cellulose cell wall that is difficult for the body's immune defenses to attack or penetrate. antibodies are ineffective against these organisms, but cell-mediated responses may be effective. protection often correlates with a positive delayed hypersensitivity skin test. once established, this immunity may be long lasting. inactivated fungal vaccines are available in some countries but it is difficult to assess their efficacy. in norway, russia, sweden, and other countries a systematic campaign against bovine ringworm that includes vaccination has been very successful. there are four commercially available vaccines against bovine ringworm in europe. three are monovalent attenuated live vaccines and one is an inactivated -way vaccine. the live vaccines appear to be more efficacious than the dead one. two doses are given to days apart and revaccination may not be necessary. these vaccines may be used prophylactically or therapeutically. dermatophytosis is of major concern in the fox, chinchilla, and rabbit fur industries because it drastically reduces the quality of their pelts. a modified live vaccine is available in eastern europe and russia for use in these species. it is of interest to note that ringworm downgrades the quality of cattle hides as well, and ringworm scars may also reduce their price. both attenuated live and inactivated vaccines against ringworm in cats and dogs are available in some european countries. unfortunately, is difficult to make an informed opinion as to their efficacy. published articles on vaccination against both canine and feline dermatophytosis have shown mixed results. three studies reported protection in dogs and foxes against microsporum canis. a killed m. canis-cell wall vaccine induced both humoral and cell-mediated immunity in experimental cats, but did not protect cats against challenge. a vaccine containing killed m. canis fractions in adjuvant was licensed in the united states for the treatment of cats rather than prevention. however, it did not prevent the establishment of a challenge infection and did not shorten the duration of disease in vaccinated cats compared with unvaccinated controls. the product is no longer available in the united states. most feline infectious diseases are of greatest significance in kittens younger than six months of age. therefore these are the primary targets of vaccination. unfortunately, maternal antibodies interfere with early vaccination by neutralizing the injected antigen and inhibiting antibody synthesis in small kittens. as noted above, current high-quality core vaccines induce high levels of maternal antibodies. as a result, maternal antibodies persist for longer and many kittens will not be primed, even by weeks of age. kittens should receive at least three doses of the core vaccines between and weeks and and weeks of age. this should successfully bridge the period until maternal antibody titers decline to nonblocking levels. one of the most significant causes of apparent vaccine failure in cats is interference by these antibodies with vaccine responses, especially if the initial vaccination series is terminated prematurely. it is uncommon for vaccines to be tested in pregnant queens. in the absence of such data, a decision to vaccinate a pregnant queen must be based on a risk assessment. the risks of abortion or birth defects must be weighed against the risk of death of the mother and kittens. additionally, vaccination of pregnant queens will generate colostral antibodies. modified live fpv vaccines should not be given to pregnant queens because this has been associated with the development of cerebellar hypoplasia in their kittens. inactivated vaccines are much less risky. the risks of acquiring infection are primarily governed by population density and the degree to which a cat encounters other cats. as a result, cats living in a multiple-cat household, in boarding facilities, breeding facilities, and in animal shelters are at substantially higher risk than housebound single cats. likewise, the introduction of a new cat into a household increases the risk of disease introduction. stress induced by changed social structures may result in feline herpesvirus recrudescing and causing clinical disease. conversely, single housebound cats are no longer exposed to other infections, and as a result will not be boosted by natural exposure. periodic housing of pets at boarding facilities while owners are away also places such cats at increased risk. in general, cats in shelters, or boarding catteries represent a random collection of cats with no known vaccination history and a high risk of infectious diseases. the most important diseases in these situations are fpv and the upper respiratory tract infections. it is essential to provide immunity rapidly and ideally cats should be vaccinated ahead of intake. the high likelihood of disease transmission demands that a comprehensive strategy be established and adhered to. all cats entering a shelter should be vaccinated with the core vaccines before entry. it would be desirable to test cats on admission for the presence of antiviral antibodies. in the absence of evidence of immunity or vaccination, as many cats as possible should be vaccinated before or on admission to the facility to establish herd immunity. unfortunately, because of animal turnover and the constant admission of susceptible cats, herd immunity may be inefficient and unreliable. remember too that onset of immunity is not immediate. vaccination is only one component of an infection control strategy for an animal shelter. managers must pay attention to many other factors such as cleaning and disinfection protocols, personal protective equipment, segregation of susceptible animals, crowding, climate management, and stressors that may influence disease resistance and viral recrudesce. most free-roaming feral cats lack protective antibodies against fpv, fhv- , and rabies. if they are vaccinated when trapped to be spayed or neutered, then those antibodies should remain elevated for several months. a single rabies dose will probably protect them for several years even although the regulatory agencies only recognize protection for one year. immunosenescence is known to suppress the immune response of old animals. however, if cats have received appropriate vaccines throughout their lives, then they will most likely be immune. it is probably most appropriate to continue with vaccination in a routine fashion. old cats have high fhv antibody levels in comparison to young animals, suggesting that revaccination at shorter intervals is not always necessary. cats are affected by two immunosuppressive retroviruses, most notably felv, and fiv. data is lacking on whether cats infected with these viruses can be successfully immunized or on how this can be accomplished. inactivated vaccines are safer in such animals than modified live virus vaccines. vaccination against these retroviruses is of no benefit if they are already infected. allergic reactions are always a possibility and the most extreme form is anaphylaxis. it occurs in to cases for every , doses of vaccine administered. in cats, the major shock organs are the lungs. cats undergoing anaphylaxis show vigorous scratching around the face and head as histamine is released into the skin. this is followed by dyspnea, salivation, vomiting, incoordination, collapse, and death. epinephrine is the specific antidote (box . ). the prevalence of vaccine-associated adverse events has been followed after the administration of , , doses of vaccine to , cats. there were adverse events reported ( . / , cats vaccinated). the risk was greatest for cats one year old. for unknown reasons, this risk was greater in neutered than in sexually intact cats. lethargy was the most commonly reported event followed by pain or swelling at the injection site, vomiting, facial edema, and pruritus ( fig. . ). the number of adverse events increased significantly when multiple vaccines were given during a single visit. revaccination should be avoided in cats that have a history of vaccine-triggered anaphylaxis. (if vaccination is considered essential then cats may be premedicated with antihistamines and corticosteroids, given a different formulation vaccine, and monitored very closely for several hours.) when cats are vaccinated, any inflammation at the injection site usually resolves rapidly and completely. in some cats however, tumors develop at the injection sites usually between three months and three years after vaccination. these tumors are mainly fibrosarcomas, malignant histiocytomas, and osteosarcomas. less common forms include rhabdomyosarcomas, hemangiosarcomas, chondrosarcomas, liposarcomas, and lymphosarcomas. these tumors are highly invasive and may metastasize. successful treatment requires a combination of radical surgical excision and adjunct therapy, including radiation, immunotherapy (such as interleukin [il]- treatment), and chemotherapy, but recurrence is common. these sarcomas were first noticed following the introduction of potent, inactivated, adjuvanted vaccines such as those directed against rabies and feline leukemia. cats with sarcomas occurring at sites where vaccines are currently administered were compared with cats that developed sarcomas at nonvaccine-injection sites. cats receiving an inactivated felv vaccine were . times more likely to develop a sarcoma at the injection site than cats that had not received a vaccine. there was a twofold increase in risk with rabies vaccination. however, the risk was not enormously high. it has been calculated that to . sarcomas develop per , vaccinated cats in the united states. (the prevalence of sarcomas in the united kingdom is somewhat lower at . per , vaccinated cats.) the risk increased with the number of doses of vaccine administered; a % increase following one dose, a % increase following two doses, and a % increase following three or four vaccines given simultaneously. vaccine-associated sarcomas tend to occur in younger animals and are larger and more aggressive than sarcomas arising at other sites. they metastasize in % to % of cases. in one study, injection site sarcomas developed on average months after rabies vaccination and months after felv vaccination. global, web-based surveys suggest a somewhat lower prevalence of sarcomas ( . sarcomas/ , cats or . sarcomas/ , doses of all vaccines, or one sarcoma from , doses administered). it must be pointed out therefore that the chances of developing a sarcoma are considerably smaller than the disease risks incurred by unvaccinated cats. in addition to rabies and felv vaccines, injection site sarcomas have also been associated with administration of inactivated vaccines against feline panleukopenia, feline herpesvirus, and feline calicivirus. similar vaccination-related injection site sarcomas have been reported in ferrets, dogs, and a horse. data from the uk's pharmacovigilance reports suggest that the prevalence of these sarcomas has been dropping progressively (fig. . ) . the pathogenesis of these sarcomas is unclear, but it has long been known that carcinogenesis and prolonged inflammation are linked. indeed, it has been estimated that % to % of cancer deaths worldwide are associated with persistent infections and chronic inflammation. when first reported it was assumed that tumor development resulted from the presence of potent adjuvants in vaccines. tumor development has however also been associated with the use of nonadjuvanted vaccines and even with injection of substances other than vaccines, including penicillin, glucocorticoids, lufenuron, cisplatin, and meloxicam, in addition to the presence of persistent suture material, a retained surgical swab, or implanted microchips. there is no evidence that feline sarcoma virus, feline immunodeficiency virus, or feline leukemia viruses cause these tumors. prolonged irritation will increase the activation state of the cells involved in inflammation and tissue repair. the repair process activates stem cells that can differentiate to replace damaged tissues. these stem cells are long lived and so have plenty of opportunities to accumulate mutations. chronic, prolonged irritation leads to an increase in stem cells and the possibility that some of these may mutate (fig. . ) . during chronic inflammation, macrophages secrete growth factors that enhance cell growth. oxidants released from activated macrophages may act as mutagens, especially in rapidly dividing cells. fibroblasts proliferate at sites of chronic inflammation and wound healing. in some of these fibroblasts, the sis oncogene may be activated. the sis oncogene codes for the platelet-derived growth factor (pdgf) receptor, and vaccine-associated sarcomas have been shown to express both pdgf and its receptor. in contrast, nonvaccine-associated tumors and normal cat lymphocytes are pdgf negative. therefore it has been suggested that lymphocytes within the vaccine-associated sarcomas secrete pdgf, which then serves as a growth factor for the fibroblasts. this combination of abnormalities could result in the loss of growth control in the fibroblasts engaged in the chronic inflammatory process. the tumor suppressor gene p encodes a protein that regulates the cell cycle. this p protein increases in response to cell damage. this in turn, delays cell division and allows dna repair before the cell divides. if the cell is severely damaged, the p protein triggers apoptosis and so prevents dna damage being transmitted to the next generation. cells in which p has mutated can, however, continue to divide, giving rise to abnormal and possibly malignant cells. as many as % of injection site sarcomas express mutated p . the key transcription factor for the innate immune system, nf-kb plays a critical role in several cancers. activation of nf-kb is rapidly induced by viral and bacterial infections, necrotic cell products, dna damage, oxidative stress, and pro-inflammatory cytokines. nf-kb is activated when its specific inhibitors are destroyed by enzymes in the ikk complex. most of the increased nf-kb activity that occurs in tumors is caused by the production of ikk-activating cytokines such as il- and tumor necrosis factor. the loss of functional p also triggers nf-kb activation. this nf-kb plays key roles in the progressive steps of tumorigenesis. it is needed for the generation of reactive oxygen species that cause dna damage and oncogenic mutations. it enhances tumor cell proliferation and survival. by chronically stimulating cancer cell proliferation, inhibiting cell death, and promoting mutagenesis, nf-kb drives malignant progression. there is no evidence to prove that injection of less irritating vaccines can reduce the incidence of sarcomas. no specific brands of vaccine, no specific manufacturers, and no other vaccinationassociated factors have been associated with an increased prevalence of sarcomas. feline injection site sarcomas have a poor prognosis. most cats die as a result of local recurrence or metastases. they must be treated by radical resection, but this is often difficult, requires a long recovery period, and may cause significant disfigurement (figs. . and . ). this is especially true of cats vaccinated subcutaneously in the interscapular region. the american association of feline practitioners recommends that the three core vaccines against feline panleukopenia (fpv ), feline herpesvirus- , and feline calicivirus be injected subcutaneously below the elbow on the right forelimb and vaccines against feline leukemia, fiv, and rabies virus be injected below the stifle joint on the left and right hind leg respectively. another possible injection site is in the distal tail. there are no significant differences in the cat's immune response. both routes elicit similar antibody responses to fpv and to rabies antigens. vaccines should be administered as distally as possible to permit amputation if required. the site of vaccine administration and the product used should be recorded for each vaccine to help in assessing risk factors. clients should be instructed to monitor injection sites for swellings or lumps so that any developing tumors are detected and excised as early as possible. a multi-national european cross-sectional study of feline calicivirus epidemiology, diversity and vaccine cross-reactivity comparative efficacy of the leucofeligen felv/rcp and purevax rcp felv vaccines against infection with circulating feline calicivirus efficacy of intranasal administration of a modified live feline herpesvirus and feline calicivirus vaccine against disease caused by bordetella bronchiseptica after experimental challenge duration of immunity in cats after vaccination or naturally acquired infection wsava guidelines for the vaccination of dogs and cats matrix vaccination guidelines: abcd recommendations for indoor/outdoor cats, rescue shelter cats and breeding catteries a dual-strain feline calicivirus vaccine stimulates broader cross-neutralization antibodies than a single-strain vaccine and lessens clinical signs in vaccinated cats when challenged with a homologous feline calicivirus strain associated with virulent systemic disease onset of immunity in kittens after vaccination with a nonadjuvanted vaccine against feline panleukopenia, feline calicivirus and feline herpesvirus three-year duration of immunity in cats vaccinated with a canarypox-vectored recombinant rabies virus vaccine. vaccine prevention of feline injection-site sarcomas: is there a scientific foundation for vaccine recommendations at this time? immunoprophylaxis of dermatophytosis in animals the domestic cat antibody response to feline herpesvirus- increases with age comparative efficacy of feline leukemia virus (felv) inactivated whole-virus vaccine and canarypox virus-vectored vaccine during virulent felv challenge and immunosuppression effects of anesthesia and surgery on serologic responses to vaccination in kittens aafp feline vaccination advisory panel report age and long-term protective immunity in dogs and cats feline leukaemia virus: a review of immunity and vaccination comparative vaccine-specific and other injectable-specific risks of injection-site sarcomas in cats key: cord- - jklwiy authors: xu, shuqin; yang, kunpeng; li, rose; zhang, lu title: mrna vaccine era—mechanisms, drug platform and clinical prospection date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: jklwiy messenger ribonucleic acid (mrna)-based drugs, notably mrna vaccines, have been widely proven as a promising treatment strategy in immune therapeutics. the extraordinary advantages associated with mrna vaccines, including their high efficacy, a relatively low severity of side effects, and low attainment costs, have enabled them to become prevalent in pre-clinical and clinical trials against various infectious diseases and cancers. recent technological advancements have alleviated some issues that hinder mrna vaccine development, such as low efficiency that exist in both gene translation and in vivo deliveries. mrna immunogenicity can also be greatly adjusted as a result of upgraded technologies. in this review, we have summarized details regarding the optimization of mrna vaccines, and the underlying biological mechanisms of this form of vaccines. applications of mrna vaccines in some infectious diseases and cancers are introduced. it also includes our prospections for mrna vaccine applications in diseases caused by bacterial pathogens, such as tuberculosis. at the same time, some suggestions for future mrna vaccine development about storage methods, safety concerns, and personalized vaccine synthesis can be found in the context. mrna, an intermediate hereditary substance in the central dogma, was first discovered in by brenner et al. [ ] . however, the concept of mrna-based drugs was not conceived until , when malone et al. demonstrated that mrna could be successfully transfected and expressed in various of eukaryotic cells under the package of a cationic lipid (n- [ -( , -dioleyloxy) propyl]-n,n,n-trimethylammonium chloride (dotma)) [ ] . in , in vitro-transcribed mrna was sufficiently expressed in mouse skeletal muscle cells through direct injection, which became the first successful attempt on mrna in vivo expression and thus proved the feasibility of mrna vaccine development [ ] . since then, mrna structure researches and other related technologies have been rapidly developed. under this condition, several development restrictions stemmed from mrna instability, high innate immunogenicity, and inefficient in vivo delivery have been mitigated, and now mrna vaccines have been widely studied in different kinds of diseases ( figure ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . mrna vaccines have demonstrated many specific advantages that conventional vaccines do not have. first of all, mrna can theoretically meet all genetic information requirements to encode and express all kinds of proteins. vaccine developing efficiency can be optimized by modifying mrna sequence, which is a more convenient way compared to other kinds of vaccine modification [ , ] . furthermore, most of the mrna vaccine production and purification processes are quite similar despite different encoded antigens, so it is potential to be retained or even standardized to develop other similar mrna vaccines [ , ] . utilizing in vitro transcription also makes mrna vaccines production easier [ ] [ ] [ ] . accordingly, it is obvious that mrna vaccines can save both time and economic costs. second of all, mrna has self-adjuvanting properties which activate strong and long-lasting adaptive immune responses through tumor necrosis factor-α (tnf-α), interferon-α (ifn-α) and other cytokines secretion by immune cells [ ] , while polypeptide and protein based vaccines need extra adjuvants to achieve a similar goal [ ] . the in vivo expression of mrna can also avoid protein and virus-derived contamination [ ] . by modifying the mrna sequence and delivery system, the expression activity and in vivo half-life of mrna can be effectively regulated [ , , ] . thirdly, in comparison with dna-based vaccines, mrna vaccines can express target proteins more efficiently because of their expression in the cytoplasm without entering the nucleus [ ] . in addition, due to the chemical constitution of the mrna sequence, which is different from dna constitution and lack of cpg islands, there is a lower possibility for mrna to integrate into host dna genome and induce a smaller immune rejection reaction [ ] . besides, mrna is only transiently active, making it easy to be completely decomposed via physiological metabolic pathways; therefore, it would not act as a burden to the host homeostasis [ ] . after the first mrna-based drug company was established in , a large number of groups began to research and develop mrna-based drugs [ ] . so far, over twenty mrna-based candidate drugs have entered the clinical trial stage. the market value for the mrna vaccine field has also increased, reaching up to tens of billions of dollars, which signifies a broad prospect for mrna-based drugs development, especially mrna vaccines. in particular, mrna vaccines have a huge potential on rapidly responding to emerging epidemics, e.g., the global explosion of the mrna vaccines have demonstrated many specific advantages that conventional vaccines do not have. first of all, mrna can theoretically meet all genetic information requirements to encode and express all kinds of proteins. vaccine developing efficiency can be optimized by modifying mrna sequence, which is a more convenient way compared to other kinds of vaccine modification [ , ] . furthermore, most of the mrna vaccine production and purification processes are quite similar despite different encoded antigens, so it is potential to be retained or even standardized to develop other similar mrna vaccines [ , ] . utilizing in vitro transcription also makes mrna vaccines production easier [ ] [ ] [ ] . accordingly, it is obvious that mrna vaccines can save both time and economic costs. second of all, mrna has self-adjuvanting properties which activate strong and long-lasting adaptive immune responses through tumor necrosis factor-α (tnf-α), interferon-α (ifn-α) and other cytokines secretion by immune cells [ ] , while polypeptide and protein based vaccines need extra adjuvants to achieve a similar goal [ ] . the in vivo expression of mrna can also avoid protein and virus-derived contamination [ ] . by modifying the mrna sequence and delivery system, the expression activity and in vivo half-life of mrna can be effectively regulated [ , , ] . thirdly, in comparison with dna-based vaccines, mrna vaccines can express target proteins more efficiently because of their expression in the cytoplasm without entering the nucleus [ ] . in addition, due to the chemical constitution of the mrna sequence, which is different from dna constitution and lack of cpg islands, there is a lower possibility for mrna to integrate into host dna genome and induce a smaller immune rejection reaction [ ] . besides, mrna is only transiently active, making it easy to be completely decomposed via physiological metabolic pathways; therefore, it would not act as a burden to the host homeostasis [ ] . after the first mrna-based drug company was established in , a large number of groups began to research and develop mrna-based drugs [ ] . so far, over twenty mrna-based candidate drugs have entered the clinical trial stage. the market value for the mrna vaccine field has also increased, reaching up to tens of billions of dollars, which signifies a broad prospect for mrna-based drugs development, especially mrna vaccines. in particular, mrna vaccines have a huge potential on rapidly responding to emerging epidemics, e.g., the global explosion of the coronavirus disease (covid- ) , stimulating more interest and research expectations from worldwide scientists [ , ] . to date, in vitro transcription technology of mrna has been mature, and the most popular method is using t , t , or sp rna polymerase and linear dna (linearized plasmid dna or synthetic dna prepared by pcr) for mrna synthesis. there are some basic structural elements of mature mrna in the eukaryocyte that are required to keep mrna functional, including five-prime cap ( cap), five-prime untranslated region ( utr) , open reading frame (orf) region, three-prime untranslated region ( utr), and poly (a) tail structure [ , ] . keeping mrna structure intact is beneficial for mrna stability and expression capability. modifying the mrna sequence based on its complete structure can further optimize the efficiency of an mrna vaccine. however, the initial product of mrna in vitro transcription is the mixture of targeted mrna, untargeted rna, nucleotides, oligodeoxynucleotides, and proteins [ ] . to purify the mrna, precipitation and extraction techniques are used to remove common impurities and chromatographic techniques are generally used to separate the target mrna from other mrna impurities in this system [ ] . mrnas from the eukaryotic and partial viral genomes have a -methylguanosine (m g) cap at the end of the mrna sequence (m gpppn structure), which connect to the first rna nucleotide through a , -triphosphate bridge (ppp) during mrna in vitro transcription. the cap can eliminate free phosphate groups in the mrna sequence so as to significantly enhance the stability of mrna, which allows the ribosome to recognize the beginning of mrna and improves translation efficiency by binding to the eukaryotic translation initiation factor e (eif e) [ , ] . so it is obvious that cap modification can be crucial to mrna property improvement. there are two common approaches in terms of in vitro mrna capping. firstly, adding a regular cap analog, m gpppg structure, to the mrna transcription system can achieve mrna capping along with in vitro transcription [ , ] . secondly, mrna capping can also be completed by capping enzyme reaction after the initial in vitro transcription [ , ] . capping with cap analog is the most common capping method of mrna in vitro transcription, but studies have found that regular cap analog can reversely bind to the mrna sequence [ ] . in this case, mrna isomers are formed and lead to low efficiency of mrna downstream translation. to avoid reverse incorporation of cap, anti-reverse cap analogs (arca) have been developed [ , ] . arca is modified at the c or c position to ensure that the methyl groups react with the hydroxyl groups at the correct site during transcription. compared to regular cap analog, arca-capped mrna has a higher translation efficiency [ ] [ ] [ ] . in recent years, further modification on the arca structure has been developed to improve mrna properties. phosphorothioate modifying based on arca, for example, would enhance the translation efficiency of mrna by increasing its affinity for eif e, and has the ability to decrease the susceptibility to decapping enzymes so as to improve the mrna stability [ ] [ ] [ ] . kuhn et al. showed that m , -o gpp s pg (β-s-arca) could significantly enhance the stability and translation efficiency of mrna in immature dendritic cells (dcs) [ ] . in , strenkowska et al. synthesized cap analogs that were composed with , -dithiodiphosphate modification, arca, and an extended polyphosphate chain, named " s analogs", the benefits of which enabled s analogs to function better than any s-arca used in clinical trials [ ] . another cap analog, a co-transcriptional capping method called "cleancap," was developed in [ ] . it utilized an initiating capped trimer to yield a naturally occurring cap structure, which increased the capping efficiency to nearly - % [ , ] . utrs are non-coding parts of mrna sequence located at the upstream ( utr) and downstream ( utr) domains of the mrna coding region. as reported, utrs are related to mrna replication and translation processes, and they can greatly alter mrna decay and translation efficiency through reactions with rna binding proteins [ , ] . in an attempt to enhance mrna stability and translation efficiency, it is essential to ensure the optimization of utrs. generally speaking, utr optimization is to increase the in vivo mrna expression level. for instance, the widely-used utr sequence derived from α-globin and β-globin contains translation and stability regulatory elements [ ] . utr is normally considered to be a concentrated region full of unstable factors in mrna, so averting unstable sequences while synthesizing utr can increase mrna stability. au-enriched sequences and gu-enriched sequences are related examples of this [ , ] . on the other hand, introducing stable elements to utr can also significantly improve the stability of mrna and expand its half-life [ , ] . orlandini von niessen et al. once connected two random utrs which contained stable elements in series, and successfully improved the translation efficiency of mrna [ ] . utr directly affects the translation of its downstream sequence orf, so the optimization of utr should not influence the normal translation process of the orf. avoiding the gene sequence in utr, which is identical to the upstream of orf, can effectively prevent false start and replacement of the reading frame during mrna translation [ ] . additionally, some particular sequences can be added to utr to enhance the stability of mrna and the accuracy of translation. for example, kozak et al. inserted sequence gcc-(a/g)-ccaugg in this region, leading to a more accurate start of translation process [ ] . study also shows that over-stabilized secondary structure of utr would hinder the binding of ribosomes to mrna, and short and loose utr is more conducive to the mrna translation processes [ ] . as the coding region of mrna, the translatable rate of orf region is definitely crucial. therefore, choosing the appropriate codons in this region can optimize the overall translation efficiency of mrna. optimized orf sequence usually incorporates synonymous frequent codons and/or codons with higher trna abundance to replace rare codons in orf, so that highly expressed genes can be translated using the same codons of the host and/or guaranteed the ampleness of trna during the expression of exogenous mrna [ ] . however, high translation rate of mrna is not all beneficial, as some proteins require a low translation rate to fold correctly, stably, and effectively; in this case, using codons with low frequency in orf can yield protein products of higher quality [ ] . therefore, for different antigens, we should use different codon optimization strategies to improve mrna translation rate and ensure the expressed antigen quality at the same time. poly (a) tail and the cap structures are both crucial elements during mrna translation. poly (a) sequence can slow down the degradation process of rna exonuclease, which increases stability, extends in vivo half-life, and enhances translation efficiency of mrna [ ] . moreover, poly (a) binding protein (pabp) can link to the cap through translational initiation factors, such as eif g and eif e, which in turn affects the closed-loop structure of mrna and synergistically regulates the stability and translation efficiency of mrna [ , , ] . however, pabp can also bind to adenylation complexes and participate in translation inhibition process mediated by microrna [ ] . the contradictory function of pabp indicates that various poly (a) sequence length can affect mrna translation efficiency differently. there are different methods to synthesize a poly (a) structure, among them, in vitro transcription process with dna template with poly (a) structure information can yield a defined poly (a) sequence length [ ] . recombinant poly (a) polymerase can also be used to add poly (a) structures by undergoing an enzymatic polyadenylation after initial mrna transcription, in which case poly (a) structural mixtures of different lengths can be obtained [ ] . early studies suggest that a long poly (a) sequence can improve mrna stability. for example, the optimal length of poly (a) sequence in dcs is roughly between - nucleotides [ , ] , and over nucleotides of poly (a) sequence length in human primary t cells can become more conducive in increasing mrna stability and translation efficiency [ ] . when poly (a) sequence length is less than nucleotides, it would reduce mrna translation efficiency [ ] . however, in , lima et al. found that mrnas with high translation efficiency generally had short poly (a) sequences through novel genome-wide research techniques, whilst short poly (a) structures were generally found in well-translated eukaryotic mrnas [ ] . therefore, it has been indicated that since the lengths of poly (a) sequences required for high translation efficiency mrna in various types of cells are different, adjustments should be made to optimize the translation efficiency of mrna. based on its self-adjuvanting effect, mrna can exhibit some properties similar to the mrna virus when it works as the vector of exogenous genes. in this case, mrna can be recognized by antigen-presenting cells (apcs), which subsequently activates pattern recognition receptors (prrs) such as toll-like receptor (tlr ), tlr , and tlr [ , , ] . the double-stranded rna (dsrna) can combine with some retinoic-acid-inducible gene i (rig-i) -like receptors (rlrs) in the cytoplasm, such as rig-i and melanoma differentiation-associated (mda ), which promotes apcs maturation, pro-inflammatory cytokines secretion, and type i interferon (ifn) secretion [ , ] . eventually this leads to strong antigen-specific humoral and cellular immune responses ( figure ). however, subunit vaccines composed of peptide or protein antigens are generally unable to activate prrs, so it is necessary to add adjuvants which can initiate and support adaptive immune responses, achieving the final result of carrying out the body's immune response of subunit vaccines [ ] . therefore, mrna's strong adaptive immune response and self-adjuvanting property can provide a huge advantage shown in mrna vaccines. single-stranded rna (ssrna) can trigger the dcs' antiviral activation state through tlr and tlr recognition during mrna in vivo transmission [ ] . the dsrna contaminants can also trigger immune activation via tlr recognition [ , ] . however, excessive immune response stimulated by mrna in the cytoplasm would stimulate cells to secrete large amounts of type i ifn and other interferons which can inhibit the translation of mrna and eventually lead to translational stagnation, rna degradation, cd (cluster of differentiation ) + t cells activation reduction, and ultimately immune response termination [ , , ] . this could leave negative effects on some mrna applications such as vaccines and protein replacement therapies. self-adjuvanting properties of mrna have both advantages and disadvantages in mrna vaccine applications, therefore, it is necessary to form mrna immunogenic regulations according to different medical demands, which in return would effectively improve the application efficacy of mrna vaccines. (c) self-adjuvant effect. various of pattern recognition receptors (prrs) can recognize mrna in vitro transcription product. ssrna can be recognized by endosomal innate immune receptors (e.g., toll-like receptor (tlr ), tlr ). dsrna can be recognized by endosomal innate immune receptors (e.g., tlr ) and cytoplasmic innate immune receptors (e.g., protein kinase rna-activated (pkr), retinoic acid-indu [ ] cible gene i protein (rig-i), melanoma differentiation-associated protein (mda ), and ′- ′-oligoadenylate synthase (oas). based on those, mrna products can stimulate the secretion of pro-inflammatory cytokines and type i interferon (ifn), which leads to antigen-presenting cells (apcs) activation and inflammatory reaction. however, they can also activate antiviral enzymes that cause stalled mrna translation and mrna degradation. mrna in vitro transcription product often contains dsrna contaminants. dsrna, which is a simulant of rna virus genome replication intermediates, can promote type i ifn production [ , ] . therefore, the purification of an mrna in vitro synthetic product can effectively reduce type i ifn immune response of mrna vaccines and increase mrna translation efficiency [ ] . studies have shown that chromatographic methods (fast protein liquid chromatography, high-performance liquid chromatography, etc.) can effectively remove dsrna from mrna products; after purification, the mrna translation level in primary cells can be increased by - times while the cytokine secretion level still remains relatively high [ , ] . ssrna can also work as a potent pathogen-associated molecular pattern (pamp) that elicits a strong immune response and stimulates type i ifn production. type i ifn can induce numerous types of ifn-stimulated genes (isgs) to inhibit mrna translation [ ] . for instance, ifn-inducible using dna with the antigen-encoding sequence as template, mrna in vitro transcription products contain single-stranded rna (ssrna), double-stranded rna (dsrna), etc. the ssrna structure normally includes five-prime cap ( cap), five-prime untranslated region ( utr), open reading frame (orf) region, three-prime untranslated region ( utr), and poly (a) tail structure. (b) rna translation and antigen presentation. through endocytosis, mrnas enter the cytoplasm. some mrnas combine with ribosomes of the host cell and translate successfully. antigen proteins can be degraded to antigenic peptides by proteasome in the cytoplasm and presented to cytotoxic t lymphocytes (ctls) via major histocompatibility complex (mhc) i pathway. or, they can be released out of the host cell and taken up by dcs. then, they are degraded and presented to helper t cells and b cells via mhc-ii pathway. b cells can also recognize released antigen proteins. (c) self-adjuvant effect. various of pattern recognition receptors (prrs) can recognize mrna in vitro transcription product. ssrna can be recognized by endosomal innate immune receptors (e.g., toll-like receptor (tlr ), tlr ). dsrna can be recognized by endosomal innate immune receptors (e.g., tlr ) and cytoplasmic innate immune receptors (e.g., protein kinase rna-activated (pkr), retinoic acid-indu [ ] cible gene i protein (rig-i), melanoma differentiation-associated protein (mda ), and - -oligoadenylate synthase (oas). based on those, mrna products can stimulate the secretion of pro-inflammatory cytokines and type i interferon (ifn), which leads to antigen-presenting cells (apcs) activation and inflammatory reaction. however, they can also activate antiviral enzymes that cause stalled mrna translation and mrna degradation. mrna in vitro transcription product often contains dsrna contaminants. dsrna, which is a simulant of rna virus genome replication intermediates, can promote type i ifn production [ , ] . therefore, the purification of an mrna in vitro synthetic product can effectively reduce type i ifn immune response of mrna vaccines and increase mrna translation efficiency [ ] . studies have shown that chromatographic methods (fast protein liquid chromatography, high-performance liquid chromatography, etc.) can effectively remove dsrna from mrna products; after purification, the mrna translation level in primary cells can be increased by - times while the cytokine secretion level still remains relatively high [ , ] . optimization of mrna sequence to regulate self-adjuvanting property ssrna can also work as a potent pathogen-associated molecular pattern (pamp) that elicits a strong immune response and stimulates type i ifn production. type i ifn can induce numerous types of ifn-stimulated genes (isgs) to inhibit mrna translation [ ] . for instance, ifn-inducible protein with tetratricoid repeats (ifit) can combine with the cap structure or interact with eif to disrupt the mrna translation process [ , ] . therefore, optimizing mrna sequence can regulate the ability to activate the immune response of mrna vaccines [ , , ] . prrs can recognize cap (m gpppn)-capped or uncapped mrna and inhibit its translation [ ] . in , kumar et al. evaluated the ability of prrs to recognize three forms of capped mrna, including cap -capped, cap (m gpppnmn)-capped, and uncapped mrna. they discovered that cap -capped mrna was still translated after being recognized by prrs, while cap -capped and uncapped mrna were not [ ] . therefore, choosing appropriate cap structure can avoid excessive immunity response. modification of the orf region can also reduce the strong immune response caused by prrs recognition, and enhance the translation level of mrna [ ] . in , anderson et al. studied the difference between unmodified mrna and pseudouridine modified mrna [ ] . the ability of mrna to be recognized by - -oligoadenylate synthetase (oas protein, induced by type i ifn) and mrna stability were assessed, and results showed that the pseudouridine modified mrna had lower efficiency in terms of oas activation, lower rate of rna degradation, and higher efficiency of mrna translation [ ] . karikó et al. intravenously injected pseudouridine modified mrna in mice, and found out that there was a higher target protein expression in the spleen and lower ifn-α concentration in serum compared with unmodified mrna treatment [ ] . uracil analog is the most common analog used in mrna modification, and some other base analogs can also be used for mrna sequence modification. kormann et al. and mays et al. used different rates of -methyl-cytidine and -thiouridine to modify mrna sequence, in which both effectively reduced the recognition rate of prrs, and increased mrna intracellular stability [ , ] . some studies need the enhancement of the immunogenicity of mrna vaccines and adding adjuvants to the mrna vaccine system can meet this requirement. formulation of self-amplified rna vaccines with the traditional adjuvant mf (made by novartis) and cationic nanoemulsion (cne) have proven to enhance the immunogenicity and efficacy of mrna vaccines in various animal models [ , ] . certain immunomodulatory molecules also have adjuvant activity. trimix, a new adjuvant strategy developed by vrije universiteit brussel, consists of mrnas that encode three immune activator proteins-cd , cd ligand (cd l) and constitutively active tlr [ , , ] . trimix mrna can increase the immunogenicity of naked, unmodified, unpurified mrna, and it is also related to the enhancement of dcs' maturation and cytotoxic t lymphocyte response [ ] . in , leal et al. adopted the trimix naked mrna strategy to treat acquired immune deficiency syndrome (aids) patients. treatment using high doses of trimix mrna showed that a high human immunodeficiency virus (hiv)-specific t cell response could be stimulated and detected [ ] . the high safety and tolerability of this strategy has been demonstrated in this research [ ] . some mrna delivery vehicles can also increase the adjuvant effect, such as cationic lipid and protamine. in , researchers used the mrna vaccine immunization strategy with cationic lipid , -dioleoyl- trimethylammonium-propane/ , -dioleoyl-sn-glycero- -phosphoethanolamine (dotap/dope) as the assigned adjuvant, and stimulated more pro-inflammatory cytokines and type i ifn secretion than naked mrna in dcs [ ] . after subcutaneous injection of this mrna vaccine in mice, large amount of type i ifn secretion and rapid aggregation of inflammatory monocytes could be detected in lymph nodes transiently [ ] . this indicates that cationic lipids can strengthen the adjuvant effect and the efficacy of mrna vaccines to a certain extent [ , ] . researches also demonstrated that mrna and protamine complexes could act as danger signal and elicit t-help cell (th ) responses via tlr and tlr involving [ , ] . the rnactive ® vaccine platform designed by curevac used co-delivered rna and protamine complex as the adjuvant to induce th t cell responses, and naked, unmodified, and sequence-optimized mrna as the antigen to develop mrna vaccines [ ] . in this technique, protamine-formulated rna only works as an adjuvant, not as a mrna carrier, enabling more rnactive ® vaccines to arouse strong immune responses in many pre-clinical models, which can successfully prevent attacks from various influenza strains [ , ] . kowalczyk et al. revealed that rnactive ® vaccine treatment in mice could initiate a balanced and strong specific immune response with intradermal immunization [ ] . this immune stimulation only existed in the stimulated site and lymphoid organs, and no pro-inflammatory factors were detected in serum. overall, rnactive ® technology is a new effective technique of mrna vaccine with high levels of safety and flexibility. mrna needs to enter the host cytoplasm to express specific antigens to remain functional; however, the mrna molecule is not small enough to pass through cell membrane by free diffusion [ , ] . additionally, mrna and cell membrane are both negatively charged, which increases the difficulty of mrna delivery. furthermore, mrna can be easily degraded by extracellular ribonucleases which exist in skin and blood [ , ] . therefore, delivering mrna into enough numbers of cells with sufficiently high translation levels is one of the most difficult application problems of mrna vaccines, as it demands highly specific and efficient mrna delivery systems [ , ] . a variety of mrna delivery methods and mrna delivery vehicles have been developed and applied currently (table ) . early study has demonstrated that naked mrna in vivo injection can provoke the immunotherapy response in mice [ ] . at present, administration strategies of mrna generally include subcutaneous injection, intradermal injection, intranodular injection, intramuscular injection, intravenous injection, intratumoral injection, etc., which are essential methods that help stimulate antigen presentation and initiate immune responses [ , , ] . in , phua et al. discovered that delivery efficiency of subcutaneous injection of naked mrna in mice was even higher than mrna nanoparticle delivery methods [ ] . van lint et al. suggested that intratumoral injection of tumor-associated mrna would elicit an appropriate immune response and believed that it could be a promising vaccination strategy for the impending future [ ] . these days, direct injection of naked mrna is mainly used to treat or prevent infectious diseases [ ] . however, even though the injection of naked mrna can cause immune response, the working effect of this delivery method is relatively weak, and the naked mrna is often rapidly degraded after injection. direct injection of naked mrna is too simple and primitive to be applied in human patients, and it is often used as an administration route to inject modified mrna vaccines with other delivery systems to achieve better vaccine effects. the efficiency of naked mrna antigen presentation can be improved with the assistance of common physical methods including electroporation, gene gun, microneedles, etc. [ ] . electroporation can increase mrna delivery efficiency without the demand of other mode receptors, which can reduce unnecessary immunoreactions [ ] . electroporation also has an adjuvant effect that it can recruit pro-inflammatory cells and induce the production of cytokines at the inoculation site, improving the immunogenicity of mrna [ ] . in , callis et al. found that electroporation could be used to transfer mrna into animal and plant cells with low transfection efficiency [ ] . however, the target intracellular expression product was high enough to reach the detection level. in , the mrna transfection efficiency in dcs had reached - % for electroporation method [ ] . the gene gun method, using compressed helium gas as an acceleration force to push mrna coated on the surface of gold particles into host cells, is an efficient method of mrna delivery [ ] . in , qiu et al. used gene gun method to transfer the human alpha- antitrypsin mrna into the mouse skin and successfully triggered the antibody response [ ] . peking et al. developed a mrna-based therapy for genetic skin diseases restoration, mrna was effectively transported to the target skin layers in mice by gene gun delivery [ ] . despite its advancements, the gene gun method is rarely used in large animals and humans. physical ways to deliver mrna may affect the physiological structure and activity of cells, even causing abnormal cell death. therefore, applying physical mrna deliveries in human is potentially hazardous [ , ] . dcs are one of the most potent apcs of immune system. they can present processed antigens to cd + , cd + t cell via the major histocompatibility complex (mhc), which triggers cellular immunity [ , ] . meanwhile, dcs can also present intact antigens to b cells, triggering humoral immunity [ ] . the common way to use dcs as mrna delivery vehicles is to transfect mrnas encoding peptides, proteins or other antigens into dcs via in vitro, and then transfer the processed dcs back into the host body to start the antigen-specific immune response [ ] . the dcs-mrna delivery system does not need to be combined with other carrier molecules and can generate high delivery efficiency. in this context, this delivery system is widely used in pre-clinical experiments, animal models and clinical researches [ , , , ] . moreover, this strategy has been mainly applied in cancer treatment because the elicitation of cellular immune response is predominant [ ] . however, the mrna transfection rate is quite low if only by dcs endocytosis, and electroporation method is often used to further improve the mrna transfection rate [ ] . gay et al. used electroporation to transfer the mrna encoding hiv antigens into dcs for hiv treatment, after intradermal injection, the number of hiv-specific cd + / cd ra-cd + factors/cytotoxic t-lymphocytes (ctls) was at least times higher than control, which enhanced the t cell immunological reactions of hiv patients [ ] . another unignorable barrier to clinical application of ex vivo-loaded dc mrna vaccines is that time-and money-consuming production process cannot meet the huge quantity demand of mrna vaccine for some treatments. besides, the immune response caused within several hours after mrna transfection can be lost during the time-consuming in vitro preparation process, leading to reduction of the therapeutic effect of mrna vaccines [ ] . out of these considerations, diseases that require large amounts of mrna vaccine treatment in the short term should give preference to delivery systems with a fast production speed. delivery systems that are able to directly target mrna to in vivo apcs can also be considered. protamine is an alkali cationic protein with resin-like structure. combining mrna with protamine in different mass ratios can yield electrostatic protamine-mrna complex particles with different diameters [ ] . this tight conjugate form can effectively protect mrna from being degraded by serum rnases, and the complex can cause a strong immune-reaction of immune cells such as dcs, monocytes, b cells, natural killer cells, and neutrophils [ , , ] . this indicates that protamine has the potential to be used not only as a mrna carrier, but also as an immune activator. in , protamine was already studied as one of the first delivery materials for long rna [ ] . fotin-mleczek et al. used protamine as the delivery material during the vaccination of mrna tumor vaccine, and successfully elicited a complete specific anti-tumor response [ ] . when the mass ratio of protamine to mrna is : , the size of the electrostatic complex formed is about nm, which is relatively stable and produces strong immune stimulation and high cytokine levels, with the downside of inhibiting protein expression significantly [ ] . however, when the mass ratio of protamine to mrna is : , compared to the previous mass ratio of : , the protein expression increased but the cytokine level decreased [ ] . hence, a common idea is that the mrna translation efficiency and immune strength are limited in the protamine-formulated mrna delivery system. and it is speculated that this defect may be related to the extremely tight electrostatic complex [ , ] . in recent years, protamine-formulated mrna delivery system has been widely used in clinical trials, and gained pretty good clinical treatment effects, such as rabies, non-small cell lung cancer, etc. [ ] [ ] [ ] . the rnactive ® vaccine platform, which use the protamine-mrna only to activate immune responses, is a prevailing technique to resolve this problem [ ] . furthermore, to use protamine as a mrna delivery and immune activator at the same time, structural optimization of protamine or searching for proteins similar to protamine in property as substitutes deserves our attention. as a commonly used gene carrier, cationic liposomes can also combine with negatively charged nucleic acids to form electrostatic complexes, improving mrna delivery efficiency [ ] . the cationic lipid-mrna complex and other preparations together can form an - nm nanoparticle called lipid nanoparticles (lnp), which can be transfected into the cytoplasm by endocytosis. lnp is one of the most advanced mrna delivery systems. this stable particle consists of ionizable cationic lipids, natural phospholipids, cholesterol and polyethylene glycol (peg) [ ] . the ionizable cationic lipid can promote the autonomous aggregation of mrnas to form a~ nm particle and release mrnas in the cytoplasm through ionization; natural phospholipids support the nanoparticles to form a lipid bilayer structure; cholesterol is used as a stabilizer to increase lnp stability; and peg can extend the half-life of lnp complex [ , ] . mrna is carried in the core of lnp which can be protected from degradation, and the lipophilicity property of lnp material allows the mrna delivery complex to fuse with the host cell membrane and deliver mrna into the cells by endocytosis [ , ] . lnp is often used as a short interfering rna (sirna) delivery system in early researches [ ] . nowadays lnp is also widely used in mrna delivery processes. geall et al. used lnp to deliver self-amplified rna vaccines, which caused the mrna expression level in mice to be significantly higher than that of naked mrna, cd + , and cd + t cell immune responses were also effectively induced. with different administration strategies, the immune-stimulation area provoked by lnp-mrna can be different [ ] , and may achieve the targeted therapy need of different diseases. pardi et al. found that injecting lnp-mrna with the appropriate dose by subcutaneous, intramuscular, and intradermal methods could mediate local gene product expression [ ] . lnp-mrna treatment with intravenous injection, intraperitoneal injection, tracheal inhalation, etc. could achieve systemic expression of gene products, as reported in , out of which intravenous injection showed the highest mrna delivery efficiency, and the target protein products were successfully expressed in the liver for days [ ] . but it is notable that escape mechanisms of mrna from complexes to free state for function in the cytoplasm are still incompletely understood. change of ionization state of lipids with in vivo environmental ph is thought to be critical to the escape process [ ] . meanwhile, further research about the toxicity reduction and immunogenicity regulation of cationic lipid-based delivery system are also urgently needed. currently, cationic polymers have been widely used as mrna delivery vectors [ , ] . commonly used polymer delivery materials include polyethylenimine (pei), poly (beta-amino esters) (pbaes), etc. among them, pei is one of the most widely used materials. pei is a kind of cationic water-soluble polymer with either dendritic, linear, or branching structure, mainly used as a dna/mrna carrier [ , ] . there is a commercial linear pei derivative called jetpei ™, which was once used for dna and sirna transfection, and currently available for mrna transfection [ , ] . however, pei is also qualified with certain cytotoxicity that is hard to be degraded, so researchers often use fatty chains to modify low-molecule-weight pei for the intention of reducing pei toxicity [ , , ] . pbaes are biodegradable polymers originally developed for dna transfection [ ] . a study in showed that pbaes could be used to deliver mrna, and higher levels of mrna transfection in vitro could be achieved when there is no serum protein in the system [ ] . this research has led to the development and application of a variety of pbaes that enhanced serum stability in vivo. there are now thousands of chemically different pbaes created thanks to the simple synthetic method of pbaes [ , , ] . in addition, pbaes and lipids can be formulated together to improve their serum stability. in , kaczmarek et al. developed a polymer-based delivery system by formulating pbaes and lipid-peg together, which had high serum stability and mrna delivery efficacy and successfully detected the target mrna product in the lungs of mice specifically by intravenous injection treatment [ ] . polymer-based materials are crucial competitors against lipids in mrna therapeutics. their toxicity, similar to cationic lipids, has been also thwarted them for broader application [ ] . apart from modification with other materials to improve the properties of polymer-based vectors, optimization for both molecular weight and branch pattern also seems to be a dependable direction. immunotherapy, especially vaccines against infectious diseases and cancers, is the core field of the mrna drug platform. investigations of other areas such as reprogramming of cell fates and genome editing based on mrna have been extensively reviewed [ , ] , therefore they are not a subject of concern in this review. mrna vaccines are generally categorized into two major types according to their construction and replication abilities: self-amplifying mrna (sam) vaccines and non-replicating mrna vaccines. the sam vaccines are developed from an alphavirus genome with its gene encoding structural proteins replaced by the sequence encoding our wanted antigen, enabling intracellular rna amplification, and abundant protein expression of the wanted antigen owing to the integrity of viral replication machinery [ ] . the full length of naked sam can be up to ~ kb. due to self-replication, a remarkable low dose of this vaccine promises a huge amount of antigen production with a considerable duration of effectiveness (up to months) [ ] . the inoculation of sam vaccines can make a simulation of the infection of acute pathogens owing to its pamp, the replication of the self-adjuvanted antigen-encoding rna and the protein expression occurring hours after the vaccination [ ] . this property of sam vaccines, nevertheless, remains controversial since it has the potential to limit the size of antigen-encoding sequence that can be accommodated, to affect the accurate regulation of induced inflammatory responses and even to elicit immune responses of the organism against those rna replication factors, thus reducing the in vivo repeated efficacy of the vaccine [ ] . non-replicating mrna vaccines have the complete structure of mature mrna which contains the orf segment that encodes our desired antigen. owing to their small length ( ~ kb), there is no size restriction for the carrier capacity on the antigen, allowing better control of triggered immune responses as well as developing more affordable approaches from synthesis to storage [ , ] . non-replicating mrna vaccines have a huge potential to become the major cure for the current epidemic outbreak. as mentioned earlier, studies regarding mrna vaccines have largely completed concept establishment and initial exploration in the s. in , martinon et al. successfully achieved in vivo induction of specific anti-influenza ctls by intravenous or subcutaneous injection of mice with liposome-entrapped mrna encoding influenza virus nuclear proteins, which was a pioneering mrna vaccine vector attempt [ ] . in , mandl et al. used the gene gun to deliver in vitro synthesized infectious rna from a flavivirus, demonstrating induced protective immunity in mice by less than ng of rna [ ] . boczkowski vaccines against infectious pathogens has always been the most effective way to prevent and limit infectious diseases, a classic example of which is the complete eradication of the smallpox virus. unfortunately, traditional strategies of vaccines, such as non-live freeze-dried vaccines and live attenuated vaccines, underperform against some chronic or recurrent pathogenic infections with a long duration of disease such as aids and tuberculosis (tb). traditional vaccines' lack of adequate speed, owing to relatively slow process of development, would not be able to address outbreaks of virulent pathogens such as zaire ebolavirus, zika virus (zikv) and coronavirus. mrna vaccines against infectious diseases have made promising accomplishments and some products have entered human clinical trials ( table ). overall development steps of those vaccines are ( ) constructing the core antigen-encoding mrna sequence optimized or combined based on selected antigen(s) from the target pathogen; ( ) trying and choosing a proper combination of mrna construction type, adjuvants, carrier materials and the route of administration; ( ) detecting in vivo expression of the encoded antigen and the level of elicited immune responses; ( ) providing research and demonstrations of immune induction mechanisms. here we have reviewed some recently published promising studies related to mrna vaccine application trials. influenza viruses have the characteristic of continuous evolution which makes them hard to be completely eradicated. the monoclonal antibody treatment targeting the conservative site of effector molecules of the influenza virus is commonly accepted as a highly specific and effective method against the virus [ ] . mrna vaccines encoding the conserved regions of influenza virus effector protein(s) are capable of provoking the generation of specific antibodies so that a better prevention or treatment effect, compared to conventional vaccines, is acheived. in addition, the rapid production process of mrna vaccines makes them easier to stand out in preventing novel influenza virus. current mrna vaccines against influenza mostly use cationic lipids-based delivery systems to effectively deliver mrna. the rnactive ® vaccine platform with the self-adjuvanting property give an impressive performance in trials of prevention of influenza, too [ ] . brazzoli et al. generated a novel oil-in-water cne as the carrier for a sam vaccine expressing influenza virus hemagglutinin (ha) antigen [ ] . the vaccination was reported to effectively induce functional neutralizing antibody and ha-specific cd + th cells and cd + cytotoxic t cells immune responses; it also defended a lethal influenza virus challenge in mice. pardi et al. successfully elicited ha stalk-specific antibody response in mice, rabbits, and ferrets by immunization with nucleoside-modified non-replicating mrna vaccine candidate encoding full-length influenza virus ha formulated in lnp [ ] . this mrna-lnp influenza vaccine partially overcome inhibition by the usage of maternal antibodies, and in turn induced a longer-lived and stronger immune protection in the mouse pups than a conventional influenza vaccine [ ] . feldman et al. reported phase i clinical trials of the first two non-replicating mrna vaccines against influenza viruses (h n and h n ) encoding full-length ha respectively from h n and h n with a : mass ratio of mrna to lnp [ ] . both vaccines used a lnp carrier that was first applied in mrna vaccines against the zika virus [ , ] ; they were proved well tolerated by healthy adults and elicited potent humoral immune responses [ ] . this research showed the potential of mrna vaccines to address highly variable pathogens. aids, a chronic and life-threatening condition owing to the infection of hiv, has not yet found a truly effective and affordable way of cure since its discovery in . defeating hiv is a significant issue of research developing mrna vaccines. at present, there are several mrna vaccines for the treatment of aids in human clinical researches. ex vivo loading of dc delivery systems seems to be a preferred delivery method which is normally used for cancer treatment. in infectious diseases, it is almost exclusively used for therapeutic research on aids, and is widely proved to safely cause antigen-specific cd + and cd + t cell immune response [ ] . however, in , gandhi et al. reported disappointing results of a clinical trial for immunization of hiv- -positive participants with autologous dcs transfected with mrna encoding hiv- structural proteins gag and nef [ ] . in that trial, merely transient and weak immune responses were detected, indicating the necessary improvement for the dc vaccination [ ] . in such a way, delivery systems that can elicit strong antigen-specific t cell immune responses are getting more attention in aids treatment. the cationic nanoparticle carrier is a promising delivery system with multiple diversity. zhao et al. developed a pei-stearic acid (psa) copolymer-based self-assembled cationic nanomicelles which delivered non-replicating mrna vaccine encoding hiv- gag [ ] . their study initially showed the potential of psa/mrna nanomicelle vaccine strategy against hiv with acceptable carrier toxicity, efficient endosomal escape and translation of mrna in dcs, and stimulated potent specific antibody secretion and pro-inflammatory cytokine expression [ ] . bogers et al. demonstrated a sam vaccine encoding a hiv- clade c envelope glycoprotein delivered by a cne system, including squalene, dotap, sorbitan trioleate and polysorbate, with a relatively mature preparation protocol [ ] . greater cellular immune responses and neutralizing antibody responses were induced by this hiv sam vaccine instead of two other sam vaccine modalities, the self-amplifying mrna of which were encapsulated by a hiv recombinant envelope protein or in an engineered viral replicon particle [ ] . hti-trimix, a combination of activation adjuvant trimix and selected mrna comprising of conservative fragments from hiv- structural proteins-gag, pol, vif, and nef, is a new mrna-based therapeutic vaccine candidate against hiv- [ ] . it encodes strong activation signals and a potent hiv recombinant antigen. the preclinical results suggested an effective induction of mature dcs, antiviral cytokine secretion (especially ifn-γ) and t cell stimulation. mice that were intranodally injected with hti-trimix generated potent antigen-specific cytotoxic t-cell responses [ ] . by the end of , phase i and phase iia clinical trials of hti-trimix have been accomplished. in phase iia, hiv- -infected participants received three vaccinations at weeks , , and detected through ultrasound-guided administration with an inguinal lymph node. although hti-trimix showed good safety and tolerance, an unexpected start codon was unfortunately found upstream of the hti recombinant antigen coding sequence which likely had a negative influence on hti protein expression [ , ] . future studies for corrected hti are not yet certain. taking into consideration of an additional translation process of mrna vaccines, pre-testing of mrna expression in vitro deserves our attention. due to the limited understanding of hiv and the unclear pathogenesis, there are still many difficulties in the treatment of aids. choosing proper antigen(s) and delivery system that can cause intense antigen-specific t cell immune response should be emphasized at mrna vaccine design in the future. in addition, mrna vaccines on aids prevention may also be a feasible field. in the last years, there have been three coronavirus infections (severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and (sars-cov- )) globally, all leading to extreme health threats and tremendous economic loss without established therapies or vaccine treatment that would cure the illness. of all the patents regarding vaccine types, most of them are related to sars and mers, only three patents have been focused on mrna vaccines as of today [ ] . in the face of the sudden new coronavirus epidemic, the speed of vaccine development determines the speed of life saving. therefore, it is inevitable that mrna vaccines with rapid product process will play an important role in the development of coronavirus vaccines. covid- , caused by sars-cov- infection, has been spreading all over the world with over . million confirmed cases and over , deaths as of august , (data from world health organization). an effective vaccine is urgently needed. lin et al. reported two non-replicating mrna vaccines respectively encoding the receptor-binding domain of the spike protein and the virus-like particles (vlps) of sars-cov- ; further optimization of antigen sequences, as well as safety and efficacy evaluations are underway [ ] . moderna first announced a mrna vaccine candidate, mrna- , against sars-cov- , and officially began phase i clinical trials for safety and immunogenicity evaluation on march , . this vaccine encodes the spike (s) protein of sars-cov- in a prefusion stabilized form. according to the interim data announced on may , , mrna- was shown generally safe and well tolerated; after two weeks following the second dose, with the vaccination dose as low as µg, the levels of both binding antibodies and neutralizing antibodies in serum were at the levels detected in samples from people having recovered from covid- . collaborative development of a new mrna vaccine against sars-cov- has been announced by sanofi pasteur and translate bio on march , . pfizer and biontech announced the positive results of the ongoing phase i/ii clinical trials of bnt b . it is a modified mrna vaccine candidate formulated by lnp, encoding trimerized sars-cov- s protein receptor binding domain. proper dose level of bnt b was initially identified between µg and µg. after two doses of µg and µg of bnt b , mean titers of specific neutralizing antibodies were . -fold and . -fold, respectively, the specific neutralizing antibody of the convalescent [ ] . cv , a prophylactic non-replicating mrna candidate vaccine combined with protamine encoding rabies virus glycoprotein (rabv-g), completed phase i clinical trial in [ ] . in pre-clinical trials, this vaccine elicited powerful functional antibody responses with a stable titer level up to one year and induced robust specific cd + and cd + t cells (higher cd + t cell induction than the induction by a licensed vaccine) when applied intradermally both in mice and pigs [ ] . although cv was shown generally safe in phase i trial, the unstable administration-dependent functional antibody titer resulted in an unclear research outlook [ ] . subsequent new preclinical studies in reported an improved humoral and cell immune response using rabv-g mrna packaged in lnp in both mice and nonhuman primates in comparison to the protamine formulated mrna candidate; corresponding human clinical trials are being followed up [ ] . in , modified mrna-lnp vaccines against zikv were reported in cell and nature respectively [ , ] . pre-membrane (prm) protein and envelope (e), two zikv structural proteins, form prm-e heterotrimers when zikv buds invade the lumen of the endoplasmic reticulum. richner et al. developed a lnp-encapsulated non-replicating mrna vaccine encoding the human ige signal sequence, which contained full-length prm and e genes (igesig-prm-e) [ ] . intramuscular inoculation of µg of igesig-prm-e lnps with a booster protected mice from severe zikv infection with remarkably high titers of neutralizing antibodies (> / , ec ) was detected [ ] . similarly, pardi et al. demonstrated a low dose ( µg) intradermal vaccination contained with mrna-lnp complex encoding prm-e glycoproteins of zikv, which sufficiently protected non-human primates from a viral challenge [ ] . against venezuelan equine encephalitis virus (veev), two synthetic cne-encapsulated venezuelan equine encephalitis sam vaccine candidates, lav-cne (carrying the rna genome of tc- , a live-attenuated investigational vaccine strain) and iav-cne (carrying tc- viral genome with the capsid gene deleted), were designed to be capable of offering immune protection [ ] . in inoculated mice, both vaccines induced robust virus-specific neutralizing antibodies and provided protection from wild-type veev aerosol challenge [ ] . in addition, mrna-based candidate vaccines have been developed and trialed against diverse viruses such as chikungunya virus, herpes simplex virus, human metapneumovirus and parainfluenza virus, all showing desirable development prospects [ ] [ ] [ ] . it's worth noting that pepini et al. reported type i ifn, which played a critical role in antiviral responses and elicited by lnp-formulated sam vaccine, could inhibit the expression of mrna-encoded antigens in mice [ ] . in line with this, zhong et al. found that a naked zikv sam vaccine encoding the zikv prm-e induced limited and unstably variable humoral immunity in wildtype mice when compared with robust response in ifnar knockout mice [ ] . those researches suggest antiviral responses, especially type i ifn response, activated by sam vaccination might have a negative effect on sam-induced immune protection and optimization of sam construction and administration should be considered. apart from viral antigens, only very few species of bacterial and parasitic antigens have been used in mrna vaccine attempts, many of which still remain at the preclinical trial stage [ ] [ ] [ ] [ ] . a wider variety of targeted antigens will represent more important issues for the next stage of mrna vaccine development. in , maruggi et al. designed two prophylactic sam vaccines mixed with cne encoding streptolysin-o (slodm) from group a (gas) streptococci and the pilus a backbone protein (bp- a) from group b (gbs) streptococci, respectively [ ] . inoculated mice succeeded in producing a large amount of fully functional antibodies which could be significantly increased by booster, and survival rate was increased for gas and gbs infections [ ] . among infectious diseases caused by a single pathogen, the tb caused by the bacterial pathogen mycobacterium tuberculosis has been ranking first in fatality rate globally for a long time. still, there is only one vaccine licensed against human tb: mycobacterium bovis bacillus calmette-guérin (bcg), an attenuated whole-cell vaccine which has been found severe limitations in numerous clinical trials [ , ] . mva a, a tb subunit vaccine expressing single antigen ag a, had no significant improved protection in phase iib trial [ ] . the letdown reminds us that, compared with the viral infections, bacterial infections tend to have more complicated stages with diverse characteristics of molecule expression, which are virtually impossible for single antigen to cover. if mrna vaccines want to be applied further into the area of prevention and treatment of bacterial infection, more optimizations should be considered, including selection and recombination of various antigens or epitopes, periodic administration for different target antigens, and even direct addition of adjuvanted passive immune compositions (e.g., kose et al. developed a chikungunya-against mrna vaccine encoding neutralizing human monoclonal antibodies [ ] ). we are all looking forward to the first successful mrna vaccine product. optimizing the primary and secondary structure of mrna and choosing the appropriate delivery system according to the characteristics of different diseases are critical steps for better application of mrna vaccines against various pathogens. as our knowledge of tumor-specific antigens gradually deepens, people are now discussing more about the possibility of developing cancer vaccines [ , ] . tumor-associated antigens (antigens preferentially expressed in cancerous cells and usually relevant to dysregulation and abnormal behaviors of cancerous cells) and tumor-specific neo-epitopes (small peptides derived from tumor-specific somatic mutation that are exposed to the surface of cancer cells and can be recognized by t cells) are now the core targets of mrna cancer vaccines [ , ] . considering the diversity and uncertainty of the cancerogenesis, cancer vaccines that are mainly therapeutic are now aimed to stimulate cellular immunity, which would potentially act as an effective cancer treatment [ , ] . there have been some clinical trials of hopeful candidates in progress (table ) . sahin et al. pioneered the concept of "mutanome," an overall detection and map of somatic mutations in individual tumors [ ] , the obtainment of which made personalized vaccination therapy possible and attractive with the help of next-generation sequencing technology [ , ] . further, they designed procedures to develop personalized mrna mutanome vaccines from mutanome identification, neo-epitopes prediction, and selection. this allowed mrna vaccines to be unique for each patient. this strategy was firstly applied on melanoma patients with inspiring results achieved. by comparing tumor biopsies and normal blood cells via exome and rna sequencing, researchers identified and selected ten mutations related to the human lymphocyte antigen (hla) function per patient. based on those mutations, core mrna were synthesized and neo-epitope vaccines (≥eight doses) were percutaneously injected into inguinal lymph nodes [ ] . robust t cell responses against multiple neo-epitopes encoded by the vaccine were detected in all patients. with pd- blockade combination therapy, complete responses to vaccination were developed in some patients [ ] . clinical trials of similar mutanome-based mrna vaccines against triple negative breast cancer are under way [ ] . co-transfecting mrna encoding immune-regulation factors into dcs, normally by electroporation, to boost immune responses elicited by dcs-mrna cancer vaccine has been an extensively studied subject [ , , ] . trimix can promote dc activation, cd + t cell phenotype shift, and cytotoxic t lymphocyte responses in numerous animal trials [ , , ] . a joint therapy, which combined the vaccination of the dc-based mrna, encoding trimix and tumor antigens, plus ipilimumab (trimixdc-mel ipi), had been applied for advanced melanoma. it successfully induced potent tumor-associated antigen specific cd + t cell responses, demonstrating excellent therapeutic effects of the tumor-specific vaccine and immune checkpoint block agents [ ] . nevertheless, there existed undetectable response after in vitro t-cell stimulation in / patients, suggesting the necessity for a further study for mechanism of action about this issue. in addition, reinhard et al. developed chimeric antigen receptor (car)-t cells targeting regulated tight junction protein claudin (cldn ) supplemented by a liposomal cldn -encoding rna vaccine which greatly boosted car-t cell stimulation and regression of large solid tumors in mice [ ] . in view of significantly uneven distribution of various lymphocytes in the whole body and different locations and attributes of different primary tumors, it is essential to select the proper carrier and administration method for optimization of the mrna vaccine effect [ , ] . general carrier systems and delivery routes are as stated above. for example, jabulowsky et al. developed a rna-lipoplex vaccine against melanoma [rna(lip)] which was injected intravenously to deliver mrna steadily to apcs in whole body for antigen expression and presentation [ ] . this vaccine is under clinical evaluation for safety and tolerance (nct ). direct intratumoral inoculation is notably an emerging method [ , , ] . shariati et al. pioneered the pressurized intraperitoneal aerosol chemotherapy (pipac) for delivering mrna complexes and demonstrated that pipac is able to apply mrna into peritoneal cavity in mice [ ] . besides, for optimization of carrier analysis and selection, a high-throughput approach to screen proper ionizable lipid-like materials as mrna delivery vehicles were developed by anderson et al. they constructed a combinatorial library of ionizable lipid-like materials using an isocyanide-mediated three-component reaction [ ] ; the screening standard was capable of facilitating in vivo mrna delivery and providing effective and specific immune activation [ ] . the best candidate chosen, heterocyclic lipids-mrna vaccine, was further demonstrated to promote apcs maturation to stimulate potent immune responses via the intracellular stimulator of interferon genes pathway [ ] . as reviewed by weissman et al., standardized in vitro good manufacturing practice of mrna production is now accessible, while barriers still exist on synthesis of some uncommon sequences as well as salable and low-cost production for some reagents [ ] . at the same time, capability of the long-term storage of mrna vaccines with invariable activity should be emphasized. it had been reported early on that purified freeze-dried rna in trehalose could maintain the activity up to months at • c, whose stability was comparable to conventional vaccines [ ] . phase i trial of mrna-based rabies virus vaccine cv demonstrated that it could be stored as a freeze-dried preparation at - • c for years and at • c for months without obvious loss of activity [ ] . in , coolen et al. developed a poly(lactic acid)-nanoparticle-based mrna vaccine, mixed with amphipathic cationic peptides, which showed stable expression efficacy after storage at • c for up to days [ ] . although further investigations are needed to study the effects of storage of mrna complexed with vector molecules under unfrozen condition, studies has suggested the potential of mrna vaccines for cold-chain free transport and storage in the future [ ] . what makes the mrna vaccines a widely recognized form compared to conventional vaccines is the non-toxic production process, its short production time and chemical nature as ribonucleic acid, in line with safety and well-tolerance of mrna vaccines shown in multiple clinical trials [ , , , , , , ] . various adverse symptoms, however, were still detected occasionally with unclear mechanism, emphasizing the importance of safety optimization [ , ] . autoimmunity triggered by type i ifn responses are suggested to play a role in adverse physical reactions [ , ] . other problems such as edema and coagulation due to excessive extracellular rna and induction of anti-mrna antibodies were also reported [ ] ; side effects owing to the vectors or administrating routes may also exist. work on both safety assessment and investigations to mechanisms of the anti-vaccine response need to be moved forward. even though no mrna vaccine product has been approved for marketing so far, development of specialized official product guidance of mrna vaccines should be taken into consideration by medical authorities, particularly in view of the momentum and potential of this field where a remarkable number of relevant preclinical and clinical trials is active or completed. the development of tools for material screening or characterization of mrna-based complexes is of utmost importance to improve the stability and protein producing efficiency of mrna vaccines. constructing a combinatorial library of ionizable lipid-like materials is a promising strategy introduced above [ ] . in , zhang et al. used the fluorescence correlation spectroscopy (fcs) to analyze the mrna-based complex stability in buffer and biological fluid such as human serum and ascitic fluid [ ] . results have shown that strong mrna binding of linear pei would likely lead to a less efficient mrna translation while a lipid-based carrier performed well in intracellular efficient release and subsequent translation of mrna. further, they applied fcs and single particle tracking to study the decay kinetics of mrna with the half-life of mrna in biological samples measured (~ - min) [ ] . single-molecule methods have a tempting application prospect of deep optimization for the construction of mrna vaccines. theoretically, mrna can be synthesized to express almost any protein antigens, which can provide a great flexibility for antigen design. for instance, a zikv vaccine from richner et al. contains mrna encoding a mutant antigen of zikv of which an immunodominant cross reactive epitope to dengue virus is deleted to minimize the induction of cross-reactive antibodies [ ] . nowadays, the application of artificial intelligence and deep learning lead to a huge progress in gene sequence-based prediction of protein structure [ , ] . the development of big data, meanwhile, immensely advances the improvement of algorithms for tumor antigen epitope prediction [ ] . with the field of data mining further, it could become a reality to optimize existing antigens much better and design unprecedented antigens independent of natural genes. optimization and personalization of mrna vaccines will become a revolutionary milestone. in conclusion, the mrna vaccine is a versatile and powerful platform. its successful development towards clinical translation will remarkably strengthen our ability to react to and control emerging communicable diseases, and prominently enrich our arsenal of treating classical and re-emerging communicable diseases and cancers from the perspective of stimulating self-immune responses. further investigations for mechanisms of action of extracellular transportation and intracellular escape and gene expression of mrna still deserve our efforts. moreover, modularization of mrna vaccine design and production targeting different application conditions seems to be a promising strategy for the clinical usage [ ] . in the next years, several critical clinical trials of mrna vaccines are going to be completed (especially those against covid- ). more extended human clinical experience will give us a more comprehensive insight into mrna vaccine platform and various delivery systems. from increasing productive capacity of mrna and various carrier materials, to screening potential carrier molecules and adjuvants, to improving the composition and construction of vaccines, to arranging a corresponding route for administration, to optimizing the core encoding mrna sequence and to demonstrating immune mechanisms of delivery and induction, the field of mrna vaccines is still far from maturity, but its potential to be the preferred vaccine pattern has been fully shown. author contributions: conceptualization, s.x. and l.z.; writing-original draft preparation, s.x. and k.y.; writing-review and editing, r.l., s.x. and k.y.; supervision, l.z. all authors have read and agreed to the published version of the manuscript. the authors declare no any commercial or financial relationships that could be construed as a potential conflict of interest in this article. an unstable intermediate carrying information from genes to ribosomes for protein synthesis cationic liposome-mediated rna transfection direct gene transfer into mouse muscle in vivo the use of basic proteins to increase the infectivity of enterovirus ribonucleic acid foreign nucleic acids as the stimulus to make interferon a blocked structure at the terminus of mrna from cytoplasmic polyhedrosis virus translation of rabbit globin mrna introduced by liposomes into mouse lymphocytes functional messenger rnas are produced by sp in vitrotranscription of cloned cdnas induction of virus-specific cytotoxic t lymphocytes in vivo by liposome-entrapped mrna characterization of a messenger rna polynucleotide vaccine vector autologous dendritic cells transfected with prostate-specific antigen rna stimulate ctl responses against metastatic prostate tumors suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna incorporation of pseudouridine into mrna yields superior nonimmunogenic vector with increased translational capacity and biological stability direct injection of protamine-protected mrna: results of a phase vaccination trial in metastatic melanoma patients intranodal vaccination with naked antigen-encoding rna elicits potent prophylactic and therapeutic antitumoral immunity protective efficacy of in vitro synthesized, specific mrna vaccines against influenza a virus infection nonviral delivery of self-amplifying rna vaccines personalized rna mutanome vaccines mobilize poly-specific therapeutic immunity against cancer three decades of messenger rna vaccine development developing mrna-vaccine technologies mrna vaccines-a new era in vaccinology tailoring mrna vaccine to balance innate/adaptive immune response the renaissance of mrna-based cancer therapy mechanism of action of mrna-based vaccines mrna-based therapeutics-developing a new class of drugs research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases towards an effective mrna vaccine against -ncov: demonstration of virus-like particles expressed from a modified mrna cocktail in vitro transcription of long rna containing modified nucleosides generating the optimal mrna for therapy: hplc purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mrna mrna transcript therapy purification of mrna guanylyltransferase and mrna (guanine- -) methyltransferase from vaccinia virions synthesis and properties of mrnas containing the novel "anti-reverse" cap analogs -methyl( -o-methyl)gpppg and -methyl ( -deoxy)gpppg. rna novel "anti-reverse" cap analogs with superior translational properties synthesis of anti-reverse cap analogs (arcas) and their applications in mrna translation and stability phosphorothioate cap analogs increase stability and translational efficiency of rna vaccines in immature dendritic cells and induce superior immune responses in vivo phosphorothioate cap analogs stabilize mrna and increase translational efficiency in mammalian cells synthesis and properties of mrna cap analogs containing imidodiphosphate moiety-fairly mimicking natural cap structure, yet resistant to enzymatic hydrolysis cap analogs modified with , -dithiodiphosphate moiety protect mrna from decapping and enhance its translational potential uridine depletion and chemical modification increase cas mrna activity and reduce immunogenicity without hplc purification a+u-rich instability elements differentially activate - and - mrna decay coordinate regulation of mrna decay networks by gu-rich elements and celf stability analysis of chemically modified mrna using micropattern-based single-cell arrays improving mrna-based therapeutic gene delivery by expression-augmenting utrs identified by cellular library screening control of translation initiation in animals at least six nucleotides preceding the aug initiator codon enhance translation in mammalian cells insertion mutagenesis to increase secondary structure within the noncoding region of a eukaryotic mrna reduces translational efficiency codon bias and heterologous protein expression the cap and poly(a) tail function synergistically to regulate mrna translational efficiency short poly(a) tails are a conserved feature of highly expressed genes modification of antigen-encoding rna increases stability, translational efficacy, and t-cell stimulatory capacity of dendritic cells a linear plasmid for generating mrna ivt templates with extended encoded poly(a) sequences regulation of poly(a) tail and translation during the somatic cell cycle delivering the messenger: advances in technologies for therapeutic mrna delivery fotin-mleczek, m. a novel, disruptive vaccination technology. hum. vaccines immunother de koker, s. type i interferons modulate cd + t cell immunity to mrna vaccines immunostimulatory rna oligonucleotides trigger an antigen-specific cytotoxic t-cell and igg a response species-specific recognition of single-stranded rna via toll-like receptor and type i interferons interfere with the capacity of mrna lipoplex vaccines to elicit cytolytic t cell responses a facile method for the removal of dsrna contaminant from in vitro-transcribed mrna inhibition of translation by ifit family members is determined by their ability to interact selectively with the -terminal regions of cap -, cap -and ppp-mrnas type i ifn counteracts the induction of antigen-specific immune responses by lipid-based delivery of mrna vaccines -triphosphate rna is the ligand for rig-i nucleoside modifications in rna limit activation of - -oligoadenylate synthetase and increase resistance to cleavage by rnase l expression of therapeutic proteins after delivery of chemically modified mrna in mice modified foxp mrna protects against asthma through an il- -dependent mechanism a cationic nanoemulsion for the delivery of next-generation rna vaccines preclinical evaluation of trimix and antigen mrna-based antitumor therapy enhancing the t-cell stimulatory capacity of human dendritic cells by co-electroporation with cd l, cd and constitutively active tlr encoding mrna phase i clinical trial of an intranodally administered mrna-based therapeutic vaccine against hiv- infection cationic lipids activate intracellular signaling pathways toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mrna self-adjuvanted mrna vaccines induce local innate immune responses that lead to a potent and boostable adaptive immunity the challenge and prospect of mrna therapeutics landscape opportunities and challenges in the delivery of mrna-based vaccines spontaneous cellular uptake of exogenous messenger rna in vivo is nucleic acid-specific, saturable and ion dependent uptake of synthetic naked rna by skin-resident dendritic cells via macropinocytosis allows antigen expression and induction of t-cell responses in mice results of the first phase i/ii clinical vaccination trial with direct injection of mrna transfection efficiency and transgene expression kinetics of mrna delivered in naked and nanoparticle format intralymphatic mrna vaccine induces cd t-cell responses that inhibit the growth of mucosally located tumours intradermal electroporation of naked replicon rna elicits strong immune responses mrna transfection of mouse and human neural stem cell cultures effective induction of anti-melanoma immunity following genetic vaccination with synthetic mrna coding for the fusion protein egfp.trp a gene gun-mediated nonviral rna trans-splicing strategy for col a repair lentacker, i. the potential of antigen and trimix sonoporation using mrna-loaded microbubbles for ultrasound-triggered cancer immunotherapy formulation, characterization and evaluation of mrna-loaded dissolvable polymeric microneedles (rnapatch) intradermal delivery of synthetic mrna using hollow microneedles for efficient and rapid production of exogenous proteins in skin immune responses and long-term disease recurrence status after telomerase-based dendritic cell immunotherapy in patients with acute myeloid leukemia long-term survival in glioblastoma with cytomegalovirus pp -targeted vaccination self-adjuvanted mrna vaccination in advanced prostate cancer patients: a first-in-man phase i/iia study safety and immunogenicity of a mrna rabies vaccine in healthy adults: an open-label, non-randomised, prospective, first-in-human phase clinical trial phase ib evaluation of a self-adjuvanted protamine formulated mrna-based active cancer immunotherapy, bi (cv ), combined with local radiation treatment in patients with stage iv non-small cell lung cancer a phase i/iia study of the mrna-based cancer immunotherapy cv in patients with stage iiib/iv non-small cell lung cancer expression kinetics of nucleoside-modified mrna delivered in lipid nanoparticles to mice by various routes systemic rna delivery to dendritic cells exploits antiviral defence for cancer immunotherapy preclinical and clinical demonstration of immunogenicity by mrna vaccines against h n and h n influenza viruses mrna vaccines against h n and h n influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase randomized clinical trials zika virus protection by a single low-dose nucleoside-modified mrna vaccination modified mrna vaccines protect against zika virus infection successful reprogramming of cellular protein production through mrna delivered by functionalized lipid nanoparticles lipid nanoparticle-delivered chemically modified mrna restores chloride secretion in cystic fibrosis safety evaluation of lipid nanoparticle-formulated modified mrna in the sprague-dawley rat and cynomolgus monkey mrna delivery for therapeutic anti-her antibody expression in vivo cationic nanoliposomes meet mrna: efficient delivery of modified mrna using hemocompatible and stable vectors for therapeutic applications monitoring translation activity of mrna-loaded nanoparticles in mice delivery of mrna vaccines with heterocyclic lipids increases anti-tumor efficacy by sting-mediated immune cell activation poly(β-amino ester)-nanoparticle mediated transfection of retinal pigment epithelial cells in vitro and in vivo polymer-lipid nanoparticles for systemic delivery of mrna to the lungs poly(glycoamidoamine) brushes formulated nanomaterials for systemic sirna and mrna delivery in vivo induction of hiv- gag specific immune responses by cationic micelles mediated delivery of gag mrna highly efficient in vivo targeting of the pulmonary endothelium using novel modifications of polyethylenimine: an importance of charge increasing lean muscle mass in mice via nanoparticle-mediated hepatic delivery of follistatin mrna inhaled nanoformulated mrna polyplexes for protein production in lung epithelium investigation of charge ratio variation in mrna-deae-dextran polyplex delivery systems kinetics of mrna delivery and protein translation in dendritic cells using lipid-coated plga nanoparticles targeted mrna therapy for ornithine transcarbamylase deficiency cationic lipid-assisted nanoparticles for delivery of mrna cancer vaccine therapeutic mrna delivery to leukocytes towards personalized, tumour-specific, therapeutic vaccines for cancer transcutaneous delivery of dna/mrna for cancer therapeutic vaccination electroporation-enhanced delivery of nucleic acid vaccines expression of mrna electroporated into plant and animal cells gene gun delivery of mrna in situ results in efficient transgene expression and genetic immunization trial watch: dendritic cell-based interventions for cancer therapy dendritic cells interact directly with naive b lymphocytes to transfer antigen and initiate class switching in a primary t-dependent response phase ii study of autologous monocyte-derived mrna electroporated dendritic cells plus ipilimumab in patients with pretreated advanced melanoma vaccination of colorectal cancer patients with cea-loaded dendritic cells: antigen-specific t cell responses in dth skin tests dendritic cells as therapeutic vaccines against cancer breckpot, k. mrna-based dendritic cell vaccines immunogenicity of ags- dendritic cell therapy in patients treated during acute hiv infection protamine enhancement of rna uptake by cultured chick cells messenger rna-based vaccines with dual activity induce balanced tlr- dependent adaptive immune responses and provide antitumor activity liposome-based cationic adjuvant formulations (caf): past, present, and future materials for non-viral intracellular delivery of messenger rna therapeutics therapeutic rnai targeting pcsk acutely lowers plasma cholesterol in rodents and ldl cholesterol in nonhuman primates tools for translation: non-viral materials for therapeutic mrna delivery non-viral vectors for gene-based therapy polyethylenimine-based non-viral gene delivery systems mrna transfection of cervical carcinoma and mesenchymal stem cells mediated by cationic carriers toxicity of cationic lipids and cationic polymers in gene delivery continuous microfluidic assembly of biodegradable poly(beta-amino ester)/dna nanoparticles for enhanced gene delivery rapid optimization of gene delivery by parallel end-modification of poly(β-amino ester)s optimization of a degradable polymer-lipid nanoparticle for potent systemic delivery of mrna to the lung endothelium and immune cells poly(β-amino ester)-co -poly(caprolactone) terpolymers as nonviral vectors for mrna delivery in vitro and in vivo an alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector rna-based vaccines in vitro-synthesized infectious rna as an attenuated live vaccine in a flavivirus model dendritic cells pulsed with rna are potent antigen-presenting cells in vitro and in vivo vaccine: induction of antitumor immunity by human glycoprotein mrna immunization. hum neutralization of influenza a viruses by insertion of a single antibody loop into the receptor binding site induction of broad-based immunity and protective efficacy by self-amplifying mrna vaccines encoding influenza virus hemagglutinin nucleoside-modified mrna immunization elicits influenza virus hemagglutinin stalk-specific antibodies nucleoside-modified mrna vaccination partially overcomes maternal antibody inhibition of de novo immune responses in mice immunization of hiv- -infected persons with autologous dendritic cells transfected with mrna encoding hiv- gag and nef: results of a randomized, placebo-controlled clinical trial potent immune responses in rhesus macaques induced by nonviral delivery of a self-amplifying rna vaccine expressing hiv type envelope with a cationic nanoemulsion preclinical evaluation of an mrna hiv vaccine combining rationally selected antigenic sequences and adjuvant signals (hti-trimix) therapeutic vaccine in chronically hiv- -infected patients: a randomized, double-blind, placebo-controlled phase iia trial with hti-trimix phase / study to describe the safety and immunogenicity of a covid- rna vaccine candidate (bnt b ) an mrna vaccine encoding rabies virus glycoprotein induces protection against lethal infection in mice and correlates of protection in adult and newborn pigs advances in rna vaccines for preventive indications: a case study of a vaccine against rabies self-amplifying rna vaccines for venezuelan equine encephalitis virus induce robust protective immunogenicity in mice nucleoside-modified mrna encoding hsv- glycoproteins c, d, and e prevents clinical and subclinical genital herpes safety and immunogenicity of a mrna-based chikungunya vaccine in a phase dose-ranging trial phase trial of an mrna-based combination vaccine against hmpv and piv induction of an ifn-mediated antiviral response by a self-amplifying rna vaccine: implications for vaccine design immunogenicity and protection efficacy of a naked self-replicating mrna-based zika virus vaccine dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h n influenza, and toxoplasma gondii challenges with a single dose immunogenicity and protective efficacy induced by self-amplifying mrna vaccines encoding bacterial antigens neutralization of the plasmodium-encoded mif ortholog confers protective immunity against malaria infection mrna as a transformative technology for vaccine development to control infectious diseases variable virulence and efficacy of bcg vaccine strains in mice and correlation with genome polymorphisms safety and efficacy of mva a, a new tuberculosis vaccine, in infants previously vaccinated with bcg: a randomised, placebo-controlled phase b trial a lipid-encapsulated mrna encoding a potently neutralizing human monoclonal antibody protects against chikungunya infection mutant mhc class ii epitopes drive therapeutic immune responses to cancer targeting the heterogeneity of cancer with individualized neoepitope vaccines neoantigens in cancer immunotherapy tumour antigens recognized by t lymphocytes: at the core of cancer immunotherapy exploiting the mutanome for tumor vaccination targeting the tumor mutanome for personalized vaccination therapy abstract ct : mutanome engineered rna immuno-therapy (merit) for patients with triple negative breast cancer (tnbc) intratumoral delivery of trimix mrna results in t-cell activation by cross-presenting dendritic cells trimix and tumor antigen mrna electroporated dendritic cell vaccination plus ipilimumab: link between t-cell activation and clinical responses in advanced melanoma an rna vaccine drives expansion and efficacy of claudin-car-t cells against solid tumors nanoparticles for nucleic acid delivery: applications in cancer immunotherapy abstract ct : a first-in-human phase i/ii clinical trial assessing novel mrna-lipoplex nanoparticles encoding shared tumor antigens for immunotherapy of malignant melanoma abstract lb- : combinatorial treatment with intratumoral cytokine mrnas results in high frequency of tumor rejection and development of anti-tumor immunity across a range of preclinical cancer models high pressure nebulization (pipac) versus injection for the intraperitoneal administration of mrna complexes long-term storage of dna-free rna for use in vaccine studies poly(lactic acid) nanoparticles and cell-penetrating peptide potentiate mrna-based vaccine expression in dendritic cells triggering their activation fluorescence correlation spectroscopy to find the critical balance between extracellular association and intracellular dissociation of mrna complexes fluorescence-based quantification of messenger rna and plasmid dna decay kinetics in extracellular biological fluids and cell extracts alphafold at casp protein structure prediction beyond alphafold personalized vaccines for cancer immunotherapy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - kfxc w authors: mcateer, john; yildirim, inci; chahroudi, ann title: the vaccines act, deciphering vaccine hesitancy in the time of covid date: - - journal: clin infect dis doi: . /cid/ciaa sha: doc_id: cord_uid: kfxc w nan a c c e p t e d m a n u s c r i p t since the covid pandemic first hit wuhan, china in december , scientists have been racing to develop and test novel vaccines to protect against sars-cov- . the speed of scientific discovery related to covid is unprecedented. with several vaccine candidates already being tested in clinical trials, we pose the question: what will the vaccine hesitant do in the face of this pandemic? vaccine hesitancy has vexed the practice of immunization ever since cowpox was used as a vaccine for smallpox by edward jenner in . skepticism about vaccines is a timeworn concept with shifting ideologies that reflect historical events and individual belief systems reflective of different societal periods. while the term "antivax" is ascribed to contemporary vaccine skeptics, "hesitant" is often a better descriptor for the majority of parents who contemplate or request delaying vaccines for their children or deviating from the recommended immunization schedule. the term hesitant avoids polarization into "pro" versus "anti" vaccination factions. hesitancy implies, however, that minds can be swayed towards acceptance, and as pediatricians armed with the latest data, we are frustrated that these facts and figures do not always convince parents in our exam rooms and those we encounter on social media that vaccination is right for their children. how and why does this hesitancy shift from physician-guided acceptance of vaccines to their refusal? vaccine hesitancy is a complex issue. one important consideration is that vaccine hesitancy has evolved in response to the complicated but ultimately successful history of vaccine science. vaccines are a victim of their own success, turning once devastating diseases into distant memories. hesitant parents, as a result, have shifted their focus to the perceived risks of vaccination as fewer people witness the consequences of forgoing vaccines in developed countries. however, some vaccine-preventable diseases are now making a comeback, which is putting our nation's children at risk. outbreaks of vaccine-preventable diseases are occurring at an alarming rate across the country, especially in geographic pockets with poor immunization rates . in alone there were , confirmed cases of measles in the united states, the greatest number reported in this country since . these outbreaks led to the possibility that the united states would lose measles elimination status, initially awarded almost years ago. despite remarkable advances in vaccine science, vaccine hesitancy has now become a recognized public health threat with potentially disastrous complications. anti-immunization sentiment is at an all-time high and the medical community has recognized the need to address it as a public health emergency, with research, action, and advocacy. the nature of hesitancy directly confronts our fundamental belief system as pediatric scientists, which is based on disease prevention and anticipatory guidance. vaccine hesitancy is one of the most challenging controversies we have faced in our history. this problem is personal to all of us. pediatricians and vaccinologists have been disbelieved and attacked. vaccine hesitancy has the potential to harm physician-patient relationships. it is difficult to comprehend that some of the greatest triumphs of medical science are being eroded by misinformation and distrust. to address vaccine hesitancy, we need to reframe our approach and focus on understanding the root causes of this complicated problem. on the surface, the idea of hesitancy is perplexing given the overwhelming scientific evidence for the safety and efficacy of vaccines. vaccine hesitancy is not a superficial issue with a universal solution, however. rather, it is a multifaceted, deeply complex construct that may be rooted in the moral composition that guides our daily decision making. research has shown that several distinct values are associated with vaccine hesitancyparticularly purity, liberty, and a c c e p t e d m a n u s c r i p t anti-authority . combined with the historical amnesia of the consequences of vaccinepreventable diseases, vaccine hesitancy becomes easier to understand as a belief system grounded in certain moral values. by considering these values, rather than relying solely on vaccine safety and efficacy data, some researchers believe we may be able to more effectively address vaccine hesitancy. although each family is unique, there are likely common drivers for vaccine hesitancy and these, we submit, are being inadequately addressed. hesitant families frequently express concern about vaccine safety, but even this issue has multiple layers, including fears regarding potential links to autism (now thoroughly refuted ), learning difficulties, and chronic illnesses, as well as a perceived lack of safety testing prior to approval for use . a famous quote from bernard shaw applies here: "the greatest problem in communication is the illusion that it has been accomplished." properly identifying and completely responding to fears regarding vaccines within the confines of a -minute well visit is next to impossible. furthermore, how we discuss and communicate about vaccines, as much as the evidence-based facts provided, may be very important. we need a systematic assessment of factors affecting vaccine uptake on a continuing basis to develop the most effective and efficient communication strategies. as a medical community, we must investigate the determinants of vaccine hesitancy and the continuously evolving challenges it entails in order to tailor evidence-based strategies to overcome the problem with sustainable interventions. in a step forward in the fight against vaccine misinformation, facebook announced on september th , that user searches for vaccine-related content will be directed to either the united states centers for disease control and prevention (cdc) or the world health organization (who) websites for accurate information regarding vaccines. in his statement of support , the who director-general dr. tedros ghebreyesus asserted that "these online efforts must be matched by tangible steps by governments and the health sector to promote trust in vaccination and respond to the needs and concerns of parents." in order to adequately respond to these needs and concerns, which differ depending on the cultural, societal, and personal beliefs of a particular region, the who recommends that each country take steps to develop an understanding of vaccine hesitancy at a local level on an ongoing basis . unfortunately, such a surveillance program in the united states (and in many other countries, for that matter) does not exist. as such, much of the information we have about vaccine hesitancy is retrospective and anecdotal. the absence of federal investment in understanding vaccine hesitancy was clear during the measles outbreaks. as cases increased nationally, cdc officials tracked cases and exposures across state lines. however, insight into the vulnerability of exposed communities was varied, and depended solely on the quality and attention state governments paid to evaluating mmr immunization rates in those communities. new york state, for example, quickly realized reluctance to immunize was centered in specific orthodox jewish communities in brooklyn and rockland counties and was rooted in concerns about potential pork products in vaccines. with local governments, the state created public awareness campaigns led by rabbis urging vaccination. other areas, absent this granular understanding, could simply react to burgeoning cases and missed opportunities that may have prevented the spread of cases to states. in recognition of these missed opportunities and in response to declining immunization rates and increasing national skepticism on the safety of vaccines, congress has introduced bipartisan legislation to expand research into vaccine hesitancy. spearheaded by congresswoman and pediatrician dr. kim schrier, the vaccine awareness campaign to m a n u s c r i p t champion immunization nationally and enhance safety (vaccines) act (h.r. ) would provide federal funding for vaccine hesitancy surveillance and authorization of a public awareness campaign to increase public confidence in vaccines . the bipartisan bill introduced in may will amend the public health service act of to support a rigorous scientific approach to learn more about the factors that drive vaccine hesitancy and will fund national surveillance of hesitant regions. while commendable, it should be noted that the requested resources in the current version of the vaccines act are inadequate to fund serious communications campaigns, given that some of the areas with the highest hesitancy also have the most expensive media markets. the bill does include a vital provision to support awareness campaigns tailored to specific communities that could prove to be an essential tool in the fight to protect children from vaccine-preventable diseases. signing this bill into law will provide a framework to better understand and respond to vaccine hesitancy in the united states. current research suggests that the drivers of vaccine hesitancy depend heavily on personal experiences and belief systems , . having an adequately federally-funded surveillance program will permit analysis of contextual influences that promote and perpetuate reluctance at the local level. high-risk populations can be identified and specifically targeted based on their societal and cultural belief systems. awareness campaigns can be tailored to specific locales to address identified concerns regarding vaccines. studying all these factors will expand our armamentarium in preventing the rise of vaccine-preventable diseases. the vaccines act is currently in the first stage of the legislative process. it has been assigned to the house energy and commerce committee where the chairs will determine if the bill will pass to the house floor . the odds of any bill becoming law at this stage are collectively low (around % since ) and even worse without strong public support . it is thus of paramount importance for the scientific community to come together in support of this legislation. vaccine hesitancy remains a persistent global threat and is a concept we are only beginning to understand. we have been lagging in our response, in part because the epidemic of vaccine hesitancy has personally challenged our motivations and values as doctors, scientists, advocates, and often, family members and friends. while we wonder if the covid pandemic will begin to change minds regarding the overwhelming value of immunization, we fear that as time elapses and memories fade, a sars-cov- vaccine may join the ranks of mmr in eliciting hesitancy from some groups. the bipartisan vaccines act is an important step in supporting evidence-based research into vaccine hesitancy. it is a call for the scientific community to shift our focus towards understanding the underlying causes of this epidemic. as this bill goes through the legislative process, we strongly urge the scientific and medical communities to contact their representatives to voice support for a fully resourced vaccines act. we believe that all children and their families deserve it. none of the authors has any potential conflicts of interest to disclose. vaccine hesitancy, history, and human nature: the stanley a. plotkin lecture association between vaccine refusal and vaccine-preventable diseases in the united states: a review of measles and pertussis increase in measles cases: united states association of moral values with vaccine hesitancy measles, mumps, rubella vaccination and autism: a nationwide cohort study countering antivaccination attitudes world health organization. immunization, vaccines and biologicals-improving vaccine demand and addressing hesitancy statistics and historical comparison a c c e p t e d m a n u s c r i p t key: cord- -mejd blb authors: lewnard, joseph a; reingold, arthur l title: emerging challenges and opportunities in infectious disease epidemiology date: - - journal: am j epidemiol doi: . /aje/kwy sha: doc_id: cord_uid: mejd blb much of the intellectual tradition of modern epidemiology stems from efforts to understand and combat chronic diseases persisting through the th century epidemiologic transition of countries such as the united states and united kingdom. after decades of relative obscurity, infectious disease epidemiology has undergone an intellectual rebirth in recent years amid increasing recognition of the threat posed by both new and familiar pathogens. here, we review the emerging coalescence of infectious disease epidemiology around a core set of study designs and statistical methods bearing little resemblance to the chronic disease epidemiology toolkit. we offer our outlook on challenges and opportunities facing the field, including the integration of novel molecular and digital information sources into disease surveillance, the assimilation of such data into models of pathogen spread, and the increasing contribution of models to public health practice. we next consider emerging paradigms in causal inference for infectious diseases, ranging from approaches to evaluating vaccines and antimicrobial therapies to the task of ascribing clinical syndromes to etiologic microorganisms, an age-old problem transformed by our increasing ability to characterize human-associated microbiota. these areas represent an increasingly important component of epidemiology training programs for future generations of researchers and practitioners. initially submitted september , ; accepted for publication november , . much of the intellectual tradition of modern epidemiology stems from efforts to understand and combat chronic diseases persisting through the th century epidemiologic transition of countries such as the united states and united kingdom. after decades of relative obscurity, infectious disease epidemiology has undergone an intellectual rebirth in recent years amid increasing recognition of the threat posed by both new and familiar pathogens. here, we review the emerging coalescence of infectious disease epidemiology around a core set of study designs and statistical methods bearing little resemblance to the chronic disease epidemiology toolkit. we offer our outlook on challenges and opportunities facing the field, including the integration of novel molecular and digital information sources into disease surveillance, the assimilation of such data into models of pathogen spread, and the increasing contribution of models to public health practice. we next consider emerging paradigms in causal inference for infectious diseases, ranging from approaches to evaluating vaccines and antimicrobial therapies to the task of ascribing clinical syndromes to etiologic microorganisms, an age-old problem transformed by our increasing ability to characterize human-associated microbiota. these areas represent an increasingly important component of epidemiology training programs for future generations of researchers and practitioners. infectious diseases; methods; modeling; surveillance abbreviation: ebov, ebola virus. the priority afforded to infectious diseases within epidemiologic research has been fluid over the past years or longer. despite the lasting prominence of early investigations into measles, cholera, plague, typhoid fever, malaria, and yellow fever ( ) ( ) ( ) ( ) ( ) ( ) , the intellectual tradition of modern epidemiology stems largely from studies of chronic diseases dating to the post-world war ii era, when such conditions came to surpass infectious diseases in morbidity and mortality in highincome countries amid improvements in living conditions and the introduction of numerous antibiotics and vaccines. this epidemiologic transition co-occurred with a shift in focus for epidemiologic research (and training programs) toward the multifactorial etiology of chronic conditions ( ) . in parallel, early th-century work on the "dependent happenings" of communicable diseases ( ) ( ) ( ) ( ) yielded to the development of today's core biostatistical methods for chronic diseases, premised on the independence of outcomes among subjects ( ) ( ) ( ) . since the late th century, the emergence of human immunodeficiency virus and acquired immunodeficiency syndrome and other infections has renewed interest in infectious diseases and their health, economic, and security implications ( ) . outbreaks of severe acute respiratory syndrome, pandemic influenza a h n , ebola virus (ebov), and zika virus have prompted international responses, whereas influenza a h n and h n , lassa fever virus, nipah virus, and middle east respiratory syndrome coronavirus, among other agents, have been a source of regional concern. the incidence and endemic range of "neglected" infections, including dengue and cholera ( , ) , have expanded, and antimicrobial resistance has threatened to derail the control of tuberculosis, typhoid, malaria, gonorrhea, yaws, and invasive bacterial infections ( ) ( ) ( ) ( ) ( ) ( ) . although highly effective vaccines are available, measles and yellow fever have resurged on multiple continents, due to gaps in vaccine coverage ( , ) , while short-lived vaccine-induced protection has facilitated unexpected resurgences in diseases once on the path to elimination, such as pertussis and mumps ( , ) . after decades of relative obscurity in the mid- th century, infectious disease epidemiology has experienced an intellectual rebirth in response to disease emergence. repopulation of this field by scientists trained not only in clinical medicine but ecology, demography, and quantitative sciences has led to the adoption of methods scarcely addressed in traditional public health training programs. cluster-randomized trial designs, for example, have become commonplace for evaluating infectious disease interventions, quantifying indirect effects resulting from contagion ( ) . classical models of ecological dynamics have been adapted to address the transmission and control of infectious agents, while our expanding ability to integrate such models with epidemiologic data through bayesian statistics has enhanced their relevance to policymaking ( ) ( ) ( ) . most recently, sequencing and phylogenetic analysis have afforded an unprecedented view into the population structure and dynamics of pathogens ( , ) . here, we review developments in infectious disease epidemiology together with their implications for research and practice, and for the training of future epidemiologists. we first consider the role of epidemiologic surveillance in the context of pathogen emergence and the integration of surveillance data into quantitative studies of transmission. next, we discuss challenges in causal inference, including the evaluation of public health interventions against emerging pathogens and the difficulties of attributing clinical syndromes to microbial agents. surveillance justifiably has been seen as a core public health function, with its important role articulated by langmuir in ( ) . in the united states and elsewhere, surveillance for diverse infectious (and noninfectious) diseases has typically relied on an essentially passive system of reporting by healthcare providers or laboratories, often mandated by public health laws. although such passive systems of disease reporting have proven invaluable, their limitations, such as incomplete, often inconsistent detection of cases and delayed detection of outbreaks, are well documented ( ) . as a result, active surveillance systems that do not depend on providers and laboratories to report have been developed and promoted. the centers for disease control and prevention-funded emerging infections program and its components (e.g., foodnet, abcs), initiated in , is notable example of such a system, as are international versions promoted through the centers for disease control and prevention's global health security initiative ( ) . such systems, while expensive to develop and maintain, are of great value, especially when they collect biological specimens (e.g., isolates of bacterial and viral pathogens) for typing and support analytic epidemiologic studies (e.g., casecontrol studies of vaccine effectiveness ( ) ). nevertheless, these systems, too, may have limitations, particularly in their ability to detect in a timely fashion outbreaks caused by novel microbial agents, prompting interest in alternative methods. in this article, we consider proposed alternatives, highlighting their benefits and challenges. beyond efforts to quantify the incidence of infectious diseases of known etiology, disease surveillance has been advocated as a means of mitigating the threat posed by novel pathogens. large-scale efforts to identify pathogens with the potential to spill over from animals to humans have received notable investment, for instance from the us agency for international development emerging pandemic threats program ( ) . more recently, metagenomic sequencing has enabled the number of known viruses to be multiplied in such studies ( ) . the aim of those working on the global virome project, launched in , to characterize within years all . million viruses thought to exist ( ) . however, the pathway for translating data sets produced by such activities into actionable threat-reduction programs remains unclear. that only approximately viruses are known to infect humans (and an even smaller handful to cause major epidemics) may constrain the value of large-scale virus discovery for identifying high-risk pathogens, as well as viral determinants of pathogenic potential ( ) . spillover events causing recent epidemics-including h n in mexico, middle east respiratory syndrome in saudi arabia, and ebov in west africa-have been poorly predicted by factors long believed to drive disease emergence ( ) . accordingly, interest in expanding our catalogue of potential pathogens should be weighed against our persisting need to enhance the detection and control of outbreaks of known pathogens ( ) . for instance, ebov epidemics in the democratic republic of the congo in took weeks to be identified, with dozens of suspected cases having already accumulated ( , ) . serological studies have received increasing enthusiasm for monitoring emerging pathogens of significance to humans ( , ) . the prevalence of antibodies indicating previous exposure may provide valuable information about the frequency of animal-human spillover events and the potential for person-toperson spread, overcoming reporting biases that favor detection of large outbreaks under traditional surveillance. moreover, the low cost of multiplex assays makes integrated surveillance of multiple pathogens plausible. although serosurveys have bolstered recent efforts to understand the geographic range and clinical spectrum of ebov and zika virus infections ( , ) , the enhancement of dengue hemorrhagic fever risk by prior exposure ( ) , and the role of immunologic history in influenza susceptibility and vaccine response ( ) , there remain few examples of public health programs undertaking serological studies for routine surveillance, at least in civilian populations ( ) . outside of laboratory-based surveillance, the increasing availability of passively collected "big data" on the health and behaviors of individuals has prompted enthusiasm about enhancing disease surveillance through alternative data streams. initiatives such as the promed-mail network and healthmap ( , ) compile and disseminate news about outbreaks from media and other sources, aiming to trigger investigation by public health organizations. data such as emergency department visits, medication sales, online search queries, and social media postings have also been suggested as real-time indicators of outbreak activity, although their integration into public health responses remains a subject of debate ( ) ( ) ( ) . the need to overcome reporting biases is a central challenge, because observations may be too nonspecific to distinguish between meaningful and spurious signals in settings with high technological capacity while also being insensitive to even high-risk events in resource-poor settings ( , ) . although nontraditional data sources have, in some applications, supported inferences about epidemic dynamics ( ) , limited information about cases from such sources remains a barrier. for instance, models fitted from news reports of recent measles and mumps outbreaks have yielded considerable underestimates of vaccine coverage ( ) ( ) ( ) , underscoring the importance of field investigations. forecasting the incidence of diseases has been a more successful application of these emerging data streams and dataanalytic approaches. although the acknowledged failure of google flu trends-a prediction approach based on internet search behavior-yielded important lessons about nonmechanistic forecasts, approaches based on machine learning and crowdsourced human judgment have provided the most accurate within-season predictions of us influenza activity in recent comparisons ( ) ( ) ( ) . given expanding interest in forecasting among researchers, funding agencies, and other stakeholders, there is a clear and compelling need to evaluate whether such forecasts can enhance the success and efficiency of public health response efforts. mathematical modeling as a means to understanding infectious disease spread dates to studies by sir ronald ross ( ) . although the use of models to connect data such as age of infection to transmission dynamics of endemic infections has longstanding precedent ( , ) , assimilation of outbreak data for near-term assessments of control priorities is a comparatively recent phenomenon. integration of modeling with the public health response to epidemics of bovine spongiform encephalopathy and foot-and-mouth disease in the united kingdom and the severe acute respiratory syndrome epidemic ( - ) has led to expectations for near real-time modeling studies during major outbreaks. in recent experience, models of the spread of pandemic influenza a h n ( ), cholera ( ), middle east respiratory syndrome ( ), ebov ( , ) , chikungunya virus ( ), zika virus ( , ) , yellow fever ( ) , and plague ( ) have all been published within weeks of the respective outbreak notifications. although the circumstances of particular epidemics dictate what data may be available and pertinent, methods for fitting models to data have generally focused on exponential growth rates in cases ( ) or the distribution of the serial interval ( ) . methods based on the latter class of data offer the advantage of illustrating real-time changes in reproductive numbers ( ) ; however, the requisite information from patient line lists is seldom available. reliance instead on ecological data exposes models to numerous vulnerabilities, notably the inability to discern individual risk factors (and thus the population meaningfully at risk). these shortcomings may prevent models from predicting reductions in transmission before depletion of the susceptible population. a challenge thus lies ahead in determining the role of models in outbreak response and the best practices for communicating modeling results. although the ability of models to evaluate prophylactic strategies may be considered a benefit, recommendations to act against remote future risks have sometimes triggered resistance among stakeholders ( ) . during the west african ebov epidemic, for example, attention to worst-case model-based projections prompted some to question the reliability of the models ( ), reflecting an important discrepancy between public understanding of modeling as a forecasting tool and the intended uses of models for scenario-based comparisons ( , ) . this use of modeling has been better understood in attempts to communicate the impact of interventions after the fact ( ). the ease of sequencing pathogen genomes has afforded a new view into transmission during outbreaks. use of sequence data to identify transmission clusters in the presence of unsure epidemiologic links dates to the early years of the human immunodeficiency virus and acquired immunodeficiency syndrome epidemic ( ) . in recent years, sequencing has aided efforts to track the sources of unexplained epidemics of cholera in haiti ( ) and ebov in west africa ( ) , and has shown increasing utility for reconstructing the geographic spread of pathogens ( , ) . a particular advantage of phylogenetic analysis is the possibility of estimating unobserved epidemiologic quantities, such as the reporting fraction ( ) and reproductive numbers for subcritical transmission ( ) , which remain difficult to assess from traditional case-notification data. beyond reconstructing the demographic history of pathogen lineages, recent years have seen progress toward joint analysis of epidemiologic and sequencing data ( ) . such "phylodynamic" approaches have shown particular relevance for emerging infections, including distinguishing the role of repeated introductions and subsequent local transmission ( ) ( ) ( ) ( ) . whereas most applications have been tailored to specific data sets and assumptions, the development of generalized methods for joint inference of epidemiologic and phylogenetic parameters remains a priority ( ) to support real-time analysis. the ability to rapidly develop and deploy countermeasures to mitigate the threat posed by emerging infections has received increasing recognition as a component of public health preparedness. however, outbreaks are difficult environments in which to evaluate interventions. during the west african ebov epidemic, the feasibility of a new paradigm for development and evaluation of interventions in emergencies was demonstrated by accelerated vaccine safety, immunogenicity, and efficacy studies ( ) . the coalition for epidemic preparedness innovations was established in , with an initial focus on vaccines against nipah virus, middle east respiratory syndrome coronavirus, and lassa fever virus, in addition to adaptable vaccine platforms for novel threats ( ) . lessons learned in ebov vaccine trials will have an influential bearing on evaluations during future emergencies. despite efforts to accelerate evaluation of candidate vaccines, incidence had reached low levels by the time phase iii efficacy trials were ready to begin, posing a threat to their statistical power: a planned trial in liberia was canceled due to declining transmission ( ) , and no cases of disease occurred in a second trial in sierra leone ( ), preventing efficacy assessments. in a stepped-wedge trial in guinea, clusters of primary and secondary contacts of ebov disease cases were randomly assigned to immediate or delayed vaccination; no cases were reported among vaccine recipients during the trial ( ) or in subsequent field deployments of the vaccine, supporting a conclusion of near % vaccine efficacy. debates surrounding design of these trials highlight methodological questions requiring additional attention. in the guinean trial, a "ring" vaccination scheme helped maximize power by enrolling contacts of known cases ( ) . however, the choice of individual-or cluster-level randomization within rings was debated. because members of a vaccinated cluster are exposed to direct protection through vaccination and indirect protection due to reduced transmission within their clusters ( ), cluster-randomized trials have weaker statistical power than individually randomized trials of the same size ( ) . moreover, the direct effect measured in individually randomized studies may be a preferred, transportable efficacy measure ( ) . uses of simulation helped in planning vaccine trials tailored to the real-world circumstances of the ebov outbreak ( ) and enabled trialists to compare alternative designs in terms of ethical mandates ( ) . simulation-guided design further presents the opportunity for applying adaptive trial methods ( ) in the context of infectious disease outbreaks, where dynamic trends in incidence may highlight the benefits of such approaches. in addition to efficacy trials for new interventions, observational studies are needed to assess licensed interventions against evolving and re-emerging pathogens. most commonly applied in evaluations of influenza vaccines, test-negative designs have become popular in routine ( ) and exploratory ( ) studies of vaccine effectiveness. by measuring vaccine effectiveness from the exposure odds ratio of vaccination among individuals seeking care who test positive or negative for a pathogen of interest, this design seeks to overcome associations of health-care seeking with vaccination status ( ) . however, it is uncertain whether health-care seeking and other sources of confounding are appropriately controlled for, and whether measures accurately capture vaccine direct effects ( , ) . uncertainty about the validity of estimates that routinely inform vaccine policy-making demonstrates the need for formal evaluations of such studies and strategies to reduce bias. time series analyses of public health surveillance data provide another approach to measuring the real-world impact of vaccination on disease incidence, with the advantage of identifying the overall effect of a vaccination program resulting from direct and indirect protection ( ) . although the ecological nature of such designs permits the introduction of biases from changes in diagnostic practices or health-care seeking, such studies nonetheless have offered important insights where other approaches failed. limited reductions in influenza-related deaths among elderly persons amid increases in influenza vaccine coverage during the s provided an important indication that the "healthy vaccinee" effect accounted for astonishing and implausible protection against all-cause mortality among elderly influenza vaccine recipients in cohort and case-control studies ( ) ( ) ( ) . newer methods continue to improve public health inferences obtained from time-series data. in a recent evaluation of invasive pneumococcal disease incidence, trends expected under continued use of -valent pneumococcal conjugate vaccine provided a counterfactual condition for measuring the impact of the switch to a -valent vaccine targeting emerging serotypes ( ) . bayesian averaging of models encoding differing pre-and postvaccination trends and change points provides a generalized strategy for defining such counterfactual comparisons ( ) . other signals of transmission intensity in surveillance data, such as age of infection and sub-or multiannual periodicity, may provide additional insights while reducing sensitivity to fluctuations in reporting effort ( , ) . the re-emergence of pathogens against which vaccines are widely deployed, such as varicella, pertussis, and mumps in the united states, poses additional challenges for conducting vaccine effectiveness studies. situational factors may undermine researchers' ability to establish the extent to which cases owe to primary or secondary vaccine failure in all or certain vaccine recipients, and whether emerging pathogen lineages are escaping vaccine-driven immune pressure. for instance, high compliance with vaccine schedules may limit variation in individuals' vaccination status and exposures, necessitating large samples to detect factors influencing vaccine performance ( ) . because re-emergence most likely reflects the expansion of or several pathogen clades, limited pathogen diversity may hinder the application of conventional approaches to identifying microbial determinants of vaccine escape ( , ) . novel methods to distinguish null from vaccine-driven mutations in antigencoding regions ( , ) may streamline efforts to identify vaccine escape, while mathematical modeling provides a basis for comparing candidate hypotheses with observations ( , ) . because vaccine studies are typically powered for primary clinical endpoints, long-term observational studies are needed to monitor for rare vaccine-attributable adverse events. such studies have been crucial to identifying safety concerns such as intussusception after rotavirus vaccination ( ) and to refuting spurious links, such as autism onset after measles-mumpsrubella vaccination ( ) . however, unique challenges arise in vaccine safety studies; at the individual level, vaccination and adverse-event detection may be confounded due to health-careseeking behavior, whereas at the population level, age-related confounding may occur when vaccine recommendations are based on the individual's age. ecological designs taking advantage of natural experiments have proven useful in numerous studies of vaccine safety ( , ) but inconclusive for certain classes of rare events, including those that also result from the vaccine-targeted infection ( , ) . recent years have seen growing interest in case-only methods offering the ability to reduce or rule out individual-level sources of confounding. case-crossover methods are common among such approaches ( , ) and resemble matched case-control studies by sampling "control" periods from the person-time contributed by case individuals before an adverse event. self-controlled case-series methods similarly benefit from the use of cases as their own controls, following a cohort logic in estimating the relative incidence of adverse events after vaccination within specified risk periods ( ) ; with adequate sample size, researchers may be able to use such analyses to eliminate or greatly reduce potential time-or age-related confounding. in response to the growing threat posed by antimicrobial resistance, the world health organization and national governments have prioritized bringing novel antimicrobial drugs to market ( ) . these plans will necessitate phase iii trials in which the efficacy of new therapeutic agents is addressed and possibly phase iv studies, in which the optimal use of new and existing drugs, either singly or in combination, can be determined. whereas patients traditionally have been enrolled in antimicrobial treatment studies on the basis of target bacterial species infections or clinical syndromes, it is uncertain within what strata such trials may yield transportable inferences. rather than merely the infecting pathogen's baseline resistance or susceptibility phenotype, strata may be defined by factors such as pathogen lineage, mutational barriers to resistance development ( ) , and presence of horizontally transferable resistance elements in cocolonizing agents or environmental sources ( ) . stratification based on interpatient and even intrapatient tumor heterogeneity is an emerging feature of cancer therapy trials and may provide a template for such designs ( ) . in addition to clinical endpoints, carriage of susceptible and resistant bacteria, including commensal agents not purposefully targeted by treatment, can inform the impact of treatment on resistance selection in targeted and bystander species ( ) . whereas between-group differences in the absolute prevalence of colonization with resistant organisms ( ) are tested for routinely, stratified measurements (see the reports of shrag et al. ( ) and feikin et al. ( ) , for example) of the effect of treatment on acquisition and clearance of susceptible and resistant pathogens are more informative of the underlying biology ( ) and may detect signals of selection masked by simpler betweengroup comparisons ( , ) . studies are also needed to address optimal deployment of new and existing antimicrobial drugs in clinical practice. the tradeoff between maximizing a drug's impact and minimizing resistance selection has led policymakers to ration certain new drugs as last-resort treatments. however, in recent experience, such decisions have ignited ethical debates ( ) . coupling mathematical modeling with field-based studies has proven useful for understanding the effectiveness of antimicrobial use policies, as highlighted in recent evaluations of the risk of resistance under population-wide access of the tuberculosis drug bedaquiline ( , ) and antimicrobial cycling to limit resistance selection in hospital settings ( , ) . whereas certain efforts we have discussed have been made to identify novel microorganisms able to cause human infection ( , ) , most epidemics caused by emerging pathogens have been recognized first by clusters of anomalous syndromes-such as cardiopulmonary syndrome caused by new world hantaviruses, severe acute respiratory syndrome caused by a coronavirus, and congenital abnormalities caused by zika virus -before the role or even existence of the etiologic microorganism had been characterized. the problem of ascribing a clinical syndrome to an etiologic agent is among the oldest in epidemiology, dating at least to the th century, when robert koch laid out criteria for such inference (i.e., koch's, or more correctly, henle-koch's, postulates ( ) ). however, these postulates have long been recognized as inadequate, particularly for illnesses caused by viruses, and thus of largely historical interest ( ) ; for instance, the notion that a pathogen should be absent from healthy individuals is incompatible with the prominence of carriage and asymptomatic infection in the natural history of numerous pathogens. a growing appreciation of the complexity of the human microbiome, and the likelihood that intricate mixtures of microorganisms at diverse body sites may be either the cause or consequence of both detrimental and beneficial physiological states, has further highlighted the difficulty of linking a given health outcome to infection by a single microorganism. the now-recognized critically important role of persistent infection and the inflammation it can produce in diverse cancers and possibly other chronic diseases has further diminished the relevance of koch's postulates and the age-old distinction between infectious and chronic diseases, as illustrated in the well-known example of human papillomavirus causing cervical, anal, and oral cancers ( ) . the best causal understanding of such relationships has come from randomized trials demonstrating that infection-preventing interventions are efficacious against downstream chronic illness, such as peptic ulcers due to helicobacter pylori and chronic wheeze due to respiratory syncytial virus ( , ) . natural experiments following the same intuition have provided additional evidence of such relationships, such as measles-induced immunosuppression ( ) , malnutrition and stunting due to enteric infection ( ) , and complex or chronic otitis media due to tissue damage from acute early-life disease ( ) . such relationships have proven difficult to probe in the absence of a randomized or natural experiment, because of the likelihood that confounding factors influence individuals' risk for initial infection as well as chronic sequelae. new paradigms for ascribing an etiologic role to microorganisms and resulting host responses are clearly needed and may prove important in efforts to quantify the health impacts of infectious disease interventions. the recognition in the s and s that infectious diseases were not, in fact, disappearing as important causes of morbidity and mortality in human populations has been followed by renewed interest in these conditions, especially in the emergence or re-emergence of diverse infectious diseases and in the role of infection in various chronic diseases. at the same time, advances in epidemiologic and statistical methods, together with the growing availability of data from diverse sources, have provided new tools and approaches for studying infectious diseases. the infectious disease epidemiologists of the future will need a solid grounding in the biology of infection and the host immune response, as well as training in the increasingly sophisticated approaches to causal inference; the manipulation and analysis of large-scale data sets, including pathogen genome sequences; and mathematical modeling, together with the behavioral and social determinants of health. integration of these elements into epidemiology training programs (e.g., through coursework in bayesian statistics and phylogenetics) represents an increasingly important consideration for academic departments. observations made during the epidemic of measles on the faroe islands in the year on the mode of communication of cholera the experimental production of plague epidemics among animals typhoid fever: its nature, mode of spreading, and prevention the prevention of malaria the etiology of yellow fever-a preliminary note life course epidemiology some a priori pathometric equations a contribution to the mathematical theory of epidemics on the statistical measure of infectiousness epidemics and crowd-diseases: measles. by major greenwood regression models and life-tables the controlled therapeutic trial an analysis of transformations factors in the emergence of infectious diseases the global burden of dengue: an analysis from the global burden of disease study updated global burden of cholera in endemic countries evolution of extensively drug-resistant tuberculosis over four decades: whole genome sequencing and dating analysis of mycobacterium tuberculosis isolates from kwazulu-natal emergence of an extensively drug-resistant salmonella enterica serovar typhi clone harboring a promiscuous plasmid encoding resistance to fluoroquinolones and third-generation cephalosporins spread of artemisinin resistance in plasmodium falciparum malaria failure of dual antimicrobial therapy in treatment of gonorrhea re-emergence of yaws after single mass azithromycin treatment followed by targeted treatment: a longitudinal study trends in antimicrobial resistance in bloodstream infection isolates at a large urban hospital in malawi ( - ): a surveillance study ongoing outbreak with well over , measles cases in italy from january to end august -what is making elimination so difficult? yellow fever in angola and beyond-the problem of vaccine supply and demand vaccine waning and mumps reemergence in the united states the impact of past vaccination coverage and immunity on pertussis resurgence direct and indirect effects in vaccine efficacy and effectiveness population biology of infectious diseases: part i population biology of infectious diseases: part ii modeling infectious disease dynamics in the complex landscape of global health beast : a software platform for bayesian evolutionary analysis supersize me: how whole-genome sequencing and big data are transforming epidemiology the surveillance of communicable diseases of national importance completeness of notifiable infectious disease reporting in the united states: an analytical literature review us centers for disease control and prevention and its partners' contributions to global health security effectiveness of seven-valent pneumococcal conjugate vaccine against invasive pneumococcal disease: a matched case-control study us agency for international development uncovering earth's virome the global virome project pandemics: spend on surveillance, not prediction global trends in emerging infectious diseases towards a genomics-informed, realtime, global pathogen surveillance system ebola virus disease: , deaths later, how far have we come? lancet world health organization regional office for africa. ebola virus disease, democratic republic of the congo: external situation report use of serological surveys to generate key insights into the changing global landscape of infectious disease mapping a filoviral serologic footprint in the democratic republic of the congo: who goes there? zika virus: history of a newly emerging arbovirus antibodydependent enhancement of severe dengue disease in humans immune history and influenza virus susceptibility description and utilization of the united states department of defense serum repository: a review of published studies promed-mail: an early warning system for emerging diseases surveillance sans frontières: internet-based emerging infectious disease intelligence and the healthmap project digital disease detection-harnessing the web for public health surveillance syndromic surveillance in public health practice if syndromic surveillance is the answer, what is the question? increased antiviral medication sales before the - influenza season productive disruption: opportunities and challenges for innovation in infectious disease surveillance social and news media enable estimation of epidemiological patterns early in the haitian cholera outbreak vaccination compliance and the us measles epidemic substandard vaccination compliance and the measles outbreak vaccine compliance and the arkansas mumps outbreak big data. the parable of google flu: traps in big data analysis flexible modeling of epidemics with an empirical bayes framework a human judgment approach to epidemiological forecasting the age distribution of whooping cough, measles, chicken pox, scarlet fever, and diphtheria in various areas in the united states derivation of rates from summation data by the catalytic curve the foot-andmouth epidemic in great britain: pattern of spread and impact of interventions foot-and-mouth disease under control in the uk transmission intensity and impact of control policies on the foot and mouth epidemic in great britain transmission dynamics and epidemiology of bse in british cattle transmission dynamics and control of severe acute respiratory syndrome transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions pandemic potential of a strain of influenza a (h n ): early findings cholera epidemic in haiti, : using a transmission model to explain spatial spread of disease and identify optimal control interventions interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk early transmission dynamics of ebola virus disease (evd) outbreak of ebola virus disease in the democratic republic of the congo local and regional spread of chikungunya fever in the americas assessing the global threat from zika virus countering the zika epidemic in latin america fractional dosing of yellow fever vaccine to extend supply: a modelling study dynamics of the pneumonic plague epidemic in madagascar avoidable errors in the modelling of outbreaks of emerging pathogens, with special reference to ebola how generation intervals shape the relationship between growth rates and reproductive numbers different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures vets asked valuable questions about foot-andmouth measures models overestimate ebola cases opinion: mathematical models: a key tool for outbreak response ebola: models do more than forecast measuring the impact of ebola control measures in sierra leone molecular epidemiology of hiv transmission in a dental practice the origin of the haitian cholera outbreak strain genomic surveillance elucidates ebola virus origin and transmission during the outbreak virus genomes reveal factors that spread and sustained the ebola epidemic zika virus evolution and spread in the americas epidemiological and viral genomic sequence analysis of the ebola outbreak reveals clustered transmission mers-cov spillover at the camel-human interface genomic analysis of emerging pathogens: methods, application and future trends genomic epidemiology reveals multiple introductions of zika virus into the united states genomic and epidemiological monitoring of yellow fever virus transmission potential co-circulating mumps lineages at multiple geographic scales genome sequencing of an extended series of ndm-producing klebsiella pneumoniae isolates from neonatal infections in a nepali hospital characterizes the extent of community-versus hospital-associated transmission in an endemic setting simultaneous inference of phylogenetic and transmission trees in infectious disease outbreaks new vaccines against epidemic infectious diseases cepi-a new global r&d organisation for epidemic preparedness and response phase placebocontrolled trial of two vaccines to prevent ebola in liberia implementing an ebola vaccine study-sierra leone efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola Ça suffit!) the ring vaccination trial: a novel cluster randomised controlled trial design to evaluate vaccine efficacy and effectiveness during outbreaks, with special reference to ebola statistical power and validity of ebola vaccine trials in sierra leone: a simulation study of trial design and analysis choices in vaccine trial design for epidemics of emerging infections real-time dynamic modelling for the design of a cluster-randomized phase ebola vaccine trial in sierra leone simulations for designing and interpreting intervention trials in infectious diseases bayesian adaptive methods for clinical trials influenza vaccine effectiveness in the united states during the - season effectiveness of a group b outer membrane vesicle meningococcal vaccine against gonorrhoea in new zealand: a retrospective casecontrol study the case testnegative design for studies of the effectiveness of influenza vaccine theoretical basis of the test-negative study design for assessment of influenza vaccine effectiveness measurement of vaccine direct effects under the test-negative design the efficacy of influenza vaccine in elderly persons. a meta-analysis and review of the literature impact of influenza vaccination on seasonal mortality in the us elderly population influenza-related mortality in the italian elderly: no decline associated with increasing vaccination coverage effect of use of -valent pneumococcal conjugate vaccine in children on invasive pneumococcal disease in children and adults in the usa: analysis of multisite, population-based surveillance bayesian model averaging with change points to assess the impact of vaccination and public health interventions the estimation of age-related rates of infection from case notifications and serological data a simple model for complex dynamical transitions in epidemics loss of vaccineinduced immunity to varicella over time sieve analysis: methods for assessing from vaccine trial data how vaccine efficacy varies with genotypic and phenotypic pathogen variation microbial genome-wide association studies: lessons from human gwas genomic analysis of isolates from the united kingdom pertussis outbreak reveals that vaccine antigen genes are unusually fast evolving intussusception among infants given an oral rotavirus vaccine autism and measles, mumps, and rubella vaccine: no epidemiological evidence for a causal association outbreak of aseptic meningitis associated with mass vaccination with a urabecontaining measles-mumps-rubella vaccine: implications for immunization programs relationship of pertussis immunization to the onset of neurologic disorders: a retrospective epidemiologic study no association between influenza a(h n )pdm vaccination and narcolepsy in south korea: an ecological study narcolepsy onset is seasonal and increased following the h n pandemic in china safety of trivalent inactivated influenza vaccine in children to months old risk analysis of aseptic meningitis after measles-mumps-rubella vaccination in korean children by using a case-crossover design relative incidence estimation from case series for vaccine safety evaluation discovery, research, and development of new antibiotics: the who priority list of antibiotic-resistant bacteria and tuberculosis compensatory mutations, antibiotic resistance and the population genetics of adaptive evolution in bacteria antibiotics and antibiotic resistance genes in natural environments next-generation sequencing to guide clinical trials estimating the proportion of bystander selection for antibiotic resistance among potentially pathogenic bacterial flora. biorxiv measuring and interpreting associations between antibiotic use and penicillin resistance in streptococcus pneumoniae effect of short-course, high-dose amoxicillin therapy on resistant pneumococcal carriage: a randomized trial increased carriage of trimethoprim/sulfamethoxazole-resistant streptococcus pneumoniae in malawian children after treatment for malaria with sulfadoxine/pyrimethamine no coexistence for free: neutral null models for multistrain pathogens impact of antimicrobial treatment for acute otitis media on carriage dynamics of penicillin-susceptible and penicillinnonsusceptible streptococcus pneumoniae a placebocontrolled trial of antimicrobial treatment for acute otitis media access to new medications for the treatment of drug-resistant tuberculosis: patient, provider and community perspectives tradeoffs in introduction policies for the anti-tuberculosis drug bedaquiline: a modelbased analysis population implications of the use of bedaquiline in people with extensively drug-resistant tuberculosis: are fears of resistance justified? ecological theory suggests that antimicrobial cycling will not reduce antimicrobial resistance in hospitals the effects of antibiotic cycling and mixing on antibiotic resistance in intensive care units: a cluster-randomised crossover trial public health. pathogen surveillance in animals causation and disease: a chronological journey. the thomas parran lecture sequence-based identification of microbial pathogens: a reconsideration of koch's postulates hpv in the etiology of human cancer effect of treatment of helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer. a randomized, controlled study respiratory syncytial virus and recurrent wheeze in healthy preterm infants long-term measlesinduced immunomodulation increases overall childhood infectious disease mortality the mal-ed study: a multinational and multidisciplinary approach to understand the relationship between enteric pathogens, malnutrition, gut physiology, physical growth, cognitive development, and immune responses in infants and children up to years of age in resourcepoor environments prevention of early episodes of otitis media by pneumococcal vaccines might reduce progression to complex disease the authors received no specific funding for this article. conflict of interest: none declared. key: cord- -nabxpyw authors: bell, sadie; clarke, richard; mounier-jack, sandra; walker, jemma l; paterson, pauline title: parents’ and guardians’ views on the acceptability of a future covid- vaccine: a multi-methods study in england date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: nabxpyw background the availability of a covid- vaccine has been heralded as key to controlling the covid- pandemic. covid- vaccination programme success will rely on public willingness to be vaccinated. methods we used a multi-methods approach - involving an online cross-sectional survey and semi-structured interviews - to investigate parents’ and guardians’ views on the acceptability of a future covid- vaccine. parents and guardians (aged + years) who reported living in england with a child aged months or under completed the survey. nineteen survey participants were interviewed. findings most survey participants reported they would likely accept a covid- vaccine for themselves (definitely . %; unsure but leaning towards yes . %) and their child/children (definitely . %; unsure but leaning towards yes . %). less than % of survey participants reported that they would definitely not accept a covid- vaccine. survey participants were more likely to accept a covid- vaccine for themselves than their child/children. participants that self-reported as black, asian, chinese, mixed or other ethnicity were almost times more likely to reject a covid- vaccine for themselves and their children than white british, white irish and white other participants. survey participants from lower-income households were also more likely to reject a covid- vaccine. in open-text survey responses and interviews, self-protection from covid- was reported as the main reason for vaccine acceptance. common concerns identified in open-text responses and interviews were around covid- vaccine safety and effectiveness, mostly prompted by the newness and rapid development of the vaccine. conclusion information on how covid- vaccines are developed and tested, including their safety and efficacy, must be communicated clearly to the public. to prevent inequalities in uptake, it is crucial to understand and address factors that may affect covid- vaccine acceptability in ethnic minority and lower-income groups who are disproportionately affected by covid- . parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england and "likely to reject" (those that answered no, definitely not or unsure but leaning towards no). of interest such as underrepresented populations in the survey (e.g. participants from ethnic minority groups or reporting a lower household income) and/or indicated they would likely refuse a covid- vaccine, for their child or themselves. we did not solely interview participants who were likely to refuse a covid- vaccine as we were keen to explore the nuances of reasons participants had for accepting or refusing a covid- vaccine. participants were emailed an information sheet, fully detailing the study objectives and explaining all aspects of participation, including the right to withdraw from the research. written informed consent was obtained from each participant. interviews lasted parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england insert table here . % of survey participants (n= ) provided their details to be contacted for a follow-on interview. in total, parents were contacted to participate. of these took part in interviews ( women and one man), did not respond to recruitment emails, two responded initially but did not follow through with an interview. the characteristics of interviewees are outlined in table ). interestingly, one interviewee (# ) that was leaning towards accepting the vaccine at the time of completing the survey discussed that she was leaning towards refusing the vaccination (for herself and her child) at the time of interview. the following reasons were given for covid- vaccine acceptance for self and for child/children, in order of how often they were mentioned by survey participants and importance to interviewees. to protect self and others of survey participants expressing positive intentions to vaccinate and leaving an open-text response, the most prevalent reason was to provide protection from covid- to the parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england protecting other people (for self: . %, n= ; for child: . %, n= ), including family members (for self: . %, n= ; for child: . %, n= ). participants also reported that they would vaccinate to protect someone known to them in a risk group for covid- (for self: . %, n= ; for child: . %, n= ). . % (n= ) of survey participants specifically mentioned that they wanted to receive the vaccine to stay healthy to look after their child/children. . this is of concern given the evidence that black, covid- ) vaccine became available would you accept the vaccine for your child/children?". a -point likert scale was used to encourage participants to take a stance on the vaccine (rather than selecting a 'do not know' answer). the likert scale options were "yes, definitely a paired samples t-test was used to compare acceptance of a covid- vaccine for self and for the participant's child. two subsequent logistic regressions were then conducted to determine the demographic factors associated with rejection of the covid- vaccine for both self-vaccination and that of the participant's child age, household income, ethnicity, location, and employment were included as predictive variables in the logistic regression models. in the logistic regression model for child vaccination to perform the logistic regressions ethnicity was dichotomised into 'white' (i -£ , ) and "high income" (>£ , ), and the vaccine acceptance variables were dichotomised into parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england around the content of the questionnaire, were used to assist the interviews. the interviews included two questions related to a covid- vaccination, the first focused on parents' and guardians' views on receiving a new covid- vaccine for themselves, should one become publicly available, and the second on their views on their child of accepting a new covid- vaccine for their child. interview participants received a £ gift voucher as a thank you for their time and contribution. the interviews took place between th april and th may and clarke [ ]: data familiarisation, coding and theme identification and refinement. the transcribed interviews were read and coded by sb. to enhance the rigour of the analysis, coding approaches and subsequent theme generation and guardians completed the survey (see participant characteristics in table ) of the survey participants parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england quantitative findings -factors associated with covid- vaccine rejection for self a forward stepwise logistic regression analysis was performed with a dichotomised version of the self-vaccination against covid- variable as the discrete variable. age, household income, ethnicity, location and employment were included as predictive variables. the final model included three predictor variables (household income, employment and ethnicity) and significantly predicted 'likely to reject' (omnibus chi-square = . , df = , p < . ) . ) as likely to reject a covid- vaccine than participants with a medium household income (£ , -£ , ) participants that self-reported as black, asian, chinese, mixed or other ethnicity were . times ( %ci: . - . ) more likely to reject a covid- vaccine than white british white irish and white other participants. there was also some indication that those that parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england a forward stepwise logistic regression analysis was performed with a dichotomised version of the child vaccination against covid- variable as the discrete variable. age, household income, ethnicity, location, employment and number of children were included as predictive variables. the final model included three predictor variables (income, ethnicity and number of children) and significantly predicted 'likely to reject age, location and employment did not significantly predict 'likely to reject', as such they were excluded from the model. the hosmer-lemeshow test demonstrates that the model adequately fits the data chi-square = . , df = , p = . mixed or other ethnicity were . times ( %ci: . - . ) more likely to reject a covid- vaccine for their child than white british, white irish and white other participants. this finding was also found for income, with participants in the lower household income bracket (<£ , ) being . times ( %ci: . - . ) as likely to reject a covid- vaccine for their child than participants with a medium household income (£ , -£ , ). participants with more than four children were also found to be around four times /or publication of this article: the research was funded by the national institute for health research health protection research unit immunisation at the london school of hygiene and tropical medicine (lshtm) in partnership with public health england (phe). the views expressed are those of the author(s) and not necessarily those of the nhs, the nihr, the department of health or european centre for disease prevention and control (ecdc) coronavirus (covid- ) in the uk. the rt hon boris johnson mp. prime minister's statement on coronavirus (covid- staying alert and safe (social distancing) government launches vaccine taskforce to combat coronavirus world health organisation. the push for a covid- vaccine millions could be vaccinated against covid- as uk secures strong portfolio of promising vaccines ensuring global access to covid- vaccines jcvi: updated interim advice on priority groups for covid- vaccination using thematic analysis in psychology parents' and guardians' views on the acceptability of a future covid- vaccine: a multi sabat, i, once we have it, will we use it? a european survey on willingness to be vaccinated against covid- determinants of covid- vaccine acceptance in the willingness to vaccinate against covid- in australia more vaccines for children? parents' views. vaccine parents with doubts about vaccines: which vaccines and reasons why politics and public trust shape vaccine risk perceptions tracking the global spread of vaccine sentiments: the global response to japan's suspension of its hpv vaccine recommendation acceptance of a covid- vaccine in southeast asia: a cross-sectional study in indonesia susceptibility to and transmission of covid- amongst children and adolescents compared with adults: a systematic review and meta-analysis. medrxiv disparities in the risk and outcomes of covid- reasons for non-vaccination: parental vaccine hesitancy and the childhood influenza vaccination school pilot programme in england childhood vaccination coverage by ethnicity within london between ethnic differences in human papillomavirus awareness and vaccine acceptability inequalities in childhood vaccination timing and completion in london patient and practice level factors associated with seasonal influenza vaccine uptake among at-risk adults in england, to : an age-stratified retrospective cohort study effect of socioeconomic deprivation on uptake of measles, mumps and rubella vaccination in liverpool, uk over years: a longitudinal ecological study. epidemiol infect managing the covid- pandemic: new results from the european survey on policy support, confidence and vaccination attitudes office for national statistics. ks uk ethnic group, local authorities in the united kingdom (excel sheet kb) average household income, uk: financial year ending parents' and guardians' views on the acceptability of a future covid- vaccine: a multimethods study in england parents' and guardians' views on the acceptability of a future covid- key: cord- -jwvz ux authors: singh, gagandeep; singh, pankaj; pillatzki, angela; nelson, eric; webb, brett; dillberger-lawson, steven; ramamoorthy, sheela title: a minimally replicative vaccine protects vaccinated piglets against challenge with the porcine epidemic diarrhea virus date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: jwvz ux porcine epidemic diarrhea virus (pedv), is an economically important enteric coronavirus, with over a % mortality rate in neonatal piglets. the virus emerged in the us in , resulting in severe production losses. effective vaccine development against pedv is a challenge. inactivated vaccines are of questionable efficacy. attenuated vaccines, while more effective, require a relatively long lead development time, are associated with safety concerns and are also unable to prevent new field outbreaks. to combine the safety and efficacy advantages of inactivated and attenuated pedv vaccines, respectively, in this study, we tested the hypothesis that subjecting pedv virions to heat treatment at °c for min to reversibly unfold structural proteins, followed by exposure to rnase to fragment the genome, would result in a vaccine preparation with intact viral structure/antigenicity but highly diminished replicative abilities. we expected the vaccine to be both safe and effective in a piglet challenge model. following the heat and rnase treatment, pedv virions had an intact electron microscopic ultrastructure and were amplified only in the rd passage in vero cells, indicating that diminished replication was achieved in vitro. strong pedv spike-protein specific and virus neutralizing antibody responses were elicited in vaccinated piglets. upon challenge, all vaccinated pigs were protected against fecal viral shedding and intestinal pathology, while the unvaccinated controls were not. the vaccine virus was not detected in the fecal matter of vaccinated pigs prior to challenge; nor did they develop intestinal lesions. thus, the described approach has significant promise in improving current approaches for pedv immunization. porcine epidemic diarrhea virus (pedv) is an enteric coronavirus which causes diarrhea, vomiting, severe dehydration, and death in pigs. neonatal pigs are particularly susceptible, with mortality rates that can be as high as - %. in older pigs, manifestation of the disease is milder but growth and production parameters are affected ( , ) . classical strains of pedv (g strains) were first detected in the uk in , and spread to asia and europe. more recently, highly virulent strains (g strains) which emerged in china have spread to other countries, with the first case in the us being recorded in ( , , ) . it is estimated that the outbreak resulted in the losses of $ . - . billion and the death of million pigs ( , ) . the availability of effective vaccines and the practice of stringent biosecurity measures are critical for the prevention of pedv. however, the development of effective vaccines has been complicated by frequent viral evolution and the fact that ped is most severe in immunologically naïve neonates. effective and safe vaccines development was also challenging because active vaccine replication in the gut is required to induce good and lasting mucosal immunity. both attenuated and inactivated pedv vaccines have been routinely used in asian countries for several years. vaccination of sows prior to farrowing induces lactogenic immunity which is transferred to neonatal piglets via colostrum. inactivated vaccines are very safe but have a low duration of immunity and appear to produce a predominantly th type immune response ( ) . attenuated vaccines, produced by serially passaging field strains between and passages, are more effective against homologous strains but have a long lead development time and have been associated with safety concerns of recombination with field strains ( ) . regardless of the type of vaccine used, viremia and transmission of pedv is not prevented in vaccinated animals. outbreaks in vaccinated herds and the periodical emergence of new, highly pathogenic strains are not uncommon in countries were vaccines have been routinely used for many years ( , ( ) ( ) ( ) . in north america, a s-protein based subunit vaccine (iped plus, harris vaccines inc.) and inactivated vaccines produced by zoetis and vido-intervac were conditionally licensed. however, their efficacy has also been questioned by independent studies, as vaccination of pedv naïve sows did not result in strong protection in neonatal piglets ( , , ) . as the strong need for effective pedv vaccines remains unmet, the practice of feeding back minced intestines from infected piglets to sows, in an attempt to induce more effective immunity against pedv, is common in the field ( , , ) . the use of autogenous vaccines, where a custom inactivated vaccine tailored to each herd is prepared using a sample provided from the production unit, is also practiced ( , , ) . both the feedback and autogenous vaccine approaches are, once again, associated with significant safety and efficacy issues but natural or intentional exposure of pigs of all ages to pedv provides stronger homologous and partial heterologous protection ( , ) . further, vaccination of naïve animals is less effective than vaccination of previously exposed pigs, indicating that current vaccines are less effective than natural infection at priming the immune response but can effectively boost the memory response ( ) . it is established that the viral spike protein is a critical protective antigen, as anti-spike protein-specific serum igg levels correlate well with protection against pedv and virus neutralizing responses ( ) . however, the s-protein based subunit vaccine (iped plus, harris vaccines inc.) is of questionable efficacy, indicating that other viral components could contribute to protection. based on the above, we hypothesized that development of a process whereby the structural integrity of the virus was maintained but viral replication was highly diminished but not abrogated, would result in a vaccine with the combined advantages of inactivated and attenuated vaccines, namely, high safety and efficacy margins. previously published data shows that the sars coronavirus capsid is metastable and can be reversibly denatured by changes in temperature or ph, with unfolding commencing at • c and complete denaturation occurring at • c ( ) . hence, in this study, our vaccine development approach consisted of exposing pedv virions to • c to unfold the capsid, followed by fragmentation or digestion of the genome with rnase to diminish viral replication and subsequent refolding of the capsid at • c. gamma-irradiated pedv virions were used as an inactivated control vaccine ( ) . the objective of this study was to evaluate the heat and rnase treated pedv vaccine for its safety, immunogenicity and ability to reduce viremia in a weanling piglet model, with the ultimate goal of developing a process which can potentially reduce lead vaccine development time, is safe and be easily applied to newly emerging strains. porcine epidemic diarrhea virus (pedv) strain pedv co [national veterinary services laboratory (nvsl), ames, ia] was cultured at a multiplicity index (moi) of . using vero cells in the presence of trypsin as previously described ( , ) . the stock virus was titrated three times to obtain the mean % tissue culture infectious dose [tcid ] using the spearman and karber formula ( ) and stored in aliquots at − • c until further use. to optimize the temperature, time of incubation, and dose of rnase treatment, the virus stock was resuspended to × tcid / /ml in media (ph . ). diluted virus culture was exposed to temperatures ranging from to • c for min for unfolding, followed by incubation at • c for min for refolding, and then moved to • c for h, as previously described for the sars coronavirus ( ) . cultures were visualized by electron microscopy to ensure structural integrity. a temperature of • c for min was selected for unfolding. similarly, to fragment the genomic rna, varying combinations of concentrations of rnase a (ameresco) and rnase t (thermo scientific) were tested by adding them to the unfolded virus cultures, followed by incubation for , , , or h at • c. treated cultures were then exposed to • c for min for refolding and cooled down on ice for h. the final optimized protocol consisted of exposing the virus culture, resuspended to tcid /ml, to • c for min, followed by . mg/ml of rnase a and µl/ml of rnase t (equivalent to units/ml rnase a or , units/ml of rnase t ), incubation at • c for h, exposure to • c for min and cooling down on ice for h before storage at − • c for further testing. the final process was tested times to ensure reproducibility. to prepare the inactivated control vaccine, × tcid / /ml of pedv was irradiated in a cesium- source gamma (γ) irradiator at time points of h to h at rad/min. an effective dose of h ( , , rad), was used to prepare the irradiated vaccine, after validation as described above. to determine the effect of the treatment on viability, the treated virus and an untreated control were serially passaged times in vero cells as described above. after each passage, flasks were subject to three freeze-thaw cycles. the culture obtained was centrifuged at , × g for min • c to remove debris. one ml of the supernatant was used to infect vero cell monolayers and also infect well chamber slides (nunc) to visualize viral replication by an indirect immunofluorescence assay (ifa) as described below. visualization of viral replication in treated and untreated cultures was achieved using an indirect immunofluorescence assay (ifa), performed essentially as described previously ( , ) . cultured and fixed cells were stained with polyclonal swine anti-pedv sera (nvsl) and examined with a fluorescent microscope for green cytoplasmic fluorescence characteristic of rna viral replication. to visualize structure, treated, and untreated viral cultures were negatively stained by standard methods ( ) . stained grids were examined with a jeol jem- cx ii transmission electron microscope (figure ). possible genetic differences between untreated and treated vaccine virions were assessed by deep sequencing. heat and rnase treated and untreated viral particles were purified from infected cells by ultra-centrifugation at , × g for . h and re-suspended in pbs. unpackaged rna and dna were removed by a rnase and dnase cocktail containing units of rnase one (promega), units benzonase (novagen), and units of turbo dnase (ambion) incubated in x buffer (ambion) for • c for . h. viral rna was then isolated by using the qiamp viral rna isolation kit (qiagen) according to the manufacturer's protocol. purified viral rna was deep sequenced by a commercial vendor (bgi genomic). the cdna library was prepared using truseq library construction kit (illumina inc., usa) with random hexamer primers. the prepared cdna library was then sequenced using hiseq pe platform (illumina inc., usa) and raw reads ( bp) were obtained. the resultant sequences reads were analyzed by bgi genomic, philadelphia, pa. the raw reads were filtered out using soapnuke to get "clean reads" by removing the reads with adaptors, reads with more than % of unknown bases (n), and low-quality reads ( ) . clean reads were mapped to reference pedv genome (genbank: kf . ) using hisat (hierarchical indexing for spliced alignment of transcripts) ( ) . the genome mapping results further analyzed using the genome analysis toolkit (gatk) to call single nucleotide polymorphism (snp) and indel (insertion and deletion of bases) ( ) . only snps with a quality score above the threshold (qpred > ) and with a snp frequency of over % were included in assembling the consensus sequences. the consensus sequences of the treated and untreated samples were compared by alignment with clustal omega ( ) to obtain changes which could be attributed to the treatment. detected changes were annotated to include the locations and proteins affected ( table ) . clean reads were mapped to the reference genome using bowtie to detect differentially expressed genes. gene expression levels were calculated with rsem version . . ( ) . differentially expressed genes were identified by the possiondis, ebseq software for samples without replicates ( ) . all animal experimentation was approved by the institutional animal care and use committee (iacuc) of s. dakota state universities (sdsu) (protocol number: - a). no other specific permissions were required for these activities. this study did not involve endangered or protected species. twenty-four, to -week-old piglets which were negative for pedv by pcr and serology were divided into groups; group -unvaccinated control group (n = ) ( ml of pbs intramuscular and oral route each), group -rnase and heat treated pedv vaccine group (pedv-vac) group (n = ) ( ml of tcid /ml, intramuscular and oral route each) and group -irradiated pedv vaccine group (n = ) ( ml of tcid /ml, intramuscular and oral route each). piglets were boosted by the same route and dose at dpv and . on dpv , small intestine, heart, liver, and spleen were collected piglets from each group (n = /group) to assess vaccine safety. the remaining piglets (n = /group) were challenged orally with tcid /ml of pedv co , as previously described ( , ) . post-challenge, the piglets were observed daily for clinical signs of ped. all piglets were euthanized -week post challenge (dpc) or at dpv and three sections of the small intestine (duodenum, jejunum, and ileum) were collected for histopathological (hp) and immunohistochemical (ihc) analysis. serum was collected from all piglets on dpv , , , , and to measure binding and neutralizing ab responses. fecal swabs were collected at dpv , , , and from all piglets to measure shedding of the vaccine virus by rt-qpcr. fecal swabs were collected on dpv and (dpc day and ) from all piglets to measure protection against shedding of the challenge virus by rt-qpcr. spike protein-specific igg responses in pigs were measured in duplicate by an indirect elisa as previously described, using the pedv s antigen or np antigen for capture ( ) . the assay format was pre-validated at the animal disease research and diagnostic laboratory (adrdl), sdsu, using serum samples from animals of known serological status. a standardized operating procedure was followed in sample analysis. the results were calculated as sample to positive (s/p) ratios as follows: s/p = optical density (od) of the sample-od of buffer/od of positive control-od of the buffer. . ± . ( / ) (p = . ) rnase + heat treated pedv/ unchallenged total number of pigs = , no. of pigs sacrificed for vaccine safety assessment prior to challenge = , no. of pigs sacrificed at day post challenge = . % total atrophic enteritis score for the ileum, jejunum, duodenum where , negative; , mild; , moderate; , severe; , sections with crypt hypertrophy. & total immunohistochemistry (ihc) for the ileum, jejunum, duodenum where , negative; , positive; ≤ %, , positive, - %; , positive, > %. * fecal score at necropsy-formed feces = , semi-formed feces = , liquid feces = . # sum of the microscopic and fecal scores. @ p < . as determined by the mann-whitney u-test, compared to the unvaccinated group. to assess the neutralizing antibody responses elicited by vaccination, a pre-validated fluorescent focus neutralization (ffn) assay was used as previously described ( ), following the standard operating procedures of the adrdl, sdsu. briefly, doubling dilutions of heat inactivated sera were incubated with foci forming units, incubated for h and cultured on vero cell monolayers. plates were stained with a pedv-specific fluorescein-labeled monoclonal antibody (sd - ) to visualize the end point, which was defined as a % reduction of foci compared to the controls. virus shedding through fecal route was assessed by a rt-qpcr performed by the ndsu veterinary diagnostic laboratory, using pre-validated standard operating procedures, and a commercial pcr kit called the swine enteric pcr panel (thermo fisher) following the manufacturer's instructions. each pig was considered a biological replicate (n = , as pigs/ group were sacrificed to assess vaccine safety prior to challenge), and each sample was assessed in duplicate. the obtained ct-values were converted to viral copy numbers using a standard curve and log transformed for representation. tissue samples, collected as described above, were fixed in neutral buffered formalin for h, trimmed, processed, and embedded in paraffin. tissues were cut into µm thick sections and stained with hematoxylin and eosin (he) or a pedv n protein-specific monoclonal antibody (sd - ) for immunohistochemistry (ihc) following the standard operating procedures of the adrdl, sdsu. scores were recorded in a blinded fashion by a board-certified veterinary pathologist. scores to measure atrophic enteritis characteristic of ped were assigned as follows: = negative, = mild, = moderate, = severe. sections with crypt hypertrophy were assigned an additional points. antigen detection in enterocytes by ihc was semi-quantitatively scored based on the following criteria: = negative, = positive, ≤ %, = positive, - %, = positive, > %. the consistency of fecal matter during necropsy was assigned scores as follows: formed feces = , semi-formed feces = , liquid feces = . total scores were calculated as the mean sum of the histology and fecal scores ( table ) . significant differences between treatments were assessed by anova and when significant (p < . ) post-hoc analysis was used to determine differences between groups. the student's ttest was used for the serology and rt-qpcr data and the mann-whitney u-test for the pathology lesion scores. the mean values of replicates, standard deviation and statistical significance are represented in the figures and tables. to achieve the targeted outcomes of maintaining structural integrity while achieving diminished viral replication, rather than complete inactivation, pedv virus cultures were first exposed to temperatures ranging from to • c for min and visualized by electron microscopy. intact structures were detected at all temperatures tested. however, increasing numbers of misshapen and fragmented virions were detected at • c and above. cultures treated at and • c remained viable as viral replication was visible by immunofluorescence (ifa) in infected vero cells using a pedv-specific antibody, without any amplification by serial passaging. virus was detected after the st passage in the cultures treated at • c. virus cultures treated at and • c were not amplified even after four serial passages in vero cells, indicating that complete inactivation occurred at these temperatures. hence a temperature of • c for min was chosen for reversible unfolding of the viral capsid ( figure b) without completely inactivating the virus. untreated control virus culture remained structurally intact as expected ( figure a) . similarly, while rnase treatment alone did not affect viability, the reduction in viral replication was proportional to the dose and time of exposure to rnase in the heat-treated virions. a dose of units of rnase a and , units of rnase t with an exposure time of h was chosen as optimal for the final vaccine preparation. while the untreated virus control showed robust replication (figure a) , following the heat and rnase treatment protocol, viral replication was detected only in the rd passage in vero cells (figure b ). for the gamma (γ) irradiated, inactivated control vaccine, typical icosahedral structures were seen in electron microscopy after h of exposure to radiation. however, the corona-like layer containing the protective spike antigens appeared to be damaged (figure c) . at this dose of radiation, the virus was not detected by the ifa with a pedv-specific ab at the third serial passage in cell culture ( figure c) . hence, a final dose of h ( , , rad) was selected to prepare the inactivated control vaccine. measurement of ab responses against the pedv spike and nucleocapsid proteins (np) by elisa ( ) showed that animals vaccinated with the heat and rnase treated virions mounted strong ab responses against the protective pedv spike antigen following the booster vaccinations on dpv and (figures a,b) . however, ab responses to non-structural nucleocapsid protein (np) remained low prior to the challenge. in pigs immunized with the irradiated vaccine, ab responses to both viral antigens were low. the mean optical density values for the elisas were significantly different between the groups (figures a,b) . to that of the spike protein-specific abs. strong virus neutralizing ab responses, were detected in animals vaccinated with the heat and rnase treated virions but not in the pigs which received the irradiated viral vaccine. the differences between the groups was statistically significant (figure c) . the spike protein-specific ab and virus neutralizing ab levels were strongly correlated in the heat and rnase treated pedv vaccinated pigs, with a correlation coefficient of . %. as expected, the unvaccinated control pigs remained sero-negative for the duration of the study. to assess the efficacy of the vaccine in protecting against challenge, shedding of the challenge viral rna in fecal matter was assessed by a pedv-specific rt-qpcr on days , , and post-challenge. all experimental animals were rt-qpcr negative on day post-challenge (dpc). at dpc and , challenge viral rna was not detected in any of the pigs vaccinated with the heat and rnase treated pedv vaccine (figure ) , while of the pigs administered the irradiated vaccine were positive by rt-qpcr on dpc . all pigs in the irradiated vaccine group turned positive by dpc (figure ) . as expected, viral rna was detected in the fecal matter of all unvaccinated pigs on both sample collection days with titers increasing between dpc and . while the viral rna loads were significantly different between the two vaccine groups at both time points, there were no significant differences between the unvaccinated controls and pigs administered the irradiated vaccine at both the time points tested, indicating that the irradiated vaccine did not provide protection against viral replication and shedding in the host. examination of the intestinal tissue of the experimental animals by histology and immunohistochemistry (ihc) showed that the heat and rnase treated pedv vaccine completely protected vaccinated pigs against the development of microscopic lesions following challenge. characteristic microscopic intestinal lesions of atrophic enteropathy and crypt hyperplasia were detected in the duodenum, jejunum, and ileum of animals in the control groups (figures d-g) . viral antigen was also detected in the enterocytes in all three sections using a pedvspecific monoclonal ab-based immunohistochemistry assay (figures a-e) . there were no significant differences between the sections, indicating the entire small intestine was affected. the total microscopic score, including the histopathology and immunohistochemistry scores was . for the unvaccinated animals and . for the pigs immunized with the irradiated vaccine and for pigs administered the heat and rnase treated vaccine. while the difference between the unvaccinated group and irradiated vaccine group was not statistically significant, the irradiated vaccine appeared to enhance intestinal pathology (table ) . similarly, the total necropsy scores, a sum of both the fecal and histology scores, were significantly different (p = . ) between the two vaccine groups but not between the unvaccinated group and the irradiated vaccine group (p = . ) ( table ). no side effects or clinical signs of ped were observed in vaccinated pigs after either the primary or booster vaccines. vaccine viral rna was not detected by rt-qpcr in the fecal matter of any of the vaccinated pigs from both groups at days after the primary vaccination or at week after the boosters. all animals remained pcr negative until the day of challenge. therefore, although the heat and rnase treated pedv virions were detected by amplification after serial passages in vero cells, replication of the vaccine virus in the host appeared to be curtailed by its immune system. in the pigs euthanized from each group prior to challenge, stools were fully formed at necropsy ( table ) . no microscopic lesions or viral antigen were detected in the small intestine sections, heart, spleen, and liver of the animals necropsied from each group prior to challenge ( table ) . representative images of the duodenum, jejunum, and ileum are depicted in figure . to identify possible mutations that could explain the highly effective attenuation observed, deep sequencing of heat and rnase treated virions from infected vero cells resulted in a total of . and . mb of raw reads were obtained by rna seq for the treated and untreated samples, respectively. clean reads obtained after trimming were . and . gb, respectively. the qphred values for the clean reads were . and . for the untreated and tread samples, respectively, indicating satisfactory quality of the data obtained. as listed in table snps and insertions or deletions (indels) were detected in the polyprotein, spike and envelope proteins ( table , s sequence file and supplementary figures - ) of heat and rnase treated virions, when compared to the untreated virions. in addition, insertions and deletions were detected in the s region for the spike protein. the n terminal signal peptide region of the spike protein had a amino acid deletion and one non-synonymous change at position , changing the sequence from igen to k-n. a conservative in-frame insertion was detected at position in the s region, changing the amino acid sequence from l----at to lkkkgat ( table and supplementary figure ) . chemical methods for inactivation of viruses have long been in use for vaccine development. while they are rapid and convenient, commonly used inactivation agents may not only affect nucleic acids but also protein structures and hence antigen presentation and vaccine efficacy. gamma irradiation has been traditionally used to inactivate viruses. the mechanisms involved include nucleic acid degradation, destruction of covalent bonds, and release of free radicals ( ) . as commercial inactivated vaccines were not available at the time of testing gamma irradiation was selected as the method of choice to prepare an inactivated control vaccine for this study. moreover, similar to the heat and rnase treated vaccine, the virus-like-particulate structure was more likely to be maintained by gamma irradiation, while achieving complete inactivation. gamma irradiation had been previously used for vaccine development with varying success, depending on the pathogen ( ) . for example, we have previously demonstrated that a gamma irradiated vaccine against neospora caninum was effective in mice ( ) . however, a gamma irradiated, lassa virus vaccine failed to protect vaccinated mice ( ) . although both approaches tested in this study targeted nucleic acids and preservation of structure, the protective outcomes varied significantly between the two vaccines tested. it is possible that release of free radicals during the irradiation process could have a deleterious effect on integrity of antigenic structures and antigen presentation in vivo. a more detailed characterization of these parameters will be the focus of future studies. similar results for the gamma irradiated vaccine in this study, it has been shown that a dendritic cell targeted spike protein-based subunit vaccine against pedv exacerbated intestinal pathology in vaccinated pigs, despite stimulating strong cd + /cd + t cell responses ( ) . while characterizing the exact physical interactions involved in the heat and rnase treatment is not within the scope of this study, our finding that exposure of pedv to temperatures below • c did not affect structure was similar to other studies showing that the sars coronavirus structure is metastable and can be reversibly denatured by exposure to varying physical conditions such ph and temperature ( , ) . although the heat and rnase treated virus culture was amplified after passages in cell culture (figure ) , the absence its detection by rt-qpcr (figure ) , or immunohistochemistry (table and figure ) and the lack of strong ab responses to the non-structural np (figure ) , in vaccinated pigs prior to challenge indicates that active vaccine viral replication was absent in the host or was undetectable by the techniques used. therefore, unlike other attenuated pedv vaccines or vaccination strategies that rely on prior exposure to field strains, it is highly improbable that reversion to virulence or recombination with field strains could occur with the heat and rnase treated vaccine. viral genomes that were identical to the untreated parental virus were not detected by deep sequencing of the heat and rnase treated virus from infected vero cells. insertions and deletions in the spike protein, especially the s region, influence pathogenicity, and immunogenicity of pedv. the core neutralizing epitope of the pedv spike protein has been localized to amino acid positions - ( , ) . the snps identified in the spike protein of the vaccine virions ( table ) did not map to these residues. while a limitation of the described method is that genetic changes induced by treatment and repair are unpredictable, repair of mutations ( ) or complementation in trans of the fragmented genome could have led to detection of a fluorescent signal in the rd passage after treatment. indeed, it has been shown that replication deficient genomes with deletions or mutations are produced during serial passaging of foot and mouth disease virus (fmdv) for attenuation. they are not infective by themselves, but when present in the same cell, the mutations in the genomes can complement each other in trans to produce plaques in vitro. when the defective-complementing virus system was used as a vaccine by rodriguez-calvo et al. vaccine virus replication was not detected but strong protection was elicited. this observation can be explained by vaccine virus replication in the host being limited by the requirement of coinfection of the same cell. even if such an unlikely coinfection event were to happen despite active host innate immunity, the recombined progeny viruses were more likely to be highly attenuated than acquire virulence, thus providing an additional vaccine safety barrier in vivo ( ) . in vivo, the presence of the host innate immune system was likely able to effectively curtail replication, despite exposure to tcid of the heat and rnase treated virus culture. more detailed studies are required to confirm these hypotheses, but they are not within the scope of this manuscript. the importance of spike protein-specific antibodies for protection against pedv is well-established ( ) . several studies describing experimental subunit and vectored vaccines or commercial attenuated and inactivated vaccines against pedv establish a strong correlation between spike protein-specific antibodies, virus neutralization titers and protection against infection ( , , , ( ) ( ) ( ) ( ) ( ) . similar to these studies, strong spikeprotein specific ab responses and virus neutralizing responses were noted in the pigs immunized with the heat and rnase treated vaccine. a commercial inactivated vaccine was able to reduce challenge viral shedding by - logs but an attenuated vaccine induced iga responses but did not affect viral shedding ( ) . testing of two attenuated pedv strains produced by serial passage in weanling pigs showed that the passaged viruses were attenuated but were not protected against challenge viral shedding or clinical signs ( ) . while direct comparisons are not possible due to differences in experimental conditions, unlike the other cited studies, intestinal lesions, or challenge virus was not detected by qpcr in the heat and rnase treated vaccine group in this study. although boosters were incorporated in the study design to minimize risk, it is likely that they were not required to achieve adequate protection as strong spike protein specific antibody responses and virus neutralizing responses were detected after the first dose of the heat and rnase treated vaccine, at dpv (figure ) . while cell mediated immunity was not assessed due to difficulties with transportation of cells, it is very likely that it was not compromised by the process used as the heat and rnase treated vaccine was very effective in preventing challenge viral replication in vaccinated pigs. while ideal for pedv, studying vaccine efficacy in pregnant sows and neonatal pigs is expensive and procedurally tedious. although clinical signs are less severe in older piglets ( ) and virulence can vary between isolates used for challenge ( , ) , pedv can infect and replicate well in pigs of all ages ( , ) . hence several researchers have used weanling piglets to screen vaccine candidates for efficacy and safety ( , , , , ( ) ( ) ( ) ( ) ( ) ( ) . this approach can help reduce animal use and cost if the candidates fall short of expectations. several swine bioassay studies in growing piglets have reported that peak pedv replication occurs between dpi and dpi after which viral loads decrease ( , , , ) . similar patterns of infectivity were observed in this study, as the uninfected control pigs had a mean fecal viral rna load of . log copy numbers at dpi (figure ) developed microscopic lesions, but not severe clinical signs. in comparison to the untreated control and irradiated vaccine groups, no fecal viral shedding or intestinal pathology was detected in the pigs immunized with the heat and rnase treated vaccine, indicating that vaccine induced immunity was highly effective against pedv challenge, within the limits of this weanling pig study model. the primary advantages of this innovative approach are safety, efficacy, convenience and a short development time. as the method can be easily adapted to newly evolving strains, provided they are readily cultured, this approach is very relevant to current field immunization practices of feedback exposure and autogenous vaccination. our future goals include testing the heat and rnase treated vaccine in pregnant sows, and improving oral and respiratory mucosal vaccine delivery systems to target improved protection. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by institutional animal care and use committee (iacuc) of s. dakota state universities (sdsu) (protocol number- - a). gs, ps, and sd-l: data collection, analysis, and manuscript editing. ap, en, and bw: data collection and manuscript editing. sr: conception, funding, and manuscript preparation and editing. this study was funded by the north dakota state agricultural products utilization committee and in part by the usda-nifa agriculture and food research initiative competitive grant no. - - , under project nd nd . the funding agencies had no role in study design, collection, analysis, and interpretation of data, in the writing of the report and in the decision to submit the article for publication. porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus porcine epidemic diarrhea: a review of current epidemiology and available vaccines new variant of porcine epidemic diarrhea virus genomic and evolutionary inferences between american and global strains of porcine epidemic diarrhea virus updated estimated economic welfare impacts of porcine epidemic diarrhea virus (pedv) assessment of the economic impacts of porcine epidemic diarrhea virus in the united states vaccines for porcine epidemic diarrhea virus and other swine coronaviruses status of vaccines for porcine epidemic diarrhea virus in the united states and canada efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs evaluation of the effects of pedv vaccine on pedv naive and previously pedv exposed sows in a challenge model comparing immune response and preweaning mortality a rapid-response humoral vaccine platform exploiting pre-existing non-cognate populations of anti-vaccine or anti-viral cd + t helper cells to confirm b cell activation the legal foundation of the production and use of herd-specific vaccines in europe an inactivated vaccine made from a u.s. field isolate of porcine epidemic disease virus is immunogenic in pigs as demonstrated by a dose-titration emerging and reemerging coronaviruses in pigs characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions low stability of nucleocapsid protein in sars virus application of radiation technology in vaccines development development of an indirect elisa, blocking elisa, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to north american strains of porcine epidemic diarrhea virus a computationally designed serological assay for porcine epidemic diarrhea virus beitrag zur kollektiven behandlung pharmakologischer reihenversuche visualizing proteins and macromolecular complexes by negative stain em: from grid preparation to image acquisition soapnuke: a mapreduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data hisat: a fast spliced aligner with low memory requirements the genome analysis toolkit: a mapreduce framework for analyzing next-generation dna sequencing data analysis tool web services from the embl-ebi rsem: accurate transcript quantification from rna-seq data with or without a reference genome ebseq: an empirical bayes hierarchical model for inference in rna-seq experiments effect of porcine epidemic diarrhea virus infectious doses on infection outcomes in naive conventional neonatal and weaned pigs pathogenicity and immunogenicity of attenuated porcine epidemic diarrhea virus pc a strain in conventional weaned pigs inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges vaccination with gamma-irradiated neospora caninum tachyzoites protects mice against acute challenge with n. caninum inactivated lassa virus elicits a non protective immune response in rhesus monkeys vaccination of sows with a dendritic cell-targeted porcine epidemic diarrhea virus s protein-based candidate vaccine reduced viral shedding but exacerbated gross pathological lesions in suckling neonatal piglets evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products distinct characteristics and complex evolution of pedv strains isolation and characterization of chinese porcine epidemic diarrhea virus with novel mutations and deletions in the s gene coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics new vaccine design based on defective genomes that combines features of attenuated and inactivated vaccines identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus display of porcine epidemic diarrhea virus spike protein on baculovirus to improve immunogenicity and protective efficacy evaluation of a ped vaccine on piglet mortality and sow immunity immunogenicity of a recombinant parapoxvirus expressing the spike protein of porcine epidemic diarrhea virus evaluation of the efficacy of a commercial inactivated genogroup b-based porcine epidemic diarrhea virus (pedv) vaccine and experimental live genogroup b exposure against b challenge genetic evolution analysis and pathogenicity assessment of porcine epidemic diarrhea virus strains circulating in part of china during assessment of the safety and efficacy of an attenuated live vaccine based on highly virulent genotype b porcine epidemic diarrhea virus in nursing piglets increased frequency of porcine epidemic diarrhea virus shedding and lesions in suckling pigs compared to nursery pigs and protective immunity in nursery pigs after homologous re-challenge lymphoproliferative responses and protection in conventional piglets inoculated orally with virulent or attenuated porcine epidemic diarrhoea virus mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus evaluation of porcine epidemic diarrhea virus transmission and the immune response in growing pigs experimental infection of a us spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original us pedv infection evaluation and comparison of the pathogenicity and host immune responses induced by a g b taiwan porcine epidemic diarrhea virus (strain pintung ) and its highly cell-culture passaged strain in conventional -week-old pigs a flagellinadjuvanted ped subunit vaccine improved protective efficiency against pedv variant challenge in pigs we thank ms. kelli mattock, marvin ssemadaali, heather vinson, scott hosselton for technical assistance and drs. michele mucciante and amanda zubke for assistance with animal experimentation. key: cord- - ns onkw authors: kusters, inca c.; matthews, james; saluzzo, jean françois title: manufacturing vaccines for an emerging viral infection–specific issues associated with the development of a prototype sars vaccine date: - - journal: vaccines for biodefense and emerging and neglected diseases doi: . /b - - - - . - sha: doc_id: cord_uid: ns onkw abstract the world was struck by surprise when a severe acute respiratory syndrome (sars) epidemic started in in china. this disease had never been observed in man before; the sars-coronavirus causing the disease was unknown. with the uncertainty about the future impact of this epidemic, an important international collaboration started spontaneously sharing scientific knowledge and reagents. resources became quickly available, and public and private efforts were undertaken to develop rapidly a vaccine. we will discuss here the importance of the international collaboration and the availability of funding. moreover, we will review the most important and challenging steps during the industrial development of the sars vaccine highlighting the difficulties in terms of safety working with such a highly pathogenic, unknown virus. we will emphasize the industrial perspectives on inactivation and decontamination experiments, the selection of the most promising vaccine candidate, the production process and the choice and use of animal models in such a pressing and difficult situation. finally, we will briefly review the unique regulatory environment created during this period for the development of a sars vaccine. with the uncertainty about the possible extent of the spread of the sars outbreak in early , there was substantial international collaboration first in the isolation and identification of the cov, sharing of sequence data, and eventually the sharing of seed virus that would be suitable for vaccine development. in the case of the sanofi pasteur vaccine, the prototype virus was supplied by the centers for disease control and prevention (cdc) (utah virus p /vero cell p ). furthermore, in this spirit of collaboration, sanofi pasteur provided stocks of the vero cells for the virus isolation to better ensure that any vaccine made from these cells would be acceptable from a regulatory perspective. these were the same vero cells as are routinely used in the commercial production of inactivated polio vaccine. in addition, there was rapid sharing of immunological reagents to ensure that the plaque-purified prototype seed virus accurately reflected the circulating epidemic sars-cov. one particular difficulty with rapidly emerging infections such as sars, of course, is that they may occur anytime of the year and are unlikely to be aligned with the annual process for assigning financial and human resources and project prioritization. in order to circumvent these problems, once it became clear that the sars-cov could grow in vero cells, the national institute for allergy and infectious diseases (niaid) in the united states focused their request on several companies that had experience in large-scale vero cell culture and had the experience to work with viruses at biosafety level (bsl ) containment. our first contact with niaid on the possibility of developing an inactivated vaccine occurred in the first quarter of . sanofi pasteur was one of only two companies that satisfied the niaid's eligibility criteria. the solicitation and funding mechanism that niaid employed was a directed request for proposals (rfp). this is sometimes called a letter contract by virtue of the fact that niaid initiated contact with the companies first by letter outlining the project objectives, infrastructure requirements, and deliverables. the advantage of this mechanism, for diseases such as sars, is that in late , several hundred cases of a severe atypical pneumonia were reported in the guangdong province of the people's republic of china. by the first quarter of , similar cases were reported in hong kong and sporadically throughout south-east asia and canada. this severe disease, with a mortality of - %, spread rapidly around the world and in april , countries on different continents had reported cases [world health organization (who), accumulative sars cases]. as a result, the who issued on march a global alert for the illness that would be known as " severe acute respiratory syndrome " (sars) (who sars alert). within the same time frame, secondary cases of sars were being identified in health care workers and family members who had close contact with patients suffering from this severe respiratory illness. by the second week of june, using the who case definition (who case description), approximately sars cases and sars-related deaths had been reported to the who. while the first wave of the sars epidemic seemed to have reached its conclusion, it was completely unclear how the spread of the virus would evolve. there was no clear understanding of the animal reservoir and the impact of virus mutation and unapparent infections. different situations could be envisaged; one scenario was that virulent sarscoronavirus (sars-cov) would persist and become endemic. another possibility was that other epidemic waves would occur or, finally, that the virus would disappear. taking into account all the uncertainties and anticipating the worst-case scenario, many laboratories and vaccine manufacturers started working on a vaccine approach against sars infection, largely based on what was known from animal covs. in this chapter, we will discuss the necessity for international cooperation and the importance of discretionary funding for rapidly developing a prototype vaccine candidate. we will review the decision-making process, the strategic choices made in terms of vaccine candidate, adjuvants, working conditions, and the safety precautions implemented at the beginning and throughout the entire production process of the sars vaccine. in addition, we will discuss the unique challenges associated with moving a vaccine such as sars through the regulatory process. with such a highly pathogenic, unknown virus. we will emphasize the industrial perspectives on inactivation and decontamination experiments, the selection of the most promising vaccine candidate, the production process and the choice and use of animal models in such a pressing and difficult situation. finally, we will briefly review the unique regulatory environment created during this period for the development of a sars vaccine. the absolute eligibility criteria, vaccine characteristics, and project objectives are clearly defined; the proposal review cycle is very short; and the necessary financial resources are immediately available. in this particular case, the rfp was received by us on may , and we submitted our project plan and budget by june . preliminary work began in july and august, and by the third week of september , we had a fully executed contract. by october , our inactivated prototype sars vaccine was filled and available for clinical assessment. another positive aspect of this mechanism is that involvement of such a large research organization provides the potential to access the expertise of many different investigators, as technical problems may arise. unlike traditional research grants, these types of contracts have very short duration and, consistent with the sense of urgency, progress against objectives is monitored on a weekly basis, perhaps, contributing to the fact that our project finished almost a month ahead of schedule. importantly, this type of mechanism does not compete with academic researchers for funding but allows these researchers to develop more basic science proposals of longer duration that can be reviewed and funded by other established mechanisms. perhaps, among others, there are three important lessons about responding to emerging threats that can be learned from the sars experience. first, information must be shared quickly and transparently. the somewhat surprising observation that the sars-cov grew well in vero cells was important in prioritizing the development of an inactivated, vero cell -derived virus. second, there should be an ongoing dialogue between the national research organizations and their potential industrial partners to understand what capacity and experience could be urgently brought to bear in a crisis situation. although there are potential issues with competitive intelligence, it remains important that information about industrial capability should be shared. third, funding organizations should have in place a mechanism for rapid proposal solicitation, review and monitoring, and also adequate discretionary funding that could support new vaccine projects in an urgent manner. as early as april , , the who and the cdc in the united states (who guidelines, cdc) recommended laboratories and vaccine manufacturers to handle sars-cov specimens using standard bsl work practices. it is not exceptional for vaccine manufacturers to work in bsl facilities for the production of certain vaccines, e.g., rabies, japanese encephalitits, or polio viruses. some of these commercial vaccines have been made for decades. for these vaccines, robust production processes and standard operating procedures have been put in place. the experience gained on the decontamination and inactivation of viral vaccines during the production process and the quality control (qc) are all important. moreover, the personnel working at the different stages of such a vaccine production are typically vaccinated, and revaccination procedures are in place in case of major accidents (i.e., boost after potential rabies contamination). in case of an emerging virus such as sars, the situation is completely different. although the bsl experience will be fully exploited, all processes and procedures need to be discussed, evaluated, validated, and implemented. for each emerging virus, this exercise needs to be repeated and specific conditions must to be adopted according to the unique characteristics of the new virus. consistent with the who and cdc recommendations, at sanofi pasteur live sars-cov was handled in a bsl facility. since no treatment or vaccine was available against sars, we decided to work in " bsl plus " laboratory conditions. essentially our basic level of containment was bsl incorporating several bsl working practices: clothing change before entering the facilities, shower on exit, and all material decontaminated before exit from the facilities. also, all steps, where the product was handled in open phase, were performed within class iii biological safety cabinets (csb). to prevent accidental contamination, the laboratory workers wore a positive pressure personal mask. these precautions were considered necessary taking into consideration the large amount of live virus handled ( - l) and finally turned out to be valid as the risk for contamination existed as was shown in the laboratory accidents in singapore, taiwan, and beijing in china ( senior, ; normile, ; orellana, ) . furthermore, as described below, the decontamination experiments demonstrated that the sars-cov is an extremely resistant virus. for the medical follow-up of the bsl -trained personal involved in the sars vaccine development, it was of critical importance to be able to distinguish the symptoms of respiratory distress caused by sars or other respiratory agents. it was therefore decided that the bsl laboratory workers should be selected in accordance with their immune status and would be immunized with streptococcus pneumoniae and influenza vaccines, if appropriate. in case a worker would present symptoms, a procedure had been put in place to isolate the worker using a high-efficiency particulate air (hepa) filter mask before being transported to prior to the start of the laboratory work since it is well known, used in many vaccines, and well accepted by regulatory authorities. when the laboratory work on the sars-cov vaccine development started, no data were available on the inactivation characteristics of the virus. one of the priorities was to identify the conditions to fully inactivate the virus for vaccine development, but also for decontamination of equipment, facilities, and waste decontamination. the results, as described in more detail below, were unexpected. the sars-cov is an extremely resistant virus and several of the routine decontamination working practices cannot be applied for this virus. these results demonstrate how important it is to immediately perform decontamination testing and adapt decontamination practices and strategies for each specific (emerging) virus. at the time the development of a vaccine against an emerging virus is initiated, it is very unlikely that routine tests and reagents are available. this was indeed the case for sars. therefore, our first experiments were dedicated to develop routine tests that are required for vaccine development and the analysis of the host response after immunization. these include neutralization tests, enzyme-linked immunosorbent assays (elisa), polymerase chain reactions (pcrs), and others. well-validated reagents are needed as reference standards for essential laboratory tests as polyclonal and monoclonal antibodies, recombinant proteins, and pcr primer pairs. one of the most sensitive issues was how to select an appropriate animal model to evaluate the candidate vaccines. for example, we observed that nmri mice gave a very heterogeneous response whereas balb-c or c bl/ j mice responded uniformly to immunization with the vaccine candidate. also, guinea pigs appeared to be a good model for the evaluation of immune responses. beyond immunogenicity, it is important to work with an animal model that is appropriate for challenge studies, as an assessment of vaccine efficacy. this is especially important for emerging infections since efficacy studies in humans may not be possible. in case of the sars, the macaca fascicularis was identified very early on ( fouchier et al., ) as a likely predictive, challenge model. all of the work was done in constant communication with the regulatory authorities. our experience with sars reinforces the idea that manufacturers should be encouraged to open and maintain an active dialogue with regulatory officials, very early in the development process. a nearby hospital where a special, dedicated negative pressure hospital room had been prepared. the very first decision concerned the choice of vaccine immunization strategy. in the intense early days of the epidemic a variety of approaches were considered. these included inactivated vaccines, subunit products, dna (either alone or in combination as part of a prime-boost strategy), vectored vaccines, and live attenuated candidates. similarly, alternative routes of vaccine administration (inoculation, aerosol, etc.) and formulation (adjuvants, etc.) were considered. in fact, several live attenuated and killed vaccines for veterinary covs are already marketed: e.g., a live attenuated and killed vaccine against the chicken infectious bronchitis virus ( ladman et al., ) , a live modified virus against canine cov ( pratelli et al., ) . ultimately, however, the crucial factor in the decision-making process was likely development timelines. at the time of this decision, the epidemic was prevalent and there was a sense of urgency that a prototype vaccine had to be developed as soon as possible . embedded in this decision-making process was the realization that we were seeking a vaccine candidate that largely used existing, conventional technology and that could be made in significant quantities, if necessary. in the context of the likelihood for rapid development and the possibility for large-scale production, the decision was taken to produce a monovalent, whole, inactivated, aluminum-adjuvanted sars vaccine for intramuscular injection. from our perspective, a whole, inactivated vaccine was a logical first choice taking into consideration our extensive experience with inactivated vaccines such as inactivated poliovirus, rabies, and hepatitis a vaccines. we were also encouraged by the fact that sars-cov grew very well in vero cells. sanofi pasteur has a long industrial experience with these particular cells and developed cell banks that are validated and registered around the world. even for such a direct experience-based approach, we realized that it would take months to make a good manufacturing practice (gmp) clinical lot for phase and clinical studies. killed vaccines need, in many cases, adjuvantation. in our case, the adjuvant of choice was aluminum hydroxide. again, in the situation where a new virus is emerging, there is no time to evaluate new adjuvants. our preference was to use aluminum hydroxide viruses can be inactivated by several methods, based on either physical or chemical mechanisms. we investigated five decontamination methods that are currently used for equipment, facilities, and waste decontamination: heat, alkaline treatment, sodium hypochlorite treatment, gaseous formol fumigation, and drying. it should be stressed that the data presented here have been obtained under our specific experimental conditions. indeed, the virus sensitivity to inactivation depends on the virus environment and concentration. thus, the methods presented here were validated with our specific suspensions and experimental conditions, and appropriate precautions should be taken when manipulating sars-cov under other laboratory conditions. inactivation experiments using different ph values gave rise to unexpected results. when the ph was adjusted to ph or ph . , a strong decrease in the viral infectivity titer can be observed. however, the virus is not totally inactivated and even after h of alkaline treatment, infectious particles can still be observed. total inactivation is observed only after h of treatment. these data are surprising as enveloped viruses are usually sensitive to such drastic alkaline conditions. the results from the experiments performed to evaluate the viral loss of the sars-cov due to drying on glass surface were also surprising: - days were necessary to inactivate the virus to the detection limit of the technique. other viruses, such as polio or rabies, can be inactivated by drying durations of approximately and h, respectively. in our experience, the sars-cov is the most resistant virus ever described in an industrial setting. the gaseous formaldehyde fumigation is a viral decontamination technique widely used throughout the world. we found that formol fumigation is totally inefficient on dried virus. however, virus in solution is efficiently inactivated. the two decontamination techniques tested here, drying and formaldehyde fumigation, reinforce the necessity to decontaminate the working areas in the laboratory, as well as the equipment, very frequently in order to avoid any drying of viral suspension onto glass or any other surface. finally, sodium hypochlorite ( ° cl) and heat treatment were evaluated. the effect of sodium hypochlorite on dried virus is very rapid and efficient, as no infectious viral particles were recovered after washing the surface with sodium hypochlorite. thermal decontamination was shown to be efficient at both and ° c. to achieve total inactivation of the sars-cov, h heating at ° c and h heating at ° c are necessary. considering the decontamination data, the following strategy was put in place. all solid waste, as well as the equipment that could resist such a drastic treatment, was autoclaved. for liquid waste, the solutions were subjected to alkaline ph treatment for at least h and then transferred into a tank for thermal decontamination at ° c for min. the laboratory facilities, and the equipment that could not be autoclaved, were decontaminated with ° cl sodium hypochlorite solutions, before being fumigated with gaseous formol. the data on decontamination of the sars-cov presented here show that existing decontamination strategies cannot be directly extrapolated to emerging viruses and that these inactivation conditions should be determined empirically for each virus. the sars-cov seed virus isolate was provided on august , , by the cdc. this isolate (the so-called utah strain) was made from the sputum from an acutely ill u.s. traveler who had apparently been exposed in hong kong. this isolate was fully sequenced by the cdc and shown to be virtually identical to the urbani strain of sars-cov. to obtain an original seed virus, in full accordance with food and drug administration (fda) requirements, sanofi pasteur provided certified vero cells to the cdc, who performed the isolation of the virus and made two passages before sending the virus to sanofi pasteur. upon receipt, the virus underwent two additional passages, and was plaque purified. this plaque purification step is of importance to limit the risk of adventitious agents during the subsequent expansion of the virus. in contrast, there is also a risk that in selecting a plaque, it may differ significantly from the uncloned vaccine. to evaluate the latter, it was decided to compare cloned vs. uncloned virus in terms of virus sequence and immunogenicity in guinea pigs. it was demonstrated that the two candidate vaccines were totally similar. following this verification, the selected clone (vvnfl ) was passaged eight additional times for adaptation. from our experience with viruses to be used to prepare an inactivated viral vaccine at industrial scale, there is a need to reach a titer of virus Ͼ . log/ml. indeed, with the sars-cov, we obtained a consistent titer of around . log tcid /ml. in vero cells, distinct cytopathic effect (cpe) is always observed at day or post-infection. these first characterization was performed using pcr testing. the pcr tests are listed in table . . all tests were performed in accordance with international requirements, showing that no adventitious agents were detected. the difficulty with inactivating viruses remains the balance between fully validated inactivation and preservation of immunogenicity or epitopes associated with protection. it is well known that reagents used to inactivate viruses [betapropiolactone (bpl), formaldehyde] can change the outer membrane antigens with the risk of a reduced immunogenicity of the vaccine. for the inactivation of sars-cov, we chose to test bpl. assays were performed to determine the best bpl concentration for inactivation while maintaining a good immune response in mice. virus experiments encouraged us that the prototype vaccine could be produced in vero cells using a single harvest totally compatible with our experience with inactivated poliovirus. raw materials used to develop the candidate vaccine (serum and trypsin) were selected in accordance with current regulation. calf serum was imported from australia, and gamma irradiated prior to use. the trypsin was from porcine origin and also gamma irradiated. extensive evaluation for adventitious agents was performed on the raw material, which included the search for cytopathogenic agents, hemadsorbing agents, and specific viral contaminants as bluetongue, reovirus, rabies, parainfluenza type , specific bovine viruses [adenovirus, parvovirus, respiratory syncytial virus (rsv)], bovine viral diarrhea, rhinotracheitis virus), and porcine viruses (parvovirus, adenovirus; transmissible gastroenteritis virus; hepatitis e virus, rabies virus, and porcine pestis virus). the viral seed lots were produced in vero cells. qc testing is a major step in the qualification of such a seed. of all the different tests performed (identification of the vero cells, sterility, mycoplasma, titer, and contaminating viruses), the research of contaminating virus(es), also called adventitious agents, represents the most crucial step. to detect adventitious agents, sensitive cell culture monolayers of cv- cells, human diploid mrc- cells, and chick embryo fibroblasts (cefs) were inoculated with the crude viral suspension and are observed for induced cpe and/or hemadsorption. cv- cells are used in these tests, as they are of the same species and the same origin as the cells used for vaccine manufacturing. mrc- cells are human diploid cells and can potentially reveal other viruses able to infect human cells. and finally, taking into consideration the origin of the specimen (pulmonary syndrome) we added cef, which are known to be sensitive to the infection of several respiratory viruses such as influenza virus and rsv. at the same time, adventitious agent testing was performed on control cells (search for hemagglutining or hemadsorbant viruses) and on the supernatant of the control cells (search for adventitious agents) by inoculation of the three cell lines: vero, mrc- , and cef. adventitious agent testing was also done in vivo by inoculation in suckling mice, mice, and guinea pigs, as well as the allanto ï c cavity and yolk sac of embryonated chicken eggs. complementary to the conventional methods to qualify viral seed, as described above, extensive inactivation was performed using three different bpl concentrations: / , / , and / (v/v). in our experience, a / dilution of bpl was the optimal concentration for the inactivation of sars-cov based on a balance between total inactivation and maintenance of antigenic properties. the / bpl dilution is similar to what is used for rabies vaccine inactivation. the usual way to measure viral inactivation is by kinetic studies, i.e., reduction in virus infectivity. this technique has a detection limit of . log tcid / ml. the kinetics of inactivation was performed at , , min, and , , , , , , and h, and it was shown that inactivation below the limit of detection was obtained after h. in order to increase the detection sensitivity, an amplification test was also performed. for this amplification, vero cells are incubated with a portion of the viral solution following inactivation for different periods of time. after days of incubation, the cells are trypsinized and cultivated for additional days. at the different stages of amplification, the cells were microscopically observed for cpe and at the end of the incubation (day ) an immunocolorimetric assay test was performed. our data demonstrated that the virus is fully inactivated by h, not h of bpl treatment, demonstrating that the amplification test is much more sensitive. to complete the validation of the amplification test, the minimum limit of detectable infectious viral particles was determined. this was done by spiking the inactivated vaccine with different concentrations of live virus and incubating with vero cells. using this approach, it was possible to establish the minimum virus detection as pfu. based on these data, it was concluded that inactivation with / bpl dilution for h fully inactivates the sars-cov batches. to ensure a very large safety margin, we adopted an inactivation period of h for the sars-cov vaccine production process. since a direct efficacy trial in humans will be impossible, because of a lack of naturally circulating sars-cov, the licensure of a sars-cov vaccine will depend on surrogate markers. recently (as described below) the fda adopted the animal efficacy rule that envisions that under such circumstances, demonstration of efficacy can be performed in two animal models. for sars-cov vaccine development, monkeys and ferrets can be used to evaluate candidate vaccine. both animal models show pathology in the lungs upon autopsy. the immunogenicity of the sars vaccine was evaluated in nonhuman primates, m. fascicularis, and ferrets. both animal models are susceptible to infection, do show some signs of disease (lethargy), and show signs of pulmonary lesions upon histological examination ( fouchier et al., ; martina et al., ; ter meulen et al., ; rowe et al., ; mcauliffe et al., ) . different doses of the sars vaccine ( or log tcid /ml) were injected in the presence or absence of aluminum hydroxide. two intramuscular injections were performed at a one month interval. regarding the humoral response, sustained levels of elisa and serum-neutralizing virus-specific antibodies were elicited in vaccinated monkeys and ferrets. a significant dose -effect relationship could be demonstrated. moreover, a strong adjuvant effect of aluminum hydroxide was evidenced for each vaccine dose and proved in most cases to be highly significant. in order to evaluate the efficacy of the sars vaccine, immunized monkeys and ferrets were challenged intratracheally with a heterologous hong kong sars-cov strain (coronovative, rotterdam, the netherlands). monkeys immunized with or log of inactivated virus were protected as measured by rt-pcr and viral titration on lung samples five days post-challenge. the ferrets were protected at the lower immunization dose of or log . based on our experience to date, the inactivated, adjuvanted sars-cov prototype vaccine seems to be a good candidate for further evaluation in phase studies. as with other vaccines, vaccines for sars and other emerging threats need to follow a structured pattern of regulatory development. the initial stages would be very similar to those followed for vaccines under development for conventional infectious diseases. in the united states, the earliest stages would include the development of sufficient preclinical information about the vaccine to allow the preparation of an investigational new drug (ind) application for submission to the fda (see chapter ). the ind may have information unique to the vaccine candidate but should include information about the rationale for the vaccine design, the source of the virus and other components, the manufacture of the active vaccine component, formulation, preliminary characterization of the vaccine to determine the effectiveness of the vaccine. for these agents, it would be too dangerous to conduct challenge studies in humans and the prevalence of the disease is either nonexistent, sporadic, or too small to allow the development of a reasonable clinical protocol. in anticipation of this problem, largely in the face of potential bioterrorist agents, in , the fda adapted the so-called animal rule [see federal register, may , (volume , number ) ]. under this guidance, new drugs or biological products that are intended to prevent serious or life-threatening conditions may be approved on evidence of effectiveness derived from appropriate studies in animals and any additional supporting data, if controlled clinical studies cannot be conducted in human volunteers and field trials are not possible. in order to satisfy this alternative mechanism, however, several criteria must be met. first, there is a reasonably well-understood pathophysiological mechanism that can be ameliorated or prevented by the product. second, the effect is demonstrated in more than one animal species, unless it is demonstrated in a single species that represents a sufficiently well-characterized animal model. third, the animal study endpoint is clearly related to the desired benefit in humans. and finally, the data are sufficiently well understood to allow selection of an effective dose in humans. it is therefore reasonable to expect that the effectiveness of the product in animal model(s) is a reliable indicator of its effectiveness in humans. obviously, it is too early to know whether the sars vaccine candidate as described in this chapter will move forward and be able to meet all of the criteria of the animal rule. in particular, sars vaccine development is hindered by relatively little information about human covs in general. until the rapid emergence of sars, most of the basic research was focused on animal covs and our inactivated sars vaccine candidate described in this chapter is exclusively based on experience with vaccines to animal covs. certainly, it is too early to conclude whether the ferret and/or m. fascicularis is/are the most appropriate model(s) for human sars infections. as a result, except for clinical cases documented during the outbreak, there is relatively little information about sars pathogenesis and correlates of immunity. another difficult aspect is that a feline infectious peritonitis (fip) vaccine was actually harmful to the health of the immunized cats upon challenge with wild-type fip virus ( weiss and scott, ) . before moving forward with approval, therefore, it will be very important to determine whether these adverse outcomes can be prompted or mimicked by any of the sars vaccine candidates. including purity and potential contaminants, immunological testing, and animal testing, including toxicology. even at this early stage, the vaccine should be made under gmp conditions and other laboratory work conducted under good laboratory practice (glp) conditions, as appropriate. the ind application should also include important information about the phase clinical design, focusing on how the safety will be monitored and a discussion of any potential adverse reactions based on the experience with vaccines that have similar components or methods of preparation. there should be an opportunity to outline the vaccine concept and phase clinical study at a pre-ind meeting that often provides the opportunity to receive the input and concerns of the regulatory agency. following approval of the ind, vaccines such as sars can progress to a conventionally designed phase study. typically, this phase clinical study is descriptive and would include a small number of healthy young adults with the emphasis on monitoring the safety of the vaccine (local and systemic reactions). often, the first immunologic assessment is part of this study. following successful completion of the phase , as with other vaccines, a sars vaccine candidate could move forward to phase . during this phase, in addition to safety monitoring, dose-ranging studies are conducted in much larger groups of individuals and the vaccine should meet predefined primary and secondary endpoints. if the phase is successful, following a pre-phase meeting, clinical studies are conducted in a greater number of subjects during which less frequent reactions can be detected and the efficacy or effectiveness of the vaccine determined. as part of this phase evaluation, the consistency of sequential lots of the vaccine are typically compared in order to ensure that the vaccine can be reproducibly manufactured. obviously, as the vaccine progresses clinically from phase to phase , the size of the lots of vaccine often increases and the manufacturing and in-process and release testing specifications become increasingly well defined, so as to guarantee that the vaccine can consistently be made at a commercially useful scale. if all three phases of clinical development are successful, the manufacturer may then submit a biologics license application (bla), which is a very extensive compilation of all of the information relating to the development and manufacture of the vaccine. as suggested in the animal models section above, the unique challenge for sars and other emerging threats, whether anthrax or ebola viruses, is that it may not be possible to conduct phase clinical studies the production of a gmp clinical lot of a monovalent, whole, inactivated, aluminum hydroxideadjuvanted sars-cov vaccine took months. in terms of vaccine development, this is extremely rapid. several factors contributed to these short timelines. the grants made available by the niaid for the development of a sars vaccine completely changed the classical environment, allowing vaccine industries to start almost immediately the development of a new vaccine. indeed, the development of a new vaccine can only be done to the detriment of other vaccine developments, mobilizing teams and facilities. from a technical point of view, the choice of a classical vaccine development strategy using conventional procedures, such as vero cell culture for viral propagation and bpl inactivation, was a decisive factor to success. importantly, we were able to quickly recruit a volunteer workforce that was both familiar with the technology and trained to work in a bsl -plus environment. a close collaboration with the reference laboratory, the cdc's influenza branch, where the sars-cov was isolated, was essential. we provided certified vero cells to the cdc, which allowed us, upon receipt of the purified strain from the cdc, to re-isolate the sars-cov under conditions making the prompt start of a vaccine development possible . when initiating vaccine development against a new emerging infectious agent, the problem of availability of reagents and routine tests to perform biological and molecular studies must be addressed. it is obvious, that at the beginning of such development, there are no such reagents or commercial kits available. as a consequence, the first step in the sars-cov project was to prepare the different reagents (antisera and monoclonal antibodies) and the appropriate tests (viral titration, pcr, elisa, immunofluorescence assay, etc.). finally, a series of preliminary experiments on monkeys (three months after the start of the project) had given guidance whether it was appropriate to use an inactivated vaccine, as well as to the choice of the adjuvant. constant communication with regulatory authorities has allowed the validation of this strategy from the beginning of the project. this communication was also very important for the qualification of the viral seed lots. the qualification of the vero cells was not an issue as several vaccines are already produced in vero cells, but this was obviously not the case for the viral seeds. two major obstacles had to be overcome: ( ) the realization that the animal testing had to be done in bsl facilities by bsl -trained personnel, and ( ) the search for adventitious agents using general classical tests (search for adventitious viruses on cells and in animals) and specific tests (pcr). for the latter, there was no list available and the final testing to be performed was under the responsibility of health authorities. this resulted in a rather exhaustive list of pcr testing. it is likely that epidemics will emerge in the future from unrecognized sources and some of these will be highly pathogenic for humans. these pathogens will be categorized as bsl or bsl pathogens needing high security level laboratories as well as specialized personnel. how to manipulate these pathogens that are highly pathogenic, in large quantities? to face the emergence of new pathogens, dedicated structures are needed with the right equipment and trained personnel. from an industrial perspective, this seems not compatible with the need and use of trained personnel and facilities that do have a constant activity to assure the production of existing vaccines and the development of new vaccines. such emergency structures could be set up and maintained by national reference centers respecting the bsl requirements as well as the gmp conditions. it would be very beneficial for industries to collaborate with such reference centers that provide purified pathogens and reagents allowing a prompt start of a vaccine development . aetiology: koch's postulates fulfilled for sars virus protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus pa/wolgemuth/ virology: sars virus infection of cats and ferrets replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys infectious diseases. second lab accident fuels fears about sars laboratory-acquired sars raises worries on biosafety safety and efficacy of a modified-live canine coronavirus vaccine in dogs macaque model for severe acute respiratory syndrome recent singapore sars case a laboratory accident human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever key: cord- -bjuvxpkc authors: neustroev, m. p.; petrova, s. g. title: developmental results of a vaccine against salmonella-induced equine abortion date: - - journal: russ agric sci doi: . /s sha: doc_id: cord_uid: bjuvxpkc an inactivated vaccine based on the sal. abortus equi bn- strain with the bac. subtilis tnp- strain filtrate used as immunomodulator has been developed in order to prevent salmonella-induced equine abortion. preclinical and clinical trials with the white mice and the horses, respectively, are carried out. the lack of toxicity is proven. the vaccine immunogenicity for mouse and mare models comprised and %, respectively. the industrial vaccine tests showed that the industrial output of foals increased by . % after immunization. cost-effectiveness of the vaccine used with the bac. subtilis tnp- strain filtrate comprised . rubles per ruble of costs, which was . -fold greater when compared to the vaccine used with a polyribonate medicine. it is ascertained that administration of the inactivated vaccine with the bac. subtilis tnp- strain is an effective method to prevent infectious abortion. scientific and technical documentation is developed based on the survey results in order to submit it for approval to the rosselkhoznadzor federal service for veterinary and phytosanitary surveillance. the instruction is compliant with the approved use. the registration certificate ( - - . - no. pvr- - -. / , as of june , ) has been issued. salmonella-induced equine abortion is most common in the asian and african countries, while some cases are reported from european countries, the united states, and argentina [ ] [ ] [ ] [ ] . a high mortality rate caused by sal. abortus equi was recorded in italy [ ] . the abortions associated with viral and bacterial infections caused by the rhinovirus and the salmonella agents of equine abortions are most severe [ ] . mixed infections are especially severe in the young horses, and the mortality rate may reach % [ ] . salmonella carriage among the healthy animals is proven [ ] [ ] . immunization through vaccination is the most effective method to prevent salmonellosis in horses. b.a. matvienko et al. [ ] developed the dry-powder tre- live rabies vaccine used in kazakhstan. complex immunization through live vaccines was suggested to prevent rhinovirus pneumonia and salmonella-induced abortion [ ] [ ] [ ] . at present, too "kaznivi" kazakh research veterinary institute produces the live attenuated vaccine against salmonella-induced abortion in mares that is based on the b- sal. abortus equi strain [ ] . an inactivated vaccine against the salmonellainduced equine abortion, which has been developed by the yakut research institute of agriculture jointly with the virology department, all-russia research institute of experimental veterinary, is used in the stud farming systems in russia [ ] . however, its high price caused by polyribonat modulator restrains the use of the vaccine. moreover, the polyribonat registration date is expired. the necessity for developing prevention means for stopping the spread of new viral infections, including covid- , is amplified. the information on the spread of coronavirus among the horses in colorado state (united states) has appeared [ ] . the objective of the surveys was to develop and test the ecologically safe and effective vaccine product for specific prevention of the salmonella-induced equine abortion. the survey was carried out at the veterinary biotechnology laboratory, safronov yakut research institute of agriculture, along with too "khotu-bakt" science production center and the horse stud farms in the sakha republic (yakutia). the sal. abortus equi bn- strain stored at the all-russia state research institute for control, standardization, and certification of veterinary preparations was used in order to develop a vaccine against the salmonella-induced equine abortion. the bac. subtilis tnp- strain-based culture liquid (cl or filtrate) stored at the all-russia state research institute for veterinary control, standardization, and certification of veterinary preparations (february , ) was used in the vaccine's composition. the analysis of vaccine toxicity after a single administration of injection through the abdomen to nonpedigreed white mice was performed according to the "sanitary regulations sp . . . - " (affirmed by decree no. of october , , rf state sanitary and epidemiological surveillance service). in order to perform the experiment, - -g mice were divided into three groups by the analog design principle. the bac. subtilis tnp- liquid, the vaccine, and the distilled water were administered to the animals in groups one, two, and three (control), respectively. the preparations were injected at a dose of ml into each in abdomen on an empty stomach in the morning. clinical observations were carried out for subsequent days, which was followed by killing the animals with alcohol ether in order to carry out the anatomical pathology tests. the anatomical pathology testing was performed according to the common methods. in order to analyze the vaccine toxicity for the mares (allocated by four animals), the vaccine at doses of ml, ml, and ml was administered with an intramuscular injection in the area of the neck of each of the horses. clinical observation was carried out for month. in order to study the stress-related immune changes, the white mice were allocated into three groups of animals each. the animals of group one were immunized with the inactivated vaccine used with the culture liquid containing the bac. subtilis strain at a dose of . ml. the animals of group two were subcutaneously injected with the inactivated vaccine used with the polyribonat modulator at a dose of . ml in the area of the animals' dorsal side. the animals of group three (control) were not administered any preparation. in days after immunization, the mice were infected with a diurnal culture of a pathogen strain. in order to analyze the vaccine-induced immune parameters, three groups of mares each in the horse stud section on the "pokrovskoe" experimental farm were formed in november-december . the animals of group one were treated with the inactivated vaccine containing the bac. subtilis strain. the animals of group two were treated with the inactivated vaccine containing the polyribonate. the animals of (control) group three were not vaccinated. the preparations at a dose of ml were administered with intramuscular injection in the upper third area of the neck. three infoal mares from each of the groups were sampled to infect them with the sal. abortus equi pathogen culture at the minimum infectious dose days after immunization. clinical observations for the animals were carried out. the mares at - months of gestation were sampled among the two stud sections on the "pokrodskoe" experimental farm in november to test them with the inactivated vaccine. the mares were treated with the "ivomek" preparation for deworming prior to the experiment. the animals were allocated into three groups. the animals of groups one and two were treated with the inactivated vaccine containing the polyribonate and the bac. subtilis tnp- strain, respectively, while the animals in (control) group three were not injected with the vaccine. the the body's condition in the white mice during the experiment was generally satisfactory. motion activity and responses to the external irritants remained common. the appetite was not lost. the hair coat was smooth. the texture of mouse droppings did not change. at the end of the experiment, the body weight gain in the mice of groups one, two, and three comprised . , . , and . g, respectively. anatomical pathology testing the dead white mice of the experimental and control groups did not reveal any organ or tissue pathology. the organ structures were not affected. according to the histological analysis, acute venous stasis in the parenchymal regions and the pulmonary edema were recorded. the pattern of changes was typical for the postmortem changes after asphyxia. changes in the gastric mucosa in the experimental mice were typical for gastritis that occurred in response to the preparation administration. the histology of the parenchymal organs was unchanged. the outcomes of the observations for the vaccinated mares could sometimes indicate a swell of pasty consistency at the injection site, which usually lasted for - days. for the whole period of observations, no refusal of mice to take forage and no symptom indicating depression were revealed. after infection of the immunized white mice with the virulent culture, two mice, four mice, and mice from groups one, two, and three (control), respectively, died. no abortion was revealed in the vaccinated mares of groups one and two after infection. unvaccinated mares of group three were aborted after infection. the sal. abortus equi pathogenic agent was isolated in the aborted fetuses. biochemical profile of mare blood tests showed that there was no change in the levels of total protein and its fractions on the th and th days after vacci- russian nation. in addition, insignificantly increased alpha globulin levels and decreased gamma globulin levels as the fractions of total protein were recorded in the mares of experimental group one. the increased levels of total protein and its albumin and gamma globulin fractions was recorded in the animals of experimental groups one and two on the th day after vaccination. this period coincides with the period of maximum synthesis of agglutinating antibodies. the outcomes of the industrial tests carried out on the "nemyugyuntsy" farm have proven that administration of the inactivated bac. subtilis tnp- strain vaccine against salmonella-induced equine abortion is an effective method to prevent infectious abortions since it contributes to a . % increase in the industrial output of foals (table ). in the animals of group one, abortions were registered; however, salmonellosis etiology was not determined. in the control group, the agent of salmonellosis causing equin abortion was isolated from three samples of abortions. the industrial tests were continued on the farms in the "taatta" and megin-kangalassy districts or raions known as uluses and the yakutsk suburbs, where in-foal mares were vaccinated. the industrial output of foals comprised . , . , and . %, which is significantly higher than the values for the previous years, which varied at the level of . - . %. the culture liquid containing the bac. subtilis tnp- strain and the vaccine against salmonellainduced equine abortion used with filtrate do not affect the body condition, do not cause any allergic reactions and pathological changes in organs and tissues, and have no toxic effect on the organisms of laboratory and target animals. the preclinical tests with the white mice and the industrial tests with the horses have proven that the inactivated bac. subtilis tnp- strain vaccine against salmonella-induced equine abortion is an effective method to prevent infectious abortions. the cost-effectiveness of the vaccine used with polyribonat comprises . rubles per ruble of costs. the cost-effectiveness of the vaccine used with the culture filtrate of bac. subtilis tnp- becomes . fold greater, comprising . rubles per ruble of costs. it should be noted that the previous surveys have determined that the use of live vaccines against salmonella-induced abortion and pneumonia caused by rhinoviruses is not reasonable because of the extreme climate environments for stud horses kept in yakutia. the vaccine developed by the authors is not inferior to the live vaccines proposed in kazakhstan in the effectiveness in the epizootic situations [ ] . therefore, the method developed by the authors for specific prevention of the salmonella-induced equine abortion with the inactivated vaccine used with the culture liquid based on the bac. subtilis tnp- strain is a cost-effective measure that should be recommended for widespread adoption in the stud farming systems. the authors consider that the high effectiveness of the inactivated vaccine may be explained by both the antigen activity of the vaccine strain and the immunomodulatory component represented by the culture liquid (filtrate) of the bac. subtilis tnp- strain. the probability to use the probiotics and the other biologically active substances as immunomodulators within animal vaccination against brucellosis [ ] , cow salmonellosis [ ] , and arctic fox salmonellosis [ ] is ascertained. the probability of using the bac. subtilis tnp- strain filtrate as a modulator to develop the inactivated vaccine against equine pneumonia caused by rhinoviruses has been proven. the bac. subtilis tnp- strain may induce the interferon synthesis and stimulate immunogenicity of inactivated bacterial and viral vaccines [ , ] . scientific and technical documentation has been developed based on the survey results to submit for approval to the "rosselkhoznadzor" federal service for veterinary and phytosanitary surveillance. the instruction is compliant with the approved use. a registration certificate ( - - . - no. pvr- - -. / of june , ) has been issued. conflict of interest. the authors declare that they have no conflict of interest. statement of welfare of animals. all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. the article does not concern any researches using animals as objects. salmonella enterica serovar abortusequi as an emergent pathogen causing equine abortion in argentine occurrence of multiple abortions due to salmonella enterica serovar abortusequi infection causes of equine abortion, stillbirth and neonatal death in central italy prevalence of salmonella in diverse environmental farm samples high mortality in foals associated with salmonella enterica subsp. enterica abortusequi infection in italy infektsionnye bolezni loshadei features of the epizootology of salmonella abortion of horses in yakutia, sel'skokhozyaistvennye nauki i agropromyshlennyi kompleks na rubezhe vekov: sbornik materialov xx mezhdunarodnoi nauchno-prakticheskoi konferentsii (agricultural sciences and agro-industrial complex at the turn of the century pathological manifestation and treatment of thrombohemorrhagic syndrome in salmonellosis in calves identifying the bacterial causes of abortion inmares characteristics of a live dry vaccine against paratyphoid abortion in mares from strain e- complex immunoprophylaxis of rhinopneumonia and salmonella abortion in mares with live vaccines biological and immunogenic properties of vaccine strains against salmonella abortion and equine rhinopneumonia infektsionnye bolezni loshadei specific prevention of salmonella abortion in mares in kazakhstan, agrarnaya nauka -sel'skokhozyaistvennomu proizvodstvu sibiri, kazakhstana, mongolii, belorussii i bolgarii: materialy xxi mezhdunarodnoi nauchno-prakticheskoi konferentsii a method for enhancing the efficacy of vaccination and reducing the frequency of infectious abortion in herd horse breeding disease features of equine coronavirus and enteric salmonellosis are similar in horses possibility of using probiotics when vaccinating animals with various antibrucellosis vaccines, materialy shestoi mezhregional'noi konferentsii, posvyashchennoi -letiyu sibnivi vniit-bzh the use of immunomodulators in the vaccination of animals against salmonellosis the use of subalin in the vaccination of polar fox puppies against salmonellosis immunomodulating properties of endogenous interferon in calves, veterinariya key: cord- - tfg h authors: dong, rong; chu, zhugang; yu, fuxun; zha, yan title: contriving multi-epitope subunit of vaccine for covid- : immunoinformatics approaches date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tfg h covid- has recently become the most serious threat to public health, and its prevalence has been increasing at an alarming rate. the incubation period for the virus is ~ – days and all age groups may be susceptible to a fatality rate of about . %. covid- is caused by a novel single-stranded, positive (+) sense rna beta coronavirus. the development of a vaccine for sars-cov- is an urgent need worldwide. immunoinformatics approaches are both cost-effective and convenient, as in silico predictions can reduce the number of experiments needed. in this study, with the aid of immunoinformatics tools, we tried to design a multi-epitope vaccine that can be used for the prevention and treatment of covid- . the epitopes were computed by using b cells, cytotoxic t lymphocytes (ctl), and helper t lymphocytes (htl) base on the proteins of sars-cov- . a vaccine was devised by fusing together the b cell, htl, and ctl epitopes with linkers. to enhance the immunogenicity, the β-defensin ( mer) amino acid sequence, and pan-hla dr binding epitopes ( aa) were adjoined to the n-terminal of the vaccine with the help of the eaaak linker. to enable the intracellular delivery of the modeled vaccine, a tat sequence ( aa) was appended to c-terminal. linkers play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable. the secondary and three-dimensional ( d) structure of the final vaccine was then predicted. furthermore, the complex between the final vaccine and immune receptors (toll-like receptor- (tlr- ), major histocompatibility complex (mhc-i), and mhc-ii) were evaluated by molecular docking. lastly, to confirm the expression of the designed vaccine, the mrna of the vaccine was enhanced with the aid of the java codon adaptation tool, and the secondary structure was generated from mfold. then we performed in silico cloning. the final vaccine requires experimental validation to determine its safety and efficacy in controlling sars-cov- infections. in december , covid- , caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) was first discovered in china and has rapidly spread across the world. as of : noon on june , a total of , , confirmed cases of covid- have been reported globally, including , deaths. the prevalence of the disease has been increasing at an alarming rate. there were , , cases in the united states, , in brazil, , in russia, , in the united kingdom, and , , in a number of other countries ( ) . the incubation period for the virus is ∼ - days, and all age groups are susceptible to a fatality rate of about . %. the most common clinical manifestations are low-grade fever, dry cough, fatigue, and gastrointestinal symptoms ( ) . about half of all patients with covid- develop shortness of breath, and severe cases may rapidly develop sars, septic shock, difficult-to-correct metabolic acidosis, and coagulation disorders ( ) . covid- may also affect other organs, most commonly the heart and kidneys ( ) ( ) ( ) . some patients may have mild symptoms, without fever, and may recover after - weeks ( ) . other patients may show signs of serious illness and some may die; however, most patients show favorable progress ( ) . male individuals with the disease and aged patients have the worst prognosis. in children, the disease is relatively mild ( ) . covid- is caused by a novel single-stranded, positive (+) sense rna beta coronavirus, which is a pathogen of the coronaviridae family, named sars-cov- ( ) . the full-length genome sequences revealed that sars-cov- has the greatest genetic similarity to bat coronavirus, ∼ - % similarity to severe acute respiratory syndromerelated coronavirus (sarsr-cov), and a smaller similarity of - % to the middle east respiratory syndrome-related coronavirus (mers-cov) ( ) . thus, a bat might be the original host of sars-cov- , but the intermediate host remains undiscovered ( ) . the genes of sars-cov- encode structural proteins and non-structural proteins. four structural proteins are absolutely vital for viral assembly and invasion of sars-cov- . spike protein homotrimers constitute the spikes on the viral surface, and these spikes are responsible for attachment to host cells by binding to their receptors ( ) . the m protein has three transmembrane domains, which determine the shape of the virion, facilitate membrane curvature, and bind to the nucleocapsid. the e protein plays an important role in virion assembly and release, as well as involved in viral pathogenesis. the n protein has two different domains, both of which bind to the viral rna genome via totally different mechanisms. in addition, some reports have shown that non-structural proteins are essential for the replication of coronaviruses ( ) . vaccination is a vital tool for the control and elimination of the virus, and the development of a vaccine for sars-cov- remains an urgent need ( ) . traditional methods of vaccine development are time-consuming and very labor-intensive ( ). the realm of immunoinformatics tools considers the mechanism of the host immune response to yield additional methodologies in the design of vaccine against diseases are cost-effective and convenient, as in silico predictions can reduce the number of experiments needed ( , ) . dozens of studies have generated epitope-based peptide vaccine of sars-cov- . baruah and bose ( ) used immunoinformatics tools to discover cytotoxic t lymphocyte (ctl) and b cell epitopes for the spike protein of sars-cov- . then, abraham et al. developed a multi-epitope vaccine that was designed using immunoinformatics tools that potentially trigger both cd + and cd + t-cell immune responses ( ) . although there are many vaccines generated by immunoinformatics tools, most of these are based on spike protein. the spike protein is responsible for attachment to host cells by binding to angiotensin-converting enzyme (ace ) ( ) . a vaccine based on the spike protein could induce antibodies to block sars-cov binding and fusion or neutralize virus infection ( ) . but there are still many obstacles, spike protein-based sars vaccine may induce harmful immune responses that cause liver damage of the vaccinated animals ( ) . other virus proteins are considered as the candidates for designing vaccine with protective and less harmful immune responses ( ) . vaccine-based on structural and non-structural proteins of the virus is revealed potential vaccine inducing protective immune responses ( , ) . pandey et al. reported the more scientifically rigorous strategy of multi-epitope subunits based on multiple proteins against parasitic and viral diseases, such as malaria, visceral leishmaniasis, and hiv ( ) ( ) ( ) . in this present, we employed immunoinformatics to predict multiple immunogenic proteins from the sars-cov- proteome and thereby design a multi-epitope vaccine. these proteins included non-structural and structural sequences of sars-cov- , their reference sequences were retrieved from the national center for biotechnology information (ncbi) database. the proteins of the sars-cov- have been reported and reference could get from ncbi ( , ) . the reference sequences of sars-cov- proteins were retrieved from ncbi protein database (https://www.ncbi.nlm.nih.gov/protein) and accession numbers in table , then we stored the reference sequences as a fasta data type. the proteins with < amino acid sequences which are too short to predict epitopes were excluded, the remaining proteins were used for further analysis. vaxijen is the first server for alignment-independent prediction of protective antigens, which overcome the limitations of alignment-dependent methods ( ) . to identify the potential antigenicity of sars-cov- proteins, an online prediction server, vaxijen v . (http://www.ddg-pharmfac.net/vaxijen/ vaxijen/vaxijen.html) was used to predict the antigenic values of each protein ( ) . this identification was applied according to the default parameters of the server. proteins having antigenicity were sorted according to an antigenic score of ≥ . (threshold for this model is . ) and were selected for further structural modeling ( ) . there are no available experimental structures of sars-cov- proteins, phyre provide model regions trough a new ab initio folding simulation with no detectable homology ( ) . the sars-cov- proteins were modeled by phyre server (http://www.sbg. bio.ic.ac.uk/phyre /). because the sars-cov- proteins with no detectable homology protein to finish the modeling, we chose the intensive search and output the accurate alignment by the alignment of hidden markov models. modrefiner was used by the galaxyrefine server (http:// galaxy.seoklab.org /cgi-bin/submit.cgi?type=refine) ( ) . the structure assessment was performed by the swiss-model workspace (https://swissmodel.expasy.org/assess) ( ) . the three dimensional ( d) models were used for the conformational (discontinuous) b-cell epitope predictions while the sequences were utilized in linear b-cell and t-cell epitope predictions. netctl- . is demonstrated to have a high predictive performance ( ) . the netctl . server (http://www.cbs. dtu.dk/services/netctl/) was applied to predict ctl epitopes for the sars-cov- at the threshold value of . with high sensitivity and specificity ( ) . to cover ∼ % of the world's population, three supertypes (a , a , and b ) were selected based on artificial neural networks, to predict mhc class i binding epitopes ( ) . the best candidates for the sars-cov- vaccine construction were sorted for further prediction, based on a half-maximal inhibitory concentration (ic ) < nm and an integrated score. the ic < nm represents epitope has a high affinity to receptor. the integrated score indicated the transporter of antigenic peptides (tap) transport efficiency, class i binding, and proteasomal cleavage prediction ( ) ( ) ( ) . then the specific treg epitopes were screened and excluded by the epitoolkit (https://epivax.com/). for mhc class ii t cell epitope predictions, the immune epitope database server predicted binders based on the percentile rank or mhc binding affinity ( ) . the immune epitope database server (iedb; http://tools.iedb.org/mhcii/) was used to predict helper t lymphocyte (htl) epitopes ( ) . we chose the combinatorial approach which recommended by iedb to predict htl epitopes. the combinatorial approach combined nn-align, smm-align, comblib, sturniolo, and netmhciipan methods ( ) ( ) ( ) ( ) ( ) . the alleles of the human leukocyte antigen (hla) were selected for the prediction at α and β chains, separately ( ) . for final construction, epitopes were selected based on their scores (low scores indicated favorable binding), the release of interferongamma (ifn-γ), induction of emergent properties, and the ic < nm. the ifn-γ cytokine makes a major contribution to antiviral mechanisms. it excites both native and specific immune responses by activating macrophages and natural killer cells ( ) . further, ifn-γ augments the response of mhc to antigens. the ifn-γ epitope server (http://crdd.osdd.net/raghava/ifnepitope/ scan.php) was used to recognize ifn-γ epitopes ( ) . we entered the htl epitopes with low scores into the ifn-γ epitope server. positive ifn-γ induction was predicted based on the support vector machine (svm) hybrid approach. the final htl epitopes were determined based on ifn-γ induction and mhc class ii binding, both of which facilitate the stimulation of t-helper cells ( ) . the abcpred (http://crdd.osdd.net/raghava/abcpred/) and bepipred linear epitope prediction (http://tools.iedb.org/bcell/ result/) servers were utilized to predict linear b cell epitopes. the abcpred server is based on an artificial neural network (ann) ( , ) . the linear b cell epitopes of the sars-cov- protein were predicted at a threshold of . . the bepipred linear epitope prediction server is based on seven methods: ( ) ( ) ( ) ( ) ( ) ( ) . we used these seven methods separately to predict the average threshold. the overlap between abcpred and bepipred severs was selected to determine the candidate epitopes for the sars-cov- vaccine construction ( ) . unlike t-cell epitopes that are linear continuous stretches of residues, b-cell epitopes are generally conformational (discontinuous) ( ) . in this study, the ellipro servers (http://tools.iedb.org/ellipro/) were applied to predict the conformational b-cells epitopes ( ) . the server predicts epitopes based on pi (protrusion index) value. the epitope with pi = . would include % of residues with % being outside the ellipsoid, discontinues b-cells epitopes with the top pi value was selected for vaccine designing ( ) . to develop the final vaccine, epitopes determined by various immunoinformatics software were linked together with the aid of separate linkers. the ctl epitopes were linked by the aay linker, htl epitopes by the gpgpg linker, and b cells were linked by the kk linker ( , , ) . to increase the vaccine immunogenicity, the β-defensin ( mer) amino acid sequence was adjoined to the n-terminal of the vaccine with the help of the eaaak linker ( ). the β-defensin peptides provoke innate immunity cells and recruit naive t cells through the chemokine receptor- (ccr- ) ( ). the pan-hla dr binding epitopes ( aa) as well as added to the n-terminal of the vaccine with the aid of the same linker ( ) . the pan-hla dr binding epitopes in vaccine construct facilitating binding to many different types of mouse and human mhc-ii alleles to induce cd -helper cell responses ( ) . to enable the intracellular delivery of the modeled vaccine, a tat sequence ( aa) was appended to c-terminal ( ) . linkers (ayy, kk, and gpgpg) play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable ( ). the allergenic proteins induce a harmful immune response, allergenicity of the vaccine should be non-allergic ( ) . the non-allergic character of the vaccine sequence was evaluated by the algpred server (http://www.imtech.res.in/raghava/algpred/) ( ) . we predicted allergenicity of vaccine sequences choosing a hybrid approach (svmc+ige epitope+arps blast+mast) with the highest accuracy and sensitivity ( ) . the vaxijen v . server (http://www.ddgpharmfac.net/ vaxijen/vaxijen/vaxijen.html) was applied to evaluate the antigenicity of the vaccine ( ) . the antigenicity prediction method was solely based on the physicochemical properties of proteins without recourse to sequence alignment. the precision rate of the server ranged from to %. to determine immune response profile of this multi-epitope vaccine, computational immune simulations were performed by the c-immsim online server at (http://kraken.iac.rm.cnr.it/ c-immsim/) ( ) . the c-immsim utilizes the celada-seiden model for describing both humoral and cellular profiles of a mammalian immune system against designed vaccine. as per the literature, three injections were administrated at different intervals of month. the simulation was performed with default parameters. the vaccine sequence was administered weeks apart. the simulation volume was , , simulation steps was , , random seed was , , and the vaccine injection with no lps ( ). the protparam tool (http://web.expasy.org/protparam/) was used to evaluate the physicochemical properties of the final vaccine protein ( ) . the physicochemical properties included the number of amino acids, molecular weight, theoretical isoelectric point (pi), amino acid composition, atomic composition, formula, extinction coefficients, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (gravy) ( ) . the molecular weight and theoretical pi were computed by user-entered sequences. the amino acid and atomic compositions were self-explanatory. the extinction coefficient of a protein was based on information about its amino acid composition. the instability index of a protein indirectly indicated the stability of the protein. if the computed instability index of protein was < , it was regarded as a stable protein, while values > were regarded as unstable. in vivo half-life evaluation of proteins was based on the principle of the "n-end rule." furthermore, gravy is a measurement of the hydrophobic nature of the protein, which is calculated by determining the total hydropathy of all amino acids divided by the number of amino acid residues in the protein. to avoid inducing pathogenic priming and autoimmunity, the sequence homology of the final vaccine to human protein was screened by blastp online server (https://blast.ncbi.nlm.nih. gov/blast.cgi) ( ) . an ideal vaccine should have non-sequence to human proteins. the designed vaccine was a reconstructed protein with no detectable homology ( ) . phyre incorporates an ab initio folding simulation to model regions of proteins with no detectable homology. the phyre server (http://www.sbg.bio. ic.ac. uk/phyre /) was used to predict the three-dimensional structure of the designed vaccine. the server generates a fulllength d model of a protein sequence by employing both multiple template modeling and simplified ab initio folding simulation ( ) . to enhance the overall and partial structural quality of the protein, the output d structure of the final vaccine from the phyre server was further refined by the galaxyrefine server (http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=refine) ( ) . the galaxyrefine server predicted five refined models of our developed vaccine construct, in which model was made by the structural perturbation based simply on the clusters of the side chains; whereas, models - were generated by deeper perturbations of loops and secondary structural elements ( ) . for the assessment of the tertiary structure of the final vaccine protein, a ramachandran plot was performed by the swiss-model workspace (https://swissmodel.expasy.org/ assess) ( ) . the ramachandran plot illuminates favored regions for backbone dihedral angles against amino acid residues in protein structure ( ) . the structure assessment page shows the most relevant scores provided by molprobity and help we easily identify where residues of low quality lie in their model or structure ( ) . then, prosa-web (https://prosa.services.came. sbg.ac.at/prosa.php) was employed in the final vaccine protein structure validation. a positive z-score commonly means an erroneous or erratic section found in the generated d protein model ( ) . to revealing the binding affinity between the vaccine construct and antigenic recognition receptors of toll-like receptor- (tlr , a z) and major histocompatibility complex (mhc-i, wuu, and mhc-ii, c j) present on the surface of immune cells ( ) . docking analysis was performed using the cluspro server (https://cluspro.bu.edu/login.php?redir/queue. php). tlr act as receptors for antigenic recognition. the cluspro server computed the models based on electrostatic interactions and desolvation energy ( ) . to reconfirm the binding affinity of the designed vaccine construct between these receptors, the patchdock server (https://bioinfo d.cs.tau.ac.il/ patchdock/) was used for docking ( ) . the server predicted the potential complex with the help of three algorithm-molecular shape representations, surface patch matching, filtering, and scoring ( ) . after the acquisition of the output from the patchdock server, the complexes were refined by the firedock algorithm, which predicted the optimal complex with the aid of energy functions ( ) . the pdb file of vaccine protein and receptor complex (tlr , mhc-i, and mhc-ii) were used to start the molecular dynamic (md) simulations. the complexes were placed in a octahedron box of water molecules represented by the threepoint charge spc model, whose boundary is at least Å from any protein atoms. the solvated protein was subsequently neutralized by chloridions. covalent bonds involving hydrogen atoms were constrained using the lincs algorithm, and longrange electrostatic interactions were treated with particle-mesh ewald employing a real-space cutoff of Å. the system was first briefly minimized with backbone atoms restrained to the initial coordinates to remove close contacts, and the restrained system was gradually heated to k under constant volume conditions in ps. each system was equilibrated for ns using the constant isothermal-isobaric ensemble at atm and k without any restraints. the parrinello-rahman barostat and a v-rescale thermostat were used with an integration time step of fs. production run md simulations were performed for ns with coordinates recorded every ps. all simulations were performed using gromacs . along with the gromos a force field ( , ) . for the purpose of cloning, codon adaptation of the designed vaccine was performed for analyzing the codon usage by the prokaryotic organism (escherichia coli, e. coli). the java codon adaptation tool (http://www.jcat.de/) was used to optimize codon ( ) . then the secondary structure of mrna was predicted by mfold (http://unafold.rna.albany.edu/? q=mfold) ( ) . for raising the expression efficiency of the final vaccine protein, the e. coli k strain was chosen. for the valid translation of the vaccine gene, we proofread and avoided rho-independent transcription termination, prokaryote ribosome binding site, and cleavage site of restriction enzymes. restriction endonuclease sites xhoi and bamhi were appended to n and c terminals of vaccine, respectively. then, it was inserted into the pet a (+) vector between the xhoi and bamhi. the flow chart of the designed work is shown in figure . the strategy of vaccine construction is presented in figure . the proteome of sars-cov- was retrieved, which comprised proteins. the reference sequences of those proteins were retrieved in the fasta format and their details are presented in table . five proteins with < amino acid sequences are too short to predict epitopes (orf protein, orf protein, orf b protein, nsp , and envelope protein) were excluded. in order to develop a subunit vaccine, it is critical to identify candidate proteins that are important for inducing a protective immune response ( ) . the remaining proteins sequence were relayed to the vaxijen server to determine their antigenicity based on the antigenic scores ( table ) . proteins with antigenic scores > . were selected for further analysis ( ) . nine proteins, namely orf a protein, orf protein, nsp , nsp , nsp , endornase, orf a protein, membrane glycoprotein, and nucleocapsid phosphoprotein were finally selected for further epitope prediction. there is no available experimental structures of these nine proteins, we predicted homology models for the nine proteins applying the normal mode of phyre online server. the most suitable templates for the nine proteins were identified to be the pbd entries ( table s ). all of the modeled structures were showed over % residues in the ramachandran favored region figure s and table s . the prediction of ctl epitopes ( mer) was performed by the netctl server. the binder sites were determined based on three supertypes (a , a , and b ), with a % coverage rate of the world's population. nine proteins were selected based on antigenicity. one epitope of each supertype was selected based on the highest score and an ic value < nm. then the specific treg-inducing epitopes were excluded by epitoolkit. a total of epitopes were selected from nine proteins as the candidates for the construction of the vaccine ( table ) . the htl epitopes ( mer) were evaluated for three hla supertypes: hla-dr (drb * : , drb * : , drb * : , drb * : , drb * : ); hla-dq (dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : ); and hla-dp (dpa * /dpb * : , dpa * : /dpb * : , dpa * : /dpb * : , dpa * : /dpb * : , dpa * : /dpb * : ). we sorted the top epitopes with the lowest scores (low scores indicated the highest binding capability) from three supertypes. the best candidate was then selected based on positive ifn-γ induction and an ic < nm. then the specific treg-inducing epitopes were excluded by epitoolkit. thus, a total of epitopes were selected for vaccine design ( table ) . we used the abcpred and bepipred servers to identify the line b cell candidate epitopes. all predicted epitopes from both servers were compared, and only the overlapping epitopes were selected for the development of the vaccine. the line epitopes identified by abcpred had prediction scores ranging from . to . , and line epitopes identified by bepipred had prediction scores ranging from . to . among these line epitopes, only ( mer) were found to be common or partly common in both servers ( table ) . these line epitopes were selected for vaccine construction ( table ) . the non-continuous b cell epitopes were predicted by the ellipro severs, a total number of non-continuous b cell epitopes were generated from ellipro. amino acid residues, sequence location, the number of residues, and the pi scores of the predicted conformational epitopes are shown in table and the graphical depiction of these epitopes can be seen in figure s . twenty-four epitopes were excluded because it added the allergenicity of vaccine, three epitopes were marked red and selected for vaccine construction. the best candidate epitopes were used for the construction of the vaccine. a total of ctl epitopes, htl epitopes, the half-maximal inhibitory concentration (ic ) value was > nm, which ensured a higher binding capability of the selected epitopes to mhc molecules. linear, and three non-continuous b cell epitopes were fused together with the aid of linker sequences. the ctl epitopes were linked by ayy (the aay liner helps the epitopes produce suitable sites for binding to tap transporter and enhances epitope presentation), the htl epitopes were combined with the aid of gpgpg (the gpgpg linker stimulate htl responses and conserve conformational dependent immunogenicity of helpers as well as antibody epitopes), and b cell epitopes were merged with the aid of kk. the final to enhance vaccine immunogenicity, the human β-defensin- sequence ( aa) and pan-hla dr binding epitopes (the pan-hla dr binding epitopes in vaccine construct facilitating binding to many different types of mouse and human mhc-ii alleles to induce cd -helper cell responses.) was added to the nterminal of the vaccine with the aid of the eaak linker. to enable the intracellular delivery of the modeled vaccine, a tat sequence ( aa) was appended to c-terminal. the vaccine was developed to be amino acids in length ( figure s ) . the sequence homology of final vaccine protein to human protein sequence shown that there were no significant alignments ( figure s ). the allergenic character of the vaccine was determined by the algpred server and was based on the hybrid approach (svmc + ige epitope + arps blast + mast) with a . % coverage. the vaccine was non-allergen with % accuracy and . % sensitivity at threshold value was − . . similarly, the antigenic the half-maximal inhibitory concentration (ic ) value was < nm, which ensured a higher binding capability of selected epitopes to mhc molecules. nature of the vaccine construct was evaluated and showed that the protein was a favorable antigen with a global prediction score of a protective antigen of . (probable antigen). the default threshold value for antigenicity was . in the virus model. moreover, the vaccine constructs contained amino acids, and its molecular weight was . kda. the theoretical pi was predicted to be . . the vaccine contained negatively charged residues and positively charged residues. the vaccine construct was composed of , atoms, and its chemical the immune response profile in silico immune simulation the immune stimulation of the final vaccine was performed using c-immsim online server, which gives the immune profiles of the designed vaccine. the proliferation in the secondary and tertian immune response were identified by igg + igg and igm, as well as, the decreasing of the antigen count igg + igm showed the proliferated (figure a) . the stimulation result revealed the development of immune response after immunization. b cell population was highly stimulated upon immunization ( figure b) . similarly, the cytotoxic and helper t cell levels were proliferated that suggested the development of secondary and tertian immune response (figures c,d) . during the exposure time, it was also observed that the production of ifn-γafter immunization ( figure e) . these results were significant for the immune response against sars-cov- . hence, the tertiary structure of the full-length vaccine sequence was predicted by phyre , and it was applied for refinement and further analysis. twenty-five templates were employing modeling as figure s shown. there were three templates from human defensin which were we added in to enhance the immunogenicity, others from virus ( figure s ) . the immune epitopes were not structural homology to human proteins that could avoid inducing autoimmune. the secondary structure of the predicted model contained % alpha-helix, %tm helices % beta-sheets, and % disordered figure s . to optimize the d structure of the modeled protein, the initial model was refined in the galaxyrefine server. the galaxyrefine server-generated five models based on the rootmean-square deviation (rmsd) and molprobity algorithm. the details of the five models are shown in table s . model with the top ramachandran favored, therefore selected for docking purposes (figure ) . a model with more residues in the ramachandran favored region, less in outliers region and rotamer region was considered as a more ideal one. the initial model generated from phyre server and refine model from galaxyrefine were evaluated with the aid of the swiss-model workspace. the initial model was . % of residues in the ramachandran favored region, . % in the ramachandran outliers region, and only . % in the rotamer region (figure ). the refine model was . % of residues in the ramachandran favored region, . % in the ramachandran outliers region, and only . % in the rotamer region (figure ) . other favorable parameters of the refined model were as follows: gdt score of . , rmsd value of . , molprobability of . , clash score of . , and poor rotamers totaling . ( table s ). the quality and potential errors in the final vaccine d model were verified by prosa-web. the z-score indicates overall model quality, the model with a lower z-score was considered as the higher quality one. the z-score of the initial model was − . , refine model is − . (figure ). to further evaluate the binding affinity between the developed vaccine construct and the relative antigenic receptors (tlr , mhc-i, and mhc-ii), molecular docking was performed. the server yielded candidate models with different binding energies. twenty-nine model complexes of tlr and covid- vaccine were determined, from which just one complex with the lower binding energy score of − . was selected to show ( table and figure ) . a total of model complexes of mhc-i and the covid- vaccine were discovered, and the lowest binding energy score was − . ( table and figure ) . a total of complex models of mhc-ii and the covid- vaccine were predicted, among which, one model complex with the lowest binding energy score of − . was chosen to show ( table and figure ) . further, the vaccine construct was evaluated using the patchdock server, which identified different models and produced a score table. the top complexes identified were refined by the firedock algorithm. among those top models, the model with the lowest binding energy was further selected to show in this paper. the refinement outcomes of tlr and the vaccine complex was solution number with global energy of − . , attractive van der waals energy (vdw) of − . , repulsive (vdw) of . , and atomic contact energy of − . ( table and figure ). the complex of mhc-i and the vaccine was ranked number nine, with global energy of − . , attractive vdw of − . , repulsive vdw of . , and atomic , g , t , t , l , k , e , p , c , s , s , g , p , h , p , l , a , d , n , k , c , c , p , d , g , v , r , s , v , s , p , k , l , f , i , r , e , e , e , t , c , g , q , q , q , t , t , l , k , g , k , k , g , v , q , i , p , c ,t , c , g , k , q , a , t , k , y , l , v , q , q , e , s , p , f . k , p , q , v , n , g , l , t , w , a , d , n ,n , c , l , s , a , p , p , a , q , y , e , l , k , h , g , t , f , t , e , y , t , g , n , y , q , c , g , h , k , t , s , k , e , t , l , y , c , i , d , g , a , l , l , t , k , s , s , e , y , k , g , p , i . d , d , t , l , v , e , f . k , e , n . endornase s , l , e , n , v , a , f , n , v , v , n , k , g , h , f , d , g , q , q , g , e , v , p , v , s , i , i , n , n , t , v , y , t , k , v , d , g , v , d , v , e , l , e , n , k , t , t , l , p , v , n . e , g , s , v , k , g , l , g , e , a , v , k . l , p , s , m , i , d , l , e , l , a , m , d , e , f , i , e , r , y , l , e , g , y , a , f , e , h , i , y , g , d , f , s , h , s , q , l , g , k , r , f , k , e , s , p , e , f , t , d , a , q , t , g , s , s , k , c , k , s , q , d , l , s , v , v , s , k , v , m , l , w , c , k , d , g , h , v , e . t , i , g , c , s , m , t , d , i , a , k , k , p , t , e , t , i , c , a , p , l , t , g , r , v , d , g , v , d , l , f , r , n , a , r , n , k , v , d , g (table and figure ). to accomplish the estimate of the stability of the vaccinereceptor complex, we performed the simulation of the docked complexes (vaccine and tlr- , mhc-i, and mhc-ii) with the help of gromacs. then, various analysis like energy minimization, pressure assessment, temperature, and potential energy calculations were performed. the temperature and pressure of the simulation system during the production run was around k and atmosphere, respectively, indicating a stable system and successful md run. the temperature and pressure of the three simulation systems (vaccine and tlr- , mhc-i, and mhc-ii complexes) during the production run were around k and atmosphere, respectively, indicating the stable systems and successful md run (figures a-f) . the complex root mean square deviation (rmsd) plot represents the structural fluctuation of the overall structure of the complex of vaccine and immune receptor. the rmsd of vaccine-tlr complex has large fluctuation during - ns simulation. after ns, the rmsd value was kept around . nm, indicating that the conformation of this complex was stable ( figure g) . otherwise, the rmsd of vaccine-mhc-i and -mhc-ii complexes has large fluctuation during - ns simulation. after ns, the rmsd value were kept around nm, indicating that the conformation of the two complexes were stable (figures h,i) has low rmsf value, indicating these residues has low structural flexibility. by contrast, residue - and - has relatively higher rmsf value, indicating the larger flexibility during those regions (figures j-l) . to fuse the final vaccine to an expression vector, codon conversion of the vaccine protein was performed by the java codon adaptation tool. restriction site xhoi and bam hi were added to n and c terminals of the codon sequence, then was inserted into the pet a (+) vector between the xhoi and bamhi (figure ) . the rna secondary structure using the mfold program was generated foldings contain , base pairs out of . % in the energy dot plot. mfold predicted an identical secondary structure of , bp formed by nucleotide fragments (figure s ). sars-cov- is characterized by high infectivity and high transmission speed; thus, a prophylactic vaccine is needed ( ) . the availability and advantages of the multi-peptide vaccine developed by immunoinformatics methods have been confirmed by previous studies ( , ) . ojha et al. used the immunoinformatics methods to develop a multiepitope subunit vaccine to epstein-barr virus-associated malignancy ( ) . in recent studies, genomics and proteomics information of sars-cov- have been retrieved, stored, and utilized ( , ) . in the present research, we tried to develop a multi-epitope subunit prophylactic vaccine of sars-cov- , with the help of immunoinformatics tools. a line of research have tried to develop the vaccine of sars-cov- by immunoinformatics tools. baruah and bose ( ) used immunoinformatics tools to discover cytotoxic t lymphocyte (ctl) and b cell epitopes for the spike protein of sars-cov- . then, abraham et al. developed a multi-epitope vaccine that was designed using immunoinformatics tools that potentially trigger both cd + and cd + t-cell immune responses ( ) . most of those research just focus on the spike protein-based vaccine. a vaccine based on the spike protein could induce antibodies to block sars-cov- binding and fusion or neutralize virus infection ( ) , as well as induce harmful immune responses that cause liver damage ( ) . other proteins should be ideal candidates for designing vaccines. in the present report, we selected nine proteins with positive antigenicity for further epitope prediction. all proteins from sars-cov- with < amino acid sequences were excluded, and the antigenic nature of the remaining proteins was evaluated. this method can facilitate the discovery of potential antigens of sars-cov- when the precise immunity mechanisms are unknown. to design an effective vaccine, we selected the sars-cov- protein through the above-mentioned methods for epitope prediction. in recently, asaf et al. reported that identify multiple epitopes for cd + and cd + t cells based on muti-protein ( ) . their protein list was the same as this in our research. in asaf 's report, they just predicted the t cell epitopes, non-b cell, b cell peptide was not predicted ( ) . the b cell epitopes are antigenic determinants from the antigen that are recognized by the b cell surface membrane receptor and evoke the production of specific antibodies. the persistent challenge in immunological prediction tools is the prediction of epitopes to a higher level of accuracy ( ) . to determine accurate linear b cell epitopes from the antigenic proteins, we used two bioinformatics tools based on different algorithms of prediction. we identified nine overlapping linear b cell epitope candidates from two different bioinformatics tools. this method was superior to the prediction of epitopes from a single tool ( ) . moreover, we also have predicted the noncontinue b-cell epitopes. the b cell immune response is preferred in the design of a vaccine. however, t cells may also elicit a strong immunoreaction. the vaccine that activates both ctls and htls should be more effective than a vaccine that only targets ctl responses ( ) . to generate a more effective vaccine, we predicted both ctl epitopes and htl epitopes. the t cell epitopes were decomposed fragments from the antigen presented by the mhc molecules of t cells and stimulated the production of effector t cells, immunological memory t cells, and ifn-γ. the cellmediated immune response induced by ctls plays a vital role in the defense against viral infections through the recognition of intracellular viral pathogens by mhc class i molecules. in the present report, mhc-i binding epitopes were predicted by choosing a , a , and b alleles, which cover ∼ % of world's population. we selected ctl epitopes. the htls play a vital role in the antiviral immune response by producing ifn-γ. moreover, htls are able to induce and maintain ctl responses. furthermore, htls epitopes were chosen based on both the binding capability and ifn-γ induction. bhattacharya et al. also used the spike protein sequence predicted for mhc-i and mhc-ii epitopes of sars-cov- , but not predicted capability of producing ifn-γ ( ). the t cell epitopes enhanced ifn-γ inducing capability, which evokes both the native and specific immune responses by activating macrophages and natural killer cells, and augmenting the response of the mhc to the antigen ( , ) . in this study, the immunogenic epitopes from b cells, ctls, and htls were chosen to develop a more valid, reliable, and effective vaccine against sars-cov- . a multiepitope approach was used by splicing together epitopes with the aid of their respective linkers. to improve the immunogenicity of this multiepitope vaccine, an adjuvant β-defensin and pan-hla dr binding epitopes ( aa) were fused to the n-terminal with the aid of an eaaak linker, then a tat sequence ( aa) was appended to c-terminal with the added of kk. the final vaccine constituted amino acids. the allergenicity, antigenicity, and stability of the designed vaccine constructs were then evaluated. the tertiary structure of the generated vaccine was predicted by using the phyre server and then refined by the galaxyrefine server. the binding affinity of complexes of the developed vaccine and receptors, in which tlr- , mhc-i, and mhc-ii (present on the surface of the immune cell) were confirmed by the cluspro server was based on molecular docking. furthermore, to ensure the translation efficiency of the designed vaccine in a specific expression system, the mrna of the vaccine was enhanced with the aid of the java codon adaptation tool. the restriction enzyme cutting sites of xho? and bamh? were then appended to the n and c terminals, respectively. the vaccine sequence was subsequently cloned in pet a (+), the expression vector. further experimental validation of the safety and efficacy of the designed vaccine for sars-cov- is warranted. all datasets presented in this study are included in the article. rd and zc performed the experiments. rd and yz wrote the paper. yz and fy edited the final version. all authors participated in the experimental design, data analysis, and agreed with the final version of the paper. available online at clinical characteristics of patients infected with sars-cov- in wuhan severe acute respiratory syndrome coronavirus from patient with novel coronavirus disease, united states a preliminary study of the relationship between novel coronavirus pneumonia and liver function damage: a multicenter study myocardial injury in patients with covid- pneumonia clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study positive rate of rt-pcr detection of sars-cov- infection in novel coronavirus patients' clinical characteristics, discharge rate, and fatality rate of meta-analysis epidemiology of covid- among children in china a novel coronavirus from patients with pneumonia in china timely development of vaccines against sars-cov- immunoinformatics: a brief review immunoinformatics and vaccine development: an overview immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of -now design of multi-epitope vaccine candidate against sars-cov- : an in-silico study the spike protein of sars-cov a target for vaccine and therapeutic development structure, function, and antigenicity of the sars-cov- spike glycoprotein evaluation of modified vaccinia virus ankara based recombinant sars vaccine in ferrets bioinformatics analysis of sars-cov m protein provides information for vaccine development respiratory syncytial virus nonstructural proteins and : exceptional disrupters of innate immune responses designing b-cell and t-cell multi-epitope based subunit vaccine using immunoinformatics approach to control zika virus infection development of multi-epitope driven subunit vaccine in secretory and membrane protein of plasmodium falciparum to convey protection against malaria infection novel immunoinformatics approaches to design a multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin identifying candidate subunit vaccines using an alignment-independent method based on principal amino acid properties vaxijen: a server for prediction of protective antigens, tumor antigens, and subunit vaccines the phyre web portal for protein modeling, prediction, and analysis galaxyweb server for protein structure prediction and refinement swiss-model: homology modeling of protein structures and complexes largescale validation of methods for cytotoxic t-lymphocyte epitope prediction an integrative approach to ctl epitope prediction: a combined algorithm integrating mhc class i binding, tap transport efficiency, and proteasomal cleavage predictions definition of supertypes for hla molecules using clustering of specificity matrices a multi-subunit based, thermodynamically stable model vaccine using combined immunoinformatics and protein structure-based approach the role of the proteasome in generating cytotoxic t-cell epitopes: insights obtained from improved predictions of proteasomal cleavage peptide binding predictions for hla dr, dp, and dq molecules nn-align. an artificial neural network-based alignment algorithm for mhc class ii peptide binding prediction prediction of mhc class ii binding affinity using smm-align, a novel stabilization matrix alignment method quantitative peptide binding motifs for human and mouse mhc class i molecules derived using positional scanning combinatorial peptide libraries generation of tissue-specific and promiscuous hla ligand databases using dna microarrays and virtual hla class ii matrices accurate pan-specific prediction of peptide-mhc class ii binding affinity with improved binding core identification development and validation of a broad scheme for prediction of hla class ii-restricted t cell epitopes the human immune response to respiratory syncytial virus infection scrutinizing, mycobacterium tuberculosis membrane and secretory proteins to formulate multiepitope subunit vaccine against pulmonary tuberculosis by utilizing immunoinformatic approaches designing of interferon-gamma inducing mhc class-ii binders prediction of continuous b-cell epitopes in an antigen using recurrent neural network bepipred- . : improving sequence-based b-cell epitope prediction using conformational epitopes improved method for predicting linear b-cell epitopes prediction of the secondary structure of proteins from their amino acid sequence induction of hepatitis a virusneutralizing antibody by a virus-specific synthetic peptide prediction of chain flexibility in proteins a semi-empirical method for prediction of antigenic determinants on protein antigens new hydrophilicity scale derived from residues with antigenicity and x-ray-derived accessible sites epitope-based peptide vaccine design and target site depiction against the middle east respiratory syndrome coronavirus: an immuneinformatics study b-cell epitopes: discontinuity and conformational analysis ellipro: a new structure-based tool for the prediction of antibody epitopes immunoinformatics approaches to design a novel multiepitope subunit vaccine against hiv infection a novel multiepitope peptide vaccine against cancer: an in silico approach modulation of hiv peptide antigenspecific cellular immune response by synthetic α-and β-defensin peptides cellular uptake of the tat protein from human immunodeficiency virus alfred: prediction of allergenic proteins and mapping of ige epitopes computational immunology meets bioinformatics: the use of prediction tools for molecular binding in the simulation of the immune system protein identification and analysis tools on the expasy server; the proteomics protocols handbook template-based protein structure modeling using the phyre web server commonly misunderstood parameters of ncbi blast and important considerations for users prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins new additions to the cluspro server motivated by capri. proteins patchdock and symmdock: servers for rigid and symmetric docking jcat: a novel tool to adapt codon usage of a target gene to its potential expression host mfold web server for nucleic acid folding and hybridization prediction contriving multiepitope subunit vaccine by exploiting structural and non-structural viral proteins to prevent epstein-barr virus-associated malignancy exploring ns / a, ns a, and ns b proteins to design conserved subunit multi-epitope vaccine against hcv utilizing immunoinformatics approaches complete genome sequence of a novel coronavirus (sars-cov- ) strain isolated in nepal the establishment of a reference sequence for sars-cov- and variation analysis sequence-based prediction of vaccine targets for inducing t cell responses to sars-cov- utilizing the bioinformatics predictor recon. biorxiv mimotope-based prediction of b-cell epitopes a probabilistic meta-predictor for the mhc class ii binding peptides development of epitope-based peptide vaccine against novel coronavirus (sars-cov- ): immunoinformatics approach the use of databases, data mining, and immunoinformatics in vaccinology: where are we? role of allograft inflammatory factor- in the pathogenesis of diseases we extend the sincerest appreciation to nhc key laboratory of pulmonary immunological diseases, and guizhou provincial people's hospital, for their technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © dong, chu, yu and zha. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -oxjd z authors: stern, zachariah; stylianou, dora c.; kostrikis, leondios g. title: the development of inovirus-associated vector vaccines using phage-display technologies date: - - journal: expert rev vaccines doi: . / . . sha: doc_id: cord_uid: oxjd z introduction: inovirus-associated vectors (iavs) are derived from bacterial filamentous viruses (phages). as vaccine carriers, they have elicited both cellular and humoral responses against a variety of pathogens causing infectious diseases and other non-infectious diseases. by displaying specific antigen epitopes or proteins on their coat proteins, iavs have merited much study, as their unique abilities are exploited for widespread vaccine development. areas covered: the architectural traits of filamentous viruses and their derivatives, iavs, facilitate the display of specific antigenic peptides which induce antibody production to prevent or curtail infection. inoviruses provide a foundation for cost-efficient large-scale specific phage display. in this paper, the development of different applications of inovirus-based phage display vaccines across a broad range of pathogens and hosts is reviewed. the references cited in this review were selected from established databases based on the authors’ knowledge of the study subject. expert commentary: the importance of phage-display technology has been recently highlighted by the nobel prize in chemistry awarded to george p. smith and sir gregory p. winter. furthermore, the symbiotic nature of filamentous viruses infecting intestinal f(+) e. coli strains offers an attractive platform for the development of novel vaccines that stimulate mucosal immunity research regarding the potential to use viruses to combat disease has been ongoing for over years [ , ] . while viruses have often been considered pernicious as they co-opt a host for their own survival, often killing the host in the process, new work with viruses that can be exploited as bacteriophages but without the harmful effects has arisen [ ] . these viruses, which can be easily manipulated and employed for phage display ability, are being increasingly used for a variety of potent biomedical tools [ ] . these filamentous bacterial viruses, which make up the genus inovirus in the family inoviridae, are thread-like viruses containing single-stranded dna genomes know as filamentous bacteriophages [ ] [ ] [ ] . over different species of filamentous viruses are known, of which a majority can infect gram-negative bacteria. although inoviruses are now being used for their phage display capabilities, these filamentous viruses have a relationship with the cell that they infect that is more similar to symbiotic non-pathogenic animal viruses than classical phages. unlike phages, which term comes from the greek word φάγος for destroyer, inoviruses do not kill their host and only slightly affect cell growth despite yielding titers of up to virions per milliliter of liquid culture. progeny virions are assembled in the host cell's membrane where single-stranded dna binding proteins are replaced by major capsid protein subunits before being released into the cell, resulting in opaque plaques on bacterial lawns [ , ] . receptor organelles in the bacterial host that are encoded by transmissible plasmids facilitate the interactions between inovirus and cell [ , ] . the functional architecture of inoviruses provides the foundation for their application in vaccine-related projects since inoviruses do not cause harm. a great number of inovirus species across the world have been isolated and characterized [ ] . despite variation by species, they have the same general physical characteristics. the virions are flexible, thin cylindrical filaments [ , ] under nm in diameter and approximately nm in length (see figure (a) for details). most of a single virion is composed of several thousand major capsid or coat protein subunits. these surround a circular single-stranded dna molecule. at the proximal end of the virion there are a few minor proteins which attach to the cell to initiate infection. at the distal end, there other minor proteins which are used for nucleation and assembly on the host membrane. the structures and life cycles are conserved across different species of inoviruses, resulting in similar functional applications. research into inovirus structure and application has been dominated by studies of ff [ ] , which infect male (f + ) strains of e. coli. the most used and best understood of the ff inoviruses are the closely related fd, f , and m types (for reviews see [ ] [ ] [ ] ) for which extensive information regarding their life cycle and genetics is known. these nearly identical viruses almost perfectly share all structural motifs, including dna and protein sequences and gene organization [ ] [ ] [ ] , although there are slight variations between the genomes. there are genes and a non-coding intergenic region which are conserved across the ff species [ ] [ ] [ ] as well as dna replication and transcriptional machinery (for a recent review see [ ] ). proteins are encoded by genes in the virion (g , g , g , g , and g ). gene proteins (gp ), the major coat protein, make up the vast majority of the virion, occurring in fivefold axial symmetry. gene proteins and (gp and gp ) are located on the proximal end of the virion and are involved in stabilization and infecting the host cell. gene proteins and (gp and gp ) are on the distal end of the virion and perform initiation assembly [ ] [ ] [ ] . as shown in the end-to-end model in figure (a), minor coat protein subunits have fivefold axial symmetry. while much research has been done into the structure of the ff virus over the past years, a conclusive structure has not been determined since the viruses cannot be crystallized. x-ray fiber diffraction studies and physiochemical measurements have revealed the five-star helical symmetry ( fold rotation axis) of the gp subunit and are referred to as class i [ ] [ ] [ ] . however, the structure of the ssdna and its relationship to the protein sheath is poorly understood, due to the small amount of dna in individual virions. however, the architecture is sufficiently understood to take advantage of the structures and capabilities of the virus throughout its life cycle. the life cycle of ff filamentous viruses begins when an adsorption structure on the proximal end of the virus is adsorbed to the tip of the f + specific pilus of e. coli. following binding between the virus and the bacterial cell (for a recent review, see [ ] ), the major coat proteins of the article highlights • inoviruses are easily manipulated viruses which coexist with their host while causing minimal harm and are highly reproducible. • simple genetic manipulation makes the insertion of random or specific oligonucleotides into the inovirus genome. these genetically modified inoviruses can display corresponding oligopeptides as fusion proteins on their surface and are referred to as inovirusassociated vectors (iavs). • iavs displaying randomly generated oligopeptides are used to identify the epitopes of specific antibodies through biopanning. by isolating recombinant inoviruses displaying mimotypes resembling an antibody discontinuous target epitope, the dna and amino acid sequence of the oligopeptides of interest can be determined. • iavs have bene used to do create vaccines against a variety of infectious and non-infectious diseases, being used both to the screen for immunogenic peptides and directly present immunogenic peptides to the organism to elicit proper antibody production. • iavs are highly immunogenic, can stimulate both humoral and cellular responses in a variety of diseases, pose no known health risks, and are cost-efficient to produce. this box summarizes key points contained in the article. virus become associated with the inner membrane of the cell [ ] [ ] [ ] . the virion's circular single-stranded dna (cssdna) is then ejected into the cytoplasm where it is converted to a parental double-stranded replicative form (rf). using a rollingcircle mechanism, ff inoviruses replicate their genome. the new virion is assembled through a complex set of interactions that binds the protein subunits to the ssdna [ ] [ ] [ ] and embeds newly synthesized coat proteins into the bacterial membrane [ ] [ ] [ ] . ssdna is passed through the mature coat protein, spanning the bacterial membrane, and additional coat proteins are added on the internal edge of the membrane. additional proteins are used to package the inovirus and release it into the cell [ , ] . inovirus assembly on the inner membrane of the bacteria is a harmonized sequential process with both viral-encoded and host proteins playing important roles. for more extensive reviews on inovirus life cycle and replication, see reviews [ , ] . importantly, this process, which requires both virus and host to complete, does not kill the host, making inovirus replication a sustainable process. inoviruses are useful for vaccine development because it is possible to insert random oligonucleotides into their genome. this easy genetic manipulation is the basis for inovirus display (phage display) technology [ , ] . inoviruses that have been genetically modified to display these oligopeptides as fusion proteins on their surface are referred to as inovirus-associated vectors (iavs). oligopeptides can be displayed on any capsid protein (gp , gp , gp , gp , and gp ) following genetic modification. a specific oligonucleotide sequence can be inserted into the viral genome to display the desired oligopeptide as a fusion with capsid proteins gp , gp , gp , or gp . this results in the oligopeptide's display on every copy of the target capsid protein. however, mosaic inovirus particles can be created where the specific capsid proteins display a mix of wild type and recombinant proteins with the desired oligopeptide [ ] . this is done using a phagemid vector which has an extra copy of a capsid protein fused to the specific oligonucleotide. a host exposed to both the phagemid vector and a wild type capsid protein from a deficient helper phage produces mosaic iavs displaying both wild type and oligopeptide fused capsid proteins. work using the capsid protein gp has resulted only in the production of mosaic iavs (for reviews see [ ] [ ] [ ] ) while both mosaic and non-mosaic iavs have been produced using gp and gp (for a review see [ ] ) and gp and gp (for reviews see [ ] [ ] [ ] ). as a result of the different locations, structures, and abundances of the capsid proteins in iavs, they have differential abilities to display different oligopeptides. figure (b) shows how each of the five capsid proteins of an iav can display antigens, as have been demonstrated in published literature. the capsid protein that can display the most copies of the desired oligopeptide is gp due to the large amount of gp in each inovirus. a non-mosaic iav can display a peptide on each of the approximately , copies of gp . however, doing so with large peptides distorts the virus; only peptides of up to amino acids can be displayed in such great quantity without affecting the structure of the inovirus. however, producing a mosaic iav with oligopeptides on far fewer gp units will not cause distortion [ ] . while it is theoretically possible to display an entire protein on gp [ ] , presenting on all copies per virion, studies have demonstrated that at most one copy of gp will display the desired protein [ ] . the ability of iavs to display different random oligopeptides on their surface has many applications, and is especially useful in for vaccine development. the creation of random peptide libraries (rpl), where random oligopeptides are fused to major capsid proteins (gp or gp ) and displayed on individual inovirus clones creating a random variety of iavs which can be used for vaccine design via epitope mapping using monoclonal or polyclonal antibodies. these random iavs, whose oligopeptide diversity increases with increased peptide length, can be used to identify the epitopes of specific antibodies through a process called biopanning. this allows for the isolation of iavs displaying mimotopes which mimic the antibody discontinuous target epitope. by isolating the recombinant inoviruses bearing mimotopes, the dna and amino acid sequence of the oligopeptides of interest can be determined. through this breakthrough technology which was the subject matter of the nobel prize in chemistry (see 'expert commentary' below), inovriuses displaying oligopeptides mimicking antigens (or specific epitopes of an antigen) can be used to vaccinate hosts thus inducing the desired antibody production. vaccines of this sort have been developed in a variety of organisms against many different diseases (see tables and in [ ] for a list of inovirus-based vaccines), as the introduction of the inoviruses can induce the production of specific antibodies. while there have already been various successes, this method can potentially open the door to the development of many novel vaccines and vaccine methodologies. iavs have been successfully used as vaccine carriers for a variety of species and diseases across a range of vaccine studies (as shown in [ ] ). these vaccines have been effective against infections agents such as viral, protozoan, and worm parasites as well as non-infectious diseases including alzheimer's and a variety of cancers. inovirus display technology has been used in two different ways to develop vaccines. the first method employs inovirus display technology to screen rpls with monoclonal antibodies to determine which peptides can be used. these immunogenic peptides are used as vaccines either with carrier proteins or in their soluble forms to elicit an immune response [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the second method not only employs inoviruses for epitope mapping, but also utilizes inoviruses to serve as the carrier for the isolated immunogenic peptide . directly using iavs to present the peptide has been more consistently successful in producing the production of the proper antibody than the introduction of soluble peptides. inovirus-bound peptides (on iavs) consistently retain their d structure and are more stable than soluble peptides, which do not always reflect the desired antigen epitope [ ] . iavs are structurally simple, which allows the immune system to respond to the fused peptides rather than the viral coat. combined with ability to display many copies of the desired peptide, iavs are highly immunogenic, creating effective vaccines [ ] . because the inoviruses replicate in e. coli cultures, the production of large numbers of vaccines is cost-efficient. iav's structural simplicity, high immunogenicity, and economical production make them an efficient and attainable system for creating a variety of effective vaccines. much of the research involving inovirus-based vaccines has been to target infectious diseases in animals. studies have shown that iavs have been constructed to protect vaccinated animals against a variety of infectious diseases by eliciting both humoral [ ] and cellular [ ] immune responses. to test the efficacy of iav vaccines against target pathogens, studies were conducted in which animals were challenged with a specific pathogen following iav vaccination. in these studies, iavs were shown to mitigate or prevent infection from viruses and parasites. in one study, mice were completely vaccinated against human respiratory syncytial virus (rsv) by binding a -mer linear epitope to gp , which induced a humoral response [ ] . monoclonal antibodies against various viral diseases have also been successfully screened against rpls to produce recombinant inovirus vaccines against herpes simplex virus type (hsv- ) using a -mer peptide [ ] and against neurotropic murine coronavirus sing a mer peptide [ ] . these inovirus vaccines induced humoral responses in mice, greatly reducing mortality and providing protection relative to the dose of the vaccine [ ] . iav vaccines have also been developed against fungal parasites in animals, providing both individual protection and large scale vaccination success. [ , ] . not only did vaccination lower the burden of infection, but it also increased the lifespan in the infected mice [ ] . in , manoutsarian et al. used iavs to vaccinate pigs against taenia solium, a parasitic worm which causes neurocysticercosis in humans but uses pigs as intermediate hosts. unlike previous studies, which used a single specific peptide fused to a inovirus, four different antigenic peptides were displayed by inoviruses in a cocktail of recombinant iavs. the induction of a cellular response completely vaccinated / of the pigs in the study and reduced the number of cysticerci in all other pigs [ ] . following the success of this study, a large-scale vaccination of pigs in mexico was undertaken in . the pigs in a natural environment were successfully immunized, reducing parasite presence in the vaccinated pigs, and providing a more costefficient alternative to vaccination with synthetic peptides [ ] . many additional vaccines have been developed that induced both humoral and cellular responses against parasites in animals providing partial protection against the infection and reducing infection burden. many of these studies were done by screening monoclonal and polyclonal antibodies against large rpl (for a review of more inovirus vaccine studies see [ ] ). in addition to their success against infectious diseases, inovirus display technology has been successfully used to design vaccines which prevent or mitigate the progression of non-infectious diseases. iavs have been used to display antigen epitopes which elicit immune responses against tumors and other non-infectious diseases. an epitope of the melanoma antigen a (mage a ) displayed on gp induced a cellular immune response against the melanoma tumor in vaccinated mice. not only did vaccination inhibit tumor growth, but it reduced mortality in the targeted mice [ ] . induction of a cellular response, decreased tumor growth, and higher survival rate was observed in mice vaccinated against murine mastocytoma p in a later study [ ] . the highly immunogenic characteristics of inovirus display technology has also been used to activate immune responses against colorectal cancer tumors, which often evade antitumor immune responses, and to reduce tumor growth [ ] . inovirus-based vaccines have also been used against diseases such as alzheimer's. the same methods have been used to induct antibodies against β-amyloid plaques by displaying a specific amino acid antigenic epitope on the surface of the inovirus. multiple studies in mice using recombinant inoviruses have induced a humoral response and reduced βamyloid plaque burden [ ] [ ] [ ] [ ] . while not used preventatively as has been done with infectious diseases, iavs can vaccinate animals against further disease progression in non-infectious diseases. these findings suggest that there will be future development of inovirus-based vaccines against non-infectious diseases that mitigate the damage done to both animals and humans. despite the advances in the use of iavs to combat disease, the consistent development of inovirus-based vaccines still faces challenges. even when inoviruses have been screened with specific antibodies to bear the desired peptide mimotopes, immunization using iavs does not always produce the expected immune response. keller et al. in were unable to induce the production of broadly neutralizing antibodies against human immunodeficiency virus type- (hiv- ) in rabbits despite having screened for the proper mimotopes [ ] . dorgham et al. in screened a -mer rpl using a broadly neutralizing antibody and, using the selected inoviruses, were able to induce antibodies in vaccinated mice. however, the induced antibodies did not exhibit neutralizing activity against hiv- [ ] . other rpls screened against monoclonal and broadly neutralizing antibodies have produced antibodies that do not have neutralizing capabilities [ , ] . in one such case, crystallization of the specific mimotope revealed that the oligopeptide borne by the inovirus was structurally different that the natural antibody epitope [ ] . this structural issue is likely at the foundation of many of the issues involving ineffectual inovirus-based vaccines. when iavs fail, it is probable that the chosen mimotopes poorly resemble the original antibody epitopes and cannot function in the desired fashion, either failing to induce antibodies or inducing of antibodies without neutralizing capabilities. despite the efficacy of these inovirus-based vaccines against a variety of infectious and non-infectious diseases, there are still many diseases for which effective vaccines have not been developed using phage display or other methods. for example as discussed above, despite extensive work with inoviruses beginning in by keller et al. [ ] , there has been very little success developing a (hiv- ) vaccine [ ] . while four major epitopes on the hiv- envelope gp and gp glycoproteins have been identified [ ] [ ] [ ] , inducing the production of effective antibodies has not been successful [ ] . while current techniques have not succeeded, both humoral and cellular responses to hiv- through inovirus-based vaccines are being researched [ , ] . however, the groundwork for future work involving hiv- and other diseases has been laid out, paving the way for a multitude of various new inovirus-based vaccines. while there have been many vaccine studies targeting hiv- using iavs, none have been completely successful. studies involving inovirus based-vaccines targeting hiv- initially only used the broadly neutralizing monoclonal antibodies f , g and b [ , [ ] [ ] [ ] [ ] [ ] . however, as discussed above, these studies have either failed to induce antibodies or induced non-neutralizing antibodies, despite often inducing a humoral response. in other studies, polyclonal sera from hiv- -infected individuals have been used to screen rpls to search for new broadly neutralizing monoclonal anti-hiv- antibodies [ ] [ ] [ ] [ ] [ ] ] . these studies have used iavs to induce the protection of neutralizing antibodies in mice [ ] and macaques (against simian-human immunodeficiency virus) [ ] . inovirusbased vaccines targeting hiv- have been able to control viral load in a variety of non-human mammals after a challenge by hiv- or induce neutralizing antibodies, but not inhibit novel infection or induce broadly neutralizing antibodies. additionally, inovirus-based hiv- vaccines have often failed due to the autoreactivity of the monoclonal antibodies that can hopefully be avoided with the new 'next generation' broadly neutralizing antibodies [ ] [ ] [ ] [ ] [ ] . inoviral vectors have been used extensively in the development of vaccines against a variety of infectious and noninfectious diseases over the past two decades. as shown above, the iavs have successfully induced a humoral or cellular response, or both. in the studies, the vaccines could provide partial or complete protection against pathogens causing infectious disease or merely lower the burden of infection. against non-infectious diseases, inovirus-based vaccines have been shown to be effective at mitigating disease development by initiating productive immune responses. there have already been many applications of vaccines against infectious and non-infectious diseases using inoviral vectors and iavs have distinct characteristics that make them more applicable for the development of new vaccines than other viral vectors. inoviruses and by extent iavs are highly immunogenic and do not require adjuvants, facilitating the ease and simplicity of usage as vaccines. they can display multiple (from few to thousands) copies of a peptide on their surface while still maintaining the desired structure and conformation as well as infectivity with their bacterial hosts. iavs, by displaying only particular peptides, allow the immune system to interact with a specific epitope rather than a larger, more complex protein, yielding a more targeted response. they are capable of stimulating humoral and cellular immune responses and do not pose known health risks to animals or humans. iavs can be administered safely in high doses through multiple routes, enhancing their usage in a variety of contexts [ ] . with their cost-efficient production, structural simplicity, and record of success in a variety of contexts, iavs have the potential to be exploited for many new vaccines in humans and animals against a variety of diseases. advances in inovirus research and phage display technology are making the development of new vaccines for a variety of diseases feasible in the near future. currently, there are many diseases without vaccines or for which treatment is highly invasive. further development involving iavs will be likely to introduce more, better, and increasingly cost-effective vaccines. as the structure and tools for genetic manipulation of inoviruses are being better developed, iavs will likely begin to be more extensively introduced into animal and human use. the current research is only revealing the tip of the iceberg of the extensive ways in which inovirus-based vaccines will be applied in future use. accordingly, there are aspects of the genetic engineering of phage-display that need to be further expanded. specifically, further studies should be conducted to evaluate the maximum antigenic load capacity of the gp coat protein without jeopardizing the architectural integrity and infectivity of the iav (ff.g , see figure ). furthermore, additional research about the potential of using the other capsid proteins (gp , gp , gp , and gp ) or combinations of any of the capsid proteins for antigen display is needed. additionally, future studies could elucidate the nuances of the uses of individual capsid proteins for specialized applications such as antigen display, penetration, targeting, etc. an additional aspect of iavs that needs to be further explored is how antigenic display on the surface of iavs induces immunogenicity. this predictive work needs to be undertaken by experimental and computational (in silico) research. further research will involve the development of new vaccines and improving the efficacy of current vaccines. additional undertakings should be done into identifying the structure of the viruses, since this will better be able to inform how the phage-display technology will best be applied as more complex work is being done using monoclonal and polyclonal antibodies to create more nuanced vaccines. the future has a dual focus: the development of vaccines for infectious diseases and the induction of immune responses in non-infectious diseases to limit the harm that they do (or eliminate them). the underlying principle of future iav-based vaccine development could be based on the unique natural symbiosis, which is known to exist between humans, non-pathogenic bacteria such as e. coli, and inoviruses. it is well established that humans or other warm-blooded animals acquire e.coli, including f+ strains, during their first few days in life or even before birth and that they are never thereafter without it. fspecific inoviruses (ff inoviruses) cannot infect humans, but they propagate at high viral loads in specific strains of e. coli including non-pathogenic strains which are symbiotic to humans. future iav-based vaccines within the next five to ten years could take advantage of the triple symbiotic nature between humans, enteric f + e. coli strains and f+-specific inoviruses to target the induction of mucosal immunity. this approach will be particularly effective against sexually transmitted pathogens such as hiv- . much future study lies the further development of iavs and how they can induce immune responses on a broad scale. research regarding the development and advantages of phage display technology was recently recognized by the nobel committee. george p. smith and gregory p. winter were awarded the nobel prize in chemistry for the 'elegant method known as phage display, where a bacteriophagea virus that infects bacteriacan be used to evolve new proteins … [this] revolution … is bringing and will bring the greatest benefit to humankind' [ ] . the development of more and better inovirus-based vaccines will continue to advance preventative treatment against diseases which have not been able to be combatted. papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers an investigation on the nature of ultra-microscopic viruses sur un microbe invisible antagoniste des bacilles dysentériques phage display as a promising approach for vaccine development beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold • a review of novel future applications of phage-display technology mahy bwj, regenmortel mhvv structure and assembly of filamentous bacteriophages dna packing in filamentous bacteriophages replication of coliphage m- . i. effects on host cells after synchronized infection filamentous bacterial viruses filamentous bacteriophage: biology, phage display and nanotechnology applications a recent comprehensive review about the molecular biology and life cycle of ff viruses (f + -specific filamentous viruses) filamentous phage: structure and biology nucleotide sequence of bacteriophage fd dna nucleotide sequence of bacteriophage f dna nucleotide sequence of the filamentous bacteriophage m dna genome: comparison with phage fd genetic analysis of the filamentous bacteriophage packaging signal and of the proteins that interact with it functional analysis of bacteriophage f intergenic region filamentous phage morphogenetic signal sequence and orientation of dna in the virion and gene-v protein complex a specific dna orientation in the filamentous bacteriophage fd as probed by psoralen crosslinking and electron microscopy morphogenesis of filamentous bacteriophage f : orientation of extrusion and production of polyphage orientation of the dna in the filamentous bacteriophage f the symmetries of filamentous phage particles helical viruses three-dimensional structure of a cloning vector. x-ray diffraction studies of filamentous bacteriophage m at a resolution chemical modification of the coat protein in bacteriophage fd and orientation of the virion during assembly and disassembly bacteriophage f infection: fate of the parental major coat protein the fate of the protein component of bacteriophage fd during infection detection of prokaryotic signal peptidase in an escherichia coli membrane fraction: endoproteolytic cleavage of nascent f pre-coat protein isolation of mutants in m coat protein that affect its synthesis, processing, and assembly into phage conserved residues of the leader peptide are essential for cleavage by leader peptidase filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short c-terminal fragment of piii architectural insight into inovirus-associated vectors (iavs) and development of iav-based vaccines inducing humoral and cellular responses: implications in hiv- vaccines a detailed review of the architecture of inoviruses and major inovirus-associated vectors (iavs) and related vaccines libraries of peptides and proteins displayed on filamentous phage phage display • an early overview of the phage-display and its applications eliminating helper phage from phage display identification of peroxisomal proteins by using m phage protein vi phage display: molecular evidence that mammalian peroxisomes contain a , -dienoyl-coa reductase phage display of cdna repertoires: the pvi display system and its applications for the selection of immunogenic ligands surface expression and ligand-based selection of cdnas fused to filamentous phage gene vi multivalent display system on filamentous bacteriophage pvii minor coat protein membrane insertion and assembly of epitopetagged gp at the tip of the m phage next generation phage display by use of pvii and pix as display scaffolds phage display: concept, innovations, applications and future vaccination with cetuximab mimotopes and biological properties of induced anti-epidermal growth factor receptor antibodies identification and characterization of protective epitope of trichinella spiralis paramyosin mimotopes selected with neutralizing antibodies against multiple subtypes of influenza a induction of humoral immune response against plasmodium falciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library high-molecular-weight melanoma-associated antigen mimotope immunizations induce antibodies recognizing melanoma cells differential immunogenicity of two peptides isolated by high molecular weight-melanoma-associated antigen-specific monoclonal antibodies with different affinities vaccination with a human high molecular weight melanoma-associated antigen mimotope induces a humoral response inhibiting melanoma cell growth in vitro targeting melanoma cells with human high molecular weight-melanoma associated antigen-specific antibodies elicited by a peptide mimotope: functional effects specificity of mimotopeinduced anti-high molecular weight-melanoma associated antigen (hmw-maa) antibodies does not ensure biological activity a mimotope peptide-based anti-cancer vaccine selected by bat monoclonal antibody peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-egfr immune response mimotope vaccination for epitope-specific induction of anti-vegf antibodies peptide mimics of the group b meningococcal capsule induce bactericidal and protective antibodies after immunization peptides selected from a phage display library with an hiv-neutralizing antibody elicit antibodies to hiv gp in rabbits, but not to the same epitope protective immune responses induced by the immunization of mice with a recombinant bacteriophage displaying an epitope of the human respiratory syncytial virus immunisation with phage displaying peptides representing single epitopes of the glycoprotein g can give rise to partial protective immunity to hsv- characterization of murine coronavirus neutralization epitopes with phage-displayed peptides prophylactic vaccination with phagedisplayed epitope of c. albicans elicits protective immune responses against systemic candidiasis in c bl/ mice protective immune responses against systemic candidiasis mediated by phage-displayed specific epitope of candida albicans heat shock protein in c bl/ j mice recombinant bacteriophage-based multiepitope vaccine against taenia solium pig cysticercosis schistosoma japonicum: isolation and identification of peptides mimicking ferritin epitopes from phage display library protective immunity induced by phage displayed mitochondrial related peptides of schistosoma japonicum induction of immunity in sheep to fasciola hepatica with mimotopes of cathepsin l selected from a phage display library trichinella spiralis: characterization of phagedisplayed specific epitopes and their protective immunity in balb/ c mice identification of hiv vaccine candidate peptides by screening random phage epitope libraries identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type antibody b immunogenicity of hiv type gp cd binding site phage mimotopes a peptide inhibitor of hiv- neutralizing antibody g is not a structural mimic of the natural carbohydrate epitope on gp constrained peptide models from phage display libraries highlighting the cognate epitope-specific potential of the anti-hiv- mab f selection of hiv-specific immunogenic epitopes by screening random peptide libraries with hiv- -positive sera collection of phage-peptide probes for hiv- immunodominant loop-epitope mimotopes selected with antibodies from hiv- -neutralizing long-term non-progressor plasma hiv- v loop crown epitope-focused mimotope selection by patient serum from random phage display libraries: implications for the epitope structural features protection of rhesus macaques against disease progression from pathogenic shiv- . pd by vaccination with phage-displayed hiv- epitopes variable epitope library-based vaccines: shooting moving targets variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type -neutralizing antibody response a general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera induction of hepatitis b virus-specific cytotoxic t lymphocytes response in vivo by filamentous phage display vaccine phage display for sitespecific immunization and characterization of high-risk human papillomavirus specific e monoclonal antibodies peptide mimotopes of rabies virus glycoprotein with immunogenic activity immunogenicity and epitope mapping of foreign sequences via genetically engineered filamentous phage multiple display of foreign peptides on a filamentous bacteriophage. peptides from plasmodium falciparum circumsporozoite protein as antigens random-peptide libraries and antigenfragment libraries for epitope mapping and the development of vaccines and diagnostics developing strategies to enhance and focus humoral immune responses using filamentous phage as a model antigen phage display of peptide epitopes from hiv- elicits strong cytolytic responses inexpensive anticysticercosis vaccine: s pvac expressed in heat inactivated m filamentous phage proves effective against naturally acquired taenia solium porcine cysticercosis the potential of phage display virions expressing malignant tumor specific antigen mage-a epitope in murine model phage display particles expressing tumor-specific antigens induce preventive and therapeutic antitumor immunity in murine p model a filamentous bacteriophage targeted to carcinoembryonic antigen induces tumor regression in mouse models of colorectal cancer active immunization against alzheimer's beta-amyloid peptide using phage display technology immunization against alzheimer's beta -amyloid plaques via efrh phage administration reduction of betaamyloid plaques in brain of transgenic mouse model of alzheimer's disease by efrh-phage immunization efrh-phage immunization of alzheimer's disease animal model improves behavioral performance in morris water maze trials hiv- vaccine strategies utilizing viral vectors including antigen-displayed inoviral vectors hiv- neutralizing antibodies: understanding nature's pathways antibodies in hiv- vaccine development and therapy broadly neutralizing antibodies and the search for an hiv- vaccine: the end of the beginning profound early control of highly pathogenic siv by an effector memory t-cell vaccine immune clearance of highly pathogenic siv infection inducing cross-clade neutralizing antibodies against hiv- by immunofocusing cardiolipin polyspecific autoreactivity in two broadly neutralizing hiv- antibodies the role of antibody polyspecificity and lipid reactivity in binding of broadly neutralizing anti-hiv- envelope human monoclonal antibodies f and e to glycoprotein membrane proximal envelope epitopes specific phospholipid recognition by human immunodeficiency virus type- neutralizing anti-gp f antibody the broadly neutralizing anti-human immunodeficiency virus type antibody g recognizes a cluster of alpha -> mannose residues on the outer face of gp antibody polyspecificity and neutralization of hiv- : a hypothesis immunocontraception: filamentous bacteriophage as a platform for vaccine development the nobel prize in chemistry we thank the university of cyprus for its support. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. key: cord- -b tndbg authors: neumann-böhme, sebastian; varghese, nirosha elsem; sabat, iryna; barros, pedro pita; brouwer, werner; van exel, job; schreyögg, jonas; stargardt, tom title: once we have it, will we use it? a european survey on willingness to be vaccinated against covid- date: - - journal: eur j health econ doi: . /s - - - sha: doc_id: cord_uid: b tndbg nan while the focus of attention currently is on developing a vaccine against the coronavirus sars-cov- to protect against the disease covid- , policymakers should prepare for the next challenge: uptake of the vaccine among the public. having a vaccine does not automatically imply it will be used. compliance with the anti-h n vaccine during the influenza pandemic, for instance, was low [ ] , and in the decade since, vaccination rates have remained an issue of concern [ ] while vaccination hesitancy has become more prevalent, leading to increases in disease outbreaks in multiple countries [ ] . it is, therefore, important to understand whether or not people are willing to be vaccinated against covid- , as this can have large consequences for the success a vaccination programme-with potentially large health and economic consequences. in this editorial, we provide some first insights into this willingness to be vaccinated, based on a multi-country european study [ ] , which hopefully result in more attention for this important issue. on april , the who counted seven covid- candidate vaccines in the clinical evaluation phase and more in the preclinical evaluation phase [ ] . this underlines the unprecedented current efforts worldwide to find an effective vaccine against the coronavirus sars-cov- . some expect that first vaccines may become available under emergency use protocols as soon as early , given the speed and scale of research and development efforts globally, while others argue it will take longer [ ] [ ] [ ] . in both cases, the development phase should be followed by large-scale vaccination programmes to attain herd immunity [ ] . that way, we can protect the lives of the most vulnerable people and reduce the social and economic burden of the current crisis. vaccination programmes can lead to herd immunity without requiring a large proportion of the population to be infected. the latter is mostly seen as an undesirable option, given the potentially high numbers of deaths as a result of infection. especially so, if the health systems are overwhelmed by a large number of patients with severe covid- symptoms [ ] . herd immunity through vaccination, however, requires a sufficient proportion of the population to be vaccinated. while vaccination is widely recognised as an effective way to reduce or eliminate the burden of infectious diseases by health authorities and the medical community [ ] , its effectiveness also depends on the individual willingness to be vaccinated. this willingness could be negatively affected by doubts and worries that exist in the population about the safety and appropriateness of vaccines. this is sometimes labelled vaccine hesitancy [ ] . if too many individuals hesitate about being vaccinated, herd immunity may not be reached. besides objective trade-offs of costs and benefits of a vaccine, risk attitude, pro-social considerations, and misinformation or misperceptions about a vaccine may play a role in this [ , , ] . at present, it is unclear whether a sufficient proportion of the population would decide to get vaccinated when a vaccine becomes available. in the eu, vaccine delays and refusals are contributing to declining immunisation rates in several countries and lead to increases in disease outbreaks [ ] . hence, and the question is whether enough europeans trust the effectiveness and safety of vaccines and the healthcare system that delivers them [ ] . to shed more light on the issue of willingness to be vaccinated, we investigated people attitudes about vaccination against covid- in an online survey among representative samples of the population (in terms of region, gender, age group and education) in seven european countries (n = . ). the sample consisted of about . respondents per country, and an additional from the highly affected region lombardy, since we expected that results might differ from the rest of italy. in this first wave of the data collection, respondents were inquired about worries and beliefs about covid- , as well as attitudes about vaccination and their willingness to be vaccinated between and april [ ] . in this editorial, we provide some first insights into the findings, to stimulate further research and policy in this area. in total, . % of the participants from denmark, france, germany, italy, portugal, the netherlands, and the uk stated that they would be willing to get vaccinated against covid- if a vaccine would be available. a further . % of respondents stated that they were not sure, and . % stated that they do not want to get vaccinated. as shown in figs. and , the willingness ranged from % in france to approx. % in denmark and the uk. the largest proportions of the population opposed to a covid- vaccination were observed in germany ( %) and france ( %), while france also has the largest group of people who were unsure about getting vaccinated ( %). looking closer, we found considerable differences in willingness to get vaccinated across genders and age groups (fig. ) . a significantly higher proportion of men were willing to get vaccinated ( . %, chi-squared, p < . ) than women ( . %). the willingness to be vaccinated is largest among men above the age of , while uncertainty ranged between and % across all age groups. males who were unwilling to get vaccinated tended to be younger with the largest share of % among the - year olds. similarly, the trend for women who were unwilling to vaccinate seems also to follow the age categories. the uncertainty among women was higher in all age groups and largest for women between the ages of and ( %). one might argue that the group who is currently unsure about getting a vaccine may be the most relevant. these are the people who potentially can be persuaded more easily to get vaccinated to achieve herd immunity. based on our results, these efforts could best be aimed at persons below the age of and at females in general, where the willingness is lower. we asked respondents who were unsure about being vaccinated about their main reasons (fig. ). more than half ( %) said they were concerned about potential side effects of a vaccine, although this concern was more frequent among women ( %) than men ( %). around % of respondents stated that a vaccine might not be safe, with no notable differences between genders. these findings are in the literature on frequent reasons for vaccine hesitancy [ ] . looking at the open text explanations given to the category "other", we saw that a common concern seems to be that a covid- vaccine might be experimental, without any studies on side effects, and that the vaccine may not be safe for specific groups, such as for pregnant woman, people with pre-existing conditions like ms, allergic persons etc. this finding highlights that while the current focus seems to be on developing a vaccine about ten times faster than [ ] , the public should also be reassured that any vaccine which becomes available that quickly is safe and effective. otherwise, there is a risk to lose the public trust in the particular vaccine, and coronavirus vaccination altogether [ ] , potentially compromising herd immunity. we find a similar trend regarding the most frequently mentioned reasons and the gender differences for the concerns about side effects among those who were not willing to get vaccinated (fig. ) . notable gender differences could also be observed among those respondents who stated that they think covid- is not dangerous to their health ( %), comprised of almost twice as many men ( %) than women ( %). furthermore, we see that an overall rejection of vaccination was more than twice as common among women ( %) than among men ( %). when looking at the open text answers of respondents who choose other reasons ( %), we found not only concerns the literature suggests multiple steps that could be taken by policymakers to decrease vaccine hesitancy and convince doubters to get vaccinated after all. one approach for vaccine advocacy suggests "vaccine adoption = access + acceptance" [ ] . looking at access, it is essential to translate the willingness to be vaccinated into actual vaccination decisions. our study measured the intention to vaccinate; this rate might differ from actual vaccination uptake (vaccination decision) depending on potential constraints, such as the price of the vaccine and the ease of access of vaccination sites. vaccines should thus be available in a timely manner and an easily accessible way to have as little attrition as possible [ ] . in the case of the coronavirus vaccine, access will prove quite challenging since, at the early stages of availability, the demand for this vaccine worldwide will be much greater than the (short-term) production capacities. currently, about billion doses of vaccine are produced yearly worldwide, of which % are seasonal flu vaccines [ ] . so even when a vaccine becomes available, access to it will probably be limited in the short run. therefore, policymakers need to prepare how access can be organised equitably and effectively. our results on acceptability suggest that substantial gains could be made among the sizeable proportion of the population (i.e. . %) that is unsure whether they want to get vaccinated. if this group needs to be convinced to be vaccinated to get to herd immunity, clear communication about safety, and potential side effects of the vaccine is especially important. this could help to stimulate the hesitant part of european citizens to get vaccinated after all. this is especially important since it is unclear whether the group of people who are willing to be vaccinated in itself is male female male female large enough to achieve herd immunity. the basic reproduction number r shows the transmission potential of diseases [ ] , i.e. to how many people the infection is expected to be passed on by one infected individual in a fully susceptible population, on average. the herd immunity threshold describes the proportion of the population that needs to be immune, so that the infectious disease is stable (r = ) and is calculated as [ ] : this means that the higher the basic reproductive number r is, the higher the herd immunity threshold becomes. a recent study estimated a covid- r of around . for europe [ ] , implying a herd immunity threshold for europe of %. for the us, it was estimated at around . , implying a herd immunity threshold of % [ ] ; while, a recent study argues these values may be lower if there is heterogeneity in the individual susceptibility to the virus [ ] . of course, these estimates are uncertain, but comparing this - % threshold range with our results indicates that the current willingness levels in france, germany and the netherlands, in particular, may prove insufficient to reach this threshold. our survey highlighted important differences between citizens from european countries in terms of willingness to be vaccinated against covid- . the levels do not follow trends that we see in other vaccination rates, e.g. against measles, which are generally higher, but in most countries below the recommended % threshold [ ] . understanding which groups in the population are not willing to be vaccinated and why remains vital for the design of policy responses to vaccination hesitancy. one of the avenues to explore could be to emphasise the social benefits of vaccination more strongly so that they weigh the public health dimension more heavily in their decision whether to vaccinate [ ] . a recent study, for example, found that people are more willing to get vaccinated when they were informed that this would protect others who are willing but unable to get vaccinated themselves [ ] . consequently, one of the communication strategies could be to emphasise how vaccination against covid- helps to protect vulnerable members of society. furthermore, the distribution of vaccinated individuals in the population matters. pockets of nonvaccinated groups could be highly problematic even when overall vaccination rates are high. unvaccinated individuals may be more often in contact with other unvaccinated individuals than with vaccinated ones [ ] . examples of measles outbreaks in the netherlands [ ] and the us [ ] , for instance, show that outbreaks in particular communities may even occur if overall vaccination rates are high, and highlight the role of religious communities and travellers in this context. alternative strategies range from restrictive measures against those who chose not to be vaccinated to mandatory vaccination schemes for certain target groups or the whole population. experimental evidence suggests that individuals under specific conditions may be willing to support mandatory vaccination policies, but this support seems very sensitive to adverse events [ ] . such a policy may be less appropriate in the context of covid- . our findings highlight that considerable policy effort may be required to come from having a vaccine to adequate vaccination rates, especially in some countries. targeting those in the population who are currently hesitant seems most promising and cost-effective, but this requires convincing evidence and clear communication on the safety and effectiveness of the vaccine. this may be at odds with the current push for having a vaccine available as soon as possible. a campaign emphasising the social benefits of vaccination could increase the willingness to be vaccinated among those amenable to such pro-social motives. finally, a sizeable proportion of the population indicates not to be open to vaccination. this group may remain at risk of spreading the virus and contracting the disease, even after herd immunity has been achieved. concluding, improving our understanding of vaccination hesitancy in the context of covid- , as well as finding and using policies to overcome it, may be as important as discovering a safe and effective vaccine. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. compliance with anti-h n vaccine among healthcare workers and general population anti-vaccine activists, web . , and the postmodern paradigm-an overview of tactics and tropes used online by the anti-vaccination movement luxembourg: publications office of the european union countering covid- : a european survey on acceptability and commitment to preventive measures world health organization: draft of the landscape of covid- candidate vaccines the race for coronavirus vaccines how can we develop a covid- vaccine quickly? in: welcome explainer covid- vaccine availability unrealistic in - month period herd immunity": a rough guide what is herd immunity and how can we achieve it with covid- ? policy and practice vaccination greatly reduces disease, disability, death and inequity worldwide strengthening vaccination programmes and health systems in the european union: a framework for action. health policy inviting free-riders or appealing to prosocial behavior? game-theoretical reflections on communicating herd immunity in vaccine advocacy social nudging: the effect of social feedback interventions on vaccine uptake s/vcp_the-state -of-vacci ne-confi dence _ don't rush to deploy covid- vaccines and drugs without sufficient safety guarantees listen, understand, engage can the world find a good covid- vaccine quickly enough ? the economist infectious disease epidemiology the effective reproduction number as a prelude to statistical estimation of time-dependent epidemic trends bt-mathematical and statistical estimation approaches in epidemiology estimating the number of infections and the impact of non-pharmaceutical interventions on covid- in european countries: technical description update the impact of changes in diagnostic testing practices on estimates of covid- transmission in the united states individual variation in susceptibility or exposure to sars-cov- lowers the herd immunity threshold fighting a global resurgence the willingness to vaccinate increases when vaccination protects others who have low responsibility for not being vaccinated positive network assortatively of influenza vaccination at a high school: implications for outbreak risk and herd immunity measles outbreak in a community with very low vaccine coverage measles cases and outbreaks individual preferences for voluntary vs. mandatory vaccination policies: an experimental analysis key: cord- -sq zsqqi authors: zaheer, tean; pal, kaushik; zaheer, iqra title: topical review on nano-vaccinology: biochemical promises and key challenges date: - - journal: process biochem doi: . /j.procbio. . . sha: doc_id: cord_uid: sq zsqqi nanomaterials have wide-ranging biomedical applications in prevention, treatment and control of diseases. nanoparticle based vaccines have proven prodigious prophylaxis of various infectious and non-infectious diseases of human and animal concern. nano-vaccines outnumber the conventional vaccines by virtue of plasticity in physio-chemical properties and ease of administration. the efficacy of nano-based vaccines may be attributed to the improved antigen stability, minimum immuno-toxicity, sustained release, enhanced immunogenicity and the flexibility of physical features of nanoparticles. based on these, the nano-based vaccines have potential to evoke both cellular and humoral immune responses. targeted and highly specific immunological pathways required for solid and long lasting immunity may be achieved with specially engineered nano-vaccines. this review presents an insight into the prevention of infectious diseases (of bacterial, viral and parasitic origin) and non-infectious diseases (cancer, auto-immune diseases) using nano-vaccinology. additionally, key challenges to the effective utilization of nano-vaccines from bench to clinical settings have been highlighted as research domains for future. 'chemistry' behind the nanoparticles and their multi-dimensional exclusive applications is quite fascinating. nanoparticles (< nm size) have been successfully applied in many fields of biomedical science including therapeutics e.g. (drug screening and targeted delivery), diagnostics, vaccine production, surgical intervention, gene delivery, theragnostic, biomarker assisted mapping, toxicity of pathogenic organisms, etc. [ ] [ ] [ ] [ ] . the nano-carriers/adjuvants e.g. liposomes, proteasomes, emulsions, synthetic polymeric nanoparticles, nano-beads, iscoms, biological polymeric nanoparticles (exosome, bacteriophage) and inorganic nanomaterials have been utilized to prevent infectious and non-infectious diseases [ , ] . the inertia of surface modification and ability to effectively co-deliver the adjuvants makes nanoparticles potential candidate for commercial vaccines. also, the nano adjuvants in vaccines protect the target antigen from degradation and enhance uptake by immune mediators of biological systems. this approach is malleable, having the ability to present the antigen in a repetitive manner leading to stable immunogenic properties. nano vaccines have been widely experimented as prophylaxis of important diseases such as: bacterial (e. coli, helicobacter sp.), viral (hiv, hpv, influenza), cancers (primary and metastatic), parasitic (malaria, toxoplasmosis, coccidiosis) and auto-immune disorders [ ] [ ] [ ] . wide variety of nanoparticles as vaccine scaffolds, enzyme, cargo have opened a new avenue towards precision medicine. these vaccines could be replicated in disease models of multi-drug resistant pathogens, which historically have j o u r n a l p r e -p r o o f presented as a great clinical challenge. deploying biological nano-polymers like proteins, peptides, dna, rna and others have improvised the immunotherapy up to folds, compared to previous clinical options [ ] . the efficacy of nano-assembled vaccines may be attributed to the improved antigen stability, minimum immuno-toxicity, sustained release, enhanced immunogenicity and the flexibility of physical features (e.g. size, morphology, surface characteristics) [ ] . nano-vaccines have a huge potential of relatively easy engineering. moreover, tailor-made personalized immune therapy is possible by harnessing the potential of nano-vaccines reveals a conceptual idea.the challenge areas of nano-vaccines are biodistribution which needs to be well-investigated and consigned.the quantitation of host immune interactions on exposure to nano-based vaccine demand in clinical trials for efficient commercialization. this paper reflects into account the novel premise, utilizations and future perspectives of nano-vaccines in human and animal diseases.brief insights and way forward for commercialization of nano-vaccines in clilnical settings have been summarized in the article. contemporary vaccine strategies employ either killed or live attenuated antigens. live attenuated vaccines may induce clinical disease arising from same or mutated genotypes [ ] . therefore, the level of desired immune response may not be achived. the nanoparticles having efficient surface properties, making them more suitable candidates for stimulating the immune system and eliciting better immunological response. the hydrophobicity of nano-sized materials enhances the expression and release of inflammatory mediators and cytokines. superior adjuvancity, owing to the exceptional surface properties of nanomaterials make them stand-out the conventional vaccine adjuvants [ ] . some of nano-based adjuvants have been officially licensed for use in making commercial antiviral vaccines j o u r n a l p r e -p r o o f [ ] . moreover, increment towards dendritic-cell mediated autophagy and presentation of antigens to the immune cells lead to a solid cellular and humoral immunity against target pathogen. first and second generation vaccines differ from nano-vaccines on the basis of lowfunctionalized plasmid dna, highly labile to degrading enzymes, lacking the smart size and hydrophobic nature. all of these properties contribute to halt efficient transfection of antigens within target cells [ ] . moreover, the difficulty in administration and development of slower immunological response based on time-taking chemical interactions at cellular levels were major issues with dna vaccines. chitosan nanoparticles have the intrinsic ability to adhere with mucosal layers of host; this cationic feature makes them efficient cargo for antigen delivery [ ] . similarly, by virtue of ionic crosslinkages, the use of biopolymers could improvise endocytosis by host cells. this internalization stimulates a sustained pattern of exposure to antigen presenting cells, resulting in a stable immunological response. this response is characterized by the interaction and front-line response by many immunomodulators of the host. the liposomal vaccine carriers, due to hydrophobic interactions, can facilitate fusion within cellular membranes [ ] . also, the cationic nature further enhances cytosolic release, which is highly desirable in dna based vaccines. most importantly, nanoparticle-based vaccines have been shown to stimulate longer immunological memory in the host [ ] . this property, combined with the ability to elicit antigen-specific (iga) mucosal immunity is the mainstay of popularity earned by nano-vaccines. brief pathway opted by nanovaccines to bring cell mediated and humoral immunity in host are being illustrated in fig. . the nucleic peptides might become very unstable within the host cells and may fail to produce desired immunological response due to proteolytic degradation inside the cells [ ] . nano-adjuvants provide a biologically compatible carrier platforms, that not only enhance antigen protection and sustained release but also enhance immune stimulation in the host for a stable and solid immunity. use of natural biomolecules such as albumin, chitosan, mannose, peptides, enzymes, chemical immunomodulators (interleukins, cytokines) or immunoglobulins as nano-carriers for vaccines have shown long span, more stable and ubiquitous peripheral tissue response to in cancer immune therapy [ ] . nano vaccines have brought a revolution in the science of small by evading cellular pathways and efficient absorption up to blood vessels [ ] . admirable performance explored in pre-clinical and clinical trials, liposomal and vlps based nano-vaccines, there are more than commercial vaccines in human practice or clinical trials. classical examples to vlp-based commercial vaccines include the porcine-circo virus vaccine, human cervical cancer and anti-hepatitis b nano-vaccines and multi-epitope anti-malarial and anti-hepatitis b vaccines [ , ] . the desired level of epitope density and costimulation is a very unique and high precision characteristic of nano-based vaccines. additionally, revamping the ability of nanomaterials to selectively enhance one of desirable, antigen specific immune responses in order to achieve optimal immunity holds huge potential in future engineered vaccines. as a case study, it is imperative to commend the most effective yet rapidly developed covid- vaccine which is based on gold nanomaterials [ - ]. the plasmonic stabilization and functionalization has made the vaccine perform fairly well in pre-clinical as well as clinical trials and safety evaluations. the world is dealing with ever rising population of super bug pathogens, the multi-drug resistant bacteria, rapidly mutating viruses, anthelmintic resistant parasites and secondary cancers. most recently, covid- pandemic has moved the major stakeholders to come up with very practical and promising candidates for prevention and therapeutic management of the virus [ ]. the first-ever, highly progressive anti-covid- vaccine is also based on nanomaterials [ ] . overview of most recent developments in nano-vaccinology during the current decade has been given in table. . nano-vaccinology has also been trialed to control many human and animal diseases of bacterial origin, major types of nano-adjuvants/ nano-carriers/ nano-scaffolds employed for vaccinology have been illustrated in fig. . viral diseases have historically caused and still posing a great threat to the integrity of entire ecosystems. limitation of availability, high cost of production and promise in emerging viral strains are major challenges to anti-viral drug production. however, vaccines have shown to almost counter these areas of concern. heterosubtypic immune protection in influenza strains (h n , h n ) is a muchneeded approach for rapidly mutating viruses [ , ] . a highly significant pathogen of humans, including hbv, hpv, hiv, denv-e have been prevented using precisely engineered nano vaccines, that have shown to offer up to - % effective immune protection [ ] . anti-aids nano-vaccines may be utlilized at clilnical settings to prevent the disease and associated complications at endemic regions of the world. the viral moieties in hiv are better functionalized and presented in a sustained manner. the nano-adjuvants to aids vaccines have shown a stable, least toxigenic and a long-lived immunological response, based on specific immunologlobulin activation. poliovirus is one of the most significant endemic pathogens of many developing countries. to this end, marsian and co-workers have proposed a plant-mediated nano-vaccine, by utilizing virus like particles drug resistance and slower development of modern anti-parasitic drugs are the major concern to widespread neglected tropical diseases. parasitic diseases of prime concern like-wise: leishmaniasis, malaria, toxoplasmosis, anaplasmosis, schistosomiasis, and coccidiosis have been treated and prevented using several forms of nanoparticles [ , ] . to date, there is no commercially available nano-vaccine against any parasite. the benefit of nanoparticles-assembled vaccines has shown highly desirable, th mediated immunological protection against leishmaniasis. recently, epitope-based nano-vaccine, using self-assembling protein nanoparticle approach (sapn) has been successfully developed against toxoplasma sp. similarly, malaria (anaplasma sp.) nano-vaccines have undergone huge development and several promising vaccine antigens have offered protective immunity in laboratory attempts [ , ] . similarly, this approach could be applied to parasitic vectors (mosquito, tick, flies) of human, animal and zoonotic diseases. there is a need to further channelize and utilize the potential of nanovaccine induced mucosal immunity for development of anti-parasitic vaccines. engineering the surfaces of nanoparticles by chemical means may alter their potential biocompatibilities [ ] . the chemical transformations therefore, indicate the necessity of developing the assays/ tests indicative of owning target set of characteristics, before functionalization with candidate antigens/proteins. similarly, the uniformity of nanomaterials and the reproducibility of experiments yielding nano-vaccines needs to be enhanced. this applies as a significant quality standard for biogenic nanomaterials, where scaling-up uniformly is a concern. vlps have shown promising performance, regarding their easy engineering, exceptionally malleable size and surface properties and potential immunogenic properties. however, there is a concern of associated ability to rapidly mutate the proteins of viral origin, being utilized for their synthesis [ ] . similar concerns may arise in the application of other nano-carriers or adjuvants used in the vaccine core antigen potentiation. closely relevant animal models may be devised to carry out the pre-clinical j o u r n a l p r e -p r o o f evaluation of such nano-vaccines [ ] . biologically mediated nanomaterials have been proven as effective carriers of chemotherapeutic and chemoprophylactic agents. proven non-pathogenic viral vectors for protective immune coverage and sustained immunological memory may be further investigated for probability of efficient commercialization [ , ] . the exact biochemical interactions and the active constitutents of nano-vaccines making them a good choice need further exploration within biological models. thorough studies upto molecular pathways are warranted to understand the dynamics of actual mechanism behind protective immunological response due to nano-vaccines. moreover, to harness the collaborative potential of computational modelling and simulation, its highly indicated to analyze and declare most promising nano-adjuvants or peptides, based on their in-silico biological structures and functions analyses. it is imperative to look up and further rationalize the potential aspects of nanomaterials, for instance the facile synthesis, requirement of lower doses yet alleviation of repetitive booster injections, easy routes of administration, etc. of nano-vaccines over conventional vaccines [ , ] . there is need to materialize the concept of nano-immunology against auto-immune diseases of idiopathic origin. for this purpose, the investigation and communication of biochemical and molecular pathways making nano-vaccines promising is imperative. the ease of administration and efficient immunogenesis has made nano-vaccines applicable at aquatic eco-systems. biological distribution of nanoparticles and uptake by excretory systems within the host need further explanation and safety evaluation. also, the commercialization of biological adjuvant-based nano-vaccines needs greater reproducibility and scaling-up of production. nano-vaccinology is the science of smaller particles, possessing huge potential. the laboratory as well as the clinical scale premise of nano-vaccines can push the boundaries towards an eco-friendly, more galal application of some nanoparticles in the field of veterinary medicine albumin nano-encapsulation of piceatannol enhances its anticancer potential in colon cancer via downregulation of nuclear p and hif- α rhim multifunctional nanocellulose/metal and metal oxide nanoparticle hybrid nanomaterials zhang nanovaccine confers dual protection against influenza a virus and porcine circovirus type albumin/vaccine nanocomplexes that assemble in vivo for combination cancer immunotherapy danquah nanosensors for better diagnosis of health chapa nanotechnology for covid- : therapeutics and vaccine research acs nano efficacy of the herpes zoster subunit vaccine in adults years of germá plasmid-dna lipid and polymeric nanovaccines: a new strategic in vaccines development ali chitosan and chitosan nanoparticles as adjuvant in local rift valley fever inactivated vaccine biotech he developing a platform system for gene delivery: amplifying virus-like particles (avlp) as an influenza vaccine npj vaccines cui intranasal nanovaccine confers homo-and hetero-subtypic influenza protection small mitter nanoparticle-based delivery of anaplasma marginale membrane proteins lomonossoff plant-made polio type stabilized vlps-a candidate synthetic polio vaccine welch titanium surface modification to enhance antibacterial and bioactive properties while retaining biocompatibility cosset towards physiologically and tightly regulated vectored antibody therapies cancers antibody gene transfer with adeno-associated viral vectors as a method for hiv prevention promise and progress of an hiv- cure by adeno-associated virus vector delivery of anti-hiv-front banerjee advancements in prophylactic and therapeutic sharif characterization of immunogenicity of avian influenza antigens encapsulated in plga nanoparticles following mucosal and subcutaneous delivery in chickens l. zhang modulating antibacterial immunity via bacterial membrane-coated nanoparticles he parainfluenza virus (piv ) amplifying virus-like particles expressing respiratory syncytial virus (rsv) antigens protect mice against rsv infection talaat a single dose polyanhydride-based nanovaccine against paratuberculosis infection npj vaccines spike nanoparticle and recombinant adenovirus vaccines induce specific antibodies against the middle east respiratory syndrome coronavirus antigen-based nano-immunotherapy controls parasite persistence, inflammatory and oxidative stress, and cardiac fibrosis, the hallmarks of chronic chagas cardiomyopathy, in a mouse model of trypanosoma cruzi infection vaccines-basel zhu application of antigen presenting cell-targeted nanovaccine delivery system in rhabdovirus disease prophylactics using fish as a model organism talebzadeh preparation of a nanovaccine against brucella melitensis m based on plga nanoparticles and oligopolysaccharide antigen ferreira nano-multilamellar lipid vesicles (nmvs) enhance protective antibody responses against shiga toxin (stx a) produced by enterohemorrhagic marques virus-like particle display of the α-gal carbohydrate for vaccination against toth polyglutamic acid-trimethyl chitosan-based intranasal peptide nano-vaccine induces potent immune responses against group a streptococcus luján efficient oral vaccination by bioengineering virus-like particles with protozoan surface proteins douglas a self-adjuvanted, modular, antigenic vlp for rapid response to influenza virus variability bouzari nanoparticle-based vaccines for brucellosis: calcium phosphate nanoparticles-adsorbed antigens induce cross protective response in mice borges development of a novel adjuvanted nasal vaccine: c / associated with chitosan nanoparticles as a path to enhance mucosal immunity irvine liposomal vaccines incorporating molecular adjuvants and intrastructural t-cell help promote the immunogenicity of hiv membrane-proximal external region peptides zhuang construction and immunological evaluation of cpg-au@hbc virus-like nanoparticles as a potential vaccine liao non-covalent glycosylated gold nanoparticles/peptides nanovaccine as potential cancer vaccines chin key: cord- -t chpsuh authors: lucas, alexander h.; apicella, michael a.; taylor, christopher e. title: carbohydrate moieties as vaccine candidates date: - - journal: clin infect dis doi: . / sha: doc_id: cord_uid: t chpsuh carbohydrate epitopes or glycotopes are structurally diverse, occur in a variety of chemical contexts, and are present on the surfaces of cells in the body and on the surfaces of pathogens. these various structures and modes of presentation affect how they are perceived and processed by the body and dictate the outcome of the immune response directed against them. this review focuses on mechanisms of carbohydrate immunity, with an emphasis on carbohydrate vaccines that have been or are being developed for protection against encapsulated bacterial pathogens. we discuss the cellular basis of carbohydrate immunity, newly identified glycotope processing pathways and recognition capabilities, and the synthetic and microarray technologies that are being developed that will permit new experimental approaches to carbohydrate vaccine development and the exploration of the interaction of the immune system with self and nonself glycans. this article focuses primarily on mechanisms of carbohydrate immunity, with an emphasis on carbohydrate vaccines that have been or are being developed for prevention of diseases caused by encapsulated bacterial pathogens. data on the use of recent technological advances in immunology, genomics, and glycomics in vaccine development are also presented. it has long been known that bactericidal and/or opsonic antibodies directed against capsular polysaccharide (ps) glycotopes protect against invasive diseases caused by encapsulated bacteria, and, accordingly, vaccine development has focused on the elicitation of these antibody specificities. purified microbial ps vaccines have been in use for years, but they have proven to be variably immunogenic and variably efficacious in protecting susceptible populations against invasive meningococcal, pneumococcal, and haemophilus influenzae type b (hib) diseases [ , ] . when administered as purified pss, their effectiveness generally is limited by the modest nature of the immune response, the lack of a memory response, and the inability to stimulate responses in young children who are at the greatest risk for serious infections due to encapsulated organisms. the demonstration that infants could produce protective anticapsular antibody responses following immunization with hib psprotein conjugates represented a major advance in public health and laid the groundwork for the subsequent development of new conjugate vaccines [ ] . an effective pneumococcal heptavalent capsular ps conjugate vaccine has been developed and is licensed in the united states for use in infants and children [ ] . use of this vaccine has led to a significant reduction in serious pneumococcal disease caused by pneumococci expressing capsular serotypes contained within the vaccine. the vaccine has also been shown to reduce the incidence of streptococcus pneumoniae otitis media [ , ] . studies from israel have indicated that this vaccine may impact favorably by reducing the number of antibiotic-resistant strains in the vaccinated population [ , ] . however, emerging evidence demonstrates that serotype replacement with nonvaccine capsular types may be a future problem necessitating changes in the vaccine spectrum. an -valent pneumococcal ps conjugate vaccine is presently undergoing evaluation. meningococcal serogroup a ps is unusual because it is immunogenic in infants as young as months of age, and administration of repeated doses elicits booster antibody responses. reimmunization at and years of age maintains a high degree of protection. older children and adults require only injection. vaccines against group a meningococci have been highly effective in preventing disease in a number of regions. in finland, herd immunity to group a meningococci was induced by vaccinating approximately one-third of the population. use of vaccines has significantly reduced the prevalence of disease in china. household contact vaccination has proven to be highly effective in africa. however, attempts at preparing protein conjugates of group a meningococcal capsular ps have not increased immunogenicity. group b meningococcus is the major cause of serious meningococcal disease in the united states [ ] . group b capsular ps is nonimmunogenic, presumably because of its identity with self-antigens [ ] . the potential for induction of autoimmunity and the lack of immunogenicity have discouraged commercial development of ps-based group b vaccines. alternative approaches have focused on other antigens, such as the outer membrane vesicle. the outer membrane-vesicle vaccine has good efficacy in older children and adults; however, the data for infants are mixed [ ] . following extensive testing, an omv vaccine prepared from an epidemic group b strain in new zealand has been provisionally licensed. this vaccine has been introduced into the population in an attempt to control the epidemic [ ] . investigators at wyeth and oxford university, england, are studying detoxified meningococcal lipopolysaccharide as a conjugate vaccine. the inner core of the meningococcal lipopolysaccharide is relatively highly conserved, and the use of appropriate conjugation procedures can result in a broadly cross-reactive meningococcal lipopolysaccharide vaccine. the meningococcal group c ps conjugate vaccine has proven to be an excellent vaccine [ ] . in the united kingdom, a decrease in the rate of group c disease was observed after vaccination with this conjugate. in children who were vaccinated during infancy at , , and months of age, the effectiveness of the vaccine decreased to very low levels after year [ ] . there was a much smaller decline in efficacy in infants who were vaccinated at - months of age, suggesting that a booster dose administered at - months of age might protect against the loss of efficacy and is now recommended. follow-up studies also demonstrated that there was no evidence of a switch of meningococcal capsular serogroup from serogroup c to other serogroups caused by vaccine pressure. as noted above, with the exception of the group a meningococcal ps, children aged ! years generally are unresponsive to vaccination with purified ps antigens. one early explanation for this anergy was that infants lacked the relevant ps-specific b cells. however, numerous studies have demonstrated that an insufficient antibody repertoire cannot account for the refractoriness of the infant to ps vaccination. infants can produce anti-ps antibodies after ps conjugate vaccination, and studies of the hib ps antibody repertoire have shown that infants can utilize the same variable (v) region genes as those used by adults [ ] . despite intensive investigation, the mechanism underlying the unresponsiveness of the infant to ps vaccination has not been precisely identified. the mechanism is likely multifactorial and may involve lacking expression of sufficient costimulatory molecules and/or their respective receptors and immaturity of dendritic cells and/or marginal zone b cells. cell surfaces in the body are replete with glycan molecules, and it is clear that self glycotopes can function as tolerogens and thus negatively shape the expressed antibody repertoire. the absence of antibodies to a and b blood-group substances in individuals expressing the respective blood groups represents a classical example. however, tolerance is imperfect, and antibodies against self-glycan structures can be present in healthy subjects. infectious agents or vaccines expressing glycotopes structurally related to self glycans can elicit antibodies reactive with self antigens. for example, neutralizing antibodies elicited by an inactivated severe acute respiratory syndrome coronavirus vaccine cross-react with the human serum glycoprotein asialoorosomucoid [ ] . what is less clear is the role of self glycans in the positive selection of the b cell repertoire. a growing body of evidence indicates that positive selection occurs during murine-b cell development [ ] and can involve selection against the self glycoprotein thy- [ ] . the recent discovery of an endogenous cd -restricted glycosphingolipid, isoglobotrihexosylceramide, suggests that self glycans may mediate the positive selection of t cells and/or nk cells [ ] . despite our lack of understanding of the mechanism, it is well established that the refractoriness of infants to pss, a class of antigens designated as thymus-independent (ti) type , can be overcome when ps glycotopes are presented in the context of an immunogenic carrier protein. coupling of protein is thought to convert glycotopes into t cell-dependent immunogens. immunization with ps protein-conjugate vaccines likely involves activation of the follicular b cell population. follicular b cells take up ps protein conjugates via their ps-specific b-cell receptor, process the carrier protein moieties, and present the resulting peptides on cell surface major histocompatibility complex class ii molecules where they may engage peptide-specific t cells. ps-specific b cells thereby receive signals via cognate t cell-b cell interactions and cd -cd l engagements that are sufficient for activation, differentiation into antibody secreting cells, germinal-center formation, class switch recombination, and somatic hypermutation. memory b cells generated during this t cell-dependent process can be activated by subsequent stimulation with either ps-protein conjugates or by multi-epitope ps. different carrier proteins have been used in the formulation of ps-conjugate vaccines, and although they are thought to convert the glycotope into a t cell-dependent form, they possess disparate immunological properties. for example, hib psconjugate vaccines utilizing different carriers elicit distinct antibody populations, as has been discerned by idiotypic [ ] and avidity analyses [ ] . furthermore, studies of humans indicate that both th and th cytokine responses are elicited following immunization with pneumococcal conjugate vaccines [ ] . the outer membrane protein complex derived from neisseria meningitidis is used as a carrier protein in some ps-conjugate vaccines. the hib ps-conjugate vaccine, having an outer membrane protein complex as the carrier, is uniquely capable of stimulating a serum anti-hib ps-antibody response in month-old infants after a single immunization. in contrast, conjugates utilizing bacterial toxoids as carriers usually do not elicit significant seroconversion until the second immunization [ ] . these properties of the outer membrane-protein complex carrier may be related to its ability to engage toll-like receptor- [ ] . thus, carrier proteins function not only as a source of peptide epitopes permitting the delivery of t cell help, but they also can possess mitogenic and adjuvant-like properties that stimulate the innate immune response. the immunological variability of different carriers and the increasing use of multivalent glycoconjugates utilizing the same carrier protein has generated concern about potential complications arising from high carrier doses, such as antigenic competition and carrier-induced epitope suppression. this concern has prompted the search for alternate carrier-protein strategies that include the synthesis of a peptide carrier that functions as a universal t helper-cell epitope by binding promiscuously to human leukocyte antigen-dr molecules [ ] and the synthesis of recombinant carrier proteins containing strings of t cell epitopes derived from several pathogen-derived antigens [ ] . pss have been classified as ti- antigens on the basis of their ability to stimulate antigen-specific b cells in the absence of t cells [ ] . ps antigens, by nature of their multivalent, repeating glycotope structure, engage a sufficient number of b cell receptors that result in b cell activation and maturation to antibody secretion. the inability of ps immunization to lead to anamnestic responses suggests that memory b cells are not generated by this type of antigenic stimulation, presumably because of failure of the immunogen to associate with mhc-ii molecules and recruit t cell help. although b cell activation by ti- antigens can occur independently of t cells, it is becoming increasingly clear that accessory signals, in addition to those generated by b cell-receptor cross-linking, are required to generate antibody responses to ti- antigens. in vitro, highly purified b cells do not respond to ti- antigens. accessory cells, such as dendritic cells and macrophages, and costimu-latory signals are required for an effective response to ti- antigens. in addition, many ti- antigens are able to fix complement, and the formation of these complexes in vivo could facilitate b cell activation by providing costimulation through complement receptors. engagement of the complement receptor lowers the antigen signaling threshold for b cell activation [ ] , and the coupling of complement c d fragments enhances ps immunogenicity [ ] . several molecules-such as the transmembrane activator and its ligand b lymphocyte stimulator, tyrosine kinase pyk- , bruton tyrosine kinase, and phospholipase c-g -have been identified as costimulatory signals and/ or receptors necessary for the generation of antibody responses to ti- antigens (reviewed in [ ] ). understanding the mechanisms by which these molecules exert their effects will not only suggest new ways to enhance responses to carbohydratebased vaccines, but also new ways to suppress autoimmune responses directed against self glycans. the immune response to glycotopes presented in the context of the microbe is important for understanding carriage, the acquisition of naturally acquired immunity, and the response to infection [ ] . the majority of older children and adults possess serum antibodies specific to a plethora of bacterial capsular glycotopes, and these antibodies mediate protective immunity. presumably, exposure to the homologous organism or to organisms expressing cross-reacting glycotopes drives the formation of these antibodies. these natural antibodies appear to derive from memory b cells, because molecular analyses of adult pneumococcal antibodies show that they have undergone both a class switch and hypermutation [ ] [ ] [ ] . these findings suggest that exposure to glycotopes presented in the context of the bacterial surface generates both ps-specific b cell antibody secretion and memory development. in mice, cd + t cells and engagement of the toll-like receptor- and its adaptor protein, myd , have been implicated in the response to pneumococcal glycotopes elicited by intact bacteria [ ] . unlike conjugate vaccination, which usually occurs by the intramuscular route, natural exposure occurs predominantly across mucosal surfaces. thus, understanding the induction of natural immunity and the maintenance of the carrier state must take into account the migration patterns between secondary lymphoid tissues and the homeostatic mechanisms operating at mucosal surfaces [ ] . it is likely that bacteria, purified ps, and glycoconjugate vaccines address overlapping as well as distinct subsets of b cells. as discussed above, follicular b cells are probably the major subset activated by ps-protein conjugates, whereas blood-borne bacteria and ps antigens are thought to stimulate marginalzone b cells (and b- b cells in the mouse) [ , ] . marginal-zone b cells differ from follicular b cells in their rapid proliferative response and elaboration of igm antibodies early in the course of bacterial infection, and it has been suggested that these b cells represent an innate-like response that mediates immediate immunity before the slower adaptive response has developed [ ] . human marginal-zone b cells express high levels of cd c, which suggests that they might be well suited for presentation of bacterially derived lipid antigens. in humans, there is a subpopulation of circulating b cells that have the phenotype of marginal-zone b cells. these cells have been designated "igm memory b cells" because they express both cell-surface igm and the memory cell marker cd . the v genes of peripheral blood igm memory b cells are mutated [ ] , and a recent report indicates that these cells are mobilized in response to ps vaccination [ ] . igm memory b cells with mutated v genes are present in patients with hyper igm syndrome who lack functional cd -cd ligand interactions [ ] . this finding has prompted the suggestion that pathways of somatic hypermutation exist in humans: the traditional pathway dependent on t cells and cd receptor-cd l receptor interactions, and another pathway independent of cd receptor-cd l receptor interactions [ ] . molecular modeling and x-ray crystallographic studies have verified the original proposal of kabat et al. [ ] that antibodycombining sites recognizing glycotopes can exist as either shallow grooves or as deep clefts, with the former binding internal epitopes expressed along the length of the glycan polymer and the latter binding to epitopes on the nonreducing ends of the glycan chain. some glycotopes are expressed by short oligomers, whereas others require longer chain lengths for expression. studies of the type pneumococcal ps have shown that the induction of osponically active antibodies is critically dependent on chain length and the expression of a conformational epitope [ ] . similarly, the protective glycotope of type iii group b streptococcal ps is a conformationally dependent, helical structure requiring extended chain lengths for expression [ ] . thus, antibodies prepared against short oligomeric glycotopes may show the requisite antigen-binding specificity, but they may not recognize native capsular glycotopes and mediate protection. this issue is particularly important in the design of vaccines using synthetic oligosaccharides. avidity has been shown to be a critical determinant of the efficacy of anticapsular antibody protection against hib [ , ] , pneumococcus [ ] and meningococcus [ ] . variations in antibody avidity occur during the course of the immune response and can be influenced by vaccine type [ ] and age [ ] . the structural basis of avidity variation has been studied in detail in the human response to hib ps, in which a struc-turally conserved, canonical combining site dominates the repertoire. regions in the combining site that drastically impact hib ps-binding avidity have been localized to areas thought to represent antigen contact sites, and they include allelic polymorphisms in complementarity region (cdr)- of the heavy chain v region [ ] and polymorphisms in the light chain cdr- that are generated by differential j-region use and utilization of different insertional residues at the v-j junction [ , ] . recently, a new mechanism of antibody glycotope recognition was described elsewhere [ ] . a monoclonal antibody that is specific for a mannose glycotope of hiv was found to have exceptionally high affinity and potent neutralizing activity against numerous viral isolates. x-ray crystallographic analysis demonstrated that the fragment antigen binding dimer possesses binding sites for the mannose determinant. two combining sites constitute the traditional site created by the association of variable heavy and light chain domains, and additional sites are created by domain exchange at the vh-vh interface. this capability for multivalent interaction provides a structural explanation for the high avidity of this antibody. thus, targeting glycotopes in addition to protein epitopes may be a worthwhile strategy in the development of an effective multivalent hiv vaccine that elicits broadly neutralizing antibody responses [ ] . molecular mimicry has been a major area of investigation in carbohydrate immunology. numerous reports have shown that peptides binding to carbohydrate combining sites can be isolated from peptide libraries [reviewed in ]. these peptides show apparent specificity in that their binding is peptide-sequence dependent but is usually limited to the combining site against which they were selected. a fundamental issue is whether these mimetics, when presented in an immunogenic form, are able to elicit carbohydrate-specific antibody responses. some studies suggest that this, indeed, is the case. peptide mimics have been shown to induce cross-reactive responses to carbohydrate antigens expressed on bacteria [ , ] , fungi [ ] , hiv envelope protein [ ] , and tumor cells [ ] , to name a few. in a mouse-tumor challenge model, immunization with a dna construct encoding a mimetic inhibited tumor growth [ ] and antimimotope antibodies block cell adhesion of hiv gp -expressing cells and human dendritic cells [ ] . one could imagine a number of advantages of using peptidyl mimics rather than glycotopes. these include the ease of synthesis and purification, the ability to be formulated in a variety of ways not amenable to glycotopes, such as multiple antigen peptides and dna vaccines [ ] , and the ability to engage cellular processing and activation pathways not normally within the capability of glycotopes. the dogma that the recognition of glycotopes is the exclusive provenance of antibody combining sites has been overturned by studies in the past decade that demonstrate that t cell receptors are able to recognize glycotopes if they are presented in the appropriate mhc-binding context [ ] [ ] [ ] . functional and structural studies of glycopeptide antigens have demonstrated that mhc molecules tether the glycopeptide by binding the peptidyl moiety in the mhc-binding pocket, such that the glycotope can directly engage the t cell receptor. cytotoxic t cell responses to carbohydrate antigens can be induced by immunization with either naturally occurring or synthetic glycopeptides [ , ] . glycolipid antigens can also be recognized by t cells and nk cells via a cd -dependent mechanism [ ] . cd molecules, which exist in multiple isoforms, are structurally related to mhc class i molecules. examples of cd -restricted antigens include mycobacterial glycolipids and the self-derived ganglioside gm . the t cells recognizing cd -presented lipid antigens may utilize a variety of t cell receptor-a and t cell receptor-b chains. in addition, nk cells expressing an invariant t cell receptor-a chain paired with various b chains (in both mouse and man) also are cd -restricted, and they recognize a glycosphingolipid known as a-galactosylceramide, a glycolipid originally identified in marine sponges. a-galactosylceramide binds with high affinity to cd d and has potent immunoregulatory properties. administration of a-galactosylceramide induces production of ifn-g and il- and activates nk cells, t cells, and b cells. a synthetic analogue of a-galactosylceramide has potent protective activities in murine models of malaria and melanoma. cd- restricted t cells may produce either th -type or th /th -type cytokine responses that could be important in antitumor and antimicrobial immune responses. although the precise role of cd -restricted t cells in adaptive immune responses requires further study, some evidence suggests that cd -restricted nkt cells are involved in immunity to mycobacterium tuberculosis, pseudomonas auruginosa, plasmodium yoelii, trypanosoma cruzi, and francisella tularensis (reviewed in [ , ] ). a recent study of the capsular ps of bacteriodes fragilis [ ] identified a new pathway of glycotope processing and presentation. this work originated from earlier studies demonstrating that ps-specific cd + t cells were involved in protection against sepsis caused by b. fragilis [ ] . the capsular ps of b. fragilis (and some pneumococcal capsules) is zwitterionic and forms a helical structure. the ps is capable of inducing protective psspecific t cells in an antigen presenting cell-dependent fashion, and this effect depends on the zwitterionic character of the ps. the b. fragilis ps is processed by antigen presenting cells via an endosomal-lysozomal pathway that involves chemical cleav-age by a nitric oxide-mediated mechanism that generates lowmolecular weight carbohydrates capable of binding to mhc-ii and being presented to cd + t cells. given the diversity of glycan structures and their chemical contexts, it is likely that additional carbohydrate-processing pathways will be discovered. for example, the c-type lectin, known as specific intracellular adhesion molecule-grabbing nonintegrin-r (sign-r ), which is expressed on splenic marginal-zone macrophages, binds with high affinity to the pneumococcal type capsular ps and mediates uptake of type pneumococci [ ] . the discoveries that foreign ps antigens and glycotopes can be processed by endosomal pathways, can be presented by mhc-i, mhc-ii, and cd- molecules, and can be recognized by t cells and endogenous lectins hold great promise for designing new glycan-based adjuvants and vaccines against tumors, bacteria, and parasites. research in carbohydrate immunology is being accelerated by the development of new technologies, such as carbohydrate microarrays, automated syntheses of oligosaccharides, and biophysical and computational methods for studying antigenantibody interactions. carbohydrate microarrays can be used to investigate lectin and antibody specificities, to identify new lectins, to screen blood for disease patterns, or to identify lead structures for vaccine design [ , ] . a consortium for functional genomics has been established and has developed a novel glycan-array format that has been demonstrated to have applicability for profiling the specificity of a wide variety of carbohydrate-binding molecules [ ] . these new technologies will contribute to the design of better-defined carbohydrate vaccines and will serve as essential tools in our understanding of the glycome and its interaction with the immune system. progress is being made in the development of chemical methods and instruments capable of efficient, high-capacity oligosaccharide synthesis [ ] , and these advances are being applied to basic research and to the development of synthetic carbohydrate vaccines. experimental vaccines utilizing synthetic carbohydrates have been developed for a number of pathogens, including s. pneumoniae [ ] , vibrio cholera [ ] , shigella flexneri [ ] , and plasmodium falciparum [ ] . a hib vaccine containing a fully synthetic oligomer of the hib capsular ps has been tested in humans and has been demonstrated to elicit functionally active antibodies [ ] . carbohydrate antigens represent a significant resource for the development of vaccines against infectious agents, particularly against pathogens for which traditional protein and/or whole organism-based approaches have not been successful. although we do not discuss it in this article, there is a large body of work exploring carbohydrate-based vaccines for the elicitation of both humoral and cellular immunity against tumors [ ] . new carbohydrate-based vaccines are being developed for pathogens such as hiv and malaria, and we might expect to see clinical trials evaluating new adjuvants, such as a-galactosylceramide. aside from the need for research, the need for a variety of resources is essential. the availability of carbohydrate microarrays and appropriate animal models, including primate models and transgenic mouse platforms, should be enhanced. also, tetramers and analytical chemistry are needed to aid in the identification of glycolipid antigens recognized by cd- . other infrastructural needs include good laboratory practice resources for the production of synthetic carbohydrate vaccines and for testing the safety of vaccine candidates in animals and more opportunities (i.e., computer capacity) to model carbohydrate antigen-antibody interactions. resources will be needed to ensure compliance with regulatory standards, such as a defined chemical composition and structure, the ability to manufacture with consistent physical and chemical characteristics, the lack of inherent toxicity, and the capability to induce protective immune responses. it is becoming increasingly apparent that the various structures and modes of glycotope presentation affect how they are perceived and processed by the body and dictate the outcome of the immune response directed against them. new glycotopeprocessing pathways and recognition capabilities are being identified, the signals and molecules involved in cell activation are being elucidated, and new synthetic and microarray technologies are being developed that will permit new experimental approaches to explore the interaction of the immune system with self and nonself glycans. these discoveries can be expected to translate into new therapeutics and vaccines to combat disease and improve human health. pneumococcal polysaccharide vaccines capsular polysaccharide and conjugate vaccines prevention of systemic infections, especially meningitis, caused by haemophilus influenzae type b: impact on public healthand implication for other polysaccharide-based vaccines safety and efficacy of the seven-valent pneumococcal conjugate vaccine: evidence from northern california community-wide vaccination with the heptavalent pneumococcal conjugate significantly alters the microbiology of acute otitis media impact of the pneumococcal conjugate vaccine on otitis media trends in antimicrobial resistance in , invasive streptococcus pneumoniae strains isolated in spanish hospitals characteristics of nasopharyngeal carriage of streptococcus pneumoniae in children during acute respiratory disease the changing epidemiology of meningococcal disease in the united states, - polysialic acid units are spatially and temporally expressed in developing postnatal rat kidney immunogenicity of serogroup b outer-membrane protein meningococcal vaccines evaluating the post-licensure effectiveness of a group b meningococcal vaccine in new zealand: a multifaceted strategy effectiveness of meningococcal serogroup c conjugate vaccine years after introduction molecular ontogeny of the human antibody repertoire to the haemophilus influenzae type b polysaccharide: expression of canonical variable regions and their variants in vaccinated infants glycan arrays lead to the discovery of autoimmunogenic activity of sars-cov b cell positive selection: road map to the primary repertoire positive selection of anti-thy- autoreactive b- cells and natural serum autoantibody production independent from bone marrow b cell development lysosomal glycosphingolipid recognition by nkt cells variable region expression in the antibody responses of infants vaccinated with haemophilus influenzae type b polysaccharide-protein conjugates: description of a new l light chainassociated idiotype and the relation between idiotype expression, avidity, and vaccine formulation avidity and bactericidal activity of antibody elicited by different haemophilus influenzae type b conjugate vaccines. collaborative vaccine study group significant variation in serotype-specific immunogenicity of the sevenvalent streptococcus pneumoniae capsular polysaccharide-crm conjugate vaccine occurs despite vigorous t cell help induced by the carrier protein differences in the immunogenicity of three haemophilus influenzae type b conjugate vaccine in infants haemophilus influenzae type b-outer membrane protein complex glycoconjugate vaccine induces cytokine production by engaging toll-like receptor (tlr ) and requires the presence of tlr for optimal immunogenicity development of experimental carbohydrate-conjugate vaccines composed of streptococcus pneumoniae capsular polysaccharides and the universal t-lymphocyte epitope (padre) rationally designed strings of promiscuous cd (+) t cell epitopes provide help to haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines t cell-independent antigens type the cd /cr /tapa- complex of b lymphocytes: linking natural to acquired immunity increased immunogenicity and induction of class switching by conjugation of complement c d to pneumococcal serotype capsular polysaccharide t-independent immune response: new aspects of b cell biology b cell activation by t-cell-independent type antigens as an integral part of the humoral response to pathogenic microorganisms combinatorial library cloning of the human antibody repertoire to streptococcus pneumoniae capsular polysaccharides: variable region primary structures and evidence for somatic mutation of fab fragments specific for capsular serotypes b, and f recurrent variable region gene usage and somatic mutation in the human antibody response to type f pneumococcal capsular polysaccharide immunogenetic analysis of the immune response to pneumococcal polysaccharide differential regulation of igg anticapsular polysaccharide and antiprotein responses to intact streptococcus pneumoniae in the presence of cognate cd + t cell help marginal zone and b b cells unite in the early response against t-independent blood-borne particulate antigens human immunoglobulinn m memory b cells controlling streptococcus pneumoniae infections are generated in the spleen human immunoglobulin (ig)m + igd + peripheral blood b cells expressing the cd cell surface antigen carry somatically mutated variable region genes: cd as a general marker for somatically mutated (memory) b cells human igm 'memory' b cells are circulating splenic marginal zone b cells harboring a prediversified immunoglobulin repertoire cd -cd l independent ig gene hypermutation suggests a second b cell diversification pathway in humans antibody complementarity and antibody structure streptococcus pneumoniae type -conjugate vaccines: stabilization of opsonophagocytic conformational polysaccharide epitopes conformational epitope of the type iii group b streptococcus capsular polysaccharide avidity as a determinant of the protective efficacy of human antibodies to pneumococcal capsular polysaccharides protective activity of group c anticapsular antibodies elicited in year-olds by an investigational quadrivalent neisseria meningitidis-diphtheria toxoid conjugate vaccine age-related disparity in functional activities of human group c serum anticapsular antibodies elicited by meningococcal polysaccharide vaccine igh v - * and its allele v - * differ in their capacity to form the canonical human antibody combining site specific for the capsular polysaccharide of haemophilus influenzae type b role of kii-a l chain cdr- junctional residues in human antibody binding to the haemophilus influenzae type b polysaccharide antibody domain exchange is an immunological solution to carbohydrate cluster recognition hiv vaccine design and the neutralizing antibody problem peptide mimotopes as surrogate antigens of carbohydrates in vaccine discovery a peptide mimotope of type pneumococcal capsular polysaccharide induces a protective immune response in mice peptide mimicry of the meningococcal group c capsular polysaccharide immunogenicity and efficacy of cryptococcus neoformans capsular polysaccharide glucuronoxylomannan peptide mimotope-protein conjugates in human immunoglobulin transgenic mice peptide mimicry of carbohydrate epitopes on human immunodeficiency virus ) • vaccines plewski z. vaccination with carbohydrate peptide mimotopes promotes anti-tumor responses a mimic of tumor rejection antigen-associated carbohydrates mediates an anti-tumor cellular response fucosylated lactosamines participate in adhesion of hiv- envelope glycoprotein to dendritic cells induction of a protective capsular polysaccharide antibody response to a multiepitope dna vaccine encoding a peptide mimic of meningococcal serogroup c capsular polysaccharide mhc interaction and t cell recognition of carbohydrates and glycopeptides glycopeptides bind mhc molecules and elicit specific t cell responses carbohydrates and antigen recognition by t cells immunization with glycosylated kb-binding peptides generates carbohydrate-specific, unrestricted cytotoxic t cells crystal structure of an mhc class i presented glycopeptide that generates carbohydratespecific ctl understanding the function of cd -restricted t cells going both ways: immune regulation via cd b-dependent nkt cells polysaccharide processing and presentation by the mhcii pathway structural features of polysaccharides that induce intrabdominal abscesses the c-type lectin sign-r mediates uptake of the capsular polysaccharide of streptococcus pneumoniae in the marginal zone of mouse spleen carbohydrate microarrays-a new set of technologies at the frontier of glycomics the use of carbohydrate microarrays to study carbohydrate-cell interactions and to detect pathogens consortium for functional genomics automated carbohydrate synthesis to drive chemical glycomics synthetic b di-, tri-and tetrasaccharide-protein conjugates contain pneumococcal type a and b common and b-specific epitopes that elicit protective antibodies in mice synthetic fragments of vibrio cholera o inaba o-specific polysaccharide bound to a protein carrier are immunogenic in mice but do not induce protective antibodies preparation of synthetic glycoconjugates as potential vaccines against shigella flexneri serotype a disease synthetic gpi as a candidate anti-toxic vaccine in a model of malaria synthetic conjugate polysaccharide vaccine against haemophilus influenzae type b on the power of chemical synthesis: immunological evaluation of models for multiantigenic carbohydrate-based cancer vaccines this review is based largely on presentations made at the carbohydrate moieties as vaccine candidates workshop held at the national institutes of health, - october . we acknowledge the valuable contributions of the workshop participants and particularly thank drs. kate rittenhouse-olson, liise-anne pirofski, and john schreiber for their critical review of the manuscript prior to submission. this workshop was sponsored by the national vaccine program office and the national institute of allergy and infectious diseases. potential conflicts of interest. all authors: no conflicts. key: cord- -yeaw nr authors: nedjadi, taoufik; el-kafrawy, sherif; sohrab, sayed s.; desprès, philippe; damanhouri, ghazi; azhar, esam title: tackling dengue fever: current status and challenges date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: yeaw nr according to recent statistics, million apparent dengue infections were estimated worldwide in . this figure is by far greater than the who prediction which indicates the rapid spread of this disease posing a growing threat to the economy and a major challenge to clinicians and health care services across the globe particularly in the affected areas. this article aims at bringing to light the current epidemiological and clinical status of the dengue fever. the relationship between genetic mutations, single nucleotide polymorphism (snp) and the pathophysiology of disease progression will be put into perspective. it will also highlight the recent advances in dengue vaccine development. thus far, a significant progress has been made in unraveling the risk factors and understanding the molecular pathogenesis associated with the disease. however, further insights in molecular features of the disease and the development of animal models will enormously help improving the therapeutic interventions and potentially contribute to finding new preventive measures for population at risk. dengue fever is a major cause of illness and death worldwide. the disease is caused by dengue virus which gets transmitted to humans by the bites of infected mosquitoes, aedes (ae.) aegypti and ae. albopictus [ ] . the disease represents a global health issue as it is endemic in around countries, most of which are in tropical and sub-tropical areas. over the last decades, the incidence rate and the geographic distribution of dengue have rapidly increased (almost -fold). data from the world health organization (who) estimates up to million cases of dengue fever each year [ ] . however, a recent published work by bhatt et al. ( ) suggested that the burden of dengue is far more than the who estimation and indicated that million infections of dengue virus could have happened every year [ ] . changes in dengue epidemiology and the increase in incidence rates (with and without co-morbidities) have led the who to propose a new dengue classification system according to disease severity ( fig. ) [ ] . dengue fever is caused by infection with dengue virus (denv). the denv is a vector-borne virus transmitted to humans primarily by bites from two mosquito species, ae. aegypti or ae. albopictus. denv is a single positivestranded rna virus belonging to flavivirus genus of the flaviviridae family and has major serotypes (denv - ) that are antigenically distinct from each other. each denv serotype is phylogenetically distinct suggesting that each serotype could be considered a separate virus [ ] . three dengue serotypes out of four (denv - ) have been found in middle eastern countries including saudi arabia and yemen. interestingly, denv- strain isolated in saudi arabia exhibited a high genetic similarity with denv- strain isolated from asian population, suggesting a widespread of the asian genotype, probably through asian pilgrims [ , ] . a recently published article has unveiled a new serotype , to be added to the existing ones [ ] . this discovery is still controversial and little-known enough to conclude how the th dengue serotype might add to the burden associated with dengue infection. mosquitoes transmit the virus by feeding on blood of infected persons. at first, the virus infects and replicates in the mid-gut epithelium of the mosquito and then spreads to other organs until it reaches the salivary glands after - days where it can be inoculated to another person during subsequent blood meal. vertical transmission of denv in mosquitoes, i.e. from mosquito to larvae has been reported by a number of research groups. in india, angel & joshi ( ) reported the detection of dengue virus by indirect fluorescence antibody test (ifat) in laboratory reared mosquitoes originating from larvae collected from urban and rural areas [ ] . a similar study was conducted in brazil by martins et al. ( ) and confirmed the isolation of denv-type in ae. albopictus larvae and denv-type in ae. aegypti larvae [ ] . similar findings were also reported in mexico [ ] and indonesia [ ] . on the other hand, mother-to-infant transmission of dengue virus via cord blood or breast milk remains controversial [ ] [ ] [ ] . based on the results from several studies, the who has launched a new dengue classification. this classification divides dengue cases into a) cases with/without warning signs and b) severe dengue cases [ ] . however, it is important to note that numerous research groups have debated the rational of this classification as it does not fit their unique local settings. the criteria for dengue case classification are presented in fig. . clinically, dengue infection has a broad spectrum of features. the vast majority of cases are asymptomatic and passes unnoticed. typically, the symptoms start to be prominent after an incubation period of - days [ ] . the severity of the clinical manifestations varies from mild symptoms to severe life threatening symptoms in the case of dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) [ ] . predicting the progression of the mild signs to a severe dhf/dss remains a challenge due to non-specificity of clinical presentation and the incomplete understanding of pathophysiology of the disease and its underlying molecular mechanisms. the early signs of the disease are non-specific. according to the who classification ( ), df is characterized by febrile episode (≥ °c for - days) frequently associated with rash, nausea, vomiting, and headache. although the disease affects people of all ages from infancy through to adulthood [ ] , epidemiological data showed that children tend to tolerate this phase of illness better than adults [ ] . the persistence of the aforementioned symptoms and appearance of other symptoms, such as abdominal pain, mucosal bleed, and lethargy and restlessness can be seen - days later. laboratory analysis of mild dengue fever cases usually shows abnormal leukocyte counts and moderate elevation of the hepatic amino-transferase enzyme activity [ ] . the emergence of these symptoms is a warning sign for disease progression to severe form (dhf/dss) if therapeutic intervention is not undertaken. at this stage clinical intervention and continuous surveillance are imperative to prevent vascular leakage, especially in an endemic area. this form of dengue infection can be attributed to any of the four known serotypes denv - . the likelihood of developing dhf/dss is high in patients who have experienced dengue infection in the past with heterogeneous serotype [ ] . about - % of patients progress to develop a severe dhf/dss which can be fatal unless treated promptly [ ] . this form develops at a late stage of df, where patients may go through defervescence phase characterized by a sudden drop of body's temperature. this phase is also distinguished by severe bleeding, particularly bleeding from the gastrointestinal tract (black, tarry stool), and thrombocytopenia (< , /mm ), which may affect up to % of dhf cases [ ] . interestingly, there was an observed negative correlation between the severity of dhf and the level of platelets in the blood. the exact mechanism of this correlation has yet to be delineated. the drop of platelet counts and the loss of their functionality lead to a vascular fragility increasing the risk of hemorrhage and plasma leakage [ ] . it has been suggested that during acute phase of the infection denv replicates quickly in platelets, as this is very critical for virus survival and dissemination [ , ] . the existence of other symptoms such as retro-orbital pain, maculopapular rash, petechiae, or bleeding from the nose or gums will help making definitive diagnosis for df [ ] . evidence of plasma leakage in various body cavities such as the pleural cavity and the peritoneal cavity, associated with profuse perspiration, adynamia, and sometimes fainting are signs of rapid progression to shock. subsidence in systolic pressure and hypotension may result in profound shock, known as dengue shock syndrome (dss). the duration of dss for a long time might predispose to further complications such as massive bleeding, disseminated intravascular coagulopathy (dic), respiratory failure, multi-organ failure, and infrequently encephalopathy leading to death [ , ] . it has been proposed that case fatality related to dhf may reach % of all cases, however, proper medical care and symptomatic management can reduce mortality rate to less than % [ ] . an early and accurate laboratory diagnosis of dengue infection is of paramount importance in the management of the disease. it has been estimated that the number of misdiagnosed dengue cases could reach a record ratio of % of all cases, mainly due to a large disparity of dengue signs and symptoms which overlap with the symptoms of other viral infections, especially for persons living in or traveling to endemic areas of tropical infectious diseases. dengue fever should be distinguished from other illnesses which share similar symptoms such as chikungunya, mayaro fever, ross river fever, west nile fever, zika fever, yellow fever and viral hemorrhagic fevers [ ] . until the antiviral vaccine becomes available, the prevention of severe cases and cut-down of the economic burden of the disease rely enormously on early and accurate diagnosis. the latter is made possible through the availability of several diagnostic laboratory and virological tests. the onset of later stage symptoms of the illness can be overwhelming and more pathognomonic. nonetheless, based on who classification schemes, the appearance of leukopenia in patients with febrile illness is a major consideration in making diagnosis of dengue infection [ ] . overall, there is an urgent need to reduce dengue morbidity and mortality by improving the diagnosis and molecular analysis of emerging dengue virus. thus far, two diagnostic modalities have been applied to detect the disease at an early stage. the first one is a direct method targeting the acute phase of dengue disease, which is based upon detection of genomic rna by rt-qpcr or soluble ns by antigen capture in blood samples from viremic patients. the second is the indirect method that relies on serological tests to detect denguerelated immunoglobulins par mac-elisa for the capture of specific igm or indirect elisa for the capture of anti-den iggs [ ] [ ] [ ] [ ] [ ] . several risk factors have been associated with dengue infection and its progression to severe dhf/dss forms. recent advances in molecular biology have revealed that the genetic makeup of the three elements of dengue infection (the virus, the vector, and the host) plays a primordial role in the pathogenesis of the disease and could potentially contribute to the dhf progression [ , , ] . hence, an in-depth analysis of genetic variability including polymorphism and mutations could be beneficial in identifying the possible factors and mechanisms of disease development [ ] . the list of host's genetic factors that confer susceptibility or resistance to dengue infection is summarized in table . like most arboviruses, denv infect different organs of the mosquito, including the salivary glands and the central nervous system. mosquito infection elicit behavioral changes including increase of the probing time which lead to host interruption that might lead to wider spread of the virus [ ] . it has been demonstrated that denv infection induced the expression of cathepsin-b, a putative cystatin, and a hypothetical ankyrin repeat-containing protein genes [ ] . the latter could alter the efficiency of virus replication in the salivary gland. this study has shown that modulation of obp and obp genes expression as well as denv infection-responsive odorant-binding protein genes increase the time length for initiation of probing before a successful blood meal, resulting in changes in the host seeking behavior of the mosquito. comparative analysis of the salivary gland transcriptomes of native and denv-infected ae. aegypti identified a number of differentially expressed genes related to sugar/protein digestion enzymes, immunity related genes and blood meal acquisition enzymes that might have an impact on the efficiency of viral replication or mosquito feeding behavior. this study showed that denv infection alter the expression of key host-seeking genes in the mosquito's main olfactory organs and the antennae [ ] . recent updates have indicated that resistance of ae. aegypti to conventional insecticides is related to different mechanisms, one of which is associated with genetic abnormalities within the vector's genome. single point mutation in the voltage-gated sodium channel gene at position (f c) resulting in phenylalanine to cysteine substitution in ae. aegypti confers resistance to permethrin. this mutation is widespread in this vector in southeast asia and latin america [ , ] . it has also been reported that a single amino acid substitution valine to glycine at position in domain ii, segment of the voltage-gated sodium channel gene was associated with less sensibility of ae. aegypti to deltamethrin in thailand [ ] . numerous multi-disciplinary studies confirmed that race, young age, virus strain, female sex and high body-mass index correlate well with increased burden of dengue [ , ] . thus, a closer consideration of human genes regulating the severity of dengue infection, especially genes associated with the immune response, might help in controlling disease spread and improve the acute symptoms of the infection. a number of studies have investigated the relationship between the host genetic polymorphisms and denv infection ( table ) . a single nucleotide polymorphism (snp) in the promoter of cd /dc-sign was associated to increased risk of developing dengue fever [ ] . association studies have successfully identified a link between polymorphisms in the human major-histocompatibility-complex (hla) class i/ii genes and non-hla host genetic factors and severity of dengue disease [ ] [ ] [ ] . polymorphisms of the tap and tap genes could be directly associated with the risk of developing dengue disease among the primary-infected individuals [ ] . both tap and tap are located within the mhc class ii region and homozygosity of the tap at position and and for tap at position , respectively, was found to protect against developing severe forms of dengue [ ] . in an independent study [ ] , the authors showed that single nucleotide polymorphism of the oligoadenylate synthetase genes (oas , and ), of the oas/rnase l antiviral immune system, enhance susceptibility to clinical outcomes of dengue infection. an association between the severity of the disease and other genes including human leukocyte antigen class i and class ii genes, tumor necrosis factor-alpha, fcgriia, vitamin d receptor, transporters associated with antigen presentation, and jak has also been proposed [ ] . the importance of vit-d in denv pathogenesis was concluded from newly-gathered data showing that vit-d impairs denv replication and polymorphism of vit-d gene increases the expression of both cd /dc-sign and fcgriia receptors that enhance denv entry in the target cells [ , ] . in another study [ ] , the authors have successfully applied genome-wide association study (gwas) approach to identify loci that confer susceptibility to severe forms of dengue disease. the investigators used samples from children affected with severe dengue infection against population control cases in vietnam. the data showed that snps at two loci, micb and plce , significantly increased the likelihood of developing dss in children. this finding was further validated in an independent cohort of cases and controls [ ] . a snp in the micb gene coding for the mhc class i polypeptide-related sequence b, an inducible activating ligand for the nkg d type ii receptor of immune cells could alter the protective role of natural killer and cd + t cells in the host responsiveness to denv at the early stage of infection [ , ] . on the other hand, plce plays a primordial role in maintaining intact vascular endothelial cell barrier function, hence, polymorphism of the plce gene may lead to blood vessels leakage and circulatory hypovolemia during dss [ ] . other host candidate genes have also been associated with early onset dengue disease. among these genes, there were receptors/attachment factors for denv linked to immune system and inflammatory response. the chemokines cxcl , cxcl and its respective chemokine receptor cxcr were reported as biomarkers for severe form of dengue infection [ ] . these results are in agreement with recent emerging data indicating strong association between cxcl , cxcl and cxcr and vascular permeability [ ] . the three genes are components of the nf-kb pathway and are involved in the pathogenesis of sars and west nile virus encephalitis [ , ] . [ ] . this process depends enormously on vector-derived salivary factors inoculated on the skin cells [ ] . till-date, there is no effective, commercially available, therapy/vaccine for dengue virus. numerous groups have already made intensive efforts and made good progress to develop a safe, affordable and effective vaccine against all serotypes for global public health [ ] [ ] [ ] [ ] [ ] [ ] [ ] . vaccines which are being developed use various approaches such as live attenuated viruses, inactivated viruses, subunit vaccines, dna vaccines, and chimeric viruses using yellow fever vaccine and attenuated dengue viruses as backbones ( table ) . currently, only one tetravalent vaccine against dengue virus, developed by sanofi-pasteur (france) has reached phase iii clinical trial and is expected to be launched in . this vaccine is based on the production of four chimeric live dengue-yellow fever viruses in which the yellow fever (yf) d vaccine sequences encoding the envelope proteins prm and e genes were substituted by the prm and e genes from dv of serotype , , , or in a molecular clone of yf- d [ ] . this vaccine was produced and tested over people using four dengue virus isolates from indonesia and thailand. this candidate vaccine was found to be attenuated and stable in animal models with respect to plaque size and yellow fever virus neurotropism [ ] . results of the clinical trials showed no adverse effects except moderate injection site pain, headache, and myalgia. another randomized, controlled trial was launched using a total of thai school children to investigate the efficacy of a recombinant, tetravalent vaccine for dengue virus and only dengue cases were reported [ ] . phase i trial of the vaccine in the philippines showed that the seropositivity increased gradually ( , & %) after - vaccinations against all four serotypes as compared to control group. the most promising results were observed in children - years old who exhibited high levels of reactivity of , , , % for denv - ; respectively [ ] . another placebo-controlled trial was conducted on , children from vietnam (vaccine, n = vs placebo, n = ) to determine the clinical efficacy and safety of cyd-tdv. the results demonstrated virologically-confirmed cases in % of the vaccine group as compared to the control group ( %). the efficacy was achieved in up to . % ( % ci . - . ). these findings indicated that the vaccine is highly efficacious with good safety profile when three injections were given to children with age group - years at , and months intervals [ ] . the data emerging from another randomized phase ii trial in india indicated that the vaccine has no serious adverse events and the immunogenicity and safety of cyd-tdv were satisfactory [ ] . a pilot study carried out in five latin american countries where more than , children aged - were recruited to receive either the cyd-tdv vaccine or placebo. the results on efficacy ( . %) and safety profiles were consistent with the previous findings [ , ] . interestingly, the vaccine efficacy ( . %) against hospitalization for dengue was promising and represented a step forward to developing an effective dengue vaccine [ ] . other candidate dengue vaccines have been developed in usa by the johns hopkins university and national institute of allergy and infectious diseases (niaid) and have reached advanced clinical trials [ ] . four liveattenuated denv/delta- were generated each containing nucleotides deletion of the '-untranslated region of genomic rna (delta- ). these vaccines efficiently impaired viral growth in human liver carcinoma cells [ ] . to improve the attenuation of denv- /delta- and denv- /delta- , chimeric denv were developed by substitution of the prm-e gene region of denv- / delta- virus with the prm-e genes of denv- and denv- [ , ] . the results from phase i clinical trial showed that all four live-attenuated denv/ delta- are safe and immunogenic with minor side effects such as faint rash and transient leucopenia only after higher dose [ , ] . dengue virus serotype- antigen was expressed in a vector based on pediatric live-attenuated schwarz measles vaccine (mv) by using the envelope domain iii (ediii) fused with the ectodomain of the membrane protein (ectom). after immunization, long-term production of denv- serotype-specific neutralizing antibodies was observed in measles virus susceptible mice [ ] . a new strategy was evaluated based on single minimal tetravalent denv antigen expression using viral vector derived from pediatric live-attenuated measles vaccine (mv). a recombinant mv vaccine construct was developed using envelope domain iii (ediii) and ectodomain of the membrane protein. the neutralizing antibodies were induced against all four serotypes of dengue virus after two injections in mice susceptible to mv infection. a strong memory neutralizing response was observed against all four serotypes in immunized mice after inoculation with live denv from each serotype [ ] . a naked dna-based candidate vaccine against denv has been developed by the naval medical research center [ , , ] . the genes encoding prm and e of denv were cloned into a shuttle vector under the transcriptional control of human cytomegalovirus (cmv) promoter. the results of phase i clinical trial showed no adverse effects except mild injection site pain, swelling, and fatigue. after second dose, strong igm and igg antibody response was observed which favors the safety profile of this vaccine. to get a better immunogenicity profile, a vaccine based on lipid adjuvant vaxfectin (vical incorporated, san diego, usa), was developed and the results demonstrated good protection profile against denv compared to dna alone [ ] . based on this technology, different groups have developed other candidate vaccines and achieved good protection in mouse models using envelope glycoproteins prm and e, the non-structural protein ns and the helicase/protease ns as vaccine antigens [ ] [ ] [ ] . the first purified inactivated vaccine was developed with aluminum hydroxide (alum) adjuvant and tested in mice and rhesus macaques in the mid- s, by walter reed army institute of research against dengue serotype and good virus protection was reported after two doses [ , ] . using similar technology, second generation japanese encephalitis (je) piv vaccine was developed [ , ] . currently, a new je vaccine (ixiaro; novartis vaccines) has been approved for use in many countries, including the usa [ ] . another dengue vaccine (dengue piv), recombinant subunit dengue e glycoprotein antigen (r e) was also developed and has entered phase i clinical trial [ ] [ ] [ ] . the centers for disease control and prevention (usa) have also developed a live-attenuated vaccine named denvax, which was found to be highly immunogenic in both children and adults and has currently entered phase i clinical trial in the united states [ , ] . recently, a novel third generation approach is being used to develop a vaccine containing recombinant subunit e domain iii (ed ) and the results of laboratory tests have shown the development of potent neutralizing antibodies in a mouse model [ ] [ ] [ ] . using the same technology, a tetravalent vaccine was developed and expressed in pichia pastoris by splicing and using flexible pentaglycyl linkers of the four ediii. the observed results showed that this antigen elicit specific antibodies against all four denv serotypes in balb/c mice [ ] . animal models are very useful for vaccine test development. the lack of animal models significantly hampered the development and efficacy testing of dengue vaccine. currently only rhesus macaques and aotus monkeys are being used for testing the vaccine before clinical trials are initiated [ ] . the d me vaccine was evaluated in both aotus monkeys and rhesus monkeys, and found to be immunogenic with - % protection against dengue infection [ , ] . porter et al. ( ) demonstrated that injection of non-human primate with three doses on day , and , with tetravalent dengue dna vaccine vaxfectin-adjuvanted, was more efficient against live dengue- virus compared to control animals. this finding support initiation of vaxfectin-adjuvanted phase i clinical trial [ ] . successful induction of immune response was obtained in mice and rhesus monkeys to the vaccines developed using dengue prm-e, dengue prm-e-nonstructural (ns) , and dengue ns antigens, and piv adjuvanted with alum [ , ] . centers for disease control and prevention (fort collins, co), hawaii biotech, and simmons developed different vaccines that showed good immunogenicity in animal models [ ] . similarly, the psoralen/uv inactivation dengue vaccine was found to be more immunogenic and protective against dengue serotype virus in aotus monkeys [ ] . thus far, there are no antiviral drugs available to treat dengue fever; therefore the community will continue to depend on the control of the mosquito vector as the main route to prevent the spread of disease. alternative approaches have been utilized against flaviviruses by targeting and inhibiting virus entry and the essential elements used in virus replication, nonstructural proteins, rna polymerase, and proteases. the most important target elements include ns helicase nucleoside triphosphatase (ntpase/ rna ' triphosphatase (rtpase), ns methyl transferase/ rna-dependent rna polymerase, and ns /ns b protease [ ] [ ] [ ] . rna interference (rnai) technology is also being used to impair virus replication against respiratory syncytial virus, hepatitis viruses, influenza virus, poliovirus and hiv [ , ] . low molecular weight phenolic compounds such as flavonoids and phytochemicals isolated from plants were previously tested and are being used for anti-dengue therapy [ , ] . an anti-viral inhibitory effect ranging from - % against denv replication was observed when methanolic extracts of momordica charantia and andrographis paniculata were used in cultured primate cells [ ] . several attempts have been made in the past to tackle dengue through elimination of ae. aegypti. the most successful experiences were related to vector control programs adopted in cuba and singapore. the programs were based on intensive insecticidal treatment and reduction of the availability of aedes larval habitats [ , ] . unfortunately, lack of sustainability of these stringent measures led to reappearance of dengue outbreaks. recently, a novel form of biological control of dengue transmission has been developed and is currently being applied. this is based on the development of genetically modified (gm) mosquitoes infected with a bacterium known as wolbachia to combat dengue infection. this bacterium blocks replication of the virus inside the mosquito and prevents its transmission to humans [ ] . in , million gm male mosquitoes were released in the wild to decrease the number of aedes mosquitoes and reduce the rate of dengue transmission. a closer monitoring of the insects revealed that over % of the eggs were wolbachia-positive which indicated that gmmosquitoes were overriding wild-mosquitoes resulting in decreased virus transmission [ ] . in an initiative to eradicate dengue fever, scientists from australia, are leading eliminate dengue (ed) program which involves community engagement as a key component in this program. since the program kicked off in , millions of wolbachia mosquitoes were released across the north queensland city-australia. based on the promising results obtained from local trial, eliminate dengue became an international research program across countries affected by dengue including australia, vietnam, indonesia, brazil and colombia [ , ] . dengue infection can be prevented by alternative approaches. the first one includes blocking virus entry into cells which is mediated by the viral envelope glycoprotein e via receptor-mediated endocytosis [ ] . dendritic cells, monocytes, and macrophages are the main targets of denv infectious entry. the second approach involves blocking virus attachment to specific cellular receptors expressed on immune cells, liver cells, and endothelial cells. small molecules and peptides targeting the hydrophobic pocket of the envelope e glycoprotein are characterized as inhibitors of virus entry. nicholson et al. ( ) explored the inhibitory effects of dn and oan , peptide entry inhibitors. the authors demonstrated that dn and oan can effectively block antibody dependent enhancement (ade) in-vitro suggesting that entry inhibitors are potential candidates to prevent development of dhf/ dss [ ] . two other compounds have also been shown to qualify as potent inhibitors of dengue virus infection are imino-sugars deoxynojirimycin and castanospermine [ ] . these compounds are natural alkaloids derived from the black bean and act as inhibitors against all dengue serotypes by disrupting the folding pathways of the envelope glycoproteins prm and e [ ] . various types of carbohydrate-binding agents, isolated from different organisms, have been shown to have antiviral activities. three plant lectins, hippeastrum hybrid agglutinin, galanthus nivalis agglutinin and urtica dioica agglutinin isolated from amaryllis, snowdrop and stinging nettle respectively were found to be potent inhibitors of denv- infection by inhibiting viral replication [ ] . heparan sulfate (hs) is a putative receptor for denv which interacts with domain iii of the e-protein. virus entry can be blocked by targeting the e-protein-hs interaction with soluble gags and other highly charged hs [ ] . fucoidan was isolated from marine algae and showed antiviral activity against denv- in bhk cells [ ] . similarly, carrageenan and dl galactan, sulfated polysaccharides from red seaweeds, exhibited strong antiviral activity against denv- and denv- but a very weak activity against denv- and denv- . furthermore, two α-d-glucans were isolated from a chinese herb and demonstrated high anti-denv- activities in bhk cells [ , ] . dengue fever represents a real economic burden especially in affected countries. extensive efforts are needed to tackle disease spread and reduce the mortality rates and the associated healthcare cost. there is a need for more scientific research which we believe is a key route to provide further insight in the pathogenesis of dengue infection and help understanding the underlying molecular mechanisms associated with progression to the severe forms of the disease (dhf/dss). this will be a step forward to develop an adequate preventive vaccine and effective treatment. the authors disclose that there is no conflict of interest. authors' contributions tn participated in the review design, coordination and helped to draft the manuscript. sk and ss participated in the review design and helped to draft the manuscript. pd participated in the article revision. gd and ea participated in the review design. read and approved the final manuscript. this project is funded by the king abdulaziz city for science and technology (kacst) under grant number ( ‫ﺍ‬ ‫ﺕ‬ - - ). the authors are also grateful to the diagnosis of dengue: an update guidelines for diagnosis, treatment prevention and control the global distribution and burden of dengue dengue viral infections outbreak of viral hemorrhagic fever caused by dengue virus type in al-mukalla complete genome sequencing and phylogenetic analysis of dengue type virus isolated from jeddah, saudi arabia surprising new dengue virus throws a spanner in disease control efforts distribution and seasonality of vertically transmitted dengue viruses in aedes mosquitoes in arid and semi-arid areas of rajasthan occurrence of natural vertical transmission of dengue- and dengue- viruses in aedes aegypti and aedes albopictus in fortaleza evidence of vertical transmission of dengue virus in two endemic localities in the state of oaxaca vertical transmission of dengue virus in aedes aegypti dengue virus infection in late pregnancy and transmission to the infants dengue infections during pregnancy: case series from a tertiary care hospital in sri lanka breast milk as a possible route of vertical transmission of dengue virus? the incubation periods of dengue viruses dengue fever new paradigms for a changing epidemiology clinical profile and outcome of hospitalized patients during first outbreak of dengue in makkah, saudi arabia dengue prevention and years of vector control in singapore early dengue infection and outcome study (eden) -study design and preliminary findings dengue in travelers. a review evaluation of diagnostic tests: dengue thrombocytopenia in dengue fever dengue virus pathogenesis. integr view a re-evaluation of the mechanisms leading to dengue hemorrhagic fever dengue myocarditis in singapore: two case reports neurological manifestations of dengue virus infection epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the st century the early clinical features of dengue in adults: challenges for early clinical diagnosis secretion of flaviviral non-structural protein ns : from diagnosis to pathogenesis duarte dos santos cn. development, characterization and application of monoclonal antibodies against brazilian dengue virus isolates a simple one-step real-time rt-pcr for diagnosis of dengue virus infection current advances in dengue diagnosis reliable classifier to differentiate primary and secondary acute dengue infection based on igg elisa human genetic susceptibility to intracellular pathogens host genetic susceptibility to severe dengue infection impact of dengue virus infection on feeding behavior of aedes aegypti dengue virus infection of the aedes aegypti salivary gland and chemosensory apparatus induces genes that modulate infection and blood-feeding behavior a novel amino acid substitution in a voltage gated sodium channel is associated with knockdown resistance to permethrin in aedes aegypti widespread distribution of newly found point mutation in voltage-gated sodium channel in pyrethroid-resistant aedes aegypti populations in vietnam detection of the v g mutation in the voltage-gated sodium channel gene of aedes aegypti (diptera: culicidae) by allele-specific pcr assay, and its distribution and effect on deltamethrin resistance in thailand race: a risk factor for dengue hemorrhagic fever asymptomatic dengue infection in a cuban population confirms the protective role of the rr variant of the fcgammariia polymorphism a variant in the cd promoter is associated with severity of the disease dengue and dengue haemorrhagic fever: implications of host genetics polymorphisms of the tap and gene may influence clinical outcome of primary dengue viral infection all serotypes of dengue virus induce hla-a major histocompatibility complex class i promoter activity in human liver cells two putative subunits of a peptide pump encoded in the human major histocompatibility complex class ii region polymorphisms in the oligoadenylate synthetase gene cluster and its association with clinical outcomes of dengue virus infection cell type specificity and host genetic polymorphisms influence antibodydependent enhancement of dengue virus infection the a, -dihydroxy-vitamin d reduces dengue virus infection in human myelomonocyte (u ) and hepatic (huh- ) cell lines and cytokine production in the infected monocytes association of vitamin d receptor gene polymorphisms with clinical outcomes of dengue virus infection genome-wide association study identifies susceptibility loci for dengue shock syndrome at micb and plce interactions of human nkg d with its ligands mica, micb, and homologs of the mouse rae- protein family natural killer cell activation enhances immune pathology and promotes chronic infection by limiting cd + t-cell immunity genetic variants of micb and plce and associations with non-severe dengue host gene expression profiling of dengue virus infection in cell lines and patients common variants of chemokine receptor gene cxcr and its ligands cxcl and cxcl associated with vascular permeability of dengue infection in peninsular malaysia early enhanced expression of interferon-inducible protein- (cxcl- ) and other chemokines predicts adverse outcome in severe acute respiratory syndrome cxcr mediates regionspecific antiviral t cell trafficking within the central nervous system during west nile virus encephalitis selective susceptibility of human skin antigen presenting cells to productive dengue virus infection role of skin immune cells on the host susceptibility to mosquito-borne viruses dengue vaccines: recent developments, ongoing challenges and current candidates vaccines against dengue: a review of current candidate vaccines at advanced development stages prospects for a dengue virus vaccine progress towards a dengue vaccine advances in dengue vaccine development dengue vaccines: progress and challenges from research to phase iii: preclinical, industrial and clinical development of the sanofi pasteur tetravalent dengue vaccine genetic stability of a dengue vaccine based on chimeric yellow fever/dengue viruses protective efficacy of the recombinant, liveattenuated, cyd tetravalent dengue vaccine in thai schoolchildren: a randomised, controlled phase b trial liveattenuated, tetravalent dengue vaccine in children, adolescents and adults in a dengue endemic country: randomized controlled phase i trial in the philippines safety and immunogenicity of recombinant, live attenuated tetravalent dengue vaccine (cyd-tdv) in healthy vietnamese adults and children clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in asia: a phase , randomised, observermasked, placebo-controlled trial cyd study group. efficacy of a tetravalent dengue vaccine in children in latin america dengue type virus mutants containing deletions in the ' noncoding region of the rna genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys a live attenuated recombinant dengue- virus vaccine candidate with restricted capacity for dissemination in mosquitoes and lack of transmission from vaccinees to mosquitoes rden delta , a live attenuated dengue virus type vaccine candidates, is safe, immunogenic, and highly infectious in healthy adult volunteers the live attenuated dengue serotype vaccine rden delta is safe and highly immunogenic in healthy adult volunteers pediatric measles vaccine expressing a dengue antigen induces durable serotype-specific neutralizing antibodies to dengue virus pediatric measles vaccine expressing a dengue tetravalent antigen elicitsneutralizing antibodies against all four dengue viruses development of dengue dna vaccines evaluation of a prototype dengue- dna vaccine in a phase clinical trial immunogenicity and protective efficacy of a vaxfectin-adjuvanted tetravalent dengue dna vaccine a dna vaccine candidate encoding the structural prm/e proteins elicits a strong immune response and protects mice against dengue- virus infection induction of a protective response in mice by the dengue virus ns protein using dna vaccines evaluation of a dna vaccine candidate expressing prm-e-ns antigens of dengue virus serotype with or without granulocyte-macrophage colony-stimulating factor (gm-csf) in immunogenicity and protection development of a purified, inactivated, dengue- virus vaccine prototype in vero cells: immunogenicity and protection in mice and rhesus monkeys immunogenic and protective response in mice immunized with a purified, inactivated, dengue- virus vaccine prototype made in fetal rhesus lung cells a purified inactivated japanese encephalitis virus vaccine made in vero cells formalin-inactivated whole virus and recombinant subunit flavivirus vaccines safety and immunogenicity of concomitant vaccination with the cell-culture based japanese encephalitis vaccine ic and the hepatitis a vaccine havrix in healthy subjects: a single-blind, randomized, controlled phase study correlation of protection against japanese encephalitis virus and je vaccine (ixiaro) induced neutralizing antibody titers the development of dengue vaccines development of a recombinant tetravalent dengue virus vaccine: immunogenicity and efficacy studies in mice and monkeys development of denvax: a chimeric dengue- pdk- -based tetravalent vaccine for protection against dengue fever immunogenicity and efficacy of chimeric dengue vaccine (denvax) formulations in interferon-deficient ag mice next-generation dengue vaccines: novel strategies currently under development next generation dengue vaccines: a review of candidates in preclinical development a tetravalent recombinant dengue domain iii protein vaccine stimulates neutralizing and enhancing antibodies in mice an envelope domain iii-based chimeric antigen produced in pichia pastoris elicits neutralizing antibodies against all four dengue virus serotypes a dengue virus serotype- dna vaccine induces virus neutralizing antibodies and provides protection from viral challenge in aotus monkeys immunogenicity of dengue virus type dna vaccines expressing truncated and full length envelope protein protection against dengue virus by non-replicating and live attenuated vaccines used together in a prime boost vaccination strategy immunogenicity and protective efficacy of a psoralen-inactivated dengue- virus vaccine candidate in aotus nancymaae monkeys ns peptide, a novel potent hepatitis c virus ns helicase inhibitor: its mechanism of action and antiviral activity in the replicon system viralns helicase activity is inhibited by peptides reproducing the arg-rich conserved motif of the enzyme (motif vi) the dengue virus ns protein as a target for drug discovery expanding small rna interference inhibition of dengue virus entry and multiplication into monocytes using rna interference antiviral activity of four types of bioflavonoid against dengue virus type- the antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell screening of anti-dengue activity in methanolic extracts of medicinal plants the risk to developed and developing countries mutual exclusion of asaia and wolbachia in the reproductive organs of mosquito vectors brazil tests gm mosquitoes to fight dengue. males with offspring-killing genes are replacing wild insects, say researchers successful establishment of wolbachia in aedes populations to suppress dengue transmission designing a community engagement framework for a new dengue control method: a case study from central vietnam dengue virus entry as target for antiviral therapy viral entry inhibitors block dengue antibody-dependent enhancement in vitro α-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum castanospermine, a potent inhibitor of dengue virus infection in vitro and in vivo antiviral activity of carbohydrate binding agents and the role of dc-sign in dengue virus infection antiviral effect of the heparan sulfate mimetic, pi- , against dengue and encephalitic flaviviruses structure and anti-dengue virus activity of sulfated polysaccharide from a marine alga structure elucidation and sulfated derivatives preparation of two α-dglucans from gastrodia elata bl. and their anti-dengue virus bioactivities submit your next manuscript to biomed central and we will help you at every step: key: cord- - g g au authors: haagmans, bart l.; osterhaus, albert d.m.e. title: sars date: - - journal: vaccines for biodefense and emerging and neglected diseases doi: . /b - - - - . - sha: doc_id: cord_uid: g g au abstract five years after the first severe acute respiratory syndrome (sars) outbreak, several candidate sars-coronavirus (cov) vaccines are at various stages of preclinical and clinical development. based on the observation that sarscov infection is efficiently controlled upon passive transfer of antibodies directed against the spike (s) protein of sars-cov, vaccines containing the s protein have been formulated. animals immunized with inactivated whole virus vaccines or live-recombinant vaccines expressing the sars-cov s protein (e.g., using rabies virus, vesicular stomatitis virus, bovine parainfluenza virus type , adenovirus, or attenuated vaccinia virus mva as a vector), as well as mice immunized with dna vaccines expressing the s protein gene all developed neutralizing antibodies to sars-cov and were protected against sars-cov challenge. although much effort has been focused on developing a sars vaccine, the commercial viability of such a vaccine for sars-cov will ultimately depend on whether the virus re-emerges in the near future. this vaccine should induce highly cross-reactive neutralizing antibodies to protect against newly emerging viruses related to sars-cov and protect both the gastrointestinal and respiratory tract in the absence of significant side effects. given the fact that in the previous outbreak mainly the elderly succumbed to the infection, special attention should be given to vaccines that are able to efficiently protect aged individuals. was successfully re-isolated from the nasal swabs and lung lesions of these animals, and a specific antibody response to the virus was shown in the infected animals, sars-cov proved to be the causative agent of this infectious disease kuiken et al., ) . sars-cov is a single-stranded, positive-sense rna virus, phylogentically related to coronaviruses from group despite the fact that it does not encode a hemagglutinin-esterase protein ( snijder et al., ) . the genome is packaged together with the nucleocapsid protein, at least five membrane proteins (m, e, a, a, and b) and the spike (s) protein ( fig. . ) . the s region within the s protein, and more specifically a -amino acid fragment of the s protein (corresponding to residues - ), has been identified as the region that interacts with the cell receptor, angiotensin-converting enzyme ( li et al., a ) . the majority of neutralizing antibodies are directed against this region of the s protein. antibodies raised severe acute respiratory syndrome coronavirus (sars-cov) first emerged in the human population in november . phylogenetic analysis of sars-cov isolates from animals indicated that this virus most probably originated from bats, was transmitted first to palm civets and subsequently to humans at the wet markets in southern china. subsequent outbreaks occurred early in hong kong, hanoi, toronto, and singapore, and could be directly traced back to one index patient who acquired the infection in guangdong and traveled to hong kong. a worldwide epidemic was halted through the efforts of the world health organization, which responded rapidly to this threat by issuing a global alert, rigorous local containment efforts, warning against unnecessary travel to affected areas, and by creating a network of international experts to combat this virus. in the end only people became ill, and people died in this first sars epidemic. because sars-cov could re-emerge and cause another epidemic at any time, development of effective vaccines remains of vital importance. although several infectious agents, including chlamydia, influenza a subtype h n , and human metapneumovirus, were considered as a possible cause of sars, three groups independently reported the isolation of a previously unrecognized cov from clinical specimens of sars patients ( peiris et al., ; rota et al., ; drosten et al., ) . through electron microscopy, serology, and reversetranscription pcr with consensus-and randomprimers, and subsequent sequencing of the replicase gene, its identity could be revealed and consistently demonstrated in clinical specimens from patients with the disease but not in healthy controls. to conclusively establish a causal role for this cov, cynomolgous macaques were inoculated with a sars-cov isolate. because the disease in macaques caused by sars-cov infection was pathologically similar to that seen in human patients with sars, and since the virus should induce highly cross-reactive neutralizing antibodies to protect against newly emerging viruses related to sars-cov and protect both the gastrointestinal and respiratory tract in the absence of significant side effects. given the fact that in the previous outbreak mainly the elderly succumbed to the infection, special attention should be given to vaccines that are able to efficiently protect aged individuals. protein also inhibit sars-cov replication in vitro but their relevance in protection remains unclear. the genome also encodes two large poly-proteins with diverse enzymatic activities needed for efficient replication and several accessory proteins with unknown function ( b, , a, b, and b). at the end of , countries reported a total of probable cases of sars ( fig. . ) . pathogenic sars-covs do not circulate in the human population at the moment, but their re-emergence from animal reservoirs may likely occur in the future. because many of the early sars patients in guandong had epidemiological links to the live-animal market trade, different animal species were tested for the presence of sars-like viruses. soon after the outbreak, a sars-like coronavirus, which had more than % homology with human sars-cov, was detected by rt-pcr in the nasal and fecal swabs of palm civets ( paguma larvata ) and a raccoon dog ( nyctereutes procyonoides ) ( guan et al., ) . more recent studies indicate that bats may potentially act as natural reservoirs for sars-like covs ( li et al., b ; lau et al., ) . however, sequence comparison of the s protein genes from bat sars-like cov and palm civet sars-like cov revealed only % genetic homology. subsequent studies by tang et al. ( ) have demonstrated that approximately % of bats sampled in china were positive for covs. interestingly, these covs are genetically diverse and many bat covs clustered with existing group viruses, while others formed a separate lineage that included only viruses from bats (putative group ). other sars-cov like viruses clustered in a putative group consisting of two subgroups, one of bat covs and another of sars-covs from humans and other mammalian hosts. however, from these studies the direct progenitor of the sars-cov isolated from palm civets has not been identified. major genetic variations in the s protein gene of these viruses from civet cats, seemed essential for the transition from animal-to-human transmission epidemiology figure . reported suspected sars cases from november , to july , (data from the world health organization, http:// www.who.int/csr/sars/country/table _ _ /en/index.html ). countries with Ͼ suspected cases are indicated. patients. in fact, none of the sars-cov-infected children aged below years in hong kong required intensive care or mechanical ventilation ( ng et al., ) . this is not totally explained by comorbid factors but similar age dependence in mortality is seen in patients with other (nonviral) causes of acute respiratory stress syndrome ( rubenfeld et al., ) . second, virus transmission is low in the first days of illness and peaks around day after disease onset ( chu et al., ) . finally, several studies revealed that high viral load in the nasopharyngeal aspirate was found to be an independent predictor of mortality ( hung et al., ; chu et al., ) . therefore, vaccine strategies aimed at reducing the viral load may suffice to provide clinical benefit. the first efforts to treat sars patients were mainly based on the use of ribavirin and corticosteroids. ribavirin, which targets imp dehydrogenase, has been known a long time as a broad-spectrum antiviral agent. however, current data do not support the use of ribavirin for sars treatment; in vitro studies did not show significant antiviral activity ( cinatl et al., ) and ribavirin enhanced the infectivity of sars-cov in mice ( barnard et al., ) . on the other hand, a protective effect of interferon (ifn)-α has been observed in a preliminary study during the sars outbreak ( loutfy et al., ) . these results are in concordance with several studies that noted antiviral activity in vitro ( cinatl et al., ; hensley et al., ) and animal studies showing that pegylated ifn-α effectively reduced sars-cov replication and excretion, viral antigen expression by type pneumocytes and the pulmonary damage in cynomolgous macaques that were infected experimentally with sars-cov ( haagmans et al., ) . however, despite an extensive literature reporting on sars treatments, it is not possible to determine whether treatments benefited patients during the sars outbreak. because of variation in treatment regimens-particularly the wide range in doses, duration of therapy, and route of administration of ribavirin and corticosteroids, no clear conclusion can be drawn regarding the efficacy of the drugs tested ( stockman et al., ) . in the event of a future outbreak of sars-cov or another novel agent, attempts should be made to develop treatment protocols, organize randomized trials and to collect and contribute information for a standardized minimum dataset that could facilitate analysis of treatment outcomes among different settings ( stockman et al., ) . to human-to-human transmission, which eventually caused the sars outbreak of - . there is at present no evidence for the virus persisting in the human population. possible options for the re-emergence of sars include the escape of the virus from laboratories, which has already occurred on three occasions. the re-emergence of the virus from its animal reservoir remains possible, given that the virus is detectable in the feces and respiratory secretions of some animals. indeed, sars-cov re-emerged in four patients in guangdong in december , although these sars-like covs caused milder clinical disease ( liang et al., ) . the us national institute of allergy and infectious diseases biodefense network classified sars-cov as a category c priority pathogen pointing out that sars-cov could be a potential biothreat agent. the clinical symptoms of sars-cov infection are those of lower respiratory tract disease and include fever, malaise, peripheral t cell lymphocytopenia, decreased platelet counts, prolonged coagulation profiles, and mildly elevated serum hepatic enzymes ( peiris et al., ; li et al., ) . chest radiography reveals infiltrates with subpleural consolidation or " ground glass " changes compatible with viral pneumonitis. around − % of individuals with sars require management in intensive care units and the overall case:fatality rate reached approximately %. although the main clinical symptoms are those of severe respiratory illness, sars-cov actually also causes a gastrointestinal and urinary tract infection; sars-cov can be detected in the feces and urine of patients, and electron microscopic studies of biopsies of the upper and lower intestinal mucosae of patients with sars confirmed the presence of the virus in these tissues ( peiris et al., ) . fecal transmission proved to be important in at least one major community outbreak in hong kong (amoy gardens), in which over patients were infected within a few days. three features of sars may be relevant for intervention strategies. first, progressive age dependence in mortality and disease severity is observed in sars the major sources of transmission in humans are droplets that deposit on the respiratory epithelium. unlike the situation in several other respiratory viral infections, viral load of sars-cov in the upper respiratory tract peaked around day after disease onset ( peiris et al., ) . therefore, virus transmission may be less efficient in the first days of illness, a finding supported by epidemiological observations. realtime pcr assays detect sars-cov during the first week in specimens of the lower respiratory tract (e.g., bronchoalveolar lavage, sputum, endotracheal aspirates), nasopharyngeal aspirate, throat swabs, and/or serum ( chan et al., ) , whereas fecal samples may show very high viral loads toward the end of the first week and second week of illness. in typical cases, which were largely confined to adult and elderly individuals, sars presented with acute respiratory distress syndrome, characterized by the presence of diffuse alveolar damage and multiorgan dysfunction upon autopsy ( nicholls et al., ) . the pathological changes in lung alveoli most likely follow a common pathway characterized by an acute phase of proteinrich alveolar fluid influx into the alveolar lumina as a consequence of the injury to the alveolar wall. subsequently type- pneumocyte hyperplasia takes place to replace the loss of infected type- pneumocytes and to cover the denuded epithelial basement membrane, resulting in restoration of the normal alveolar architecture. severe alveolar injury may lead to fibrosis with loss of alveolar function in more protracted cases ( fig. . ). it has been hypothesized that the pathological changes observed in the lungs are initiated by a disproportional innate immune response, illustrated by elevated levels of inflammatory cytokines and chemokines, such as cxcl (ip- ), ccl (mcp- ), il- , il- , il- , il- β , and ifn-γ ( huang et al., ; wong et al., ) . these in vivo data have been confirmed in vitro, demonstrating that sars-cov infection induces a range of cytokines and chemokines in diverse cell types law et al., ) . although in vitro studies argued that production of type i ifns is inhibited or delayed by sars-cov, in sars-covinfected macaques, and also early during sars in humans, type i ifns can be readily demonstrated (de lang et al., ) . because prophylactic treatment of macaques with pegylated ifn-α reduces sars-cov replication in the lungs, regulation of the production of ifns may be important in controlling sars ( haagmans et al., ) . based on the observation that sars-cov infection is efficiently controlled upon passive transfer of antibodies directed against the s protein of sars-cov, a range of vaccines containing the s protein/gene has been developed. animals immunized with inactivated whole virus vaccines or live-recombinant vaccines expressing the sars-cov s protein (e.g., using rabies virus, vesicular stomatitis virus, bovine parainfluenza virus type , adenovirus, or attenuated vaccinia virus mva as a vector), as well as mice immunized with dna vaccines expressing the s protein gene all developed neutralizing antibodies to sars-cov and were protected against sars-cov challenge. table . displays an overview of vaccines that have been tested for efficacy in animal models. inactivated sars vaccines have been reported to elicit high titers of s protein-specific neutralizing antibodies. few studies, however, have addressed whether inactivated whole sars-cov virions confer protection from virus challenge. mice that were immunized twice with a candidate sars-cov vaccine, produced through a two-step inactivation procedure involving sequential formaldehyde and u.v. inactivation developed high-antibody titers against the sars-cov s protein and high levels of neutralizing antibodies ( spruth et al., ) (see chapter ). moreover, the vaccine conferred protective immunity as demonstrated by prevention of sars-cov replication in the respiratory tract of mice after intranasal challenge with sars-cov. protection of mice was correlated to the antibody titer against the sars-cov s protein and neutralizing antibody titer. similar results have been obtained using a beta-propiolactone inactivated sars-cov vaccine in mice . in addition, two chinese groups have demonstrated protective efficacy of inactivated sars vaccines in rhesus monkeys ( qin et al., ; zhou et al., ) . a soluble recombinant polypeptide containing the n-terminal segment of the s glycoprotein may suffice to induce neutralizing antibodies and protective immunity in mice ( bisht et al., ) . in addition, a trimeric recombinant s protein was able to elicit an efficacious protective immune response in hamsters ( kam et al., ) . one of the most promising vaccine candidates is based on the combination of recombinant s protein with the protollin adjuvant. in both young and aged mice, an intranasal protollin-formulated s protein seroconversion usually occurs in weeks or of illness and virus-neutralizing antibodies can be detected in convalescent human serum. in patients who had recovered from sars-neutralizing antibody titers peaked at month but were undetectable in % of patients at month ( cao et al., ) . neutralizing antibodies may be directed against different regions of the s protein (s and s ) as monoclonal antibodies against these epitopes exert potent neutralization of sars-cov in vitro ( sui et al., ; lip et al., ) . conversely, peptides which are located in these regions were able to induce neutralizing antibodies ( bisht et al., ; keng et al., ; zhang et al., ) . although experiments in diverse animal models have revealed that relatively low levels of neutralizing antibodies exert potent protection against lower respiratory tract infection, the neutralizing antibody titer necessary to achieve protection in humans exposed to sars-cov is not known. a concern in case of reemergence of sars is the possible absence of cross protection against these viruses. however, recent studies by he et al. ( ) have shown that the some neutralizing epitopes of sars-cov have been maintained during cross-species transmission, suggesting that receptor binding domain-based vaccines may induce broad protection against both human and animal sars-cov variants. in most sars autopsies, extensive necrosis of the spleen and atrophy of the white pulp with severe lymphocyte depletion have been observed. on the other hand, rapid phase in peripheral lymphocyte recovery usually coincided with improved clinical conditions of sars patients (peiris et al., ) . long-lived memory t cell responses against sars-cov nucleocapsid and s protein have been demonstrated in recovered sars patients, although their relevance in antiviral protection is not well understood peng et al., ; li et al., ) . however, despite potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients were not restored to normal levels . interestingly, mice that lack nk-t cells, or nk cells, or t and b cells all cleared the virus by day after infection ( glass et al., ) . these data argue that cell-mediated immune responses are not essential to control virus clearance. vaccine elicited high levels of antigen-specific igg in serum and significant levels of antigen-specific lung iga ( hu et al., ) . in contrast, mice immunized intramuscularly with alum absorbed s protein did not develop detectable iga responses. following virus challenge of the aged mice, no virus was detected in the lungs of mice vaccinated intranasally, whereas intramuscularly immunized mice did not show significant control of virus replication compared to controls ( hu et al., ) . a dna vaccine encoding the s glycoprotein of the sars-cov induces t cell, neutralizing antibody responses, and protective immunity in a mouse model . these authors also demonstrated that antibody responses in mice vaccinated with an expression vector encoding a form of s protein that includes its transmembrane domain elicited neutralizing antibodies. viral replication was reduced by more than six orders of magnitude in the lungs of mice vaccinated with these s protein plasmid dna expression vectors, and protection was mediated by a humoral, but not a t-cell-dependent, immune mechanism. subsequent studies using a prime-boost combination of dna and whole killed sars-cov vaccines elicited higher antibody responses than dna or whole killed virus vaccines alone . apart from this study, several other groups have analyzed the immunogenicity of sars dna vaccines but none of these challenged the vaccinated animals with sars-cov. adenovirus-vector based vaccination strategies against sars-cov were employed early on after the sars outbreak to demonstrate that vaccinated rhesus macaques developed virus-neutralizing antibody responses against fragment s of the s protein and t cell responses against the nucleocapsid ( gao et al., ) . more recently, see et al. ( ) demonstrated that vaccination of c b/l mice with adenovirus type -expressing s and nucleocapsid administered intranasally, but not intramuscularly, significantly limited sars-cov replication in the lungs. vaccines reduction in pulmonary sars-cov titer compared with control animals ( dinapoli et al., ) . finally, venezuelan equine encephalitis virus based vaccines have been tested extensively in young and aged mice. most importantly, different recombinant sars-cov bearing epidemic and zoonotic s protein variants were used to challenge the vaccinated mice. venezuelan equine encephalitis virus replicon particles expressing the epidemic urbani sars-cov strain s glycoprotein but not particles containing the nucleocapsid protein from the same strain provided complete short-and long-term protection against homologous strain challenge in young and senescent mice ( deming et al., ) . although the s protein encoding vaccine provided complete short-term protection against heterologous (strain gd ) challenge in young mice, only limited protection was seen in vaccinated senescent animals. interestingly, nucleocapsid-encoding vaccines not only failed to protect from homologous or heterologous challenge but also resulted in enhanced immunopathology with eosinophilic infiltrates within the lungs of sars-covchallenged mice ( deming et al., ) . a new generation of vaccines may be obtained from manipulating the full-length infectious cdna clone of sars-cov. one approach would be to delete the orfs a, b, , a, b, a, b, or b similar to other coronavirus mutants generated previously; some mouse hepatitis or feline infectious peritonitis deletion viruses replicate to the same extent as wild-type viruses in vitro but are severely attenuated in vivo making them potential vaccine candidates. however, sars-cov deletion mutants lacking orfs a, b, , a, or b, grew similar to that of the parental wild-type virus in the mouse model ( yount et al., ) . on the other hand, a recombinant sars-cov that lacks the e gene was attenuated both in vitro and in vivo . viable recombinant virus with the e gene deleted was recovered in vero cells with a titer around pfu/ml but titers in the respiratory tract of hamsters were - -fold reduced compared to wild-type sars-cov replication, suggesting that this mutant is attenuated. multiplication of these viruses in packaging cell lines would provide the missing protein in trans and would make a promising sars-cov vaccine candidate that has the e gene deleted. live attenuated virus vaccines may revert to wildtype and recombine with other circulating human or the highly attenuated modified vaccinia virus ankara (mva) has been used to express the s glycoprotein of sars-cov in vaccination experiments using mouse, ferret, and rhesus monkey models ( bisht et al., ; chen et al., ; weingartl et al., ) . intranasal and intramuscular administration of mva encoding the sars-cov s protein led to the induction of a humoral immune response in balb/c mice, as well as reduced viral titers in the respiratory tract ( bisht et al., ) . recombinant bovine-human parainfluenza virus type vector (bhpiv ) is being developed as a live attenuated, intranasal pediatric vaccine against human parainfluenza virus type . immunization of african green monkeys with a single dose of bhpiv expressing sars-cov s protein administered via the respiratory tract induced the production of sars-cov neutralizing antibodies . a recombinant bhpiv expressing sars-cov structural protein (s, m, and n) individually or in combination has been evaluated for immunogenicity and protective efficacy in hamsters . in the absence of s protein, expression of m, n, or e did not induce a detectable serum sars-cov-neutralizing antibody response and no protection against sars-cov challenge in the respiratory tract, whereas the vectors expressing the s protein induced neutralizing antibody responses and protection. recombinant rabies virus expressing the s protein of sars-cov induced a neutralizing antibody response in mice ( faber et al., ) . similarly, an attenuated vesicular stomatitis virus vector that encodes the sars-cov s protein may be used to induce neutralizing antibody responses ( kapadia et al., ) . mice vaccinated with recombinant vesicular stomatitis virus expressing s protein developed sars-cov-neutralizing antibody and were able to control a challenge with sars-cov performed at either month or months after a single vaccination. in addition, by passive antibody transfer experiments these authors demonstrated that the antibody response induced by the vaccine was sufficient to control sars-cov infection. the efficacy of these vectors was further demonstrated in studies using aged mice. in aged mice, vaccinated with recombinant vesicular stomatitis virus expressing s protein, antibody titers induced were sufficient to protect them against subsequent challenge with sars-cov ( vogel et al., ) . african green monkeys immunized via the respiratory tract with two doses of a recombinant newcastle disease virus encoding the s protein developed a relatively high titer of sars-cov neutralizing antibodies and upon challenge demonstrated a -fold zoonotic coronaviruses. in order to prevent this, it has been proposed to delete an essential gene, located in a position distant from gene e, and the relocation of the deleted gene to the position previously occupied by gene e ( enjuanes et al., ) . a potential recombination leading to the rescue of gene e would lead to the loss of the essential gene. alternatively, the transcriptional regulatory sequences (trs) of a vaccine virus could be genetically manipulated to a sequence incompatible with the trs of any known circulating coronavirus as described by yount et al. ( ) . this virus could be further modified by building attenuating mutations on the genetic backbone of the recombination resistant trs rewired virus either for use as a safe high titer seed stock for making killed vaccines or as a live virus vaccine. one such attenuating mutation could be targeted to the nonstructural protein . recombinant mouse hepatitis virus (mhv) encoding a deletion in the nsp -coding sequence grew normally in tissue culture but was severely attenuated in vivo ( züst et al., ) . low doses of nsp mutant mhv elicited potent cytotoxic t cell responses and protected mice against homologous and heterologous virus challenge. this attenuation strategy provides a new paradigm for the development of highly efficient coronavirus vaccines. although several types of vectored vaccines have been developed, several companies favored the classical approach using inactivated whole virus to develop a vaccine to be used for preclinical testing (see chapter ). methods in place for the production of available vaccines could be easily used using wellestablished technologies. in the preclinical development stage, it is preferable that different animal species are used to evaluate the safety and efficacy of candidate vaccines. overall, a wide range of animal species, including rodents (mice and hamsters), carnivores (ferrets and cats), and nonhuman primates (cynomolgus and rhesus macaques, common marmosets, and african green monkeys) can be experimentally infected with sars-cov roberts et al., b ; martina et al., ; kuiken et al., ; mcauliffe et al., ; haagmans and osterhaus, ) . most species show no clinical signs of disease, although the virus replicates efficiently in respiratory tissues. aged mice and ferrets on the other hand, show signs of clinical disease, albeit in the absence of the typical lung lesions seen in humans with sars ( roberts et al., a ) . in contrast, inoculation of sars-cov in the respiratory tract of cynomolgus macaques causes infection of bronchial epithelial cells and type- pneumocytes - days postinfection, followed by extensive type- pneumocyte hyperplasia in the lungs at - days postinfection haagmans and osterhaus, ) . the lesions, consisting of multiple foci of acute diffuse alveolar damage and characterized by flooding of alveoli with protein-rich edema fluid mixed with variable numbers of neutrophils, are quite similar to those observed in humans in the acute stages of sars. remarkably, vaccine candidates tested in ferrets showed reduced efficacy. vaccination with mva encoding the s protein induced only moderate antibody responses and consequently did not protect against intranasal sars-cov infection and even resulted in an inflammatory response in the livers of the vaccinated ferrets (weingartl et al., ) . whether these aberrant responses resulted from immunopathological mechanisms, like antibody-dependent enhancement of infection, or represented recall responses to viral antigen in the liver is not clear at the moment but deserves further investigation. in addition, limited protection from sars-cov challenge was observed in ferrets vaccinated with inactivated whole virus ( darnell et al., ) . two out of four ferrets showed little or no neutralizing antibody even after the second immunization and none of the vaccinated ferrets were able to reduce virus excretion at day after challenge but subsequently cleared the virus more rapidly compared to control animals. adenovirus-based vaccines tested in ferrets seemed more powerful as they protected the lower respiratory tract efficiently but had less effect on virus excretion in the upper respiratory tract ( kobinger et al., ) . five years after the first sars outbreak a range of candidate vaccines have been developed. early in some companies in china and the us initiated phase trials. the first clinical trial has been initiated by a chinese company, sinovac biotech of beijing in collaboration with the chinese academy of medical sciences using an inactivated whole virus vaccine. to evaluate the safety and immunogenicity of this vaccine, subjects received two doses of vaccine or placebo control. on day , all individuals showed seroconversion and peak titers of neutralizing antibodies were reached weeks after the second vaccination followed clinical trials iii. virus vaccines with a type i coronavirus, feline infectious peritonitis virus (fipv), when subsequently exposed to fipv infection ( weiss and scott, ) . macrophages are able to take up feline coronavirus-antibody complexes more efficiently causing the virus to replicate to higher titers. interestingly, one study also demonstrated that antibodies against human sars-cov isolates enhance entry of pseudo-typed viruses expressing the civet cat sars-like cov s protein into cells, but not replication ( yang et al., ) . to date, there is no evidence for enhanced replication following sars-cov challenge in previously immunized animals. one other problem which may arise after vaccination with whole inactivated virus when absorbed with certain adjuvants such as alum, could relate to the induction of skewed th recall responses similar to what has been observed in children vaccinated with inactivated respiratory syncytial and measles virus vaccines. although much effort has been focused on developing a sars vaccine, the commercial viability of developing a vaccine for sars-cov will ultimately depend on whether the virus re-emerges in the near future. it is questionable whether possible future outbreaks will cause major outbreaks but vaccines, antivirals, or passive immunization would be relevant in the context of protecting high-risk individuals such as laboratory and health-care workers. • five years after the first sars outbreak, several candidate sars-cov vaccines are at various stages of preclinical and clinical development. • based on the observation that sars-cov infection is efficiently controlled upon passive transfer of antibodies directed against the s protein of sars-cov, vaccines encoding this protein have been developed. • animals immunized with diverse vaccines containing the s protein or s protein gene developed sars-cov-neutralizing antibodies and were protected against sars-cov challenge. • future challenges for the development of sars-cov vaccines are linked to the potential re-emergence of this virus. vaccines able to induce highly cross-reactive antibodies which efficiently protect the gastrointestinal and respiratory tract are needed. given the fact that in the previous outbreak mainly elderly humans succumbed to the infection, special attention should be given to protect specifically these individuals. by a significant decline weeks later ( lin et al., ) . several other candidate sars vaccines are at various stages of preclinical and clinical development. in sars patients who recover, high levels of neutralizing antibody responses are observed, suggesting that antibody responses play a role in determining the ultimate disease outcome of sars-cov-infected patients . although attempts have been made to test the efficacy of serum preparations from seroconvalescent sars patients in the acute phase of sars, no conclusive evidence has been obtained regarding their efficacy. in mice, on the other hand, sars-cov infection is efficiently controlled upon passive transfer of convalescent immunoglobulines . the concept that antibodies protect against sars has been further explored through the generation of human monoclonal antibodies against sars-cov. prophylactic administration of a human monoclonal antibody reduced replication of sars-cov in the lungs of infected ferrets by -fold, completely prevented the development of sars-cov-induced macroscopic lung pathology, and abolished shedding of virus in pharyngeal secretions ( ter meulen et al., ) . in subsequent studies, several other monoclonal antibodies were evaluated for their efficacy in mouse and hamster models ( sui et al., ; traggiai et al., ) . the importance of assessing immunogenicity of candidate sars-cov vaccines using virus neutralization assays is well acknowledged, but the variety of these tests in use is a significant problem since there is at this time no consensus on the most sensitive, specific, and reproducible assay system. to compare data from each of the candidate vaccines requires international standardization of the immunological assays and the availability of an antibody standard used for the evaluation of these vaccines. to test cross reactivity of antibodies generated by vaccination, murine leukemia virus was used to generate infectious particles containing different s proteins ( giroglou et al., ) . enhanced disease and mortality have been observed in kittens immunized against or infected enhancement of the infectivity of sars-cov in balb/c mice by imp dehydrogenase inhibitors, including ribavirin severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice neutralizing antibody and protective immunity to sars coronavirus infection of mice induced by a soluble recombinant polypeptide containing an n-terminal segment of the spike glycoprotein contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity mucosal immunisation of african green monkeys ( cercopithecus aethiops ) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars disappearance of antibodies to sars-associated coronavirus after recovery detection of sars coronavirus in patients with suspected sars nucleocapsid protein as early diagnostic marker for sars recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis initial viral load and the outcomes of sars treatment of sars with human interferons severe acute respiratory syndrome coronavirus infection in vaccinated ferrets a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov-infected macaques vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens identification of a novel coronavirus in patients with severe acute respiratory syndrome vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease a single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (sars-cov) s protein results in the production of high levels of sars-cov-neutralizing antibodies aetiology: koch's postulates fulfilled for sars virus effects of a sars-associated coronavirus vaccine in monkeys retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice isolation and characterization of viruses related to the sars coronavirus from animals in southern china pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques nonhuman primate models for sars cross-neutralization of human and palm civet severe acute respiratory syndrome coronaviruses by antibodies targeting the receptor-binding domain of spike protein interferon-beta a and sars coronavirus replication intranasal protollinformulated recombinant sars s-protein elicits respiratory and serum neutralizing antibodies and protection in mice an interferon gammarelated cytokine storm in sars patients viral loads in clinical specimens and sars manifestations antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine amino acids to in the s region of severe acute respiratory syndrome coronavirus s protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans severe acute respiratory syndrome coronavirus infection of golden syrian hamsters characterization of a novel coronavirus associated with severe acute respiratory syndrome incidence and outcomes of acute lung injury comparative evaluation of two severe acute respiratory syndrome (sars) vaccine candidates in mice challenged with sars coronavirus unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage a doubleinactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses sars vaccine protective in mice sars: systematic review of treatment effects prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association evaluation of human monoclonal antibody r for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants prevelance and genetic diversity of coronaviruses in bats from china human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus utility of the aged balb/c mouse model to demonstrate prevention and control strategies for severe acute respiratory syndrome coronavirus (sars-cov) immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome modulation of the immune response to the severe acute respiratory syndrome spike glycoprotein by gene-based and inactivated virus immunization newly discovered coronavirus as the primary cause of severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells structure of sars coronavirus spike receptor-binding domain complexed with receptor significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome long-term persistence of robust antibody and cytotoxic t cell responses in recovered patients infected with sars coronavirus bats are natural reservoirs of sars-like coronaviruses laboratory diagnosis of four recent sporadic cases of community-acquired sars, guangdong province safety and immunogenicity from a phase i trial of inactivated severe acute respiratory syndrome coronavirus vaccine monoclonal antibodies targeting the hr domain and the region immediately upstream of the hr of the s protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus natural mutations in the receptor binding domain of spike glycoprotein determine the reactivity of cross-neutralization between palm civet coronavirus and severe acute respiratory syndrome coronavirus interferon alfacon- plus corticosteroids in severe acute respiratory syndrome: a preliminary study virology: sars virus infection of cats and ferrets replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys sars in newborns and children lung pathology of fatal severe acute respiratory syndrome severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine long-lived effector/central memory t-cell responses to severe acute respiratory syndrome coronavirus (sars-cov) s antigen in recovered sars patients a dna vaccine induces sars coronavirus neutralization and protective immunity in mice evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses rewiring the severe acute respiratory syndrome coronavirus (sars-cov) transcription circuit: engineering a recombination-resistant genome severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice identification of an antigenic determinant on the s domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies antibody responses against sars coronavirus are correlated with disease outcome of infected individuals immunogenicity, safety, and protective efficacy of an inactivated sars-associated coronavirus vaccine in rhesus monkeys prognostic factors for severe acute respiratory syndrome: a clinical analysis of cases coronavirus nonstructural protein is a major pathogenicity factor: implications for the rational design of coronavirus vaccines key: cord- -hp s ebh authors: petráš, marek; lesný, petr; musil, jan; limberková, radomíra; pátíková, alžběta; jirsa, milan; krsek, daniel; březovský, pavel; koladiya, abhishek; vaníková, Šárka; macková, barbora; jírová, dagmar; krijt, matyáš; králová lesná, ivana; adámková, věra title: early immune response in mice immunized with a semi-split inactivated vaccine against sars-cov- containing s protein-free particles and subunit s protein date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: hp s ebh the development of a vaccine against covid- is a hot topic for many research laboratories all over the world. our aim was to design a semi-split inactivated vaccine offering a wide range of multi-epitope determinants important for the immune system including not only the spike (s) protein but also the envelope, membrane and nucleocapsid proteins. we designed a semi-split vaccine prototype consisting of s protein-depleted viral particles and free s protein. next, we investigated its immunogenic potential in balb/c mice. the animals were immunized intradermally or intramuscularly with the dose adjusted with buffer or addition of aluminum hydroxide, respectively. the antibody response was evaluated by plasma analysis at days after the first or second dose. the immune cell response was studied by flow cytometry analysis of splenocytes. the data showed a very early onset of both s protein-specific antibodies and virus-neutralizing antibodies at % inhibition regardless of the route of vaccine administration. however, significantly higher levels of neutralizing antibodies were detected in the intradermally (geometric mean titer - gmt of . ± . ) than in the intramuscularly immunized mice (gmt of . ± . ). in accordance with this, stimulation of cellular immunity by the semi-split vaccine was suggested by elevated levels of b and t lymphocyte subpopulations in the murine spleens. these responses were more predominant in the intradermally immunized mice compared with the intramuscular route of administration. the upward trend in the levels of plasmablasts, memory b cells, th and th lymphocytes, including follicular helper t cells, was confirmed even in mice receiving the vaccine intradermally at a dose of . μg. we demonstrated that the semi-split vaccine is capable of eliciting both humoral and cellular immunity early after vaccination. our prototype thus represents a promising step toward the development of an efficient anti-covid- vaccine for human use. the covid pandemic has made the development of a vaccine an emergency priority. hectic research was enabled by recent major advances in sequencing, protein structure applied into the right caudal thigh muscle, the intradermal one was applied into the auricle under the guidance of a microscope. one week after the first or second dose, the mice were anesthetized with isoflurane and blood was intracardially withdrawn using a ml syringe, transferred to anticoagulant tubes (k edta), and mixed. whole blood was centrifuged ( rpm, minutes, °c), plasma aliquoted into µl microtubes and frozen at - ° c. the spleens were collected in chilled rpmi medium on ice and transported to laboratory within hours for flow cytometry examination. anti-s specific igg antibodies as well as virus-neutralizing antibodies were expressed as the titer. the value had to be log-transformed to pass a normality test (d'agostino & pearson or shapiro-wilk test) followed by a parametric t-test or analysis of variance (anova). as the lymphocyte populations were measured from pooled spleens of each group, the traditional statistical approach could not be employed; hence, the change in lymphocyte subsets using linear regression was determined. if the slope of lines exhibited values significantly different from null, then the observed change was considered to be confirmed. merged lymphocyte subpopulations were analyzed with parametric tests after log- transformation to pass a normality test (shapiro-wilk test). the power of each test was insufficient since the sample size was small. a > % power of the test was only achieved when comparing the geometric mean titers of antibodies between immunized and unimmunized mice. the correlation between igg antibodies and virus-neutralizing antibodies was assessed with the pearson correlation coefficient after the log-transformation of their values. continuous data were summarized using standard descriptive statistics, i.e., median including range or geometric mean with standard deviation or % confidence interval. all tests were two-tailed, and the level of significance was set at . . statistical analyses were performed using prism (graphpad software, inc., san diego, ca) and stata version software (statcorp, college station, tx). results course of the culture, inactivation and purification process to obtain a viral stock for vaccine production, the virus strain was first purified by passages in vero e cells. subsequently, another passages were performed to generate a sufficient stock of virus for the production of the master seed as the source of a virus bank for vaccine production through the seed lot system. growth kinetics analysis of the second passage showed a sufficient virus replication rate reaching a titer of . - . log tcid /ml in the supernatant within - days ( figure ). the peak titers of . and . log tcid /ml from the supernatant and lysate, respectively, were at hours post-infection. moreover, multiplicities of infection (moi) of . - . at a culture temperature of °c and % co were confirmed. confluent monolayers of vero e cells, grown in optipro serum-free medium (life technologies europe bv, the netherlands) with % fetal bovine serum (fbs), were infected by the strain from the second passage of the stock and incubated in a serum-free medium at °c and % co for hours. the first viral suspension of ml was obtained by centrifugation of the supernatant harvest at g for minutes. the second suspension was harvested from infected cells by adding an approx. ml of medium, overnight freezing and thawing at - °c followed by centrifugation under the same conditions. both live viral suspensions containing viral particles with typical corona spikes as documented by electron- microscopic inspection ( figure a ) were stored at °c for at least days. the suspensions were subsequently inactivated by adding beta-propiolactone diluted : , , and continuously shaken up at a temperature of °c for hours. the beta-propiolactone hydrolysis at °c lasted hours. inactivated suspensions were centrifuged and the supernatants were stored at °c for one week. the second inactivation was performed with beta-propiolactone diluted : , at °c for hours. after that, the pellets were centrifuged at , g for minutes and both inactivated suspensions were subsequently placed in water baths to hydrolyze beta-propiolactone with continuous stirring at °c for hours with the temperature gradually decreasing to °c overnight. the inactivated viral suspension contained of . μ g/ml of the s protein for the supernatant harvest and . μ g/ml for the lysate harvest. while the first inactivation did not influence the virion structure, the second one showed partial changes, i.e., complete and incomplete particles with disrupted s protein ( figure b and c). the inactivation including hydrolysis decreased the ph to . - . that disrupted s protein binding to the viral envelope as documented by a record from the electron microscope ( figure c). both suspensions were stored at °c for days to be followed by vaccine purification and thickening to obtain μ g/ μ l the for harvested supernatant and μ g/ μ l for the harvested lysate. this was achieved by a multiple ultra/diafiltration process utilizing amicon ® ultra centrifugal filter units (amicon ultracel) with a volume of ml and a membrane molecular weight cutoff (mwco) of kda (amicon ® ultra- centrifugal filter unit - kda cutoff, merck millipore ltd., darmstadt, germany). the suspensions were centrifuged several times in amicon ultracel at , g for at least minutes to exchange the culture medium and to remove cellular debris and low-molecular substances. the suspension was washed several times with phosphate buffer saline (pbs) of ph . . within a dilution range of - to - . the result of purification and thickening were suspensions containing both virion particles free of the spike and separated s protein as documented by electron microscopy ( figure d ). the vaccine dose for the test of immunogenicity in mice was adjusted in pbs to concentrations of . significantly increasing proportions not only of follicular th lymphocytes but also th and th lymphocytes with an increasing number of doses ( figure a ). in addition, the same effect was observed in these mice for subsets of plasmablasts and memory b cells. discussion the above laboratory procedure generated a semi-split inactivated vaccine, i.e., a vaccine with the s protein separated from the viral particle exhibiting an early, both humoral and cellular, immune response. the design of this prototype vaccine was based on the usual procedures [tang , spruth , gao ]. we selected a strain showing higher infectivity with lower virulence, i.e., one that showed signs of attenuation. overall, we have tested several candidate strains for both immunogenicity through convalescent plasma and propagation capacity by viral titration. viral growth kinetics analysis was in line with the published data [gao , manenti . furthermore, we devised a technique to increase the virus concentration in suspension about to , -fold (note: this finding requires validation). to inactivate the virus properly, beta-propiolactone was chosen because it can alkylate the viral genome and keeps the virus capable of inducing a protective immune response [delrue ] . the process of viral suspension double inactivation was designed in accordance with the procedures of other vaccine manufacturers [pu , xia . design of a multiepitope-based peptide vaccine against the e protein of human covid- : an immunoinformatics approach guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition) inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind, randomised controlled trial development of an inactivated vaccine candidate for sars-cov- an in-silico approach to develop of a multi-epitope vaccine candidate against sars-cov- envelope (e) protein. res sq cov- cell entry depends on ace and tmprss and is blocked by a beigel jh; mrna- study group vaccine against sars-cov- -preliminary report evaluation of sars-cov- neutralizing antibodies using a cpe- based colorimetric live virus micro-neutralization assay in human serum samples epub ahead of print rna vaccine bnt b in adults immune and bioinformatics identification of t cell and b cell epitopes in the protein structure of sars-cov- : a systematic review an in-depth investigation of the safety and immunogenicity of an inactivated sars-cov- vaccine determination of % endpoint titer using a simple formula convergent antibody responses to sars-cov- in convalescent individuals concurrent human antibody and th type t-cell responses elicited by a covid- rna vaccine structural insight into the role of novel sars-cov- e protein: a potential target for vaccine development and other therapeutic strategies a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses. vaccine preparation, characterization and preliminary in vivo studies of inactivated sars-cov vaccine immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus rna-based covid- vaccine bnt b selected for a pivotal efficacy study. medrxiv inactivated vaccine against sars-cov- on safety and immunogenicity outcomes: interim analysis of randomized clinical trials immunogenicity and safety of a sars-cov- double-blind, and placebo-controlled phase clinical trial augmentation of immune responses to sars coronavirus by a combination of dna and whole killed virus vaccines. vaccine immunogenicity and safety of a recombinant adenovirus type- -vectored covid- vaccine in healthy adults safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation non-randomised, first-in-human trial key: cord- - gbe qo authors: loomba, s.; de figueiredo, a.; piatek, s.; de graaf, k.; larson, h. j. title: measuring the impact of exposure to covid- vaccine misinformation on vaccine intent in the uk and us date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: gbe qo the successful development and widespread acceptance of a sars-cov- vaccine will be a major step in fighting the pandemic, yet obtaining high uptake will be a challenging task, worsened by online misinformation. to help inform successful covid- vaccination campaigns in the uk and us, we conducted a survey to quantify how online misinformation impacts covid- vaccine uptake intent and identify socio-economic groups that are most at-risk of non-vaccination and most susceptible to online misinformation. here, we report findings from nationally representative surveys in the uk and the us conducted in september . we show that recent misinformation around a covid- vaccine induces a fall in vaccination intent among those who would otherwise 'definitely' vaccinate by . ( . , . ) percentages points in the uk and . ( . , . ) in the us, with larger decreases found in intent to vaccinate to protect others. we find evidence that socio-econo-demographic, political, and trust factors are associated with low intent to vaccinate and susceptibility to misinformation: notably, older age groups in the us are more susceptible to misinformation. we find evidence that scientific-sounding misinformation relating to covid- and vaccines covid- vaccine misinformation lowers vaccination intent, while corresponding factual information does not. these findings reveal how recent covid- misinformation can impact vaccination rates and suggest pathways to robust messaging campaigns. of existing protective immunity-though it is unclear whether post-infection immunity confers long-term immunity (altmann )-and the rapidly evolving nature of misinformation surrounding the pandemic (pennycook , who , it is unclear whether we will reach vaccination levels required for herd immunity. although studies have examined the effect of covid- misinformation on public pandemic perceptions (geldsetzer ; islam ; kim ) and the tendency of certain socio-political groups to believe misinformation (kreps ; murphy ) , we lack a quantitative understanding of the link between exposure to misinformation surrounding covid- and intent to receive a future vaccination. as a viable vaccine comes closer to reality, it is essential to understand this link, how it differentially impacts sociodemographic groups, and whether groups at high risk of developing severe complications from are vulnerable to misinformation. to fill this gap, we developed a covid- questionnaire to measure vaccine intent pre-and postexposure to online sources of recent misinformation relating to covid- and vaccines. this questionnaire was used to survey , respondents across the uk and us via nationally representative sampling. , respondents in each country were exposed to misinformation (see figure ), while the remaining , were shown information about a covid- vaccine that was factual to serve as a randomised control (see figure and methods). a large suite of complementary data were collected for each individual including socioecono-demographic status (age, gender, highest education level, employment type, religious affiliation, ethnicity, income level), sources of trust for information about covid- , political affiliation, social media usage, and reasons for being unsure about taking a covid- vaccine (see table and questionnaire, appendix e). to assist policymakers and stakeholders in the design of communication and messaging campaigns, we calculate the determinants of covid- vaccine intent both pre-exposure and post-exposure, allowing public health messaging campaigns (nhsa ; nhsb ; cdca ; cdcb ). moreover, an assessment of the impact of different types of misinformation presented to respondents yields an understanding of the semantic and stylistic content of misinformation that has the largest impact in lowering intent to vaccinate in this study. our findings are interpreted in light of vaccination levels required for herd immunity and we discuss messaging strategies that may help mitigate or counter the impact of online vaccine misinformation. throughout this study, misinformation refers to information that is inadvertently false, but no harm is meant in sharing it and that "[is] considered incorrect based on the best available evidence from relevant experts at the time" (wardle , vraga . the questionnaire was fielded between and september, to a total of , respondents. all respondents were recruited via an online panel by orb (gallup) international (www.orb-international.com). respondent quotas were set according to national demographic distributions for gender, age, and sub-national region (state in the us and first level of nomenclature of territorial units in the uk (ons ). a total of , participants were surveyed in the uk and , in the us, and all respondents were aged or over. of these , respondents, , ( , ) respondents were exposed to misinformation relating to covid- and vaccines (treatment group) in the uk (us) and , were exposed to factual covid- vaccine information (control group). all information (misinformation and factual) was identified using meltwater ® (www.meltwater.com) via a boolean search string eliciting (mis)information around a covid- vaccine. a systematic selection approach was used to identify the covid- vaccine information on social media with high circulation and engagement between june, , and august, (see methods). a final set of five pieces of misinformation comprising non-overlapping messaging and themes were selected to represent the diverse messaging found in covid- vaccine misinformation (such as information questioning the importance or safety of a vaccine, see figure ). as misinformation can be highly country and context-dependent, it was decided to expose uk and us respondents to different sets of misinformation to reflect the different audiences targeted by the sources of misinformation, while factual information was the same for both. each respondents were asked about their intent to receive a covid- vaccine to protect themselves and, to explore altruistic behaviours, to protect others: "if a new coronavirus vaccine became available, would you accept the vaccine for yourself?" (self) and "if a new coronavirus vaccine became available, would you accept the vaccine if it meant protecting friends, family, or at-risk groups?" (other). responses were collected on a four-point scale: "yes, definitely," "unsure, but leaning towards yes," "unsure, but leaning towards no," and "no, definitely not." this scale is chosen to remove subjective ambiguity involved with likert scales and to allow respondents to detail explicitly their intent, thereby allowing a more meaningful interpretation of results. both questions (self and other) were asked to respondents before and after exposure to either the treatment or control information set. we first investigated individuals' intent to vaccinate before and after information exposure. before any exposure, . % ( % percentile interval [pi], . to . ) of respondents in the uk and . % ( . , . ) in the us report that they will "definitely" accept a covid- vaccine to protect themselves (table ) . higher intent to accept a covid- in the uk than the us has been recently reported (mcandrew ). exposure to misinformation induces significant decreases in the number of respondents reporting that they would "definitely" accept a vaccine: a . ( . , . ) percentage point (pp) fall in the uk and a . pp ( . , . ) fall in the us. there are corresponding increases in the percentages of respondents either now unsure whether to vaccinate or who would now definitely not vaccinate (table ) . we find that factual information induces no "significant" shifts ( % pi of difference distribution includes zero) in the proportion of respondents falling into each response category before and after exposure (table ) . we find two results that may be of particular interest to policymakers harnessing altruistic messaging devices to boost public compliance with recommended interventions. firstly, more respondents in both countries would accept a vaccine if it meant protecting family, friends, or at-risk groups (than if the vaccine was for themselves): . % ( . , . ) of respondents in the uk and . % ( . , . ) in the us say that they would "definitely" get vaccinated to protect others (table ) . secondly, misinformation induces a disproportionately larger percentage point fall in vaccination intent to protect others than to protect oneself: a . pp fall in the uk (a . % percentage drop compared to . % for the drop associated with intent to vaccinate themselves) and a . pp fall in the us ( . % versus . %). these results suggest that all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . https://doi.org/ . / misinformation may have a damaging effect on messaging drives that leverage individual altruistic behaviours. while the above analysis reveals exposure to misinformation impacts overall vaccine intent, we cannot casually attribute these changes to the misinformation shown to respondents without accounting for the control group. we therefore estimate the risk difference of transitioning from one vaccine acceptance category to another upon exposure to misinformation, relative to exposure to factual information-see figures s , s and tables s , s in appendix d. these risk differences (as defined in equation of appendix c) reveal that there is still an overall transition of people from categories of higher to lower vaccine acceptance when controlling for exposure to factual information. bayesian ordinal logistic regressions (see methods) were used to find the factors associated with rejecting a covid- vaccine and the factors associated with a susceptibility to misinformation-that is, a tendency for individuals to lower their vaccination intent after seeing misinformation. there are some key similarities and differences between the determinants in the uk and the us of relevance to country-specific public health policy, which we outline here. effects are reported as odds ratios (or), where odds ratios larger than represent a factor that is associated with increased chance of rejecting the vaccine compared to the baseline group (male, - , highest education, employed, christian, white, conservative (uk)/republican (us), highest income, and no social media usage). in both countries, females are more likely than males to refuse a covid- vaccine, with a larger effect-size in the us (odds ratio . , % percentage interval (pi): . to . ) than the uk (or . [ . , . ]). (all odds ratios and credible intervals are shown in figure for uk and us pre-exposure determinants (a, c) and determinants of susceptibility (b, d).) in both countries, individuals with highest educational attainment below postgraduate degrees, low income groups, and non-whites are more likely to reject a covid- vaccine. trends in uptake intent determinants are found to differ along age, employment status, political affiliation, and social media use. most notable among these differences are that - year-olds and over s are less likely to reject a covid- vaccine than - year-olds in the uk, whereas all age groups except over s are more likely to reject a vaccine than - year-olds in the us. in the us, democrats are less likely to reject the vaccine than republicans (or . [ . , . ] ) whereas those who do not affiliate with any of the four major parties in the uk (conservative, labour, lib dems, snp) are far more likely to all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint refuse a vaccine. these results mirror recent surveys in ireland, where covid- vaccination intent is associated with non-mainstream political affiliation (murphy et al. ) , and in the us, where males and those with degrees were more likely to accept the vaccine (malik ) , and where democrat voters are "more likely to correctly identify a covid-related headline as true or false than were independents or republicans" (kreps ) . individuals in the us with no social media use are less likely to accept a vaccine than individuals who use social media. in the us, individuals with low social media usage (and high legacy media usage) have higher vaccine intent than those with low consumption of both (mcandrew in the uk, the impact of misinformation on intent appears to have no differential effect across socioecono-demographic groups, but those not supporting one of the four main political parties are more susceptible to misinformation . [ . , . ] (compared to the conservatives). respondents who would not "definitely" take a covid- vaccine were asked for their reasons, which were included as explanatory factors in the ordinal logistic regressions used previously, while controlling for sociodemographic factors (see methods). in this section the log odds ratio (log or) is used: log ors larger than represent a factor that is associated with increased chance of rejecting the vaccine, and of being more susceptible to misinformation, compared to the baseline group (same as previously stated with no social media baseline). all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . in the uk and us, vaccine rejection is associated with a belief that covid- does not pose a risk or that they will not be ill if they contracted the disease ( figure a , b). such barriers fall within the "complacency" vaccine hesitancy under who's strategic advisory group of experts (sage) on immunisation's " c" hesitancy model (who ) . respondents who claimed that they wanted to "wait until others" received the vaccine, were more likely to lean towards vaccinating ("unsure, but leaning towards yes") than respondents who said they were "unsure, but leaning towards no" or would "definitely not" vaccinate (log odds ratio , - . [- . , - . ] for the uk and log or - . [- . , - . ] for the us). respondents who report that "vaccine approval may be rushed" tent to be significantly more vulnerable to misinformation in the uk (log or . [ . , . ]) ( figure a ), while, in the us, respondents who are unsure whether the vaccine is safe are more susceptible to misinformation (log or . [ . , . ]) ( figure b ). these two responses are both related to explicit "confidence" barriers (who ). respondents across both countries who trust television news, government briefings, health authorities, and (perhaps surprisingly) celebrities tend to have higher pre-exposure inclination to vaccinate. respondents who indicate that they trust family and friends have lower pre-exposure intent than those who do not trust family or friends in the uk (log or . [ . , . ]). respondents in both countries who report trusting no sources ("none of the above", see table s , appendix d) have lower pre-exposure vaccination intent (see figure c , d). those who do not trust any typical sources remain significantly more susceptible to misinformation exposure in both the uk and us ( figure c and d), suggesting that a lack of trust in mainstream authorities is indicative of being influenced by misinformation exposure. trust in social media and scientists also contributes to susceptibility for the us, but not in the uk. after exposure to misinformation or factual information, respondents were asked to report whether, for each image: it raised their vaccination intent; they agreed with the information presented; they found the information to be trustworthy; they were likely to fact-check ; and they were likely to share the image with friends or followers. across both countries, sizeable proportions (between % and % of those who would not definitely accept the vaccine) either agreed with the misinformation or found it trustworthy, though the all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint majority of respondents did not agree and did not find it trustworthy ( figure a , b). to investigate the impact that each individual image had on vaccine intent, weights were assigned to each image while regressing image characteristics-self-reported by respondents-against changes in vaccine intent upon exposure, thus quantifying the contribution of each piece of (mis)information to the change (see methods). this analysis revealed that the images with the largest contribution to a loss in vaccination intent in the uk were image ("scientists have expressed doubts [...] over the coronavirus vaccine [...] after all of the monkeys used in initial testing contracted coronavirus") and image , which claimed the new covid- vaccine will "literally alter your dna" (appendix d, table s and s ). this latter image was the most impactful in lowering vaccination intent in the us, followed by image "yale university and the u.s. government are running clinical trials to develop propaganda messaging to persuade americans to take [...] vaccines [...]" (appendix d, table s and s ). in the control set-which was shown to respondents in both countries-the image that contributed the least to fall in vaccine intent was image (figure ), in which the university of oxford announced that their vaccine "produces a good immune response" and that the "teams @vaccinetrials and @oxfordvacgroup have found there were no safety concerns". while other images arguably used some scientific messaging (such as image in figure a , "big pharma whistleblower: ' % of corona vaccine recipients will become infertile'"), the images identified as having the most impact on lowering vaccination intent stated a direct link between the covid- vaccine and adverse effects and cited articles and scientific imagery or links to articles purporting to be reputable to strengthen their claim. this contrasted to more memetic imaging (for example, "striking images with text superimposed on top" (wardle )) which was far less impactful (images and , figure a and images and , figure b ). the manner of information exposure in our study is a simplification of how people are naturally exposed to information online social media platforms. we asked respondents if they had encountered similar images to the ones they were exposed to on social media in the past one month, to explore the relationship between their vaccination intents and pre-study exposure to misinformation or factual information. we find evidence of misinformation and factual information "filter bubbles", which is the phenomenon of people seeing more of the content that agrees with their prior beliefs (pariser , bakshy . in the uk, respondents who reported that they would definitely not vaccinate to protect themselves before exposure to misinformation were . % ( . , . ) more likely than those who would "definitely" get vaccinated to have seen similar misinformation. in both the uk and us, respondents in the control group who reported that they all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint would "definitely not" vaccinate were significantly less likely to have seen similar factual information (see appendix d, table s and figure s ). using individual-level survey data collected from nationally representative samples of , respondents in each of the uk and us, we reveal a number of key findings of importance to policymakers and stakeholders engaged in either public health communication or the design of vaccine-rollout programmes. we find that, as of september , only . % of the public in the uk and . % in the us would "definitely" accept a covid- vaccine to protect themselves. the main barriers to reporting certainty over vaccinating were concerns over vaccine safety or a belief that they would not be at risk of contracting covid- or would not be ill if they did (that is, vaccine-importance related confidence barriers). individuals who wanted to "wait until others" had been vaccinated were less likely to outright reject a vaccine. currently, these values are below those required to achieve the anticipated herd immunity levels, however, higher proportions of individuals in both countries would "definitely" vaccinate to protect family, friends, and at-risk groups, suggesting that effective altruistic messaging may be required to boost uptake. however, we have also shown that exposure to misinformation not only lowers intent to vaccinate, but that it disproportionately lowers intent to vaccinate to protect others, which could complicate messaging campaigns focusing on altruistic behaviours. campaigns may also have to battle with misinformation purporting to be based in science or medicine, which appears to be particularly damaging on vaccination intentions. these findings are, however, unlikely to be representative of the effect of misinformation on uptake rates in real world social-media settings. individuals are unlikely to experience misinformation in the same manner as implemented in this survey, and there will be differences in the volume and rate of misinformation people will be exposed to, depending on their online social media preferences and demographics. a demographic re-weighting would be required to obtain more robust estimates of anticipated covid- vaccine rejection at sub-national or national levels. misinformation may have also already embedded itself in the public's consciousness, and studies have shown that brief exposure to misinformation can embed itself into long-term memory (zhu ) . policymakers may therefore find challenges ahead to "undo" the impact all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint it may have already had and to clearly communicate messages surrounding the safety, effectiveness, and importance of the vaccine. willingness to accept a covid- vaccine and susceptibility to misinformation is found to depend on a number of socio-economic factors (these findings are likely to be more robust since the survey ensures consistent misinformation exposure across different demographics). females, ethnic minority groups, those without university degrees, and low-income groups were less willing to accept a vaccine in both the uk and us (with respect to the baseline, see table ), in alignment with recent studies (sherman ; mcandrew & allington ; murphy ) . given that covid- incidence and mortality rates are higher in some black and minority ethnic groups as well as lower-income groups in the uk and us (aldridge ; pan ; patel ; price-haywood ), it is vital that vaccination rollout campaigns not only ensure sufficient access to these communities, but that confidence in the vaccine is built before rollout. the groups most susceptible to misinformation are older age groups in the us. given that age is an important risk factor for covid- , an increase in misinformation targeted at these groups could prove detrimental to efforts in protecting those who are most at-risk. in the uk, older age groups appear to be more confident in the vaccine-consistent with previous studies into both covid- perceptions (murphy ) and longer-term general vaccine confidence trends in the uk (larson ) . respondents who did not report even a single source of trust were significantly more susceptible to misinformation than those who did, echoing research from the uk and ireland which found that "those who were resistant to a covid- vaccine were less likely to obtain information about the pandemic from traditional and authoritative sources and had similar levels of mistrust in these sources" (murphy et al. ). in the uk, individuals who believed the vaccine may be rushed were the most negatively impacted by vaccine misinformation. although our study indicates the possible impact of covid- misinformation campaigns on vaccination intent, this study does not replicate a real-world social media platform environment-where information exposure is a complex combination of what is shown to a person via the platform's algorithms and what is shared by their friends or followers (bakshy ) . online social network structures, governed by social homophily, can lead to selective exposure and creation of homogeneous "echo-chambers" (del vicario ) which may amplify (or dampen) the spread of misinformation among certain demographics. indeed, we do find some evidence in this study of filter bubbles with regards to information on covid- all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint vaccine-those who would definitely not accept the vaccine had seen more misinformation (in the uk) and less factual information (in both the uk and us) recently online when compared to those who definitely would accept the vaccine. these network effects cannot be replicated via a questionnaire. therefore, our estimates for the losses in vaccination intent due to misinformation must be placed in the context of this study: caution must be exercised in generalising these findings to a real-world setting, which may see greater or lesser drops in vaccination intent depending on the wider context of influencing factors. moreover, we are limited in the type and volume of misinformation presented to respondents and there may exist other types of misinformation which may be far more impactful on vaccination intent. addressing the spread of misinformation will likely be a major component of a successful covid- vaccination campaign, especially as misinformation on social media has been shown to spread faster than factually correct information (vosoughi ) and that, even after a brief exposure, misinformation can result in long-term attitudinal and behavioural shifts (pluviano ; zhu ) that pro-vaccination messaging may find hard to overcome (pluviano ) . with regards to covid- , misinformation has even been shown to lead to information avoidance and less systematic processing of covid- information (kim ) , however, the amplification of "questionable" sources of covid- misinformation is highly platform dependent, with some platforms amplifying questionable content less than reliable content (cinelli ) . the analysis reveals that, in both the uk and us, fewer people would "definitely" take a vaccine than is required for herd immunity, and that misinformation could push these levels further away from herd immunity targets. this analysis provides a platform to help us test and understand how more effective public health communication strategies can be designed and on whom these strategies would have the most positive impact in countering covid- vaccine misinformation. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint figure widely circulating misinformation on social media surrounding a covid- vaccine between june and august . for each of the uk and us, five images were selected (see methods) to expose to respondents. these "treatment" image sets were shown to , respondents in the uk (a) and the us (b). for reference, each image is numbered. urls for all images are provided in appendix b. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint figure widely circulating factual information on social media surrounding a covid- vaccine between june and august . the same five images were selected (see methods) for exposure to respondents in the uk and us. these "control" image sets were shown to , respondents in the uk and the us. urls for all images are provided in appendix b. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in table exposure to misinformation reduces intent to vaccinate. percentage of respondents in the uk and us reporting that they would definitely accept, definitely not accept, or were unsure about accepting a covid- vaccine for themselves (self) or others (other) pre-and postexposure to factually incorrect information about covid- (treatment) or factually correct information (control). numbers in parentheses denote the upper and lower % percentile interval. only significant pre-and post-exposure changes (Δ) are shown. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . https://doi.org/ . / figure determinants of vaccination intent before exposure and of susceptibility to misinformation contribution of socio-demographic and social-media-use characteristics to preexposure vaccine intent (columns a, c) and susceptibility to misinformation exposure (columns b, d) for the uk (left) and us (right). red bars depict odds-ratios (or) of being more reluctant to vaccinate or more susceptible to misinformation relative to the reference category of male, - , highest education, employed, christian, white, conservative, highest income, and no social media usage. odds ratios above indicate the group is more likely to reject a covid- vaccine than the reference group, and more likely to be susceptible to misinformation relating to covid- and vaccines. black bars indicate % percentile intervals and starred (*) bars show effects whose % pi excludes zero (which we term "significant"). numbers on the right indicate sample sizes of the corresponding demographic. see table s in appendix d for more details. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . https://doi.org/ . / contribution of reasons that respondents provide for not being "definitely" sure of taking a covid- vaccine (a, b) and contribution of sources of information that people trust (c, d), to the pre-exposure vaccine hesitancy (left of every sub- figure) and susceptibility to vaccine misinformation (right of every sub-figure) as measured by drop in vaccine intent-after controlling for socio-demographics. values depict odds-ratios (or) of being more hesitant (for pre-exposure) or more susceptible (for susceptibility)-as measured relative to when the reason was not indicated for hesitancy, or when the source was not indicated as being trusted. or> indicates the group is more likely to not accept a covid- vaccine if they indicated that reason for hesitancy (a, b) or that source of information as trustworthy (c, d). bars indicate % percentile intervals and * indicate "statistical significance" based on them. numbers on the right indicate sample sizes that had indicated the corresponding reason/source, within both the total (n= for uk and n= for us) and treatment respondent set (n= for uk and n= for us). since reasons for hesitancy were only asked to those who did not choose "yes, definitely" when asked if they would vaccinate to protect themselves, that analysis is conditioned to those who did not indicate "yes, definitely" for the self question. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . doi: medrxiv preprint figure breakdown of views to information displayed to respondents the percentage of respondents providing a given response to each follow-up question to explore their perceptions of each image. respondents were asked whether each image raises their vaccine intent (column ); contains information they agree with (column ); contains information they find trustworthy (column ); is likely to be fact-checked by them (column ); and is something they will likely share with others (column ). rows represent images shown to (a) uk treatments, (b) us treatments, (c) uk controls and (d) us controls. note that those responding with "do not know" were grouped with those saying "neither/nor". % percentile intervals have been excluded here for clarity. note that the response scale has been inverted for column for direct comparison across all questions (see section of appendix e for the relevant questionnaire subsection). all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . https://doi.org/ . / a total of , participants were recruited via an online panel by orb (gallup) international (www.orbinternational.com) and surveyed between and september, and who obtained informed consent was obtained from all participants. a total of , participants were surveyed in the uk and , in the us. of these respondents, , ( , ) respondents were exposed to covid- misinformation around vaccines (the treatment group) in the uk (us), while , in each country exposed to factual information relating to covid- vaccines (the control group). each group was shown a total of five pieces of information: all of these five were either misinformation or factual. respondents were sampled to match proportions of national demographic breakdowns for gender, age, and sub-national region (state in the us and second administrative level in the uk (ons )). survey weights were provided to account for mis-matches between expected national sex, age, and regional distributions and those obtained via online panels. survey sizes for both the exposure and control exceed those typically conducted as national-level surveys (n= ) and therefore summary variables contain at most a ~ % error. respondents are asked about their intent to vaccinate to protect themselves and (separately) their intent to vaccinate themselves to protect friends, family, and at-risk groups contracting covid- . to deduce changes in vaccination intent, respondents are asked for their intent to vaccinate both before and after exposure. the questions were as follows, these two questions are answered on a four-level scale: "yes, definitely", "unsure, but leaning towards yes", "unsure, but leaning towards no", and "no, definitely not". this scale is chosen to remove ambiguity involved with likert scales and to provide a meaningful interpretation of results with regards to vaccination intent. if all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . / . . . doi: medrxiv preprint respondents do not assert that they would "yes, definitely" take a vaccine, then reasons for hesitancy are explored (see figure a , b). the information exposed to respondents are screenshots of social media posts, which contain a mixture of text and images (see figures and ) . a different set of five treatment images are shown to respondents in the uk and us to reflect country-specific differences in online social media sources and content, while the control images are a set of five images fixed for both countries (see methods: selection of intervention images for further information). for each exposure image, respondents are asked to rate the extent that: ) they agree with the information displayed; ) they are inclined to be vaccinated; ) they believe the information to be trustworthy; ) they will fact-check the information; and ) they would share the image. after exposure, the respondents were also asked if they had seen "similar" content on social media in the last one month. (see appendix e for the full questionnaire.) respondents' socio-demographic information is collected, including gender, age, education, employment status, religious affiliation, ethnicity, income and political affiliation. to probe relationships with social media use, respondents are asked to self-report the amount of time spent daily on social media platforms (ernala ) . raw counts for these characteristics are shown in table (see appendix a for details on variable recoding). these characteristics are used to determine the groups most reluctant to take a covid- vaccine and the groups most impacted by misinformation. in addition, respondents are asked to report the sources they trust for information surrounding covid- . in order to elicit responses that can be most readily interpreted in light of the current state of online misinformation in both countries, the information shown to respondents should satisfy a number of criteria, it should: ) be recent and relevant to a covid- vaccine; ) have a high engagement, either through user reach or other publicity, and thus represent information that respondents are not unlikely to be exposed to through social media use; ) include posts shared by organisations or people with whom respondents are familiar (so that, for example, us/uk audiences are not shown information from people with whom they are unfamiliar); ) form a distinct set, not replicating content or core messaging, allowing us to probe the most impactful types of misinformation. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . to this end, we followed a principled approach to selecting five images, combining both quantitative and qualitative methods. the procedures for selecting treatment and control image sets are as follows: (treatment set) we used a covid- vaccine-specific boolean search query-corona* or coronavirus or covid* or "wuhan virus" or wuhanvirus or "chinese virus" or "china virus" or chinavirus or "ncov*" or sars-cov*) and vaccin* and (gates or g or microchip or "new world order" or cabal or globali*)-to extract covid- vaccine related online images from june, , to august, using meltwater ® (www.meltwater.com), an online social media listening platform. this boolean search term was based on previous research which used similar search terms which obtained the highest levels of user engagement with covid- media and social media articles containing misinformation. this boolean search string returned over thousand social media posts which were initially filtered by user engagement and reach to provide the most widely shared and viewed posts. two independent coders (sp and kdg) screened top posts and excluded posts that failed criteria ( - ) above. some posts had relatively low levels of engagement,yet were included because they repeatedly appeared in different formats across different outlets and were thus deemed to be influential on social media. a set of five final posts were obtained for the us and the uk. for instance, misinformation selected to be shown to the us sample included a post falsely claiming that a covid- vaccine will alter dna in humans, while that in the uk included a post falsely claiming that covid- vaccine will cause % of recipients to become infertile. (control set) the aim of exposing people to factual coronavirus vaccine information is to serve as a control against the treatment exposure of misinformation since (a) exposure to any information can in principle cause respondents to change their vaccine inclination (control information therefore controls for other elements of our survey), and (b) respondents may misreport post-exposure vaccine intent due to recall bias or other between-conditions difference. factual information was also obtained via meltwater ® using the same boolean search term above, but excluding the last clause containing misinformation-specific search keys. over posts were returned that were filtered to a final set of five. information was often from authoritative sources (or otherwise referenced to authoritative sources) such as vaccine groups and scientific organisations. we ensured that these five posts were not overtly pro-vaccination, and did not reference anti-vaccination all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . https://doi.org/ . / campaigns or materials. for instance: information presented included information on: the current state of coronavirus vaccine trials; the importance of a vaccine to get out of the covid- pandemic; and how a candidate vaccine generates good immune response. see appendix b for more details regarding image selection for both treatment and control image sets. these final image sets are shown in figure (misinformation) and figure (factual information). bayesian statistical modelling is used to answer all quantitative research questions. relevant statistics for parameters of interest (percentages, odds-ratios and log odds-ratios) are reported as a mean estimate (the effect size) with corresponding % percentile intervals (pi) (that is, values at . % and . % percentiles) to indicate credible values of the statistic. a percentage (%) or log odds ratio (log or) is deemed "significant" if the % pi excludes zero, and an odds ratio (or) is deemed "significant" if the % pi exclude one. exposure to misinformation on an online social media platform is a complex system to model. there are many confounding factors regarding: psychological, socio-economic and cultural characteristics of consumers of information-traits that determine consumption of both offline and online media; characteristics of platforms serving this information including the algorithms which deliver information on social media can be opaque black-boxes; and characteristics of the information itself, such as style and semantic content, or the identity of the entity sharing the information, such as friends, family, celebrities, or organisations. these factors make it difficult to determine the "causal" impact of exposure to any piece of information (bursztyn et al. ). however, we can control for some of these effects by pursuing a randomised-controlled-trial strategy to make some progress towards measuring causal impact (hornsey ) . (see figure c in appendix c which shows a causal diagram that simplifies the online information dynamics, and how they may influence personal beliefs, such as vaccine intent. the figure also depicts how conducting an "exposure", where respondents are divided into a treatment group that is shown covid- vaccine misinformation and a control group that is shown factual information, allows for a causal measurement of the drop in vaccine intent in response to the exposure to misinformation.) throughout the analysis, individuals' responses to the vaccination intent survey questions (see ( ) and ( )) are modelled as ordinal variables. vaccine intent of a respondent before all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . (after) any intervention is modelled as !"# ∈ { , , , } ( !$%& ∈ { , , , }), where corresponds to the survey response "yes, definitely" and corresponds to "no, definitely not" so that we have the ordering < < < . individuals are assigned an intervention group ∈ { , }: treatment (t) or control (c). to judge the impact of exposure to factual information or misinformation, we define a statistic, , that measures the net change of respondents providing a specific response ∈ { , , , }, given their specific exposure, ] when = ( ), then this statistic measures the effect of exposure to misinformation (factual information). vaccination intent: individual variation in vaccine intent !"# and !$%& is modelled via ordered logistic regression models (mcelreath ). that is, ∼ ( , ) where is an ordered vector of length that encodes the intercepts of the cumulative distribution for ( !"# , !$%& ), and is a placeholder for the set of predictor variables included in the regression. !"# as a predictor variable-for susceptibility models with outcome !$%& -is treated as an ordinal variable containing categories indexed { , , , }, where the overall slope is separated from the contribution of every additional category level by considering the parameter ' such that ' > and ∑ ' = ( ')* , and the total slope for category !"# ( ) can be written as since is a -simplex (the first category has a reference contribution of ) we place a weak dirichlet prior on it ∼ ℎ ( , , ). are modelled as categorical data. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . all model specifications, including likelihood and prior choices are outlined below-with sensibly regularising priors chosen to prevent overestimation of effects. table illustrates the selection of explanatory variables used in each model. modelling the impact of information exposure on vaccination intent tables s , s table bayesian ordinal logistic regression models for vaccine intent in the main study. the full specification of each model, and of models with outcomes other than vaccine intent, can be found in the main text. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . distributions on priors: results: figures , ; tables s , s , s likelihood distribution: the analysis is conditioned on the groups (treatment or control) modeled independently. for each of the images shown, respondents were asked to report perceptions of the image across dimensions (see methods: survey design) on a -level likert scale. this model treats the response for each image and image-metric as an ordinal variable. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . likelihood distribution: the analysis is conditioned on the groups (treatment or control), images and imagemetrics modelled independently. after exposure to (mis)information, respondents were asked if they had seen "similar" information on social media recently (see methods: survey design), to which they could respond with "yes", "no", or "do not know". this model treats this response as an ordinal variable. likelihood distribution: the analysis is conditioned on the groups (treatment or control) modeled independently. here, we do not treat !"# as ordinal, to capture any u-shaped effects. let ∈ { , }refer to whether the respondents had "seen similar content online in the last month on social media," where implies "yes", implies "no" and "do not know"s were ignored. iterations (i.e. samples excluding warm-up) after ensuring model convergence, with the potential scale reduction factor satisfying ≤ . for all model parameters (gelman & rubin ) . relevant statistics for parameters of interest (coefficients, contrasts, odds-ratios and percentages) were extracted from the samples, and all results report the mean estimate-to judge magnitude of the effect-alongside % percentile intervals (pi) (i.e. values at . % and . % percentiles) to indicate credible values of the statistic-to judge significance of the effect. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; model calculations and inference and created all publication figures. sp, kdg, and sl performed image selection. adf and sl wrote the final manuscript with input from all authors. all authors contributed to the interpretation of the results. adf and hl supervised the project. the data are not public, but researchers can apply to use the resource. the code for this project will be made available at https://github.com/sloomba/covid -misinfo/. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted october , . ; https://doi.org/ . asian and minority ethnic groups in england are at increased risk of death from covid- : indirect standardisation of nhs mortality data what policy makers need to know about covid- protective immunity the proximal origin of sars-cov- exposure to ideologically diverse news and opinion on facebook communication and miscommunication of risk: understanding uk parents' attitudes to combined mmr vaccination beyond (mis)representation: visuals in covid- vaccine misinformation and social media wear a mask to protect yourself and your friends do it for yourself and your friends the covid- social media infodemic misinformation during a pandemic a probabilistic programming language a future vaccination campaign against covid- at risk of vaccine hesitancy and politicisation mapping global trends in vaccine confidence and investigating barriers to vaccine uptake: a large-scale retrospective temporal modelling study how well do people report time spent on facebook? an evaluation of established survey questions with recommendations covid- : the deadly threat of misinformation. the lancet. infectious diseases knowledge and perceptions of covid- among the general public in the united states and the united kingdom: a cross-sectional online survey inference from iterative simulation using multiple sequences donald trump and vaccination: the effect of political identity, conspiracist ideation and presidential tweets on vaccine hesitancy covid- -related infodemic and its impact on public health: a global social media analysis effects of covid- misinformation on information seeking, avoidance, and processing: a multicountry comparative study medical misinformation in the covid- pandemic. available at ssrn herd immunity-estimating the level required to halt the covid- epidemics in affected countries the state of vaccine confidence in the eu public health and economic consequences of vaccine hesitancy for measles in the united states determinants of covid- vaccine acceptance in the us mode and frequency of covid- information updates, political values, and future covid- vaccine attitudes statistical rethinking: a bayesian course with examples in r and stan even covid- can't kill the anti-vaccination movement preparing for a covid- vaccine: identifying and psychologically profiling those who are vaccine hesitant or resistant in two general population samples link nhs(a) coronavirus resources ( ) protect your loved ones with the official covid- tracing app the filter bubble: how the new personalized web is changing what we read and how we think the impact of ethnicity on clinical outcomes in covid- : a systematic review poverty, inequality and covid- : the forgotten vulnerable fighting covid- misinformation on social media: experimental evidence for a scalable accuracy-nudge intervention hospitalization and mortality among black patients and white patients with covid- misinformation lingers in memory: failure of three provaccination strategies the effect of opinion clustering on disease outbreaks high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus covid- vaccination intention in the uk: results from the covid- vaccination acceptability study (covaccs), a nationally representative cross-sectional survey. medrxiv pystan: the python interface to stan public now divided over whether to get covid- vaccine ( ) pew research centre the spread of true and false news online correction as a solution for health misinformation on social media information disorder: toward an interdisciplinary framework for research and policy making report of the sage working group on vaccine hesitancy a pneumonia outbreak associated with a new coronavirus of probable bat origin brief exposure to misinformation can lead to long-term false memories this project was funded by the un's verified initiative. the funders had no role in data collection or study design. the vaccine confidence project™ would like to acknowledge its long partnership and collaboration with orb (gallup) international who collected all data for this study. the funders had no role in data collection, questionnaire design, data analysis, data interpretation, or writing of this report. the corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.ethical approval: approval for this study was obtained by the lshtm ethics committee on june with reference . all authors contributed to questionnaire design with adf obtaining ethical approval for the study via the lshtm ethics committee. sl and adf designed the statistical analyses. sl performed all likelihood distributions: the analysis is conditioned on the groups (treatment or control) modelled independently.where ∼ ( , ( * , * , ⋯ ;/* )) implies thatdistributions on priors:results: table likelihood distribution: the analysis is not conditioned on groups. analysing reasons of hesitancy, since we still control for socio-demographics, %$a@ >(-) is replaced by "# %$ (-) , and similarly for sources of information that are trusted, %$a@ >(-) is replaced by &"<%&(-) . distributions on priors: . * , , ( ∼ ( , ) key: cord- - xorq ce authors: naz, anam; shahid, fatima; butt, tariq tahir; awan, faryal mehwish; ali, amjad; malik, arif title: designing multi-epitope vaccines to combat emerging coronavirus disease (covid- ) by employing immuno-informatics approach date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: xorq ce a recent pandemic caused by a single-stranded rna virus, covid- , initially discovered in china, is now spreading globally. this poses a serious threat that needs to be addressed immediately. genome analysis of sars-cov- has revealed its close relation to sars-coronavirus along with few changes in its spike protein. the spike protein aids in receptor binding and viral entry within the host and therefore represents a potential target for vaccine and therapeutic development. in the current study, the spike protein of sars-cov- was explored for potential immunogenic epitopes to design multi-epitope vaccine constructs. the s and s domains of spike proteins were analyzed, and two vaccine constructs were prioritized with t-cell and b-cell epitopes. we adapted a comprehensive predictive framework to provide novel insights into immunogenic epitopes of spike proteins, which can further be evaluated as potential vaccine candidates against covid- . prioritized epitopes were then modeled using linkers and adjuvants, and respective d models were constructed to evaluate their physiochemical properties and their possible interactions with ace , hla superfamily alleles, tlr , and tlr . a rapid increase in the human population and its mobility has led to urbanization and subsequent climate and ecological changes, catering to emerging infectious diseases that galvanize an implacable threat to human health around the world ( ) . the human race has encountered multiple bacterial and viral pathogens, some being inconsequential while others causing global chaos. interestingly, before the twenty-first century, human coronaviruses were thought to be trivially harmful, causing only common cold in healthy individuals ( ) . coronaviruses have an enveloped positive-sense rna genome comprising about - kilobases. they have been identified in multiple mammalian hosts, including dogs, cats, bats, camels, pigs, and civets ( ) . according to centers for disease control and prevention (cdc), common human infecting coronaviruses include e coronavirus, nl coronavirus, oc beta coronavirus, hku coronavirus, mers-cov, sars-cov, and the recently emerged deadly coronavirus disease . the first four account for - % of upper respiratory tract infections in human adults. while the latter three have emerged as perpetual challenge for the scientific community. in november , an outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in guangdong, china, led to the deaths of around out of ∼ , infected individuals from different countries ( ) . common symptoms in sarsinfected individuals were documented as cough, fever, dyspnea, and occasional diarrhea. although sequence analysis of the virus depicted that bats were its hosts, human-to-human transmission was also observed ( , ) . likewise, in , the emergence of middle east respiratory syndrome coronavirus (mers-cov) was reported in saudi arabia ( ) . the symptoms included atypical pneumonia along with gastrointestinal problems and kidney failure. as a result, out of , reported cases, patients have died to date as of november (world health organization report). in december , covid- was initially encountered in wuhan, china, and has now rapidly spread to multiple countries. the affected individuals exhibit mild symptoms that turn into pneumonia as the illness progresses ( ) . according to nature news, as of february th, this virus is responsible for infecting about , humans in china, leading to the death of patients. the majority of the cases tend to have some connection to the seafood and animal market, which indicates the virus is zoologically transmitted. this situation has gained the attention of authorities at both a local and state level and has highlighted an urgent need to devise a method for rapid treatment of the deadly pathogen ( , ) . recent research has established that the rna genome of recently discovered sars-cov comprises of , amino acids. it features two untranslated regions at both flanking ends while only a single polyprotein encoding open reading frame is present between them. the genome is organized in a sequential manner starting from ' replicase, and it is followed by structural proteins: the spike, envelope, and nucleocapsid at the n terminal ( ) . reportedly, the spike protein acts as multifunctional molecular machinery to mediate viral entry into host cells and is involved in viral transmission. initially, it binds the host cell-surface receptor via the s subunit domain and afterwards carries out the fusion of host and viral cell membranes with the help of the s domain. a wide variety of host receptors can be recognized by two subsequent domains in s region of sars-cov- , leading to viral attachment. the n-terminal peptide domain (ranges from amino acid - in the sequence) as well as the c-terminal peptide domain (the receptor binding domain ranging from amino acid number to ) of the s zone have the ability to bind host cell receptors. it has been suggested that sars-cov- exploits angiotensin-converting enzyme (ace ) as a cell receptor ( , , ) . outbreaks of infectious disease like covid- poses a serious challenge to the scientific community since they usually arise from unrecognized zoonotic sources or due to scarcity data. viruses can emerge by evolving from their animal-restricted form to another form that can infect humans by attainment of their receptors and biosynthetic machinery. a majority of the recently emerging pathogens are difficult to treat due to the lack of specific therapeutic options ( ) . so far, no therapeutic vaccine for either sars-cov, mers-cov, or sars-cov- currently exists in the market, although some clinical trials are in progress ( ) . innovative computational biology approaches have enabled us to obtain immunogenic and highly conserved epitopes from bacterial and viral antigens ( ) ( ) ( ) ( ) . both cd + and cd + epitopes can be used separately or in combination to construct broad spectrum vaccine candidates. the proposed vaccines can combat a wide variety of pathogens and possess the ability to elicit cellular and humoral responses in human hosts. once administered, the mock epitopes from the vaccine are presented by mhc. the presented epitopes are recognized by their corresponding t-cell receptors that proliferates and generates suitable immune responses. considering this, tcell epitopes from deadly pathogens can facilitate t-cell-based vaccine development (cd + and cd +) . more precisely, a cd + -based subunit vaccine usually deals with exogenous antigens that are phagocytosed by apcs and subsequently bind to mhc-ii, which presents them to cd + t cells. accordingly, a cd + -based t-cell vaccine encompasses endogenous antigens that are degraded by apcs and later presented via mhc-i to cd + t cells ( , , ) . epitope-based chimeric/subunit vaccines have many advantages when compared to vaccines produced via conventional vaccinology. for instance, they are cheaper to develop, do not require microbial culturing, and can surpass many wet lab experiments, saving time. they are a safer option, as they do not contain the entire pathogen and are highly specific and stable ( ) . nevertheless, due to the presence of mutable hla variants, epitope-based vaccines targeting limited hla alleles usually do not produce the required/equal effect among the human population. hence highly promiscuous epitopes can bind multiple alleles at a time and can ensure the desired immune response among a heterogeneous human population ( ) . the current study focuses on finding promiscuous cd + and cd t + cell epitopes for chimeric covid- vaccine development using a variety of web-based tools. the proposed potential vaccine is then checked for its binding affinity with suitable receptors. the surface glycoprotein sequence of the pneumonia virus discovered at the wuhan seafood market (qhd . from mn . reference genome) was retrieved from ncbi ( ) . to scrutinize required hla binding epitopes, a tepitool from iedb was used ( ) . a set of mhc class i super-types (a * : , a * : ,a * : , a * : ,a * : , b * : , b * : , b * : , b * : , b * : , b * : , and b * : ) were used, and the two highest-scoring epitopes (based on percentile rank and ic values) for each allele were selected. a percentile rank is calculated by the comparison of the peptide's predicted bindingaffinity against a panel of a variety of peptides randomly selected from the swiss-prot. hence, a lower percentile rank numerical value depicts better binders. additionally, all the predicted peptides were checked for their ic value, and those with ic ≤ nm were taken into account. specific immune responses are based on cd + and cd + t cells, and protective vaccines should thus induce specific t-cell responses based on peptides represented by mhc-i and mhc-ii alleles. the rationale behind prioritizing hla binding epitopes is to ensure the specific immune response in infected macrophages. for mhc-ii-binding peptide epitopes, the sevenallele method was used. this selection is based on the median of consensus percentile ranks among the seven commonly encountered dr alleles, namely, hla-drb * : , hla-drb * : , hla-drb * : , hla-drb * : , hla-drb * : , hla-drb * : , and hla-drb * : . epitopes with a median consensus percentile rank ≤ . were designated as good binders. the scrutiny of promiscuous peptide epitopes was established based on the median of the consensus percentile rank of the seven preselected alleles. for b-cell epitope prediction, bepipred . from immune epitope database analysis resource (iedb-ar) was used ( ) . iedb-ar is linked to iedb and offers computational analysis regarding both b and t cell epitope prediction and their subsequent analysis. bepipred . works on the basis of a randomly chosen forest algorithm that has been trained on epitopes acquired from antibody-antigen models obtained from interactive protein structures ( ) . owing to the significance of spike protein, the selected epitopes were manually screened for their presence in this zone. the epitopes were further examined for antigenic potential via vaxijen version . ( ) . a threshold value of . was taken into account. non-antigenic peptides (having vaxijen score < . ) were discarded, while antigenic epitopes (with threshold value > . ) were further prioritized for their immunogenicity. the immune epitope database (iedb) tool for immunogenicity score calculation was used to predict immunogenicity scores for all mhc-i predicted epitopes ( ) . this tool is designed to predict immunogenicity of the peptide based on amino-acid position and properties. immunogenic epitopes were then verified for their presence in iedb database. shortlisted top-scoring epitopes were checked for their binding affinity with each other for determining the final sequence of the chimeric vaccine. the epitopes were analyzed using a haddock web server (guru interface) ( ) . clusters representing two epitopes, which possessed the highest interaction scores, depicting their maximum interaction, were refined by removing the water molecules, which may hinder their interaction, and then having them dock to the third epitope. likewise, evaluation of clusters with three epitopes was done. the refined and the highest-scoring cluster was docked to the fourth epitope to obtain the final sequence. to facilitate the process of vaccine development, a flexible linker ggggs was added between each epitope. this helps to restore protein folding by allowing interaction between different domains ( , ) . additionally, another linker eaaak was added at the n terminal to separate bi-functional domains. designed vaccines were then tested with different epitopes, including truncated ov-asp- protein (residues - ) and beta defensin ( residues long), and constructs having higher antigenicity and that are predicted to produce high antibody titers were added with the multi epitope vaccine construct to the enhance immune response ( ) . three different constructs were designed in this study, one comprising the top-scoring cd and cd epitopes lying in the s domain, while another is formed by taking two epitopes from the s domain and two from the s domain, representing mhc-i and mhc-ii binders. finally, the third one is formed by adding a b-cell epitope to the second one but with a different adjuvant. the final sequences of the chimeric vaccine constructs were screened for its antigenic potential and solubility using antigenpro and solpro ( ) . allertop version . was used to check the probability of the construct to cause an allergic reaction ( ) . sequence of the finalized vaccine candidate in fasta format was given as an input to expasy server, in order to calculate various parameters like molecular weight, theoretical pi, half-life of the protein, instability index, amino acid composition, aliphatic index, and gravy ( ). secondary structures of the vaccine constructs were predicted using pdbsum ( ) . this step was executed to better understand the structures of predicted vaccines. pdbsum is a database that is exclusively designed to show the molecules that build dna or proteins, ligands, and metal ions along with the illustration of graphical representation of their interactions with each other. to generate d structures of the vaccine candidates, dpro was used ( ) . the predicted models were then refined using galaxy refine server ( ) . this server is responsible for subjecting the predicted d model to structural perturbations and subsequent structural relaxations. it generates five different models. all five models for each vaccine construct were screened for gdt-ha, rmsd, and poor rotamers, and the finest predicted models were taken to the next step. the finalized models were further evaluated using errat scores and ramachandran plot analysis for verification. in order to obtain stabilized vaccine constructs, energy minimization was carried out using online yasara server. yasara deals with molecular-dynamics simulations of the given models in solvent, using an exclusive forcefield that has been derived from amber, whose constraints have been improved to minimalize the impairment done to protein structure during the process of energy minimization ( ) . in order to study the binding affinity of the putative vaccine candidates with immune receptors, molecular docking technique was adopted. prioritized vaccine constructs were docked to ace receptor (pdb id: sci), tlr (pdb id: z x), and tlr (pdb id: g a). vaccine , having a b-cell epitope, was also checked for its interaction with a b-cell receptor (bcr) cd (pdb id: kg ). for this protein-protein docking validation process, the haadock server (guru interface and refinement interface) was used ( ) . additionally, to obtain a graphical illustration of the interactions between vaccine and receptor, pdbsum was used ( ) . moreover, in order to verify the binding affinity of our multiepitope peptide vaccines with hla alleles, all our vaccine constructs were docked with class i and class ii superfamily alleles to reveal the interaction of epitopes with mhc alleles when combined as well. hence, for this purpose, class i [hla a * (pdb id u y), hla b * (pdb id mji)] and class ii [hla-drb * (pdb id atf)] were used; they represent broad-spectrum peptide-binding repertoires. population coverage of epitopes was determined using iedb for prioritized epitopes, as it helps to determine the percentage population that can respond to the particular epitope and can elicit an immune response against it. initially, , hla class i epitopes have been predicted within spike glycoprotein of covid- . scrutiny on the basis of percentile rank filtered peptide epitopes. each of them had a considerable binding affinity for the superfamily alleles. all of these epitopes, along with their features and respective binding alleles, are reported in table s . further analysis revealed that predicted epitopes lie within the s domain of the spike protein, eight epitopes lie in the n terminal domain ( - aa), and three epitopes are in the receptor-binding domain ( - aa). vaxijen antigenic score prediction at a threshold of . was used to detect the antigenicity of peptide epitopes. antigenic epitopes tend to trigger a large number of antibody titers to fight the infection. among predicted epitopes of covid- virus, six epitopes showed considerable antigenic potential, including five from the n-terminal domain and one from the receptor binding domain. an immunogenicity analysis was then carried out for further filtration, and, consequently, five epitopes were screened out; one of the epitopes lying within n-terminal domain showed relatively less immunogenicity value. out of these five mhc-i epitopes, two epitopes from s domain with high antigenic and immunogenicity score were further selected for multiepitope vaccine construction. these were gvyfastek and stqdlflpf . gvyfastek is a part of the n-terminal binding domain with antigenicity and immunogenicity scores of . and . , respectively. epitope stqdlflpf also lies within the n-terminal domain and has an antigenicity and immunogenicity score of . and . , respectively. moreover, another hla class i epitope ktsvdctmy from the s domain of the spike proteins was also screened to be potential candidates for multi-epitope vaccine construction. a total of , unique epitopes against seven drb alleles were identified. twenty ( -mer epitopes) epitopes were screened out via filtration on the basis of median percentile rank < ( table s ). the major portion of binding energy between a peptide epitope and mhc class ii receptor molecule is delivered through the basic peptide core, comprising ∼ amino acids in length. nevertheless, the existence of extra amino acids around the basic binding core seems to play a significant role in stable binding even if they do not precisely bind the peptide-bindinggroove of the mhc receptor. the -mer epitopes for binding with mhc-ii are thus usually recommended ( ) . ten of mhc class ii epitopes (three in the receptorbinding area and seven in the n-terminal domain) were found to be a part of the s domain. while considering a total of s epitopes, four were found to be highly antigenic (threshold > . ). among these, three belonged to the nterminal domain of s while was a part of the receptorbinding domain. two epitopes efvfknidgyfkiys and qpyrvvvlsfellha were selected for vaccine construction on the basis of their high antigenic potential. the former belonged to the n-terminal domain with an antigenicity score of . , while the later was a part of the receptorbinding domain and had an antigenicity score of . . an epitope mtktsvdctmyicgd from the s domain was also prioritized and was used along with the s epitope qpyrvvvlsfellha for vaccine construction. to the best of our knowledge, none of the epitopes reported in this study have been previously added to the iedb database. table shows the final epitopes picked for vaccine development. an iedb server was used to identify b cell epitopes. out of these, were found to be antigenic in nature (threshold > . ). they were further checked for their allergenicity, and the highly antigenic epitope, found to be non-allergenic in nature ( ynsasfstfkcygvsptklndlcft ), was picked. this epitope was conjugated with the s and s epitopes along with a beta defensin adjuvant to design the vaccine construct. envelope-affixed spike protein of coronaviruses plays an important role in receptor recognition. several virology studies have been carried out to discover the exact mechanism of receptor binding and subsequent entry into the host cells. the sars-cov- spike protein has been found to be % identical to the sars-cov urbani stains' spike protein and % identical to the bat sarsr-cov zxc and zc spike protein ( ) . the shortlisted epitopes have also been subjected to conservation analysis, hence manifesting cross protection against other species. conservation analysis revealed the high similarity between the prioritized epitopes of the sars-cov- spike protein with mers and sars spike protein epitopes ( table ). all our seven epitopes were found to be a part of at least eight viral sequences present on ncbi, while one of the prioritized epitopes, ktsvdctmy, was found to be % identical in available coronavirus sequences (table s ) . the finalized epitopes in table were examined for their interactive ability with one another using haddock. all possible combinations of epitopes along with a flexible linker ggggs between them were explored. for vaccine , the e s -e s combination had the highest haddock refinement score. the binding affinity of the e s -e s combination with the other two epitopes was determined to find the best combination of three epitopes. e s -e s -e s was thus formed. finally, the vaccine construct obtained after combination analysis was e s -e s -e s -e s (table ) . similarly, for vaccine , e -s -e s was the first combination, and it was followed by e s -e s -e s and e s -e s -e s -e s . each best combinations haddock refinement score vaccine e s -e s − . +/- . e s -e s -e s − . +/- . e s -e s -e s -e s − . +/- . vaccine e s -e s − . +/- . e s -e s -e s − . +/- . e s -e s -e s -e s − . +/- . vaccine e s -e s − . +/- . e s -e s -e s − . +/- . e s -e s -e s -e s − . +/- . e s -e s -e s -e s -e s − . +/- . probable combination lined up for putative vaccine design along with their corresponding haddock scores is present in table . moreover, truncated ov-asp (ivvavtgyncpgg kltalerkkivgqnnkyrsdlingklknrngtymprgk nmleltwdcklessaqrwanqcifghsprqqregvgen vyaywssvsveglkktagtdagkswwsklpklyennpsn nmtwkvagqgvlhftq) was attached to the n terminal of both the putative vaccines using another linker, eaaak. the finalized vaccines together with the linkers and adjuvant were amino acids long. ov-asp- reportedly has ability to activate antigen-processing cells (apcs) which define its good adjuvanticity for a number of vaccines and antigens ( ) . they are thus added in vaccine constructs to improve the efficacy of these new generation subunit vaccines. in order to ensure both cell and humoral mediated responses, a potent b-cell epitope was added to vaccine based on the best docking scores predicting the combination pattern of epitopes. vaccine was created with an order; e s -e s -e s -e s -e s and the corresponding docking score are enlisted in table . for comparison purposes, another adjuvant betadefensin (giintlqkyycrvrggrcavlsclpkeeqigkcstr grkccrrkk) was added to this combination. beta defensin has previously been reported as a potent adjuvant when conjugated with mers-cov antigens ( ) . vaccines containing defensins as adjuvants have been shown, both in vivo and in vitro, to activate the primary innate antiviral immune response and mediate other immunomodulatory activities against a number of viruses, including coronaviruses ( , ) . vaccine , after addition of this adjuvant at the n terminal along with eaaaak and ggggs linkers, consisted of residues. the final combination of epitopes of all three vaccine constructs have been shown in figure . various physiochemical properties were examined for both the constructs. the molecular weight of vaccine is . g/mol while the theoretical pi is . , depicting the basic nature of the peptide construct. the instability index ii showed that the construct is stable with a score of . . the gravy (grand average of hydropathy) index was calculated to be − . , validating the hydrophilic nature of the construct that can form interactions with surrounding water molecules. the aliphatic index . illustrated that the construct is thermostable in nature. vaccine has a molecular weight of . g/mol, and its theoretical pi is . . hence, this construct was also found to be basic in nature. likewise, instability analysis showed that the protein is stable with a score of . . the gravy index testified the hydrophilic nature of this construct as well (− . ). the thermostable nature of the construct was established by the value of aliphatic index, . . the predicted values of antigenicity for both the vaccines were found to be . and . , respectively. this ensured highly antigenic nature of the constructs. similarly, the solubility upon overexpression was predicted to be . and . . furthermore, both vaccine constructs designed in this study were designated as non-allergenic by allergenpro. vaccine has a molecular weight of . g/mol and its theoretical pi is . . therefore, this vaccine construct was also found to be basic in nature. the gravy index testified the hydrophilic nature of this construct as well (− . ). the thermostable nature of the construct was established by the value of aliphatic index, . . the predicted values of antigenicity for this particular the vaccine was . . this ensured highly antigenic nature of the construct. similarly, the solubility upon overexpression was predicted to be . . furthermore, like both the previous vaccine constructs designed in this study, this vaccine was also found to be non-allergenic by allergenpro. the secondary structure of vaccine includes six helices, beta turns, seven gamma turns, and nine helix-helix interactions. the secondary structure of vaccine has eight helices, beta turns, gamma turns, and nine helix-helix interactions. for vaccine , secondary structure consisted of two beta strand, one hairpin, one sheet, four helices, beta turns, gamma turns, and one helix-helix interaction. helix-helix interaction presents facts about different pairs of helices, interacting with each other with the vicinity of the protein structure, whereas beta turns depict frontiers in immunology | www.frontiersin.org four consecutive residues. these four residues are represented by i, i + , i + , and i + . this is possible when the measured distance between the alpha carbon atom of the first residue (i) and alpha carbon atom of the fourth residue (i + ) is < Å plus the two residues between them are not helical. a gamma turn comprises of three residues i, i + , and i + . this is possible when a hydrogen bond is present between the two residues (i.e., i and i + ). moreover, the phi angle and the psi angle of the second residue i.e., i + lies within a range of degrees in one of the next two cases: ( ) classic [phi i + ( ), psi i + (− )] or ( ) inverse [phi i + (− ), psi i + (− )]. the dpro tool, which works on the basis of ab initio method for predicting tertiary structure, was used to predict three dimensional structures of proposed vaccine constructs. this strategy was adopted due to the lack of fine homolog proteins that could be exploited for homology modeling. the obtained models were then refined via several structure perturbations and subsequent structure relaxations using glaxyrefine server. the obtained best models are shown in figure . the errat score for d models of three vaccines were calculated as . , . , and . , respectively. while ramachandran plot analysis showed . % residues in favored region for vaccine , . % residues in the favored region for vaccine and . % for vaccine (figure ) . these analyses authenticated the reliability and stability of the predicted structures. energy minimization by a yasara server was performed. for vaccine , the yasara force field was applied to , atoms. a total of , water molecules were found. the initial energy was − . kj/mol (z score − . ), which was minimized to − . kj/mol (− . ). for vaccine , the yasara force field was applied to , atoms while the water molecules were , . initial energy was − . kj/mol (z score − . ); however, the final energy was . kj/mol (z score − . ). for vaccine , the yasara force field was applied to , atoms while the water molecules were , . initial energy was − . kj/mol (z score − . ); however, the final energy was − . kj/mol (z score − . ). sars-cov spike protein has been studied previously for its exceptional binding affinity with human ace- . it should be noted that, structurally, sars-cov- and sars-cov spike proteins are highly homologous in nature, sharing . % identical amino acids. atomic level studies between sars-cov and ace- show promising interactions between the two, and therefore, owing to the structural and sequence similarity, it is anticipated that an ace- blocker might be handy in curbing sar-cov- ( ) . for vaccine and the ace- receptor, therefore, docking was carried out. a haddock server clustered probable structures into seven different clusters, which represented a total of . % of the water-refined models. the top-scoring cluster had a score of . +/- . and a z score of − . . similarly, for vaccine and the ace- receptor, haddock clustered structures in three clusters, which represented . % of the water-refined models. the topscoring cluster had a value of . +/- . and a z score of − . . likewise, for vaccine , haddock clustered structures into five clusters, which depicted % of the water refined models generated by haddock. here, the best cluster had a score of . +/- . and a z score of − . (figure ) . tlr and tlr are well-studied toll-like receptors that identify both structural and non-structural proteins of the virus and subsequent cytokine production and inflammation. they are present on the surface of cells and are triggered by viral glycoproteins. tlr agonists have the potential to initiate an immune response and actively participate in viral clearance ( ) . the prioritized vaccine constructs were therefore also explored for their interaction with toll-like receptors tlr and tlr . vaccine and tlr interaction revealed structures in a total of six clusters that represented . % of the water-refined models. the model with the highest score, − . +/- . had a z value of − . . likewise, for vaccine and tlr , haddock clustered structures in clusters, which represented . % of the water-refined models. here, the highest-scoring model had a score of − . +/- . with a z-value of − . . for vaccine and tlr , haddock clustered structures in clusters, which represented . % of the water-refined models. the highest scoring model had a score of − . +/- . with a z-value of − . . moreover, haddock clustered structures in clusters to determine vaccine and tlr interaction, which represented . % of the water-refined models. the top-scoring model had a score of . +/- . and a z-value of − . , whereas the interaction of vaccine and tlr is determined by structures in nine cluster(s), which represents . % of the water-refined models. the top-scoring model had a score of − . +/- . (z-value − . ). similarly, haddock clustered structures in eight clusters to determine vaccine and tlr interaction, which represented . % of the water-refined models. the top-scoring model had a score of . +/- . and a z-value of − . . models from top clusters were refined using haddock refinement interface. this server was used to cluster structures, obtained via haddock, into one cluster. this final cluster symbolized % of water-refined models that were generated by haddock. the statistics observed in interactions of vaccine , vaccine , and vaccine from their refined clusters can be seen in table , and complexes are shown in figure . the pdbsum analysis of vaccine with ace showed hydrogen bonds and one salt bridge. additionally, interface residues of vaccine , representing an interface area of , (a ), were found while the corresponding ace had interface residues covering an area of , (a ). for vaccine and ace , there were two salt bridges and seven hydrogen bonds predicted by pdbsum. additionally, and residues from vaccine and ace interacted with each other covering an area of , and , , respectively. likewise, for vaccine there was one salt bridge and hydrogen bonds predicted by pdbsum. additionally, and residues from vaccine and ace interacted with each other, covering an area of , and , , respectively. an interaction analysis of vaccine with the tlr interacting complex via pdbsum exhibited hydrogen bonds and one salt bridge. furthermore, interface residues of vaccine , representing an interface area of , (a ), were found while a corresponding tlr had interface residues encompassing an area of , (a ). for vaccine and tlr , there were two salt bridges and hydrogen bonds predicted by pdbsum. additionally, and residues from vaccine and tlr interacted with each other, covering an area of , and , , respectively. lastly, for vaccine and tlr pdbsum, hydrogen bonds and five salt bridges were found. furthermore, interface residues of vaccine , representing an interface area of , (a ), were found while corresponding a tlr had interface residues, encompassing an area of , (a ). similarly, the interaction of vaccine with tlr exhibited eight hydrogen bonds and interface residues of vaccine , representing an interface area of , (a ) while a corresponding tlr had interface residues, encompassing an area of , (a ). for vaccine and tlr , there were three salt bridges and hydrogen bonds predicted by pdbsum. additionally, and residues from vaccine and tlr here, a lower haddock score indicates the higher strength of interaction between the proteins. z-score of all docking complexes came out to be . interacted with each other, covering an area of , and , , respectively. in case of vaccine and tlr , hydrogen bonds and three salt bridges were found, while interface residues of vaccine represented an interface area of , (a ) and a corresponding tlr had interface residues, encompassing an area of , (a ). for the interaction analysis of vaccine and bcr (cd ), the haddock server clustered probable structures into different clusters, which represented a total of % of the water-refined models. the top scoring cluster had a score of − . +/- . and a z score of − . . models from top clusters were refined using haddock refinement interface. this server was used to cluster structures, obtained via haddock, into one cluster. this final cluster symbolized % of waterrefined models that were generated by haddock. the statistics observed in interactions of vaccine and bcr from its particular refined clusters can be seen in table . pdbsum analysis showed that and residues from vaccine and bcr interacted with each other covering an interface area (a ) of , and , , respectively. they formed one salt bridge and hydrogen bonds. for interaction analysis of vaccine and hla a allele, the haddock server clustered probable structures into different clusters, which represented a total of . % of the waterrefined models. the top scoring cluster had a score of − . +/- . and a z score of − . . similarly, for vaccine and the hla a allele, haddock clustered structures in clusters, which represented . % of the water-refined models. the top scoring cluster had a value − . +/- . and a z score of − . . likewise, for vaccine haddock clustered structures into three clusters, which depicted . % of the water refined models generated by haddock. here the best cluster had a score of − . +/- . and a z score of − . . for vaccine and hla b allele, probable structures were clustered by haddock into different clusters, which represented a total of . % of the water-refined models. the top scoring cluster had a score of − . +/- . and a z score of − . . similarly, for vaccine and the hla b allele, haddock clustered structures into nine clusters, which represented % of the water-refined models. the top scoring cluster had score of − . +/- . and z score of − . . likewise, for vaccine haddock clustered structures into clusters, which depicted % of the water refined models were generated. here, the best cluster had a score of − . +/- . and a z score of − . . furthermore, for vaccine and the hla drb allele docking, the haddock server clustered probable structures into different clusters, which represented a total of . % of the waterrefined models. the top-scoring cluster had a score of − . +/- . and a z score of − . . similarly, for vaccine and hla drb allele, haddock clustered structures in clusters, which represented % of the water-refined models. the topscoring cluster had score of − . +/- . and z score of − . . likewise, for vaccine , haddock clustered structures into clusters, which depicted . % of the water refined models generated by haddock. here the best cluster had a score of − . +/- . and a z score of − . . models from top clusters were refined using haddock refinement interface. this server was used to cluster structures, obtained via haddock, into one cluster. this final cluster symbolized % of waterrefined models that were generated by haddock. the statistics observed in interactions of vaccine , vaccine , and vaccine from their particular refined clusters can be seen in table s . epitope population coverage was checked by iedb population coverage tool. resultantly, all epitopes had a combined class i and class two average coverage score of %. this step was performed by using the entire world population datasets and the mhc restricted alleles used in this case were (a * : , coronavirus can reportedly spread from person to person via droplet transmission. however, there is currently no available fda-approved vaccine against covid- ( , ) . a vaccination regime, if successfully developed against covid- , has the ability to improve global human health statistics. the advent of immuno-informatics approaches has revolutionized the area of vaccine development. antibody response as well as cell mediated immunity can be established by using proper protein antigens ( ) . notably, the natural infections elicit a minimal immune response that can be enhanced by developing epitope-based vaccines. therefore, rational selections are done to separate the constituents required for the desired immune response. efforts to identify suitable tcell epitopes as well as the design of effective strategies in order to deliver those epitopes are under consideration. the benefits of epitope-based vaccine construction includes improved safety levels, time saving, and, additionally it can provide the opportunity to specifically attach/engineer combinations of epitopes for augmented potency. this also facilitates to emphasize the required immune responses on antigenic/ conserved epitopes ( ) . spike proteins of coronaviruses are responsible for selection and entry into the target cells. any therapeutic approach to target the spike protein can prove to be fruitful to curb the deadly pathogen. moreover, it has been reported that like sars-cov, sars-cov- uses the ace human receptor to bind and enter the cells ( ) . peptides that potentially interact with the functional domain of the coronavirus spike protein, can be designated as viral entry inhibitors. in our study, the chosen cd + and cd + t cell epitopes are predicted to be antigenic and immunogenic, and they can thus play a vital role in viral clearance mechanisms. to further validate the authenticity of our proposed vaccines, more detailed docking analysis and experimentation has to be performed. nevertheless, it might take months to years to actually derive a vaccine against covid- , we believe that our contribution in this case might be a useful to initiate the process. for vaccine , four epitopes from the s domain were picked. the s domain, which comprises of amino acids from to , is further divided into the n-terminal domain and receptor-binding domain. analysis showed that three of our chosen epitopes lied in the n-terminal domain of the s protein while one " qpyrvvvlsfellha " was a part of receptorbinding domain ( - ). viral infections are prompted by the interaction of the spike-protein with the receptor, present on the surface of the target cell. this process is mediated by the receptor binding portion of the s domain. hence, it plays a significant role in the attachment, and subsequent fusion and entry of the virus into the host cell. hence this particular portion can be targeted for designing antiviral agents ( ) . vaccine is comprised of a combination of strong and weak epitopes. it had two epitopes (mhc-i and mhc-ii) that were found to be the best epitopes for s domain. regardless of the fact that efvfknidgyfkiys had a higher antigenicity score compared to qpyrvvvlsfellha ( . and . ), the latter was used in vaccine construction due to its presence in receptor binding domain. additionally, another experimental strategy was applied; comparatively weak epitopes from s were selected and their binding affinity was checked. docking with tlrs and ace showed that they bind effectively; from mtktsvdctmyicgd , thr , val , lys , thr , and ser bound to tlr ; lys had affinity for ace receptor. the other s epitope ktsvdctmy completely overlapped with mtktsvdctmyicgd . vaccine is a modified form of vaccine with an additional mer b-cell epitope ynsasfstfkcygvsptklndlcft integrated. the whole idea to include b-cell epitopes along with was to ensure both cellular and humoral defense responses ( ) . b-cell epitopes are precisely amino acids clusters present at the cell surfaces that are identified by certain antibodies or bcell receptors, that in turn elicit cellular or hormonal immune response ( ) . antibodies released by b-cells can neutralize toxins and thus label them for destruction ( , ) . in this case, in addition to considerable interactions with tlrs and hla superfamily alleles, notable interactions were observed between cys and phe from b cell epitope and arg and glu from bcr, respectively. designed vaccines have been tested against different receptors to identify their potential to induce immune response within the host. results revealed that proposed vaccines are likely to be presented by mhc-i and mhc-ii, as that was the prime objective of this study. also, they may interact with human tlr and tlr to induce innate immune response, as these receptors have been revealed to play a key role in the induction of immune responses ( ) . moreover, the spike protein of sars-cov has been reported to play a significant role in the induction of neutralizing-antibodies and t-cell responses as well as protective immunity during the infection ( ) . therefore, keeping in view the importance of spike proteins in immunity, we applied this predictive framework to identify potential vaccine candidates in spike protein of sars-cov- against its potential host receptor ace as well as against tlr and tlr . recent studies have strongly suggested that covid- uses angiotensin-converting enzyme (ace ) as its potential receptor. several critical residues in covid- receptor binding motif (rbm) of s domain particularly gln provide favorable interactions with human ace ( ) . thus, it has been proposed in several studies that spike-protein-based vaccines can be potential therapeutic targets against sars-cov- , as they may block the viral interaction with ace and may thus prevent the downregulation of ace and ultimately the pulmonary vascular permeability ( ) . vaccines designed in this study may also interact with ace resulting interrupted interaction of the receptor with the viral spike protein and thus can be a potential therapeutic target against covid- . the overall effect of all these interactions within the host is still unknown and requires further experimental studies for their clear role in the immune regulation and virus clearance. concisely, we have combined several immuno-informatics tools to propose a set of potentially antigenic and immunogenic peptide epitopes that can facilitate vaccine design. the predicted vaccine constructs consist of distant epitopes. the authenticity of these constructs must be validated via further experimentation. however, further experimental authentication is required to verify this study. we anticipate promising outcomes from the predicted peptide epitopes to curb the deadly covid- pandemic. all datasets presented in this study are included in the article/supplementary material. an, fs, and fa conceptualized the study, validated the study, and wrote the original draft. fs performed the data curation and developed the software. an and fs performed the formal analysis, methodology, and visualized the study. tb, aa, and am performed the funding acquisition. an performed the investigation and supervised the study. an and am performed the project administration. an, tb, fa, aa, and am wrote, reviewed, and edited the manuscript. all authors contributed to the article and approved the submitted version. does urbanization make emergence of zoonosis more likely? evidence, myths and gaps coronavirus infections-more than just the common cold genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding can we contain the covid- outbreak with the same measures as for sars? severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia first case of novel coronavirus in the united states return of the coronavirus: -ncov a novel coronavirus from patients with pneumonia in china genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan the novel coronavirus ( -ncov) uses the sars-coronavirus receptor ace and the cellular protease tmprss for entry into target cells fusion mechanism of -ncov and fusion inhibitors targeting hr domain in spike protein coronaviruses: important emerging human pathogens emerging threats from zoonotic coronaviruses-from sars and mers to -ncov in silico design of a dnabased hiv- multi-epitope vaccine for chinese populations prediction of promiscuous t-cell epitopes in the zika virus polyprotein: an in silico approach t-cell epitope vaccine design by immunoinformatics identification of putative vaccine candidates against helicobacter pylori exploiting exoproteome and secretome: a reverse vaccinology based approach redirecting soluble antigen for mhc class i cross presentation during phagocytosis a bioinformatics tool for epitope-based vaccine design that accounts for human ethnic diversity: application to emerging infectious diseases database resources of the national center for biotechnology information tepitool: a pipeline for computational prediction of t cell epitope candidates iedb-ar: immune epitope database-analysis resource in bepipred- . : improving sequence-based b-cell epitope prediction using conformational epitopes vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines the immune epitope database (iedb) . haddock: a proteinprotein docking approach based on biochemical or biophysical information design of novel multi-epitope vaccines against severe acute respiratory syndrome validated through multistage molecular interaction and dynamics a truncated fragment of ov-asp- consisting of the core pathogenesis-related- (pr- ) domain maintains adjuvanticity as the full-length protein scratch: a protein structure and structural feature prediction server allertop v. -a server for in silico prediction of allergens protein identification and analysis tools on the expasy server. the proteomics protocols handbook pdbsum: a web-based database of summaries and analyses of all pdb structures galaxy: a platform for interactive large-scale genome analysis yasara: a tool to obtain structural guidance in biocatalytic investigations structure, function, and antigenicity of the sars-cov- spike glycoprotein human β-defensin plays a regulatory role in innate antiviral immunity and is capable of potentiating the induction of antigen-specific immunity towards the application of human defensins as antivirals angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target toll-like receptors in antiviral innate immunity clinical features of patients infected with novel coronavirus in wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia genome-based approaches to develop vaccines against bacterial pathogens epitope-based vaccines: an update on epitope identification, vaccine design and delivery receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets for development of antiviral vaccines in silico screening of antigenic bcell derived t-cell epitopes and designing of a multi-epitope peptide vaccine for acinetobacter nosocomialis current progress of immunoinformatics approach harnessed for cellular-and antibody-dependent vaccine design fundamentals and methods for t-and b-cell epitope prediction toll-like receptors: the swiss army knife of immunity and vaccine development the spike protein of sars-cov-a target for vaccine and therapeutic development receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars a review of sars-cov- and the ongoing clinical trials we want to acknowledge institute of molecular biology and biotechnology (imbb) at the university of lahore for providing the facilities and confidence to publish this article. we also want to acknowledge atta-ur-rehman school of applied biosciences (asab) at the national university of sciences and technology for collaboration and support. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © naz, shahid, butt, awan, ali and malik. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - qlk y h authors: friedrich, brian m.; trefry, john c.; biggins, julia e.; hensley, lisa e.; honko, anna n.; smith, darci r.; olinger, gene g. title: potential vaccines and post-exposure treatments for filovirus infections date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qlk y h viruses of the family filoviridae represent significant health risks as emerging infectious diseases as well as potentially engineered biothreats. while many research efforts have been published offering possibilities toward the mitigation of filoviral infection, there remain no sanctioned therapeutic or vaccine strategies. current progress in the development of filovirus therapeutics and vaccines is outlined herein with respect to their current level of testing, evaluation, and proximity toward human implementation, specifically with regard to human clinical trials, nonhuman primate studies, small animal studies, and in vitro development. contemporary methods of supportive care and previous treatment approaches for human patients are also discussed. the family filoviridae includes two accepted genera, ebolavirus and marburgvirus. the genus ebolavirus includes five species (each represented by a single virus): zaire ebolavirus (ebola virus, ebov), sudan ebolavirus (sudan virus, sudv), reston ebolavirus (reston virus, restv), taï forest ebolavirus (tai forest virus, tafv), and bundibugyo ebolavirus (bundibugyo virus, bdbv). the genus marburgvirus includes a single species, marburg marburgvirus, which has two members: marburg virus (marv) and ravn virus (ravv) [ , ] . in , the first cases of filoviral infection were documented in three simultaneous outbreaks in west germany and yugoslavia. the virus responsible for the outbreaks was named "marburg virus" after the german city of marburg in which it was first recognized [ , ] . from documented instances of infection, it seems that the members of the filovirus genera may exist in quite opposite climates of africa with marburgvirus infections occurring more frequently in the dry woodlands, while ebolavirus infections occur more frequently in rain forests [ ] . more than years of effort have focused on the search for the reservoir of these viruses in africa, and while the search is still ongoing, recent evidence indicates that bats may be reservoirs for both marburgviruses and ebolaviruses [ , [ ] [ ] [ ] [ ] [ ] [ ] . however, the recent outbreak of restv in domestic pigs in the philippines demonstrated the potential for animals other than primates and bats to be infected and potentially spread or amplify outbreaks [ ] . filoviruses are named for their long, filamentous shape which can been seen on the order of micrometers in length, while their width is more narrow (usually around nm) with little fluctuation [ ] . contained within this filamentous virus is a single, -kb negative-sense rna genome that encodes seven proteins [ , ] . the seven filoviral proteins are the glycoprotein (gp), the polymerase (l), the nucleoprotein (np), a secondary matrix protein (vp ), the transcriptional activator (vp ), the polymerase cofactor (vp ), and the matrix protein (vp ) [ , ] . homotrimers of the viral gp cover the surface of the virion, and this viral gp is believed to be the sole host attachment factor for filoviruses [ , ] . candidates for filoviral receptor and co-factors include transferrin, dc-sign, tim- , and npc [ , [ ] [ ] [ ] [ ] . after entry, filoviruses replicate their genomes and viral proteins in the cytoplasm using a rna-dependent rna-polymerase which is carried in with the virus. wild-type filovirus infection has been associated with severe case fatality rates in humans, as high as % [ ] . in humans, filovirus infection is characterized by an abrupt onset of flu-like illness, after an initial incubation period of - days. following this initial illness, signs and symptoms of disease include anorexia, nausea, vomiting, chest pain, cough, edema, postural hypotension, neurologic complications, petechiae, and mucosal hemorrhage. there have also been several observed wild-type filovirus outbreaks among great apes in africa that have demonstrated similarly high mortality rates [ ] . in an effort to create cost and time-effective models of filoviral disease for the development of vaccines and therapeutics, small animals, such as mice and guinea pigs, are often used. however, these animals usually demonstrate significant resistance to wild-type filovirus infection, and only demonstrate mortality rates similar to primates when the filovirus in question has been adapted to the model species [ ] . due to the difficulties in evaluating wild-type filovirus infection in small animals and the generally high level of immune protection correlates derived from non-human primate (nhp) models of infection, therapeutics and vaccines are ultimately evaluated in nhp species for efficacy against filovirus. of the nhp models available for filovirus study, rhesus and cynomolgus macaques have been the most highly characterized and utilized for therapeutic and vaccine development, respectively. however, the starting point of vaccine and therapeutic development remains small animal models due to the cost, ethical, and time-associated benefits [ ] . this review will highlight the current research into filovirus vaccines and therapeutics. the current clinical standard for filoviral infection is supportive care as there are currently no fda-approved treatment strategies. supportive care consists of oral fluid rehydration, oral medication, nutritional supplementation, and psychosocial support [ ] . nasogastric feeding tubes and i.v. administration of both fluids and medication are increasingly considered supportive care where possible during outbreak scenarios to prevent dehydration and facilitate support of blood pressure [ , ] . however, given the limited equipment and laboratory support during outbreaks, care must be taken to prevent overaggressive fluid administration [ ] . fluid replacement was evaluated briefly in rhesus macaques, and while there was no significant benefit to survival, a less severe renal compromise was observed [ ] . while supportive care may (or may not) reduce the overall case fatality rate in humans, the true impact of simple interventions such as fluid management has yet to be fully evaluated and the potential for benefit in combination with direct antiviral measures has yet to be assessed [ ] . treatment of filovirus infection with passive transfer of antibodies is an attractive therapy. while there have been conflicting results in vitro, in animals, and in humans, recent breakthroughs have solidified the potential for this strategy of intervention. in addition, there have been a number of immunotherapies developed for other agents, such as respiratory syncytial virus (rsv), which can provide potential roadmaps or precedence to facilitate the advancement of these products through the necessary regulatory hurdles [ , ] . transfer of immune serum for the treatment of filovirus infection in humans has previously been attempted. however, interpretation of these results has been cautious due to the study conditions as well as the uncertainty of the disease stage at which the individuals were treated [ ] . as a result, much attention has focused on animal studies evaluating candidate products. while many of the early studies in mice and guinea pigs were successful, these successes did not translate to nhp studies and tempered the enthusiasm for further evaluation of candidate products. [ , [ ] [ ] [ ] [ ] . when similar passive transfer strategies were attempted in nhps, viremia onset and outward signs of disease were reduced, but the treatment did not affect survivorship [ ] [ ] [ ] . in addition, the suggestion that antibodies could enhance filovirus infections in vitro caused further concern [ ] [ ] [ ] . however, recently a series of experiments have made researchers, developers, and funding agencies reconsider the potential of this category of products for filoviruses. both polyclonal and monoclonal passive therapies have been shown to be efficacious in rodents for filovirus infection [ ] [ ] [ ] . furthermore, evidence of enhancing antibodies exists in the antibody response to ebov [ ] . more recent studies have demonstrated protection in macaques with polyclonal and monoclonal passive therapy [ ] [ ] [ ] . these sources of monoclonal antibodies have ranged from murine monoclonal antibodies to recombinant-derived cloned human monoclonal antibodies from survivors of filovirus infection [ , ] . development of new antibodies to be used for post-exposure treatment is on-going. in one study, an antibody ( f ) targeting the ebov gp mucin-like domain was generated and subsequently shown to protect % of mice against a lethal ebov challenge when given days post-exposure [ ] . this antibody was then modified to generate h- f , a human recombinant antibody. this human recombinant antibody also significantly protected mice against a lethal challenge of ebov [ ] . in another method, a recombinant vsvΔg/ebovgp was used to generate a total of monoclonal antibodies which were subsequently characterized. all monoclonal antibodies improved survival rate of mice ( %- %) against a high-dose lethal challenge by mouse-adapted ebov [ ] . another antibody, kz , was isolated from the bone marrow of a human survivor of ebov infection and is specific for the complex of gp and gp [ ] . kz neutralized ebov in vitro and offered protection from lethal ebov challenge in a rodent model [ ] , but was non-protective in nhps [ ] . table . . . dna vaccines the first clinical trials involving filovirus vaccines were based off of plasmids expressing ebov np and gp as well as sudv gp [ ] . this strategy proved safe in subjects involved with phase i testing. however, the prime/boost dna vaccine strategy covering four separate plasmid doses administered three times each was ineffective at creating durable immunity as evidenced by the near non-existent antibody titer in these subjects after year [ ] . while clinical trials with this vaccine have halted, it may be possible that this strategy can supplement another vaccine technology in a prime/boost capacity. while no vaccines or therapeutics are currently licensed for use by the fda, phase i clinical trial safety tests have been performed on one particular platform for an ebov vaccine. this vaccine is based on a replication deficient, recombinant adenovirus serotype (rad ) vector genetically engineered to carry the genes for ebov glycoprotein (gp (z)) and sudv glycoprotein (gp (s/g)). as a common human pathogen, this vector vaccine utilized the broad-tropism of the adenovirus vector to infect cells and once inside the inserted ebolavirus glycoproteins are expressed. upon expression of these inserted ebolavirus genes, the host immune system will recognize them as foreign and mount a response against them. the advancement of this vaccine technology to phase i trials manifested from its ability to provide % protection among cynomolgus macaques vaccinated - days prior to challenge and its ability to generate potent humoral and cell-mediated immune responses [ , ] . in the clinical trial participants remained asymptomatic after a single vaccination with either × or × viral particles [ ] . furthermore, for both doses of the vaccine significant antibody titers were observed at weeks post-vaccination with % and % of participants receiving × viral particles being positive for gp (s/g) and gp (z), respectively. significant antibody titers were observed again at weeks post-vaccination and, while decreased from weeks, demonstrated the potential durability of this vaccine over time [ ] . t-cell activation was also examined for these individuals and found to directly correlate with the dose administered, but to a lesser extent than the previously mentioned antibody response. cd + activation was observed with greater frequency than cd + activation in those receiving the vaccine. importantly, these results were obtained in the context of significant pre-existing immunity to ad as pre-entry evaluation of antibody titers revealed that % of participants were positive against ad , showing that pre-existing immunity to ad still resulted in protection against ebov [ ] . while this study shows great promise, further development and additional studies in nhps and humans are needed. table . in addition to the adenovirus platform in clinical trials, additional variations of the rad vaccine are also in development and have been evaluated in nhp models. based on the adenovirus vector platform, the complex adenovirus (cadvax) technology substantially increased the genetic payload capacity of the vector, up to kb. additionally, this strategy involved the blending of four separate vectors expressing the glycoproteins of ebov, sudv, and marv along with the nucleoproteins of ebov and marv. when administered in a prime/boost strategy, this technology offered % protection against ebov, sudv, and marv [ ] . another variation of the cadvax system designed to express modified ebov glycoprotein and sudv glycoprotein was effective in protecting against both parenteral and aerosol challenge when administered in a prime/boost strategy [ ] . both implementations of the cadvax technology demonstrated significant antibody titers. further improvement upon the adenovirus-based ebov vaccine technology is ongoing. richardson et al. reformatted the genetic insert for the vector which included the addition of a cytomegalovirus (cmv)-chicken β-actin hybrid promoter, optimized codons and a consensus kozak sequence [ ] . these improvements led to three-to seven-fold increases in ebov glycoprotein expression. neutralizing antibody titers were found at doses as low as infectious forming units (ifu) with comparable titers requiring ifu of the unmodified vaccine in mice. these modifications demonstrated % protection of mice at doses two orders of magnitude lower than the unmodified vaccine. interestingly, at min post-challenge, the modified ad-cmvzgp/ad-cagoptzgp offered % protection compared to the % protection of mice offered from the original vaccine [ ] . this vector format has also recently showed promise when administered sublingually in mice, therefore eliminating the complexities of parenteral administration such as the necessity for sterile tools, aseptic chemicals, and the risks of potential blood-borne pathogen exposure [ ] . while this adenovirus vector vaccine technology is promising, demonstrations that pre-existing immunity to the ad vector depressed the desired immune response may impede its implementation. in efforts to circumvent issues of pre-existing immunity to ad , geisbert et al. sought out a less prevalent serovariation [ ] . in their study, a heterologous prime/boost strategy with recombinant adenovirus serotypes and carrying gp (z) and gp (s/g) demonstrated complete protection among nhps. each of these vectors was capable of stimulating humoral and cell-mediated immune responses in the context of nhps pre-vaccinated with rad as evidenced by antibody titers reaching an order of magnitude above those achieved in rad vaccinated subjects ( : , compared to : , ), and cd + intracellular cytokine staining was . -fold greater among heterologous prime/boosted subjects ( . % compared to . %) [ ] . rhabdoviruses have recently offered unique vaccine platforms to generate both genus/species specific immunity as well as potential for cross-protective immunity for filoviruses. for example, based on an attenuated recombinant vesicular stomatitis virus (rvsv), the replication-competent virus expresses the glycoprotein of the target filovirus in place of its wild-type membrane glycoprotein. as this virus is primarily an agricultural pathogen, pre-existing immunity interfering with the desired immune response and subsequent protection is unlikely [ ] . several studies have begun to address the safety of the filovirus vsv platforms. evaluation of this platform in immunocompromised nhps has suggested that this technology may be safe among similarly immunocompromised humans [ ] . further encouragement for the safety of this live-attenuated vaccine came recently from mire et al. who showed that ebov and marv rvsv showed no signs of neurovirulence associated with vsv [ ] . the utility of the vsv-based vaccine for protection against filoviral hemorrhagic fever was highlighted by geisbert et al. [ ] . using a blended vaccine consisting of equal amounts of three different vsv vectors each carrying the ebov, sudv or marv glycoprotein, they were able to generate % protection of nhps against challenges with ebov, sudv, tafv, and marv with no observed ill effects from this replication-competent vaccine. of all vaccinated nhps, only one showed signs of viremia as assayed by rt-pcr. each of the vaccinated nhps also demonstrated elevated antibody responses after vaccination, with titers ranging from : to : for all three glycoprotein components of the blended vaccine [ ] . in addition to providing such high levels of protection as a prophylactic vaccine strategy, the vsv-based technology has demonstrated post-exposure protection for both ebov and marv when administered via intramuscular (i.m.) injection [ ] . when marv-rvsv was administered i.m. - min post-challenge with marv, % of nhps survived. in this study viral rna was observed in the blood on day three post-challenge when assayed by rt-pcr, but active virus was unobservable by traditional plaque assay. clinical chemistry results demonstrated that these surviving nhps experienced significant rises in aspartate aminotransferase, gamma-glutamyl transferase, total bilirubin, and blood urea nitrogen indicating that, while protective, the post-exposure treatment did not completely prevent typical pathogenic events associated with marv infection. similar experiments demonstrated sudv-rvsv delivered - min after challenge offered % protection [ ] . post-exposure protection for ebov-rvsv was less effective at - min but still afforded % protection to eight nhps [ ] . as a post-exposure treatment, ebov glycoprotein rvsv was used recently h after a suspected human exposure via needlestick in the laboratory. while there is no direct evidence the laboratory worker was indeed exposed, that person survived the experience with no discernible sequelae from the treatment outside of a transient fever occurring h after an injection of × plaque forming units (pfu) [ ] . also of note for rhabdovirus-based filoviral vaccines was a recent report that generated dual immunity for both ebov and rabies virus infection. ebov gp was efficiently expressed from an attenuated vaccine used for wildlife against rabies virus in place of the wild-type rabies envelope glycoprotein, g [ ] . this vaccine vector was capable of inducing protective immunity to ebov infection as well as to rabies virus infection in both live-attenuated format and β-propriolactone inactivated vaccine. neurovirulence of the recombinant vector was unobserved in suckling mice when compared to the unaltered vaccine [ ] . this versatility offers increased storage options with an inactivated vaccine as well as the opportunity to vaccinate for each disease where they are both endemic. alphavirus particle-based vaccines also provide high levels of protection to nhps against lethal filovirus challenge. these vaccines for ebov, sudv, and marv are composed of venezuelan equine encephalitis virus (veev)-based replicon particles (vrps) that express the viral glycoprotein of interest [ ] . replicon particles are replication-defective, single-cycle vectors which cannot spread from cell-to-cell. the vrps are composed of an attenuated veev replicon that contains veev non-structural genes and the filovirus glycoprotein. vrps are generated when the replicon is co-transfected into cells with helper plasmids containing the veev structural genes. the resulting single-cycle propagation defective particles are then administered to the appropriate animal model for efficacy testing [ , ] . this technology offers several advantages, including high expression levels of heterologous genes, dendritic cell tropism, induction of robust cellular and antibody immune responses, rapid construction into single and multivalent vaccines, and relative resistance to anti-vector immunity [ , ] . in vivo studies with the marv vrp offered the first demonstration of a fully protective filovirus vaccine. nhps exhibited % survival after vaccination with focus forming units (ffu) of vrp in three consecutive doses spaced at day intervals prior to challenge with marv [ ] . this protection was offered when the vrps were constructed to express gp alone or gp + np; however, the nhps vaccinated with np alone all exhibited clinical symptoms of illness and only two out of three survived the challenge. substantial antibody titers were found in each of the vaccinated nhps. additionally, no conspicuous elevations in clinical chemistries were observed in nhps throughout the experiment. experiments performed on mice and guinea pigs supported the ability of vrps expressing gp to mediate complete protection from lethal marv and ebov challenge [ ] . in mice, adoptive transfer of cd + cells, but not cd + cells or passive antibody transfer, from vrp-np-immunized mice was protective, suggesting this vaccine may be most protective by stimulating the host cell-mediated immune response [ ] . additionally, adoptive transfer of cd + t-cells after activation via specific ebov peptides provided mice complete protection indicating a mechanism for vrp-based immunity [ ] . recent studies indicate that a vrp-based vaccine is fully protective in cynomolgus macaques against ebov, sudv, and marv parenteral and aerosol virus challenges (unpublished observations, olinger). paramyxovirus-based vectors for vaccination against filoviral threats have recently demonstrated the capacity to protect nhps from infection and stimulate strong immune responses. paramyxoviruses have a natural tropism for the respiratory tract and, as filoviruses are both emerging diseases and potential weaponized threats, the idea of targeting vaccines to this area is ideal. two candidates for this category of vaccines have been investigated to date: human parainfluenza virus (hpiv- ) and newcastle disease virus (ndv). of these two systems, the hpiv- system has been evaluated in nhp models of ebov infection. combinations of ebov gp alone, ebov gp + np, and ebov gp + human granulocyte macrophage colony stimulating factor (gm-csf) were inserted into the genome of hpiv- . each of these vaccine vectors was used to vaccinate nhps both intranasally (i.n.) and intratracheally (i.t.) as initial studies offered complete protection of guinea pigs vaccinated via the respiratory route [ ] . at least one nhp in all three vaccine groups receiving × tcid (median tissue culture infective dose) of their respective vector displayed clinical signs of illness after challenge during the study. each group held two nhps and out of the three groups only one vaccinated animal from the ebov gp + np succumbed to the disease. immune responses from these subjects, prior to challenge, revealed antibody titers ranging from : to : , [ ] . by manipulating dose and administration strategies, bukreyev et al. were able to achieve complete protection of nhps after two successive doses of × tcid given at day zero and again at day with challenge occurring on day [ ] . the two-dose strategy produced igg titers ranging between : , and : , , much higher than the single dose. each of these experiments highlights the potential of the hpiv- platform for ebov vaccination but the known prevalence of pre-existing immunity to hpiv- in humans could hinder the generation of targeted immunity [ ] . to address these concerns, bukreyev et al. compared the immunogenicity of ebov gp expressing hpiv- vector among naïve and pre-immune nhps [ ] . in these experiments ebov-specific igg levels were substantially decreased among hpiv- pre-immune nhps; however, this hindrance was overcome when the nhps were vaccinated with two doses of recombinant vector which was previously shown to offer complete protection against ebov challenge [ ] . in efforts to diversify the paramyxovirus-based vectors and avoid issues surrounding the pre-existing immunity found for hpiv- , a new vector design based on ndv was established. ndv is an avian paramyxovirus that infects the respiratory tract. this virus has been shown to be highly attenuated in nhps due to natural host restriction processes [ ] . additionally, this vector system has proven successful as a vaccine platform for severe acute respiratory syndrome-associated coronavirus (sars-cov) and influenza h n in nhps [ ] . although this system has yet to be evaluated in the context of nhp models of ebov infection and disease, it was recently shown to be immunogenic in nhps. single vaccination with ndv expressing ebov gp produced lower titers than the hpiv- platform, demonstrating this vector is less immunogenic; however, in a homologous prime/boost vaccination strategy ebov-specific mucosal iga levels reached those similar to the hpiv- homologous prime boost vaccination strategy [ ] . igg specific for ebov did not reach levels comparable to the previous hpiv- platform. these reports support the potential use of paramyxoviruses as vaccine candidates, but further examination of immunostimulatory effects and pre-existing immunity will require investigation. the vlp technology works by generating non-replicating virus particles that do not contain any filoviral genetic material. immune responses generated in response to exposure to vlps are derived from the filoviral protein shell that is the vlp itself. vlps are constructed through the matrix protein vp 's ability to drive the budding process. by simple transfection and expression of vp into target cells, filamentous structures can be generated [ ] . co-expression of additional filoviral proteins, such as gp and np, can dramatically enhance and stabilize the production of vlps from target cells [ ] . when traditional target/production cells were swapped out for insect cells and filoviral protein expression was driven from a baculovirus vector, vlps were generated and found to have filamentous structures resembling wild-type virus. vlps have demonstrated immunogenicity and the ability to protect nhps from lethal challenge. the first such study involving nhps utilized the baculovirusproduced vlps containing ebov gp, np, and vp [ ] . five cynomolgus macaques were vaccinated three times at -day intervals with µg of vlp with ribi adjuvant. after the full vaccination schedule, % survived a lethal ebov challenge, and there was a three-to ten-fold increase in ebov specific antibodies which possessed an % plaque reduction at titers between : and : [ ] . additionally, these antibodies were shown to have both compliment-mediated and antibody-dependent cell-mediated cytotoxic properties [ ] . using this same vlp technology, swenson et al. were able to show a similar effect for marv. three i.m. injections of mg of vlp in . ml of qs- adjuvant spaced days apart offered % protection against marv and ravv; however, one animal challenged with ravv did exhibit slight morbidity [ ] . all animals had no detectable viremia as determined by plaque assay at days , , , , , , and post-exposure. additionally, two injections of the vlps correlated with a peak in homologous antibody titer while three injections correlated to peak heterologous antibody titer [ ] . the ability of vlps to generate protective immunity against filoviral challenge has been demonstrated in guinea pigs as well [ ] . table . immunogenic virus-like particles (vlps) of ebov and marv can be easily generated in mammalian systems. ebov-like particles (vlps) can be efficiently generated in mammalian cells after expression of vp alone, but other filovirus proteins can be co-expressed as well [ ] [ ] [ ] . baculovirus-derived vlps have also been successfully generated in insect cells and stimulated both cellular and antibody immune responses against hepatitis e virus, human papilloma virus, rotavirus, and simian immunodeficiency virus [ ] [ ] [ ] [ ] . several groups have made and tested baculovirus-generated filovirus-vlps as vaccines in small animal models. warfield et al. generated ebola-vlps (evlp) and marburg-vlp (mvlp) containing vp , np, and gp. this vaccine had up to % survival following a lethal infection in mice (vaccine dose-dependent) [ ] . sun et al. produced an evlp containing vp and gp. this vaccine was also up to % effective following a lethal ebov infection in mice (vaccine dose-dependent) [ , ] . many reports document the ability of filoviral gp to act as a potent immunogen, and as such, viral vectors are a popular method of vaccinating through the expression of gp by the host and subsequent immune recognition. however, a recent report sought to utilize gp itself as the vaccination agent. in order to efficiently produce the protein, an expression vector was constructed such that the fc portion of a human igg was fused to ebov-gp [ ] [ ] [ ] . this gp-fc fusion protein induced both cell-mediated and humoral immune responses, and mice vaccinated with zebovgp-fc demonstrated % protection against a lethal ebov challenge. [ ] . while further studies are required, these results show that vaccination with ebovgp-fc alone is effective against a lethal ebov infection, and thus fusion proteins could potentially be used as an effective vaccine against filoviruses [ ] . the use of plants to produce vaccine antigens and antibodies has been demonstrated previously and is attractive for several reasons, including low manufacturing cost, efficient production, minimal risk of contamination with human pathogens or toxins, and the fact that plants have similar secretory pathways and endosomal systems as mammalian cells [ ] [ ] [ ] [ ] . recently, a bean yellow dwarf virus (beydv)-derived replicon system was developed and shown to efficiently produce antibodies against ebov in nicotiana benthamiana leaves [ ] . poolcharoen et al. used this system to develop and fuse ebov-gp to the c-terminus of an igg heavy chain [ ] . these ebov immune complexes (eic) were then used as a vaccine to immunize mice. serum antibody response tests showed that this eic was highly immunogenic in mice and produced antibody levels similar to mice protected from a lethal ebov challenge [ ] . while antibody titers alone do not fully correlated with protection from lethal challenge, there is potential for further development of this vaccine. a replicating vaccine that could spread through the target population after initial inoculation would be an attractive, alternative approach to filovirus development. this approach could provide high coverage with minimal initial vaccinations. a cmv-based vaccine would allow for this type of spread [ ] . cmv, a member of the family betaherpesvirinae, induces a high "effector" memory t-cell (t em ) response before establishing a low-level persistent infection [ ] [ ] [ ] . this high immunogenicity makes cmv a good potential vaccine vector, and the cmv vector has been previously shown to be effective as a vaccine for siv in rhesus macaques [ , ] . tsuda et al. constructed a mouse cmv vector expressing a cd + t cell epitope from the nucleoprotein (np) of ebov (mcmv/ebov-npctl). this vaccine induced high levels of ebov-specific cd + t cells, and subsequently, protected % of mice against a lethal ebov challenge. this shows a proof-ofconcept for cmv as a potential vaccine vector for ebov [ ] . as discussed previously, paramyxovirus-based vectors have demonstrated the capacity to induce strong immune responses and protect animals from infection. as such, a chimeric hpiv was developed where all the hpiv surface markers/receptors were deleted and replaced with ebov-gp [ ] . this chimeric virus can be amplified and recovered easily. additionally, this chimeric virus has -fold greater incorporation of ebov-gp into the virion due to the lack of competition with hpiv surface proteins [ ] . testing in guinea pigs showed that hpiv /Δf-hn/ebogp is highly attenuated, as compared to both hpiv and hpiv /ebogp, and immunogenic, as % developed neutralizing antibodies against ebov. finally, a × pfu i.n. inoculation of hpiv /Δf-hn/ebogp was able to protect % of guinea pigs against a lethal ebov challenge [ ] . table . . . rnapc severe coagulation disorders are one of the most prominent features of filoviral infection. in the event of a breach in vascular integrity a strict balance of pro-and anti-coagulant host factors must be maintained to successfully clot the breach and to prevent too much or too little clot formation. in the instance of filovirus infection, sustained microvascular injury in effected organs results in host coagulation inhibitor depletion which results in disseminated intravascular coagulation (dic). in dic, tissue factor (tf), a clotting factor normally present on cells not exposed to blood, complexes with circulating factor vii leading to clot formation and fibrin deposition through the extrinsic pathway [ , ] . as numerous studies have demonstrated a clear link with dic and resultant organ failure, geisbert et al, sought to eliminate potential tf pathogenesis during filovirus infection by using recombinant nematode anticoagulant protein c (rnapc ) [ ] . they demonstrated that rnapc , an inhibitor of the tf pathway, provided partial post-exposure protection to rhesus macaques during filovirus infection [ ] . previous studies with rnapc have already gone through phase ii trials in orthopedic surgery [ ] and coronary revascularization [ ] . geisbert et al. showed a % survival rate, in addition to a . -day increase in mean time-to-death, for ebov-infected rhesus macaques and a % survival rate, with a . -day increase in mean time-to-death, in marv-infected rhesus macaques, when treated with rnapc post-exposure [ , ] . in a normally % lethal model of filovirus infection, rnapc demonstrated a clear benefit to survival as an increase in the mean time-todeath was observed. rnapc has completed phase ii clinical trials with a good safety record. primates. comparison of current drug candidates at the highest level of development, either in human clinical trials or those that have shown promise in nhps. also listed are the afforded levels of protection in nhps, the type of drug used to induce immunity and the dosing paradigm used to achieve the listed results. phosphorodiamidate morpholino oligomers (pmos) exert their anti-translation effects through steric hindrance of the translation machinery. this steric hindrance is possible due to a morpholino group, similar to a ribose base in rna, and a methylene phosphorodiamidate linking moiety that physically bind to mrna and prevent the translation machinery from accessing the mrna [ ] . once the antisense pmos bind to their target mrna, they are highly stable and highly soluble which would allow high levels of translation inhibition and predictably low levels of potential cytotoxicity [ ] [ ] [ ] [ ] . pmos have previously demonstrated effective antiviral activity against coronaviruses and flaviviruses [ , ] . swenson et al. initially utilized pmos targeting ebov vp and ebov vp to highly protect mice and guinea pigs against a lethal challenge with ebov and marv [ , ] . subsequently, avi- (a combination of pmos against both ebov vp and vp ) and avi- (a combination of pmos against both marv vp and np) were developed and tested in a nhp post-exposure scenario. these pmos, delivered - min post-exposure, protected > % of rhesus macaques against lethal ebov infection and % of cynomolgus macaques against marv infection [ ] . both the ease of controlled manufacture and their efficacy in nhp models to combat filovirus infection, pmos represent a viable therapeutic strategy [ ] . currently, avi- and avi- are in phase i clinical trials. table . . . recombinant human activated protein c ebolavirus disease (evd) and severe sepsis (or septic shock) share many clinical features including fever, hypotension, increased production of tissue factor, elevated levels of nitric oxide, and elevated levels of d-dimers [ ] [ ] [ ] . in addition, the most prominent and consistent finding in severe sepsis is severe protein c deficiency [ , ] . it was shown that treatment of patients with severe sepsis with recombinant human activated protein c (rhapc) resulted in improved survival [ ] . later experiments in nhps demonstrated that ebov infection results in rapid reduction of circulating protein c levels [ , ] . therefore, it was tested if treatment with rhapc could protect against lethal ebov infection in rhesus macaques. fourteen rhesus macaques were infected with a lethal dose of ebov; eleven were then treated with iv rhapc - min after challenge, continuing for days. all control animals died on day post-exposure; however, of the rhapc-treated animals survived (~ % survival). additionally, the mean time-to-death for rhapc-treated animals was . days, which is significant compared to the . days observed in placebo and historical controls [ ] . this product was pulled as a single post-exposure treatment, but given that this intervention is not directly targeting the virus, there may be additional merit in assessing this product in conjunction with a direct antiviral. rna interference (rnai) represents a powerful, naturally occurring biological strategy for inhibiting gene expression. rnai interferes with the translation of mrna to protein products by either sterically blocking mrna or by triggering rnase h-mediated cleavage of the dna/rna duplex, resulting in inhibition of gene expression [ ] . for many years rnai as demonstrated clear efficacy in preventing viral replication in vitro against a number of viruses, including coxsackieviruses, hepatitis b virus (hbv), hepatitis c virus (hcv), herpesviruses, human immunodeficiency virus (hiv), human papillomavirus, rsv, influenza a virus, lymphocytic choriomeningitis virus, polioviruses, and sars-cov [ ] [ ] [ ] [ ] . fowler et al. demonstrated that small-interfering rna (sirna) downregulation of various marv mrna transcripts was able to significantly decrease viral protein production and subsequent viral release in cell culture [ ] . unfortunately, efficient delivery vehicles providing effective drug targeting and stability have hindered the application of rnai in the clinical setting. however, recent developments in the field of nanotechnology have made nanoparticles the solution to increasing pharmacokinetic profiles for rnai therapies [ ] . additionally, tekmira, inc. developed proprietary lipid encapsulation as a means of improving the pharmacology of sirna targeting the ebola rna polymerase l protein, as demonstrated by geisbert et al. [ , ] . to efficiently deliver the sirna to target cells, a mixture of lipids forming a bilayered liposome, or stable nucleic acid-lipid particles (snalp), was designed. the snalp ensures cell entry by preferential fusing with the endosomal membrane upon exposure to the decreasing ph of the endosome. the snalps were further modified by conjugation with polyethylene glycol (peg) ensuring stability and efficient delivery of the sirna payload by neutralizing surface charges and presenting a hydrophilic exterior. this encapsulation was initially demonstrated to significantly increase the stability, half-life, and effectiveness of sirna directed against hbv [ ] . the snalp-encapsulation of sirna targeting the ebov l protein was initially shown to completely protect guinea pigs when administered shortly after a lethal ebov challenge [ ] . this treatment was then assessed for efficacy in rhesus macaques. snalp-encapsulated sirnas targeting ebov l polymerase, vp , and vp were given to rhesus monkeys either four or seven times following a lethal challenge of ebov. two of the three monkeys given four doses survived lethal infection, while all four monkeys given seven doses survived infection [ ] . the enhanced survivorship among the snalp-treated group highlights the efficacy of this potential therapeutic. additionally, there was little to no evidence of side effects associated with the treatment group, aside from mildly altered liver enzyme levels (which could have been an artifact separate from the challenge course) [ ] . table . innate immunity is often the first line of defense against invading pathogens. one mechanism by which innate immunity functions relies on the identification of pathogen-associated molecular patterns (pamps). pamps consist of unique carbohydrate moieties on the external surfaces of foreign microbes, such as hexose and mannose, which are not expressed on the surfaces of the host cells. these pamps are then recognized by host proteins such as mannose-binding lectins (mbl), which recognize these high hexose and mannose contents [ ] . filoviruses have large amounts of mannose comprising their glycoproteins and thus are a target of mbl [ ] . upon exposure, host mbl targets filoviruses for compliment-dependent virus neutralization through the lectin pathway of the compliment cascade [ ] . when administered in supraphysiological doses before or after lethal challenge with ebov, recombinant mbl treatment protected % of mice [ ] . these studies showed that mbl may be a potential post-exposure prophylactic. table . post-exposure treatments effective in small animal models. comparison of current treatment candidates at the small animal model level of development, specifically mouse models. also listed are the afforded levels of protection, the type of drug used to induce immunity and the dosing paradigm used to achieve the listed results. high-throughput screening (hts) is a significant tool for novel drug discovery. hts involves screening libraries consisting of thousands to hundreds of thousands of unique molecules against specific targets. available libraries used in hts have included natural compounds [ , ] , peptides [ ] , drugs [ ] , and synthetic compounds [ , ] . recently, compound fgi- was identified during a screen with an ebov-gfp pseudotyped virus and has shown strong antiviral activity in vitro against high doses of ebov and marv. fgi- was subsequently shown to protect mice against lethal challenges of both ebov and marv [ ] . additionally, compound fgi- was initially identified in a similar manner and was shown to exhibit strong antiviral activity in vitro against ebov, rift valley fever virus (rvfv), all four types of dengue virus (denv), hcv, and hiv- . fgi- also protected mice against a lethal challenge of ebov when given postexposure [ ] . taken together, this suggests that fgi- probably acts on a conserved pathway common to these four viruses, and potentially makes for a very intriguing antiviral. a second screen was done using a collection of , small molecule compounds obtained from the nci and the ebov-gfp pseudotyped virus [ ] . as a result, nsc , a reactive oxygen species scavenger, was identified and shown to have high antiviral activity against ebov, marv, lassa virus, rvfv, and veev. in vivo studies demonstrated that this compound protected mice against a lethal challenge of ebov and marv when given either pre-or post-exposure [ ] . metal ion-based therapeutics are a new potential class of drugs because they differ from carbon-based compounds due to the charged central ion which determines the molecular geometry of the compound. through these unique molecular geometries, specific compounds can be isolated that inhibit biological processes, and are unlike traditional carbon-based compounds because of their unique geometry. this difference allows these compounds to form octahedral and square planar molecular geometries. hexamminecobalt (iii) chloride (cohex) is a complex of a cobalt (iii) ion surrounded by six ammonia ligands in a full octahedral coordination. cohex was initially reported to have antiviral activity against adenovirus and sindbis virus, and was subsequently thought to have potential broad-spectrum antiviral activities [ ] . cohex was shown to be well-tolerated in mice with no apparent toxicity. mice were treated with cohex daily and infected with a lethal dose of ebov. cohex-treated mice had a significant increase in mean time-to-death, and the highest concentration treatment group had a % survival rate [ ] . this suggests that cohex has the potential to be an effective treatment against ebov infection. niemann-pick c (npc ), a cholesterol transporter found in endosomes and lysosomes, was recently identified as being required for ebov replication during a gene trap screen in hap cells using a replication-competent vsv bearing the ebov glycoprotein (rvsv-gp-ebov). in these experiments, cells with non-functional npc demonstrated decreased infectivity by rvsv-gp-ebov; however, expression of a functional npc rescued the normal infectivity of these viruses [ ] . npc is known to affect calcium homeostasis as well as endosomal and lysosomal fission and fusion [ ] [ ] [ ] . it has also been shown to be involved in hiv- release [ ] . loss of npc causes a neurological disorder called niemann-pick disease, which is characterized by cholesterol accumulation in lysosomes [ ] . while heterozygous npc knockout mice (npc +/− ) do not show evidence of niemann-pick disease, most npc +/− knockout mice were protected against a lethal challenge of mouse adapted ebov ( % survival) and marv ( % survival) [ ] . additionally, small molecules, such as u a, have been identified that interfere with npc and cause a cellular phenotype similar to npc deficiency [ ] . as such, u a was subsequently shown to block infection of ebov in vitro [ ] . heat-shock protein (hsp ) is a molecular chaperone that aids in the folding, trafficking, and proteolytic processing of many proteins [ , ] . as a result of their many functionalities, hsp inhibitors have been designed to combat diseases such as cancer, and there are currently several drugs now in phase i and ii clinical trials [ , ] additionally, hsp has shown to be important for replication of negative-strand viruses, as well as hcv, hbv, and polio [ ] [ ] [ ] [ ] . the effects of several natural and synthetic hsp inhibitors on ebov replication were tested in vitro. results of this study demonstrated that three hsp inhibitors significantly inhibited the replication of ebov in vero cells and primary human monocytes, suggesting their use as a potential therapeutic [ ] . ebolaviruses express two secreted glycoproteins, soluble gp (sgp) and small soluble gp (ssgp) [ ] . sgp has been associated with stabilization of the endothelial barrier function and reduction of endothelial barrier permeability by opposing the effects of tnf-α. these effects are in direct opposition to the observed roles for gp, which has been associated with endothelial cell destruction [ ] . ssgp has yet to have a clear role during infection [ ] . each of the gp forms contains identical n-termini but differ in the structure of their c-termini. during the differentiation process of the c-termini, the homodimer sgp is cleaved by furin to yield the mature sgp and a Δ-peptide which is retained in cells longer as compared to sgp. when these Δ-peptides from ebov, sudv, and tafv were fused with fc tags and transfected into cells before infection, they were capable of inhibiting both ebov and marv infection in a dose dependent fashion [ ] . these observations indicated that Δ-peptides might play an important role in filovirus pathogenesis, and could be exploited as a novel anti-filoviral therapeutic [ ] . while many events in filovirus entry remain undiscovered, a fusion mechanism similar to hiv and sars-cov is thought to occur such that a conformational rearrangement of glycoproteins on the viral surface results in viral fusion with the cellular membrane. inhibitors of viral fusion have been developed for hiv- and sars-cov. these inhibitors prevent the c-terminal heptad repeat in the envelope protein from interacting with the cellular membrane proteins by directly competing and blocking its interaction with the n-terminal heptad repeat, which normally would result in the formation of the six-helix bundle required for membrane fusion. c-peptides, which are inhibitors of viral envelope fusion, have had limited success against filoviruses in the past, most likely due to the suggestive evidence that filovirus fusion occurs far along in the endosomal maturation process [ ] [ ] [ ] [ ] [ ] . however, when these c-peptides were conjugated with an argenine-rich segment of hiv- 's tat protein (a protein known for its endosomal localization) the conjugated c-peptide exhibited marked antiviral effects, up to % inhibition of ebov and marv in vitro [ ] [ ] [ ] . unfortunately, the concentrations used to generate this inhibition in vitro were not possible in the clinical setting, but this report indicates that future c-peptide research may result in filovirus entry prevention for future therapeutics. a recent report that screened , molecules demonstrated that chlorophyllide was able to decrease the section of hbv dna in a hbv antiviral assay. these results were obtained at compound concentrations which exhibited no cytotoxic effects. this molecule is an alkylated porphyrin containing copper and as such this compound is carries a charge at neutral phs [ ] . during these screens, the chlorin e compound, a metal-free chlorophyllide-like molecule, was found to be the most potent and was subsequently tested against other viruses, including marv. during testing, the chlorine e compound showed significant antiviral activity in vitro against marv. this compound also inhibited junin virus, denv, hcv, and hiv- [ ] . another recent study that examined a library of , compounds yielded candidates capable of generating ≥ % inhibition of a hiv- /ebov-gp pseudotyped virus, while not interfering with the hiv- /vsv-g control virus. from these candidates, compound , a benzodiazepine derivative, showed an ability to inhibit both ebov and marv to a similar degree in vitro [ ] . results from these experiments suggested that compound acts at an early stage of viral entry, preventing infection by an unknown mechanism [ ] . lj , an aryl methyldiene rhodanine derivative, was identified during a high-throughput screening of inhibitors for nipah virus entry in the context of a vsv-pseudotyped vector [ ] . this compound was subsequently shown to inhibit a variety of enveloped viruses, including marv, ebov, nipah, rvfv, hiv- , hcv, wnv, etc. [ ] . however, it did not inhibit nonenveloped viruses such as adenovirus and reovirus. further testing demonstrated that lj binds to the viral membrane and prevents virus-cell fusion. while initial testing in mice did not show antiviral efficacy, further development of this compound to improve pharmacokinetics and potency is distinctly possible [ ] . development of medical countermeasures for ebov and marv remain a high priority and substantial progress has been made over the past decade. we have moved from the inability to protect from infection in various animal models of disease to a realm of medical countermeasures that protect prophylactically and more recently successful treatments that can be employed following known exposure to the viruses. initial efforts, focused on preventing the disease with vaccination strategies, ranged from subunit vaccines to vlps, vectored systems, dna vaccines, and live-attenuated virus systems that express the ebov or marv glycoproteins. to that aim, vaccine efficacy has been achieved by multiple vaccines against parenteral and aerosol routes of exposure. with the success of these new vaccine platforms, the attention of the past years has focused on the ability to treat infected patients. in the animal models, success has been demonstrated with traditional small molecules and antibodies directed against the virus or critical host proteins or pathways associated with pathogenesis. the ability to utilize various rna silencing technologies has been a focus for therapeutics that could be beneficial for filovirus infection, other infectious diseases and cancer therapy. despite these successes, there is much work to do to adequately prepare for this infectious threat. the ability to provide a beneficial therapeutic impact at a point when patients experience clinical symptoms and seek relief from caregivers remains a hurdle for the medical countermeasure development. moreover, the quality of life of patients following infection and treatment may require additional development efforts or the combination of multiple therapeutic approaches. as seen in outbreaks, the clinical sequelae observed in patients that survive infection are severe and life changing. these observations emphasize the need for medical countermeasures that not only provide survival but also decrease morbidity and long-term pathological outcomes following infection. lastly, the funding resources fortitude and the ability to navigate regulatory pathways will be essential to reaching either emergency use authorization (eua) status or licensed drug status for therapeutics and vaccines. however, the field remains optimistic that medical countermeasure solutions for human use are possible. proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations virus taxonomy-ninth report of the international committee on taxonomy of viruses a hitherto unknown infectious disease contracted from monkeys filoviridae: a taxonomic home for marburg and ebola viruses? a compendium of years of epidemiological, clinical, and laboratory studies drug targets in infections with ebola and marburg viruses ebolavirus and other filoviruses human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo spatial and temporal patterns of zaire ebolavirus antibody prevalence in the possible reservoir bat species large serological survey showing cocirculation of ebola and marburg viruses in gabonese bat populations, and a high seroprevalence of both viruses in rousettus aegyptiacus marburg virus infection detected in a common african bat discovery of swine as a host for the reston ebolavirus differentiation of filoviruses by electron microscopy virion nucleic acid of ebola virus marburg and ebola viruses recent advances in ebolavirus vaccine development filovirus vaccines. hum. vaccin processing of the ebola virus glycoprotein by the proprotein convertase furin c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus recovery potential of a western lowland gorilla population following a major ebola outbreak: results from a ten year study correlates of immunity to filovirus infection filovirus hemorrhagic fever outbreak case management: a review of current and future treatment options the medecins sans frontieres intervention in the marburg hemorrhagic fever epidemic, uige, angola, . i. lessons learned in the hospital dengue and dengue hemorrhagic fever: management issues in an intensive care unit real-time monitoring of cardiovascular function in rhesus macaques infected with zaire ebolavirus progress towards the treatment of ebola haemorrhagic fever a phase , randomized, double-blind safety and pharmacokinetic assessment of respiratory syncytial virus (rsv) prophylaxis with motavizumab and palivizumab administered in the same season antibodies for prevention and treatment of respiratory syncytial virus infections in children ebola virus: from discovery to vaccine treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee the marburg virus outbreak of and subsequent episodes an infectious disease transmitted by cercopithecus aethiops. ("green monkey disease") evaluation of immune globulin and recombinant interferon-alpha b for treatment of experimental ebola virus infections neutralizing antibody fails to impact the course of ebola virus infection in monkeys protective efficacy of neutralizing antibodies against ebola virus infection antibody-dependent enhancement of marburg virus infection epitopes required for antibody-dependent enhancement of ebola virus infection antibody-dependent enhancement of ebola virus infection recombinant human monoclonal antibodies to ebola virus pre-and postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody epitopes involved in antibody-mediated protection from ebola virus postexposure antibody prophylaxis protects nonhuman primates from filovirus disease protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of ebola hemorrhagic fever successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies enhanced potency of a fucose-free monoclonal antibody being developed as an ebola virus immunoprotectant characterization of zaire ebolavirus glycoprotein-specific monoclonal antibodies neutralizing ebolavirus: structural insights into the envelope glycoprotein and antibodies targeted against it a dna vaccine for ebola virus is safe and immunogenic in a phase i clinical trial immune protection of nonhuman primates against ebola virus with single low-dose adenovirus vectors encoding modified gps development of a preventive vaccine for ebola virus infection in primates a replication defective recombinant ad vaccine expressing ebola virus gp is safe and immunogenic in healthy adults vaccine to confer to nonhuman primates complete protection against multistrain ebola and marburg virus infections protection of nonhuman primates against two species of ebola virus infection with a single complex adenovirus vector enhanced protection against ebola virus mediated by an improved adenovirusbased vaccine a single sublingual dose of an adenovirus-based vaccine protects against lethal ebola challenge in mice and guinea pigs recombinant adenovirus serotype (ad ) and ad vaccine vectors bypass immunity to ad and protect nonhuman primates against ebolavirus challenge rhabdoviridae: the viruses and their replication vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates single-injection vaccine protects nonhuman primates against infection with marburg virus and three species of ebola virus cross-protection against marburg virus strains by using a live, attenuated recombinant vaccine recombinant vesicular stomatitis virus vector mediates postexposure protection against sudan ebola hemorrhagic fever in nonhuman primates effective post-exposure treatment of ebola infection inactivated or liveattenuated bivalent vaccines that confer protection against rabies and ebola viruses replicon-helper systems from attenuated venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo recombinant rna replicons derived from attenuated venezuelan equine encephalitis virus protect guinea pigs and mice from ebola hemorrhagic fever virus marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates vaccine potential of ebola virus vp , vp , vp , and vp proteins protective cytotoxic t-cell responses induced by venezuelan equine encephalitis virus replicons expressing ebola virus proteins a single intranasal inoculation with a paramyxovirus-vectored vaccine protects guinea pigs against a lethal-dose ebola virus challenge successful topical respiratory tract immunization of primates against ebola virus parainfluenza viruses mucosal parainfluenza virus-vectored vaccine against ebola virus replicates in the respiratory tract of vector-immune monkeys and is immunogenic recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of h n highly pathogenic avian influenza virus protect against virus challenge in monkeys respiratory tract immunization of non-human primates with a newcastle disease virus-vectored vaccine candidate against ebola virus elicits a neutralizing antibody response ebola virus vp -induced particle formation and association with the lipid bilayer contribution of ebola virus glycoprotein, nucleoprotein, and vp to budding of vp virus-like particles ebola virus-like particle-based vaccine protects nonhuman primates against lethal ebola virus challenge monovalent virus-like particle vaccine protects guinea pigs and nonhuman primates against infection with multiple marburg viruses filovirus-like particles produced in insect cells: immunogenicity and protection in rodents ebola virus vp -induced particle formation and association with the lipid bilayer ebola virus vp drives the formation of virus-like filamentous particles along with gp immunogenicity and protective efficacy of rotavirus / -virus-like particles produced by a dual baculovirus expression vector and administered intramuscularly, intranasally, or orally to mice expression and self-assembly of empty virus-like particles of hepatitis e virus enhanced mucosal and systemic immune responses following intravaginal immunization with human papillomavirus l virus-like particle vaccine in thermosensitive mucoadhesive delivery systems intranasal immunization with siv virus-like particles (vlps) elicits systemic and mucosal immunity protection against lethal challenge by ebola virus-like particles produced in insect cells ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies immunogenicity of the outer domain of a hiv- clade c gp increased potency of fc-receptor-targeted antigens cross-reactive hiv- -neutralizing activity of serum igg from a rabbit immunized with gp fused to igg fc: possible role of the prolonged half-life of the immunogen ebola virus glycoprotein fc fusion protein confers protection against lethal challenge in vaccinated mice transgenic plants as protein factories monoclonal antibody manufacturing in transgenic plants--myths and realities high-level rapid production of full-size monoclonal antibodies in plants by a single-vector dna replicon system recombinant pharmaceuticals from plants: the plant endomembrane system as bioreactor expression of an immunogenic ebola immune complex in nicotiana benthamiana a replicating cytomegalovirus-based vaccine encoding a single ebola virus nucleoprotein ctl epitope confers protection against ebola virus effector memory t cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge broadly targeted human cytomegalovirus-specific cd + and cd + t cells dominate the memory compartments of exposed subjects human cytomegalovirus tropism for endothelial cells: not all endothelial cells are created equal profound early control of highly pathogenic siv by an effector memory t-cell vaccine chimeric human parainfluenza virus bearing the ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against ebola virus challenge disseminated intravascular coagulation (dic) coli septic shock is prevented by blocking tissue factor with monoclonal antibody treatment of ebola virus infection with a recombinant inhibitor of factor viia/tissue factor: a study in rhesus monkeys dose-response study of recombinant factor viia/tissue factor inhibitor recombinant nematode anticoagulant protein c in prevention of postoperative venous thromboembolism in patients undergoing total knee replacement recombinant nematode anticoagulant protein c , an inhibitor of the tissue factor/factor viia complex, in patients undergoing elective coronary angioplasty marburg virus angola infection of rhesus macaques: pathogenesis and treatment with recombinant nematode anticoagulant protein c cell penetrating peptide conjugates of steric block oligonucleotides phosphorodiamidate morpholino oligomers: favorable properties for sequencespecific gene inactivation morpholino antisense oligomers: design, preparation, and properties stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells inhibition of dengue virus serotypes to in vero cell cultures with morpholino oligomers inhibition, escape, and attenuated growth of severe acute respiratory syndrome coronavirus treated with antisense morpholino oligomers vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice chemical modifications of antisense morpholino oligomers enhance their efficacy against ebola virus infection advanced antisense therapies for postexposure protection against lethal filovirus infections ebola hemorrhagic fever and septic shock recombinant human activated protein c for the postexposure treatment of ebola hemorrhagic fever marburg and ebola hemorrhagic fevers: does the primary course of infection depend on the accessibility of organ-specific macrophages? severe protein c deficiency predicts early death in severe sepsis protein c concentrations in severe sepsis: an early directional change in plasma levels predicts outcome efficacy and safety of recombinant human activated protein c for severe sepsis mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event silencing viruses by rna interference. microbes infect rnai, a new therapeutic strategy against viral infection rna interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus inhibition of sars-cov replication by sirna inhibition of marburg virus protein expression and viral release by rna interference lipidic systems for in vivo sirna delivery postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by rna interference postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference: a proof-of-concept study potent and persistent in vivo anti-hbv activity of chemically modified sirnas high-dose mannose-binding lectin therapy for ebola virus infection phase i safety, tolerability, and pharmacokinetic study of recombinant human mannanbinding lectin mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dcsign and complement-mediated virus neutralization marine natural product libraries for high-throughput screening and rapid drug discovery identification of upregulators of bmp expression via high-throughput screening of a synthetic and natural compound library in vitro system for high-throughput screening of random peptide libraries for antimicrobial peptides that recognize bacterial membranes high-content assay to identify inhibitors of dengue virus infection drugs for bad bugs: confronting the challenges of antibacterial discovery antiviral activity of a small-molecule inhibitor of filovirus infection development of a broad-spectrum antiviral with activity against ebola virus development of high-content imaging assays for lethal viral pathogens identification of an antioxidant small-molecule with broad-spectrum antiviral activity hexamminecobalt (iii) chloride as a broad-spectrum antiviral complex niemann-pick c disease gene: homology to mediators of cholesterol homeostasis niemann-pick c functions independently of niemann-pick c in the initial stage of retrograde transport of membrane-impermeable lysosomal cargo niemann-pick disease type c is a sphingosine storage disease that causes deregulation of lysosomal calcium deficiency of niemann-pick type c- protein impairs release of human immunodeficiency virus type and results in gag accumulation in late endosomal/lysosomal compartments cholesterol synthesis inhibitor u a and the role of sterol metabolism and trafficking in numerous pathophysiological processes inhibition of heat-shock protein reduces ebola virus replication regulation of signaling protein function and trafficking by the hsp /hsp -based chaperone machinery phase i trial of -allylamino- -demethoxygeldanamycin in patients with advanced cancer hsp and the chaperoning of cancer antiviral activity and rna polymerase degradation following hsp inhibition in a range of negative strand viruses evolutionary constraints on chaperonemediated folding provide an antiviral approach refractory to development of drug resistance molecular chaperone hsp is important for vaccinia virus growth in cells heat-shock protein is essential for stabilization of the hepatitis c virus nonstructural protein ns ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry effects of ebola virus glycoproteins on endothelial cell activation and barrier function heptad repeat-derived peptides block protease-mediated direct entry from the cell surface of severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type gp are potent inhibitors of virus infection evidence that a prominent cavity in the coiled coil of hiv type gp is an attractive drug target capture of an early fusion-active conformation of hiv- gp inhibition of ebola virus entry by a c-peptide targeted to endosomes tat transduction: the molecular mechanism and therapeutic prospects cellular uptake of unconjugated tat peptide involves clathrin-dependent endocytosis and heparan sulfate receptors alkylated porphyrins have broad antiviral activity against hepadnaviruses, flaviviruses, filoviruses, and arenaviruses identification of a small-molecule entry inhibitor for filoviruses a broad-spectrum antiviral targeting entry of enveloped viruses the authors declare no conflict of interest. key: cord- - xsbpid authors: condit, richard c; kim, denny; robertson, james s.; excler, jean-louis; gurwith, marc; monath, thomas p.; pavlakis, george; fast, patricia e.; smith, jonathan; smith, emily r.; chen, robert t.; kochhar, sonali title: the brighton collaboration standardized template for collection of key information for benefit-risk assessment of viral vector vaccines date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xsbpid many of the vaccines under development for covid- involve the use of viral vectors. the brighton collaboration benefit-risk assessment of vaccines by technology (bravato, formerly the viral vector vaccine safety working group, v swg) working group has prepared a standardized template to describe the key considerations for the benefit-risk assessment of viral vector vaccines. this will facilitate key stakeholders to anticipate potential safety issues and interpret or assess safety data. this would also help improve communication and public acceptance of licensed viral vector vaccines. in , the speed of vaccine development for covid- is unprecedented [ ] . keeping in mind the volume and pace of vaccine development, a systematic and deliberate approach to vaccine safety that is understandable and accessible to diverse stakeholders is of considerable importance. several viral vectored vaccines are among the covid- vaccines in development. the brighton collaboration (www.brightoncollaboration.us) was launched in to improve the science of vaccine safety [ ] . the brighton collaboration formed the viral vector vaccines safety working group (v swg) in october to improve the ability to anticipate potential safety issues and meaningfully assess or interpret safety data, thereby facilitating greater public acceptance when a viral vector vaccine is licensed [ ] . pursuant to this goal, the v swg developed a standardized template that the coalition for epidemic preparedness innovations (cepi) and other key stakeholders could use to evaluate and communicate key considerations for the benefit-risk assessment of viral vectors and viral vector vaccines. the information in the template will help in the communication of technical and complex data among key stakeholders, and increase the comprehension, transparency and comparability of essential information (see table ). the viral vector vaccine template and the mission of the v swg has evolved over time. the first version of the template (v . ) was used for the standardized benefit-risk assessment of several new viral vectors or viral vector vaccines [ ] [ ] [ ] , including a vaccine targeting ebola. the who global advisory committee on vaccine safety (gacvs) endorsed the use of the viral vector template for other new candidate ebola vaccines ''as it is a structured approach to vaccine safety" [ ] . a second version of the template (v . ) was used to describe viral vectors based on adenovirus and modified vaccinia virus ankara (in preparation). experience with earlier versions of the viral vector template and with other vaccine platform templates under development by the v swg inspired improvements included with the template presented here. a detailed history of the development of the viral vector vaccine template is archived on the brighton collaboration website (https://brightoncollaboration.us/bravato/). the v wsg has recently been renamed to the benefit-risk assessment of vaccines by technology (bra-vato) working group to reflect its expanded role in development of templates for additional vaccine platforms, namely nucleic acid based, live attenuated, inactivated, and protein based vaccines [ ] . viral vector vaccines are laboratory-generated, chimeric viruses that are based upon replicating or non-replicating virus vectors into which have been spliced genes encoding antigenic proteins for a target pathogen. consideration of safety issues associated with viral vector vaccines requires a clear understanding of the agents used for construction of the vaccine. these include ( ) the wild type virus from which the vector is derived, referred to in the template as ''wild type virus"; ( ) the vector itself before incorporation of the foreign antigen, referred to in the template as ''viral vector"; and ( ) the final recombinant viral vector vaccine, referred to in the template as ''vaccine". wild type viruses used as vectors may originate from human or animal hosts and may have low or high pathogenic potential in humans regardless of species of origin. understanding the characteristics of the wild type virus as directed in the template is critical in anticipating the potential behavior of any vector adapted from the wild type virus. viral vectors can originate from attenuated viral vaccines used in humans (e.g. yellow fever, modified vaccinia virus ankara); from attenuated human or animal viruses (e.g. human adenovirus, vesicular stomatitis virus); or from human or animal viruses with low pathogenic potential (e.g. adeno associated virus, chimp adenovirus). viral vectors can be replicating (e.g. vesicular stomatitis virus) or non-replicating (e.g. modified vaccinia virus ankara). viral vectors usually, but not always, have properties in a human host that differ from the wild type virus from which they were derived. incorporation of a target antigen into a viral vector to create a vaccine may alter the properties of the vector such that the vaccine may have properties that differ from the vector. this updated version of the brighton collaboration vaccine vector template is designed for dual use. it may be used to describe exclusively viral vectors into which transgenes may be incorporated to create vaccines, or it may be used to describe viral vector vaccines for specific pathogens. thus, the template has two main parts. part i is used to describe a viral vector and part ii is used additionally to describe a specific vaccine, where this is the intent. pursuant to understanding completely the characteristics of a given vector, part i considers the wild type virus from which the vector is derived (section ) in addition to characteristics of the vector itself (sections and ). pursuant to understanding completely the characteristics of a vaccine, part ii additionally considers the target pathogen (section ) and the potential impact of transgene insertion to create a vaccine (section ). each part contains its own sections evaluating the toxicology, adverse effects and overall assessment of either the vector alone or a vaccine. when the template is being used to characterize a viral vector vaccine, it is understood that there may be limited information concerning the vector itself, especially concerning toxicology and potency of the vector (section ), and section on adverse events may not be relevant. vaccine developers should nevertheless complete section i to what extent this is feasible. bravato intends that this template focuses on key questions related to the essential safety and benefit-risk issues relevant for the intrinsic properties of the vaccine components. we recognize that there are many other aspects of manufacturing, quality, and implementation that can play an important role in the safety of a vaccine, but we have chosen to keep some of those issues out of scope for the template in order to summarize information that is the most useful to the most stakeholders. the latest version of the template can be accessed on https:// brightoncollaboration.us/bravato/. vaccine developers are encouraged to complete the relevant templates for their vaccine candidate platform or vaccine candidate and collaborate with bravato. the draft templates would be shared for review by bra-vato and submitted for publication. similarly, updates to the templates by the vaccine developers should be submitted to the brighton collaboration website for bravato review. see supplementary material for definitions and additional guidance for completing this template. please read these instructions before you complete the thirteen sections. send questions to:brightoncollaborationv swg@gmail.com the first section entitled ''authorship and affiliation" should include your name, your affiliation and the latest date completing the form. if you are working with someone else to complete this form, their name and affiliation should be provided as well. if you are updating the form, please provide the updated date. these co-authors will be included in the final published template in vaccine once reviewed and approved by bravato and in subsequent wiki updates on the bravato website. part i collects information regarding a viral vector alone, while part ii collects information regarding a vaccine based on the viral vector. if the template is being used to describe a vector only, then complete part i only. if the template is being used to describe a vector vaccine, then complete both parts i and ii. within part i, sections - collect information regarding the wild type virus (section ) and the vector (sections and - ). within part ii, section collects information regarding the target pathogen and population while sections - collect information regarding the vaccine based on the vector. depending on the circumstances, some sections may be redundant, for example if a vector is in fact identical to the wild type virus. in cases of redundancies, an answer may simply refer to the answer in another section. furthermore, some sections may not be applicable, for example if safety evaluations have been conducted only in the context of a vaccine and not with an empty viral vector alone. in such cases the answer should include ''not applicable" or ''not tested", whichever is relevant. whether competing only part i or both parts i and ii, any supplementary information should be added in section . answer questions by responding in the column entitled 'information.' if you have any comments or concerns regarding the question or your answer to the question, note these in the 'comments/concerns' column. finally, please provide references wherever possible in both the ''information" and ''comments/concerns" columns. referencing should use the vaccine journal format, with references numbered sequentially in the text and full citations listed in sequence at the end of the form. more than one reference can be used per question. sections , , and have column titles that differ from preceding sections intended to provide a summary assessment of adverse effects and toxicity of the vector. please summarize adverse effect and toxicities as requested and rate the risk in the following fashion: none, minimal, low, moderate, high, or unknown. if there is insufficient data for use of the vector in humans to accurately make these assessments, please state so in response to the questions. when completing information on adverse effects in sections and , please provide as many details as possible based on the brighton collaboration guidelines for collection, analysis and presentation of vaccine safety data in pre-and postlicensure clinical studies [ ] . in the references, unpublished data and non-peer reviewed published data are acceptable, though we do wish that you include the source and contact information. if a literature search was conducted to complete any of the sections (strongly encouraged), please provide the following information in the reference section: ( ) the findings, opinions, conclusions, and assertions contained in this consensus document are those of the individual members of the working group. they do not necessarily represent the official positions of any participant's organization (e.g., government, university, or corporations) and should not be construed to represent any agency determination or policy. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper the covid- vaccine development landscape the brighton collaboration: addressing the need for standardized case definitions of adverse table (continued) events following immunization (aefi) the brighton collaboration viral vector vaccines safety working group (v swg) live virus vaccines based on a yellow fever vaccine backbone: standardized template with key considerations for a risk/benefit assessment live virus vaccines based on a vesicular stomatitis virus (vsv) backbone: standardized template with key considerations for a risk/benefit assessment rvsvdg-zebov-gp (also designated v ) recombinant vesicular stomatitis virus pseudotyped with ebola zaire glycoprotein: standardized template with key considerations for a risk/benefit assessment global advisory committee on vaccine safety the brighton collaboration standardized templates for collection of key information for benefit-risk assessment of vaccines by technology (bravato; formerly v swg). vaccine guidelines for collection, analysis and presentation of vaccine safety data in pre-and post-licensure clinical studies we thank the following colleagues for their helpful advice: ( ) margaret liu, brighton collaboration members; and ( ) other members of bravato during the preparation of this document: david wood, karin bok, najwa khuri-bulos, bettina klug, and merita kucuku. we acknowledge the financial support provided by the coalition for epidemic preparedness innovations (cepi) for our work under a service order entitled safety platform for emergency vaccines (speac) project with the brighton collaboration, a program of the task force for global health, decatur, ga. key: cord- -owcapg h authors: dietrich, jes; andreasen, lars vibe; andersen, peter; agger, else marie title: inducing dose sparing with inactivated polio virus formulated in adjuvant caf date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: owcapg h the development of new low cost inactivated polio virus based vaccines (ipv) is a high priority, and will be required to eradicate polio. in addition, such a vaccine constitutes the only realistic polio vaccine in the post-eradication era. one way to reduce the cost of a vaccine is to increase immunogenicity by use of adjuvants. the caf adjuvant has previously been shown to be a safe and potent adjuvant with several antigens, and here we show that in mice ipv formulated with caf induced increased systemic protective immunity measured by binding and neutralization antibody titers in serum. caf also influenced the kinetics of both the cellular and humoral response against ipv to produce a faster, as well as a stronger, response, dominated by igg a, igg b, and igg c isotypes as well as ipv specific t cells secreting ifn-γ/il- . finally, as intestinal immunity is also a priority of polio vaccines, we present a vaccine strategy based on simultaneous priming at an intradermal and an intramuscular site that generate intestinal immune responses against polio virus. taken together, the ipv-caf formulation constitutes a new promising vaccine against polio with the ability to generate strong humoral and cellular immunity against the polio virus. poliomyelitis is caused by the polio virus, an rna virus that can colonize the gastroenteral tract which may lead to an acute, viral, infectious disease that spreads from person to person, primarily via the fecal-oral route. in , the world health assembly resolved to globally eradicate poliomyelitis (polio) [ ] . the initial objective, the end of polio by , has proven more difficult than originally envisioned and polio still exist in countries such as afghanistan, nigeria and pakistan. however, due to great efforts the number of polio cases has decreased to a level where full eradication within a decade or two is a realistic goal. two vaccines exist against polio; inactivated polio vaccine (ipv) and trivalent live oral polio virus (topv). topv with attenuated sabin strains of poliovirus types , and , has been the vaccine of choice for polio vaccination in most countries because it induces both systemic and intestinal immunity, can immunize or boost immunity of close contacts through secondary spread, and is inexpensive and easy to administer. however, one problem with opv is that on rare occasions opv can cause vaccine-associated paralytic poliomyelitis (vapp) and/or can revert to a neurovirulent form of poliovirus which is believed to be as transmissible and virulent as wild polioviruses [ ] [ ] [ ] . therefore, steps have been taken to discontinue opv as a vaccine against polio, rendering ipv the only realistic polio vaccine in the post-eradication era. when opv is withdrawn, several challenges concerning ipv have to be dealt with. one such challenge is that the high purchase costs for ipv potentially can lead to limited supplies of ipv in many countries. another challenge concerns the immunity induced by ipv, and how to achieve intestinal immunity with this vaccine. ipv protects the vaccine recipient from paralysis, but compared to opv it provides less protection against re-infection. furthermore ipv does not reduce fecal excretion following reinfection as much as opv because it provides weaker intestinal immunity [ ] [ ] [ ] [ ] [ ] . there are however studies that demonstrated that ipv can induce some intestinal immunity [ ] [ ] [ ] . one way to reduce the cost of a vaccine is to use adjuvants [ ] . in the field of pandemic influenza vaccines the use of adjuvants has permitted dose reduction, increased the availability and reduced cost of the vaccine [ ] [ ] [ ] [ ] [ ] . therefore, it has been speculated that an adjuvanted vaccine formulation of ipv would reduce cost and also increase the number of available ipv doses worldwide. in support of this, it was recently shown that the potency of sabin inactivated polio vaccines is increased when adjuvanted with aluminium hydroxide or cpg [ , ] . caf is a novel adjuvant composed of cationic liposomes dda (dimethyldioctadecylammonium) stabilized with the synthetic immunomodulator tdb (trehalose , -dibehenate) [ ] . caf has proven to enhance both humoral and cell-mediated memory immune responses to a number of different experimental vaccine candidates [ ] [ ] [ ] in preclinical models. caf has furthermore already been tested in three phase-i trials with an excellent safety and immunogenicity profile (eag, personal communications and [ ] [ ] [ ] ). additionally, caf was also found to provide dose-sparing when used in a combination with the trivalent influenza split vaccine in ferrets [ ] . we therefore tested the suitably of the caf adjuvant for inducing protective immunity against an infection with polio virus. it is well known that mucosal immunity can be achieved through vaccination at the mucosal site in question and that a vaccine given by the intramuscular route primarily produce systemic immunity [ , ] . mucosal sites are not isolated immunological sites, and priming of an immune response at one mucosal site may also induce protection at another mucosal site [ ] . although the skin is not traditionally regarded as a mucosal site, previous results from other groups have nevertheless indicated that an intradermal administration may confer intestinal/mucosal protection [ , ] . in addition, intradermal administration is a well-known delivery route for non-adjuvanted ipv vaccines in humans, and has been shown to be a safe delivery route with no adverse effects [ , ] . we therefore decided to test different vaccine strategies involving both intradermal and intramuscular administration of caf adjuvanted ipv in order to achieve intestinal immunity with an ipv vaccine. taken together, the aim for the present work was twofold: ) to achieve dose sparing with caf , measured by neutralization titers, and ) to induce intestinal immunity against ipv with an ipv-caf vaccine. studies were performed with -to -wk-old female cb f c bl/ xbalb/c mice from harlan, scandinavia. animals were housed in appropriate animal facilities at statens serum institut. the ipv vaccines applied throughout this study was commercial gmp grade material manufactured at statens serum institut (ssi). the trivalent ipv was formulated in the ratio : : for polio virus type (brunhilde strain, ipv ), type (mef- , ipv ) and type (saukett, ipv ), respectively. the trivalent ipv for the vaccines was in this study always formulated to a trivalent stock, ipv tp ( : : du/ml), from individual concentrated bulk materials of ipv , ipv and ipv . monovalent ipvs were stored in m buffer. dilution of ipv to the respective concentration used for immunization was performed with mm tris buffer, ph . . the trivalent stock ipv tp of : : du/ml was diluted to the appropriate concentration by adding the ipv stock solution to a given volume of buffer. indicated dosage units in the all experiments correspond to ipv d antigen units and all vaccine are trivalent unless otherwise stated. the caf adjuvant suspension was manufactured at two lipid concentrations: mg/ml (caf / mg of dda and tdb) and mg/ml (caf / ), respectively. dda and tdb were manufactured by niels clauson-kaas a/s, farum, denmark. in brief, a lipid film for ml caf / was formed by dissolving dda ( . g) in ml chloroform/ methanol ( : , v/v) and mixing it with tdb ( . g) dissolved in ml chloroform/methanol ( : , v/v) in a ml bluecap flask. the organic solvent was removed using a gentle stream of n forming a thin lipid film at the bottom of the flask. the lipid film was dried under vacuum over night to remove trace amounts of the organic solvent. the lipid film was hydrated ml mm tris-buffer, ph . by high speed high-shear mixing (heidolph silent crusher m) at - uc for minutes. adjuvants were stored at - uc until use. for the formulation of caf / , the same procedure is applied, with smaller amounts of dda and tdb, . g and . , respectively in ml. all adjuvants were stored at - c until use. the ipv-caf vaccines were formulated by add-mixing : v/v of an ipv solution into a solution of caf under moderate stirring using magnetic stirring bars. the vaccines were rested for minutes to allow for adsorption of ipv to the cationic caf liposomes. for ipv-caf vaccines intended for mice, the caf / mg was applied in order to formulate a mouse dose caf ( / mg) into ml dose volume for im vaccination after : v/v mixing with ipv antigen solution. caf / was applied to formulate vaccines for physic-chemical analyses. all vaccine formulations were performed in laf units. all vaccines were stored at - c until use. manufacturing of both the caf adjuvants, ipv vaccines and ipv-caf vaccines was performed in laf units. the final concentration of caf in all the mouse studies was / ug dda/tdb. mice were immunized two or three times at two-week intervals intramuscularly (im in the caudal thigh) with ml ipv or ipv formulated with caf . alternatively the mice were immunized with ml via the intradermal (id, on the back) route. the ipv doses used in the experiments are indicated in the figures. two weeks after the second or third vaccination the immune response against ipv was analyzed. in some experiments the immune response was also analyzed and weeks after the second vaccination. lymphocytes were cultured and purified as described previously [ ] . briefly, pbmcs were purified on a density gradient and splenocyte cultures were obtained by passage through a cell strainer (bd pharmingen). ipv for stimulation were all used at mg/ml. supernatants from triplicate cultures ( cells per well) were harvested from cultures after h of incubation for the investigation of cytokines. a sandwich elisa was used to determine the concentration of ifn-c in culture supernatants as previously described [ ] . alternatively, the cytokines were analyzed by multiplex cytokine assay. the th /th cytokine -plex assay and the il- singleplex assay (meso scale discovery, gaithersburg, md) were performed according to the manufacturer's instructions. the plates were read on the sector imager system, and calculation of cytokine concentrations in unknown samples was determined by parameter logistic non-linear regression analysis of the standard curve. ipv d-antigen elisa (du elisa) ipv d-antigen was quantified using a sandwich elisa. the du elisa detects the d-unit activity of ipv. in brief, immuno plates (maxisorp, nunc) were coated with polyclonal antibodies (raised in rabbits by immunization with inactivated poliovirus) of type (brunhilde strain), type (mef strain), or type (saukett strain)) against polio type , , or diluted in pbs with phenol-red for - k hours in a humidified atmosphere at room temperature. after washing and blocking of the wells, standards and samples diluted in pbs containing nacl, kcl, triton x- , bsa and phenol-red were added to the wells followed by an overnight incubation in a humidified atmosphere at - uc. the wells were washed and subsequently hrp-conjugated polyclonal antibodies against polio type , , or diluted in fbs with phenol-red were added to the wells and incubated for hours in a humidified atmosphere at room temperature. the wells were washed and opd substrate was added followed by a minute incubation period in the dark at room temperature. the reaction was stopped by the addition of h so and plates were read at od nm with non-specific absorption at nm subtracted. briefly, neutralizing antibody titers to the three poliovirus serotypes , and were measured using brunhilde, mef- , and saukett virus stocks on a vero cells. dilutions of the sera in ml were made in duplicate starting at : with serial -fold dilutions. ml of approximately ccid (cell culture infectious dose %) of poliovirus type , or was added to each well (in a well plate), and incubated for k- hours at ( ) ( ) ( ) uc and % co , and at - uc till the next day. vero cells were prepared as a cell suspension of cells/ml and ml are added to each well containing the polio virus/serum mixture. plates were incubated at uc and % co for ( - ) days. the wells showing neutralizing/cytopathogenic activity are recorded. the result from a single dilution series is given as /! times the lowest dilution factor with dead vero-cells and the final titre calculated as the geometric mean of the results from two independent dilution series. samples were tested in triplicate using a standard microneutralization assay for antibodies to poliovirus types , , and according to established protocols at the global polio specialized laboratory, centers for disease control and prevention (cdc). briefly, - ccid of each poliovirus serotype and two-fold serial dilutions of serum were combined and pre-incubated at uc for hours before addition of hep- (c) cells. after incubation for days at uc and % co , plates were stained with crystal violet and cell viability measured by optical density in a spectrophotometer. each specimen was run in triplicate, with parallel specimens from one study subject tested in the same assay run, and the neutralization titers estimated by the spearman-kä rber method and reported as the reciprocal of the calculated % endpoint. each run contained multiple replicates of a reference antiserum pool starting at a : dilution to monitor performance variation. a serum sample was considered positive if antibodies were present at $ : dilution. specimens with antibody titers , : were considered seronegative and specimens with titers . : were considered seropositive. mice were bled for the collection of serum following the vaccination - . maxisorp micro titer plates (nunc, maxisorp, roskilde, denmark) were coated with ipv tp (trivalent inactivated polio virus, see materials and methods) or ipv type , or ( ug/ml) in pbs over night at uc. free binding sites were blocked with % skimmed milk in pbs. individual mouse sera were analyzed in duplicate in five-fold dilutions in pbs containing bovine serum albumin starting with a -fold dilution. horseradish peroxidase (hrp)-conjugated secondary antibodies (rabbit anti-mouse igg, igg , igg a, igg b, igg c and iga; zymed) diluted / in pbs with % bovine serum albumin were added. after h of incubation, antigen-specific antibodies were detected by tmb substrate as described by the manufacturer (kem-en-tec, copenhagen, denmark). to stop the reaction, ul of n h so was added, and the optical density (od) was measured at nm. the absorbance values were plotted as a function of the reciprocal dilution of serum samples. fecal pellets were collected from mice two weeks following each immunization. the mice were placed in individual cages and fresh fecal pellets ( - pellets per mouse) were collected into microfuge tubes containing ml of ice-cold buffer: pbs with soybean trypsin inhibitor (sigma; . mg/ml), bovine serum albumin (bsa; % w/v), ethylenediaminetetraacetic acid (edta; mm), glycerol ( % v/v) and phenylmethylsulfonylfluoride (pmsf; mm). the fecal pellets were broken up to form a suspension and then incubated on ice for hours. after incubation, fecal pellet solutions were clarified by centrifugation at , g for minutes at uc, and the supernatants transferred to microfuge tubes that had been blocked overnight with pbs containing % (w/v) bsa. supernatants were then frozen and analysed at a later date by elisa. the z-average diameter, z avg , of the liposomes was determined by dynamic light scattering using the photon correlation spectroscopy (pcs) technique. the measurements were performed at uc using a malvern nanozs (malvern instruments, worcestershire, uk) equipped with a nm laser and u detection optics. malvern dts v. . software was used for data acquisition and analysis. nanospheretm size standards nm (duke scientific corp., duke, nc) was used to verify the performance of the instrument. the samples were diluted with mm trisbuffer at ph . to achieve the optimal vesicle concentration. surface charge on the vesicles was measured indirectly via analysis of zeta-potential at uc using a malvern nanozs (using monomodal analysis model) (malvern instruments, worcestershire, uk) of a / - / dilution in milli-q water. a significant difference in immune responses measured by elisa, or a difference in neutralization titers, was evaluated using a one-way anova, and tukey's multiple comparison test for multiple comparisons. a value of p, . was considered significant. prism version software (graphpad) was used for analysis. the trivalent ipv was analyzed with or without caf . the trivalent ipv vaccine was formulated from individual ipv , ipv and ipv materials. both ipv and the stock ipv tp showed a z avg (hydrodynamic diameter) of nm. the z avg of nm is in accordance with values for polio virus size determined by electron microscopy ( nm) indicating that the size did not change upon mixing of three ipv monovalents into a trivalent ipv (fig. a) . furthermore, the analysis of ipv content measured as d-unit activity confirmed that ipv d-unit activity was maintained after mixing of ipv - into ipv tp (fig. b) . on formulation with caf , the particle size distribution for an ipv-caf vaccine (z avg, d.nm ) was found to be nm compared to nm for the caf adjuvant. this indicated a slight increase in particle size on ipv binding to the caf , which however was within an acceptable range. we also measured the zeta potential of ipv, caf and ipv-caf formulations (fig. c) . the zeta potential for the cationic caf of mv was in accordance with previous observations [ ] . monovalent ipv showed a slightly negative zeta potential ( mv), whereas the adjuvanted ipv-caf vaccine showed a positive zeta potential of mv. therefore, in ipv vaccines formulated with caf , a binding between antigen and adjuvant is electrostatically favorable. to measure the degree of association between caf and ipv we assumed that since caf is a suspension of cationic liposomes, it is possible to separate an ipv-caf vaccine formulation into a vaccine pellet fraction and a supernatant fraction by centrifugation, and subsequently measure ipv (by du elisa) in both fractions. ipv bound to caf will be expected to be located in the vaccine pellet. three mono-ipv-caf vaccines were formulated to evaluate the binding of each ipv type - . the results of the du elisa from centrifuged samples are shown in fig. d . no ipv was found in the supernatant fraction of the three mono-ipv-caf formulations, which indicated that all ipv serotypes are indeed bound to caf . this was in agreement with the zeta potential analysis that suggested a favorable interaction of caf and ipv. on analyzing the resuspended vaccine pellet a recovery rate of - % ipv was obtained (fig. d) . the reason for a recovery of less than % is most probably due to steric hindrance (caused by caf ) of the binding of the detecting antibodies in the ipv du elisa to ipv. however, we cannot fully exclude that some ipv was partly inactivated in the caf formulation. we next examined the ipv-caf formulation in mice. the first objective was to determine the ability of caf to increase the neutralizing antibody titer against polio virus in the blood of vaccinated animals. based on neutralization titers obtained from mice vaccinated with a range of doses from du (d-units) to . du (data not shown) we choose du as a full mouse dose and du as the dose formulated into the caf adjuvant (indicated dose units in the all experiments correspond to polio virus type- d antigen units). thus, the aim was to achieve times dose sparing with caf . mice were immunized with du ipv or with the lower du ipv, the latter with or without caf . the mice received two immunizations spaced by two weeks and neutralizing antibody titers were determined in the sera two weeks after the last immunization. three independent experiments were performed and two assays (performed at statens serum institut, ssi, and center for disease control, atlants, cdc) were used to determine the neutralization titers. figure represents all three experiments, whereas the individual experiments are shown in figure s . the results showed that caf induced a significant increase in neutralization titers to all serotypes (type , type and type , p, . ) (fig. ) . neutralization titers to type increased from . +/ . (log transformed titers) in the du ipv group to (fig. ) . however, two vaccinations with ipv du + caf gave neutralization titers that were not statistically different from vaccinations with ipv du, although there was a trend towards higher titers against type in the ipv alone group that received three vaccinations (fig. ) . in conclusion, addition of the adjuvant caf allowed for a times dose sparing. moreover, only two vaccinations with the caf formulated vaccine were required to achieve the same neutralization titers as observed after three vaccinations with nonadjuvanted ipv. we also examined if the observed increase in neutralization titers correlated with an increase in the antibody binding titer. two weeks after the second vaccination, sera from individual mice were analyzed for ipv specific antibody titers measuring both the different ipv specific igg isotypes (igg , igg a, igg b, igg c), as well as the total igg level against the three virus serotypes. in the ipv du group we observed higher igg titers than in sera from the ipv du against trivalent ipv, or against serotypes and . binding titers against type were not different between the ipv and du groups. importantly, the titers in sera from mice vaccinated with ipv du + caf were higher than both the ipv du group against all the serotypes, and at least as high as the ipv du group (fig. a) . the igg isotypes responsible for the binding titer observed in figure a was also examined. the results showed that caf led to increased antibody titers for the igg isotypes igg a, igg b, and igg c (fig. b) . the increased igg titer observed in the caf group was also observed and weeks following the second vaccination, demonstrating that it was not merely a transient increase (fig. c ). to further characterize the ipv-caf formulation we also analyzed the cellular response against ipv. cellular immunity towards polio virus has received less attention due to compelling evidence that humoral immunity is the most important effector mechanism against polio. however, there are numerous examples that cellular immunity, besides being an effector mechanism in itself, can be important for humoral immunity [ ] [ ] [ ] [ ] [ ] [ ] [ ] , even humoral immunity against polio virus [ , ] . the cellular response was characterized by analyzing the cytokine profile of ipv-specific t cells induced by vaccination with ipv alone or ipv adjuvanted with caf . pbmcs were obtained from mice vaccinated with or du ipv alone or ipv du + caf two and five weeks following the booster vaccination. non-vaccinated mice were included as control. thereafter, the cells were stimulated in vitro with ipv for hours and the supernatant was collected for multiplex cytokine analysis. at week post vaccination, in the ipv non-adjuvanted group, multiplex cytokine analysis showed very low responses for both doses (fig. a) . the highest response was observed with il- ( and pg/ml in the ipv du and du groups, respectively). in contrast, in the ipv du + caf group we observed a significant increase for the cytokines ifn-c, il- , il- , il- , il- and tnf-a but not il . in particular, at week post vaccination caf led to strong induction of ifn-c ( +/ pg/ml versus +/ pg/ml in the ipv du group) and il- ( +/ pg/ml versus +/ in the ipv du group). at week post vaccination, in the ipv alone groups we observed an increased secretion of cytokines ifn-c (to +/ pg/ml in ipv du mice and +/ pg/ml in the ipv du group) and il- ( +/ pg/ml in the ipv du mice and +/ pg/ml mice). in the ipv du + caf we observed an increase in ifn-c (to +/ pg/ml) and il- (to +/ pg/ml), but a significant (p, . ) decrease in secretion of il- , il- , il- , and tnf-a compared to week post vaccination. thus, at week the major secretion of cytokines was observed with ifn-c and il- in all the groups with the strongest responses observed in the caf -adjuvanted group (fig. ) . taken together, the use of caf induced a faster and stronger cellular response compared to non-adjuvanted ipv. interestingly, the cytokine profile of the caf group changed from week to week to resemble a th profile similar to the profile induced by ipv alone (fig. ) . shedding of polio virus is considered the principal marker for protection and studies have implicated intestinal iga as one of the effector mechanisms to reduce shedding of virus [ ] . we therefore examined different vaccine strategies with caf with the aim of generating intestinal iga. more specifically, as previous experiments have indicated that an intradermal vaccine administration may induce intestinal iga [ , ] we decided to first compare intramuscular (im) and intradermal (id) administration. we first examined the iga production in the intestine two weeks following two vaccinations with either ipv du + caf given either id or im. to avoid the risk of blood contamination of the intestinal samples we only examined fecal pellets taken from the colon of sacrificed animals. neither of the administration forms induced intestinal iga (fig. a) . in contrast, both vaccine strategies induced igg in the serum with the im administration being the highest igg inducer (fig. b) . this correlated well with the serum neutralization titers (fig. c) . thus, ipv-caf induced a significant increase in titers compared to both the ipv id group and the ipv im group. this was observed with all the virus serotypes. furthermore, the id administration gave a reduced binding and neutralization titer compared to im administration, suggesting that the id administration is less efficient at priming a protective humoral immune response compared to the im administration. we next decided to test a strategy combining im administration and id administration. of practical importance the animals received ml of the same vaccine in the im and id dose (in contrast to two individually formulated vaccines). as booster vaccination the mice were given an im administration (or another id+im co-administration, data not shown). the results showed that supplementing an id administration with an im administration in the first vaccination induced a strong increase in the iga titer in the feces (fig. a) . in contrast, we did not observe increased fecal igg levels (fig. b) . we next examined if the id+im administration also affected the systemic humoral or cellular response. at week post the final vaccination the animals were sacrificed and blood was analyzed for igg binding titers, neutralization titers, and for cellular immunity. igg titers in both the adjuvanted groups were increased compared to the non-adjuvanted ipv du group and reached levels comparable to the ipv du group (fig. c) . cellular immunity was measured by ifn-c elisa on supernatants from pbmcs stimulated with ipv in vitro. the results showed that the ifn-c response in the ipv du + caf id+im group was as high ( +/ pg/ml) as in the ipv du + caf im group ( +/ pg/ml) and significantly higher than in the non-adjuvanted group (fig. d) . finally, the neutralization titers in the ipv du + caf id+im group was as high as in sera from the ipv du + caf im group, and significantly higher that the non-adjuvanted ipv du group (fig. e) caf id+im group were even significantly higher than titers from the ipv du group demonstrating a dose sparing effect of more than for the ipv du + caf id+im group. taken together, compared to im administration a side by side id+im administration with a caf -adjuvanted vaccine followed by an im administration did not negatively affect systemic immunity, measured by antibody binding and neutralization titers and t cell ifn-c secretion (fig. ) . importantly, in contrast to the ipv du id or ipv du im groups, in the ipv du id+im group we observed a significant increase in fecal iga whereas fecal igg was not observed in either of the vaccine groups. as the id+im group received twice the antigen amount in the first vaccination, we also tested a group that received two im vaccinations at the same time (at two different sites). however, this did not lead to fecal iga (data not shown). finally, we did not observe any significant adverse effects at the id site of administration. the primarily goal of the present study was to examine if the adjuvant caf could be used to achieve dose sparing with ipv, and a subsequent goal was to achieve intestinal immunity with an ipv-caf vaccine. it is well known that a prerequisite for worldwide replacement of opv with ipv is the availability of affordable ipv in low and middle-income countries. lower cost options being pursued include reducing the number of doses and the amount of antigen per dose [ , , , ] . in addition, lowering the manufacturing cost for ipv in developing countries is a priority. our results showed that with the adjuvant caf we were able to achieve a times dose sparing effect in mice measured by the virus neutralization titers in the blood (fig. ) . importantly, the results also showed that two vaccinations with ipv du + caf gave neutralization titers that either equaled those induced by vaccinations with ipv du (type and titers), or were slightly reduced (type titers) (fig. ) . thus, addition of the adjuvant caf induced a significant dose sparing effect, or allowed for a vaccine schedule with one less dose. the potential of applying only two vaccinations compared to three will have significant economical and practical impact. presently, ipv vaccination schedules involve up to doses commonly administered at week , , and at month and . a recent report concluded that children in resource-poor settings probably are protected against paralytic poliomyelitis with as few as two doses of ipv in primary vaccines [ ] . the study was based on ipv vaccines given at month and , i.e. when maternal transferred antibodies have declined and consequently were not expected to inhibit the ipv vaccines. in addition, children receiving a previous opv vaccination were protected with just a single ipv dose most probably due to boosting of a prominent opv induced memory b cell response [ ] . thus, at least with a booster vaccine, the study indicated that one or two vaccinations should protect children in resource-poor settings against paralytic poliomyelitis [ ] . our results indicated that the inclusion of an adjuvant will affect both the required dose (allowing for a reduced ipv dose) and the number of vaccinations (allowing for a vaccine schedule with less vaccine doses) required to induce proper protection against infection with polio virus. the response to ipv and several other vaccines are highly sensitive to the maternal antibody level [ ] [ ] [ ] [ ] [ ] [ ] . there were significantly lower rates of seroconversion for all three poliovirus types in a study performed in thailand and for types and in an oman study including babies from birth and weeks onwards and with children with baseline maternal antibody titres of $ . in addition, previous studies have shown that ipv appears to be more sensitive than opv to the effect of high maternal antibody titers on reducing the neutralizing antibody response to vaccine given in the first months of life [ ] [ ] [ ] [ ] [ ] . however, previous studies have highlighted two important points concerning the antibody response against ipv. one study showed that maternal antibodies inhibited vaccine-induced antibody responses but not t cell responses, and that the latter allowed for high antibody titers following a booster vaccine [ ] . in addition, krishnan et al. [ ] showed that the negative effect of maternal antibodies on the response to ipv may be reduced by increasing the intervals between doses. taken together, and in combination with our results, we suggest that with the inclusion of an adjuvant, such as caf , combined with a booster vaccine given at a longer interval than the commonly used weeks (e.g. weeks as proposed previously [ ] ), a - -fold reduction of the ipv vaccine dose is indeed feasible, even in the presence of maternal antibodies. we also characterized the influence of caf on the immune response against ipv. firstly, the antibody binding titers at week post vaccination were increased to levels that in most cases were higher than the levels induced by a times higher ipv dose ( du) (fig. ) . this was observed against all three polio virus types (fig. a) . interestingly, in contrast to for igg a, igg b, and igg c, we only observed a minor igg response in both adjuvanted and non-adjuvanted groups (fig. b) . in contrast, previous observations with the caf adjuvant formulated with recombinant proteins showed a strong igg response [ ] . this suggested that even in the presence of a strong adjuvant, ipv can influence the igg response. as the igg isotype profile of the ipv-caf vaccine resembled that of ipv alone, this suggested that it is ipv that determines the igg profile even in the presence of caf . the increased antibody response induced by caf also correlated with an increased cellular response measured by multiplex cytokine analysis. although the cellular immune response is not considered a direct correlate of protection for polio vaccines it may play an indirect role by supporting a memory b cell response [ , ] . our analysis of anti-ipv t cell immunity revealed that caf induced both a faster and stronger response against ipv. at week post vaccination only a minor response was observed in the non-adjuvanted ipv groups in contrast to the caf -adjuvanted groups that showed increased levels against all the cytokines tested (fig. a) . the caf adjuvanted group also showed elevated cytokine responses at week post vaccination . interestingly our results also showed that the cytokine profile of the ipv-caf group changed significantly from week to week to resemble the profile induced by ipv alone, a profile dominated by ifn-c and il- secretion (fig. b) . this indicates that ipv as an antigen can also influence the type of cellular immunity induced against it. furthermore, as ifn-c is known to induce isotype switch to igg a, these results also explains the observed induction of igg a, and the lack of igg . thus, both regarding the igg response and the cellular t cell response the role of caf in the formulation with ipv is to induce a response that is quantitatively different from the response induced by ipv but not qualitatively different. the observed t cell profile presented in our study is in agreement with a previous study showing the same cytokine profile following immunization with sabin ipv +/ alum [ ] . interestingly, in that study it was also demonstrated that the t cells were required for the protection against a polio infection, most probably by acting as helper t cells for b cells, and through the stimulation of neutralizing antibody production of the igg a type. in addition, cd t cells have also been shown to be required for the generation of optimal antibody responses following infection with coronavirus [ ] , vaccinia virus [ , ] , yellow fever virus [ ] or vesicular stomatitis virus (vsv) [ ] , supporting that the role of t cells against a viral infection such as polio virus should not be underestimated. we speculate that t cell immunity mediated by the ifn-c/il- expressing th t cells may be important for the protection against the polio virus due to their ability to ) induce isotype switching to igg a and ) induce innate immunity to contributes to antiviral immunity [ , , ] . it is accepted that the major port of entry for the polio virus is the intestinal tract, and that fecal iga is important in preventing entry of the pathogen [ , ] . therefore, inducing intestinal iga is a priority of any polio vaccine. several publications indicate an immunological connection between the intradermal and intestinal site [ , ] . however, in humans, the results obtained with id administration of ipv are not entirely clear. thus, one large-scale pediatric study conducted in cuba by the who initiative for vaccine research [ ] failed to meet non-inferiority criteria, whereas another showed that three immunizations with one-fifth of the dose of ipv given id resulted in similar seroconversion rates compared to three full doses of ipv given im in infants [ ] although the antibody were significantly lower in infants given the decreased id dose of ipv [ ] . thus, the usefulness of the id administration route for ipv is still being debated. the results presented in this paper indicated that as an injection site to prime an immune response, the intradermal administration is not as efficient as the im administration in mice. this was based on experiments showing reduced binding/neutralization titers in the id group compared to the im group, receiving the same vaccine (fig. ). in addition, the id administration did not induce iga in the intestine or in fecal samples ( fig. and data not shown) . however, compared to im or id alone administration, a side-byside id+im administration with a caf adjuvanted vaccine followed by an im administration induced significant levels of fecal iga and, of importance, without compromising serum binding and neutralization titers. although a subject for future investigations, it may be suggested that t cells primed systemically by ipv-caf can be recruited to other sites, e.g. the intestine, and act as helper t cells to potentiate the response at these sites. this may be of some importance as it suggests that a strong intestinal priming may not be entirely required for intestinal protection as long as an intestinal challenge can effectively boost a pre-existing minor intestinal immunity and/or lead to fast recruitment of immunity residing in other (systemic) locations. in fact, in this respect it is of particular interest that some studies have shown that ipv (administered via the im route) may indeed 'prime' substantial protective intestinal immunity [ ] . furthermore, in tropical developing countries, a supplemental dose of ipv administered to children previously exposed to opv boosts humoral and intestinal immunity more effectively than a supplemental dose of opv [ ] [ ] [ ] , ] , which is supported by a study performed in the netherlands [ ] . thus, it seems reasonable to assume that an im vaccination with ipv will induce immunity with some ability to interact with the intestinal site and that a simultaneous priming at both the im and id site with the same vaccine will increase the intestinal response. in conclusion, we have for the first time characterized the formulation of ipv with the caf adjuvant. the formulation proved to constitute a stable formulation where the majority of ipv was associated with caf and maintained its d-unit activity. ipv-caf induced a strong systemic protective immunity in mice and also influenced the kinetics of the ipv immune response to produce a faster response. finally, a simultaneous priming at an intradermal and an intramuscular site with the ipv-caf vaccine led to intestinal immunity. caf has successfully completed several phase i studies with other vaccine candidates, and further studies are now ongoing to support the development the ipv-caf towards a human trial. figure s virus neutralization titers from individual experiments. virus neutralization antibody titers from mice (n = / group) days following the second or the third immunization with inactivated polio vaccine (with or without adjuvant). neutralizing antibody titers against poliovirus serotype - are shown. the individual titers for each mouse are plotted and the bar represents the mean neutralizing antibody titer. sem of poliovirus vaccination options for achieving eradication and securing the endgame vaccine-derived polioviruses and the endgame strategy for global polio eradication implications of a circulating vaccine-derived poliovirus in nigeria oral and inactivated poliovirus vaccines in the newborn: a review a study of poliovaccination in infancy: excretion following challenge with live virus by children given killed or living poliovaccine limitation of fecal and pharyngeal poliovirus excretion in salk-vaccinated children. a family study during a type poliomyelitis epidemic randomized, placebo-controlled trial of inactivated poliovirus vaccine in cuba mucosal immunity and poliovirus vaccines: impact on wild poliovirus infection and transmission adjuvants and inactivated polio vaccine: a systematic review safety and immunogenicity of a subvirion inactivated influenza a/h n vaccine with or without aluminum hydroxide among healthy elderly adults safety and immunogenicity of an inactivated subvirion influenza a (h n ) vaccine dose ranging of adjuvant and antigen in a cell culture h n influenza vaccine: safety and immunogenicity of a phase / clinical trial a vaccine manufacturer's approach to address medical needs related to seasonal and pandemic influenza viruses priming with as a-adjuvanted h n influenza vaccine improves the kinetics, magnitude and durability of the immune response after a heterologous booster vaccination: an open non-randomised extension of a double-blind randomised primary study antigen sparing with adjuvanted inactivated polio vaccine based on sabin strains cpg oligodeoxynucleotides are a potent adjuvant for an inactivated polio vaccine produced from sabin strains of poliovirus cationic liposomes formulated with synthetic mycobacterial cordfactor (caf ): a versatile adjuvant for vaccines with different immunological requirements protection against chlamydia promoted by a subunit vaccine (cth ) compared with a primary intranasal infection in a mouse genital challenge model enhanced humoral and cell-mediated immune responses after immunization with trivalent influenza vaccine adjuvanted with cationic liposomes therapeutic vaccination using cationic liposome-adjuvanted hiv type peptides representing hla-supertype-restricted subdominant t cell epitopes: safety, immunogenicity, and feasibility in guinea-bissau adjuvanted hla-supertype restricted subdominant peptides induce new t-cell immunity during untreated hiv- -infection development and preclinical safety evaluation of a new therapeutic hiv- vaccine based on t-cell minimal epitope peptides applying a novel cationic adjuvant caf caf potentiates immune responses and efficacy of an inactivated influenza vaccine in ferrets vaccines against mucosal infections new advances in mucosal vaccination mucosal immunity and vaccines intradermal delivery of recombinant vaccinia virus vector dis induces gutmucosal immunity the induction of systemic and mucosal immune responses to antigen-adjuvant compositions administered into the skin: alterations in the migratory properties of dendritic cells appears to be important for stimulating mucosal immunity intradermal delivery for vaccine dose sparing: overview of current issues intradermal delivery of vaccines: potential benefits and current challenges the hyvac subunit vaccine efficiently boosts bcg-primed anti-mycobacterial protective immunity characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from m. tuberculosis (trehalose , -dibehenate)-a novel adjuvant inducing both strong cmi and antibody responses cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd + t cells are important in control of sars-cov infection cd + t cells provide intermolecular help to generate robust antibody responses in vaccinia virus-vaccinated humans selective cd + t cell help for antibody responses to a large viral pathogen: deterministic linkage of specificities yellow fever virus encephalitis: properties of the brain-associated t-cell response during virus clearance in normal and gamma interferon-deficient mice and requirement for cd + lymphocytes cooperation of b cells and t cells is required for survival of mice infected with vesicular stomatitis virus functional analysis of influenza-specific helper t cell clones in vivo. t cells specific for internal viral proteins provide cognate help for b cell responses to hemagglutinin antibody production to the nucleocapsid and envelope of the hepatitis b virus primed by a single synthetic t cell site poliovirus-specific cd + th clones with both cytotoxic and helper activity mediate protective humoral immunity against a lethal poliovirus infection in transgenic mice expressing the human poliovirus receptor cellular and humoral immune responses to poliovirus in mice: a role for helper t cells in heterotypic immunity to poliovirus preexisting poliovirus-specific iga in the circulation correlates with protection against virus excretion in the elderly increased potency of an inactivated trivalent polio vaccine with oil-in-water emulsions ) , -dihydroxyvitamin d enhances systemic and mucosal immune responses to inactivated poliovirus vaccine in mice immunogenicity of supplemental doses of poliovirus vaccine for children aged - months in moradabad, india: a community-based, randomised controlled trial inactivated polio vaccine and global polio eradication immunization against hepatitis a in the first year of life: priming despite the presence of maternal antibody hepatitis a vaccination of infants: effect of maternal antibody status on antibody persistence and response to a booster dose influence of maternal antibodies on efficacy of a subunit vaccine: transmission of classical swine fever virus between pigs vaccinated at weeks of age effect of maternally derived antibodies on the clinical signs and immune response in pigs after primary and secondary infection with an influenza h n virus the influence of maternal immunization on light chain response to haemophilus influenzae type b vaccine priming of immune responses to hepatitis b surface antigen in young mice immunized in the presence of maternally derived antibodies influence of maternal antibodies on vaccine responses: inhibition of antibody but not t cell responses allows successful early prime-boost strategies in mice efficacy of inactivated poliovirus vaccine in india cationic liposomes formulated with synthetic mycobacterial cordfactor (caf ): a versatile adjuvant for vaccines with different immunological requirements elisa as a possible alternative to the neutralization test for evaluating the immune response to poliovirus vaccines how to predict the immune status of poliovirus vaccinees? a comparison of virus neutralization at a very low serum dilution versus elisa in a cohort of infants expanding roles for cd (+) t cells in immunity to viruses systematic review of mucosal immunity induced by oral and inactivated poliovirus vaccines against virus shedding following oral poliovirus challenge randomized controlled clinical trial of fractional doses of inactivated poliovirus vaccine administered intradermally by needle-free device in cuba fractional doses of inactivated poliovirus vaccine in oman immunogenicity of a supplemental dose of oral versus inactivated poliovirus vaccine induction of mucosal immunity by inactivated poliovirus vaccine is dependent on previous mucosal contact with live virus the excellent technical assistance provided by lene rasmussen and janne rabech and as well as the animal technicians at the statens serum institut is gratefully acknowledged. we also acknowledge dr. gitte stawski for providing neutralization titers. thanks to dr morten ruhwald for critically reading the manuscript. we thank center for disease control, atlanta (cdc) for performing polio virus neutralization assays. key: cord- -nc v s authors: margolin, emmanuel; burgers, wendy a.; sturrock, edward d.; mendelson, marc; chapman, rosamund; douglass, nicola; williamson, anna-lise; rybicki, edward p. title: prospects for sars-cov- diagnostics, therapeutics and vaccines in africa date: - - journal: nat rev microbiol doi: . /s - - - sha: doc_id: cord_uid: nc v s the emergence of severe acute respiratory syndrome coronavirus (sars-cov- ) has resulted in a global pandemic, prompting unprecedented efforts to contain the virus. many developed countries have implemented widespread testing and have rapidly mobilized research programmes to develop vaccines and therapeutics. however, these approaches may be impractical in africa, where the infrastructure for testing is poorly developed and owing to the limited manufacturing capacity to produce pharmaceuticals. furthermore, a large burden of hiv- and tuberculosis in africa could exacerbate the severity of infection and may affect vaccine immunogenicity. this review discusses global efforts to develop diagnostics, therapeutics and vaccines, with these considerations in mind. we also highlight vaccine and diagnostic production platforms that are being developed in africa and that could be translated into clinical development through appropriate partnerships for manufacture. coronaviruses are ubiquitous rna viruses that are responsible for endemic infections in humans and other animals, and sporadic outbreaks of potentially fatal respiratory disease in humans. four human coronaviruses, namely hcov- e, hcov-oc , hcov-nl and hcov-hku , circulate in the human population, causing the common cold, with some causing potentially life-threatening disease in infants, young children, older individuals and individuals who are immunocompromised . in the recent past, two additional coronaviruses have crossed the species barrier from other animals to infect humans. these are severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), which emerged in and , respectively , . in december , a novel betacoronavirus, subsequently named sars-cov- , was implicated in an outbreak of respiratory disease in wuhan, china . the first cases to be reported presented as atypical pneumonia and were traced to the huanan seafood wholesale market, although cases without any association with the market, and predating the putative index cases, were subsequently recognized. following these first reports, community transmission rapidly ensued, culminating in a global pandemic , . the virus is speculated to have originated in bats and possibly to have passed through another host before infecting humans, but an intermediate host or intermediate hosts have yet to be defined. this remains the subject of considerable debate, and recent work suggests that the host receptor-binding motif of sars-cov- was acquired through recombination with a pangolin coronavirus [ ] [ ] [ ] , but further work is needed to establish the origin of the virus. infection with sars-cov- in humans manifests as coronavirus disease- (covid- ) , a spectrum of disease that ranges from asymptomatic infection to acute respiratory distress syndrome with multisystem involvement. older individuals and individuals with co-morbidities are at greatest risk . in individuals who are symptomatic, fever and cough are most commonly reported, although sore throat, shortness of breath, fatigue, anosmia, dysgeusia and gastrointestinal involvement are also frequently observed , . extrapulmonary manifestations of covid- are being increasingly recognized. among adults with preexisting diabetes mellitus, diabetic ketoacidosis may be a common complication and is associated with a poor prognosis , . according to the international diabetes federation, africa has an estimated . million adults aged between and years living with diabetes and is the region with the highest proportion of undiagnosed diabetes . neurological and neuropsychiatric complications have also been recognized as presenting or complicating factors . children generally have a milder course of disease and are more likely to be asymptomatic, although recent reports have described hyperinflammatory shock in children who were previously asymptomatic that seems similar to kawasaki disease [ ] [ ] [ ] . further studies are required to determine the prevalence of this phenomenon and to define the immunological the first documented cases in a disease outbreak. a rare condition associated with inflammation in blood vessels that most commonly presents in children under years of age. drivers of the illness. however, the contrasting presentation of covid- in children and adults suggests that the immune responses of children and adults to sars-cov- may be different. the virus continued to spread globally, prompting the implementation of radical travel restrictions and social distancing measures . at the time of writing this article, the virus has resulted in over million confirmed infections and has claimed the lives of over , people . as of august , there have been over . million confirmed cases of covid- in africa, with , deaths reported (africa cdc) there is concern that the pandemic may pose an even greater risk to countries in africa owing to their weak health-care infrastructure, large burden of co-infections, including hiv- and tuberculosis, and ongoing outbreaks of emerging and re-emerging infections such as ebola virus (democratic republic of congo) and lassa haemorrhagic fever (nigeria) that will divert much-needed resources away from the fight against covid- (ref. ) ( fig. ). differences in global population demographics and health status are also likely to affect the severity of the pandemic in different regions and are a major concern in africa ( fig. ). in addition to the health-care infrastructure, the general infrastructure throughout africa is also highly variable, and thus access to appropriate medical care is an important determinant of covid- disease outcome. the number of hospital beds in a population of , individuals varies from as low as in mali to in libya. however, libya is an exception for the region, and many central and west african countries are at the lower end of this range and generally report fewer than beds per , individuals. this is in stark contrast to other developed countries such as germany and the usa, where and . beds per , individuals are available, respectively. a similar trend is also seen for the number of doctors per , individuals. in more than african countries, less than doctor is available (per , individuals), whereas germany and the usa report and doctors (per , individuals), respectively . concerns have been raised regarding the impact of the pandemic on other diseases and access to essential medicines . for example, according to a newspaper article, the ministry of health in zimbabwe reported a % increase in malaria infections compared with (ref. ). many african countries lack the capacity to implement widespread testing, including the identification of asymptomatic and mild infections that are major drivers of the pandemic . although it is difficult to determine the number of tests conducted in many africa countries, the publicly available data clearly highlight the limited testing capacity on the continent. south africa is currently conducting the largest number of tests per , individuals ( . / , individuals), whereas many other countries, including ethiopia, nigeria, zimbabwe, tunisia, senegal and rwanda, perform fewer than . tests/ , individuals. this is markedly less than in the usa ( . / , individuals), the uk ( . / , individuals), italy ( . / , individuals) or germany ( . / , individuals) . similar infrastructure limitations constrain the development of prophylactic vaccines and therapeutic interventions, which results in a concerning reliance on developed countries. another important consideration in the response to the pandemic in africa will be to limit the impact of the virus on vulnerable economies where prolonged lockdowns may not be feasible. the first case of covid- in africa was reported in egypt on february ; subsequently, infections - ) pandemic. this is worsened by the high burden of infectious diseases, which may worsen disease outcome and compete for the available resources. a further challenge is the dire economic consequences of prolonged lockdowns in countries with weak economies. www.nature.com/nrmicro have been documented in other african countries, with south africa reporting the highest number of cases . interestingly, in spite of the obvious challenges in combatting the growing pandemic, african countries have observed a delay in the exponential growth trajectory that has been described by countries in the developed world . this may be partly attributable to lower testing capacity in the region and the impact of implementing lockdowns in the early phase of the pandemic. the warmer climate has also been proposed to influence the spread of covid- , which could explain the delayed pandemic in africa compared with the rest of the world, although this is largely speculative (box ). the transition into winter in southern africa has been accompanied by an increase in sars-cov- infections, further complicated by seasonal influenza and limited influenza vaccine availability. in this review, we discuss the global efforts to develop diagnostic tests and therapeutic options to treat covid- , as well as the vaccine platforms for immunization, with a focus on the opportunities and challenges for africa. the diagnosis of sars-cov- poses a major challenge owing to the prevalence of asymptomatic infections, pre-symptomatic infections with high viral loads in the upper airways (probably at peak infectivity) and the range of non-specific symptoms that manifest in individuals who are symptomatic , . widespread testing is therefore critical to identify infected individuals who are asymptomatic, pre-symptomatic and symptomatic, and to enable contact tracing and isolation . whereas this has been highly successful in countries such as germany and south korea, it is not generally possible in most african countries where the infrastructure is weak. indeed, in countries such as south africa, where widespread community testing was attempted, this has resulted in a very large backlog of tests and delays of weeks for returning test results, which are then rendered meaningless for quarantining of cases and containment . in many african countries, testing is only available for severe cases of presumed covid- , and self-isolation is recommended for less severe cases. therefore, reported cases and true prevalence do not equate. accordingly, the capacity provided by academic laboratories and pharmaceutical companies is being leveraged to increase testing capacity further, as has been necessary even in developed countries . diagnosis of acute infection is by pcr with reverse transcription of respiratory tract specimens, which is generally performed in central laboratories with specialized equipment . scale-up of testing is a major challenge for countries in africa, owing to laboratory infrastructure, costs and availability of test reagents that are largely imported and currently stretched global supply chains. a recently launched, continent-wide initiative, population demographics and prevalence of known co-morbidities for each of the six world health organization regions. although africa reports a lower average age compared with other regions, the burden of infectious disease is disproportionately high. both hiv and tuberculosis are associated with an increase in coronavirus disease- (covid- ) disease severity, and their prevalence in africa will increase the risk of fatal infection for a large number of people. there is also a large proportion of individuals in africa with raised blood pressure, which is a known risk factor for severe disease. other known co-morbidities, including raised cholesterol, raised glucose and obesity, are less prevalent in africa compared with the other reported regions. raised blood pressure (systolic blood pressure ≥ mm/hg or diastolic blood pressure ≥ mmhg), raised fasting blood glucose levels (≥ mmol/l or taking medication), raised total cholesterol levels (≥ mmol/l) and body mass index (bmi) > are reflected as age-standardized estimates. all data shown reflect the latest available data from the world health data platform (global health observatory). the number of people living with hiv- /aids (in millions) reflects the population of individuals who were infected in , tuberculosis cases shown reflect the number of incident cases in and malaria cases reflect the estimated number of cases in . nature reviews | microbiology the africa medical supplies platform, seeks to leverage collective purchasing for procurement of testing supplies, personal protective equipment, medical equipment and even, potentially, future vaccines . in addition, repurposing of rapid, automated molecular diagnostics platforms such as genexpert® (cepheid), which is widely used for the diagnosis of tuberculosis in south africa, has the potential to decentralize and accelerate testing in certain countries, including using mobile testing centres, although test kits are also in limited supply. however, this has to be understood in the light of the potential for unintended consequences on the management of tuberculosis, with fewer diagnostic platforms being available as a result of increased sars-cov- testing. testing for tuberculosis in south africa has reportedly decreased by % during the lockdown period and, concurrently, the weekly average of microbiologically confirmed tuberculosis cases decreased by % (ref. ). recently, a rapid method of heating samples prior to quantitative pcr with reverse transcription has shown promise to improve the turnaround time for testing and bypasses the need to order rna extraction reagents or kits . furthermore, a rapid crispr-cas -based test has also been developed to diagnose infection from respiratory sample-derived rna . the test yields a result within h and is less reliant on sophisticated laboratory infrastructure and test reagents that are in limited supply. implementing this test in africa could be a useful way of expanding the current testing capacity and could offer a faster turnaround time for high-priority cases. serology-based testing approaches have been proposed, but the delay between infection and the development of detectable antibodies (within days) renders this approach impractical for the diagnosis of acute infection , . nonetheless, these tests are critical for seroprevalence studies and to identify appropriate donors for convalescent sera, and potentially for the isolation of monoclonal antibodies that can be developed as therapeutics. serology studies are also crucial for understanding the longevity of the antibody response after infection, with the key caveat that it is not known whether humoral responses are a correlate of immunity against the virus. in addition, preliminary data suggest that not all individuals who are infected may seroconvert , and early evidence is emerging that antibody levels may wane rapidly during the convalescent phase . several serological assays have already been developed, and binding antibodies against the spike and nucleocapsid proteins are both indicative of past sars-cov- infection , . many of these assays are also commercially available, but their specificity and sensitivities seem to be variable . a major outstanding question is which antigen, or region of the antigen, is most appropriate for serology testing. most assays have favoured the spike glycoprotein for the detection of an immune response against the virus, although it is worth noting that the nucleocapsid is the most abundant viral antigen . recent work has suggested that the receptor-binding domain alone may be sufficient to detect antibody responses to sars-cov- , and given that it is not conserved between coronaviruses, its use may limit cross-reactivity arising from other coronavirus infections . nonetheless, a nucleocapsid-based elisa (enzyme-linked immunosorbent assay) may be the easiest to implement in an african context as the antigen could easily be produced locally at low cost. nucleocapsid could be produced in escherichia coli, pichia pastoris or even in plants, as has been reported for the nucleocapsid proteins of three bunyaviruses, two of which were used successfully in validated assays [ ] [ ] [ ] . moreover, the biovac institute in south africa has the capacity for bacterial fermentation and the required infrastructure for downstream processing, and a new plant-based production facility (cape bio pharms) is currently generating s protein derivatives as reagents. although the spike glycoprotein is heavily glycosylated and needs to be expressed in a more complex expression host to ensure appropriate post-translational modifications, both mammalian cells and plants would be suitable to produce both spike and nucleocapsid, and novel approaches to enhance recombinant glycoprotein production in plants have also been developed in south africa . given the optimistic development timeline of - months before any vaccines could be available for widespread use, it is clear that these efforts will not box | potential impact of climate on sars-cov- dissemination the comparatively low incidence of coronavirus disease- (covid- ) in africa has raised the possibility that climate could influence the spread of severe acute respiratory syndrome coronavirus (sars-cov- ). there is some circumstantial evidence describing a possible association between higher temperatures and lower severity of covid- to support this hypothesis; however, outbreaks in malaysia, hong kong, australia and south africa seem to be inconsistent with this theory as large numbers of infections have been reported despite higher temperatures [ ] [ ] [ ] . the influence of climate could potentially account for the severity of the pandemic in central china and northern italy, where winter may have been particularly conducive to the spread of the virus . these cold conditions are reminiscent of the environment in which sars-cov first emerged in china in november (ref. ). although these observations are compelling, it is noteworthy that many of these studies have yet to undergo formal peer review, and the accuracy of species distribution models is constrained by variability in global testing capacity . for example, infections in many african countries are expected to be an underestimate that reflects the lower number of tests conducted. it is also acknowledged that numerous other variables could influence the spread of the virus and may confound interpretations of the impact of climate. these variables may include variation in population density and age distribution, timely lockdown measures, adherence to social distancing protocols or even childhood vaccination with mycobacterium bovis bacille calmette-guérin as examples . the impact of differing behaviour, with increased social mixing, in the winter months also cannot be discounted . as with many respiratory pathogens, both middle east respiratory syndrome (mers-cov) and sars-cov exhibit decreased viability in the laboratory following exposure to increasing temperature and humidity , . similar observations have also been reported for influenza virus and respiratory syncytial virus, for which the incidence of infection is highest under cold and dry conditions, which results in seasonal cycles of infection , . a similar seasonality has also been observed for other endemic human coronaviruses, which led to the speculation that sars-cov- may also conform to a seasonal cycle of infection . however, although all four endemic human coronaviruses (hcov- e, hcov-oc , hcov-nl and hcov-hku ) exhibit a marked winter seasonality , the pathogenic human coronaviruses (mers-cov and sars-cov) do not conform to such a defined infection cycle. for example, mers-cov generally occurs mostly during summer months in the middle east despite temperatures often exceeding °c . by contrast, the highest incidence of sars-cov was reported during the winter months, although the outbreak continued to spread throughout spring in hong kong , . therefore, more research is needed to define the impact of climate on the spread of sars-cov- . a diagnostic test that measures the presence of antibodies in blood to determine exposure to pathogens or to diagnosis autoimmune diseases. sera obtained from individuals who have recovered from an infectious disease and contain antibodies against the pathogen. www.nature.com/nrmicro affect the first wave of the pandemic . more importantly, the lack of manufacturing capacity in africa and the global demand for immunization against the virus will further delay the availability of vaccines in the region. repurposing existing drugs presents a feasible short-term strategy to manage the pandemic, especially given that some of the drug candidates are already available and have an established safety profile in humans . these drugs would face lower regulatory barriers for approval and, in addition to being used for treating active infections, may have potential to be used as prophylactics for individuals at high risk, such as health-care workers or those who have been in contact with documented cases of infection. currently, two treatments have been shown to have an effect on the outcome of covid- . the broad-spectrum antiviral drug remdesivir has been shown to shorten the recovery time in adults admitted to hospital with severe covid- in a publication of preliminary results from a double-blind, randomized, placebo-controlled trial in the usa . however, remdesivir did not reduce mortality. by contrast, initial data from the recent recovery trial in the uk suggest that daily oral or intravenous doses of dexamethasone ( mg for days) reduced mortality by one-fifth in hospitalized patients with proven covid- requiring oxygen therapy, and that mortality was reduced by one-third in patients who needed mechanical ventilation . it had no effect on patients hospitalized with covid- who were not requiring oxygen. the reductions in mortality were seen in patients whose symptoms started > days before receipt of the drug. the fact that a commonly used corticosteroid could reduce mortality in this trial is promising, as numerous other corticosteroids such as prednisolone and hydrocortisone (which were options in the recovery trial in pregnant women) are equally available, and some of them are manufactured in africa, which means that access may be less of an issue than for other more novel medicines. more commonly available medicines have been, and some continue to be, used in investigational treatments for covid- . the commonly available antimalarials chloroquine and hydroxychloroquine were among the first to be investigated. initial studies were small and underpowered, and some combined hydroxychloroquine with azithromycin and some proved highly controversial in relation to their conduct, leading to retraction . one of the arms of the recovery trial included hydroxychloroquine, and on june the independent data monitoring committee review of the data concluded that there was no beneficial effect of hydroxychloroquine in patients hospitalized with covid- (ref. ). shortly after, the world health organization (who) announced that recruitment for the hydroxychloroquine arm of the solidarity trial was being stopped , . all experimental treatments should either be introduced into properly conducted clinical trials or, if a country decides to use such a medicine outside a trial, then it should be controlled according to the who's monitored emergency use of unregistered interventions (meuri) framework, whereby it can be ethically appropriate to offer individuals investigational interventions on an emergency basis, in the context of an outbreak characterized by high mortality . large-scale adaptive studies such as the recovery and solidarity trials continue, and such trials will reduce the time taken for randomized clinical trials . several african countries, including south africa, burkina faso and senegal, are in the process of joining the solidarity study. similarly, small studies are ongoing in several countries, looking at the utility of convalescent plasma from patients who recently recovered from covid- as potential prophylaxis or treatment . the need for randomized control trials using this treatment modality has been stressed . unlike other investigational medicines, convalescent plasma can be readily produced, even in low and middle-income countries, through the national blood transfusion service, making it an attractive option for study. however, scaling production for use is the rate-limiting step for this intervention. preliminary studies identified monoclonal antibodies with the ability to neutralize sars-cov- , which may also be important candidates for both treatment and prophylaxis, although similar issues with manufacture are a challenge , . there is increasing recognition that pathophysiology of severe covid- includes an appreciable component of hyperactivation of inflammatory responses, manifesting as a cytokine storm and secondary haemophagocytic lymphocytic histiocytosis. in addition to the findings relating to dexamethasone detailed above, various immune-modulating drugs have been proposed as treatment options for covid- . the il- inhibitors tocilizumab (actemra; roche) and sarilumab (kevzara; sanofi and regeneron), which are used to treat arthritis, are already being used in patients with covid- (nct ) . their mechanism of action involves the prevention and the inhibition of the overactive inflammatory responses in the lungs. both drugs have entered phase iii clinical trials for sars-cov- . a late-stage clinical trial with another il- inhibitor, siltuximab (sylvant; eusa), started in italy in mid-march (nct ). anti-inflammatory drugs used in combination with an antiviral drug such as remdesivir may increase the potential of the drug to improve disease outcome . genentech has recently initiated a phase iii trial (remdecta) to study the efficacy and safety of tocilizumab and remdesivir in patients hospitalized with severe covid- pneumonia (nct ). additionally, the covacta study (nct ) will evaluate tocilizumab and standard of care versus standard of care alone in a similar cohort . patients who have chronic medical conditions may be at higher risk for serious illness from covid- , including those with pulmonary fibrosis . the antifibrotic drug pirfenidone (genentech) has already entered a study to evaluate its efficacy and safety (nct ). recombinant angiotensin-converting enzyme (ace ; apn ) that lacks the transmembrane region of the protein was developed by apeiron biologics for the treatment of acute lung injury and pulmonary artery hypertension. the soluble ace has the potential to reduce lung injury by activating the anti-fibrotic and a disproportionately large cytokine response that promotes inflammation and is harmful to the host. nature reviews | microbiology anti-inflammatory angiotensin ( - )-mas receptor axis of the renin-angiotensin-aldosterone system, and by acting as a decoy and preventing infection by binding to the sars-cov- virus and inactivating it. apn is being tested in a phase i trial in china, and approval has been secured to carry out phase ii trials in austria, germany and denmark (nct ). currently, there are no targeted therapies for covid- . however, numerous drug discovery programmes are in progress, and a recent study reported a structure-based drug design strategy, as well as virtual and high-throughput screening to identify lead compounds that bind to the main protease of the virus (m pro ; also known as cl pro ) . the active site of the protease is highly conserved among coronaviruses, making a strong case for pursuing an m pro -targeting drug. the organoselenium drug ebselen, which is an anti-inflammatory and antioxidant, showed high affinity for m pro and showed promising antiviral activity (concentration that gives half-maximal response ec = . μm). thus, the presented approach may greatly accelerate the discovery of drug leads with potential in the clinic. the drug discovery and development centre (h d) based at the university of cape town is the only fully integrated drug discovery centre in africa that has taken a drug into a phase ii clinical trial. the centre has very strong collaborations with the pharmaceutical industry and mmv, a leading product development partnership, as well as the infrastructure and expertise to find potential therapies against covid- . h d has assembled chemical libraries for its malaria and tuberculosis projects that could be screened to identify possible drug leads against sars-cov- ; however, this will require additional resources and funding because the centre is contractually focused on antimalarial and anti-tuberculosis drug development. the infrastructure for large-scale, high-volume vaccine manufacturing is largely absent in africa, and the rapidly escalating covid- pandemic highlights the urgent need for capital investment in the region to lessen reliance on developed countries. the few facilities that are available are specialized, and are not well-suited to produce vaccines for sars-cov- (table ) . it is also anticipated that it would take a minimum of months to build a suitable manufacturing plant under ideal conditions, and therefore to contribute to the global covid- vaccine initiative, african developers will need to outsource large-scale manufacturing in the short term. the african vaccine manufacturing initiative, which aims to develop local manufacturing capacity in africa, has established a working group and is actively engaged with key stakeholders to meet the local need for a vaccine. innovative biotech (nigeria) has already partnered with medigen (usa) and merck (germany) to apply their insect cell production platform to producing virus-like particles with the intention of initiating a clinical trial in nigeria. similarly, the ethiopian public health institute (ephi) is planning to partner with techinvention (india) to produce the sars-cov- spike protein in a yeast-based fermentation system, although limited details are available (personal communication, s. agwale, ceo of innovative biotech). last, biovac (south africa) has modern facilities at a modest scale and has initiated a feasibility study for a large-scale facility with an annual minimum production capacity of million vaccine doses for covid- and future pandemic vaccines, as well as vaccines for routine immunization use (personal communication, p. tippoo, head of science and innovation, biovac). given the global demand for a covid- vaccine, it is likely that even when a suitable candidate is approved for human use, there will be a considerable delay before it is available in africa. this is not unprecedentedduring the h n influenza pandemic, a global shortage of influenza vaccines resulted in limited supplies being provided for countries in the region, and, in fact, the vaccines only became generally available after (ref. ). this unfortunate, but entirely plausible, scenario may necessitate prioritizing high-risk groups, such as health-care workers and older individuals, to receive the first sars-cov- vaccines to reach africa. more than vaccine candidates are currently in preclinical development around the world, and vaccines are already being tested in clinical trials , (table ) . these vaccines are mostly focused on eliciting immunity against the spike glycoprotein, although other viral antigens may also have a role in vaccine-mediated protection (box ). the speed of clinical deployment of these vaccines is unprecedented, but there are concerns regarding the longevity of immune responses and the potential although the rapid progress to clinical testing is encouraging, it is still too early to determine whether they will confer immunity against sars-cov- infection or whether they will ameliorate the disease course following infection. the only peer-reviewed report of a sars-cov- vaccine in clinical trial to date is for cansino biologics' ad -ncov vaccine, which recently completed phase i testing. encouragingly, the vaccine elicited both binding antibodies and antigen-specific t cells, although, disappointingly, only % of volunteers developed neutralizing antibodies in the low ( × viral particles) and medium ( × viral particles) dose regimens. however, % of the high-dose group ( . × viral particles) developed neutralizing antibodies. perhaps unsurprisingly, the high-dose group also reported a higher incidence of adverse effects following vaccination and only the low and intermediate doses will be pursued in phase ii trials . despite the absence of suitable facilities for current good manufacturing practice (cgmp)-compliant vaccine or therapeutics manufacturing in most of africa, considerable expertise in preclinical vaccine development is also available in academic institutes, and vaccines could be manufactured on contract for clinical trials as was the case for the south african aids vaccine initiative . accordingly, groups at the university of cape town (south africa), the national research centre (egypt) and the kenya aids vaccine initiative (kavi) have all confirmed that early-stage research on sars-cov- vaccine development is underway -although further details have not been disclosed . important considerations for these vaccines will be the cost, their safety in individuals who box | sars-cov- virus structure and targets for vaccine development severe acute respiratory syndrome coronavirus (sars-cov- ) comprises pleomorphic virions, ranging from to nm in diameter, with prominent glycoprotein spike proteins projecting from the virus surface . the virion also contains the membrane, envelope and nucleocapsid proteins, which encapsulate the viral genome and accessory proteins (see the figure, left). the spike protein is a glycosylated type fusion protein that mediates infection by binding the host membrane-anchored angiotensin-converting enzyme (ace ) . the glycoprotein is organized into extracellular (s ) and membrane-spanning (s ) subunits, which mediate receptor binding and membrane fusion, respectively (not shown). binding of the spike protein to ace results in a conformational change that enables the dissociation of the s subunit and the insertion of the fusion peptide into the host membrane . the spike glycoprotein is the primary target of vaccine development, based on the premise that neutralizing antibodies against spike will prevent viral entry into susceptible cells (see the figure, right). this is supported by preclinical immunogenicity studies, for the related middle east respiratory syndrome coronavirus (mers-cov) and sars-cov, for which immunization with spike-based vaccines elicited protective antibody responses , . more recently, neutralizing antibodies against the sars-cov- spike have been reported in natural infection; these are readily elicited and frequently target the receptor-binding domain in s (ref. ). the potential role of cell-mediated immunity in coronavirus vaccines generally has not been as well explored. it is reasonable to expect that cellular immune responses would contribute to viral clearance and ameliorate the severity of the disease, as well as support the development of antibody responses. accordingly, robust and durable cellular responses have been observed against the spike, membrane, envelope and nucleocapsid proteins in patients who recovered from sars coronavirus infection [ ] [ ] [ ] . ultimately, both cell-mediated and humoral responses are desirable in a vaccine, especially given the observation that cellular responses are longer lived than antibodies following infection with sars coronaviruses , . mhc, major histocompatibility complex. humoral immunity: • neutralizing antibodies prevent interaction of spike with ace • antibody effector functions can contribute to viral clearance • b cell memory for durable immunity genetic immunization with plasmid dna is perhaps the easiest vaccine modality to develop for clinical trials as the manufacturing process is well established, the incumbent costs are low compared with other platforms and multiple clinical trials have shown their safety. technological advances have also substantially reduced the time from identifying the viral sequence to initiating immunizations in humans . accordingly, dna vaccines have been advanced into the clinic in response to several emerging pathogens, including mers-cov, and inovio pharmaceuticals (usa) have already completed recruiting participants to initiate a phase i trial with a candidate dna vaccine against sars-cov- (nct ) . recent preclinical data demonstrated that the vaccine elicited neutralizing antibodies in both mice and guinea pigs, and an unrelated study reported that immunization with a dna vaccine protected against viral challenge in macaques , . genetic immunization is well-suited to clinical development for africa, and candidate vaccines could be manufactured to cgmp standards using one of the contract manufacturers offering this service. however, there are no licensed human vaccines based on this platform, and the current delivery methods are not suitable for large-scale immunization. host-restricted viral vectors are another promising vector platform for immunization in africa . replication-deficient chimpanzee adenovirus-based vaccines have shown promise for several emerging viruses, and given their simian origin, they circumvent concerns for vector-specific immunity as was observed when using human adenoviral vectors for immunization . a single dose of a mers-cov- vaccine using this platform was reported to elicit protective immunity in non-human primates . more recently, a single immunization with chadox encoding the sars-cov- spike protected against pneumonia and lowered viral loads in both bronchoalveolar lavage and respiratory tract samples in macaques following challenge . this effect was observed in the absence of high titres of neutralizing antibodies and the impact of the vaccine was to ameliorate severe disease rather than to prevent infection. although it is disappointing that the vaccine did not confer sterilizing immunity in monkeys, it is noteworthy that the monkeys only received a single immunization and that the inoculum used for challenge was high. it should be noted that the high-challenge inoculum was conceived to determine whether immunization resulted in vaccine-mediated enhancement of infection, and that there was no evidence to suggest that this would be a concern . this is the vaccine being pursued by the university of oxford in collaboration with astrazeneca that is now in phase ii testing. a clinical trial for this vaccine has recently been initiated in johannesburg (south africa), and this is the first vaccine for sars-cov- to be tested in africa. the manufacturing cost of chadox would be far less than for a subunit vaccine and, moreover, no adjuvant is needed for immunization. poxvirus-based vectors are similarly attractive: they elicit strong humoral and cellular immune responses, can be manufactured at low cost and are stable in the absence of a sustained cold chain , . in addition, they can accommodate larger genetic insertions, which could be exploited to encode multiple sars-cov- genes (such as the spike, nucleocapsid, membrane and envelope antigens) and could potentially produce virus-like particles. suitable examples of candidate poxvirus vectors include the attenuated orthopoxviruses modified vaccinia ankara (mva) and nyvac , the avipoxviruses canarypox virus (alvac) and fowlpox virus (fwpv) , and the capripoxvirus lumpy skin disease virus (lsdv) . mva is the most widely explored of these vectors. having been attenuated by more than passages in chick embryo fibroblast cells, mva has a well established safety record, including in individuals who are immunocompromised, and has recently been approved as a vaccine against smallpox , . nyvac was engineered by the purposeful deletion of genes involved in host range and pathogenicity; it causes no disseminated disease in immunodeficient mice, like mva, and is unable to replicate in humans . several mva-vectored vaccines of particular relevance to africa have shown promise in clinical trials, usually in prime-boost regimens together with other vectors such as dna or adenovirus. these include vaccines against hiv- (ref. ), mycobacterium tuberculosis and box | immunological challenges for sars-cov- vaccine development two concerns have been raised that could undermine the vaccines against severe acute respiratory syndrome coronavirus (sars-cov- ) in clinical testing: the longevity of immunity, and the potential for adverse effects following sars-cov- infection in immunized volunteers. the durability of antibody responses has implications for vaccine development, as immunization may need to induce stronger immunity than natural infection. this concern is partly due to observations of waning neutralizing antibody titres after sars coronavirus infection, and a lack of knowledge regarding the potential for sars-cov- re-infection [ ] [ ] [ ] . encouragingly, preliminary data suggest that rhesus macaques may be resistant to challenge with sars-cov- after clearing the primary infection . the duration of this protection remains unclear, as do the correlates of immunity. low neutralizing antibody titres were recently reported in % of patients who recovered from mild infection with sars-cov- , which suggests that cellular responses may have an important role in viral clearance. however, it is plausible that neutralizing antibody titres correlate with disease severity and merely reflect the extent of antigenic stimulation . another concern is vaccine-induced enhancement of infection. this can manifest as either antibody-dependent enhancement or cell-mediated inflammatory responses that result in pathology following exposure to the virus. accordingly, type t helper cell-mediated lung pathology with eosinophilic infiltrates has been observed in vaccinated and challenged animals for both middle east respiratory syndrome coronavirus (mers-cov) and sars-cov [ ] [ ] [ ] . the potential impact of antibodydependent enhancement in the context of coronavirus vaccines has not been as well defined, although the phenomenon has been described for a monoclonal antibody targeting the mers coronavirus spike glycoprotein . preliminary data suggest that antibody-dependent enhancement may account for the severity of covid- in some cases, where previous exposure to other coronaviruses may have elicited responses that enhanced infection, although this remains to be determined . www.nature.com/nrmicro ebola virus . lsd, a notifiable disease of cattle worldwide, is prevalent in most african countries, and the live-attenuated neethling vaccine strain is widely used to control the disease on the continent . lsdv is being developed both as a multivalent cattle vaccine vector , and as a host-restricted hiv- vaccine vector . it has been shown to have no adverse effects in immunodeficient mice, and although this vector could not be used in countries free of lsdv, it has potential as a human vaccine in sub-saharan africa . together with mva and nyvac, the avipoxvirus vectors alvac (attenuated canarypox virus) and fwpv are probably more realistic targets for rapid clinical development, as they have also undergone testing in humans, and alvac is already licensed for several veterinary applications plant-based vaccine protein production is an emerging technology that is well-suited to resource-limited areas given the capacity of the system for rapidly scalable production, the low manufacturing costs and the less sophisticated infrastructure requirements than mammalian expression systems . the platform is well established to produce diverse classes of recombinant proteins, and recent advances in expression technologies and molecular engineering have also enabled improvements in glycoprotein production in plants , . encouragingly, a preliminary pilot study suggests that these appro aches can be applied to produce the sars-cov- spike in nicotiana benthamiana plants, warranting further testing of the recombinant antigen in preclinical vaccine immunogenicity models . three leading plant biotechnology companies, medicago inc. (canada), ibio inc. (usa) and kentucky bioprocessing inc. (usa), have already announced the successful production of candidate virus-like particle vaccines against sars-cov- . although plant-based manufacturing of recombinant protein antigens may be the most suitable solution for africa, it may also pose a challenge for manufacturing. the major advantages of plant-based vaccine production for sars-cov- in africa are the lower costs and the potential for rapid production scale-up to accommodate the large demand for a vaccine. this is best demonstrated in the context of influenza vaccine development, as a fully formulated virus-like particle vaccine was produced within weeks following release of the viral sequence . this rapid development timeline supported the production of million doses of the vaccine within month . however, despite the costs to establish a gmp-compliant plant-based manufacturing facility being considerably less than those for the equivalent mammalian platform (for example, us$ - million versus us$ - million, respectively), they are not insignificant, and the capital investment required has been prohibitive for africa . furthermore, there are few suitable contract manufacturing organizations worldwide, and these are already invested in their own sars-cov- vaccine development programmes. several recent preliminary data have suggested a possible correlation between bacille calmette-guérin (bcg) vaccination and lower prevalence and mortality due to covid- (refs [ ] [ ] [ ] [ ] ). the bcg vaccine is one of the most widely used vaccines worldwide and has been used to vaccinate against tuberculosis for nearly years. the vaccine comprises a live, attenuated form of mycobacterium bovis, which provides protection against disseminated forms of tuberculosis in infants but gives variable protection against pulmonary tuberculosis in adults , . non-specific cross-protection against other pathogens, including those causing respiratory tract infections, has also been documented . this effect may be attributable to altered expression of host cytokines and pattern-recognition receptors, as well as the reprogramming of different cellular metabolic pathways that, in turn, increases the innate immune response to other pathogens , . however, potential correlation between bcg vaccination and covid- severity should be interpreted with caution. first, it is unlikely that bcg vaccination at birth will still provide non-specific cross-protection against viral pathogens in older individuals. second, the correlation could be influenced by numerous unknown confounding factors, including variation in testing between countries, which leads to differences in the recorded case numbers; differences in average population age, ethnic and genetic backgrounds; the stage of the pandemic in each country; and different approaches to mitigating the spread of the disease in different countries. numerous clinical trials are presently underway to determine whether bcg vaccination reduces the incidence and severity of covid- in health-care workers and older individuals (supplementary table) . a trial has also started in egypt (nct ), where disease severity and mortality in patients with covid- will be compared between those with positive and negative tuberculin tests. in brazil, the bcg vaccine will be given to patients with covid- as a therapeutic vaccine to evaluate the impact on the rate of elimination of sars-cov- , the clinical evolution of covid- and the seroconversion rate and titres of anti-sars-cov- antibodies. as the bcg vaccine has been administered to most neonates in south africa and france until recently, these trials will also investigate the effect of revaccination with the bcg vaccine. in addition, a new modified version of bcg, namely vpm , which expresses listeriolysin instead of urease c, will be tested in health-care workers and older individuals in germany . securing a reliable supply of bcg vaccine doses could be a challenge in africa if re-immunization shows promise, as there is limited manufacturing capacity for the vaccine on the continent. historically, shortages of the vaccines were documented in % of countries on the continent between and (ref. ). this was largely due tuberculosis diagnostic tests that involve the intradermal injection of bacterial antigens to determine whether the recipient mounts an immune response at the site of injection. nature reviews | microbiology to lack of supply, but the limited availability of financing, procurement shortcomings and ineffective vaccine management also contributed to the shortage. the low price for a bcg vaccine and limited investment has also reduced the incentive for manufacturers to redesign and improve production processes in the region. from to , the -tokyo bcg strain was produced at the state vaccine institute in cape town, south africa; however, this was discontinued as the cost of importing the vaccine was lower than that of local manufacture. the potential impact of co-infections africa shoulders a considerable burden of co-infections. although hiv- and tuberculosis may be the most important infections when considering potentially enhanced covid- disease severity, the high incidence of malaria and helminth infections as well as multiple ongoing outbreaks of ebola virus disease, lassa fever, cholera, measles, yellow fever, hepatitis e and chikungunya virus all represent infections with unknown interactions with sars-cov- . the high prevalence of hiv- and tuberculosis in sub-saharan africa presents an important but largely unknown challenge for the continent with regard to covid- . the urgent question that needs answering is whether individuals with hiv- , or those with past or current tuberculosis, have a higher risk of infection or greater morbidity and mortality from covid- . of the . million people living with hiv- globally, . million live in sub-saharan africa, and it is estimated that % are accessing antiretroviral therapy and % are virally suppressed . although individuals who are immunocompetent with well-controlled hiv- infections may be at no greater risk for covid- , there remains a considerable number of individuals with low cd counts and uncontrolled hiv- viraemia who may be at risk of severe disease. to date, there have been two published reports of concurrent covid- and hiv- infection , . although the cohort was an extremely limited group of patients predominantly established on antiretroviral therapy, the pattern of clinical disease did not differ from that observed in the general population, but more research is needed to confirm this result. the severity of other respiratory infections concomitant with hiv- may provide some clues: although the immunopathogenesis of sars-cov- is probably distinct in several aspects from influenza viruses, there are some shared clinical features. hiv- infection is associated with a greater susceptibility to influenza virus infection, increased severity of influenza-related disease and poorer prognosis in patients who are severely immunocompromised . a large south african study observed an eightfold higher incidence of influenza virus infection and a fourfold greater risk of death in the case of hiv- co-infection . paradoxically, there is also evidence that lower inflammatory responses in individuals who are immunocompetent and infected with hiv- may lead to milder influenza-related disease . in addition to altering the clinical course of disease, hiv- infections may result in poorer antibody responses that may lead to prolonged viral shedding, thereby influencing disease transmission . tuberculosis, a disease that causes chronic lung damage, may also present a challenge in the covid- era. there were approximately . million new cases of tuberculosis in africa in (ref. ). in a south african study of patients who were hospitalized for severe respiratory illness, those with influenza virus infection together with laboratory-confirmed tuberculosis had a . -fold greater risk of death . hiv- largely drives the tuberculosis epidemic in sub-saharan africa, and the 'triple-hit' of hiv- , tuberculosis and sars-cov- infection is consequently of considerable concern. a preliminary study suggests that hiv- infection increases the risk of mortality from covid- by . -fold, and this increased risk seemed to be independent of suppressed hiv- viral load due to antiretroviral therapy. individuals with current tuberculosis had a . -fold greater risk of death . these figures represent a modest increased risk compared with older age and co-morbidities such as diabetes in the same population, which suggests that hiv- and tuberculosis may not be considered major risk factors for covid- . although this would be considered good news, further studies are awaited to confirm these initial observations. the two main potential issues for using sars-cov- vaccines in individuals infected with hiv- are safety and efficacy. however, potential safety issues are likely to be restricted to use of certain vaccine modalities, such as live-attenuated or replicating vaccines, in individuals who are highly immunosuppressed. when considering vaccine efficacy, the magnitude and durability of immunity in individuals infected with hiv- for both vaccination against and natural infection with sars-cov- is unknown. to date, there are no reports describing immune responses to sars-cov- in individuals infected with hiv- . it is possible that individuals with hiv- may have incomplete immune reconstitution and impaired immunity that may influence vaccine safety and efficacy, even if they are receiving antiretroviral therapy, owing to persistent immune activation and incomplete recovery of t cell and b cell immunity , . suboptimal neutralizing antibody responses have been described following immunization against influenza virus or other pathogens in individuals infected with hiv- (ref. ). weaker antibody responses and lower influenza virus-specific memory b cell responses in individuals infected with hiv- were directly related to cd counts . it will be important to test candidate vaccines for their ability to generate immune responses in a range of high-risk groups, including patients with hiv- . several strategies may improve the magnitude and durability of vaccine responses in individuals infected with hiv- , such as higher doses, booster immunizations and/or the use of adjuvants . substantive data on the clinical and immunological interaction of hiv- , tuberculosis and covid- will emerge from africa in time for improved strategies to guide clinical management of patients who are co-infected and the vaccine regimens. finally, an important additional point to note is the indirect effects of covid- on health in africa within the setting of a high burden of infectious diseases. the who estimates that the disruption in vaccination due to www.nature.com/nrmicro disruption in supply could put million infants at risk of contracting vaccine-preventable diseases . several countries have reported reduced uptake of tuberculosis testing, and patients failing to collect tuberculosis medication or antiretroviral therapy owing to overwhelmed health-care systems, lockdown interventions and public fear of contracting covid- (ref. ). mitigating these interruptions in prevention, diagnosis and treatment, and ensuring that essential health services continue, will ultimately lower the overall impact of the covid- pandemic in africa. the ongoing covid- pandemic presents an unprecedented global humanitarian and medical challenge. although this has prompted unparalleled progress in the development of vaccines and therapeutics in many countries, it has also highlighted the vulnerability of resource-limited countries in africa. not only do these countries have limited testing capacity but the infrastructure to manufacture tests, vaccines and therapeutic drugs is largely absent, and few clinical trials are underway on the continent to combat sars-cov- . clearly, there is an urgent need for capacity development and the available resources should focus on solutions that are specific to the needs of the continent. for example, there is an urgent need to inexpensively manufacture viral antigens for serological testing: this will determine the seroprevalence of the virus where pcr-based testing is not available for mild infections. therapeutics development should focus on repurposing existing drugs, or using convalescent plasma that can rapidly be used to treat infection and could be prioritized for individuals who are at high risk. appropriate manufacturing partnerships need to be established to produce vaccines that could be tested and licensed on the continent, to limit reliance on global initiatives that may be overwhelmed by the global demand for a vaccine. in fact, this may present an opportunity for governments to finally invest in much-needed cgmp-compliant vaccine manufacturing facilities. although the situation is unquestionably dire, africa has an important role in the global fight against covid- , and the resilience and resourcefulness of the people are not to be underestimated. published online xx xx xxxx epidemiology, genetic recombination, and pathogenesis of coronaviruses identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a novel coronavirus from patients with pneumonia in china epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study an interactive web-based dashboard to track covid- in real time a pneumonia outbreak associated with a new coronavirus of probable bat origin probable pangolin origin of sars-cov- associated with the covid- outbreak emergence of sars-cov- through recombination and strong purifying selection clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of coronavirus disease in china covid- infection may cause ketosis and ketoacidosis diabetic ketoacidosis in covid- : unique concerns and considerations neurological and neuropsychiatric complications of covid- in patients: a uk-wide surveillance study hyperinflammatory shock in children during covid- pandemic kawasaki-like disease: emerging complication during the covid- pandemic sars-cov- infection in children the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak looming threat of covid- infection in africa: act collectively, and fast hospital beds (per population covid- : keep essential malaria services going during pandemic, urges who zimbabwe under strain as malaria cases surge during covid- fight substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) coronavirus (covid- ) testing. global change data lab covid- in europe: the italian lesson feasibility of controlling covid- outbreaks by isolation of cases and contacts south africa's coronavirus testing strategy is broken and not fit for purpose: it's time for a change let africa into the market for covid- diagnostics detection of novel coronavirus ( -ncov) by real-time rt-pcr african countries unite to create 'one stop shop' to lower cost of covid- tests and ppe covid- lockdowns in low-and middle-income countries: success against covid- at the price of greater costs an alternative workflow for molecular detection of sars-cov- -escape from the na extraction kit-shortage crispr-cas -based detection of sars-cov- antibody responses to sars-cov- in patients with covid- antibody tests for identification of current and past infection with sars-cov- dynamics of igg seroconversion and pathophysiology of covid- infections rapid decay of anti-sars-cov- antibodies in persons with mild covid- sars-cov- seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease patients evaluation of nine commercial sars-cov- immunoassays developing antibody tests for sars-cov- the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov- patients plant-produced crimean-congo haemorrhagic fever virus nucleoprotein for use in indirect elisa expression of rift valley fever virus n-protein in nicotiana benthamiana for use as a diagnostic antigen the expression of sars-cov m gene in p. pastoris and the diagnostic utility of the expression product co-expression of human calreticulin significantly improves the production of hiv gp and other viral glycoproteins in plants vaccines: status report a sars-cov- protein interaction map reveals targets for drug repurposing remdesivir for the treatment of covid- -preliminary report dexamethasone in hospitalized patients with covid- -preliminary report hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial retraction-hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis no clinical benefit from use of hydroxychloroquine in hospitalised patients with covid- . nuffield department of population health solidarity" clinical trial for covid- treatments hydroxychloroquine use against sars-cov- infection in non-human primates world health organization guidance for managing ethical issues in infectious disease outbreaks race to find covid- treatments accelerates effectiveness of convalescent plasma therapy in severe covid- patients deployment of convalescent plasma for the prevention and treatment of covid- neutralizing antibody responses to sars-cov- in a covid- recovered patient cohort and their implications human neutralizing antibodies elicited by sars-cov- infection effective treatment of severe covid- patients with tocilizumab covid- : combining antiviral and anti-inflammatory treatments phase iii trial to study combination of genentech's actemra, gilead's remdesivir versus severe covid- pulmonary fibrosis and covid- : the potential role for antifibrotic therapy structure of m pro from covid- virus and discovery of its inhibitors setting up a platform for plant-based influenza virus vaccine production in south africa the covid- vaccine development landscape covid- vaccine development pipeline gears up safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial subtype c gp vaccine boosts immune responses primed by the south african aids vaccine initiative dna-c and mva-c hiv vaccines after more than a -year gap african nations missing from coronavirus trials novel vaccine technologies: essential components of an adequate response to emerging viral diseases new vaccine technologies to combat outbreak situations immunogenicity of a dna vaccine candidate for covid- dna vaccine protection against sars-cov- in rhesus macaques the evolution of poxvirus vaccines chimpanzee adenoviral vectors as vaccines for outbreak pathogens a single dose of chadox mers provides protective immunity in rhesus macaques chadox ncov- vaccine prevents sars-cov- pneumonia in rhesus macaques vaccinia-based vaccines to biothreat and emerging viruses the smallpox vaccination strain mva: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defence mechanism the poxvirus vectors mva and nyvac as gene delivery systems for vaccination against infectious diseases and cancer applications of canarypox (alvac) vectors in human and veterinary vaccination fowlpox virus as a recombinant vaccine vector for use in mammals and poultry immunogenicity of a recombinant lumpy skin disease virus (neethling vaccine strain) expressing the rabies virus glycoprotein in cattle vaccination against pox diseases under immunosuppressive conditions phase efficacy trial of modified vaccinia ankara as a vaccine against smallpox nyvac: a highly attenuated strain of vaccinia virus aiming for protective t cell responses: a focus on the first generation conserved-region hiv consv vaccines in preventive and therapeutic clinical trials a phase iia trial of the new tuberculosis vaccine, mva a, in hiv-and/or mycobacterium tuberculosis-infected adults safety and immunogenicity of a -dose heterologous vaccine regimen with ad .zebov and mva-bn-filo ebola vaccines: -month data from a phase randomized clinical trial in nairobi, kenya review: capripoxvirus diseases: current status and opportunities for control evaluation of lumpy skin disease virus, a capripoxvirus, as a replication-deficient vaccine vector a novel candidate hiv vaccine vector based on the replication deficient capripoxvirus, lumpy skin disease virus (lsdv) development and registration of recombinant veterinary vaccines. the example of the canarypox vector platform molecular pharming for low and middle income countries when plant virology met agrobacterium: the rise of the deconstructed clones calreticulin co-expression supports high level production of a recombinant sars-cov- spike mimetic in nicotiana benthamiana the production of hemagglutininbased virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza darpa's blue angel -pentagon prepares millions of vaccines against future global flu correlation between universal bcg vaccination policy and reduced morbidity and mortality for covid- : an epidemiological study connecting bcg vaccination and covid- : additional data differential covid- -attributable mortality and bcg vaccine use in countries association of bcg vaccination policy with prevalence and mortality of covid- systematic review and meta-analysis of the current evidence on the duration of protection by bacillus calmette-guérin vaccination against tuberculosis the efficacy of bacillus calmette-guérin vaccination of newborns and infants in the prevention of tuberculosis: meta-analyses of the published literature non-specific effects of bcg vaccine on viral infections bcg-induced cross-protection and development of trained immunity: implication for vaccine design safety and immunogenicity of the recombinant mycobacterium bovis bcg vaccine vpm in hiv-unexposed newborn infants in south africa bacillus calmette-guérin (bcg) vaccine: a global assessment of demand and supply balance outbreaks and emergencies bulletin covid- in patients with hiv: clinical case series co-infection of sars-cov- and hiv in a patient in wuhan city impact of hiv on the severity of influenza severe influenza-associated respiratory infection in high hiv prevalence setting influenza viral shedding in a prospective cohort of hiv-infected and uninfected children and adults in provinces of south africa who. global tuberculosis report the impact of influenza and tuberculosis interaction on mortality among individuals aged ≥ years hospitalized with severe www.nature.com/nrmicro respiratory illness in south africa hiv and risk of covid- death: a population cohort study from the western cape province, south africa effect of antiretroviral therapy on the memory and activation profiles of b cells in hiv-infected african women residual t cell activation and skewed cd + t cell memory differentiation despite antiretroviral therapy-induced hiv suppression immunization for hiv-positive individuals compromised b cell responses to influenza vaccination in hiv-infected individuals vaccination in hiv-infected adults at least million children under one at risk of diseases such as diphtheria, measles and polio as covid- disrupts routine vaccination efforts, warn gavi, who and unicef potential impact of the covid- pandemic on hiv, tuberculosis, and malaria in low-income and middle-income countries: a modelling study evidence that higher temperatures are associated with lower incidence of covid- in pandemic state, cumulative cases reported up to distribution of the sars-cov- pandemic and its monthly forecast based on seasonal climate patterns role of meteorological temperature and relative humidity in the coronavirus as a possible cause of severe acute respiratory syndrome species distribution models are inappropriate for covid- spatial modeling cannot currently differentiate sars-cov- coronavirus and human distributions on the basis of climate in the united states seasonality of respiratory viral infections stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions the effects of temperature and relative humidity on the viability of the sars coronavirus absolute humidity and the seasonal onset of influenza in the continental united states epidemic dynamics of respiratory syncytial virus in current and future climates epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study molecular evolution of the sars coronavirus during the course of the sars epidemic in china epidemiology, transmission dynamics and control of sars: the - epidemic cryo-em structure of the -ncov spike in the prefusion conformation tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion a dna vaccine induces sars coronavirus neutralization and protective immunity in mice evaluation of candidate vaccine approaches for mers-cov persistent memory cd + and cd + t cell responses in recovered severe acute respiratory syndrome (sars) patients to sars coronavirus m antigen human memory t cell responses to sars-cov e protein long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients the host immune response in respiratory virus infection: balancing virus clearance and immunopathology virus-specific memory cd t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection disappearance of antibodies to sars-associated coronavirus after recovery two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome positive rt-pcr test results in patients recovered from covid- primary exposure to sars-cov- protects against reinfection in rhesus macaques immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge molecular mechanism for antibodydependent enhancement of coronavirus entry medical countermeasures analysis of -ncov and vaccine risks for antibody-dependent enhancement (ade) the authors acknowledge support from the south african medical research council with funds received from the south african department of science and technology, core funding from the wellcome trust ( /z/ /z) and funding from the south african research chairs initiative of the department of science and technology and national research foundation (grant number ). the authors contributed equally to all aspects of the article. the authors declare no competing interests. nature reviews microbiology thanks m. baylis, g. dougan, s. jiang and l. f. p. ng for their contribution to the peer review of this work. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. supplementary information is available for this paper at https://doi.org/ . /s - - - . key: cord- - sctqkwr authors: alcamí, josé; joseph munné, joan; muñoz-fernández, maría Ángeles; esteban, mariano title: current situation in the development of a preventive hiv vaccine date: - - journal: enfermedades infecciosas y microbiología clínica doi: . /s - x( ) - sha: doc_id: cord_uid: sctqkwr the uncontrolled progression of the aids epidemic has made the development of an efficacious human immunodeficiency virus (hiv) vaccine a major objective of scientific research. no effective preventive vaccine against hiv is currently available and sterilizing immunity has not yet been achieved in animal models. this review analyses the major challenges in developing an aids vaccine, in particular the mechanisms involved in viral escape from the immune response, and summarizes the results obtained with the different prototypes of therapeutic and preventive vaccines. finally, social, economic and healthcare aspects of research into hiv vaccines and current controversies regarding the development of clinical trials are discussed. in the year , the aids pandemic was responsible for more than three million deaths, and five million people are calculated to have contracted the virus during this period. this takes the number of infected people throughout the world to million, with million deaths registered since the origin of the pandemic was identified . the geographical and economic differences in this disease are obvious, with more than % of cases and deaths by aids occurring in the third world ( % in africa), especially among young adults, with a gradual increase in women. it is dramatic to see how, in sub-saharian africa, the epidemic continues to spread and, in many countries, the high percentage of people infected with aids has devastating effects on families and on the productive economy. the explosion of this epidemic in developing countries has raised the need to take urgent preventive measures and provide expanded access to antiretroviral therapy. however, in some parts of the world, these measures, although essential, are probably insufficient to curb the epidemic, so that the development of a vaccine is the only available possibility of controlling it. therefore, the development of an efficacious hiv vaccine is not only an area of aids research which has yet to be resolved but also an urgent need for developing countries. this awareness has led to a considerable increase in financing the search for an aids vaccine. to obtain an effective hiv vaccine represents a major challenge and priority scientific objective for public and private institutions, governments and ngos [ ] [ ] [ ] (table ) . this chapter analyses prototype vaccines being developed and the results obtained, the scientific difficulties in developing a vaccine, especially with regard to the mechanisms of viral escape from the immune response. furthermore, as this research is set in the social, economic and healthcare context of the aids pandemic, current controversies surrounding clinical trials with the different types of vaccine are addressed in this review. situación actual en el desarrollo de una vacuna preventiva frente al vih el avance de la epidemia de sida ha convertido la obtención de una vacuna eficaz frente al virus de la inmunodeficiencia humana (vih) como un objetivo científico prioritario. en el momento actual no disponemos de una vacuna preventiva frente a la infección por el vih y en ningún modelo animal se ha conseguido la protección frente a la infección. en esta revisión se analizan las dificultades existentes en el desarrollo de una vacuna contra el sida, en especial los mecanismos de escape viral a la respuesta inmunitaria y se describen los prototipos de vacunas preventivas y terapéuticas en desarrollo y los resultados obtenidos. por otra parte se sitúa esta investigación en el contexto sanitario, económico y social de la pandemia de sida y se analizan las polémicas in order to develop a vaccine, it is necessary to know the genes of the pathogen involved in the induction of a specific immune response, and to use experimental models to test the efficacy of the virus. despite the fact that, in the past, there have been important advances in the control of infectious diseases by vaccination, many of the mechanisms used by the pathogen to take over cell machinery are unknown. the same applies to mechanisms of escape from the immune system and ways of boosting an immune response capable of eliminating the infected cell. in scientific terms, obtaining an efficacious vaccine capable of preventing hiv infection faces a series of challenges: the immune response begins with the recognition by cd lymphocytes via their receptor (tcr) of foreign antigens in the class-ii hla groove presented by cells specialized in antigen processing. antigen recognition activates the different effector mechanisms of the immune system: cytokine and chemokine synthesis, production of antibodies by b lymphocytes, activation of cd lymphocytes and generation of cytotoxic cd lymphocytes. these last events represent the main mechanism involved in killing of virus-infected cells and to initiate this process cytotoxic lymphocytes must recognize the antigenic determinants of the virus lodged in the class-i hla groove of infected cells . as a consequence of the polymorphism of the hla system both in antigen presenting cells and target cells different peptides (that are anchored in class ii and class i hla molecules respectively) are selected according to individual hla haplotypes. in many viruses there are "major immunodominant determinants" (epitopes) which induce a potent response by the immune system. the efficacy of this response depends on two characteristics: first, they correspond to epitopes or domains of the viral proteins which are conserved among the different isolates, even in the context of highly variable viruses; second, these major determinants are capable of binding to the grooves of most hla haplotypes. the existence and identification of these major immunogenic determinants is crucial when developing a vaccine, since they make up viral targets par excellence by being "universal" in two senses: as epitopes conserved in the viral protein among different isolates and as epitopes susceptible to antigenic presentation by most subjects regardless of their hla haplotype. however, in the case of hiv, no similar dominant epitopes have been found to date, which represents a very important limitation when designing a vaccine. the presence of these major immunogenicity determinants in viruses with tremendous genetic variability, such as hiv- , makes it practically impossible to define these epitopes empirically and experimentally. nevertheless, bioinformatics can provide us with predictive methods which make identification easier . the objective of a vaccine is to induce an efficacious, memory-type immune response which allows the immune system to react against the infectious agent by preventing its spread. for this objective to be reached, it is essential to know which immunological effectors are efficacious in the control of the infection in order to define a series of surrogate immunological parameters which enable us to evaluate whether a vaccine preparation is efficient or not. the intense immune response of hiv-infected patients is reported to encompass practically all the effector mechanisms of the immune system. this response is relatively wide, since it is developed against numerous epitopes, and practically all the viral proteins, both structural and regulatory, are recognized as foreign antigens. however, controversy still surrounds the role of "protection" played by each of these components of the antiviral response. below, we describe the type of immune response generated against hiv infection. hiv infection induces an intense antibody response against practically all the regulatory and structural proteins of hiv . some of these antibodies have neutralizing capacity in vitro and in in vivo adoptive immunotherapy experiments , . however, the production of antibodies with neutralizing capacity is scarce and viral escape from these antibodies is rapid . furthermore, in the immunization models developed to date, high levels of neutralizing antibodies are not consistently obtained and their presence is not systematically associated with protection. these data raise severe concerns about the role of the humoral response in the control of hiv infection , . nevertheless, almost all preventive vaccines induce neutralizing antibodies and their role as a surrogate protection marker is clearly demonstrated in other diseases. therefore, "a priori", a preventive hiv vaccine should induce broad-spectrum neutralizing antibodies and this is one of the huge challenges currently facing the development of an aids vaccine. recent studies which define the loca- tion of neutralization epitopes, antibody structure and mechanism of action represent an important advance to define the characteristics that a given vaccine must have to induce neutralizing antibodies. most studies agree that the combined response of cd and cd is probably the most important protective mechanism against hiv . study of the cellular response has shown that in seropositive patients there is a clonal expansion of cd and cd lymphocytes which are active against hiv. this expansion is particularly intense in patients with primary infection and correlates with the control of viral replication , . there are also reports showing an intense cd and cd response in some patients during immune reconstitution after antiretroviral therapy, especially in those with a good immunological status before starting therapy , . the same phenomenon has been described in patients with structured treatment interruptions who spontaneously control viral replication . although it is difficult to draw conclusions as to a cause-effect relationship between the appearance of a specific type of immune response and the control of viral replication, all the data seem to suggest that both helper and cytotoxic immune responses are essential to contain viral replication in the early stages of the disease, when the immune system is relatively undamaged. the most conclusive experimental data on the role of the cellular response in the control of viral replication come from studies in which selective depletion of cd lymphocytes in macaques infected with siv leads to a huge increase in viremia and accelerated evolution to aids . hiv transmission occurs mainly via the mucosa. the large quantity of cd + lymphocytes in genito-rectal lymphoid tissue represent a major reservoir for the replication of hiv or siv, even when the infection is contracted intravenously. the gut-associated lymphoid tissue system (galt) is set up by activated b and t lymphocytes and dendritic cells which migrate through the lymphatic system and bloodstream to distant lymph nodes where they become resident. thus, the induction of a strong immune response in mucosa is probably a necessary requirement for a vaccine to be efficient against hiv. each family of viruses develops different escape mechanisms to avoid elimination by the immune system. a vaccine must face the same escape mechanisms and, to be successful, must induce a series of immune responses capable of overcoming them. the rate of variability of hiv is due to the high error rate of reverse transcriptase (one substitution per - nucleotides and round of copy). in consequence there is wide intersubtype and intrasubtype variability but the immunological relevance for vaccine design of this genetic disparity is a matter of debate. several investigations have shown that genetic differences among hiv subtypes do not correlate with immunotypes. in fact, several genetic subtypes could share common protective epitopes and more than one immunotype can be found in the same genetic subtype. in general, neutralizing antibodies seem to be more strain-specific, whereas cellular immune responses have a broader spectrum. this lack of fidelity generates a high diversity in viral proteins which allows escape from the control of specific immune response. therefore, hiv shares with other rna viruses a common escape mechanism related to their high variability that allow the virus finding holes in the immune repertoire. together with the variability generated by the high error rate of reverse transcriptase other mechanisms such as genetic recombination, which produces new subtypes and "mosaic" viruses among different subtypes, are also involved in the generation of hiv variants. several molecular epidemiology studies have stressed the rapid dissemination of hiv variants and have described the distribution of several subtypes or recombinant viruses in different parts of the world. this could be an obstacle to the development of a universal vaccine . one central aspect of hiv infection which is not totally understood is the reason why viral replication is not controlled despite the potent immune responses elicited in primary infection. although several explanations have been put forward, the most widely reported is viral escape through mutations in the viral epitopes recognized by the different effector mechanisms of the immune system . escape from ctl response is due to ad hoc mutations of the viral epitopes which interact with the groove of the hla molecules. it has been shown that mutations in critical residues generate viral escape in both animal models and patients with primary infection and this event results in loss in the ctl response and parallel increase in viremia , . however, in the chronic phase of infection, there is no clear correlation between the presence of specific ctl and the elimination or persistence of a given viral variant . in addition to merely quantitative data, functional analysis have shown qualitative differences between ctl from progressors and non-progressors, such as the expression of perforins , production of cytokines and chemokines, and reduced activity of the t-cell receptor against viral epitopes presented in the hla complex . these data suggest that qualitative aspects of ctl may be also important in the control of viral replication. a general strategy for maximizing the efficacy of a vaccine would be to obtain a cytotoxic response against a large number of epitopes from several proteins. however, recent studies suggest that a more targeted approach can be more effective. thus, ctl against non-structural proteins (tat, nef) are more efficient in controlling infection but more prone to viral escape and do not last as long as ctl against the structural proteins gag and pol . for a sterilizing vaccine, the objective would be to induce an intense ctl response against early proteins, whereas immunization against structural proteins would generate a response which would control viral replication thus attenuating infection. another problem which may be a serious genetic resistance barrier is the adaptation of the virus to the hla haplotype of the infected patient. in this situation peptides from viral mutants generated would reduce their affinity to hla thus decreasing recognition by tcr and generating a greater resistance to ctl response . the structure of the viral envelope in its native form hides the domains of interaction with viral coreceptors due to the trimeric structure and folding of the protein (oligomeric exclusion and entropic masking) . exposure of these conserved epitopes which are identified by neutralizing antibodies takes place at the moment of interaction between the viral and cellular membranes, a setting in which antibodies efficacy is lower given their low accessibility to neutralization epitopes. a second, more classic escape mechanism is epitopic mutation in the hypervariable regions found in the external domain of the viral envelope. nevertheless, recent studies show that escape from these antibodies does not always require mutation in amino acid residues but takes place by glycosylation of the residues and formation of carbohydrate structures on viral gp known as "glycan shields" that represent authentic barriers to the action of neutralizing antibodies . one of the most spectacular studies published during the last year shows how, during evolution in a specific patient, the viral envelopes gradually become resistant to all types of neutralization by neutralizing antibodies via accumulation of the previously mentioned escape mechanisms . both in animal models and in patients with primary infection through sexual contact the establishment of hiv infection is a very rapid process . in a few hours, the lymphoid cells of the rectal and vaginal submucosa become infected and, in seven days, the infection spreads to systemic lymph nodes where it reaches viral and proviral loads similar to levels found in chronic infection . the speed at which these reservoirs appear, before a specific immune response is triggered, represents a major obstacle to the control of viral replication since once established hiv infection will "persists" in lymphocytes despite immune response . hiv can infect target cells in a latent form. in this situation no viral proteins are expressed on the membrane of infected cells thus allowing escape from immune surveillance. furthermore, reactivation-reinfection processes take place in lymphoid organs, which provide an ideal cellular microenvironment for the process of infection: dendritic cells express in their membrane a lectin (dc-sign) which interacts with the virions and lymphocytes and enhances hiv infection . antigenic recognition by lymphocytes and the presence of cytokines in this microenvironment in turn increase infection of target cells and promote viral replication. as confirmation of these data, hiv-specific lymphocyte clones have been shown to be infected at higher proportion, which implies a preferential immunosuppression of the hiv-specific responses . it must be stressed that the continuous generation of new, latently infected cells from the active viral replication compartment generates a "continuous archive" of changes produced in the virus throughout the disease, by including treatment-resistant mutated genomes and variants of the immune escape. the latent compartment is therefore not static and in some way hiv stores its "history" in latently infected cells thus contributing to viral diversity as a mechanism of escape from antiretroviral therapy and vaccines. attenuated virus vaccines are without doubt the most efficacious because the germ carries out a limited series of replication cycles and simulates a low-level infection which induces the whole spectrum of antiviral response in a physiological setting. in the case of lentiviruses, one of the most spectacular findings was that which showed that a defective nef-deleted siv virus induced a protective response against the challenge with highly pathogenic aggressive viable viruses . these experimental data had a natural correlate in the "sydney cohort", made up of patients infected through blood transfusion from a seropositive donor and who, after years of infection, had an excellent clinical and immunological status. the cloning and characterization of the virus in these patients and the donor showed that it presented deletions in the nef gene and in critical regulatory sequences of the ltr region . these findings led to the proposal of attenuated hiv vaccines similar to defective siv mutants. however, it must be stressed that attenuated vaccines are usually used against viruses which do not persist or, alternatively, the attenuated virus used as vaccine is not capable of persisting in the host. this is not the case for nef-defective viruses which not only infect, but also replicate and persist in the host, with the risk of drifting towards more aggressive variants in the vaccinated subject. the first alarming data came from vaccination of newborn maca ques in which, in contrast with was found in adults, the innocuous virus rapidly induced aggressive infection and death by immunodeficiency . furthermore, prolonged follow-up of patients from the sydney cohort enabled us to observe an immunological deterioration and blips of viremia in some subjects . similarly, some adult maca ques vaccinated with the defective siv virus developed aids from the virus they had been vaccinated with, which suffered reversions of the mutant phenotype . therefore, the use of vaccines from defective viruses has been ruled out, and this approach has been explicitly excluded in guidelines and recommendations of regulatory agencies. inactivated viruses have scarcely been used as preventive vaccines. on the contrary, this is the most widely used model in therapeutic vaccines of which remune ® is the prototype. these viral preparations are composed of complete virions or particles whose envelope has been eliminated, which are then inactivated using different chemical methods and administered in conjunction with freund's incomplete adjuvant . the first hiv vaccines were based on the hepatitis b immunization model. they were composed of recombinant proteins gp and gp produced by genetic engineer-ing or using vaccinia virus as expression vectors. in preclinical studies and in phase i and phase ii clinical trials, the preparation was safe and induced antibody synthesis against the viral envelope , but these antibodies were incapable of neutralizing wild-type variants "in vitro" . in spite of the evidence against the efficacy of this prototype, phase iii trials were continued (see below). other trials have used the regulatory protein tat in toxoid form, which has provided good protection results in macaque studies, although its role remains controversial . peptide vaccines have little immunogenic capacity, since, in many cases, the antibodies do not recognize the primary structure of the aminoacid sequence, but rather secondary and tertiary structures in the target proteins which are not simulated by the peptides. therefore, peptides are generally used in combination with other vaccine preparations such as viral vectors or dna in order to induce complementary immunization . the advantages of these combinations are low toxicity, the possibility of preparing peptide "cocktails" which cover a wide range of viral isolates in proteins presenting high variability, and the use of "mixed peptides" which, by including t and b immunodominant epitopes induce cellular and humoral responses. these systems use viruses or bacteria into whose genome hiv genes are inserted in such a way that their proteins are expressed during the course of replication of the vectors in the host cell. the most developed models are those which use poxvirus (vaccinia, canarypox, modified ankara virus/mva) and adenovirus . other experimental approaches use bacteria (bcg, salmonella) and rna viruses (coronavirus, vsv, sfv, reovirus, poliovirus, influenza) including also lentivirus . some of these systems are limited by the risk that exogenous genetic information from the vector can be integrated in the host genome. the advantage of these viral and bacterial systems lies in the possibility of inserting several viral genes in their genomes and their capacity to express high levels of viral proteins. strong antigen expression can in turn induce a potent and prolonged immune stimulation, particularly of cellular immune responses, against these proteins. the vaccine prototypes currently being developed include the genes gag, pol, env and nef in different combinations , , different priming-boosting strategies and vaccine doses. these types of preparation have failed as preventive vaccines in animal models, since they have not achieved protective immunity, probably due to the fact that the humoral response induced against hiv proteins is erratic and of reduced potency. they do, however, induce a potent cellular response which makes viral load stabilize at low levels , . in the most optimistic scenario it has been suggested that this response could be enough to "attenuate" the infection and transforming vaccinated patients who become infected into "long-term survivors". new vectors, such as bcg, salmonella and poliovirus are expected to induce greater humoral and cellular immunity in the mucosa by means of oral administration, thus improving the efficacy of these vaccines. the observation that "naked dna" is capable of inducing an immune response against several viruses and in different animal models broke new ground in the development of vaccines . in infection models with siv and shiv, it has been observed that, as with microbial vectors, immunization with dna is capable of inducing an immune response which, although it does not protect against infection, can often attenuate viral replication and clinical symptoms . the main limitation of dna vectors is that the intensity of the immune response induced is low, therefore they are generally used in combination with viral vectors. a disadvantage of this type of vaccine is the potential long-term secondary effects owing to chromosomal integration processes. adjuvants are preparations which boost the immune response of vaccine antigens by different mechanisms. traditional adjuvants, such as freund's, are bacterial lysates which, by inducing a non-specific inflammatory response, "recruit" immune cells at the injection site. others, such as iscom or liposomes improve the presentation of antigens. recent studies have demonstrated the efficacy of interleukins, especially these activating th responses (interleukins and ) or chemokines, in boosting the response induced by attenuated-vectors or naked-dna vaccines . successive inoculation with an interval of some weeks using two different vectors expressing the same hiv antigen (prime/booster) has been shown to induce a stronger cellular immune response against hiv antigens than when the same vector is administered in two doses. these procedures, which boost specific cd t cell induction, were developed in the murine malaria system by showing that this increase correlates with protection against the pathogen . one of the best systems reported is based on recombinant poxviruses, especially the attenuated vaccinia virus ankara (mva). this vector must be administered at the second immunization (booster), whereas in the first inoculation (priming), dna, capsids and other viral protein-expressing vectors can be used indiscriminately. the most promising prime/booster combinations include dna/pox, sfv/pox, and adeno/pox. there is currently no available preventive hiv vaccine. in fact, previously described strategies have failed because no one single animal has been protected from infection in any experimental model. table . this trial has raised controversy over whether it should be carried out, since both the experimental results and the immune response induced by these vaccine preparations have been very limited , . the most advanced pre-clinical protocols of the new vaccine prototypes include that being developed by aventis pasteur in uganda, which uses a canarypox vector for the expression of viral structural proteins . also in uganda, january saw the start of a phase i trial combining dna + mva (strain a) . a similar phase i clinical trial sponsored by iavi and kavi is being carried out in kenya. unfortunately, it seems unlikely these assays go on due to the low proportion of individuals in which a relevant immune response has been elicited with the vaccine preparations tested. in europe, through the eurovacs initiative a clinical phase i trial using poxviral-based vaccine ny-vav has been completed by juin . another phase i trial based on the combination dna/nyvac expressing gp , gag, pol and nef of subtype c was launched also in juin . a comparative assay between nyvac-c and mva-c is planned in . this latter immunogen will be generated in spain at the national centre of biotechnology. complete and updated information on the situation of existing vaccines and clinical trials is available at: www. hvtn.org/trials. obtaining an aids vaccine has become a global enterprise - . this is very positive because it has increased social awareness of the disease and financial support. it is also important to note that priority given to vaccine research and the development of new approaches appears at a time when we know much more about the pathogenesis of aids than ten years ago. nevertheless, it must be remembered that demonstrating the usefulness of a vaccine is a long and expensive process. therefore, a critical aspect of the problem is to define strategies and criteria for the different phases in vaccine development: type of vaccine, objectives of the vaccine, experimental design, animal models, pre-clinical and laboratory analysis, phase i and ii trials and requirements and infrastructure for phase iii trials. given the healthcare, social and political priorities, this subject is sometimes affected by serious concerns outside the scientific world. these include the social pressure from international organizations and countries devastated by the epidemic and financial pressure from the pharmaceutical companies. although some of these motives are understandable, given the severity of the situation, these attitudes can also distort the scientific process. below, we summarize the key questions in the search for an "aids vaccine". some scientists doubt that an efficacious aids vaccine can be found . the reason is the difficulty in obtaining what has been defined as "sterilizing immunity" against retroviruses. if we analyze the mechanisms of action of vaccines, in most cases they do not achieve "sterilizing im-munity", since they do not prevent infection but rather the persistence of the microorganism and development of disease: the germ infects, but the immune response prevents it from spreading and destroying new infected cells, thus helping to eradicate the infection. in the case of siv and hiv infection, we know that, after the first inoculum, infection takes place in a short period of time and an important reservoir of cells from the lymphoid system become infected. in some of these initially infected cells, the virus replicates actively, but in others it remains in a state of latency as an integrated provirus in the genome of the host cell. therefore, despite the immune response induced by vaccines, the virus can "persist" in the reservoirs from where it can replicate continuously. a particularly controversial area is the "final objective" of the vaccine: some people argue that if it is not possible to induce "sterilizing immunity" to prevent infection, will it be enough to have an immune response capable of controlling the level of viral replication to sufficiently low levels that allow the immune system to escape from huge destruction. the objective would not be so much to prevent infection but to attenuate it, in such a way that the infected patients become "long-term survivors" capable of living with the virus. another area of debate is the level of protection which must be "reached" by an aids vaccine. in contrast with the high efficacy of protection in most vaccines (above % of vaccinated patients) different authors propose that a partial protection rate of - % should be considered as "sufficient". this reduction in the final objectives to be attained by an hiv vaccine is arguable. on one hand, it is doubtful that "attenuation of the infection" will be a definitive phenomenon in the medium-long term. both in animal models and in isolated cases in which an infection has been caused by a defective virus, this virus increases its virulence in the long-term. on the other hand, although it is true that the establishment of a low viral load after primary infection is a good prognostic factor in the medium term, this does not guarantee that patients who present low levels of viral load after vaccination will behave as long-term survivors. the fact that some scientists set the "sufficient" efficacy of a vaccine at - % protection level can also be criticized. this could, perhaps, be considered a realistic stance and, even in specific prevalence rates in specific risk groups, a vaccine of this type could be efficacious, but we do not know its real impact on the evolution of the epidemic in the medium term. we must not forget that one of the mechanisms of vaccine efficacy is due to the "population or epidemiological impact" in the decrease in the prevalence of the infection and the consequent reduced possibility of transmitting the germ among the general population. this epidemiological impact of the vaccine would not exist with the proposed protection rates. some authors suggest that variability among subtypes represents an important obstacle for the development of a universal vaccine and that "ad hoc" vaccines should be manufactured based on the subtypes circulating in each region . however, the new vaccine prototypes use other viral genes (env, nef, gag, pol, tat) as targets which have a much lower variability than the envelope. in fact, different studies show that the immune response induced by vaccination against a specific hiv subtype is capable of acting against other subtypes , . how, when and where is the efficacy of the different vaccines to be evaluated? the efficacy of an aids vaccine must be evaluated in populations with a high rate of infection in order to obtain significant differences between the control group and the vaccinated group in the shortest time possible. this means that almost all the trials are carried out in africa and southeast asia, where annual seroconversion rate in the most affected areas is approximately % of the population . carrying out trials in developing countries raises a series of ethical issues: . it is essential that the studies comply with all ethical requirements and that patients' rights are guaranteed . . the vaccines tried must have satisfied the scientific and medical requisites of potency and safety which are necessary in any medicine tried on humans. . vaccine trials need a wide-ranging follow-up infrastructure which can guarantee patient follow-up. therefore, it is essential to develop healthcare structures and reference centers with the following objectives: recruitment and follow-up of volunteers, extraction, freeze and storage of blood according to standard procedures, and assessment of immunological parameters such as lymphoid populations, cytokine production and neutralizing antibodies. if this requisite is not met, the analysis of results could be skewed and/or incomplete, thus making it impossible to draw conclusions . . one demand by governments is that if a vaccine is efficacious, free access to the vaccine must be guaranteed to the country where the evaluation was carried out. . according to the ethical guidelines of unaids, lifelong antiretroviral therapy must be administered to any person infected during the clinical trial . an important problem, now the center of social and scientific controversy, is to define the requirements a vaccine preparation must fulfill to start a phase iii clinical trial. the journal "science" has been the forum for a series of letters from prestigious scientists criticizing investment strategy in the development of an aids vaccine and the initiation of phase iii clinical trials , . the strict scientific position defends that there are no consistent data on the efficacy of current vaccine prototypes to carry out phase iii clinical trials. consequently, such investment should be concentrated in basic research in order to get a better understanding on the mechanisms of protective immune responses and to develop new relevant animal models. faced with this stance, a more humanist position bases the start of phase iii trials on the catastrophic situation in developing countries and on the counterargument that, if there are no adequate animal models, it will be anyway necessary to carry out all the phases of the studies, including phase iii, in humans to obtain a definitive response. despite the reticence and pessimism of a large part of the scientific community, the general impression is that phase iii trials will be carried out. it is important to remember the cost and effort involved in these trials, which require the follow-up of , patients for at least five years to obtain conclusive results. therefore, with regard to aids vaccines, we are living in difficult times in which a huge economic investment will be necessary so that the scientific community can generate, develop and evaluate all the vaccine prototypes imaginable in animal models in order to find the holy grail of vaccines. as a reference, in case the european union decided to start a program of phase i and ii clinical trials with a reduced number of vaccine prototypes already generated in european laboratories an investment of . billions euros in the following years should be required. with this objective in mind, the development of vaccine research centers has been proposed . these centers would combine: (i) a critical mass of investigators, (ii) their sole dedication to the development of prototype hiv vaccines, (iii) a long-term commitment by academic, governmental and private institutions, (iv) sufficient resources and (v) continuous exchange of information and collaboration with the private sector. as a consequence of this policy the main leader organizations (nih, iavi, anrs, eu, gates foundation...) should finance vaccine development centers and would coordinate their work. the prototypes considered interesting would be prepared under the conditions of good manufacturing practice for use in humans and would enter a previously defined process of pre-clinical studies and phase i, ii and iii clinical trials. all the prototypes would meet the minimum requirements for clinical application, which would mean not only defining these criteria but also involving the regulatory authorities (fda, emea) in their development. the evaluation of prototypes also requires the definition of those immunological markers which must be used to evaluate their potential efficacy. this in turn would mean developing standardized and reproducible trials to evaluate the humoral and cellular responses to hiv and the approval of laboratories which would carry out these immunological determinations. lastly, the necessary healthcare structures should be set up to carry out the trials in clinical phases in developing countries, and the ethical criteria to be fulfilled in these trials should be defined. given the large number of current prototypes (table ), the application of homogeneous evaluation criteria is the only way to reach consistent conclusions which can be extrapolated to all situations. nevertheless, it is important to be aware that this search is full of unanswered questions and that it can fail despite all the efforts made. as it may not be possible to develop a vaccine it may be time to convey this terrible possibility to society. the history of vaccines is defined by the words "empiricism" and "success". no intervention has saved as many lives throughout the history of medicine as vaccines. these successes were often the fruit of the most basic empiricism. however, at present, empiricism cannot serve as the basis of success in the scientific development of an aids vaccine. to conclude, in recent years, the development of an aids vaccine has changed radically due to different factors: the devastating growth of the epidemic, social awareness, fi-nancial investment and, in particular, a better understanding of the pathogenesis. all these new elements enable us to face this challenge rationally and with adequate resources. only scientific effort combined with unprecedented solidarity will allow us to decide whether it is possible to find a vaccine against hiv and whether its application will be sufficient to curb the current aids pandemic. genève: who the global alliance for vaccines and immunization: a millennial challenge accelerating the development and future availability of hiv- vaccines: why, when, where, and how? challenges and opportunities for development of an aids vaccines hiv vaccines - mhc-restricted cytotoxic t-cells: studies on the biological role of major transplantation antigens determining t-cell restriction-specificity, function and responsiveness genomewide conserved epitope profiles of hiv- predicted by biophysical properties of mhc binding peptides inmunidad humoral en la infección por el vih antibodies and resistance to natural hiv infection protection of macaques against vaginal transmission of a pathogenic hiv- /siv chimeric virus by passive infusion of neutralizing antibodies rapid evolution of the neutralizing antibody response to hiv type infection neutralizing antibody-independent containment of immunodeficiency virus challenges by dna priming and recombinant pox virus booster immunizations hiv- neutralizing antibodies: how full is the bottle? antibodies, viruses and vaccines hiv vaccine design and the neutralizing antibody problem cellular immune responses to hiv. nature vigorous hiv- -specific cd + t cell responses associated with control of viremia quantitation of hiv- -specific cytotoxic t lymphocytes and plasma load of viral rna immune control of hiv- after early treatment of acute infection hiv- -specific cd + t cells are detectable in most individuals with active hiv- infection, but decline with prolonged viral suppression the challenge of immune control of immunodeficiency virus effects of in vivo cd + t cell depletion on virus replication in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine molecular epidemiology of hiv- genetic forms and its significance for vaccine development and therapy immunopathogenesis and immunotherapy in aids virus infections antiviral pressure exerted by hiv- -specific cytotoxic t lymphocytes (ctls) during primary infection demonstrated by rapid selection of ctl escape virus acute phase citotoxic t lymphocyte escape is a hallmarg of simian immunodeficiency virus infection lack of strong immune selection pressure by the immunodominant, hla-a* -restricted cytotoxic t lymphocyte response in chronic human immunodeficiency virus- infection hiv-specific cd + t cell proliferation is coupled to perforin expression and is maintained in nonprogressors ctl ontogeny and viral escape: implications for hiv- vaccine design hiv-tailored to fit hiv- evades antibody-mediated neutralization through conformational masking of receptor-binding sites antibody neutralization and escape by hiv- rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus population biology of hiv- infection: viral and cd + t cell demographics and dynamics in lymphatic tissues the challenge of viral reservoirs in hiv- infection dc-sign, a dendritic cell-specific hiv- -binding protein that enhances trans-infection of t cells hiv preferentially infects hiv-specific cd + t cells protective effects of a live attenuated siv vaccine with a deletion in the nef gene genomic structure of an attenuated quasi species of hiv- from a blood transfusion donor and recipients live-attenuated, multiply deleted siv causes aids in infants and adult macaques declining cd t-cell counts in a person infected with nef-deleted hiv- pathogenic conversion of live attenuated siv vaccine is associated with expression of truncated evaluation of hiv- immunogen, an immunologic modifier, administered to patients infected whith hiv having to × /l cd cell counts: a randomized controlled trial advancing aidsvax to phase . safety, immunogencity, and plans for phase immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type tat as one key to hiv-induced immune pathogenesis and tat toxoid as an important component of a vaccine neutralization of hiv- by secretory iga induced by oral immunization with a new macromolecular multicomponent peptide vaccine candidate immunization with a modified vaccinia virus expressing simian immunodeficiency virus (siv) gag-pol primes for an anamnestic gag-specific cytotoxic t-lymphocyte response and is associated with reduction of viremia after siv challenge replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity protective immune responses induced by secretion of a chimeric soluble protein from a recombinant mycobacterium bovis bacillus calmette-guérin vector candidate vaccine for human immunodeficiency virus type in small animals lentiviral vectors: turning a deadly foe into a therapeutic agent hiv- dna vaccines and chemokines augmentation of immune responses to hiv- and simian immunodeficiency virus dna vaccines by il- /ig plasmid administration in rhesus monkeys il- and il- act in synergy to clear vaccinia virus infection: involvement of innate and adaptive components of the immune system priming with recombinant influenza virus followed by administration of recombinant vaccinia virus induces cd + t-cell-mediated protective immunity against malaria preventive hiv vaccine development in thailand aidsvax flop leaves vaccine field unscathed public health. a sound rationale needed for phase iii hiv- vaccine trials policy rebuttal. hiv vaccine trial justified a canarypox vaccine expressing multiple human immunodeficiency virus type genes given alone or with rgp elicits broad and durable cd + cytotoxic t lymphocyte responses in seronegative volunteers effective induction of simian immunodeficiency virus-specific cytotoxic t lymphocytes in macaques by using a multiepitope gene and dna primemodified vaccinia virus ankara boost vaccination regimen why an hiv vaccine is not currently within our grasp. xi croi clade b-based hiv- vaccines elicit cross-clade cytotoxic t lymphocyte reactivities in uninfected volunteers a human immunodeficiency virus prime-boost immunization regimen in humans induces antibodies that show interclade cross-reactivity and neutralize several x -, r -, and dualtropic clade b and c primary isolates the highest attainable standard: ethical issues in aids vaccines some important issues in the planning of phase iii hiv vaccine efficacy trials unaids. ethical considerations in hiv preventive vaccine research the needfor a global hiv vaccine enterprise our laboratories are funded by redes temáticas cooperativas de investigación en sida (ris g / ) y de genética clínica y molecular (rig / ), "fundación para la investigación y la prevención del sida en españa" (fipse / ; / ; / ; / ), "programa nacional de salud" (saf - y - - ) and comunidad de madrid. we are grateful to florencia etcheverry for her help in revising the bibliography of this manuscript. key: cord- -mo mvwch authors: huang, jiechen; wang, juan; xia, chengyi title: role of vaccine efficacy in the vaccination behavior under myopic update rule on complex networks date: - - journal: chaos solitons fractals doi: . /j.chaos. . sha: doc_id: cord_uid: mo mvwch how to effectively prevent the diffusion of infectious disease has become an intriguing topic in the field of public hygienics. to be noted that, for the non-periodic infectious diseases, many people hope to obtain the vaccine of epidemics in time to be inoculated, rather than at the end of the epidemic. however, the vaccine may fail as a result of invalid storage, transportation and usage, and then vaccinated individuals may become re-susceptible and be infected again during the outbreak. to this end, we build a new framework that considers the imperfect vaccination during the one cycle of infectious disease within the spatially structured and heterogeneous population. meanwhile, we propose a new vaccination update rule: myopic update rule, which is only based on one focal player’s own perception regarding the disease outbreak, and one susceptible individual makes a decision to adopt the vaccine just by comparing the perceived payoffs vaccination with the perceived ones of being infected. extensive monte-carlo simulations are performed to demonstrate the imperfect vaccination behavior under the myopic update rule in the spatially structured and heterogeneous population. the results indicate that healthy individuals are often willing to inoculate the vaccine under the myopic update rule, which can stop the infectious disease from being spread, in particular, it is found that the vaccine efficacy influences the fraction of vaccinated individuals much more than the relative cost of vaccination on the regular lattice, meanwhile, vaccine efficacy is more sensitive on the heterogeneous scale-free network. current results are helpful to further analyze and model the choice of vaccination strategy during the disease outbreaks. over the past two decades, the outbreak of infectious diseases has been threatening the safety of human lives and properties, such as the severe acute respiratory syndrome sars [ ] , h n [ ] , ebola [ ] and so on. thus, how to prevent the extensive outbreaks of epidemics has become a challenging topic in the field of public health [ ] [ ] [ ] . meanwhile, the difference of population distribution, religious belief and regional differences may greatly affect the spread of infectious diseases, for example, refs. [ ] [ ] [ ] [ ] explore the impact of various topological structures within the population on the infectious diseases spread, and it is convincingly found that heterogeneous networks may quicken the disease spreading within the population, even lead to the absence of epidemic threshold [ ] . meanwhile, the individual reactions to infectious diseases may also substantially influence the diffusion processes of epidemics. one of the most striking cases regarding the outbreaks was h n pandemic in [ ] , which induced around , deaths. during the outbreaks of h n , the suppression of epidemic processes can not only be attributed to the public measures, but also through personal and uncoordinated responses, that is, the human behavior has noticeably interfered with the epidemic spreading. in the long run, human behavior has been intricately correlated with the contagion of infectious diseases. in medieval ages, the deadly bubonic plague rendered many people to avoid and flee away from the sick and their close contacts so that their own immunity can be secured [ ] . similarly, the villagers of yorkshire in eyam tried to voluntarily quarantine themselves to stop the spread of the plague from that village [ ] . in , during the outbreak of sars, many citizens spontaneously wear the face masks, some schools are temporarily closed and the students are imperatively required to stay at home so as to avoid the further epidemic infection as much as possible [ ] . in addition, protective behavior when confronting the epidemics has also been observed in many other contexts, such as measles-mumps-rubella(mmr) [ ] , tuberculosis(tb) [ ] and hiv [ ] etc. while the impact of human behaviors on the epidemic spreading process has often been mentioned anecdotally, the accurate modeling or quantitative models are relatively fewer regarding their nature, property, or the effect they may have on the spread of the disease. at present, mathematical models have been put forward to study the role of human behavior in the context of social population, such as escape panic [ ] , pedestrian trails [ ] , but effort s to quantitatively explore the role of human behavior in the large-scale epidemics generally focus on assessing the effectiveness of various public health measures including the social distancing, school closure etc. in the recent years, there are many fields and methods to help us to study infectious disease [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , however, there is an increasing attention about the effect of spontaneous individual action or response strategy on the progression of an infectious disease, in which this kind of spontaneous actions may highly restrain from the further diffusion of epidemics and even change the fate of outbreaks. thus, it is of great significance to fully understand the interacting mechanisms between the human behavior and disease dynamics within the specified population. it is worth mentioning that funk et al. [ ] systematically summarized the related works and provided a taxonomy framework of behavior-disease models. on the one hand, they classify these models according to source and type of information that individuals base their neighbors on, in which source of information may be local or global and the type of information that individuals change their behaviors are prevalence-based or belief-based; on the other hand, they classify the previous works based on the impact of individual behavior changes on the disease dynamics, which include the following three aspects: (i) the disease state; (ii) model parameters (infection or recovering rate); and (iii) the network contact structure relevant for the spread of epidemics. in particular, for some preventable infectious diseases with the help of vaccines, the epidemic outbreaks are intricately linked with the individual vaccination behavior since the vaccines can help the vaccinators not to be infected by a specific disease. meanwhile, these vaccinators may indirectly protect their nearest neighbors with whom they contact, and then these neighbors may choose not to vaccinate again (that is, free-ride the vaccinators) so as to avoid the necessary vaccine fees or other potential risk and side effects. henceforth, the vaccination behavior may dominate the evolutionary process of vaccine preventable diseases. among them, bauch and earn [ ] seminally utilized the game theory to model the dilemmatic situation for an individual facing with the epidemics, and they proposed a class of vaccination game to denote the individual decision making and found that, for the well-mixed population, the nash equilibrium is never to vaccinate if the vaccination cost is higher than that of being infected; but there exists a nash equilibrium yielding a suboptimal vaccinated fraction if the vaccine cost is lower than that of being infected. as a further step, complex networks, beyond the well-mixed topology, provide a unified platform to characterize the topology of real-world populations, where the nodes represent individuals and links mimic the contacts among them [ ] . thus, under framework of game theory, many works are devoted to exploring the interplay between contact patterns, behavioral responses and disease dynamics. as an example, fu et al. [ ] found that heterogeneous networks, such as scale-free ones, can induce a broad range of vaccinating actions of many individuals since highdegree hubs with many neighbors become voluntary vaccinators more probably in order to reduce the risk of being infected. after that, zhang et al. [ ] demonstrated that the hubs may largely inhibit the outbreaks of infectious diseases under the voluntary vaccination policy. in the meantime, various subsidy policies on controlling the epidemic spreading have been determined from the socioeconomic perspectives within the well-mixed and networked population [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, most previous works [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] often assume that the vaccine has a perfect efficacy, which will endow the complete immunity for the inoculated individuals, but an interesting topic is on how the epidemic spreads when the vaccine is not fully effective for the disease (i.e., % efficacy)? besides, in the vaccination game, whether an individual decides to vaccinate the vaccine or not will be often determined by the estimated payoff, and the vaccinating decision may be transferred from one player to another one inside the population according to a specific role model during the outbreaks. nevertheless, under some real-world scenarios, some individuals are not willing to imitate the behaviors of others as a result of special belief, religion, opinion and even awareness of a disease. according to the above description, in the real world, when a new epidemic starts to outbreak, people want to take some measures to protect themselves immediately. generally, inoculating the vaccine is considered as an effective measure, but the vaccine may fail as result of invalid storage, transportation and usage, and then rendered that the vaccinated individuals may confront the risk of being infected. henceforth, some individuals make a decision to vaccinate just judge by themselves and don't consider their neighbors. thus, in order to deeply analyze the role of vaccine efficacy and spontaneous individual decision mechanism in the outbreak of vaccine preventable diseases, we propose a vaccination game model to explore the impact of imperfect vaccine efficacy and myopic update rule in the spatially structure and heterogeneous populations. the rest of this paper is structured as follows. firstly, we depict the new vaccination game model in section in detail. then, section provides extensive numerical simulation results, which are obtained in the regular lattice and heterogeneous scalefree networks, respectively. lastly, in section , we end this paper with some conclusions and point out the potential works in the future. as mentioned above, we consider the vaccination game for a class of emerging epidemics within the structured population, where the vaccine can be obtained after the epidemic spreads. thus, in the current model, all individuals have no chances to vaccinate at the initial time step ( t = ). after that, each time step ( t ≥ ) is divided into two elementary sub-steps: one step is for the decision of inoculating the vaccine; the other one is used to model the process of epidemic spreading. among them, for the epidemic sub-steps including t = , we leverage the frequently used susceptible-infective-recovery (sir) compartment model to characterize the evolution of epidemics, where each individual may lie in the susceptible (s), infective (i) or recovery (r) state. during the vaccination decision sub-steps, each susceptible individual needs to assess the risk of being infected, and then make the decision whether he will vaccinate or not. regarding the sir model, any susceptible individual may be infected through the contact with infective neighbors and the transmission rate along each infective link is assumed to be β. meanwhile, the infected individual can be cured with the recovering rate μ, and the recovered one will not be infected again or infect any other healthy ones. hence, the probability that the susceptible player i without inoculating the vaccine will be infected by all possible infective neighbors can be written as follows, where k i in f denotes the total number of infected neighbors of the focal player i . the epidemic continues until there are no more newly infected individuals. as for the individual vaccination decision, each susceptible one will evaluate the risk of being infected and compare the difference between the vaccine cost ( c v ) and the potential expenses once he has been infected. without loss of generality, we fix the infection cost c i = , while the vaccine cost is usually lower than c i and then its relative cost can be re-scaled as < c = c v /c i < . in order to quantitatively perform the decision, by borrowing from the terms in game theory, we assume that the decision process is based on the comparison between the perceived payoffs of vacci-nation i v and the perceived payoffs of being infected i nv if he is not vaccinated, which can be expressed as follows, respectively, where λ i denotes the potential infection probability that can be calculated according to eq. ( ) . then, the susceptible agent i will independently decide to inoculate the vaccine with the following fermi-like probability, where k represents the impact of the noise or its reverse / k means the strength of strategy selection, which reflects the uncertainty of vaccination strategy adoption. here, we term this vaccination decision as the myopic update rule just based on one player's own perception, which is different from imitating the vaccination strategy of others in many previous works [ , , [ ] [ ] [ ] [ ] [ ] . when k → , agent i is totally rational and whether he will vaccinate or not is fully determined by comparing i v and i nv , on the contrary, when k → ∞ , agent i will randomly perform the vaccination choice. in addition, we consider the imperfect vaccination program, that is, the vaccine efficacy is not % and the vaccination may fail as a result of incorrect transportation, storage, and usage of vaccine. thus, we introduce an independent parameter θ to characterize the vaccine failure rate, which implies that the susceptible individual to choose the vaccination is still kept in the susceptible state with the probability θ , while the probability changing from s into v ( s → v ) state is set to be ( − θ ). once the healthy individual decides to vaccinate, he will have a chance to enter into the vaccinated ( v ) state, the sir epidemic dynamics will evolve into the sirv model as illustrated in fig. , in which the successfully vaccinated individual will be equivalent to the r-type one at the next time step, that is, the successfully vaccinated (v-type) agent does not get the infection or infect others, either. we do not know when does vaccine fails, so that at each propagating time step, inoculation individuals are judged whether the vaccine is fails or not. to be noted, the vaccinated healthy individuals will not be vaccinated again during this epidemics even if the vaccine lost its effect. in order to further explore the impact of network topology on the evolutionary process under the imperfect vaccination, we simulate the current mechanism on l × l regular lattices and scale-free networks with n = l nodes, respectively. initially, we stochastically choose i = individuals as the infective seeds within the population, and other ( n − i ) individuals are kept in the susceptible state, and thus there have no vaccinators. at t = step, the system starts the evolution according to the sir model. after that, from t = , each susceptible agent has the opportunity to receive the vaccination and then the system carries out the evolution of epidemics based on the above-mentioned two elementary steps. the system continuously evolves until there are no infective individuals. in addition, the current results are averaged over independent runs so that the large fluctuations can be removed. in all the numerical simulations, the population size is fixed to be n = . in the homogenous topology, we use the regular lattice satisfying the periodic boundary with the size l = as the underlying networks, and each individual has nearest neighbors (that is, the von-neumann neighborhood). as for the heterogeneous topology, we generate the scale-free network by using the configuration model, in which the average degree is fixed to be < k > = and the power exponent is set to be γ = . in this section, we conduct extensive numerical simulations to demonstrate the vaccination behavior on the regular lattices and scale-free networks, respectively. among them, we mainly discuss the influence of vaccine cost c and failure rate θ on the collective vaccination level within the population. without loss of generality, we set the value of parameters in the sir model as β = . and γ = / , which are identical with those in ref. [ ] . first, we investigate the equilibrium fraction of both vaccinated ( ρ v ) and recovered ( ρ r ) state individual size for different values of relative cost of vaccination c and vaccine failure rate θ . fig. plots . (yellow triangle), respectively. on the one hand, for a specific vaccine failure rate θ , ρ v declines with the increase of the relative vaccination cost c , while ρ r increases as c augments, which means that the vaccination cost will markedly affect the willingness of individuals to inoculate the vaccine. as an example, when c ≤ . , ρ v and ρ r can almost keep the similar vaccination level as c increases; however, c > . leads to the substantial reduction of ρ v and the continuous rising of ρ r since the vaccination cost is comparable to the infection cost; in particular, ρ v will be dramatically reduced when c is up to . , even tends to zero as the vaccination cost is too high, especially for c = . . on the other hand, under the same vaccination cost c, ρ v decreases as the vaccine failure rate becomes higher, for instance, the fraction of adopting the vaccination strategy under c = . is much less than that with c = . , which implies that the vaccinated fraction within the whole population will be a little more sensitive to the vaccine failure rate. then, we discuss the influence of the noise factor k on the vaccination behavior within the population in fig. , where we set σ = k , termed as the strength of selection ( < σ < ∞ ), as and . , which are slightly different from that in fig. . likewise, it can be clearly shown that ρ v declines and ρ r increases slowly when the relative cost of vaccination c lies between and . . afterwards, when the relative cost of vaccination c is more than . , the greater the noise selection strength, the more rapidly the varying trend of ρ v and ρ r . in fact, as the strength of selection increases, individuals become much more rational and will not tend to take the vaccination strategy since they will take their own economic cost and the related interests, say, free-riding behavior, into account. in particular, the relative cost c is beyond . , or the vaccine loss rate is higher (i.e., θ = . ), the un-vaccination behavior of rational individuals become much more prominent, and thus it is unable to prevent the outbreaks of epidemics, which can be observed from the larger ρ r as c > . or θ = . . next, in order to fully check the impact of relative vaccination cost c and the vaccine failure rate θ on the vaccination behavior, fig. illustrates the evolution of ρ v and ρ r within the broader ranges of c and θ . it is clearly indicated that at the lower vaccine failure rates (say, θ < . ), the fraction of vaccinated individuals is often more than half of the total population, even if relative cost of vaccination c is large (e.g., . ); meanwhile, a plethora of vacci- thus, creating the high quality vaccine is significant, which greatly determines the individual vaccination inclination. furthermore, to deeply understand individual state change in the lattice as sirv model evolves, we record the evolutionary snapshots of individual states at various time steps for θ = . , c = . and θ = . , c = . in fig. . among them, the upper eight panels denote the snapshots under θ = . and c = . , while the lower eight panels represent the ones for θ = . and c = . . at time step t = , there are no vaccinated individuals on the lattice and only i = randomly infected seeds, and then the epidemic starts to propagate at this time. after that ( t ≥ ), the susceptible individuals have the opportunity to determine whether they will inoculate the vaccine or not. it is clearly observed that most individuals choose to vaccinate under these two cases when epidemic begins to spread. however, when θ = . is lower, there is fewer vaccinated individuals to become susceptible, and then most of vaccinated individuals are immunized, in which the epidemic is hard to spread and finally tends to be extinct. reversely, for the higher vaccine failure rate (i.e., θ = . ), the vaccine is easy to be invalid, many vaccinated individuals become susceptible due to the loss of vaccine efficacy. therefore, the epidemic can be pandemic and then most of individuals enter the recovered state in the end. all these results again demonstrate that the vaccine efficacy plays the significant role in the evolution of vaccination behavior of epidemic outbreaks within the structured population. in the real world, many networks are often heterogeneous, and thus it is necessary to understand the mechanics of myopic update rule better on heterogeneous topology. to this end, we formulated the game of taking the vaccine on the scale-free network. here, we generate the scale-free network with , node under the configuration model, where the average degree of the whole network is equal to and the power exponent . after the fundamental networks are created, the system evolves according to the sirv model, which is identical with the iteration procedure on regular lattices, and the epidemic continues until there are no more newly infected individuals. first of all, we plot the time courses of fraction of susceptible, vaccinated and recovered individuals for different the relative cost of vaccination c and vaccine failure rate θ in fig. . in all panels, the red, blue and yellow lines denote the evolution of susceptible, recovered and vaccinated individuals, respectively. it can be found that the vaccinated individuals increase rapidly in a very short time, and then reach a peak. vaccinated individuals are rarely become susceptible because of the vaccine failure θ is lower (as shown in fig. a,c) so that the number of recovered ones increases a little and arrives at the equilibrium quickly, which states clearly that the epidemic is eliminated and has not become pandemic. due to the vaccine failure rate, the fraction of vaccinated individuals goes down and then tends to be zero after reaching the peak. however, when the value of vaccine failure rate θ is raised (as shown in fig. b,d) , even though the vaccinated individuals increase rapidly, they become susceptible quickly due to the high vaccine failure rate θ , it can't prevent the epidemic spread so that the number of recovered individuals increases. additionally, we found that for the value of vaccine failure rate θ = . , whatever the values of relative cost of vaccination c , the number of recovered agents is the same as that at the equilibrium. generally, when the epidemic starts to spread, many susceptibles take the vaccine in the population at a short due to the perception of infection risk. also, this vaccination behavior is almost widespread regardless of the values of relative cost of vaccination c and vaccine failure rate θ . at the lower vaccine failure rate, vaccinated individuals are hard to become susceptible, which leads to the disease propagates difficultly and be eliminated as soon as possible. these results are also consistent with the work of zhang et al. [ ] , since the hub nodes are often vaccinated immediately after the disease starts to spread. but for the higher vaccine failure rate, the vaccinated individuals become susceptible quickly, the disease can outbreak. we also consider the equilibrium fraction of both vaccinated ( ρ v ) and recovered ( ρ r ) state individual size for different values of it can be found that in the figs. and , whatever the ways of vaccination, the epidemic can outbreak and vaccinated individuals are more sensitive to the vaccine failure rate. therefore, except β = . , we consider the epidemic evolution of sirv model under the lower transmission rate β = . . meanwhile, we set the i = initial infective seeds as the top largest degree nodes, which is here termed as the hub infection scheme. correspondingly, we call the randomly selecting one for the generous case as the random infection. in summary, based on the sir epidemic dynamics, we investigate the imperfect vaccine immunity under the myopic update rule in different foundation topology including the regular lattice and scale-free networks, where the focal player makes the vaccination decision just according to his own judgement about the epidemic situation. extensive numerical simulations show that most unvaccinated susceptible individuals are willing to inoculate the vaccine under the myopic update rule, whatever the type of network is, in particular for the lower vaccine cost ( c ≤ . ) and failure rate ( θ ≤ . ). after the epidemic starts to propagate, and most of individuals change their strategies to adopt the vaccine in a short time since the individual can estimate the infection risk at the early stage, which leads the epidemics to be hard to spread within the population. however, due to the failure of vaccine or the free-riding behavior of susceptible individuals, vaccinated individuals become susceptible again and then confront the risk of being infected, which creates the potential epidemics situation. to be of great interest, we find that the impact of vaccine failure rate on the vaccination coverage becomes much higher, when compared to the role of the relative cost of vaccine. for example, the value of vaccine failure rate θ is usually assumed to be no more than %, or else most vaccinated individuals become re-susceptible again. at a fixed θ , the fraction of vaccinated individuals almost keep unchanged when the relative vaccine cost is not beyond c = . , but this value will become lower and lower after c is more than . , which is basically consistent with the reality of vaccine usage. on the contrary, on the scale-free networks, the number of vaccinated individuals is more sensitive to the effect on vaccine failure rate θ . hub nodes have a stronger inclination to adopt the vaccine under the myopic update rule, which can effectively prevent the diffusion of epidemics. hence, the disease will be eliminated quickly in heterogeneous topology. however, when the values of vaccine failure rate increase a little bit, hub nodes become susceptible and cannot prevent the epidemic spread, which leads to the epidemic outbreak. meanwhile, when the relative cost of vaccination c increases, unvaccinated susceptible individuals are not willing to choose the vaccination strategy, which can not stop the outbreak of infectious diseases. when the vaccine failure rate further increases, such as . , the number of vaccinated individuals decays to zero before the epidemic is eliminated, which is almost equivalent with the classic sir model. anyway, current results are conducive to better understanding the individual vaccination behaviors when confronting the real epidemics. the authors declare that they do not have any financial or nonfinancial conflict of interests. transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions novel influenza a (h n ) pregnancy working group. h n influenza virus infection during pregnancy in the usa an outbreak of ebola in uganda infectious diseases of humans: dynamics and control evolving public perceptions and stability in vaccine uptake vaccination greatly reduces disease, disability, death and inequity worldwide modelling disease outbreaks in realistic urban social networks dynamic social networks and the implications for the spread of infectious disease when individual behavior matters: homogeneous and network models in epidemiology a high-resolution human contact network for infectious disease transmission capturing human behaviour early assessment of anxiety and behavioral response to novel swine-origin influenza a(h n ) plagues and peoples biology of plagues: evidence from historical populations sars-related perceptions in hong kong institute of medicine (us) immunization safety review committee. immunization safety review: measles-mumps-rubella vaccine and autism immunology of tuberculosis the responsiveness of the demand for condoms to the local prevalence of aids simulating dynamical features of escape panic modelling the evolution of human trail systems dynamic cluster formation game for attributed graph clustering fast and accurate mining the community structure: integrating center locating and membership optimization detecting prosumer-community groups in smart grids from the multiagent perspective enhance the performance of network computation by a tunable weighting strategy blockchain-based medical records secure storage and medical service framework diabetic complication prediction using a similarity-enhanced latent dirichlet allocation model smart electronic gastroscope system using a cloud-edge collaborative framework social and juristic challenges of artificial intelligence the impact of awareness diffusion on sirlike epidemics in multiplex networks a new coupled disease-awareness spreading model with mass media on multiplex networks improved centrality indicators to characterize the nodal spreading capability in complex networks interplay between sir-based disease spreading and awareness diffusion on multiplex networks modelling the influence of human behavior on the spread of infectious diseases: a review vaccination and the theory of games epidemic dynamics on complex networks imitation dynamics of vaccination behavior on social networks hub nodes inhibit the outbreak of epidemic under voluntary vaccination policy evaluation for the subsidy for influenza vaccination in elderly does subsidy work? price elasticity of demand for influenza vaccination among the elderly in japan impacts of subsidy policies on vaccination decisions in contact networks simulation analysis of vaccination subsidy with abm approach preferential imitation can invalidate targeted subsidy policies on seasonal-influenza diseases subsidy strategy based on history information can stimulate voluntary vaccination behaviors on seasonal diseases effects of behavioral response and vaccination policy on epidemic spreading -an approach based on evolutionary-game dynamics a susceptible-infected epidemic model with voluntary vaccinations group interest versus self-interest in smallpox vaccination policy imitation dynamics predict vaccinating behavior impact of committed individuals on vaccination behavior the impact of imitation on vaccination behavior in social contact networks the influence of social norms on the dynamics of vaccinating behavior for paediatric infectious diseases risk assessment for infectious disease and its impact on voluntary vaccination behavior in social networks can influenza epidemics be prevented by voluntary vaccination? this project is financially supported by the national natural science foundation of china (nsfc) (grant nos. and ). key: cord- - k ej l authors: saiz, juan-carlos title: vaccines against rna viruses date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: k ej l rna viruses cause animal, human, and zoonotic diseases that affect millions of individuals, as is being exemplified by the devastating ongoing epidemic of the recently identified sars-cov- [...]. rna viruses cause animal, human, and zoonotic diseases that affect millions of individuals, as is being exemplified by the devastating ongoing epidemic of the recently identified sars-cov- . for years vaccines have had an enormous impact on overcoming the global burden of diseases. nowadays, a vast number of different approaches, from purified inactivated and live attenuated viruses, nucleic acid (dna or rna) based candidates, virus-like particles, subunit elements, and recombinant viruses are been employed to combat diverse diseases caused by rna viruses. however, although for some of them efficient vaccines are available, in most instances, and for different reasons (technologic o economic restrictions, etc.), they are scarcely used in the field, and even for many of them no licensed vaccines exist. this will probably change dramatically with the current covid- pandemic, as a vast variety of vaccinology approaches are being tested against it, with hundreds of candidates under development, dozens of them already in clinical trials phase iii and iv, a fact that is breaking records in vaccine development and implementation. this is becoming possible thanks to the enormous work carried out during years to have the bases for a quick response, even against unknown pathogens, in an impressive short time. in this vaccines special issue "vaccines against rna viruses", results obtained with different vaccine methodological approaches against human, animal, and zoonotic viruses are presented by field experts. human immunodeficiency virus (hiv), for which no vaccine is available, continues to be a major global public health issue that has already cost almost million lives so far, and with an estimated million people living with it. even though antiretroviral therapy considerably prolongs the lifespan of patients making the disease a manageable chronic health condition, treatment is expensive and is not available for all infected patients. thus, vaccines are needed to control hiv spread. so, in this issue, rudometov et al. [ ] describe the development of a human immunodeficiency virus- (hiv- ) immunogen using a polypeptide recombinant non transferrin bound iron (ntbi) protein carrying four t-and five b-cell epitopes from hiv- env and gag proteins recognized by broadly neutralizing hiv- antibodies, combined with th-epitopes. immunization of rabbits with this ntbi protein elicited antibodies that recognize hiv- proteins, supporting its possible use as immunogen. another relevant human virus is the human rotavirus (hrv) that is a leading cause of severe, dehydrating gastroenteritis, mainly in children. current live rotavirus vaccines are efficient, but costly, and they may present increased risk of intussusception due to vaccine replication in the gut of vaccinated children. here, ramesh and coworkers [ ] assess the immunogenicity and protective capacity of a novel p -vp * nanoparticle vaccine using the gnotobiotic (gn) pig model of human rotavirus. three doses ( µg/dose) of the vaccine candidate intramuscularly administered with al(oh) ( µg) as an adjuvant conferred significant protection against infection and diarrhea after challenged with virulent heterologous rotavirus strains. vaccinated animals showed a significant reduction in the mean duration of diarrhea, virus shedding in feces, and significantly lower fecal cumulative consistency scores in comparison with mock immunized control animals. vaccinated animals also elicited strong vp *-specific serum neutralizing antibody responses, but, consistent with the methodological approach (immunization route, adjuvant, and lack of replication), no serum or intestinal iga antibody responses, or strong effector t cell responses, were induced. authors indicate that their results open the option of the initiation of clinical trials using the new designed p -vp * nanoparticle vaccine candidate. for his part, duncan and coworkers [ ] update the current status of the hepatitis c vaccination (hcv), describing and discussing the different ongoing research in the field, and emphasizing that, despite the great advances in hcv therapeutics achieved lately, treatments sometime fail to prevent reinfection and are quite expensive and, thus, efficient prophylactic hcv vaccines are still needed. pigs are one of the main livestock species, comprising % of the worldwide meat consumption, with hundreds of tons produced yearly. pigs can be infected by a wide variety of rna viruses that cause huge losses for the food industry. in addition, they can serve as bridge between wild host animals and humans. thereby, availability of efficient vaccines is crucial. among the viral infections affecting pigs, a very important one is that of the porcine reproductive and respiratory syndrome virus (prrsv) that causes reproductive failure in pregnant sows and respiratory disease in young pigs, accounting for huge economic losses to industry across the world. one critical drawback for vaccine development against prrsv is its high genetic diversity. here, ciu and coworkers [ ] show their results after testing a glycoprotein gp -mosaic dna vaccine and a recombinant gp -mosaic vaccinia virus (rgp -mosaic vacv) vaccine in pigs, and compare their immunogenicity and protective capacity against heterologous virulent strains (vr or mn c) with that of a rgp -wyld-type, wt (vr ) dna and a rgp -wt (vr ) vacv vaccine, using as controls groups those inoculated with empty vector dna or empty vacv. the authors show that vaccination with the gp -mosaic-based vaccines resulted in cellular reactivity and higher levels of neutralizing antibodies to both vr and mn c prrsv strains, whilst vaccination with the gp -wt vaccines only induced response against the heterologous challenging virus (vr ). after infection with either strain, viral titers in sera and tissues, as well as lung lesions, were lower in the gp -mosaic vaccinated pigs. these results indicate that, using a dna-prime/vacv boost regimen, the developed gp -mosaic candidates confer protection in pigs against heterologous viruses and, thus, are feasible vaccine candidates. another important disease in pigs is that caused by the porcine epidemic diarrhea virus (pedv), a coronavirus responsible of highly contagious intestinal infections that may result in the death of newborn piglets and weight loss in pigs of all ages, and that seriously damages the swine industry. from , novel highly virulent pedv genotype variants have spread through china, resulting in high mortality of newborn piglets and huge economic losses. current vaccines against the classical cv strain of genotype do not provide effective protection against chinese highly virulent pedv variant infections. thus, shi and colleagues [ ] using a rna interference (rnai) approach, show that three short hairpin rna (shrna)-expressing plasmids directed against the viral nucleocapsid (n) present antiviral activities in intestine epithelial cells infected with a both classical cv and lnct strains, and that these shrnas markedly reduce viral replication of both of strains upon downregulation of n protein production; thus, these strategies, based on targeting viral processivity factors, may be feasible vaccine alternatives. by a different approach, cañas-arranz et al. [ ] deepen their many years of previous research with peptides as vaccine candidates against foot-and mouth disease virus (fmdv). the virus is responsible for a highly contagious transmissible infection of cloven-hoofed animals (mainly pigs and cows) that may cause huge economic impact and whose control relies on efficient vaccination using a conventional chemically inactivated virus. however, these current vaccines present limitations, as the need for a strict maintenance of the cold chain and for a constant update of vaccine strains due to the virus's antigenic diversity, as well as the fact that the manufacturing process poses significant biosafety concerns, as it has been related to occasional escape episodes. therefore, vaccines incorporating specific outbreak-relevant epitopes capable of eliciting protective and quick responses can become an invaluable emergency resource for fmd containments during outbreaks. with this background, the spanish group go one step further by testing in pigs a dendrimer peptide b t with two copies of a b-cell vp epitope linked through maleimide units to a t-cell a, and show that a single dose of b t evokes a specific protective immune response with high neutralizing titers, activates the t cell response, induces ifn production, and fully protects % of vaccinated pigs that did not present clinical signs of the disease. their results strengthen the potential of b t as a safe, cost-effective candidate vaccine, which can be of particular interest in emergency scenarios. rift valley fever virus (rvfv), a mosquito-borne bunyavirus widely distributed in sub-saharan countries, egypt, and the arabian peninsula, is another important pathogen causing disease in humans, in which severe cases can end in encephalitis or hemorrhagic fever, and in ruminant livestock, characterized by an increased incidence of abortion or fetal malformation. at present, there are a few veterinary vaccines available for use in endemic areas, but there is no licensed human vaccine. lópez-gil and coworkers [ ] , by using an approach based on the modified vaccinia ankara (mva) encoding the rvfv glycoproteins (rmvagngc), extend their previous observations that a single inoculation was sufficient to induce a protective immune response in mice after a lethal viral challenge, which was related to the presence of glycoprotein specific cd + cells and a low-level detection of in vitro neutralizing antibodies. they tested the efficacy and immune response in mice immunized with recombinant mva viruses expressing either glycoprotein gn (rmvagn) or gc (rmvagc) and suggest that, in the absence of serum neutralizing antibodies, protection is strain-dependent and mainly due to the activation of the cellular response against gc epitopes. even more, their data point to the induction of a suboptimal humoral immune response, since disease was exacerbated upon the virus challenge in the presence of rmvagngc or rmvagn immune serum. these results support that gc-specific cellular immunity is an important component that contributes to efficient protection against rvfv infection. the fishing industry is increasingly relevant, and the number of farms has grown exponentially, being a very important source of food. viral hemorrhagic septicemia virus (vhsv), a novirhabdovirus, is one of the worst viral threats to fish farming. vhsv has been isolated from more than fish species across the world, including farmed and free-living marine species. detection of even a single positive sample in a farm has to be notified to the office international des epizooties, and implies the sacrifice of all the farmed fish, thus leading to serious economic losses. non-virion (nv) gene-deleted vhsv (dnv-vhsv) has been postulated as an attenuated virus, as its absence leads to lower induced pathogenicity. in the study by chinchilla et al. [ ] the immune transcriptome profiling in trout infected with dnv-vhsv and wt-vhsv and the pathways involved in immune responses were analyzed in the context of infection. the authors show that dnv-vhsv upregulates more trout-signaling immune related genes and pathways, whereas wt-vhsv maintains more non-regulated genes. therefore, wt-vhsv impairs the activation at short stages of infection of pro-inflammatory, antiviral, proliferation, and apoptosis pathways, delaying innate humoral response and cellular crosstalk, whereas dnv-vhsv promotes the opposite effects, supporting the use of dnv-vhsv as a potential live vaccine candidate. finally, jiménez de oya and coworkers [ ] deeply update current knowledge and available data about the vaccination of birds against the west nile virus (wnv), the worldwide most distributed mosquito-borne flavivirus. although humans and equids can sporadically be infected, birds are the natural host of wnv. when clinical signs arise in birds it is due to multi-organ invasion, mainly in the central nervous system, which can lead to death - h later. nowadays, vaccines are only available for use in equids; thus, availability of avian vaccines would benefit bird populations, both domestic and wild ones. such vaccines could be used in endangered species housed in rehabilitation and wildlife reserves, and in animals located at zoos and other recreational installations, but also in farm birds, and in those that are grown for hunting and restocking activities. even more, controlling wnv infection in birds can also be useful to prevent its spread and limit outbreaks. in their review, jimenez de oya and colleagues comprehensively present the results obtained with commercial and experimental vaccines in domestic and wild avian species, and the possible benefits and drawbacks of bird vaccination against wnv are discussed. the world remains burdened by high morbidity and mortality diseases and, as exemplified by the current devastating pandemic of sars-cov- , new emerging or re-emerging pathogens are likely to spread in the future. hereby, a more comprehensive understanding of the current trends in vaccine development and assessment of the molecular mechanisms and immune responses involved in the elicited responses are essential. in this line, the articles in this issue highlight recent advances in the development of efficient vaccines against rna viruses infecting animals and humans, some of which are zoonotic. different approaches are described from attenuated and recombinant viruses, to peptides, and dna and rna-based candidates, which hopefully will contribute to a better and quick preparedness against rna virus infections. artificial anti-hiv- immunogen comprising epitopes of broadly neutralizing antibodies f , e , and a peptide mimic of vrc discontinuous epitope parenterally administered p -vp * nanoparticle vaccine conferred strong protection against rotavirus diarrhea and virus shedding in gnotobiotic pigs hepatitis c virus vaccine: challenges and prospects broad protection of pigs against heterologous prrsv strains by a gp -mosaic dna vaccine prime/gp -mosaic rvaccinia (vacv) vaccine boost significant interference with porcine epidemic diarrhea virus pandemic and classical strain replication in small-intestine epithelial cells using an shrna expression vector a single dose of dendrimer b t peptide vaccine partially protects pigs against foot-and-mouth disease virus infection mva vectored vaccines encoding rift valley fever virus glycoproteins protect mice against lethal challenge in the absence of neutralizing antibody responses differential immune transcriptome and modulated signalling pathways in rainbow trout infected with viral haemorrhagic septicaemia virus (vhsv) and its derivative non-virion (nv) gene deleted. vaccines , , current progress of avian vaccines against west nile virus funding: this research received no external funding. the author declares no conflict of interest.